Mol Cell Biol, 1990 Feb, 10(2), 859 - 62 Efficient transcription of the glycolytic gene ADH1 and three translational component genes requires the GCR1 product, which can act through TUF/GRF/RAP binding sites; Santangelo GM et al.; Glycolytic gene expression in Saccharomyces cerevisiae is thought to be activated by the GCR and TUF proteins . We tested the hypothesis that GCR function is mediated by TUF/GRF/RAP binding sites (UASRPG elements) . We found that UASRPG-dependent activation of a heterologous gene and transcription of ADH1, TEF1, TEF2, and RP59 were sensitive to GCR1 disruption . GCR is not required for TUF/GRF/RAP expression or in vitro DNA-binding activity. Mol Cell Biol, 1990 Feb, 10(2), 752 - 9 Complex modes of heat shock factor activation; Zimarino V et al.; Eucaryotic organisms respond to elevated environmental temperatures by rapidly activating the expression of heat shock genes . The transcriptional activation of heat shock genes is mediated by a conserved upstream regulatory sequence, the heat shock element (HSE) . Using an HSE-binding assay, we show that a cellular factor present in a range of vertebrate species binds specifically to the HSE . This factor is presumably the transcriptional activator of heat shock genes, heat shock factor (HSF) . In vertebrates, the binding of HSF to the HSE was induced when cells were subjected to heat shock at high temperatures, even in the absence of protein synthesis . Under mild heat shock conditions, HSF binding was induced to a lesser extent, but this induction required protein synthesis, suggesting that synthesis of HSF itself, or an activating factor, is necessary for response to heat shock at intermediate temperatures . The inducibility of HSF binding in higher eucaryotes is contrasted with constitutive HSF binding activity in fungi . It appears that despite conservation of the HSE in evolution, the means by which HSF is activated to bind DNA in higher and lower eucaryotes may have diverged. EMBO J, 1990 Feb, 9(2), 543 - 50 Ubiquitin-conjugating enzymes UBC4 and UBC5 mediate selective degradation of short-lived and abnormal proteins; Seufert W et al.; Ubiquitin-conjugating enzymes catalyse the covalent attachment of ubiquitin to target proteins . Members of this enzyme family are involved in strikingly diverse cellular functions: UBC2 (RAD6) is central to DNA repair, UBC3 (CDC34) is involved in cell cycle control . We have cloned the genes for two novel ubiquitin-conjugating enzymes, UBC4 and UBC5, from the yeast Saccharomyces cerevisiae . These enzymes mediate selective degradation of short-lived and abnormal proteins . UBC4 and UBC5 are closely related in sequence and complementing in function . Expression of UBC4 and UBC5 genes is heat inducible . UBC4 and UBC5 enzymes generate high mol . wt ubiquitin-protein conjugates in vivo consistent with previous studies which suggested that attachment of multiple ubiquitin molecules to proteolytic substrates is required for their selective degradation . UBC4 and UBC5 enzymes comprise a major part of total ubiquitin-conjugation activity in stressed cells . Turnover of short-lived proteins and canavanyl-peptides but not of long-lived proteins is markedly reduced in ubc4ubc5 mutants . Loss of UBC4 and UBC5 activity impairs cell growth, leads to inviability at elevated temperatures or in the presence of an amino acid analog, and induces the stress response. Bioessays, 1990 Feb, 12(2), 53 - 9 Mating type and mating strategies in Neurospora; Metzenberg RL et al.; In the heterothallic species Neurospora crassa, strains of opposite mating type, A and a, must interact to give the series of events resulting in fruiting body formation, meiosis, and the generation of dormant ascospores . The mating type of a strain is specified by the DNA sequence it carries in the mating type region; strains that are otherwise isogenic can mate and produce ascospores . The DNA of the A and a regions have completely dissimilar sequences . Probing DNA from strains of each mating type with labelled sequences from the A and the a regions has shown that, unlike in Saccharomyces cerevisiae, only a single copy of a mating type sequence is present in a haploid genome . The failure to switch is explainable by the physical absence of DNA sequences characteristic of the opposite mating type . While the mating type sequences must be of the opposite kind for mating to occur in the sexual cycle, two strains of opposite mating type cannot form a stable heterokaryon during vegetative growth; instead, they fuse abortively to give a heterokaryon incompatibility reaction, which results in death of the cells along the fusion line . The DNA sequences responsible for this reaction are coextensive with those sequences in the A and a regions which are necessary to initiate fruiting body formation . The genus Neurospora also includes homothallic species--ones in which a single haploid nucleus carries all the information necessary to form fruiting bodies, undergo meiosis, and produce new haploid spores . One such species, N . terricola, contains one copy each of the A and the a sequences within each haploid genome.(ABSTRACT TRUNCATED AT 250 WORDS) Appl Biochem Biotechnol, 1990 Feb, 23(2), 155 - 70 Enzyme-catalyzed organic synthesis of sucrose and trehalose with in situ regeneration of UDP-glucose; Haynie SL et al.; This paper describes cell-free enzymatic syntheses of sucrose and trehalose using partially-purified preparations of sucrose and trehalose synthetase . The coupling of the regeneration of uridine-5'-diphosphoglucose (UDP-Glc) with synthesis of the disaccharide offers a practical route to millimol quantities of these carbohydrates . The syntheses used pyruvate kinase, UDP-Glc pyrophosphorylase, and inorganic pyrophosphatase, and the regenerated UDP-Glc was cycled approximately 10 times. Semin Cancer Biol, 1990 Feb, 1(1), 47 - 58 The SRE: a growth factor responsive transcriptional regulator; Treisman R; Cellular immediate-early genes are rapidly and transiently activated when cells are stimulated with many different growth factors . This review examines the function of a short immediate-early gene regulatory sequence, the Serum Response Element (SRE), that is sufficient for this transient transcriptional activation . The structures of SREs and SRE-containing promoters are presented, followed by a summary of SRE regulatory properties . The roles of other SRE-like regulatory sequences in mammalian and yeast cells are then considered . The properties of SRE binding proteins are reviewed, followed by a discussion of the effects of mutations on both SRE function and protein binding . Finally, possible models for SRE function are discussed. Proc Natl Acad Sci U S A, 1990 Feb, 87(3), 1238 - 42 ADP-ribosylation factor is functionally and physically associated with the Golgi complex; Stearns T et al.; ADP-ribosylation factor (ARF) is a ubiquitous, highly conserved 21-kDa GTP-binding protein, first identified in animal cells as the cofactor required for the in vitro ADP-ribosylation of the stimulatory regulatory subunit of adenylate cyclase, Gs, by cholera toxin . As the relevance of this activity to in vivo function is unknown, we have taken advantage of the conserved nature of ARF to study its function in Saccharomyces cerevisiae . Yeast cells bearing an arf1 null mutation display a number of phenotypes suggesting a defect in the secretory pathway . Secreted invertase is only partially glycosylated, and there is a small internal accumulation of invertase . Genetic experiments revealed interactions between ARF1 and other genes known to be involved in the secretory pathway, including YPT1, which encodes a different GTP-binding protein . In accord with these genetic results, immunofluorescence and immunoelectron microscopy show that ARF protein is localized to the Golgi apparatus in mammalian cells, in particular to the cytosolic surface of predominantly cis-Golgi membranes . Together, these results indicate that ARF functions in intracellular protein transport to or within the Golgi apparatus, a role not predicted by the previous in vitro biochemical studies. Enzyme Microb Technol, 1990 Feb, 12(2), 86 - 9 Production of 5'-ribonucleotides by enzymatic hydrolysis of RNA; Benaiges MD et al.; Study of optimal operational conditions for RNA enzymatic hydrolysis to obtain 5'-ribonucleotides has been carried out . RNA from brewer's yeasts, obtained by ammonium extraction, was hydrolysed by a partially purified 5'-phosphodiesterase from barley rootlets . Temperature of 60 degrees C and pH 7 have been determined as the best operational conditions . Low RNA initial concentration (approximately 0.1%) and reaction time (approximately 1 h) have been identified as necessary to obtain a good yield of 5'-ribonucleotides. Enzyme Microb Technol, 1990 Feb, 12(2), 95 - 103 Magnetic aqueous two-phase separation in preparative applications; Flygare S et al.; Magnetic aqueous two-phase separation is a new technique to speed up the separation of aqueous two-phase systems (Anal . Biochem . 1987, 167, 331-339) . It is based on the addition of magnetically susceptible material (e.g . 1-micron iron oxide particles) which induces rapid phase separation when a mixed system is placed in a magnetic field . The technique has been applied to a number of two-phase systems . The time for phase separation was decreased by a factor of 5-240,000, with the largest improvement for systems containing high concentrations of protein and for systems with viscous or nearly isopycnic phases . An apparatus for preparative multistage extraction with magnetic separation was constructed and tested on glycolytic enzymes present in a yeast extract using a dextran/Cibacron blue-polyethylene glycol system . The presence of iron oxide particles did not adversely affect the extracted enzymes . An electromagnet-based apparatus for continuous phase separation on a larger scale was also designed . A phase system containing crude dextran and unpurified cell homogenate was effectively processed . The apparatus also allowed effective separation when the phase containing iron oxide particles was only a small fraction (4%) of the total phase system. Science, 1990 Jan 26, 247(4941), 467 - 70 Two distinct transcription factors that bind the immunoglobulin enhancer microE5/kappa 2 motif; Henthorn P et al.; Activity of the immunoglobulin heavy and kappa light chain gene enhancers depends on a complex interplay of ubiquitous and developmentally regulated proteins . Two complementary DNAs were isolated that encode proteins, denoted ITF-1 and ITF-2, that are expressed in a variety of cell types and bind the microE5/kappa 2 motif found in both heavy and kappa light chain enhancers . The complementary DNAs are the products of distinct genes, yet both ITF-1 and ITF-2 are structurally and functionally similar . The two proteins interact with one another through their putative helix-loop-helix motifs and each possesses a distinct domain that dictates transcription activation. Nature, 1990 Jan 25, 343(6256), 387 - 9 Nucleosome positioning can affect the function of a cis-acting DNA element in vivo; Simpson RT; Positioning of nucleosomes has been proposed as one mechanism whereby the activity of DNA is regulated: cis-acting elements located in linker DNA might be more accessible for interaction with trans-acting protein factors than they would be if they were directly associated with histones in nucleosome core particles . The eleven base pairs constituting the autonomously replicating sequence (ARS) of the high-copy-number TRP1ARS1 plasmid of Saccharomyces cerevisiae are located in a linker region near the edge of a positioned nucleosome and form an origin of replication . Could nucleosome positioning render the ARS accessible for interaction with the proteins necessary for its function? I have tested this hypothesis by making deletions and an insertion to move the ARS into the nucleosome DNA and then examining the effects on ARS function . There is a marked decrease in copy number when the ARS is moved into the central DNA region of the nucleosome core particle, a region known to differ in structure and stability from the peripheral segments of nucleosome DNA. Nature, 1990 Jan 25, 343(6256), 383 - 6 Reverse self-splicing of group II intron RNAs in vitro; Augustin S et al.; Group II introns, which are classed together on the basis of a conserved secondary structure, are found in organellar genes of lower eukaryotes and plants . Like introns in nuclear pre-messenger RNA, they are excised by a two-step splicing reaction to generate branched circular RNAs, the so-called lariats . A remarkable feature of group II introns is their self-splicing activity in vitro . In the absence of a nucleotide cofactor, the intron RNAs catalyse two successive transesterification reactions which lead to autocatalytic excision of the lariat IVS from pre-mRNA and concomitantly to exon ligation . By virtue of its ability to specifically bind the 5' exon, the intron can also catalyse such reactions on exogenous RNA substrates . This sequence-specific attachment could enable group II introns to integrate into unrelated RNAs by reverse splicing, in a process similar to that described for the self-splicing Tetrahymena group I intron . Here we report that group II lariat IVS can indeed reintegrate itself into an RNA composed of the ligated exons in vitro . This occurs by a process of self-splicing that completely reverses both transesterification steps of the forward reaction: it involves a transition of the 2'-5' phosphodiester bond of the lariat RNA into the 3'-5' bond of the reconstituted 5' splice junction. Biochemistry, 1990 Jan 16, 29(2), 340 - 6 Electrical potential of transfer RNAs: codon-anticodon recognition; Sharp KA et al.; Calculations of the electrostatic potentials were made around yeast elongator phenylalanine, aspartate tRNAs, and yeast initiator methionine tRNA in aqueous solution at physiological ionic strength . The calculations were carried out with a finite difference algorithm for solving the nonlinear Poisson-Boltzmann equation that incorporates the screening effects of the electrolyte, the exclusion of ions by the molecule, the molecular shape, and the different polarizabilities of the solvent and the tRNA . The initiator tRNA is surrounded by uniformly spaced contours of negative potential . The elongator tRNAs are also surrounded by a similar contour pattern except in the anticodon region where there is a pronounced "hole" in the potential surface . This hole is caused by an invagination of the potential contours, which also results in an increase in the local field strength . The effect of this hole is that the anticodon region in the elongator tRNAs is the least negative, or conversely the most positive, region of the molecule . This hole, which is not found when simple Coulombic potentials are used, is due both to the structure of the elongator tRNA anticodon loops and to the different polarizabilities of the solvent and tRNA . The existence of the potential hole in elongator tRNAs may account in part for their ability to associate with other negatively charged macromolecules, in particular mRNA . Moreover, it suggests that the anticodon loop of elongator tRNAs is the energetically most favorable point of approach of mRNA to tRNA. Biochem J, 1990 Jan 15, 265(2), 519 - 24 Phosphoglucoisomerase-catalysed interconversion of hexose phosphates . Kinetic study by 13C n.m.r . of the phosphoglucoisomerase reaction in 2H2O; Willem R et al.; The fate of D-{2-13C}glucose 6-phosphate exposed to phosphoglucoisomerase (glucose-6-phosphate isomerase, EC 5.3.1.9) in 2H2O was monitored by 13C-n.m.r . spectroscopy . The generation of the anomers of both D-{2-13C}fructose 6-phosphate and D-{2-13C,2-2H}glucose 6-phosphate followed a single-exponential pattern . The rate constant, which was proportional to the enzyme concentration, was about 14 times higher, however, in the former than in the latter case . The disappearance of D-{2-13C,2-1H}glucose 6-phosphate occurred in a bi-exponential manner, the rate constants for the fast and the slow processes being in fair agreement with those obtained for the generation of D-{2-13C}fructose 6-phosphate and D-{2-13C,2-2H}glucose 6-phosphate respectively . These findings indicate that the process of equilibration of D-{2-13C}glucose 6-phosphate and D-{2-13C}fructose 6-phosphate is at least one order of magnitude faster than the intermolecular proton transfer involving the deuterons from the solvent . Such a difference provides strong support to the view that the inverconversion of hexose phosphates in the reaction catalysed by phosphoglucoisomerase proceeds in two distinct steps, the second of which occurs according to two competing modalities with either an intramolecular or an intermolecular proton transfer. Mol Cell Biol, 1990 Jan, 10(1), 235 - 42 Characterization of Holliday structures in FLP protein-promoted site-specific recombination; Meyer-Leon L et al.; Holliday structures are formed in the course of FLP protein-promoted site-specific recombination . Here, we demonstrate that Holliday structures are formed in reactions involving wild-type substrates and that they are kinetically competent with respect to the overall reaction rate . Together with a previous demonstration of chemical competence (L . Meyer-Leon, L.-C . Huang, S . W . Umlauf, M . M . Cox, and R . B . Inman, Mol . Cell . Biol . 8:3784-3796, 1988), Holliday structures therefore meet all criteria necessary to establish that they are obligate reaction intermediates in FLP-mediated site-specific recombination . In addition, kinetic evidence suggests that two distinct forms of the Holliday intermediate are present in the reaction pathway, interconverted in an isomerization process that is rate limiting at 0 degree C. Chirality, 1990, 2(2), 67 - 73 Stereoselective arylpropionyl-CoA thioester formation in vitro; Knadler MP et al.; The inversion from R- to S-enantiomer that occurs for some arylpropionic acids may have both toxicological and therapeutic implications . To characterize some properties of this inversion, arylpropionyl-CoA thioester formation was studied in rat tissue homogenates and subcellular fractions for the enantiomers of fenoprofen, ibuprofen, and flurbiprofen . Thioesters were formed from (R)-fenoprofen (64%) and (R)-ibuprofen (33%) but not from the corresponding S-enantiomers or the enantiomers of flurbiprofen . This correlates with the extensive inversion of fenoprofen and ibuprofen and lack of inversion of flurbiprofen in vivo . Subcellular fractions from rat liver showed thioester formation to occur in mitochondria and microsomes but not cytosol . Once formed, the thioesters were readily racemized by whole rat liver homogenate, mitochondria, and cytosol, but only partially inverted (S:R = 0.3) in microsomes . Thioester formation from fenoprofen and ibuprofen was studied in tissue homogenate obtained from liver, diaphragm, kidney, lung, skeletal muscle, smooth muscle, fat, caecum, and intestines . The liver was at least 50-fold more efficient than the other tissues studied and would be expected to be a major organ of enantiomeric inversion . Our data support the hypothesis that R- to S-enantiomeric inversion of arylpropionic acids proceeds via the stereoselective formation of CoA thioesters followed by enzymatic racemization and hydrolysis of the thioesters to regenerate free acid. Ann Nutr Metab, 1990, 34(4), 244 - 51 Recovery study in Mg-deficient rats given an organic source of Mg; Aranda P et al.; Recovery from Mg deficiency was studied in rats given an organic source of Mg derived from yeast (Saccharomyces cerevisiae) cultured in an Mg gluconate-enriched medium, as the only source of Mg . Magnesium was given either at a rate above the RDA or as a supplement to the regular laboratory diet at the rate of 70 mg Mg/100 g food, of which 40 mg Mg was in the form of MgCO3 and the remaining 30 mg Mg was in the form of the organic product under study . The results showed a good degree of digestive and metabolic utilization of the organic compound, which led to the recovery of normal Mg levels in blood and bone by the end of the 1st week of treatment, and in muscle by the 3rd week of treatment . Muscle Mg content appears to be a good indicator of deficiency . Supplementation above the RDA failed to improve recovery rates in deficient animals. Biomed Biochim Acta, 1990, 49(6), 465 - 71 Cross-linking of pyruvate decarboxylase . Characterization of the native and substrate-activated enzyme states; Konig S et al.; In order to demonstrate the role of the protein component of pyruvate decarboxylase in the mechanism of substrate activation, we have isolated and characterized two states of the enzyme, the non-activated and the substrate-activated state, by covalent linking with bifunctional reagents . Because of the fact that modification of the reactive amino groups by 2,4,6-trinitobenzenesulfonic acid or methyl propionimidate influences neither the catalytic nor the regulatory properties of pyruvate decarboxylase, we used bisimidates of different chain length in the modification experiments . Both the non-activated and the substrate-activated enzyme states could be characterized separately . The lag phase of product formation as a typical property of the native enzyme disappeared completely when the enzyme had been cross-linked in the presence of the substrate . The permanently activated enzyme state shows 85% of the activity of native pyruvate decarboxylase and is exclusively stabilized by intra-subunit links . Elimination and subsequent reincorporation of the cofactors thiamine pyrophosphate and magnesium ions resulted in a complete regaining of the properties of the permanently activated enzyme form . An inactive enzyme form was obtained after cross-linking of non-activated pyruvate decarboxylase at low ionic strength (less than 0.01) . Using a disulfide-containing linker we could prove that the inactivity of the obtained enzyme preparation was only the result of the incorporated cross-links and not that of denaturation. Folia Microbiol (Praha), 1990, 35(4), 353 - 62 The effect of soluble glucan derivates on spleen colony-forming units in sublethally irradiated mice; Liskova A et al.; We examined the effects of 5 soluble derivatives of yeast glucan on the formation of exogenous (CFU-S) and endogenous (E-CFU) colony-forming units in the spleens of sublethally irradiated (60Co, 6.5-7.0 Gy) mice of two inbred strains . For the estimation of CFU-S, glucans were administered intravenously either to donors or recipients of spleen cells 24 h prior to irradiation or removal of the spleen . The number of CFU-S was increased when both the donors and recipients were treated with glucan; the highest increase was obtained with glucans S, P and K . All glucan preparations increased significantly also the number of E-CFU even when administered 90 min after irradiation . There exist differences in the response to the stimulatory effect of glucans among individual mouse strains . Thus, for example, the stimulatory effect of glucan KM on the E-CFU number was significantly more pronounced in strain A/Ph than in strain C57Bl/6. Arch Microbiol, 1990, 154(2), 175 - 8 Killer toxin of Hanseniaspora uvarum; Radler F et al.; The yeast Hanseniaspora uvarum liberates a killer toxin lethal to sensitive strains of the species Saccharomyces cerevisiae . Secretion of this killer toxin was inhibited by tunicamycin, an inhibitor of N-glycosylation, although the mature killer protein did not show any detectable carbohydrate structures . Culture supernatants of the killer strain were concentrated by ultrafiltration and the extracellular killer toxin was precipitated with ethanol and purified by ion exchange chromatography . SDS-PAGE of the electrophoretically homogenous killer protein indicated an apparent molecular mass of 18,000 . Additional investigations of the primary toxin binding sites within the cell wall of sensitive yeast strains showed that the killer toxin of Hanseniaspora uvarum is bound by beta-1, 6-D-glucans. Prog Clin Biol Res, 1990, 344, 683 - 99 Molecular evolution from argininosuccinate lyase to delta-crystallin; Mori M et al.; cDNA clones for rat argininosuccinate lyase, a urea cycle enzyme, were cloned and amino acid sequence of the enzyme was predicted . The rat enzyme is 54% identical with the yeast enzyme, which is involved in arginine biosynthesis, thereby indicating that this urea cycle enzyme evolved from the arginine biosynthetic enzyme . A striking similarity (64% identity) was found between amino acid sequences of rat argininosuccinate lyase and chicken delta-crystallin, a major structural protein of the eye lens . The gene for the rat argininosuccinate lyase was cloned and its structure was determined . This gene is a single-copy gene about 14 kilobases long and is split into 16 exons . A comparison with chicken delta-crystallin genes revealed that all introns interrupt the protein-coding regions at homologous positions . This close similarity in structural organization provides strong evidence for the view that the chicken delta 1- and delta 2-crystallin genes evolved by recruitment and duplication of the preexisting argininosuccinate lyase gene and that delta 2-crystallin is probably the direct homologue of argininosuccinate lyase. Mol Biol (Mosk), 1990 Jan-Feb, 24(1), 163 - 72 {Insertion of (dA-dT)n sequences into the regulatory region of the pho5 gene inhibits its expression}; Sidorova IuM et al.; We have studied the effect of some regular sequences namely (dA-dT)n, (dA)n and (dG-dC)n on the Saccharomyces cerevisiae PHO5 gene expression . These sequences were inserted into the ClaI site, located between two phosphate-responsible upstream activating sequences (UAS's) . A modified PHO5 gene cloned in vector plasmids was used to transform the pho3, pho5 yeast strain and the level of acid phosphatase expression in various conditions was measured . We show that the insertion of (dA-dT)n blocks, but not (dA)n or (dG-dC)n, leads to the dramatic decrease of PHO5 gene expression in a derepressed state . (dA-dT)n inserts also inhibit the expression of PHO5 gene which was put under the control of heterologous UASgal. Mutagenesis, 1990 Jan, 5(1), 51 - 4 Strategies for optimization of short-term genotoxicity tests: the synergistic effect of NADPH and NADH on P450 function in processing pre-mutagens; Paolini M et al.; The synergistic effect of NADPH and NADH on P450 functions upon pre-mutagens requiring metabolism during the incubation conditions used in the liver microsomal assay (LMA) was studied . The mean specific activity (Asp) during 1 h of pre-incubation (LMA) of some microsomal mono-oxygenases (i.e . ethylmorphine N-demethylase, p-nitroanisole O-demethylase and aminopyrine N-demethylase) examined with S9 fractions from sodium phenobarbital and beta-naphthoflavone pre-treated mice, was doubled when both NADPH and NADH were present . In contrast, when lipid peroxidation was used as the main enzymatic inactivation index, there was no appreciable change . In agreement with biochemical data, in vitro DNA binding of the pre-mutagenic agent {14C}-1,1,1,2-tetrachloroethane ({14C}TTCE), mediated by mouse hepatic enzymes, showed a significant enhancement (4.4-fold) of specific activity in the presence of both pyridine nucleotides . Mutagenesis experiments using TTCE in the diploid D7 strain of Saccharomyces cerevisiae (from stationary growth phase) as a biological test system, showed a significant enhancement of mitotic gene conversion and reverse point mutation frequencies when using NADPH plus NADH in the medium . Conversely, no positive results without NADH were seen . These findings lead us to suggest the routine use of both NADPH and NADH in order to increase the 'sensitivity' of in vitro mutagenicity screens. Trends Biochem Sci, 1990 Jan, 15(1), 23 - 6 Processing of precursors by dipeptidylaminopeptidases: a case of molecular ticketing; Kreil G; Stepwise cleavage of dipeptides by dipeptidylaminopeptidases as a late step in the liberation of peptides from their precursors has been observed in diverse organisms such as yeast, insects and frogs. Mol Biochem Parasitol, 1990 Jan 1, 38(1), 141 - 50 The TOP2 gene of Trypanosoma brucei: a single-copy gene that shares extensive homology with other TOP2 genes encoding eukaryotic DNA topoisomerase II; Strauss PR et al.; A mixed oligonucleotide probe containing sequences encoding a septapeptide found in yeast, Drosophila and human DNA topoisomerase II was used to screen a genomic library of Trypanosoma brucei . A positive was obtained, and nucleotide sequencing shows that the entire gene encoding DNA topoisomerase II of this organism, TbrTOP2, resides within the T . brucei insert of the clone . A single open reading frame of 1221 triplet codons starting from the first ATG was identified; the amino acid sequence deduced from it is highly homologous to other eukaryotic DNA topoisomerase II and corresponds to a 137-kDa polypeptide . Analysis of restriction endonuclease digests of T . brucei DNA by blot hybridization following gel electrophoresis indicates that TbrTOP2 is a single-copy gene. Food Chem Toxicol, 1990 Jan, 28(1), 29 - 33 Effect of patulin on microbicidal activity of mouse peritoneal macrophages; Bourdiol D et al.; Patulin, a fungal metabolite shown previously to exert immunosuppressive effects on the cellular and humoral immune systems, was examined for its in vitro effects on some functions of murine peritoneal macrophages . The cells were pre-incubated for 2 hr with mycotoxin concentrations of 0.01-2 micrograms/ml . Phagocytosis and phagosome-lysosome fusion were diminished above 0.1 microgram patulin/ml and lysosomal enzymes and microbicidal activity above 0.5 microgram/ml, whereas O2- production was inhibited only above 2 micrograms/ml . This indicated that the killing mechanism did not depend on products of the oxidative burst . The concentrations used did not decrease the cell viability . Under natural circumstances, patulin may constitute a health risk for animals. Mol Pharmacol, 1990 Jan, 37(1), 7 - 10 Rat homologs of the Drosophila dunce gene code for cyclic AMP phosphodiesterases sensitive to rolipram and RO 20-1724; Henkel-Tigges J et al.; The dunce locus of Drosophila melanogaster codes for a low Km, cAMP phosphodiesterase . The correct function of this gene is required for normal learning and memory activity in flies, because dunce mutants fail in tests of behavioral conditioning . These observations have indicated that cAMP regulation is an important aspect of the biochemistry underlying learning and memory processes in insects . To determine whether the locus is functionally conserved in mammals, we have expressed dunce gene homologs from the rat in a yeast expression system . We find that the rat homologs encode low Km, cAMP phosphodiesterases similar to that coded for by the Drosophila dunce+ gene and, more importantly, that the mammalian enzymes are inhibited by rolipram and RO 20-1724, drugs with antidepressant properties . Surprisingly, the dunce-encoded phosphodiesterase was not inhibited by rolipram or RO 20-1724 . These findings suggest that the phosphodiesterases, through their regulation of cAMP levels, influence learning and memory in insects and mood in mammals. Life Sci, 1990, 47(13), 1135 - 9 Arginine and lysine product inhibition of bovine adrenomedullary carboxypeptidase H, a prohormone processing enzyme; Hook VY; Carboxypeptidase H (CPH) is one of the later enzymes in the cascade of proteolytic steps required for the posttranslational processing of peptide hormone precursors, including processing of proenkephalin . In this study, CPH activity in the soluble and membrane fractions of enkephalin-containing bovine chromaffin granules was competitively inhibited by its products arginine and lysine . Ki values for arginine and lysine were 4.6 +/- 1.3 and 7.6 +/- 1.9 mM, respectively, indicating that arginine was a more effective inhibitor than lysine . Other amino acids (at 10 mM) had no effect . The in vivo intragranular concentrations of lysine and arginine are similar to the measured Ki values, indicating that product inhibition of CPH by basic amino acids may occur in vivo. Exp Gerontol, 1990, 25(6), 533 - 43 Body temperature rhythms, cold tolerance, and fever in young and old rats of both genders; Refinetti R et al.; The circadian rhythm of body temperature (CTR) of male and female rats living at 23 degrees C, as well as their body temperature response to a yeast injection or to a 2-h exposure to 0 degree C, was investigated by telemetry . Young rats had a clear CTR with a mean nocturnal peak of 38.0 +/- 0.1 degree C and diurnal trough of 36.2 +/- 0.1 degree C . Older rats, starting at about 18 months of age, tended to have poor (that is, lower amplitude) rhythms . Mean daily body temperature was 37.1 +/- 0.2 degree C at all ages . After exposure to the cold, the body temperature of young rats, old rats with a strong CTR, and old rats with a poor CTR changed in the ranges of -0.3 to +1.5 degree C, -3.1 to +0.7 degree C, and -5.2 to +0.4 degree C, respectively . This indicates that old animals, especially but not exclusively those with poor CTRs, are less resistant to cold stress . On the other hand, the capacity to develop a fever in response to a yeast injection was equivalent in the three groups of animals, although females had a smaller response than males . It is concluded that the process of aging does not have a generalized debilitating effect on temperature regulation in rats . Rather, aging seems to affect individual components of the thermoregulatory system differentially. Dev Genet, 1990, 11(5-6), 410 - 7 Nonsense suppression in Dictyostelium discoideum; Dingermann T et al.; We describe the generation of Dictyostelium discoideum cell lines that carry different suppressor tRNA genes . These genes were constructed by primer-directed mutagenesis changing a tRNA(Trp)(CCA) gene from D . discoideum to a tRNA(Trp)(amber) gene and changing a tRNA(Glu)(UUC) gene from D . discoideum to a tRNA(Glu)(ochre) as well as a tRNA(Glu)(amber) gene . These genes were stably integrated into the D . discoideum genome together with a reporter gene . An actin 6::lacZ gene fusion carrying corresponding translational stop signals served as a reported . Active beta-galactosidase is expressed only in D . discoideum strains that contain, in addition to the reporter, a functional suppressor tRNA . Both amber suppressors are active in D . discoideum without interfering significantly with cell growth and development . We failed, however, to establish cell lines containing a functional tRNA(Glu)(ochre) suppressor . This may be due to the fact that nearly every message from D . discoideum known so far terminates with UAA . Therefore a tRNA capable of reading this termination codon may not be compatible with cell growth. Int J Rad Appl Instrum B, 1990, 17(8), 745 - 9 Influence of the presence of a methyl group on the myocardial metabolism of 15-(paraiodophenyl)-3 methyl pentadecanoic acid (IMPPA); Humbert T et al.; The objective of the present study was to determine the mechanism of accumulation of myocardial activity following i.v . injection of 15-(paraiodophenyl)-3 methyl pentadecanoic acid (IMPPA) . IMPPA and 15 phenyl-3 methyl pentadecanoic acid (MPPA) were labeled with 14C at position 1 and used to perfuse isolated rat hearts in a closed system . After 5 min of perfusion, IMPPA reached 2/3 of its value at 45 min . 14CO2 production was low . Most of the myocardial activity was in the form of free IMPPA . Analysis of IMPPA activation by CoA SH revealed that it was very strongly inhibited . The retention of myocardial activity is thus due to intracellular accumulation of free IMPPA following inhibition of activation . Comparison of results obtained with IMPPA and MPPA showed that the presence of iodine in the molecule accentuates the inhibition of activation. Crit Rev Oncog, 1990, 1(4), 409 - 36 Molecular analysis of the ets genes and their products; Watson DK et al.; Organisms from human to Drosophila have been found to contain cellular sequences and transcripts that are homologous to the ets region of the avian retrovirus, E26 . Ets-related sequences are present on at least two distinct functional loci in chickens and mammals, and have been designated ets-1 and ets-2 . The E26 virus transduced sequences from the chicken ets-1 locus, which encompasses over 60 kb of DNA . The ets genes characterized so far from sea urchin and Drosophila are most closely related to the 3' end of the known ets genes . The predicted viral and avian ets proteins are very similar, except at the termini . The similarity between the predicted ets proteins so far described is discussed . The ets proteins have been identified and localized by immunoprecipitation and immunofluorescence . While the ets-1 proteins are found in the nuclear and cytoplasmic fractions, the viral gag-myb-ets protein (p135) and the ets-2 proteins are nuclear . The ets-1 and ets-2 genes are differentially regulated in different cell types, probably reflecting unique controlling elements . Because chromosomal translocations have been associated with different human leukemias, studies addressing the possible association with the ETS1 (11q23) or ETS2 (21q22.3) loci are reviewed. Appl Theor Electrophor, 1990, 1(4), 189 - 92 Acid depurination after field inversion agarose gel electrophoresis reduces transfer of large DNA molecules; Van Devanter DR et al.; Field-inversion gel electrophoresis (FIGE) is an economical method for the resolution of DNA fragments 20 to 1000 kilobase pairs (kbp) in length, sizes beyond the resolving capabilities of normal agarose gel electrophoresis . A theoretical limitation to FIGE is the subsequent transfer of large DNA molecules to membrane supports for hybridization . After normal electrophoresis, uniform and rapid capillary transfer of DNA fragments from agarose gels has been previously reported to be facilitated by brief depurination of DNA with 0.25 N HCl . However, after FIGE we have found that brief treatment of large (greater than 200 kbp) linear DNA fragments with 0.25 N HCl reduces the extent of subsequent transfer by capillary methods . After FIGE and transfer, ethidium bromide staining of DNA suggests that acid treatment causes trapping of DNA within the agarose matrix. Dev Genet, 1990, 11(5-6), 369 - 76 Heterodimeric capping proteins constitute a highly conserved group of actin-binding proteins; Hartmann H et al.; The two subunits of the heterodimeric protein cap32/34, an actin-binding protein, are encoded by separate single-copy genes . We have established the genomic structure of both genes . A sequence comparison of cap32/34 with capZ from chicken skeletal muscle and two partially known sequences from Saccharomyces cerevisiae and Xenopus laevis show that heterodimeric capping proteins belong to a highly conserved group of actin-binding proteins . This conclusion is supported by the cross-reaction of polyclonal antibodies against cap32 and cap34 with proteins from lower and higher eukaryotes . In addition, a system is presented that allows the expression of truncated cap34 polypeptides under the control of the cap34 promoter. Methods Enzymol, 1990, 181, 97 - 121 Affinity chromatography with biotinylated RNAs; Ruby SW et al.; Small, reversibly biotinylated RNAs as described here are versatile ligands for affinity chromatography of RNA-binding components . These RNAs can be attached to a solid support by binding to avidin and used as ligands, or they may be hybridized to another RNA which acts as the ligand . The incorporation of a disulfide bond in the linker arm connecting biotin to the RNA makes it possible to dissociate the RNA from avidin under mild conditions . Our results regarding the binding and elution of the biotinylated RNA may be applied to other, reversibly biotinylated molecules. Biophys J, 1990 Jan, 57(1), 23 - 31 Interaction between the VDAC channel and a polyanionic effector . An electron microscopic study; Mannella CA et al.; The conductance of the voltage-dependent mitochondrial outer membrane channel is modulated by a synthetic anionic polymer . When added to suspensions of membrane crystals of the channel, the polyanion caused disordering of the usual parallelogram array and increased occurrence of a contracted form of the array . Correlation averages obtained from electron microscopic images of the channel crystals indicated a narrowing of the projected channel lumen in the presence of the polyanion and the appearance of new, narrow zones of stain exclusion on the outside of the channel . These effects are interpreted in terms of possible conformational changes induced in the channel by binding of the polyanion. Gene, 1989 Dec 28, 85(2), 461 - 9 Definition of T-cell specific DNA-binding factors that interact with a 3'-silencer in the CD4+ T-cell gene Rpt-1; Patarca R et al.; Analysis of the region 3' to the CD4+ T-cell gene Rpt-1 (encoding regulatory protein T-lymphocyte 1) led to the definition of a silencer element that inhibits heterologous gene expression in certain CD4+ T-cell lines but not in B-cell or non-lymphoid cell lines . Functional silencer activity in vivo was associated with the presence of a specific silencer-DNA-protein complex in electrophoretic mobility shift assays with T-cell extracts . Formation of this complex was selectively inhibited by the region in HIV-1 containing a silencer element . We discuss the possibility that DNA-binding factors may coregulate HIV-1 and Rpt-1 gene expression through a common transcriptional silencer element. Biochim Biophys Acta, 1989 Dec 28, 987(2), 165 - 70 Developmental patterns in rat brain of phosphatidylinositol synthetic enzymes and phosphatidylinositol transfer protein; Nyquist DA et al.; Phosphatidylinositol synthetic and intermembrane transfer activities were studied in rat in the developing whole brain and isolated cerebellum . Specific activities of CTP:phosphatidate cytidylyltransferase and CDPdiacylglycerol:inositol phosphatidyltransferase were found to have similar developmental patterns . Levels of phosphatidyltransferase seen in fetal animals (whole brain only) and neonatal (whole brain and cerebellum) were maintained through approximately postnatal day 15, peaked at day 28, and then declined to somewhat higher than fetal levels at day 60 . Cytidylyltransferase activity varied from the phosphatidylinositol synthesizing enzyme in that specific activity continued to increase up to day 60 . Whole brain phosphatidylinositol transfer specific activity showed a sharp peak at postnatal day 9 after which activity was maintained at or above the fetal levels to day 60 . Cerebellum phosphatidylinositol transfer specific activity had a similar peak which was delayed 7-10 days compared to the whole brain . Phosphatidylinositol transfer protein was also determined immunologically: whole brain levels increased dramatically from fetal day 16 to 18 and then remained relatively constant, while cerebellum levels (measured from postnatal day 7) displayed a variable profile between days 7 and 28 . The developmental pattern of CTP:phosphatidate cytidylyltransferase in rat brain is reported here for the first time. J Biol Chem, 1989 Dec 25, 264(36), 21857 - 64 Defective H(+)-ATPase of hygromycin B-resistant pma1 mutants fromSaccharomyces cerevisiae; Perlin DS et al.; Mutations in the plasma membrane H(+)-ATPase gene (PMA1) of Saccharomyces cerevisiae that confer growth resistance to hygromycin B have been shown recently to cause a marked depolarization of whole cell membrane potential (Perlin, D . S., Brown, C . L., and Haber, J . E . (1988) J . Biol . Chem . 263, 18118-18122) . In this report, the biochemical and genetic properties of H+-ATPases from four prominent hygromycin B-resistant pma1 mutants, pma1-105, pma1-114, pma1-147, and pma1-155, are described . Single base pair changes were identified in pma1-105, pma1-114, and pma1-147 that resulted in amino acid substitutions of Ser-368----Phe, Gly-158----Asp, Pro-640----Leu, respectively . An A----G transition mutation at -39 in the 5'-untranslated region of the mRNA of pma1-155 was also found . This mutation creates an out-of-Frame upstream AUG initiation codon that apparently reduces normal translation of PMA1 . DNA sequence analysis of PMA1 from strain Y55 identified 9 base pair substitutions that resulted in 6 amino acid changes in nonconserved regions when compared to the published sequence for strain S288C . Plasma membranes of three of the four pma1 mutants contained normal amounts of H(+)-ATPase; membranes from pma1-155 contained enzyme at 62% of the wild-type level . The kinetics of ATP hydrolysis were most strongly altered for enzymes from pma1-105 and pma1-147 which showed changes in both Km and Vmax . A striking pH dependence for these parameters was found for enzyme from pma1-105 which resulted in a precipitous decline in Km and Vmax below pH 6.5 . ATP hydrolysis by enzymes from pma1-105 and pma1-147 was insensitive to inhibition by vanadate . These enzymes, in contrast to wild-type and vanadate-sensitive mutant enzymes, were poorly protected from trypsin-induced inactivation by MgATP and vanadate or Pi alone . These results are pertinent to the mechanism of vanadate-induced enzyme inhibition and suggest that Ser-368 and Pro-640 influence the affinity of the phosphate-binding site for Pi . All mutant enzymes catalyzed ATP-induced pH gradient formation following purification and reconstitution into liposomes . Finally, these results further demonstrate the usefulness of hygromycin B as a generalized screening tool for isolating diverse plasma membrane ATPase mutants. Biochemistry, 1989 Dec 12, 28(25), 9834 - 9 Replacement of a conserved proline and the alkaline conformational change in iso-2-cytochrome c; Nall BT et al.; Although point mutations usually lead to minor localized changes in protein structure, replacement of conserved Pro-76 with Gly in iso-2-cytochrome c induces a major conformational change . The change in structure results from mutation-induced depression of the pK for transition to an alkaline conformation with altered heme ligation . To assess the importance of position 76 in stabilizing the native versus the alkaline structure, the equilibrium and kinetic properties of the pH-induced conformational change have been compared for normal and mutant iso-2-cytochrome c . The pKapp for the conformational change is reduced from 8.45 (normal iso-2) to 6.71 in the mutant protein (Gly-76 iso-2), suggesting that conservation of Pro-76 may be required to stabilize the native conformation at physiological pH . The kinetics of the conformational change for both the normal and mutant proteins are well-described by a single kinetic phase throughout most of the pH-induced transition zone . Over this pH range, a minimal mechanism proposed for horse cytochrome c {Davis, L . A., Schejter, A., & Hess, G . P . (1974) J . Biol . Chem . 249, 2624-2632} is consistent with the data for normal and mutant yeast iso-2-cytochromes c: NH KH----N + H+ kcf in equilibrium kcb A NH and N are native forms of cytochrome c with a 695-nm absorbance band, A is an alkaline form that lacks the 695-nm band, KH is a proton dissociation constant, and kcf and kcb are microscopic rate constants for the conformational change . The Gly-76 mutation increases kcf by almost 70-fold, but kcb and KH are unchanged.(ABSTRACT TRUNCATED AT 250 WORDS) Eur J Biochem, 1989 Dec 8, 186(1-2), 71 - 7 Analysis of magnesium, europium and lead binding sites in methionine initiator and elongator tRNAs by specific metal-ion-induced cleavages; Ciesiolka J et al.; The specificity of cleavages in yeast and lupin initiator and elongator methionine tRNAs induced by magnesium, europium and lead has been analysed and compared with known patterns of yeast tRNA(Phe) hydrolysis . The strong D-loop cleavages occur in methionine elongator tRNAs at similar positions and with comparable efficiency to those found in tRNA(Phe), while the sites of weak anticodon loop cuts, identical in methionine elongator tRNAs, differ from those found in tRNA(Phe) . Methionine initiator tRNAs differ from their elongator counterparts: (a) they are cleaved in the D-loop with much lower efficiency; (b) they are cleaved in the variable loop which is completely resistant to hydrolysis in elongator tRNAs; (c) cleavages in the anticodon loop are stronger in initiator tRNAs and they are located mostly at the 5' side of the loop whereas in elongator tRNAs they occur mostly at the opposite, 3' side of the loop . The distinct pattern of the anticodon loop cleavages is considered to be related to different conformations of the anticodon loop in the two types of methionine tRNAs. J Biol Chem, 1989 Dec 5, 264(34), 20206 - 15 The mitochondrial targeting function of randomly generated peptide sequences correlates with predicted helical amphiphilicity; Lemire BD et al.; A pool of oligonucleotides encoding a start methionine and nine random amino acids was inserted at the 5'-end of the gene for the yeast cytochrome oxidase subunit IV lacking its own mitochondrial targeting sequence . Approximately one-quarter of the randomly generated sequences targeted subunit IV to its correct intramitochondrial location in vivo . Sequence analysis of 89 randomly generated sequences showed that their efficiencies as mitochondrial targeting signals correlated with the potential to fold into an amphiphilic alpha-helix . Functional targeting sequences were enriched in arginine and isoleucine residues but contained few aspartate, glutamate, and proline residues . Nonfunctional sequences predicted to have significant helical amphiphilicity often had at least one acidic or multiple helix-breaking residues that would be expected to interfere with targeting functioning . These results support the hypothesis that the signal for targeting a protein into the mitochondrial matrix is usually a positively charged amphiphilic helix. Diabetes Res, 1989 Dec, 12(4), 165 - 8 Non-enzymatic glycation of phosphoglucoisomerase; Zahner D et al.; Incubation of yeast phosphoglucoisomerase for 14 days at a high concentration (100 mM) of D-glucose was found to cause the non-enzymatic glycation of the enzyme . The kinetic properties of the glycated and control enzymes were similar in terms of specific activity, affinity for D-glucose 6-phosphate, isotopic discrimination between D-(U-14C) glucose 6-phosphate and D-(2-3H) glucose 6-phosphate, intramolecular 3H transfer from the latter substrate, and anomeric specificity . It is proposed that the quantitation of glycated phosphoglucoisomerase in distinct cell types may be used as an index for the non-enzymatic glycation of cytosolic proteins. Anal Biochem, 1989 Dec, 183(2), 220 - 4 Identification of covalently bound fatty acids on acylated proteins immobilized on nitrocellulose paper; Callahan FE et al.; A general method for identification of fatty acids covalently bound to acylated proteins following their electrophoretic transfer onto nitrocellulose paper is described . As demonstrated for {3H}palmitoylated RAS1 protein of Saccharomyces cerevisiae and the acylated acyl carrier protein of Spirodela oligorrhiza, this procedure alleviates the need for elution of proteins from polyacrylamide gel slices . Fatty acid ligands of such proteins are hydrolyzed directly from their immobilized state on the nitrocellulose paper, then derivatized with p-nitrophenacyl bromide, and finally resolved by reversed-phase high-performance liquid chromatography . The amount of acylated protein required for identification of acyl groups is minimized compared to that required for more conventional approaches by coupling a radioactive flow detector with the HPLC system. J Reprod Immunol, 1989 Dec, 16(3), 269 - 84 Inhibition of macrophage phagocytic activity by SV-IV, a major protein secreted from the rat seminal vesicle epithelium; Galdiero F et al.; The protein SV-IV, one of the major secretory proteins produced by the rat seminal vesicle epithelium, has been found to possess a marked ability to inhibit in vitro the phagocytic properties of activated peritoneal rat macrophages, by a mechanism that apparently involves phagocytes and target cells . Although SV-IV is a substrate for transglutaminase (TGase), an enzyme secreted by activated macrophages, TGase does not seem to play any significant role either in the binding of the protein to the cells participating in the phagocytic process or in the inhibition of macrophage phagocytosis by SV-IV . The significance of the findings in relation to the reproductive process and their possible clinical implications are discussed. Curr Genet, 1989 Dec, 16(5-6), 315 - 21 Mutations in ADE3 reduce the efficiency of the omnipotent suppressor sup45-2; Song JM et al.; Mutations in a known yeast gene, ADE3, were shown to act as an antisuppressor, reducing the efficiency of the omnipotent suppressor, sup45-2 . The ADE3 locus encodes the trifunctional enzyme C1-tetrahydrofolate synthase, which is required for the biosynthesis of purines, thymidylate, methionine, histidine, pantothenic acid and formylmethionyl-tRNA(fMet . The role of this enzyme in translational fidelity had not previously been suspected. Mol Cell Biol, 1989 Dec, 9(12), 5750 - 3 The C-terminal domain of the largest subunit of RNA polymerase II and transcription initiation; Moyle M et al.; Monoclonal antibodies specific for the evolutionarily conserved C-terminal heptapeptide repeat domain of the largest subunit of RNA polymerase II inhibited the initiation of transcription from mammalian promoters in vitro . Since these antibodies did not inhibit elongation and randomly initiated transcription, the heptapeptide repeats may function by binding class II transcription initiation factor(s). Mol Cell Biol, 1989 Dec, 9(12), 5387 - 94 RNA polymerase II subunit RPB3 is an essential component of the mRNA transcription apparatus; Kolodziej P et al.; To improve our understanding of RNA polymerase II, the gene that encodes its third-largest subunit, RPB3, was isolated from a lambda gt11 DNA library by using antibody probes . The RPB3 DNA sequence predicts a 318-amino-acid protein whose sequence was confirmed, in part, by microsequence analysis of the gel-purified RNA polymerase II subunit . RPB3 was found to be an essential single-copy gene that is tightly linked to HIS6 on chromosome IX . An RPB3 temperature-sensitive mutant that arrested growth after three to four generations at the restrictive temperature was isolated . When the mutant was shifted to the restrictive temperature, RNA polymerase II could no longer assemble, previously assembled functional enzyme was depleted, and mRNA levels were consequently reduced . These results demonstrate that RPB3 is an essential component of the mRNA transcription apparatus . Finally, the RPB3 protein is similar in sequence and length to RPC5, a subunit common to RNA polymerases I and III, suggesting that these subunits may play similar roles in RNA polymerases I, II, and III. Mol Cell Biol, 1989 Dec, 9(12), 5373 - 86 Identification of the DNA damage-responsive element of RNR2 and evidence that four distinct cellular factors bind it; Elledge SJ et al.; The RNR2 gene encodes the small subunit of ribonucleotide reductase, the enzyme that catalyzes the first step in the pathway for the production of the deoxyribonucleotides needed for DNA synthesis . Transcription of this gene is induced approximately 20-fold in response to environmental stimuli that damage DNA or block DNA replication . Deletion and subcloning analysis identified two, and possibly three, upstream activating sequences (UAS) and one repressing (URS) element in the RNR2 regulatory region . A 42-base-pair (bp) fragment from this region was found to be necessary for proper regulation of RNR2 and to be capable of conferring DNA damage inducibility upon a heterologous promoter . This fragment contained both positively and negatively acting sequences . Four DNA-binding factors interacted with the RNR2 regulatory region . One factor was identified as the GRF1 protein, the product of the RAP1 gene . GRF1 bound to the UAS2 element of RNR2, which was found to be directly adjacent to the 42-bp fragment . UAS2 activity was repressed by the 42-bp fragment . Three other factors bound to the 42-bp fragment; one of these factors, RRF3, had a second binding site in the RNR2 promoter . These factors are likely to mediate the response of RNR2 to DNA damage. Biol Chem Hoppe Seyler, 1989 Dec, 370(12), 1265 - 78 {Identification of human porins . II . Characterization and primary structure of a 31-lDa porin from human B lymphocytes (Porin 31HL).}; Kayser H et al.; We characterize and describe for the first time the primary structure of a human porin with the molecular mass of 31 kDa derived from the plasmalemm of B-lymphocytes (Porin 31HL) . Porin 31HL is shown to be a basic, channel forming membrane protein . The protein chain is composed of 282 amino acids with a relative molecular mass of 30641 Da without derivatisation . It is not a glycoprotein . The N-terminus is acetylated . Altogether the amino-acid sequence shows 56% hydrophilic or charged amino acids arranged in alternating regions of hydrophilic or hydrophobic character as it is typical for porins . In addition the 18 N-terminal amino acids of Porin 31HL can be arranged to an amphilic alpha-helix like in other porins . Porin 31HL shows approx . 29% or 24% identity to the primary structure of mitochondrial porins of Neurospora crassa and Saccharomyces cerevisiae . Partial data on mitochondrial porins from rat kidney and beef heart show sequence identity of about 90% to the human B cell porin elaborated here. J Cell Biol, 1989 Dec, 109(6 Pt 1), 2603 - 16 Protein import into mitochondria: ATP-dependent protein translocation activity in a submitochondrial fraction enriched in membrane contact sites and specific proteins; Pon L et al.; To identify the membrane regions through which yeast mitochondria import proteins from the cytoplasm, we have tagged these regions with two different partly translocated precursor proteins . One of these was bound to the mitochondrial surface of ATP-depleted mitochondria and could subsequently be chased into mitochondria upon addition of ATP . The other intermediate was irreversibly stuck across both mitochondrial membranes at protein import sites . Upon subfraction of the mitochondria, both intermediates cofractionated with membrane vesicles whose buoyant density was between that of inner and outer membranes . When these vesicles were prepared from mitochondria containing the chaseable intermediate, they internalized it upon addition of ATP . A non-hydrolyzable ATP analogue was inactive . This vesicle fraction contained closed, right-side-out inner membrane vesicles attached to leaky outer membrane vesicles . The vesicles contained the mitochondrial binding sites for cytoplasmic ribosomes and contained several mitochondrial proteins that were enriched relative to markers of inner or outer membranes . By immunoelectron microscopy, two of these proteins were concentrated at sites where mitochondrial inner and outer membranes are closely apposed . We conclude that these vesicles contain contact sites between the two mitochondrial membranes, that these sites are the entry point for proteins into mitochondria, and that the isolated vesicles are still translocation competent. Arch Biochem Biophys, 1989 Dec, 275(2), 371 - 8 Role of lysine methylation in the activities of elongation factor 1 alpha; Sherman M et al.; Previous work in our laboratory has demonstrated that 19% of the lysine residues in the protein synthesis elongation factor (EF-1 alpha) are methylated when the factor is purified from the mycelial form of the fungus Mucor racemosus . However, the same factor, when purified from spores of M . racemosus, is largely unmethylated . Despite its wide-spread occurrence in a great number of basic proteins, the functional significance of lysine N-methylation remains poorly understood . Spore and mycelial forms of EF-1 alpha were therefore compared in a series of assays to determine their relative affinities for various substrates and cofactors known to interact with the factor during the elongation cycle . The results suggested that hypomethylated and fully methylated EF-1 alpha had equal affinities for GTP, aminoacyl-tRNA, and ribosomes . Also, methylation did not appear to affect the accuracy of translation in an in vitro system . However, experiments did suggest that methylation may affect the ability of the factor to form complexes with other subunits (EF-1 beta gamma) which are known to enhance the overall rate of protein synthesis. J Virol, 1989 Dec, 63(12), 5483 - 8 Infection of peritoneal macrophages in vitro and in vivo with feline immunodeficiency virus; Brunner D et al.; Macrophages were harvested from the peritoneal cavities of healthy specific-pathogen-free cats by saline lavage . Three days before collection, the peritoneal cavities were stimulated with glutaraldehyde-fixed Saccharomyces cerevisiae cells to induce greater numbers of macrophages and to begin the activation sequence . Peritoneal macrophages from cats stimulated once with yeast consisted mainly of small macrophages and a smaller number of larger activated macrophages . After several days in culture, many of the small macrophages became activated and a portion of the activated macrophages developed into multinucleated giant cells . Peritoneal cells from cats that were stimulated twice or three times with yeast at 3-week intervals consisted of a higher proportion of activated macrophages initially and produced more and larger multinuclear giant cells with time . Cultures of peritoneal cells stimulated once with yeast were easily infected in vitro with feline immunodeficiency virus (FIV) and produced a transient burst of reverse transcriptase activity . After the initial burst of virus replication, the infection became latent . Even more multinucleated giant cells appeared after infection, and many of these cells fused with each other . Replicating virus could be rescued from the latently infected macrophages after 2 to 3 weeks of phorbol myristate acetate stimulation and cocultivation with T-lymphocyte-enriched peripheral blood mononuclear cells . Multiply stimulated peritoneal cells, which contained a much higher proportion of activated macrophages, could also be infected in vitro with FIV . The infection usually became latent, however, without going through an initial replicative stage . Peritoneal cells from chronically FIV-infected specific-pathogen-free cats contained a higher proportion of activated macrophages and were latently infected with FIV from the outset. Biochem Biophys Res Commun, 1989 Nov 30, 165(1), 118 - 24 Investigation on glyoxalase I inhibitors; Barnard JF et al.; Several classes of compounds - flavones, coumarins, S- and N-substituted glutathione analogs, transition state analogs, porphyrins, nucleotides and nucleosides - have been reported to inhibit the enzyme gloxalase I . In the current study, examination of some of the aforementioned compounds has revealed that squaric acid does not function as an inhibitor of glyoxalase I and several other compounds are much less effective in this regard than previously reported . Several new potent inhibitors of yeast glyoxalase I have been identified . Compounds containing the tropolone structure were especially inhibitory . Glutathione adducts of benzoquinone and naphthoquinone were also inhibitory and may be of particular interest with regard to the toxicology of normal aromatic metabolites in vivo. Gene, 1989 Nov 30, 83(2), 387 - 93 Sequence analysis of the URA1 gene encoding orotidine-5'-monophosphate decarboxylase of Schizophyllum commune; Froeliger EH et al.; The URA1 gene (encoding orotidine-5'-monophosphate decarboxylase) of the basidiomycete fungus Schizophyllum commune was mapped to a 1.4-kb BglI-BamHI fragment of two independent phage lambda clones previously isolated from a Schizophyllum genomic library . The fragment was identified by its ability to complement Schizophyllum ura1 mutants via transformation . The complete nucleotide sequence of the fragment containing the URA1 gene was determined . Sequence analysis revealed that the coding region of the URA1 gene encompasses a polypeptide of 279 amino acids (aa) interrupted by two small introns . The deduced aa sequence corresponds to 30.3 kDa and is substantially similar to the sequences of analogous polypeptides from other organisms . No canonical 5'-TATA sequence nor 3'-AATAAA polyadenylation signal are evident in the flanking regions of the URA1 gene. J Immunol Methods, 1989 Nov 30, 124(2), 231 - 4 Assessment of phagocytic activity of granulocytes using laser flow cytometry; Buschmann H et al.; A laser flow cytometer assay for assessing two functions of polymorphonuclear leucocytes is described . The technique permits the quantitation of phagocytosis and intracellular killing of yeast cells by leucocytes and is illustrated with results obtained using porcine polymorphonuclear leucocytes. Biochim Biophys Acta, 1989 Nov 28, 1006(2), 209 - 13 The 3-hydroxy group of lanosterol is essential for orienting the substrate site of cytochrome P-450(14DM) (lanosterol 14 alpha- demethylase); Aoyama Y et al.; Interaction of lanosterol, 3-epilanosterol, 3-oxolanosta-8,24-diene, 3-methylenelanost-8-ene and lanosterol acetate with cytochrome P-450(14DM) were studied . The cytochrome mediated the 14alpha-demethylation of 3-epilanosterol with nearly the same activity as lanosterol but could not mediate the 14alpha-demethylation of the 3-methylene derivative and the 3-acetate . The cytochrome catalyzed the 14alpha-demethylation of the 3-oxo derivative with low rate . Based on these and some additional observations the hydrogen bond formation between the 3-hydroxy group of lanosterol and the specific amino acid residue in the substrate site is assumed to be essential for orienting the substrate in the substrate site of the cytochrome. Lancet, 1989 Nov 25, 2(8674), 1236 - 9 Association of low levels of mannan-binding protein with a common defect of opsonisation; Super M et al.; Failure to opsonise bakers' yeast (Saccharomyces cerevisiae) is a defect found in 5-7% of the general population . In this study, the presence of the defect was linked with low levels of mannan-binding protein (MBP), a calcium-dependent serum lectin . Purified MBP corrected the defect in a dose-dependent way in an in-vitro assay measuring the deposition of complement moieties on a mannan-coated surface . There was a highly significant correlation between the serum MBP level and the generation of C3b opsonins in a population of healthy blood donors . The median MBP level of ten children previously shown to have the functional opsonic defect was 4.9 micrograms/l (range 2.5-35.0 micrograms/l) compared with 143 micrograms/l (range 2.5-880 micrograms/l) for a paediatric control group. Science, 1989 Nov 24, 246(4933), 1034 - 8 Similarity between the transcriptional silencer binding proteins ABF1 and RAP1; Diffley JF et al.; The yeast ARS binding factor 1 (ABF1)--where ARS is an autonomously replicating sequence--and repressor/activator protein 1 (RAP1) have been implicated in DNA replication, transcriptional activation, and transcriptional silencing . The ABF1 gene was cloned and sequenced and shown to be essential for viability . The predicted amino acid sequence contains a novel sequence motif related to the zinc finger, and the ABF1 protein requires zinc and unmodified cysteine residues for sequence-specific DNA binding . Interestingly, ABF1 is extensively related to its counterpart, RAP1, and both proteins share a region of similarity with SAN1, a suppressor of certain SIR4 mutations, suggesting that this region may be involved in mediating SIR function at the silent mating type loci. Science, 1989 Nov 17, 246(4932), 931 - 5 BAS1 has a Myb motif and activates HIS4 transcription only in combination with BAS2; Tice-Baldwin K et al.; The BAS1 and BAS2 proteins are both required for activation of GCN4-independent (basal) HIS4 transcription in yeast . BAS1 has an NH2-terminal region similar to those of the myb proto-oncogene family . BAS1 and BAS2, which contains a homeo box, bound to adjacent sites on the HIS4 promoter . The joint requirement of BAS1 and BAS2 for activation is probably not due to cooperative binding or the transcriptional control of one of the genes by the other . Although BAS1 and BAS2 were both required for activation of HIS4 transcription, BAS1 was not required for BAS2-dependent expression of the secreted acid phosphatases . The transcriptional activators of HIS4 have DNA binding domains that are conserved in evolution (BAS1 = Myb, BAS2 = homeo box, GCN4 = Jun) . Their interactions, therefore, may be relevant to the control of gene expression in more complex systems. Biochem J, 1989 Nov 15, 264(1), 175 - 84 An artificial-intelligence technique for qualitatively deriving enzyme kinetic mechanisms from initial-velocity measurements and its application to hexokinase; Garfinkel L et al.; We have developed a computer method based on artificial-intelligence techniques for qualitatively analysing steady-state initial-velocity enzyme kinetic data . We have applied our system to experiments on hexokinase from a variety of sources: yeast, ascites and muscle . Our system accepts qualitative stylized descriptions of experimental data, infers constraints from the observed data behaviour and then compares the experimentally inferred constraints with corresponding theoretical model-based constraints . It is desirable to have large data sets which include the results of a variety of experiments . Human intervention is needed to interpret non-kinetic information, differences in conditions, etc . Different strategies were used by the several experimenters whose data was studied to formulate mechanisms for their enzyme preparations, including different methods (product inhibitors or alternate substrates), different experimental protocols (monitoring enzyme activity differently), or different experimental conditions (temperature, pH or ionic strength) . The different ordered and rapid-equilibrium mechanisms proposed by these experimenters were generally consistent with their data . On comparing the constraints derived from the several experimental data sets, they are found to be in much less disagreement than the mechanisms published, and some of the disagreement can be ascribed to different experimental conditions (especially ionic strength). Biochem Pharmacol, 1989 Nov 15, 38(22), 3969 - 79 Alkylthio acetic acids (3-thia fatty acids)--a new group of non-beta-oxidizable peroxisome-inducing fatty acid analogues--II . Dose-response studies on hepatic peroxisomal- and mitochondrial changes and long-chain fatty acid metabolizing enzymes in rats; Berge RK et al.; The activity of key enzymes involved in oxidation and esterification of long-chain fatty acids was investigated after male Wistar rats were treated with different doses of sulfur substituted fatty acid analogues, 1,10-bis(carboxymethylthiodecane) (BCMTD, non-beta-oxidizable and non-omega-oxidizable), 1-mono(carboxymethylthiotetradecane) (CMTTD, trivial name, alkylthio acetic acid, non-beta-oxidizable) and 1-mono(carboxyethylthiotetradecane) (CETTD trivial name, alkylthio propionic acid, beta-oxidizable) . The sulfur substituted dicarboxylic acid and the alkylthio acetic acid induced in a dose-dependent manner the mitochondrial, microsomal and especially the peroxisomal palmitoyl-CoA synthetase activity, the mitochondrial and cytosolic palmitoyl-CoA hydrolase activity, the mitochondrial and especially the microsomal glycerophosphate acyltransferase activity and the peroxisomal beta-oxidation, especially revealed in the microsomal fraction . Morphometric analysis of randomly selected hepatocytes revealed that BCMTD and CMTTD treatment increased the number, size and volume fraction of peroxisomes and mitochondria . Thus, the observed changes in the specific activity of fatty acid metabolizing enzymes with multiple subcellular localization can partly be explained as an effect of changes in the s-values of the organelles as proliferation of mitochondria and peroxisomes occurred . The most striking effect of the alkylthio propionic acid was the formation of numerous fat droplets in the liver cells and enhancement of the hepatic triglyceride level . This was in contrast to BCMTD treatment which decreased the hepatic triglyceride content . In conclusion, the results provide evidence that administration of non-beta-oxidizable fatty acid analogues had much higher in vivo potency in inducing hepatomegaly and key enzymes involved in fatty acid metabolism, including proliferation of peroxisomes and mitochondria than is exhibited in the beta-oxidizable, alkylthio propionic acid . Moreover, the dicarboxylic acid was apparently three to six times more potent than the alkylthio acetic acid in inducing peroxisomal beta-oxidation and peroxisome proliferation when considered on a mumol/day basis . As palmitic acid and hexadecanedioic acid only marginally affected these hepatic responses, it is conceivable that the potency of the selected compounds as proliferators of peroxisomes and inducers of the associated enzymes depends on their accessibility for beta-oxidation. Biochem Biophys Res Commun, 1989 Nov 15, 164(3), 1170 - 5 Defective sterol C5-6 desaturation and azole resistance: a new hypothesis for the mode of action of azole antifungals; Watson PF et al.; Two azole resistant isolates of Saccharomyces cerevisiae carried mutations allelic to erg 3 and were blocked to differing degrees at the C5-6 desaturation step of ergosterol biosynthesis . When treated with the sterol 14 alpha-demethylation inhibitor fluconazole the wild-type sensitive strain accumulated lanosterol and 14 alpha-methyl-erogosta-8,24(28)-dien-3 beta, 6 alpha-diol (14-methyl-3,6 diol) . The stringent desaturase mutant, A2, accumulated 14 alpha-methyl-8,24(28)-dien-3 beta-ol (14-methyl fecosterol) and lanosterol as the major sterol components when treated with fluconazole . Resistant isolate A3 accumulated 14-methyl-3,6-diol, 14-methyl fecosterol, and lanosterol and was only partially blocked at sterol C5-6 desaturation . We conclude that functional sterol C5-6 desaturase is required for the synthesis of 14-methyl-3,6-diol under conditions of azole inhibition . We present a new hypothesis for the mode of action of azole antifungals based on the inability of 14-methyl-3,6-diol to support growth, and suggest that growth can occur through utilisation of 14-methyl fecosterol, produced by a combination of azole inhibition and defective sterol C5-6 desaturation. Biochemistry, 1989 Nov 14, 28(23), 9047 - 52 Purification of recombinant pp60v-src protein tyrosine kinase and phosphorylation of peptides with different secondary structure preference; Radziejewski C et al.; The expression of the transforming gene product of Rous sarcoma virus (pp60v-src) in Saccharomyces cerevisiae has recently been reported (Kornbluth et al., 1987; Brugge et al., 1987) . To carry out biochemical and structural studies of this enzyme, a facile purification was developed . The purification was accomplished in four chromatographic steps: Q-Sepharose, Affi-Gel Blue, phosphoagarose, and hydroxylapatite chromatography . The tyrosine kinase was isolated in milligram quantities as two highly active proteolytic fragments (52 and 54 kDa) . Three model tyrosine kinase substrates with propensities to adopt helical or omega-loop conformations were synthesized and characterized . The peptides were based on the sites of phosphorylation of pp60v-src, lipocortin I, and lipocortin II . Circular dichroism spectroscopy was used to study the conformation of the helix-forming peptides in 50 mM Tris and in 50% trifluoroethanol/Tris . Peptide 1, which was designed to form an amphiphilic alpha-helix, displayed 24.2% helicity in buffer and 40.2% helicity in 50% TFE/buffer . Similar experiments for peptide 3, the other helix former, showed a lower helicity (8.1% helical and 26.0% helical in buffer and in 50% TFE/buffer, respectively) . All three peptides were shown to be substrates for the recombinant tyrosine kinase . Kinetic measurements using high-voltage paper electrophoresis indicated that the helix-forming peptides exhibited low KM values (approximately 450 microM) for the purified src gene product, consistent with the notion that elements of secondary structure may be important in substrate recognition by tyrosine kinases. Eur J Biochem, 1989 Nov 6, 185(2), 419 - 23 Study of the fast-reacting cysteines in phosphoglycerate kinase using chemical modification and site-directed mutagenesis; Minard P et al.; Horse muscle phosphoglycerate kinase, like other mammalian phosphoglycerate kinases, contains seven cysteine residues of which two react rapidly with 5,5'-dithio-bis(2-nitrobenzoate) (Nbs2) following second-order kinetics (k = 640 M-1.s-1) . Selective cyanylation of the fast-reacting cysteines, followed by chemical cleavage and subsequent sodium dodecyl sulfate/polyacrylamide gel electrophoresis analysis of the resulting polypeptides, suggested that these cysteines are at positions 378 and 379 . Cysteine residues were introduced into yeast phosphoglycerate kinase by site-directed mutagenesis . Mutant enzymes, each containing only one cysteine residue at position 364, 376, or 377, were constructed from a mutant devoid of cysteine (Cys97----Ala) . In the last two mutants, the cysteines were at positions corresponding to Cys378 and Cys379, respectively, in the horse muscle enzyme . The chemical reactivity of the cysteine groups in these latter two yeast mutant enzymes was similar to that of the fast-reacting cysteines in the horse muscle enzyme . Furthermore, they were similarly modified upon substrate binding . All these data demonstrate unambiguously that the fast-reacting cysteines in the horse muscle enzyme are Cys378 and Cys379. FEBS Lett, 1989 Nov 6, 257(2), 247 - 50 Prothymosin alpha is an evolutionary conserved protein covalently linked to a small RNA; Makarova T et al.; A 13 kDa protein, covalently linked to a small RNA from the cytoplasm of mouse cells, was studied . Sequence analysis of its tryptic peptides revealed that the RNA-linked protein is identical to prothymosin alpha . Very similar RNA-protein complexes were identified in human, bovine and yeast cells . Tryptic peptide maps of 125I-labelled RNA-linked proteins of diverse origin demonstrated their marked similarity, thus indicating high evolutionary conservation of prothymosin alpha from yeast to man. J Med Virol, 1989 Nov, 29(3), 204 - 14 Mutations that change the immunological subtype of hepatitis B virus surface antigen and distinguish between antigenic and immunogenic determination; Ashton-Rickardt PG et al.; The molecular basis of the d or y immunological subtype of hepatitis B virus (HBV) surface antigen (HBsAg) has been investigated by mutation of specific amino acid residues . When combined with substitution of serine 113 by threonine, replacement of arginine 122 by lysine or of tyrosine 134 by phenylalanine, or both of these changes, altered the antigenic subtype of HBsAg from y+d- to y+d+ . These same mutations had a more dramatic effect on the subtype of antibodies induced by the antigens, a combination of all three mutations completely changing the subtype from y to d . Our study thus identifies residues in HBsAg that not only affect the subtype but discriminate between changes in antigenic and immunogenic behaviour . It also shows how the y and d subtypes may be manifest by the same molecule. J Med Virol, 1989 Nov, 29(3), 196 - 203 Mutants of the hepatitis B virus surface antigen that define some antigenically essential residues in the immunodominant a region; Ashton-Rickardt PG et al.; The nature of the immunodominant a region of hepatitis B virus surface antigen (HBsAg) has been examined by mutation of specific amino acid residues . Proline 142 was required for the exhibition of full antigenicity . Replacement of cysteines 124 and 147 by serines drastically reduced or eliminated reactivity with antibodies to HBsAg, which implicates these two residues in stabilising the conformation of the antigen . The a region of HBsAg has also been shown to influence both the immunoreactivity of the adjacent subtype antigenic region, despite being immunologically distinct from it, and the ability of the antigen to interact with a subtype-specific monoclonal antibody . These results emphasise the importance of the polypeptide region between cysteine residues 124 and 147 in determining a antigenicity, as well as manifestation of the subtype of HBsAg, and reinforce the view that these are conformational epitopes. DNA, 1989 Nov, 8(9), 623 - 34 Characterization and sequence analysis of the human ornithine decarboxylase gene; Fitzgerald MC et al.; This report describes the characterization and complete sequence of the human ornithine decarboxylase (ODC) gene . Genomic Southern blot analysis shows only a single gene hybridizing at high stringency, in contrast to the murine multigene family . A Pst I restriction fragment length polymorphism was identified and an allele of the human ODC gene containing the polymorphic Pst I site was cloned and sequenced . The ODC gene is divided into 12 exons and spans 8 kb . Comparison of the human, rat, and mouse ODC genes shows striking conservation of genomic organization, as well as 82% identity in the first 148 bp of the 5'-flanking region . This region contains a TATA box, cAMP-responsive element, CCAAT box, and AP-2 binding site and is consistent with induction of ODC gene expression by both the cAMP and protein kinase C-mediated signaling pathways . The first intron of the human gene is 2,849 bp in length, and contains two putative Sp1 binding sites, as well as an Ap1 binding site, suggesting a role for the first intron in transcriptional regulation . The 5' noncoding region of the predicted mRNA contains regions of virtual identity with that of mouse and rat ODC mRNA, suggesting sequences involved in translational regulation . In addition, it was found that the exon segments corresponding to the amino and carboxyl termini of Saccharomyces cerevisiae and Trypanosoma b . brucei are unrelated to their mammalian counterparts, whereas the middle segments of the protein are conserved . These differences may influence the difference in protein half-life seen between T . b . brucei and mammalian ODC. Chromosoma, 1989 Nov, 98(5), 309 - 16 SINEs and LINEs cluster in distinct DNA fragments of Giemsa band size; Chen TL et al.; By in situ hybridization, short interspersed repeated DNA elements (SINEs), exemplified by Alu repeats, are located principally in Giemsa-light human metaphase chromosome bands . In contrast, the L1 family of long interspersed repeats (LINEs) preferentially cluster in Giemsa-dark bands . These SINE/LINE patterns also generally correspond to early and later replication band patterns . In order to provide a molecular link between structurally visible chromosome bands and a framework of interspersed repeats, we investigated patterns of SINE and LINE hybridization using pulse-field gel electrophoresis (PFGE) . Interspersed SINEs and LINEs hybridize with high intensity to specific size fragments of 0.2-3 megabase pairs (Mb) . Using appropriate restriction enzymes and pulse-field conditions, a number of fragments were delineated that were either SINE or LINE rich, and were mutually exclusive . Control studies with a human endogenous retroviral repeat that is related in sequence to the major LINE family, delineated a subset of fragments of 0.07-0.4 Mb with unequal intensity . Thus these less numerous repeats also appear to cluster selectively in DNA domains that are larger than a chromosome loop (60-120 kb) . In summary, PFGE studies independently confirm the clustering of interspersed repeats on contiguous DNA loops . Selective clustering of repeat motifs may contribute to special structural or functional properties of large chromosome domains, such as chromatin extension/condensation or replication characteristics . In some cases the DNA fragments defined by these repeats approach the size of tandem satellite arrays. Appl Biochem Biotechnol, 1989 Nov, 22(2), 119 - 27 Removal of RNA during protein concentration by means of enzyme membrane; Rucka M et al.; Immobilization of RNase in PVC ultrafiltration membranes was carried out . The obtained membranes were used for concentration of BSA solution, RNA being simultaneously removed . The yield of RNA hydrolysis was found to be controlled by the initial concentration of RNA in feed solution . The protein affected enzyme action as a result of its adsorption on the membrane surface at the beginning of ultrafiltration, whereas it did not inhibit RNase activity during the process. Anal Biochem, 1989 Nov 1, 182(2), 452 - 6 Assay for phosphatidylserine decarboxylase utilizing DEAE-cellulose column chromatography; Overmeyer JH et al.; A simple assay for phosphatidylserine decarboxylase is described . Following incubation of a mitochondrial fraction from Saccharomyces cerevisiae with purified, exogenous phosphatidyl{3H}serine, the lipid extract is applied to a small DEAE-cellulose column equilibrated in CHCI3-CH3OH (1:1) . The unreacted substrate, phosphatidyl{3H}serine, is quantitatively bound by the ion-exchange column while the product, phosphatidyl{3H}ethanolamine, is eluted by sequential washing with CHCI3-CH3OH (1:1) and CH3OH . The organic solvents are evaporated, and the amount of radiolabeled phosphatidyl{3H}ethanolamine formed by enzymatic decarboxylation is determined by liquid scintillation spectrometry . The reliability of this assay was established by showing that several enzymatic properties of the yeast enzyme, defined by the new assay, were essentially identical to the properties characterized by a more tedious paper chromatographic assay described previously . Virtually identical rates of enzymatic decarboxylation of phosphatidyl{3H}serine were also obtained for mitochondrial fractions from pig brain and rat liver when the activities were compared by the column and paper chromatographic methods. Biochem J, 1989 Nov 1, 263(3), 849 - 53 The role of the C-terminal tail of flavocytochrome b2; White SA et al.; A flavocytochrome b2 (L-lactate dehydrogenase) mutant was constructed in which the C-terminal tail (23 amino acid residues) had been deleted (Gly-489----Stop) . This tail appears to form many intersubunit contacts in the tetrameric wild-type protein, and it was expected that its removal might lead to the formation of monomeric flavocytochrome b2 . The isolated tail-deleted mutant enzyme (TD-b2), however, was found to be tetrameric (Mr 220,000) . TD-b2 shows Km and kcat . values (at 25 degrees C and pH 7.5) of 0.96 +/- 0.06 mM and 165 +/- 6 s-1 respectively compared with 0.49 +/- 0.04 mM and 200 +/- 10 s-1 for the wild-type enzyme . The kinetic isotope effect with {2-2H}lactate as substrate seen for TD-b2, with ferricyanide as electron acceptor, was essentially the same as that observed for the wild-type enzyme . TD-b2 exhibited loss of activity during turnover in a biphasic process . The rate of the faster of the two phases was dependent on L-lactate concentration and at saturating concentrations showed a first-order deactivation rate constant, kf(deact.), of 0.029 s-1 (at 25 degrees C and pH 7.5) . The slower phase, however, was independent of L-lactate concentration and gave a first-order deactivation rate constant, ks(deact.), of 0.01 s-1 (at 25 degrees C and pH 7.5) . This slower phase was found to correlate with dissociation of FMN, which is one of the prosthetic groups of the enzyme . Thus fully deactivated TD-b2, which was also tetrameric, was found to be completely devoid of FMN . Much of the original activity of TD-b2 could be recovered by re-incorporation of FMN . Thus the C-terminal tail of flavocytochrome b2 appears to be required for the structural integrity of the enzyme around the flavin active site even though the two are well separated in space. Pept Res, 1989 Nov-Dec, 2(6), 381 - 8 Total chemical synthesis of ubiquitin using BOP reagent: biochemical and immunochemical properties of the purified synthetic product; Briand JP et al.; The complete ubiquitin molecule (76 residues) has been synthesized by the solid-phase method of Merrifield by using BOP as the coupling reagent . The crude product was purified by gel filtration, middle-pressure liquid chromatography and ion-exchange chromatography . Seven mg of a ca . 95% pure peptide was finally obtained; the overall yield of the synthesis was 1% . The final product was controlled by amino acid analysis and sequencing, HPLC and FAB-Mass spectrometry . Synthetic ubiquitin was found to be antigenically active in immunoblotting experiments and in an enzyme-linked immunosorbent assay with anti-ubiquitin antibodies as well as with anti-ubiquitin autoantibodies from autoimmune patients . Its activity was controlled in a conjugation enzymatic system and was found similar to that of commercial ubiquitin . The successful total synthesis of ubiquitin opens the way to the preparation of various stable analogs that should be useful for studying the intracellular metabolism of this molecular and its involvement in the protein degradation pathway. New Biol, 1989 Nov, 1(2), 181 - 91 Jun DNA-binding is modulated by mutations between the leucines or by direct interaction of fos with the TGACTCA sequence; Hirai S et al.; Mutations between the leucines of the "leucine zipper" domain of Jun D can either decrease (Asn 301 to Ala) or increase (Thr 307, Ala 308, to Glu, Val) homodimer formation and specific binding to DNA even though such changes do not modify the predicted alpha-helical structure of this region . As shown previously, addition of Fos strongly increases the affinity of Jun for DNA by forming a heterodimer . The jun down mutation (Asn 301 to Ala) also diminishes DNA binding by the Fos-Jun D heterodimer . These data strongly support the coiled coil conformation of this region where residues adjacent to the leucines are also important for dimer formation . Ultraviolet cross-linking experiments have shown that both Fos and Jun directly contact the TGACTCA palindromic sequence defined as a TPA (12-O-tetradecanoyl phorbol-13-acetate) response element or TRE . Both Jun homodimers and Jun-Fos heterodimers bind this TRE as well as the cAMP responsive element (CRE or TGACGTCA) with comparable affinities . While strong c-Jun or Jun D binding requires a perfect palindrome, Jun-Fos complexes can also efficiently recognize sequences where the right half of the palindrome is less conserved (TGACTAA or TGACGCA). Proc Natl Acad Sci U S A, 1989 Nov, 86(21), 8432 - 6 Translocation of proteins across the mitochondrial inner membrane, but not into the outer membrane, requires nucleoside triphosphates in the matrix; Hwang ST et al.; Work in several laboratories has established that import of proteins across the mitochondrial inner membrane requires an electrochemical potential across that membrane and cleavage of nucleoside triphosphate . We now show that nucleoside triphosphate must be present inside the inner membrane . In contrast, the potential-independent insertion of porin into the outer membrane requires ATP only outside the inner membrane. J Virol, 1989 Nov, 63(11), 4913 - 8 In vitro mutagenesis of the herpes simplex virus type 1 DNA polymerase gene results in altered drug sensitivity of the enzyme; Matthews JT et al.; A mutation (asparagine 815 to serine 815) was introduced into the herpes simplex virus type 1 (HSV-1) DNA polymerase (pol) . The HSV-1 pol enzyme in lysates of Saccharomyces cerevisiae cells expressing the mutant protein showed increased resistance to acyclovir triphosphate and increased sensitivity to phosphonoacetate but was not substantially altered with respect to sensitivity to phosphonoformate or aphidicolin . These results directly demonstrate that both resistance to acyclovir triphosphate and sensitivity to phosphonoacetate can be conferred by this mutation in the absence of other viral factors and that the yeast expression system can be used for structure-function studies on HSV-1 pol. Mol Cell Biol, 1989 Nov, 9(11), 4645 - 52 Regulation of the nuclear genes encoding the cytoplasmic and mitochondrial leucyl-tRNA synthetases of Neurospora crassa; Chow CM et al.; We show that the nuclear genes for the cytoplasmic and mitochondrial leucyl-tRNA synthetase (LeuRS) of Neurospora crassa are distinct in their encoded proteins, codon usage, mRNA levels, and regulation . The 4.2-kilobase-pair region representing the structural gene for cytoplasmic LeuRS and flanking regions has been sequenced . The positions of the 5' and 3' ends of mRNA and of a single 62-base-pair intron have been mapped . The methionine-initiated open reading frame encoded a protein of 1,123 amino acids and displayed a strong codon bias . Although cytoplasmic LeuRS shares with mitochondrial LeuRS some general features common to most aminoacyl-tRNA synthetases, there is little amino acid sequence similarity between them, mRNA levels for cytoplasmic LeuRS were much higher than those for mitochondrial LeuRS . This observation and the strong codon bias in the cytoplasmic LeuRS gene may contribute to a greater abundance of cytoplasmic LeuRS than mitochondrial LeuRS . The genes for cytoplasmic and mitochondrial LeuRS are regulated independently . The cytoplasmic LeuRS gene is regulated by the cross-pathway control system in N . crassa, which is analogous to general amino acid control in Saccharomyces cerevisiae . The cytoplasmic LeuRS mRNA levels are induced by amino acid starvation resulting from the addition of aminotriazole . Part of this increase is due to utilization of new transcription start sites . In contrast, the mitochondrial LeuRS gene is not induced by amino acid limitation . However, the mitochondrial LeuRS mRNA levels did increase dramatically upon inhibition of mitochondrial protein synthesis by chloramphenicol or ethidium bromide or in the temperature-sensitive strain leu-5 carrying a mutation in the mitochondrial LeuRS structural gene. Proc Natl Acad Sci U S A, 1989 Nov, 86(22), 8708 - 11 Vacuolar ATPases, like F1,F0-ATPases, show a strong dependence of the reaction velocity on the binding of more than one ATP per enzyme; Kasho VN et al.; Recent studies with vacuolar ATPases have shown that multiple copies catalytic subunits are present and that these have definite sequence homology with catalytic subunits of the F1,F0-ATPases . Experiments are reported that assess whether the vacuolar ATPases may have the unusual catalytic cooperativity with sequential catalytic site participation as in the binding change mechanism for the F1,F0-ATPases . The extent of reversal of bound ATP hydrolysis to bound ADP and Pi as medium ATP concentration was lowered was determined by 18O-exchange measurements for yeast and neurospora vacuolar ATPases . The results show a pronounced increase in the extent of water oxygen incorporation into the Pi formed as ATP concentration is decreased to the micromolar range . The F1,F0-ATPase from neurospora mitochondria showed an even more pronounced modulation, similar to that of other F1-type ATPases . The vacuolar ATPases thus appear to have a catalytic mechanism quite analogous to that of the F1,F0-ATPases. Science, 1989 Oct 27, 246(4929), 482 - 6 Intracellular targeting and structural conservation of a prohormone-processing endoprotease; Fuller RS et al.; The prohormone-processing endoprotease (KEX2 gene product) of the yeast Saccharomyces cerevisiae is a membrane-bound, 135,000-dalton glycoprotein, which contains both asparagine-linked and serine- and threonine-linked oligosaccharide and resides in a secretory compartment . Analysis of mutant kex2 genes truncated at their 3' end indicates that carboxyl terminal domains of the enzyme are required for its proper localization within the cell . A human gene product, "furin," shares 50% identity with the catalytic domain of Kex2 protease and is, therefore, a candidate for a human prohormone-processing enzyme. Cell, 1989 Oct 20, 59(2), 349 - 58 Identification of functional U1 snRNA-pre-mRNA complexes committed to spliceosome assembly and splicing; Seraphin B et al.; Although both U1 and U2 snRNPs have been implicated in the splicing process, their respective roles in the earliest stages of intron recognition and spliceosome assembly are uncertain . To address this issue, we developed a new strategy to prepare snRNP-depleted splicing extracts using Saccharomyces cerevisiae cells conditionally expressing U1 or U2 snRNP . Complementation analyses and chase experiments show that a stable complex, committed to the splicing pathway, forms in the absence of U2 snRNP . U1 snRNP and a substrate containing both a 5' splice site and a branchpoint sequence are required for optimal formation of this commitment complex . We developed new gel electrophoresis conditions to identify these committed complexes and to show that they contain U1 snRNA . Chase experiments demonstrated that these complexes are functional intermediates in spliceosome assembly and splicing . Our results have implications for the process of splice site selection. Biochemistry, 1989 Oct 17, 28(21), 8588 - 96 A simple model for proteins with interacting domains . Applications to scanning calorimetry data; Brandts JF et al.; A simple thermodynamic model is formulated for the purpose of interpreting scanning calorimetry data on proteins that have interacting domains . Interactions are quantified by inclusion of an interface free energy, delta GAB, in the thermodynamics of unfolding for multidomain proteins . The assumption is made that delta GAB goes to zero with the unfolding of either domain involved in pairwise interaction, so the interaction term appears to stabilize only the domain with the lower TM . Application of the model to calorimetric data leads to an estimate of -25,000 cal/mol for interactions between the regulatory and catalytic subunits of native aspartate transcarbamoylase and to a value of 0 for delta GAB between the transmembrane and cytoplasmic domains of band 3 of the human erythrocyte membrane . Estimates of changes in delta GAB are also obtained for mutant forms of yeast phosphoglycerate kinase that have been altered in the hinge region between amino-terminal and carboxy-terminal domains . The model is also applied to ligand binding to proteins having domains that communicate through pairwise interaction . It is shown that whenever the delta GAB term is ligand-dependent, then attachment of the ligand to the binding domain will be partially controlled by the other (regulatory) domain . This situation can sometimes be recognized and quantified when calorimetric scans are carried out at varying ligand concentrations . According to the model, the binding of MgATP to the carboxy-terminal domain of phosphoglycerate kinase is strongly stabilized (ca . 20% of the unitary free energy of binding) by participation of the amino-terminal domain, which acts to increase the binding constant 25-fold.(ABSTRACT TRUNCATED AT 250 WORDS) Biochim Biophys Acta, 1989 Oct 17, 1005(3), 289 - 95 Interactions of thiophosphatidic acid with enzymes which metabolize phosphatidic acid . Inhibition of phosphatidic acid phosphatase and utilization by CDP-diacylglycerol synthase; Bonnel SI et al.; Thiophosphatidic acid (1,2-diacyl-sn-glycero-3-phosphorothioate; thioPA) was chemically synthesized from egg phosphatidylcholine-derived 1,2-diacylglycerol and PSCl3 and tested for its effects on enzymes which utilize phosphatidic acid (PA) in phospholipid biosynthesis . The compound was not a substrate for rat liver cytosolic PA phosphatase and strongly inhibited this enzyme activity . ThioPA was also a potent inhibitor of purified membrane-associated PA phosphatase from Saccharomyces cerevisiae in a competitive manner and exhibited an apparent Ki = 60 microM . In contrast, purified CDPdiacylglycerol synthase (PA:CTP cytidylyltransferase) from this organism was able to convert thioPA to CDP-diacylglycerol . The apparent Vmax for thioPA was 7-fold lower than that for PA, whereas the apparent Km for thioPA (70 microM) was 4-fold lower than that for PA . Calculation of the specificity constant (Vmax/Km) demonstrated that PA was the preferred substrate . These properties of thioPA indicate that this substance may prove useful in studies of phospholipid metabolism and function. Gene, 1989 Oct 15, 82(1), 91 - 114 Group I introns as mobile genetic elements: facts and mechanistic speculations--a review; Dujon B; Group I introns form a structural and functional group of introns with widespread but irregular distribution among very diverse organisms and genetic systems . Evidence is now accumulating that several group I introns are mobile genetic elements with properties similar to those originally described for the omega system of Saccharomyces cerevisiae: mobile group I introns encode sequence-specific double-strand (ds) endoDNases, which recognize and cleave intronless genes to insert a copy of the intron by a ds-break repair mechanism . This mechanism results in: the efficient propagation of group I introns into their cognate sites; their maintenance at the site against spontaneous loss; and, perhaps, their transposition to different sites . The spontaneous loss of group I introns occurs with low frequency by an RNA-mediated mechanism . This mechanism eliminates introns defective for mobility and/or for RNA splicing . Mechanisms of intron acquisition and intron loss must create an equilibrium, which explains the irregular distribution of group I introns in various genetic systems . Furthermore, the observed distribution also predicts that horizontal transfer of intron sequences must occur between unrelated species, using vectors yet to be discovered. Nucleic Acids Res, 1989 Oct 11, 17(19), 7581 - 90 Vaccinia virus encodes a thymidylate kinase gene: sequence and transcriptional mapping; Smith GL et al.; The nucleotide sequence and deduced amino acid sequence of a vaccinia virus gene from the SalI F fragment are shown . The predicted polypeptide shares 42% amino acid identity over a 200 amino acid region with Saccharomyces cerevisiae thymidylate kinase (TmpK) and has low homology with herpes simplex virus deoxypyrimidine kinase . Northern blotting and S1 nuclease protection showed that the TmpK gene is transcribed early during infection and mapped the mRNA 5' end to immediately upstream of the second inframe ATG codon of the open reading frame (ORF) . The encoded polypeptide is predicted to be 204 amino acids long (23.2 kD) and is almost colinear with yeast TmpK . Vaccinia virus possesses genes for TK and TmpK, separated by 57 kilobases of DNA, which are co-ordinately expressed and the encoded enzymes perform sequential steps in the same biochemical pathway. FEBS Lett, 1989 Oct 9, 256(1-2), 4 - 10 The number of molecules taken up by electroporated cells: quantitative determination; Bartoletti DC et al.; Fluorescent and fluorescent-labeled molecules were used with calibrated flow cytometric fluorescence measurements of electrically pulsed cells (intact yeast: Saccharomyces cerevisiae) to demonstrate a method for determining the net number of molecules transported into electroporated cells . For the conditions used, a single pulse of width 50 microseconds and magnitude 8.0 +/- 0.5 kV/cm resulted in an average net molecular uptake which is large, n = 1.4 x 10(5) molecules of 70 kDa FITC-dextran (supplied extracellular concentration of 500 microM), and n = 1.0 x 10(8) molecules of 660 Da propidium iodide (PI; 80 microM) . Both molecules were present in pulsed cells at less than equilibrium values, consistent with a transient uptake mechanism . Intracellular FITC-dextran is present in soluble form, while PI is predominantly bound to nucleic acids . A broad, statistically significant distribution of molecular uptake was also observed . Such quantitative determinations should be important for guiding applications of electroporation, and for testing models of electroporation mechanisms . Further, the use of PI, which is well established as a membrane exclusion dye, provides additional support for the interpretation that both PI and FITC-dextran were internalized as a result of an electrical pulse. J Biol Chem, 1989 Oct 5, 264(28), 16557 - 64 Isolation and sequence of cDNA clones encoding rat phosphatidylinositol transfer protein; Dickeson SK et al.; Phosphatidylinositol (PtdIns) transfer protein is a cytosolic protein that catalyzes the transfer of PtdIns between membranes . It is expressed in organisms from yeast to man, and activity has been found in all animal tissues examined . Using antibodies prepared against bovine brain PtdIns transfer protein, lambda gt11 rat brain cDNA libraries were screened and several clones isolated . DNA sequence analysis showed that the cDNAs encoded a polypeptide of 271 amino acids with a mass of 31,911 Da . Comparison of the deduced amino acid sequence with N-terminal sequence data obtained for the intact purified bovine brain protein and rat lung phospholipid transfer protein verified that the cDNAs were PtdIns transfer protein clones . The predicted protein shows no significant sequence similarity to other known (phospholipid)-binding proteins . DNA blot hybridization suggests that the rat genome may contain more than one gene encoding PtdIns transfer protein . RNA blot hybridization reveals that the PtdIns transfer protein gene is expressed at low levels in a wide variety of rat tissues; all tissues examined showed a major mRNA component of 1.9 kilobases and a minor component of 3.4 kilobases . The isolation of clones encoding rat PtdIns transfer protein will greatly facilitate studies of the structure and function of PtdIns transfer proteins and their role in lipid metabolism. J Mol Biol, 1989 Oct 5, 209(3), 423 - 32 An amino-terminal fragment of GAL4 binds DNA as a dimer; Carey M et al.; GAL4 is a yeast transcriptional activator protein that binds to specific 2-fold rotationally symmetric sites on DNA and stimulates transcription of the genes required for galactose catabolism . The DNA binding region of the protein is located within the first 74 amino acids and contains a "zinc finger" sequence motif . We show that a polypeptide comprising the first 147 amino acids of GAL4, designated GAL4 (1-147), binds DNA as a dimer in vitro . Although a protein containing only the first 74 amino acids, designated GAL4 (1-74), binds DNA specifically, its affinity is reduced relative to GAL4 (1-147) . Addition of the strong dimerization domain of lambda repressor to GAL4 (1-74) generates a protein that binds as tightly as GAL4 (1-147) . GAL4 (1-147) makes rotationally symmetric contacts with its recognition site when assayed by DNase I, exonuclease III and hydroxyl radical footprinting and by phosphate ethylation interference . Binding of GAL4 (1-147) in vitro requires either zinc or cadmium. Biochemistry, 1989 Oct 3, 28(20), 8108 - 16 Glucosylation of glycoproteins by mammalian, plant, fungal, and trypanosomatid protozoa microsomal membranes; Trombetta SE et al.; An assay for UDP-Glc:glycoprotein glucosyltransferase was developed . Incubation of rat liver microsomes with UDP-{14C}Glc led to the formation of hot trichloroacetic acid insoluble material identified as protein-linked Glc1Man7-9GlcNAc2 . Addition of 8 M urea-denatured thyroglobulin to the incubation mixtures stimulated up to 10-12-fold the formation of the same compounds but only in the presence of detergents . Native thyroglobulin was ineffective . Several experiments indicated that the stimulation was due to the transfer of glucose residues from UDP-Glc to high-mannose oligosaccharides in urea-denatured thyroglobulin and that this transfer reaction did not involve dolichol mono- or diphosphate derivatives as intermediates . The glycoprotein glucosylating activity was mainly located in the endoplasmic reticulum and could glucosylate glycopeptides derived from the digestion of thyroglobulin with an unspecific protease . Glucosylation of oligosaccharides in those glycopeptides occurred, however, at a rate at least 2 orders of magnitude slower than that of the same compounds in urea-denatured thyroglobulin . Tryptic digestion of urea-denatured thyroglobulin did not affect its glucosylation rate . The structure of Glc1Man9GlcNAc2 linked to urea-denatured thyroglobulin was identical with that of Glc1Man9GlcNAc2-P-P-dolichol . The assay of UDP-Glc:glycoprotein glucosyltransferase allowed detection of the activity in microsomal membranes in which endogenous acceptors appeared to be absent or almost absent, such as those derived from mung bean, Mucor rouxii, Crithidia fasciculata, and Trypanosoma cruzi cells.(ABSTRACT TRUNCATED AT 250 WORDS) J Cell Biol, 1989 Oct, 109(4 Pt 1), 1439 - 44 Protein transport from endoplasmic reticulum to the Golgi complex can occur during meiotic metaphase in Xenopus oocytes; Ceriotti A et al.; We have previously shown that Xenopus oocytes arrested at second meiotic metaphase lost their characteristic multicisternal Golgi apparati and cannot secrete proteins into the surrounding medium . In this paper, we extend these studies to ask whether intracellular transport events affecting the movement of secretory proteins from the endoplasmic reticulum to the Golgi apparatus are also similarly inhibited in such oocytes . Using the acquisition of resistance to endoglycosidase H (endo H) as an assay for movement to the Golgi, we find that within 6 h, up to 66% of the influenza virus membrane protein, hemagglutinin (HA), synthesized from injected synthetic RNA, can move to the Golgi apparati in nonmatured oocytes; indeed after longer periods some correctly folded HA can be detected at the cell surface where it distributes in a nonpolarized fashion . In matured oocytes, up to 49% of the HA becomes endo H resistant in the same 6-h period . We conclude that movement from the endoplasmic reticulum to the Golgi can occur in matured oocytes despite the dramatic fragmentation of the Golgi apparati that we observe to occur on maturation . This observation of residual protein movement during meiotic metaphase contrasts with the situation at mitotic metabphase in cultured mammalian cells where all movement ceases, but resembles that in the budding yeast Saccharomyces cerevisiae where transport is unaffected. Mutat Res, 1989 Oct, 224(2), 287 - 303 Induction of chromosome loss by mixtures of organic solvents including neurotoxins; Zimmermann FK et al.; Twenty-three aprotic polar solvents - 3 nitriles, 8 organic esters, 10 ketones and 2 lactones - and LiCl were tested in combination with propionitrile alone or a mixture of ethyl acetate and propionitrile for the induction of mitotic chromosome loss in the D61.M strain of the yeast Saccharomyces cerevisiae . Propionitrile and ethyl acetate are very potent inducers of chromosome loss . Mixtures of propionitrile and ethyl acetate induced chromosome loss at much higher frequencies than was observed with the pure chemicals . To test the potentiating effects of propionitrile or mixtures of propionitrile with ethyl acetate on other chemicals, they were used in concentrations that were at or below the level for induction of chromosome loss . Twenty chemicals when tested in pure form were negative or only marginally active in the test for chromosome loss . Except for amyl propionate and benzyl acetate, the same chemicals showed strong induction in combination treatments with the potentiating chemicals . All the ketones including the neurotoxic methyl ethyl ketone, 2-hexanone and 2.5-hexanedione induced high frequencies of chromosome loss . Only methyl ethyl ketone is capable of inducing high levels of chromosome loss when tested in the pure form at much higher concentrations . 1-Methyl-2-pyrrolidinone and gamma-valerolactone had previously been shown to induce chromosome loss only when the treatment at a growth-supporting temperature was interrupted by a cold shock within a narrow range of low temperatures which prevented growth . Both gave very strong induction in combination treatment performed at a continuous growth-supporting temperature . LiCl is a weak inducer of chromosome loss: strong induction can be achieved in combination treatments. J Clin Microbiol, 1989 Oct, 27(10), 2369 - 72 DNA probe for the identification of Histoplasma capsulatum; Keath EJ et al.; A 1.85-kilobase HindIII nuclear DNA probe from Histoplasma capsulatum G217B detected polymorphic restriction fragments within whole-cell DNA from different clinical isolates of H . capsulatum, consistent with the previous system of classification . The probe failed to hybridize to DNA from Blastomyces dermatitidis, Candida spp., Saccharomyces cerevisiae, Sepedonium chrysospermum, and Chrysosporium keratinophilum under low-stringency conditions and therefore may have value as a diagnostic reagent to identify H . capsulatum. J Protein Chem, 1989 Oct, 8(5), 679 - 88 Conformational energy analysis of the leucine repeat regions of C/EBP, GCN4, and the proteins of the myc, jun, and fos oncogenes; Brandt-Rauf PW et al.; It has been recently proposed that certain DNA binding proteins (including C/EBP, GCN4 and the myc, jun, and fos oncogene proteins) share a common structural motif based on helix-promoting regions containing heptad repeat sequences of leucines . It has been suggested that this structure is critical to the biological activity of these proteins, since it facilitates the formation of functional dimers held together by interdigitating leucine side-chains along the hydrophobic interfaces between long alpha-helical regions of the polypeptide chains in a configuration termed the "leucine zipper." In this paper, conformational energy analysis is used to deterrmine the preferred three-dimensional structures of the leucine repeat regions of these proteins . The results indicate that, in all cases, the global minimum energy conformation for these regions is an amphipathic alpha-helix with the leucine side-chains arrayed on one side in such a way to favor "leucine zipper" dimerization . Furthermore, amino acid substitutions in these regions (such as Pro for Leu), that are known to inhibit dimer formation and prevent DNA binding, are found