specific nuclease $1, polyadenylated and used as a template for the oligo(dT) ... 50 % ; the $1 nuclease resistance of the cDNA after self-annealing was approx.
649
J. gen. Virol. 0979), 43, 649-662 Printed in Great Britain
I n vitro S y n t h e s i s a n d C h a r a c t e r i z a t i o n Complementary
of DNA
to Cadang-cadang-associated
RNA
By J. W. R A N D L E S AND P. P A L U K A I T I S
Department of Plant Pathology, Waite Agricultural Research Institute, and Department of Biochemistry, University of Adelaide, South Australia (Accepted 7 December I978)
SUMMARY
The anomalous viroid-like RNA associated with cadang-cadang disease of coconut palms (ccRNA-I) has been cleaved by treatment with the single-strand specific nuclease $1, polyadenylated and used as a template for the oligo(dT) primed synthesis of complementary DNA (cDNA) by the avian myeloblastosis virus reverse transcriptase. The efficiency of synthesis was low, with only 3 to 4"5 ng of cDNA synthesized from 2 #g of RNA. Most of the cDNA was in the 4S size class. A Rot½ value of I x lO-3 mol. s/l was obtained when this cDNA was hybridized with ccRNA-l, consistent with ccRNA-t representing a unique species of mol. wt. approx. IOOOOO. The maximum hybridization value obtained with ccRNA-I was approx. 50 % ; the $1 nuclease resistance of the cDNA after self-annealing was approx. 7 %The melting behaviour of the homologous hybrids provided evidence for the specificity of base-pairing with no evidence of mismatching. The cDNA has been shown to be a specific probe for cadang-cadang associated RNA. It has been used to demonstrate that ccRNA-I and ceRNA-2 have common nucleotide sequences, that ccRNA-I is uniquely associated with diseased and not healthy palms and that it has no significant homology with high moI. wt. RNA or DNA from diseased palms. The value of the cDNA as a diagnostic probe for ccRNA-I in crude nucleic acid extracts has been demonstrated.
INTRODUCTION
Cadang-cadang disease of coconut palm is suspected of having a viroid aetiology (Randles, I975; Randles et al. I976 , T977). However, direct proof of the viroid hypothesis awaits the demonstraton of the pathogenicity of the viroid-like anomalous RNA (ccRNA-I) associated with the disease. Progress in this and other aspects of the nature and biology ofcadang-cadang depend upon being able to transmit and recognize the disease under experimental conditions. The experimental transmission of the diagnostic marker ccRNA-I and hence probably also the disease has been achieved recently by inoculating unfractionated nucleic acids from diseased palms (Randles et al. I977). Thus a means of testing the pathogenicity ofccRNA-I is now available. Nevertheless, progress is expected to be slow because the incubation period of the disease is long (Price, I97I), despite having been reduced to I½ to 2 years by assaying for the presence of ccRNA-I (Randles, I975; Randles et al. 1977). In an attempt to increase the sensitivity of the assay for ccRNA-I, we have synthesized a single-stranded DNA complementary to ccRNA-I (cDNA). The method oo22-I317/79/oooo-3383 $o2.oo~)I979 SGM
650
J.w.
RANDLES
AND
P. P A L U K A I T I S
of synthesis is described, the cDNA characterized and it is used to confirm the unique association of ccRNAq and -2 with diseased palms. The value of the cDNA as a probe for detecting ccRNA-I in palm extracts is demonstrated and its potential for speeding up the assay of the disease in transmission, epidemiological and replication studies is discussed.
METHODS
Preparation of total coconut leaf nucleic acids. Leaflets were chopped, then blended with 5 vol. (w/v) of o'o5 M-tris-C1 buffer, pH 7"3, containing 1% diethylpyrocarbonate. The extract was strained and blended with one-half volume of aqueous 9° % phenol-o.i ~o 8-OH quinoline and one-tenth volume of 4% sodium dodecyl sulphate (SDS) for 3 rain. The aqueous phase was recovered by centrifugation and re-extracted by adding I vol. of phenol and stirring for 30 rain. After centrifugation, nucleic acids in the aqueous phase were precipitated with 3 vol. of ethanol. Approximately IOO/~g of nucleic acids were extracted per gram of tissue. Preparation of DNA. DNA was extracted from the folded spear leaf of a healthy and a diseased palm using a technique devised by R. Francki and G. Boccardo (personal communication). Eighty g of tissue were blended with 20o ml TNE buffer (o.I M-tris-C1, pH 7, o.I M-NaC1, o.oI M-EDTA) containing o.I M-Na2SQ. The extract was strained, SDS powder was added to 1% and the pH was adjusted to 7"5 with :z N-KOH. Protease powder was added to I mg/ml, stirred and left z 4 h at room temperature. An equal vol. of 9o ~o phenol - o. 1 ~o g-OH quinoline was added, the mixture stirred for 4 h, centrifuged and the supernatant mixed with an equal vol. of chloroform:isoamylalcohol (zo: I) for 2 min. The upper phase was removed and mixed with ethanol to precipitate nucleic acids. The precipitate was dissolved in SSC (o-I 5 M-NaC1, o-oi5 M-sodium citrate) treated sequentially with 5 #g/ml heated ribonuclease A (5o °C, 3o min), I mg/ml each of amylase 0c and/5' (37 °C, 60 min), then Ioo/zg/ml protease in o'5% SDS (37 °C, I6 h). DNA was spooled out after adding ethanol. Before hybridization, the dissolved DNA was incubated with o.3y-NaOH at 37 °C for I6h, neutralized with HC1, and recovered by ethanol precipitation. Preparation ofccRNA-I and ccRNA-2. Nucleic acids were extracted from diseased palms by the following modification of the polyethylene glycol (PEG) method of Randles et al. (I976). Sodium sulphite (o.I M) was substituted for the NPTD blending medium, sometimes also with the addition of diethyl pyrocarbonate to 1%, and the first phenol-SDS supernatant was extracted for 5 min with an equal vol. of chloroform (W. D. O. Hamilton, personal communication), before continuing with the protease digestion and second phenol extraction. ccRNA-I was isolated from the nucleic acids by three cycles of electrophoresis. The nucleic acids were dissolved in tris-borate-EDTA (TBE) buffer (Peacock & Dingman, I968) containing 8 M-urea, heated at 65 °C for 5 min, then subjected to electrophoresis in r x Io cm cylindrical 5 ~o polyacrylamide gels buffered with TBE. Gels were stained with o.oi % ethidium bromide in a % acetic acid and bands were detected by u.v. fluorescence. ccRNA-~ bands were excised, macerated by passage through an I8-gauge hypodermic needle, mixed with 5 to IO ml of TBE and eluted by shaking continuously for I6 h. Phenol (90 ~o aqueous) was added to a final concentration of2o ~o, extracts were shaken for another 4 h, centrifuged and the aqueous supernatant mixed with 3 vol. of ethanol. The resulting precipitate was dried, dissolved in the E buffer of Bailey & Davidson (I976), containing 8 M-urea, heated and subjected to electrophoresis on 2% agarose gels (I x to cm). Bands were detected, eluted and ccRNA-r was precipitated as above. The third cycle of electrophoresis was on Io% polyacrylamide slab gels. ccRNA-I was again heated in TBE buffer,
Cadang-cadang cDNA
65
containing 8 M-urea, before loading. After detection, excision and elution, the supernatant was chilled on ice for to min before clarification at ~5ooog for 15 min. ccRNA-I was precipitated with ethanol, washed with ethanol, dried and dissolved in water. Concentration was estimated assuming A~°~~* = 25. Samples of 2 #g were lyophilized and stored below -
-
20 °C.
The ccRNA-2 preparation was separated by electrophoresis on 5 % polyacrylamide gels. Bands were excised and ccRNA-2 was extracted by maceration and elution and recovered by ethanol precipitation. A second fractionation was achieved by centrifugation of this ccRNA-2 through to to 4o% linear sucrose density gradients containing o.~ × SSC for I6 h at 37oo0 rev/min in the Beckman 41 rotor. The purity of the RNA species was checked by re-electrophoresis (Fig. 3a) and the identity of ccRNA-I was confirmed by its characteristic thermal denaturation behaviour in o.I x SSC buffer (Randles et al. I976). Treatment of ccRNA-~ with $1 nuclease. $1 nuclease from Aspergillus oryzae was prepared up to and including the DEAE-cellulose step of Vogt 0973). Enzyme activity was assayed and the unit of activity was as described by Vogt (I973). ccRNA-1 (2 #g) was dissolved in l o / d of 'low' salt Sx assay buffer (o.o3i-sodium acetate, o'o5 M-NaC1, I mM-ZnSO4, 5% glycerol, pH 4'6; Vogt, I973). Two units of S~ nuclease (r #1) were added and the mixture was incubated for 3o rain at 45 °C, before adding I #1 of o.2 M-Na2 EDTA and 3 vol. of ethanol to terminate the reaction. The ethanol precipitate was collected by centrifugation, washed once with ethanol and dried. Polyadenylation and preparation of cDNA to $1 nuclease-treated ccRNA-I. Dried Sx nuclease-treated ccRNA-I samples were dissolved in 4o #1 of water, heated at IOO °C for I min, chilled and used directly for polyadenylation as described by Gould et al. (I978). The polyadenylated $1 nuclease-treated RNA was isolated and used as a template for cDNA synthesis (Gould et al. I978) using either ~z-32P-dATP (2o Ci/mmol; Symons, I977) or 5-3H-dCTP (I5"5 Ci/mmol; Radiochemical Centre, Amersham) as the labelled deoxyribonucleotide precursor. Hybridization studies. Nucleic acid preparations (I to 2 #1) were added to 5o #1 of hybridization buffer (o.I8 M-NaCI, o'o5% SDS, o-oi i-tris-Cl, pH 7'o; Gould & Symons, I977) in siliconized glass tubes, approx. 7 × 5o ram. cDNA (6 to 15 pg, 4oo to IOOOct/min) and a layer of paraffin oil were added, samples were immersed in boiling water for 3 rain, then incubated at 65 to 66 °C. Hybridization was terminated by chilling the tubes, removing 25 to 4o/~1 aliquots and adding them to 300 #1 of a 'high' salt Sx assay buffer (o'o3 Msodium acetate, o'3 M-NaC1, I mM-ZnSO4, 5 % glycerol, pH 4"6) containing 4 ° #g/ml of denatured calf thymus DNA. Two samples, each of 15° #l, were taken and to one was added 2 units of $1 nuclease; the other was left as a control. After incubation'of both samples at 45 °C for 3° to 4o rain, nuclease resistance was determined by comparing the duplicates incubated either with or without enzyme, as described by Gould & Symons (1977). Values were corrected for self-annealing of the cDNA in the absence of RNA (approx. 7 % in our assays) on the same basis as described by Gonda & Symons (1978) and expressed as percentage hybrid formation. Percent hybridization was plotted against Rot (concentration of ribonucleotides at zero time in moles per litre multiplied by time of hybridization in seconds). Formamide gel electrophoresis. The mol wt. of cDNA was estimated by electrophoresis in 5% polyacrylamide-98% formamide gels (Staynov et al. 1972). Mol. wt. markers were Escherichia coli 4S RNA (mol. wt. o.26 to o'36 x 1@) and 16S RNA (tool. wt. 5"6 x I@), and cucumber mosaic virus satellite RNA (mol. wt. i'I5 x IO5; D. W. Mossop, personal communication) and RNAa (moI. wt. 3"5 x io5; Peden & Symons, 1973). Tm of DNA-RNA hybrid. A homologous hybridization mixture in a total vol. of loo td
65 z
J. W . R A N D L E S 1.3
I
AND
I
P. P A L U K A I T I S
i
I
I
I
Io~0.0_0_o-o-o-o-o-0-0 0-05 ra /o
/o
B
= 1.2
0
cq
/o/°to
0
/
0.3 M
jO
o
/
P N 1-1
~,o~o-o'-o
/°
/"
/o.o_o.O-~ )
/
°/°
o~o~O~ 0
P 1.0
/
-,--,~0"?'~0/" 20 40 I
t I
I
60 80 Time (min)
I
I
100
120
Fig. t. Effect o f salt concentration on the kinetics of the sequential digestion of ccRNA-x with St nuclease and ribonuclease A. Subs'trate was at a concentration o f I'5 # g / m l in $1 nuclease assay buffer containing either 0'o5 or 0"3 M-NaCI. $1 nuclease (5"5 units/ml) was added at zero time, R N a s e A (['4/zg/ml) was added at the time shown by the arrow.
was set up and incubated at 66 °C to an Rot value exceeding o.I tool. s/l. The mixture was chilled and placed in a water bath in which the temperature was raised by approx. I °C per min. At the appropriate temperatures, 5/zl portions were transferred to I5O #1 of cold $1 assay buffer and the percentage of hybrids remaining at each temperature was determined by $1 nuclease resistance. RESULTS
S 1 nuclease digestion o f c c R N A - 1
Preliminary attempts to synthesize cDNA by the polyadenylation-reverse transcription procedure (Hell et al. 1976) using purified ccRNA-I as a template were largely unsuccessful. Because the unusual molecular conformation of ccRNA-I (Randles et al. t976) may have prevented or greatly inhibited the attachment of either poly(A) polymerase or reverse transcriptase to initiating sites, $1 nuclease was used to cleave ccRNA-I at its presumed single-stranded regions (Randles et al. 1976). The enzyme has been shown to cleave singlestranded regions of tRNA (Harada & Dahlberg, I975; Rushizky & Mozejko, 1977). It has already been shown that ccRNA-I is partially digested by $1 nuclease in the presence o f o ' I 5 M-NaC1 (Randles et al. x976). Fig. ~ shows that in o'3 M-N~CI, ccRNA-I was much more resistant to $1 than in o'o5 M-NaCI, probably reflecting the influence of secondary and tertiary structure on the availability to the enzyme of single-stranded regions. Incomplete hydrolysis occurred at both salt concentrations, as shown by the residual hyperchromicity upon the addition of ribonuclease A. The products of S~ nuclease hydrolysis in low salt
Cadang-cadang c D N A
653
)
,!
2
,;:;i*#
:ii M
1
i:
Fig. 2. Progressive hydrolysis of unfractionated nucleic acids from a diseased palm with S1 nuclease. Ten/*1 samples in low salt $1 assay buffer were incubated at 45 °C with 2 units of $1 nuclease for o, io or 30 min, Incubation was terminated by adding 4 vol. of chilled electrophoresis buffer and products were compared with an untreated extract ( - ) by electrophoresis on 3"3 % polyacrylamide gels. Positions of ccRNA-I and -2 are indicated by numbers I and 2, respectively, that of the bromnphenol blue marker is shown with an ink mark. Gels were stained with toluidine blue. medium were analysed on polyacrylamide gels. Fig. 2 shows the time sequence o f digestion o f c c R N A - I in an unfractionated nucleic acid extract. On the 3"3 ~o gel, a faster moving component o f approx. 6S appeared as the c c R N A - t component disappeared. Analysis o f the St nuclease product o f purified c c R N A - I on ~O~o gels (Fig. 3 a, I + ) showed two very lightly stained components migrating ahead o f c c R N A - I . These bands were more diffuse than c c R N A - t and the low intensity o f staining may be partially due to the greater dispersion o f St products than o f c c R N A - I . The second cadang-cadang associated R N A , c c R N A - z , which has a tool. wt. estimated to be approximately twice that o f c c R N A - I (Randles, I975), was undetectable after incubation with St nuclease (Fig. 2 and 3a) and no lower mol. wt. products were detected by staining the polyacrylamide gels (Fig. 3 a).
654
J, W. R A N D L E S
A N D P. P A L U K A I T I S
(a) 1-
1+
(b) 2-
2+
12
14
47
10
42
32
33
15
41
43
26
13
39
47
33
24
32
40
23
20
37
38
18
33
Fig. 3. (a) Analysis on a Io % polyacrylamide slab gel of ccRNA-I ( ! - ) and ccRNA-2 (2--), and St nuclease-treated ccRNA-I (x + ) and ccRNA-2(2+). Samples (2/zg) of each RNA, prepared as described in Methods, were dissolved in IO/zl of ' low' salt St assay buffer. Two units (I #1) of St nuclease were added to the treated samples. All samples were incubated at 45 °C for 3o min, then mixed with 5Old of TBE and subjected to electrophoresis for 18 h at 45 V. Brackets show the position of bands detectable by u.v. fluorescence after staining with ethidium bromide. These same bands stained very faintly when gels were post-stained with toluidine blue for photography. (b) Distribution of nucleotide sequences homologous with ccRNA-I in the gel. The gel was subdivided as shown, pieces were crushed with a glass rod, 28o #1 of hybridization buffer were added and the slurry was shaken gently for 48 h. Gel pieces were sedimented at loooo g (for 2o min) and 4o #1 samples were hybridized with cDNA to an Rot value (calculated for the ccRNA- I band and assuming complete extraction of RNA from gels) of 7 x io -1. Values given are the percentage hybridizations obtained for extracts from each gel piece (no analysis was done where no value is given).
Polyadenylation of ccRNA-I and preparation of eDNA Between 3 and 9 % of the cc82P-ATP in the polyadenylation reaction mixture was incorporated into ccRNA-I. This is equivalent to an average of between I5 and 45 AMP residues per molecule of ccRNA-I. This proportion was not significantly affected either by melting
655
Cadang-cadang c D N A
16S CMV4
I
I
CMV SAT
I
4S I--1
o 3(
E
*d
.//,'// /,.¢.~ ,,°/*\'~/
!
1
0
1
I
l
10
20 30 40 50 Gel slice number Fig. 4. Size distribution of zzP-cDNA on a denaturing 5 % polyacrylamide-98% formamide gel, shown in relation to marker RNA species (see Methods for tool. wt.). Slices (z ram) were counted by Cerenkov radiation.
and chilling the ccRNA-I, or by 81 nuclease pre-treatment with or without subsequent melting and chilling immediately before polyadenylation. (In a parallel experiment, untreated E. coli tRNA incorporated 69 % of the input ATP, equivalent to an average of approx. 5o AMP residues per molecule.) However, only the ccRNA-I which had been treated with $1 nuclease, melted and chilled prior to polyadenylation yielded a cDNA transcript which gave the homologous hybridization kinetics expected for a R N A the size of ccRNA-I (Hell et al. I976; Gould et al. I978). Incorporation of radioactivity into cDNA was 2 to 3 x io 5 ct/min per 2/zg of ccRNA-r (i.e. 3 to 4"5 ng c D N A / 2 #g ccRNA-I). Characteristics of the cDNA
The majority of the cDNA fell into the 4S size class (Fig. 4) with smaller amounts of higher mol. wt. material also present. The size distribution resembles that of cDNA synthesized from chrysanthemum stunt viroid (CSV; Palukaitis & Symons, 1978), potato spindle tuber viroid (PSTV; Owens, I978) and CMV-satellite RNA (Gould et al. I978). Although the mol. wt. of ccRNA-I is near that of CMV-satellite RNA, it has not been accurately determined and because of its circularity (unpublished data) ccRNA-I is not included as a mol. wt. marker with the other linear RNAs.
656
J. W. R A N D L E S AND P. P A L U K A I T I S T a b l e I. Hybridization specificity of cDNA to ccRNA-I
Nucleic acid
RNA concentration (/zg/ml)
R 0t
% hybridization
eDNA alone eDNA + ccR.NA- I ccRNA-I (St treated) ccR.NA-2 ccR.NA-2 ($1 treated) Chrysanthemum stunt viroid CMV satellite RNA CMV RNA Tobacco ringspot virus RNA Tobacco mosaic virus RNA Yeast soluble RNA
-0-9 I* I t* I I 1 5 14"4 zo
-o'z o'z 0'7 o'7 0'2 o'2 o'2 Po 3"0 4"I
6"7 43'9 46"0 51"9 35"7 5"2 3"6 5"5 5.4 4-I 3"6
* Treated with $1 nuclease as described in Methods - concentration is that of RNA before $1 treatment.
T a b l e 2. Hybridization of cDNA to ccRNA-I with nucleic acids O[ diseased
and healthy palms Palm Diseased Diseased Healthy Diseased No. 1 Diseased No. z Diseased No. 3 Diseased No. 4 Healthy Diseased Healthy
Nucleic acid preparation
% hybridization*
Purified ccRNA- I t PEG procedure, unfractionated$ PEG procedure, unfractionated Total, phenol extracted§ Total, phenol extracted Total, phenol extracted Total, phenol extracted Total, phenol extracted Total DNA LI Total DNA
46 56 io 45 46 31 6I 3 o o
* Hybridization percentages corrected for self-hybridization of cDNA. t Heterologous ccRNA-I, Rot = 0"48 mol. s/l. Nucleic acids from I g leaf tissue, hybridization time I5"5 h. § Nucleic acids from oq g leaf tissue, hybridization time 68 h. II Cot = I85 mol. s/1. The specificity o f the e D N A for c c R N A - I (with a n d w i t h o u t Sx nuclease p r e - t r e a t m e n t ) a n d c c R N A - 2 is shown in T a b l e L O t h e r small R N A species, virus R N A a n d nucleic acids extracted f r o m h e a l t h y c o c o n u t p a l m s ( T a b l e z) show no significant h o m o l o g y with the e D N A when h y b r i d i z e d to Rot values at least twenty times g r e a t e r t h a n the R0t~ value for h o m o l o g o u s h y b r i d i z a t i o n . A l t h o u g h St nuclease digests c c R N A - 2 (Fig. 3 a), $1 nucleaseresistant sequences r e m a i n which hybridize to a m a x i m u m value o f a r o u n d 36 % (Table I ; see also Fig. 3b). Fig. 5(a) shows the kinetics o f h y b r i d i z a t i o n with h o m o l o g o u s c c R N A - I in a typical analysis. Similar kinetics were o b t a i n e d using $1 nuclease-treated c c R N A - I ( d a t a n o t shown). Curves h a d the expected sigmoid shape, b u t m a x i m u m h y b r i d i z a t i o n values rarely exceeded 5o %. It is possible t h a t this low m a x i m u m value m a y be due to c c R N A - I retaining c o n s i d e r a b l e s e c o n d a r y structure at the t e m p e r a t u r e - s a l t c o n c e n t r a t i o n regime used for hybridization. Nevertheless, v a r y i n g the t e m p e r a t u r e a n d salt c o n c e n t r a t i o n a n d including f o r m a m i d e in the h y b r i d i z a t i o n buffer, failed to increase the m a x i m u m h y b r i d i z a t i o n values obtained. The Rot ~ value o f a p p r o x . I x i o -3 mol. s / l for the h y b r i d i z a t i o n o f the e D N A with c c R N A - t is close to t h a t o b t a i n e d for CMV-satellite R N A in a parallel e x p e r i m e n t a n d
Cadang-cadang eDNA 60
I
50
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I
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657
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40 30 20 50 lO o
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Temperature (°C) Rot (tool s/l) Fig. 6 Fig. 5 Fig. 5. Kinetics of hybridization of 32P-cDNA to (a) homologous ccRNA-I, and (b) ccRNA-l and (c) CC RNA-z extracted from another diseased palm. Blank values have been subtracted from all samples before calculating percentage St nuclease resistance. Fig. 6. Thermal denaturation of the cDNA ccRNA-l hybrid in hybridization buffer, expressed as percentage of hybrid remaining resistant to St nuclease. is also very close to t h a t o b t a i n e d at the same salt c o n c e n t r a t i o n by G o u l d e t al. (~978) for CMV-satellite R N A a n d by Palukaitis & S y m o n s 0 9 7 8 ) for c h r y s a n t h e m u m stunt viroid ( b o t h I × IO -3 tool. s/l). c c R N A - I therefore has a nucleotide c o m p l e x i t y close to that o f these two s t a n d a r d R N A species, a n d close to its e s t i m a t e d tool. wt. o f a p p r o x , r × IO -5 (Randles, I975). H o m o l o g o u s h y b r i d s melted s h a r p l y in h y b r i d i z a t i o n buffer with a T m o f 87 o C (Fig. 6). The melting profile closely resembles t h a t o b t a i n e d for other h o m o l o g o u s h y b r i d s in the same buffer ( G o u l d & Symons, I977; G o u l d et al. t978) a n d h y b r i d f o r m a t i o n was therefore a p p a r e n t l y specific with no evidence o f mismatching.
100
658
J. W. RANDLES AND P. PALUKAITIS
Homology between ccRNA-I and ccRNA-2 Hybridization analysis was used to examine the relationship between ccRNA-I and -2. The hybridization kinetics of eDNA with the homologous ccRNA-I preparation from which it was transcribed (Fig. 5a) were compared with those obtained when the cDNA was hybridized with 'heterologous' ccRNA-I (Fig. 5b) and ccRNA-2 (Fig. 5 e) extracted from another diseased palm. The similarities of the shapes of the curves, the rates of hybridization and the maximum hybridization values indicate that there is a high degree of homology between these RNAs. The Rot½ of the cDNA-ccRNA-2 hybridization was very close to that for the c D N A eeRNA-I hybridization. It seems improbable that contamination of ccRNA-2 with -I could account for such homology because more than 5o ~o contamination would be required to give such an R0t~ value. Re-analysis of ccRNA-2 on polyacrylamide both with and without (Fig. 3a) prior treatment in 4 M-urea at 6o °C for 15 min and staining in both ethidium bromide and toluidine blue, showed no detectable ccRNA-I at a threshold of detection of around 5 % of the ecRNA-2 concentration. Because the hybridization assay has the limitation that a maximum of approx. 5o % of the e D N A has hybridized in all eases, it cannot be concluded that ccRNA-I and -2 have identical nucleotide sequences. Nevertheless, the ccRNA-I sequences to which the c D N A hybridizes are found in both ccRNA-I and -z.
Demonstration of the unique nature of ceRNA-I and -2 The eDNA was used to determine whether ceRNA-I was a species of RNA unique to diseased palms and whether its nucleotide sequences were represented in higher mol. wt. R N A species from diseased palms. Nucleic acids were fractionated by electrophoresis on agarose gels; regions of the gels were excised, nucleic acids extracted, concentrations equalized and percentage hybridization with e D N A determined (Fig. 7). Components from the healthy palm showed no significant nucleotide sequence homology with ccRNA-I. The higher percentage of hybridization in the Hoo region could be due to random sequence homology between small oligonucleotides and the cDNA, but it is well below that for the corresponding component from the diseased palm. The ccRNA-I zone gave around the maximum value expected for homologous saturation hybridization and ~cRNA-2 hybridized to about the same extent as ccRNA-I. The higher tool. wt. components CC3 and CCT showed negligible hybridization. The lower but significant values in the CC0 (4S) and CCoo zones suggests that they probably contain fragments of ccRNA-I or -2. The size distribution of the RNA components homologous with, but smaller than, ccRNA-I or -2 was studied by hybridizing eDNA with RNA extracted from the gel shown in Fig. 3(a). Hybridization was to an Rot value which would be expected to detect sequences at an approx. Ioo times lower concentration than in the ceRNA-I component (assuming equivalent effieiencies of extraction of R N A from gel). Fig. 3(b) shows the percentage hybridization obtained for each gel piece analysed. ccRNA-I showed low hybridization in the ccRNA-z region, indicating negligible contamination with ccRNA-2. At and below the ccRNA-I region values near or at the maximum were obtained for all pieces, whether or not they contained nucleic acids detectable by staining. Thus, this more sensitive assay shows that the ccRNA-I preparation contains a small amount of lower mol. wt. nucleic acids which comprise the sequences represented in the eDNA. While S 1 nuclease treatment makes the ccRNA-I band undetectable by staining, sufficient remains to be detected by hybridization analysis. Smaller components remain which show maximum homology with the cDNA.
Cadang-cadang eDNA
~
659 CCT -- 2"2%
I CCa - 7.3%
H3 - 1"5%
] CC2 - 55.9%
H2 - 0,1% I ] CC - 48.9%
~
CCo -25"1%
H0 - 3.0% I
Hoo -
l CCoo - 42"7%
9.2 % I
H
CC
Fig. 7. Distribution of ccRNA-I sequences in nucleic acids from healthy (H) and diseased (CC) palms, fractionated by electrophoresis on 4 % agarose gels. Nucleic acids were extracted from stained gel pieces by blending in equal vol. of oq M-sodium acetate and 90 % phenol containing o'4 % SDS, shaking for 16 h, separating phases by centrifugation and precipitating nucleic acids from the aqueous phase with ethanol. Acetyltrimethyl-ammonium bromide precipitation step (Randles et al. 1976) followed. Nucleic acid concentrations from each zone were adjusted to I t~g/ml in hybridization buffer and hybridization with eDNA proceeded to an Rot of 1-8 x xo-1 mol. s/1. ccRNA-I and -2 are marked CC~ and CC~. Percentage hybridization is shown for each zone. Ink marks show dye marker.
ccRNA-2 hybridized to around the maximum expected value (Table I; Fig. 5c), while lower values were obtained for parts of the gel expected to contain smaller molecules. These low values were raised to around 4o % by increasing the Rot value tenfold, indicating that the low values were due to low concentration rather than incomplete homology, ccRNA-2 was undetectable after $1 nuclease treatment and although some homology was detected near the bottom of the gel, maximum values did not exceed those obtained in other experiments (Table I) even at higher Rot values. Although ecRNA-2 contains sequences which are homologous with ccRNA-I they appear not to show the same degree of resistance to S1 nuclease as they do in ccRNA-I.
Utilization of cDNA as a probe for ccRNA-I Results summarized in Table 2 show that ccRNA-I can be detected in unfractionated nucleic acids from diseased palms, prepared either by the PEG procedure, or by the total nucleic acid extraction procedure. The differences between the values obtained for healthy and diseased plant extracts are a clear indication of the value of the eDNA as a diagnostic
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P. P A L U K A I T I S
probe. The presence of sequences complementary to PSTV in host DNA has been shown by Hadidi et al. (I976). In our study the absence of homology with DNA from healthy or diseased palms at high Cot values (Table 2) precludes the possibility that ccRNA-I copies in DNA might interfere with the detection of ccRNA-I or -2 in total nucleic acid extracts. Unsuitability o f alternative procedures.]'or the synthesis o f c D N A
The technique of Taylor et al. (1976), utilizing random oligodeoxynucleotidesas internal primers for the reverse transcriptase catalysed synthesis of cDNA, yielded a cDNA which gave sigmoid homologous hybridization kinetics with a Rob value of 2 × IO-8 mol. s/1. However, transcription was inefficient with approx. 8 x io 4 ct/min incorporated ( I q z ng cDNA) per 2 #g of untreated ccRNA-I template. Furthermore, the cDNA had high $1 nuclease resistance (29 to 55 %) and gave saturation hybridization values of only 32 to 36 %. The method of Kacian & Myers (~976) for synthesizing cDNA to polyadenylated RNA, gave more efficient incorporation of radioactivity into cDNA (I2 x lOs ct/min per z/~g ccRNA-1 ; equivalent to 14.4 ng cDNA per 2 #g RNA) and hybridization gave a R0t~ value of I x io -a mol. s/1. However, $1 nuclease resistance of the cDNA was high (45 to 55 %) and maximum homologous hybridization values of only I6 to 23 % were obtained. The high $1 nuclease resistance is apparently due to 'snap back' because melting and rapidly chilling the cDNA had no effect on subsequent Sa resistance.
DISCUSSION
A complementary DNA probe specific for ccRNA-I has been synthesized using an $1 nuclease-treated, heat-denatured template for the in vitro polyadenylation and reverse transcription reactions (Hell et aL I976). Other methods for synthesizing probes were less satisfactory (Taylor et aL I976; Kacian & Myers, [976) or were considered inappropriate for the studies which we envisage (Hadidi et al. 1977; Owens & Diener, I977). Our preliminary attempts to synthesize a cDNA probe demonstrated the necessity for using highly purified RNA as a template. Minor host RNA components were transcribed much more efficiently than the ccRNA-I. Because ccRNA-I is extracted in the presence of host nucleic acids, to achieve high purity we took advantage of the high stability of ccRNA-I to thermal denaturation (ccRNA-I withstands at least three cycles of melting-cooling with no change in hyperchromicity at each cycle; J. W. Randles, unpublished data). The RNA was melted before each electrophoresis step to dissociate host RNA contaminant: from ccRNA-~. A range o f gel concentrations was used to exploit possible differences in relative electrophoretic mobilities which might arise from structural differences between ccRNA-1 and host RNA. A possible additional advantage of using S~ nuclease treatment of the ccRNA-I was that residual single-stranded contaminants may have been hydrolysed. Hybridization analyses indicate that the ccRNA-I was sufficiently pure to give a cDNA which could be used as a specific probe. The S~ nuclease-heat denaturation procedure appears to be important for the synthesis of a cDNA probe for ccRNA-I. This procedure is thought to cleave the circular (Randles & Hatta, I979) ccRNA-1 molecule, possibly at one or more sites yielding free ends available for polyadenylation, priming and transcription. The specific nature of the template for cDNA transcription is not known, as a range of homologous RNA molecules is present in $1 nuclease-treated preparations (Fig. 3b). The yields of cDNA were low compared with other systems using plant virus RNA (Gould & Symons, I977; Gould et al. I978), potato spindle tuber viroid (Owens, 1978) and chrysanthemum stunt viroid (Palukaitis & Symons, 1978). Insufficient cDNA was synthe-
Cadang-cadang e D N A
66I
sized to allow calculation of its representivity. Unusual features of the hybridization kinetics, such as the somewhat broader transition from minimum to maximum values and low maximum hybridization percentages, may be due to the viroid-like circular base-paired structure of the ccRNA-I, cDNAs to PSTV (Hadidi et al. I977) and CDV (Palukaitis & Symons, I978) have similar hybridization characteristics, but c D N A which was further purified by hybridization with the PSTV template showed higher maximum values and a sharp transition from maximum to minimum values (Owens, 1978). R0t~ values obtained for c c R N A - I were equivalent to those obtained for homologous systems with R N A of a size similar to c c R N A - t and this, in association with the demonstrated specificity of the c D N A for the cadang-cadang associated RNA, allows us to conclude that: ( 0 c c R N A - I is a unique R N A species of tool. wt. approx. I × IOn; (2) ccRNA-I and -z have common nucleotide sequences; (3) both ccRNA-I and -2 are uniquely associated with cadangcadang-affected coconut palms; (4) sequences of c c R N A - I are not significantly represented in the high tool. wt. nucleic acid component of diseased or healthy palms, hence c c R N A - I and -2 do not arise as breakdown products of high molecular weight R N A species; and (5) no evidence exists for homology between ccRNA-~ and host plant DNA. This evidence supports that already obtained (Randles et al. I976) which suggests a viroid aetiology for cadang-cadang disease. It is expected that the sensitivity and specificity of the molecular hybridization analysis will shorten the effective incubation period in transmission trials designed to test the pathogenicity of c c R N A - t and -2, that it will provide a means of seeking alternative hosts and vectors in epidemiological studies and allow investigations of the nature and replication of the cadang-cadang associated RNA. We thank Dr R. H. Symons for his encouragement, suggestions and helpful discussion throughout this study, Dr A. R. Gould for his ready co-operation and advice on the synthesis of cDNA, Mr C. Davies for technical assistance and Mrs L. Wichman for figure preparation. We are grateful to Dr J. R. E. Wells for avian myeloblastosis virus reverse transcriptase (kindly provided by the Office of Program Resources and Logistics, Viral Cancer Program, Division of Cancer Cause and Prevention, National Cancer Institute, Bethesda, Maryland 2oo 14, U.S.A.). Part of this work was done by J. W. R. during a consultancy with FAO. Support was provided by the Australian Research Grants Committee.
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(Received r3 July 1978)
