Differentially regulated malate synthase genes participate in carbon and nitrogen metabolism of S. cerevisiae.

We have isolated a second gene (MLS1), which in addition to DAL7, encodes malate synthase from S. cerevisiae. Expression of the two genes is specific for their physiological roles in carbon and nitrogen metabolism. Expression of MLS1, which participates in the utilization of non-fermentable carbon sources, is sensitive to carbon catabolite repression, but nearly insensitive to nitrogen catabolite repression. DAL7, which participates in catabolism of the nitrogenous compound allantoin, is insensitive to carbon catabolite repression, but highly sensitive to nitrogen catabolite repression. Results obtained with null mutations in these genes suggest that S. cerevisiae contains at least one and perhaps two additional malate synthase genes.     

Metabolism of nitric oxide and nitrous oxide during nitrification and denitrification in soil at different incubation conditions

Abstract NO production and consumption rates as well as N₂O accumulation rates were measured in a loamy cambisol which was incubated under different conditions (i.e. soil moisture content, addition of nitrogen fertilizer and/or glucose, aerobic or anaerobic gas phase). Inhibition of nitrification with acetylene allowed us to distinguish between nitrification and denitrification as sources of NO and N₂O. Under aerobic conditions untreated soil showed very low release of NO and N₂O but high consumption of NO. Fertilization with NH₄⁺ or urea stimulated both NO and N₂O production by nitrification. Addition of glucose at high soil moisture contents led to increased N₂ and N₂O production by denitrification, but not to increased NO production rates. Anaerobic conditions, however, stimulated both NO and N₂O production by denitrification. The production of NO and N₂O was further stimulated at low moisture contents and after addition of glucose or NO₃⁻. Anaerobic consumption of NO by denitrification followed Michaelis-Menten kinetics and was stimulated by addition of glucose and NO₃⁻. Aerobic consumption of NO followed first-order kinetics up to mixing ratios of at least 14 ppmv NO, was inhibited by autoclaving but not by acetylene, and decreased with increasing soil moisture content. The high NO-consumption activity and the effects of soil moisture on the apparent rates of anaerobic and aerobic production and consumption of NO suggest that diffusional constraints have an important influence on the release of NO, and may be a reason for the different behaviour of NO release vs N₂O release.     

The role of CDC28 and cyclins during mitosis in the budding yeast S. cerevisiae.

cdc28-1N is a conditional allele that has normal G1 (Start) function but confers a mitotic defect. We have isolated seven genes that in high dosage suppress the growth defect of cdc28-1N cells but not of Start-defective cdc28-4 cells. Three of these (CLB1, CLB2, and CLB4) encode proteins strongly homologous to G2-specific B-type cyclins. Another gene, CLB3, was cloned using PCR, CLB1 and CLB2 encode a pair of closely related proteins; CLB3 and CLB4 encode a second pair. Neither CLB1 nor CLB2 is essential; however, disruption of both is lethal and causes a mitotic defect. Furthermore, the double mutant cdc28-1N clb2::LEU2 is nonviable, whereas cdc28-4 clb2::LEU2 is viable, suggesting that the cdc28-1N protein may be defective in its interaction with B-type cyclins. Our results are consistent with CDC28 function being required in both G1 and mitosis. Its mitotic role, we believe, involves interaction with a family of at least four G2-specific cyclins.     

Structure and function of disk aggregates of the coat protein of tobacco mosaic virus

Experiments have been carried out on the coat protein of tobacco mosaic virus (TMVP) to test for the occurrence of the previously postulated RNA-induced direct switching, during in vitro assembly of tobacco mosaic virus (TMV), of the subunit packing from the cylindrical bilayer disk to the virus helical arrangement. No evidence was found for such RNA-induced switching and no evidence for the direct participation of the bilayer disk in either the nucleation or elongation phases of the in vitro virus assembly. Instead, virus assembly proceeds by an initiation step involving the binding of the RNA to the previously characterized two-plus turn helical aggregate that is formed from small oligomers of subunits. However, a bilayer disk, which has been characterized in high ionic strength crystals, has been observed in low ionic strength virus assembly solutions only as a transient species upon depolymerization of dimers of bilayer disks formed in solution at high ionic strength, and not as an equilibrium species of TMVP.     

Temperature dependence of anion transport in the human red blood cell

Arrhenius plots of chloride and bromide transport yield two regions with different activation energies (Ea). Below 15 or 25 degrees C (for Cl- and Br-, respectively), Ea is about 32.5 kcal/mol; above these temperatures, about 22.5 kcal/mol (Brahm, J. (1977) J. Gen. Physiol. 70, 283-306). For the temperature dependence of SO4(2-) transport up to 37 degrees C, no such break could be observed. We were able to show that the temperature coefficient for the rate of SO4(2-) transport is higher than that for the rate of denaturation of the band 3 protein (as measured by NMR) or the destruction of the permeability barrier in the red cell membrane. It was possible, therefore, to extend the range of flux measurements up to 60 degrees C and to show that, even for the slowly permeating SO4(2-) in the Arrhenius plot, there appears a break, which is located somewhere between 30 and 37 degrees C and where Ea changes from 32.5 to 24.1 kcal/mol. At the break, the turnover number is approx. 6.9 ions/band 3 per s. Using 35Cl- -NMR (Falke, Pace and Chan (1984) J. Biol. Chem. 259, 6472-6480), we also determined the temperature dependence of Cl- -binding. We found no significant change over the entire range from 0 to 57 degrees C, regardless of whether the measurements were performed in the absence or presence of competing SO4(2-). We conclude that the enthalpy changes associated with Cl- - or SO4(2-)-binding are negligible as compared to the Ea values observed. It was possible, therefore, to calculate the thermodynamic parameters defined by transition-state theory for the transition of the anion-loaded transport protein to the activated state for Cl-, Br- and SO4(2-) below and above the temperatures at which the breaks in the Arrhenius plots are seen. We found in both regions a high positive activation entropy, resulting in a low free enthalpy of activation. Thus the internal energy required for carrying the complex between anion and transport protein over the rate-limiting energy barrier is largely compensated for by an increase of randomness in the protein and/or its aqueous environment.     

A Chinese hamster cell cycle mutant arrested at G2 phase has a temperature-sensitive ubiquitin-activating enzyme, E1

The effect of restrictive temperature on ubiquitin conjugation activity has been studied in cells of ts20, a temperature-sensitive cell cycle mutant of the Chinese hamster cell line E36. Ts20 is arrested in early G2 phase at nonpermissive temperature. Immunoblotting with antibodies to ubiquitin conjugates shows that conjugates disappear rapidly at restrictive temperatures in ts20 mutant but not in wild type E36 cells. The incorporation of 125I-ubiquitin into permeabilized ts20 cells is temperature-sensitive. Addition of extracts of another G2 phase mutant, FM3A ts85, with a temperature-sensitive ubiquitin activation enzyme (E1), to permeabilized ts20 cells at restrictive temperatures fails to complement their ubiquitin ligation activity. This indicates that the lesions in the two mutants are similar. Purified E1 from reticulocytes restores the conjugation activity of heat-inactivated permeabilized ts20 cells. Ubiquitin conjugation activity of cell-free extracts of ts20 cells was temperature-sensitive and could be restored by adding purified reticulocyte E1. Purified reticulocyte E2 or E3, on the other hand, did not restore the ubiquitin conjugation activity of heat-treated ts20 extracts. These results are consistent with the conclusion that ts20 has temperature-sensitive ubiquitin-activating enzyme (E1). The fact that two E1 mutants (ts20 and ts85) derived from different cell lines are arrested at the S/G2 boundary at restrictive temperatures strongly indicates that ubiquitin ligation is necessary for passage through this part of the cell cycle. The temperature thresholds of heat shock protein synthesis of ts20 and wild type E36 cells were identical. The implications of these findings with respect to a suggested role of ubiquitin in coupling between protein denaturation and the heat shock response are discussed.     

Improved procedure for the isolation of a double-strand-specific ribonuclease and its application to structural analysis of various 5S rRNAs and tRNAs

An improved method for the isolation of a double-strand-specific RNase from snake venom is presented. This RNase, called CSV, was used to cleave yeast tRNAPhe and tRNA2Glu and tRNAfMet from Escherichia coli. In addition these RNAs and E. coli tRNAPhe were examined with the single-strand-specific nuclease S1. The results are discussed in terms of the specificity of CSV RNase and the structure of tRNAs. S1 nuclease digestions at increasing temperatures allowed the melting of tertiary and secondary structure to be monitored. 5S rRNA from E. coli, Thermoplasma acidophilum and the chloroplasts of Spinacia oleracea were digested with CSV and S1. The information these results give on the secondary-structural differences between different classes of 5S rRNA are discussed. Supporting evidence is found for tertiary interactions between hairpin loop c and internal loop d of eubacterial 5S rRNA.     

Interaction of azide with beef heart mitochondrial ATPase

This study examined the inhibition of azide as a probe of the magnesium regulation of beef heart mitochondrial ATPase (F1) catalysis. Azide elicited a slow hysteretic effect on both ATP and ITP hydrolysis of F1. This hysteretic effect was shown to be due to the consecutive binding of magnesium and azide, and to be independent of catalytic turnover. The azide binding site was also shown to be separate from the anion binding HCO3- site on F1. The results presented indicate that metal binding is important in the inhibition of the hydrolytic activity and regulation of F1. A model is presented which is consistent with the hysteretic inhibition of F1 by azide, in which there is a slow equilibration between free enzyme and the enzyme-magnesium-azide complex.     

Hirsutinolides,glaucolides and sesquiterpene lactone from Vernonia species

The investigation of nine Vernonia species afforded in addition to known sesquiterpenes 28 new ones. The structures were elucidated by high field ¹H NMR spectroscopy and the configurations were determined by NOE difference spectroscopy and, in one case, by X-ray analysis. The results indicated that configurations of several previously reported sesquiterpene lactones have had to be revised. In addition to known types two new ones, the jalcaguaianolides and the vernojalcanolides, are described. Furthermore some unusual reaction products are presented which, in part, led to some natural occurring lactones.