Asp537, Asp812 are essential and Lys631, His811 are catalytically significant in bacteriophage T7 RNA polymerase activity.

To define catalytically essential residues of bacteriophage T7 RNA polymerase, we have generated five mutants of the polymerase, D537N, K631M, Y639F, H811Q and D812N, by site-directed mutagenesis and purified them to homogeneity. The choice of specific amino acids for mutagenesis was based upon photoaffinity-labeling studies with 8-azido-ATP and homology comparisons with the Klenow fragment and other DNA/RNA polymerases. Secondary structural analysis by circular dichroism indicates that the protein folding is intact in these mutants. The mutants D537N and D812N are totally inactive. The mutant K631M has 1% activity, confined to short oligonucleotide synthesis. The mutant H811Q has 25% activity for synthesis of both short and long oligonucleotides. The mutant Y639F retains full enzymatic activity although individual kinetic parameters are somewhat different. Kinetic parameters, (kcat)app and (Km)app for the nucleotides, reveal that the mutation of Lys to Met has a much more drastic effect on (kcat)app than on (Km)app, indicating the involvement of K631 primarily in phosphodiester bond formation. The mutation of His to Gln has effects on both (kcat)app and (Km)app; namely, three- to fivefold reduction in (kcat)app and two- to threefold increase in (Km)app, implying that His811 may be involved in both nucleotide binding and phosphodiester bond formation. The ability of the mutant T7 RNA polymerases to bind template has not been greatly impaired. We have shown that amino acids D537 and D812 are essential, that amino acids K631 and H811 play significant roles in catalysis, and that the active site of T7 RNA polymerase is composed of different regions of the polypeptide chain. Possible roles for these catalytically significant residues in the polymerase mechanism are discussed.     

Influence of Nitrate and Sulfate on the Anaerobic Treatment of Pharmaceutical Wastewater

Anaerobic treatment of wastewater from the pharmaceutical industry, which contained about 3.2 g/L of sulfate, was carried out in an Upflow Anaerobic Sludge Blanket (UASB) reactor. After a startup period of 120 days, a chemical oxygen demand (COD) removal efficiency of more than 90 % was obtained along with an organic loading rate (OLR) of 1.5 g COD/(L day). During the same period, the sulfate removal was about 90 %. However, the performance of the reactor was affected when the loading rate was increased to 2.09 g COD/(L day). It was found that the accumulation of sulfides, combined with a decrease in the pH, affected the reactor performance. In batch reactor studies with pharmaceutical wastewater it was observed that methane production began only after the initiation of nitrate consumption. The denitrification process can inhibit sulfate reduction at high nitrate concentrations, but compared to reactors without nitrate, the sulfate reduction process and sulfide formation were quickly initiated at low nitrate concentrations. The methanogenic activity was however affected by the presence of more than 2 g/L of sulfate.     

Germination of concentrated suspensions of spores from Aspergillus niger

Summary Germination of condiospores fromA.niger in very concentrated suspension was required to inoculate solid fermentation media, but a germination self-inhibitor was detected in spores. It was found that the inhibitory effect depended on the composition of the medium and was reduced when glucose was used as a carbon source. The effect of the self-inhibitor was eliminated by washing the spores and using glucose and a protein nitrogen source in the germination medium. By this method it was possible to increase about 100 times (10⁶ to 10⁸) spore concentration, keeping more than 90% germination.     

Yeast Intrachromosomal Recombination: Long Gene Conversion Tracts Are Preferentially Associated with Reciprocal Exchange and Require the Rad1 and Rad3 Gene Products

A yeast intrachromosomal recombination system based on an inverted repeat has been designed to examine mitotic gene conversion tract length and the association of crossing over with gene conversion as a function of the conversion tract length. Short conversion tracts are found to be preferentially noncrossover while conversion tracts longer than 1.16 kb show a 50% association with crossover. Mutation in the excision repair gene RAD1 leads to a reduction in conversion tracts of at least 1.16 kb and a reduction in crossovers associated with conversion, regardless of the length of the conversion tract. Mutation in the excision repair gene RAD3, which encodes a DNA helicase, also leads to a reduction in conversion tracts of at least 1.16 kb, but has no effect on the frequency of associated crossovers. The roles of RAD1 and RAD3 in recombination are discussed.     

Genetic Control of Intrachromosomal Recombination in Saccharomyces Cerevisiae. I. Isolation and Genetic Characterization of Hyper-Recombination Mutations

Eight complementation groups have been defined for recessive mutations conferring an increased mitotic intrachromosomal recombination phenotype (hpr genes) in Saccharomyces cerevisiae. Some of the mutations preferentially increase intrachromosomal gene conversion (hpr4, hpr5 and hpr8) between repeated sequences, some increase loss of a marker between duplicated genes (hpr1 and hpr6), and some increase both types of events (hpr2, hpr3 and hpr7). New alleles of the CDC2 and CDC17 genes were recovered among these mutants. The mutants were also characterized for sensitivity to DNA damaging agents and for mutator activity. Among the more interesting mutants are hpr5, which shows a biased gene conversion in a leu2-112::URA3::leu2-k duplication; and hpr1, which has a much weaker effect on interchromosomal mitotic recombination than on intrachromosomal mitotic recombination. These analyses suggest that gene conversion and reciprocal exchange can be separated mutationally. Further studies are required to show whether different recombination pathways or different outcomes of the same recombination pathway are controlled by the genes identified in this study.     

Gonadotropin release and redistribution of calcium-activated, phospholipid-dependent protein kinase in phorbol-stimulated rat pituitary cells

The effect of phorbol esters on calcium-activated, phospholipid-dependent kinase (protein kinase C) and luteinizing hormone (LH) secretion was examined in cultured rat anterior pituitary cells. The potent tumor promoter 12-O-tetra-decanoylphorbol-13-acetate (TPA) stimulated LH secretion and activated pituitary protein kinase C in the presence of calcium and phosphatidylserine. The enzyme activity present in cytosol and particulate fractions was eluted at about 0.05 M NaCl during DE52-cellulose chromatography. Preincubation of pituitary cells with TPA markedly decreased cytosolic protein kinase C activity and increased enzyme activity in the particulate fraction. The maximal TPA-induced change in enzyme activity, with a 76% decrease in cytosol and a 4.3-fold increase in the particulate fraction, occurred within 10 min. The dose-dependent changes in protein kinase C redistribution in TPA-treated cells were correlated with the stimulation of LH release by the phorbol ester. These results suggest that activation of protein kinase C by TPA is associated with intracellular redistribution of the enzyme and is related to the process of secretory granule release from gonadotrophs.     

Hormone-induced redistribution of calcium-activated phospholipid-dependent protein kinase in pituitary gonadotrophs

The distribution of calcium-activated, phospholipid-dependent protein kinase (protein kinase C) between cytosol and membrane fractions was analyzed in cultured pituitary gonadotrophs during treatment with gonadotropin-releasing hormone (GnRH). In pituitary cells purified by centrifugal elutriation, the extent of protein kinase C redistribution during GnRH stimulation was correlated with the enrichment of gonadotrophs. GnRH-stimulated release of luteinizing hormone (LH) from gonadotroph-enriched cells was accompanied by a rapid and dose-dependent decrease in cytosolic protein kinase C and by a corresponding increase in protein kinase C activity in the particulate fraction. Retinal directly inhibited the activity of cytosolic protein kinase C and also attenuated the release of LH from GnRH-stimulated gonadotrophs. These findings, and the ability of GnRH to cause rapid translocation of cytosolic protein kinase C to a membrane-associated form, suggest that hormonal activation of protein kinase C is an intermediate step in the stimulation of pituitary LH secretion by GnRH.     

Ethanol-sensitive mutants of Saccharomyces cerevisiae

Saccharomyces cerevisiae mutants unable to grow at ethanol concentrations at which the wild type strain S288C does grow, have been isolated. Some of them show additional phenotypic alterations in colony size, temperature sensitivity and viability in ethanol, which cosegregate with the growth sensitivity in ethanol. 21 selected monogenic ethanol-sensitive mutants define 20 complementation groups, denominated ETA1 to ETA20, which indicates that there is a high number of genes involved in the ethanol tolerance/sensitivity mechanism.Out of 21 selected monogenic mutants, 20 are not altered in the glycolytic pathway since, when maintained in glucosesupplemented medium, they can produce as much ethanol as the wild type and at about the same velocity. Nor do any of the mutants seem to be altered in the lipid biosynthetic pathway since, whether grown in the absence or in the presence of ethanol, their concentration of fatty acids and ergosterol is similar to that of the wild type under the same conditions. Therefore growth sensitivity to ethanol does not seem necessarily to be related to carbohydrate or lipid metabolism.Non-common abbreviations YP yeast extract peptone medium - YPD yeast extract peptone dextrose agar or medium - YPG yeast extract peptone glycerol agar - YPDE yeast extract peptone dextrose ethanol agar or medium - SD yeast nitrogen base dextrose agar - SPO yeast extract potassium acetate glucose agar - PD parental ditype - NPD non-parental ditype - TT tetratype