Article available at http://www.parasite-journal.org or http://dx.doi.org/10.
1051/parasite/199401s1057
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 TAI TZ I). : Hypervariability of simple sequences as a general source
Genet., 1991. 20. 391-396.
DUKE B O X . : Geographical aspects o f onchocerciasis. Ann. Soc.
BelgeMed. Trop., 1981, 61, 179-186.
WEBER J.L. and MAY P.E. : Abundant class of human DNA polymor­
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
WELSH J . and Mc CLELLAND M. : Fingerprinting genomes using PCR
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. : Onchocerciasis 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, 1994, J, I S
PARASITE B I O L O G Y AND BIOCHEMISTRY
individualize ungulates. Appl Theor. Electrophoresis, 1992, 2, 193-
200.
for polymorphic DNA markers. Nucl. Ac. Res., 1989, 17, n° 16,
6463-6471.
phisms which can be typed using the polymerase chain reaction.
Am. J. Hum. Genet., 1989, 44, 388-396.
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.
CUTICLIN 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 protects t h e animal, serves as an
e x o s k e l e t o n a n d provides t h e surface o v e r which inter­
actions with the external e n v i r o n m e n t occur. 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 host
and, as such, antigens e x p r e s s e d on/in the cuticle are
within reach o f both the 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 layered structure,
the c o m p o n e n t s o f which a r e classified according to their
solubility : lipids, s o m e proteins a n d other readily soluble,
non-structural c o m p o n e n t s a r e mostly localized o n the sur­
face b u t a r e also distributed to a lesser extent throughout
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 the
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 well-characterized g e n e family, a r e n o t e x p o s e d
o n the cuticle surface a n d c a n b e solubilized with SDS 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,
insoluble a n d c o m p l e x mixture o f proteins present throu­
ghout 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 impossible to determine 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 allow either molecular 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.
57
 PARASITE 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 and 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 the vitelline
m e m b r a n e o f the egg. cut-l mRNA is 1 4 2 2 nt long, has four
e x o n s c o d i n g for 4 2 3 a m i n o acids and is transpliced to SL1,
the s p l i c e d leader present at the 5' e n d o f m a n y mRNA's in
most n e m a t o d e s ; cut-2 mRNA is 8 4 7 nt long, contains only
two e x o n s c o d i n g for 2 3 7 a m i n o acids, and is not 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 dauer larvae
stage, cut-2 mRNA is transcribed 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 have b e e n used to raise specific antibodies.
T h e s e h a v e b e e n used 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, and to localize the products
within the cuticles o f w o r m s at different stages b y i m m u n o ­
f l u o r e s c e n c e and 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 stage specifity o f C U T - 1 , and
t h e fact that C U T - 2 is a c o m p o n e n t o f the c u t i c l e at all
stages. B o t h proteins are localized o n cuticle residues after
treatment with strong reducing agents, s h o w i n g t h e m to b e
definitively m e m b e r s o f the 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 peptides and share a short motif r e p e a t e d 5 times in
CUT-1 and 12 times in CUT-2. Each repetition is characteri­
z e d b y the p r e s e n c e o f the a m i n o a c i d s e q u e n c e AAPA.
This s a m e motif 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. These
proteins are all involved in the formation o f insoluble, pro­
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 the c o n s e r v e d
d o m a i n s may h a v e an important functional role.
Interestingly, the a m i n o acid s e q u e n c e o f CUT-2 shows
tyrosine residues that c o u l d participate in dityrosine bridge
formation (dityrosine is present in the insoluble residue o f
parasitic n e m a t o d e cuticles). W e have s h o w n that soluble
r e c o m b i n a n t CUT-2, p r o d u c e d in E. coli, c a n b e polymeri­
z e d in vitro into high 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
ducts 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 tyrosine
and the reaction is inhibited b y the p r e s e n c e o f free tyro­
sine. This clearly b e g s the question 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 has 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
has also b e e n isolated from the plant parasitic n e m a t o d e ,
Meloidogyne artiella, demonstrating 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 insoluble cuticlin residue is present in the cuticles 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, plus the 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 , has p r o m p t e d the search for g e n e s h o m o l o g o u s to
cut-l and 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 library 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 antibodies raised against the r e c o m b i n a n t CUT-1 and
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 pBluescript p h a g e m i d system and are
at present 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 cuticlin g e n e h o m o l o g u e s have b e e n found the
a p p r o a c h to their study will follow 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
be studied using r e v e r s e transcriptase a n d PCR. And
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 resultant
r e c o m b i n a n t proteins used in b i o c h e m i c a l and i m m u n o l o g i ­
cal studies. Antibodies c o u l d also 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 ­
ments, and 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 and cut-2
h a v e b e e n fully c h a r a c t e r i z e d , the roles a n d functions o f
t h e s e clearly important proteins will b e better u n d e r s t o o d
and 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 control o f the diseases c a u s e d by t h e s e parasitic n e m a ­
todes.
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., BAZZICAUIPO 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 RISO L.,
SEBASTIANO 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 signals from the 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 their life c y c l e and 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.