Substrate specificity and adenosine triphosphatase activity of the ATP-dependent deoxyribonuclease of Bacillus subtilis

Studies on the specificity of the ATP-dependent DNase of Bacillus subtilis 168, carried out with pure enzyme at the optimal conditions for its action, have shown that the substrate is double-stranded linear DNA. Linear single-stranded DNA (separated strands of B. subtilis DNA and linear phage fd DNA) is not attacked, neither are there any circular forms (supercoiled or nicked simian virus 40 and circular single-stranded fd DNAs). The double-stranded DNA can be completely hydrolysed, the limit products being, almost exclusively, mononucleotides. The presence of terminal phosphate residues in the substrate (either at the 3' or the 5' end) is not necessary for enzyme action. This DNase appears therefore to be an exonuclease processively liberating mononucleotides from both strands of the native linear DNA. ATP (indispensable for the DNase reaction) is also hydrolysed by the enzyme, to ADP and inorganic orthophosphate (Pi) in the presence of DNA. The apparent Km for ATP, in the ATPase reaction, is 0.15 mM. At high ATP concentrations, which inhibit the DNase activity, there is activation of the ATPase reaction. Three molecules of ATP are consumed for each DNA phosphodiester bond split, at optimal conditions for DNase activity.     

Preparation of metaphase chromatin of Physarumpolycephalum without the loss of repressed RNA synthesis

A novel method for the preparation of intact chromatin from the slime mold $\text{Physarum}\text{polycephalum>}$ which retains the $\text{in}\text{vivo}$ property of RNA synthesis is described. Preparations from G₂-cells were highly active, while those from metaphase-cells were inactive. The plasmodial cells were disrupted by gentle homogenization on a polyethylene sieve in a neutral isotonic sucrose medium containing Mg⁺⁺, deoxycholate and EGTA, a Ca⁺⁺-chelating agent. The nuclei were lysed in a hypotonic buffer without use of EDTA and chromatin was precipitated by centrifugation after addition of Mg⁺⁺.     

Regulation of nitrogenase activity in aerobes by MG2+ availability: An hypothesis

Nitrogenase functions at or near its maximum capacity $\text{in}\text{vivo}$, despite a reported energy charge in the cell that should severely inhibit the enzyme. Deenergizing cellular membranes, which is postulated to release magnesium in mitochondria, has been reported to produce rapid inhibition of nitrogenase activity while giving only small changes in energy charge and $\text{NAD}{}^{\mathrm{+}}\text{NADH}$ ratio. It is proposed that the level of magnesium available for complexation by the potent inhibitor ADP is the rate controlling variable for nitrogenase activity.     

Isolation of mutants of Caulobacter crescentus deficient in ATP-dependent deoxyribonuclease activity

Caulobacter crescentus mutants sensitive to UV radiation, mitomycin C and methyl methane-sulfonate were isolated and tested for ATP-dependent deoxyribonuclease activity. Two mutants were identified of which one had less than 5 per cent of the wild-type level of the nuclease activity. This mutant in cross with another auxotrophic partner gave highly reproducible two-fold reduction in number of recombinants compared to a control cross. The suggested causes for reduced recombination frequency when one of the partners has residual ATP-dependent deoxyribonuclease activity are discussed.     

Isolation, subunit structure and properties of the ATP-dependent deoxyribonuclease of Bacillus subtilis. State of the protein in a mutant devoid of activity.

A prodcedure was developed for the purification of the ATP-dependent deoxyribonuclease of Bacillus subtilis 168. It comprises ammonium sulphate fractionation, Sephadex gel filtration, DEAE-cellulose chromatography and gel electrophoresis on a discontinuous polyacrylamide gradient. The enzyme has been obtained in a homogeneous state. Its molecular weight was estimated to be 270000 by disc electrophoresis. Dodecylsulfate-polyacrylamide gel electrophoresis showed the presence of five nonidentical subunits of the following molecular weights: 81000, 70000, 62000, 52500 and 42500. These values give 308000 as the molecular weight of the native enzyme. The pH optimum of the purified enzyme is 9.6. The optimal concentrations of Mg2+ and ATP for exonuclease activity on native B. subtilis DNA were determined. ATP-requirement for hydrolysis of single-stranded DNA is less strigent. The enzyme also possesses high DNA-dependent ATPase activity. The purification procedure was applied to extracts of a mutant devoid of activity for this enzyme (strain GSY 1290). A protein was isolated which is very similar to the active DNAase as regards electrophoretic mobility, reaction with specific antisera and size of four of the subunits. One subunit is missing (Mr 70000) and is replaced by a smaller polypeptide (Mr 565000). The latter results suggest that the mutant is affected in the genetic locus coding for the 70000-Mr subunit.     

Inhibition of repair of UV-damaged DNA by caffeine and mutation induction in Chinese hamster cells

The effect of caffeine on UV-irradiated Chinese hamster cells in vitro was studied on the cellular and molecular levels. Caffeine (1 mM) was shown to decrease the colony-forming ability and the frequencies of spontaneous and UV-induced mutations in Chinese hamster cells. The effect of caffeine in reducing the frequency of UV-induced mutations was demonstrated only if caffeine was present in the culture medium during the first post-irradiation cell division. Using alkaline sucrose gradient centrifugation, both parental and newly synthesized DNA in UV-irradiated and unirradiated cells were studied in the presence and absence of caffeine. Caffeine affected the sedimentation profile of DNA synthesized in UV-irradiated cells but not in unirradiated cells. Caffeine had no apparent effect on the incorporation of [³H]-thymidine into DNA of control or UV-irradiated cells, nor on the small amount of excision of UV-induced pyrimidine dimers. These results may be interpreted by a hypothesis that caffeine inhibits a certain S-phase specific, post-replication, dark-repair mechanism. The hamster and perhaps other rodent cells exposed to low doses of UV are capable of DNA replication, by-passing the non-excised pyrimidine dimers. This postulated repair process probably involves de novo DNA synthesis to seal the gaps in the nascent strand. This repair may be also responsible for the enzymatic production of mutations.     

Cell cycle regulation of DNA repair in normal and repair deficient human cells

The regulation of nucleotide excision repair and base excision repair by normal and repair deficient human cells was determined. Synchronous cultures of WI-38 normal diploid fibroblasts and Xeroderma pigmentosum fibroblasts (complementation group D) (XP-D) were used to investigate whether DNA repair pathways were modulated during the cell cycle. Two criteria were used: (1) unscheduled DNA synthesis (UDS) in the presence of hydroxyurea (HU) after exposure to UV light or after exposure to N-acetoxy-acetylaminofluorene (N-AcO-AAF) to quantitate nucleotide excision repair or UDS after exposure to methylmethane sulfonate (MMS) to measure base excision repair; (2) repair replication into parental DNA in the absence of HU after exposure to UV light. Nucleotide excision repair after UV irradiation was induced in WI-38 fibroblasts during the cell cycle reaching a maximum in cultures exposed 14–15 h after cell stimulation. Similar results were observed after exposure to N-AcO-AAF. DNA repair was increased 2–4-fold after UV exposure and was increased 3-fold after N-AcO-AAF exposure. In either instance nucleotide excision repair was sequentially stimulated prior to the enhancement of base excision repair which was stimulated prior to the induction of DNA replication. In contrast XP-D failed to induce nucleotide excision repair after UV irradiation at any interval in the cell cycle. However, base excision repair and DNA replication were stimulated comparable to that enhancement observed in WI-38 cells. The distinctive induction of nucleotide excision repair and base excision repair prior to the onset of DNA replication suggests that separate DNA repair complexes may be formed during the eucaryotic cell cycle.     

Nucleotide sequence of the Bacillus subtilis trpE and trpD genes

Several overlapping portions of the tryptophan (trp) operon of Bacillus subtilis have been cloned into plasmid pBR322. The nucleotide sequence of the region comprising the trpE and trpD genes and a portion of the trpC gene has been determined. When the deduced amino acid sequences of these genes are compared with their counterparts in Escherichia coli, several regions of striking homology are seen. The probable initiation codons for the trpE, D and C genes are each preceded by a recognizable Shine-Dalgarno sequence. The coding sequences for the trpE and trpD genes and for the trpD and trpC genes overlap slightly, leaving no intercistronic regions between the genes.     

Tissue distribution,binding properties and lesions produced by dehydroretronecine in the nonhuman primate

Four-month-old rhesus monkeys were injected with 65 mg of [³H]dehydroretronecine per kg body weight and sacrificed at 6, 12 and 24 h following injection. By the 24th h 13% of the dose had been eliminated in the urine. Although there were no feces, the extremely high radioactivity of the bile and intestinal contents suggested this route was a major one for the excretion of this compound. The³H was distributed throughout the body by the 6th h with the greatest percentage being in the skin and muscle. However, per gram of tissue the gastric mucosa and bile showed the highest radioactivity. Likewise, it was in the gastric mucosa that the lesions produced by dehydroretronecine were the most severe. High levels of radioactivity persisted in the gastric mucosal lysates after washings with trichloroacetic acid (TCA) while only a small percentage of the³H remained in the washed liver lysate. It was determined that over 20% of the³H was bound to mucosal protein and less than 1% to the nucleic acids.     

Influence of dietary cadmium on the distribution of the essential metals copper,zinc and iron in tissues of the rat

The effect of long-term dietary cadmium treatment upon the distribution of the metals copper, iron and zinc has been compared in various organs of male and female rats. The renal accumulation of cadmium was similar in both sexes without a plateau being reached. In contrast, the hepatic accumulation of cadmium was higher in the female than in the male rat and a plateau was observed after 30–35 weeks of dietary cadmium treatment. Most of the cadmium which accumulated in these organs was recovered in the metallothionein fraction and the concentration of hepatic cadmiumthionein in the female rat was correspondingly higher than in the male rat. Accumulation of cadmium was associated with an increased zinc concentration in the liver and an increased copper concentration in the kidney; these increases were correlated with increases in liver and kidney metallothioneins induced by cadmium. Uptake of cadmium into organs other than liver and kidney occurred to a small extent but was not associated with changes in the concentration of copper and zinc. Cadmium also accumulated in the intestinal mucosa where it could be recovered in a fraction corresponding to metallothionein. A loss of iron from the liver and kidney was also observed following dietary cadmium treatment and involved mainly a loss of iron from ferritin.     

A method of DNA quantitation for localization of DNA in metrizamide gradients.

A quantitative assay for DNA is described that involves the binding of the cytologicalstain, methyl green, to DNA. At pH 7.9, solutions containing free methyl green undergo complete fading whereas solutions containing DNA-bound methyl green retain color in proportion to the amount of DNA present. The procedure allows for the quantitation of DNA in the presence of urea, sucrose, EDTA, protein, dithiothreitol, metrizamide, and low-concentration salts. This method is also applicable to the quantitation of DNA in chromatin.     

Affinity labelling of alpha-adrenoceptors in intact liver cells by [3H]phenoxybenzamine.

[³H]phenoxybenzamine of high specific activity (5.3 Ci/mmol) was synthesized and its binding to isolated, viable rat liver cells was studied. Phentolamine suppressible binding of [³H]phenoxybenzamine was irreversible and saturable (EC50: 10 nM, b_max: 200 fmol/mg wet cell weight). Competition-inhibition studies showed structural and stereoselectivity compatible with α-receptors. The IC50 of unlabelled phenoxybenzamine to reduce specific binding (9 nM) or to block adrenaline-induced phosphorylase activation in the same cells (2 nM) was similar, whereas the IC50 of agonists to suppress binding was higher than their EC50's for phosphorylase activation. The results represent the first example of labelling α-adrenoceptors in intact liver cells. The sites labelled by [³H]phenoxybenzamine mediate the block of phosphorylase activation by α-adrenoceptor antagonists. However, the relationship of these sites to receptors that mediate responses to physiological, low concentrations of catecholamines remains to be clarified.     

The Golgi complex. III. The effects of puromycin on ultrastructure and glycoprotein synthesis.

The effect of puromycin has been investigated on protein and glycoprotein synthesis and on ultrastructure of the Golgi complex from rat liver. Incorporation of [14C]leucine into protein in Golgi fractions and into serum proteins was depressed rapidly after puromycin treatment. In the serum proteins, incorporation returned to normal levels at 2 h whereas in Golgi fractions it continued to rise to 200% of the control levels at 3 h and was still elevated at 24 h after puromycin treatment. Incorporation of [14C]glucosamine into glycoprotein was depressed in Golgi and serum fractions in a similar manner but slightly later than that of leucine. Leucine labelled material found at 3 h was a poor acceptor for carbohydrate, since [14C]glucosamine incorporation was not elevated above control values. Galactosyl transferase activity was not depressed in the Golgi membranes and, at 3 h, was elevated implying that an adequate supply of enzyme was available at all times. The activity of the galactosyl transferase in serum appeared to be depressed suggesting that transport of enzyme from Golgi complex to serum was defective. Ultrastructural changes in the Golgi complex were observed to occur rapidly after puromycin treatment. The cisternae became irregular, compressed, and degenerated progressively from central region towards the periphery. Irregular tubular structures formed at the expense of cisternal membrane and showed accumulation of low density lipoprotein. Vesiculation and degenerative changes of the Golgi membranes continued from 2-12 h while more typical arrangements of the Golgi complex were observed between 24-48 h. The morphological changes correlated with changes in glycoprotein synthesis.     

Altered regulation of macromolecular synthesis in methionine-inhibited cultures of Pseudomonas fluorescens UK1.

Effects of high methionine concentrations on growth of Pseudomonas fluorescens UK1 are reported. The following phenomena were observed: (i) Immediate inhibition of growth for a period corresponding to approximately half a generation. Steady-state conditions of growth were no more attained. (ii) In spite of stringency of the macromolecular synthesis in this organism, simultaneously with the growth inhibition, the rate of labelled leucine incorporation into trichloroacetic acid (TCA)-insoluble material was reduced 60% while the rate of labelled uracil incorporation remained constant. (iii) The organism began to liberate methanethiol half a generation after the methionine supplement. Demethiolating activity increased linearly with the cell mass. It is concluded that the inhibition of growth is not due to the liberation of methanethiol from methionine but the amino acid is able to uncouple the mechanism coordinating protein and RNA synthesis in P. fluorescens UK1.     

Variations in activity of aminoacyl-tRNA synthetases as a function of development in Bacillus subtilis

Extracts from Bacillus sublilis cells at various stages of growth and spores were assayed for aminoacyl-tRNA synthetase and methionyl-tRNA transformylase activity. There was no major change in any synthetase activity or in methionyl-tRNA transformylase activity during the sporulation cycle, which implies that these are not sporulation induced enzymes. However, extracts from B. subtilis cultures showed a burst of activity of aminoacyl-tRNA synthetases during exponential growth.Preparations from dormant spores possessed the same kinds of aminoacyl-tRNA synthetase activities as vegetative cells for all the amino acids which were studied. Spores also contained methionyl-tRNA transformylases. These findings suggest that spores ought to be able to aminoacylate tRNA and formylate the initiator. N-formylmethionyl-tRNA, immediately upon germination.     

A protein-bound form of thioacetamide in liver nucleoli

The cellular distribution of ³⁵S from ³⁵S- thioacetamide was determined in rabbit liver subcellular fractions following its $\text{in vivo}$ administration. Of the various fractions isolated, only the nucleolar fraction contained ³⁵S counts that were insoluble in 10% trichloroacetic acid but soluble in trichloroacetic acid if the fraction was treated with trypsin but not RNase or DNase. These results demonstrate that a protein bound form of thioacetamide is present in the nucleolus following $\text{in vivo}$ administration of this drug.