The moderately repetitive DNA sequences of Caenorhabditis elegans do not show short-period interspersion

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Biochimica et Biophysica Acta, 520 ( 1 9 7 8 ) 6 8 8 - - 6 9 2 © E l s e v i e r / N o r t h - H o l l a n d Biomedical Press

BBA Report BBA 9 1 4 7 6

THE M O D E R A T E L Y REPETITIVE DNA SEQUENCES OF CAENORHABDITIS ELEGANS DO NOT SHOW SHORT-PERIOD INTERSPERSION

F R E D S C H A C H A T * , D E B O R A H J. O ' C O N N O R a n d H E N R Y F. E P S T E I N * *

Department of Pharmacology, Stanford University Medical Center, Stanford, Calif. 94305 (U.S.A.) ( R e c e i v e d J u n e 2 9 t h , 1978)

Summary In these studies we show that the moderately repetitive DNA sequences of Caenorhabditis elegans are n o t arranged in the characteristic short-period interspersion pattern of most eukaryotes. Rather, the moderately repetitive sequences are arranged in long arrays as in Drosophila and Apis. These findings indicate that this t y p e of arrangement is more phylogenetically diverse and hence less exceptional than previously believed.

The arrangement of moderately repetitive DNA sequences in the eukaryotic genome has been the focus of considerable interest since Britten and Davidson [1] proposed a model in which moderately repetitive DNA sequences were involved in the regulation of gene expression. The DNA of almost all eukaryotes is characterized by what has been called a short-period interspersion or "Xenopus p a t t e r n " [2,3] in which a block of 300--400 nucleotides of moderately repetitive sequences is interspersed in 1000--2000 nucleotides of single copy sequences. Only in two insects, the dipteran Drosophila melanogaster [4,5] and the hymenopteran Apis mellifera [2], has a different arrangement been observed. In this arrangement, called the "Drosophila p a t t e r n " [3], there is very little interspersion of moderately repetitive and single c o p y sequences, both are contiguous over several to tens of thousands of nucleotides. This "Drosophila pattern" has been considered "exceptional" or "atypical" because it is u n c o m m o n [3]. We show here that moderately repetitive sequence in the nematode, Caenorhabditis *Present address: D e p a r t m e n t of Anatomy, Duke University Medical Center, Durham, N.C.

27710 (U.S.A.) **Present address: Departments of Neurology and Biochemistry, Baylor College of Medicine,

Houston, Texas 77030 (U.S.A.)

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elegans, are arranged in a "Drosophila pattern" indicating that this arrangement is phylogenetically more diverse and hence less "exceptional" than previously believed. DNA of greater than 25 000 nucleotides in length was isolated from C. elegans by a modification of the procedure of Schachat and Hogness [ 6 ] . Animals from an asynchronously growing culture were lysed by incubation in 0.25M EDTA, 3.3% sarkosyl, pH 9.5 at 60°C for 3--4 h and then digested with 0.5 mg/ml Proteinase K (Beckman) at 37°C for 12 h. Solid CsC1 (Harshaw, optical grade) was added to a density of 1.70 g/cm 3 , and the solution was centrifuged to equilibrium at 40 000 rev./min in a 60 Ti rotor. The DNA-containing fractions were further purified by chromatography on hydroxyapatite (Bio-Rad) to remove non-DNA contaminants [7]. 32p-labelled DNA was prepared in the same way from animals grown on 32p-labelled bacteria [8]. DNA purified by this technique exhibits a single thermal transition with a Tm of 83.5°C and a hyperchromicity of 26% at 260 nm in 0.12 M sodium phosphate, pH 6.8, indicating 95--100% purity. When analyzed by analytical ultracentrifugation in CsC1, a main band is found with a density of 1.694 g/cm 3 (relative to Micrococcus xanthus with a density of 1.727 g/cm 3 ) and a satellite peak at 1.709 g/cm 3 . The DNA was fragmented for reassociation studies either by shearing in a Virtis homogenizer or by sonication and was sized by sedimentation in alkaline sucrose gradients or by electrophoresis in agarose gels under denaturing conditions [9]. Unlabelled 400 nucleotide moderately repetitive DNA fragments were prepared by two cycles of reassociation to a Co t of 15 followed by fractionation of the reassociated DNA on hydroxyapatite. Two types of experiments were done to determine the relationship between moderately repetitive and single c o p y sequences in C. elegans. In the first, the kinetics of reassociation of short and long fragments were compared and, in the second, the fraction of long fragments containing moderately repetitive DNA sequences was determined by reassociation with excess short, moderately repetitive fragments. Both studies indicate that at least 80% of the single copy sequences of C. eIegans are n o t involved in a short-period interspersion arrangement and that as few as 2500 transitions between moderately repetitive and single copy sequences in the C. elegans genome are sufficient to account for the observed results. Fig. 1 shows the reassociation kinetics of short (380 nucleotide) and long (3850 nucleotide) DNA fragments. The results were analyzed and the second-order rate constants and the fraction of single copy and moderately repetitive sequences determined by a least squares fit (Table I). From the reassociation of the short fragments we determined that 15% of the C. elegans DNA is composed of moderately repetitive sequences and 80% composed of single copy sequences. These values agree with previous determinations, and the reassociation rates agree within a factor of 2 [8]. Although the ' single copy sequences within long fragments from C. elegans renature more rapidly than those in the short fragments, the difference is n o t significant (Table I) if the rates are corrected for length [ 1 0 ] . In organisms with shortperiod interspersion the rate of long fragments is much greater than that of short fragments. By comparison of the fraction of fragments containing

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I

20--

--

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80--

0.1

I

1.0

I

10 EOU[VkLENT Cot

I

100

1000

Fig.1. Rea~oeiation kinetics of long and short C. elega~ D N A fragments. D N A fragments of lengths 3 8 0 (m) and 3 8 5 0 (o) were prepared from a mixture of labelled and unlabelled D N A . T h e reassociat i o n k i n e t i c s w e r e a s s a y e d b y b i n d i n g to h y d r o x y a p a t i t e a n d t h e z e r o - t i m e b i n d i n g c o m p o n e n t ( 0 . 0 4 9 f o r t h e 3 8 0 n u c l e o t i d e l o n g f r a g m e n t s a n d 0 . 1 5 f o r t h e 3 8 5 0 n u c l e o t i d e l o n g f r a g m e n t s ) corr e c t e d f o r b y t h e p r o c e d u r e o f D a v i d s o n e t al. [ 1 1 ] . T h e c u r v e s are t h e least s q u a r e s fit of t h e d a t a . TABLE I K I N E T I C A N A L Y S I S OF C A E N O R H A B D I T I S Fragment length

Component*

380 nucleotide

3850 nucleotide

ELEGANS

DNA

F r a c t i o n of fragments

K**

C o tb~

Length corrected K* *, ***

Moderately repetitive Single c o p y

0.15 0.80

4 0.013

0.25 77

4.8 0.015

Moderately repetitive Single c o p y

0.32 0.64

4 0.051

0.25T 20

1,44T 0.018

Root mean s q u a r e (%) 1.4

1.2

* T h e h i g h l y r e p e t i t i v e s e q u e n c e s w e r e n o t i n c l u d e d in t h e c o m p u t a t i o n as t h e y a c c o u n t e d f o r less t h a n 2% o f the D N A o n t h e basis of S I -studies at s m a l l C Ot values. * * R a t e c o n s t a n t in w h o l e D N A , M - I .s - I . ***Rate constant corrected to 500 nucleotide long fragments. t N o t a c c u r a t e l y d e t e r m i n e d d u e t o p a u c i t y of d a t a a t l o w C Ot values.

only single c o p y sequences at long and short fragment lengths, we estimate that 80% (0.64/0.80) of the single copy sequences of C. elegans are n o t associated with moderately repetitive sequences at fragment lengths of 3850 nucleotides. A similar conclusion can be drawn from the second experiment in which 32P-labelled, long (2470 nucleotide) fragments were mixed with a 50-fold excess of unlabelled, moderately repetitive, short (400 nucleotide) fragments and reassociated to a C0 t of 7.2. Under these conditions greater than 95% of the moderately repetitive sequences, but less than 0.4% of the single copy sequences of the labelled DNA would have been renatured given the rate constants in Table I. In two determinations, 26% and 30% of the long fragments b o u n d to hydroxyapatite, indicating that they contained moderately repetitive sequences. Since 15% of the C. elegans genome is composed of moderately repetitive sequences,

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it follows that at the long length 81% to 86% of the single c o p y sequences are n o t involved in a short period interspersion (1--((fraction b o u n d - - 0.15)/0.80} ). This arrangement is in marked contrast to the "Xenopus pattern", where typically only 35% or less of the~single copy DNA is not interspersed with moderately repetitive sequences at the long fragment lengths we have considered [ 1 1 ] . In fact, the pattern is very similar to D. meIanogaster where at fragment lengths of 1850 nucleotides and 2200 nucleotides, 91 and 85%, respectively of the single copy DNA is n o t interspersed with moderately repetitive sequences [5]. So it appears that C. elegans has a "Drosophila pattern". The lack of substantial interspersion of moderately repetitive and single copy sequences observed in D. melanogaster, A. mellifera and, now, C. elegans, although rare, does n o t seem necessarily to be "exceptional". The proposed regulatory functions of moderately repetitive sequences may be compatible with this arrangement. These is no reason for each gene sized single copy sequence to be next to a moderately repetitive sequences, especially since most single copy sequences are not transcribed at any stage of development [ 1 2 ] . Indeed the number of transitions between moderately repetitive and single copy sequences necessary to account for our observations (2500) is approximately the number of genes estimated by formal genetic methods (about 2000) [ 1 3 ] . A similar situation exists in D. melanogaster [4]. Also, even in eukaryotes characterized by the "Xenopus pattern", there is a significant fraction of the moderately repetitive sequences which are n o t interspersed in single copy sequences [14]. The "Drosophila pattern", therefore, may represent an arrangement comm o n to all eukaryotes. Long arrays of moderately repetitive sequences may play roles similar to short-period interspersed sequences, or they may have a role in chromosome structure. Such a role is suggested by in situ hybridization studies locate one such array to Drosophila teleomeres [ 15]. One result of this and the other studies [4,5] is that an exclusive pattern of short-period interspersion is n o t necessary for the regulation of gene expression in all eukaryotes and the different arrangements need n o t be considered exclusive. We thank Drs. E.H. Davidson and R.J. Britten for allowing one of us (F.S.) to visit their laboratories and Dr. Douglas Brutlag for his advice and the use of his program for the least-square fit of the reassociation data. This work was supported by a grant from the National Institute of Aging, a research career development award from the National Institute of Child Health and Human Development to H.F.E., a National Science Foundation predoctoral fellowship to D.J.O'C., and a Muscular D y s t r o p h y Association postdoctoral fellowship to F.S.

References

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Manning, J.E., Schmid, C.W. and Davidson, N. (1975) Cell 4, 141 Crain, W.R., Eden, F.C., Pearson, W.R., Davidson, E.H. and Britten, R.J. (1976) C h r o m o s o m a

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Schachat, F.H. and Hogness, D.S. (1973) Cold Spring Harb. Symp. Quant. Biol. 38, 371 Britten, R.J., Graham, D.E. and Neufeld, B.R. (1974) Methods in Enz ymol ogy, 29, 363 Sulston, J.E. and Brenner, S. (1974) Genetics 77, 95 McDonnell, M.W., Simon, M.N. and Studier, W.F. (1977) J. Mol. Biol. 110, 119 Wetmur, J.G. and Davidson, N. (1968) J. Mol. Biol. 3 1 , 3 4 9 Davidson, E.H., Hough, B.R., Amenson, C.S. and Brittcn, R.J. (1973) J. Mol. Biol. 77, 1 Davidson, E.H. (1976) Gene Activity in Early Development, 2nd ed., Academic Press, New Y ork Brenner, S. (1974) Genetics 77, 71 Goldberg, R.B., Crain, W.R., Ruderman, J.V., Moore, G.P., Barnett, T.R., Higgins, R.C., Gelfand, R.A., Galau, G.A., Britten, R.J. and Davidson, E.H. (1975) C h r o m o s o m a 51, 225 Rubin, G. (1977) Cold Spring Harb. Syrup. Quant. Biol. 4, in the press