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the study on the PMase mutant. Thanks are also due to Professor D. MIZUNO and Professor Y. IKEDA for their interest throughout the work.

Institute o/ Applied Microbiology, HIUGA SAITO University o[ Tokyo, Tokyo (Japan)

Faculty o/Pharmaceutical Sciences, YUKITO !~¢[ASAMUNE University o/ Tokyo, Tokyo (Japan)

x j . D. MANDELL AND A. D. HERSHEY, Anal. Biochem., I (196o) 66. 2 N. SUEOKA AND T.-Y. CHENG, J. Mol. Biol., 4 (1962) 161.

T.-Y. CHENG AND N. SUEOKA, Science, 141 (1962) 1194. 4 H. SAITO AND K. MIURA, Biochim. Biophys. Acta, 72 (1963) 619.

G. CERRIOTTI, J. Biol. Chem., 214 (1955) 59. e E. W. NESTER AND J. LEDERBERG, Proc. Natl. Acad. Sci. U.S., 47 (1961) 52.

E. EPHRATI-ELIZUR, P. R. SRINIVASAN AND S. ZAMENHOF, Proc. Natl. Acad. Sci. U.S., 47 (1961) 56.

8 C. ANAGNOSTOPOULOS AND I. P. CRAWFORD, Proc. Natl. Acad. Sci. U.S., 47 (1961) 378. J. SPIZlZEN, Proc. Natl. Acad. Sci. U.S., 44 (1958) lO72.

x0 H. SAITO, M. KOHIYAMA AND Y. IKEDA, J. Gen. Appl. Microbiol. Tokyo, 7 (1961) 243. 11 M. IROGER AND t~-. D. HOTCHKISS, Proc. Natl. Acad. Sci. U.S., 47 (1961) 653. 1~ p. DOTY, J. MARMUR AND N. SUEOKA, Brookhaven Syrup. Biol., 12 (1959) I. 18 R. ROLFE AND M. MESELSON, Proc. Natl. Acad. Sci. U.S., 45 (1959) lO39. 14 W. R. GUILD, J. Mol. Biol., 6 (1963) 214.

Received April I7th, 1964

Biochim. Biophys. Acta, 91 (1964) 344-347

SC 93008

Retention of transforming activity on filtrotion of D N A solutions

I t has recently been demonstrated 1 that solutions of native T2 and T 7 DNA can be filtered through small-pore cellulose ester filters without any breakage of the mole- cules by hydrodynamic shear stresses. The intrinsic viscosity of the filtered material was the same as of the original unfiltered sample and there was no change in the light-scattering properties. To study the biological effects on DNA of filtration through small-pore membrane filters, the transforming activity of Bacillus subtilis DNA which had been subjected to such filtration has now been investigated.

Breakage of DNA by hydrodynamic shear decreases the transforming activity of the DNA. GUILD AND DEFILIPPES ~ found that pneumoccocal transforming prin- ciple is readily inactivated by exposure to ultrasonic vibration. In a study of the effect of sonic vibration at 9 kcycles on pneumococcal transforming principle, LITT et al. 8 found a direct dependence of the transforming activity on the molecular weight of the DNA. KAISER 4 has shown that hydrodynamic shear destroys the in- fectivity of the DNA of 2 phage, and that one of the two half molecules produced is capable of transforming certain markers but not others.

Donor DNA was prepared from strain 23 of B. subtilis grown in Penassay broth containing 0.5 % glucose (18 h, 37 °, with vigorous shaking). The DNA was isolated by the method of SAITO AND )]IIURA 5 and no RNA was detectable by the orcinol reaction 6. For all experiments, this DNA was dissolved in saline-citrate buffer

Biochim. Biophys. Acta, 91 (1964) 347-349

348 SHORT COMMUNICATIONS

(o.15 M NaC1, O.Ol 5 M trisodium citrate, pH 7.o). The DNA concentration was determined by the diphenylamine reaction ~.

Viscosities were determined in a low-shear rotating-cylinder viscometer 7 as previously described 1. Solutions were filtered through a 0.22-/, (type GS) pore diameter cellulose ester Millipore membrane (47 mm diameter) supported in a stain- less steel pressure holder at a pressure of ~-~ 20 mm Hg (flow rate ~ 2 ml per rain). The shear rate at this rate of flow is less than the critical shear rate where molecular breakage begins.

Transformation experiments were carried out as described by SPIZIZEN 8 and EPHRATI-ELIZUR et al. 9 using strain 30 of B. subt i l i s as recipient. This strain requires tryptophan or indole (ind-) and histidine (his-) for growth. These two markers are unlinked 9 in this strain. For transformation, the filtered and the unfiltered DNA from strain 23 were precipitated with two volumes of ethanol and dissolved in sterile saline-citrate buffer. After 2-h contact with transforming DNA, the recipient cells were plated on minimal-agar plates and on plates supplemented with either trypto- phan or histidine.

Table I summarizes the results on the effect of filtration on the intrinsic viscocity of B. subt i l i s DNA. Within experimental error, the viscosity is unchanged by filtering, suggesting that denaturation and molecular breakage are apparently insignificant. As found previously for filtered T2 and T 7 DNA (ref. I), the filtered material is less concentrated than the unfiltered solution and this could be due to partial ultrafiltration

T A B L E I

EFFI~CT OF FILTRATION ON CONCENTRATION AND VISCOSITY OF ]~. sub t i l i s D N A

Unfiltered Filtered

Concentrat ion before and after f i l t rat ion (/~g/ml) 116 7 o.5

Concentrat ion of solution used for viscosity nleasurenlent (#g/ml) 49.3 49.9

Relative viscosity, 71r~.l. 1.773 1.8o6

In ~ r e l . E~]j ( d l / g ) 116 118 c

or some absorption of the DNA by the membrane. The intrinsic viscosity of un- filtered B , subt i l i s DNA determined in a separate experiment by measuring relative viscosity versus DNA concentration was 115.

Fig. I shows the transforming activity of both the filtered and unfiltered DNA with respect to the two markers his + and ind + and to the double transformant his+ind +. The number of double transformants is about 1.5-4.o % of the single transformants 9. Within the limits of experimental error, there is no change in the transforming activity of the DNA on filtration through a 0,22-/, membrane filter. Breakage of the DNA by hydrodynamic shear would be expected to decrease the transforming ac- tivity for some of these markers.

The fact that filtering DNA did not cause any change in the intrinsic viscosity or in the transforming activity of the DNA can be taken as evidence that the DNA is unaffected by this procedure. Therefore, filtration of DNA through small-pore cellulose ester filters would be an easy and quick method for the removal of dust particles from DNA solutions for light-scattering measurements.

Biochim. ]diophys. Acta, 91 (19~4) 347 349

SHORT COMMUNICATIONS 349

I his "l- t }

i / in d +

4 . 0

3,2

-- 2.4 o

K ],6

~ 0.8

~ o z 4 . 0

5 , 2

~ 2.4

1.6

0.8

0 %

F 3.2 h /$ ÷ i

2.4 u_

o.8 I

O ~ I I I I I I I I t I [ O I 2 3 4 5 6 7 8 9 I 0

C O N C N - O F D N A ( , u g / m l )

Fig. I . Transforming act iv i ty of filtered and unfiltered B. subtilis DNA. Both filtered and un- filtered DNA were tested at the same concentrat ions for the two markers, his + and ind +, and for the double t ransformant , his+ind +. The data are plot ted as per cent cells t ransformed. O,

filtered DNA; S , unfiltered DNA.

The author wishes to acknowledge the able technical assistance of Miss R. SAMPOLLO. He is indebted to Dr. S. ZAMENHOF and Miss L. HELDENMUTH for helpful discussions and advice. This investigation was supported by research grant NSF-GB- 273 from the National Science Foundation.

Department o/ Biochemistry, College o~ Physicians and Surgeons

Columbia University, New York, N.Y. (U.S.A.)

ALVIN I. KRASNA

1 A. I. KRASNA AND J . A. HARPST, Proc. Natl. Acad. Sci. U.S., 51 (1964) 36. 2 W. R. GUILD AND F. M. DEFILIPPES, Biochim. Biophys. Acta, 26 (1957) 241. 3 M. LITT, J. MARMUR, H. EPHRUSSI-TAYLOR AND P. DUTY, Proc. Natl. Acad. Sci. U.S., 44 (1958)

144. 4 A. D. KAISER, J. Mol. Biol., 4 (1962) 275. s H . SAITO AND K . MIURA, Biochim. Biophys. Acta, 72 (1963) 619. 6 W . C. SCHNEIDER, i n S. P . COLOWICK AND N. O. t{APLAN, Methods in Enzymology, Vol . 3,

Academic Press, New York, 1957, p. 680. 7 B. H . ZIMM AND D . 1VL CROTHERS, Proc. Natl. Acad. Sci. U.S., 48 (1962) 9 o 5. s j . SPIZlZEN, Proc. Natl. Acad. U.S., 44 (1958) lO72. 9 E . EPHRATI-ELIZUR, P . R . SRINIVASAN AND S. ZAMENHOF, Proc. Natl. Acad. Sci. U.S., 47

(1961) 56.

Received April iDth , 1964

Biochim. Biophys. Acta, 91 (1964) 347-349