Note: Descriptions are shown in the official language in which they were submitted.
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INSECTICIDAL GENE AND PEPTIDE.
The present invention relates to FMRFamide type peptides and their non-
amidated
precursors, to recombinant nucleic acids encoding for such peptides, to the
use of these
nucleic acids, in forms substantially isolated from other nucleic acid or when
incorporated
into vectors, to transform organisms in order to render them capable of
producing these
peptides, to the use of such peptides, nucleic acid and vectors as pesticidal
agents, eg.
insecticidal, acaricidal and helminthecidal agents and to compositions
comprising one or
more of these.
The FMRFamide family of peptides are known to be implicated in invertebrate
neurohormonal control and have been proposed for use as insect control agents.
The
cloning of Drosophila FMRF gene into baculovirus has been reported (Keeley et
al., (1988)
Biotechnol. Biol. Pestic. ,99-106) while WO 9517515 (US Serial No. 08/172,653)
describes
a method for incapacitating insects by infecting them with an active
recombinant
baculovirus containing an FMRFamide like peptide precursor. The latter
document
describes the linking of a gene encoding for the FMRFamide precursor to a
silkworm
cytoplasmic actin promoter and enhancer from Bombyx mori. Vectors using the
baculovirus
1 E-1 gene are particularly favoured.
The present inventors now report the isolation of the octapeptide, Ile-Pro-Pro-
Gln-
Phe-Met-Arg-Phe.amide (IPPQFMRFamide) from acidified ethanol extracts of the
skin of
the African rhacophorid frog, Hylambates (Kassina) maculata, commonly known as
the
red-legged pan frog. This peptide represents the first authentic member of the
FMRFamide
family of peptides isolated from a vertebrate source and the C-terminal
tetrapeptide is
identical to the original peptide isolated from the brain of the bivalve
mollusc,
Macrocallista nimbosa. This newly identified and isolated vertebrate FMRFamide-
like
peptide fulfills the criteria of activation of receptors on invertebrate
tissues and the mode
of action of this frog venom component is consistent with its role in
protection from
invertebrate predators.
The identification and isolation of this peptide provides the first evidence
that
vertebrates produce anti-invertebrate peptides in their defensive secretions
and that by
extrapolation, the peptidergic regulatory system of these invertebrates is an
appropriate and
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available target for peptidic insecticides/anthelmintics.
Thus in a first aspect of the present invention there is provided a peptide or
salt
thereof comprising an amino acid sequence Pro-Pro-(Xaa')n-Phe-Xaa2-Arg-Xaa3
or a conservatively substituted analogue thereof
wherein the N terminal of said peptide may be free amino or protected amino
and the C-
terminal of said peptide, which is provided by the C-terminal Xaa3 in the
sequence above,
may consist of that residue having a free carboxylic acid group or having an
amidated
carboxyl of formula CONR'RZ wherein R' and RZ are independently selected from
hydrogen, alkyl, alkenyl, hydroxyalkyl, alkoxyalkyl and aminoalkyl or together
with the
nitrogen to which they are attached form a C~_8 heterocylic ring; wherein
Xaa' is any amino acid, which is independently selected at each repeat n and
n is an integer from 0 to 10 ;
Xaa'- is Met, Val, Ile, Leu, Nle or Phe or a conservatively substituted
analogue
thereof and .
Xaa3 is Phe, Asp or Glu.
provided that when the peptide is Ile-Pro-Pro-Gln-Phe-Met-Arg-Phe it is in a
form not
associated with, eg. when not in the form of, skin of the frog Hylambates
(Kassina)
maculata.
Where FMRFamide agonist activity is desired, Xaa~ is preferably Phe.
Where FMRFamide antagonist activity is desired, Xaa3 is preferably Asp or Glu.
Preferably n is from 1 to 3 and most preferably is 1.
Preferably Xaa' is an alpha amino acid, particularly a naturally occurring
amino
acid but may be of D- or L- configuration, more preferably being glutamine or
a
conservative substitution thereof , most preferably being L-glutamine. Use of
D-amino
acids will be expected to give resistance to enzymolysis where used and may be
beneficial.
Preferably Xaaz is Met, Ile, Leu or Nle.
Preferably R' and R'- are independently selected from hydrogen, C,~ n- or iso-
alkyl
or C3_$ cycloalkyl, eg. cyclohexyl. Alternatively R' and RZ together with the
nitrogen to
which they are attached may form a C3_8 heterocyclic ring.
Preferably the N-terminal, when protected, is protected with conventionally
employed peptide chemistry N-terminal protecting goups, but more preferably
such groups
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1 . _.._..__ T.. ___.._._.__. _.___._. T
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are pharmaceutically, agriculturally or horticulturally acceptable.
Preferably the peptide is of 6 to 20 amino acids long, most preferably 7 to
10.
For the purpose of production of the peptides of the invention it may be
preferred
to produce them as repeating polymers wherein the peptide sequence described
above is
repeated a number of times linked together by a specific enzyme lysable bond.
Such
principle is described in PCT/LTS93/0651 (WO 94/01451 ) where multicopy
peptides are
disclosed together with methods for producing amidated peptides. Such peptides
may be
provided by synthesising cDNA for the peptide and ligating numbers of copies
of this
together such that it encodes for eg. polypeptide [-Spacer-Pro-Pro-(Xaa')"-Phe-
Xaa2-Arg-
Xaa~] P where p is an integer from 2 to several thousand, eg. 10,000,
preferably 10 to 100.
The cleavable spacer group may equally be on the C-terminal for this purpose.
Suitable nucleic acid encoding for specifically cleavable spacer petides which
can
be cleaved without cleaving the peptide units of the invention will occur to
those skilled
in the art, particularly in the light of the aforesaid published PCT, and will
offer more
efficient expression targets for recombinant production of the present
peptides.
Preferably the amino acids are L-configuration amino acids such as those which
are
naturally occurring in biological systems. This is particularly preferred for
the end four C-
terminal amino acids, whereas the N-terminal may employ one or more D-
configuration
acids.
When the peptide is in the form of a peptide salt, this may comprise the
peptide
together with any anion or cation conventionally used in pesticidal or
pharmaceutical
preparations as counterion in salt preparations.
Thus the amino acid sequence above, when of the preferred 6 to 20 amino acid
residues long, has up to 12 amino acid residues appended to its N-terminal.
Most preferred
peptides are those wherein n is 1 and/or where only 1 further amino acid
residue is
appended to the N-terminal. Preferred peptides of the first aspect of the
present invention
comprise an amino acid sequence Ile-Pro-Pro-Gln-Phe-Xaa-Arg-Phe or a
conservatively
substituted analogue thereof wherein Xaa represents Met, Val, Ile, Leu, Nle or
Phe or a
conservatively substituted analogue thereof.
The sequence Phe-Xaa-Arg-Phe is that shared by the FMRF.amide family of
invertebrate peptides while the sequence Ile-Pro-Pro-Gln is that determined as
specific to
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the vertebrate peptide isolated as reported above. Thus a preferred such
peptide of the
invention is provided comprising an amino acid sequence Ile-Pro-Pro-Gln-Phe-
Met-Arg-
Phe or a conservatively substituted analogue thereof.
Most preferred peptides of the invention are amide terminated at their C-
terminal
ends. Particular novel forms of the peptide, particularly of the native Ile-
Pro-Pro-Gln-Phe-
Met-Arg-Phe.amide and its precursor ile-Pro-Pro-Gln-Phe-Met-Arg-Phe are
characterised
in that they are forms not encountered in nature, ie. substantially isolated
or in novel or
formulated form, eg. together with pharmaceutically, agriculturally or
horticulturally
acceptable excipients. Novel forms may include, inter alia, those in solid
state, in
substantially pure state, or in aqueous solution in a form not combined with
other
Hylambates (Kassina) maculata frog proteins or not combined with its skin.
The preferred peptides of the invention have an amino acid sequence consisting
of
Ile-Pro-Pro-Gln-Phe-Met-Arg-Phe and particularly preferred is the wild type
peptide.amide
Ile-Pro-Pro-Gln-Phe-Met-Arg-Phe.amide. Also preferred are those peptides where
the MetS
in this sequence is replaced by an amino acid that is less susceptible to
oxidation, whether
in use or in preparation, eg. such as Ile.
The present inventor has determined that the various amino acid sequences
described above and their amidated forms will best retain the activity of the
naturally
occuring octapeptide with varying degrees of resistance to degredation by
biological
systems such as those including peptidase enzymes.
It will be realised that the present peptides may include or be conjugated to
a variety
of other molecules such as labelling agents or other bioactives. Particularly
advantageous
will be fusion peptides incorporating a cytolytic peptide sequence which can
aid penetration
of the peptide of the invention through pest tissues.
In a second aspect of the invention there is provided a nucleic acid encoding
for a
peptide as described above. For the preferred peptides of the invention this
is characterised
in that the nucleic acid is in recombinant form or a form free of skin of the
frog Hylambates
(Kassina) maculata.
Preferred nucleic acids comprise a base sequence of SEQ ID No 1
AUH CCN CCN CAR UUY AUG MGN UUY
whereinHisA,CorU;RisAorG;YisCorU;MisAorCandNisA,C,GorU
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_..~~._._.._ . T ___
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provided that when M is A, N is selected from A or G only and when the nucleic
acid is an
RNA, U is uracil and when it is a DNA U is thymine (T);
or a sequence complementary thereto.
The nucleic acid is most preferably in the form of recombinant nucleic acid,
preferably in the form of a vector, but may be in the form of DNA suitable for
direct
incorporation into the genome of an organism using techniques such as
electroporation or
by use of agents such as lipofectin (see WO 9517515).
Where the nucleic acid is in the form of a vector, that vector is preferably a
virus
or virus derived vector; particularly a baculovirus or baculovirus derived
vector. Preferably
the vector is one such as those described in WO 9517515 in so far as it is
capable of early
expression of the peptide once it has infected a pest, eg. insect, cell.
Bacterial vectors
suitable for plant protection or plant transformation will also occur to those
skilled in the
art and are provided by the present invention.
A third aspect of the present invention provides the use of a peptide of the
invention
as defined as a pesticide to treat loci other than Hylambates (Kassina)
maculata, eg. plants
or animals, whether adminstered internally or externally. Preferred such use
is of peptides
of amino acid sequence Ile-Pro-Pro-Gln-Phe-Xaaz-Arg-Phe or a conservatively
substituted
analogue thereof. Again, it is preferred that the peptide has an amidated C-
terminal, but this
is not essential as the FMRFamide family is widespread in invertebrates and
the necessary
enzymes for specific or non-specific amidation of the FMRF C-terminal end will
be present
in many target pest tissues. Preferably Xaa is Met, Leu, Ile or Nle or a
conservative
substitution thereof.
The preferred use of the pesticide is against invertebrates possessing FMRF or
related tetrapeptide sequence receptors. Preferably the invertebrates are
insects, acarids,
helminthe-worms, eg. platyhelminthes, or nematodes. Most preferably the
invertebrates are
insects and helminthes.
In a fourth aspect of the present invention there are provided compositions
comprising a peptide or nucleic acid of the invention together with a carrier
and/or
excipients that are physiologically acceptable to one or more plants or
vertebrates, ie. they
are pharmaceutically, agriculturally or horticulturally acceptable. Preferably
the carrier will
be suitable for application to all commercially and aesthetically and
ecologically desirable
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plants, but this will not be essential where the target area is not sensitive
to such action.
Carriers suitable for the application of peptide or vector agents will be well
known to those
skilled in the art. WO 9517515 discloses the use of suspensions of baculovirus
occlusion
bodies applied to leaves. Compositions suitable for use in plant protection
will occur to
those skilled in the art in the light of the present disclosure.
Also provided are compositions for protecting or treating animals, eg. humans,
against or for internal or external pests, eg. as topical compositions for
application to skin,
eg. as creams, lotions or aerosols, or for internal administration as is
conventional in the
pharmaceutical art. For treatment of gut parasites it may be preferred to use
enterically
coated compositions, with the advantage that the peptides of the invention
will not be
hydrolysed in the stomach. For treatment of blood borne parasites parenteral
administration
of peptide may be appropriate.
All the compositions of the invention will be likely to benefit from inclusion
of
inhibitors of one or more enzymes, eg. amidases, capable of cleaving any of
the peptide
bonds present. These inhibitors may be non-peptide in nature but may be as
simple as
glycine amide or some similar amino acid or peptide based molecule.
In a fifth aspect the present invention provides transgenic cells and
organisms
which express one or more peptides of the invention. Such organisms or cells
may be
invertebrate, eg. insect, in nature and be useful in production of the
peptides of the
invention for the purpose of their use as pesticides, or may be plant cells or
plants which,
besides this peptide production utility are capable of combatting their own
infestation with
pests by use of the expressed peptide as a defence mechanism.
In a sixth aspect of the present invention there are provided antibodies and
vaccines
for their production comprising . The vaccines are characterised in that they
comprise
peptides of formula -Phe-Xaa2-Arg-Xaa', preferably -Pro-Pro-(Xaa')"-Phe-Xaaz-
Arg-Xaa~,
that are antigenic in character. The present inventor has determined that by
conjugating the
-Phe-Xaaz-Arg-Xaa3 residues to a suitable macromolecule, eg. ovalbumen or a
similar
hapten, eg by glutaraldehyde treatment, specific antibodies may be raised in
animals. Eg.
when Ile-Pro-Pro-Gln-Phe-Met-Arg-Phe.amide is linked by glutaraldehyde
crosslinking to
ovalbumen and emulsified in adjuvant, particularly Freund's incomplete
adjuvant, it
induces a specific antibody response in rabbits. Such approach will readily
lend itself to the
-6-
_____... ._... .._.. T .._._...... _.. . __.__...
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production of protective antibodies in animals and allow production of
monoclonal
antibodies, using conventional techniques, which optionally may be engineered
in known
to fashion to produce humanised or otherwise optimised antibodies.
A seventh aspect of the present invention provides the vaccines and antibodies
of
the invention for use in therapy, eg. of invertebrate parasitic conditions and
for prophylaxis
thereof.
Methods of producing transgenic plant and animal cells will be well known to
those
skilled in the art. In addition to use of viral vectors and direct
incorporation of nucleic acid
into the genome of an organism using techniques such as electroporation and
lipofectin as
referred to above, bacterial vectors such as agrobacterium may be used to
deliver the
nucleic acid. For plants such incorporation may be into pollen or seeds.
The choice of a particular nucleic acid sequence of the invention for
transforming
the cells of a particular organism will depend on that cells codon
preferences. The use of
degenerative substitutions is thus advantageous where the cell or vector
expressing the
protein is of such different type to the DNA source organism cell that it has
different codon
preferences for transcriptionltranslation to that of the cDNA source cell.
Such degenerative
substitutions will thus be host specific.
The expression 'conservative substitutions' as used with respect to amino
acids
relates to the substitution of a given amino acid by an amino acid having
physicochemical
characteristics in the same class. Thus where an amino acid in the SEQ ID No 2
has a
hydrophobic characterising group, a conservative substitution replaces it by
another amino
acid also having a hydrophobic characterising group; other such classes are
those where the
characterising group is hydrophilic, cationic, anionic or contains a thiol or
thioether. Such
substitutions are well known to those of ordinary skill in the art, ie. see US
5380712.
A still further aspect of the present invention provides methods for synthesis
of the
peptides of the present invention. Such methods include the production of such
peptides by
expression of the DNA of the invention in host cells such as will occur to
those skilled in
the art of recombinant DNA expression systems, see eg. WO 9517515, or may eg.
comprise
conventional solid or liquid phase peptide synthetic techniques, automated or
otherwise.
For synthesis and subsequent conversion of the peptides of the invention
having free
carboxylic acid C-terminals to their amidated forms standard peptide
techniques will also
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occur to those skilled in the art, such as those employed JP 1050896 and JP
2257890 in
FMRF.amide chemistry.
The present invention will now be described further by way of illustration
only by
reference to the following non-limiting Examples and Sequence listing and
Figures. Further
embodiments falling within the scope of the invention will occur to those
skilled in the art
in the light of these.
FIGURES
Figure 1. Muscle tension recording showing the effect of 1 p.M VF 1 on an
innervated
muscle preparation of Ascaris suum. The addition of the peptide is indicated
by the
arrow head. Horizontal bar represents 2 min while vertical bar represents 0.25
g.
Figure 2. Physiograph showing the effect of 10 pM VF1 on the contractility of
insect
hindgut. Peptide addition is marked by the arrow head while peptide removal is
indicated
by the inverted arrow head. Horizontal bar represents 1 min while vertical bar
represents 10 mg.
Figure 3. Physiograph displaying the effects of 10 ~,M VF 1 on the
contractility of
the uterus of A.scaris suum. Peptide addition is indicated by an arrow head
and peptide
removal by an inverted arrow head. Horizontal bar represents 1 min and
vertical bar
represents 10 mg.
SEQUENCE LISTING
SEQ ID No 1, that of hypothetical mRNA encoding for the naturally occurring
peptide
from frog skin.
SEQ IDs No 2 to 14: those of peptides of the invention.
EXAMPLES
EXAMPLE 1: Isolation of octapeptide
The peptide Ile-Pro-Pro-Gln-Phe-Met-Arg-Phe.amide was confirmed to be present
in the venom of Hylambates (Kassina) maculata species obtained from a number
of living
specimens thus proving that it is a secreted product from different
individuals of the same
species. The peptide was identified by radioimmunoassay of HPLC fractions of
the extract
using an antiserum raised against invertebrate FMRFamide. This detection
system was
_g-
t ..__ _..._ ? _.._ . _._._
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used in conjunction with a series of HPLC fractionations, to isolate the
peptide in pure
form. The primary structure was established by a combination of
microsequencing and
mass spectroscopy. The molecular mass of the peptide was 1034 Da and the
presence of
a C-terminal amide was established by radioimmunoassay cross-reactivity and by
methylation followed by mass spectroscopy. The peptide was synthesised to >95%
purity
using conventional peptide synthesis techniques and the identity of the
synthetic replicate
was confirmed using HPLC and microsequencing.
I~XAMPLE 2: Ascaris somatic muscle assay.
The synthetic replicate was initially tested on a sensitive preparation for
this family
of peptide, the somatic muscle of the parasitic roundworm, Ascaris suum. At a
concentration of 1 micromolar, the peptide caused rapid and prolonged
relaxation of the test
muscle strip (Figure 1 ). This established that the vertebrate peptide could
in fact interact
with endogenous invertebrate FMRFamide like peptide receptors.
EXAMPLE 3: Insect hindgut assay.
The presence of this peptide in a vertebrate defensive secretion was a mystery
until
we determined that one of the main groups of predators of this frog are giant
carnivorous
aquatic insects. The peptide was thus proposed to perform a defensive function
against
these insect predators, an action which would depend on its interaction with
endogenous
insect receptors for endogenous FMRFamide-related peptides. To test this
hypothesis, the
synthetic peptide was subjected to biological testing on an insect tissue, the
hindgut. which
is a standard insect bioassay for bioactive peptides including FMRFamide-
related peptides.
The results are shown in Figure 2. Application of the frog peptide to this
preparation at
a concentration of 10 micromolar caused a rapid increase in contraction
frequency and
magnitude. The effect was prolonged and only partially reversible with time.
These data
provided evidence that this vertebrate peptide was interactive with endogenous
insect FaRP
receptors and was highly stimulatory.
EXAMPLE 4: Roundworm uterus assay.
The peptide was applied to a novel physiological preparation, the roundworm
uterus
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preparation as described in the legend to Fig 3. As shown in that Figure, the
peptide was
excitatory at a concentration of 10 micromolar. These data are consistent with
activation
of endogenous invertebrate FMRFamide-like peptide receptors.
EXAMPLE 5: Antifeedant assay.
Fifth instar larave of Schistocerca gregaria were injected into the hemolymph
with
saline or various amounts of peptide.amide of Example 1. Each test involved
injection of
ten controls and ten treated larvae. In table 1 below numbers represent the
reduction
grammes weight of a cole leaf during the test period, being the amount eaten
plus
evaporation.
TABLE 1
Control averageInmole 5nmole lOnmole
0.122 0.122
0. I 45 0.055
0.112 0.040
0.124 0.099
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SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: BRITISH TECHNOLOGY GROUP LIMITED
(B) STREET: 10 FLEET PLACE
(C) CITY: LONDON
(E) COUNTRY: UNITED KINGDOM (GB)
(F) POSTAL CODE (ZIP): EC4M 7SB
(A) NAME: SHAW CHRISTOPHER
(B) STREET: 3 SWALLOW CLOSE
(C) CITY: COMBER
(E) COUNTRY: UNITED KINGDOM (GB)
(F) POSTAL CODE (ZIP) : BT23 5UJ
(ii) TITLE OF INVENTION: INSECTICIDAL GENE AND PEPTIDE
(iii) NUMBER OF SEQUENCES: 14
(iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #I.30 (EPO)
(vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: GB 9702189.3
(B) FILING DATE: 04-FEB-1997
(vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: GB 9725149.0
(B) FILING DATE: 27-NOV-1997
(2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: mRNA
(iii) HYPOTHETICAL: YES
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Hylambates (Kassina) maculates
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:1..24
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
AUHCCNCCNC ARUUYAUGMG NUUY 24
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: C-terminal
(vi) ORIGINAL SOURCE:
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(A) ORGANISM: Hylambates (Kassina) maculates
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Pro Pro Xaa Phe Xaa Arg Xaa
1 5
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE : C-terminal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Hylambates (Kassina) maculates
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
Pro Pro Xaa Phe Xaa Arg Phe
1 5
(2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGL~NT TYPE: C-terminal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Hylambates (Kassina) maculates
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
Pro Pro Phe Xaa Arg Xaa
1 5
(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: YES
(v) FRAGL~NT TYPE: C-terminal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Hylambates (Kassina) maculates
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:1..24
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
Pro Pro Xaa Xaa Phe Xaa Arg Xaa
1 5
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....__ T _......_.
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(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: C-terminal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Hylambates (Kassina) maculates
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
Pro Pro Xaa Xaa Xaa Phe Xaa Arg Xaa
1 5
(2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: C-terminal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Hylambates (Kassina) maculates
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
Pro Pro Xaa Xaa Xaa Xaa Phe Xaa Arg Xaa
1 5 10
(2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: C-terminal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Hylambates (Kassina) maculates
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
Pro Pro Xaa Xaa Xaa Xaa Xaa Phe Xaa Arg Xaa
1 5 10
(2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
-13-
CA 02279579 1999-08-03
WO 98/33906 PCT/GB98/00308
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: C-terminal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Hylambates (Kassina) maculates
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
Pro Pro Xaa Xaa Xaa Xaa Xaa Xaa Phe Xaa Arg Xaa
1 5 10
(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ia.) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: C-terminal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Hylambates (Kassina) maculates
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
Pro Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Phe Xaa Arg Xaa
1 5 10
(2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: C-terminal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Hylambates (Kassina) maculates
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
Pro Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Phe Xaa Arg Xaa
1 5 10
(2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: C-terminal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Hylambates (Kassina) maculates
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
Pro Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Phe Xaa Arg Xaa
1 5 10 15
-14-
__~ . ~ ._
CA 02279579 1999-08-03
WO 98/33906 PCT/GB98100308
(2) INFORMATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: C-terminal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Hylambates (Kassina) maculates
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
Pro Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Phe Xaa Arg Xaa
1 5 10 15
(2) INFORMATION FOR SEQ ID NO: 14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ia.) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: C-terminal
vi) ORIGINAL SOURCE:
(A) ORGANISM: Hylambates (Kassina) maculates
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
Ile Pro Pro Gln Phe Met Arg Phe
1 5
-15-
