Polyadenylic acid synthesis activity of purified DNA-dependent RNA polymerase from Caulobacter

Characterization of purified DNA-dependent RNA polymerase (EC 2.7.7.6) of Caulobacter crescentus, strain CB15 has led to the conclusion that this enzyme catalyzes poly(A) synthesis in the absence of template. Poly(A) synthetase activity co-purifies with both holoenzyme and core polymerase on DNA-cellulose columns, and core polymerase purified to 98% homogeneity by glycerol gradient centrifugation is still capable of catalyzing poly(A) polymerization. Both RNA synthesis and poly(A) polymerization activities are sensitive to rifampicin. In addition, RNA polymerase purified from partially rifampicin-sensitive mutants exhibits the same partial sensitivity in vitro to the drug in the synthesis of RNA and poly(A). The enzyme used in these studies was prepared by a simple method which allows a high yield of pure RNA polymerase from large batches of exponential cells. The procedure includes high speed centrifugation of cell extracts, DEAE-cellulose column, DNA-affinity chromatography, and low salt glycerol gradient centrifugation. Holoenzyme can be resolved into core and sigma subunit by either DNA-cellulose chromatography or glycerol gradient centrifugation, and the latter step allows recovery of pure sigma factor.     

Site-directed mutagenesis of glutathione S-transferase YaYa. Important roles of tyrosine 9 and aspartic acid 101 in catalysis.

The roles of tyrosine 9 and aspartic acid 101 in the catalytic mechanism of rat glutathione S-transferase YaYa were studied by site-directed mutagenesis. Replacement of tyrosine 9 with phenylalanine (Y9F), threonine (Y9T), histidine (Y9H), or valine (Y9V) resulted in mutant enzymes with less than 5% catalytic activity of the wild type enzymes. Kinetic studies with purified Y9F and Y9T mutants demonstrated poor catalytic efficiencies which were largely due to a drastic decrease in kcat. The estimated pK alpha values of the sulfhydryl group of glutathione bound to Y9F and Y9T mutant enzymes were 8.5 to 8.7, similar to the chemical reaction, in contrast to the estimated pK alpha value of 6.7 to 6.8 for the glutathione enzyme complex of wild type glutathione S-transferase. These results indicate that tyrosine 9 is directly responsible for the lowering of the pKa of the sulfhydryl group of glutathione, presumably due to the stabilization of the thiolate anion through hydrogen bonding with the hydroxyl group of tyrosine. To examine the role of aspartic acid in the binding of glutathione to YaYa, 4 conserved aspartic acid residues at positions 61, 93, 101, and 157 were changed to glutamic acid and asparagine. All mutant enzymes retained either full or partial activity except D157N, which was virtually inactive. Kinetic studies with four mutant enzymes (D93E, D93N, D101E, and D101N) indicate that only D101N exhibited a 5-fold increase in Km toward glutathione. Also, the binding of this mutant to the affinity column was greatly reduced. These results demonstrate that aspartic acid 101 plays an important role in glutathione interaction to YaYa. The role of aspartic acid 157 in catalysis remains to be determined.     

Differing macrophage and lymphocyte roles in resistance to Legionella pneumophila infection.

Similar to guinea pig macrophages and human monocytes, macrophages from the peritoneal cavity of thioglycolate pretreated A/J mice are permissive for growth of Legionella pneumophila. In contrast, macrophages from BDF1 mice are not permissive for L. pneumophila. Lymphocytes from A/J and BDF1 mice proliferated in response to Legionella Ag but guinea pig lymphocytes did not. Also, splenocyte cultures from A/J mice treated with either Con A or Legionella vaccine produced supernatants which induced A/J macrophages to restrict Legionella growth, but guinea pig splenocyte culture supernatants obtained after stimulation with L. pneumophila vaccine did not induce Legionella growth restriction activity by guinea pig macrophages. Murine rIFN-gamma but not rIFN-alpha markedly inhibited growth of Legionella in A/J mouse macrophages and monoclonal anti-IFN-gamma antibody neutralized the anti-Legionella activity of culture supernatants from A/J mouse splenocytes responding to Legionella Ag. From these data, IFN-gamma appears to be an important factor in anti-Legionella activity of Ag-activated mouse splenocyte culture supernatants. Cyclosporin A, when given to either A/J or BDF1 mice, reduced the proliferation responses of splenocytes to T cell mitogens and also decreased the IFN production of A/J spleen cells to Legionella Ag. In addition, drug treatment decreased the resistance of A/J mice to Legionella infection as shown by an increase in the number of viable bacteria in the liver. However, injection of drug treated mice with lymphokine-rich splenocyte culture supernatant reconstituted the resistance of these animals. These results suggest an important role for lymphocyte activation and lymphokine production in the resistance of A/J mice to Legionella infection. The greater resistance of BDF1 mice, however, may result from nonpermissive macrophages and responsive lymphocytes. In the case of guinea pigs, susceptibility to Legionella infections may result from both the permissive nature of the macrophages and the relatively unresponsive nature of the lymphocytes in these animals.     

Resolution of Holliday junction analogs by T4 endonuclease VII can be directed by substrate structure

Endonuclease VII is an enzyme from bacteriophage T4 capable of resolving four-arm Holliday junction intermediates in recombination. Since natural Holliday junctions have homologous (2-fold) sequence symmetry, they can branch migrate, creating a population of substrates that have the branch point at different sites. We have explored the substrate requirements of endonuclease VII by using immobile analogs of Holliday junctions that lack this homology, thereby situating the branch point at a fixed site in the molecule. We have found that immobile junctions whose double-helical arms contain fewer than nine nucleotide pairs do not serve as substrates for resolution by endonuclease VII. Scission of substrates with 2-fold symmetrically elongated arms produces resolution products that are a function of the particular arms that are lengthened. We have confirmed that the scission products are those of resolution, rather than nicking of individual strands, by using shamrock junction molecules formed from a single oligonucleotide strand. A combination of end-labeled and internally labeled shamrock molecules has been used to demonstrate that all of the scission is due to coordinated cleavage of DNA on opposite sides of the junction, 3' to the branch point. Endonuclease VII is known to cleave the crossover strands of Holliday junctions in this fashion. The relationship of the long arms to the cleavage direction suggests that the portion of the enzyme which requires the minimum arm length interacts with the pair of arms containing the 3' portion of the crossover strands on the bound surface of the antiparallel junction.     

Importance of domain closure for homotropic cooperativity in Escherichia coli aspartate transcarbamylase

The importance of the interdomain bridging interactions observed only in the R-state structure of Escherichia coli aspartate transcarbamylase between Glu-50 of the carbamoyl phosphate domain with both Arg-167 and Arg-234 of the aspartate domain has been investigated by using site-specific mutagenesis. Two mutant versions of aspartate transcarbamylase were constructed, one with alanine at position 50 (Glu-50----Ala) and the other with aspartic acid at position 50 (Glu-50----Asp). The alanine substitution totally prevents the interdomain bridging interactions, while the aspartic acid substitution was expected to weaken these interactions. The Glu-50----Ala holoenzyme exhibits a 15-fold loss of activity, no substrate cooperativity, and a more than 6-fold increase in the aspartate concentration at half the maximal observed specific activity. The Glu-50----Asp holoenzyme exhibits a less than 3-fold loss of activity, reduced cooperativity for substrates, and a 2-fold increase in the aspartate concentration at half the maximal observed specific activity. Although the Glu-50----Ala enzyme exhibits no homotropic cooperativity, it is activated by N-(phosphonoacetyl)-L-aspartate (PALA). As opposed to the wild-type enzyme, the Glu-50----Ala enzyme is activated by PALA at saturating concentrations of aspartate. At subsaturating concentrations of aspartate, both mutant enzymes are activated by ATP, but are inhibited less by CTP than is the wild-type enzyme. At saturating concentrations of aspartate, the Glu-50----Ala enzyme is activated by ATP and inhibited by CTP to an even greater extent than at subsaturating concentrations of aspartate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electrochemical cells for voltammetry, coulometry, and protein activity assays of small-volume biological samples

Cell designs, experimental protocols, and results for electrochemical investigation of small quantitites of biological materials under anaerobic conditions are reported. Three types of electrochemical experiments are considered: (i) cyclic voltammetry of 20- to 100-microliters samples; (ii) direct coulometry of 0.5- to 1.5-ml samples; and (iii) an electrochemically initiated protein activity assay which includes provision for analysis of gaseous reaction products and correlation with electron flux. The first two procedures are illustrated by measurement of the formal electrode potential (E0') and number of electrons transferred (n) in redox reactions of small quantities of biological and inorganic materials. The third procedure is illustrated by assaying the activity of the MoFe protein plus Fe protein complex from Azotobacter vinelandii nitrogenase for reduction of C2H2 to C2H4.     

Pattern of growth in weight of alate and apterous nymphs of the English grain aphid, Sitobion avenae

Daily increase in fresh weight was recorded for apterous and alate nymphs of S. avenae at 20°C. Comparison with a control group indicated that daily disturbance and weighing of nymphs did not affect significantly their growth, developmental time or survival. The increase in fresh weight of apterous and alate virginoparae at 20°C was best described by logistic equations. Alate virginoparae were significantly heavier than apterous virginoparae at birth and throughout most of their nymphal life, but they experienced a weight loss at the final ecdysis. The relative growth rate did not remain constant, but declined during development. The decline is associated with a decline in honeydew production per unit body weight. The implications of an inconstant relative growth rate and the marked loss in weight at the adult moult in alates are discussed.

---

Résumé L'enregistrement de l'augmentation quotidienne du poids frais à 20°C des larves ailées et aptères de S. avenae a montré que des perturbations quotidiennes n'affectent pas significativement la croissance, la durée du développement et la survie. Les équations logistiques décrivent plus exactement l'augmentation de poids frais des aptères et des ailés virginipares à 20°C. Les virginipares ailés étaient significativement plus lourds que les virginipares aptères à la naissance et pendant la plus grande partie de la vie larvaire, mais présentaient une perte de poids à la mue finale. Le taux de croissance relative ne restait pas constant, mais diminuait au cours du développement. La diminution était associée à une diminution de la production de miellat par unité de poids du corps. La discussion porte sur les conséquences de la variation de l'augmentation du poids relatif et de la perte marquée de poids à la mue imaginale.

     

Red-coloured snow algae in svalbard — Some environmental factors determining the distribution of Chlamydomonas nivalis (Chlorophyta volvocales)

Summary Studies of colonization of snow slopes by the snow algae Chlamydomonas nivalis (Chlorophyta volvocales) show that various environmental factors can be correlated with regions of algal colonization. All colonized sites discovered faced west, and when transects were made across the slopes it was found that areas of maximum colonization showed minima in temperature and pH and maxima of conductivity and chloride ion concentration.Svalbard Expedition (1980) Perse School, Cambridge     

Iron-molybdenum cofactor from nitrogenase. Modified extraction methods as probes for composition

Five modifications of the preparative procedure for isolating iron-molybdenum cofactor (FeMoco) from the molybdenum-iron (MoFe) protein of Azotobacter vinelandii nitrogenase have been developed. This variety of isolation methods has established that no single component of the original isolation protocol, i.e. Tris, Cl-, citrate, HPO4(2-), N,N-dimethylformamide, and N-methylformamide, is essential for the effective isolation and/or structural stability of FeMoco, although any of them may act as ligands to FeMoco when present. The acid-bse status (effective pH) of the extracting solvent is a key adjustable parameter in the isolation procedure. The new procedures produced FeMoco with yields, metal analysis, charge, EPR spectrum, and specific activity (after reconstituting crude extracts from A. vinelandii UW45 mutant cells) essentially identical with FeMoco isolated by the original procedure. After purification, FeMoco apparently contains molybdenum, iron, and sulfide in a 1:7:4 ratio with N-methylformamide as a ligand but no amino acid residues, common sugars, coenzyme A, or lipoic acid. Reaction with o-phenanthroline allows quantitation of both adventitious and FeMoco-associated iron. Correlations of total activity after UW45 reconstitution with molybdenum, total iron, and o-phenanthroline-resistant iron contents show that only the last gives a consistent relationship of 35 +/- 5 nmol of C2H4/min/ng atom of Fe. Both o-phenanthroline and EDTA interact with FeMoco to abolish its EPR signal in reactions reversible by additions of Fe2+ or Zn2+, respectively. These and related reactions point against the presence of an endogenous organic component in FeMoco and toward the presence of exogenous ligands and imply a relatively labile coordination sphere whose nature may be determinable by a systematic investigation.