- Email: [email protected]
Differentiation of Schistosoma haematobium from related species using cloned ribosomal RNA gene probes
Molecular and Biochemical Parasitology, 20 (1986) 123-131 Elsevier
123
MBP 00683
Differentiation of Schistosoma haematobium from related species using cloned ribosomal R N A gene probes Tina K. Walker 1, David Rollinson 1 and Andrew J.G. Simpson 2 lDepartment of Zoology, British Museum (Natural History), Cromwell Road, London SW7 5BD and 2parasitology Division, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, U.K. (Received 14 January 1986; accepted 17 March 1986)
The ribosomal RNA (rRNA) gene units of Schistosoma mansoni (lateral spined eggs) and six species of schistosomes with terminal spined eggs (S. haematobium, S. curassoni, S. bovis, S. intercalatum, S. margrebowiei and S. mattheei) have been studied. The schistosome rRNA gene unit consists of a regular interspersion of the two genes encoding the large and small rRNA units with two spacers. The large spacer is not transcribed while the small spacer is part of the transcription unit. Variation in the rRNA gene unit of the species studied is demonstrated and takes three forms: First, variation in DNA sequence leads to both reduced homology in the spacer regions between species and loss or gain of restriction sites. Second, variation in the length of the transcribed spacer is demonstrated and DNA insertions of 0.2 kilobases (kb) and 0.1 kb are observed in S. mattheei and S. margrebowiei, respectively. Third, the length of the non-transcribed spacer region varies between species. S. haematobium has a 0.5 kb deletion in this region, while that of S. margrebowiei contains varying numbers of a 0.4 kb DNA insert. These interspecific variations have been shown to be conserved within species. Analysis of the rRNA genes by DNA hybridisation techniques therefore serves as a means of species identification, whereby it is possible to differentiate S. haematobium from other schistosome species with terminal spined eggs. Similarly, S. margrebowiei and S. mattheei may be clearly distinguished, although no major variation has been detected between S. curassoni, S. bovis and S. intercalatum. All these species differ from S. mansoni by the absence of certain restriction sites in the non-transcribed spacer. Key words: Schistosomes; Ribosomal RNA gene; Species differentiation
Introduction Schistosomes are parasitic helminths responsib l e for t h e d e b i l i t a t i n g t r o p i c a l d i s e a s e k n o w n as Bilharzia or Schistosomiasis. Of the eighteen or so species of Schistosoma currently recognised five a r e i m p o r t a n t p a r a s i t e s o f m a n : S. mansoni, S. h a e m a t o b i u m , S. intercalatum, S. j a p o n i c u m a n d S. m e k o n g i , w h e r e a s o t h e r s p e c i e s c a u s e d i s e a s e in wild a n d d o m e s t i c a n i m a l s . It is c o m m o n p r a c tice to arrange the species into three groups which reflect b o t h t h e m o r p h o l o g y o f t h e egg a n d t h e i n t e r m e d i a t e h o s t r e s p o n s i b l e for t h e i r t r a n s m i s sion; t h o s e w i t h a l a t e r a l s p i n e to t h e egg as S. mansoni, t h o s e w i t h a t e r m i n a l s p i n e to t h e egg
Abbreviations: cpm, counts per minute; EDTA, ethylenediaminetetraacetic acid; kb, kilobases; rRNA, ribosomal RNA; SDS, sodium dodecyl sulphate; SSC, standard saline citrate.
as S. h a e m a t o b i u m a n d t h o s e with r o u n d minu t e l y s p i n e d eggs as S. ]aponicum. A v a r i e t y o f a n a t o m i c a l , d e v e l o p m e n t a l a n d h o s t - r a n g e characteristics a r e u s e d for t h e i d e n t i f i c a t i o n o f species. I n a d d i t i o n , e l e c t r o p h o r e t i c studies h a v e c o m p l e m e n t e d t r a d i t i o n a l a p p r o a c h e s to c h a r a c t e r i s a t i o n a n d h a v e s o u g h t to p r o v i d e a l t e r n a t i v e m e t h o d s for i n v e s t i g a t i n g intraspecific v a r i a t i o n [1]. M o r e recently, the use of cloned D N A p r o b e s to distinguish strains o f S. m a n s o n i has b e e n rep o r t e d [2] a n d it h a s b e e n s h o w n t h a t v a r i a t i o n o f t h e r R N A g e n e f a m i l y o f s c h i s t o s o m e s can b e used to differentiate S. mansoni, S. japonicum and S. haematobium [3]. Similar techniques have b e e n u t i l i s e d for t h e i d e n t i f i c a t i o n o f s e v e r a l o t h e r helminths. F o r example, Echinococcus [4], Brugia [5] a n d Trichinella ( A . E . C h a m b e r s , m a n u s c r i p t s u b m i t t e d ) , r e v i e w e d b y R o l l i n s o n et al. [6]. R i b o s o m a l R N A ( r R N A ) g e n e o r g a n i s a t i o n is
124 very similar in all eukaryotes [7]. A tandemly repeating unit consisting of the following sequences occurs m a n y times throughout the genome: 5'non-transcribed spacer, external transcribed spacer, small r R N A gene (18S), 5.8S ribosomal R N A gene flanked by transcribed spacers, large ribosomal R N A gene (28S)-3'. The main size variation of this repeating unit observed between organisms is usually due to the presence of long or short non-transcribed spacer D N A . The spacer D N A within an organism m a y be either homogeneous (yeast) or heterogeneous ( X e n o p u s )
analysis may be utilised to identify both schistosome species as well as strains of S. mansoni [2]. This p a p e r presents restriction maps of the r R N A genes of various schistosome species of the terminal spined egg group from Africa, including S. haematobium, S. rnattheei, S. margrebowiei, S. bovis, S. curassoni and S. intercalatum and compares them with that of S. mansoni. Variation in the transcribed and non-transcribed spacer regions of the r R N A gene has been identified and the possibility of utilising these genetic variations to differentiate species has been explored.
[7,8]. The r R N A genes of S. m a n s o n i have been assigned to a unit of similar organisation although the presence of a gene corresponding to 5.8S R N A has not been confirmed. The unit, approximately 10 kilobases (kb) in length, is estimated to be repeated tandemly some 100 times throughout the g e n o m e [9,10]. B a m H 1 fragments containing the r R N A genes of S. m a n s o n i have b e e n cloned [11] and used as probes for mapping the corresponding genes of various schistosome strains and species. This previous work d e m o n s t r a t e d that direct genome
Materials and Methods Parasites. Schistosome species were collected from a n u m b e r of geographical locations to ensure that results were consistent between isolates. During the laboratory passaging procedure no attempt was made to clone strains from individual eggs. In each case, the parasite material used for D N A extraction was a group of worms originating from a single geographic location. The origin and the number of laboratory passages performed for each species yet analysed is presented in Table I.
TABLE I Origin of schistosome isolates analysed to date and the number of laboratory passages performed Species
Origin
Number laboratory passages
Figure number in which isolates are shown
S. rodhaini S. mansoni
Burundi Laboratory NIMR Puerto Rico Senegal Senegal Tanzania Senegal Senegal Kenya Gambia Sudan Durban, S. Africa Senegal Nigeria Zambia S. Africa Malawi Zambia Cameroun Zaire Gabon
4th Numerous
4 1, 3, 4, 6
Collected from sheep 3rd passage Unknown 4th passage Collected from cattle Unknown 1st passage 1st passage 1st passage 1st passage 1st passage 1st passage 40th passage 1st passage 23rd passage Unknown Unknown Unknown
1, 3, 4, 6 6 1, 3, 4 3 6 1, 4 1, 3, 4, 6 1, 3, 4, 6 1, 3, 4, 6
S. curassoni S. bovis S. haematobium
S. mattheei S. margrebowiei S. intercalatum
125 Approximately 50--100 adult schistosome worms were gently homogenised in a small volume (100 ~1/50 worms) of extraction buffer (50 mM Tris-HCl, 50 mM ethylenediaminetetraacetic acid (EDTA), 100 mM NaC1, pH 8). An equal volume of extraction buffer containing 1% sodium dodecyl sulphate (SDS) was added, together with proteinase K to a final concentration of 1%, and the mixture incubated at 37°C for 2-3 h. DNA was extracted three times with equal volumes of phenol, phenol/chloroform (1:1) and chloroform. Finally, the DNA was precipitated by the addition of 2.5 volumes of absolute redistilled ethanol. DNA was washed in 70% ethanol, vacuum dried, redissolved in Tris-EDTA buffer (pH 7.4) and stored at -70°C.
Preparation o f D N A .
kb
Filter hybridisation. Genomic schistosome DNA was digested with restriction endonucleases obtained from either Boehringer or BRL and utilised according to the supplier's recommendations. Restriction fragments were separated by 0.6% agarose flat-bed electrophoresis and transferred to nitrocellulose filters according to the technique of Southern [13]. Filters were prehybridised in 4x Denhardt's solution [14]; 4× standard saline citrate (SSC) and 0.1% SDS at 65°C for 4-5 h. At least 107 cpm of DNA probe was denatured by boiling for 5 min with sheared calf thymus DNA and hybridised overnight in 30 ml 4x Denhardt's solution; 4x SSC and 0.1% SDS at 65°C with the nitrocellulose filters. Hybridised filters were washed in 0.1x SSC and 0.05% SDS at 52°C for 3-4 h with at least 3 buffer changes. Finally, the filters were dried in air and exposed for autoradiography using flashed Fuji Xray film backed by a 'Cronex' intensifying screen.
5-0
Results
3-2
D e m o n s t r a t i o n o f variation in the r R N A g e n e o f various s c h i s t o s o m e species. Adult worm genomic
EcoR1 Digest
kb 23-1-
9'4-6"74-4-
-
-4.5 -
R a d i o l a b e l l i n g o f D N A . DNA probes pSM890, pSM889 and pSM389 are BamH1 fragments of the rRNA gene of S. m a n s o n i as prepared by Simpson et al. [11]. DNA was labelled with [32p]dCTP by Nick translation [12] using the kit supplied by BRL and according to their instructions. Incorporation was generally to an activity of 1 × 107 to 2 × 107 cpm i~g-1.
2'3-2-0-
-1"6
Fig. 1. Autoradiograph of the hybridisationof 32p-labeUed pSM889with EcoR1 digestedgenomicDNA fromS. mansoni and six species of the terminal spirted egg group. Band sizes are marked, int = S. intercalatum, mat = S. mattheei, marg = S. margrebowiei, haem = S. haematobium, bov = S. bovis, cura = S. curassoni, man = S. mansoni.
DNA was Southern blotted and hybridised with radiolabelled rRNA gene probes. An example of the results obtained by this method is shown in Fig. 1, where the DNA from several terminal spined egg group species has been digested with EcoRI and hybridised with the radiolabelled probe pSM889. Variations in the rRNA gene of the various schistosomes are identified. For each species, three major bands, or groups of bands are produced, of which the large and small bands vary between species, while the middle band remains constant. Such results suggest that the variation observed may be confined to particular regions of the rDNA. Probes pSM389, pSM890 and pSM889 were
126
HindlII and Xbal hybridised with pSM889 (Fig. 3). Five rRNA gene fragments possessing the ability to hybridise with pSM889 were generated (see Fig. 2). Two of these fragments were of equal size and electrophoresed as one band, while a 0.4 kb fragment is not shown. Hence, hybridisation with probe pSM889 generates three bands, of which the larger two bands represent regions of the rRNA gene encoding the large and small rRNA subunits. The smallest band (see arrow) encompasses the transcribed spacer which is 0.75 kb for S. haematobium, S. bovis, S. curassoni and S. mansoni, but 0.85 kb for S. margrebowiei and 0.95 kb for S. mattheei. Double digests using restriction enzymes EcoR1
each hybridised with adult schistosome DNA cut by several restriction enzymes in turn. Comparison of the banding patterns obtained with the restriction map already constructed for S. mansoni rDNA [11] allowed restriction maps for each of the terminal spined egg group schistosomes to be constructed, Fig. 2. Some details of the mapping procedure are described below. Confirmation that interspecific variation occurs in the spacer regions. The areas of the rRNA gene unit of most interest are those exhibiting interspecific variation. The variable regions of the rRNA gene were identified using double digests of genomic D N A using restriction enzymes SPACER S.MANSONI
•
pSM889
E
P
REGION
AND
VARIATION
TERMINAL
4.4kb
SPINED
:,,
IN T H E EGG
rRNA
GROUP
pSM890 2.4kb
E
GENE
OF
SCHISTOSOMES
:,,
pSM389
3.1kb
E H I
B S.mansoni I
Transcribed Spacer
B
X I
H I
Non-transcribed Spacer
Large rRNA
P B I I
Small rRNA ,(
S.bovis c~'~c~ag-s . o ni
~H
P b
H
P I
S.haematobium
~H
PI
S.margrebowiei
E I H
P I
S.intercalatum
S.matthQei
E q
I
I
kb
0
1 ,
2 ,
3 ,
4 ,
5 ,
6 ,
7 ,
8 ,
9 h
10 ,
Fig. 2. Restriction map of schistosome rRNA gene units. Regions of the rRNA gene unit of schistosomes within the terminal spined egg group that differ from that of S. m a n s o n i are presented. Homologous regions are omitted. E=EcoR1; H=HindlII; P=Pstl; X = X b a l ; B=BamH1.
127 Hind m / x b a
1 Digest
t
3
3
Kb
2 a. , -
9.4kb
6.74.4
sisting of several bands differing in length by 0.4 kb. Marked differences in the degree of binding were observed between the larger fragments, containing the non-transcribed spacer for each species, analysed. The intensity of the larger band is much greater for S. m a n s o n i and S. r o d h a i n i (lateral spined egg group species) than for the terminal spined egg group schistosomes. Intensity variation of the 2.0 kb band was due to differing amounts of D N A loaded on the gel. The two bands were analysed on a Joyce-Loebl chromoscan 3, and the ratio of the intensity of the upper band to the lower band was plotted for each species (Fig. 5). This ratio was greater than 2.5 for S. m a n s o n i and S. r o d h a i n i , while for species
Xba I / E c o R 1
2.3 2.0
Digest
:3"
3
- 0 95 - 0 . 8• 5
~1~
'~g
-0.75
and X b a l were also carried out for several schistosome species which demonstrated differences in length of the non-transcribed spacer (Fig. 4). Hybridisation with p r o b e pSM389 generated two bands. A band of 2.0 kb is consistent in each species analysed and represents the part of the r R N A gene encoding the small r R N A unit. The second band encompasses the whole non-transcribed spacer region and 0.5 kb of the region encoding the large r R N A unit. For S. i n t e r c a l a t u m , S. m a t theei, S. b o v i s , S. c u r a s s o n i and S. m a n s o n i , this second band is 3.5 kb while for S. h a e m a t o b i u m it is 3.0 kb in length. S. m a r g r e b o w i e i produces the characteristic multiple banding pattern con-
M
Kb
0.6-
Fig. 3. Autoradiograph of the hybridisation of 32p-labelled pSM889 with HindIII/Xbal digested genomic DNA from S. mansoni and six species of the terminal spined egg group. The size and position of bands representing DNA fragments containing the transcribed spacer region of the rRNA gene unit are marked, int = S. intercalatum; mat = S. mattheei; mar = S. margrebowiei; haem = S. haematobium; bov = S. bovis; cur = S. curassoni; man = S. mansoni.
'¢
23.1-
P •
9.4-
kb
6.74.4-3.0 2.3-
2 . 0 - ~D
I
-2.0
Fig. 4. Autoradiograph of hybridisati0n of 32P-labelled pSM389 with EcoR1/Xbal digested genomic DNA from S. mansoni and S. rodhaini (lateral spined eggs) and six species of the terminal spined egg group, int = S. intercalatum; mat = S. mattheei; mar = S. margrebowiei; haem =S. haematobium; bov= S. bovis; cur = S. curassoni; man = S. mansoni; rod = S. rodhaini. Band sizes are marked.
128
of the terminal spined egg group it was less than 1.0.
Differentiation of schistosome species by hybridisation of digested genomic DNA with rRNA gene probes. Hybridisation patterns generated by restriction digests and hybridisation with rRNA gene probes may be used to differentiate schistosome species. Of particular use are the EcoR1 digests hybridised with pSM889 (Fig. 1). Alternatively, the species can be distinguished by a restriction enzyme/probe combination generating a single, variable band of hybridisation (Fig. 6). BamH1 digestion of genomic DNA and its sub-
sequent hybridisation with radiolabelled probe pSM890 produces a major band of 2.4 kb with S. mansoni and major bands of between 5.0 and 5.5 kb with the schistosomes of the terminal spined egg group. This hybridisation pattern again illustrates the non-transcribed spacer deletion of S. haematobium and the characteristic multiple banding pattern of S. margrebowiei. To date, no variation within the rRNA genes has been found between S. intercalatum, S. bovis and S. curassoni. To demonstrate that the hybridisation patterns are consistent within each species, several isolates of each terminal spined egg group species
RATIO
B a m H1 D i g e s t
3.0
3
o
er
ID
C
0
Kb
~
3
3 _
~
~D
....
23.1-
2.0
kb .4
.
.
.
.
6.7-5.5 -5.0
i,~gI 4.4-
• w
1.0
-2.4 2.3-
2.0-
> Z
0 0
c
=0
0 <
> m 3:
•
•
=o
Fig. 5. Histogram representing relative intensities of the upper and lower bands in Fig. 4. The ratio of the intensity of the upper band to the lower band was calculated for each species. M A N = S. m a n s o n i ; R O D = S. r o d h a i n i ; C U R = S. c u r a s s o n i ; B O V = S. b o v i s ; H A E M = S. h a e m a t o b i u m ; M A R = S. m a r g r e b o w i e i ; M A T = S. rnattheei; I N T = S. i n t e r c a l a t u m .
Fig. 6. Autoradiograph of the hybridisation of 3Zp-labelled pSM890 with B a m H 1 digested genomic D N A from S. m a n s o n i and six species within the terminal spined egg group. Band sizes are marked, m a n = S. r n a n s o n i ; cur = S. c u r a s s o n i ; bov = S. b o v i s ; h a e m = S. h a e m a t o b i u m ; m a r = S. m a r g r e b o w i e i ; mat = S. m a n h e e i ; int = S. i n t e r c a l a t u m .
129 have been analysed. The figure in which each isolate has been shown is presented in Table I. Some experiments are not shown; however, no differences in any of the major bands of hybridisation have been observed within a species. In each case, the D N A was extracted from a group of worms originating from one geographical location, which further emphasises the lack of variability. Several of the schistosomes examined were isolated from, or passaged through, different hosts and host DNA contamination has not been shown to affect hybridisation patterns. Discussion
Restriction maps have been constructed of the rRNA genes of S. mansoni and various terminal spined egg group schistosomes including S. haematobium, S. bovis, S. curassoni, S. margrebowiei, S. mattheei and S. intercalatum. These restriction maps are summarised in Fig. 2. The rRNA genes of the terminal spined egg group of schistosomes differ from that of S. mansoni in three ways. First, changes in the length of the transcribed spacers occur. This was verified by a double digest using the HindIII/Xbal restriction enzyme combination (Fig. 3) demonstrating DNA insertions of 0.1 and 0.2 kb in the transcribed spacers of S. rnargrebowiei and S. mattheei, respectively. The second difference that occurs between the rRNA gene of S. mansoni and that of the terminal spined egg group of schistosomes is variation in the length of the non-transcribed spacer as was demonstrated by an EcoR1/Xbal double digest. This cuts out the non-transcribed spacer region with only 0.5 kb of the adjacent 5' D N A sequence (Fig. 4) and demonstrates that the nontranscribed spacer of S. haematobiurn has a 0.5 kb deletion, while that of S. margrebowiei hybridises to form several bands each differing in length by 0.4 kb. Similar analysis of individual S. margrebowiei worms should be carried out, in order to determine how the non-transcribed spacer varies between, and possibly within, individuals. This experiment demonstrates the non-transcribed spacer to be of identical length for S. intercalatum, S. mattheei, S. bovis, S. curassoni, S. rodhaini and
S. mansoni. The third way in which the rRNA genes of the terminal spined egg group of schistosomes differ from that of S. mansoni is by DNA sequence variation. Such variation may be demonstrated by the study of the overall homology between regions of DNA for various species. The intensity of binding and hence the homology that exists between Southern blotted DNA and a bound probe, is proportional to the intensity of the band on the autoradiograph. The binding intensity of the large bands in Fig. 4 which represent the region of the non-transcribed spacer, is much reduced with terminal spined egg group species compared with that of S. mansoni. The probe hybridised with this filter was pSM389, encompassing the non-transcribed spacer region of S. rnansoni from which it was derived. Therefore, homology between the non-transcribed spacer region of S. mansoni and of species within the terminal spined egg group is greatly reduced. S. rodhaini, which belongs to the lateral spined egg group along with S. mansoni, binds strongly with pSM389. The comparable intensities of the 2.0 kb band encompassing the small rRNA unit coding sequence, which is homologous between species, is consistent with the general finding that the 18S and 28S RNA gene sequences are highly conserved during animal evolution [15]. This is due to the necessity for maintaining functional ribosome units. The observation that the spacer regions exhibit interspecific heterology, while being homologous within species is also consistent with previous findings [16-18]. These observations imply that the spacer regions have evolved rapidly relative to the adjacent coding regions that give rise to the rRNAs [81. DNA sequence alterations may additionally lead to loss or gain of single restriction sites exemplified by a BamH1 digestion of the DNA from various schistosome species (Fig. 6). Hybridisation with pSM890 produces a band of 2.4 kb with S. mansoni, while bands approximately twice this size are produced with the terminal spined egg group schistosomes. The probes utilised for these studies were derived by BamH1 digestion of the S. mansoni rRNA gene unit and probes pSM890 and pSM389 represent adjacent regions 2.4 kb and 3.1 kb in length, respectively. Loss of the
130 BamH1 site separating these regions of the rDNA in the terminal spined egg group results in pSM890 hybridising with a band approximately 5.4 kb in length (equivalent to both pSM890 and pSM389 together). The differences in the r R N A gene presented in this report, as well as being of genetic and evolutionary interest in relation to schistosomes, could potentially also be a useful tool for their identification and differentiation. Major differences occur in the r R N A gene unit of S. haematobium, S. margrebowiei and S. mattheei. In particular, the 0.5 kb deletion in the non-transcribed spacer of S. haematobium means that any r R N A gene probe hydridising with a r D N A fragment encompassing this spacer will produce a band 0.5 kb smaller than other species within the group that lack the deletion. In this way, S. haematobium may be differentiated from other species within the terminal spined egg group. S. haematobium strains representing a wide geographical distribution and with dependence on different snail hosts have all been characterised by this deletion. It should be possible, therefore, to utilise this character for the identification of S. haematobium in areas of Africa where this human pathogen overlaps with other schistosome species. Where several isolates of each species have been analysed, no variation in the major bands of hybridisation has been observed. For some of the species very few isolates have been examined and the possibility of strain specific variation between the major bands of hybridisation cannot be ruled out. This is unlikely, however, as where many strains and cloned organisms have been analysed, as for S. mansoni [2] and S. haematobium, no major band variation has been observed. McCutchan et al. [2] demonstrated polymorphism in low copy number r R N A genes of S. mansoni and successfully utilised variation in the resulting minor bands of hybridisation for discrimination of a large number of S. mansoni strains. Preliminary evidence suggests that minor bands may differ between strains of species within the terminal, spined egg group and further experimentation is in progress to confirm these observations. Similarly, such variation may prove to be of use for discriminating between S. bovis, S. curassoni and S. intercalatum. No attempts were made to clone
any strains from eggs or to obtain D N A from single worms. Since D N A from groups of worms was used for each analysis it is not known whether the minor band heterogeneity observed in many of the digests is due to differences within or between individual worms. Analysis of individual S. mansoni worms [2] has demonstrated variation between individuals which collectively produce the minor banding pattern from the D N A of the entire worm population. No differences in the degree of complexity of the overall hybridisation patterns have been observed between parasites passaged a varying number of times through laboratory animals, suggesting consistency of the r R N A gene. The major band hybridisation patterns obtained from parasites passaged through different laboratory hosts are identical, suggesting no effects arise from host D N A contamination.
Acknowledgements We are grateful to the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases for financial assistance. We are indebted to the many people who have helped in supplying schistosome isolates, especially Dr. F. Frandsen, Dr. C.H.J. Schutte, Dr. T. Jackson, Dr. C. Teesdale, Professor J. Vercruysse, Dr. C. Betterton, Dr. P. Hagan and Professor G.W. Howard. We would like to thank R.J. Knowles, M.A. Anderson and G.J. Johnson for technical assistance.
References 1 Rollinson, D. (1984) Recent advances in the characterisation of schistosomes and their intermediate hosts. Trop. Dis. Res. Ser. 5,401-441. 2 McCutchan, T.F., Simpson, A.J.G., Mullins, J.A., Sher, A., Nash, T.E., Lewis, F. and Richards, C. (1984) Differentiation of schistosomes by species, strain and sex by using cloned DNA markers. Proc. Natl. Acad. Sci. U.S.A. 81,889-893. 3 Simpson, A.J.G. and McCutchan, T.F. (1984) The use of cloned DNA to distinguish strains and species of schistosome. Trop. Dis. Res. Ser. 5,442-451. 4 McManus, D.P. and Simpson, A.J.G. (1985) Identification of the Echinococctts (hydatid disease) organisms using cloned DNA markers. Mol. Biochem. Parasitol. 17, 171-178.
131 5 McReynolds, L.A., De Simone, S. and Williams, S. (1986) Cloning and comparison of repeated DNA sequences from the human filarial parasite Brugia malayi and the animal parasite Brugia pahangi. Proc. Natl. Acad. Sci. U.S.A. 83, 797-801. 6 Rollinson, D., Walker, T.K. and Simpson, A.J.G. (1986) The application of recombinant DNA technology to problems of helminth identification. Parasitology 91,553-571. 7 Long, E.O. and Dawid, I.B. (1980) Repeated genes in eukaryotes. Annu. Rev. Biochem. 49, 727-764. 8 Fedoroff, N.V. (1979) On Spacers (Review). Cell 16, 697-710. 9 Simpson, A.J.G., Sher, A. and McCutchan, T.F. (1982) The genome of Schistosoma mansoni: Isolation of DNA, its size, bases and repetitive sequences. Mol. Biochem. Parasitol. 6, 125-137. 10 Keulen, H.V., LoVerde, P.T., Bobek, L.A. and Rekosh, D.M. (1985) Organisation of the ribosomal RNA genes in Schistosoma rnansoni. Mol. Biochem. Parasitol. 15, 215-230. 11 Simpson, A.J.G., Dame, J.B., Lewis, F.A. and McCutchan, T.F. (1984) The arrangement of ribosomal RNA genes in Schistosorna mansoni. Identification of polymorphic structural variants. Eur. J. Biochem. 139, 41-45. 12 Rigby, P.W.J., Dieckman, M., Rhodes, C. and Berg, P.
13
14
15
16
17
18
(1977) Labelling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J. Mol. Biol. 113,237-251. Southern, E.M. (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. ]. Mol. Biol. 98, 503-517. Denhardt, D.T. (1966) A membrane-filter technique for the detection of complementary DNA. Biochem. Biophys. Res. Commun. 23,641-646. Sinclair, J.H. and Brown, D.D. (1971) Retention of common nucleotide sequences in the ribosomal deoxyribonucleic acid of eukaryotes and some of their physical characteristics. Biochemistry 10 (14), 2761. Hori, H. and Osawa, S. (1979) Evolutionary change in the 5S RNA secondary structure and a phylogenic tree of 54 5S RNA species. Proc. Natl. Acad. Sci. U.S.A. 76, 381-385. Dover, G., Brown, S., Coen, E., Dallas, J., Strachan, T. and Trick, M. (1982) The dynamics of genome evolution and species differentiation. In: Genome Evolution (Dover, G.A. and Flavell, R.B., eds.), pp. 344-372, Academic Press, New York. Dover, G.A. and Flavell, R.B. (1984) Molecular coevolution: DNA divergence and the maintenance of function. Cell 38,622-623.
123
MBP 00683
Differentiation of Schistosoma haematobium from related species using cloned ribosomal R N A gene probes Tina K. Walker 1, David Rollinson 1 and Andrew J.G. Simpson 2 lDepartment of Zoology, British Museum (Natural History), Cromwell Road, London SW7 5BD and 2parasitology Division, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, U.K. (Received 14 January 1986; accepted 17 March 1986)
The ribosomal RNA (rRNA) gene units of Schistosoma mansoni (lateral spined eggs) and six species of schistosomes with terminal spined eggs (S. haematobium, S. curassoni, S. bovis, S. intercalatum, S. margrebowiei and S. mattheei) have been studied. The schistosome rRNA gene unit consists of a regular interspersion of the two genes encoding the large and small rRNA units with two spacers. The large spacer is not transcribed while the small spacer is part of the transcription unit. Variation in the rRNA gene unit of the species studied is demonstrated and takes three forms: First, variation in DNA sequence leads to both reduced homology in the spacer regions between species and loss or gain of restriction sites. Second, variation in the length of the transcribed spacer is demonstrated and DNA insertions of 0.2 kilobases (kb) and 0.1 kb are observed in S. mattheei and S. margrebowiei, respectively. Third, the length of the non-transcribed spacer region varies between species. S. haematobium has a 0.5 kb deletion in this region, while that of S. margrebowiei contains varying numbers of a 0.4 kb DNA insert. These interspecific variations have been shown to be conserved within species. Analysis of the rRNA genes by DNA hybridisation techniques therefore serves as a means of species identification, whereby it is possible to differentiate S. haematobium from other schistosome species with terminal spined eggs. Similarly, S. margrebowiei and S. mattheei may be clearly distinguished, although no major variation has been detected between S. curassoni, S. bovis and S. intercalatum. All these species differ from S. mansoni by the absence of certain restriction sites in the non-transcribed spacer. Key words: Schistosomes; Ribosomal RNA gene; Species differentiation
Introduction Schistosomes are parasitic helminths responsib l e for t h e d e b i l i t a t i n g t r o p i c a l d i s e a s e k n o w n as Bilharzia or Schistosomiasis. Of the eighteen or so species of Schistosoma currently recognised five a r e i m p o r t a n t p a r a s i t e s o f m a n : S. mansoni, S. h a e m a t o b i u m , S. intercalatum, S. j a p o n i c u m a n d S. m e k o n g i , w h e r e a s o t h e r s p e c i e s c a u s e d i s e a s e in wild a n d d o m e s t i c a n i m a l s . It is c o m m o n p r a c tice to arrange the species into three groups which reflect b o t h t h e m o r p h o l o g y o f t h e egg a n d t h e i n t e r m e d i a t e h o s t r e s p o n s i b l e for t h e i r t r a n s m i s sion; t h o s e w i t h a l a t e r a l s p i n e to t h e egg as S. mansoni, t h o s e w i t h a t e r m i n a l s p i n e to t h e egg
Abbreviations: cpm, counts per minute; EDTA, ethylenediaminetetraacetic acid; kb, kilobases; rRNA, ribosomal RNA; SDS, sodium dodecyl sulphate; SSC, standard saline citrate.
as S. h a e m a t o b i u m a n d t h o s e with r o u n d minu t e l y s p i n e d eggs as S. ]aponicum. A v a r i e t y o f a n a t o m i c a l , d e v e l o p m e n t a l a n d h o s t - r a n g e characteristics a r e u s e d for t h e i d e n t i f i c a t i o n o f species. I n a d d i t i o n , e l e c t r o p h o r e t i c studies h a v e c o m p l e m e n t e d t r a d i t i o n a l a p p r o a c h e s to c h a r a c t e r i s a t i o n a n d h a v e s o u g h t to p r o v i d e a l t e r n a t i v e m e t h o d s for i n v e s t i g a t i n g intraspecific v a r i a t i o n [1]. M o r e recently, the use of cloned D N A p r o b e s to distinguish strains o f S. m a n s o n i has b e e n rep o r t e d [2] a n d it h a s b e e n s h o w n t h a t v a r i a t i o n o f t h e r R N A g e n e f a m i l y o f s c h i s t o s o m e s can b e used to differentiate S. mansoni, S. japonicum and S. haematobium [3]. Similar techniques have b e e n u t i l i s e d for t h e i d e n t i f i c a t i o n o f s e v e r a l o t h e r helminths. F o r example, Echinococcus [4], Brugia [5] a n d Trichinella ( A . E . C h a m b e r s , m a n u s c r i p t s u b m i t t e d ) , r e v i e w e d b y R o l l i n s o n et al. [6]. R i b o s o m a l R N A ( r R N A ) g e n e o r g a n i s a t i o n is
124 very similar in all eukaryotes [7]. A tandemly repeating unit consisting of the following sequences occurs m a n y times throughout the genome: 5'non-transcribed spacer, external transcribed spacer, small r R N A gene (18S), 5.8S ribosomal R N A gene flanked by transcribed spacers, large ribosomal R N A gene (28S)-3'. The main size variation of this repeating unit observed between organisms is usually due to the presence of long or short non-transcribed spacer D N A . The spacer D N A within an organism m a y be either homogeneous (yeast) or heterogeneous ( X e n o p u s )
analysis may be utilised to identify both schistosome species as well as strains of S. mansoni [2]. This p a p e r presents restriction maps of the r R N A genes of various schistosome species of the terminal spined egg group from Africa, including S. haematobium, S. rnattheei, S. margrebowiei, S. bovis, S. curassoni and S. intercalatum and compares them with that of S. mansoni. Variation in the transcribed and non-transcribed spacer regions of the r R N A gene has been identified and the possibility of utilising these genetic variations to differentiate species has been explored.
[7,8]. The r R N A genes of S. m a n s o n i have been assigned to a unit of similar organisation although the presence of a gene corresponding to 5.8S R N A has not been confirmed. The unit, approximately 10 kilobases (kb) in length, is estimated to be repeated tandemly some 100 times throughout the g e n o m e [9,10]. B a m H 1 fragments containing the r R N A genes of S. m a n s o n i have b e e n cloned [11] and used as probes for mapping the corresponding genes of various schistosome strains and species. This previous work d e m o n s t r a t e d that direct genome
Materials and Methods Parasites. Schistosome species were collected from a n u m b e r of geographical locations to ensure that results were consistent between isolates. During the laboratory passaging procedure no attempt was made to clone strains from individual eggs. In each case, the parasite material used for D N A extraction was a group of worms originating from a single geographic location. The origin and the number of laboratory passages performed for each species yet analysed is presented in Table I.
TABLE I Origin of schistosome isolates analysed to date and the number of laboratory passages performed Species
Origin
Number laboratory passages
Figure number in which isolates are shown
S. rodhaini S. mansoni
Burundi Laboratory NIMR Puerto Rico Senegal Senegal Tanzania Senegal Senegal Kenya Gambia Sudan Durban, S. Africa Senegal Nigeria Zambia S. Africa Malawi Zambia Cameroun Zaire Gabon
4th Numerous
4 1, 3, 4, 6
Collected from sheep 3rd passage Unknown 4th passage Collected from cattle Unknown 1st passage 1st passage 1st passage 1st passage 1st passage 1st passage 40th passage 1st passage 23rd passage Unknown Unknown Unknown
1, 3, 4, 6 6 1, 3, 4 3 6 1, 4 1, 3, 4, 6 1, 3, 4, 6 1, 3, 4, 6
S. curassoni S. bovis S. haematobium
S. mattheei S. margrebowiei S. intercalatum
125 Approximately 50--100 adult schistosome worms were gently homogenised in a small volume (100 ~1/50 worms) of extraction buffer (50 mM Tris-HCl, 50 mM ethylenediaminetetraacetic acid (EDTA), 100 mM NaC1, pH 8). An equal volume of extraction buffer containing 1% sodium dodecyl sulphate (SDS) was added, together with proteinase K to a final concentration of 1%, and the mixture incubated at 37°C for 2-3 h. DNA was extracted three times with equal volumes of phenol, phenol/chloroform (1:1) and chloroform. Finally, the DNA was precipitated by the addition of 2.5 volumes of absolute redistilled ethanol. DNA was washed in 70% ethanol, vacuum dried, redissolved in Tris-EDTA buffer (pH 7.4) and stored at -70°C.
Preparation o f D N A .
kb
Filter hybridisation. Genomic schistosome DNA was digested with restriction endonucleases obtained from either Boehringer or BRL and utilised according to the supplier's recommendations. Restriction fragments were separated by 0.6% agarose flat-bed electrophoresis and transferred to nitrocellulose filters according to the technique of Southern [13]. Filters were prehybridised in 4x Denhardt's solution [14]; 4× standard saline citrate (SSC) and 0.1% SDS at 65°C for 4-5 h. At least 107 cpm of DNA probe was denatured by boiling for 5 min with sheared calf thymus DNA and hybridised overnight in 30 ml 4x Denhardt's solution; 4x SSC and 0.1% SDS at 65°C with the nitrocellulose filters. Hybridised filters were washed in 0.1x SSC and 0.05% SDS at 52°C for 3-4 h with at least 3 buffer changes. Finally, the filters were dried in air and exposed for autoradiography using flashed Fuji Xray film backed by a 'Cronex' intensifying screen.
5-0
Results
3-2
D e m o n s t r a t i o n o f variation in the r R N A g e n e o f various s c h i s t o s o m e species. Adult worm genomic
EcoR1 Digest
kb 23-1-
9'4-6"74-4-
-
-4.5 -
R a d i o l a b e l l i n g o f D N A . DNA probes pSM890, pSM889 and pSM389 are BamH1 fragments of the rRNA gene of S. m a n s o n i as prepared by Simpson et al. [11]. DNA was labelled with [32p]dCTP by Nick translation [12] using the kit supplied by BRL and according to their instructions. Incorporation was generally to an activity of 1 × 107 to 2 × 107 cpm i~g-1.
2'3-2-0-
-1"6
Fig. 1. Autoradiograph of the hybridisationof 32p-labeUed pSM889with EcoR1 digestedgenomicDNA fromS. mansoni and six species of the terminal spirted egg group. Band sizes are marked, int = S. intercalatum, mat = S. mattheei, marg = S. margrebowiei, haem = S. haematobium, bov = S. bovis, cura = S. curassoni, man = S. mansoni.
DNA was Southern blotted and hybridised with radiolabelled rRNA gene probes. An example of the results obtained by this method is shown in Fig. 1, where the DNA from several terminal spined egg group species has been digested with EcoRI and hybridised with the radiolabelled probe pSM889. Variations in the rRNA gene of the various schistosomes are identified. For each species, three major bands, or groups of bands are produced, of which the large and small bands vary between species, while the middle band remains constant. Such results suggest that the variation observed may be confined to particular regions of the rDNA. Probes pSM389, pSM890 and pSM889 were
126
HindlII and Xbal hybridised with pSM889 (Fig. 3). Five rRNA gene fragments possessing the ability to hybridise with pSM889 were generated (see Fig. 2). Two of these fragments were of equal size and electrophoresed as one band, while a 0.4 kb fragment is not shown. Hence, hybridisation with probe pSM889 generates three bands, of which the larger two bands represent regions of the rRNA gene encoding the large and small rRNA subunits. The smallest band (see arrow) encompasses the transcribed spacer which is 0.75 kb for S. haematobium, S. bovis, S. curassoni and S. mansoni, but 0.85 kb for S. margrebowiei and 0.95 kb for S. mattheei. Double digests using restriction enzymes EcoR1
each hybridised with adult schistosome DNA cut by several restriction enzymes in turn. Comparison of the banding patterns obtained with the restriction map already constructed for S. mansoni rDNA [11] allowed restriction maps for each of the terminal spined egg group schistosomes to be constructed, Fig. 2. Some details of the mapping procedure are described below. Confirmation that interspecific variation occurs in the spacer regions. The areas of the rRNA gene unit of most interest are those exhibiting interspecific variation. The variable regions of the rRNA gene were identified using double digests of genomic D N A using restriction enzymes SPACER S.MANSONI
•
pSM889
E
P
REGION
AND
VARIATION
TERMINAL
4.4kb
SPINED
:,,
IN T H E EGG
rRNA
GROUP
pSM890 2.4kb
E
GENE
OF
SCHISTOSOMES
:,,
pSM389
3.1kb
E H I
B S.mansoni I
Transcribed Spacer
B
X I
H I
Non-transcribed Spacer
Large rRNA
P B I I
Small rRNA ,(
S.bovis c~'~c~ag-s . o ni
~H
P b
H
P I
S.haematobium
~H
PI
S.margrebowiei
E I H
P I
S.intercalatum
S.matthQei
E q
I
I
kb
0
1 ,
2 ,
3 ,
4 ,
5 ,
6 ,
7 ,
8 ,
9 h
10 ,
Fig. 2. Restriction map of schistosome rRNA gene units. Regions of the rRNA gene unit of schistosomes within the terminal spined egg group that differ from that of S. m a n s o n i are presented. Homologous regions are omitted. E=EcoR1; H=HindlII; P=Pstl; X = X b a l ; B=BamH1.
127 Hind m / x b a
1 Digest
t
3
3
Kb
2 a. , -
9.4kb
6.74.4
sisting of several bands differing in length by 0.4 kb. Marked differences in the degree of binding were observed between the larger fragments, containing the non-transcribed spacer for each species, analysed. The intensity of the larger band is much greater for S. m a n s o n i and S. r o d h a i n i (lateral spined egg group species) than for the terminal spined egg group schistosomes. Intensity variation of the 2.0 kb band was due to differing amounts of D N A loaded on the gel. The two bands were analysed on a Joyce-Loebl chromoscan 3, and the ratio of the intensity of the upper band to the lower band was plotted for each species (Fig. 5). This ratio was greater than 2.5 for S. m a n s o n i and S. r o d h a i n i , while for species
Xba I / E c o R 1
2.3 2.0
Digest
:3"
3
- 0 95 - 0 . 8• 5
~1~
'~g
-0.75
and X b a l were also carried out for several schistosome species which demonstrated differences in length of the non-transcribed spacer (Fig. 4). Hybridisation with p r o b e pSM389 generated two bands. A band of 2.0 kb is consistent in each species analysed and represents the part of the r R N A gene encoding the small r R N A unit. The second band encompasses the whole non-transcribed spacer region and 0.5 kb of the region encoding the large r R N A unit. For S. i n t e r c a l a t u m , S. m a t theei, S. b o v i s , S. c u r a s s o n i and S. m a n s o n i , this second band is 3.5 kb while for S. h a e m a t o b i u m it is 3.0 kb in length. S. m a r g r e b o w i e i produces the characteristic multiple banding pattern con-
M
Kb
0.6-
Fig. 3. Autoradiograph of the hybridisation of 32p-labelled pSM889 with HindIII/Xbal digested genomic DNA from S. mansoni and six species of the terminal spined egg group. The size and position of bands representing DNA fragments containing the transcribed spacer region of the rRNA gene unit are marked, int = S. intercalatum; mat = S. mattheei; mar = S. margrebowiei; haem = S. haematobium; bov = S. bovis; cur = S. curassoni; man = S. mansoni.
'¢
23.1-
P •
9.4-
kb
6.74.4-3.0 2.3-
2 . 0 - ~D
I
-2.0
Fig. 4. Autoradiograph of hybridisati0n of 32P-labelled pSM389 with EcoR1/Xbal digested genomic DNA from S. mansoni and S. rodhaini (lateral spined eggs) and six species of the terminal spined egg group, int = S. intercalatum; mat = S. mattheei; mar = S. margrebowiei; haem =S. haematobium; bov= S. bovis; cur = S. curassoni; man = S. mansoni; rod = S. rodhaini. Band sizes are marked.
128
of the terminal spined egg group it was less than 1.0.
Differentiation of schistosome species by hybridisation of digested genomic DNA with rRNA gene probes. Hybridisation patterns generated by restriction digests and hybridisation with rRNA gene probes may be used to differentiate schistosome species. Of particular use are the EcoR1 digests hybridised with pSM889 (Fig. 1). Alternatively, the species can be distinguished by a restriction enzyme/probe combination generating a single, variable band of hybridisation (Fig. 6). BamH1 digestion of genomic DNA and its sub-
sequent hybridisation with radiolabelled probe pSM890 produces a major band of 2.4 kb with S. mansoni and major bands of between 5.0 and 5.5 kb with the schistosomes of the terminal spined egg group. This hybridisation pattern again illustrates the non-transcribed spacer deletion of S. haematobium and the characteristic multiple banding pattern of S. margrebowiei. To date, no variation within the rRNA genes has been found between S. intercalatum, S. bovis and S. curassoni. To demonstrate that the hybridisation patterns are consistent within each species, several isolates of each terminal spined egg group species
RATIO
B a m H1 D i g e s t
3.0
3
o
er
ID
C
0
Kb
~
3
3 _
~
~D
....
23.1-
2.0
kb .4
.
.
.
.
6.7-5.5 -5.0
i,~gI 4.4-
• w
1.0
-2.4 2.3-
2.0-
> Z
0 0
c
=0
0 <
> m 3:
•
•
=o
Fig. 5. Histogram representing relative intensities of the upper and lower bands in Fig. 4. The ratio of the intensity of the upper band to the lower band was calculated for each species. M A N = S. m a n s o n i ; R O D = S. r o d h a i n i ; C U R = S. c u r a s s o n i ; B O V = S. b o v i s ; H A E M = S. h a e m a t o b i u m ; M A R = S. m a r g r e b o w i e i ; M A T = S. rnattheei; I N T = S. i n t e r c a l a t u m .
Fig. 6. Autoradiograph of the hybridisation of 3Zp-labelled pSM890 with B a m H 1 digested genomic D N A from S. m a n s o n i and six species within the terminal spined egg group. Band sizes are marked, m a n = S. r n a n s o n i ; cur = S. c u r a s s o n i ; bov = S. b o v i s ; h a e m = S. h a e m a t o b i u m ; m a r = S. m a r g r e b o w i e i ; mat = S. m a n h e e i ; int = S. i n t e r c a l a t u m .
129 have been analysed. The figure in which each isolate has been shown is presented in Table I. Some experiments are not shown; however, no differences in any of the major bands of hybridisation have been observed within a species. In each case, the D N A was extracted from a group of worms originating from one geographical location, which further emphasises the lack of variability. Several of the schistosomes examined were isolated from, or passaged through, different hosts and host DNA contamination has not been shown to affect hybridisation patterns. Discussion
Restriction maps have been constructed of the rRNA genes of S. mansoni and various terminal spined egg group schistosomes including S. haematobium, S. bovis, S. curassoni, S. margrebowiei, S. mattheei and S. intercalatum. These restriction maps are summarised in Fig. 2. The rRNA genes of the terminal spined egg group of schistosomes differ from that of S. mansoni in three ways. First, changes in the length of the transcribed spacers occur. This was verified by a double digest using the HindIII/Xbal restriction enzyme combination (Fig. 3) demonstrating DNA insertions of 0.1 and 0.2 kb in the transcribed spacers of S. rnargrebowiei and S. mattheei, respectively. The second difference that occurs between the rRNA gene of S. mansoni and that of the terminal spined egg group of schistosomes is variation in the length of the non-transcribed spacer as was demonstrated by an EcoR1/Xbal double digest. This cuts out the non-transcribed spacer region with only 0.5 kb of the adjacent 5' D N A sequence (Fig. 4) and demonstrates that the nontranscribed spacer of S. haematobiurn has a 0.5 kb deletion, while that of S. margrebowiei hybridises to form several bands each differing in length by 0.4 kb. Similar analysis of individual S. margrebowiei worms should be carried out, in order to determine how the non-transcribed spacer varies between, and possibly within, individuals. This experiment demonstrates the non-transcribed spacer to be of identical length for S. intercalatum, S. mattheei, S. bovis, S. curassoni, S. rodhaini and
S. mansoni. The third way in which the rRNA genes of the terminal spined egg group of schistosomes differ from that of S. mansoni is by DNA sequence variation. Such variation may be demonstrated by the study of the overall homology between regions of DNA for various species. The intensity of binding and hence the homology that exists between Southern blotted DNA and a bound probe, is proportional to the intensity of the band on the autoradiograph. The binding intensity of the large bands in Fig. 4 which represent the region of the non-transcribed spacer, is much reduced with terminal spined egg group species compared with that of S. mansoni. The probe hybridised with this filter was pSM389, encompassing the non-transcribed spacer region of S. rnansoni from which it was derived. Therefore, homology between the non-transcribed spacer region of S. mansoni and of species within the terminal spined egg group is greatly reduced. S. rodhaini, which belongs to the lateral spined egg group along with S. mansoni, binds strongly with pSM389. The comparable intensities of the 2.0 kb band encompassing the small rRNA unit coding sequence, which is homologous between species, is consistent with the general finding that the 18S and 28S RNA gene sequences are highly conserved during animal evolution [15]. This is due to the necessity for maintaining functional ribosome units. The observation that the spacer regions exhibit interspecific heterology, while being homologous within species is also consistent with previous findings [16-18]. These observations imply that the spacer regions have evolved rapidly relative to the adjacent coding regions that give rise to the rRNAs [81. DNA sequence alterations may additionally lead to loss or gain of single restriction sites exemplified by a BamH1 digestion of the DNA from various schistosome species (Fig. 6). Hybridisation with pSM890 produces a band of 2.4 kb with S. mansoni, while bands approximately twice this size are produced with the terminal spined egg group schistosomes. The probes utilised for these studies were derived by BamH1 digestion of the S. mansoni rRNA gene unit and probes pSM890 and pSM389 represent adjacent regions 2.4 kb and 3.1 kb in length, respectively. Loss of the
130 BamH1 site separating these regions of the rDNA in the terminal spined egg group results in pSM890 hybridising with a band approximately 5.4 kb in length (equivalent to both pSM890 and pSM389 together). The differences in the r R N A gene presented in this report, as well as being of genetic and evolutionary interest in relation to schistosomes, could potentially also be a useful tool for their identification and differentiation. Major differences occur in the r R N A gene unit of S. haematobium, S. margrebowiei and S. mattheei. In particular, the 0.5 kb deletion in the non-transcribed spacer of S. haematobium means that any r R N A gene probe hydridising with a r D N A fragment encompassing this spacer will produce a band 0.5 kb smaller than other species within the group that lack the deletion. In this way, S. haematobium may be differentiated from other species within the terminal spined egg group. S. haematobium strains representing a wide geographical distribution and with dependence on different snail hosts have all been characterised by this deletion. It should be possible, therefore, to utilise this character for the identification of S. haematobium in areas of Africa where this human pathogen overlaps with other schistosome species. Where several isolates of each species have been analysed, no variation in the major bands of hybridisation has been observed. For some of the species very few isolates have been examined and the possibility of strain specific variation between the major bands of hybridisation cannot be ruled out. This is unlikely, however, as where many strains and cloned organisms have been analysed, as for S. mansoni [2] and S. haematobium, no major band variation has been observed. McCutchan et al. [2] demonstrated polymorphism in low copy number r R N A genes of S. mansoni and successfully utilised variation in the resulting minor bands of hybridisation for discrimination of a large number of S. mansoni strains. Preliminary evidence suggests that minor bands may differ between strains of species within the terminal, spined egg group and further experimentation is in progress to confirm these observations. Similarly, such variation may prove to be of use for discriminating between S. bovis, S. curassoni and S. intercalatum. No attempts were made to clone
any strains from eggs or to obtain D N A from single worms. Since D N A from groups of worms was used for each analysis it is not known whether the minor band heterogeneity observed in many of the digests is due to differences within or between individual worms. Analysis of individual S. mansoni worms [2] has demonstrated variation between individuals which collectively produce the minor banding pattern from the D N A of the entire worm population. No differences in the degree of complexity of the overall hybridisation patterns have been observed between parasites passaged a varying number of times through laboratory animals, suggesting consistency of the r R N A gene. The major band hybridisation patterns obtained from parasites passaged through different laboratory hosts are identical, suggesting no effects arise from host D N A contamination.
Acknowledgements We are grateful to the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases for financial assistance. We are indebted to the many people who have helped in supplying schistosome isolates, especially Dr. F. Frandsen, Dr. C.H.J. Schutte, Dr. T. Jackson, Dr. C. Teesdale, Professor J. Vercruysse, Dr. C. Betterton, Dr. P. Hagan and Professor G.W. Howard. We would like to thank R.J. Knowles, M.A. Anderson and G.J. Johnson for technical assistance.
References 1 Rollinson, D. (1984) Recent advances in the characterisation of schistosomes and their intermediate hosts. Trop. Dis. Res. Ser. 5,401-441. 2 McCutchan, T.F., Simpson, A.J.G., Mullins, J.A., Sher, A., Nash, T.E., Lewis, F. and Richards, C. (1984) Differentiation of schistosomes by species, strain and sex by using cloned DNA markers. Proc. Natl. Acad. Sci. U.S.A. 81,889-893. 3 Simpson, A.J.G. and McCutchan, T.F. (1984) The use of cloned DNA to distinguish strains and species of schistosome. Trop. Dis. Res. Ser. 5,442-451. 4 McManus, D.P. and Simpson, A.J.G. (1985) Identification of the Echinococctts (hydatid disease) organisms using cloned DNA markers. Mol. Biochem. Parasitol. 17, 171-178.
131 5 McReynolds, L.A., De Simone, S. and Williams, S. (1986) Cloning and comparison of repeated DNA sequences from the human filarial parasite Brugia malayi and the animal parasite Brugia pahangi. Proc. Natl. Acad. Sci. U.S.A. 83, 797-801. 6 Rollinson, D., Walker, T.K. and Simpson, A.J.G. (1986) The application of recombinant DNA technology to problems of helminth identification. Parasitology 91,553-571. 7 Long, E.O. and Dawid, I.B. (1980) Repeated genes in eukaryotes. Annu. Rev. Biochem. 49, 727-764. 8 Fedoroff, N.V. (1979) On Spacers (Review). Cell 16, 697-710. 9 Simpson, A.J.G., Sher, A. and McCutchan, T.F. (1982) The genome of Schistosoma mansoni: Isolation of DNA, its size, bases and repetitive sequences. Mol. Biochem. Parasitol. 6, 125-137. 10 Keulen, H.V., LoVerde, P.T., Bobek, L.A. and Rekosh, D.M. (1985) Organisation of the ribosomal RNA genes in Schistosoma rnansoni. Mol. Biochem. Parasitol. 15, 215-230. 11 Simpson, A.J.G., Dame, J.B., Lewis, F.A. and McCutchan, T.F. (1984) The arrangement of ribosomal RNA genes in Schistosorna mansoni. Identification of polymorphic structural variants. Eur. J. Biochem. 139, 41-45. 12 Rigby, P.W.J., Dieckman, M., Rhodes, C. and Berg, P.
13
14
15
16
17
18
(1977) Labelling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J. Mol. Biol. 113,237-251. Southern, E.M. (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. ]. Mol. Biol. 98, 503-517. Denhardt, D.T. (1966) A membrane-filter technique for the detection of complementary DNA. Biochem. Biophys. Res. Commun. 23,641-646. Sinclair, J.H. and Brown, D.D. (1971) Retention of common nucleotide sequences in the ribosomal deoxyribonucleic acid of eukaryotes and some of their physical characteristics. Biochemistry 10 (14), 2761. Hori, H. and Osawa, S. (1979) Evolutionary change in the 5S RNA secondary structure and a phylogenic tree of 54 5S RNA species. Proc. Natl. Acad. Sci. U.S.A. 76, 381-385. Dover, G., Brown, S., Coen, E., Dallas, J., Strachan, T. and Trick, M. (1982) The dynamics of genome evolution and species differentiation. In: Genome Evolution (Dover, G.A. and Flavell, R.B., eds.), pp. 344-372, Academic Press, New York. Dover, G.A. and Flavell, R.B. (1984) Molecular coevolution: DNA divergence and the maintenance of function. Cell 38,622-623.