Studies on the control of enzyme synthesis during the early embryonic development of the sea urchins

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PN 91001 Studies on the control of enzyme synthesis during the early embryonic development of the sea urchins The amount of deoxycytidylate anainohydrolase I per embryo decreases during early embryonic development of the sea urchin2, a. Furthermore, it has been shown that iv abnormal embryos, in which development stops at gastrula stage, the , ' n z y m . content remains nearly constant". These findings seem to offer art .:xperimenr:tl approach for investigating tim control mechanis,ns . f enzt me "._'~cls in animal (:ells, and furthermore, they indicate that the control ol synthesis of an enzyme for D N A biosynthesis m a y be an important parameter of normal d e w d o p m - n t and differep. tiation. Repression of tho structural genes of deCMP aminohydrolase migh) cxt)i~tl,-. the d e c r e a s - i n the enzyme level during development; the reptt':sor 4 c,,uld be inactive during ovogenesis, whil,' (hiring developnlent an active ret;res~ion tnechaniam might explain the decrease in enzvm,, level, notwithst:',nding the increaao in .-tructural genes. It is pertinent to ment ion that the plut,'vs contains about 2ooo cells and consequent!y about 2ooo pairs of structural gc::c,. The effect of sub.,iances i n t v r f e r i n / w i t h tilt" transfer of genetic inf, n matio,a fr,,m D N A to R N A and tu proteir, s on lho !~.vel ,,f deCMl' aminohydrola,;e dm;l~g , m bryogenesis m a y give some clue to ~he mechanisnas for the control of enzyme h:vels in animal cells. It has been shown that actin,,mycin~ C an;1 D inhibit the D N A - d e p e n d c n t R N A synthesis 5-r. In sea-urchin embryos actinomycin 1) stops R N : \ synthesis s and the embryonic development at blastula or gastrula stage'% ~°. Tt,is paper reports tilt- effect of actinornycin C on the content uf deCMP ammohydrolase in embryos of the sea urchin Sflhaerechinus granularis at various stares. The embryos were grown in .~,.a water (apl)ro.':. 5 ooo ooo en~bryt)s..l) at ~,'; 2 o ' , containing actinontycin C at variou.~ concentrations. At different s t a r t s ,,f ch \ el(:pment the embryos were collected I,x centrifugati~m, homogenized in o i a,I phosl,h:~te bulfcl (pH 7.3) and centrifuged at J.'5'ooo'hotc,m,:'tric m¢~)hod ,1,,scribed else.where 2, except that the i,u:':bation ;,i x ture contained 2.5 lllNI deCMP, o.o 4 mM de(.TFP, o. 4 n:M MgCI a, and Ioo mgI phosphate buffer (pH 7-3). th'CTl> was added to the incubation mixture, to (.i,nlillattt' the inhibition of the ehzvnm by the possible l)rcsence of varying amounts ol inhibitory allosteric effectors ~ :.tF,t to prot(.<~: the enzyme fiom inactivation during the assay. The proteins w e r e nlcasl.n't.,t witi~ the ]:olin Ciocalteu method la. The specific activity of the enzynae was measured ast~moles ot deCMP deaminated per io lnin per mg of proteins. Actinomycin t" has no effect on the enzyme assa}. Since the ratio protein to total nitrogen and the total content of protein per embryo up to the pluteus stage aro constant both during normal development and in tile presence ot actinomycin t;, th," specific activity referred to mg of proteins is directly proportional to the content ol tile enzyme per embryo. [t has been observed, as alruady described by others u,~U, that In the presence ol actinomycin tile ernbryonic development is stopped at blastula or gastrula stage tCzoch:~:, thophys..t~t,z. '~7 (i'~t'-t.) t74-17U

175

PRELIMINARY NOTES

depending upon the actinomycin concentration, and that afterwards the embryos disaggregate and free cells remain in the cultivation mediumIL Actinomycin C changes the pattern of deCMP aminohydrolase levels in the developing embryos. As shown in Figs.. I and 2, after a decrease in the first 20 h

0.5

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0.3

0.2

TIME AFTER FERTILIZATION (h)

Fig. z. Action of actinomycin C on deCMP aminohydrolase activity during the early embryonic development of Sphaevechinus gyanMa~is. On the ordinate are plotted the/~M of deCMP deamihated per mg of proteins and per to min. Incubation mixture: 2 mM deCMP, too mM phosphate buffer (pH 7.3). The treated embryos remained at blastula stage. C), controls; A, embryos grown in 5 mg/1 of actinomycin C; O, embryos grown in xo mg/1 of actinomycin C; ~ , embryos grown in 20 mg/l of actinomycin C; A, embryos grown in 4 ° mg/1 of actinomycin C.

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TIME AFTER FERTILIZATION (h)

Fig. 2. Action of actinomycin C on deCMP aminohydrolase activity during the early embryonic development of Spkaevechinus gvanularis. On the ordinate are plotted the pM of deCMP deamihated per mg of proteins and per io rain. Incubation mixture: 2.5 mM deCMP, o.o 4 mM deCTP, o. 4 mM MgCIa, zoo mM phosphate buffer (pH 7-3). The development of the treated embryos is stopped a t blastula stage. ©, controls; A, embryos grown in t5 mg/1 of actinomycin C; n , embryos grown in 3 ° mg/1 of actinomycin C.

Biochim. Biophys. Acta, 87 (x964) x74-176

176

PRELIMINARY NOTES

of development, similar to the controls, the enzyme remains constant or increases to values even higher than those of unfertilized eggs. These experiments indicate that DNA-dependent RNA synthesis is required for the control of the normal pattern of deCMP aminohydrolase during early embryonic development of Sphaerechinus granularis. An attractive hypothesis is that actinomycin interferes with the synthesis of a specific repressor. Further experiments are in course in this laboratory to inw~stigate the effect of inhibitors of prote.in synthesis and of analogs of purine and pyrimidine bases, and of amino acids on the. pattern of levels of dcCMP aminohydrolase in embryonic development. This work has been supported by Euratom-CNR-.CNEN Contract No. oI2-0I-Z2 BIAI, and by Research Grant No. CA-o68o 9 from the National Cancer Institute of the U.S.A.

International Laboratory o/ Genetics and Biophysics, and Institute o~ Human Physiology, University o/Naples, Naples (Italy)

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E D U A R D O ~CARANO B E N I T A DE ~)ETROCELLIS GABRIEI.LA A U G U S r I - T o c c o

E. SCARANO, Biochim. Biophys. Acta, 29 (x9581 459. E. SCARANO AND 1¢.. MAGGIO, Exptl. Cell Res., 18 (I959) 333. E. SCARANO, Syrup. on Germ Cells and Development, Inst. Intern. d'Embryologzc and Fondaalone A. Baselli, I96I, p. 4o2. F. JACOB AND J. MONOD, J. Mol. Biol., 3 (196.i) 318. j . HURWITZ, G. G. FI;RTH, M. LALANV aND M. ALEXANDER, Proe. Natl. Acad. Sci. U.S., 48 (z962) 1 2 2 2 . G. M. KIRK, Biochim. Biophys. Acta, 42 (I96O) I67. 1. tl. GOLDBERG AND M. RABINOWITZ,Science, 136 (1962) 315 . i). R. GROSS AND G. A. COU$INEAU, Biochim. Biophys. Res. Commun., io (19631 32I. 1'~. LALLIER, Experientia, 19 (1963) 572.

to j . BRACFIET, M. DECROLY, A. FICQ AND J. QUERTIER, Biochim. Biophys. Acta, 72 (I9631 660. 11 E. SCARANO, (;. GERACI, A. POLZELLA AND E. CA.~IPANILE, J. Biol. Chem., 238 (i9631 PC I556. 12 O. H. LOWRY, H. J. I{OSEBROUGIt, A. L. FARR AND R. J. IIANDALL, J. Biol. Chem., 193 (I9511 13

265 . B. MARKMAN,Arkiv Zool., x6 (19631 2o 7.

Received March 3xst, I964 Biochim. Biophys. Acta, 87 (19o41 174-176