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SOBOSLAY P.T.. DREWECK CM., TAYLOR HR., BROTMAN B . , WENK P .
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76, 23-24.
ISOLATION O F N E W M A R K E R S T O DETECT GENETIC VARIATION I N ONCHOCERCA VOLVULUS
HERDER S.*, BELLEC CH* AND CUNY G.**
KEYWORDS : Onchocerca volvulus, genetic variability. RAPD. microsatellite.
PCR.
SUMMARY
Newly developed techniques (RADP : Random Amplified Polymorphic DNA and microsatellite DNA sequences) were used to study genetic
* Laboratoire d'Epidemiologie de Maladies ä Vecteurs, Centre ORS- TOM d e M o n t p e l l i e r , 9 1 1 a v . A g r o p o l i s , BP 5 0 4 5 , 3 4 0 3 2 Montpellier, France.
** Laboratoire Retrovirus-Parasites, Centre ORSTOM de Montpellier, 911 av. Agropolis. B P 5045. 34032 Montpellier. Fiance.
variation in Onchocerca volvulus. RAPD technique, derived from the Polymerase Chain Reaction (PCR), allow clear distinction between the different species of the genus Onchocerca.
Microsatellite DNA has been shown to be useful as polymorphic mar
kers for populations and even for Individuals. Short repeated sequences (CA repeats) have been isolated, and a PCR assay using such microsatellite DNA sequences to generate polymorphisms Is cur
rently being experimented.
In W e s t A f r i c a , p a r t i c u l a r l y i n t h e O C P a r e a ( O n c h o c e r c i a s i s Control P r o g r a m m e ) , at least t w o strains o f the parasite a r e k n o w n t o exist. T h e s o - c a l l e d "savannah'' strain a s s o c i a t e d with high b l i n d n e s s level, a n d t h e "forest"
s t r a i n a s s o c i a t e d w i t h a m i l d e r f o r m o f o n c h o c e r c i a s i s w h i c h c a u s e s b l i n d n e s s in less than 1 % o f t h e population (Prost et al., 1 9 8 0 ) . H o w e v e r , Sierra L e o n e is e x c e p t i o n a l in t h a t h i g h l e v e l s o f b l i n d n e s s a r e f o u n d i n f o r e s t a r e a s ( M c M a h o n et al., 1 9 8 6 ) .
V a r i o u s Onchocerca specific DNA p r o b e s h a v e b e e n d e v e l o p e d (Meredith et al., 1 9 8 9 ) a n d also strain specific p r o b e s ( E r t t m a n n et al., 1 9 8 7 a n d 1 9 9 0 ) . T h e s e D N A p r o b e s s e q u e n c e s a r e b a s e d o n t h e 150 b p repeat family. Recently, s t u d i e s c o n d u c t e d in W e s t Africa h a v e s h o w n that DNA p r o b e c l a s s i f i c a t i o n c o r r e l a t e s with t h e e p i d e m i o l o g y o f b l i n d n e s s ( Z i m m e r m a n et al., 1 9 9 2 ) .
In C a m e r o o n , t h e situation is m o r e c o m p l e x , n o t a b l y t h e p a t h o l o g y o f b l i n d n e s s is distributed without apparent rela
tion t o b i o c l i m a t i c z o n e s ( D u k e , 1981 ; B o u s s i n e s q et al., 1 9 9 3 ) . T o date, n o studies similar t o t h o s e carried o u t in W e s t Africa h a v e b e e n d o n e in C a m e r o o n .
W e a r e investigating o t h e r m o l e c u l a r m a r k e r s that m a y b e u s e f u l f o r s t u d i e s o n g e n e t i c v a r i a t i o n in O. volvulus, n a m e l y RAPD a n d microsatellite s e q u e n c e s .
P o l y m o r p h i s m in g e n o m i c f i n g e r p r i n t s g e n e r a t e d b y R a n d o m Amplified P o l y m o r p h i c DNA ( R A P D ) is a recently d e v e l o p e d assay that is b a s e d o n t h e amplification b y t h e P o l y m e r a s e C h a i n R e a c t i o n ( P C R ) o f r a n d o m DNA frag
ments. In RAPD m a p p i n g , d e c a m e r o l i g o n u c l e o t i d e primers o f arbitrary' s e q u e n c e b u t with a G C c o n t e n t o f 5 0 % o r h i g h e r a r e u s e d t o a m p l i f y f r a g m e n t s o f g e n o m i c D N A (Williams etal. 1 9 9 0 ; W e l s h a n d McClelland, 1 9 9 0 ) . R A P D h a s b e e n s u c c e s s f u l l y a p p l i e d t o t h e a n a l y s i s o f g e n o m i c DNA variation o f several o r g a n i s m s (Williams et al, 1 9 9 0 ; Hadrys et al, 1 9 9 2 ; Klein-Lankhorst et al., 1991 ; Crowhurst et al.. 1991 ; Mazurier et al.. 1 9 9 2 ) . Strains c a n b e distinguished b y c o m p a r i n g p o l y m o r p h i s m s in g e n o m i c fingerprints ( W e l s h et al. 1 9 9 1 ) .
T h e particular a d v a n t a g e s o f RAPD t e c h n o l o g y is that small a m o u n t s ( n a n o g r a m s ) o f g e n o m i c DNA a r e n e e d e d a n d , u n l i k e PCR, n o prior s e q u e n c e k n o w l e d g e is n e e d e d . Microsatellite DNA s e q u e n c e s are short (from 2 t o 5 b p ) , tan- demly repeated s e q u e n c e s that have b e e n s h o w n t o b e use
ful a s p o l y m o r p h i c m a r k e r s for p o p u l a t i o n s a n d e v e n for individuals. T h e y have b e e n reported from a large panel o f eukaryotic s p e c i e s such as human, m o u s e , cattle, w h a l e s a n d insects (Tautz, 1 9 8 9 ; Love etal. 1 9 9 0 ; Swaiger etal, 1 9 9 2 ; W e b e r a n d May, 1 9 8 9 ; H u g h e s a n d Queller, 1 9 9 3 ) . T h e s e d i n u c l e o t i d e r e p e a t s a r e referred t o as microsatellites (Litt and Luty, 1 9 8 9 ) . Most o f t h e s e regions are less than 2 0 0 b p in length a n d PCR is used t o detect length polymorphisms.
W e d e s c r i b e h e r e t h e u s e o f t h e RAPD assay for t h e d e t e c tion o f variation in t h e g e n u s Onchocerca, using a set o f 2 0
Parasite, 1994, 7, I S 55
Article available athttp://www.parasite-journal.orgorhttp://dx.doi.org/10.1051/parasite/199401s1055
PARASITE B I O L O G Y AND BIOCHEMISTRY
o l i g o n u c l e o t i d e d e c a m e r p r i m e r s a n d t h e a d a p t a t i o n o f microsatellite DNA s e q u e n c e s t o study g e n e t i c variability in O. volvulus p o p u l a t i o n s .
METHODS
R A P D
N
odules containing O. volvulus w e r e e x c i s e d and c o l l a g e - nase-digested ( S c h u l z - K e y et ai, 1 9 7 7 ) a n d t h e w o r m s preserved in absolute a l c o h o l . T h e nodules w e r e from savannah a n d d e g r a d e d forest areas o f C a m e r o o n ( T o u b o r o , Poli a n d Bafia, S a ' a ) a n d from forest r e g i o n s o f Sierra L e o n e (Lunsar and Njala).
O. gutturosa, O. lienalis, O. gibsoni a n d O. ochengi DNA w a s p r o v i d e d b y Dr. Meredith. O. volvulus DNA w a s e x t r a c ted from w o r m s a s d e s c r i b e d b y Meredith et al. ( 1 9 9 1 ) . PCR r e a c t i o n s w e r e p e r f o r m e d in 2 5 ul o f lOmM T r i s - H C l ( p H 9 . 0 ) , 5 0 niM KC1, 0.1 % Triton X - 1 0 0 , 1.5 m M M g C l2, 0 . 2 m M o f e a c h d N T P , 2 0 p i c o m o l e s o f d e c a m e r p r i m e r ( s ) ( O p e r o n T e c h n o l o g i e s I n c . ) , 2 5 n g t e m p l a t e DNA a n d 1 unit T a q p o l y m e r a s e ( P r o m e g a ) . Amplification w a s carried out in a t h e r m o c y c l e r ( T e c h n e P H C - 3 ) p r o g r a m m e d for 4 5 c y c l e s o f 1 m i n u t e at 9 2 ° C, 1 m i n u t e at 3 6 ° C a n d 2 m i n u t e s at 7 2 ° C. A m p l i f i c a t i o n p r o d u c t s w e r e r e s o l v e d e l e c t r o p h o r e t i c a l l y o n 1.4 % a g a r o s e gel.
MICROSATELLITE D N A SEQUENCES
Nodules c o n t a i n i n g O. volvulus w o r m s from 14 patients ori
ginating from 4 different foci in C a m e r o o n w e r e e x c i s e d and c o l l a g e n a s e digested in t h e field. Total g e n o m i c DNA from the w o r m s w a s digested t o c o m p l e t i o n with t h e restriction e n z y m e s HaelW, Alul, Taql, Hinfi a n d Sauik a n d shotgun c l o n e d u n d e r standard c o n d i t i o n s into a M 1 3 B M 2 0 v e c t o r c o n t a i n i n g an EcdRV site ( H a n a h a n , 1 9 8 3 ) . P o l y ( C A )n/ ( G T )n and p o l y ( G A )n/ ( C T )n p r o b e s for d e t e c t i o n o f r e s p e c t i v e l y ( C A )n/ ( G T )n a n d ( G A )n/ ( C T )n m i c r o s a t e l l i t e s s e q u e n c e s w e r e l a b e l l e d b y r a n d o m p r i m i n g in t h e p r e s e n c e o f [oc- 3 2 P ] d C T P ( F e i n b e r g a n d V o g e l s t e i n , 1 9 8 3 ) . T h e g e n o m i c M 1 3 library w a s s c r e e n e d b y transfering t h e p l a q u e s o n t o nylon m e m b r a n e s . Prehybridization a n d hybridization w e r e p e r f o r m e d at 6 5 ° C in a solution c o n t a i n i n g 0 . 5 % nonfat p o w d e r e d milk, 5 X SSPE, 1 % S D S , 100 u g / m l d e n a t u r e d s a l m o n s p e r m DNA, f o l l o w e d b y t w o w a s h e s o f 15 minutes e a c h in 2 X SSPE, 0.1 % S D S , o n e o f 15 minutes in 0 . 5 X SSPE, 0.1 % S D S and t h e stringent w a s h o f 15 minutes in 0.1 X SSPE, 0.1 % SDS at 6 5 ° C. Dried filters w e r e then autora- d i o g r a p h e d w i t h a n i n t e n s i f y i n g s c r e e n o n H y p e r f i l m ( A m e r s h a m ) at - 7 0 ° C for 2 t o 16 h o u r s . S i n g l e - s t r a n d e d template DNA w a s isolated from positive p l a q u e s (Messing, 1 9 8 3 ) a n d s e q u e n c e d ( S a n g e r , 1 9 7 7 ) o n a n a u t o m a t i c s e q u e n c e r (Applied B i o s y s t e m s , I n c . ) .
PCR primers w e r e c h o s e n from t h e r e g i o n immediatly flan
king t h e microsatellite s e q u e n c e . T h e s e primers w e r e desi
g n e d with c o m p u t e r a s s i s t a n c e t o m i n i m i z e self annealing.
P C R c o n d i t i o n s w e r e o p t i m i z e d b y c o m b i n i n g d i f f e r e n t M g 2 + c o n c e n t r a t i o n s , a n n e a l i n g t e m p e r a t u r e s a n d n u m b e r o f a m p l i f i c a t i o n c y c l e s . P C R p r o d u c t s w e r e r e s o l v e d o n n o n - d e n a t u r i n g a c r y l a m i d e g e l s (from 7 t o 10 % ) a n d visua
l i s e d b y e t h i d i u m b r o m i d e ( 1 m g / m l ) o r s i l v e r s t a i n i n g
( 0 . 2 % s i l v e r n i t r a t e for 3 0 m i n u t e s a n d r e d u c t i o n w i t h 0 . 7 5 M N a O H ; 0.1M f o r m a l d e h y d e for 10 m i n u t e s ) .
RESULTS AND DISCUSSION
1
- ; irst, w e investigated t h e potential o f RAPD in differentiating at t h e s p e c i e s level. G e n o m i c DNA from different s p e c i e s w a s amplified b y u s e o f a single d e c a m e r primer, 2 0 primers w e r e tested in t h e s e e x p e r i m e n t s a n d in e a c h c a s e , at l e a s t o n e s p e c i f i c f r a g m e n t w a s o b t a i n e d f o r t h e
Onchocerca D N A ' s u s e d . F o r e a c h s a m p l e , s o m e o f t h e bands (ranging from 1.5 K b and 1 5 0 b p ) a r e c o m m o n for the g e n u s Onchocerca a n d s o m e a r e u n i q u e t o o n e s p e c i e s d e p e n d i n g o n t h e primer u s e d . B y c o m p a r i n g fingerprints g e n e r a t e d b y t h e RAPD m e t h o d . Onchocerca volvulus, O.
gutturosa, O. lienalis, O. gibsoni and O. ochengi c a n b e iden
tified a n d distinguished from e a c h o t h e r b y the specific poly
m o r p h i c patterns.
S e c o n d l y , RAPD profiles o f different O. volvulus i s o l a t e s h a v e b e e n c o m p a r e d using t h e s a m e primer a s d e s c r i b e d earlier. Different p a t t e r n s a r e o b s e r v e d b e t w e e n different isolates a n d e v e n b e t w e e n w o r m s from t h e s a m e n o d u l e . Using t h e s e primers, w e c o u l d o b s e r v e differences b e t w e e n s p e c i e s a n d a l s o b e t w e e n O. volvulus i s o l a t e s . H o w e v e r w h e n amplification is r e p e a t e d , w e often find t h e profiles to vary slightly. F u r t h e r m o r e , t h e p r e s e n c e o f DNA c o n t a m i nation c a n s e r i o u s l y affect t h e results. T h u s , in this c a s e , this t e c h n i q u e s e e m s m o r e s u i t a b l e for i s o l a t i o n o f n e w DNA p r o b e s rather than for studies o n p o p u l a t i o n g e n e t i c . M i c r o s a t e l l i t e loci c a n b e e a s i l y s c o r e d u s i n g p o l y m e r a s e c h a i n reaction ( P C R ) f o l l o w e d b y e l e c t r o p h o r e s i s t o s e p a rate alleles w h i c h differ in length as a result o f differences in t h e n u m b e r o f r e p e a t units. T h e microsatellite s e q u e n c e s isolated from O. volvulus h a v e b e e n u s e d t o m a k e primers (microsatellite flanking r e g i o n s ) t o identify different strains o f t h e parasite. T h e s e particular s e q u e n c e s c o n s i s t o f CA r e p e a t s . Initial results using O. volvulus m i c r o s a t e l l i t e pri
m e r s s h o w s o n l y slight differences b e t w e e n isolates. T h i s l a c k o f r e s o l u t i o n o b s e r v e d b e t w e e n b a n d s r e p r e s e n t i n g a l l e l e s ( i n s o m e c a s e s s e p a r a t e d o n l y b y 2 b p ) c a n b e e x p l a i n e d b y t h e u s e o f n o n denaturing a c r y l a m i d e g e l t o visualize a m p l i f i c a t i o n p r o d u c t s . In o r d e r t o a l l o w b e t t e r resolution o f DNA fragments, this t e c h n i q u e c a n b e impro
v e d b y using denaturing s e q u e n c e a c r y l a m i d e g e l s .
In c o n c l u s i o n , w e b e l i e v e that t h e s e n e w t o o l s d e v e l o p e d (particularly m i c r o s a t e l l i t e s ) will b e useful for investigating the g e n e t i c variation in O. volvulus.
ACKNOWLEDGEMENTS
e a r e very grateful t o Dr. M. B o u s s i n e s q a n d Dr. J . P . W C h i p p a u x for providing us with material a n d e p i d e m i o l o g i c a l data. W e a l s o t h a n k s Dr. J . P . Aussel for e x c e l l e n t t e c h n i c a l assistance.
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PARASITE B I O L O G Y AND BIOCHEMISTRY
C U T I C L I N G E N E S O F N E M A T O D E S
LEWIS E.*, SEBASTIANO M.*, NOLA M.*, ZEI F.*, LASSANDRO F.*, RISTORATORE F.,* CERMOLA M.*, FAVRE R.* AND BAZZICALUPO P.*
KEYWORDS : parasitic nematodes, cuticle, cuticlin. dityrosine. cross-linking.
SUMMARY
Two genes coding for cuticlin components of Caenorhabditis elegans have been cloned and their structure is described. Recombinant pro
teins have been produced in E. coli and antibodies raised against them. Nucleic acid and specific antibodies are being used to isolate the homologues from the parasitic species Ascaris lumbricoides and Brugia pahangi.
T h e n e m a t o d e cuticle p r o t e c t s t h e animal, s e r v e s as an e x o s k e l e t o n a n d p r o v i d e s t h e surface o v e r w h i c h inter
a c t i o n s with t h e e x t e r n a l e n v i r o n m e n t o c c u r . In t h e c a s e o f parasitic n e m a t o d e s t h e e x t e r n a l e n v i r o n m e n t is t h e h o s t a n d , a s s u c h , a n t i g e n s e x p r e s s e d o n / i n t h e c u t i c l e a r e within r e a c h o f b o t h t h e humoral a n d cellular c o m p o n e n t s o f t h e i m m u n e system. T h e cuticle is a l a y e r e d structure, the c o m p o n e n t s o f w h i c h a r e classified a c c o r d i n g t o their solubility : lipids, s o m e p r o t e i n s a n d o t h e r readily s o l u b l e , non-structural c o m p o n e n t s a r e mostly l o c a l i z e d o n t h e sur
face b u t a r e a l s o distributed t o a lesser e x t e n t t h r o u g h o u t the l o w e r layers o f t h e cuticle ; t h e c o l l a g e n s m a k e u p t h e structural bulk o f t h e cuticle, a r e c o d e d for b y a large a n d relatively w e l l - c h a r a c t e r i z e d g e n e family, a r e n o t e x p o s e d o n t h e cuticle surface a n d c a n b e solubilized with S D S a n d m e r c a p t o e t h a n o l ; a n d finally there is a highly cross-linked, i n s o l u b l e a n d c o m p l e x mixture o f p r o t e i n s present throu
g h o u t t h e cuticle a n d k n o w n a s t h e cuticlins. U p until n o w it h a s b e e n virtually i m p o s s i b l e t o d e t e r m i n e t h e roles a n d i m p o r t a n c e o f t h e cuticlins b e c a u s e their insolubility d o e s not a l l o w either m o l e c u l a r o r b i o c h e m i c a l analysis o f indivi
dual proteins.
* International Institute of Genetics and Biophysics. CNR. via G.
Marconi 10, 80125 Napoli, Italy.
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