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The effect of phytohemagglutinin on the DNA-dependent RNA polymerase activity of nuclei isolated from human lymphocytes
BIOCHIMICA ET BIOPHYSICA ACTA
95
BBA 96406
T H E E F F E C T OF P H Y T O H E M A G G L U T I N I N ON T H E D N A - D E P E N D E N T RNA P O L Y M E R A S E ACTIVITY OF NUCLEI ISOLATED FROM HUMAN LYMPHOCYTES STANLEY D. HANDMAKER ° AND JOHN W. GRAEF** National Institutes o[ Health, Cell Biology Section, Laboratory o[ Biochemistry, and Human Genetics Branch, National Institute o¢ Dental Research. Bethesda, Md. (U.S.A.)
(Received August 2Ist, I969)
SUMMARY I. DNA-dependent RNA polymerase activity was studied in nuclei from human peripheral blood lymphocytes, incubated with and without phytohemagglutinin. Bivalent cation and (NH4)zSO 4 requirements fur maximum activity were the same in both resting and phytohemagglutinin-stimulated cells. 2. Nuclei obtained from lymphoeytes incubated with phytohemagglutinin for 2- 4 h showed an increased RNA polymerase activity in low salt (without (NH4)2S04) but no increase over controls in high salt (0.4 M (NH4)zS04). 3. Lymphocytes incubated for 20, 46, and 72 h with phytohemagglutinin had a progressive increase in RNA polymerase activity per nucleus in both high and low salt conditions. 4. The ratio of activity in high/low salt was consistently higher in resting than in phytohemagglutinin-stimulated cells. 5. The pattern of increased RNA polymerase activity in phytohemagglutininstimulated lymphocytes was essentially similar to that of regenerating liver ('ells and hormone-activated target cells in a number of systems.
INTRODUCTION Human peripheral blood lymphocytes incubated under standard cell culture conditions synthesize only small amounts of RNA and protein and remain in a resting, non-dividing statO. Following the addition of any of a number of agents, these small lymphocytes are stimulated to enlarge and divide 2. One of the most effective and best known of these agents is phytohemagglutinin a, an extract of the red kidney bean, Phaseolus vulgaris (review by NASPITZ AND RICHTER4). The transformation of a lymphocyte from a small, resting cell to a rapidly growing, proliferating cell is accompanied by an acceleration of overall RNA synthesis 5. The synthesis of all classes of RNA is enhanced, but there is a proportionately greater increase in the synthetic rates of ribosomal RNA 6-9 and transfer RNA 1° than of * Present address: Sir William l)unn School of Pathology, University of Oxford, Oxford, England. "* Present address: Children's ttospital Medical Center, Boston, Mass., U.S.A. Biochim. Biophys. Acta, 199 (197o) 95-1o2
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S. D. HANDMAKER, J. W. GRAEF
heterogeneous RNA. The latter, however, continues to comprise the largest fraction of rapidly-synthesized RNA. The increase in RNA synthesis is reported to follow a prior alteration in the deoxyribonucleoprotein, as measured by enhanced binding of acridine orange n, increased acetylation of histones t*, enhanced rate of phosphorylation and dephosphorylation of nuclear proteins ~3, and greater ability of isolated phytohemagglutinin-stimulated lymphocyte nuclei to serve as template for exogenous (bacterial) RNA polymerase 14. No detailed studies of the effect of phytohemagglutinin stimulation on endogenous RNA polymerase (nucleosidetriphosphate : RNA nucleotidyl transferase, EC 2.7.7.6 ) have been reported, although there have been two preliminary reports with contradictory findings ~5,16. In the present study we have examined the effects of phytohenmgglutininstimulation on endogenous RNA polymerase activity of nuclei isolated from human peripheral lymphocytes. MATERIALS AND METHODS
Lymphocyte cultures Peripheral blood was obtained by venipuncture of normal human subjects and collected in heparin. Lymphocytes were purified using a nylon fiber column, as described by COOPERIL Cells were cultured at a concentration of 2 • IoS/ml in Eagle's minimal essential medium, spinner modification ts, with penicillin (IO U/ml) and streptomycin (Io/lg/ml) and IO % autologous plasma added. They were incubated at 37 ° in loose-capped 32-oz precription bottles in a humidified atmosphere of 5 o~,, CO2 in air. Phytohemagglutinin (Burroughs-Wellcome, Lot No .CTM 1553) was added to replicate cultures at a concentration of 5/,g/ml for long term (18-72 h) studies and IO ~g/ml for short term experiments. The short term experiments were performed on cultures which were preincubated overnight and then aliquoted into sterile 5o-ml plastic centrifuge tubes (Falcon). Isolation o/nuclei Nuclei were isolated from lymphocytes essentially as described by WOI.FI: el alJL The cells were washed once in Eagle's medium and then resuspended in a hypotonic solution of 2 mM MgCI2 and o. 4 mM potassium phosphate (pH 6.7) for 2 rain to swell the lymphocytes. Isotonicity was restored by adjusting the sucrose concentration to o.32 M. The cells were centrifuged and resuspended in o.3z M sucrose solution containing 2 mM MgCI2 and o. 4 mM potassium phosphate (standard solution) at a concentration of Io7/ml. Isolated nuclei were prepared by 15 passes/2 ml in a tightfitting glass Dounce homogenizer. The nuclei were centrifuged, resuspended in 3-5 ml of standard solution, and counted in a hemocytometer. In general, the yield of nuclei was approx. 5 ° °/o of the original number of cells. The final preparation of nuclei contained approx. 2o % intact cells, as determined by phase contrast microscopy. The concentration of nuclei was adjusted to I • IC/ml for most experiments. In all cases the final concentration of nuclei from phytohemagglutinin-stinmlated cells was made equivalent to the concentration of nuclei from resting cells. Determination o / R N A polymerase activity DNA-dependent R N A polymerase activity was assayed using our modification 2° of the method described by Worn:l," et al. ~9. Except where indicated, the assay solution ttiochim. Biophys. Acta, 199 (I97 o) 9 5 - I o 2
PHYTOHEMAGGLUTININEFFECT ON RNA POLYMERASE
97
contained the following in #moles in a volume of 0.5 ml: Tris-HC1 (pH 7.4), IOO; MnCl 2, 1.25; (NH,)SO,, 200; ATP, 0.6; UTP, 0.6; GTP, 0.6; CTP, 0.06 with 1.253.33/tC [5-3H]CTP (13.o C/mmole). Unlabelled nucleoside triphosphates and [5-3H] CTP were obtained from Schwarz BioRes., Orangeburg, N. Y. Enzyme activity was assayed by adding o.2-ml aliquots of nuclei to 0.5 ml of assay solution and incubating at 37 ° for 60 rain. The reaction was stopped by addition of 1.5 ml of cold IO % trichloroacetic acid containing 0.02 M sodium pyrophosphate. Precipitation of the product was facilitated by addition of 0. 5 ml (62. 5/zg) carrier RNA (yeast tRNA, Schwarz BioRes.). All subsequent steps were performed at 4 °. After standing for IO min, the tubes were centrifuged and precipitates were washed successively in 3-ml aliquots of 5 O/,o trichloroacetic acid with 0.02 M sodium pyrophosphate, 5 %,, trichloroacetic acid (twice), 95 % ethanol, and chloroform-ethanolether (2 : 2 : I, by vol.). Tile precipitates were disolved in 0. 4 ml Nuclear Chicago solibilizer and rinsed into counting vials with 12 ml toluene scintillation fluid containing 2,5-diphenyloxazole and 1,4-bis-(5-phenyloxazolyl-2)benzene (New England Nuclear Corp.). Radioactivity was measured in a Packard TriCarb liquid scintillation spectrometer with an absolute 3H counting efficiency of approx. 20 %. All assays were performed at lea.st in duplicate, and when numbers of nuclei permitted, in quadruplicate. Each experiment was repeated at least once with lymphocytes from a different donor. Values given in the table and figures represent the means of experimental values for incorporation of -5-3H ]CTP after 60 rain incubation at 37 ° minus appropriate zero time incorporation in each case. Statistical analysis of the data was performed using the Student's t-test.
RESULTS Characteristics o/the reaction ALLFREY et al. 21 working with calf thymus cells, showed that lymphocyte nuclei
isolated in sucrose solutions retained their capacity to synthesize RNA. The enzyme catalyzing the reaction, DNA-dependent RNA polymerase, was first described in isolated mammalian nuclei by WEISS 2z. Activity was found to depend on the presence of all four nucleoside triphosphates, a bivalent cation, and a DNA template. H u m a n peripheral lymphocytes were incubated 18-2o h with and without 5/,g/ml phytohemagglutinin. Nuclei were isolated and RNA polymerase activity was determined. Table I shows the general characteristics of the reaction carried out by nuclei obtained from resting and phytohemagglutinin-stimulated human peripheral lymphocytes. Phytohemagglutinin produced no signifiant alteration in the requirements, which were typical of RNA polymerase reactions. Removal of salt ((NH4)zSO,) bivalent cation, or one or more nucleoside triphosphates as well as incubation with actinomycin resulted in loss of activity. Nuclei of phytohemagglutinin-stimulated cells incorporated between 4.28 and 8.64 in nmoles CTP per mg DNA, as compared with nuclei from unstimulated cells which incorporated 3.28-4.24 nmoles CTP per mg DNA. The assays were performed in substrate excess since incorporation of [5-sH]CTP was proportional to number of nuclei from phytohemagglutinin-treated and from unstimulated cells (Fig. I). Nuclei from phytohemagglutinin-stimulated cells gave a proportionately greater response than those from resting cells, at all Biochim. Biophys. Acta, 199 (x97o) 95-Io2
98
S. D . H A N D M A K E R ,
J. W . G R A E F
TABI.E I CHARACTERISTICS LYMPHOCYTES
OF THE REACTION
INCUBATED
PERFORMED
BY NUCLEI
I8--20 h WITII AND WITHOUT
ISOLATED
#g/ml
5
FROM HUMAN PERIPHERAL
PHYTOHEMAGGLUTININ
Each assay was performed on 2-4 replicates, and the values given represent the m e a n s of incorporation after 6o min minus zero time incorporation in each case. The complete assay solution contained the following in #moles in o. 5 ml: T r i s - H C l (pH 7.4), ioo; MnC1v 1.25; (NH4)3SO ,, 2oo; ATP, o.6; UTP, o.6; GTP, 0.6; CTP, o.o6 with 1.25-3.33 #tC i5-3H]CTP (I3.o C/mmole). D N A c(mtent was obtained from d e t e r m i n a t i o n of lO 3 l y m p h o c y t e nuclei having 6.25/*g DNA I'. The actinomycin D was gift from Merck, S h a r p and D o h m e to Dr. Robert M. Friedman.
Incubation medium
(5-3H]CTP incorporated (nmoles/mg DNA ) phytohemagglutinin
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+ phytohemagglutinin .
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Complete - - (NH4)tSO ,
3.9 2 0.6 4
6. r 6
Complete _ Mn'~+ - - M n ~+, + 2 0 m M
Mg ~
3.28 0.32 ~92
4.96 0.6 4 3.76
Complete + a c t i n o m y c i n D, 5 #g incubated at 4 °
3.84 0.32 o.16
8.64 1.28 o.48
Complete -GTP -- UTP, - - G T P - - A T P . - UTP, - G T P
4.24 o.32 o 24 0.08
4.28 0.88 0.72 o.48
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1.28
..............................
8O
7o
!0o ,~ 3o
tO 0
0
I 2 N U M B E R OF NUCLEI x 10"e
I 4
Fig. L Effect of concentration of nuclei on incorporation of [5-3H~CTP. Incubation conditions were as given in MATERIALS AND METHODS, O - - O , nuclei from resting lymphocytes; e - e nuclei from p h y t o h e m a g g l u t i n i n - s t i m u l a t e d lymphocytes.
c o n c e n t r a t i o n s studied. Most of t h e e x p e r i m e n t s r e p o r t e d here were p e r f o r m e d using 2 • IO e nuclei per assay. The p H o p t i m u m for the a s s a y of R N A p o l y m e r a s e in nuclei from p h y t o h e m a g g l u t i n i n - t r e a t e d cells was the s a m e as for resting cells, p H 7.3-7.4. W h e n cells were t r e a t e d w i t h p h y t o h e m a g g l u t i n i n for 18-2o h, their nuclei showed greater R N A p o l y m e r a s e a c t i v i t y t h a n control nuclei at all c o n c e n t r a t i o n s t e s t e d of both Mn ~+ a n d Mg ~+ (Fig. 2). B o t h tile shape of tile curves a n d the o p t i m a l c o n c e n t r a t i o n of 2.5 mM Mn 2+ were the s a m e in p h y t o h e m a g g l u t i n i n - t r e a t e d as in resting cells. A t the Biochim. Biophys. Acta, t99 U97 o) 95--xo2
PHYTOHEMAGGLUTININ EFFECT OX RNA POLYMERASE
ioc IP'.o.
/
A ,#
,~5c
"PHA-~
f
//¢PHA
~ pH .7.4
pH-Z4
d ¢c
o.o-- - - -o
,.o-
"''"'0..
99
I
0
21.5 5 I; II~ 20 Mg2*CONCENTRATION (mM )
Mn 2¢ CONCENTRATION(mM)
Fig. 2. Effect of b i v a l e n t cation c o n c e n t r a t i o n on i n c o r p o r a t i o n of [5-sH]CTP. I n c u b a t i o n cond i t i o n s were as given in MATERIALS AND METHODS. A. C o n c e n t r a t i o n of MnC1 t (the m a x i m a l v a l u e w a s 4.96 n m o l e s [ 5 - s H ] C T P i n c o r p o r a t e d per m g D N A per 6o rain). B. C o n c e n t r a t i o n of m a g n e s i u m - a c e t a t e (the m a x i m a l v a l u e was 3.71 n m o l e s [5-sH~CTP i n c o r p o r a t e d / m g D N A per 60 m i n ) . C ) - - - C ) , nuclei from r e s t i n g l y m p h o c y t e s ; O - O , nuclei from p h y t o h e m a g g l u t i n i n - s t i m u l a t e d lymphocytes. PHA, phytohemagglutinin.
concentrations tested, we found no difference between Mg2+ and Mn z+ in facilitating the reaction in low salt in either case. WIDNELL AND T A T A z3, working with isolated rat liver nuclei, found Mn 2~ to give maximal activity in high salt, but they reported Mg2+ to be more effective than Mn z+ at low ionic strength. However, PEGG AND KORNER~ demonstrated that rat liver nuclei incubated in a hypotonic medium were maximally activated by Mn *+ in both high and low salt. The assay conditions used in the present experiments were hypotonic in the absence of (NH4)zSO,, and our results were in agreement with those of PEGG AND K O R N E R ~ .
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0.1 02 0.3 0.4 0.~ O,I JuraMONIUMSULFATI[CONC~NTRAT~(M)
I
0.?
0
I
2
TIME AFTER PHA (it)
4
oI
Fig. 3- E f f e c t of ( N H , ) t S O , c o n c e n t r a t i o n on i n c o r p o r a t i o n of [5-sH]CTP. I n c u b a t i o n condit i o n s were as given in MATERIALS AND METHODS. O - C ) , nuclei from r e s t i n g l y m p h o c y t e s ; 0 - 0 , nuclei from p h y t o h e m a g g l u t i n i n - s t i m u l a t e d l y m p h o c y t e s . Fig. 4. E f f e c t of s h o r t - t e r m i n c u b a t i o n w i t h p h y t o h e m a g g l u t i n i n ( P H A ) on [ 5 - s H ] C T P incorporation. I n c u b a t i o n c o n d i t i o n s were as given in MATERIALS AND METHODS. E a c h p o i n t r e p r e s e n t s t h e m e a n of 4 replicate d e t e r m i n a t i o n s , t h e i n d i v i d u a l v a l u e s being w i t h i n + x 5 % of t h e m e a n . C)-C), nuclei a s s a y e d in low s a l t ( w i t h o u t (NH,)ISO4); O - O , nuclei a s s a y e d in h i g h salt (o. 4 M (NH,),SO,).
Biochim. Biophys. dcta, I99 (I97o) 9 5 - x o 2
I00
S . D . HANDMAKF.R,
j. w . GRAEF
The RNA polymerase activity of nuclei from both resting and phytohemagglutinin-stimulated lymphocytes was markedly enhanced by high salt (Fig. 3). This was true when Mg*+ was the bivalent cation as well as when Mn 2~ was present, as shown here. Optimal activity was achieved in 0.5 M (NH4)~SO 4, although the optimal concentration ranged from 0. 4 to 0.6 M in other experiments. The proportionate effect of (NH4)2SO , was greater in unstimulated nuclei than in nuclei from phytohemagglutinin-treated cells.
E//ect o/short-term incubation ve,ith phytohemagglutinin After incubation overnight, lymphocytes from the same donor were divided into several equal portions, and IO/,g/ml phytohemagglutinin was added to replicate cultures at 2-h intervals. Nuclei were prepared from each culture, and RNA polymerase activity was determined. An increased activity in low salt was observed within 2- 4 h after the addition of phytohemagglutinin (Fig. 4); however, no elevation of the reaction in high salt occurred during this period. The activity in low salt after 4 h phytohemagglutinin was increased by 52 % and was statistically significant (P -~. o.o25). The ratio of low/high salt activity was 43 % greater in nuclei from phytohemagglutinin-treated cells than those from resting cells when assayed at both 2 and 4 h after phytohemagglutinin, and the difference in both cases was significant (P < 0.05). The observed small decrease in activity in high salt after 2 h phytohemagglutinin probably reflects an error in counting nuclei, as the ratio of activity in low/high salt was exactly the same (43 %) after z and 4 h phytohemagglutinin. The addition of phytohemagglutinin directly to unstimulated nuclei had no effect on activity in high or low salt.
E//ect o/ long-term incubation with phytohemagglutinin Lymphocyte cultures from the same donor were incubated with and without 5/zg/ml phytohemagglutinin for zo, 46, and 72 h. Nuclei were isolated from each culture, and [5-3HICTP incorporation was determined. In the nuclei from phytohemag~= oI= 5(~ 0--
i
RATIO (high/lows(lit) 20h 46 h 72h Unstimulated PHA- st imuloted
6.2 4.7
G.6 4.2
/~,o j./ /
9.7 43
/ /
4(:
// //
,~.
+{NH4 )zS04
Z ~
I(
~-- ..... 24
~
( NH4|/ S04 48
72
TIME OF INCUBATION {h)
Fig. 5. E(fect of l o n g - t e r m i n c u b a t i o n w i t h p h y t o h e m a g g l u t i n i n on [5-sHJC'I'P incorporation. I n c u b a t i o n c o n d i t i o n s were as given in raArERIALS AND METHODS. E a c h p o i n t r e p r e s e n t s t h e m e a n of 4 replicate d e t e r m i n a t i o n s , t h e i n d i v i d u a l v a l u e s being w i t h i n + xo % of t h e m e a n . O - O , nuclei from r e s t i n g l y m p h o c y t e s ; Q)- - -Q), nuclei from p h y t o h e m a g g l u t i n i n - s t i m u l a t e d l y m p h o cytes. P H A , p h y t o h e m a g g l u t i n i n .
13iochirn. 13iophys. Acta, I99 (x97 o) 9 5 - I o z
PHYTOHEMAGGLUTINI.-'q EFFECT ON
RNA POLYMERASE
IOI
glutinin-stimulated cells there was a progressive increase in RNA polymerase activity in both low salt and high salt conditions (Fig. 5). Incubation for 2o h with phytohemagglutinin resulted in a doubling (207 %, P < 0.005) of the low salt activity and a 57 % increase (P < o.ooI) in the activity of the enzyme in high salt. By 72 h, there was 5- times as much (5oI %, P << o.oox) activity in low salt and more than double (220 °/o, P << o.ooI) the activity in high salt. The enhancement of RNA polymerase activity by high salt was consistently greater in nuclei of unstimulated cells. A sharp rise was found in the high/low salt activity of resting cells incubated for 7 2 h. The reason for this is not readily apparent.
DISCUSSION
Phytohemagglutinin-stimulated lymphocytes have been shown to contain more RNA than unstimulated cultures 7,16..5,.6. The rate of incorporation of labelled uridine into RNA after zo h incubation with phytohemagglutinin is IO-2O times greater than in resting cells. As reported here, nuclei from lymphocytes incubated with phytohemagglutinin for 2o h showed an increase in endogenous RNA polymerase activity. However, the enhancement was much less than the increase in uridine incorporation, a doubling of RNA synthetic capacity assayed in low salt and 1.5-times as much activity in high salt. Studies comparing the level of uridine incorporation with uridine kinase (ATP : uridine 5'-phosphotransferase, EC 2.7.I.48 ) activity x5,~7'~8have suggested that uridine incorporation is not a quantitative index of the rate of RNA synthesis. Furthermore, the rather small increase in RNA synthetic capacity of nuclei from phytohemagglutinin-treated cells and the demonstration from COOPER29 of reduced wastage of I8 S ribosomal RNA within I h after the addition of phytohemagglutinin suggest that enhanced survival of new RNA may be a significant factor in the phytohemagglutinin-induced increase in total cellular RNA, which is predominantly ribosomal. In the experiments reported here, we found that nuclei which were isolated within 2- 4 h after the addition of phytohemagglutinin showed an increase in RNA synthetic capacity only when assayed ill IOW salt. In the presence of 0.4 M (NH,),SO, there was no significant difference in activity at this time between phytohemagglutinin-treated and control cells. When nuclei were isolated after 20, 46, and 72 h incubation with phytohemagglutinin, there was an increase in RNA polymerase activity in both high and low salt. In every case the ratio of high/low salt activity was greater in nuclei from unstimulated cells. These findings are basically similar to those obtained in other mammalian systems in which extensive gene activation or derepression is believed to occur, regenerating liver following partial hepatectomy of young adult rats 3° as well as a number of systems after hormone stimulation ~,a~-3~ (review by TATAae).
The basis for the difference in activity of RNA polymerase in high and low salt is not clear. Since the initial description of GOLDBERG~7 of the enhancement of RNA polymerase activity by increased ionic strength, there has been speculation as to its meaning. The activity in high salt ha.q been variously explained as being due to (I) unmasking of a second enzyme ~, (2) dissociation of histones from DNA, thereby increasing template availability u,38, (3) inhibition of degradative enzymes a9,4°, (4) an Biochim. Biophys. Acta, I99 (I97 o) 95-xo2
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S. D. HANDMAKER, J. w . GRAEF
effect on the enzyme itself 41. The product of the low salt reaction in liver cell preparations has been found to be ribosomal-like in base composition, in contrast to the DNAlike product of the high salt reaction 29,42,.3. These findings as well as our own, are compatible with any of the above interpretations.
ACKNOWLEDGMENTS We thank Mrs. Thelma Prather for her excellent technical assistance and Drs. Herbert L. Cooper, John E. K a y and Robert Stern for their interest in this work and for their critical review of the manuscript. REFERENCES I L. B. El'STEIN AND F. STOHLMAN, Blood, 24 (1964) 69. 2 N. R. LING, Lymphocyte Stimulation, N o r t h Holland, A m s t e r d a m , r968. 3 P. C. NOWELL, Cancer Res., 20 (196o) 462. 4 C. K. NASPITZ AND M. RICHTER, Progr. Allergy, 12 (1968) 1. 5 0 . R. MClSTYRE AND F. G. EBAUGH, JR., Blood, 19 (1962) 4436 H. I.. COOPER, in R. BASERGA, Biochemistry o/ Cell Division, Charles C. Thomas, Springfield, Ill., 1969, p. 91. 7 J. E. KAY, in M. W. ELVES, The Biological E//ects o/ Phytohemagglutinin, The R o b e r t J o n e s and Agnes H u n t Hospital M a n a g e m e n t Committee, Oswestry, 1966. 8 I~'. I,. TORELLI, P. l-I. HENRY AND S. -'~. WEISSMAN, J. Clin. Invest., 47 (1968) 1°83. o A. l). RUBIN, Nature, 22o (I968) 196. 1o J. E. KAY AND H. L. COOPER, Biochim. Biophys. Acta, 186 (I969) 62. 1i D. KILI.ANDER AND R. RIGLER, Exptl. Cell Res., 39 (1965) 71o. I2 B. G. T. POGO. V. G. ALLFREY AND A. E. MIRSKV, Proc. Natl. Acad. Sci. U.S.. 55 (1966) 805. 13 l.. J. KLEINSMITH, V. G. ALI.FREY AND A. E. MIRSKY, Science, 154 (1966) 780. 14 R. HIRSCHHORN, W. TROLL, G. BRITTINGER AND G. WEISSMANN, Nature, 222 (1969) 1247. I 5 iv). -}-[AUSEN AND H. STEIN, EuroOean J. Biochem., 4 (1968) 4Ol. I6 Z. J. LUCAS, in W. O. RIECKE, l~roc. 3rd Ann. Con/. Leukocyte Cult., Appleton Century Crofts, New York, 1969, p. 655. 17 H. L. COOPER, J. Biol. Chem., 243 (1968) 3418 H. EAGLE, Science, 13o (1949) 432. 19 J. S. WOLFF, I l l , J. A. LANGSTAFF, G. WEINBERG AND G. ~V. ABELL, Biochem. Biophys. Res. Commun., 26 (1967) 366. 20 J. w . GRAEF AND S. O. HANDMAKER, in preparation. 2i V. G.. ALLFREY, A. E. MIRSKY AND S. OSAWA, j . Gen. Physiol., 4 ° (1957) 45 I. 22 S. B. WEiss, Proc. Natl. Acad. Sci. U.S., 46 (196o) lO2O. 23 C. (" WIDNELL AND J. R. TATA, Biochim. Biophys. Acta, 87 (1964) 531. 24 A. E. ])EGG AND A. KORNER, Nature, 2o 5 (19651 904. 25 D. R. FORSDYKE, Biochem. J., lO 5 (1967) 679. 26 S. D. HANDMAKER, B. G. LEVENTHAL AND H. L. COOPER, in W. O. I{IECKE, Proc. 3rd Ann. Con]. Leukocyte Cult., Appleton C e n t u r y Croffs, New York, 1969, p. 53. 27 Z. J. LUCAS, Science, I56 (1967) 1237. 28 J. E. KAY AND S. D. HANDMAKER, in preparation. 29 H. L. COOPER, J. Biol. Chem., (1969) in the press. 3 ° A. O. I~OGO, V. G. ALLFREY AND A. E. MIRSKY, Proc. Natl. Acad. Nci. U.S., 56 (1966) 550. 31 J. (;ORSKL J. Biol. Chem., 234 (1964) 889 . 32 J. R. TATA AND C. C. WIDNELL, Biochem. J., 98 (I966) 604. 33 ('. B. BREt1R AND J. R. FLORINI, Biochemistry, 5 (1966) 3857 • 34 ('- C. V~rID.~ELL AND J. R. TA'rA, Biochem. J., 98 (1966) 621. 35 T. H. HAMILTON, C. C.'\VIDNELL AND J. R. TATA, J. Biol. Chem., 243 (1968) 4o8. 36 d. R. TATA, Progr. Nucleic Acad. Res., 5 (1966) 2Ol. 37 I. H. GOLDBERG, Biochim. Biophys. Acta, 51 (196I) 2Ol. 38 I'. CHAMBON, M. RAMUZ AND J. DOLY, Biochem. Biophys. Res. Commun., 19 (1965) 156. 39 D. D. CUNNINGHAM AND D. F. STEINER, Federation Proc., 25 (1966) 788. 4 ° A. I. MEISLER AND 13. E. Tt~OPP, Biochim. Biophys. Acta, 174 (1969) 476. 41 S. T. JAcon, E. M. SAJDEL AND N. H. MUNRO, Biochem. Biophys. Res. Commun., 32 (1968) 831. 42 K. MARUSHIGE AND J. BONNER, J. Mol. Biol., 15 (1966) 16o. 43 P. CHAMBON, H. KARON, M. RAMUZ AND P. MANDEL, Biochim. Biophvs. Acta, ~57 (1968) 52o.
Biochim. Biophys. Acta, 199 (197 o) 95-IO2
95
BBA 96406
T H E E F F E C T OF P H Y T O H E M A G G L U T I N I N ON T H E D N A - D E P E N D E N T RNA P O L Y M E R A S E ACTIVITY OF NUCLEI ISOLATED FROM HUMAN LYMPHOCYTES STANLEY D. HANDMAKER ° AND JOHN W. GRAEF** National Institutes o[ Health, Cell Biology Section, Laboratory o[ Biochemistry, and Human Genetics Branch, National Institute o¢ Dental Research. Bethesda, Md. (U.S.A.)
(Received August 2Ist, I969)
SUMMARY I. DNA-dependent RNA polymerase activity was studied in nuclei from human peripheral blood lymphocytes, incubated with and without phytohemagglutinin. Bivalent cation and (NH4)zSO 4 requirements fur maximum activity were the same in both resting and phytohemagglutinin-stimulated cells. 2. Nuclei obtained from lymphoeytes incubated with phytohemagglutinin for 2- 4 h showed an increased RNA polymerase activity in low salt (without (NH4)2S04) but no increase over controls in high salt (0.4 M (NH4)zS04). 3. Lymphocytes incubated for 20, 46, and 72 h with phytohemagglutinin had a progressive increase in RNA polymerase activity per nucleus in both high and low salt conditions. 4. The ratio of activity in high/low salt was consistently higher in resting than in phytohemagglutinin-stimulated cells. 5. The pattern of increased RNA polymerase activity in phytohemagglutininstimulated lymphocytes was essentially similar to that of regenerating liver ('ells and hormone-activated target cells in a number of systems.
INTRODUCTION Human peripheral blood lymphocytes incubated under standard cell culture conditions synthesize only small amounts of RNA and protein and remain in a resting, non-dividing statO. Following the addition of any of a number of agents, these small lymphocytes are stimulated to enlarge and divide 2. One of the most effective and best known of these agents is phytohemagglutinin a, an extract of the red kidney bean, Phaseolus vulgaris (review by NASPITZ AND RICHTER4). The transformation of a lymphocyte from a small, resting cell to a rapidly growing, proliferating cell is accompanied by an acceleration of overall RNA synthesis 5. The synthesis of all classes of RNA is enhanced, but there is a proportionately greater increase in the synthetic rates of ribosomal RNA 6-9 and transfer RNA 1° than of * Present address: Sir William l)unn School of Pathology, University of Oxford, Oxford, England. "* Present address: Children's ttospital Medical Center, Boston, Mass., U.S.A. Biochim. Biophys. Acta, 199 (197o) 95-1o2
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S. D. HANDMAKER, J. W. GRAEF
heterogeneous RNA. The latter, however, continues to comprise the largest fraction of rapidly-synthesized RNA. The increase in RNA synthesis is reported to follow a prior alteration in the deoxyribonucleoprotein, as measured by enhanced binding of acridine orange n, increased acetylation of histones t*, enhanced rate of phosphorylation and dephosphorylation of nuclear proteins ~3, and greater ability of isolated phytohemagglutinin-stimulated lymphocyte nuclei to serve as template for exogenous (bacterial) RNA polymerase 14. No detailed studies of the effect of phytohemagglutinin stimulation on endogenous RNA polymerase (nucleosidetriphosphate : RNA nucleotidyl transferase, EC 2.7.7.6 ) have been reported, although there have been two preliminary reports with contradictory findings ~5,16. In the present study we have examined the effects of phytohenmgglutininstimulation on endogenous RNA polymerase activity of nuclei isolated from human peripheral lymphocytes. MATERIALS AND METHODS
Lymphocyte cultures Peripheral blood was obtained by venipuncture of normal human subjects and collected in heparin. Lymphocytes were purified using a nylon fiber column, as described by COOPERIL Cells were cultured at a concentration of 2 • IoS/ml in Eagle's minimal essential medium, spinner modification ts, with penicillin (IO U/ml) and streptomycin (Io/lg/ml) and IO % autologous plasma added. They were incubated at 37 ° in loose-capped 32-oz precription bottles in a humidified atmosphere of 5 o~,, CO2 in air. Phytohemagglutinin (Burroughs-Wellcome, Lot No .CTM 1553) was added to replicate cultures at a concentration of 5/,g/ml for long term (18-72 h) studies and IO ~g/ml for short term experiments. The short term experiments were performed on cultures which were preincubated overnight and then aliquoted into sterile 5o-ml plastic centrifuge tubes (Falcon). Isolation o/nuclei Nuclei were isolated from lymphocytes essentially as described by WOI.FI: el alJL The cells were washed once in Eagle's medium and then resuspended in a hypotonic solution of 2 mM MgCI2 and o. 4 mM potassium phosphate (pH 6.7) for 2 rain to swell the lymphocytes. Isotonicity was restored by adjusting the sucrose concentration to o.32 M. The cells were centrifuged and resuspended in o.3z M sucrose solution containing 2 mM MgCI2 and o. 4 mM potassium phosphate (standard solution) at a concentration of Io7/ml. Isolated nuclei were prepared by 15 passes/2 ml in a tightfitting glass Dounce homogenizer. The nuclei were centrifuged, resuspended in 3-5 ml of standard solution, and counted in a hemocytometer. In general, the yield of nuclei was approx. 5 ° °/o of the original number of cells. The final preparation of nuclei contained approx. 2o % intact cells, as determined by phase contrast microscopy. The concentration of nuclei was adjusted to I • IC/ml for most experiments. In all cases the final concentration of nuclei from phytohemagglutinin-stinmlated cells was made equivalent to the concentration of nuclei from resting cells. Determination o / R N A polymerase activity DNA-dependent R N A polymerase activity was assayed using our modification 2° of the method described by Worn:l," et al. ~9. Except where indicated, the assay solution ttiochim. Biophys. Acta, 199 (I97 o) 9 5 - I o 2
PHYTOHEMAGGLUTININEFFECT ON RNA POLYMERASE
97
contained the following in #moles in a volume of 0.5 ml: Tris-HC1 (pH 7.4), IOO; MnCl 2, 1.25; (NH,)SO,, 200; ATP, 0.6; UTP, 0.6; GTP, 0.6; CTP, 0.06 with 1.253.33/tC [5-3H]CTP (13.o C/mmole). Unlabelled nucleoside triphosphates and [5-3H] CTP were obtained from Schwarz BioRes., Orangeburg, N. Y. Enzyme activity was assayed by adding o.2-ml aliquots of nuclei to 0.5 ml of assay solution and incubating at 37 ° for 60 rain. The reaction was stopped by addition of 1.5 ml of cold IO % trichloroacetic acid containing 0.02 M sodium pyrophosphate. Precipitation of the product was facilitated by addition of 0. 5 ml (62. 5/zg) carrier RNA (yeast tRNA, Schwarz BioRes.). All subsequent steps were performed at 4 °. After standing for IO min, the tubes were centrifuged and precipitates were washed successively in 3-ml aliquots of 5 O/,o trichloroacetic acid with 0.02 M sodium pyrophosphate, 5 %,, trichloroacetic acid (twice), 95 % ethanol, and chloroform-ethanolether (2 : 2 : I, by vol.). Tile precipitates were disolved in 0. 4 ml Nuclear Chicago solibilizer and rinsed into counting vials with 12 ml toluene scintillation fluid containing 2,5-diphenyloxazole and 1,4-bis-(5-phenyloxazolyl-2)benzene (New England Nuclear Corp.). Radioactivity was measured in a Packard TriCarb liquid scintillation spectrometer with an absolute 3H counting efficiency of approx. 20 %. All assays were performed at lea.st in duplicate, and when numbers of nuclei permitted, in quadruplicate. Each experiment was repeated at least once with lymphocytes from a different donor. Values given in the table and figures represent the means of experimental values for incorporation of -5-3H ]CTP after 60 rain incubation at 37 ° minus appropriate zero time incorporation in each case. Statistical analysis of the data was performed using the Student's t-test.
RESULTS Characteristics o/the reaction ALLFREY et al. 21 working with calf thymus cells, showed that lymphocyte nuclei
isolated in sucrose solutions retained their capacity to synthesize RNA. The enzyme catalyzing the reaction, DNA-dependent RNA polymerase, was first described in isolated mammalian nuclei by WEISS 2z. Activity was found to depend on the presence of all four nucleoside triphosphates, a bivalent cation, and a DNA template. H u m a n peripheral lymphocytes were incubated 18-2o h with and without 5/,g/ml phytohemagglutinin. Nuclei were isolated and RNA polymerase activity was determined. Table I shows the general characteristics of the reaction carried out by nuclei obtained from resting and phytohemagglutinin-stimulated human peripheral lymphocytes. Phytohemagglutinin produced no signifiant alteration in the requirements, which were typical of RNA polymerase reactions. Removal of salt ((NH4)zSO,) bivalent cation, or one or more nucleoside triphosphates as well as incubation with actinomycin resulted in loss of activity. Nuclei of phytohemagglutinin-stimulated cells incorporated between 4.28 and 8.64 in nmoles CTP per mg DNA, as compared with nuclei from unstimulated cells which incorporated 3.28-4.24 nmoles CTP per mg DNA. The assays were performed in substrate excess since incorporation of [5-sH]CTP was proportional to number of nuclei from phytohemagglutinin-treated and from unstimulated cells (Fig. I). Nuclei from phytohemagglutinin-stimulated cells gave a proportionately greater response than those from resting cells, at all Biochim. Biophys. Acta, 199 (x97o) 95-Io2
98
S. D . H A N D M A K E R ,
J. W . G R A E F
TABI.E I CHARACTERISTICS LYMPHOCYTES
OF THE REACTION
INCUBATED
PERFORMED
BY NUCLEI
I8--20 h WITII AND WITHOUT
ISOLATED
#g/ml
5
FROM HUMAN PERIPHERAL
PHYTOHEMAGGLUTININ
Each assay was performed on 2-4 replicates, and the values given represent the m e a n s of incorporation after 6o min minus zero time incorporation in each case. The complete assay solution contained the following in #moles in o. 5 ml: T r i s - H C l (pH 7.4), ioo; MnC1v 1.25; (NH4)3SO ,, 2oo; ATP, o.6; UTP, o.6; GTP, 0.6; CTP, o.o6 with 1.25-3.33 #tC i5-3H]CTP (I3.o C/mmole). D N A c(mtent was obtained from d e t e r m i n a t i o n of lO 3 l y m p h o c y t e nuclei having 6.25/*g DNA I'. The actinomycin D was gift from Merck, S h a r p and D o h m e to Dr. Robert M. Friedman.
Incubation medium
(5-3H]CTP incorporated (nmoles/mg DNA ) phytohemagglutinin
- -
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+ phytohemagglutinin .
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Complete - - (NH4)tSO ,
3.9 2 0.6 4
6. r 6
Complete _ Mn'~+ - - M n ~+, + 2 0 m M
Mg ~
3.28 0.32 ~92
4.96 0.6 4 3.76
Complete + a c t i n o m y c i n D, 5 #g incubated at 4 °
3.84 0.32 o.16
8.64 1.28 o.48
Complete -GTP -- UTP, - - G T P - - A T P . - UTP, - G T P
4.24 o.32 o 24 0.08
4.28 0.88 0.72 o.48
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1.28
..............................
8O
7o
!0o ,~ 3o
tO 0
0
I 2 N U M B E R OF NUCLEI x 10"e
I 4
Fig. L Effect of concentration of nuclei on incorporation of [5-3H~CTP. Incubation conditions were as given in MATERIALS AND METHODS, O - - O , nuclei from resting lymphocytes; e - e nuclei from p h y t o h e m a g g l u t i n i n - s t i m u l a t e d lymphocytes.
c o n c e n t r a t i o n s studied. Most of t h e e x p e r i m e n t s r e p o r t e d here were p e r f o r m e d using 2 • IO e nuclei per assay. The p H o p t i m u m for the a s s a y of R N A p o l y m e r a s e in nuclei from p h y t o h e m a g g l u t i n i n - t r e a t e d cells was the s a m e as for resting cells, p H 7.3-7.4. W h e n cells were t r e a t e d w i t h p h y t o h e m a g g l u t i n i n for 18-2o h, their nuclei showed greater R N A p o l y m e r a s e a c t i v i t y t h a n control nuclei at all c o n c e n t r a t i o n s t e s t e d of both Mn ~+ a n d Mg ~+ (Fig. 2). B o t h tile shape of tile curves a n d the o p t i m a l c o n c e n t r a t i o n of 2.5 mM Mn 2+ were the s a m e in p h y t o h e m a g g l u t i n i n - t r e a t e d as in resting cells. A t the Biochim. Biophys. Acta, t99 U97 o) 95--xo2
PHYTOHEMAGGLUTININ EFFECT OX RNA POLYMERASE
ioc IP'.o.
/
A ,#
,~5c
"PHA-~
f
//¢PHA
~ pH .7.4
pH-Z4
d ¢c
o.o-- - - -o
,.o-
"''"'0..
99
I
0
21.5 5 I; II~ 20 Mg2*CONCENTRATION (mM )
Mn 2¢ CONCENTRATION(mM)
Fig. 2. Effect of b i v a l e n t cation c o n c e n t r a t i o n on i n c o r p o r a t i o n of [5-sH]CTP. I n c u b a t i o n cond i t i o n s were as given in MATERIALS AND METHODS. A. C o n c e n t r a t i o n of MnC1 t (the m a x i m a l v a l u e w a s 4.96 n m o l e s [ 5 - s H ] C T P i n c o r p o r a t e d per m g D N A per 6o rain). B. C o n c e n t r a t i o n of m a g n e s i u m - a c e t a t e (the m a x i m a l v a l u e was 3.71 n m o l e s [5-sH~CTP i n c o r p o r a t e d / m g D N A per 60 m i n ) . C ) - - - C ) , nuclei from r e s t i n g l y m p h o c y t e s ; O - O , nuclei from p h y t o h e m a g g l u t i n i n - s t i m u l a t e d lymphocytes. PHA, phytohemagglutinin.
concentrations tested, we found no difference between Mg2+ and Mn z+ in facilitating the reaction in low salt in either case. WIDNELL AND T A T A z3, working with isolated rat liver nuclei, found Mn 2~ to give maximal activity in high salt, but they reported Mg2+ to be more effective than Mn z+ at low ionic strength. However, PEGG AND KORNER~ demonstrated that rat liver nuclei incubated in a hypotonic medium were maximally activated by Mn *+ in both high and low salt. The assay conditions used in the present experiments were hypotonic in the absence of (NH4)zSO,, and our results were in agreement with those of PEGG AND K O R N E R ~ .
I~
15
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I
I
0.1 02 0.3 0.4 0.~ O,I JuraMONIUMSULFATI[CONC~NTRAT~(M)
I
0.?
0
I
2
TIME AFTER PHA (it)
4
oI
Fig. 3- E f f e c t of ( N H , ) t S O , c o n c e n t r a t i o n on i n c o r p o r a t i o n of [5-sH]CTP. I n c u b a t i o n condit i o n s were as given in MATERIALS AND METHODS. O - C ) , nuclei from r e s t i n g l y m p h o c y t e s ; 0 - 0 , nuclei from p h y t o h e m a g g l u t i n i n - s t i m u l a t e d l y m p h o c y t e s . Fig. 4. E f f e c t of s h o r t - t e r m i n c u b a t i o n w i t h p h y t o h e m a g g l u t i n i n ( P H A ) on [ 5 - s H ] C T P incorporation. I n c u b a t i o n c o n d i t i o n s were as given in MATERIALS AND METHODS. E a c h p o i n t r e p r e s e n t s t h e m e a n of 4 replicate d e t e r m i n a t i o n s , t h e i n d i v i d u a l v a l u e s being w i t h i n + x 5 % of t h e m e a n . C)-C), nuclei a s s a y e d in low s a l t ( w i t h o u t (NH,)ISO4); O - O , nuclei a s s a y e d in h i g h salt (o. 4 M (NH,),SO,).
Biochim. Biophys. dcta, I99 (I97o) 9 5 - x o 2
I00
S . D . HANDMAKF.R,
j. w . GRAEF
The RNA polymerase activity of nuclei from both resting and phytohemagglutinin-stimulated lymphocytes was markedly enhanced by high salt (Fig. 3). This was true when Mg*+ was the bivalent cation as well as when Mn 2~ was present, as shown here. Optimal activity was achieved in 0.5 M (NH4)~SO 4, although the optimal concentration ranged from 0. 4 to 0.6 M in other experiments. The proportionate effect of (NH4)2SO , was greater in unstimulated nuclei than in nuclei from phytohemagglutinin-treated cells.
E//ect o/short-term incubation ve,ith phytohemagglutinin After incubation overnight, lymphocytes from the same donor were divided into several equal portions, and IO/,g/ml phytohemagglutinin was added to replicate cultures at 2-h intervals. Nuclei were prepared from each culture, and RNA polymerase activity was determined. An increased activity in low salt was observed within 2- 4 h after the addition of phytohemagglutinin (Fig. 4); however, no elevation of the reaction in high salt occurred during this period. The activity in low salt after 4 h phytohemagglutinin was increased by 52 % and was statistically significant (P -~. o.o25). The ratio of low/high salt activity was 43 % greater in nuclei from phytohemagglutinin-treated cells than those from resting cells when assayed at both 2 and 4 h after phytohemagglutinin, and the difference in both cases was significant (P < 0.05). The observed small decrease in activity in high salt after 2 h phytohemagglutinin probably reflects an error in counting nuclei, as the ratio of activity in low/high salt was exactly the same (43 %) after z and 4 h phytohemagglutinin. The addition of phytohemagglutinin directly to unstimulated nuclei had no effect on activity in high or low salt.
E//ect o/ long-term incubation with phytohemagglutinin Lymphocyte cultures from the same donor were incubated with and without 5/zg/ml phytohemagglutinin for zo, 46, and 72 h. Nuclei were isolated from each culture, and [5-3HICTP incorporation was determined. In the nuclei from phytohemag~= oI= 5(~ 0--
i
RATIO (high/lows(lit) 20h 46 h 72h Unstimulated PHA- st imuloted
6.2 4.7
G.6 4.2
/~,o j./ /
9.7 43
/ /
4(:
// //
,~.
+{NH4 )zS04
Z ~
I(
~-- ..... 24
~
( NH4|/ S04 48
72
TIME OF INCUBATION {h)
Fig. 5. E(fect of l o n g - t e r m i n c u b a t i o n w i t h p h y t o h e m a g g l u t i n i n on [5-sHJC'I'P incorporation. I n c u b a t i o n c o n d i t i o n s were as given in raArERIALS AND METHODS. E a c h p o i n t r e p r e s e n t s t h e m e a n of 4 replicate d e t e r m i n a t i o n s , t h e i n d i v i d u a l v a l u e s being w i t h i n + xo % of t h e m e a n . O - O , nuclei from r e s t i n g l y m p h o c y t e s ; Q)- - -Q), nuclei from p h y t o h e m a g g l u t i n i n - s t i m u l a t e d l y m p h o cytes. P H A , p h y t o h e m a g g l u t i n i n .
13iochirn. 13iophys. Acta, I99 (x97 o) 9 5 - I o z
PHYTOHEMAGGLUTINI.-'q EFFECT ON
RNA POLYMERASE
IOI
glutinin-stimulated cells there was a progressive increase in RNA polymerase activity in both low salt and high salt conditions (Fig. 5). Incubation for 2o h with phytohemagglutinin resulted in a doubling (207 %, P < 0.005) of the low salt activity and a 57 % increase (P < o.ooI) in the activity of the enzyme in high salt. By 72 h, there was 5- times as much (5oI %, P << o.oox) activity in low salt and more than double (220 °/o, P << o.ooI) the activity in high salt. The enhancement of RNA polymerase activity by high salt was consistently greater in nuclei of unstimulated cells. A sharp rise was found in the high/low salt activity of resting cells incubated for 7 2 h. The reason for this is not readily apparent.
DISCUSSION
Phytohemagglutinin-stimulated lymphocytes have been shown to contain more RNA than unstimulated cultures 7,16..5,.6. The rate of incorporation of labelled uridine into RNA after zo h incubation with phytohemagglutinin is IO-2O times greater than in resting cells. As reported here, nuclei from lymphocytes incubated with phytohemagglutinin for 2o h showed an increase in endogenous RNA polymerase activity. However, the enhancement was much less than the increase in uridine incorporation, a doubling of RNA synthetic capacity assayed in low salt and 1.5-times as much activity in high salt. Studies comparing the level of uridine incorporation with uridine kinase (ATP : uridine 5'-phosphotransferase, EC 2.7.I.48 ) activity x5,~7'~8have suggested that uridine incorporation is not a quantitative index of the rate of RNA synthesis. Furthermore, the rather small increase in RNA synthetic capacity of nuclei from phytohemagglutinin-treated cells and the demonstration from COOPER29 of reduced wastage of I8 S ribosomal RNA within I h after the addition of phytohemagglutinin suggest that enhanced survival of new RNA may be a significant factor in the phytohemagglutinin-induced increase in total cellular RNA, which is predominantly ribosomal. In the experiments reported here, we found that nuclei which were isolated within 2- 4 h after the addition of phytohemagglutinin showed an increase in RNA synthetic capacity only when assayed ill IOW salt. In the presence of 0.4 M (NH,),SO, there was no significant difference in activity at this time between phytohemagglutinin-treated and control cells. When nuclei were isolated after 20, 46, and 72 h incubation with phytohemagglutinin, there was an increase in RNA polymerase activity in both high and low salt. In every case the ratio of high/low salt activity was greater in nuclei from unstimulated cells. These findings are basically similar to those obtained in other mammalian systems in which extensive gene activation or derepression is believed to occur, regenerating liver following partial hepatectomy of young adult rats 3° as well as a number of systems after hormone stimulation ~,a~-3~ (review by TATAae).
The basis for the difference in activity of RNA polymerase in high and low salt is not clear. Since the initial description of GOLDBERG~7 of the enhancement of RNA polymerase activity by increased ionic strength, there has been speculation as to its meaning. The activity in high salt ha.q been variously explained as being due to (I) unmasking of a second enzyme ~, (2) dissociation of histones from DNA, thereby increasing template availability u,38, (3) inhibition of degradative enzymes a9,4°, (4) an Biochim. Biophys. Acta, I99 (I97 o) 95-xo2
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S. D. HANDMAKER, J. w . GRAEF
effect on the enzyme itself 41. The product of the low salt reaction in liver cell preparations has been found to be ribosomal-like in base composition, in contrast to the DNAlike product of the high salt reaction 29,42,.3. These findings as well as our own, are compatible with any of the above interpretations.
ACKNOWLEDGMENTS We thank Mrs. Thelma Prather for her excellent technical assistance and Drs. Herbert L. Cooper, John E. K a y and Robert Stern for their interest in this work and for their critical review of the manuscript. REFERENCES I L. B. El'STEIN AND F. STOHLMAN, Blood, 24 (1964) 69. 2 N. R. LING, Lymphocyte Stimulation, N o r t h Holland, A m s t e r d a m , r968. 3 P. C. NOWELL, Cancer Res., 20 (196o) 462. 4 C. K. NASPITZ AND M. RICHTER, Progr. Allergy, 12 (1968) 1. 5 0 . R. MClSTYRE AND F. G. EBAUGH, JR., Blood, 19 (1962) 4436 H. I.. COOPER, in R. BASERGA, Biochemistry o/ Cell Division, Charles C. Thomas, Springfield, Ill., 1969, p. 91. 7 J. E. KAY, in M. W. ELVES, The Biological E//ects o/ Phytohemagglutinin, The R o b e r t J o n e s and Agnes H u n t Hospital M a n a g e m e n t Committee, Oswestry, 1966. 8 I~'. I,. TORELLI, P. l-I. HENRY AND S. -'~. WEISSMAN, J. Clin. Invest., 47 (1968) 1°83. o A. l). RUBIN, Nature, 22o (I968) 196. 1o J. E. KAY AND H. L. COOPER, Biochim. Biophys. Acta, 186 (I969) 62. 1i D. KILI.ANDER AND R. RIGLER, Exptl. Cell Res., 39 (1965) 71o. I2 B. G. T. POGO. V. G. ALLFREY AND A. E. MIRSKV, Proc. Natl. Acad. Sci. U.S.. 55 (1966) 805. 13 l.. J. KLEINSMITH, V. G. ALI.FREY AND A. E. MIRSKY, Science, 154 (1966) 780. 14 R. HIRSCHHORN, W. TROLL, G. BRITTINGER AND G. WEISSMANN, Nature, 222 (1969) 1247. I 5 iv). -}-[AUSEN AND H. STEIN, EuroOean J. Biochem., 4 (1968) 4Ol. I6 Z. J. LUCAS, in W. O. RIECKE, l~roc. 3rd Ann. Con/. Leukocyte Cult., Appleton Century Crofts, New York, 1969, p. 655. 17 H. L. COOPER, J. Biol. Chem., 243 (1968) 3418 H. EAGLE, Science, 13o (1949) 432. 19 J. S. WOLFF, I l l , J. A. LANGSTAFF, G. WEINBERG AND G. ~V. ABELL, Biochem. Biophys. Res. Commun., 26 (1967) 366. 20 J. w . GRAEF AND S. O. HANDMAKER, in preparation. 2i V. G.. ALLFREY, A. E. MIRSKY AND S. OSAWA, j . Gen. Physiol., 4 ° (1957) 45 I. 22 S. B. WEiss, Proc. Natl. Acad. Sci. U.S., 46 (196o) lO2O. 23 C. (" WIDNELL AND J. R. TATA, Biochim. Biophys. Acta, 87 (1964) 531. 24 A. E. ])EGG AND A. KORNER, Nature, 2o 5 (19651 904. 25 D. R. FORSDYKE, Biochem. J., lO 5 (1967) 679. 26 S. D. HANDMAKER, B. G. LEVENTHAL AND H. L. COOPER, in W. O. I{IECKE, Proc. 3rd Ann. Con]. Leukocyte Cult., Appleton C e n t u r y Croffs, New York, 1969, p. 53. 27 Z. J. LUCAS, Science, I56 (1967) 1237. 28 J. E. KAY AND S. D. HANDMAKER, in preparation. 29 H. L. COOPER, J. Biol. Chem., (1969) in the press. 3 ° A. O. I~OGO, V. G. ALLFREY AND A. E. MIRSKY, Proc. Natl. Acad. Nci. U.S., 56 (1966) 550. 31 J. (;ORSKL J. Biol. Chem., 234 (1964) 889 . 32 J. R. TATA AND C. C. WIDNELL, Biochem. J., 98 (I966) 604. 33 ('. B. BREt1R AND J. R. FLORINI, Biochemistry, 5 (1966) 3857 • 34 ('- C. V~rID.~ELL AND J. R. TA'rA, Biochem. J., 98 (1966) 621. 35 T. H. HAMILTON, C. C.'\VIDNELL AND J. R. TATA, J. Biol. Chem., 243 (1968) 4o8. 36 d. R. TATA, Progr. Nucleic Acad. Res., 5 (1966) 2Ol. 37 I. H. GOLDBERG, Biochim. Biophys. Acta, 51 (196I) 2Ol. 38 I'. CHAMBON, M. RAMUZ AND J. DOLY, Biochem. Biophys. Res. Commun., 19 (1965) 156. 39 D. D. CUNNINGHAM AND D. F. STEINER, Federation Proc., 25 (1966) 788. 4 ° A. I. MEISLER AND 13. E. Tt~OPP, Biochim. Biophys. Acta, 174 (1969) 476. 41 S. T. JAcon, E. M. SAJDEL AND N. H. MUNRO, Biochem. Biophys. Res. Commun., 32 (1968) 831. 42 K. MARUSHIGE AND J. BONNER, J. Mol. Biol., 15 (1966) 16o. 43 P. CHAMBON, H. KARON, M. RAMUZ AND P. MANDEL, Biochim. Biophvs. Acta, ~57 (1968) 52o.
Biochim. Biophys. Acta, 199 (197 o) 95-IO2