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Comparison of the activities of nuclear DNA-dependent RNA polymerases isolated from livers of several strains of mice
Itrr. J. Bbrhem., 0 Pergamon
Vol. IO. pp. 713 lo 721 Press Ltd 1979. Printed in Great Britain
0020.71
IX/79/0801-0713102.00/0
COMPARISON OF THE ACTIVITIES OF NUCLEAR DNA-DEPENDENT RNA POLYMERASES ISOLATED FROM LIVERS OF SEVERAL STRAINS OF MICE D. G. R. BLAIR Department of Biochemistry, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N OWO (Received 1 February 1979)
Abstract-l. Nuclear DNA-dependent RNA polymerases from liver of mouse, Mus musculus, were separated chromatographically on DEAE-Sephadex A-25. 2. Five major enzyme peaks were obtained from each sample of liver. The sensitivity of the enzymes to a-amanitin was as follows: Ia and Ib, insensitive; Ha and IIb, highly sensitive; and III, slightly sensitive. 3. The percentage distributions, activities per mg DNA, specific activities and purifications of the enzymes from BDF,, C3H/HeJ, C57BL/6J and Swiss albino mice were compared and discussed. 4. Statistically significant differences (P < 0.05) were found between some strains of mice for percentage distribution, activity per mg DNA, and purification of some RNA polymerases.
INTRODUCTION
Searle Corp. Calf thymus “native” DNA (highly polymerized) was a product of Worthington Biochemical Corp.
Multiple nuclear DNA-dependent RNA polymerases Bovine serum albumin (BSA; fraction V) and the disodium [nucleosidetriphosphate:RNA nucleotidyltransferase salts of GTP and CTP were supplied by Schwarz/Mann. (DNA dependent), EC 2.7.7.61 have now been isolated Yeast total RNA, phosphoenolpyruvate trisodium salt, from numerous eukaryotic tissues or cells (Biswas et rabbit muscle pyruvate kinase (type III; 500 units/mg), the al., 1975; Chambon, 1975) including the liver of the disodium salt ii ATP, the trisod&m salt of UTP and dithcommon mouse, Mus musculus. The enzymes are iothreitol (DTT) were obtained from Sinma Chemical Co. DEAE-hphahex A-25 (Pharmaciay 4&120 PC) was classified according to their order of elution from DEAE-Sephadex A-25 (Roeder & Rutter, 1969) or swollen in TGMED buffer [SOmM Tris-HCI, pH 7.9; 25% (vol/vol) glycerol; 5 mM MgCl*; 0.1 mM EDTA; 0.5 mM according to their sensitivity to cc-amanitin (Kedinger DTT] containing 0.5 M (NH&SO, and 0.002% Hibitane, et al., 1971; Chambon, 1975). Type A (or I) mam- and equilibrated with TGMED buffer containing 0.05 M malian RNA polymerases are insensitive to a-amani(NH&SO, prior to use by repeated washing. tin whereas type B (or II) enzymes are highly sensitive a-Amanitin was a gift from Dr T. Wieland, Max Planck and type C (or III) are inhibited only at very high Institut, Heidelberg, West Germany. All other chemicals were reagent grade, and all solutions were prepared with concentrations of the mushroom toxin (Chambon, double-distilled water. 1975). Animals. Specific-pathogen-free Swiss albino mice (ConMultiple RNA polymerases have been isolated from livers of BALB/c (Schwartz et al., 1974), CBA naught stock) were obtained from the Animal Resources Center, University of Saskatchewan. C3H/HeJ and (Smuckler & Hadjiolov, 1972), CFl (Versteegh & C57BL/6J strain mice and B6D2FI/J (BDF1) hybrids were Warner, 1973), Swiss albino (Blair, 1975), and Swiss- purchased from the Jackson Laboratory, Bar Harbor, Webster (Bagshaw, 1974) mice, but a systematic com- Maine. All animals were adult males weighing 20-25 g. parison of activities and other parameters of the enzymes has not been made. Two mouse liver type Methods Preparation of nuclei from mouse liver. Livers were I (or A) enzymes (Smuckler & Hadjiolov, 1972; Versteegh & Warner, 1973; Blair, 1975), two type II (or excised from 5 mice following etherization of the animals B) enzymes (Smuckler & Hadjiolov, 1972; Blair, 1975) and placed in ice-cold Hanks’ balanced salt solution and at least one type III (or C) enzyme (Blair, 1975; (HBSS) (Hanks & Wallace, 1949). They were cleaned of extraneous tissue, blotted dry, weighed and chopped finely Schwartz et al., 1974) have been isolated. In conjuncwith scissors in 4 vol of SMEP (0.25 M sucrose, 5 mM tion with studies on nuclear RNA polymerases from MgCII, 4 mM EDTA and 10mM potassium phosphate a series of murine transplantable tumors and tissues buffer, pH 7.0) (Read & Mauritzen, 1970). The chopped of normal Swiss albino mice (D. G. R. Blair, manutissue was then homogenized briefly in a Dounce glass script in preparation), RNA polymerases were also homogenizer with teflon pestle. Following the addition of isolated from livers of several strains of normal mice, sapon& (lo%),the homogdnate was shaken in an ice-water which were used as the tumor hosts. The latter studies bath for 30 min. diluted 1:2 with SMEP and centrifuged for 10min at i2OOg (average). The saponin treatment are the subject of this communication. Preliminary (Read & Mauritzen, 1970) was necessary in order to treat reports of some of the studies have been presented the liver nuclei in the same way as nuclei from murine (Blair et al., 1977; Blair, 1978). tumors (Blair, 1975). The sedimented nuclei were resusMATERIALS AND
METHODS
Materials Chemicals. Tritiated uridine 5’-triphosphate (3H-5-UTP) of 15. 17.8 or 24 Ci/mmol was obtained from Amersham-
pended in SMEP and mixed thoroughly with 6~01 of 2.4 M sucrose-5 mM MgC12. This suspension was centrifuged for 1 hr at 30,000~ (average) to sediment the nuclei (Chauveau et al., 1956; Read & Mauritzen, 1970). The supernatant fraction was discarded, the walls of the tubes 713
714
D. G. R. BLAIR
were wiped clean and the pellet of nuclei was resuspended and washed twice in SMEP to remove excess sucrose. Purity of the nuclei was checked by phase contrast microscopy. The purified nuclei were used immediately for solubilization of RNA polymerase (o.i.) after suspension in TSMD buffer CO.01 M Tris-HCI (pH 7.9), 1.0 M sucrose, 5 mM MgC12, 5 mM DTT]. Samples df nuclei were taken for determination of DNA and protein and RNA polymerase activity (o.i.). Based on DNA content the recovery of nuclei was 60-650/b. Solubilization of RNA polymerase. RNA polymerase activity was solubilized from liver nuclei according to the method of Roeder & Rutter (1970) and in the same manner as from nuclei of Ehrlich ascites cells (Blair & Mukherjee, 1973; Blair, 1975) by sonic disruption of the nuclei in TSMD (LX) containing 0.3 M (NH&SO,. The enzymes were precipitated with (NH&SO,, collected by centrifugation, dissolved in TGMED buffer (pH 7.9) containing 0.05 M (NH&SO4 and dialyzed versus the same buffer for 6-8 hr at 4°C (Blair & Mukherjee, 1973). The diffusate was centrifuged at 105,OOOg (average) for 1 hr and the resultant supernatant fraction (F4) was stored at -70°C for chromatography the next day (u.i.). The solubilization of RNA polymerase was monitored at each step by determination of protein and DNA and of RNA polymerase activity. Chromatographic separation ofnuclear RNA polymerases. RNA polymerases of the F4 fraction were separated by chromatography on DEAE-Sephadex A-25 (Roeder & Rutter, 1969) with a nine-step gradient of (NH&SO., in TGMED buffer as described for the enzymes from mouse tumors and normal tissues (Blair, 1975). Columns of DEAE-Sephadex A-25 (0.9 x 1.2cm) were prepared and equilibrated with TGMED buffer (pH 7.9) containing 0.05 M (NH&SO4 in the cold room (4°C). A 5-ml sample of solubilized RNA polymerase (F4) (616 mg protein) was layered on top of the resin and washed into it with 5 ml of TGMED buffer (pH 7.9) containing 0.05 M (NH&SO,. Five-ml portions of buffer containing 0.10, 0.15. 0.20, 0.25, 0.30, 0.35, 0.40, 0.45 or 0.50 M (NH&SO4 were then added in succession. One-ml fractions were collected with a Buchler Fractomat fraction collector at 4°C. Protein was estimated from the absorption of the eluates at 280 nm with BSA (fraction V) as a standard which has a specific absorbance of 0.65/mg/ml at 280 nm, similar to that for RNA polymerase from Escherichia coli (Richardson, 1966) and Pseudomonas putida (Johnson et al., 1971). The fractions (0.1 ml aliquo&) were assayed immediately for RNA polvmerase activity, and, after the addition of BSA (1 mgjmi) as an enzyme stabilizer (Roeder & Rutter, 1969). were stored at -70°C. RNA polymerase assays. (1) Standard assay. RNA polymerase activity of nuclear and chromatographic fractions was determined by the incorporation of C3H]UMP from C3H]UTP into acid-precipitable RNA in the same manner as for murine tumors and normal tissues (Blair & Mukherjee, 1973; Blair, 1975). A unit of enzyme activity is defined as the amount of enzyme which catalyzes the incorporation of 1 pmol of [3H]UMP into an acid-insoluble product in 1 min at 37°C. The standard assay medium (Blair, 1975) was essentially the same as that employed by Roeder & Rutter (1970) for enzymes from rat liver and sea urchin. The reaction medium (0.25 ml) included 0.1 ml of enzyme preparation in TGMED buffer (pH 7.9) plus 0.05 M (NH&SO+ The final concentrations of components were: “native” calf thymus DNA, 160pg/ml; pyruvate kinase, 6.8 I.U./ml; phosphoenolpyruvate, 4 mM; 2-mercaptoethanol, 1.6 mM; NaF, 6 mM; KCI, 8 mM; MnCI,, 1.6 mM; ATP, CTP and GTP, 0.6mM each; [“HIUTP. O.lOmM (4pCi/ml);
(NH&SO,, 0.05 M; Tris-HCI buffer (pH 7.9), 76 mM; MgCIZ, 2 mM; EDTA, 0.04 mM; DTT, 0.2 mM; and glycerol, 10%.
After an incubation period of 10 min at 37-C. the reaction mixtures were placed in ice. and 1 ml of Na,P,Ocontaining 2mg of both yeast RNA and BSA was added immkdiately to each. followed sequentially by 1 ml of 5”,, (wt/vol) sodium dodecyl sulfate and 1 ml of cold 20“” trichloracetic acid (TCA) plus 0.04 M Na4P,0,. The precipitates were collected on glass fiber filters (Whatman GF/C) and processed for determination of radioactivity, which was measured in a Nuclear-Chicago Unilux I spectrometer at a counting efficiency of about 45”/, (Blair & Mukherjee, 1973; Blair, 1975). Enzyme incubations were done in duplicate. Values for enzyme-free controls were subtracted from those of samples containing enzyme to calculate net incorporation of [‘H]UMP into RNA. (2) Assay of activity of isolated nuclei. RNA polymerase activity of samples of intact nuclei was measured as described previously (Blair & Mukheriee, 1973; Blair, 1975) essentially according to the method of Roeder & Rutter (1970) with the standard assay medium for solubilized activity which lacked calf thymus DNA, but contained 0.32 M (NH.&S04. 2 mM MgCI, and 1.6 mM MnCI,. Inhibition of RNA po/ymerases by a-amanitin. The inhibitory capaccty of a-amanitin versus the isolated RNA polymerases was tested by adding the inhibitor to the standard assay medium just before adding the enzyme. Protein und DNA assays. The protein content of nuclei and the nuclear fractions was determined by the method of Lowry et al. (1951) with BSA (fraction V) as a standard. DNA was estimated by the diphenylamine reaction (Disthe, 1930) after isolation by the method of Herbert et al. (1957). RESULTS Solubilization liver
of nuclear
RNA polymerase from mouse
Representative purification data for RNA polymerase obtained during solubilization of the enzyme activity from nuclei from livers of four strains of mice are presented in Table 1. Large variation between mouse strains is seen in regard to enzyme specific activity and to yield and purification from the suspension of nuclei or from the nuclear sonicate. The large variability was also found between samples for the same strain as will be shown by the large standard deviations of the means in the tables to follow (Tables 2-7). The purifications from nuclei found for the mouse liver solubilized RNA polymerases (F4 fractions, Table 1) are similar to those found by Versteegh & Warner (1973) for CFl mouse liver and by Blair (1975) for Swiss mouse kidney, liver and spleen. Mean data for the partial purification of the solubilized RNA polymerase (F4 fraction) from four samples of liver from each strain of mouse are presented in Table 2. Much lower mean specific activities were found for liver RNA polymerase from C3H, C57BL and BDF, mice than from Swiss mice, but only values for BDFI and C57BL were significantly different (P < q.05 > 0.025) from each other. The mean specific activity of Swiss mouse liver RNA polymerase (50.7 units/mg protein) is superior to that (38.5 units/mg protein) reported for the enzyme from CFl mouse liver (Versteegh & Warner, 1973) and to that (16.5 units/mg protein) which we previously found for Swiss mouse liver (Blair, 1975). Although mean yields of activity from the suspensions of liver nuclei were similar for the four strains of mice, yields for C57BL and Swiss were significantly different
105,000g supexnatant of diffusate (F4)
BDF, C3H/HeJ C57BL/6J Swiss
227 676 192 719 181 601 184 835
0.4 1.0 0 2.7 0 0.7 0 1.0
17.8 18.6 24.0 41.1 13.9 13.7 21.4 23.7
BDF, C3H/HeJ C57BL/6J Swiss
precipitate
(NH&SO* (F3)
210 698 222 997
359 1112 307 2438
Total activity (units)
224 708 209 1073
82.0 79.0 87.1 108.7
BDF, C3H/HeJ C57BL/6J Swiss
105,000g supernatant (F2)
11.3 20.8 13.9 56.0
11.3 20.8 13.9 56.0
Total DNA (m8)
8.4 11.2 3.3 51.6
102.3 90.8 113.5 126.7
BDF, C3H/HeJ C57BLJ6J Swiss
Nuclear sonicate (Fl)
102.3 90.8 113.5 126.7
BDF, C3H/HeJ C57BL/6J Swiss
Total protein (m8)
Nuclear suspension
Fraction
Mouse strain
13.0 43.9 8.6 35.2
12.8 36.3 8.0 17.5
2.7 9.0 2.4 9.9
2.1 7.7 2.0 7.9
3.5 12.3 2.7 19.2
Specific activity (units/mg protein)
50.4 54.1 59.9 34.3
63.2 60.8 62.5 29.5
62.4 63.7 68.0 44.0
58.5 62.8 61.8 40.8
100 100 100 100
86.2 86.1 82.8 83.8
108.1 96.8 86.5 72.1
106.7 101.4 94.1 107.6
100 100 100 100
-
Yield From From nuclear nuclear suspension sonicate (%) (%)
Table 1. Representative purification of nuclear RNA polymerase from livers of various strains of mice
3.71 3.57 3.19 1.83
3.65 2.95 2.96 0.91
0.77 0.73 0.89 0.52
0.60 0.62 0.41 0.41
1.00 1.00 1.00 1.00
8
1 !
1.17 2.18 1.25 6.09 4.71 4.00 2.22 6.19 5.70 4.30 4.46
5 B
1.29
a 2 !! W
z 1.00 1.00 1.00 1.00
-
-
Purification From From nuclear nuclear sonicate suspension (fold) (fold)
D. G. R. BLAIR
716 Table
2. Purification
of solubihzed
nuclear
RNA polymerase
(F4 fraction)
from mouse
Purification From From nuclear nuclear suspension sonicate (fold) (fold)
,Yield
No. of samples
Mouse strain BDF, C3H/HeJ C57BL/6J Swiss * Values
Specific activity (units/mg protein)
4 4 4 4 are means
10.69 8.25 4.75 50.72
f. k 5 f
3.71* 17.92 1.84 56.21
47.24 30.48 33.43 53.79
+ k + +
sonicate was approximately 50% higher for Swiss than for the other strains of mice, but yields were not significantly different one from the other. The degree of enzyme purification, from either the nuclear suspension or the sonicate, was also higher for the Swiss strain than for the others, but differences were not significant. Total RNA polymerase activity per amount of DNA The total units of liver RNA polymerase activity per mg of DNA, based on endogenous nuclear activity (nuclear suspension) or on solubilized activity (F4 fraction), were quite similar for all strains of mice (Table 3). Differences between strains for either parameters were not significant statistically. Both parameters were highest for C3H liver, but the remaining ranking order differed, with BDFi being lowest for one and C57BL for the other. These data indicate that similar amounts of enzyme activity were obtained for similar amounts of tissue prior to and after enzyme solubilization. Purification of mouse liver RNA DEAE-Sephadex A-2S
polymerases on
Five peaks of RNA polymerase activity solubilized from mouse liver nuclei were eluted from columns of DEAE-Sephadex A-25 with a stepwise gradient of (NH&SO, (Blair, 1975). Similar chromatograms were obtained for liver enzymes from all four strains Table
3. Total
BDF, C3H/HeJ C57BL/6J Swiss * Values
4 4 4 4 are means
Table 4. Percentage
* Values
62.77 65.00 60.38 93.62
f k + k
29.66 24.04 23.41 67.50
3.11 1.62 1.88 6.60
f f * 1
0.79 1.13 0.43 7.11
4.21 2.98 3.36 7.63
+ k f. k
1.42 1.52 1.26 6.46
of mice (Figs l-4). RNA polymerase forms Ia, Ib and III were resistant to 0.3 pg a-amanitin/ml whereas the activities of enzyme forms IIa and IIb were inhibited completely. Enzyme activity designated III was inhibited by about 50% at 5Opg a-amanitin/ml whereas activity designated I was not. Note that the enzyme activity scale for BDFi mouse liver (Fig. 1) and C3H/HeJ mouse liver (Fig. 2) is twice that for C57BL/6 mouse liver (Fig. 3) and ten times that for Swiss mouse liver (Fig. 4), thus rendering some peaks of activity comparatively insignificant in the latter case. The percentage recoveries of enzyme activity from the chromatographic columns were 95, 89, 100 and 93, respectively, for BDFi, C3H/HeJ, C57BL/6 and Swiss mouse liver samples (Figs l-4). The mean percentage recoveries of enzyme activities from the chromatographic columns for 4 samples each of BDFi, C3H/HeJ, C57BL/6 and Swiss mouse liver RNA polymerase were 105, 93, 100 and 101, respectively. No activity was eluted at an ammonium sulfate concentration below 0.1 M. There were two or three peaks of activity preceding RNA polymerase Ha, one peak each of Ha and IIb, and one or more peaks designated RNA polymerase III following the IIb peak. BDF, (Fig. 1) and C57BL/6J (Fig. 3) chromatograms usually had the pre-Ia peak. The post-Ib peaks on the C57BL/6J (Fig. 3) and Swiss (Fig. 4) chromatograms were type I activity according to their resistance to a-amanitin. Although enzyme peak Ib is activity
33.54 48.32 36.56 43.19
k + k +-
9.65* 27.59 16.50 33.63
of RNA polymerase Percentage
k SD.
f & + +
DNA
Solubilized (F4 fraction) activity (units/mg DNA)
Endogenous nuclear activity (units/mg DNA)
Ia 14.71 25.72 20.29 11.62
per mg of nuclear
16.58 20.72 11.78 13.51
& + + k
10.11 14.17 4.39 9.21
+ SD
distribution
N 3 4 4 4
are means
RNA polymerase
No. of samples
Mouse strain
BDF, C3H/HeJ C57BL/6J Swiss
24.72 17.45 5.30 11.44
+ SD.
(P < 0.05 > 0.025). The mean yield from the nuclear
Mouse strain
From nuclear sonicate (%)
From nuclear suspension (X)
liver nuclei
Ib 4.00, 14.33 5.21 4.44
4.80 11.72 8.35 8.70
+ + + k
1.74 3.46 5.10 4.49
activity
among
distribution Ha 26.14 21.93 32.68 47.21
+ + + f
3.86 12.05 8.55 7.04
the multiple
liver enzymes
of activity IIb 31.98 25.76 20.71 20.35
+ + + +
III 9.20 9.32 9.75 9.85
8.31 5.08 7.61 1.98
f f f +
2.65 1.01 1.46 0.71
DNA dependent RNA polymerases
717
Table 5. Activities of mouse liver RNA polymerases Mouse strain BDF, C3H/HeJ C57BLf6J Swiss
N
la
3 4 4 4
2.72 k 3.49 * 2.45 f 1.57 *
Units of RNA polymerase/mg DNA Ha IIb Ib 0.30* 3.05 1.11 0.52
0.87 * 1.66 * 1.01 k 1.05 k
0.12 1.51 0.72 0.24
5.10 + 1.93 k 4.16 f 7.73 f
1.21 1.06 2.23 4.47
III
7.24 + 4.54 3.99 f 3.97 2.06 rf: 0.30 3.86 k 3.35
1.65 f 0.65 0.54 + 0.30 0.95 f 0.49 0.27 If: 0.09
* Values are means k SD.
Table 6. Specific activities of mouse liver RNA polymerases Mouse strain BDF, C3H/HeJ C57BL/6J Swiss
N
Ia
3 4 4 4
57.1 + 62.7 k 53.7 k 40.3 +
Specific activities (units/mg protein) IIa
Ib 39.0* 55.5 42.3 23.4
23.2 + 82.3 + 107.2 f 38.7 k
15.8 68.0 76.8 22.5
93.3 + 58.3 + 205.1 f 703.6 k
34.0 16.6 125.0 986.9
III
IIb 1144.0 k 120.3 k 301.4 f 646.4 f
1365.1 67.1 304.8 974.1
98.9 + 137.2 f 109.4 + 56.9 +
27.2 124.7 44.8 27.3
* Mean values + SD. Table 7. Degree of purification of mouse liver RNA polymerases
Mouse strain BDF, C3H/HeJ C57BLJ6J Swiss
N
Ia
3 4 4 4
4.7 * 5.5 * 9.7 f 1.6 +
Purification from solubilized enzyme (fold) IIa IIb Ib 3.1* 5.5 6.4 0.9
1.9 f 7.0 + 20.5 + 1.6 f
1.2 6.8 8.9 1.1
7.5 f 4.9 * 43.0 f 15.5 f
2.7 2.1 14.8 8.4
82.8 * 9.9 + 67.0 f 12.6 f
III 104.0 7.0 49.1 8.9
8.0 f 10.2 f 28.3 f 5.8 +
3.2 12.0 14.6 8.7
* Mean values + SD. I
29 BDF,
(NH,)$30,
(MB
MOUSE
:.I
LIVER
I 0.1,
1 0.2
FRACTION ,NUMBER I I 0.25 0.3 0.31
I 0.4
I 0.4,
I 0.5
Fig. 1. DEAE-Sephadex A-25 chromatography of solubilized nuclear RNA polymerase from BDF, mouse liver. Solubilixed enzyme (F4 fraction) (106 units/l.1 mg protein) was applied to a 0.1 x 12 cm column of DEAE-Sephadex A-25 in 5 ml of TGMED buffer, pH 7.9, containing 0.05 M (NH&SO., and washed into the resin with 5 ml of the application buffer. RNA polymerases were eluted by the stepwise application of successive 5-ml portions of TGMED buffer, pH 7.9, containing 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45 and 0.50 M (NH&SO.+, beginning at the fractions indicated by the arrows. Fractions of l.Oml were collected, and aliquots of 0.1 ml of each were assayed for protein and RNA polymerase activity. B.C. IO/S-D
718
D. G. R. BLAIR
C3HIH.J
MOUSE
LIVER
lb
IO (NH&SO,
(M)
20
d.,
O.IJ ’
30 FRACTION NUMBER
0.2 ’
0.2, ’
0.3 ’
40
0.1, ’
50
0.4 ’
0.4, ’
,
01’
Fig. 2. DEAE-Sephadex A-25 chromatography of solubilized nuclear RNA polymerase from C3H/HeJ mouse liver. Solubilized enzyme (F4 fraction) (117 units/lOmg protein) was applied to a 0.9 x 12cm column of DEAE-Sephadex A-25 in 5 ml of TGMED buffer, pH 7.9, containing 0.05 M (NH&S04 and washed into the resin with 5 ml of the application buffer. Enzymes were eluted and assayed as described in the legend to Fig. 1. larger than peak Ia in the particular
chromatograms
for C3H/HeJ and Swiss mouse livers (Figs 2 and 4) shown here, in other cases the reverse was true. Similarly, there was not complete consistency in the relative sizes of peaks IIa and IIb. These variations are reflected in the mean percentage distributions of activity between the multiple RNA polymerases which have large standard deviations (o,i., Table 4). Consequently, enzyme peaks Ia and Ib, as well as IIa and IIb, are regarded as being closely related forms of
type I and type II RNA polymerase, respectively. No studies have been done to purify these enzymes further or to determine their subunit structure since our primary purpose was to compare elution patterns of the mouse liver enzymes from DEAE-Sephadex A-25 with those of mouse tumors and other tissues from normal Swiss mice (D. G. R. Blair, manuscript in preparation), and to show the variation obtained even for RNA polymerases of the same tissue from different strains of mice.
D-
C!57BL/6
MOUSE
LIVER
II0 I lib
D-
O-
Q-
30
(NH,),SO,
IO (Lo
do
30 FRACTION ,NlJMfl:R 1 I 0.2 0.25 0.3 0,s
20 cd,
SO
40 I 0.4
I 0.4,
I 0.5
Fig. 3. DEAE-Sephadex A-25 chromatography of solubilized nuclear RNA polymerase from C57BL/6 mouse liver. Solubilized enzyme (F4 fraction) (239 unitsjl3.0 mg protein) was applied to a 0.9 x 12 cm column of DEAE-Sephadex A-25 in 5 ml of TGMED buffer, pH 7.9, containing 0.05 M (NH&SO4 and washed into the resin with Sml of the application buffer. Enzymes were eluted and assayed as described in the legend to Fig. 1. .
DNA dependent RNA polymerases
f RA~TION,NUWBIER (NH&SO,
(M)
o!
0.15 ’
1
1 0.1
0.25
03
0.35
0.4
t 045
I 0.5
Fig. 4. DEAE-Sephadex A-25 chromatography of solubilized nuclear RNA polymerases from Swiss mouse liver. Solubilized enzyme (F4 fraction) (564 units/i6mg protein) was applied to a 0.9 x 12cm column of DEAE-Sephadex A-25 in 5 ml of TGMED buffer, pH 7.9, containing 0.05 M (NH&JO4 and washed into the resin with 5 ml of the application buffer. Enzymes were eluted and assayed as described in the legend to Fig. 1. Percentage distribution of RNA polymerase activity among the nuclear enzymes isolated on DEAESephadex A-25
The percentage distribution of activity among the multiple peaks of mouse liver RNA polymerase was not the same for each strain of mouse (Table 4). Whereas the mean percentage of activity in peak III was greater than in peak Ib in the case of BDFi, it was smaller than in peak Ib in all other cases. For BDFr and C3H/HeJ the percentage of activity in IIb was greater than in IIa, but the opposite was found for the other two mouse strains. Also, whereas similar mean percentages of activity were found in peak IIb for all mouse strains, greater variation between strains was found for the other peaks of activity. Statistical analysis (Student’s t-test) showed that differences between Swiss and C3H/HeJ for Ia, BDF, and C3H/HeJ for Ib and C3H/HeJ and C57BL/6J for III were probably significant (P < 0.025 > O.Ol), while the difference between Swiss and C3H for III was significant (P < 0.005 > 0.001) and that between Swiss and C57BL/6J for III was highly significant (P < 0.001). Activities of isolated RNA polymerases per amount of DNA
Table 5 presents data on the total activity present in each chromatographic peak of RNA polymerase per amount of DNA. These mean values reflect the mean percentage distributions of activity (Table 4) and also have relatively large standard deviations. Statistical analysis (t-test) revealed probably significant differences only between Swiss and BDFi for enzyme Ia (P < 0.05 > 0.025) and between BDFi and C3H/HeJ for enzyme IIa (P < 0.025 > 0.01). Specific activities of isolated RNA polymerases The specific activity of the most active fraction
from each chromatographic peak of RNA polymerase was calculated, and the means with standard deviations are presented in Table 6. The same degree of purification was not obtained for the same RNA polymerase from each of the strains of mice and standard deviations were relatively large for values for any strain. Generally RNA polymerase Ia or Ib had the lowest specific activity of the five major RNA polymerase forms and RNA polymerase IIb had the highest. However, in Table 6 C3H/HeJ is an exception for which enzyme III had the highest mean specific activity and IIa the lowest. The enzymes eluting with the lowest concentrations of ammonium sulfate generally had the lowest specific activities because the bulk of the protein was also eluted at these concentrations. Statistical analysis (t-test) of the data showed that the differences between mouse strains for each RNA polymerase were not significant. The best single specific activities (units/mg protein) obtained were 155.7 (C3H), 232.8 (C57BL), 2664.4 (Swiss), 3074.1 (BDFt) and 324.1 (C3H) for enzymes Ia, Ib, IIa, IIb and III, respectively. These specific activities compare favorably with values of 503 units/mg protein for enzyme B and 87.4 units/mg protein for enzyme A from CBA mouse liver recalculated from the data of Smuckler & Hadjiolov (1973). Degree of purijication of isolated RNA polymerases
The degree of purification of the chromatographitally isolated liver RNA polymerases from the partially purified mixture of enzymes (F4 fraction) is indicated in Table 7. The same degree of purification was not obtained for the same enzyme from different strains of mice, and, as for the mean specific activities, standard deviations were relatively large. The highest purifications were generally obtained for enzyme IIb and the lowest for enzyme Ia, but C3H, as in the case of specific activity, was an exception for which
D. G.
720
enzyme III was purified the most and enzyme IIa the least. Statistical analysis (t-test) revealed probably significant differences (P < 0.05 > 0.01) in purification between Swiss and C57BL livers for enzymes la, IIa and III, and between BDF, and C57BL livers for enzymes Ib and IIa. There were significant differences (P < 0.01 > 0.001) between Swiss and C57BL livers for enzyme Ib and between C3H and C57BL for enzyme IIa. The best single purifications (fold) from the F4 fraction which were obtained were 18.7 (C57BL). 35.9 (C57BL). 60.1 (C57BL), 234.7 (BDF,) and 48.0 (C57BL) for enzymes Ia, Ib, Ha, IIb and III, respectively. These represent overall purifications (fold) from nuclei of 39.8, 66.9, 112.0, 876.0 and 57.0. respectively. DISCUSSION
Comparison of the hepatic RNA polymerase activity of Swiss albino, C3H/HeJ, C57BL/6J and BDF, mice showed that considerable variation exists between samples from each strain of mouse and between samples from different strains in regard to all parameters measured. C3H/HeJ, C57BL/6J and Swiss albino mice each have separate origins and therefore are not closely related genetically (Staats, 1966), but BDFi mice, being Fi hybrids from the C57BL/6J female x DBA/ZJ male cross, are closely related to the C57BL/6J male mice used in these experiments. If the variation between samples is genetically based, it should be less between samples from BDF, and C57BL/6J mice. However, the fact that the specific activities of the solubilized RNA polymerase (F4 fractions) were probably significantly different (P -L0.05> 0.025) only for these two strains suggests that the variation is not genetically based. This is further supported by probably significant differences between the two strains in regard to degree of purification of enzyme forms Ib and IIa. Parameters measured were not consistently highest for one specific mouse strain and lowest for another. Thus, variation appears to be usually random and probably due to the large number of procedures and manipulations required for purifying the RNA polymerases. Since, to our knowledge, no similar statistical data have been presented in the literature we do not know if our data show excessive variation. The mean purifications of the solubilized mouse liver RNA polymerase preparations (F4) from nuclei which we obtained were similar to those found previously by us (Blair, 1975) for Swiss mouse liver and by Versteegh & Warner (1973) for CFl mouse liver. Although the mean specific activity of the solubilized RNA polymerase from Swiss mouse liver was superior to previously published values for Swiss (Blair, 1975) and CFl (Versteegh & Warner, 1973) mouse livers, specific activities for the solubilized enzyme from livers of the other three strains of mice were lower. There is no apparent reason for the differences in specific activities, and similar amounts of solubilized activity were obtained per mg of DNA, indicating that similar amounts of enzyme are obtained from similar amounts of tissue. We have demonstrated previously (Blair, 1975) that RNA polymerases designated Ia and Ib are very
R.
BLAIR
closely related type A forms and, similarly, that polymerases IIa and IIb are very nearly the same. If forms a, and b are interconvertable, then random or uncontrolled interconversion during solubilization from nuclei and/or during chromatographic separation could account for the variability found in the percentage distributions of RNA polymerases. Data are lacking for mouse liver RNA polymerases but Matsui er al. (1976) and Kellas et al. (1977) have presented evidence that the two class I (or A) RNA polymerases isolated from rat liver nuclei represent “free” and “template-bound” or “engaged” forms, and Kellas et al. (1977) have shown that sonication results in conversion of the latter form to the former one. However, we found previously (Blair. 1975) that forms IIa and IIb were not converted one to the other during chromatography, and statistically significant differences appeared to be non-random because they were usually between enzymes from Swiss mouse liver and those from livers from other mouse strains and usually for enzyme III. The percentage distribution of enzymes from Swiss mouse liver was similar to that reported previously (Blair, 1975). It is also similar to the distribution found by Schwartz et al. (1974) for BALB/c mouse liver but differs from that reported for CFl mouse liver (Versteegh & Warner, 1973) and CBA mouse liver (Smuckler & Hadjiolov, 1972) in that no RNA polymerase III (or C) was reported in the latter studies. The finding of significant differences in regard to units of activity per DNA amount only for two enzymes (la and IIa) and between only three strains of mice (Swiss and BDF, and BDF, and C3H, respectively) indicates that, notwithstanding the extensive variability between samples, there is substantial uniformity between mouse strains. Uniformity is further indicated by the lack of statistically significant differences in regard to the specific activities of the individual RNA polymerases. However, there were significant differences in regard to degree of purification. As indicated above (see “Results”) the best specific activities which we obtained for mouse liver RNA polymerases were several fold greater than those previously reported for CBA mouse liver (Smuckler & Hadjiolov, 1972). The best overall purifications of the mouse liver enzymes from nuclei of 4&876-fold compare favorably with the purifications of 12-179-fold previously reported for enzymes from the murine Ehrlich ascites carcinoma (Blair, 1975). Percentage distributions for RNA polymerases similar to those found for mouse liver in the present investigation have also been found by us for Swiss mouse heart, lung, kidney and spleen (Blair, 1975; Blair, 1978; Blair et al.. 1977). Only nuclear RNA polymerases I and II were isolated from spleens from ICR/Ha Swiss mice (Babcock & Rich, 1973) and enzymes I and II (heterogeneous) from spleens from NIH Swiss mice (Sethi & Gallo, 1975), but nuclear enzymes I, II and III were obtained from B6AF1/J mouse testes (Farrell & Hecht, 1975) and from 3T6 mouse fibroblasts (Mauck. 1977). Acknowledgements-The author thanks the following persons for technical assistance: Mrs E. Davis. Mrs M. Dommasch and Miss M. Hrynchak. This research was sup-
DNA dependent RNA polymerases ported in part by a grant from the Medical Research Council of Canada.
REFERENCES BABCOCK D.
F. & RICH M. A. (1973) Deoxyribonucleic acid-dependent ribonucleic acid polymerases from murine spleen cells. Increased amounts of the nucleolar species in leukaemic tissue. Biochem. J. 133, 797-804. BAGSHAW J. C. (1974) New evidence that release of RNA polymerase in isolated nuclei requires transcription. Bio-
ihem. biophys. Res. Commun. 57; 177-182. BISWAS B. B.. GANGULY A. & DAS A. (1975)
_
Eukarvotic RNA polymerases and factors that control them. drogr. Nucleic Acid Res. Molec. Biol. 15, 145-184. BLAIRD. G. R. (1975) DNA-dependent RNA polymerases of Ehrlich carcinoma, other murine ascites tumors, and murine normal tissues. J. natn. Cancer Inst. 55, 397-412. BLAIR D. G. R. (1978) Nuclear RNA polymerases from murine transplantable tumors and homologous normal tissues. Proc. Third Int. Co@. Difirentiation. Minneapolis, MN, U.S.A. p. 30. BLAIRD. G. R. & MUKHERJEE A. K. (1973) Partial purification and characterization of RNA polymerase from nuclei of Ehrlich ascites tumor cells. Can. J. Biochem. 51, 586596. BLAIRD. G. R., D~MMASCHM. DAVISE. (1977) Comparison of nuclear RNA polymerases from mouse ascites tumors and normal tissues. Proc. Am. Assoc. Cancer Res. 18, 11. CHAUVEAUJ., MOULBY. & ROUILLIERC. (1956) Isolation of pure and unaltered liver nuclei. Morphology and biochemical comnosition. ExnI. Cell Res. 11. 317-321. CHAMBONP. (19;s) Eukaryoiic nuclear RNA polymerases. A. Rev. Biochem. 44, 613-638. DISCHEZ. (1930) Uber einige neue charakteristische farbenreaktionen der thymonukleinsaure und eine mikromethode zur bestimmung derselben in tierischen organen mit helfe dieser reactionen.
Mikrochemie
8, 432.
FARRELLD. & HECHTN. B. (1975) DNA-dependent RNA polymerases from murine testis. Expl. Cell Res. 93, 12G126.
HANKSJ. H. & WALLACER. E. (1949) Relation of oxygen and temperature in the preservation of tissues by refrigeration. Proc. Sot. exp. Biol. Med. 71, 196200. HERBERTE., POTTERV. R. & HECHTL. I. (1957) Nucleotide metabolism. VII. The incorporation of radioactivity from erotic acid-6-CL4 into ribonucleic acid in cell-free systems from rat liver. J. biol. Chem. 225, 65%674. JOI~NSON J. C., DEBACKERM. & Bonzr J. A. (1971) Deoxy-
721
ribonucleic acid-dependent ribonucleic acid polymerase of Pseudomonas putida. J. biol. Chem. 244, 1222-1232. KEDINGERC., NURET P. & CHAMLION P. (1971) Structural evidence for two a-amanitin sensitive RNA polymerases in calf thymus. FEBS Lett. 15, 169174. KELLASB., AUSTOKERJ. L.,. BEEBEET. J. C. & BUTTERWORTHP. H. W. (1977) Forms AI and AI1 DNA-dependent RNA polymerases as components of two defined pools of polymerase activity in mammalian cells. Eur. J. Biochem. 72, 583-594.
LOWRY0. H., ROSEBROUGH M. J., FARR A. L. & RANDALL A. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. MATSUIT., ONISHIT. & MURAMATSU M. (1976) Nucleolar DNA-dependent RNA polymerase from rat liver. 2. Two forms and their physiological significance. Eur. J. Biothem. 71, 361-368. MAUCK J. C. (1977) Solubilized DNA-dependent RNA polymerase activities in resting and growing fibroblast. Biochemistry 16, 793-797.
READR. S. D. & MAURITZENC. M. (1970) Isolation and preservation of cell nuclei for studies on RNA polymerase activity. Can. J. Biochem. 48, 559-565. ’ RICHARDSON J. P. (1966) Some physical properties of RNA polymerase. Proc. narn. Acad. Sci. U.S.A. 55, 16161623. ROEDERR. G. & RUTTERW. J. (1969) Multiple forms of DNA-dependent RNA polymerase in eukaryotic organisms. Nature 224, 234-237. ROEDERR. G. & RUTTERW. J. (1970) Specific nucleolar and nucleoplasmic RNA polymerases. Proc. narn. Acad. Sci. U.S.A. 65, 675-682. SCHWARTZ L.
B., SKLARV. E. F., JAEHNINGJ. A., WEINMANNR. & ROEDERR. G. (1974) Isolation and partial characterization of the multiple forms of deoxyribonucleic acid-dependent ribonucleic acid polymerase in the mouse myeloma, MOPC 315. J. biol. Chem. 249, 5889-5897.
SETHIV. S. 8c GALLOR. C. (1975) Deoxyribonucleic aciddependent ribonucleic acid polymerases from spleen of uninfected and Rauscher murine leukemia virus-infected NIH Swiss mice. Biochim. biophys. Acra 269, 269-281. SMUCKLERE. A. SC HADJIOLOVA. A. (1972) Inhibition of hepatic deoxyribonucleic acid-dependent ribonucleic acid polymerases by the exotoxin of Bacillus rhtrringiensis in comparison with the effects of a-amanitin and cordycepin. Biochem. J. 129, 153-166. STAATSJ. (1966) The laboratory mouse. In Biology of the Laboratory Mouse. (Edited by GREEN E. L.) 2nd Edn, pp. l-9. McGraw-Hill, New York. VERSTEEGH L. R. & WARNERC. (1973) DNA-dependent RNA polymerases from normal mouse liver. Biochem. biophys. Res. Commun. 53, 838-844.
Vol. IO. pp. 713 lo 721 Press Ltd 1979. Printed in Great Britain
0020.71
IX/79/0801-0713102.00/0
COMPARISON OF THE ACTIVITIES OF NUCLEAR DNA-DEPENDENT RNA POLYMERASES ISOLATED FROM LIVERS OF SEVERAL STRAINS OF MICE D. G. R. BLAIR Department of Biochemistry, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N OWO (Received 1 February 1979)
Abstract-l. Nuclear DNA-dependent RNA polymerases from liver of mouse, Mus musculus, were separated chromatographically on DEAE-Sephadex A-25. 2. Five major enzyme peaks were obtained from each sample of liver. The sensitivity of the enzymes to a-amanitin was as follows: Ia and Ib, insensitive; Ha and IIb, highly sensitive; and III, slightly sensitive. 3. The percentage distributions, activities per mg DNA, specific activities and purifications of the enzymes from BDF,, C3H/HeJ, C57BL/6J and Swiss albino mice were compared and discussed. 4. Statistically significant differences (P < 0.05) were found between some strains of mice for percentage distribution, activity per mg DNA, and purification of some RNA polymerases.
INTRODUCTION
Searle Corp. Calf thymus “native” DNA (highly polymerized) was a product of Worthington Biochemical Corp.
Multiple nuclear DNA-dependent RNA polymerases Bovine serum albumin (BSA; fraction V) and the disodium [nucleosidetriphosphate:RNA nucleotidyltransferase salts of GTP and CTP were supplied by Schwarz/Mann. (DNA dependent), EC 2.7.7.61 have now been isolated Yeast total RNA, phosphoenolpyruvate trisodium salt, from numerous eukaryotic tissues or cells (Biswas et rabbit muscle pyruvate kinase (type III; 500 units/mg), the al., 1975; Chambon, 1975) including the liver of the disodium salt ii ATP, the trisod&m salt of UTP and dithcommon mouse, Mus musculus. The enzymes are iothreitol (DTT) were obtained from Sinma Chemical Co. DEAE-hphahex A-25 (Pharmaciay 4&120 PC) was classified according to their order of elution from DEAE-Sephadex A-25 (Roeder & Rutter, 1969) or swollen in TGMED buffer [SOmM Tris-HCI, pH 7.9; 25% (vol/vol) glycerol; 5 mM MgCl*; 0.1 mM EDTA; 0.5 mM according to their sensitivity to cc-amanitin (Kedinger DTT] containing 0.5 M (NH&SO, and 0.002% Hibitane, et al., 1971; Chambon, 1975). Type A (or I) mam- and equilibrated with TGMED buffer containing 0.05 M malian RNA polymerases are insensitive to a-amani(NH&SO, prior to use by repeated washing. tin whereas type B (or II) enzymes are highly sensitive a-Amanitin was a gift from Dr T. Wieland, Max Planck and type C (or III) are inhibited only at very high Institut, Heidelberg, West Germany. All other chemicals were reagent grade, and all solutions were prepared with concentrations of the mushroom toxin (Chambon, double-distilled water. 1975). Animals. Specific-pathogen-free Swiss albino mice (ConMultiple RNA polymerases have been isolated from livers of BALB/c (Schwartz et al., 1974), CBA naught stock) were obtained from the Animal Resources Center, University of Saskatchewan. C3H/HeJ and (Smuckler & Hadjiolov, 1972), CFl (Versteegh & C57BL/6J strain mice and B6D2FI/J (BDF1) hybrids were Warner, 1973), Swiss albino (Blair, 1975), and Swiss- purchased from the Jackson Laboratory, Bar Harbor, Webster (Bagshaw, 1974) mice, but a systematic com- Maine. All animals were adult males weighing 20-25 g. parison of activities and other parameters of the enzymes has not been made. Two mouse liver type Methods Preparation of nuclei from mouse liver. Livers were I (or A) enzymes (Smuckler & Hadjiolov, 1972; Versteegh & Warner, 1973; Blair, 1975), two type II (or excised from 5 mice following etherization of the animals B) enzymes (Smuckler & Hadjiolov, 1972; Blair, 1975) and placed in ice-cold Hanks’ balanced salt solution and at least one type III (or C) enzyme (Blair, 1975; (HBSS) (Hanks & Wallace, 1949). They were cleaned of extraneous tissue, blotted dry, weighed and chopped finely Schwartz et al., 1974) have been isolated. In conjuncwith scissors in 4 vol of SMEP (0.25 M sucrose, 5 mM tion with studies on nuclear RNA polymerases from MgCII, 4 mM EDTA and 10mM potassium phosphate a series of murine transplantable tumors and tissues buffer, pH 7.0) (Read & Mauritzen, 1970). The chopped of normal Swiss albino mice (D. G. R. Blair, manutissue was then homogenized briefly in a Dounce glass script in preparation), RNA polymerases were also homogenizer with teflon pestle. Following the addition of isolated from livers of several strains of normal mice, sapon& (lo%),the homogdnate was shaken in an ice-water which were used as the tumor hosts. The latter studies bath for 30 min. diluted 1:2 with SMEP and centrifuged for 10min at i2OOg (average). The saponin treatment are the subject of this communication. Preliminary (Read & Mauritzen, 1970) was necessary in order to treat reports of some of the studies have been presented the liver nuclei in the same way as nuclei from murine (Blair et al., 1977; Blair, 1978). tumors (Blair, 1975). The sedimented nuclei were resusMATERIALS AND
METHODS
Materials Chemicals. Tritiated uridine 5’-triphosphate (3H-5-UTP) of 15. 17.8 or 24 Ci/mmol was obtained from Amersham-
pended in SMEP and mixed thoroughly with 6~01 of 2.4 M sucrose-5 mM MgC12. This suspension was centrifuged for 1 hr at 30,000~ (average) to sediment the nuclei (Chauveau et al., 1956; Read & Mauritzen, 1970). The supernatant fraction was discarded, the walls of the tubes 713
714
D. G. R. BLAIR
were wiped clean and the pellet of nuclei was resuspended and washed twice in SMEP to remove excess sucrose. Purity of the nuclei was checked by phase contrast microscopy. The purified nuclei were used immediately for solubilization of RNA polymerase (o.i.) after suspension in TSMD buffer CO.01 M Tris-HCI (pH 7.9), 1.0 M sucrose, 5 mM MgC12, 5 mM DTT]. Samples df nuclei were taken for determination of DNA and protein and RNA polymerase activity (o.i.). Based on DNA content the recovery of nuclei was 60-650/b. Solubilization of RNA polymerase. RNA polymerase activity was solubilized from liver nuclei according to the method of Roeder & Rutter (1970) and in the same manner as from nuclei of Ehrlich ascites cells (Blair & Mukherjee, 1973; Blair, 1975) by sonic disruption of the nuclei in TSMD (LX) containing 0.3 M (NH&SO,. The enzymes were precipitated with (NH&SO,, collected by centrifugation, dissolved in TGMED buffer (pH 7.9) containing 0.05 M (NH&SO4 and dialyzed versus the same buffer for 6-8 hr at 4°C (Blair & Mukherjee, 1973). The diffusate was centrifuged at 105,OOOg (average) for 1 hr and the resultant supernatant fraction (F4) was stored at -70°C for chromatography the next day (u.i.). The solubilization of RNA polymerase was monitored at each step by determination of protein and DNA and of RNA polymerase activity. Chromatographic separation ofnuclear RNA polymerases. RNA polymerases of the F4 fraction were separated by chromatography on DEAE-Sephadex A-25 (Roeder & Rutter, 1969) with a nine-step gradient of (NH&SO., in TGMED buffer as described for the enzymes from mouse tumors and normal tissues (Blair, 1975). Columns of DEAE-Sephadex A-25 (0.9 x 1.2cm) were prepared and equilibrated with TGMED buffer (pH 7.9) containing 0.05 M (NH&SO4 in the cold room (4°C). A 5-ml sample of solubilized RNA polymerase (F4) (616 mg protein) was layered on top of the resin and washed into it with 5 ml of TGMED buffer (pH 7.9) containing 0.05 M (NH&SO,. Five-ml portions of buffer containing 0.10, 0.15. 0.20, 0.25, 0.30, 0.35, 0.40, 0.45 or 0.50 M (NH&SO4 were then added in succession. One-ml fractions were collected with a Buchler Fractomat fraction collector at 4°C. Protein was estimated from the absorption of the eluates at 280 nm with BSA (fraction V) as a standard which has a specific absorbance of 0.65/mg/ml at 280 nm, similar to that for RNA polymerase from Escherichia coli (Richardson, 1966) and Pseudomonas putida (Johnson et al., 1971). The fractions (0.1 ml aliquo&) were assayed immediately for RNA polvmerase activity, and, after the addition of BSA (1 mgjmi) as an enzyme stabilizer (Roeder & Rutter, 1969). were stored at -70°C. RNA polymerase assays. (1) Standard assay. RNA polymerase activity of nuclear and chromatographic fractions was determined by the incorporation of C3H]UMP from C3H]UTP into acid-precipitable RNA in the same manner as for murine tumors and normal tissues (Blair & Mukherjee, 1973; Blair, 1975). A unit of enzyme activity is defined as the amount of enzyme which catalyzes the incorporation of 1 pmol of [3H]UMP into an acid-insoluble product in 1 min at 37°C. The standard assay medium (Blair, 1975) was essentially the same as that employed by Roeder & Rutter (1970) for enzymes from rat liver and sea urchin. The reaction medium (0.25 ml) included 0.1 ml of enzyme preparation in TGMED buffer (pH 7.9) plus 0.05 M (NH&SO+ The final concentrations of components were: “native” calf thymus DNA, 160pg/ml; pyruvate kinase, 6.8 I.U./ml; phosphoenolpyruvate, 4 mM; 2-mercaptoethanol, 1.6 mM; NaF, 6 mM; KCI, 8 mM; MnCI,, 1.6 mM; ATP, CTP and GTP, 0.6mM each; [“HIUTP. O.lOmM (4pCi/ml);
(NH&SO,, 0.05 M; Tris-HCI buffer (pH 7.9), 76 mM; MgCIZ, 2 mM; EDTA, 0.04 mM; DTT, 0.2 mM; and glycerol, 10%.
After an incubation period of 10 min at 37-C. the reaction mixtures were placed in ice. and 1 ml of Na,P,Ocontaining 2mg of both yeast RNA and BSA was added immkdiately to each. followed sequentially by 1 ml of 5”,, (wt/vol) sodium dodecyl sulfate and 1 ml of cold 20“” trichloracetic acid (TCA) plus 0.04 M Na4P,0,. The precipitates were collected on glass fiber filters (Whatman GF/C) and processed for determination of radioactivity, which was measured in a Nuclear-Chicago Unilux I spectrometer at a counting efficiency of about 45”/, (Blair & Mukherjee, 1973; Blair, 1975). Enzyme incubations were done in duplicate. Values for enzyme-free controls were subtracted from those of samples containing enzyme to calculate net incorporation of [‘H]UMP into RNA. (2) Assay of activity of isolated nuclei. RNA polymerase activity of samples of intact nuclei was measured as described previously (Blair & Mukheriee, 1973; Blair, 1975) essentially according to the method of Roeder & Rutter (1970) with the standard assay medium for solubilized activity which lacked calf thymus DNA, but contained 0.32 M (NH.&S04. 2 mM MgCI, and 1.6 mM MnCI,. Inhibition of RNA po/ymerases by a-amanitin. The inhibitory capaccty of a-amanitin versus the isolated RNA polymerases was tested by adding the inhibitor to the standard assay medium just before adding the enzyme. Protein und DNA assays. The protein content of nuclei and the nuclear fractions was determined by the method of Lowry et al. (1951) with BSA (fraction V) as a standard. DNA was estimated by the diphenylamine reaction (Disthe, 1930) after isolation by the method of Herbert et al. (1957). RESULTS Solubilization liver
of nuclear
RNA polymerase from mouse
Representative purification data for RNA polymerase obtained during solubilization of the enzyme activity from nuclei from livers of four strains of mice are presented in Table 1. Large variation between mouse strains is seen in regard to enzyme specific activity and to yield and purification from the suspension of nuclei or from the nuclear sonicate. The large variability was also found between samples for the same strain as will be shown by the large standard deviations of the means in the tables to follow (Tables 2-7). The purifications from nuclei found for the mouse liver solubilized RNA polymerases (F4 fractions, Table 1) are similar to those found by Versteegh & Warner (1973) for CFl mouse liver and by Blair (1975) for Swiss mouse kidney, liver and spleen. Mean data for the partial purification of the solubilized RNA polymerase (F4 fraction) from four samples of liver from each strain of mouse are presented in Table 2. Much lower mean specific activities were found for liver RNA polymerase from C3H, C57BL and BDF, mice than from Swiss mice, but only values for BDFI and C57BL were significantly different (P < q.05 > 0.025) from each other. The mean specific activity of Swiss mouse liver RNA polymerase (50.7 units/mg protein) is superior to that (38.5 units/mg protein) reported for the enzyme from CFl mouse liver (Versteegh & Warner, 1973) and to that (16.5 units/mg protein) which we previously found for Swiss mouse liver (Blair, 1975). Although mean yields of activity from the suspensions of liver nuclei were similar for the four strains of mice, yields for C57BL and Swiss were significantly different
105,000g supexnatant of diffusate (F4)
BDF, C3H/HeJ C57BL/6J Swiss
227 676 192 719 181 601 184 835
0.4 1.0 0 2.7 0 0.7 0 1.0
17.8 18.6 24.0 41.1 13.9 13.7 21.4 23.7
BDF, C3H/HeJ C57BL/6J Swiss
precipitate
(NH&SO* (F3)
210 698 222 997
359 1112 307 2438
Total activity (units)
224 708 209 1073
82.0 79.0 87.1 108.7
BDF, C3H/HeJ C57BL/6J Swiss
105,000g supernatant (F2)
11.3 20.8 13.9 56.0
11.3 20.8 13.9 56.0
Total DNA (m8)
8.4 11.2 3.3 51.6
102.3 90.8 113.5 126.7
BDF, C3H/HeJ C57BLJ6J Swiss
Nuclear sonicate (Fl)
102.3 90.8 113.5 126.7
BDF, C3H/HeJ C57BL/6J Swiss
Total protein (m8)
Nuclear suspension
Fraction
Mouse strain
13.0 43.9 8.6 35.2
12.8 36.3 8.0 17.5
2.7 9.0 2.4 9.9
2.1 7.7 2.0 7.9
3.5 12.3 2.7 19.2
Specific activity (units/mg protein)
50.4 54.1 59.9 34.3
63.2 60.8 62.5 29.5
62.4 63.7 68.0 44.0
58.5 62.8 61.8 40.8
100 100 100 100
86.2 86.1 82.8 83.8
108.1 96.8 86.5 72.1
106.7 101.4 94.1 107.6
100 100 100 100
-
Yield From From nuclear nuclear suspension sonicate (%) (%)
Table 1. Representative purification of nuclear RNA polymerase from livers of various strains of mice
3.71 3.57 3.19 1.83
3.65 2.95 2.96 0.91
0.77 0.73 0.89 0.52
0.60 0.62 0.41 0.41
1.00 1.00 1.00 1.00
8
1 !
1.17 2.18 1.25 6.09 4.71 4.00 2.22 6.19 5.70 4.30 4.46
5 B
1.29
a 2 !! W
z 1.00 1.00 1.00 1.00
-
-
Purification From From nuclear nuclear sonicate suspension (fold) (fold)
D. G. R. BLAIR
716 Table
2. Purification
of solubihzed
nuclear
RNA polymerase
(F4 fraction)
from mouse
Purification From From nuclear nuclear suspension sonicate (fold) (fold)
,Yield
No. of samples
Mouse strain BDF, C3H/HeJ C57BL/6J Swiss * Values
Specific activity (units/mg protein)
4 4 4 4 are means
10.69 8.25 4.75 50.72
f. k 5 f
3.71* 17.92 1.84 56.21
47.24 30.48 33.43 53.79
+ k + +
sonicate was approximately 50% higher for Swiss than for the other strains of mice, but yields were not significantly different one from the other. The degree of enzyme purification, from either the nuclear suspension or the sonicate, was also higher for the Swiss strain than for the others, but differences were not significant. Total RNA polymerase activity per amount of DNA The total units of liver RNA polymerase activity per mg of DNA, based on endogenous nuclear activity (nuclear suspension) or on solubilized activity (F4 fraction), were quite similar for all strains of mice (Table 3). Differences between strains for either parameters were not significant statistically. Both parameters were highest for C3H liver, but the remaining ranking order differed, with BDFi being lowest for one and C57BL for the other. These data indicate that similar amounts of enzyme activity were obtained for similar amounts of tissue prior to and after enzyme solubilization. Purification of mouse liver RNA DEAE-Sephadex A-2S
polymerases on
Five peaks of RNA polymerase activity solubilized from mouse liver nuclei were eluted from columns of DEAE-Sephadex A-25 with a stepwise gradient of (NH&SO, (Blair, 1975). Similar chromatograms were obtained for liver enzymes from all four strains Table
3. Total
BDF, C3H/HeJ C57BL/6J Swiss * Values
4 4 4 4 are means
Table 4. Percentage
* Values
62.77 65.00 60.38 93.62
f k + k
29.66 24.04 23.41 67.50
3.11 1.62 1.88 6.60
f f * 1
0.79 1.13 0.43 7.11
4.21 2.98 3.36 7.63
+ k f. k
1.42 1.52 1.26 6.46
of mice (Figs l-4). RNA polymerase forms Ia, Ib and III were resistant to 0.3 pg a-amanitin/ml whereas the activities of enzyme forms IIa and IIb were inhibited completely. Enzyme activity designated III was inhibited by about 50% at 5Opg a-amanitin/ml whereas activity designated I was not. Note that the enzyme activity scale for BDFi mouse liver (Fig. 1) and C3H/HeJ mouse liver (Fig. 2) is twice that for C57BL/6 mouse liver (Fig. 3) and ten times that for Swiss mouse liver (Fig. 4), thus rendering some peaks of activity comparatively insignificant in the latter case. The percentage recoveries of enzyme activity from the chromatographic columns were 95, 89, 100 and 93, respectively, for BDFi, C3H/HeJ, C57BL/6 and Swiss mouse liver samples (Figs l-4). The mean percentage recoveries of enzyme activities from the chromatographic columns for 4 samples each of BDFi, C3H/HeJ, C57BL/6 and Swiss mouse liver RNA polymerase were 105, 93, 100 and 101, respectively. No activity was eluted at an ammonium sulfate concentration below 0.1 M. There were two or three peaks of activity preceding RNA polymerase Ha, one peak each of Ha and IIb, and one or more peaks designated RNA polymerase III following the IIb peak. BDF, (Fig. 1) and C57BL/6J (Fig. 3) chromatograms usually had the pre-Ia peak. The post-Ib peaks on the C57BL/6J (Fig. 3) and Swiss (Fig. 4) chromatograms were type I activity according to their resistance to a-amanitin. Although enzyme peak Ib is activity
33.54 48.32 36.56 43.19
k + k +-
9.65* 27.59 16.50 33.63
of RNA polymerase Percentage
k SD.
f & + +
DNA
Solubilized (F4 fraction) activity (units/mg DNA)
Endogenous nuclear activity (units/mg DNA)
Ia 14.71 25.72 20.29 11.62
per mg of nuclear
16.58 20.72 11.78 13.51
& + + k
10.11 14.17 4.39 9.21
+ SD
distribution
N 3 4 4 4
are means
RNA polymerase
No. of samples
Mouse strain
BDF, C3H/HeJ C57BL/6J Swiss
24.72 17.45 5.30 11.44
+ SD.
(P < 0.05 > 0.025). The mean yield from the nuclear
Mouse strain
From nuclear sonicate (%)
From nuclear suspension (X)
liver nuclei
Ib 4.00, 14.33 5.21 4.44
4.80 11.72 8.35 8.70
+ + + k
1.74 3.46 5.10 4.49
activity
among
distribution Ha 26.14 21.93 32.68 47.21
+ + + f
3.86 12.05 8.55 7.04
the multiple
liver enzymes
of activity IIb 31.98 25.76 20.71 20.35
+ + + +
III 9.20 9.32 9.75 9.85
8.31 5.08 7.61 1.98
f f f +
2.65 1.01 1.46 0.71
DNA dependent RNA polymerases
717
Table 5. Activities of mouse liver RNA polymerases Mouse strain BDF, C3H/HeJ C57BLf6J Swiss
N
la
3 4 4 4
2.72 k 3.49 * 2.45 f 1.57 *
Units of RNA polymerase/mg DNA Ha IIb Ib 0.30* 3.05 1.11 0.52
0.87 * 1.66 * 1.01 k 1.05 k
0.12 1.51 0.72 0.24
5.10 + 1.93 k 4.16 f 7.73 f
1.21 1.06 2.23 4.47
III
7.24 + 4.54 3.99 f 3.97 2.06 rf: 0.30 3.86 k 3.35
1.65 f 0.65 0.54 + 0.30 0.95 f 0.49 0.27 If: 0.09
* Values are means k SD.
Table 6. Specific activities of mouse liver RNA polymerases Mouse strain BDF, C3H/HeJ C57BL/6J Swiss
N
Ia
3 4 4 4
57.1 + 62.7 k 53.7 k 40.3 +
Specific activities (units/mg protein) IIa
Ib 39.0* 55.5 42.3 23.4
23.2 + 82.3 + 107.2 f 38.7 k
15.8 68.0 76.8 22.5
93.3 + 58.3 + 205.1 f 703.6 k
34.0 16.6 125.0 986.9
III
IIb 1144.0 k 120.3 k 301.4 f 646.4 f
1365.1 67.1 304.8 974.1
98.9 + 137.2 f 109.4 + 56.9 +
27.2 124.7 44.8 27.3
* Mean values + SD. Table 7. Degree of purification of mouse liver RNA polymerases
Mouse strain BDF, C3H/HeJ C57BLJ6J Swiss
N
Ia
3 4 4 4
4.7 * 5.5 * 9.7 f 1.6 +
Purification from solubilized enzyme (fold) IIa IIb Ib 3.1* 5.5 6.4 0.9
1.9 f 7.0 + 20.5 + 1.6 f
1.2 6.8 8.9 1.1
7.5 f 4.9 * 43.0 f 15.5 f
2.7 2.1 14.8 8.4
82.8 * 9.9 + 67.0 f 12.6 f
III 104.0 7.0 49.1 8.9
8.0 f 10.2 f 28.3 f 5.8 +
3.2 12.0 14.6 8.7
* Mean values + SD. I
29 BDF,
(NH,)$30,
(MB
MOUSE
:.I
LIVER
I 0.1,
1 0.2
FRACTION ,NUMBER I I 0.25 0.3 0.31
I 0.4
I 0.4,
I 0.5
Fig. 1. DEAE-Sephadex A-25 chromatography of solubilized nuclear RNA polymerase from BDF, mouse liver. Solubilixed enzyme (F4 fraction) (106 units/l.1 mg protein) was applied to a 0.1 x 12 cm column of DEAE-Sephadex A-25 in 5 ml of TGMED buffer, pH 7.9, containing 0.05 M (NH&SO., and washed into the resin with 5 ml of the application buffer. RNA polymerases were eluted by the stepwise application of successive 5-ml portions of TGMED buffer, pH 7.9, containing 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45 and 0.50 M (NH&SO.+, beginning at the fractions indicated by the arrows. Fractions of l.Oml were collected, and aliquots of 0.1 ml of each were assayed for protein and RNA polymerase activity. B.C. IO/S-D
718
D. G. R. BLAIR
C3HIH.J
MOUSE
LIVER
lb
IO (NH&SO,
(M)
20
d.,
O.IJ ’
30 FRACTION NUMBER
0.2 ’
0.2, ’
0.3 ’
40
0.1, ’
50
0.4 ’
0.4, ’
,
01’
Fig. 2. DEAE-Sephadex A-25 chromatography of solubilized nuclear RNA polymerase from C3H/HeJ mouse liver. Solubilized enzyme (F4 fraction) (117 units/lOmg protein) was applied to a 0.9 x 12cm column of DEAE-Sephadex A-25 in 5 ml of TGMED buffer, pH 7.9, containing 0.05 M (NH&S04 and washed into the resin with 5 ml of the application buffer. Enzymes were eluted and assayed as described in the legend to Fig. 1. larger than peak Ia in the particular
chromatograms
for C3H/HeJ and Swiss mouse livers (Figs 2 and 4) shown here, in other cases the reverse was true. Similarly, there was not complete consistency in the relative sizes of peaks IIa and IIb. These variations are reflected in the mean percentage distributions of activity between the multiple RNA polymerases which have large standard deviations (o,i., Table 4). Consequently, enzyme peaks Ia and Ib, as well as IIa and IIb, are regarded as being closely related forms of
type I and type II RNA polymerase, respectively. No studies have been done to purify these enzymes further or to determine their subunit structure since our primary purpose was to compare elution patterns of the mouse liver enzymes from DEAE-Sephadex A-25 with those of mouse tumors and other tissues from normal Swiss mice (D. G. R. Blair, manuscript in preparation), and to show the variation obtained even for RNA polymerases of the same tissue from different strains of mice.
D-
C!57BL/6
MOUSE
LIVER
II0 I lib
D-
O-
Q-
30
(NH,),SO,
IO (Lo
do
30 FRACTION ,NlJMfl:R 1 I 0.2 0.25 0.3 0,s
20 cd,
SO
40 I 0.4
I 0.4,
I 0.5
Fig. 3. DEAE-Sephadex A-25 chromatography of solubilized nuclear RNA polymerase from C57BL/6 mouse liver. Solubilized enzyme (F4 fraction) (239 unitsjl3.0 mg protein) was applied to a 0.9 x 12 cm column of DEAE-Sephadex A-25 in 5 ml of TGMED buffer, pH 7.9, containing 0.05 M (NH&SO4 and washed into the resin with Sml of the application buffer. Enzymes were eluted and assayed as described in the legend to Fig. 1. .
DNA dependent RNA polymerases
f RA~TION,NUWBIER (NH&SO,
(M)
o!
0.15 ’
1
1 0.1
0.25
03
0.35
0.4
t 045
I 0.5
Fig. 4. DEAE-Sephadex A-25 chromatography of solubilized nuclear RNA polymerases from Swiss mouse liver. Solubilized enzyme (F4 fraction) (564 units/i6mg protein) was applied to a 0.9 x 12cm column of DEAE-Sephadex A-25 in 5 ml of TGMED buffer, pH 7.9, containing 0.05 M (NH&JO4 and washed into the resin with 5 ml of the application buffer. Enzymes were eluted and assayed as described in the legend to Fig. 1. Percentage distribution of RNA polymerase activity among the nuclear enzymes isolated on DEAESephadex A-25
The percentage distribution of activity among the multiple peaks of mouse liver RNA polymerase was not the same for each strain of mouse (Table 4). Whereas the mean percentage of activity in peak III was greater than in peak Ib in the case of BDFi, it was smaller than in peak Ib in all other cases. For BDFr and C3H/HeJ the percentage of activity in IIb was greater than in IIa, but the opposite was found for the other two mouse strains. Also, whereas similar mean percentages of activity were found in peak IIb for all mouse strains, greater variation between strains was found for the other peaks of activity. Statistical analysis (Student’s t-test) showed that differences between Swiss and C3H/HeJ for Ia, BDF, and C3H/HeJ for Ib and C3H/HeJ and C57BL/6J for III were probably significant (P < 0.025 > O.Ol), while the difference between Swiss and C3H for III was significant (P < 0.005 > 0.001) and that between Swiss and C57BL/6J for III was highly significant (P < 0.001). Activities of isolated RNA polymerases per amount of DNA
Table 5 presents data on the total activity present in each chromatographic peak of RNA polymerase per amount of DNA. These mean values reflect the mean percentage distributions of activity (Table 4) and also have relatively large standard deviations. Statistical analysis (t-test) revealed probably significant differences only between Swiss and BDFi for enzyme Ia (P < 0.05 > 0.025) and between BDFi and C3H/HeJ for enzyme IIa (P < 0.025 > 0.01). Specific activities of isolated RNA polymerases The specific activity of the most active fraction
from each chromatographic peak of RNA polymerase was calculated, and the means with standard deviations are presented in Table 6. The same degree of purification was not obtained for the same RNA polymerase from each of the strains of mice and standard deviations were relatively large for values for any strain. Generally RNA polymerase Ia or Ib had the lowest specific activity of the five major RNA polymerase forms and RNA polymerase IIb had the highest. However, in Table 6 C3H/HeJ is an exception for which enzyme III had the highest mean specific activity and IIa the lowest. The enzymes eluting with the lowest concentrations of ammonium sulfate generally had the lowest specific activities because the bulk of the protein was also eluted at these concentrations. Statistical analysis (t-test) of the data showed that the differences between mouse strains for each RNA polymerase were not significant. The best single specific activities (units/mg protein) obtained were 155.7 (C3H), 232.8 (C57BL), 2664.4 (Swiss), 3074.1 (BDFt) and 324.1 (C3H) for enzymes Ia, Ib, IIa, IIb and III, respectively. These specific activities compare favorably with values of 503 units/mg protein for enzyme B and 87.4 units/mg protein for enzyme A from CBA mouse liver recalculated from the data of Smuckler & Hadjiolov (1973). Degree of purijication of isolated RNA polymerases
The degree of purification of the chromatographitally isolated liver RNA polymerases from the partially purified mixture of enzymes (F4 fraction) is indicated in Table 7. The same degree of purification was not obtained for the same enzyme from different strains of mice, and, as for the mean specific activities, standard deviations were relatively large. The highest purifications were generally obtained for enzyme IIb and the lowest for enzyme Ia, but C3H, as in the case of specific activity, was an exception for which
D. G.
720
enzyme III was purified the most and enzyme IIa the least. Statistical analysis (t-test) revealed probably significant differences (P < 0.05 > 0.01) in purification between Swiss and C57BL livers for enzymes la, IIa and III, and between BDF, and C57BL livers for enzymes Ib and IIa. There were significant differences (P < 0.01 > 0.001) between Swiss and C57BL livers for enzyme Ib and between C3H and C57BL for enzyme IIa. The best single purifications (fold) from the F4 fraction which were obtained were 18.7 (C57BL). 35.9 (C57BL). 60.1 (C57BL), 234.7 (BDF,) and 48.0 (C57BL) for enzymes Ia, Ib, Ha, IIb and III, respectively. These represent overall purifications (fold) from nuclei of 39.8, 66.9, 112.0, 876.0 and 57.0. respectively. DISCUSSION
Comparison of the hepatic RNA polymerase activity of Swiss albino, C3H/HeJ, C57BL/6J and BDF, mice showed that considerable variation exists between samples from each strain of mouse and between samples from different strains in regard to all parameters measured. C3H/HeJ, C57BL/6J and Swiss albino mice each have separate origins and therefore are not closely related genetically (Staats, 1966), but BDFi mice, being Fi hybrids from the C57BL/6J female x DBA/ZJ male cross, are closely related to the C57BL/6J male mice used in these experiments. If the variation between samples is genetically based, it should be less between samples from BDF, and C57BL/6J mice. However, the fact that the specific activities of the solubilized RNA polymerase (F4 fractions) were probably significantly different (P -L0.05> 0.025) only for these two strains suggests that the variation is not genetically based. This is further supported by probably significant differences between the two strains in regard to degree of purification of enzyme forms Ib and IIa. Parameters measured were not consistently highest for one specific mouse strain and lowest for another. Thus, variation appears to be usually random and probably due to the large number of procedures and manipulations required for purifying the RNA polymerases. Since, to our knowledge, no similar statistical data have been presented in the literature we do not know if our data show excessive variation. The mean purifications of the solubilized mouse liver RNA polymerase preparations (F4) from nuclei which we obtained were similar to those found previously by us (Blair, 1975) for Swiss mouse liver and by Versteegh & Warner (1973) for CFl mouse liver. Although the mean specific activity of the solubilized RNA polymerase from Swiss mouse liver was superior to previously published values for Swiss (Blair, 1975) and CFl (Versteegh & Warner, 1973) mouse livers, specific activities for the solubilized enzyme from livers of the other three strains of mice were lower. There is no apparent reason for the differences in specific activities, and similar amounts of solubilized activity were obtained per mg of DNA, indicating that similar amounts of enzyme are obtained from similar amounts of tissue. We have demonstrated previously (Blair, 1975) that RNA polymerases designated Ia and Ib are very
R.
BLAIR
closely related type A forms and, similarly, that polymerases IIa and IIb are very nearly the same. If forms a, and b are interconvertable, then random or uncontrolled interconversion during solubilization from nuclei and/or during chromatographic separation could account for the variability found in the percentage distributions of RNA polymerases. Data are lacking for mouse liver RNA polymerases but Matsui er al. (1976) and Kellas et al. (1977) have presented evidence that the two class I (or A) RNA polymerases isolated from rat liver nuclei represent “free” and “template-bound” or “engaged” forms, and Kellas et al. (1977) have shown that sonication results in conversion of the latter form to the former one. However, we found previously (Blair. 1975) that forms IIa and IIb were not converted one to the other during chromatography, and statistically significant differences appeared to be non-random because they were usually between enzymes from Swiss mouse liver and those from livers from other mouse strains and usually for enzyme III. The percentage distribution of enzymes from Swiss mouse liver was similar to that reported previously (Blair, 1975). It is also similar to the distribution found by Schwartz et al. (1974) for BALB/c mouse liver but differs from that reported for CFl mouse liver (Versteegh & Warner, 1973) and CBA mouse liver (Smuckler & Hadjiolov, 1972) in that no RNA polymerase III (or C) was reported in the latter studies. The finding of significant differences in regard to units of activity per DNA amount only for two enzymes (la and IIa) and between only three strains of mice (Swiss and BDF, and BDF, and C3H, respectively) indicates that, notwithstanding the extensive variability between samples, there is substantial uniformity between mouse strains. Uniformity is further indicated by the lack of statistically significant differences in regard to the specific activities of the individual RNA polymerases. However, there were significant differences in regard to degree of purification. As indicated above (see “Results”) the best specific activities which we obtained for mouse liver RNA polymerases were several fold greater than those previously reported for CBA mouse liver (Smuckler & Hadjiolov, 1972). The best overall purifications of the mouse liver enzymes from nuclei of 4&876-fold compare favorably with the purifications of 12-179-fold previously reported for enzymes from the murine Ehrlich ascites carcinoma (Blair, 1975). Percentage distributions for RNA polymerases similar to those found for mouse liver in the present investigation have also been found by us for Swiss mouse heart, lung, kidney and spleen (Blair, 1975; Blair, 1978; Blair et al.. 1977). Only nuclear RNA polymerases I and II were isolated from spleens from ICR/Ha Swiss mice (Babcock & Rich, 1973) and enzymes I and II (heterogeneous) from spleens from NIH Swiss mice (Sethi & Gallo, 1975), but nuclear enzymes I, II and III were obtained from B6AF1/J mouse testes (Farrell & Hecht, 1975) and from 3T6 mouse fibroblasts (Mauck. 1977). Acknowledgements-The author thanks the following persons for technical assistance: Mrs E. Davis. Mrs M. Dommasch and Miss M. Hrynchak. This research was sup-
DNA dependent RNA polymerases ported in part by a grant from the Medical Research Council of Canada.
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SETHIV. S. 8c GALLOR. C. (1975) Deoxyribonucleic aciddependent ribonucleic acid polymerases from spleen of uninfected and Rauscher murine leukemia virus-infected NIH Swiss mice. Biochim. biophys. Acra 269, 269-281. SMUCKLERE. A. SC HADJIOLOVA. A. (1972) Inhibition of hepatic deoxyribonucleic acid-dependent ribonucleic acid polymerases by the exotoxin of Bacillus rhtrringiensis in comparison with the effects of a-amanitin and cordycepin. Biochem. J. 129, 153-166. STAATSJ. (1966) The laboratory mouse. In Biology of the Laboratory Mouse. (Edited by GREEN E. L.) 2nd Edn, pp. l-9. McGraw-Hill, New York. VERSTEEGH L. R. & WARNERC. (1973) DNA-dependent RNA polymerases from normal mouse liver. Biochem. biophys. Res. Commun. 53, 838-844.