Note: Descriptions are shown in the official language in which they were submitted.
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i
DNA coding for 13-tubulin and its use
The invention relates to DNA which codes for (i-tubulin from nematodes of the
family of the Strongylidae, the polypeptide encoded by this DNA, the use of
the
DNA for the diagnosis of anthelmintic resistance of these nematodes and for
the
identification of the species of these nematodes, the use of the (3-tubulin as
a
constituent of a vaccine, and a process for the identification of new
anthelmintic or
antibiotic compounds.
Parasitic helminths (worms) are a health problem for humans and animals and
cause
significant economic damage. In 1996, the expenditure for anthelmintics
worldwide
was more than 2 billion US dollars. The most important anthelmintics which are
presently used can be divided into three groups according to their mechanism
of
action:
1. The cyclic amidines pyrantel and morantel act, together with the
imidazothiazoles tetramisole and levamisole, as cholinergic compounds for
the parasitic nervous system.
2. The benzimidazoles are inhibitors of the polymerization of microtubules and
lead to the degradation of tubulin, followed by the loss of a number of cell
functions such as transport within cells and cell division.
3. The macrocyclic lactones bind and open glutaminergic chloride channels and
thus act as inhibitors of the nervous system of nematodes and arthropods.
Microtubules are built up from tubulin subunits. Tubulin is a dimeric protein
which
consists of a- and 13-tubulin and is in a dynamic equilibrium between tubulin
and
microtubules. This equilibrium can be influenced by exogenous substances,
which are
designated as microtubule inhibitors. Some of these inhibitors, such as, for
example,
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the benzimidazoles, act by their binding to tubulin, whereby they prevent the
self
association of these subunits to growing microtubules, while at the opposite
end the
dissociation of the microtubules is continued. On account of this,
dysfunctions occur
in processes within the cell which are important to life and finally the death
of the
cell and of the entire organism occurs (Lacey, E. ( 1990) Mode of action of
benz-
imidazoles. Parasitology Today 6, 112-115). Such microtubule inhibitors
include
various classes of compound which are synthetically prepared or produced by
various
organisms.
The binding of microtubule inhibitors to tubulin from various organisms shows
great
differences with respect to the affinity of the binding. Thus the
anthelmintics
oxfendazole and thiabendazole show a high affinity for tubulin from Ascaridia
galli
and an only slight affinity for tubulin from mammals such as the sheep (Dawson
et
al. (1983) Purification and characterisation of tubulin from the parasitic
nematode,
Ascaridia galli, Molecular and Biochemical Parasitology 7, 267-277). The
selective
toxicity of benzimidazoles can be explained by means of this selective
affinity
(Lacey, E. (1988) The role of the cytoskeletal protein, tubulin, in the mode
of action
and mechanism of drug resistance to benzimidazoles, International Journal for
Parasitology 18, 885-936).
The widespread use of these anthelmintics has led to considerable resistance
problems against all three classes, especially in livestock (Bauer et al.
(1994)
Anthelmintic resistance in nematodes of farm animals. A seminar organised for
the
European Commission, Brussels, Belgium, 8 to 9 November 1993, pp. 17-24). The
most widespread class of anthelmintics are the benzimidazoles. Resistance to
benzimidazoles has been described worldwide in the case of parasites of sheep,
cattle, pigs and horses. Benzimidazoles are broad-spectrum anthelmintics with
action
against nematodes, cestodes and trematodes. Investigations in German stud
farms
showed a resistance of small strongylids to benzimidazoles in more than 80% of
the
cases (Ullrich et al. (1988) Benzimidazole resistance in small strongylids
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(Cyathostominae): distribution in horse stock in Northrhine-Westphalia,
Berliner
Miinchner Tierarztliche Wochenschrift 101, 406-408).
For an effective treatment with anthelmintics, it is therefore of great
importance to
obtain information about possible resistance of worms in a horse or within a
herd of
horses. In order to check the possible resistance of a worm population of
small
stronglids, diagnostic procedures have already been developed which are based
on
the activity of anthelmintics in the stages of development of the parasites
(eggs,
larvae) (Coles et al. (1992) World Association for the Advancement of
Veterinary
Parasitology (W.A.A.V.P.) Methods for the detection of anthelmintic resistance
in
nematodes of veterinary importance, Veterinary Parasitology, 44, 35-44). The
"larval
development assay" (LDA) describes the inhibitory effect of anthelmintics in
the
nonparasitic first larval stage as a function of the anthelmintic
concentration
employed. In a similar manner, the second approach, the "egg hatch assay"
(EHA)
uses the inhibitory action of the anthelmintics on the hatching of the larvae
as a
function of the anthelmintic concentration employed. A disadvantage of both
approaches lies in the low reproducibility of the results. Moreover, both
approaches
are time-consuming and labor-intensive.
The sensitivity of both approaches is moreover low. An existing resistance is
only
detected if more than 25% of the population are resistant (Roos et al. (1995)
New
genetic and practical implications of selection for anthelmintic resistance in
parasitic
nematodes, Parasitology Today 11. 148-150).
There is therefore an urgent need for diagnostic procedures which are
sensitive, rapid
and reproducible. For the sheep parasites, Haemonchus contortus and
Teladorsagia
circumcincta, a procedure based on the PCR technique has been described. This
is
based on at least one point mutation in codon 200 of the 13-tubulin isotype 1
gene
(Elard et al. (1999) PCR diagnosis of benzimidazole - susceptibility or -
resistance in
natural populations of the small ruminant parasite, Teladorsagia circumcincta,
Veterinary Parasitology 80, 231-237). The point mutations in codon 200 result
in an
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amino acid replacement of phenylalanine by tyrosine and correlates with a
benzimidazole resistance of the mutated protein (Kwa et al. (1995) t3-tubulin
genes
from parasitic nematode Haemonchus contortus modulate drug resistance in
Caenorhabditis elegans, Journal of Molecular Biology 246, S00-510).
From studies on Haemonchus contortus, it is known that the sequences of
nematodes
coding for tubulin are species-specific (WO 92/03549).
The various species of the small strongylids of the horse differ in their
epidemiological frequency. The species which are most important and found most
frequently worldwide include Cylicocyclus nassatus, Cyathosto»ium coronatum
and
Cyathostomum catinatum. Resistances of these species to benzimidazoles have
been
described in various countries, inter alia also in Germany (Burger, H.-J. and
Bauer,
C. (1987) Efficacy of four anthelmintics against benzimidazole - resistant
cyathostomes of horses, Veterinary Record 120, 293-296).
The nucleic acid sequencing of !3-tubulin cDNAs from the species of small
strongylids according to the invention had an identity of over 95%. The
identity with
the known, abovementioned 13-tubulin sequences of sheep parasites, however, is
only
75.4-82.6%.
The derived amino acid sequences are very similar within the sequences
according to
the invention. This is also true of the derived 13-tubulin amino acid
sequences of the
sheep parasites. The identity here is between 95 and 99.8%. Only very few
positions
result in which amino acid exchange occurs. To be emphasized here is codon
200, in
which a change from phenylalanine to tyrosine results.
However, the noncoding 13-tubulin sequences from various helminth species
published hitherto show no significant identity. It is accordingly surprising
that not
only the coding sequences, but also parts of the noncoding sequences, of the
various
species of small strongylids of the application present here have a high
identity.
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These regions are therefore also suitable to be able to differentiate various
species of
small strongylids and other nematode species from one another. PCR primers
which
are derived from these intron regions can be used for the specific detection
of small
strongylids within a sample which also contains genetic material from other
helminth
organisms.
13-Tubulin from nematodes or parts thereof are known for having a protective,
immunological potential (Bughio et al. (1993) Characterisation and biological
activities of anti-Brugia pahangi tubulin monoclonal antibodies, International
Journal for Parasitology, 7, 913-924). The 13-tubulin of the small strongylids
encoded
by the abovementioned DNA can be used as a vaccine just as monoclonal
antibodies
can be used against the 13-tubulin.
Inhibitors of the interaction of the tubulin or of its subunits, such as
benzimidazoles,
1 S colchizine and taxol, are important lead structures of a series of
therapeutics which
are directed against human, animal or plant diseases. The importance of
tubulin as
the target of these compounds is far-reaching and underlines its potential for
the
search for new active compounds for the control of these diseases.
The invention relates to the DNA coding for the 13-tubulin from nematodes of
the
family of the Strongylidae, particularly of the subfamily of the
Cyathostominae, or
fragments of this DNA. The 13-tubulin DNA can in this case be genomic DNA or
cDNA. The DNA sequences which this invention relates to can be considered as
new
members of the tubulin gene family of parasitic nematodes of the order
Strongylida,
particularly of the subfamily of the Cyathostominae. The invention relates
very
particularly to the DNA sequences which code for 13-tubulin from parasitic
nematodes of the genera Cyathostomum and Cylicocyclus.
The invention likewise relates to DNA sequences which have an identity of more
than 85% to a polynucleotide coding for one of the amino acid sequences as set
forth
inSEQIDN0.2,4,6,8or10.
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The invention likewise relates to preferred DNA sequences which have an
identity of
more than 95% to a polynucleotide coding for one of the amino acid sequences
as set
forth in SEQ m NO. 2, 4, 6, 8 or 10.
S
The invention relates in particular to DNA sequences coding for 13-tubulin,
which
originate from parasitic nematodes of the genera Cylicocyclus and
Cyathostomum,
very particularly those sequences which originate from parasitic nematodes of
the
species Cylicocyclus nassatus, preferably DNA as set forth in SEQ m NO. 3, 5,
7, 9
or 11 or from Cyathostonum coronatum, preferably DNA according to SEQ ID NO.
1.
The invention likewise relates to DNA sequences as described above, which in
contrast to these sequences have at least one point mutation or one nucleotide
replacement in codon 200. These point mutations result in a change in the
amino acid
sequence encoded by this DNA, e.g. a replacement of the amino acid
phenylalanine
by tyrosine, and correlate with the resistance of tubulin with appropriate
mutations to
benzimidazoles.
The invention likewise relates to DNA sequences which are complementary to the
DNA described above or fragments of this DNA, and to fragments of these DNA
sequences. These DNA sequences or these fragments comprise oligonucleotides
which are derived from one of the DNA sequences mentioned above or described
under SEQ )D NO. 1, 3, 5, 7, 9 or 11 or sequences identical to 85% thereto,
preferably sequences identical to 95%, and are derived from strands
complementary
thereto and can hybridize to these.
The invention in this case relates preferably to oligonucleotides consisting
of or
comprising one of the sequences as set forth in SEQ 1D NO. 12 to SEQ ID NO.
51,
which hybridize to abovementioned DNA sequences, preferably in the region of
noncoding sequence sections of the f3-tubulin genes.
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The invention likewise preferably relates to oligonucleotides consisting of or
comprising one of the sequences as set forth in SEQ )D NO. 12 to SEQ >D NO.
51,
which hybridize to coding regions of the abovementioned sequences.
The invention likewise relates to RNA sequences which are complementary to the
DNA described above or fragments of this DNA, and fragments of these RNA
sequences. These RNA sequences or these fragments comprise
ribooligonucleotides
which correspond to a region of one of the DNA sequences mentioned above or
described under SEQ )D NO. 1, 3, 5, 7, 9 or 11, sequences complementary
thereto or
DNA sequences 85% identical, preferably 95% identical, thereto, and can
hybridize
to these.
The invention likewise relates to an expression construct which comprises one
of the
DNA sequences described above, and to a DNA sequence linked thereto which
makes possible the expression of the DNA. These include, for example, at least
one
promoter for constitutive or inducible expression or alternatively enhancers.
Suitable
promoters for expression in E. coli are natural hybrid or bacteriophage
promoters,
preferably promoters of the group of ~,-phages, hsp, omp or synthetic
promoters such
as mentioned, for example, in WO 98/5625, DE 3 430 683 or EP 0 173 149.
The invention likewise relates to vectors which comprise one of the DNA
sequences
described above and make possible the expression of the Q-tubulin according to
the
invention or fragments thereof in a host cell.
The invention likewise relates to host cells which contain the abovementioned
DNA,
an expression construct as mentioned above, or a vector and allow the
expression of
the 13-tubulin or fragments thereof.
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The invention likewise relates to polypeptides which are encoded by one of the
abovementioned DNA sequences or fragments of these DNA sequences, and to
fragments of these polypeptides.
The invention in this case relates preferably to polypeptides which are
encoded by a
DNA sequence comprising SEQ m NO. l, 3, 5, 7, 9 or 1 l, by DNA sequences which
have an identity of 85% to these sequences, preferably of 95%, or of fragments
of
this DNA.
The invention also relates to polypeptides which are encoded by the DNA
sequence
described above, which contain at least one point mutation in codon 200 as
described
above and show resistance to benzimidazoles, and to fragments of these
polypeptides.
The invention in this case relates very particularly preferably to
polypeptides
comprising one of the amino acid sequences described in SEQ ID NO. 2, 4, 6, 8
or 10
or fragments thereof.
The invention in this case relates to polypeptides, particularly to purified
polypeptides or polypeptides prepared recombinantly.
The invention relates to polypeptides of full length and also to corresponding
fragments of these polypeptides, e.g. certain motifs or domains. These
fragments can
be of different length and comprise, for example, 5, 10, 25, 50, 100, 150,
200, 250 or
300 amino acids.
This invention likewise relates to fusion proteins which comprise a
polypeptide as
described above. The fusion protein can in this case contain a further
polypeptide
component which is not connected to the 13-tubulin (e.g. LexA, B42,
glutathione S-
transferase, a His tag, a polypeptide having enzymatic activity such as
alkaline
phosphatase or an epitope tag).
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The invention also relates to a process for the preparation of a polypeptide
as
described above in suitable prokaryotic or eukaryotic expression systems. The
expression can in this case be carried out permanently or transiently as
described
above in a corresponding cell line in each case or corresponding host cells.
Suitable
prokaryotic expression systems are known host-vector systems such as bacteria
(e.g.
Streptomyces spp., Bacillus subtilis, Salmonella typhimurium, Serratia
marcescens
and particularly Escherichia coli).
Expression in a eukaryotic system is preferably carned out in the baculovirus
system,
particularly in a ~ system which allows the introduction of post-translational
modifications.
This invention likewise relates to the use of DNA as mentioned above for the
detection of DNA from nematodes of the family Strongylidae, preferably of the
subfamily Cyathostominae, particularly preferably of the genera Cyathostomum
and
Cylicocyclus, very particularly preferably of the species Cyathostomum
coronatum
and Cylicocyclus nassatus. The invention in this case relates to
oligonucleotides as
mentioned above which are complementary to DNA coding for 13-tubulin or
strands
complementary thereto and can hybridize to this DNA. Preferably, these
oligonucleotides hybridize to the intron regions, i.e. the noncoding DNA
sequences.
The invention relates to the use of these oligonucleotides or parts thereof as
a) samples in Northern or Southern blot assays,
b) PCR primers in a diagnostic procedure for the detection of the
abovementioned nematodes, the DNA of the nematodes concerned being
specifically identified and amplified with the aid of the primer and the PCR
technique.
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The invention in this case relates preferably to oligonucleotides consisting
of or
comprising one of the sequences as set forth in SEQ ID NO. 12 to SEQ ID NO.
51.
The invention likewise relates to the use of DNA as mentioned above for the
detection of DNA from nematodes of the family of the Strongylidae, preferably
csf the
subfamily of the Cyathostominae, particularly preferably of the genera
Cylicocyclus
and Cyathostomum, very particularly preferably of the species Cylicocyclus
nassatus
and Cyathostomum coronatum, which codes for 13-tubulin or fragments thereof,
which are resistant to benzimidazoles. The invention in this case relates to
oligonucleotides as mentioned above which are complementary to DNAs which code
for f3-tubulin with a resistance to benzimidazoles or to the complementary
strands of
this DNA and which can specifically hybridize to this DNA.
The invention also relates to the use of these oligonucleotides or parts
thereof as
a) samples in Northern or Southern blot assays,
b) PCR primers in a diagnostic procedure for the detection of the
abovementioned nematodes with a resistance to benzimidazoles, the DNA of
the nematodes concerned being specifically identified and amplified with the
aid of the primer and the PCR technique.
The invention in this case preferably relates to oligonucleotides consisting
of or
comprising one of the sequences as set forth in SEQ ID NO. 12 to SEQ >D NO.
51.
The invention also relates to a procedure for the detection of nematodes of
the family
of the Strongylidae, preferably of the subfamily of the Cyathostominae,
particularly
preferably of the genera Cylicocyclus and Cyathostomum, very particularly
preferably of the species Cylicocyclus nassatus and Cyathostomum coronatum,
where
oligonucleotides as described above specifically hybridize to DNA sequences
which
originate from the organisms mentioned, and which can be amplified with the
aid of
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the PCR technique. The hybridization is preferably carried out in the
noncoding
regions of the ~i-tubulin gene (introns).
The detection of organisms as mentioned above can be carried out, for example,
by
a) making available an oligonucleotide probe or primer which can hybridize to
the abovementioned DNA coding for 13-tubulin or strands complementary
thereto or to the 5'- or 3'-flanking regions thereof,
b) bringing the oligonucleotide probe or the primers into contact with an
appropriately prepared probe containing DNA,
c) detecting the hybridization of the oligonucleotide or primer (e.g. with the
aid
of the polymerase chain reaction),
d) sequencing the detected sequence of the 13-tubulin gene, and
e) comparing this sequence with the DNA sequences according to the invention
which were described above, preferably with DNA sequences as set forth in
SEQ ID NO. 1, 3, 5, 7, 9 or 11.
The invention also relates to a process for the detection of nematodes of the
family of
the Strongylidae, preferably of the subfamily of the Cyathostominae,
particularly
preferably of the genera Cylicocyclus and Cyathostomum, very particularly
preferably of the species Cylicocyclus nassatus and Cyathostomum coronatum,
which
are resistant to benzimidazoles, where oligonucleotides as described above
specifically hybridize to DNA sequences which originate from the organisms
mentioned and which can be amplified with the aid of the PCR technique. The
hybridization is preferably carried out in the noncoding regions of the (3-
tubulin gene
(introns).
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The detection of organisms as mentioned above can be carried out, for example,
by
a) making available an oligonucleotide probe or primer which can hybridize to
the abovementioned DNA coding for f3-tubulin or strands complementary
thereto or to the 5'- or 3'-flanking regions thereof,
b) bringing the oligonucleotide probe or the primers into contact with an
appropriately prepared probe containing DNA,
c) detecting the hybridization of the oligonucleotide or primer (e.g. with the
aid
of the polymerase chain reaction),
d) sequencing the detected sequence of the f3-tubulin gene, and
e) comparing this sequence with the DNA sequences according to the invention
which were described above, preferably with DNA sequences as set forth in
SEQ >D NO. l, 3, 5, 7, 9 or 11, having at least one point mutation in codon
200, which leads to a resistance of the /3-tubulin encoded by these sequences
to benzimidazoles.
The invention likewise relates to a diagnostic test kit for the detection and
identification of nematodes of the family of the Strongylidae, preferably of
the
subfamily of the Cyathostominae, particularly preferably of the genera
Cylicocyclus
and Cyathostomum, very particularly preferably of the species Cylicocyclus
nassatus
and Cyathostomum coronatum, which, inter alia, makes available
oligonucleotides as
described above, which can be used in procedures for the detection of the
species
mentioned. The present invention likewise makes available oligonucleotides
which
specifically hybridize to sequences as set forth in SEQ )D NO. 1, 3, 5, 7, 9
or 11,
sequences complementary thereto, sequences having at least one point mutation
in
codon 200, or fragments of these sequences. In this case, oligonucleotides
consisting
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of or including sequences as set forth in SEQ )D NO. 12 to SEQ m NO. 51 are
particularly preferred.
The invention likewise relates to a diagnostic test kit for the detection of
nematodes
of the family of the Strongylidae, preferably of the subfamily of the
Cyathostominaes
particularly preferably of the genera Cylicocyclus and Cyathostomum, very
particularly preferably of the species Cylicocyclus nassatus and Cyathostomum
coronatum, having a resistance to benzimidazoles which, inter alia, makes
available
oligonucleotides as described above, which can be used in procedures for the
detection of the species mentioned. The present invention likewise makes
available
oligonucleotides which hybridize specifically to sequences as set forth in SEQ
>D
NO. l, 3, 5, 7, 9 or 11, sequences complementary thereto, sequences having at
least
one point mutation in codon 200, or fragments of these sequences. In this
case,
oligonucleotides consisting of or comprising sequences as set forth in SEQ m
NO.
12 to SEQ ID NO. 51 are particularly preferred.
The invention likewise relates to a diagnostic test kit as described above,
where the
oligonucleotides made available in this kit are provided with a detectable
marker.
Such detectable markers can include, inter alia, enzymes, enzyme substrates,
coenzymes, enzyme inhibitors, fluorescence markers, chromophores, luminescent
markers and radioisotopes.
This invention likewise relates to antibodies which react specifically with an
epitope
of a 13-tubulin from nematodes of the family of the Strongylidae, preferably
of the
subfamily of the Cyathostominae, particularly preferably of the genera
Cylicocyclus
and Cyathostomum, very particularly preferably of the species Cylicocyclus
nassatus
and Cyathostomum coronatum.
This invention likewise particularly relates to monoclonal antibodies which
react
specifically with an epitope of a 13-tubulin from nematodes of the family of
the
Strongylidae, preferably of the subfamily of ~ the Cyathostominae,
particularly
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preferably of the genera Cylicocyclus and Cyathostomum, very particularly
preferably of the species Cylicocyclus nassatus and Cyathostomum coronatum.
This invention likewise relates to the use of the abovementioned antibodies as
nematicides.
This invention also relates to the use of the abovementioned 13-tubulin
polypeptides
or fragments thereof from nematodes of the family of the Strongylidae,
preferably of
the subfamily of the Cyathostominae, particularly preferably of the genera
Cylicocyclus and Cyathostomum, very particularly preferably of the species
Cylicocyclus nassatus and Cyathostomum coronatum, for the preparation of
vaccines
which contain at least one of the 13-tubulin polypeptides mentioned or
fragments
thereof. The vaccine is in this case able to produce an immune response which
is
specific for a 13-tubulin described above.
In a preferred embodiment, the vaccine contains an antigenic determinant, e.g.
an
individual determinant of a polypeptide having an amino acid sequence as set
forth in
SEQ ID NO. 2, 4, 6, 8 or 10 or of a polypeptide which is encoded by one of the
abovementioned DNAs or fragments thereof.
The invention likewise relates to a procedure for the preparation of an
immunogenic
composition for the immunization of mammals, consisting of at least one of the
abovementioned (3-tubulin polypeptides according to the invention or fragments
thereof or of at least one of the abovementioned antibodies.
The invention likewise relates to the use of the expression vectors described
above
containing a nucleic acid coding for a (3-tubulin according to the invention,
preferably
a sequence as set forth in SEQ 1D NO. 1, 3, 5, 7, 9 or 11, fragments thereof
or
sequences homologous thereto for the preparation of an immunogenic composition
for administration to a host for the activation of a protective immune
response in this
host, which is directed at ~3-tubulin from parasitic nematodes.
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The invention likewise relates to an immunogenic composition comprising a
vector
(comprising a nucleic acid coding for the ~i-tubulin according to the
invention,
preferably a sequence as set forth in SEQ >D NO. 1, 3, S, 7, 9 or 11,
fragments
thereof or sequences homologous thereto and to a promoter sequence which is
linked
functionally to said nucleotide sequence and which controls the expression of
the ~i-
tubulin according to the invention producing an immune response) and a vehicle
suitable for pharmaceutical purposes.
This invention likewise relates to a process for the identification of
substances which
modulate the interaction of tubulin or the interaction of subunits of tubulin.
The
process is based on the use of tubulin, preferably on tubulin from parasitic
nematodes, particularly preferably on 13-tubulin from parasitic nematodes of
the order
strongylida, very particularly preferably on 13-tubulin from parasitic
nematodes of the
family of the Strongylidae, most preferably on 13-tubulin from parasitic
nematodes of
the subfamily of the Cyathostominae. A particularly preferred group of the 13-
tubulin
used in this procedure is 13-tubulin from parasitic nematodes of the genera
Cylicocyclus and Cyathostomum.
The invention relates to the identification of substances, e.g. small organic
molecules,
which are able to modulate the interaction of tubulin protein molecules or its
subunits
with one another. Preferably, the invention relates to the identification of
compounds
which inhibit the interaction.
The invention likewise relates to a procedure as described above, which is
based on
a) bringing the substance to be tested into contact with the tubulin, the
conditions selected allowing the interaction of the tubulin molecules with one
another and the binding of the test substance to tubulin,
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b) detecting the binding which has taken place by determining the ability of
the
tubulin protein molecules to interact with one another and
c) comparing the ability of the tubulin protein molecules to interact with one
another in the presence of a test substance to the ability to interact with
one
another in the absence of a test substance.
The invention likewise relates to a process for the identification of
substances which
modulate the ability of tubulin molecules to interact with one another.
Particularly
preferably, the invention in this case relates to a procedure which uses one
of the
polypeptides described above which are coded by the DNAs described above or
fragments thereof, particularly by DNAs consisting of or comprising sequences
as set
forth in SEQ >D NO. 1, 3, 5, 7, 9 or 11, and by sequences which have an
identity of
85% thereto, preferably of 95% and code for 13-tubulin which has an amino acid
sequence as set forth in SEQ 2, 4, 6, 8 or 10.
The invention likewise relates to a procedure for the identification of
substances
which modulate the ability of tubulin to interact with one another as
described above,
the procedure used being based on detecting a modulation of the tubulin
interaction
in the presence of a test substance with the aid of a test system based on
cells. A
preferred embodiment of such a test system is the "two hybrid system"
(US 5 283 317, Zervos et al. (1993) Cell 72, 223-232; WO 94/10300). This
system is
suitable for documenting or describing the interaction of two proteins by the
interaction leading to a detectable signal. Such a system can also be adapted
to test
systems having high throughput numbers.
The invention likewise relates to a procedure for the identification of
substances
which modulate the ability of tubulin to interact with one another, the
procedure used
being based on detecting a modulation of the tubulin interaction in the
presence of a
test substance with the aid of a cell-free test system. A particularly
preferred
embodiment of such a test system is the "scintillation proximity assay" (SPA)
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(EP Ol S 473). This test system is based on the detection of an interaction of
a
receptor bound to microspheres or beads, e.g. of a tubulin molecule with a
ligand, the
microspheres or beads being provided with a scintillating molecule. A signal
is
detected if the receptor-ligand complex decomposes.
The invention likewise relates to substances which have hitherto still not
been
described, which are identified with the aid of the procedure described above
and are
suitable to modulate, preferably to inhibit, the interaction of tubulin
molecules.
The invention likewise relates to the use of substances which have hitherto
still not
been described, which were identified using one of the procedures described
above,
for the preparation of an agent which is used for the prophylactic or
therapeutic
treatment of animals or humans which can be attacked or have been attacked by
nematodes. The agents according to the invention contain at least one of the
substances identified by one of the procedures described above and can be
administered nasally, dermally, parenterally or enterally.
For better understanding, the meaning of certain words and terms which are
used in
the description, the examples and attached claims are explained in greater
detail
below.
The term "fragments" with respect to proteins and DNA describes parts of the
nucleic acids or amino acid sequences described under SEQ >D NO. 1 to 11,
sequences complementary thereto or sequences identical thereto to 85%,
preferably
to 95%. The fragments of the DNA and polypeptide sequences comprise at least
five
nucleotides or amino acids, but can likewise comprise up to 447 amino acids or
up to
1343 nucleotides, or up to 2565 nucleotides in the case of the sequence as set
forth in
SEQ m NO. 11.
The terms "homology", "identity" or "similarity" relate to sequence
similarities
between two peptides or between two nucleic acid molecules. Homology can be
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determined by in each case comparing a position in each sequence with one
another.
If a position in the compared sequence is occupied by the same base or amino
acid,
the two molecules in this position are homologous. The measure of homology
between sequences is a function of the number of the corresponding or
homologous
positions which the sequences share with one another. A "nonhomologous"
sequence
has an identity of less than 40%, but preferably of less than 25% identity.
The term "homology" in particular means that DNA segments of a length of at
least
base pairs or strands complementary to the DNA agree to at least 85%,
preferably
10 95%, with the nucleotides with the corresponding DNA. A homology can be
determined, inter alia, with the aid of computer programs such as the GCG
program
(Devereux et al. (1983), Nucleic Acids Res. 12, 387-395).
A "homology" also exists if a DNA segment can hybridize to the DNA strand
15 concerned or its complementary strand.
The term "hybridize" or "hybridization" describes the process in which a
single-
stranded nucleic acid molecule undergoes base pairing with a complementary DNA
strand, the ability of a single-stranded nucleic acid molecule depending on
the
stringency of the hybridization conditions.
The term "stringency" relates to the hybridization conditions. "High
stringency" is
imparted if base pairing is rendered difficult. "Low stringency" is imparted
if base
pairing is facilitated.
The term "complementary" relates to the ability of purine and pyrimidine
nucleotides
to form base pairs with one another via hydrogen bridges. Complementary base
pairs
are, inter alia, guanine and cytosine, adenine and thymine and adenine and
uracil.
The person skilled in the art realizes that, on account of the degenerate
genetic code
(i.e. 64 codons code for 20 amino acids), numerous "silent" substitutions of
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nucleotide base pairs can be introduced into the sequence coded therefor
without
changing the identity of the protein products encoded thereby. All such
substitutions
are to be contained in the scope of the invention.
The term "specifically hybridize" relates to the ability of a nucleic acid
molecule of
the present invention to hybridize to at least approximately 6, 12, 20, 30,
50, 100,
150, 200, 300, 350, 400 or 440 successive nucleotides of one of the 13-tubulin
genes
described above, preferably to one of the sequences as set forth in SEQ ID NO.
1, 3,
5, 7, 9 or 11 or sequences homologous or complementary thereto, namely in such
a
way that ten times more molecules hybridize, preferably 100 times more
molecules
hybridize, and particularly preferably more than 100 times more molecules
hybridize
than to a cellular nucleic acid (e.g. mRNA or genomic DNA), which codes for a
protein other than the 13-tubulin described above.
The term "plasmid" relates to an extrachromosomal genetic element. The
original
plasmids used for the present invention are either commercially obtainable,
freely
accessible or can be derived from such plasmids according to known processes.
The term "vector" describes a DNA element which is used for the introduction
of
exogenous DNA into host cells. A vector contains a nucleotide sequence which
codes
for one or more polypeptides. Vectors which are able to control the expression
of the
genes which they contain are designated as "expression vectors".
The term "gene" relates in the scope of the present invention to a nucleic
acid
comprising an open reading frame which codes for one of the f3-tubulin
polypeptides
described above. Exon and possible intron sequences are additionally included
here.
The term "interact" or "interaction" describes detectable interactions between
molecules. The term "binding" is additionally included here.
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The term "modulate" relates both to a stimulation and to a suppression or
inhibition
of a biochemical process. In the context of the present invention,
"modulation"
means an inhibition or suppression of the interaction between tubulin
polypeptides or
fragments or subunits thereof, or a stimulation of this interaction which can
be
shown, for example, in an irreversible binding of tubulin polypeptides to one
another.
The term "nucleic acid" relates to polynucleotides such as deoxyribonucleic
acids
(DNA) or, if appropriate, to ribonucleic acids (RNA). In an equivalent manner,
the
term also includes analogs of RNA or DNA which are prepared from nucleotide
analogs, and, in the case concerned, single-stranded ("sense" or "antisense")
and
double-stranded polynucleotides.
The term "promoter" relates to DNA sequences which regulate the expression of
a
specific DNA, which are functionally linked to the promoter. The term also
includes
"tissue-specific" promoters, i.e. promoters which control the expression of
the
specific DNA only in certain cells (e.g. cells of a certain tissue). Likewise
included
are "tissue-nonspecific" promoters and promoters which lead to a constitutive
expression or are inducible.
The ten~ns "protein", "polypeptide" and "peptide" are exchangeable in their
use in the
context of the present application if they relate to a gene product.
A "fusion protein" is a fusion of a first amino acid sequence coding for one
of the (3-
tubulin polypeptides described above having a second amino acid sequence which
has no commonality or fundamental homology to the tubulin sequence. The second
amino acid sequence can in this case originate from the same organism as the
first, or
alternatively can originate from another organism (intergenic). In general, a
fusion
protein can be represented by means of the formula X-tubulin-Y, where
"tubulin"
represents one of the polypeptides described above, and X and Y represent a
polypeptide which is not connected to a tubulin amino acid sequence. X or Y
can in
each case independently of one another be absent.
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The terms "cell" or "host cell" can be used in the same sense in the context
of the
application present here. It is understood that these terms relate not only to
an
individual cell, but also to the descendants of such a cell. On account of
certain
modifications in the course of following generations (e.g. mutations), such
descendants are possibly not identical to the stem cell, but are additionally
included
by the present invention.
The term "intron" describes those sequences of the described, preferably
genomic,
DNA which are transcribed, but are then removed from the transcript by
"splicing",
the adjacent sequences (exons) being linked.
Nucleic acids
As already described, one aspect of the invention relates to nucleic acids
from
nematodes of the family of the Strongylidae, particularly of the subfamily of
the
Cyathostominae, very particularly to the genera Cyathostomum and Cylicocyclus,
especially of the species Cylicocyclus nassatus and Cyathostomum coronatum,
which
code for 13-tubulin polypeptides or fragments thereof or nucleic acids
homologous
thereto which are homologous to 85% to the [lacuna] in SEQ ID NO. 1, 3, 5, 7,
9 and
11, preferably to 95%, and code for a 13-tubulin according to one of the
sequences as
set forth in SEQ ID NO. 2, 4, 6, 8 or 10 or fragments thereof. SEQ ID NO. 3
represents the degenerated sequence of the nucleic acid from Cylicocyclus
nassatus
coding for [i-tubulin, where "r" represents a purine (guanine or adenine), "y"
represents a pyrimidine (thymine, or uracil or cytosine) and "w" represents an
adenine or a thymine, or a uraciI. SEQ ID NO. 3 thus includes a number of
sequences
which can be present in the organisms of the species Cylicocyclus nassatus.
The
sequences as set forth in SEQ 1D NO. 5, 7 and 11 show three defined sequences
coding for 13-tubulin, which are exemplary and preferred embodiments of DNA as
set
forth in SEQ ID NO. 3.
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Likewise part of the invention are oligonucleotides which optionally code for
13-
tubulin polypentides which comprise a length of at least 2, S, 10, 25, 50,
100, 150,
200, 250, 300, 350 or 400 amino acids. Such oligonucleotides can be used as
primers
or antisense molecules (i.e. as noncoding nucleic acids) and comprise at least
approximately 6, 12, 24, 30, 60, 100, 120, 150 or 210 base pairs, while coding
nucleic acids comprise approximately 200, 300, 400, 500, 600, 700, 800, 900,
1000,
1100, 1200 or 1300 base pairs.
The invention also describes those oligonucleotides which specifically
hybridize
under stringent conditions to nucleic acids which are represented by one of
the
sequences as set forth in SEQ ID NO. 1, 3, 5, 7, 9 or 11. Correspondingly
stringent
conditions are, for example, 6 x sodium chloride/sodium citrate (SSC) at
approximately 45°C, followed by a washing step with 2 x SSC at
50°C, and are
familiar to the person skilled in the art (see, for example, Current protocols
in
Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6). Thus the salt
concentrations in the washing step can be chosen such that the stringency is
lower
(2 x SSC, 50°C) or is higher (0.2 x SSC, 50°C). Furthermore, the
temperature in the
washing step can be varied, from conditions for a low stringency (e.g. about
22°C),
up to conditions of higher stringency (e.g. about 65°C). Both salt
concentration and
temperature can be varied and tailored to one another.
Particularly preferred oligonucleotides which can be used as primers or for
hybridization for the identification and characterization of an existing, e.g.
genomic,
DNA, are described in SEQ m NO. 12-51. However, other oligonucleotides can
also
be derived from the sequences as set forth in SEQ m NO. l, 3, 5, 7, 9 or 11,
which
can then be used as primers or for hybridization. Particularly preferred for
the
identification of the species or genus to which an existing DNA can be
assigned are
those oligonucleotides which hybridize in the region of the intron of an
existing
(genomic) DNA. The introns of a genomic DNA coding for (3-tubulin, which can
be
isolated from the abovementioned nematodes, are described by way of example of
Cylicocyclus nassatus in SEQ ID NO. 11. The introns are thus those sequences
which
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are located between the coding exons. In a preferred embodiment, the described
primers or hybridization probes contain a labeled group which makes possible
the
detection of the oligonucleotides, i.e. for example, radioisotopes,
fluorescent groups,
enzymes or enzyme cofactors.
Such oligonucleotides can be employed in diagnostic test kits in order to
determine
the origin (i.e. the organism) of an existing DNA. The oligonucleotides are
suitable
due to their specific hybridization with the DNAs mentioned under SEQ ID NO.
1, 3,
5, 7, 9 and 11 [lacuna] their fragments, sequences homologous thereto and
sequences
complementary thereto for recognizing defined sequences. Thus they also make
possible the recognition and identification of those sequences coding for 13-
tubulin
which, on account of one or more point mutations in codon 200, lead to the
expression of benzimidazole-resistant 13-tubulin. These oligonucleotides
derived from
the sequences SEQ >D NO. 1, 3, 5, 7, 9 and 11, preferably the embodiments
described under SEQ ID NO. 12 to S 1, are thus suitable for the identification
of the
frequently occurring nematode species of the subfamily of the cyathostominae,
and
for the recognition of existing resistances to benzimidazoles.
The oligonucleotides according to the present invention can be prepared using
standard methods which are familiar to the person skilled in the art, e.g. by
de novo
DNA synthesis.
The nucleic acids mentioned here can be present in partially purified or
biologically
pure form in complete cells or in cell lysates, i.e. if other cell components
or
chemical precursors and by-products have been separated off in the case of a
chemical synthesis of the DNA.
Nucleic acids coding for 13-tubulin, as described above, can be obtained
starting from
mRNA which is present in a number of eukaryotic cells. It is also possible to
obtain
the DNA according to the invention starting from genomic DNA from the nematode
cells concerned (see also the following examples). A gene coding for 13-
tubulin can
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be obtained, for example, from a cDNA library or a genomic DNA library. cDNA
can be obtained by isolating the total mRNA of a cell, e.g. of a nematode
cell.
Starting from the mRNA, double-stranded cDNA can then be prepared and inserted
into a suitable plasmid or a suitable vector. The DNA according to the
invention can
S also be obtained by amplification with the aid of the known polymerase chain
reaction (PCR) or alternatively by de novo DNA synthesis (see also J. Sambrook
et
al. (1989) Molecular Cloning, 2nd Edition, Chap. 14).
Vectors and plasmids
The present invention also comprises expression vectors which contain one of
the
nucleic acid sequences according to the invention, which are functionally
linked to a
transcription-regulatory sequence. "Functionally linked" means that the
nucleic acid
sequence is linked to the regulatory sequence in a manner such that the
expression of
1 S the protein encoded by the nucleic acid sequence can be controlled.
"Transcription-
regulatory sequences" include, for example, promoters, enhancers and other
control
elements. The expression vectors contain, for example, a gene coding for a 13-
tubulin
according to the invention or fragments thereof. These vectors can be used for
incorporation into cells where the corresponding polypeptides or alternatively
fusion
proteins are then formed. Suitable promoters for the expression of the protein
according to the invention in E.coli include natural hybrid or bacteriophage
promoters. Preferably, they are promoters from the group of the phage ~,
promoters,
omp promoters or synthetic promoters (see also WO 98/15625, DE 3 430 683,
EP 0 173 149). Suitable vectors are commercially obtainable, e.g. the
expression
vectors of the pET series (e.g. pET3a, pET23a, pET28a and His tag or pET32a
with
His tag) or pGEX with glutathion synthetase fusion. The expression vectors can
then
be transformed, for example, into DE3-lysogenic E.coli strains, e.g. BL21
(DE3),
HM S 174(DE3) or AD494(DE3).
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Expression of the 13-tubulinpolypeptides
The present invention also comprises cells which contain the nucleic acid
sequences
according to the invention (e.g. inserted into a vector or into the genome).
These host
cells can be prokaryotic or eukaryotic.
Suitable prokaryotic expression systems are, for example, bacterial systems
such as
Streptomyces spp., Bacillus subtilis, Salmonella typhimurium, Serratia
marcescens
and preferably E.coli.
A preferred eukaryotic expression system is the baculovirus system,
particularly
preferably that which allows post-translational modifications.
1 S Other eukaryotic expression systems (e.g. yeast, insect cells) can
likewise be used.
Pol~rpentides
The present invention likewise comprises f3-tubulin polypeptides which are
encoded
by the DNAs according to the invention, preferably the DNA sequences as set
forth
in SEQ m NO. 1, 3, 5, 7, 9 and 11, by fragments thereof or by homologous DNA
sequences as described above. Preferred embodiments of these 13-tubulin
polypeptides are described in the sequences as set forth in SEQ )D NO. 2, 4,
6, 8 and
10. In a preferred embodiment, the described polypeptides are purified
polypeptides
which are free of contaminating proteins of those cells in which the
polypeptides
according to the invention have been produced.
The polypeptides described are proteins of full length or fragments, motifs or
domains thereof which comprise lengths of at least 5, 10, 25, 50, 75, 100,
125, 150,
200, 250, 300, 350 or 400 amino acids. Polypeptide fragments can be obtained
and
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are selected by the testing of polypeptides which are encoded by nucleic acid
fragments derived from the sequences as set forth in SEQ ID NO. 1, 3, 5, 7, 9
and 11.
Polypeptide fragments can also be chemically synthesized in a known manner.
The invention also comprises polypeptides which are encoded by the degenerate
sequence as set forth in SEQ D7 NO. 3. Owing to the various possible bases in
defined positions of the DNA sequence, various polypeptides result having an
amino
acid encoded by the codon resulting in each case. The polypeptides encoded by
DNA
as set forth in SEQ 1D NO. 3 are described in SEQ ID NO. 4, the variable amino
acids being marked by "Xaa".
Preferred embodiments of the polypeptides are described in SEQ ID NO. 2, 4, 6,
8
and 10.
The present invention comprises all processes for the preparation of the
polypeptides
according to the invention.
It is known to the person skilled in the art that the polypeptides of the
present
invention can be obtained in various ways, e.g. by chemical methods such as
the
solid-phase method. For the obtainment of larger amounts of protein, the use
of
recombinant methods is recommended.
The underlying steps for the preparation of the recombinant D-tubulin are:
1. Obtainment of a natural, synthetic or semisynthetic DNA which codes for the
13-tubulin according to the invention.
2. Incorporation of this DNA into an expression vector which is suitable for
expressing the 13-tubulin according to the invention, either on its own or as
a
fusion protein.
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3. Transformation of a suitable, preferably prokaryotic, host cell using this
expression vector.
4. Growth of this transformed host cell in a manner which is suitable for
expressing the f3-tubulin according to the invention.
5. Harvesting of the cells and purification of the 13-tubulin by means of
suitable,
known methods.
For example, the expression vectors can be transformed in hDE3-lysogenic E.
coli
strains, e.g. BL21(DE3), HM 5174(DE3) or AD494(DE3). After the growth of the
cells under the standard conditions familiar to the person skilled in the art,
expression
is induced using IPTG. After induction of the cells, incubation is carried out
at
temperatures of 18 to 37°C for 3 to 24 hours. The cells are disrupted,
the expressed
protein is purified by means of chromatographic methods, in the case of
protein
expressed with His tag by means of FPLC on an Ni-NTA column, and also by ion
exchange chromatography, gel filtration chromatography, ultrafiltration,
electrophoresis or alternatively immunoaffinity purification, which are
specific for
the polypeptides according to the invention.
Homologs or fragments of the polypeptides according to the invention can be
generated by mutagenesis, such as, for example, by directed (point)
mutagenesis, or
by deletions.
The polypeptides according to the invention can also be chemically modified,
e.g.
with glycosyl groups, lipids, phosphates, acetyl groups or similar groups.
Covalent
derivatives can be obtained by the linkage of the modifying group with
functional
groups of the amino acid side chains or the N terminus or C terminus of the
polypeptide.
;..
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In the expression of the polypeptides according to the present invention, it
may be
advantageous to change certain codons in order to make possible optimum
expression. This is true if the use of certain codons ("codon usage") in the
heterologous expression system is different than in one of the organisms
according to
the invention. Furthermore, the deletion of the 5'- or 3'-untranslated region
is
possible, e.g. if several destabilizing sequence motifs (e.g. ATTTA) are
present in the
3' region of the cDNA.
Fusion proteins
The polypeptides according to the invention can also be present as part of a
fusion
protein. Such fusion proteins are embraced in full by the present invention.
Fusion
proteins can be useful under conditions where it is desirable to obtain an
immunogenic fragment of the 13-tubulin (see, for example, EP 0 259 149;
Schlienger
et al. ( 1992) J. Virol. 66, 2). Under certain circumstances, fusion proteins
facilitate
the expression of a polypeptide. For example, the polypeptides according to
the
invention can be prepared as glutathione S-transferase (GST) fusion proteins.
Such
GST fusion proteins facilitate easy purification of the polypeptide (see, for
example,
Current Protocols in Molecular Biology, eds. Ausubel et al. (John Wiley &
Sons,
N.Y. 1991). Fusion proteins can contain, for example, a "leader" sequence
which is
used for the purification, e.g. allows a poly-His sequence at the N terminus
(but also
at the C terminus) of the protein whose purification [lacuna] by means of
chromatography on an Ni2+ NTA column (see, for example, Hachuli et al. (1987)
J.
Chromatography 411, 177).
Techniques for preparing such fusion proteins are familiar to the person
skilled in the
art.
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Antibodies
Another aspect of the present invention relates to antibodies which react
specifically
with the 13-tubulin polypeptides according to the invention.
For example, anti-protein or anti-peptide antisera or monoclonal antibodies
can be
prepared according to standard protocols by the use of immunogens which have
been
derived from (3-tubulin polypeptides according to the invention (see, for
example,
Antibodies: A. Laboratory Manual ed. by Harlow and Lane (Cold Spring Harbor
Press, 1988)).
Mammals such as mice, hamsters or rabbits can be immunized using an
immunogenic form or an immunogenic fraction of the polypeptide according to
the
invention, e.g. using a polypeptide which is able to produce an antibody
response
(see also "fusion proteins" above). The appropriate techniques are familiar to
the
person skilled in the art. Thus an immunogenic fraction of the 13-tubulin can
be
administered in the presence of an adjuvant. The course of the immunization
can be
observed by checking the antibody titer in the plasma or serum, e.g. by
customary
ELISA assays or other immunoassays.
In a preferred embodiment, the antibodies according to the invention are
immunospecific for an antigenic determinant of a 13-tubulin polypeptide
according to
the invention, e.g. of a polypeptide as set forth in SEQ 1D NO. 2, 4, 6, 8 or
10 or
those polypeptides which are encoded by DNAs as set forth in SEQ ID NO. 1, 3,
S, 7,
9 or 11 or sequences identical to 85% therewith, preferably sequences
identical to
95%.
After the immunization of a mammal, polyclonal anti-Q-tubulin antibodies can
be
isolated from the serum. For the production of monoclonal antibodies, antibody-
producing cells (lymphocytes) can be obtained from an immunized~animal and
fused
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according to known methods with immortal cells such as myeloma cells in order
to
obtain hybridoma cells (see, for example, Kohler and Milstein (1975) Nature
256.
495-497; Kozbar et al. (1983) Immunology Today 4, 72; Cole et al. (1985)
Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. pp. 77-96).
The "antibodies" mentioned here should also include fragments of antibodies
which
react specifically with f3-tubulin according to the invention. Antibodies can
be
fragmented using conventional techniques and the fragments tested.
A preferred embodiment relates to antibodies such as described above, which
carry a
detectable label (e.g. radioisotopes, fluorescent groups, enzymes or enzyme
cofactors).
Antibodies which bind specifically to 13-tubulin polypeptides according to the
invention can also be used for the immunohistochemical staining of tissue
samples in
order to detect the expression of a specific [3-tubulin. The anti-13-tubulin
antibodies
can likewise be used for diagnostic purposes, e.g. for immunoprecipitation or
for
immunoblotting.
Diagnostic test procedures
The present invention likewise makes available nucleic acid molecules which
can be
used for diagnostic purposes.
These include nucleic acid molecules such as described above, the fragments of
the
DNA sequences described under SEQ ID NO. 1, 3, 5, 7, 9 or 11 or DNA sequences
complementary thereto. For example, oligonucleotides as set forth in SEQ m NO.
12
to 51 are made available, which are able to hybridize to sense or antisense
sequences
coding for !3-tubulin, and to intron sequence sections which are described by
way of
example in SEQ ID NO. 11.
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In this procedure, the nucleic acid of a cell is rendered accessible to
hybridization, the
DNA probe is brought into contact with the oligonucleotides, and the
hybridization
of the sample is detected using the oligonucleotide.
In this manner, a method is made available which makes it possible to
differentiate
between various species of small strongylids and/or other nematode species by
means
of the specific hybridization of the oligonucleotides according to the
invention to a
DNA probe, preferably with the aid of those oligonucleotides which hybridize
to the
intron regions of the DNA coding for 13-tubulin. In a particularly preferred
embodiment, the oligonucleotides according to the invention allow the
identification
of resistances in small (Cyathostaminae) e.g. in horses, especially
resistances of the
species Cylicocyclus nassatus, Cyathostomum coronatum and Cyathostomum
catinatum.1n this procedure, the fact can be used that resistant forms of the
13-tubulin
carry at least one .point mutation in the DNA according to the invention
coding
therefor; which can be detected by means of PCR, such as described, for
example, in
an analogous manner in Elard et al. (1998) PCR diagnosis of benzimidazole-
susceptibility or -resistance in natural populations of the small ruminant
parasite,
Teladorsagia circumcincta. Veterinary Parasitology 80, 231-237.
The method described is particularly helpful for the assessment of possible
treatment
strategies in humans and animals affected with nematodes, such as, for
example,
horses, sheep, pigs, goats, camels, buffalo, donkeys, hares, roe deer, fur-
bearing
animals, birds (e.g. chickens, turkeys, ducks), fresh- and salt-water fish
(e.g. trout,
carp). It makes possible the identification and differentiation of the
parasitic
nematodes and the recognition of resistant populations thereof, and avoid a
treatment
with inactive nematicides.
The methods described here can be made available, for example, in the form of
prefabricated diagnostic test kits which contain at least one of the
abovementioned
nucleic acid molecules or an antibody such as described above, which is
prepared in
ready-to-use form.
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Procedure for the discovery of nematicidal substances
The invention relates to a procedure in which, with the aid of tubulin or
fragments
thereof, novel, specific anthelmintic substances can be identified.
In a preferred embodiment, ~i-tubulin polypeptides according to the present
invention
are used for this. However, the procedure can also be carried out using
tubulin from
species other than those mentioned here. Procedures which use (3-tubulin
polypeptides other than those according to the invention are included in full
by the
present invention. Particularly preferably, /3-tubulin polypeptides as set
forth in SEQ
m NO. 2, 4, 6, 8 or 10 are used for the procedure mentioned. In the present
invention, recombinant /3-tubulin polypeptides from frequently occurnng
parasitic
nematodes are thus additionally made available. These can be used in various
test
systems for the identification of new inhibitors of tubulin interaction, or
the
interaction of tubulin subunits.
Cell-free test systems
Many test systems which have the testing of compounds and natural extracts as
their
aim are aimed at high throughput numbers in order to maximize the number of
substances investigated in a given period of time. Test systems which are
based on
cell-free studies use purified or semi-purified protein. They are suitable for
a "first"
test, which primarily aims at detecting a possible influence of a substance on
the
target protein.
Effects such as cell toxicity are as a rule ignored in these in vitro systems.
The test
systems in this case check both inhibitory and suppressive effects of the
substances,
and stimulatory effects. The effectiveness of a substance can be checked by
means of
concentration-dependent test series. Control batches without test substances
can be
used for the assessment of the effects.
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One possibility for the identification of substances which modulate the
interaction of
tubulin or of its subunits is the "scintillation proximity assay" (SPA), see
EP 015
473. This test system uses the interaction of a receptor (e.g. tubulin) with a
radiolabeled ligand (e.g. a small organic molecule or a second, radiolabeled
protein
molecule). The receptor is in this case bound to small spheres
("microspheres") or
beads which are provided with scintillating molecules. In the course of the
decay of
the radioactivity, the scintillating substance in the microsphere is excited
by the
subatomic particles of the radioactive label and a detectable photon is
emitted. The
test conditions are optimized such that only those particles emanating from
the ligand
lead to a signal which emanate from a ligand bound to the receptor or the
tubulin.
In one possible embodiment, tubulin is bound to the beads, either together or
without
interacting or binding test substances. a- or (3-Tubulin subunits could be
used here. A
radiolabeled ligand could be, for example, a labeled benzimidazole or a
further,
labeled (3-tubulin molecule. In the case of binding of the ligand to the
immobilized
tubulin, this ligand must inhibit or abolish an existing interaction between
immobilized and free tubulin in order to bind itself in the region of the
contact
surface. Binding to the immobilized tubulin which has taken place can then be
detected by means of a flash of light. Correspondingly, an existing complex
between
an immobilized and a free, labeled tubulin is destroyed by the binding of a
test
substance, which leads to a decrease in the detected intensity of the flash of
light. The
test system then corresponds to a complementary inhibition system.
Test svstem based on cells
The ~i-tubulin available by means of the present invention, but also tubulin
from
other species, makes possible the development of test systems, which are based
on
cells, for the identification of substances which inhibit the tubulin
interaction.
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An example of such a test system is the "two hybrid system". A specific
example of
this is the "interaction trap". This is a genetic selection of interacting
proteins in yeast
(see, for example, Gyuris et al. (1993) Cdi 1, a human G1 and S phase protein
phosphatase that associates with Cdk 2. Cell 75, 791-803). The test system is
designed to detect and to describe the interaction of two proteins in that an
interaction which has taken place leads to a detectable signal.
Such a test system can also be adapted to the testing of large numbers of test
substances in a given period of time.
The system is based on the construction of two vectors, the "bait" vector and
the
"prey" vector. A gene coding for tubulin, preferably a gene coding for a ~i-
tubulin
according to the invention, is cloned in the bait vector and then expressed as
a fusion
protein with the LexA protein, a DNA-binding protein. A second gene, coding
for
1 S tubulin, preferably for a (i-tubulin according to the invention, is cloned
in the prey
vector, where it is expressed as a fusion protein with the B42 prey protein.
Both
vectors are present in a Saccharomyces cerevisiae host, which contains copies
of a
LexA-binding DNA on the 5'-side of a lacZ or HIS3 reporter gene. If an
interaction
takes place between the two tubulin (fusion) proteins, activation of the
transcription
of the reporter gene occurs. If the presence of a test substance leads to the
inhibition
or disturbance of the tubulin interaction, the two tubulin (fusion) proteins
can no
longer interact and the product of the reporter gene is no longer prepared.
With the aid of tubulin, particularly of the ~i-tubulin according to the
invention or
fragments thereof, and the processes described above, it is possible to
identify new
and specific antiparasitic compounds.
Compounds which are found with the aid of the processes and polypeptides
described
are valuable for the treatment of humans and animals which are infected with
pathogenic endoparasites of the human or of agricultural animals, pets, zoo
animals,
and laboratory and experimental animals.
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The compounds are active against all stages of development of normal,
sensitive
strains and also resistant strains. By treatment with agents which contain one
or more
of these compounds, both economic losses in the case of agricultural animals
and
diseases in humans and animals can be avoided or treated. The following
parasites
are in this case of particular interest as targets of the active compounds
found:
En~lida, e.g. Trichuris spp., Capillaria spp., Trichomosoides spp.,
Trichinella spp.
Rhabditia, e.g. Micronema spp., Strongyloides spp.
Strongylida, e.g. Strongylus spp., Triodontophorus spp., Oesophagodontus spp.,
Trichonema spp., Gyalocephalus spp., Cylindropharynx spp., Poteriostomum spp.,
Cyclococercus spp., Cylicostephanus spp., Oesophagostomum spp., Chabertia
spp.,
Stephanurus spp., Ancylostoma spp., Uncinaria spp., Bunostomum spp., Globoce-
phalus spp., Syngamus spp., Cyathostomum spp., Cylicocyclus spp.,
Neostrongylus
spp., Cystocaulus spp., Pneumostrongylus spp., Spicocaulus spp.,
Elaphostrongylus
spp., Parelaphostrongylus spp., Crenosoma spp., Paracrenosoma spp., Angiostron
gylus spp., Aelurostrongylus spp., Filaroides spp., Paraftlaroides spp.,
Tricho
strongylus spp., Haemonchus spp., Ostertagia spp., Marshallagia spp., Cooperia
spp., Nematodirus spp., Hyostrongylus spp., Obeliscoides spp., Amidostomum
spp.,
Ollulanus spp.
Oxyurida, e.g. Oxyuris spp., Enterobius spp., Passalurus spp., Syphacia spp.,
Aspi-
culuris spp., Heterakis spp.
Ascaridia, e.g. Ascaris spp., Toxascaris spp., Toxocara spp., Parascaris spp.,
Anisakis
spp., Ascaridia spp.
Spirurida, e.g. Gnathostoma spp., Physaloptera spp., Thelazia spp.,
Gongylonema
spp., Habronema spp., Parabronema spp., Draschia spp., Dracunculus spp.
Filariida, e.g. Stephanofilaria spp., Parafilaria spp., Setaria spp., Loa
spp.,
Dirofilaria spp., Litomosoides spp., Brugia spp., Wuchereria spp., Onchocerca
spp.
Gigantorhynchida, e.g. Filicollis spp., Moniliformis spp., Macracanthorhynchus
spp.,
Prosthenorchis spp.
Mastigophora (Fla;~llata)
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Trypanosomatidae. e.g. Trypanosoma b. brucei, T. b. gambiense, T. b.
rhodesiense,
T. congolense, T. cruzi, T. evansi, T. equinum, T. lewisi, T. percae, T.
simiae, T.
vivax, Leishmania brasiliensis, L. donovani, L. tropica
Trichomonadidae , e.g. Giardia lambilia, G. cams.
Sarcomastigophora (Rhizopoda), e.g. Entamoeba histolytica
Hartmanellidae, e.g. Acanthamoeba sp., Hartmanella spp.
Ayicom~lexa (Sporozoa), e.g. Eimeria acervulina, E. adenoides, E.
alabahmensis, E.
anatis, E. anseris, E. arloingi, E. ashata, E. auburnensis, E. bovis, E.
brunetti, E.
canis, E. chinchillae, E. clupearum, E. columbae, E. contorta, E. crandalis,
E.
debliecki, E. dispersa, E. ellipsoidales, E. falciformis, E. faurei, E.
labbeana, E.
leucarti, E. magna, E. maxima, E. media, E. meleagridis. E. meleagrimitis, E.
mitis;
E. necatrix, E. ninakohlyakimovae, E. ovis, E. parva, E. pavonis, E.
perforans, E.
phasani, E. piriformis, E. praecox, E. residua, E. scabra, E. spec., E.
stiedai, E. suis,
E. tenella, E. truncata, E. truttae, E. zuernii, Globidium spec., Isospora
belli, I.
canis, I. fells, 1. ohioensis, I. rivolta, 1. spec., 1. suis, Neospora
caninum,
Cystisospora spec. Cryptosporidium spec.
Toxoplasmadidae, e.g. Toxoplasma gondii
Sarcocystidae, e.g. Sarcocystis bovicanis, S. bovihominis, S. neuvona, S.
ovicanis, S.
ovifelis, S. spec., S suihominis
Leucozoide, e.g. Leucozytozoon simondi
Plasmodiidae, e.g. Plasmodium berghei, P. falciparum, P. malariae, P. ovate,
P.
vivax, P. spec.
Piroplasmea, e.g. Babesia argentina, B. bovis, B. canis, B. spec., Theileria
parva, T.
spec.
Adeleina, e.g. Hepatozoon canis, H. spec.
The following are furthermore of importance
Myxospora and Microspora, e.g. Glugea spec. and Nosema spec., and Pneumocystis
carinii, Ciliophora (Ciliata), e.g. Balantidium toll, Ichthiophthirius spec.,
Trichondina spec. or E is lis spec.
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The compounds and agents found are likewise effective against protozoa of
insects,
such as those of the strain Microsporidia, particularly those of the order
Nosema,
very particularly those of the species Nosema apis, which are parasites of
honeybees.
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Examples
Example 1
Obtainment of~i-tubulin cDNA and ,~enomic DNA
mRNA was obtained with the aid of the Quick Prep~ Micro mRNA kit (Pharmacia
Biotech, Freiburg, Germany) from C. nassatus worms and from C. coronatum and
C.catinatum worms using the Dynal~ mRNA Direct Kit (Dynal, Hamburg,
Germany). The worms were isolated from the large intestine of horses and
differentiated microscopically according to the characterized structure of
head and
tail (see R. S. Lichterlfeld (1975), Helminths of domestic equids. Proceedings
of the
Helminthological Society, Washington, 42 (Special issue), 1-92).
The synthesis of the cDNA was carried out with the aid of the "reverse
transcription
system" (Promega, Madison, USA) in the case of the mRNA from C. nassatus and
using the superscript RTII reverse transcriptase (Gibco BRL Life Technologies)
in
the case of the mRNA from G coronatum and C. catinatum. In the cases
mentioned,
oligonucleotides having a length of 15 base pairs were used. Incubation was
carried
out for one hour at 42°C.
Genomic DNA was obtained from 4 to 40 adult worms using the QIA Amp-tissue kit
(Qiagen, Hilden, Germany). In this procedure, the worms were digested for 2
hours at
55°C with proteinase K and the genomic DNA extracted using "spin
columns".
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Example 2
Amplification of Jai-tubulin sequences
S The amplification of ~i-tubulin sequences of full length or of fragments can
be carried
out, for example, using AmpliTaq GoIdTM polymerase (Perkin Elmer, Foster City,
California, USA).
An amplification of the /3-tubulin sequences as set forth in SEQ 1D NO. 1, 3,
S, 7, 9
or 11 or fragments thereof can be carned out with the aid of the primers as
set forth
in SEQ m NO. 12 - 51.
For the amplification of the cDNA from C. coronatum, for example, the
sequences as
set forth in SEQ m NO. 43 and 44 are suitable, for the amplification of the
cDNA
from C. catinatum, the sequences as set forth in SEQ ID NO. 40 and 42 are
particularly suitable.
The amplification of the C. nassatus cDNA and of the genomic DNA of all
species
according to the present invention was carned out in a total volume of 50 p1,
containing 5 p1 of 10 x buffer, 2.5 p1 of MgCl2 (25 mM), 2 p1 of dNTP mix (2mM
for each NTP), 1 p1 of each specific primer (SEQ )D NO. 12 - 47) (50 p moUpl),
0.5
p1 (2.5 U) of polymerase and 1 - S p1 of DNA template. When using degenerate
primers (SEQ >D NO. 48 - 51 ), 1 p1 of each primer of a conc. of 500 pmol/p.l
was
employed. The annealing was carried out in the case of degenerate primers at
46°C,
in the case of specific primers the temperature was varied according to the
calculated
melting temperature. The PCR cycles were chosen as follows:
95°C for 10 min, then 35 - 40 cycles with 1 min denaturation at
94°C, 1 min
annealing, 1 min at 72°C and a final step at 72°C for 10 min. In
the amplification of
cDNA from C. coronatum and C. catinatum, a "touchdown" PCR temperature
program was carned out, which has the following profile:
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firstly 15 cycles at 94°C for 30 sec, then 1 min at 60°C and 1
min at 72°C, followed
by 1 S cycles with 30 sec at 95°C, 55°C for 1 min and
72°C for 1 min and finally 10
cycles at 95°C for 30 sec, then 45°C for 1 min and 72°C
for 1 min. For the
amplification of larger fragments (>1000 base pairs), the elongation phase at
72°C
was lengthened to 2.30 min.
Example 3
PCR products from the amplification of cDNA or genomic DNA from C. nassatus,
C. coronatum and C. catinatum were cloned with the aid of the "Original TA
Cloning Kit" /Invitrogen, Leek, Netherlands), namely in the "Original TA
Cloning~"
vector.
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SEQUENZPROTOKOLL
<110> Bayer AG
S
<120> DNA kodierend fur Beta-Tubulin and deren Verwendung
<130> Le A 33 759
<140>
<141>
<160> 51
IS <170> PatentIn Ver. 2.1
<210> 1
<211> 1380
<212> DNA
<213> Cyathostomum coronatum
<220>
<221> CDS
<222> (1)..(1344)
<400> 1
atg cgt gag atc gtg cat gta caa get gga caa tgt gga aac caa att 48
Met Arg Glu Ile Val His Val Gln Ala Gly Gln Cys Gly Asn Gln Ile
1 5 10 15
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-2-
ggt tcc aag ttt tgg gaa gtg atc tct gac gag cat ggc att aag ccc 96
Gly Ser Lys Phe Trp Glu Val Ile Ser Asp Glu His Gly Ile Lys Pro
20 25 30
S gat ggc aca tac cac gga gaa tct gat cta caa tta gaa cga atc aat
144
Asp Gly Thr Tyr His Gly Glu Ser Asp Leu Gln Leu Glu Arg Ile Asn
35 40 45
gtg tac tat aat gaa gca cat gga ggc aaa tat gtc cca cgt gca gtt
192
Val Tyr Tyr Asn Glu Ala His Gly Gly Lys Tyr Val Pro Arg Ala Val
50 55 60
ctt gtt gat ctc gag ccc gga act atg gat tcc gtc cgt tcc ggg cca
240
Leu Val Asp Leu Glu Pro Gly Thr Met Asp Ser Val Arg Ser Gly Pro
65 70 75 80
tac ggg caa ttg ttc cgc cct gat aac tac gtg ttt gga cag tct ggc
288
Tyr Gly Gln Leu Phe Arg Pro Asp Asn Tyr Val Phe Gly Gln Ser Gly
85 90 95
gca gga aat aac tgg gca aaa ggt cac tac act gaa ggc get gaa ctt
336
Ala Gly Asn Asn Trp Ala Lys Gly His Tyr Thr Glu Gly Ala Glu Leu
100 105 110
gtc gac aat gta cta gat gta gtg cga aaa gaa gca gaa gga tgt gac
384
Val Asp Asn Val Leu Asp Val Val Arg Lys Glu Ala Glu Gly Cys Asp
115 120 125
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-3-
tgt ctg cag ggc ttc cag cta act cac tca ctt gga gga ggt acc ggt
432
Cys Leu Gln Gly Phe Gln Leu Thr His Ser Leu Gly Gly Gly Thr Gly
$ 130 135 140
tcg ggt atg ggc act ctc ctc atc tcc aaa att cgg gag gag tat cct
480
Ser Gly Met Gly Thr Leu Leu Ile Ser Lys Ile Arg Glu Glu Tyr Pro
1~ 145 150 155 160
gat aga atc atg tcc tcg ttc tcc gtt gtc ccc tca cca aag gtc tcc
528
Asp Arg Ile Met Ser Ser Phe Ser Val Val Pro Ser Pro Lys Val Ser
1$ 165 170 175
gac act gtt gtg gag cct tac aat get acc cta tcc gtt cat cag ttg
576
Asp Thr Val Val Glu Pro Tyr Asn Ala Thr Leu Ser Val His Gln Leu
180 185 190
gtt gaa aat aca gac gag act tat tgt att gac aat gaa gcc ctg tat
624
Val Glu Asn Thr Asp Glu Thr Tyr Cys Ile Asp Asn Glu Ala Leu Tyr
2$ 195 200 205
gat att tgc ttc cgc act ttg aaa ctc acg aac cca act tat gga gat
672
Asp Ile Cys Phe Arg Thr Leu Lys Leu Thr Asn Pro Thr Tyr Gly Asp
210 215 220
ctg aat cat ctt gtg tct gta aca atg tct ggt gtc acc aca tgt ctt
720
WO 01/04281 CA 02378407 2002-O1-04 pCT/EP00/06104
-4-
Leu Asn His Leu Val Ser Val Thr Met Ser Gly Val Thr Thr Cys Leu
225 230 235 240
cgc ttc cct ggc caa ttg aat gcc gat cta cgc aaa cta get gtt aac
$ 768
Arg Phe Pro Gly Gln Leu Asn Ala Asp Leu Arg Lys Leu Ala Val Asn
245 250 255
atg gtt cca ttc cct cgt ctt cac ttc ttc atg cct ggt ttt get cct
816
Met Val Pro Phe Pro Arg Leu His Phe Phe Met Pro Gly Phe Ala Pro
260 265 270
ctt tct get aaa ggt get cag get tac cgt get ctt acc gta gcc gag
1$ 864
Leu Ser Ala Lys Gly Ala Gln Ala Tyr Arg Ala Leu Thr Val Ala Glu
275 280 285
ctt aca cag cag atg ttt gat get aag aat atg atg get get tgc gac
912
Leu Thr Gln Gln Met Phe Asp Ala Lys Asn Met Met Ala Ala Cys Asp
290 295 300
cct cga cat gga cgt tat ctc acc gtc gca gcc atg ttc cga gga aga
2$ 960
Pro Arg His Gly Arg Tyr Leu Thr Val Ala Ala Met Phe Arg Gly Arg
305 310 315 320
atg agc atg agg gaa gta gac gac cag atg atg tca gtg cag aac aag
1008
Met Ser Met Arg Glu Val Asp Asp Gln Met Met Ser Val Gln Asn Lys
325 330 335
WO 01/04281 CA 02378407 2002-O1-04 pCT/EP00/06104
-$-
aac tcc tca tac ttc gta gag tgg atc ccg aac aac gtg aag acc get
1056
Asn Ser Ser Tyr Phe Val Glu Trp Ile Pro Asn Asn Val Lys Thr Ala
340 345 350
$
gta tgc gac atc ccg cca cga gga ctg aag atg gcc get acc ttc gtt
1104
Val Cys Asp Ile Pro Pro Arg Gly Leu Lys Met Ala Ala Thr Phe Val
355 360 365
gga aac tca act gcc atc caa gag ctg ttc aag cgc att tca gaa caa
1152
Gly Asn Ser Thr Ala Ile Gln Glu Leu Phe Lys Arg Ile Ser Glu Gln
370 375 380
1$
ttt aca gcc atg ttc cgc cgc aaa gcg ttc ttg cat tgg tac act ggt
1200
Phe Thr Ala Met Phe Arg Arg Lys Ala Phe Leu His Trp Tyr Thr Gly
385 390 395 400
gaa ggt atg gac gag atg gag ttc act gaa gca gag tcc aac atg aat
1248
Glu Gly Met Asp Glu Met Glu Phe Thr Glu Ala Glu Ser Asn Met Asn
405 410 415
2$
gat ctc atc tcc gag tac caa cag tac cag gaa gcc acc get gac gac
1296
Asp Leu Ile Ser Glu Tyr Gln Gln Tyr Gln Glu Ala Thr Ala Asp Asp
420 425 430
atg ggc gat ctt gat gcg gaa ggc get gaa gag get tat cct gag gaa
1344
Met Gly Asp Leu Asp Ala Glu Gly Ala Glu Glu Ala Tyr Pro Glu Glu
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-6-
435 440 445
taaaccagca gatcgtgttg cgttgttcgt ttctct
1380
<210> 2
<211> 448
<212> PRT
<213> Cyathostomum coronatum
<400> 2
Met Arg Glu Ile Val His Val Gln Ala Gly Gln Cys Gly Asn Gln Ile
1 5 10 15
1$
Gly Ser Lys Phe Trp Glu Val Ile Ser Asp Glu His Gly Ile Lys Pro
25 30
Asp Gly Thr Tyr His Gly Glu Ser Asp Leu Gln Leu Glu Arg Ile Asn
20 35 40 45
Val Tyr Tyr Asn Glu Ala His Gly Gly Lys Tyr Val Pro Arg Ala Val
50 55 60
Leu Val Asp Leu Glu Pro Gly Thr Met Asp Ser Val Arg Ser Gly Pro
65 70 75 80
Tyr Gly Gln Leu Phe Arg Pro Asp Asn Tyr Val Phe Gly Gln Ser Gly
85 90 95
WO 01/04281 CA 02378407 2002-O1-04 PCT/EP00/06104
_ '7 _
Ala Gly Asn Asn Trp Ala Lys Gly His Tyr Thr Glu Gly Ala Glu Leu
100 105 110
Val Asp Asn Val Leu Asp Val Val Arg Lys Glu Ala Glu Gly Cys Asp
$ 115 120 125
Cys Leu Gln Gly Phe Gln Leu Thr His Ser Leu Gly Gly Gly Thr Gly
130 135 140
Ser Gly Met Gly Thr Leu Leu Ile Ser Lys Ile Arg Glu Glu Tyr Pro
145 150 155 160
Asp Arg Ile Met Ser Ser Phe Ser Val Val Pro Ser Pro Lys Val Ser
165 170 175
1$
Asp Thr Val Val Glu Pro Tyr Asn Ala Thr Leu Ser Val His Gln Leu
180 185 190
Val Glu Asn Thr Asp Glu Thr Tyr Cys Ile Asp Asn Glu Ala Leu Tyr
195 200 205
Asp Ile Cys Phe Arg Thr Leu Lys Leu Thr Asn Pro Thr Tyr Gly Asp
210 215 220
2$ Leu Asn His Leu Val Ser Val Thr Met Ser Gly Val Thr Thr Cys Leu
225 230 235 240
Arg Phe Pro Gly Gln Leu Asn Ala Asp Leu Arg Lys Leu Ala Val Asn
245 250 255
WO 01/04281 CA 02378407 2002-O1-04 PCT/EP00/06104
_g_
Met Val Pro Phe Pro Arg Leu His Phe Phe Met Pro Gly Phe Ala Pro
260 265 270
Leu Ser Ala Lys Gly Ala Gln Ala Tyr Arg Ala Leu Thr Val Ala Glu
$ 275 280 285
Leu Thr Gln Gln Met Phe Asp Ala Lys Asn Met Met Ala Ala Cys Asp
290 295 300
Pro Arg His Gly Arg Tyr Leu Thr Val Ala Ala Met Phe Arg Gly Arg
305 310 315 320
Met Ser Met Arg Glu Val Asp Asp Gln Met Met Ser Val Gln Asn Lys
325 330 335
IS
Asn Ser Ser Tyr Phe Val Glu Trp Ile Pro Asn Asn Val Lys Thr Ala
340 345 350
Val Cys Asp Ile Pro Pro Arg Gly Leu Lys Met Ala Ala Thr Phe Val
355 360 365
Gly Asn Ser Thr Ala Ile Gln Glu Leu Phe Lys Arg Ile Ser Glu Gln
370 375 380
2$ Phe Thr Ala Met Phe Arg Arg Lys Ala Phe Leu His Trp Tyr Thr Gly
385 390 395 400
Glu Gly Met Asp Glu Met Glu Phe Thr Glu Ala Glu Ser Asn Met Asn
405 410 415
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-9-
Asp Leu Ile Ser Glu Tyr Gln Gln Tyr Gln Glu Ala Thr Ala Asp Asp
420 425 430
Met Gly Asp Leu Asp Ala Glu Gly Ala Glu Glu Ala Tyr Pro Glu Glu
$ 435 440 445
<210> 3
1~ <211> 1429
<212> DNA
<213> Cylicocyclus nassatus
<220>
1$ <221> CDS
<222> (1) . . (1362)
<400> 3
aag ttc tct act gca ata atg cgt gag atc gtg cat gta caa get gga 48
Lys Phe Ser Thr Ala Ile Met Arg Glu Ile Val His Val Gln Ala Gly
1 5 10 15
car tgt gga aac caa att ggy tcc aag tty tgg gaa gtg atc tct gac 96
Gln Cys Gly Asn Gln Ile Xaa Ser Lys Phe Trp Glu Val Ile Ser Asp
2$ 20 25 30
gag cac ggc att aag ccy gay ggc aca tac cay gga gaa tct gay yta
144
Glu His Gly Ile Lys Xaa Asp Gly Thr Tyr His Gly Glu Ser Asp Xaa
35 40 45
WO 01/04281 CA 02378407 2002-O1-04 pCT/EP00/06104
- 10-
caa tta gaa cga atc aat gtg tac tat aat gaa gca cat gga ggc aar
192
Gln Leu Glu Arg Ile Asn Val Tyr Tyr Asn Glu Ala His Gly Gly Lys
50 55 60
tat gtc ccg cgt gca gtt ctt gtt gat ctc gag ccc gga act atg gat
240
Tyr Val Pro Arg Ala Val Leu Val Asp Leu Glu Pro Gly Thr Met Asp
65 70 75 80
to gtc cgy tcy ggg cca tac ggg caa ttg ttc cgc cct gat aac tac
288
Xaa Val Xaa Xaa Gly Pro Tyr Gly Gln Leu Phe Arg Pro Asp Asn Tyr
1$ 85 90 95
gtg ttt gga cag tct ggc gca gga aat aac tgg gca aaa ggt cac tac
336
Val Phe Gly Gln Ser Gly Ala Gly Asn Asn Trp Ala Lys Gly His Tyr
loo l05 llo
act gaa ggy get gaa ctt gtc gac aat gta cta gat gta gtg cga aaa
384
Thr Glu Xaa Ala Glu Leu Val Asp Asn Val Leu Asp Val Val Arg Lys
2$ 115 120 125
gaa get gaa gga tgt gac tgt ctg cag ggc ttc cag cta act cac tca
432
Glu Ala Glu Gly Cys Asp Cys Leu Gln Gly Phe Gln Leu Thr His Ser
130 135 140
ctt gga gga ggt acc gga tcg rgt atg ggc acw ctc ctc atc tyc aaa
480
W~ 01/04281 CA 02378407 2002-O1-04 pCT/EP00/06104
-11-
Leu Gly Gly Gly Thr Gly Ser Xaa Met Gly Xaa Leu Leu Ile Xaa Lys
150 155 160
145
att cgg gag gag tat cct gat aga atc atr tcc tcg ttc tyc gtt gtt
528
Ile Arg Glu Glu Tyr Pro Asp Arg Ile Xaa Ser Ser Phe Xaa Val Val
165 170 175
ccc tca cca aag gtc tyc gay acy gtt gtg gag ccg tac aat get acc
576
Pro Ser Pro Lys Val Xaa Asp Xaa Val Val Glu Pro Tyr Asn Ala Thr
180 185 190
cta tcc gtt cat cag ttg gtt gaa aat aca gac gar act twc tgt att
1$ 624
Leu Ser Val His Gln Leu Val Glu Asn Thr Asp Glu Thr Xaa Cys Ile
195 200 205
gac aat gaa get ctt tat gat att tgc ttc cgc acy ytg aaa ctc acs
67z
Asp Asn Glu Ala Leu Tyr Asp Ile Cys Phe Arg Xaa Xaa Lys Leu Xaa
210 215 220
aac cca act tat gga gat ctg aat cat ctt gtg tct gta aca atg tct
720
Asn Pro Thr Tyr Gly Asp Leu Asn His Leu Val Ser Val Thr Met Ser
230 235 240
225
ggy gtc act aca tgy ctt cgc ttc cct ggc caa ttg rry gcc gat ctw
768
Xaa Val Thr Thr Cys Leu Arg Phe Pro Gly Gln Leu Xaa Ala Asp Xaa
245 250 255
WO 01/04281 CA 02378407 2002-O1-04 PCT/EP00/06104
-12-
cgt aaa cta get gtt aac atg gyt cca~ttc cct cgt ctt cac tty tty
816
Arg Lys Leu Ala Val Asn Met Xaa Pro Phe Pro Arg Leu His Phe Phe
260 265 270
$
atg cct ggc ttt get ccc ctc tct gcy aaa ggc gcy cag get tac cgt
864
Met Pro Gly Phe Ala Pro Leu Ser Xaa Lys Gly Xaa Gln Ala Tyr Arg
275 280 285
get ctt act gta gcc gag ctw acy caa yag atg ttc gat gcc aaa aat
912
Ala Leu Thr Val Ala Glu Xaa Xaa Gln Xaa Met Phe Asp Ala Lys Asn
290 295 300
1$
atg atg gcc get tgc gac cct cga cat gga crt tat ctc acc gty gca
960
Met Met Ala Ala Cys Asp Pro Arg His Gly Xaa Tyr Leu Thr Xaa Ala
305 310 315 320
gcc atg ttc cga gga cga atg agc ayg agg gar gta gac gac cag atg
1008
Ala Met Phe Arg Gly Arg Met Ser Xaa Arg Glu Val Asp Asp Gln Met
325 330 335
2$
atg tca gtg cag aac aag aac tcc tca tac ttc gta gag tgg att ccg
1056
Met Ser Val Gln Asn Lys Asn Ser Ser Tyr Phe Val Glu Trp Ile Pro
340 345 350
aac aac gtc aar acc gcy gta tgc gac att ccg ccr aga gga ctg aaa
1104
Asn Asn Val Lys Thr Xaa Val Cys Asp Ile Pro Xaa Arg Gly Leu Lys
CA 02378407 2002-O1-04
WO 01/04281 PCT/EP00/06104
-13-
355 360 365
atg gcc get acc ttc gtt gga aac yca act gcc aty caa gag ctg tty
1152
$ Met Ala Ala Thr Phe Val Gly Asn Xaa Thr Ala Xaa Gln Glu Leu Phe
370 375 380
aag cgc att tca gaa caa tty aca get atg ttc cgc cgc aaa gcg tty
1200
1~ Lys Arg Ile Ser Glu Gln Phe Thr Ala Met Phe Arg Arg Lys Ala Phe
385 390 395 400
ttg cat ygg tay act ggw gaa ggt atg gay gag atg gag ttc act gaa
1248
1$ Leu His Xaa Tyr Thr Xaa Glu Gly Met Asp Glu Met Glu Phe Thr Glu
405 410 415
gcc gag tcc aac atg aat gat ctc atc tcc gaa tac car caa tac cag
1296
Ala Glu Ser Asn Met Asn Asp Leu Ile Ser Glu Tyr Gln Gln Tyr Gln
420 425 430
gaa get acm get gac gat atg ggc gat ctc gat gcg gaa ggc get gaa
1344
2$ Glu Ala Xaa Ala Asp Asp Met Gly Asp Leu Asp Ala Glu Gly Ala Glu
435 440 445
gag get tac cct gar gaa tagamcagca gaytgtgttg cgttgttcgt
1392
3~ Glu Ala Tyr Pro Glu Glu
450
WO 01/04281 CA 02378407 2002-O1-04 pCT/EP00/06104
-14-
ttctctrtgt caatgcgaaa tacacattga ttgcgtt
1429
$ <210> 4
<211> 454
<212> PRT
<213> Cylicocyclus nassatus
<400> 4
Lys Phe Ser Thr Ala Ile Met Arg Glu Ile Val His Val Gln Ala Gly
1 5 10 15
Gln Cys Gly Asn Gln Ile Xaa Ser Lys Phe Trp Glu Val Ile Ser Asp
1$ 20 25 30
Glu His Gly Ile Lys Xaa Asp Gly Thr Tyr His Gly Glu Ser Asp Xaa
35 40 45
Gln Leu Glu Arg Ile Asn Val Tyr Tyr Asn Glu Ala His Gly Gly Lys
50 55 60
Tyr Val Pro Arg Ala Val Leu Val Asp Leu Glu Pro Gly Thr Met Asp
65 70 75 80
Xaa Val Xaa Xaa Gly Pro Tyr Gly Gln Leu Phe Arg Pro Asp Asn Tyr
85 90 95
Val Phe Gly Gln Ser Gly Ala Gly Asn Asn Trp Ala Lys Gly His Tyr
3~ 100 105 110
CA 02378407 2002-O1-04
WO 01/04281 PCT/EP00/06104
-15-
Thr Glu Xaa Ala Glu Leu Val Asp Asn Val Leu Asp Val Val Arg Lys
115 120 125
$ Glu Ala Glu Gly Cys Asp Cys Leu Gln Gly Phe Gln Leu Thr His Ser
130 135 140
Leu Gly Gly Gly Thr Gly Ser Xaa Met Gly Xaa Leu Leu Ile Xaa Lys
145 150 155 160
1~
Ile Arg Glu Glu Tyr Pro Asp Arg Ile Xaa Ser Ser Phe Xaa Val Val
165 170 175
Pro Ser Pro Lys Val Xaa Asp Xaa Val Val Glu Pro Tyr Asn Ala Thr
1$ 180 185 190
Leu Ser Val His Gln Leu Val Glu Asn Thr Asp Glu Thr Xaa Cys Ile
195 200 205
20 Asp Asn Glu Ala Leu Tyr Asp Ile Cys Phe Arg Xaa Xaa Lys Leu Xaa
210 215 220
Asn Pro Thr Tyr Gly Asp Leu Asn His Leu Val Ser Val Thr Met Ser
225 230 235 240
Xaa Val Thr Thr Cys Leu Arg Phe Pro Gly Gln Leu Xaa Ala Asp Xaa
245 250 255
Arg Lys Leu Ala Val Asn Met Xaa Pro Phe Pro Arg Leu His Phe Phe
260 265 270
WO 01/04281 CA 02378407 2002-O1-04 pCT~P00/06104
-16-
Met Pro Gly Phe Ala Pro Leu Ser Xaa Lys Gly Xaa Gln Ala Tyr Arg
275 280 285
$ Ala Leu Thr Val Ala Glu Xaa Xaa Gln Xaa Met Phe Asp Ala Lys Asn
290 295 300
Met Met Ala Ala Cys Asp Pro Arg His Gly Xaa Tyr Leu Thr Xaa Ala
305 310 315 320
1~
Ala Met Phe Arg Gly Arg Met Ser Xaa Arg Glu Val Asp Asp Gln Met
325 330 335
Met Ser Val Gln Asn Lys Asn Ser Ser Tyr Phe Val Glu Trp Ile Pro
15 340 345 350
Asn Asn Val Lys Thr Xaa Val Cys Asp Ile Pro Xaa Arg Gly Leu Lys
355 360 365
20 Met Ala Ala Thr Phe Val Gly Asn Xaa Thr Ala Xaa Gln Glu Leu Phe
370 375 380
Lys Arg Ile Ser Glu Gln Phe Thr Ala Met Phe Arg Arg Lys Ala Phe
385 390 395 400
Leu His Xaa Tyr Thr Xaa Glu Gly Met Asp Glu Met Glu Phe Thr Glu
405 410 415
Ala Glu Ser Asn Met Asn Asp Leu Ile Ser Glu Tyr Gln Gln Tyr Gln
420 425 430
CA 02378407 2002-O1-04
WO 01/04281 PCT/EP00/06104
-17-
Glu Ala Xaa Ala Asp Asp Met Gly Asp Leu Asp Ala Glu Gly Ala Glu
435 440 445
$ Glu Ala Tyr Pro Glu Glu
450
<zlo> 5
<211> 1428
<212> DNA
<213> Cylicocyclus nassatus
1$ <220>
<221> CDS
<222> (1)..(1362)
<400> 5
2~ aag ttc tct act gca ata atg cgt gag atc gtg cat gta caa get gga 48
Lys Phe Ser Thr Ala Ile Met Arg Glu Ile Val His Val Gln Ala Gly
1 5 10 15
cag tgt gga aac caa att ggc tcc aag ttt tgg gaa gtg atc tct gac 96
2$ Gln Cys Gly Asn Gln Ile Gly Ser Lys Phe Trp Glu Val Ile Ser Asp
25 30
gag cac ggc att aag cct gat ggc aca tac cac gga gaa tct gat tta
144
Glu His Gly Ile Lys Pro Asp Gly Thr Tyr His Gly Glu Ser Asp Leu
WO 01/04281 CA 02378407 2002-O1-04 pCT~P00/06104
-18-
35 40 45
caa tta gaa cga atc aat gtg tac tat aat gaa gca cat gga ggc aaa
192
$ Gln Leu Glu Arg Ile Asn Val Tyr Tyr Asn Glu Ala His Gly Gly Lys
50 55 60
tat gtc ccg cgt gca gtt ctt gtt gat ctc gag ccc gga act atg gat
240
Tyr Val Pro Arg Ala Val Leu Val Asp Leu Glu Pro Gly Thr Met Asp
65 70 75 80
tcg gtc cgt tcc ggg cca tac ggg caa ttg ttc cgc cct gat aac tac
288
1$ Ser Val Arg Ser Gly Pro Tyr Gly Gln Leu Phe Arg Pro Asp Asn Tyr
85 90 95
gtg ttt gga cag tct ggc gca gga aat aac tgg gca aaa ggt cac tac
336
Val Phe Gly Gln Ser Gly Ala Gly Asn Asn Trp Ala Lys Gly His Tyr
100 105 110
act gaa ggc get gaa ctt gtt gac aat gta cta gat gta gtg cga aaa
384
2$ Thr Glu Gly Ala Glu Leu Val Asp Asn Val Leu Asp Val Val Arg Lys
115 120 125
gaa get gaa gga tgt gac tgt ctg cag ggc ttc cag cta act cac tca
432
Glu Ala Glu Gly Cys Asp Cys Leu Gln Gly Phe Gln Leu Thr His Ser
130 135 140
WO 01/04281 CA 02378407 2002-0l-04 PCT/EP00/06104
-19-
ctt gga gga ggt acc gga tcg ggt atg ggc act ctc ctc atc tcc aaa
480
Leu Gly Gly Gly Thr Gly Ser Gly Met Gly Thr Leu Leu Ile Ser Lys
145 150 155 160
att cgg gag gag tat cct gat aga atc atg tcc tcg ttc tcc gtt gtt
528
Ile Arg Glu Glu Tyr Pro Asp Arg Ile Met Ser Ser Phe Ser Val Val
165 170 175
ccc tca cca aag gtc tcc gac acc gtt gtg gag ccg tac aat get acc
576
Pro Ser Pro Lys Val Ser Asp Thr Val Val Glu Pro Tyr Asn Ala Thr
180 185 190
cta tcc gtt cat cag ttg gtt gaa aat aca gac gag act ttc tgt att
624
Leu Ser Val His Gln Leu Val Glu Asn Thr Asp Glu Thr Phe Cys Ile
195 200 205
gac aat gaa get ctt tat gat att tgc ttc cgc act ttg aaa ctc acg
672
Asp Asn Glu Ala Leu Tyr Asp Ile Cys Phe Arg Thr Leu Lys Leu Thr
210 215 220
aac cca act tat gga gat ctg aat cat ctt gtg tct gta aca atg tct
720
Asn Pro Thr Tyr Gly Asp Leu Asn His Leu Val Ser Val Thr Met Ser
225 230 235 240
ggt gtc act aca tgt ctt cgc ttc cct ggc caa ttg aat gcc gat cta
768
Gly Val Thr Thr Cys Leu Arg Phe Pro Gly Gln Leu Asn Ala Asp Leu
WO 01/04281 CA 02378407 2002-O1-04 pCT~P00/06104
-20-
245 250 255
cgt aaa cta get gtt aac atg gtt cca ttc cct cgt ctt cac ttc ttt
816
Arg Lys Leu Ala Val Asn Met Val Pro Phe Pro Arg Leu His Phe Phe
260 265 270
atg cct ggc ttt get ccc ctc tct get aaa ggc get cag get tac cgt
864
Met Pro Gly Phe Ala Pro Leu Ser Ala Lys Gly Ala Gln Ala Tyr Arg
275 280 285
get ctt act gta gcc gag cta act caa cag atg ttc gat gcc aaa aat
912
1$ Ala Leu Thr Val Ala Glu Leu Thr Gln Gln Met Phe Asp Ala Lys Asn
290 295 300
atg atg gcc get tgc gac cct cga cat gga cgt tat ctc acc gtc gca
960
Met Met Ala Ala Cys Asp Pro Arg His Gly Arg Tyr Leu Thr Val Ala
305 310 315 320
gcc atg ttc cga gga cga atg agc atg agg gag gta gac gac cag atg
1008
Ala Met Phe Arg Gly Arg Met Ser Met Arg Glu Val Asp Asp Gln Met
325 330 335
atg tca gtg cag aac aag aac tcc tca tac ttc gta gag tgg att ccg
1056
Met Ser Val Gln Asn Lys Asn Ser Ser Tyr Phe Val Glu Trp Ile Pro
340 345 350
WO 01/04281 CA 02378407 2002-O1-04 PCT/EP00/06104
-21-
aac aac gtc aag acc get gta tgc gac att ccg ccg aga gga ctg aaa
1104
Asn Asn Val Lys Thr Ala Val Cys Asp Ile Pro Pro Arg Gly Leu Lys
355 360 365
atg gcc get acc ttc gtt gga aac tca act gcc atc caa gag ctg ttc
1152
Met Ala Ala Thr Phe Val Gly Asn Ser Thr Ala Ile Gln Glu Leu Phe
370 375 380
aag cgc att tca gaa caa ttc aca get atg ttc cgc cgc aaa gcg ttc
1200
Lys Arg Ile Ser Glu Gln Phe Thr Ala Met Phe Arg Arg Lys Ala Phe
385 390 395 400
ttg cat tgg tat act ggt gaa ggt atg gac gag atg gag ttc act gaa
1248
Leu His Trp Tyr Thr Gly Glu Gly Met Asp Glu Met Glu Phe Thr Glu
405 410 415
gcc gag tcc aac atg aat gat ctc atc tcc gaa tac cag caa tac cag
1296
Ala Glu Ser Asn Met Asn Asp Leu Ile Ser Glu Tyr Gln Gln Tyr Gln
420 425 430
gaa get aca get gac gat atg ggc gat ctc gat gcg gaa ggc get gaa
1344
Glu Ala Thr Ala Asp Asp Met Gly Asp Leu Asp Ala Glu Gly Ala Glu
435 440 445
gag get tac cct gaa gaa tagacagcag attgtgttgc gttgttcgtt
1392
Glu Ala Tyr Pro Glu Glu
WO 01/04281 CA 02378407 2002-O1-04 pCT~P00/06104
-22-
450
tctctgtgtc aatgcgaaat acacattgat tgcgtt
1428
<210> 6
<211> 454
<212> PRT
<213> Cylicocyclus nassatus
<400> 6
Lys Phe Ser Thr Ala Ile Met Arg Glu Ile Val His Val Gln Ala Gly
1 5 10 15
Gln Cys Gly Asn Gln Ile Gly Ser Lys Phe Trp Glu Val Ile Ser Asp
25 30
Glu His Gly Ile Lys Pro Asp Gly Thr Tyr His Gly Glu Ser Asp Leu
20 35 40 45
Gln Leu Glu Arg Ile Asn Val Tyr Tyr Asn Glu Ala His Gly Gly Lys
50 55 60
2$ Tyr Val Pro Arg Ala Val Leu Val Asp Leu Glu Pro Gly Thr Met Asp
65 70 75 80
Ser Val Arg Ser Gly Pro Tyr Gly Gln Leu Phe Arg Pro Asp Asn Tyr
85 90 95
CA 02378407 2002-O1-04
WO 01/04281 PCT/EP00/06104
-23-
Val Phe Gly Gln Ser Gly Ala Gly Asn Asn Trp Ala Lys Gly His Tyr
100 105 110
Thr Glu Gly Ala Glu Leu Val Asp Asn Val Leu Asp Val Val Arg Lys
$ 115 120 125
Glu Ala Glu Gly Cys Asp Cys Leu Gln Gly Phe Gln Leu Thr His Ser
130 135 140
Leu Gly Gly Gly Thr Gly Ser Gly Met Gly Thr Leu Leu Ile Ser Lys
145 150 155 160
Ile Arg Glu Glu Tyr Pro Asp Arg Ile Met Ser Ser Phe Ser Val Val
165 170 175
Pro Ser Pro Lys Val Ser Asp Thr Val Val Glu Pro Tyr Asn Ala Thr
180 185 190
Leu Ser Val His Gln Leu Val Glu Asn Thr Asp Glu Thr Phe Cys Ile
2.0 195 200 205
Asp Asn Glu Ala Leu Tyr Asp Ile Cys Phe Arg Thr Leu Lys Leu Thr
210 215 220
Asn Pro Thr Tyr Gly Asp Leu Asn His Leu Val Ser Val Thr Met Ser
225 230 235 240
Gly Val Thr Thr Cys Leu Arg Phe Pro Gly Gln Leu Asn Ala Asp Leu
245 250 255
CA 02378407 2002-O1-04
WO 01/04281 PCT/EP00/06104
-24-
Arg Lys Leu Ala Val Asn Met Val Pro Phe Pro Arg Leu His Phe Phe
260 265 270
Met Pro Gly Phe Ala Pro Leu Ser Ala Lys Gly Ala Gln Ala Tyr Arg
$ 275 280 285
Ala Leu Thr Val Ala Glu Leu Thr Gln Gln Met Phe Asp Ala Lys Asn
290 295 300
Met Met Ala Ala Cys Asp Pro Arg His Gly Arg Tyr Leu Thr Val Ala
305 310 315 320
Ala Met Phe Arg Gly Arg Met Ser Met Arg Glu Val Asp Asp Gln Met
325 330 335
1$
Met Ser Val Gln Asn Lys Asn Ser Ser Tyr Phe Val Glu Trp Ile Pro
340 345 350
Asn Asn Val Lys Thr Ala Val Cys Asp Ile Pro Pro Arg Gly Leu Lys
355 360 365
Met Ala Ala Thr Phe Val Gly Asn Ser Thr Ala Ile Gln Glu Leu Phe
370 375 380
2$ Lys Arg Ile Ser Glu Gln Phe Thr Ala Met Phe Arg Arg Lys Ala Phe
385 390 395 400
Leu His Trp Tyr Thr Gly Glu Gly Met Asp Glu Met Glu Phe Thr Glu
405 410 415
WO 01/04281 CA 02378407 2002-O1-04 pCT/EP00/06104
-25-
Ala Glu Ser Asn Met Asn Asp Leu Ile Ser Glu Tyr Gln Gln Tyr Gln
420 425 430
Glu Ala Thr Ala Asp Asp Met Gly Asp Leu Asp Ala Glu Gly Ala Glu
$ 435 440 445
Glu Ala Tyr Pro Glu Glu
450
<210> 7
<211> 1429
<212> DNA
<213> Cylicocyclus nassatus
<220>
<221> CDS
<222> (1)..(1362)
<400> ~
aag ttc tct act gca ata atg cgt gag atc gtg cat gta caa get gga 48
Lys Phe Ser Thr Ala Ile Met Arg Glu Ile Val His Val Gln Ala Gly
1 5 10 15
cag tgt gga aac caa att ggt tcc aag ttt tgg gaa gtg atc tct gac 96
Gln Cys Gly Asn Gln Ile Gly Ser Lys Phe Trp Glu Val Ile Ser Asp
20 25 30
WO 01/04281 CA 02378407 2002-O1-04 PCT/EP00/06104
-26-
gag cac ggc att aag ccc gat ggc aca tac cac gga gaa tct gac tta
144
Glu His Gly Ile Lys Pro Asp Gly Thr Tyr His Gly Glu Ser Asp Leu
35 40 45
caa tta gaa cga atc aat gtg tac tat aat gaa gca cat gga ggc aaa
192
Gln Leu Glu Arg Ile Asn Val Tyr Tyr Asn Glu Ala His Gly Gly Lys
50 55 60
tat gtc ccg cgt gca gtt ctt gtt gat ctc gag ccc gga act atg gat
240
Tyr Val Pro Arg Ala Val Leu Val Asp Leu Glu Pro Gly Thr Met Asp
65 70 75 80
tcg gtc cgt tcc ggg cca tac ggg caa ttg ttc cgc cct gat aac tac
288
Ser Val Arg Ser Gly Pro Tyr Gly Gln Leu Phe Arg Pro Asp Asn Tyr
85 90 95
gtg ttt gga cag tct ggc gca gga aat aac tgg gca aaa ggt cac tac
336
Val Phe Gly Gln Ser Gly Ala Gly Asn Asn Trp Ala Lys Gly His Tyr
100 105 110
act gaa ggc get gaa ctt gtc gac aat gta cta gat gta gtg cga aaa
384
Thr Glu Gly Ala Glu Leu Val Asp Asn Val Leu Asp Val Val Arg Lys
115 120 125
gaa get gaa gga tgt gac tgt ctg cag ggc ttc cag cta act cac tca
432
Glu Ala Glu Gly Cys Asp Cys Leu Gln Gly Phe Gln Leu Thr His Ser
WO 01/04281 CA 02378407 2002-O1-04 pCT/EP00/06104
-27-
130 135 140
ctt gga gga ggt acc gga tcg agt atg ggc act ctc ctc atc ttc aaa
480
$ Leu Gly Gly Gly Thr Gly Ser Ser Met Gly Thr Leu Leu Ile Phe Lys
145 150 155 160
att cgg gag gag tat cct gat aga atc ata tcc tcg ttc ttc gtt gtt
528
Ile Arg Glu Glu Tyr Pro Asp Arg Ile Ile Ser Ser Phe Phe Val Val
165 170 175
ccc tca cca aag gtc tcc gac acc gtt gtg gag ccg tac aat get acc
576
1$ Pro Ser Pro Lys Val Ser Asp Thr Val Val Glu Pro Tyr Asn Ala Thr
180 185 190
cta tcc gtt cat cag ttg gtt gaa aat aca gac gag act ttc tgt att
624
Leu Ser Val His Gln Leu Val Glu Asn Thr Asp Glu Thr Phe Cys Ile
195 200 205
gac aat gaa get ctt tat gat att tgc ttc cgc act ttg aaa ctc acg
672
2$ Asp Asn Glu Ala Leu Tyr Asp Ile Cys Phe Arg Thr Leu Lys Leu Thr
210 215 220
aac cca act tat gga gat ctg aat cat ctt gtg tct gta aca atg tct
720
Asn Pro Thr Tyr Gly Asp Leu Asn His Leu Val Ser Val Thr Met Ser
225 230 235 240
CA 02378407 2002-O1-04
WO 01/04281 PCT/EP00/06104
-28-
ggt gtc act aca tgt ctt cgc ttc cct ggc caa ttg agt gcc gat cta
768
Gly Val Thr Thr Cys Leu Arg Phe Pro Gly Gln Leu Ser Ala Asp Leu
245 250 255
cgt aaa cta get gtt aac atg gtt cca ttc cct cgt ctt cac ttc ttt
816
Arg Lys Leu Ala Val Asn Met Val Pro Phe Pro Arg Leu His Phe Phe
260 265 270
atg cct ggc ttt get ccc ctc tct get aaa ggc get cag get tac cgt
864
Met Pro Gly Phe Ala Pro Leu Ser Ala Lys Gly Ala Gln Ala Tyr Arg
275 280 285
get ctt act gta gcc gag cta act caa cag atg ttc gat gcc aaa aat
912
Ala Leu Thr Val Ala Glu Leu Thr Gln Gln Met Phe Asp Ala Lys Asn
290 295 300
atg atg gcc get tgc gac cct cga cat gga cgt tat ctc acc gtc gca
960
Met Met Ala Ala Cys Asp Pro Arg His Gly Arg Tyr Leu Thr Val Ala
305 310 315 320
gcc atg ttc cga gga cga atg agc atg agg gag gta gac gac cag atg
1008
Ala Met Phe Arg Gly Arg Met Ser Met Arg Glu Val Asp Asp Gln Met
325 330 335
atg tca gtg cag aac aag aac tcc tca tac ttc gta gag tgg att ccg
1056
Met Ser Val Gln Asn Lys Asn Ser Ser Tyr Phe Val Glu Trp Ile Pro
CA 02378407 2002-O1-04
WO 01/04281 PCT/EP00/06104
-29-
340 345 350
aac aac gtc aag acc get gta tgc gac att ccg ccg aga gga ctg aaa
1104
$ Asn Asn Val Lys Thr Ala Val Cys Asp Ile Pro Pro Arg Gly Leu Lys
355 360 365
atg gcc get acc ttc gtt gga aac tca act gcc att caa gag ctg ttc
1152
1~ Met Ala Ala Thr Phe Val Gly Asn Ser Thr Ala Ile Gln Glu Leu Phe
370 375 380
aag cgc att tca gaa caa ttt aca get atg ttc cgc cgc aaa gcg ttc
1200
1$ Lys Arg Ile Ser Glu Gln Phe Thr Ala Met Phe Arg Arg Lys Ala Phe
385 390 395 400
ttg cat tgg tat act ggt gaa ggt atg gac gag atg gag ttc act gaa
1248
2~ Leu His Trp Tyr Thr Gly Glu Gly Met Asp Glu Met Glu Phe Thr Glu
405 410 415
gcc gag tcc aac atg aat gat ctc atc tcc gaa tac caa caa tac cag
1296
2$ Ala Glu Ser Asn Met Asn Asp Leu Ile Ser Glu Tyr Gln Gln Tyr Gln
420 425 430
gaa get acc get gac gat atg ggc gat ctc gat gcg gaa ggc get gaa
1344
3~ Glu Ala Thr Ala Asp Asp Met Gly Asp Leu Asp Ala Glu Gly Ala Glu
435 440 445
WO 01/04281 CA 02378407 2002-O1-04 PCT/EP00/06104
-30-
gag get tac cct gag gaa tagaacagca gattgtgttg cgttgttcgt
1392
Glu Ala Tyr Pro Glu Glu
450
ttctctgtgt caatgcgaaa tacacattga ttgcgtt
1429
<210> 8
<211> 454
<212> PRT
<213> Cylicocyclus nassatus
<400> 8
Lys Phe Ser Thr Ala Ile Met Arg Glu Ile Val His Val Gln Ala Gly
1 5 10 15
Gln Cys Gly Asn Gln Ile Gly Ser Lys Phe Trp Glu Val Ile Ser Asp
20 25 30
Glu His Gly Ile Lys Pro Asp Gly Thr Tyr His Gly Glu Ser Asp Leu
35 40 45
Gln Leu Glu Arg Ile Asn Val Tyr Tyr Asn Glu Ala His Gly Gly Lys
50 55 60
Tyr Val Pro Arg Ala Val Leu Val Asp Leu Glu Pro Gly Thr Met Asp
65 70 75 80
Ser Val Arg Ser Gly Pro Tyr Gly Gln Leu Phe Arg Pro Asp Asn Tyr
WO 01/04281 CA 02378407 2002-O1-04 pCT/EP00/06104
-31 -
85 90 95
Val Phe Gly Gln Ser Gly Ala Gly Asn Asn Trp Ala Lys Gly His Tyr
100 105 110
Thr Glu Gly Ala Glu Leu Val Asp Asn Val Leu Asp Val Val Arg Lys
115 120 125
Glu Ala Glu Gly Cys Asp Cys Leu Gln Gly Phe Gln Leu Thr His Ser
130 135 140
Leu Gly Gly Gly Thr Gly Ser Ser Met Gly Thr Leu Leu Ile Phe Lys
145 150 155 160
i$ Ile Arg Glu Glu Tyr Pro Asp Arg Ile Ile Ser Ser Phe Phe Val Val
165 170 175
Pro Ser Pro Lys Val Ser Asp Thr Val Val Glu Pro Tyr Asn Ala Thr
180 185 190
Leu Ser Val His Gln Leu Val Glu Asn Thr Asp Glu Thr Phe Cys Ile
195 200 205
Asp Asn Glu Ala Leu Tyr Asp Ile Cys Phe Arg Thr Leu Lys Leu Thr
2$ 210 215 220
Asn Pro Thr Tyr Gly Asp Leu Asn His Leu Val Ser Val Thr Met Ser
225 230 235 240
Gly Val Thr Thr Cys Leu Arg Phe Pro Gly Gln Leu Ser Ala Asp Leu
CA 02378407 2002-O1-04
WO 01/04281 PCT/EP00/06104
-32-
245 250 255
Arg Lys Leu Ala Val Asn Met Val Pro Phe Pro Arg Leu His Phe Phe
260 265 270
Met Pro Gly Phe Ala Pro Leu Ser Ala Lys Gly Ala Gln Ala Tyr Arg
275 280 285
Ala Leu Thr Val Ala Glu Leu Thr Gln Gln Met Phe Asp Ala Lys Asn
290 295 300
Met Met Ala Ala Cys Asp Pro Arg His Gly Arg Tyr Leu Thr Val Ala
305 310 315 320
Ala Met Phe Arg Gly Arg Met Ser Met Arg Glu Val Asp Asp Gln Met
325 330 335
Met Ser Val Gln Asn Lys Asn Ser Ser Tyr Phe Val Glu Trp Ile Pro
340 345 350
Asn Asn Val Lys Thr Ala Val Cys Asp Ile Pro Pro Arg Gly Leu Lys
355 360 365
Met Ala Ala Thr Phe Val Gly Asn Ser Thr Ala Ile Gln Glu Leu Phe
370 375 380
Lys Arg Ile Ser Glu Gln Phe Thr Ala Met Phe Arg Arg Lys Ala Phe
385 390 395 400
Leu His Trp Tyr Thr Gly Glu Gly Met Asp Glu Met Glu Phe Thr Glu
WO 01/04281 CA 02378407 2002-O1-04 pCT/EP00/06104
-33-
405 410 415
Ala Glu Ser Asn Met Asn Asp Leu Ile Ser Glu Tyr Gln Gln Tyr Gln
420 425 430
Glu Ala Thr Ala Asp Asp Met Gly Asp Leu Asp Ala Glu Gly Ala Glu
435 440 445
Glu Ala Tyr Pro Glu Glu
450
<210> 9
1$ <211> 1428
<212> DNA
<213> Cylicocyclus nassatus
<220>
<221> CDS
<222> (1)..(1362)
<400> 9
aag ttc tct act gca ata atg cgt gag atc gtg cat gta caa get gga 48
25 Lys Phe Ser Thr Ala Ile Met Arg Glu Ile Val His Val Gln Ala Gly
1 5 10 15
cag tgt gga aac caa att ggt tcc aag ttc tgg gaa gtg atc tct gac 96
Gln Cys Gly Asn Gln Ile Gly Ser Lys Phe Trp Glu Val Ile Ser Asp
3~ 20 25 30
CA 02378407 2002-O1-04
WO 01/04281 PCT/EP00/06104
-34-
gag cac ggc att aag ccc gac ggc aca tac cat gga gaa tct gat cta
144
Glu His Gly Ile Lys Pro Asp Gly Thr Tyr His Gly Glu Ser Asp Leu
$ 35 40 45
caa tta gaa cga atc aat gtg tac tat aat gaa gca cat gga ggc aag
192
Gln Leu Glu Arg Ile Asn Val Tyr Tyr Asn Glu Ala His Gly Gly Lys
50 55 60
tat gtc ccg cgt gca gtt ctt gtt gat ctc gag ccc gga act atg gat
240
Tyr Val Pro Arg Ala Val Leu Val Asp Leu Glu Pro Gly Thr Met Asp
1$ 65 70 75 80
tca gtc cgt tct ggg cca tac ggg caa ttg ttc cgc cct gat aac tac
288
Ser Val Arg Ser Gly Pro Tyr Gly Gln Leu Phe Arg Pro Asp Asn Tyr
2~ 85 90 95
gtg ttt gga cag tct ggc gca gga aat aac tgg gca aaa ggt cac tac
336
Val Phe Gly Gln Ser Gly Ala Gly Asn Asn Trp Ala Lys Gly His Tyr
2$ loo l05 llo
act gaa ggc get gaa ctt gtc gac aat gta cta gat gta gtg cga aaa
384
Thr Glu Gly Ala Glu Leu Val Asp Asn Val Leu Asp Val Val Arg Lys
115 120 125
gaa get gaa gga tgt gac tgt ctg cag ggc ttc cag cta act cac tca
432
WO 01/04281 CA 02378407 2002-O1-04 pCT~P00/06104
-3$-
Glu Ala Glu Gly Cys Asp Cys Leu Gln Gly Phe Gln Leu Thr His Ser
130 135 140
ctt gga gga ggt acc gga tcg ggt atg ggc aca ctc ctc atc tcc aaa
$ 4so
Leu Gly Gly Gly Thr Gly Ser Gly Met Gly Thr Leu Leu Ile Ser Lys
145 150 155 160
att cgg gag gag tat cct gat aga atc atg tcc tcg ttc tcc gtt gtt
528
Ile Arg Glu Glu Tyr Pro Asp Arg Ile Met Ser Ser Phe Ser Val Val
165 170 175
ccc tca cca aag gtc ttc gat act gtt gtg gag ccg tac aat get acc
1$ 576
Pro Ser Pro Lys Val Phe Asp Thr Val Val Glu Pro Tyr Asn Ala Thr
180 185 190
cta tcc gtt cat cag ttg gtt gaa aat aca gac gag act ttc tgt att
624
Leu Ser Val His Gln Leu Val Glu Asn Thr Asp Glu Thr Phe Cys Ile
195 200 205
gac aat gaa get ctt tat gat att tgc ttc cgc acc ttg aaa ctc acg
2$ 672
Asp Asn Glu Ala Leu Tyr Asp Ile Cys Phe Arg Thr Leu Lys Leu Thr
210 215 220
aac cca act tat gga gat ctg aat cat ctt gtg tct gta aca atg tct
720
Asn Pro Thr Tyr Gly Asp Leu Asn His Leu Val Ser Val Thr Met Ser
225 230 235 240
WO 01/04281 CA 02378407 2002-O1-04 pCT/EP00/06104
-36-
ggt gtc act aca tgc ctt cgc ttc cct ggc caa ttg aat gcc gat cta
768
Gly Val Thr Thr Cys Leu Arg Phe Pro Gly Gln Leu Asn Ala Asp Leu
245 250 255
cgt aaa cta get gtt aac atg gtt cca ttc cct cgt ctt cac ttc ttc
816
Arg Lys Leu Ala Val Asn Met Val Pro Phe Pro Arg Leu His Phe Phe
260 265 270
atg cct ggc ttt get ccc ctc tct gcc aaa ggc gcc cag get tac cgt
864
Met Pro Gly Phe Ala Pro Leu Ser Ala Lys Gly Ala Gln Ala Tyr Arg
275 280 285
get ctt act gta gcc gag cta act caa cag atg ttc gat gcc aaa aat
912
Ala Leu Thr Val Ala Glu Leu Thr Gln Gln Met Phe Asp Ala Lys Asn
290 295 300
atg atg gcc get tgc gac cct cga cat gga cgt tat ctc acc gtc gca
960
Met Met Ala Ala Cys Asp Pro Arg His Gly Arg Tyr Leu Thr Val Ala
305 310 315 320
gcc atg ttc cga gga cga atg agc atg agg gag gta gac gac cag atg
1008
Ala Met Phe Arg Gly Arg Met Ser Met Arg Glu Val Asp Asp Gln Met
325 330 335
atg tca gtg cag aac aag aac tcc tca tac ttc gta gag tgg att ccg
1056
Met Ser Val Gln Asn Lys Asn Ser Ser Tyr Phe Val Glu Trp Ile Pro
CA 02378407 2002-O1-04
WO 01/04281 PCT/EP00/06104
-37-
340 345 350
aac aac gtc aaa acc get gta tgc gac att ccg ccg aga gga ctg aaa
1104
$ Asn Asn Val Lys Thr Ala Val Cys Asp Ile Pro Pro Arg Gly Leu Lys
355 360 365
atg gcc get acc ttc gtt gga aac tca act gcc att caa gag ctg ttc
1152
Met Ala Ala Thr Phe Val Gly Asn Ser Thr Ala Ile Gln Glu Leu Phe
370 375 380
aag cgc att tca gaa caa ttc aca get atg ttc cgc cgc aaa gcg ttc
1200
1$ Lys Arg Ile Ser Glu Gln Phe Thr Ala Met Phe Arg Arg Lys Ala Phe
385 390 395 400
ttg cat tgg tat act ggt gaa ggt atg gac gag atg gag ttc act gaa
1248
2~ Leu His Trp Tyr Thr Gly Glu Gly Met Asp Glu Met Glu Phe Thr Glu
405 410 415
gcc gag tcc aac atg aat gat ctc atc tcc gaa tac cag caa tac cag
1296
2$ Ala Glu Ser Asn Met Asn Asp Leu Ile Ser Glu Tyr Gln Gln Tyr Gln
420 425 430
gaa get acc get gac gat atg ggc gat ctc gat gcg gaa ggc get gaa
1344
Glu Ala Thr Ala Asp Asp Met Gly Asp Leu Asp Ala Glu Gly Ala Glu
435 440 445
WO 01/04281 CA 02378407 2002-O1-04 PCT/EP00/06104
-38-
gag get tac cct gaa gaa tagacagcag attgtgttgc gttgttcgtt
1392
Glu Ala Tyr Pro Glu Glu
450
tctctgtgtc aatgcgaaat acacattgat tgcgtt
1428
<210> to
<211> 454
<212> PRT
<213> Cylicocyclus nassatus
<400> to
Lys Phe Ser Thr Ala Ile Met Arg Glu Ile Val His Val Gln Ala Gly
1 5 10 15
Gln Cys Gly Asn Gln Ile Gly Ser Lys Phe Trp Glu Val Ile Ser Asp
20 25 30
Glu His Gly Ile Lys Pro Asp Gly Thr Tyr His Gly Glu Ser Asp Leu
35 40 45
Gln Leu Glu Arg Ile Asn Val Tyr Tyr Asn Glu Ala His Gly Gly Lys
50 55 60
Tyr Val Pro Arg Ala Val Leu Val Asp Leu Glu Pro Gly Thr Met Asp
65 70 75 80
Ser Val Arg Ser Gly Pro Tyr Gly Gln Leu Phe Arg Pro Asp Asn Tyr
WO 01/04281 CA 02378407 2002-O1-04 pCT/EP00/06104
-39-
85 90 95
Val Phe Gly Gln Ser Gly Ala Gly Asn Asn Trp Ala Lys Gly His Tyr
100 105 110
Thr Glu Gly Ala Glu Leu Val Asp Asn Val Leu Asp Val Val Arg Lys
115 120 125
Glu Ala Glu Gly Cys Asp Cys Leu Gln Gly Phe Gln Leu Thr His Ser
130 135 140
Leu Gly Gly Gly Thr Gly Ser Gly Met Gly Thr Leu Leu Ile Ser Lys
145 150 155 160
1$ Ile Arg Glu Glu Tyr Pro Asp Arg Ile Met Ser Ser Phe Ser Val Val
165 170 175
Pro Ser Pro Lys Val Phe Asp Thr Val Val Glu Pro Tyr Asn Ala Thr
180 185 190
Leu Ser Val His Gln Leu Val Glu Asn Thr Asp Glu Thr Phe Cys Ile
195 200 205
Asp Asn Glu Ala Leu Tyr Asp Ile Cys Phe Arg Thr Leu Lys Leu Thr
2$ 210 215 220
Asn Pro Thr Tyr Gly Asp Leu Asn His Leu Val Ser Val Thr Met Ser
225 230 235 240
Gly Val Thr Thr Cys Leu Arg Phe Pro Gly Gln Leu Asn Ala Asp Leu
WO 01/04281 CA 02378407 2002-O1-04 PCT/EP00/06104
-40-
245 250 255
Arg Lys Leu Ala Val Asn Met Val Pro Phe Pro Arg Leu His Phe Phe
260 265 270
$
Met Pro Gly Phe Ala Pro Leu Ser Ala Lys Gly Ala Gln Ala Tyr Arg
275 280 285
Ala Leu Thr Val Ala Glu Leu Thr Gln Gln Met Phe Asp Ala Lys Asn
290 295 300
Met Met Ala Ala Cys Asp Pro Arg His Gly Arg Tyr Leu Thr Val Ala
305 310 315 320
1$ Ala Met Phe Arg Gly Arg Met Ser Met Arg Glu Val Asp Asp Gln Met
325 330 335
Met Ser Val Gln Asn Lys Asn Ser Ser Tyr Phe Val Glu Trp Ile Pro
340 345 350
Asn Asn Val Lys Thr Ala Val Cys Asp Ile Pro Pro Arg Gly Leu Lys
355 360 365
Met Ala Ala Thr Phe Val Gly Asn Ser Thr Ala Ile Gln Glu Leu Phe
2$ 370 375 380
Lys Arg Ile Ser Glu Gln Phe Thr Ala Met Phe Arg Arg Lys Ala Phe
385 390 395 400
Leu His Trp Tyr Thr Gly Glu Gly Met Asp Glu Met Glu Phe Thr Glu
WO 01/04281 CA 02378407 2002-O1-04 pCT~P00/06104
-41-
405 410 415
Ala Glu Ser Asn Met Asn Asp Leu Ile Ser Glu Tyr Gln Gln Tyr Gln
420 425 430
Glu Ala Thr Ala Asp Asp Met Gly Asp Leu Asp Ala Glu Gly Ala Glu
435 440 445
Glu Ala Tyr Pro Glu Glu
450
<210> 11
1$ <211> 2655
<212> DNA
<213> Cylicocyclus nassatus
<220>
<221> intron
<222> (1) . . (18)
<220>
<221> intron
<222> (1)..(18)
<220>
<221> intron
<222> (76)..(358)
WO 01/04281 CA 02378407 2002-O1-04 pCT/EP00/06104
-42-
<220>
<221> intron
<222> (469)..(637)
$ <220>
<221> intron
<222> (865)..(1374)
<220>
<221> intron
<222> (1666) .. (1723)
<220>
<221> intron
1$ <222> (1915)..(1966)
<220>
<221> intron
<222> (2064)..(2119)
<220>
<221> intron
<222> (2306)..(2354)
<220>
<221> intron
<222> (2475)..(2523)
<220>
<221> intron
CA 02378407 2002-O1-04
WO 01/04281 PCT/EP00/06104
- 43 -
<222> (2592)..(2655)
<400> 11
aagttctcta ctgcaataat gcgtgagatc gtgcatgtac aagctggaca rtgtggaaac 60
caaattggtt ccaaggtrcg gtagtttyrr twktytrytg atcgtaattc sggmgytytr
120
dagtrryttt ttycgytgyy ratgttgcat yrtgttgcga taaascyraa aawtcawwag
180
rcgaggctgt aaaagsactt ytacttttra atmcrytgta gcagcatgag tcatcrgcat
240
gtttgcagtg sgttttttat gcgcwgawcc yctcagaaga tgagaatgcg wtccaytgag
300
cwtagartct grctttctct cgttawctaa ratcaamtta carcrytyca ttttkcagtt
360
ytgggaagtg atctctgacg agcacggcat taagccygay ggcacatacc ayggagaatc
420
tgatytacaa ttagaacgaa tcaatgtgta ctataatgaa gcacatggtt agtcgtayat
480
ccgcttcgtt gtytcccmat gcagrccyct tcagttttta taactgycga aatatcgatc
540
gggctctttt gcagcggccw ygattacgca ataccayygc ygcygcagtg gcrgtcgaaa
600
ttaatgtggt caracgtgaa aatgtggtgc tttyaggagg caartatgtc ccgcgtgcag
660
ttcttgttga tctcgagccc ggaactatgg attcggtccg ytccgggcca tacgggcaat
720
tgttccgccc tgataactac gtgtttggac agtctggcgc aggaaataac tgggcaaaag
780
gtcactacac tgaaggygct gaacttgtcg acaatgtact agatgtagtg cgaaaagaag
840
ctgaaggatg tgactgtctg caggtaaatt tccaagtagt agcaggaaat ggtwtgtgra
900
tagcataaca aaagtcatag aaggaatatg gacgctagtc aaaacaaagw tggacgttar
960
tcggtcgtcc gggacarttt ggaagtcatg ggtcasccaa cacgcttttt tamaagtaca
1020
tcatactctt ttcccacgaa aagctatttt gcgtattacg gggtacaggg gaggggtcaa
1080
WO 01/04281 CA 02378407 2002-O1-04 PCT/EP00/06104
aatcacagat tgctgaaaty tggttcactg ragttattgr tgaaaatcat attgattttg
1140
cttgctactg ccttcttttr aggctatgct ttacaatctt ggggcctgga taaccgaatt
1200
$ gtcygaagtt tttcggtcat cacggacggg gaaggggcat artatcgtta kttcttgkta
1260
tttcgcagca tatggcaatc tytccacttc tgacaagttt tcygtagaaa atatwcttca
1320
aggtstcaag aacyttgctg ctagrgctgt aaaccaayct gtatcycttt cagggcttcc
1380
agctaactca ctcacttgga ggaggtaccg gatcgggtat gggcactctc ctcatctcca
1440
aaattcggga ggagtatcct gatagaatca tgtcctcgtt ctccgttgtt ccctcaccaa
1500
1$ aggtctccga caccgttgtg gagccgtaca atgctaccct atccgttcat cagttggttg
1560
aaaatacaga cgaracttwc tgtattgaca atgaagctct ttatgatatt tgcttccgca
1620
cyytgaaact cacsaaccca acttatggag atctgaatca tcttggtrrg yrayatkcsa
1680
ytgctgagct tdgtrgaatt tvctaattwt ktyhamtdty yagtgtctgt aacaatgtct
1740
ggygtcacta catgycttcg cttccctggc caattgrayg ccgatctwcg taaactagct
1800
2$ gttaacatgg ytccattccc tcgtcttcac ttyttyatgc ctggctttgc tcccctctct
1860
gcyaaaggcg cycaggctta ccgtgctctt actgtagccg agctwacyca rcaggtgcgt
1920
ctgcttatcr ttgwtgayrt gtgtttattc kttgtrtatt ttayagatgt tcgatgccaa
1980
aaatatgatg gccgcttgcg accctcgaca tggacrttat ctcaccgtyg cagccatgtt
2040
ccgaggacga atgagcayga gggtaagtgg mtkmttggyc ytytaryaya rctcrgacga
2100
3$ awtgctgtta tgtcmtagga rgtagacgac cagatgatgt cagtgcagaa caagaactcc
2160
tcatacttcg tagagtggat tccgaacaac gtcaaraccg cygtatgcga cattccgccr
2220
agaggactga aaatggccgc taccttcgtt ggaaacycaa ctgccatcca agagctgtty
2280
WO 01/04281 CA 02378407 2002-O1-04 pCT/EP00/06104
- 45 -
aagcgcattt cagaacaatt yacaggttdg tttgtgcaya ttatggtgaa agcagattar
2340
ttgcgaygtt gcagctatgt tccgccgcaa agcgttyttg catyggtaya ctggwgaagg
2400
tatggaygag atggagttca ctgaagccga gtccaacatg aatgatctca tctccgaata
2460
ccarcaatac caggttcggc tgtytttcwt rgayactgtr tttaataatt wttyttgtct
2520
aggaagctac cgctgacgat atgggcgatc tcgatgcgga aggcgctgaa gaggcttacc
25ao
ctgargaata gamcagcaga ytgtgttgcg ttgttcgttt ctctrtgtca atgcgaaata
2640
cacattgatt gcgtt
2655
<210> 12
<211> 23
<212> DNA
<213> Kunstliche Sequenz
<220>
<223> Beschreibung der kunstlichen
Sequenz:Hybridisierungssonde/Primer
<400> 12
ggtttaatta tcccaagttt gag 23
<210> 13
<211> 20
<212> DNA
<213> Kunstliche Sequenz
WO 01/04281 CA 02378407 2002-O1-04 PCT/EP00/06104
-46-
<220>
<223> Beschreibung der kiinstlichen
Sequenz:Primer/Hybridisierungssonden
<400> 13
ggccacgcgt cgactagtac 20
1~ <210> 14
<211> 37
<212> DNA
<213> Kiinstliche Sequenz
1$ <220>
<223> Beschreibung der kunstlichen
Sequenz:Primer/Hybridisierungssonden
<400> 14
2~ ggccacgcgt cgactagtac tttttttttt ttttttt 37
<210> 15
<211> 22
2,5 <212> DNA
<213> Kiinstliche Sequenz
<220>
<223> Beschreibung der kunstlichen
30 Sequenz:Primer/Hybridisierungssonden
CA 02378407 2002-O1-04
WO 01/04281 PCT/EP00/06104
-47-
<400> 15
gaccgctgta tgcgacattc cg 22
<210> 16
<211> 22
<212> DNA
<213> Kiinstliche Sequenz
<220>
<223> Beschreibung der kiinstlichen
Sequenz:Primer/Hybridisierungssonden
<400> 16
aactcaactg ccatccaaga gc 22
<210> 17
<211> 21
<212> DNA
<213> Kiznstliche Sequenz
<220>
<223> Beschreibung der kiinstlichen
Sequenz:Primer/Hybridisierungssonden
<400> 17
gctatgttcc gccgcaaagc g 21
WO 01/04281 CA 02378407 2002-O1-04 pCT/Ep00/06104
-48-
<210> la
<211> 25
<212> DNA
$ <213> Kiinstliche Sequenz
<220>
<223> Beschreibung der kiinstlichen
Sequenz:Primer/Hybridisierungssonden
<400> 1e
acgagcacgg cattaagcct gatgg 25
1$ <210> 19
<211> 25
<212> DNA
<213> Kiinstliche Sequenz
<220>
<223> Beschreibung der kiinstlichen
Sequenz:Primer/Hybridisierungssonden
<400> 19
ccatcaggct taatgccgtg ctcgt 25
<210> 20
<211> 25
<212> DNA
CA 02378407 2002-O1-04
WO 01/04281 PCT/EP00/06104
-49-
<213> Kunstliche Sequenz
<220>
<223> Beschreibung der kiinstlichen
$ Sequenz:Primer/Hybridisierungssonden
<400> 20
ccgaatccat agttccgggc tcgag 25
<210> 21
<211> 24
<212> DNA
<213> Kiinstliche Sequenz
1$
<220>
<223> Beschreibung der kiinstlichen
Sequenz:Primer/Hybridisierungssonden
2~ <400> 21
ccgacaccgt tgtggagccg taca 24
<210> 22
25 <211> 2s
<212> DNA
<213> Kunstliche Sequenz
<220>
30 <223> Beschreibung der kiinstlichen
WO 01/04281 CA 02378407 2002-O1-04 pCT/EP00/06104
-50-
Sequenz:Primer/Hybridisierungssonden
<400> 22
gcgaccctcg acatggacgt tatct 25
<210> 23
<211> 25
<212> DNA
<213> Kunstliche Sequenz
<220>
<223> Beschreibung der kiinstlichen
Sequenz:Primer/Hybridisierungssonden
<400> 23
agataacgtc catgtcgagg gtcgc 25
<210> 24
<211> 25
<212> DNA
<213> Kiinstliche Sequenz
2$ <220>
<223> Beschreibung der kunstlichen
Sequenz:Primer/Hybridisierungssonden
<400> 24
tgttccgagg acgaatgagc atgag 25
CA 02378407 2002-O1-04
WO 01/04281 PCT/EP00/06104
-51-
<210> 25
<211> 25
$ <212> DNA
<213> Kunstliche Sequenz
<220>
<223> Beschreibung der kiinstlichen
Sequenz:Primer/Hybridisierungssonden
<400> 25
ctcatgctca ttcgtcctcg gaaca 25
<210> 26
<211> 26
<212> DNA
<213> Kiinstliche Sequenz
<220>
<223> Beschreibung der kiinstlichen
Sequenz:Primer/Hybridisierungssonden
<400> 26
aggtagacga ccagatgatg tcagtg 26
<210> 27
<211> 26
WO 01/04281 CA 02378407 2002-O1-04 pCT~P00/06104
-$2-
<212> DNA
<213> Kunstliche Sequenz
<220>
$ <223> Beschreibung der kiinstlichen
Sequenz:Primer/Hybridisierungssonden
<400> 27
cactgacatc atctggtcgt ctacct 26
<210> 28
<211> 24
<212> DNA
1$ <213> Kiinstliche Sequenz
<220>
<223> Beschreibung der kiinstlichen
Sequenz:Primer/Hybridisierungssonden
<400> 28
ggcggaatgt cgcatacagc ggtc 24
2$ <210> 29
<211> 26
<212> DNA
<213> Kiinstliche Sequenz
<220>
CA 02378407 2002-O1-04
WO 01/04281 PCT/EP00/06104
-53-
<223> Beschreibung der kunstlichen
Sequenz:Primer/Hybridisierungssonden
<400> 29
cggagatgag atcattcatg ttggac 26
<210> 30
<211> 24
<212> DNA
<213> Kiinstliche Sequenz
<220>
<223> Beschreibung der kiinstlichen
Sequenz:Primer/Hybridisierungssonden
<400> 30
ctctcctcat ctccaaaatt cggg 24
<210> 31
<211> 25
<212> DNA
<213> Kiinstliche Sequenz
<220>
<223> Beschreibung der kiinstlichen
Sequenz:Primer/Hybridisierungssonden
<400> 31
CA 02378407 2002-O1-04
WO 01/04281 PCT/EP00/06104
-54-
cagctaactc actcacttgg aggag 25
<210> 32
$ <211> 25
<212> DNA
<213> Kiinstliche Sequenz
<220>
<223> Beschreibung der kiinstlichen
Sequenz:Primer/Hybridisierungssonden
<400> 32
aagttctcta ctgcaataat gcgtg 25
<210> 33
<211> 24
<212> DNA
<213> Kiinstliche Sequenz
<220>
<223> Beschreibung der kunstlichen
Sequenz:Primer/Hybridisierungssonden
<400> 33
ggttgaaaat acagacgaga cttt 24
<210> 34
CA 02378407 2002-O1-04
WO 01/04281 PCT/EP00/06104
-SS-
<211> 24
<212> DNA
<213> Kiinstliche Sequenz
$ <220>
<223> Beschreibung der kunstlichen
Sequenz:Primer/Hybridisierungssonden
<400> 34
ggttgaaaat acagacgaga ctta 24
<210> 35
<211> 24
1$ <212> DNA
<213> Kiinstliche Sequenz
<220>
<223> Beschreibung der kiinstlichen
Sequenz:Primer/Hybridisierungssonden
<400> 35
aaagagcttc attgtcaata Gaga 24
<210> 36
<211> 23
<212> DNA
<213> Kiinstliche Sequenz
CA 02378407 2002-O1-04
WO 01/04281 PCT/EP00/06104
-$6-
<220>
<223> Beschreibung der kunstlichen
Sequenz:Primer/Hybridisierungssonden
$ <400> 36
caattggcca gggaagcgaa gac 23
<210> 37
<211> 23
<212> DNA
<213> Kiinstliche Sequenz
<220>
1$ <223> Beschreibung der kunstlichen
Sequenz:Primer/Hybridisierungssonden
<400> 37
gtcttcgctt ccctggccaa ttg 23
<210> 38
<211> 24
<212> DNA
2$ <213> Kiinstliche Sequenz
<220>
<223> Beschreibung der kunstlichen
Sequenz:Primer/Hybridisierungssonden
WO 01/04281 CA 02378407 2002-O1-04 PCT/EP00/06104
-57-
<400> 38
cctggctttg ctcccctctc tgct 24
$ <210> 39
<211> 24
<212> DNA
<213> Kunstliche Sequenz
<220>
<223> Beschreibung der kiinstlichen
Sequenz:Primer/Hybridisierungssonden
<400> 39
agcagagagg ggagcaaagc cagg 24
<210> 40
<211> 23
<212> DNA
<213> Kiinstliche Sequenz
<220>
<223> Beschreibung der kunstlichen
Sequenz:Primer/Hybridisierungssonden
<400> 40
aacgcaatca atgtgtattt cgc 23
CA 02378407 2002-O1-04
WO 01/04281 PCT/EP00/06104
-58-
<210> 41
<211> 18
<212> DNA
<213> Kiinstliche Sequenz
<220>
<223> Beschreibung der kiinstlichen
Sequenz:Primer/Hybridisierungssonden
<400> 41
cccgacggca cataccat 18
<210> 42
1$ <211> 24
<212> DNA
<213> Kiinstliche Sequenz
<220>
20 <223> Beschreibung der kunstlichen
Sequenz:Primer/Hybridisierungssonden
<400> 42
gaaacgaaca acgcaacaca atct 24
<210> 43
<211> 22
<212> DNA
<213> Kunstliche Sequenz
CA 02378407 2002-O1-04
WO 01/04281 PCT/EP00/06104
-59-
<220>
<223> Beschreibung der kunstlichen
Sequenz:Primer/Hybridisierungssonden
<400> 43
caagctggac aatgtggaaa cc 22
<210> 44
<211> 22
<212> DNA
<213> Kiinstliche Sequenz
IS <220>
<223> Beschreibung der kiinstlichen
Sequenz:Primer/Hybridisierungssonden
<400> 44
20 yagagaaacg aacaacgcaa ca 22
<210> 45
<211> 23
2$ <212> DNA
<213> Kiinstliche Sequenz
<220>
<223> Beschreibung der kunstlichen
30 Sequenz:Primer/Hybridisierungssonden
WO 01/04281 CA 02378407 2002-O1-04 pCT/EP00/06104
-60-
<400> 45
ttgatctcga gcccggaact atg 23
<210> 46
<211> 22
<212> DNA
<213> Kiinstliche Sequenz
<220>
<223> Beschreibung der kiinstlichen
Sequenz:Primer/Hybridisierungssonden
1$ <400> 46
tctcgagccc ggaactatgg at 22
<210> 47
<211> 24
<212> DNA
<213> Kiinstliche Sequenz
<220>
2$ <223> Beschreibung der kiinstlichen
Sequenz:Primer/Hybridisierungssonden
<400> 47
cccgaatttt ggagatgagg agag 24
CA 02378407 2002-O1-04
WO 01/04281 PCT/EP00/06104
-61 -
<210> 48
<211> 20
<212> DNA
$ <213> Kiinstliche Sequenz
<220>
<223> Beschreibung der kiinstlichen
Sequenz:Primer/Hybridisierungssonden
<400> 48
ggncartgyg gnaaycarat 20
1$ <210> 49
<211> 20
<212> DNA
<213> Kiinstliche Sequenz
<220>
<223> Beschreibung der kunstlichen
Sequenz:Primer/Hybridisierungssonden
<400> 49
gaytgyytnc arggnttyca 20
<210> 50
<211> 20
<212> DNA
WO 01/04281 CA 02378407 2002-O1-04 pCT~P00/06104
-62-
<213> Kunstliche Sequenz
<220>
<223> Beschreibung der kunstlichen
$ Sequenz:Primer/Hybridisierungssonden
<400> 50
tgraanccyt gnarrcartc 20
<210> 51
<211> 20
<212> DNA
<213> Kunstliche Sequenz
<220>
<223> Beschreibung der kunstlichen
Sequenz:Primer/Hybridisierungssonden
<400> 51
tcytgrtayt gytgrtaytc 20
