Structure of the 5 s rRNA genes in birch (Betula papyrifera) and alder (Alnus incana)

D. A. JOHNSON,' C. C-Y. CHAN, S. G. GOTTLOB-MCHUGH, K. MACKENZIE, L. MARENGERE, AND M. C. PRLTD'HOMME

Ottawa-Carleton Institute of Biology, University of Ottawa, Ottawa, Ont., Canada KIN 6N5 Corresponding Editor: P. B. Moens

Received July 4, 1991 Accepted October 18, 1991

JOHNSON, D. A., CHAN, C. C-Y., GO-ITLOB-MCHUGH, S. G., MACKENZIE, K., MARENGERE, L., and PRUD'HOMME, M. C. 1992. Structure of the 5s rRNA genes in birch (Betulapapyriferq) and alder (Alnus incana). Genome, 35: 337-341.

Hybridization of a 5s rDNA probe to Southern transfers of birch (Betula papyrifera) or alder (Alnus incana) DNA digested with BamHl reveals similar triple-band "ladder-like" patterns. The sizes of sequenced 5s repeat units from both plants ranges only from 471 to 490 base pairs, suggesting that the complexity detected by Southern analysis is not due to different size classes of 5s repeats as found in other species. Within the intercistronic spacer region, conserva- tion of large blocks of sequence between birch and alder 5s is observed implying a close evolutionary relationship between these two species. In both species, a duplication of part of the coding sequence including a restriction site for BamHl introduces a second BamHl site into the repeat unit. Differential methylation of the two BamHI restriction sites can account for the observed triple-band pattern.

Key words: 5s rDNA repeat, sequence, methylation, birch, alder.

JOHNSON, D. A., CHAN, C. C-Y., GO'ITLOB-MCHUGH, S. G., MACKENZIE, K., MARENGERE, L., et PRUD'HOMME, M. C. 1992. Structure of the 5s rRNA genes in birch (Betulapapyrifera) and alder (Alnus incana). Genome, 35 : 337-341.

L'hybridation d'une sonde d'ADNr 5s a des transferts Southern d'ADN de bouleau (Betula papyrifera) et d'aulne (Alnus incana) digeres par l'enzyme BamH1 a rCvC1C des profils similaires en bandes triples d'aspect scalaire. Les dimen- sions d'unitCs de rCpCtition 5s sCquencCes de ces deux espkces n'ont variC que de 471 a 490 paires de bases, ce qui suggkre que la complexitC dCcelCe par les analyses Southern ne serait pas due a des classes diffbrentes de dimension des rCpCtitions 5S, comme cela a CtC avancC pour d'autres espkces. A l'intbieur des rCgions d'espacement intercistro- niques, de larges blocs sont conservCs entre les 5s du bouleau et de l'aulne, ce qui implique une Ctroite relation Cvolu- tive entre les deux espkces. De plus, chez les deux espkces, une duplication d'une partie de la sequence codante, incluant un site de restriction pour l'enzyme BamHl, introduit un second site de restriction pour cette enzyme dans l'unitC de rCpCtition. Une mkthylation differentielle de deux sites de restriction de BamH1 peut expliquer le profil en bandes triples observe.

Mots elks : rCpCtition d'ADNr 5S, sCquence, methylation, bouleau, aulne. [Traduit par la rCdaction]

Introduction The 5 s rDNA genes of higher plants, as in most higher

eucaryotes (Long and Dawid 1980), are organized into clusters of tandemly repeated units. Each repeat consists of a highly conserved 5 s rRNA coding region of approximately 120 base pairs (bp) in length (Wolters and Erdmann 1988) separated by an intercistronic spacer sequence that, in general, lacks sequence homology between different species. This organization has been confirmed by sequence analysis of 5 s repeats from a variety of plants including wheat (Gerlach and Dyer 1980), corn (Mascia et al. 198 l) , flax (Goldsbrough et al. 1982), lupin (Rafalski et al. 1982), rice (Hariharan et al. 1987), barley (Khvyrleva et al. 1988), pea (Ellis et al. 1988), petunia (Frasch et al. 1989), rye (Reddy and Appels 1989), and soybean (Gottlob-McHugh et al. 1990). Furthermore, analysis of Southern hybridization data suggests that the same organization is present in over 30 species of higher plants (Gottlob-McHugh et al. 1990).

Plant DNAs are known to be highly methylated at C res- idues within the sequences CpG and CpNpG (Gruenbaum et al. 1981). When plant DNA is digested with a methylation-sensitive restriction enzyme such as BamHI or Mspl and hybridized with a 5 s rDNA probe, complex pat- terns resembling ladders are observed. These patterns are due to differential methylation of the enzyme's recognition

' ~ u t h o r to whom all correspondence should be addressed. Printed in Canada / lmprime au Canada

sites within the tandemly repeated 5 s rDNA units. In addi- tion, the presence of two size classes of repeat within a single species, resulting from differences in the sizes of the spacer regions, may result in double-band hybridization patterns (Gerlach and Dyer 1980; Khvyrleva et al. 1988; Reddy and Appels 1989). In a recent survey of over 30 plants, several examples of double- and triple-band hybridization patterns were observed (Gottlob-McHugh et al. 1990), suggesting that repeat unit heterogeneity was being detected. We have cloned and sequenced 5 s rDNA repeats from the two species that had demonstrated a triple-band pattern, namely, speckled alder (Alnus incana) and paper birch (Betula papyrifera). In contrast with most plant species, alder and birch share large blocks of highly conserved sequence within the intercistronic spacer region. Furthermore, our data sug- gests that the triple-band pattern is not due to multiple size classes within the 5 s repeat units of these species, as found in olher plants. Instead the complex Southern patterns can be explained by differential methylation of the two BamHl sites located within the 5 s rDNA repeat units.

Material and methods DNA was prepared by the method of Hattori et al. (1987) from

the leaves of either speckled alder (Alnus incana) grown from seed purchased from F. W. Schumacher Co. Inc., Sandwich, Mass., U.S.A., or paper birch (Betula papyrifera), which was collected locally.

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GENOME, VOL. 35, 1992

FIG. 1. Southern hybridization analysis of the birch 5 s repeat structure. Birch DNA (1 pg) was digested with the appropriate enzyme, electrophoresed, transferred to BIOTRANS membrane, and hybridized to the soybean 5 s rDNA repeat probe. DNA was restricted with the following enzymes: lane 1, BamHl; lane 2, Sau3A; lane 3, Bstl . The molecular weight standards are lambda DNA digested with Hind111 and pBR322 DNA digested with TaqI.

5 s rDNA repeats were cloned by ligating BamHl digested, purified DNA to BamHl digested, calf intestinal alkaline phos- phatase (Boehringer Mannheirn) treated pGEM4Z plasmid (Promega). The ligated DNA was transformed into E. coli strain DH5a mcr (BRL) (Maniatis et al. 1982). 5 s rDNA sequences were detected by colony hybridization using a labelled soybean 5 s rDNA probe

and subcloned into M13mp18 or M13mp19 (Yannish-Perron et al. 1985) for sequencing. The DNA techniques for restriction anal- ysis, Southern hybridization (Southern 1979), and sequencing (Sanger et al. 1977) have been described previously (Gottlob- McHugh et al. 1990). DNA analysis was performed using PCGene (IntelliGenetics) or Pustell (IBI) software.

Results and discussion When birch or alder DNA was digested with BamH1 and

hybridized to the soybean 5s probe, a ladder pattern similar to that seen in other plants is observed; however, each rung on the ladder could be resolved into three bands (Fig. 1; see also Gottlob-McHugh et al. 1990). For birch the sizes of these bands calculated from Southern transfers are 388, 512, and 632 bp for the first rung; 875, 1000, and 1060 bp for the second rung; 1375, 1500, and 1625 bp for the third rung; and so on. In wheat (Gerlach and Dyer 1980), barley (Khvyrleva et al. 1988), and rye (Reddy and Appels 1989) sequence analysis has demonstrated that double-band pat- terns are due to the presence of two repeat classes arising from differences in the sizes of the spacer regions. How- ever, the values obtained with birch DNA are not easily accommodated by a model proposing the presence of three different size classes of repeat unit, since the calculated sizes are not multiples of the monomer sizes. In addition a band with an estimated size of 125 bp can be seen on some transfers (Fig. 1). Digestion with BstI, an isoschizomer of BamH1 that is not methylation sensitive (Kessler and Manta 1990), did not reveal the ladder pattern (Fig. 1). This result confirms that methylation of the BamHl recognition site is important for the production of the ladder pattern and that frequent mutation of BamH1 restriction sites within the birch 5s repeat has not occurred. The presence of Bstl frag- ments with sizes of 388 bp (strong hybridization) and 512 bp (less intense band) does suggest that there is some sequence heterogeneity within BamH 1 recognition sites. Similarly, restriction with Sau3A that is methylation sensitive (Kessler and Manta 1990) resulted in a ladder pattern (Fig. 1); how- ever, digestion with Mbol, its isoschizomer that is not sen- sitive to methylation of C residues (Kessler and Manta 1990), produced bands of 390 and 121 bp (data not shown). Restriction of alder DNA resulted in patterns similar to those of birch DNA (Fig. 1) with bands of identical sizes. These Southern hybridization results suggest that in birch and alder the 5 s genes are clustered, tandemly repeated, and highly methylated at sites recognized by BamHl, Sau3A, and also HpaII and MspI (data not shown).

To understand the molecular basis for the observed South- ern hybridization patterns and to investigate repeat hetero- geneity, several repeat units from birch and alder were cloned and sequenced. These were aligned with a birch clone that contained one entire repeat unit of 488 bp (clone BIRCH

of Fig. 2). By comparison to previously sequenced plant 5 s genes, we infer that the coding region starts with GGG for birch and AGG for alder (labelled position + 1). The 3 ' end of the coding region is likely located within a stretch of

- -

C residues between + 116 and + 121 for a maximum 5s rRNA size of 121 bp. A putative promoter sequence similar to the AT-rich sequence required for transcription in Neurospora (Selker et al. 1986) and found in many other plants (Hemleben and Werts 1988; Rafalski et al. 1982; Ellis et al. 1988; Scoles et al. 1988; Gottlob-McHugh et al. 1990)

(the 330-bp fragment described in Gottlob-McHugh et al. 1990) was identified at - 29 to - 24 (ATATAA for birch and

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340 GENOME, VOL. 35, 1992

TTATAA for alder). Clusters of T residues were found 3 ' to the coding region (+ 123 to + 155). Such T-rich sequences have been implicated in transcription termination (Korn 1982). Blocks of T residues seen in alder and birch are also found in many plant 5 s repeats (Hemleben and Werts 1988; Scoles et al. 1988). In birch, a C-rich block ( + 139 to + 143) is found within this region as has been previously observed for 5 s rDNA repeats from wheat (Gerlach and Dyer 1980) and rice (Hariharan et al. 1987). This C-rich block is not found in alder.

As expected, the 5 s coding sequences are highly conserved within and between the two species. The coding regions are highly homologous to other plant 5 s rRNA gene sequences and can be folded into the typical secondary structures proposed for 5 s rRNAs (Vandenberghe et al. 1984; Wolters and Erdmann 1988). The alder repeats are almost identical in sequence, varying at only four positions. Of interest is the G residue within the 5 s coding sequence at + 71. Some secondary structure models of plant 5s rRNA have an unusual AC base pair at this position (Wolters and Erdmann 1988), although alternative arrangements of this region have been proposed (Nalaskowska et al. 1991). In alder the normal GC base pairing is found. In birch, repeats are more variable than in alder; however, the observed variability among birch repeats is largely localized to a region of the spacer between 260 and 302 bp of the sequence BIRCH in which many substitutions and insertions/deletions can be found. Out- side of this block, the birch spacer regions have long stretches that are almost identical in sequence. For example, between 303 and 466 bp there are only five positions with substitu- tions. This variability is comparable with the variation seen within the 5 s coding sequence (4/ 120). Spacer regions show little sequence homology between species and, outside of regulatory signals, do not code for any known function. Therefore, gene conversion may play a significant role in maintaining the sequence homogeneity found within large regions of the birch 5 s rDNA spacer. In five species of the Triticeae, duplications of the coding sequence near the 3 ' end were observed (Gerlach and Dyer 1980; Scoles et al. 1988). In both birch and alder a 16-bp region near the 5 ' end of the coding sequence (GCACCGGATCCCATCA from + 26 to + 4 I), including the underlined BamH 1 restriction site, is duplicated within the spacer region 5 ' to the gene (sequence BIRCH of Fig. 2 from 484 to 11 in the adjacent repeat). As a result of this duplication there are two BamHl sites per repeat unit. The presence of the duplication in both birch and alder suggests that it arose prior to the divergence of the two species and has subsequently accumulated muta- tions in different isolates. In addition, the similarity between birch and alder spacer sequences (regions 303-348 and 460-15 in the adjacent unit) also indicates a close evolu- tionary relationship between the two species.

The size of the birch 5s rDNA repeat ranged from 471 to 490 bp. The sizes of the alder 5 s repeats were 479 and 480 bp. This level of heterogeneity is insufficient to explain the observed triple-band pattern. However, the presence of two BamHl sites within the repeat unit could account for this pattern. Unlike other 5 s repeat sequences that contain a single BamHl site within the coding region ( + 3 1) (Wolters and Erdmann 1988), the birch and alder sequences contain a second site ( - 73) (see Fig. 2). Digestion of unmethylated

DNA would result in BamHl fragments of 102 bp (small fragment, "S") and 386 bp (large fragment, "L") for isolate "BIRCH"; however, partial methylation of both BamHl sites would give rise to a series of bands with sizes of 102 bp (S), 386 bp (L), 488 bp (S + L), 590 bp (2s + L), 874 bp (S + 2L), 976 bp (2s + 2L), 1078 bp (3s + 2L), and so on. These sizes closely agree with those obtained from Southern hybridization data.

Previously, complex Southern patterns were thought to be due to the presence of different size class of 5 s repeat, e.g., the "long" and "short" repeats in rye (Reddy and Appels 1989). However as demonstrated above, .the pres- ence of two partially methylated BamHl sites within the birch or alder repeat unit can explain the observed ladder pattern seen in Fig. 1 and the presence of different size classes of repeat need not be invoked. In addition, the com- plexity of the 5 s rDNA BamHl patterns observed in some other species may in part be due to the presence of addi- tional restriction sites within the 5s repeat units.

Acknowledgements The authors thank P. Brunon and J. Helie for process-

ing of material for figures. We are indebted to Dr. J. Hattori for aid with computer programs and helpful discussions. This work was supported by a grant from the Natural Sciences and Engineering Research Council of Canada (NSERC). S.G. Gottlob-McHugh was a holder of an NSERC scholarship. These sequence data will appear in the EMBL/GenBank/DDBJ nucleotide sequence databases under the accession numbers M68868 sdsald and M68869 sdsbir .

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