Human ribosomal RNA gene spacer sequences are found interpersed elsewhere in the genome

Gene, 15 (1981) 177-186 Elsevier/North-HollandBiomedical Press

177

H u m a n ribosomal R N A gene spacer sequences are found interspersed elsewhere in the genome (Recombinant DNA; restriction endonuclease mapping; gel-blot hybridization; Alu family sequences)

Russell liiguchi *, Howard D. Stang, Jeffrey K. Browne, Mark O. Martin *, Marie Huot, Janice Lipeles and Winston Saber ** Department of Biology and Molecular Biology Institute, Universityof California, Los Angeles, CA 90024 fU.S.A.)

(Received January 12th, 1981) (Accepted June 1st, 1981)

SUMMARY A cloned EcoRI fragment containing human 18 S rRNA gene sequences was used to screen a gene library to obtain a set of 8 overlapping cloned DNA segments extending into the non-transcribed spacer region of the human ribosomal RNA gene cluster. 19.4 kb of the approx. 43.kb rDNA repeat was obtained in cloned form and mapped with restriction endonucleases. None of the clones obtained extended into 28 S rRNA sequences. A 7-kb region of non-transcribed spacer DNA shared in common between five independent clones was subjected to com. parative restriction digests. It w&~ estimated that sequences among the five different spacer isolates varied by no more than 1.0%, if all the obaerved differences are assumed due to point mutation. HaelI.restriction fragwents from within this same 7-kb region contain sequences carried not only within the tandem repeats of the gene cluster but interspersed elsewhere in the genome. Some of these sequences correspond to the Alu family of highly repeated interspersed sequences.

INTRODUCrlON The hut.ran 18 S and 28 S rRNA genes exist within tandem repeat units located in the nucleolus organizer region of five chromosome pairs (Tantravahi * Present addresses (R.H,): Department of Biochemistry, University of California at Berkeley, Berkeley, CA 94720 (U.S.A.); (M,O,~) Departm~t of Biological Sciences,Stanford University,Stanford, CA 94305 (U,S.A,). ** To whom repot requests sho~ be sent, Abbreviations: huh'b, h n m riboso~;,kb, kilobases or kilobase ~ s ; rDNA, ~ o ~ DNA;rRNA, ~osomal RNA; SDS, sodiumd~ecyl sulfate; 1 x SSC,150 mM NaCI, 15 Na citrate,pH 7J).

et al., 1976). There are from 150 to 300 repeat unitg per haploid genome (Gaubatz et al., 1976), or on average from 30 to 60 repeats per chromosomal location. Using human rDNA purified by densitygradient centrifugation, Wellauer and Dawid (1979) have, by R.loop mapping, delineated the repeat unit structure with regard to EcoRl cleavage sites and 18 S, 28 S and precursor 45 S rRNA regions of transcription. As shown in Fig, 1, this basic repeat structure can be described in terms of four EcoRI fragments; with two containing most of the transcribed sequences and two from the non-transcribed region. In all, about 31 kb of the 43-kb repeat length is devoted to non.tran~ribed spacer.

0378-1119/81/0000-0000/$02.75 © 1981 Elsevier/North-HollandBiomedicalPress

178 Wilson et al. (1978) have purified by recombinant DNA techniques the 6-kb EcoRI fragment containing most of the 18 S rRNA sequences. The start of 45 S precursor rRNA transcription has been mapped near one end of this fragment (WeUauer and Dawid, 1979). In this paper we report the purification of 13.5 kb of non-transcribed spacer DNA on the 5' side of this fragment and the preliminary analysis of this region.

MATERIALS AND METHODS

37°C for 2 mitt, mixed with 3 ml of 1.0% tryptone afar at 4T'C and plated onto 1.0% tryptone agar 100 mm plates. A partial Haelll, Alul human gene library (Maniatiset al., 1978; Lawn et al., 1978) cloned in CH4A was the gift of T. Maniatis (this gene library was cloned with DNA from a single human placenta). Screening of gene libraries was performed using the plaque hybridization method of Benton and Davis (1978). For screenings using rRNA (labeled with 32p using polynucleotide kinase), h 1/2-11 wash at room temperature cont~n~ng 2 mg/ml pancreatic ~2qase (Worthington A-grade) in 2 X SSC was introduced.

(a) Enzymes (c) Restriction mapping Restriction endonucleases EeoRI, Sstl, and Xbei were gifts of M. Komaromy. T4 DNA ligase was a gift from Forrest Fuller. BamHl was prepared by S. Hendricks. Other enzymes were commercial preparations. Restriction digests were performed under standard conditions. (b) Recombinant DNA human gene library construction Hi?l, Mr human DNA was isolated from the sperm of ap?rox. 20 donors (Wallace and Salser, 1979). This DNA was subjected to partial endonuclease EcoRl tigesr.ion, size selected to between 10 to 15 kb on preparative Seaplaque ® agarose gel electrophoresis (see below), mixed with the purified "arms" of the EK-2 phage vector XgtWES in equal molar amounts and ligated using T4 ligase at a total DNA concentration of 25/2g/ml in 100 mM Tris- HCI (pH 7.9), 12 mM MgCIz, 0.1 mM ATP, 50 mM NaCI, 0.1 mM EDTA, 10 mM dithiothreitol, 50 gg/ml bovine serum albumin. Transfection into E. coli LE392 was perforraed as described in Tiemeier et al. (I 977) with the foll,~wing modifications: after the cells reached an Asg0 = 0.6 the whole culture w~s rapidly chilled by swhling for 2 min in an ice-water bath and mainrained on ice at 0°C for 3 h. "[his gave a consistent 3-fcld increase in transfection efficiency. The final resL~spension of cells was in a I/I0 vol. of 25 mM Tris • HCI (pH 7.5), I0 mM NaCl, 50 mM CaCl2. For each plating, 0.5 ml of competent cells was mixed with 2 /al (50 ng of DNA) of the ligation mixture. This mixture was kept on ice for I h, transferred to

Mapping was performed using single and double enzyme digests of 32P.labeled DNA from the clone designated hurib 8 (see Fig. 1), and by ethidium bromide fluorescence on the cloned 6-kb EcoRl fragment containing 18 S rRNA sequences (hurib 1). Digests were electrophoresed on 6% acrylamide and 3% and 0.7% agarose gels. Some mapping was also performed using partial restriction endonuclease digestion (Smith and Bimstiel, 1976).

(d) Extraction of DNA from gels Gels for isolating specific size classes of DNA were prepared using low-melting temperature Seaplaque ® agarose (Marine Colloids, Rockland, MN) in hori. 7ontal gels. Slices of the gel containing DNA fragm ~ t s stained with ethidium bromide were held at 65°C for 10 rain past apparent liquefaction and then transferred to 47°C. The NaCI concentration was adjusted to 0.1 M and the agarose concentration, if greater than 1.0%, down to 1.0%. Room-temperature phenol (RNA grade) freshly saturated with electrophoresis buffer (brought to 0.1 M NaCI) was added to this melted gel solution in a ratio of 3 : 4 and the mixture was immediately vortexed 30 s. Use of a clinical centrifuge was adequate to separate the two phases. If a larger proportion o f phenol was used a tight interface could not be formed from the extracted agarose and this interfered with DNA recovery. The aqueous phase was again extracted with 3/4 vol. of phenol and then twice with 2 X vols. of n-butanol to remove ethidium bron~de and concentrate the

179 solution. DNA is pre~pitated from the aqueous solution with 2.5 vols, of ethanol. Recoveries have ranged from 50 to 90%. Carrier RNA may be used at any stage. (e) Gel blot hybridizations G e l blot hybridizations were performed according to the method of Southern (1975) and the dextran sulfate modification of Wahl et al. (1979) using DNA probe 32P-labeled by nick translation (Rigby et al., 1977). Wash salt concentrations and temperatures were as indicated for the figures.

RESULTS (a) Human ribosomal RNA gene clones Human gene library (from a partial gcoRl digestion), representing approximately the length of the

haploid human genome in the form of cloned inserts, was screened with 32P-labeled human 18 S and 28 S rRNJL Two positive signals were obtained from recombinant phage that each contained a single 6-kb EcoRl insertion. The BamHI digestion pattern of this insert fragment matches that reported by Wilson et al. (1978) (see Fig. 1) with the addition of a sin~e site dividing the smallest fragment. This 6-kb insert fragment was labeled with 32p and used as a probe to screen 5 × 104 plaques from a HaelII/Alul human gene library obtained from Lawr, et al. (1978). The 8 clones isolated are displayed in Fig. 1. As shown, no cloned inserts were obtained extending into the 7-kb EcoRI fragment described by WeUauer and Dawid (1979) as containing 28 S rRNA sequences, and most of the cloned inserts were short relative to the 18-kb size selection used on DNA that was cloned into this library (Lawn et al., 1978). It is not clear why 28 S rDNA was not represented in any of the 8 clones obtained. There should have been

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Fig. 1. Restriction mapping of inserted human rDNA. (1) Cloned human zDNA insertions obtained from the partial Haelll/AluI gene fibrary. (2) The t~*peating unit structure determined by Wellauer and Dawid (1979) with respect to EcoRl cleavage sites. (3) Restrietion sites for cloned inserts from khmib 1 and 8. Alignment of khufib 8 to the repeating unit was determined by its failure to hybridize 18 S rRNA and 28 S zRNAa,~dby hybridization of its 2.5-kbEcoRl fragment to the 6-kb insert of ~,huzib 1. Alignment of other clonedinserts was in relationship to 7,hurib 8. The 7-kb Xbal/EcoRl fragment compared between clones in Table I is indicated for the group of cloned inserts. (4) HaelI fragments 1-5 of this same fragment that were used in the experiments described in Fig. 5. R, EcoRI; X, Xbal; S, $ali; B, BamHI; Hi, H/ndlll; H, Haell.

180

phoresis. When digested with BamHl t l ~ DNA is cleaved into five fragments. Identical fragments are obtained from each o f the five clones indicating that there are no large deletions or insertions o f DNA. There are published methods for estimating the degree o f difference in sequence between any two related DNAs by differences in the patterns obtained upon restriction endonuclease cleavage and electrophoresis (Upholt, 1977; Nei and Li, 1979). Consequently these fragments were also digested with Alul, Taql and Sau3A and compared with respect to subfragment electrophoretic mobility. Digestions were electrophoresed on both 6% acryiamide and 3.0% agarose gels. Between any two o f these clones the bands sI~red in common could be counted and the formulas o f Nei and Li (1979) and Upholt (1977) applied in order to approximate the amount o f base substitution necessary to account for the observed differences in band pattern. This is compiled for all possible comparisons between the five clones ia Table I. The average o f these calculated base substitu-

an equal probability o f obtaining cloned inserts extending in either direction f r o m the 18 S rDNA fragment used as probe, yet all inserts extend only into the spacer region. Also, the total fraction o f rDNA clones in the library was roughly l O-fold less than expected. The longest insert was in ~hurib 8, which conrained 16 kb o f ribosomal DNA including sequences extending 14 kb into the spacer region. This insert and the 6-kb 18 S r R N A fragment were subjected to the restriction enzyme mapping shown in Fig. 1. (b) Spacer region sequence variation A 7-kb fragment o f human rDNA created by cleavage~with the enzymes Xbal and EcoRI was shared in common among five cloned inserts, as shown in Fig. I. This fragment should contain non-transcribed spacer sequences near the start o f 45 S rRNA transcription. The fragment was isolated from these five clones by preparative Seaplaque ® agarose gel electro-

TABLE 1 Analysis of human ribosomal spacer sequence variants "Ihe corresponding Xbal/EcoRI-generated 7-kb fragments from five different human rDNA clones (see Fig. I. area 1) were subjected to restriction enzyme digestion followed by both 6~ acrylamide and 3% agarose-gel electrophoresis° For any two DNA doles. X and Y, an estimate of sequence divergence can be obtained by counting the number of fragments, nxy, which share the same electrophoretic mobility between digests of the two clones with the same enzyme (in this case Taql, ~u3A and Aiui). The method of Nei and Li (1979) can be used to calculate F, the fraction of fragments shared in common between the two DNA clones. This is done in this table by tabulating nxy, n x and ny (the total number of fragments generated in a single enzyme digestion of clone X and clone Y, respectively), and the sums of each for all three enzymes used. F can be used to calculate S, the fraction of restriction sites conserved (Upholt, 1977) and f'mally P, the level of nucleotide substitution between any two isolates.

Taql x

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44 44 44 47 47 44 44 47 44 47

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1 0.977 0.977 0.923 0.923 0.977 1 0.901

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181

tions gives an idea of the level of heterogeneity among the different repeats of this spacer sequence from a single individual (this gene library was cloned with DNA from a single human placenta; Lawn et al., 1978). As shown in Table I, the average of the differences obtained was 0.36% with a range of 0% to 0.9%. If all differences are assumed to be due to base substitution, one non-transcribed spacer probably varies from one to another locus by no more than 1.0%. If there are differences due to small rearrangements this figure will be lower. We believe that there are very few spacer variants with deletions or insertions greater than 300 bp in this 7-kb region. If such variants were common they would have been seen as additional bands or smears in track 5 of Fig. 3 or tracks 3 and 5 of Fig. 4 (vide infra). As noted above, the efficiency of cloning obtained for the human ribosomal genes was only 10% of that expected. Therefore it is formally possible that the

ribosomal repeats studied here represent a subgroup with limited sequence heterogeneity. We consider it more likely that all of the spacer fragments from this region clone with equal efficiency. (c) The spacer region contains sequences interspersed in the genome The 7-kb XbaI/EcoRI spacer region fragment used above, when 32p-labeled and used as probe in a Southern-type gel blot hybridization'against EcoRI ci~aved human DNA, hybridized as expected to the 12-kb EcoRl fragment of the spa~er region. Hybridization was also obtained, however, to a continuum of bands forming a smear from high to low M r. This was true for any of the different clonal isolates used as probe. This suggested that the 7-kb fragment contained sequences which were not only maintained within the tandem repeats of the rRNA gene cluster

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182

but interspersed elsewhere in the genome as well. The phenomenon of sLvch continuum hybridization pe_.-sists when the 7-kb fragment is subdivided w i ~ HaeIl it.to five proI~e fragments ranging from 0.7 to 1.8 kb. These 32P-labeled fragments, numbered 1 to 5 erom lar~st to ~ U e s t , were purified by gel electrophoresis (Fig. 2) and used to hybridize EcoRlcleaved human DNA blots as before. In Fig. 3 are shown the results after a wash of the blots at 42°C in 5(Y/~ formamide, 2 X SSC, 0 ! % SDS. All probes except for fragment 5 hybridize to the continuum of hume~a DNA fragment sizes. In Fig. 4 are the same blots ~fter x~ashing at 65°C and 0.1 X SSC, 0.1% SDS. The background continuum can be washed off for the ~ragrnent 3 probe while persisting for fragments 1, 2 ar;d 4, although for 1,2 and 4 the relative intensity of continuum hybridization has been reduced relative to the hybridization to the 12-kb repeated

'1

12

2

spacer fragment. Thus the interspersed repeats with homology to fragment 3 are apparently somewhat divergent from this fragment. From Fig. 1, note that the HaeH fragment order 5' to 3' towards the start of rRNA transcription is 5, 4, 1, 2 and 3, with the fragments 1, 2~ and 4 showing strong homology to interspersed sequences grouped together. It is important to note that the rDNA sequences themselves are not distributed throughout the gel in a wa.v which can explain the continuum of hybridization obtained: the starting material was high Mr (approx. 100 kb) DNA and, most important, two of the probes (5 and 3) hybridize cleanly to a single 12-kb EcoRl band when the hybridization is carried out at high stringency. To assess the frequency in the genome of these putative interspersed sequences, labeled spacer DNA was used to screen a lambda phage human gene library (Lawn et ai., 1978) in a plaque hybridization.

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183

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Fig. 4. The same blots as in Fig. 3 except after washing for 0.5 h in 0.2 X SSC, i% SDS at 55°C, followed by a 0.5 h wash in 0.1 × SSC, 1% SDS at 65°C.

The frequency in the library of responding plaques should give a rough idea of the repetition level. Using Haell fragment four (Fig. 1) as probe, fully one-half of all plaques hybridized (only one out of 3 X 104 plaques respond to 18 S rRNA probe). Since only the highly repetitive, Alu family of 300 nucleotide interspersed repeat sequences should appear ~ s frequently among clones (Rubin et al., 1980), this result suggested that the spacer region contains sequences of this family. To verify this possibility, a cloned Alu sequence, plasmid Blur8 (Rubin et al., 1980), was used to hybridize a Southern blot of HaeH digested 7-kb EcoRl/ Xbal spacer fragment (Fig. 5). As shown this probe hybridizes strongly to fragments 2 and 4, weakly, ff at all, to fragments 1 and 3 and show~ no hybridization to fragment 5. When labeled HaeII fragment 4 was used as the probe we observed that it hybridized strongly to a blot of plasmid Blur8 but not to

the control sequence, a blot of the parent vector pBR322.

DISCUSSION

(a) Spacer sequence homogeneity Arnheim et al. (1981) obtained evidence for the concerted evolution of rDNA repeats among the primates. By restriction endonuclease mapping and southern gel blot hybridization, it was shown that much more variation in spacer sequences occurs between species than within the different repeats from the same species or individual of a species. Thus, all the copies seem to evolve it, t,arallel". Mechanisms proposed to explain this phenomenon have included unequal crossover between homologous

184

heterogeneity was seen in three sequencing studies of primate satellite DNA (Rosenberg et al., 1978; Manuelidis and Wu, 1978; Wallace, 1979) obtained from non~lonal-satellite DNA restriction fragments. (b) Repeated interspersed spacer sequences

Fig. 5. Haell rDNA fragments 1-5 (as in Fig. 2) were elec~:rophoresed ~right) and blotted. The blot was probed with the cloned human Alu family interspersed repeat sequence. Bl.~Jr8 (Rubin et al., 1980), with a final wash in 5 × SSC, 50% formamide at 42°C. As shown (left) fragments 2 and 4 respond strong'~y to this probe. A partial digestion product (containing fragments 4 and 5 and seen above fragment 1) 'also responds strongly, presumably due to the presence of fragment 4 sequences.

chromosomes (Smith, 1973) and "master/slave" correction (Callan, 1967). "We have made an estimate of the level of homogeneity maintained (Table I). On the average there is probably less than 1% divergence between the different repeats of the rRNA gene nontranscribed spacer regions examined. This is somewhat less than reported levels of homology between members of other repe~t families. About 12 bp out of 661 (1.8%) were pre~ously seen to vary between two cloned and sequenced copies of Xenopus laevis 5 S DNA containhlg gene, pseudogene and spacer regions ~tiller and Brownlee, 1978). Less than 2-3%

The human rDNA spacer region appears to contain sequences both repeated several hun&ed times as part of the tandem repeats of the rRNA gene cluster and frequently repeated interspersed elsewhere in the genome. Amheim et al. (1980) have found the same to be true for mouse ribosomal gene spacer sequences. Sequences repeated both as part of tandem repeats and interspersed have been reported before in Drosophila melanogaster, in whose genome a 5-kb sequence interrupts the 28 S r ~ A coding region in approx. 35% of the ribosomal gNA genes on the X chromosome (Dawid and Botc"han, 1977). Sequences complementary to this 5-kb insertion occur at about 80 other loci within the genome as well. These genes with interwpted coding regions are inactive. The human non-transcribed spacers with interspersed repeats are apparently part of the normal ribosomal RNA gene arrangement. The A iu family of sequences are suspected to be involved in DNA replication, perhaps as universal origin sequences (Jelinek et al., 1980). It is consistent with this idea that Alu sequences could be present somewhere within rDNA clusters, since otherwise the arrays of tandem repeats of this gene would be without an origin for great lengths of DNA. More precisely, each repeat, and there may be some 30 to 60 repeats on average per chromosomal location, is 43 kb in length (Wellauer and Dawid, 1979); but in Chinese hamster ovary cells replication appears to start at 50 to 100 kb-spacings (Huberman and Riggs, 1968). It is ~:ue, however, that Yurov and Hapunova (1977) have reported that replication units as large as 700 kb exist and Painter and Young (1976) have shown that DNA replication can proceed for great lengths subsequent to inactivation of initiation.

ACKNOWLEDGEMENTS

We would like to thank T. Maniatis for the generous gift of his human gene library, C. Schmid for plas.

185 mid Blur8, M. K o m a r o m y and F. Fuller for enzymes and N. A m h e i m for providing us with data before publication. We t h a n k Susan Salser a n d Jocyndra Wright for help with this manuscript. This work was supported by USPHS grants HL-21831 and GM-18586 to W.S. and a USPHS grant in Hematology 1 ROL CA 27682-10 to H . S . J . K . B . was supported b y a National Institutes o f Health Predoctoral Training Grant in Genetic Mechanisms 5T32GM07104.

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Manuefidis, L. and Wu, J.C.: Homology between human and simian repeated DNA. Nature L76 (1978) 92-94. Miller, J.R. and Brownlee~,G.G.: Is there a correction mechanism in the 5 S m~ltigene system? Nature 275 (1978) 556-558. Nei, M. and Li, W.: MJ*~ematical model for studying genetic variation in term: ~, restriction endonucleases. Proc. Natl. Acad. ScL USA 76 (1779) 5 269-5 273. Painter, R.B. and Young, B.R.: Formation of nascent DNA molecules during inhibition of repficon initiation in mammalian cells. Biochhn. Biophys. Acta 418 (1976) 146153. Rigby, P.W.J., Dieckmann, M., Rhodes, C. and Berg, P.: Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J. Mol. Biol. 113 (1977) 237-251. Rosenberg, H., Singer, M. and Rosenberg, M.: Highly reiterated sequences of SIMIANSIMIANSIMIANSIMIANSIMIAN. Science 200 (1978) 394-396. Rubin, C.M., Houck, C.M., Deininger, P.L., Friedmann, T. and Schmld, L.W.: Partial nucleotide sequence for the 300-nucleotide interspersed repeated human DNA sequences. Nature 284 (1980) 372-374. Smith, G.P.: Unequal crossover and the evolution of multigene femilies. Cold Spring Harbor Symp. Quant. Biol. 38 (1973) 507-513. Smith, H.O. and Bitnstiel, M.L.: A simple method for DNA restriction site mapping. Nucl. Acids Res. 3 (1976) 2 3~,7-2 398. Southern, E.M.: Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98 (1975) 503-517. Tantravahi, R., Miller, D.A., Dev, V.G. and Miller, O.J.: Detection of nucleolus organizer regions in chromosomes of human, chimpanzee, gorilla, orangutan and gibbon. Chromosoma 56 (1976) 15-27. Tiemeier, D.C., Tilshman, S.M. and Leder, P.: Purification and cloning of a mouse ribosomal gene fragment in coliphage lambda. Gene 2 (1977) 173-191. Upholt, W.B.: Estimation of DNA se~;t~ence divergence from comparison of restriction endonuclease digests. Nucl. Acids Res. 4 (1977) 1 257-1 265. Wahl, G.M., Stern, M. and Stark, G.R.: Efficient transfer of large DNA fragments from agazo~ gels to diazobenzyloxymethyl-paper and r~pid ~ybridization by using dextran sulfate. Proc. Natl. Acad. Sci. USA 76 (1979) 3 6 8 3 3 687. Wallace, B.: Studies on human repeated DNAs involving sequence analysis and recombinant DNA technology. Ph.D. Thesis, UCLA, Los Angeles, CA (1979). Wallace, B.E. and Saiser, W.: Isolation of human germ-line DNA suitable for recombinant DNA studies. Gene 7 (1979) 343-347. Wellauer, P.K. and Dawid, I.B.: Isolation and sequence organization of human ribosomal DNA. J. Mol. Biol. 128 (1979) 289-303.

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Yurov, Y,B. and Liapunova, N,A.: The units of DNA ~plica.. tion in the mmumalian chromosomes: evidence for a lm'ge size of n~plication units. Ch_romosoma 60 (1977) 2.53267. Communicated by' J. Carbon.