Ribonucleic acid synthesis in relation to precursor pools in regenerating rat liver

BIOCHIMICA

ET BIOPHYSICA

491

ACTA

BBA 96098

RIBONUCLEIC

ACID SYNTHESIS

IN REGENERATING

N. L. R. BUCHER

AND

IN RELATION

August z6th,

POOLS

RAT LIVER

M. N. SWAFFIELD

John Collins Warren Laboratories of the Huntington Memorial the Massachusetts General Hospital, Boston, Mass. (U.S.A.) (Received

TO PRECURSOR

Hospital of Harvard University at

1968)

SUMMARY

The rate of RNA synthesis during the early stages of hepatic regeneration has been evaluated with radioactive precursor techniques. Since RNA labeling is influenced by alterations in effective liver mass, size of endogenous precursor pools, and possibly other factors as well as by regenerative activity, the experimental procedures were modified to permit estimation of RNA biosynthetic rates in terms of m,umoles of nucleoside triphosphate incorporated - conditions which permitted more meaningful comparisons than heretofore among rats differing in nutritional status, size of liver cell complement, or dose of labeled precursor. When regenerating liver was examined under these new conditions, we found that the endogenous UTP and CTP pools expanded by 50 y0 or more within 3 h after partial hepatectomy, and the rate of RNA synthesis, after a negligible depression at time zero, rose by 50-100 “i; at 3-6 h and then remained level for an additional 6 h. RNA content followed similar trends. Sham operation caused minor transient rises in pool size, biosynthetic rate and RNA content.

INTRODUCTION

In rat liver induced to regenerate by partial hepatectomy, there is an early increase in the rate of incorporation of labeled precursors into RNA1-8. The extent of the rise may or may not be proportional to the actual rate of RNA synthesis, depending upon other factors that also affect the rate at which labeled precursors enter RNA. These include especially (I) reduction of effective liver mass by the surgery (gradually reversed by the regeneration), and (2) enlargement of the endogenous precursor pools, the former tending to increase and the latter to decrease the amount of radioactivity incorporateds-‘I. Prior studies in our own and other laboratories conducted without due regard for these factors require reappraisal. The necessity for this was underscored by unresolved discrepancies arising when we attempted to compare fed and fasting rats and those with whole or partially resected livers. The present study reexamines RNA biosynthesis under conditions tailored to permit adjustment for changes in liver mass and in the quantity of endogenous RNA Biochim.

Biophys.

Ada,

174 (1969) 49r-502

N. I,. I<. BUCHEK,

402

M. S. SWAFFIELI~

precursors. Although additional sources of variability may remain, the results s11ow greater consistency under varied conditions than heretofore, and hence may more nearly approximate the actual rates of RNA synthesis in 7!1110.Only the rates of total RNA synthesis are dealt with here, but we assume that the same principles will apply to individual RNA species.

MATERIALS

AND

METHODS

[6J4C]Orotic acid was obtained from Nuclear Chicago Corp., Des Plaines,Ill.; [2J4C]cytidine from New England Nuclear Corp., Boston, Mass.; and Cab-O-!%1 from the Cabot Corp., Boston, Mass. Female CD rats, supplied at 21 days of age by the Charles River Breeding Laboratories, Inc., N. Wilmington, Mass., were maintained under standard environmental conditions4 until used at 32-35 days of age. For 12.h before receiving radioactive compounds, animals were either denied food or continued on ad lib&m feeding as indicated. Experimental animals were subjected to partial hepatectomy with resection of approx. 68 oh of the liver, and given radioactive precursors at specified intervals thereafter. Control animals were either (I) normal or (2) sham hepatectomized, with livers intact, given the labeled precursors at times corresponding to the experimental animals or (3) newly hepatectomized, given the labeled precursor immediately after partial hepatectomy at “time zero,” presumably before regeneration could get under way. All operative procedures, including partial hepatectomies, injections, infusions, and taking of liver samples, were carried out while the rats were anesthetized in an ether-100 o/o0, atmosphere lo. Labeled compounds in physiological saline solution were either injected or continuously infused into the tail vein. For the latter procedure, a motor-driven syringe (Harvard Apparatus Co., Millis, Mass. infusion pump) connected to a scalp vein infusion set (No. 23 gauge needle) delivered 0.08 ml/min. At the end of a 15 min infusion the liver samples were instantly frozen in sit% by compression between flat-ended metal tongs precooled in liquid nitrogen1°,12.

Homogenates

The frozen liver was weighed, pulverized and then homogenized in ice cold 0.8 M HClO,, 7 ml/g (ref. IO). Duplicate 300 &and zoo-$ aliquants of homogenate were then set aside for nucleic acid determinations, and the remainder centrifuged. A known volume of the supernatant was adjusted to neutrality with r/roth volume of 7 M KOH, and the resulting KClO, precipitate discarded, leaving a clear supernatant fraction which contained the acid-soluble components. UTP and CTP were isolated by thin-layer chromatography either directly from the supernatant or after it was concentrated by lyophilization as described below.

DNA content and radioactivity

in RNA

per mg DNA

The acid-insoluble material in the 300~~1 aliquants of homogenate was extracted with 1.0 ml of 5 o/ohot trichloroacetic acid after several washings with cold trichloroacetic acid, and the DNA content determined by the diphenylamine procedure13. In Biochim.

Biophys.

Acta,

174

(1969)

4g1-502

RNA

SYNTHESIS

AND

PRECURSOR

POOLS

493

addition, separate zoo-p1 portions of each trichloroacetic acid extract were assayed for radioactivity in vials containing a mixture of Cab-O-S1 and scintillation fluid14J5 @us 200 ,d of 4 M NH,OH in a Model 314 Packard TriCarb liquid scintillation spectrometer. With the short incorporation times employed no radioactive label was detectable in DNA, and therefore the results could be expressed as disintegrations/min in RNA relative to the DNA content of the liver (see discussion). RNA

content and specific activity

The acid-insoluble material in the zoo-,ul aliquants of homogenate was washed with cold HClO, and with 0.1 M sodium acetate in g5 o/0 ethanol, then subjected to alkaline hydrolysis, acidified16, and the soluble fraction assayed for RNA by the orcino1 methodl’. The RNA content relative to DNAwas thus obtained (RNA/DNAratio), and from this and the previous data the disintegrations/min per mg RNA (RNA spe cific activity). Radioactivity

in UTP OYCTP

per

mg

DNA

Duplicate IOO-~1aliquants of KClO, supernatant were spotted on a poly(ethyleneimine)-impregnated cellulose thin-layer plastic plate and chromatographed in I M LiCl saturated with boric acid (pH 7) as described previouslylO*ls. In rats fed ad lib&m, with livers rich in glycogen, it was necessary to run 0.2 M LiCl to 2 cm above the origin and spot only 50-~1 aliquants, applying the extract very slowly to the wet platels. After chromatography the plates were desalted with methanol, and the nucleoside triphosphates located by radioautographylO,r*. The UTP and CTP spots were cut out with scissors and eluted quantitatively by floating face down in 500,ul of 4 M NH,OH for 45 min at room temperature in a sealed container. Aliquants of eluate were withdrawn after gentle mixing and counted as above; counting vials always contained a total volume of 400 ~1 of sample in 4 M NH,OH and II ml of scintillation fluid mixture. Since the volumes of aliquants were all known, the disintegrations/min in UTP and CTP relative to DNA could be calculated.

Specific activity of UTP and CTP

Lyophilized residue from 1-1.5 ml of KClO, supernatant was taken up in 300 ~1 of water and streaked in duplicate for 7 cm along the origin of a thin-layer plate (50 ~1 superimposed 3 times for each streak) and chromatographed as above. The bands containing UTP or CTP were cut out and eluted for 45 min in ice cold I M triethylammonium carbonate. The eluate was filtered with suction in a micro Buchner funnel through W’hatman No. I paper and lyophilized until all of the triethylammonium carbonate was eliminated. The residue from each streak was taken up in water, spotted on a second thin-layer plate and chromatographed in two dimensions; the solvent for the first dimension was LiCl-boric acid (pH 7) for UTP and 1.0 M LiCl1.5 M acetate buffer (pH 4.4) for CTP. The respective plates were desalted with methanol and chromatographed in the second dimension in 0.6 M (NH,),SO, (ref. 18). The UTP and CTP spots were cut out and eluted in 4 M NH,OH, aliquants assayed spectrophotometrically as previously describedlO and counted in scintillation vials as above, giving the values for disintegrations/min per pmole of UTP and CTP. Biochim.

Biophys.

Acta,

174

(1969)

4g1-=joz

h’. L.

494

Ii. BUCHER,

hf. N. SWAFFIELD

UTP and CTP pooh The sizes of the endogenous in UTP

(or CTP)/mg

pools were calculated

DNA and the UTP

(or CTP)

from the disintegrations/min specific

activity

as described

previouslyiO.

RESULTS

AND

LlISCUSSION

The endogenous order to become thereby

pools through

incorporated

which initial

into RNA

diluting the tracer molecules

enlarge

labeled

and diminishing

difficulty

we determined

the specific activities

constitute

the immediate

labeled precursors

precursors

as regeneration

must pass in

gets under

RNA labelingll.

of the nucleoside

way,

To avoid this

triphosphates

that

of RNA - in the present study, UTP and

CTP. If it is assumed that the labeled molecules entering RNA during the incorporation period remain in RNA, and if the specific activity of the UTP (or CTP) during this time rises linearly, disintegrationslmin + UTP

we can estimate

in RNA/unit

the biosynthetic

of tissue

,umoles of UTP --- into RNA/unit

(or CTP) disintegrations/min/,umole

The initial

precursors

rate as follows:

used in the present

study

(or CTP) incorporated of tissue/unit of time

were either

[6-l*C]orotic

acid or

[G4C]cytidine. With the [14C]orotic acid, it sufficed to examine only the UTP pools because, although the liver readily converts this precursor into both UTP and CTP, the latter

labels far more slowly, and when incorporation

only negligible

activity4.

When

[14C]cytidine

alone is labeled.

Since normally

as triphosphates,

it is often satisfactory

mono- plus di- ply In the present

70-80

triphosphate

instance,

however,

is the initial

yO of the endogenous

times are short, precursor,

nucleotides

to measure only the activity

pools after hydrolysis we carried

acquires

the CTP pool are present

of the combined

to the monophosphate

out the more troublesome

form.

alternative

of determining the activity of the triphosphates alone because of the possibility that the rapid readjustments of metabolism during regeneration might cause disproportionate expansion of the mono- or diphosphate pools (as, for example, through creased breakdown of UDP sugars or RNAls). Special precautions were required measuring instability,

infor

the triphosphate pools as they exist in vivo because of their biological leading to extraordinarily rapid breakdown under hypoxic conditions.

This could be minimized

by supplemental

oxygen

during anesthesia

and by freezing

the liver in situ, measures previously shown to be effectivelO. The liver deficit created by partial hepatectomy results in an increased incorporation of labeled erotic acid into RNA, observable immediately after operation. This is attributable not to regenerative activity, which has scarcely begun, but rather to the reduced complement of liver cells in the partially hepatectomized animal, so that more labeled precursor is available per cell sJ”. The effect is especially pronounced in the case of erotic acid which is taken up by the liver with great avidity. The “newly hepatectomized” controls were introduced in the present study to show that the modified methods now employed do yield similar biosynthetic rates in animals with different amounts of liver. To determine the appropriate means of administering the labeled precursor we compared injection and continuous infusion of [14C]orotic acid (Figs. IA and IB). Biochim.

Biophys.

Acta,

174

(1969)

4g1-5oz

RNA SYNTHESIS AND PRECURSOR POOLS

495

Fig. r. In vavo rate of UTP labeling in liver relative to DNA content following either intravenous injection or continuous intravenous infusion of [6-%]orotic acid. All rats denied food for 12 h before erotic acid administration. A. Dose injected during approx. IO set: 0.12 ,umole = 0.5 ,& (specific activity 4.2 @/@mole). Data previously reportedO: each point is an analysis of pooled livers of 3 rats, except the “newly hepatectomized” point which is an average value from IO rats. B. Dose infused during 15 min: 0.1 pmole = 3.0,uC (specific activity, 30$/pmole). 1-3 rats per point. O- - -0, Normal controls; 0. . .V, sham-hepatectomized controls, rats subjected to surgery 45 min before receiving erotic acid; o-0, newly hepatectomized controls: rats partially hepatectomized immediately before receiving erotic acid; +-+, regenerating livers: rats partially hepatectomized 45 min before receiving erotic acid.

Fig. IB shows that a IS-mm infusion resulted in a steady linear rate of conversion of the precursor into UTP. This contrasts with the non-linear rate of UTP labeling that occurred during the 15 min when the same total dose of erotic acid (0.1 ,umole) was given within a few seconds by injection (Fig. IA). In the latter case, the UTPlabeling curves were linear only during the first I-Z min, a period when the endogenous pools were flooded with an excess of erotic acid. The similarity of the curves for control and 45-min regenerating livers during this critical linear phase implies that these livers actually differ little from each other in rate of UTP formation (Fig. IA). The subsequent divergence of the curves is attributable to the more rapid exhaustion of the supply of labeled precursor by the animals with intact livers which have a several-fold larger complement of liver cells to utilize it (Fig. IA). The greater UTP labeling in the newly hepatectomized rats in Figs. IA and B is likewise attributable to the reduction of effective liver mass by the partial hepatectomy, resulting in a more abundant supply of labeled erotic acid for each liver cells. Since the means of correcting for this source of error require a linear rate of UTP labeling, and since incorporation times of I-Z min are technically difficult to manage with requisite precision following an injection, we chose the more feasible alternative of prolonging the linear labeling rate by continuous infusion of the tagged precursor (Fig. IB). Adequate labeling of UTP and RNA was thus obtained under conditions suitable for calculating the rate of RNA synthesis on the basis of average specific activity of UTP during the incorporation period, as described above. The results of labeling experiments are generally expressed in either of two ways: as rate of increase in specific activity of the product (e.g., disintegrationslmin per mg of RNA), or as amount of radioactivity entering RNA per unit of liver (e.g., counts/min in RNA per g of liver, or per mg of protien or DNA). We have previously Biochim.

Biophys.

Acta,

174 (1969)

4g1-502

S. L. R. RUCHER,

490 expressed

a preference

for DNA as a standard

of reference

because

M. X. SWAFFIELD

during the first

half day of regeneration it remains constant while other constituents change2. The expression “counts/min in RNA per mg DNA” is essentially like “counts/min in RNA per average liver cell.” Further ing the rate of RNA synthesis

information

can be derived from the data by express-

in terms of the amount

of UTP incorporated

as men-

tioned at the outset. The main parameters that affect the several ways of expressing RNA formation are (I) the amount of endogenous RNA present and (2) the specific activity

of its immediate

labeled

precursor,

UTP.

The UTP

specific

activity

is in

turn affected by (a) the size of the endogenous UTP pool, (b) the amount of liver present, and (c) its capacity for taking up and converting exogenous erotic acid to UTP. These effects are illustrated in Fig. 2, which shows data from 6 rats fed ad UTP,

I

A

I

C

B

RNA

D

E

F

-,

G

H

Fig. z. Incorporation of [W*C]orotic acid into UTP and RNA, and UTP and RNA content of livers of normal or partially hepatectomized rats fed ad Zibitum. Orotic acid dosage as in Fig. IB; liver samples taken after 15 min of infusion. Solid bars represent 2 normal, finely striped bars 2 newly hepatectomized, and segmented bars 2 12-h regenerating livers. Labeling data are expressed in various ways (see text) as follows: (A) Specific activity of UTP (disint./min x IO-~ per pmole); rate at which radioactivity enters a unit of UTP. (B) Rate at which a unit cf liver tissue converts [Wlorotic acid into UTP (disint./min x IO+ per mg DNA). (C) Size of endogenous UTP pool: UTP content (nmoles/mg DNA). (D) Specific activity of RNA (disint./min x IO-~ per mg); rate at which radioactivity enters a unit of RNA. (E) Rate at which UTP molecules enter a unit of RNA (nmoles UTP --f RNA per mg RNA). (F) Rate at which a unit of liver tissue incorporates radioactivity into its RNA (RNA disint./min x IO-~ per mg DNA). (G) Rate at which a unit of liver tissue incorporates UTP molecules into its RNA (nmoles UTP --f RNA per mg DNA). (H) RNA content of a unit of liver tissue (RNA/DNA).

libitztlri - z normal, 2 newly hepatectomized, and 2 with 12-h regenerating livers. The behavior of their UTP pools is shown in Columns A, B, and C. In these animals the rate at which liver cells produced labeled UTP (Column B) was above normal in both newly hepatectomized and 12-h regenerating livers because of their reduced tissue mass (more [14C]orotic acid available per cell). The specific activity of the UTP (Column A) reflected the same phenomenon, except that in the 12-h regenerating livers, the value was slightly lower than in the newly hepatectomized animals due B&him.

Biophys.

Acta.

174

(1969)

491-502

RNA

SYNTHESIS AND PRECURSOR POOLS

497

to the small increase in size of the endogenous UTP pool at 12 h of regeneration (Column C). The rate of RNA labeling, whether expressed as RNA specific activity (disintegrations/min per mg RNA, Column D) or as radioactivity in RNA in average liver cells (disintegrations/min in RNA per mg DNA, Column F), reflected the specific activity of the precursor UTP in the normal and newly hepatectomized controls, but was disproportionately higher in the 12-h regenerating livers - an indication that the rate of synthesis was actually increased. These values when adjusted for the corresponding UTP specific activities yielded estimated rates of RNA synthesis expressed respectively as m,umoles of UTP entering RNA per mg RNA per 15 min (Column E), or as mpmoles of UTP entering RNA per mg DNA per 15 min (Column G). This calculation eliminated the variable effects of differences in amount of liver, rate of UTP formation and size of its pool; the rates of RNA formation were then shown to be nearly alike in the 2 controls (normal and newly hepatectomized) and increased by about 1.5 or 1.7 times normal in the 12-h regenerating livers (Columns E and G, respectively). Since we expect the normal and newly hepatectomized controls to be similar, Columns E and G are the preferred ways to express rate of RNA synthesis. In these normally fed animals, the regenerating livers exhibited a negligibly small net gain in RNA content (Column H). In the following groups of rats, it will be seen that when there is a significant RNA increment, the method of Column G has an advantage over Column E. Since some experimental conditions interfere with nutrition, we examined a group of rats differing from the above only in having been denied food for 12 h before the infusion of [i4C]orotic acid (Fig. 3). It is reported that a fast of even this brief duration can lead to discrepancies in UTP and RNA labelingzO. The most obvious results of this treatment were the pronounced enlargement of the UTP pool and modest rise in RNA content in the 12-h regenerating livers compared with the newly hepatectomized controls (cf. Figs. 2 and 3, Columns C and H). In these regenerating livers

UTPl

A

0

IRNA(

C

D

E

F

G

H

Fig. 3. Incorporation of [6-%]orotic acid into UTP and RNA, and UTP and RNA content of livers of normal or partially hepatectomized rats denied food for 12 h before taking of liver samples. Conditions and units otherwise as in Fig. 2. B&him.

Biophys.

.4&z,

174 (1969)

491-501

S. L. Ii. BUCHER,

498

M. N. SWAFFIELD

of fasting rats, the uncorrected rate of RNA labeling per mg DNA (Column F), and the RNA specific activity (Column D) even when adjusted for the altered UTP specific activity (Column E), suggested no increase in RNA biosynthetic rate; Column G alone, incorporating adjustments for both changes in UTP specific activity and RNA content, showed that there actually was a rise in RNA synthesis in the 12-h regenerating livers comparable to the fed group. In Fig. 4 are data from a similar group of fasting animals, differing from those in Fig. 3 only in that they were infused with [z-%]cytidine instead of [6-%]orotic

IRNA-----l

T---Tpl

TO-

Y’-

Fig. 4. Incorporation of [z-Wlcytidine into CTP and RNA, and CTP and RNA content of livers of normal or partially hepatectomized rats denied food for 12 h before taking of liver samples. Solid bars represent normal, finely striped bars newly hepatectomized, and segmented bars 12-h regenerating livers from rats receiving 0.1 fimole = 2.5 PC (specific activity 25 yC/j_4mole) of cytidine, and wavy bars normal liver from a rat given I .o pmole = I ,uC (specific activity I ,uC/,umole). Conditions and units otherwise as in Figs. 2 and 3.

acid. Utilization of cytidine seemed to differ from that of erotic acid in being less dependent upon the effective liver mass, as evidenced by the formation of CTP at more nearly similar rates in newly hepatectomized and normal controls (compare Column B in Figs. z-4). Both CTP and UTP pools were enlarged to a similar extent in the 12-h regenerating livers when rats were denied food during that period (Figs. 3 and 4 Column C), although the CTP pool was only about 1/3 as large as the UTP pool in all instances. There was also reasonable agreement in relative rates of RNA synthesis as determined with the two precursors (Figs. 3 and 4 Column G). The wavy lines in Fig. 4 record data from a normal rat given cytidine of only 1/25th the specific activity received by the others, but the estimated rate of RNA synthesis was similar (Column G). It is known that extensive enlargement of the UTP pool results from feeding I o/o erotic acid in the diet21,22.To examine doses in the range used in the present experiments, we gave 12-h fasting rats (comparable to those in Figs. 3 and 4) an intravenous injection of either 0.1 ,umole or 1.0 pmole of nonlabeled erotic acid 45 Biochim.

Biophys.

Acta.

174 (1969) 4g1-502

RNA SYNTHESIS AND PRECURSOR POOLS

499

min before the usual r5-min infusion of [14C]orotic acid (Fig. 5). The principal result of the preinjection was enlargement of the UTP pool in both the newly hepatectomized rats and in those hepatectomized 12 h previously, but not in the normal animals (Figs. 3 and 5 Column C). The lack of response in the latter group could be ,-

UTPs-1

A

B

,-

C

RNA

0

E

------l

F

G

H

Fig. 5. Incorporation of [V4C]orotic acid into UTP and RNA, and UTP and RNA content of livers of normal or partially hepatectomized rats denied food for 12 h and preinjected with nonlabeled erotic acid. Solid bars represent normal, finely striped bars newly hepatectomized, and segmented bars 12-h regenerating liver from rats preinjected with 0.1 ,umole of erotic acid I h before liver samples were taken. Diagonally striped bars represent 12-h regenerating liver from rats preinjected with 1.0 pmole. Conditions and units otherwise as in Fig. 3.

accounted for by the sharing of the erotic acid among more cells (therefore relatively less per cell); an insufficiency of the requisite UTP-forming enzymes seems less likely, since they appear to be present in excess immediately after hepatectomy, when regeneration has scarcely begun. I pmole was only slightly more effective than 0.1 pmole. Our previous findings did not suggest that a significant increase in pool size was induced by the single injection of 0.1 pmole, since we got essentially similar values for pools measured by the present method and one in which no erotic acid was administeredlO. It now appears that when this dose is given twice, a pool expansion does occur, at least in liver-depleted animals. The effect is probably insignificant when only the usual 0.1 pmole is infused, since we find little or no difference between normal and newly hepatectomized controls under these conditions (Fig. 3). A more important consideration is that the estimated rate of RNA synthesis appears to be unaffected by the erotic acid dosage, the values in preinjected and non-preinjected rats being in reasonable agreement (cf. Figs. 3 and 5 Column G). Biochinz. Biophys.

Acta,

174 (1969)

z+gI-502

?T. I.. R. BUCHER,

500

XI. N. SWAFFIELD

The data are presented in the above manner to bring out the difficulties encountered when only the rate of RNA labeling is considered, without regard for precursor pools. The salient feature of Figs. 2-5 is the erratic relation between normal and newly hepatectomized and rz-h regenerating livers in all parameters except those expressed in Columns G and H. Column G shows that in normally fed or 12-h fasting animals, regardless of whether erotic acid or cytidine of varied specific activity is the initial

labeled

precursor,

the relative

rate of RNA

synthesis

as estimated

in this

manner is nearly the same in the two kinds of controls, and increased 1.5~z-fold in the rz-h regenerating livers. Column H demonstrates the same tendencies in net gains in RNA content.

Although

the rates of RNA synthesis

determined

in this way are

probably acceptable approximations of true in viva rates, they remain estimates because other sources of variability cannot entirely be ruled out - e.g., possible compartmentation

of endogenous

precursor

for technical reasonsiO. On the basis of the foregoing, were examined

pools which we have been unable to evaluate

the changes taking place in UTP,

during the early hours after partial hepatectomy

CTP and RNA

or a sham operation.

Expansion of the UTP pool was found to be an early event in the regenerative sequence, an increment of 50 %, or more occurring in both fed and fasting rats by 3 h after partial hepatectomy

(Fig. 6). The pool was initially

smaller,

and the increment

Fig. 6. Size of endogenous UTP and CTP pools in normal liver (N), and livers at intervals after partial hepatectomy (solid lines) or a sham operation (dotted lines). A A, UTP in rats fed ad libitum; 0 0, UTP in rats denied food for 12 h; 0 ?? , CTP in rats denied food for 12 h. Open Small numbers symbols rats with whole livers, closed symbols rats partially hepatectomized. beside each point indicate number of rats. Fig. 7. Estimated

rates of RNA synthesis

in same livers shown in Fig. 6.

relatively greater in the fasting animals. CTP was 113 as abundant as UTP and increased more gradually; in the fasting animals both pools were doubled by IZ h of regeneration. A minor transient rise was elicited in the UTP pool by a sham operation. In Fig. 7 are the estimated rates of RNA synthesis in the same animals as shown in Fig. 6. An initial small depression at time zero in all groups was followed by a rise that occurred earlier in the fed than in the fasting animals, and then by a plateau. The increase in biosynthetic rate lagged behind the expansion of the UTP B&him.

Biophys.

Acta,

174

(1969)

491-502

RNA

SYNTHESIS

AND PRECURSOR

POOLS

501

pool in the fasting but not in the fed rats (Figs. 6 and 7); presumably to additional

functions

besides

RNA

synthesis.

Sham

operation

pool sizes relate affected

both

the

biosynthetic rate and the pool size, in parallel fashion. The RNA content in the same livers followed a course similar to the rate of RNA synthesis in each group - rising sooner in the fed than the fasting animals, and also increasing temporarily in the sham-hepatectomized short duration of the period of fasting should be emphasized food deprivation

is reported to have an opposite effect, I.c. to reduce the RNA content

below the level in fed rats, at least in non-regenerating

3 N

0

Hours

Fig.

8. RNA

1

I

1

/

3

6

9

I2

porm tlepoteckmly

after

The number

of rats per point are too few to permit

conclusions

fully elsewhere”,

but the overall consistency cited.

These

but a few additional

to verify the shrinkage at a slightly

livers23.

content of ssme livers shown in Figs. 6 and 7,

cance to small differences, the general

controls (Fig. 8). The since more prolonged

and other features comments

of signifi-

have been discussed

to

more

may be in order. We have not sought

of UTP and CTP pools found by

later stage of regeneration

the assigning

of the data lends support

MANDEL

and coworkers25,26

than that studied by us, but an expansion

of these pools such as we have observed is in line w-ith their expressed concept that nucleoside triphosphate levels are higher in rapidly growing organs and tumor cells than in tissues with a low mitotic synthesis

during hepatic

we need only affirm

index.

regeneration

that our results

The numerous

divergent

have been repeatedly are in general

studies

summarized,

of RNA and here

accord with the RNA labeling

rates originally obtained in 1963 by LIEBERMAN and coworkersl. Perhaps in the fed rats which they studied, a fortuitous counterbalancing of variables obviated the necessity of adjusting for immediate precursor specific activity. We have consolidated their findings by confirmatory experiments in both fed and fasting animals, using modified techniques that circumvent previous difficulties relating to restrictions in food intake and variations in tissue mass. reduce divergences in future investigations, development,

aging, and neoplasia

It is hoped that these procedures will and enable RNA synthesis in normal

to be examined Biochinz.

with added insight. Biophys.

Acta,

174

(1969)

491-502

N.

502

L. R.

BUCHER, M. N. SWAFFIELD

ACKNOWLEDGMEKTS The authors are grateful to Drs. E. RANDERATH and K. RANDERATH for advice about thin-layer chromatography to Dr. F. L. MOOLTEN for helpful suggestions and for critically reviewing the manuscript. This work was supported by American Cancer Society, Inc. Grant E-5oD and U. S. Public Health Service Grant CAoz146-15. This is publication No. 1341 of the Cancer Commission of Harvard University.

REFERENCES I M. FUJIOKA, M. KOGA AND I. LIEBERMAN, J. Biol. Chem., 238 (1963) 3401. 2 N. L. R. BUCHER, Intern. Rev. Cytol.. 15 (1963) 245.

3 I. LIEBERMAN, P. KANE AND J. SHORT, /. Biol. Chem., 240 (1965) 3140. 4 N. L. R. BUCHER AND M. N. SWAFFIELD, Biochim. Biophys. Acta, 108 (1965) 551. 5 T. UCHIYAMA, N. FAUSTO AND J. L. VAN LANCKER, J. Biol. Chem., 241 (1966) 991. 6 A. H. GORDON AND G. S. HODGSON, Biochim. Biofihys. Acta, 119 (1966) 427. 7 N. L. R. BUCHER, New England J. Med., 277 (1967) 686 and 738. 8 E. BRESNICK, S. S. WILLIAMS AND H. Moss&, Cancer Res., 27 (1967) 469. 9 N. L. R. BUCHER AND M. N. SWAFFIELD, Exptl. Mol. Pathol., 5 (1966) 443. IO N. L. R. BUCHER AND M. N. SWABFIELD, Biochim. Biophys. Actn, Izg (1966) 445. 1 I N. L. R. BUCHER AND M. N. SWAFFIELD, J. Cell. Biol., 31 (1966) 17A. 12 A. WOLLENBERGER, 0. RISTAU AND G. SCHOFFA, Arch. Ges. Physiol. 270 (1960) 399. 13 F. B. SEIBERT, J. Biol. Chem., 133 (1940) 593. 14 C. F. GORDON AND A. L. WOLFE, Anal. Chem., 32 (1960) 574. 15 F. E. KINARD, Rev. Sci. Ilzstr., 28 (1957) 293. 16 A. FLECK AND H. N. MUNRO, Biochim. Biophys. Acta, 55 (1962) 571. 17 W. MEJBAUM, Z. Physiol. Chem., 259 (1939) 117. 18 K. RANDERATH AND E. RANDERATH, in S. P. COLOWICK AND N. 0. KAPLAN, Methods in Enzymology, Vol. 12, Academic Press, New York, 1st edition, 1967, p. 323. 19 D. P. NIERLICH AND W. VIELMETTER, J. Mol. Biol., 32 (1968) 135. 20 P. OVE, R. L. P. ADAMS, R. ABRAMS AND I. LIEBERMAN, Biochim. Biophys. Acta, 123 (1966)

421.

PUDDU AND C. M. CALDERERA, Biochim. Biophys. Acta, 61 (1962) 826. R. J. RUBIN AND R. E. HANDSCHUMACHER, J. Biol. Chem., 238 (1963) 2464. R. POTTER, J. Mol. Biol., 26 (1967) 279. M. N. SWAFFIELD, F. L. MOOLTEN AND T. R. SCHROCK, in R. BASERGA, of Cell DilJision, C. C. Thomas, Springfield, Ill., in the press. M. WINTZERITH, N. KLEIN-PETE AND L. MAND~L, Nature, 198 (1963) 1000.

21 M. MARCHETTI, P. 22 L. H. VON EULER, 23 G. BLOBEL AND V. 24 N. L. R. BUCHER,

Biochemistry

25 P. MANDEL, 26 P. MANDEL, Bull. Sot. Chim. Biol., 49 (1967) 1491.

Biochim.

Biophys.

Acta,

174 (1969) 491-502