S. cerevisiae

Genome, 1989, 31(2), 1055 - 8
Evolving strategies for making physical maps of mammalian chromosomes; Smith CL et al.; Two types of physical maps are described: restriction maps made by top down approaches using enzymes that cut the genome infrequently, and complete libraries, made by bottom up approaches using fingerprinting of randomly selected cloned DNA . Construction of such maps for mammalian chromosomes is complicated by the mosaic nature of mammalian genomes, and extensive polymorphisms at the cleavage sites of most enzymes that yield large DNA fragments . However, it appears that both of these potential difficulties can be turned into advantages by new mapping strategies . When combined with yeast artificial chromosome cloning and polymerase chain reaction amplification methods, these approaches should soon yield complete maps of many human chromosomes.

Exp Gerontol, 1989, 24(5-6), 423 - 36
Replication control and cellular life span; Jazwinski SM et al.; Cell proliferation involves both control of progress through the current cell cycle and coordination of successive cell cycles . We have focused our attention on the events that trigger traversal of the G1/S boundary of the cell cycle . A protein kinase activity was found in preparations of the DNA-replicative complex from the budding yeast Saccharomyces cerevisiae . The activity phosphorylated only a few of the proteins present in the replicative fraction, and it displayed a marked preference for a 48-kDa polypeptide . Most importantly, the protein kinase activity was heat-sensitive in replicative fractions from cdc7 cells, a mutant that arrests at the G1/S boundary at restrictive temperature . The results suggest that phosphorylation of components of the replication machinery may play a role in control of initiation of DNA replication during the cell cycle . We have also begun an analysis of cellular aging in yeast, as a means of addressing the problem of coordination of successive cell cycles . Yeast cells have a finite life span defined by reproductive capacity . With age, the generation time of yeast cells lengthened . The cell cycle of the daughter cell was under the control of the mother . This control was transient, and the daughter cell began dividing at the rate characteristic of its own age within three divisions of its birth . This suggests that the senescent phenotype, as manifested by lengthened generation time, is a dominant feature in yeast cells, and that it is determined by a diffusible cytoplasmic molecule(s) that undergoes turnover in young cells . In a search for this putative senescence factor(s), we are cloning genes that differentially expressed during the yeast life span . Several such genes have been isolated and partially characterized . Our goals are to determine whether the expression of one or more of these genes is casually associated with cell longevity . We propose the Cell Spiral model to describe the relationship between the cell cycle and cellular aging.

Prog Clin Biol Res, 1989, 318, 195 - 203
Genetic approaches to microtubule function; Solomon F; To date, the experiments in yeast have been successful primarily in distinguishing what is not important . Divergent tubulin sequences are functionally interchangeable, interesting domains of tubulins can undergo dramatic alteration without affecting substantially the function of the protein, and the cells can assemble sufficient microtubule organelles from half the normal amount of tubulin . These results are with a few genes, in one organism, and the extent to which they can be extrapolated to other organisms with more complex complements of tubulin genes--in particular, metazoa--is not known . It is extremely unlikely that all of the suggestions that tubulin sequences and tubulin quantities participate in regulating microtubule function are erroneous . But the yeast system does offer us the opportunity to look past these obvious regulators, and to detect more subtle interactive elements that may be crucial for normal function . That is the immediate goal of our next experiments.

Scanning Microsc Suppl, 1989, 3, 201 - 10; discussion 211
Improved representation of cell surface structures by freeze substitution and backscattered electron imaging; Walther P et al.; A surface preparation method for the SEM based on cryofixation is presented suitable for the demonstration of membrane particles on whole cells . LLC-PK1 cells (a renal epithelial cell line in culture) were fast frozen, freeze substituted, critical point dried, shadowed with 2 nm of platinum carbon and stabilized with a carbon backing layer . Membrane bound particles are visualized by the material dependent backscattered electron image mainly originating from the platinum shadow . The surface of the LLC-PK1 cells is almost free of precipitated material indicating that the culture medium is removed during freeze substitution or critical point drying . The apical plasma membrane with microvilli and ciliae is well preserved and differences in particle density can be detected . The feasibility of the coating technique for backscattered electron imaging was tested on the well known hexagonally arranged intramembranous particles of fractured and partially freeze dried yeast . This 16.5 nm periodic structure is clearly demonstrable on the bulk SEM-specimen stablized with the carbon backing layer . Without a carbon layer severe shrinking artifacts occurred.

Tsitol Genet, 1989 Jan-Feb, 23(1), 9 - 13
{Comparative study of the mutagenic activity of dicurin on five test-objects}; Kurinnyi AI et al.; Mutagenic activity of dicourol belonging to coumarol derivatives has been studied on 5 test-objects (mice, cultivated lymphocytes of human blood, yeast-saccharomycetes, Tradescantia, Crepis) . Results of investigations allowed attributing dicourol to highly hazardous mutagens . In this connection it is admitted unadvisable to enforce it into agricultural practice . The tested objects differ in their dicourol mutagenic sensibility as follows: cultivated lymphocytes of human blood - Tradescantia - yeast - Crepis - mice.

Enzyme, 1989, 41(1), 1 - 5
Characterization of the inhibition effect induced by nickel on glucose-6-phosphate dehydrogenase and glutathione reductase; Cartana J et al.; Kinetic characterization of the inhibition effect of nickel on glucose-6-phosphate dehydrogenase (EC 1.1.1.49) (G-6-PD) and glutathione reductase (GR; EC 1.6.4.2) from Saccharomyces cerevisiae was made . The effect of nickel on G-6-PD activity is consistent with a mixed-type inhibition pattern, with a competitive character, since the inequality ki,int greater than ki,slope shows an inverse relation between varied substrate concentrations and fractional inhibition . An inhibition effect of nickel on GR activity, when NADPH is the varied substrate, is also consistent with a mixed-type inhibition pattern . However, pure competitive inhibition is found on GR reaction when oxidized glutathione is the varied substrate . This investigation shows the highest sensibility of GR before the inhibitory effect of nickel, in agreement with the experimental values of inhibition constants found in this study, where constants related to the GR system are lower than the ones of the G-6-PD system.

Cell Mol Biol, 1989, 35(1), 89 - 95
Presence of translational inhibitory activity in partially purified extracts from two Petrocoptis species; Ferreras JM et al.; The presence of translational inhibitory activity in partially purified extracts from several paleoendemisms from Spain was investigated . The precipitates from 40-80% (NH4)2SO4 fraction from Petrocoptis glaucifolia and Petrocoptis grandiflora displayed a strong inhibitory activity on the protein synthesis of cell-free extracts from rat liver, rabbit reticulocytes and yeast and to a much lower extent on the protein synthesis in isolated rat liver cells . The inhibitors seem to be proteins since they were precipitated by high salt concentrations, were non-dialysable and were inactivated by heat . Since the partially purified extracts did not show unspecific RNA-A or protease activities, the active compounds can be considered to belong to the plant ribosome-inactivating proteins.

Genomics, 1989 Jan, 4(1), 53 - 9
cDNA sequence, interspecies comparison, and gene mapping analysis of argininosuccinate lyase; Todd S et al.; A cDNA clone of the argininosuccinate lyase gene (ASL) was isolated from an adult human liver library by probing with synthetic oligonucleotide probes . This clone and a yeast genomic DNA fragment containing the ASL gene were sequenced using the M13-dideoxynucleotide method . Comparison of the yeast and human clones at the nucleotide and putative amino acid sequence levels indicated identities of 50 and 54%, respectively . The most conserved region of the yeast gene was used to detect human clones in the liver cDNA library to test phylogenetic screening capabilities of conserved genes . ASL was mapped to human chromosome 7pter----q22 using human-mouse somatic cell hybrid DNA and further mapped by in situ hybridization to chromosome 7cen----q11.2 on human metaphase chromosomes . The probe also detected a sequence on chromosome 22 . Somatic cell hybrid DNA digested with PvuII revealed a mouse polymorphism between Balb/c and C3H mice in the ASL gene.

Teratog Carcinog Mutagen, 1989, 9(6), 349 - 57
Tetrachloroethane, pentachloroethane, and hexachloroethane: genetic and biochemical studies; Bronzetti G et al.; Tetrachloroethane (TTCE), pentachloroethane (PCE), and hexachloroethane (HCE) were tested in diploid strain (D7) of the yeast Saccharomyces cerevisiae in suspension test with and without mammalian metabolic activation (S9) . TTCE, PCE, and HCE gave positive results on cells harvested from logarithmic growth phase; only PCE induced a significant increase (P less than or equal to .01) of mitotic gene conversion and point reverse mutation on cells from stationary growth phase with metabolic activation (S9) . The in vivo effects on cytochrome P450 content (cyt . P450), pentoxyresorufin O-dealkylase (P450-like, class IIB, PROD), and ethoxy-resorufin O-deethylase (P448-like, class IA, EROD) activities were examined in hepatic microsomes from mice 24 h after acute intoxication . All the halogenated hydrocarbons displayed a marked toxic effect as shown by the significant decrease in cyt . P450 levels (maximum of 76% decrease, with TTCE 753.2 mg/kg) and EROD (maximum of 69% decrease, with PCE 925.4 mg/kg), and to a lesser extent in PROD (maximum of 52.4% decrease, with HCE 3150 mg/kg) . Although a general decrease of P450 functions was observed, the toxic effects of TTCE and PCE seem to be preferentially related to P448 forms.

Int J Biochem, 1989, 21(12), 1407 - 14
Effect of enzymatic methylation of apocytochrome c on holocytochrome c formation and proteolysis; Frost B et al.; 1 . Methylation of the lysine at residue 72 of yeast apocytochrome c increases its import into mitochondria . 2 . Using methylated and unmethylated apocytochrome c as substrate and intact yeast mitochondria and a solubilized mitochondrial fraction as a source of cytochrome c heme lyase, the results show that the methylation state of the apoprotein has no significant effect on its conversion to holoprotein . 3 . The above result suggests that the import mechanism is separate from the heme-attaching activity . 4 . Unmethylated apocytochrome c was less resistant to a yeast homogenate fraction that methylated apocytochrome c, suggesting that methylation of apocytochrome c alters the conformation of the whole protein.

EMBO J, 1989 Jan, 8(1), 127 - 32
Epstein-Barr virus BZLF1 trans-activator specifically binds to a consensus AP-1 site and is related to c-fos; Farrell PJ et al.; Two regions of the Epstein-Barr virus BZLF1 trans-activator protein have sequence similarity to the c-fos protein . Part of the similarity corresponds to the region of c-fos which is similar to the DNA binding domain of c-jun and GCN-4 . The structure of the exon which contains this region in c-fos and BZLF1 is also highly conserved between the two genes . Complete BZLF1 protein and a C terminal fragment were prepared either as purified fusion proteins or by in vitro translation from a BZLF1 cDNA . Gel retardation and DNase footprinting assays using these proteins show that BZLF1 is a sequence specific DNA binding protein capable of binding to a target sequence which contains a consensus AP-1 site.

Mol Cell Biol, 1989 Jan, 9(1), 159 - 68
DNA topoisomerase II must act at mitosis to prevent nondisjunction and chromosome breakage; Holm C et al.; The hypothesis that DNA topoisomerase II facilitates the separation of replicated sister chromatids was tested by examining the consequences of chromosome segregation in the absence of topoisomerase II activity . We observed a substantial elevation in the rate of nondisjunction in top2/top2 cells incubated at the restrictive temperature for one generation time . In contrast, only a minor increase in the amount of chromosome breakage was observed by either physical or genetic assays . These results suggest that aneuploidy is a major cause of the nonviability observed when top2 cells undergo mitosis at the restrictive temperature . In related experiments, we determined that topoisomerase II must act specifically during mitosis . This latter observation is consistent with the hypothesis that the mitotic spindle is necessary to allow topoisomerase II to complete the untangling of sister chromatids.

Exp Lung Res, 1989, 15(1), 49 - 62
Intraphagosomal pH in alveolar macrophages studied with fluorescein-labeled amorphous silica particles; Nyberg K et al.; Intraphagosomal pH in rabbit alveolar macrophages was studied using amorphous silica particles (FSP) and yeast particles (FYP) labeled with fluorescein . The pH was estimated from the quotient between the fluorescence intensity at wavelength 519 nm with excitation at wavelengths 495 and 450 nm . Within a factor of 10, pH was independent of the number of FSP added to macrophages in vitro . In macrophages cultured for 24 h, the pH obtained with FSP and FYP was about 5 . Three hours after lavage, pH was the same as after 24 h for the FSP but significantly higher for the FYP, 5.8 . Both 3 and 24 h after lavage, more lysosomes were in contact with the FYP-containing phagosomes than with the FSP-containing ones . Most FSP were in tight contact with the phagosomal membrane, while there was a clear zone between most FYP and the phagosomal membrane . The differences in pH and morphology between cells containing FSP and FYP might be explained by the assumptions that the macrophages disintegrate the FYP, which results in higher pH, and that the disintegration of FYP is more efficient at 3 than at 24 h after the lavage . The intraphagosomal pH was lower when the macrophages were allowed to phagocytize the FSP in vivo . The pH values were 5.1 in vitro, 4.9 at 24 h, and 4.5 at 1 week after the FSP had been instilled via trachea . The FSP should be a useful tool for estimation of intraphagosomal pH at basic conditions, i.e., the milieu to which many inhaled inorganic particles will be exposed.

J Cell Sci Suppl, 1989, 12, 77 - 97
The role of cyclin synthesis, modification and destruction in the control of cell division; Minshull J et al.; This paper reviews our current knowledge of the cyclins based on observations of the oocytes and eggs of sea urchins, clams and frogs . Cyclins are proteins found in all eukaryotes whose special property is rapid destruction at specific stages in the cell cycle . The cyclins fall into three families . A-type cyclins have been found in clams, flies and frogs . B-type cyclins have been found in clams, flies, frogs, sea urchins and fission yeast . A more distantly related family of three genes is found in Saccharomyces cerevisiae . B-type cyclins appear to be required for cells to enter mitosis, and their destruction is thought to be necessary for exit from mitosis . We describe evidence in support of these ideas, and describe various conditions under which cyclin destruction is delayed or deranged . We conclude with a discussion of the relationship between the cyclins and maturation- (or M phase-) promoting factor and some ideas on how the cyclins may work.

Adv Exp Med Biol, 1989, 251, 83 - 98
The application of molecular biology to the development of novel vaccines; Kniskern PJ et al.; In summary, we have shown that yeast is the preferred host for the expression of recombinant-derived hepatitis B vaccines, and that a yeast expression system which is productive, stable and scaleable can be developed for each of the three HBV envelope proteins . The versatility of regulated and integrated yeast expression systems in the production of foreign polypeptides with biomedical utility also has been highlighted . We also have shown that careful attention to the development of recombinant clones helps to optimize the entire production process leading to highly purified products which share many biochemical properties with the plasma-derived vaccine . Furthermore, immunization with PreS2 sequences is capable of protecting chimpanzees from HBV infection . The availability of PreS2 + S and PreS1 + PreS2 + S proteins expressed in yeast now provides the opportunity for establishing the relevance of such candidate vaccines in preventing human disease, thereby highlighting the utility of molecular biology in modern vaccine development.

Biochem J, 1989 Jan 1, 257(1), 221 - 9
Subcellular distribution and characteristics of trihydroxycoprostanoyl-CoA synthetase in rat liver; Schepers L et al.; The subcellular distribution and characteristics of trihydroxycoprostanoyl-CoA synthetase were studied in rat liver and were compared with those of palmitoyl-CoA synthetase and choloyl-CoA synthetase . Trihydroxycoprostanoyl-CoA synthetase and choloyl-CoA synthetase were localized almost completely in the endoplasmic reticulum . A quantitatively insignificant part of trihydroxycoprostanoyl-CoA synthetase was perhaps present in mitochondria . Peroxisomes, which convert trihydroxycoprostanoyl-CoA into choloyl-CoA, were devoid of trihydroxycoprostanoyl-CoA synthetase . As already known, palmitoyl-CoA synthetase was distributed among mitochondria, peroxisomes and endoplasmic reticulum . Substrate- and cofactor- (ATP, CoASH) dependence of the three synthesis activities were also studied . Cholic acid and trihydroxycoprostanic acid did not inhibit palmitoyl-CoA synthetase; palmitate inhibited the other synthetases non-competitively . Likewise, cholic acid inhibited trihydroxycoprostanic acid activation non-competitively and vice versa . The pH curves of the synthetases did not coincide . Triton X-100 affected the activity of each of the synthetases differently . Trihydroxycoprostanoyl-CoA synthetase was less sensitive towards inhibition by pyrophosphate than choloyl-CoA synthetase . The synthetases could not be solubilized from microsomal membranes by treatment with 1 M-NaCl, but could be solubilized with Triton X-100 or Triton X-100 plus NaCl . The detergent-solubilized trihydroxycoprostanoyl-CoA synthetase could be separated from the solubilized choloyl-CoA synthetase and palmitoyl-CoA synthetase by affinity chromatograpy on Sepharose to which trihydroxycoprostanic acid was bound . Choloyl-CoA synthetase and trihydroxycoprostanoyl-CoA synthetase could not be detected in homogenates from kidney or intestinal mucosa . The results indicate that long-chain fatty acids, cholic acid and trihydroxycoprostanic acid are activated by three separate enzymes.

Psychiatr Neurol Med Psychol (Leipz), 1989 Jan, 41(1), 50 - 3
{Phagocytic reactivity of granulocytes in the peripheral blood of patients with multiple sclerosis and normal probands}; Lehmitz R et al.; For estimation of phagocytic activity the uptake by granulocytes of heat-inactivated, opsonised yeast particles (Saccharomyces cerevisiae) was measured . Peripheral granulocytes of multiple sclerosis patients revealed an enhanced ability for phagocytosis in comparison to normal healthy controls, if tested with normal serum as opsonic source . An activated state of cells in multiple sclerosis is supposed . The selective influence on granulocyte phagocytosis of treatment by ultraviolet-irradiated blood in multiple sclerosis patients supports the view of altered reactivity of these cells in comparison to normal controls.

Gene, 1988 Dec 30, 74(2), 347 - 56
Vacuum blotting: a simple method for transferring DNA from sequencing gels to nylon membranes; Gross DS et al.; We describe a vacuum blotting procedure for transferring DNA fragments from conventional polyacrylamide sequencing gels to nylon membranes . The method employs a combination of vacuum-assisted diffusion (effected by a standard gel drier) and an osmotic gradient (effected by over- and underlying filters presoaked in ammonium acetate) . Fragments up to 310 nucleotides in length transfer at 40-60% efficiency within 90 min . When combined with indirect end-labelling, the method allows genomic sequencing of a single-copy gene of Saccharomyces cerevisiae employing as little as 5 micrograms DNA per lane.

Biochemistry, 1988 Dec 27, 27(26), 9216 - 21
High-resolution separation and accurate size determination in pulsed-field gel electrophoresis of DNA . 3 . Effect of electrical field shape; Cantor CR et al.; The resolution of pulsed-field gel electrophoresis is dramatically affected by the number and configuration of the electrodes used, because these alter the shape of the applied electrical fields . Here we present calculations and experiments on the effect of electrode position in one of the most commonly used pulsed-field gel electrophoresis configurations . The goal was to explore which aspects of the electrical field shape correlate with improved electrophoretic resolution . The most critical variable appears to be the angle between the alternate electrical fields . The most effective electrode configurations yield angles of more than 110 degrees . A continually increasing angle between the fields produces band sharpening that greatly enhances the resolution.

Gene, 1988 Dec 20, 73(2), 373 - 84
A family of non-allelic tRNA(ValGUU) genes from the cellular slime mold Dictyostelium discoideum; Dingermann T et al.; A haploid genome of the cellular slime mold Dictyostelium discoideum contains at least 14 non-allelic gene copies coding for a tRNA(ValGUU) . The structure, genomic organization, and expression of these genes have been analyzed in relation to stages of the developmental cycle . So far, 13 tRNA(ValGUU) genes have been isolated and characterized . All genes contain identical mature tRNA-coding regions, and consequently identical gene internal promoter elements . However, different genes differ with respect to their 5'- and 3'-flanking regions, although a certain degree of sequence conservation seems apparent . Different members of this tRNA gene family appear to be randomly dispersed along the seven D . discoideum chromosomes, and not clustered at any one genomic location . In vivo expression of individual genes was studied in yeast . All but one tRNA(ValGUU) gene are actively transcribed, though with different efficiencies . There is also evidence that not all of these tRNA genes are constitutively transcribed in Dictyostelium throughout the developmental cycle . One characteristic primary transcript can only be detected in cells of the late preaggregation phase, whereas growing cells, cells in the stationary phase or cells harvested 4 h after the onset of development do not seem to carry this transcript . This product seems to be transcribed from a gene of an unusual structure . Although this particular gene has not yet been isolated, it can be predicted from the sequence of the cDNA synthesized from primary transcription products of this putative gene, that it is composed of nt 1-54 of a 3'-truncated tRNA(ValGUU) gene linked to a bona fide tRNA(ValGUU) gene.

Biochim Biophys Acta, 1988 Dec 16, 963(3), 509 - 14
Acyl-CoA ligases from rat brain microsomes: an immunochemical study; Singh I et al.; Acyl-CoA ligase activities, solubilized from rat brain microsomes, were fractionated into three different peaks by hydroxyapatite chromatography . Based on physical and chemical properties, we suggested that peak A (pamitoyl-CoA ligase) and peak C (lignoceroyl-CoA ligase) were two different enzymes (A . Bhushan, R . P . Singh, and I . Singh (1986) Arch . Biochem . Biophys . 246, 374-380) . We raised antibodies against purified liver microsomal palmitoyl-CoA ligase (EC 6.2.1.3) and examined the effect of this antibody on acyl-CoA ligase activities for palmitic, arachidonic and lignoceric acids in microsomal enzyme extract and different acyl-CoA ligase peaks from the hydroxyapatite column . In an enzyme activity assay system in microsomal extract, the antisera inhibited the palmitoyl-CoA ligase activity but had very little effect on the acyl-CoA ligase activities for arachidonic and lignoceric acids . This antisera inhibited the acyl-CoA ligase activities for these three fatty acids in peak A and had no effect on these activities in peak B or peak C . Western blot analysis demonstrated that antibody to liver microsomal palmitoyl-CoA ligase cross-reacted with only peak A (palmitoyl-CoA ligase), but not with peak B or peak C . This immunochemical study demonstrates that palmitoyl-CoA ligase does not share immunological determinants with acyl-CoA ligases in peaks B or C, thus demonstrating that palmitoyl-CoA ligase (peak A) is different from the arachidonoyl-CoA and lignoceroyl-CoA ligase activities in peaks B or C.

Eur J Biochem, 1988 Dec 15, 178(2), 329 - 33
Probing the active site of flavocytochrome b2 by site-directed mutagenesis; Reid GA et al.; The three-dimensional structure of flavocytochrome b2 (L-lactate dehydrogenase) from bakers' yeast (Saccharomyces cerevisiae) has recently been solved at 0.24-nm resolution {Mathews & Xia (1987) in Flavins and flavoproteins, Walter de Gruyter, Berlin, pp . 123-131} . We have used this structural information to investigate the roles of particular amino acid residues likely to be involved in the oxidation of L-lactate by kinetic analysis of mutant enzymes generated by site-directed mutagenesis of the isolated gene . The hydroxyl group of Tyr254 was expected to be important for the abstraction of the hydroxyl proton of L-lactate in the oxidation to pyruvate . Replacement of this tyrosine by phenylalanine reduced kcat from 190 +/- 3 s-1 (25 degrees C, pH 7.5) to 4.3 +/- 0.1 s-1 . This substitution had, however, no discernable effect on Km for lactate (0.54 +/- 0.03 mM for the mutant compared with 0.49 +/- 0.03 mM for the wild-type enzyme) . Arg376 was expected to be essential for productive binding and orientation of L-lactate . Replacing Arg376 with lysine abolished all detectable activity . A total loss of enzymic activity was also observed when Lys349, thought likely to stabilize the anionic form of the flavin hydroquinone, was replaced by arginine . An amino acid residue replacement at a distance from the active site, Ala306 to serine, had a minor but significant effect on kcat (reduced from 190 s-1 to 160 s-1) and Km (increased from 0.49 mM to 0.83 mM) presumably arising from small conformational effects . The implications of these results are discussed in relation to the mechanism of L-lactate oxidation.

Nucleic Acids Res, 1988 Dec 9, 16(23), 11303 - 17
Evolution of the herpes thymidine kinase: identification and comparison of the equine herpesvirus 1 thymidine kinase gene reveals similarity to a cell-encoded thymidylate kinase; Robertson GR et al.; We have identified the equine herpesvirus 1 (EHV-1) thymidine kinase gene (TK) by DNA-mediated transformation and by DNA sequencing . Alignment of the amino acid sequence of the EHV-1 TK with the TKs from 3 other herpesviruses revealed regions of homology, some of which correspond to the previously identified substrate binding sites, while others have as yet, no assigned function . In particular, the strict conservation of an aspartate within the proposed nucleoside binding site suggests a role in ATP binding for this residue . Comparison of 5 herpes TKs with the thymidylate kinase of yeast revealed significant similarity which was strongest in those regions important to catalytic activity of the herpes TKs, and, therefore we propose that the herpes TK may be derived from a cellular thymidylate kinase . The implications for the evolution of enzyme activities within a pathway of nucleotide metabolism are discussed.

Biochim Biophys Acta, 1988 Dec 8, 946(1), 119 - 28
Tissue distribution, purification and characterization of rat phosphatidylinositol transfer protein; Venuti SE et al.; Phosphatidylinositol transfer activity is measured in cytosol fractions prepared from 13 rat tissues; specific activity is highest in brain and lowest in adipose and skeletal muscle . Based upon electrophoretic analysis phosphatidylinositol transfer protein is purified to homogeneity from whole rat brain . The protein has a molecular weight of 36,000 and exists as a mixture of species having isoelectric points of 4.9 and 5.3 . In a vesicle-vesicle assay system, the intermembrane transfer rate is greatest for phosphatidylinositol and less by a factor of 2 for phosphatidylcholine; transfer of phosphatidylethanolamine, phosphatidylserine or sphingomyelin is not observed . Using a polyclonal rabbit antibody against bovine phosphatidylinositol transfer protein, immunologic cross-reactivity is noted between the rat protein and other mammalian phosphatidylinositol transfer proteins . A strong correlation is established between a tissue's capacity for phosphatidylinositol transfer and the amount of immunoreactive transfer protein seen in that tissue . Purified phosphatidylinositol transfer protein is capable of transporting newly synthesized phosphatidylinositol molecules from rat brain microsomes to small unilamellar phospholipid vesicles . The results are discussed within the context of cellular phosphoinositide metabolism and the maintenance of the metabolically responsive pool of phosphatidylinositol in the plasma membrane.

Carcinogenesis, 1988 Dec, 9(12), 2265 - 70
Improvement of short-term tests for mutagenicity: on the optimal pH for the liver microsomal assay; Paolini M et al.; The aim of this study was to optimize the pH in the liver microsomal assay (LMA) in processing short-term mutagenicity tests . pH optimization would increase the sensitivity (i.e . decrease the presence of false negatives) and increase the specificity (decrease false positives) . Such optimization is a function of the relative activities and stabilities of the liver microsomal cytochrome P-450- and FAD-containing monooxygenase-dependent biotransformation enzymes present in the incubation mixtures used . The enzyme activities ethoxyresorufin O-deethylase, dinemorphan N-demethylase, aminopyrine N-demethylase, p-nitroanisole O-demethylase and thiobenzamide S-oxidase (as phase-I markers), were examined in terms of their exact incubation conditions for the LMA during a period of pre-incubation (1 h) over the pH range 6-9 . As a comparison, the behaviours of glutathione S-transferase and epoxide hydrase activities (as phase-II markers) were also studied . Lipid peroxidation was also determined . Experiments were carried out on S9 fractions derived from Na-phenobarbital and beta-naphthoflavone induced mouse liver . The maximal value of the mean specific activity (Asp) was found at pH 7.8 for the phase-I drug metabolizing enzymes considered (30-45% increase) . On the contrary, a lower increase of Asp for epoxide hydrase and glutathione S-transferase (approximately 14%), was observed between pH 7.4 and 7.8 . Lipid peroxidation was not changed appreciably by varying pH . In vitro DNA binding of the well-known pre-mutagenic agent {14C}dimethylnitrosamine ({14C}DMNA), mediated by mouse hepatic microsomal enzymes, showed a significant increase of specific activity at pH 7.8 (2.8-fold) compared to the usual pH (7.4) employed . Additional support for the above results has come from mutagenesis experiments using DMNA on the diploid D7 strain of Saccharomyces cerevisiae as a biological test system . In fact, a significant enhancement of mitotic gene conversion (1.7-fold), mitotic cross-over (2.6-fold) and reverse point mutation (2.3-fold) frequencies were observed at pH 7.8 compared to pH 7.4 . These data indicate that pH 7.8 provides a more favourable condition for in vitro mutagenesis tests resulting in greater rates of biotransformation (as measured by an increased Asp phase-I/Asp phase-II ratio), DNA binding and genotoxic response.

Mol Biol Med, 1988 Dec, 5(3), 173 - 84
Accuracy and limitations of pulsed field gel electrophoresis in sizing partial deletions of the factor VIII gene; Cutting GR et al.; Pulsed field gel electrophoresis (PFGE) has been recently used to separate DNA fragments ranging from 100 to 2000 kb in size . In order to assess the accuracy of the sizes of the DNA fragments and the resolving capability of this technique, we used PFGE combined with Southern blotting and probe hybridization techniques to determine the size and approximate location of four partial deletions of the factor VIII gene . This gene was chosen because of its large size (186 kb) and the availability of hemophiliac patients with well-characterized partial deletions . The sizes of three deletions estimated by PFGE (55 kb, 60 kb and 133 to 145 kb) were within 10% of the sizes calculated from conventional restriction analysis . Therefore, concatemers of lambda DNA and intact chromosomal DNA of Saccharomyces cerevisiae provide a relatively accurate system (within 10%) for sizing mammalian DNA fragments that are 100 to 550 kb in size . However, analysis of the fourth deletion (9 to 12 kb) revealed that it is difficult to detect changes in the size of mammalian DNA fragments of 8% or less using Southern blotting and PFGE.

DNA, 1988 Dec, 7(10), 701 - 11
Genetically engineered modification of P450 monooxygenases: functional analysis of the amino-terminal hydrophobic region and hinge region of the P450/reductase fused enzyme; Yabusaki Y et al.; Modified constructions of a microsomal cytochrome P450, of NADPH-cytochrome P450 reductase, and of a P450/reductase fused enzyme were prepared to analyze the function of the amino-terminal hydrophobic regions of these enzymes and the hinge region of the fused enzyme . Expression plasmids for delta P450c, delta reductase, and the delta P450/reductase fused enzyme, all of which lacked their amino-terminal hydrophobic regions, were constructed by inserting each of the corresponding cDNAs between the yeast alcohol dehydrogenase I promoter and the terminator of the expression vector pAAH5 . Yeast transformed with plasmids encoding delta P450 and the delta P450/reductase fused enzyme produced smaller amounts of the respective enzymes and showed lower monooxygenase activity toward 7-ethoxycoumarin than did yeast transformed with plasmids encoding the complete enzymes . Both delta P450 and delta P450/reductase were found in the microsomal fraction of the yeast cells . Yeast transformed with the expression plasmid for delta reductase produced 20 times more enzyme than did yeast transformed with the plasmid for the complete enzyme . delta Reductase was present in the soluble fraction and was 33 times more active in reducing cytochrome c than was the complete enzyme . The results suggest that the amino-terminal hydrophobic regions of P450c and the P450/reductase fused enzyme play an important role in their stability and function in the yeast microsomes . By contrast, the amino-terminal-containing P450 reductase appears to be unstable in yeast cells . Altering the size of the hinge regions does not affect the activity of the P450/reductase fused enzyme significantly, but some amino acid changes in this region increase the stability of the fused enzyme slightly.

Proc Natl Acad Sci U S A, 1988 Dec, 85(23), 8795 - 9
Heteroatom-substituted fatty acid analogs as substrates for N-myristoyltransferase: an approach for studying both the enzymology and function of protein acylation; Heuckeroth RO et al.; Myristoyl-CoA:protein N-myristoyltransferase (NMT), the enzyme that transfers the myristoyl (14:0) moiety from myristoyl CoA thioester to nascent proteins, is remarkably specific for both peptide and fatty acyl CoA substrates . To investigate the interaction of NMT with fatty acyl CoA substrates, we have synthesized 10 oxygen- or sulfur-substituted fatty acid analogs . These analogs differ dramatically in hydrophobicity from naturally occurring fatty acids of similar length . As acylpeptides, sulfur-substituted myristic acid analogs migrate on reverse-phase HPLC like 11:0 or 12:0 fatty acids, while oxygen-substituted analogs migrate like 9:0 to 11:0 fatty acids . CoA thioesters of several of these analogs serve as good NMT substrates in vitro, implying that NMT selects fatty acyl substrates primarily on the basis of chain length rather than hydrophobicity . Myristelaidoyl (14:1, delta 9,10-trans) CoA is also a significantly better substrate than myristoleoyl (14:1, delta 9,10-cis) CoA . The fatty acyl group bound to NMT profoundly influences the rate of acylpeptide formation and the affinity of NMT for peptide substrates . However, the peptide substrate bound to NMT does not produce significant alterations in the enzyme's affinity for myristoyl CoA . In vitro characterization of these heteroatom substituted analogs suggests that they will be efficiently incorporated into proteins in vivo and may markedly alter acylprotein targeting and function.

Mol Cell Biol, 1988 Dec, 8(12), 5439 - 47
The first and fourth upstream open reading frames in GCN4 mRNA have similar initiation efficiencies but respond differently in translational control to change in length and sequence; Mueller PP et al.; The third and fourth AUG codons in GCN4 mRNA efficiently repress translation of the GCN4-coding sequences under normal growth conditions . The first AUG codon is approximately 30-fold less inhibitory and is required under amino acid starvation conditions to override the repressing effects of AUG codons 3 and 4 . lacZ fusions constructed to functional, elongated versions of the first and fourth upstream open reading frames (URFs) were used to show that AUG codons 1 and 4 function similarly as efficient translational start sites in vivo, raising the possibility that steps following initiation distinguish the regulatory properties of URFs 1 and 4 . In accord with this idea, we observed different consequences of changing the length and termination site of URF1 versus changing those of URFs 3 and 4 . The latter were lengthened considerably, with little or no effect on regulation . In fact, the function of URFs 3 and 4 was partially reconstituted with a completely heterologous URF . By contrast, certain mutations that lengthen URF1 impaired its positive regulatory function nearly as much as removing its AUG codon did . The same mutations also made URF1 a much more inhibitory element when it was present alone in the mRNA leader . These results strongly suggest that URFs 1 and 4 both function in regulation as translated coding sequences . To account for the phenotypes of the URF1 mutations, we suggest the most ribosomes normally translate URF1 and that the mutations reduce the number of ribosomes that are able to complete URF1 translation and resume scanning downstream . This effect would impair URF1 positive regulatory function if ribosomes must first translate URF1 in order to overcome the strong translational block at the 3'-proximal URFs . Because URF1-lacZ fusions were translated at the same rate under repressing and derepressing conditions, it appears that modulating initiation at URF1 is not the means that is used to restrict the regulatory consequences of URF1 translation to starvation conditions.

Mol Cell Biol, 1988 Dec, 8(12), 5292 - 8
Gene conversion adjacent to regions of double-strand break repair; Orr-Weaver TL et al.; The repair of double-strand breaks and gaps can be studied in vegetative yeast cells by transforming the DNA with restriction enzyme-cut plasmids . Postulated models for this repair process require the formation of heteroduplex DNA on either side of the region of break or gap repair . We describe the use of restriction site mutations in the his3 gene to detect conversion events flanking but outside of a region of a double-strand break repair . The frequency with which a mutation was converted declined with increasing distance between the mutation and the edge of the gap repair region . The data are consistent with heteroduplex DNA tracts of at least several hundred base pairs adjacent to regions of double-strand break repair.

Curr Genet, 1988 Dec, 14(6), 561 - 6
An efficient cell-free translation system from Aspergillus nidulans and in vitro translocation of prepro-alpha-factor across Aspergillus microsomes; Devchand M et al.; We describe the preparation of an in vitro translation system from heat shock-treated Aspergillus nidulans, capable of supporting efficient and faithful synthesis of proteins from natural and in vitro transcribed eukaryotic messages . In vitro synthesized prepro-alpha-factor was translocated across Aspergillus nidulans microsomal membranes in either the homologous A . nidulans or a yeast cell-free system . The translocated prepro-alpha-factor was protected from digestion by protease and glycosylated to higher MW forms.

Curr Genet, 1988 Dec, 14(6), 553 - 60
Structure of the Aspergillus nidulans pyruvate kinase gene; de Graaff L et al.; The complete nucleotide sequence of the Aspergillus nidulans pyruvate kinase gene, including its flanking sequences, is presented . The gene has a 1,578 bp coding sequence that encodes a protein of 526 amino acids; the latter is strongly homologous to the pyruvate kinases found in Saccharomyces cerevisiae (66%) and mammals (53%) . The gene is interrupted by seven introns, three of which are in a conserved position compared to those present in the mammalian pyruvate kinase genes sequenced thus far . A fourth intron within the mononucleotide binding fold domain is in a conserved position with respect to the position of an intron within the NAD+ binding region of maize ADH I . The transcription start site has been determined; a major site of transcription was found 80 bp before the translation initiation codon . The promoter region of the A . nidulans pyruvate kinase gene contains no direct homologies with the TATA or CCAAT sequences in the expected region (30-70 bp) before the transcription initiation site . However, extended CT-enriched regions are found in the promotor region, similar to what has been observed in genes that are highly expressed in Saccharomyces cerevisiae and filamentous fungi.

J Cell Biol, 1988 Dec, 107(6 Pt 1), 2051 - 7
70-kD heat shock-related protein is one of at least two distinct cytosolic factors stimulating protein import into mitochondria; Murakami H et al.; We have developed an in vitro system in which the posttranslational import of Put2 (delta-pyrroline-5-carboxylate dehydrogenase), into yeast mitochondria is dependent on the addition of yeast postribosomal supernatant (PRS) . When mRNA for a nuclear-encoded yeast mitochondrial matrix protein, Put2, was translated in a wheat germ cell-free system, import into posttranslationally added yeast mitochondria was negligible . However, when a yeast PRS was added, significant import was observed . The import stimulating activity of the yeast PRS was shown to consist of at least two distinct factors . One of these is the recently purified 70-kD heat shock-related protein Ssalp/Ssa2p, two proteins that are 98% homologous . The other factor is an N-ethylmaleimide-sensitive protein(s) . Both factors act synergistically.

J Cell Biol, 1988 Dec, 107(6 Pt 1), 2037 - 43
A chimeric mitochondrial precursor protein with internal disulfide bridges blocks import of authentic precursors into mitochondria and allows quantitation of import sites; Vestweber D et al.; Bovine pancreatic trypsin inhibitor (which contains three intramolecular disulfide bridges) was chemically coupled to the COOH terminus of a purified artificial mitochondrial precursor protein . When the resulting chimeric precursor was presented to energized isolated yeast mitochondria, its trypsin inhibitor moiety prevented the protein from completely entering the organelle; the protein remained stuck across both mitochondrial membranes, with its NH2 terminus in the matrix and its trypsin inhibitor moiety still exposed on the mitochondrial surface . The incompletely imported protein appeared to "jam" mitochondrial protein import sites since it blocked import of three authentic mitochondrial precursor proteins; it did not collapse the potential across the mitochondrial inner membrane . Quantification of the inhibition indicated that each isolated mitochondrial particle contains between 10(2) and 10(3) protein import sites.

Mol Cell Biol, 1988 Dec, 8(12), 5299 - 309
A Ty1 cell-type-specific regulatory sequence is a recognition element for a constitutive binding factor; Company M et al.; Ty transposable-element insertion mutations of Saccharomyces cerevisiae can cause cell-type-dependent activation of adjacent-gene expression . Several cis-acting regulatory regions within Ty1 are responsible for the effect of Ty1 on adjacent-gene expression . One of these is the block II sequence that was defined by its homology to mammalian enhancers and to the yeast a1-alpha 2 control site . Tandem copies of a 57-base-pair region encompassing block II caused an additive increase in expression of the CYC7 reporter gene in the absence of other Ty1 sequences . The activation of gene expression by the multiple repeats was abolished in a/alpha diploid cells . A specific complex between a constitutive factor in whole-cell extracts and the DNA regulatory element was observed . The protein-binding site for the constitutive factor coincided with the block II element . Base-pair substitutions within the binding site abolished the ability of the block II element to function as a component of the Ty1 activator and to form the factor-DNA complex . The correlation between complex formation and reporter gene expression indicates that factor binding to the cis-acting element is essential for this element to function as a component of the Ty1 activator.

Genes Dev, 1988 Dec, 2(12B), 1764 - 78
Activation of the U2 snRNA promoter by the octamer motif defines a new class of RNA polymerase II enhancer elements; Tanaka M et al.; The recent discovery that the activation domains of transcriptional activators (e.g., GAL4) from a number of species are interchangeable has led to the concept of a general mechanism for activation of RNA polymerase II genes . We have examined the different activities of the SV40 octamer motif ATGCAAAG in B cells and in HeLa cells in the context of either the beta-globin promoter, a TATA-box-containing mRNA promoter, or the U2 snRNA promoter, which contains a snRNA-specific proximal element . In the context of the beta-globin promoter, the octamer motif is a B-cell-specific enhancer element, whereas it is a ubiquitous enhancer element for the U2 snRNA promoter . The U2 promoter is unique in that it is not activated by enhancer elements that activate the beta-globin promoter, and a hybrid U2 promoter containing the upstream activating sequence UASG is not stimulated by a yeast GAL4 trans-activator . Together, these observations suggest that in the context of the U2 promoter, the octamer motif defines a new class of RNA polymerase II enhancer elements, which bind transcription factors that trans-activate gene expression by a different mechanism than the general mechanism mentioned above . These results are discussed in light of the possibility that the ubiquitous octamer binding protein Oct-1 and the B-cell-specific octamer binding protein Oct-2 are involved in the activation of the U2 and beta-globin promoters, respectively.

J Cell Biol, 1988 Dec, 107(6 Pt 1), 2045 - 9
Mitochondria can import artificial precursor proteins containing a branched polypeptide chain or a carboxy-terminal stilbene disulfonate; Vestweber D et al.; A purified, artificial precursor protein was used as a transport vehicle to test the tolerance of the mitochondrial protein import system . The precursor was a fusion protein consisting of mouse dihydrofolate reductase linked to a yeast mitochondrial presequence; it contained a unique cysteine as its COOH-terminal residue . This COOH-terminal cysteine was covalently coupled to either a stilbene disulfonate derivative or, with the aid of a bifunctional cross-linker, to one of the free amino groups of horse heart cytochrome c . Coupling to horse heart cytochrome c generated a mixture of branched polypeptide chains since this cytochrome lacks a free alpha-amino group . Both adducts were imported and cleaved by isolated yeast mitochondria . The mitochondrial protein import machinery can thus transport more complex structures and even highly charged "membrane-impermeant" organic molecules . This suggests that transport occurs through a hydrophilic environment.

Eur J Epidemiol, 1988 Dec, 4(4), 395 - 9
From interferon induction to fungal viruses; Buck KW; Viruses of fungi (mycoviruses) were first discovered in diseased mushrooms . However the finding that the antiviral and interferon-inducing activities of extracts of apparently healthy isolates of a number of Penicillium species were due to the presence of double-stranded (ds) RNA arising from mycovirus infections sparked off an explosion of interest in what has now become a distinct area of virology . Two families of dsRNA mycoviruses are now established: the Totiviridae and the Partitiviridae which comprise isometric viruses with genomes of one or two dsRNA segments respectively . Virus isolates in both families often contain additional satellite dsRNAs, which may in some fungi (e.g . Saccharomyces cerevisiae, Ustilago maydis) code for "killer" proteins which are toxic to other sensitive strains of the same or closely related species . In Endothia parasitica, which causes chestnut blight disease, dsRNA is associated with hypovirulence and is enclosed in lipid-rich vesicles . In Ophiostoma (Ceratocystis) ulmi, which causes Dutch elm disease, dsRNA is associated with the mitochondria and, in some diseased isolates of the fungus, specific dsRNA segments are associated with reduction of cytochrome oxidase and respiratory deficiency, resulting in slow growth and abnormal morphology.

Biochem Biophys Res Commun, 1988 Nov 30, 157(1), 154 - 9
A simple and rapid method for the preparation of homologous DNA oligonucleotide hybridization probes from heterologous gene sequences and probes; Maxwell ES et al.; We describe a simple and rapid method for the preparation of homologous DNA oligonucleotide probes for hybridization analysis and/or cDNA/genomic library screening . With this method, a synthetic DNA oligonucleotide derived from a known heterologous DNA/RNA/protein sequence is annealed to an RNA preparation containing the gene transcript of interest . Any unpaired 3'-terminal oligonucleotides of the heterologous DNA primer are then removed using the 3' exonuclease activity of the DNA Polymerase I Klenow fragment before primer extension/dideoxynucleotide sequencing of the annealed RNA species with AMV reverse transcriptase . From the determined RNA sequence, a completely homologous DNA oligonucleotide probe is then prepared . This approach has been used to prepare a homologous DNA oligonucleotide probe for the successful library screening of the yeast hybRNA gene starting with a heterologous mouse hybRNA DNA oligonucleotide probe.

J Biol Chem, 1988 Nov 25, 263(33), 17360 - 5
Characterization of dolichol diphosphate oligosaccharide: protein oligosaccharyltransferase and glycoprotein-processing glucosidases occurring in trypanosomatid protozoa; Bosch M et al.; We have previously reported that the oligosaccharides transferred in vivo from dolichol-P-P derivatives in protein N-glycosylation in trypanosomatids are devoid of glucose residues and contain 2 N-acetylglucosamine and 6, 7, or 9 mannose units depending on the species . In this respect trypanosomatids differ from wild type mammalian, plant, insect, and fungal cells in which Glc3Man9GlcNAc2 is transferred . We are now reporting that incubation of Glc1-3Man9GlcNAc2-P-P-dolichol and Man7-9GlcNAc2-P-P-dolichol with membranes of Trypanosoma cruzi, Leptomonas samueli, Crithidia fasciculata, and Blastocrithidia culicis and an acceptor hexapeptide leads to the transfer of the six above mentioned lipid-linked oligosaccharides at the same rate . Control experiments performed under similar conditions but with rat liver and Saccharomyces cerevisiae membranes showed that, as already known, Glc3Man9GlcNAc2 is preferentially transferred in the latter systems . We have also previously reported that, once transferred to protein, the oligosaccharides become transiently glucosylated in trypanosomatids . Depending on the species, protein-linked Glc1Man5-9GlcNAc2 have been transiently detected in cells incubated with {14C} glucose . We are now reporting that glucosidase activities degrading both Glc1Man9GlcNAc2 and Glc2Man9GlcNAc2 were detected in T . cruzi, L . samueli, and C . fasciculata . The enzymatic activities were associated with a membrane fraction; they had a neutral optimum pH value, and similarly to mammalian glucosidase II, the enzyme acting on the monoglucosylated substrate showed a decreased affinity when the latter contained fewer mannose residues . No glucosidase I-like enzyme acting on Glc3Man9GlcNAc2 was detected in any of the three above-mentioned protozoan species . This result is consistent with the fact that no oligosaccharides containing 3 glucose units occur in trypanosomatids.

Biochim Biophys Acta, 1988 Nov 25, 963(2), 349 - 58
Ethionine-induced alterations of enzymes involved in lipid metabolism and their possible relationship to induction of fatty liver; Aarsaether N et al.; Changes of enzymes involved in the hepatic metabolism of long-chain fatty acids (palmitoyl-CoA synthetase (EC 6.2.1.3), carnitine palmitoyltransferase (EC 6.2.1.3), glycerophosphate acyltransferase (EC 2.3.1.15)) in the liver of male rats were examined after ethionine exposure . Ethionine administration resulted in a dose- and time-dependent enhancement of the palmitoyl-CoA synthetase activity both in the mitochondrial, peroxisomal and microsomal fractions . The total carnitine palmitoyltransferase activity in the mitochondrial fraction was enhanced . Ethionine administration was also associated with dose- and time-dependent changes of the microsomal glycerophosphate acyltransferase activity, whereas the mitochondrial enzyme activity was marginally affected . The hepatic triacylglycerol content of the ethionine-treated animals was increased . Hepatic lipids were accumulated in large droplets . Serum triacylglycerol and cholesterol were decreased . In particular, the serum HDL-cholesterol level was lowered . The concentration of ATP in the liver decreased . Accumulation of the metabolic product S-adenosylethionine (AdoEth) was observed for the first 2 days of exposure followed by a fall in S-adenosylmethionine (Ado-Met) during the next 10 days . Linear regression analysis of ATP content versus AdoEth and AdoMet showed highly significant correlations . A significant correlation between the hepatic triacylglycerol and AdoEth content was also observed upon ethionine treatment . The data show that ethionine perturbs the hepatic lipid metabolism . Enhanced esterification of long-chain fatty acids, but not a simple reduction of their oxidation, might contribute to ethionine-induced fatty liver in addition to a block in secretion of lipoproteins and decreased protein synthesis.

Cell, 1988 Nov 18, 55(4), 705 - 17
Copper activates metallothionein gene transcription by altering the conformation of a specific DNA binding protein; Furst P et al.; Copper homeostasis in yeast involves a copper binding protein, metallothionein, and a trans-acting regulatory protein that activates transcription of the metallothionein gene in response to copper ions . We show that the regulatory protein specifically binds to the metallothionein gene control sequences in the presence, but not in the absence, of copper . Both the DNA binding and metalloregulatory functions of the transacting factor are contained within its aminoterminal domain, and partial proteolysis experiments show that copper activates this domain by causing a major switch in its conformation . Silver also activates the DNA binding domain in vitro and induces metallothionein gene transcription in vivo . We propose a novel copper cluster model for the DNA binding domain based on its surprising structural similarities to metallothionein itself.

Science, 1988 Nov 18, 242(4881), 1028 - 35
An early hierarchic role of U1 small nuclear ribonucleoprotein in spliceosome assembly; Ruby SW et al.; Splicing of nuclear precursor messenger RNA (pre-mRNA) occurs on a large ribonucleoprotein complex, the spliceosome . Several small nuclear ribonucleoproteins (snRNP's) are subunits of this complex that assembles on the pre-mRNA . Although the U1 snRNP is known to recognize the 5' splice site, its roles in spliceosome formation and splice site alignment have been unclear . A new affinity purification method for the spliceosome is described which has provided insight into the very early stages of spliceosome formation in a yeast in vitro splicing system . Surprisingly, the U1 snRNP initially recognizes sequences at or near both splice junctions in the intron . This interaction must occur before the other snRNP's (U2, U4, U5, and U6) can join the complex . The results suggest that interaction of the two splice site regions occurs at an early stage of spliceosome formation and is probably mediated by U1 snRNP and perhaps other factors.

Cell, 1988 Nov 18, 55(4), 663 - 71
Gene overlap results in a viral protein having an RNA binding domain and a major coat protein domain; Fujimura T et al.; L-A double-stranded RNA (dsRNA) replicates in vivo in yeast in a conservative, asynchronous (first {+} strand then {-} strand), intraviral process . New particles are formed by packaging (+) strands . Added viral (+) single-stranded RNA (ssRNA) is specifically bound by empty virus-like particles (VLPs) and, in a reaction requiring a host factor, is converted in vitro to dsRNA . We find that the isolated binding complex replicates only if it was formed in the presence of the host factor . The VLP minor 180 kd protein, but not the major coat protein, has ssRNA binding activity on Western blots . The 180 kd protein shares a common antigenic domain with the major coat protein, the latter known to be encoded by L-A dsRNA . The 180 kd protein, but not the major coat protein, also shares an antigenic domain with a sequence encoded by the 3' end of the L-A (+) strand . Thus the 180 kd protein is also encoded by L-A dsRNA and consists of a major coat protein domain and a ssRNA binding domain.

Gene, 1988 Nov 15, 71(1), 225 - 30
A cautionary note on the use of blot hybridization for RNA size determination; Hauge BM; The ease with which RNA blot hybridization analysis can be performed makes it among the most widely used analytical tools in molecular biology . Hybridization with a labeled probe, subsequent to size fractionation and immobilization on a filter, allows one to approximate both the size and abundance of a given RNA in a single experiment . This communication demonstrates that dramatic differences in the electrophoretic mobility of the yeast GCN2 transcript are observed when size fractionation on formaldehyde gels is compared to fractionation of glyoxalated RNA . Both routinely used systems are considered to be fully denaturing . The anomalous mobilities therefore pose a potential problem when size determination is performed using a single gel system.

Biochemistry, 1988 Nov 15, 27(23), 8562 - 8
Replacement of a conserved proline eliminates the absorbance-detected slow folding phase of iso-2-cytochrome c; Wood LC et al.; As a test of the proline isomerization model, we have used oligonucleotide site-directed mutagenesis to construct a mutant form of iso-2-cytochrome c in which proline-76 is replaced by glycine {Wood, L . C., Muthukrishnan, K., White, T . B., Ramdas, L., & Nall, B . T . (1988) Biochemistry (preceding paper in this issue)} . For the oxidized form of Gly-76 iso-2, an estimate of stability by guanidine hydrochloride induced unfolding indicates that the mutation destabilizes the protein by 1.2 kcal/mol under standard conditions of neutral pH and 20 degrees C (delta G degrees u = 3.8 kcal/mol for normal Pro-76 iso-2 versus 2.6 kcal/mol for Gly-76 iso-2) . The kinetics of folding/unfolding have been monitored by fluorescence changes throughout the transition region using stopped-flow mixing . The rates for fast and slow fluorescence-detected refolding are unchanged, while fast unfolding is increased in rate 3-fold in the mutant protein compared to normal iso-2 . A new kinetic phase in the 1-s time range is observed in fluorescence-detected unfolding of the mutant protein . The presence of the new phase is correlated with the presence of species with an altered folded conformation in the initial conditions, suggesting assignment of the phase to unfolding of this species . The fluorescence-detected and absorbance-detected slow folding phases have been monitored as a function of final pH by manual mixing between pH 5.5 and 8 (0.3 M guanidine hydrochloride, 20 degrees C).(ABSTRACT TRUNCATED AT 250 WORDS)

Biochem Biophys Res Commun, 1988 Nov 15, 156(3), 1063 - 9
Evidence of gene amplification in tunicamycin-resistant Chinese hamster ovary cells; Scocca JR et al.; Restriction digests of genomic DNA from tunicamycin-resistant Chinese hamster ovary cells, 3E11, were probed with the yeast transferase gene, ALG7 . The data presented suggest moderate amplification of the N-acetylglucosaminyl-1-phosphate transferase gene occurred in these cells, consistent with the previously observed chromosomal translocations and increased enzymatic activity . This is the first example of gene amplification as a mechanism for aberrations in N-linked glycosylation.

Biochim Biophys Acta, 1988 Nov 10, 951(1), 71 - 7
Reaction of nucleic acid bases with alpha-acetylenic esters . Part IV . Preparation of an alkylating derivative of tRNA(Phe) by conformation-specific chemical modification; Roques P et al.; The reaction of yeast tRNA(Phe) with methyl chlorotetrolate, ClCH2-C identical to C-COOCH3, was studied . This reagent converts adenine and cytosine rings into derivatives in which an additional heterocycle bearing the alkylating chloromethyl group is fused to the original base; these derivatives can exist in two isomeric forms . Modified nucleosides of this type can be easily identified by reverse-phase HPLC . It was found that under native conditions, the modification of tRNA involves the anticodon loop and the 3'-end . The isomers of adenine derivatives formed in the anticodon loop were different from those formed in the 3'-end . It is suggested that the isomeric structure of the derivatives is related to the fine conformational differences between these two regions of tRNA(Phe) . Methyl chlorotetrolate could thus be used as a conformational probe of single-stranded nucleic acids . Preliminary assays showed that modified tRNA(Phe) binds irreversibly to yeast phenylalanyl-tRNA synthetase.

J Biol Chem, 1988 Nov 5, 263(31), 16388 - 94
Metabolite regulation of argininosuccinate synthetase in cultured human cells; Jackson MJ et al.; We have studied the transcription of the argininosuccinate synthetase gene in cultured RPMI 2650 cells under conditions where the enzyme is subject to metabolite regulation and in canavanine-resistant variants (Canr1 cells) which overproduce the enzyme greater than 200-fold . When grown continuously in medium with citrulline substituted for arginine, the argininosuccinate synthetase activity of RPMI 2650 cells increases 5- to 10-fold . In these cells, expression of a transfected minigene containing the 5'-flanking region of the argininosuccinate synthetase gene was increased 20-fold by short term starvation for arginine and 10-fold by short term starvation for leucine . Levels of nuclear RNA from the first intron of the gene correlated with enzyme activity; i.e . RPMI 2650 cells cultured in arginine medium less than RPMI 2650 cells cultured in citrulline medium less than Canr1 cells . Run-off transcription experiments showed that the transcription of argininosuccinate synthetase increased in RPMI 2650 cells starved for either arginine or leucine . While expression of the minigene and the transcription rate for argininosuccinate synthetase were increased during 48 to 72 h of starvation, the endogenous enzyme activity did not increase in RPMI 2650 cells . Amino acid starvation did not affect the rate of transcription of argininosuccinate synthetase in Canr1 cells . The results indicate that the steady state levels of argininosuccinate synthetase expression in Canr1 cells and in citrulline-adapted RPMI 2650 cells are largely determined by the rate of transcription . The failure of increased transcription rate to correlate with increased enzyme activity during acute starvation for arginine or leucine may suggest the involvement of post-transcriptional regulatory mechanisms for argininosuccinate synthetase or may merely be due to amino acid deprivation . The finding that leucine starvation has effects similar to arginine starvation raises the question of whether mammalian cells have general control mechanisms which are similar to the general control of amino acid biosynthesis in Saccharomyces cerevisiae.

J Biol Chem, 1988 Nov 5, 263(31), 16209 - 17
Proteasomes (multi-protease complexes) as 20 S ring-shaped particles in a variety of eukaryotic cells; Tanaka K et al.; Latent multicatalytic protease complexes, named proteasomes, were purified to apparent homogeneity from various eukaryotic sources, such as human, rat, and chicken liver, Xenopus laevis ovary, and yeast (Saccharomyces cerevisiae), and their functional and structural properties were compared . They showed latency in breakdown of {methyl-3H}casein, but were greatly activated in various ways, such as by addition of polylysine . They all degraded three types of fluorogenic oligopeptides at the carboxyl side of basic, neutral, and acidic amino acids, and the three cleavage reactions showed different spectra for inhibition, suggesting that they had three distinct active sites . The proteasomes all seemed to be seryl endopeptidases with similar pH optima in the weakly alkaline region . Their physiochemical properties, such as their sedimentation coefficients (19 S to 22 S), diffusion coefficients (2.0-2.6 X 10(-7) cm2 s-1), molecular masses (700-900 kDa), and circular dichroic spectra, were similar . Their amino acid compositions were also very similar . Electron microscopy showed that they had similar well-defined symmetrical morphology, appearing to be ring-shaped particles with a small hole in the center . All the proteasomes seemed to be multisubunit complexes consisting of 15-20 polypeptides with molecular masses of 22-33 kDa and isoelectric points of pH 3-10, but they showed species-specific differences in subunit multiplicity . Moreover, they differed immunologically, as shown by Ouchterlony tests and immunoblotting analyses, although cross-immunoreactivities of some subunits or domains were observed . These results indicate that the sizes and shapes of these proteasomes have been highly conserved during evolution, but that they show species-specific differences in immunoreactivities and subunit structures . Thus proteasomes with similar structure and function seem to be ubiquitously distributed in eukaryotic organisms ranging from man to yeast . This distribution implies the general importance of these proteasomes for proteolysis.

J Biol Chem, 1988 Nov 5, 263(31), 15946 - 50
Primary structure of a human trifunctional enzyme . Isolation of a cDNA encoding methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase; Hum DW et al.; A DNA clone complementary to the messenger RNA encoding the human trifunctional enzyme 5,10-methylenetetrahydrofolate dehydrogenase-5,10-methenyl-tetrahydrofolate cyclohydrolase-10-formyltetrahydrofolate synthetase has been isolated from a lambda gt10 library . In vitro transcription-translation of the 3.1-kilobase cDNA clone yields a protein of 101 kDa, which is of identical size and exhibits the same immunoreactivity as the enzyme purified from human liver . A coding region of 2805 base pairs in the cDNA encodes a protein of 935 amino acids . The initiator methionine is absent from the purified enzyme and the amino-terminal 30 amino acids derived by automated sequence analysis are identical (arginine at position 18 was not identified) with that deduced from the nucleotide sequence . The amino acid sequence of the human enzyme shows extensive homology with that of the yeast enzyme, although the amino-terminal bifunctional dehydrogenase-cyclohydrolase domain is less homologous than the carboxyl-terminal synthetase domain . A region was identified which probably serves as a link between these two major domains of the human enzyme . The synthetase domain contains two regions that are homologous to consensus sequences for an ATP-binding site.

Arch Biochem Biophys, 1988 Nov 1, 266(2), 486 - 95
Distinct long chain and very long chain fatty acyl CoA synthetases in rat liver peroxisomes and microsomes; Singh H et al.; Mitochondria, peroxisomes, and microsomes were isolated from rat liver homogenates, and stearic acid and lignoceric acid beta-oxidation, as well as stearoyl CoA synthetase and lignoceroyl CoA synthetase activities in the three organelles, were compared . Stearic acid beta-oxidation in peroxisomes was sixfold greater compared to the oxidation in mitochondria . Lignoceric acid beta-oxidation, observed only in peroxisomes, was fivefold lower compared to stearic acid beta-oxidation . Stearoyl CoA synthetase was present whereas lignoceroyl CoA synthetase was absent in mitochondria . Stearoyl CoA synthetase and lignoceroyl CoA synthetase activities were present in microsomes and peroxisomes, but the activity of stearoyl CoA synthetase was several-fold greater compared to lignoceroyl CoA synthetase in both organelles . The differing responses to detergents and phospholipids of stearoyl CoA and lignoceroyl CoA synthetase activities in microsomes as well as peroxisomes indicated that each activity was catalyzed by a separate enzyme . Differences in detergent and phospholipid response were also noted when either stearoyl CoA or lignoceroyl CoA synthetase activity in one organelle was compared with the corresponding activity in the other organelle, suggesting that the same activity in different organelles may be catalyzed by separate enzyme proteins.

Mol Cell Biol, 1988 Nov, 8(11), 4808 - 20
Molecular analysis of GCN3, a translational activator of GCN4: evidence for posttranslational control of GCN3 regulatory function; Hannig EM et al.; GCN4 encodes a transcriptional activator of amino acid biosynthetic genes in Saccharomyces cerevisiae . The GCN3 product is a positive regulator required for increased synthesis of GCN4 protein in amino acid-starved cells . GCN3 appears to act indirectly by antagonizing GCD-encoded negative regulators of GCN4 expression under starvation conditions; however, GCN3 can also suppress the effects of gcd12 mutations under nonstarvation conditions . These results imply that the GCN3 product can promote either repression or activation of GCN4 expression depending on amino acid availability . We present a complete physical description of the GCN3 gene and its transcript, plus measurements of GCN3 expression at the transcriptional and translational levels under different growth conditions . GCN3 encodes a 305-amino-acid polypeptide with no significant homology to any other known protein sequence . GCN3 mRNA contains no leader AUG codons, and no potential GCN4 binding sites were found in GCN3 5' noncoding DNA . In accord with the absence of these regulatory sequences found at other genes in the general control system, GCN3 mRNA and a GCN3-lacZ fusion enzyme are present at similar levels under both starvation and nonstarvation conditions . These data suggest that modulation of GCN3 regulatory function in response to amino acid availability occurs posttranslationally . A gcn3 deletion leads to unconditional lethality in a gcd1-101 mutant, supporting the idea that GCN3 is expressed under normal growth conditions and cooperates with the GCD1 product under these circumstances to carry out an essential cellular function . We describe a point mutation that adds three amino acids to the carboxyl terminus of GCN3, which inactivates its positive regulatory function required under starvation conditions without impairing its ability to promote functions carried out by GCD12 under nonstarvation conditions.

J Cell Biol, 1988 Nov, 107(5), 1669 - 75
Alcohol oxidase expressed under nonmethylotrophic conditions is imported, assembled, and enzymatically active in peroxisomes of Hansenula polymorpha; Distel B et al.; We have introduced into Hansenula polymorpha an extra copy of its alcohol oxidase gene . This gene which is under the control of the Saccharomyces cerevisiae phosphoglycerate kinase promoter is integrated in a chromosome different from the one containing the endogenous gene . Cells with the extra alcohol oxidase gene, grown on glucose or ethanol as the sole carbon source, express enzymatically active alcohol oxidase . However, other enzymes characteristic for methylotrophic growth conditions are absent or present at low levels . Most of the alcohol oxidase occurs in the octameric state and immuno- and cytochemical evidence shows that it is located in a single enlarged peroxisome per cell . Such peroxisomes show crystalloid inclusions which are lacking in the peroxisomes present in glucose grown control cells . Our results suggest that import into peroxisomes of H . polymorpha, assembly and activation of alcohol oxidase is not conditionally dependent on adaptation to methylotrophic growth conditions and that proliferation of peroxisomes is a well-programmed process that is not triggered solely by overproduction of a peroxisomal protein.

Biochem Biophys Res Commun, 1988 Oct 31, 156(2), 987 - 94
Cloning and sequencing of a cDNA for human mitochondrial ubiquinone-binding protein of complex III; Suzuki H et al.; The ubiquinone-binding protein (QP-C) is a nuclear-encoded component of ubiquinol-cytochrome c oxidoreductase in the mitochondrial respiratory chain and plays an important role in electron transfer as a ubiquinone-QP-C complex . We obtained a partial cDNA for rat liver QP-C by screening a lambda gt11 rat liver cDNA library using antiserum directed against bovine heart QP-C . Using this cDNA as a probe, a cDNA clone was isolated from a human fibroblast cDNA library by colony hybridization . The total length of the cloned cDNA was 518 base pairs with an open reading frame of 333 base pairs . The 111-amino acid sequence deduced from the nucleotide sequence of the cDNA is 85% homologous to that of bovine QP-C and contains only a single additional amino-terminal methionine . This implies that the human QP-C is synthesized without a presequence which is required for import of most nuclear-encoded mitochondrial proteins into mitochondria.

Nucleic Acids Res, 1988 Oct 25, 16(20), 9677 - 86
Novel non-templated nucleotide addition reactions catalyzed by procaryotic and eucaryotic DNA polymerases; Clark JM; DNA polymerases catalyze the addition of deoxyribonucleotides onto DNA primers in a template-directed manner . The requirement for template instruction distinguishes these enzymes from other nucleotidyl transferases, such as terminal deoxynucleotidyl transferase, that do not utilize a template . An oligonucleotide substrate was used to characterize a novel, non-templated nucleotide addition reaction carried out by DNA polymerases from a variety of procaryotic and eucaryotic sources . The products of the reaction, in which a deoxyribonucleotide was added to the 3' hydroxyl terminus of a blunt-ended DNA substrate, were analyzed by electrophoresis on high resolution, denaturing polyacrylamide gels . DNA polymerase from Thermus aquaticus, polymerase alpha from chick embryo, rat polymerase beta, reverse transcriptase from avian myeloblastosis virus, and DNA polymerase I from Saccharomyces cerevisiae all carried out the blunt-end addition reaction . The reaction required a duplex DNA substrate but did not require coding information from the template strand . These results demonstrate that template instruction is not an absolute requirement for the catalysis of nucleotidyl transfer reactions by DNA polymerases.

Biochemistry, 1988 Oct 18, 27(21), 8074 - 81
Crystal structure of nitric oxide inhibited cytochrome c peroxidase; Edwards SL et al.; We have collected X-ray diffraction data from a crystal of cytochrome c peroxidase (CCP) complexed with the inhibitor nitric oxide to a resolution of 2.55 A . A difference Fourier map shows density indicating the NO ligand is bound to the heme iron at the sixth coordination site in a bent configuration . Structural adjustments were determined by least-squares refinement that yielded an agreement residual of R = 0.18 . The orientation of the ligand, tilting toward Arg-48, causes adjustment in the position of this nearby polar side chain . As a model for the substrate hydrogen peroxide, this geometry is consistent with the suggestion that Arg-48 serves to polarize the O-O peroxide bond to promote heterolytic cleavage of the bond {Poulos, T . L., & Kraut, J . (1980) J . Biol . Chem . 255, 8199-8205} . Strong difference density is also observed near residues 190-194, especially around the indole ring of Trp-191 . The density indicates movement of the indole ring away from the proximal His-175 imidazole ring by about 0.25 A, which appears to cause perturbation of the neighboring residues . The response of Trp-191 on the proximal side of the heme to binding nitric oxide on the distal side probably results from delocalization of the electron density of the ligand . Relevant to this is the recent finding that a mutant in which Trp-191 is replaced by phenylalanine has dramatically reduced activity, less than 0.05% of the parent activity {Mauro, J . M., Fishel, L . A., Hazzard, J . T., Meyer, T . E., Tollin, G., Cusanovich, M . A., & Kraut, J . (1988) Biochemistry 27, 6243-6256}.(ABSTRACT TRUNCATED AT 250 WORDS)

Eur J Biochem, 1988 Oct 15, 177(1), 179 - 85
Two-dimensional NMR as a probe of structural similarity applied to mutants of cytochrome c; Pielak GJ et al.; Using site-directed mutagenesis, it is possible to prepare many mutants of a protein in a short time, and to uncover differences in function . To understand the changes in function, it is essential to understand the effect(s) of the mutation in terms of structural and dynamic changes . It is particularly important to establish a rapid method for comparing the structure of the mutants with that of the wild-type protein . We propose that a combination of overlayed and difference two-dimensional NOE spectra between the wild-type and mutant protein provide a rapid method for determination of structural similarity . The observation of differences other than those due directly to the field effects of the exchanged side chain allow both local and distant conformational changes to be assessed . Here we compare NOESY spectra from a mutant of yeast iso-1-ferrocytochrome c in which the invariant residue Phe-82 has been changed to a Tyr . We conclude that NMR can show subtle changes in protein structure . Specifically, we show the change must involve the reorientation of the side chain of Leu-85 which is proximal to the mutation . The dynamics of the aromatic side chain at position 82 are shown not to give rise to measurable differences between the wild-type and mutant protein . Structural changes are not propagated to a measurable degree in other parts of the protein.

Science, 1988 Oct 7, 242(4875), 93 - 7
tRNAi(met) functions in directing the scanning ribosome to the start site of translation; Cigan AM et al.; The mechanism by which the scanning ribosome recognizes the first AUG codon nearest the 5' end of eukaryotic messenger RNA has not been established . To investigate this an anticodon change (3'-UCC-5') was introduced into one of the four methionine initiator (tRNAi(met) genes of Saccharomyces cerevisiae . The ability of the mutant transfer RNA to restore growth properties to his4 initiator codon mutant yeast strains in the absence of histidine was then assayed . Only the complementary codon, AGG, at the his4 initiator region supported His+ growth . The mutant transfer RNA also directed the ribosome to initiate at an AGG placed in the upstream region of the his4 message . Initiation at this upstream AGG precluded initiation at a downstream AGG in accordance with the "scanning" model . Therefore, an anticodon: codon interaction between tRNAi(met) as part of the scanning ribosome and the first AUG must function in directing the ribosome to the eukaryotic initiator region.

Nature, 1988 Oct 6, 335(6190), 563 - 4
GAL4-VP16 is an unusually potent transcriptional activator; Sadowski I et al.; Recent work has defined a class of transcriptional activators, members of which activate transcription in yeast, plant, insect and mammalian cells . These proteins contain two parts: one directs DNA binding and the other, called the activating region, presumably interacts with some component of the transcriptional machinery . Activating regions are typically acidic and require some poorly-understood aspect of structure, probably at least in part an alpha-helix . Here we describe a new member of this class, formed by fusing a DNA-binding fragment of the yeast activator GAL4 to a highly acidic portion of the herpes simplex virus protein VP16 (ref . 11; also called Vmw65) . VP16 activates transcription of immediate early viral genes by using its amino-terminal sequences to attach to one or more host-encoded proteins that recognise DNA sequences in their promoters . We show that the hybrid protein (GAL4-VP16) activates transcription unusually efficiently in mammalian cells when bound close to, or at large distances from the gene . We suggest that the activating region of VP16 may be near-maximally potent and that it is not coincidental that such a strong activator is encoded by a virus.

J Biol Chem, 1988 Oct 5, 263(28), 14267 - 75
Reduction of cytochrome c peroxidase compounds I and II by ferrocytochrome c . A stopped-flow kinetic investigation; Summers FE et al.; The oxidation of yeast cytochrome c peroxidase by hydrogen peroxide produces a unique enzyme intermediate, cytochrome c peroxidase Compound I, in which the ferric heme iron has been oxidized to an oxyferryl state, Fe(IV), and an amino acid residue has been oxidized to a radical state . The reduction of cytochrome c peroxidase Compound I by horse heart ferrocytochrome c is biphasic in the presence of excess ferrocytochrome c as cytochrome c peroxidase Compound I is reduced to the native enzyme via a second enzyme intermediate, cytochrome c peroxidase Compound II . In the first phase of the reaction, the oxyferryl heme iron in Compound I is reduced to the ferric state producing Compound II which retains the amino acid free radical . The pseudo-first order rate constant for reduction of Compound I to Compound II increases with increasing cytochrome c concentration in a hyperbolic fashion . The limiting value at infinite cytochrome c concentration, which is attributed to the intracomplex electron transfer rate from ferrocytochrome c to the heme site in Compound I, is 450 +/- 20 s-1 at pH 7.5 and 25 degrees C . Ferricytochrome c inhibits the reaction in a competitive manner . The reduction of the free radical in Compound II is complex . At low cytochrome c peroxidase concentrations, the reduction rate is 5 +/- 3 s-1, independent of the ferrocytochrome c concentration . At higher peroxidase concentrations, a term proportional to the square of the Compound II concentration is involved in the reduction of the free radical . Reduction of Compound II is not inhibited by ferricytochrome c . The rates and equilibrium constant for the interconversion of the free radical and oxyferryl forms of Compound II have also been determined.

Mol Cell Biol, 1988 Oct, 8(10), 4314 - 21
Ribosomal protein S14 is encoded by a pair of highly conserved, adjacent genes on the X chromosome of Drosophila melanogaster; Brown SJ et al.; We describe a Drosophila DNA clone of tandemly duplicated genes encoding an amino acid sequence nearly identical to human ribosomal protein S14 and yeast rp59 . Despite their remarkably similar exons, the locations and sizes of introns differ radically among the Drosophila, human, and yeast (Saccharomyces cerevisiae) ribosomal protein genes . Transcripts of both Drosophila RPS14 genes were detected in embryonic and adult tissues and are the same length as mammalian S14 message . Drosophila RPS14 was mapped to region 7C5-9 on the X chromosome . This interval also encodes a previously characterized Minute locus, M(1)7C.

Acta Chem Scand B, 1988 Oct, 42(9), 635 - 9
Effects of anions on the fluorescence emission of the 1-anilino-8-naphthalenesulfonate-phosphoglycerate kinase complex; Khamis MM et al.; When 1-anilino-8-naphthalenesulfonate (ANS) interacts with phosphoglycerate kinase (ATP:3-phospho-D-glycerate 1-phosphotransferase, EC 2.7.2.3) its fluorescence is enhanced and a blue shift occurs . There is evidence that ANS binds to the site of the nucleotide substrate . The work described herein shows that when various anion inhibitors are added to the ANS-enzyme solution, de-enhancement of the fluorescence occurs . Extrapolation to infinite anion concentration shows that pyruvate ions are the most effective quenchers (ca . 90%) and nitrate ions the least effective, sulfate and phosphate ions being intermediate . The results are consistent with earlier enzymes kinetic findings suggesting that pyruvate ions and ANS, both competing with the nucleotide substrate, are able to bind to the enzyme simultaneously and that sulfate, phosphate and nitrate ions can, to various extents, affect the properties at the active centre of phosphoglycerate kinase via conformational changes without sharing ligands with the nucleotide substrate.

Genes Dev, 1988 Oct, 2(10), 1268 - 76
The role of the mammalian branchpoint sequence in pre-mRNA splicing; Reed R et al.; We show that base substitutions in the mammalian branchpoint sequence (BPS) YNCUGAC dramatically reduce the efficiency of pre-mRNA splicing in vitro and alter 3' splice-site selection in vivo . Contrary to current dogma that an adenine residue at the appropriate distance from the 3' splice site is the primary determinant of lariat formation, we find that many mutations in the BPS virtually abolish splicing even though the position of this adenine is unchanged . Comparison of the analogous single-base changes in the mammalian and yeast BPSs revealed similar relative effects on splicing efficiency . However, in contrast to yeast, mammalian branchpoint mutations that decrease splicing efficiency severely do not prevent spliceosome assembly . Thus, mutations in the mammalian BPS appear to uncouple spliceosome assembly from cleavage at the 5' splice site and lariat formation.

Proc Natl Acad Sci U S A, 1988 Oct, 85(19), 7279 - 83
Evidence for regulation of reinitiation in translational control of GCN4 mRNA; Hinnebusch AG et al.; Translational control of the GCN4 gene of Saccharomyces cerevisiae is mediated by four upstream open reading frames (URFs) present in the leader of GCN4 mRNA . URFs 3 and 4 efficiently repress GCN4 expression in normal growth conditions; URFs 1 and 2 are required to overcome this repression in amino acid-starved cells . lacZ fusions to URFs 3 and 4 were used to determine the translational event that is regulated at these sequences by URFs 1 and 2 . URF3-lacZ, URF4-lacZ, and GCN4-lacZ fusions are affected similarly by URFs 1 and 2 when no other URFs are present in the leader: expression from all three fusions is reduced by an amount slightly greater in repressing than in derepressing conditions . These results are inconsistent with models that postulate a differential effect of URFs 1 and 2 on initiation or elongation rates at URFs 3 and 4 versus the GCN4 coding sequences . We propose that the efficiency of reinitiation at the GCN4 AUG codon after translation of URFs 3 and 4 is the translational event that is stimulated in derepressing conditions by URFs 1 and 2.

Proc Natl Acad Sci U S A, 1988 Oct, 85(19), 7221 - 5
Proteins from eight eukaryotic cytochrome P-450 families share a segmented region of sequence similarity; Kalb VF et al.; Proteins from eight eukaryotic families in the cytochrome P-450 superfamily share one region of sequence similarity . This region begins 275-310 amino acids from the amino terminus of each P-450, continues for approximately 170 residues, and ends 35-50 amino acids before the carboxyl terminus . The region can be divided into four domains of sequence similarity, each possessing its own pattern of invariant, conserved, and variable amino acids . The four domains are 56, 20, 59, and 28 residues long and are connected by three shorter segments of limited sequence similarity . The number of residues in these short segments varies with the P-450 protein but ranges from 0 to 20 residues . Consensus sequences based on these similarities can be used to determine whether the sequence of an unidentified peptide resembles that expected for a P-450 . Sequence similarities between proteins sometimes reflect constraints imposed by the requirements of a common function . The fourth domain of the P-450s, for example, contains an invariant cysteine that provides the axial thiolate ligand to the heme iron . Other relationships between the four domains and P-450 function can be examined by in vitro mutagenic procedures that alter the conserved amino acids or modify the distance between domains.

Mol Gen Genet, 1988 Oct, 214(2), 224 - 31
Molecular organisation of the quinic acid utilization (QUT) gene cluster in Aspergillus nidulans; Hawkins AR et al.; The functional integrity of the QUTB gene (encoding quinate dehydrogenase) has been confirmed by transformation of a qutB mutant strain . The DNA sequence of the contiguous genes QUTD (quinate permease), QUTB and QUTG (function unknown) has been determined and analysed, together with that of QUTE (catabolic 3-dehydroquinase) . The QUTB sequence shows significant homology with the shikimate dehydrogenase function of the complex AROM locus of Aspergillus nidulans, and with the QA-3 quinate dehydrogenase and QA-1S (repressor) genes of Neurospora crassa . The QUTD gene shows strong homology with the N . crassa QA-Y gene and QUTG with the QA-X gene . QUTD, QUTB, and QUTG, QUTE form two pairs of divergently transcribed genes, and conserved sequence motifs identified in the two common 5' non-coding regions show significant homology with UASGAL and UASQA sequences of the Saccharomyces cerevisiae and N . crassa Gal and QA systems . In addition, conserved 5' sequences homologous to the mammalian CAAT box are noted and a previously unreported conserved 22 nucleotide motif is presented.

Scand J Immunol, 1988 Oct, 28(4), 397 - 402
Ligand-specific cross-inhibition of monocyte phagocytosis; Kavai M et al.; Human monocytes exposed to particles reacting with receptors of one specific type demonstrate a markedly reduced phagocytosis of particles reacting with receptors of another type . The phagocytosis of yeast particles (Saccharomyces cerevisiae) coated with C3 fragments and of uncoated ones by monocytes can inhibit the subsequent endocytosis of sheep erythrocytes sensitized with IgG antibody and vice versa . The erythrocytes were labelled with 51Cr and the yeast particles with 125I . The dependence of the receptor-ligand reaction on temperature and time of preincubation, as well as on ratio of cell and particle, suggests that this cross-inhibition may be associated with plasma membrane modulations beginning with the attachment of ligands to a receptor and followed by their endocytosis . The differences between the functions of temperature, time, and concentration in inhibition caused by different receptor-ligand reactions suggest that these membrane modulations are probably ligand-specific processes.

FEBS Lett, 1988 Sep 26, 238(1), 205 - 10
The maximum of the histone acetyltransferase activity precedes DNA-synthesis in regenerating rat liver; Weiss G et al.; A pronounced transitory increase of histone acetyltransferase (EC 2.3.1.48) activity before the onset of DNA replication is shown to occur in regenerating rat liver . This effect is followed by an increase in the H1/H1(0) ratio at the start of DNA synthesis; H1(0) de is replaced by H1 . Histone acetylation and H1/H1(0) exchange are discussed as steps in the signal transduction for DNA replication.

Nucleic Acids Res, 1988 Sep 26, 16(18), 9017 - 26
Conservation of short patches of amino acid sequence amongst proteins with a common function but evolutionarily distinct origins: implications for cloning genes and for structure-function analysis; Reichardt JK et al.; Small patches of identical amino acid sequences commonly occur in proteins that have the same function but are derived from evolutionarily distant organisms . Reverse translation of such patches into degenerate pools of oligonucleotides provide useful hybridization probes for cloning the gene for the corresponding protein from other organisms . Since the conserved patches of identical amino acid sequence are probably important for the protein's biological function, they are preferred targets for reverse genetic studies aimed at defining structure-function relationships.

Biochemistry, 1988 Sep 20, 27(19), 7371 - 5
Equilibrium of 5,6-hydration of NADH and mechanism of ATP-dependent dehydration; Acheson SA et al.; At equilibrium, water addition to the 5,6 double bond of NADH was observed to favor the hydrate by a factor of approximately 100 . Hydration generates two epimers of NADHX (beta-6-hydroxy-1,4,5,6-tetrahydronicotinamide adenine dinucleotide) . Only the 6S epimer of the hydrate was found to serve as a true substrate for an ATP-dependent dehydratase from yeast that regenerates NADH . Yet enzymatic conversion of both epimers of the hydrate to NADH was found to proceed essentially to completion in the presence of ATP and dehydratase . This is explained by the observed ability of the epimers to undergo rapid spontaneous equilibration, so that it is unnecessary to postulate a lack of stereospecificity in the dehydratase.

Biochemistry, 1988 Sep 20, 27(19), 7310 - 4
pH dependence of folding of iso-2-cytochrome c; Nall BT et al.; Starting from a standard unfolded state (3.0 M guanidine hydrochloride, pH 7.2), the kinetics of refolding of iso-2-cytochrome c have been investigated as a function of final pH between pH 3 and pH 10 . Absorbance in the ultraviolet and visible spectral regions and tryptophan fluorescence are used to monitor folding . Over most of the pH range, fast and slow folding phases are detected by both fluorescence and absorbance probes . Near neutral pH, the rate of fast folding appears to be the same when monitored by absorbance and fluorescence probes . At higher and lower pH, there are two fast folding reactions, with absorbance-detected fast folding occurring in a slightly faster time range than fluorescence-detected fast folding . The rates of both fast folding reactions pass through broad minima near neutral pH, indicating involvement of ionizable groups in rate-limiting steps . The rates of slow folding also depend on the final pH . At acid pH, there appears to be a single slow folding phase for both fluorescence and absorbance probes . At neutral pH, the absorbance-detected and fluorescence-detected slow folding phases separate into distinct kinetic processes which differ in rate and relative amplitude . At high pH, absorbance-detected slow folding is no longer observed, while fluorescence-detected slow folding is decreased in amplitude . In contrast, the equilibrium and kinetic properties of proline imide bond isomerization, believed to be involved in the slow folding reactions, are largely independent of pH . The results suggest that the pH dependence of slow folding involves coupling of pH-sensitive structure to proline imide bond isomerization.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochim Biophys Acta, 1988 Sep 14, 935(2), 208 - 16
The oxidation-reduction kinetics of cytochromes b, c1 and c in initially fully reduced mitochondrial membranes are in agreement with the Q-cycle hypothesis; de Vries S et al.; Stopped-flow experiments were performed to distinguish between two hypotheses, the Q-cycle and the SQ-cycle, each describing the pathway of electron transfer in the QH2:cytochrome c oxidoreductases . It was observed that, when mitochondrial membranes from the yeast Saccharomyces cerevisiae were poised at a low redox potential with appropriate amounts of sodium dithionite to completely reduce cytochrome b, the kinetics of oxidation of cytochrome b showed a lag period of maximally 100 ms . Under the same experimental conditions, the oxidation-reduction kinetics of cytochromes c + c1 showed transient behaviour . These results do not support the presence of a mobile species of semiquinone in the QH2:cytochrome c oxidoreductases, as envisaged in the SQ-cycle, but are consistent with a Q-cycle mechanism in which the two quinone-binding domains do not exchange electrons directly on the timescale of turnover of the enzyme.

Nucleic Acids Res, 1988 Sep 12, 16(17), 8213 - 31
Structure, evolution and properties of a novel repetitive DNA family in Caenorhabditis elegans; La Volpe A et al.; We have identified a moderately repeated DNA sequence in Caenorhabditis elegans present at least at twenty different locations in the genome . Elements of this intermingled repetitive DNA family are made up of tandem subreapeats whose smaller unit is ten base pairs long . The occurrence of single base changes between units is reminiscent of mammalian satellite DNA . Sequence analysis has shown that the consensus of these repeats is identical to the consensus of the heat-shock element (HSE) common to all eukaryotes (C--GAA--TTC--G) . This consensus in our sequences is repeated in tandem with an overlap of four bases (C--GAA--TTC--GAA--TTC...) . We studied in detail one cloned element of the family and we were unable to detect transcription in the flanking regions either under normal growth or after heat induction . Nevertheless a 242 bp sequences out of this same element was sufficient, when located on a multicopy plasmid in Saccharomyces cerevisiae, to drive transcription from a downstream gene under heat shock conditions.

FEBS Lett, 1988 Sep 12, 237(1-2), 208 - 12
Primary and post-irradiation inactivation of the sulfhydryl enzyme malate synthase: correlation of protective effects of additives; Durchschlag H et al.; The presence of additives during X-irradiation of malate synthase led to radioprotective effects against primary and post-irradiation inactivation . Pronounced effects were provided by typical scavengers, sulfhydryl reagents and specific ligands (substrates, products, analogues) . The results show that scavenging and specific protection are responsible for the protective efficiency of additives . Scavengers delete noxious species formed during irradiation or post-radiationem . Sulfhydryl reagents may act as repair substances . Specific ligands protect the active site of the enzyme and the essential sulfhydryls; specific protection is more pronounced post-radiationem . Ligands and sulfhydryl reagents may additionally act as scavengers . A cumulative index for the protective power of additives against both sorts of inactivation was established.

J Biol Chem, 1988 Sep 5, 263(25), 12698 - 704
Ubiquitin genes in trypanosomatidae; Kirchhoff LV et al.; A ubiquitin encoding cDNA from Trypanosoma cruzi, the protozoan cause of Chagas' disease, was isolated by immunoscreening a lambda gt11 expression library with serum from a mouse chronically infected with this parasite . The cDNA encodes a precursor protein consisting of four tandem repeats of ubiquitin differing from that of Saccharomyces cerevisiae in four positions, followed by an unrelated 52-amino acid tail containing a putative metal and nucleic acid binding domain . Southern and Northern blots of DNA and RNA from various strains of T . cruzi and from several African trypanosome and Leishmania isolates revealed dramatic differences in the numbers and sizes of ubiquitin genes and transcripts . One of the T . cruzi isolates examined has as many as 10 ubiquitin genes, while a strain of Leishmania donovani appears to have only 1 . Forty or more tandemly arranged ubiquitin coding repeats are present in some genes, while others have only two or three . Evidence for stage-specific expression of a ubiquitin transcript was found in one strain, but no stress-induced changes in the pattern of transcripts were detected in the one isolate examined . Thus the cellular requirements for ubiquitin in trypanosomatids can be supplied by diversely organized genes containing highly variable numbers of ubiquitin coding repeats.

Science, 1988 Sep 2, 241(4870), 1203 - 5
A novel instrument for separating large DNA molecules with pulsed homogeneous electric fields; Clark SM et al.; A new instrument has been developed for the electrophoretic separation of large DNA molecules that can independently regulate the voltage of each of 24 electrodes and allow the magnitude, orientation, homogeneity, and duration of the electric field to be precisely controlled . Each parameter can be varied at any time during the electrophoretic process . Thus distinct sets of conditions can be combined to optimize the separation of various fragment sizes in a single run . Independent control of electrode voltage allows all of the fields to be generated with electrodes arranged in a closed contour, independent of a particular geometry . This device increases both the resolution in any size range and the speed of separation, especially for DNA molecules larger than 3 megabases.

Mol Gen Genet, 1988 Sep, 214(1), 85 - 8
Expression of MF alpha 1 in MATa cells supersensitive to alpha-factor leads to self-arrest; Whiteway M et al.; MATa cells of Saccharomyces cerevisiae defective in both the SST1 and SST2 gene products exhibit self-arrest when they express the MF alpha 1 gene under the control of the GAL1 promoter . This response to endogenously produced pheromone can be alleviated by mutations which prevent the production of, or response to, alpha-factor . Suppressors of the self-arrest phenotype include a class of mutants which remain responsive to low levels of pheromone, but are resistant to high levels of alpha-factor . One of these mutants has been mapped to chromosome X, 31 cM distal to SUP4, and defines a new locus designated STE18.

Mol Cell Biol, 1988 Sep, 8(9), 3827 - 36
The positive regulatory function of the 5'-proximal open reading frames in GCN4 mRNA can be mimicked by heterologous, short coding sequences; Williams NP et al.; Translational control of GCN4 expression in the yeast Saccharomyces cerevisiae is mediated by multiple AUG codons present in the leader of GCN4 mRNA, each of which initiates a short open reading frame of only two or three codons . Upstream AUG codons 3 and 4 are required to repress GCN4 expression in normal growth conditions; AUG codons 1 and 2 are needed to overcome this repression in amino acid starvation conditions . We show that the regulatory function of AUG codons 1 and 2 can be qualitatively mimicked by the AUG codons of two heterologous upstream open reading frames (URFs) containing the initiation regions of the yeast genes PGK and TRP1 . These AUG codons inhibit GCN4 expression when present singly in the mRNA leader; however, they stimulate GCN4 expression in derepressing conditions when inserted upstream from AUG codons 3 and 4 . This finding supports the idea that AUG codons 1 and 2 function in the control mechanism as translation initiation sites and further suggests that suppression of the inhibitory effects of AUG codons 3 and 4 is a general consequence of the translation of URF 1 and 2 sequences upstream . Several observations suggest that AUG codons 3 and 4 are efficient initiation sites; however, these sequences do not act as positive regulatory elements when placed upstream from URF 1 . This result suggests that efficient translation is only one of the important properties of the 5' proximal URFs in GCN4 mRNA . We propose that a second property is the ability to permit reinitiation following termination of translation and that URF 1 is optimized for this regulatory function.

Mol Cell Biol, 1988 Sep, 8(9), 3784 - 96
Holliday intermediates and reaction by-products in FLP protein-promoted site-specific recombination; Meyer-Leon L et al.; Holliday structures are formed and resolved by FLP protein during site-specific recombination . These structures have been isolated and are visualized in both native and partially denatured states by electron microscopy . No single-strand breaks are found within the junction, indicating that the structure results from a reciprocal exchange of strands . These structures have properties consistent with being reaction intermediates . Double-strand cleavage products and "Y structures" are also detected and appear to be by-products of the reaction . The Y structures are three-armed branched molecules with a covalently closed junction located at the FLP recombination target site . Models are discussed, suggesting that both of these novel structures are made by aberrant cleavages during formation and resolution of the Holliday intermediate.

Genetika, 1988 Sep, 24(9), 1572 - 8
{Characteristics of detection of mutants on selective media}; Chepurnoi AI et al.; The regularities of formation and appearance of mutants after transfer or plating yeast cells on selective media are analysed . The knowledge of these patterns is necessary for quantitative evaluation of mutants' content in the culture . Mutants formed in a cell culture, prior to transfer onto selective medium, are revealed on this medium as colonies and form a wave in time that is being observed during several days . Due to residual growth of cells, new mutants are formed and revealed as the second wave . Following two waves, an increasing front may approach which is formed from mutants of unknown origin . For evaluation of the number of mutants in the culture, prior to transfer onto selective medium, it is necessary to count up only the "first wave" of mutant colonies' appearance.

Curr Genet, 1988 Sep, 14(3), 211 - 23
A reexamination of the role of the RAD52 gene in spontaneous mitotic recombination; Malone RE et al.; The RAD52 gene is required for much of the recombination that occurs in Saccharomyces cerevisiae . One of the two commonly utilized mutant alleles, rad52-2, increases rather than reduces mitotic recombination, yet in other respects appears to be a typical rad52 mutant allele . This raises the question as to whether RAD52 is really necessary for mitotic recombination . Analysis of a deletion/insertion allele created in vitro indicates that the null mutant phenotype is indeed a deficiency in mitotic recombination, especially in gene conversion . The data also indicate that RAD52 is required for crossing-over between at least some chromosomes . Finally, examination of the behavior of a replicating plasmid in rad52-1 strains indicates that the frequency of plasmid integration is substantially reduced from that in wild type, a conclusion consistent with a role for RAD52 in reciprocal crossing-over . Analysis of recombinants arising in rad52-2 strains suggests that this allele may result in the increased activity of a RAD52-independent recombinational pathway.

Genetics, 1988 Sep, 120(1), 75 - 81
a/Alpha-specific repression by MAT alpha 2; Strathern J et al.; The product of the MAT alpha 2 gene is a DNA-binding protein that acts as a repressor of two different sets of cell type-specific genes . In alpha cells, the alpha 2 protein represses the transcription of several a-specific genes . In a/alpha cells, the alpha 2 protein acts together with the product of the MATa1 gene, the a1 protein, to repress several genes used by haploids in the mating process . In addition to the mat alpha 2 mutations that result in defects in both types of regulation, other mat alpha 2 alleles have been described that result in defects in the repression of a-specific genes but that do not affect the ability of the alpha 2 and a1 proteins to interact to repress the haploid-specific genes . We report here the isolation of a new class of mat alpha 2 mutations that do not affect the ability of the alpha 2 protein to repress a-specific genes, but that interfere with the ability of the alpha 2 protein to interact with the a1 protein to repress the haploid-specific genes and establish the a/alpha cell type . These mutations may help determine the means by which the a1 protein interacts with alpha 2 to expand the set of genes under its control.

Biochem Int, 1988 Sep, 17(3), 395 - 403
Modulation of the catalytic activity of pyridoxal kinase by metallothionein; Churchich JE et al.; Pyridoxal kinase displays high catalytic activity in the presence of metallothionein . The apoprotein of metallothionein as well as the peptide LYS-CYS-THR-CYS-CYS-ALA exert a strong inhibitory effect upon pyridoxal kinase by sequestering free Zn ions . Several steps intervene in the process of pyridoxal kinase activation, i.e . binding of Zn ions by ATP and interaction of Zn-ATP with the enzyme; but direct interaction between metallothionein and pyridoxal kinase (protein association) could not be detected by emission anisotropy measurements . Since the concentration of free Zn++ in mammalian tissues is lower than 10(-9)M, it is postulated that the concentration of metallothionein regulates the catalytic activity of pyridoxal kinase . The mechanism of reconstitution of the metalloenzyme yeast aldolase in the presence of metallothionein was also investigated.

Nature, 1988 Sep 1, 335(6185), 90 - 3
Cloning of bovine GAP and its interaction with oncogenic ras p21; Vogel US et al.; The plasma membrane-bound mammalian ras proteins of relative molecular mass 21,000 (ras p21) share biochemical and structural properties with other guanine nucleotide-binding regulatory proteins (G-proteins) . Oncogenic ras p21 variants result from amino acid substitutions at specific positions that cause p21 to occur predominantly complexed to GTP in vivo . Recently, a GTPase activating protein (GAP) with cytosolic activity has been discovered that stimulates the GTPase activity of normal but not of oncogenic ras p21 . GAP might be either a negative regulatory agent which acts further upstream in the regulatory pathway or the downstream target of ras p21 . We have identified a protein from bovine brain with apparent relative molecular mass 125,000 that has GAP activity . Here, using pure GAP in a kinetic competition assay, we show that GAP interacts preferentially with the active GTP complexes of both normal and oncogenic Harvey (Ha) ras p21 compared with the inactive GDP complexes . We also report the cloning and sequencing of the complementary DNA for bovine GAP . Regions of GAP share amino acid similarity with the noncatalytic domain of adenylate cyclase from the yeast Saccharomyces cerevisiae and with regions conserved between phospholipase C-148, the crk oncogene product and the nonreceptor tyrosine kinases.

FEBS Lett, 1988 Aug 29, 236(2), 345 - 51
Reconstitution of the active rat liver 60 S ribosomal subunit from different preparations of core particles and split proteins; Lavergne JP et al.; Proteins extracted from the 60 S rat liver ribosomal subunit with 50% ethanol/0.5 M K Cl produced only a partial reactivation of the corresponding core particles . In contrast, the same split proteins were able to reactivate the core particles prepared with dimethyl-maleic anhydride (DMMA) to the same level as that observed using the DMMA-split proteins, i.e . 60-80% of the control according to the catalytic activities tested . Comparative analysis of the two split protein fractions showed only four common proteins: P1-P2, which alone restored part of the activities, especially the EF-2-dependent GTPase one, and L10a, L12, which must be responsible for the additional reactivation . The poor ability of the ethanol/KCl core particles to be reactivated was shown to be probably related to a conformational alteration which destabilized the 5 S RNA-protein complex . Proteins present in the ethanol/KCl wash of Saccharomyces cerevisiae 60 S subunits were found to be partly active in subunit reconstitution using rat liver DMMA core particles.

Cell, 1988 Aug 26, 54(5), 633 - 9
Structure of the beta subunit of translational initiation factor eIF-2; Pathak VK et al.; A human liver cDNA encoding the beta subunit of protein synthesis initiation factor 2 (eIF-2) was isolated and sequenced . The 1416 bp cDNA encodes a protein of 333 amino acids (38,404 daltons) with characteristics that resemble authentic purified eIF-2 beta . De novo synthesized eIF-2 beta from cDNA transcripts incorporates into endogenous rabbit eIF-2 complexes . The protein possesses putative GTP-binding sites, a zinc finger motif, and a highly charged N-terminal region composed of three basic polylysine blocks separated by acidic domains . The polylysine blocks and the zinc finger motif suggest that eIF-2 beta interacts with RNA . A yeast protein, Sui3, isolated as an extragenic suppressor of his4 initiation codon mutations, exhibits extensive sequence identity with human eIF-2 beta, especially in the polylysine and zinc finger domains, thereby reinforcing the view that these elements are important for function.

Nature, 1988 Aug 25, 334(6184), 721 - 4
Negative effect of the transcriptional activator GAL4; Gill G et al.; The yeast transcriptional activator GAL4 binds specific sites on DNA to activate transcription of adjacent genes . The distinct activating regions of GAL4 are rich in acidic residues and it has been suggested that these regions interact with another protein component of the transcriptional machinery (such as the TATA-binding protein or RNA polymerase II) while the DNA-binding region serves to position the activating region near the gene . Here we show that various GAL4 derivatives, when expressed at high levels in yeast, inhibit transcription of certain genes lacking GAL4 binding sites, that more efficient activators inhibit more strongly and that inhibition does not depend on the DNA-binding domain . We suggest that this inhibition, which we call squelching, reflects titration of a transcription factor by the activating region of GAL4.

Biochemistry, 1988 Aug 23, 27(17), 6208 - 14
Properties of the binding sites for the sn-1 and sn-2 acyl chains on the phosphatidylinositol transfer protein from bovine brain; van Paridon PA et al.; We have studied the properties of the fatty acyl binding sites of the phosphatidylinositol transfer protein (PI-TP) from bovine brain, by measuring the binding and transfer of pyrenylacyl-containing phosphatidylinositol (PyrPI) species and pyrenylacyl-containing phosphatidylcholine (PyrPC) species as a function of the acyl chain length . The PyrPI species carried a pyrene-labeled acyl chain of variable length in the sn-2 position and either palmitic acid {C(16)}, palmitoleic acid {C(16:1)}, or stearic acid {C(18:1)} in the sn-1 position . Binding and transfer of the PI species increased in the order C(18) less than C(16) less than C(16:1), with a distinct preference for those species that carry a pyrenyloctanoyl {Pyr(8)} or a pyrenyldecanoyl {Pyr(10)} chain . The PyrPC species studied consisted of two sets of positional isomers: one set contained a pyrenylacyl chain of variable length and a C(16) chain, and the other set contained an unlabeled chain of variable length and a Pyr(10) chain . The binding and transfer experiments showed that PI-TP discriminates between positional isomers with a preference for the species with a pyrenylacyl chain in the sn-1 position . This discrimination is interpreted to indicate that separate binding sites exist for the sn-1 and sn-2 acyl chains . From the binding and transfer profiles it is apparent that the binding sites differ in their preference for a particular acyl chain length . The binding and transfer vs chain length profiles were quite similar for C(16)Pyr(x)PC and C(16)Pyr(x)PI species, suggesting that the sn-2 acyl chains of PI and PC share a common binding site in PI-TP.

Biochemistry, 1988 Aug 23, 27(17), 6282 - 7
Isolation of bovine angiogenin using a placental ribonuclease inhibitor binding assay; Bond MD et al.; Angiogenin, which induces the formation of new blood vessels, was isolated previously from two human sources--HT-29 tumor conditioned media and normal plasma . By use of a newly developed binding assay, a similar protein has now been purified from bovine plasma at levels of 30-80 micrograms/L . This protein has the structural, enzymatic, and biological characteristics expected for an angiogenin molecule . Its amino acid composition is similar to that of the human protein, and 22 of 31 residues in the amino-terminal sequences are identical, including a block of 11 consecutive residues . Like human angiogenin, the bovine protein binds placental ribonuclease inhibitor, is inactive toward conventional RNase A substrates, and displays selective ribonucleolytic activity toward some rRNAs . In addition, the bovine protein induces angiogenesis in vivo in the chick embryo chorioallantoic membrane assay at levels as low as 44 fmol per egg . Thus, angiogenin is present in bovine sera at levels similar to those observed in man, and its enzymatic and biological activities are identical with those of the human protein.

Mol Cell Biol, 1988 Aug, 8(8), 3332 - 7
Pseudouridine modification in the tRNA(Tyr) anticodon is dependent on the presence, but independent of the size and sequence, of the intron in eucaryotic tRNA(Tyr) genes; Choffat Y et al.; In Saccharomyces cerevisiae, pseudouridine formation in the middle position of the tRNA(Tyr) anticodon (psi 35) is dependent on the presence of the intron in the tRNA(Tyr) gene (Johnson and Abelson, Nature 302:681-687, 1983) . Drosophila melanogaster tRNA(Tyr) genes contain introns of three size classes: 20 or 21 base pairs (bp) (six genes), 48 bp (one gene), and 113 bp (one gene) . As in yeast, removal of the intron led to loss of psi 35 in the anticodon when transcription was assayed in Xenopus laevis oocytes . All Drosophila intron sizes supported psi 35 formation . The same results were obtained with the homologous X . laevis tRNA(Tyr) genes containing introns of 12 or 13 bp or with a deleted intron . The introns of yeast (Nishikura and DeRobertis, J . Mol . Biol . 145:405-420, 1981), D . melanogaster, and X . laevis tRNA(Tyr) wild-type genes, while they all supported psi 35 synthesis, did not share any consensus sequences . As discussed, these results, taken together, suggest that for appropriate function the psi 35 enzyme in the X . laevis oocyte needs the presence of an unqualified intron in the tRNA gene and a tRNA(Tyr)-like structure in the unprocessed tRNA precursor.

Vaccine, 1988 Aug, 6(4), 328 - 30
Immunogenicity of low doses of recombinant hepatitis B vaccine in young males; Tsakalakis C et al.; A trial of low doses (1.25 and 0.625 micrograms) of recombinant hepatitis B vaccine in 110 males aged 17-19 years is reported . Follow-up was extended to one year and further data are reported for participants from earlier trials of 10, 5 and 2.5 microgram doses for comparison . Seroconversion rates after the booster dose were 94% at 1.25 micrograms and 89% at 0.625 micrograms, compared to 100% at higher doses . Geometric mean titres (GMT) of antibody to hepatitis B surface antigen (anti-HBs) were significantly lower with the 0.625 microgram dose than with 1.25 micrograms at all times, reaching a GMT of 42.6 IU l-1 and 152.9 l-1, respectively, after the booster dose . GMT values at 12 months were 513.9, 510.0, 320.7, 46.5 and 13.3 IU l-1 in the 10, 5, 2.5, 1.25 and 0.625 microgram dose groups, respectively . Increases in GMT after the booster dose were parallelled in all groups . GMT decreased by approximately equal to 65% between 7 and 12 months in the three lowest dose groups, significantly less than the fall of 80% at 5 and 10 micrograms . However, to secure variability among vaccinees, the minimum effective dose for young adults should be greater than 5 micrograms per dose.

Biol Reprod, 1988 Aug, 39(1), 76 - 80
Evidence for the regulation of fatty acid utilization in human sperm by docosahexaenoic acid; Jones RE et al.; The effects of stearic (18:0), linolenic (18:3), and docosahexaenoic (22:6) acids on palmitoyl coenzyme A (CoA) formation by a long-chain fatty acid:CoASH ligase (adenosine monophosphate) (E . C . 6.2.1.3-enriched fraction from human spermatozoa were studied . Both 18:0 and 18:3 were competitive inhibitors for palmitic (16:0) acid activation with Kis of 17.7 and 5.7 microM, respectively . In contrast, 22:6 was a noncompetitive inhibitor demonstrating a Ki of 9.5 microM . These data coupled with previous studies support the conclusion that 16:0, 18:0, and 18:3 and other saturated and unsaturated fatty acids are activated by the same ligase enzyme in sperm . Although the kinetics and interactions of 22:6 are unique compared to the other fatty acids found in sperm phospholipids, we cannot discern from our data if it is activated by a separate enzyme . We propose that 22:6, or a metabolite of 22:6, may regulate free fatty acid utilization in human sperm and that this hypothesis may provide an enzymatic explanation for the changes observed in phospholipid-bound fatty acids during the epididymal maturation of sperm.

Proc Natl Acad Sci U S A, 1988 Aug, 85(16), 6022 - 6
Recognition and cleavage site of the intron-encoded omega transposase; Colleaux L et al.; The optional group I intron of the mitochondrial 21S rRNA gene of Saccharomyces cerevisiae contains a 235-codon-long open reading frame the translation product of which (the omega transposase) catalyzes the formation of a double-strand break within the intron-minus (omega-) copies of the same gene . Purified omega transposase generates in vitro a 4-base-pair staggered cut with 3' hydroxyl overhangs at the exact position where the intron eventually inserts in the gene . Using randomly mutagenized synthetic oligonucleotides, single-base mutants were produced at 21 positions around the cleavage site . Experiments with these oligonucleotides show that the recognition site extends over an 18-base pair-long sequence within which minimal sequence degeneracy is tolerated . The intron-encoded omega transposase is, therefore, one of the most specific restriction endonucleases known to date.

Cell, 1988 Jul 29, 54(3), 335 - 44
Reconstitution of SEC gene product-dependent intercompartmental protein transport; Baker D et al.; Transport of alpha-factor precursor from the endoplasmic reticulum to the Golgi apparatus has been reconstituted in gently lysed yeast spheroplasts . Transport is measured through the coupled addition of outer-chain carbohydrate to {35S}methionine-labeled alpha-factor precursor translocated into the endoplasmic reticulum of broken spheroplasts . The reaction is absolutely dependent on ATP, stimulated 6-fold by cytosol, and occurs between physically separable sealed compartments . Transport is inhibited by the guanine nucleotide analog GTP gamma S . sec23 mutant cells have a temperature-sensitive defect in endoplasmic reticulum-to-Golgi transport in vivo . This defect has been reproduced in vitro using sec23 membranes and cytosol . Transport at 30 degrees C with sec23 membranes requires addition of cytosol containing the SEC23 (wild-type) gene product . This demonstrates that an in vitro inter-organelle transport reaction depends on a factor required for transport in vivo . Complementation of sec mutations in vitro provides a functional assay for the purification of individual intercompartmental transport factors.

Science, 1988 Jul 29, 241(4865), 570 - 3
DNAs of the two mating-type alleles of Neurospora crassa are highly dissimilar; Glass NL et al.; The mating-type alleles A and a of Neurospora crassa control mating in the sexual cycle and function in establishing heterokaryon incompatibility in the vegetative cycle . The A and a alleles were cloned, and they were shown to encode both the sexual functions and vegetative incompatibility . The mating-type clones contain nonhomologous DNA segments that are flanked by common DNA sequences . Neurospora crassa and all heterothallic and pseudohomothallic Neurospora species contain a single copy of one mating-type sequence or the other within each haploid genome . The six known self-fertile homothallic isolates contain an A homolog, but only one species also contains a homologous sequences . Homothallism in these species is not due to mating-type switching, as it is in Saccharomyces cerevisiae.

Biochemistry, 1988 Jul 26, 27(15), 5486 - 92
Cytochrome c peroxidase mutant active site structures probed by resonance Raman and infrared signatures of the CO adducts; Smulevich G et al.; Vibrational frequencies associated with FeC and CO stretching and FeCO bending modes have been determined via resonance Raman (RR) and infrared (IR) spectroscopy for cytochrome c peroxidase (CCP) mutants prepared by site-directed mutagenesis . These include the bacterial "wild type", CCP(MI), and mutations involving groups on the proximal (Asp-235----Asn; Trp-191---Phe) and distal (Trp-51----Phe; Arg-48----Leu and Lys) side of the heme . The data were analyzed with the aid of a recently established correlation between nu FeC and nu CO, which can be used to distinguish between back-bonding and axial ligand donor effects . At high pH all adducts showed essentially the same vibrational pattern (form I') with nu FeC approximately 505 cm-1, nu CO approximately 1948 cm-1, and delta FeCO (weak RR band) approximately 576 cm-1 . These frequencies are very similar to those shown by the myoglobin CO adduct and imply a "normal" H-bond of the proximal histidine . At pH 7 (pH 6 for Asn-235 and Leu-48), different forms are seen for different proteins: form I (nu FeC approximately 500 cm-1, nu CO = 1922-1941 cm-1, and delta FeCO approximately 580 cm-1, very weak) in the case of CCP(MI) and Phe-191, as well as bakers' yeast CCP, or form II (nu FeC approximately 530 cm-1, nu CO = 1922-1933 cm-1, and delta FeCO = 585 cm-1, moderately strong) for Asn-235 and Phe-51.(ABSTRACT TRUNCATED AT 250 WORDS)

Experientia, 1988 Jul 15, 44(7), 586 - 93
Regulatory aspects of low intensity photon emission; Van Wijk R et al.; Photon emission from unicellular and multicellular organisms has been a subject of study for many decennia . In contrast to the well-known phenomenon of bioluminescence originating in luciferin-luciferase reactions, low intensity emission in the visible region of the electromagnetic spectrum has been found in almost every species studied so far . At present, the nomenclature of this phenomenon has not crystallized and it is referred to by a variety of names, such as mitogenetic radiation 29, dark luminescence 7, low-level chemiluminescence 20,36, and biophotons 57 . Particular attention has been focussed on the relationship between photon emission and the regulation of various aspects of cellular metabolism, although in many cases quantitative data are still lacking . Throughout the history of this field of research the question of a functional biological role of the low intensity emission has been repeatedly raised; this is reflected, for instance, in the heterogeneity of the terms used to describe it . The discussion concerns the possible participation of photons of low intensity in intra- and intercellular communication . This paper reviews literature on the metabolic regulation of low intensity emission, as well as the regulation of photon emission initiated by external light . Furthermore, recent data are discussed with respect to a possible biocommunicative function of low intensity photon emission.

Biochem J, 1988 Jul 15, 253(2), 351 - 5
Changes in activities of some enzymes of glycerolipid synthesis in brown adipose tissue of cold-acclimated rats; Darnley AC et al.; 1 . Measurements were made of the activities of the following enzymes of glycerolipid synthesis in homogenates of interscapsular brown adipose tissue obtained from rats subjected to a 4 degrees C environment for time periods of 6 h up to 12 days: fatty acyl-CoA synthetase (FAS), mitochondrial and microsomal forms of glycerolphosphate acyltransferase (GPAT), monoacylglycerolphosphate acyltransferase (MGPAT) and Mg2+-dependent phosphatidate phosphohydrolase (PPH) . 2 . Relative to tissue DNA content, the activities of mitochondrial GPAT, MGPAT and Mg2+-dependent PPH were significantly increased after 1 day of exposure to cold, and continued to increase thereafter . By contrast, FAS and microsomal GPAT activities were unchanged relative to tissue DNA . 3 . The time profile of the increase in MGPAT activity correlated well with a concomitant increase in the microsomal marker NADP+-cytochrome c reductase . Changes in mitochondrial GPAT and in Mg2+-dependent PPH activities were larger in amplitude than that of MGPAT . 4 . It is proposed that these selective changes in enzyme activity may be associated with the onset of brown-adipose-tissue hyperplasia or possibly with an increase in triacylglycerol synthesis during cold-acclimation.

Eur J Biochem, 1988 Jul 1, 174(4), 699 - 705
Post-translational translocation of polypeptides across the mammalian endoplasmic reticulum membrane is size and ribosome dependent; Roitsch T et al.; The translation and translocation of two yeast glycoproteins, invertase and carboxypeptidase Y, were studied in a heterologous cell-free translation system from reticulocytes supplemented with dog pancreas microsomes . Using in vitro synthesized mRNA transcripts, encoding complete or truncated invertase forms, the influence of polypeptide size and ribosome dependence was studied . It was found that C-terminal truncated fragments of 25 kDa, i.e . a size larger than the average size of a domain structure, are translocated and processed post-translationally with a similar efficiency to the cotranslational events . Post-translational import decreases with increasing peptide chain, mature polypeptide (60 kDa) being no longer translocated . Post-translational competence is only maintained as long as the peptide remains associated with ribosomes . Translocation of invertase depends on the presence of the leader peptide and requires energy independent of protein synthesis . Size dependence of post-translational import could also be demonstrated for carboxypeptidase Y.

Proc Natl Acad Sci U S A, 1988 Jul, 85(13), 4628 - 32
FLP recombinase is an enzyme; Gates CA et al.; The FLP protein of the yeast 2-microns plasmid catalyzes intermolecular site-specific recombination with a turnover number of approximately equal to 0.12 min-1 (per FLP monomer) for relaxed DNA substrates . Under conditions that enhance its stability, the protein can be used in catalytic rather than stoichiometric amounts . The reaction rate exhibits a strong dependence on FLP protein concentration even when the protein is present in excess relative to available recombination sites.

Mutat Res, 1988 Jul, 208(3-4), 189 - 94
EDTA affects cytochrome P450-dependent biotransformation reactions during incubations for the liver microsomal assay; Paolini M et al.; In order to optimize the condition of the liver microsomal assay (LMA), studies were carried out to determine the effects of EDTA on mixed-function oxidase activity and its stability under the exact incubation conditions for the LMA . Aminopyrine N-demethylase (APD) and p-nitroanisole O-demethylase (p-NAD) activities as well as lipid peroxidation development (LP) in S9 liver fractions from beta-naphthoflavone and sodium phenobarbital (beta-NF + PB)- or Aroclor 1254 (AC)-treated mice were examined during a period of preincubation with EDTA ranging from 1 to 40 mM . At 5 mM EDTA, we obtained a strong inhibition of the microsomal LP as well as the greatest value of the mean specific activity (Asp) for both APD and pNAD activities . In agreement with the biochemical data, the presence of 5 mM EDTA in the incubation mixtures for the LMA significantly increased the mitotic gene conversion, mitotic crossing-over and point-reverse mutation of the well-known premutagen cyclophosphamide (30 mM) on the diploid D7 strain of Saccharomyces cerevisiae as the outcome of a greater metabolic activity . We concluded that the systematic use of 5 mM EDTA in LMA mixtures could improve the reliability and sensitivity of such a test.

Protein Eng, 1988 Jul, 2(2), 157 - 66
Soluble, prolonged-acting insulin derivatives . III . Degree of protraction, crystallizability and chemical stability of insulins substituted in positions A21, B13, B23, B27 and B30; Markussen J et al.; It was previously demonstrated that insulins to which positive charge has been added by substituting B13 glutamic acid with a glutamine residue, B27 threonine with an arginine or lysine residue, and by blocking the C-terminal carboxyl group of the B-chain by amidation, featured a prolonged absorption from the subcutis of rabbits and pigs after injection in solution at acidic pH . The phenomenon is ascribed to a low solubility combined with the readiness by which these analogs crystallize as the injectant is being neutralized in the tissue . However, acid solutions of insulin are chemically unstable as A21 asparagine both deamidates to aspartic acid and takes part in formation of covalent dimers via alpha-amino groups of other molecules . In order to circumvent the instability, substitutions were introduced in position A21, in addition to those in B13, B27 and B30, challenging the fact that A21 asparagine has been conserved in this position throughout the evolution . Biological potency was retained when glycine, serine, threonine, aspartic acid, histidine and arginine were introduced in this position, although to a varying degree . In the crystal structure of insulin a hydrogen bond bridges the alpha-nitrogen of A21 with the backbone carbonyl of B23 glycine . In order to investigate the importance of this hydrogen bond for biological activity a gene for the single-chain precursor B-chain(1-29)-Ala-Ala-Lys-A-chain(1-21) featuring an A21 proline was synthesized . However, this single-chain precursor failed to be properly produced by yeast, pointing to the formation of this hydrogen bond as an essential step in the folding process . The stability of the A21-substituted analogs in acid solutions (pH 3-4) with respect to deamidation and formation of dimers was approximately 5-10 times higher than that of human insulin in neutral solution . The rate of absorption of most insulins is decreased by increasing the Zn2+ concentration of the preparation . However, one analog with A21 glycine showed first-order absorption kinetics in pigs with a half-life of approximately 25 h, independent of the Zn2+ concentration . The day-to-day variation of the absorption of this analog was significantly lower than that of the conventional insulin suspensions, a property that might render such an insulin useful in the attempts to improve glucose control in diabetics by a more predictable delivery of basal insulin.

Z Naturforsch {C}, 1988 Jul-Aug, 43(7-8), 601 - 5
Biochemical and chemical characterization of trifluoromethylglyoxal bis(guanylhydrazone), a close analog of the antileukemic drug mitoguazone; Elo H et al.; In order to study the structure-activity relationships of bis(guanylhydrazone) type polyamine antimetabolites, trifluoromethylglyoxal bis(guanylhydrazone) (CF3-GBG), a close analog of the antileukemic drug methylglyoxal bis(guanylhydrazone) (mitoguazone, MGBG) was synthesized according to a novel modification of previous methods, yielding single crystals . Single-crystal X-ray crystallography revealed the presence of an isomer different from the one detected in the case of MGBG and all other bis(guanylhydrazones) so far studied . In contrast to MGBG, CF3-GBG was shown to be a very weak inhibitor of yeast adenosylmethionine decarboxylase, being thus devoid of value as a polyamine antimetabolite . In addition, the compound did not have antiproliferative activity against mouse L1210 leukemia cells in vitro . As long as analogous isomers of the two compounds are not available, no conclusions can be drawn about the reasons lying behind the drastical differences between their biological properties.

Am J Physiol, 1988 Jul, 255(1 Pt 1), E1 - 8
Effect of short-term DHEA administration on liver metabolism of lean and obese rats; Mohan PF et al.; Lean and obese Zucker rats, 7-8 wk of age, were treated with dehydroepiandrosterone (DHEA) for either 3 or 7 days to determine the initial cellular event(s) that might be responsible for the antiobesity activity of DHEA . Epididymal, retroperitoneal, and brown adipose tissue weights were unaltered by either 3 or 7 days of DHEA treatment . Liver weight was not affected by 3 days of treatment but was 13 and 18% higher in 7-day DHEA-treated lean and obese rats, respectively, compared with their corresponding control group . Mitochondrial state 3 respiration rates with glutamate-malate and succinate as substrates were elevated by an average of 35% in 3- and 7-day DHEA-treated obese rats and by 15-20% in 7-day DHEA-treated lean rats compared with rates obtained in the corresponding control groups . State 3 respiration was not affected in 3-day DHEA-treated lean rats compared with control lean rats . The specific activities of long-chain fatty acyl-coenzyme A synthase and hydrolase and the levels of free CoA were increased by severalfold in cellular fractions of both DHEA-treated lean and obese rats compared with their respective control group . Hepatic malic enzyme activity, which was shown earlier to be elevated with long-term DHEA treatment, was unaltered by either 3 or 7 days of DHEA administration . The above results suggest the involvement of mitochondrial respiration and fatty acid deacylation/reacylation in the antiobesity mechanism of action of DHEA.

Nucleic Acids Res, 1988 Jun 24, 16(12), 5407 - 26
Plant small nuclear RNAs . V . U4 RNA is present in broad bean plants in the form of sequence variants and is base-paired with U6 RNA; Kiss T et al.; U4 RNA, which is known to play an indispensable role in pre-mRNA splicing, is present in plant nuclei, has a canonical m3 2,2,7 G cap at its 5' end and is associated with U6 RNA in snRNP particles . It occurs in broad bean in the form of a number of sequence variants . Two of these were sequenced: U4A RNA is 154 and U4B RNA is 152 nucleotides long . Sequence similarity of broad bean U4B RNA is 94 per cent to broad bean U4A RNA, 65 per cent to rat U4A RNA, 61 per cent to Drosophila U4A RNA and 50 per cent to snR14, the U4 RNA equivalent of the yeast Saccharomyces cerevisiae . Sequence conservation is much more pronounced in the 5' half of the molecule than in its 3' half . The secondary structure of both variants of broad bean U4 RNA perfectly fits with that of all other U4 RNAs sequenced so far . Nucleotide changes between broad bean U4A and U4B RNAs are restricted to molecular regions that affect the thermodynamic stability of these molecules . A model is proposed for the base pairing interaction of broad bean U4 RNA with broad bean U6 RNA . This is the first report on the structure of a plant U4 RNA.

FEBS Lett, 1988 Jun 20, 233(2), 379 - 82
31P NMR investigations on free and enzyme bound thiamine pyrophosphate; Flatau S et al.; Pyruvate decarboxylase (PDC) contains thiamine pyrophosphate (TPP) and Mg2+ as cofactors . 31P NMR studies with PDC in the presence of added Mn2+ reveal the pyrophosphate moiety of TPP to be a nonaccessible area for the external Mn2+ and thus proving the Mg-P-complex (taking part in the binding of the coenzyme to the protein) to be a nonaccessible area for the medium . Glyoxylic acid, acting as an inhibitor of PDC by forming a noncleavable bond with the catalytic center of TPP causes a steric immobilization of the coenzyme indicated by a line broadening of the pyrophosphate moiety.

J Biol Chem, 1988 Jun 15, 263(17), 8017 - 21
The effect of water on enzyme action in organic media; Zaks A et al.; Three model, unrelated enzymes (yeast alcohol oxidase, mushroom polyphenol oxidase, and horse liver alcohol dehydrogenase) were found to be catalytically active in a variety of organic solvents . For all enzymes and solvents tested, the enzymatic activity greatly increased upon an increase in the water content in the solvents (which always remained below the solubility limit) . Much less water was required to reach the maximal activity in hydrophobic solvents than in their hydrophilic counterparts . However, when the catalytic activity was plotted versus the amount of water bound to the enzymes, a common pattern emerged for different solvents . These data suggest that the effect of organic solvents on an enzyme is primarily due to interactions with the enzyme-bound, essential layer of water rather than with the enzyme itself . At optimal water contents, enzymatic activities in organic solvents were in the range from 20 to 40% of those in aqueous solutions . From experiments on (i) replacement of water with other hydrogen bond-forming additives and (ii) titration of enzyme amino groups in an organic medium, as well as the literature data on dehydrated enzymes, it is concluded that the water required by enzymes in nonaqueous solvents provides them with sufficient conformational flexibility needed for catalysis.

Biochim Biophys Acta, 1988 Jun 15, 960(3), 417 - 26
Rapid stimulation of liver palmitoyl-CoA synthetase, carnitine palmitoyltransferase and glycerophosphate acyltransferase compared to peroxisomal beta-oxidation and palmitoyl-CoA hydrolase in rats fed high-fat diets; Berge RK et al.; Key enzymes involved in oxidation and esterification of long-chain fatty acids were investigated in male rats fed different types and amounts of oil in their diet . A diet with 20% (w/w) fish oil, partially hydrogenated fish oil (PHFO) and partially hydrogenated soybean oil (PHSO) was shown to stimulate the mitochondrial and microsomal palmitoyl-CoA synthetase activity (EC 6.2.1.3) compared to soybean oil-fed animals after 1 week of feeding . Rapeseed oil had no effect . Partially hydrogenated oils in the diet resulted in significantly higher levels of mitochondrial glycerophosphate acyltransferase compared to unhydrogenated oils in the diet . Rats fed 20% (w/w) rapeseed oil had a decreased activity of this mitochondrial enzyme, whereas the microsomal glycerophosphate acyltransferase activity was stimulated to a comparable extent with 20% (w/w) rapeseed oil, fish oil or PHFO in the diet . Increasing the amount of PHFO (from 5 to 25% (w/w)) in the diet for 3 days led to increased mitochondrial and microsomal palmitoyl-CoA synthetase and microsomal glycerophosphate acyltransferase activities with 5% of this oil in the diet . The mitochondrial glycerophosphate acyltransferase was only marginally affected by increasing the oil dose . Administration of 20% (w/w) PHFO increased rapidly the mitochondrial and microsomal palmitoyl-CoA synthetase, carnitine palmitoyltransferase and microsomal glycerophosphate acyltransferase activities almost to their maximum value within 36 h . In contrast, the glycerophosphate acyltransferase and palmitoyl-CoA hydrolase (EC 3.1.2.2) activities of the mitochondrial fraction and the peroxisomal beta-oxidation reached their maximum activities after administration of the dietary oil for 6.5 days . This sequence of enzyme changes (a) is in accordance with the proposal that an increased cellular level of long-chain acyl-CoA species act as metabolic messages for induction of peroxisomal beta-oxidation and palmitoyl-CoA hydrolase, i.e., these enzymes are regulated by a substrate-induced mechanism, and (b) indicates that, with PHFO, a greater part of the activated fatty acids are directed from triacylglycerol esterification and hydrolysis towards oxidation in the mitochondria . It is also conceivable that the mitochondrial beta-oxidation is proceeding before the enhancement of peroxisomal beta-oxidation.

J Biol Chem, 1988 Jun 15, 263(17), 8420 - 9
On the nature of cysteine coordination to CuA in cytochrome c oxidase; Martin CT et al.; The resolution of new features in the 1H electron nuclear double resonance (ENDOR) spectrum of the oxidized CuA site in beef heart cytochrome c oxidase is presented . In a previous study, we assigned resonances in the CuA ENDOR spectrum to hyperfine interactions of methylene protons on one or two cysteine ligands to CuA (Stevens, T.H., Martin, C.T., Wang, H., Brudvig, G.W., Scholes, C.P., and Chan, S.I . (1982) J . Biol . Chem . 257, 12106-12113) . In this work, a new 1H ENDOR resonance in the beef heart CuA ENDOR spectrum is reported and can be assigned to either anisotropy in a previously resolved cysteine methylene proton hyperfine interaction (Aiso = 12 MHz, Aaniso = 2.5 MHz) or to a third isotropic hyperfine coupling (A = 13.6 MHz) to a cysteine methylene proton of a second cysteine ligand to copper . In either case, the 1H ENDOR results require the delocalization of approximately 50% of the unpaired spin from copper onto either one or two cysteine ligands to CuA . To characterize further the CuA site, we have prepared yeast cytochrome c oxidase incorporating isotopically substituted {beta-13C}cysteine . The CuA ENDOR spectrum of this species shows only one clearly resolved 13C hyperfine interaction (A = 3.6 MHz) . This result confirms the assignment of at least one strongly interacting cysteine ligand to CuA and suggests that if the assignment of two cysteine ligands to CuA is correct, the two cysteines interact with copper in a highly symmetric manner . A recent extended x-ray absorption fine structure study of native and modified forms of cytochrome c oxidase indicates the coordination of two sulfur ligands to CuA (Li, P.M., Gelles, J., Chan, S.I., Sullivan R.J., and Scott, R.A . (1987) Biochemistry 26, 2091-2095) . In light of the new possibility of two symmetrically coordinated cysteine ligands to CuA, we propose a molecular orbital description of the oxidized CuA site which is characterized by a high degree of delocalization of unpaired spin away from copper and onto a pair of symmetrically coordinated cysteine sulfur ligands . We also present a protein model for the CuA site in which two cysteine ligands derived from subunit II lie on the face of an alpha-helix . This structure would allow the unprecedented stable coordination of two cysteine thiolate sulfurs to copper and may provide a mechanism for the redox-linked proton pumping by cytochrome c oxidase.

FEBS Lett, 1988 Jun 6, 233(1), 105 - 8
Amino acid sequence template useful for alpha-helix-turn-alpha-helix prediction; Shestopalov BV; Necessary stereochemical requirements for an amino acid sequence segment to fold into an alpha-helix-turn-alpha-helix supersecondary structure are presented in sequence template form . The usefulness of the template is illustrated by alpha-helix-turn-alpha-helix predictions consistent with experimental data from the large T antigens of two polyoma viruses, simian virus 40 (segment 143-165) and mouse polyoma virus (segment 297-319), and the yeast transcription activator GCN4 (segment 256-278).

Proc Natl Acad Sci U S A, 1988 Jun, 85(11), 3946 - 50
Nuclear fusion-defective phenocopies in Chlamydomonas reinhardtii: mating-type functions for meiosis can act through the cytoplasm; Dutcher SK; Nuclear fusion in newly formed Chlamydomonas reinhardtii zygotes can be inhibited by drugs that affect microtubule stability, which include colchicine, amiprophosmethyl, oryzalin, and taxol . This inhibition can be monitored genetically by the production of haploid meiotic products from conjugations between haploid and diploid parents . Such zygotes would normally produce aneuploid progeny . Inhibition of nuclear fusion by colchicine requires treatment of gametic cells both before conjugation and after formation of the zygotes . These results suggest that nuclear fusion requires dynamic microtubules . Treated zygotes formed from a haploid-diploid mating can produce six spores, but only four spores germinate to form viable haploid colonies . No contribution from the nuclear genome of the haploid parent is recovered, whereas all loci from the diploid parent are recovered . The four viable products from the diploid parent of inhibited zygotes show normal segregation of loci located on linkage groups segregating according to Mendelian laws . Levels of meiotic recombination were tested for pairs of loci on linkage groups XVIII and XIX and found to be unchanged by inhibition of nuclear fusion . Thus, similar to Saccharomyces cerevisiae, C . reinhardtii mating-type functions necessary for nuclear fusion are not nuclear limited and can act through the cytoplasm . Inhibition of nuclear fusion can be used to analyze diploid Chlamydomonas that cannot enter meiosis . This technique permits direct analysis of dominant mutations, dominant suppressors and enhancers, and new alleles of identified loci that have been isolated in diploid strains.

Immunology, 1988 Jun, 64(2), 201 - 5
Characterization of the IgA receptor from human polymorphonuclear leucocytes; Albrechtsen M et al.; Human polymorphonuclear leucocytes (PMNs) will phagocytose yeasts opsonized with specific affinity-purified human serum IgA . PMNs also bind to Sepharose beads coated with IgA or IgG, but not to beads coated with bovine serum albumin (BSA) or horseradish peroxidase (HRP) . Binding to IgA-Sepharose stimulates the cells to release lysozyme . Affinity chromatography of 125I-labelled PMN membrane proteins on IgA-Sepharose results in isolation of a polypeptide of apparent 60,000 MW . The protein, which is not bound to IgG-Sepharose under the same conditions, appears as a diffuse band on SDS-PAGE, suggesting it is heavily glycosylated.

J Cell Biol, 1988 Jun, 106(6), 1997 - 2010
Diverse effects of beta-tubulin mutations on microtubule formation and function; Huffaker TC et al.; We have used in vitro mutagenesis and gene replacement to construct five new cold-sensitive mutations in TUB2, the sole gene encoding beta-tubulin in the yeast Saccharomyces cerevisiae . These and one previously isolated tub2 mutant display diverse phenotypes that have allowed us to define the functions of yeast microtubules in vivo . At the restrictive temperature, all of the tub2 mutations inhibit chromosome segregation and block the mitotic cell cycle . However, different microtubule arrays are present in these arrested cells depending on the tub2 allele . One mutant (tub2-401) contains no detectable microtubules, two (tub2-403 and tub2-405) contain greatly diminished levels of both nuclear and cytoplasmic microtubules, one (tub2-104) contains predominantly nuclear microtubules, one (tub2-402) contains predominantly cytoplasmic microtubules, and one (tub2-404) contains prominent nuclear and cytoplasmic microtubule arrays . Using these mutants we demonstrate here that cytoplasmic microtubules are necessary for nuclear migration during the mitotic cell cycle and for nuclear migration and fusion during conjugation; only those mutants that possess cytoplasmic microtubules are able to perform these functions . We also show that microtubules are not required for secretory vesicle transport in yeast; bud growth and invertase secretion occur in cells which contain no microtubules.

Proc Natl Acad Sci U S A, 1988 Jun, 85(12), 4426 - 30
Dominant negative protein kinase mutations that confer a G1 arrest phenotype; Mendenhall MD et al.; The CDC28 gene of Saccharomyces cerevisiae encodes a protein kinase that is required for passage through the G1 phase of the cell cycle . We have used an inducible promoter fused to the CDC28 coding sequence to isolate conditionally dominant mutant alleles of CDC28 . Overexpression of these dominant alleles causes arrest in the G1 phase of the cell cycle but permits the distinctive asymmetric growth that is characteristic of recessive temperature-sensitive cdc28 mutants . The dominant alleles encode products with no detectable protein kinase activity, and their phenotypic effects can be suppressed by simultaneous overproduction of the wild-type protein . DNA sequence analysis showed that the mutant site in at least one of the dominant alleles is in a residue that is highly conserved among protein kinases . These properties are best understood if the dominant mutation results in the catalytic inactivation of the protein kinase but still allows the binding of another component needed for CDC28 function . By this model, high levels of the mutant protein arrest cell division by denying the wild-type protein access to this other component . Suppressors that may encode this other component have been isolated on high-copy-number plasmids.

EMBO J, 1988 Jun, 7(6), 1763 - 8
Post-translational transport of proteins into microsomal membranes of Candida maltosa; Wiedmann M et al.; We have isolated from the yeast Candida maltosa microsomal membranes that are active in the translocation of proteins synthesized in cell-free systems derived from C . maltosa, Saccharomyces cerevisiae or wheat germ . Translocation and core glycosylation of prepro-alpha-factor, a secretory protein, were observed with yeast microsomes added during or after translation . The signal peptide is cleaved off . Cytochrome P-450 from C . maltosa, the first integral membrane protein studied in a yeast system, is also inserted both co- and post-translationally into Candida microsomal membranes . Its insertion into canine microsomes occurs efficiently only in a co-translational manner and is dependent on the function of the signal recognition particle.

Biochimie, 1988 Jun, 70(6), 791 - 802
Peptide enzymatic microsynthesis, using carboxypeptidase Y as the catalyst: application to stepwise synthesis of Leuenkephalin; Hellio F et al.; In order to develop the use of carboxypeptidase Y (CPD-Y, EC 3.4.12.1) as a catalyst for radioactive hormonal synthesis, the stepwise synthesis of a pentapeptide, Leu-enkephalin, was studied on a microscale . Each peptide bond was formed by enzymatic catalysis, using microquantities of the precursors (amino acid or peptide esters as acyl-components and amino acid ester or amide as nucleophiles) . The high condensation yields obtained suggests that CPD-Y can be a useful tool for preparation of radioactive hormonal peptides.

Mol Cell Biol, 1988 Jun, 8(6), 2638 - 46
A new means of inducibly inactivating a cellular protein; Carlson JR; This paper presents a general means of eliminating the function of a single protein without relying on genetic alterations in its structure or level of synthesis . The strategy is based on the inducible cellular expression of neutralizing antibody to inactivate the protein selectively . The feasibility of this approach is illustrated by using alcohol dehydrogenase I (ADH I) in Saccharomyces cerevisiae as a model . Heavy- and light-chain cDNAs were isolated from a hybridoma secreting an antibody which neutralizes yeast ADH I . The cDNAs were characterized with respect to their length and identity, their signal sequences were removed, and synthetic translation initiation codons were joined to them . These truncated sequences were then inserted into an inducible expression vector and shown to be expressed as stable heavy and light chains, which assemble and bind antigen . The sequences were introduced into yeast mutants containing different levels of ADH activity, and evidence is provided that the antibodies produce limited neutralization of enzyme activity in vivo . In principle, the approach can be used for any cell type in which functional antibody can be inducibly expressed.

Proc Natl Acad Sci U S A, 1988 Jun, 85(12), 4383 - 6
Evolution of competitive ability in Drosophila by density-dependent natural selection; Mueller LD; The theory of density-dependent natural selection predicts that populations kept at extreme densities should evolve different competitive abilities for limited resources . These predictions have been tested with laboratory populations of Drosophila melanogaster . Six independent populations were maintained in two environments, called r and K, for 128 generations . In the r environment, population sizes were small and resources for larvae and adults were abundant . In contrast the populations in the K environment were large and crowded, and resources, such as food and space, were in short supply . The relative competitive ability for food has been estimated for each population . Populations from the K environment consume food at a rate that is 58% greater than the average rate for the r population . The differentiation of competitive abilities in these populations is due to natural selection and is consistent with predictions from the theory of evolutionary ecology.

Virus Genes, 1988 Jun, 1(3), 243 - 53
Construction of full-length cDNA copies of viral double-stranded RNA; Nemeroff ME et al.; A method is described for the construction of full-length cDNA clones of dsRNAs . All dsRNA viruses have a capsid-associated transcriptase that is responsible for synthesis of the plus strand that is then extruded from viral particles . We have used in vitro transcripts synthesized by the segmented Saccharomyces cerevisiae virus (ScV) as templates for first-strand cDNA synthesis . Synthesis was primed by a 33-base synthetic oligonucleotide . This contained 27 nucleotides complementary to the 3' end of the plus strand from one ScV viral dsRNA segment (S14), and 6 additional nucleotides encoding an XbaI restriction site at the 5' end . The second cDNA strand was synthesized using a similar XbaI linker-synthetic oligonucleotide and the ds cDNA was cloned by standard ligation techniques . All four cDNA plasmid isolates characterized by sequence analysis contained the complete 5' end sequence of S14 . Two of these were complete at the 3' end, and one lacked a single base here . Of these four clones, one also retained the XbaI sites at either end . Preparing full-length cDNA clones with unique restriction-site linkers by the use of synthetic oligonucleotides allows for easier screening for complete cDNA clones if neither the vector nor the cDNA has the chosen restriction site . It also provides for easier sequence analysis and manipulation of the genome for later studies, such as cloning into expression vectors . This method is more efficient than any previously described for production of full-sized cDNA clones.

Anal Biochem, 1988 Jun, 171(2), 256 - 65
Formation and characterization of precise eucaryotic transcription complexes using a semisynthetic DNA template and specific oligoribonucleotide primers; Mougey EB et al.; An artificial template of defined sequence which supports specific in vitro initiation and elongation by yeast RNA polymerase II has been constructed . This template is a pBR322 derivative which contains a synthetic oligonucleotide inserted into the BamHI cloning site . The sequence of this oligonucleotide is such that when the plasmid is restricted with SacI the two ends obtained are identical . The addition of an oligodeoxycytidylate chain to the 3' hydroxy termini produces a DNA template, (poly dC-p+22), with the sequence: 3'(C)nTCGA-GAGTCTCCTA . . . . The underlined position denotes the beginning of the duplex region . When initiation is primed with the diribonucleotide GpC the predicted sequence of the transcript obtained is: 5'GCUCUCAGAGGAU . . . . Kinetic and product analyses indicate that a ternary complex containing a precise length of transcript can be produced which is subsequently resistant to heparin inactivation . Initiation can also be directed to a specific position dictated by a tri or tetraribonucleotide primer.

Mol Cell Biol, 1988 Jun, 8(6), 2545 - 54
Identification of a Ty1 regulatory sequence responsive to STE7 and STE12; Company M et al.; Ty1 activation of gene expression observed in haploid cell types of Saccharomyces cerevisiae requires the STE7 and STE12 gene products . An activator sequence within Ty1 that is responsive to these two regulators has been defined . Complex formation between a factor in whole-cell extracts and the DNA regulatory element showed the same dependence on the STE7 and STE12 gene products as did reporter gene expression . Base pair substitutions within the binding site abolished the ability to form the factor-DNA complex and to activate gene expression . The correlation between complex formation and reporter gene expression indicates that factor binding to the cis-acting element is essential for gene activation . Because the predicted protein for the STE7 gene product is homologous to protein kinases, we suggest that protein phosphorylation may directly or indirectly regulate formation of this DNA-protein complex.

Arch Biol Med Exp (Santiago), 1988 Jun, 21(1), 117 - 21
Amino-terminal sequence of spinach chloroplast fructose-1,6-bisphosphatase; Marcus F et al.; The sequence of the NH2-terminal 25-amino acid residues of purified spinach chloroplast fructose-1,6-bisphosphatase was determined by automated Edman degradation . The amino acid sequence is as follows: Ala-Ala-Val-Gly-Glu-Ala-Ala-Thr-Gln-Thr-Lys-Ala- Arg-Thr-Arg-Ser-Lys-Tyr-Glu-Ile-Glu-Thr-Leu-Thr-Gly . A comparison of this sequence with the corresponding region of pig kidney and yeast (Saccharomyces cerevisiae) fructose-1,6-bisphosphatases shows that the sequence of residues 1-19 of the chloroplast enzyme has no homology with the other fructose-1,6-bisphosphatases, but homology is evident after residue 20 . The dissimilar sequence contains a region (residues (8-17) rich in basic and hydroxylated amino acids, a structure which is typical of presequences of mitochondrial and chloroplast proteins . Since chloroplast fructose-1,6-bisphosphatase is nuclear in origin, these results suggest that the chloroplast targeting region may have been retained within the amino acid sequence of the mature protein.

Genetics, 1988 Jun, 119(2), 237 - 47
Analysis of the mechanism for reversion of a disrupted gene; Schiestl RH et al.; A positive selection system for intrachromosomal recombination in Saccharomyces cerevisiae has been developed . This was achieved by integration of a plasmid containing an internal fragment of the HIS3 gene into its chromosomal location . This resulted in two copies of the HIS3 gene one with a terminal deletion at the 3' end and the other with a terminal deletion at the 5' end . Reversion of the gene disruption could be brought about by plasmid excision, unequal sister chromatid exchange or sister chromatid conversion . The purpose of this study was to define the mechanisms involved in reversion of the gene disruption . The frequency of plasmid excision could be determined by placing a yeast sequence bearing an origin of replication onto the plasmid that was subsequently integrated into the yeast genome . Unequal sister chromatid exchange and conversion could be distinguished by determining the nature of the reciprocal product by Southern blotting . The results indicate that reversion might occur mainly by conversion between sister chromatids . This is because the frequency of plasmid excision was about two orders of magnitude lower than the overall frequency of reversion and no reciprocal product indicative of sister chromatid exchange was found . The findings of this presentation suggest that conversion might be an important mechanism for recombination of sister chromatids and possibly for repair of damaged DNA in S or G2.

Proc Natl Acad Sci U S A, 1988 Jun, 85(12), 4232 - 6
Potential positive and negative autoregulation of p60c-src by intermolecular autophosphorylation; Cooper JA et al.; The product of the protooncogene c-src is a protein-tyrosine kinase, p60c-src, that is normally inhibited by phosphorylation at a tyrosine residue close to the C terminus (Tyr-527) . If activated by dephosphorylation of Tyr-527, or by other means, p60c-src becomes phosphorylated at a tyrosine residue in the catalytic domain (Tyr-416) . To test whether either or both of these tyrosines can be phosphorylated by p60c-src itself, we have created four mutations in c-src . One mutant product can receive but cannot donate phosphate, and other mutants are capable of catalysis but lack phosphorylation sites . The mutant genes were expressed singly or in combination in yeast . Analysis of the phosphorylation of mutant p60c-src in the yeast cells and in immunoprecipitates showed that p60c-src molecules can phosphorylate each other at Tyr-416 and -527 . Prohibiting intramolecular phosphorylation had little effect on reaction rates and extents, suggesting that intermolecular phosphorylation predominates . If the same situation pertains in the milieu of the vertebrate fibroblast, phosphorylation of one p60c-src by another at Tyr-416 or -527 could permit positive or negative autoregulation.

Biochemistry, 1988 May 31, 27(11), 4138 - 41
Site-directed mutageneses of rat liver cytochrome P-450d: axial ligand and heme incorporation; Shimizu T et al.; By oligonucleotide-directed mutageneses, 13 substitutions of amino acids at the carboxy-terminal region of rat liver cytochrome P-450d were done as follows: (A) Phe-449----Tyr; (B) Gly-450----Ser; (C) Leu-451----Ser; (D) Gly-452----Glu; (E) Lys-453----Glu; (F) Arg-454----Leu; (G) Arg-455----Gly; (H) Cys-456----Tyr; (I) Cys-456----His; (J) Ile-457----Ser; (K) Gly-458----Glu; (L) Glu-459----Ala; (M) Ile-460----Ser . The CO-bound reduced forms of the wild type and mutants B-G, J, L, and M gave Soret peaks at 448 nm . The CO complex of mutant A gave a Soret peak at 445 nm . The intensities of the CO-bound forms of mutants A, C, D, and J were very small compared with that of the wild-type complex . The CO-reduced forms of mutants H, I, and K did not give a Soret peak around 450 nm at all . The 448-nm peak of mutant F was unstable and quickly disappeared with the concomitant appearance of a peak at 420 nm.(ABSTRACT TRUNCATED AT 250 WORDS)

Philos Trans R Soc Lond B Biol Sci, 1988 May 31, 319(1193), 85 - 95
Mitochondrial biogenesis: recent developments and insights; Grivell LA et al.; Biosynthesis of a functional mitochondrion requires the coordinate expression of genes in both mitochondrial and nuclear DNAs . In yeast, three mitochondrial genes are split and RNA splicing plays a pivotal role in their expression . The recent finding that some introns are capable of self-splicing activity in vitro has permitted analysis of the mechanisms involved in RNA catalysis and may eventually shed light on the evolution of splicing mechanisms in general . Most mitochondrial proteins are encoded by nuclear genes, synthesized in the cytoplasm and imported by the organelle . The availability of cloned genes coding for several constituent subunits of the ubiquinol-cytochrome c reductase, which are imported by mitochondria, has allowed study of selected steps in the addressing of proteins to mitochondria and their intercompartmental sorting within the organelle . Recent developments are discussed.

Biochemistry, 1988 May 31, 27(11), 3955 - 61
Isolation and characterization of a complementary DNA for the nuclear-coded precursor of the beta-subunit of bovine mitochondrial F1-ATPase; Breen GA et al.; We have isolated a cDNA clone encoding the precursor of the beta-subunit of the bovine heart mitochondrial F1-ATPase . Two probes were used to isolate this precursor from a bovine heart cDNA library . One probe was a mixed-sequence oligonucleotide directed against a portion of the amino acid sequence of the mature protein, and the other probe was the F1-ATPase beta-subunit gene from Saccharomyces cerevisiae . Determination of the nucleotide sequence of this cDNA reveals that it contains a 1584-nucleotide-long open reading frame that encodes the complete mature beta-subunit protein and a 48 amino acid long NH2-terminal extension . This amino-terminal presequence contains four basic arginine residues, one acidic glutamic acid residue, four polar uncharged serine residues, and five proline residues . Southern blot hybridization analyses suggest that the bovine F1-ATPase beta-subunit precursor is encoded by a single genetic locus . RNA blot hybridization analyses reveal a single mRNA species of approximately 1.9 kilobases from both bovine liver and heart.

Gene, 1988 May 15, 65(1), 31 - 9
Isolation and characterization of cloned cDNAs that code for human ribosomal protein S6; Lott JB et al.; Ribosomal protein (rp) S6 is the major substrate of protein kinases in eukaryotic ribosomes . To facilitate the identification of cloned cDNAs for human rpS6, we used published amino acid (aa) sequence data for rat liver rpS6 and yeast (Saccharomyces carlsbergensis) rpS10 to design mixed oligodeoxynucleotide probes . Screening of several human cDNA libraries with these probes permitted the isolation of plasmids which encompass the entire coding sequence of rpS6 (249 aa residues), 27 bp of the 5'-untranslated leader and all 39 bp of the 3'-untranslated region . A comparison of the predicted human rpS6 amino acid sequence and the yeast rpS10 amino acid sequence shows highly conserved areas separated by regions of divergence.

J Immunol Methods, 1988 May 9, 109(2), 157 - 60
Heat production as a quantitative parameter of phagocytosis; Hayatsu H et al.; Microcalorimetry was applied to measure phagocytosis by human peripheral blood neutrophils and monocytes . Heat production was 9.1 +/- 2.6 microW by 1 X 10(6) unstimulated neutrophils and increased to 28.4 +/- 3.2 microW in association with phagocytosis . The increase in heat production was directly proportional to the number of Saccharomyces cerevisiae particles phagocytosed as well as to the concentration of opsonizing serum . No heat increase was observed in the absence of phagocytosis . An increase in heat production by monocytes was also observed in association with phagocytosis, but it was much less obvious than that by neutrophils . Heat production can thus be used as a quantitative measure of phagocytosis.

DNA, 1988 May, 7(4), 269 - 73
Primary structure of rat ribosomal protein L36a; Gallagher MJ et al.; The amino acid sequence of rat ribosomal protein L36a, which may form part of the peptidyl transferase center, was deduced from the sequence of nucleotides in a recombinant cDNA and confirmed from the amino-terminal amino acid sequence of the protein . Ribosomal protein L36a contains 105 amino acids (the amino-terminal methionine is removed after translation of the mRNA) and has a molecular weight of 12,311 . Hybridization of the cDNA to digests of nuclear DNA suggests that there are multiple copies of the L36a gene . Rat ribosomal protein L36a is homologous to a protein HL44 present in ribosomes of humans and protein 44 from Saccharomyces cerevisiae ribosomes.

Mutat Res, 1988 May-Aug, 205(1-4), 385 - 92
The equivalence of assays within individual guidelines for the testing of the potential mutagenicity of chemicals: problems associated with battery selection; Parry JM; Many individual Mutagenicity Guidelines contain suggested test systems with choices of such parameters as strains, cell types and even endpoint assayed . Comparisons have been made of data obtained from variants of yeast assays for the induction of mitotic recombination, in vitro assays for the induction of chromosome aberrations and assays for the induction of cell transformation . Individual test variants included in guidelines of the EEC and OECD show considerable qualitative and quantitative variability of response to potential mutagens and carcinogens . Such variability between assays within the same guideline raises considerable problems in the selection of test batteries chosen from published Mutagenicity Guidelines . Improved battery selection is dependent upon the reduction of choice within guidelines to those assays which produce consistent and reproducible results.

Farmaco {Sci}, 1988 May, 43(5), 395 - 407
Some photochemical and photobiological properties of 8,8-desmethylxanthyletine (homopsoralen), a potential photochemotherapeutic agent; Vedaldi D et al.; In this paper the photochemical and photobiological properties of 8,8-desmethyl-xanthyletine (homopsoralen) have been studied . This drug forms a molecular complex with DNA undergoing intercalation between two base pairs of the macromolecule . By subsequent irradiation (365 nm) the compound photobinds covalently to the macromolecule showing an initial DNA-photobinding rate higher than that of 8-methoxypsoralen . In the photoreaction with DNA the drug effectively forms inter-strand cross-linkages . Its ability to generate singlet oxygen when irradiated with ultraviolet light is markedly higher than that of 8-MOP . Homopsoralen when irradiated in water solution undergoes effective photolysis . The drug shows a lower antiproliferative activity on diploid yeast (D 7) than 8-MOP and an almost parallel mutagenic activity . Also the gene convertogenic activity, determined on the same yeast strain, is lower than that of 8-MOP . Homopsoralen does not show any skin phototoxicity on guinea-pig skin . Taking into account its DNA-photobinding capacity, its antiproliferative activity, its reduced genotoxicity and lack of skin-phototoxicity homopsoralen may deserve further studies for evaluating its photochemotherapeutic activity.

Z Naturforsch {C}, 1988 May-Jun, 43(5-6), 476 - 8
Demonstrating evolutionary relationships between macromolecular sequences through mutual relationships with a third sequence; Staves MP et al.; Corresponding sites of the Euglena chloroplast and yeast small subunit ribosomal RNAs (rRNAs) show only an insignificant match with each other but show extensive matches with Euglena chloroplast tRNA(arg) . The match with the tRNA extends farther toward the 5' end of the Euglena rRNA and toward the 3' end of the yeast rRNA . The expected number of such configurations given the number of RNAs searched is about 1 in 100,000 . Comparison of two sequences with a third sequence frequently reveals relationships where pairwise comparisons fail to do so.

Mutagenesis, 1988 May, 3(3), 239 - 43
Isolation of S9 fractions from mouse and rat with increased enzyme activities after repeated administration of cytochrome P-450 and P-448 inducers; Paolini M et al.; Cytochrome P-450 (cyt P-450), NADPH cytochrome P-450 reductase and various microsomal monooxygenase activities {e.g . aminopyrine N-demethylase, p-nitroanisole O-demethylase, dinemorphan N-demethylase, ethoxycoumarin O-deethylase and ethoxyresorufin O-deethylase (ERD)}, were determined in hepatic post-mitochondrial supernatant from mice and rats . Experiments were performed on male and female animals treated with a combination of sodium phenobarbital and beta-naphthoflavone according to the standard protocol schedule for short-term genotoxicity testing . A second inductive treatment after 2, 3, 4 or 5 weeks was provided . The increase in cyt P-450 and in all enzymatic activities measured was enhanced in both species by a second induction treatment, particularly when given after 4 weeks . ERD activity was the only monooxygenase activity which was sex-dependent, being more active in female than in male animals . To extend the biochemical data, experiments were performed with the proposed S9 fractions on styrene, which previously has proved difficult to detect in short-term in vitro mutagenicity tests . Using the new induction conditions positive results were obtained with the D7 strain of Saccharomyces cerevisiae . It was concluded that a simple pre-induction of the animals 3-4 weeks before the main induction treatment leads to a more active S9 fraction for in vitro genotoxicity studies.

Virology, 1988 May, 164(1), 114 - 20
The histidine-221 to tyrosine substitution in v-mos abolishes its biological function and its protein kinase activity; Singh B et al.; The viral mos gene encodes a cytoplasmic transforming protein termed p37mos . Evidence gathered from a number of experimental approaches is consistent with p37mos having a serine/threonine protein kinase activity . To gain further understanding of the p37mos-associated biochemical activity, we constructed a mutation in the v-mos gene by oligonucleotide-directed mutagenesis yielding a histidine to tyrosine substitution at residue 221 in p37mos . Based upon nucleotide sequences, the histidine residue at the corresponding position is conserved in all the serine/threonine protein kinases from yeast to man, and is absent in protein-tyrosine kinases . The mutant p37mos (Tyr-221) was expressed in yeast and assayed for kinase activity . The mutant protein was inactive as judged by a loss of autophosphorylation activity in vitro, thus providing further support for the conclusion that p37mos is a protein kinase . When the mutant v-mos gene was introduced into a retroviral vector, pDD102, and assayed for focus-forming ability on NIH/3T3 cells, it was found to be inactive at both 37 and 30 degrees . In contrast, the wild-type v-mos had transforming activity at both temperatures . These results extend our earlier findings on the correlation between transforming ability and protein kinase activity . A histidine to tyrosine substitution at the corresponding position of the v-mos protein and the yeast CDC28 gene product causes a similar effect on the kinase activity . Therefore, this residue and/or the sequence near the N-terminal side of the conserved predicted phosphate transfer domain, near the middle of the complete catalytic domain, might be specifically involved in the catalytic activity of serine/threonine protein kinases in general.

Biochem Biophys Res Commun, 1988 Apr 29, 152(2), 916 - 20
Reaction of S-nitrosoglutathione with sulfhydryl groups in protein; Park JW; The covalent modification of sulfhydryl groups by S-nitrosoglutathione has been examined using model compounds . S-Nitrosoglutathione and thiol compounds causing extremely fast transnitrosation reaction and subsequent production of mixed disulfide . Yeast alcohol dehydrogenase is rapidly inactivated by S-nitrosoglutathione . The reversibility and Ellman test demonstrate that the inactivation is the result of covalent modification of sulfhydryl groups in this enzyme.

Nature, 1988 Apr 28, 332(6167), 805 - 10
70K heat shock related proteins stimulate protein translocation into microsomes; Chirico WJ et al.; A yeast cytosol is shown to contain two distinct activities that stimulate protein translocation across microsomal membranes . One activity was purified . It consists of two constitutively expressed 70K heat shock related proteins that increase the rate of translocation . Possible mechanisms of action of these proteins are discussed.

Nature, 1988 Apr 28, 332(6167), 800 - 5
A subfamily of stress proteins facilitates translocation of secretory and mitochondrial precursor polypeptides; Deshaies RJ et al.; Depletion of a subset of 70K stress proteins in yeast mutants shows that they are involved in the post-translational import of precursor polypeptides into both mitochondria and the lumen of the endoplasmic reticulum . The identification of such a basic function may explain the remarkable evolutionary conservation of the gene family encoding these proteins.

Nature, 1988 Apr 28, 332(6167), 853 - 6
GAL4 activates transcription in Drosophila; Fischer JA et al.; GAL4 is a yeast regulatory protein that binds to specific sites within a DNA sequence called UASG (galactose upstream activating sequence) and activates transcription of linked genes . This activation requires two functions of the protein: a DNA binding domain located near the amino terminus, and one or more 'activating regions' . The 'activating regions' are highly acidic (see also ref . 12) and can be replaced, for example, by a short peptide designed to form a negatively charged, amphipathic alpha-helix . GAL4, as well as deletion derivatives bearing one or more 'activating regions' attached to the DNA binding domain, activates transcription in cultured mammalian cells from mammalian promoters linked to a UASG (refs 14, 15) . Here we show that GAL4, when expressed in particular tissues of Drosophila larvae, stimulates tissue-specific transcription of a Drosophila promoter linked to GAL4 binding sites.

Biochim Biophys Acta, 1988 Apr 28, 954(1), 95 - 107
Methionine modification in cytochrome-c peroxidase; Kim K et al.; Hydrogen peroxide oxidizes Met-119, Met-230 and Met-231 to the sulfoxide derivatives with equal initial rates in apocytochrome-c peroxidase at pH 4 in 0.1 M sodium acetate buffer . No detectable oxidation of Met-163 and Met-172 occurs under these conditions . Apoenzyme, in which up to two residues of methionine have been oxidized, binds heme stoichiometrically . Heme-reconstituted modified enzyme has an absorption spectrum with the Soret maximum red-shifted compared to that of the native enzyme, indicating a perturbation of the heme environment in the modified enzyme . Heme-reconstituted modified enzyme can bind cyanide with an affinity nearly identical to that of the native enzyme . The heme-reconstituted enzyme loses its ability to react with hydrogen peroxide to form Compound I . The loss of the ability to form Compound I is correlated with the modification of at least one of the residues in the Met-230/Met-231 pair.

Biochem Biophys Res Commun, 1988 Apr 15, 152(1), 9 - 14
A supersensitive dot-hybridization method: rapid and quantitative detection of host-derived DNA in recombinant products at the one picogram level; Kuroda S et al.; We have developed a highly sensitive method of DNA dot-blotting hybridization to detect host-derived DNA (nuclear DNA and plasmid DNA) in a recombinant product . This method has two distinctive features compared to the conventional hybridization method: firstly, a highly specific radioactive probe is prepared by using ultrasonicated DNA, instead of untreated DNA, as a template for the oligo-labeling reaction; secondly, the signal to noise ratio is increased by the use of lambda phage DNA as non-homologous DNA . This method enabled us to detect host-derived DNA at the one picogram level without using a radioisotope of high specific activity and long exposure times.

Biochem Biophys Res Commun, 1988 Apr 15, 152(1), 264 - 9
Bovine liver cDNA clones encoding a precursor of the alpha-subunit of the mitochondrial ATP synthase complex; Breen GA; cDNA clones encoding a precursor of the alpha-subunit of the mitochondrial ATP synthase complex have been isolated from a bovine liver cDNA library using the alpha-subunit gene from Saccharomyces cerevisiae as a probe . Analyses of the nucleotide sequence of these cDNA clones reveal that the bovine liver ATP synthase alpha-subunit is initially synthesized as a precursor with an aminoterminal extension 43 amino acids in length . This aminoterminal presequence contains seven basic residues, no acidic residues, and seven polar uncharged serine and threonine residues . A single mRNA species, approximately 1.85 kb in length, was detected for the ATP synthase alpha-subunit precursor in both bovine liver and heart.

J Biol Chem, 1988 Apr 5, 263(10), 4704 - 11
The isolation and purification of a specific "protector" protein which inhibits enzyme inactivation by a thiol/Fe(III)/O2 mixed-function oxidation system; Kim K et al.; Mixed-function oxidation systems comprised of Fe3+, O2, and electron donors such as thiol compounds, ascorbate, NAD(P)H/NAD(P)H oxidase, and xanthine oxidase/hypoxanthine, catalyze the inactivation of many enzymes . This report describes the isolation and purification of a soluble protein from Saccharomyces cerevisiae, which specifically inhibits the inactivation of various enzymes by a nonenzymatic Fe3+/O2/thiol mixed-function oxidase system . When thiol is replaced with another electron donor (e.g . ascorbate), this specific protein no longer protects against iron (or copper)/O2-dependent radical-induced enzyme inactivation . Purification steps included a polyethylene glycol precipitation followed sequentially by a chromatography on DE52 and high pressure liquid chromatography on phenyl, DEAE, and gel-filtrated columns . The final gel filtration step yielded two protein peaks exhibiting protector activity and possessing a Mr of 500,000 and 90,000 . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of these two fractions gave a single band at 27 kDa suggesting that these protein species simply represent different oligomeric structures . The protector protein did not possess catalase, glutathione peroxidase, superoxide dismutase, or iron chelation activities . Since the protection activity reported herein is specific for mixed-function oxidation systems containing thiols, we propose that the protector protein functions as a sulfur radical scavenger.

Biochemistry, 1988 Apr 5, 27(7), 2490 - 6
Calorimetric studies of the thermal denaturation of cytochrome c peroxidase; Kresheck GC et al.; Two endotherms are observed by differential scanning calorimetry during the thermal denaturation of cytochrome c peroxidase at pH 7.0 . The transition midpoint temperatures (tm) were 43.9 +/- 1.4 and 63.3 +/- 1.6 degrees C, independent of concentration . The two endotherms were observed at all pH values between 4 and 8, with the transition temperatures varying with pH . Precipitation was observed between pH 4 and 6, and only qualitative data are presented for this region . The thermal unfolding of cytochrome c peroxidase was sensitive to the presence and ligation state of the heme . Only a single endotherm was observed for the unfolding of the apoprotein, and this transition was similar to the high-temperature transition in the holoenzyme . Addition of KCN to the holoenzyme increases the midpoint of the high-temperature transition whereas the low-temperature transition was increased upon addition of KF . Binding of the natural substrate ferricytochrome c to the enzyme increases the low-temperature transition by 4.8 +/- 1.3 degrees C but has no effect on the high-temperature transition at pH 7 . The presence of cytochrome c peroxidase decreases the stability of cytochrome c, and both proteins appear to unfold simultaneously . The results are discussed in terms of the two domains evident in the X-ray crystallographic structure of cytochrome c peroxidase.

J Cell Biol, 1988 Apr, 106(4), 1017 - 26
Micronuclei and the cytoplasm of growing Tetrahymena contain a histone acetylase activity which is highly specific for free histone H4; Richman R et al.; Salt extracts prepared from purified micronuclei and the cytoplasm of growing Tetrahymena contain a histone acetylase (also referred to as histone acetyltransferase) activity which is highly specific for H4 when tested as a free histone . With both extracts, H4 is acetylated first at position 4 (monoacetylated) or positions 4 and 11 (diacetylated), sites diagnostic of deposition-related acetylation of newly synthesized H4 in vivo . As the concentration of cytosolic extract is decreased in the in vitro reactions, acetylation of H3 is also observed . Neither activity acetylates histone in a chromatin form . These activities are distinct from a macronuclear acetylase which acetylates H3 and H4 (macro- or micronuclear) equally well as free histones and which acetylates all four core histones when mononucleosomes are used as substrate . As well, the micronuclear and cytoplasmic activities give similar thermal-inactivation profiles which are different from that of the macronuclear activity . In situ enzyme assays demonstrate a macronuclear-specific activity which acetylates endogenous macronuclear chromatin and an independent micronuclear-cytosolic activity which is able to act upon exogenously added free H4 . These results argue strongly that an identical acetylase is responsible for the micronuclear and cytoplasmic activity which is either modified or altogether distinct from that in macronuclei.

Proc Natl Acad Sci U S A, 1988 Apr, 85(8), 2696 - 700
Mitotic sectored colonies: evidence of heteroduplex DNA formation during direct repeat recombination; Ronne H et al.; In yeast meiosis, ascosporal colonies are sometimes sectored for a marker--i.e., half the colony has one allele and half has the other . This is interpreted as replicative resolution of heteroduplex DNA (hDNA) formed as a recombination intermediate . We have looked for similar evidence of hDNA formation during mitotic recombination between two repeated sequences on the same chromosome . The two repeats, an ochre suppressor and a wild-type tRNA gene, are separated by plasmid DNA and the URA3 marker . Recombination between the repeats excises the URA3 gene and one copy of the repeat, leaving either the wild-type tRNA or the suppressor on the chromosome . A red/white color assay is used to distinguish between the two . We find that some colonies that have lost the URA3 gene are sectored for the suppressor . This suggests that hDNA is formed across the anticodon during the recombination event and then resolved by replication . The disruption of either of two genes involved in recombination and repair, RAD1 and RAD52, does not significantly alter the frequency of sectored colony formation during plasmid excision.

Radiat Res, 1988 Apr, 114(1), 54 - 63
Exponential or shouldered survival curves result from repair of DNA double-strand breaks depending on postirradiation conditions; Frankenberg-Schwager M et al.; The yeast mutant rad54-3 is temperature conditional for the rejoining of DNA double-strand breaks, but cells do proliferate at both the restrictive and permissive temperatures . Thus, after irradiation with 30 MeV electrons, survival curves can be obtained which may or may not involve double-strand break rejoining under certain experimental conditions . Because of this special property of rad54-3 cells, it was possible to demonstrate that rejoining of radiation-induced double-strand breaks under nongrowth conditions yields exponential survival curves the slopes of which decrease as a function of the rejoining time . These survival data suggest that, under nongrowth conditions, the rejoining of double-strand breaks is an unsaturated process and lacks binary misrepair . In contrast, whenever rejoining of double-strand breaks occurs under growth conditions, shouldered survival curves are observed . This is true for immediate plating as well as for delayed plating survival curves . It is proposed that it is the unsaturated rejoining of double-strand breaks under nongrowth conditions, lacking binary misrepair, which is responsible for potentially lethal damage repair.

J Virol, 1988 Apr, 62(4), 1278 - 85
A deletion mutant of L-A double-stranded RNA replicates like M1 double-stranded RNA; Esteban R et al.; X double-stranded RNA (dsRNA) is a 0.52-kilobase dsRNA molecule that arose spontaneously in a nonkiller strain of Saccharomyces cerevisiae originally containing L-A and L-BC dsRNAs (L-BC is the same size as L-A but shares no homology with it) . X hybridized with L-A, and direct RNA sequencing of X showed that the first 5' 25 base pairs (of the X positive strand) and at least the last 110 base pairs of the 3' end were identical to the ends of L-A dsRNA . X showed cytoplasmic inheritance and, like M1, was dependent on L-A for its maintenance . X was encapsidated in viruslike particles whose major coat protein was provided by L-A (as is true for M1), and X was found in viruslike particles with one to eight X molecules per particle . This finding confirms our "head-full replication" model originally proposed for M1 and M2 . Like M1 or M2, X lowers the copy number of L-A, especially in a ski host . Surprisingly, X requires many chromosomal MAK genes that are necessary for M1 but not for L-A.

Proc Natl Acad Sci U S A, 1988 Apr, 85(8), 2623 - 7
Molecular cloning and expression of the human homologue of the murine gene encoding myeloid leukemia-inhibitory factor; Gough NM et al.; A human homologue of the recently cloned murine leukemia-inhibitory factor (LIF) gene was isolated from a genomic library by using the murine cDNA as a hybridization probe . The nucleotide sequence of the human gene indicated that human LIF has 78% amino acid sequence identity with murine LIF, with no insertions or deletions, and that the region of the human gene encoding the mature protein has one intervening sequence . After oligonucleotide-mediated mutagenesis, the mature protein-coding region of the LIF gene was introduced into the yeast expression vector YEpsec1 . Yeast cells transformed with the resulting recombinant could be induced with galactose to produce high levels of a factor that induced the differentiation of murine M1 leukemic cells in a manner analogous to murine LIF . This factor competed with 125I-labeled native murine LIF for binding to specific cellular receptors on murine cells, compatible with a high degree of structural similarity between the murine and human factors.

J Immunol, 1988 Apr 1, 140(7), 2288 - 95
Isolation and characterization of an expressible cDNA encoding human IL-3 . Induction of IL-3 mRNA in human T cell clones; Otsuka T et al.; We have isolated cDNA clones encoding human IL-3 from libraries constructed in a modified pcD mammalian expression vector by using mRNA prepared from activated human T cell clones . Amino acid sequence of human IL-3 deduced from DNA sequence of these cDNA clones agrees with that predicted from genomic sequence except at amino acid position 27 . Northern blotting analysis and S1 nuclease analysis show that almost all activated T cell clones express IL-3 mRNA with kinetics similar to that observed in mouse T cell clones . However, striking difference was found in the level of granulocyte-macrophage-CSF and IL-3 mRNA expressed in activated human T cells . In contrast to mouse T cell clones, granulocyte-macrophage-CSF mRNA is expressed at least two orders of magnitude more abundant than IL-3 mRNA . Yeast Saccharomyces cerevisiae carrying human IL-3 cDNA fused downstream to alpha-factor leader sequence expressed and secreted biologically active IL-3 . Several different rat anti-peptide antisera have been used to confirm the presence of human rIL-3 immunochemically . The immunoreactive human IL-3 expressed in transiently transfected COS7 cells or in yeast was observed to be heterogeneous . Human rIL-3 expressed in COS7 cells has multipotential CSF activity in semisolid cultures of bone marrow cells, and selectively induced the proliferation of My-10+ marrow or cord blood cells in liquid cultures.

J Ultrastruct Mol Struct Res, 1988 Apr, 99(1), 48 - 58
Electron spectroscopic imaging of DNA; Bazett-Jones DP et al.; Electron spectroscopic imaging (ESI) was carried out using a fixed beam electron microscope equipped with a parallel electron energy filter to form micrographs of purified plasmid DNA without the use of heavy metal stains and shadows . Inelastically scattered electrons that have ionized the phosphorus LII,III shell electrons were used to form phosphorus distribution maps of DNA and deoxyribonucleoprotein complexes . Signal-to-noise values of the net phosphorus content over single DNA molecules compared to two and four interwound DNA strands directly reflect the known stoichiometric levels of phosphorus content, illustrating that ESI can be used to determine the relative levels of nucleic acid in nucleoprotein complexes . An initial attempt to characterize nucleosomal and transcriptionally active chromatin from Saccharomyces cerevisiae with this technique reveals three distinct ultrastructural classes of the basic chromatin fiber.

EMBO J, 1988 Apr, 7(4), 1153 - 8
Latent membrane perturbation activity of a mitochondrial precursor protein is exposed by unfolding; Endo T et al.; We have purified milligram amounts of an importable mitochondrial precursor protein {the presequence of yeast cytochrome oxidase subunit IV fused to mouse dihydrofolate reductase (DHFR)} . This has made it possible, for the first time, to perform detailed studies on the conformation of a precursor protein and its interaction with lipid membranes . The precursor protein closely resembled authentic mouse DHFR with respect to secondary structure (measured by CD spectra) and stability towards urea (measured by tryptophan fluorescence and enzyme activity) . With this precursor protein, the presequence thus does not significantly alter the folding of the attached 'passenger protein' . In contrast to the corresponding presequence peptide, the native precursor exhibited only weak ability to disrupt vesicles with a low mol% of negatively charged lipids, suggesting that the passenger protein masks the amphiphilic properties of the presequence . The membrane-perturbing properties of the precursor were greatly enhanced by increasing the vesicles' content of negatively charged lipid or by denaturing the precursor in 5 M urea . Interaction with vesicles rich in acidic phospholipid was accompanied by partial unfolding of the precursor, suggesting that such a conformational change may also be involved in the interaction of the precursor with the mitochondrial membranes.

EMBO J, 1988 Apr, 7(4), 1139 - 45
Unfolding and refolding of a purified precursor protein during import into isolated mitochondria; Eilers M et al.; A purified mitochondrial precursor protein unfolds to a protease-sensitive conformation at the surface of isolated mitochondria before being imported into the organelles . This unfolding is stimulated by a potential across the mitochondrial inner membrane, but does not require ATP . In contrast, import of the surface-bound unfolded precursor requires ATP, but no potential; it is accompanied by a refolding inside the mitochondria.

EMBO J, 1988 Apr, 7(4), 1121 - 7
The genomic organization and transcription of the ubiquitin genes of Trypanosoma cruzi; Swindle J et al.; We describe here the organization of the ubiquitin genes of the parasitic protozoan Trypanosoma cruzi . T . cruzi contains greater than 100 ubiquitin coding sequences all of which are clustered into a 27 kb segment of the genome . Two types of ubiquitin coding sequences were found . There are five fusion genes (FUS1-5) consisting of a ubiquitin coding sequence fused to a basic non-ubiquitin sequence . The T . cruzi ubiquitin fusion protein is 84% homologous to the product of the UBI gene of Saccharomyces cerevisiae . The non-ubiquitin domains of the two proteins are 67% homologous . There are five polyubiquitin coding genes (PUB) each consisting of varying lengths of polyubiquitin coding sequence and terminating with a single copy of the larger fusion gene . Transcription of the ubiquitin genes results in the generation of six major poly(A)+ mRNAs . The pattern of transcription accurately reflects the genomic organization, in that the transcripts consist of either a single copy of the ubiquitin fusion coding sequence or varying lengths of polyubiquitin (up to 52 copies of the ubiquitin coding unit) each ending with a single copy of the ubiquitin fusion sequence . Finally, there are heat shock elements 5' to the PUB genes and transcription patterns are altered under conditions of stress.

Biochem J, 1988 Apr 1, 251(1), 41 - 6
The effects of pH and various salts upon the activity of a series of superoxide dismutases; O'Neill P et al.; The CuZn superoxide dismutases (SODs) from ox, sheep, pig and yeast were investigated by pulse radiolysis in order to evaluate the role of electrostatic interactions between O2.- and SOD proteins in the mechanism of action of the SOD enzymes . The protein net charge in this series varies, as evaluated by the protein pI values spanning over a large range of pH: 8.0 (sheep), 6.5 (pig), 5.2 (ox) and 4.6 (yeast) . The amino acid sequences are largely conserved, with the three mammalian proteins being highly homologous and the yeast protein having some distinct variations in the region surrounding the active site . At pH 8.0 the activities of the SODs from various sources are similar, though the minor differences observed suggest that in the highly homologous mammalian series the most acidic protein is the most enzymically efficient one . The pH-dependences of the various activities in the pH range 7-12 are similar, and the related curves are best fitted by two pK values, which are approx . 9.2 and 11.0 for the mammalian enzymes and 9.1 and 11.4 for the yeast enzyme . The activities of the proteins at I 0.1 are decreased by approx . 20% when compared with the activity at I 0.02 at pH 8.5, whereas at pH above 10 the pH-dependence of the activity approaches that determined at I 0.02 and at pH 11.9 the activity is essentially independent of ionic strength . The dependence upon ionic strength also depends on the salt used, with perchlorate being more effective than phosphate or borate or Mops and still effective at pH above 10.5, where the effect of other salts becomes negligible . The dual and concerted dependence of the activities of different SODs on pH and salt concentration is explained with the encounter of O2.- with the active-site copper being governed by the protonation of two positively charged groups in the vicinity of the active site . The gradient between these localized charges and the rest of the protein may explain the different activities of the mammalian proteins at lower pH . On the basis of the sequence variation of the SODs examined it is not possible to definitely identify these groups . Likely candidates are conserved basic amino acid side chains in the vicinity (less than or equal to 1.2 nm) of the active site, i.e . Lys-134 and Arg-141, but co-ordination of OH- in the first copper co-ordination sphere may be an additional factor accounting for the higher pK.(ABSTRACT TRUNCATED AT 400 WORDS)

Anal Biochem, 1988 Apr, 170(1), 68 - 72
Detection of biospecific interactions using amplified ellipsometry; Mandenius CF et al.; Amplified detection of biomolecules and biological interactions using an optical surface technique, ellipsometry, is demonstrated for two biosystems--immunoglobulin G with anti-immunoglobulin G (IgG) and the lectin concanavalin A (Con A) with yeast cells . In order to improve the sensitivity of the ellipsometer signal, an amplifier conjugate is formed by binding the affinity ligand to a 12-nm silica particle which is readily detected by the ellipsometer . Thus by using conjugates of IgG-silica and Con A-silica, amplifications of five to seven times have been obtained enabling detection of less than 20 pg/mm2 of biomolecular material.

J Cell Biol, 1988 Apr, 106(4), 1043 - 8
Full-length prepro-alpha-factor can be translocated across the mammalian microsomal membrane only if translation has not terminated; Garcia PD et al.; We have previously shown that fully synthesized prepro-alpha-factor (pp alpha F), the precursor for the yeast pheromone alpha-factor, can be translocated posttranslationally across yeast rough microsomal (RM) membranes from a soluble, ribosome-free pool . We show here that this is not the case for translocation of pp alpha F across mammalian RM . Rather we found that a small amount of translocation of full-length pp alpha F is observed, but is solely due to polypeptide chains that were still ribosome bound and covalently attached to tRNA, i.e., not terminated . In addition, both signal recognition particle (SRP) and SRP receptor are required, i.e., the same targeting machinery that is normally responsible for the coupling between protein synthesis and translocation . Thus, the molecular requirements for targeting are distinct from posttranslational translocation across yeast RM . As termination is generally regarded as part of translation, the translocation of full-length pp alpha F across mammalian RM does not occur "posttranslationally," albeit independent of elongation . Most other proteins for which posttranslational translocation across mammalian RM was previously claimed fall into the same category in that ribosome attachment as peptidyl-tRNA is required . To clearly separate these two distinct processes, we suggest that the term posttranslational be reserved for those processes that occur in the complete absence of the translational machinery . We propose the term "ribosome-coupled translocation" for the events described here.

Proc Natl Acad Sci U S A, 1988 Apr, 85(8), 2543 - 7
cDNA cloning of human DNA topoisomerase I: catalytic activity of a 67.7-kDa carboxyl-terminal fragment; D'Arpa P et al.; cDNA clones encoding human topoisomerase I were isolated from an expression vector library (lambda gt11) screened with autoimmune anti-topoisomerase I serum . One of these clones has been expressed as a fusion protein comprised of a 32-kDa fragment of the bacterial TrpE protein linked to 67.7 kDa of protein encoded by the cDNA . Three lines of evidence indicate that the cloned cDNA encodes topoisomerase I . (i) Proteolysis maps of the fusion protein and human nuclear topoisomerase I are essentially identical . (ii) The fusion protein relaxes supercoiled DNA, an activity that can be immunoprecipitated by anti-topoisomerase I serum . (iii) Sequence analysis has revealed that the longest cDNA clone (3645 base pairs) encodes a protein of 765 amino acids that shares 42% identity with Saccharomyces cerevisiae topoisomerase I . The sequence data also show that the catalytically active 67.7-kDa fragment is comprised of the carboxyl terminus.

Science, 1988 Apr 1, 240(4848), 68 - 70
A mutation in the catalytic subunit of cAMP-dependent protein kinase that disrupts regulation; Levin LR et al.; A mutant catalytic subunit of adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase has been isolated from Saccharomyces cerevisiae that is no longer subject to regulation yet retains its catalytic activity . Biochemical analysis of the mutant subunit indicates a 100-fold decreased affinity for the regulatory subunit . The mutant catalytic subunit exhibits approximately a threefold increase in Michaelis constant for adenosine triphosphate and peptide cosubstrates, and is essentially unchanged in its catalytic rate . The nucleotide sequence of the mutant gene contains a single nucleotide change resulting in a threonine-to-alanine substitution at amino acid 241 . This residue is conserved in other serine-threonine protein kinases . These results identify this threonine as an important contact between catalytic and regulatory subunits but only a minor contact in substrate recognition.

Gene, 1988 Mar 31, 63(2), 297 - 308
Molecular cloning and characterization of the met2 gene from Ascobolus immersus; Goyon C et al.; We have cloned the met2 gene from Ascobolus immersus by heterologous hybridization with the MET2 gene of Saccharomyces cerevisiae . This gene codes for the homoserine O-transacetylase, one of the methionine biosynthetic enzymes . The complete nucleotide sequence of a 2910-bp DNA fragment carrying the met2 gene has been determined . The gene contains a 165-bp intron which is similar in structure to other fungal introns . The deduced amino acid (aa) sequence (518 aa residues; Mr of 57726) shows three domains with a significant level of homology with the corresponding yeast protein . Northern-blot analysis reveals at least two transcripts (2.4 and 2.1 kb) probably due to transcription termination heterogeneity, as suggested by S1-mapping experiments . Polymorphism has been observed in the met2 gene flanking regions of Ascobolus strains from two different stocks.

Toxicol Appl Pharmacol, 1988 Mar 30, 93(1), 118 - 26
Biochemical evidence that high concentrations of the antidepressant amoxapine may cause inhibition of mitochondrial electron transport; Roberton AM et al.; Overdosage with the antidepressant amoxapine causes metabolic acidosis and may lead to brain damage and death . To better understand the metabolic disturbances caused by amoxapine overdose, its effects on three simple systems were studied: growth of Saccharomyces cerevisiae, mitochondrial energy metabolism, and an electron transport system in microsomal membranes . Growth of yeast on all substrates except lactate was inhibited by amoxapine at 50-100 micrograms ml-1 . Growth on lactate was observed at 200 micrograms ml-1 of amoxapine . In beef heart mitochondria, amoxapine at 100 micrograms ml-1 inhibited reactions involving large sections of the electron transport chain . Energy-linked reactions in submitochondrial particles were also inhibited . Electron microscopy showed some disruption of the mitochondrial internal structure by amoxapine and a change from orthodox to condensed conformation . Microsomal NADH-cytochrome b5 reductase was inhibited by amoxapine, but at higher amoxapine concentrations than mitochondrial reactions . The results suggest amoxapine disrupts reactions of membrane-associated enzyme complexes, and mitochondrial energy conservation may be one of the first systems affected . We speculate that lactic acid accumulation in patients with amoxapine overdose may be caused by loss of electron acceptor activity in tissues.

J Biol Chem, 1988 Mar 25, 263(9), 4139 - 44
The primary structure of the human ribosomal protein S6 derived from a cloned cDNA; Heinze H et al.; Polyclonal antibodies directed against a synthetic octapeptide of the cAMP-dependent phosphorylation site of the ribosomal protein S6 of rat liver were used to screen a lambda gt11 cDNA expression library of human lymphoblasts . An S6 specific clone was isolated . It consists of the complete coding sequence of 747 base pairs and the 3' noncoding region of 40 base pairs . The sequence of 249 amino acids was deduced from the nucleotide sequence . The amino- and carboxyl-terminal regions are almost identical to the reported partial peptide sequences of rat liver S6 . The yeast protein S10 is homologous to the human S6 with the exception of 3 amino acid insertions and a carboxyl-terminal extension of 10 amino acids within the human S6 . The only two phosphorylation sites at the carboxyl terminus of yeast S10 are homologous to the two cAMP-dependent sites in human S6 . Since there are additional phosphorylation sites in mammalian S6, one can assume that they are located in the cluster of 5 serines within the carboxyl-terminal extension . The sequence comparison of these two ribosomal proteins from evolutionarily distant eucaryotes, such as man and yeast, indicates that the structure and probably the function of the phosphoprotein S6 of the small ribosomal subunit has been highly conserved . The expression of the S6 gene has been investigated in proliferating lymphocytes stimulated by concanavalin A . During all stages of lymphoblast development the level of S6 mRNA appeared to be similar . Southern blot analysis of human genomic DNA suggests that multiple genes exist for the S6 protein.

Biochim Biophys Acta, 1988 Mar 23, 953(2), 134 - 41
Evidence by NMR for mobility of the cytochrome domain within flavocytochrome b2; Labeyrie F et al.; According to a model proposed by Gervais, M, Groudinsky, O., Risler, Y . and Labeyrie, F . ((1977) Biochem . Biophys . Res . Commun . 77, 1543-1551) flavocytochrome b2 is composed of a central flavodehydrogenase entity of 4 X 45 kDa to which are attached four cytochrome b2 globules of approx . 11 kDa that are released after proteolysis of the connective loops . A possible inherent mobility of the latter with functional significance was suspected . Proton NMR spectra at 400 MHz of the isolated and of the flavodehydrogenase-bound ferricytochrome b2 units have been compared . In the ranges downfield of +12 ppm and upfield from -4 ppm, where hyperfine-shifted heme proton resonances reside, the chemical shifts are identical for the two forms, but the linewidths are markedly broader for flavocytochrome b2 . The linewidths of three heme resonances, a methyl at +19 ppm, two single protons at -6 and -8 ppm (most probably from one vinyl) and an unassigned line at -2.4 ppm, all increase by a factor of about 4 . Since, in the present case, linewidths are controlled mainly by proton/proton dipolar relaxations which are caused by molecular tumbling, a change in linewidths of about 15 would be expected if the cytochrome b2 globule had no free motion relative to the flavodehydrogenase domain . The present results thus support the previous hypothesis that such a relative mobility, of unknown correlation time and amplitude, actually exists.

FEBS Lett, 1988 Mar 14, 229(2), 273 - 8
Complete cDNA sequence of a Dictyostelium ubiquitin with a carboxy-terminal tail and identification of the protein using an anti-peptide antibody; Muller-Taubenberger A et al.; The complete sequence of a Dictyostelium discoideum cDNA is presented that codes for monoubiquitin extended at its C-terminus by a 52 amino acid tail . The sequence of both the ubiquitin portion and the tail is highly homologous to the one of Saccharomyces cerevisiae and to a partial mouse sequence . The highly basic tail sequence contains a putative metal and nucleic acid-binding motif . The gene encoding the 0.6 kb mRNA of the C-terminally extended ubiquitin is represented only once in the haploid genome . The 0.6 kb mRNA as well as its translation product, a 15 kDa protein, is expressed in exponentially growing cells and remains present for at least 5 h of development . Using antibodies against a synthetic peptide that corresponds to the C-terminal amino acid sequence, a 15 kDa protein containing the extension a synthetic peptide that corresponds to the C-terminal amino acid sequence, a 15 kDa protein containing the extension was also detected in yeast.

Nucleic Acids Res, 1988 Mar 11, 16(5), 1715 - 28
A backtranslation method based on codon usage strategy; Pesole G et al.; This study describes a method for the backtranslation of an aminoacidic sequence, an extremely useful tool for various experimental approaches . It involves two computer programs CLUSTER and BACKTR written in Fortran 77 running on a VAX/VMS computer . CLUSTER generates a reliable codon usage table through a cluster analysis, based on a chi 2-like distance between the sequences . BACKTR produces backtranslated sequences according to different options when use is made of the codon usage table obtained in addition to selecting the least ambiguous potential oligonucleotide probes within an aminoacidic sequence . The method was tested by applying it to 158 yeast genes.

J Biol Chem, 1988 Mar 5, 263(7), 3220 - 4
The complete amino acid sequence of the catalytic domain of rat brain hexokinase, deduced from the cloned cDNA; Schwab DA et al.; The complete amino acid sequence of the catalytic domain of rat brain hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) has been deduced from the nucleotide sequence of cloned cDNA . Extensive similarity in sequence, taken to indicate similarity in secondary and tertiary structure, is seen between the mammalian enzyme and yeast hexokinase isozymes A and B . All residues critical for binding glucose to the yeast enzyme are conserved in brain hexokinase . A location for the substrate ATP binding site is proposed based on relation of structural features in the yeast enzyme to characteristics commonly observed in other nucleotide binding enzymes; sequences in regions proposed to be important for binding of ATP to the yeast enzyme are highly conserved in brain hexokinase.

J Biol Chem, 1988 Mar 5, 263(7), 3432 - 9
Group II intron self-splicing . Alternative reaction conditions yield novel products; Jarrell KA et al.; Reaction parameters were modified to enhance the in vitro reaction rate and to reveal partial and novel reactions of the group II intron 5g of the mitochondrial gene from Saccharomyces cerevisiae encoding cytochrome c oxidase subunit I . One alteration yields separate 5'- and 3'-exons plus linear excised intron as the main products . A linear reaction intermediate, containing intron and 3'-exon, and products resulting from cleavages at two unexpected sites were identified . Spliced exon "reopening," a novel reaction between excised intron and spliced exons, appears responsible for separate 5'- and 3'-exon products.

Scanning Microsc, 1988 Mar, 2(1), 33 - 40
3-dimensional imaging of biological structures by high resolution confocal scanning laser microscopy; Brakenhoff GJ et al.; Imaging in confocal microscopy is characterized by the ability to make a selective image of just one plane inside a specimen, virtually unaffected -within certain limits- by the out-of-focus regions above and below it . This property, called optical sectioning, is accompanied by improved imaging transverse to the optical axis . We have coupled a confocal microscope to a computer system, making the combination of both an excellent instrument for mapping the 3-dimensional structure of extended specimens into a computer memory/data array . We measured that the volume element contributing to each data point has, under typical fluorescence conditions, a size of 0.2 X 0.2 X 0.72 micron . The data can be analysed and represented in various ways, i.e., stereoscopical views from any desired angle . After a description of the experimental arrangement, we show various examples of biological and food-structural studies . The microscope can be operated either in reflection or in fluorescence . In the latter mode a spectral element allows selection of the wavelength band of fluorescence light contributing to the image . In this way, we can distinguish various structures inside the cell and study their 3-dimensional relationships . Various applications in biology and the study of food structure are presented.

Mol Cell Biol, 1988 Mar, 8(3), 1247 - 52
Transforming growth factor alpha: mutation of aspartic acid 47 and leucine 48 results in different biological activities; Lazar E et al.; To study the relationship between the primary structure of transforming growth factor alpha (TGF-alpha) and some of its functional properties (competition with epidermal growth factor (EGF) for binding to the EGF receptor and induction of anchorage-independent growth), we introduced single amino acid mutations into the sequence for the fully processed, 50-amino-acid human TGF-alpha . The wild-type and mutant proteins were expressed in a vector by using a yeast alpha mating pheromone promoter . Mutations of two amino acids that are conserved in the family of the EGF-like peptides and are located in the carboxy-terminal part of TGF-alpha resulted in different biological effects . When aspartic acid 47 was mutated to alanine or asparagine, biological activity was retained; in contrast, substitutions of this residue with serine or glutamic acid generated mutants with reduced binding and colony-forming capacities . When leucine 48 was mutated to alanine, a complete loss of binding and colony-forming abilities resulted; mutation of leucine 48 to isoleucine or methionine resulted in very low activities . Our data suggest that these two adjacent conserved amino acids in positions 47 and 48 play different roles in defining the structure and/or biological activity of TGF-alpha and that the carboxy terminus of TGF-alpha is involved in interactions with cellular TGF-alpha receptors . The side chain of leucine 48 appears to be crucial either indirectly in determining the biologically active conformation of TGF-alpha or directly in the molecular recognition of TGF-alpha by its receptor.

Arch Biochem Biophys, 1988 Mar, 261(2), 405 - 8
How a soluble enzyme can be forced to work as a transport system: description of an experimental design; Vincent JC et al.; The cellular transport systems which have been studied up to now have been found to be based on the functioning of specialized proteins anchored asymmetrically in cell membranes . In the present paper we show that a single soluble enzyme inserted at random in a gel slab can drive an uphill transport, provided that asymmetrical boundary conditions force the reversible reaction catalyzed by this enzyme to work forward on one face of the gel slab and backward on the other face . Experimentally, we have used a yeast alcohol dehydrogenase to induce an uphill transport of NADH . It cannot be excluded that comparable structurally symmetrical transport systems also exist in living cells . Such systems would be particularly well suited to preserving cell homeostasis with regard to small solutes.

Mol Cell Endocrinol, 1988 Mar, 56(1-2), 123 - 31
Long-chain fatty acyl-CoA synthetase of rat adrenal microsomes . Effect of ACTH and epinephrine; Mandon EC et al.; Acyl-CoA synthetase activity with various long-chain fatty acid substrates and its kinetic properties were measured in rat adrenal microsomes . The apparent Michaelis constants (Km) for substrate fatty acids increased in the order eicosa-8,11,14-trienoic acid less than alpha-linolenic acid less than linoleic acid less than palmitic acid . The maximum velocities with these fatty acids decreased in the order linolenic greater than eicosa-8,11,14-trienoic acid greater than palmitic acid . The synthesis of radioactivity palmitoyl-CoA, linoleyl-CoA, alpha-linolenyl-CoA and eicosa-8,11,14-trienoyl-CoA from the respective radioactive substrates decreased in the presence of all the other fatty acids mentioned above . These effects were inversely correlated with their apparent Km values . These results support the idea of a single long-chain fatty acyl-CoA synthetase in the adrenal microsomal fraction for the acid tested . After testing the influence of different hormones, it was shown that the administration of epinephrine, ACTH and dexamethasone caused a significant decrease in the activity of the long-chain fatty acid-CoA synthetase . This inhibition is independent of the one produced by the same hormones on the desaturation of linoleic to gamma-linolenic acid.

FEBS Lett, 1988 Feb 29, 229(1), 167 - 70
Calcium-dependent KEX2-like protease found in hepatic secretory vesicles converts proalbumin to albumin; Brennan SO et al.; The yeast KEX2 protease is the only enzyme that has a proven role in the activation of polypeptide hormones through cleavage at pairs of basic residues . The enzyme that fulfils this role in higher eukaryotes has yet to be unequivocally identified . In this investigation, a KEX2-like calcium-dependent protease has been identified in rat hepatic microsomes . The enzyme is membrane-bound, has a pH optimum of 5-6 and converts proalbumin to albumin . More importantly, like the KEX2 protease, it meets two other exacting criteria defined by specific mutations in humans . Namely, it does not process proalbumin Christchurch (-1 Arg----Gln) which lacks one of the requisite basic residues and, whilst not itself a serine protease, it is inhibited by the reactive center variant, alpha 1-antitrypsin Pittsburgh (358 Met----Arg) but not by normal alpha 1-antitrypsin.

Biochemistry, 1988 Feb 23, 27(4), 1316 - 20
Structure of phenylalanine-accepting transfer ribonucleic acid and of its environment in aqueous solvents with different salts; Zaccai G et al.; Yeast tRNA(Phe) was studied in different salt-containing solvents by UV absorbance and small-angle neutron scattering (SANS) . This extends results obtained previously in NaCl and KCl solutions {Li, Z.-Q., Giege, R., Jacrot, B., Oberthur, R., Thierry, J . C., & Zaccai, G . (1983) Biochemistry 22, 4380-4388} . As expected, at low concentrations of all salts studied, the tRNA molecule is unfolded . The importance of specific counterion interactions and the flexibility of the macromolecule are emphasized by the observation that it cannot take up its folded structure in N(CH3)4Cl solvents, even when that salt concentration is increased to 1 M, in the absence of Mg ions . In CsCl solvents, on the other hand, the folded conformation is obtained in salt concentrations above about 0.2 M, similar to NaCl or KCl . By a comparison of SANS results in CsCl H2O and CsCl 2H2O solvents with the data from NaCl and KCl solvents, thermodynamic and structural parameters were derived for the solvated macromolecule . All the data are accounted for, quantitatively, by a model for the particle in NaCl, KCl, or CsCl solution made up of tRNA76-, closely associated with 76 positive hydrated counterions, surrounded by an aqueous solvent layer that excludes salt (and, therefore, of density different from that of bulk solvent) . The mass of water in that layer depends on salt concentration, and the values found are consistent with those predicted by the Donnan effect.

J Biol Chem, 1988 Feb 15, 263(5), 2513 - 7
3-Hydroxy-3-methylglutaryl coenzyme A reductase in the sea urchin embryo is developmentally regulated; Woodward HD et al.; The activity of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, an enzyme which plays a regulatory role in the synthesis of cholesterol, dolichol, and coenzyme Q, has been measured in the developing embryo of the sea urchin . Enzyme activity increased at least 200-fold during development from the unfertilized egg to the pluteus stage embryo . Mixing experiments suggested that the low level of enzyme activity found at early stages was not due to the presence of inhibitor(s) in the egg or zygote . The enzyme in the sea urchin embryo exhibited properties different from that found in mammals: only a fraction of the activity could be solubilized from microsomes, and mild trypsinization inactivated the enzyme without releasing any of it from the microsomes in soluble form . To further study the sea urchin HMG-CoA reductase, a genomic clone was identified by hybridization to a cDNA encoding hamster HMG-CoA reductase . Sequence analysis of this clone revealed a coding region that shares a high degree of homology with the carboxyl-terminal domain of hamster HMG-CoA reductase . Analysis of sea urchin embryo HMG-CoA reductase mRNA levels using a restriction fragment derived from the genomic clone revealed a 5.5-kilobase poly(A)+ mRNA that increased 15-fold during development from the egg to the gastrula stage and then decreased 1.5-fold at the pluteus stage . Since the relative increase in HMG-CoA reductase mRNA was less than the increase in enzyme activity (15-fold versus 200-fold) factors in addition to the level of mRNA may control the activity of this enzyme during embryogenesis.

Curr Genet, 1988 Feb, 13(2), 129 - 35
Nuclear omnipotent suppressors of premature termination codons in mitochondrial genes affect the 37S mitoribosomal subunit; Boguta M et al.; nam3 and R705, yeast nuclear omnipotent suppressors of mitochondrial mit- mutations, reverse the superimposed spectrum of trans-recessive splicing defects by affecting the protein composition of the small mitoribosomal subunit . Analysis of the suppressor's interaction suggests that suppression results from mutations in the mitoribosomal polypeptides . These data indicate an obligatory connection between mitoribosome function and splicing of introns bI2, bI4 and aI1 in yeast mitochondria.

Arch Biochem Biophys, 1988 Feb 1, 260(2), 622 - 7
Purification to homogeneity and some properties of squalene synthetase; Sasiak K et al.; Squalene synthetase has been purified to homogeneity from yeast . It is a single polypeptide of Mr 47,000 . This enzyme catalyzes the synthesis of squalene from farnesyl diphosphate via presqualene diphosphate . In the presence of reduced pyridine nucleotides, presqualene diphosphate and squalene are produced in a ratio of 6:1 from either the purified protein or the crude microsomal fraction.

Arch Biochem Biophys, 1988 Feb 1, 260(2), 532 - 9
Substrate as a source of thermodynamic nonideality in enzyme kinetic studies: invertase-catalyzed hydrolysis of sucrose; Shearwin KE et al.; Expressions for the effects of thermodynamic nonideality arising from the use of high concentrations of small substrate in enzyme kinetic studies are derived . Their application to experimental results for the hydrolysis of sucrose by yeast invertase (pH 4.9, 37 degrees C) signifies that the progressive decrease in initial velocity at high sucrose concentration is consistent with the occurrence of isomeric expansion during the transition of an enzyme-substrate complex to its activated state . Ultracentrifuge studies on the yeast enzyme preparation are then used to establish the physical acceptability of the volume change required to account for the kinetic effects in these terms: the postulated expansion of 1.3 liter/mol would represent a mere 0.16% increase in hydrated volume (or a corresponding increase in extent of asymmetry) . Finally, although originally interpreted to signify an effect of sucrose on water concentration, published results for the invertase-sucrose system {J . M . Nelson and M . P . Schubert (1928) J . Amer . Chem . Soc . 50, 2188-2193} also find a rational explanation in terms of the present analysis based on effects of thermodynamic nonideality in enzyme kinetic studies.

Immunology, 1988 Feb, 63(2), 319 - 24
Induction of a beta-1,3-D-glucan receptor in P388D1 cells treated with retinoic acid or 1,25-dihydroxyvitamin D3; Goldman R; Retinoic acid (RA) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) induce the capability to phagocytose heat-killed yeast (Y) (Saccharomyces cerevisiae) in P388D1 cells . Y phagocytosis is specifically inhibited (100%) by particulate and soluble beta-1,3-D-glucan . Other polysaccharides, such as agarose, dextran and dextran sulphate, are not inhibitory . The inhibitory capacity of mannan was totally abrogated by treatment with beta-glucanase, suggesting that its activity is derived from a residual beta-glucan structure . Partial hydrolysis of glucan particles with formic acid yielded soluble glucan that was fractionated according to size . Glucan1, glucan2 and glucan3 had an average chain length of 34, 23.5 and 15.5 glucose units, respectively . Fifty percent inhibition of Y phagocytosis by RA-P388D1 cells was attained at less than 0.02 microgram/ml (approximately 2 nM) glucan1 and at 1.1 micrograms/ml glucan3 . A further decrease in chain length (less than or equal to 12.6) resulted in oligomers of marginal inhibitory activity . Preincubation of RA- and 1,25(OH)2D3-P388D1 cells with glucan1 for 30 seconds to 5 min, at 4 degrees or 37 degrees, followed by washes with buffer, sufficed to bring about 85-95% inhibition of Y phagocytosis . Recovery of the phagocytic capability was time dependent and required protein synthesis, suggesting a glucan1-induced removal of membrane receptors . The results suggest that recognition and ingestion of Y by RA- or 1,25(OH)2D3-treated P388D1 cells depends almost exclusively on a beta-glucan-specific receptor.

Exp Cell Res, 1988 Feb, 174(2), 481 - 90
Characteristics of the beta-glucan receptor of murine macrophages; Goldman R; Phagocytosis of heat-killed yeast (HK-yeast), zymosan, and glucan particles by thioglycollate-elicited mouse peritoneal macrophages (Tg-macrophages) was inhibited by soluble glucan polymers/oligomers . The inhibitory capacity of soluble glucans decreased steeply with the decrease in the degree of polymerization (DPn); i.e., the concentration at which 50% inhibition of phagocytosis was attained was 0.23 microgram/ml for glucan 1 (DPn 24.8), 0.8 microgram/ml for glucan 2 (DPn 21.9), and greater than 40 micrograms/ml for glucan 3 (DPn 13.8) . The glucan polymers were obtained by partial hydrolysis of glucan particles with formic acid (90%, 95 degrees C, 20 min) and fractionation according to solubility in ethanol water mixtures . A short preincubation (5 min, 4 or 37 degrees C) of Tg-macrophages with glucan 1 led to a subsequent inhibition of HK-yeast phagocytosis . Recovery of the phagocytic function was slow (27% in 3 h; 68% in 5 h) and required protein synthesis . beta-Glucan receptor expression was also suppressed by dexamethasone treatment . Mannan exerted at high concentrations (5 mg/ml) a partial inhibitory activity which was totally abrogated by beta-glucanase treatment . Treatment of macrophages with glucan together with mannan did not enhance the inhibitory capacity of glucan beyond the component abrogated by enzyme treatment . Contribution of local opsonization of HK-yeast to the phagocytic response (involvement of complement receptors) was indirectly negated; (a) glucan 1 which inhibits HK-yeast phagocytosis by up to 95% is not an activator of complement and therefore could not compete for the opsonizing proteins; (b) cycloheximide treatment in itself inhibited only partially HK-yeast phagocytosis whereas it inhibited the reexpression of the glucan receptors; (c) glucan 1 did not affect the phagocytosis of serum opsonized HK-yeast . Thus under the experimental conditions described, phagocytosis of HK-yeast by murine macrophages is mediated by and large by the beta-glucan receptors, while the mannose receptors and complement receptors do not contribute to the process.

Cell, 1988 Jan 29, 52(2), 161 - 7
GAL4 activates gene expression in mammalian cells; Kakidani H et al.; GAL4, a protein that activates transcription in yeast, is shown to activate the mouse mammary tumor virus promoter in mammalian cells . Activation depends upon a GAL4-binding sequence inserted upstream of the gene . Deletion mutants of GAL4 bearing one or both of the "activating regions" required for activation in yeast also activate transcription in mammalian cells . A derivative of GAL4 that binds to DNA but cannot activate transcription in yeast also fails to activate transcription in mammalian cells . We also show that GAL4 and the glucocorticoid receptor activate the mouse mammary tumor virus promoter synergistically.

J Biol Chem, 1988 Jan 25, 263(3), 1467 - 75
The MAK11 protein is essential for cell growth and replication of M double-stranded RNA and is apparently a membrane-associated protein; Icho T et al.; MAK11 is a gene necessary for the maintenance of killer M1 double-stranded RNA, but not for other cellular double-stranded RNAs (L-A, L-BC, T, W) . The DNA sequence of this gene revealed a 1407-base pair open reading frame, which corresponds to a 54-kDa protein . The C-terminal region is lysine-rich and is necessary for mak11-complementing activity . The N-terminal 24 amino acids of the open reading frame include 16 hydrophobic amino acids, 4 basic residues, and 4 neutral amino acids; this sequence could span a membrane . We constructed a MAK11-lacZ fusion that includes the entire MAK11 protein and complements the mak11-1 mutation . The fusion protein was localized in a membrane fraction as shown by centrifugation in Percoll gradients . The fusion protein could be released from the membrane fraction by salt washing . Western blotting of protein, isolated from the membrane fraction and purified by p-aminophenyl-beta-D-thiogalactoside-agarose column chromatography, revealed a fusion protein monomer of 170 kDa which agrees with the predicted molecular weight . While the mak11-1 mutation results in specific loss of M1 double-stranded RNA without any apparent growth defect, replacing a 792-base pair internal EcoRV fragment of MAK11 with the URA3 gene (gene disruption) resulted in a lethal mutation.

Biochim Biophys Acta, 1988 Jan 19, 958(1), 70 - 80
Effect of methotrexate on long-chain fatty acid metabolism in liver of rats fed a standard or a defined, choline-deficient diet; Aarsaether N et al.; The effect of methotrexate on lipids in serum and liver and key enzymes involved in esterification and oxidation of long-chain fatty acids were investigated in rats fed a standard diet and a defined choline-deficient diet . Hepatic metabolism of long-chain fatty acids were also studied in rats fed the defined diet with or without choline . When methotrexate was administered to the rats fed the standard diet there was a slight increase in hepatic lipids and a moderate reduction in the serum level . The palmitoyl-CoA synthetase activity and the microsomal glycerophosphate acyltransferase activity in the liver of rats were increased by methotrexate . The data are consistent with those where the liver may fail to transfer the newly formed triacylglycerols into the plasma with a resultant increase in liver triacylglycerol content and a decrease in serum lipid levels . Fatty liver of methotrexate-exposed rats can not be attributed simply to a reduction of fatty acid oxidation as the carnitine palmitoyltransferase activity was increased . The methotrexate response in the rats fed the defined choline-deficient diet was different . There was a reduction in both serum and hepatic triacylglycerol and the glycerophosphate acyltransferase and palmitoyl-CoA synthetase activities . The carnitine palmitoyltransferase activity was unchanged . Hepatomegaly and increased hepatic fat content, but decreased serum triacylglycerol, total cholesterol and HDL cholesterol were found to be related to the development of choline deficiency as the pleiotropic responses were almost fully prevented by addition of choline to the choline-deficient diet . Addition of choline to the choline-deficient diet normalized the total palmitoyl-CoA synthetase and carnitine palmitoyltransferase activities . In contrast to methotrexate exposure, choline deficiency increased the mitochondrial glycerophosphate acyltransferase activity . The data are consistent with those of where fatty liver induction of choline deficiency may be related to an enhanced esterification of long-chain fatty acids concomitant with a reduction of their oxidation.

Cell, 1988 Jan 15, 52(1), 27 - 37
Autoregulation of 2 micron circle gene expression provides a model for maintenance of stable plasmid copy levels; Som T et al.; The yeast plasmid 2 micron circle actively maintains high but stable copy levels in the cell, even though the plasmid confers no selective advantage to its host . To address the mechanism by which stable copy control is achieved, we have examined the level of expression of the genes resident on the yeast plasmid 2 micron circle as a function of the presence of proteins encoded by the plasmid . We find that transcription of the site-specific recombinase gene, FLP, is repressed at least 100-fold by the concerted action of the products of two other plasmid genes, REP1 and REP2 . In addition, these products repress transcription of the REP1 gene itself . These results can be formulated into a consistent model for plasmid copy control.

Biochemistry, 1988 Jan 12, 27(1), 183 - 93
Carbanicotinamide adenine dinucleotide: synthesis and enzymological properties of a carbocyclic analogue of oxidized nicotinamide adenine dinucleotide; Slama JT et al.; The dinucleotide carbanicotinamide adenine dinucleotide (carba-NAD), in which a 2,3-dihydroxycyclopentane ring replaces the beta-D-ribonucleotide ring of the nicotinamide ribonucleoside moiety of NAD, has been synthesized and characterized enzymologically . The synthesis begins with the known 1-aminoribose analogue (+/-)-4 beta-amino-2 alpha,3 alpha-dihydroxy-1 beta-cyclopentanemethanol . The pyridinium ring is first introduced and the resultant nucleoside analogue specifically 5'-phosphorylated . Coupling the racemic carbanicotinamide 5'-mononucleotide with adenosine 5'-monophosphate produces two diastereomeric carba-NAD analogues which are chromatographically separable . Only one diastereomer is a substrate for alcohol dehydrogenase and on this basis is assigned a configuration analogous to D-ribose . The reduced dinucleotide carba-NADH was characterized by fluorescence spectroscopy and found to adopt a "stacked" conformation similar to that of NADH . The analogue is reduced by both yeast and horse liver alcohol dehydrogenase with Km and Vmax values for the analogue close to those observed for NAD . Carba-NAD is resistant to cleavage by NAD glycohydrolase, and the analogue has been demonstrated to noncovalently inhibit the soluble NAD glycohydrolase from Bungarus fasciatus venom at low concentrations (less than or equal to 100 microM).

FEBS Lett, 1988 Jan 4, 226(2), 343 - 6
Early reduction of cytochrome c in hypoxia; Chance B; Shifts of the steady state of cytochromes a, a3 and c at high pO2 values are cited as evidence of the low O2 affinity of cytochrome oxidase in vivo {(1971) Brain Res . 108, 143-154; (1985) Adv . Exp . Med . Biol . 191, 833-842; (1987) in: Int . Soc . Oxygen Transp . Tissue, Sapporo, p . 84} . Highly aerobic, small diploid yeast cells show less than 0.44% change of steady state in the interval prior to an abrupt reduction of cytochrome c . Thus, 'pre-reduction' seems falsified as a physiological response of metabolizing yeast cells that are free of hemoglobin, do not aggregate and maintain a steady state . Pre-reduction may be due to spectroscopic interference, for example hemoglobin deoxygenation, to oxygen gradients in aggregated cells and tissues or to non-steady states of substrate and metabolic controls, as contrasted to an altered cytochrome oxidase oxygen affinity in vivo.

Comp Biochem Physiol B, 1988, 89(2), 433 - 7
Homology of delta crystallin and argininosuccinate lyase; Yeh LS et al.; 1 . Delta crystallin, a major lens protein characteristic of birds and reptiles, is homologous to argininosuccinate lyase; 57% of the residues in chicken delta crystallin and human lyase are identical . 2 . Even more similar (62% identical residues) to the human lyase is the sequence translated from the presumably inactive delta-2 gene of the delta crystallin locus . 3 . As both delta crystallin and lyase are synthesized in birds only during the embryonic and juvenile stages, the persistence of delta crystallin in the adult lens appears to be paedomorphic . 4 . Possible correlations of the origins of delta crystallin with other events in sauropsid evolution are proposed.

Mol Cell Biol, 1988 Jan, 8(1), 330 - 9
Genetic analysis of the repetitive carboxyl-terminal domain of the largest subunit of mouse RNA polymerase II; Bartolomei MS et al.; The carboxyl-terminal domain (CTD) of the mouse RNA polymerase II largest subunit consists of 52 repeats of a seven-amino-acid block with the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser . A genetic approach was used to determine whether the CTD plays an essential role in RNA polymerase function . Deletion, insertion, and substitution mutations were created in the repetitive region of an alpha-amanitin-resistant largest-subunit gene . The effects of these mutations on RNA polymerase II activity were assayed by measuring the ability of mutant genes to confer alpha-amanitin resistance after transfection of susceptible rodent cells . Mutations that resulted in CTDs containing between 36 and 78 repeats had no effect on the transfer of alpha-amanitin resistance, whereas mutations with 25 or fewer repeats were inactive in this assay . Mutations that contained 29, 31, or 32 repeats had an intermediate effect; the number of alpha-amanitin-resistant colonies was lower and the colonies obtained were smaller, indicating that the mutant RNA polymerase II was defective . In addition, not all of the heptameric repeats were functionally equivalent in that repeats that diverged in up to three amino acids from the consensus sequence could not substitute for the conserved heptamer repeats . We concluded that the CTD is essential for RNA polymerase II activity, since substantial mutations in this region result in loss of function.

Dev Genet, 1988, 9(4-5), 483 - 93
DNA repair in Dictyostelium; Deering RA; Recent approaches to the study of DNA repair in Dictyostelium discoideum are reviewed . Thymidine auxotrophs facilitate the uptake of labeled thymidine into DNA during its replication and repair . The tmpA600 mutation leads to a loss of thymidylate synthase activity, and tdrA600 results in increased transport of thymidine into the cell . In the HPS401 double mutant (tmpA600tdrA600), thymidine is taken up uniformly into the nuclear and mitochondrial DNAs at levels up to 50-fold that in the wild type . tmpA maps on linkage group III . tdrA is on IV or VI, which cosegregate in strains containing this mutation . Alkaline sucrose gradients of nuclei from HPS401 pulsed for 15 min with {3H}thymidine in axenic medium show that the initially labeled single-strand DNA is about 7 x 10(6) daltons, which may be the size of the replicon . This nascent DNA matures in about 45 minutes to 2 x 10(8) daltons . Ultraviolet light (254 nm) decreases the size of the nascent DNA and delays its maturation . In addition to studies of DNA repair utilizing repair-proficient and -deficient mutants of thymidine auxotrophs, we are currently using two approaches for cloning genes involved in repair: 1) genes are sought that can functionally complement repair defects in Saccharomyces cerevisiae following transformation with a D . discoideum DNA library in YEp 24(URA); 4-NQO is used for the selection of RAD transformants; and 2) we have characterized and purified to near-homogeneity two repair enzymes from D . discoideum--uracil-DNA glycosylase and AP-endonuclease . An N-terminal sequence has been determined for the glycosylase, and a synthetic oligonucleotide probe derived from this sequence will be used to screen for this gene . A similar approach is in progress for the AP-endonuclease.

Folia Microbiol (Praha), 1988, 33(6), 462 - 5
Multiple populations of double-stranded RNA in two virus-harbouring strains of Trichomonas vaginalis; Flegr J et al.; The existence of six dsRNA segments of Trichomonas vaginalis virus was confirmed and the molar mass and relative abundance of these segments were determined by agarose gel electrophoresis with reovirus dsRNA serving as a standard . The M's were 3.5, 3.4, 3.2, 2.5, 1.4 and 0.34 Mg/mol for the two strains studied, the relative abundances, however, were 1.0, 1.4, 3.0, 0.3, 2.7, 4.2 and 1.0, 0.6, 1.7, 0.5, 3.4 1.0 for these strains, respectively . Cell homogenate fractionation showed that all dsRNA segments were associated with viral particles . The data appeared to support the hypothesis of a relationship between viruses of the protozoan T . vaginalis and of the yeast Saccharomyces cerevisiae.

Physiol Behav, 1988, 44(6), 709 - 15
Fever alters characteristics of sleep in rats; Kent S et al.; This study examined the effects of a fungal infection on body temperature (Tb) and sleep states . Tb and sleep were recorded in male rats for 24 hr after a saline injection and 48 hr after a subcutaneous injection of live brewer's yeast, at ambient temperatures (Ta's) of 20 degrees and 30 degrees C . Peak fevers of 1.6-3.1 degrees C occurred within 6-10 hr at both Ta's . The rats remained febrile for the next 12-24 hr . For the first 24 hr postyeast, amounts of SWS increased by 19 +/- 3% at 20 degrees C and 12 +/- 2% at 30 degrees C . Specifically, SWS was significantly increased from hr 5-8 (lights-on) and 13-24 (lights-off) at 20 degrees, and from hr 5-8 and 17-24 at 30 degrees C . Ta did not affect the changes in Tb or the changes in SWS after either saline or yeast . Duration of REMS varied with Ta after saline . After yeast, REMS increased by 21 +/- 12% at 20 degrees and decreased by 28 +/- 6% at 30 degrees C, with the net result that REMS at the two Ta's was equal during the fever . Furthermore, while the rats were febrile the normal diurnal variation in REMS was eliminated . Sleep and Tb returned to control values during the second fever day . These results suggest that an activated immune system both increases SWS and overrides the diurnal and thermoregulatory modulations of REMS.

Ann N Y Acad Sci, 1988, 550, 360 - 73
Mitochondrial function in normal and genetically altered cells and tissues; Chance B et al.; The impact upon oxidative metabolism of normal and pathological variations of oxidative capability is just beginning to be understood, based upon the few examples of human and animal subject survivals and the relatively few cell systems in which the impact of molecular pathologies on function has been studied . On the one hand, difficulties of isolation of systems containing altered oxidases are significant because of ineffective assembly or small amounts of surviving isoenzymes, and on the other hand, unexpected fragilities of the oxidase system may lead to low yields when subjected to the preparative stresses appropriate to the wild types . To circumvent these problems, this paper describes the application, in vivo, of noninvasive, nondestructive techniques to study the function of cytochrome oxidase and other components of the respiratory chain, particularly cytochromes b-c1 in human subjects on the one hand, and in isolated cells on the other, principally mutants of Saccharomyces cerevisiae in which the subunit content is varied . Two principal spectroscopic approaches are employed: optical and phosphorus magnetic resonance spectroscopy (P MRS) . Optical spectroscopy of the near red region of the spectrum provides effective analysis of brain and muscle, as does the surface coil of space-resolved phosphorus magnetic resonance . Both techniques are applicable to suspensions of single cells such as yeast . The optical method yields essential information on oxygen delivery to tissues by hemoglobin and myoglobin and oxygen utilization by cytochrome oxidase . P MRS affords essential information on the efficiency of ATP generation and the extent to which oxidative metabolism meets the needs of cell function in terms of the ratio of phosphocreatine to inorganic phosphate (PCr/Pi) . This in turn enables the calculation of the velocity of oxidative metabolism, V, in relation to its maximum capability, Vm, according to a Michaelis-Menten relationship that involves control not only by ADP (Pi/PCr) and Pi, but also by oxygen and substrate deliveries . Thus, an overview of the functionality of mitochondria in cells and tissues is uniquely provided by this combined approach and thereby deficiencies of components of the respiratory chain are quantified.

Scanning Microsc Suppl, 1988, 2, 303 - 14
Three-dimensional reconstruction from serial section images by computer graphics; Baba N et al.; A method is described which allows a three-dimensional object to be reconstructed from micrographs of serial sections using computer graphic techniques . The reconstructed object, which can be rotated three-dimensionally, is displayed on a color CRT and the surface of the object is shaded in order that it can be observed to provide an illusion of a three-dimensional structure . Using biological samples, some useful representation methods are demonstrated, which are a transparent display, smoothing of a reconstructed surface, cuts through a reconstructed model and a three-dimensional distribution representation of particles, and so forth . Furthermore, an example measuring the volume and surface area of reconstructed objects is shown.

Proteins, 1988, 4(1), 56 - 62
Structure-function relationships in 3-phosphoglycerate kinase: role of the carboxy-terminal peptide; Mas MT et al.; Yeast 3-phosphoglycerate kinase (PGK) is a monomeric enzyme (Mr approximately 45,000) composed of two globular domains . Each domain corresponds approximately to the amino- and carboxy-terminal halves of the polypeptide chain . The carboxy-terminal end extends over the interdomain "hinge" region and packs against the amino-terminal domain . It has been proposed that domain movement, resulting in closure of the active site cleft, is essential for the catalytic function of PGK . Large-scale conformational changes have also been postulated to explain activation of the enzyme by sulfate ions . Using site-specific mutagenesis, we have removed a 15-amino-acid carboxy-terminal fragment, in order to probe its role in the substrate- and sulfate-induced conformational changes . The truncated enzyme exhibited approximately 1% of the activity of native PGK and lost the ability to undergo sulfate-induced activation . The Km for ATP was essentially unchanged (Km = 0.23 mM) in comparison to the native enzyme (Km = 0.30 mM), whereas the Km value for 3-phosphoglycerate was increased about eightfold (Km = 3.85 mM and 0.50 mM, respectively) . These results suggest that the carboxy-terminal segment is important for the mechanism of the substrate- and sulfate-induced conformational transitions . CD spectra and sedimentation velocity measurements indicate that the carboxy-terminal peptide is essential for structural integrity of PGK . The increased susceptibility of the truncated enzyme to thermal inactivation implies that the carboxy-terminal peptide also contributes to the stability of PGK.

Acta Biochim Pol, 1988, 35(1), 7 - 17
Associations between a fluorescent DNA ligand, 4',6-diamidine-2-phenylindole.2HCl (DAPI), and RNA; Skoczylas B; The experiments performed in vitro have shown that DAPI and RNA form insoluble and indigestible complexes . This seems to explain the earlier observed retardation of drug accumulation in the nucleus of a living cell in the presence of RNA.

Acta Med Hung, 1988, 45(1), 53 - 61
Function of monocytes in patients with systemic sclerosis; Czirjak L et al.; Functions of monocytes from the peripheral blood of 23 patients with systemic sclerosis were investigated in vitro . The yeast phagocytosis, opsonized yeast phagocytosis and binding of EA (erythrocyte-antibody) particles were found to be normal . A depressed chemotactic response was demonstrated against a zymosan-activated, complement-derived chemotactic factor . In 12 cases, monocytes were cultured for 168 hours . By the 5th and 7th days, the initially depressed chemotactic activity of monocytes returned to normal as compared to controls . This fact supports the speculation that the decreased chemotaxis cannot be caused by an intrinsic abnormality of monocytes/macrophages in systemic sclerosis.

Comp Biochem Physiol B, 1988, 90(2), 335 - 9
Comparative studies of glutathione reductase and lipoamide dehydrogenase; Tsai CS et al.; 1 . Glutathione reductase and lipoamide dehydrogenase are structurally and mechanistically related flavoenzymes catalyzing various one and two electron transfer reactions between NAD(P)H and substrates with different structures . 2 . The two enzymes differ in their coenzyme and functional specificities . Lipoamide dehydrogenase shows higher coenzyme preference while glutathione reductase displays greater functional specificity . 3 . Binding preference of the two flavoenzymes for nicotinamide coenzymes is demonstrated by 31P-NMR spectroscopy . 4 . The presence of arginines in glutathione reductase which is inactivated by phenyl glyoxal, is likely to be responsible for the NADPH-activity of glutathione reductase . 5 . The substrate binding sites of the two enzymes are similar, though their functional details differ . 6 . The active-site histidine of glutathione reductase functions primarily as the proton donor during catalysis . While the active-site histidine of lipoamide dehydrogenase stabilizes the thiolate anion intermediate and relays a proton in the catalytic process.

Life Sci, 1988, 43(5), 437 - 44
Stable analogs of acyl adenylates . Inhibition of acetyl- and acyl-CoA synthetase by adenosine 5'-alkylphosphates; Grayson NA et al.; Adenosine 5'-alkylphosphates are potent inhibitors of acetyl- and acyl-CoA synthetase . In each case, the most effective inhibitor in the series is homologous with the tightly bound acyl adenylate intermediate . Adenosine 5'-ethylphosphate (Ki = 33 nM) is 88-fold more potent than adenosine 5'-methylphosphate (Ki = 2900 nM) as a competitive inhibitor of acetyl-CoA synthetase; the contribution of a single carbon to the observed binding energy (-11 kJ/mol) is much larger than is typically observed.

J Mol Evol, 1988, 27(2), 109 - 13
Informational parameters and randomness of mitochondrial DNA; Granero-Porati MI et al.; The informational content of genomes of nuclear and mitochondrial origin is examined . By using the parameters of Shannon's information theory the language of mitochondrial DNA is shown to be more similar to the language of bacterial DNA than to that of nuclear DNA in more evolutionarily advanced animals . Moreover, using the parameters of Kolmogorov's theory on randomness, genes of different organisms (Neurospora crassa and Saccharomyces cerevisiae) coding for the same protein (subunit 9 of ATPase) are shown to have, if both of mitochondrial origin, a similar degree of randomness, whereas genes coding for the same protein, both belonging to the same organisms, exhibit a quite different degree of randomness when one is of mitochondrial origin and the other of nuclear origin . These results are in favor of the symbiotic origin of mitochondria.

Prog Clin Biol Res, 1988, 274, 463 - 75
Where is the radical in compound I of cytochrome c peroxidase? Clues from crystallography and mutagenesis; Edwards SL et al.; A difference Fourier map shows small structural perturbations on oxidation of cytochrome c peroxidase (CCP) to its semi-stable intermediate, compound I . Least-squares refinement of both CCP and compound I quantifies these perturbations and suggests that the radical site may be on the distal side of the heme, since that is where most of the small movements occur . Several engineered mutants of CCP were created in an attempt to assess the function of various side chains, among them Trp-51----Phe, Trp-191----Phe and His-181----Gly . X-ray structures of the mutant CCP's confirm that only minimal changes are caused by these substitutions . Preliminary examination of the mutants' kinetic properties show that Trp-51 is not the radical site; that Trp-191 has an important enzymic function; and that His-181 is not essential for electron transfer, but probably has some more indirect role . The locus of the radical in compound I, however, remains to be established.

J Biochem (Tokyo), 1988 Jan, 103(1), 177 - 81
Amino acid sequence of rat argininosuccinate lyase deduced from cDNA; Amaya Y et al.; Argininosuccinate lyase {EC 4.3.2.1} is an enzyme of the urea cycle in the liver of ureotelic animals . The enzymes of the urea cycle, including argininosuccinate lyase, are regulated developmentally and in response to dietary and hormonal changes, in a coordinated manner . The nucleotide sequence of rat argininosuccinate lyase cDNA, which was isolated previously (Amaya, Y., Kawamoto, S., Oda, T., Kuzumi, T., Saheki, T., Kimula, S., & Mori, M . (1986) Biochem . Int . 13, 433-438), was determined . The cDNA clone contained an open reading frame encoding a polypeptide of 461 amino acid residues (predicted Mr = 51,549), a 5'-untranslated sequence of 150 bp, and a 3'-untranslated sequence of 41 bp . The amino acid composition of rat liver argininosuccinate lyase predicted from the cDNA sequence is in close agreement with that determined on the purified enzyme . The predicted amino acid sequences of the human and yeast enzymes along the entire sequences (94 and 39%, respectively), except for a region of 66 residues of the human enzyme near the COOH terminus . However, the sequence of this region of the human enzyme predicted from another reading frame of the human enzyme cDNA is homologous with the corresponding sequences of the rat and yeast enzymes . Therefore, the human sequence should be re-examined . Lysine-51, the putative binding site for argininosuccinate, and the flanking sequences are highly conserved among the rat, steer, human, and yeast enzymes.

Nucleic Acids Symp Ser, 1988, (19), 57 - 60
Two-dimensional gel electrophoresis to separate large RNA molecules such as messenger RNAs; Ikemura T; This paper describes a two-dimensional gel electrophoresis method for separating large RNA molecules such as messenger RNAs . RNAs were at first separated on a polyacrylamide plus agarose composite gel and subjected to a second dimension electrophoresis on a polyacrylamide gel containing urea . This method is illustrated by analyses of poly(A)+ yeast RNAs . About 80 discrete spots were detected on the gel, when RNAs from 1000 to 3500 nucleotides in size were examined.

Biol Cell, 1988, 63(3), 307 - 17
A 70 kDa microtubule-associated protein in NIL8 cells comigrates with the 70 kDa heat shock protein; Weller NK; When eukaryotic cells are exposed to environmental stress such as elevated temperature, the synthesis of heat shock proteins (HSP) is stimulated . We have raised a monoclonal antibody to a 70 kDa cytoskeleton-associated protein; this antibody also appears to recognize HSPs 68, 70 and 90, as well as an additional 40 kDa non-heat shock protein . We have used this monoclonal antibody to study the localization of the 70 kDa protein in the cytoskeletons of NIL8 hamster fibroblasts . By selective sequential solubilization of the components of NIL8 cells and analysis of the resulting cytoskeletal preparations by Western blot technique and indirect immunofluorescence, we have shown that the 70 kDa protein is associated with microtubules in mitotic and interphase cells and comigrates with HSP70 on 2-dimensional gel electrophoretigrams.

Hepatology, 1988 Jan-Feb, 8(1), 82 - 7
A candidate vaccine for hepatitis B containing the complete viral surface protein; Kniskern PJ et al.; The entire surface protein of hepatitis B virus serotype ayw containing the preS (preS1+preS2) and S domains has been expressed in the yeast Saccharomyces cerevisiae . Yeast containing a recombinant plasmid utilizing a constitutive promoter did not express this gene successfully due to the toxicity of the protein . A plasmid using a regulatable promoter directed expression which initiated late in the exponential phase of growth and resulted in the accumulation of high intracellular levels of the complete surface protein . The purified polypeptide aggregates into a form which, although not comprised of typical 20 nm particles, displays antigenic determinants encoded by the preS1, preS2 and S domains . Immunization of rabbits elicited the formation of antibodies directed against all three domains . This candidate vaccine will be useful for studying the contributions to viral immunity of the host response to the preS1 and preS2 domains.

Biochem Biophys Res Commun, 1987 Dec 31, 149(3), 1172 - 8
Properties of recombinant hepatitis B vaccine; Ohmura T et al.; A large-scale purification method for hepatitis B surface antigen produced in a recombinant yeast (Saccharomyces cerevisiae) was established . The resulting HBsAg was greater than 99% pure and suitable for vaccine use . The yeast-derived HBsAg was structurally and biochemically similar to plasma-derived HBsAg . The anti-HBs antibody producing potency of the yeast-derived vaccine in mice was significantly higher than that of the plasma-derived vaccine . The yeast-derived vaccine induced protective antibody against hepatitis B virus of either adr or ayw subtype in a chimpanzee efficacy study . These observations demonstrate the usefulness of the yeast-derived vaccine as a second-generation hepatitis B vaccine.

Eur J Biochem, 1987 Dec 30, 170(1-2), 165 - 71
A synthetic functional metabolic compartment . The role of propinquity in a linked pair of immobilized enzymes; Fossel ET et al.; A system was created to model the influence of microcompartments on linked enzymatic reactions . Creatine kinase and hexokinase were covalently attached to Sepharose beads . The gel could be perfused in a specially constructed chamber inside a 360-MHz NMR spectrometer at different flow rates with solutions containing various concentrations of substrates . 31P NMR studies were carried out on the linked enzymatic reaction, creatine phosphate + glucose----creatine + glucose 6-phosphate in two enzyme gels differing in only one aspect, the average distance between hexokinase and creatine kinase . At a distance on the order of 0.1 mm between the enzymes, the average bulk concentrations of substrates and products in the perfusate determined the overall function of the linked system . At an average distance of the order of 10 nm, flux through the linked pair was much higher and much less dependent on the concentration of the intermediate substrate/product ADP/ATP . Even at adenine nucleotide concentrations far below the Km of hexokinase, substantial amounts of glucose 6-phosphate were produced when the enzymes were near but not when they were distant . From saturation transfer measurements and turnover calculations, the lifetime of ATP in the system is estimated to be 0.14-0.5 s when the enzymes are near . This compares to 6 s for distant enzymes . From this it appears that the pair of linked enzymes comprise a functional compartment supported by propinquity in which hexokinase has preferential access to ATP produced by creatine kinase, and creatine kinase to ADP from the hexokinase reaction.

Eur J Biochem, 1987 Dec 30, 170(1-2), 351 - 6
Chemical modification of phenol hydroxylase by ethoxyformic anhydride; Sejlitz T et al.; Phenol hydroxylase was inactivated by ethoxyformic anhydride . Part of the inactivation was related to modification of histidyl residues . The remaining part of the inactivation is proposed to be due to the modification of a lysyl residue which, we suggest, is identical with the one previously described, being essential for the binding of NADPH {Neujahr, H . Y . and Kjellen, K . G . (1980) Biochemistry 19, 4967-4972} . The overall inactivation reaction is biphasic and follows pseudo-first-order kinetics . Numerical analysis of kinetic data was applied to discriminate between simultaneous reactions at different sites . It is proposed that phenol hydroxylase contains two essential histidyl residues, located in or near the NADPH-binding sites . Ethoxyformylation of the lysyl residue(s) caused tightening of the binding of phenol and perturbation of the FAD spectrum of phenol hydroxylase, similar to that caused by phenolic effectors.

Cell, 1987 Dec 24, 51(6), 1027 - 37
Successive translocation into and out of the mitochondrial matrix: targeting of proteins to the intermembrane space by a bipartite signal peptide; Hartl FU et al.; We investigated the import and sorting pathways of cytochrome b2 and cytochrome c1, which are functionally located in the intermembrane space of mitochondria . Both proteins are synthesized on cytoplasmic ribosomes as larger precursors and are processed in mitochondria in two steps upon import . The precursors are first translocated across both mitochondrial membranes via contact sites into the matrix . Processing by the matrix peptidase leads to intermediate-sized forms, which are subsequently redirected across the inner membrane . The second proteolytic processing occurs in the intermembrane space . We conclude that the hydrophobic stretches in the presequences of the intermediate-sized forms do not stop transfer across the inner membrane, but rather act as transport signals to direct export from the matrix into the intermembrane space.

EMBO J, 1987 Dec 20, 6(13), 4083 - 94
A Xenopus laevis gene encodes both homeobox-containing and homeobox-less transcripts; Wright CV et al.; A cDNA clone (p52) that contains all the protein-coding region from the maternally expressed XlHbox 2 locus of the frog Xenopus laevis has been isolated and sequenced . A probe containing the exon preceding the homeobox detected transcripts which arise from a splicing event in which the homeobox-containing exon is replaced by another exon lying 5' to it in the genome . Both the homeobox-containing and homeobox-less splicing event occur in the same tissues, with the homeobox-less RNA representing the minority of mRNA from this gene . There may therefore be a function for two types of transcript, and hence protein, from this locus . This phenomenon may not be exclusive to the XlHbox 2 gene of Xenopus, but might occur more generally in other homeobox-containing genes . The protein deduced from the homeobox-containing cDNA is significantly similar to the yeast mating type factor a1 (MAT-a1) gene product . In addition to the previously described homology of the homeodomains, the amino-terminal domains of XlHbox 2 and MAT-a1 are similar to each other; thus essentially all of the MAT-a1 protein corresponds to some part of the XlHbox 2 protein . In the case of XlHbox 2, the protein coded for by the homeoboxless mRNA would contain all of the non-homeobox homology to yeast MAT-a1.

Biochim Biophys Acta, 1987 Dec 14, 922(3), 270 - 7
7-Oxo-24,25-dihydrolanosterol: a novel lanosterol 14 alpha-demethylase (P-45014DM) inhibitor which blocks electron transfer to the oxyferro intermediate; Aoyama Y et al.; 7-Oxo-24,25-dihydrolanosterol (3 beta-hydroxy-8-lanosten-7-one, 7-oxo-HDL) was a potent competitive inhibitor for lanosterol 14 alpha-demethylase (cytochrome P-45014DM) of Saccharomyces cerevisiae . Affinity of 7-oxo-DHL for the enzyme was more than 50-times higher than those of the inherent substrates, lanosterol and 24,25-dihydrolanosterol . 7-Oxo-DHL accelerated NADPH-dependent reduction of cytochrome P-45014DM in the reconstituted system consisting of the cytochrome and NADPH-cytochrome P-450 reductase . These observations indicated that 7-oxo-DHL interacted with the substrate site of cytochrome P-45014DM . However, 7-oxo-DHL was not metabolized by the reconstituted system . Incubation of 7-oxo-DHL with the reconstituted system caused accumulation of oxyferro intermediate of cytochrome P-45014DM . It can thus be concluded that 7-oxo-DHL interfered with electron transfer to the oxyferro intermediate of the cytochrome, though it stimulated reduction of the heme iron . So far as we know, 7-oxo-DHL is the first example of a cytochrome P-450 inhibitor which selectively interferes with the electron transfer to oxyferro intermediate . 7 alpha-Hydroxy-24,25-dihydrolanosterol was also a competitive inhibitor of cytochrome P-45014DM . However, this compound was metabolized by the reconstituted system and could not block the electron transfer to oxyferro intermediate . 11-Oxo-24,25-dihydrolanosterol, an isomer of 7-oxo-DHL, did not have such inhibitory effects . These lines of evidence suggest a possibility that the keto group at C-7 of lanost-8-ene skeleton may interact with a certain site of cytochrome P-45014DM which has an important role in the electron transfer to oxyferro intermediate.

Zentralbl Bakteriol Mikrobiol Hyg {A}, 1987 Dec, 267(2), 217 - 27
Development of mathematical models for an in vitro-phagocytosis test system; Petri I et al.; Phagocytosis tests have been carried out by many authors using different methods under different conditions . The results have been interpreted in different ways, as well sometimes with conflicting notations . In order to get to a more systematic data analysis and to separate intrinsic from methodic influences, the possibility to apply mathematical models to a phagocytosis test has been studied . In agreement with previous experiences that phagocytosis can well be represented by a mathematical treatment as Michaelis-Menten-type enzyme kinetics concerning its initial rate and by an exponential function under in vivo conditions, in vitro-phagocytosis was phenomenologically described as an analogon of an irreversible bimolecular chemical reaction . In this way, rate and capacity of phagocytosis may be quantified separately . On the basis of systematic deviations of the data from this model, modifications have been developed which could be connected with pertinent observations . The design of further experiments from preliminary results on the basis of our models is discussed.

Proc Natl Acad Sci U S A, 1987 Dec, 84(24), 8763 - 7
Comparative mutational analysis of wild-type and stretched tRNA3(Leu) gene promoters; Fabrizio P et al.; We demonstrate that, when the yeast tRNA(3Leu) gene is stretched so that the distance between the two portions of the intragenic promoter is increased to 365 base pairs, the A and B blocks remain functional . Mutations in the A block, which show a weak phenotype when inserted in the wild type, exert a dramatic effect when inserted into the stretched gene . Experiments with extensively purified transcription factor tau indicate that the tau B-B block interaction is not influenced by A-B distance; only the ability of tau A to interact with A block sequences is affected, possibly because of the additional free-energy cost of forming a large loop of the intervening DNA.

J Biomol Struct Dyn, 1987 Dec, 5(3), 669 - 87
Importance of conserved residues for the conformation of the T-loop in tRNAs; Romby P et al.; The conformation of the T-loop of yeast tRNA(Asp) was studied by structural mapping techniques using chemical and enzymatic probes and by three-dimensional graphics modeling with the known crystallographic structures of tRNAs as references . The structural importance of C61 (conserved in the T-stem of all tRNAs) for the loop conformation was directly checked by ethylnitrosourea phosphate alkylation, either on the 3'-half tRNAAsp molecule or on a variant in which C61 was replaced by U61 . The reactivity of P60 against ethylnitrosourea alkylation in the variant emphasizes the role of the hydrogen bond between this phosphate and position N4 of C61 for stabilizing the conformation of the T-loop . Experiments on several tRNA variants, containing C61 but altered in the sequence or in the length of the T-loop, indicate that other structural features help to stabilize the hydrogen bond network around P60 . Evidence is presented that the reverse Hoogsteen base pair T54-A58 contributes to this stabilization by maintaining the hydrogen bonding between the 2'OH of ribose 58 and P60 . Using graphics modeling and based on the chemical data . T-loops of several variants were constructed . It appears that both the constant length of the T-loop and the presence of psi 55 are crucial for the correct interaction between the T- and D-loops . The conclusion of this study is that the T-loop in tRNA possesses an intrinsic conformation (mainly governed by the constant residues) existing primarily without the structural context of the entire tRNA molecule.

Eur J Biochem, 1987 Dec 1, 169(2), 289 - 93
Mitochondrial precursor proteins are imported through a hydrophilic membrane environment; Pfanner N et al.; We have analyzed how translocation intermediates of imported mitochondrial precursor proteins, which span contact sites, interact with the mitochondrial membranes . F1-ATPase subunit beta (F1 beta) was trapped at contact sites by importing it into Neurospora mitochondria in the presence of low levels of nucleoside triphosphates . This F1 beta translocation intermediate could be extracted from the membranes by treatment with protein denaturants such as alkaline pH or urea . By performing import at low temperatures, the ADP/ATP carrier was accumulated in contact sites of Neurospora mitochondria and cytochrome b2 in contact sites of yeast mitochondria . These translocation intermediates were also extractable from the membranes at alkaline pH . Thus, translocation of precursor proteins across mitochondrial membranes seems to occur through an environment which is accessible to aqueous perturbants . We propose that proteinaceous structures are essential components of a translocation apparatus present in contact sites.

EMBO J, 1987 Dec 1, 6(12), 3827 - 31
Mutations at the lariat acceptor site allow self-splicing of a group II intron without lariat formation; van der Veen R et al.; The fifth intron in the gene for cytochrome c oxidase subunit I in yeast mitochondrial DNA is of the group II type and is capable of self-splicing in vitro . The reaction results in lariat formation, concomitant with exon-exon ligation and does not require a guanosine nucleotide for its initiation . It is generally assumed, but not formally proven, that the first step in splicing is a nucleophilic attack of the 2'-hydroxyl of the branchpoint nucleotide (A) on the 5'-exon-intron junction . To investigate the role of intron sequences in recognition of the 5'-splice junction and the ensuing event of cleavage and lariat formation, mutations have been introduced at and around the branchsite . Results obtained show that although branchpoint attack and subsequent lariat formation are strongly preferred events under conditions normally used for self-splicing, addition of a single T residue at intron position 856, a mutation which brings the branchpoint adenosine into a basepair, leads to a conditionally active intron, which at high ionic strength catalyses exon-exon ligation in the absence of lariat formation . Comparable behaviour is also observed with the branchpoint A deletion mutant . The implications of these findings for the mechanism of self-splicing of group II introns are discussed.

Immunology, 1987 Dec, 62(4), 593 - 8
Cellular immunity to nucleocapsid and pre-S determinants in asymptomatic carriers of hepatitis B virus; Vento S et al.; Previous studies of cellular immunity in asymptomatic HBV carriers have been limited to evaluation of responses to plasma-derived HBsAg preparations . We have explored the specificity of cellular immune responses to HBV antigens in these subjects using an indirect T-lymphocyte migration inhibitory factor assay and three antigen preparations (recombinant nucleocapsid antigen (HBcAg), plasma-derived HBsAg with or without pre-S2, and Saccharomyces cerevisiae-synthesized HBsAg without pre-S2 region) . T cells from 10 asymptomatic chronic HBV carriers with normal liver function tests were responsive to nucleocapside determinants (mean migration index = 0.55 +/- SD 0.07) and to pre-S2-positive plasma-derived HBsAg (MI = 0.62 +/- 0.05) . However, none responded to HBsAg devoid of pre-S2 sequences (MI = 0.98 +/- 0.04) . In further experiments, T cells from three HBV carriers, cultured with six different HBsAg preparations, exhibited responsiveness only to those preparations containing significant pre-S2 activities . Our results show that T-cell immunity to nucleocapsid determinants of the virus and HBsAg in present in asymptomatic HBV carriers; the latter is restricted to antigenic preparations containing significant pre-S2 activities . Hence, T-cell immunity to pre-S determinants may not always be associated with HBV clearance.

Biochemistry, 1987 Nov 17, 26(23), 7493 - 500
31P NMR saturation-transfer and 13C NMR kinetic studies of glycolytic regulation during anaerobic and aerobic glycolysis; Campbell-Burk SL et al.; 31P NMR saturation-transfer techniques have been employed in glucose-grown derepressed yeast to determine unidirectional fluxes in the upper part of the Embden-Meyerhof-Parnas pathway . The experiments were performed during anaerobic and aerobic glycolysis by saturating the ATP gamma resonances and monitoring changes in the phosphomonoester signals from glucose 6-phosphate and fructose 1,6-bis-phosphate . These experiments were supplemented with 13C NMR measurements of glucose utilization rates and 13C NMR label distribution studies . Combined with data obtained previously from radioisotope measurements, these 31P and 13C NMR kinetic studies allowed estimation of the net glycolytic flow in addition to relative flows through phosphofructokinase (PFK) and Fru-1,6-P2ase during anaerobic and aerobic glycolysis . The 31P NMR saturation-transfer results are consistent with previous results obtained from measurements of metabolite levels, radioisotope data, and 13C NMR studies {den Hollander, J.A., Ugurbil, K., Brown, T.R., Bednar, M., Redfield, C., & Shulman, R.G . (1986a) Biochemistry 25, 203-211}, providing additional support for in vivo measurement of the flows during glycolysis.

J Biol Chem, 1987 Nov 15, 262(32), 15598 - 604
The role of protein structure in the mitochondrial import pathway . Analysis of the soluble F1-ATPase beta-subunit precursor; Chen WJ et al.; A series of proteins containing defined internal and presequence deletions in the F1-ATPase beta-subunit precursor have been synthesized in vitro using a linked transcription-translation system . These different forms of the protein have been analyzed by the combination of gel filtration and in vitro mitochondrial import studies . These studies reveal that the soluble F1 beta-subunit precursor (55 kDa) forms a homooligomeric assembly of apparent molecular weight 230,000 on gel filtration analysis . The formation of this tetrameric beta-protein was dependent on the sequence between residues 122 and 144 of the precursor and was independent of the presence of a mitochondrial presequence within the first 19 residues of the precursor . When the tetrameric F1 beta-precursor was partially purified from the translation reaction it was incompetent for import into mitochondria . However, import of the partially purified beta-subunit could be restored by addition of reticulocyte lysate protein . In the absence of the tetramer-forming sequence, the protein behaved as an aggregate complex approximately 400 kDa in size . Formation of the high molecular weight aggregate and import into mitochondria was dependent upon a functional presequence at the amino terminus of the precursor . These studies are discussed in terms of the maintenance of an import competent structure for mitochondrial precursors and role of soluble factors in this process.

Mol Cell Biol, 1987 Nov, 7(11), 3929 - 36
Expression of a gene for mouse eucaryotic elongation factor Tu during murine erythroleukemic cell differentiation; Roth WW et al.; The eucaryotic elongation factor Tu (eEF-Tu) is a single polypeptide with an approximate Mr of 53,000 . During protein synthesis eEF-Tu promotes the binding of aminoacyl-tRNA to the ribosome . To study the expression of the gene(s) for this factor, a genomic clone was isolated that contains a mouse eEF-Tu gene . We screened a phage genomic library with a synthetic oligonucleotide probe complementary to a region of the Saccharomyces cerevisiae and Artemia sp . eEF-Tu genes which codes for an area that is highly conserved between both yeast and Artemia sp . eEF-Tu . From approximately 75,000 phage plaques we obtained five isolates with apparently identical inserts . All five clones contained a 3.8-kilobase EcoRI fragment that hybridized to additional oligonucleotide probes corresponding to different conserved regions of eEF-Tu . We sequenced the 5' end of one genomic clone and determined the length of the cloned fragment that was protected by eEF-Tu mRNA in S1 nuclease protection assays . A quantitative S1 nuclease protection assay was used to compare the relative steady-state levels of eEF-Tu mRNA in total mRNA in total RNA isolated from hexamethylene-bisacetamide-induced murine erythroleukemia cells . The results show a dramatic reduction in the steady-state level of eEF-Tu mRNA as differentiation proceeds . A similar reduction in transcription of eEF-Tu mRNA was observed in isolated nuclei . Finally, we examined the in vivo synthesis of eEF-Tu during differentiation and found that it declined in a manner parallel to the decline in the steady-state level of eEF-Tu mRNA . In addition, we have isolated and sequenced a cDNA clone for mouse eEF-Tu . The derived amino acid sequence is compared with sequences from other eucaryotes.

Am J Med Sci, 1987 Nov, 294(5), 294 - 300
Immunochemotherapy of bladder carcinoma with glucan and cyclophosphamide; Thompson IM et al.; Recent evidence suggests a role for both immunotherapy and chemotherapy in the treatment of transitional cell carcinoma . Glucan, a derivative of the cell wall of Saccharomyces cerevisiae and a potent immunostimulant, was used in combination with cyclophosphamide for treatment of implanted murine transitional cell carcinoma (MBT 2) . Cyclophosphamide prevented tumor appearance when tumor burden was low and decreased tumor growth rate in larger tumor volumes, but was unable to eradicate established tumors . Glucan did not reduce tumor incidence but decreased animal mortality . These experimental observations may correlate well with clinical evidence and suggest future clinical use of these agents.

Arzneimittelforschung, 1987 Nov, 37(11), 1276 - 81
Comparison of antitumor properties of nitracrine and amsacrine analogs; Mazerska Z et al.; For seven new methoxy and/or nitro derivatives of acridine antitumor drugs, nitracrine and amsacrine, biological activity in a few in vitro tests, as well as activity against experimental murine tumors Sarcoma-180 and Leukemia L1210 were investigated . Acute toxicity on mice (LD50) was also determined . High activity in vitro and specific activity against Sa-180 were found to be characteristic features of nitracrine, whereas amsacrine was characterized by high antileukemic activity . Methoxylation of position 2 of the acridine ring in both drugs suppressed their characteristic activity . Besides, substitution of the aminoalkyl side chain in nitracrine by methanesulfon-m-anisidine group suppressed its high antitumor activity, and the presence of a nitro group in position 1 of amsacrine suppressed its antileukemic activity . Comparison of biological properties of nitracrine, amsacrine and their analogs indicated differences in some steps of their mode of action.

Proc Natl Acad Sci U S A, 1987 Nov, 84(22), 8035 - 9
Isolation, DNA sequence, and regulation of a meiosis-specific eukaryotic recombination gene; Atcheson CL et al.; The SPO11 gene, required for meiotic recombination in Saccharomyces cerevisiae, has been cloned by direct selection for complementation of the spo11-1 phenotype: lack of meiotic recombination and low spore viability . DNA sequencing indicates that the gene encodes a 398-amino acid protein having a predicted molecular mass of 45.3 kDa . There is no significant similarity between the SPO11 protein and other protein sequences, including those from genes known to be involved in DNA recombination or repair . Strains bearing a disruption allele are viable, indicating that SPO11 is dispensable for mitotic growth . RNA analyses demonstrate that SPO11 produces a 1.5-kilobase transcript that is developmentally regulated and expressed early in the sporulation process.

Proc Natl Acad Sci U S A, 1987 Nov, 84(22), 8011 - 5
Pulsed-field electrophoresis: application of a computer model to the separation of large DNA molecules; Lalande M et al.; The biased reptation theory has been applied to the pulsed-field electrophoresis of DNA in agarose gels . A computer simulation of the theoretical model that calculates the mobility of large DNA molecules as a function of agarose pore size, DNA chain properties, and electric field conditions has been used to generate mobility curves for DNA molecules in the size range of the larger yeast chromosomes . Pulsed-field electrophoresis experiments resulting in the establishment of an electrophoretic karyotype for yeast, where the mobility of the DNA fragments is a monotonic function of molecular size for the entire size range that is resolved (200-2200 kilobase pairs), has been compared to the theoretical mobility curves generated by the computer model . The various physical mechanisms and experimental conditions responsible for band inversion and improved electrophoretic separation are identified and discussed in the framework of the model.

J Bacteriol, 1987 Nov, 169(11), 4991 - 4
Parasexual genetics of Torulopsis glabrata; Whelan WL et al.; Prototrophic hybrids were generated in the asexual yeast Torulopsis glabrata by the fusion of spheroplasts derived from parent strains which bore complementing auxotrophic markers . The DNA content (per cell) of two hybrids was essentially that predicted by summing the corresponding parental values . UV irradiation of these two hybrids resulted in the formation of sectored colonies with genetic properties consistent with their origin by either mitotic recombination or chromosomal nondisjunction.

J Biochem (Tokyo), 1987 Nov, 102(5), 1311 - 20
On the structure of old yellow enzyme studied by specific limited proteolysis; Miura R et al.; Limited proteolysis of brewer's yeast old yellow enzyme (OYE) was carried out with bovine pancreatic alpha-chymotrypsin . The reaction proceeded with a decrease of the NADPH oxidase activity, generating specifically two peptides (designated as 34K and 14K fragments) with apparent molecular weights of 34,000 and 14,000, respectively . The same proteolytic treatment of apo OYE resulted in rapid and complete digestion of the protein . The 34K and 14K fragments are so intimately associated with each other that the isolation of each peptide from the other in the native form was unsuccessful . However, the complex of the two fragments was separated from the intact OYE and termed "nicked OYE." Nicked OYE still retained FMN and showed a visible-absorption spectrum slightly modified from that of intact OYE . Nicked OYE showed decreased affinity toward rho-bromophenol as compared to intact OYE . Nicked OYE exhibited lower Km and Vmax values than intact OYE in the NADPH oxidase reaction . The 34K and 14K fragments could be separated from each other by reversed-phase HPLC under denaturing conditions and the amino acid sequences of the two fragments and intact OYE in the amino terminal regions were determined . The N-terminal sequence of the 34K fragment coincided with that of intact OYE, indicating that the 34K fragment lies in the N-terminal side of OYE . The N-terminal sequence of the 14K fragment was found to show homology with the site of flavodoxin where it forms an electron-transfer complex with cytochrome c . The characteristic feature of this region is the presence of acidic residues and is shared by the FMN domain of NADPH-cytochrome P-450 reductase . We interpret these findings as indicating that OYE has a physiological role as an electron transfer component.

Proc Natl Acad Sci U S A, 1987 Nov, 84(21), 7478 - 82
Identification of a vaccinia virus gene encoding a type I DNA topoisomerase; Shuman S et al.; Vaccinia virus encapsidates a type I DNA topoisomerase (EC 5.99.1.2) . The enzyme was purified from virus cores to apparent homogeneity, yielding a protein of Mr 32,000 . The amino-terminal sequence of the isolated Mr 32,000 polypeptide was determined and used to map the putative structural gene for the vaccinia topoisomerase to the H7r open reading frame of the vaccinia genome . This gene encodes a 314-amino acid polypeptide containing a region homologous to a region of the type I topoisomerase from the yeast Saccharomyces cerevisiae.

Mol Gen Genet, 1987 Nov, 210(1), 145 - 52
Nuclear suppression of a mitochondrial RNA splice defect: nucleotide sequence and disruption of the MRS3 gene; Schmidt C et al.; A mitochondrial RNA splice defect in the first intron of the COB gene (bI1) can be suppressed by a dominant nuclear mutation SUP-101 . Starting with a gene bank of yeast nuclear DNA from a SUP-101 suppressor strain cloned in the YEp13 plasmid, we have isolated a recombinant plasmid which exerts a suppressor activity similar to the SUP-101 allele . The N3(2) insert of this plasmid contains an open reading frame (ORF) of 1014 bp which is transcribed to a 12 S RNA . Deletion of the 5' end of this ORF and its upstream sequences abolishes the suppressor activity . The N3(2) insert thus carries a functional gene (called MRS3) which can suppress a mitochondrial splice defect . The chromosomal equivalent of the cloned gene has been mapped to chromosome 10 . Disruption of this chromosomal gene has no phenotypic effect on wild-type cells.

Nucleic Acids Res, 1987 Oct 26, 15(20), 8387 - 98
Plasmid migration using orthogonal-field-alternation gel electrophoresis; Hightower RC et al.; The migration properties of a series of supercoiled plasmids ranging in size from 4 to 16 kilobases (kb) have been analyzed by orthogonal-field-alternation gel electrophoresis (OFAGE) . These circular DNAs enter the gel and are well resolved . Unlike linear DNA molecules, the relative mobilities of these plasmids are constant over a wide range of pulse times, from 10 to 120 seconds, as well as over a broad range of total running times, from 6 to 24 hours . Electrophoresis of supercoiled, relaxed, and nicked open circular forms as well as topoisomers of pBR322 shows that the extent of supercoiling has a dramatic effect on plasmid migration on OFAGE . Several practical applications for exploiting the different migration properties of circular and linear DNA molecules on OFAGE are presented.

Biochim Biophys Acta, 1987 Oct 15, 915(3), 393 - 8
Affinity and stability modifications of immobilized alcohol dehydrogenase through multipoint copolymerization; Bille V et al.; Yeast alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1), a potentially useful enzyme for cofactor regeneration processes, was covalently immobilized in a multipoint fashion by activation with acryloyl chloride and subsequent copolymerization in a polyacrylamide gel . Several properties such as the activity and stability were systematically studied for the free enzyme, the acryloate-enzyme and the immobilized enzyme . The activation energy was significantly lowered upon immobilization . The thermal stability of the immobilized enzyme was, however, greatly increased . But its maximum activity was observed at a lower temperature . These results suggest an important effect of the diffusional restrictions and of the mode of activation and immobilization on the activity and the stability of the enzyme.

FEBS Lett, 1987 Oct 5, 222(2), 322 - 6
Histone acetyltransferase activity during the cell cycle; Golderer G et al.; Histone acetyltransferase activity was measured in isolated nuclei during the synchronous cell cycle of the myxomycete Physarum polycephalum . Nuclei were incubated with {14C}acetyl-coenzyme A and an excess of exogenous calf thymus histones . The activity is periodic during the cell cycle; it rises during the S-phase to reach a maximum in the early G2-period with a decline in mid and late G2 . Comparison of the pattern of enzyme activity with the in vivo acetylation of histones during the cell cycle reveals that the enzyme activity does not wholly determine the acetylation state, indicating that other factors, including possibly the structural state of chromatin, are responsible for the observed cell cycle pattern of in vivo histone acetylation.

Mol Cell Biol, 1987 Oct, 7(10), 3799 - 805
Insertions of up to 17 amino acids into a region of alpha-tubulin do not disrupt function in vivo; Schatz PJ et al.; Microtubules in yeasts are essential components of the mitotic and meiotic spindle and are necessary for nuclear movement during cell division and mating . The yeast Saccharomyces cerevisiae has two alpha-tubulin genes, TUB1 and TUB3, either of which alone is sufficient for these processes when present in a high enough copy number . Comparisons of sequences from several species reveals the presence of a variable region near the amino terminus of alpha-tubulin proteins . We perturbed the structure of this region in TUB3 by inserting into it 3, 9, or 17 amino acids and tested the ability of these altered proteins to function as the only alpha-tubulin protein in yeast cells . We found that each of these altered proteins was sufficient on its own for mitotic growth, mating, and methods of yeast . We conclude that this region can tolerate considerable variation without losing any of the highly conserved functions of alpha-tubulin . Our results suggest that variability in this region occurs because it can be tolerated, not because it specifies an important function for the protein.

Mol Cell Biol, 1987 Oct, 7(10), 3446 - 51
Interaction of GAL4 and GAL80 gene regulatory proteins in vitro; Lue NF et al.; The GAL80 protein of Saccharomyces cerevisiae, synthesized in vitro, bound tightly to GAL4 protein and to a GAL4 protein-upstream activation sequence DNA complex, as shown by (i) coimmunoprecipitation of GAL4 and GAL80 proteins with anti-GAL4 antiserum, (ii) an electrophoretic mobility shift of a GAL4 protein-upstream activation sequence DNA complex upon the addition of GAL80 protein, and (iii) GAL4-dependent binding of GAL80 protein to upstream activation sequence DNA immobilized on Sepharose beads . Anti-GAL4 antisera were raised against a GAL4-URA3 fusion protein, which could be purified to homogeneity in a single step with the use of an affinity chromatographic procedure for the URA3 gene product.

Anal Biochem, 1987 Oct, 166(1), 85 - 9
A rapid method for assay of glycosidases involved in glycoprotein biosynthesis; Herscovics A et al.; A rapid procedure to measure processing glycosidases with labeled oligosaccharide as substrate is described, using assay of the specific processing alpha-mannosidase from Saccharomyces cerevisiae as an example . After incubation of {3H}mannose-labeled Man9GlcNAc with the mannosidase, a solution of concanavalin A is added, followed by polyethylene glycol to precipitate the oligosaccharide-lectin complex . The radioactivity present in the supernatant after centrifugation is then measured to determine the amount of labeled mannose released . It is shown that the results of this procedure are similar to those obtained previously using small columns of concanavalin A-Sepharose (B . Saunier, R . D . Kilker, Jr., J . S . Tkacz, A . Quaroni, and A . Herscovics (1982) J . Biol . Chem . 257, 14155-14161) . The precipitation procedure, which can be applied to the assays of other processing enzymes, is much more convenient when a large number of samples must be analyzed.

Mol Cell Biol, 1987 Oct, 7(10), 3792 - 8
Domains of beta-tubulin essential for conserved functions in vivo; Fridovich-Keil JL et al.; The relationship between the primary sequence of tubulins and their properties in cells was studied by gene transfection experiments . Previously, we studied a chimeric beta-tubulin formed from chicken beta-tubulin-2 sequences in the amino-terminal portion and the highly divergent Saccharomyces cerevisiae TUB2 sequences in the carboxy-terminal 25% of the molecule . In the cytoplasm of cultured animal cells, this protein incorporates into all microtubule structures and assembles with the same efficiency as endogenous tubulin . We show that the protein products of chimeric genes with an increasing proportion of yeast sequence, extending 5' of the carboxy-terminal 25%, are abnormal in two ways . First, they assemble with a significantly lower efficiency than the original chimeric protein or the endogenous tubulins . Second, they are less stable in the cytoplasm . The results suggest that the position of the yeast sequences is crucial in determining the properties of the molecule . Results of analyses of 1 deletion mutation and 10 linker insertions in the original chimeric tubulin suggest that those changes made outside the carboxyl terminus completely disrupt assembly activity, while those made in the carboxyl terminus do not.

Biochim Biophys Acta, 1987 Sep 18, 903(1), 68 - 77
On the relationship between the dual specificity of the bovine brain phosphatidylinositol transfer protein and membrane phosphatidylinositol levels; Van Paridon PA et al.; The phosphatidylinositol transfer protein from bovine brain has a remarkable specificity pattern with a distinct preference for phosphatidylinositol (PI) and a low affinity for phosphatidylcholine (PC) . In this study we have determined the affinity of PI-transfer protein for PI relative to that for PC by measuring the binding of the fluorescent pyrene-labeled analogs of these phospholipids . From competition binding experiments it was estimated that the transfer protein has a 16-fold higher affinity for PI than for PC . This relative affinity together with the relative abundance of PI and PC, determines what proportion of the protein contains PI (e.g . 65% of the PI-transfer protein in the case of bovine brain) . From measuring lipid transfer between donor vesicles consisting of equimolar amounts of PC and PI, and an excess of acceptor vesicles consisting of various ratios of PC and PI, we have observed that the relative rates of the PI-transfer protein-mediated transfer of PI and PC varies between 5 and 20 . Kinetic analysis has indicated that PI-transfer protein carrying a PI molecule has different kinetic properties than the PI-transfer protein carrying a PC molecule . It will be discussed that because of the dual specificity, PI-transfer protein is ideally suited for maintaining PI levels in intracellular membranes.

Biochem J, 1987 Sep 15, 246(3), 611 - 7
Cell-free acylation of rat brain myelin proteolipid protein and DM-20; Yoshimura T et al.; Incubation of rat brain myelin with {3H}palmitic acid in the presence of ATP, CoA and MgCl2 or {14C}-palmitoyl-CoA in a cell-free system resulted in the selective labelling of 'PLP' {proteolipid protein; Folch & Lees (1951) J . Biol . Chem . 191, 807-817} and 'DM-20' {Agrawal, Burton, Fishman, Mitchell & Prensky (1972) J . Neurochem . 19, 2083-2089} which, after polyacrylamide-gel electrophoresis in SDS, were revealed by fluorography . These results provide evidence of the association of fatty acid-CoA ligase and acyltransferase in isolated myelin . Palmitic acid is covalently bound to PLP and DM-20, because 70 and 92% of the radioactivity was removed from proteolipid proteins after treatment with hydroxylamine and methanolic NaOH respectively . Incubation of myelin with {3H}palmitic acid in the absence of ATP, CoA, MgCl2, or all three, decreased incorporation of fatty acid into PLP to 3, 55, 18 and 2% respectively . The cell-free system exhibits specificity with respect to the chain length of the fatty acids, since myristic acid is incorporated into PLP at a lower rate when compared with palmitic and oleic acids . The acylation of PLP is an enzymic reaction, since (1) maximum incorporation of {3H}palmitic acid into PLP occurred at physiological temperatures and decreased with an increase in the temperature; (2) acylation of PLP with {3H}palmitic acid and {14C}palmitoyl-CoA was severely inhibited by SDS (0.05%); and (3) the incorporation of fatty acid and palmitoyl-CoA into PLP was substantially decreased by the process of freezing-thawing and freeze-drying of myelin . We have provided evidence that all of the enzymes required for acylation of PLP and DM-20 are present in isolated rat brain myelin . Acylation of PLP in a cell-free system with fatty acids and palmitoyl-CoA suggests that a presynthesized pool of non-acylated PLP and DM-20 is available for acylation.

Cell, 1987 Sep 11, 50(6), 909 - 15
Functional redundancy and structural polymorphism in the large subunit of RNA polymerase II; Nonet M et al.; The RNA polymerase II large subunit contains tandem copies of the sequence Pro Thr Ser Pro Ser Tyr Ser at its carboxyl terminus, the number of which varies from 26 in yeast to 52 in mice . Our results indicate that the heptapeptide repeat sequence is unique and essential to RNA polymerase II . We have determined that a portion of the heptapeptide repeat domain is essential for viability by constructing and analyzing unidirectional deletions of the carboxy-terminal coding sequence in yeast . Cells containing an RNA polymerase II large subunit with less than 10 complete heptapeptide repeats are inviable, those containing 10-12 complete repeats are conditionally viable, and those with 13 or more complete repeats are unconditionally viable . The inviable deletion mutants studied here have truncated RNA polymerase subunits that are stable, but functionally deficient . Finally, the number of repeat units is polymorphic in wild-type yeast strains . These results have implications for the function of this unusual sequence in transcription.

Cell, 1987 Sep 11, 50(6), 937 - 43
Enzymatically inactive p60c-src mutant with altered ATP-binding site is fully phosphorylated in its carboxy-terminal regulatory region; Jove R et al.; Cellular src protein, p60c-src, is phosphorylated on tyrosine 527 in chicken embryo fibroblasts, and this phosphorylation is implicated in suppressing the protein-tyrosine kinase activity and transforming potential of p60c-src . To determine whether tyrosine 527 phosphorylation is dependent on p60c-src kinase activity, the ATP-binding site of chicken p60c-src was destroyed by substitution of lysine 295 with methionine . The resultant protein, p60c-src(M295), expressed either in chicken cells or in yeast, lacked detectable kinase activity . Nevertheless, tyrosine and serine phosphorylation of p60c-src(M295) overproduced in chicken cells were indistinguishable from that of authentic p60c-src . By contrast, p60c-src(M295) was not phosphorylated on tyrosine in yeast . These results suggest that a protein kinase present in chicken cells but not in yeast phosphorylates tyrosine 527 in trans, and are consistent with the possibility that this kinase is distinct from p60c-src.

Biochemistry, 1987 Sep 8, 26(18), 5890 - 6
A physical-chemical model for cellular uptake of fatty acids: prediction of intracellular pool sizes; Cooper R et al.; If the uptake of fatty acids by liver is a physical, not a biological, process, then the size and location of the intrahepatic pool of fatty acids can be predicted from uptake rates and thermodynamic data . The purpose of the experiments in this paper was to test the accuracy of this idea . Rat livers were perfused with palmitate bound to albumin, and the total amounts of palmitate removed from the perfusate were measured at 3-s intervals . The intrahepatic pools of palmitate calculated from these data were 13.8 and 23.0 nmol/g of liver at ratios of palmitate/albumin (mol/mol) (afferent side) of 2/1 and 4/1, respectively, in the steady state . The intrahepatic pools of palmitate calculated from the distributions of palmitate between membranes, H2O, albumin, and fatty acid binding protein and the measured first-order rate constants for acyl-CoA ligases in mitochondria and microsomes were 12.1 and 34.6 nmol/g for perfusate ratios of palmitate/albumin of 2/1 and 4/1, in the steady state . Intrahepatic pools of palmitate measured after establishment of a steady-state rate of uptake were 15.0 and 31.8 nmol/g for these ratios of palmitate/albumin of 2/1 and 4/1.

EMBO J, 1987 Sep, 6(9), 2781 - 4
GCN4, a eukaryotic transcriptional activator protein, binds as a dimer to target DNA; Hope IA et al.; The eukaryotic transcriptional activator protein, GCN4, synthesized in vitro from the cloned gene, binds specifically to the promoters of yeast amino acid biosynthetic genes . Previous analysis of truncated GCN4 derivatives localized the DNA binding domain to the C-terminal 60 amino acids and revealed that the size of the GCN4 derivative and the electrophoretic mobility of the protein-DNA complex were inversely related . This observation was utilized here to develop a novel method for determining the subunit structure of DNA binding proteins . A mixture of wild-type GCN4 protein and a smaller GCN4 derivative generated three complexes with DNA, two corresponding to those observed when the proteins are present individually and one new complex of intermediate mobility . This extra complex results from the heterodimer of the two GCN4 proteins of different sizes, demonstrating that GCN4 binds DNA as a dimer . The contacts sufficient for dimerization were localized to the 60 C-terminal amino acid, DNA binding domain, suggesting that dimerization of GCN4 is a critical aspect of specific DNA binding . Furthermore, stable GCN4 dimers were formed in the absence of target DNA . These observations suggest a structural model of GCN4 protein in which a dimer binds to overlapping and non-identical half-sites, explaining why GCN4 recognition sites act bidirectionally in stimulating transcription.

Mol Cell Biol, 1987 Sep, 7(9), 3212 - 20
Comparison of tRNA gene transcription complexes formed in vitro and in nuclei; Huibregtse JM et al.; The nucleoprotein structure of single-copy tRNA genes in yeast nuclei was examined by DNase I footprinting and compared with that of complexes formed in vitro between the same genes and transcription factor C . Transcription factor C bound to both the 5' and 3' intragenic promoters of the tRNA(SUP53Leu) gene in vitro, protecting approximately 30 base pairs at the 3' promoter (B block) and 40 base pairs at the 5' promoter (A block) and causing enhanced DNase I cleavages between the protected regions . Binding to the two sites was independent of the relative orientation of the two sites on the helix and was eliminated by a single point mutation in the 3' promoter . The chromosomal tRNA(SUP53Leu) and tRNA(UCGSer) genes showed a pattern of protection and enhanced cleavages similar to that observed in vitro, indicating that the stable complexes formed in vitro accurately reflect at least some aspects of the nucleoprotein structure of the genes in chromatin.

Proc Natl Acad Sci U S A, 1987 Sep, 84(17), 6215 - 9
Recombinational substrates designed to study recombination between unique and repetitive sequences in vivo; Fasullo MT et al.; Three recombination events, reciprocal recombination, sister-chromatid recombination, and gene conversion, were studied using substrates designed in vitro . Each type of recombination event can be monitored at any chromosomal location . We have shown that sister-chromatid recombination is induced mitotically by DNA damaging agents, such as methyl methanesulfonate and gamma-rays, but is decreased mitotically in strains defective in rad52 . Reciprocal recombination by which circular plasmids integrate into the genome is unaffected by rad52 defective alleles and occurs by a different recombination pathway . Mechanisms are suggested by which gene conversion between sister chromatids can generate chromosome rearrangements.

Eur J Biochem, 1987 Sep 1, 167(2), 327 - 38
Amino acid sequence of endothiapepsin . Complete primary structure of the aspartic protease from Endothia parasitica; Barkholt V; The amino acid sequence of endothiapepsin, the aspartic protease from Endothia parasitica has been determined . The enzyme consists of 330 residues . The sequence determination was performed exclusively at the protein level . The homology of this fungal milk-clotting enzyme with aspartic proteases is demonstrated by alignment with pepsin, chymosin, gastricsin, renin, and cathepsin D from various vertebrates and proteinase A from Saccharomyces cerevisiae showing 25-30% identity . The identity with mucor rennin from Mucor pucillus was 21% and with penicillopepsin from Penicillium janthinellum 53%, the fungal enzymes thus representing the lowest as well as the highest degree of homology.

Mol Gen Genet, 1987 Sep, 209(2), 283 - 9
Correlation between restriction map, genetic map and catalytic functions in the gene complex URA2; Potier S et al.; We replaced the URA2 gene by six different deleted alleles constructed in vitro by Bg/II digestion in order to correlate the genetic map with the restriction map and to define the regions coding for the different functions of the carbamylphosphate synthetase--aspartate transcarbamylase complex (CPSase-ATCase) . We also enlarged the collection of ura2 point mutations by using a positive selection method based on resistance to the toxic accumulation of ureidosuccinic acid (USA) . Of the new independent mutations nine mapped in the intermediary zone, a previously defined mutationless region localized between regions coding for CPSase and ATCase . This shows that the former definition resulted from analysis of a limited number of mutants (40) . The study of an allele deleted in the intermediary zone shows that this sequence codes for a protein region necessary for the feedback inhibition of the CPSase-ATcase enzyme complex . The CPSase- ATCase- phenotype of 26 mutants resistant to USA accumulation shows the importance of the in vivo channelling of carbamylphosphate in the CPSase-ATCase complex for USA and subsequent pyrimidine biosynthesis . Finally, our results confirm that the CPSase and ATCase activities are separate functions.

J Biol Chem, 1987 Aug 25, 262(24), 11578 - 83
Proton hyperfine resonance assignments in cyanide-ligated cytochrome c peroxidase using the nuclear Overhauser effect; Satterlee JD et al.; The development of the proton nuclear Overhauser effect (NOE) for hyperfine shifted resonances of cyanide-ligated cytochrome c peroxidase (Saccharomyces cerevisiae) has been studied . In the pre-steady state regime, the major effects are due to primary NOEs to nearest neighbor protons . This has been used to advantage in making assignments of all of the remaining unassigned, resolved, downfield hyperfine shifted resonances . This work also determined the relative orientation of the heme pyrrole II substituents which is the cis configuration with the 4 alpha-vinyl proton pointing away from the 3CH3 . In addition to heme protons, resonances of histidine 175, threonine 180, and histidine 52 have been assigned . These results indicate some structural rearrangement of the distal amino acids accompanying ligation.

J Biol Chem, 1987 Aug 15, 262(23), 11182 - 7
Examination of the role of arginine-143 in the human copper and zinc superoxide dismutase by site-specific mutagenesis; Beyer WF Jr et al.; The active site arginine-143 of human Cu,Zn superoxide dismutase has been replaced by lysine or by isoleucine . The mutant proteins were expressed at high levels in yeast, purified, and the amino acid substitution explored through the use of group specific reagents . The specific activities of these enzymes, measured by the xanthine oxidase/cytochrome c method and by using dry weight determination to establish protein concentration, were: native enzyme, 6570 units/mg; Lys-substituted enzyme, 2840 units/mg, Ile-substituted enzyme, 708 units/mg . The active site arginine thus plays an important, but not an essential, role in the catalytic process.

Biochim Biophys Acta, 1987 Aug 5, 914(2), 162 - 9
Substituent effects during the rat liver aldehyde dehydrogenase catalyzed oxidation of aromatic aldehydes; Rietveld EC et al.; The influence of the steric hindrance of halogen substituents was investigated in vitro by measuring the activity of yeast aldehyde dehydrogenase (aldehyde: NAD(P)+ oxidoreductase, EC 1.2.1.5) and of aldehyde dehydrogenases in subcellular rat liver fractions with a series of ortho- and para-halo-substituted benzaldehydes as substrates . Upon an increase in the size of the halogen substituent (F, Cl, Br), the reactivity of yeast aldehyde dehydrogenase to ortho-substituted benzaldehydes decreased drastically . The same phenomenon was observed with the unspecific aldehyde dehydrogenases in three rat liver fractions; cytoplasm, mitochondria and microsomes . The corresponding para-halobenzaldehydes (F, Cl, Br, I) did not reveal large differences in reactivity to the various rat liver aldehyde dehydrogenases . The aldehyde dehydrogenases in the rat liver microsomal fraction exhibited most clearly the regiospecificity . Enzymatic oxidation of 4-bromobenzaldehyde was more than 30-times faster then the ortho-isomer . The findings in this investigation confirm the suggestion that the steric hindrance of bulky ortho-substituents of benzaldehydes account for the slowing down of the aldehyde dehydrogenase-catalyzed oxidation of benzaldehydes to corresponding benzoic acids . The enzymatic oxidation of microsomal aldehyde dehydrogenase is strongly influenced by steric effects of benzaldehydes, bearing a halogen in ortho-position . We think that the microsomal aldehyde dehydrogenase might be the principal enzyme responsible for oxidation of halobenzaldehydes in rat liver.

Oncogene Res, 1987 Aug, 1(3), 243 - 53
The complete primary structure of the rat A-raf cDNA coding region: conservation of the putative regulatory regions present in rat c-raf; Ishikawa F et al.; A-raf gene was first detected by low-stringent hybridization with v-raf and was reported as a possible oncogene . We have cloned rat A-raf cDNA and determined the complete nucleotide sequence over its long open reading frame . Alignment of rat A-raf- and c-raf-deduced amino acid sequences shows 59.4% homology . Both the ATP binding site and the kinase domain are conserved in A-raf as in c-raf . Furthermore, three regions highly conserved between A-raf and c-raf were identified in the 5'-half region of c-raf which has been shown to be deleted in the activated form of c-raf . These regions were speculated to be the candidates of the regulatory domain controlling the protein kinase activity of raf products . Southern blot analysis shows that genes homologous to A-raf and c-raf are present as independent loci in Drosophila melanogaster, Caenorhabditis elegans, Saccharomyces cerevisiae and Candida boidinii . A-raf was expressed in several tissues and cell line at various amounts.

Strahlenther Onkol, 1987 Aug, 163(8), 552 - 6
{Electrophoretic studies on the effect of different types of radiation (60Co, electrons, x-ray, photons, UV) on low-molecular ribonucleic acids}; Lickl E et al.; Electron irradiation (45 MeV) with the chosen doses modifies the molecule composition of isolated dry yeast RNA . High-energy irradiation will crack the RNA which then forms new chains of macromolecular nucleic acids . Other radiation types (60Co, photons, X-rays) do not modify dry RNA, but when irradiated in aqueous solution, these macromolecular bands will be built, too . After UV irradiation with 254 nm delivered over 24 hours these macromolecular bands disappear completely in the electrophoretic diagram . This wavelength corresponds to the absorption maximum of purines, thus stimulating them and reducing their stability.

Radiat Res, 1987 Aug, 111(2), 192 - 200
Potentially lethal damage repair is due to the difference of DNA double-strand break repair under immediate and delayed plating conditions; Frankenberg-Schwager M et al.; Cells plated immediately after irradiation on nutrient agar (immediate plating) exhibit a lower survival than cells which are kept under nongrowth conditions before plating (delayed plating) . The difference between the survival curves obtained after immediate plating and delayed plating is considered to exhibit the cell's capacity to repair potentially lethal damage . In yeast evidence has been presented previously for the DNA double-strand break (DSB) as the molecular lesion involved in the repair of potentially lethal damage observed at the cellular level . Radiation-induced DSB are repaired in cells plated on nutrient agar, i.e., under growth conditions, as well as in cells kept under nongrowth conditions . In this paper DSB repair under growth and nongrowth conditions is studied with the help of the yeast mutant rad54-3 which is temperature conditional for DSB repair . It is shown that the extent of repair of potentially lethal damage can be varied by shifting the relative fractions of repair of DSB under growth conditions versus nongrowth conditions . Repair of DSB in cells plated on nutrient agar is promoted when glucose is substituted by Na-succinate as an energy source . As a result the immediate plating survival curve approaches the delayed plating survival curve, thus reducing the operationally defined repair of potentially lethal damage . We show that this reduced potentially lethal damage repair is caused, however, by a higher amount of DSB repair in cells immediately plated on succinate agar as compared to glucose agar.

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