Repeated DNA sequences found in the large spacer of Vicia faba rDNA

Biochimica et Biophysica Acta 825 (1985) 411-415

411

Elsevier BBA 91502

R e p e a t e d D N A s e q u e n c e s f o u n d in the large spacer of Vicia faba r D N A A t s u s h i K a t o a, K i m i t a k a Y a k u r a b a n d S h i g e y u k i T a n i f u j i ~ Department of Botany, Faculty of Science, Hokkaido University, Sapporo 060, and b Department of Biology, Faculty of Education, Kanazawa University, Kanazawa 920 (Japan) (Received May 3rd, 1985)

Key words: rDNA heterogeneity; rDNA spacer; repetitive DNA; (V. faba)

In the large spacer of the rDNA of Vicia faba, multiples of a 0.32 kilobasepair (kb) sequence reiterate to various degrees. We sequenced the repetitious region consisting of the repeating sequences and its flanking regions using two cloned plasmids, which contain V. faba rDNA segments encompassing the whole region of the large spacer. The repetitious region was found to consist of multiple complete copies and one truncated copy of a 325 bp repeat unit and to be flanked by direct repeat sequences of about 150 bp. The set of direct repeats located at either side of the repetitious region differed from each other with about 10% sequence heterogeneity. However, nucleotide sequences of the direct repeats were well conserved between the two clones examined. Southern blot hybridization indicated a widespread distribution within the whole V. faba genome of some related sequences with high homologies to the 325 bp repeat unit and to the direct repeats.

Introduction The high degree of length heterogeneity of the repeating unit of rDNA observed in Drosophila [1-3], Xenopus [4,5] and mouse [6] is caused by repetition of certain repeated sequences. Considerable heterogeneity has been observed also in plants such as radish [7], barley [8,9], wheat [10], Trillium [11] and Vicia faba .[12-15]. Analysis of cloned wheat rDNA fragments indicated that a different degree of amplification of a 130 bp sequence caused the size variation of the nontranscribed spacer of rDNA [10]. In V. faba, a 325 bp [14] or 334 bp [15] repeat unit was characterized as a repetitive sequence causing the length heterogeneity of the large spacer. We previously obtained 16 cloned plasmids containing EcoRI-fragments of V. faba rDNA which encompassed the large spacer regions of various lengths. These clones fell into five classes differing from each other in the lengths of their inserts. Detailed analyses were carried out on the

structural organization of five clones, one from each size class: VER 1, VER 6, VER 14, VER 15 and VER 16. The 325 bp repeat unit found in clone VER 15, which was defined by MluI or AccI digestion, had already been sequenced [14]. This repeat unit consisted of a doublet of an approx. 155 bp sequence and another 14 bp short sequence containing the target sequence of AccI. In the present study, both distal portions of the repetitious region consisting of tandemly repeating 325 bp unit sequences and its flanking regions in the clones, VER 1 and VER 6, were sequenced, thereby revealing the organization pattern of the entire sequence of the repetitious region. Further, the possible existence of some sequences homologous to the 0.32 kb rDNA repeats in the genomic portion outside of the rDNA was explored.

0167-4781/85/$03.30 ('~ 1985 Elsevier Science Publishers B.V. (Biomedical Division)

412 Materials and Methods

rDNA clones The origin of the cloned plasmids VER 1 and VER 6 used in the present study has already been described [13,14]. VER 1 and VER 6 contain 5.5 and 6.7 kb inserts of EcoRI fragments encompassing the whole large spacer of different lengths of V. faba rDNA. Plasmid D N A was isolated as described by Kahn et al. [16]. DNA preparation and analysis V. faba seeds were soaked for 24 h at 27°C, and embryonic axes were collected. Then a nuclei-enriched fraction was prepared, from which D N A was extracted and purified by CsCl-ethidium bromide centrifugation according to Bedbrook et al. [17]. D N A digestion with restriction endonucleases, gel electrophoresis on agarose or polyacrylamide gels, transfer of D N A fragments to nitrocellulose filters and labeling of 5'-ends of D N A fragments and rRNA with [T-32P]ATP were carried out as previously described [11A4,17]. Southern blot hybridization using 32p-labeled r R N A probes was carried out in a hybridization solution containing 5 × SSC and 50% formamide. Prehybridization and hybridization solutions for DNA/DNA hybridization contained 4 × SSC, 0.1% SDS, 1 0 × D e n h a r d t ' s solution, 50% formamide and 100 ~ g / m l denatured E. coli DNA; hybridization was carried out at 37°C according to Southern [19]. Sequence analysis D N A fragments were labeled at their 5'-ends, using [7-3-~P]ATP and T4 polynucleotide kinase [20,21]. Two labeled 5'-ends were separated by cleaving the fragments with certain restriction endonucleases or separating two strands on neutral polyacrylamide gel after heating in dimethyl sulfoxide or after alkaline treatment. DNA was sequenced by the method of Maxam and Gilbert [22]. Results and Discussion

We previously reported [14] that complete digestion with MluI of r D N A segments inserted in cloned plasmids of five sizes, VER 1, VER 6, VER

14, VER 15 and VER 16, yielded multiple 0.32 kb fragments and probably one copy of a 0.35 kb fragment, in addition to 0.5, 0.7, 3.4 and 5.8 kb fragments. Physical maps were also constructed on the basis of results of Southern blot hybridization with MluI partial digests of these cloned plasmids [14]. However, the position of the 0.35 kb Mlul fragment relative to the multiple 0.32 kb Mlul fragments had not been clearly indicated. Similarly, complete digestion with Accl of the rDNA inserts of these plasmids yielded 0.3, 0.6, 1.0 and 2.9 kb fragments. The relative positions of these fragments had been determined by the Southern hybridization with partial digestion products. However, subsequently the Accl fragments appearing as a broad 0.3 kb band on 1% agarose gel were shown by electrophoresis on 5% polyacrylamide gel to consist of multiple 0.32 kb fragments and probably one copy of a 0.27 kb fragment. Alignmerit of the 0.35 kb Mlul fragment or 0.27 kb Accl fragment with the 0.32 kb Mlul fragments or 0.32 kb AccI fragments was achieved in the present study. Fig. 1 shows the whole nucleotide sequence of the repetitious region in the large spacer and its flanking regions, which had been cloned in pBR 325 as the clone VER 1. The data indicated that the repetitious region of VER 1 consisted of one truncated and two complete copies of the 325 bp sequence: this repetitious region was flanked by about 150 bp incomplete direct repeat sequences. It remains undetermined whether the two- and three-nucleotide sequences, TG and CCG respectively, near the borders between the repetitious region and the two flanking direct repeats are distal portions of the former or those of the latter. Therefore, it is impossible to indicate the precise positions of the 5'- and 3'-termini of the direct repeats. The nucleotide sequences of a pair of direct repeats in VER 1 differed from each other in having single-base changes at twelve sites and single-base deletions at five ~ites (Fig. 1C). The results of sequence analysis with VER 6 indicated that the repetitious region in VER 6 consisted of six tandem copies of the 325 bp repeat unit and a copy of the truncated sequence with a size of about 190 bp. This longer repetitious region of VER 6 was also flanked by the 150 bp direct repeats.

413

A

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AGACAAGTCC

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CTATGTGTTA

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GTTTGCCTCC CGAAACTCCT

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Fig. 1. A. Nucleotide sequence of the repetitious region containing 325 bp repeat units and its flanking regions inserted in a cloned plasmid, VER 1. Arrows indicate direct repeat sequences. Triangles represent possible 5'- and 3'-ends of the direct repeats. M l u l and Accl cleavage sites are marked M and A. B. Sequencing strategies and symbolized structure. Restricted fragments were labeled at their 5'-ends and sequenced along the directions indicated by arrows. Solid black boxes indicate portions of the rRNA coding regions, bold lines represent direct repeat sequences and dotted areas represent the repetitious region, As described previously [14]. the 325 bp repeat unit consists of a doublet of an approx. 155 bp sequence and another short sequence including an Accl target sequence. Therefore, these two 155 bp subrepeats are distinguished by the different sizes of dots, and the 13 bp short sequences are indicated by narrow hatched areas. The sequence of this short segment had been previously determined to be 14 bp in VER 15. In the case of VER 15, there was a C between nucleotides 2(G) and 3(A) after the end of the Accl target sequence (GTAGAC) in the repetitious region: in VER 1 and VER 6 there was a T between nucleotides 8(G) and 9(G) after the end of the Mlul sequence. Moreover, an ambiguous region in VER 15, 5'-G***C-3', was now determined to be 5'-GGGG(]CC-3' in VER 1 and VER 6. Therefore, the number of nucleotides in the 0.32 kb repeat unit is identical among these 3 clones. Again, M and A are the sites cleaved by Mlul and Accl. Numerals represent molecular lengths expressed in kb. C. Comparison of nucleotide sequences between direct repeats in VER 1 and VER 6. The upper and lower sequences correspond to the direct repeats contained in the 0.27 kb Accl segment and the 0.35 kb Mlul segment (of. B). Unclear 5'- and 3'-distal portions of the direct repeats are boxed. Hyphens indicate the same nucleotides. Plusses indicate deletions of nucleotides. Percent values indicate the degree of sequence homology between the sequences.

414 The nucleotide sequence of the 325 bp repeat unit was identical in VER 1 and VER 6, but different at three sites from that of VER 15, which had been previously determined [14] (see the legend for Fig. 1). The nucleotide sequence of the 150 bp direct repeat located in the 0.27 kb AccI fragment was identical in VER 1 and VER 6. Similarly, the direct repeat located in the 0.35 kb Mlul fragment of the opposite side was again identical in both clones. The structural organization of the repetitious region may be identical also in other cloned plasmids of various sizes, such as VER 15 and VER 16, because digestion with MluI consistently yielded the 0.35 kb fragments, in addition to the 0.32 kb ones. The former 0.35 kb MluI fragments hybridized efficiently to 32P-labeled 0.27 kb Accl fragments obtained from VER 1 or VER 6. Since the 0.27 kb AccI fragments contain one sequence of the 150 bp direct repeats, the 0.35 kb Mlul fragments from other cloned plasmids each may have also the direct repeat sequence. This compound structure, consisting of a repetitious region and its flanking regions of 150 bp direct repeats, resembles the long terminal repeats of retrovirus, copia-like elements in Drosophila and Tys of yeast transposable elements. However, the 150 bp direct repeats found in the V. faba r D N A spacer differ from the long terminal repeat sequences in lacking inverted sequences within each direct repeat and also in lacking short direct repeats of the target D N A sequence for insertion. However, a recent study with Staphylococcus aureus transposon indicated that its transposition does not generate any short direct repeats of the target sequence [22]. Therefore, the absence of such short direct repeats at both termini of the V. faba transposon-like structure would not disprove our hypothesis that the structure consisting of the repetitious region and the 150 bp direct repeats might be a transposed D N A sequence. Fig. 2 shows the Southern blot hybridization that was carried out with V. faba or wheat DNAs digested with some restriction endonucleases using 32p-labeled probes of the 325 bp repeat sequence and V. faba 18 S rRNA. When the 325 bp repeat sequence was used as the probe, very broad distributions of hybridized D N A fragments were observed with V. faba D N A digested with EcoRI, HindIlI and BamHI. By contrast, wheat DNA

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4.6 3.7-

1

2

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4

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Fig. 2. Southern blot hybridization of V. faba and wheat nuclear DNA digested with restriction endonucleases to 32P-labeled probes of the 325 bp repeat sequence and l~i faba rRNA. The 325 bp repeat sequence was hybridized to V. faba DNA digested with EcoRI (lane 2), HindIII (lane 3) t)r BamHl (lane 4) or to wheat DNA digested with EcoRl (lane 5). l~faba 18 S rRNA was hybridizedto V. faba DNA digested with EcoRl (lane 1). Numerals on the left-hand side indicate the molecular lengths of the rDNA fragments expressed in kb.

digested with EcoRl, Hindlll or B a m H I did not hybridize to the 32p-labeled V. faba 325 bp sequence. Therefore, the hybridization patterns obtained with V. faba D N A digests are not due to nonspecific hybridization. D N A fragments of V. faba rDNA cleaved by EcoRI appeared in the Southern blot hybridization using 32P-labeled 18 S rRNA as a 3.7 kb discrete band and a somewhat broad band ranging from 4.6 to 9.2 kb, due to the length heterogeneity of the nontranscribed spacer [14]. Therefore, extensive broad distribution of D N A fragments hybridized with the labeled 325 bp sequence indicates a widespread occurrence of the 325 bp repeat sequence or some related sequences within the whole genome of V. faba. Similar hybridization patterns were obtained in the Southern blot hybridization using the 32p_ labeled 0.27 kb AccI fragment which contains the 150 bp direct repeat (data not shown). These resuits support the possibility of transposition of the

415

325 bp repeat sequences within the genome and from the extranucleolar nucleoplasmic chromatin into the nucleolus-associated, rDNA-containing chromatin, though the alternative possibility of their transposition from the rDNA-chromatin to other genomic portions still remains. The phenomenon of transposition of some repetitious sequences into the nontranscribed spacer of r D N A has been reported in Chironornus thummi thummi [23], in which the repetitive sequence, 120 bp ClaI-fragments, were shown to be closely related to the highly repeated centromeric sequence. The sequencing data with VER 1 and VER 6 further revealed the following noticeable features: the 77 80 bp sequence from the cleaved end of the first AccI site up to the 5'-terminus of the 150 bp direct repeat and the 16-18 bp sequence from the next base of the 3'-terminus of another direct repeat up to the first base of the last MluI target sequence were identical in their nucleotide sequence at least between VER 1 and VER 6 (Fig. 1). This suggests that an initial transposon-like structure, consisting of a certain number of copies of the 325 bp repeat unit and one truncated sequence of the unit, had been inserted at a certain site in the large spacer of r D N A and then descendant transposon-like structures with the 325 bp repeat units of various copy numbers appeared. An alternative explanation is the transposition of transposon-like structures with various copy numbers of the repeat unit into the large spacers of m a n y r R N A genes. However, it seems less probable that all the transposon-like structures had been inserted at certain specific sites in the large spacer regions of various r R N A genes. As mentioned earlier, the nucleotide sequences of the 325 bp repeat units and the remaining portions of the transposon-like structures were essentially identical between VER 1 and VER 6. These facts allow us to speculate that initially a type of transposonlike structure was inserted into the large spacer of rDNA, followed by generation of such structures with varying numbers of the 325 bp sequence, probably via unequal crossing over events.

Acknowledgements This study was supported by Grants-in-Aids from the Ministry of Education, Science and Culture of Japan.

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