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Incorporation of 32P into isolated nuclei of rabbit appendix: The role of deoxyribonucleic acid
.~ ~
,x. F. s. a. H:mEE~ el aL
voL 34 (I959)
REFERENCES
i S. j . SIN~E~, Prod. ,Vat. A~ad. Sci. U.S., 4 r (1955) I o 4 t . S, J . S~NGER, J . Cell. Co~np. PhysioL, 50, s u p p l . I ( J 9 5 7 ) 5 I . A. F. S. A. HABI~Ee, If. G. CASSltJ~" AND S. J . STNGEIL 13iochim. Biophys. Acre, 29 ( [ 9 5 8 ) 587. * S, I, EvsX~zN A.~t> S. J. S t a G e R , J. A m . Chem. Soc., 80 ( I 9 5 8 ) x274. 5 I). PRESSMAN. A. L . GROSSBERG, L. H . PENCtg AND L , P A U L I N G , J . A ~71.Chem. Sot., 68 ( i 9 4 6 ) 250. [-I. F R A E N l i I ~ L - C o N R A T , R . S. B E A N AND H . LINEVCEAVI~R, ,l. BiOl. Chem., 177 ( t 9 4 9 ) 385. r ~A'. L. HUGely:s, H, A. SAROFF AND A. L. CARNEY, .l. AIR, Chgm. Sot., 71 (1949) 2476. 8 A, N I S b ~ ' O F F AND i ) , PRESfl~,IAN, Science, x28 ( t 9 5 8 ) 659. R. A. KEKW]eX< ANt) R . I<. ('AN~AN, Biochem. J., 3o ( t 9 3 6 ) 227. lt~ St. C. B a y : c a , D. H . Ca~t eg~LL, S. I. Epsrr:~.~ ANt> S. J. S ~ N ~ , J . A m. Chem. See.. 78 (~ 956) 3 ~ 2. ~ D. H . CA.~teB~LL, R . H. BLAKER AND A. B. P A R O ~ , J . A m . Chem. Sot., 70 ( I 0 4 8 ) 2496. ~z H . FRA~r~KEL-Co~RA*, J . I. H A R R m ANI) A. L. L~VY, i n D, GL~CK, 2t4ethods o[ Biochemical A~zalysis, Vol. z, I n t e r s c i e n c e , N . Y , , 1955, p. 37 o. 18 j . l{. MARRACK .~.ND E . S. ORLAN,~, BF]I. J , E x p l L l~athol,, 35 (I954) 389. 14 I r. H . ~][AURER, J , SRI R A M AND S. E H R E N P R E I S , AVCli. Biochem. #iophys., 67 (x957) 1 9 6 . ~ \V. A. KLEV_: AND F. M. R l C n A r t o s , ]. Biol. Chem., ~29 (~957) 489.
INCORPORATION
OF
OF
THE
ROLE
a-~p I N T O
RABBIT
ISOLATED
APPENDIX:
OF DEOXYRIBONUCLEIC
~. SEKIGUCH[
NUCLEI'
ACID
° AND A. S I B . X T A N I
C3qo~hamistvy Laboratory, Ya~nagl~ti 3ledicai School, t:be (Japan) ( R e c e i v e d O c t o b e r 22rid, "/958)
SUMMARY
I. Nuclei isolated from r a b b i t a p p e n d i x could incorporate asp into DNA, R N A a n d organic acid-soluble phosphorus fraction (OASP) i~ vitro. 2. The removal of tile bulk of DNA from th~ nucleus b y p a n c r e a t i c D N a s e abolished the incorporation into DNA a n d R N A almost completely, while t h a t i n t o OA~qP was r e d u c e d o n l y to a b o u t 6o %. 3. Appreciable restoration of the i m p a i r e d incorporation a c t i v i t y was o b s e r v e d when salmon sperm DNA or y e a s t R N A was a d d e d to the nuclei t r e a t e d w i t h DNase. However, if an extensivc r e m o v a l of D N A h a d been achieved, the restoration occurred o n l y in the case of RNA, a n d n o t DNA, of the nucleus on a d d i t i o n of exogenous nucleic acids. 4. Some indication was o b t a i n e d t h a t certain polyanionic compounds, such as c h o n d r o i t i n s u l p h a t e or h y a l u r o n a t c , could replace tile nucleic ac:ds in restoring t h e imi)aired a c t i v i t y of D N a s e - t r e a t e d nuclei, p r o v i d e d tile action of DNase w a s not v e r y extensive. 5. It was suggested t h a t in the observed restoration a d d e d nucleic acids a c t e d m e r e l y as polyanions which m i g h t eliminate tl~e imbalance of electrostatic charges resulting from the r e m o v a l of the bulk of DNA from the nucleus. A b b r e v i a t i o n s : A'I'I', a d e n o s i n e t r i p h o s p h a t e ; R N A , r i b o n u c l e i c a c i d ; D N A , d e o x y r i b o , nucleic acid; DNase, de(ix),rib~nuclease; ()ASP, orgatlic acid-soluble phosphate fraction, " G n l e a v e fr[~irt t h e D e p a r t m e n t o[ B i , l o g y , F a c u l t y of S c i e n c e , O s a k a U n i v e r s i t y , Oz~ka0 Japan.
Relerences p. 435/456.
VOL. 34 (X959)
ROLF. OV D N A [.~" a2p
445
INCORPOR.kTION
iNTRODUCTION
l~Iuch information has been collected in recent years a b o u t the synthetic activities of isolated nuclei of animal c~,ls. T h e y can p h o s p h o r y l a t e adenosine m o n o p h o s p h a t e to adenosine triphosphate ~, at.4 incorporate various labeIled precursors into proteins a, fibonucleic acids 2-6, and d~oxyribonucleic acidS-L The removal of the bulk of D N A b y d e o x y r i b o n u c l e a s e impairs the incorporation of the precursors into R N A a n d protein, a n d the lost a c t i v i t y is restored b y various nucleotide c o m p o u n d s of relatively high molecular weight, including D N A a n d R N A L The f o r m a t i o n of A T P in the nucleus is also d e p e n d e n t on the presence of D N A , which can likewise be replaced b y exogenous nucleic acids s. I t has t h u s been p r e s u m e d t h a t one of the roles of D N A is to p r o v i d e energy-rich nucleotides needed for the s y n t h e t i c a c t i v i t y of the nucleusL E n z y m e s catalysing the incorporation of t h y m i d i n v or d e o x y r i b o n u c l e o t i d e t r i p h o s p h a t e s into D N A were found in the s u p e r n a t a n t fraction of certain animal cells; t h e y require D N A for their full a c t i v i t y g, m. I n t h e present s t u d y we have a t t e m p t e d to determine (x) whether the preformed D N A in the nucleus is indispensable for the incorporation of inorganic a2p into D N A in isolated nuclei, a n d (2) w h e t h e r the incorporation a c t i v i t y of isolated nuclei depleted of their D N A can be rest,~rcd b y some anionic c o m p o u n d s of non-nueleotide nature. In doing this we are looking for some relevant information coucerning the possible role of D N A in t h e anabolic processes exhibited b y the isolated nuclei, either as a carrier of information needed for fabrication of macromolecules, as a potential source of high-energy p h o s p h a t e c o m p o u n d s , or as a polyaniordc c o m p o u n d the removal" of which will cause s o m e d i s t u r b a n c e in the electrostatic charge distribution within t h e nucleus. A p r e l i m i n a r y n o t e on this work haz been published~L
EXPERIMI'-'NTAL
-"
Chemical compounds Y e a s t R N A was a gift of Dr. KVROIWA, Kirin Research Institute, Kirin B r e w e r y Co., I~td., Amagasaki. H i g h l y polymerized salmon s p e r m I ) N A was received from Prof. I. WATANABE, U n i v e r s i t y of T o k y o . A sample of chondroitiu sulphate was o b t a i n e d from Dr. T. MAKI, Minophagen P h a r m a c e u t i c a l Co., T o k y o , a n d another s a m p l e p u r c h a s e d from W a k o Pure Chemical Industries, Ltd., together with a sample of heparin. A highly purified p r e p a r a t i o n of h y a t u r o n a t e was a generous gift of Dr. M. ISHIMOTO, U n i v e r s i t y of Tokyo. A crystalline preparation of egg albumin was kindly supplied b y Dr. Y. FUJISAKI, Y a m a g u t i Medical School. The mixed 2', 3'-mononucleotides were o b t a i n e d b y hydroiysing y e a s t R N A in o.I N N a O H at 80 ° for 20 Iain a n d neutralising with HCI, the c o n t e n t of NaCl was calculated a n d s u b s t i t u t e d for the NaCI in the incubation medium, asp was received from the Radiochemicai Cenh~, A m e r s h a m , England, on allocation of J a p a n Isotope Association. Preparations of pancreatic D N a s e , once crystallized for t r e a t m e n t of nuclei, a n d n o t crystallized for hydrolysing a D N A preparation, c a m e from the W o r t h i n g t o n Biochemical Corporation. A p a r t of t h e m was m a d e available t h r o u g h the courtesy of Dr. K. SuzuKI a n d Dr. H. UCHLPA of t h e U n i v e r s i t y of T o k y o . Re/eyences p. 4551456.
,t46
,~I. sv.lltGUCrtX, a. SmAT.~NI
w)L. 114 (I959)
I>rcparaliott, o/ mtclei and lymphatic, cells Ap p en d ices and P e y e r ' s p a t c h e s of a d u l t r a b b i t s served a,> the s t a r t i n g m at eri al . Sucrose mtclci were p r e p a r e d essentially accordi ng to ALLFREV el al.a; t h e y were tinnily s u s p en d ed in 0.25 3 f sucrose-o.oo2 3 [ CaCI= at a delasity of 1.5" Io ~ nuelei/ml. Lyml¢,latic cells were o b t a i n e d as described in a s e p a r a t e c o m m u n i c a t i o n le.
l~wubati,.n aith a'.,p l_:sually the final i n c u b a t i o n m i x t u r e consisted of i ml of a i :i m i x t u r e of 0.25 ~ir s u c ro s e- o .o o 2 3 I CaCla c o n t a i n i n g 3 ' r o ~ nuclei a n d Ca-free T y r o d e solution, T hi s s u s pen d in g m e d i u m will h e n c e f o r w a r d be called the s u c r o s e - ' F y r o d e m e d i u m . T h e su.qpension also c o n t a i n e d 5 tzg of s t r e p t o m y c i n , IO I~C of ~ " P - o r t h o p h o s p h a t e a n d o t h e r a p p r o p r i a t e a d d i t i o n s w he r e i n d i c a t e d . Tim m i x t u r e was e q u i l i b r a t e d in a IS-ml centrifl:ge l u b e at 37* for 5 rain a nd t h e n shaken at tim slime t e m p e r a t u r e . I f not specified, the inc~ibatio:i lasted I. 5 h. l';xpcrime1~ts w i t h l y m p h a t i c cells were c o n d u c t e d as d e s c r i b e d e l s e w h e r e ~. hMividxlal tests were rtm in d u p l i c a t e tubes. In m o s t e x p e r i m e n t s , where t o t a l a c t i v i t y of mlcleic acids p e r t u b e h a d to be d e t e r m i n e d , a n o t h e r set of d u p l i c a t e t u b e s :vitht,ut ~zP was r u n for each of t h e var i e d , ; x p e r i m e n t a l condi t i ons, a n d s u b j e c t e d to the determination" " ~ of nucleic acids b y the S c h n e i d e r m e t h o d at t h e end of i n c u b a t i o n withot~t s e p a r a t i n g the nuclei from the i n c u b a t i o n m e d i u m .
Dit¢cstion. o/~mt:lci a i¢/~ DNase Th e nuclei (o.2 ml of t h e original suspension) were m i x e d with 0.2 ml of T y r o d e solution c o n t a i n i n g o.o125 M MgCla w i t h a n d withotlt an a p p r o p r i a t e a m o u n t of c r y s t a l l i n e I)?-;a.~c_ T he m i x t u r e was inc~lbated at 37 ° for I5 rain, chilled in ice, a n d a d d ed to 5 ml of ice-cold x :r m i x t u r e of o.25 3 I s u c m s e - o . o o 2 .lI CaCI 2 aucl Ca- a n d Mg-free T y r o d c solutic,'n c o n t a i n i n g o.oz 3~/sodium ci t rat e. T h e nuclei were s e d i m e n t e d b y centrifuging, and rcsu~pendcd in t h e final i n c u b a t i o n m i x t u r e as d e s c r i b e d a b o v e .
l'r~'paralim, el mwlcic acids a~ul organic acid-soluble phosphale [raetio~ Incub:.dion was t e r m i n a t e d by chilling in ice; t h e c o n t e n t s of i n d i v i d u a l t u b e s (nuc|ei plus m e d i u m ) along with the a p p r o p r i a t e z e r o - t i m e cont rol were i m m e d i a t e l y s u b j e c t e d to the p r o c e d u r e described e l s e w h e r e e for th~ p r e p a r a t i o n of nucleic acids a n d OASP.
Deter~nD:ation o/mtcleic avids and total phosphor~ts D N A - P a n d t o t a l P were d e t e r m i n e d as in a previ ous r e p o r t ~s. R N A - P was m e a s u r e d b y the phloroglucinol test accorr!ing to HAItN AND VON EUI,ER 14.
3ice~urvmc'l~i o~ r.dft,.~ticdy This was m a d e a~ in a previou~ r e p o r t ~a using an e n d - w i n d o w Geiger-Miiller eemnter. In each e x p e r i m e n t t h e r a d l o a e t l w t y of t h e sam pl es of z e r o - t i m e c o n t r o l were m e a s u r e d , and when not ~legligible, s u b t r a c t e d f r o m t he values of t h e tr~sts. This was n e c e s s a r y because t h e activities o b t a i n e d were r a t h e r low, especially w i t h DNA, in m a n y of t h e e xpe r i m c r t t s r e p o r t e d here, a l t h o u g h t he c o n t a m i n a t i o n f o u n d in zeio-tim~ samples was usuMly v e r y slight, if any. Specific a c t i v i t y was e x p r e s s e d l'¢e#rr,wes p. 455/ ~56.
VOL. 3 4 (t959)
ROLE oF D N A
I x .~2p tNCORPOR.VrlON
447
in c o u n t s / m i n / / ~ g P. I n m o s t cases, h o w e v e r , ~he t o t a l r a d i t ~ a c t i v i t y ( c o u n t s f r n i n / t u b e ) w a s e m p l o y e d t o e x p r e s s t h e e x t e n t of i n c o r p o r a t i o n into nucleic acids. T h i s w a s c a l c u l a t e d f r o m t h e s p e e i f i , a c t i v i t y of p u r i f i e d samp!e.~ a n d t h e t o t a l a m o u n t of r e s p e c t i v e nucleic a c i d P (/.g/tube}.
Cl,romatographio proae~h~re A l a r g e - s c a l e i n c u b a t i o n of t h e nuclei wa:; c o n d u c t e d u s i n g I o o m l of n u c l e a r s u s p e n s i o n w i t h t h e s a m e c o m p o s i t i o n a s a l r e a d y d e s c r i b e d . T h e m i x e d nucleic a c i d s w e r e o b t a i n e d b y t h e r o u t i n e p r o c e d u r e ~2. T h e y wen> f r a c t i o n a t e d i n t o D N A a n d R N A in t h e u s u a l w a y b y a l k a l i n e h y d r o l y s i s w i t h o.3 N K O H a t 37 ° a n d a c i d i f i c a t i o n with io N perchloric acidlL The RNA fraction was adjusted to pH 3 and treated w i t h c h a r c o M , a n d t h e a d s o r b e d m o n o n u c l e o t i d e s w e r e e l u t e d w i t h 20 %; a q u e o u s p y r i d i n e a c c o r d i n g t o V,x.,," Ill.;l~iCu.-,it~, in o r d e r to e l i m i n a t e the e v e n t u a l c o n t a m i n a t i o n of i n o r g a n i c ~-P. T h e y w e r e t h e n chr0m.:togral,J~ed on a D o w e x - i c o l u m n ( X - x o , f o m n a t e f o r m ) a c c o r d i n g t o Os.xw.x et al.tL "With i n t a c t l y m p h a t i c cells, 40 m l of cell s u s p e n s i o n (4" lOS c e l i s / m l of C a - f r e e T y r o d e s o l u t i o n ) w a s u s e d a n d t h e c h a r c o a l t r e a t m e n t w a s o m i t t e d becau'se t h e ~zp c o n t a m i n a t i o n c o u l d b e n e g l e c t e d in t h i s case. T h e D N A p r e c i p i t a t e d f r o m t h e a c i d i f i e d s o l u t i o n of t h e "alkaline d i g e s t of c r u d e • n u c l e i c a c i d s w a s w a s h e d t w i c e w i t h c o l d i o % "tr'.'-~hloroacetic acid, r e d i s s o l v e d in a d i l u t e N a O H , n e u t r a l i s e d w i t h HC1, p r e c i p i t a t e d will,_ e t h a n o l , w a s h d d in e t h a n o l a n d e t h e r , a n d dried. T w e n t y m g of D N A t h u s o b t a i n e d w a s d i s s o l v e d in 4 m l of o.oo~ M MgCI,, s o l u t i o n c o n t a i n i n g 4 m g D N a s e a n d i n c u b a t e d a t 37 ~ for 8o rnin a n d t h e d i g e s t w a s a c i d i f i e d w i t h c o l d p e r c M o r i c a c i d a n d c e n t r i f u g e d . T h e m i x t u r e of n u c l e o t i d e s of ~,afious sizes in t h e s u p e r n a t a n t w a s c h r o m a t o g r a p h e d orL a D o w e x - I c o l u m n (X-Io, f o r m a t e f o r m ) a c c o r d i n g t o t h e m e t h o d of SAK.XK[ls, u s i n g O.3¢>--7.0 N f o r m i c a c i d in t h e reser~-oir of a g r a d a a t e d e l u t i o n s y s t e m .
RESULTS
Inaorporation aativity o/tlw isolated m~cloi ~ q ~ e n t h e nuclei w e r e i n c u b a t e d w i t h zap in t h e s u c r o s e ~ T y r o d e m e d i u m , z2p w a s i n c o r p o r a t e d i n t o D N A , R N A a n d OASP, t h e i r specific a c t i v i t i e s i n c r e a s i n g in t h e o r d e r m e n t i o n e d ( T a b l e I), A s s h o w i t in Fig. I , t h e i n c o r p o r a t i o n i n t o t h o s e f r a c t i o n s p r o c e e d e d l i n e a r l y f o r a b o u t I h asxd t h e n t e n d e d t o level off. I n s u b s e q u e n t o x p e r i m e n t s t h e p e r i o d o f i n c u b a t i o n w a s f i x e d a t 9 ° rain. TABLE I f~lg 71~t¥0 I N C O R P O R A T I O N
OF 52p
INTO LYXfPHATIC CELLS AND SUCROSE
NUCLEI
OF RABBIT
APFENDIX
Cells ~,+ndnucle'." w e r e obt-.xined from the same source of pooled t~szucs ~nd inc.tlbated for i .5 h at 3 7" + :llal,t, rial
3" i o s cells 6.1o s nuclei
/llCuGrillorl ln',edi.:im
2 m[ Ca-free Tyrode 2 ml Suerose-Tyrode
Retercnces p. 4.55]456.
Cvnce~anl,'i,m o1 a~l" (,~d-'. ~lil,r
O:Lx,/:.
5 Jo
455o t42o
.q~-": :,'i~
3-'2 50,2
48-5 2.15
6.6 26.2
448
M.
SEKIGUC}II,
h. SIItATANI
VOL. 34 (~959)
C o m p a r e d with the results on l y m p h a t i c cells prepare d from the s a m e tissue, the incorporation into the nuclei was considecably less (Table I). The specific a c t i v i t y ratio R N A / D N A was m u c h higher in the nuclei t h a n in the whole cells, a n d this was in a g r e e m e n t with the current view t h a t the isotopic u p t a k e b y nuclear R N A w a s much higher than t h a t b y c y t o p l a s m i c R N A TM. These observations, t o g e t h e r with the fact t h a t D N a s e t r e a t m e n t reduced the D N A c o n t e n t a n d z2p u p t a k e of the nuclear preparation (Table VI} b u t not of the intact ceils, indicate t h a t the o b s e r v e d incorporation was due to the real a c t i v i t y of isolated nuclei rather t h a n of contAminzting i n t a c t cells. In the course of the w o r k it was noticed t h a t the incorporation a c t i v i t y of t h e nucleus was subject to seasonal variation, being highest in s u m m e r a n d decreasing gradually to re~ch a c o m p l e t e loss of a c t i v i t y in midwinter. A similar t r e n d w a s alsc. noticed with i y m p h a t i e cells of r a b b i t a p p e n d i x ta. mP,+a laNAI
O.~SP 2000 DN,X
.~ 5@ ! L5
1500
.~ ./. . . . . ~3o
""6ase
./.t
I.o
1OO0
,~ 2o .~
0+5
, /,'7"Y
500
Incubati.c,n t i m e (h)
l ' i g . +. [ ' i m e c o u r s e , f f a a l '
incorporation
into I)NA, RNA appendix.
and OASP
~I i s o l a t e d
nuclei of rabbit:
Labelling o~ m+cl~ia acids The specific a c t i v i t y p a t t e r n s of individual 2 ' , 3 ' - m o n o n u e l e o t i d e s o b t a i n e d b y alkaline hydrolysis of R N A ' s of isolated nuclei a n d i n t a c t cells were v e r y similar (Table II). It m a y be noted t h a t the results closely paralleled those o b t a i n e d w i t h RNA of the rat liver i.~ vivo (I and 2 4 h) ~°. The fractions o b t a i n e d c h r o m a t o g r a p h i c a l l y from a D N a s e - d i g e s t of D N A of t h e i n c u b a t e d nuclei showed a m u c h more diverse d i s t r i b u t i o n of specific activities t h a n t h a t observed in ~,ivols,za, b u t appreciable quantities of r a d i o a c t i v i t y were r e c o v e r e d TABLE LAI}I~LLING
lI
OF INI}IVIDUAL 2 / , 3 ~ - . % I O N O N U C L I r ~ O T l t . ) E N OlV R N A LYMPI4ATIC CI£I.L,~ ()F RAI~Iti'I" A P P I r . h ' I ) ] X I N C U F I A ' I ' ] , : I )
N u c l e i a n d cells ¢)rigill&tt'd f r o n t S e p a r a t e s o u r c e s , ] ) u r a t i o n
IN NUCLF.I ~;Jt l,,~'tY9
ot i n c u b a t i o I ] :
3,p¢¢i[ic aeffvdy (¢om:t~ pnin /,~ I')
I+ymphatic
ceils
IsoJated quc]ci
R e : .~re~:ca~ ~. 4551456.
:t denyti¢ .eat-ul
Gmo=vtfc a c &l
Cytfdyli, a ~ td
7.5- ~
53.o
54.5
7.°5
-1.2 .5
4.7 °
! "ridvl& .rid
Oz,D 7-84
.%NI)
I. 5 h
VOL. 34 (1959)
l~OXr: OP D N A iN a~p INCORPtHiATION
44"9
from all the ultraviolet-absorbing fractions e x a m i n e d (Table II[). The observed radioaetivities of the nucleic acid fractions o b t a i n e d from the nuclei incubated w i t h a~p can therefore be t a k e n to indicate t+he incorporation of amp into polynueleotides of DNA and RNA. "I'..~ D I . E
Ill
D I S T R I B U T I O N el-" R A D I O A C T I V I T I E S IN IHb'FIgRI-;N'T FRAC'IIC)N'.; ¢]P" A I)NA.qI-;-I>IGI';ST el-" ] ) N : X OF R-~BFII'I" APPb;,~I>IX N U C L E I I N C U B ~ T I ' ; | ) i~$ Z/i~rO I)NA
was
obtained
Ori~,i n a t DN, I
Specific activity (counts[min/H ~ t')
from
the
same
m>tterial
as I{.NA :~f'Fable
F m r l fim * ............................................................... • 3 -I .'~ "
o.3,'~" +
Relative P content {°/o o f t o t a l }
II,
y
+
+,,
I,,
o.,~-
O. Tt
o,3t'
:~-3:
o,57
;*-3 t
o-37
o.54
1.56
->,43
o.0
I +5
:¢. 7
2.o
4..5
4 t.~)
23.0
~-7
o.tb
7.1,.
* Fraction.~ I S were obtained cbro.~ato~raphica!ly; fraction O was extracted wit!-, b.+~t 5 ",~ trichlor+mcetic a c i d fr, m l t h e r e s + n , ' f f t e r t e r m i n a t i + m of elution w i t h 7 .V f o r m i c i t c l c l : I r a c t i ~ m t+~ was the acid-m.~oh~ble residue of ONase-digest. " * V ~ , ' e i R h t e d m e a n ,>.¢ s p e c i f i c a e t i v i t i e ~ o f f r a c t i < m s t - t +~: o.3<~.
Use el the total activity as a r~aasurc el i3~¢orporatio;v In e x p e r i m e n t s involving the removaI of DNA from the nucleas b y DNase a n d / o r the a d d i t i o n of nucleic acids, the use of the specific a c t i v i t y to express the m a g n i t u d e of ipcorpora*.ion is obviously misleading, hecaltse the a m o u n t of nucleic acids in different tubes m a y v a r y considerably and c a n n o t be used as the c o m m o n reference s t a n d a r d for the incorporated radioactivity. It was therefore decided to express the m a g n i t u d e of incorporation b y total a c t i v i t y . For OASP this wa.~ determined directly+ F o r DNA and R N A it was calculated from the c o n t e n t of respectiv'e nucleic acids per tube a n d the specific ,mtivities of the purified samples u n d e r the a s s u m p t i o n tha~ .'he lattm were representative: sar:~_ple.s of the nucleic acids present in respective tubes. Such an a s s u m p t i o n m a y be ju~¢ificd because the relative yield of nucleic acids in our f r a c t i o n a t i o n procedure was largely independent of the absolute a m o u n t of the acids (Table iV). It was also found t h a t the m a g n i t u d e o~ contaminatit~g a~P was independent el the a m o u n t of nucleic acids present. E~e,;t o/D.~ras~ treatmem In order to examine the effect of the removal of DNA on the a2p uptake, the nude;, were i n c u b a t e d for 15 rain w i t h varying concentrations of D,Nase in s u c r o s e - T y r o d e m e d i u m fortified with magnesium. The control tubes were also i n c u b a t e d with the same m e d i u m lacking DNase. As shown i~l Table V, a selective d e s t r u c t i o n of DNA in DNa_~e-treated nuclei could be detected. However, during the subsequent incubation a considerable mltount of DNA was reJ.e~.sed from the control a n d t r e a t e d nuclei. I n subnequent experiments, the e x t e n t of tl~e removal of DNA b y DNase was d e t e r m i n e d (somewhat arbitrarily) from the whole c o n t e n t of the tubes at stage C of Tabte V0 i.e., at the end of incubation for ,2p incorporation, the possible release of DNA from the nuclei into the medium during this incubation being disregarded. References
p.
45.~]456.
450
M. GEKtt~UCHI, A. SIBATANI "["\ H I_ E
rOE 3 4 (I959)
.IV
ASH)t:NT AND ItAI~ll)ACTtVII"T" (;1: I)~'N-.'k I"RO*I T/IN NUCLEI .qUBJI~CTED TO ])~"%'ASE TREATMIgNT OR AIIDITION OF SALMON S;PIgRM DN;Sk N u , c l e i w e r e d ~ g c s t e d w i t h D N a s e { 123 j t g ] m l ) , w a s h e d , : r o d s u s p e n d e d w i t h a n d w i t h b u t I ) N & (4 m g ] r n l ) . .'| r/l~l'//'[,¢l
IT.N',t.,t'
trcd/~/roll
.
.
.
.
.
.
.
[ ~*t'~t'nal
. . . . . . . . . I)NA -7" .... I).%~*:~.
.
.
.
.
.
.
.
.
I£ rtr:tctcd
I lO 342 "
.
.
I)¢,
42. 323
" "l't~.c r e ; t s o n w h y t h e c f f e c t of [)N~Lse t r e a t r n e n t in the tubes supple,uented with DNA is not clear 7A13LE
(Uo'¢/P~,~.'~tttl¢/?l /¢~"I~I
Total activilj, (c~)lOIt8~?l[~tfl~')
57
t -~:3
! NO
~)l 30 (2'3
0.40 O.O2 O. 4 [
z 30 I 1 3 I"
.
3ptltl]llt~activity
.
lt'equlivc vicld ("o1
2o9 21 202
in sncrose. -Tyrode mediura
was not
reflected
by the umount
of I . ) N A
V
CHANGE IN Q t r A N T [ ' I ' y OF I.'Ik~N~.-k A N D 1~%~'_3k DURING THE TR~;ATMI.JNT '4VITtl ].)~%'AS~ A N I ) •qUI~.~E~UENT INCURATION OF NUI:LE[
Samples were taken at ~the following stages. A: Original nuclear s-spension. B: Af~:er incubation w i t h o r w i t h , ) u t l h N a s e ( 1 2 5 p g / r t t l ) f o r i 5 n l i n ; t h e w h o l e c o n t e n t o f t h e t u t , e s w a s subjected t o analysis. Then the quclei were separated by centrifugation, resuspended in sucrose-Tyrode m e d i n m w i t h o r w i t h o u t a d d i t i o n s ~tnd i n c u b a . t e d f o r 9 0 n l i n . C : T h e w h o l e c o n t e n t o f the tubes as well as the txuclei separated from the medium by centrifugation was analysed. tfg P.lu(,c iu,rubated: Siege
A.
.4dditton
Initin!
-
13. A f t e r f i r s t incubation
--
Frarti~l
with DNage (test]
witho~ 1)Nase (contmO
Total
D,\'.t
RNA
~7 °
33
DNA
R.VA
70
33
*
Total
t 57
29
4"
3x
-
Total S:- ~ i m e n t
zi 6 89
3o i8
42 4z
29 29
S~tlmon sperm 1)NA
Total Sed~atent
342 " --
-26, ---
323 67
36 2.5
V c , ~ s t ft.','>.
"rotal Sediment
~ 21 {)7
44
235 ~)O
(1)Nose)
C, A f t e r s e c o n d
incubation (z:,p)
• Owing to the high viscesity ordinary centrifugatior~.
of I)NA
solution
added
the
2zs
38 nuclei could
22
not
be separated
by
Table VI shows the e x t e n t of removM of D N A and the c h a n g e in t o t a l a c t i v i t y of O A S P . R N A and D N A of the nuclei digested w i t h different c o n c e n t r a t i o n s o f DNa.se. The incorporation into D N A and R N A was progressively impaired b y t h e increase in the e x t e n t of ,+he r e m o v a l of D N A . A p p a r e n t l y the incorporation into D N A was more sensitive to the removal of D N A t h a n t h a t i n t o RNA. The i n c o r p o r a t i o a i n t o O A S P was m u c h less sensitive in this respect; it decreased o n l y t o 60 % of the control when the incorporation into nuclei~ acids was abolished almost completely Re[erences
p.
4~fi/456.
VOL. 3 4 (1959)
ROLE OF D N A
IN s e p IXCort'OR.VrlON
451
+r.&l+l .F+ v1 IEEFECT
Of
I¢|~.',IOV.-'t.L t ) [ : ].)~-'N",~ O N : | 2 I ) 1 N C O R P O I < A ' I ' I O N
T . t , d ,+cti+']h' F¢ou++ts rain trd~e)
.-/mot+hi ~./D.X':t P;.+pt, ,%'
of l),X'+tse (1+~ +ntJ
3123
(+!~ ,'~tt ~'+;~l/~,~]J
O.'L",I"
I(it+ 07
I 3,q3o ! ,'t,f~oo
O 10
63
~p2 04
z5 .3,12 t
~ I : r~(~L:%qrp;D N U C L I ~ I
I, t()O t,l~O
137 I 1.5
I (),t~oo
7~2
.S3
t !..5oo
45"
I ~,,-% l ()
()4~ [ ,5<)
4-
0
I+DO
12'5
.] I
Z -i,Ol (-'
d St>
20
z 2,4(~t,
7~
.5 t}~)
27 2[)
<1,5o+,
o
I +()f|l)
f~_%++'!
fq.%'.'~
, ,%[ tr¢l(+
20.~ 2i"
"
' 7
!8 [)
I Z
b y t h e r e m o v a l o f a b o u t 7 0 % o f t h e D N A , a n d e v e n a s l i g h t s t i m u l a t i o n of i n c o r p o r ; t t i o n w a s o b s e r v e d w i t h O A S P w h e n s m a U arr, ount.~ of D N A w e r e r e m o v e d . D u r i n g t h e c o u r s e o f e x p e r i m e n t s i t w a s n o t i c e d t h a t it w a s t h e c o n e e v t t r : t t i o , : o f D N a s e u s e d r a t h e r t h a n t h e e x t e n t of t h e r e m o v a l of D N A t h a t d e t e r m i n e d t h e e x t e n t o f i m p a i r m e n t of t h e i n c o r p o r a t i o n , A l s o t h e d i f f e r e n t b a t c h e s of l ) N a s e c o u l d differ markedly in their effect on the nuclei. "
Restoration+ o/th~ impaired .incorporation activity o/ D.V..~c-trcate
e l ~" ~ N U U L E I I t : ON
.-'t..CIDS A N D
SO~IE
OTIIER
-PHI+ I . ' - : ( . ' O R P O I ~ A T I { ) N
V II C(),~lPOl.'Nll:q
O1" ; 1 2 | ) [ N ' Y ( )
IX
Till';
Cd}N'I'ROL
IN~-t,_ l + . ~ r p j ~
.%HX rLrJNl-;
.",L'('LIC.[
'l'+,t,d ~lcti:'z'lY ( " , ,
.¢ C+#~tt,',D
..Idditi,,n ~p c t trd.'l
K.rpt, A'o.
L,;J" ,:"
I,L,+ 4
I),V.'~
7° 133
8~ 14(7
72 z 19
I ]2 ~ 51
1_, 7 7;-I
~ i~ ,"e,-i
96 ,~ I [3 ,S~. 33 4! t tz
zq, -~~' 07 ] n4 22 30 ~o
.......................
-%I24 M- 5 ~[3~
.'t,lz6 .M3o ~1138
Re#fences p. 45.~1456.
4 rrtg I~N.\ 1 mg R N . \ z nag tCNA 4 nt~ I-~NA z p.lg [;~N.~
2 2 4 z -' ;
mg -",3'-nxol~mtncleotitte.~ n~g c h o n d r o i t i n .~ttlphate mg c h o n d r o i t m sulpli,-tte rt;g hyah+r
,';4
3{)6 Io7 t IT 4 ¢;' 01 ¢11
452
~ . sm~tc, v c m ,
a . Sm,~TaNt
VOL. 3 4 (Z959)
Usually the presence of nucleic acids in the incubation medium did not very much affect the total activity of the control nuclei ('fable VI I), but occasionally there was a remarkable inexplicable diminution of the incorporation activity (Table VII, M38). In experiments involving the D N a s e digestion of, and addition of nucleic acidg to, the nuclei, the total activity of individual fractions was usually expressed in % of the undigested controls receiving the same addition, but in some experiments such "addition controls" were not run: the undigested control without addition served ~,s the ,',~¢~rence standard (values in parentheses in TaMe VIII). TABLE
VIII
1,2.FFICCT O F N ' U C L E I C A C I D S z~.ND S O N I E O T H E R C O M P t ~ U N I ) R I N I~HI~ I N C U B A T I O N O N TME: I N C O R P O R A T I O N O F gZ[, ][*','TO.~[JCI.EI TRIf, A T F . I ) 'I,'~ITM D N A ~
F rpL .Vo.
3Ia. t
Fraction o/
(.'O*|fC~ltrafi¢~lt O# I~Ncs¢ (p~ 'm#)
J),N'A rt'#/toved
iz 5
0, t
,4d*h'.Qo/t (per tube)
('+'o]
4 l~i~ I.)NA
4 irig I~N.\ Mzo
3I 25
Iooo
: 25
....
84
/,.lie)
Iooo
74
M39
M3S
25 o* ~
6 3 **
1 -'.5" *
25
~7 i ':6
t rsl,
B~
07
44
(, 1 o)
--
I~N..k
--z nag ]¢.NA 2 Irt~ 2 " , 3 " - r n o n o n u c l e o t i d e s 2 n'~g e h, m d r o i t i n s u l p h a t ~
6(,
3s
97 i o7-
2. 5 n t g I£N..K
-
2.5 m/4" c h o n d r o i t i n 314o
DY.'.4
7"
'-"nlg I~N..i 4 mg RNA
I -'5
~N,q
I ~9)
z nzg
3I2 ~
Total a~livity ( ~ o# ¢onlret*) O,q,Np
2.5 m g D N A
63
--2 mg RNA 2 mg chondroitin -o 2 illg RiNA 2 rnp. c h o n d r o i t i n 4 mg ehondroitin
'7
-2 ntg
R',:
~
z mg heparm 2 mg hya|uronate 2 mg egg albumin
sulphate
sulphate
sulphate .~ulphate
~IED1U,%I
o
( ~ O 6) (46)
S
( ~ e>) (9)
0 (J
~ .3
c,
el
4 r
Oo 57
57 83
35 54 U4
64 (I 1,1) t25 69
Io 13o) I5 "~7
t r (33) 42 44
15o
3
*
(184t
(9)
('}
I3S J l5 i i7
z8
2a
48 16
tI8 58
,o7
io7 ~ -'4 7q
87 113 83 96
75 l.;~, 13z tor
~r ]3 t
43 '~7
, .'o 34(.1 95
83 157
fJ7
57
I9t 51~ 2 I5
~4
" Undigested contro|s also received addiiions except for the cases in p0rentheses, whore undigestr:d controls did not receive additions. • " I n t h e s e e x p e r i m e n t s a d i t t e r c n t b;~.tch ,'*f I ) N a s e f r o n t t h a t t ps e d i n o t h e r ¢ x p e r i m e , ~ n w a s e m p l o y e d . T h e re~',d t s w i t h t i f f s b a t c h ~vere m u c h i n f e r i o r t[> t h o s e o b t a i n e d w i t h t i l e p r e v i o u s b a t c h a s r e g a r d s r e m ~ , v a l o f I ) N A a n d i m p a i r n l e n t of il)co~ p o r a t i o n .
Re]erence~ p. 4551456.
v o L . 8 4 (r959)
rOLE oF D N A
;r~- 3~p INCORIJORATION
453
T h e r e s u l t s s u m m a f i s e d in T a b l e V I I I n o w c l e a r l y i n d i c a t e t h e L'~!!o;;-ing p o i n t s . (I) B o t h D N A a n d R N A , w h e n p r e s e n t in t h e i n c u b a t i o n m i x t u r e of l ) N : ~ e t r e a t e d nuclei, w e r e e f f e c t i v e in r e s t o r i n g t h e lost i n c o r p o r a t i o r t a c t i v i t y , p r o v i d e d t h e d i g e s t i o n w i t h D N a s e was n o t v e r y e x t e n s i v e (M24). (2) T h i s e f f e c t of nucleic a c i d s i n c r e a s e d w i t h t h e i r c o n c e n t r a t i o n ~ (Mz5). (3) W h e n a h i g h e r c o n c e n t r a t i o n of D N a s e was u s e d a n d t h e r e m o v a l of D N A was exhaustive, hoth RNA and DNA :estored the incorporation into RNA, but not i n t o D N A (M',o).
Attempts to rvstorv tlw lost in~orporatio~ avtiz'ily by ad,.litio~t o[ compvuruts olhtw t',~.a~ medci¢ avhts T h e n e x t s t e p was t o e x a m i n e n o n - n u c l e o t i d e p o l y a n i o n s a n d s o m e ot[mr c o m p o u n d s for t h e a b i l i t y t o r e s t o r e t h e t~-st i n c o r p o r a t i o n a c t i v i t y of t h e D N a s e - t r e a t e d nuclei. U n f o r t u n a t e l y t h e r e s u l t s were in m o s t cases not c l e a r - c u t . F o r o n e t h i n g , n e w b a t c h of D N a s c was u s e d in a few e x p e r i m e n t s , a n d this was fourtd t o be n o t so e f f e c t i v e in r e m o v i n g t h e D N A f r o m t h e n u c l e i as t h a t used in p r e c e d i n g e x p e r i m e n t s . F u r t h e r , s o m e c o m p o u n d s t e s t e d b r o u g h t a b o u t a p r o f o u n d m o d i f i c a t i o n of t h e inc o r p o r a t i o n a c t i v i t y i n t h e c o n t r o l nuclei, m a k i n g t h e r c s u t t s a m b i g u o u s (Tables V I I an.2 V I I I ) . Chondroitin sulphate did not significantly affect the incorporation into the c o n t r o l nuclei, I t s effect o n t h e D N a s e - t r e a t e d nuclei was e i t h e r a h n o s t equ'a_l (M26, M39 ) or i n f e r i o r t o (Mr 9, M4o) t h a t of R N A . H y a l u r o n a t e i n h i b i t e d t h e i n c o r p o r a t i o n i n t o t h e c o n t r o l n u c l e i r a t h e r s e v e r e l y . B u t t h e a c t i v i t y o f t h e DNase-tree~ted nucIei was h i g h e r t h a n t h a t ot t h e c o n t r o l n u c l e i in t h e p r e s e n c e of h y M u r o n a t e . I t seems t h e r e f o r e t h a t t h e d e p r e s s i o n of i n c o r p o r a t i o r t c a u s e d b y t b e d e p l e t i o n of D N A was r e l i e v e d b y h y a l u r o n a t e " *. H e p a r i n also h a d a n i n h i b i t o r y e f f e c t o n t h e c o n t r o l nuclei a n d i~s e f f e c t o n t h e D N a s e - t r e a t e d n u c l e i was n o t v e r y c o n s p i c u o u s . E g g a l b u m i n , as a n e x a m p l e of n o n - a n i o n i c c o m p o u n d s h a v i n g no significant e'ffect o n t h e c o n t r o l nuclei, m i g h t h a v e a slight r e s t o r i n g a b i l i t y , b u t c o m p a r e d w i t h R N A a n d h y a l u r o n a t e , its e f f e c t was not significant. T h e effect of a m i x t u r e o f 2 ' , 3 ' - m o n o n u c l e o t i d e s was also a m b i g u o u s , b e c a u s e it c a u s e d a m o d e r a t e i n h i b i t i o n ot t h e i n c o r p o r a t i o n i n t o D N A of t h e c o n t r o l nuclei t o g e t h e r w i t h a c o n s i d e r a b l e e n h a n c e m e n t of t h e i n c o r p o r a t i o n i n t o O A S P in b o t h c 0 n t r o l a n d D N a s e - t r e a t e d nuclei. I)IS(:USSIOS
T h e r e s u l t s of t h e p r e s e n t i n ' z e s t i g a t i o n c o n f i r m e d in essentials t h e findings of t h e p r e v i o u s i n v e s t i g a t o r s . As was i n d i c a t e d b y FRIEDI~IN .k.ND WOOD 7, BREITS[)kN AND W E B S T E I k 6 o r FI(; 9 AND E R R E R A ''a w i t h calf t h y m u s nuclei, D N a s e i m p a i r e d t h e i n c o r p o r a t i o n of t h e p r e c u r s o r irtto D N A in i s o l a t e d nuclei. I t also a f f e c t e d t h e i n c o r p o r a t i o n i n t o R N A , a n d t o a les.~ier e x t e n t , t h a t i n t o O A S P . S u c h a n i m p a i r m e n t c o u l d be r e l i e v e d b y exoger, o u s nucleic acids. T h e s e r e s u l t s closely parallel t h e " However, higher concentration o.~ N N A might atiect the hmorpor~ttion into tile nuc!eic acids of tile control nut|el (Table VII, M25). " In this particular experhuent (M38,1 e~ en added t~N.k inb.ibited the incorporation into I)NA of the control nuclei (]'able X'II).
Re]erences p. 45M456.
454
M. S/SKIGUCHI, A. $IBATAN[
VOL. ~z~.(I959)
pioneering findings of ALLFREY e t a l . I concerning the effect of various nucleotide c o m p o u n d s on the restoration of i m p a i r e d incorporation of labelled precursors into protein a n d R N A of the D N a s e - t r e a t e d nuclei. H o w e v e r , an extensive r e m o v a l of DiqA u l t i m a t e l y resulted in a different response of nuclear D N A a n d R N A with regard to their ability to recover t h e incorporation a c t i v i t y on addition of exogenous nucleic acids; while incorporation into R N A w a s partially or fully restored, t h a t into D N A was scarcely regained. This indicates t h a t the bulk of the s2p incorporation into nuclear R N A can proceed w i t h o u t an a m o u n t of pre-existent D N A w:~icb is indispensable for the a2p i n c o r p o r a t i o n into D N A . Such findings m a y at first sight seem to be irt line with the s y n t h e s i s of d e o x y p o l y nucleotide b y the polymerising e n z y m e in the s u p e r n a t a n t fraction of m a m m a l i a n cells ~, ~o, which, like t h e corresponding bacterial e n z y m e 2~, requires p r e f o r m e d D N A as the primer. Howe*cer, since even t h e addition of heterologous D N A failed t o restore the incorporation into D N A in t h e nuclei t r e a t e d e x t e n s i v e l y with D N a s e , th~ significance of pre-existing D N A in t h e incorporation into D N A in isolated nuclei m u s t be something more c o m p l i c a t e d t h a n its function s i m p l y as a " p r i m e r " . Although some of the results of the present investigation were n o t v e r y conclusive, the main b o d y of results w o u l d suggest t h a t the f a v o u r a b l e action of the cxogeneous D N A and R N A on the D N a s e - t r e a t e d nuclei is n o t peculiar t o nuclcotide compounds. The hypothesis previously proposed b y ALLFR:EY AND MIRSK~ ascribing the action of a d d e d nucleic acids to their function as the potential source of highenergy nucleotides becomes thus r a t h e r unlikely. In animal cells the D N A is " n e u t r a l i s e d " b y basic proteins such as p r o t a m i n e s a n d histones, a n d the r e m o v a l of D N A w i t h o u t t o u c h i n g the basic proteins will leave the nuclei positively charged, a n d this will p r o b a b l y b e t h e cause of the reduction of the ability of D N a s e - t r o a t e d nuclei to form A T P a n d of t h e c o n s e q u e n t b l o c k a d e e l s y n t h e t i c a c i t i v i t y s. Polyanions of either nucleotide or non-nucleotide n a t u r e m a y then "'neutralise . . . . basic" nuclei depleted of D N A . SPIEGEr--~tA,~"~* s h o w e d t h a t in l y s e d p r o t o p l a s t s of B. megatariu~n t h e depletion of D N A did n o t abolish the a b i l i t y to synthesise D N A as well as R N A or protein. Tiffs was confirmed b y OTSUJ[ AND TAKAGI ~ w i t h l y s e d protoplastc ,~: E. aoli. T h e difference b e t w e e n animal cells a n d bacterial cells m a y p a r t l y be explained b y t h e fact t h a t D N A in bacterial cells is, unlike in animal ceils, a p p a r e n t l y c o m p l e x e d w i t h some amines of relatively ]ow molectdar weight such as putrescine or spermidine m, which, on elimination of DNA, m a y easily be l o s t from t h e p r o t o p l a s t a n d wi!I n o t cause a serious d i s t u r b a n c e of electrostatic charge distribution a r o u n d the s y n t h e t i c mechanisms still o p e r a t i v e on the lysod protoplasts. Considerable incorporation of vzp into O A S P was noticed in o u r present s t u d y , a n d it was not as sensitive to the r e m o v a l of D N A as asp incorporation into nucleic acids. H o w e v e r , tim n a t u r e of this incorporation into O A S P was n o t studied, and w h a t fraction of it represented t h e formation of nuclear A T P therefore remains obscure, The results r e p o r t e d here are consistent with t h e idea t h a t for D N A s y n t h e s i s in the nucleus preformed specific D N A is n e e d e d for t r a n s m i t t i n g the genetic information to the p r o g e n y molecules, b u t it is n o t required for the synthesis of nuclear R N A for which the genetic information is t r a n s m i t t e d f r o m D N A on;,y indirectly via some sort of macromolecules o t h e r than D N A , B u t it m u s t be emphasized t h a t t h e present s t u d y c a n n o t be t a k e n even as a partial proof of this. Firstly, o n l y t h e incorporation Re[erences p. 455[456.
voL. ~4 (x959)
goI.E OF DNA tN a~-p INCORPORATION
455
Of s--p, not the net .oy . . . .". .' -. .-."~s, was foUowed i~ vitro; secondly, no i n f o r m a t i o n was collected a b o u t the f o r m a t i o n of specific macromolecules. F u r t h e r , t h e r e m o v a l of D N A was n o t absoh:te!y complete, a n d one does n o t k n o w w h a t role t h e DzNA remMning after DNase t r e a t m e n t played in the observed anabolic processes of t h e nuclei which were restored b y the polyanions. NOTF, A D D E D
IN I ' R O O F
After this paper h a d been s u b m i t t e d for publication, ALLFREY AND MiRSI~.X"~7 r e p o r t e d an extensive investig~ttion on the effects of various non-nucleotide poly,anions, including chondroitin sulphate a n d p o l y e t h y l e n e .~ttlphonaee, upon the DNase~ t r e a t e d nuclei. Their results v e r y clearly confirmed our suggestion as to the role of D N A as a p o l y a n i o n in the anabolic processes of isolated nuclei. T h a n k s t o the imp r o v e m e n t of technique indicated b y the American authors, it has also been demons t r a t e d in this l;tboratory e* t h a t in the presence of chondroitin sulphate the nuclei cart be digested with DNase w i t h o u t their a b i l i t y to incorporate azp into R N A a n d DNA being ~fffected; b u t in tiffs case also, an e x h a u s t i v e removal of D N A completely abolished t h e incorporation into DNA, whereas t h a t into R N A was fully retained u n d e r the s a m e conditions. Thus, a m b i g u o u s points of the present paper h a v e now largely been e l i m i n a t e d (Received March 3Ist, 1959) ACKNO~,VLEI)GJ~MENffS
The advice a n d help of Dr. S. OsAw:x, N a g o y a U n i v e r s i t y , a n d Dr. Y. Sucdxo, T a k e d a Pharmaceutical" Industries, Ltd., Osaka, on the technical aspects of tiffs s t u d y are deeply appreciated. We f u r t h e r express our g r a t i t u d e to those who were m e n t i o n e d above as h a v i n g supplied materials. R EFERF.NC ES S. OsAWA, V. G. ALLFRE~" A.XD A. E . 3ItKsKY, j . Ge~,. Physiol., 4o {t957) 4 9 t . V. G . ALLFRF.Y, A. E . R IIRSKY AND ~. ()SA~.VA, J. ~e~t. Physial.. 4 ° {tc~57 ) 45~R . LOGAN, Biochim. ICiophys. Acta, 26 0 9 5 7 } 227. V . G . A L L F R B Y AND A . E . ~][RSKY, P y o c . 2~?'i,tl..-[clrd. Sc~. U. S., 4 3 (I957) 8 2 z . T . R . BblEtTMAN AND G. ('. ~,V~;BSTER, B~ochim. Biophys. ,-; ta..27 ( r 9 5 8 ) 4o8. 8 T . R . BREHMAN AND G. C. ~VEBsTER, l-'ederatioJt Prec., t 7 ( [ 0 . ~ ] 0 4 . 7 M. FRIEDKIN .AND H . V~rooD. ]V, J. Biol. Chem., 2.20 [ [ 9 5 6 ) 039. 8 V. (.7,. ALLFREY AND A. E . ~[IRSKY, Proc. Natl..'lead..b'ci. U. S , 43 (i957} 580. I~. MANTSAVINOS A~;D E . S. CAN'ELLAKIS, Biochim. Bicpt;ys. Acta, 27 (I958) 66~. to /~. j . BOLLUM AND V . R . P O T T E R , J. t3t0l. Che~., 2 3 3 (I958} 478. 11 M . Sa~.KIGUCHI AND A . StrBATANI, J~Jiochi~n. Bio#hys. Acta, 28 {[958) 4 5 5 la A. SIBAZANI. I£xptl. Cell Reset~rcl~, f i n t h e p r e s s ) . t3 ]-t. K . I
45(j
.'VI, S E K I G U C H I , A0 SIBATANI
VOL. ~14
(1959)
e.'2 M. J , BESSMAN, I. R . Lt~HMAN, E , S. SI.~I~IS AND A. KORNBERG, J . 13iOL C]~e1~t., 233 (t958) X7X. ~1 .~. FICQ AND 1"~{. ERRI'RA, .E.gp[l. Cell l{ese~vvtt, x4 (x958) I8'2. 2~ S. SI'IEGEL~tAr¢, i n XV. D . MCl/;LnOY a ~ n I3. GLASs, The Chemical Basis el Hevedily, J o h n s H o p k i n s P r e s s , B a l t i m o r e , r 9 5 7 , p. 232. 25 N, OTSIJJI 2~ND Y. "]~AKAI~;I,.l'~J'Oa.7lk Symposittm on Nuc, leic /tc~ds, ~Vagoya, J a p a n , 2958, p. 6. 16 D. L. IO~ISa'ER, Fedevatio~t .Pvoc,, 17 (1058) 84. =¢ V. G. ALLFRE~r AND A. E, ~{rastcv. Prec, ,Nratl. Arad. Sci. U.S., 44 0 9 5 8 ) 9 8 I . ll~ Y.-T. "['CHOIg .~.NI) a . SIBATANI, Nurture, in t h e p r e s s .
STUDIES THE
ON THE LIVER
CARBOHYDRATE FLUKE
METABOLISM
OF
FASCIOLA HEPATICA
"FAG E . . X l A N S O U l t
DcpaYtment o/ Ph:*rmcc¢ology, Louisitz~a Stale UJ~ioeysily, Hvhool o[ 2~ledici~te, .V~;ro Orleans, La. ([7.S..4.) ( R e c e i v e d O c t o b e r 3 x st, 1058)
SUM,MARY
P'asciol~ ha~at@a utilizes c a r b o h y d r a t e a n a e r o b i c a l l y at a high rate. W h e n glucose is o m i t t e d from t h e m e d i u m , tim a m o u n t of glycogen utilized varies from 8 4 - 9 7 t~moles of glucose units/g wet wt. :6 h. The r a t e of glucose utilization from t h e o u t s i d e medium varies from i i o - i 8 o / x m o l e s f g w e t wt./6 h. P r o d u c t i o n of volatile f a t t y acids accounts for a l m o s t all of the c a r b o h y d r a t e utilized anaerobically. These acids were identified as propionic and acetic acids in an a p p r o x i m a t e ratio of 3:x. O n l y 4 - 9 % of the metabolized c a r b o h y d r a t e is c o n v e r t e d to lactic acid. The .rates of glucose utilization, lactic acid a n d volatile f a t t y acid p r o d u c t i o n are o n l y slightly decreased in the presence of atmospheric oxygen. O x y g e n is utilized b y these organisms when available. The r e s p i r a t o r y q u o t i e n t varies from x.56-2.2. Anaerobic m e t a b o l i s m of flukes with ligatured oral openings is identical with i n t a c t organisms. This : a d i c a t e s that neither the absorption of glucose from outside m e d i u m , n o r the excretion of the metabolic p r o d u c t s are carried o u t t h r o u g h the g u t of t h e organism.
INTRODUCTION
Previous studies indicated t h a t c a r b o h y d r a t e is the major, if n o t the exclusive source of energy for m a n y parasitic helminths 1. a. T h e e n d p r o d u c t s of c a r b o h y d r a t e m e t a b o lism v a r y from one species to another. As e a r l y as 1926, WIt-INLAND AND VON 13RAND 3 o b s e r v e d t h a t the utilization of c a r b o h y d r a t e b y l:asciola hepatiaa in vitro is associated with the production of higher f a t t y acids and possibly b u t y r i c acid. Their experim e n t s were carried o u t on the E u r o p e a n v a r i e t y of these organisms. The e x p e r i m e n t s reported in this p a p e r were e a n i e d o u t on a v a r i e t y of Fasciola hepatica which is p r e d o m i n a n t on the Gulf Coast of this country. I n these experiments, t h e r a t e of endogenous c a r b o h y d r a t e utilization a n d of glucose u p t a k e were m e a s u r e d u n d e r Relerenaes p. 464.
,x. F. s. a. H:mEE~ el aL
voL 34 (I959)
REFERENCES
i S. j . SIN~E~, Prod. ,Vat. A~ad. Sci. U.S., 4 r (1955) I o 4 t . S, J . S~NGER, J . Cell. Co~np. PhysioL, 50, s u p p l . I ( J 9 5 7 ) 5 I . A. F. S. A. HABI~Ee, If. G. CASSltJ~" AND S. J . STNGEIL 13iochim. Biophys. Acre, 29 ( [ 9 5 8 ) 587. * S, I, EvsX~zN A.~t> S. J. S t a G e R , J. A m . Chem. Soc., 80 ( I 9 5 8 ) x274. 5 I). PRESSMAN. A. L . GROSSBERG, L. H . PENCtg AND L , P A U L I N G , J . A ~71.Chem. Sot., 68 ( i 9 4 6 ) 250. [-I. F R A E N l i I ~ L - C o N R A T , R . S. B E A N AND H . LINEVCEAVI~R, ,l. BiOl. Chem., 177 ( t 9 4 9 ) 385. r ~A'. L. HUGely:s, H, A. SAROFF AND A. L. CARNEY, .l. AIR, Chgm. Sot., 71 (1949) 2476. 8 A, N I S b ~ ' O F F AND i ) , PRESfl~,IAN, Science, x28 ( t 9 5 8 ) 659. R. A. KEKW]eX< ANt) R . I<. ('AN~AN, Biochem. J., 3o ( t 9 3 6 ) 227. lt~ St. C. B a y : c a , D. H . Ca~t eg~LL, S. I. Epsrr:~.~ ANt> S. J. S ~ N ~ , J . A m. Chem. See.. 78 (~ 956) 3 ~ 2. ~ D. H . CA.~teB~LL, R . H. BLAKER AND A. B. P A R O ~ , J . A m . Chem. Sot., 70 ( I 0 4 8 ) 2496. ~z H . FRA~r~KEL-Co~RA*, J . I. H A R R m ANI) A. L. L~VY, i n D, GL~CK, 2t4ethods o[ Biochemical A~zalysis, Vol. z, I n t e r s c i e n c e , N . Y , , 1955, p. 37 o. 18 j . l{. MARRACK .~.ND E . S. ORLAN,~, BF]I. J , E x p l L l~athol,, 35 (I954) 389. 14 I r. H . ~][AURER, J , SRI R A M AND S. E H R E N P R E I S , AVCli. Biochem. #iophys., 67 (x957) 1 9 6 . ~ \V. A. KLEV_: AND F. M. R l C n A r t o s , ]. Biol. Chem., ~29 (~957) 489.
INCORPORATION
OF
OF
THE
ROLE
a-~p I N T O
RABBIT
ISOLATED
APPENDIX:
OF DEOXYRIBONUCLEIC
~. SEKIGUCH[
NUCLEI'
ACID
° AND A. S I B . X T A N I
C3qo~hamistvy Laboratory, Ya~nagl~ti 3ledicai School, t:be (Japan) ( R e c e i v e d O c t o b e r 22rid, "/958)
SUMMARY
I. Nuclei isolated from r a b b i t a p p e n d i x could incorporate asp into DNA, R N A a n d organic acid-soluble phosphorus fraction (OASP) i~ vitro. 2. The removal of tile bulk of DNA from th~ nucleus b y p a n c r e a t i c D N a s e abolished the incorporation into DNA a n d R N A almost completely, while t h a t i n t o OA~qP was r e d u c e d o n l y to a b o u t 6o %. 3. Appreciable restoration of the i m p a i r e d incorporation a c t i v i t y was o b s e r v e d when salmon sperm DNA or y e a s t R N A was a d d e d to the nuclei t r e a t e d w i t h DNase. However, if an extensivc r e m o v a l of D N A h a d been achieved, the restoration occurred o n l y in the case of RNA, a n d n o t DNA, of the nucleus on a d d i t i o n of exogenous nucleic acids. 4. Some indication was o b t a i n e d t h a t certain polyanionic compounds, such as c h o n d r o i t i n s u l p h a t e or h y a l u r o n a t c , could replace tile nucleic ac:ds in restoring t h e imi)aired a c t i v i t y of D N a s e - t r e a t e d nuclei, p r o v i d e d tile action of DNase w a s not v e r y extensive. 5. It was suggested t h a t in the observed restoration a d d e d nucleic acids a c t e d m e r e l y as polyanions which m i g h t eliminate tl~e imbalance of electrostatic charges resulting from the r e m o v a l of the bulk of DNA from the nucleus. A b b r e v i a t i o n s : A'I'I', a d e n o s i n e t r i p h o s p h a t e ; R N A , r i b o n u c l e i c a c i d ; D N A , d e o x y r i b o , nucleic acid; DNase, de(ix),rib~nuclease; ()ASP, orgatlic acid-soluble phosphate fraction, " G n l e a v e fr[~irt t h e D e p a r t m e n t o[ B i , l o g y , F a c u l t y of S c i e n c e , O s a k a U n i v e r s i t y , Oz~ka0 Japan.
Relerences p. 435/456.
VOL. 34 (X959)
ROLF. OV D N A [.~" a2p
445
INCORPOR.kTION
iNTRODUCTION
l~Iuch information has been collected in recent years a b o u t the synthetic activities of isolated nuclei of animal c~,ls. T h e y can p h o s p h o r y l a t e adenosine m o n o p h o s p h a t e to adenosine triphosphate ~, at.4 incorporate various labeIled precursors into proteins a, fibonucleic acids 2-6, and d~oxyribonucleic acidS-L The removal of the bulk of D N A b y d e o x y r i b o n u c l e a s e impairs the incorporation of the precursors into R N A a n d protein, a n d the lost a c t i v i t y is restored b y various nucleotide c o m p o u n d s of relatively high molecular weight, including D N A a n d R N A L The f o r m a t i o n of A T P in the nucleus is also d e p e n d e n t on the presence of D N A , which can likewise be replaced b y exogenous nucleic acids s. I t has t h u s been p r e s u m e d t h a t one of the roles of D N A is to p r o v i d e energy-rich nucleotides needed for the s y n t h e t i c a c t i v i t y of the nucleusL E n z y m e s catalysing the incorporation of t h y m i d i n v or d e o x y r i b o n u c l e o t i d e t r i p h o s p h a t e s into D N A were found in the s u p e r n a t a n t fraction of certain animal cells; t h e y require D N A for their full a c t i v i t y g, m. I n t h e present s t u d y we have a t t e m p t e d to determine (x) whether the preformed D N A in the nucleus is indispensable for the incorporation of inorganic a2p into D N A in isolated nuclei, a n d (2) w h e t h e r the incorporation a c t i v i t y of isolated nuclei depleted of their D N A can be rest,~rcd b y some anionic c o m p o u n d s of non-nueleotide nature. In doing this we are looking for some relevant information coucerning the possible role of D N A in t h e anabolic processes exhibited b y the isolated nuclei, either as a carrier of information needed for fabrication of macromolecules, as a potential source of high-energy p h o s p h a t e c o m p o u n d s , or as a polyaniordc c o m p o u n d the removal" of which will cause s o m e d i s t u r b a n c e in the electrostatic charge distribution within t h e nucleus. A p r e l i m i n a r y n o t e on this work haz been published~L
EXPERIMI'-'NTAL
-"
Chemical compounds Y e a s t R N A was a gift of Dr. KVROIWA, Kirin Research Institute, Kirin B r e w e r y Co., I~td., Amagasaki. H i g h l y polymerized salmon s p e r m I ) N A was received from Prof. I. WATANABE, U n i v e r s i t y of T o k y o . A sample of chondroitiu sulphate was o b t a i n e d from Dr. T. MAKI, Minophagen P h a r m a c e u t i c a l Co., T o k y o , a n d another s a m p l e p u r c h a s e d from W a k o Pure Chemical Industries, Ltd., together with a sample of heparin. A highly purified p r e p a r a t i o n of h y a t u r o n a t e was a generous gift of Dr. M. ISHIMOTO, U n i v e r s i t y of Tokyo. A crystalline preparation of egg albumin was kindly supplied b y Dr. Y. FUJISAKI, Y a m a g u t i Medical School. The mixed 2', 3'-mononucleotides were o b t a i n e d b y hydroiysing y e a s t R N A in o.I N N a O H at 80 ° for 20 Iain a n d neutralising with HCI, the c o n t e n t of NaCl was calculated a n d s u b s t i t u t e d for the NaCI in the incubation medium, asp was received from the Radiochemicai Cenh~, A m e r s h a m , England, on allocation of J a p a n Isotope Association. Preparations of pancreatic D N a s e , once crystallized for t r e a t m e n t of nuclei, a n d n o t crystallized for hydrolysing a D N A preparation, c a m e from the W o r t h i n g t o n Biochemical Corporation. A p a r t of t h e m was m a d e available t h r o u g h the courtesy of Dr. K. SuzuKI a n d Dr. H. UCHLPA of t h e U n i v e r s i t y of T o k y o . Re/eyences p. 4551456.
,t46
,~I. sv.lltGUCrtX, a. SmAT.~NI
w)L. 114 (I959)
I>rcparaliott, o/ mtclei and lymphatic, cells Ap p en d ices and P e y e r ' s p a t c h e s of a d u l t r a b b i t s served a,> the s t a r t i n g m at eri al . Sucrose mtclci were p r e p a r e d essentially accordi ng to ALLFREV el al.a; t h e y were tinnily s u s p en d ed in 0.25 3 f sucrose-o.oo2 3 [ CaCI= at a delasity of 1.5" Io ~ nuelei/ml. Lyml¢,latic cells were o b t a i n e d as described in a s e p a r a t e c o m m u n i c a t i o n le.
l~wubati,.n aith a'.,p l_:sually the final i n c u b a t i o n m i x t u r e consisted of i ml of a i :i m i x t u r e of 0.25 ~ir s u c ro s e- o .o o 2 3 I CaCla c o n t a i n i n g 3 ' r o ~ nuclei a n d Ca-free T y r o d e solution, T hi s s u s pen d in g m e d i u m will h e n c e f o r w a r d be called the s u c r o s e - ' F y r o d e m e d i u m . T h e su.qpension also c o n t a i n e d 5 tzg of s t r e p t o m y c i n , IO I~C of ~ " P - o r t h o p h o s p h a t e a n d o t h e r a p p r o p r i a t e a d d i t i o n s w he r e i n d i c a t e d . Tim m i x t u r e was e q u i l i b r a t e d in a IS-ml centrifl:ge l u b e at 37* for 5 rain a nd t h e n shaken at tim slime t e m p e r a t u r e . I f not specified, the inc~ibatio:i lasted I. 5 h. l';xpcrime1~ts w i t h l y m p h a t i c cells were c o n d u c t e d as d e s c r i b e d e l s e w h e r e ~. hMividxlal tests were rtm in d u p l i c a t e tubes. In m o s t e x p e r i m e n t s , where t o t a l a c t i v i t y of mlcleic acids p e r t u b e h a d to be d e t e r m i n e d , a n o t h e r set of d u p l i c a t e t u b e s :vitht,ut ~zP was r u n for each of t h e var i e d , ; x p e r i m e n t a l condi t i ons, a n d s u b j e c t e d to the determination" " ~ of nucleic acids b y the S c h n e i d e r m e t h o d at t h e end of i n c u b a t i o n withot~t s e p a r a t i n g the nuclei from the i n c u b a t i o n m e d i u m .
Dit¢cstion. o/~mt:lci a i¢/~ DNase Th e nuclei (o.2 ml of t h e original suspension) were m i x e d with 0.2 ml of T y r o d e solution c o n t a i n i n g o.o125 M MgCla w i t h a n d withotlt an a p p r o p r i a t e a m o u n t of c r y s t a l l i n e I)?-;a.~c_ T he m i x t u r e was inc~lbated at 37 ° for I5 rain, chilled in ice, a n d a d d ed to 5 ml of ice-cold x :r m i x t u r e of o.25 3 I s u c m s e - o . o o 2 .lI CaCI 2 aucl Ca- a n d Mg-free T y r o d c solutic,'n c o n t a i n i n g o.oz 3~/sodium ci t rat e. T h e nuclei were s e d i m e n t e d b y centrifuging, and rcsu~pendcd in t h e final i n c u b a t i o n m i x t u r e as d e s c r i b e d a b o v e .
l'r~'paralim, el mwlcic acids a~ul organic acid-soluble phosphale [raetio~ Incub:.dion was t e r m i n a t e d by chilling in ice; t h e c o n t e n t s of i n d i v i d u a l t u b e s (nuc|ei plus m e d i u m ) along with the a p p r o p r i a t e z e r o - t i m e cont rol were i m m e d i a t e l y s u b j e c t e d to the p r o c e d u r e described e l s e w h e r e e for th~ p r e p a r a t i o n of nucleic acids a n d OASP.
Deter~nD:ation o/mtcleic avids and total phosphor~ts D N A - P a n d t o t a l P were d e t e r m i n e d as in a previ ous r e p o r t ~s. R N A - P was m e a s u r e d b y the phloroglucinol test accorr!ing to HAItN AND VON EUI,ER 14.
3ice~urvmc'l~i o~ r.dft,.~ticdy This was m a d e a~ in a previou~ r e p o r t ~a using an e n d - w i n d o w Geiger-Miiller eemnter. In each e x p e r i m e n t t h e r a d l o a e t l w t y of t h e sam pl es of z e r o - t i m e c o n t r o l were m e a s u r e d , and when not ~legligible, s u b t r a c t e d f r o m t he values of t h e tr~sts. This was n e c e s s a r y because t h e activities o b t a i n e d were r a t h e r low, especially w i t h DNA, in m a n y of t h e e xpe r i m c r t t s r e p o r t e d here, a l t h o u g h t he c o n t a m i n a t i o n f o u n d in zeio-tim~ samples was usuMly v e r y slight, if any. Specific a c t i v i t y was e x p r e s s e d l'¢e#rr,wes p. 455/ ~56.
VOL. 3 4 (t959)
ROLE oF D N A
I x .~2p tNCORPOR.VrlON
447
in c o u n t s / m i n / / ~ g P. I n m o s t cases, h o w e v e r , ~he t o t a l r a d i t ~ a c t i v i t y ( c o u n t s f r n i n / t u b e ) w a s e m p l o y e d t o e x p r e s s t h e e x t e n t of i n c o r p o r a t i o n into nucleic acids. T h i s w a s c a l c u l a t e d f r o m t h e s p e e i f i , a c t i v i t y of p u r i f i e d samp!e.~ a n d t h e t o t a l a m o u n t of r e s p e c t i v e nucleic a c i d P (/.g/tube}.
Cl,romatographio proae~h~re A l a r g e - s c a l e i n c u b a t i o n of t h e nuclei wa:; c o n d u c t e d u s i n g I o o m l of n u c l e a r s u s p e n s i o n w i t h t h e s a m e c o m p o s i t i o n a s a l r e a d y d e s c r i b e d . T h e m i x e d nucleic a c i d s w e r e o b t a i n e d b y t h e r o u t i n e p r o c e d u r e ~2. T h e y wen> f r a c t i o n a t e d i n t o D N A a n d R N A in t h e u s u a l w a y b y a l k a l i n e h y d r o l y s i s w i t h o.3 N K O H a t 37 ° a n d a c i d i f i c a t i o n with io N perchloric acidlL The RNA fraction was adjusted to pH 3 and treated w i t h c h a r c o M , a n d t h e a d s o r b e d m o n o n u c l e o t i d e s w e r e e l u t e d w i t h 20 %; a q u e o u s p y r i d i n e a c c o r d i n g t o V,x.,," Ill.;l~iCu.-,it~, in o r d e r to e l i m i n a t e the e v e n t u a l c o n t a m i n a t i o n of i n o r g a n i c ~-P. T h e y w e r e t h e n chr0m.:togral,J~ed on a D o w e x - i c o l u m n ( X - x o , f o m n a t e f o r m ) a c c o r d i n g t o Os.xw.x et al.tL "With i n t a c t l y m p h a t i c cells, 40 m l of cell s u s p e n s i o n (4" lOS c e l i s / m l of C a - f r e e T y r o d e s o l u t i o n ) w a s u s e d a n d t h e c h a r c o a l t r e a t m e n t w a s o m i t t e d becau'se t h e ~zp c o n t a m i n a t i o n c o u l d b e n e g l e c t e d in t h i s case. T h e D N A p r e c i p i t a t e d f r o m t h e a c i d i f i e d s o l u t i o n of t h e "alkaline d i g e s t of c r u d e • n u c l e i c a c i d s w a s w a s h e d t w i c e w i t h c o l d i o % "tr'.'-~hloroacetic acid, r e d i s s o l v e d in a d i l u t e N a O H , n e u t r a l i s e d w i t h HC1, p r e c i p i t a t e d will,_ e t h a n o l , w a s h d d in e t h a n o l a n d e t h e r , a n d dried. T w e n t y m g of D N A t h u s o b t a i n e d w a s d i s s o l v e d in 4 m l of o.oo~ M MgCI,, s o l u t i o n c o n t a i n i n g 4 m g D N a s e a n d i n c u b a t e d a t 37 ~ for 8o rnin a n d t h e d i g e s t w a s a c i d i f i e d w i t h c o l d p e r c M o r i c a c i d a n d c e n t r i f u g e d . T h e m i x t u r e of n u c l e o t i d e s of ~,afious sizes in t h e s u p e r n a t a n t w a s c h r o m a t o g r a p h e d orL a D o w e x - I c o l u m n (X-Io, f o r m a t e f o r m ) a c c o r d i n g t o t h e m e t h o d of SAK.XK[ls, u s i n g O.3¢>--7.0 N f o r m i c a c i d in t h e reser~-oir of a g r a d a a t e d e l u t i o n s y s t e m .
RESULTS
Inaorporation aativity o/tlw isolated m~cloi ~ q ~ e n t h e nuclei w e r e i n c u b a t e d w i t h zap in t h e s u c r o s e ~ T y r o d e m e d i u m , z2p w a s i n c o r p o r a t e d i n t o D N A , R N A a n d OASP, t h e i r specific a c t i v i t i e s i n c r e a s i n g in t h e o r d e r m e n t i o n e d ( T a b l e I), A s s h o w i t in Fig. I , t h e i n c o r p o r a t i o n i n t o t h o s e f r a c t i o n s p r o c e e d e d l i n e a r l y f o r a b o u t I h asxd t h e n t e n d e d t o level off. I n s u b s e q u e n t o x p e r i m e n t s t h e p e r i o d o f i n c u b a t i o n w a s f i x e d a t 9 ° rain. TABLE I f~lg 71~t¥0 I N C O R P O R A T I O N
OF 52p
INTO LYXfPHATIC CELLS AND SUCROSE
NUCLEI
OF RABBIT
APFENDIX
Cells ~,+ndnucle'." w e r e obt-.xined from the same source of pooled t~szucs ~nd inc.tlbated for i .5 h at 3 7" + :llal,t, rial
3" i o s cells 6.1o s nuclei
/llCuGrillorl ln',edi.:im
2 m[ Ca-free Tyrode 2 ml Suerose-Tyrode
Retercnces p. 4.55]456.
Cvnce~anl,'i,m o1 a~l" (,~d-'. ~lil,r
O:Lx,/:.
5 Jo
455o t42o
.q~-": :,'i~
3-'2 50,2
48-5 2.15
6.6 26.2
448
M.
SEKIGUC}II,
h. SIItATANI
VOL. 34 (~959)
C o m p a r e d with the results on l y m p h a t i c cells prepare d from the s a m e tissue, the incorporation into the nuclei was considecably less (Table I). The specific a c t i v i t y ratio R N A / D N A was m u c h higher in the nuclei t h a n in the whole cells, a n d this was in a g r e e m e n t with the current view t h a t the isotopic u p t a k e b y nuclear R N A w a s much higher than t h a t b y c y t o p l a s m i c R N A TM. These observations, t o g e t h e r with the fact t h a t D N a s e t r e a t m e n t reduced the D N A c o n t e n t a n d z2p u p t a k e of the nuclear preparation (Table VI} b u t not of the intact ceils, indicate t h a t the o b s e r v e d incorporation was due to the real a c t i v i t y of isolated nuclei rather t h a n of contAminzting i n t a c t cells. In the course of the w o r k it was noticed t h a t the incorporation a c t i v i t y of t h e nucleus was subject to seasonal variation, being highest in s u m m e r a n d decreasing gradually to re~ch a c o m p l e t e loss of a c t i v i t y in midwinter. A similar t r e n d w a s alsc. noticed with i y m p h a t i e cells of r a b b i t a p p e n d i x ta. mP,+a laNAI
O.~SP 2000 DN,X
.~ 5@ ! L5
1500
.~ ./. . . . . ~3o
""6ase
./.t
I.o
1OO0
,~ 2o .~
0+5
, /,'7"Y
500
Incubati.c,n t i m e (h)
l ' i g . +. [ ' i m e c o u r s e , f f a a l '
incorporation
into I)NA, RNA appendix.
and OASP
~I i s o l a t e d
nuclei of rabbit:
Labelling o~ m+cl~ia acids The specific a c t i v i t y p a t t e r n s of individual 2 ' , 3 ' - m o n o n u e l e o t i d e s o b t a i n e d b y alkaline hydrolysis of R N A ' s of isolated nuclei a n d i n t a c t cells were v e r y similar (Table II). It m a y be noted t h a t the results closely paralleled those o b t a i n e d w i t h RNA of the rat liver i.~ vivo (I and 2 4 h) ~°. The fractions o b t a i n e d c h r o m a t o g r a p h i c a l l y from a D N a s e - d i g e s t of D N A of t h e i n c u b a t e d nuclei showed a m u c h more diverse d i s t r i b u t i o n of specific activities t h a n t h a t observed in ~,ivols,za, b u t appreciable quantities of r a d i o a c t i v i t y were r e c o v e r e d TABLE LAI}I~LLING
lI
OF INI}IVIDUAL 2 / , 3 ~ - . % I O N O N U C L I r ~ O T l t . ) E N OlV R N A LYMPI4ATIC CI£I.L,~ ()F RAI~Iti'I" A P P I r . h ' I ) ] X I N C U F I A ' I ' ] , : I )
N u c l e i a n d cells ¢)rigill&tt'd f r o n t S e p a r a t e s o u r c e s , ] ) u r a t i o n
IN NUCLF.I ~;Jt l,,~'tY9
ot i n c u b a t i o I ] :
3,p¢¢i[ic aeffvdy (¢om:t~ pnin /,~ I')
I+ymphatic
ceils
IsoJated quc]ci
R e : .~re~:ca~ ~. 4551456.
:t denyti¢ .eat-ul
Gmo=vtfc a c &l
Cytfdyli, a ~ td
7.5- ~
53.o
54.5
7.°5
-1.2 .5
4.7 °
! "ridvl& .rid
Oz,D 7-84
.%NI)
I. 5 h
VOL. 34 (1959)
l~OXr: OP D N A iN a~p INCORPtHiATION
44"9
from all the ultraviolet-absorbing fractions e x a m i n e d (Table II[). The observed radioaetivities of the nucleic acid fractions o b t a i n e d from the nuclei incubated w i t h a~p can therefore be t a k e n to indicate t+he incorporation of amp into polynueleotides of DNA and RNA. "I'..~ D I . E
Ill
D I S T R I B U T I O N el-" R A D I O A C T I V I T I E S IN IHb'FIgRI-;N'T FRAC'IIC)N'.; ¢]P" A I)NA.qI-;-I>IGI';ST el-" ] ) N : X OF R-~BFII'I" APPb;,~I>IX N U C L E I I N C U B ~ T I ' ; | ) i~$ Z/i~rO I)NA
was
obtained
Ori~,i n a t DN, I
Specific activity (counts[min/H ~ t')
from
the
same
m>tterial
as I{.NA :~f'Fable
F m r l fim * ............................................................... • 3 -I .'~ "
o.3,'~" +
Relative P content {°/o o f t o t a l }
II,
y
+
+,,
I,,
o.,~-
O. Tt
o,3t'
:~-3:
o,57
;*-3 t
o-37
o.54
1.56
->,43
o.0
I +5
:¢. 7
2.o
4..5
4 t.~)
23.0
~-7
o.tb
7.1,.
* Fraction.~ I S were obtained cbro.~ato~raphica!ly; fraction O was extracted wit!-, b.+~t 5 ",~ trichlor+mcetic a c i d fr, m l t h e r e s + n , ' f f t e r t e r m i n a t i + m of elution w i t h 7 .V f o r m i c i t c l c l : I r a c t i ~ m t+~ was the acid-m.~oh~ble residue of ONase-digest. " * V ~ , ' e i R h t e d m e a n ,>.¢ s p e c i f i c a e t i v i t i e ~ o f f r a c t i < m s t - t +~: o.3<~.
Use el the total activity as a r~aasurc el i3~¢orporatio;v In e x p e r i m e n t s involving the removaI of DNA from the nucleas b y DNase a n d / o r the a d d i t i o n of nucleic acids, the use of the specific a c t i v i t y to express the m a g n i t u d e of ipcorpora*.ion is obviously misleading, hecaltse the a m o u n t of nucleic acids in different tubes m a y v a r y considerably and c a n n o t be used as the c o m m o n reference s t a n d a r d for the incorporated radioactivity. It was therefore decided to express the m a g n i t u d e of incorporation b y total a c t i v i t y . For OASP this wa.~ determined directly+ F o r DNA and R N A it was calculated from the c o n t e n t of respectiv'e nucleic acids per tube a n d the specific ,mtivities of the purified samples u n d e r the a s s u m p t i o n tha~ .'he lattm were representative: sar:~_ple.s of the nucleic acids present in respective tubes. Such an a s s u m p t i o n m a y be ju~¢ificd because the relative yield of nucleic acids in our f r a c t i o n a t i o n procedure was largely independent of the absolute a m o u n t of the acids (Table iV). It was also found t h a t the m a g n i t u d e o~ contaminatit~g a~P was independent el the a m o u n t of nucleic acids present. E~e,;t o/D.~ras~ treatmem In order to examine the effect of the removal of DNA on the a2p uptake, the nude;, were i n c u b a t e d for 15 rain w i t h varying concentrations of D,Nase in s u c r o s e - T y r o d e m e d i u m fortified with magnesium. The control tubes were also i n c u b a t e d with the same m e d i u m lacking DNase. As shown i~l Table V, a selective d e s t r u c t i o n of DNA in DNa_~e-treated nuclei could be detected. However, during the subsequent incubation a considerable mltount of DNA was reJ.e~.sed from the control a n d t r e a t e d nuclei. I n subnequent experiments, the e x t e n t of tl~e removal of DNA b y DNase was d e t e r m i n e d (somewhat arbitrarily) from the whole c o n t e n t of the tubes at stage C of Tabte V0 i.e., at the end of incubation for ,2p incorporation, the possible release of DNA from the nuclei into the medium during this incubation being disregarded. References
p.
45.~]456.
450
M. GEKtt~UCHI, A. SIBATANI "["\ H I_ E
rOE 3 4 (I959)
.IV
ASH)t:NT AND ItAI~ll)ACTtVII"T" (;1: I)~'N-.'k I"RO*I T/IN NUCLEI .qUBJI~CTED TO ])~"%'ASE TREATMIgNT OR AIIDITION OF SALMON S;PIgRM DN;Sk N u , c l e i w e r e d ~ g c s t e d w i t h D N a s e { 123 j t g ] m l ) , w a s h e d , : r o d s u s p e n d e d w i t h a n d w i t h b u t I ) N & (4 m g ] r n l ) . .'| r/l~l'//'[,¢l
IT.N',t.,t'
trcd/~/roll
.
.
.
.
.
.
.
[ ~*t'~t'nal
. . . . . . . . . I)NA -7" .... I).%~*:~.
.
.
.
.
.
.
.
.
I£ rtr:tctcd
I lO 342 "
.
.
I)¢,
42. 323
" "l't~.c r e ; t s o n w h y t h e c f f e c t of [)N~Lse t r e a t r n e n t in the tubes supple,uented with DNA is not clear 7A13LE
(Uo'¢/P~,~.'~tttl¢/?l /¢~"I~I
Total activilj, (c~)lOIt8~?l[~tfl~')
57
t -~:3
! NO
~)l 30 (2'3
0.40 O.O2 O. 4 [
z 30 I 1 3 I"
.
3ptltl]llt~activity
.
lt'equlivc vicld ("o1
2o9 21 202
in sncrose. -Tyrode mediura
was not
reflected
by the umount
of I . ) N A
V
CHANGE IN Q t r A N T [ ' I ' y OF I.'Ik~N~.-k A N D 1~%~'_3k DURING THE TR~;ATMI.JNT '4VITtl ].)~%'AS~ A N I ) •qUI~.~E~UENT INCURATION OF NUI:LE[
Samples were taken at ~the following stages. A: Original nuclear s-spension. B: Af~:er incubation w i t h o r w i t h , ) u t l h N a s e ( 1 2 5 p g / r t t l ) f o r i 5 n l i n ; t h e w h o l e c o n t e n t o f t h e t u t , e s w a s subjected t o analysis. Then the quclei were separated by centrifugation, resuspended in sucrose-Tyrode m e d i n m w i t h o r w i t h o u t a d d i t i o n s ~tnd i n c u b a . t e d f o r 9 0 n l i n . C : T h e w h o l e c o n t e n t o f the tubes as well as the txuclei separated from the medium by centrifugation was analysed. tfg P.lu(,c iu,rubated: Siege
A.
.4dditton
Initin!
-
13. A f t e r f i r s t incubation
--
Frarti~l
with DNage (test]
witho~ 1)Nase (contmO
Total
D,\'.t
RNA
~7 °
33
DNA
R.VA
70
33
*
Total
t 57
29
4"
3x
-
Total S:- ~ i m e n t
zi 6 89
3o i8
42 4z
29 29
S~tlmon sperm 1)NA
Total Sed~atent
342 " --
-26, ---
323 67
36 2.5
V c , ~ s t ft.','>.
"rotal Sediment
~ 21 {)7
44
235 ~)O
(1)Nose)
C, A f t e r s e c o n d
incubation (z:,p)
• Owing to the high viscesity ordinary centrifugatior~.
of I)NA
solution
added
the
2zs
38 nuclei could
22
not
be separated
by
Table VI shows the e x t e n t of removM of D N A and the c h a n g e in t o t a l a c t i v i t y of O A S P . R N A and D N A of the nuclei digested w i t h different c o n c e n t r a t i o n s o f DNa.se. The incorporation into D N A and R N A was progressively impaired b y t h e increase in the e x t e n t of ,+he r e m o v a l of D N A . A p p a r e n t l y the incorporation into D N A was more sensitive to the removal of D N A t h a n t h a t i n t o RNA. The i n c o r p o r a t i o a i n t o O A S P was m u c h less sensitive in this respect; it decreased o n l y t o 60 % of the control when the incorporation into nuclei~ acids was abolished almost completely Re[erences
p.
4~fi/456.
VOL. 3 4 (1959)
ROLE OF D N A
IN s e p IXCort'OR.VrlON
451
+r.&l+l .F+ v1 IEEFECT
Of
I¢|~.',IOV.-'t.L t ) [ : ].)~-'N",~ O N : | 2 I ) 1 N C O R P O I < A ' I ' I O N
T . t , d ,+cti+']h' F¢ou++ts rain trd~e)
.-/mot+hi ~./D.X':t P;.+pt, ,%'
of l),X'+tse (1+~ +ntJ
3123
(+!~ ,'~tt ~'+;~l/~,~]J
O.'L",I"
I(it+ 07
I 3,q3o ! ,'t,f~oo
O 10
63
~p2 04
z5 .3,12 t
~ I : r~(~L:%qrp;D N U C L I ~ I
I, t()O t,l~O
137 I 1.5
I (),t~oo
7~2
.S3
t !..5oo
45"
I ~,,-% l ()
()4~ [ ,5<)
4-
0
I+DO
12'5
.] I
Z -i,Ol (-'
d St>
20
z 2,4(~t,
7~
.5 t}~)
27 2[)
<1,5o+,
o
I +()f|l)
f~_%++'!
fq.%'.'~
, ,%[ tr¢l(+
20.~ 2i"
"
' 7
!8 [)
I Z
b y t h e r e m o v a l o f a b o u t 7 0 % o f t h e D N A , a n d e v e n a s l i g h t s t i m u l a t i o n of i n c o r p o r ; t t i o n w a s o b s e r v e d w i t h O A S P w h e n s m a U arr, ount.~ of D N A w e r e r e m o v e d . D u r i n g t h e c o u r s e o f e x p e r i m e n t s i t w a s n o t i c e d t h a t it w a s t h e c o n e e v t t r : t t i o , : o f D N a s e u s e d r a t h e r t h a n t h e e x t e n t of t h e r e m o v a l of D N A t h a t d e t e r m i n e d t h e e x t e n t o f i m p a i r m e n t of t h e i n c o r p o r a t i o n , A l s o t h e d i f f e r e n t b a t c h e s of l ) N a s e c o u l d differ markedly in their effect on the nuclei. "
Restoration+ o/th~ impaired .incorporation activity o/ D.V..~c-trcate
e l ~" ~ N U U L E I I t : ON
.-'t..CIDS A N D
SO~IE
OTIIER
-PHI+ I . ' - : ( . ' O R P O I ~ A T I { ) N
V II C(),~lPOl.'Nll:q
O1" ; 1 2 | ) [ N ' Y ( )
IX
Till';
Cd}N'I'ROL
IN~-t,_ l + . ~ r p j ~
.%HX rLrJNl-;
.",L'('LIC.[
'l'+,t,d ~lcti:'z'lY ( " , ,
.¢ C+#~tt,',D
..Idditi,,n ~p c t trd.'l
K.rpt, A'o.
L,;J" ,:"
I,L,+ 4
I),V.'~
7° 133
8~ 14(7
72 z 19
I ]2 ~ 51
1_, 7 7;-I
~ i~ ,"e,-i
96 ,~ I [3 ,S~. 33 4! t tz
zq, -~~' 07 ] n4 22 30 ~o
.......................
-%I24 M- 5 ~[3~
.'t,lz6 .M3o ~1138
Re#fences p. 45.~1456.
4 rrtg I~N.\ 1 mg R N . \ z nag tCNA 4 nt~ I-~NA z p.lg [;~N.~
2 2 4 z -' ;
mg -",3'-nxol~mtncleotitte.~ n~g c h o n d r o i t i n .~ttlphate mg c h o n d r o i t m sulpli,-tte rt;g hyah+r
,';4
3{)6 Io7 t IT 4 ¢;' 01 ¢11
452
~ . sm~tc, v c m ,
a . Sm,~TaNt
VOL. 3 4 (Z959)
Usually the presence of nucleic acids in the incubation medium did not very much affect the total activity of the control nuclei ('fable VI I), but occasionally there was a remarkable inexplicable diminution of the incorporation activity (Table VII, M38). In experiments involving the D N a s e digestion of, and addition of nucleic acidg to, the nuclei, the total activity of individual fractions was usually expressed in % of the undigested controls receiving the same addition, but in some experiments such "addition controls" were not run: the undigested control without addition served ~,s the ,',~¢~rence standard (values in parentheses in TaMe VIII). TABLE
VIII
1,2.FFICCT O F N ' U C L E I C A C I D S z~.ND S O N I E O T H E R C O M P t ~ U N I ) R I N I~HI~ I N C U B A T I O N O N TME: I N C O R P O R A T I O N O F gZ[, ][*','TO.~[JCI.EI TRIf, A T F . I ) 'I,'~ITM D N A ~
F rpL .Vo.
3Ia. t
Fraction o/
(.'O*|fC~ltrafi¢~lt O# I~Ncs¢ (p~ 'm#)
J),N'A rt'#/toved
iz 5
0, t
,4d*h'.Qo/t (per tube)
('+'o]
4 l~i~ I.)NA
4 irig I~N.\ Mzo
3I 25
Iooo
: 25
....
84
/,.lie)
Iooo
74
M39
M3S
25 o* ~
6 3 **
1 -'.5" *
25
~7 i ':6
t rsl,
B~
07
44
(, 1 o)
--
I~N..k
--z nag ]¢.NA 2 Irt~ 2 " , 3 " - r n o n o n u c l e o t i d e s 2 n'~g e h, m d r o i t i n s u l p h a t ~
6(,
3s
97 i o7-
2. 5 n t g I£N..K
-
2.5 m/4" c h o n d r o i t i n 314o
DY.'.4
7"
'-"nlg I~N..i 4 mg RNA
I -'5
~N,q
I ~9)
z nzg
3I2 ~
Total a~livity ( ~ o# ¢onlret*) O,q,Np
2.5 m g D N A
63
--2 mg RNA 2 mg chondroitin -o 2 illg RiNA 2 rnp. c h o n d r o i t i n 4 mg ehondroitin
'7
-2 ntg
R',:
~
z mg heparm 2 mg hya|uronate 2 mg egg albumin
sulphate
sulphate
sulphate .~ulphate
~IED1U,%I
o
( ~ O 6) (46)
S
( ~ e>) (9)
0 (J
~ .3
c,
el
4 r
Oo 57
57 83
35 54 U4
64 (I 1,1) t25 69
Io 13o) I5 "~7
t r (33) 42 44
15o
3
*
(184t
(9)
('}
I3S J l5 i i7
z8
2a
48 16
tI8 58
,o7
io7 ~ -'4 7q
87 113 83 96
75 l.;~, 13z tor
~r ]3 t
43 '~7
, .'o 34(.1 95
83 157
fJ7
57
I9t 51~ 2 I5
~4
" Undigested contro|s also received addiiions except for the cases in p0rentheses, whore undigestr:d controls did not receive additions. • " I n t h e s e e x p e r i m e n t s a d i t t e r c n t b;~.tch ,'*f I ) N a s e f r o n t t h a t t ps e d i n o t h e r ¢ x p e r i m e , ~ n w a s e m p l o y e d . T h e re~',d t s w i t h t i f f s b a t c h ~vere m u c h i n f e r i o r t[> t h o s e o b t a i n e d w i t h t i l e p r e v i o u s b a t c h a s r e g a r d s r e m ~ , v a l o f I ) N A a n d i m p a i r n l e n t of il)co~ p o r a t i o n .
Re]erence~ p. 4551456.
v o L . 8 4 (r959)
rOLE oF D N A
;r~- 3~p INCORIJORATION
453
T h e r e s u l t s s u m m a f i s e d in T a b l e V I I I n o w c l e a r l y i n d i c a t e t h e L'~!!o;;-ing p o i n t s . (I) B o t h D N A a n d R N A , w h e n p r e s e n t in t h e i n c u b a t i o n m i x t u r e of l ) N : ~ e t r e a t e d nuclei, w e r e e f f e c t i v e in r e s t o r i n g t h e lost i n c o r p o r a t i o r t a c t i v i t y , p r o v i d e d t h e d i g e s t i o n w i t h D N a s e was n o t v e r y e x t e n s i v e (M24). (2) T h i s e f f e c t of nucleic a c i d s i n c r e a s e d w i t h t h e i r c o n c e n t r a t i o n ~ (Mz5). (3) W h e n a h i g h e r c o n c e n t r a t i o n of D N a s e was u s e d a n d t h e r e m o v a l of D N A was exhaustive, hoth RNA and DNA :estored the incorporation into RNA, but not i n t o D N A (M',o).
Attempts to rvstorv tlw lost in~orporatio~ avtiz'ily by ad,.litio~t o[ compvuruts olhtw t',~.a~ medci¢ avhts T h e n e x t s t e p was t o e x a m i n e n o n - n u c l e o t i d e p o l y a n i o n s a n d s o m e ot[mr c o m p o u n d s for t h e a b i l i t y t o r e s t o r e t h e t~-st i n c o r p o r a t i o n a c t i v i t y of t h e D N a s e - t r e a t e d nuclei. U n f o r t u n a t e l y t h e r e s u l t s were in m o s t cases not c l e a r - c u t . F o r o n e t h i n g , n e w b a t c h of D N a s c was u s e d in a few e x p e r i m e n t s , a n d this was fourtd t o be n o t so e f f e c t i v e in r e m o v i n g t h e D N A f r o m t h e n u c l e i as t h a t used in p r e c e d i n g e x p e r i m e n t s . F u r t h e r , s o m e c o m p o u n d s t e s t e d b r o u g h t a b o u t a p r o f o u n d m o d i f i c a t i o n of t h e inc o r p o r a t i o n a c t i v i t y i n t h e c o n t r o l nuclei, m a k i n g t h e r c s u t t s a m b i g u o u s (Tables V I I an.2 V I I I ) . Chondroitin sulphate did not significantly affect the incorporation into the c o n t r o l nuclei, I t s effect o n t h e D N a s e - t r e a t e d nuclei was e i t h e r a h n o s t equ'a_l (M26, M39 ) or i n f e r i o r t o (Mr 9, M4o) t h a t of R N A . H y a l u r o n a t e i n h i b i t e d t h e i n c o r p o r a t i o n i n t o t h e c o n t r o l n u c l e i r a t h e r s e v e r e l y . B u t t h e a c t i v i t y o f t h e DNase-tree~ted nucIei was h i g h e r t h a n t h a t ot t h e c o n t r o l n u c l e i in t h e p r e s e n c e of h y M u r o n a t e . I t seems t h e r e f o r e t h a t t h e d e p r e s s i o n of i n c o r p o r a t i o r t c a u s e d b y t b e d e p l e t i o n of D N A was r e l i e v e d b y h y a l u r o n a t e " *. H e p a r i n also h a d a n i n h i b i t o r y e f f e c t o n t h e c o n t r o l nuclei a n d i~s e f f e c t o n t h e D N a s e - t r e a t e d n u c l e i was n o t v e r y c o n s p i c u o u s . E g g a l b u m i n , as a n e x a m p l e of n o n - a n i o n i c c o m p o u n d s h a v i n g no significant e'ffect o n t h e c o n t r o l nuclei, m i g h t h a v e a slight r e s t o r i n g a b i l i t y , b u t c o m p a r e d w i t h R N A a n d h y a l u r o n a t e , its e f f e c t was not significant. T h e effect of a m i x t u r e o f 2 ' , 3 ' - m o n o n u c l e o t i d e s was also a m b i g u o u s , b e c a u s e it c a u s e d a m o d e r a t e i n h i b i t i o n ot t h e i n c o r p o r a t i o n i n t o D N A of t h e c o n t r o l nuclei t o g e t h e r w i t h a c o n s i d e r a b l e e n h a n c e m e n t of t h e i n c o r p o r a t i o n i n t o O A S P in b o t h c 0 n t r o l a n d D N a s e - t r e a t e d nuclei. I)IS(:USSIOS
T h e r e s u l t s of t h e p r e s e n t i n ' z e s t i g a t i o n c o n f i r m e d in essentials t h e findings of t h e p r e v i o u s i n v e s t i g a t o r s . As was i n d i c a t e d b y FRIEDI~IN .k.ND WOOD 7, BREITS[)kN AND W E B S T E I k 6 o r FI(; 9 AND E R R E R A ''a w i t h calf t h y m u s nuclei, D N a s e i m p a i r e d t h e i n c o r p o r a t i o n of t h e p r e c u r s o r irtto D N A in i s o l a t e d nuclei. I t also a f f e c t e d t h e i n c o r p o r a t i o n i n t o R N A , a n d t o a les.~ier e x t e n t , t h a t i n t o O A S P . S u c h a n i m p a i r m e n t c o u l d be r e l i e v e d b y exoger, o u s nucleic acids. T h e s e r e s u l t s closely parallel t h e " However, higher concentration o.~ N N A might atiect the hmorpor~ttion into tile nuc!eic acids of tile control nut|el (Table VII, M25). " In this particular experhuent (M38,1 e~ en added t~N.k inb.ibited the incorporation into I)NA of the control nuclei (]'able X'II).
Re]erences p. 45M456.
454
M. S/SKIGUCHI, A. $IBATAN[
VOL. ~z~.(I959)
pioneering findings of ALLFREY e t a l . I concerning the effect of various nucleotide c o m p o u n d s on the restoration of i m p a i r e d incorporation of labelled precursors into protein a n d R N A of the D N a s e - t r e a t e d nuclei. H o w e v e r , an extensive r e m o v a l of DiqA u l t i m a t e l y resulted in a different response of nuclear D N A a n d R N A with regard to their ability to recover t h e incorporation a c t i v i t y on addition of exogenous nucleic acids; while incorporation into R N A w a s partially or fully restored, t h a t into D N A was scarcely regained. This indicates t h a t the bulk of the s2p incorporation into nuclear R N A can proceed w i t h o u t an a m o u n t of pre-existent D N A w:~icb is indispensable for the a2p i n c o r p o r a t i o n into D N A . Such findings m a y at first sight seem to be irt line with the s y n t h e s i s of d e o x y p o l y nucleotide b y the polymerising e n z y m e in the s u p e r n a t a n t fraction of m a m m a l i a n cells ~, ~o, which, like t h e corresponding bacterial e n z y m e 2~, requires p r e f o r m e d D N A as the primer. Howe*cer, since even t h e addition of heterologous D N A failed t o restore the incorporation into D N A in t h e nuclei t r e a t e d e x t e n s i v e l y with D N a s e , th~ significance of pre-existing D N A in t h e incorporation into D N A in isolated nuclei m u s t be something more c o m p l i c a t e d t h a n its function s i m p l y as a " p r i m e r " . Although some of the results of the present investigation were n o t v e r y conclusive, the main b o d y of results w o u l d suggest t h a t the f a v o u r a b l e action of the cxogeneous D N A and R N A on the D N a s e - t r e a t e d nuclei is n o t peculiar t o nuclcotide compounds. The hypothesis previously proposed b y ALLFR:EY AND MIRSK~ ascribing the action of a d d e d nucleic acids to their function as the potential source of highenergy nucleotides becomes thus r a t h e r unlikely. In animal cells the D N A is " n e u t r a l i s e d " b y basic proteins such as p r o t a m i n e s a n d histones, a n d the r e m o v a l of D N A w i t h o u t t o u c h i n g the basic proteins will leave the nuclei positively charged, a n d this will p r o b a b l y b e t h e cause of the reduction of the ability of D N a s e - t r o a t e d nuclei to form A T P a n d of t h e c o n s e q u e n t b l o c k a d e e l s y n t h e t i c a c i t i v i t y s. Polyanions of either nucleotide or non-nucleotide n a t u r e m a y then "'neutralise . . . . basic" nuclei depleted of D N A . SPIEGEr--~tA,~"~* s h o w e d t h a t in l y s e d p r o t o p l a s t s of B. megatariu~n t h e depletion of D N A did n o t abolish the a b i l i t y to synthesise D N A as well as R N A or protein. Tiffs was confirmed b y OTSUJ[ AND TAKAGI ~ w i t h l y s e d protoplastc ,~: E. aoli. T h e difference b e t w e e n animal cells a n d bacterial cells m a y p a r t l y be explained b y t h e fact t h a t D N A in bacterial cells is, unlike in animal ceils, a p p a r e n t l y c o m p l e x e d w i t h some amines of relatively ]ow molectdar weight such as putrescine or spermidine m, which, on elimination of DNA, m a y easily be l o s t from t h e p r o t o p l a s t a n d wi!I n o t cause a serious d i s t u r b a n c e of electrostatic charge distribution a r o u n d the s y n t h e t i c mechanisms still o p e r a t i v e on the lysod protoplasts. Considerable incorporation of vzp into O A S P was noticed in o u r present s t u d y , a n d it was not as sensitive to the r e m o v a l of D N A as asp incorporation into nucleic acids. H o w e v e r , tim n a t u r e of this incorporation into O A S P was n o t studied, and w h a t fraction of it represented t h e formation of nuclear A T P therefore remains obscure, The results r e p o r t e d here are consistent with t h e idea t h a t for D N A s y n t h e s i s in the nucleus preformed specific D N A is n e e d e d for t r a n s m i t t i n g the genetic information to the p r o g e n y molecules, b u t it is n o t required for the synthesis of nuclear R N A for which the genetic information is t r a n s m i t t e d f r o m D N A on;,y indirectly via some sort of macromolecules o t h e r than D N A , B u t it m u s t be emphasized t h a t t h e present s t u d y c a n n o t be t a k e n even as a partial proof of this. Firstly, o n l y t h e incorporation Re[erences p. 455[456.
voL. ~4 (x959)
goI.E OF DNA tN a~-p INCORPORATION
455
Of s--p, not the net .oy . . . .". .' -. .-."~s, was foUowed i~ vitro; secondly, no i n f o r m a t i o n was collected a b o u t the f o r m a t i o n of specific macromolecules. F u r t h e r , t h e r e m o v a l of D N A was n o t absoh:te!y complete, a n d one does n o t k n o w w h a t role t h e DzNA remMning after DNase t r e a t m e n t played in the observed anabolic processes of t h e nuclei which were restored b y the polyanions. NOTF, A D D E D
IN I ' R O O F
After this paper h a d been s u b m i t t e d for publication, ALLFREY AND MiRSI~.X"~7 r e p o r t e d an extensive investig~ttion on the effects of various non-nucleotide poly,anions, including chondroitin sulphate a n d p o l y e t h y l e n e .~ttlphonaee, upon the DNase~ t r e a t e d nuclei. Their results v e r y clearly confirmed our suggestion as to the role of D N A as a p o l y a n i o n in the anabolic processes of isolated nuclei. T h a n k s t o the imp r o v e m e n t of technique indicated b y the American authors, it has also been demons t r a t e d in this l;tboratory e* t h a t in the presence of chondroitin sulphate the nuclei cart be digested with DNase w i t h o u t their a b i l i t y to incorporate azp into R N A a n d DNA being ~fffected; b u t in tiffs case also, an e x h a u s t i v e removal of D N A completely abolished t h e incorporation into DNA, whereas t h a t into R N A was fully retained u n d e r the s a m e conditions. Thus, a m b i g u o u s points of the present paper h a v e now largely been e l i m i n a t e d (Received March 3Ist, 1959) ACKNO~,VLEI)GJ~MENffS
The advice a n d help of Dr. S. OsAw:x, N a g o y a U n i v e r s i t y , a n d Dr. Y. Sucdxo, T a k e d a Pharmaceutical" Industries, Ltd., Osaka, on the technical aspects of tiffs s t u d y are deeply appreciated. We f u r t h e r express our g r a t i t u d e to those who were m e n t i o n e d above as h a v i n g supplied materials. R EFERF.NC ES S. OsAWA, V. G. ALLFRE~" A.XD A. E . 3ItKsKY, j . Ge~,. Physiol., 4o {t957) 4 9 t . V. G . ALLFRF.Y, A. E . R IIRSKY AND ~. ()SA~.VA, J. ~e~t. Physial.. 4 ° {tc~57 ) 45~R . LOGAN, Biochim. ICiophys. Acta, 26 0 9 5 7 } 227. V . G . A L L F R B Y AND A . E . ~][RSKY, P y o c . 2~?'i,tl..-[clrd. Sc~. U. S., 4 3 (I957) 8 2 z . T . R . BblEtTMAN AND G. ('. ~,V~;BSTER, B~ochim. Biophys. ,-; ta..27 ( r 9 5 8 ) 4o8. 8 T . R . BREHMAN AND G. C. ~VEBsTER, l-'ederatioJt Prec., t 7 ( [ 0 . ~ ] 0 4 . 7 M. FRIEDKIN .AND H . V~rooD. ]V, J. Biol. Chem., 2.20 [ [ 9 5 6 ) 039. 8 V. (.7,. ALLFREY AND A. E . ~[IRSKY, Proc. Natl..'lead..b'ci. U. S , 43 (i957} 580. I~. MANTSAVINOS A~;D E . S. CAN'ELLAKIS, Biochim. Bicpt;ys. Acta, 27 (I958) 66~. to /~. j . BOLLUM AND V . R . P O T T E R , J. t3t0l. Che~., 2 3 3 (I958} 478. 11 M . Sa~.KIGUCHI AND A . StrBATANI, J~Jiochi~n. Bio#hys. Acta, 28 {[958) 4 5 5 la A. SIBAZANI. I£xptl. Cell Reset~rcl~, f i n t h e p r e s s ) . t3 ]-t. K . I
45(j
.'VI, S E K I G U C H I , A0 SIBATANI
VOL. ~14
(1959)
e.'2 M. J , BESSMAN, I. R . Lt~HMAN, E , S. SI.~I~IS AND A. KORNBERG, J . 13iOL C]~e1~t., 233 (t958) X7X. ~1 .~. FICQ AND 1"~{. ERRI'RA, .E.gp[l. Cell l{ese~vvtt, x4 (x958) I8'2. 2~ S. SI'IEGEL~tAr¢, i n XV. D . MCl/;LnOY a ~ n I3. GLASs, The Chemical Basis el Hevedily, J o h n s H o p k i n s P r e s s , B a l t i m o r e , r 9 5 7 , p. 232. 25 N, OTSIJJI 2~ND Y. "]~AKAI~;I,.l'~J'Oa.7lk Symposittm on Nuc, leic /tc~ds, ~Vagoya, J a p a n , 2958, p. 6. 16 D. L. IO~ISa'ER, Fedevatio~t .Pvoc,, 17 (1058) 84. =¢ V. G. ALLFRE~r AND A. E, ~{rastcv. Prec, ,Nratl. Arad. Sci. U.S., 44 0 9 5 8 ) 9 8 I . ll~ Y.-T. "['CHOIg .~.NI) a . SIBATANI, Nurture, in t h e p r e s s .
STUDIES THE
ON THE LIVER
CARBOHYDRATE FLUKE
METABOLISM
OF
FASCIOLA HEPATICA
"FAG E . . X l A N S O U l t
DcpaYtment o/ Ph:*rmcc¢ology, Louisitz~a Stale UJ~ioeysily, Hvhool o[ 2~ledici~te, .V~;ro Orleans, La. ([7.S..4.) ( R e c e i v e d O c t o b e r 3 x st, 1058)
SUM,MARY
P'asciol~ ha~at@a utilizes c a r b o h y d r a t e a n a e r o b i c a l l y at a high rate. W h e n glucose is o m i t t e d from t h e m e d i u m , tim a m o u n t of glycogen utilized varies from 8 4 - 9 7 t~moles of glucose units/g wet wt. :6 h. The r a t e of glucose utilization from t h e o u t s i d e medium varies from i i o - i 8 o / x m o l e s f g w e t wt./6 h. P r o d u c t i o n of volatile f a t t y acids accounts for a l m o s t all of the c a r b o h y d r a t e utilized anaerobically. These acids were identified as propionic and acetic acids in an a p p r o x i m a t e ratio of 3:x. O n l y 4 - 9 % of the metabolized c a r b o h y d r a t e is c o n v e r t e d to lactic acid. The .rates of glucose utilization, lactic acid a n d volatile f a t t y acid p r o d u c t i o n are o n l y slightly decreased in the presence of atmospheric oxygen. O x y g e n is utilized b y these organisms when available. The r e s p i r a t o r y q u o t i e n t varies from x.56-2.2. Anaerobic m e t a b o l i s m of flukes with ligatured oral openings is identical with i n t a c t organisms. This : a d i c a t e s that neither the absorption of glucose from outside m e d i u m , n o r the excretion of the metabolic p r o d u c t s are carried o u t t h r o u g h the g u t of t h e organism.
INTRODUCTION
Previous studies indicated t h a t c a r b o h y d r a t e is the major, if n o t the exclusive source of energy for m a n y parasitic helminths 1. a. T h e e n d p r o d u c t s of c a r b o h y d r a t e m e t a b o lism v a r y from one species to another. As e a r l y as 1926, WIt-INLAND AND VON 13RAND 3 o b s e r v e d t h a t the utilization of c a r b o h y d r a t e b y l:asciola hepatiaa in vitro is associated with the production of higher f a t t y acids and possibly b u t y r i c acid. Their experim e n t s were carried o u t on the E u r o p e a n v a r i e t y of these organisms. The e x p e r i m e n t s reported in this p a p e r were e a n i e d o u t on a v a r i e t y of Fasciola hepatica which is p r e d o m i n a n t on the Gulf Coast of this country. I n these experiments, t h e r a t e of endogenous c a r b o h y d r a t e utilization a n d of glucose u p t a k e were m e a s u r e d u n d e r Relerenaes p. 464.