Genetic analysis and molecular cloning of the Escherichia coli ruv gene

Summary The genetic organisation of the ruv gene, a component of the SOS system for DNA repair and recombination in Escherichia coli, was investigated. New point mutations as well as insertions and deletions were generated using transposon Tn10 inserted in eda as a linked marker for site specific mutagenesis, or directly as a mutagen. The mutations were ordered with respect to one another and previously isolated ruv alleles by means of transductional crosses. The direction of chromosome mobilization from ruv:: Mud(Ap^R lac)strains carrying F42lac ⁺ established that ruv is transcribed in a counterclockwise direction. Recombinant phages able to restore UV resistance to ruv mutants were identified, and the ruv ⁺ region was subcloned into a low copy number plasmid. The ruv ⁺ plasmid was able to correct the extreme radiation sensitivity and recombination deficiency of ruv recBC sbcB strains.     

Rapid Activation of Tyrosine Hydroxylase in Response to Nerve Growth Factor

Abstract: Nerve growth factor protein (NGF) was found to rapidly promote the activation of tyrosine hydroxylase in cultured rat PC 12 pheochromocytoma cells. PC 12 cultures were exposed to NGF for periods of less than 1 h and the soluble contents of homogenates prepared from the cells were assayed for tyrosine hydroxylase activity. Under these conditions, the specific enzymatic activity was increased by 60 ± 10% (n = 13) in comparison with that in untreated sister cultures. The increase was half maximal by 2–5 min of exposure and at NGF concentrations of about 10 ng/ml (0.36 n M ). Antiserum against NGF blocked the effect. Tyrosine hydroxylase activity could also be rapidly increased by NGF in cultures of PC12 cells that had been treated with the factor for several weeks in order to produce a neuron-like phenotype. This was achieved by withdrawing NGF for about 4 h and then readding it for 30 min. The NGF-induced increase of tyrosine hydroxylase activity in PC12 cultures was not affected by inhibition of protein synthesis and therefore appeared to be due to activation of the enzyme. Kinetic experiments revealed that NGF brought about no change in the apparent K_m of the enzyme for tyrosine or for co-factor (6-methyltetrahydropteridine), but that it did significantly increase the apparent maximum specific activity of the enzyme. These observations suggest that NGF (perhaps released by target organs) could promote a rapid and local enhancement of noradrenergic transmission in the sympathetic nervous system.     

Effects of fire,topography and year-to-year climatic variation on species composition in tallgrass prairie

Native unploughed tallgrass prairie from Konza Prairie, Kansas, USA is described with respect to plant species compositional changes over a five year period in response to fire and topography. The principal gradient of variation in the vegetation is related to time since burning. Species show an individualistic response in terms of relative abundance to this gradient. Both the percentage of and cover of C₄ species and all grasses decrease as the prairie remains unburnt. Forb and woody plant species numbers and abundance increase along this gradient. A secondary gradient of variation reflects topography (i.e. upland versus lowland soils). Upland soils support a higher species richness and diversity. Upland and lowland plant assemblages are distinct except on annually burnt prairie. The interaction between burning regime, topography and year-to-year climatic variation affects the relative abundance of the plant species differentially. The most dominant species overall, Andropogon gerardii, was affected only by year-to-year variation (i.e. climate). Its position at the top of the species abundance hierarchy was unaffected by burning regime or soil type. The other dominant species showed a suite of varying responses to these factors.Deceased May, 1986.     

Methanogenesis from methanol in Methanosarcina barkeri studied using membrane inlet mass spectrometry

Abstract A mass spectrometer with membrane inlet was used to study methanol metabolism by Methanosarcina barkeri strain MS. The addition of methanol to methanol grown culture samples in the mass spectrometer vessel stimulated methanogenesis and hydrogen production. The apparent K _s for methanol was determined as 0.5 mM and the V _max as 8.14 mmol g (dry weight) h⁻¹. The V _max for methane production was fairly constant during growth of the culture on methanol implying that growth is tightly coupled to methanogenesis. The addition of methanol to culture samples in the mass spectrometer vessel stimulated methanogenesis with no lag which indicated that methanogenesis can be uncoupled from growth. Exposure of the culture sample in the mass spectrometer vessel to an atmosphere of 2 kPa oxygen for 80 min resulted in a decrease in the rate of methanogenesis from methanol but on returning the atmosphere to nitrogen the addition of further methanol stimulated methanogenesis. The effect of other inhibitors of methanogenesis (2-bromoethane sulphonate and monensin); K _j values 21.5 μM and 0.3 mM, respectively) were also studied.     

Murine epidermal growth factor: heterogeneity on high resolution ion-exchange chromatography

We have shown that epidermal growth factor (EGF) purified either by the classical method of Savage and Cohen, or solely by h.p.l.c. techniques can be resolved into two species, EGF alpha and EGF beta. However, despite the apparent purity of such materials, as determined both chromatographically and by amino acid analysis, they failed to give homogeneous products on radioiodination. Analysis by isoelectric focusing on agarose gels followed by transfer to nitrocellulose and silver staining showed that EGF alpha could be further resolved into three sub-species which focused at pH 4.6, 4.3 and 4.1. EGF beta (which also focused at pH 4.6) contained very small amounts of the species with isoelectric points of 4.1 and 4.3, probably due to slight contamination of this preparation by EGF alpha. Preparative separation of the sub-species of EGF alpha was achieved by high performance anion-exchange chromatography at pH 6.5 on a Pharmacia Mono Q column. Radioiodination of these purified sub-species did not produce significant charge heterogeneity. However, two slightly different forms of [125I]EGF alpha 1 (pH 4.6 species) were separable by anion-exchange chromatography on the Mono Q column. All of the EGF species competed for binding to EGF receptors on A431 cells and were active mitogens for BALB/c 3T3 fibroblasts.     

New Class of Streptomycin-Resistant Mutants Incompatible with supX Suppressor Mutations in Salmonella typhimurium

Streptomycin-resistant colonies of Salmonella typhimurium appearing in platings of supX suppressors of strain leu-500 are less variegated in size than are those derived from strain leu-500 counterparts. Several of the streptomycin-resistant leu-500 clones, furthermore, yield suppressors and revertants of the leu-500 auxotrophy at unusually low rates, suggesting that they provide a genetic background inimicable to supX suppression. Two such "suppression-restrictive" leu-500 streptomycin-resistant (str) mutants, designated strains M(1) and M(4), were characterized as to their ability to receive the trp-supX-cysB linkage region by transduction. Coentry of a donor supX deletion mutation with the selected trp(+) marker was not observed even though these sites display more than 10% linkage in control experiments. This was demonstrably the result of nonviability of the combined supX mutant, M(1) or M(4) streptomycin-resistant genotype, rather than the lack of suppression of the leu-500 imparted auxotrophy. Both M(1)- and M(4)-type resistance was accompanied by pleiotropic effects resembling those caused by strB (nonribosomal)- rather than strA (ribosomal)-type resistance, but both restrictive mutants had a high upper limit of resistance corresponding to that of strA-type mutants. Transduction analyses indicated that the str character of neither the M(1) nor the M(4) strain was linked to the strA or the strB gene. These mutations define a previously undescribed locus, which we propose to designate strC, apparently related to streptomycin uptake rather than its intracellular action. Mutation at this locus is evidently incompatible with the inactivation or removal of the supX site, suggesting a functional association between products of the genes.     

Some novel flavin-nicotinamide biscoenzymes and their reactivities

The present study was undertaken to investigate flavin-nicotinamide reactions and interactions. A series of novel flavin-nicotinamide biscoenzymes have been synthesized by a general three-step procedure. The structures of these compounds were confirmed by nuclear magnetic resonance spectra, absorption spectra and elemental analysis. These compounds consist of short linear hydrocarbon chains interconnecting the N-1 of nicotinamide and the N-10 of the 7,8-dimethyl-isoalloxazine ring. The compounds were reduced with sodium dithionite (Na₂S₂O₄) and the flavin portion was reoxidized with ferricyanide. Re-reduction of the flavin portion by the nicotinamide portion of the molecule was followed anaerobically at 442 nm. When the interconnecting hydrocarbon chain was unsaturated, a second order reaction was observed with a rate equal to that of lumiflavin and 1-propyl-1,4-dihydronicotinamide (NprNicH₂) under the same conditions. When the two halves of the biscoenzymes were connected by saturated three- and four-carbon chains, the expected unimolecular reaction was not observed. Instead, the reduced biscoenzyme, after separation from excess sodium dithionite, was shown to have a strong absorption at 298 nm. This absorption is characteristic of hydration of dihydronicotinamides at the 5,6-double bond.In further studies, the C₃-biscoenzyme exhibited an absorption at 600 nm due to a complex between the reduced flavin and oxidized nicotinamide portions of the molecule. Absorbance at 600 nm increased linearly with the C₃-biscoenzyme concentration, clearly indicating that this is an intramolecular complex. When the C₃-biscoenzyme was at 0°C in 60–75% dimethylformamide buffer solution, no absorption at 600 nm was observed. When excess dithionite was removed, the spectrum under these conditions showed definite peaks at 297 and 357 nm. These respective peaks were attributed to hydrated dihydronicotinamide and dihydronicotinamide species present in the reaction mixture.The reduced flavin was postulated to be a catalyst for the hydration of dihydronicotinamide. This hypothesis was tested by incubating 1-propyl-1,4-dihydronicotinamide alone and with several concentrations of reduced riboflavin under basic anaerobic conditions. The results show that the reduced flavin increases the rate of disappearance of the dihydronicotinamide species and that the product shows an absorption near 298 nm. These results indicate that a reduced form of the flavin nucleus catalyzes the hydration of dihydronicotinamides.     

Isolation of nuclear acidic proteins from rat tissues. Characterization of acetylated liver nuclear acidic proteins

Nuclear acidic proteins isolated from rat brain, heart, kidney and liver showed similar, complex patterns on electrophoresis in sodium dodecyl sulphate-polyacrylamide gels. The contamination of nuclear acidic proteins by nuclear-membrane acidic proteins was found to the extent of 11%. Incorporation of [(3)H]acetate into the various nuclear acidic proteins in vivo, which were fractionated by polyacrylamide-gel electrophoresis, differed from tissue to tissue. Hydrolysis of these acetylated nuclear acidic proteins with 6m-HCl at 110 degrees C released 70% of the radioactivity, which indicated that labile acetyl groups had been incorporated into these proteins. Analysis of [(3)H]acetate-labelled nuclear acidic proteins revealed two acetylated amino acid residues, N(2)-acetylserine and N(2)-acetyl-lysine. The significance of the role played by nuclear acidic proteins in relation to gene regulation is discussed.