Preparations and properties of deamino-deoxyribonucleic acid

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The rapid incorporation in the smooth vesicles of the renoprival kidney is of interest because the increase in absolute number of ribosomes provoked b y nephrect o m y is reflected mainly b y an increase in the number of membrane-bound ribosomes (MALT, unpublished observations) in contrast to the increase in free ribosomes found after hepatectomy is. One might consider that a precursor-product relationship exists between the smooth and rough forms of this cellular component, although the d a t a are not sufficient to indicate in which direction the conversion might go. Electron micrographs will, of course, be necessary to confirm the absence of ribosomes in the harvested vesicles, but the enrichment of radioactive RNA of the vesicles in the early pulses while the specific activity of the ribosomes is low and the fall at 47 h while the specific activity of the ribosomes is high, indicate that this source of error is not present. The widely different patterns of synthesis of microsomal membrane lipid and of membrane RNA suggests that there might be at least two phases in the synthesis of membranes, a fast process during which lipid and some of the RNA are made and a much slower one during which most of the RNA is added. This investigation was supported b y an Established Investigatorship of the American H e a r t Association (R.A.M.) and b y grants from the Shriners Burns Institute, Massachusetts Heart Association (No. 696 ), and the Damon Runyon Memorial Fund (No. 796-A).

Surgical Services, Massachusetts General Hospital, and Department o/ Surgery, Harvard Medical School, Boston, Mass. (U.S.A.) I 2 3 4 5 6 7 8 9 IO ii 12 13 14 15

RONALD A. MALT S. KAY STODDARD

P. MANDEL, L. MANDEL AND M. JACOB, Compt. Rend. Soc. Biol., 144 (195 o) 1548. L. VEGNI, Arch. Sci. Biol., 37 (1953) 454. N. M. SULKIN, Anat. Record., lO6 (1955) 29o. N. B. KURNICK, J. Histochem. Cytochem., 3 (1955) 290. I. LIEBERMAN, R. ABRAMS AND P. OvE, J. Biol. Chem., 238 (1963) 2141. R. A. MALT, J. Surg. Res., 6 (1966) 152. H. FRAENKEL-CONRAT, B. SINGER AND A. TSUGITA, Virology, 14 (1961) 54. Y. MOUL~ AND G. DELHUMEAU DE ONGAY, Biochim. Biophys. Acta, 91 (1964) 113. W. !V[EJBAUM, Z. Physiol. Chem., 258 (1939) 117, G. DALLNER, P. SIEKEVITZ AND G. E. PALADE, Biochem. Biophys. Res. Commun., 2o (1965) 135. G. DALLNER, P. SIEKEVITZ AND G. E. PALADE, Biochem. Biophys. Res. Commun., 20 (1965) 142. C. H. FISKE AND Y. SUBBAROW, J. Biol. Chem., 66 (1925) 375. J. CHAUVEAU, Y. IV[OUL~, C. ROUILLER AND J. SCHNEEBELI, J. Cell Biol., 12 (1962) 17. C. BOOVET AND Y. MOULI~, Exptl. Cell Res., 33 (1964) 33 o. G. DELHUMEAU DE ONGAY, Y. MOULI~ AND C. FRAYSSlNET, Exptl. Cell Res., 38 (1965) 187.

Received December 3rd, 1965 Biochim. Biophys. Aeta, 119 (1966) 2o 7 21o BBA 93122

Preparations and properties of deamino-deoxyribonucleic acid When infective RNA from tobacco mosaic virus was treated with HNO 2, it was found that the inactivation and mutation rate both followed a one-hit curve, showing that the deamination of only one of the amino bases adenine, guanine or cytosine in the RNA was lethal or mutagenic, respectively 1,2. Several investigators have studied the lethal and mutagenic effect of HNOg. on biologically active material containing DNA ~. Biochi~n. Biophys. Acla, 119 (1966) 21o-212

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In this paper we describe a method by which DNA is completely deaminated, and report the various properties of the deaminated DNA. A solution of IOO mg of calf thymus DNA (prepared b y GULLAND'S method 4) in 0.4 ml of water was neutralized with o.I M N a O H and mixed with 0.24 ml of 43 ~o NaNO~. The reaction mixture was adjusted to p H 4.2 by gradually adding glacial acetic acid and then incubating for 12 h at 25 °. The mixture was poured into 4 vol. of ethanol and centrifuged. The precipitated solid was dissolved in 2 ml of water, immediately transferred to a cellophane tube and dialyzed at 4 ° against running t a p water for 20 h. Insoluble material was removed by centrifugation and the product precipitated from the supernatant solution b y the addition of 4 vol. of ethanol and one drop of I M HC1. The precipitated solid was washed with 7 ° ~o and Ioo °,o ethanol, then with ether, and was dried under vacuum at room temperature. The product was a pure white fluff, weighing 80 mg. The base composition of the product was determined spectrophotometrically after perchloric acid hydrolysis and separation of the bases b y paper chromatography. The ultraviolet-absorbing areas were cut out and eluted b y soaking in 4 ml of o.I M HC1 overnight. From the ultraviolet spectra of the eluates, the four spots on the chromatogram were shown to be thymine, uracil, hypoxanthine and xanthine, respectively. No adenine, guanine or cytosine were detected and the recovery of the pyrimidine, purine and the phosphorus is almost quantitative (Table I). From these results it m a y be concluded that the product is almost free of amino bases. TABLE

I

T H E COMPOSITIONS OF D N A

Base

Thymine Cytosine Uracil Adenine Hypoxanthine Guanine Xanthine

T R E A T E D W I T H N a N O 2 AND P A R E N T D N A

Moles per

Ioog

atoms o[ P

Parent DNA

Treated DNA

28.2 21,6 -28.2 -2 i.o --

29.0 -2 I. 2 -26.9 -18.2

The absorption m a x i m u m of the deaminated DNA was at 258 m# with an eM(P) of 71oo, the minimum at 230 m/~ with an eM(P) of 3600 in phosphate buffer of p H 5.4 (Fig. I). An average molecular weight of the deaminated DNA was estimated at about IOO ooo from sedimentation and diffusion constant values. In order to know whether the deaminated DNA was double-stranded, it was dissolved in a solution of NaC1 containing sodium citrate, and the absorbance of the solution at 260 m/~ at various temperatures (t) was measured. The relationship between the increase in the ratio A,/A25o and t showed that the deaminated DNA belonged to the single-stranded type. The action of pancreatic deoxyribonuclease I(EC 3.1.4.5) on the deaminated DNA was then investigated. To a solution of 20 mg of the deaminated DNA or Biochim. Biophys. Acta,

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(i966)

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8000 ,

/ \

,

\ \

600C

(

o.6I

}°41

400C IE

200C

C5

Wavelength(rnp)

Jo

@o

Fig. i. Ultraviolet absorption curves of deaminated DNA at pH 5.4 ( - - ) Fig. 2. Action of deoxyribonuclease I on parent DNA ( - - )

~o

~o

Time (rain)

and 12. 3 (- - -).

and deaminated DNA (---).

p a r e n t D N A in 1.8 ml of w a t e r was a d d e d 0.I m l of o.4 M MgSO 4, o.o 3 m l of o.0I o/ /o phenol red, a t rac e of 0,02 M N a 0 H t o p r o d u c e t h e same eolour as t h a t shown b y t h e s t a n d a r d (pH 7.5) buffer solution, a n d finally o.I m l of 0.02 % d e o x y r i b o n u c l e a s e I solution. T h e r e a c t i o n m i x t u r e was i n c u b a t e d at 37 ° an d t i t r a t e d at various i n t er v al s w i t h o.02 M N a O H to show th e same colour w i t h t h e standard. As shown in Fig. 2, t h e d e a m i n a t e d D N A was c o m p l e t e l y r e s is t a n t to t h e action of d e o x y r i b o n u c l e a s e I. This fact seems to i n d i c a t e t h a t th e presence in D N A of t h e am i n o g r o u p of t h e base is necessary for t h e c a t a l y t i c a c t i v i t y of t h e d e o x y r i b o n u c l e a s e I. T h e au t h o rs wish to t h a n k Prof. K. MAKINO for his e n c o u r a g e m e n t .

Department o[ Biochemistry, The Jikei University School o/ Medicine, Atago-cho, Shiba, Minato-ku, Tokyo (Japan) i 2 3 4

MAKOTO MATSUDA HEIHACHIRO OGOSHI

H. SCHUSTERAND G. SCHRAMM, Z. Natur/orsch., IDb (1957) 697, A. GIERER AND I~. W. 1V[UNDRY,Nature, 189 (1958) 1457. W. VIELMETTERAND H. SCHUSTER,Biochem. Biophys. Res. Commun., 2 (196o) 324 . J. M. GULLAND, D. O. JORDAN AND C. J. TI-IRELFALL,J. Chem. Soc., (195o) 1129.

R e c e i v e d S e p t e m b e r I 6 t h , 1965

Biochim. Biophys. Acta, 119 (1966) ZLO-212