RNA polymerase activity of erythroblasts isolated from regenerating or erythroblastosis-infected avian bone marrow

BIOCHIMICA ET BIOPHYSICA ACTA

75

BBA 96484

RNA P O L Y M E R A S E A C T I V I T Y OF E R Y T H R O B L A S T S I S O L A T E D FROM R E G E N E R A T I N G OR E R Y T H R O B L A S T O S I S - I N F E C T E D AVIAN B O N E MARROW R O B E R T L. S T A I N E S * AND E S T H E R W. Y A M A D A

Department of Biochemistry, University of Manitoba, Winnipeg 3, Manitoba (Canada) (Received December i s t h , 1969)

SUMMARY

I. DNA-dependent RNA polymerase (nucleoside triphosphate: RNA nucleotidyltransferase, EC 2.7.7.6) activity of nuclei isolated from six erythroblast-rich fractions of regenerating ("regenerating") avian bone marrow was compared to that of six comparable preparations obtained from erythroblastosis-infected ("viral") bone marrow. 2. Both initial rates of RNA synthesis and net RNA synthesis were measured since it was found that the effect of (NH4),SO 4 (0.35 M) on each was different. Both were found to be correlated with the per cent erythroblasts present in "viral" or "regenerating" fractions, in the presence or absence of (NH,)2SO 4. 3- Under the standard conditions of assay the initial rates of RNA synthesis catalysed by nuclei of "viral" erythroblasts was twice that of nuclei of "regenerating" erythroblasts (P < o.ooi). This difference was even more pronounced in the presence of (NH4)2SO 4 and became 3-fold; although (NH4)2SO 4 had a negligible effect on the initial rates of "viral" nuclei it decreased those of "regenerating" nuclei. 4- The net amount of RNA synthesized b y nuclei of "viral" erythroblasts was 1. 3 times that synthesized by nuclei of "regenerating" erythroblasts (P ~, 0.0I). (NH4)2S04 had no effect on the net synthesis of RNA of "regenerating" nuclei but resulted in a 2.4-fold increase in the net synthesis of "viral" nuclei. Thus, in the presence of (NH4)2S04, net synthesis of RNA of "viral" nuclei was 2.4 times that of "regenerating" nuclei (P < 0.00I). 5. These data indicate that increases in DNA-dependent RNA polymerase activity occur after infection of erythroblasts of avian bone marrow with erythroblastosis virus.

INTRODUCTION

Several investigators have found marked alterations in the rate of RNA synthesis after infection of mammalian cells with RNA-containing viruses. Thus, recent reports indicate that inhibition of the activity of host cell RNA polymerase b y virus-specific proteins m a y be a general phenomenon associated with infection b y * S u b m i t t e d in partial fulfillment of the r e q u i r e m e n t s for the degree of Master of Science.

Biochim. Biophys. Acta, 209 (197 ° ) 75-85

76

R.L. STAINES, E. W. YAMADA

picornaviruses 1-a. These viruses are capable of replication in the absence of host cell DNA synthesis or DNA-dependent RNA synthesis. Another group of viruses, including the tumorigenic avian leukosis viruses, require the replication of DNA to proceed in host cells in order for their own replication to proceed 4,5. The infection of mouse spleen cells by a virus of the latter group, viz. murine leukemia virus, was found to result in an 8-fold increase in the activity of DNA-dependent RNA polymerase activity of nuclei isolated from infected cells 6. It was the purpose of present studies to see if changes in DNA-dependent RNA polymerase (nucleoside triphosphate :RNA nucleotidyltransferase, EC 2.7.7.6) activity could be detected after infection of bone marrow cells with erythroblastosis virus, one of the avian leukosis viruses. The kinetics of the reaction catalysed by RNA polymerase of nuclei isolated from erythroblast-rich fractions obtained from either erythroblastosis-infected ("viral") or regenerating ("regenerating") bone marrow, the control tissue of choice, were studied. The activity of "viral" nuclei was always greater than that of "regenerating" nuclei both in the presence or absence of (NH4)2SO ~. These results and those of earlier workers 6 are in accord with the view that infection by tumorigenic viruses results in increases in the activity of DNAdependent RNA polymerase of host cells.

MATERIALSAND METHODS

Chemicals 14C-labelled nucleoside triphosphates were purchased from Schwarz Bioresearch. Unlabelled nucleoside triphosphates were purchased from the Sigma Chemical Co. Highly polymerized calf thymus DNA, crystalline deoxyribonuclease (ribonuclease-free) and ribonuclease IA were products of Mann Research Laboratories. Actinomycin D was a gift from Merck, Sharp and Dohme Co.

Animals Purified preparations of erythroblastosis virus, Strain R, were gifts from Professor B. Thorell, Stockholm and Dr. R. Bather, Saskatoon. White Leghorn chickens, Line 15 I, East Lansing, were obtained from Dr. R. B. Burmester and were used in all experiments. Livers from chickens in the terminal stages of erythroblastosis were used as the source of virus. They were homogenized in 5 volumes of 0. 9 % NaC1 in ice. The homogenate was centrifuged for io min at IOOO×g at o ° and the supernatant was decanted into sterile tubes. I-ml aliquots were injected per kg of body weight into the wing vein of chickens. The course of the infection was followed by examination of blood smears. Normally 8- 9 days elapsed between the time of injection of the virus preparation and morbidity 7. Control chickens were treated with phenylhydrazine as described previously s.

Separation o/erythroblasts Chickens were decapitated and their leg bones removed and placed immediately in ice. All subsequent operations were carried out at 2 °. In a cold room the leg bones were cut and the bone marrow was extruded into a preweighed beaker by means of a pressure pump. It was then suspended in 4 volumes of Ca~+-free Hanks'

Biochim. Biophys. Acta, 2o9 (I97o) 75-85

RNA

P O L Y M E R A S E A C T I V I T Y OF E R Y T H R O B L A S T N U C L E I

77

balanced salt solution (Medium A) containing IO % sterile chicken serum (Colorado Serum Co.). The cells were dispersed by forcing the suspension through a syringe and then through stainless wire gauze (60 mesh). The resultant suspension was filtered through four layers of cotton gauze (28 x 2 4 mesh). 4 ml of the cell suspension were then layered on top of 25 ml of 5 o/ ~o (w/w) Dextran T-8o (Pharmacia Co.), dissolved in Medium A, and the tubes were centrifuged in a swing-out bucket rotor (HB 4) in a Sorvall refrigerated centrifuge for 5 rain at 12o ×g and then for IO rain at 1465 ×g. The erythroblasts were pelleted at the bottom of the centrifuge tube. The erythroblast-rich pellet was washed 2 3 times in Medium A. Aliquots were removed routinely for differential cell counts.

Isolation o/ nuclei The washed erythroblast-rich pellets were suspended in 2 vol. of IO mM Tris (pH 7.4)-1.5 mM MgC12-IO mM KC1- 5 mM 2-mercaptoethanol buffer containing I 9/0 saponin. The cells were homogenized in a Potter-Elvehjem homogenizer with about 18 passes of a loosely-fitting teflon pestle. The homogenate was then diluted 3 4fold with io mM Tris (pH 7.4)-1.5 mM MgC12-IO mM KC1-5 mM 2-mercaptoethanol buffer and centrifuged at 900 ×g for 8 rain. The pellet, containing intact nuclei, was washed twice in IO mM Tris (pH 7.4)-1.5 mM MgC12-IO mM KC1-5 mM 2-mercaptoethanol buffer. It was then suspended in 2 volumes of 50 mM Tris buffer (pH 7.0) containing 5 mM 2-mercaptoethanol. The nuclei when stained with crystal violet appeared intact in the light microscope and virtually free of cytoplasmic contamination. They could be stored at --20 ° until use without loss of enzymic activity or change in appearance even after 12 days. Nuclei were prepared from twelve erythroblast-rich suspensions from "viral" or "regenerating" bone marrow. The differential cell counts of these samples are given in Table I. Some variation in the number of erythroblasts was found particularly in the "viral" samples. This was because the samples contained the bone marrow of three or more chickens and the chickens showed some differences in susceptibility to virus and in rate of regeneration after phenylhydrazine injection. Since erythrocytes were the major contaminants of the suspensions additional samples containing only erythrocytes were prepared from whole chicken blood. The blood was centrifuged for 30 min at 2o00 x g at 2 °. The erythrocytes were removed from the bottom layer and washed several times with Medium A. Nuclei were then isolated and tested for RNA polymerase activity. In agreement with the findings of others 9,1° nuclei from mature erythrocytes had little or no activity in the presence or absence of (NH4)2SO4.

Assay o / R N A polyrnerase activity Enzyme activity was determined b y measuring the rate of incorporation of nucleotide from E8A4C]ATP into acid-insoluble RNA. Unless otherwise specified the reaction mixture contained the following in a final volume of 0.25 ml: 25/,moles oi Tris buffer (pH 9.o), 1.75 #moles of magnesium acetate, 2.5/*moles of 2-phosphoenolpyruvate, 5/*moles of NaF, 1.25/*moles of 2-mereaptoethanol, 25/*g of DNA, 2.5/,g of pyruvate kinase, 30 nmoles each of UTP, GTP and CTP, 25 nmoles of E8-14C]ATP (2.22. IO5 disint./min) and nuclear fraction (o.75-1.o nag of protein). The p H oi the reaction mixture was 8.6. The reaction was started by the addition of nucleoside triphosphates (labelled and unlabelled) and incubation was continued at 37 °. RouBiochim. Biophys. Acta, 2o9 (197 ° ) 7 5 - 8 5

H

,,0

I

* Includes

"Viral",

71.5 62.9 51-o 4 5 .2 3 °.0 28. 5

84.1 77.5 71.7 68.6 5 7 .1 55.9

forms

23.2 31.2 45.2 4 9 .8 65-5 66.1

ii.o 18.2 24.9 26.5 39.3 39.3

Erythrocytes

2. 4 2. 5 2.2 2.8 1.5 2. 7

1.8 1.2 0.4 1.5 0.6 1.5

of red blood cells; Giemsa

Erythroblasts"

stain was used.

o.o o.o o.I 0. 4 o.o o.o

o.o o.o o.o o.o o.o o.o

Thromboblasts Metagranu+ thrombocytes loblasts

USED IN THE COMPARISON OF ACTIVITIES OF "VIRAL"

Di//erenlial cell count (%)

all immature

I 2 3 4 5 6

2 3 4 5 6

"Regenerating",i

Sample

C E L L COMPOSITION OF S U S P E N S I O N S

TABLE

1.8 2.0 1.2 0.8 1. 5 1. 9

0. 9 2.1 3.o 0.9 2. 4 2.5

Polymorphonuclear leucocytes

NUCLEI

0.8 1. 4 1. 3 0. 4 o.9 0.8

1.8 0.6 o.o 1.9 0.6 0.8

Lymphocytes

AND "REGENERATING"

o. 3 o.o o.o 0.6 0.6 o.o

0. 4 0. 4 o.o 0.6 o.o o.o

Broken cells

"-1 O0

RNA

79

POLYMERASE ACTIVITY OF ERYTHROBLAST NUCLEI

tinely, only 5 min were allowed to elapse between the addition of nuclear fraction and the beginning of incubation. During this time and during the addition of all components except substrate the reaction mixture was kept at o ° in an ice-bath. Frozen preparations of nuclei were thawed out just before use. Repeated freezing and thawing resulted in loss of enzyme activity. Where specified in the text (NH4)2SOa, adjusted to p H 8.3 with NH4OH, was added to the reaction mixture. The reaction was terminated by the addition of 2 ml of IO % trichloroacetic acid and I m g of carrier bovine albumin. The precipitate was collected on Millipore filters (0.45 # pore size) in a cold room. I t was washed 7 times with 5 % trichloroacetic acid containing 0.02 M tetrasodimn pyrophosphate, dried in an oven at 9O-lOO °, placed in 15 ml of BBOT scintillation solution* and counted in a Packard Tri-Carb spectrometer. Later, glass membrane filters (Whatman GF/C) were used with comparable results. All tests were performed in duplicate with appropriate zero time controls to which trichloroacetic acid was added immediately after the addition of labelled ATP. Protein was determined by the method of LOWRY et al. n. RNA polymerase activity is expressed in terms of either the initial rate of incorporation of labelled nucleotide into acid-insoluble RNA after incubation for 5 min or the m a x i m u m incorporation (net RNA synthesis) after incubation for 15-25 rain. In both cases activity is expressed as the pmoles of E14C]AMP incorporated per incubation time per mg protein.

RESULTS

Time relationship The time-course of the reaction is illustrated in Fig. I. For all nuclear preparations tested the reaction proceeded linearly with time up to 5 rain; incorporation of labelled nucleotide into RNA was maximal between 15 and 60 rain. 24

(B) "Viral" ~20

(A)"Rege~nerating '' '

•

16 E

o a .c_ I

O -~ -

-0

<

~=~T'0

2'0 4b Time (rain)

6b

o

o

2b

Time [mln)

1)

20

2J

3'

6'o

Fig. I. 1RNA p o l y m e r a s e a c t i v i t y as a function of time. I n c u b a t i o n conditions were as given in MATERIALS AND METHODS. A. A c t i v i t y of " r e g e n e r a t i n g " nuclei. I n c u b a t i o n in the absence ( O ) or presence ( 0 ) of 0.35 M (NH4)2SO ~. B. Activity of " v i r a l " nuclei in the absence (17) or presence (m) of 0.35 M (NH4)2SO 4. 4 g of B B O T (2,5-bis-[2-(5-tert.-butylbenzoxazolyl)]thiophene, 4oo ml of methylcellosolve, 80 g of n a p h t h a l e n e and 6oo ml of xylene.

Biochim. Biophys. Acta, 209 (I97 o) 75-85

~O

R.L.

S T A I N E S , E. W. Y A M A D A

The presence of o.35 M (NH4)2SO 4 resulted in a slight but reproducible decrease in the initial rate of incorporation of "regenerating" nuclei but did not alter the time-course of the reaction between 15 and 6o min (Fig. IA). With "viral" nuclei, however, the initial rate of incorporation was increased in the presence of o.35 M (NH4)2SO4; the rate did not fall off as for "regenerating" nuclei but rather a second linear rate was established between 5 and 15 min (Fig. IB). Maximum incorporation, more than 2-fold that without (NH4)~SO4, was obtained after 15 min. The results for "viral" nuclei are similar to those reported by WIDNELL AND TATA12Aa for rat liver nuclei.

Enzyme concentration There was a linear relationship between initial rates of incorporation (5-min activity) catalysed by "regenerating" or "viral" nuclei and concentration of protein, up to 4 mg/ml. The ultimate amount of RNA synthesized (I5-min activity) with both preparations also increased in proportion to enzyme concentration (up to 4 mg of protein/ml) in essential agreement with the results of others 14 who showed that these kinetics reflect the ability of each enzyme molecule to synthesize one RNA molecule which remains bound to the enzyme-DNA complex. Since both the 5-min activity of "viral" and "regenerating" nuclei and net RNA synthesis catalysed by "viral" nuclei were altered t o different extents in the presence of (NH4)~SO 4 both parameters were used in present studies.

Ionic strength Fig. 2 shows that the 5-min activity of "regenerating" nuclei was inhibited at concentrations of (NH4)2SO 4 greater than 0.2 M in the presence of Mg2+. Surprisingly enough, the I5-min activity of "regenerating" nuclei was increased but little by (NH4)2SO 4 in the presence of either Mg2+ or Mn e+ even after preincubation at 37 ° for 15 min. The 5-rain activity of "viral" nuclei was increased slightly when (NH4)2SO 4 was included in the reaction mixture (Fig. 2); the I5-min activity of "viral" nuclei 30

.

.

.

.

.

(A) "Regenerating "

i

2O

~o<./-~ 8 .g

"----o

<

O

oh5 ( N H 4 ) 2 SO4

a~e o!

o.~5

0.50

c o n c h . (K4)

F i g . 2. E f f e c t of c o n c e n t r a t i o n of ( N H 4 ) 2 S O 4 o n R N A p o l y m e r a s e a c t i v i t y . T h e s t a n d a r d c o n d i t i o n s of a s s a y w e r e u s e d . A. A c t i v i t y o f " r e g e n e r a t i n g " n u c l e i . B . A c t i v i t y of " v i r a l " n u c l e i . I n c u b a t i o n f o r 5 r a i n w i t h 7 m M M g ~+ ( O ) - I n c u b a t i o n f o r 15 r a i n w i t h 7 m M M g 2+ ( 0 ) o r 1. 4 m M M n 2+ ( I ) .

Biochim. Biophys. Acta, 2 0 9 ( i 9 7 o) 7 5 - 8 5

RNA POLYMERASEACTIVITYOF ERYTHROBLASTNUCLEI

81

was increased more than 2-fold. Maximum incorporation was found with o.35 M (NH~)2SO 4 (ionic strength of o.7) , well within the concentration range required to give maximum stimulation of the activity of "aggregate" preparations 15-~v, intact nuclei 12,13 or nucleoli ~s isolated from other types of animal cells. These increases occurred with optimal concentrations of either Mg2+ or Mn 2+, in essential agreement with the findings of others for rat liver nuclei ~9-21. Divalent cations Both Mg 2+ and Mn 2+, at optimal concentrations of 7 and 1. 4 raM, respectively, increased the activity of "regenerating" nuclei. There was negligible change in the divalent cation requirements of the 5- or I5-min activities resulting from the addition of (NH4)2SO 4 to the reaction mixture. In media lacking (NH4)2SO a Mn 2+ increased the 5-rain activity of "viral" nuclei slightly more than did Mg2+; in media containing this salt the 5-rain activity with Mn 2+ was about the same as that with Mg2+. In addition, the I5-min activity of "viral" nuclei was increased by about the same amount by Mg 2+ and Mn 2+, at optimal concentrations of each, in the presence or absence of (NH4)2SO a. Earlier, the (NH4)2SO4-dependent activity of rat liver nuclei was thought to be Mn2+-specific 12,13. More recently, it has been found that (NHa)2SO 4 stimulation occurs in the presence of either Mg2+ or Mn 2+ (refs. 19-21 ) although the divalent cation does dictate the type of product RNA formed 2°,21. Substrates, inhibitors and polyamines Optimal rates of RNA synthesis were obtained with the addition of 3o mnoles each of UTP, CTP and GTP when phosphoenolpyruvate and pyruvate kinase were also present. Little activity was measurable when these components were omitted from the reaction mixture. NaF enhanced the rate of nucleotide incorporation but the effect of 2-mercaptoethanol was variable. The addition of DNA led to only slight increases in the rate of RNA synthesis but that the reaction was DNA-dependent was apparent from the drastic reduction in rates of 8o-9o O/~owhen IO/~g of deoxyribonuclease or IO #g of actinomycin D were included in the reaction mixture. When Io #g of ribonuclease was added there was again ahnost complete loss of synthesis. Similar results were obtained for "viral" and "regenerating" nuclei after incubation for 5 or 15 rain in the presence or absence of (NH4)2S Q . Neither spermine nor spermidine (I raM) altered the activities of either type of nuclear preparation significantly. Several polyamines at concentrations greater than 30 mM were found to prevent lysis of rat liver nuclei but even when lysis of the nuclei occurred no decrease in the rate of RNA synthesis was detected 19. H + concentration Both 5- and I5-min activities of "regenerating" nuclei were maximal at pH values of 8.6-9.0. The optimum pH values were not altered when 0.35 M (NH4)2SO 4 was added to the reaction mixture. The 5- and I5-min activities of "viral" nuclei were also maximal at pH values of 8.6-9.0 in the absence of (NH4)2SO a. (NH4)2SO 4 had a negligible effect on the pH optimum after incubation for 5 rain. However, after incubation for 15 rain in reaction media containing (NH4)2SO 4 and either Mn 2+ or Mg2+ a shift in optimum pH Biochira. Biophys. Acta, 2o9 (197o) 75-85

82

R.L.

S T A I N E S , E. W. Y A M A D A

values to 8.4-8.6 occurred. This shift, albeit of only half a p H unit, was of the same order of magnitude as that reported b y CHAMBON et al. is for "aggregate" preparations of rat liver. Activity and per cent erythroblasts Fig. 3 shows the relationship between initial rates of RNA synthesis and per cent erythroblasts. The lines of best fit and correlation coefficients (r) were calcu•~-~ 6 E 'Regenerating' +(Nt-14) 2 SO4

6. Viral"+ (NN4)2S04J | / ~ m

0-

.£ o 3 ~

r :0.9880t O.Ol

o

O~

100

6

i

o~°+/

3L/

k

~

.o'l /, /

r

o

5o

6

"Viral

I'

e~* o/"o

/

3k

"O00A r :U'IY~bU ±u'ur 50

,oP'/

0~/

100

100

i

[" Regeneratlng"

~L~ O ~ 0

=0~927~ 0 - 0 6

r =0i9725._ O.02

0

100

50

Erythroblasts (%) Fig. 3. Initial ratesof RNA synthesiscatalysedby nuclearfractionsof "regenerating"or "viral" b o n e m a r r o w p l o t t e d a g a i n s t p e r c e n t e r y t b r o b l a s t s . I n i t i a l r a t e s w e r e m e a s u r e d a s d e s c r i b e d in MATERIALS AND METHODS. I n c u b a t i o n s w e r e c o n t i n u e d f o r 5 m i d a t 37 °. y = a+bx d e s c r i b e s t h e l i n e of b e s t fit; r is t h e c o r r e l a t i o n c o e f f i c i e n t 4- s t a n d a r d e r r o r of r.

A

.c E

6C "Regenerating"÷ (NI44) 2 SO4

60

!

~ / "Viral" + (NH4)2S04 / / m , ~

c~

30

.c

- , 0 9-g9 ~ •.% %A~ "

•

~

Is /

o.o;

5O

r o0.9745_*0.02

100

60. ~}" i'Regeneratlng"

100 60/'viral'' 301/

I ~

J -

.~,0 .30~ -

.o,. 4 4 ao4 100

0

50

100

Erythr~blasts (%) F i g . 4. N e t R N A s y n t h e s i s c a t a l y s e d b y n u c l e a r f r a c t i o n s o f " r e g e n e r a t i n g " ~ o r " v i r a l " b o n e m a r r o w p l o t t e d a g a i n s t p e r c e n t e r y t h r o b l a s t s . N e t R N A s y n t h e s i s w a s m e a s u r e d as d e s c r i b e d i n MATERIALS AND METHODS. I n c u b a t i o n s w e r e c o n t i n u e d f o r 25 m i d a t 37 °. y = a+bx d e s c r i b e s t h e l i n e o f b e s t fit; t h e c o r r e l a t i o n c o e f f i c i e n t (r) is g i v e n i s t a n d a r d e r r o r o f r.

Biochim. Biophys. Acta, 2 0 9 (197 o) 7 5 - 8 5

RNA POLYMERASE ACTIVITY OF ERYTHROBLAST NUCLEI

83

lated as described b y STANLEY22. A high correlation between the initial rates and per cent erythroblasts of "regenerating" preparations as well as of "viral" preparations, in the presence or absence of (NH4)2SO 4, is apparent from this figure. Fig. 4 shows that there is a high correlation between net RNA synthesis and per cent erythroblasts of "regenerating" as well as of "viral" preparations both in the presence of (NH4)2SO * and in its absence.

T A B L E II COMPARISON OF INITIAL RATES A N D " V I R A L '* P R E P A R A T I O N S

AND

NET

SYNTHESIS

OF

RNA

CATALYSED

BY

"REGENERATING"

R e g r e s s i o n coefficients (b) are t h e slopes of t h e lines of b e s t fit s h o w n in Figs. 3 a n d 4 w h e r e b = RNA polymerase activity/per cent erythroblasts.

Sample

"Regenerating" " R e g e n e r a t i n g " + (NH~)2SO a "Viral" " V i r a l " + (NH4)2SO 4

R N d polymerase activity (regression coe]/icient (b) ± S.E.) Initial rates

Net synthesis

o.o34 ± o.ooo 3 o.o23 ~:o.ooi 4 o.o63 ~: o.oo6o o.o7o~o.olio

o.234 :[- o.o33o o.299io.o42o o.3oi :[:o.o38o o.714io.o63o

Comparison o~ activities o/"regenerating" and "viral" nuclei This comparison (Table II) was done on the basis of regression coefficients (slopes) of the lines of best fit which give a measure of initial rates of RNA synthesis (Fig. 3) and net RNA synthesis (Fig. 4) in terms of the number of erythroblasts. Of interest was the finding that the initial rates of nuclei of "viral" erythroblasts were about 2-fold those of nuclei of "regenerating" erythroblasts, in the absence of (NH4)~SO 4 (P < 0.00I). The initial rates of nuclei of "regenerating" erythroblasts were decreased b y (NH4)2SO 4 (P < 0.0i). In contrast, although (NH4)2SO 4 increased the initial rates of nuclei of "viral" erythroblasts, these increases were not statistically significant (P > 0.05). Nevertheless, in the presence of (NH4)2S04, the initial rates of "viral" nuclei were about 3-fold those of "regenerating" (P < 0.00I). (NH~)2SO 4 had no effect on net RNA synthesis catalysed b y "regenerating" nuclei (P > 0.I) but stimulated that eatalysed b y "viral" nuclei more than 2-fold (P < 0.00i). In the absence of (NH4)2SO l net RNA synthesis catalysed by nuclei of "viral" erythroblasts was 1.3 times t h a t of nuclei of "regenerating" erythroblasts (P < 0.0I) and in the presence of (NH4)2SO 4 became 2.4 times greater (P < 0.00i).

DISCUSSION

Which of the activities of intact erythroblast nuclei correspond to the "soluble ''23-2e, "aggregate ''15,16,27 or chromatin-bound forms 9,2s,29 of RNA polymerase is not clear at present. In the absence of (NHa)2SO 4 the initial rates of RNA synthesis catalysed b y "viral" erythroblast nuclei were 2-fold those of "regenerating" eryBiochim. Biophys. Xcta, 2o9 (197 o) 7 5 - 8 5

84

R.L.

S T A I N E S , E. W. Y A M A D A

throblast nuclei. Pertinent to this finding are those of FURTH el al? ° who found that soluble RNA polymerase activity was greater in lymphosarcoma cells than in cells of normal lymph nodes. These increases might reflect mitotic rate, alone 3°. Correspondingly, BRODYet al. 31 found the mitotic index of erythroblasts of erythroblastosisinfected avian bone marrow to be greater than that of erythroblasts of regenerating bone marrow. W h y (NHa)2SO 4 at ionic strengths greater than 0. 4 inhibited the initial activity of "regenerating" nuclei but had negligible effect on this activity of "viral" nuclei is not known at present. It is known, however, that the activity of "soluble" enzymes isolated from other tissues 24,26,32 and Escherichia coli 33,34 are inhibited by ionic strengths exceeding o.3. In direct contrast, however, to its effect on initial rates, high concentrations of (NH4)2SO 4 did not affect net RNA synthesis catalysed by "regenerating" nuclei but stimulated that of "viral" nuclei 2-fold. Possibly "viral" and "regenerating" nuclei contain different proportions of free and bound enzyme forms of RNA polymerase making kinetic studies with (NH4)2SO 4 complex. The results of PEGG AND KORNER35 and POGO2° are consistent with the view that (NH4)2SO 4 selectively removes "repressor" proteins from the DNA genome. Whether this occurs with "viral" nuclei but not with "regenerating" nuclei is not clear at present. It is not possible to distinguish between the virogenic and tumorigenic aspects of erythroblastosis infection at this time. Previous workers reported a suppression of RNA-synthesizing activity of "aggregate" enzyme complexes prepared from L cells after infection with the non-tumorigenic virus, mengovirus. The activity of these preparations was measured in the presence of high concentrations of (NH4)2SO 4 (ref. 36). In subsequent studies of RNA polymerase of chromatin isolated from Ehrlich ascites cells infected with Maus-Elberfeld virus a7 a decrease in template activity, after infection, to levels normally found in the repressed chromatin of mature avian erythrocytes was found. The template activity of the chromatin from infected cells was increased after treatment with trypsin. In investigations of RNA polymerase activity of nuclei obtained from mouse spleen cells infected with murine leukemia virus, although increases in RNA polymerase activity were found, the activities in the presence of (NHa)2SO 4 were not reported*. It is nevertheless apparent from these studies that there are significant differences in the effect on RNA polymerase activity of host cells as a result of infection by tumorigenic as compared to non-tumorigenic RNA-containing viruses. In addition, the dependency of avian leukosis viruses 4,5 and other tumorigenic viruses 6 on replication of host cell DNA would suggest that increases in host cell RNA polymerase activity are related to the tumorigenic process.

ACKNOWLEDGEMENTS

This work was supported by the National Cancer Institute of Canada and the Medical Research Council of Canada. The authors are indebted to Mr. Bert Grift and Mr. Spencer Silver for skillful technical assistance. The cooperation of Professors P. A. Kondra and G. C. Hodgson of the Department of Animal Science, University of Manitoba in caring for and breeding the chickens used in this research is gratefully acknowledged. Biochim. Biophys. Acta, 209 (197° ) 75 85

R N A POLYMERASE ACTIVITY OF ERYTHROBLAST NUCLEI

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