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The effect of mitomycin C on deoxyribonucleic acid and messenger ribonucleic acid in Escherichia coli
254
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 95357
T H E E F F E C T OF MITOMYCIN C ON D E O X Y R I B O N U C L E I C ACID AND MESSENGER R I B O N U C L E I C ACID IN E S C H E R I C H I A
COLI
INGRID S M I T H - K I E L L A N D
Department o] Biochemistry, University o/ Oslo, Blindern (Norway) (Received May 2oth, 1965)
SUMMARY
I. The presence of mitomycin C (Io #g/ml) in a growing culture of a laboratory strain of Escherichia coli arrests the synthesis of DNA. No degradation of DNA seems to take place. 2. Messenger RNA's labelled with E2-14C]uracil after pulses of 15 and 30 sec respectively, were isolated from mitomycin C-inhibited (Io/zg/ml) and control cultures. These were less efficient in forming hybrids with denatured DNA (no mitomycin in culture) compared with control messenger RNA. However, when RNA's which had been pulse-labelled for 60 sec and 12o sec respectively, were used, there was negligible difference in hybridization between the RNA's from the mitomycin C-treated culture and the control culture. 3. DNA isolated from the mitomycin C-treated culture was less efficient in forming hybrids with messenger RNA (no mitomycin in culture) than was denatured DNA isolated from cells which had been grown in the absence of mitomycin C.
INTRODUCTION
It is well known that the antibiotic Mitomycin C in growing bacteria arrests the synthesis of DNA within a few minutes after its addition, whereas a corresponding inhibition of total RNA (as measured b y the oreinol reaction) and of protein synthesis (as measured b y the biuret method) does not occur 1. Experiments by IYER AND SZYBALSKI2, 3 a n d MATSUMOTO AND LARK4 suggest that in bacteria the primary action of Mitomycin C is on DNA and that mitomycin C causes the formation of crosslinks between the DNA strands. Recent results suggest that cellular RNA such as messenger RNA 5, soluble RNA6, 7 and ribosomal RNA s, is copied from DNA. The introduction of mitomycin C crosslinks in the bacterial DNA could affect the formation of some of the RNA Abbreviations: DNAMc and DNAc, DNA isolated from mitomycin C treated and control cultures respectively; RNAMc and 1RNAc, RNA isolated from mitomycin C treated and control cultures respectively.
Biochi~. Biophys. Acta, 114 (1966) 254 263
EFFECT OF MITOMYCIN C ON DNA AND m - R N A IN E. coli
255
molecular species being synthesized through the participation of DNA. In order to study the effect of mitomycin C on RNA synthesis in exponentially growing cultures of Escherichia coli, a more detailed examination of messenger RNA, soluble RNA and ribosomal RNA synthesis has been undertaken. Preliminary results to this effect have previously been published 9,1°. In the present report the effect of mitomycin C on the ability of messenger RNA to hybridize with DNA has been studied. It has been found that RNA, pulselabelled with E2-14C~uracil (up to 6o-sec pulse) in a mitomycin C culture is less efficient in forming hybrids with DNA from a control culture (no mitomycin C) than is the corresponding RNA from the control culture. Furthermore, DNA~c is less efficient in forming hybrids with pulse labelled RNA c than is DNA c. Thus, the mitomycin C crosslinks seem to interfere with the synthesis of messenger RNA.
MATERIALS AND METHODS
Adenosine 5'-phosphate, deoxyadenosine and crystalline samples of deoxyribonuclease and ribonuclease were obtained from Mann Research Laboratories Inc., New York, N.Y., U.S.A. Crystalline lysozyme was purchased from Worthington Biochemical Sales Co., New Jersey (U.S.A.) and albumin from Sigma Chemical Co., St. Louis, Mo. (U.S.A.). The crystalline Mitomycin C was obtained from Kyowa H a k k o Kogyo Co. Ltd., Tokyo (Japan). Sodium dodecylsulfate was purchased from L. Light & Co. Ltd., Bucks. (Great Britain). E2-1~C]Uracil (27.8 mC/mmole) from New England Nuclear Corp., Mass. (U.S.A.) and nitrocellulose membranefilters (group 2, 29 m m in diameter) were obtained from Membranfilter Gesellschaft GmbH, G6ttingen (Germany).
Growth o/ the organism The bacterium used was a laboratory strain of E. coli, which was grown under aeration in a Gyrotory incubator shaker at 37 ° in a liquid medium (minimal medium) prepared as follows: NH4C1, 2 g; N a 2 H P Q . 2 H 2 0 , 7-5 g; KH~P04, 3 g; NaC1, 3 g; MgC12" 6H20, 85.6 mg and Na2SO 4. ioH20 , 260 mg were dissolved in 980 ml of water. After autoclaving, a sterile glucose solution was added to a final concentration of 4 %. The absorbances of the culture were measured in a Beckman DU spectrophotometer at 650 m/z with a I-cm light path.
Estimation o/ nucleic acids and proteins RNA was estimated using the orcinol reaction u and adenosine 5'-phosphate as a standard. DNA was determined b y the procedure of BURTONlz with deoxyadenosine as standard. The protein was estimated b y the method of LowRY et al. 13 with serum albumin as a standard. Extraction of bacterial nucleic acids and protein was carried out as follows. Aliquots (IO ml) of the bacterial culture were chilled to o ° and rapidly centrifuged. Cold aqueous 5 ~ (w/v) trichloroacetic acid (2 ml) was added to the sediment, and the mixture centrifuged after standing at o ° for 30 rain. The sediment Biochim. Biophys. Acta, 114 (1966) 254-263
256
I. SMITH-KIELLAND
was then heated with 5 o/ , 0 trichloroacetic acid (2 ml) at 9 °o for 3o rain in order to extract nucleic acids. To dissolve protein, the remaining residue was treated with N N a O H (I ml).
Radioactivity measurements All samples were counted in a Frieseke and Hoepfner flow-counter on nitrocellulose filters or after direct plating.
Spectrophotometry A Beckman DU Spectrophotometer employing I-cm cuvettes was used.
Preparation o/ pulse-labelled RNA Pulse-labelled culture (I volume) was added to frozen (--4 o°) minimal medium (0.5 volume), the cells were harvested b y centrifugation at 2 °, washed twice with cold o.oI M Tris-HC1 buffer (pH 7-3) containing o.oi M KC1 (0.03 volume each time) and finally frozen. RNA was subsequently isolated b y the following method 14. The frozen cells were suspended in the Tris-HC1 buffer (o.03 volume), a solution of 20 °/o sodium dodecylsulfate was added to a final concentration of 0.5 %, the mixture was heated to 50 ° and was then shaken for 3 min. The pH of the suspension was adjusted to 5.6~5 by the addition of 0.3 N acetic acid and the mixture shaken for 5 rain at 60 ° with an equal volume of phenol previously equilibrated with o.oi M sodium acetate buffer (pH 5.2). This was followed by centrifugation for 2o rain in a Wifug centrifuge at 6000 rev./min. The operation was repeated three or four times (until absence of interphase). The aqueous RNA solution was purified on a Sephadex G-25 column (where phenol separates from RNA). After passage through the Sephadex column, the RNA was precipitated b y addition of two volumes of ethanol (96 °/o) and collected b y centrifugation. The precipitate was dissolved (i mg/ml) in 0.o6 M KC1 and frozen at --2o ° in I-ml portions. All the RNA preparations, which were used, contained less than I °/o of DNA and protein. A comparison has been made between the samples of RNA which were obtained from cells which were lysed in different ways. A culture of cells was divided into two equal parts, one of which was lysed with sodium dodecylsulfate, the other which had been disrupted for I min in a Mullard ultrasonic disintegrator, at + 2 ° prior to treatment with sodium dodecylsulfate. Both batches yielded the same amount of RNA. However, the RNA prepared after ultrasonic treatment contained 2 o(, of DNA in contrast to the other preparation which contained none.
Preparation o/ DNA All samples of DNA from E. coli were isolated and purified after treatment of the cells with lysozyme and sodium dodecylsulfate according to the method of MARMUR15. Biochim. Biophys, Acta, 114 (1966) 254-263
EFFECT OF MITOMYCIN C ON DNA AND m-RNA IN E. coli
257
Hybridization The procedure of NYGAARD AND HALL14 was used. Nitrocellulose membrane filters were soaked in o.oi M Tris-HC1 buffer (pH 7-3) containing o.5 M KC1 for 30 rain and each filter was washed with 25 ml of the buffer before application of the samples of nucleic acid. The filter was mounted on a stainless steel grid with a stainless steel cylinder placed on top. Suction was provided by a water pump. The DNA used was denatured by heating at a concentration of Ioo#g/ml at 9°° for 15 rain in o.oi M Tris-HC1 buffer (pH 7.3) containing 0.06 M KC1 and rapidly cooled by pouring the solution on to half its volume of frozen buffer. KC1 was added to a final concentration of 0.5 M, and I-ml samples were frozen at --20 °. Samples of RNA solutions were made 0.5 M with respect to KC1 and filtered through the membrane filter prior to complex formation (to reduce the 'background' caused by the trapping of free RNA). Desired amounts of RNA and DNA were mixed, diluted with o.oi M Tris-HC1 buffer (pH 7.3) containing 0. 5 M KCI and heated in a waterbath to a given temperature. At intervals, samples varying from lOO-25o #1 depending upon the radioactivity of the RNA, were diluted into 15 ml of the same buffer at room temperature. In the diluted state at this temperature no further reaction occurs and the hybrid is stable for several hours. The samples were filtered through the membrane filters and the filters washed with 60 ml of the KC1 solution at room temperature, airdried and counted. To determine the total radioactivity in hybridization mixtures an aliquot (5o/*1) was mixed with 25/~g of thymus-DNA dissolved in 25 #I of water, and trichloroacetic acid solution was added to the mixture to a final concentration of IO % (w/v). The samples were allowed to stand in an ice bath for 3 ° rain, filtered through a nitrocellulose filter (previously washed with water) and washed with IO ml of IO % trichloroacetic acid. The filter was air-dried and counted. All analyses were carried out in duplicate.
RESULTS It is well known that the amount of mitomycin C required to stop the synthesis of DNA varies from bacterium to bacterium1,2, 4 and even in different strains of the same bacteriumt, 1~. Thus, in the present case it was necessary to determine the concentration of mitomycin C required to stop DNA synthesis. It is seen from Fig. I that I0 #g/ml of mitomycin C is sufficient to stop DNA synthesis completely, whereas in the presence of 0.I #g/ml and I #g/ml some synthesis takes place. Thus, in all subsequent experiments the concentration of mitomycin C used was I0/~g/mt. In addition, its effect on RNA and protein synthesis is reported in Figs. 2a and 2b. It is interesting to note that upon addition of mitomycin C there is an increase in RNA synthesis over the first 3 ° min.
DNA isolated/rom mitomycin C treated and control cultures An exponentially-growing culture with a density reading of 0.35 was divided into two halves and mitomycin C (I0/~g/ml) was added to only one of the flasks.
Biochim. Biophy. Acta, 1I 4 (1966) 254-263
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Fig. i. T h e e f f e c t of m i t o m y c i n C on D N A - s y n t h e s i s . A n e x p o n e n t i a l l y g r o w i n g c u l t u r e of E. coli ( d e n s i t y r e a d i n g o.35 ) w a s d i v i d e d into four e q u a l portions. To t h r e e of t h e m were a d d e d o.I, I.O a n d I O / , g / m l of m i t o m y c i n C respectively, a n d t h e f o u r t h w a s u s e d as a control. T h e c u l t u r e s w e r e i n c u b a t e d a n d a n a l y z e d a t i n t e r v a l s for t h e i r c o n t e n t of D N A . M i t o m y c i n C w a s a d d e d a t zero t i m e . O - O , c o n t r o l culture. M i t o m y c i n C c u l t u r e s : Z~-&, o.I # g / m l ; r q - [ ] , I ffg/ml; © - © , loffg/ml. lOOO
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Figs. 2a a n d 2b. T h e e f f e c t of m i t o m y c i n C (io/~g/ml) o n R N A a n d p r o t e i n s y n t h e s i s . E x p e r i m e n t a l c o n d i t i o n s as described in Fig. i. M i t o m y c i n C w a s a d d e d at zero t i m e . O - O , c o n t r o l culture; O - O , m i t o m y c i n C t r e a t e d culture.
Both were incubated for further 3o min after which the control and mitomycin Ctreated cultures had reached densities of o.42 and o.41, respectively. DNA was isolated from each culture and used in all subsequent experiments. The yields of DNAMc and DNA c per culture was 2.85 mg and 3.54 mg, respectively. The appearance of DNAMc differed from that of DNA c. While the DNA c formed long threads upon precipitation with ethanol, most of the DNAMc was precipitated as shorter threads. The ultraviolet absorption spectra of the two DNA's were determined in o.oI M Tris-HC1 buffer (pH 7.3)- The spectra were identical. Biochim. Biophys. Acta, i14 (1966) 254 263
EFFECT OF MITOMYCIN C ON DNA AND m-RNA IN E. coli
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9O0 ~._ 0.8
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Fig. 3. Melting, and slow cooling, curves for DNAMc and D N A o Equal a m o u n t s (5o/~g) of DNAMc and DNAc were dissolved ill 2.5 ml o.oo15 M t r i s o d i u m citrate buffer (pH 7.5) containing O.Ol 5 M NaC1 and heated up to 82 ° (i°/min) followed by slow cooling (I.5°/min). A heating block m o u n t e d directly to a Zeiss recording thermospectrophotometer was used. O - Q , DNAc; O - Q , DNAMo Fig. 4. The effect of t e m p e r a t u r e and time u p o n D N A - R N A hybrid formation. The R N A used was isolated from a culture of optical reading 0.45 after a pulse-labelling of 2 m i n with I5/~C of [2-14Cluracil/1. A n u m b e r of identical m i x t u r e s each containing 80/~g of h e a t - d e n a t u r e d DNAc and 2oo/~g of R N A c (162 counts/min]/~g of RNA) were incubated at different temperatures. Samples were taken out at intervals.
The effect of deoxyribonuclease on the two types of DNA was studied. Equal amounts (25/~g) of DNAMc and DNA c were each dissolved in 3 ml of o.oi M Tris-HC1 buffer (pH 7.3) containing o.oi M magnesium acetate and 0.05 mg of deoxyribonuclease. The mixtures were incubated at 37 ° and the maximal increase in absorbance at 260 m~ was determined. In the case of DNAMc the increase was 30 %, whereas in the case of DNA c it was 42 %. To characterize the two DNA types further, a comparison between the changes in ultraviolet absorption upon heating and slow cooling was carried out. It is seen from Fig. 3 that the transition point (Tm) for the DNAMc and DNA c is very nearly the same (75.7 ° and 75.2 ° respectively). However, the extent of increase in absorbance is lower for DNAMc (25 %) than for the control (32 %). Upon slow cooling, the DNAMc showed a decrease in hyperchromicity at a higher temperature than that of DNA c.
Hybridization experiments To determine the most suitable conditions for the formation of hybrid between DNA c and RNA o hybridization at different temperatures was carried out. It is seen from Fig. 4 that maximal hybrid formation, stable for 24 h, is obtained at a hybridization temperature of 65 °. Thi~ temperature has been used in all subsequent experiments. Some experiments have been carried out to study the initial rate and extent of hybridization with increasing concentration of RNA. It is seen from Fig. 5 that the initial rate and the extent of complex formation is proportional to the concentration of RNA. Thus, even with a 13 fold higher concentration of RNA (than of DNA) maximum complex formation occurs. Biochim. Biophys. Acta, 114 (1966) 254-263
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I. SMITH-KIELLAND
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Fig. 5. H y b r i d - f o r m a t i o n as a f u n c t i o n of IRNA-concentration. T h e R N A w h i c h w a s u s e d w a s isolated f r o m a c u l t u r e of optical r e a d i n g o.45 after p u l s e labelling for 3 ° sec w i t h 15 ffC of [2-14C] uracil/l. M i x t u r e s , each c o n t a i n i n g 6 o f f g of h e a t - d e n a t u r e d D N A c a n d 250, 500 or 8oo/~g of R N A c ( 6 o c o u n t s / m i n / / ~ g of R N A ) b u t o t h e r w i s e identical, were i n c u b a t e d a n d s a m p l e s were taken out at intervals (×-×). Inset: Initial rate (O-O).
To examine the complex formation between DNA c and messenger RNA from mitomycin C and control cultures, RNA preparations which have been pulse-labelled for I5, 30, 60 and 12o sec have been used. It is seen from Fig. 6a that in the case of RNA pulse-labelled for 15 sec, the extent of complex formation between DNA c and RNAMc is about 30 % less than what it is for RNA c. It is apparent from Fig. 6b that this difference (16 %) although significant, is less pronounced when RNA pulse-labelled for 30 sec was used. In two additional experiments this difference was found to be 17 and 20 % respectively. Fig. 6c shows that in the case of RNA pulselabelled for 60 sec the difference is less. RNA pulse-labelled for 12o sec forms slightly more hybrid with DNA c than does RNA c (Fig. 6d). An examination of the ability of DNAMc to form complexes with pulse-labelled RNA c compared to that of DNA c has also been carried out. Fig. 7 shows that the ability of DNAMc to form complexes is 17 % lower than that of DNA c. In identical experiments with samples of DNA isolated from two different batches of E. coli, the hybrid formation between DNAMc and RNA c was 17 and 2I O//olower than the controls.
DISCUSSION
The concentration of mitomycin C (IO #g/ml) required to stop DNA synthesis in the present strain is similar to that found for other strains 15.A number of reports 16-1s have appeared stating that the primary effect of mitomycin C is to depotymerize DNA. On the other hand IYER AND SZYBALSKI~ suggest that the breakdown is an accessory phenomenon only secondarily related to the lethal effect of the antibiotic. The present result (Fig. I) supports SZYBALSKI'Sview since the DNA concentration of the mitomycin C-inhibited culture remains constant over a 2-h period. Recent Biochim. Biophys. Acta, 114 (1966) 254 263
ON DNA AND m-RNA in E. coli
EFFECT OF MITOMYCIN C
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Fig. 6. T i m e - c o u r s e of h y b r i d i z a t i o n b e t w e e n D N A c a n d pulse-labelled R N A M c a n d R N A c respectively. T h e R N A s a m p l e s u s e d were e x t r a c t e d f r o m c u l t u r e s as follows: A g r o w i n g c u l t u r e ( a b s o r b a n c e of o.35) w a s d i v i d e d into two e q u a l p a r t s a n d IO/~g/ml of m i t o m y c i n C w a s a d d e d to one of t h e m . A f t e r 3o m i n , 15/~C of E2-14C~uracil]l w a s a d d e d to each c u l t u r e a n d t h e g r o w t h t e r m i n a t e d 15, 3 o, 6o, or 12o sec later b y p o u r i n g 1/4 of each c u l t u r e after t h e a p p r o p r i a t e t i m e i n t e r v a l o n to frozen m e d i u m k e p t a t - - 4 o°. R N A M c or R N A c (4oo/~g) w a s i n c u b a t e d w i t h 6 0 / , g of h e a t - d e n a t u r e d D N A , a n d s a m p l e s were r e m o v e d a t i n t e r v a l s . Figs. 6a, b, c, d describe exp e r i m e n t s w i t h R N A p r e p a r e d a f t e r p u l s e s of 15, 3 o, 6o a n d 12o sec. 0 - O , R N A c ; O - O , RNAMc.
work suggests 3 that in a mitomycin C-inhibited culture the two strands in the DNA molecule are crosslinked. That the DNAMc examined in the present work also has different physico-chemical properties from those of DNA c is supported by several findings such as the smaller increase in absorbance after the action of deoxyribonuclease and after heating to 80 °. Upon slow cooling, the decrease in hyperchromicity occurs at a higher temperature than that at which the hyperchromicity of the DNA c begins to decrease. This is in agreement with the observation of MATSUMOTOAND LARK4. The difference in hybrid-formation between messenger RNA c and DNAMc and DNA c respectively (Fig. 7), demonstrates the structural differences between the two DNA's. RNA isolated after I5-sec pulse with L2A4Cluracil in the mitomycin C-treated culture differed from messenger RNA from a control culture in its reduced ability Biochim. Biophys. Acta, 114 (1966) 254-263
262
I. SMITH-KIELLAND 2400 2200 2000 1800 1600
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Fig. 7. Time-course of hybridization between DNAMc and DNAc and pulse-labelled 1RNAC. The preparation of R N A which was used was identical to t h a t described under Fig. 5. R N A (5oo/tg) was incubated with i 2 o / , g of h e a t - d e n a t u r e d DNAMc and D N A c respectively, a n d samples were taken out at intervals. 0 - 0 , DNAc: Q-C), DNAMc.
to form hybrids with heat-denatured DNA c. This difference decreases with the duration of the pulse (after a pulse of 12o sec the degree of hybridization with DNA is somewhat higher for RNAMc than for RNAc) and m a y be due to a faster turnover of messenger RNA c than that of messenger RNAMc. On the other hand it is conceivable t h a t the presence of radioactive impurities in the messenger RNA from the mitomycin C culture could explain the lower percentage of hybridization. Since uracil is the labelled substance and because of the extensive purification of RNA, the presence of any impurities does not seem very likely. Since crosslinks in the DNA molecule prevent a separation of the two strands at the crosslinked sites, this would be the most plausible explanation for the reduced transcription. The recent reports that messenger RNA is synthesized from one strand of the DNA molecule~°, 21, are not contradictory to this view.
ACKNOWLEDGEMENTS
I wish to thank Professor A. NYGAARD, Department of Biochemistry, University of Bergen Medical School, Bergen, for m a n y helpful discussions and Miss S. SCHAU for excellent technical assistance. I am grateful to Mr. E. JELLUM, Central Laboratory, Rikshospitalet, Oslo (Norway) for the use of a Zeiss Spectrophotometer. Financial support from Nansenfondet and The Norwegian Research Council for Science and the Humanities is gratefully acknowledged. Biochim. Biophys. Acta, 114 (1966) 254 263
EFFECT
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REFERENCES I 2 3 4 5 6 7 8 9 io ii 12 13 14 15 16 17 18 19 20 21
S. SHIBA, A. TERA~VAKI, T. TAGUCHI AND J. KA'vVAMATA,Nature, 183 (1959) lO56. V. N. IY~R AND W. SZYBALSKI, Proc. Natl. Acad. Sci. U.S., 50 (1963) 355. V. N. IYER AND W. SZYBALSKI, Science, 145 (1964) 55I. MATSUMOTO AND K. G. LARK, Exptl. Cell Res., 32 (1963) 192. B. D. HALL AND S. SPIEGELMAN, Proc. Natl. Acad. Sci. U.S., 47 (1961) 137. D. GIACOMONI AND S. SPIEGELMAN, Science, 138 (1962) 1328. H. M. GOODMAN AND A. RICH, Proc. Natl. Acad. Sci. U.S., 48 (1962) 21Ol, S. A. YANKOFSKY AND S. SPIEGELMAN, Proc. Natl. Acad. Sci. U.S., 49 (1963) 538. I. SMITH-K1ELLAND, Biochim. Biophys. Acta, 91 (1964) 360. I. SMITH-KIELLAND, Biochem. J., 92 (1964) 26P. W. I~¢[EJBAOM, Z. Physiol. Chem., 258 (1939) 117. K. BURTON, Biochem. J., 62 (1956 ) 315O. H. LOWRY, lXT.J, ROSEBROUGH, A. L. FARR AND R. J. RANDALL, J. Biol. Chem., 193 (I95I) 265. A. P. NYGAARD AND B. D. HALL, Biochem. Biophys. Res. Commun., 12 (1963) 98. J. MARMUR, J. Mol. Biol., 3 (1961) 208. M. SEKIGUCHI AND Y. TAKAGI, Biochim. Biophys. Acta 41 (196o) 434. H. I~ERSTEN AND W . I~ERSTEN, Z. Physiol. Chem., 334 (1963) 141" Y. •AKATA, K. NAKATA AND Y. SAKAMOTO, Biochem. Biophys. Res. Commun., 6 (1961) 339. E. REICH, A. J. SHATKIN AND E. L. TATUM, Biochim. Biophys. Acta, 45 (196o) 6o8. ~¢[. HAYASHI, M. N, HAYASHI AND S. SPIEGEl_MAN, Proc. Nat. Acad. Sci. U.S., 5 ° (1963) 664. J. MARMUR AND C. GREENSPAN, Science, 142 (1963) 387 •
Biochim. Biophys. Acta, 114 (1966) 254-263
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 95357
T H E E F F E C T OF MITOMYCIN C ON D E O X Y R I B O N U C L E I C ACID AND MESSENGER R I B O N U C L E I C ACID IN E S C H E R I C H I A
COLI
INGRID S M I T H - K I E L L A N D
Department o] Biochemistry, University o/ Oslo, Blindern (Norway) (Received May 2oth, 1965)
SUMMARY
I. The presence of mitomycin C (Io #g/ml) in a growing culture of a laboratory strain of Escherichia coli arrests the synthesis of DNA. No degradation of DNA seems to take place. 2. Messenger RNA's labelled with E2-14C]uracil after pulses of 15 and 30 sec respectively, were isolated from mitomycin C-inhibited (Io/zg/ml) and control cultures. These were less efficient in forming hybrids with denatured DNA (no mitomycin in culture) compared with control messenger RNA. However, when RNA's which had been pulse-labelled for 60 sec and 12o sec respectively, were used, there was negligible difference in hybridization between the RNA's from the mitomycin C-treated culture and the control culture. 3. DNA isolated from the mitomycin C-treated culture was less efficient in forming hybrids with messenger RNA (no mitomycin in culture) than was denatured DNA isolated from cells which had been grown in the absence of mitomycin C.
INTRODUCTION
It is well known that the antibiotic Mitomycin C in growing bacteria arrests the synthesis of DNA within a few minutes after its addition, whereas a corresponding inhibition of total RNA (as measured b y the oreinol reaction) and of protein synthesis (as measured b y the biuret method) does not occur 1. Experiments by IYER AND SZYBALSKI2, 3 a n d MATSUMOTO AND LARK4 suggest that in bacteria the primary action of Mitomycin C is on DNA and that mitomycin C causes the formation of crosslinks between the DNA strands. Recent results suggest that cellular RNA such as messenger RNA 5, soluble RNA6, 7 and ribosomal RNA s, is copied from DNA. The introduction of mitomycin C crosslinks in the bacterial DNA could affect the formation of some of the RNA Abbreviations: DNAMc and DNAc, DNA isolated from mitomycin C treated and control cultures respectively; RNAMc and 1RNAc, RNA isolated from mitomycin C treated and control cultures respectively.
Biochi~. Biophys. Acta, 114 (1966) 254 263
EFFECT OF MITOMYCIN C ON DNA AND m - R N A IN E. coli
255
molecular species being synthesized through the participation of DNA. In order to study the effect of mitomycin C on RNA synthesis in exponentially growing cultures of Escherichia coli, a more detailed examination of messenger RNA, soluble RNA and ribosomal RNA synthesis has been undertaken. Preliminary results to this effect have previously been published 9,1°. In the present report the effect of mitomycin C on the ability of messenger RNA to hybridize with DNA has been studied. It has been found that RNA, pulselabelled with E2-14C~uracil (up to 6o-sec pulse) in a mitomycin C culture is less efficient in forming hybrids with DNA from a control culture (no mitomycin C) than is the corresponding RNA from the control culture. Furthermore, DNA~c is less efficient in forming hybrids with pulse labelled RNA c than is DNA c. Thus, the mitomycin C crosslinks seem to interfere with the synthesis of messenger RNA.
MATERIALS AND METHODS
Adenosine 5'-phosphate, deoxyadenosine and crystalline samples of deoxyribonuclease and ribonuclease were obtained from Mann Research Laboratories Inc., New York, N.Y., U.S.A. Crystalline lysozyme was purchased from Worthington Biochemical Sales Co., New Jersey (U.S.A.) and albumin from Sigma Chemical Co., St. Louis, Mo. (U.S.A.). The crystalline Mitomycin C was obtained from Kyowa H a k k o Kogyo Co. Ltd., Tokyo (Japan). Sodium dodecylsulfate was purchased from L. Light & Co. Ltd., Bucks. (Great Britain). E2-1~C]Uracil (27.8 mC/mmole) from New England Nuclear Corp., Mass. (U.S.A.) and nitrocellulose membranefilters (group 2, 29 m m in diameter) were obtained from Membranfilter Gesellschaft GmbH, G6ttingen (Germany).
Growth o/ the organism The bacterium used was a laboratory strain of E. coli, which was grown under aeration in a Gyrotory incubator shaker at 37 ° in a liquid medium (minimal medium) prepared as follows: NH4C1, 2 g; N a 2 H P Q . 2 H 2 0 , 7-5 g; KH~P04, 3 g; NaC1, 3 g; MgC12" 6H20, 85.6 mg and Na2SO 4. ioH20 , 260 mg were dissolved in 980 ml of water. After autoclaving, a sterile glucose solution was added to a final concentration of 4 %. The absorbances of the culture were measured in a Beckman DU spectrophotometer at 650 m/z with a I-cm light path.
Estimation o/ nucleic acids and proteins RNA was estimated using the orcinol reaction u and adenosine 5'-phosphate as a standard. DNA was determined b y the procedure of BURTONlz with deoxyadenosine as standard. The protein was estimated b y the method of LowRY et al. 13 with serum albumin as a standard. Extraction of bacterial nucleic acids and protein was carried out as follows. Aliquots (IO ml) of the bacterial culture were chilled to o ° and rapidly centrifuged. Cold aqueous 5 ~ (w/v) trichloroacetic acid (2 ml) was added to the sediment, and the mixture centrifuged after standing at o ° for 30 rain. The sediment Biochim. Biophys. Acta, 114 (1966) 254-263
256
I. SMITH-KIELLAND
was then heated with 5 o/ , 0 trichloroacetic acid (2 ml) at 9 °o for 3o rain in order to extract nucleic acids. To dissolve protein, the remaining residue was treated with N N a O H (I ml).
Radioactivity measurements All samples were counted in a Frieseke and Hoepfner flow-counter on nitrocellulose filters or after direct plating.
Spectrophotometry A Beckman DU Spectrophotometer employing I-cm cuvettes was used.
Preparation o/ pulse-labelled RNA Pulse-labelled culture (I volume) was added to frozen (--4 o°) minimal medium (0.5 volume), the cells were harvested b y centrifugation at 2 °, washed twice with cold o.oI M Tris-HC1 buffer (pH 7-3) containing o.oi M KC1 (0.03 volume each time) and finally frozen. RNA was subsequently isolated b y the following method 14. The frozen cells were suspended in the Tris-HC1 buffer (o.03 volume), a solution of 20 °/o sodium dodecylsulfate was added to a final concentration of 0.5 %, the mixture was heated to 50 ° and was then shaken for 3 min. The pH of the suspension was adjusted to 5.6~5 by the addition of 0.3 N acetic acid and the mixture shaken for 5 rain at 60 ° with an equal volume of phenol previously equilibrated with o.oi M sodium acetate buffer (pH 5.2). This was followed by centrifugation for 2o rain in a Wifug centrifuge at 6000 rev./min. The operation was repeated three or four times (until absence of interphase). The aqueous RNA solution was purified on a Sephadex G-25 column (where phenol separates from RNA). After passage through the Sephadex column, the RNA was precipitated b y addition of two volumes of ethanol (96 °/o) and collected b y centrifugation. The precipitate was dissolved (i mg/ml) in 0.o6 M KC1 and frozen at --2o ° in I-ml portions. All the RNA preparations, which were used, contained less than I °/o of DNA and protein. A comparison has been made between the samples of RNA which were obtained from cells which were lysed in different ways. A culture of cells was divided into two equal parts, one of which was lysed with sodium dodecylsulfate, the other which had been disrupted for I min in a Mullard ultrasonic disintegrator, at + 2 ° prior to treatment with sodium dodecylsulfate. Both batches yielded the same amount of RNA. However, the RNA prepared after ultrasonic treatment contained 2 o(, of DNA in contrast to the other preparation which contained none.
Preparation o/ DNA All samples of DNA from E. coli were isolated and purified after treatment of the cells with lysozyme and sodium dodecylsulfate according to the method of MARMUR15. Biochim. Biophys, Acta, 114 (1966) 254-263
EFFECT OF MITOMYCIN C ON DNA AND m-RNA IN E. coli
257
Hybridization The procedure of NYGAARD AND HALL14 was used. Nitrocellulose membrane filters were soaked in o.oi M Tris-HC1 buffer (pH 7-3) containing o.5 M KC1 for 30 rain and each filter was washed with 25 ml of the buffer before application of the samples of nucleic acid. The filter was mounted on a stainless steel grid with a stainless steel cylinder placed on top. Suction was provided by a water pump. The DNA used was denatured by heating at a concentration of Ioo#g/ml at 9°° for 15 rain in o.oi M Tris-HC1 buffer (pH 7.3) containing 0.06 M KC1 and rapidly cooled by pouring the solution on to half its volume of frozen buffer. KC1 was added to a final concentration of 0.5 M, and I-ml samples were frozen at --20 °. Samples of RNA solutions were made 0.5 M with respect to KC1 and filtered through the membrane filter prior to complex formation (to reduce the 'background' caused by the trapping of free RNA). Desired amounts of RNA and DNA were mixed, diluted with o.oi M Tris-HC1 buffer (pH 7.3) containing 0. 5 M KCI and heated in a waterbath to a given temperature. At intervals, samples varying from lOO-25o #1 depending upon the radioactivity of the RNA, were diluted into 15 ml of the same buffer at room temperature. In the diluted state at this temperature no further reaction occurs and the hybrid is stable for several hours. The samples were filtered through the membrane filters and the filters washed with 60 ml of the KC1 solution at room temperature, airdried and counted. To determine the total radioactivity in hybridization mixtures an aliquot (5o/*1) was mixed with 25/~g of thymus-DNA dissolved in 25 #I of water, and trichloroacetic acid solution was added to the mixture to a final concentration of IO % (w/v). The samples were allowed to stand in an ice bath for 3 ° rain, filtered through a nitrocellulose filter (previously washed with water) and washed with IO ml of IO % trichloroacetic acid. The filter was air-dried and counted. All analyses were carried out in duplicate.
RESULTS It is well known that the amount of mitomycin C required to stop the synthesis of DNA varies from bacterium to bacterium1,2, 4 and even in different strains of the same bacteriumt, 1~. Thus, in the present case it was necessary to determine the concentration of mitomycin C required to stop DNA synthesis. It is seen from Fig. I that I0 #g/ml of mitomycin C is sufficient to stop DNA synthesis completely, whereas in the presence of 0.I #g/ml and I #g/ml some synthesis takes place. Thus, in all subsequent experiments the concentration of mitomycin C used was I0/~g/mt. In addition, its effect on RNA and protein synthesis is reported in Figs. 2a and 2b. It is interesting to note that upon addition of mitomycin C there is an increase in RNA synthesis over the first 3 ° min.
DNA isolated/rom mitomycin C treated and control cultures An exponentially-growing culture with a density reading of 0.35 was divided into two halves and mitomycin C (I0/~g/ml) was added to only one of the flasks.
Biochim. Biophy. Acta, 1I 4 (1966) 254-263
258
I. S M I T H - K I E L L A N D
30(
20( 3
0
a
100 I
o©
I
75
go
Time (rain)
Fig. i. T h e e f f e c t of m i t o m y c i n C on D N A - s y n t h e s i s . A n e x p o n e n t i a l l y g r o w i n g c u l t u r e of E. coli ( d e n s i t y r e a d i n g o.35 ) w a s d i v i d e d into four e q u a l portions. To t h r e e of t h e m were a d d e d o.I, I.O a n d I O / , g / m l of m i t o m y c i n C respectively, a n d t h e f o u r t h w a s u s e d as a control. T h e c u l t u r e s w e r e i n c u b a t e d a n d a n a l y z e d a t i n t e r v a l s for t h e i r c o n t e n t of D N A . M i t o m y c i n C w a s a d d e d a t zero t i m e . O - O , c o n t r o l culture. M i t o m y c i n C c u l t u r e s : Z~-&, o.I # g / m l ; r q - [ ] , I ffg/ml; © - © , loffg/ml. lOOO
5000
b
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600
2 5u
4000 3000
9
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200
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.
.
.
,
.
,
0 1 5 3 4 0 5 6075 90
,
,
,
,
,
,
0 15 30 45 60 75 90
Time (min)
Time (rain)
Figs. 2a a n d 2b. T h e e f f e c t of m i t o m y c i n C (io/~g/ml) o n R N A a n d p r o t e i n s y n t h e s i s . E x p e r i m e n t a l c o n d i t i o n s as described in Fig. i. M i t o m y c i n C w a s a d d e d at zero t i m e . O - O , c o n t r o l culture; O - O , m i t o m y c i n C t r e a t e d culture.
Both were incubated for further 3o min after which the control and mitomycin Ctreated cultures had reached densities of o.42 and o.41, respectively. DNA was isolated from each culture and used in all subsequent experiments. The yields of DNAMc and DNA c per culture was 2.85 mg and 3.54 mg, respectively. The appearance of DNAMc differed from that of DNA c. While the DNA c formed long threads upon precipitation with ethanol, most of the DNAMc was precipitated as shorter threads. The ultraviolet absorption spectra of the two DNA's were determined in o.oI M Tris-HC1 buffer (pH 7.3)- The spectra were identical. Biochim. Biophys. Acta, i14 (1966) 254 263
EFFECT OF MITOMYCIN C ON DNA AND m-RNA IN E. coli
259
9O0 ~._ 0.8
o ~50C O7 ~a 30C o 10C
0.6 50
I
60
¢
,
O 80 Temperoture
,
80
¢
0
,
60
,
50
0
t
t
1
2
Time of hybridization
t 3
i 4
i ~ l 5
24
(h)
Fig. 3. Melting, and slow cooling, curves for DNAMc and D N A o Equal a m o u n t s (5o/~g) of DNAMc and DNAc were dissolved ill 2.5 ml o.oo15 M t r i s o d i u m citrate buffer (pH 7.5) containing O.Ol 5 M NaC1 and heated up to 82 ° (i°/min) followed by slow cooling (I.5°/min). A heating block m o u n t e d directly to a Zeiss recording thermospectrophotometer was used. O - Q , DNAc; O - Q , DNAMo Fig. 4. The effect of t e m p e r a t u r e and time u p o n D N A - R N A hybrid formation. The R N A used was isolated from a culture of optical reading 0.45 after a pulse-labelling of 2 m i n with I5/~C of [2-14Cluracil/1. A n u m b e r of identical m i x t u r e s each containing 80/~g of h e a t - d e n a t u r e d DNAc and 2oo/~g of R N A c (162 counts/min]/~g of RNA) were incubated at different temperatures. Samples were taken out at intervals.
The effect of deoxyribonuclease on the two types of DNA was studied. Equal amounts (25/~g) of DNAMc and DNA c were each dissolved in 3 ml of o.oi M Tris-HC1 buffer (pH 7.3) containing o.oi M magnesium acetate and 0.05 mg of deoxyribonuclease. The mixtures were incubated at 37 ° and the maximal increase in absorbance at 260 m~ was determined. In the case of DNAMc the increase was 30 %, whereas in the case of DNA c it was 42 %. To characterize the two DNA types further, a comparison between the changes in ultraviolet absorption upon heating and slow cooling was carried out. It is seen from Fig. 3 that the transition point (Tm) for the DNAMc and DNA c is very nearly the same (75.7 ° and 75.2 ° respectively). However, the extent of increase in absorbance is lower for DNAMc (25 %) than for the control (32 %). Upon slow cooling, the DNAMc showed a decrease in hyperchromicity at a higher temperature than that of DNA c.
Hybridization experiments To determine the most suitable conditions for the formation of hybrid between DNA c and RNA o hybridization at different temperatures was carried out. It is seen from Fig. 4 that maximal hybrid formation, stable for 24 h, is obtained at a hybridization temperature of 65 °. Thi~ temperature has been used in all subsequent experiments. Some experiments have been carried out to study the initial rate and extent of hybridization with increasing concentration of RNA. It is seen from Fig. 5 that the initial rate and the extent of complex formation is proportional to the concentration of RNA. Thus, even with a 13 fold higher concentration of RNA (than of DNA) maximum complex formation occurs. Biochim. Biophys. Acta, 114 (1966) 254-263
200
I. SMITH-KIELLAND
2400 2400 t
2100
2ooo, 800/J£ RNA c~ 1600-
1800 E 1500 u~
~ 1200 0 U
>., 9 0 0 >
"5 600
X
_~lt
250 U~ RN
300 t',"
o
o Time of hybridization (h)
Fig. 5. H y b r i d - f o r m a t i o n as a f u n c t i o n of IRNA-concentration. T h e R N A w h i c h w a s u s e d w a s isolated f r o m a c u l t u r e of optical r e a d i n g o.45 after p u l s e labelling for 3 ° sec w i t h 15 ffC of [2-14C] uracil/l. M i x t u r e s , each c o n t a i n i n g 6 o f f g of h e a t - d e n a t u r e d D N A c a n d 250, 500 or 8oo/~g of R N A c ( 6 o c o u n t s / m i n / / ~ g of R N A ) b u t o t h e r w i s e identical, were i n c u b a t e d a n d s a m p l e s were taken out at intervals (×-×). Inset: Initial rate (O-O).
To examine the complex formation between DNA c and messenger RNA from mitomycin C and control cultures, RNA preparations which have been pulse-labelled for I5, 30, 60 and 12o sec have been used. It is seen from Fig. 6a that in the case of RNA pulse-labelled for 15 sec, the extent of complex formation between DNA c and RNAMc is about 30 % less than what it is for RNA c. It is apparent from Fig. 6b that this difference (16 %) although significant, is less pronounced when RNA pulse-labelled for 30 sec was used. In two additional experiments this difference was found to be 17 and 20 % respectively. Fig. 6c shows that in the case of RNA pulselabelled for 60 sec the difference is less. RNA pulse-labelled for 12o sec forms slightly more hybrid with DNA c than does RNA c (Fig. 6d). An examination of the ability of DNAMc to form complexes with pulse-labelled RNA c compared to that of DNA c has also been carried out. Fig. 7 shows that the ability of DNAMc to form complexes is 17 % lower than that of DNA c. In identical experiments with samples of DNA isolated from two different batches of E. coli, the hybrid formation between DNAMc and RNA c was 17 and 2I O//olower than the controls.
DISCUSSION
The concentration of mitomycin C (IO #g/ml) required to stop DNA synthesis in the present strain is similar to that found for other strains 15.A number of reports 16-1s have appeared stating that the primary effect of mitomycin C is to depotymerize DNA. On the other hand IYER AND SZYBALSKI~ suggest that the breakdown is an accessory phenomenon only secondarily related to the lethal effect of the antibiotic. The present result (Fig. I) supports SZYBALSKI'Sview since the DNA concentration of the mitomycin C-inhibited culture remains constant over a 2-h period. Recent Biochim. Biophys. Acta, 114 (1966) 254 263
ON DNA AND m-RNA in E. coli
EFFECT OF MITOMYCIN C
26I
10
"e
8
8
~6
6
6
o 4 Q~
-6
2 ~S d i
I
'
I
3
5
Time of
~0
I
0
hyboidizetion (h)
I
1 3 Time of hybridization (h)
I
5
8
~8
6
o 4
o
I
0
I
1 3 Time of hybridization (h)
2
J
5
I
0
I
I 3 Time Of hybridization (h)
I
b
Fig. 6. T i m e - c o u r s e of h y b r i d i z a t i o n b e t w e e n D N A c a n d pulse-labelled R N A M c a n d R N A c respectively. T h e R N A s a m p l e s u s e d were e x t r a c t e d f r o m c u l t u r e s as follows: A g r o w i n g c u l t u r e ( a b s o r b a n c e of o.35) w a s d i v i d e d into two e q u a l p a r t s a n d IO/~g/ml of m i t o m y c i n C w a s a d d e d to one of t h e m . A f t e r 3o m i n , 15/~C of E2-14C~uracil]l w a s a d d e d to each c u l t u r e a n d t h e g r o w t h t e r m i n a t e d 15, 3 o, 6o, or 12o sec later b y p o u r i n g 1/4 of each c u l t u r e after t h e a p p r o p r i a t e t i m e i n t e r v a l o n to frozen m e d i u m k e p t a t - - 4 o°. R N A M c or R N A c (4oo/~g) w a s i n c u b a t e d w i t h 6 0 / , g of h e a t - d e n a t u r e d D N A , a n d s a m p l e s were r e m o v e d a t i n t e r v a l s . Figs. 6a, b, c, d describe exp e r i m e n t s w i t h R N A p r e p a r e d a f t e r p u l s e s of 15, 3 o, 6o a n d 12o sec. 0 - O , R N A c ; O - O , RNAMc.
work suggests 3 that in a mitomycin C-inhibited culture the two strands in the DNA molecule are crosslinked. That the DNAMc examined in the present work also has different physico-chemical properties from those of DNA c is supported by several findings such as the smaller increase in absorbance after the action of deoxyribonuclease and after heating to 80 °. Upon slow cooling, the decrease in hyperchromicity occurs at a higher temperature than that at which the hyperchromicity of the DNA c begins to decrease. This is in agreement with the observation of MATSUMOTOAND LARK4. The difference in hybrid-formation between messenger RNA c and DNAMc and DNA c respectively (Fig. 7), demonstrates the structural differences between the two DNA's. RNA isolated after I5-sec pulse with L2A4Cluracil in the mitomycin C-treated culture differed from messenger RNA from a control culture in its reduced ability Biochim. Biophys. Acta, 114 (1966) 254-263
262
I. SMITH-KIELLAND 2400 2200 2000 1800 1600
'F- 1 4 0 0 u~ 1200 o
1ooo
>, 800 60o
o ~o O~
400 200 I
0'"
I
I
1 2 3 Time of hybr'idizotion
I
4 (h)
l
I
5
6
Fig. 7. Time-course of hybridization between DNAMc and DNAc and pulse-labelled 1RNAC. The preparation of R N A which was used was identical to t h a t described under Fig. 5. R N A (5oo/tg) was incubated with i 2 o / , g of h e a t - d e n a t u r e d DNAMc and D N A c respectively, a n d samples were taken out at intervals. 0 - 0 , DNAc: Q-C), DNAMc.
to form hybrids with heat-denatured DNA c. This difference decreases with the duration of the pulse (after a pulse of 12o sec the degree of hybridization with DNA is somewhat higher for RNAMc than for RNAc) and m a y be due to a faster turnover of messenger RNA c than that of messenger RNAMc. On the other hand it is conceivable t h a t the presence of radioactive impurities in the messenger RNA from the mitomycin C culture could explain the lower percentage of hybridization. Since uracil is the labelled substance and because of the extensive purification of RNA, the presence of any impurities does not seem very likely. Since crosslinks in the DNA molecule prevent a separation of the two strands at the crosslinked sites, this would be the most plausible explanation for the reduced transcription. The recent reports that messenger RNA is synthesized from one strand of the DNA molecule~°, 21, are not contradictory to this view.
ACKNOWLEDGEMENTS
I wish to thank Professor A. NYGAARD, Department of Biochemistry, University of Bergen Medical School, Bergen, for m a n y helpful discussions and Miss S. SCHAU for excellent technical assistance. I am grateful to Mr. E. JELLUM, Central Laboratory, Rikshospitalet, Oslo (Norway) for the use of a Zeiss Spectrophotometer. Financial support from Nansenfondet and The Norwegian Research Council for Science and the Humanities is gratefully acknowledged. Biochim. Biophys. Acta, 114 (1966) 254 263
EFFECT
OF MITOMYCIN
C ON D N A
AND m - R N A
IN E . coli
263
REFERENCES I 2 3 4 5 6 7 8 9 io ii 12 13 14 15 16 17 18 19 20 21
S. SHIBA, A. TERA~VAKI, T. TAGUCHI AND J. KA'vVAMATA,Nature, 183 (1959) lO56. V. N. IY~R AND W. SZYBALSKI, Proc. Natl. Acad. Sci. U.S., 50 (1963) 355. V. N. IYER AND W. SZYBALSKI, Science, 145 (1964) 55I. MATSUMOTO AND K. G. LARK, Exptl. Cell Res., 32 (1963) 192. B. D. HALL AND S. SPIEGELMAN, Proc. Natl. Acad. Sci. U.S., 47 (1961) 137. D. GIACOMONI AND S. SPIEGELMAN, Science, 138 (1962) 1328. H. M. GOODMAN AND A. RICH, Proc. Natl. Acad. Sci. U.S., 48 (1962) 21Ol, S. A. YANKOFSKY AND S. SPIEGELMAN, Proc. Natl. Acad. Sci. U.S., 49 (1963) 538. I. SMITH-K1ELLAND, Biochim. Biophys. Acta, 91 (1964) 360. I. SMITH-KIELLAND, Biochem. J., 92 (1964) 26P. W. I~¢[EJBAOM, Z. Physiol. Chem., 258 (1939) 117. K. BURTON, Biochem. J., 62 (1956 ) 315O. H. LOWRY, lXT.J, ROSEBROUGH, A. L. FARR AND R. J. RANDALL, J. Biol. Chem., 193 (I95I) 265. A. P. NYGAARD AND B. D. HALL, Biochem. Biophys. Res. Commun., 12 (1963) 98. J. MARMUR, J. Mol. Biol., 3 (1961) 208. M. SEKIGUCHI AND Y. TAKAGI, Biochim. Biophys. Acta 41 (196o) 434. H. I~ERSTEN AND W . I~ERSTEN, Z. Physiol. Chem., 334 (1963) 141" Y. •AKATA, K. NAKATA AND Y. SAKAMOTO, Biochem. Biophys. Res. Commun., 6 (1961) 339. E. REICH, A. J. SHATKIN AND E. L. TATUM, Biochim. Biophys. Acta, 45 (196o) 6o8. ~¢[. HAYASHI, M. N, HAYASHI AND S. SPIEGEl_MAN, Proc. Nat. Acad. Sci. U.S., 5 ° (1963) 664. J. MARMUR AND C. GREENSPAN, Science, 142 (1963) 387 •
Biochim. Biophys. Acta, 114 (1966) 254-263