Fluorinated Pyrimidines, A New Class of Tumour-Inhibitory Compounds

The Determination of Nucleic Acids

The properties of an extremely thermophilic bacterium isolated from water at a Japanese hot spring and previously named Flavobacterium thermophilum are described. The cells are gram-negative, nonsporulating, aerobic rods containing yellow pigment. The optimum temperature for growth is between 65 and 72 C, the maximum being 85 C and the minimum being 47 C. The guanine plus cytosine content of the deoxyribonucleic acid of the thermophile is 69 mol %. This microorganism is sensitive to various antibiotics including those which are known to be rather ineffective against gram-negative bacteria. Spheroplast-like bodies are formed upon treating intact cells with egg-white lysozyme at 60 C. The spheres are osmotically more stable than mesophile protoplasts, and their rupture under hypotonic conditions is not complete unless 0.5% Brij 58 is added to the suspension. Bulk protein extracted from this thermophile is much more stable to heat than mesophile proteins, and only about 10% of the total protein is denatured by heating at 110 C for 5 min. Nevertheless, the amino acid composition of the bulk protein is similar to that of mesophile proteins. As the properties of this organism are similar to those of Thermus aquaticus (Brock and Freeze) and inasmuch as Flavobacterium is a poorly defined genus, this thermophilic microorganism is transferred to the genus Thermus as T. thermophilus (Yoshida and Oshima) comb. nov. The type strain is HB8 (=ATCC 27634).

Description of Thermus thermophilus (Yoshida and Oshima) comb. nov., a Nonsporulating Thermophilic Bacterium from a Japanese Thermal Spa

A single-stranded deoxyribonucleic acid from bacteriophage φX174

The synthesis of methidiumpropyl-EDTA (MPE) is described. The binding affinities of MPE, MPE•Ni(II), and MPE•Mg(II) to calf thymus DNA are 2.4 X 10^4 M^(-1), 1.5 X 10^5 M^(-1), and 1.2 X 10^5 M^-1), respectively, in 50 mM NaCl, pH 7.4. The binding site size is two base pairs. MPE•Mg(II) unwinds PM2 DNA 11 ± 3° per bound molecule. MPE•Fe(II) in the presence of O_2 efficiently cleaves DNA and with low sequence specificity. Reducing agents significantly enhance the efficiency of the cleavage reaction in the order sodium ascorbate > dithiothreitol > NADPH. At concentrations of 0.1-0.01 µM in MPE•Fe(II) and 10 µM in DNA base pairs, optimum ascorbate and dithiothreitol concentrations for DNA cleavage are 1-5 mM. Efficient cleavage of DNA (10 µM in base pairs) with MPE•Fe(II) (0.1-0.01 µM) occurs over a pH range of 7-10 with the optimum at 7.4 (Tris-HC1
buffer). The optimum cleavage time is 3.5 h (22 °C). DNA cleavage is efficient in a Na^+ ion concentration range of 5 mM to 1 M, with the optimum at 5 mM NaCl. The number of single-strand scissions on supercoiled DNA per MPE•Fe(II) under optimum conditions is 1.4. Metals such as Co(II), Mg(II), Ni(II), and Zn(II) inhibit strand scission by MPE. The released products from DNA cleavage by MPE•Fe(II) are the four nucleotide bases. The DNA termini at the cleavage site are 5'-phosphate and roughly equal proportions of 3'-phosphate and 3'-(phosphogiycolic acid). The products are consistent with the oxidative degradation of the deoxyribose ring of the DNA backbone, most likely by hydroxy radical.

Cleavage of DNA with methidiumpropyl-EDTA-iron(II): reaction conditions and product analyses

Microdetermination of purines and pyrimidines in biological materials.

Evidence for a nuclear precursor of ribo- and desoxyribonucleic acid

Specific activity of P32 in cell constituents of rabbit liver.

Purine and pyrimidine content of the nucleic acids of nuclei and cytoplasm.

Dubos and Davis (1946) and Davis and Dubos (1947) have shown that Mycobacterium tuberculosis gives uniform growth throughout a liquid nutrient medium in the presence of a wetting agent, sorbitan monooleate, and albumin. It has become possible, therefore, to use turbidimetry for the quantitative study of the growth of this bacillus. The experiments to be reported here show that the growth of Mycobacterium tuberculosis in the Dubos medium (Dubos and Middlebrook, 1947) can be followed quantitatively by turbidimetry. With the procedures described it has been possible to analyze the role of glucose as a carbon source and of ammonium chloride as a nitrogen source for the growth of tubercle bacilli. It has been found that under certain conditions, i.e., with glucose available but with ammonium chloride limiting, autolysis of these bacteria occurs, and also that, when the rapidly growing bacteria are deprived of ammonium chloride, the turbidity of the culture will continue to increase for a time in the absence of an external supply of nitrogen. The human tubercle bacillus, strain H37Rv,' was used in all these experiments.

Alfred Marshak

Papers

Microdetermination of purines and pyrimidines in biological materials.

Evidence for a nuclear precursor of ribo- and desoxyribonucleic acid

Specific activity of P32 in cell constituents of rabbit liver.

Purine and pyrimidine content of the nucleic acids of nuclei and cytoplasm.

The growth of mycobacterium tuberculosis as a function of its nutrients