DEVELOPMENTAL GENETICS 13:143-150 (1992)
In Vitro Transcription in Isolated Nucleoli of Tetrahymena pyriformis TADASHI MATSUURA AND TORU HIGASHINAKAGAWA Department of Developmental Biology, Mitsubishi Kasei Institute of Life Sciences, Machida, Tokyo, Japan
ulation of gene expression [Mitchell and Tjian, 1989; Sawadogo and Sentenac, 19901. However, many questions remain unanswered due to the complex composition and structural arrangement of DNA and proteins which constitute eukaryotic chromatin. A simple model system is needed for analysis of the molecular mechanisms underlying the control of chromatin transcription. With this goal in mind, we began studying rDNA in the ciliated protozoan, Tetrahymena. Within the macronucleus of Tetrahymena, rDNAs exist as free palindromic molecules of about 19 kbp that are packaged in a number of extrachromosomal nucleoli [Engberg et al., 1976; Karrer and Gall, 19761. This biological peculiarity represents one of the advantages of the Tetrahymena rDNA system. Tetrahymena nucleoli are organelles that contain copies of a single gene for rRNA, and isolated nucleoli provide a unique source of chromatin for transcription studies. Previously, we reported a procedure for isolating nucleoli [Higashinakagawa et al., 19791, and we significantly improved it recently [Higashinakagawa et al., 19921. We also cloned a rDNA fragment and determined the transcription initiation site [Higashinakagawa et al., 1981; Saiga et al., 19821. Initiation of transcription was reproduced in vitro with a homologous Tetrahymena cell extract and cloned rDNA fragment as a template Key words: Extrachromosomal nucleoli, in vitro [Matsuura et al., 19861. As a n extension of these expertranscription, chromatin, ribosomal DNA (rDNA), iments, in vitro transcription of isolated nucleoli was Tetrahymena, P1 nuclease mapping examined in order to determine the role of chromosomal proteins in the transcription process. Here we report that transcription was initiated from the in vivo INTRODUCTION initiation site with a nucleolar template and that the In eukaryotic cells, about one-half the mass of the efficiency of transcription of the nucleolar template chromatin composing the transcriptional apparatus was much higher than that of a purified rDNA clone. consists of histones and other chromosomal proteins [van Holde, 19891. Gene expression during development and differentiation is known to be greatly affected by structural alterations in chromatin resulting from nonrandom interactions between DNA and these chromosomal proteins [Grunstein, 19901. Historically, Received for publication September 30, 1991; accepted November 13, the concept of “open” and “closed” conformations of 1991. chromatin was proposed based on several kinds of experiments, most notably the assessment of the nuclease Address reprint requests to Dr. Toru Higashinakagawa, Department of Developmental Biology, Mitsubishi Kasei Institute of Life Sciences, sensitivity of chromatin DNA [Gross and Garrard, Machida, Tokyo 194, Japan. 1988; Elgin, 19881. Recently, various transcription factors and DNA binding proteins have been isolated and Dr. Matsuura is now a t the Department of Parasitology, Shinshu found to act at specific sites in chromatin during reg- University School of Medicine, 3-1-1Asahi, Matsumoto 390, Japan. An in vitro transcription system ABSTRACT was established using extrachromosomal nucleoli from Tetrahymena pyriformis macronuclei as a template. Ribosomal precursor RNA (pre-rRNA) and nascent pre-rRNA chains were removed from isolated nucleoli by treatment with RNase T1 and Sarkosyl. Nucleoli were then incubated in a RNA synthetic cocktail containing a cellular extract from Tetrahymena thermophila. The transcription product was examined for the presence of transcripts from T. pyriformis ribosomal DNA (rDNA) by P1 nuclease protection mapping using a DNA probe from a T. pyriformis rDNA clone. A sequence difference between T. pyriformis and T. thermophila in the 5‘ region of their 35s pre-rRNAs permitted exclusive detection of T. pyriformis transcripts. The results showed that faithful transcription initiation occurred in vitro from the in vivo initiation site of the nucleolar template and that the nucleolar template had a much higher efficiency of transcription than that of the purified rDNA clone. This system may offer unique advantages for future studies of transcriptional control during development and differentiation. 0 1992 Wiley-tiss, Inc.
~~~~~
0 1992 WILEY-LISS, INC.
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a 1
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Fig. 1. Elimination of background derived from pre-rRNA in isolated nucleoli. a. Isolated nucleoli from 2'. pyriformis were incubated at 37°C. b. Isolated nucleoli were digested with RNase T1 a t 37°C. RNA was extracted from digested nucleoli and subjected to P1 nu-
clease mapping with the 5'-end labeled coding strand ofKpnI-Hind11 fragment of pTprl4S [Matsuura et al., 19861 as a probe. Lanes 1 and 4,0 hr; lanes 2 and 5, 1hr; lanes 3 and 6, 2 hr.
MATERIALS AND METHODS RNase T1 Digestion and sarkOsY1 Treatment Of Isolated Nucleoli Nucleoli were isolated from Tetrahymena pyriformis in logarithmic growth phase as described [Higashinakagawa et al., 19921. Nucleoli derived from 4 to 8 x 10' cells were suspended in 800 p1 of 60 mM NaCl, 10 mM Tris-HC1 (pH 7.51, 7 mM MgCl,, and 5 mM dithiothreitol. The suspension was digested with 3,360 U of RNase T1 for 1 h r a t 37°C. Digested nucleoli were washed with the same buffer without RNase T1 by centrifugation a t 100,OOOg for 2 h r at 4°C. Nucleoli were then resuspended in 1ml of borate buffer (0.5 mM boric acid, 0.5 mM KCl, pH 8.0). Sarkosyl NL-30 was added to a final concentration of 0.5% (v/v). Sarkosyl NL-30 was a 30% (w/v) aqueous solution of n-lauroylsarcosine sodium salt. After mixing, the suspension was centrifuged at 100,OOOg for 2 h r a t 4°C. The pellet was resuspended in 800 pl of borate buffer, and it was digested again with 3,360 U of RNase T1. Then the digest was washed by centrifugation in buffer as before.
In Vitro Transcription RNase "1 and Sarkosyl treated nucleoli were incubated in a RNA synthetic cocktail as described [Matsuura et al., 19861: The standard reaction mixture was composed of treated nucleoli as a template, macronuclear lysate (ML), total cell extract (CE), and other necessary components. The ML and CE were prepared from T. thermophila essentially as described [Sutiphong et al., 19841. The reaction mixture was incubated a t 30°C for 30 min, after which it was extracted twice with phenol-chloroform ( l : l , v/v), twice with chloroform isoamyl alcohol (34:1, v/v), and three times with ether. DNA and RNA in the aqueous phase were precipitated with 2.5 vol of ethanol and redissolved in 100 ~1 of Tris-HC1 (pH 7.5), 10 mM MgCl,. DNA within the nucleolar template was digested with 1 pg of DNasz I a t 37°C for 30 min. Thereafter, the mixture was extracted with a n equal volume of phenol-chloroform followed by chloroform-isoamyl alcohol, and RNA was precipitated with ethanol containing 0.1 M NaCl.
IN VITRO TRANSCRIPTION OF TETRAHYMENA NUCLEOLI Nuclease P1 Protection Mapping Nuclease P1 protection mapping was performed as described [Financsek et al., 19821 with some modifications. The DNA probe was a KpnI-Hind11 fragment prepared from a rDNA clone, pTprl4S [Matsuura et al., 19861. The in vitro transcription product and the DNA probe were co-precipitated, dried in vacuo, and redissolved in 20 pl of 70% deionized formamide buffered with 100 mM PIPES (pH 7.8) and 10 mM EDTA. After heating at 65°C for 10 min, the hybridization mixture was incubated at 465°C for 15 hr. Then 300 p1 of P1 buffer (150 mM CH,COONa, pH 6.0,400 mM NaC1,O.l mM ZnSO,) and 6 pg of heat-denatured calf thymus DNA were added, and the solution was digested with 0.5-5 pg of single strand nucleic acid specific nuclease P1 at 37°C for 1hr. The reaction was stopped by adding phenol and 5 pg of carrier tRNA. After phenol extraction, nucleic acids were precipitated with ethanol. DNNRNA hybrids were electrophoresed on 4% polyacrylamide urea-gels, and autoradiography was performed with Kodak X-Omat S film using a Du Pont Cronex Lightning Plus Screen at -80°C.
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RESULTS Removal of Endogenous Nucleolar RNA Isolated nucleoli are known to contain 35s pre-rRNA or at least its 5' region in the form of nascent RNA chains or degradation products. These RNA sequences are not distinguishable from the in vitro transcription product from a nucleolar template and may prevent detection of new transcripts during P1 nuclease mapFig. 2. RNase activity of the nucleoli digested with RNase T1.RNA ping. Another potential source of RNA sequences that was extracted and analyzed by P1 nuclease mapping with the KpnImay interfere with P1 nuclease mapping is the cellular Hind11 fragment as a probe. Lane 1, nucleoli were digested with extract if it is prepared from homologous T.pyriformis RNase T1 and then washed; lane 3, nucleolar RNA from 2'. pyriformis cells. In fact, the CE contained sequences corresonding was added to RNase-treated and washed nucleoli; lane 2, the mixture in lane 3 was incubated for 10 min at 30°C under transcription conto the 5' region of 35s pre-rRNA (data not shown). The ditions hut without nucleotides. former obstacle was removed by RNase T1 and Sarkosyl treatment of isolated nucleoli, the latter by using a cellular extract from a different Tetrahymena strain, T. for the use of cellular extract from T. thermophila is thermophila. Initially, isolated nucleoli were simply incubated a t discussed in the next section. RNA extracted from this 37°C with the expectation that endogenous nucleases incubation mixture protected the probe (Fig. 2, lane 2)) would digest endogenous precursor RNA sequences. showing that RNase T 1 was effectively removed by the After incubation, RNA was extracted and probed with washing procedure. This indicated that the detection of a KpnI-Hind11 coding strand from a rDNA clone, new transcripts was possible even if RNase treated nupTprl4S, labeled with a 32Pat its H i n d I I restriction cleoli were used as a template. One explanation may be site. Lanes 1-3 in Figure 1 show t h a t autodigestion that the presence of a vast excess of RNA in the extract was not effective in removing endogenous sequences effectively compete for the remaining RNase T1. even after 2 h r of incubation. Consequently, isolated In Vitro Transcription Using nucleoli were digested with RNase T1. In lanes 4-6 of T.thermophila Extract Figure 1, the RNA sequence that protected the probe was not detectable in RNA fractions after 1h r of RNase Figure 3 compares rDNA sequences of T. pyriformis treatment. In order to monitor removal of exogenously to those of T. thermophila. Close inspection reveals a added RNase, the nucleoli were washed once, and they single base difference 8 nucleotides upstream from the were incubated at 30°C under transcription conditions H i n d I I site. In addition, differences of two bases and 7 with T. pyriformis nucleolar RNA plus a cellular ex- bases are found 38 nucleotides and 260 nucleotides uptract from a related strain, T. thermophila. The reason stream from the H i n d I I site, respectively. Accord-
146
MATSUURA AND HIGASHINAKAGAWA II
IAGCG, i I i i t A A A A i A i G i - , t i A A i r A i T G A i , A , T i i 1 i i i t t i 1 i I ,-GiTTGA, t 1 iGGi tGt I T , t t t t 8 I T , i I i i t TCAAAAAAATGAGTGGAGTCATAAAGTTGGAGAGAATCAGAAAAATACAAGGAAACCAGAAATTTTCTCAAAGACTTTTTTGTGGCAA I
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I
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C I I I I I 1 1 i I I I I I I I I I ~I I Ii 1 1 i i I i I I i i t 1 1 i I i 1 I i 1 I C i I I i , T i i i G i T i i i I 1 I I i 1 I T 1 1 1 1 I i t i 1 i c i i I i I I i 1 1 i A i t t i I 1 I I I i i ATTTAAGAAGGGGAAACATCTCCGGATAAAAATAAAATATCAGTTCGATCTGAACATCAGTAAGATGTCCTTTTGGGAAACCAAGGATGACGATAATGAA I
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I I I I I I I I I t i I I i 1 1 1 1 I I I I 1 1 I I A i i t i I iAAA----i I I A I I 1 i i i t i C - i I i i i i - i i i i ~ T i i i iiI i i i i i C i C r i i I I I I I O I I I I I I I A I AAATGGCCTGACTGAAATTTTCATGATGGCTGTGACGTCGTTCGCTGCAAAGGATTCGCAAGCCTTTCCCAGTGAGAGTTGTTGTATCGATATCTTT I
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I I I I I I I I 1 1 I I I I I I II I I 1-1 I A i i II i i 1 I t ii i i ii1 i i i i i i i i i 1 i i i1 i i t i A i i i G i I 1 1 i i1 iiI i 1 I 1 ii ii it 1 iii i iii A i ii I 1-1 GCAGATATTGTTACAAATAACAGCGACACGCTAGTACTGTTATAAATCGGTGTCGCTGATATTATTAACAGCTAGCAACAAAGTTGACTTGAGTCCG
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+4;0
A Hind 111
Fig. 3. Comparison of nucleotide sequence of rDNA. The transcription initiation site and the region surrounding it in T. pyriformis rDNA [lower line; Saiga et al., 19821 were compared with the corre-
sponding region in T. thermophila rDNA [upper line; Engberg et al., 19841. Dots above the T. pyriformis sequence indicate identity of nucleotides and dashes indicate a deletion.
ingly, P1 nuclease was expected to digest the nucleolar RNA from T. thermophila if the KpnI-Hind11 fragment of T. pyriformis was used as a probe, due to the mismatches just mentioned. This prediction was confirmed experimentally (Fig. 4). Homologous T. pyriformis nucleolar RNA gave the expected signal (lane P), while the nucleolar RNA from T. thermophila gave no signal within the range of the gel used (lane TI. Another point to be considered is whether or not the extract from T. thermophila transcribes the T.pyriformis rDNA clone. As shown in Figure 5, when pTprl4S was transcribed using the T. thermophila extract, a protected fragment 500 bases in length was detected. The result makes it highly likely that the T. thermophila extract was faithfully transcribing the T. pyriformis rDNA clone. The signals corresponding to 520 and 550 base fragments could be due to incomplete digestion of the probe. The ability of the T. thermophila extract to substitute for T. pyriformis may correspond to the similarity of transcription factors observed between rat and mouse [Mishima et al., 19821 as well as between Xenopus laevis and Xenopus borealis [Moss, 19821.
In Vitro Transcription of RNase-Treated Nucleoli Based on the experiments described in the previous sections, RNase treated nucleoli from T. pyriformis were incubated in the presence of ML and CE prepared from T. thermophila and other components necessary for transcription. Transcription products were subjected to P1 nuclease mapping with a KpnI-Hind11 fragment of pTprl4S as a probe. Figure 6, lane 2, shows that the level of endogenous nucleolar RNA in the template was substantially reduced. However, in this particular experiment, removal was not complete. When these nucleoli were incubated with four kinds of nucleoside triphosphates (4 NTPs), a signal corresponding to a 500-base fragment appeared after P1 nuclease mapping (lane 3). The addition of excess T . thermophila RNA polymerase or cellular extracts had no effect on the signal intensity (lanes 4-7). The origin of this signal may be nascent RNA chains in the transcription initiation region that survived RNase treatment, elongated, and traversed the Hind11 site by addition of 4 NTPs. Protection of these nascent chains may result from steric hindrance of the RNase molecule by some
IN VITRO TRANSCRIPTION OF TETRAwy2MENA NUCLEOLI
M
P
T
147
M
P+T
51 7 506 5177=
506
Fig. 4. P1 nuclease mapping of nucleolar RNA. The 5’-end labeled coding strand DNA of KpnI-Hind11 fragment was hybridized with nucleolar RNA from two species of Tetruhymenu, treated with P1 nuclease, and electrophoresed on a 4% polyacrylamide-urea gel. Lane P, RNA extracted from T. pyriformis; lane T, RNA extracted from T. thermophilu; lane P + T, mixture of RNA from both species. Arrowhead indicates the position of the predicted protection fragment.
macromolecular structure. Such a structure may also block entry of RNA polymerase or the necessary factors in the extract into the transcription site, thereby preventing initiation of new RNA chains. Based upon this hypothesis, efforts were made to destroy the presumed higher order structure. Treatment of nucleoli with Sarkosyl after RNase digestion was ineffective (Fig. 7 ) ,but another cycle of RNase T1 treatment did remove these residual sequences and the resultant spurious signal.
In Vitro Transcription of RNase-Sarkosyl-Treated Nucleoli When isolated nucleoli were treated sequentially with RNase T1, 0.5% Sarkosyl, and RNase T1, the background was completely removed (Fig. 8). Even after the 4 NTPs were added, no signal was detected (lane 2). The addition of T. thermophila extract resulted in the appearance of a n expected signal 500
In Vitro Transcription in Isolated Nucleoli of Tetrahymena pyriformis TADASHI MATSUURA AND TORU HIGASHINAKAGAWA Department of Developmental Biology, Mitsubishi Kasei Institute of Life Sciences, Machida, Tokyo, Japan
ulation of gene expression [Mitchell and Tjian, 1989; Sawadogo and Sentenac, 19901. However, many questions remain unanswered due to the complex composition and structural arrangement of DNA and proteins which constitute eukaryotic chromatin. A simple model system is needed for analysis of the molecular mechanisms underlying the control of chromatin transcription. With this goal in mind, we began studying rDNA in the ciliated protozoan, Tetrahymena. Within the macronucleus of Tetrahymena, rDNAs exist as free palindromic molecules of about 19 kbp that are packaged in a number of extrachromosomal nucleoli [Engberg et al., 1976; Karrer and Gall, 19761. This biological peculiarity represents one of the advantages of the Tetrahymena rDNA system. Tetrahymena nucleoli are organelles that contain copies of a single gene for rRNA, and isolated nucleoli provide a unique source of chromatin for transcription studies. Previously, we reported a procedure for isolating nucleoli [Higashinakagawa et al., 19791, and we significantly improved it recently [Higashinakagawa et al., 19921. We also cloned a rDNA fragment and determined the transcription initiation site [Higashinakagawa et al., 1981; Saiga et al., 19821. Initiation of transcription was reproduced in vitro with a homologous Tetrahymena cell extract and cloned rDNA fragment as a template Key words: Extrachromosomal nucleoli, in vitro [Matsuura et al., 19861. As a n extension of these expertranscription, chromatin, ribosomal DNA (rDNA), iments, in vitro transcription of isolated nucleoli was Tetrahymena, P1 nuclease mapping examined in order to determine the role of chromosomal proteins in the transcription process. Here we report that transcription was initiated from the in vivo INTRODUCTION initiation site with a nucleolar template and that the In eukaryotic cells, about one-half the mass of the efficiency of transcription of the nucleolar template chromatin composing the transcriptional apparatus was much higher than that of a purified rDNA clone. consists of histones and other chromosomal proteins [van Holde, 19891. Gene expression during development and differentiation is known to be greatly affected by structural alterations in chromatin resulting from nonrandom interactions between DNA and these chromosomal proteins [Grunstein, 19901. Historically, Received for publication September 30, 1991; accepted November 13, the concept of “open” and “closed” conformations of 1991. chromatin was proposed based on several kinds of experiments, most notably the assessment of the nuclease Address reprint requests to Dr. Toru Higashinakagawa, Department of Developmental Biology, Mitsubishi Kasei Institute of Life Sciences, sensitivity of chromatin DNA [Gross and Garrard, Machida, Tokyo 194, Japan. 1988; Elgin, 19881. Recently, various transcription factors and DNA binding proteins have been isolated and Dr. Matsuura is now a t the Department of Parasitology, Shinshu found to act at specific sites in chromatin during reg- University School of Medicine, 3-1-1Asahi, Matsumoto 390, Japan. An in vitro transcription system ABSTRACT was established using extrachromosomal nucleoli from Tetrahymena pyriformis macronuclei as a template. Ribosomal precursor RNA (pre-rRNA) and nascent pre-rRNA chains were removed from isolated nucleoli by treatment with RNase T1 and Sarkosyl. Nucleoli were then incubated in a RNA synthetic cocktail containing a cellular extract from Tetrahymena thermophila. The transcription product was examined for the presence of transcripts from T. pyriformis ribosomal DNA (rDNA) by P1 nuclease protection mapping using a DNA probe from a T. pyriformis rDNA clone. A sequence difference between T. pyriformis and T. thermophila in the 5‘ region of their 35s pre-rRNAs permitted exclusive detection of T. pyriformis transcripts. The results showed that faithful transcription initiation occurred in vitro from the in vivo initiation site of the nucleolar template and that the nucleolar template had a much higher efficiency of transcription than that of the purified rDNA clone. This system may offer unique advantages for future studies of transcriptional control during development and differentiation. 0 1992 Wiley-tiss, Inc.
~~~~~
0 1992 WILEY-LISS, INC.
144
MATSUURA AND HIGASHINAKAGAWA
a 1
2
3
M
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i
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Fig. 1. Elimination of background derived from pre-rRNA in isolated nucleoli. a. Isolated nucleoli from 2'. pyriformis were incubated at 37°C. b. Isolated nucleoli were digested with RNase T1 a t 37°C. RNA was extracted from digested nucleoli and subjected to P1 nu-
clease mapping with the 5'-end labeled coding strand ofKpnI-Hind11 fragment of pTprl4S [Matsuura et al., 19861 as a probe. Lanes 1 and 4,0 hr; lanes 2 and 5, 1hr; lanes 3 and 6, 2 hr.
MATERIALS AND METHODS RNase T1 Digestion and sarkOsY1 Treatment Of Isolated Nucleoli Nucleoli were isolated from Tetrahymena pyriformis in logarithmic growth phase as described [Higashinakagawa et al., 19921. Nucleoli derived from 4 to 8 x 10' cells were suspended in 800 p1 of 60 mM NaCl, 10 mM Tris-HC1 (pH 7.51, 7 mM MgCl,, and 5 mM dithiothreitol. The suspension was digested with 3,360 U of RNase T1 for 1 h r a t 37°C. Digested nucleoli were washed with the same buffer without RNase T1 by centrifugation a t 100,OOOg for 2 h r at 4°C. Nucleoli were then resuspended in 1ml of borate buffer (0.5 mM boric acid, 0.5 mM KCl, pH 8.0). Sarkosyl NL-30 was added to a final concentration of 0.5% (v/v). Sarkosyl NL-30 was a 30% (w/v) aqueous solution of n-lauroylsarcosine sodium salt. After mixing, the suspension was centrifuged at 100,OOOg for 2 h r a t 4°C. The pellet was resuspended in 800 pl of borate buffer, and it was digested again with 3,360 U of RNase T1. Then the digest was washed by centrifugation in buffer as before.
In Vitro Transcription RNase "1 and Sarkosyl treated nucleoli were incubated in a RNA synthetic cocktail as described [Matsuura et al., 19861: The standard reaction mixture was composed of treated nucleoli as a template, macronuclear lysate (ML), total cell extract (CE), and other necessary components. The ML and CE were prepared from T. thermophila essentially as described [Sutiphong et al., 19841. The reaction mixture was incubated a t 30°C for 30 min, after which it was extracted twice with phenol-chloroform ( l : l , v/v), twice with chloroform isoamyl alcohol (34:1, v/v), and three times with ether. DNA and RNA in the aqueous phase were precipitated with 2.5 vol of ethanol and redissolved in 100 ~1 of Tris-HC1 (pH 7.5), 10 mM MgCl,. DNA within the nucleolar template was digested with 1 pg of DNasz I a t 37°C for 30 min. Thereafter, the mixture was extracted with a n equal volume of phenol-chloroform followed by chloroform-isoamyl alcohol, and RNA was precipitated with ethanol containing 0.1 M NaCl.
IN VITRO TRANSCRIPTION OF TETRAHYMENA NUCLEOLI Nuclease P1 Protection Mapping Nuclease P1 protection mapping was performed as described [Financsek et al., 19821 with some modifications. The DNA probe was a KpnI-Hind11 fragment prepared from a rDNA clone, pTprl4S [Matsuura et al., 19861. The in vitro transcription product and the DNA probe were co-precipitated, dried in vacuo, and redissolved in 20 pl of 70% deionized formamide buffered with 100 mM PIPES (pH 7.8) and 10 mM EDTA. After heating at 65°C for 10 min, the hybridization mixture was incubated at 465°C for 15 hr. Then 300 p1 of P1 buffer (150 mM CH,COONa, pH 6.0,400 mM NaC1,O.l mM ZnSO,) and 6 pg of heat-denatured calf thymus DNA were added, and the solution was digested with 0.5-5 pg of single strand nucleic acid specific nuclease P1 at 37°C for 1hr. The reaction was stopped by adding phenol and 5 pg of carrier tRNA. After phenol extraction, nucleic acids were precipitated with ethanol. DNNRNA hybrids were electrophoresed on 4% polyacrylamide urea-gels, and autoradiography was performed with Kodak X-Omat S film using a Du Pont Cronex Lightning Plus Screen at -80°C.
M
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RESULTS Removal of Endogenous Nucleolar RNA Isolated nucleoli are known to contain 35s pre-rRNA or at least its 5' region in the form of nascent RNA chains or degradation products. These RNA sequences are not distinguishable from the in vitro transcription product from a nucleolar template and may prevent detection of new transcripts during P1 nuclease mapFig. 2. RNase activity of the nucleoli digested with RNase T1.RNA ping. Another potential source of RNA sequences that was extracted and analyzed by P1 nuclease mapping with the KpnImay interfere with P1 nuclease mapping is the cellular Hind11 fragment as a probe. Lane 1, nucleoli were digested with extract if it is prepared from homologous T.pyriformis RNase T1 and then washed; lane 3, nucleolar RNA from 2'. pyriformis cells. In fact, the CE contained sequences corresonding was added to RNase-treated and washed nucleoli; lane 2, the mixture in lane 3 was incubated for 10 min at 30°C under transcription conto the 5' region of 35s pre-rRNA (data not shown). The ditions hut without nucleotides. former obstacle was removed by RNase T1 and Sarkosyl treatment of isolated nucleoli, the latter by using a cellular extract from a different Tetrahymena strain, T. for the use of cellular extract from T. thermophila is thermophila. Initially, isolated nucleoli were simply incubated a t discussed in the next section. RNA extracted from this 37°C with the expectation that endogenous nucleases incubation mixture protected the probe (Fig. 2, lane 2)) would digest endogenous precursor RNA sequences. showing that RNase T 1 was effectively removed by the After incubation, RNA was extracted and probed with washing procedure. This indicated that the detection of a KpnI-Hind11 coding strand from a rDNA clone, new transcripts was possible even if RNase treated nupTprl4S, labeled with a 32Pat its H i n d I I restriction cleoli were used as a template. One explanation may be site. Lanes 1-3 in Figure 1 show t h a t autodigestion that the presence of a vast excess of RNA in the extract was not effective in removing endogenous sequences effectively compete for the remaining RNase T1. even after 2 h r of incubation. Consequently, isolated In Vitro Transcription Using nucleoli were digested with RNase T1. In lanes 4-6 of T.thermophila Extract Figure 1, the RNA sequence that protected the probe was not detectable in RNA fractions after 1h r of RNase Figure 3 compares rDNA sequences of T. pyriformis treatment. In order to monitor removal of exogenously to those of T. thermophila. Close inspection reveals a added RNase, the nucleoli were washed once, and they single base difference 8 nucleotides upstream from the were incubated at 30°C under transcription conditions H i n d I I site. In addition, differences of two bases and 7 with T. pyriformis nucleolar RNA plus a cellular ex- bases are found 38 nucleotides and 260 nucleotides uptract from a related strain, T. thermophila. The reason stream from the H i n d I I site, respectively. Accord-
146
MATSUURA AND HIGASHINAKAGAWA II
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-1 00
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I
+1
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I
I
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+loo
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I I I I I I I I I t i I I i 1 1 1 1 I I I I 1 1 I I A i i t i I iAAA----i I I A I I 1 i i i t i C - i I i i i i - i i i i ~ T i i i iiI i i i i i C i C r i i I I I I I O I I I I I I I A I AAATGGCCTGACTGAAATTTTCATGATGGCTGTGACGTCGTTCGCTGCAAAGGATTCGCAAGCCTTTCCCAGTGAGAGTTGTTGTATCGATATCTTT I
+zoo
1
I
I
I
I
I
I
I
I
+250
I I I I I I I I 1 1 I I I I I I II I I 1-1 I A i i II i i 1 I t ii i i ii1 i i i i i i i i i 1 i i i1 i i t i A i i i G i I 1 1 i i1 iiI i 1 I 1 ii ii it 1 iii i iii A i ii I 1-1 GCAGATATTGTTACAAATAACAGCGACACGCTAGTACTGTTATAAATCGGTGTCGCTGATATTATTAACAGCTAGCAACAAAGTTGACTTGAGTCCG
I
I
I
I
I
I
+300
I
+3;0
I I 1 1 1 1I I i 1 1 i A i i 1 i i i i-i t i i iii i G i i iii iii ii i i T i A i i 1 1 t i ii i i t i i i i T A i t i i t i t i 1 1 1 1 iiiii i i i t i iTi i i i i i A i i i 1 1 1 1 AAGAGATGC GATTGAGTTTTTCTCATTGTACCTTCGAAGATTCTTGCAACTAGAAGAAACAGATAGTAAACGAAACGATGCGG~CTATGTG~AAAGCT I I I I I
+400
+4;0
A Hind 111
Fig. 3. Comparison of nucleotide sequence of rDNA. The transcription initiation site and the region surrounding it in T. pyriformis rDNA [lower line; Saiga et al., 19821 were compared with the corre-
sponding region in T. thermophila rDNA [upper line; Engberg et al., 19841. Dots above the T. pyriformis sequence indicate identity of nucleotides and dashes indicate a deletion.
ingly, P1 nuclease was expected to digest the nucleolar RNA from T. thermophila if the KpnI-Hind11 fragment of T. pyriformis was used as a probe, due to the mismatches just mentioned. This prediction was confirmed experimentally (Fig. 4). Homologous T. pyriformis nucleolar RNA gave the expected signal (lane P), while the nucleolar RNA from T. thermophila gave no signal within the range of the gel used (lane TI. Another point to be considered is whether or not the extract from T. thermophila transcribes the T.pyriformis rDNA clone. As shown in Figure 5, when pTprl4S was transcribed using the T. thermophila extract, a protected fragment 500 bases in length was detected. The result makes it highly likely that the T. thermophila extract was faithfully transcribing the T. pyriformis rDNA clone. The signals corresponding to 520 and 550 base fragments could be due to incomplete digestion of the probe. The ability of the T. thermophila extract to substitute for T. pyriformis may correspond to the similarity of transcription factors observed between rat and mouse [Mishima et al., 19821 as well as between Xenopus laevis and Xenopus borealis [Moss, 19821.
In Vitro Transcription of RNase-Treated Nucleoli Based on the experiments described in the previous sections, RNase treated nucleoli from T. pyriformis were incubated in the presence of ML and CE prepared from T. thermophila and other components necessary for transcription. Transcription products were subjected to P1 nuclease mapping with a KpnI-Hind11 fragment of pTprl4S as a probe. Figure 6, lane 2, shows that the level of endogenous nucleolar RNA in the template was substantially reduced. However, in this particular experiment, removal was not complete. When these nucleoli were incubated with four kinds of nucleoside triphosphates (4 NTPs), a signal corresponding to a 500-base fragment appeared after P1 nuclease mapping (lane 3). The addition of excess T . thermophila RNA polymerase or cellular extracts had no effect on the signal intensity (lanes 4-7). The origin of this signal may be nascent RNA chains in the transcription initiation region that survived RNase treatment, elongated, and traversed the Hind11 site by addition of 4 NTPs. Protection of these nascent chains may result from steric hindrance of the RNase molecule by some
IN VITRO TRANSCRIPTION OF TETRAwy2MENA NUCLEOLI
M
P
T
147
M
P+T
51 7 506 5177=
506
Fig. 4. P1 nuclease mapping of nucleolar RNA. The 5’-end labeled coding strand DNA of KpnI-Hind11 fragment was hybridized with nucleolar RNA from two species of Tetruhymenu, treated with P1 nuclease, and electrophoresed on a 4% polyacrylamide-urea gel. Lane P, RNA extracted from T. pyriformis; lane T, RNA extracted from T. thermophilu; lane P + T, mixture of RNA from both species. Arrowhead indicates the position of the predicted protection fragment.
macromolecular structure. Such a structure may also block entry of RNA polymerase or the necessary factors in the extract into the transcription site, thereby preventing initiation of new RNA chains. Based upon this hypothesis, efforts were made to destroy the presumed higher order structure. Treatment of nucleoli with Sarkosyl after RNase digestion was ineffective (Fig. 7 ) ,but another cycle of RNase T1 treatment did remove these residual sequences and the resultant spurious signal.
In Vitro Transcription of RNase-Sarkosyl-Treated Nucleoli When isolated nucleoli were treated sequentially with RNase T1, 0.5% Sarkosyl, and RNase T1, the background was completely removed (Fig. 8). Even after the 4 NTPs were added, no signal was detected (lane 2). The addition of T. thermophila extract resulted in the appearance of a n expected signal 500
