THE JOURNAL OF BIOLOGICAL CHEMISTRY 266, No. 7, Issue of 5, … · 2001. 6. 8. · THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1991 by The American Society for Biochemistry and Molecular

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1991 by The American Society for Biochemistry and Molecular Biology, Inc

. 266, , No. 7, Issue o f March 5, pp. 4409-4416,1991 Printed in U. S. A.

Transcriptional Regulation of Ribosomal RNA Synthesis during Growth of Cardiac Myocytes in Culture*

(Received for publication, July 27, 1990)

Paul J. McDermottS, Lois L. Carl, Kimberly J. Conner, and Simon N. A110 From the Sigfried and Janet Weis Center for Research, Geisinger Clinic, Danuille, Pennsylvania 17822

The mechanism(s) by which rRNA accumulates dur- ing the growth of cardiac myocytes was investigated. The rates of rDNA transcription were measured in contracting myocytes and compared with nonbeating myocytes depolarized with 50 mM KCl. After 3 days of contraction the absolute rate of rDNA transcription was accelerated by 2-fold as measured by incorpora- tion of [‘HIUTP into the external transcribed spacer of preribosomal RNA. Corresponding increases in transcription were observed in isolated nuclei of con- tracting myocytes as measured by either hybridization of run-on transcripts of preribosomal RNA or activity of RNA polymerase I. The extent to which transcrip- tion was stimulated in contracting myocytes accounted for the previously observed acceleration of rRNA syn- thesis rates. The steady-state levels of preribosomal RNA relative to rRNA were unchanged in contracting myocytes, but the total amount of preribosomal RNA was 1.3-fold greater as a result of increased rRNA content. The increase of preribosomal RNA in propor- tion to rRNA in contracting myocytes demonstrated that the rate of preribosomal RNA processing was unchanged and that rRNA synthesis is regulated by an accelerated rate of rDNA transcription.

Ribosomes are major components of the translational ap- paratus for protein synthesis and are the key component that regulates growth of the cell (1, 2). The biogenesis and subse- quent accumulation of ribosomes are tightly coupled to both hyperplastic and hypertrophic growth (2,3). Upon stimulation of hypertrophic growth of cardiac myocytes, the rate of protein synthesis is accelerated by an increase in ribosome content (3-5). Net accumulation of ribosomes has been shown to occur in response to growth stimuli in vivo and in myocytes main- tained in cell culture (3, 4, 6). In most models of cardiac hypertrophy, ribosome accumulation either precedes or occurs concomitantly with accelerated rates of protein synthesis and cell growth. It has been concluded that an increase in ribosome content is the primary mechanism by which the rate of protein synthesis is regulated because 80-90% of the ribosome pool is found in polysomes of both growing and nongrowing myo- cytes, thus reducing the potential for translational regulation at the level of peptide chain initiation and elongation (4, 7). These facts suggest that putative intracellular signals gener-

* This work was supported by American Heart Association Grant- in-aid 890707 and by the Geisinger Foundation. The costs of publi- cation of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertise- ment” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$Present address: Dept. of Cardiology, Medical University of South Carolina, Veterans Administration Medical Center, 109 Bee St., Charleston, SC 29403. To whom correspondence should be sent.

ated in response to a growth stimulus accelerate the rate of protein synthesis in cardiac myocytes by affecting specific reactions involved in ribosome formation.

Several models of cardiac hypertrophy have correlated changes in ribosomal RNA (rRNA) synthesis or ribosome formation with the application of a growth stimulus. In pres- sure overload hypertrophy in uiuo, increases in 28 S rRNA synthesis and in the activity of RNA polymerase I, the enzyme that transcribes rDNA, have been demonstrated (8, 9). An increased rate of ribosome formation in the rapidly growing left ventricle of the newborn pig occurs by 18 h after birth (10). Stretch of the ventricular wall in response to elevated intraventricular pressure has been demonstrated to increase ribosome formation in isolated adult heart preparations (11). During growth of synchronously contracting myocytes in cul- ture, rRNA accumulated as a result of accelerated rRNA synthesis (6). The transcription of rDNA by RNA polymerase I was stimulated in nuclei isolated from contracting myocytes and after norepinephrine treatment (6, 12). A number of intracellular signaling mechanisms, induced by mechanical or hormonal means, have been implicated in models of develop- mental and adaptive cardiac growth (11,13-15). These include activation of protein kinases through CAMP or phosphatidyl- inositol turnover coupled to diacylglycerol production, and transient expression of gene products usually associated with mitogenic growth. Although largely unexplored, the rate-lim- iting reactions of rRNA synthesis are a logical target for any signaling mechanism that accelerates the rate of protein syn- thesis.

The synthesis of rRNA involves a number of reactions including transcription of rDNA genes, processing of the preribosomal RNA (pre-rRNA)’ transcript, and assembly with ribosomal proteins into ribosomes prior to transport from the nucleus into the cytoplasm (1, 2). The regulation of ribosome formation, in most instances, is at the level of rDNA transcription (2, 16, 17). In skeletal muscle cultures, ribosome formation is regulated by rDNA transcription (18-20). In contrast, the processing of pre-rRNA is rapid and may begin before transcription of the full-length precursor transcript is completed (2, 17). Because of the proficiency of the reactions involved in pre-rRNA processing, there is minimal evidence to suggest that pre-rRNA processing is a regulatory site for rRNA synthesis in cardiac myocytes. Ribosomal protein syn- thesis and assembly are coordinately regulated with rRNA synthesis during ribosome formation, but a substantial role in regulating the rate of ribosome accumulation has not been demonstrated (19-21). Furthermore, ribosomal proteins not assembled into ribosomes are rapidly degraded, and a signifi-

The abbreviations used are: pre-rRNA, preribosomal RNA; HPLC, high pressure liquid chromatography; ETS, external tran- scribed spacer; MOPS, 4-morpholinepropanesulfonic acid; SDS, so- dium dodecyl sulfate.

4409

by guest on Decem

ber 21, 2020http://w

ww

.jbc.org/D

ownloaded from

4410 Transcriptional Regulation of Ribosomal RNA Synthesis

cant pool of free ribosomal proteins does not exist in muscle cells (7, 21, 22).

In this report, the specific reactions that accelerate the rate of rRNA synthesis during stimulation of myocyte growth by contraction were investigated. During growth of synchro- nously contracting myocytes, there was net accumulation of rRNA as a result of accelerated rates of rRNA synthesis in excess of rRNA degradation (6). Thus, absolute rates of rDNA transcription were measured during growth of synchronously contracting heart cells and compared with nonbeating cells depolarized with 50 mM KC1 in the medium. Previously meas- ured rates of 18 and 28 S rRNA synthesis were utilized as reference values to determine the relative extent to which transcription of rDNA was involved in regulating ribosome accumulation during growth. Knowledge of the rate-limiting reactions for rRNA synthesis, such as rDNA transcription, provides a useful approach for evaluating intracellular signals that transduce a growth stimulus into changes in the rate of protein synthesis.

MATERIALS AND METHODS

Myocyte Culture-Primary cultures were prepared from minced ventricular myocardium of 2-4-day-old rats as described previously (4). The cells were plated at a concentration of 4 X 106/60-mm dish, as measured with a Coulter Counter (Coulter Electronics, Inc.), and incubated overnight in minimal Eagle's medium containing 10% newborn calf serum and 0.1 mM 5-bromo-2'-deoxyuridine at 37 "C in an atmosphere of 5% COz, 95% room air. Thereafter, the cells were rinsed and maintained in serum-free medium. Potassium chloride- depolarized cells (KC1-depolarized) were prepared by adding 2.5 M KC1 dissolved in medium to a final concentration of 50 mM KC1 in the same serum-free medium.

Radiolabeling of Myocyte RNA-The synthesis of pre-rRNA in myocytes was determined by pulse labeling. [5,6-3H]Uridine (35-40 Ci/mmol) was taken to dryness in a vacuum centrifuge and resus- pended in serum-free medium to yield a final concentration of 200 pCi/ml. For most experiments, six dishes/time point were incubated for 5 min in 1.5 ml of medium containing [3H]uridine followed by incubation in 2 ml of the same medium without uridine. The cells were rinsed three times with ice-cold phosphate-buffered saline, and the RNA was immediately extracted from four of the dishes by adding 1 ml of RNAzol (Cinna/Biotecx)/dish. The cells were scraped in RNAzol, homogenized by drawing the extract up and down repeatedly with a Pasteur pipette, and centrifuged. The RNA was precipitated with an equal volume of 2-propanol and centrifuged. The pellet was washed twice with 70% ethanol and resuspended in H20. An aliquot was used to measure RNA concentration and incorporation of [3H] uridine into total RNA. The remaining two companion dishes were employed to measure the specific radioactivity of the cellular UTP pool (dpm/pmol). Briefly, after rinsing with phosphate-buffered sa- line, the cells were scraped into l ml of 0.5 N HC10, with several rinses. The extract was centrifuged and the supernatant neutralized by the addition of 2 M KHCOB to a final concentration of 0.5 M. The neutralized extract was centrifuged, and the supernatant was filtered and taken to dryness in a vacuum centrifuge. UTP was purified by reverse phase HPLC (Beckman Instruments) using a 4.6-mm X 25- cm column as described previously (6). The corresponding peak of radioactivity was integrated directly with an, on-line radioisotope detector (Beckman) and the specific radioactivity of UTP calculated. The HC10,-precipitable pellet was used to calculate total RNA con- centration/dish as described previously (6).

rDNA Clones-The rDNA clones used for these studies were derived from the primary genomic clone XCHR-B4 (23). Two clones for the external transcribed spacer region (ETS) of rat pre-rRNA were used and were subcloned into pBR322 and pGEM-3Z+ vectors (Promega Biotec). p2.0 contained the rDNA sequence from -167 base pairs upstream of the transcription start site to approximately the first 2 kilobases of the ETS (24). The second clone, 5.1E/E, extended from -286 base pairs upstream to the first 300 base pairs of the ETS (25). Sense and antisense cRNA transcripts were synthesized in vitro using the pGEM system. Transcripts were of the predicted size as assessed by agarose gel electrophoresis and visualization by either fluorography (for 3H- or 14C-labeled transcripts) or autoradiography (for "P-labeled transcripts). The specific radioactivity of UTP in the

transcription reactions was measured by reverse phase HPLC and used to quantitate the amount of cRNA.

Hybridization of Preribosornal RNA-The recombinant rDNA sub- clone p2.0 was heated at 95 "C for 5 min in 10 X standard sodium citrate buffer (SSC), cooled on ice, and immobilized on Hybond N (Amersham Corp.) by use of a Minifold I1 slot-blotting apparatus (Schleicher & Schuell). Generally, 1.5 pg of recombinant DNA was added per slot. The slots were rinsed three times with 1.5 M NaCl, 0.5 N NaOH and neutralized with three rinses of 1.5 M NaC1, 10 mM Tris, pH 7.2, 1 mM EDTA. The DNA was cross-linked to the mem- brane by UV irradiation, and individual slots were cut out and transferred to microcentrifuge tubes containing 200 p1 of hybridiza- tion solution. The hybridization solution was 50% (v/v) deionized formamide, 0.2% (w/v) polyvinylpyrrolidone, 0.2% (w/v) Ficoll, 5 X SSC, 10 mM MOPS, pH 7.0, 2 mM EDTA, 10 pg of yeast tRNA/ml, and 0.2% (w/v) sodium dodecyl sulfate (SDS). Prehybridization pro- ceeded at 52 "C for at least 1 h. For hybridization, 10 pg of radiolabeled RNA/tube was taken to dryness in microcentrifuge tubes by vacuum centrifugation and resuspended in 200 p1 of hybridization buffer to which 25-50 pmol of sense 14C-labeled p2.0 transcript/ml was added. Three or four reaction tubes were set up for each time point. A filter containing the prehybridized rDNA probe was added to each hybrid- ization tube, and the reaction proceeded for 60 h at 52 "C. The slots were individually washed three times with 2 X SSC, 0.1% SDS at 52 "C for 30 min followed by two washes in 0.1 X SSC at 65 "C. Slots from each time point were combined and incubated in 400 p1 of 0.3 N NaOH at 37 "C for 3 h. The hydrolysate was neutralized with H3P04, and the 3H/14C ratio of hybridized RNA was determined by liquid scintillation counting (Beckman). A dual label program was used in which the spill and counting efficiencies for quenched standards of 3H and "C were determined in both channels. The total incorporation of [3H]UTP into pre-rRNA (dpm/mg input RNA) was calculated after multiplying the 'H/"C ratio of hybridized RNA by the dpm of sense 14C-labeled cRNA added to the reaction.

The following experiments were performed to verify the hybridi- zation procedure for isolation of pre-rRNA and corresponding meas- urements of [3H]UTP incorporation by the dual label method.

1. Hybridization of RNA to pBR322 alone did not result in recovery of radioactivity.

2. The recombinant p2.0 rDNA clone hybridized specifically to electrophoretically resolved pre-rRNA as assessed by Northern blot- ting. Hybridization to 18 or 28 S rRNA was not observed.

3. Hybridization reactions, in which sucrose gradient-purified 28 S [3H]rRNA was added in amounts comparable to its mass in total RNA (6.5 pg of 28 S rRNA/tube), failed to recover significant dpm of hybridized 28 S rRNA. The 28 S [3H]rRNA added was purified after labeling myocytes for 6 h and had a specific radioactivity that was greater than 28 S rRNA in total RNA samples that were labeled for 60 min.

4. Increasing the input RNA concentration in the reaction resulted in a proportional increase in the dpm recovered by hybridization. As expected, the 3H/14C ratio recovered after hybridization remained constant if the mass ratio of 13H]RNA to [14C]cRNA transcript was the same at each input RNA concentration.

5. After alkaline hydrolysis of hybridized pre-rRNA, all of the measurable radioactivity was present in 3'-UMP as determined by HPLC with an on-line radioisotope detector using carrier 3'-UMP and 3'-CMP.

The dual label hybridization method was used to calculate the rate of rDNA transcription (pmol of UTP/mg of input RNA) .min" using the following formula:

Rate of [3H]UTP incorporation dpm, - dpm, mg input RNA

UTP specific radioactivity - (zl) where dpm, and dpm, represent [3H]UTP incorporation into pre- rRNA at time t and after completion of the 5-min pulse with [3H] uridine ( t o ) , respectively.

Preribosomal RNA Pools-Myocytes were rinsed three times with ice-cold phosphate-buffered saline and extracted with RNAzol as described above. RNA was subjected to horizontal gel electrophoresis on 1% agarose gels as described previously (6). The gels were run for 18 h at 40 V and rinsed with several changes of H20. The gels were incubated at room temperature in 50 mM NaOH, 10 mM NaCl for 45 min followed by neutralization in 0.1 M Tris, pH 7.5, for 45 min. The

by guest on Decem

ber 21, 2020http://w

ww

.jbc.org/D

ownloaded from

Transcriptional Regulation of Ribosomal RNA Synthesis 441 1

gels were soaked for a t least 1 h in 20 X standard sodium phosphate huffer (SSPE) and the RNA transferred to Hybond N. The blots were dried, UV cross-linked, and hybridized to the antisense ['"PI cRNA for the ETS region of pre-rRNA. The hybridization and washing conditions were similar to those described above. The cRNA probes were denatured in hybridization buffer by incubation at 65 "C for 10 min. In some experiments, the blots were hybridized to nick- translated recombinant plasmid IRYP]DNA prepared using a commer- cial nick translation k i t (Amersham Corp.).

Autoradiograms were prepared by exposure of the blots to Kodak X-Omat AR film a t -70 "C with intensifying screens. The intensity of the bands was measured by computer-generated digital image processing with a Microscan 1000 (Technology Resources, Inc.) gel analysis system. In some experiments, the radioactivity of hyhridized RNA on the blots was measured directly with an Amhis radioanal.ytic image system (Ambis Systems).

Transcription in Isolated M-yocyte NucCi-Transcriptionally active nuclei were isolated by the method of Rowman with modifications (20). Generally, six to eight dishes were used per experimental group. Each dish was rinsed two times with phosphate-buffered saline fol- lowed hy one rinse and incubation on ice for 10 min with 0.5 ml of buffer containing 10 mM Tris, pH 8.0, 10 mM NaCI, 5 mM dithiothre- itol, 2.5 mM MgCI?. The cells were scraped from the dishes after adding an equal volume of the same buffer containing 0.6 M sucrose and 0.6% (v/v) Triton X-100. The cells were homogenized hy means of a Dounce homogenizer and layered on an equal volume of homog- enization buffer containing 0.6 M sucrose. The nuclei were pelleted hy centrifugation a t 1,500 X g for 10 min and resuspended in 100 pl/ dish of a buffer containing 50 mM Tris, pH 8.0, 0.1 mM EDTA, 20% glycerol, 5 mM MgC12.

Transcription was assayed in buffer containing 50 mM Tris, pH 8.0; 5 mM MgCIz; 5 mM dithiothreitol; 50 mM (NH,)2S04; 0.5 mM CTP, GTP, and ATP; 25 pM ["HIUTP (5 pCi/assay tuhe, 35 Ci/ mmol); 5 units of RNasin/ml; and 1 pg of n-amanitin/ml. The transcription reaction was initiated by adding the nuclear suspension. After 5 min, the reaction was terminated by adding an equal volume of 20% trichloroacetic acid containing 2 mM UTP. Rackground activ- ity was measured in parallel tubes by immediately terminating the reaction after the addition of the nuclei with the acid (time 0). The pellets were washed three times with 10% trichloroacetic acid con- taining 10 mM Na4P207 and hydrolyzed overnight in 0.3 N NaOH. Concentrated perchloric acid was added to the assay tubes at a final concentration of 1.5 N, and the tubes were centrifuged. Radioactivity in the supernatant was measured by liquid scintillation counting. Aliquots of the nuclear suspension were solubilized in a solution containing 1 X SSC, 0.25% SDS, frozen a t -20 "C, and used to measure DNA concentration. The specific radioactivity of UTP (dpm/pmol) in the assay buffer was measured by reverse phase HPLC. Data are expressed as UTP incorporation into trichloroacetic acid-precipitable material over 5 min minus the zero time point and normalized to the amount of DNA in the assay tuhe.

Transcription of rDNA was measured in nuclear preparations by hybridization of "P-labeled run-on transcripts to the p2.0 rDNA clone for the ETS region. The transcription reactions proceeded as described above except that 50 pCi of ["'P]UTP/tuhe was used instead of "H, and n-amanitin was omitted. Transcription proceeded for 15 min after the addition of the nuclear suspension. The RNA in each reaction tube was purified extensively by the method of McKnight and Palmiter (26) using 50 pg of carrier tRNA/tube. Hybridization was performed by adding filter-immobilized recombinant p2.0 rDNA to the purified RNA of each reaction tuhe as described above. A sense "H-labeled p2.0 transcript was added as a reference to monitor recov- ery of RNA and hybridization efficiency of pre-rRNA.

Filter-immobilized pRR322 was added to each hybridization reac- tion to monitor background radioactivity. The filter-hybridized RNA was washed as described above, hydrolyzed in 0.3 N NaOH, neutral- ized by adding HaPO, to pH 6.5, and counted for radioactivity. The :"P/'H ratios of hybridized RNA were multiplied by the input dpm of "H to calculate total cpm of "2P-radiolabeled transcript hybridized per pg of nuclear DNA in each reaction.

RESULTS

Pre-rRNA Pools in KC1-depolarized and Contracting Myo- cytes-In previous studies, net rRNA accumulation resulted from accelerated rates of 18 and 28 S rRNA synthesis (6). To investigate the link between accelerated rRNA synthesis and

transcription of rDNA genes, the size of the 45 S pre-rRNA pool in contracting myocytes was compared with KCI-depo- larized cells. As in previous studies, myocytes were maintained in a serum-free medium containing 50 mM KC1 until dav 3 in culture. On day 8, the cells were either maintained in high K' medium or allowed to contract by switching to the same medium containing 4 mM KCI. Comparisons were performed 3 days after the onset of contraction, the time at which changes in the rates of rRNA synthesis were maximal. No detectable differences were observed, as measured bv digital image processing, in the pool of 45 S pre-rRNA relative to total RNA in three separate experiments (Fig. 1). The cRNA probe hybridized to 28 S rRNA, which provided an internal standard for the amount of RNA applied to the gel and blotted to the hybridization membrane. In addition, potential differ- ences in the pre-rRNA pool were evaluated at 24-h intervals after the onset of contraction by direct measurement from the blots of radioactivity hybridized to 45 S pre-rRNA and 28 S rRNA utilizing the Ambis radioanal-ytic imaging system (Table I). At each time point over the duration oft he stimulus, the ratios of cpm in 45 S pre-rRNA to 28 S rKNA were not significantly different. These results were confirmed by digital image analysis of corresponding autoradiograms. Further- more, the relative size of the pre-rRNA pool did not change appreciably between days 4 and 6 in culture for either voup.

The first processing site for 45 S pre-rRNA occurs approx- imately a t nucleotide +650 of the ETS region (2). Therefore, the relative size of the pre-rRNA pool WAS measured with an antisense transcript complementary to the first 800 bases of the ETS region (p.E.lE/E) to determine whether the pool contained the full-length transcript of 45 S pre-rRNA. Results similar to those employing the p2.0 cRNA transcript were obtained, confirming that the size of the pre-rRNA pool was

A B

45s - 28s -

K C K C K C K C K C K C M P : 1 1 2 2 3 3 1 1 2 2 3 3

FIG. 1. P re - rRNA pools in KCI-depolarized and contracting myocytes. Total RNA WAS extracted frnm KCl-depolarized (h') and contracting (C) myocytes 3 days after the onset o f contraction Ida? 6 in culture). Total RNA was suhjected to Northern blotting and probed with an antisense"'P-labeled c R S A transcript for the externrtl transcribed spacer region of 45 S pre-rRNA (p2.01. The results o f three separate experiments are indicated on the autoradiogram. A. 2 pg of total RNA; N. 4 pg of total ItNA.

by guest on Decem

ber 21, 2020http://w

ww

.jbc.org/D

ownloaded from

4412 Transcriptional Regulation of Ribosomal RNA Synthesis

TABLE I Pre-rRNA pool size in KC1-depolarized and contracting myocytes Total RNA was extracted from myocytes at the indicated time

points after the onset of contraction, subjected to Northern blotting, and probed as described in Fig. 1. The cpm of 32P hybridized to 45 and 28s were measured directly from the blots with an Ambis radioan- alytic imaging system and used to calculate the corresponding ratios. Values at each point are the mean and range for two separate experiments.

Onset of contraction

h 24 KC1-depolarized 0.18

Contracting 0.15 f 0.02 48 KC1-depolarized 0.12 f 0.02

Contracting 0.12 f 0.02 72 KC1-depolarized 0.13 & 0.01

Contracting 0.13 & 0.01

Myocytes 45 SI28 S

unchanged in contracting as compared with KC1-depolarized myocytes (data not shown).

Transcription of rDNA in Contracting and KCI-depolarized Myocytes-Steady-state levels of pre-rRNA were unchanged in contracting myocytes. However, the absolute amount of pre-rRNA was approximately 1.3-fold greater after 3 days of contraction because the RNA content was increased signifi- cantly by 32% (KC1-depolarized, 26.6 & 1.9 pg of RNA/dish versus contracting, 35.2 & 2.2 pg RNA/dish, p < 0.05), an increase similar to previous studies (4,6). These observations suggested that flux of nascent rRNA into the ribosome may be regulated by the rate of rDNA transcription. Transcription rates of rDNA in myocytes were measured by incorporation of [3H]UTP into pre-rRNA after hybridization to an rDNA clone for the ETS region. The pool of pre-rRNA is small as compared with cellular rRNA and is processed rapidly. Thus, in contrast to previous studies of rRNA synthesis in which the specific radioactivity of 3'-UMP in hybridized 18 and 28 S rRNA was measured, it was not practical to recover enough 3'-UMP in hybridized pre-rRNA (6). The rate of transcription of pre-rRNA was measured by dual label hybridization.

In this approach, input [3H]RNA containing pre-rRNA sequences was mixed with a sense 14C-labeled cRNA tran- script synthesized in vitro from p2.0 template subcloned into the pGEM transcription system. This resulted in a fixed mass ratio between 3H- and I4C-labeled pre-rRNA sequences in the hybridization reaction. The mixture of radiolabeled RNA was hybridized to p2.0 rDNA immobilized on hybridization filters. The 'H/14C rat.io of hybridized pre-rRNA was multiplied by the dpm of input sense 14C-labeled cRNA to calculate the total dpm of 3H-labeled pre-rRNA hybridized per mg of input RNA. In Fig. 2, the 3H/14C ratio of hybridized pre-rRNA was plotted as a function of the specific radioactivity of input 3H- labeled pre-rRNA. The 'H/I4C ratio was directly proportional to the specific radioactivity of equivalent amounts of 3H- labeled pre-rRNA added to the reaction. As demonstrated in the table in the lower section of Fig. 2, the 3H/14C ratio recovered after hybridization can be used to determine the total dpm of pre-rRNA added to the reaction.

In Fig. 3A the incorporation of [3H]UTP into myocyte pre- rRNA is demonstrated as a function of time using the dual label hybridization method. The total dpm of 3H incorporated into pre-rRNA at each time point was divided by the corre- sponding specific radioactivity of the cellular UTP pool (dpm/ pmol) to calculate the number of pmol of UTP incorporated per mg of input RNA. In previous work, it was demonstrated that the cellular UTP pool served as the immediate precursor for rRNA synthesis. By using the labeling protocol described

[ qIp2.0 Specific Radioactivity (dpm/pmol)

DUAL LABEL HYBRIDIZATION

(3W"C mi01 Input RNA Input ['H)RNA Hybridized RNA Hybridized [3H]RNA

(dpml ____ _ _ _ _ _ _ . ~ ~ ('HI'% n l o ) (dpml.

0.23 5342 0.23 5253

0.47 10684 0.49 11192

1.17 2671 0 1.24 28322

2.34 53420 2.52 57553

labeled pre-rRNA by dual label hybridization. Radiolabeled FIG. 2. Measurement of specific radioactivity of input 'H-

sense transcripts of p2.0 were synthesized in uitro, mixed, and probed with p2.0 rDNA as described under "Materials and Methods." The 3H/14C ratios of the hybridized RNA are plotted as a function of the specific radioactivity of the 3H-labeled p2.0 transcript. In the table in the lower portion, the recovery of hybridized 3H-labeled pre-rRNA (*) was calculated by multiplying the 3H/'4C ratios recovered by hybrid- ization by the input dpm of the sense I4C-labeled reference transcript (22,840 dpm).

under "Materials and Methods," the specific radioactivity of the cellular UTP pool remained relatively constant over the period during which incorporation was measured. Over the 1- h labeling period, the specific radioactivity of the UTP pool averaged 1,259 dpm/pmol with a standard deviation of 13.7% (Fig. 3C). The incorporation of [3H]UTP into total RNA was linear over the same time period (Fig. 3B). The relative constancy of UTP specific radioactivity simplified the precur- sor-product relationship of incorporation from the UTP pool into RNA which provides the basis for pre-rRNA synthesis measurements.

In contrast to total RNA, the incorporation of [3H]UTP into the pre-rRNA pool was linear for approximately 15 min (Fig. 3, A versus B). Thereafter, an equilibrium was achieved in which the specific radioactivity of UTP in pre-rRNA equil- ibrated with the specific radioactivity of the cellular UTP pool. Consistent with these data, the ratio of dpm incorpo- rated into pre-rRNA to the dpm of total RNA added to the dual label hybridization reactions increased for the first 20 min of labeling. Once equilibrium was achieved, this ratio decreased as expected because incorporation into total RNA remained linear (data not shown). The kinetics of labeling of the pre-rRNA pool, as measured by the dual label method, agreed with other studies demonstrating that equilibrium was achieved after approximately 15 min (16).

Because the percentage of pre-rRNA in total RNA was the

by guest on Decem

ber 21, 2020http://w

ww

.jbc.org/D

ownloaded from

Transcriptional Regulation of Ribosomal RNA Synthesis 4413

200 250 > 150

100

50

0 14000 -

h

.; I 12000 1 cur S m L C 10000 m a

-

5 8000 -

z E 6000 - a 0

+ s ' E 4000 -

B

2ooo 0 5 5 2000 z E IC

U

0 5 10 15202530354045505560 Time (min)

FIG. 3. RNA labeling with [3H]uridine in contracting myo- cytes. RNA was labeled by incubation in medium containing 200 pCi of [3H]uridine/ml (35-40 Ci/mmol) for 5 min followed by incubation in medium without label. At the indicated time points after the 5- min pulse (time 0), the following were measured A, incorporation of ['HIUTP into pre-rRNA by dual label hybridization; B, incorporation of ['HIUTP into total RNA; C, specific radioactivity of the cellular UTP pool by reverse phase HPLC. Values represent the average of two individual experiments. The incorporation of pmol of UTP plotted in A and B was calculated using the average UTP specific radioactivity demonstrated in C over each measured time point.

same for both KC1-depolarized and contracting myocytes, direct comparisons of [3H]UTP incorporation into pre-rRNA could be made by the dual label hybridization method. In Fig. 4, the transcription rates of rDNA in KC1-depolarized and contracting myocytes were compared 3 days after the onset of contraction. Transcription rates were measured after 10 min, a time point at which incorporation was linear, and compared with measurements made after 60 min, the time at which labeling of the pre-rRNA pool was essentially com- pleted (Fig. 3). As in Fig. 3, equivalent amounts of 3H-labeled total RNA at each time point were added in a fixed mass ratio

250 r

10 min 60 min Labeling Time

FIG. 4. Effect of contraction on rates of rDNA transcrip- tion. Rates of rDNA transcription in KC1-depolarized and contract- ing myocytes were measured at 10 and 60 min by dual label hybridi- zation as described under "Materials and Methods." Values are the mean +- S.E. of six experiments, each representing an individual preparation, performed 3 days after the onset of contraction (day 6 in culture). *, significant difference as determined by Student's t test, p < 0.005.

with the sense 14C-labeled reference transcript of p2.0. The number of pmol of UTP incorporated was calculated from the corresponding measurements of UTP specific radioactivity. In contracting myocytes, the rate of transcription/mg of input RNA was accelerated by 85% as measured after 10 min (Fig. 4). In absolute terms, the rate of rDNA transcription/cell was accelerated 2.4-fold because of the proportional 32% increase in pre-rRNA and total RNA of contracting myocytes. By 60 min, a labeling time at which the pre-RNA pool equilibrated with the precursor pool of cellular UTP, the values for rDNA transcription were similar. These data demonstrated that the relative size of the pre-rRNA pool was the same in contracting and KC1-depolarized myocytes and confirmed the results ob- tained for direct measurements of the pre-rRNA pool (Fig. 1 and Table I).

The increased rate of transcription in contracting myocytes as measured after 10 min of labeling reflected the fact that the pre-rRNA pool approached equilibrium with the cellular UTP pool at least %fold faster than in KC1-depolarized cells. To test these results further, fractional rates of rDNA tran- scription (KJ were calculated for the same experiments shown in Fig. 4 using the formula for equilibrium labeling: R', = R',,, (1 - e-Ks(c)) where R', and R',,, are the incorporation of [3H]UTP into pre-rRNA at 10 and at 60 min (equilibrium), respectively. These calculations did not require the corre- sponding measurements of UTP specific radioactivities. The K, of contracting myocytes was 0.075. min" +- 0.012, a value that was 2.1-fold greater than the K, for KC1-depolarized of 0.035 .min" f 0.003, p < 0.01. Calculated values for K. using the UTP specific radioactivities were identical.

Transcription of rDNA in Isolated Myocyte Nuclei-To confirm the observation that contraction stimulated rDNA transcription, run-on transcription was measured employing isolated myocyte nuclei. Isolated myocyte nuclei were used to measure the activity of RNA polymerase I. RNA polymerase I activity was determined by measuring the incorporation of ['HIUTP into RNA in the presence of a-amanitin. In agree- ment with previous studies using cardiac myocyte nuclei (9), complete a-amanitin-insensitive activity to 55% of total tran- scriptional activity was achieved at a concentration of 1 pg/ ml (data not shown). In Fig. 5, the activity of RNA polymerase I is plotted as a function of assay time. RNA polymerase I activity was linear for approximately 5 min and ceased ab-

by guest on Decem

ber 21, 2020http://w

ww

.jbc.org/D

ownloaded from

Transcriptional Regulation of Ribosomal RNA Synthesis

/'

0 2.5 5 7.5 10

Time (min) FIG. 5. RNA polymerase I activity in isolated myocyte nu-

clei. Run-on transcription in isolated myocyte nuclei was measured in the presence of 1 pg of or-amanitinlml by incorporation of [3H] UTP into RNA. Incorporation at each time point was normalized to the number of nuclei added to the reaction as assessed by fluorometric determinations of DNA (27). Similar results were obtained in an experiment in which transcription was measured over 30 min.

TABLE I1 Effects of contraction on RNA polymerase I activity

Nuclei were isolated from myocytes 3 days after the onset of contraction (day 6 in culture). Incorporation of [3H]UTP into RNA was measured as described in Fig. 5. Values are the mean f S.E. of five separate experiments.

Myocytes [3H]UTP Incorporation

pnollmg DNA15 rnin KC1-depolarized 146 f 20

Contracting 236 f 28" Significant difference as determined by Student's t test. p < 0.05.

ruptly, presumably because run-on transcription was com- pleted. Thus, RNA polymerase I activity as measured by 5 min reflected the number of initiated transcription complexes prior to nuclear isolation, and reinitiation did not occur.

RNA polymerase I activity in nuclei isolated from contract- ing and KC1-depolarized myocytes was compared (Table 11). Contraction stimulated RNA polymerase I activity by 62% as measured after 5 min in the presence of 1 pg of a-amanitin/ ml. Similar numbers of nuclei were added as determined by direct measurements of DNA (27). RNA polymerase I activity was linear in the range of DNA added to the assay (data not shown).

Run-on transcription in isolated myocyte nuclei was also determined by hybridization of pre-rRNA transcripts to the recombinant p2.0 rDNA clone. In three separate experiments, there were significant increases in the number of run-on transcripts produced by nuclei derived from contracting myo- cytes (Table 111). The percentage increase in transcription as monitored using the 3H-labeled reference transcript was con- sistent with the increased RNA polymerase I activity observed in contracting myocytes.

DISCUSSION

It was observed previously that synchronous contraction accelerated the rate of rRNA synthesis and was responsible for net rRNA accumulation during myocyte growth. As an extension of these studies, specific rate-limiting reactions for

TABLE I11 Transcription of rDNA in myocyte nuclei

Run-on transcription was measured in myocyte nuclei isolated after 3 days of contraction (day 6 in culture). The recovery of 32P- labeled pre-rRNA/pg of nuclear DNA was monitored by adding a sense 3H-labeled cRNA transcript of p2.0. The 32P/3H ratio measured after hybridization was multiplied by the input cpm of the [3H]cRNA transcript to calculate total cpm of 32P-labeled pre-rRNA. Each experiment represents the mean f S.E. of four run-on reactions and corresponding hybridizations.

Experiments Recovery Difference

c w l f i g DNA % Expt. 1

KC1-depolarized 21 f 1 Contracting 33 f 4" 50

KC1-depolarized 70 f 3 Contracting 105 f 10" 50

KC1-depolarized 50 f 1 Contracting 96 f 15" 92

Expt. 2.

Expt. 3.

" p < 0.05 as determined by Student's t test.

rRNA synthesis such as rDNA transcription were assessed during growth of contracting myocytes. The major finding of this report was that the rate of rRNA synthesis was regulated at the level of transcription of rDNA. The rate of rDNA transcription was accelerated approximately 2-fold by con- traction, identical to the 2-fold acceleration of 18 and 28 S rRNA synthesis rates which reflected the end product of the reactions involved in rRNA synthesis (6). A %fold accelera- tion of the rDNA transcription rate was sufficient to facilitate ribosome accumulation in growing myocytes. This conclusion is supported by the observations that 1) the constitutive rate of rRNA synthesis is high even in nongrowing cardiac myo- cytes (6); and 2) the pool of rRNA is relatively large, with a half-life of approximately 4.5 days in the ribosome (28, 29). Furthermore, in most models of myocyte hypertrophy RNA content was increased 20-50% over 2-3 days presumably because the rate of rRNA degradation increased as rRNA accumulated during myocyte growth (3,5-7).

Information regarding transcription of rDNA upon stimu- lation of myocyte growth could not be ascertained from changes in pre-rRNA levels because of the efficiency of pre- rRNA processing. A dual label hybridization procedure was developed to measure rates of rDNA transcription. The in- corporation of [3H]UTP from the precursor pool into pre- rRNA was measured relative to a sense 14C-labeled cRNA reference transcript of pre-rRNA. This method took advan- tage of the fact that the amount of pre-rRNA relative to total RNA was the same in KC1-depolarized and contracting myo- cytes. The fixed mass ratio between the 3H- and 14C-labeled pre-rRNA in the reaction was used to determine the specific radioactivity of 3H-labeled pre-rRNA, as reflected in the 3H/ 14C ratios measured after hybridization. The [14C]cRNA tran- script also functioned as a recovery marker to calculate the total dpm of [3H]UTP incorporated into pre-rRNA per mg of input RNA. A similar approach of hybridizing radiolabeled RNA relative to an internal standard has been employed to calculate absolute rates of transcription in isolated nuclei and to measure fractional rates of transcription in vivo (20, 26, 30). The dual label method used in these studies provided two additional advantages. 1) The absolute rate of transcription was determined without using total transcription rates. Total transcription rates, particularly when measured by short pulse labeling times, reflect rates of rapidly transcribed mRNA and tRNA genes in addition to the approximate 50% that is attributable to transcription of rDNA genes. 2) The cellular

by guest on Decem

ber 21, 2020http://w

ww

.jbc.org/D

ownloaded from

Transcriptional Regulation of Ribosomal RNA Synthesis 4415

pool of UTP, established as the immediate precursor for rRNA synthesis in cardiac myocytes, was relatively constant over the period that incorporation into pre-rRNA was meas- ured. This simplified the precursor-product relationship for rRNA synthesis measurements.

The nuclear run-on data in isolated nuclei confirmed that contraction stimulated rDNA transcription. The extent of stimulation (approximately 65%) was also similar to that observed for cardiac myocytes after 24 h of norepinephrine treatment (12). Additionally, changes in the rate of rDNA transcription as measured by RNA polymerase I activity have been demonstrated in myocyte nuclei isolated after pressure overload hypertrophy (9, 31). However, run-on transcription reflects only the number of initiated transcripts prior to nuclei isolation (Fig. 5). This distinction may explain the approxi- mately 30% difference between the extent to which the rDNA transcription rate was accelerated in myocytes and in isolated nuclei. Although run-on assays have been used to calculate absolute rates of transcription, these calculations depended upon total transcription rates and also assumed that the elongation rate of RNA polymerase was not modified (20,26).

The rate of rDNA transcription was measured by isolation of entire full-length 45 S pre-rRNA transcripts that contained the sequence complementary to the ETS region. Hybridiza- tion of the recombinant rDNA clone to electrophoretically resolved RNA on Northern blots revealed, in addition to the 45 S molecule, two minor bands that probably represented 43 and 41 S processing intermediates. These intermediates re- tained some of the ETS sequence after processing of the 45 S transcript. The relative amounts of these intermediates were the same in KC1-depolarized and contracting myocytes, and qualitative differences in the processing patterns or intensities of the minor bands were not observed (data not shown). However, a potential rate-limiting processing reaction that regulates the overall synthesis rate of rRNA cannot be ruled out entirely because an intermediate devoid of the ETS se- quence may not have been detected. This possibility has been investigated by probing for more distal processing interme- diates using rDNA clones for regions encoding 18 and 28 S rRNA. To date, no quantitative or qualitative differences in processing have been observed.' Thus, in these studies, rates of transcription were for the full-length molecule of 45 S and any additional intermediate molecules containing a portion of the ETS sequence.

The conclusion that transcription of rDNA regulates ribo- some formation is in agreement with other studies. In skeletal myoblast to myotube transformation, rDNA transcription declines to one-half the rate and is accompanied by a corre- sponding decline in the rate of rRNA formation (19, 20). The decline in transcription was associated with a corresponding decrease in pre-rRNA, and it was concluded that the process- ing rate did not change (20). In contracting myocytes, the amount of pre-rRNA increased in proportion to rRNA con- tent, and the rate of rDNA transcription was accelerated. Based upon these observations, it is concluded that the rate of pre-rRNA processing was unchanged in contracting myo- cytes. Because relative pre-rRNA levels were unchanged by contraction, pre-rRNA processing was not rate limiting for rRNA synthesis. It has not been determined whether the transcription rate eventually declined in contracting myocytes or if changes in pre-rRNA processing rates occurred simul- taneously.

Two primary questions remain in regard to the relationship between rDNA transcription and contractile activity. First, what is/are the specific intracellular signal(s) in contracting

P. J. McDermott and L. L. Carl, unpublished observations.

myocytes responsible for accelerating rDNA transcription? Second, how does an intracellular signal regulate rDNA tran- scription? A specific intracellular signal that mediates rRNA accumulation and growth during contraction has not been identified. The relevant stimulus for growth of contracting myocytes could be derived from changes in membrane poten- tial or, alternatively, physical deformation of the sarcolemma during contraction or expansion of the myocyte in the culture medium. Ion movements may occur through activation of voltage-sensitive Na+ or Ca'+ channels, stretch activated ion channels, Na+-Ca2+ exchange, or release of Ca'+ from intra- cellular sources such as the sarcoplasmic reticulum (32-34). During contraction, activation of protein kinase C could me- diate growth as demonstrated for other models (13). In skel- etal muscle, membrane depolarization and contraction stim- ulated phophatidylinositol turnover and membrane-associ- ated protein kinase C activity (35, 36). In isolated rat heart preparations, phosphatidylinositol turnover in response to stretch has been demonstrated recently (37). Passive stretch of cardiac myocytes in culture stimulated amino acid incor- poration into protein (38, 39). Furthermore, activation of CAMP-dependent protein kinase has been implicated in the regulation of cardiac growth. Stretch of the ventricle in re- sponse to increased intraventricular pressure accelerated the rates of ribosome formation and protein synthesis through a CAMP-dependent mechanism in isolated rat hearts (11). Growth was observed in cultured myocytes after elevating cAMP levels and associated contractility by treatment with adrenergic agonists (40, 41). However, a role for cAMP in mediating growth of spontaneously contracting myocytes is questionable because the activity of CAMP-dependent protein kinase did not change after the onset of contraction in 4 mM KC1 m e d i ~ m . ~ The ability to measure rDNA transcription rates in myocytes using specific growth stimuli should prove useful for evaluation of putative intracellular signals involved in regulating the rate of protein synthesis.

The regulation of rDNA transcription has not been inves- tigated in cardiac muscle. In the upstream region of mam- malian rDNA genes, cis-regulatory elements, including the core promoter, upstream promoter, and enhancer sequences, have been identified (2, 24, 25). Trans-activation of rDNA genes is facilitated through binding of transcription factors to these elements (25,42). The regulation of rDNA transcription may occur by regulating the levels or activities of these factors (2,43). Furthermore, RNA polymerase I activity can be mod- ulated either by direct association with rDNA transcription factors or interaction with additional proteins (44, 45). For example, phosphorylation of a subunit or factor associated with RNA polymerase I may be involved in regulating the transcription rate of rDNA. Other proteins such as nucleolin or DNA topoisomerase I may also be regulatory components for RNA polymerase I activity (46, 47). Further studies are needed to determine how an intracellular signal generated in response to a stimulus for myocyte growth accelerates the rate of rDNA transcription and in turn, ribosome accumula- tion.

Acknowledgments-We thank Drs. Howard Morgan, Cathy Bein- lich, and Lawrence Rothblum for helpful discussions and critical reading of the manuscript and Theresa Nestico and Brian Shoop for preparation of the manuscript.

REFERENCES 1. Hadjiolov, A. A., and Nikolaev, N. (1976) Prog. Biophys. Mol.

Biol. 31,95-144

X. P. Xenophontos and P. J. McDermott, unpublished observa- tions.

by guest on Decem

ber 21, 2020http://w

ww

.jbc.org/D

ownloaded from

4416 Transcriptional Regulation 2. Sollner-Webb, B., and Tower, J. (1986) Annu. Reu. Biochem. 55,

3. Morgan, H. E., Gordon, E. E., Kira, Y., Chua, B. H. L., Russo, L. A., Peterson, C. J., McDermott, P. J., and Watson, P. A. (1987) Annu. Reu. Physiol. 49,533-543

4. McDermott, P. J., and Morgan, H. E. (1989) Circ. Res. 64, 542- 553

5. Peterson, C. J., Whitman, V., Watson, P. A., Schuler, H. G., and Morgan, H. E. (1989) Circ. Res. 64,360-369

6. McDermott, P. J., Rothblum, L. I., Smith, S. D., and Morgan, H. E. (1989) J. Biol. Chem. 264, 18220-18227

7. Siehl, D., Chua, B. H. L., Lautensack-Belser, N., and Morgan, H. E. (1985) Am. J. Physwl. 248, C309-C319

8. Rossi, A., Aussedat, J., Olivaris, J., Ray, A., and Verdys, M. (1984) Eur. Heart J. 5, Suppl. F, 155-162

9. Cutilletta, A. F., Rudnik, M., and Zak, R. (1978) J. Mol. Cell. Cardwl. 10,677-687

10. Camacho, J. A., Peterson, C. J., White, G. W., and Morgan, H. E. (1990) Am. J. Physwl. 268, C86-C91

11. Watson, P. A., Haneda, T., and Morgan, H. E. (1989) Am. J.

12. Long, C. S., Ordahl, C. P., and Simpson, P. C. (1989) J. Clin.

13. Henrich, C. J., and Simpson, P. C. (1988) J. Mol. Cell. Cardwl.

14. Izumo, S., Nadal-Ginard, B., and Mahdavi, V. (1988) Proc. Natl.

15. Simpson, P. C. (1988) Am. J. Cardwl. 62, 13G-19G 16. Wolf, S., Sameshima, M., Leibhaber, S. A., and Schlessinger, D.

17. Veinot-Drebot, L. M., Singer, R. A., and Johnston, G. C. (1988)

18. Krauter, K. S., Soeiro, R., and Nadal-Ginard, B. (1979) J. Mol.

19. Jacobs, F. A., Bird, R. C., and Sells, B. H. (1985) Eur. J. Biochem.

20. Bowman, L. H. (1987) Deu. Biol. 119,152-163 21. Warner, J. R. (1977) J. Mol. Biol. 115, 315-333 22. Tsurugi, K., and Ogata, K. (1979) Eur. J. Biochem. 101, 205-

23. Rothblum, L. I., Parker, D. L., and Cassidy, B. (1982) Gene

24. Cassidy, B. G., Yang-Yen, H., and Rothblum, L. I. (1987) Mol.

801-830

Physiol. 256, C1257-C1261

Znuest. 83, 1078-1082

20, 1081-1085

Acad. Sci. U. S. A. 85, 339-343

(1980) Biochemistry 19,3484-3490

J. Mol. Biol. 199, 107-113

Biol. 134, 727-741

150,255-263

213

(Amst.) 17, 75-77

Cell. Biol. 7, 2388-2396

of Ribosomal RNA Synthesis 25. Smith, S. D., Oriahi, E., Yang-Yen, H., Xie, W., Chen, C., and

Rothblum, L. I. (1990) Nucleic Acids Res. 18, 1677-1685 26. McKnight, G. S., and Palmiter, R. D. (1979) J. Biol. Chem. 254,

27. Cesarone, C. F., Bolognesi, C., and Santi, L. (1979)Anal. Biochem.

28. Ashford, A. J., and Pain, V. M. (1986) J. Biol. Chem. 261,4059-

29. Lardeux, B. R., Heydrick, S. J., and Mortimore, G. E. (1987) J.

30. Watson, G., and Paigen, K. (1988) Mol. Cell. Biol. 8, 2117-2124 31. Schreiber, S. S., Oratz, M., and Rothschild, M. A. (1969) Am. J.

32. Blaustein, M. P. (1988) J. Cardiovasc. Pharmacol. 12, Suppl. 5,

33. Morris, C. E. (1990) J. Membr. Biol. 112,93-107 34. Kent, R. L., Hoober, K., and Cooper, G., IV (1989) Circ. Res. 64,

35. Vergara, J., Tsien, R. Y., and Delay M. (1985) Proc. Natl. Acad.

36. Richter, E. A,, Cleland, P. J. F., Rattigan, S., and Clark, M. G.

37. von Harsdorf, R., Lang, R. E., Fullerton, M., and Woodcock, E. A. (1989) Circ. Res. 65,494-501

38. Komuro, I., Kaida, T., Shibazaki, Y., Kurabayashi, M., Katoh, Y., Hoh, E., Takaku, F., and Yazaki, Y. (1990) J. Biol. Chem.

39. Mann, D. L., Kent, R. L., and Cooper, G., IV (1989) Circ. Res.

40. Rochna-Singh, K. J., Simpson, P. C., and Karliner, J. S. (1989)

41. Dubus, I., Samuel, J.-L., Marotte, F., Delcayre, C., and Rappa-

42. Bell, S. P., Learned, R. M., Jantzen, H., and Tjian, R. (1988)

43. Zahradka, P., and Sells, B. H. (1988) Eur. J. Biochem. 171, 37-

44. Tower, J., and Sollner-Webb, B. (1987) Cell 50,873-883 45. Bateman, E., and Paule, M. R. (1986) Cell 47,445-450 46. Belenguer, P., Baldin, V., Mathian, C., Prats, H., Bensaid, M.,

Bouche, G., and Amalric, F. (1989) Nucleic Acids Res. 17,

47. Zhang, H., Wang, J. C., and Liu, L. F. (1988) Proc. Natl. Acad.

9050-9058

100,188-197

4065

Biol. Chem. 262,14507-14513

Physiol. 271,1305-1309

S56-S68

74-85

Sci. U. S. A. 82,6352-6356

(1987) FEBS Lett. 217, 232-236

265,3595-3598

64,1079-1090

Circulation 80, Suppl. 2, 93 (abstr.)

port, L. (1990) Circ. Res. 66,867-874

Science 241, 1192-1197

43

6625-6636

Sci. U. S. A. 85,1060-1064

by guest on Decem

ber 21, 2020http://w

ww

.jbc.org/D

ownloaded from

P J McDermott, L L Carl, K J Conner and S N Allomyocytes in culture.

Transcriptional regulation of ribosomal RNA synthesis during growth of cardiac

1991, 266:4409-4416.J. Biol. Chem. 

  http://www.jbc.org/content/266/7/4409Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/266/7/4409.full.html#ref-list-1

This article cites 0 references, 0 of which can be accessed free at

by guest on Decem

ber 21, 2020http://w

ww

.jbc.org/D

ownloaded from