558 PRELIMINARY NOTES

The authors are grateful to Dr. M. DAY for supplying the mastocytoma cells and to Dr. J. JAFFE for supplying the Ehrlich ascites cells.

Department of Pharmacology, Yale University School of Medicine, S. SCHANBERG* New Haven, Conn. (U.S.A.) N . J . GIARMAN

1 V. ERSPAMER, X X I Intern. Congr. Physiol. Sciences, 1959. 2 M. A. CEOSSLAND, J. Pharm. and Pharmacol., 12 (196o) i .

J. I~. COOPER, personal communica t ion . 4 C. E. DALGLIESCH, in Advances in Clinical Chemistry, Academic Press, New York, 1958, ¥ol . I,

p. 193. 5 R. ~RoDNIGHT AND H. MclLWAIN, Biochem. J., 57 (1954) 649.

G. V. R. BORN AND J. ]3RICKNELL, J. Physiol., 147 (1959) 153. 7 N. J. GIARMAN, unpubl i shed results. 8 j . H. GADDUM AND N. J. GIARMAN, Brit. J. Pharmacol., I i (1956) 88.

V. ]~RSPAMI~R, Rend. Sci. Farmilalia, I (1954) I.

Received May 9th, 196o

* Pre-doctora l Publ ic Hea l th Service Research Fel low (National i n s t i t u t e of Neurological Diseases and Blindness, BF-6489).

Biochim. Biophys. Acta, 41 (196o) 556-558

Regulatory mechanisms in the synthesis of deoxyribonucleic acid in vitro

In this communication we wish to describe some of the conditions for the synthesis of DNA in vitro, using a ribonucleotide as the labeled precursor. Such a system should contain the enzymes required for the conversion of the ribonucleotide to the corresponding deoxyribonucleotide, as well as the enzymes required for the in- corporation of the latter into DNA. An extract of 5-day old chick embryos obtained by centrifugation of a homogenate at 25,ooo /~ g for 45 rain was used as a source of the combined enzyme systems.

The conversion of cytidylic acid to deoxycytidylic acid seems to occur at the diphosphate leveP, whereas the deoxyribonucleoside triphosphates 2-4 are the pre- cursors of DNA. Thus, it was expected that the degree of phosphorylation of the ribonucleosides would be critical in defining the best conditions for the overall reaction. Fig. I shows that the addition of ATP alone was better than the presence of an ATP-regenerating system in maintaining an optimal balance between di- and tri- phosphates.

A maximal rate of DNA synthesis from deoxyribonucleotides occurs when all four deoxyribonucleotides are present 2-4. In the present system, using [l~Cldeoxy- CMP as the labeled precursor, the synthesis of DNA was stimulated by the addition of deoxyATP, deoxyGTP and TTP. In contrast, DNA formation from ~3H~CMP was strongly inhibited by the addition of these deoxynucleoside triphosphates. As is shown in Fig. 2, this inhibition occurred during the transformation of the ribosyl to the deoxyribosyl compound. Effective inhibition was observed at deoxyribonu-

Abbrevia t ions : DNA, deoxyribonucleic acid; ATP, adenosine 5 ' - t r iphosphate ; GTP, gua- nosine 5"-tr iphosphate; TTP, thymid ine t r iphospha te ; CMP and CTP, cyt idine 5 ' -mono- and t r i -phosphate , respectively.

Biochim. Biophys. Acta, 41 (196o) 558-559

PRELIMINARY NOTES 559

cleotide levels, which were very low when compared with the levels of CMP and ATP. Fig. 2 shows that in the presence of 155 m/~moles CMP and I/zmole ATP about 3 m/zmoles deoxyGTP cause a 50 % inhibition of the reaction.

CMP ~ dCMP

l C M P ~ DNA

30 rain

Fig, i . E n e r g y r e q u i r e m e n t for t he overal l convers ion of CMP ~ d e o x y C M P ~ DNA. The incuba t ion m i x t u r e con ta ined 114 m # m o l e s [~2p] CMP (4.6" I o s counts / ra in) , i .o #mo l e s ATP, 5.0 / ,moles MgC12, ioo #g hea t ed calf t h y m u s D N A ~, 15 # m o l e s t r i s ( h y d r o x y m e t h y l ) a m i n o - m e t h a n e - H C 1 buffer, p H 7-4, o-4 # m o l e mer- cap toe thano l , a n d o. 3 m l of e n z y m e (14 m g pro te in /ml) in a final v o l u m e of o.41 ml ( × - - × ) ; in addi t ion , 4.5 # m o l e s c rea t ine p h o s p h a t e a n d ioo #g crea t ine k inase were added to t he s a m p l e s ind ica ted b y + Cr ~ P ( O - - O ) . The s a m p l e s were i ncuba t ed a t 37 °, D N A was ex-

I f + dCTP 2.0" • ~ ~

= , . o ~ \ ~--<2y ~

5 ~o ,5' 3~,~3 ~, M x l O 5 Deoxyribonucleosidetriphos phote

Fig. 2. Effect of deoxyr ibonuc leos ide t r iphos- p h a t e s on t he convers ion of CMP to deoxyCMP. T he incuba t ion m i x t u r e con ta ined 155 r e#moles ~sH]CMP (2 .85 . io e counts / ra in) , i .o # m o l e ATP, 15 # m o l e s t r i s ( h y d r o x y m e t h y l ) a m i n o - m e t h a n e - H C 1 buffer, p H 8.0, 0. 4 # m o l e mercap, toe thano l ,deoxyr ibonuc leos ide t r i p h o s p h a t e s a s indicated, and 0. 3 ml of e n z y m e (13 m g pro te in /ml) in a final v o l u m e of o.38 ml . The s amp le s were i n c u b a t e d for 15 m i n a t 37 ° . D e o x y C M P was isola ted f rom the acid-soluble f rac t ion af te r acid hyd ro lys i s a n d ana lyzed ~.

t r ac t ed and a s sayed 6 and t h e resu l t s are expressed as r e#mo le s rad ioac t ive precursor incorpora ted in to DNA. Ana lys i s of t he ex t r ac t ed [32P]DNA identif ied deoxy-CMP b o u n d in in te rnuc leo t ide

l ink as t h e rad ioac t ive c o m p o n e n t of DNA.

In summary, the results indicate that optimal rates of DNA synthesis from CMP were obtained in vitro when a critical level of phosphorylated intermediates existed. It has also been shown that the conversion of CMP to deoxyCMP (and indirectly the synthesis of DNA) was strongly inhibited by the presence of deoxyATP, deoxyGTP and to a lesser extent by TTP; in contrast, deoxyCTP showed no such inhibition. These phenomena may be components of a homeostatic mechanism governing the synthesis of DNA.

This investigation was supported by a personal grant to one of us (P.R.) from the Jane Coffin Childs Memorial Fund and by a grant (CY 3076 C2) from the National Institutes of Health, U.S. Public Health Service.

Department of Pharmacology, Yale University School of Medicine, PETER REICHARD* New Haven, Conn. (U.S.A.) Z.N. CANELLAKIS

E . S. CANELLAKIS

1 p . REICHARD AND L. RUTBERG, Biochim. Biophys. Acta, 37 (196o) 554. 2 M. J . BESSMAN, I. 1~.. LEHMAN, E. S. SIMMS AND A. KORNBERG/J. Biol. Chem., 233 (1958) 171

F. J. BOLLOM, J. Am. Chem. Soe., 80 (1958) 1766. 4 1~. MANTSAVINOS AND E . S. CANELLAKIS, J. Biol. Chem., 234 (1959)628 . 5 F. J. BOLLOM, J. Biol. Chem., 234 (1959) 2733. 6 I~. MANTSAVINOS AND E. S. CANELLAKIS, Cancer Research, 19 (1959) 1239. 7 p. REICHARD, Acta Chem. Scand., 12 (1958) 2048.

Received May 2nd, !96o

* P e r m a n e n t address : D e p a r t m e n t of C h e m i s t r y I, Ka ro l in ska In s t i t u t e t , S tockho lm (Sweden).

Biochim. Bioph~s. Acta, 41 (196o) 558-559