Gene, 15 (1981) 7-20 Elsevier/North-Holland Biomedical Press

Localization o f the 5S rRNA genes and evidence for diversity in the 5S rDNA region of maize

(Recombinant DNA; Charon 27 vector; sequence divergence; post-replication modification; cloning of plar, t DNA; gene repeats; restriction analysis)

P.N. Mascia a,b,*, I. Rubenstein a,**, R.L. Phillips b, A.S. Wang b and Lu Zhen Xiang a

Departments of a Genetics and Cell Biology and b Agronomy and Plant Genetics, University of Minnesota, St. Paul. MN Y.5108 (USA/

(Received January 17th, 1981) (Accepted February 14th, 1981)

SUMMARY

The 5S rRNA genes of maize are located in the long arm of chromosome 2 (88% of the distance from the cen- tromere to the end) and organized in a 320-bp repeat. Genomic blots of maize DNA digested with the restriction enzymes BamHI and MspI reveal ladders of bands in multiples of 320 bp. Analysis of 5S rDNA from genomic clones reveals that the ladders are due to both modification and divergence of the 5S rDNA nucleotide sequence.

INTRODUCTION

Repeated DNA makes up a large portion of the genomes of eukaryotes. These repeated DNAs can be divided into two broad categories. (1)Repeated sequences of which there are no transcripts in the cytoplasm and whose function is unknown. These include the highly and middle repetitive DNA sequences. (2) Repeated genes whose sequence is transcribed and whose products are readily identi- fied. This class includes (17, 5.8 and 26S) rRNA,

* Present address: Monsanto Company, 800 N. Lindbergh Blvd., St. Louis, MO 63166 (USA). ** To whm z e s t r e q ~ sho~ be addressed. Abbreviations: bp, base ~ ; DTT, dithiotlu~tol; NOR, nueleolus organizer region; zDNA, DNA coding for ribo- somal RNA; 8DS, sodium dodecyl sulfate.

58 rRNA, histones, and multigene systems. Struc- turally, the highly repetitive satellite.type sequences share several features with the rRNA and 5S rRNA genes. First, the highly repetitive sequences tend to be clustered at one or several sites. Second, such DNA may be of a buoyant density distinct from the majority of the genome. Third, the sequences are generally tandemly arranged without major stretches of interspersed non.repetitive sequences. Fourth, the sequences are subject to divergence of their nucleotide sequences. The major difference between these two classes of sequence is the necessity of repeated genes to encode a functional product.

The 5S rRNA genes have been localized in several organisms by in situ hybridization. In some cases they are sequestered at one site, in others they are distn'buted among several chromosomes (Long and Dawid, 1980; Appels et al., 1980). Wimber et al.

0378-1119181/0000-0000/$02.50 O 1981 Elsevier/North-Holland Biomedical Press

(1974) suggested that the 5S rRNA genes of maize are lucated in either chromosome 2 or 3.

The nucleotide sequence organization of 5S rRNA genes has been described in a number of organisms including Drosophila melanogaster (Artavanls" -Tsako- nas et al., 1977), Xenopus/aevis (Miller et al., 1978; Fedoroff and Brown, 1978), sea urchin (Bird et al., 1979) and wheat and rye (Appels et al., 1980). These organisms exhibit two somewhat different modes of 5S rRNA gene organization. A tandem gene-spacer mode occurs in Drosophila, sea urchin, wheat and rye; a complex repeat including a spacer, gene, spacer and a pseudogene is found in Xenopus. In these reports comments were made concerning the exis- tence of divergence, but the extent of tl~s divergence was not detailed. Methylation of perhaps all the CG dinucleotides occurs in Xenopus and this accounts for the methylation in the Xenopus 5S rDNA repeat (Miller et al., 1o78). Bird et al. (1979) suggested that the 5S rDNA of the sea urchin is in a nonmethyl- ated compartment of the genome; evidene.e for diver- gence of sequences was also noted.

When repetitive DNA is detected in restriction endonuclease-digested repeated DNA, a "ladder" is often found, representing a series of DNA fragments having molecular weights that differ by a unit amount (Southern, 1975b; Bedbrook et al., 1980; Mate et al., 1977). It has been proposed that these ladders arise as a consequence of nucleotide sequence divergence in a given repeat, fellowed by unequal crossing over gener- ating higher levels of organization in the tandem repeats (Smith, 1973). Several additional models for hP~hly repeated DNA amplification have been pro- posed (Mate et al., 1977). However, divergence in nu- cleotide sequences to the extent seen in highly repeated DNA would not seem likely in a repeated gene constrained to produce a functional product. Blockage of restriction sites by methylation is an additional possibility. Methylation is extensive in maize DNA (Kemp and Sutton, 1976); 27% of the cytosine residues in maize DNA are methylated. In Xenopus laevis approx. 99% of CG dinucleotides are methylated (Bird and Southern, 1978; Bird, 1978) and as evidenced in this report an extremely high per- centage of methylated CG (meCG) is present in maize.

The da~ presented in this paper confirm the loca- tion of the 5S rRNA gene system of maize the long ann of chromosome 2. Genomic Southern blots are

used to analyze the 5S rDNA repeat structure, and analysis of generate clones of the 5S rRNA genes are used to demonstrate that the repeats contain both post-re?fication modification (methylation) and divergence of nucleotide sequences.

MATERIALS AND METHODS

(a) Plant material

Plants were grown either in the field or greenhouse until the sporocyte stage (6 -8 weeks) when the inner whorl of leaves (sometimes including tassel and nodes) were removed. Inner leaves were sliced into 1 cm pieces, placed in liquid nitrogen, and stored at -70 ° to -90°C until used. Plant strains used m this investigation were A188, W22 and Black Mexican sweet corn.

(b) DNA isolation

Nuclei were isolated by a modification of the method of Kislev and Rubenstein (1980). Frozen tis- sue was ground in liquid nitrogen in a Waring blender for 30 s. The tissue was then infused for 5 min with MNIB (4 mM n-octyl alcohol, 2% gum arabic, 0.4 M sucrose, 2 mM CaCl2, 0.02 M Tris pH 7.4, 400 ~tg/ ml ethidium bromide, 2 mg/ml polyvinylpyrolidone) under vacuum, and then ground for 30 s at top speed using a Brinkmann polytron. The homogenate was then f'dtered through a 60 ttm Nitex f'dter (Tetko Inc., Elmsford, NY) under vacuum. The material remaining on the filter was reground for 30 s in MNIB and filtered through a 60/lm Nitex filter. The nuclei were pelleted by centrifugation at 800-1 000 X g for 10 rain and resuspended in NSB (0.2 M sucrose, 2 mM CaCl2, 10 mM Tris pH 7.4) containing 0.15% Triton X-100 to lyse chloroplasts and mitochondria, and pelleted at 800-1000 X g for 10 rain. The pellet was resuspended by vortexing in NSB with 0.15% Triton X-100 and repelleted. The purified nuclei were then resuspended in NSB without Triton and lysed by adding 2 vols. PDM (0J31 M Tris pH 8.6, 0.5% Sarkosyl, 0.01 M EDTA, 0.01 M NaCl)and 1 vol. PDM + 1 mg/ml proteinase K. Digestion pro- ceeded overnight in the dark at 37°C on a Labin- dustries Labquake rocker. The material was centri- fuged at 20 000 X g for 20 min to remove starch and

cell wall material. The supernatant was then extract- ed 3 times with phenol (0.1 M NaCI, 10 mM Tris pH 8.6, 1 mM EDTA saturated)-chloroform-isoamyl alcohol (25 : 24 : 1) followed by 2 extractions with chloroform-isoamyl alcohol (24 : 1) and two extrac- tions with ether. The ether was then blown off with nitrogen gas. The purified RNA and DNA solution was digested with 40 ~g/ml RNase A [that had been heated in acetate buffer (pH 5) to 80°C for 10 rain]

for 30 rain on ice. Three additional phenol-chloro- form-isoamy! alcohol (25 : 24 : 1) extractions were carried out, followed by two chloroform-isoamyl alcohol (24 : 1) extractions. The DNA was then pre- cipitated at room temperature by bringing the salt concentration to 0.3 M with sodium acetate (pH 7) and adding 0.54 vol. isopropanol. The DNA was spooled on a glass rod and resuspended in 1 mM Tris pH 7.4, 0.1 mM EDTA and stored at 4~C.

(c) RNA isolation

Total RNA was isolated from Black Mexican maize tissue culture cells developed by C.E. Green (Univer. sity of Minnesota) and grown at 28°C in a modified Murashige and Skoog (1962) medium. The culture was f'fltered through a 25/~m Nitex filter. Cells were then ground to a powder in a Waring blender with hq- uid nitrogen for 3 rain.

For in situ hybridization 5S rRNA was isolated as follows: The powder was resuspended in a buffer con- tabling 50 mM Tricine pH 6.8, 1% SDS, I% mercapto- ethanol and 0.1% diethyl pyrocarbonate (Sigma). To this was added 1 vol. of phenol saturated with 0.1 M NaCI, 10 mM "Iris pH 8.6, 1 mM EDTA. The mix. tare was shaken for 10 rain and centrifuged at 9 000 rev./min for 30 rain in a Sorvall RC-2B. The aqueous phase was re-extracted two times with equal volumes of saturated phenol-chloroform-isoamyl alcohol (25 : 24 : 1). The RNA was precipitated with 2 vols. of ethanol, centrifuged at 20000 Xg for 20 rain, resuspended in distilled water and purified by centri- fugation on a 5-30% sucrose gradient using a SW27 for 17 h at 26000 rev./min. The small RNAs, 5S, 5.8S and 4S found near the top of the gradient were pooled, ethanol-precipitated, and centrifuged at 20 000 X g for 20 rain. The pellet was resuspended in distilled water.

5S rRNA was isolated from ribosomes, labeled and used as hybrid'w.ation probes on Southern genomic

blots to screen for genomic clones of 5S rDNA. The ribosomes were isolated by suspending the powdered cells in 50 mM Tricine pH 6.8, 1 rnM EDTA, 0.1%/3 mercaptoethanol, 0.25 M sucrose, 10 mM MgCl2,

'0.1 M NaCI, 0.1% diethyl pyrocarbonate; the large particles were removed by centrifugation at 8000 rev./min for 10 min. The ribosomes in the superna- tant were then pelleted through a 2 ml, 50% sucrose cushion at 50 000 rev./min in a Beckman 40 rotor for 3 h at 4°C. The ribosome pellet was suspended in 50 mM Tris pH 7.4, 100 mM NaCl, 10 mM EDTA, 0.5% SDS and digested with 0.1 mg/ml proteinase K for 1 h at 37°C. The RNA was extracted with phenol-chloroform-isoamyl alcohol ( 2 5 : 2 4 : 1) and precipitated by adding 2 vols. of 95% ethanol. The pellet was resuspended in 0.3 M Na • acetate pH 5 and the large RNA precipitated at room tempera- ture by addition of 0.54 vol. of isopropanol. The supernatant fraction contained 5S and a small amount of 5.8S and tRNA. This was precipitated by addition Of 1 vol. of isopropanol. The small RNAs were resuspended in water and run preparatively in a 10% Tris-glycine SDS-polyacrylamide gel (LaemmLi, 1970). The 5S band was cut out, the polyacrylamide t~ulverized by passage through a 16-gauge needle, and staked in water overnight. The RNA was alcohol- p,ecipitated, pelleted, resuspended in water and run over 5-30% sucrose gradient at 50 000 rev./min in a Beckman SW50.1 at 4°C for 18 h. A sample of this material was tested in a polyacrylamide gel for purity. The RNA remained intact and showed few contain- inating tRNAs. Hybridization to genomic blots dem. onstrated the presence of slight 5.8S, 17 and 26S rRNA contaminants. These were competed out in our hybridization experiments by addition of a 500-fold excess of mixed 17, 26 and 5.8S rRNAs. We found it necessary to obtain probes for the genomic blots from purified ribosomes; the 5S rRNA isolated for the in situ hybridization contained minor RNA con- taminants that could not be competed out with 17, 26 and 5.8S rRNAs.

(d) Labeling of RNA and nick translation of cloned DNA

12sI-labeling of the RNA was carried out accord- ing to the procedure of Commerford (1971). The reaction consisted of 1/~g RNA, 8 mM T1CI~, 50 raM Na. acetate pH 4.7 and 1 mCi 12sl (New England Nu-

10

clear, NEZ033L). The reaction was allowed to pro- ceed for 30 rain at 65°C. The reaction was stopped by placing in i ~ for 30 min; then brought to 10 mM Tris, 1 mM EDFA pH 7.9 and placed at 65°C for 30 min. ~ mi.~ture was chromatographed through Sephadex G-2~ to separate RNA-bound 12Sl from free 12Sl, and the 12SHabeled RNA was centrifuged through 5-30% sucrose in an SW50.1 Spinco rotor at 50000 rev./min for 18 h. The peak of radioactiv- ity was precipitated with ethanol and resuspended in a small volume of distilled water. The resulting RNA was intact at this time and had a specific activ- ity of greater than l0 s cpm//~g. The RNA degraded ram~y and los~. specificity within 1 week after prep- ar~tion.

32P-end-labeling was performed by treating 1/~g of 5S rRNA with bacterial alkaline phosphatase (Worthington) in 10 mM Tris pH 7.9 for 90 rain at 37°C. This was followed by extraction 5 times with phenol (saturated as above) and ethanol precip- itation. The kinase reaction was performed by the method of Donnis-Keller et al. (1977). The RNA was precipitated with 2 vols. of ethanol, centrifuged and resuspended in 50 mM Tris pH 7.6, 10 mM MgCI:, 5 mM DTT, 0.1 mM spermidine, 0.01 mM EDTA. The RNA was added to a molar amount of [~/.32p]. ATP equal to the number of 5' ends in the 5S rRNA. Two units of T4 polynucleotide kinase (New England Biolabs) were added and the reaction allowed to pro- ceed at 37°C for 60 min. The 32P.labeled 5S rRNA was then separated from 32P-labeled ATP by chro- matography through Sephadex G.50. The specific activity ranged from 5 X 107 to 2 X 10 s cpm/~ of RNA.

Cloned DNA was nick-translated according to the procedure of Rigby et al. (1977).

(e) Restriction endonuclease digestion

Restriction enzymes were purchased from either Bethesda Research Laboratories (BRL) or New Eng- land Biolabs. Appropriate amounts of enzyme were added to the DNA in the buffer recommended by the supplier. Reactions were allowed to proceed for 1-5 h.

(f) Agarose gel electrophoresis

Horizontal gels were prepared and run in 90 mM Tris.base, 3 n ~ EDTA with 90 mM boric acid, pH

i

8.0 (Peacock and Dingman, 1967) and 0.5 /Jg/ml ethidium bromide. The 5'fl2p-labeled Z DNA restric- tion fragments were run as internal size standards.

(g) Transfer of DNA and hybridization

The Southern (1975) method was used to trans- fer DNA from agarose to nitrocellulose. Following .the transfer, the fdters were washed with 2 × SSCP (1XSSCP= 120 mM NaCI, 15 mM sodium citrate, 13 mM KH2PO4, 1 mM EDTA, pH 7.2) and baked at 80°C for 1.5 h in vacuum. Hybridizations were car- ried out in 50% formamide (MCB or Eastman) 5 X SSC? at 43°C for 4-18 h. The falters were then washed twice in either 50% formamide-5 X SSCP at 43°C or in 2 X SSCP at 65°C, 30 min each, washed once in 2 × SSCP at room temperature, blotted dry and ~xposed. Nick-translated probes were hybridized according to the method of Wahl et al. (1979)but no dext~an sulfate was added. Filters were exposed to preflashed Kodak XR5 film and exposed at -80°C with a Dupont lightning fast intensifying screen (Laskey and Mills, 1977).

(h) In situ hybridization

In situ hybridization was carried out as described by Phillips et al. (1979), with certain modifications. The translocation stocks T2-6(8786), T2-6(5419)and T3~(030-8) were grown in the field. Sporocytes were collected, fixed in 95% ethanol-acetic acid (3 : 1) and stored for 1 week. The hybridization was carried out in 50% formamide 5 × SSCP for 4 -6 h at 43°C. The RNA concentration was 1/~g/ml with 400/~g/ml unlabeled 17 and 26S rRNA as competitor. Expo- sures were for 1 week.

(i) Cloning in 0t27 vector

Lambda Charon 27 bacteriophage (Ch27) was pro- vided by Fred Blattner (University of Wisconsin). This bacteriophage was used to obtain a library of maize (W22) DNA fragments with a size of less than 9 kb. Maize (W22) DNA was digested to completion with BamHI, the fragments sedimented on a 10--40% sucrose gradient, and the DNA in the range of 0 - 9 kb was selected and used to prepare an equimolar mix- ture. These DNA fragments were ligated (BRL, T4- ligase) to Ch27 DNA cut with BamHI. The DNA was packaged into a X-vector library as described by Holm and Murray (1977) and Collins and Holm (1978).

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200000 phage plaques from an unamplified library were screened as described by Benton and Davis (1977). Sixteen clones were isolated and 25-ml cul- tu~s of each were prepared. The phage were purified by one round of CsC! banding; the partially pufiiied phage were treated with micrococcal nuclease, and the DNA was isolated. The DNA was precipitated with isopropanol az room temperature and resus- pended in 1.0 mM Tris pH 7.4, 0,1 mM EDTA and stered at 4°C.

RESULTS

(a) In situ hybridization

Conventional cytological procedures allow idemi- fication of each of the maize chromosomes. Proce- dures used for in situ hybridization, however, distort

chromosome morphology and make identification difficult. The report by Wimber et al. (1974)con- cerning the 5S rRNA gene location left doubt as to whether the genes were in chromosome 2 or 3. These authors did not attempt to pinpoint the genes specifi- caUy. We have used homozygous translocations to precisely determine the 5S rRNA gene location and readily identify the chromosome.

Fig. l a illustrates the chromosome configuration of T24~(8786). Breakpoints are at 2S,97 and 6S NOR thus bringing most of the short arm and all of the long ann of chromosome 2 into association with the NOR. Hybridization should therefore be at the distal tip of the chromosome associated with the nucleolus if the 5S rRNA genes are located in the long arm of chromosome 2. Fig. l b is a photomicrograph of a pachytene preparation of T2-6(8786). Silver grains are ~en near the end of the chromosome. Fig. I c is a photomicrograph of a whole cell at early diakinesis. Ten bivalents are clearly seen with most of the silver

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Fig. 2. In situ ~ybridization of [12si]5S zRNA to T2-6(5419). (A) Diagramatic representation of normal and tzanslocated chromosomes 2 ~ind 6 indicating the site of the 5S zRNA genes zelative to the breakpoint. (B) Pachytene preparation of the 62 chromosome shelving a silver grain cluster on the translocated piece of chromosome 2. (C) Diakinesis cell showing the 10 paired maize chromosomes with an aggregation of grains over the 65 bivalent associated with the nucleolus. (D) Higher magnitification of 62 at diakinesi~

Fig. 3. In situ hybridization of [12sI]5S rRNA to T3-6 (030-8) showing an aggregation of grains associated with a bivalent independent of the nucleolus.

grains at the distal tip of the bivalent associated with the nucleolus. The homozygous interchange T2.6 (5419) is illustrated in Fig. 2a. This chromosomal aberration brings the distal 18% of chromosome 2L into association with the NOR. A pachytene prepara- tion of this interchange (Fig. 2b) demonstrates that

13

the 5S rRNA genes are located approximately half- way between the breakpoint and the tip of t, he chro- mosome. Our measurements place the genes in the long arm of chromosome 2 at site 0,88 distal to the centromere (2L.88). Early diakinesis preparations confirm the location of the 5S genes near the tip of 2L and demonstrate no other conspicuous chromo- somal sites of 5S gene localization (Fig. 2C). Fig. 3 illustrates a pachytene preparation of T3-6(030-8). In this photomicrograph a normal chromosome 2 con- taining a site of hybridization is seen independent of the nucleolus and an aggregation of chromosomes.

(b) 5S rRNA gene repeat length

Genomic Southern blots allow direct visualization of gene organization. Knowledge of the DNA sequence modifications that inhibit certain enzymes allows some inferences regarding modification in the maize genome. Fig. 4 is a genomic blot of A188 maize DNA generated with several restriction enzymes and hybridized to [32p]5S rRNA. The TaqI restriction endonuclease defines the repeat, generat- ing one size of fragment approx. 320 bp long. The Mbol digest also generates only one visible fragment size class. In 2% gels of partially digested DNA (Fig. 5) it is clear that the sn,all Mbol fragment seen in Fig, 4 is 285 bp long. An additional fragment must be

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Fig. 4. Southern blot of maize rDNA exhaustively digested with the various indicated restriction enzymes and hybridized to [3~P]5'-end-tabeled 55 rRNA. The agarose gel was 0.5% in 90 mM Tris-bor~te pH 8.3 and run at 0.5 V/cm on a 30 cm horizontal

geL Direction of electrophoresis was from right to left.

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Fig. 5. Southern blot of maize DNA hybridized to [a2p] nick-translated X(W22)SR40 carrying inserts of the 5S rRNA gene of maize. The Mbol and Taql lanes represent partially digested DNA. The agarose gel was an 11 cm 2% gel in 90 mM Tris-borate pH 8.3 and run at 3 V/cm for 4 h. The frag- ment lengths are specified in bp on the left margin.

generated but is too small to bind to nitrocellulose. The enzyme BamHl generates a ladder of bands start- ing with the 320-bp monomer; the ladder contains all the multimers up to 20 times this size. To insure that the digest was complete the same DNA digest was

also hybridized to maize 32P-labeled 17 and 26S

rRNA (not shown). No ladders for the 17 and 26S rDNA were seen indicating that complete digestion of the maize DNA had occurred. The restriction enzymes EcoRl, HindIII and Xbal have no periodic site in the 5S rDNA repeat (Fig. 4). Alul and HaellI, although having no regular site in the 5S rDNA repeat, do generate several bands. Alul generates three conspicuous bands in register with the 8amHl ladder at the pentamer, hexamer and heptamer level. In addition to these, however, Alul and HaelH gen- erate some bands that do not seem to be in register with the 5S rDNA ladders (Fig. 4).

HpaH and Mspl recognize the same DNA sequence (CCGG). Hpall does not cut when the internal C is methylated (Roberts, 1980); Mspl does not cut when the external C is methylated (Van der Ploeg and Flavell, 1980). Hpall does not recognize a site in the 5S rDNA of maize. A ladder in register with the BamHl ladder (but generally higher in Mr than the 8amHl ladder) is generated by Mspl. The monomer band is almost totally missing in the Mspl digest. In the digestion with Hpall and Mspl, the maize DNA was restricted to completion; when additional enzyme was added, no change in pattern was seen. Several maize lines have been examined with respect to the size of their BamHl and Mspl 5S rDNA frag- ments. The lines Black Mexican and W22 are indis- tinguishable from A188. Thus, we presume that the results reported here may be extended to the 5S rDNA region of other maize strains.

(c) 5S rDNA in cloned fragments

120 clones containing 8amHl fragments homol- ogous to 5S rRNA were found in 200000 plaques examined from an unamplified lambda C'h27 library. The DNAs isolated from 16 of these clones indi- cated the presence of 53 rDNA and were examined.

11 contain 5S rDNA monomers, 7 of these 11 also contain DNA sequences in addition to 5S rDNA. 5 of the 16 clones contain multiples of the 5S rDNA repeat. In 3 of these a complete 8amHl digest pro- duces monomers; in one a dimer and in another a trimer are seen. Fig. 6a is a blot of three clones hy- bridized to 32p-labeled 5S rRNA. 8amHl cleaves at the site of insertion into Ch27. Clones ~(W22) 5R20 and ~0V22)5R40 generate only the monomers

when restricted with BamHl. Clone ~.(W22)SR59 generates a prominent monomer and trimer as well as a dimer, tetramer and pentamer. In addition, a small fragment (about 285 bp) was homologous to the probe. Clone X(W22)SR12 (not shown) contains a monomer and a dimer.

Double digestion of these clones with EcoRI and Hindlll (Fig. 6B) produces an intact insert plus 1944 bp of Ch27. This allows determination of the insert

15

size. Thus, excision in a monomer-containing clone by EcoRI and HindllI would produce a 2264-bp frag- ment. This is the observation with clone ~,(W22) 5R20. Clones ;k(W22)5R40 and ~,(W22)SR59 exhibit prominent heptameric and decameric insert frag- ments, respectively. Other fragments that differ in molecular weight in multiples of the 320-bp 5S rDNA repeat length are also clearly seen. We assume that un- equal crossing over in the Ch27 infected cells has led

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Fig. 6. Southern blot hybridization of the gel-fractionated restriction digests of ?~(W22)5R59, MW22)5R40 and 7,(W22)5R20 DNAs hybridized to [32P]5'-end-labeled 5S rRNA. (A) The DNAs were digested with BamHl and run on a 2% agarose gel in 90 mM Tris-borate pH 8.3. (B) The DNAs were double digested with EcoRI and HindllI and run on a 0.5% agarose gel in 90 mM Tris-borate pH 8.3. The fragment lengths are specified in bp on the left margins.

16

to this variation, since each fragment cloned in Ch27 was a discrete size, yet the inserts seen in the 5S rDI~ZA clones containing multimers of the 5S rDNA are variable in size. Thus, the size of the original insert is not known. In clone 3,(W22)5R59 we assume that ~ e original cloned sequence contained a region from which two BamHI sites were absent resulting in a trimer after BamHl digestion. By unequal crossing over in this region during its replication in E. coil, dimers, tetrame~s and pentamers were generated.

To confirm that the sequences in the clones repre- sent the ladders seen in the genome, ~,(W22)5R40 was nick-translated and hybridized to W22 DNA digested with BamHl, Mbol, Mspl or Taql (Fig. 5).

DISCUSSION

(a) Location

Genes coding for 5S rRNA are located at one or multiple sites in higher eukaryotes. In maize the ma- jority of sequences homologous to 5S rRNA is located in the long arm of chromosome 2, 88% of the distance out from the centromere. This is consistent with previous results (Wimber et al., 1974; Mascia et al., 1979; Steffensen and Patterson, 1979).

(b) Genomic blots

(1) Repeat length A simple 320-bp repeat of the 5S rDNA prevails in

corn. In genomic DNA, Taql digestion serves to define the repeat with most 5S rDNA restricted to the monomer size. Mbol also completely restricts the 5S rDNA region. Endonuelease BamHl recognizes a site in some, but not all 5S rDNA repeats. Thus, a ladder of bands in multiples of the 320-bp monomer is generated. Maize DNA restricted with HpalI remains largely uncut. Similarly, the 5S rDNA repeat has virtually no recognizable Hpall sites;MsFl, which recognizes the same sequence, generates a ladder of bands similar to, but higher in molecular weight on the average than, the BamHl ladder. Such observa- tions have not been reported in Drosophila (Artavanis- Tsakonas et al., 1977) or Xenopus (Miller et al., 1978). Bird et al. (1979) observed ladders in the 5S rRNA genes of sea urchin with both Hpall and

Mspl. Three explanations for the ladders of bands are possible: (1) partial digestion, (2) divergence, or (3) post-replication modificition of bases. Controls ruling out partial digestion gere discussed in our results. The latter two will be considered further.

(2] [h'vergence The ladders seen in the BamHl and Mspl digests

are similar to those seen in highly repetitive (satellite) DNAs (Southern, 197.%; Bedbrook, 1980; Maio et al., 1977). In satellites, the phenomenon is hypothesized to be due to divergence of sequences followed by un- equal crossing over (Smith, 1973). The presence of several Alul and Haelll bands in Fig. 4 suggests that some divergence has occurred in the 5S rDNA region. The absence of ladders in the Mbol and Taql digests suggests that some sites are highly conserved. The structural region-spacer region arrangement of the 5S rRNA genes may account in part for these obser- vations. The rare AIuI(AGCT) and HaeIII(GGCC) sites may occur due to divergence in the 200-bp spacer region. The conserved MboI(GATC) site is known in rye, tomato, s~mflower, dwarf bean, broad bean and maize to be located within the 120-bp structural region at the BamHI(GGATCC) site (Hori and Osawa, 1979; and unpublished sequence data of D. Geraghty and I. Rubenstein). The conserved Taql (TCGA) site, however, maps in the spacer region (data not shown). Thus, not all the constant sites.are within the structural region or (as will be seen below) all the divergent sites within the spacer region.

The existence of highly repeated DNA that encodes an RNA product can be perceived from two viewpoints: (i) many copies of the gene may be need- ed to provide a large amount of product required dur- ing some phase of development. This hypothesis has been discussed extensively but has not been rigor- ously demonstrated (Finnegan et al., 1977; Brown, 1981); (ii) a certain number of genes may be required for optimal function, but these can be amplified and diverge with little negative selective effect on the organism. If the number of normal genes becomes low, compensation at the cellular level may occur. Orgel and Crick (1980) suggests that repeated DNA is similar to an innocuous parasite within its host. The relationship of repeated genes, such as 5S rDNA, to the genome may be considered more accurately as symbiotic, each insuring the survival of the other. A

17

substantial amount of divergence may be tolerable within this framework.

(3} Modifications Lack of digestion of maize DNA with Hpall dem-

onstrates that in virtually all 5S rDNA repeats the internal C of the CCGG site is modified; this is sup- ported by the fact that a double HpaH[Mspl digestion (not shown) does not restrict any further than the Mspl digestion alone. This is similar to the result reporeted by Miller ct ai (1978) in the Xenopus 5S rDNA repeat and is omsislent with reports that meCG is the predominant modi"ication pattern in animals (Bird and Southern, 1978) and plants (Gerlach and Bedbrook, 1979) and leads us to conclude that the modifications in the 5S rDNA of maize are meth- ylations. The meCG pattern does not, however, explain the results observed with Mspl [an isoschizo- mer of Hpall that recognizes the site when the inter- nal, but net the external, C is methylated (Van der Ploeg and Flavell, 1980)] and BamHl [that recog- nizes GGATCC except when the internal C is methyl. ated (Roberts, 1980)]. Analysis of the sequences of 5S rRNAs from 5 plant species (Hori and Osawa, 1979) and preliminary data on the sequence of a maize 5S rDNA clone (unpublished data of D. Geraghty and I. Rubenstein) reveal the presence of an overlapping Mspl, BamHl site in the 5S rRNA coding region (CCGGATCC). Double digests with BamHl and Mspl (not shown) suggest that this site is also present in maize, i.e., the ladder generated by the double digest is in register with either single digest. Symmetrical modification of either the BamHl or Mspl site would block both sites simultaneously. BamHI sites, however, are more frequent than MspI sites (Fig. 4). The simplest explanation for these results is that a modifying system modifies C's other than those of the CG doublet but with lower effi- ciency. This is presumably the situation in mouse- cell satellite DNA where 40% of 5-methylcytosine is in purine-SmeC-purine and the rest is in CT, CT3, C2T4, C2Ts, C3Ts oligonucleotides (l-larbers et al., 1975). Another possibility is that methylation at this site is not symmetrical.

An indirect effect of methylation is that spontane- ous mutation might be expected to occur at a higher frequency at sites where cytosines are methylated. In E. cell, Coulon&e et al. (1978) reported that divergence results from spontaneous deamination of

5-methylcytosine to thymine. This change cannot be corrected by DNA-uracil glycosidase and results in conversion of GC to AT. Such a system could have greater affect on the HpaII site than the BamHI site.

The function of modification is not clear. Regula- tory roles have been suggested (Holliday and Pugh, 1975; Riggs, 1975; Razin and Riggs, 1980). In this regard, the frequency of 5-meC has been observed to be higher in mouse satellite sequences than in main band DNA (Harbers et al., 1975). The DNA of the sez urchin can be divided into a methylated and a non- methylated class. Ribosomal and 5S rDNA are not methylated while satellite DNA is methylated (Bird et al., 1979). Van der Ploeg and Flavell (1980)have shown that the ~,5~ globin genes are not highly modi- fied in cells expressing the genes, while cells not expressing the genes are generally highly modified. On the other hand, methylation does not seem to im- pair correct transcription. Miller et al. (1978)report- ed that virtually all of the CG doublets in 5S rDNA are methylated in Xenopus laevis. This methylation does not inhibit transcription of the 5S rRNA genes in oocyte nuclei (Brown and Gurdon, 1977). In sum- mary, these findings suggest that methylation occurs to a greater extent on DNA that is not actively trans. crib~d, whether this proves to be true for the 5S rRNA genes remains to be shown.

(c) Cloned fragments

Maize DNA was restricted to completion with BamHl generating 5S rDNA fragments of 320 bp and multiples. Lambda Charon 27 bacteriophage was selected as a cloning vector since it accepts fragments less than 9 000 bp in size and because it has a BamHI site into which such fragments can be inserted. An unamplified library of approximately 2 X 106 clones was generated; 2 X l0 s plaques were screened for sequences homologous to 5S rRNA. Amplification can distort the frequencies of particular sequences since all clones may not have the same replication efficiency. In our screen of the unamplified library all clones were presumably different and all were present in the frequency at which they were packaged. We obtained 119 clones with homology to 5S rRNA and purified 16 of these to homogeneity. In bacteria and phage, tandemly repeated DNA sequences undergo unequal crossing over generating variation in sequence reiteration (Anderson and Roth, 1977). Thus, in the

genome the copy number of any given repeat can

18

vary up or down. When the copy number reaches one, the phage genome size would become stable. Since we sometimes inserted multiple copies of a gene, this type of repeat variation was able to occur in the 5S rDNA clones. When DNA was isolated from the 16 pure clones and restricted with EcoRI and HindIH (~_ese sites flank the BamHI site generating a 1944 bp fragment of Ch27 DNA) an intact 5S rDNA insert was excised. Thus, a clone containing a monomer (320 bp) yields a single 2264-bp fragment. Clones containing multiples of the 320-bp 5S rDNA mono- mer apparently are unstable. In gels, ladders of bands spanning upwards and downwards from a prominent fre4~nent are seen. Due to this instability it is not pos- sible to determine the size of the original insert. Clones containing monomers, for example, may be derived from an original clone of a multimer frag-

ment. By cloning fragments containing multiples of the

5S rDNA repeat the question of divergence vs. modi- fication could readily be resolved. If the cloned frag- ment contained a diverged site this would be reflected in the digest in the form of fragments larger than 320 bp. If the ladders were due to modification of the site in the maize DNA, however, all of the repeats should be restricted to the monomer level after growth in E. coil. DNA from the 16 purified clones was restricted with BamHl to excise precisely the insert. A 320-bp fragment homologous to maize 5S rRNA was com- mon to all clones. Seven clones had fragments in addi- tion to the 320-bp fragment that were not homolo- gous to 5S rRNA. We believe that in these clones two originally independent fragments were ligated together and inserted into Ch27. In two clones con- taining multiples of the 5S rDNA repeat, all repeats were restricted to the monomer level with BamHI. Thus, the BamHl sites must have been modified when they were present in the genome and replication of the maize DNA in E. coil gave rise to unmodified sites. Two clones containing multimers yielded mono- mers as weL as multilners of the 5S rDNA repeat. In these clone~ both divergence and modification appar- ently were present in the original maize fragment. Divergence ~s indicated by the absence of BamHI sites in certain tandem repeats (Fig. 6A); modification is indicated by the uncovering o~" BamHI sites by repli- cation of the cloned maize DNA in E. coil; presum- abl:~, post-replicational modification does not occur in F coli as it does in the maize cell. One of the

clones had a fragment smaller than 320 bp indicating that either a second BamHl site is present in some repeats or some repeats are shorter. This smaller frag- ment was not seen in the whole genome Southern blots, probably due to its low frequency at the mono- mer level. In clones containing multimers no diver- genre was seen at the Hpall site (not shown). We can- not infer anything concerning spontaneous muta- tional events at the HpaII site due to the small num- ber of repeats examined.

To confirm that the cloned fragments represented 5S rDNA repeats, nick-translated ?,(W22)5S40 was hybridized to a Southern blot of maize DNA digested with BamHl, Mbol, Mspl or Taql. This ?~ probe gave precisely the same result for these partial and com- plete digests as was seen using 32p-end-labeled 5S rRNA as a probe. The experiment also demonstrated that there was no significant homology between 5S rDNA sequences and other regions of the maize genome.

We conclude that the 5S rRNA genes are located in chromosome 2 of maize and represent a repeated gene system composed of 320-bp units which con- tain nucleotide divergence and are also subject to post.replicational modification.

ACKNOWLEDGEMENTS

This work was supported by grants from the Na- tional Science Foundation (PCM 76-02600 and PCM 79-12069) and the National Institutes of Health (GM 24756). Paper No. 11674, Scientific Journal Series, Minnesota Agricultural Experiment Station, St. Paul, Minnesota.

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Communicated by N. Fedoroff.