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Methylation pattern of nuclear ribosomal RNA genes from rice (Oryza sativa)
Plant Science Letters, 37 (1984) 123--127
123
Elsevier Scientific Publishers Ireland Ltd.
M E T H Y L A T I O N P A T T E R N O F N U C L E A R R I B O S O M A L R N A G E N E S F R O M RICE ( O R Y Z A SA TIVA )
ADELA OLMEDILLA*, DOMINIQUE DELCASSO and MICHEL DELSENY** Laboratoire de Physiologie Vdgdtale, U.A. 565 du CNRS, Universitd de Perpignan, Avenue de Villeneuve, 66025 Perpignan (France)
(Received July 27th, 1984) (Revision received September 25th, 1984) (Accepted September 25th, 1984) A restriction map of rice (Oryza sativa) rDNA was established for EcoRI, BamHI and BglII. The methylation pattern of these genes was studied using HpaII and MspI. Surprisingly, rice rDNA was found much more sensitive to these enzymes than the rDNA of the other plant species studied so far. No difference was observed between the methylation pattern of rDNA prepared from embryos grown in aerobic or anaerobic conditions although there is a marked difference between the rates of rRNA synthesis in these two situations. Key words: ribosomal genes; methylation; anaerobiosis; rice
Introduction D N A m e t h y l a t i o n is s u p p o s e d t o play a k e y role in regulating gene e x p r e s s i o n in eukaryotes [ 1 ] . Much i n f o r m a t i o n is available f o r animal and fungal genes b u t d a t a c o n c e r n i n g h i g h e r plants are still r a t h e r scarce. R i b o s o m a l genes seem t o be r a t h e r heavily m e t h y l a t e d in t h e few p l a n t species w h i c h have b e e n investigated so far [ 2 - - 7 ] . H o w e v e r specific sites are regularly h y p o m e t h y l a t e d [ 5 - - 7 ] . Up t o n o w we have very little i n f o r m a t i o n a b o u t changes in m e t b y l a t i o n p a t t e r n s . F e w discrete modifications in r D N A m e t h y l a t i o n have been n o t i c e d in radish during d e v e l o p m e n t a l changes [7]. A n o t h e r change, also c o r r e l a t e d with gene expression, has been d e t e c t e d in s t u d y i n g a zein gene in maize [8]. Obviously, o t h e r physiological situations n e e d to be studied b e f o r e conclusions can be d r a w n a b o u t the f u n c t i o n o f D N A m e t h y l a t i o n in higher plants. As p a r t o f a s t u d y o n the a d a p t a t i o n o f r R N A synthesis in rice d u r i n g g r o w t h in anaerobic *Permanent address: CSIC, Instituto de Biologia Celular, Velasquez 144, Madrid 6 (Spain). **To whom correspondence should be sent.
c o n d i t i o n s [9] we have analysed the m e t h y l a t i o n p a t t e r n o f rice r D N A genes. In this r e p o r t we characterize t h e general o r g a n i z a t i o n of rice r D N A genes and show t h a t in this p l a n t these genes are surprisingly m u c h less m e t h y l a t e d t h a n in o t h e r species. We find n o d i f f e r e n c e b e t w e e n the m e t h y l a t i o n p a t t e r n s o f r D N A f r o m seedlings grown in aerobic and a n a e r o b i c c o n d i t i o n s . Materials and m e t h o d s Rice seeds (cultivar Cigalon I N R A ) were g r o w n u n d e r aerobic o r anaerobic c o n d i t i o n s and h a r v e s t e d as previously described [9]. D N A was purified f r o m nuclei-enriched pellets, lysed in t h e presence o f e t h i d i u m b r o m i d e and sarkosyl [ 1 0 ] . It was f u r t h e r p u r i f i e d t h r o u g h an e t h i d i u m b r o m i d e CsC1 gradient and e n r i c h e d in r D N A by a s e c o n d CsC1 gradient. T h e h e a v y part o f the DNA p e a k in this s e c o n d gradient was t a k e n as r D N A [ 1 1 ] . R e s t r i c t i o n e n z y m e digestions, gel electrophoresis, n i c k t r a n s l a t i o n and h y b r i d i z a t i o n were carried o u t as previously described [7, 12]. Plasmid pTA 71 was used as a h e t e r o l o g o u s p r o b e f o r r D N A : it c o n t a i n s a c o m p l e t e w h e a t
0304-4211/84/$03.00 © 1984 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
124 unit inserted at the Eco RI site o f the plasmid vector pACYC 184 [2]. Results
General organization of the rice rDNA units DNA enriched for ribosomal genes was digested with Eco RI, Bgl II and Barn HI, run on agarose gels, transferred o n t o nitrocellulose filters and hybridized with a cloned wheat rDNA unit. Eco RI and Bgl II cut the rice rDNA unit once and Barn HI twice. Fr om these experiments we estimated the size of the rice rDNA unit to be ~ 7 8 0 0 - - 8 0 0 0 bp. The respective positions of these sites were established by analysing double digestions with these enzymes. The map which can be derived (Fig. 1) turns o u t to be very similar to those published for wheat and barley [2]. This was co n f ir med by comigration of genomic rice rDNA fragments with wheat cloned rDNA (pTA 71) digested with Eco RI, Barn HI and Eco RI + Barn HI. We have n o t e d t w o differences in comparing rice and wheat rDNAs: The external spacer of wheat is somewhat longer than the rice one and wheat genes have t w o Bgl II sites instead o f one. The 4 sites which have been m a p p e d in rice are conserved in wheat and barley. In a previous study [13] it has been noticed that rice rDNA has two different unit types differing in size. We have confirmed this observation. In the Cigalon cultivar the size difference between the t w o types of units is ~ 2 0 0 bp and the larger units represent less than 20% o f total rDNA.
BamHI
EcoRI
Methylation pattern of rDNA The m e t h y l a t i o n pattern was analysed after digestion with the restriction enzymes Hpa I! and Msp I. Both enzymes recognize the sequence CCGG but Hpa II is unable to cleave CmCGG or mCmCGG whereas Msp I is unable t o cleave mCCGG or mCmCGG [14]. Figure 2A shows t hat most of the DNA is digested by Hpa II into fragments the size of the repeat unit or smaller. A very low a m o u n t of rDNA is resistant to digestion, a situation which contrasts with that observed in other plants in which a large p r o p o r t i o n of rDNA is completely resistant to Hpa II. A radish rDNA Hpa II digest is shown for comparison in Fig. 2B. As in ot her plant species a n u m b e r of diffuse bands smaller than the unit size are clearly visible in the rice patterns. This indicates that some specific sites are cleaved more frequently than others and are t herefore preferentially undermethylated. That these sites are modified rather than absent is proved by the digestion with the isoschizomer Msp 1. We have also digested this rDNA with Hha I, a n o t h e r e n z y m e sensitive to CpG methylation. All the rDNA is digested into fragments smaller than the unit size (not shown). Double digestion with Hpa II and a n o t h e r e n z y m e allows one to map some of the hypom e t h y l a t e d sites. In the Eco RI + Hpa lI pattern two major new bands corresponding to fragments of 5 3 0 0 - 5 8 0 0 and 2500 bp show up {arrows). This indicates two possible regions for the corresponding major hypom e t h y l a t e d Hpa II sites: either in the larger Bam H1 fragments or in the smaller. Since
BamHI
BamHI B g I II
0 18s 1 i
kbp i
Fig. 1. Restriction map of rice rDNA units.
5.8 S
25s
EcoRI
125
1
2
3
4
5
6
7
8
9
kbp _
unit size
kbp
21.8
_
9.46
-
6.67
21,8 iiiiiiii
-
5.25
-
4.21
-
3.38
:ii
8,3 5.25
J
_
1.96
1.96
1.33 0.92
m
A
B
i ;~*~ ~
Fig. 2. Methylation patterns of rice and radish rDNA. (A) Rice DNA was digested with Eco RI (lane 1), Eco RI + Hpa II (lane 2), Hpa II (lane 3), Barn HI + Hpa II (lane 4), Barn HI (lane 5), and Msp I (lane 6). Digests were analyzed on a 1% agarose gel, transferred onto a nitrocellulose filter and hybridized with 32P-labelled pTA 71 plasmid. Eco RI digestion was partial in order to show both the size of undigested DNA and the size of the rDNA units. (B) Radish DNA was digested with Hind III (lane 8) and Hpa II (lane 9). Non-digested DNA was loaded in slot 7. After electrophoresis and transfer the filter was hybridized with pBG 35 (a flax rDNA unit [5 ] cloned in pAT 153) as a probe. Size markers were k Hind III and k Hind II! + Eco RI fragments.
m o s t o f the smaller f r a g m e n t is resistent t o Hpa II h y d r o l y s i s in t h e Barn H I + Hpa II d o u b l e digestion we have t o c o n c l u d e t h a t t h e m a j o r h y p o m e t h y l a t e d site is in the large B am H1 f r a g m e n t in t h e e x t e r n a l spacer. A Barn H1 + Hpa II f r a g m e n t ~-1000 bp long c o u l d corresp o n d to this site. Several o t h e r n e w bands (small arrows) indicate t h a t o t h e r sites are available in o t h e r units. It is n o t y e t possible t o m a p t h e m a c c u r a t e l y a l t h o u g h the Barn H1
+ Hpa II d o u b l e digestion suggests t h a t m o s t o f t h e m are also l o c a t e d in the larger Barn H1 fragment.
rDNA methylation patterns are identical in seedlings grown in aerobic and anaerobic conditions In o r d e r to investigate if some changes have o c c u r r e d during t h e a d a p t a t i o n to a n a e r o b i c c o n d i t i o n s we have analysed r D N A
126
1
2
A
A
3
4
5
A
N2
Discussion kbp
-- 21.8 _ --
i
9.46 6.67 5.25 4.21 3.38
1.96
1.33 •
. . . . .
0.92
iiii!~ii~iiiiiii!~!~i:iiiii
Fig. 3. M e t h y l a t i o n patterns o f rice r D N A f r o m embryos grown under aerobic (A) or anaerobic conditions (N2). Rice D N A was digested with Barn HI (lane 1), Barn HI + Hpa II (lane 2 and 3) and Eco R I + Hpa II (lanes 4 and 5). Lanes 1, 2 and 4 correspond to r D N A f r o m e m b r y o s grown in aerobic conditions and lanes 3 and 5 to e m b r y o s grown in anaerobic conditions.
extracted f~om seedlings grown in the two conditions. The results are shown in Fig. 3 which represents Barn H1 + Hpa II and Eco RI + Hpa II patterns of seedlings grown under aerobic and anaerobic conditions. There is virtually no difference between the two patterns. We derive a similar conclusion from the analysis of the Hha I patterns.
In this report, we have presented a restriction map of the rice rDNA unit. This map is very similar to that of wheat, the main difference being in the length of the external spacer. While this paper was being written, a restriction map of a cloned rice rDNA unit as well as part of its sequence have been published [15] confirming the mapping results reported here. The most surprising observation is that virtually all rDNA can be digested into fragments the size of the repeat unit or smaller by enzymes such as Hpa II or Hha I. This indicates that most units have at least one hypom e t h y l a t e d Hpa II site and many others have several such sites. This situation is different from that reported for several other plants. In flax about 30% of the units were completely resistant to Hpa II [ 5]. In radish we estimated the proportion to be about 50% [7] and the percentage is higher in wheat [2] and tobacco [4]. In pumpkin a number of units have one or two h y p o m e t h y l a t e d sites but the percentage of completely resistant units has not been reported [6]. We do not know the reason for this higher percentage of h y p o m e t h y l a t e d units in rice. This may perhaps be related to the copy number of ribosomal genes: in rice, it has been estimated to be ~-850 copies, which is one of the lowest numbers reported for plants [13]. It is n o t e w o r t h y that one of the major regions for h y p o m e t h y l a t e d Hpa II sites is in the presumed promoter region as already observed for flax [5]. Very few variations have been observed in the methylation patterns of higher plant genes [7,8] so that it is presently difficult to correlate gene activity and hypomethylation. In this study we have analysed two physiological situations in which we knew that the transcription of ribosomal genes was very difficult [9]. However no change in the methylation pattern could be detected. This observation confirms our previous conclusion that there is no obvious relationship between the activity of plant ribosomal genes and their degree of methylation.
127
Acknowledgements Adela Olmedilla was recipient of a shortterm EMBO fellowship. The authors wish to thank Drs. Bernard Mocquot and Alain Pradet (I.N.R.A., Pont de la Maye, France) for providing rice seedlings grown in aerobic and anaerobic conditions, and for stimulating discussions. They also thank Dr. R.B. Flavell {Plant Breeding Institute, Cambridge UK) for a generous gift of plasmid pTA 71. References 1 A. Razin and A.D. Riggs, Science, 210 (1980) 604. 2 W.L. Gerlach and J.R. Bedbrook, Nucleic Acids Res., 7 (1979) 1869. 3 P.B. Goldsbrough, T.H.N. Ellis and C.A. Cullis, Nucleic Acids Res., 9 (1981) 5695. 4 H. Uchimiya, H. Kato, T. Ohgawara, H. Harada and M. Sugiura, Plant Cell Physiol., 23 (1982) 1129.
5 T.H.N. Ellis, P.B. Goldsbrough and J.A. Castleton, Nucleic Acids Res., 11 (1983) 3047. 6 A. Siegel and K. Kolacz, Plant Physiol., 72 (1983) ~166. '7 M. Delseny, M. Laroche and P. Penon, Plant Physiol., 76 (1984) in press. 8 A. Spena, A. Viotti and V. Pirrotta, J. Mol. Biol., 169 (1983) 799. 9 L. Aspart, A. Got, M. Delseny, B. Mocquot and A. Pradet, Plant Physiol., 72 (1983) 115. 10 A.J. Bendich, R.S. Anderson and B.L. Ward, in C.J. Leaver (Ed.), Genome organization and expression in plants, Plenum Press, New-York, pp. 31--33. 11 M. Delseny, L. Aspart, R. Cooke, F. Grellet and P. Penon, Biochem. Biophys. Res. Commun., 91 (1979) 540. 12 M. Delseny, R. Cooke and P. Penon, Plant Sci. Lett., 30 (1983) 107. 13 K. Oono and M. Sugiura, Chromosoma, 76 (1980) 85. 14 M. Mac Cleland, Nucleic Acids Res., 11 (1983) r169. 1 5 F. Takaiwa, K. Oono and M. Sugiura, Nucleic Acids Res., 12 (1984) 5441.
123
Elsevier Scientific Publishers Ireland Ltd.
M E T H Y L A T I O N P A T T E R N O F N U C L E A R R I B O S O M A L R N A G E N E S F R O M RICE ( O R Y Z A SA TIVA )
ADELA OLMEDILLA*, DOMINIQUE DELCASSO and MICHEL DELSENY** Laboratoire de Physiologie Vdgdtale, U.A. 565 du CNRS, Universitd de Perpignan, Avenue de Villeneuve, 66025 Perpignan (France)
(Received July 27th, 1984) (Revision received September 25th, 1984) (Accepted September 25th, 1984) A restriction map of rice (Oryza sativa) rDNA was established for EcoRI, BamHI and BglII. The methylation pattern of these genes was studied using HpaII and MspI. Surprisingly, rice rDNA was found much more sensitive to these enzymes than the rDNA of the other plant species studied so far. No difference was observed between the methylation pattern of rDNA prepared from embryos grown in aerobic or anaerobic conditions although there is a marked difference between the rates of rRNA synthesis in these two situations. Key words: ribosomal genes; methylation; anaerobiosis; rice
Introduction D N A m e t h y l a t i o n is s u p p o s e d t o play a k e y role in regulating gene e x p r e s s i o n in eukaryotes [ 1 ] . Much i n f o r m a t i o n is available f o r animal and fungal genes b u t d a t a c o n c e r n i n g h i g h e r plants are still r a t h e r scarce. R i b o s o m a l genes seem t o be r a t h e r heavily m e t h y l a t e d in t h e few p l a n t species w h i c h have b e e n investigated so far [ 2 - - 7 ] . H o w e v e r specific sites are regularly h y p o m e t h y l a t e d [ 5 - - 7 ] . Up t o n o w we have very little i n f o r m a t i o n a b o u t changes in m e t b y l a t i o n p a t t e r n s . F e w discrete modifications in r D N A m e t h y l a t i o n have been n o t i c e d in radish during d e v e l o p m e n t a l changes [7]. A n o t h e r change, also c o r r e l a t e d with gene expression, has been d e t e c t e d in s t u d y i n g a zein gene in maize [8]. Obviously, o t h e r physiological situations n e e d to be studied b e f o r e conclusions can be d r a w n a b o u t the f u n c t i o n o f D N A m e t h y l a t i o n in higher plants. As p a r t o f a s t u d y o n the a d a p t a t i o n o f r R N A synthesis in rice d u r i n g g r o w t h in anaerobic *Permanent address: CSIC, Instituto de Biologia Celular, Velasquez 144, Madrid 6 (Spain). **To whom correspondence should be sent.
c o n d i t i o n s [9] we have analysed the m e t h y l a t i o n p a t t e r n o f rice r D N A genes. In this r e p o r t we characterize t h e general o r g a n i z a t i o n of rice r D N A genes and show t h a t in this p l a n t these genes are surprisingly m u c h less m e t h y l a t e d t h a n in o t h e r species. We find n o d i f f e r e n c e b e t w e e n the m e t h y l a t i o n p a t t e r n s o f r D N A f r o m seedlings grown in aerobic and a n a e r o b i c c o n d i t i o n s . Materials and m e t h o d s Rice seeds (cultivar Cigalon I N R A ) were g r o w n u n d e r aerobic o r anaerobic c o n d i t i o n s and h a r v e s t e d as previously described [9]. D N A was purified f r o m nuclei-enriched pellets, lysed in t h e presence o f e t h i d i u m b r o m i d e and sarkosyl [ 1 0 ] . It was f u r t h e r p u r i f i e d t h r o u g h an e t h i d i u m b r o m i d e CsC1 gradient and e n r i c h e d in r D N A by a s e c o n d CsC1 gradient. T h e h e a v y part o f the DNA p e a k in this s e c o n d gradient was t a k e n as r D N A [ 1 1 ] . R e s t r i c t i o n e n z y m e digestions, gel electrophoresis, n i c k t r a n s l a t i o n and h y b r i d i z a t i o n were carried o u t as previously described [7, 12]. Plasmid pTA 71 was used as a h e t e r o l o g o u s p r o b e f o r r D N A : it c o n t a i n s a c o m p l e t e w h e a t
0304-4211/84/$03.00 © 1984 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
124 unit inserted at the Eco RI site o f the plasmid vector pACYC 184 [2]. Results
General organization of the rice rDNA units DNA enriched for ribosomal genes was digested with Eco RI, Bgl II and Barn HI, run on agarose gels, transferred o n t o nitrocellulose filters and hybridized with a cloned wheat rDNA unit. Eco RI and Bgl II cut the rice rDNA unit once and Barn HI twice. Fr om these experiments we estimated the size of the rice rDNA unit to be ~ 7 8 0 0 - - 8 0 0 0 bp. The respective positions of these sites were established by analysing double digestions with these enzymes. The map which can be derived (Fig. 1) turns o u t to be very similar to those published for wheat and barley [2]. This was co n f ir med by comigration of genomic rice rDNA fragments with wheat cloned rDNA (pTA 71) digested with Eco RI, Barn HI and Eco RI + Barn HI. We have n o t e d t w o differences in comparing rice and wheat rDNAs: The external spacer of wheat is somewhat longer than the rice one and wheat genes have t w o Bgl II sites instead o f one. The 4 sites which have been m a p p e d in rice are conserved in wheat and barley. In a previous study [13] it has been noticed that rice rDNA has two different unit types differing in size. We have confirmed this observation. In the Cigalon cultivar the size difference between the t w o types of units is ~ 2 0 0 bp and the larger units represent less than 20% o f total rDNA.
BamHI
EcoRI
Methylation pattern of rDNA The m e t h y l a t i o n pattern was analysed after digestion with the restriction enzymes Hpa I! and Msp I. Both enzymes recognize the sequence CCGG but Hpa II is unable to cleave CmCGG or mCmCGG whereas Msp I is unable t o cleave mCCGG or mCmCGG [14]. Figure 2A shows t hat most of the DNA is digested by Hpa II into fragments the size of the repeat unit or smaller. A very low a m o u n t of rDNA is resistant to digestion, a situation which contrasts with that observed in other plants in which a large p r o p o r t i o n of rDNA is completely resistant to Hpa II. A radish rDNA Hpa II digest is shown for comparison in Fig. 2B. As in ot her plant species a n u m b e r of diffuse bands smaller than the unit size are clearly visible in the rice patterns. This indicates that some specific sites are cleaved more frequently than others and are t herefore preferentially undermethylated. That these sites are modified rather than absent is proved by the digestion with the isoschizomer Msp 1. We have also digested this rDNA with Hha I, a n o t h e r e n z y m e sensitive to CpG methylation. All the rDNA is digested into fragments smaller than the unit size (not shown). Double digestion with Hpa II and a n o t h e r e n z y m e allows one to map some of the hypom e t h y l a t e d sites. In the Eco RI + Hpa lI pattern two major new bands corresponding to fragments of 5 3 0 0 - 5 8 0 0 and 2500 bp show up {arrows). This indicates two possible regions for the corresponding major hypom e t h y l a t e d Hpa II sites: either in the larger Bam H1 fragments or in the smaller. Since
BamHI
BamHI B g I II
0 18s 1 i
kbp i
Fig. 1. Restriction map of rice rDNA units.
5.8 S
25s
EcoRI
125
1
2
3
4
5
6
7
8
9
kbp _
unit size
kbp
21.8
_
9.46
-
6.67
21,8 iiiiiiii
-
5.25
-
4.21
-
3.38
:ii
8,3 5.25
J
_
1.96
1.96
1.33 0.92
m
A
B
i ;~*~ ~
Fig. 2. Methylation patterns of rice and radish rDNA. (A) Rice DNA was digested with Eco RI (lane 1), Eco RI + Hpa II (lane 2), Hpa II (lane 3), Barn HI + Hpa II (lane 4), Barn HI (lane 5), and Msp I (lane 6). Digests were analyzed on a 1% agarose gel, transferred onto a nitrocellulose filter and hybridized with 32P-labelled pTA 71 plasmid. Eco RI digestion was partial in order to show both the size of undigested DNA and the size of the rDNA units. (B) Radish DNA was digested with Hind III (lane 8) and Hpa II (lane 9). Non-digested DNA was loaded in slot 7. After electrophoresis and transfer the filter was hybridized with pBG 35 (a flax rDNA unit [5 ] cloned in pAT 153) as a probe. Size markers were k Hind III and k Hind II! + Eco RI fragments.
m o s t o f the smaller f r a g m e n t is resistent t o Hpa II h y d r o l y s i s in t h e Barn H I + Hpa II d o u b l e digestion we have t o c o n c l u d e t h a t t h e m a j o r h y p o m e t h y l a t e d site is in the large B am H1 f r a g m e n t in t h e e x t e r n a l spacer. A Barn H1 + Hpa II f r a g m e n t ~-1000 bp long c o u l d corresp o n d to this site. Several o t h e r n e w bands (small arrows) indicate t h a t o t h e r sites are available in o t h e r units. It is n o t y e t possible t o m a p t h e m a c c u r a t e l y a l t h o u g h the Barn H1
+ Hpa II d o u b l e digestion suggests t h a t m o s t o f t h e m are also l o c a t e d in the larger Barn H1 fragment.
rDNA methylation patterns are identical in seedlings grown in aerobic and anaerobic conditions In o r d e r to investigate if some changes have o c c u r r e d during t h e a d a p t a t i o n to a n a e r o b i c c o n d i t i o n s we have analysed r D N A
126
1
2
A
A
3
4
5
A
N2
Discussion kbp
-- 21.8 _ --
i
9.46 6.67 5.25 4.21 3.38
1.96
1.33 •
. . . . .
0.92
iiii!~ii~iiiiiii!~!~i:iiiii
Fig. 3. M e t h y l a t i o n patterns o f rice r D N A f r o m embryos grown under aerobic (A) or anaerobic conditions (N2). Rice D N A was digested with Barn HI (lane 1), Barn HI + Hpa II (lane 2 and 3) and Eco R I + Hpa II (lanes 4 and 5). Lanes 1, 2 and 4 correspond to r D N A f r o m e m b r y o s grown in aerobic conditions and lanes 3 and 5 to e m b r y o s grown in anaerobic conditions.
extracted f~om seedlings grown in the two conditions. The results are shown in Fig. 3 which represents Barn H1 + Hpa II and Eco RI + Hpa II patterns of seedlings grown under aerobic and anaerobic conditions. There is virtually no difference between the two patterns. We derive a similar conclusion from the analysis of the Hha I patterns.
In this report, we have presented a restriction map of the rice rDNA unit. This map is very similar to that of wheat, the main difference being in the length of the external spacer. While this paper was being written, a restriction map of a cloned rice rDNA unit as well as part of its sequence have been published [15] confirming the mapping results reported here. The most surprising observation is that virtually all rDNA can be digested into fragments the size of the repeat unit or smaller by enzymes such as Hpa II or Hha I. This indicates that most units have at least one hypom e t h y l a t e d Hpa II site and many others have several such sites. This situation is different from that reported for several other plants. In flax about 30% of the units were completely resistant to Hpa II [ 5]. In radish we estimated the proportion to be about 50% [7] and the percentage is higher in wheat [2] and tobacco [4]. In pumpkin a number of units have one or two h y p o m e t h y l a t e d sites but the percentage of completely resistant units has not been reported [6]. We do not know the reason for this higher percentage of h y p o m e t h y l a t e d units in rice. This may perhaps be related to the copy number of ribosomal genes: in rice, it has been estimated to be ~-850 copies, which is one of the lowest numbers reported for plants [13]. It is n o t e w o r t h y that one of the major regions for h y p o m e t h y l a t e d Hpa II sites is in the presumed promoter region as already observed for flax [5]. Very few variations have been observed in the methylation patterns of higher plant genes [7,8] so that it is presently difficult to correlate gene activity and hypomethylation. In this study we have analysed two physiological situations in which we knew that the transcription of ribosomal genes was very difficult [9]. However no change in the methylation pattern could be detected. This observation confirms our previous conclusion that there is no obvious relationship between the activity of plant ribosomal genes and their degree of methylation.
127
Acknowledgements Adela Olmedilla was recipient of a shortterm EMBO fellowship. The authors wish to thank Drs. Bernard Mocquot and Alain Pradet (I.N.R.A., Pont de la Maye, France) for providing rice seedlings grown in aerobic and anaerobic conditions, and for stimulating discussions. They also thank Dr. R.B. Flavell {Plant Breeding Institute, Cambridge UK) for a generous gift of plasmid pTA 71. References 1 A. Razin and A.D. Riggs, Science, 210 (1980) 604. 2 W.L. Gerlach and J.R. Bedbrook, Nucleic Acids Res., 7 (1979) 1869. 3 P.B. Goldsbrough, T.H.N. Ellis and C.A. Cullis, Nucleic Acids Res., 9 (1981) 5695. 4 H. Uchimiya, H. Kato, T. Ohgawara, H. Harada and M. Sugiura, Plant Cell Physiol., 23 (1982) 1129.
5 T.H.N. Ellis, P.B. Goldsbrough and J.A. Castleton, Nucleic Acids Res., 11 (1983) 3047. 6 A. Siegel and K. Kolacz, Plant Physiol., 72 (1983) ~166. '7 M. Delseny, M. Laroche and P. Penon, Plant Physiol., 76 (1984) in press. 8 A. Spena, A. Viotti and V. Pirrotta, J. Mol. Biol., 169 (1983) 799. 9 L. Aspart, A. Got, M. Delseny, B. Mocquot and A. Pradet, Plant Physiol., 72 (1983) 115. 10 A.J. Bendich, R.S. Anderson and B.L. Ward, in C.J. Leaver (Ed.), Genome organization and expression in plants, Plenum Press, New-York, pp. 31--33. 11 M. Delseny, L. Aspart, R. Cooke, F. Grellet and P. Penon, Biochem. Biophys. Res. Commun., 91 (1979) 540. 12 M. Delseny, R. Cooke and P. Penon, Plant Sci. Lett., 30 (1983) 107. 13 K. Oono and M. Sugiura, Chromosoma, 76 (1980) 85. 14 M. Mac Cleland, Nucleic Acids Res., 11 (1983) r169. 1 5 F. Takaiwa, K. Oono and M. Sugiura, Nucleic Acids Res., 12 (1984) 5441.