Transforming Activity of Deoxyribonucleic Acid labelled with 5-Bromouracil

Abstract

Ephrati-Elizur and Zamenhof¹ demonstrated that deoxyribonucleic acid from Bacillus subtilis grown in 5-bromouracil-supplemented media contained this analogue and retained its transforming activity. It thus became important to determine the specific transforming activity for purified fractions of each of the three molecular variants of deoxyribonucleic acid which can be extracted from organisms treated with 5-bromouracil : (1) unlabelled deoxyribonucleic acid molecules, and those in. which 5-bromouracil has been substituted for thymine in (2) one strand (‘unifilar’ labelling) or in (3) both strands (‘bifilar’ labelling) of the double helix. Two strains of Bacillus subtilis, wild type² and uracil-requiring strain 265 (kindly supplied by Dr. R. Guthrie), served as the donors of deoxyribonucleic acid. These were first grown with aeration at 37° C. for 4 hr. in nutrient broth supplemented with thymidine (10 µgm. per ml.), and afterwards incubated for 2–3 generations in minimal broth² containing 2–5 µgm. 5-fluorodeoxyuridine (serving as the thymidylic acid synthetase inhibitor³), 25 µgm. uridine (to suppress the conversion of 5-fLuorodeoxyuridine to 5-fluorouracil and 5-fluorouridine) and 50–250 µgm. 5-bromodeoxyuridine per ml. Under these conditions, similar to those first employed for Escherichia coli by Lorkiewicz and Szybalski⁴, the molar ratio of 5-bromouracil to 5-bromouracil + thymine (5-bromouracil substitution) in non-fractionated deoxyribonucleic acid varied from 35 to 61 per cent as assessed by Chromatographic determination⁵.