CROWHURST R.N., HAWTHORNE B . T . , RIKKERINK E . H . and TEMPEETON M.D. : Differentiation of Fusarium solanii. sp. Cucurbitae races 1 and 2 by random amplification o f polymorphic DNA. Current Genet., 1991. 20. 391-396.
DUKE B O X . : Geographical aspects o f onchocerciasis. Ann. Soc.
BelgeMed. Trop., 1981, 61, 179-186.
ERTTMANN K . D . , UNNASCH T.R., GREENE B.M., ALBIEZ E J . , BOATENG J . , DENKE A.M., FERRARONI J . J . , KARAM M., SCHULZ-KEY H . and WILLIAMS P.N. : A DNA sequence specific for forest form Onchocerca vol
vulus. Nature, 1987, 327, 415-417.
ERTTMANN K . D . , MEREDITH S.E.O., GREENE B.M. and UNNASCH T.R. : Isolation and characterisation of form specific DNA sequences of O. volvulus. Acta Leidensia. 1990, 1-2, 253-260.
FEINBERG A.P. and VOLGELSTEIN B . : A technique for radio labelling DNA restriction endonucleases fragments to high specific activity.
Anal. Biochem., 1983, 137, 6-13.
HADRYS H , BAUCK M. and SCHIERWATER B . : Applications of random amplified polymorphic DNA (RAPD) in molecular ecology. Mol.
Ecol, 1992, /, 55-63.
HANAHAN D . : Studies on transformation of Escherichia coli with Plasmids../. Mol. Biol.. 1983, 166, 557-580.
HUGUES C R . and QUELLER D.C. : Detection of highly polymorphic microsatellite loci in a species with little allozyme polymorphism.
Mol. Ecol. 1993. 2, 131-137.
KLEIN-LANKHORST R.M.. VERMUNT A., WEIDE R„ LIHARSKA T. and ZABEL P. : Isolation o f molecular markers for tomato (I. esculentum) using random amplified polymorphic DNA (RAPD). Theor. Appl.
Genet.. 1991, 83. 108-114.
LITT M. and LUTY J.A. : A hypervariable microsatellite revealed by in vitro amplification o f a dinucleotide repeat within the cardiac muscle actin gene. Am. J. Hum. Genet., 1989, 44, 397-401.
LOVE J.M., KNIGHT A.M., MCALEER M.A. and TODD J.A. : Towards construction o f a high resolution map o f the mouse genome using PCR-analysed microsatellites. Nucl. Ac. Res., 1990, 18, n° 14, 4123-4130.
MAZURIER S . , VAN DE GIESSEN A., HEUVELMAN K . and WERNARS K . : RAPD analysis of Campylobacter isolates : DNA fingerprinting without the need to purify DNA. Letters in Appl. Microbiol, 1992,
14, 260-262.
MCMAHON J . E . , DAMES J . В . , WHITE M.D., GODDARD J . M . , BEECH- GARWOOD P.A. and KIRKWOOD B.R. : O n c h o c e r c i a s i s in Sierra Leone. I. Studies on the prevalence and transmission at Gbaiima village. Trans. Roy. Soc. Trop. Med. Hyg., 1986, 80, 802-809.
MEREDITH S.E.O., UNNASCH T.R., KARAM M., PIESSENS W.F. and WIRTH D.F. : Cloning and characterisation of an Onchocerca volvulus specific DNA sequence. Mol. Biochem. Parasitol, 1989, 36, 1-10.
MEREDITH S.E.O., LANDO G., GBAKIMA A.A., ZIMMERMAN P.A. and UNNASCH T.R. : Onchocerca volvulus • application of polymerase chain reaction to identification and strain differentiation of the para
site. Exp. Parasitol. 1991. 73, 335-344.
MESSING J . : New M13 v e c t o r s for c l o n i n g . In M e t h o d s in Enzymology. Wu R., Grossman L. and Moldave K . (eds), New York. Academic Press, 1983. vol 101. 10-89.
PROST A., ROUGEMONT A. and OMAR M.S. : Caracteres E P I D E M I O L O -
giques, cliniques et biologiques des onchocercoses de savane et de forc't en Afrique occidentale. Ann. Parasitol. 1980, 55, 347- 355.
SANGER F . , NICKLEN S. and COULSON A.R. : DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Scl, 1977, 74.
5463-5467.
SCHULZ-KEY H . , ALBIEZ E.J., BUTTNER D.W. : Isolation of living adult Onchocerca volvulus from nodules. Tropenmed. Parasitol, 1977, 28, 428-430.
SWAIGER F.W., GOMOLKA M., GELDERMANN H . , ZISCHLER H . , BUITKAMP J., EPPI.EN J.T. and AMMER H . : Oligonucleotide fingerprinting to
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.
Parasite, 1994, J, I S
57individualize ungulates. Appl Theor. Electrophoresis, 1992, 2, 193- 200.
TAI TZ I). : Hypervariability of simple sequences as a general source for polymorphic DNA markers. Nucl. Ac. Res., 1989, 17, n° 16, 6463-6471.
WEBER J.L. and MAY P.E. : Abundant class of human DNA polymor
phisms which can be typed using the polymerase chain reaction.
Am. J. Hum. Genet., 1989, 44, 388-396.
WELSH J . and Mc CLELLAND M. : Fingerprinting genomes using PCR with arbitrary primers. Nucl. Ac. Res., 1990, 18, n° 24, 7213-7218.
WELSH J . , PETERSEN C. and Mc CLELLAND M. : Polymorphisms genera
ted by arbitrarily primed PCR in the mouse : application to strain identification and genetic mapping. Nucl. Ac. Res.. 1991. 19, n° 2, 303-306.
WILLIAMS J . G . K . , KUBELIK A.R., LIVAK K . J . , RAFALSKI J.A. and TINGEY S . V . : DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucl. Ac. Res., 1990, 19, n° 2, 303-306.
ZIMMERMAN P.A., DADZIE K . Y . , DE SOLE G., REMME J . , SOUMBEY ALLEY E. and UNNASCH T.R. : Onchocerca volvulus DNA probe classification correlates with epidemiologic patterns of blindness.
j. Inf. Diseases, 1992, 165, 964-968.
Article available athttp://www.parasite-journal.orgorhttp://dx.doi.org/10.1051/parasite/199401s1057
P A R A S I T E B I O L O G Y A N D B I O C H E M I S T R Y
H o w e v e r , t w o cuticlin g e n e s , cut-l a n d cut-2, h a v e n o w b e e n isolated from C. elegans in this laboratory, using a D.
melanogaster p r o b e c o d i n g for a c o m p o n e n t o f t h e vitelline m e m b r a n e o f the egg. cut-l mRNA is 1 4 2 2 nt long, h a s four e x o n s c o d i n g for 4 2 3 a m i n o acids a n d is transpliced to SL1, t h e s p l i c e d l e a d e r p r e s e n t at the 5' e n d o f m a n y mRNA's in m o s t n e m a t o d e s ; cut-2 mRNA is 8 4 7 nt long, c o n t a i n s o n l y t w o e x o n s c o d i n g for 2 3 7 a m i n o acids, a n d is n o t transpli
ced. Northern analysis indicates that w h i l e cut-l is transcri
b e d stage-specifically b y w o r m s entering the d a u e r larvae stage, cut-2 mRNA is t r a n s c r i b e d during cuticle synthesis, immediatly b e f o r e e a c h moult.
Parts o f b o t h g e n e s h a v e b e e n e x p r e s s e d as fusion proteins in E. coli and h a v e b e e n u s e d to raise s p e c i f i c a n t i b o d i e s . T h e s e h a v e b e e n u s e d to study the e x p r e s s i o n pattern of the t w o g e n e s b y w e s t e r n blot, a n d to l o c a l i z e the p r o d u c t s within the c u t i c l e s o f w o r m s at different s t a g e s b y i m m u n o f l u o r e s c e n c e a n d i m m u n o - e l e c t r o n m i c r o s c o p y . T h e results o b t a i n e d confirm the partial s t a g e specifity o f C U T - 1 , a n d t h e fact that C U T - 2 is a c o m p o n e n t o f t h e c u t i c l e at all stages. B o t h proteins are l o c a l i z e d o n cuticle residues after treatment with strong reducing a g e n t s , s h o w i n g t h e m to b e definitively m e m b e r s o f t h e cuticlin residue.
T h e proteins deriving from the c o n c e p t u a l translation o f the g e n e s differ substantially, a l t h o u g h t h e y b o t h b e g i n with signal p e p t i d e s a n d s h a r e a short motif r e p e a t e d 5 t i m e s in CUT-1 a n d 12 t i m e s in C U T - 2 . E a c h repetition is characteri
z e d b y t h e p r e s e n c e o f t h e a m i n o a c i d s e q u e n c e AAPA.
T h i s s a m e m o t i f c a n also b e found in s o m e o f the vitelline m e m b r a n e p r o t e i n s o f Drosophila a n d in t h e p r o t e i n s w h i c h m a k e up the c u t i c l e o f Locusta migratoria. T h e s e p r o t e i n s are all i n v o l v e d in the formation o f i n s o l u b l e , p r o t e c t i v e e x t r a c e l l u l a r l a y e r s , i m p l y i n g that t h e c o n s e r v e d d o m a i n s m a y h a v e an important functional role.
I n t e r e s t i n g l y , t h e a m i n o a c i d s e q u e n c e o f C U T - 2 s h o w s tyrosine residues that c o u l d participate in dityrosine bridge formation (dityrosine is p r e s e n t in the i n s o l u b l e residue o f parasitic n e m a t o d e c u t i c l e s ) . W e h a v e s h o w n that s o l u b l e r e c o m b i n a n t C U T - 2 , p r o d u c e d in E. coli, c a n b e p o l y m e r i z e d in vitro i n t o h i g h m o l e c u l a r w e i g h t s p e c i e s b y t h e action o f HR p e r o x i d a s e in the p r e s e n c e o f H 2 O 2 . T h e pro
d u c t s o f the r e a c t i o n b e c o m e i n s o l u b l e , c o n t a i n t y r o s i n e a n d the r e a c t i o n is inhibited b y the p r e s e n c e o f free tyro
sine. T h i s clearly b e g s the q u e s t i o n w h e t h e r CUT-2 is res
p o n s i b l e for the insolubility o f the cuticle.
A s e c o n d g e n e s h o w i n g significant h o m o l o g y ( > 8 0 % ) to cut-l h a s b e e n isolated for C. elegans, confirming the p o s sible e x i s t e n c e o f a cuticlin g e n e family. A cut-l h o m o l o g u e h a s a l s o b e e n isolated from the plant parasitic n e m a t o d e , Meloidogyne artiella, d e m o n s t r a t i n g the strongly c o n s e r v e d nature o f the s e q u e n c e a m o n g s t n e m a t o d e s .
An i n s o l u b l e cuticlin residue is p r e s e n t in the c u t i c l e s o f all n e m a t o d e s s t u d i e d s o far. T h i s fact, p l u s t h e a p p a r e n t l y c o n s e r v e d nature o f the g e n e and the protein for w h i c h it c o d e s , h a s p r o m p t e d t h e s e a r c h for g e n e s h o m o l o g o u s to cut-l a n d cut-2 in t w o parasitic n e m a t o d e s , Ascaris lumbri- coides and Brugiapahangi.
T w o parallel a p p r o a c h e s h a v e b e e n u s e d : the first involves s c r e e n i n g a g e n o m i c l i b r a r y w i t h l a b e l l e d D N A p r o b e s m a d e from C. elegans cuticlin g e n e s ; the s e c o n d involves s c r e e n i n g a parasite c D N A e x p r e s s i o n library with the s p e
cific a n t i b o d i e s raised against the r e c o m b i n a n t C U T - 1 a n d C U T - 2 purified p r o t e i n s . T h e p o s i t i v e c l o n e s h a v e b e e n s u b - c l o n e d into the p B l u e s c r i p t p h a g e m i d s y s t e m a n d are at p r e s e n t b e i n g s e q u e n c e d . T h e s e q u e n c e s will b e c h e c k e d for h o m o l o g y against the C. elegans cuticlin g e n e s a n d the S e q u e n c e s Data B a s e .
O n c e c u t i c l i n g e n e h o m o l o g u e s h a v e b e e n f o u n d t h e a p p r o a c h to their study will f o l l o w t w o b r o a d p a t h w a y s . Firstly transcription patterns in the parasite life-cycles will b e s t u d i e d u s i n g r e v e r s e t r a n s c r i p t a s e a n d P C R . A n d s e c o n d l y t h e inserts will b e e x p r e s s e d a n d t h e r e s u l t a n t r e c o m b i n a n t p r o t e i n s u s e d in b i o c h e m i c a l a n d i m m u n o l o g i cal studies. A n t i b o d i e s c o u l d a l s o b e raised against t h e s e p r o t e i n s for u s e in l o c a l i z a t i o n a n d t i m e - c o u r s e e x p e r i m e n t s , a n d hopefully to s c r e e n the c D N A libraries o f o t h e r parasitic n e m a t o d e s .
O n c e parasitic n e m a t o d e h o m o l o g u e s o f cut-l a n d cut-2 h a v e b e e n fully c h a r a c t e r i z e d , t h e r o l e s a n d f u n c t i o n s o f t h e s e clearly important p r o t e i n s will b e b e t t e r u n d e r s t o o d a n d hopefully the k n o w l e d g e c a n in s o m e w a y b e u s e d in the c o n t r o l o f the d i s e a s e s c a u s e d by t h e s e parasitic n e m a t o d e s .
REFERENCES
LASSANDRO F . , SKBASTIANO M . , ZEI F . and BAZZICAUIPO P. : cut-2, a second cuticlin gene of Caenorbabditis elegans. The role of dity
rosine formation in the crosslinking of its product. Mot. Biocbem.
Parasitol, 1993, submitted.
POLITZ S . M . and PHILIPI- M . : Caenorbabditis elegans as a model for parasitic nematodes : A focus on the cuticle. Parasitol. Today, 1992, 8, 6-12.
SEBASTIA.NO M . , LASSANDRO F . , B A Z Z I C A U I P O P . : cut-l, a Caenorbabditis elegans gene coding for a dauer-specific non col
lagenous component of the cuticle. Dev. Biol., 1991, 146, 519- 530.
N E U R O N S A N D G E N E S I N V O L V E D I N CHE
MICAL S E N S I T I V I T Y I N N E M A T O D E S
BAZZICALUPO P.*, HILLIARD M., LEWIS E.*, D E R I S O L., S E B A S T I A N O M.*, R I S T O R A T O R E F.*
KEYWORDS : chemoreception. nematodes, behavior, avoidance, amphid.
SUMMARY
Organelles and neurons of nematodes involved in sensing chemical signals present in the environment are described. Laser ablation of neurons has helped assign them a specific function. Genetic mutatio
nal analysis has led to the identification of genes controlling the beha
viour of the worms and/or some cellular properties of the
chemosensory neurons. Some conclusions on the general organization and functioning of chemoreception in nematodes can be drawn.
h e m i c a l s i g n a l s from t h e e n v i r o n m e n t a r e t h e m o s t important s e n s o r y inputs for n e m a t o d e s . T h e ability to r e c e i v e a n d interpret c h e m i c a l s i g n a l s from t h e e n v i r o n m e n t is essential for parasitic n e m a t o d e s to find the host, c o m p l e t e t h e i r life c y c l e a n d r e p r o d u c e . I f b e t t e r u n d e r -
* International Institute of Genetics and Biophysics, CNR, via G.
Marconi 10, 80125 Napoli, Italy.
