S. cerevisiae

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 lead to the establishment of functional chiasmata . The MER2 gene product is essential for meiosis.

J Biol Chem, 1990 Sep 5, 265(25), 15224 - 30
Cloning, sequence analysis, and expression of ligninolytic phenoloxidase genes of the white-rot basidiomycete Coriolus hirsutus; Kojima Y et al.; Two cDNAs and two genomic DNAs coding for the allelic forms of the ligninolytic phenoloxidase were isolated from the white-rot fungus Coriolus hirsutus . The cloned genes were identified in genetic libraries by hybridization screening using four deoxyoligonucleotide probes which corresponded to the partial amino acid sequence of the purified enzyme . Each cDNA encoded the full-length of the phenoloxidase, a protein consisting of 499 amino acid residues, and its putative signal peptide of 21 amino acid residues . The nucleotide sequences of the two alleles differed by 18 single base changes within the open reading frames resulting in one amino acid substitution . Ten small introns interrupted both genomic DNAs as indicated by direct comparison with the corresponding cDNAs . Putative eukaryotic regulatory sequences, "CAAT" and "TATA," were observed in the 5'-flanking region of both genomic DNAs . Each of the phenoloxidase cDNAs was successfully expressed in an active form in Saccharomyces cerevisiae using the useful yeast expression vector YEp51.

Biochemistry, 1990 Sep 4, 29(35), 8020 - 4
Thermal unfolding studies of a leucine zipper domain and its specific DNA complex: implications for scissor's grip recognition; Weiss MA; A newly recognized class of eukaryotic transcription factors is characterized by a bipartite sequence motif, consisting of a C-terminal dimerization region (the leucine zipper) and an N-terminal basic region (which mediates DNA binding) . In studies of isolated leucine zipper peptides, the dimerization region has been characterized as a coiled coil of parallel alpha-helices . To extend these studies to a functional DNA-binding domain, we describe CD studies of the thermal unfolding and refolding of a 58-residue fragment of GCN4, the yeast homologue of the c-Jun protooncoprotein . This fragment, which contains the complete leucine zipper and basic region, retains the DNA-binding properties of the intact protein . The GCN4 DNA-binding domain exhibits two independent helix-coil unfolding transitions . The major transition (midpoint 65 degrees C) is due to dissociation of the dimer in accord with previous studies of an isolated leucine zipper . A novel pretransition in the temperature range 0-40 degrees C is also observed, which reflects partial stabilization of the nascent helix in the basic region . Remarkably, complete folding of the basic region as an alpha-helix requires specific DNA binding, and the protein-DNA complex exhibits a single cooperative unfolding transition . These results support a major feature of the recently proposed "scissor's grip" model of DNA recognition, in which the basic regions extend from the leucine zipper as bifurcating alpha-helical arms.

Biochim Biophys Acta, 1990 Sep 3, 1040(2), 211 - 6
Resonance Raman study on mutant cytochrome P-450 obtained by site-directed mutagenesis; Egawa T et al.; Resonance Raman spectra were observed for the threonine-301 to serine or valine mutant as well as the wild type of rabbit liver microsomal cytochrome P-450 {laurate(omega-1)-hydroxylase} (P-450(omega-1}, which were prepared through site-directed mutagenesis . The high-spin marker resonance Raman (RR) bands became similarly stronger for all the P-450s examined in the oxidized form upon addition of laurate, and the RR spectra in the higher frequency region of the oxidized, reduced and CO-adduct forms did not distinctly differ among the P-450s examined . Nevertheless, the Fe-CO stretching mode (vFe-CO) of the CO adduct exhibited an upshift for the valine mutant, suggesting positional proximity of Thr-301 to bound CO like Thr-252 of P-450cam, in agreement with the expectation from the sequence analysis . The vFe-CO band was shifted to higher frequency upon binding of normal alkyl fatty acids with C10 or longer alkyl chain but little affected by binding of shorter fatty acids.

J Neurol Sci, 1990 Sep, 98(2-3), 185 - 93
Antimitochondrial autoantibodies of primary biliary cirrhosis as a novel probe in the study of 2-oxo acid dehydrogenases in patients with mitochondrial myopathies; Sudoyo H et al.; Autoantibodies present in the autoimmune disease primary biliary cirrhosis react by immunoblotting with four human skeletal muscle mitochondrial antigens of 70 kDa, 52 kDa, 50 kDa and 45 kDa, identified as the lipoate acetyl transferases (E2) of the pyruvate dehydrogenase, component X of E2 pyruvate dehydrogenase, E2 of 2-oxo glutarate dehydrogenase and E2 of branched-chain 2-oxo acid dehydrogenase complexes respectively . These autoantibodies have been employed as a novel probe to study whether there is a defect in the synthesis of the 2-oxo acid dehydrogenase complexes in patients with mitochondrial respiratory chain disorders . The reactive antigens are present normally in four patients with oculomyopathy in whom partial deletions of the mtDNA have been detected, and in two patients with MERRF and MELAS encephalomyopathy . Thus, unlike in the yeast Saccharomyces cerevisiae, there appear to be no regulatory interactions which coordinate the assembly of the mitochondrial respiratory chain with the development of the pyruvate dehydrogenase complex, which plays an important role in regulating the flow of metabolic intermediates to oxidative energy metabolism.

Trends Biochem Sci, 1990 Sep, 15(9), 351 - 4
Self-splicing group II and nuclear pre-mRNA introns: how similar are they?
Jacquier A.
The splicing pathway of pre-mRNA introns bears similarities to that of the group II introns, some members of which undergo self-splicing . The snRNAs may provide the pre-mRNA introns with RNA structures in trans comparable to those available in cis in group II introns . This article examines the available evidence for the hypothesis that the catalysis of these two splicing pathways is fundamentally equivalent.

Yeast, 1990 Sep-Oct, 6(5), 441 - 50
The omnipotent suppressor SUP45 affects nucleic acid metabolism and mitochondrial structure; Pocklington MJ et al.; Yeast (Saccharomyces cerevisiae) strains sensitive to a variety of drugs were used to select for novobiocin-resistant mutants that were simultaneously temperature-sensitive . The mutants remained as sensitive as the parent strains to a wide range of drugs other than novobiocin, and did not exhibit any suppression of suppressible auxotrophic markers . At the non-permissive temperature, the mutant cells arrested mainly as unbudded cells, and were instantly defective in DNA and RNA synthesis, but not protein synthesis . The cloned wild-type gene was identified as SUP45, which has been previously implicated in the translation process . Our results suggest that SUP45 may have a function in addition to, or different from, the one that has been assigned to it previously.

Mutat Res, 1990 Sep, 232(1), 37 - 43
Genotoxic, mutagenic and recombinogenic effects of rauwolfia alkaloids; von Poser G et al.; In the last decade, the possible correlation between the use of reserpine and rauwolfia drugs as antihypertensive agents and breast cancer incidence has been investigated . For the purpose of evaluating the mutagenic and genotoxic effects of these drugs, reserpine and ajmalicine were studied using the SOS Chromotest and the induction of gene conversion, crossing-over and reverse mutation in the yeast diploid strain XS2316 . The results indicated a lack of genotoxic, mutagenic and recombinogenic effects.

Mol Cell Biol, 1990 Sep, 10(9), 4623 - 9
Phosphorylated forms of GAL4 are correlated with ability to activate transcription; Mylin LM et al.; GAL4I, GAL4II, and GAL4III are three forms of the yeast transcriptional activator protein that are readily distinguished on the basis of electrophoretic mobility during sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Phosphorylation accounts for the reduced mobility of the slowest-migrating form, GAL4III, which is found to be closely associated with high-level GAL/MEL gene expression (L . Mylin, P . Bhat, and J . Hopper, Genes Dev . 3:1157-1165, 1989) . Here we show that GAL4II, like GAL4III, can be converted to GAL4I by phosphatase treatment, suggesting that in vivo GAL4II is derived from GAL4I by phosphorylation . We found that cells which overproduced GAL4 under conditions in which it drove moderate to low levels of GAL/MEL gene expression showed only forms GAL4I and GAL4II . To distinguish which forms of GAL4 (GAL4I, GAL4II, or both) might be responsible for transcription activation in the absence of GAL4III, we performed immunoblot analysis on UASgal-binding-competent GAL4 proteins from four gal4 missense mutants selected for their inability to activate transcription (M . Johnston and J . Dover, Proc . Natl . Acad . Sci . USA 84:2401-2405, 1987; Genetics 120;63-74, 1988) . The three mutants with no detectable GAL1 expression did not appear to form GAL4II or GAL4III, but revertants in which GAL4-dependent transcription was restored did display GAL4II- or GAL4III-like electrophoretic species . Detection of GAL4II in a UASgal-binding mutant suggests that neither UASgal binding nor GAL/MEL gene activation is required for the formation of GAL4II . Overall, our results imply that GAL4I may be inactive in transcriptional activation, whereas GAL4II appears to be active . In light of this work, we hypothesize that phosphorylation of GAL4I makes it competent to activate transcription.

J Neurochem, 1990 Sep, 55(3), 989 - 1000
Metabolic stability of 3-O-methyl-D-glucose in brain and other tissues; Jay TM et al.; 3-O-Methyl-D-glucose (methylglucose) is often used to study blood-brain barrier transport and the distribution spaces of hexoses in brain . A critical requirement of this application is that it not be chemically converted in the tissues . Recent reports of phosphorylation of methylglucose by yeast and heart hexokinase have raised questions about its metabolic stability in brain . Therefore, we have re-examined this question by studying the metabolism of methylglucose by yeast hexokinase and rat brain homogenates in vitro and rat brain, heart, and liver in vivo . Commercial preparations of yeast hexokinase did convert methylglucose to acidic products, but only when the enzyme was present in very large amounts . Methylglucose was not phosphorylated by brain homogenates under conditions that converted 97% of {U-14C}glucose to ionic derivatives . When {14C}methylglucose, labeled in either the methyl or glucose moiety, was administered to rats by an intravenous pulse or a programmed infusion that maintained the arterial concentration constant and total 14C was extracted from the tissues 60 min later, 97-100% of the 14C in brain, greater than 99% of the 14C in plasma, and greater than 90% of that in heart and liver were recovered as unmetabolized {14C}methylglucose . Small amounts of 14C in brain (1-3%), heart (3-6%), and liver (4-7%) were recovered in acidic products . Plasma glucose levels ranging from hypoglycemia to hyperglycemia had little influence on the degree of this conversion . The distribution spaces for methylglucose were found to be 0.52 in brain and heart and 0.75 in liver.

Mol Cell Biol, 1990 Sep, 10(9), 4837 - 45
Transcription stimulates homologous recombination in mammalian cells; Nickoloff JA et al.; Transcription stimulates homologous recombination in Saccharomyces cerevisiae and has been implicated in the control of recombinational events during the development of mammalian immune systems . Here, we describe a plasmid-based system in which an inducible promoter from the mouse mammary tumor virus is located upstream of heteroallelic neomycin genes carried on two plasmids . Pairs of plasmids are introduced into Chinese hamster ovary cells by electroporation, and recombination is monitored by scoring colonies resistant to the aminoglycoside G418 . When transcription is induced with dexamethasone, a synthetic glucocorticoid hormone, and double-strand breaks are introduced at mutation sites, recombination is stimulated sixfold over noninduced levels . Inducing transcription in circular substrates or in substrates cleaved at sites distant from the mutations has no detectable effect on recombination between neomycin genes . Results are presented that are consistent with the observed stimulation of recombination occurring before plasmids integrate into the cellular DNA . Our results are discussed in relation to molecular models for extrachromosomal recombination in mammalian cells.

J Virol, 1990 Sep, 64(9), 4169 - 79
Incorporation of chimeric gag protein into retroviral particles; Weldon RA Jr et al.; The product of the Rous sarcoma virus (RSV) gag gene, Pr76gag, is a polyprotein precursor which is cleaved by the viral protease to yield the major structural proteins of the virion during particle assembly in avian host cells . We have recently shown that myristylated forms of the RSV Gag protein can induce particle formation with very high efficiency when expressed in mammalian cells (J . W . Wills, R . C . Craven, and J . A . Achacoso, J . Virol . 63:4331-4343, 1989) . We made use of this mammalian system to examine the abilities of foreign antigens to be incorporated into particles when fused directly to the myristylated Gag protein . Our initial experiments showed that removal of various portions of the viral protease located at the carboxy terminus of the RSV Gag protein did not disrupt particle formation . We therefore chose this region for coupling of iso-1-cytochrome c from Saccharomyces cerevisiae to Gag . This was accomplished by constructing an in-frame fusion of the CYC1 and gag coding sequences at a common restriction endonuclease site . Expression of the chimeric gene resulted in synthesis of the Gag-cytochrome fusion protein and its release into the cell culture medium . The chimeric particles were readily purified by simple centrifugation, and transmission electron microscopy of cells that produced them revealed a morphology similar to that of immature type C retrovirions.

Genet Anal Tech Appl, 1990 Sep, 7(5), 119 - 25
YAC cloning of telomeres; Cheng JF et al.; Human telomeres have been successfully cloned in Saccharomyces cerevisiae by complementing deficient yeast artificial chromosomes (YACs) . This technique allows cloning of DNA sequences that can recognize particular chromosomal ends, and therefore facilitates the mapping of eukaryotic genomes . Although the biology of adopting foreign telomeres in yeast is not fully understood, the cloning system itself seems to be a useful tool for constructing telomeric DNA libraries from higher eukaryotes . Here we describe the techniques that are currently being used in cloning of telomeric DNA.

EMBO J, 1990 Sep, 9(9), 2877 - 84
An early embryonic product of the gene shaggy encodes a serine/threonine protein kinase related to the CDC28/cdc2+ subfamily; Bourouis M et al.; The product(s) of the gene shaggy (sgg) is required for seemingly unrelated events during the development of Drosophila melanogaster . In embryos, maternal and zygotically derived sgg products are required initially to construct a normal syncytial blastoderm and later for normal segmentation . Furthermore, in mutant animals a process of intercellular communication that is required for the segregation of the neural and epidermal lineage during the formation of the central nervous system and the adult peripheral nervous system is disrupted . Here we describe a transcription unit of approximately 40 kb lying within the cloned chromosomal interval 3B1, and provide evidence that it encodes the sgg+ function . Of seven developmentally regulated transcripts that are partially generated by alternative splicing, two seem to be responsible for early sgg activity . Sequence analysis of corresponding cDNA(s) predicts a protein of 514 amino acids with a canonical catalytic domain found in serine/threonine specific protein kinases, linked to an unusual region rich in Gly, Ala and Ser . A search for homologies as well as a comparative study of the kinase catalytic domain with that of other proteins, revealed that the protein kinase domain of sgg is distantly related to the members of the CDC28/cdc2+ subfamily of protein kinases, all of which play cardinal roles in the regulation of the yeast and mammalian cell cycles . Ubiquitous expression of sgg transcripts was found during embryonic stages . A possible role of the sgg protein in a signal transduction pathway necessary for intercellular communication at different stages of development is discussed.

EMBO J, 1990 Sep, 9(9), 2811 - 8
Role of the two activating domains of the oestrogen receptor in the cell-type and promoter-context dependent agonistic activity of the anti-oestrogen 4-hydroxytamoxifen; Berry M et al.; Various oestrogen responsive reporter genes and vectors expressing truncated or chimeric human oestrogen receptors (hER) containing either of the two independent hER transcriptional activation functions (TAF-1 and TAF-2) have been transfected into HeLa cells, chicken embryo fibroblast (CEF) or yeast cells to investigate the agonistic activity of the anti-oestrogen 4-hydroxytamoxifen (OHT) . We demonstrate that the agonistic effect of OHT on the whole hER is due to the cell-type and promoter-context dependent activity of TAF-1 . In similar experiments, we show that the anti-oestrogen, ICI 164,384, does not exhibit any oestrogenic activity and, therefore, acts always as a pure antagonist, even though it does not inhibit the activity of the isolated TAF-1 . We also confirm that the wild type human oestrogen receptor has no ligand independent transcriptional activity . The implications of our results for the variable antagonist/agonist activity of anti-oestrogens in vivo are discussed.

Genomics, 1990 Sep, 8(1), 119 - 26
Spatial normalization of one-dimensional electrophoretic gel images; Drury HA et al.; A strategy for using processed, digitized images of one-dimensional electrophoretic gels to facilitate the analysis of large sets of overlapping clones is described . The images are acquired from fluorescently stained gels or from transilluminated gel photographs using a cooled, solid-state charge-coupled device camera . By employing sets of bands in the size-standard lanes as reference points, all the gel images are spatially normalized to a common reference template . After normalization, lane images from different gels can be compared as though the gels had been electrophoresed under identical, uniform-field conditions . Applications of this procedure to the analysis of a large set of overlapping lambda clones from chromosome VII of Saccharomyces cerevisiae and to the estimation of fragment sizes are illustrated.

Biochem Int, 1990 Sep, 21(6), 1081 - 7
Multiple forms of transketolase; Kuimov A et al.; The multiplicity of transketolase forms, differing in thermostability and separated by phosphocellulose chromatography, has been shown . Using SDS-PAGE, it was found that the mobility of yeast enzyme forms in all cases was identical . Certain forms of transketolase from yeast, rat or pig liver and from some organs of the rabbit were similar with regard to their chromatographic behaviour and thermostability . The form ratio seems to be determined by the physiological state of the organism.

EMBO J, 1990 Sep, 9(9), 2765 - 73
CBP7 codes for a co-factor required in conjunction with a mitochondrial maturase for splicing of its cognate intervening sequence; Muroff I et al.; Maturases are required for the processing of their cognate intervening sequences in the mitochondrial cytochrome beta pre-mRNA . In this paper we characterize a nuclear gene, CBP7, already known to be required for the translation of the cytochrome b transcript; we present further evidence that it is also required as a co-factor in conjunction with the maturases for the efficient processing of their intervening sequences . Results are based on Northern blot analyses which show that in cbp7 mutants with the short cytochrome b gene the maturase encoding intervening sequence fails to be excised although, at the same time, at least wild-type levels of the maturase are present as indicated by antibodies specific to it in Western blot analyses.

Biotechnol Prog, 1990 Sep-Oct, 6(5), 311 - 8
Effect of transcription promoters on the optimal production of secreted protein in fed-batch reactors; Park S et al.; Production of heterologous proteins by yeast secretion imposes additional factors that need to be considered, which do not appear with production by direct expression . These include additional intracellular polypeptide processing dynamics through the secretory organelles and the protein concentration in the culture medium, which is the usual final destination of the product . Optimal control theory is applied to optimize fed-batch production of secreted protein . We maximize an objective function that includes both total production rate and product concentration . A mutant invertase is chosen as the model heterologous secretory protein . Optimal control control strategies have been obtained for the use of two different promoters for the gene transcription, a dere-pressible SUC2 promoter and a strong glycolytic GPD promoter . With the use of the strong GPD promoter, achieving maximum production occurs on the singular arc of maximum specific growth rate . As the object switches to maximum product concentration, operation occurs for longer periods of time at a slow glucose singular arc condition . The optimal control for maximizing protein production with the weak SUC2 promoter requires transitions between high and low glucose concentrations associated with multiple distinct singular arc conditions . For maximum product concentration, the high concentration branches of the singular arc supporting maximum growth rate and maximum secretion rate disappear . Operation stays essentially on the low glucose concentration branch of the singular arc, which maximizes the protein production rate and minimizes the dilution of the broth product concentration.

Science, 1990 Aug 31, 249(4972), 1046 - 9
Presence of a potent transcription activating sequence in the p53 protein; Fields S et al.; The p53 gene is frequently mutated in a wide variety of human cancers . However, the role of the wild-type p53 gene in growth control is not known . Hybrid proteins that contain the DNA binding domain of yeast GAL4 and portions of p53 have been used to show that the p53 protein contains a transcription-activating sequence that functions in both yeast and mammalian cells . The NH2-terminal 73 residues of p53 activated transcription in mammalian cells as efficiently as the herpes virus protein VP16, which contains one of the strongest known activation domains . Combined with previous data that showed p53 is localized to the nucleus and can bind to DNA, these results support the idea that one function of p53 is to activate the transcription of genes that suppress cell proliferation.

Biochim Biophys Acta, 1990 Aug 27, 1050(1-3), 51 - 5
The acidic ribosomal proteins as regulators of the eukaryotic ribosomal activity; Saenz-Robles MT et al.; The acidic proteins, A-proteins, from the large ribosomal subunit of Saccharomyces cerevisiae grown under different conditions have been quantitatively estimated by ELISA tests using rabbit sera specific for these polypeptides . It has been found that the amount of A-protein present in the ribosome is not constant and depends on the metabolic state of the cell . Ribosomes from exponentially growing cultures have about 40% more of these proteins than those from stationary phase . Similarly, the particles forming part of the polysomes are enriched in A-proteins as compared with the free 80 S ribosomes . The cytoplasmic pool of A-protein is considerably high, containing as a whole as much protein as the total ribosome population . These results are compatible with an exchanging process of the acidic proteins during protein synthesis that can regulate the activity of the ribosome . On the other hand, cells inhibited with different metabolic inhibitors produce a very low yield of ribosomes that contain, however, a surprisingly high amount of acidic proteins while the cytoplasmic pool is considerably reduced, suggesting that under stress conditions the ribosome and the A-protein may aggregate, forming complex structures that are not recovered by the standard preparation methods.

Biochim Biophys Acta, 1990 Aug 27, 1050(1-3), 179 - 85
Search of essential parameters for the aminoacylation of viral tRNA-like molecules . Comparison with canonical transfer RNAs; Giege R et al.; Comparative structural and functional results on the valine and tyrosine accepting tRNA-like molecules from turnip yellow mosaic virus (TYMV) and brome mosaic virus (BMV), and the corresponding cognate yeast tRNAs are presented . Novel experiments on TYMV RNA include design of variant genes of the tRNA-like domain and their transcription in vitro by T7 RNA polymerase, analysis of their valylation catalyzed by yeast valyl-tRNA synthetase, and structural mapping with dimethyl sulfate and carbodiimide combined with graphical modelling . Particular emphasis is given to conformational effects affecting the valylation capacity of the TYMV tRNA-like molecule (e.g., the effect of the U43----C43 mutation) . The contacts of the TYMV and BMV RNAs with valyl- and tyrosyl-tRNA synthetases are compared with the positions in the molecules affecting their aminoacylation capacities . Finally, the involvement of the putative valine and tyrosine anticodons in the tRNA-like valylation and tyrosylation reactions is discussed . While an anticodon-like sequence participates in the valine identity of TYMV RNA, this seems not to be the case for the tyrosine identity of BMV RNA despite the fact that the tyrosine anticodon has been shown to be involved in the tyrosylation of canonical tRNA.

Biochim Biophys Acta, 1990 Aug 27, 1050(1-3), 151 - 4
cis-acting sequences involved in the translational control of GCN4 expression; Miller PF et al.; Four short upstream open reading frames (uORFs) in the mRNA leader are required for the translational control of GCN4 expression in response to amino acid availability . Data are reviewed demonstrating that the fourth (3' proximal) uORF is sufficient to establish the repressed levels of GCN4 expression, while the first uORF functions as a positive regulatory element under starvation conditions to stimulate GCN4 translation . Furthermore, positive and negative trans-acting regulatory factors, the activities of which appear to be modulated according to amino acid availability, exert their effects on GCN4 expression through the uORFs . Direct comparison of the uORFs indicates that there are important nucleotide sequence differences between uORF1 and 4, and that these are located primarily around the termination codons of these elements . Recent findings suggest that the sequences that mediate repression of GCN4 expression are complex, but can be overcome under starvation conditions by ribosomes that have previously translated uORF1.

Nucleic Acids Res, 1990 Aug 25, 18(16), 4737 - 42
Transcript levels of the Saccharomyes cerevisiae DNA repair gene RAD23 increase in response to UV light and in meiosis but remain constant in the mitotic cell cycle; Madura K et al.; The RAD23 gene of Saccharomyces cerevisiae is required for excision-repair of UV damaged DNA . In this paper, we determine the location of the RAD23 gene in a cloned DNA fragment, identify the 1.6 kb RAD23 transcript, and examine RAD23 transcript levels in UV damaged cells, during the mitotic cell cycle, and in meiosis . The RAD23 mRNA levels are elevated 5-fold between 30 to 60 min after 37 J/m2 of UV light . RAD23 mRNA levels rise over 6-fold during meiosis at a stage coincident with high levels of genetic recombination . This response is specific to sporulation competent MATa/MAT alpha diploid cells, and is not observed in asporogenous MATa/MATa diploids . RAD23 mRNA levels, however, remain constant during the mitotic cell cycle.

J Biol Chem, 1990 Aug 25, 265(24), 14250 - 5
Cloning, sequence, and expression of a cDNA encoding hamster UDP-GlcNAc:dolichol phosphate N-acetylglucosamine-1-phosphate transferase; Zhu XY et al.; UDP-GlcNAc:dolichol phosphate N-acetylglucosamine-1-phosphate transferase (GPT) catalyzes the initial reaction required for synthesis of dolichol-P-P-oligosaccharides . We report here on the sequence and expression of a full-length cDNA clone encoding hamster GPT . The cDNA predicts a protein of 408 amino acid residues including 10 hydrophobic segments . Several portions of the hamster GPT sequence constituting one-third of the protein have 60% or greater identity with yeast GPT, and one-half of the conserved sequence falls within the hydrophobic segments . In addition, hamster GPT has two copies of a putative dolichol recognition sequence recently identified in three yeast enzymes that interact with dolichol . The protein lacks KDEL or DEKKMP-type carboxyl-terminal ER sorting sequences . When expressed in COS-1 cells, the cDNA causes a 5-7-fold increase of GPT activity in membrane fractions . The activity was completely inhibitable by tunicamycin, and the primary product was shown to be GlcNAc-pyrophosphoryldolichol . This cDNA represents the first enzyme of the dolichol-oligosaccharide biosynthetic pathway to be cloned from a vertebrate source and demonstrates structural homology between the enzymes of the yeast and mammalian pathways.

Science, 1990 Aug 17, 249(4970), 769 - 71
Sequence-specific DNA binding by a short peptide dimer; Talanian RV et al.; A recently described class of DNA binding proteins is characterized by the "bZIP" motif, which consists of a basic region that contacts DNA and an adjacent "leucine zipper" that mediates protein dimerization . A peptide model for the basic region of the yeast transcriptional activator GCN4 has been developed in which the leucine zipper has been replaced by a disulfide bond . The 34-residue peptide dimer, but not the reduced monomer, binds DNA with nanomolar affinity at 4 degrees C . DNA binding is sequence-specific as judged by deoxyribonuclease I footprinting . Circular dichroism spectroscopy suggests that the peptide adopts a helical structure when bound to DNA . These results demonstrate directly that the GCN4 basic region is sufficient for sequence-specific DNA binding and suggest that a major function of the GCN4 leucine zipper is simply to mediate protein dimerization . Our approach provides a strategy for the design of short sequence-specific DNA binding peptides.

Nature, 1990 Aug 16, 346(6285), 663 - 5
Transcriptional activation domain of the muscle-specific gene-regulatory protein myf5; Braun T et al.; The human muscle determination factor myf5, like MyoD and other members of the family of skeletal muscle-specific regulatory proteins, contains a highly conserved putative helix-loop-helix domain . In MyoD this motif is required for the initiation of myogenesis in C3H mouse 10T1/2 fibroblasts and other non-muscle cells as well as for transcriptional activation of muscle genes . High affinity DNA binding of MyoD to regulatory DNA elements in muscle genes requires the formation of heterodimers with ubiquitous helix-loop-helix proteins such as E12 or E47 . To investigate the potential of myf5 as a transcription factor, we have fused the GAL4 DNA-binding domain to various parts of the myf5 protein and analysed the transactivation of a GAL4 reporter plasmid . Here we report that myf5 contains an intrinsic transcriptional activation domain which is distinct from the helix-loop-helix motif . The predominant transactivating effect is associated with the C-terminal half of the myf5 molecule . High-affinity sequence-specific DNA binding of myf5 also requires hetero-oligomeric association with the enhancer-binding protein E12 to confer muscle-specific transactivation.

Nucleic Acids Res, 1990 Aug 11, 18(15), 4453 - 61
Random-breakage mapping, a rapid method for physically locating an internal sequence with respect to the ends of a DNA molecule; Game JC et al.; We describe a method for determining the position of a cloned internal sequence with respect to the ends of a DNA molecule . The molecules are randomly broken at low frequency and the fragments are subjected to electrophoresis . Southern hybridization using the cloned DNA as a probe identifies only those fragments containing the sequence . The size distribution of these fragments is such that two threshold changes in intensity of signal are seen in the smear pattern below the unbroken molecules . The positions of the changes represent the distances from the sequence to each molecular end . The intensity changes arise because the natural ends of the molecules influence the fragment distribution obtained . From once-broken molecules, no fragments can arise that contain a given sequence and are shorter than the distance between that sequence and the nearest molecular end . We tested the method by using x-rays to induce breakage in yeast DNA . Genes of independently known position were mapped within whole chromosomes or Not I restriction fragments using Southern blots from gels of irradiated molecules . We present equations to predict fragment distribution as a function of break-frequency and position of the probed sequence.

Biochemistry, 1990 Aug 7, 29(31), 7303 - 9
Catalytic and conformational changes induced by limited subtilisin cleavage of bovine carboxypeptidase A; Solomon BM et al.; Limited proteolysis of carboxypeptidase A from bovine pancreas with subtilisin Carlsberg generates a stable intermediate, carboxypeptidase S, whose esterase and peptidase activities are increased and decreased, respectively, under standard assay conditions . Carboxypeptidase S was isolated by affinity chromatography . Sequence analysis shows that it is cleaved solely at the Ala154-Gly155 bond . Its enzymatic properties were determined under stopped-flow conditions with Dns-Gly-Ala-Phe and its ester analogue Dns-Gly-Ala-OPhe . For both substrates, the Km values are increased 30-40-fold . The kcat value for peptide hydrolysis is virtually unaffected whereas that for ester hydrolysis is increased 10-fold . The magnitude of the Km effect is equivalent to a loss of 9 kJ/mol of binding energy and likely reflects a disruption of the network of hydrogen bonds that links Tyr-248 and Arg-145 to the backbone carbonyls of Ala-154 and Gly-155 . The difference in kcat effects for the two substrate classes is related to differences in the chemical nature of the rate-determining step . Product release is rate determining for catalytic hydrolysis of ester substrates, and hence, the increase in kcat indicates that dissociation of products is facilitated as a result of the Ala154-Gly155 bond scission . The changes in enzymatic activity accompanying limited proteolysis are due to conformational alterations in the vicinity of the active center of the molecule . The affinity of a monoclonal antibody, mAb 100, directed toward the antigenic determinant located between residues 209 and 218 in carboxypeptidase A is diminished considerably for carboxypeptidase S.(ABSTRACT TRUNCATED AT 250 WORDS)

Science, 1990 Aug 3, 249(4968), 546 - 9
Antibody-mediated activation of Drosophila heat shock factor in vitro; Zimarino V et al.; Eukaryotic cells respond to elevated temperatures by rapidly activating the expression of heat shock genes . Central to this activation is heat shock-inducible binding of the transcriptional activator, termed heat shock factor (HSF), to common regulatory elements, which are located upstream of all heat shock genes . The DNA binding activity of the inactive form of Drosophila HSF was induced in vitro by treatment with polyclonal antibodies to the purified, in vivo-activated factor . This finding, together with observations that high temperature and low pH activate HSF binding in vitro, suggests that the inactive form of HSF can directly recognize and transduce the heat shock signal without undergoing a covalent modification of protein structure.

J Bacteriol, 1990 Aug, 172(8), 4522 - 8
sti35, a stress-responsive gene in Fusarium spp; Choi GH et al.; A stress-induced mRNA was identified in the phytopathogenic fungus Fusarium oxysporum f . sp . cucumerinum . Treatment of the fungus with ethanol resulted in the induction of a major mRNA species encoding a protein of approximate Mr 37,000 . A full-length cDNA clone of the induced message was obtained . RNA blot analysis indicated that the mRNA was induced by various other stresses, including treatment with copper(II) chloride and heat (37 degrees C) . However, it was not greatly induced by treatment with phaseollinisoflavan, an antifungal isoflavonoid produced by Phaseolus vulgaris (French bean) . In contrast, phaseollinisoflavan induced the homologous mRNA in the related bean pathogen Fusarium solani f . sp . phaseoli . A genomic clone of the F . solani f . sp . phaseoli gene was obtained, and both this and the cDNA clone from F . oxysporum f . sp . cucumerinum were sequenced . The latter indicated an open reading frame of 320 codons encoding a 34,556-dalton polypeptide . The corresponding reading frame in F . solani f . sp . phaseoli was 324 codons, 89% identical to the F . oxysporum f . sp . cucumerium sequence, and was interrupted by a short intron . The gene was designated sti35 (stress-inducible mRNA) . Although computer homology searches were negative, the cloned gene was observed to cross-hybridize to DNAs of other filamentous fungi, Saccharomyces cerevisiae, and soybean . Thus, sti35 appears to be a common gene among a variety of eucaryotes.

Indian J Exp Biol, 1990 Aug, 28(8), 762 - 5
Microcalorimetric studies on cell survival and repair after UV induced damage; Sharma M et al.; Rate of heat production during cell proliferation following UV-irradiation of respiratory-deficient yeast cells was measured as a function of time (p-t curve) in a batch microcalorimeter . Following observations were made: (a) All growing cell cultures showed 3 distinct phases of heat production namely lag, exponential and declining phases of rate of heat production . (b) Duration of the lag phase is inversely proportional to the number of cells capable of proliferation . (c) After UV-irradiation, lag phase increased in a dose dependent manner . (d) Liquid-holding reactivation increased the surviving fraction and reduced the lag phase in p-t curves . Presence of 2-deoxy-D-glucose during liquid-holding prevented the reduction in lag phase due to the inhibition of repair processes.

J Parasitol, 1990 Aug, 76(4), 577 - 8
Inhibition of leukocyte function by serum from patients with trichinellosis; Bruschi F et al.; Modification of leukocytic function has been reported in only a few human parasitic diseases . In this study we evaluated the effects of the sera from patients infected with Trichinella on chemotactic and phagocytic responses in leukocytes . Leukocyte chemotaxis was tested by the agarose method and phagocytosis by the technique of Yamamura, modified for Saccharomyces cerevisiae . Sera were acquired from patients during a trichinellosis outbreak that occurred in northern Italy in 1986 . The parasite was isolated from 1 patient and isoenzymatically typed as Trichinella sp . 3, a new taxon, previously considered Trichinella nelsoni . The results indicated that sera from Trichinella-infected humans inhibited both chemotaxis and phagocytic responses in leukocytes . These findings suggest the existence of serum factor(s) in trichinellosis patients that modify host leukocytic functions . The source and nature of active serum components and the mechanism by which they modulate leukocyte function remain to be clarified.

Mol Cell Biol, 1990 Aug, 10(8), 4420 - 3
Meiotic recombination between dispersed repeated genes is associated with heteroduplex formation; Nag DK et al.; In Saccharomyces cerevisiae, recombination events occurring between allelic genes located on homologous chromosomes are often associated with heteroduplex formation . We found that recombination events between repeated genes on nonhomologous chromosomes (ectopic events) are also associated with the formation of heteroduplexes, indicating that classical and ectopic recombination events involve similar mechanisms.

Mol Cell Biol, 1990 Aug, 10(8), 4375 - 8
Translational activation of GCN4 mRNA in a cell-free system is triggered by uncharged tRNAs; Krupitza G et al.; Translation of GCN4 mRNA is activated when yeast cells are grown under conditions of amino acid limitation . In this study, we established the conditions through which translation of the GCN4 mRNA could be activated in a homologous in vitro system . This activation paralleled the in vivo situation: it required the small open reading frames located in the 5' untranslated region of the GCN4 mRNA, and it was coupled with reduced rates of 43S preinitiation complex formation . Translational derepression in vitro was triggered by uncharged tRNA molecules, demonstrating that deacylated tRNAs are more proximal signals for translational activation of the GCN4 mRNA.

Mol Cell Biol, 1990 Aug, 10(8), 4256 - 65
A nucleosome-positioning sequence is required for GCN4 to activate transcription in the absence of a TATA element; Brandl CJ et al.; In the gal-his3 hybrid promoter his3-GG1, the yeast upstream activator protein GCN4 stimulates transcription when bound at the position normally occupied by the TATA element . This TATA-independent activation by GCN4 requires two additional elements in the gal enhancer region that are distinct from those involved in normal galactose induction . Both additional elements appear to be functionally distinct from a classical TATA element because they cannot be replaced by the TFIID-binding sequence TATAAA . One of these elements, termed Q, is essential for GCN4-activated transcription and contains the sequence GTCAC CCG, which overlaps (but is distinct from) a GAL4 binding site . Surprisingly, relatively small increases in the distance between Q and the GCN4 binding site significantly reduce the level of transcription . The Q element specifically interacts with a yeast protein (Q-binding protein {QBP}) that may be equivalent to Y, a protein that binds at a sequence that forms a constraint to nucleosome positioning . Analysis of various deletion mutants indicates that the sequence requirements for binding by QBP in vitro are indistinguishable from those necessary for Q activity in vivo, strongly suggesting that QBP is required for the function of this TATA-independent promoter . These results support the view that transcriptional activation can occur by an alternative mechanism in which the TATA-binding factor TFIID either is not required or is not directly bound to DNA . In addition, they suggest a potential role of nucleosome positioning for the activity of a promoter.

Mol Cell Biol, 1990 Aug, 10(8), 3994 - 4006
Identification and characterization of the poly(A)-binding proteins from the sea urchin: a quantitative analysis; Drawbridge J et al.; Poly(A)-binding proteins (PABPs) are the best characterized messenger RNA-binding proteins of eucaryotic cells and have been identified in diverse organisms such as mammals and yeasts . The in vitro poly(A)-binding properties of these proteins have been studied intensively; however, little is known about their function in cells . In this report, we show that sea urchin eggs have two molecular weight forms of PABP (molecular weights of 66,000 and 80,000) . Each of these has at least five posttranslationally modified forms . Both sea urchin PABPs are found in approximately 1:1 ratios in both cytoplasmic and nuclear fractions of embryonic cells . Quantification in eggs and embryos revealed that sea urchin PABPs are surprisingly abundant, composing about 0.6% of total cellular protein . This is 50 times more than required to bind all the poly(A) in the egg based on the binding stoichiometry of 1 PABP per 27 adenosine residues . We found that density gradient centrifugation strips PABP from poly(A) and therefore underestimates the amount of PABP complexed to poly(A)+ RNA in cell homogenates . However, large-pore gel filtration chromatography could be used to separate intact poly(A)-PABP complexes from free PABP . Using the gel filtration method, we found that the threefold increase in poly(A) content of the egg after fertilization is paralleled by an approximate fivefold increase in the amount of bound PABP . Furthermore, both translated and nontranslated poly(A)+ RNAs appear to be complexed to PABP . As expected from the observation that PABPs are so abundant, greater than 95% of the PABP of the cell is uncomplexed protein.

Anal Biochem, 1990 Aug 1, 188(2), 366 - 73
Covalent immobilization of proteins and peptides for solid-phase sequencing using prepacked capillary columns; Liang SP et al.; The use of prepacked capillary columns for immobilizing proteins and peptides for solid-phase Edman degradation is described . Capillary tubes with an internal volume of about 30 microliters are filled with glass beads bearing isothiocyanato groups (DITC-glass), aminophenyl groups (AP-glass), or aminoethylaminopropyl groups (AEAP-glass) and are sealed with porous plugs . Proteins or peptides in appropriate buffers are introduced into the columns by capillary action and are covalently coupled to the glass beads, either by reaction of lysine side-chain amino groups with DITC-glass, by carbodi-imide-mediated reaction of carboxyl groups with AP-glass, or by reaction of homoserine lactone groups with AEAP-glass . Optimization of attachment conditions is described . The capillary columns are loaded into the sequencer and, when sequencing has been completed, are discarded . This technique greatly simplifies polypeptide immobilization and is suitable for microsequencing (less than 50-1000 pmol) or macrosequencing (1-50 nmol).

Mol Gen Genet, 1990 Aug, 223(1), 49 - 57
Deletion analysis of domain independence in the TRP1 gene product of Neurospora crassa; Walker MS et al.; The trifunctional TRP1 gene from Neurospora crassa (N-TRP1) was subcloned into the yeast-Escherichia coli shuttle vector YEp13 and expressed in Saccharomyces cerevisiae . The three activities of the N-TRP1 gene product were detected in yeast mutants that lacked either N-(5'-phosphoribosyl) anthranilate (PRA) isomerase or both the glutamine amidotransferase function of anthranilate synthase and indole-3-glycerol phosphate (InGP) synthase . The protein was detected on immunoblots only as the full length 83 kda product indicating that the trifunctional gene product was expressed in yeast primarily in a fully active, undegraded form . By placing the subcloned N-TRP1 gene under the control of the inducible PHO5 promoter from yeast, the expression of all three activities was increased to more than ten fold that of wild-type yeast and the overproduced protein could be visualized by SDS-polyacrylamide gel electrophoresis of crude extract and Coomassie Blue staining . Using the expression system described the effect of selective deletion of regions of the coding sequence of the N-TRP1 gene on expression of the three activities was tested . Expression of either the F- or C-domains, catalyzing respectively the PRA isomerase or InGP synthase activities, did not depend on the presence of the other domain in the active polypeptide . Furthermore, normal dimer formation occurred with a protein active for InGP synthase in a deletion derivative lacking most of the PRA isomerase domain, ruling out the hypothesis that interaction between the active site regions for PRA isomerase and InGP synthase accounted for dimer formation in the trifunctional product.

EMBO J, 1990 Aug, 9(8), 2555 - 61
Unexpected flexibility in an evolutionarily conserved protein-RNA interaction: genetic analysis of the Sm binding site; Jones MH et al.; Human autoantibodies of the Sm specificity recognize a conserved set of proteins found in the U class small nuclear ribonucleoproteins (U snRNPs), key trans-acting factors involved in the splicing of mRNA precursors . The Sm protein binding site in U snRNAs is unusual because of its single-stranded nature and its simple sequence motif (AU5-6GPu) . Here we use genetics to probe this specific protein-RNA interaction by saturation mutagenesis of the Sm binding site of the Saccharomyces cerevisiae U5 snRNA . The assay system used to analyze these mutations takes advantage of a conditionally expressed U5 gene which does not support growth under non-permissive conditions; U5 genes containing Sm site mutations were tested for their ability to complement this lethal phenotype . Our results indicate that the Sm binding site is remarkably tolerant to mutation despite its high degree of conservation, suggesting that relatively few or redundant specific contacts can determine recognition of single-stranded RNA by protein . A complementary biochemical analysis of these mutants demonstrates that integrity of the Sm site is necessary for snRNP stability in vivo and in vitro.

Dev Biol, 1990 Aug, 140(2), 281 - 90
Vitellogenesis in Drosophila: sequestration of a yolk polypeptide/invertase fusion protein into developing oocytes; Yan YL et al.; The mechanism of yolk deposition into developing oocytes of Drosophila was investigated by following the fate of a reporter protein fused to a vitellogenin, or yolk polypeptide (YP) . Embryos were transformed with a hybrid gene consisting of the promotor and amino terminal 430 codons of the Yp2 gene fused to the cytoplasmic form of the invertase gene from the yeast Saccharomyces cerevisiae . RNA hybridization experiments with established lines of transformed flies showed that the hybrid gene was expressed in female fat bodies and ovaries but not in any male cells . Immunoblotting and endoglycosidase digestion showed that the hybrid protein was secreted from fat body cells via the secretory pathway, transported in hemolymph, and sequestered into developing oocytes . Transfusion experiments with hemolymph and pure invertase showed that sequestration of invertase depended on its attachment to YP . Immunocytochemistry demonstrated that the hybrid protein became localized in yolk granules as oocytes developed . Females homozygous for the fusion gene are generally sterile; their eggs containing the hybrid protein often collapse and their embryos fail to develop, suggesting that the structure of the yolk polypeptides is important for embryonic development . These experiments show that YP2 carries structural information sufficient to direct a reporter protein from fat body cells, through the hemolymph, and into the yolk granules of developing oocytes . This work provides a means of identifying the features of yolk polypeptides that are responsible for their deposition into yolk during oogenesis.

Biochemistry, 1990 Jul 31, 29(30), 7089 - 94
Isolation and characterization of a novel eukaryotic monofunctional NAD(+)-dependent 5,10-methylenetetrahydrofolate dehydrogenase; Barlowe CK et al.; An NAD(+)-dependent 5,10-methylenetetrahydrofolate (THF) dehydrogenase has been purified to homogeneity from the yeast Saccharomyces cerevisiae . The purified enzyme exhibits a final specific activity of 5.4 units mg-1 and is represented by a single protein of apparent Mr = 33,000-38,000 as determined by sodium dodecyl sulfate gel electrophoresis . A native Mr = 64,000 was determined by gel filtration, suggesting a homodimer subunit structure . Cross-linking experiments with dimethyl suberimidate confirmed the dimeric structure . The enzyme is specific for NAD+ and is not dependent on Mg2+ for activity . The forward reaction initial velocity kinetics are consistent with a sequential reaction mechanism . With this model, Km values for NAD+ and (6R,S)-5,10-methylene-THF are 1.6 and 0.06 mM, respectively . In contrast to all other previously described eukaryotic 5,10-methylene-THF dehydrogenases, the purified enzyme is apparently monofunctional, with undetectable 5,10-methenyl-THF cyclohydrolase and 10-formyl-THF synthetase activities . Subcellular fractionation of yeast indicates the enzyme is cytoplasmic, with no NAD(+)-dependent 5,10-methylene-THF dehydrogenase detectable in mitochondria . The activity was found in all yeast strains examined, at all stages of growth from the lag phase through the stationary phase.

Nature, 1990 Jul 26, 346(6282), 390 - 4
Arabidopsis thaliana contains two genes for TFIID; Gasch A et al.; The general transcription initiation factor TFIID plays a primary part in the activation of eukaryotic genes transcribed by RNA polymerase II . Binding of TFIID to the TATA box initiates the assembly of other general transcription factors as well as RNA polymerase II at the promoter resulting in a preinitiation complex capable of accurate transcription initiation in vitro . Human TFIID has been shown to interact with various regulatory factors . The observation that stimulation of transcription by different trans-acting factors is mediated through distinct TATA elements led to the suggestion that different types of TFIID may exist in yeast, humans and plants . Here we report the cloning and characterization of two distinct TFIID complementary DNA clones from Arabidopsis thaliana . Furthermore, we have found that TFIID from Arabidopsis and other organisms shows homology to helix-loop-helix proteins.

Biochim Biophys Acta, 1990 Jul 25, 1018(2-3), 229 - 33
The role of contact sites between inner and outer mitochondrial membrane in energy transfer; Nicolay K et al.; Three functions have been suggested to be localized in contact sites between the inner and the outer membrane of mitochondria from mammalian cells: (i) transfer of energy from matrix to cytosol through the action of peripheral kinases; (ii) import of mitochondrial precursor proteins; and (iii) transfer of lipids between outer and inner membrane . In the contact site-related energy transfer a number of kinases localized in the periphery of the mitochondrion play a crucial role . Two examples of such kinases are relevant here: (i) hexokinase isoenzyme I which is capable of binding to the outer aspect of the outer membrane; and (ii) the mitochondrial isoenzyme of creatine kinase which is localized in the intermembrane space . Recently, evidence was presented that both hexokinase and creatine kinase are preferentially localized in contact sites (Adams, V . et al . (1989) Biochim . Biophys . Acta 981, 213-225) . The aim of the present experiments was two-fold . First, to establish methods which enable the bioenergetic aspects of energy transfer mediated by kinases in contact sites to be measured . In these experiments emphasis was on hexokinase, while 31P-NMR was the major experimental technique . Second, we wanted to develop methods which can give insight into factors playing a role in the formation of contact sites involved in energy transfer . In the latter approach, mitochondrial creatine kinase was studied using monolayer techniques.

Biochim Biophys Acta, 1990 Jul 25, 1018(2-3), 185 - 9
Control processes in oxidative phosphorylation: kinetic constraints and stoichiometry; Rigoulet M; Control processes in oxidative phosphorylation have been studied in three experimental models . (1) In isolated yeast mitochondria, external ATP is a regulatory effector of cytochrome-c oxidase activity . In phosphorylating or uncoupling states, the relationships between respiratory rate and delta mu H+, and the respiratory rate and cytochrome-c oxidase reduction level are dependent on this kinetic regulation . (2) In rat liver mitochondria, the response of the respiratory rate to uncoupler addition is age-dependent: liver mitochondria isolated from young rats maintain a greater delta mu H+ than liver mitochondria isolated from adults, with the same respiratory rate obtained with the same concentration of uncoupler . This behaviour is linked to redox proton pump properties, i.e., to the degree of intrinsic uncoupling induced by uncoupler addition . (3) The effect of almitrine, a new kind of ATPase/ATPsynthase inhibitor, was studied in mammalian mitochondria . (i) Almitrine inhibits oligomycin-sensitive ATPase - it decreases the ATPase/O value without any change in delta mu H+; (ii) almitrine increased the mechanistic H+/ATP stoichiometry of ATPase/ATPsynthase; (iii) almitrine-induced changes in H+/ATPase stoichiometry depend on the flux magnitude through ATPase . These results are discussed in terms of the following interdependent parameters; flux value, force, pump efficiency and control coefficient.

Biochemistry, 1990 Jul 17, 29(28), 6619 - 25
Crystallographic analysis of the complex between triosephosphate isomerase and 2-phosphoglycolate at 2.5-A resolution: implications for catalysis; Lolis E et al.; The binding of the transition-state analogue 2-phosphoglycolate to triosephosphate isomerase from yeast has been investigated crystallographically . An atomic model of the enzyme-inhibitor complex has been refined against data to 2.5-A resolution to a final R factor of 0.18 . The interactions between the inhibitor and enzyme have been analyzed . The inhibitor forms hydrogen bonds to the side chains of His 95 and Glu 165 . The latter hydrogen bond confirms that Glu 165 is protonated upon PGA binding . The structure of the complexed enzyme has been compared to that of the unbound form of the enzyme, and conformational changes have been observed: the side chain of Glu 165 moves over 2 A and a 10-residue flexible loop moves over 7 A to close over the active site . Spectroscopic results of phosphoglycolic acid binding to triosephosphate isomerase that have been amassed over the years are also explained in structural terms . The implications for catalysis are noted.

Biochem Biophys Res Commun, 1990 Jul 16, 170(1), 187 - 93
Primary structure of the non-transcribed spacer region and flanking sequences of the ribosomal DNA of Neurospora crassa and comparison with other organisms; Dutta SK et al.; The non-transcribed spacer (NTS) region of the rDNA of Neurospora crassa contains the transcription regulatory sequences . We isolated a 3.4 kb EcoRI fragment from wild type N.crassa rDNA and cloned in the plasmid pBR325 at the EcoRI site . The insert contains the entire NTS region along with the flanking sequences . Nucleotide sequencing of 3592 nt shows many interesting features like: the NTS region is rich in G+C content (65% G+C); it contains the conserved rRNA processing site 6 (with the nucleotide sequence motif GGTGCGAGAACCCGG, from nt residue 226 to 240, a characteristic feature of most eukaryotic rDNA nontranscribed spacer region); and the NTS region also contains the transcription termination site with the representative Sal I box (from nt residue 1469 to 1477) . The potential sequences of transcription termination site are located 288 nt downstream from the end of 26S rRNA gene, and another sequence motif CTTCCT (from nt residue 512 to 517) shows similarity with the human transcription termination site T-2 of its pre-rRNA . Nucleotide sequence homology matrix analysis suggests its relatedness to Saccharomyces cerevisiae and not to human, mouse, rat, Drosophila, Xenopus, wheat, rice and cucumber NTS region . The phylogenetic implication of the NTS region and exploitation of N.crassa NTS rDNA clone to correlate the otherwise indistinguishable species of Neurospora and the correlation with other organisms has been discussed . To the best of our knowledge this is the first report where the nucleotide sequence of the entire NTS region of a filamentous fungus has been determined.

Nature, 1990 Jul 12, 346(6280), 147 - 52
Evidence for interaction of different eukaryotic transcriptional activators with distinct cellular targets; Martin KJ et al.; The adenovirus E1a protein (E1a), a potent transcription activator, contains a transcriptional activating region . Compared with previously described cellular and viral activators, E1a's activating region has unusual structural properties . It seems that E1a's activating region interacts with a cellular target not required for the function of transcriptional activators with 'acidic' activating regions . By contrast, the target of an acidic activating region is required both by acidic activators and by E1a . It is proposed that the cellular target of E1a's activating region is an 'adaptor' that allows E1a to interact with the basic transcriptional machinery.

Biochemistry, 1990 Jul 10, 29(27), 6393 - 400
Substitution of Tyr254 with Phe at the active site of flavocytochrome b2: consequences on catalysis of lactate dehydrogenation; Dubois J et al.; A role for Tyr254 in L-lactate dehydrogenation catalyzed by flavocytochrome b2 has recently been proposed on the basis of the known active-site structure and of studies that had suggested a mechanism involving the initial formation of a lactate carbanion {Lederer, F., & Mathews, F.S . (1987) in Flavins and Flavoproteins, Proceedings of the Ninth International Symposium, Atlanta, GA, 1987 (Edmondson, D.E., & McCormick, D.B., Eds.) pp 133-142, Walter de Gruyter, Berlin} . This role is now examined after replacement of Tyr254 with phenylalanine . The kcat is decreased about 40-fold, Km for lactate appears unchanged, and the mainly rate-limiting step is still alpha-hydrogen abstraction, as judged from the steady-state deuterium isotope effect . Modeling studies with lactate introduced into the active site indicate two possible substrate conformations with different hydrogen-bonding partners for the substrate hydroxyl . If the hydrogen bond is formed with Tyr254, as was initially postulated, the mechanism must involve removal by His373 of the C2 hydrogen, with carbanion formation . If, in the absence of the Tyr254 phenol group, the hydrogen bond is formed with His373 N3, the substrate is positioned in such a way that the reaction must proceed by hydride transfer . Therefore the mechanism of the Y254F enzyme was investigated so as to distinguish between the two mechanistic possibilities . 2-Hydroxy-3-butynoate behaves with the mutant as a suicide reagent, as with the wild-type enzyme . Similarly, the mutant protein also catalyzes the reduction and the dehydrohalogenation of bromopyruvate under transhydrogenation conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

J Mol Biol, 1990 Jul 5, 214(1), 55 - 72
Synaptic intermediates promoted by the FLP recombinase; Amin AA et al.; We have devised a novel assay to trap nucleoprotein synaptic intermediates of the FLP recombination reaction . DNase I footprinting analysis of these intermediates indicates that synapsis is mediated by protein-protein interactions between FLP molecules bound to each FLP recombination target (FRT) site . Under certain conditions we have observed a synaptic structure in which the FRT sites have come together in an aberrant arrangement . Although our analysis shows that homology between the core sequences of the sites is not a prerequisite for synapsis, the data suggest that homology between cores dictates the directionality of the reaction . Many of the intermediates contain a Holliday junction indicating that the FLP protein has catalysed strand exchanges between the FRT sites . The general scheme of the assay should prove useful to analyse nucleoprotein intermediates in other site-specific recombination systems, and to investigate protein-protein and protein-DNA interactions in intermediates important for DNA replication and transcription.

Gene, 1990 Jul 2, 91(1), 131 - 4
Activity and thermal stability of genetically truncated forms of Aspergillus glucoamylase; Evans R et al.; Glucoamylase (GA) from Aspergillus awamori (EC 3.2.1.3) is a secreted starch hydrolase with a large catalytic domain (aa 1-440), a starch-binding domain (aa 513-616), and a highly O-glycosylated region of 72 aa of unknown function that links the catalytic and starch-binding domains . We have genetically engineered a series of truncated forms of GA to determine how much of the highly O-glycosylated region is necessary for the activity or stability of GAII, a fully active form of the enzyme that lacks the starch-binding domain . Mutations were made by inserting stop-codon linkers into restriction sites within the coding region of the GA gene, and mutated genes were expressed in Saccharomyces cerevisiae for analysis of the truncated enzymes . Our results show that up to 30 aa from the C-terminal end of GAII can be deleted with little effect on the activity, thermal stability, or secretion of the enzyme . Further deletions resulted in diminution or loss of enzyme activity on starch plates, and loss of detectable enzyme in culture supernatants, indicating that these residues are essential for GAII function.

J Histochem Cytochem, 1990 Jul, 38(7), 957 - 63
Immunocytochemical visualization of the Golgi apparatus in several species, including human, and tissues with an antiserum against MG-160, a sialoglycoprotein of rat Golgi apparatus; Croul S et al.; We used a monoclonal antibody (10A8), derived from mice immunized with fractions enriched in Golgi apparatus of rat brain neurons, to isolate an intrinsic membrane sialoglycoprotein of 160 KD from rat brain . By immunoelectron microscopy the sialoglycoprotein, named MG-160, was localized in medical cisternae of the Golgi apparatus of neurons, glia, adenohypophysis, and cultured rat pheochromocytoma (PC 12) . The monoclonal antibody (MAb) reacted only with rat tissues . Because the epitope(s) recognized by a monoclonal antibody may be restricted, localization of an antigen by a single MAb may not reflect the extent of the distribution of antigen in various species and tissues . Therefore, to further investigate the presence and localization of MG-160 or of an antigenically related protein in several species and tissues, we used a polyclonal antiserum raised against MG-160 purified by antibody (10A8) affinity chromatography . Immunoblots of crude microsomal fractions from rat brain probed with the antiserum against MG-160 showed two to three prominent bands of approximately 160, 150, and 68 KD . Immunoblots of crude microsomal fractions from human, chicken, and frog brains showed prominent bands of 130-140 and 68 KD . Immunoblots of crude membrane fractions from Saccharomyces cerevisiae showed prominent bands of approximately 110-120 and 80 KD . Light microscopic immunocytochemical studies with frog, chicken, mouse, rat, rabbit, bovine, and human brains and with several other rat and human tissues showed a staining pattern consistent with the Golgi apparatus . Immunoelectron microscopy with rat and human brain and with rat myocardium and pituitary showed prominent and exclusive staining of cis, medial, and occasionally trans cisternae of the Golgi apparatus . The cisternae of the trans Golgi network were not stained . These findings are consistent with the hypothesis that a polypeptide related to MG-160 is present in the Golgi apparatus of several tissues in human, rodents, chicken, and frog and possibly in Saccharomyces cerevisiae . The antiserum to MG-160 represents a reliable reagent for immunohistochemical visualization of the Golgi apparatus in brain and several other human tissues obtained at autopsy, fixed with Bouin's, and embedded in paraffin.

DNA Cell Biol, 1990 Jul-Aug, 9(6), 415 - 24
cDNA sequence of two distinct pituitary proteins homologous to Kex2 and furin gene products: tissue-specific mRNAs encoding candidates for pro-hormone processing proteinases; Seidah NG et al.; Based on the concept of sequence conservation around the active sites of serine proteinases, polymerase chain reaction applied to mRNA amplification allowed us to obtain a 260-bp probe which was used to screen a mouse pituitary cDNA library . The primers used derived from the cDNA sequence of active sites Ser* and Asn* of human furin . Two cDNA sequences were obtained from a number of positive clones . These code for two similar but distinct structures (mPC1 and mPC2), each being homologous to yeast Kex2 and human furin . In situ hybridization (mPC1) and Northern blots (mPC1 = 3.0 kb and mPC2 = 2.8 and 4.8 kb) demonstrated tissue and cellular specificity of expression, only within endocrine and neuroendocrine cells . These data suggest that mPC1 and mPC2 represent prime candidates for tissue-specific pro-hormone converting proteinases.

Yeast, 1990 Jul-Aug, 6(4), 331 - 43
The hydrophilic and acidic N-terminus of the integral membrane enzyme phosphatidylserine synthase is required for efficient membrane insertion; Sperka-Gottlieb C et al.; The product of the yeast CHO 1 gene, phosphatidylserine synthase (PSS), is an integral membrane protein that catalyses a central step in cellular phospholipid biosynthesis . A 1.2 kb fragment containing the regulatory and structural components of the CHO 1 gene was sequenced . Transcription initiation in wild-type cells was found to occur between -1 and -15 relative to the first ATG of a large open reading frame capable of encoding a 30,804 molecular weight protein . This translation initiation site was active in vivo and in vitro in a heterologous system . In both cases it supported production of a protein of approximately 30,000 molecular weight . A second potential translation initiation site was detected 225 or 228 bases downstream from the first ATG . This second site was active in vitro where it supported production of a protein of 22,400 molecular weight . A subclone, lacking the 5' regulatory region and the sequence encoding the first 12 amino acids of the large open reading frame, allowed translation in vivo starting at the second ATG . The resulting protein was 22,000 molecular weight, lacked the 74 N-terminal amino acids and was capable of complementing the choline auxotrophy of a cho 1 null-mutant . In transformants carrying this construct, PSS activity and 22 kDa protein was found to be associated with membrane fractions corresponding to mitochondria and endoplasmic reticulum . However, most of the truncated PSS protein accumulated in the cytosol in an inactive form . A hybrid-protein containing the 63 N-terminal amino acids of PSS fused to mouse dihydrofolate reductase was found exclusively in the cytosol when expressed in wild-type yeast . Thus, the hydrophilic, highly acidic N-terminus of PSS is required for efficient membrane insertion but does not appear to contain sequences required for a targeting to the membrane compartment.

Patol Fiziol Eksp Ter, 1990 Jul-Aug, (4), 11 - 3
{Low-molecular peptides in food as factors altering the resistance of rats to emotional stress}; Dmitrieva NV et al.; The authors studied the resistance of animals to emotional stress when they were given in the diet, as a source of protein, casein and a protein fraction composed of a mixture of amino acids and low-molecular peptides (18 and 2.7%) obtained by reprocessing Saccharomyces cerevisiae yeasts, biomass . The ECG, rheovasogram, systolic arterial pressure, and respiratory rate were studied in rats given a diet containing 18 and 2.7% low-molecular peptides (controls) and in the same animals after 24-hour immobilization . Addition of low-molecular peptides in the indicated concentration to the diet reduced the resistance of animals to emotional stress.

Biotechnol Prog, 1990 Jul-Aug, 6(4), 283 - 5
Precipitation of nucleic acids with poly(ethyleneimine); Cordes RM et al.; Removal of nucleic acids from cell extracts is a common early step in downstream processing for protein recovery . We report on the precipitation of nucleic acids from a homogenate of Saccharomyces cerevisiae by addition of the cationic polyelectrolyte poly(ethyleneimine) (PEI), focusing on the effect of PEI dosage on particle size, protein loss, and extent of nucleic acid removal in both batch and continuous mode . Better than 95% removal of nucleic acids from yeast homogenates was achieved by means of precipitation with PEI with protein losses of approximately 15% with or without previous removal of cell debris . The coprecipitated protein is predominately large molecular weight material and exhibits both low and high isoelectric points . Such treatment does not aggregate the cell debris; size distribution of the precipitated particles from a continuous precipitator is very similar to that for protein precipitation.

Enzyme Microb Technol, 1990 Jul, 12(7), 539 - 45
Mechanical stability and diffusional resistance of a polymeric gel used for biocatalyst immobilization; de Alteriis E et al.; The mechanical strength of gelatin gels insolubilized by crosslinking with formaldehyde was measured at various gelatin percentages and formaldehyde-to-gelatin ratios . This property was shown to be related to the characteristic sponge-like structure of the insolubilized gelatin gel, a structure that unexpectedly is also responsible for the resistance to substrate and product diffusion . A comparison between immobilizates of invertase and invertase-active yeast cells prepared with different gelatin concentrations showed that the enzyme, in contrast to cells, is deeply involved in the gel insolubilization process . The catalytic behavior of agar, kappa-carrageenan, alginate, and gelatin immobilizates was compared under the same conditions of cell loading.

Cell, 1990 Jun 29, 61(7), 1187 - 97
Mechanism of transcriptional activation by Sp1: evidence for coactivators; Pugh BF et al.; In reconstituted reactions, Sp1 stimulates transcription at TATA-containing promoters in the presence of semipurified TFIID fractions from either human or Drosophila cells, but is unable to do so when these fractions are replaced by purified, cloned Drosophila or yeast TFIID . Our findings with Sp1 and CTF suggest that partially purified TFIID fractions from human and Drosophila cells contain coactivators that are dispensable for basal transcription but are required as molecular adaptors between trans-activators and the general transcription initiation machinery . Experiments using cloned TFIID proteins suggest that these coactivators function through the amino-terminal portion of TFIID and that coactivator-TFIID interactions are species specific . At promoters lacking a TATA box, an additional activity distinct from coactivators is required for Sp1 activation of transcription.

Cell, 1990 Jun 29, 61(7), 1179 - 86
Isolation and characterization of the Drosophila gene encoding the TATA box binding protein, TFIID; Hoey T et al.; To investigate the biochemical mechanisms involved in interactions between regulatory factors and the general transcription complex, we have cloned, expressed, and characterized the Drosophila gene encoding the TATA binding protein, dTFIID . Comparison of the protein sequences of the Drosophila and yeast TATA binding proteins reveals a bipartite organization consisting of a highly conserved, basic carboxy-terminal domain and a nonconserved amino-terminal region rich in Gln, Gly, Ser, and Met residues . Purified dTFIID protein binds specifically to the TATA sequence and activates basal-level transcription, and the conserved carboxy-terminal half of the molecule is sufficient for both activities . Partially purified TFIID from Drosophila cells mediates activation by the transcription factor Sp1 . In contrast, purified dTFIID expressed from the cloned gene is unable to support Sp1-dependent activation, suggesting that other factors may be required to mediate interactions between upstream activators like Sp1 and the TATA binding protein.

Cell, 1990 Jun 29, 61(7), 1209 - 15
A novel mediator between activator proteins and the RNA polymerase II transcription apparatus; Kelleher RJ 3rd et al.; One gene activator protein may interfere with the effects of another in eukaryotic cells . We report here that a hybrid yeast-herpes gene activator protein inhibits transcriptional activation by a thymidine-rich DNA element in yeast . This example of activator interference can be faithfully reproduced in vitro . Interference is reversed by a partially purified yeast component, but not by RNA polymerase II or various polymerase II transcription factors . We conclude that the partially purified yeast component is a novel factor, and we suggest this factor mediates the transcriptional activation process.

Cell, 1990 Jun 29, 61(7), 1217 - 24
A specific member of the ATF transcription factor family can mediate transcription activation by the adenovirus E1a protein; Liu F et al.; The adenovirus E1a protein stimulates transcription of viral early genes . Recent experiments indicate that E1a contains a transcriptional activating region, which functions when directed to a promoter . Because E1a is not a sequence-specific DNA binding protein, how it targets to viral promoters has been a question . Several of the viral early promoters contain one or more binding sites for ATFs, a family of cellular transcription factors . Here we show that E1a can function through a specific ATF protein, designated ATF-2 . We provide evidence that E1a interacts with a discrete region of promoter-bound ATF-2, thereby positioning the E1a activating region at a viral promoter.

Nature, 1990 Jun 28, 345(6278), 783 - 6
Direct and selective binding of an acidic transcriptional activation domain to the TATA-box factor TFIID; Stringer KF et al.; The potent transactivation domain of the herpes simplex virion protein VP16 was used as a column ligand for affinity chromatography . VP16 binds strongly and highly selectively to the human and yeast TATA box-binding factors . Our results imply that the principal target for acidic activation domains is the TATA-box factor TFIID.

J Biol Chem, 1990 Jun 25, 265(18), 10300 - 8
Regulation of inorganic sulfate activation in filamentous fungi . Allosteric inhibition of ATP sulfurylase by 3'-phosphoadenosine-5'-phosphosulfate; Renosto F et al.; ATP sulfurylases from Penicillium chrysogenum, Penicillium duponti, Aspergillus nidulans, and Neurospora crassa are strongly inhibited by 3'-phosphoadenosine-5'-phosphosulfate (PAPS), the product of the second (adenosine-5'-phosphosulfate kinase-catalyzed) reaction in the two-step activation of inorganic sulfate . The v versus {PAPS} plots are sigmoidal . At physiological concentrations of MgATP (0.17-3 mM) and SO4(2-) (0.4-10 mM), the {I}0.5 for PAPS inhibition of the P . chrysogenum enzyme is 35-200 microM; {I}0.9 is 68-310 microM . In the presence of PAPS, the {S}0.5 values for both substrates are increased and the v versus {MgATP} and v versus {SO4(2-)} or {MoO4(2-)} plots are sigmoidal . Fluorosulfonate (FSO3-) and thiosulfate (S2O3(2-} (non-reactive sulfate analogs) inhibit the enzyme at subsaturating substrate concentrations in the absence of PAPS, but low concentrations of the analogs activate the enzyme when PAPS is present . Thus, PAPS behaves as an allosteric inhibitor of ATP sulfurylase . In contrast, adenosine-5'-phosphosulfate (APS = product Q), the immediate product of the SO4(2-)-dependent reaction, is a linear inhibitor of the P . chrysogenum enzyme, competitive with both MgATP and MoO4(2-) (Kiq = 36-73 nM) . FSO3- or S2O3(2-) does not activate the enzyme in the presence of APS . The effect of PAPS on fungal ATP sulfurylase is very similar to that observed when a single highly reactive cysteinyl SH group/subunit (SH-1) is covalently modified (Renosto, F., Martin, R . L., and Segel, I . H . (1987) J . Biol . Chem . 262, 16279-16288) . The results suggest that in vitro SH-1 modification induces a conformational change in the enzyme that mimics the change induced in vivo by the reversible binding of PAPS . No evidence was obtained to suggest that PAPS covalently modifies SH-1 . ATP sulfurylases from rat liver (Yu, M., Martin, R . L., Jain, S., Chen, L . T., and Segel, I . H . (1989) Arch . Biochem . Biophys . 269, 156-174), spinach leaf, cabbage leaf, and Saccharomyces cerevisiae are not strongly inhibited by PAPS, do not display sigmoidal initial velocity plots in the presence of PAPS, and do not contain a highly reactive cysteinyl residue whose modification induces increased {S}0.5 values and sigmoidal velocity curves . The allosteric effect of PAPS on the fungal ATP sulfurylase may be part of a sequential feedback process unique to a group of organisms that use PAPS for two diverging pathways, reductive assimilation and sulfate ester formation.

Eur J Biochem, 1990 Jun 20, 190(2), 407 - 14
The roles of ATP4- and Mg2+ in control steps of phosphoglycerate kinase; Fairbrother WJ et al.; 1H-NMR measurements were made of solutions of yeast phosphoglycerate kinase containing the nucleotide substrate, ATP, and Mg2+ in varying concentrations in order to investigate the affect that the metal ion has on the mode of ATP binding to the enzyme . From the change in the chemical shifts of the 'basic-patch' histidine resonances (His62, His167 and His170) and the nucleotide C8H, C2H and C1'H resonances it is apparent that there are at least two ATP-binding sites on the enzyme . Downfield shifts observed for the above histidine resonances at low nucleotide/enzyme molar ratios indicates that the primary binding site involves electrostatic interactions between the nucleotide triphosphate chain and the basic-patch region of the N-terminal domain . The secondary binding site is shown to involve predominantly hydrophobic interactions between the adenosine moiety and the protein . Evidence from previous two-dimensional NMR experiments {Fairbrother et al . (1990) Eur . J . Biochem . 190, 161-169} suggests that the secondary site is equivalent to the crystallographically observed catalytic site . The affinity of the catalytic site is increased relative to the primary electrostatic site with increasing Mg2+ concentration . The possible importance of these observations in the regulation of this enzyme in vivo are discussed.

J Biol Chem, 1990 Jun 15, 265(17), 10109 - 17
Purification of three related peripheral membrane proteins needed for vesicular transport; Clary DO et al.; We report conditions under which Golgi membranes depleted of peripheral membrane proteins can be reconstituted for intra-cisternal vesicular transport . Analysis of the reconstitution reveals requirements for N-ethylmaleimide-sensitive fusion protein, a purified peripheral protein involved in the fusion stage of vesicular transport, as well as other peripheral protein activities which can be provided by mammalian cytosol but not yeast cytosol . The restorative activity in bovine brain cytosol is found in two broad and complementing fractions, of average native molecular masses of about 500 and 40 kDa, termed Fr1 and Fr2, respectively . This resolved transport system was used to develop a purification scheme for Fr2 . Three proteins of apparent molecular masses of 35, 36, and 39 kDa (Fr2-alpha, -beta, and -gamma, respectively) were found to be responsible for Fr2 activity and were purified to homogeneity . Each Fr2 protein has activity by itself in the reconstituted in vitro Golgi transport assay, although each exhibits a different specific activity and plateau value . No synergy of the three Fr2 proteins was observed during mixing experiments . The three Fr2 proteins seem to be closely related based on size, in vitro activities, chromatographic properties, and peptide maps and may comprise a new family of proteins involved in vesicular transport.

Nature, 1990 Jun 14, 345(6276), 642 - 6
Structure of the fibronectin type 1 module; Baron M et al.; The rapid accumulation of sequence data has provided insight into the evolution of proteins and led to the identification of 'mosaic proteins' . These proteins have evolved by duplication, insertion and deletion of a common pool of structural units or modules, yet their biological functions are diverse . They are involved in cell adhesion and migration, embryogenesis and the pathways of blood clotting, fibrinolysis and complement . The modular units are defined by 'consensus sequences' which often include conserved disulphide bonds . Despite the available sequence information, little is known of the tertiary structure of mosaic proteins . If, however, the 'consensus structure' of the modules were known, valuable structural information could be inferred about a wide variety of proteins and biological systems . An important mosaic protein is fibronectin, an extracellular matrix protein that consists of three types of module (see refs 3, 7 for reviews) . Here we describe the structure of the fibronectin type 1 module which appears twelve times in fibronectin and is also found in factor XII and tissue plasminogen activator . The module was produced using a yeast expression system and the structure was determined in solution using 1H NMR . This methodology promises to be extremely powerful in the investigation of modules from a wide range of mosaic proteins.

Nature, 1990 Jun 7, 345(6275), 553 - 6
Small GTP-binding protein associated with Golgi cisternae; Goud B et al.; Eukaryotic cells seem to use GTP hydrolysis to regulate vesicular traffic in exocytosis and endocytosis . The best evidence for this comes from studies on the yeast Saccharomyces cerevisiae that have identified two small Ras-related GTP-binding proteins, Sec4p and Ypt1p, which control distinct stages of the secretory pathway . In mammalian cells the effects of a non-hydrolysable GTP analogue, GTP-gamma S, on different transport events have suggested that they also have proteins functionally related to yeast Sec4p and Ypt1p . The rab genes have recently been cloned and sequenced for rat and human and their proteins have highly conserved domains in common with Sec4p and Ypt1p (including a putative effector binding site) . They are therefore good candidates for GTP-binding proteins involved in intracellular transport in mammalian cells . One of the Rab proteins (Rab1p) is the mammalian counterpart of Ypt1p (ref . 13) . Here we report the localization of the protein Rab6p to the Golgi apparatus in several cell types . By immunolabelling and electron microscopy, Rab6p appears to be concentrated predominantly on the medial and trans cisternae and distributed over their entire surface.

J Biol Chem, 1990 Jun 5, 265(16), 9170 - 5
Peroxisome proliferators enhance linoleic acid metabolism in rat liver . Increased biosynthesis of omega 6 polyunsaturated fatty acids; Kawashima Y et al.; The alterations by peroxisome proliferators of metabolism of linoleic acid in rat liver were studied . Administration of P-chlorophenoxyisobutyric acid (clofibric acid) enhanced in vivo conversion of linoleic acid to its desaturated and/or elongated metabolites, 6,9,12-octadecatrienoic acid, 8,11,14-eicosatrienoic acid, and arachidonic acid, whereas the formation of 11,14-eicosadienoic acid was decreased . These changes observed in vivo were confirmed in vitro to be due to the increases in activities of delta 6 desaturation of linoleic acid to 6,9,12-octadecatrienoic acid (18.4 times), delta 8 desaturation of 11,14-eicosadienoic acid to 8,11,14-eicosatrienoic acid (3.4 times), and delta 5 desaturation of 8,11,14-eicosatrienoic acid to arachidonic acid (4.1 times) . No considerable changes in activities of chain elongation of either linoleic acid or 6,9,12-octadecatrienoic acid were observed . The increases in the activities of three desaturations by clofibric acid were prevented by the treatment of rats with cycloheximide . The inductions of delta 6 and delta 5 desaturations were brought about by the treatment of rats with 2,2'-(decamethylenedithio)diethanol or di-(2-ethylhexyl)-phthalate, peroxisome proliferators structurally unrelated to clofibric acid, as well . These changes in metabolism of linoleic acid by clofibric acid were consistent with the changes in mass proportion of omega 6 fatty acids in hepatic lipid . Physiological significance of the marked changes in linoleic acid metabolism by peroxisome proliferators was discussed.

Z Naturforsch {C}, 1990 Jun, 45(6), 645 - 54
X-ray induced inactivation of the sulfhydryl enzyme malate synthase in the presence of various additives . Probing the extent of primary and post-irradiation inactivation and repair by rapid screening on the microlevel; Durchschlag H et al.; The sulfhydryl enzyme malate synthase was inactivated by X-irradiation in air-saturated aqueous solution, in the absence or presence of a variety of additives (thiols, antioxienzymes, typical radical scavengers, inorganic salts, buffer components, substrates, products, analogues) . Radiation-induced changes of enzymic activity were registered immediately after stop of irradiation and in the post-irradiation period . Repair experiments were initiated by post-irradiation addition of dithiothreitol . Additionally, post-irradiation inactivation was modulated by some further additives . Probing the extent of primary and post-irradiation inactivation and repair was accomplished effectively by screening experiments on the microlevel, and by derivation of normalized efficiency parameters which allowed quick comparisons of the various additives with respect to their protective and repair-promotive efficiencies . Correlations between the efficiency parameters were studied by means of binary and ternary diagrams . Most of the substances added before irradiation were found to protect the enzyme against primary and post-irradiation inactivation and to increase the reparability of the enzyme by dithiothreitol, the extent of the effects depending on the nature (and concentration) of the additives used . Our results indicate that both specific protection (by substrates, products, analogues, and by sulfhydryl agents) and scavenging are responsible for the radioprotective efficiencies of the additives.

Science, 1990 Jun 1, 248(4959), 1112 - 5
HSP104 required for induced thermotolerance; Sanchez Y et al.; A heat shock protein gene, HSP104, was isolated from Saccharomyces cerevisiae and a deletion mutation was introduced into yeast cells . Mutant cells grew at the same rate as wild-type cells and died at the same rate when exposed directly to high temperatures . However, when given a mild pre-heat treatment, the mutant cells did not acquire tolerance to heat, as did wild-type cells . Transformation with the wild-type gene rescued the defect of mutant cells . The results demonstrate that a particular heat shock protein plays a critical role in cell survival at extreme temperatures.

Mol Cell Biol, 1990 Jun, 10(6), 2916 - 23
GAL4 protein: purification, association with GAL80 protein, and conserved domain structure; Chasman DI et al.; Expression of the yeast Saccharomyces cerevisiae GAL4 protein under its own (galactose-inducible) control gave 5 to 10 times the level of protein observed when the GAL4 gene was on a high-copy plasmid . Purification of GAL4 by a procedure including affinity chromatography on a GAL4-binding DNA column yielded not only GAL4 but also a second protein, shown to be GAL80 by its reaction with an antipeptide antibody . Sequence comparisons of GAL4 and other members of a family of proteins sharing homologous cysteine finger motifs identified an additional region of homology in the middle of these proteins shown by genetic analysis to be important for GAL4 function . GAL4 could be cleaved proteolytically at the boundary of the conserved region, defining internal and carboxy-terminal folded domains.

Mol Cell Biol, 1990 Jun, 10(6), 2820 - 31
Identification of positive-acting domains in GCN2 protein kinase required for translational activation of GCN4 expression; Wek RC et al.; GCN4 is a transcriptional activator of amino acid-biosynthetic genes in the yeast Saccharomyces cerevisiae . GCN2, a translational activator of GCN4 expression, contains a domain homologous to the catalytic subunit of eucaryotic protein kinases . Substitution of a highly conserved lysine residue in the kinase domain abolished GCN2 regulatory function in vivo and its ability to autophosphorylate in vitro, indicating that GCN2 acts as a protein kinase in stimulating GCN4 expression . Elevated GCN2 gene dosage led to derepression of GCN4 under nonstarvation conditions; however, we found that GCN2 mRNA and protein levels did not increase in wild-type cells in response to amino acid starvation . Therefore, it appears that GCN2 protein kinase function is stimulated posttranslationally in amino acid-starved cells . Three dominant-constitutive GCN2 point mutations were isolated that led to derepressed GCN4 expression under nonstarvation conditions . Two of the GCN2(Con) mutations mapped in the kinase domain itself . The third mapped just downstream from a carboxyl-terminal segment homologous to histidyl-tRNA synthetase (HisRS), which we suggested might function to detect uncharged tRNA in amino acid-starved cells and activate the adjacent protein kinase moiety . Deletions and substitutions in the HisRS-related sequences and in the carboxyl-terminal segment in which one of the GCN2(Con) mutation mapped abolished GCN2 positive regulatory function in vivo without lowering autophosphorylation activity in vitro . These results suggest that sequences flanking the GCN2 protein kinase moiety are positive-acting domains required to increase recognition of physiological substrates or lower the requirement for uncharged tRNA to activate kinase activity under conditions of amino acid starvation.

Mol Cell Biol, 1990 Jun, 10(6), 2801 - 8
Mutant alcohol dehydrogenase (ADH III) presequences that affect both in vitro mitochondrial import and in vitro processing by the matrix protease; Mooney DT et al.; Point mutations in the presequence of the mitochondrial alcohol dehydrogerase isoenzyme (ADH III) have been shown to affect either the import of the precursor protein into yeast mitochondria in vivo or its processing within the organelle . In the present work, the behavior of these mutants during in vitro import into isolated mitochondria was investigated . All point mutants tested were imported with a slower initial rate than that of the wild-type precursor . This defect was corrected when the precursors were treated with urea prior to import . Once imported, the extent of processing to the mature form of mutant precursors varied greatly and correlated well with the defects observed in vivo . This result was not affected by prior urea treatment . When matrix extracts enriched for the processing protease were used, this defect was shown to be due to failure of the protease to efficiently recognize or cleave the presequence, rather than to a lack of access to the precursor . The rate of import of two ADH III precursors bearing internal deletions in the leader sequence was similar to those of the point mutants, whereas a deletion leading to the removal of the 15 amino-terminal amino acids was poorly imported . The mature amino terminus of wild-type ADH III was determined to be Gln-25 . Mutant m01 (Ser-26 to Phe), which reduced the efficiency of cleavage in vitro by 80%, was cleaved at the correct site.

EMBO J, 1990 Jun, 9(6), 1907 - 13
Transcriptional activation by the papillomavirus E6 zinc finger oncoprotein; Lamberti C et al.; The introduction of the bovine (BPV) or human papillomavirus E6 gene into susceptible cells can result in their transformation, but there are few clues to the mechanism of action of the E6 gene . The characteristic features of E6 proteins are their small size (approximately 150 amino acids) and the potential to form two large zinc fingers . To determine if E6 can function as a transcription factor, the BPV E6 gene was fused to the sequence specific DNA binding peptide encoded by the BPV E2 gene . This chimeric E6-E2 protein trans-activated promoters that incorporated E2 binding elements in both rodent cells and Saccharomyces cerevisiae . In the absence of E6-E2 localization to the target promoter, trans-activation did not occur . Alteration of the cysteine residues at the base of each finger abrogated the transcriptional activity of the E6-E2 hybrids . These data demonstrated that the BPV E6 gene encodes a transcription activation domain and imply that a specific structure of the protein, most likely the zinc fingers, is critical for this function . Since these cysteine mutants are also transformation defective, E6 transcriptional functions may be required for its oncogenic activity.

Mol Cell Biol, 1990 Jun, 10(6), 3163 - 73
Efficiency and diversity of protein localization by random signal sequences; Kaiser CA et al.; Three randomly derived sequences that can substitute for the signal peptide of Saccharomyces cerevisiae invertase were tested for the efficiency with which they can translocate invertase or beta-galactosidase into the endoplasmic reticulum . The rate of translocation, as measured by glycosylation, was estimated in pulse-chase experiments to be less than 6 min . When fused to beta-galactosidase, these peptides, like the normal invertase signal sequence, direct the hybrid protein to a perinuclear region, consistent with localization to the endoplasmic reticulum . The diversity of function of random peptides was studied further by immunofluorescence localization of proteins fused to 28 random sequences: 4 directed the hybrid to the endoplasmic reticulum, 3 directed it to the mitochondria, and 1 directed it to the nucleus.

J Virol, 1990 Jun, 64(6), 2599 - 607
Characterization of a transpositionally active Ty3 element and identification of the Ty3 integrase protein; Hansen LJ et al.; Ty3 is a Saccharomyces cerevisiae retrotransposon associated with tRNA genes . Two Ty3 elements have been cloned and characterized . The complete nucleotide sequence for one element, Ty3-2, was reported previously (L . J . Hansen, D . L . Chalker, and S . B . Sandmeyer, Mol . Cell . Biol . 9:5245-5256, 1988) . However, this element is incapable of autonomous transposition . The complete DNA sequence of a transpositionally competent Ty3 element, Ty3-1, is presented here . Its sequence translates into two overlapping open reading frames, TYA3-1 and TYB3-1, which encode proteins with homology to the proteins specified by the retroviral gag and pol genes, respectively . Comparison of the Ty3-1 nucleotide sequence to Ty3-2 suggests that the TYB3-2 open reading frame of Ty3-2 is truncated by the deletion of a single nucleotide, which causes a frameshift mutation . Restoration of the reading frame with insertion of a single adenine by site-directed mutagenesis converted Ty3-2 into a transpositionally active element, Ty3-2(+ A) . Western blot analysis with antibodies made against synthetic peptides identified integrase (IN) proteins in viruslike particle preparations from cells expressing Ty3 elements . Cells expressing Ty3-1 and Ty3-2 (+A) produce antibody-reactive proteins with approximate molecular masses of 61 and 58 kilodaltons (kDa), while cells expressing Ty3-2 produce reactive proteins of approximately 52 and 49 kDa . Together, these data show that the 61- or 58-kDa protein, or both, provides the integrase function of Ty3.

J Med Virol, 1990 Jun, 31(2), 109 - 11
Diminished response to recombinant hepatitis B vaccine in homosexual men with HIV antibody: an indicator of poor prognosis; Loke RH et al.; Three doses of a recombinant DNA HBV vaccine (MSD) were given to healthy male homosexuals . Seventy-eight out of 104 (77.6%) participants had detectable antibody (anti-HBs) two months after the third dose . Seroconversion occurred in only 9 out of 27 subjects (33.3%) who were anti-HIV positive compared with 69 out of 77 (89.6%) who were negative (chi 2 = 30.8; P less than .001) . Fifteen of the 18 anti-HIV positive who did not mount an antibody response to the hepatitis B vaccine (anti-HBs) later progressed to persistent generalised lymphadenopathy syndrome (5), AIDS-related complex (5), and AIDS (5) . Only one of the nine anti-HIV positive anti-HBs responders developed PGL (chi 2 = 10.14; P less than .005) . Our results show that anti-HIV positive homosexuals are poor responders to the recombinant hepatitis B vaccine and anti-HIV positive non-responders are more likely to develop clinically apparent HIV infection.

Proc Natl Acad Sci U S A, 1990 Jun, 87(12), 4509 - 13
Transcriptional activation by Sp1 as directed through TATA or initiator: specific requirement for mammalian transcription factor IID; Smale ST et al.; Transcription of mammalian genes by RNA polymerase II often begins at a specific nucleotide, whose location is determined either by an upstream DNA element known as a TATA box or by an element positioned at the transcription start site called an initiator (Inr) . By in vitro analysis of synthetic promoters, we demonstrate here that the TATA and Inr elements are functionally similar and that the Inr is contained between nucleotides -3 and +5 relative to the initiation site . Moreover, we found that a mammalian transcription factor IID (TFIID) protein fraction is required for transcriptional stimulation by an Sp1-dependent activating element placed upstream of either TATA or Inr elements . However, in these assays, the yeast TATA-binding protein, which previously was shown to function similarly to mammalian TFIID, could not efficiently substitute for the mammalian TFIID fraction . These results demonstrate that mammalian TFIID is functionally distinct from the yeast TATA-binding protein and may contain additional subunits or domains that are important for transcriptional activation from some promoters.

Pediatr Clin North Am, 1990 Jun, 37(3), 585 - 601
Vaccination of infants and children against hepatitis B; West DJ et al.; Hepatitis B infection and its sequelae constitute a significant public health problem in the United States . It is estimated that 300,000 acute hepatitis B infections occur each year, with about 25% accompanied by both clinical illness and jaundice . Some infections become chronic and ultimately may cause development of liver cirrhosis or primary hepatocellular carcinoma . The risk of chronicity is especially great with infections that occur in infancy . Highly effective vaccines comprised of purified hepatitis B surface antigen (HBsAg) particles are now available for the prevention of hepatitis B infection . A first-generation vaccine utilized HBsAg derived from the plasma of infected persons; this has now been replaced by two similar vaccines that incorporate HBsAg produced by genetically engineered strains of the common bakers' yeast, Saccharomyces cerevisiae . Improved use of vaccine is needed to reduce and ultimately eliminate hepatitis B infection . Vaccination already is recommended for persons recognized to be at increased risk of exposure to virus-containing blood or other body fluids (e.g., infants born to carrier mothers, household or sexual contacts of carriers); however, mass vaccination of adolescents and infants is needed to interdict effectively a majority of all exposures to the hepatitis B virus.

EMBO J, 1990 Jun, 9(6), 1757 - 67
An abundant and ubiquitous homo-oligomeric ring-shaped ATPase particle related to the putative vesicle fusion proteins Sec18p and NSF; Peters JM et al.; We have discovered a ring-shaped particle of 12.5 nm diameter, 14.5S and apparent molecular weight of approximately 570,000 that displays 6-fold radial symmetry and is composed of a single kind of an acidic (pI approximately 5.5) polypeptide of Mr 97,000 (p97) . Using antibodies to this protein we have detected its occurrence in a wide range of cells and tissues of diverse species from frog to man, including highly specialized cells such as mammalian erythrocytes and spermatozoa . In Xenopus laevis oocytes, the particle is found in both isolated nuclei and in manually enucleated ooplasms, which corresponds to immunofluorescence staining dispersed over both nucleoplasm and cytoplasm . The particle has a N-ethylmaleimide (NEM)-inhibitable Mg2(+)-ATPase activity, and its amino acid sequence, as deduced from cDNA clones, displays considerable homology to the mammalian NEM-sensitive fusion protein (NSF) and yeast Sec18p believed to be essential for vesicle fusion in secretory processes, indicating that these three proteins belong to the same multigene family.

Biochimie, 1990 Jun-Jul, 72(6-7), 417 - 29
Flexibility and folding of phosphoglycerate kinase; Yon JM et al.; Flexibility and folding of phosphoglycerate kinase, a two-domain monomeric enzyme, have been studied using a wide variety of methods including theoretical approaches . Mutants of yeast phosphoglycerate kinase have been prepared in order to introduce cysteinyl residues as local probes throughout the molecule without perturbating significantly the structural or the functional properties of the enzyme . The apparent reactivity of a unique cysteine in each mutant has been used to study the flexibility of PGK . The regions of larger mobility have been found around residue 183 on segment beta F in the N-domain and residue 376 on helix XII in the C-domain . These regions are also parts of the molecule which unfold first . Ligand binding induces conformational motions in the molecule, especially in the regions located in the cleft . Moreover, the results obtained by introducing a fluorescent probe covalently linked to a cysteine are in agreement with the helix scissor motion of helices 7 and 14 assumed by Blake to direct the hinge bending motion of the domains during the catalytic cycle . The folding process of both horse muscle and yeast phosphoglycerate kinases involves intermediates . These intermediates are more stable in the horse muscle than in the yeast enzyme . In both enzymes, domains behave as structural modules capable of folding and stabilizing independently, but in the horse muscle enzyme the C-domain is more stable and refolds prior to the N-domain, contrary to that which has been observed in the yeast enzyme . A direct demonstration of the independence of domains in yeast phosphoglycerate kinase has been provided following the obtention of separated domains by site-directed mutagenesis . These domains have a native-like structure and refold spontaneously after denaturation by guanidine hydrochloride.

J Photochem Photobiol B, 1990 Jun, 6(1-2), 221 - 36
Repair of furocoumarin-plus-UVA-induced damage and mutagenic consequences in eukaryotic cells; Averbeck D et al.; In the presence of near-UV radiation (UVA) furocoumarins (psoralens) photoinduce defined lesions in DNA, i.e . monoadducts and interstrand crosslinks . Their use in photochemotherapy (psoralen plus UVA (PUVA) treatment) and cosmetics raises questions concerning the repairability of these lesions and their genotoxic consequences . We have analysed the repair of psoralen photoadducts in cultured eukaryotic cells, such as yeast and mammalian cells, for furocoumarins of photochemotherapeutic interest . In yeast, the interaction of repair pathways differs in exogenous (plasmid) and endogenous (chromosomal) DNA . The order of mutagenic activity is 4,5',8-trimethylpsoralen greater than 5-methoxypsoralen greater than 8-methoxypsoralen greater than 7-methylpyrido{3,4-c}psoralen greater than 3-carbethoxypsoralen . The mutagenicity is dependent on psoralen functionality, concentration and bioavailability, maximal UVA dose, wavelength, dose (fluence) rate and presence or absence of chemical filters . It probably involves an inducible component . Chromosome breakage occurs during the repair period after PUVA treatment . It appears that the genotoxic effects of psoralens are produced by a specific arrangement of induced photolesions and the interaction of different repair systems.

New Biol, 1990 Jun, 2(6), 544 - 55
The DNA and Cu binding functions of ACE1 are interdigitated within a single domain; Hu S et al.; We present genetic and biochemical evidence that the amino-terminal region of ACE1, the activator of yeast Cu-metallothionein gene transcription, is composed of a single domain in which the DNA- and Cu-binding residues are interdigitated . Analysis of truncation mutants showed that both the DNA and Cu interactions functions of ACE1 are contained within an amino-terminal 101 amino acid peptide that can fold into a protease-resistant domain structure . Studies of point mutants revealed that two basic residues within this domain are required for efficient DNA binding although not for productive interaction with Cu . Mutations at these sites alter the specificity of ACE1 for two binding sites in the upstream activation region, both of which are shown to be necessary for efficient transcription in vivo . Systematic mutagenesis of the 12 cysteine residues in ACE1 showed that all 11 cysteines within the minimal DNA-binding domain are required for ACE1 to undergo a Cu-induced conformational switch into an active DNA-binding protein . A twelfth cysteine, located outside the DNA-binding domain, is not required for proper folding . The critical basic and cysteine residues of ACE1 are interdigitated, thereby providing an unusual example of overlapping small molecule and DNA binding functions within a directly regulated transcription factor . In contrast, the carboxyl-terminal region of ACE1 is shown to contain a constitutive trans-activation domain that is spatially distinct and functionally dissociable from the DNA- and Cu-binding domain.

J Biomol Struct Dyn, 1990 Jun, 7(6), 1237 - 49
Flexibility difference between double-stranded RNA and DNA as revealed by gel electrophoresis; Livshits MA et al.; A systematic study of agarose gel electrophoresis of double-stranded RNA in the kilobase range of sizes was performed . The dsRNA to dsDNA relative mobility was found to depend on gel concentration: in low density gels RNA moves slower and in high density gels - faster than DNA of the same molecular size . The electrophoretic differences were interpreted within the reptation theory to be mainly due to the molecular stiffness differences . The dsRNA persistence length was roughly estimated to be about twice as great as that of DNA.

J Cell Biol, 1990 Jun, 110(6), 1897 - 909
Sec2 protein contains a coiled-coil domain essential for vesicular transport and a dispensable carboxy terminal domain; Nair J et al.; SEC2 function is required at the post-Golgi apparatus stage of the yeast secretory pathway . The SEC2 sequence encodes a protein product of 759 amino acids containing an amino terminal region that is predicted to be in an alpha-helical, coiled-coil conformation . Two temperature-sensitive alleles, sec2-41 and sec2-59, encode proteins truncated by opal stop codons and are suppressible by an opal tRNA suppressor . Deletion analysis indicates that removal of the carboxyl terminal 251 amino acids has no apparent phenotype, while truncation of 368 amino acids causes temperature sensitivity . The amino terminal half of the protein, containing the putative coiled-coil domain, is essential at all temperatures . Sec2 protein is found predominantly in the soluble fraction and displays a native molecular mass of greater than 500 kD . All phenotypes of the temperature-sensitive sec2 alleles are partially suppressed by duplication of the SEC4 gene, but the lethality of a sec2 disruption is not suppressed . The sec2-41 mutation exhibits synthetic lethality with the same subset of the late acting sec mutants as does sec4-8 and sec15-1 . The Sec2 protein may function in conjunction with the Sec4 and Sec15 proteins to control vesicular traffic.

Agric Biol Chem, 1990 Jun, 54(6), 1531 - 6
Syntheses and biological activities of (+/-)-streptovitacin A and E-73; Kondo H et al.; (+/-)-Streptovitacin A (1) and its stereoisomers were synthesized by an aldol reaction of (+/-)-2,4-dimethyl-4-trimethylsiloxy-1-cyclohexanones (4b and 9) with 4-(2-oxoethyl)-2,6-piperidinedione (5) . E-72 (2) was derived from synthetic 1 . (+/-)-1 showed moderate growth inhibition against fungi and lettuce seeds.

J Biol Chem, 1990 May 25, 265(15), 8681 - 5
Structure and regulation of rat long-chain acyl-CoA synthetase; Suzuki H et al.; Complementary DNAs encoding rat long-chain acyl-CoA synthetase have been isolated . The cDNAs were identified using synthetic oligonucleotide probes based on partial amino acid sequences of lysyl endopeptidase peptides of the purified enzyme . Rat long-chain acyl-CoA synthetase is predicted to contain 699 amino acid residues and to have a calculated molecular weight of 78,177 . Significant sequence similarity was found between parts of long-chain acyl-CoA synthetase and firefly luciferase . Based on the similarity of the reaction mechanisms of the two enzymes, we propose a function for the similar region . The long-chain acyl-CoA synthetase mRNA is expressed in liver, heart, and epididymal adipose tissues and, to a much lesser extent, in brain, small intestine, and lung . The level of long-chain acyl-CoA synthetase mRNA is increased 7-8-fold in rat liver by feeding a diet high in carbohydrate or fat, consistent with the physiological significance of the enzyme in fatty acid metabolism.

J Biol Chem, 1990 May 25, 265(15), 8420 - 5
The functional efficiency of a mammalian signal peptide is directly related to its hydrophobicity; Bird P et al.; We have previously shown that the signal sequence of the Saccharomyces cerevisiae vacuolar protein carboxypeptidase Y (CPY) does not function in mammalian cells unless a glycine residue in the central core is replaced by leucine . Additional mutants were constructed to investigate the features of this hydrophobic core (h) region that are important for signal sequence function in mammalian cells . We find that the degree of hydrophobicity of the h region of any particular mutant signal is directly related to the efficiency with which it directs the translocation of CPY . A minimal h region in a functional signal appears to consist of five hydrophobic residues interrupted by 1 glycine . Analysis of potential secondary structures suggests that a functional mutant signal is more likely than the nonfunctional CPY signal to adopt either a beta strand or an alpha-helical conformation.

J Biol Chem, 1990 May 25, 265(15), 8400 - 3
The amino acid sequence of the human RNA polymerase II 33-kDa subunit hRPB 33 is highly conserved among eukaryotes; Pati UK et al.; We have cloned and sequenced a cDNA of 1766 base pairs in length encoding the 275 amino acids of hRPB 33, the third largest subunit of human RNA polymerase II . The DNA was isolated by screening of a human lambda gt11 cDNA library with oligonucleotides designed on the basis of the amino acid residue analysis of the bovine material . The hRPB 33 amino acid sequence is highly conserved between Saccharomyces cerevisiae and human . Overall, 45% of the amino acid residues are identical with the yeast homologue RPB 3, and 65% of the amino acids are identical in the two major conserved regions at residues 0-103 and 151-197 . hRPB 33 is also homologous to yeast RPC 5 . The amino acid sequence of hRPB 33 showed no obvious homology with bacterial RNA polymerase or with any of its sigma factors.

J Biol Chem, 1990 May 25, 265(15), 8817 - 22
Localization of a synthetic presequence that blocks protein import into mitochondria; Glaser SM et al.; In the accompanying paper (Glaser, S . M., and Cumsky, M . G . (1990) J . Biol . Chem . 265, 8808-8816) we demonstrated that pL4-(1-22), a synthetic peptide corresponding to the N-terminal 22 residues of the cytochrome c oxidase subunit IV presequence, blocked protein import into mitochondria . Import inhibition was reversible and occurred at a step subsequent to the initial recognition and binding of precursor proteins to the mitochondrial surface . In the present work we have studied the nature of the association between the peptide and mitochondria, as well as determined its intramitochondrial location . We found that pL4-(1-22) was imported into mitochondria in a manner that was dependent upon the delta psi and that the majority of the mitochondrially associated peptide was in the membrane fraction . Density gradient analysis of total membranes indicated that pL4-(1-22) cofractionated with the inner membrane, although the possibility that it was present in both membranes could not be ruled out . It appeared to be inserted within the bilayer since it could not be extracted with salts, chaotropic agents, or high pH . We observed a steady decrease in the amount of pL4-(1-22) found within peptide-treated mitochondria over time . Coincident with this decrease was an increase in the ability of those mitochondria to import and process precursor proteins, suggesting that the peptide was ultimately turned over . The results presented here correlate well with those of the accompanying paper . Together they suggest that pL4-(1-22) blocks import at the level of the mitochondrial membranes, although the exact nature of the import block is not yet clear.

Biochim Biophys Acta, 1990 May 22, 1044(2), 193 - 200
Uptake and phosphorylation of phosphatidylinositol by rat liver nuclei . Role of phosphatidylinositol transfer protein; Capitani S et al.; The incorporation of phosphatidyl{2-3H}inositol ({3H}PI) from vesicles or microsomal membranes into rat liver nuclei is greatly stimulated by phosphatidylinositol transfer protein (PI-TP) . The nuclei are able to phosphorylate {3H}PI, with the production of phosphatidylinositol 4-phosphate (PIP) . Recovery of tritiated inositol trisphosphate, inositol phosphate, glycerophosphoinositol and inositol, suggests that in isolated nuclei a large set of enzymes of the PI cycle is present, similar to the enzymes involved in the plasma membrane PI cycle . Incubation with {gamma-32P}ATP shows that isolated nuclei are able to phosphorylate endogenous PI to PIP and phosphatidylinositol 4,5-bisphosphate (PIP2) . In the presence of exogenous PI and detergent the synthesis of PIP is increased, indicating that in nuclei the PI pool is suboptimal for the PI-kinase activity . The present study suggests that PI-TP may be involved in providing substrates for PI metabolism at the nuclear level.

Biochim Biophys Acta, 1990 May 22, 1044(2), 211 - 21
Fatty acid metabolism in liver of rats treated with hypolipidemic sulphur-substituted fatty acid analogues; Asiedu D et al.; The purpose of this study was to investigate early biochemical changes and possible mechanisms via which alkyl(C12)thioacetic acid (CMTTD, blocked for beta-oxidation), alkyl(C12)thiopropionic acid (CETTD, undergo one cycle of beta-oxidation) and a 3-thiadicarboxylic acid (BCMTD, blocked for both omega- (and beta-oxidation) influence the peroxisomal beta-oxidation in liver of rats . Treatment of rats with CMTTD caused a stimulation of the palmitoyl-CoA synthetase activity accompanied with increased concentration of hepatic acid-insoluble CoA . This effect was already established during 12-24 h of feeding . From 2 days of feeding, the cellular level of acid-insoluble CoA began to decrease, whereas free CoASH content increased . Stimulation of {1-14C}palmitoyl-CoA oxidation in the presence of KCN, palmitoyl-CoA-dependent dehydrogenase (termed peroxisomal beta-oxidation) and palmitoyl-CoA hydrolase activities were revealed after 36-48 h of CMTTD-feeding . Administration of BCMTD affected the enzymatic activities and altered the distribution of CoA between acid-insoluble and free forms comparable to what was observed in CMTTD-treated rats . It is evident that treatment of peroxisome proliferators (BCMTD and CMTTD), the level of acyl-CoA esters and the enzyme activity involved in their formation precede the increase in peroxisomal and palmitoyl-CoA hydrolase activities . In CMTTD-fed animals the activity of cyanide-insensitive fatty acid oxidation remained unchanged when the mitochondrial beta-oxidation and carnitine palmitoyltransferase operated at maximum rates . The sequence and redistribution of CoA and enzyme changes were interpreted as support for the hypothesis that substrate supply is an important factor in the regulation of peroxisomal fatty acid metabolism, i.e., the fatty acyl-CoA species appear to be catabolized by peroxisomes at high rates only when uptake into mitochondria is saturated . Administration of CETTD led to an inhibition of mitochondrial fatty acid oxidation accompanied with a rise in the concentration of acyl-CoA esters in the liver . Consequently, fatty liver developed . The peroxisomal beta-oxidation was marginally affected . Whether inhibition of mitochondrial beta-oxidation may be involved in regulation of peroxisomal fatty acid metabolism and in development of fatty liver should be considered.

Cell, 1990 May 18, 61(4), 723 - 33
Distinct sets of SEC genes govern transport vesicle formation and fusion early in the secretory pathway; Kaiser CA et al.; A vesicular intermediate in protein transport from the endoplasmic reticulum is detected in a subset of temperature-sensitive mutants blocked early in the yeast secretory pathway . By electron microscopy three of the mutants, sec18, sec17, and sec22, accumulate 50 nm vesicles at the nonpermissive temperature . Vesicle accumulation is blocked by the mutations sec12, sec13, sec16, and sec23 as shown by analysis of double-mutant strains . Thus the early SEC genes can be divided into vesicle forming and vesicle fusion functions . Synthetic lethal interactions between sec mutations define two groups of SEC genes, corresponding to the groups involved in vesicle formation or fusion . Mutations in two of the genes involved in vesicle fusion, SEC17 and SEC18, are lethal in combination, and five of six possible pairwise combinations of mutations in genes required for vesicle formation, SEC12, SEC13, SEC16, and SEC23, are lethal . These interactions suggest cooperation between different SEC gene products in vesicle budding and vesicle fusion processes.

Science, 1990 May 18, 248(4957), 847 - 50
Structural motif of the GCN4 DNA binding domain characterized by affinity cleaving; Oakley MG et al.; The NH2-terminal locations of a dimer containing the DNA binding domain of the yeast transcriptional activator GCN4 have been mapped on the binding sites 5'-CTGACTAAT-3' and 5'-ATGACTCTT-3' . Affinity cleaving was effected by synthetic GCN4 proteins with Fe.EDTA moieties at the NH2-terminus . Analysis of the DNA cleavage patterns for dimers of the Fe.EDTA-proteins corresponding to GCN4 residues 222 to 281 and 226 to 281 revealed that the NH2-termini were in the major groove nine to ten base pairs apart and were symmetrically displaced four to five base pairs from the central C of the recognition site . This result is consistent with the Y-shaped scissor grip-leucine zipper model recently proposed for a class of DNA binding proteins important in the regulation of gene expression.

Biochem Pharmacol, 1990 May 15, 39(10), 1529 - 36
The incorporation of 3-phenoxybenzoic acid and other xenobiotic acids into xenobiotic lipids by enzymes of the monoacylglycerol pathway in microsomes from adult and neonatal tissues; Moorhouse KG et al.; The incorporation of 3-phenoxybenzoic acid (3PBA) into xenobiotic lipids by enzymes of the monoacylglycerol (MG) pathway was measured using microsomes prepared from rat liver as an enzyme source . The mean activities of the three enzymes involved were: acyl-CoA synthetase, 1.1 nmol/min/mg protein; MG acyltransferase, 75 pmol/min/mg protein; and diacylglycerol acyltransferase, 11.4 pmol/min/mg protein . MG and DG acyltransferase also showed activity with benzoyl-CoA or 1-naphthylacetyl-CoA as acyl donor but none with clofibryl-CoA or 2,4-dichlorophenoxyacetyl-CoA . MG acyltransferase activity, using 3PBA-CoA, was higher in microsomes from rat intestinal mucosa and pig liver, and lower in rat adipose tissue, rat liver and mouse liver . This ranking of activities corresponds to published activities using natural substrates . There was a large increase in MG acyltransferase, using either 3PBA-CoA or palmitoyl-CoA as substrate, in microsomes from the livers of rats 16-18 days old . Lysophosphatidic acid (lyso-PA) and lysophosphatidylethanolamine (lyso-PE), but not other phospholipids or detergents, stimulated MG acyltransferase activity more than two-fold . Lyso-PA (5 microM) increased the Vmax but had little effect on the Km for 2-hexadecylglycerol, whereas 100 microM lyso-PE decreased the Km and had a smaller effect on the Vmax . These results illustrate that the incorporation of xenobiotic acids into diacyl- and triacylglycerol by enzymes of the MG pathway may be a more general phenomenon than was previously suspected and that it may be subject to a variety of developmental and physiological controls.

Biochem Pharmacol, 1990 May 15, 39(10), 1505 - 12
Participation of the peroxisomal beta-oxidation system in the chain-shortening of PCA16, a metabolite of the cytosine arabinoside prodrug, YNKO1, in rat liver; Yoshida Y et al.; When PCA16, a metabolite of the cytosine arabinoside prodrug YNKO1, was incubated with isolated rat hepatocytes, time-dependent H2O2 generation was found . When the hepatocytes obtained from clofibrate-treated rat liver were used as an enzyme source, PCA16-dependent production of H2O2 was increased by around 6-fold . The activity of peroxisomal beta-oxidation for PCA16 assayed by H2O2 generation was 3-fold higher than that for palmitic acid, whereas the activity of mitochondrial beta-oxidation for PCA16 assayed by ketone body production was much less than that for palmitic acid . A subcellular distribution study revealed that the distribution of the activities of beta-oxidation and fatty acyl-CoA oxidase for PCA16-CoA coincided with those of cyanide-insensitive palmitoyl-CoA-dependent beta-oxidation and catalase, a marker enzyme of peroxisomes . The profile of the cofactor requirement for beta-oxidation of PCA16-CoA in isolated peroxisomes was similar to that for palmitoyl-CoA oxidation, and the reaction was not inhibited by KCN . The formation of CoA derivative prior to beta-oxidation reaction was essential . HPLC analysis of metabolites after incubation of PCA16-CoA with isolated peroxisomes demonstrated the production of four metabolites, two of which were identified as PCA14 and PCA12 by fast atomic bombardment-mass spectrometry . These results indicate that peroxisomal beta-oxidation participates in the shortening of the alkyl-side chain of PCA16 and plays an important role in the formation of antileukemic cytosine arabinoside from YNKO1.

Biochemistry, 1990 May 8, 29(18), 4312 - 7
Proton diffusion in the active site of triosephosphate isomerase; Rose IA et al.; The current model for hydrogen flow in the aldose-ketose isomerases is probably incorrect . Enzymes of this class are characterized by both hydrogen transfer and proton exchange in the interconversion of substrate and product . The transfer is believed to be due to the action of a unique basic residue in the active site . Exchange is presumed to occur by dissociation of the abstracted proton and reassociation from the medium prior to its transfer to the intermediate enediol on the way to product . Dissociation of a necessary proton from the intermediate state imposes limits on the overall catalytic rate depending on the pKa of the protonated base and the pH of the medium . A case in point is triose-P isomerase (TIM), where kcat is approximately 10(4) s-1 . T-Labeled substrate is found to lose approximately 95% of its T to the medium when totally converted to product . Although the active site base is believed to be a glutamate of pKa = 3.9, the pH dependence of maximum velocity is known to be flat up to pH 10 . The loss of hydrogen required to form product as indicated by isotope exchange must be restored completely at this high pH, requiring a base of very high pKa, or there must be some other explanation for the loss of isotope . The present study demonstrates the existence of a single proton on human and rabbit TIM and three protons on yeast TIM that rapidly exchange with the abstracted proton at the E.enediol state internal exchange . Exchange with the medium external exchange occurs from the enzyme after substrate or product has dissociated.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1990 May 8, 29(18), 4283 - 8
pH-dependent spectral and kinetic properties of cytochrome c peroxidase: comparison of freshly isolated and stored enzyme; Vitello LB et al.; The effect of long-term storage on the electronic absorption spectrum and the kinetic properties of cytochrome c peroxidase has been investigated . No detectable differences were observed between freshly isolated enzyme and enzyme stored below -20 degrees C, in the crystalline state, for up to 41 months . The electronic absorption spectrum and the rate of the enzyme-hydrogen peroxide reaction are essentially independent of pH in 0.1 M potassium phosphate buffers for both fresh and stored enzyme . In buffers containing KNO3, the absorption spectrum and the kinetic properties of both fresh and stored enzyme vary with pH, consistent with the titration of an ionizable group with an apparent pKa of 5.5 +/- 0.1 . The differences between phosphate- and nitrate-containing buffers are attributed to specific ion effects . In KNO3-containing buffers, the high-pH form of the enzyme reacts rapidly with hydrogen peroxide while the low-pH form is unreactive . Evidence is presented which indicates that both the low-pH and high-pH forms of the enzyme in KNO3-containing buffers are 5-coordinate, high-spin Fe(III) species.

FEBS Lett, 1990 May 7, 264(1), 141 - 4
Replication-linked histone acetylation in rat liver tissue is sensitive to alkylating agents; Talasz H et al.; The effect of alkylating agents on histone acetyltransferase (EC 2.3.1.48) activity and thymidine incorporation was investigated in benign and malignant proliferating rat liver tissue and compared with the effect in normal non-proliferating rat liver tissue . In both, benign and malignant proliferating tissue, but not in quiescent tissue, the histone acetylation is depressed by alkylating agents and this depression correlates with the inhibition of the thymidine incorporation . This effect suggests that the depression of the replication associated histone acetylation may be an important factor for the antiproliferative activity of alkylating agents.

J Biol Chem, 1990 May 5, 265(13), 7570 - 5
Evidence for difference in the roles of two cysteine residues involved in disulfide bond formation in the folding of human lysozyme; Taniyama Y et al.; Human lysozyme is made up of 130 amino acid residues and has four disulfide bonds at Cys6-Cys128, Cys30-Cys116, Cys65-Cys81, and Cys77-Cys95 . Our previous results using the Saccharomyces cerevisiae secretion system indicate that the individual disulfide bonds of human lysozyme have different functions in the correct in vivo folding and enzymatic activity of the protein (Taniyama, Y., Yamamoto, Y., Nakao, M., Kikuchi, M., and Ikehara, M . (1988) Biochem . Biophys . Res . Commun . 152, 962-967) . In this paper, we report the results of experiments that were focused on the roles of Cys65 and Cys81 in the folding of human lysozyme protein in yeast . A mutant protein (C81A), in which Cys81 was replaced with Ala, had almost the same enzymatic activity and conformation as those of the native enzyme . On the other hand, another mutant (C65A), in which Cys65 was replaced with Ala, was not found to fold correctly . These results indicate that Cys81 is not a requisite for both correct folding and activity, whereas Cys65 is indispensable . The mutant protein C81A is seen to contain a new, non-native disulfide bond at Cys65-Cys77 . The possible occurrence of disulfide bond interchange during our mapping experiments cannot be ruled out by the experimental techniques presently available, but characterization of other mutant proteins and computer analysis suggest that the intramolecular exchange of disulfide bonds is present in the folding pathway of human lysozyme in vivo.

Mutagenesis, 1990 May, 5(3), 293 - 5
Vanadium: genetical and biochemical investigations; Bronzetti G et al.; Ammonium metavanadate was studied for its ability to induce mitotic gene conversion and reverse point mutation in the D7 strain of Saccharomyces cerevisiae . Metavanadate increased the convertant and revertant frequencies; the highest activity was observed without metabolic activation . This indicated that the S9 hepatic fraction and yeast cells in logarithmic phase (and containing a high level of cytochrome P450) biotransform vanadate, probably reducing it to vanadyl . In addition, the effect of ammonium metavanadate on the hepatic monooxygenase system was studied in mice by measuring the level of cytochrome P450 and determining the activities of aminopyrine N-demethylase, p-nitroanisole O-demethylase and 7-ethoxycoumarin O-deethylase in mouse liver microsomal fraction . The results indicated that this compound reduced mono-oxygenase activity and also the level of cytochrome P450.

Genes Dev, 1990 May, 4(5), 740 - 51
Two genes differentially regulated in the cell cycle and by DNA-damaging agents encode alternative regulatory subunits of ribonucleotide reductase; Elledge SJ et al.; Ribonucleotide reductase activity is essential for progression through the cell cycle, catalyzing the rate-limiting step for the production of deoxyribonucleotides needed for DNA synthesis . The enzymatic activity of the enzyme fluctuates in the cell cycle with an activity maximum in S phase . We have identified and characterized two Saccharomyces cerevisiae genes encoding the regulatory subunit of ribonucleotide reductase, RNR1 and RNR3 . They share approximately 80% amino acid identity with each other and 60% with the mammalian homolog, M1 . Genetic disruption reveals that the RNR1 gene is essential for mitotic viability, whereas the RNR3 gene is not essential . A high-copy-number clone of RNR3 is able to suppress the lethality of rnr1 mutations . Analysis of mRNA levels in cell-cycle-synchronized cultures reveals that the RNR1 mRNA is tightly cell-cycle regulated, fluctuating 15- to 30-fold, and is coordinately regulated with the POL1 mRNA, being expressed in the late G1 and S phases of the cell cycle . Progression from the alpha-factor-induced G1 block to induction of RNR1 mRNA is blocked by cycloheximide, further defining the requirement for protein synthesis in the G1- to S-phase transition . Both RNR1 and RNR3 transcripts are inducible by treatments that damage DNA, such as 4-nitroquinoline-1-oxide and methylmethanesulfonate, or block DNA replication, such as hydroxyurea . RNR1 is inducible 3- to 5-fold, and RNR3 is inducible greater than 100-fold . When MATa cells are arrested in G1 by alpha-factor, RNR1 and RNR3 mRNA is still inducible by DNA damage, indicating that the observed induction can occur outside of S phase . Inhibition of ribonucleotide reductase activity by hydroxyurea treatment results in arrest of the cell cycle in S phase as large budded, uninucleate cells . This specific cell-cycle arrest is independent of the RAD9 gene, defining a separate pathway for the coordination of DNA synthesis and cell-cycle progression.

Oncogene, 1990 May, 5(5), 657 - 61
Transcriptional activation by human c-myb and v-myb genes; Kalkbrenner F et al.; The human c-myb proto-oncogene is the cellular progenitor of the viral v-myb oncogene and codes for a 75 kD protein involved in growth regulation and differentiation in a number of cells . Fusion proteins in which human c-myb sequences are linked to the DNA binding domain of the yeast transcriptional activator GAL4 can activate transcription from a reporter gene which carries the chloramphenicol acetyl transferase (CAT) gene linked in cis to a repeat of the GAL4 binding site . Deletions of carboxyterminal sequences allowed the identification of the domain responsible for transcriptional activation, which is located between amino acid residues 275 to 327 . Deletion of this activator domain results in abrogation of the transcriptional activation . The GAL4-v-myb fusion protein can also activate transcription whereas no transactivation by GAL4-c-myb is observed, indicating that a carboxyterminal domain of c-myb which is absent from v-myb apparently negatively regulates transcriptional activation . Dimer formation which is required for transactivation by GAL4 fusion proteins can, when GAL4 is truncated, be mediated by a region of the c-myb protein upstream of the transactivator domain possibly including the transactivator domain itself but not a putative leucine zipper located downstream of this region.

Mol Cell Biol, 1990 May, 10(5), 2390 - 401
Transcription factor requirements for in vitro formation of transcriptionally competent 5S rRNA gene chromatin; Felts SJ et al.; The Saccharomyces cerevisiae 5S rRNA gene was used as a model system to study the requirements for assembling transcriptionally active chromatin in vitro with purified components . When a plasmid containing yeast 5S rDNA was assembled into chromatin with purified core histones, the gene was inaccessible to the yeast class III gene transcription machinery . Preformation of a 5S rRNA gene-TFIIIA complex was not sufficient for the formation of active chromatin in this in vitro system . Instead, a complete transcription factor complex consisting of TFIIIA, TFIIIB, and TFIIIC needed to be formed before the addition of histones in order for the 5S chromatin to subsequently be transcribed by RNA polymerase III . Various 5S rRNA maxigenes were constructed and used for chromatin assembly studies . In vitro transcription from these assembled 5S maxigenes revealed that RNA polymerase III was readily able to transcribe through one, two, or four nucleosomes . However, we found that RNA polymerase III was not able to efficiently transcribe a chromatin template containing a more extended array of nucleosomes . In vivo expression experiments indicated that all in vitro-constructed maxigenes were transcriptionally competent . Analyses of protein-DNA interactions formed on these maxigenes in vivo by indirect end labeling indicated that there are extensive interactions throughout the length of these maxigenes . The patterns of protein-DNA interactions formed on these genes are consistent with these DNAs being assembled into extensive nucleosomal arrays.

Mol Cell Biol, 1990 May, 10(5), 1915 - 20
RNA polymerase II subunit composition, stoichiometry, and phosphorylation; Kolodziej PA et al.; RNA polymerase II subunit composition, stoichiometry, and phosphorylation were investigated in Saccharomyces cerevisiae by attaching an epitope coding sequence to a well-characterized RNA polymerase II subunit gene (RPB3) and by immunoprecipitating the product of this gene with its associated polypeptides . The immunopurified enzyme catalyzed alpha-amanitin-sensitive RNA synthesis in vitro . The 10 polypeptides that immunoprecipitated were identical in size and number to those previously described for RNA polymerase II purified by conventional column chromatography . The relative stoichiometry of the subunits was deduced from knowledge of the sequence of the subunits and from the extent of labeling with {35S}methionine . Immunoprecipitation from 32P-labeled cell extracts revealed that three of the subunits, RPB1, RPB2, and RPB6, are phosphorylated in vivo . Phosphorylated and unphosphorylated forms of RPB1 could be distinguished; approximately half of the RNA polymerase II molecules contained a phosphorylated RPB1 subunit . These results more precisely define the subunit composition and phosphorylation of a eucaryotic RNA polymerase II enzyme.

Mol Cell Biol, 1990 May, 10(5), 1873 - 81
Removal of a hydrophobic domain within the mature portion of a mitochondrial inner membrane protein causes its mislocalization to the matrix; Glaser SM et al.; We have examined the import and intramitochondrial localization of the precursor to yeast cytochrome c oxidase subunit Va, a protein of the mitochondrial inner membrane . The results of studies on the import of subunit Va derivatives carrying altered presequences suggest that the uptake of this protein is highly efficient . We found that a presequence of only 5 amino acids (Met-Leu-Ser-Leu-Arg) could direct the import and localization of subunit Va with wild-type efficiency, as judged by several different assays . We also found that subunit Va could be effectively targeted to the mitochondrial inner membrane with a heterologous presequence that failed to direct import of its cognate protein . The results presented here confirmed those of an earlier study and showed clearly that the information required to "sort" subunit Va to the inner membrane resides in the mature protein sequence, not within the presequence per se . We present additional evidence that the aforementioned sorting information is contained, at least in part, in a hydrophobic stretch of 22 amino acids residing within the C-terminal third of the protein . Removal of this domain caused subunit Va to be mislocalized to the mitochondrial matrix.

Biochimie, 1990 May, 72(5), 323 - 6
Effects of substrates on the thermal stability of nuclear histone acetyltransferase; Sharpe DJ et al.; Calf thymus nuclear histone acetyltransferase was found to have an optimal activity at about 30 degrees C with an energy of activation of 13.2 +/- 0.4 kcal/mol . The enzyme, however, is thermally unstable . At 38, 42, and 46 degrees C, the enzyme was inactivated with rate constants of 0.01, 0.033 and 0.097 min-1, respectively . High salt concentrations and histones accelerated the rate of thermal inactivation . Bovine serum albumin while alone having no effect on the inactivation process, decreased the exacerbation of heat inactivation caused by histones . Acetyl-CoA, on the other hand, protected the enzyme from heat denaturation . The acetyl-enzyme intermediate also underwent heat inactivated but at a much slower rate . The first order rate constant for the process at 42 degrees C decreased from 0.033 min-1 for the native enzyme to 0.013 min-1 for the acetyl-enzyme, corresponding to an increase in Arrhenius energy of inactivation from 55 kcal/mol to 82 kcal/mol . The distinct effects of acetyl-CoA and histones on the thermal stability of the enzyme suggest that interactions of the enzyme with these two substrates are different and thereby result in different enzyme substrate complexes . This observation further supports our previously proposed two-site ping-pong kinetic reaction mechanism which suggests that the two structurally and electrostatically different substrates bind to separate sites on the enzyme.

Biotechnol Prog, 1990 May-Jun, 6(3), 171 - 7
Structural and functional repetition in a marine mussel adhesive protein; Filpula DR et al.; The DOPA-rich polyphenolic protein secreted by the marine mussel Mytilus edulis establishes key chemical linkages in a water-resistant adhesive . Molecular cloning of the gene for this remarkable protein reveals its primary structure as one of the most repetitive proteins identified in the animal kingdom . Expression and purification of polyphenolic proteins from recombinant yeast have provided sufficient material to demonstrate adhesivity of these polypeptides in the laboratory . Adhesive tests reveal a water-resistant bonding capacity of the protein that is dependent on in vitro modification of tyrosine residues to DOPA and the subsequent oxidation to quinone.

Biochem Biophys Res Commun, 1990 Apr 30, 168(2), 747 - 55
Characterization of the 70KDA component of the human Ku autoantigen expressed in insect cell nuclei using a recombinant baculovirus vector; Allaway GP et al.; The Ku autoantigen is a human nuclear, DNA-binding heterodimer of 70kDa and 86kDa proteins . It is the target of autoantibodies in several autoimmune diseases . We now report the expression of a cDNA encoding the 70kDa Ku protein . Large amounts of protein were obtained using a recombinant baculovirus vector, in contrast with earlier unsuccessful attempts using other expression systems . We demonstrate that the 70kDa Ku protein is targeted to the nucleus and is associated with the nuclear matrix when expressed in the absence of the 86kDa Ku component . No post-translational modifications were observed . The 70kDa protein binds double and single-stranded DNA with very high affinity . Our results suggest that the baculovirus expression system may be of widespread use in the production and characterization of human autoantigens.

Nucleic Acids Res, 1990 Apr 25, 18(8), 1951 - 6
A 3.5 genome equivalent multi access YAC library: construction, characterisation, screening and storage; Anand R et al.; The construction of a yeast artificial chromosome (YAC) primary gridded library of 35,000 clones from human lymphoblastoid (48,XXXX) cell line DNA is described . The average YAC size is approximately 350kb representing a greater than 3.5 times coverage of the genome . The library is stored at -70 degrees C as gridded clones on nylon filters impregnated with 20% glycerol and as glycerol suspensions of individual clones in microtitre plates providing a prolonged multi-user potential . To date we have used 14 single copy probes to screen this library by colony hybridisation as well as PCR and have isolated between 1 and 5 YAC clones for every probe.

Biochemistry, 1990 Apr 24, 29(16), 3981 - 6
Topographical localization of peroxisomal acyl-CoA ligases: differential localization of palmitoyl-CoA and lignoceroyl-CoA ligases; Lazo O et al.; We found that peroxisomal lignoceroyl-CoA ligase, like palmitoyl-CoA ligase, is present in the peroxisomal membrane whereas the peroxisomal beta-oxidation enzyme system is localized in the matrix . To further define the role of peroxisomal acyl-CoA ligases (membrane component) in providing acyl-CoA for peroxisomal beta-oxidation, we examined the transverse topographical localization of enzymatic sites of palmitoyl-CoA and lignoceroyl-CoA ligases in the peroxisomal membranes . The disruption of peroxisomes by various techniques resulted in the release of a "latent" pool of lignoceroyl-CoA ligase activity while palmitoyl-CoA ligase activity remained the same . Proteolytic enzyme treatment inhibited palmitoyl-CoA ligase activity in intact peroxisomes but had no effect on lignoceroyl-CoA ligase activity . Lignoceroyl-CoA ligase activity was inhibited only if peroxisomes were disrupted with detergent before trypsin treatment . Antibodies to palmitoyl-CoA ligase and to peroxisomal membrane proteins (PMP) inhibited palmitoyl-CoA ligase in intact peroxisomes, and no pool of "latent" activity appeared when antibody-treated peroxisomes were disrupted with detergent . On the other hand, disruption of PMP antibody-treated peroxisomes with detergent resulted in the appearance of a "latent" pool of lignoceroyl-CoA ligase activity . These results demonstrate that the enzymatic site of palmitoyl-CoA ligase is on the cytoplasmic surface whereas that for lignoceroyl-CoA ligase is on the luminal surface of peroxisomal membranes . This implies that palmitoyl-CoA is synthesized on the cytoplasmic surface and is then transferred to the matrix through the peroxisomal membrane for beta-oxidation in the matrix.(ABSTRACT TRUNCATED AT 250 WORDS)

J Biol Chem, 1990 Apr 15, 265(11), 5950 - 1
Crystallization of recombinant rat cathepsin B; Lee X et al.; A glycosylation-minus mutant of rat cathepsin B expressed in yeast has been purified and crystallized . X-ray diffraction data have been collected and molecular replacement for solving the structure is in progress . The space group for the recombinant rat cathepsin B was determined to be P2(1) with unit cell dimensions alpha = 62.2 A, b = 90.19 A, c = 47.07 A, and beta = 97.43 degrees . A unit cell contains 4 molecules and 2 molecules per asymmetric unit.

J Biol Chem, 1990 Apr 5, 265(10), 5349 - 52
A new class of lysosomal/vacuolar protein sorting signals; Klionsky DJ et al.; A number of inherited lysosomal diseases are known to result from missorting of lysosomal proteins . Considerable attention has been directed toward an understanding of this sorting pathway, and it has become apparent that different mechanisms are used for the sorting of lysosomal membrane and soluble proteins . Protein sorting to the yeast vacuole/lysosome provides a simple model system to study this process . We have mapped the first sorting signal in a vacuolar membrane protein, repressible alkaline phosphatase, and have shown it to be both necessary and sufficient for vacuolar delivery of this enzyme . The sorting information is confined to the transmembrane and cytoplasmic tail region of this type II integral membrane protein . The location of this sorting signal provides an explanation for some of the differences observed between membrane and soluble vacuolar protein sorting.

Chromosoma, 1990 Apr, 99(2), 102 - 10
The presence of an antigen reactive with a human autoantibody in Trichosia pubescens (Diptera: Sciaridae) and its association with certain transcriptionally active regions of the genome; Amabis JM et al.; The antigens in HeLa and Trichosia pubescens cells, recognized by sera from patients with rheumatic diseases containing anti-Ku antibodies, were compared by means of immunoprecipitation of labeled cell extracts . The autoantibodies present in the tested sera precipitate at least two polypeptides of approximately Mr = 70,000 and Mr = 80,000 in HeLa cell extracts and a polypeptide of approximately Mr = 72,000 in Trichosia salivary gland cell extracts . The distribution of the insect antigen in chromatin was studied in salivary gland polytene chromosomes by indirect immunofluorescent staining with sera from two different patients . Both sera react with certain transcriptionally active chromosomal sites . The presence of the antigen in polytene chromosomes is strictly dependent on transcription, as no reaction is observed in the same sites before or after gene activation . Other sites, such as the nucleolar organizing region, are very active in transcription but never reacted with the anti-Ku positive sera . These results show that the insect antigen is associated with transcription-related processes of a subset of the chromosomal loci of T . pubescens . The anti-Ku positive sera react with a highly conserved antigen, which may serve a very important and similar role in the cellular metabolism of both insect and mammalian cells.

Electrophoresis, 1990 Apr, 11(4), 304 - 9
Free flow electrophoresis for the purification of proteins: I . Zone electrophoresis and isotachophoresis; Hoffstetter-Kuhn S et al.; The principles and some applications of free flow zone electrophoresis and isotachophoresis are described . The influence of (i) carrier electrolyte conductivity on the migration velocity and (ii) band shape on zone electrophoresis was investigated . The technique was found convenient for studying the effect of pH on the mobility of proteins to create a mobility curve . The purification of alcohol dehydrogenase from a crude yeast extract revealed the separation power of zone electrophoresis for complex protein mixtures . Without additional steps, a purification factor of 5.4, with a recovery of 97% alcohol dehydrogenase, was achieved . Free flow isotachophoresis was applied to the purification of immunoglobulins from human serum . Disadvantages of this technique are the time-consuming development of an optimized separation system and the empirical search for suitable spacers . Also, reaching of the steady state becomes increasingly difficult as the number of sample components increases.

Trends Biochem Sci, 1990 Apr, 15(4), 148 - 52
Involvement of an initiation factor and protein phosphorylation in translational control of GCN4 mRNA; Hinnebusch AG; Regulation of the GCN4 gene of Saccharomyces cerevisiae is one of the best-documented instances of gene-specific translational control in an eukaryote . Upstream open reading frames (uORFs) in GCN4 mRNA modulate the flow of scanning ribosomes to the GCN4 start codon according to the availability of amino acids . Recent results suggest that sequences at the termination codons of the uORFs, a general initiation factor, and a protein kinase all make important contributions to the proper functioning of this interesting translational-control element.

Chromosoma, 1990 Apr, 99(2), 138 - 42
Cloning a fragment from the telomere of the long arm of human chromosome 9 in a YAC vector; Guerrini AM et al.; The construction of a yeast artificial chromosome containing a human DNA insert is reported . This molecule of about 200 kb behaves as a native yeast chromosome since it has a very high mitotic stability and is present in the yeast transformant clone at a copy number similar to that of the resident chromosomes . Hybridization with the TTAGGG sequence demonstrates that this chromosome contains human telomeric sequences . In situ hybridization of the biotin-labelled artificial chromosome to metaphase human chromosomes shows that the insert occupies a telomeric position on the long arm of chromosome 9 . Since the fragment was cloned as an EcoRI insert and not as a telomere, it is situated medially to the telomeric sequences and harbours telomere-associated sequences, that have been shown to contain the TTAGGG sequence . The fragment represents the end of the genetic map of chromosome 9 and thus can be used to characterize the sequence and the structure of the chromosomal region that runs from the end of the chromosome to the first gene.

Fundam Appl Toxicol, 1990 Apr, 14(3), 542 - 53
Screening for immunomodulators: effects of xenobiotics on macrophage chemiluminescence in vitro; Tam PE et al.; Macrophage chemiluminescence (CL) was evaluated as a primary screening assay by assessing the modulatory activity of 17 different chemicals . The chemicals were either known immunomodulatory drugs or environmental toxicants with reported immunomodulatory activity . Elicited mouse peritoneal macrophages were exposed to the chemicals in vitro, and CL was measured in response to an opsonized yeast stimulus . Ten chemicals (hydrocortisone, dextran sulfate, di-n-octyltin dichloride, dimethyltin dichloride, azathioprine, lambda carrageenan (l-carrageenan), lead, N-propyl gallate, gallic acid, and indomethacin) were identified as effective modulators of CL . The polyanions dextran sulfate and l-carrageenan either suppressed or enhanced CL, depending on the experimental conditions, while the remaining modulators were inhibitory . A series of secondary assays was used to verify this modulatory activity and to explore different mechanisms of action . Each effective modulator altered only a few specific components of the more complex CL response, and the following general mechanisms were apparent . At least 2 chemicals showed distinct antioxidant activity and thus probably did not alter functional aspects of macrophage CL . Chemicals which blocked Fc receptor function delayed the peak CL of macrophages stimulated by opsonized yeast . Nine of the 10 modulators inhibited hydrogen peroxide release, but only 3 inhibited the release of superoxide . Finally, some effective modulators were chemicals known to interact with cell membranes or specific membrane receptors, and these were able to directly induce a CL response without the addition of opsonized yeast as a stimulus . Thus, macrophage CL was a simple, quantitative, yet sensitive immunotoxicologic screening assay capable of identifying many known immunomodulatory drugs . Supplementary assays were useful both in confirming the functional effects of these chemicals on CL and in delineating potential mechanisms by which modulation of CL can occur.

Proc Natl Acad Sci U S A, 1990 Apr, 87(8), 2872 - 6
Molecular cloning of mevalonate kinase and regulation of its mRNA levels in rat liver; Tanaka RD et al.; Mevalonate kinase {ATP:(R)-mevalonate 5-phosphotransferase, EC 2.7.1.36} may be a regulatory site in the cholesterol biosynthetic pathway, and a mutation in the gene coding for this enzyme is thought to cause the genetic disease mevalonic aciduria . To characterize this enzyme, a rat liver cDNA library was screened with a monospecific antibody, and a 1.7-kilobase cDNA clone coding for mevalonate kinase was isolated . The complete DNA sequence was determined, and the longest open reading frame coded for a protein containing 395 amino acids with a deduced molecular weight of 41,990 . Identification of the cDNA clone was confirmed by expression of enzyme activity in yeast and by protein sequence data obtained from sequencing purified rat mevalonate kinase . The deduced amino acid sequence of mevalonate kinase contained a motif for the ATP-binding site found in protein kinases, and it also showed sequence homology to the yeast RAR1 protein . The size of mevalonate kinase mRNA in rat liver was approximately 2 kilobases . Treatment with diets containing cholesterol-lowering agents caused an increase in both mevalonate kinase activity and mRNA levels, whereas diets containing 5% cholesterol lowered the levels of both enzyme activity and mRNA . These data indicate that long-term regulation of enzyme activity in rat liver is controlled by changes in the levels of mevalonate kinase mRNA.

FASEB J, 1990 Apr 1, 4(6), 1598 - 605
The sodium pump needs its beta subunit; McDonough AA et al.; The sodium pump Na,K-ATPase, located in the plasma membrane of all animal cells, is a member of a family of ion-translocating ATPases that share highly homologous catalytic subunits . In this family, only Na,K-ATPase has been established to be a heterodimer of catalytic (alpha) and glycoprotein (beta) subunits . The beta subunit has not been associated with the pump's transport or enzymatic activity, and its role in Na,K-ATPase function has been, until recently, a puzzle . In this review we describe what is known about the structure of beta and summarize evidence that expression of both alpha and beta subunits is required for Na,K-ATPase activity, that inhibition of glycosylation causes a decrease in accumulation of both alpha and beta subunits, and we provide evidence that pretranslational up-regulation of beta alone can lead to increased abundance of sodium pumps . These findings are all consistent with the hypothesis that the beta subunit regulates, through assembly of alpha beta heterodimers, the number of sodium pumps transported to the plasma membrane.

J Lipid Res, 1990 Apr, 31(4), 583 - 95
Cellular oxidation of lignoceric acid is regulated by the subcellular localization of lignoceroyl-CoA ligases; Lazo O et al.; The acyl-CoA ligases convert free fatty acids to acyl-CoA derivatives, and these enzymes have been shown to be present in mitochondria, peroxisomes, and endoplasmic reticulum . Because their activity is obligatory for fatty acid metabolism, it is important to identify their substrate specificities and subcellular distributions to further understand the cellular regulation of these pathways . To define the role of the enzymes and organelles involved in the metabolism of very long chain (VLC) fatty acids, we studied human genetic cell mutants impaired for the metabolism of these molecules . Fibroblast cell lines were derived from patients with X-linked adrenoleukodystrophy (X-ALD) and Zellweger's cerebro-hepato-renal syndrome (CHRS) . While peroxisomes are present and morphologically normal in X-ALD, they are either greatly reduced in number or absent in CHRS . Palmitoyl-CoA ligase is known to be present in mitochondria, peroxisomes, and endoplasmic reticulum (microsomes) . We found enzyme-dependent formation of lignoceroyl-CoA in these same organelles (specific activities were 0.32 +/- 0.12, 0.86 +/- 0.12, and 0.78 +/- 0.07 nmol/h per mg protein, respectively) . However, lignoceroyl-CoA synthesis was inhibited by an antibody to palmitoyl-CoA ligase in isolated mitochondria while it was not inhibited in peroxisomes or endoplasmic reticulum (ER) . This suggests that palmitoyl-CoA ligase and lignoceroyl-CoA are different enzymes and that mitochondria lack lignoceroyl-CoA ligase . This conclusion is further supported by data showing that oxidation of lignoceric acid was found almost exclusively in peroxisomes (0.17 nmol/h per mg protein) but was largely absent from mitochondria and the finding that monolayers of CHRS fibroblasts lacking peroxisomes showed a pronounced deficiency in lignoceric acid oxidation in situ (1.8% of control) . In spite of the observation that lignoceroyl-CoA ligase activity is present on the cytoplasmic surface of ER, our data indicate that lignoceroyl-CoA synthesized by ER is not available for oxidation in mitochondria . This organelle plays no physiological role in the beta-oxidation of VLC fatty acids . Furthermore, the normal peroxisomal oxidation of lignoceroyl-CoA but deficient oxidation of lignoceric acid in X-ALD cells indicates that cellular VLC fatty acid oxidation is dependent on peroxisomal lignoceroyl-CoA ligase . These studies allow us to propose a model for the subcellular localization of various acyl-CoA ligases and to describe how these enzymes control cellular fatty acid metabolism.

Biochimie, 1990 Apr, 72(4), 299 - 302
The primary structure of rat ribosomal protein S19; Suzuki K et al.; The covalent structure of rat ribosomal protein S19 was deduced from the sequence of nucleotides in a recombinant cDNA and confirmed from the NH2-terminal amino acid sequence of the protein . Ribosomal protein S19 contains 144 amino acids (the NH2-terminal methionine is removed after translation of the mRNA) and has a molecular weight of 15,944 . Hybridization of the cDNA to digests of nuclear DNA suggests that there are 15-18 copies of the S19 gene . The mRNA for the protein is about 640 nucleotides in length . Rat S19 is related to Saccharomyces cerevisiae S16A and to Halobacterium marismortui S12.

J Biotechnol, 1990 Apr, 14(1), 71 - 9
Flow injection analysis and biosensors: applications for biotechnology and environmental control; Ludi H et al.; Our experience in industrial bioprocess monitoring and environmental control let us develop a concept for biosensor research which distinguishes itself from other, more popular, approaches . Biosensors must improve and/or simplify existing state-of-the-art analysis systems . Only the parallel development of biosensors and their complementary metrology leads to industrially sound solutions . The combination of flow injection analysis with immobilized enzymes in the form of enzyme columns is already used today for the solution of on-line analytical problems in bioprocesses and environmental control.

Biochemistry, 1990 Mar 27, 29(12), 2891 - 4
Secondary structure of a leucine zipper determined by nuclear magnetic resonance spectroscopy; Oas TG et al.; Previous work has shown that a synthetic peptide corresponding to the leucine zipper region of the yeast transcriptional activator GCN4 forms a stable dimer of alpha-helices and that the helices are oriented in a parallel manner . Two-dimensional nuclear magnetic resonance spectroscopy (NMR) is used here to demonstrate that the helix is continuous for at least 32 of the 33 residues in the peptide . The results also indicate that the dimer is symmetric . It is therefore unlikely that the interdigitation model for the structure of leucine zippers is correct, since interdigitation of leucine residues in a parallel dimer would lead to an asymmetric structure . The data are consistent with a coiled-coil structure.

Cell, 1990 Mar 23, 60(6), 1009 - 17
Segregation of recombined chromosomes in meiosis I requires DNA topoisomerase II; Rose D et al.; To understand better the similarities and differences between meiosis and mitosis, we examined the meiotic role of DNA topoisomerase II, an enzyme that is required mitotically to disentangle sister chromatids at the time of chromosome segregation . In meiosis, we found that topoisomerase II is required only at the time of nuclear division . When cold-sensitive top2 mutants are induced to sporulate at the restrictive temperature, they undergo premeiotic DNA synthesis and commitment to meiotic levels of recombination but fail to complete the first meiotic nuclear division . The introduction of a mutation blocking recombination relieves the requirement for topoisomerase II in meiosis I . These results suggest that topoisomerase II is required at the time of chromosome segregation in meiosis I for the resolution of recombined chromosomes.

Biochem Biophys Res Commun, 1990 Mar 16, 167(2), 498 - 503
Replacing the carboxy-terminal 28 residues of rabbit liver P-450 (laurate (omega-1)-hydroxylase) with those of P-450 (testosterone 16 alpha-hydroxylase) produces a new stereospecific hydroxylase activity; Uno T et al.; cDNA for chimeric P-450 consisting of the amino-terminal 462 residues of P-450 (laurate (omega-1)-hydroxylase) and the remaining 28 residues of P-450 (testosterone 16 alpha-hydroxylase) was constructed and expressed in yeast cells . The resulting chimera could catalyze laurate (omega-1)-hydroxylation and benzphetamine N-demethylation at much higher rates than the parental P-450s, but exhibited the same specificity towards fatty acid substrates as the wild-type laurate hydroxylase . When testosterone was examined as a substrate, the 16 beta-hydroxylated product, which cannot be formed by either of the parental P-450s, was detected, suggesting that the laurate hydroxylase contains a structure that is capable of binding testosterone at a proper orientation so that it can be hydroxylated at the 16 beta position.

Biochem Biophys Res Commun, 1990 Mar 16, 167(2), 471 - 6
Subcellular localization of casein kinase I; Grankowski N et al.; An anti-yeast CKI antiserum was shown to cross-react with CKI isolated from Krebs II mouse ascites tumour cells . The mammalian CKI showed virtually the same molecular mass (app . 45 kDa) as the yeast enzyme . By immunofluorescence it could be shown that CKI is preferably located in the nucleolus.

Biochem Biophys Res Commun, 1990 Mar 16, 167(2), 673 - 9
Stabilization and activation of recombinant human immunodeficiency virus-1 reverse transcriptase-P66; Rowley GL et al.; Human immunodeficiency virus-1 reverse transcriptase-p66 is surprisingly unstable at 4 degrees C in a typical reverse transcriptase buffer that provides complete stability when enzyme is frozen at -70 degrees C . Incorporation of (rA)n(dT)12-18 template-primer in the buffer vastly improved solution stability of dilute enzyme . Incorporation of 1.0 M ammonium phosphate in the buffer promoted an unexpected and reproducible approximately 260% activation of enzyme . In addition, even enzyme that had been inactivated to 13% of its initial activity could be reactivated to the same approximately 260% higher activity level indicating a reversible interconversion of two forms of the enzyme . The effects of chaotropic and antichaotropic salts coupled with a prior observation of p66 monomer-dimer equilibrium provide suggestive evidence that these two forms of enzyme are monomeric and dimeric p66.

Gene, 1990 Mar 15, 87(2), 233 - 42
Sequence similarities among monkey ori-enriched (ors) fragments; Rao BS et al.; Nucleotide sequences have been determined for eight ors (ori-enriched sequence) fragments isolated from monkey DNA by a method that was designed to enrich for origins of DNA replication {Kaufmann et al., Mol . Cell . Biol . 5 (1985) 721-727} . Evidence has been presented that some or possibly all of these sequences can serve, albeit inefficiently, as oris in vivo {Frappier and Zannis-Hadjopoulos, Proc . Natl . Acad . Sci . USA 84 (1987) 6668-6672} . Two of the fragments were found to contain the long terminal repeat-like elements of the 'O-family' of moderately repetitive sequences that are present in human DNA as a transposon-like element {Paulson et al., Nature 315 (1985) 359-361} . Extensive pair-wise comparisons of the sequences failed to detect any statistically significant common sequences, except for long asymmetrically distributed A + T-rich stretches . Nonetheless, when the ors fragments were examined for the presence of published consensus sequences, seven of eight were found to contain the control sequence described by Dierks et al . {Cell 32 (1983) 695-706}, and the same seven of eight were found to contain both the scaffold attachment region T consensus {Gasser and Laemmli, Cell 46 (1986) 521-530} and the minimal Saccharomyces cerevisiae autonomously replicating sequence consensus {e.g., Palzkill and Newlon, Cell 53 (1988) 441-450}.

Eur J Biochem, 1990 Mar 10, 188(2), 247 - 52
Evidence for an endogenous ATPase inhibitor protein in plant mitochondria . Purification and characterization; Norling B et al.; An endogenous ATPase inhibitor protein has been identified and isolated for the first time from plant mitochondria . The inhibitor protein was isolated from potato (Solanum tuberosum) tuber mitochondria and purified to homogeneity . The isolated inhibitor is a heat-stable, trypsin-sensitive, basic protein, with a molecular mass approximately 8.3 kDa . Amino acid analysis reveals a high content of glutamic acid, lysine and arginine and the absence of proline; threonine and leucine . The interaction of the inhibitor with F1-ATPase requires the presence of Mg2(+)-ATP in the incubation medium . The ATPase activity of isolated F1 is inhibited to 50% in the presence of 14 micrograms inhibitor/mg F1 . A stoichiometry of 1.3 mol inhibitor/mol F1 for complete inhibition can be calculated from this value . The potato ATPase inhibitor is also a potent inhibitor of the ATPase activity of the isolated yeast F1 . The inhibitor resembles the ATPase inhibitors of yeast and mammalian mitochondria, and does not seem to be related to the inhibitory peptide, epsilon subunit, of chloroplast ATPase.

Science, 1990 Mar 9, 247(4947), 1233 - 6
Selectivity changes in site-directed mutants of the VDAC ion channel: structural implications; Blachly-Dyson E et al.; The gene encoding the yeast mitochondrial outer membrane channel VDAC was subjected to site-directed mutagenesis to change amino acids at 29 positions to residues differing in charge from the wild-type sequence . The mutant genes were then expressed in yeast, and the physiological consequences of single and multiple amino acid changes were assessed after isolation and insertion of mutant channels into phospholipid bilayers . Selectivity changes were observed at 14 sites distributed throughout the length of the molecule . These sites are likely to define the position of the protein walls lining the aqueous pore and hence, the transmembrane segments . These results have been used to develop a model of the open state of the channel in which each polypeptide contributes 12 beta strands and one alpha helix to form the aqueous transmembrane pathway.

J Biol Chem, 1990 Mar 5, 265(7), 3949 - 55
Purification and characterization of two porcine liver nuclear histone acetyltransferases; Attisano L et al.; Two forms of porcine histone acetyltransferase (types I and II) have been purified to apparent homogeneity from liver nuclei . Both activities are extracted from nuclei by 0.5 M NaCl and display a native Mr of 110,000 as determined by gel filtration . Saline enzyme extracts were subject to ammonium sulfate precipitation and sequential chromatography on Q-Sepharose, Sephacryl S-200, hydroxylapatite, and Mono Q supports . The histone acetyltransferase type I fraction contains three polypeptide chains with apparent Mr values of 105,000, 62,000, and 45,000, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Cyanogen bromide peptide mapping and immunoblotting suggest that the Mr 62,000 and 45,000 polypeptides are derived by cleavage of the Mr 105,000 polypeptide . Histone acetyltransferase type II contains two different subunits with apparent Mr values of 50,000 and 40,000, respectively . The amino acid composition, heat inactivation profiles, and Michaelis constants with respect to both acetyl coenzyme A and histones were indistinguishable for types I and II . However, affinity-purified polyclonal antibodies to both forms of the enzyme do not cross-react; cyanogen bromide-derived in situ cleavage digest patterns show few similarities; and the turnover number for type I is approximately 15-fold lower than that for type II . We estimate that there is one enzyme molecule for every 500 nucleosomes . The existence of two distinct forms of nuclear histone acetyltransferase in pig liver suggests that they may have separate functions in vivo.

Science, 1990 Mar 2, 247(4946), 1077 - 9
Translation initiation and ribosomal biogenesis: involvement of a putative rRNA helicase and RPL46; Sachs AB et al.; Cold-sensitive mutations in the SPB genes (spb1-spb7) of Saccharomyces cerevisiae suppress the inhibition of translation initiation resulting from deletion of the poly(A)-binding protein gene (PAB1) . The SPB4 protein belongs to a family of adenosine triphosphate (ATP)-dependent RNA helicases . The aberrant production of 25S ribosomal RNA (rRNA) occurring in spb4-1 mutants or the deletion of SPB2 (RPL46) permits the deletion of PAB1 . These data suggest that mutations affecting different steps of 60S subunit formation can allow PAB-independent translation, and they indicate that further characterization of the spb mutations could lend insight into the biogenesis of the ribosome.

Proc Natl Acad Sci U S A, 1990 Mar, 87(5), 1889 - 93
Genomic subtraction for cloning DNA corresponding to deletion mutations; Straus D et al.; We have developed a technique, called genomic subtraction, for isolating the DNA that is absent in deletion mutants . The method removes from wild-type DNA the sequences that are present in both the wild-type and the deletion mutant genomes . The DNA that corresponds to the deleted region remains . Enrichment for the deleted sequences is achieved by allowing a mixture of denatured wild-type and biotinylated mutant DNA to reassociate . After reassociation, the biotinylated sequences are removed by binding to avidin-coated beads . This subtraction process is then repeated several times . In each cycle we hybridize the unbound wild-type DNA from the previous round with fresh biotinylated deletion mutant DNA . The unbound DNA from the final cycle is ligated to adaptors and amplified by using one strand of the adaptor as a primer in the polymerase chain reaction . The amplified sequences can then be used to probe a genomic library . We applied genomic subtraction to a yeast strain that has a 5-kilobase deletion, corresponding to 1/4000th of the genome . In the experiment reported here, three rounds of subtraction were sufficient to accurately identify genomic clones containing sequences that are missing in the deletion mutant . We discuss the limitations and some potential applications of the method.

Mol Cell Biol, 1990 Mar, 10(3), 1270 - 5
Conditional mutations occur predominantly in highly conserved residues of RNA polymerase II subunits; Scafe C et al.; Conditional mutations in the Saccharomyces cerevisiae RNA polymerase II large subunit, RPB1, were obtained by introducing a mutagenized RPB1 plasmid into yeast cells, selecting for loss of the wild-type RPB1 gene, and screening the cells for heat or cold sensitivity . Sequence analysis of 10 conditional RPB1 mutations and 10 conditional RPB2 mutations revealed that the amino acid residues altered by these distinct mutations are nearly always invariant among eucaryotic RPB1 and RPB2 homologs . These results suggest that RNA polymerase mutants might be obtained in other eucaryotic organisms by alteration of these invariant residues.

Mol Cell Biol, 1990 Mar, 10(3), 1010 - 6
RNA polymerase II mutants defective in transcription of a subset of genes; Scafe C et al.; Saccharomyces cerevisiae RNA polymerase II conditional mutants that selectively disrupt the synthesis of specific mRNAs were isolated . At the permissive temperature, several of the mutants were inositol auxotrophs as a result of inadequate induction of INO1 transcription . The transcriptional defects exhibited by one of these Ino- mutants (rpb2-2) were further investigated . The induction of GAL10 and HIS4 transcription in rpb2-2 strains was similar to that of wild-type strains, in contrast to the lack of induction of INO1 transcription . When shifted to the nonpermissive temperature, cells containing rpb2-2 continued to accumulate some mRNAs but not others . Together, these results indicate that transcription of specific genes can be disrupted by RNA polymerase II mutations . The rpb2-2 allele alters an amino acid residue that occurs in a highly conserved segment of the RPB2 protein and that is shared by homologous subunits in other species.

Proc Natl Acad Sci U S A, 1990 Mar, 87(5), 1777 - 81
Isolation and characterization of cDNA encoding the 80-kDa subunit protein of the human autoantigen Ku (p70/p80) recognized by autoantibodies from patients with scleroderma-polymyositis overlap syndrome; Mimori T et al.; Anti-Ku (p70/p80) autoantibodies in patients with scleroderma-polymyositis overlap syndrome recognize a 70-kDa/80-kDa protein heterodimer which binds to terminal regions of double-stranded DNA . In the present study, we isolated full-length cDNAs that encode the 80-kDa Ku subunit . Initial screening of a human spleen cDNA library with anti-Ku antibodies yielded a cDNA of 1.0 kilobase (kb) (termed K71) encoding a portion of the 80-kDa Ku polypeptide (identification based on immunological criteria) . In RNA blots, this cDNA hybridized with two mRNAs of 3.4 and 2.6 kb . In rescreening of a cDNA library constructed from simian virus 40-transformed human fibroblast mRNA with the K71 cDNA as a hybridization probe, three positive clones were isolated, and that bearing the longest insert (termed Ku80-6) was selected for further characterization . In vitro transcription and translation experiments produced an immunoprecipitable polypeptide which comigrated with the 80-kDa Ku subunit . The Ku80-6 cDNA proved to be 3304 nucleotides in length, with an additional poly(A) tail, closely approximating the size of the larger mRNA . It contains a single long open reading frame encoding 732 amino acids (Mr = 82,713) . The putative polypeptide has a high content of acidic amino acids and a region with periodic repeat of leucine in every seventh position which may form the "leucine zipper" structure . In genomic DNA blots, probes derived from the opposite ends of cDNA Ku80-6 hybridized with several nonoverlapping restriction fragments from human leukocyte DNA, indicating that the gene encoding the 80-kDa Ku polypeptide is divided into several exons by intervening sequences.

Mol Cell Biol, 1990 Mar, 10(3), 939 - 46
Capping of mammalian U6 small nuclear RNA in vitro is directed by a conserved stem-loop and AUAUAC sequence: conversion of a noncapped RNA into a capped RNA; Singh R et al.; The cap structure of U6 small nuclear RNA (snRNA) is gamma-monomethyl phosphate and is distinct from other known RNA cap structures (R . Singh and R . Reddy, Proc . Natl . Acad . Sci . USA 86:8280-8283, 1989) . Here we show that the information for capping the U6 snRNA in vitro is within the initial 25 nucleotides of the U6 RNA . The capping determinant in mammalian U6 snRNA is a bipartite element--a phylogenetically conserved stem-loop structure and an AUAUAC sequence, or a part thereof, following this stem-loop . Wild-type capping efficiency was obtained when the AUAUAC motif immediately followed the stem-loop and when the gamma-phosphate of the initiation nucleotide was in close proximity to the capping determinant . Incorporation of a synthetic stem-loop followed by an AUAUAC sequence is sufficient to covert a noncapped heterologous transcript into a capped transcript . Transcripts with the initial 32 nucleotides of Saccharomyces cerevisiae U6 snRNA are accurately capped in HeLa cell extract, indicating that capping machinery from HeLa cells can cap U6 snRNA from an evolutionarily distant eucaryote . The U6-snRNA-specific capping is unusual in that it is RNA sequence dependent, while the capping of mRNAs and other U snRNAs is tightly coupled to transcription and is independent of the RNA sequence.

Genes Dev, 1990 Mar, 4(3), 313 - 23
Subunits shared by eukaryotic nuclear RNA polymerases; Woychik NA et al.; RNA polymerases I, II, and III share three subunits that are immunologically and biochemically indistinguishable . The Saccharomyces cerevisiae genes that encode these subunits (RPB5, RPB6, and RPB8) were isolated and sequenced, and their transcriptional start sites were deduced . RPB5 encodes a 25-kD protein, RPB6, an 18-kD protein, and RPB8, a 16-kD protein . These genes are single copy, reside on different chromosomes, and are essential for viability . The fact that the genes are single copy, corroborates previous evidence suggesting that each of the common subunits is identical in RNA polymerases I, II, and III . Furthermore, immunoprecipitation of RPB6 coprecipitates proteins whose sizes are consistent with RNA polymerase I, II, and III subunits . Sequence similarity between the yeast RPB5 protein and a previously characterized human RNA polymerase subunit demonstrates that the common subunits of the nuclear RNA polymerases are well conserved among eukaryotes . The presence of these conserved and essential subunits in all three nuclear RNA polymerases and the absence of recognizable sequence motifs for DNA and nucleoside triphosphate-binding indicate that the common subunits do not have a catalytic role but are important for a function shared by the RNA polymerases such as transcriptional efficiency, nuclear localization, enzyme stability, or coordinate regulation of rRNA, mRNA, and tRNA synthesis.

Genetics, 1990 Mar, 124(3), 561 - 72
A chromosome containing HOT1 preferentially receives information during mitotic interchromosomal gene conversion; Voelkel-Meiman K et al.; The recombination-stimulating sequence, HOT1, is derived from yeast ribosomal DNA and corresponds to the sequences required for promotion of transcription by RNA polymerase I . The effect of HOT1 on mitotic interchromosomal gene conversion was examined in diploid strains carrying his4 heteroalleles . When HOT1 is inserted adjacent to both copies of HIS4, the frequency of His+ recombinants is increased approximately 10-fold . When HOT1 is present on only one of the two homologs, recombination is enhanced and the his4 gene on the HOT1-containing chromosome is preferentially converted . In both pairs of his4 heteroalleles examined, HOT1 stimulates conversion of the his4 mutation which is further from the site of HOT1 insertion more than it stimulates conversion of the HOT1-proximal his4 allele . Compared to recombinants isolated from control strains that lack HOT1, HOT1-promoted His+ recombinants are more often homozygous for sequences distal to HIS4 . The preferential conversion of sequences on the HOT1-containing chromosome is consistent with the double-strand-gap repair model of recombination and suggests that HOT1-promoted gene conversion initiates with a double-strand break in HOT1-adjacent sequences.

Genes Dev, 1990 Mar, 4(3), 324 - 30
A specific terminal structure is required for Ty1 transposition; Eichinger DJ et al.; Yeast retrotransposon Ty1 directs the synthesis of virus-like particles (VLPs) consisting of Ty1-encoded proteins, RNA, and reverse transcripts . Ty1 reverse transcripts, tagged with a selectable marker and found within VLPs, are capable of transposing into naked target DNA in vitro . Cassettes consisting of a Ty long terminal repeat (LTR), or delta, marked with supF, and flanked by appropriate restriction sites were constructed . These artificial substrates, whose termini resemble those of linear, full-length Ty1 reverse transcripts, can be coincubated with VLPs (containing unmarked reverse transcripts), resulting in the very efficient integration of the artificial substrate . The results suggest that Ty DNA is limiting for transposition in vivo, suggesting that inefficient reverse transcription regulates Ty1 transposition . Analysis of the transposition of these model substrates, which resemble in vivo Ty1 transposition intermediates or differ from them in subtle ways, shows that Ty transposition proceeds by the linkage of the 3' hydroxyl residue of the reverse transcript to target DNA.

Proc Natl Acad Sci U S A, 1990 Mar, 87(6), 2077 - 81
GAL4 transcription factor is not a "zinc finger" but forms a Zn(II)2Cys6 binuclear cluster; Pan T et al.; The DNA-binding domain of the transcription factor GAL4, consisting of the 62 N-terminal residues and denoted GAL4(62*), contains a Cys-Xaa2-Cys-Xaa6-Cys-Xaa6-Cys-Xaa2-Cys-Xaa6+ ++-Cys motif, which has been shown previously to bind two Zn(II) or Cd(II) ions . Binding of Zn(II) or Cd(II) is essential for the recognition by GAL4 of the specific palindromic DNA sequence to which it binds upstream of genes for galactose-metabolizing enzymes, the UASG sequence . On the basis of the 113Cd NMR chemical shifts of the two bound 113Cd(II) ions, we propose a binuclear cluster model for this Zn(II)-binding subdomain . 1H-113Cd heteronuclear multiple-quantum NMR spectroscopy and phase-sensitive double-quantum filtered 1H correlation spectroscopy of the 112Cd(II)- and 113Cd(II)-substituted GAL4(62*) derivatives provide direct evidence that the two bound 113Cd(II) ions are coordinated only by the six cysteine residues, two of which form bridging ligands between the two 113Cd(II) ions . The latter can be identified from the pattern of 1H-113Cd J coupling . Thus a binuclear metal ion cluster rather than a "zinc finger" is formed by the six cysteine residues of the GAL4 DNA-binding domain . This model can be directly applied to eight other fungal transcription factors which have been shown to contain similarly spaced Cys6 clusters . 1H NMR spectra of apo-GAL4(62*) suggest conformational fluctuation of the metal-binding subdomain upon removal of Zn(II) or Cd(II) . Both Cd(II)2- and Zn(II)2-containing species of GAL4 can be formed, and the similar 1H NMR spectra suggest similar conformations.

Biochim Biophys Acta, 1990 Mar 1, 1037(3), 307 - 12
Cooperative effect of fructose bisphosphate and glyceraldehyde-3-phosphate dehydrogenase on aldolase action; Neuzil J et al.; The combination of binding and kinetic approaches is suggested to study (i) the mechanism of substrate-modulated dynamic enzyme associations; (ii) the specificity of enzyme interactions . The effect of complex formation between aldolase and glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) on aldolase catalysis was investigated under pseudo-first-order conditions . No change in kcat but a significant increase in KM of fructose 1,6-bisphosphate for aldolase was found when both enzymes were obtained from muscle . In contrast, kcat rather than KM changed if dehydrogenase was isolated from yeast . Next, the conversion of fructose 1-phosphate was not affected by interactions between enzyme couples isolated from muscle . The influence of fructose phosphates on the enzyme-complex formation was studied by means of covalently attached fluorescent probe . We found that the interaction ws not perturbed by the presence of fructose 1-phosphate; however, fructose 1,6-bisphosphate altered the dissociation constant of the enzyme complex . A molecular model for fructose 1,6-bisphosphate-modulated enzyme interaction has been evaluated which suggests that high levels of fructose bisphosphate would drive the formation of the 'channelling' complex between aldolase and glyceraldehyde-3-phosphate dehydrogenase.

Enzyme Microb Technol, 1990 Mar, 12(3), 214 - 7
Hydrolysis of concentrated sucrose syrups by invertase immobilized on anion exchanger waste cotton thread; Godbole SS et al.; Yeast invertase was immobilized on polyethyleneimine-coated cotton thread by adsorption followed by crosslinking with glutaraldehyde . The thread-bound invertase was used as an easily retrievable system for the hydrolysis of 80% w/v commercial sucrose syrups . The immobilized enzyme was stable for over 90 days to a temperature of 50 degrees C, only when stored in 80% sucrose solution . Above this temperature, inactivation of enzyme was observed . The cotton threads were used in a batch reactor for hydrolysis of sucrose in about 30 batches carried out over a period of 50 days without loss in activity . The threads could also be used in a packed bed reactor (1.51) for 97% hydrolysis of 80% sucrose syrups at 50 degrees C at a rate of about 360 kg per month for a period of 3 months.

FEBS Lett, 1990 Feb 26, 261(2), 335 - 8
Stimulatory effect of ammonium sulfate at high concentrations on the aminoacylation of tRNA and tRNA-like molecules; Florentz C et al.; The influence of various salts on the aminoacylation of tRNA(Val) and the tRNA-like structure from turnip yellow mosaic virus RNA by yeast valyl-tRNA synthetase has been studied . As expected, increasing the concentration of salts inhibits the enzymatic reaction . However, in the presence of high concentration of ammonium sulfate, and only this salt, the inhibitory effect is suppressed . Under such conditions, the aminoacylation becomes comparable to that measured in the absence of salt . It was shown that ammonium sulfate affects both the catalytic rate of the reaction and the affinity between valyl-tRNA synthetase and the RNAs . Because the affinity between the partners in the complex is increased when the concentration of the salt is high, it is suggested that hydrophobic effects are involved in tRNA/synthetase interactions.

Cell, 1990 Feb 23, 60(4), 649 - 64
FUS3 encodes a cdc2+/CDC28-related kinase required for the transition from mitosis into conjugation; Elion EA et al.; FUS3 is required for both the arrest of cells in G1 and mating . Upon exposure to mating pheromone, fus3-1 and fus3-2 mutants fail to arrest in G1 and continue to divide while undergoing the transcription induction and morphological changes typical of mating cells . The G1 arrest defect of these fus3 mutants is suppressed by a daf1/whi1 null mutation (also called cln3, a putative cyclin) . FUS3 has a positive role in conjugation, because overexpression of FUS3 increases the pheromone sensitivity of wild-type cells, while the absence of FUS3 causes sterility . The suppression of a gpa1 null (G alpha subunit) by a fus3 null also suggests FUS3 is in the signal transduction pathway . The predicted FUS3 protein is 35% identical to the cdc2+/CDC28 kinases and 52% identical to the KSS1 predicted kinase.

Science, 1990 Feb 16, 247(4944), 841 - 5
Genetic analysis of histone H4: essential role of lysines subject to reversible acetylation; Megee PC et al.; The nucleosome is the fundamental unit of assembly of the chromosome and reversible modifications of the histones have been suggested to be important in many aspects of nucleosome function . The structure-function relations of the amino-terminal domain of yeast histone H4 were examined by the creation of directed point mutations . The four lysines subject to reversible acetylation were essential for histone function as the substitution of arginine or asparagine at these four positions was lethal . No single lysine residue was completely essential since arginine substitutions at each position were viable, although several of these mutants were slower in completing DNA replication . The simultaneous substitution of glutamine for the four lysine residues was viable but conferred several phenotypes including mating sterility, slow progression through the G2/M period of the division cycle, and temperature-sensitive growth, as well as a prolonged period of DNA replication . These results provide genetic proof for the roles of the H4 amino-terminal domain lysines in gene expression, replication, and nuclear division.

Anal Biochem, 1990 Feb 15, 185(1), 118 - 24
Measurement of 2-deoxyglucose and 2-deoxyglucose 6-phosphate in tissues; Manchester JK et al.; The enzymatic methods previously described for 2-deoxyglucose (DG) and 2-deoxyglucose 6-phosphate have been refined and adapted to measurements of brain samples ranging from 50 mg wet weight to less than a microgram dry weight . Procedures for preparing such samples for assay are described . Analytical properties of the enzymes employed are given together with means for overcoming their possible short comings . Emphasis is placed on information useful for employing DG to assess rapid changes in glucose metabolism.

Biochem J, 1990 Feb 15, 266(1), 301 - 4
The carbanion of nitroethane is an inhibitor of, and not a substrate for, flavocytochrome b2 {L-(+)-lactate dehydrogenase}; Genet R et al.; Although nitroethane does not bind to the active site of flavocytochrome b2, its anion, ethane nitronate, behaves as a competitive inhibitor, with a Ki of 2.2 mM . No electron transfer can be detected between the nitronate and the enzyme, in contrast with the observations of other workers on D-amino acid oxidase . Propionate is a competitive inhibitor, with a Ki of 28 mM . The significance of these results with respect to the proposed carbanion mechanism and the putative existence of a covalent enzyme-substrate intermediate is discussed.

J Biol Chem, 1990 Feb 15, 265(5), 2588 - 95
Ligand binding and structural perturbations in cytochrome c peroxidase . A crystallographic study; Edwards SL et al.; Crystal structures of the complexes formed between cytochrome c peroxidase and cyanide, nitric oxide, carbon monoxide, and fluoride have been determined and refined to 1.85 A . In all four complexes significant changes occur in the distal heme pocket due to movement of Arg-48, His-52, and a rearrangement of active site water molecules . In the cyanide, nitric oxide, and carbon monoxide complexes, Arg-48 moves away from the ligand while in the fluoride complex Arg-48 moves in toward the ligand to form a hydrogen bond or ion pair with the fluoride . More subtle changes occur on the proximal side of the heme . In an earlier study at lower resolution (Edwards, S . L., Kraut, J., and Poulos, T . L . (1988) Biochemistry 27, 8074-8081), we found that nitric oxide binding causes perturbations in the proximal domain involving Trp-191 which has been confirmed by the present study . Trp-191 is stacked parallel to and in contact with the proximal ligand, His-175 . Nitric oxide binding results in a slight movement of Trp-191 away from His-175 and a large increase in crystallographic temperature factors indicating increased mobility of these residues on the proximal side of the heme . These proximal-side changes are unique to nitric oxide and are not related strictly to spin-state or oxidation state of the iron atom since similar changes were not observed in the cyanide (low-spin ferric), carbon monoxide (low-spin ferrous), or fluoride (high-spin ferric) complexes.

Experientia, 1990 Feb 15, 46(2), 146 - 53
The biogenesis and function of eukaryotic porins; Dihanich M; Like most other mitochondrial proteins porin is synthesized in the cytosol and imported posttranslationally into the outer mitochondrial membrane . This transport follows the general rules for mitochondrial protein import with a few aberrations: a) porin contains an uncleaved NH2-terminal signal sequence, b) also its carboxyterminus might be involved in the import process, and c) this transport does not seem to require a membrane potential delta psi, although it is ATP-dependent . Most likely the actual import step occurs at contact sites between the outer and the inner mitochondrial membrane and involves at least one receptor protein . Although porin is known to be the major gate through the outer mitochondrial membrane, its absence only causes transient respiratory problems in yeast cells . This could mean a) that there is a bypass for some mitochondrial functions in the cytosol and/or b) that there are alternative channel proteins in the outer membrane . The first idea is supported by the overexpression of cytosolic virus-like particles in yeast cells lacking porin and the second by the occurrence of residual pore activity in mitochondrial outer membrane purified from porinless mutant cells.

Nucleic Acids Res, 1990 Feb 11, 18(3), 647 - 52
Ambiguities in results obtained with 2D gel replicon mapping techniques; Linskens MH et al.; Recently, two 2-dimensional (2D) gel techniques, termed neutral/neutral and neutral/alkaline, have been developed and employed to map replication origins in eukaryotic plasmids and chromosomal DNA (1-11) . The neutral/neutral technique, which requires less DNA for analysis, has been preferentially used in recent studies . We show here that the signal predicted for an origin is not detected using the neutral/neutral technique if the origin is located near the end of the analyzed restriction fragment . We also demonstrate that analysis of the same batch of DNA by the two different mapping techniques can generate apparently contradictory results: in some situations where neutral/alkaline 2D analysis indicates that a certain origin is always used, neutral/neutral 2D analysis suggests that the origin is not always used . Several possible explanations for this type of disagreement between the two techniques are discussed, and we conclude that it is important to use both techniques in combination in order to minimize possible misinterpretations.

Nucleic Acids Res, 1990 Feb 11, 18(3), 569 - 75
Molecular detrapping and band narrowing with high frequency modulation of pulsed field electrophoresis; Turmel C et al.; In high electric fields, megabase DNA fragments are found to be trapped, i.e . to enter or migrate in the gel only very slowly, if at all, leading to very broad electrophoretic bands and loss of separation . As a consequence, low electric fields are usually used to separate these molecules by pulsed field electrophoretic methods . We report here that high-frequency pulses eliminate the molecular trapping found in continuous fields . When high frequency pulses are used to modulate the longer pulses used in pulsed field electrophoresis, narrower bands result, and higher fields can be used . We suggest that this is due to effects that occur on the length scale of a single pore.

Mol Cell Biochem, 1990 Feb 9, 92(2), 159 - 67
Reconstitution of rat liver 60S ribosomal subunits following disassembly by dimethylmaleic anhydride; Garrido S et al.; Modification of 60S ribosomal subunits from rat liver with dimethylmaleic anhydride (60 mumols/ml) is accompanied by release of 35% of the protein . The acidic ribosomal proteins, as well as 9 basic proteins, are selectively liberated from the ribosomal subunits . Reconstitution of the protein-deficient particles with the corresponding split proteins is accompanied by substantial recovery of the original polyphenylalanine synthetic activity . The described reconstitution procedure can be used to investigate the roles played by the released proteins and the functional similarities of proteins from different sources . Hybrid reconstitution of residual ribosomal particles from rat liver or yeast with the corresponding heterologous split proteins produces subunits which have incorporated heterologous proteins but are inactive in polyphenylalanine synthesis.

Science, 1990 Feb 9, 247(4943), 710 - 2
A potent GAL4 derivative activates transcription at a distance in vitro; Carey M et al.; Transcription of a typical eukaryotic gene by RNA polymerase II is activated by proteins bound to sites found near the beginning of the gene as well as to sites, called enhancers, located a great distance from the gene . According to one view, the primary difference between an activator that can work at a large distance and one that cannot is that the former bears a particularly strong activating region; the stronger the activating region, the more readily the activator interacts with its target bound near the transcriptional start site, with the intervening DNA looping out to accommodate the reaction . One alternative view is that the effect of proteins bound to enhancers might require some special aspect of cellular or chromosome structure . Consistent with the first view, an activator bearing an unusually potent activating region can stimulate transcription of a mammalian gene in a HeLa nuclear extract when bound as far as 1.3 kilobase pairs upstream or 320 base pairs downstream of the transcriptional start site.

Biochemistry, 1990 Feb 6, 29(5), 1342 - 7
Degradation of DNA and structure-activity relationship between bleomycins A2 and B2 in the absence of DNA repair; Moore CW; The contribution of DNA repair to the net number of DNA breaks produced during chemical degradation of DNA was determined by using temperature-sensitive mutant cells deficient in ATP-dependent DNA ligase {poly(deoxyribonucleotide):poly(deoxyribonucleotide) ligase, EC 6.5.1.1} . In a very sensitive assay for determining lesions introduced into Saccharomyces cerevisiae DNAs, 2-14C- and 6-3H-prelabeled DNAs from ligase-proficient and ligase-deficient cells were sedimented together through precalibrated, isokinetic alkaline sucrose gradients . DNA ligation was slower after chemical degradation of DNA by bleomycin than after gamma irradiation . DNA breaks increased approximately linearly with drug concentrations, and were approximately equivalent for ligase-proficient and ligase-deficient cells . These results were unexpected because ligase-deficient, but not ligase-proficient, cells lacked the capacity to eliminate DNA breaks produced by bleomycin . The results indicated that DNA repair did not occur during the chemical degradation of DNA under the experimental conditions . Bleomycin B2 produced considerably more DNA breaks than bleomycin A2 over a range of concentrations in ligase-proficient cells, which tolerated higher numbers of DNA breaks in general than ligase-deficient cells . The chemical analogues are structurally identical except for their cationic C-terminal amine . The actual number of DNA breaks produced by bleomycin A2 or bleomycin B2, and not the concentration of bleomycin A2 or bleomycin B2 per se, determined the amount of cell killing . DNA repair is critical in quantitating DNA breaks produced by chemicals, but was ruled out as a factor in the higher DNA breakage by bleomycin B2 than bleomycin A2.

EMBO J, 1990 Feb, 9(2), 475 - 80
The first EGF-like domain from human factor IX contains a high-affinity calcium binding site; Handford PA et al.; It has been suggested that epidermal growth factor-like (EGF-like) domains, containing conserved carboxylate residues, are responsible for the high-affinity calcium binding exhibited by a number of vitamin K-dependent plasma proteins involved in the control of the blood coagulation cascade . These include the procoagulant factors IX and X, and the anticoagulants protein C and protein S . To test this hypothesis we have expressed the first EGF-like domain from human factor IX (residues 46-84) using a yeast secretion system, and examined calcium binding to the domain . Using 1H-NMR to measure a calcium-dependent shift assigned to Tyr69 we have detected a high-affinity calcium binding site (Kd = 200-300 microM) . We suggest that other EGF-like domains of this type may have similar calcium binding properties . In addition, we have completely assigned the aromatic region of the NMR spectrum by NOESY and COSY analysis, and have used these data to discuss the effect of calcium and pH on the conformation of the domain with reference to a model based on the structure of human EGF.

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