72
Biochimica et Biophysica Acta, 563 (1979) 72--81 © Elsevier/North-Holland Biomedical Press
BBA 99465
METHYLATION OF MOSQUITO DNA
R.L.P. ADAMS, E.L. McKAY, L.M. CRAIG and R.H. BURDON
Department of Biochemistry, University of Glasgow, Glasgow G12 8QQ (U.K.) (Received October 12th, 1978)
Key words: DNA methylation; Methyl nucleotide; (Mosquito)
Summary Cells of the mosquito Aedes albopictus have 5.6 pg DNA/cell. This DNA is 58 tool% (A + T) and has about 0.03 tool% each of 5-methylcytosine and 6-methylaminopurine. The DNA is largely degraded by the restriction enzyme HpaII showing the virtual absence of the sequence CMeCGG. Small amounts of enzymic activity recovered largely in non-nuclear fractions transfer methyl groups from S-adenosylmethionine to cytosine and adenine.
Introduction In the D N A from all higher eukaryotes which have been studied there is one minor base, namely 5-methylcytosine. The frequency of occurrence of this minor base varies dramatically. Thus, in higher plants the proportion of cytosines methylated varies between 18% and 3 3 % [1]. Most other species from the Coelentera, Mollusa, Annelida, Echinoderrnata and Chordata [2--4] have from 2 % to 8 % of their cytosines methylated. The Arthropoda, however, as far as they have been investigated have a lower level of methylation [2,3]. In Drosophila this is associated with a very low content of DNA/cell [5]. The role of D N A methylation in eukaryotes is not known. Various suggestions have been made that it m a y play a part in the control of differentiation [6,7]. If methylation has a c o m m o n role in eukaryotes it is surprisingthat the extent of methylation varies so widely. One possibility is that the lower the amount of DNA/cell the smaller the proportion of this DNA is methylated. To investigate this possibility we have looked at the level of methylation of mosquito DNA. We report here that, .although mosquito cells are comparable to mammalian cells in their DNA content, the 5-methylcytosine content of this DNA is extremely low. Enzymic activity capable of methylating DNA in
73 vitro can be recovered from insect cells. Furthermore trace amounts of methyladenine are found in Aedes DNA and the enzyme preparation methylates both cytosine and adenine. Materials and Methods
Cells and nuclei. Aedes albopictus cells were obtained from Flow Laboratories, Irvine, U.K., and maintained as monlayer cultures at 27°C in Mitsuhashi and Maramorosch medium supplemented with 20% foetal bovine serum, nonessential amino acids, glutamine and penicillin and streptomycin. The medium was adjusted to pH 7.2 with NaOH. The cultures were tested for mycoplasmal contamination using PPLO agar and for bacterial contamination on blood agar plates. All tests were negative. Cells were harvested by scraping them off the glass and washed with phosphate-buffered saline. Nuclei were prepared either by homogenesing the cells in buffer M (50 mM Tris-HC1, pH 7.8, 10% glycerol, 1 mM EDTA, 1 mM dithiothreitol) or in 1% Tween 80. The nuclei were then sedimented at 800 × g for 10 min. Ascites cells and nuclei were prepared as described previously [8]. When appropriate cells were labelled with [U-14C]deoxycytidine (1 gCi/ 30 ml); deoxy[G-3H]adenosine (300 uCi!30 ml) or [Me-3H]methionine (500 gCi/30 ml) (The Radiochemical Centre, Amersham, U.K.). Methylase preparation and assay. The method used was the same as that for obtaining DNA methylase from mouse ascites cells [8] and involved preparing nuclei and treating them in buffer M containing 0.4 M NaC1. The resulting extract was dialysed against buffer M. In addition, to test for non-nuclear enzymic activity, cells were homogenised in buffer M as described above and both the supernatant fraction and a nuclear extract were assayed for DNA methylase activity as previously described [8]. The standard assay mixture (70 gl) contained 20 pg single stranded Escherichia coli DNA, 1.7 pCi S-adenosyl-L-[Me-3H]methionine ( l p C i / n m o l ; The Radiochemical Centre, Amersham, U.K.) 0.1 M NaC1 and 30--50 ~l buffered enzyme solution. After incubation the DNA was purified and incorporation measured as described previously [8]. In studies of nuclear incorporation 70 ~1 of buffer M nuclei were incubated with 1.7 uCi S-adenosyl-L-[Me-3H]methionine as above and incorporation into endogenous DNA measured as previously described [9]. The DNA methylase activity could be partially purified and concentrated by ammonium sulphate fractionation. The activity from a pooled supernatant fraction and nuclear extract was recovered in the 30--45% saturation fraction on precipitation with ammonium sulphate and it was this material which was centrifuged on glycerol gradients following dialysis against buffer M containing 0.2 M NaC1 and 5% glycerol. Glycerol gradients were prepared by layering 1.3 ml each of 10%, 20% and 30% glycerol in buffer M containing 0.2 M NaCI into tubes for the SW 50.1 Spinco rotor. The gradients were allowed to form at room temperature for 5 h and then cooled on ice. Enzyme fractions (0.3 ml) were layered onto the gradients which were spun at 144 000 × g for 16 h. Markers of alcohol
74 dehydrogenase and catalase were used. In the assay for enzymic activity in fractions from the glycerol gradients the assay volume was doubled enabling 70-/~1 samples to be assayed and the specific activity of the S-adenosyl-L[Me-3H]methionine was increased to 3.4 pCi/nmol. DNA preparation and base analysis. DNA was prepared from cultured mosquito cells by the method previously described [7]. For use as substrate in the methylase assay it was dissolved in 50 mM KC1. The DNA solution had E26o/E28o = 1.9 and on analysis of the spectrum [10] the DNA was found to have an A + T content of 58 mol%. Commercial preparations of calf thymus DNA, E. coli DNA and a preparation of mouse L929 cell DNA made by the same method as the Aedes DNA had A + T contents of 60, 50 and 60 mol%, respectively. For base analysis the DNA was hydrolysed either using 12 N perchloric acid as previously described [8] or using 98% formic acid at 180°C for 30 min. The formic acid was then evaporated and the bases dissolved in 0.1 N HCI for chromatography on Whatman No. 1 paper using butanol/HC1 as previously described [8]. The proportion of the radioactivity in the various spots was analysed by shredding them into toluene-based scintillation fluid. Alternatively, after hydrolysis of the DNA, the bases were dissolved in ammonium carbonate buffer (20 mM; pH 10.0 at room temperature} and applied to a column of Aminex A6 (Bio Rad Labs; 27 cm × 1.2 cm) which was eluted with the same buffer at 50°C with a flow rate of about 1 ml/min. E. coli B, salmon testis and calf thymus DNA were purchased from Sigma, London, Chemical Co., k-DNA from Miles Laboratories, Slough, U.K. Restriction enzyme treatment. For treatment with the restriction enzyme HpaII duplicate samples of ~4C-labelled DNA were dissolved in 100 pl buffer containing 20 mM Tris-HC1, pH 7.4 20 mM MgC12, 12 mM KC1, 2 mM dithiothreitol, and 0.2 mg/ml gelatin, and to one sample was added 2 pl HpaII (Boehringer Corp. Ltd.). The samples were incubated at 37°C for 90 min and electrophoresed in 1.5% agarose tube gels as described by Tegtmeyer [11]. When the bromophenol blue marker had run 8 cm the gels were sliced; the slices incubated in 0.3 N NaOH at 60°C overnight and the neutralised samples counted using a Triton/toluene scintillator. Results
DNA content o f Aedes cells Three independent suspensions of exponentially growing A. albopictus cells were prepared by trypsinisation and counted in a Coulter counter model B. After washing the cells in balanced salt solution and cold 0.5 N perchloric acid, D N A was extracted into 0.5 N perchloric acid at 70°C for 30 rain. Duplicate samples were assayed for DNA by the method of Burton [12]. The value obtained for the D N A content of an exponentially growing population o f A. aibopictus cells was 5.62 _+0.18 pg/cell. This compares with a value of 2.9 pg/Aedes cell obtained by Spradling et al. [13] and a figure of 13 pg/L929 cell obtained by us. It is very much higher than the 0.2 pg/cell recorded for Drosophila [ 5 ].
75
Presence of minor bases in mosquito DNA Two different approaches were used to determine the presence of minor bases in the insect DNA. These were (1) incubation of cells with a radioactive nucleoside followed by base analysis to search for conversion to a minor base or, (2) incubation o f cells with [Me-SH]methionine followed by base analysis to search for specific methylated bases. Insect cells were grown for three days in the presence o f [ 14C]deoxycytidine and the DNA purified from these cells was subjected to base analysis. Fig. l a shows the section o f the paper chromatogram where cytosine and 5-methylcytosine are separated. For comparison a similar result for mouse L929 cells is shown. No 5-methylcytosine is detectable in the insect DNA and we feel the level o f sensitivity would enable us to detect if 0.2% of the cytosine had been methylated. However, analysis of this material using ion exchange in Aminex A6 which gives a much better separation o f cytosine and 5-methylcytosine reveals a very small shoulder on the curve which shows that 0.17% of the cytosine are methylated (Fig. lb). Similar base analyses following incubation of cells with [Me-3H]methionine reveal a number o f tritiated species. Most radioactivity is found in thymine and (a)
(b) C
16
21
14
12
MC
10r
. ~-Aedes
Ib
18 G
10
!
15
I
A
C
MC
46
I I e~
'
8
4
M
B
6
0~ 25
30
35 cm
40
20
30 40 Fraction No.
50
60
Fig. 1. I n vivo c o n v e r l t o n o f d e o x y [ 1 4 C ] c y t i d i n e t o 5 - m e t h y l d e o x y e y t i d i n e . Early l o g p h a s e A e d e s cells w e r e g ~ o w n for 3 d a y s in t h e p r e s e n c e o f d e o x y [ U - 1 4 C ] c y t i d i n e ( 1 p C i / 3 0 m l ) after w h i c h t i m e t h e D N A w u i s o l a t e d a n d h y d r o l y s e d t o t h e b a s e s as d e s c r i b e d in Materials and M e t h o d s . (a) H y d r o l y s i s w i t h 1 2 N perchioric acid a n d c h r o m a t o g r a p h y o n paper, o, A e d e s cells; e , m o u s e L 9 2 9 cells. (b) H y d r o l y s i s w i t h formic acid a n d s e p a r a t i o n in A m i n c x A 6 . T h e a r r o w s indicate t h e p o r t i o n o f e l u t i o n o f m a r k e r s o f t h y m i n e ( T ) , guanine (G), a d e n i n e ( A ) , c y t o s i n e (C) and 5omethylcytol~-le (MC). T h e insert s h o w s t u b e s 4 0 - - 5 6 o n a r e d u c e d scale.
76 (a)
(b)
MAP MC
5°o
2D-T?
T
?
4O0 15300
,~
~-oo
II
100
:
~ I
5 -
O --I~--~-~" I0
20
30
cm
15
25
35
45
Fraction
Fig. 2. In vivo i n c o r p o r a t i o n of m e t h y l groups from [Me-3H ]me t ht oni ne i n t o Aede8 DNA. Early log phase A e d e s cells were grown for 2 days in the presence of [ M e - 3 H ] m e t h i o n i n e (500 # C i / 3 0 ml ) after which t i m e the DNA was isolated, h y d r o l y s e d with formic acid a nd t he bases separated. ( a ) C h r o m a t o g r a p h y on paper; o, A e d e s cells; o, m o u s e L929 cells on 1/10 scale. (b) Separation on A m i n e x AS. The arrows indicate the p o s i t i o n of elution of markers of t h y m i n e (T), guanine (G), adenine (A), cytosine (C), purine (Pu), 5 - m e t h y l c y t o s i n e (MC) and 6 - m e t h y l a m i n o p u r i n e (MAP).
significant amounts are found in the purines, particularly adenine. Two other species are found which, both on paper chromatography and ion exchange run together with 5-methylcytosine and 6-methylaminopurine (Fig.2a and b). A similar experiment with mouse L929 cells reveals 5-methylcytosine as the only TG A
C
MAP
20 i 12 -
20
~20
30 40 Fraction
"
50
Fig. 3. In vivo e o n v e n i o n of deoxy[G-3H]adeno4dnc to 6-methylaminopultxm. Early log phase A e d e s cells were grown for 2 d a y s in t h e presence of d e o x y [ G - 3 H ] a d e n o s / n e (300 ~Ci~30 mD a f t ~ w hi c h t i m e t he DNA was isolated, h y d r o l y s e d w i t h fonnde aoAd and the bases s e pa ra t e d on A m l n e x A6. The arrows indicare t h e poldtdon o f elut/on of m a r k e r s t h y m i n e (T), guanine (G), adenine (A), eytos/ne (C) a nd 6-methyla m l n o p u r l n e (MAP). The insert shows t u b e s 35---50 on a reduc e d scale.
77 minor base. In an attempt to confirm the presence of a methylated adenine Aedes cells were incubated with deoxy[G-3H]adenosine for two days and the DNA isolated, hydrolysed and the bases analysed using ion-exchange chromatography. Fig. 3 shows a shoulder of radioactivity in the position of 6-methylaminopurine corresponding to about 0.1% of the adenine present.
Treatment with HpalI Treatment of 14C-labelled Aedes DNA with the restriction enzyme HpaII leads to total degradation of the high molecular weight DNA to fragments ranging in length from less than 100 nucleotide pairs to several thousand nucleotide pairs (Fig. 4). HpaII cleaves unmodified double-stranded DNA in the sequence CCGG; but methylation of a cytosine in the CG doublet prevents cleavage [14,15]. Although a regular distribution of HpaII sites would lead to production of fragments of around 330 nucleotide pairs long the results of Fig. 4 are not inconsistent with a random distribution of HpaII sites, few if any of which are modified. Similar studies with mammalian DNA in which a con-
2000
-
1500 -
~1000 -
500
[ I f
C°ntr°l
-
2
4
6 crn
8
10
12
Fig. 4. T r e a t m e n t o f 1 4 C o l a b e l l e d A e d e s D N A w i t h HpaII. D N A w a s i s o l a t e d f r o m Ae de s cells g r o w n i n the p r e s e n c e o f d e o x y [ 1 4 C ] e y t i d i n e . T h e D N A w a s i n c u b a t e d w i t h t h e r e s t r i c t i o n e n z y m e H p a I I f o r 0 ( e ) o r 9 0 m i n (o) a n d s u b j e c t e d t o e l e e t r o p h o r e s t s o n a g a r o s e gels as d e s c r i b e d i n M a t e r i a l s a n d M e t h o d s . T h e p o s i t i o n o f t h e r a d i o a c t i v e D N A f r a g m e n t s w a s a s c e r t a i n e d b y slicing t h e gel a n d c o u n t i n g t h e s a m pies u s i n g a T r i t o n / t o l u e n e s c i n U l l a t o r .
78 siderable number of cytosines are methylated show only slight degradation by
HpaII [16]. DNA rnethylase activity In order to help reinforce the finding of trace amounts of minor bases in
Aedes DNA a search was made for an enzyme or enzymes present in insect cells which could methylate DNA. When nuclei, prepared from mouse L929 or Krebs II ascites cells by homogenising the cells in buffer M, are incubated with S-adenosyl[Me-3H]methionine then some methylation of endogenous DNA occurs (unpublished results and Ref. 9). Under similar conditions nuclei prepared from growing mosquito cells failed to incorporate detectable radioactivity into endogenous DNA. In view of the possibility that in cells of A. albopictus the DNA methylase activity may be located in the cytoplasm or only very loosely bound within the nuclei, we homogenised Aedes cells in a very small volume of buffer M and after removing the nuclei by sedimentation at 800 X g for 10 min we assayed the supernatant fraction for DNA methylase activity. Using denatured E. coli DNA as acceptor a low level of activity was present in this supernatant fraction (0.17 pmol/30 pl per h) and even less (0.06 pmol/30 pl per h) was found in the 0.4 M NaC1 nuclear extract. The specific activity of this enzyme is only about 0.6 unit/mg compared with the 0.4 M NaC1 extract of Ascites nuclei which has a 70-fold greater specific activity (Table I). The incorporation of methyl groups continues almost linearly for 3 h and is approximately the same at 22 and 37°C. DNA prepared from Aedes cells is an equally good or better substrate than DNA from E. coli or calf thymus. It makes little difference whether the DNA is single or double-stranded but in both cases the reaction is inhibited by 0.1 M NaC1. An enzyme preparation made by combining the 800 X g supernatant fraction from a homogenate with the dialysed 0.4 M NaC1 nuclear extract was subjected to ammonium sulphate fractionation. Over 60% of the activity was recovered in the 30--45% saturation cut with the remainder precipitating between 45% and 60% saturation.
Glycerol gradients In order to see whther only one or multiple species of enzyme are present active fractions were subjected to gel filtration and glycerol gradient centrifuga-
tion. TABLE I ACTIVITY AND DISTRIBUTION
OF Aedes DNA METHYLASE
T h e e n z y m e s w e r e p r e p a r e d as d e s c r i b e d in t h e t e x t a n d 3 0 ~1 w a s i n c u b a t e d foz 3 h a t 3 7 ° C w i t h 1 5 ~ g d e n a t u r e d E. coU D N A in a s t a n d a r d i n c u b a t i o n ( t o t a l v o l u m e 0 7 0 /~1), b y w ~ t/me 165 cpm had been incorporated with the 0.4 M extract of Aedes nuclei.
Enzyme
pmol 'Me' incorporated
Protein (mg/m/)
Ulmg
Aedes, s u p e r n a t a n t Aedes, 0 . 4 M e x t r a c t Krebs II, 0 . 4 M e x t r a c t
0.22 0.09 15.15
4.18 1.43 4.30
0.58 0.7 39.3
79
The enzyme shows one major peak on gel filtration, eluting with alcohol dehydrogenase (160 000 tool. wt.) and on sedimentation through a glycerol gradient again one major peak is seen giving an s value of 6 S and an apparent molecular weight of about 130 000 (Fig. 5). This higher apparent molecular weight on gel filtration is also found with the methylase from Krebs II ascites cells which on sedimentation through a glycerol gradient gives an apparent molecular weight of 184 000 [7]. A second peak of activity is reproducibly seen at 8.7 S. Product of the enzyme reaction The product of the enzymic reaction was investigated by isolation of the methylated DNA, hydrolysis with formic acid, and separation of the bases using either paper chromatography or ion exchange on Aminex A6. Two methylated bases are produced (Fig. 6) which cochromatograph with 5-methylcytosine and 6-methylaminopurine. In different experiments the relative amounts of the two methylated bases varied but, with one particular enzyme
2o
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(a)
(b)
15 TG
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~
5
4o
50
,.~lh
t
2O
10
1 5 20
F raction No.
L
0
10
20
cm
so
4o
L
15 20
30 35 Fraction
25
40 45
Fig. 5. Glycerol gradient e e n t r i f u g a t i o n o f A e d e s D N A m e t h y l a s e . A combined supernatant and nuclear extract w a s s u b j e c t e d to a m m o n i u m s u l p h a t e fractionation and the 30---45% saturation cut was centrifuged on a glycerol gradient as described in Materials and M e t h o d s . Fig. 6. Product o f A e d e s D N A m e t h y l a s c . 50 #1 combined supernatant and nuclear extract w e r e incubated for 0, 1 or 3 h with 2 #g native A e d e s DNA and S-adenosyl[3H]methionine (5.5 Ci/mmol). A f t e r p h e n o l extraction, e t h a n o l p r e c i p i t a t i o n and alkali digestion t h e p r o d u c t w a s acid w a s h e d and hydrolyscd t o t h e bases. (a) H y d r o l y s i s w i t h 12 N perchloric acid and chromatography on paper; e0 1 h incubation; o, 3 h incubation. (b) H y d r o l y s i s w i t h formic acid and separation in Arninex A6; o, 3 h incubation; e, z e r o t i m e incubation. T h e a r r o w s indicate t h e p o s i t i o n o f markers o f t h y m i n e (T), guanine (G), adenine (A), c y t o sine (C)0 5-methylcytostne (MC) and 6-methylamtnopurine (MAP).
80 preparation incubated for 1 h or 3 h with either Aedes or calf thymus DNA as substrate the proportion of the two bases methylated remained the same (Fig. 6a). Unfortunately insufficient radioactivity was incorporated by the fractions from the glycerol gradient to enable us to determine whether the two peaks of activity corresponded to two enzymes with distinct specificities. Discussion
We have confirmed that the DNA content of mosquito cells is similar to that of mammalian cells. A similar, thought slight lower, result was obtained by Sprading et al. [13]. The standard techniques in use in our laboratory failed to detect the presence of any 5-methylcytosine in the Aedes DNA (Fig. la). A value of twice the background count over the 5-methylcytosine peak would indicate 0.2% of the cytosines were methylated. It is possible to add more DNA hydrolysate to the ion-exchange column, and together with the better separation achieved an obvious peak of 5-methylcytosine is visible in Fig. l b indicating that this base contributes 0.036% of the total bases of Aedes DNA. Incubations with [3H]deoxyadenosine and [Me-3H]methionine indicate the presence of a slightly lesser amount of a second minor methylated base. Although it cochromatographs with 6-methylaminopurine in both systems used the identification of this compound has not been proved beyond doubt. The question arises as to whether the small amounts of minor bases detected are really present in the insect DNA or whether they arise from some bacterial or mycoplasmal contaminant. Regular tests failed to indicate any contamination in the cultures and only a marked contamination would be expected to lead to the observed findings. Nonetheless, the finding of methyladenine in eukaryotes is unusual although it is the only minor methylated base (contributing 2.5 tool%) in the nuclear DNA of Paramecium aurelia [17] and is also present in DNA from Tetrahymena pyriformis Villadsen, I.S. and Vrang, A., personal communication). DNA methylase activity can be recovered from insect cells, but in contrast with mammalian cells the greater part of the activity is not associated with the nucleus following homogenisation in hypotonic buffer. There is, however, no reason to believe that this reflects a cytoplasmic location for the enzyme(s). As Aedes DNA is a good substrate for the enzyme preparation it is possible that the low level of methylation of the DNA in vivo is a result of the very low specific activity of the enzyme(s). Alternatively the DNA in the nucleus of Aedes cells may be protected from the action of the methylase by other proteins. The enzyme preparation probably contains at least two species of enzyme capable of transferring methyl groups from S-adenosylmethionine to DNA. Major and minor peaks of activity are recovered from glycerol gradients and two different bases are methylated by the enzyme preparation. These methylated bases cochromatograph with 5-methylcytosine and 6-methylaminopurine though because of the small amounts available exhaustive tests have not been carried out to unequivocally prove their identity. We have failed to find a correlation between the DNA content of a cell and
81 the amount of 5-methylcytosine present in the DNA. Thus Aedes resembles Drosophila in having very low levels of minor methylated DNA bases but differs in having 28-times as much DNA/cell. In mammalian DNA the most highly methylated fraction is that which renatures fastest, i.e. the highly reiterated DNA [16] but it has been shown that this fraction represents 10--20% of the DNA of Aede8, mammalian [13] and Drosophila cells [18]. It is, therefore, not a defiency of this class of DNA which leads to the low level of methylation of insect DNA.
Acknowledgements The authors gratefully acknowledge the encouragement of Professor R.M.S. SmeUie. This work was partly supported by funds provided by the Medical Research Council.
References 1 Thomas, A.J. and She~att, H.S.A. (1956) Biochem. J. 62, 1--4 2 Antonov, A.C., Favorova, O.O. and Belozerski, A.N. (1962) Dokl. Akad. Nauk. SSSR 147, 1480-1483. 3 Wyatt, G.R. (1951) Biochem. J. 48, 584--590 4 Chargaff, E., Lipshitz, R. and Green, C. (1952) J. Biol. Chem. 195, 155--160 5 Kurnick, N.B. and Herskowitz, I.H. (1952) J. Cell Comp. Physiol. 39, 281 299 6 Holliday, R. and Pugh, J.E. (1975) Science 187, 226--232 7 Adams, R.L.P., McKay, E.L., Craig, L.M. and Burdon, R.H. (1978) Biochim. Biophys. Acta, in the press
8 9 10 11 12 13 14 15 16 17 18
Turnbull, J.F. and Adams, R.L.P. (1976) Nucleic Acids Res. 3 , 6 7 7 - - 6 9 5 Adams, R.L.P. and Hogarth, C. (1973) Biochim. Biophys. Aeta 3 3 1 , 2 1 4 - - 2 2 0 Hirsehman, S.Z. and Felsenfeld, G. (1966) J. Mol. Biol. 16, 347--358 Tegtmeyer, P. (1972) J. Virol. 10, 599--604 B u r t o n , K. ( 1 9 5 6 ) B i o c h e m . J. 62, 315---323 Spradl/rlg, A., Penman, S., Campo, M.S. and Bishop, J.O. (1974) Cell 3, 23--30 Nathans, D. and Smith, H,O. (1975) Annu. Rev. Bioehem. 44, 273--293 Bird, A. (1978) J. Mol. Biol. 118, 49--60 Browne, M.J. Cato, A.C.B. and Bugdon, R.H. (1978) FEBS Lett. 91, 69--73 Cummings, D.J., TaR, A. and Goddard, J.M. (1974) Bioehim. Biophys. Acta 374, 1--11 Manning, J.E., Schmid, C.W. and Davidson, N. (1975) Cell 4, 141--155
Biochimica et Biophysica Acta, 563 (1979) 72--81 © Elsevier/North-Holland Biomedical Press
BBA 99465
METHYLATION OF MOSQUITO DNA
R.L.P. ADAMS, E.L. McKAY, L.M. CRAIG and R.H. BURDON
Department of Biochemistry, University of Glasgow, Glasgow G12 8QQ (U.K.) (Received October 12th, 1978)
Key words: DNA methylation; Methyl nucleotide; (Mosquito)
Summary Cells of the mosquito Aedes albopictus have 5.6 pg DNA/cell. This DNA is 58 tool% (A + T) and has about 0.03 tool% each of 5-methylcytosine and 6-methylaminopurine. The DNA is largely degraded by the restriction enzyme HpaII showing the virtual absence of the sequence CMeCGG. Small amounts of enzymic activity recovered largely in non-nuclear fractions transfer methyl groups from S-adenosylmethionine to cytosine and adenine.
Introduction In the D N A from all higher eukaryotes which have been studied there is one minor base, namely 5-methylcytosine. The frequency of occurrence of this minor base varies dramatically. Thus, in higher plants the proportion of cytosines methylated varies between 18% and 3 3 % [1]. Most other species from the Coelentera, Mollusa, Annelida, Echinoderrnata and Chordata [2--4] have from 2 % to 8 % of their cytosines methylated. The Arthropoda, however, as far as they have been investigated have a lower level of methylation [2,3]. In Drosophila this is associated with a very low content of DNA/cell [5]. The role of D N A methylation in eukaryotes is not known. Various suggestions have been made that it m a y play a part in the control of differentiation [6,7]. If methylation has a c o m m o n role in eukaryotes it is surprisingthat the extent of methylation varies so widely. One possibility is that the lower the amount of DNA/cell the smaller the proportion of this DNA is methylated. To investigate this possibility we have looked at the level of methylation of mosquito DNA. We report here that, .although mosquito cells are comparable to mammalian cells in their DNA content, the 5-methylcytosine content of this DNA is extremely low. Enzymic activity capable of methylating DNA in
73 vitro can be recovered from insect cells. Furthermore trace amounts of methyladenine are found in Aedes DNA and the enzyme preparation methylates both cytosine and adenine. Materials and Methods
Cells and nuclei. Aedes albopictus cells were obtained from Flow Laboratories, Irvine, U.K., and maintained as monlayer cultures at 27°C in Mitsuhashi and Maramorosch medium supplemented with 20% foetal bovine serum, nonessential amino acids, glutamine and penicillin and streptomycin. The medium was adjusted to pH 7.2 with NaOH. The cultures were tested for mycoplasmal contamination using PPLO agar and for bacterial contamination on blood agar plates. All tests were negative. Cells were harvested by scraping them off the glass and washed with phosphate-buffered saline. Nuclei were prepared either by homogenesing the cells in buffer M (50 mM Tris-HC1, pH 7.8, 10% glycerol, 1 mM EDTA, 1 mM dithiothreitol) or in 1% Tween 80. The nuclei were then sedimented at 800 × g for 10 min. Ascites cells and nuclei were prepared as described previously [8]. When appropriate cells were labelled with [U-14C]deoxycytidine (1 gCi/ 30 ml); deoxy[G-3H]adenosine (300 uCi!30 ml) or [Me-3H]methionine (500 gCi/30 ml) (The Radiochemical Centre, Amersham, U.K.). Methylase preparation and assay. The method used was the same as that for obtaining DNA methylase from mouse ascites cells [8] and involved preparing nuclei and treating them in buffer M containing 0.4 M NaC1. The resulting extract was dialysed against buffer M. In addition, to test for non-nuclear enzymic activity, cells were homogenised in buffer M as described above and both the supernatant fraction and a nuclear extract were assayed for DNA methylase activity as previously described [8]. The standard assay mixture (70 gl) contained 20 pg single stranded Escherichia coli DNA, 1.7 pCi S-adenosyl-L-[Me-3H]methionine ( l p C i / n m o l ; The Radiochemical Centre, Amersham, U.K.) 0.1 M NaC1 and 30--50 ~l buffered enzyme solution. After incubation the DNA was purified and incorporation measured as described previously [8]. In studies of nuclear incorporation 70 ~1 of buffer M nuclei were incubated with 1.7 uCi S-adenosyl-L-[Me-3H]methionine as above and incorporation into endogenous DNA measured as previously described [9]. The DNA methylase activity could be partially purified and concentrated by ammonium sulphate fractionation. The activity from a pooled supernatant fraction and nuclear extract was recovered in the 30--45% saturation fraction on precipitation with ammonium sulphate and it was this material which was centrifuged on glycerol gradients following dialysis against buffer M containing 0.2 M NaC1 and 5% glycerol. Glycerol gradients were prepared by layering 1.3 ml each of 10%, 20% and 30% glycerol in buffer M containing 0.2 M NaCI into tubes for the SW 50.1 Spinco rotor. The gradients were allowed to form at room temperature for 5 h and then cooled on ice. Enzyme fractions (0.3 ml) were layered onto the gradients which were spun at 144 000 × g for 16 h. Markers of alcohol
74 dehydrogenase and catalase were used. In the assay for enzymic activity in fractions from the glycerol gradients the assay volume was doubled enabling 70-/~1 samples to be assayed and the specific activity of the S-adenosyl-L[Me-3H]methionine was increased to 3.4 pCi/nmol. DNA preparation and base analysis. DNA was prepared from cultured mosquito cells by the method previously described [7]. For use as substrate in the methylase assay it was dissolved in 50 mM KC1. The DNA solution had E26o/E28o = 1.9 and on analysis of the spectrum [10] the DNA was found to have an A + T content of 58 mol%. Commercial preparations of calf thymus DNA, E. coli DNA and a preparation of mouse L929 cell DNA made by the same method as the Aedes DNA had A + T contents of 60, 50 and 60 mol%, respectively. For base analysis the DNA was hydrolysed either using 12 N perchloric acid as previously described [8] or using 98% formic acid at 180°C for 30 min. The formic acid was then evaporated and the bases dissolved in 0.1 N HCI for chromatography on Whatman No. 1 paper using butanol/HC1 as previously described [8]. The proportion of the radioactivity in the various spots was analysed by shredding them into toluene-based scintillation fluid. Alternatively, after hydrolysis of the DNA, the bases were dissolved in ammonium carbonate buffer (20 mM; pH 10.0 at room temperature} and applied to a column of Aminex A6 (Bio Rad Labs; 27 cm × 1.2 cm) which was eluted with the same buffer at 50°C with a flow rate of about 1 ml/min. E. coli B, salmon testis and calf thymus DNA were purchased from Sigma, London, Chemical Co., k-DNA from Miles Laboratories, Slough, U.K. Restriction enzyme treatment. For treatment with the restriction enzyme HpaII duplicate samples of ~4C-labelled DNA were dissolved in 100 pl buffer containing 20 mM Tris-HC1, pH 7.4 20 mM MgC12, 12 mM KC1, 2 mM dithiothreitol, and 0.2 mg/ml gelatin, and to one sample was added 2 pl HpaII (Boehringer Corp. Ltd.). The samples were incubated at 37°C for 90 min and electrophoresed in 1.5% agarose tube gels as described by Tegtmeyer [11]. When the bromophenol blue marker had run 8 cm the gels were sliced; the slices incubated in 0.3 N NaOH at 60°C overnight and the neutralised samples counted using a Triton/toluene scintillator. Results
DNA content o f Aedes cells Three independent suspensions of exponentially growing A. albopictus cells were prepared by trypsinisation and counted in a Coulter counter model B. After washing the cells in balanced salt solution and cold 0.5 N perchloric acid, D N A was extracted into 0.5 N perchloric acid at 70°C for 30 rain. Duplicate samples were assayed for DNA by the method of Burton [12]. The value obtained for the D N A content of an exponentially growing population o f A. aibopictus cells was 5.62 _+0.18 pg/cell. This compares with a value of 2.9 pg/Aedes cell obtained by Spradling et al. [13] and a figure of 13 pg/L929 cell obtained by us. It is very much higher than the 0.2 pg/cell recorded for Drosophila [ 5 ].
75
Presence of minor bases in mosquito DNA Two different approaches were used to determine the presence of minor bases in the insect DNA. These were (1) incubation of cells with a radioactive nucleoside followed by base analysis to search for conversion to a minor base or, (2) incubation o f cells with [Me-SH]methionine followed by base analysis to search for specific methylated bases. Insect cells were grown for three days in the presence o f [ 14C]deoxycytidine and the DNA purified from these cells was subjected to base analysis. Fig. l a shows the section o f the paper chromatogram where cytosine and 5-methylcytosine are separated. For comparison a similar result for mouse L929 cells is shown. No 5-methylcytosine is detectable in the insect DNA and we feel the level o f sensitivity would enable us to detect if 0.2% of the cytosine had been methylated. However, analysis of this material using ion exchange in Aminex A6 which gives a much better separation o f cytosine and 5-methylcytosine reveals a very small shoulder on the curve which shows that 0.17% of the cytosine are methylated (Fig. lb). Similar base analyses following incubation of cells with [Me-3H]methionine reveal a number o f tritiated species. Most radioactivity is found in thymine and (a)
(b) C
16
21
14
12
MC
10r
. ~-Aedes
Ib
18 G
10
!
15
I
A
C
MC
46
I I e~
'
8
4
M
B
6
0~ 25
30
35 cm
40
20
30 40 Fraction No.
50
60
Fig. 1. I n vivo c o n v e r l t o n o f d e o x y [ 1 4 C ] c y t i d i n e t o 5 - m e t h y l d e o x y e y t i d i n e . Early l o g p h a s e A e d e s cells w e r e g ~ o w n for 3 d a y s in t h e p r e s e n c e o f d e o x y [ U - 1 4 C ] c y t i d i n e ( 1 p C i / 3 0 m l ) after w h i c h t i m e t h e D N A w u i s o l a t e d a n d h y d r o l y s e d t o t h e b a s e s as d e s c r i b e d in Materials and M e t h o d s . (a) H y d r o l y s i s w i t h 1 2 N perchioric acid a n d c h r o m a t o g r a p h y o n paper, o, A e d e s cells; e , m o u s e L 9 2 9 cells. (b) H y d r o l y s i s w i t h formic acid a n d s e p a r a t i o n in A m i n c x A 6 . T h e a r r o w s indicate t h e p o r t i o n o f e l u t i o n o f m a r k e r s o f t h y m i n e ( T ) , guanine (G), a d e n i n e ( A ) , c y t o s i n e (C) and 5omethylcytol~-le (MC). T h e insert s h o w s t u b e s 4 0 - - 5 6 o n a r e d u c e d scale.
76 (a)
(b)
MAP MC
5°o
2D-T?
T
?
4O0 15300
,~
~-oo
II
100
:
~ I
5 -
O --I~--~-~" I0
20
30
cm
15
25
35
45
Fraction
Fig. 2. In vivo i n c o r p o r a t i o n of m e t h y l groups from [Me-3H ]me t ht oni ne i n t o Aede8 DNA. Early log phase A e d e s cells were grown for 2 days in the presence of [ M e - 3 H ] m e t h i o n i n e (500 # C i / 3 0 ml ) after which t i m e the DNA was isolated, h y d r o l y s e d with formic acid a nd t he bases separated. ( a ) C h r o m a t o g r a p h y on paper; o, A e d e s cells; o, m o u s e L929 cells on 1/10 scale. (b) Separation on A m i n e x AS. The arrows indicate the p o s i t i o n of elution of markers of t h y m i n e (T), guanine (G), adenine (A), cytosine (C), purine (Pu), 5 - m e t h y l c y t o s i n e (MC) and 6 - m e t h y l a m i n o p u r i n e (MAP).
significant amounts are found in the purines, particularly adenine. Two other species are found which, both on paper chromatography and ion exchange run together with 5-methylcytosine and 6-methylaminopurine (Fig.2a and b). A similar experiment with mouse L929 cells reveals 5-methylcytosine as the only TG A
C
MAP
20 i 12 -
20
~20
30 40 Fraction
"
50
Fig. 3. In vivo e o n v e n i o n of deoxy[G-3H]adeno4dnc to 6-methylaminopultxm. Early log phase A e d e s cells were grown for 2 d a y s in t h e presence of d e o x y [ G - 3 H ] a d e n o s / n e (300 ~Ci~30 mD a f t ~ w hi c h t i m e t he DNA was isolated, h y d r o l y s e d w i t h fonnde aoAd and the bases s e pa ra t e d on A m l n e x A6. The arrows indicare t h e poldtdon o f elut/on of m a r k e r s t h y m i n e (T), guanine (G), adenine (A), eytos/ne (C) a nd 6-methyla m l n o p u r l n e (MAP). The insert shows t u b e s 35---50 on a reduc e d scale.
77 minor base. In an attempt to confirm the presence of a methylated adenine Aedes cells were incubated with deoxy[G-3H]adenosine for two days and the DNA isolated, hydrolysed and the bases analysed using ion-exchange chromatography. Fig. 3 shows a shoulder of radioactivity in the position of 6-methylaminopurine corresponding to about 0.1% of the adenine present.
Treatment with HpalI Treatment of 14C-labelled Aedes DNA with the restriction enzyme HpaII leads to total degradation of the high molecular weight DNA to fragments ranging in length from less than 100 nucleotide pairs to several thousand nucleotide pairs (Fig. 4). HpaII cleaves unmodified double-stranded DNA in the sequence CCGG; but methylation of a cytosine in the CG doublet prevents cleavage [14,15]. Although a regular distribution of HpaII sites would lead to production of fragments of around 330 nucleotide pairs long the results of Fig. 4 are not inconsistent with a random distribution of HpaII sites, few if any of which are modified. Similar studies with mammalian DNA in which a con-
2000
-
1500 -
~1000 -
500
[ I f
C°ntr°l
-
2
4
6 crn
8
10
12
Fig. 4. T r e a t m e n t o f 1 4 C o l a b e l l e d A e d e s D N A w i t h HpaII. D N A w a s i s o l a t e d f r o m Ae de s cells g r o w n i n the p r e s e n c e o f d e o x y [ 1 4 C ] e y t i d i n e . T h e D N A w a s i n c u b a t e d w i t h t h e r e s t r i c t i o n e n z y m e H p a I I f o r 0 ( e ) o r 9 0 m i n (o) a n d s u b j e c t e d t o e l e e t r o p h o r e s t s o n a g a r o s e gels as d e s c r i b e d i n M a t e r i a l s a n d M e t h o d s . T h e p o s i t i o n o f t h e r a d i o a c t i v e D N A f r a g m e n t s w a s a s c e r t a i n e d b y slicing t h e gel a n d c o u n t i n g t h e s a m pies u s i n g a T r i t o n / t o l u e n e s c i n U l l a t o r .
78 siderable number of cytosines are methylated show only slight degradation by
HpaII [16]. DNA rnethylase activity In order to help reinforce the finding of trace amounts of minor bases in
Aedes DNA a search was made for an enzyme or enzymes present in insect cells which could methylate DNA. When nuclei, prepared from mouse L929 or Krebs II ascites cells by homogenising the cells in buffer M, are incubated with S-adenosyl[Me-3H]methionine then some methylation of endogenous DNA occurs (unpublished results and Ref. 9). Under similar conditions nuclei prepared from growing mosquito cells failed to incorporate detectable radioactivity into endogenous DNA. In view of the possibility that in cells of A. albopictus the DNA methylase activity may be located in the cytoplasm or only very loosely bound within the nuclei, we homogenised Aedes cells in a very small volume of buffer M and after removing the nuclei by sedimentation at 800 X g for 10 min we assayed the supernatant fraction for DNA methylase activity. Using denatured E. coli DNA as acceptor a low level of activity was present in this supernatant fraction (0.17 pmol/30 pl per h) and even less (0.06 pmol/30 pl per h) was found in the 0.4 M NaC1 nuclear extract. The specific activity of this enzyme is only about 0.6 unit/mg compared with the 0.4 M NaC1 extract of Ascites nuclei which has a 70-fold greater specific activity (Table I). The incorporation of methyl groups continues almost linearly for 3 h and is approximately the same at 22 and 37°C. DNA prepared from Aedes cells is an equally good or better substrate than DNA from E. coli or calf thymus. It makes little difference whether the DNA is single or double-stranded but in both cases the reaction is inhibited by 0.1 M NaC1. An enzyme preparation made by combining the 800 X g supernatant fraction from a homogenate with the dialysed 0.4 M NaC1 nuclear extract was subjected to ammonium sulphate fractionation. Over 60% of the activity was recovered in the 30--45% saturation cut with the remainder precipitating between 45% and 60% saturation.
Glycerol gradients In order to see whther only one or multiple species of enzyme are present active fractions were subjected to gel filtration and glycerol gradient centrifuga-
tion. TABLE I ACTIVITY AND DISTRIBUTION
OF Aedes DNA METHYLASE
T h e e n z y m e s w e r e p r e p a r e d as d e s c r i b e d in t h e t e x t a n d 3 0 ~1 w a s i n c u b a t e d foz 3 h a t 3 7 ° C w i t h 1 5 ~ g d e n a t u r e d E. coU D N A in a s t a n d a r d i n c u b a t i o n ( t o t a l v o l u m e 0 7 0 /~1), b y w ~ t/me 165 cpm had been incorporated with the 0.4 M extract of Aedes nuclei.
Enzyme
pmol 'Me' incorporated
Protein (mg/m/)
Ulmg
Aedes, s u p e r n a t a n t Aedes, 0 . 4 M e x t r a c t Krebs II, 0 . 4 M e x t r a c t
0.22 0.09 15.15
4.18 1.43 4.30
0.58 0.7 39.3
79
The enzyme shows one major peak on gel filtration, eluting with alcohol dehydrogenase (160 000 tool. wt.) and on sedimentation through a glycerol gradient again one major peak is seen giving an s value of 6 S and an apparent molecular weight of about 130 000 (Fig. 5). This higher apparent molecular weight on gel filtration is also found with the methylase from Krebs II ascites cells which on sedimentation through a glycerol gradient gives an apparent molecular weight of 184 000 [7]. A second peak of activity is reproducibly seen at 8.7 S. Product of the enzyme reaction The product of the enzymic reaction was investigated by isolation of the methylated DNA, hydrolysis with formic acid, and separation of the bases using either paper chromatography or ion exchange on Aminex A6. Two methylated bases are produced (Fig. 6) which cochromatograph with 5-methylcytosine and 6-methylaminopurine. In different experiments the relative amounts of the two methylated bases varied but, with one particular enzyme
2o
MAP MC
(a)
(b)
15 TG
A
C MC MAP
ADH Cat 200
10
180
3h
150
3h~-~
-I
100
!
~
5
4o
50
,.~lh
t
2O
10
1 5 20
F raction No.
L
0
10
20
cm
so
4o
L
15 20
30 35 Fraction
25
40 45
Fig. 5. Glycerol gradient e e n t r i f u g a t i o n o f A e d e s D N A m e t h y l a s e . A combined supernatant and nuclear extract w a s s u b j e c t e d to a m m o n i u m s u l p h a t e fractionation and the 30---45% saturation cut was centrifuged on a glycerol gradient as described in Materials and M e t h o d s . Fig. 6. Product o f A e d e s D N A m e t h y l a s c . 50 #1 combined supernatant and nuclear extract w e r e incubated for 0, 1 or 3 h with 2 #g native A e d e s DNA and S-adenosyl[3H]methionine (5.5 Ci/mmol). A f t e r p h e n o l extraction, e t h a n o l p r e c i p i t a t i o n and alkali digestion t h e p r o d u c t w a s acid w a s h e d and hydrolyscd t o t h e bases. (a) H y d r o l y s i s w i t h 12 N perchloric acid and chromatography on paper; e0 1 h incubation; o, 3 h incubation. (b) H y d r o l y s i s w i t h formic acid and separation in Arninex A6; o, 3 h incubation; e, z e r o t i m e incubation. T h e a r r o w s indicate t h e p o s i t i o n o f markers o f t h y m i n e (T), guanine (G), adenine (A), c y t o sine (C)0 5-methylcytostne (MC) and 6-methylamtnopurine (MAP).
80 preparation incubated for 1 h or 3 h with either Aedes or calf thymus DNA as substrate the proportion of the two bases methylated remained the same (Fig. 6a). Unfortunately insufficient radioactivity was incorporated by the fractions from the glycerol gradient to enable us to determine whether the two peaks of activity corresponded to two enzymes with distinct specificities. Discussion
We have confirmed that the DNA content of mosquito cells is similar to that of mammalian cells. A similar, thought slight lower, result was obtained by Sprading et al. [13]. The standard techniques in use in our laboratory failed to detect the presence of any 5-methylcytosine in the Aedes DNA (Fig. la). A value of twice the background count over the 5-methylcytosine peak would indicate 0.2% of the cytosines were methylated. It is possible to add more DNA hydrolysate to the ion-exchange column, and together with the better separation achieved an obvious peak of 5-methylcytosine is visible in Fig. l b indicating that this base contributes 0.036% of the total bases of Aedes DNA. Incubations with [3H]deoxyadenosine and [Me-3H]methionine indicate the presence of a slightly lesser amount of a second minor methylated base. Although it cochromatographs with 6-methylaminopurine in both systems used the identification of this compound has not been proved beyond doubt. The question arises as to whether the small amounts of minor bases detected are really present in the insect DNA or whether they arise from some bacterial or mycoplasmal contaminant. Regular tests failed to indicate any contamination in the cultures and only a marked contamination would be expected to lead to the observed findings. Nonetheless, the finding of methyladenine in eukaryotes is unusual although it is the only minor methylated base (contributing 2.5 tool%) in the nuclear DNA of Paramecium aurelia [17] and is also present in DNA from Tetrahymena pyriformis Villadsen, I.S. and Vrang, A., personal communication). DNA methylase activity can be recovered from insect cells, but in contrast with mammalian cells the greater part of the activity is not associated with the nucleus following homogenisation in hypotonic buffer. There is, however, no reason to believe that this reflects a cytoplasmic location for the enzyme(s). As Aedes DNA is a good substrate for the enzyme preparation it is possible that the low level of methylation of the DNA in vivo is a result of the very low specific activity of the enzyme(s). Alternatively the DNA in the nucleus of Aedes cells may be protected from the action of the methylase by other proteins. The enzyme preparation probably contains at least two species of enzyme capable of transferring methyl groups from S-adenosylmethionine to DNA. Major and minor peaks of activity are recovered from glycerol gradients and two different bases are methylated by the enzyme preparation. These methylated bases cochromatograph with 5-methylcytosine and 6-methylaminopurine though because of the small amounts available exhaustive tests have not been carried out to unequivocally prove their identity. We have failed to find a correlation between the DNA content of a cell and
81 the amount of 5-methylcytosine present in the DNA. Thus Aedes resembles Drosophila in having very low levels of minor methylated DNA bases but differs in having 28-times as much DNA/cell. In mammalian DNA the most highly methylated fraction is that which renatures fastest, i.e. the highly reiterated DNA [16] but it has been shown that this fraction represents 10--20% of the DNA of Aede8, mammalian [13] and Drosophila cells [18]. It is, therefore, not a defiency of this class of DNA which leads to the low level of methylation of insect DNA.
Acknowledgements The authors gratefully acknowledge the encouragement of Professor R.M.S. SmeUie. This work was partly supported by funds provided by the Medical Research Council.
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8 9 10 11 12 13 14 15 16 17 18
Turnbull, J.F. and Adams, R.L.P. (1976) Nucleic Acids Res. 3 , 6 7 7 - - 6 9 5 Adams, R.L.P. and Hogarth, C. (1973) Biochim. Biophys. Aeta 3 3 1 , 2 1 4 - - 2 2 0 Hirsehman, S.Z. and Felsenfeld, G. (1966) J. Mol. Biol. 16, 347--358 Tegtmeyer, P. (1972) J. Virol. 10, 599--604 B u r t o n , K. ( 1 9 5 6 ) B i o c h e m . J. 62, 315---323 Spradl/rlg, A., Penman, S., Campo, M.S. and Bishop, J.O. (1974) Cell 3, 23--30 Nathans, D. and Smith, H,O. (1975) Annu. Rev. Bioehem. 44, 273--293 Bird, A. (1978) J. Mol. Biol. 118, 49--60 Browne, M.J. Cato, A.C.B. and Bugdon, R.H. (1978) FEBS Lett. 91, 69--73 Cummings, D.J., TaR, A. and Goddard, J.M. (1974) Bioehim. Biophys. Acta 374, 1--11 Manning, J.E., Schmid, C.W. and Davidson, N. (1975) Cell 4, 141--155
