Transcription of spacer sequences flanking the rat 45S ribosomal DNA ...

Vol. 7, No. 1

MOLECULAR AND CELLULAR BIOLOGY, Jan. 1987, p. 314-325

0270-7306/87/010314-12$02.00/0 Copyright © 1987, American Society for Microbiology

Transcription of Spacer Sequences Flanking the Rat 45S Ribosomal DNA Gene CHRISTINA A. HARRINGTON* AND DONA M. CHIKARAISHI Neuroscience Program, Tufts University Medical School, Boston, Massachusetts 02111 Received 9 June 1986/Accepted 20 October 1986 The transcriptional activity of spacer sequences flanking the rat 45S ribosomal DNA (rDNA) gene were studied. Nascent RNA labeled in in vitro nuclear run-on reactions hybridized with both 5' and 3' spacer regions. The highest level of hybridization was seen with an rDNA fragment containing tandem repeats of a 130-base-pair sequence upstream of the 45S rRNA initiation site. Synthesis of RNA transcripts homologous to this internally repetitious spacer region was insensitive to high levels of a-amanitin, suggesting that it is mediated by RNA polymerase I. Analysis of steady-state RNA showed that these transcripts were present at extremely low levels in vivo relative to precursor rRNA transcripts. In contrast, precursor and spacer run-on RNAs were synthesized at similar levels. This suggests that spacer transcripts are highly unstable in vivo; therefore, it may be the process of transcription rather than the presence of spacer transcripts that is functionally important. Transcription in this upstream rDNA region may be involved in regulation of 45S rRNA synthesis in rodents, as has been suggested previously for frog rRNA. In addition, the presence of transcriptional activity in other regions of the spacer suggests that some polymerase I molecules may transcribe through the spacer from one 45S gene to the next on rodent rDNA.

Mammalian ribosomal DNA (rDNA) is organized in tandem repeats at a few chromosomal loci for a total of 100 to 250 copies per haploid genome (reviewed in reference 23). Each repeat consists of approximately 14 kilobases (kb) of DNA encoding the 45S precursor rRNA and 20 to 30 kb of spacer region termed nontranscribed spacer (NTS). Internally repetitious elements in the NTS flank both the 5' and 3' ends of the 45S gene in rodent rDNA (1, 8, 12, 21). The 3' repeat pattern has been identified by the presence of multiple Sall restriction sites within 1 kb of the 28S rRNA coding region. It has recently been shown that rDNA transcription in mice terminates adjacent to this downstream repetitious region and that this region appears to contain information required for polymerase I (pol I) transcription termination (22). The 5' repeating element in both rats (12) and mice (28) is approximately 130 base pairs (bp) long and is bounded by variable-length tracts of T's. This repetitious region terminates approximately 210 bp upstream from the initiation site for 45S rRNA synthesis. Variation in the number of repeats in this region is responsible for major polymorphisms in both species (1-3, 12). No function has yet been attributed to the 130-bp upstream elements, although it has been suggested that such repetitious regions may arise during recombination of tandemly repeated genes and, perhaps, facilitate this process (16; also see reference 48 for a discussion of NTS function). Repetitious regions flanking the initiation site for precursor rRNA synthesis have also been extensively described in nonmammals. In Xenopus spp., the repeated elements contain functional duplications of the RNA pol I promoter, as well as multiple copies of a 60- or 81-bp element that is partially homologous to the promoter region (5, 6, 35, 36, 45). Serial repeats of a 240-bp element in Drosophila melanogaster also contain sequence homology to the pol I promoter and can support in vitro transcription (14, 26). Low-level transcription from these regions in both frogs (18, 34, 36, 40) and D. melanogaster (32) can be detected in vivo.

Moss (35) has suggested that transcription in the Xenopus NTS is functionally related to precursor rRNA synthesis. In his model, ribosomal spacer serves as a loading site for pol I molecules that are then delivered to the rDNA gene promoter by the process of transcription. At the same time, Reeder and his colleagues (10, 29, 38, 39) have evidence that indicates that the 60/81-bp repeated elements adjacent to the pol I promoters act as orientation-independent enhancers. In a recent report, De Winter and Moss (15) presented evidence that a functional spacer promoter is required for maximal pre-rRNA synthesis but also suggested that enhancement of transcription by the 60/81-element is equally important. Analysis of the in vitro template activity of deletion or point mutations of mouse rDNA has delineated the rodent pol I promoter. A DNA sequence from -39 to +9 (with +1 being the first base of 45S rRNA) is sufficient for pol I promoter function (20, 33, 44, 48, 51). However, maximal promoter activity requires sequences up to -149 when transcription is assayed under suboptimal conditions or in competition with wild-type templates (20, 33). Analysis of the 130-bp repeating elements that extend upstream from approximately -210 in the 5' NTS revealed no extensive sequence homology to the rodent pol I promoter (28; unpublished data), unlike the situation in Xenopus spp. and D. melanogaster. Nonetheless, we found that transcripts homologous to the 130-bp repeat are present in rat RNA. These transcripts are present in relatively high concentrations in nascent RNA but apparently are rapidly degraded in vivo. These results suggest that transcription of tandemly repetitious sequences upstream of the precursor rRNA promoter may also play a role in mammalian rDNA transcription. MATERIALS AND METHODS Cell culture. H35 cells, a gift of John Koontz, are a clone of the H4-EII-C3 rat hepatoma cell line designated Reuber H35, clone KRC-7 (27). RT4-D1 cells are a diploid glioma line isolated from a chemically induced rat neurotumor (25). Cells were grown in Dulbecco modified Eagle medium plus 50 U of penicillin per ml and 50 jig of streptomycin per

* Corresponding author. 314

VOL. 7, 1987

ml. H35 cells were supplemented with 5% newborn calf serum and 5% fetal calf serum. RT4-D1 cells were supple'nented with 4% newborn calf serum and 1% fetal calf serum. Phage and plasmids. All rDNA recombinants used in this work with the exception of M13-SS1.1 have been previously described (12, 24) (see Fig. 1). To generate SS1.1+ and SS1.1-, the 1.1-kb SstI-SalI fragment (-2.86 to -1.76) of pEH 5.0 was gel purified and ligated into the SstI-SalI sites of M13mplO and M13mpll. rDNA recombinants were identified by hybridization with 32P nick-translated pEH5.0. Plasmid and single-stranded M13 phage DNAs were prepared as previously described (24). Isolation of nuclei. Tissue culture cells were washed twice with phosphate-buffered saline, and the cells were scraped from the plate in buffered sucrose (SB; 0.22 M sucrose, 50 mM Tris [pH 7.6], 10 mM NaF, 1 mM MgCl2, 2 mM P-mercaptoethanol). Cells were pelleted by low-speed centrifugation and suspended in 1 to 2 ml of SB per 107 cells. Nonidet P-40 and Triton X-100 were added to 0.5% each. Cells were lysed by 10 to 20 strokes in a Dounce homogenizer. The suspension was layered over a 0.35 M sucrose cushion containing 25 mM Tris (pH 7.9), 6 mM NaF, 6 mM ,-mercaptoethanol, and 1 mM MgC12. Nuclei were pelleted by centrifugation at 4,100 x g for 4 to 5 min. Nuclei were prepared from Sprague-Dawley (SD) rat liver or brain by two cycles of motor-driven homogenization of chopped tissue in SB plus detergents, followed centrifugation through a sucrose cushion as described above. RNA preparation. RNA was prepared by the lithium chloride-urea precipitation method (4). Phosphate-buffered saline-washed cells or isolated nuclei were suspended in 3 M LiCl-6 M urea-10 mM vanadyl ribonucleoside (Bethesda Research Laboratories or Life Sciences) at a concentration of approximately 107 cells per ml. To reduce viscosity, samples were sonicated briefly with a Branson sonifier with a microtip. The suspensions were kept on ice for 24 to 48 h and then centrifuged for 10 min at maximum speed (11,600 x g) in a Beckman Microfuge 11 or at 10,000 rpm (16,490 x g) in a Sorvall centrifuge with an HB-4 rotor. Supernatants were removed, and the pellets were suspended in 10 mM Tris (pH 7.5)-10 mM EDTA-0.2% NaDodSO4 (SDS). After three extractions with phenol-chloroform-isoamyl alcohol (24:24:1) plus 0.1% hydroxyquinoline, sodium acetate was added to 0.1 M and the RNA samples were precipitated with 2.5 volumes of ethanol. The precipitated pellets were suspended in 50 RI of 10 mM Tris (pH 7.5)-0.5 mM EDTA (TE) and then diluted with 10 to 20 volumes of 3 M LiCl-6 M urea. Samples were again kept on ice overnight and then centrifuged as described above. After one or two phenolchloroform extractions, the samples were ethanol precipitated. The pellets were suspended in TE, and concentrations were determined by measuring A260Brain nuclear RNA was prepared by lysing nuclei in 6 M guanidinium thiocyanate and pelleting RNA through a 5.7 M CsCl solution (13). Labeling of nascent RNA (nuclear run on). Freshly isolated nuclei from H35 cells were suspended at approximately 108 nuclei per ml in 100 ,u of transcription buffer containing 20 mM Tris (pH 7.9), 50 mM (NH4)2SO4, 5 mM MgCI2, 6 mM NaF, 6 mM P-mercaptoethanol, 0.25 mM aurin tricarboxylic acid, and 0.075% Sarkosyl (in some reactions, MnCl2 was included at 2 mM and heparin was included at 1 mg/ml). a-amanitin (Sigma) was added to 100 or 200 ,ug/ml to inhibit polymerase II and polymerase III activities (42), or an equal volume of H20 was added, and samples were briefly vortexed. [a-32P]CTP or [a-32P]UTP (700 to 800 Ci/mmol;

rDNA UPSTREAM TRANSCRIPTION

315

New England Nuclear Corp.) plus cold ribonucleoside triphosphates were added to a final concentration of 17,uM [32P]CTP or [32P)]UTP (120 to 125 Ci/mmol) and 300 ,uM (each) ATP, GTP, and either UTP or CTP. Elongation reactions were incubated in a 37°C water bath for 10 min. The reaction was terminated by addition of an equal volume of quench buffer (0.2 M NaCl, 20 mM Tris [pH 7.5], 1% SDS, 0.25 mM aurin tricarboxylic acid, 50 mM EDTA) plus 125 to 150 ,Ig of proteinase K (Sigma) per ml. Samples were incubated for 10 min at 37°C, phenol-chloroform extracted twice, and ethanol precipitated at -20°C. Dried ethanol precipitates were suspended in 200 ,u of 10 mM NaCl-10 mM MgC12-10 mM Tris (pH 7.5)-0.5 mM aurin tricarboxylic acid. Affinity-purified (31) DNase I (Worthington; 5 to 10 ,uig) was added, and samples were incubated at 37°C for 15 min. Then 5 ,lI of 10% SDS and 25 ,ug of proteinase K were added and incubation was continued for 10 min. The samples were extracted twice with phenol-chloroform and applied to a Sephadex G50 column equilibrated in 0.4 M NaCl-10 mM Tris (pH 7.5)-i mM EDTA-0.1% SDS. The excluded peak fractions were pooled, ethanol precipitated, and suspended in TE. Incorporation in the presence of ot-amanitin ranged from 0.7 to 1.5 cpm per cell. Incorporation in the absence of a-amanitin was approximately 1.5 to 3-fold greater depending on whether or not MnCl2 was included in the reactions. MnCl2 stimulates polymerase II and polymerase III activities (42). Hybridization with labeled, nascent RNA. Plasmid DNA was cleaved with the indicated restriction enzymes and transferred to nitrocellulose by the method of Southern (46). After baking and prehybridization, the filter was annealed with 0.5 x 106 to 1.0 x 106 cpm of run-on RNA in 10 ml of 3x SSPE (lx SSP; 0.18 M NaCl, 10 mM NaPO4 [pH 7.7], 1 mM EDTA)-150 jig of heparin per ml-0.1% sodium pyrophosphate-100 jig of tRNA per ml-0.2% SDS at 63°C for 48 h. After hybridization, the filter was washed briefly in lx SSC (0.15 M NaCl, 0.015 M sodium citrate, pH 7.0)-0.1% SDS at room temperature, once in 1 x SSC-0.1% SDS for 40 min at 65°C, and once in 0.2x SSC-0.1% SDS for 40 min at 65°C. After a brief rinse in 2x SSC, the filter was treated with 20 jig of RNase A (Boehringer Mannheim Biochemicals) in 200 ml of 2x SSC at room temperature for 30 min. The filter was then treated with 50 jig of proteinase K and rinsed briefly in lx SSC-0.1% SDS followed by 0.2x SSC-0.1% SDS. The filter was blotted dry and exposed to Kodak XAR-5 film with an intensifying screen. Solution hybridizations were performed with individual single-stranded M13-rDNA recombinants and the indicated amount of labeled nascent RNA. Hybridizations were calculated to be in DNA excess. Two micrograms of each

recombinant DNA was incubated with in vitro run-on RNA in 50 to 75 jIl of 2.8 x SSPE plus 3H-labeled M13 cRNA as an internal control for hybridization efficiency. Samples were incubated overnight at 63 to 64°C. Hybridized samples were processed as follows. (i) Cold 2x SSC (1 ml) was added to each Microfuge sample tube; (ii) this solution was transferred to a larger tube containing 3 ml of 2 x SSC; (iii) the samples were slowly filtered through Schleicher & Schuell 24-mm BA85 nitrocellulose filters prewet in 2 x SSC by using a Hoefer filtering apparatus; (iv) filters were washed with 10 ml of 2x SSC and then transferred to glass scintillation vials containing 3 ml of 2x SSC; (v) approximately 100 jil of an RNase solution containing 200 jig of RNase A-150 U of RNase Ti per ml was added, and the samples were incubated at 37°C for several hours; (vi) the RNase solution was removed, and the filters were washed with 2x SSC-0.1%

316

HARRINGTON AND CHIKARAISHI

MOL. CELL. BIOL.

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FIG. 1. (A) Restriction map of the regions surrounding the 5' and 3' termini of the rat DNA gene. The hatched box represents rDNA transcribed into 45S precursor rRNA. The arrow at zero corresponds to the initiation site for the 45S molecule. Approximately 15 to 20 kb of additional spacer extends beyond the EcoRI site at + 17.2 to the EcoRI site at -4.88 kb of the next rDNA repeat. pEH5.0 and pEE4.6 are pBR322 and pBR325 recombinants, respectively, containing the indicated regions of rDNA. (B) Region surrounding the 5' terminus of the rDNA gene. M13 recombinants are shown below the restriction map. Single-stranded recombinants with the same strandedness as 45S rRNA are designated plus (+); opposite strands (coding strand) are designated minus (-).

SDS followed by 0.2x SSC-0.1% SDS; and (vii) filters were dried overnight and counted in 5 ml of Omnifluor (New England Nuclear). RNA dot hybridizations. Single-stranded M13 recombinants were digested with micrococcal nuclease and end labeled with [y-32P]ATP as previously described (12). RNA samples were denatured in formaldehyde, serially diluted by one-third in 15x SSC, and applied to nitrocellulose (49) by using a Bethesda Research Laboratories hybridot manifold. Filters were baked and prehybridized in 3 x SSPE-0.2% SDS-100 ,ug of heparin per ml-0.1% sodium pyrophosphate-50% formamide-50 p.g of tRNA per ml. Labeled rDNA recombinants (4 x 106 cpm) were hybridized to the RNA dots in 5 to 6 ml of hybridization buffer for 48 h at 50°C. The filters were washed once at room temperature in 2 x SSC-0.1% SDS, once in 0.1 x SSC-0.1% SDS, and twice for 30 min each time in 0.1x SSC-0.1% SDS at 65°C. Transcription mapping with single-stranded DNA. M13rDNA recombinants were digested with HaeIII, end labeled by exchange reaction with [y-32P]ATP (ICN Pharmaceuticals, Inc.; 7,000 Ci/mmol), and annealed with the indicated amount of cold RNA. Hybridization and processing were as previously described (9) except that approximately 10 times as much probe (20 to 40 ng) was used. Genomic Southern blots. EcoRI- or SstI-HindIII-digested H35 DNA (approximately 3 ,ug per lane) was fractionated on 1.1% agarose gels and transferred to Zetabind (AMF) as described by Southern (46). Filters were prehybridized and hybridized in 3x SSPE-100 gxg of heparin per ml-0.1% sodium pyrophosphate-0.2% SDS at 65°C. End-labeled M13 single-stranded DNA (107 cpm) was added to each hybrid-

ization. Filters were washed as described above for RNA dot hybridizations. RESULTS Southern hybridizations with nascent transcripts labeled in vitro. We investigated nascent transcripts from the rDNA gene prior to the processing events that produce the stable rRNA species (28S, 18S, and 5.8S) and degrade the remaining precursor transcript. For this purpose, nascent RNA from a cultured hepatoma line, H35, was labeled by incubating isolated nuclei in the presence of [a-32P]UTP or [o32P]CTP and cold ribonucleoside triphosphates as described in Materials and Methods. The presence of Sarkosyl (19) and aurin tricarboxylic acid (41) in the nuclear lysate ensured that only previously initiated RNA polymerases were active in this system. After in vitro elongation, RNA was purified from DNase I-treated nuclear preparations. Labeled RNA was hybridized with Southern blots of ribosomal DNA recombinants pEH5.0 and pEE4.6, which contain the 5' and 3' termini, respectively, of the 45S precursor and adjacent spacer regions (Fig. 1A). In Fig. 2A, results of hybridization with [32P]UTP-labeled run-on RNA are shown. Lane 1 shows the ethidium bromide-stained gel of pEH5.0 cleaved with Sall and SstI. The DNA fragment at 0.92 kb contains 290 bp of rDNA (-0.168 to +0.125) of which 125 bases are transcribed into 45S precursor RNA (24); the remainder of the fragment is vector DNA. The 1.6-kb fragment contains the tandemly repeated 130-bp element of the spacer (-1.76 to -0.168), and the 1.1-kb fragment contains the adjacent spacer region from


VOL. 7, 1987

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3 4 2 6 5 1 FIG. 2. Southern blot hybridization of pEH5.0 and pEE4.6 with 32P-labeled run-on H35 RNA. Panel A shows the results of hybridization with [ac-32P]UTP-labeled RNA. Lane 1 is the ethidium bromide-stained gel pattern of 3 jig of pEH5.0 cleaved with SstI and SalI that corresponds to the Southern blots shown in lanes 2, 3, and 4. Lane 2, pEH5.0 hybridized with 106 cpm of run-on H35 nuclear RNA. Lane 3, pEH5.0 hybridized with 4 x 105 cpm of run-on H35 nuclear RNA synthesized in the presence of 100 ,ug of a-amanitin per ml. Panel B shows the results of hybridization with [a-32P]CTPlabeled RNA elongated in the presence of 200 jig of a-amanitin per ml. Lanes 4 and 5 contain pEH5.0 and pEE4.6, respectively, hybridized with 6 x 105 cpm of run-on H35 nuclear RNA. Lane 6 is the ethidium bromide-stained gel pattern of 3 ,ug of pEE4.6 cleaved with HindlIl and PvuII that corresponds to the Southern blot in lane 5. The numbers indicate molecular size in kilobases.

-2.86 to -1.76. The large 5.6-kb fragment contains the rDNA region from -4.88 to -2.86 plus 3.7 kb of pBR322. (The faint band between 5.6 and 1.6 kb is due to incomplete Sall digestion.) Lane 2 shows the hybridization pattern obtained when RNA from nuclei labeled in the absence of a-amanitin was annealed with a Southern blot of the DNA seen in lane 1. All of the rDNA fragments hybridized to the run-on RNA, with the 1.6-kb band exhibiting the strongest signal. Hybridizations with the 1.1- and 0.92-kb bands were weak but clearly detectable in the original autoradiogram. When a-amanitin was added to the nuclear run-on reaction at 100 ,ug/ml (lane 3), the pattern of hybridization remained essentially the same except for the region between -4.88 and -2.86. This latter region contains DNA sequences homologous to highly repeated sequences found interspersed elsewhere in the genome (7, 8, 12, 17, 37, 52, 53) that are transcribed by polymerases II and III. When these two polymerase activities were inhibited by a-amanitin, hybridization with the -4.88 to -2.86 region was reduced to a level comparable to that seen with the 1.1-kb region from -2.86 to -1.76 which does not contain highly repeated DNA. In a separate experiment, nascent RNA that was labeled in the presence of 200 jig of a-amanitin per ml and [32P]CTP was hybridized to the same restriction fragments of pEH5.0. The 1.6-kb fragment hybridized strongly with the run-on RNA even at this high a-amanitin concentration, indicating that it is transcribed by RNA pol I (Fig. 2B, lane 4). Figure 2B, lane 5, shows the hybridization pattern obtained when pEE4.6 cleaved with PvuII and Hindlll was annealed with the nuclear run-on RNA labeled in the presence of 200 gig of a-amanitin per ml. The restriction fragments generated by this cleavage can be seen in lane 6. The

317

2.3-kb fragment contains rDNA sequence from + 12.6 to + 14.8 (approximately 1 kb of precursor rRNA encoding region) plus a small amount of vector DNA. As expected, it hybridized with nascent RNA. The 1.05- and 0.95-kb fragments contain rDNA from +14.8 to +15.85 and +15.85 to + 16.8, respectively. Positive hybridization signals were observed with both of these fragments. A very faint signal was seen with the 1.7-kb fragment, which contains the sequence from +16.8 to +17.2 of rDNA plus pBR325 sequence. The 2.6- and 2.0-kb fragments were derived solely from pBR325 and did not hybridize with the labeled RNA. The intensity of the signal seen with the 2.3-kb fragment was greatly reduced by the high levels of unlabeled nuclear ribosomal RNA that competed in these hybridizations. When solution hybridizations (as described below) were performed such that the rDNA recombinants were in excess over nuclear precursor rRNA, the counts bound to the 3' precursor region were around 100-fold greater than those bound to an equivalent length of spacer downstream of + 14.8 in pEE4.6 (unpublished data). Solution hybridization with nascent transcripts labeled in vitro. To quantitate spacer region transcripts more accurately and determine which strand of the rDNA was being transcribed, radioactive nascent RNA, prepared as described above, was annealed with single-stranded M13 recombinants containing various rDNA fragments. After solution hybridization in DNA excess at 63°C overnight, samples were filtered through nitrocellulose filters. Single-stranded M13 molecules bind the nitrocellulose, and RNA is retained by virtue of its hybridization with complementary regions of the rDNA recombinants. After washing and RNase treatment, counts bound to M13 recombinants and attached to the filters were determined by scintillation counting. Solution hybridization was used for these experiments instead of filter hybridization (which has a lower background) because of the difficulty of maintaining high enough rDNA concentrations to drive all of the precursor and stable rRNA species into hybrid molecules. In actively growing cells, rRNA can account for as much as 40% of the total RNA in the nucleus. The transcriptional activities of rDNA sequences encoding 45S rRNA and of regions upstream of the initiation site for precursor rRNA synthesis were analyzed. Table 1 shows the results for hybridizations of run-on nuclear RNA from two different H35 nuclear preparations with a set of M13rDNA recombinants; HX.51 encodes 510 bases of 45S rRNA, and SS1.6 contains the upstream, repeated 130-base elements (Fig. 1B). The data are expressed in three ways. Column 3 shows disintegrations per minute bound after a control filter containing nonrecombinant M13 DNA had been subtracted out; column 4 presents disintegrations per minute bound normalized to the length of the rDNA fragment; and column 5 lists disintegrations per minute per kilobase divided by the number of cells giving rise to the input amounts of labeled RNA. In this latter column, hybridization is standardized to cell number rather than to total transcriptional activity because it permits direct comparison of hy-

bridized RNA synthesized in the presence or absence of a-amanitin, since in the former case a much larger fraction of the labeled RNA is actually ribosomal. The final column shows the ratio of counts bound to SS1.6- and HX.51-. As expected, nascent RNA hybridized with HX.51-, which contains the coding strand for 45S rRNA, and the absolute levels of hybridization were similar in both the presence and absence of a-amanitin. Considerable hybridization was also seen with the SS1.6- recombinant, which would hybridize to RNA synthesized from the same strand

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MOL. CELL. BIOL.


TABLE 1. Hybridization of 32P-labeled nascent RNA with rDNA-M13 recombinants No. of dpm/kb No of No. of dpm Input dpm DNAa Labeling conditions per 101 cells bound dpmlkb Lablincndiis(104) Expt 1b [32P]UTP

[32P]UTP + a-amanitin

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[32P]CTP + a-amanitin

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0 52 384 256

0 102 240 502

0 88 207 433

0.48

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17 12 350 243

11 24 219 476

9 20 189 410

0.46

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0 0 394 228

0 0

246 447

0 0 129 235

0.55

2 3 323 187

1 6 202 367

1 4 106 192

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A minus (-) denotes a DNA strand that anneals to rRNA; a plus (+) denotes a DNA strand with the same polarity as rRNA. The RNA hybridized in experiment 1 was labeled with [32P]UTP in the presence of 2 mM MnCl2 in addition to the standard in vitro elongation buffer. c The RNA hybridized in experiment 2 was labeled with [32P]CTP, and no MnCl2 was added.

a

b

as 45S RNA. The level of hybridization with SS1.6- was similar to that seen with HX.51-; when normalized to rDNA fragment length, approximately half as many counts were bound to SS1.6- as to HX.51-. The number of counts bound to the opposite strand of each of these rDNA fragments was low or undetectable. Significantly, the ratio of SS1.6- to HX.51- in either the presence or absence of 100 ,ug of a-amanitin per ml stayed the same. This suggests that transcription from SS1.6, as well as HX.51, is mediated by pol I. Solution hybridization experiments were also performed with SS1.1, which contains spacer DNA between -2.86 and -1.76 (Fig. 1B), but the results were difficult to interpret because of low levels of hybridization and the sequence characteristics of this region. SS1.1 is partly composed of simple repeating sequences such as (CT), and (TCCC), (17) that can cross-hybridize to similar simple repeats in the 3' flanking region of the precursor rRNA gene (37, 52, 53; unpublished data) and presumably to such repeats in other regions of the genome. Therefore, hybridization results are complicated by the potential for cross-hybridization between any of these simple repeats that may be transcribed. Nonetheless, at high a-amanitin levels, hybridization to regions containing these sequences can be attributed to RNA pol I activity even though the precise template in rDNA cannot be determined. Preliminary results from high-stringency filterbound DNA hybridizations with labeled nascent RNA from untreated nuclei indicated that very low levels of nascent transcript hybridization can be seen with SS1.1+ and SS1.1-. However, in the presence of a-amanitin the majority of the hybridization signal with SS1.1+ was lost, whereas hybridization with SS1.1- remained the same. This suggests that the RNAs complementary to SS1.1- are produced by pol I transcription and that the RNAs that anneal to SS1.1+ are largely (or solely) produced by polymerase II, and polymerase III, or both. Overall, the results of solution and Southern blot hybrid-

izations of labeled nascent RNA with rDNA fragments flanking the precursor gene indicated that there is pol I transcriptional activity outside of the 45S coding region. The highest levels of ot-amanitin-resistant hybridization with the spacer regions examined were seen with the SS1.6 fragment that extends from -1.76 to -0.168. Since other workers have reported the absence of spacer transcripts from this internally repetitious region in steady-state RNA from a murine lymphosarcoma (50), we also examined in-vivosynthesized cellular RNA for detectable spacer transcripts. Hybridization of labeled rDNA recombinants to RNA dots. Single-stranded M13 DNAs containing rDNA sequences were digested with micrococcal nuclease and end labeled with [32P]ATP as previously described (12). Total RNA from H35 cells or nuclear RNA from rat brain (containing a predominantly nondividing cell population) was prepared and dotted onto nitrocellulose. Figure 3 shows the results of hybridizations with recombinants encoding portions of the 45S gene and upstream spacer sequences. BH.41- and HX.51-, which encode 125 and 510 bases of 45S rRNA, respectively, hybridized strongly with H35 RNA. The intensity of the signals approximately reflected the relative proportion of 45S coding sequence (1:4, BH:41-HX.51). SS1.6+ and SS1.1- had weak signals that could not be distinguished above the background. SS1.6- hybridization produced a slightly stronger signal which, though difficult to see in Fig. 3, was detectable in the original autoradiogram. SS1.1+ hybridized with H35 RNA to the same extent as did BH.41-. The H35 total-cell RNA dots contained 220 ng of RNA in the highest-concentration dot. Approximately 10% of this amount was dotted for brain nuclear RNA, which roughly corresponds to the amount of nuclear RNA mass per totalcell RNA. A strong hybridization signal to brain nuclear RNA was seen with HX.51- and also with SS1.1+. A weaker signal was seen with BH.41-, and no detectable signals were seen with the other probes. The relatively lower


VOL. 7, 1987

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FIG. 3. Hybridization of labeled M13-rDNA to RNA dots. Dot 1 contains 220 ng of total H35 RNA, dot 2 contains 71 of ng H35 RNA, dot 3 contains 23 ng of H35 RNA, and dot 4 contains 20 ng of brain nuclear RNA. RNA dots were hybridized as described in Materials and Methods with the indicated M13 recombinants containing 5' precursor 45S sequences (HX.51 and BH.41) and upstream spacer flanking regions (SS1.6 and SS1.1).

signals for precursor rRNA in the brain probably reflect reduced ribosomal synthesis in nongrowing tissue compared with that in rapidly growing tissue culture cells. Transcription mapping with spacer and precursor rDNA recombinants. Because of the low signal to the SS1.6region, we used a more sensitive technique to investigate the presence of stable spacer transcripts. Transcription mapping was performed with single-stranded M13 recombinants by minor modification of the procedure described by Brilliant et al. (9). BH.41+, BH.41-, SS1.6+, SS1.6-, SS1.1+, and SS1.1- were digested with HaeIII, end labeled, and hybridized with nuclear or total-cell RNAs from SD rat livers, H35 cells, and RT4-D1 cells. After incubation overnight at 63°C, the hybridized samples were treated with S1 nuclease at 32°C to remove all unhybridized probe. Rat liver and RT4-D1 cell RNAs were analyzed in these experiments in addition to H35 RNA to ensure that any spacer transcripts detected were not limited to the hepatoma cell line. Figure 4A shows the results of this analysis with endlabeled BH.41 -, which is complementary to 125 bases at the 5' end of the 45S precursor RNA and contains rDNA from -283 to + 125. Hybridizations were performed with 10 p.g of H35 (lane b) or RT4-D1 (lane d) total-cell RNA and with 5 pug of RT4-D1 nuclear RNA (lane c). There was a major band at 109 bases that corresponded to protection by RNA initiated at the previously mapped transcription initiation site (24) and extending to the HaeIII site at + 109. The numerous other bands seen below the 109 band were presumably due to S1 digestion at mismatches due to minor heterogeneities of the multiple rDNA repeats. When five times more RNA was hybridized to the opposite (RNA sense) strand, BH.41 +, no protection of end-labeled DNA fragments was observed (Fig. 4B). The internal rDNA fragments of BH.41 generated by cleavage with HaeIII at -239, -203, -75, and +109 (36, 128, and 183 bases) are indicated by italicized numbers on either side of panels A and B. Very faint bands were seen at

319

183 and 128 (Fig. 4A, lanes b, c, and d). No protection of the opposite strands of these fragments was seen (Fig. 4B), nor were any bands seen in a control hybridization with tRNA and labeled BH.41- DNA (data not shown; control hybridizations were completely blank with all rDNA probes). This suggests that protection of the 183- and 128-base fragments may be due to the presence of RNA transcripts that extend at least 203 bases upstream from the 45S precursor initiation site. SS1.6 consists of approximately 11 repeats of an element that varies between 125 and 135 bases in length and also overlaps the left-hand end of BH.41 by 167 bases that do not include the transcription initiation site (-283 to -164). Cleavage with HaeIII within the repeats for which sequence information is available (12) produced three fragments at 17, 23, and 84 or 94 bases. The sequence of one repeat is given in Fig. 4G (-414 to -286). Cleavage within the other repeats presumably produces fragments of similar size (or 17 or 23 bases longer if a GGCC site is missing). Hybridization of these labeled fragments in SS1.6- with 50 ,ug of total H35 or RT4-D1 RNA or 25 ,ug of Dl or liver nuclear RNA produced protection of the 23-base fragment and both partial and complete protection of larger fragments 82 to 109 bases long (Fig. 4C). The 17-base fragment was not efficiently recovered in our procedure, nor was it well resolved in our gels. It does appear, however, that there may be some protection of this fragment in SS1.6- (unpublished data). The diffuse band at approximately 50 bases was most likely due to cleavage within the poly(U)-poly(dA) region of the hybrid (see the sequence in Fig. 4G). There is also protection of the 36-base fragment that comes from the region of SS1.6 that overlaps BH.41 (-239 to -203) and an 82-base fragment (-321 to -239) immediately adjacent to it. Complete analysis of the pattern of end-labeled fragments and their protection is not possible at present because of the lack of complete sequence information on the internal repeats of SS1.6 and because of the heterogeneity of this region in genomic DNA. When identical nuclease mapping experiments were performed with labeled HaeIII digests of SS1.6+, only a small amount of protection of a band at approximately 109 kb was seen (Fig. 4D). We speculate that this may be due to the presence of a few 130-bp repeats in a reverse orientation in the rat genome. Transcription through such a repeat in the direction of the 45S precursor gene would produce a transcript complementary to SS1.6+. The sequence of the region between -2.86 and - 1.76 that corresponds to SS1.1 was recently reported by Financzek et al. (17). Relative to the +1 initiation site in our rDNA genomic clone, there are HaeIII sites at approximately -1.92, -2.22, -2.27, and -2.86. Cleavage of SS1.1 with HaeIII, therefore, produced internal fragments at 297, 53, and 590 bases. Hybridization of cellular RNA with HaeIIIcut SS1.1- showed little or no protection of these fragments (Fig. 4E) except for faint signals at 53 and 297 bases with H35 nuclear RNA (lane e). Partial protection of SS1.1+ fragments, however, was observed with all of the RNAs analyzed (Fig. 4F). The multiple bands seen with SS1.1+ are presumably due to heterogeneity of the hybridizing RNA species. Since the simple repeats discussed earlier do not contain the HaeIII recognition sequence (GGCC), hybridization solely to these regions would not be detected in this assay. In summary, the transcription mapping experiments showed that transcription of the 130-bp elements contained in rDNA recombinant SS1.6 occurs in vivo and is in the same direction- as 45S precursor rRNA synthesis. At least


320

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FIG. 4. Transcription mapping with end-labeled, HaeIII-digested M13-rDNA recombinants. Each autoradiogram shown is a 4-day exposure with XAR film and an intensifying screen. Panel A, hybridization with 2 x io1 cpm of BH.41-. Lanes: a, 103 cpm of undigested BH.41-; b, hybridization with 10 jig of H35 total RNA; c, 5 jig of nuclear RT4-D1 RNA; d, 10 jig of total RT4-D1 RNA. Panel B, hybridization with 2 x io5 cpm of BH.41+. Lanes: e, 25 jig of tRNA; f, 50 jig of total liver RNA; g, 50 jig of total RT4-D1 RNA; h, i03 CpM of undigested BH.41+. Panel C, hybridization with 6 x i0' cpm of SS1.6-. Lanes: a, 2 x i03 cpm of undigested SS1.6-; b, 50 jig of H35 total RNA; c, 25 jig of RT4-D1 nuclear RNA; d, 50 jig of RT4-D1 total RNA; e, 25 jig of liver nuclear RNA; f, 25 jig of tRNA. Panel D,


VOL. 7, 1987

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some of this transcription may continue downstream past the HaeIII site at -203 and possibly even onto the 45S gene. The levels of these transcripts are very low compared with sequences contained in the 5' end of the rRNA precursor. RNAs homologous to sequences upstream of the 130-bp repeats that would be transcribed from the same DNA strand as 45S rRNA and overlap the HaeIII sites in SS1.1 were extremely low or undetectable. There was, however, a heterologous set of cellular RNAs that hybridized to the opposite strand of SS1.1; i.e., transcription would proceed in the direction opposite to 45S rRNA synthesis. Genomic Southern hybridization with SS1.l, SS1.6, and HX.51. Previous work has shown that the DNA region containing SS1.6 is the site of a major polymorphism in the rDNA repeats of individual SD and BD-9 rats (12). Variations in the length of restricted genomic DNA hybridizing with SS1.6 are presumably due to different numbers of the 130-bp elements. In the SD and BD-9 rat DNAs examined, the major EcoRI fragments of rDNA containing the 130-bp element were found to be approximately 10.9 and 11.8 kb long. Genomic Southern blots of H35 DNA were analyzed in the course of the present research to determine the nature of the rDNA polymorphism in these cells and to examine in more detail the genomic distribution of sequences homologous to SS1.1 and SS1.6. Purified DNA was digested with EcoRI or SstI-HindIII, and the Southern blots were probed with SS1.1, SS1.6, and HX.51. In the original X-rDNA recombinant (12), each of these sequences was found on the same EcoRI fragment (10.9 kb long). Cleavage with SstI-HindIII, however, gener-

ated a 3,000-bp fragment that contains SS1.1 and SS1.6 but not HX.51, which is found on a 6,100-bp fragment (Fig. 1). The EcoRI digest of H35 DNA contained a band at approximately 10,900 bp that hybridized to each probe (Fig. 5A, B, and C, lanes 1). There was also a minor band at 1,300 bp seen only in the hybridization with SS1.6. A minor band of similar size was also seen in EcoRI digests of SD and BD-9 rat DNAs (unpublished data). The faint band at 6,400 bp in the EcoRI digest may represent a junction fragment flanking the rDNA repeat. In SstI-HindIII digests, HX.51 hybridized with a fragment at 6,100 bp as would be predicted. The minor band at 9,000 bp was probably due to the absence of a HindIII site at + 125 in some rDNA repeats, although it could possibly be due to incomplete digestion at this site. Other faint bands may represent minor polymorphisms or junction fragments. Digests probed with SS1.6 and SS1.1 showed strong hybridization at 3,000 bp. There was also a signal at 2,850 bp that could be discerned in shorter exposures of the autoradiogram shown in Fig. 5, which is roughly four- to five-fold less intense than the 3,000-bp band. These two fragments accounted for the majority of the rDNA repeats in H35 cells. Faint bands at 2,325 and 3,300 bp may be due to additional polymorphisms in the number of 130-bp elements. The band at 5,200 may be a junction fragment as it was similar in intensity to the 6,400-bp band in the EcoRI digest. The slightly stronger signal at 2,300, only seen in the hybridization with SS1.6, probably corresponds to the 1,300-bp fragment seen in the EcoRI digest. These results indicate that the majority of the rDNA genes

hybridization with 6 x 10i cpm of SS1.6+. Lanes: g, 2 x 103 cpm of undigested SS1.6+; h, 25 p.g of tRNA; i, 25 jig of liver nuclear RNA; j, 50 ,ug of RT4-D1 total RNA; k, 25 ji.g of RT4-D1 nuclear RNA; 1, 25 jig of H35 nuclear RNA. Panel E, hybridization with 2 x 10i cpm of SS1.1-. Lanes: a, 25 ,ig of liver nuclear RNA; b, 50 jLg of RT4-D1 total RNA; c, 25 ,ug of RT4-D1 nuclear RNA; d, 50 jLg of H35 total RNA; e, 25 jig of H35 nuclear RNA; f, 103 cpm of undigested SS1.1-. Panel F, hybridization with 2 x 10i cpm of SS1.1+. Lanes: g, 25 ,ug of liver nuclear RNA; h, 50 ,ug of RT4-D1 total RNA; i, 25 jig of RT4-D1 nuclear RNA; j, 25 ,ug of H35 nuclear RNA. Panel G, sequence of promoter-proximal end of SS1.6, -414 to -164, relative to the initiation site for 45S rRNA synthesis at +1. Poly(T) stretches flanking representative repeat elements are underlined. HaeIII sites are indicated by arrowheads. The numbers in panels A through F indicate molecular size in bases.

322


in H35 cells contain an EcoRI fragment of 10.9 kb that encodes SS1.1, SS1.6, and HX.51. There was a variant present in approximately 20% of the genes that presumably carries one less 130-bp element than the major repeat. It could also be observed that SS1.1 hybridized to DNA outside of the rDNA repeat as demonstrated by the light background smear in Fig. SA. This is consistent with the interpretation that DNA homologous to repetitive or simple sequences found elsewhere in the genome is present in this region of the spacer. Previous work with both Southern blot (12, 37) and sequence (17) analyses has shown that highly repeated DNA sequences such as Alu-type II and identifier elements are not found downstream of the SstI site at -2.86 and therefore do not account for the low level of crosshybridization observed in Fig. SA. DISCUSSION Nascent RNA transcripts labeled in in vitro nuclear run-on reactions in the presence of high levels of a-amanitin contain molecules that hybridize to the so-called NTS of rat rDNA. We found that rDNA regions extending several kilobases upstream of the 45S rRNA coding region and several kilobases downstream of it hybridized to labeled nascent RNA in Southern blots. The strongest hybridization signal was observed with the SS1.6 (-1.76 to -0.168) fragment of genomic clone pEH5.0, which is predominantly composed of approximately 11 copies of a 130-bp repeat (12). Solution hybridization with labeled nascent RNA and single-stranded M13-rDNA recombinants showed that all or most of the SS1.6 transcription is from the same strand as that for 45S rRNA. The extent of hybridization with HX.51, which encodes 510 bases of precursor rRNA near the 45S initiation site (24), is approximately 60% that of SS1.6 on an absolute basis and approximately two-fold greater when disintegrations per minute bound are normalized to the length of the rDNA fragment assayed. The resistance of SS1.6 homologous transcription to high levels of at-amanitin suggests that it is mediated by RNA pol I. The detection of pol I transcription that extends beyond the 3' end of the rat 45S precursor gene and possibly through the entire spacer is consistent with the recent report of transcription of spacer sequences in Xenopus rDNA in nuclear run-on reactions (30). Readthrough spacer transcription has also been observed in Drosophila rDNA both in vivo and in nuclear run-on assays (47). Pol I molecules may transcribe through the termination sites mapped by Grummt et al. (22) at very low efficiency. Some of these polymerases that read through may subsequently dissociate from the spacer rDNA at other sites since the spacer transcription outside of the 130-bp repeat region appears highest near the 3' end of the precursor gene. Although we do not know whether termination of the 45S transcript is accurate in our run-on reactions, the measured incorporation in a 10-min incubation (200 to 300 ribonucleotides per nascent chain; unpublished data) is not sufficient to account for transcription more than a few hundred bases into the 3' spacer. We were surprised to find high levels of spacer transcription upstream of the rat rRNA initiation site in the region containing the tandemly repetitious 130-bp elements. Although spacer transcription 5' to the precursor rRNA promoter has been documented in Xenopus spp. (18, 34, 35, 36, 40) and D. melanogaster (14, 26, 32), the analogous spacer regions in mammals have thought to be transcriptionally inert. Wood et al. (50) were unable to detect transcription of the analogous mouse spacer region containing the internally

MOL. CELL. BIOL.

repetitious DNA (-1894 to -169). Their experiments, however, relied on analyses of steady-state RNA and RNA transcripts from rDNA-plasmid templates transcribed in vitro. We found that when total-cell RNA labeled in vivo was analyzed, it did not detectably hybridize with the 1.6-kb fragment (-1.6 to -0.168) in Southern blots (unpublished data). This in-vivo-labeled RNA does, however, hybridize with a fragment containing the first 125 bases of 45S precursor RNA and with regions further upstream of the SalI site at -1.76 that carry highly repeated DNA sequences found in other areas of the genome (7, 8, 37, 52, 53). These results suggested that SS1.6 homologous transcripts are very unstable and, although detectable as nascent transcripts, they are too quickly degraded to be detected in steady-state RNA. Indeed, when RNA dot hybridizations were performed with labeled M13-rDNA recombinants, whereas strong hybridization signals were achieved with precursor-containing fragments BH.41- and HX.51-, little hybridization was detected with SS1.6-, and none was seen with SS1.6+ or SS1.1-. There was, however, hybridization to SS1.1+, which is likely due to DNA components in this region annealing to transcripts from elsewhere in the genome that are products of polymerase II or polymerase III transcription. (Interestingly, the levels of SS1.1 homologous transcripts were higher in nondividing SD brain tissue than in tissue-cultured H35 cells. The significance of this difference is not known). In our laboratory, we have elaborated transcription mapping techniques to detect very low levels of RNA, i.e., on the order of 0.1 copy of RNA per cell (9). Using a modification of this technique in which single-stranded rDNA recombinants are cleaved with HaeIII, end labeled, and hybridized with total-cell and nuclear RNAs, we analyzed the region from + 125 in the rDNA precursor gene to the SstI site at -2.86 in the spacer. Transcription mapping with end-labeled probes requires that any transcribed RNA extend up to or beyond the restriction site at which the label is introduced. Since we do not yet know the actual sequences protected by these RNAs, it is possible that some transcripts cannot be detected with our present set of end-labeled DNAs. Therefore, the data presented here show that there is in vivo transcription of SS1.6 (-1.76 to -0.168) from the same DNA strand as 45S rRNA synthesis. This transcription includes both the 130-bp repeat element and sequence between the HaeIII sites at -239 and -203. The presence of faint bands larger than the 45S protected fragment at 109 bases in the BH.41- transcription mapping experiments also suggests that SS1.6 transcription continues through to the precursor rRNA gene at a low level. RNAs complementary to regions upstream of the SalI site at -1.76 that would be transcribed in the same direction as the 45S gene may be present at a very low level in vivo, but they are generally not detectable. Transcription mapping experiments with SS1.1 show that RNA transcripts are present that hybridize to SS1.1+ (the opposite strand relative to rDNA transcription) at levels similar to that seen with SS1.6- mapping. However, we know from the dot hybridization results that overall there is more steady-state RNA complementary to SS1.1+ than to SS1.6-. Presumably the use of discrete end-labeled probes requiring protection of the HaeIII sites in SS1.1 for detection does not show the presence of RNAs that anneal to the simple repeat sequences or are very heterogeneous. Our results show that RNA homologous to regions upstream of the 45S precursor rDNA region is produced in vivo. Some of this RNA could be due to readthrough of pol

VOL. 7, 1987

I molecules from one rDNA repeat on to the next as discussed above. However, the level of nascent RNA transcripts hybridizing to SS1.6 is greater than other spacer regions examined. In addition, experiments with both nascent and steady-state RNAs indicate little transcription complementary to SS1.1-, which is the upstream neighbor of SS1.6-. It appears that there is unique transcription of the 130-bp repeat sequences found in the SS1.6 fragment. This transcription may originate in the 130-bp repeat or it may initiate slightly upstream and continue through the repetitious region. Alternatively, pol I molecules that read through from upstream rDNA genes may be concentrated in the 130-bp tandem repeat region. The RNA synthesized from these sequences appears to be turned over very rapidly, more rapidly than the externally transcribed spacer regions of 45S precursor rRNA. This may mean that it is the process of transcription that is important for the function of these spacer pol I molecules rather than the spacer transcripts themselves. Transcription of spacer sequences may simply be a means of propelling the pol I molecules to the precursor promoter, as Moss has suggested (35), or of stabilizing the association of polymerase with the rDNA gene. It is also possible that the conditions of in vitro nuclear elongation reactions release pol I molecules that have initiated in the tandem repeat region in vivo from a transcriptional block. If this is the case, it may mean that a similar mechanism operates in vivo to release pol I molecules for precursor rRNA synthesis when needed. One caveat for the above discussion should be mentioned in light of the genomic Southern blot data obtained with SS1.6 hybridization. Restriction enzyme cleavage of rat genomic DNA with EcoRI produces two major hybridizing bands at 10.9 and 11.8 kb for SD rat DNA and one major band at 10.9 kb for H35 DNA. In both DNAs there is also a minor band at 1.3 kb that hybridizes to the SS1.6 probe. The larger bands co-map with 45S precursor DNA. The smaller band may represent a minor variant linked to the rDNA repeat or it could be due to 130-bp repeat sequences elsewhere in the genome. If the latter is true, then SS1.6 transcription may arise outside the nucleolus. Nonetheless, the insensitivity of this transcription to high ax-amanitin levels indicates that pol I is responsible for its synthesis. In Xenopus spp. (5, 6, 45) and D. melanogaster (14, 26, 43), regions upstream from the primary rDNA promoter have been shown to contain repeated sequences that are homologous to the pol I promoter region. Low-level in vivo transcription of the sequences has been observed both at the electron microscope level (18, 34) and biochemically (32, 35, 36, 40). These upstream regions can function as promoters or as competitors for transcription factor binding in vivo and in vitro, and in Xenopus spp. they have been shown to be required for maximal enhancement of rDNA transcription (10, 15, 29, 35, 38, 39). The upstream repeats in rodents bear no obvious sequence homology to the pol I promoter region mapped for rDNA. Miller et al. (33) have demonstrated that the boundaries for maximal activity of the rDNA promoter extend from approximately -149 to +9 under suboptimal in vitro transcription conditions. There is no extensive homology to the 130-bp repeat in this region. Attempts to detect pol I promoter activity in upstream regions in vitro have also been unsuccessful (50). It has recently been observed,

however, that elements upstream of -150 in rodent rDNA serve to enhance pol I transcription in vitro (11). It is possible that these repeated elements serve the same enhancer-regulator function in rodents as has be'en reported for Xenopus spp. Moreover, the results reported here suggest


323

that this regulation may be mediated through transcriptional activity in the 5' internally repetitious region. The absence of in vitro promoter activity in this region in mouse rDNA (50) may be due to the lack of required transcription factors or chromatin conformation present in the spacer in vivo. Alternatively, sequences upstream of the Sall site at -1.76 kb may be required for promoter activity or the polymerases may come from the readthrough of upstream rDNA genes. Recently Henderson and Sollner-Webb (Cell, in press) have noted that the sequence surrounding the Sail site at -168 in mouse rDNA is highly homologous to the repeated Sail element that Grummt et al. (22) have implicated in pol I transcription termination, and they have found that it can function as a terminator in vitro. We have found that rRNA precursor synthesis in rats also terminates in the vicinity of the downstream Sail repeats (unpublished data) and that there is a sequence surrounding the SalI site at -168 in rats that is homologous to the terminator sequence found in mice. These findings suggest analogy to the presence of a functional pol I terminator in the spacer upstream of the Xenopus 40S rRNA gene that was described by Moss (35). He found that Xenopus spacer transcription appeared to terminate at approximately -213 relative to the initiation site of frog 40S precursor RNA. This region corresponds to the T3 termination site described by Labhart and Reeder (30). In Moss's model, pol I molecules that had initiated at upstream promoter elements terminated transcription at this site before reinitiating at the primary rDNA promoter to produce precursor rRNA. The transcriptional activity that we detected in the internally repetitious spacer of rat rDNA suggests that a similar model may apply to rodents. Perhaps binding of pol I molecules in these regions is a way of sequestering polymerase, and the subsequent transcription is a mechanism for loading polymerase molecules onto the primary rDNA promoter downstream, as has been suggested for Xenopus rDNA transcription. The presence of low levels of pol I activity in other regions of the rDNA spacer suggests that some readthrough of these regions occurs in the tandemly arranged rDNA genes. Thus, some or all of the pol I molecules that appear to be present on the 130-bp repeat elements could originate from rDNA genes upstream. The quantitative differences in transcripts from the tandem repeat region and other regions of the spacer could reflect new initiations in the 130-bp elements in addition to readthrough polymerases or may be due to inherent differences in the stability of spacer transcripts downstream of -1.76 as compared with transcripts upstream of this site. It is also interesting that there is a sequence surrounding the Sail site at -1.76 in rats that is homologous to the terminator sequence. Perhaps, additional regulation occurs in this region that affects the distribution or activity of pol I molecules delivered to the 130-bp elements. ACKNOWLEDGMENTS We are grateful to Elaine Lewis for critical reading of the manuscript and to Barbara D'Angelo for excellent work in typing and preparing it. This work was supported by Public Health Service grants GM29986 and GM33990 from the National Institutes of Health. LITERATURE CITED 1. Arnheim, N., and M. Kuehn. 1979. The genetic behavior of a cloned mouse ribosomal DNA segment mimics mouse ribosomal gene evolution. J. Mol. Biol. 134:743-756. 2. Arnheim, N., and E. M. Southern. 1977. Heterogeneity of the ribosomal genes in mice and men. Cell 11:363-370.

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3. Arnheim, N., D. Treco, B. Taylor, and E. M. Eicher. 1982. Distribution of ribosomal gene length variants among mouse chromosomes. Proc. Natl. Acad. Sci. USA 79:4677-4680. 4. Auffrey, C., and F. Rougeon. 1980. Purification of mouse immunoglobin heavy-chain messenger RNAs from total myeloma tumor RNA. Eur. J. Biochem. 107:303-314. 5. Bach, R., B. Ailet, and M. Crippa. 1981. Sequence organization of the spacer in the ribosomal gene of Xenopus clivii and Xenopus borealis. Nucleic Acids Res. 9:5311-5330. 6. Boseley, P., T. Moss, M. Machler, R. Portmann, and M. Birnstiel. 1979. Sequence organization of the spacer DNA in a ribosomal gene unit of X laevis. Cell 17:19-31. 7. Braga, E. A., T. A. Avdonina, V. B. Zhurkin, and V. V. Nosikov. 1985. Structural organization of rat ribosomal RNA genes: interspersed sequences and their putative role in the alignment of nucleosomes. Gene 36:249-262. 8. Braga, E. A., N. Yussifov, and V. V. Nosikov. 1982. Structural organization of rat ribosomal genes: restriction endonuclease analysis of genomic and cloned ribosomal DNAs. Gene 20:145-156. 9. Brilliant, M. H., N. Sueoka, and D. M. Chikaraishi. 1984. Cloning of DNA corresponding to rare transcripts of rat brain: evidence of transcriptional and post-transcriptional control and of the existence of nonpolyadenylated transcripts. Mol. Cell. Biol. 4:2187-2197. 10. Busby, S. J., and R. H. Reeder. 1983. Spacer sequences regulate transcription of ribosomal gene plasmids injected into Xenopus embryos. Cell 34:989-996. 11. Cassidy, B. G., H.-F. Yang-Yen, and L. I. Rothblum. 1986. Transcriptional role for the nontranscribed spacer of rat ribosomal DNA. Mol. Cell. Biol. 6:2766-2773. 12. Chikaraishi, D. M., L. Buchanan, K. J. Danna, and C. A. Harrington. 1983. Genomic organization of rat rDNA. Nucleic Acids Res. 11:6437-6452. 13. Chirgwin, J. M., A. E. Przybyla, R. J. MacDonald, and W. J. Rutter. 1979. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry

14. 15. 16. 17.

18. 19.

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