BIOCHEMICAL
MEDICINE
13, 127-137 (1975)
Comparisons from
of RNA
the
Breast
and
Dystrophic
Populations
Muscle
of Normal
Chickens
MICHAEL W. NEAL' AND JAMES R. FLORINI Biology
Department, Syracuse University, Syracuse, New York 13210
Received March 24. 1975
Progressive loss of muscle mass is a principal symptom of muscular dystrophy; it might reasonably be inferred that reduced synthesis of RNA and a resultant decrease in protein synthesis could cause this loss. Several years ago, Baieve and Florini (1) reported a lesser activity of the Mn2+-activated RNA polymerase in nuclei from dystrophic (compared with normal) chicken muscle. They suggested that a depressed rate of mRNA synthesis could decrease protein synthesis in dystrophic muscle. However, Battelle and Florini (2) subsequently found that the lowered activity of ribosomes from muscle of very young dystrophic chicks did not result from a deficiency in mRNA content of the preparation; indeed, RNA synthesis has been reported to be elevated when studied in dystrophic chickens in vivo (3). Measurement of RNA synthesis in dystrophic muscle is complicated by changes in levels of nucleases associated with the disease (4-6). Interpretation of the results is complicated by necrosis and the invasion of macrophages and other cell types (7, 8) in dystrophic muscle. Thus it is not possible to attribute a causal role to nuclease changes observed late in the progress of muscular dystrophy. The experiments reported here were done in an attempt to explain the apparent discrepancy between the earlier reports (1, 2) from this laboratory. In addition, we wished to compare populations of RNA formed in muscle to assess the possible role of abnormalities in RNA synthesis or degradation during the early stages of muscular dystrophy in chickens. We conclude that muscular dystrophy in chickens does not result from a deficiency in synthesis of messenger RNA or a detectable change in other populations of RNA, even at early stages of the disease when decreased protein synthesis is observed (2). * Current address: Pathology Department, New York 13210.
SUNY Upstate Medical Center, Syracuse,
127 Copyright Ail rights
@ 1975 by Academic Press, Inc. of reproduction in any form reserved.
128
NEAL
AND
FLORINI
MATERIALS Normal white Leghorn chickens were hatched from fertilized eggs supplied by a local distributor (Ray Sachs Farms, Camillus, NY). Dystrophic fertilized eggs were supplied by Dr. Louis Pierro of the Department of Animal Genetics, University of Connecticut, Storrs, Connecticut. Normal and dystrophic eggs were incubated in a Petersime #1 incubator under carefully controlled temperature (37”) and humidity (55%) until hatching. Chickens were kept in the incubator for two days after hatching, after which they were removed to a brooder. [14C]Uridine, [3H]uridine, and [3H]uridine triphosphate were purchased from New England Nuclear or Schwartz BioResearch. The PPO (2,5-diphenyl-oxazole) and POPOP (1,4-bis[2-(-4-dimethyl-5-phenyloxazolyl)] used for scintillation fluid was obtained from Fisher Scientific, and NCS from Amersham Searle. The RNase inhibitors PVS (polyvinylsulfate) and DEOF (diethyloxydoformate) were purchased from General Biochemicals and Eastman Chemicals, respectively. Medium 199 was obtained from Grand Island Biological Company, and oligo-dT-cellulose from Research Associates, Inc. Phenol was purchased from Baker and purified by distillation under vacuum and stored frozen at -20” as a 90% solution. METHODS Labeling
of RNA
in Vitro
Muscle from 3 day old chickens was obtained from animals killed by decapitation. The breast muscle was removed quickly and placed in icecold Medium 199 until all animals were killed. The muscle was sliced perpendicular to the fibers on a bed of razor blades 1.0 mm apart, and the minced muscle placed in a flask (prewarmed to 37”) containing 5 ml of Medium 199 per breast muscle and 5 $Zi labeled uridine. The muscle was incubated in a CO2 incubator (95% air, 5% CO, with saturated H,O atmosphere) at 37” for 2 hr. The flasks were agitated every 30 min to insure equilibration of nutrients. At the end of the incubation the muscles were removed, placed on a stainless steel screen, and washed with 10 ml cold phosphate buffered saline (0.01 M phosphate and 0.14 M NaCl pH 7.6). Following the wash the muscle was immediately placed in a test tube and frozen in a dry ice-ethanol bath. The muscle was stored at -20” until used in RNA extraction. Using these procedures, muscle minces from both normal and dystrophic chickens exhibited linear incorporation of radioactive uridine for at least 2 hr. RNA Isolation
Frozen muscle was weighed and combined with 9 vol of 0.1 M TrisHCl, pH 9.0 and 100 pg/ml PVS. The muscle was homogenized for 90 set with a Polytron 10 OD homogenizer at setting 5. To the homo-
RNA
OF
NORMAL
AND
DYSTROPHIC
CHICKS
129
genate 0.1 vol of 1% sodium dodecylsulfate (SDS) in the above buffer was added. The SDS was added after homogenization to avoid foaming. The homogenate was allowed to stir at room temperature for 5 min followed by the addition of an equal volume of 90% redistilled phenol, and vigorous stirring continued for 25 min at room temperature. The solution was then cooled to 0” by stirring in an ice bath for 15 min. The phenol and aqueous phases were separated by centrifugation at 7,OOOg for 10 min at 4”. The aqueous phase was removed and made 0.2 M NaCl by the addition of 0.05 vol of 4 M NaCl followed by extraction with 0.5 vol of 90% phenol for 10 min at room temperature. The aqueous phases were combined and precipitated by the addition of 2 vol cold ethanol. The phenol phase from the first extraction was reextracted with 0.5 vol 0.1 M Tris, pH 9.0 by stirring for 10 min at room temperature. After phase separation, the aqueous layer was combined with the phenol phase from the second phenol extraction and stirred for an additional 10 min at room temperature. The phases were separated and the RNA precipitated with 2 vol cold ethanol. The ethanol precipitates were combined and allowed to stand at -20” overnight before proceeding. RNA was collected from the ethanol by centrifugation and dissolved in 1.5 ml of 10 mM Tris, pH 7.6 and 1.0 mM MgClz to which was added an equal volume of 40 pg/ml electrophoretically pure DNase I (RNase free) dissolved in the same buffer. The reaction mixture was incubated for 15 min at 4” followed by an additional incubation for 5 min at 37”. The reaction was stopped by adding 0.1 vol of a solution containing 1.0 M Tris. pH 9.0, and 1% SDS followed immediately by an equal volume of 90% phenol. The mixture was stirred for 10 min at room temperature, phases were separated by centrifugation, and the aqueous phase then reextracted with 0.5 volumes 90% phenol. After centrifugation, the aqueous phase was made 0.2 M NaCl as above and the RNA precipitated with 2 vol cold ethanol. The RNA was allowed to stand overnight at -20”. RNA obtained by this procedure had no detectable protein or DNA contamination. The Az6,,/Azsoratio was always between 1.8 and 2.0 with yields of approximately 1 mg/g muscle. The RNA was stored as the ethanol precipitate at -20” until used. Analytical
Procedures
The ethanol-precipitated RNA was collected by centrifugation, and the pellet dissolved in 1.5 to 2.0 ml of 10 mM Tris, pH 7.5. The sample was chromatographed on 1.5 ml of oligo-dT-cellulose by the procedure of Aviv and Leder (9). In order to precipitate with ethanol the poly (A)containing RNA eluted from the column, the solution was made 50 pg/ml with carrier tRNA isolated by the method of Bloemendal (10). This concentration of tRNA was shown to give maximal precipitation without causing detectable distortion in the subsequent sedimentation analyses.
130
NEAL
AND
FLORINI
Sucrose gradients (lO-50% w/v) were prepared and centrifuged at 40,000 rpm for 14 hr in an SB 283 rotor with an International B-60 ultracentrifuge as described previously (11). For the total and oligo-dTcellulose excluded RNA, 150 pg samples were layered on the gradients; approximately 50 pug of poly(A)-containing RNA (eluted from oligo-dTcellulose) containing 4 mg carrier tRNA was layered on gradients. Following centrifugation, gradients were fractionated on an ISCO 180 gradient fractionator and continuously scanned at 254 nm with an ISCO UA-2 ultraviolet analyzer; 10 drop fractions were taken. To each fraction 1 mg carrier RNA was added, and the sample precipitated with 10% TCA. After washing with an additional 5 ml 10% TCA, the RNA pellets were hydrolyzed in 0.2 ml NCS mixed with toluene phosphor and counted in a Nuclear-Chicago Unilux II liquid scintillation counter equipped with an external standard. Standard curves of counting efficiency vs channels ratio were prepared for each experiment, and the efficiency of counting for each sample determined by the standard channels ratio technique. RNase activity was measured by the RNA agar gel diffusion procedure described by Tan (12). In this method the RNase activity was related to the hydrolysis of RNA trapped in agar gels. Following incubation for 2 hr at 37” the undigested RNA was fixed with 1.0 N HCI and the gels photographed. Tracings were made of the negatives on a Beckman film densitometer. From these tracings the diameter of the circle created by the diffusing RNase (x) and the diameter of the well (y) were measured. The quantity of RNase in the well was proportional to x2-y”. Isolation
of Muscle
Nuclei
and Assay
of RNA
Polymerase
Activity
Nuclei were isolated from 4 day old chickens by the modification of the procedure of Nair et al. (13) described by Baieve and Florini (1). The nuclear preparations were examined under a compound microscope after staining with 1% (w/v) crystal violet/acetic acid and appeared to have little contamination and few damaged nuclei. There was no apparent difference between nuclei of normal and dystrophic muscle. Following isolation the nuclei were assayed immediately for RNA polymerase activity. Each assay tube contained (in a final volume of 0.4 ml) 50 pmoles Tris, pH 8.0; 25 pmoles sucrose; 0.4 pmoles each ATB, CTP, and GTE; 3 pmoles NaF; 10 pmoles KCl; 5 pmoles creatine phosphate; 10 pg creatine phosphokinase; 2 &i[3H]UTP and either 1 pmole MgCl, or 8 pmoles MnCl,, 0.4 M (NHJ,SO,. The reaction solution was warmed to 37” and the reaction started by the addition of 0.1 ml of the suspension of nuclei. At the times indicated, the reaction was stopped by adding 2 ml of cold 10% TCA with concommitant vigorous mixing on a Vortex. The samples were allowed to precipitate in the cold for 2 hr and KOH-hydrolyzable radioactivity determined. The DNA con-
RNA OF NORMAL
tent of the nuclear reaction (14).
suspension
AND
DYSTROPHIC
was determined
CHICKS
131
by the diphenylamine
RESULTS RNase Activity Muscle was homogenized in 0.1 M Tris, pH 9.0 and RNase activity measured in order to obtain not only an estimate of the RNase levels in muscle, but also activity of RNase under the ionic and pH conditions used for RNA isolation. The level of RNase in the dystrophic muscle homogenate was more than twice that of normal muscle (Fig. 1). Although this was only a crude estimate of RNase levels (optimal conditions for the enzyme were not determined), it was apparent that differences in degradation of RNA during isolation could cause difficulties in interpretation of the results. Two frequently used RNase inhibitors DEOF and PVS were examined for their effectiveness with muscle homogenized in 0.1 M Tris, pH 9.0 (Fig. 1). DEOF was not effective in this system, and actually produced a slight stimulation of RNase activity, possibly by releasing membranebound enzyme. On the other hand, PVS reduced the RNase level of the homogenate of both the normal and dystrophic muscle to an undetectable level. PVS at 100 pg/ml was included in the RNA isolation medium to minimize the effects of RNase during the isolation of RNA. Comparisons of Labeled RNA Preparations Double-labeled mixed-fractionation analysis was used to detect any qualitative or relative quantitative differences between RNA isolated
Cmhd
PVS
DEOF
1. RNase activity and effectiveness of RNase inhibitors in muscle homogenates. The breast muscle of normal and dystrophic chickens were removed and homogenized in RNA isolation buffer (0.1 M Tris, pH 9.0) or buffer containing RNase inhibitors (either 100 pg/ml PVS or 0.5% DEOF). RNA agar gels were prepared with 4 ~1 of homogenate in each well and the slides incubated and analyzed as described in Methods. Protein concentration in the homogenates were determined by the method of Lowry, and all data expressed as RNase activity per mg protein (mm2/mg protein). The solid bars represent RNase activity in normal muscle, and the hatched bars represent RNase activity in dystrophic muscle. FIG.
132
NEAL
AND
FLORINI
from normal and dystrophic chickens. When this procedure was applied to RNA isolated from normal and dystrophic muscle, there was no detectable difference in the 3H to 14C ratio in comparisons among total RNA and two major fractions, one rich in poly(A)-containing RNA and the other rich in non-poly(A)-containing RNA, obtained from the oligodT-cellulose column. In our determinations of radioactivity, enough counts were accumulated to obtain an accurcy of at least +-2%. Subsequent analysis of the RNA fractions by centrifugation through sucrose gradients revealed no substantial differences between the RNA from normal or dystrophic animals. The sucrose gradient of total RNA (Fig. 2A) showed a typical optical density profile with major peaks attributed to tRNA and rRNA. In the area of the tRNA peak, a slight decrease in the relative labeling of dystrophic RNA occurred. Although this difference persisted with duplicate gradients of the same RNA and in repeated experiments, it does not seem large enough to be considered a major effect of the disease.
Fmction
No.
Fraction
Fraction
No.
No.
FIG. 2. Sucrose gradient analysis of RNA from normal and dystrophic chickens. Brease muscle from 3 day old normal chickens was incubated with [‘4C]uridine and muscles from 3 days old dystrophic chickens with [3H]uridine as described in Methods. The muscles were combined and the RNA extracted and analyzed on l&50% sucrose gradients and the radioactivity determined as described in Methods. Three fractions were analyzed: A. Total cellular RNA, B. RNA excluded from an oligo-dT-cellulose column and C. RNA which bound to an oligo-dT-cellulose column. The broad solid line represents the absorbancy at 254 nm and the dotted line the percent of the total disintegrations of 14Cin each fraction, and the narrow solid line the percent of the total disintegrations of 3H in each fraction. The direction of sedimentation was from left to right.
RNA OF NORMAL
AND DYSTROPHIC
133
CHICKS
In order to determine that the tRNA and rRNA, which comprise 97% of the isolated RNA, were not masking differences in the poly(A)-contaming RNA’s sedimentation analyses of the oligo-dT-cellulose excluded (Fig. 2B) and bound (Fig. 2C) fractions were performed. The optical density profile of the excluded RNA consisted of 4 S, 18 S and 28 S peaks, while the bound RNA showed the predominant peak at the top of the gradient due to the carrier tRNA added (see Methods), followed by a broad peak of heterogeneous RNA. The radioactivity profile of the excluded RNA was very similar to that obtained with the total RNA, showing no differences except for a small depression of the dystrophic RNA in the 4 S region. The profiles of radioactivity for the poly(A)-containing RNA’s of normal and dystrophic muscle were virtually identical. There appear to be no relative quantitative differences between normal and dystrophic RNA’s labeled for 2 hr under our conditions. To establish that the small differences between normal and dystrophic RNA were within experimental variation, RNA from two normal chickens was compared. The breast muscle of one normal animal was labeled with r3H]uridine while the breast muscle of another normal chicken was labeled with [14C]uridine. The muscles were combined and extracted as before. The optical density and radioactivity profiles obtained from sucrose gradient analyses of the oligo-dT-cellulose excluded and bound RNA’s (Fig. 3) were similar to those observed in the comparison of normal and dystrophic RNA’s. This demonstrates that the observed variation between normal and dystrophic RNA was no greater than the usual variation due to individual animals and counting procedures.
Fraction
No.
FIG. 3. Sucrose gradient analysis of RNA from normal muscle. RNA from normal muscles labeled with [3H] and [14C]uridine was extracted and fractionated on oligodT-cellulose as described in Methods. The RNA was analyzed on a lO- 50% sucrose gradient and the radioactivity determined as described in Methods. Two fractions were analyzed: A. RNA excluded from an oligo-dT-cellulose column, and B. RNA which bound to an oligo-dTcellulose column. The broad solid line represents the absorbancy at 254 nm and the dotted line the percentage of the total disintegrations of 14Cin each fraction, and the narrow solid line the percent of the total disintegrations of 3H in each fraction. The direction of sedimentation was from left to right.
134
NEAL
IJ-jq 5 10
Time
AND
FLORINI
I~ 15
(min)
12 Time
3 (min)
4
5
FIG. 4. Time course of [3H]UTP incorporation into RNA by isolated nuclei from chicken breast muscle. Nuclei were prepared from 4 day old chickens and the RNA polymerase activity assayed as described in Methods. The nuclei were assayed over long (A) and short (B) time courses. The solid line indicates normal and the broken line dystrophic muscle. The circles indicate nuclei assayed in the presence of 16 mM MnCl,, and the triangles indicated nuclei assayed in the presence of 2 mM MnCI, and 0.4 M (NH&SO,.
RNA Polymerase Activity in Isolated Nuclei Nuclei from normal and dystrophic chickens were isolated and assayed following the procedure of Baieve and Florini (2); the results are summarized in Fig. 4A. The Mg2+ dependent activities were the same in normal and dystrophic nuclei, while the Mn2+, (NH&SO, dependent activity of dystrophic nuclei was 45% lower than the same RNA polymerase activity in normal nuclei at 5 min. However, the rate of incorporation of [3H]UTP into RNA began to decrease between 5 and 10 min and reached a plateau by 15 min in the case of both the Mg2+ and the Mn2+, (NH&SO, dependent activities. To determine whether incorporation was linear between zero and 5 min, the RNA polymerase activities were measured at shorter times (Fig. 4B). When these shorter time periods were used, the Mg 2+ dependent activities of normal and dystrophic nuclei were still identical and the incorporation appeared linear up to 5 min. The differences in the MI+‘+, (NH&SO4 dependent activity observed above and in previous studies (1) were not detected at the initial stages of the reaction, but only began to appear after 2.5 min incubation. At that time the rate of incorporation of L3H]UTP into RNA in both normal and dystrophic nuclei began to decrease with a greater rate decrease occurring in the dystrophic nuclei. During the first 2.5 min of incubation when both the normal and dystrophic nuclei showed linear incorporation there was no perceptible difference in the Mn2+, (NH,),SO, dependent RNA polymerase activity of normal and dystrophic nuclei. DISCUSSION
Although RNase activity in homogenates from dystrophic muscle was twice that in normal muscle, there was no observable shift to slower sedimenting RNA on sucrose gradients. This lack of degradation sup-
RNA
OF
NORMAL
AND
DYSTROPHIC
CHICKS
135
ports the suggestion of Pennington (8) that these increases in catabolic enzymes are due to invading macrophages which are in the process of catabolizing dead cells. Moosa (7) and Zellweger (15) have observed necrotic cells in human dystrophy; thus the heightened levels of catabolism in dystrophic muscle might be the secondary result of removal of dead cells, rather than a primary cause of the disease. Measurements of catabolic enzymes in such a heterogeneous system as dystrophic muscle must be interpreted with caution until it can be ascertained what cell types are producing the enzyme. We found no difference in the RNA populations labeled in muscle minces in vitro. With oligo-dT-cellulose, two fractions of RNA were produced: A) RNA which did not bind to the column and did not contain poly(A) segments (i.e., rRNA, tRNA and their precursors (16)), and B) RNA which bound to the column and contains poly(A) segments (i.e., mRNA, except histone message, and some heterogeneous nuclear RNA (17-20)). The suggestion of Baieve and Florini (1) that RNA polymerase II activity was decreased in dystrophic muscle led us to expect a decrease in the amount of label in the poly(A)-containing RNA fraction of RNA isolated from this muscle. However, we did not observe any difference in the ratio of poly(A)-containing RNA to non-poly(A)-contaming RNA between normal and dystrophic animals. Furthermore, sucrose gradient centrifugation revealed no qualitative differences greater than those observed when comparing RNA samples from two normal chickens. This lack of qualitative differences in the RNA agrees with preliminary studies of Battelle and Florini (2) which showed no qualitative difference in the electrophoretic profile of in vitro labeled proteins; similarly, Oppenheimer and Markiewicz (21) found no differences in the RNA of isolated ribosomes from normal and dystrophic chickens. It appears that in dystrophic muscle the RNA’s observed by pulse labeling were quantitatively and qualitatively similar to those in normal chicken muscle. Even the later increase in protein synthesis in dystrophic muscle cannot be attributed to changes in RNA; Weinstock and Markiewicz (22) have recently demonstrated that this change is attributable to increases in activity of elongation factors. The results presented in Fig. 4 provide a possible explanation of the discrepancy between the observations of Baieve and Florini (l), which indicated a decrease in mRNA synthesis, and those of Battelle and Florini (2), who found no decrease in mRNA content of ribosome preparations from young dystrophic chickens. We now believe that the apparent depression of activity of the Mn2+-stimulated RNA polymerase in muscle of dystrophic animals can be attributed to an unusually rapid loss of activity of the enzyme upon incubation of the isolated nuclei. Our present results demonstrate that there is no difference in this activity between nuclei from normal and dystrophic muscle during the first few
136
NEAL
AND
FLORINI
minutes of the incubation, but there is a more rapid loss of activity in nuclei from diseased animals. This more rapid loss might be explained by one or more of the following phenomena. Blobel and Potter (23) observed by electron microscopy that approximately 75% of purified liver nuclei showed membrane damage, while Cox et al. (24) showed that homogenization of the tissue caused extensive damage to the DNA in intact nuclei in the form of single strand nicks. These nicks were probably caused by the action of DNase during homogenization and isolation. It is highly likely that DNA in muscle nuclei is being degraded during isolation and assay of RNA polymerase. A recent report by Flint et al. (2.5) indicates that the isolated RNA polymerase activities of liver were considerably altered by changes in the integrity of the DNA template. Although double stranded breaks had the greatest inhibitory effect, it was suggested that factors not coisolated with the purified enzyme might further limit the activity of RNA polymerase to intact DNA in a similar manner as shown for sigma factor and core enzyme of Escherichia coli (26). Eukaryotic initiation and termination factors are presently being studied for the RNA polymerase of coconut (27 and 28) and elongation factors for the enzyme from rat liver (29), but little is yet known of how these factors can alter the activity of the RNA polymerase. Although it is difficult to draw conclusions from such diverse biological sources, it appears that the RNA polymerase assay system of isolated nuclei is very complex; there are many processes which may alter the observed activity of RNA polymerase. Although the results of RNA polymerase measurements in isolated nuclei must be interpreted with caution, the similarity in normal and dystrophic chicken nuclei of both Mn2+ and Mg2+ RNA polymerase activities during the early portion of the time course agrees with the observed similarity of the RNA produced by these cells. Differences in catabolic enzymes (such as DNase) coupled with possible differential mechanical damage to the nuclei during isolation may have contributed to the more rapid decrease in RNA polymerase activity in the nuclei from dystrophic chickens. As far as could be detected, it appeared that the 3 day old dystrophic chicken breast muscle synthesized the same types and relative quantities of RNA as muscle from normal chickens. SUMMARY
RNA isolated from 3 day old normal and dystrophic chickens showed no qualitative or relative quantitative differences, when compared by oligo-dT-cellulose chromatography and sedimentation analysis. These two RNA populations were similar even though the RNase activity was double normal values in muscle homogenates from dystrophic animals. Measurements of the Mn2+ and Mg2+ RNA polymerase activities were made in isolated nuclei from both normal and dystrophic chickens.
RNA
OF
NORMAL
AND
DYSTROPHIC
CHICKS
137
Measurements made at short time periods showed no difference in the activity between diseased and control animals, while measurements at incubation times of 5 min or greater showed a preferential decrease of approximately 55% in the Mn2+ RNA polymerase activity of the nuclei from dystrophic chickens. These results explain differences in previous experiments; we now conclude that there are no significant differences in the RNA polymerase activities of normal and dystrophic chicken muscle. ACKNOWLEDGMENT This investigation was supported by a grant from the Muscular Dystrophy Associations of America.
REFERENCES 1. Baieve, D., and Florini. J. R., Arch. Biochem. Biophys. 139, 393 (1970). 2. Battelle, B., and Florini. J. R., Biochemistry 12, 635 (1973). 3. Weinstock, I. M., Bondar, M., Blanchard, K. R.. and Arslan-Contin, P.. Biochim. Biophys. Acta 277, 96 (1972). 4. Tappel, A. L., Zalkin, H., Caldwell. K. A., Desai. I. D., and Shibko, S.. Arch. Biothem. Biophys. 96, 340 (1962). 5. Mey, W. L., Little, B. W., Faussner, J. R., and Mayer, D. M., in “Research in Muscle Development and Muscle Spindle” (B. Q. Banker, R. J. Przybylski. J. P. Vander Meulen and M. Victor, Eds.), pp. 195-217. Exerpta Medica Foundation, New York, 1971. 6. Berlinquet, L. and Srivastava, U., Can. J. Biochem. 44, 613 (1966). 7. Moosa, A., Dev. Med. Child. Neurol. 16, 97 (1974). 8. Pennington, R. J., Biochem. J. 88, 64 (1963). 9. Aviv. J., and Leder, P.. Proc. Nat. Acad. Sci. USA 69, 1408 (1972). 10. Bloemendal. H.. Huizinga, F., DeVries, M., and Bosh, L.. Biochim. Biophys. Actu 61, 209 (1962). 11. Neal, M. W., and Florini, J. R., Anal. Biochem. 45, 271 (1972). 12. Tan, C. H., Biochim. Biophys. Acta 247, 463 (1971). 13. Nair, K. G.. Rabinowitz, M., and Chen Tu, M.. Biochemistry 6, 1898 (1967). 14. Burton, K. A.. Biochem. J. 65, 315 (1956). 15. Zellweger, H., Dumin, R., and Simpson, J., Acta Neural. Stand. 48, 87 (1972). 16. Eiden, J. J., and Nichols, J. L., Biochemisrry 12, 3951 (1973). 17. Adesnik, M.. Salditt, M., Thomas, W.. and Darnell. J. E., 1. Mol. Biol. 71, 21 (1972). 18. Brawerman, G., Ann. Rev. Biochem. 43, 621 (1974). 19. Sheldon, R.. Kates, J., Kelley, D. E., and Perry, R. P., Biochemistr?/ 11, 3829 (1972). 20. Adesnik, M.. and Darnell. J. E., J. Mol. Biol. 67, 397 (1972). 21. Oppenheimer, H., and Markiewicz, L., B&hem. Med. 7, 479 (1973). 22. Weinstock. I. M., and Markiewicz, L.. B&him. Biophys. Actu 374, 197 (1974). 23. Blobel, G., and Potter, V. R., Science 154, 1662 (1966). 24. Cox. R., Damjanov, I., Abanobi, S. E., and Sarma. D. S. R.. Cancer Res. 33, 2114 (1973). 25. Flint. S. J., DePormerai, D. I., Chesterton, C. J., and Butterworth. P. H. W., Eur. J. B&hem. 42, 567 (1974). 26. Dausse, J., Sentenac, A., and Fromageot. P., Eur. J. Biochem. 26, 43 (1972). 27. Mondal, H., Mandal, R. K., and Biswas. B. B., Eur. J. Biochem. 25, 463 (1972). 28. Mondal, H., Ganguly, A., Das, A., Mandal, R. K., and Biswas. B. B.. Eur. J. Biochem. 28, 463 (1972). 29. Seifart, K. H.. Juhasz, P. P.. and Benecke, B. J.. Eur. J. Biochem. 33, 181 (1973).
MEDICINE
13, 127-137 (1975)
Comparisons from
of RNA
the
Breast
and
Dystrophic
Populations
Muscle
of Normal
Chickens
MICHAEL W. NEAL' AND JAMES R. FLORINI Biology
Department, Syracuse University, Syracuse, New York 13210
Received March 24. 1975
Progressive loss of muscle mass is a principal symptom of muscular dystrophy; it might reasonably be inferred that reduced synthesis of RNA and a resultant decrease in protein synthesis could cause this loss. Several years ago, Baieve and Florini (1) reported a lesser activity of the Mn2+-activated RNA polymerase in nuclei from dystrophic (compared with normal) chicken muscle. They suggested that a depressed rate of mRNA synthesis could decrease protein synthesis in dystrophic muscle. However, Battelle and Florini (2) subsequently found that the lowered activity of ribosomes from muscle of very young dystrophic chicks did not result from a deficiency in mRNA content of the preparation; indeed, RNA synthesis has been reported to be elevated when studied in dystrophic chickens in vivo (3). Measurement of RNA synthesis in dystrophic muscle is complicated by changes in levels of nucleases associated with the disease (4-6). Interpretation of the results is complicated by necrosis and the invasion of macrophages and other cell types (7, 8) in dystrophic muscle. Thus it is not possible to attribute a causal role to nuclease changes observed late in the progress of muscular dystrophy. The experiments reported here were done in an attempt to explain the apparent discrepancy between the earlier reports (1, 2) from this laboratory. In addition, we wished to compare populations of RNA formed in muscle to assess the possible role of abnormalities in RNA synthesis or degradation during the early stages of muscular dystrophy in chickens. We conclude that muscular dystrophy in chickens does not result from a deficiency in synthesis of messenger RNA or a detectable change in other populations of RNA, even at early stages of the disease when decreased protein synthesis is observed (2). * Current address: Pathology Department, New York 13210.
SUNY Upstate Medical Center, Syracuse,
127 Copyright Ail rights
@ 1975 by Academic Press, Inc. of reproduction in any form reserved.
128
NEAL
AND
FLORINI
MATERIALS Normal white Leghorn chickens were hatched from fertilized eggs supplied by a local distributor (Ray Sachs Farms, Camillus, NY). Dystrophic fertilized eggs were supplied by Dr. Louis Pierro of the Department of Animal Genetics, University of Connecticut, Storrs, Connecticut. Normal and dystrophic eggs were incubated in a Petersime #1 incubator under carefully controlled temperature (37”) and humidity (55%) until hatching. Chickens were kept in the incubator for two days after hatching, after which they were removed to a brooder. [14C]Uridine, [3H]uridine, and [3H]uridine triphosphate were purchased from New England Nuclear or Schwartz BioResearch. The PPO (2,5-diphenyl-oxazole) and POPOP (1,4-bis[2-(-4-dimethyl-5-phenyloxazolyl)] used for scintillation fluid was obtained from Fisher Scientific, and NCS from Amersham Searle. The RNase inhibitors PVS (polyvinylsulfate) and DEOF (diethyloxydoformate) were purchased from General Biochemicals and Eastman Chemicals, respectively. Medium 199 was obtained from Grand Island Biological Company, and oligo-dT-cellulose from Research Associates, Inc. Phenol was purchased from Baker and purified by distillation under vacuum and stored frozen at -20” as a 90% solution. METHODS Labeling
of RNA
in Vitro
Muscle from 3 day old chickens was obtained from animals killed by decapitation. The breast muscle was removed quickly and placed in icecold Medium 199 until all animals were killed. The muscle was sliced perpendicular to the fibers on a bed of razor blades 1.0 mm apart, and the minced muscle placed in a flask (prewarmed to 37”) containing 5 ml of Medium 199 per breast muscle and 5 $Zi labeled uridine. The muscle was incubated in a CO2 incubator (95% air, 5% CO, with saturated H,O atmosphere) at 37” for 2 hr. The flasks were agitated every 30 min to insure equilibration of nutrients. At the end of the incubation the muscles were removed, placed on a stainless steel screen, and washed with 10 ml cold phosphate buffered saline (0.01 M phosphate and 0.14 M NaCl pH 7.6). Following the wash the muscle was immediately placed in a test tube and frozen in a dry ice-ethanol bath. The muscle was stored at -20” until used in RNA extraction. Using these procedures, muscle minces from both normal and dystrophic chickens exhibited linear incorporation of radioactive uridine for at least 2 hr. RNA Isolation
Frozen muscle was weighed and combined with 9 vol of 0.1 M TrisHCl, pH 9.0 and 100 pg/ml PVS. The muscle was homogenized for 90 set with a Polytron 10 OD homogenizer at setting 5. To the homo-
RNA
OF
NORMAL
AND
DYSTROPHIC
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129
genate 0.1 vol of 1% sodium dodecylsulfate (SDS) in the above buffer was added. The SDS was added after homogenization to avoid foaming. The homogenate was allowed to stir at room temperature for 5 min followed by the addition of an equal volume of 90% redistilled phenol, and vigorous stirring continued for 25 min at room temperature. The solution was then cooled to 0” by stirring in an ice bath for 15 min. The phenol and aqueous phases were separated by centrifugation at 7,OOOg for 10 min at 4”. The aqueous phase was removed and made 0.2 M NaCl by the addition of 0.05 vol of 4 M NaCl followed by extraction with 0.5 vol of 90% phenol for 10 min at room temperature. The aqueous phases were combined and precipitated by the addition of 2 vol cold ethanol. The phenol phase from the first extraction was reextracted with 0.5 vol 0.1 M Tris, pH 9.0 by stirring for 10 min at room temperature. After phase separation, the aqueous layer was combined with the phenol phase from the second phenol extraction and stirred for an additional 10 min at room temperature. The phases were separated and the RNA precipitated with 2 vol cold ethanol. The ethanol precipitates were combined and allowed to stand at -20” overnight before proceeding. RNA was collected from the ethanol by centrifugation and dissolved in 1.5 ml of 10 mM Tris, pH 7.6 and 1.0 mM MgClz to which was added an equal volume of 40 pg/ml electrophoretically pure DNase I (RNase free) dissolved in the same buffer. The reaction mixture was incubated for 15 min at 4” followed by an additional incubation for 5 min at 37”. The reaction was stopped by adding 0.1 vol of a solution containing 1.0 M Tris. pH 9.0, and 1% SDS followed immediately by an equal volume of 90% phenol. The mixture was stirred for 10 min at room temperature, phases were separated by centrifugation, and the aqueous phase then reextracted with 0.5 volumes 90% phenol. After centrifugation, the aqueous phase was made 0.2 M NaCl as above and the RNA precipitated with 2 vol cold ethanol. The RNA was allowed to stand overnight at -20”. RNA obtained by this procedure had no detectable protein or DNA contamination. The Az6,,/Azsoratio was always between 1.8 and 2.0 with yields of approximately 1 mg/g muscle. The RNA was stored as the ethanol precipitate at -20” until used. Analytical
Procedures
The ethanol-precipitated RNA was collected by centrifugation, and the pellet dissolved in 1.5 to 2.0 ml of 10 mM Tris, pH 7.5. The sample was chromatographed on 1.5 ml of oligo-dT-cellulose by the procedure of Aviv and Leder (9). In order to precipitate with ethanol the poly (A)containing RNA eluted from the column, the solution was made 50 pg/ml with carrier tRNA isolated by the method of Bloemendal (10). This concentration of tRNA was shown to give maximal precipitation without causing detectable distortion in the subsequent sedimentation analyses.
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Sucrose gradients (lO-50% w/v) were prepared and centrifuged at 40,000 rpm for 14 hr in an SB 283 rotor with an International B-60 ultracentrifuge as described previously (11). For the total and oligo-dTcellulose excluded RNA, 150 pg samples were layered on the gradients; approximately 50 pug of poly(A)-containing RNA (eluted from oligo-dTcellulose) containing 4 mg carrier tRNA was layered on gradients. Following centrifugation, gradients were fractionated on an ISCO 180 gradient fractionator and continuously scanned at 254 nm with an ISCO UA-2 ultraviolet analyzer; 10 drop fractions were taken. To each fraction 1 mg carrier RNA was added, and the sample precipitated with 10% TCA. After washing with an additional 5 ml 10% TCA, the RNA pellets were hydrolyzed in 0.2 ml NCS mixed with toluene phosphor and counted in a Nuclear-Chicago Unilux II liquid scintillation counter equipped with an external standard. Standard curves of counting efficiency vs channels ratio were prepared for each experiment, and the efficiency of counting for each sample determined by the standard channels ratio technique. RNase activity was measured by the RNA agar gel diffusion procedure described by Tan (12). In this method the RNase activity was related to the hydrolysis of RNA trapped in agar gels. Following incubation for 2 hr at 37” the undigested RNA was fixed with 1.0 N HCI and the gels photographed. Tracings were made of the negatives on a Beckman film densitometer. From these tracings the diameter of the circle created by the diffusing RNase (x) and the diameter of the well (y) were measured. The quantity of RNase in the well was proportional to x2-y”. Isolation
of Muscle
Nuclei
and Assay
of RNA
Polymerase
Activity
Nuclei were isolated from 4 day old chickens by the modification of the procedure of Nair et al. (13) described by Baieve and Florini (1). The nuclear preparations were examined under a compound microscope after staining with 1% (w/v) crystal violet/acetic acid and appeared to have little contamination and few damaged nuclei. There was no apparent difference between nuclei of normal and dystrophic muscle. Following isolation the nuclei were assayed immediately for RNA polymerase activity. Each assay tube contained (in a final volume of 0.4 ml) 50 pmoles Tris, pH 8.0; 25 pmoles sucrose; 0.4 pmoles each ATB, CTP, and GTE; 3 pmoles NaF; 10 pmoles KCl; 5 pmoles creatine phosphate; 10 pg creatine phosphokinase; 2 &i[3H]UTP and either 1 pmole MgCl, or 8 pmoles MnCl,, 0.4 M (NHJ,SO,. The reaction solution was warmed to 37” and the reaction started by the addition of 0.1 ml of the suspension of nuclei. At the times indicated, the reaction was stopped by adding 2 ml of cold 10% TCA with concommitant vigorous mixing on a Vortex. The samples were allowed to precipitate in the cold for 2 hr and KOH-hydrolyzable radioactivity determined. The DNA con-
RNA OF NORMAL
tent of the nuclear reaction (14).
suspension
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was determined
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by the diphenylamine
RESULTS RNase Activity Muscle was homogenized in 0.1 M Tris, pH 9.0 and RNase activity measured in order to obtain not only an estimate of the RNase levels in muscle, but also activity of RNase under the ionic and pH conditions used for RNA isolation. The level of RNase in the dystrophic muscle homogenate was more than twice that of normal muscle (Fig. 1). Although this was only a crude estimate of RNase levels (optimal conditions for the enzyme were not determined), it was apparent that differences in degradation of RNA during isolation could cause difficulties in interpretation of the results. Two frequently used RNase inhibitors DEOF and PVS were examined for their effectiveness with muscle homogenized in 0.1 M Tris, pH 9.0 (Fig. 1). DEOF was not effective in this system, and actually produced a slight stimulation of RNase activity, possibly by releasing membranebound enzyme. On the other hand, PVS reduced the RNase level of the homogenate of both the normal and dystrophic muscle to an undetectable level. PVS at 100 pg/ml was included in the RNA isolation medium to minimize the effects of RNase during the isolation of RNA. Comparisons of Labeled RNA Preparations Double-labeled mixed-fractionation analysis was used to detect any qualitative or relative quantitative differences between RNA isolated
Cmhd
PVS
DEOF
1. RNase activity and effectiveness of RNase inhibitors in muscle homogenates. The breast muscle of normal and dystrophic chickens were removed and homogenized in RNA isolation buffer (0.1 M Tris, pH 9.0) or buffer containing RNase inhibitors (either 100 pg/ml PVS or 0.5% DEOF). RNA agar gels were prepared with 4 ~1 of homogenate in each well and the slides incubated and analyzed as described in Methods. Protein concentration in the homogenates were determined by the method of Lowry, and all data expressed as RNase activity per mg protein (mm2/mg protein). The solid bars represent RNase activity in normal muscle, and the hatched bars represent RNase activity in dystrophic muscle. FIG.
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from normal and dystrophic chickens. When this procedure was applied to RNA isolated from normal and dystrophic muscle, there was no detectable difference in the 3H to 14C ratio in comparisons among total RNA and two major fractions, one rich in poly(A)-containing RNA and the other rich in non-poly(A)-containing RNA, obtained from the oligodT-cellulose column. In our determinations of radioactivity, enough counts were accumulated to obtain an accurcy of at least +-2%. Subsequent analysis of the RNA fractions by centrifugation through sucrose gradients revealed no substantial differences between the RNA from normal or dystrophic animals. The sucrose gradient of total RNA (Fig. 2A) showed a typical optical density profile with major peaks attributed to tRNA and rRNA. In the area of the tRNA peak, a slight decrease in the relative labeling of dystrophic RNA occurred. Although this difference persisted with duplicate gradients of the same RNA and in repeated experiments, it does not seem large enough to be considered a major effect of the disease.
Fmction
No.
Fraction
Fraction
No.
No.
FIG. 2. Sucrose gradient analysis of RNA from normal and dystrophic chickens. Brease muscle from 3 day old normal chickens was incubated with [‘4C]uridine and muscles from 3 days old dystrophic chickens with [3H]uridine as described in Methods. The muscles were combined and the RNA extracted and analyzed on l&50% sucrose gradients and the radioactivity determined as described in Methods. Three fractions were analyzed: A. Total cellular RNA, B. RNA excluded from an oligo-dT-cellulose column and C. RNA which bound to an oligo-dT-cellulose column. The broad solid line represents the absorbancy at 254 nm and the dotted line the percent of the total disintegrations of 14Cin each fraction, and the narrow solid line the percent of the total disintegrations of 3H in each fraction. The direction of sedimentation was from left to right.
RNA OF NORMAL
AND DYSTROPHIC
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In order to determine that the tRNA and rRNA, which comprise 97% of the isolated RNA, were not masking differences in the poly(A)-contaming RNA’s sedimentation analyses of the oligo-dT-cellulose excluded (Fig. 2B) and bound (Fig. 2C) fractions were performed. The optical density profile of the excluded RNA consisted of 4 S, 18 S and 28 S peaks, while the bound RNA showed the predominant peak at the top of the gradient due to the carrier tRNA added (see Methods), followed by a broad peak of heterogeneous RNA. The radioactivity profile of the excluded RNA was very similar to that obtained with the total RNA, showing no differences except for a small depression of the dystrophic RNA in the 4 S region. The profiles of radioactivity for the poly(A)-containing RNA’s of normal and dystrophic muscle were virtually identical. There appear to be no relative quantitative differences between normal and dystrophic RNA’s labeled for 2 hr under our conditions. To establish that the small differences between normal and dystrophic RNA were within experimental variation, RNA from two normal chickens was compared. The breast muscle of one normal animal was labeled with r3H]uridine while the breast muscle of another normal chicken was labeled with [14C]uridine. The muscles were combined and extracted as before. The optical density and radioactivity profiles obtained from sucrose gradient analyses of the oligo-dT-cellulose excluded and bound RNA’s (Fig. 3) were similar to those observed in the comparison of normal and dystrophic RNA’s. This demonstrates that the observed variation between normal and dystrophic RNA was no greater than the usual variation due to individual animals and counting procedures.
Fraction
No.
FIG. 3. Sucrose gradient analysis of RNA from normal muscle. RNA from normal muscles labeled with [3H] and [14C]uridine was extracted and fractionated on oligodT-cellulose as described in Methods. The RNA was analyzed on a lO- 50% sucrose gradient and the radioactivity determined as described in Methods. Two fractions were analyzed: A. RNA excluded from an oligo-dT-cellulose column, and B. RNA which bound to an oligo-dTcellulose column. The broad solid line represents the absorbancy at 254 nm and the dotted line the percentage of the total disintegrations of 14Cin each fraction, and the narrow solid line the percent of the total disintegrations of 3H in each fraction. The direction of sedimentation was from left to right.
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IJ-jq 5 10
Time
AND
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I~ 15
(min)
12 Time
3 (min)
4
5
FIG. 4. Time course of [3H]UTP incorporation into RNA by isolated nuclei from chicken breast muscle. Nuclei were prepared from 4 day old chickens and the RNA polymerase activity assayed as described in Methods. The nuclei were assayed over long (A) and short (B) time courses. The solid line indicates normal and the broken line dystrophic muscle. The circles indicate nuclei assayed in the presence of 16 mM MnCl,, and the triangles indicated nuclei assayed in the presence of 2 mM MnCI, and 0.4 M (NH&SO,.
RNA Polymerase Activity in Isolated Nuclei Nuclei from normal and dystrophic chickens were isolated and assayed following the procedure of Baieve and Florini (2); the results are summarized in Fig. 4A. The Mg2+ dependent activities were the same in normal and dystrophic nuclei, while the Mn2+, (NH&SO, dependent activity of dystrophic nuclei was 45% lower than the same RNA polymerase activity in normal nuclei at 5 min. However, the rate of incorporation of [3H]UTP into RNA began to decrease between 5 and 10 min and reached a plateau by 15 min in the case of both the Mg2+ and the Mn2+, (NH&SO, dependent activities. To determine whether incorporation was linear between zero and 5 min, the RNA polymerase activities were measured at shorter times (Fig. 4B). When these shorter time periods were used, the Mg 2+ dependent activities of normal and dystrophic nuclei were still identical and the incorporation appeared linear up to 5 min. The differences in the MI+‘+, (NH&SO4 dependent activity observed above and in previous studies (1) were not detected at the initial stages of the reaction, but only began to appear after 2.5 min incubation. At that time the rate of incorporation of L3H]UTP into RNA in both normal and dystrophic nuclei began to decrease with a greater rate decrease occurring in the dystrophic nuclei. During the first 2.5 min of incubation when both the normal and dystrophic nuclei showed linear incorporation there was no perceptible difference in the Mn2+, (NH,),SO, dependent RNA polymerase activity of normal and dystrophic nuclei. DISCUSSION
Although RNase activity in homogenates from dystrophic muscle was twice that in normal muscle, there was no observable shift to slower sedimenting RNA on sucrose gradients. This lack of degradation sup-
RNA
OF
NORMAL
AND
DYSTROPHIC
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135
ports the suggestion of Pennington (8) that these increases in catabolic enzymes are due to invading macrophages which are in the process of catabolizing dead cells. Moosa (7) and Zellweger (15) have observed necrotic cells in human dystrophy; thus the heightened levels of catabolism in dystrophic muscle might be the secondary result of removal of dead cells, rather than a primary cause of the disease. Measurements of catabolic enzymes in such a heterogeneous system as dystrophic muscle must be interpreted with caution until it can be ascertained what cell types are producing the enzyme. We found no difference in the RNA populations labeled in muscle minces in vitro. With oligo-dT-cellulose, two fractions of RNA were produced: A) RNA which did not bind to the column and did not contain poly(A) segments (i.e., rRNA, tRNA and their precursors (16)), and B) RNA which bound to the column and contains poly(A) segments (i.e., mRNA, except histone message, and some heterogeneous nuclear RNA (17-20)). The suggestion of Baieve and Florini (1) that RNA polymerase II activity was decreased in dystrophic muscle led us to expect a decrease in the amount of label in the poly(A)-containing RNA fraction of RNA isolated from this muscle. However, we did not observe any difference in the ratio of poly(A)-containing RNA to non-poly(A)-contaming RNA between normal and dystrophic animals. Furthermore, sucrose gradient centrifugation revealed no qualitative differences greater than those observed when comparing RNA samples from two normal chickens. This lack of qualitative differences in the RNA agrees with preliminary studies of Battelle and Florini (2) which showed no qualitative difference in the electrophoretic profile of in vitro labeled proteins; similarly, Oppenheimer and Markiewicz (21) found no differences in the RNA of isolated ribosomes from normal and dystrophic chickens. It appears that in dystrophic muscle the RNA’s observed by pulse labeling were quantitatively and qualitatively similar to those in normal chicken muscle. Even the later increase in protein synthesis in dystrophic muscle cannot be attributed to changes in RNA; Weinstock and Markiewicz (22) have recently demonstrated that this change is attributable to increases in activity of elongation factors. The results presented in Fig. 4 provide a possible explanation of the discrepancy between the observations of Baieve and Florini (l), which indicated a decrease in mRNA synthesis, and those of Battelle and Florini (2), who found no decrease in mRNA content of ribosome preparations from young dystrophic chickens. We now believe that the apparent depression of activity of the Mn2+-stimulated RNA polymerase in muscle of dystrophic animals can be attributed to an unusually rapid loss of activity of the enzyme upon incubation of the isolated nuclei. Our present results demonstrate that there is no difference in this activity between nuclei from normal and dystrophic muscle during the first few
136
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minutes of the incubation, but there is a more rapid loss of activity in nuclei from diseased animals. This more rapid loss might be explained by one or more of the following phenomena. Blobel and Potter (23) observed by electron microscopy that approximately 75% of purified liver nuclei showed membrane damage, while Cox et al. (24) showed that homogenization of the tissue caused extensive damage to the DNA in intact nuclei in the form of single strand nicks. These nicks were probably caused by the action of DNase during homogenization and isolation. It is highly likely that DNA in muscle nuclei is being degraded during isolation and assay of RNA polymerase. A recent report by Flint et al. (2.5) indicates that the isolated RNA polymerase activities of liver were considerably altered by changes in the integrity of the DNA template. Although double stranded breaks had the greatest inhibitory effect, it was suggested that factors not coisolated with the purified enzyme might further limit the activity of RNA polymerase to intact DNA in a similar manner as shown for sigma factor and core enzyme of Escherichia coli (26). Eukaryotic initiation and termination factors are presently being studied for the RNA polymerase of coconut (27 and 28) and elongation factors for the enzyme from rat liver (29), but little is yet known of how these factors can alter the activity of the RNA polymerase. Although it is difficult to draw conclusions from such diverse biological sources, it appears that the RNA polymerase assay system of isolated nuclei is very complex; there are many processes which may alter the observed activity of RNA polymerase. Although the results of RNA polymerase measurements in isolated nuclei must be interpreted with caution, the similarity in normal and dystrophic chicken nuclei of both Mn2+ and Mg2+ RNA polymerase activities during the early portion of the time course agrees with the observed similarity of the RNA produced by these cells. Differences in catabolic enzymes (such as DNase) coupled with possible differential mechanical damage to the nuclei during isolation may have contributed to the more rapid decrease in RNA polymerase activity in the nuclei from dystrophic chickens. As far as could be detected, it appeared that the 3 day old dystrophic chicken breast muscle synthesized the same types and relative quantities of RNA as muscle from normal chickens. SUMMARY
RNA isolated from 3 day old normal and dystrophic chickens showed no qualitative or relative quantitative differences, when compared by oligo-dT-cellulose chromatography and sedimentation analysis. These two RNA populations were similar even though the RNase activity was double normal values in muscle homogenates from dystrophic animals. Measurements of the Mn2+ and Mg2+ RNA polymerase activities were made in isolated nuclei from both normal and dystrophic chickens.
RNA
OF
NORMAL
AND
DYSTROPHIC
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137
Measurements made at short time periods showed no difference in the activity between diseased and control animals, while measurements at incubation times of 5 min or greater showed a preferential decrease of approximately 55% in the Mn2+ RNA polymerase activity of the nuclei from dystrophic chickens. These results explain differences in previous experiments; we now conclude that there are no significant differences in the RNA polymerase activities of normal and dystrophic chicken muscle. ACKNOWLEDGMENT This investigation was supported by a grant from the Muscular Dystrophy Associations of America.
REFERENCES 1. Baieve, D., and Florini. J. R., Arch. Biochem. Biophys. 139, 393 (1970). 2. Battelle, B., and Florini. J. R., Biochemistry 12, 635 (1973). 3. Weinstock, I. M., Bondar, M., Blanchard, K. R.. and Arslan-Contin, P.. Biochim. Biophys. Acta 277, 96 (1972). 4. Tappel, A. L., Zalkin, H., Caldwell. K. A., Desai. I. D., and Shibko, S.. Arch. Biothem. Biophys. 96, 340 (1962). 5. Mey, W. L., Little, B. W., Faussner, J. R., and Mayer, D. M., in “Research in Muscle Development and Muscle Spindle” (B. Q. Banker, R. J. Przybylski. J. P. Vander Meulen and M. Victor, Eds.), pp. 195-217. Exerpta Medica Foundation, New York, 1971. 6. Berlinquet, L. and Srivastava, U., Can. J. Biochem. 44, 613 (1966). 7. Moosa, A., Dev. Med. Child. Neurol. 16, 97 (1974). 8. Pennington, R. J., Biochem. J. 88, 64 (1963). 9. Aviv. J., and Leder, P.. Proc. Nat. Acad. Sci. USA 69, 1408 (1972). 10. Bloemendal. H.. Huizinga, F., DeVries, M., and Bosh, L.. Biochim. Biophys. Actu 61, 209 (1962). 11. Neal, M. W., and Florini, J. R., Anal. Biochem. 45, 271 (1972). 12. Tan, C. H., Biochim. Biophys. Acta 247, 463 (1971). 13. Nair, K. G.. Rabinowitz, M., and Chen Tu, M.. Biochemistry 6, 1898 (1967). 14. Burton, K. A.. Biochem. J. 65, 315 (1956). 15. Zellweger, H., Dumin, R., and Simpson, J., Acta Neural. Stand. 48, 87 (1972). 16. Eiden, J. J., and Nichols, J. L., Biochemisrry 12, 3951 (1973). 17. Adesnik, M.. Salditt, M., Thomas, W.. and Darnell. J. E., 1. Mol. Biol. 71, 21 (1972). 18. Brawerman, G., Ann. Rev. Biochem. 43, 621 (1974). 19. Sheldon, R.. Kates, J., Kelley, D. E., and Perry, R. P., Biochemistr?/ 11, 3829 (1972). 20. Adesnik, M.. and Darnell. J. E., J. Mol. Biol. 67, 397 (1972). 21. Oppenheimer, H., and Markiewicz, L., B&hem. Med. 7, 479 (1973). 22. Weinstock. I. M., and Markiewicz, L.. B&him. Biophys. Actu 374, 197 (1974). 23. Blobel, G., and Potter, V. R., Science 154, 1662 (1966). 24. Cox. R., Damjanov, I., Abanobi, S. E., and Sarma. D. S. R.. Cancer Res. 33, 2114 (1973). 25. Flint. S. J., DePormerai, D. I., Chesterton, C. J., and Butterworth. P. H. W., Eur. J. B&hem. 42, 567 (1974). 26. Dausse, J., Sentenac, A., and Fromageot. P., Eur. J. Biochem. 26, 43 (1972). 27. Mondal, H., Mandal, R. K., and Biswas. B. B., Eur. J. Biochem. 25, 463 (1972). 28. Mondal, H., Ganguly, A., Das, A., Mandal, R. K., and Biswas. B. B.. Eur. J. Biochem. 28, 463 (1972). 29. Seifart, K. H.. Juhasz, P. P.. and Benecke, B. J.. Eur. J. Biochem. 33, 181 (1973).
