Volumew 7 NJumber 4 1979

Nucleic Acids Research

Research Acids Nucleic 41979 7 Number Volume

Trnscription initiation of Xenopus SS ribosonial RNA genes in vitro

Laurence J.Kom*, Edward H.Birkenmeier and Donald D.Brown

Carnegie Institution of Washington, Department of Embryology, 1 5 West University Parkway, Baltimore, MD 21210, USA Received 16 August 1979

ABSTRACT We have studied initiation of transcription of 5S RNA genes in extracts of Xenopus laevis oocyte nuclei. To aid in this study we developed a general assay for specificity of transcription initiation that does not require termination of transcription. Following in vitro transcription with accurai gaimia- P-labeled nucleoside triphosphates, the RNA is digested with pancreatic RNAase and fingerprinted by two dimensional chromatography. A 5S RNA gene with a variant sequence, in which the G residue at position +1 is replaced by a C, initiates transcription at an A residue one nucleotide preceding the C. Although Xenopus RNA polymerase form III can initiate transcription at many sites on plasmid DNA, all of the transcripts start with purines. The majority of these purines are triphosphorylated. When a repeating unit of Xenopus 5S DNA is inserted into the plasmid, initiations at the vector start sites are suppressed and the major labeled 5' oligonucleotide is derived from 5S RNA.

INTRODUCTION When Xenopus 5S ribosomal RNA genes (5S DNA) purified from genomic DNA or cloned into plasmids are injected into nuclei (germinal vesicles or GVs) of X. laevis oocytes, they direct the synthesis of 5S RNA (1, 2). Recently, similar results have been described when 5S DNA is added to a cell-free extract of Xenopus oocyte nuclei (3). Inhibition of transcription by intermediate concentrations of a-amanitin indicates that RNA polymerase III transcribes at least 90% of all RNA synthesized. In previous experiments faithful transcription has been assayed by the appearance of a band of radioactive RNA in an electrophoretic gel. The production of these full length transcripts requires accurate initiation and termination of RNA synthesis or processing of incorrect transcription products to the proper size. The gel assay per se cannot distinguish between these two possibilities. We have argued against processing of 5S RNA (3) from two kinds of evidence. Synthesis of mature 5S RNA occurs without a lag and one mole of a polyphosphorylated nucleotide is obtained for each mole of the radioactive C Information Retrieval Umited 1 Falconberg Court London Wl V 5FG England

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Nucleic Acids Research 5S RNA synthesized. In this paper we examine the metabolism of y-32P-ATP in extracts of nuclei, from Xenopus oocytes in order to develop a simple assay for accurate initiation of transcription that does not require proper termination. We have used this assay to study the specificity of initiation by RNA polymerase III in vitro. METHODS RNAase Ti and pancreatic RNAase were purchased from Sankyo and Sigma, respectively; Si nuclease was a gift from W. Walhi and U. Schibler and purified according to the method of Vogt (4). Polyethyleneiminecellulose thin-layer (PEI MN 300) 20 x 20 cm sheets were purchased from Brinkmann Instruments. DEAE-Sephadex A-25 and Sephadex G-50 were purchased from Pharmacia Fine Chemicals. y- 32P-ATP (-3000 Ci/mmole) was purchased from New Eigland Nuclear. a- 32P-CTP and a-32P-GTP (300 to 400 Ci/mnole) were purchased from Amersham. Preparation of Recombinant DNA The recombinant DNA used in these experiments was produced by ligating a Hind III digest of Xenopus 5S DNA with the plasmids pMB9 or pBR322 (5). pXlo31 is pMB9 containing one repeating unit of X. laevis oocyte-specific 5S DNA. pXbsi is pBR322 containing one repeating unit of X. borealis somatic type 5S DNA. pXbol/502 is pBR322 containing a fragment of X. borealis oocyte-specific 5S DNA that includes two genes. The construction and sequence of pXbol/502 has been previously described (6). All work with recombinant DNA was carried out according to the NIH Guidelines. In Vitro Transcription Reactions

Vector DNA or vector containing Xenopus 5S DNA was transcribed in nuclear extracts (GV supernate) of X. laevis oocytes (3). DNA (0.05 to 0.20 9g/jil extract) was preincubated in the nuclear extract for 30 min at 220C before the addition of labeled nucleotides. Unlabeled nucleoside triphosphates were present at 0.02 to 0.10 nM in y-32P-ATP experiments and 0.02 mM for the radioactive and 0.10 mMl for the unlabeled XTPs in a-32P-XTP experiments. 3 to 10 pmols of y-32P-ATP or 7 to 10 pmols of a-32P-XTPs were added for each 10 ul of the reaction mixture. The reaction was teminated after 1 to 2 hr at 220C and the RNA was extracted (3). Where indicated, some of the reactions used purified RNA polymerase III rather than GV supernate to transcribe the DNA. Samples of purified RNA 948

Nucleic Acids Research polymerase III were gifts of Steve McKnight and Carl Parker. The enzyme was dialyzed against J buffer 170 mM NH4C1, 7 nM MgCl2, 0.1 nmM EDTA, 2 mM dithiothreitol, 6% (v/v) glycerol and 10 mM HEPES pH 7.4] and used imnediately in the transcription reaction. Unlabeled XTPs and y-32P-ATP were incubated with GV supernate at 220C for 5 min to synthesize all four y-labeled XTPs. The GV supernate was then heat inactivated at 900C for 2-4 min before addition of the purified RNA polymerase and DNA to the y-32P-XTPs. Nuclease Treatment RNA was digested at 37°C in a volume of 5-10 il with nuclease as indicated: pancreatic RNAase (0.1 pg/kl) in 10 mM Tris pH 8.0 and 1 mM EDTA for 30 min; Ti RNAase (2 xig/pl) in H20 for 15 min; and S1 nuclease (0.2 units) in 0.05 M sodium acetate pH 4.75, 0.15 NaCl and 0.5 mM ZnSO4 for 1.5 to 2 hr. RNA was hydrolyzed in 0.3 N NaOH for 16 hr at 370C. Polyacrylamide Gel Electrophoresis

Electrophoresis of RNA was in 12% polyacrylamide gels (7) or thin 12% polyacrylamide 8 M urea gels (8). The gels were examined by autoradiography, the bands of 5S RNA were cut from the gels, the radioactivity measured, and the RNA extracted (1). Thin Layer Chromatography on Polyethyleneiminecellulose (PEI) Sheets In vitro transcription reactions to be analyzed by two dimensional thin layer chromatography were stopped by the addition of 0.5% SDS, 0.15 M NaCl, 5 mlM EDTA, 50 mM Tris (pH 7.8) (SDS-SET). The sample was extracted with phenol to remove protein and applied to a Sephadex G-50 column to remove acid-soluble material. The column was eluted with 0.3 M sodium acetate or SDS-SET and the void volume collected. The radioactivity in transcripts obtained by this procedure resides in the polyphosphorylated S' termini of the RNA molecules. However, unidentified radioactive contaminants in the commercial - 32P-ATP are also present. The sample was ethanol precipitated twice in the presence of 3 pg carrier tRNA, washed once with ethanol and digested with pancreatic RNAase. The products of this digestion were separated by two dimensional chromatographyon PEI thin layer plates as described by Miller and Burgess (9). Migration of oligonucleotides varied with different lots of PEI plates. When oligonucleotides migrated slowly, a wick of Whatman 3 F1 paper (6 x 20 cm in the first dimension and 3 x 20 cm in the second dimension) was attached with paper clips to the top of the PEI plates to draw up additional solvent. The 949

Nucleic Acids Research first dimension uses a solvent of 2.0 M LiCl and 0.01 M1 EDTA (added from a stock solution of 0.2 M EDTA, pH 7.0), with a final pH of 6.5. The solvent for the second dimension is 1.5 M LiCl, 0.005 EDTA, 1.8 M formic acid, adjusted to pH 2.0 with solid LiOH. In some cases, the RNA digested with pancreatic RNAase was further digested with Si nuclease. The dried sample was resuspended in 1 to 3 ]PI 10 mM XTPs or 10 mM XTPs, 10 mM XDPs and 10 mM XMPs and applied to PEI plates. Ascending chromatography in one dimension was then performed using an anmnonium sulphate solvent (0.4 M to 4 cm above the origin, 0.7 M until the solvent front reaches the top of the sheet). Unlabeled XTP, XDP and XMP markers applied with the sample were located on the plate by ultraviolet absorption.

DEAE-Sephadex Chromatography Samples to be fractionated by DEAE-Sephadex chromatography were hydrolyzed in alkali (see above) and then diluted in 100 mM NaCl, 10 mM Tris pH 7.5, and 7 M urea. 5 to 15 mg wheat germ RNA digested with Ti RNAase was added. Sephadex A-25 chromatography was as described by Tener (10). Selected fractions were pooled and desalted for further analysis (11). RESULTS The Metabolism of y-32P-ATP in Extracts of Nuclei from Xenopus Oocytes To study transcription initiation, we studied the incorporation of y-labeled nucleoside triphosphates (XTPs) into the 5' termini of RNA molecules. When y-32P-ATP was incubated with extracts of Xenopus oocyte nuclei, with or without exogeneous DNA, the label was rapidly transferred to the other nucleoside triphosphates as shown in Figure 1. At 220C in less than one min after the addition of y-32P-ATP, the label equilibrated among the four nucleoside triphosphates in the reaction mix. After a one hr reaction, the label remained equally distributed among the nucleoside triphosphates, but some radioactive inorganic phosphate was generated. The label was not transferred to the a-phosphate position since even in a two hr transcription reaction with 5S DNA, only the 5' terminal nucleotide of the newly synthesized 5S RNA was labeled (see below). We infer that the label was not transferred to the B position of XDPs or XTPs by the following observations: First, a-labeled XTPs gave rise to their corresponding XDP and XMP in the nuclear extract (not shown), and second, no radioactive XDPs were detected when y-32P-ATP was in the reaction mix. 950

Nucleic Acids Research

b

Fig, 1. Transfer of 32P from Y- P-ATP to unlabeled nucleoside triphosphates in

c

Pi

*

-U-UTP

^ ^

------CTP

extracts

of Xenopus oocyte nuclei. y32P-ATP and unlabeled nucleoside triphosphates were added to extracts of Xenopus oocyte nuclei. Aliquots of the reaction were removed after various times of incubation at 220C and chromatographed with unlabeled XTPs as markers on PEI plates as described in Methods. (a) 0 min, (b) 1 min, (c) 1 hr.

-ATP GTP -Origin

Therefore, the label is transferred only to the gamnma position of the XTPs. To substantiate the use of y-labeled RNA to study transcription initiation it was necessary to show that most 5S RNA molecules can be isolated with a 5'-triphosphate terminus. An in vitro transcription reaction was run with a--32P-GTP and the plasmid pXbsl (pBR322 ligated to a single repeating unit of X. borealis somatic 5S DNA). The 5S RNA was isolated by electrophoresis in a 12% polyacrylamide gel. The 5S RWA was eluted from the gel and hydrolyzed with alkali. The nucleotides were separated on a DEAE-Sephadex A-25 colunm, which fractionates oligonucleotides on the basis of their charge (10). The 5' terminal nucleotide would have a charge of -6 if it were a triphosphate end (pppXp), -5 if it were a diphosphate end (ppXp), or -4 if it were a monophosphate end (pXp). However, Schibler and Perry (12) observed that pppXp and ppXp elute together in a single peak as if they had a charge between -5 and -6. No radioactivity eluted from the colunm with a charge between -3 and -4, where pXp 951

Nucleic Acids Research

is expected to run. The radioactive nucleotides eluting in a peak between -5 and -6 were desalted, treated with Si nuclease to generate 3' hydroxymononucleotides and-chromatographed on PEI thin layer sheets. 85% of the termini were pppG and 15% were ppG.

The Initiation Assay A sensitive assay for the accurate initiation of 5S RNA synthesis is the appearance of a single 5' terminal oligorucleotide containing the first two nucleotides of 5S RNA (pppGpCp) following digestion with pancreatic RNAase. This oligonucleotfde can be detected independent of correct termination. For this assay, total RNA synthesized in the in vitro transcription system in the presence of y labeled nucleoside triphosphates was digested with pancreatic RNAase and the products'separated by two dimensional chromatography on PEI thin layer sheets. The result of such an analysis is shown in Figure 2 for the RNA transcripts from the pihasnsid alone (pBR322) and the plasmid with two different SS DNA inserts (pXlOM. and pXbsl). About 5% and 80% of the total RNA synthesized in one hour from pXlo31 and pXbsl, respectively, migrated coincidently with 5S RNA. There were few radioactive bands in the pBR322 transcripts, and several more discrete bands representing RNAs of different sizes were transcribed from pXlo31. The complex transcription pattern for pXlo31 was characteristic of single repeating units of X. laevis oocyte 5S DNA. Most of the radioactive RNA was too large to enter these gels. Pancreatic RtiAase digests of the y- 32p labeled -RNA transcribed in vitro from pBR322- gave 4 or 5 major and several minor 5' labeled oligonucleotides (Figure 2). A very similar pattern was seen for the plasmid pMB9 (data not shown). Despite the heterogeneous size distribution of the RNA transcribed from pXlo31 there was one major 5' terminal oligonucleotide fonred from pancreatic RNAase digestion just as was the case with pXbsl transcripts. Complete digestion of the 5' terminal oligonucleotide to mononucleotides with S1 nuclease followed by PEI chromatography has identified the first nucleotide residue as GTP. Based on its migration relative to known markers (9),-we have tentatively identified this oligonucleotide as pppGpCp, the oligonucleotide expected to be cleaved from 5S RNA by pancreatic RNAase. A Variant Transcription Start Site for a 5S RNA Gene

All eucaryotic 5S RNAs examined start with guanine or adenine (13). A sequenced, cloned fragment of X. borealis oocyte 5S DNA (Xbol) contains a gene (designated gene 3) that along with its 5' flanking sequence differs 952

Nucleic Acids Research d

_ .~~~

l

If

I

+12

e

ar m

P

&.

Fig. 2. Polyacrylamide gel and two dimensional thin layer chromatography of y-32P-labeled RNA. DNA was preincubated in extracts of Xenopus oocyte nuclei for 30 min at 220C. y-32p-ATP was added and the reaction allowed to continue for 1 hr. The RNA was extracted, and an aliquot was electrophoresed on a 12% polyacrylamide gel (a) pBR322, (b) pXbsl, (c) pXlo31. The rest of the sample was column purified, digested with pancreatic RNAase and chromatographed in two dimensions on PEI sheets (d) pBR322, (e) pXbsl, (f) pXl o31.

2

by many residues from the genes coding for the dominant X. borealis oocyte 5S RNA (Figure 3). One of the base differences within gene 3 is the first nucleotide of the gene (G to C). We wanted to determine whether initiation of transcription of gene 3 begins at the usual location (+1), which is a pyrimidine, or if a preference for purine starts causes it to initiate at a different nucleotide. We previously showed that gene 3 was transcribed about half as efficiently in the in vitro system as other 5S RNA genes in the same 5S DNA fragment (6). An analysis of the gene 3 transcript in polyacrylamide urea gels showed it to be indistinguishable in size from that of gene 2. A fingerprint of the transcript of gene 3 labeled with a-32P-GTP contained only the labeled RNAase Ti oligonucleotides predicted from the gene sequence (6) thus indicating that initiation of transcription must occur to the right of the first G residue in 953

Nucleic Acids Research Gene 2

AGTCTTCACTCCGATGCCTACGGCCACACC 51 3' Gene 3 AAGTCAGCAAACCTACCCTGCGGCTACACC

Fig. 3. The sequence surrounding the transcription initiation sites of two Xenopus borealis oocyte-type 5S ribosomal RNA genes. The top line shows the sequence (noncoding strand) of the 15 nucleotides preceding Xbol gene 2 and the first 15 nucleotides of the 5S RNA gene (6). Transcription initiates at position +1 shown by the arrow. The second line shows the same region for Xbol gene 3; the sequences are aligned by the homology between the gene sequences.

the 5' flanking sequence (Figure 3). To determine the site of transcription initiation for gene 3, we have analyzed the 5S RNA synthesized in an in vitro transcription reaction containing pXbol/502 DNA and y-32P-ATP. The plasmid pXbol/502 contains Xbol genes 2 and 3 inserted in the Hind III site of pBR322. The gene 2 and gene 3 transcripts were separated by electrophoresis in a polyacrylamide gel (6). They separate under these conditions presumably due to differences in secondary structure. The two 5S RNA bands were eluted from the gel and digested with RNAase Ti and Si nuclease to obtain the 5' terminal nucleotide. The sample was mixed with unlabeled marker XTPs and chromatographed on PEI plates (Figure 4). As expected, the 5' terminal nucleotide from gene 2 RNA comigrated with GTP. The major 5' terminal nucleotide of the slower migrating gene 3 RNA was ATP, indicating that gene 3 started with an A. A small amount of GTP was also present; this results from contamination with gene 2 RNA as determined by fingerprint analysis (data not shown). The two spots (X, Y) in Figure 4 didnotcomigrate with unlabeled XTP or XDP markers. They are not related to transcription initiation; they were seen even when the in vitro transcription reaction was run in the absence of added DNA and no RNA was synthesized. They were not investigated further. We conclude from the size of the gene 3 transcript, the fingerprint of internally labeled gene 3 RNA (6), and the observation that gene 3 RNA starts with an adenine residue that initiation of transcription is shifted one nucleotide to the position -1 of the 5S RNA gene and the transcript is probably 121 nucleotides long; one more than 5S RNA.

Xenopus RNA Polymerase III Initiates Transcription with Purines To determine whether Xenopus RNA polymerase III always initiates transcription with a purine, total RNA transcribed from pBR322 in extracts of oocyte nuclei was digested with pancreatic RNAase followed by Si nuclease, 954

Nucleic Acids Research

a

Fig. 4. The y- P-labeled 5' nucleotides of RNA transcribe from gene 2 and gene 3 of pXbol/502. y- 2P-labeled 5S RNAs transcribed from genes 2 and 3 were eluted from a 12% polyacrylamide gel and digested with RNAase Ti and Si nuclease. The digested RNA from each gene was suspended in 10 nM XTPs and 10 mM XDPs, and 1 to 3 Ol chromatographed on PEI plates (a) gene 2, (b) gene 3. The positions of the unlabeled ATP and GTP are indicated.

b

-x

g

---ATP

--Origiln

which cleaves RNA to 3' hydroxy-mononucleotides. Since y-32P-ATP was used in the transcription reaction, only the 5' nucleotide of each transcript was labeled. The nucleotides were chromatographed and detected by autoradiography (Figure 5). The two labeled spots comigrated with the purines ATP and GTP. The two pyrimidine triphosphate spots accounted for less than 2% of the radioactivity. The large radioactive spot at the origin was a contaminant in the ATP preparation. In extracts of Xenopus oocyte nuclei, transcription of 5S DNA by endogenous RNA polymerase III resulted in synthesis of 5S RNA. When purified X. laevis RNA polymerase III was incubated with 5S DNA, however, transcription did not result in a band of 5S RNA. Even though there was a lack of specificity, the purified enzyme continued to initiate transcription of pBR322 or pXbsl with purines exclusively (data not shown). DISCUSSION We previously described an in vitro transcription system from extracts of Xenopus laevis oocyte nuclei that accurately transcribes 5S DNA and other genes that are transcribed by RNA polymerase III. These other genes include 955

Nucleic Acids Research

UTP

-

CTP

ATP

GTP

Origin 2

Fig. 5. Two-dimensional chromatography of y-32P-labeled 5' nucleotides from RNA transcripts of pBR322. pBR322 DNA was transcribed and the RNA extracted. The RNA was digested with pancreatic RNAase and Si nuclease. Two-dimensional chromatography was on PEI sheets. The anmnonium sulfate solvent system was used in the first dimension; chromatography in the second dimension was with 1.5 M LiCl, 0.005 M EDTA, 1.8 M formic acid, adjusted to pH 2.0 with solid LiOH. The positions of unlabeled nucleoside triphosphates added as markers are indicated. the VA RNA, gene from adenovirus (3) and tRNA genes from yeast and Drosophila (14). Previously, faithful transcription in the in vitro system has been assayed by the production of an RNA band of the correct size in a gel that was then fingerprinted to confirm its identity. A positive result by this assay assumes initiation of transcription at the correct site, chain elongation and proper transcription termination. In the case of tRNA genes the transcript can also undergo processing of 5', 3' and intervening sequences in the nuclear extract (15). We have developed an assay for accurate initiation of transcription that requires transcription initiation and chain elongation. The initiation assay also depends upon the transfer of 32P from y-32P-ATP to the y-position of all unlabeled nucleoside triphosphates jn the reaction mixture. Using the initiation assay, we examined transcription in vitro of the plasmid pBR322 with and without inserted Xenopus 5S DNA fragments. RNA synthesized from pBR322 yields 4 or 5 major and several minor initiation

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Nucleic Acids Research 5' oligonucleotides following pancreatic RNAase treatment. When X. borealis somatic 5S DNA is inserted into the plasmid, initiation within the vector DNA is suppressed and the labeled 5' oligonucleotide of 5S RNA (pppGpCp) is observed. These results suggest that Xenopus RNA polymerase III transcription at eight or more sites on vector DNA. (The can initiate number of distinct 51 labeled oligonucleotides is a minimum estimate of the number of initiation sites.) When a preferred initiation site for transcription by RNA polymerase III, such as a 5S RNA gene, is inserted into the vector, virtually all initiation events occur at the 5S RNA gene. Thus, both the suppression of transcription initiation on pBR322 DNA and the enhancement of a single diagnostic 5' oligonucleotide are indicative of accurate initiation of 5S RNA synthesis. pXlo31 is a plasmid containing a single X. laevis oocyte 5S RNA gene. In vitro transcription of pXlo31 produces a heterogeneous population of RNA molecules (Figure 2); only a small fraction of the RNA synthesized is 5S RNA. The initiation assay, however, shows that most of the transcripts begin with pppGpCp, implying that the majority of these heterogeneous transcripts initiate at the 5S RNA gene but continue past the normal termination site. The reason for this is unknown. We showed that a 5S RNA gene with a variant sequence, including a C residue replacing the G at the first nucleotide of the gene, initiates transcription at an A residue inmnediately preceding the normal initiation site for 5S RNA. The discovery of an altered initiation site in this gene suggests that RNA polymerase III has some flexibility in the distance between its DNA binding site and its transcription initiation site. of pXbol initiates transcription with a The finding that gene 3 purine at a new site rather than initiate with a pyrimidine at position +1 of the 5S RNA gene suggests that RNA polymerase III has a strong preference for initiating with purines. We have shown that Xenopus RNA polymerase III initiates transcription exclusively with purines even on the procaryotic DNA templates pBR322 and pMB9. This result was obtained with extracts of Xenopus oocyte nuclei and with the purified enzyme, suggesting that the requirement for initiation with purines is an inherent property of the polymerase and does not depend on other factors in the nuclear extract. From an examination of published DNA sequences we observed that not only does RNA polymeraseIII initiate transcription with a purine but that the initiation sites are often preceded and followed by a pyrimidine (6). The sequence surrounding the initiation site for the Xenopus 5S RNA genes 957

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sequenced in our laboratory is Py I I and the Xbol variant gene 3 has -1 +1+2 1 +1 +2 T A C. Perhaps, the arrangment Py Pu Py helps to specify the precise site of transcription initiation. Alternatively, by surrounding the first purine of the transcript by pyrimidines, the ambiguity of start sites may be reduced. ACKNOWLEDGEMENT The authors thank E. Jordan for expert technical assistance. Drs. Daniel Bogenhagen, Ronald Peterson and Barbara Sollner-Webb made helpful criticisms on the manuscript. This research was aided in part by grant GM22395 awarded to DDB.

*Present address: Medical Research Council, Laboratory of Molecular Biology, University Postgraduate Medical School, HiI1s Road, Cambridge, CB2 2QH, England. REFERENCES 1.

2. 3. 4.

5. 6. 7. 8.

9. 10. 11. 12. 13. 14. 15.

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Brown, D. D. and Gurdon, J. B. (1977) Proc. Natl. Acad. Sci. USA 74, 2064-2068. Brown, D. D. and Gurdon, J. B. (1978) Proc. Natl. Acad. Sci. USA 75, 2849-2853. Birkenmeier, E. H., Brown, D. D. and Jordan, E. (1978) Cell 15, 10771086. Vogt, V. M. (1973) Eur. J. Biochem. 33, 192-200. Brown, D. D. and Jordan, E. (1976) Carnegie Inst. Washington Yearbook 75, 12-13. Korn, L. J. and Brown, D. D. (1978) Cell 15, 1145-1156. Peacock, A. C. and Dingman, C. W. (1968) Biochemistry 7, 668-674. Maxam, A. M. and Gilbert, W. (1977) Proc. Natl. Acad. Sci. USA 74, 560-564. Miller, J. S. and Burgess, R. R. (1978) Biochemistry 17, 2054-2059. Tener, G. M. (1967) Methods Enzymol. Vol. 12A, 398-401, Academic Press, New York. Brownlee, G. G. (1972) Determination of Sequences in RNA, American Elsevier, New York. Schibler, U. and Perry, R. P. (1976) Cell 9, 121-130. Erdmann, V. A. (1978) Nucleic Acids Res. 5, rl-r13. Schmidt, O., Mao, J., Silverman, S., Hovemann, B., and Soll, D. (1978) Proc. Natl. Acad. USA 75, 4819-4823. Ogden, R. C., Beckmann, J. S., Abelson, J., Kang, H. W., Soll, D. and Schmidt, 0. (1979) Cell 17, 399-406.

Transcription initiation of Xenopus 5S ribosomal RNA genes in vitro.

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