Organisation and properties of repeated DNA sequences in rice genome

Plant Science, 55 (1988) 4 3 - 52

43

Elsevier Scientific Publishers Ireland Ltd.

O R G A N I S A T I O N A N D P R O P E R T I E S OF R E P E A T E D DNA SEQUENCES IN RICE GENOME*

MADHU S. DHAR, MADHAVI M. DABAK, VIDYA S. GUPTA and P.K. RANJEKAR** Division of Biochemical Sciences, National Chemical Laboratory, Pune -- ~11 008 (India}

(Received August 14th, 1987) (Revision received November 16th, 1987) (Accepted December 3rd, 1987)

Reassociation of high molecular weight rice DNA has revealed the occurrence of long stretches of repeated DNA which are not interrupted by single copy DNA even at a fragment length as high as 20 kilo base pairs (kbp). Majority of these repeated sequences are unusually G + C rich and show significant variations in their thermal stability. Homology studies indicate that short repeats may have evolved from long repeats in total repetitive DNA while they may be of different origin in highly repetitive DNA fraction. Restriction enzyme analysis shows the occurrence of A v a / a n d EcoR Vrepeat families. K e y words: rice; repetitive DNA; long repeats; short repeats; homology

Introduction

The arrangement of repeated and single copy DNA sequences has been studied in plants and these data have been reviewed extensively by Thompson and Murray [1], Walbot and Goldberg [2] and Flavell [3]. During the study of genome structure and genome organisation in 18 higher plants [4-8], we have shown that plants exhibit a great diversity in genome organisation patterns and that this diversity arises mainly due to variations in the length of interspersed repeat DNA sequences. We have further shown that rice and cucumber are unique genomes in consisting of long stretches of repeat and single copy DNA sequences which are not interrupted by each other [4,8]. We were intrigued by this novel type of genome organisation and hence wished to characterise the repeat DNA sequences in detail. Since, cucumber satellite DNA which accounts for a major portion of repetitive DNA, is being studied by others [9,10], we decided to carry out this work in rice. *NCL Communication No.: 4330. **To whom all correspondence should be addressed.

In the present work, reassociation of high molecular weight rice DNA (>20 kbp), was determined at the limit Cot of 50 (a Cot value by which most of the repeat DNA sequences undergo reassociation). Rice repetitive DNA was then fractionated into long and short repeats and these were characterised by determination of their thermal stability. Significant variations in G + C content were noticed in different repeated DNA sequences. The homology studies indicated that long and short repetitive DNA sequences reassociating by Cot 0.1 may belong to different family of repeats, as compared to those reassociating between Cot 0 . 1 50. Materials and methods

Isolation of nuclear DNA Native, high molecular weight DNA was isolated from the shoots of 8-day-old plants of rice (Oryza sativa var. Basmati) as described earlier [11]. Isolation of highly repetitive and total repetitive DNA Highly repetitive (Cot 1.0 x 10 -1 M.s) and

0618-9452/88/$03.50 © 1988 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

44

total repetitive (Cot 5.0 × 101 M.s) DNA fractions were isolated from native, unsheared rice DNA (>20 kbp). The latter was dissolved in 0.18M NaC1 containing 0.006 M P I P E S buffer, (pH 6.8) and was denatured for 10 rain followed by reassociation at 62°C to 1.0 × 10 -1 M.s and 5.0 x 101 i . s . After the reassociation, DNA samples were adjusted to 25 mM sodium acetate (pH 4.5), 0.1 mM ZnSO 4 and 25 mM 2-mercaptoethanol [12]. $1 nuclease (0.10 units/~g of DNA was then added and the DNA samples were incubated for 15 min at 37°C to digest single-stranded DNA. The reaction was terminated by treatment with chloroform/isoamyl alcohol (24:1 v/v) mixture and S1 nuclease resistant repetitive DNA duplexes were precipitated with 2 volumes of chilled ethanol. Cot 0.1 and Cot 50 repetitive DNA fractions were resolved into long and short repeats by agarose gel electrophoresis [13]. In brief, the highly repetitive (Cot 0.1) and total repetitive (Cot 50) DNAs were loaded on 0.7% agarose gels. After electrophoresis, the gel was stained with ethidium bromide at 1 ~g/ml, for 30 min and visualised on a ultraviolet (UV)~transilluminator. Two regions corresponding to long ( 4 20 kbp) and short repeats (about 0.5 kbp) were cut and the agarose pieces were submerged in phenol equilibrated with tris buffer and frozen at - 7 0 ° C for 2 h. Then the frozen gel pieces and phenol were thawed out gradually to 4 10 °C and aqueous layer was collected by centrifugation. Extraction of ethidium bromide from the aqueous layer and precipitation of DNA were carried out according to Maniatis et al. [14]. Prior to using these fractions for further studies, their size distribution was confirmed by both neutral and alkaline gel electrophoresis according to Maniatis et al. [14].

Restriction enzyme analysis Restriction enzyme digestions of repetitive DNAs of rice were carried out according to Maniatis et al. [14]. The digestion mixtures were analysed on 1% agarose gels using )J-lind III digest as a molecular weight marker. Southern blotting and hybridisation The short repeats of highly repetitive and of total repetitive DNAs were used as probes for Southern hybridisation. These were labelled by nick translation according to Rigby et al. [16] and the unincorporated dNTPs were removed by Sephadex G-50 column chromatography. Transfer of DNA from agarose gels to nitrocellulose membrane, prehybridisation, hybridisation and washing of the blots to remove unhybridised radioactive material were essentially according to Maniatis et al. [14]. Results

Reassociation of high molecular weight rice DNA We have earlier reported that rice had a novel genome organisation as there is no interspersion of repeat and single copy DNA sequences at a DNA fragment length of 6.5 kbp [4]. In order to find out if this unique pattern persists even at a higher DNA fragment length, reassociation of high molecular weight rice DNA (> 20 kbp) was examined. From Table I, it can be seen that the extent of reassociation

Table I. Reassociation of rice DNA of different f r a g m e n t lengths. Figures in parenthesis indicate the n u m b e r of experiments carried out and independent DNA preparations used. F r a g m e n t size in bp

Thermal denaturation studies These experiments were carried out in Beckmann DU-8B UV-visible spectrophotometer equipped with a T programme cassette. The melting temperatures ( T ) and G + C contents were calculated according to Mandel and Martour [15].

550 (7) 6500 (5) 20000 (5)

% reassociation' + S1 nuelease

- S1 nuclease

48 -+ 0.40 48 -+ 0.52 46 -+ 0.57

52 _+ 0.15 55 _+ 0.1 55 _+ 0.26

aReassociation is m e a s u r e d at limit Cot-value of 50, i.e. the Cot-value at which most of the repeated sequences reassociate.

45

r o s e gel e l e c t r o p h o r e s i s , it r e v e a l s t h e p r e s e n c e of b o t h long (4-- 20 kbp) and s h o r t ( 2 0 0 - 300 bp) D N A s e q u e n c e s . T h e s e a c c o u n t for 6 0 % and 4 0 % , r e s p e c t i v e l y , of t h e t o t a l Cot 50 D N A (Fig. 1A,1B). T h e Cot 0.1 fraction which is cons i d e r e d to consist of m a i n l y highly r e p e t i t i v e D N A s e q u e n c e s also s h o w s t h e o c c u r r e n c e of t h e a b o v e t w o t y p e s of r e p e a t s e x c e p t t h a t t h e s h o r t r e p e a t s r e p r e s e n t 6 0 % of t h e t o t a l Cot 0.1 D N A (Fig. 1A, 1C).

of rice D N A at t h e limit Cot of 50 does not i n c r e a s e a p p r e c i a b l y w i t h i n c r e a s e in t h e D N A f r a g m e n t length. F u r t h e r m o r e , t h e d e c r e a s e in p e r c e n t r e a s s o c i a t i o n a f t e r S1 n u c l e a s e is 5 5 46% in case of > 2 0 k b p D N A as a g a i n s t 55-480/0 in case of 6.5 k b p D N A This d e c r e a s e cannot b e c o n s i d e r e d v e r y a p p r e c i a b l e and hence it can be concluded t h a t m a j o r p a r t s of the r e p e a t e d and single copy D N A s e q u e n c e s a r e u n i n t e r s p e r s e d at a D N A f r a g m e n t l e n g t h of > 20 k b p in rice g e n o m e .

Thermal stability of rice repetitive D N A T h e m e l t i n g d a t a of t h e t o t a l r e p e t i t i v e D N A s and t h e i r r e p e a t f r a c t i o n s a r e t a b u l a t e d in T a b l e II. T h e high m o l e c u l a r w e i g h t rice

Occurrence of long and short repeats in rice repetitive D N A W h e n rice Cot 50 D N A is a n a l y s e d b y agae

b

c

d

e

o

b

c

d

a

b

c

d

i (A)

(B)

(C)

Fig. 1. Electrophoresis of high molecular weight rice DNA along with highly repetitive (Cot 0.1) and total repetitive (Cot 50) DNAs and their long and short repeats. (A) High molecular weight rice DNA (lane a), Cot 0.1 DNA (lane c), Cot 50 DNA (lane d), )J-lindIII digest (lane b) and ®X 174 RF DNA Hae III digest (lane e) as molecular weight markers. (B) Long and short repeats isolated from Cot 50 DNA. Long repeats (lane b), intermediate repeats (lane c) and ),HindIII digest as high molecular weight marker (lane a). (C) Long and short repeats isolated from Cot 0.1 DNA. Long repeats (lane a), intermediate repeats (lane c), short repeats (lane d) and ~I-IindIll digest as high molecular weight marker (lane b). Electrophoresis was carried out on 1.0% (A) and 1.4o/0 (B and C) neutral agarose slab gels in TAE buffer (pH 8.1) at a constant current of 30 mA.

46 Table II. Thermal denaturation properties of rice repetitive DNAs. LMF and HMF stand for low melting fraction and high melting fraction, respectively. Figures in parenthesis indicate the number of experiments carried out. Fraction

Calf thymus DNA (4) Total rice DNA (5) Highly repetitive DNA (5) (Cot 1.0 x 10-~M.s) Cot 0.1 Long repeat (4) Short repeat (4) Total repetitive DNA (5) (Cot 5.0 x 101 M.s) Cot 50 Long repeat (4) Short repeat (4)

Proportion in the genome %

T °C'

Hyperchromicity b %

G + C content c %

15.0

85.8 _+ 0.75 85.1 __. 0.8 95.5 ___1.38

27.8 ___1.18 24.4 +__1.1 20.0 __ 1.5

40.2 38.5 63.9

6.0 9.0 50.0

94.8 + 0.21 92.9 _+ 1.06 88.9 _+ 1.4

27.0 _+ 2.6 21.5 _+ 1.6 17.7 +_ 1.7

62.2 57.5 47.8

27.0 20.0 LMF 3.0 HMF

97.5 ___0.5 83.7 ___0.35 LMF 98.0 _ 0.35 HMF

16.8 _+ 0.21 11.4 _+ 0.9

68.8 35.1 LMF 70.0 HMF

•Temperature corresponding to 50% hyperchromicity. bCalculated using the formula H = Am (98°C) - A 2 6 o (62°C)/As~0(98°C) × 100. ¢Calculated using the f o r m u l a % G + C = ( T - 69.3) x 2.44. D N A has a T of 85.1°C. T h e Cot 0.1 and Cot 50 D N A s on t h e o t h e r hand h a v e h i g h e r T m -values of t h e o r d e r of 95.5 °C and 88.9 °C, r e s p e c t i v e l y . This i n d i c a t e s t h a t r e p e a t D N A s e q u e n c e s in rice a r e G + C rich. I t is i n t e r e s t i n g to note t h a t highly r e p e t i t i v e (Cot 0.1) D N A in particular is v e r y t h e r m o s t a b l e . T h e long and s h o r t r e p e a t s of Cot 0.1 D N A s h o w a m o n o p h a s i c c u r v e with a T -value of 94.8°C and 92.9°C, r e s p e c t i v e l y . The long r e p e a t s of Cot 50 D N A h a v e a T - v a l u e of 97.5 °C while its s h o r t r e p e a t s show a biphasic c u r v e with a m a j o r low m e l t i n g fraction of T = 83.7 °C and a m i n o r high m e l t i n g fraction of T = 98°C. T h e s e r e s u l t s a r e s u m m a r i s e d in T a b l e II.

D N A sequence homology between long and short repeats F r o m t h e a g a r o s e gel e l e c t r o p h o r e s i s and t h e r m a l s t a b i l i t y d a t a of Cot 0. 1 and Cot 50 D N A fractions, it is clear t h a t rice r e p e t i t i v e D N A consists of d i f f e r e n t s t r e t c h e s of r e p e a t families s h o w i n g v a r i a t i o n s in t h e i r length and t h e r m a l stability. To a s s e s s w h e t h e r t h e s e r e p e a t families b e l o n g to s a m e / s i m i l a r t y p e of r e p e a t s e q u e n c e s or t h e y a r e i n d e p e n d e n t of

each other, S o u t h e r n h y b r i d i s a t i o n e x p e r i m e n t w e r e u n d e r t a k e n u s i n g s h o r t r e p e a t s of Cot 0.1 and Cot 50 D N A s as p r o b e s . F r o m Fig. 2A and B, it is o b s e r v e d t h a t s h o r t r e p e a t s of Cot 0.1 s h o w v e r y little h y b r i d i s a t i o n to its long r e p e a t s w h e r e a s s t r o n g hybridisation is s e e n with itself. H o w e v e r , t h e s a m e p r o b e shows s t r o n g h y b r i d i s a t i o n to long r e p e a t s of Cot 50 D N A , while v e r y little hybridisation is s e e n w i t h its s h o r t r e p e a t s . This s u g g e s t s t h a t t h e long and s h o r t r e p e a t s r e a s s o c i a t i n g b y Cot 0.1 a r e i n d e p e n d e n t and b e l o n g to d i f f e r e n t r e p e a t families. W h e n s h o r t r e p e a t s of Cot 50 w e r e u s e d as a probe, h y b r i d i s a t i o n is s e e n with its own long r e p e a t s , while v e r y little h y b r i d i s a t i o n is seen with s h o r t as well as long r e p e a t s of Cot 0.1 (Fig. 3A,B). This indicates t h a t t h e long and s h o r t r e p e a t s r e a s s o c i a t i n g b e t w e e n Cot 0.1 and 50 m a y belong to s i m i l a r / s a m e t y p e of r e p e a t families which h a v e u n d e r g o n e v e r y little s e q u e n c e d i v e r g e n c e d u r i n g the course of evolution.

Restriction enzyme analysis of rice repetitive DNA Since,

the

thermal

denaturation

studies

47

o

b

d

0

Kbp

Kbp 23.1 N

-25.1

9-6--

-

9"6

6.6--

-

6.6

4.4--

--

4-4.

--

2"2 2"0

.~ 2"2-2"0--

o

" *.

j

-!

.

o

0

Q

(A)

(B)

Fig. 2. Autoradiograms showing the homology of Cot 0.1 short repeats with the long and short repeats of Cot 0.1 and Cot 50 DNAs. (A) Southern hybridisation of Cot 0.1 short repeats to long and short repeats of itself. Cot 0.1 long repeats (lane a), Cot 0.1 intermediate repeats (lane c), Cot 0.1 short repeats (lane d), and iHind III digest as high molecular weight marker (lane b). (B) Southern hybridisation of Cot 0.1 short repeats to long and short repeats of Cot 50 DNA. Cot 50 long repeats (lane a), Cot 50 intermediate repeats (lane b), Cot 50 short repeats (lane c).

revealed the occurrence of G + C rich sequences in the rice genome, restriction enzyme analysis of the latter was undertaken to provide additional evidences for the presence of these sequences. Detection of sequence specific methylation and specific repeat families were the additional aims in this study. From Fig. 4A and 4B, it is clear that enzymes

specific for GC rich sequences like Msp I, Hpa II, Hae III and Hha I show extensive digestion of rice repetitive DNA thus confirming the presence of GC rich sequences. The above figures also show the digestion patterns of other enzymes like Barn H1, Alu I and Taq /. It is observed that Hpa H digests rice repetitive DNA more than Msp I. Hpa H

48

Q

b

c

a

b

c

Kbp

Kbp

23.1 "" 9-6-6"6-

25.1 u

9.6 6.6

4,-4--

4.4

2,2

m

2.0-I

(A)

.

,

2"2 2"0

(B)

Fig. 3. Autoradiograms showing the homology of Cot 50 short repeats with the long and short repeats of Cot 0.1 and Cot 50 DNAs. (A) Southern hybridisation of Cot 50 short repeats to long and short repeats of Cot 0.1 DNA. Cot 0.1 long repeats (lane a), Cot 0.1 intermediate repeats (lane b), Cot 0.1 short repeats (lane c). (B) Southern hybridisation of Cot 50 short repeats to long and short repeats of itself. Cot 50 long repeats (lane a), Cot 50 intermediate repeats (lane b), Cot 50 short repeats (lane c).

does not cleave the DNA when internal C residue of the sequence 5'-CCGG-3' is methylated [17,18]; while Msp I cleaves it when internal C is [17-20]. Thus, the digestion pattern suggets the abundance of mCCGG type of sequences rather than CmCGG type. This also indicates a high frequency of CpC methylation as compared to CpG methylation. The enzymes

Hae I I I and Hha I also give information about

methylation status of sequences of 5'-GGCC-3' and 5'-GCGC-3', respectively. H h a I recognising 5'-GCGC-3' gives appreciable digestion. Since, methylation of either of C residues in Hha I site can prevent digestion [21,22], it can be inferred that there is very little GpC methylation. As compared to this, Hae I I I shows slightly less

b

c

d

(A)

e

f

g

h

i

a

b

c

d

(B)

e

f

g

h

i

a

b

(C)

c

d

•

Fig. 4. Electrophoresis pattern of rice repetitive DNA fractions with different restriction enzymes. (A and B) Cot 0.1 and Cot 50 DNA digested with restriction enzymes; Msp I (lane b), Hpa I I (lane c), Hae I l l (lane d), Hha I (lane e), A l u I (lane f), Bam H I (lane g), Taq I (lane i) and Attind I I I digest as high molecular weight marker (lanes a and h). (C) Cot 0.1 and Cot 50 DNA digested with restriction enzymes; A v a I (lanes b and d), EcoR V (lanes c and e) and A/-/ind I I I digest as high molecular weight marker (lane a). Electrophoresis was carried out on 1.0O/o (A, B and C) neutral agarose slab gels in TAE buffer (pH 8.1) at a constant current of 30 mA.

a

50 digestion. Hae I I I digests 5'-GGCmC-3 ' type of sequences [21,22], thus indicating 5-methylcytosine to be a part of CpC dinucleotides. When the rice repetitive DNA fractions are digested with A v a I, two bands of about 4.3 kbp and 4.9 kbp are observed. Also, digestion with E c o R V gives a single band of about 4.6 kbp. This indicates that 5'-GPyCGPuG-3' and 5'GATATC-3' type of sequences are present at regularly spaced intervals in rice genome. Thus, these data suggest the presence of A v a I and E c o R V repeat families in rice DNA. Discussion

The characterisation of rice repetitive DNA by restriction enzymes has been reported earlier [23], and a basic repeat unit of about 3 0 0 800 bp is detected after E c o R I digestion. The r R N A genes have also been analysed in rice and it is shown that the size of the rice rDNA unit is about 7 8 0 0 - 8 0 0 0 bp [24]. The present work describes the occurrence of different long and short repeat sequences in rice genome and attempts have been made to assess their homology with each other. One of the unique features of our study is about the organisation of rice repetitive DNA. These sequences occur in very long stretches (4--20 kbp) without the interspersion of single copy DNA sequences. This is a novel arrangement and has not been reported so far in higher eukaryotes. The second important feature of rice repetitive DNA is its high thermal stability. As shown in Table II, most of the repetitive classes have T - v a l u e s much higher than the native rice DNA. The occurrence of a substantial amount of high melting fraction (about 30O/o of the total DNA) is particularly very striking. Its presence has been reported earlier in some millets [25]. However, it accounts for a very small portion of the total DNA in millets. The high thermal stability of rice repetitive DNA can be interpreted to indicate a very low rate of sequence divergence or a recent origin of repeats. Thirdly, based on the hybridisation and melt.

ing data, rice repetitive DNA consists of the following classes of repeat sequences: (1) Short repeats reassociating by Cot 0.1 ( T 92.9°C) with no homology to the long repeats reassociating by Cot 0.1. (2) Long repeats reassociating by Cot 0.1 ( T 94.8°C). (3) Short repeats reassociating between Cot 0.1 and 50 with homology to long repeats reassociating between Cot 0.1 and 50; and consisting of a major low melting fraction ( T 83.7 °C) and a minor high melting fraction ( T 98 °C). (4) Long repeats reassociating between Cot 0.1 and 50 ( T 97.5 °C). From the above discussion, it is clear that long and short repeats reassociating between Cot 0.1 and 50 show homology to each other, indicating that they constitute same or similar type of repeat families. Moreover, these repeats appear to have undergone a very low degree of sequence divergence. As reported in a few plant species [26,27], it is found that short repeats are more diverged than long repeats. Hence, it is generally assumed that short repeats evolve from long repeats. If this is so, then the long repeats should exhibit higher thermal stability because they are relatively recent in origin and have got little time to diverge. This is exactly the case in the repeats reassociating between Cot 0.1 and 50 in the rice genome. As against this, there are evidences which show that short repeats evolve from shorter sequences such as tRNA or 5S RNA [28,29]. The long and short repeats reassociating by Cot 0.1 do not show any hybridisation with each other, suggesting that they belong to different repeat families or have independently diverged extensively. The Cot 0.1 short repeats do not show any hybridisation with short repeats of Cot 0.1 - 50. This is rather an unexpected observation. It suggests that the Cot 0.1 short repeats may be interspersed with other repeat families which do not reassociate rapidly at Cot 0.1. This indicates that rapidly reassociating sequences are not interspersed with very slow or single copy sequences. Similar observation of inter-

51 s p e r s i o n of r e p e t i t i v e s e q u e n c e s b e l o n g i n g t o different kinetic classes among themselves h a v e b e e n r e p o r t e d e a r l i e r [6,7]. F r o m t h e a b o v e o b s e r v a t i o n s , it is c l e a r t h a t we have got some information about the gross i n t e r r e l a t i o n s h i p s of l o n g a n d s h o r t r e p e a t s e q u e n c e s in the rice genome. F i n e r analysis can be achieved by cloning and sequence a n a l y s i s of a specific p r o b e f r o m t h e s e r e p e t i tive classes. Finally, the rice r e p e t i t i v e D N A does not s h o w a n y o r g a n i s e d s t r u c t u r e w i t h m o s t of t h e e n z y m e s u s e d e x c e p t A v a I a n d E c o R V. A s c o m p a r e d to the available d a t a on t a n d e m and d i s p e r s e d r e p e a t f a m i l i e s i n p l a n t s , it is for t h e f i r s t t i m e t h a t t h e o c c u r r e n c e of f a m i l i e s is shown with these two enzymes.

7

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12 Acknowledgements T w o of u s (M. D h a r a n d M. D a b a k ) t h a n k t h e C o u n c i l of S c i e n t i f i c a n d I n d u s t r i a l R e s e a r c h for t h e a w a r d of S e n i o r R e s e a r c h F e l l o w s h i p . The a u t h o r s are i n d e b t e d to P r o f e s s o r J o h n B a r n a b a s , H e a d , D i v i s i o n of B i o c h e m i c a l S c i e n c e s , N C L for his s u p p o r t a n d e n c o u r a g e ment.

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W.F. Thompson and M.G. Murray, The nuclear genome: structure and function, in: A. Marcus (Ed.),The Biochemistry of Plants: A Comprehensive Treatise, Vol. 6, Proteins and Nucleic Acids, Academic Press, 1981, pp. 1-82. V. Walbot and R. Goldberg, Plant genome organisation and its relationship to classicalplant genetics, in: T.C. Hall and J.W. Davies {Eds.), Nucleic Acids in Plants, Vol. I,C R C Press, Florida, 1979, pp. 3--41. R. Flavell,The molecular characterisation and organisation of plant chromosomal D N A sequences. Annu. Rev. Plant Physiol.,31 (1980)569--596. V.S. Gupta, S.R. Gadre and P.K. Ranjekar, Novel D N A sequence organisation in rice genome. Biochim. Biophys. Acta, 656 (1981) 147-- 154. V.S. Gupta and P.K. Ranjekar, D N A sequence organisation in finger millet (Eleusine coracana). J. Biosci. 3 (1981) 417 -- 430. M.R. Bhave, V.S. Gupta and P.K. Ranjekar, Arrangement and size distribution of repeat and single copy DNA sequences in four species of Cucurbitaceae. Plant Syst. Evol., 152 (1986) 133-- 151.

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