Volume 6 Number 5 1979
Nucleic Acids Research
Properties of in vitro transcription by isolated Xenopus oocyte nucleoli
Hidetoshi Saiga I and Toru Higashinakagawa1 5 Laboratory of Developmental Biology, Mitsubishi-Kasei Institute of life Sciences, 11 Minamiooya, Machida, Tokyo, Japan Received 8 March 1979 ABSTRACT Some properties of in vitro transcription by isolated Xenopus oocyte nucleoli were described. When incubated with labeled RNA precursors, Xenopus oocyte nucleoli exhibited prolonged incorporation of radioactivity into RNA. The synthetic activity was exclusively due to type I RNA polymerase as revealed by its insensitivity to low and high doses of a-amanitin. The size of the in vitro transcript was mQstly larger than 28S at 10 minute incubation and became smaller as incubation proceeded. When [Y-32P]ATP was included in the reaction mixture, 32P radioactivity was incorporated into RNA suggesting the possible initiation of transcription in this system. However, analysis of the terminal nucleotide of the transcript revealed that the incorporation of radioactivity from [y-32P]ATP was not due to the initiation of transcription but due to polynucleotide kinase activity in the nucleolar preparation. These results demonstrate that the incorporation of radioactivity from [y_32p] labeled nucleoside triphosphates cannot necessarily be regarded as an index of the initiation of transcription. INTRODUCTION One way to define the components involved in the control of transcription in eukaryotes would be to construct a faithful in vitro system which displays an accurate transcription as compared with events in vivo. Nucleoli of Xenopus oocytes are especially suited for this approach, since these nucleoli, in contrast to the nucleoli from other somatic cells, contain mostly ribosomal RNA genes and are regarded as a single gene organelle. These features may greatly facilitate the identification of the components which control the transcription of the ribosomal gene. From these standpoints, we have examined the in vitro transcription by isolated Xenopus oocyte nucleoli. There are several criteria to be satisfied for accurate transcription. The faithful system should possess stable RNA synthesizing activity, permit RNA polymerase to select proper DNA strand and allow precise RNA chain initiation and termination. In studies with isolated nuclei or nucleoli as an in vitro transcription system, the most crucial test is whether or not the initiation of transcription can be detected by an appropriate method.
C) Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England
1929
Nucleic Acids Research In this paper some properties of the in vitro transcription by isolated Xenopus oocyte nucleoli are presented. Although the oocyte nucleoli exhibited incorporation of radioactivity from [y-3 P] labeled nucleoside triphosphates into RNA, the incorporation was exclusively due to other enzymatic activity rather than the initiation of transcription.
MATERIALS AND METHODS Isolation of Xenopus oocyte nucleoli The procedure for the isolation of oocyte nucleoli has already been described by Higashinakagawa et al. (1). In this paper we have modified and simplified the procedure, omitting the centrifugation in Metrizamide (described elsewhere). The DNA composition of the nucleolar preparation was examined by analytical ultracentrifugation in CsCl density gradient. About 70 % of the total DNA was found to be ribosomal gene (data not shown). Conditions for in vitro transcription The standard reaction mixture for in vitro transcription consisted of 50 % (v/v) glycerol, 0.15 M NaCl, 10 mM MgCl2, 10 mM Tris-HCl (pH 8.0), 0.1 mM EDTA, 5 mM dithiothreitol, 10 - 125 pg/ml of a-amanitin, 0.6 mM each of ATP, UTP, and GTP, 0.1 pCi of [14 CCTP or 1 pCi of [3H]CTP and nucleoli from 4 frogs. Incubations were performed at 25°C. In the reaction to test the initiation of transcription, the composition of the reaction mixture was the same as described above except that cold ATP (or GTP) was substituted with [y- PIATP (or[y- P]GTP). After the incubation, an aliquot was spotted onto the glass fiber disc (Whatman GF/C), washed in cold 5 % TCA-1 % Na4P207 and in ethanol. The discs were dried and counted for radioactivity. The discs were marked by an ink for drawing use so that many discs were washed batchwise simultaneously. Nucleic acid extraction Incubations were terminated by the addition of equal volume of 0.6 % SDS-0.28 M NaCl-0.1 M CH3COONa (pH 5.1), followed by extraction with phenolm-cresol mixture (phenol:m-cresol:H20, 7:1:2 v/v, containing 0.1 % 8-hydroxyquinoline). The aqueous phase was applied onto a Sephadex G-25 column (1.5 x 20 cm) equilibrated with 0.3 % SDS-0.14 M NaCl-0.05 M CH3COONa (pH 5.1). The excluded fraction was collected and the nucleic acid was precipitated with 2.5 volumes of ethanol containing 10 % (v/v) m-cresol and 2 % (w/v) CH3COOK at -20°C. The pellet was washed in 75 % (v/v) ethanol-l % (w/v) CH3COOK and in 75 % (v/v) ethanol and dissolved in an appropriate buffer.
1930
Nucleic Acids Research Determination of RNase sensitive radioactivity The nucleic acid fraction was dried in vacuo, and dissolved in 0.15 M NaCl-l mM EDTA-0.05 M Tris-HCl (pH 8.0). RNase digestion was performed with 50 tg/ml of pancreatic RNase and 5 units/ml of RNase T1 at 37°C for 30 minutes. The reaction was terminated by the addition of equal volume of cold 10 % TCA-2 % Na4P207 and 100 ig of bovine serum albumin as carrier. The acid-insoluble material was collected onto glass fiber disc (Whatman GF/C). The discs were washed with cold 5 % TCA-1 % Na4P207 and counted for radioactivity. Control incubation without RNases was performed and radioactivity was determined as described above. RNase sensitive fraction was defined as the difference between control and the RNase treated fraction. Sedimentation on sucrose gradient RNA fraction was dissolved in a small volume of 0.1 M CH3COONa-l mM EDTA-0.01 % sodium lauroyl sarcosinate (pH 6.0) and was layered on the top of 13 ml of 10 - 30 % (w/w) sucrose gradient in 0.1 M CH 3COONa-1 mM EDTA-0.01 % sodium lauroyl sarcosinate (pH 6.0) together with 10 OD260 units of rat liver cytoplasmic RNA as size marker. The gradient was centrifuged in a SW41Ti rotor (Beckman) at 30,000 rpm for 17 hours at 4°C. The gradient was fractionated with an Auto-Densiflow, monitoring at 254 nm with an ISCO UV monitor. To each fraction, equal volume of cold 10 % TCA-2 % Na P207 was added. Acid-insoluble material was collected onto glass fiber disc (Whatman GF/C) and counted for radioactivity.
Analysis of 5'-end of RNA synthesized in vitro RNA synthesized in the presence of [y- P]ATP was dissolved in 0.05 M CH3COOK (pH 4.6) and was hydrolyzed with 20 units/ml of RNase T2 at 37°C for 5 hours. The hydrolysate was applied, together with charge marker (RNase T1 digest of E. coli tRNA), onto a column of DEAE-Sephadex A-25 (0.4 x45 cm) equilibrated with 7 M urea-0.05 M Tris-HCl (pH 7.8). Fractionation was performed with a linear gradient of 0.1 - 0.5 M NaCl in the same buffer. Absorbancy at 254 nm was recorded with an ISCO UV monitor, and each fraction was counted for radioactivity by Cerenkov radiation. The pooled peak fractions at -4 charge were passed through a small DEAE-Sephadex column in order to remove NaCl and urea and hydrolyzed with 500 ig/ml of nuclease P1. The hydrolysate was applied onto a DEAE-Sephadex A-25 column (0.4 x 20 cm) equilibrated with 7 M urea-0.05 M Tris-HCl (pH 7.8) together with charge markers (5'-AMP and 5'-ATP). Fractionation and counting for radioactivity were performed as described above. The peak fractions of the second DEAE-Sephadex column chromatography 1931
Nucleic Acids Research were diluted to 10 fold with distilled water and applied together with 5'XMPs (X=A,C,G or U) onto a Dowex 1 x 2 column (0.4 x 15 cm) equilibrated with 0.0075 N HR. Elution was performed with a linear gradient of 0 - 0.05 M
NaCl in 0.0075 N HCl, monitoring with an ISCO UV monitor at 254 nm. radioactivity was counted directly by 6erenkov radiation.
32P
Assay for polynucleotide kinase activity Polynucleotide kinase activity in the isolated nucleoli was assayed according to the method of Ichimura and Tsukada with some modifications (2). The reaction mixture consisted of 0.083 M sodium acetate buffer (pH 5.5),
0.01 M1 MgCl2, 0.0167 M 0-mercaptoethanol, 15 ig of 5'-hydroxyl-terminated calf thymus DNA, 2 nmoles of [y-3 P]ATP and isolated nucleoli. Incubation was performed at 37°C for 30 minutes. After the reaction, NaOH was added to give final concentration of 0.1 M. The mixture was then heated at 80°C for 20 minutes. After neutralization with HR, the mixture was treated with 50 pg/ml of pancreatic RNase and acid-insoluble radioactivity was counted. When the assay was performed under the conditions for in vitro transcription, the extracted nucleic acid was treated with alkali and RNase and subjected to alkaline CsCl density gradient centrifugation. Centrifugation was performed in a RP65T rotor (Hitachi) at 33,000 rpm for 60 hours at 20°C. The gradient was fractionated with an Auto-Densiflow and radioactivity was counted by &erenkov radiation. 5'-Hydroxyl-terminated calf thymus DNA was prepared according to the
method of Richardson (3). Materials
[3HICTP
(20.1 Ci/mmole), [y- P]ATP (2 - 10 Ci/mmole) were purchased from New England Nuclear, Boston, U.S.A. and [14CCTP (494 mCi/mmole) from Radiochemical Center, Amersham, England. [y-3 P]ATP and [y-3 P]GTP were also enzymatically synthesized according to the method of Glynn and Chappell (4). The sources of enzymes and chemicals were as follows; RNase T1 and T2 from Sankyo, pancreatic RNase from Sigma, nuclease P1 from Yamasa Shoyu, micrococcal nuclease from Worthington, a-amanitin from Boehlinger Mannheim. All other reagents were of analytical grade.
RESULTS
RNA synthetic activity of isolated oocyte nucleoli When incubated with RNA precursors, Xenopus oocyte nucleoli exhibited 1932
Nucleic Acids Research remarkable synthetic activity. As shown in Fig. 1, the incorporation of radioactivity into acid-insoluble fraction continued linearly at least up to 240 minute period. The radioactivity in acid-insoluble fraction was ascribed to RNA in terms of its sensitivity to RNases (discussed later). Table 1 summarizes
the
effect
of
various
concentrations
of
a-amanitin.
The
activity
hardly inhibited by low and high doses of this drug. These data indicate that the observed RNA synthetic activity of isolated Xenopus oocyte nucleoli was exclusively due to type I RNA polymerase. Also these data corroborate the estimation of nucleolar purity in CsCl gradient centrifugation in that the nucleoli isolated in the present procedure are not seriously contaminated by bulk DNA containing structures. Removal of ATP or GTP from the reaction mixture reduced the incorporation to a level of as low as 20 %of the control,
was
which eliminates the possibility of homopolymer formation (data not shown). The RNA synthesized in vitro was subjected to sedimentation analysis on sucrose gradient (Fig. 2). At 10 minutes of incubation, the size of the product was mostly larger than 28S and became smaller as incubation proceeded, which may probably suggest the processing of a larger precursor RNA.
cpm (x
10')
20
IL
S0
0
10
0
120
160
200
240
Incubation time (min)
Fig. 1. Kinetics of [3H]CMP incorporation into acid-insoluble fraction by isolated Xenopus oocyte nucleoli. 1933
Nucleic Acids Research Table 1.
Effect of a-amanitin on RNA synthesis by isolated Xenopus oocyte nucleoli
Concentration of a-amanitin
Incorporation relative to control
0 pg/ml 2 20 200
100 90 89 86
The data were average values of 4 experiments. Typical incubation exhibited the incorporation of 0.36 pmole of [3H]CTP in 80 minutes.
Incorporation of [y-3 P] labeled purine nucleoside triphosphate When incubated with [y-3 P]GTP in addition to [3H]CTP, both radioactivities were incorporated into acid-insoluble fraction in a time-dependent fashion (Fig. 3). Almost all the 3H radioactivity was shown to be RNase sensitive while a small fraction of the original acid-insoluble 32P radioactivity was recovered as RNA after phenol extraction. Major fraction of cpm
Ss
lss
28s
10
15
150 [
100 I
50
F
o top
5
20
bottom
Fraction number
Sucrose density gradient analysis of RNA synthesized in 0-O 10 minute incubation; *---4 20 minute incubation; fr.....u 30 minute incubation.
Fig. 2. vitro.
1934
Nucleic Acids Research cpm
Fig. 3. Kinetics of incorporation of radioactivity from [y-32P]GTP and [3H] CTP into acid-insoluble fraction.
i
I. a.
I U
z
C
0 C6
0
20 60 40 Incubation time (min)
80
acid-insoluble 32P radioactivity seems to account for protein phosphorylation. The amount of 32p incorporation was comparable to that of 3H radioactivity which represents the RNA chain elongation (Table 2). Furthermore, the incorporation was found to be totally insensitive to 12.5 ig/ml of actino-
Table 2.
Incorporation of radioactivity from
[3HICTP
and [y- P]GTP or ATP
Incorporation of radioactivity into RNase sensitive nucleic acid acid-insoluble fraction fraction fraction 3
3H]CTP
Exp. 1
[y- 32 P]GTP
Exp. 2 [Y-
Exp. 3 [y-
4.0 pmoles 12.2
3.4 0.4
pmoles
3.4
pmoles
0.3
[ H]CTP
-
P]ATP
0.87 0.26
0.87
-
[ H]CTP
-
0.58
0.58
P]ATP
-
0.49
0.48
0.23
The number of frogs used in Exp. 1, 2 and 3 was 10, 6 and 6, respectively. Incubations were performed for 80 minutes. 1935
Nucleic Acids Research mycin D which inhibited as much as 96 % of the incorporation of radioactivity from [ HICTP (Table 3). These data made us suspect that the incorporation of 32p radioactivity might not be due to RNA chain initiation even if it was sensitive to RNase. Analysis of 5'-end of RNA synthesized in vitro In order to see whether the P incorporation into RNA was due to the initiation of transcription, 5'-end of RNA synthesized in the presence of [y-3 P]ATP was analyzed. The transcript was hydrolyzed with RNase T2 and the terminal mononucleotides were separated on a DEAE-Sephadex A-25 column as shown in Fig. 4. No significant radioactivity was detected over the mononucleotide peak at -6 charge which corresponds to the position of pppXp. The only prominent 32p peak was at -4 charge, which is the position for a monophosphorylated 5'-terminus, pXp. This result was further confirmed by the hydrolysis of the pooled -4 charge fractions with nuclease Pl which is known to act on mononucleotides as a 3'-phosphatase (5). Rechromatography on a DEAE-Sephadex column of the hydrolysate exhibited a single peak eluted at -2 charge (Fig. 5), which is consistent with pX structure and corroborates the structure before Pl treatment to be pXp. Fig. 6 shows the chromatogram on a Dowex column of the fractions recovered from the peak shown in Fig. 5. Out of total 32p radioactivity recovered, 50 % was AMP; CMP, UMP and GMP were 5 %, 10 % and 20 %, respectively. The remaining 15 % was found to be inorganic phosphate. These data show that all four kinds of mononucleotides were labeled during incubation to produce 5'-3 P labeled terminal mononucleoside diphosphate, 3 pXp. Therefore, the incorporation of P radioactivity from [y- ]ATP into RNA is not the reflection of the initiation of transcription, but due to other activity of the nucleolar preparation which transfers phosphate residue from y-position of ATP to 5'-hydroxyl group of RNA. The most probable candidate for such an activity would be polynucleotide kinase, which we have
Table 3.
Effect of actinomycin D on the incorporation of radioactivity from [3H]CTP and [y-32P]ATp
Control Actinomycin D (12.5 ug/ml)
[3H]CTP
[y- 32P]ATP
0.33 pmole 0.014
0.11 pmole 0.11
Incubations were performed for 80 minutes. 1936
Nucleic Acids Research Fig. 4. lom-
-2
-3
-4
-5
-6
s
s
s
s
s
-7
RNase T2
E
DEAE-Sephadex
A-25 column chromatography of
terminal nucleo-
tides.
RNA synthesized
in vitro in the
presence
tdf [yT632P]ATP was dil l gested with RNase T2 and
E
5X 500-
X
|
;
the nucleotides were on a DEAE-
|
1
separated
Sephadex A-25 column. Arrows indicate the position of respective charge markers.
10
20
30
Fraction
40
50
60
number
detected in the isolated oocyte nucleoli.
Polynucleotide kinase activity in the Xenopus oocyte nucleoli The activity of polynucleotide kinase was assayed using 5'-hydroxylterminated calf thymus DNA as a phosphate acceptor. Under the conditions described in METHODS, 18.6 pmoles of P were transferred from [y- P]ATP to DNA by the nucleolar preparation from 5 frogs. The assay was also carried out under the conditions for the in vitro RNA synthesis. As shown in Fig. 7, 32P radioactivity could be recovered in DNA fraction as revealed by centrifugation in alkaline CsCl gradient. These results strongly suggest that the incorporation of 3 P radioactivity from [y-3 P]ATP into RNA to produce 3 pXp residues could be explained by the in the isolated nucleoli.
presence of
polynucleotide kinase activity
DISCUSSION
The amplified nucleoli of Xenopus oocytes afford
-2
200
-4 Nuclease Pm
a
l
a.
1io-
A 10
Fraction
special opportunity
RechromatograDEAE-Sephadex A-25 column of the -4 peak fractions of Fig. 4 after digestion with nuclease P1. Arrows indicate the positions of charge markers. Fig. 5.
phy
E
a
on a
20
number
1937
Nucleic Acids Research
2.0
400 E a
1.0
1-
I
E
200
UN
0.0 20 Fraction
number
Fig. 6. Terminal mononucleotide analysis. The -2 peak fractions were collected and subjected to Dowex 1 x 2 column chromatography together with 5'-XMPs (X=A,C,G or U) as position markers.
for the study of transcription. They can be isolated as organelle containing only one class of genes which codes for ribosomal RNA. This situation resembles the nucleoli in the macronuclei of Tetrahymena pyriformis (6) and the nucleoli of Physarum polycepharum (7). The approach using these nucleoli as an in vitro transcription system might answer the questions as to the nature and the mechanism of the components involved in the control of transcription in a more precise way than other more complicated systems. To this end, it is most crucial to see whether or not the system used exhibits faithful transcription with respect to several criteria. In the present study we have detected continued incorporation of radio-
Alkaline CsCl centrifugation of calf thymus DNA incubated in the presence of nucleoli and [y-32p]ATP.
Fig. 7. 400
Ea. U
200
44
a
a.
0
44
10
Fraction
1938
20
Nucleic Acids Research active precursors into RNA due to type I RNA polymerase. The evidence suggestive of processing of the transcript was also obtained. However, we were unable to detect the initiation of transcription with [y- PIATP as a probe. The P radioactivity found in RNA was present in the form of pXp (X=A,C,U or G), and not in the form of pppAp. This reflects the transfer of y-phosphate residue from [y- P]ATP to 5'-hydroxyl group at the terminus or at the site of the hidden scission of the pre-existing or newly transcribed RNA chain, possibly due to the action of polynucleotide kinase. Polynucleotide kinase activity has been reported to reside in the nuclei (8,9), but not in the nucleoli per se. We have detected this activity in the isolated nucleoli under the conditions for the in vitro transcription. However, judging from the purity of the nucleolar preparation, the possibility may still exsist that the activity resides in the contaminating follicle cell nuclei or in the bulk DNA chromatin of the oocytes. Taken altogether, our results show that the nucleoli as isolated possess RNA chain elongation activity and some processing activity although the activity which degrades exogenous RNA into acid-soluble form is relatively low (Tashiro and Higashinakagawa, unpublished). The present procedure did not allow us to detect the initiation of transcription. With the aid of other appropriate probe such as [y-S] nucleoside triphosphate (10), we might be able to detect RNA chain initiation in this system. It should also be stressed from our results, coupled with other previous suggestions (9,10,11), that the incorporation of radioactivity from [y-3 P] nucleoside triphosphate into RNA cannot necessarily be regarded as an index of the initiation of transcription. In this connection, studies of in vitro transcription which adopt this criterion as an index of chain initiation should seriously be reconsidered in the light of these findings (12,13,14).
ACKNOWLEDGMENTS We thank Dr. Yoshihiro Kato for his continuous supports throughout this work. We also thank Drs. Akira Kuninaka and Toshio Onishi for valuable suggestions. The excellent technical assistance of Miss Mariko Sezaki is gratefully acknowledged. REFERENCES 1 Higashinakagawa, T., Wahn, H., and Reeder, R.H. (1977) Develop. Biol. 55, 375-386 2 Ichimura, M., and Tsukada, K. (1971) J. Biochem. 58, 297-302 3 Richardson, C.C. (1965) Proc. Nat. Acad. Sci. U.S.A. 54, 158-165 1939
Nucleic Acids Research 4 Glynn, J.M., and Chappell, J.B. (1964) Biochem. J. 90, 147-149 5 Fujimoto, M., Kuninaka, A., and Yoshino, H. (1974) Agr. Biol. Chem. 38 1555-1561 6 Higashinakagawa, T., Sezaki, M., and Kondo, S. (1979) Develop. Biol. (in press) 7 Grainger, R.M., and Ogle, R.C. (1978) Chromosoma 65, 115-126 8 Teraoka, H., Mizuta, K., Sato, F., Shimoyagi, M., and Tsukada, K. (1975) Eur. J. Biochem. 58, 297-302 9 Winicov, I. (1977) Biochemistry 16, 4233-4237 10 Smith, M.M., Reeve, A.E., and Huang, R.C.C. (1978) Cell 15, 615-626 11 Gilboa, E., Soreq, H., and Aviv, H. (1977) Eur. J. Biochem. 77, 393-400 12 Hallick, R.B., Lipper, C., Richards, O.C., and Rutter, W.J. (1976) Biochemistry 15, 3039-3045 13 Fodor, E.J.B. and Doty, P. (1977) Biochem. Biophys. Res. Commun. 77, 1478-1485 14 Dierks-Ventling, C., Stalder, J., and Gautschi, J. (1978) Nucleic Acids Res. 5, 2643-2655 15 Present address: Department of Molecular Biology, Faculty of Medicine, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yawata Nishi-Ku, Kitakyushu, Fukuoka 807, Japan
1940
Nucleic Acids Research
Properties of in vitro transcription by isolated Xenopus oocyte nucleoli
Hidetoshi Saiga I and Toru Higashinakagawa1 5 Laboratory of Developmental Biology, Mitsubishi-Kasei Institute of life Sciences, 11 Minamiooya, Machida, Tokyo, Japan Received 8 March 1979 ABSTRACT Some properties of in vitro transcription by isolated Xenopus oocyte nucleoli were described. When incubated with labeled RNA precursors, Xenopus oocyte nucleoli exhibited prolonged incorporation of radioactivity into RNA. The synthetic activity was exclusively due to type I RNA polymerase as revealed by its insensitivity to low and high doses of a-amanitin. The size of the in vitro transcript was mQstly larger than 28S at 10 minute incubation and became smaller as incubation proceeded. When [Y-32P]ATP was included in the reaction mixture, 32P radioactivity was incorporated into RNA suggesting the possible initiation of transcription in this system. However, analysis of the terminal nucleotide of the transcript revealed that the incorporation of radioactivity from [y-32P]ATP was not due to the initiation of transcription but due to polynucleotide kinase activity in the nucleolar preparation. These results demonstrate that the incorporation of radioactivity from [y_32p] labeled nucleoside triphosphates cannot necessarily be regarded as an index of the initiation of transcription. INTRODUCTION One way to define the components involved in the control of transcription in eukaryotes would be to construct a faithful in vitro system which displays an accurate transcription as compared with events in vivo. Nucleoli of Xenopus oocytes are especially suited for this approach, since these nucleoli, in contrast to the nucleoli from other somatic cells, contain mostly ribosomal RNA genes and are regarded as a single gene organelle. These features may greatly facilitate the identification of the components which control the transcription of the ribosomal gene. From these standpoints, we have examined the in vitro transcription by isolated Xenopus oocyte nucleoli. There are several criteria to be satisfied for accurate transcription. The faithful system should possess stable RNA synthesizing activity, permit RNA polymerase to select proper DNA strand and allow precise RNA chain initiation and termination. In studies with isolated nuclei or nucleoli as an in vitro transcription system, the most crucial test is whether or not the initiation of transcription can be detected by an appropriate method.
C) Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England
1929
Nucleic Acids Research In this paper some properties of the in vitro transcription by isolated Xenopus oocyte nucleoli are presented. Although the oocyte nucleoli exhibited incorporation of radioactivity from [y-3 P] labeled nucleoside triphosphates into RNA, the incorporation was exclusively due to other enzymatic activity rather than the initiation of transcription.
MATERIALS AND METHODS Isolation of Xenopus oocyte nucleoli The procedure for the isolation of oocyte nucleoli has already been described by Higashinakagawa et al. (1). In this paper we have modified and simplified the procedure, omitting the centrifugation in Metrizamide (described elsewhere). The DNA composition of the nucleolar preparation was examined by analytical ultracentrifugation in CsCl density gradient. About 70 % of the total DNA was found to be ribosomal gene (data not shown). Conditions for in vitro transcription The standard reaction mixture for in vitro transcription consisted of 50 % (v/v) glycerol, 0.15 M NaCl, 10 mM MgCl2, 10 mM Tris-HCl (pH 8.0), 0.1 mM EDTA, 5 mM dithiothreitol, 10 - 125 pg/ml of a-amanitin, 0.6 mM each of ATP, UTP, and GTP, 0.1 pCi of [14 CCTP or 1 pCi of [3H]CTP and nucleoli from 4 frogs. Incubations were performed at 25°C. In the reaction to test the initiation of transcription, the composition of the reaction mixture was the same as described above except that cold ATP (or GTP) was substituted with [y- PIATP (or[y- P]GTP). After the incubation, an aliquot was spotted onto the glass fiber disc (Whatman GF/C), washed in cold 5 % TCA-1 % Na4P207 and in ethanol. The discs were dried and counted for radioactivity. The discs were marked by an ink for drawing use so that many discs were washed batchwise simultaneously. Nucleic acid extraction Incubations were terminated by the addition of equal volume of 0.6 % SDS-0.28 M NaCl-0.1 M CH3COONa (pH 5.1), followed by extraction with phenolm-cresol mixture (phenol:m-cresol:H20, 7:1:2 v/v, containing 0.1 % 8-hydroxyquinoline). The aqueous phase was applied onto a Sephadex G-25 column (1.5 x 20 cm) equilibrated with 0.3 % SDS-0.14 M NaCl-0.05 M CH3COONa (pH 5.1). The excluded fraction was collected and the nucleic acid was precipitated with 2.5 volumes of ethanol containing 10 % (v/v) m-cresol and 2 % (w/v) CH3COOK at -20°C. The pellet was washed in 75 % (v/v) ethanol-l % (w/v) CH3COOK and in 75 % (v/v) ethanol and dissolved in an appropriate buffer.
1930
Nucleic Acids Research Determination of RNase sensitive radioactivity The nucleic acid fraction was dried in vacuo, and dissolved in 0.15 M NaCl-l mM EDTA-0.05 M Tris-HCl (pH 8.0). RNase digestion was performed with 50 tg/ml of pancreatic RNase and 5 units/ml of RNase T1 at 37°C for 30 minutes. The reaction was terminated by the addition of equal volume of cold 10 % TCA-2 % Na4P207 and 100 ig of bovine serum albumin as carrier. The acid-insoluble material was collected onto glass fiber disc (Whatman GF/C). The discs were washed with cold 5 % TCA-1 % Na4P207 and counted for radioactivity. Control incubation without RNases was performed and radioactivity was determined as described above. RNase sensitive fraction was defined as the difference between control and the RNase treated fraction. Sedimentation on sucrose gradient RNA fraction was dissolved in a small volume of 0.1 M CH3COONa-l mM EDTA-0.01 % sodium lauroyl sarcosinate (pH 6.0) and was layered on the top of 13 ml of 10 - 30 % (w/w) sucrose gradient in 0.1 M CH 3COONa-1 mM EDTA-0.01 % sodium lauroyl sarcosinate (pH 6.0) together with 10 OD260 units of rat liver cytoplasmic RNA as size marker. The gradient was centrifuged in a SW41Ti rotor (Beckman) at 30,000 rpm for 17 hours at 4°C. The gradient was fractionated with an Auto-Densiflow, monitoring at 254 nm with an ISCO UV monitor. To each fraction, equal volume of cold 10 % TCA-2 % Na P207 was added. Acid-insoluble material was collected onto glass fiber disc (Whatman GF/C) and counted for radioactivity.
Analysis of 5'-end of RNA synthesized in vitro RNA synthesized in the presence of [y- P]ATP was dissolved in 0.05 M CH3COOK (pH 4.6) and was hydrolyzed with 20 units/ml of RNase T2 at 37°C for 5 hours. The hydrolysate was applied, together with charge marker (RNase T1 digest of E. coli tRNA), onto a column of DEAE-Sephadex A-25 (0.4 x45 cm) equilibrated with 7 M urea-0.05 M Tris-HCl (pH 7.8). Fractionation was performed with a linear gradient of 0.1 - 0.5 M NaCl in the same buffer. Absorbancy at 254 nm was recorded with an ISCO UV monitor, and each fraction was counted for radioactivity by Cerenkov radiation. The pooled peak fractions at -4 charge were passed through a small DEAE-Sephadex column in order to remove NaCl and urea and hydrolyzed with 500 ig/ml of nuclease P1. The hydrolysate was applied onto a DEAE-Sephadex A-25 column (0.4 x 20 cm) equilibrated with 7 M urea-0.05 M Tris-HCl (pH 7.8) together with charge markers (5'-AMP and 5'-ATP). Fractionation and counting for radioactivity were performed as described above. The peak fractions of the second DEAE-Sephadex column chromatography 1931
Nucleic Acids Research were diluted to 10 fold with distilled water and applied together with 5'XMPs (X=A,C,G or U) onto a Dowex 1 x 2 column (0.4 x 15 cm) equilibrated with 0.0075 N HR. Elution was performed with a linear gradient of 0 - 0.05 M
NaCl in 0.0075 N HCl, monitoring with an ISCO UV monitor at 254 nm. radioactivity was counted directly by 6erenkov radiation.
32P
Assay for polynucleotide kinase activity Polynucleotide kinase activity in the isolated nucleoli was assayed according to the method of Ichimura and Tsukada with some modifications (2). The reaction mixture consisted of 0.083 M sodium acetate buffer (pH 5.5),
0.01 M1 MgCl2, 0.0167 M 0-mercaptoethanol, 15 ig of 5'-hydroxyl-terminated calf thymus DNA, 2 nmoles of [y-3 P]ATP and isolated nucleoli. Incubation was performed at 37°C for 30 minutes. After the reaction, NaOH was added to give final concentration of 0.1 M. The mixture was then heated at 80°C for 20 minutes. After neutralization with HR, the mixture was treated with 50 pg/ml of pancreatic RNase and acid-insoluble radioactivity was counted. When the assay was performed under the conditions for in vitro transcription, the extracted nucleic acid was treated with alkali and RNase and subjected to alkaline CsCl density gradient centrifugation. Centrifugation was performed in a RP65T rotor (Hitachi) at 33,000 rpm for 60 hours at 20°C. The gradient was fractionated with an Auto-Densiflow and radioactivity was counted by &erenkov radiation. 5'-Hydroxyl-terminated calf thymus DNA was prepared according to the
method of Richardson (3). Materials
[3HICTP
(20.1 Ci/mmole), [y- P]ATP (2 - 10 Ci/mmole) were purchased from New England Nuclear, Boston, U.S.A. and [14CCTP (494 mCi/mmole) from Radiochemical Center, Amersham, England. [y-3 P]ATP and [y-3 P]GTP were also enzymatically synthesized according to the method of Glynn and Chappell (4). The sources of enzymes and chemicals were as follows; RNase T1 and T2 from Sankyo, pancreatic RNase from Sigma, nuclease P1 from Yamasa Shoyu, micrococcal nuclease from Worthington, a-amanitin from Boehlinger Mannheim. All other reagents were of analytical grade.
RESULTS
RNA synthetic activity of isolated oocyte nucleoli When incubated with RNA precursors, Xenopus oocyte nucleoli exhibited 1932
Nucleic Acids Research remarkable synthetic activity. As shown in Fig. 1, the incorporation of radioactivity into acid-insoluble fraction continued linearly at least up to 240 minute period. The radioactivity in acid-insoluble fraction was ascribed to RNA in terms of its sensitivity to RNases (discussed later). Table 1 summarizes
the
effect
of
various
concentrations
of
a-amanitin.
The
activity
hardly inhibited by low and high doses of this drug. These data indicate that the observed RNA synthetic activity of isolated Xenopus oocyte nucleoli was exclusively due to type I RNA polymerase. Also these data corroborate the estimation of nucleolar purity in CsCl gradient centrifugation in that the nucleoli isolated in the present procedure are not seriously contaminated by bulk DNA containing structures. Removal of ATP or GTP from the reaction mixture reduced the incorporation to a level of as low as 20 %of the control,
was
which eliminates the possibility of homopolymer formation (data not shown). The RNA synthesized in vitro was subjected to sedimentation analysis on sucrose gradient (Fig. 2). At 10 minutes of incubation, the size of the product was mostly larger than 28S and became smaller as incubation proceeded, which may probably suggest the processing of a larger precursor RNA.
cpm (x
10')
20
IL
S0
0
10
0
120
160
200
240
Incubation time (min)
Fig. 1. Kinetics of [3H]CMP incorporation into acid-insoluble fraction by isolated Xenopus oocyte nucleoli. 1933
Nucleic Acids Research Table 1.
Effect of a-amanitin on RNA synthesis by isolated Xenopus oocyte nucleoli
Concentration of a-amanitin
Incorporation relative to control
0 pg/ml 2 20 200
100 90 89 86
The data were average values of 4 experiments. Typical incubation exhibited the incorporation of 0.36 pmole of [3H]CTP in 80 minutes.
Incorporation of [y-3 P] labeled purine nucleoside triphosphate When incubated with [y-3 P]GTP in addition to [3H]CTP, both radioactivities were incorporated into acid-insoluble fraction in a time-dependent fashion (Fig. 3). Almost all the 3H radioactivity was shown to be RNase sensitive while a small fraction of the original acid-insoluble 32P radioactivity was recovered as RNA after phenol extraction. Major fraction of cpm
Ss
lss
28s
10
15
150 [
100 I
50
F
o top
5
20
bottom
Fraction number
Sucrose density gradient analysis of RNA synthesized in 0-O 10 minute incubation; *---4 20 minute incubation; fr.....u 30 minute incubation.
Fig. 2. vitro.
1934
Nucleic Acids Research cpm
Fig. 3. Kinetics of incorporation of radioactivity from [y-32P]GTP and [3H] CTP into acid-insoluble fraction.
i
I. a.
I U
z
C
0 C6
0
20 60 40 Incubation time (min)
80
acid-insoluble 32P radioactivity seems to account for protein phosphorylation. The amount of 32p incorporation was comparable to that of 3H radioactivity which represents the RNA chain elongation (Table 2). Furthermore, the incorporation was found to be totally insensitive to 12.5 ig/ml of actino-
Table 2.
Incorporation of radioactivity from
[3HICTP
and [y- P]GTP or ATP
Incorporation of radioactivity into RNase sensitive nucleic acid acid-insoluble fraction fraction fraction 3
3H]CTP
Exp. 1
[y- 32 P]GTP
Exp. 2 [Y-
Exp. 3 [y-
4.0 pmoles 12.2
3.4 0.4
pmoles
3.4
pmoles
0.3
[ H]CTP
-
P]ATP
0.87 0.26
0.87
-
[ H]CTP
-
0.58
0.58
P]ATP
-
0.49
0.48
0.23
The number of frogs used in Exp. 1, 2 and 3 was 10, 6 and 6, respectively. Incubations were performed for 80 minutes. 1935
Nucleic Acids Research mycin D which inhibited as much as 96 % of the incorporation of radioactivity from [ HICTP (Table 3). These data made us suspect that the incorporation of 32p radioactivity might not be due to RNA chain initiation even if it was sensitive to RNase. Analysis of 5'-end of RNA synthesized in vitro In order to see whether the P incorporation into RNA was due to the initiation of transcription, 5'-end of RNA synthesized in the presence of [y-3 P]ATP was analyzed. The transcript was hydrolyzed with RNase T2 and the terminal mononucleotides were separated on a DEAE-Sephadex A-25 column as shown in Fig. 4. No significant radioactivity was detected over the mononucleotide peak at -6 charge which corresponds to the position of pppXp. The only prominent 32p peak was at -4 charge, which is the position for a monophosphorylated 5'-terminus, pXp. This result was further confirmed by the hydrolysis of the pooled -4 charge fractions with nuclease Pl which is known to act on mononucleotides as a 3'-phosphatase (5). Rechromatography on a DEAE-Sephadex column of the hydrolysate exhibited a single peak eluted at -2 charge (Fig. 5), which is consistent with pX structure and corroborates the structure before Pl treatment to be pXp. Fig. 6 shows the chromatogram on a Dowex column of the fractions recovered from the peak shown in Fig. 5. Out of total 32p radioactivity recovered, 50 % was AMP; CMP, UMP and GMP were 5 %, 10 % and 20 %, respectively. The remaining 15 % was found to be inorganic phosphate. These data show that all four kinds of mononucleotides were labeled during incubation to produce 5'-3 P labeled terminal mononucleoside diphosphate, 3 pXp. Therefore, the incorporation of P radioactivity from [y- ]ATP into RNA is not the reflection of the initiation of transcription, but due to other activity of the nucleolar preparation which transfers phosphate residue from y-position of ATP to 5'-hydroxyl group of RNA. The most probable candidate for such an activity would be polynucleotide kinase, which we have
Table 3.
Effect of actinomycin D on the incorporation of radioactivity from [3H]CTP and [y-32P]ATp
Control Actinomycin D (12.5 ug/ml)
[3H]CTP
[y- 32P]ATP
0.33 pmole 0.014
0.11 pmole 0.11
Incubations were performed for 80 minutes. 1936
Nucleic Acids Research Fig. 4. lom-
-2
-3
-4
-5
-6
s
s
s
s
s
-7
RNase T2
E
DEAE-Sephadex
A-25 column chromatography of
terminal nucleo-
tides.
RNA synthesized
in vitro in the
presence
tdf [yT632P]ATP was dil l gested with RNase T2 and
E
5X 500-
X
|
;
the nucleotides were on a DEAE-
|
1
separated
Sephadex A-25 column. Arrows indicate the position of respective charge markers.
10
20
30
Fraction
40
50
60
number
detected in the isolated oocyte nucleoli.
Polynucleotide kinase activity in the Xenopus oocyte nucleoli The activity of polynucleotide kinase was assayed using 5'-hydroxylterminated calf thymus DNA as a phosphate acceptor. Under the conditions described in METHODS, 18.6 pmoles of P were transferred from [y- P]ATP to DNA by the nucleolar preparation from 5 frogs. The assay was also carried out under the conditions for the in vitro RNA synthesis. As shown in Fig. 7, 32P radioactivity could be recovered in DNA fraction as revealed by centrifugation in alkaline CsCl gradient. These results strongly suggest that the incorporation of 3 P radioactivity from [y-3 P]ATP into RNA to produce 3 pXp residues could be explained by the in the isolated nucleoli.
presence of
polynucleotide kinase activity
DISCUSSION
The amplified nucleoli of Xenopus oocytes afford
-2
200
-4 Nuclease Pm
a
l
a.
1io-
A 10
Fraction
special opportunity
RechromatograDEAE-Sephadex A-25 column of the -4 peak fractions of Fig. 4 after digestion with nuclease P1. Arrows indicate the positions of charge markers. Fig. 5.
phy
E
a
on a
20
number
1937
Nucleic Acids Research
2.0
400 E a
1.0
1-
I
E
200
UN
0.0 20 Fraction
number
Fig. 6. Terminal mononucleotide analysis. The -2 peak fractions were collected and subjected to Dowex 1 x 2 column chromatography together with 5'-XMPs (X=A,C,G or U) as position markers.
for the study of transcription. They can be isolated as organelle containing only one class of genes which codes for ribosomal RNA. This situation resembles the nucleoli in the macronuclei of Tetrahymena pyriformis (6) and the nucleoli of Physarum polycepharum (7). The approach using these nucleoli as an in vitro transcription system might answer the questions as to the nature and the mechanism of the components involved in the control of transcription in a more precise way than other more complicated systems. To this end, it is most crucial to see whether or not the system used exhibits faithful transcription with respect to several criteria. In the present study we have detected continued incorporation of radio-
Alkaline CsCl centrifugation of calf thymus DNA incubated in the presence of nucleoli and [y-32p]ATP.
Fig. 7. 400
Ea. U
200
44
a
a.
0
44
10
Fraction
1938
20
Nucleic Acids Research active precursors into RNA due to type I RNA polymerase. The evidence suggestive of processing of the transcript was also obtained. However, we were unable to detect the initiation of transcription with [y- PIATP as a probe. The P radioactivity found in RNA was present in the form of pXp (X=A,C,U or G), and not in the form of pppAp. This reflects the transfer of y-phosphate residue from [y- P]ATP to 5'-hydroxyl group at the terminus or at the site of the hidden scission of the pre-existing or newly transcribed RNA chain, possibly due to the action of polynucleotide kinase. Polynucleotide kinase activity has been reported to reside in the nuclei (8,9), but not in the nucleoli per se. We have detected this activity in the isolated nucleoli under the conditions for the in vitro transcription. However, judging from the purity of the nucleolar preparation, the possibility may still exsist that the activity resides in the contaminating follicle cell nuclei or in the bulk DNA chromatin of the oocytes. Taken altogether, our results show that the nucleoli as isolated possess RNA chain elongation activity and some processing activity although the activity which degrades exogenous RNA into acid-soluble form is relatively low (Tashiro and Higashinakagawa, unpublished). The present procedure did not allow us to detect the initiation of transcription. With the aid of other appropriate probe such as [y-S] nucleoside triphosphate (10), we might be able to detect RNA chain initiation in this system. It should also be stressed from our results, coupled with other previous suggestions (9,10,11), that the incorporation of radioactivity from [y-3 P] nucleoside triphosphate into RNA cannot necessarily be regarded as an index of the initiation of transcription. In this connection, studies of in vitro transcription which adopt this criterion as an index of chain initiation should seriously be reconsidered in the light of these findings (12,13,14).
ACKNOWLEDGMENTS We thank Dr. Yoshihiro Kato for his continuous supports throughout this work. We also thank Drs. Akira Kuninaka and Toshio Onishi for valuable suggestions. The excellent technical assistance of Miss Mariko Sezaki is gratefully acknowledged. REFERENCES 1 Higashinakagawa, T., Wahn, H., and Reeder, R.H. (1977) Develop. Biol. 55, 375-386 2 Ichimura, M., and Tsukada, K. (1971) J. Biochem. 58, 297-302 3 Richardson, C.C. (1965) Proc. Nat. Acad. Sci. U.S.A. 54, 158-165 1939
Nucleic Acids Research 4 Glynn, J.M., and Chappell, J.B. (1964) Biochem. J. 90, 147-149 5 Fujimoto, M., Kuninaka, A., and Yoshino, H. (1974) Agr. Biol. Chem. 38 1555-1561 6 Higashinakagawa, T., Sezaki, M., and Kondo, S. (1979) Develop. Biol. (in press) 7 Grainger, R.M., and Ogle, R.C. (1978) Chromosoma 65, 115-126 8 Teraoka, H., Mizuta, K., Sato, F., Shimoyagi, M., and Tsukada, K. (1975) Eur. J. Biochem. 58, 297-302 9 Winicov, I. (1977) Biochemistry 16, 4233-4237 10 Smith, M.M., Reeve, A.E., and Huang, R.C.C. (1978) Cell 15, 615-626 11 Gilboa, E., Soreq, H., and Aviv, H. (1977) Eur. J. Biochem. 77, 393-400 12 Hallick, R.B., Lipper, C., Richards, O.C., and Rutter, W.J. (1976) Biochemistry 15, 3039-3045 13 Fodor, E.J.B. and Doty, P. (1977) Biochem. Biophys. Res. Commun. 77, 1478-1485 14 Dierks-Ventling, C., Stalder, J., and Gautschi, J. (1978) Nucleic Acids Res. 5, 2643-2655 15 Present address: Department of Molecular Biology, Faculty of Medicine, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yawata Nishi-Ku, Kitakyushu, Fukuoka 807, Japan
1940
