The intramolecular heterogeneity of Bacillus subtilis DNA studied by isopycnic CsCl gradient

BIOCHIMICA ET BIOPHYSICA ACTA

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SHORT C O M M U N I C A T I O N S BBA 93530

The intramolecular heterogeneity of Bacillus subtilis D N A studied by isopycnic CsCI 9radient The intramolecular heterogeneity of Bacillus subtilis DNA or chromosome has been studied from various aspects. MUNAKATAet al. 1 compared the relative sensitivities of several genetic markers to ultraviolet irradiation, and SAITO AND MASAMUNE2 compared the relative elution profiles of several genetic markers from methylated albumin-kieselguhr columns. The heterogeneity was also studied by the buoyant density method s-5. Although the last is the most direct method for solving the heterogeneity problem, it appeared to us that the information obtained from these studies is still insufficient for the reasons that the markers examined are still limited and the resolvability of the analytical method used is not sharp. This communication describes the distribution of various marker activities in the profile of CsC1 density gradient centrifugation. To increase the resolving ability, an angle rotor was used for the isopycnic CsC1 gradient analysis, and to enable an accurate evaluation of obtained results, an auxotrophic strain with seven genetic markers was used as the recipient in transforming experiments. 14C-labeled DNA was prepared from B. subtilis 168 thy str-r grown in the enriched minimal medium e supplemented with o.oi #C of [uC~thymidine per ml and I #g of unlabeled thymidine per ml according to the method of SAITO AND MIURA7. 25 #g of the DNA per ml were dissolved in standard saline citrate and passed through a No. 27 hypodermic needle 7 times for shearing. The DNA pieces thus prepared were highly uniform in their size and had an average molecular weight of 0. 9 • lOT (ref. 8). For pycnographic fractionation, 5O-lOO #g of the sheared DNA were dissolved in 3.3 ml of o.oi M Tris-HC1 buffer plus o.oi M NaC1 solution (pH 7.5) and discharged into a tube, to which 4.20 g of CsC1 were added so that the final solution might show a refractive index of 1.399 ° at 25 °. The centrifuge tube was spun in a Spinco Model L-2 ultracentrifuge using a No. 40 fixed-angle rotor at 33 ooo rev./min for 7 ° h at 25 °. Usually, o.o5-ml fractions were collected from the tube and mixed with I.O ml of standard saline citrate. Then the absorbance at 260 nm was recorded using a spectrophotometer. For transforming experiments, an aliquot was diluted Io-fold with standard saline citrate. Transforming experiments were carried out using o.I ml of the diluted DNA solution as the donor and a strain (HLL-3g) carrying aden, leus, rnet~, indl~ 8, his 1, thrs, lys21 and ery-r markers 9 as the recipient, according to the method of TANOOKAAND SAKAKIBARA10. In Fig. I, each marker activity in a o.o5-ml fraction is expressed as a percent of the total transformants. Since the DNA concentration (below 0.02/,g/ml) of the linear dose response in our system was used, the percentages can be taken as the relative amounts of marked DNA pieces in each fraction. Fig. I shows that the marked DNA pieces can be arranged in terms of buoyant density of CsC1 in the following heavy to light order: ade6, (thrs, his1) ,str, leu s (lys2z , indz68), mets. Biochim. Biophys. Acta, 213 (197 o) 5 2 3 - 5 2 5

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Fig. I. Fractionation of B. subtilis D N A on CsC1 density gradients. Sheared D N A of B. sublilis was fractionated b y the CsC1 density gradient m e t h o d using an angle rotor. (2)- - - O, distribution p a t t e r n of 14C radioactivity. Distribution p a t t e r n s of m a r k e r activities assayed b y the t r a n f o r m a tion method: (2) O , ade6; + - + , thrs; [~-[N, hiSl; 0 - 0 , str; ~7-~7, indl,8; / x - & , mets; 0 - 0 , r R N A cistron. The total recovery of adeo, leu v mete, ind188, his 1, thrs, lys,1 and str was o.75. lO s, o.32.IO*, o . 3 o . l o e, o.7o. Io*, o.97.1o 8, o.96.IO*, o.67-1o8 and I . I . i O 6 per ml of t r a n s f o r m i n g culture, respectively. Fig. 2. Transforming activity of each m a r k e r relative to leu v The t r a n s f o r m i n g activity of each m a r k e r was divided b y the t r a n s f o r m i n g activity of leu s in the same fraction. Reproduced from Fig. I. S y m b o l s as in Fig. I except 0 - 0 here stands for lys2v

Fig. 2 illustrates the activity of each marked DNA piece normalized to that of leu 8 pieces. The coincidence of the observed results and the positions of tested markers on the chromosome (Fig. 3) suggest that there m a y be an inclination of guanine plus cytosine content from the origin of the replication to the terminus. But we don't know whether the inclination is continuous or not. A similar observation has been presented by O'SULLIVAN et al3. : y [ s ~ t r e]ry ! \ aq

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One of the authors (H.T.) has previously reported 1° that the rRNA cistrons in B. subtilis are located in the heavy region of the chromosome. Distribution of rRNA

cistrons in the centrifugation profile was studied by hybridization with [3H]uridinelabeled rRNA. The experimental procedures were described previously 1°. Again, the DNA pieces carrying rRNA cistrons were found in the heaviest fractions (Fig. I). Biochim. Biophys. Acta, 213 (197 ° ) 523-525

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The amount of rRNA cistrons in Fraction 32 was compared with that in Fraction 44, the mean fraction, and a concentration effect of about 4o-fold was obtained. It is interesting to note that the distribution of rRNA cistrons differed significantly from that of the str gene. These two cistrons might reside on different DNA pieces which have molecular weights as large as 0. 9 • lO T.

Institu/e o/ Applied Microbiology, University o/Tokyo, Tokyo (Japan) I 2 3 4 5 6 7 8 9 IO

HIDEO TAKAHASHI YONOSUKE I~EDA

N. MUNAK&TA, H. SAITO AND Y. IKEDA, Mvtation Res., 3 (1966) 93. H. SAITO AND Y. MASAMISNE, Biochim. Biophys. Acta, 91 (1964) 344. A. T. GANESAN AND J. LEDERBERG, J. Mol. Biol., 9 (1964) 683A. O'SULLIVAN, H. YOSHIKAWA AND N. SUEOKA, Bacteriol. Proc., 65 (1965) 13. C. STEWART, ./. Bacteriol., 98 (1969) 1239. I. SMITH, D. DUBNAU, P. ]VIORELL AND J. 1V[ARMUR,.[. Mol. Biol., 33 (1968) 123. H. SAITO AND K. MIURA, Biochim. Biophys. Acta, 72 (1963) 619. H. TAI~AHASHI, Biochim. Biophys. _/tcta, 19o (1969) 214. H. TAKAHASHI AND Y. IKEDA, J. Gen..dppl. Microbiol., 14 (1968) 451. I~. TANOOKA AND ~r. SAKAKIBARA, Biochim. Biophys. Acta, 155 (1968) 13o.

Received April 27th, 197o Biochim. Biophys. Acta, 213 (197 o) 523-525

BBA 93526 A purification procedure for smoll amounts of rodiooctive Escherichia coli R N A polymerose Radioactive RNA polymerase is useful for studies on the interaction of the enzyme with DNA 1 and could also be used to explore the possibility that protein factors exchange between polymerase molecules. Great difficulty was encountered in attempting to purify small quantities of radioactive polymerase b y conventional procedures2, s. The procedure described here will yield highly purified radioactive RNA polymerase, all of which is initially capable of binding to DNA. Escherichia coli cells were grown in a I 1 volume in S medium containing 20 mC 35S. S medium contains per 1:2 g NH4C1, 6 g N a 2 H P Q , 3 g K H 2 P Q , 3 g NaC1, 2 ~o glycerol, o.3 mM MgC12 and 60 #M MgSO~. Without added MgSO 4 cells grew to about I.IoS/ml. S042- was no longer limiting above 0.4 raM. Cells were harvested at an A650 nm of 0.8, shortly before the growth rate of the cells begins to decrease. The cells were collected by centrifugation and washed with R buffer (o.oi M Tris buffer, p H 7.9, 5 mM MgC12, 0.2 mM EDTA, 4 mM 2-mercaptoethanol, 5 % glycerol) containing 0.25 M KC1. The cells were suspended in 2 ml of R buffer containing 0.25 M KC1 and broken open by sonicating them 3 times for 30 sec each with an MSE ultrasonic disintegrator. Debris and ribosomes were removed b y centrifugation in the Spinco SW 39 rotor for I h at 39 ooo rev./min. The supernatant was fractionated with (NH4)2SO 4 and the protein precipitating between 35 and 50 9o of saturation was collected b y centrifugation. The (NH4)2SO 4 precipitate was dissolved in 50 ml of R buffer lacking KC1 and adsorbed immediately onto a 5 ml DEAE-cellulose column. The column was then Biochim. Biophys. Acta, 213 (197 o) 525-528