A study of the synthesis and interrelationships of ribonucleic acids in duck erythrocytes

280

BIOCHIMICA ET BIOPHYSICA ACTA

BBA 95153

A STUDY OF T H E SYNTHESIS AND I N T E R R E L A T I O N S H I P S OF R I B O N U C L E I C ACIDS IN DUCK E R Y T H R O C Y T E S

GAIL P. BRUNS', SIEGMUND FISCHER'* AND BERTRAM A. LO'WY Departments o/ Medicine and Biochemistry, Albert Einstein College o/ Medicine, Yeshiva University, New York, N . Y . (U.S.A,)

(Received July 28th, 1964)

SUMMARY

Duck erythrocytes have served as a source for a number of R N A fractions. In addition to ribosomal and soluble RNA, five R N A fractions have been prepared from a 15 000 ×g residue, containing nuclei and cell membranes. Three of the fractions, constituting about I0 % of the total cellular RNA, exhibited a rapid uptake of labeled precursors. Studies to evaluate the possible role of the rapidly labeled fractions as cytoplasmic RNA precursors by chase and actinomycin-inhibition experiments suggest that a simple nuclear to cytoplasmic relationship may not exist in the duck erythrocyte.

INTRODUCTION The mammalian erythrocyte loses its nucleus and DNA during an early stage of development in the bone marrow 1 and loses its I~NA during maturation of the reticulocyte ~. The mammalian reticulocyte which can synthesize heine 3 and globin 4 and purine nucleotides s is unable to synthesize R N A 6,7, presumably because of the loss of DNA or of R N A polymerase (EC 2.7.7.6), or of both. The avian erythrocyte, in contrast to the mammalian erythrocyte, retains its nuclear structure and DNA throughout its life span in the peripheral circulation yet does not undergo mitosis after the polychromatic normoblast stage of development s . Although amino acid incorporation into protein occurs in normal circulating avian erythrocytes 9, this incorporation is a reflection of the presence of immature cells in the peripheral circulation 1°. This paper describes a study of R N A synthesis in nonmitotic, DNA-containing duck erythrocytes n. * Present address: Laboratory of Physiological Chemistry, University of Louvain, Louvain (Belgium). ** Present address: Department of Virology, University of Chile, School of Medicine, Santiago (Chile). Biochim. Biophys. Acta, 95 (1965) 28o-29o

R1BONUCLEIC ACIDS IN DUCK ERYTHROCYTES

281

MATERIALS

[6-14C]Orotic acid and sodium [l~C]formate were purchased from Volk Radiochemical Company. [8-14C]Adenine was obtained from Schwarz Bioresearch and Calbiochem. Unlabeled purines, pyrimidines, nucleosides and amino acids were obtained from Schwarz Bioresearch and Pabst Laboratories. Pancreatic ribonuclease (EC 2.7.7.16) and spleen phosphodiesterase (EC 3.1.4.1 ) were supplied b y Worthington Biochemical Corporation. Actinomycin D was generously provided b y Merck Sharp and Dohme Research Laboratories.

METHODS

White Long Island ducks, weighing about 2.5 kg each, were injected intramuscularly with acetylphenylhydrazine (15 mg/kg of body weight) on each of four successive days and exsanguinated b y the jugular vein on the sixth day. Blood from the anemic animals was stained b y the new methylene blue procedure and 70-80 % of the cells were observed to contain reticulum or Heinz bodies. The immature erythrocytes with this staining characteristic were predominantly reticulocytes. Blood was collected into sodium heparin solution and centrifuged at 15oo ×g for 15 min at 4 °. Plasma was removed and the cells resuspended in an equal volume of plasma or isotonic Tris-saline buffer as described b y BORSOOK12 but minus amino acids and incubated as described in the tables. In several experiments, blood from normal, untreated ducks was used. Nucleic acids were isolated from the erythrocytes following incubation by one of three procedures.

Procedure A (Fig. I). The incubation suspension was centrifuged at 15oo ×g for 15 min at 4 °. The cells were washed three times with equal volumes of a solution containing o.13 M NaC1, o.oo52 M KC1, o.oo75 MgCI~. The cells were lysed by the addition of one volume of o.005 M MgCl2 containing o.oo6 M fl-mercaptoethanol and b y 3 cycles of freezing and thawing. The lysate was centrifuged at 15 ooo × g for 15 min at 4 ° and the 15 ooo ×g supernatant solution was recentrifuged at lO5 ooo ×g for 2 h in a Spinco Ultracentrifuge Model L. Ribosomal R N A and soluble R N A were isolated from the lO5 ooo × g pellet and from the supernatant solution respectively by the procedure of NIRENBERG AND IV[ATTHAE113.Nucleic acid fractions were prepared from the 15 ooo ×g residue, which contained nuclei and cell membranes, by a modification of the procedure of SIBATANI et al. 1~. The 15 ooo ×g pellet was homogenized in a Waring Blendor for 5 sec in IO volumes of o.I M Tris buffer (pH 7.6) containing 0.005 M MgCI~ and 0.o03 M CaC12. After centrifugation at 15oo ×g for 15 min, the supernatant solution was recentrifuged at lO5 ooo ×g for 2 h. R N A 1 was prepared from the lO5 ooo ×g pellet as described above. The 15oo x g pellet, resuspended in 20 volumes of I IV[NaC1, was homogenized in a Waring Blendor for IO sec, and stirred for 15 h at 4 °. The I ?¢[ NaC1 suspension was centrifuged at 78 ooo ×g for 2 h. DNA could be Biochim. Biophys. Acta, 95 (1965) 28o--29o

282

G. P. BRUNS, S. FISCHER, B. A. I.OWY

RETICULOCYTES (lysale) 15000

x

I RESIDUE

I

SUPERNATANT

I ...... I RESIDUE

I ....... l SUPEiATANT

I

RESIDUE

I

I PHENOLLAYER

AQUEOUS PHASE

RESIDUE

RNA2

I

RESIDUE

I--,o,°

PELLET

, rRNA

SUPERINATANT

.L sRNA

RNA i

I

+ INTERPHASE

I I

i,o.oo.

• I SUPERNATANT PELLET

DNA

$

1

SUPERNATANT

j.

RNA3 RESIDUE F

SUPER~dATANT

•

$

RNA+ RES

$

ATANT

RNA5 Fig. I. Fractionation of nucleic acids of duck erythrocytes.

prepared from the 78 o o o x g supernatant solution. The 78 o o o × g pellet was homogenized in o.oi M Tris buffer (pH 7.8) containing o.oi M magnesium acetate, 0.06 M KC1 and 0.006 M/~-mercaptoethanol and was shaken with an equal volume of 88 % phenol for 30 rain at 25 °. :RNA 2 was precipitated from the aqueous phase obtained after centrifugation at 15oo ×g for 15 rain by the addition of ethanol. The phenol layer plus the interphase was extracted three times with ethanol-ether (4:I), and the resultant pellet, suspended in water, was shaken for 30 rain at 25 °. The suspension was centrifuged at 15oo ×g for 15 rain and RNA a was precipitated from the aqueous phase b y ethanol addition. The 15oo x g pellet was resuspended in water, heated at 65 ° for 5 rain, cooled slowly to 25 ° and centrifuged at 15oo x g for 15 rain. RNA 4 was precipitated from the aqueous phase. The pellet was resuspended in water, heated at 85 ° for 15 rain, cooled slowly to 25 ° and centrifuged at 15oo ×g for 15 min. :RNA 5 was precipitated from the aqueous phase.

Procedure B

Incubation suspensions were washed, the cells were lysed and the lysates were centrifuged at 15 ooo x g as described in Procedure A. :Ribosomal :RNA and soluble RNA were prepared from the 15 ooo x g supernatant solution as described above. The 15 ooo x g pellet, which contained nuclei and cell membranes, was suspended in o.oi M sodium acetate buffer (pH 5.I), and sodium dodecyl sulfate and bentonite were added to a final concentration of 0.5 % and 50 rag% respectively. :RNA was prepared b y the 60 ° phenol extraction procedure described b y SCHERRER AND DARNELL 15.

Biochim. Biophys. Acta, 95 (1965) 28o-29o

RIBONUCLEIC ACIDS IN DUCK ERYTHROCYTES

283

Procedure C Incubation suspensions were washed and the washed cells were lysed with o.oi M sodium acetate buffer (pH 5.I), containing 0.5 % sodium dodecyl sulfate and bentonite (50 rag%). Total RNA was prepared by the method of SCHERRER AND DARNELL 15.

Analysis o[ RNA /ractions RNA concentrations were determined spectrophotometrically (Assom#, 21. 4 cm~mg-1). The samples contained less than 1 % protein as determined by ultraviolet absorption ratios at 260 m#:28o m# of 1.8-2.o. For assay of radioactivity, the samples were precipitated with 7 % perchloric acid and were washed three times with 1 % perchloric acid. The samples were dissolved in 0.2 M NH4OH, and the solutions were assayed with a Nuclear-Chicago gas flow counter with micromil window. Corrections for self-absorption were applied. Sucrose-density-gradient centrifugations were performed in a SW 25.1 rotor using a 5 % to 20 % linear gradient. Sucrose solutions were prepared in o.oI M sodium acetate buffer (pH 5.I), containing 0.05 M NaC1 and o.oooi MgC12. Centrifugation was performed at 22 500 rev./min for 11.25 h. Ultraviolet absorption at 260 m/~ was determined on I-ml fractions. Base ratios of nucleic acid samples were determined on formic acid hydrolysates or on hydrolysates following pancreatic ribonuclease and spleen phosphodiesterase digestion. Hydrolysates prepared in the same way were employed for localization of the label, which was found to be present in the anticipated purine or pyrimidine bases or nucleotides. RESULTS

Base analyses of ribosomal and soluble RNA and of the several RNA fractions derived from the 15 ooo ×g pellet indicated that they were all of a high guanine plus cytosine type (Table I). None of the fractions derived from the 15 o o o × g residue resembled the base composition of the DNA of the duck erythrocyte. The percentage yields indicate that the RNA derived from the phenol-interphase in Procedure A contains 9.8 % of the total isolated RNA. In Procedure B the yield of RNA obtained from the 15 ooo ×g pellet was approx. 33 % of the total RNA isolated. Sucrose-density-gradient centrifugation profiles of ribosomal and soluble RNA fractions showed ultraviolet absorption in regions expected for 23-S, I6-S and 4-S components of ribosomal RNA and for a 4-S component in soluble RNA. The RNA fractions isolated from the I5 ooo ×g pellet by Procedure A showed one broad peak of about lO-2O S. No stimulation of amino acid incorporation into acid precipitable material could be demonstrated with these fractions in a rabbit-reticulocyte cell free system le. RNA derived from the 15 ooo ×g pellet by Procedure B showed 23-S, I6-S, and 4-S components. Total RNA isolated from the cells by Procedure C showed similar 23-S, I6-S, and 4-S components. Biochim. Biophys. Acta, 95 (1965) 28o-29o

284

G.P.

TABLE

I

PURINE IMMATURE

AND

Average RNA.

per cent yield based on 14 experiments;

PYRIMIDINE BASE DUCK ERYTHROCYTES

Fraction

RATIOS

AND

PERCENTAGE

Number of Percentage samples Guanine Adenine

Ribosomal RNA Soluble R N A RNA1 RNAz RNA a RNA 4 RNAs DNA

YIELD

BRUNS, S. FISCHER, B. A. I,OWV

OF

I~NA

FRACTIONS

ioo ml cells per experiment;

Cytosine

PREPARED

average

FROM

total yield -- 84. 4 nlg

Uracil

Percentage Purine Guanine Pyri~nidine +cytosine

Yield ( °~o)

41.8 26.5 9-4 12. 4 3.8 2.8 3.2

18 18 4 4 I2 14 9

27.4 26.0 27.8 28.4 29.7 28. 5 28.2

20.4 20. 5 20.8 20.5 22-3 21.6 22.9

28-3 29.4 28. i 29.2 26.7 28. I 25. 4

24-3 24.0 23-3 21.9 21.3 21.8 23. 5

55.7 55.4 55.9 57.6 56.4 56.6 53.6

0.91 0.87 0.95 o.96 I.IO I.OO 1.o6

3

21.2

28. 5

22.3

28.1

43.5

0-99

The specific activities of RNA fractions isolated b y Procedure A from immature duck erythrocytes incubated with sodium E14C]formate for several time periods are shown in Table II. The specific activities fall into three groups. Ribosomal RNA and R N A 1 have low specific activities while the specific activities of soluble RNA and ~RNA2 are higher. The fractions obtained from the phenol plus interphase TABLE INCORPORATION

II OF

SODIUM

~I4C]FORMATE

INTO

RNA

FRACTIONS

OF

IMMATURE

DUCK

ERYTHROCYTES in vitro Cells were i n c u b a t e d in p l a s m a at 3°° w i t h s o d i u m E14C]formate (2o/zC), 20 #moles; glycine, 20/*moles; NaHCOa, 20/,moles; L-aspartic acid, 20/,moles; L-glutamine, 4 °/~moles; and glucose, 35 m g % / h .

Fraction

Incubation time Experiment I 2o rain

Experiment I I 60 rain

2h

4h

17 89 26 84 4 °0 900 3 °0

37 230 37 14o 650 1490 112o

Counts/rain~rag R N A Ribosomal RNA Soluble R N A RNA1 RNA2 RNAa RNA 4 RNA5

6 16 3 9 51 lO 5 --

27 45 8 28 245 460 533

layer were consistently of highest specific activity. There was a lO-2O fold increase in the specific activities of all fractions between the 2o-min and 4-h incubation periods. In an experiment with ES-14C]adenine a similar pattern of incorporation was found in the several R N A fractions as shown in Table I I I . At each time period the RNA obtained from the phenol plus interphase layer was ~ound to be most highly Biochim. Biophys. Acta, 95 (1965) 28o-29o

285

RIBONUCLEIC ACIDS IN DUCK ERYTHROCYTES TABLE III INCORPORATION

OF

[8-14C]ADENINE

INTO

RNA

FRACTIONS

OF

IMMATURE

DUCK

ERYTHROCYTES

in vitro

Cells were incubated in plasma at 3°0 with ~8-~4C]adenine (20/zC), 1.4/zmoles; guanosine, uracil, cytosine, thymine 2.8 #moles each; and glucose, 35 mg%/h. Fraction

Incubation time Expt. 1

Ribosomal RNA Soluble RNA RNA 1 RNA2 RNA 3 RNA 4 RNA s

Expt. I I

20 rain

60 rain

Counts/rain/rag

RNA

14 46 18 53 500 480 426

33 84 18 95 655 127o 658

Expt. II1

2h

4h

18 h

33 176 48 191 185o 2065 lO8O

53 4o8 56 233 lO95 321o 1995

172 57° 62 162 735 1328 2246

labeled. A f t e r i n c u b a t i o n for 18 h, the ribosomal an d soluble R N A fractions were m o r e h i g h l y labeled r e l a t i v e to th e phenol plus i n t e l p h a s e fractions t h a n at earlier t i m e periods. T h e I8-h sample was found to be free of bacterial c o n t a m i n a t i o n . T h e effect of cell i m m a t u r i t y u p o n t h e p a t t e r n of R N A synthesis in duck e r y t h r o c y t e s was e x a m i n e d b y c o m p a r i n g th e specific activities of R N A fractions d e r i v e d from e r y t h r o c y t e s of n o r m a l blood w i t h those of i m m a t u r e cells p r o d u c e d b y a c e t y l p h e n y l h y d r a z i n e t r e a t m e n t . T h e d a t a of Tab l e I V indicate t h a t t h e specific TABLE IV INCORPORATION OF [8-14C]ADENINE INTO RNA FRACTIONS OF IMMATURE BLOOD CELLS INCUBATED I N P L A S M A A N D B U F F E R in vitro

AND NORMAL

DUCK-RED

Cells were incubated in plasma or Tris-saline buffer (pH 7.6) (ref. 12) at 3°° for 2 h with [8-14C]adenine, (2o#C) o.37#moles; guanosine, uracil, cytosine, o.74/~mole each; and glucose, 35 mg%/h. Fraction

d cetylphenylhydrazine A cetylphenylhydrazine Normal plasma bu[#r plasma

Normal buffer

Counts]min/mg R N A

Ribosomal RNA Soluble RNA RNA 1 RNA 2 RNAs RNA4 RNA5

13 55 17 IOI 235 683 1132

9 49 27 II I 206 838 lO72

13 41 83 71 64 603 968

Ii 23 84 81 49 496 363

activities of t h e several R N A fractions o b t a i n e d from b o t h t y p e s of cell p r e p a r a t i o n s were similar, a l t h o u g h the a m o u n t of R N A isolated from t h e e r y t h r o c y t e s of n o r m a l blood was considerably less t h a n t h a t isolated from t h e i m m a t u r e cells. T h e d a t a also i n d i c a t e t h a t t h e i n c o r p o r a t i o n of [8-14CJadenine occurs e q u a l l y well in a buffer a n d in plasma. Biochim. Biophys. Acta, 95 (1965) 28o-29o

286

G.P.

BRUNS, S. FISCHER, 13. A.

L()WY

T h e c o n t r i b u t i o n of w h i t e b l o o d cells t o t h e e x t e n t of l a b e l i n g of t h e R N A w a s also i n v e s t i g a t e d ( T a b l e V). T h e a d d i t i o n of a t h r e e - f o l d e x c e s s of w h i t e b l o o d cells t o e i t h e r t y p e of cell p r e p a r a t i o n d i d n o t s i g n i f i c a n t l y a l t e r t h e s p e c i f i c a c t i v i t i e s of t h e s e v e r a l R N A f r a c t i o n s . TABLE V EFFECT OF ADDED WHITE BLOOD CELLS ON INCORPORATION OF ~8-14C]ADENINE T I O N S O F I M M A T U R E A N D N O R M A L D U C K R E D B L O O D C E L L S in vitro

INTO R~A

FRAC-

Cells were incubated in plasma at 4 °° for 2 h with E8-14C]adenine (20/~C), 1.4/zmoles; guanosine, uridine, cytidine, 2.8/zmoles each; and glucose, 35 mg%/h. Fraction

d cetylphenylhyclrazine, i.o ~o white blood cells

d cetylphenylhydrazine, 2.8 % white blood cells

Normal, 0. 4 % white blood cells

Normal, 1. 4 % white blood cells

13 47 45 63 7° 568 676

I6 5° 38 74 117 727 --

Counts~miD~rag R N A

Ribosomal RNA Soluble RNA RNA 1 RNA 2 RNA 8 RNA 4 RNA5

15 57 12 89 IIO 466 97 °

16 47 ii 93 152 647 795

T h e c o n s i s t e n t l y h i g h s p e c i f i c a c t i v i t i e s of t h e p h e n o l p l u s i n t e r p h a s e f r a c t i o n s suggested a possible precursor relationship to other RNA fractions. To investigate this relationship, chase experiments were performed. A representative chase exp e r i m e n t is s h o w n i n T a b l e V I . h n m a t u r e d u c k cells w e r e i n c u b a t e d for 3o miD i n t h e p r e s e n c e of [6-14C]orotic acid. T h e i n c u b a t i o n w a s t h e n e i t h e r t e r m i n a t e d ( C o l u m n I ) , a l l o w e d t o c o n t i n u e i n t h e p r e s e n c e of g l u c o s e ( C o l u m n 2), or c o n t i n u e d T A B L E VI ATTEMPTED INCUBATION

C H A S E O F L A B E L E D RNA F R A C T I O N S WITH [6-1*CDOROTIC ACID

IN IMMATURE

DUCK

ERYTHROCYTES

in vitro:

Cells were incubated in plasma at 3 °0 with: (a) [6A4C]orotic acid (20/zC), 6. 7/zmoles; guanosine and adenine, 3.3 t tm°les each; glucose, 35 mg%/h, or (b) glucose, 35 mg%/h, or (c) cytosine, uracil, guanosine, adenine, 3.3/~moles each; glucose, 35 mg%/h/incubation. (I)

(2)

(3)

(4)

Initial contains Final contains

3 ° mid (a ) --

3 ° miD (a ) 9o miD (b)

3 ° miD (a ) 9o miD (c)

3 ° miD (b ) 3 ° mid (a)

Fraction

Counts/miD/rag R N A

Ribosomal RNA Soluble RNA RNA 1 RNA~ RNA~ RNA 4 RNA 5

i io i 38 146 37 °

5 33 13 202 1076 1627

i 7 2 46 142 325

.

3 36 16 204 995 1473 .

.

Biochim. Biophys. Acta, 95 (1965) 28o-29o

.

287

RIBONUCLE1C ACIDS IN DUCK ERYTHROCYTES

in the presence of a IO fold excess of cytosine, uracil, adenine and guanosine (Column 3). The data of Columns 2 and 3 show that incorporation continued during the final incubation period. No detectable increase in the specific activities of ribosomal or soluble R N A or of RNA 1 or RNA~ was observed as a consequence of the chase conditions nor was a decrease in the specific activities observed in the phenol plus interphase RNA fractions. Additional evidence for the viability of the erythrocytes during at least a portion of the final incubation period is shown in Column 4 where an aliquot of cells was incubated initially for 30 min in the absence of a labeled precursor, followed b y a short incubation period with labeled precursor. Failure to observe a transfer of label during the chase period in this experiment m a y have been due to the continued presence of labeled orotic acid and of labeled pyrimidine nucleotides formed from the orotic acid. In order to reduce the amount of labeled orotic acid in the plasma during the final incubation period, another experiment was carried out in which the cells were centrifuged after the initial incubation period. Fresh plasma containing glucose or glucose plus uracil, cytosine, adenine and guanosine was added prior to the final incubation. Although this procedure removed 73 % of the initial label, an effective chase was not observed. The antibiotic actinomycin D has been shown to inhibit DNA-primed RNA synthesis17,18. The effect of this antibiotic on R N A synthesis in the immature duck erythrocyte was investigated. Total RNA was prepared following incubation with [6J4C]orotic acid. I t can be seen from Fig. 2 that extensive incorporation of [6-~4C]-

o

Actinomycin conc.

- -

JJg/ml

tOO0 800

~

400 ~ 200

20.

0 INCUBATION TIME (hours)

Fig. 2. T h e effect of a c t i n o m y c i n c o n c e n t r a t i o n on t h e i n c o r p o r a t i o n of [6-14C]orotic acid i n t o t o t a l R N A of i m m a t u r e d u c k e r y t h r o c y t e s .

orotic acid into total RNA occurred during an 8-h incubation period in the absence of actinomycin D. Actinomycin D, at a concentration of 20 #g/ml of cells, resulted in a rapid and extensive inhibition of RNA labeling. Higher concentrations of actinomycin D were slightly more inhibitory. The inhibition of R N A synthesis b y actinomycin D has been a useful tool for the study of possible precursor relationships among the several RNA fractions of the cell. The results of such a study are indicated in Table VII. I m m a t u r e duck erythrocytes were incubated with [6-14C]orotic acid for 4 h. The incubation was then either terminated (Column I), continued for 12 h in the presence of glucose (Column 2), or continued in the presence of glucose and actinomycin D at a concentration of 50/~g/ml of cells (Column 3)- Ribosomal and soluble RNA were isolated from the Biochim. Biophys. Acla, 95 (1965) 28o-29o

2~

G.P.

BRUNS, S. FISCHER, 13. A. I,()WY

TABLE VII EFFECT OF ACTINOMYCIN ON INCORPORATION OF [6-14CIOROTIC ACID INTO R N A OF DUCKRED BLOOD CELLS in vilro Cells were i n c u b a t e d in p l a s m a a t 37 ° w i t h [6-14C]orotic a c i d (2o/zC), 6 . 7 # m o l e s ; g u a n o s i n e , a d e n i n e , c y t i d i n e , 6. 7 / z m o l e s each; glucose, 35 m g % / h i n c u b a t i o n ; a n d penicillin, i o mg. Act i n o m y c i n (5 ° / z g / m l of ceils) w a s p r e s e n t in Vessel 4 d u r i n g t h e e n t i r e i n c u b a t i o n p e r i o d a n d i n Vessel 3 d u r i n g t h e final 12-h i n c u b a t i o n period.

Initial

(i) 4h

(2) 4 h

(3) 4 h

(4) 4h

d ctinomycin Final

--

12 h

12 h

12 h

A clinomycin Fraction

Counts/min/mg R N A

Ribosomal Soluble Nuclear

23 123 56

26 186 lO 3

18 99 56

i 24 14

cytoplasm and total RNA was isolated from the 15 ooo ×g fraction. The data of Column 2 indicate that RNA synthesis continued during the final incubation in the absence of actinomycin D. The data of Column 3 show that in the presence of actinomycin D no further labeling of R N A occurred during the final incubation period. Neither a significant increase in the specific activities of ribosomal or soluble RNA, nor significant decrease in the specific activity of nuclear RNA was observed following the incubation in the presence of actinomycin (Column 3 vs. Column I). These results do not support a simple direct precursor relationship between nuclear and cytoplasmic R N A fractions in the duck erythrocyte.

DISCUSSION

By applying a modification of the procedure of SIBATANI et al. 14 to cluck red blood cells, it was possible to prepare, in addition to ribosomal and soluble RNA fractions from the cell cytoplasm, five additional RNA fractions from the 15 ooo x g residue. Three of the five fractions prepared from the residue were present in the phenol plus interphase layer formed during the extraction procedure and these fractions exhibited rapid uptake of labeled precursors. Although the 15 o o o × g residue m a y have contained some undisrupted cells, tile bulk of the residue consisted of nuclei and cell membranes. Similar labeling patterns were obtained in an alternate procedure in which a nuclei-enriched fraction was prepared from immature duck erythrocytes b y centrifugation at 8 o o × g and subsequent recentrifugation in an isotonic sucrose-salt solutionlt Although three discrete fractions were prepared from the phenol plus interphase material, their individualities m a y be fortuitous. Indeed, similarities are suggested b y their specific activities and base compositions. I t is possible, however, t h a t each fraction is itself heterogenous, and in fact, that the heterogeneity conceals a small DNA-like IRNA fraction. Alternatively, the inability to detect a comparable fraction in immature duck cells m a y be related to the predominantly non-mitotic Biochim. Biophys. Mcta, 95 (1965) 28o-29 o

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nature of this cell population. In other species, heterogeneity of nuclear R N A 19-22 and the isolation of an R N A fraction resembling DNA in its base composition have been r e p o r t e d 14,~°,23,~4. The lack of observed transfer of labeled R N A from the cell nucleus to the cytoplasm in the nucleated duck erythrocyte m a y indicate that the relationship of nuclear RNA to cytoplasmic R N A is not a simple one in these non-mitotic cells. Evidence for the role of nuclear RNA as a cytoplasmic RNA precursor has been presented b y a number of investigators in cell culture experiments based on the transfer of radioautographically detectable grain counts from the nucleus to the cytoplasm, in chase experiments 25-27 or in experiments with actinomycin 27. SCHERRER et al.15, ~° have presented evidence for the transfer of newly synthesized 45-S and 35-S RNA to 23-S and I6-S forms in H e L a cells, both in the presence and absence of actinomycin. Similar data have been presented by PERRY27 and by TAMAOKI AND 1V[UELLER2s. In more recent investigations GIRARD et al. 29 have shown that although transfer of labeled RNA from the H e L a cell nucleus to cytoplasmic polysomes does occur, the amount of R N A transferred is limited in the presence of actinomycin. The data of LEVY3° also suggest that actinomycin m a y inhibit the transfer of R N A from nucleus to cytoplasm. DNA-like RNA has been found in the cytoplasm of mammalian liver by HOYER et al. al and GEORGIEV et al. 24. These observations are consistent with the concept of a transfer of 1RNA from the nucleus to the cytoplasm. HARRIS et al. 32 have presented data suggesting that the rapidly labeled nuclear RNA m a y not be transferred intact to cytoplasmic RNA during an extended chase period in H e L a cells, in culture, but that the rapidly labeled nuclear RNA m a y be degraded in the nucleus b y a polynucleotide phosphorylase-like enzyme, with reutilization of the degradation products a3. ~I-IIATT19, in studies of RNA metabolism in regenerating rat liver, found that while the newly synthesized nuclear RNA was transferred from 45-S to 23-S and I6-S forms in the nucleus, incorporation of labeled precursors into cytoplasmic RNA was first detectable in 4-S form and subsequently appeared in I6-S and 23-S forms. A number of investigators have suggested that the transfer of nuclear IRNA to the cytoplasm m a y be associated with alterations in size distribution and base composition of the RNA transferred26,29, 32. In a rapidly dividing cell in which a doubling of the nucleic acid content must occur every few hours, transfer of ribosomal, soluble and informational RNA molecules from the nucleus to the cytoplasm m a y be detectable. It has been suggested that transfer of nuclear R N A to the cytoplasm m a y occur continuonsly~, ~5 or m a y be associated with the dissolution of the nuclear membrane during mitosis35, 3s. In a non-mitotic cell, however, new ribosomal and soluble R N A synthesis m a y not be required and the amount of informational R N A transfer m a y be too small to be detected by these techniques. In the rabbit reticulocyte, a cell in which the messenger RNA for hemoglobin synthesis appears to be stable6, 7, messenger RNA molecules m a y become components of the cytoplasm prior to the loss of the cell nucleus a7. In the type of immature duck cell utilized here, in which actinomycin has no effect on protein synthesis ", the messenger RNA for hemoglobin synthesis m a y also be stable. If so, transfer of * Unpublished observation of A. GRAYZELAND I. M. LONDON. Biochim. B i o p h y s . . d c t a ,

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messenger RNA from the nucleus to the cytoplasm may be minimal. The rapidly labeled nuclear RNA in the duck erythrocyte may, therefore, be concerned with some as yet unknown role or vestigial capacity of the nucleus.

ACKNOWLEDGEMENTS

The authors wish to acknowledge the technical assistance of T. KOSTOMAJ and J. FUHR. The work was supported in part by a United States Public Health Service Career Development Award (K3-GM-3o59) to B. A. LowY, and by research grants from the U.S.P.H.S. (A-28o3, H-2655) and the Office of Naval Research (Nonr 4094). REFERENCES I 2 3 4 5 6 7 8 9 IO II 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 3° 31 32 33 34 35 36 37

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