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(in vitro transcription/cloned rDNA/Southern analysis). RONALD REICHEL*, HANS-JURG MONSTEINt, HANS-WILLI JANSEN*, LENNART PHILIPSONt,.
Proc. Natli Acad. Sci. USA Vol. 79, pp. 3106-3110, May 1982

Biochemistry

Small nuclear RNAs are encoded in the nontranscribed region of ribosomal spacer DNA (in vitro transcription/cloned rDNA/Southern analysis)

RONALD REICHEL*, HANS-JURG MONSTEINt, HANS-WILLI JANSEN*, LENNART PHILIPSONt, AND BERND-JOACHIM BENECKE** *Department of Biochemistry, Ruhr-University, D-463 Bochum, Federal Republic of Germany; and tDepartment of Microbiology, Biomedical Center, Uppsala

University, Uppsala, Sweden Communicated by James E. Darnell, February 8, 1982

nuclease T1 oligonucleotide pattern analysis. The results show that snPI RNA and these snRNA species are not related to each other. Furthermore, the genes coding for snPI RNA molecules have been localized within sequences of the nontranscribed ribosomal spacer region, immediately adjacent to the 5' end of the 45S transcription unit.

ABSTRACT The structure ofin vitro synthesized mouse small nuclear RNA transcribed by RNA polymerase I (snPI RNA) was studied by T1 RNase digestion pattern analysis. The patterns of four different snPI RNA species were different from those of the Ul and U2 RNA species. In addition, the four different snPI RNA species, ranging from 130 to 240 nucleotides in length, yielded almost identical patterns. The snPI RNA molecules hybridized to cloned mouse ribosomal DNA containing the nontranscribed spacer DNA and 45S ribosomal precursor RNA molecules did not compete with this hybridization. Southern blot analysis of fragments from the ribosomal DNA confirmed that snPI RNA species exclusively hybridized to sequences corresponding to the so-called nontranscribed ribosomal spacer region.

MATERIALS AND METHODS In Vitro RNA Synthesis. Nuclei were isolated from mouse 3T6 fibroblasts by lysis of the cells in hypotonic buffer (6 mM KCV1.6 mM MgCl2/i mM dithiothreitol/10 mM Tris HCI, pH 8.0) in the presence of 0.5% Nonidet P-40. The nuclei scraped off the dishes were washed in incubation buffer [1 mM magnesium acetate/i mM dithiothreitoV0.2 mM EDTA/10 mM Tris HCI, pH 7.9/25% (vol/vol) glycerol], resuspended, and preincubated with a-amanitin for 10 min on ice. The final reaction mixture contained 1 mM ATP, 1 mM GTP, 1 mM CTP, 2 uM UTP, 2 mM MnCl2, 5 mM magnesium acetate, 50 mM (NH4)2SO4, 1 mM dithiothreitol, and 25 ,Ci (1 Ci = 3.7 X 1010 becquerels) of [3H]UTP (40-60 Ci/mmol) or [a-32P]UTP (>350 Ci/mmol) (Amersham) in a total volume of 280 ,ul. Incubation was for 30 min at 25°C, and the RNA was isolated after disruption ofthe nuclei with 1 ml of HSB (0.5 M NaCl/5 mM MgCl2/ 0.01 M Tris HCI, pH 7.4) and digestion ofthe DNA with RNasefree (4) DNase I (50 ,ug/ml; Boehringer Mannheim). The phenol-extracted RNA was analyzed in 6-15% polyacrylamide gels as decribed (5). Individual RNA bands were eluted from the gel pieces electrophoretically in sealed dialysis bags coated with tRNA. T1 RNase products were separated by electrophoresis on cellulose acetate in the first dimension and homochromatography on DEAE-cellulose in the second dimension, as described (6). Hybridization to Ribosomal DNA (rDNA). The cloning of the mouse rDNA in bacteriophage A and the characterization of the AgtWES Mr 974 clone (obtained from I. Grummt, Wurzburg) have been described in detail (7, 8). In vitro synthesized snPI RNA was hybridized to the cloned rDNA under the following conditions: 4X SET (1 x SET is 0.15 M NaCl/l mM EDTA/0.03 M Tris HCl, pH 8.0), 5X Denhardt's reagent (9), 50% (vol/vol) formamide, 0.1% NaDodSO4, and Escherichia coli nucleic acids at 75 Ag/ml for 18 hr at 42°C. The filters were washed twice in 4X SET/0. 1% NaDodSO4, twice in 2x SET/0.1% NaDodSO4, and twice in 0.5X SET/ 0.1% NaDodSO4 at 42°C for 45 min, respectively. Elution of the RNA hybridized was at 800C for 3 min with sterilized water

Initially, the existence of a class oflow molecular weight nuclear RNA species that differ from the small nuclear RNA (snRNA) molecules by number and size was described in HeLa cells (1). These RNA molecules can be labeled in isolated nuclei in vitro. Their synthesis is resistant to high concentrations of a-amanitin (200 Ag/ml) and they have therefore been called "small nuclear polymerase I RNA (snPI RNA)." HeLa cell snPI RNA represents a large number of distinct RNA molecules in the size range 6 S to 12 S with major products around 8 S. In contrast to ribosomal precursor RNA, the synthesis of snPI RNA is not inhibited by pretreatment of intact cells with low doses of actinomycin D, and subfractionation of nuclei seemed to indicate that the synthesis of snPI RNA is in the nucleoplasm rather than in the nucleolar fraction. Cell fractionation studies in combination with short-term labeling kinetics demonstrated that snPI RNA molecules also accumulate in vivo (1). snPI RNA molecules have been detected in all mammalian cell lines studied. In contrast to other low molecular weight RNA species, these molecules reveal a pronounced species specificity with an RNA pattern unique to each animal species but similar in different cell types within a species (2). This remarkable species specificity may be used to separate evolutionarily related organisms such as mouse and rat or the closely related species gorilla and man. Because no detailed information is available with respect to snRNA biosynthesis, including the question of whether or not these RNA species are synthesized via precursor molecules, we wanted to investigate a possible relationship between snPI RNA molecules and snRNA species. Indirect evidence also suggested that RNA polymerase I might be involved in snRNA transcription (3). In the present report we compare in vitro synthesized mouse snPI RNA species and two species of snRNA (Ul and U2) by

Abbreviations: snRNA, small nuclear RNA; snPI RNA, small nuclear polymerase I RNA; rDNA, DNA containing the genes for ribosomal RNA; kb, kilobase(s).

The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

* To whom reprint requests should be addressed.

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containing 0.5% NaDodSO4. Competition hybridization consisted ofprehybridization ofthe filters with unlabeled 45S rRNA precursor (35 pug/ml) for 24 hr followed by incubation with labeled snPI RNA for 18 hr as described above. To identify the sequences coding for snPI RNA, the rDNA ofclone Mr 974 was digested with Sal I and the fragments were separated in a 0.8% agarose gel. Transfer of the DNA fragments to nitrocellulose filters was as described by Southern (10) and hybridization with 32P-labeled snPI RNA was as above. The dried filters were exposed on Kodak X-Omat R films for 4 days with a Cronex intensifier screen. Experiments involving recombinant DNA were carried out under L2/B2 conditions in accordance with the German guidelines for recombinant DNA research.

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terns (Fig. 2 A and B) clearly differed from those obtained with snPI RNA species 1, 2, 3, and 5, indicating that both RNA populations do not represent common RNA sequences. The snRNA U1 and U2 patterns were similar to those published by other authors (12, 13) but different from those obtained with snPI RNA species. The simpler oligoribonucleotide pattern with snPI RNA is explained by the fact that snPI RNA molecules contain only one labeled nucleotide species (UMP). Furthermore, although differing in length by more than 100 nucleotides, the four snPI RNA species analyzed all reveal identical T1 RNase patterns, suggesting that they are composed of identical RNA sequences. The mouse snPI RNA species probably do not represent aggregates of the same molecules because reelectrophoresis of species 1-5 under denaturating conditions in gels containing 7 M urea (Fig. 1, lane 5) still resulted in distinct RNA bands with similar size differences. The patterns of longer snPI RNA molecules did not reveal additional oligonucleotides, suggesting that snPI RNA molecules are composed of repetitive nucleotide sequences. Because the snPI RNA species are transcribed by the same RNA polymerase that is involved in rRNA transcription and because rDNA is known to contain clusters of repetitive sequences associated with the nontranscribed spacer DNA separating the individual rDNA transcription units (14, 15), in vitro synthesized mouse snPI RNA molecules were hybridized to cloned mouse rDNA. The mouse rDNA clone AgtWES Mr 974, known to contain about two-thirds of the 18S rRNA gene, the external transcribed spacer, and about 6.7 kilobases (kb) of the nontranscribed spacer of mouse rDNA (7, 8), was used as a DNA source. snPI RNA molecules hybridized efficiently to this rDNA clone (Table 1). The hybridization of snPI RNA was not inhibited by an excess of unlabeled 45S rRNA precursor, although 45S rRNA hybridization was inhibited effectively, suggesting that hybridization occurs outside the region corresponding to the 45S RNA. It is interesting to note that HeLa cell snPI RNA molecules did not hybridize to the mouse rDNA clone. This is in good agreement with the finding that the "nontranscribed" ribosomal spacer shows extensive heterogeneity not only in length (14-17) but also in sequence (18-20) between

RESULTS Isolated mouse 3T6 fibroblast nuclei were used for in vitro transcription studies in the presence of various concentrations of aamanitin. Low molecular weight RNA species labeled in the presence of a-amanitin (200 jig/ml) (Fig. 1, lane 3) after pretreatment of the cells with low doses of actinomycin D to suppress transcription of ribosomal precursor RNA selectively (11) have been designated "snPI RNA molecules" (1, 2). These snPI RNA molecules do not arise as a consequence of the pretreatment with actinomycin D because they also were observed in the absence of this inhibitor (Fig. 1, lane 4), although the drug treatment resulted in enhanced synthesis of snPI RNA molecules. When compared to the corresponding RNA molecules obtained with HeLa cell nuclei (lanes 6 and 7), the mouse snPI RNA pattern clearly revealed prominent RNA species considerably smaller than the HeLa snPI RNA species. The mouse snPI RNA species migrated slightly differently from mouse snRNA species labeled in vivo (lane 1). In order to determine whether the in vitro synthesized snPI RNA molecules are related to the snRNA species and therefore might represent the corresponding snRNA precursors, T1 RNase digestion patterns of individual RNA species from both RNA populations were compared. The snRNA U1 and U2 pat-

FIG. 1. Electrophoresis of in vitro

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FIG. 2. T1 RNase digestionpatterns analysis of mouse snRNA and snPI RNA. (A and B) snRNA species U1 and U2 were isolated from nuclei after labeling of the cells with [32P]orthophosphate (25 pCi/ml) in "low-phosphate" (6 mg/liter) Dulbecco's medium for 36 hr. (C-F) snPI RNA species 1, 2, 3, and 5 synthesized in vitro in the presence of a-amanitin (200 ,ug/ml) with 25 jCi of [a-32P]UTP per assay. Individual bands were cut from the gels, eluted electrophoretically, and analyzed after T1 RNase digestion.

different animal species, analogous to results obtained with various mammalian snPI RNA species (2). When 32P-labeled snPI RNA was hybridized to DNA isolated from AgtWES Mr 974 clone and the RNA recovered from the hybrids was analyzed by gel electrophoresis, a similar snPI RNA pattern was obtained (Fig. 1, lanes 8 and 9). In order to determine the localization of sequences coding for snPI RNA within the Mr 974 clone, 32P-labeled snPI RNA was hybridized to DNA fragments generated by Sal I. The restriction map of AgtWES Mr 974 as elaborated by Grummt et aL (7, 8) is presented in Fig. 3. The two Sal I sites in the right arm and the four sites in the Table 1. Hybridization of snPI RNA to cloned mouse rDNA Hybridization probe cpm Hybridization probe cpm Mouse snPI RNA 3,069 Mouse 45S rRNA precursor 335 Mouse snPI RNA + Mouse 45S rRNA precursor unlabeled 45S + unlabeled 45S rRNA rRNA precursor precursor (35 pg/ml) 77 Mouse 45S rRNA precursor (35 ,ug/ml) 2,978 HeLa snPI RNA 17 (blank filter, loaded with Mouse snPI RNA E. coli DNA) 58 (blank filter) 14 3H-Labeled snPI RNA (50,000 cpm in a total volume of 300 /4) or 32P-labeled 45S rRNA (30,000 cpm) was hybridized to duplicate filters loaded with 5 gg of mouse Mr 974 DNA.

insert will lead to a shortened right arm (12.6 kb), the left arm (23.25 kb) carrying about 1,950 nucleotides of the 18S gene, a 0.48-kb fragment from the right arm, and four additional fragments-A (4.7 kb), B (3.3 kb), D (1.75 kb), and E (0.57 kb)representing external transcribed and nontranscribed ribosomal spacer sequences. In Fig. 4, lane A shows the expected Sal I restriction fragments A, B, and D in addition to the left and right arms. The two smaller fragments, 0.57 and 0.48 kb, were not detectable because this region of the gel is obscured by a smear of low molecular weight material. This material originates from contaminating E. coli nucleic acids because in this experiment the A phages were purified by only one cesium chloride centrifugation step. snPI RNA hybridized only to the 3.3-kb Sal I fragment B consisting of about 600 nucleotides of nontranscribed ribosomal spacer and about 2.7 kb ofexternal transcribed spacer (Fig. 4, lane B). Nucleotide, sequence analysis of the initiation region of the ribosomal transcription unit from mouse has revealed a Pvu II site upstream from the 5' end of the 45S rRNA precursor at position -157 (8). Accordingly, if purified Sal I fragment B is digested with Pvu II, two fragments can be resolved, a smaller one of 461 nucleotides consisting entirely of nontranscribed spacer sequences and a larger one ofabout 2.8 kb including most of the external transcribed spacer and the preceding 156 nucleotides of nontranscribed spacer DNA (Fig. 5, lane C). Hybridization to the cor-

Biochemistry: Reichel et aL

Proc. Natl. Acad. Sci. USA 79 (1982) 0,57

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responding Southern blot demonstrated that both fragments contain DNA sequences complementary to snPI RNA (Fig. 5, lane D). Hybridization was not due to some random base pairing because treatment of the hybrid filters with pancreatic RNase

(Fig. 5, lane E) had no effect. In order to demonstrate that snPI RNA does not hybridize to the external transcribed spacer sequence, blots were prehybridized with a large excess (35 ,tg/ ml) of 45S rRNA precursor. This did not prevent hybridization of snPI RNA to either fragment (Fig. 5, lane F). Therefore, hybridization to the 2.8-kb fragment containing the external transcribed spacer must be due to the 156-nucleotide sequence of nontranscribed spacer DNA within this fragment. Together, these results demonstrate that snPI RNA molecules are transcribed from the so-called nontranscribed ribosomal spacer of the mouse rDNA. M

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DISCUSSION The structure of in vitro synthesized mouse snPI RNA molecules has been analyzed by T1 RNase digestion patterns and these patterns were compared to the oligonucleotide patterns obtained with snRNA species U1 and U2 ofabout the same size. It is shown that these classes of snRNA molecules are not related to each other. This is also true for snRNA U3 because HeLa cell U3 RNA does not hybridize to a HeLa rDNA clone containing snPI RNA genes (unpublished data). Conversely, the failure of HeLa cell snPI RNA to hybridize to cloned snRNA genes isolated from a human gene library in our laboratory (unpublished data) also rules out a relationship between these classes of low molecular weight RNA. To date, snPI RNA molecules are characterized by three remarkable findings: (i) these molecules are synthesized by RNA polymerase I, the enzyme involved in ribosomal precursor RNA synthesis; (ii) snPI RNA molecules are transcribed from repetitive DNA sequences; and (iii) these low molecular weight nuclear RNA species reveal an extensive divergence between different animal species, even among evolutionarily related organisms. The latter two properties are shared by the external

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FIG. 4. Separation of restrictionfragments of clone Mr 974 in 0.8% gels and hybridization with snPI RNA. Lanes: M, A marker digested with Hindu; A, Sal I restriction fragments of Mr 974; B, hybridization of 32P-labeled in vitro synthesized mouse snPI RNA to Southern blots of A. agarose

FIG. 5. Hybridization of snPI RNA to Southern blots of the 3.3-kb Sal I fragment digested with Pvu H. Lanes: A, A marker digested with HindI; B, reelectrophoresis of the 3.3-kb Sal I fragment of Mr 974; C, 3.3-kb Sal I fragment digested with Pvu II; D, hybridization of snPI RNA to Southern blots of the Pvu II fragments; E, as D but the hybridized filters were treated with pancreatic RNase (50 ,ug/ml) for 30 min at 37°C; F, as E but hybridization was in the presence of 45S rRNA precursor (35 yg/ml).

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nontranscribed spacer sequences associated with the ribosomal genes (14-22). In addition, the involvement of RNA polymerase I further points to a possible relationship of snPI RNA transcription and ribosomal genes. These findings prompted us to look for sequence complementarity between snPI RNA and rDNA including nontranscribed spacer sequences. An appropriate mouse rDNA clone (AgtWES Mr 974) has been isolated and extensively characterized by Grummt et al (7, 8). The results presented here demonstrate that nontranscribed spacer sequences immediately adjacent to the 5' end of the 45S transcription unit indeed code for snPI RNA molecules. The DNA sequence of this region has been determined recently (8). From this it can be predicted that the predominant spots 5 and 6 in Fig. 2 correspond to the sequences [C-U]G and [C-U-U]G, respectively, which are present in 9 and 7 mol per 300 nucleotides on the 5' side of the 45S rRNA gene. The patterns thus support our idea that the snPI RNA is unrelated to other snRNA genes including the gene for U3 RNA (23) which contains only 2 mol of each of these oligonucleotides. The snPI RNA molecules must be rapidly exported from the nucleolus because nuclear fractionation studies as well as electron microscopic autoradiography showed an almost exclusive association of snPI RNA with the nucleoplasmic fraction of HeLa cells (1). Our finding that parts of the so-called nontranscribed ribosomal spacers are actively transcribed is in good agreement with electron microscopic studies by Scheer et aL (24, 25) who observed transcription complexes and nascent RNA chains associated with nontranscribed spacer regions of Xenopus oocytes. In addition, by using cloned rDNA exclusively containing nontranscribed spacer sequences, Rungger et aL (26) identified RNA transcripts complementary to the central parts of the nontranscribed rDNA spacer in cultured Xenopus epithelial cells. These transcripts were found to represent a heterogeneous RNA population with a size between 5 and 23 S. HeLa cell snPI RNA and mouse snPI RNA molecules have similar size distributions when analyzed in sucrose gradients (ref. 1; unpublished data). However, it is not clear at present whether the RNA molecules identified in Xenopus cells and the snPI RNA decribed here belong to the same class of RNA molecules because, up to now, the synthesis and structure of the former RNA population have not been characterized biochemically. In addition, in contrast to the results obtained by Rungger et aL, even with the larger snPI RNA molecules we do not observe any hybridization to sequences of the nontranscribed rDNA spacer further away from the 45S transcription unit-for example, Sal I fragment D (not shown). The most intriguing question remaining concerns the physiological role of snPI RNA molecules. One possible explanation could be that, instead of using an initiation site at the 5' end of the 45S transcription unit, initiation of RNA polymerase I also takes place at various sites upstream from this position and snPI RNA molecules are generated by cleavage of such a putative rRNA pre-precursor molecule during a very early processing step. The postulation of new initiation sites for polymerase I is consistent with the failure to detect a 5'-terminal triphosphate on mammalian 45S rRNA precursor molecules (27, 28). In addition, Bach et aL (8) found only a minor fraction of 45S rRNA precursor (about 10-15%) capable of accepting a cap structure supplied by the vaccinia virus capping enzyme in vitro. This indicates that only a limited amount of rRNA precursor molecules are bearing a polyphosphate at the 5' terminus. The additional initiation sites for RNA polymerase I postulated here might explain why, after inactivation of the ribosomal genes by low doses of actinomycin D, increased amounts of snPI RNA molecules accumulate, possibly as a result of a shift of the major

initiation site to a position (or positions) located upstream in the nontranscribed ribosomal spacer. Another possibility could be that snPI RNA molecules are transcribed independently and are involved in rRNA precursor processing, comparable to the role of snRNA in the processing of heterogeneous nuclear RNA. Finally, it should be mentioned that snPI RNA genes are not representing a class of reiterated sequences dispersed throughout the genome-for example, like theAlu family sequences-because restriction ofgenomic DNA and hybridization of snPI RNA to the DNA fragments shows only a single DNA band hybridizing to snPI RNA. This is the same band that also hybridizes with 45S rRNA precursor (data not shown). After submission ofthis paper it has been reported from several laboratories (29-31) that the initiation site for 45S rRNA transcription is located in the nontranscribed region within the Pvu II fragment. We thank Dr. I. Grummt (Wtirzburg) for kindly providing the mouse

rDNAclone Mr 974 and Prof. 0. Pongs (Bochum)forprovidingresearch facilities. This work was supported by a grant from the Stiftung Volkswagenwerk. The work in Uppsala was supported by research grants from the Swedish Natural Science Research Council, the Swedish Society against Cancer, and a European Molecular Biology Organization Short-Term Fellowship to R.R. 1. Benecke, B. J. & Penman, S. (1977) Cell 12, 939-946. 2. Benecke, B. J. & Penman, S. (1979) J. Cell Biol 80, 778-783. 3. Zieve, G., Benecke, B. J. & Penman, S. (1977) Biochemistry 16, 4520-4525.

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4239-4250. 7. Grummt, I., Soellner, C. & Scholz, I. (1979) Nucleic Acids Res. 6, 1351-1369. 8. Bach, R., Grummt, I. & Allet, B. (1981) Nucleic Acids Res. 9, 1559-1569. 9. Denhardt, D. T. (1966) Biochem. Biophys. Res. Commun. 23, 641-646. 10. Southern, E. M. (1975) J. Mot. Biot 98, 503-518. 11. Perry, R. P. (1962) Proc. Natl Acad. Sci. USA 48, 2179-2183. 12. Reddy, R., Ro-Choi, T. S., Henning, D. & Busch, H. (1974) J. Biol. Chem. 249, 6486-6494. 13. Lerner, M. R. & Steitz, J. A. (1979) Proc. Natl Acad. Sci. USA 76, 5495-5499. 14. Wellauer, P. K., Dawid, I. B., Brown, D. D. & Reeder, R. H. (1976) J. Molt Biot 105, 461-486. 15. Wellauer, P. K., Reeder, R. H., Dawid, I. B. & Brown, D. D. (1976) J. Mot Biot 105, 487-505. 16. Wellauer, P. K. & Dawid, I. B. (1977) Cell 10, 193-212. 17. Krystal, M. & Arnheim, N. (1978)J. Mot Biol 126, 91-104. 18. Arnheim, N. & Southern, E. M. (1977) Cell 11, 363-370. 19. Cory, S. & Adams, J. M. (1977) Cell 11, 795-05. 20. Wellauer, P. K. & Dawid, I. B. (1979)J. Mol Biot 128, 289-303. 21. Wellauer, P. K. & Dawid, I. B. (1978)J. Mot Biot 126, 769-782. 22. Fedoroff, N. & Brown, D. D. (1978) Cell 13, 701-716. 23. Reddy, R., Henning, D. & Busch, H. (1979) J. Biot Chem. 254, 11097-11105. 24. Scheer, U., Trendelenburg, M. F. & Franke, W. W. (1973) Exp. Cell Res. 80, 175-190. 25. Scheer, U., Trendelenburg, M. F. & Franke, W. W. (1977) Chromosoma 60, 147-167. 26. Rungger, D., Achermann, H. & Crippa, M. (1979) Proc. Natt Acad. Sci. USA 76, 3957-3961. 27. Choi, Y. C. & Busch, H. (1970)J. Biot Chem. 245, 1954-1961. 28. Kominami, R. & Muramatsu, M. (1977) Nucleic Acids Res. 4, 229-240. 29. Grummt, I. (1981) Nucleic Acids Res. 9, 6093-6102. 30. Mishima, Y., Yamamoto, O., Kominami, R. & Muramatsu, M. (1981) Nucleic Acids Res. 9, 6773-6785. 31. Miller, K. G. & Sollner-Webb, B. (1981) Cell 27, 165-174.