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Organization and sequence analysis of the 5S rRNA genes in Brassica campestris
plan ience ELSEVIER SCIENTIFIC PUBLISHERS IRELAND
Plant Science92 (1993) 47-55
Organization and sequence analysis of the 5S rRNA genes in Brassica campestris S a b h y a t a B h a t i a a, K a p i l S i n g h b, V. J a g a n n a t h a n a, M a l a t h i L a k s h m i k u m a r a n *a aBiotechnology Division, Tata Energy Research Institute, 90 Jor Bagh, New Delhi 110 003 India hCentre for Plant Molecular Biology, Tamil Nadu Agricultural University, Coimbatore 641 003 India
(Received 16 December 1992; revision received 24 March 1993; accepted 28 April 1993)
Abstract
The 5S rRNA gene from Brassica campestris has been cloned, sequenced and characterized. The 5S rDNA repeat unit is 495 bp in length. It consists of a highly conserved, 119 bp coding region and a variable non-coding spacer region which separates it from the coding region of the next repeat unit. The 5S rDNA units are organized into clusters of tandem repeats. Sequences responsible for initiation and termination of transcription of the 5S rRNA are present within the repeat unit. Hybridization data with isoschizomers reveals a high amount of 'C' methylation. The size of the repeat unit and the organization of the 5S rRNA genes in other closely related crucifers has been investigated and found to be quite similar to that of B. campestris. Key words: 5S rDNA; Tandem repeat; Brassica; Methylation
1. Introduction
The 5S rRNA genes in higher eukaryotes are present in multiple copies per genome and are generally organized into clusters of tandem arrays. Each repeat unit consists of a highly conserved coding region, approx. 120 base pairs long, which is transcribed into the 5S rRNA. Each repeat unit also contains a non-transcribed spacer region which separates it from the coding region of the next repeating unit [1-7]. This non-transcribed spacer varies from species to species both in sequence and length. Also, more than one variant * Corresponding author.
form of the 5S rDNA may exist within the same species as in flax [5], wheat [7] and Eruca sativa (unpublished results). These clusters of tandem arrays may be localized on either one or several chromosomes and are generally separate from the genes encoding the large ribosomal RNAs [1,8,9]. The 5S rRNA genes have been isolated and characterized from some plant species such as pea [1], yellow lupin [4], flax [5], mung bean [6], Matthiola incana [6], wheat [7], maize [10], tobacco [11], rice [12], soybean [13], rapeseed [14], barley [15], birch and alder [16] and Secale [17]. The transcription of the 5S rRNA genes and its regulation has been studied in organisms such as yeast [18], Drosophila [19] and Xenopus [20].
0168-9452/93/$06.00 © 1993 ElsevierScientificPublishers Ireland Ltd. All rights reserved. SSDI 0168-9452(93)03648-F
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The organization and chromosomal localization of the 5S rDNA repeats has been investigated by means of in situ hybridization and PFGE in plants like pea [1], wheat [9], rye [17] and tomato [21]. In these plant species it has been shown that the 5S rDNA exists as a cluster at one to three sites and are not interspersed with other ribosomal genes. In the present study, the 5S rRNA gene of Brassica campestris has been cloned and sequenced. The organization and methylation pattern of the 5S rDNA, in this species, has been analysed. Hybridization studies have also been carried out to investigate the organization of the 5S rDNA in related crucifers.
S. Bhatia cta/.
P[anl .~'ci 92
1993J 47-55
C-extra membranes (Amersham, UK) according to the method of Southern [24]. Hybridizations were carried out according to the procedure of Lakshmikumaran et al. [25]. The filters were hybridized to probes labelled with [c~-~-~P]dATP, [c~-32p]dGTP or [c~-32p]dCTP which were obtained from the Bhabha Atomic Research Center, Bombay, India. The DNA was labelled according to the procedure of Rigby et al. [26]. Hybridization probes used were 32p-labelled B. campestris DNA and pTA 665 (the 500 bp 5S rDNA from wheat cloned into pBR 322 [7]). Gel purified DNA fragments were labelled by the random primer method of Feinberg and Vogelstein [27] using the multiprime labelling kit (Amersham, UK).
2. Materials and methods
Leaf material from B. campestris (cv. YsPb-24), Brassica oleracea (cv. Early Kuwari), Brassica nigra (cv. IC257), Brassiea juncea (cv. Pusa bold), Brassica carinata (cv. BEC 218), Brassica napus (cv. B015), Brassica tournefortii, Eruca sativa (cv. Itsa) and other wild relatives was obtained from plants grown at Gwal Pahari, Delhi (TERI's field station). Seeds of Sinapis arvensis (cv. SA-6) and Diplotaxis erucoides were obtained from Dr. K. Hinata, Tohoku University, Japan. Seeds of Raphanus sativus, Brassica oxyrrhina, Brassica fruticulosa, Brassica barrelieri, Brassica adpressa and Erucastrum varium were obtained from Dr. Shyam Prakash, Indian Agricultural Research Institute, New Delhi.
2.1. DNA preparation Nuclear DNA from different Brassica species was prepared from frozen leaf tissue as described by Malmberg et al. [22] and purified on CsC1 density gradient. Total DNA was extracted from frozen leaves by the method of Dellaporta et al. [23]. 2.2. Restriction endonuclease digestion and hybridization analysis Restriction endonuclease digestion of the DNA was performed with restriction enzymes such as BamHI, EcoRI, HindIII, AluI, HaeIII, MspI, MboI, Hinfl, Sau3AI, DpnI and HpaII as recommended by the manufacturers. The digests were fractionated by electrophoresis on 1% or 1.5% agarose gels. The DNA was transferred onto Hybond-
2.3. Molecular cloning of the 5S rRNA genes A partial library of BamHI restricted nuclear B. campestris (cv. YsPb-24) DNA (fragment size 0.4-1.5 kb) was made in the phagemid pGem7zf(+) (Promega, USA) using the Escherichia coli strain NM522 for transformation. Positive recombinants were detected by colony hybridization using labelled B. campestris DNA. Plasmid DNAs isolated from the positive recombinants [28] were restricted with BamHI and hybridized to the labelled 500 bp insert of pTA 665 [7]. The inserts of many recombinants produced a hybridization signal, some of which were selected for further analysis. 2.4. DNA sequencing Single and double stranded DNAs [29] from the recombinant clones were sequenced by Sanger's dideoxy chain termination method [30] to obtain sequence data in both orientations. [35S]dATP and Taq polymerase (Promega, USA) or Sequenase (USB, USA) were used for the sequencing reaction. Sequence comparisons and analysis was carried out using the DNASIS software (LKB Pharmacia, USA). 3. Results and discussion
3.1. Identification and characterization of the 5S rRNA genes A partial BamHI library of nuclear B. campestris DNA was constructed and screened for the 5S rRNA genes as described in Materials and
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S. Bhatia et al. / Plant Sci. 92 (1993) 47-55
methods. On screening, a number of clones gave positive hybridization signals, of which four clones, namely pBC5S-I*, pBC5S-2, pBC5S-3 and pBC5S-4~ were subsequently characterized. B. campestris genomic DNA was digested with different restriction enzymes such as BamHI, EcoRI, HindlII, AluI, Hinfl and HaelII, fractionated on a 1.5% agarose gel, Southern blotted and hybridized to the cloned B. campestris 5S rDNA repeat clone, pBC5S-I (Fig. 1). BamHI restricted DNA produced a ladder consisting of bands whose sizes were multiples of 500 bp, which is the monomeric unit size of the 5S rDNA (Fig. 1). The ladder pattern obtained indicated that the 5S rDNA units were tandemly repeated and that the BamHI sites in these units were variably methylated. A similar ladder pattern was also obtained with HaelII restriction but after prolonged exposure (data not shown). The hybridization pattern obtained with BamHI restriction of B. campestris DNA, is in agreement with the earlier reports from some higher plants investigated [4,5,7,10,13,16] where similar ladder patterns were produced, consisting of bands whose sizes were multiples of the monomeric 5S rDNA repeat unit of that plant. The ladder patterns obtained were not due to partial digestion because addition of more enzyme or digestion for a longer time did not change the pattern of hybridization. However, restriction of genomic DNA with EcoRI or HindlII did not produce a ladder; instead, most of the signal remained at the top of the gel. This indicated that sites for these enzymes may not exist in the 5S rDNA repeat units (Fig. 1). On restriction with AluI a smear was obtained, whereas with Hinfl restriction bands were obtained at 500 bp, 350 bp and 250 bp indicating that more than one Hinfl site is present per unit of the 5S rDNA. (Fig. 1, lane e).
3.2. Sequence analysis of the 5S rRNA gene Four clones, pBC5S-I, pBC5S-2, pBC5S-3 and pBC5S-4, identified as the putative 5S rDNA clones, were sequenced and characterized. The nucleotide sequences of the four clones are shown in Fig. 2. All the four sequences are nearly similar ex*This sequence data will appear in the EMBL data bank under the accession number X 60723.
Kb
abcde
f
1"51,-
Fig. 1. Southern blot analysis of the B. campestris (cv. YsPb-24) 5S rDNA repeat. DNA was digested, electrophoresed on a 1.5% agarose gel, Southern blotted and hybridized with the clone pBC5S-1 of B. campestris. Lane a, BamHI; lane b, EcoRI; lane c, HindllI; lane d, AluI; lane e, Hinfl; lane f, Haelll.
cept for minor differences as indicated in Fig. 2. Comparison of these sequences with those from other plant species [1,3-7,13,16,17,31] reveals that the coding region is 119 bp in size (positions 35-153; Fig. 2) and is highly conserved amongst plants and animals except for a few base pair changes. The remaining 376 bp region is the noncoding spacer region which separates the coding region of one repeat unit from that of the next. The A + T content of the 5S rDNA unit in pBC5S-I is 52.9%. Comparison with other plant 5S rDNA sequences [1,3-7,13,16,17] tentatively assigns 'GGG' as the start and 'CCC' as the end of the coding region of the B. campestris 5S rDNA (positions 35 and 153; Fig. 2). Some other plants that also have the same start and end sequence as B. campestris are flax [5], Matthiola incana [6] and
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,% Bhdtta et al. ,' l'lan! ,%'ci. 92 f 1993 J 4 7-55
5'
8 TAGAAACGAG
TGACGGGTGC
AGTGATGTAC
GATCATACCA
GCACTAATGC
pBC5S-I
AAAGGATATA
pBCSS-2
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
60
pBC5S-3
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
60
pBCSS-4
G ..... C-A-
-G---T--C-
-C-C ....................................
pBC58-1
H BS ACCGGATCCC
ATCAGAACTC
CGCAGTTAAG
pBC5S-2
............................................................
pBC58-3
................................
C ...........................
120
pBC5S-4
................................
G ...........................
120
pBC5S-I
H GTGACCTCCC
pBC5S-2
.............................................
pBC58-3
.......................................................
pBC5S-4
......................................
GGGAAGTCCT
CGTGTTGCAC
T . . . . . . . . . . . . . . . . . . . . . . . . .
CGTGCTTGGG
CCCTTTTTAT
CGAGAGTAGT
60
ACTAGGATGG
[20 120
TTTTTTATTT
TTTTT*AGCC
A ........ TA
60
A*CC-T T ....
.... G-T-A-
-**+C*GAT-
179 179 180 176
8
pBC5S-I
TAAAACGAGT
pBC5S-2
...................
CTAAACTTG*
* G .........................
G .............
pBC5S-3
...................
* ..........................
* .............
238
pBCSS-4
........
*--T
234
T ....
AAAACCTCAT
GCT---G
......
AACTTTTGAA
* ..........
CCGTGA*GAA
T ........
CTACGTCGCC
---T-C
....
237 238
8
pBC5S-I
CATAGCATCA
pBCSS-2
....................................
TTTCGTAAAG
CCCCAGAAAC
CATAATGGGC
pBC58-3
............................................................
pBC5S-4
--C .... C .......
pBC5S-I
TTTCGGGCTC
pBC58-2
..........................
pBC5S-3
............................................................
pBCSS-4
......
pBC5S-I
AAAAAGAATC
pBC5S-2
..... AG .....
* ..............
pBC5S-3
............
* ...............
pBCSS-4
* .... T-G--
A-G-T---TT
pBC5S-i
AACCTCGATG
CACTTTTTCG
pBC5S-2
..................
pBC58-3
...........................................
pBC5S-4
--T---**C-
T ..... GGTT
-AG .... G ....
pBC5S-i
CCTTACACTT
GGATTGGCCG
CGA
G .................
AAATTCAGCC
GTTTGGACCC
C-A ....
TCAAACGGGC
GGGCGCAATT
T ................. C-T---G---
294
TGCGGAAAGT
TATGGCACGT
357
**
ATGCT*TTCT
* .............. * .............
357 358
T .... T-
*GTGTTTTTG
£-**
297
A-TT--CG-A
T ..................
TTCTCAAG**
297 298
T ..................................
C-C ...............
GA*AAGCGGA
GGGGAATAGG
* .....
--C--AG---
354
TAACGCCGTT
412
* ..............
412
* ...............
413
--T--G--TG
TT--G-G---
T--T-G--G-
GTT-TA-A-C
413
CTCGAAACAA
AACGGCAAAA
AAAATCATGT
GCGCCCACGG
472
A .................
*--G
........... TCT .......
*T--**C
.......
T ....
* ........ *T ..... G-T--CAC-
470 472 468
3'
* ....
* ......
495
pBCSS-2
--* .....
G--
490
pBCSS-3
....................
G--
495
pBC5S-4
-AA-C .... C
AAC
491
-A .... T-GA
Fig. 2. Nucleotide sequences of the B. campestris 5S r D N A repeat clones: pBC5S-I, pBC5S-2, pBC5S-3, pBC5S-4 in the 5' to 3' orientation. The sequences are aligned to give maximum homology. Differences of nucleotides are indicated. An asterix (*) indicates a deletion and a hyphen (-), the same nucleotide as in the first row. Underlined sequences are possible regulatory regions as described in the text. The coding region is shown in bold print. The sites for the various restriction enzymes are indicated as follows: B, BamHh H, Hpall and M~;ol; S, S a u 3 A l , M b o i and Dpnl.
S. Bhatia et al./ Plant Sci. 92 (1993) 47-55
rapeseed [14] whereas plants such as pea [1], mungbean [6] and soybean [13] have 'AGG' as the start and 'CTC' as the end of their coding regions. The cluster of 'T' residues required for the termination of transcription of the 5S rRNA genes [1,2,6,13] are present after position 153 in the B. campestris sequence (pBC5S-1; Fig. 2). The highly conserved AT rich promoter sequence (also known as the 'TATA' box) is found 5' to the coding region and is critical in the initiation of transcription of the 5S rRNA genes [32,33]. In B. campestris the 'TATA' box is present 29 bases upstream of the start of the coding region (positions 6-12 in pBC5S-1; Fig. 2), as in other plant species [1,2,4,6,13,16]. The well-known 'GC' motif, with a conserved hexanucleotide core sequence and a larger consensus sequence, (G/T) GGGCGG (G/A) (G/A) (C/T) [34], is present upstream of the 5S rRNA coding regions both in humans [35] and in hamsters [36] where it is known to be involved in regulation of transcription, but in B. campestris it is present downstream of the coding region (position 274 to 283 in pBC5S-1; Fig. 2). Hence it is difficult to determine whether this 'GC' motif has any regulatory role in B. campestris as has been reported in humans or whether it is just a chance sequence similarity. To investigate the heterogeneity, if any, present in the B. campestris 5S rDNA, four different clones were sequenced and their sequences compared (Fig. 2). Sequence comparison shows that pBC5S-I, pBC5S-2 and pBC5S-3 are very similar to each other, especially in the coding regions which have nearly 100% homology. One striking difference is that the coding region of clone pBC5S-3 begins 5' with 'TGG' instead of 'GGG' which is present in the other three clones sequenced (position 35; Fig. 2). The clone pBC5S-4 differs from the other three clones mainly in the non-coding spacer region. It shows only about 60% homology to the noncoding region of pBC5S-1. Even though the sequences that mark the start and the end of the coding region in pBC5S-4 are the same as in pBC5S-1, the highly conserved 'TATA' box is very different in pBC5S-4 and has the sequence 'ACAAATG'. Also, the 'GC' motif mentioned
51
above has a different sequence in pBC5S-4 (position 270 to 280 in pBC5S-4; Fig. 2). Another region in pBC5S-4 which shows variation from the other three sequences is the 'T' rich region which marks the end of the coding region. The sequence differs from position 159 to 169 as compared with the other three clones. There is also a three base deletion in pBC5S-4 after nucleotide position 171 (Fig. 2). As a result of the above mentioned differences, pBC5S-4 may be a variant form of the 5S rRNA genes present. Due to the differences in the 'TATA' box region and the 'GC' motif, this variant may not be expressed as well as the other nonvariant forms. Variants have been reported in pea [1], flax [5], wheat [7], Secale [171, human [351, mouse [37] and Eruca sativa (M. Lakshmikumaran, unpublished results).
3.3. Methylation pattern Plant DNAs, especially the ribosomal RNA genes, are known to be highly methylated at the 'C' residues in the sequences CpG and CpNpG [5,38,39]. Hence, the ladder pattern observed in Fig. 1 may be due to the methylation of the B. campestris 5S rRNA genes. To investigate the pattern of methylation, isoschizomers were used which are differentially sensitive to methylation of bases within the recognition sequence. Southern blot analysis of B. campestris DNA digested with the isoschizomers MspI and HpalI is shown in Fig. 3 (lanes e and f). These two enzymes are known to be sensitive towards 'C' methylation in the sequence 5'-CCGG-3', which occurs twice in the clone pBC5S-1, at positions 62 and 129 (Fig. 2). MspI does not cut when the external 'C' is methylated (5'-mCCGG-3'), whereas it is insensitive to internal 'C' methylation. On the other hand, HpalI is sensitive both to internal and external 'C' methylation and does not cut the sequence 5 '-mCCGG-3' or 5'-CmCGG-3 '. Fig. 3 shows that MspI digests B. campestris DNA producing a ladder with a unit size of approx. 500 bp whereas HpalI restriction shows a faint ladder starting from 1.5 kb, superimposed on a smear. HpalI restriction pattern shows that in most of the 5S rDNA units the sequence 5' CCGG-3' may be 'C' methylated. Complete restriction with MspI should have produced bands
52
S. Bhatia et al. / Phmt S('i. (]2 ;1993) 47-55
Kb
abcdef
1"51D,-
1.0J,0.51,,-
Fig. 3. Methylation pattern of the B. campestris 5S rDNA repeat by Southern hybridization. B. campestris (cv. YsPb-24) DNA was digested and electrophoresed on a 1.5% agarose gel, Southern blotted and hybridized to a 32p-labelled nick translated clone pBC5S-I of B. campestris. Lane a, BamHI; lane b, Mbol; lane c, Sau3Al; lane d, Dpnl; lane e, Mspl; lane f, Hpall.
of 67 bp and 428 bp as there are two MspI sites per unit of the 5S rDNA (positions 62 and 129; Fig. 2), but since only a single ladder of unit size 500 bp was obtained, it clearly indicated that MspI cuts at only one of the two sites present. To determine which site(s) is susceptible, double digestion with BamHI and MspI was performed as there is an overlapping BamHI/MspI site at position 62 (Fig. 2). Double digestion also produced a ladder pattern of 500 bp (similar to that obtained with BamHI above) and not signals at 67 bp and 428 bp as expected. This clearly indicates that the overlapping site at position 62 is variably methylated and
that the MspI site at position 129 is methylated at the external 'C' in most of the 5S rDNA units. If this site did not have external 'C' methylation at position 129 then bands of 67 bp and 428 bp would have been produced. Fig. 3 also shows the Southern blot analysis of B. campestris DNA digested with the isoschizomers MboI, Sau3A1 and DpnI (lanes b, c and d). All these enzymes recognize the sequence 5' GATC-3' but MboI does not cut the sequence 5' GATC-3' if 'A' is methylated whereas Sau3AI does not restrict when 'C' is methylated, while DpnI cleaves only if 'A' is methylated. The 5'GATC-3' sequence is present at positions 41 and 65 in pBC5S-1 (Fig. 2). This sequence is also likely to be present at more sites in some of the 5S rDNA units as observed at positions 173 and 222 in pBC5S-4 (Fig. 2) which accounts for the presence of the bands at 360 bp and 285 bp (lane b, Fig. 3). Genomic DNA on restriction with MboI produces a very faint band at 450 bp, a dark band at 360 bp and a faint one at 285 bp. On the other hand, Sau3AI produces bands of the same sizes as those obtained with MboI, but of different intensities. For example, the 450 bp band with Sau3Al is very intense as compared with the very faint one obtained with MboI while the 360 bp band with Sau3AI is weak as compared with the corresponding one with MboI. This pattern demonstrates that there is extensive 'C' methylation as compared with 'A' methylation. Hybridization data (lane d; Fig. 3) shows that as genomic DNA does not get restricted with DpnI (which cuts only when 'A' is methylated), the 'A' residues may not be methylated. Thus, the hybridization data with isoschizomers MboI, Sau3AI and DpnI demonstrates that 'A' residues are not methylated while 'C' residues are methylated, as was also indicated by the results with MspI and HpaII. Double digestion with BamHI and Sau3AI gives a pattern similar to that obtained with Sau3AI alone. This indicates that the sites are differentially methylated but it could not distinguish between the methylation patterns of the different Sau3AI sites. Extensive methylation at 'C' residues o f 5S rDNA have been reported in pea [1], maize [10], soybean [13] and barley [15], which is consistent with our results obtained above.
53
S. Bhatia et al./ Plant Sci. 92 (1993) 47-55
Kbp
abc
d • f gh
6.5--
4.5-
0.5-
Fig. 4. Organization of the 5S rDNA repeats in related crucifers by Southern hybridization. DNA from different crucifers was digested with BamHl, fractionated on a 1.5% agarose gel, Southern blotted and hybridized to 32p-labelled, nick translated clone pBC5S-I of B. campestris. Lane a, B. campestris; lane b, B. nigra; lane c, B. oleracea; lane d, B. juncea; lane e, E. sativa; lane f, D. erucoides; lane g, R. sativus; lane h, S. arvensis.
3.4. Organization of the 5S rRNA genes in related crueifers DNA analysis of the crucifers B. nigra, B. oleracea, B. juncea, Eruca sativa, Diplotaxis erucoides, Raphanus sativus and Sinapis arvensis was performed in order to investigate the organization of the 5S rRNA genes (Fig. 4). The DNA from the different crucifers was digested with BamHI, fractionated on a 1.5% agarose gel, Southern blotted and hybridized with pBC5S-1 (Fig. 4). All of them produced a ladder of bands which were multiples of the monomeric unit size of the 5S rDNA repeat of the respective plant. The unit size of the 5S rDNA of these crucifers is approx. 500
bp. The pattern of hybridization obtained indicates that the organization of the 5S rRNA genes in these crucifers is the same as in B. campestris and in the other higher plant species investigated [1,2,4-6,13]. Hybridization data also reveals that the 5S rDNA in these crucifers is present in clusters of tandem repeats and are extensively methylated, thus giving rise to a ladder of bands. Organization of the 5S rDNA in B. oleracea is interesting because it shows an additional band at 250 bp (Fig. 4, lane c). This indicates that there is perhaps another class of 5S rDNA in B. oleracea with additional BamHI sites in these units. The organization of the 5S rDNA in some wild crucifers was also investigated (data not shown). Studies showed ladder patterns consisting of bands which were multiples of the monomeric unit sizes. In some other species investigated such as B. oxyrrhina, B. fruticalosa, B. barrelieri, B. adpressa, B. tournefortii and Erucastrum varium the monomeric size of the 5S rDNA repeats is approx. 500 bp (data not shown). The organization of the 5S rDNA in B. tournefortii was noteworthy as it showed additional bands at 400 bp and 100 bp (data not shown). This indicated that more than one class of the 5S rRNA genes exist in B. tournefortii containing more than one BamHI site per unit. The occurrence of more than one form of the 5S rDNA has previously been reported from flax [51, wheat [7], Secale [17] and Eruca sativa (M. Lakshmikumaran, unpublished results).
4. Acknowledgements The authors gratefully acknowledge the Technical Assistance of Mr. Madan Singh Negi. The award of Senior Research Fellowship by the Council for Scientific and Industrial Research, India to S.B. is gratefully acknowledged. This work was supported by a grant from the Department of Biotechnology, Government of India.
5. References T.H.N. Ellis, D. Lee, C.M. Thomas, P.R. Simpson, W.G. Cleary, M.A. Newman and K.W.G. Burcham, 5S rRNA genes in Pisum: Sequence, long range and chromosomal organization. Mol. Gen. Genet., 214 (1988) 333-342.
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E.U. Selker, C. Yanofsky, K. Driftmier, R.L. Metzenberg, B. DeWeerd and U.L. RajBhandary, Dispersed 5S RNA in N. erassa: structure, expression and evolution. Cell, 24 (1981) 819-828. N. Junakovic, Variability in the molecular organization of the 5S RNA genes among strains of Drosophila melanogaster. Nucleic Acids Res., 8 (1980) 3611-3622. R.C. Peterson, J.L. Doering and D.D. Brown, Characterization of two Xenopus somatic 5S DNAs and one minor oocyte-specific 5S DNA. Cell, 20 (1980) 131-141. N.L.V. Lapitan, M.W. Ganat and S.D. Tanskley, Organisation of the 5S ribosomal RNA genes in the genome of tomato. Genome, 34 ( 1991 ) 509-514. R. Malmberg, J. Messing and I. Sussex, Molecular Biology of Plants: A Laboratory Course Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1985. S.L. Dellaporta, J. Wood and J.B. Hicks, Maize DNA mini-prep, in: Molecular Biology of Plants: A Laboratory Course Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1984, pp. 36-37. E.M. Southern, Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol., 98 (1975) 503-517. M.S. Lakshmikumaran, E. D'Ambrosio, L.A. Laimins, D.T. Lin and A.V. Furano, Long interspersed repeated DNA (LINE) causes polymorphism at the rat insulin 1 locus. Mol. Cell. Biol., 5 (1985) 2197-2203. P.W.J. Rigby, M. Dieckman, C. Rhodes and P. Berg, Labelling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J. Mol. Biol., 113 (1977) 237-251. A.P. Feinberg and B. Vogelstein, A technique for radio labelling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem., 132 (1983) 6-13. H.C. Birnboim, A rapid alkaline extraction procedure for screening of plasmid DNA. Methods Enzymol., 100 (1983) 243-255. T. Maniatis, E.F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1982. F. Sanger, S. Nicklen and A.R. Coulson, DNA sequencing with chain terminating inhibitors. Proc~ Natl. Acad. Sci. USA, 74 (1977) 5463-5467. A. Vandenberghe, M.W. Chen, E. Dams, R. De Baere, E. De Roeck, E. Huysmans and R. De Wachter, The corrected nucleotide sequences of 5S RNAs from six angiosperms. Fed. Eur. Biochem. Soc. Lett., 171 (1984) 17-23. L.J. Korn, Transcription of Xenopus 5S ribosomal RNA genes. Nature (London), 295 (1982) 101-105. E.U. Selker, E. Morzycha-Wroblewska, J.N. Steven and R.L. Metzenberg, An upstream signal is required for in vitro transcription of Neurospora 5S RNA genes. Mol. Gen. Genet., 205 (1986) 189-192. J.T. Kadonaga, K.A. Jones and R. Tjian, Promoter specific activation of RNA polymerase II transcription by Spl. Trends Biochem. Sci., 11 (1986) 20-23. P.D. Sorensen and S. Frederiksen, Characterization of
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Plant Science92 (1993) 47-55
Organization and sequence analysis of the 5S rRNA genes in Brassica campestris S a b h y a t a B h a t i a a, K a p i l S i n g h b, V. J a g a n n a t h a n a, M a l a t h i L a k s h m i k u m a r a n *a aBiotechnology Division, Tata Energy Research Institute, 90 Jor Bagh, New Delhi 110 003 India hCentre for Plant Molecular Biology, Tamil Nadu Agricultural University, Coimbatore 641 003 India
(Received 16 December 1992; revision received 24 March 1993; accepted 28 April 1993)
Abstract
The 5S rRNA gene from Brassica campestris has been cloned, sequenced and characterized. The 5S rDNA repeat unit is 495 bp in length. It consists of a highly conserved, 119 bp coding region and a variable non-coding spacer region which separates it from the coding region of the next repeat unit. The 5S rDNA units are organized into clusters of tandem repeats. Sequences responsible for initiation and termination of transcription of the 5S rRNA are present within the repeat unit. Hybridization data with isoschizomers reveals a high amount of 'C' methylation. The size of the repeat unit and the organization of the 5S rRNA genes in other closely related crucifers has been investigated and found to be quite similar to that of B. campestris. Key words: 5S rDNA; Tandem repeat; Brassica; Methylation
1. Introduction
The 5S rRNA genes in higher eukaryotes are present in multiple copies per genome and are generally organized into clusters of tandem arrays. Each repeat unit consists of a highly conserved coding region, approx. 120 base pairs long, which is transcribed into the 5S rRNA. Each repeat unit also contains a non-transcribed spacer region which separates it from the coding region of the next repeating unit [1-7]. This non-transcribed spacer varies from species to species both in sequence and length. Also, more than one variant * Corresponding author.
form of the 5S rDNA may exist within the same species as in flax [5], wheat [7] and Eruca sativa (unpublished results). These clusters of tandem arrays may be localized on either one or several chromosomes and are generally separate from the genes encoding the large ribosomal RNAs [1,8,9]. The 5S rRNA genes have been isolated and characterized from some plant species such as pea [1], yellow lupin [4], flax [5], mung bean [6], Matthiola incana [6], wheat [7], maize [10], tobacco [11], rice [12], soybean [13], rapeseed [14], barley [15], birch and alder [16] and Secale [17]. The transcription of the 5S rRNA genes and its regulation has been studied in organisms such as yeast [18], Drosophila [19] and Xenopus [20].
0168-9452/93/$06.00 © 1993 ElsevierScientificPublishers Ireland Ltd. All rights reserved. SSDI 0168-9452(93)03648-F
48
The organization and chromosomal localization of the 5S rDNA repeats has been investigated by means of in situ hybridization and PFGE in plants like pea [1], wheat [9], rye [17] and tomato [21]. In these plant species it has been shown that the 5S rDNA exists as a cluster at one to three sites and are not interspersed with other ribosomal genes. In the present study, the 5S rRNA gene of Brassica campestris has been cloned and sequenced. The organization and methylation pattern of the 5S rDNA, in this species, has been analysed. Hybridization studies have also been carried out to investigate the organization of the 5S rDNA in related crucifers.
S. Bhatia cta/.
P[anl .~'ci 92
1993J 47-55
C-extra membranes (Amersham, UK) according to the method of Southern [24]. Hybridizations were carried out according to the procedure of Lakshmikumaran et al. [25]. The filters were hybridized to probes labelled with [c~-~-~P]dATP, [c~-32p]dGTP or [c~-32p]dCTP which were obtained from the Bhabha Atomic Research Center, Bombay, India. The DNA was labelled according to the procedure of Rigby et al. [26]. Hybridization probes used were 32p-labelled B. campestris DNA and pTA 665 (the 500 bp 5S rDNA from wheat cloned into pBR 322 [7]). Gel purified DNA fragments were labelled by the random primer method of Feinberg and Vogelstein [27] using the multiprime labelling kit (Amersham, UK).
2. Materials and methods
Leaf material from B. campestris (cv. YsPb-24), Brassica oleracea (cv. Early Kuwari), Brassica nigra (cv. IC257), Brassiea juncea (cv. Pusa bold), Brassica carinata (cv. BEC 218), Brassica napus (cv. B015), Brassica tournefortii, Eruca sativa (cv. Itsa) and other wild relatives was obtained from plants grown at Gwal Pahari, Delhi (TERI's field station). Seeds of Sinapis arvensis (cv. SA-6) and Diplotaxis erucoides were obtained from Dr. K. Hinata, Tohoku University, Japan. Seeds of Raphanus sativus, Brassica oxyrrhina, Brassica fruticulosa, Brassica barrelieri, Brassica adpressa and Erucastrum varium were obtained from Dr. Shyam Prakash, Indian Agricultural Research Institute, New Delhi.
2.1. DNA preparation Nuclear DNA from different Brassica species was prepared from frozen leaf tissue as described by Malmberg et al. [22] and purified on CsC1 density gradient. Total DNA was extracted from frozen leaves by the method of Dellaporta et al. [23]. 2.2. Restriction endonuclease digestion and hybridization analysis Restriction endonuclease digestion of the DNA was performed with restriction enzymes such as BamHI, EcoRI, HindIII, AluI, HaeIII, MspI, MboI, Hinfl, Sau3AI, DpnI and HpaII as recommended by the manufacturers. The digests were fractionated by electrophoresis on 1% or 1.5% agarose gels. The DNA was transferred onto Hybond-
2.3. Molecular cloning of the 5S rRNA genes A partial library of BamHI restricted nuclear B. campestris (cv. YsPb-24) DNA (fragment size 0.4-1.5 kb) was made in the phagemid pGem7zf(+) (Promega, USA) using the Escherichia coli strain NM522 for transformation. Positive recombinants were detected by colony hybridization using labelled B. campestris DNA. Plasmid DNAs isolated from the positive recombinants [28] were restricted with BamHI and hybridized to the labelled 500 bp insert of pTA 665 [7]. The inserts of many recombinants produced a hybridization signal, some of which were selected for further analysis. 2.4. DNA sequencing Single and double stranded DNAs [29] from the recombinant clones were sequenced by Sanger's dideoxy chain termination method [30] to obtain sequence data in both orientations. [35S]dATP and Taq polymerase (Promega, USA) or Sequenase (USB, USA) were used for the sequencing reaction. Sequence comparisons and analysis was carried out using the DNASIS software (LKB Pharmacia, USA). 3. Results and discussion
3.1. Identification and characterization of the 5S rRNA genes A partial BamHI library of nuclear B. campestris DNA was constructed and screened for the 5S rRNA genes as described in Materials and
49
S. Bhatia et al. / Plant Sci. 92 (1993) 47-55
methods. On screening, a number of clones gave positive hybridization signals, of which four clones, namely pBC5S-I*, pBC5S-2, pBC5S-3 and pBC5S-4~ were subsequently characterized. B. campestris genomic DNA was digested with different restriction enzymes such as BamHI, EcoRI, HindlII, AluI, Hinfl and HaelII, fractionated on a 1.5% agarose gel, Southern blotted and hybridized to the cloned B. campestris 5S rDNA repeat clone, pBC5S-I (Fig. 1). BamHI restricted DNA produced a ladder consisting of bands whose sizes were multiples of 500 bp, which is the monomeric unit size of the 5S rDNA (Fig. 1). The ladder pattern obtained indicated that the 5S rDNA units were tandemly repeated and that the BamHI sites in these units were variably methylated. A similar ladder pattern was also obtained with HaelII restriction but after prolonged exposure (data not shown). The hybridization pattern obtained with BamHI restriction of B. campestris DNA, is in agreement with the earlier reports from some higher plants investigated [4,5,7,10,13,16] where similar ladder patterns were produced, consisting of bands whose sizes were multiples of the monomeric 5S rDNA repeat unit of that plant. The ladder patterns obtained were not due to partial digestion because addition of more enzyme or digestion for a longer time did not change the pattern of hybridization. However, restriction of genomic DNA with EcoRI or HindlII did not produce a ladder; instead, most of the signal remained at the top of the gel. This indicated that sites for these enzymes may not exist in the 5S rDNA repeat units (Fig. 1). On restriction with AluI a smear was obtained, whereas with Hinfl restriction bands were obtained at 500 bp, 350 bp and 250 bp indicating that more than one Hinfl site is present per unit of the 5S rDNA. (Fig. 1, lane e).
3.2. Sequence analysis of the 5S rRNA gene Four clones, pBC5S-I, pBC5S-2, pBC5S-3 and pBC5S-4, identified as the putative 5S rDNA clones, were sequenced and characterized. The nucleotide sequences of the four clones are shown in Fig. 2. All the four sequences are nearly similar ex*This sequence data will appear in the EMBL data bank under the accession number X 60723.
Kb
abcde
f
1"51,-
Fig. 1. Southern blot analysis of the B. campestris (cv. YsPb-24) 5S rDNA repeat. DNA was digested, electrophoresed on a 1.5% agarose gel, Southern blotted and hybridized with the clone pBC5S-1 of B. campestris. Lane a, BamHI; lane b, EcoRI; lane c, HindllI; lane d, AluI; lane e, Hinfl; lane f, Haelll.
cept for minor differences as indicated in Fig. 2. Comparison of these sequences with those from other plant species [1,3-7,13,16,17,31] reveals that the coding region is 119 bp in size (positions 35-153; Fig. 2) and is highly conserved amongst plants and animals except for a few base pair changes. The remaining 376 bp region is the noncoding spacer region which separates the coding region of one repeat unit from that of the next. The A + T content of the 5S rDNA unit in pBC5S-I is 52.9%. Comparison with other plant 5S rDNA sequences [1,3-7,13,16,17] tentatively assigns 'GGG' as the start and 'CCC' as the end of the coding region of the B. campestris 5S rDNA (positions 35 and 153; Fig. 2). Some other plants that also have the same start and end sequence as B. campestris are flax [5], Matthiola incana [6] and
50
,% Bhdtta et al. ,' l'lan! ,%'ci. 92 f 1993 J 4 7-55
5'
8 TAGAAACGAG
TGACGGGTGC
AGTGATGTAC
GATCATACCA
GCACTAATGC
pBC5S-I
AAAGGATATA
pBCSS-2
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
60
pBC5S-3
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
60
pBCSS-4
G ..... C-A-
-G---T--C-
-C-C ....................................
pBC58-1
H BS ACCGGATCCC
ATCAGAACTC
CGCAGTTAAG
pBC5S-2
............................................................
pBC58-3
................................
C ...........................
120
pBC5S-4
................................
G ...........................
120
pBC5S-I
H GTGACCTCCC
pBC5S-2
.............................................
pBC58-3
.......................................................
pBC5S-4
......................................
GGGAAGTCCT
CGTGTTGCAC
T . . . . . . . . . . . . . . . . . . . . . . . . .
CGTGCTTGGG
CCCTTTTTAT
CGAGAGTAGT
60
ACTAGGATGG
[20 120
TTTTTTATTT
TTTTT*AGCC
A ........ TA
60
A*CC-T T ....
.... G-T-A-
-**+C*GAT-
179 179 180 176
8
pBC5S-I
TAAAACGAGT
pBC5S-2
...................
CTAAACTTG*
* G .........................
G .............
pBC5S-3
...................
* ..........................
* .............
238
pBCSS-4
........
*--T
234
T ....
AAAACCTCAT
GCT---G
......
AACTTTTGAA
* ..........
CCGTGA*GAA
T ........
CTACGTCGCC
---T-C
....
237 238
8
pBC5S-I
CATAGCATCA
pBCSS-2
....................................
TTTCGTAAAG
CCCCAGAAAC
CATAATGGGC
pBC58-3
............................................................
pBC5S-4
--C .... C .......
pBC5S-I
TTTCGGGCTC
pBC58-2
..........................
pBC5S-3
............................................................
pBCSS-4
......
pBC5S-I
AAAAAGAATC
pBC5S-2
..... AG .....
* ..............
pBC5S-3
............
* ...............
pBCSS-4
* .... T-G--
A-G-T---TT
pBC5S-i
AACCTCGATG
CACTTTTTCG
pBC5S-2
..................
pBC58-3
...........................................
pBC5S-4
--T---**C-
T ..... GGTT
-AG .... G ....
pBC5S-i
CCTTACACTT
GGATTGGCCG
CGA
G .................
AAATTCAGCC
GTTTGGACCC
C-A ....
TCAAACGGGC
GGGCGCAATT
T ................. C-T---G---
294
TGCGGAAAGT
TATGGCACGT
357
**
ATGCT*TTCT
* .............. * .............
357 358
T .... T-
*GTGTTTTTG
£-**
297
A-TT--CG-A
T ..................
TTCTCAAG**
297 298
T ..................................
C-C ...............
GA*AAGCGGA
GGGGAATAGG
* .....
--C--AG---
354
TAACGCCGTT
412
* ..............
412
* ...............
413
--T--G--TG
TT--G-G---
T--T-G--G-
GTT-TA-A-C
413
CTCGAAACAA
AACGGCAAAA
AAAATCATGT
GCGCCCACGG
472
A .................
*--G
........... TCT .......
*T--**C
.......
T ....
* ........ *T ..... G-T--CAC-
470 472 468
3'
* ....
* ......
495
pBCSS-2
--* .....
G--
490
pBCSS-3
....................
G--
495
pBC5S-4
-AA-C .... C
AAC
491
-A .... T-GA
Fig. 2. Nucleotide sequences of the B. campestris 5S r D N A repeat clones: pBC5S-I, pBC5S-2, pBC5S-3, pBC5S-4 in the 5' to 3' orientation. The sequences are aligned to give maximum homology. Differences of nucleotides are indicated. An asterix (*) indicates a deletion and a hyphen (-), the same nucleotide as in the first row. Underlined sequences are possible regulatory regions as described in the text. The coding region is shown in bold print. The sites for the various restriction enzymes are indicated as follows: B, BamHh H, Hpall and M~;ol; S, S a u 3 A l , M b o i and Dpnl.
S. Bhatia et al./ Plant Sci. 92 (1993) 47-55
rapeseed [14] whereas plants such as pea [1], mungbean [6] and soybean [13] have 'AGG' as the start and 'CTC' as the end of their coding regions. The cluster of 'T' residues required for the termination of transcription of the 5S rRNA genes [1,2,6,13] are present after position 153 in the B. campestris sequence (pBC5S-1; Fig. 2). The highly conserved AT rich promoter sequence (also known as the 'TATA' box) is found 5' to the coding region and is critical in the initiation of transcription of the 5S rRNA genes [32,33]. In B. campestris the 'TATA' box is present 29 bases upstream of the start of the coding region (positions 6-12 in pBC5S-1; Fig. 2), as in other plant species [1,2,4,6,13,16]. The well-known 'GC' motif, with a conserved hexanucleotide core sequence and a larger consensus sequence, (G/T) GGGCGG (G/A) (G/A) (C/T) [34], is present upstream of the 5S rRNA coding regions both in humans [35] and in hamsters [36] where it is known to be involved in regulation of transcription, but in B. campestris it is present downstream of the coding region (position 274 to 283 in pBC5S-1; Fig. 2). Hence it is difficult to determine whether this 'GC' motif has any regulatory role in B. campestris as has been reported in humans or whether it is just a chance sequence similarity. To investigate the heterogeneity, if any, present in the B. campestris 5S rDNA, four different clones were sequenced and their sequences compared (Fig. 2). Sequence comparison shows that pBC5S-I, pBC5S-2 and pBC5S-3 are very similar to each other, especially in the coding regions which have nearly 100% homology. One striking difference is that the coding region of clone pBC5S-3 begins 5' with 'TGG' instead of 'GGG' which is present in the other three clones sequenced (position 35; Fig. 2). The clone pBC5S-4 differs from the other three clones mainly in the non-coding spacer region. It shows only about 60% homology to the noncoding region of pBC5S-1. Even though the sequences that mark the start and the end of the coding region in pBC5S-4 are the same as in pBC5S-1, the highly conserved 'TATA' box is very different in pBC5S-4 and has the sequence 'ACAAATG'. Also, the 'GC' motif mentioned
51
above has a different sequence in pBC5S-4 (position 270 to 280 in pBC5S-4; Fig. 2). Another region in pBC5S-4 which shows variation from the other three sequences is the 'T' rich region which marks the end of the coding region. The sequence differs from position 159 to 169 as compared with the other three clones. There is also a three base deletion in pBC5S-4 after nucleotide position 171 (Fig. 2). As a result of the above mentioned differences, pBC5S-4 may be a variant form of the 5S rRNA genes present. Due to the differences in the 'TATA' box region and the 'GC' motif, this variant may not be expressed as well as the other nonvariant forms. Variants have been reported in pea [1], flax [5], wheat [7], Secale [171, human [351, mouse [37] and Eruca sativa (M. Lakshmikumaran, unpublished results).
3.3. Methylation pattern Plant DNAs, especially the ribosomal RNA genes, are known to be highly methylated at the 'C' residues in the sequences CpG and CpNpG [5,38,39]. Hence, the ladder pattern observed in Fig. 1 may be due to the methylation of the B. campestris 5S rRNA genes. To investigate the pattern of methylation, isoschizomers were used which are differentially sensitive to methylation of bases within the recognition sequence. Southern blot analysis of B. campestris DNA digested with the isoschizomers MspI and HpalI is shown in Fig. 3 (lanes e and f). These two enzymes are known to be sensitive towards 'C' methylation in the sequence 5'-CCGG-3', which occurs twice in the clone pBC5S-1, at positions 62 and 129 (Fig. 2). MspI does not cut when the external 'C' is methylated (5'-mCCGG-3'), whereas it is insensitive to internal 'C' methylation. On the other hand, HpalI is sensitive both to internal and external 'C' methylation and does not cut the sequence 5 '-mCCGG-3' or 5'-CmCGG-3 '. Fig. 3 shows that MspI digests B. campestris DNA producing a ladder with a unit size of approx. 500 bp whereas HpalI restriction shows a faint ladder starting from 1.5 kb, superimposed on a smear. HpalI restriction pattern shows that in most of the 5S rDNA units the sequence 5' CCGG-3' may be 'C' methylated. Complete restriction with MspI should have produced bands
52
S. Bhatia et al. / Phmt S('i. (]2 ;1993) 47-55
Kb
abcdef
1"51D,-
1.0J,0.51,,-
Fig. 3. Methylation pattern of the B. campestris 5S rDNA repeat by Southern hybridization. B. campestris (cv. YsPb-24) DNA was digested and electrophoresed on a 1.5% agarose gel, Southern blotted and hybridized to a 32p-labelled nick translated clone pBC5S-I of B. campestris. Lane a, BamHI; lane b, Mbol; lane c, Sau3Al; lane d, Dpnl; lane e, Mspl; lane f, Hpall.
of 67 bp and 428 bp as there are two MspI sites per unit of the 5S rDNA (positions 62 and 129; Fig. 2), but since only a single ladder of unit size 500 bp was obtained, it clearly indicated that MspI cuts at only one of the two sites present. To determine which site(s) is susceptible, double digestion with BamHI and MspI was performed as there is an overlapping BamHI/MspI site at position 62 (Fig. 2). Double digestion also produced a ladder pattern of 500 bp (similar to that obtained with BamHI above) and not signals at 67 bp and 428 bp as expected. This clearly indicates that the overlapping site at position 62 is variably methylated and
that the MspI site at position 129 is methylated at the external 'C' in most of the 5S rDNA units. If this site did not have external 'C' methylation at position 129 then bands of 67 bp and 428 bp would have been produced. Fig. 3 also shows the Southern blot analysis of B. campestris DNA digested with the isoschizomers MboI, Sau3A1 and DpnI (lanes b, c and d). All these enzymes recognize the sequence 5' GATC-3' but MboI does not cut the sequence 5' GATC-3' if 'A' is methylated whereas Sau3AI does not restrict when 'C' is methylated, while DpnI cleaves only if 'A' is methylated. The 5'GATC-3' sequence is present at positions 41 and 65 in pBC5S-1 (Fig. 2). This sequence is also likely to be present at more sites in some of the 5S rDNA units as observed at positions 173 and 222 in pBC5S-4 (Fig. 2) which accounts for the presence of the bands at 360 bp and 285 bp (lane b, Fig. 3). Genomic DNA on restriction with MboI produces a very faint band at 450 bp, a dark band at 360 bp and a faint one at 285 bp. On the other hand, Sau3AI produces bands of the same sizes as those obtained with MboI, but of different intensities. For example, the 450 bp band with Sau3Al is very intense as compared with the very faint one obtained with MboI while the 360 bp band with Sau3AI is weak as compared with the corresponding one with MboI. This pattern demonstrates that there is extensive 'C' methylation as compared with 'A' methylation. Hybridization data (lane d; Fig. 3) shows that as genomic DNA does not get restricted with DpnI (which cuts only when 'A' is methylated), the 'A' residues may not be methylated. Thus, the hybridization data with isoschizomers MboI, Sau3AI and DpnI demonstrates that 'A' residues are not methylated while 'C' residues are methylated, as was also indicated by the results with MspI and HpaII. Double digestion with BamHI and Sau3AI gives a pattern similar to that obtained with Sau3AI alone. This indicates that the sites are differentially methylated but it could not distinguish between the methylation patterns of the different Sau3AI sites. Extensive methylation at 'C' residues o f 5S rDNA have been reported in pea [1], maize [10], soybean [13] and barley [15], which is consistent with our results obtained above.
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S. Bhatia et al./ Plant Sci. 92 (1993) 47-55
Kbp
abc
d • f gh
6.5--
4.5-
0.5-
Fig. 4. Organization of the 5S rDNA repeats in related crucifers by Southern hybridization. DNA from different crucifers was digested with BamHl, fractionated on a 1.5% agarose gel, Southern blotted and hybridized to 32p-labelled, nick translated clone pBC5S-I of B. campestris. Lane a, B. campestris; lane b, B. nigra; lane c, B. oleracea; lane d, B. juncea; lane e, E. sativa; lane f, D. erucoides; lane g, R. sativus; lane h, S. arvensis.
3.4. Organization of the 5S rRNA genes in related crueifers DNA analysis of the crucifers B. nigra, B. oleracea, B. juncea, Eruca sativa, Diplotaxis erucoides, Raphanus sativus and Sinapis arvensis was performed in order to investigate the organization of the 5S rRNA genes (Fig. 4). The DNA from the different crucifers was digested with BamHI, fractionated on a 1.5% agarose gel, Southern blotted and hybridized with pBC5S-1 (Fig. 4). All of them produced a ladder of bands which were multiples of the monomeric unit size of the 5S rDNA repeat of the respective plant. The unit size of the 5S rDNA of these crucifers is approx. 500
bp. The pattern of hybridization obtained indicates that the organization of the 5S rRNA genes in these crucifers is the same as in B. campestris and in the other higher plant species investigated [1,2,4-6,13]. Hybridization data also reveals that the 5S rDNA in these crucifers is present in clusters of tandem repeats and are extensively methylated, thus giving rise to a ladder of bands. Organization of the 5S rDNA in B. oleracea is interesting because it shows an additional band at 250 bp (Fig. 4, lane c). This indicates that there is perhaps another class of 5S rDNA in B. oleracea with additional BamHI sites in these units. The organization of the 5S rDNA in some wild crucifers was also investigated (data not shown). Studies showed ladder patterns consisting of bands which were multiples of the monomeric unit sizes. In some other species investigated such as B. oxyrrhina, B. fruticalosa, B. barrelieri, B. adpressa, B. tournefortii and Erucastrum varium the monomeric size of the 5S rDNA repeats is approx. 500 bp (data not shown). The organization of the 5S rDNA in B. tournefortii was noteworthy as it showed additional bands at 400 bp and 100 bp (data not shown). This indicated that more than one class of the 5S rRNA genes exist in B. tournefortii containing more than one BamHI site per unit. The occurrence of more than one form of the 5S rDNA has previously been reported from flax [51, wheat [7], Secale [17] and Eruca sativa (M. Lakshmikumaran, unpublished results).
4. Acknowledgements The authors gratefully acknowledge the Technical Assistance of Mr. Madan Singh Negi. The award of Senior Research Fellowship by the Council for Scientific and Industrial Research, India to S.B. is gratefully acknowledged. This work was supported by a grant from the Department of Biotechnology, Government of India.
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