Nucleic Acids Research

We have sequenced one complete rDNA tandem repeat from the nematode C. elegans. By comparative analysis we derive secondary structures for the 18s, 5.8s, and 26s rRNA molecules, and comment on other important features of the sequence. We also present the sequence of a junction between the rDNA and non-ribosomal DNA. Finally, we use our data to quantify the evolutionary relationships among several organisms currently studied in developmental biology.


INTRODUCTION
All higher eukaryotes carry a set of tandem repeated units which contain the genes for the 18s, 5.8s, and 26s rRNA molecules (for review see 1). Because of its small size and low level of variability, we have determined the complete sequence of one of these units, isolated from the nematode Caenorhabditis elegans and characterized by J. Files and D. Hirsh (2). We present models for the secondary structure of each of the encoded rRNAs, based on comparative analysis.
We have also isolated and sequenced one region of the chromosome where the rDNA repeating units are joined to non-ribosomal DNA. The rDNA here begins abruptly in the middle of the 26s rRNA gene, and from that point on matches perfectly with the rDNA we had previously sequenced, indicating a comparatively recent rearrangement. Finally, we use selected portions of the rDNA sequence to characterize more precisely the evolutionary relationship between C. elegans and other eukaryotic organisms now being used for developmental studies.

MATERIALS AND METHODS rDNA Clone
We sequenced a single copy of the C. elegans rDNA tandem repeat, carried on the plasmid pCe7, which was given to us by D. Hirsh (2). This fragment is cloned into the Bam HI site of the plasmid, and so runs from a position just inside the start of one 18s rRNA gene to the same site on an adjacent rDNA unit. DNA Sequence Analysis The dideoxy chain termination procedure (3) was used to sequence DNA cloned in bacteriophage M13. We used several different methods to subclone fragments of the rDNA unit into an M13 vector. Blunt-end fragments were prepared either by sonication of the plasmid pCe7, followed by end repair with T4 DNA Polymerase (4), or by digestion with one of the following enzymes: Fnu D2, Alu I, Hae III, or Rsa I. We then used T4 DNA Ligase to clone these fragments into the Sma I site of M13mp8 (5). After plating the phage, only plaques which contained rDNA were selected, as determined by hybridization of nick-translated rDNA (6) to nitrocellulose replicas of the plates. Some blunt-end fragments were also generated by sonication of specific Hind III fragments of the rDNA, obtained by subcloning in pUC 9 (7). Finally, pCe7 fragments generated by Sau 3A digestion were cloned into the Bam HI site of M13mp7 and screened as above.
We also prepared specific fragments selected with reference to a restriction map of the rDNA (2), by digestion of pCe7 with pairs of enzymes: Bam HI and Xba I, Pst I and Xba I, Pst I and Hind III, Sal I and Hind III, and Eco RI and Hind III. The fragments were isolated on LGT agarose gels, and cloned into the appropriate sites on M13mplO and M13mpll (8).
Occasionally, we used the clone turn-around procedure (9), or the reverse sequencing procedure of Hong (10), to sequence an rDNA fragment in its reverse orientation. Furthermore, we screened many of our M13 clones using only ddTTP reactions (11) in order to avoid sequencing redundant clones.
The phage M13 template for the sequencing reactions was prepared essentially as in (11). The sequencing reactions were performed according to the procedure of Biggin et al. (12) using dATP[alpha-35S]; we also followed their protocol for use of gradient gels. Non-gradient gels and reactions using [alpha-3 P]dATP were according to (11). We used programs by R. Staden (13,14) on a Dec VAX computer to compile and analyze the sequence data, and determined secondary structures by inspection and comparison with published rDNA sequences.
Isolation of rDNA/non-rDNA Junction DNA was isolated from 1 gram of C. elegans and purified in a cesium chloride gradient (cf. Sulston and Brenner (15)). We used strain CB2769, which has let-209(e2000) balanced by eDf3 (16). The let-209 chromosome carries a defiency that covers most of the ribosomal gene cluster (17). Following the procedure of Karn (18), we constructed a genomic library of strain CB2769 in lambda 2001 (19). A total of 8.4 ug of nematode DNA was partially digested with three different concentrations of Sau 3A; the combined digests were fractionated on a 0.5% LGT agarose gel, and a size fraction of about 17 kb was isolated. Approximately 0.2 ug of this fraction was ligated with 0.3 ug of purified Bam HI cut lambda 2001 arms. After in vitro packaging we titered the library on E. coli Q358 and Q359 (20), but did not amplify it.
To screen the library we followed the procedure of Maniatis et al. (6).
The plates were replicated on nitrocellulose filters, hybridized with nick-translated pCe7 and autoradiographed. A modified column procedure of M. Olson (21) was used to prepare minipreparations of DNA from those plaques containing rDNA inserts. In order to rapidly identify clones carrying a set of restriction sites not characteristic only of the rDNA, we followed the following "fingerprinting" procedure (Brenner, Coulson, Karn, and Sulston, in preparation). By digesting a clone with a restriction enzyme recognizing a hexanucleotide sequence, end labeling the fragments, and then redigesting with a restriction enzyme recognizing a tetranucleotide sequence, a small group of labeled fragments is generated which taken together are characteristic of the clone. rDNA carried in lambda 2001 has a simple and distinct pattern of fragments for several combinations of enzymes, and we searched for clones which carried additions to this set.
Briefly, we digested each of the lambda 2001/rDNA clones with Hind III and simultaneously end labeled the resulting fragments with [alpha-32P]dATP by using AMV Reverse Transcriptase. We added ddGTP to the reaction to drive it to completion. After heat inactivation of the reverse transcriptase we digested the fragments to completion with Sau 3A, denatured the DNA, and ran the samples on a 4% acrylamide : bisacrylamide (19:1) 8 M urea 1X TBE sequencing gel. After autoradiography, the positions of the labeled fragments were measured and recorded with respect to a known standard. We also repeated this procedure using Eco RI, Sal I, Xba I, and Xho I together, in place of the Hind III. In this case the end labeling was done after the initial digestion had been completed, and [alpha-32P]dCTP was used together with [alpha-32P]dATP. Southern OCOOAAAT § T6OAGTAAT ACATOCAACT ATACCCCAAC OCAAGOOCSO OTOCAATTAT TAGAACAGAC CAAACO1IITT I8S   COGACOTTOT TT6TI6ACTC T6AATAAA6 ATTTACTOT CA6TTTCOAC TGACTCTATC COOAAAOOOT OTCTOCCCTT TCAACTAOAT GOGTAGTTTAT  116  itS   126  146  156  166  176  166   196   266   TOOACTAjC^TOOTTGTTAC OGOTAACO 6AATAA6T4 TCOACTCCOO AGAGOOAOCC TTAOAAACOG CTACCACGTC CAAOOAAQ^OC Figure 1 : The sequence of one rDNA tandem repeat from C. elegans. The non-transcribed strand is pictured, and is numbered from the start of the Bam HI site into which the rDNA was cloned. The 18s, 5.8s, and 26s rRNA genes are underlined. Their endpoints are based only on comparative analysis.
carrying an rDNA/non-rDNA junction. Those identified from the Hind III screen were digested with Hind III, Xba I, or Xho I, and run on a 0.6% HGT agarose gel overnight at 25 mA. Clones identified from the other screen were digested with Eco RI, Sal I, Xba I, or Xho I and treated similarly. After blotting, we hybridized the nitrocellulose reproduction of each gel with nick-translated pCe7 and used the autoradiograms to construct restriction maps of the clones.
After subcloning into pUC9 individual Hind III frag;nents which might Figure 2(a,b) Secondary structure model of C. elegans 18s rRNA. Helical stems are numbered from the 5' end of the molecule, following the system of Nelles et al. (23), and nucleotides are numbered as in figure 1. All compensatory base changes in previously invariant regions are marked by a line over each nucleotide involved, and highly variable regions are circled.
contain a junction between the rDNA and non-rDNA, the procedures described above were used to sequence the relevant portions of these fragments.

RESULTS rDNA Sequence
We have determined the complete sequence of the C. elegans rDNA tandem repeat contained in pCe7 ( fig. 1). All bases except for 1-53, 923-993, and 7166-7204 have been sequenced on both strands of the DNA. Furthermore, we have confirmed 2447 bases by partially sequencing three clones isolated during our search for an rDNA/non-rDNA junction. These contained no detectable alterations from the pCe7 rDNA.
Based on sequence homology with the rDNA of other eukaryotes, we have mapped the locations of the three rRNA genes ( fig. 1). Because of the limitations of this method, the exact ends of the rRNA genes are not certain, but we have indicated the most likely sites. By comparative analysis of the C. elegans sequences with those of other eukaryotes we have also developed models for the secondary structures of the different rRNAs (figs. 2,3).

rDNA/non-rDNA Junction
The proportion of rDNA clones increases almost tenfold during amplification of a wild type C. elegans library, making it very difficult to screen for those few clones containing a junction between the rDNA and non-rDNA. We therefore constructed an unamplified library; to further decrease the background, we used C. elegans strain CB2769, since it has a large deletion in the rDNA region on one chromosome (17).
The library contained approximately 330 pfu/ul, and plating on both Q358 and Q359 verified that most of the phage carried inserts. We screened about 20,000 plaques and from this recovered 121 phage containing rDNA. This is a frequency of about 0.6%, much closer to the theoretical value of 0.3% than was found in the amplified library. Of the 121 lambda 2001/rDNA clones, 13 were selected by the "fingerprinting" procedures for examination with Southern blots, and of these 13 three were analyzed further.
Two of these three were shown by subcloning and sequencing to be clones from the rDNA tandem repeat cluster, with a small alteration at the polylinker site of the lambda 2001 vector (data not shown). The third, as shown by sequence analysis, contains a rDNA/non-rDNA junction (fig. 4). The rDNA begins abruptly in the D10 region of the 26s rRNA gene and continues from that point in perfect homology with the pCe7 rDNA for the 162 bases sequenced. Southern blotting indicates that approximately 90% of one rDNA repeat unit is contained in this clone, and the rDNA is adjacent to the short arm of the vector.
To confirm the existence of the putative junction a 4 kb Hind III fragment from the non-rDNA region of our lambda 2001 clone was isolated and used to probe a wild type (N2) C. elegans genomic library in lambda 2001 (also made from Sau 3A partial digestions). Six of the seven positives found were linked both to each other and to the rDNA, and connect to a set of clones which cover a total length of approximately 50 kb just outside of the rDNA. By using this same probe on a Southern blot containing both genomic N2 DNA and genomic CB2769 DNA cut with Hind III, XbaI, or Eco RI, we confirmed that both of these C. elegans strains are identical in this region (data not shown). Finally, DNA sequence analysis of the independently isolated junction demonstrated its identity to the one obtained from the CB2769 bank. Helical stems are numbered from the 5' end of the molecule, following the system of Michot et al. (38),and nucleotides are numbered as in figure   1. All compensatory base changes in previously invariant regions are marked by a line over each nucleotide involved, and highly variable regions are circled. Part a depicts the 5.8s rRNA/26s rRNA interaction, and parts be depict the rest of the 26s rRNA structure. The 5.8s rRNA nucleotides appear in lower case letters.  (23). Because of this we have followed their system of helix nomenclature. We present our model of the C. elegans 18s rRNA secondary structure in figure 2, with the warning that helices in the highly variable regions should be regarded as tentative.
The following points are of special interest. First, compensatory base changes provide the first evidence for the existence of both helices 0 and 1 in eukaryotes. This creates the possibility of a series of tightly stacked helices at the 5' end of the molecule. However, cytosine methylation and kethoxal reaction studies indicate that the base pairing in helix 1 may be weak (24,25).
C. elegans is also the only eukaryote with a variant sequence in the area of helix 3. Two different structural models exist for this region (23,25,26), and non-compensatory base changes in our sequence cast doubt on both. Though it is possible to construct shortened and modified forms of helix 3 for C. elegans, we agree with Atmadja et al. (27) that in the absence of any positive evidence, helix 3 probably does not exist.
Several compensatory base changes provide evidence for the existence of previously invariant helices 15 (though with a small deletion) and 18. Non-compensatory base changes also shorten several helices in C. elegans from those observed in other eukaryotes. Most notably, two base pairs at the end of the otherwise invariant helix 18-1 are impossible in C. elegans. Furthermore, one of the G residues in this helix has been shown to be strongly reactive with kethoxal (25). This renders the existence of this newly proposed (23), eukaryotic specific structure problematical. However, the bacterial structure in this region fits the C. elegans sequence even more poorly, and only definitive physical studies can truly resolve these problems.
We have noted other compensatory base changes in figure 2. The final point of interest is that our sequence accords well with Nelles proposals for structure in the variable regions (except for helix 17), though further comparative evidence is needed here. It is interesting that only V4 approaches the level of diversity seen in several of the 26s rRNA variable regions. (For further discussion of variable regions see 26s rRNA below).

5.8s rRNA
Our model of the structure of C. elegans 5.8s rRNA and its interaction with the 26s rRNA agrees closely with that proposed by Walker et al. (28) and Michot et al. (29) for mouse, especially in those areas where it differs from other, "universal" models (30,31,32). Both 5.8s rRNA/26s rRNA helices I and III are now well established (28)(29)(30)(31)(32)(33). Most authors (29)(30)(31)(32)(33) have also proposed the existence of helix I', which includes a relatively invariant region. There is little comparative evidence for this helix in eukaryotes, and this entire region in the mouse is very susceptible to Si nuclease cleavage (28). Furthermore, C. elegans contains a noncompensatory base change which destabilizes the center of the helix. It is thus only likely to exist in a very weakly paired form, or not at all.
We also find that 5.8s rRNA/26s rRNA helix II (28,29,32,33) is strongly confirmed. Of special interest is the fact that the C. elegans sequence also contains a compensatory base change which supports the extension of this helix (note the underlined bases in fig.3). This extension had been suggested earlier (29,33), but until now remained unsupported. The region of helix II is hypothesized by Ursi et al. (30,31) to form an internal 5.8s rRNA helix. However, though it is possible to draw an internal helix involving this region for C. elegans, such a structure appears much less stable, differs in detail from that proposed by Ursi, and involves a conserved base (#2206) which has been shown to be unpaired in mouse (28). The additional 5.8s rRNA/26s rRNA helix proposed by Ursi but absent in other models is also refuted by the absence of suitable base pairing in the C. elegans sequence.
The other feature to note is that the newly proposed helix E of Vaughn (32) can only be formed with a two base pair stem and three base pair loop for C. elegans. This argues against its existence. Finally, we suggest that in C. elegans, helix C, which is weakly structured in many organisms, may contain an internal loop as shown in figure 3. This allows the more highly conserved end loop to be constricted in the same manner as it is in rRNAs with a more structured helix D, as, for example, in mouse (28,29). 26s rRNA Since Veldman et al. (33) proposed a structure for the yeast 26s rRNA based on homology with that of E. coli, the Xenopus laevis (34), rat (35), mouse (36), and Physarum polycephalum (37) large rRNAs have all been sequenced. This new information has confirmed most of the yeast model, but also led to some new proposals for specifically eukaryotic structures (38)(39)(40).
The C. elegans sequence provides strong evidence for the existence of one of these: helix 13, recently hypothesized by Michot et al. (38). Four directly compensatory base changes in the center of the helix confirm that it is a conserved structure among eukaryotes, serving to tie together two regions nearly 800 bases apart. Helix 22, and the entire region of helices 40-45, (38) are also supported by our sequence, although alternative models for helix 22 (33,39) remain possible.
A noncompensatory base change prevents pairing at the end of helix 49, and so indicates that this region may be unpaired in other eukaryotes as well, as proposed in (39).
We have had considerable difficulty developing a consistent model for the 3' end of the molecule. In particular, helix 78 is poorly conserved in C. elegans, and alternative pairings for this helix involving the D12 region are possible. Furthermore, although several bases in helix 84 are invariant among both prokaryotes and eukaryotes, the secondary structures in which they are presented seem to vary greatly. We present a general structure for this helix similar to that suggested for X. laevis (39), but recognize that it does not fit the mouse sequence well. It is probable that tertiary interactions or stabilizing protein interactions are very important here.
Our sequence also provides the first comparative evidence in eukaryotes for helices 25, 34, and 61, and additional important evidence for helices 7, 39, 51, 70, and 79. We have noted these and other new compensatory basechanges in figure 3.
Finally, we wish to note that while some of the secondary structures in the variable regions seem very different in different organisms, others, such as D3 and D5, share many common features. The work of Michot et al. (38) in analyzing the structure of these regions has been confirmed by the C. elegans sequence in many cases, which emphasizes the fact that many of the variable regions are subject to specific structural constraints. It thus appears likely that some of them may be involved in forming functionally important structures in the eukaryotic ribosome. Others, however, may serve no function, instead being constrained only by the need to avoid damage to the ribosome. The long, GC rich helices in such areas as D2 and D8 in vertebrates may function merely to form tightly bound structures which project from the surface of the ribosomes and thus avoid interference with their function. It is possible that in creatures such as C. elegans and yeast this inefficiency is too strongly selected against to occur. Alternatively, these variable regions could play a role as sites for recognition and control of ribosomes by various species specific proteins. rDNA Gene Organization The C. elegans rDNA tandem repeat is only 7200 bp long, and so provides a system where the essential features of rRNA gene organization can be easily studied.
Starting with the non-transcribed spacer (NTS) and external transcribed spacer (ETS), we first notice a very strong homology between the sequence to the 3' side of the 26s rRNA gene (non-transcribed strand) in C. elegans and in yeast (41). However, though it is attractive to postulate that these T rich sequences serve as a transcription termination signal, they are not present in P. polycephalum (37). The next 120 bases contain several long tracts of A's and T's. Though we have not mapped the start site of the primary rRNA transcript, this area is almost certainly part of the NTS. Although C. elegans DNA is 65% AT rich, we feel these tracts are not merely accidental, both because the rest of the ribosomal DNA is consistently about 50% AT, even in spacer regions, and because the probability of finding such long tracts even in 65% AT rich DNA is very low. In many other eukaryotes studied (41)(42)(43)(44)(45)(46) there are also long tracts of A's or T's found 5' to the start site of transcription. Some of  Table 1 Each region of the rDNA tandem repeat treated in the Discussion is listed, togather with its predicted length, location and nucleotide composition. The locations are numbered as in figure 1.
these tracts lie in repeated regions hypothesized to serve as enhancers (42,47). Humans and some Xenopus species, however, do not seem to contain such tracts (48,49), which argues against their being general RNA polymerase I enhancers. The NTR/ETS region is also rich in sequences which could serve as TATA boxes.
Because only 940 bases separate the end of one 26s rRNA gene and the start of the next 18s rRNA gene, and much of this must lie in the ETS, all those functions which the NTS must discharge are coded for in a region only a few hundred nucleotides long. Thus the great length of the NTS in many other eukaryotes (see 1 for review) is not an essential feature of rDNA gene organization.
The essential statistics of the rRNA genes and the internal transcribed spacers (ITSs) are given in table 1. The GC composition is 47-49% throughout, unlike that of some vertebrates, where the ITS's have a much higher GC content than the rRNA genes (50). This ITS composition must be actively selected for, since the GC content in the rest of the genome is only 35% (15).
We have located two conserved features in the second ITS. the first is the sequence PyCAAT located shortly after the end of the 5.8s rRNA gene. The second is the 3' terminal region of the 7s partially processed transcript, found 100-110 bp 3' of the 5.8s rRNA. This site was first proposed by Reddy et al. (51), and has been observed in several organisms (52). A computer search has not uncovered any tRNA genes in either the ETS or the ITS's.

rDNA/non-rDNA Junction
The junction between the rDNA and non-rDNA occurs in the D10 region of the 26s rRNA gene (figure 4). Although this gene is thus not functional, the  Small areas where certain alignment is impossible were omitted, and the second set of figures was normalized to that of the first by linearly transforming its values so that its maximum and minimum were the same as for the first set of figures.
rDNA sequence contains no alterations from that found in pCe7, indicating that the rearrangement must be comparatively recent, or possibly that the incomplete sequence has been subject to correction by a gene conversion mechanism. Smith has proposed (53), and work in yeast has supported (54-56), the idea that the population of rRNA genes in a species is kept relatively homogeneous by frequent unequal crossing-over among the repetitive units. Several cases have also been observed of apparently aberrant rearrangements within one or two of the rDNA tandem repeating units, caused possibly by deletions or duplications (see (2) for example in C. elegans) or insertions (45). In the D. melanogaster case cited (45), the insertion of a large element into the 26s rRNA gene has occured, but the gene sequence has not been altered, indicating a fairly recent event. These observations, together with our own, indicate that besides homologous unequal crossing-over, aberrant cross-overs or rearrangements in the rDNA can occur often, and occasionally involve non-rDNA sequences as well.
Finally, we wish to note that the non-rDNA near the junction contains long poly dA:poly dT tracts. Though this may have been a chance event, it is likely that such tracts both here and in the NTR of the rDNA hinder nucleosomes from packaging this region (57,58). This would allow better access of other proteins to nearby sequences. This system could help keep the rRNA genes more homogeneous by increasing the rate of unequal homologous crossing-over. The ability of these sequences to remain unpackaged is also a prerequisite for their possible role as enhancers in the NTR region. Molecular Evolution of C. elegans In table 2 we present information on the degree of divergence among the rRNA's of C. elegans and five other eukaryotes. We have examined both 93 bases from the highly conserved 5' portion of the 5.8s rRNA, and 156 bases from the region of helices 40-45 in the 26s rRNA, where much more divergence has occured. Small areas within these regions where certain alignment is impossible were omitted, and the percent divergence in the second case was normalized to that of the first for ease of comparison.
Two points are important. First, these results indicate that the error caused by a small sample size and other factors is very significant, and can influence the construction of phylogenetic trees by comparative analysis of 5.8s rRNA sequences. Notice especially the artificially small percent difference between D. melanogaster and the vertebrates in the 5.8s comparison. For this reason we present our data only in tabular form.
Second, it is interesting that both C. elegans and D. melanogaster have diverged greatly both from each other and from the vertebrates, as much or even more so than yeast has. This emphasizes the very large number of cycles of reproduction and selection each of these organisms has undergone since both diverged from a common ancestor.