Biological activity of β-phenylethanol and its derivates

506

BIOCHIMICA ET BIOPHYSICAACTA

BBA 96914

BIOLOGICAL ACTIVITY OF /5-PHENYLETHANOL AND ITS D E R I V A T E S V. I N F L U E N C E ON DNA AND RNA SYNTHESIS IN D I F F E R E N T I N V I T R O SYSTEMS

W. E. G. M U L L E R , B. H E I C K E AND R. K. Z A H N

Physiologisch-Chemisches Institut d~r Universitd!, 65 Mainz, Saarstrasse 2I (Germany) (Received F e b r u a r y 26th, 197 t)

SUMMARY

I. /5-Phenylalcohol was found to be an inhibitor of DNA and RNA synthesis in vitro; thus in the presence of the drug the activities of DNA polymerases (deoxynucleoside triphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7) of Escherichia coli, of Paracentrotus lividus, of mouse lymphoma cells as well as of DNAdependent RNA polymerases (nucleoside triphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) of mouse lymphoma, of pig spleen and of E. coli are reduced. 2. The inhibition of mammalian DNA polymerase is of the non-competitive type. The inhibitor constant KI has been found to be in the same range as the /5phenylethanol concentration, which in vivo reduces the cell division rate of mouse lymphoma cells to 5° %. Therefore the in vivo inhibition is attributed to the inhibitory effect of fl-phenylethanol on the DNA-polymerizing system. Further studies indicate that the enzyme protein is affected, /5-phenylethanol neither acting as chelating agent (for the required Mg 2+) nor as substrate (base) analogue. 3- The data in this paper show considerable variation of inhibitory fl-phenylethanol concentrations for the polymerases from different organisms.

INTRODUCTION

/5-Phenylethanol, an autoantibiotic produced by the fungus Candida albicans 1, inhibits cell proliferation in microorganisms, plants and animals (survey ref. 2). About the major sites of actions of/5-phenylethanol in the different test systems, some conflicting views have been presented in literature. It has been assumed that fl-phenylethanol changes permeability and transport properties of cell membranes and thereby influences cell proliferation 3. This hypothesis at least does not apply for some mammaliar cells2, whereas it seems to be valid for microorganisms 4,5. Further mechanisms of action have been proposed and consist of the blocking of different steps of informationdirected synthesis, e.g. DNA synthesis (in bacterial cells °, in mammalian cells 7) or RNA synthesis (in bacterial cells8,9, in mammalian cells1°). Testing in vivo mouse lymphoma cells for/5-phenylethanol inhibition, shows 2,11 DNA synthesis among the most sensitive activities which are observed. Biochim. Biophys. Acta, 240 (I97 t) 5o6-514

PHENYLETHANOLAND DNA AND RNA SYNTHESIS

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In this paper we report on the influence of fl-phenylethanol on different isolated enzyme preparations, considering the inhibitor concentrations effective on Escherichia coli versus metazoan systems TM. MATERIALSAND METHODS Materials

Materials were obtained as follows: dATP, dCTP, dGTP, dTTP, E3HldATP (specific activity 15.1 C/mmole) and ~14C]dATP (specific activity, 53 mC/mmole) from Schwarz Bioresearch, Orangeburg, N.Y. (U.S.A.). ATP, CTP, GTP, UTP, creafine phosphate and creatine phosphokinase (ATP:creatine phosphotransferase, EC 2.7.3.2 ) from Boehringer Mannheim, Tutzing (Germany). EnCIUTP (specific activity, 56 mC/mmole) from The Radiochemical Centre, Amersham (England). E. eoli DNA polymerase (deoxynucleoside triphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7) (specific activity, 5000 units/rag protein), isolated according to the method of RICHARDSON et al. 13, Fraction VII and E. coli DNA-dependent RNA polymerase (nucleoside triphosphate:RNA nucleotidyltransferase, EC 2.7.7.6 ) (specific activity, 2000 units/rag protein), according to CHAMBERLIZ~AND BERG14 from Biopolymers, Ohio (U.S.A.). Bovine pancreas deoxyribonuclease (12 500 units/rag) from Worthington Biochemical Corp., Freehold, N.J. (U.S.A.). Dialysis tubing (diameter, 21 ram) from Visking Comp., Chicago, Ill. (U.S.A.). The herring DNA, isolated according to ZAHN et al. ~5, was a gift of H. Mack, Illertissen, Bayern (Germany). Further sources of enzymes were: (I) Calf thymus, deep frozen directly after killing the animal. (2) Female sea urchins of the species Paracentrotus lividus Lain., collected at the Center for Marine Research, Rovinj (Yugoslavia). The medium and growth conditions of the L51~sv mouse lymphoma cells were as described previously~. Isolation o/polymerases

The DNA polymerases from calf thymus glands were isolated according to BOLLUMTM, with modifications according to HEICKE17. The isolation of the DNA polymerase of gonades from female sea urchins of the species Paracentrotus lividus was performed by a method described by MOLLERetal. TM. For DNA polymerase of mouse lymphoma the isolation procedure from Lsl,Sy cells has been used as described by us TM. For extraction of DNA-dependent RNA polymerase of L51,sy mouse lymphoma cells, a clean preparation of cell nuclei was isolated by the procedure of WANG19. The subsequent steps of lysis, DNA precipitation and extraction of the soluble enzyme were essentially those of SEIFART AND SEKERIS ~-°. Polymerase assays

The assays for the nucleotidyltransferase activities were based on the measurement of the incorporation of the nucleoside triphosphates into the acid-insoluble form when DNA or RNA wele added to the system. In the test for replicative deoxynucleotidyltransferase of calf thymus the reaction mixture TM of 0.8 ml contained: o.I mM [3H~dATP (4" lO6 counts/rain per/,mole), o.I mM dGTP, dCTP, and dTTP, 40 mM potassium phosphate (pH 7.0), 8 mM MgCI~, I mM 2-mercaptoethanol and Biochim. Biophys. Acta, 240 (1971) 5o6-514

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200/~g denatured DNA (heated io min in a IOO° water bath and chilled). The reaction was started b y the addition of 0.2 ml enzyme preparation. After an incubation time of o, 60 or 12o min in some cases at 37 °, 20/,1 were withdrawn, applied to a filter disc (Whatman No. I) and processed as described by BOLLUM21. For the testing of the terminal deoxynucleotidyltransferase from calf thymus the incubation mixture contained in a volume of 0.8 ml: 4 ° mM potassium phosphate buffer (pH 6.8), 8 mM MgC12, i mM [14C]dATP (7" lO5 counts/rain per /,mole), o.03 mM deoxyribooligonucleotides from herring sperm and 0.2 ml enzyme preparation. Further processing was as described earlier 17. In the case of the DNA polymerase of Paracentrotus lividus the assay was the same as for replicative deoxynucleotidyltransferase of calf thymus, except for the use of 0. 5 M (NH4)2CO 3 (pH 8.0) as buffer. Incubation at 37 ° was terminated after 60 rain. In the test for DNA polymerase of E. coli the reaction mixture (0. 9 ml) consists of o.I M E3H~dATP (8 • lO5 counts/min per/~mole), dCTP, d T T P and dGTP, 30 mM potassium phosphate buffer (pH 7.4), 8 mlV[ MgC12, I mM 2-mercaptoethanol, 200 #g native DNA (in 30 mM potassium phosphate buffer, p H 7.4), respectively, 200/~g denatured DNA, and o.I ml enzyme preparation. Further processing was as described earlier. For the testing of DNA-dependent RNA polymerase from L51~sy cells or from pig spleen the reaction mixture contained in 0.5 ml, 4.5 mM ATP, GTP, CTP, and [14C]UTP (6 • lO 5 counts/min per #mole), 12 my[ MgC12, I8 mY[ 2-mercaptoethanol, 241 mM (NH,)2SO 4, 12o mM Tris-HC1 (pH 7-9), IO mM creatine phosphate, 15/,g creatine phosphokinase, 18o/zg native DNA (in 12o mM Tris-HC1 buffer, p H 7.9) and o.2ml enzyme preparation. IOO#1 samples are taken at o up to 2omin and treated as described earlier. In the case of DNA-dependent RNA polymerase of E. coli the incubation mixture (0.8 ml) contained 4.5 mM ATP, GTP, CTP, and U T P (4" lO5 counts/min per ~mole), I2 mM MgC12, 18 mM 2-mercaptoethanol, 241 mM (NH,)~SO,, I2o mM TrisHC1 (pH 7.9), 4o/~g native DNA (in 12o mM Tris-HC1 buffer, p H 7.9) and o.I ml enzyme preparation. 2o-/,1 samples are taken at o and 15 rain and treated as described earlier. Oligodeoxynucleotides to serve as primer were obtained by enzymatic degradation of unbuffered herring sperm DNA in a pH-stat (Radiometer-Kopenhagen) at p H 7.0. The reaction mixture contained 500/~g DNA, 50/~g deoxyribonuclease I and o.I Mm MgC121~. For protein determination we used the biuret reagent of GORNALL et al. 22. In inhibitor assays varying amounts of fl-phenylethanol were added in buffered reaction mixtures. For controls the buffer solution~was added under equal conditions. The incubation was started with the addition of the enzyme preparation. The final p-phenylethanol concentrations causing 50 % inhibition (EDs0) and their standard deviation were calculated from the initial rate of 8 different p-phenylethanol concentrations between o and IOO % inhibition under the condition of template saturation b y logit regression 2'a. The inhibitor constants (Ki) were obtained by running tests at different non-saturating template levels against different inhibitor concentrations; the graphs are based on linear regression .3. The data of the DNA polymerase isolated from L51~sy cells came from the published K i values 1~. Biochim. Biophys. Acta, 240 (I97 I) 5o6-514

PHENYLETHANOL AND D N A AND R N A SYNTHESIS

509

RESULTS AND DISCUSSION P o s s i b l e inter/erence o / f l - p h e n y l e t h a n o l w i t h nucleoside t r i p h o s p h a t e s T h e question as to w h e t h e r f l - p h e n y l e t h a n o l inhibition comes a b o u t b y interference w i t h D N A precursors has been t a c k l e d b y t e s t i n g t h e 50 % i n h i b i t o r y conc e n t r a t i o n (EDs0) in t h e presence of t w o t r i p h o s p h a t e concentrations, each with its proper controls (Table.I). A s a t u r a t i n g t r i p h o s p h a t e c o n c e n t r a t i o n is used in t h e assay, the o t h er concentration, IO times higher exerts a m o d e r a t e inhibition. I n b o t h cases fl-phenylethanol inhibition is just th e same, i n d i c a t i n g t h a t there is no d e t e c t a b l e interference of f l - p h e n y l e t h a n o l w i t h D N A precursors.

TABLE 1 P O S S I B L E I N T E R F E R E N C E OF ~ - P H E N Y L E T H A N O L W I T H N U C L E O S I D E T R I P H O S P H A T E S U B S T R A T E S IN E N Z Y M A T I C D N A S Y N T H E S I S B Y CALF T H Y M U S R E P L I C A T I V E D E O X Y N U C L E O T I D Y L T R A N S F E R A S E W I T H D E N A T U R E D D N A AS T E M P L A T E

Incubation was 60 rain at 37°. The results of three experimental series are given with their standard deviations. Assay No.

Deoxyribonucleoside triphosphate conch. (raM)

fl-Phenylethanol concn, (mg/ml) (raM)

Incorporation rate ( ! S.D.) nmoles dA T P • rn1-1 • h -1 ( × to_l)

%

ia ib

o.i o.i

7,2olo.12 3.51±o.o4

lOO.O11. 7 48.7t1.1

2a 2b

i.o i.o

-0.53 4.34 -0.53 4.34

6.92to.o8 3.37!o.o 4

lOO.Otl.2 48.7tl.2

Possible inter[erence o / ~ - j b h e n y l e t h a n o l w i t h activating M g ~+ T h e Mg 2+ c o n t e n t of t h e i n c u b a t i o n m i x t u r e has been changed so t h a t a decrease in t h e incorporation rate for the precursors occurred which is j u s ~ i g n i f i c a n t (Table II). A d d i n g t h e EDs0 of ~ - p h e n y l e t h a n o l causes t h e same inhibition in b o t h cases. I t seems impossible t h a t /~-phenylethanol inhibition should come a b o u t b y interference w i t h t h e a c t i v a t i n g Mg ~+.

TABLE 1I P O S S I B L E I N T E R F E R E N C E OF f l - P H E N Y L ~ T H A N O L W I T H T H E ACTIVATING Mg~+ IN T H E E N Z Y M A T I C D N A ASSAY W I T H CALF T H Y M U S R E P L I C A T I V E D E O X Y N U C L E O T I D Y L T R A N S F E R A S E

Denatured DNA was used as template. Incubation was for 6o rain at 37 °. ]Results come from three independent experimental series. Assay No.

MgCl 2 conch. (raM)

~-Phenylethanol conch. (mg/ml) (mM)

Ia ib

4 4

2a 2b

8 8

-0.53 4-34 -0.53 4,34

Incorporation rate ( ± S . D . ) nmoles dA T P . m1-1 • h -1 ( × zo_l )

°/o

4.22to.19 2.251O.lO

lOO.O14.5 53.3±4.5

4.52!o.2o 2,32!o.19

IOO.O~2.2

51.3±8.2

Biochim. Biophys. Acta, 240 (1971) 5o6-514

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al.

Reversibility o] possible action o/fl-phenylethanol on the DNA template LEACH el alY published experimental results on a shift in the hyperchromicity in DNA upon addition of fl-phenylethanol, pointing to an alteration of template properties. Such a change should not easily be reversible as long as the denaturing conditions are sustained, while fl-phenylethanol is dialysed away from the DNA. Yet even using the highest fl-phenylethanol concentration possible (approx. 18 times the ED60 ) for interaction with denatured DNA or with native DNA there is no detectable trace (TablelII). This is in accordance with other investigations which have not shown any modification of DNA by fl-phenylethanols,23,25. Possibly, the mentioned alterations of template DNA had been caused by impurities 26, i.e. phenethylaldehyde that might have entered into reaction with the DNA. TABLE III REVERSIBILITY OF POSSIBLE ACTION OF fl-PHENYLETHANOL ON D N A TEMPLATE IN MOUSE LYMPHOMA D N A POLYMERASE REACTION (a) IO ml denatured D N A (2 mg]ml) were dialyzed against 200 ml of a s a t u r a t e d fl-phenylethanol solution in distilled w a t e r at 2 ° (approx. i8 times ED~o ) for 4 h. T h e n the solution was transferred into two changes of ~ 1 distilled w a t e r for 6 h each. (b) io ml native D N A (2 mg/ml) were treated with fl-phenylethanol as before. T h e n the D N A was heat denatured and tested. (c) F o r controls d e n a t u r e d D N A was dialyzed for 16 h against water. The p r e p a r a t i o n s were tested in n o n - s a t u r a t ing t e m p l a t e conditions using a concentration of 80 • lO -6 g/ml DNA. Pretreatment o/

Incorporation rate ( i S.D.)

D N A template

nmoles dA T P • m1-1 • h -1

%

( × lo-~) None Effect of fl-phenylethanol on denatured D N A Effect of fl-phenylethanol on native D N A

1.93 ± o. 16

IOO.OzL 8.29

1.82=/=o.17

94.o~8.78

1.98io.I9

IO2.7~9.85

Reversibility o/fl-phenylethanol action on the enzyme The same kind of procedure as in the preceding investigation is applied for testing the reversibility of enzyme inhibition at 1. 7 times the EDs0 concentration in fl-phenylethanol (Table IV). Terminal transferase was taken because of its higher TABLE IV REVERSIBILITY OF fl-PHENYLETHANOL ACTION ON CALF THYMUS D N A - T E R M I N A L TRANSFERASE

(a) F o r controls 0.5 ml of the enzyme p r e p a r a t i o n were dialyzed for 16 h against a 4 mM p o t a s s i u m p h o s p h a t e buffer (pH 6.8) with I mM 2-mercaptoethanol at 2 °, (b) 0. 5 ml enzyme were dialyzed in parallel for 4 h against i 1 of equally buffered fl-phenylethanol solution (18 mM ~ 1. 7 times EDs0 ). Subsequently, the enzymes were dialyzed against two changes of the fl-phenylethanolfree buffer for 6 h each. The two enzyme p r e p a r a t i o n s were t h e n adjusted to equal volumes and tested for terminal transferase activity using t e m p l a t e s a t u r a t i n g conditions. The values come from three independent experimental series. Enzyme

Without fl-phenylethanol t r e a t m e n t After fl-phenylethanol t r e a t m e n t

Incorporation rate ( ~ S.D.) nmoles d A T P • m1-1 . h -1

%

4.o6io.98

IOO.O-~ 24.I

3.9o~=o.9I

96.ot22.4

Biochim. Biophys. Acta, 24 ° (I97 I) 5o6-514

PHENYLETHANOL AND

DNA AND RNA SYNTHESIS

5I~I

stability towards dialysis. As a consequence of the treatment, standard deviations were observed to increase. No significant inhibitory effect following dialysis is discernible. This in vitro reversibility compares well with the in vivo reversibility*, making repair mechanisms unnecessary.

Quantitative comparison o/fl-phenylethanol action on di//erent polymerases If it should turn out that a DNA-synthesizing enzyme, (acting in replicating or in repair synthesis) should be the primary target, this could be recognized by its highest sensitivity towards fl-phenylethanol inhibition e' lX,2a. The inhibitory concentrations in the enzyme assay should be comparable to the inhibitory concentration in the cell culture, unless it is shown that fl-phenylethanol is actively transported into and accumulated in the cells, which we consider quite unlikely on the evidence available at present. For comparison of the fl-phenylethanol sensitivity we plotted the EDso concentrations and some K, values for 9 enzymes (Table V). The most sensitive enzyme is

TABLE V Q U A N T I T A T I V E COMPARISON OF f l - P H E N Y L E T H A N O L ACTION ON D I F F E R E N T POLYMERASES

The fl-phenylethanol concentration t h a t causes a 5 ° % reduction in incorporation rate has been determined under the conditions of template saturation. In K, determinations template concentration was parameter. Enzymes

Source of the enzyme

I. DNA polymerases

Mouse lymphoma cells Ls~sy Calf thymus (enzyme fraction : replicative deoxynucleotidyltransferase) Calf thymus (enzyme fraction: terminal deoxynucleotidyltransferase) Escherichia coli (template: denatured DNA) Escherichia coli (template: native DNA) Paracentrotus lividus

fl-Phenylethanol concn. (4- S.D) causing 50 °/o inhibition (mg/ml)

Inhibitor constant, K i (4- S.D.) (mg/ml) (raM)

0.864-0.35 7.054-2.86 o.714-o.3o 5.824-2.46

0,824-0.25 6,724-2.o5 o,534-o.I9 4.344-1.56

1.514-0.69 12.514-5.65

1.42-4-o.63 11.63±5.16

16.924-5.34 138.5 4-43.4 I7.434-4.68 142.6 +38.5 20.6 4-7.2 168.8 -4-59.0

II. D N A -

dependent RNA polymerases

Mouse lymphoma cells L51~a~ Pig spleen Escherichia coli

8.1 66.4 9.3 76.4 13.2 lO8.2

4-3-4 4-27.9 4-3.9 4-31.7 4-6.7 4-54.9 Biochim. Biophys. Acta, 240 {i97 I) 5o6--514

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al.

the calf thymus replicative nucleotidyltransferase. In case it should turn out that this is not the DNA-synthesizing enzyme 27 of the calf thymus cells, its function could be tested b y means of/%phenylethanol. The mouse lymphoma DNA polymerase, which has not been purified to the same extent shows about the same sensitivity. The calf thymus terminal transferase falls into about the same range, the difference between EDs0 of replicative to terminal transferase being significant at the level of P < 0.03. Enzymes as sensitive as these extracted polymerases in our opinion are also likely to be strongly affected b y comparable ~-phenylethanol levels in the cell. Indeed as a comparison in intact L cells in culture a reduction of the 72-h cell count to 50 ~o of the control value was brought about by o.55±o.o3mg/~-phenylethanol per ml and this is in perfect agreement with the above in vitro figures 2. In addition it has been shown that over this concentration range of/~-phenylethanol intracellular thymidine incorporation into the DNA fraction was seriously impaired, whereas uracil incorporation into the RNA fraction was not affected measurably 2. On the other hand the E. coli DNA polymerase and a DNA polymerase from Paracentrotus lividus were found to be less sensitive to/~-phenylethanal b y one to two orders of magnitude and so are all three DNA-dependent RNA polymerases.

Type o/$-phenylethanol inhibition It has not been decided whether the reversible action of/%phenylethanol is on the template or on the enzyme itself. Eventuallythe type of inhibition could provide

15

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20"

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// •

/x//~

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/"

/ ~

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Fig. I. I n h i b i t i o n of calf t h y m u s t e r m i n a l d e o x y n u c l e o t i d y l t r a n s f e r a s e b y f l - p h e n y l e t h a n o l . Plot according to LINEWEAVER AND BURK 2s. x-axis: I/[S], reciprocal v a l u e s of t h e p r i m e r concent r a t i o n in m l / m g oligonucleotides, y-axis: I/v, reciprocal v a l u e s of t h e initial r e a c t i o n velocity in lO 9 × h . m l . n m o l e s -1 d A T P . ( ~ o n - s a t u r a t i n g p r i m e r c o n c e n t r a t i o n s . ) L i n e a r regressions: 0 - 0 , control; × - - - × , i n h i b i t o r c o n c e n t r a t i o n , i.o m g / m l ; A - . - A , inhibitor c o n c e n t r a t i o n , 1.6 m g ] m l ; A - . - Z x , i n h i b i t o r c o n c e n t r a t i o n , 2.2 m g / m l f l - p h e n y l e t h a n o l . K~ ~ 1,42 ( ~ o . 6 3 ) m g fl-phenyle t h a n o l ; Km ~ 29 ( i 5 ) " IO-e g oligonucleotides p e r ml. Fig. 2. I n h i b i t i o n of calf t h y m u s replicative d e o x y n u c l e o t i d y l t r a n s f e r a s e b y f l - p h e n y l e t h a n o l . P l o t according to DIXON AND WEBB 2~. X-axis: [I], i n h i b i t o r c o n c e n t r a t i o n s in m g f l - p h e n y l e t h a n o l p e r ml; y-axis: i/v, reciprocal v a l u e s of t h e initial r e a c t i o n v e l o c i t y in i o * × h • m l . n m o l e s -1 d A T P . ( N o n - s a t u r a t i n g t e m p l a t e c o n c e n t r a t i o n s . ) L i n e a r regressions: Q - Q , t e m p l a t e c o n c e n t r a t i o n , 16o/~g D N A per ml; X - - - × , t e m p l a t e c o n c e n t r a t i o n , 8 o / , g D N A per ml; A - . - & , t e m p l a t e c o n c e n t r a t i o n , 4 °/~g D N A p e r ml; K , = o.53 ( ± o . I 9 ) m g f l - p h e n y l e t h a n o l p e r ml; K m = 76 ( ~ 8 ) • lO -6 g D N A p e r ml.

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PHENYLETHANOL AND D N A A N D R N A s Y N T H E S I S

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us with some indication. In the case of L5178ynucleotidyl transferase a non-competitive inhibition type has been found with a K~ = o.82 mg fl-phenylethanol per ml (Table V) 11. In the case of the terminal transferase and the replicase of calf thymus the type of inhibition is the same (Figs. I and 2). In both cases an undisturbed non-competitive-type inhibition emerges quite clearly. Comparing the K, values with the EDs0 values the agreement in the case of terminal transferase is perfect (Table V). We know that this enzyme is quite stable and its preparation is highly reproducible. This is different with the replicase. In this case the initial velocities used in the case of K, determination are not strictly proportionate to the 6o-min measurements used for the EDs0 values run at saturating template conditions. Thus we find the agreement rather satisfying and we feel that the values in Table V may be accepted as fair estimates for the K, values of the enzyme. Regarding the good agreement of our in vitro results in isolated mammalian enzyme systems with data obtained from intact mammalian cells 2, we assume that fl-phenylethanol is exerting its inhibitory effect primarily by acting on the cellular DNA-polymerizing enzymes. The investigation will be continued.

ACKNOWLEDGEMENTS

We thank H. Mack GmbH, Illertissen, Germany, for gifts of highly purified fl-phenylethanol. The authors express their gratitude to Miss Eva Kruse, Christa Meyer, Ingeborg Walter, and Mr. R. Beyer for their excellent technical assistance. Some loans of equipment by Deutsche Forschungsgemeinschaft und b y Fraunhofer Gesellschaft are gratefully acknowledged.

REFERENCES I B. T. LINGUPPA, M. PRASAD AND J. LINGUPPA, Science, 163 (I969) 192. 2 W . E. G. MULLER, B. HEICKE, W . HANSKE, W . HOLLSTEIN AND R. K. ZAHN, s u b m i t t e d for publication. 3 H. L. HIGGINS, T. J. SHAW, M. C. TILLMANN AND F. R, L]~ACH, Exp. Cell Res., 56 (1969) 24. 4 S. SILVER AND L. WENDT, J. Bacleriol., 93 (1967) 560. 5 A. G-. RICHARDSON, D. L. PIERSON AND F. R. LEACH, Biochim. Biophys. Acta, 174 (1969) 276. 6 R. W . TREICK AND W . A. KONETZKA, J. Bacteriol., 88 (1964) 158o. 7 F. R. LEACH, N. H. BEST, E. M. DAVIS, D. C. SAI~DERS AND D. M. GI~LIN, Exp. Cell Res., 36 (1964) 524. 8 H. S. ROSENKRANZ, H. S. CARR AND H. M. R o s E , J. Bacteriol., 89 (1965) 1354. 9 C. PREVOST AND V. MOSES, J. Bacteriol., 91 (1966) 1446. IO P. G. W. PLAGEMANN, Biochim. Biophys. Acta, 155 (I968) 2o2. I I R. K. ZAHN, B. HEICKE, H. G. OCHS, E. TIESLER, W . FORSTER, W . HANSKE AND H. HOLLSTEIN, Nature, 212 (1966) 297. 12 W . E. G. MOLLER, B. HEICKE, A. MAIDHOF, W . FORSTER AND R. K. ZAHN,F E B S Left., 8 (197 ° ) 116. 13 C. C. RICHARDSON, C. L. SCHILDKRAUT, A. V. APOSHIAN AND A. I'~ORNBERG, J. Biol. Chem., 239 (1964) 222. 14 M. CHAMBERLIN AND P. BERG, Proc. Natl. Acad. Sci. U.S., 48 (1962) 81. 15 R. K. ZAHN, E. TIESLER, A. K. KLEINSCHMIDT AND D. LAI~G, Biochem. Z., 336 (1962) 281. 16 F. J. BOLLUM, in G. Z. CANTONI AND D. R. DAvis, Procedures in Nucleic Acid Research, H a r p e r a n d Row, N e w Y o r k - L o n d o n , 1966, p. 284. 17 B. HEICKE, Arch. Pharmakol., 266 (197 o) i4o. 18 W . MOLLER AND R. K. ZAHN, Tkalassia (Jugosl. Akad. Wiss.), 6 (197 o) lO 7.

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