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INSECZZCIDAL TOXWS FROM PHOTORHABDUS
The invention relates to novel toxins from Photorhabdus luminescens, nucleic
acid
sequences whose expression results in said toxins, and methods of making and
methods of
using the toxins and corresponding nucleic acid sequences to control insects.
Insect pests are a major cause of crop losses. Solely in the US, about $7.7
billion
are lost every year due to infestation by various genera of insects. In
addition to losses in
field crops, insect pests are also a burden to vegetable and fruit growers, to
producers of
ornamental flowers, and they are a nuisance to gardeners and home owners.
Insect pests are mainly controlled by intensive applications of chemical
insecticides,
which are active through inhibition of insect growth, prevention of insect
feeding or
reproduction, or death of the insects. Good insect control can thus be
reached; but these
chemicals can sometimes also affect other, beneficial insects. Another problem
resulting
from the wide use of chemical pesticides is the appearance of resistant insect
varieties.
This has been partially alleviated by various resistance management
strategies, but there is
an increasing need for alternative pest control agents. Biological insect
control agents,
such as Bacillus thuringiensis strains expressing insecticidal toxins like d-
endotoxins, have
also been applied with satisfactory results, offering an alternative or a
complement to
chemical insecticides. Recently, the genes coding for some of these d-
endotoxins have
been isolated and their expression in heterologous hosts have been shown to
provide
another tool for the control of economically important insect pests. In
particular, the
expression of insecticidal toxins in transgenic plants, such as Bacillus
thuringiensis d-
endotoxins, has provided efficient protection against selected insect pests,
and transgenic
plants expressing such toxins have been commercialized, allowing farmers to
reduce
applications of chemical insect control agents. Yet, even in this case, the
development of
resistance remains a possibility and only a few specific insect pests are
controllable.
Consequently, there remains a long-felt but unfulfilled need to discover new
and effective
insect control agents that provide an economic benefit to farmers and that are
environmentally acceptable.
The present invention addresses the need for novel insect control agents.
Particularly needed are control agents that are targeted to economically
important insect
pests and that efficiently control insect strains resistant to existing insect
control agents.
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Furthermore, agents whose application minimizes the burden on the environment
are
desirable.
In the search of novel insect control agents, certain classes of nematodes
from the
genera Hei'erorhabdus and Steinernema are of particular interest because of
their
insecticidal properties. They kill insect larvae and their offspring feed in
the dead larvae.
Indeed, the insecticidal activity is due to symbiotic bacteria living in the
nematodes. These
symbiotic bacteria are Photorhabdus in the case of Heterorhabdus and
Xenorhabdus in the
case of Steinernema.
The present invention is drawn to nucleic acid sequences isolated from
Photorhabdus
luminescens, and sequences substantially similar thereto, whose expression
results in
toxins that are highly toxic to economically important insect pests,
particularly insect pests
that infest plants. The invention is further drawn to the toxins resulting
from the expression
of the nucleic acid sequences, and to compositions and formulations containing
the toxins,
which are capable of inhibiting the ability of insect pests to survive, grow
or reproduce, or of
limiting insect-related damage or loss in crop plants. The invention is
further drawn to a
method of making the toxins and to methods of using the nucleic acid
sequences, for
example in microorganisms to control insects or in transgenic plants to confer
insect
resistance, and to a method of using the toxins, and compositions and
formulations
comprising the toxins, for example applying the toxins or compositions or
formulations to
insect-infested areas, or to prophylactically treat insect-susceptible areas
or plants to confer
protection or resistance to the insects.
The novel toxins are highly active against insects. For example, a number of
economically important insect pests, such as the Lepidopterans Plutella
xylostella
(Diamondback Moth), Trichoplusia ni (Cabbage Looper), Ostrinia nubilalis
(European Corn
Borer), Heliothis virescens (Tobacco Budworm), Helicoverpa zea {Corn Earworm),
Manduca
sexta (Tobacco Hornworm), Spodoptera exigua (Beet Armyworm), and Spodoptera
frugiperda (Fall Armyworm), as well as the Coleopterans Diabrotica virgifera
virgifera
(Western Corn Rootworm), Diabrotica undecimpunctata howardi (Southern Corn
Rootworm), and Leptinotarsa decimlineata (Colorado Potato Beetle) can be
controlled by
one or more of the toxins. The toxins can be used in multiple insect control
strategies,
resulting in maximal efficiency with minimal impact on the environment.
According to one aspect, the present invention provides an isolated nucleic
acid
molecule comprising: (a) a nucleotide sequence substantially similar to a
nucleotide
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sequence selected from the group consisting of: nucleotides 412-1665 of SEQ ID
N0:1,
nucleotides 1686-2447 of SEQ ID N0:1, nucleotides 2758-3318 of SEQ ID N0:1,
nucleotides 3342-4118 of SEQ ID N0:1, nucleotides 4515-9269 of SEQ ID N0:1,
nucleotides 15,171-18,035 of SEQ ID N0:11, and nucleotides 31,393-35,838 of
SEQ ID
N0:11; (b) a nucleotide sequence comprising nucleotides 23,768-31,336 of SEQ
ID N0:11;
or (c) a nucleotide sequence isocoding with the nucleotide sequence of (a) or
(b); wherein
expression of the nucleic acid molecule results in at least one toxin that is
active against
insects.
In one embodiment of this aspect, the nucleotide sequence is isocoding with a
nucleotide sequence substantially similar to nucleotides 412-1665 of SEQ ID
N0:1,
nucleotides 1686-2447 of SEQ ID N0:1, nucleotides 2758-3318 of SEQ ID N0:1,
nucleotides 3342-4118 of SEQ ID N0:1, or nucleotides 4515-9269 of SEQ ID N0:1.
Preferably, the nucleotide sequence is substantially similar to nucleotides
412-1665 of SEQ
ID N0:1, nucleotides 1686-2447 of SEGl ID N0:1, nucleotides 2758-3318 of SEQ
ID N0:1,
nucleotides 3342-4118 of SEQ ID N0:1, or nucleotides 4515-9269 of SEQ ID N0:1.
More
preferably, the nucleotide sequence encodes an amino acid sequence selected
from the
group consisting of SEQ ID NOs:2-6. Most preferably, the nucleotide sequence
corriprises
nucleotides 412-1665 of SEQ ID N0:1, nucleotides 1686-2447 of SEQ ID N0:1,
nucleotides
2758-3318 of SEQ ID N0:1, nucleotides 3342-4118 of SEQ ID N0:1, or nucleotides
4515-
9269 of SEQ ID N0:1.
In another embodiment of this aspect, the nucleotide sequence is isocoding
with a
nucleotide sequence substantially similar to nucleotides 15,171-18,035 of SEQ
ID N0:11.
Preferably, the nucleotide sequence is substantially similar to nucleotides
15,171-18,035 of
SEQ ID N0:11. More preferably, the nucleotide sequence encodes the amino acid
sequence set forth in SEQ ID N0:12. Most preferably, the nucleotide sequence
comprises
nucleotides 15,171-18,035 of SEQ ID N0:11.
In still another embodiment of this aspect, the nucleotide sequence is
isocoding with
a nucleotide sequence substantially similar to nucleotides 31,393-35,838 of
SEQ ID N0:11.
Preferably, the nucleotide sequence is substantially similar to nucleotides
31,393-35,838 of
SEQ ID N0:11. More preferably, the nucleotide sequence encodes the amino acid
sequence set forth in SEQ ID N0:14. Most preferably, the nucleotide sequence
comprises
nucleotides 31,393-35,838 of SEQ ID N0:11.
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In yet another embodiment of this aspect, the nucleotide sequence encodes the
amino acid sequence set forth in SEQ ID N0:13, and preferably comprises
nucleotides
23,768-31,336 of SEQ ID N0:11.
In one embodiment, the nucleotide sequence of the invention comprises the
approximately 9.7 kb DNA fragment harbored in E. coli strain DHSa, designated
as NRRL
accession number B-21835.
In another embodiment, the nucleotide sequence of the. invention comprises the
approximately 38 kb DNA fragment harbored in E. coli strain DHSa, designated
as NRRL
accession number B-30077.
In still another embodiment, the nucleotide sequence of the invention
comprises the
approximately 22.2 kb DNA fragment harbored in E. coli strain DHSa, designated
as NRRL
accession number B-30078.
According to one embodiment of the invention, the toxins resulting from
expression of
the nucleic acid molecules of the invention have activity against Lepidopteran
insects.
Preferably, according to this embodiment, the toxins have activity against
Plutella xylostella
(Diamondback Moth), Trichoplusia ni (Cabbage Looper), Ostrinia nubilalis
{European Corn
Borer), Heliothis virescens (Tobacco Budworm), Helicoverpa zea (Corn
Earvvorm),
Spodoptera exigua (Beet Armyworm), and Spodopi'era frugiperda (Fall Armyworm).
According to another embodiment of the invention, the toxins resulting from
expression of the nucleic acid molecule of the invention have activity against
Lepidopteran
and Coleopteran insects. Preferably, according to this embodiment, the toxins
have
insecticidal activity against Plutella xylostella (Diamondback Moth), Ostrinia
nubilalis
(European Com Borer), and Manduca sexta (Tobacco Hornworm), Diabrotica
virgifera
virgifera (Western Corn Rootworm), Diabrotica undecimpunctata howardi
(Southern Com
Rootworm), and Lepfinotarsa decimlineata (Colorado Potato Beetle).
In another aspect, the present invention provides an isolated nucleic acid
molecule
comprising a 20 base pair nucleotide portion identical in sequence to a
consecutive 20 base
pair nucleotide portion of a nucleotide sequence selected from the group
consisting of:
nucleotides 412-1665 of SEQ ID N0:1, nucleotides 1686-2447 of SEQ ID N0:1,
nucleotides
2758-3318 of SEO ID N0:1, nucleotides 3342-4118 of SEO ID N0:1, nucleotides
4515-
9269 of SEQ ID N0:1, nucleotides 15,171-18,035 of SEQ ID N0:11, and
nucleotides
31,393-35,838 of SEQ ID N0:11, wherein expression of the nucleic acid molecule
results in
at least one toxin that is active against insects.
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In one embodiment of this aspect, the isolated nucleic acid molecule of the
invention
comprises a 20 base pair nucleotide portion identical in sequence to a
consecutive 20 base
pair nucleotide portion of nucleotides 412-1665 of SEQ ID N0:1, nucleotides
1686-2447 of
SEQ ID N0:1, nucleotides 2758-3318 of SEQ ID N0:1, nucleotides 3342-4118 of
SEQ ID
N0:1, or nucleotides 4515-9269 of SEQ ID N0:1.
In another embodiment of this aspect, the isolated nucleic acid molecule of
the
invention comprises a 20 base pair nucleotide portion identical in sequence to
a
consecutive 20 base pair nucleotide portion of nucleotides 15,171-18,035 of
SEQ ID
NO: i 1.
in still another embodiment of this aspect, the isolated nucleic acid molecule
of the
invention comprises a 20 base pair nucleotide portion identical in sequence to
a
consecutive 20 base pair nucleotide portion of nucleotides 31,393-35,838 of
SEQ ID
N0:11.
In a further aspect, the present invention provides an isolated nucleic acid
molecule
comprising a nucleotide sequence from Photorhabdus luminescens selected from
the group
consisting of: nucleotides 412-1665 of SEQ ID N0:1, nucleotides 1686-2447 of
SEQ ID
N0:1, nucleotides 2758-3318 of SEQ (D N0:1, nucleotides 3342-4118 of SEQ ID
N0:1,
nucleotides 4515-9269 of SEQ ID N0:1, nucleotides 66-1898 of SEQ ID N0:11,
nucleotides 2416-9909 of SEQ ID N0:11, the complement of nucleotides 2817-3395
of
SEQ ID N0:11, nucleotides 9966-14,633 of SEGO ID N0:11, nucleotides 14,699-
15,007 of
SEQ ID N0:11, nucleotides 15,171-18,035 of SEQ ID N0:11, the complement of
nucleotides 17,072-17,398 of SEQ ID N0:11, the complement of nucleotides
18,235-
19,167 of SEt~ ID N0:11, the complement of nucleotides 19,385-20,116 of SEQ ID
N0:11,
the complement of nucleotides 20,217-20,963 of SEQ ID N0:11, the complement of
nucleotides 22,172-23,086 of SEQ ID N0:11, nucleotides 23,768-31,336 of SEQ ID
N0:11,
nucleotides 31,393-35,838 of SEQ ID N0:11, the complement of nucleotides
35,383-
35,709 of SEQ ID N0:11, the complement of nucleotides 36,032-36,661 of SEQ ID
N0:11,
and the complement of nucleotides 36,654-37,781 of SEQ ID N0:11.
The present invention also provides a chimeric gene comprising a heterologous
promoter sequence operatively linked to the nucleic acid molecule of the
invention. Further,
the present invention provides a recombinant vector comprising such a chimeric
gene. Still
further, the present invention provides a host cell comprising such a chimeric
gene. A host
cell according to this aspect of the invention may be a bacterial cell, a
yeast cell, or a plant
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cell, preferably a plant cell. Even further, the present invention provides a
plant comprising
such a plant cell. Preferably, the plant is maize.
In yet another aspect, the present invention provides toxins produced by the
expression of DNA molecules of the present invention.
According to one embodiment, the toxins of the invention have activity against
Lepidopteran insects, preferably against Plutella xylostella (Diamondback
Moth),
Trichoplusia ni (Cabbage Looper), Ostrinia nubilalis (European Corn Borer),
Heliothis
virescens (Tobacco Budworm), Helicoverpa zea (Com Earworm), Spodoptera exigua
(Beet
Armyworm), and Spodoptera frugiperda (Fall Armyworm).
According to another embodiment, the toxins of the invention have activity
against
Lepidopteran and Coleopteran insects, preferably against Plutella xylostella
(Diamondback
Moth), Ostrinia nubilalis (European Corn Borer), and Manduca sexta (Tobacco
Homworm),
Diabrotica virgifera virgifera (Western Corn Rootworm), Diabrotica
undecimpunctata
howardi (Southern Com Rootworm), and Leptinotarsa decimlineata (Colorado
Potato
Beetle).
In one embodiment, the toxins are produced by the E. coli strain designated as
NRRL accession number B-21835.
In another embodiment, the toxins are produced by E. coli strain designated as
NRRL accession number B-30077.
!n still another embodiment, the toxins are produced by E. coli strain
designated as
NRRL accession number B-30078.
In one embodiment, a toxin of the invention comprises an amino acid sequence
selected from the group consisting of: SE4 ID NOs:2-6.
In another embodiment, a toxin of the invention comprises an amino acid
sequence
selected from the group consisting of: SEGO ID NOs:l2-14.
The present invention also provides a composition comprising an insecticidally
effective amount of a toxin according to the invention.
In another aspect, the present invention provides a method of producing a
toxin that is
active against insects, comprising: (a) obtaining a host cell comprising a
chimeric gene,
which itself comprises a heterologous promoter sequence operatively linked to
the nucleic
acid molecule of the invention; and (b) expressing the nucleic acid molecule
in the cell,
which results in at least one toxin that is active against insects.
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In a further aspect, the present invention provides a method of producing an
insect-
resistant plant, comprising introducing a nucleic acid molecule of the
invention into the
plant, wherein the nucleic acid molecule is expressible in the plant in an
effective amount to
control insects. According to one embodiment, the insects are Lepidopteran
insects,
preferably selected from the group consisting of: Plutella xylostella
(Diamondback Moth),
Trichoplusia ni (Cabbage Looper), Ostrinia nubilalis (European Corn Borer),
Heliothis
virescens (Tobacco Budworm), Helicoverpa zea (Corn Earworm), Spodoptera exigua
(Beet
Armyworm), and Spodoptera frugiperda (Fall Armyworm). According to another
embodiment, the insects are Lepidopteran and Coleopteran insects, preferably
selected
from the group consisting of: Plutella xylostella (Diamondback Moth), Ostrinia
nubilalis
(European Corn Borer), and Manduca sexta (Tobacco Homworm), Diabrotica
virgifera
virgitera (Western Corn Rootworm), Diabrotica undecimpunctata howardi
(Southern Com
Rootworm), and Leptinotarsa. decimlineata (Colorado Potato Beetle).
In still a further aspect, the present invention provides a method of
controlling insects
comprising delivering to the insects an effective amount of a toxin according
to the present
invention. According to one embodiment, the insects are Lepidopteran insects,
preferably
selected from the group consisting of: Plutella xylostella (Diamondback Moth),
Trichoplusia
ni (Cabbage Looper), Ostrinia nubilalis (European Corn Borer), Heliothis
virescens
(Tobacco Budworm), Helicoverpa zea (Corn Earworm), Spodoptera exigua (Beet
Armyworm), and Spodoptera frugiperda (Fall Armyworm). According to another
embodiment, the insects are Lepidopteran and Coleopteran insects, preferably
selected
from the group consisting of: Plutella xylostella (Diamondback Moth), Ostrinia
nubilalis
(European Corn Borer), and Manduca sexta (Tobacco Hornworm), Diabrotica
virgifera
virgifera (Western Com Rootworm), Diabrotica undecimpunctata howardi (Southern
Com
Rootworm), and Leptinotarsa decimlineata (Colorado Potato Beetle). Preferably,
the toxin
is delivered to the insects orally.
Yet another aspect of the present invention is the provision of a method for
mutagenizing a nucleic acid molecule according to the present invention,
wherein the
nucleic acid molecule has been cleaved into population of double-stranded
random
fragments of a desired size, comprising: (a) adding to the population of
double-stranded
random fragments one or more single- or double-stranded oligonucleotides,
wherein the
oligonucleotides each comprise an area of identity and an area of heterology
to a doubie-
stranded template polynucleotide; (b) denaturing the resultant mixture of
double-stranded
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random fragments and oligonucleotides into single-stranded fragments; (c)
incubating the
resultant population of single-stranded fragments with a polymerase under
conditions which
result in the annealing of the single-stranded fragments at the areas of
identity to form pairs
of annealed fragments, the areas of identity being sufficient for one member
of a pair to
prime replication of the other, thereby forming a mutagenized double-stranded
polynucleotide; and (d) repeating the second and third steps for at least two
further cycles,
wherein the resultant mixture in the second step of a further cycle includes
the mutagenized
double-stranded polynucleotide from the third step of the previous cycle, and
wherein the
further cycle forms a further mutagenized double-stranded polynucleotide.
Other aspects and advantages of the present invention will become apparent to
those
skilled in the art from a study of the following description of the invention
and non-limiting
examples.
DEFINITIONS
"Activity" of the toxins of the invention is meant that the toxins function as
orally
active insect control agents, have a toxic effect, or are able to disrupt or
deter insect
feeding, which may or may not cause death of the insect. When a toxin of the
invention is
delivered to the insect, the result is typically death of the insect, or the
insect does not feed
upon the source that makes the toxin available to the insect.
"Associated with / operatively linked" refer to two nucleic acid sequences
that are
related physically or functionally. For example, a promoter or regulatory DNA
sequence is
said to be "associated with" a DNA sequence that codes for an RNA or a protein
if the two
sequences are operatively linked, or situated such that the regulator DNA
sequence will
affect the expression level of the coding or structural DNA sequence.
A "chimeric gene" is a recombinant nucleic acid sequence in which a promoter
or
regulatory nucleic acid sequence is operatively linked to, or associated with,
a nucleic acid
sequence that codes for an mRNA or which is expressed as a protein, such that
the
regulator nucleic acid sequence is able to regulate transcription or
expression of the
associated nucleic acid sequence. The regulator nucleic acid sequence of the
chimeric
gene is not normally operatively linked to the associated nucleic acid
sequence as found in
nature.
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A "coding sequence" is a nucleic acid sequence that is transcribed into RNA
such as
mRNA, rRNA, tRNA, snRNA, sense RNA or antisense RNA. Preferably the RNA is
then
translated in an organism to produce a protein.
To "control" insects means to inhibit, through a toxic effect, the ability of
insect pests
to survive, grow, feed, and/or reproduce, or to limit insect-related damage or
loss in crop
plants. To "control" insects may or may not mean killing the insects, although
it preferably
means killing the insects.
To "deliver' a toxin means that the toxin comes in contact with an insect,
resulting in
toxic effect and control of the insect. The toxin can be delivered in many
recognized ways,
e.g., orally by ingestion by the insect or by contact with the insect via
transgenic plant
expression, formulated protein composition(s), sprayable protein
composition(s), a bait
matrix, or any other art-recognized toxin delivery system.
"Expression cassette" as used herein means a nucleic acid sequence capable of
directing expression of a particular nucleotide sequence in an appropriate
host cell,
comprising a promoter operably linked to the nucleotide sequence of interest
which is
operably linked to termination signals. It also typically comprises sequences
required for
proper translation of the nucleotide sequence. The expression cassette
comprising the
nucleotide sequence of interest may be chimeric, meaning that at least one of
its
components is heterologous with respect to at least one of its other
components. The
expression cassette may also be one which is naturally occurring but has been
obtained in
a recombinant form useful for heterologous expression. Typically, however, the
expression
cassette is heterologous with respect to the host, i.e., the particular
nucleic acid sequence
of the expression cassette does not occur naturally in the host cell and must
have been
introduced into the host cell or an ancestor of the host cell by a
transformation event. The
expression of the nucleotide sequence in the expression cassette may be under
the control
of a constitutive promoter or of an inducible promoter which initiates
transcription only when
the host cell is exposed to some particular external stimulus. In the case of
a multicellular
organism, such as a plant, the promoter can also be specific to a particular
tissue, or organ,
or stage of development.
A "gene" is a defined region that is located within a genome and that, besides
the
aforementioned coding nucleic acid sequence, comprises other, primarily
regulatory, nucleic
acid sequences responsible for the control of the expression, that is to say
the transcription
and translation, of the coding portion. A gene may also comprise other 5' and
3'
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untranslated sequences and termination sequences. Further elements that may be
present
are, for example, introns.
"Gene of interest" refers to any gene which, when transferred to a plant,
confers upon
the plant a desired characteristic such as antibiotic resistance, virus
resistance, insect
resistance, disease resistance, or resistance to other pests, herbicide
tolerance, improved
nutritional value, improved performance in an industrial process or altered
reproductive
capability. The "gene of interest" may also be one that is transferred to
plants for the
production of commercially valuable enzymes or metabolites in the plant.
A "heterologous" nucleic acid sequence is a nucleic acid sequence not
naturally
associated with a host cell into which it is introduced, including non-
naturally occurring
multiple copies of a naturally occurring nucleic acid sequence.
A "homologous" nucleic acid sequence is a nucleic acid sequence naturally
associated with a host cell into which it is introduced.
"Homologous recombination" is the reciprocal exchange of nucleic acid
fragments
between homologous nucleic acid molecules.
"Insecticidal" is defined as a toxic biological activity capable of
controlling insects,
preferably by killing them.
A nucleic acid sequence is "isocoding with" a reference nucleic acid sequence
when
the nucleic acid sequence encodes a polypeptide having the same amino acid
sequence as
the polypeptide encoded by the reference nucleic acid sequence.
An "isolated" nucleic acid molecule or an isolated enzyme is a nucleic acid
molecule
or enzyme that, by the hand of man, exists apart from its native environment
and is
therefore not a product of nature. An isolated nucleic acid molecule or enzyme
may exist in
a purffied form or may exist in a non-native environment such as, for example,
a
recombinant host cell.
A "nucleic acid molecule" or "nucleic acid sequence" is a linear segment of
single- or
double-stranded DNA or RNA that can be isolated from any source. In the
context of the
present invention, the nucleic acid molecule is preferably a segment of DNA.
"ORF" means open reading frame.
A "plant" is any plant at any stage of development, particularly a seed plant.
A "plant cell" is a structural and physiological unit of a plant, comprising a
protopiast
and a cell wall. The plant cell may be in form of an isolated single cell or a
cultured cell, or
as a part of higher organized unit such as, for example, plant tissue, a plant
organ, or a
whole plant.
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"Plant cell culture° means cultures of plant units such as, for
example, protoplasts,
cell culture cells, cells in plant tissues, pollen, pollen tubes, ovules,
embryo sacs, zygotes
and embryos at various stages of development.
"Plant material" refers to leaves, stems, roots, flowers or flower parts,
fruits, pollen,
egg cells, zygotes, seeds, cuttings, cell or tissue cultures, or any other
part or product of a
plant.
A "plant organ" is a distinct and visibly structured and differentiated part
of a plant
such as a root, stem, leaf, flower bud, or embryo.
"Plant tissue" as used herein means a group of plant cells organized into a
structural
and functional unit. Any tissue of a plant in planfa or in culture is
included. This term
includes, but is not limited to, whole plants, plant organs, plant seeds,
tissue culture and
any groups of plant cells organized into structural and/or functional units.
The use of this
term in conjunction with, or in the absence of, any specific type of plant
tissue as listed
above or otherwise embraced by this definition is not intended to be exclusive
of any other
type of plant tissue.
A "promoter" is an untranslated DNA sequence upstream of the coding region
that
contains the binding site for RNA polymerase II and initiates transcription of
the DNA. The
promoter region may also include other elements that act as regulators of gene
expression.
A "protoplast" is an isolated plant cell without a cell wall or with only
parts of the cell
wall.
"Regulatory elements" refer to sequences involved in controlling the
expression of a
nucleotide sequence. Regulatory elements comprise a promoter operably linked
to the
nucleotide sequence of interest and termination signals. They also typically
encompass
sequences required for proper translation of the nucleotide sequence.
In its broadest sense, the term "substantially similar", when used herein with
respect
to a nucleotide sequence, means a nucleotide sequence corresponding to a
reference
nucleotide sequence, wherein the corresponding sequence encodes a polypeptide
having
substantially the same structure and function as the polypeptide encoded by
the reference
nucleotide sequence, e.g. where only changes in amino acids not affecting the
polypeptide
function occur. Desirably the substantially similar nucleotide sequence
encodes the
polypeptide encoded by the reference nucleotide sequence. The percentage of
identity
between the substantially similar nucleotide sequence and the reference
nucleotide
sequence desirably is at least 80%, more desirably at least 85%, preferably at
least 90%,
more preferably at least 95%, still more preferably at least 99%. A nucleotide
sequence
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"substantially similar" to reference nucleotide sequence hybridizes to the
reference
nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaP04, 1 mM EDTA
at
50°C with washing in 2X SSC, 0.1 % SDS at 50°C, more desirably
in 7% sodium dodecyl
sulfate (SDS), 0.5 M NaP04, 1 mM EDTA at 50°C with washing in 1 X SSC,
0.1 % SDS at
50°C, more desirably still in 7% sodium dodecyl sulfate (SDS), 0.5 M
NaP04, 1 mM EDTA at
50°C with washing in 0.5X SSC, O.i% SDS at 50°C, preferably in
7% sodium dodecyl
sulfate (SDS), 0.5 M NaP04, 1 mM EDTA at 50°C with washing in 0.1 X
SSC, 0.1 % SDS at
50°C, more preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaP04,
1 mM EDTA at
50°C with washing in 0.1 X SSC, 0.1 % SDS at 65°C.
"Synthetic" refers to a nucleotide sequence comprising structural characters
that are
not present in the natural sequence. For example, an artificial sequence that
resembles
more closely the G+C content and the normal codon distribution of dicot and/or
monocot
genes is said to be synthetic.
'Transformation° is a process for introducing heterologous nucleic acid
into a host
cell or organism. In particular, "transformation" means the stable integration
of a DNA
molecule into the genome of an organism of interest.
'Transformed / transgenic / recombinant" refer to a host organism such as a
bacterium or a plant into which a heterologous nucleic acid molecule has been
introduced.
The nucleic acid molecule can be stably integrated into the genome of the host
or the
nucleic acid molecule can also be present as an extrachromosomal molecule.
Such an
extrachromosomal molecule can be auto-replicating. Transformed cells, tissues,
or plants
are understood to encompass not only the end product of a transformation
process, but
also transgenic progeny thereof. A "non-transformed", "non-transgenic", or
"non-
recombinant" host refers to a wild-type organism, e.g., a bacterium or plant,
which does not
contain the heterologous nucleic acid molecule.
Nucleotides are indicated by their bases by the following standard
abbreviations:
adenine (A), cytosine (C), thymine (T), and guanine (G). Amino acids are
likewise indicated
by the following standard abbreviations: alanine (Ala; A), arginine (Arg; R),
asparagine (Asn;
N), aspartic acid (Asp; D), cysteine (Cys; C), glutamine (Gln; Q), glutamic
acid (Glu; E),
glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L),
lysine (Lys; K),
methionine {Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser;
S), threonine
(Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).
Furthermore, (Xaa; X)
represents any amino acid.
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BRIEF DESCRIPTION OF THE SEQUENCES IN THE SE4UENCE LISTING
SEQ ID N0:1 is the sequence of the approximately 9.7 kb DNA fragment comprised
in
pCIB9359-7 which comprises the following ORFs at the specified nucleotide
positions:
Name Start End
orf1 412 1665
orf2 1686 2447
orf3 2758 3318
o rf4 3342 4118
orf5 4515 9269
SEA ID N0:2 is the sequence of the -46.4 kDa protein encoded by orf1 of SEQ ID
N0:1.
SE4 ID N0:3 is the sequence of the --28.1 kDa protein encoded by orf2 of SEQ
ID N0:1.
SEQ ID N0:4 is the sequence of the -20.7 kDa protein encoded by orf3 of SEQ ID
N0:1.
SEQ ID N0:5 is the sequence of the -28.7 kDa protein encoded by orf4 of SEG1
ID N0:1.
SEQ ID N0:6 is the sequence of the --176 kDa protein encoded by orf5 of SEQ ID
N0:1.
SE4 ID NOs:7-10 are oligonucleotides.
SE4 ID N0:11 is the sequence of the approximately 38 kb DNA fragment comprised
in
pNOV2400, which comprises the following ORFs at the specified nucleotide
positions
(descending numbers and "C" indicates that the ORF is on the complementary
strand):
Name S_~art End
orf7 66 1898 (partial sequence)
hph3 2416 9909
orfl8 3395 2817 C
orf4 9966 14,633
orf19 14,699 15,007
orf5 15,i 71 18,035
orf22 17,398 17,072 C
orfl0 19,167 18,235 C
orfl4 20,116 19,385 C
orfl3 20,963 20,217 C
orfll 23,086 22,172 C
hph2 23,768 31,336
orf2 31,393 35,838
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orf21 35,709 35,383 C
orfl6 36,661 36,032 C
orf8 37,781 36,654 C
SEQ ID N0:11 also includes
the following restriction
sites, some of which
are used in the
subcloning steps set ple 17:
forth in Exam
Restriction Site Nucleotide Position(sl
Acdll 2835
BamHl 18,915
BsmB) 11,350
Bstt 1071 29,684
Eagl 13,590; 31,481
Eco721 34,474
Mlul 2444; 5116; 9327; 26,204
Nod 13,589
Pad 9915; 23,353; 37,888
Pvul 8816
Sapl 35,248
SexAl 28,946
Sgl1 8815
Spel 2157; 3769; 7831; 11,168
Sphl 755
Stul 35,690
Tth111 I 21,443
SE4 ID N0:12 is the sequence of the protein encoded by orf5 of SEQ ID N0:11.
SEQ ID N0:13 is the sequence of the protein encoded by hph2 of SEQ ID N0:11
SEQ ID N0:14 is the sequence of the protein encoded by orf2 of SEGl ID N0:11.
SEQ ID NOs:l5-22 are oligonucleotides.
DEPOSITS
The following material has been deposited with the Agricultural Research
Service,
Patent Culture Collection (NRRL), 1815 North University Street, Peoria,
Illinois 61604, under
the terms of the Budapest Treaty on the International Recognition of the
Deposit of
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Microorganisms for the Purposes of Patent Procedure. All restrictions on the
availability of
the deposited material will be irrevocably removed upon the granting of a
patent.
Clone Accession Number Date of Deposit
pCIB9359-7 NRRL B-21835 September 17, 1997
pNOV2400 NRRL B-30077 December 3, 1998
pNOV1001 NRRL B-30078 December 3, 1998
Novel Nucleic Acid Sequences whose Expression Results in Insecticidal Toxins
This invention relates to nucleic acid sequences whose expression results in
novel
toxins, and to the making and using of the toxins to control insect pests. The
nucleic acid
sequences are derived from Photorhabdus luminescens, a member of the
Enterobacteriaceae family. P. luminescens is a symbiotic bacterium of
nematodes of the
genus Heterorhabditis. The nematodes colonize insect larva, kill them, and
their offspring
feed on the dead larvae. The insecticidal activity is actually produced by the
symbiotic P.
luminescens bacteria. The inventors are the first to isolate the nucleic acid
sequences of
the present invention from P. luminescens (ATCC strain number 29999). The
expression of
the nucleic acid sequences of the present invention results in toxins that can
be used to
control Lepidopteran insects such as Plutella xylostella (Diamondback Moth),
Trichoplusia ni
(Cabbage Looper), Osfrinia nubilalis (European Corn Borer), Heliothis
virescens (Tobacco
Budworm), Helicoverpa zea (Corn Earworm); Manduca sexta (Tobacco Hornworm),
Spodoptera exigua (Beet Armyworm), and Spodoptera frugiperda (Fall Armyworm),
as well
as Coleopteran insects such as Diabrotica virgifera virgifera (Western Corn
Rootworm),
Diabrotica undecimpunctata howardi (Southern Com Rootworm), Diabrotica
longicomis
barberi (Northern Corn Rootworm), and Leptinotarsa decimlineata (Colorado
Potato
Beetle).
In one preferred embodiment, the invention encompasses an isolated nucleic
acid
molecule comprising a nucleotide sequence substantially similar to the
approximately 9.7 kb
nucleic acid sequence set forth in SEQ ID N0:1, whose expression results in
insect control
activity (further illustrated in Examples 1-11). Five open reading frames
(ORFs) are present
in the nucleic acid sequence set forth in SECI ID N0:1, coding for proteins of
predicted sizes
45 kDa, 28 kDa, 21 kDA, 29 kDa, and 176 kDa. The five ORFs are arranged in an
operon-
like structure. When expressed in a heterologous host, the - 9.7 kb DNA
fragment from P.
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Iuminescens results in insect control activity against Lepidopterans such as
Plutella
xylostella (Diamondback Moth), Trichoplusia ni (Cabbage Looper), Ostrinia
nubilalis
(European Com Borer), Heliothis virescens (Tobacco Budworm), Helicoverpa zea
(Com
Earworm), Spodoptera exigua (Beet Armyworm), and Spodoptera frugiperda (Fall
Armyworm), showing that expression of the - 9.7 kb nucleotide sequence set
forth in SEQ
ID N0:1 is necessary and sufficient for such insect control activity. In a
preferred
embodiment, the invention encompasses a DNA molecule, whose expression results
in an
insecticidal toxin, which is deposited in the E. coli strain pC189359-7 (NRRL
accession
number B-21835).
In another preferred embodiment, the invention encompasses an isolated nucleic
acid molecule comprising a nucleotide sequence substantially similar to the
approximately
38 kb nucleic acid fragment set forth in SEQ ID N0:11 and deposited in the E.
coli strain
pNOV2400 (NRRL accession number B-30077), whose expression results in insect
control
activity (see Examples 12-18). In a more preferred embodiment, the invention
encompasses
an isolated nucleic acid molecule comprising a nucleotide sequence
substantially similar to
the - 22 kb DNA fragment deposited in the E. coli strain pNOV1001 (NRRL
accession
number B-30078), whose expression results in insect control activity. In a
most preferred
embodiment, the invention encompasses isolated nucleic acid molecules
comprising
nucleotide sequences substantially similar to the three ORFs corresponding to
nucleotides
23,768-31,336 (hph2), 31,393-35,838 {orf2), and 15,171-18,035 (orf5) of the
DNA fragment
set forth in SEGt ID N0:11, as well as the proteins encoded thereby. When co-
expressed in
a heterologous host, these three ORFs result in insect control activity
against Lepidopterans
such as Plutella xylostella {Diamondback Moth), Ostrinia nubilalis (European
Corn Borer),
and Manduca sexta (Tobacco Hornworm), as well as against Coleopterans such as
Diabrotica virgifera virgifera (Western Corn Rootworm), Diabrotica
undecimpunctata
howardi (Southern Corn Rootworm), and Leptinotarsa decimlineata (Colorado
Potato
Beetle), showing that co-expression of these three ORFs (hph2, orf2, and orf5)
is necessary
and sufficient for such insect control activity.
The present invention also encompasses recombinant vectors comprising the
nucleic
acid sequences of this invention. In such vectors, the nucleic acid sequences
are preferably
comprised in expression cassettes comprising regulatory elements for
expression of the
nucleotide sequences in a host cell capable of expressing the nucleotide
sequences. Such
regulatory elements usually comprise promoter and termination signals and
preferably also
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comprise elements allowing efficient translation of polypeptides encoded by
the nucleic
acid sequences of the present invention. Vectors comprising the nucleic acid
sequences
are usually capable of replication in particular host cells, preferably as
extrachromosomal
molecules, and are therefore used to amplify the nucleic acid sequences of
this invention in
the host cells. In one embodiment, host cells for such vectors are
microorganisms, such as
bacteria, in particular E.coli. In another embodiment, host cells for such
recombinant vectors
are endophytes or epiphytes. A preferred host cell for such vectors is a
eukaryotic cell,
such as a yeast, a plant cell, or an insect cell. Plant cells such as maize
cells are most
preferred host cells. In another preferred embodiment, such vectors are viral
vectors and
are used for replication of the nucleotide sequences in particular host cells,
e.g. insect cells
or plant cells. Recombinant vectors are also used for transformation of the
nucleotide
sequences of this invention into host cells, whereby the nucleotide sequences
are stably
integrated into the DNA of such host cells. In one, such host cells are
prokaryotic cells. In a
preferred embodiment, such host cells are eukaryotic cells, such as yeast
cells, insect cells,
or plant cells. In a most preferred embodiment, the host cells are plant
cells, such as maize
cells.
In preferred embodiments, the insecticidal toxins of the invention each
comprise at
least one polypeptide encoded by a nucleotide sequence of the invention. In
another
preferred embodiment, the insecticidal toxins are produced from a purified
strain of P.
luminescens, such the strain with ATTC accession number 29999. The toxins of
the
present invention have insect control activity when tested against insect
pests in bioassays;
and these properties of the insecticidal toxins are further illustrated in
Examples 1-18. The
insecticidal toxins desribed in the present invention are further
characterized in that their
molecular weights are larger than 6,000, as found by size fractionation
experiments. The
insecticidal toxins retain full insectidical activity after being stored at
4°C for 2 weeks. One
is also shown to retain its full insecticidal activity after being freeze-
dried and stored at 22°C
for 2 weeks. However, the insecticidal toxins of the invention lose their
insecticidal activity
after incubation for 5 minutes at 100°C.
In further embodiments, the nucleotide sequences of the invention can be
modified
by incorporation of random mutations in a technique known as in-vitro
recombination or
DNA shuffling. This technique is described in Stemmer et al., Nature 370: 389-
391 (1994)
and US Patent 5,605,793, which are incorporated herein by reference. Millions
of mutant
copies of a nucleotide sequence are produced based on an original nucleotide
sequence of
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this invention and variants with improved properties, such as increased
insecticidal activity,
enhanced stability, or different specificity or range of target insect pests
are recovered. The
method encompasses forming a mutagenized double-stranded polynucleotide from a
template double-stranded polynucleotide comprising a nucleotide sequence of
this
invention, wherein the template double-stranded polynucleotide has been
cleaved into
double-stranded-random fragments of a desired size, and comprises the steps of
adding to
the resultant population of double-stranded random fragments one or more
single or
double-stranded oligonucleotides, wherein said oligonucleotides comprise an
area of
identity and an area of heterology to the double-stranded template
polynucleotide;
denaturing the resultant mixture of double-stranded random fragments and
oligonucleotides
into single-stranded fragments; incubating the resultant population of single-
stranded
fragments with a poiymerase under conditions which result in the annealing of
said single-
stranded fragments at said areas of identity to form pairs of annealed
fragments, said areas
of identity being sufficient for one member of a pair to prime replication of
the other, thereby
forming a mutagenized double-stranded polynucleotide; and repeating the second
and third
steps for at least two further cycles, wherein the resultant mixture in the
second step of a
further cycle includes the mutagenized double-stranded polynucleotide from the
third step
of the previous cycle, and the further cycle forms a further mutagenized
double-stranded
polynucleotide. In a preferred embodiment, the concentration of a single
species of double-
stranded random fragment in the population of double-stranded random fragments
is less
than 1 % by weight of the total DNA. In a further preferred embodiment, the
template
double-stranded polynucleotide comprises at least about 100 species of
polynucleotides. In
another preferred embodiment, the size of the double-stranded random fragments
is from
about 5 by to 5 kb. In a further preferred embodiment, the fourth step of the
method
comprises repeating the second and the third steps for at least 10 cycles.
Expression of the Nucleotide Seauences in Heteroloaous Microbial Hosts
As biological insect control agents, the insecticidal toxins are produced by
expression
of the nucleotide sequences in heterologous host cells capable of expressing
the nucleotide
sequences. In a first embodiment, P. luminescens cells comprising
modifications of at least
one nucleotide sequence of this invention at its chromosomal location are
described. Such
modifications encompass mutations or deletions of existing regulatory
elements, thus
leading to altered expression of the nucleotide sequence, or the incorporation
of new
regulatory elements controlling the expression of the nucleotide sequence. !n
another
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embodiment, additional copies of one or more of the nucleotide sequences are
added to P.
luminescens cells either by insertion into the chromosome or by introduction
of
extrachromosomally replicating molecules containing the nucleotide sequences.
In another embodiment, at least one of the nucleotide sequences of the
invention is
inserted into an appropriate expression cassette, comprising a promoter and
termination
signals. Expression of the nucleotide sequence is constitutive, or an
inducible promoter
responding to various types of stimuli to initiate transcription is used. In a
preferred
embodiment, the cell in which the toxin is expressed is a microorganism, such
as a virus, a
bacteria, or a fungus. In a preferred embodiment, a virus, such as a
baculovirus, contains a
nucleotide sequence of the invention in its genome and expresses large amounts
of the
corresponding insecticidal toxin after infection of appropriate eukaryotic
cells that are
suitable for virus replication and expression of the nucleotide sequence. The
insecticidal
toxin thus produced is used as an insecticidal agent. Alternatively,
baculoviruses
engineered to include the nucleotide sequence are used to infect insects in-
vivo and kill
them either by expression of the insecticidal toxin or by a combination of
viral infection and
expression of the insecticidal toxin.
Bacterial cells are also hosts for the expression of the nucleotide sequences
of the
invention. In a preferred embodiment, non-pathogenic symbiotic bacteria, which
are able to
live and replicate within plant tissues, so-called endophytes, or non-
pathogenic symbiotic
bacteria, which are capable of colonizing the phyllosphere or the rhizosphere,
so-called
epiphytes, are used. Such bacteria include bacteria of the genera
Agrobacterium,
Alcaligenes, Azospirillum, Azotobacfer, Bacillus, Clavibacter, Enterobacter,
Erwinia,
Flavobacter, Klebsiella, Pseudomonas, Rhizobium, Serratia, Streptomyces and
Xanthomonas. Symbiotic fungi, such as Trichodem~a and Gliocladium are also
possible
hosts for expression of the inventive nucleotide sequences for the same
purpose.
Techniques for these genetic manipulations are specific for the different
available
hosts and are known in the art. For example, the expression vectors pKK223-3
and
pKK223-2 can be used to express heterologous genes in E, coli, either in
transcriptional or
translational fusion, behind the tac or trc promoter. For the expression of
operons encoding
multiple ORFs, the simplest procedure is to insert the operon into a vector
such as pKK223-
3 in transcriptional fusion, allowing the cognate ribosome binding site of the
heterologous
genes to be used. Techniques for overexpression in gram-positive species such
as Bacillus
are also known in the art and can be used in the context of this invention
(Quax et al. In.:
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Industrial Microorganisms: Basic and Applied Molecular Genetics, Eds. Baltz et
aL,
American Society for Microbiology, Washington (1993)). Alternate systems for
overexpression rely for example, on yeast vectors and include the use of
Pichia,
Saccharomyces and Kluyveromyces (Sreekrishna, In: Industrial microorganisms:
basic and
applied molecular genetics, Baltz, Hegeman, and Skatrud eds., American Society
for
Microbiology, Washington (1993); Dequin & Barre, Biotechnology 12:173-177
(1994); van
den Berg et al., Biotechnology 8:135-139 (1990)).
In another preferred embodiment, at least one of the described nucleotide
sequences is transferred to and expressed in Pseudomonas fluorescens strain
CGA267356
(described in the published application EU 0 472 494 and in WO 94/01561 )
which has
biocontrol characteristics. In another preferred embodiment, a nucleotide
sequence of the
invention is transferred to Pseudomonas aureofaciens strain 30-84 which also
has
biocontrol characteristics. Expression in heteroiogous biocontrol strains
requires the
selection of vectors appropriate for replication in the chosen hast and a
suitable choice of
promoter. Techniques are well known in the art for expression in gram-negative
and gram-
positive bacteria and fungi.
Exaression of the Nucleotide Seguencg~ in Plant Tissue
In a particularly preferred embodiment, at least one of the insecticidal
toxins of the
invention is expressed in a higher organism, e.g., a plant. In this case,
transgenic plants
expressing effective amounts of the toxins protect themselves from insect
pests. When the
insect starts feeding on such a transgenic plant, it also ingests the
expressed toxins. This
will deter the insect from further biting into the plant tissue or may even
harm or kill the
insect. A nucleotide sequence of the present invention is inserted into an
expression
cassette, which is then preferably stably integrated in the genome of said
plant. In another
preferred embodiment, the nucleotide sequence is included in a non-pathogenic
self-
replicating virus. Plants transformed in accordance with the present invention
may be
monocots or dicots and include, but are not limited to, maize, wheat, barley,
rye, sweet
potato, bean, pea, chicory, lettuce, cabbage, cauliflower, broccoli, turnip,
radish, spinach,
asparagus, onion, garlic, pepper, celery, squash, pumpkin, hemp, zucchini,
apple, pear,
quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape,
raspberry,
blackberry, pineapple, avocado, papaya, mango, banana, soybean, tomato,
sorghum,
sugarcane, sugarbeet, sunflower, rapeseed, clover, tobacco, carrot, cotton,
alfalfa, rice,
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potato, eggplant, cucumber, Arabidopsis, and woody plants such as coniferous
and
deciduous trees.
Once a desired nucleotide sequence has been transformed into a particular
plant
species, it may be propagated in that species or moved into other varieties of
the same
species, particularly including commercial varieties, using traditional
breeding techniques.
A nucleotide sequence of this invention is preferably expressed in transgenic
plants,
thus causing the biosynthesis of the corresponding toxin in the transgenic
plants. In this
way, transgenic plants with enhanced resistance to insects are generated. For
their
expression in transgenic plants, the nucleotide sequences of the invention may
require
modification and optimization. Although in many cases genes from microbial
organisms can
be expressed in plants at high levels without modification, low expression in
transgenic
plants may result from microbial nucleotide sequences having codons that are
not preferred
in plants. It is known in the art that all organisms have specific preferences
for codon
usage, and the codons of the nucleotide sequences described in this invention
can be
changed to conform with plant preferences, while maintaining the amino acids
encoded
thereby. Furthermore, high expression in plants is best achieved from coding
sequences
that have at least 35% about GC content, preferably more than about 45%, more
preferably
more than about 50%, and most preferably more than about 60%. Microbial
nucleotide
sequences which have low GC contents may express poorly in plants due to the
existence
of ATTTA motffs which may destabilize messages, and AATAAA motifs which may
cause
inappropriate polyadenylation. Although preferred gene sequences may be
adequately
expressed in both monocotyledonous and dicotyledonous plant species, sequences
can be
modified to account for the specific codon preferences and GC content
preferences of
monocotyledons or dicotyledons as these preferences have been shown to differ
(Murray ef
al. Nucl. Acids Res. 17: 477-498 (1989)). In addition, the nucleotide
sequences are
screened for the existence of illegitimate splice sites that may cause message
truncation.
All changes required to be made within the nucleotide sequences such as those
described
above are made using well known techniques of site directed mutagenesis, PCR,
and
synthetic gene construction using the methods described in the published
patent
applications EP 0 385 962 (to Monsanto), EP 0 359 472 {to Lubrizol, and WO
93/07278 (to
Ciba-Geigy).
For efficient initiation of translation, sequences adjacent to the initiating
methionine
may require modification. For example, they can be modified by the inclusion
of sequences
known to be effective in plants. Joshi has suggested an appropriate consensus
for plants
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(NAR 15: 6643-6653 (1987)) and Clontech suggests a further consensus
translation
initiator (1993/1994 catalog, page 210). These consensuses are suitable for
use with the
nucleotide sequences of this invention. The sequences are incorporated into
constructions
comprising the nucleotide sequences, up to and including the ATG (whilst
leaving the
second amino acid unmodified), or alternatively up to and including the GTC
subsequent to
the ATG (with the possibility of modifying the second amino acid of the
transgene).
Expression of the nucleotide sequences in transgenic plants is driven by
promoters
shown to be functional in plants. The choice of promoter will vary depending
on the
temporal and spatial requirements for expression, and also depending on the
target
species. Thus, expression of the nucleotide sequences of this invention in
leaves, in ears, in
inflorescences (e.g. spikes, panicles, cobs, etc.), in roots, and/or seedlings
is preferred. In
many cases, however, protection against more than one type of insect pest is
sought, and
thus expression in multiple tissues is desirable. Although many promoters from
dicotyledons have been shown to be operational in monocotyledons and vice
versa, ideally
dicotyledonous promoters are selected for expression in dicotyledons, and
monocotyledonous promoters for expression in monocotyledons. However, there is
no
restriction to the provenance of selected promoters; it is sufficient that
they are operational
in driving the expression of the nucleotide sequences in the desired cell.
Preferred promoters that are expressed constitutively include promoters from
genes
encoding actin or ubiquitin and the CaMV 35S and 19S promoters. The nucleotide
sequences of this invention can also be expressed under the regulation of
promoters that
are chemically regulated. This enables the insecticidal toxins to be
synthesized only when
the crop plants are treated with the inducing chemicals. Preferred technology
for chemical
induction of gene expression is detailed in the published application EP 0 332
104 (to Ciba-
Geigy) and US patent 5,614,395. A preferred promoter for chemical induction is
the
tobacco PR-1 a promoter.
A preferred category of promoters is that which is wound inducible. Numerous
promoters have been described which are expressed at wound sites and also at
the sites of
phytopathogen infection. Ideally, such a promoter should only be active
locally at the sites
of infection, and in this way the insecticidal toxins only accumulate in cells
which need to
synthesize the insecticidal toxins to kill the invading insect pest. Preferred
promoters of this
kind include those described by Stanford ef aI. Mol. Gen. Genet. 215: 200-208
(1989), Xu et
al. Plant Molec. Biol. 22: 573-588 (1993), Lo'gemann et al. Plant Cell 1: 151-
158 (1989),
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Rohrmeier 8~ Lehle, Plant Molec. Biol. 22: 783-792 (1993), Firek et al. Plant
Molec. Biol. 22:
129-142 (1993), and Wamer et al. Plant J. 3: 191-201 (1993).
Preferred tissue specific expression patterns include green tissue specific,
root
specific, stem specific, and flower specific. Promoters suitable for
expression in green
tissue include many which regulate genes involved in photosynthesis and many
of these
have been cloned from both monocotyledons and dicotyledons. A preferred
promoter is the
maize PEPC promoter from the phosphoenol carboxylase gene (Hudspeth & Grula,
Plant
Molec. Biol. 12: 579-589 (1989)). A preferred promoter for root specific
expression is that
described by de Framond (FEES 290: 103-106 (1991 ); EP 0 452 269 to Ciba-
Geigy). A
preferred stem specific promoter is that described in US patent 5,625,136 (to
Ciba-Geigy)
and which drives expression of the maize trpA gene.
Especially preferred embodiments of the invention are transgenic plants
expressing
at least one of the nucleotide sequences of the invention in a root-preferred
or root-specific
fashion. Further preferred embodiments are transgenic plants expressing the
nucleotide
sequences in a wound-inducible or pathogen infection-inducible manner.
In addition to the selection of a suitable promoter, constructions for
expression of an
insecticidal toxin in plants require an appropriate transcription terminator
to be attached
downstream of the heterologous nucleotide sequence. Several such terminators
are
available and known in the art (e.g. tml from CaMV, E9 from rbc57. Any
available
terminator known to function in plants can be used in the context of this
invention.
Numerous other sequences can be incorporated into expression cassettes
described in this invention. These include sequences which have been shown to
enhance
expression such as intron sequences (e.g. from Adh1 and bronzel) and viral
leader
sequences (e.g. from TMV, MCMV and AMV).
It may be preferable to target expression of the nucleotide sequences of the
present
invention to different cellular iocalizations in the plant. In some cases,
localization in the
cytosol may be desirable, whereas in other cases, localization in some
subcellular organelle
may be preferred. Subcellular localization of transgene encoded enzymes is
undertaken
using techniques well known in the art. Typically, the DNA encoding the target
peptide from
a known organelle-targeted gene product is manipulated and fused upstream of
the
nucleotide sequence. Many such target sequences are known for the chloroplast
and their
functioning in heterologous constructions has been shown. The expression of
the
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nucleotide sequences of the present invention is also targeted to the
endoplasmic reticulum
or to the vacuoles of the host cells. Techniques to achieve this are well-
known in the art.
Vectors suitable for plant transformation are described elsewhere in this
specification. For Agrobacterium-mediated transformation, binary vectors or
vectors
carrying at least one T-DNA border sequence are suitable, whereas for direct
gene transfer
any vector is suitable and linear DNA containing only the construction of
interest may be
preferred. In the case of direct gene transfer, transformation with a single
DNA species or
co-transformation can be used (Schocher et al. Biotechnology 4: 1093-1096
(1986}}. For
both direct gene transfer and Agrobacterium-mediated transfer, transformation
is usually
(but not necessarily) undertaken with a selectable marker which may provide
resistance to
an antibiotic (kanamycin, hygromycin or methotrexate) or a herbicide (basta).
The choice of
selectable marker is not, however, critical to the invention.
In another preferred embodiment, a nucleotide sequence of the present
invention is
directly transformed into the plastid genome. A major advantage of plastid
transformation is
that plastids are generally capable of expressing bacterial genes without
substantial
modification, and plastids are capable of expressing multiple open reading
frames under
control of a single promoter. Plastid transformation technology is extensively
described in
U.S. Patent Nos. 5,451,513, 5,545,817, and 5,545,818, in PCT application no.
WO
95/16783, and in McBride et al. (1994) Proc. Natl. Acad. Sci. USA 91, 7301-
7305. The basic
technique for chloroplast transformation involves introducing regions of
cloned plastid DNA
flanking a selectable marker together with the gene of interest into a
suitable target tissue,
e.g., using biolistics or protoplast transformation (e.g., calcium chloride or
PEG mediated
transformation). The 1 to 1.5 kb flanking regions, termed targeting sequences,
facilitate
homologous recombination with the plastid genome and thus allow the
replacement or
modification of specific regions of the plastome. Initially, point mutations
in the chloroplast
16S rRNA and rpsl2 genes conferring resistance to spectinomycin and/or
streptomycin are
utilized as selectable markers for transformation (Svab, Z., Hajdukiewicz, P.,
and Maliga, P.
(1990) Proc. Natl. Acad. Sci. USA 87, 8526-8530; Staub, J. M., and Maliga, P.
(1992) Plant
Cell 4, 39-45). This resulted in stable homopiasmic transformants at a
frequency of
approximately one per 100 bombardments of target leaves. The presence of
cloning sites
between these markers allowed creation of a plastid targeting vector for
introduction of
foreign genes {Staub, J.M., and Maliga, P. (1993) EMBO J. 12, 601-606).
Substantial
increases in transformation frequency are obtained by replacement of the
recessive rRNA
or r-protein antibiotic resistance genes with a dominant selectable marker,
the bacterial
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aadA gene encoding the spectinomycin-detoxifying enzyme aminoglycoside-3'-
adenyltransferase (Svab, Z., and Maliga, P. (1993) Proc. Natl. Acad. Sci. USA
90, 913-917).
Previously, this marker had been used successfully for high-frequency
transformation of the
plastid genome of the green alga Chlamydomonas reinhardtii (Goldschmidt-
Clermont, M.
(1991 ) Nucl. Acids Res. 19: 4083-4089). Other selectable markers useful for
plastid
transformation are known in the art and encompassed within the scope of the
invention.
Typically, approximately 15-20 cell division cycles following transformation
are required to
reach a homoplastidic state. Plastid expression, in which genes are inserted
by homologous
recombination into all of the several thousand copies of the circular plastid
genome present
in each plant cell, takes advantage of the enormous copy number advantage over
nuclear-
expressed genes to permit expression levels that can readily exceed 10% of the
total
soluble plant protein. In a preferred embodiment, a nucleotide sequence of the
present
invention is inserted into a plastid targeting vector and transformed into the
plastid genome
of a desired plant host. Plants homoplastic for plastid genomes containing a
nucleotide
sequence of the present invention are obtained, and are preferentially capable
of high
expression of the nucleotide sequence.
Formulation of Insecticidal Compositions
The invention also includes compositions comprising at least one of the
insecticidal
toxins of the present invention. In order to effectively control insect pests
such compositions
preferably contain sufficient amounts of toxin. Such amounts vary depending on
the crop to
be protected, on the particular pest to be targeted, and on the environmental
conditions,
such as humidity, temperature or type of soil. In a preferred embodiment,
compositions
comprising the insecticidal toxins comprise host cells expressing the toxins
without
additional purification. In another preferred embodiment, the cells expressing
the
insecticidal toxins are lyophilized prior to their use as an insecticidal
agent. In another
embodiment, the insecticidal toxins are engineered to be secreted from the
host cells. In
cases where purification of the toxins from the host cells in which they are
expressed is
desired, various degrees of purification of the insecticidal toxins are
reached.
The present invention further embraces the preparation of compositions
comprising
at least one insecticidal toxin of the present invention, which is
homogeneously mixed with
one or more compounds or groups of compounds described herein. The present
invention
also relates to methods of treating plants, which comprise application of the
insecticidal
toxins or compositions containing the insecticidal toxins, to plants. The
insecticidal toxins
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can be applied to the crop area in the form of compositions or plant to be
treated,
simultaneously or in succession, with further compounds. These compounds can
be both
fertilizers or micronutrient donors or other preparations that influence plant
growth. They
can also be selective herbicides, insecticides, fungicides, bactericides,
nematicides,
molluscicides or mixtures of several of these preparations, if desired
together with further
carriers, surfactants or application-promoting adjuvants customarily employed
in the art of
formulation. Suitable carriers and adjuvants can be solid or liquid and
correspond to the
substances ordinarily employed in formulation technology, e.g. natural or
regenerated
mineral substances, solvents, dispersants, wetting agents, tackifiers, binders
or fertilizers.
A preferred method of applying insecticidal toxins of the present invention is
by
spraying to the environment hosting the insect pest like the soil, water, or
foliage of plants.
The number of applications and the rate of application depend on the type and
intensity of
infestation by the insect pest. The insecticidal toxins can also penetrate the
plant through
the roots via the soil (systemic action) by impregnating the locus of the
plant with a liquid
composition, or by applying the compounds in solid form to the soil, e.g. in
granular form
(soil application). The insecticidal toxins may also be applied to seeds
(coating) by
impregnating the seeds either with a liquid formulation containing
insecticidal toxins, or
coating them with a solid formulation. In special cases, further types of
application are also
possible, for example, selective treatment of the plant stems or buds. The
insecticidal
toxins can also be provided as bait located above or below the ground.
The insecticidal toxins are used in unmodified form or, preferably, together
with the
adjuvants conventionally employed in the art of formulation, and are therefore
formulated in
known manner to emulsifiable concentrates, coatable pastes, directly sprayable
or dilutable
solutions, dilute emulsions, wettable powders, soluble powders, dusts,
granulates, and also
encapsulations, for example, in polymer substances. Like the nature of the
compositions,
the methods of application, such as spraying, atomizing, dusting, scattering
or pouring, are
chosen in accordance with the intended objectives and the prevailing
circumstances.
The formulations, compositions or preparations containing the insecticidal
toxins
and, where appropriate, a solid or liquid adjuvant, are prepared in known
manner, for
example by homogeneously mixing and/or grinding the insecticidal toxins with
extenders,
for example solvents, solid carriers and, where appropriate, surface-active
compounds
(surfactants).
Suitable solvents include aromatic hydrocarbons, preferably the fractions
having 8 to
12 carbon atoms, for example, xylene mixtures or substituted naphthalenes,
phthalates
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such as dibutyl phthalate or dioctyl phthalate, aliphatic hydrocarbons such as
cyclohexane
or paraffins, alcohois and glycols and their ethers and esters, such as
ethanol, ethylene
glycol monomethyl or monoethyl ether, ketones such as cyclohexanone, strongly
polar
solvents such as N-methyl-2-pyrrolidone, dimethyl sulfoxide or dimethyl
formamide, as well
as epoxidized vegetable oils such as epoxidized coconut oil or soybean oil or
water.
The solid carriers used e.g. for dusts and dispersible powders, are normally
natural
mineral fillers such as calcite, talcum, kaolin, montmorillonite or
attapulgite. In order to
improve the physical properties it is also possible to add highly dispersed
silicic acid or
highly dispersed absorbent polymers. Suitable granulated adsorptive carriers
are porous
types, for example pumice, broken brick, sepiolite or bentonite; and suitable
nonsorbent
carriers are materials such as calcite or sand. In addition, a great number of
pregranulated
materials of inorganic or organic nature can be used, e.g. especially dolomite
or pulverized
plant residues.
Suitable surface-active compounds are nonionic, cationic and/or anionic
surfactants
having good emulsifying, dispersing and wetting properties. The term
"surfactantsN will also
be understood as comprising mixtures of surfactants. Suitable anionic
surfactants can be
both water-soluble soaps and water-soluble synthetic surface-active compounds.
Suitable soaps are the alkali metal salts, alkaline earth metal salts or
unsubstituted
or substituted ammonium salts of higher fatty acids (chains of 10 to 22 carbon
atoms), for
example the sodium or potassium salts of oleic or stearic acid, or of natural
fatty acid
mixtures which can be obtained for example from coconut oil or tallow oil. The
fatty acid
methyltaurin salts may also be used.
More frequently, however, so-called synthetic surfactants are used, especially
fatty
suifonates, fatty sulfates, sulfonated benzimidazole derivatives or
alkylarylsulfonates.
The fatty sulfonates or sulfates are usually in the form of alkali metal
salts, alkaline
earth metal salts or unsubstituted or substituted ammonium salts and have a 8
to 22 carbon
alkyl radical which also includes the alkyl moiety of alkyl radicals, for
example, the sodium
or calcium salt of lignonsulfonic acid, of dodecylsulfate or of a mixture of
fatty alcohol
sulfates obtained from natural fatty acids. These compounds also comprise the
salts of
sulfuric acid esters and sulfonic acids of fatty alcohol/ethyiene oxide
adducts. The
sulfonated benzimidazole derivatives preferably contain 2 sulfonic acid groups
and one fatty
acid radical containing 8 to 22 carbon atoms. Examples of alkylarylsulfonates
are the
sodium, calcium or triethanolamine salts of dodecylbenzenesulfonic acid,
dibutylnapthalenesulfonic acid, or of a naphthalenesulfonic acid/formaldehyde
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condensation product. Also suitable are corresponding phosphates, e.g. salts
of the
phosphoric acid ester of an adduct of p-nonylphenol with 4 to 14 moles of
ethylene oxide.
Non-ionic surfactants are preferably polyglycol ether derivatives of aliphatic
or
cycloaliphatic alcohols, or saturated or unsaturated fatty acids and
alkylphenols, said
derivatives containing 3 to 30 glycol ether groups and 8 to 20 carbon atoms in
the (aliphatic)
hydrocarbon moiety and 6 to 18 carbon atoms in the alkyl moiety of the
alkylphenols.
Further suitable non-ionic surfactants are the water-soluble adducts of
polyethylene
oxide with polypropylene glycol, ethylenediamine propylene glycol and
alkylpolypropylene
glycol containing 1 to 10 carbon atoms in the alkyl chain, which adducts
contain 20 to 250
ethylene glycol ether groups and 10 to 100 propylene glycol ether groups.
These
compounds usually contain 1 to 5 ethylene glycol units per propylene glycol
unit.
Representative examples of non-ionic surfactants are
nonylphenolpolyethoxyethanols, castor oil polyglycol ethers,
polypropylene/polyethylene
oxide adducts, tributylphenoxypolyethoxyethanol, polyethylene glycol and
octylphenoxyethoxyethanol. Fatty acid esters of polyoxyethylene sorbitan and
polyoxyethylene sorbitan trioleate are also suitable non-ionic surfactants.
Cationic surfactants are preferably quaternary ammonium salts which have, as N-
substituent, at feast one C8-C22 alkyl radical and, as further substituents,
lower
unsubstituted or halogenated alkyl, benzyl or lower hydroxyalkyl radicals. The
salts are
preferably in the form of halides, methylsulfates or ethylsulfates, e.g.
stearyltrimethylammonium chloride or benzyldi(2-chloroethyl)ethylammonium
bromide.
The surfactants customarily employed in the art of formulation are described,
for
example, in "McCutcheon's Detergents and Emulsifiers Annual," MC Publishing
Corp.
Ringwood, New Jersey, 1979, and Sisely and Wood, "Encyclopedia of Surface
Active
Agents," Chemical Publishing Co., Inc. New York, 1980.
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EXAMPLES
The invention will be further described by reference to the following detailed
examples. These examples are provided for purposes of illustration only, and
are not
intended to be limiting unless otherwise specified. Standard recombinant DNA
and
molecular cloning techniques used here are well known in the art and are
described by
Ausubel (ed.), Current Protocols in Molecular Biology, John Wiley and Sons,
Inc. (1994); T.
Maniatis, E. F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory
Manual, Cold
Spring Harbor laboratory, Cold Spring Harbor, NY (1989); and by T.J. Silhavy,
M.L. Berman,
and L.W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor
Laboratory, Cold
Spring Harbor, NY (1984).
A. Isolation Of Nucleotide Seauences Whose Exaression Results In Toxins Active
Against Lepidoateran Insects
Example 1: Construction of Cosmid Library from Photorhabdus luminescens
Photorhabdus luminescens strain ATCC 29999 is grown in nutrient broth at
25°C for
three days as described in the ATCC protocol for bioassay. The culture is
grown for 24
hours for DNA isolation. Total DNA is isolated by treating freshly grown cells
resuspended
in 100 mM Tris pH 8, 10 mM EDTA with 2 mg/ml lysozyme for 30 minutes at
37°C.
Proteinase K is added to a final concentration of 100 mg/ml, SDS is added to a
final
concentration of 0.5% SDS and the sample is incubated at 45°C. After
the solution
becomes clear and viscous, the SDS concentration is raised to 1 %, and 300 mM
NaCI and
an equal volume of phenol-chloroform-isoamyl alcohol are added, mixed gently
for 5
minutes and centrifuged at 3K. The phenol-chloroform-isoamyl alcohol
extraction is
repeated twice. The aqueous phase is mixed with 0.7 volumes isopropanol, and
the sample
is centrifuged. The pellet is washed 3 times with 70% ethanol and the nucleic
acids are
gently resuspended in 0.5X TE.
The DNA is treated with 0.3 units of Sau3A per mg DNA at 37°C for 3.5
minutes in
100 ml volume containing a total of 6 mg DNA. The reaction is then heated for
30 minutes
at 65°C to inactivate the enzyme. Then 2 units of Calf Intestinal
Alkaline Phosphatase are
added and incubated for 30 minutes at 37°C. The sample is mixed with an
equal volume of
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phenol-chloroform-isoamyl alcohol and centrifuged. The aqueous phase is
removed,
precipitated with 0.7 volume isopropanol and centrifuged. The supernatant is
transferred to
a fresh tube, precipitated with ethanol, and the nucleic acids are resuspended
in 0.5X TE at
a concentration of 100 hgiml.
SuperCos cosmid vector (Stratagene, La Joila, CA) is prepared as described by
the
supplier utilizing the BamHl cloning site. Prepared SuperCos at 100 hg/ml is
figated with the
Sau3A digested P.luminescens DNA at a molar ratio of 2:1 in a 5 ml volume
overnight at
6°C. The figation mixture is packaged using Gigapack XL III
(Stratagene), as described by
the supplier. Packaged phages are used to infect XL-1 MR (Stratagene) cells as
described
by the supplier. The cosmid library is plated on L-agar with 50 mgiml
kanamycin and
incubated 16 hours at 37°C. 500 colonies are patched onto fresh L-kan
plates at 50
colonies per plate. From the other plates the cells are washed off with L
broth and mixed
with 20% glycerol and frozen at -80°C.
Example 2: Insect Bioassays
Plutella xylostella bioassays are performed by aliquoting of 50 wl of the E.
coil culture
on the solid artificial Plutella xylostella diet (Biever and Boldt, Annals of
Entomological
Society of America, 1971; Shelton et al., J. Ent. Sci. 26:17). 4 ml of the
diet is poured into 1
oz. clear plastic cups (Bioserve product #9051 ). 5 neonate P. xylostella from
a diet adapted
lab colony are placed in each diet-containing cup and then covered with a
white paper lid
(Bioserve product #9049). 10 larvae are assayed per concentration. Trays of
cups are
placed in an incubator for 3 days at 72°F with a 14:10 (hours)
light:dark cycle. Then, the
number of live larvae in each cup is recorded. Bioassays for other insects are
performed as
described for Plutella xylostella, but using the diet required by the insect
to be tested.
The broth of P. luminescens undiluted and diluted 1:100 gives 100% mortality
against P. xylosfella. The broth of P. luminescens also gives 100% mortality
against
Diabrotica virgifera virgifera. Three clones with activity against P.
xylosfella and Heliothis
virescens are obtained after screening 500 E. coil clones by insect bioassay.
These cosmid
clones are given the numbers pCIB9349, pCIB9350, and pCIB9351.
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Example 3: Isolation of the Nucleotide Sequence Responsible for Insect Control
Activity from Clones pCIB9349, pCIB9350, and pCIB9351
The three clones pCIB9349, pCIB9350 and pC189351 are found to be overlapping
cosmids by restriction enzyme mapping. After digestion with Pad, clones
pCIB9349 and
pCIB9351 give two DNA fragments each, and pCIB9350 gives three DNA fragments.
Each
fragment is isolated and is self-ligated. The enzyme Pad does not cut the
SuperCos vector;
therefore, only fragments linked to it are re-isolated. The ligation mixtures
are transformed
into DHSa E. coli cells. Isolated transformed bacterial colonies are grown in
L broth with 50
p.g/ml kanamycin, and plasmid DNA is isolated by using the alkaline miniprep
protocol as
described in Sambrook, et al. DNA is digested with NotllPacl and two clones,
pCIB9355
and pCIB935fi, are found by bioassay to still contain the insecticidal
activity. Clone
pCIB9355 is digested with Notl and a 17 kb and a 4 kb DNA fragment are
generated. The
17 kb fragment is isolated and ligated into Bluescript vector previously cut
with Noil and
transformed into DHSa E. coli cells. The isolated transformed bacterial
colonies are grown
as described and plasmid DNA is isolated by the alkaline miniprep protocol. A
clone
containing the 17 kb insert is named pC189359 and tested by bioassay. The
results are
shown in Example 5. 3 pg of the 17 kb insert is isolated and treated with 0.3
unit of Sau3A
per wg DNA for 4, 6, and 8 minutes at 37°C, heated at 75°C for
15 minutes. The samples
are pooled and ligated into pUCl9 previously cut with BamHl and treated with
calf intestinal
alkaline phosphatase. The ligation is transformed into DHSa cells and plated
on L agar with
XgallAmp as described in Sambrook et al. and grown overnight at 37°C:
White colonies
are picked and grown in L broth with 100 N.g/ml and plasmid DNA is isolated as
previously
described. DNA is digested with EcoRllHindlll and novel restriction patterns
are sequenced.
Sequencing primers are ordered from Genosys Biotechnologies (Woodlands, TX).
Sequencing is performed using the dideoxy chain-termination method. Sequencing
is
completed using Applied Biosystems Inc. model 377 automated DNA sequencer
(Foster
City, CA). Sequence is assembled using 3.0 from Gene Codes Corporation (Ann
Arbor,
MI).
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Example 4: Subcloning of the 9.7 kb EcoRllXbal Fragment From pCIB9359
pCIB9359 is digested with EcoRl and Xbal and the DNA is run on a 0.8%
Seaplaque/TBE gel. The 9.7 kb fragment (SEQ ID NO:1 ) is isolated and ligated
into pUCl9
previously digested with EcoRl and Xbal. The ligation mixture is transformed
into DHSa E.
coli cells. Transformed bacteria are grown and plasmid DNA is isolated as
previously
described. The vector containing the 9.7 kb fragment in pUCl9 is designated
pCIB9359-7
and bioassay results are shown in Example 5.
Example 5: Bioassay Results for Cosmid Clones pCIB9359 and pCIB9359-7
Cultures of E. coli strains 9359 and 9359-7 containing clones pCIB9359 and
pCIB9359-7, respectively, are tested for insecticidal activity against the
following insects in
insect bioassays:
Insects - Clones
pCIB9359 and pCIB9359-7
Plutella xylostella (Diamondback Moth +++
(DBM))
Heliofhis virescens (Tobacco Budworm ++
(TBW))
Helicoverpa zea (Corn Earworm (CEW)) +++
Spodoptera exigua (Beet Armyworm (BAW))+
Spodoptera frugiperda (Fall Armyworm +
(FAW))
Trichoplusia ni (Cabbage Looper (CL)) +++
Ostrinia nubilalis (European Com Boreri-+
(ECB))
Manduca sexta (Tobacco Hornworm (THW) na
Diabrotica virgifera (Western Corn na
Rootworm (WCR))
Agrotis ipsilon (Black Cutworm (BCW)) na
na = not active
+ = significant growth inhibition
++ _ >40% mortality, but less than 100%
+++ = 100% mortality
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The clones show insecticidal activity against P, xylostella, H, virescens, H.
zea, T. ni,
and O. nubilalis, and significant insect control activity against S. exigua
and S. frugiperda.
Example 6: Identification of Active Region of pCIB9359-7 By Subcloning
Cultures of E. coli strains containing subclones of pCIB9359-7 are tested for
insecticida! activity in insect bioassays against P. xylosfella.
Restriction Nucleotide Position Relative to 9.7 kb Insecticidal Activity
Against
Fragment EcoRUXbaI fragment (SEO ID N0:1 ) Plutella xylosfella
from pC1B9539-7 and Size in kb
EcoRllXbal 1 to 9712 9.7 kb +++
EcoRV (-912) to 23093.2 kb na
Hindlll 665 to 5438 4.7 kb na
Kpnl 1441 to 8137 6.9 kb na
SacllXbal 2677 to 9712 7.0 kb na
na = not active
+ = significant growth inhibition
++ _ >40% mortality, but less than 100%
+++ = 100% mortality
Example 7: Characterization of pCIB9359-7 Insect Control Activity By Titration
Dilutions of a culture of E.coli strain 9359-7 containing pCIB9359-7 are
tested for
insecticidal activity in insect bioassays. Dilutions are prepared in a culture
of E.coli XL-1 in a
total volume of 100 ul and are transferred to diet cups with 5 insects per
cup. The results
show the percentage (%) of insect mortality.
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p.l 9359-7 CulturePx Hv Hz Tn
100 i00 72 48 100
50 100 84 68 92
25 100 52 32 100
12.5 96 52 36 68
6.25 88 20 4 32
0 36 20 24 0
Px = P. xylostella, Hv = H. virescens, Hz = N. zea, Tn = T. ni.
Cultures of E. coli 9359-7 still show substantial insecticidal activity after
dilution.
Example 8: Stability of pCIB9359-7 Activity
The stability of the toxins is tested after storage for 2 weeks at different
temperatures and conditions. 300 ml of Luria broth containing 100 (p.g/ml
ampicillin is
inoculated with E. coli strain 9359-7 and grown overnight at 37°C.
Samples are placed in
sterile 15 ml screw cap tubes and stored at 22°C and 4°C.
Another sample is centrifuged;
the supernatant is removed, freeze dried and stored at 22°C. The
samples are stored under
these conditions for 2 weeks and then a bioassay is conducted against P.
xylostella. The
freeze dried material is resuspended in the same volume as before. All samples
are
resuspended by vortexing.
Conditions Results
22°C (2 weeks) +++
4°C (2 weeks) +++
Freeze Dried (2 weeks) +++
na = not active; + = significant growth inhibition; ++ _ >40% mortality, but
less than 100%;
+++ = 100% mortality
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This demonstrates that the toxins retain their activity for at least two weeks
at 22°C,
4°C, and freeze-dried, and are therefore very stable.
Example 9: Size Fraction of pCIB9359-7 Activity
The approximate sizes of the insecticidal toxins are determined. P.
luminescens
cosmid clones pCIB9359-7 and pUCl9 in E. coli host DHSa are grown in media
consisting
of 50% Terrific broth and 50% Luria broth, supplemented with 50 ~g/ml
ampicillin. Cultures
(three tubes of each strain) are inoculated into 3 ml of the above media in
culture tubes and
incubated on a roller wheel overnight at 37°C. Cultures of each strain
are combined and
sonicated using a Branson Model 450 Sonicator, micro tip, for approximately
six 10 second
cycles with cooling on ice between cycles. The sonicates are centrifuged in a
Sorvall SS34
rotor at 6000 RPM for 10 minutes. The resultant supernatants are filtered
through a 0.2 N
filter. The 3 ml fractions of the filtrates are applied to Bio-Rad Econo-Pac
10DG columns
that have been previously equilibrated with 10 ml of 50mM NaCI, 25 mM Tris
base, pH 7Ø
The flow through collected during sample loading is discarded. The samples are
fractionated with two subsequent additions of 4 ml each of the NaCI - Tris
equilibration
buffer. The two four ml fractions are saved for testing. The first fraction
contains all
material above about 6,000 mot. wt; the second fraction contains material
smaller than
6,000 mol. wt. A sample of the whole culture broth, the sonicate, and the
filtered
supernatant on the sonicate are tested along with the three fractions from the
10DG column
for activity on P. xylostella neonates in bioassays.
The culture, the sonicate, and the filtered supernatant of the sonicate, and
the first
column fraction from the 9359-7 sample are highly active on P. xylostella. The
second
column fraction from 9359-7 is slightly active (some stunting only). No
activity is found in
the third fraction from 9359-7. The sample from DH5-pUCl9 does not have any
activity.
This indicates that the molecular weights of the toxins are above 6,000.
Example 10: Heat Inactivitation of pCIB9359-7 Activity
The heat stability of the toxins is determined. Overnight cultures of the E.
coli strain
pCIB9359-7 are grown in a 50:50 mixture of Luria broth and Terrific broth.
Cultures are
grown at 37°C in culture tubes on a tube roller. A one ml sample of the
culture is placed in
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a 1.5 ml eppendorf tube and placed in a boiling water bath. The sample is
removed after
five minutes and allowed to cool to room temperature. This sample along with
an untreated
portion of the culture is assayed on P. xylostella. 50N1 of sample of sample
is spread on
diet, allowed to dry and neonate larvae P. xylostella applied to the surface.
The assay is
incubated for 5 days at room temperature.
The untreated sample causes 100% mortality. The heat treated sample and a diet
alone control do not cause any observable mortality, showing the toxins are
heat sensitive.
Example 11: Leaf Dip Bioassay of pCIB9359-7
Insecticidal activity of the toxins is tested in a leaf dip bioassay. Six
leaves
approximately 2cm in diameter each are cut from seedlings of turnip and placed
in a 1 oz.
plastic cup (Jet Plastica) with 4m1-5ml of the resuspended toxin, covered
tightly, and shaken
until thoroughly wetted. The treated leaves are placed in 50mm petri dishes
(Gelman
Sciences) on absorbent pads moistened with 300w1 of water. The dish covers are
left open
until the leaf surface appears dry and then placed on tightly so that the
leaves do not dry
out.
Ten neonate P. xylostella larvae are placed in each petri dish arena. Also, a
treatment of 0.1 % Bond spreader/sticker with no toxin is set up as a control.
The arenas
are monitored daily for signs of drying leaves, and water is added or leaves
replaced if
necessary. After 3 days the leaves and arenas are examined under a dissecting
microscope, and the number of live larvae in each arena is recorded.
100% mortality is found for 9359-7 and none in the no-toxin control, showing
that the
toxins are also insecticidal in a leaf dip assay.
B. Isolation Of Nucleic Acld Seauences Whose Expression Results In Toxins
Active
Acrainst Lepldoateran and Coleopteran Insects
Example 12: Total DNA Isolation from Photorhabdus luminescens
Photorhabdus luminescens strain ATCC 29999 is grown 14-18 hours in L broth.
Total DNA is isolated from 1.5 mls of culture resuspended in 0.5% SDS,
100pg/ml
proteinase K, TE to a final volume of 600 ~I. After a 1 hour incubation at
37°C, 1001 5M
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NaCI and 80111 CTAB/NaCI are added and the culture is incubated at 65°C
for 10 minutes.
An equal volume of chloroform is added; the culture is mixed gently and spun.
The
aqueous phase is extracted once with phenol and once with chloroform. The
nucleic acids
are treated with 10 wg RNase A for 30 minutes at room temperature. The aqueous
phase is
mixed with 0.6 volumes isopropanoi and the sample is centrifuged. The pellet
is washed
once with 70% ethanol and the nucleic acids are gently resuspended in 100-
200u1 TE.
Example 13: PCR Amplification of Probes
Two probes are PCR amplified from Photorhabdus luminescens strain ATCC 29999
genomic DNA using oligos 5'-ACACAGCAGGTTCGTCAG-3' (SEQ ID N0:7) and 5'-
GGCAGAAGCACTCAACTC-3' (SEGO ID N0:8) to amplify probe #1 and oligos 5'-
ATTGATAGCACGCGGCGACC-3' (SEQ ID N0:9) and 5'-
TTGTAACGTGGAGCCGAACTGG-3' (SEQ ID N0:10) to amplify probe #2. The oligos are
ordered from Genosys Biotechnologies, Inc. (Texas). Approximately 10-50 ng of
genomic
DNA is used as the template. 0.8u.M of oligos, 200p.M of dNTPs, 1 X Taq DNA
Polymerase
buffer and 2.5 units of Taq DNA Polymerase are included in the reaction. The
reaction
conditions are as follows:
94°C - 1 minute
94°C - 30 seconds / 60°C - 30 seconds / 72°C - 30 seconds
(25 cycles)
72°C - 5 minutes
4°C - indefinite soak
The reactions are preferably carried out in a PCR System 9600 (Perkin Elmer)
thermocycler.
Exampie 14: Probing a Phoforhabdus luminescens Library
600 clones from the P. luminescens cosmid library described in Example f are
patched to L-amp plates in duplicate. The colonies are grown overnight then
moved to 4°C.
The colonies are lifted onto Colony/Plaque Screen Hybridization Transfer
Membranes
(Biotechnology Systems NEN Research Products). The membranes are incubated 2-3
minutes in 0.75m1 0.5N NaOH twice. The membranes are then incubated 2-3
minutes in
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0.75m1 1.OM Tris-HCI, pH 7.5 twice. The membranes are allowed to dry at room
temperature.
Probe #1 and probe #2 described in Example 13 are labeled using the DECAprime
II
Kit as described by the manufacturer (Ambion cat# 1455). Unincorporated
nucleotides are
removed from the labeled probes using Quick Spin Columns as described by the
manufacturer (Boehringer Mannheim cat #1273973). The labeled probes are
measured for
incorporated radioactivity and the specific activity is 10,000,000 cpm.
Membranes are
prewetted with 2X SSC and hybridized with the probes for 12-16 hours at
65°C. One set of
colony lifts is hybridized with probe #1 and the other set is hybridized with
probe #2. The
membranes are washed with wash CHURCH solutions 1 and 2 (Church and Gilbert,
Proc.
Natl. Acad. Sci. USA 81:1991-1995 (1984)) and exposed to Kodak film.
Twenty one clones are identified that hybridize to probe #1 and seven clones
are
identified that hybridize to probe #2. The gene in the clones isolated with
probe #1 is
named hph i and the gene in the clones isolated with probe #2 is named hph2.
Example 15: Insect Bioassays
The clones identified in Example 14 are tested for insecticidal activity
against the
following insects in insect bioassays: Diabrotica virgifera virgifera (Western
Com Rootworm
(WCR)), Diabrotica undecimpunctata howardi (Southern Com Rootworm (SCR)),
Ostrinia
nubilalis (European Corn Borer (ECB)), and Plutella xylostella (Diamondback
Moth (DBM)).
Diabrotica virgifera virgifera (Western Corn Rootworm) and Diabrofica
undecimpunctata howardi (Southern Com Rootworm) assays are performed using a
diet
incorporation method. 500p.1 of an overnight culture of the cosmid library in
XL-1 Blue MR
cells (Stratagene) is sonicated and then mixed with 500w1 of diet. Once the
diet solidifies, it
is dispensed in a petri dish and 20 larvae are introduced over the diet. Trays
of dishes are
placed in an incubator for 3-5 days, and percent mortality is recorded at the
end of the
assay period.
Ostrinia nubilalis (European Corn Borer) and Plufella xylostella (Diamondback
Moth)
assays are performed by a surface treatment method. The diet is poured in the
petri dish
and allowed it to solidify. The E. coli culture of 200 -300p1 volume is
dispensed over the diet
surface and entire diet surface is covered to spread the culture with the help
of bacterial
loop. Once the surface is dry, 10 larvae are introduced over the diet surface.
Trays of
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dishes are placed in an incubator for 3-5 days. The assay with European Com
Borer is
incubated at 30°C in complete darkness; the assay with Diamondback Moth
is incubated at
72°F with a 14:10 (hours} light:dark cycle. Percent mortality is
recorded at the end of the
assay period.
Cosmids containing hph2 are identified with a range of activities, including:
WCR
only; SCR only; WCR and SCR; SCR and ECB; WCR, SCR, and ECB; or WCR, SCR, ECB,
and DBM activity.
In addition to probing the P. luminescens cosmid library with DNA probes, 600
clones are screened by Western Com Rootworm bioassay. A clone is identified
with activity
against Western Com Rootworm. This clone hybridizes with probe #2.
From these bioassays, cosmid 514, having activity against WCR, SCR, ECB, and
DBM, is selected for sequencing.
Example 16: Sequencing of Cosmid 514
Cosmid 514 is sequenced using dye terminator chemistry on an ABI 377
instrument.
The nucleotide sequence of cosmid 514 is set forth as SEQ ID N0:11. Cosmid 514
is
designated pNOV2400 and deposited with the NRRL in E. coil DHSa and assigned
accession no. B-30077.
Example 17: Subcloning Insecticidal Regions of Cosmid 514
An 9011 base pair fragment within cosmid 514 (SEA ID N0:11 ) is removed by
digesting the cosmid with the restriction endonuclease Spel (New England
Biolabs
(Massachusetts), and ligating (T4 DNA Ligase, NEB) the remainder of 514.
Subclone 514a
consists of cosmid 514 DNA from base pairs 1-2157 ligated to base pairs 11,169-
37,948.
H202/pET34
hph2 and orf2 (SEQ ID N0:11, base pairs 23,768-35,838) are cloned into pET34b
(Novagen, Wisconsin). Restriction sites are engineered on both ends of each
gene to
facilitate cloning. PCR is used to add the restriction sites to the genes. A
BamHl site is on
the 5' end of hph2 immediately upstream of the ATG of hph2, and a Sad site is
added to
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the 3' end of hph2 immediately following the DNA triplet encoding the stop
codon. A
guanidine is added between the BamHl site and the start codon of hph2 to put
the hph2
gene in frame with the Cellulose Binding Domain tag in pET34b. Orf2 has a Sad
site
upstream of the 56 base pairs between the stop codon of hph2 and the start
codon of orf2.
The 56 base pairs are included in the hph2-orf2 construct to mimic their setup
in the 514
cosmid. Orf2 has an Xhol site on the 3' end immediately following the stop
codon. The
oligos used to add the restriction sites to hph2 and orf2 are as follows:
hph2-A 5'-CGGGATCCGATGATTTTAAAAGG-3' {SEQ ID N0:15)
hph2-B 5'-GCGCCATTGATTTGAG-3' (SEQ ID N0:16)
hph2-C 5'-CATTAGAGGTCGAACGTAC-3' (SEGO ID N0:17)
hph2-D 5'-GAGCGAGCTCTTACTTAATGGTGTAG-3' (SEGl ID N0:18)
orf2-A3 5'-CAGCGAGCTCCATGCAGAATTCACAGAC-3' (SEQ ID N0:19)
orf2-B 5'-GGCAATGGCAGCGATAAG-3' (SEQ ID N0:20)
orf2-C 5'-CATTAACGCAGGAAGAGC-3' (SEQ ID N0:21 )
orf2-D 5'-GACCTCGAGTTACACGAGCGCGTCAG-3' (SEA ID N0:22)
The BamHl-Sad 7583 base pair fragment, corresponding to the hph2 gene, and the
Sad-Xhol 4502 base pair orf2 (including the 56 base pairs between hph2 and
orf2 open
reading frames), corresponding to orf2, are ligated with BamHl-Xhol-digested
vector DNA
p ET34b.
Orf5/nBS (Nod-BamHl)
The 5325 base pair Nod-BamHl fragment of cosmid 514 is cloned into pBS-SK
using
AfAll-Notl (415 bp) and BamHl-Afllll {2530 bp) fragments of pBS-SK.
05-H2-02
The 12,031 base pair BamHl-Xhol fragment of H202/pET34 is cloned into the 8220
base pair Xhol-BamHl fragment of Orf5/pBS.
051011 H2O2
A 7298 base pair BamHl-Mlul fragment from subclone 514a is ligated {T4 DNA
Ligase, NEB} with 9588 by Mlul-Xhol and 8220 by Xhol-BamHl fragments of
subclone 05-
H2-02. The resulting ~ 22 kb subclone 051011 H2O2, which has activity against
WCR and
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ECB, is designated pNOV1001 and deposited with the NRRL in E, coli DHSa and
assigned
accession no. B-30078.
AKH2O2
A 12,074 base pair BamHl-Avrll fragment of H202/pET34 is ligated (T4 DNA
Ligase,
NEB) into pK184 Nhel-BamHl fragment (2228 bp), generating a clone containing
hph2 and
orf2 in a pl5a origin of replication, kanamycin-resistant vector.
Example 18: Insecticidal Activity of Subclones
Bioassays as described above are performed with E. coli cultures that express
the
above subclones, both singly and in combination. Coexpressing AKH2O2 and
OrfS/pBS in
E. coli, for example in DHSa or HB101, is found to give insecticidal activity
against the
Lepidopterans Plutella xylostella (Diamondback Moth), Ostrinia nubilalis
(European Corn
Borer), and Manduca sexta (Tobacco Homworm), as well as against the
Coleopterans
Diabroiica virgifera virgifera (Western Corn Rootworm), Diabrotica
undecimpunctata
howardi (Southern Com Rootworm), and Leptinotarsa decimlineata (Colorado
Potato
Beetle). Thus, coexpression of hph2 (SEQ ID N0:11, base pairs 23,768-31,336),
orf2
(SEQ ID N0:11, base pairs 31,393-35,838), and orf5 (SEQ ID N0:11, base pairs
15,171-
18,035) is sufficient to control these insects. In addition, expression of
each of these three
ORFs on separate plasmids gives insect control activity, demonstrating that
they do not
have to be genetically linked to be active, so long as all three gene products
are present.
C. Expression of the Nucleic Acid Seouences of the Invention in HeteroloQOUs
Mlcroblal Hosts
Microorganisms which are suitable for the heterologous expression of the
nucleotide
sequences of the invention are all microorganisms which are capable of
colonizing plants or
the rhizosphere. As such they will be brought into contact with insect pests.
These include
gram-negative microorganisms such as Pseudomonas, Enterobacter and Serratia,
the
gram-positive microorganism Bacillus and the fungi Trichoderma, Gliocladium,
and
Saccharomyces cerevisiae. Particularly preferred heterologous hosts are
Pseudomonas
fluorescens, Pseudomonas putida, Pseudomonas cepacia, Pseudomonas
aureofaciens,
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Pseudomonas aurantiaca, Enterobacter claacae, Serratia marscesens, Bacillus
subtilis,
Bacillus cereus, Trichoderma wide, Trichodenna harzianum, Gliocladium wens,
and
Saccharomyces cerevisiae.
Example 19: Expression of the Nucleotide Sequences in E. coli and Other Gram-
Negative Bacteria
Many genes have been expressed in gram-negative bacteria in a heterologous
manner. Expression vector pKK223-3 (Pharmacia catalogue # 27-4935-01 ) allows
expression in E. coli. This vector has a strong tac promoter (Brosius, J. et
al., Proc. Natl.
Acad. Sci. USA 81) regulated by the lac repressor and induced by IPTG. A
number of other
expression systems have been developed for use in E. coli. The thermoinducible
expression vector pP~ (Pharmacia #27-4946-01 ) uses a tightly regulated
bacteriophage ~,
promoter which allows for high level expression of proteins. The lac promoter
provides
another means of expression but the promoter is not expressed at such high
levels as the
tac promoter. With the addition of broad host range replicons to some of these
expression
system vectors, expression of the nucleotide sequence in closely related gram
negative-
bacteria such as Pseudomonas, Enterobacter, Serratia and Erwinia is possible.
For
example, pLRKD211 (Kaiser & Kroos, Proc. Natl. Acad. Sci. USA 81: 5816-5820
(1984))
contains the broad host range replicon on T which allows replication in many
gram-negative
bacteria.
In E. coli, induction by IPTG is required for expression of the tac (i.e. trp-
lac)
promoter. When this same promoter (e.g. on wide-host range plasmid pLRKD211)
is
introduced into Pseudomonas it is constitutively active without induction by
IPTG. This irp-
lac promoter can be placed in front of any gene or operon of interest for
expression in
Pseudomonas or any other closely related bacterium for the purposes of the
constitutive
expression of such a gene. Thus, a nucleotide sequence whose expression
results in an
insecticidal toxin can therefore be placed behind a strong constitutive
promoter, transferred
to a bacterium which has plant or rhizosphere colonizing properties turning
this organism to
an insecticidal agent. Other possible promoters can be used for the
constitutive expression
of the nucleotide sequence in gram-negative bacteria. These include, for
example, the
promoter.from the Pseudomonas regulatory genes gafA and IemA (WO 94/01561) and
the
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Pseudomonas savastanoi IAA operon promoter (Gaffney et al., J. Bacteriol. 172:
5593-5601
(1990).
Example 20: Expression of the Nucleotide Sequences in Gram-Positive Bacteria
Heterologous expression of the nucleotides sequence in gram-positive bacteria
is
another means of producing the insecticidal toxins. Expression systems for
Bacillus and
Streptomyces are the best characterized. The promoter for the erythromycin
resistance
gene {ermR) from Streptococcus pneumoniae has been shown to be active in gram-
positive
aerobes and anaerobes and also in E.coli (Trieu-Cuot et al., Nucl Acids Res
18: 3660
(1990)). A further antibiotic resistanc$ promoter from the thiostreptone gene
has been used
in Streptomyces cloning vectors (Bibb, Mol Gen Genet 199: 26-36 (1985)). The
shuttle
vector pHT3101 is also appropriate for expression in Bacillus {Lereclus, FEMS
Microbiol
Lett 60: 211-218 (1989)). A significant advantage of this approach is that
many gram-
positive bacteria produce spores which can be used in formulations that
produce
insecticidal agents with a longer shelf life. Bacillus and Streptomyces
species are
aggressive colonizers of soils
Example 21: Expression of the Nucleotide Sequences in Fungi
Trichoderma harzianum and Gliocladium wens have been shown to provide varying
levels of biocontrol in the field (US 5,165,928 and US 4,996,157, both to
Cornell Research
Foundation). A nucleotide sequence whose expression results in an insecticidal
toxin could
be expressed in such a fungus. This could be accomplished by a number of ways
which are
well known in the art. One is protoplast-mediated transformation of the fungus
by PEG or
electroporation-mediated techniques. Alternatively, particle bombardment can
be used to
transform protoplasts or other fungal cells with the ability to develop into
regenerated
mature structures. The vector pAN7-1, originally developed for Aspergillus
transformation
and now used widely for fungal transformation (Curragh et al., Mycol. Res.
97(3): 313-317
(1992); Tooley et al., Curr. Genet. 21: 55-60 (1992); Punt et al., Gene 56:
117-124 (1987))
is engineered to contain the nucleotide sequence. This plasmid contains the E.
coli the
hygromycin B resistance gene flanked by the Aspergillus nidulans gpd promoter
and the
trpC terminator (Punt et al., Gene 56: 117-124 (1987)).
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In a preferred embodiment, the nucleic acid sequences of the invention are
expressed in the yeast Saccharomyces cerevisiae. Each of the three ORF's of
SEQ ID
N0:11 (hph2, orf2 and orf5), which together confer insecticidal activity, are
cloned into
individual vectors with the GAL1 inducible promoter and the CYC1 terminator.
Each vector
has ampicillin resistance and the 2 micron replicon. The vectors differ in
their yeast growth
markers. hph2 is cloned into p424 (TRP1, ATCC 87329), orf2 into p423 (HIS3,
ATCC
87327), and orf5 into p425 (LEU2, ATCC 87331 ). The three constructs are
transformed
into S. cerevisiae independently and together. The three ORFs are expressed
together and
tested for protein expression and insecticidal activity.
D. Exnnession of the Nucleotide Seouences in Transaenic Plants
The nucleic acid sequences described in this application can be incorporated
into
plant cells using conventional recombinant DNA technology. Generally, this
involves
inserting a coding sequence of the invention into an expression system to
which the coding
sequence is heterologous (i.e., not normally present) using standard cloning
procedures
known in the art. The vector contains the necessary elements for the
transcription and
translation of the inserted protein-coding sequences. A large number of vector
systems
known in the art can be used, such as plasmids, bacteriophage viruses and
other modified
viruses. Suitable vectors include, but are not limited to, viral vectors such
as lambda vector
systems ~,gtll, 7~,gt10 and Charon 4; plasmid vectors such as pB1121, pBR322,
pACYC177,
pACYC184, pAR series, pKK223-3, pUCB, pUC9, pUCl8, pUCl9, pLG339, pRK290,
pKC37, pKC101, pCDNAII; and other similar systems. The components of the
expression
system may also be modified to increase expression. For example, truncated
sequences,
nucleotide substitutions or other modifications may be employed. The
expression systems
described herein can be used to transform virtually any crop plant cell under
suitable
conditions. Transformed cells can be regenerated into whole plants such that
the
nucleotide sequence of the invention confer insect resistance to the
transgenic plants.
Example 22: Modification of Coding Sequences and Adjacent Sequences
The nucleotide sequences described in this application can be modified for
expression in transgenic plant hosts. A host plant expressing the nucleotide
sequences and
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which produces the insecticidal toxins in its cells has enhanced resistance to
insect attack
and is thus better equipped to withstand crop losses associated with such
attack.
The transgenic expression in plants of genes derived from microbial sources
may
require the modification of those genes to achieve and optimize their
expression in plants.
In particular, bacterial ORFs which encode separate enzymes but which are
encoded by the
same transcript in the native microbe are best expressed in plants on separate
transcripts.
To achieve this, each microbial ORF is isolated individually and cloned within
a cassette
which provides a plant promoter sequence at the 5' end of the ORF and a plant
transcriptional terminator at the 3' end of the ORF. The isolated ORF sequence
preferably
includes the initiating ATG codon and the terminating STOP codon but may
include
additional sequence beyond the initiating ATG and the STOP codon. In addition,
the ORF
may be truncated, but still retain the required activity; for particularly
long ORFs, truncated
versions which retain activity may be preferable for expression in transgenic
organisms. By
"plant promoter" and "plant transcriptional terminator" it is intended to mean
promoters and
transcriptional terminators which operate within plant cells. This includes
promoters and
transcription terminators which may be derived from non-plant sources such as
viruses (an
example is the Cauliflower Mosaic Virus).
In some cases, modification to the ORF coding sequences and adjacent sequence
is not required. It is sufficient to isolate a fragment containing the ORF of
interest and to
insert it downstream of a plant promoter. For example, Gaffney et al. (Science
261: 754-
756 (1993)) have expressed the Pseudomonas nahG gene in transgenic plants
under the
control of the CaMV 35S promoter and the CaMV tml terminator successfully
without
modification of the coding sequence and with x by of the Pseudomonas gene
upstream of
the ATG still attached, and y by downstream of the STOP codon still attached
to the nahG
ORF. Preferably as little adjacent microbial sequence should be left attached
upstream of
the ATG and downstream of the STOP codon. In practice, such construction may
depend
on the availability of restriction sites.
In other cases, the expression of genes derived from microbial sources may
provide
problems in expression. These problems have been well characterized in the art
and are
particularly common with genes derived from certain sources such as Bacillus.
These
problems may apply to the nucleotide sequence of this invention and the
modification of
these genes can be undertaken using techniques now well known in the art. The
following
problems may be encountered:
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1. Codon Usage.
The preferred codon usage in plants differs from the preferred codon usage in
certain microorganisms. Comparison of the usage of codons within a cloned
microbial ORF
to usage in plant genes (and in particular genes from the target plant) will
enable an
identification of the codons within the ORF which should preferably be
changed. Typically
plant evolution has tended towards a strong preference of the nucleotides C
and G in the
third base position of monocotyledons, whereas dicotyledons often use the
nucleotides A or
T at this position. By modifying a gene to incorporate preferred codon usage
for a particular
target transgenic species, many of the problems described below for GC/AT
content and
illegitimate splicing will be overcome.
2. GC/AT Content.
Plant genes typically have a GC content of more than 35%. ORF sequences which
are rich in A and T nucleotides can cause several problems in plants. Firstly,
motifs of
ATTTA are believed to cause destabilization of messages and are found at the
3' end of
many short-lived mRNAs. Secondly, the occurrence of pofyadenylation signals
such as
AATAAA at inappropriate positions within the message is believed to cause
premature
truncation of transcription. In addition, monocotyledons may recognize AT-rich
sequences
as splice sites (see below).
3. Sequences Adjacent to the Initiating Methionine.
Plants differ from microorganisms in that their messages do not possess a
defined
ribosome binding site. Rather, it is believed that ribosomes attach to the 5'
end of the
message and scan for the first available ATG at which to start translation.
Nevertheless, it
is believed that there is a preference for certain nucleotides adjacent to the
ATG and that
expression of microbial genes can be enhanced by the inclusion of a eukaryotic
consensus
translation initiator at the ATG. Clontech (1993/1994 catalog, page 210,
incorporated
herein by reference) have suggested one sequence as a consensus translation
initiator for
the expression of the E. coil uidA gene in plants. Further, Joshi (NAR 15:
6643-6653
(1987), incorporated herein by reference) has compared many plant sequences
adjacent to
the ATG and suggests another consensus sequence. In situations where
difficulties are
encountered in the expression of microbial ORFs in plants, inclusion of one of
these
sequences at the initiating ATG may improve translation. In such cases the
last three
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nucleotides of the consensus may not be appropriate for inclusion in the
modified sequence
due to their modification of the second AA residue. Preferred sequences
adjacent to the
initiating methionine may differ between different plant species. A survey of
14 maize
genes Located in the GenBank database provided the following results:
Position Before the lnitiatin_c1 ATG in 14 Maize Genes:
--10 -9 -8 -7 -6 -5 -4 -3 -2
-1
C 3 8 4 6 2 5 6 0 10
7
T 3 0 3 4 3 2 1 1 1 0
A 2 3 1 4 3 2 3 7 2 3
G 6 3 6 0 6 5 4 6 1 5
This analysis can be done for the desired plant species into which the
nucleotide sequence
is being incorporated, and the sequence adjacent to the ATG modified to
incorporate the
preferred nucleotides.
4. Removal of Illegitimate Splice Sites.
Genes cloned from non-plant sources and not optimized for expression in plants
may also contain motifs which may be recognized in plants as 5' or 3' splice
sites, and be
cleaved, thus generating truncated or deleted messages. These sites can be
removed
using the techniques well known in the art.
Techniques for the modification of coding sequences and adjacent sequences are
well known in the art. In cases where the initial expression of a microbial
ORF is low and it
is deemed appropriate to make alterations to the sequence as described above,
then the
construction of synthetic genes can be accomplished according to methods well
known in
the art. These are, for example, described in the published patent disclosures
EP 0 385
962 (to Monsanto), EP 0 359 472 (to Lubrizol) and WO 93/07278 (to Ciba-Geigy),
all of
which are incorporated herein by reference. In most cases it is preferable to
assay the
expression of gene constructions using transient assay protocols (which are
well known in
the art) prior to their transfer to transgenic plants.
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Example 23: Construction of Plant Expression Cassettes
Coding sequences intended for expression in transgenic plants are first
assembled in
expression cassettes behind a suitable promoter expressible in plants. The
expression
cassettes may also comprise any further sequences required or selected for the
expression
of the transgene. Such sequences include, but are not restricted to,
transcription
terminators, extraneous sequences to enhance expression such as introns, vital
sequences,
and sequences intended for the targeting of the gene product to specific
organelles and cell
compartments. These expression cassettes can then be easily transferred to the
plant
transformation vectors described below. The following is a description of
various
components of typical expression cassettes.
1. Promoters
The selection of the promoter used in expression cassettes will determine the
spatial
and temporal expression pattern of the transgene in the transgenic plant.
Selected
promoters will express transgenes in specific cell types (such as leaf
epidermal cells,
mesophyll cells, root cortex cells) or in specific tissues or organs (roots,
leaves or flowers,
for example) and the selection will reflect the desired location of
accumulation of the gene
product. Alternatively, the selected promoter may drive expression of the gene
under
various inducing conditions. Promoters vary in their strength, i.e., ability
to promote
transcription. Depending upon the host cell system utilized, any one of a
number of suitable
promoters can be used, including the gene's native promoter. The following are
non-
limiting examples of promoters that may be used in expression cassettes.
a. Constitutive Expression, the Ubiquitin Promoter:
Ubiquitin is a gene product known to accumulate in many cell types and its
promoter
has been cloned from several species for use in transgenic plants (e.g.
sunflower - Binet et
aG Plant Science 79: 87-94 (1991 ); maize - Christensen ef al. Plant Molec.
Biol. 12: 619-
632 (1989); and Arabidopsis - Norris ef al., Plant Mol. Biol. 21:895-906
(1993)). The maize
ubiquitin promoter has been developed in transgenic monocot systems and its
sequence
and vectors constructed for monocot transformation are disclosed in the patent
publication
EP 0 342 926 (to Lubrizol) which is herein incorporated by reference. Taylor
et al. (Plant
Cell Rep. 12: 491-495 (1993)) describe a vector (pAHC25) that comprises the
maize
ubiquitin promoter and first intron and its high activity in cell suspensions
of numerous
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monocotyledons when introduced via microprojectile bombardment. The
Arabidopsis
ubiquitin promoter is ideal for use with the nucleotide sequences of the
present invention.
The ubiquitin promoter is suitable for gene expression in transgenic plants,
both
monocotyledons and dicotyledons. Suitable vectors are derivatives of pAHC25 or
any of
the transformation vectors described in this application, modified by the
introduction of the
appropriate ubiquitin promoter and/or intron sequences.
b. Constitutive Expression, the CaMV 35S Promoter:
Construction of the plasmid pCGN1761 is described in the published patent
application EP 0 392 225 (Example 23), which is hereby incorporated by
reference.
pCGN1761 contains the "double" CaMV 35S promoter and the tml transcriptional
terminator
with a unique EcoRl site between the promoter and the terminator and has a pUC-
type
backbone. A derivative of pCGN1761 is constructed which has a modified
polylinker which
includes Not! and Xhol sites in addition to the existing EcoRl site. This
derivative is
designated pCGN1761 ENX. pCGN1761 ENX is useful for the cloning of cDNA
sequences
or coding sequences (including microbial ORF sequences) within its polylinker
for the
purpose of their expression under the control of the 35S promoter in
transgenic plants. The
entire 35S promoter-coding sequence-tml terminator cassette of such a
construction can be
excised by Hindlll, Sphl, Sall, and Xbal sites 5' to the promoter and Xbal,
BamHl and Bgll
sites 3' to the terminator for transfer to transformation vectors such as
those described
below. Furthermore, the double 35S promoter fragment can be removed by 5'
excision with
Hindlll, Sphl, Sall, Xbal, or Pstl, and 3' excision with any of the polylinker
restriction sites
(EcoRl, Notl or Xhol) for replacement with another promoter. If desired,
modifications
around the cloning sites can be made by the introduction of sequences that may
enhance
translation. This is particularly useful when overexpression is desired. For
example,
pCGN1761 ENX may be modified by optimization of the translational initiation
site as
described in Example 37 of U.S. Patent No. 5,639,949, incorporated herein by
reference.
c. Constitutive Expression, the Actin Promoter:
Several isoforms of actin are known to be expressed in most cell types and
consequently the actin promoter is a good choice for a constitutive promoter.
In particular,
the promoter from the rice Actl gene has been cloned and characterized
(McElroy et al.
Plant Cell 2: 163-171 (1990)). A l.3kb fragment of the promoter was found to
contain all
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the regulatory elements required for expression in rice protoplasts.
Furthermore, numerous
expression vectors based on the Actl promoter have been constructed
specifically for use in
monocotyledons (McElroy et al. Mol. Gen. Genet. 231: 150-160 (1991 )). These
incorporate
the Actl-intron 1, Adhl 5' flanking sequence and Adhl intron 1 (from the maize
alcohol
dehydrogenase gene) and sequence from the CaMV 35S promoter. Vectors showing
highest expression were fusions of 35S and Actl intron or the Actl 5' flanking
sequence and
the Actl intron. Optimization of sequences around the initiating ATG (of the
GUS reporter
gene) also enhanced expression. The promoter expression cassettes described by
McElroy
et al. (Mol. Gen. Genet. 231: 150-160 (i 991 )) can be easily modified for
gene expression
and are particularly suitable for use in monocotyledonous hosts. For example,
promoter-
containing fragments is removed from the McElroy constructions and used to
replace the ,
double 35S promoter in pCGN1761 ENX, which is then available for the insertion
of specific
gene sequences. The fusion genes thus constructed can then be transferred to
appropriate
transformation vectors. In a separate report, the rice Actl promoter with its
first intron has
also been found to direct high expression in cultured barley cells (Chibbar et
al. Plant Cell
Rep. 12: 506-509 (i 993)).
d. Inducible Expression, the PR-1 Promoter:
The double 35S promoter in pCGN1761 ENX may be replaced with any other
promoter
of choice that will result in suitably high expression levels. By way of
example, one of the
chemically regulatable promoters described in U.S. Patent No. 5,614,395 may
replace the
double 35S promoter. The promoter of choice is preferably excised from its
source by
restriction enzymes, but can alternatively be PCR-amplified using primers that
carry
appropriate terminal restriction sites. Should PCR-amplification be
undertaken, then the
promoter should be re-sequenced to check for amplification errors after the
cloning of the
amplified promoter in the target vector. The chemically/pathogen regulatable
tobacco PR-
1 a promoter is cleaved from plasmid pCIB1004 (for construction, see example
21 of
EP 0 332 104, which is hereby incorporated by reference) and transferred to
piasmid
pCGN1761 ENX (Uknes et al., 1992). pCIB1004 is cleaved with Ncol and the
resultant 3'
overhang of the linearized fragment is rendered blunt by treatment with T4 DNA
polymerase. The fragment is then cleaved with Hindlll and the resultant PR-1 a
promoter-
containing fragment is gel purified and cloned into pCGN1761 ENX from which
the double
35S promoter has been removed. This is done by cleavage with Xhol and blunting
with T4
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polymerase, followed by cleavage with Hindlll and isolation of the larger
vector-terminator
containing fragment into which the pCIB1004 promoter fragment is cloned. This
generates
a pCGN1761 ENX derivative with the PR-1 a promoter and the tml terminator and
an
intervening polylinker with unique EcoRl and Notl sites. The selected coding
sequence can
be inserted into this vector, and the fusion products (i.e. promoter-gene-
terminator) can
subsequently be transferred to any selected transformation vector, including
those
described infra. Various chemical regulators may be employed to induce
expression of the
selected coding sequence in the plants transformed according to the present
invention,
including the benzothiadiazole, isonicotinic acid, and salicylic acid
compounds disclosed in
U.S. Patent Nos. 5,523,311 and 5,614,395.
e. lnducible Expression, an Ethanol-Inducible Promoter:
A promoter inducible by certain alcohols or ketones, such as ethanol, may also
be
used to confer inducible expression of a coding sequence of the present
invention. Such a
promoter is for example the alcA gene promoter from Aspergillus nidulans
(Caddick et al.
(1998) Nat. Biotechnol 16:177-i 80). In A. nidulans, the alcA gene encodes
alcohol
dehydrogenase I, the expression of which is regulated by the AIcR
transcription factors in
presence of the chemical inducer. For the purposes of the present invention,
the CAT
coding sequences in plasmid paIcA:CAT comprising a alcA gene promoter sequence
fused
to a minimal 35S promoter (Caddick et al. {1998) Nat. Biotechnol 16:177-180)
are replaced
by a coding sequence of the present invention to form an expression cassette
having the
coding sequence under the control of the alcA gene promoter. This is carried
out using
methods well known in the art.
f. Inducible Expression, a Glucocorticoid-Inducible Promoter:
Induction of expression of a nucleic acid sequence of the present invention
using
systems based on steroid hormones is also contemplated. For example, a
glucocorticoid-
mediated induction system is used (Aoyama and Chua (1997) The Planf Journal
11: 605-
612) and gene expression is induced by application of a glucocorticoid, for
example a
synthetic glucocorticoid, preferably dexamethasone, preferably at a
concentration ranging
from 0.1 mM to 1 mM, more preferably from lOmM to 1 OOmM. For the purposes of
the
present invention, the luciferase gene sequences are replaced by a nucleic
acid sequence
of the invention to form an expression cassette having a nucleic acid sequence
of the
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invention under the control of six copies of the GAL4 upstream activating
sequences fused
to the 35S minimal promoter. This is carried out using methods well known in
the art. The
trans-acting factor comprises the GAL4 DNA-binding domain (Keegan et al.
(1986) Science
231: 699-704) fused to the transactivating domain of the herpes viral protein
VP16
(Triezenberg et ai. (1988) Genes Devel. 2: 718-729) fused to the hormone-
binding domain
of the rat glucocorticoid receptor (Picard et al. (1988) Cell 54: 1073-1080).
The expression
of the fusion protein is controlled by any promoter suitable for expression in
plants known in
the art or described here. This expression cassette is also comprised in the
plant comprising
a nucleic acid sequence of the invention fused to the 6xGAL4/minimal promoter.
Thus,
tissue- or organ-specificity of the fusion protein is achieved leading to
inducible tissue- or
organ-specificity of the insecticidal toxin.
g. Root Specific Expression:
Another pattern of gene expression is root expression. A suitable root
promoter is
described by de Framond (FEBS 2~0: 103-106 (1991 )) and also in the published
patent
application EP 0 452 269, which is herein incorporated by reference. This
promoter is
transferred to a suitable vector such as pCGN1761 ENX for the insertion of a
selected gene
and subsequent transfer of the entire promoter-gene-terminator cassette to a
transformation
vector of interest.
h. Wound-Inducible Promoters:
Wound-inducible promoters may also be suitable for gene expression. Numerous
such promoters have been described (e.g. Xu et al. Plant Molec. Biol. 22: 573-
588 (1993),
Logemann ef al. Plant Cell 1: 151-158 (1989), Rohrmeier & Lehle, Plant Molec.
Biol. 22:
783-792 (1993), Firek et al. Plant Molec. Biol. 22: 129-i42 (1993), Warner et
al. Plant J. _3:
191-201 (1993)) and all are suitable for use with the instant invention.
Logemann et al.
describe the 5' upstream sequences of the dicotyledonous potato wunl gene. Xu
ef al.
show that a wound-inducible promoter from the dicotyledon potato (pink is
active in the
monocotyledon rice. Further, Rohrmeier & Lehle describe the cloning of the
maize Wipl
cDNA which is wound induced and which can be used to isolate the cognate
promoter using
standard techniques. Similar, Firek et al. and Warner et aJ. have described a
wound-
induced gene from the monocotyledon Asparagus officinalis, which is expressed
at local
wound and pathogen invasion sites. Using cloning techniques well known in the
art, these
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promoters can be transferred to suitable vectors, fused to the genes
pertaining to this
invention, and used to express these genes at the sites of plant wounding.
i. Pith-Preferred Expression:
Patent Application WO 93/07278, which is herein incorporated by reference,
describes the isolation of the maize trpA gene, which is preferentially
expressed in pith
cells. The gene sequence and promoter extending up to -1726 by from the start
of
transcription are presented. Using standard molecular biological techniques,
this promoter,
or parts thereof, can be transferred to a vector such as pCGN1761 where it can
replace the
35S promoter and be used to drive the expression of a foreign gene in a pith-
preferred
manner. In fact, fragments containing the pith-preferred promoter or parts
thereof can be
transferred to any vector and modified for utility in transgenic plants.
j. Leaf-Specific Expression:
A maize gene encoding phosphoenol carboxylase (PEPC) has been described by
Hudspeth & Grula (Plant Molec Biol 12: 579-589 (1989)). Using standard
molecular
biological techniques the promoter for this gene can be used to drive the
expression of any
gene in a leaf-specific manner in transgenic plants.
k. Pollen-Specific Expression:
WO 93/07278 describes the isolation of the maize calcium-dependent protein
kinase
(CDPK) gene which is expressed in pollen cells. The gene sequence and promoter
extend
up to 1400 by from the start of transcription. Using standard molecular
biological
techniques, this promoter or parts thereof, can be transferred to a vector
such as
pCGN1761 where it can replace the 35S promoter and be used to drive the
expression of a
nucleic acid sequence of the invention in a pollen-specific manner.
2. Transcriptional Terminators
A variety of transcriptional terminators are available for use in expression
cassettes.
These are responsible for the termination of transcription beyond the
transgene and its
correct polyadenylation. Appropriate transcriptional terminators are those
that are known to
function in plants and include the CaMV 35S terminator, the tml terminator,
the nopaline
synthase terminator and the pea rbcS E9 terminator. These can be used in both
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monocotyledons and dicotyledons. In addition, a gene's native transcription
terminator may
be used.
3. Sequences for the Enhancement or Regulation of Expression
Numerous sequences have been found to enhance gene expression from within the
transcriptional unit and these sequences can be used in conjunction with the
genes of this
invention to increase their expression in transgenic plants.
Various intros sequences have been shown to enhance expression; particularly
in
monocotyledonous cells. For example, the introns of the maize Adhl gene have
been found
to significantly enhance the expression of the wild-type gene under its
cognate promoter
when introduced into maize cells. Intros 1 was found to be particularly
effective and
enhanced expression in fusion constructs with the chloramphenicol
acetyltransferase gene
(Callis et al., Genes Develop. 1: 1183-1200 (1987)). In the same experimental
system, the
intros from the maize bronze! gene had a similar effect in enhancing
expression. Intros
sequences have been routinely incorporated into plant transformation vectors,
typically
within the non-translated leader.
A number of non-translated leader sequences derived from viruses are also
known to
enhance expression, and these are particularly effective in dicotyledonous
cells.
Specifically, leader sequences from Tobacco Mosaic Virus (TMV, the "W-
sequence"), Maize
Chlorotic Mottle Virus (MCMV), and Alfalfa Mosaic Virus (AMV) have been shown
to be
effective in enhancing expression (e.g. Gallie et al. Nucl. Acids Res. 15:
8693-8711 (1987);
Skuzeski et al. Plant Molec. Biol. 15: 65-79 (1990)).
4. Targeting of the Gene Product Within the Cell
Various mechanisms for targeting gene products are known to exist in plants
and the
sequences controlling the functioning of these mechanisms have been
characterized in
some detail. For example, the targeting of gene products to the chloroplast is
controlled by
a signal sequence found at the amino terminal end of various proteins which is
cleaved
during chloroplast import to yield the mature protein (e.g. Comai et al. J.
Biol. Chem. 263:
15104-15109 (1988)). These signal sequences can be fused to heterologous gene
products to effect the import of heterologous products into the chloropiast
(van den Broeck,
et al. Nature 313: 358-363 (1985)). DNA encoding for appropriate signal
sequences can be
isolated from the 5' end of the cDNAs encoding the RUBISCO protein, the CAB
protein, the
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EPSP synthase enzyme, the GS2 protein and many other proteins which are known
to be
chloroplast localized. See also, the section entitled "Expression With
Chloroplast Targeting"
in Example 37 of U.S. Patent No. 5,639,949.
Other gene products are localized to other organelles such as the
mitochondrion and
the peroxisome (e.g. Unger et al. Plant Molec. Biol. 13: 411-418 (1989)). The
cDNAs
encoding these products can also be manipulated to effect the targeting of
heterologous
gene products to these organelles. Examples of such sequences are the nuclear-
encoded
ATPases and specific aspartate amino transferase isoforms for mitochondria.
Targeting
cellular protein bodies has been described by Rogers et al. (Proc. Natl. Acad.
Sci. USA 82:
6512-6516 (1985)).
In addition, sequences have been characterized which cause the targeting of
gene
products to other cell compartments. Amino terminal sequences are responsible
for
targeting to the ER, the apoplast, and extracellular secretion from aleurone
cells (Koehler &
Ho, Plant Cell 2: 769-783 (1990)). Additionally, amino terminal sequences in
conjunction
with carboxy terminal sequences are responsible for vacuolar targeting of gene
products
(Shinshi et al. Plant Molec. Biol. 14: 357-368 (1990)).
By the fusion of the appropriate targeting sequences described above to
transgene
sequences of interest it is possible to direct the transgene product to any
organelle or cell
compartment. For chloroplast targeting, for example, the chloroplast signal
sequence from
the RUBISCO gene, the CAB gene, the EPSP synthase gene, or the GS2 gene is
fused in
frame to the amino terminal ATG of the transgene. The signal sequence selected
should
include the known cleavage site, and the fusion constructed should take into
account any
amino acids after the cleavage site which are required for cleavage. In some
cases this
requirement may be fulfilled by the addition of a small number of amino acids
between the
cleavage site and the transgene ATG or, alternatively, replacement of some
amino acids
within the transgene sequence. Fusions constructed for chloroplast import can
be tested
for efficacy of chloroplast uptake by in vitro translation of in vitro
transcribed constructions
followed by in vifro chloroplast uptake using techniques described by Bartlett
et al. In:
Edelmann et al. (Eds.) Methods in Chloropiast Molecular Biology, Elsevier pp
1081-1091
(1982) and Wasmann et al. Mol. Gen. Genet. 205: 446-453 (1986). These
construction
techniques are well known in the art and are equally applicable to
mitochondria and
peroxisomes.
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The above-described mechanisms for cellular targeting can be utilized not only
in
conjunction with their cognate promoters, but also in conjunction with
heterologous
promoters so as to effect a specific cell-targeting goal under the
transcriptional regulation of
a promoter that has an expression pattern different to that of the promoter
from which the
targeting signal derives.
Example 24: Construction of Plant Transformation Vectors
Numerous transformation vectors available for plant transformation are known
to
those of ordinary skill in the plant transformation arts, and the genes
pertinent to this
invention can be used in conjunction with any such vectors. The selection of
vector will
depend upon the preferred transformation technique and the target species for
transformation. For certain target species, different antibiotic or herbicide
selection markers
may be preferred. Selection markers used routinely in transformation include
the nptll
gene, which confers resistance to kanamycin and related antibiotics (Messing &
Vierra.
Gene 19: 259-268 (1982); Bevan et al., Nature 304:184-187 (1983)), the bar
gene, which
confers resistance to the herbicide phosphinothricin (White et al., Nucl.
Acids Res 18: 1062
(1990), Spencer et al. Theor. Appl. Genet 79: 625-631 (1990)), the hph gene,
which confers
resistance to the antibiotic hygromycin (Blochinger & Diggelmann, Mol Cell
Biol 4: 2929-
2931 ), and the dhfr gene, which confers resistance to methatrexate (Bourouis
et al., EMBO
J. 2 7 : 1099-1104 (1983)), and the EPSPS gene, which confers resistance to
glyphosate
(U.S. Patent Nos. 4,940,935 and 5,188,642).
1. Vectors Suitable for Agrobacterium Transformation
Many vectors are available for transformation using Agrobacterium tumefaciens.
These typically carry at least one T-DNA border sequence and include vectors
such as
pBINl9 (Bevan, Nucl. Acids Res. (1984)) and pXYZ. Below, the construction of
two typical
vectors suitable for Agrobacterium transformation is described.
a. pCIB200 and pCIB2001:
The binary vectors pc18200 and pCIB2001 are used for the construction of
recombinant vectors for use with Agrobacterium and are constructed in the
following
manner. pTJS75kan is created by Narl digestion of pTJS75 (Schmidhauser &
Helinski, J.
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Bacteriol. 164: 446-455 (1985)) allowing excision of the tetracycline-
resistance gene,
followed by insertion of an Accl fragment from pUC4K carrying an NPTII
(Messing & Vierra,
Gene 19: 259-268 (1982): Bevan et al., Nature 304: 184-187 (1983): McBride et
al., Plant
Molecular Biology 14: 266-276 (1990)). Xhol linkers are ligated to the EcoRV
fragment of
PCIB7 which contains the left and right T-DNA borders, a plant selectable
noslnptll chimeric
gene and the pUC polylinker (Rothstein et al., Gene 53: 153-161 (1987)), and
the XhoH
digested fragment are cloned into Sall-digested pTJS75kan to create pCIB200
(see also EP
0 332 104, example 19). pCIB200 contains the following unique polylinker
restriction sites:
EcoRl, Sstl, Kpnl, Bglll, Xbal, and Sall. pCIB2001 is a derivative of pCIB200
created by the
insertion into the polylinker of additional restriction sites. Unique
restriction sites in the
polylinker of pCIB2001 are EcoRl, Ssil, Kpnl, Bglll, Xbal, Sall, Mlul, Bcll,
Avrll, Apal, Hpal,
and Sful. pCIB2001, in addition to containing these unique restriction sites
also has plant
and bacterial kanamycin selection, left and right T-DNA borders for
Agrobacterium-mediated
transformation, the RK2-derived trfA function for mobilization between E. coli
and other
hosts, and the OriT and OriV functions also from RK2. The pCIB2001 polylinker
is suitable
for the cloning of plant expression cassettes containing their own regulatory
signals.
b. pCIBlO and Hygromycin Selection Derivatives thereof:
The binary vector pCIBlO contains a gene encoding kanamycin resistance for
selection in plants and T-DNA right and left border sequences and incorporates
sequences
from the wide host-range plasmid pRK252 allowing it to replicate in both E.
coli and
Agrobacterium. Its construction is described by Rothstein et al. (Gene 53: 153-
161 (1987)).
Various derivatives of pCIBlO are constructed which incorporate the gene for
hygromycin B
phosphotransferase described by Gritz et al. (Gene 25: 179-188 (1983)). These
derivatives
enable selection of transgenic plant cells on hygromycin only (pCIB743), or
hygromycin and
kanamycin (pCIB715, pCIB717).
2. Vectors Suitable for non-Agrobacterium Transformation
Transformation without the use of Agrobacterium fumefaciens circumvents the
requirement for T-DNA sequences in the chosen transformation vector and
consequently
vectors lacking these sequences can be utilized in addition to vectors such as
the ones
described above which contain T-DNA sequences. Transformation techniques that
do not
rely on Agrobacterium include transformation via particle bombardment,
protoplast uptake
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(e.g. PEG and electroporation) and microinjection. The choice of vector
depends largely on
the preferred selection for the species being transformed. Below, the
construction of typical
vectors suitable for non-Agrobacterium transformation is described.
a. pCIB3064:
pCIB3064 is a pUC-derived vector suitable for direct gene transfer techniques
in
combination with selection by the herbicide basta (or phosphinothricin). The
plasmid
pCIB246 comprises the CaMV 35S promoter in operational fusion to the E. coli
GUS gene
and the CaMV 35S transcriptional terminator and is described in the PCT
published
application WO 93/07278. The 35S promoter of this vector contains two ATG
sequences 5'
of the start site. These sites are mutated using standard PCR techniques in
such a way as
to remove the ATGs and generate the restriction sites Sspl and Pvull. The new
restriction
sites are 96 and 37 by away from the unique Sall site and 101 and 42 by away
from the
actual start site. The resultant derivative of pCIB246 is designated pCIB3025.
The GUS
gene is then excised from pCIB3025 by digestion with Sall and Sacl, the
termini rendered
blunt and relegated to generate plasmid pCIB3060. The plasmid pJIT82 is
obtained from the
John Innes Centre, Norwich and the a 400 by Smal fragment containing the bar
gene from
Streptomyces viridochromogenes is excised and inserted into the Hpal site of
pCIB3060
(Thompson et al. EMBO J 6_: 2519-2523 (1987)). This generated pCIB3064, which
comprises the bar gene under the control of the CaMV 35S promoter and
terminator for
herbicide selection, a gene for ampicillin resistance (for selection in E.
coh) and a poiylinker
with the unique sites Sphl, Pstl, Hindlll, and BamHl. This vector is suitable
for the cloning
of plant expression cassettes containing their own regulatory signals.
b. pSOGl9 and pSOG35:
pSOG35 is a transformation vector that utilizes the E. coli gene dihydrofolate
reductase (DFR) as a selectable marker conferring resistance to methotrexate.
PCR is
used to amplify the 35S promoter (-800 bp), intron 6 from the maize Adh1 gene
(-550 bp)
and 18 by of the GUS untranslated leader sequence from pSOGlO. A 250-by
fragment
encoding the E. coli dihydrofolate reductase type II gene is also amplified by
PCR and
these two PCR fragments are assembled with a Sacl-Psfl fragment from pB1221
(Clontech)
which comprises the pUCl9 vector backbone and the nopaline synthase
terminator.
Assembly of these fragments generates pSOGl9 which contains the 35S promoter
in fusion
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with the intron 6 sequence, the GUS leader, the DHFR gene and the nopaline
synthase
terminator. Replacement of the GUS leader in pSOGl9 with the leader sequence
from
Maize Chlorotic Mottle Virus (MCMV) generates the vector pSOG35. pSOGl9 and
pSOG35
carry the pUC gene for ampicillin resistance and have Hindlll, Sphl, Pstl and
EcoRl sites
available for the cloning of foreign substances.
Example 25: Transformation
Once a nucleic acid sequence of the invention has been cloned into an
expression
system, it is transformed into a plant cell. Methods for transformation and
regeneration of
plants are well known in the art. For example, Ti plasmid vectors have been
utilized for the
delivery of foreign DNA, as well as direct DNA uptake, liposomes,
electroporation, micro-
injection, and microprojectiles. In addition, bacteria from the genus
Agrobacierlum can be
utilized to transform plant cells. Below are descriptions of representative
techniques for
transforming both dicotyledonous and monocotyledonous plants.
1. Transformation of Dicotyledons
Transformation techniques for dicotyledons are well known in the art and
include
Agrobacterium-based techniques and techniques that do not require
Agrobacterium. Non-
Agrobacierium techniques involve the uptake of exogenous genetic material
directly by
protoplasts or cells. This can be accomplished by PEG or electroporation
mediated uptake,
particle bombardment-mediated delivery, or microinjection. Examples of these
techniques
are described by Paszkowski et al., EMBO J _3: 2717-2722 (1984), Potrykus et
aL, Mol. Gen.
Genet. 199: 169-177 (1985), Reich et al., Biotechnology 4: 1001-1004 (1986),
and Klein et
aL, Nature ~: 70-73 (1987). In each case the transformed cells are regenerated
to whole
plants using standard techniques known in the art.
Agrobacterlum-mediated transformation is a preferred technique for
transformation of
dicotyledons because of its high efficiency of transformation and its broad
utility with many
different species. Agrobacrerium transformation typically involves the
transfer of the binary
vector carrying the foreign DNA of interest (e.g. pCIB200 or pCIB2001 ) to an
appropriate
Agrobacferium strain which may depend of the complement of vir genes carried
by the host
Agrobacferium strain either on a co-resident Ti plasmid or chromosomally (e.g.
strain
CIB542 for pCIB200 and pCIB2001 (Uknes et al. Plant Cell _5: 159-169 (1993)).
The
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transfer of the recombinant binary vector to Agrobacterium is accomplished by
a triparental
mating procedure using E. coli carrying the recombinant binary vector, a
helper E. coli strain
which carries a plasmid such as pRK2013 and which is able to mobilize the
recombinant
binary vector to the target Agrobacferium strain. Alternatively, the
recombinant binary
vector can be transferred to Agrobacterium by DNA transformation (Hofgen &
Willmitzer,
Nucl. Acids Res. 16: 9877 (1988)).
Transformation of the target plant species by recombinant Agrobacterium
usually
involves co-cultivation of the Agrobacterium with explants from the plant and
follows
protocols well known in the art. Transformed tissue is regenerated on
selectable medium
carrying the antibiotic or herbicide resistance marker present between the
binary plasmid T-
DNA borders.
Another approach to transforming plant cells with a gene involves propelling
inert or
biologically active particles at plant tissues and cells. This technique is
disclosed in U.S.
Patent Nos. 4,945,050, 5,036,006, and 5,100,792 all to Sanford et al.
Generally, this
procedure involves propelling inert or biologically active particles at the
cells under
conditions effective to penetrate the outer surface of the cell and afford
incorporation within
the interior thereof. When inert particles are utilized, the vector can be
introduced into the
cell by coating the particles with the vector containing the desired gene.
Alternatively, the
target cell can be surrounded by the vector so that the vector is carried into
the cell by the
wake of the particle. Biologically active particles (e.g., dried yeast cells,
dried bacterium or a
bacteriophage, each containing DNA sought to be introduced) can also be
propelled into
plant cell tissue.
2. Transformation of Monocotyledons
Transformation of most monocotyledon species has now also become routine.
Preferred techniques include direct gene transfer into protoplasts using PEG
or
electroporation techniques, and particle bombardment into callus tissue.
Transformations
can be undertaken with a single DNA species or multiple DNA species (i.e. co-
transformation) and both these techniques are suitable for use with this
invention. Co-
transformation may have the advantage of avoiding complete vector construction
and of
generating transgenic plants with unlinked loci for the gene of interest and
the selectable
marker, enabling the removal of the selectable marker in subsequent
generations, should
this be regarded desirable. However, a disadvantage of the use of co-
transformation is the
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less than 100% frequency with which separate DNA species are integrated into
the genome
(Schocher et al. Biotechnology 4: 1093-1096 (1986)).
Patent Applications EP 0 292 435, EP 0 392 225, and WO 93/07278 describe
techniques for the preparation of callus and protoplasts from an elite inbred
line of maize,
transformation of protoplasts using PEG or electroporation, and the
regeneration of maize
plants from transformed protoplasts. Gordon-Kamm et aL (Plant Cell _2: 603-618
(1990))
and Fromm et al. (Biotechnology 8_: 833-839 (1990)) have published techniques
for
transformation of A188-derived maize line using particle bombardment.
Furthermore,
WO 93/07278 and Koziel et al. (Biotechnology 11: 194-200 (1993)) describe
techniques for
the transformation of elite inbred lines of maize by particle bombardment.
This technique
utilizes immature maize embryos of 1.5-2.5 mm length excised from a maize ear
14-15 days
after pollination and a PDS-1000He Biolistics device for bombardment.
Transformation of rice can also be undertaken by direct gene transfer
techniques
utilizing protoplasts or particle bombardment. Protoplast-mediated
transformation has been
described for Japonica-types and Indica-types (Zhang et aL Plant Cell Rep 7:
379-384
(1988}; Shimamoto et al. Nature 338: 274-277 (1989); Datta et al.
Biotechnology 8: 736-740
(1990)). Both types are also routinely transformable using particle
bombardment (Christou
et aG Biotechnology 9: 957-962 (1991 )). Furthermore, WO 93/21335 describes
techniques
for the transformation of rice via electroporation.
Patent Application EP 0 332 581 describes techniques for the generation,
transformation and regeneration of Pooideae protoplasts. These techniques
allow the
transformation of Dacfylis and wheat. Furthermore, wheat transformation has
been
described by Vasii et al. (Biotechnology 10: 667-674 (1992)) using particle
bombardment
into cells of type C long-term regenerable callus, and also by Vasil et al.
(Biotechnology 11:
1553-1558 (1993)) and Weeks et al. (Plant Physiol. 102: 1077-1084 (1993))
using particle
bombardment of immature embryos and immature embryo-derived callus. A
preferred
technique for wheat transformation, however, involves the transformation of
wheat by
particle bombardment of immature embryos and includes either a high sucrose or
a high
maltose step prior to gene delivery. Prior to bombardment, any number of
embryos (0.75-1
mm in length) are plated onto MS medium with 3% sucrose (Murashiga & Skoog,
Physiologia Plantarum 15: 473-497 (1962)) and 3 mg/I 2,4-D for induction of
somatic
embryos, which is allowed to proceed in the dark. On the chosen day of
bombardment,
embryos are removed from the induction medium and placed onto the osmoticum
(i.e.
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induction medium with sucrose or maltose added at the desired concentration,
typically
15%). The embryos are allowed to plasmolyze for 2-3 h and are then bombarded.
Twenty
embryos per target plate is typical, although not critical. An appropriate
gene-carrying
plasmid (such as pCIB3064 or pSG35) is precipitated onto micrometer size gold
particles
using standard procedures. Each plate of embryos is shot with the DuPont
Biolistics~
helium device using a burst pressure of -1000 psi using a standard 80 mesh
screen. After
bombardment, the embryos are placed back into the dark to recover for about 24
h (still on
osmoticum). After 24 hrs, the embryos are removed from the osmoticum and
placed back
onto induction medium where they stay for about a month before regeneration.
Approximately one month later the embryo explants with developing embryogenic
callus are
transferred to regeneration medium (MS + 1 mg/liter NAA, 5 mg/liter GA),
further containing
the appropriate selection agent (10 mgll basta in the case of pCIB3064 and 2
mg/I
methotrexate in the case of pSOG35). After approximately one month, developed
shoots
are transferred to larger sterile containers known as °GA7s' which
contain half-strength MS,
2% sucrose, and the same concentration of selection agent.
Tranformation of monocotyledons using Agrobacterium has also been described.
See, WO 94/00977 and U.S. Patent No. 5,591,616, both of which are incorporated
herein
by reference.
E. Hreedin4 and Seed Production
Example 26: Breeding
The plants obtained via tranformation with a nucleic acid sequence of the
present
invention can be any of a wide variety of plant species, including those of
monocots and
divots; however, the plants used in the method of the invention are preferably
selected from
the list of agronomically important target crops set forth supra. The
expression of a gene of
the present invention in combination with other characteristics important for
production and
quality can be incorporated into plant lines through breeding. Breeding
approaches and
techniques are known in the art. See, for example, Welsh J. R., Fundamentals
of Plant
Genetics and Breeding, John Wiley 8~ Sons, NY (1981 ); Crop Breeding, Wood D.
R. (Ed.)
American Society of Agronomy Madison, Wisconsin (1983); Mayo O., The Theory of
Plant
Breeding, Second Edition, Clarendon Press, Oxford (1987); Singh, D.P.,
Breeding for
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Resistance to Diseases and Insect Pests, Springer-Verlag, NY (1986); and
Wricke and
Weber, Quantitative Genetics and Selection Plant Breeding, Walter de Gruyter
and Co.,
Berlin (1986).
The genetic properties engineered into the transgenic seeds and plants
described
above are passed on by sexual reproduction or vegetative growth and can thus
be
maintained and propagated in progeny plants. Generally said maintenance and
propagation
make use of known agricultural methods developed to fit specific purposes such
as tilling,
sowing or harvesting. Specialized processes such as hydroponics or greenhouse
technologies can also be applied. As the growing crop is vulnerable to attack
and damages
caused by insects or infections as well as to competition by weed plants,
measures are
undertaken to control weeds, plant diseases, insects, nematodes, and other
adverse
conditions to improve yield. These include mechanical measures such a tillage
of the soil or
removal of weeds and infected plants, as well as the application of
agrochemicals such as
herbicides, fungicides, gametocides, nematicides, growth regulants, ripening
agents and
insecticides.
Use of the advantageous genetic properties of the transgenic plants and seeds
according to the invention can further be made in plant breeding, which aims
at the
development of plants with improved properties such as tolerance of pests,
herbicides, or
stress, improved nutritional value, increased yield, or improved structure
causing less loss
from lodging or shattering. The various breeding steps are characterized by
well-defined
human intervention such as selecting the lines to be crossed, directing
pollination of the
parental lines, or selecting appropriate progeny plants. Depending on the
desired
properties, different breeding measures are taken. The relevant techniques are
well known
in the art and include but are not limited to hybridization, inbreeding,
backcross breeding,
multiline breeding, variety blend, interspecific hybridization, aneuploid
techniques, etc.
Hybridization techniques also include the sterilization of plants to yield
male or female
sterile plants by mechanical, chemical, or biochemical means. Cross
pollination of a male
sterile plant with pollen of a different line assures that the genome of the
male sterile but
female fertile plant will uniformly obtain properties of both parental lines.
Thus, the
transgenic seeds and plants according to the invention can be used for the
breeding of
improved plant lines, that for example, increase the effectiveness of
conventional methods
such as herbicide or pestidice treatment or allow one to dispense with said
methods due to
their modified genetic properties. Alternatively new crops with improved
stress tolerance can
be obtained, which, due to their optimized genetic "equipment", yield
harvested product of
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better quality than products that were not able to tolerate comparable adverse
developmental conditions.
Example 27: Seed Production
In seed production, germination quality and uniformity of seeds are essential
product
characteristics, whereas germination quality and uniformity of seeds harvested
and sold by
the farmer is not important. As it is difficult to keep a crop free from other
crop and weed
seeds, to control seedbome diseases, and to produce seed with good
germination, fairly
extensive and wail-defined seed production practices have been developed by
seed
producers, who are experienced in the art of growing, conditioning and
marketing of pure
seed. Thus, it is common practice for the farmer to buy certified seed meeting
specific
quality standards instead of using seed harvested from his own crop.
Propagation material
to be used as seeds is customarily treated with a protectant coating
comprising herbicides,
insecticides, fungicides, bactericides, nematicides, molluscicides, or
mixtures thereof.
Customarily used protectant coatings comprise compounds such as captan,
carboxin,
thiram (TMTD'), methalaxyl (Apron~), and pirimiphos-methyl (Actellic~. If
desired, these
compounds are formulated together with further carriers, surfactants or
application-
promoting adjuvants customarily employed in the art of formulation to provide
protection
against damage caused by bacterial, fungal or animal pests. The protectant
coatings may
be applied by impregnating propagation material with a liquid formulation or
by coating with
a combined wet or dry formulation. Other methods of application are also
possible such as
treatment directed at the buds or the fruit.
It is a further aspect of the present invention to provide new agricultural
methods,
such as the methods examplified above, which are characterized by the use of
transgenic
plants, transgenic plant material, or transgenic seed according to the present
invention.
The seeds may be provided in a bag, container or vessel comprised of a
suitable
packaging material, the bag or container capable of being closed to contain
seeds. The
bag, container or vessel may be designed for either short term or long term
storage, or both,
of the seed. Examples of a suitable packaging material include paper, such as
kraft paper,
rigid or pliable plastic or other polymeric material, glass or metal.
Desirably the bag,
container, or vessel is comprised of a plurality of layers of packaging
materials, of the same
or differing type. In one embodiment the bag, container or vessel is provided
so as to
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-65-
exclude or limit water and moisture from contacting the seed. In one example,
the bag,
container or vessel is sealed, for example heat sealed, to prevent water or
moisture from
entering. In another embodiment water absorbent materials are placed between
or
adjacent to packaging material layers. In yet another embodiment the bag,
container or
vessel, or packaging material of which it is comprised is treated to limit,
suppress or
prevent disease, contamination or other adverse affects of storage or
transport of the seed.
An example of such treatment is sterilization, for example by chemical means
or by
exposure to radiation. Comprised by the present invention is a commercial bag
comprising
seed of a transgenic plant comprising a gene of the present invention that is
expressed in
said transformed plant at higher levels than in a wild type plant, together
with a suitable
carrier, together with label instructions for the use thereof for conferring
broad spectrum
disease resistance to plants.
CA 02320801 2000-08-14
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- 66 -
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N~ AND 7lDDRSae OY T>iE plIRTY TO fi~011
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CA 02320801 2000-08-14
WO 99/42589 PCT/EP99/01015
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7~ddress~ 1815 N. 9niverrity
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CA 02320801 2000-08-14
WO 99/42589 PCT/EP99/01015
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CA 02320801 2000-08-14
WO 99/42589 PCT/EP99/01015
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4.'1 ~
Addrsee~ 1815 N. university street~ ~-3-~d
Indiaato the deco n! tM ert'inrl deposit os vn~n a nev deporit hh~ 1wn wow.
~ Hurt vlch ~ ereNr tAa ~0~1111Y1e has.
Ih the eseee retaend to In ftNl~ l0.ifslriil end 11111, refer co the aoee
Meant viability ease.
~ riis sn rt tM tntoa~clen nee been soqw~cad.
CA 02320801 2000-08-14
WO 99/42589 PC'T/EP99/01015
- 70 -
bDpAPRBT TA~ITx ON TIDE =TID~I.
~ooc~rrloar o8 Txs Darosrr os azcaoo~a~I~
t~oa Tine poRpoaa oa aATaaT pROCaoaase
z~rsRanTIO~ ~o~
To Rscai~r irr THS rass os ~ oRial~ Daposrr
povaztla Cosp. istwd pursuant to Rule 7.1 by the
c/o Novartis JIG Z~~tATiObZIL DShOSZTAR7f AnTHORITY
P. O. Dox 13Ib7 ideatitied at the bottom of thin page
Reuarch Triangle Bark, NC 27709
mlla J1ND J1DDRBSB
OF DB?OSITOR
I. ID~PICIITiON OF TFIa ~dICRO0R0~71NISM
Identification referaaae given Jloaession number given by the
by the
DRP09ZTDR : Ilfl'IR~DITIOR71L Du?OSiTARY A~tORITx
8aetsria sp. pCIB 9359-7 NRRL 8-11135
II. SCIRliTIPIC DRBCRIPTION AND/OR
PROP08aD TAXON01~C DS8I01NATi0IQ
The microosgaaism idsatilied
under I, above ran soaompaaied
by:
~ a scientific description
a proposed taxonomic designation
lllarJc with a cross where licablel
III. RaCaIPT 71N0 ACCBI~'
This International Dopositary
Authority acoepts the microorgassiam
identified under I.
above, which wan received by
it on Septemoer 17, 1999 (date
of the original deposit)'
IV. RBCaIPT OF RS 9T FOR CONV&ABION
Ths microosganism identified
under I. nbovs ras received
by thin Interaatiaaal
Depoaitary Authority on tdsts
of the original depositf and
a reqwst
to convert the original deposit
to a deposit under the Budapest
Treaty was reaeivsd by
it on Idste of receipt of request
for conversion).
V . IIiTSR~ITZOtdTAL DRP09ITJt,RY
ADTHORIT7C
Name: Agricultural Research Culture8ignaturela) o! personls) having
the porsr
Collection INRRL) to represent she International
Depoeitary
Intesssntional Dspoaitasy AuthorityAuthority or of au~orized officialls):
llddrsas: isl5 N. Oniveraity
Street
Peoria. Illinois 6160a n.S.A. Date: ~t~13 "~/~
' lihere Rule s.ald) appliaa, such data is the date on whidi the status of
international
depodtary authority was acquired.
CA 02320801 2000-08-14
WO 99/42589 PC'T/EP99/01015
- 71 -
aao~sls~r TR~T~r oN TH$ =err=~u. x>rcoo~cT:oN os ~ as~srr
os »cpooat3«Ier~ voR ~ »olrt>aosas ov vu~r pRO~aR~s
IIiTIi!>101TI~L 10R~
'fo ~aaat'~T
Ifavartis t:ozp.
c/o Norastis 110 isswd pussuaat to hula 10.3 by the
p. o. eax iiia7 Ia~rsx»TZO~ a~ross~R~r AaTxoRrrY
Muarcb TriaaQle park, NC 27709 ideatitied at the bottom of this page
~ ,AND AOO~BB Op 'f~ PRRTY TO Ni~I
one vrasrzx~r s~rJ,~ xs xsgeen
I. D8>Z08ITOR _ ., ..~ II. IDlITIFICATIOIO OF Tli! D~CItO0R~I81~
t(asw: Norastis Cosp Depoaitor~s tasxxsomic deaiqaation
sad
c/o Novartis JIO aaoessioa number gives by the
Address: P. 0. Bauc iZ=57 I!1'TliRIIIATIONAL DE?09ZTARY AvTHORITY:
Research Triangle psrk, 8aotesia sp. NR>tL H-21835
AC Z~709
ot:9sptambas 17, 1997
t Origitsal Deposit
' New Deposit
'
Rapsopsgati.oa of Original Deposit
III. (a) Vf7t8ILZZ'7C 9TJlTlI~SiT
Deposit vas fouled: ~ Viable
0 Na~nviable oa September 18,
1997(Datal
Iataraatiaaal Dspositary Authosity~s
preparation was found viable
oa September Z5,
1997tDatel'
III. (b) D$POSITOR~B E CY D$CTJ1RATION
Depaaitor daterlsined the International
Depoeitasy Authority~s preparation
was
$quivaleat 0 ' Nat equivalent
co deposit oa (Date)
Signsture of Depositor
Iv. CONDITIONS CLiDSR WHICH T!t&
VIABILITY T89T t018 PBRgOR~D
(De aitors/De oait )'
V . IRTIOI'~AL DSP08ITARY ACTI(ORITY
peals: Agricultural Reaearcts Sigaaturets) of persoate) having
Culture the power
Colleotioa ttiRRLI to repraaeat tho Iaterisational
Depoaitsty
Iates~lational Depositsry AuthorityAuthority or of authorised officialla):
~ddsssa: 1815 N. Uaiwrsity Street
II11~'f7
tndlcau the data of tho otiqlnal dapoalt os than n nwv arposit nLU boon
air~fe.
Matt with a atoas tAw ~pplln.~~l~ h.~s.
In thw oases dtvervd to In Nulw 1lf.2fn1111.1 an4 IIIII, rn Mr ~n chu nwrc
castor vlrbtltty tact.
f~itl In it tM lntosaatlon has e~~n c~quoat~d.
CA 02320801 2000-08-14
WO 99/42589 PCT/EP99/01015
-1 -
SF~~ LISTING
<110> Novartis AG
<120> Novel Zb~dns And Uses Ztaereof
<130> PI/5-30421/A/C~c3C 1963
<140>
<141>
<160> 22
<170> Patentln Ver. 2.0
<210> 1
<211> 9717
<212> LNA
<213> Photos huninesc~s
<220>
<221> CD6
<222> (412)..(1665)
<223> orfl 46.4 kDa
<220>
<221> (21S
<222> (1686)..(2447)
<223> orf2 -28.1kIa3
<220>
<221> C'D6
<222> (2758)..(3318)
<223> orf3 -20.7 kDa
<220>
<221> CDS
<222> (3342)..(4118)
<223> orf4 -28.7 kDa
<220>
<221> CAS
<222> (4515)..(9269)
<223> orf5 -176 kDa
<400> 1
gaattcatat gctatgaaat aaacagttgg cgcaataatt aaagctatta tttttatttt 60
gtttttatac aatgatatgc tttattaaac agaataatga gttaatgata aataaatcct 120
cgggatttat catgatatta tggccgaatg tgatgtgaac aattatttta taattagatt 180
aataatataa tggtattaaa ataacaatat atttattcat gggtatttat catcggtttt 240
attacatggg gaataatcta taaattagtt ttacataatt cacaaatagc gattccatta 300
accaggaata tCaaaaatac ttatttatga ttatggtgat atatcttcat tagcctactt 360
ttataactag aaaaattgac attttcaatc catgtataaa tggtaaccaa t atg cag 417
Met Gln
1
aga get caa cga gtt gtt att aca ggt atg ggt gcc gta aca ccg att 465
Arg Ala Gln Az~g Val Val Ile Thr Gly Met Gly Ala Val 'rtw Pro Ile
10 15
CA 02320801 2000-08-14
WO 99/42589 PCT/EP99/01015
_2_
ggt gaa gat gtt gaa tca tgt tgg caa agt att 513
att gaa aaa caa cat
Gly Glu Asp Val Glu Ser Cys Trp Gln Ser Ile
Ile Glu Lys Gln His
20 25 30
cga ttt cac aga att gaa ttt cct gac tca ttc 561
att aat tcg cgt ttc
Arg Phe His Arg Ile Glu Phe Pro Asp Ser Phe
Ile Asn Ser Arg Phe
35 40 45 50
ttt tct ttc ctt gca cca aac cca tcc cgc tat 609
cag tta tta cca aaa
Phe Ser Phe Leu Ala Pro Asn Pro Ser Arg Tyr
Gln Leu Leu Pro Lys
55 60 65
aag ttg act cat aca ctt tct gac tgc gga aaa 657
gca gca ttg aag gcg
Lys Leu Thr His 2hr Leu Ser Asp Cars Gly
Lys Ala Ala Leu Lys Ala
70 75 80
act tat caa get ttt acc caa gca ttc gge gtg 705
aat ata tca cet gtt
2hr Zyr Gln Ala Phe Thr Gln Ala Phe Gly Val
A.sn Ile Ser Pro Val
85 90 95
gaa tat tac gat aaa tac gaa tgt ggc gta att 753
ctt ggc agt ggt tgg
Glu 'i~rr 'tyr Asp Lys Zyr Glu Cars Gly Val
Ile Leu Gly Ser Gly 'I~p
100 105 110
gga get att gat aat gce gga gat cat get tgc 801
caa tat aag caa gca
Gly Ala Ile Asp Asn Ala Gly Asp His Ala Cps
Gln Tyr Lys Gln Ala
115 120 125 130
aaa tta get cat cct atg agt aat ctt att acc 849
atg cea agc tcc atg
Lys Leu Ala His Pro Met Ser Asn Leu Ile Thr
Met Pro Ser Ser Meet
135 140 145
acg get gca tgt tcg att atg tat gga cta cgt 897
ggt tat caa aat acc
Thr Ala Ala Cys Sex Ile Met 'Iyr Gly Leu
Arg Gly Zyr Gln Asn Thr
150 155 160
gtt atg get gcc tgc gca acg ggc aca atg gcg 945
ata ggc gat gcc ttt
Val Met Ala Ala Cps Ala 'Phr Gly Thr Met
Ala Ile Gly Asp Ala Phe
165 170 175
gaa att att egc tca ggg cgg gca aaa tgt atg 993
att gec gga gcc get
Glu Ile Ile Arg Ser Gly Arg Ala Lys Cps Met
Ile Ala Gly Ala Ala
180 185 190
gaa tca ctc acg cgg gaa tgt aat att tgg agt 1041
att gat gta ctg aat
Glu Ser Leu Thr Arg Glu Cps Asn Ile Txp Ser
Ile Asp Val Leu Asn
195 200 205 210
gca tta tcg aaa gaa caa gcg gac cca aat ctt 1089
gca tgt tgt cca ttt
Ala Leu Ser Lys Glu Gln Ala Asp Pro Asn Leu
Ala (.ys Cys pro Phe
215 220 225
agc ctt gat cgc tct gga ttt gta tta gcc gaa 1137
gga gcg gcg gta gtt
Ser Leu Asp Arg Ser Gly Phe Val Leu Ala Glu
Gly Ala Ala Val Val
230 235 240
tgt ctg gaa aat tat gat tca gcc atc gcg cgt 1185
ggt gca acg att tta
Cys Leu Glu Asn 'iyr Asp Ser Ala Ile Ala
Arg Gly Ala Thr Ile Leu
245 250 255
gcg gaa att aaa ggt tac gcc caa tat tca gat 1233
gcc gtt aat tta acc
Ala Glu Ile Lys Glyyz~ Ala Gln Tyr Ser Asp
Ala Val Asn Leu 'I~r
260 265 270
CA 02320801 2000-08-14
WO 99/42589 PCT/EP99/01015
-3-
cgg cca aca gaa gat att gaa cct aaa ata tta 1281
gcg ata act aaa gcc
Arg Pro ~Ihr Glu Asp Ile Glu Pro Lys Ile Leu
Ala Ile 'Ii~r Lys Ala
275 280 285 290
att gag cag gca cag att tcg ccg aaa gat att 1329
gac tac att aat get
Ile Glu Gln Ala Gln Ile Ser Pro Lys Asp Ile
Asp Tyr Ile Asn Ala
295 300 305
cat ggt act tct aca ccg tta aat gat ctt tat 1377
gaa act cag gca att
His Gly Zlzr Ser 2lir Pro Leu Asn Asp Leu
Tyr Glu Thr Gln Ala Ile
310 315 320
aaa gca gca ctg ggc caa tat get tat cag gta 1425
cct ata tca agc aca
Lys Ala Ala Leu Gly Gln Tyr Ala err Gln Val
Pro Ile Ser Ser I'hr
325 330 335
aaa tct tat acc ggc cac ctt att get gcc gcc 1473
ggt agt ttt gaa acg
Lys Ser Tyr Thr Gly His Leu Ile Ala Ala Ala
Giy Ser Phe Glu Thr
340 345 350
att gta tgt gtg aaa gca tta get gaa aat tgc 1521
ttg cca gca aca ttg
Ile Val Cys Val Lys Ala Leu Ala Glu Asn Cars
Leu pro Ala Zhr Leu
355 360 365 370
aat tta cac cgg gcc gat cca gat tgc gat ctc 1569
aat tat ttg cct aat
Asn Leu His Arg Ala Asp Pro Asp Cys Asp Leu
Asn 'iyr Leu Pro Asn
375 380 385
caa cat tgc tac acc get caa cca gag gtg aca 1617
ctc aat att agc gca
Gln His Cars Tyr Thr Ala Gln Pro Glu Val Zhr
Leu Asn Ile Ser Ala
390 395 400
ggt ttc ggc ggg cat aac get gcg ttg gtt atc 1665
get aag gta agg taa
Gly Phe Gly Gly His Asn Ala Ala Leu Val Ile
Ala Lys Val Arg
405 410 415
ctgatatgtt gatttttgca atg gaa gat att gaa 1718
cat tgg tcg aat ttc tct
Met Glu Asp Ile Glu His Trp Ser Asn Phe Ser
420 425
ggg gat ttt aac ccc atc cat tat tcg gcg aaa 1766
agc gag tct ttg cgc
Gly Asp Phe Asn Pro Ile His Zyr Ser Ala Lys
Ser Glu Ser Leu Arg
430 435 440 445
aat ata cag caa cac cxg gtg cag gga atg ttg 1814
agt ttg ctc tat gta
Asn Ile Gln Gln His Pro Val Gln Gly Met Lieu
Ser Leu Leu Tyr Val
450 455 460
cgg caa cag ttt tct caa tta act tcc get ttt 1862
aca acg gga ata ttg
Arg Gln Gln Phe Ser Gln Leu Zhr Ser Ala Phe
Thr Zhr Gly Ile r!~,
465 470 475
aac att gat gcc tct ttc cgc cag tat gtt tat 1910
acc gca tta ccc cat
Asn Ile Asp Ala Ser Phe Arg Gln Zyr Val ~r
'Ihr Ala Leu Pro His
480 485 490
caa ctg agg att aat act aaa aac aaa acg ttt 1958
aaa tta gaa aat ccc
Gln L~eu Arg Ile Asn Thr Lys Asn Lys 'IZzr
Phe Lys Leu Glu Asn Pro
495 500 505
agt aaa gaa aac acg ttg ttc ggc aat acc agc 2006
gta gag aat aca atg
Ser Lys Glu Asn Thr Leu Phe Gly Asn 'I'hr
Ser Val Glu Asn ~'hr Met
510 515 520 525
gag tca att gaa gat tgg atc gtt cag gat aat tgt caa aaa cta acg 2054
CA 02320801 2000-08-14
WO 99!42589 PCT/EP99/01015
-4-
Glu Ser Ile Glu Asp Trp Ile Val Gln Asp Asn
Cys Gln Lys Leu Thr
530 535 540
ata aca ggg gag gaa gtt tgt c~-aa aag tat get 2102
gtc ttt aga tac tat
Ile Thr Gly Glu Glu Val Cars Glu Lys 'Iyr Ala
Val Phe Arg err Zyr
545 550 555
ttc cca agt gtc act tct att gga tgg ttc ctg 2150
gat gcg ctt get ttt
Phe Pro Ser Val 'Ihr Ser Ile Gly '1'rp Phe
Leu Asp Ala Leu Ala Phe
560 565 570
cat ctt att att aat tcg aca gga ttt ctt aat 2198
ttt gag cac tac cat
His Leu Ile Ile Asn Ser Ztir Gly Phe Leu Asn
Phe Glu His Zyr His
575 580 585
ttt aac caa tta cag gat tat ctg agt caa tct 2246
ttt act ttg cat act
Phe Asn Gln Leu Gln Asp 'Iyr I~u Ser Gln Ser
Phe ~r Leu His Zfir
590 595 600 605
ggg caa gcg att aaa atc agg aag gag att gtt 2294
aat agt aca gta tta
Gly Gln Ala Ile Lys Ile Arg Lys Glu Ile Val
Asn Ser Thr Val Leu
610 615 620
tta tct tca ccg gat atc tgt gtt gaa tta aat 2342
cct cct tta ttg att
Leu Ser Ser Pro Asp Ile Cys Val Glu Leu Asn
Pro Pro Leu Leu Ile
625 630 635
aag aat ggc gat aaa gat tat att cgt att ttc 2390
tat tat cga tgt tta
Lys Asn Gly Asp Lys Asp 'I~rr Ile Arg Ile Phe
Tyr ~I~rz~ Arg Cys Leu
640 645 650
tat gat aaa aaa cct att ttt gta tca aag act 2438
tca att atc tct aag
Tyr Asp Lys Lys Pro Ile Phe Val Ser Lys Thr
Ser Ile Ile Ser Lys
655 660 665
atg aaa taa aaggaaagcg aaatgccaac acaaagtgat 2487
attttcactg
Met Lys
670
aaataaagaa tagaatatta atgatgaagg atatagaaga
tgaagaaata acaccagagt 2547
cctcttttgt ttcgcttgaa tttgatagtc ttgactatgt
ggaaatccaa gtttttgtgt 2607
tggaagcgta tggtattgtg cttaaagccg aacttttttc
aaatcattct atttcaacat 2667
taaatgagct cactgactat ttaaaatcaa aattgtaatc
tgaattttta cttaattatg 2727
ttttttcacc attaacatta agaggttata atg aac gtt 2781
tta gaa caa ggt aag
Met Asn Val Reu Glu Gln Gly Lys
675 680
gtt get get tta tat tca gcc tat tcg gaa aca 2829
gaa ggt tct tcg tgg
Val Ala Ala Leu Zyr Ser Ala 'I~r Ser Glu Thr
Glu Gly Ser Ser Trp
685 690 695
gtg gga aac ttg tgc tgt ttt tca agt gat cgg 2877
gag cat ttg cct att
Val Gly Asn Leu Cys Cys Phe Ser Ser Asp Arg
Glu His Leu Pro Ile
700 705 710
atc gtg aat ggg cgt cgt ttc ttg att gaa ttt 2925
gtt att cca gat cat
Ile Val Asn Gly Axg Arg Phe Leu Ile Glu Phe
Val Ile Pro Asp His
715 720 725
tta ctt gat aaa acg gtt aaa ccc aga gta ttc 2973
gat ttg gat atc aat
Leu Leu Asp Lys 'Ihr Val Lys Pro Arg Val Phe
Asp Leu Asp Ile Asn
CA 02320801 2000-08-14
WO 99/42589 PCT/EP99/01015
-5-
730 735 740
aaa caa ttt tta ctg cgt cgt gac cat cgt gag 3021
ata aat att tat ctt
Lys Gln Phe Leu Leu Arg Arg Asp His Arg Glu
Ile Asn Ile Tyr Leu
745 750 755 760
tta ggt gaa gga aat ttt atg gat agg acg acg 3069
aca gat aaa aat cta
Leu Gly Glu Gly Asn Phe Met Asp Arg Thr Thr
Zlzr Asp Lys Asn Leu
765 770 775
ttc gag tta aat gag gat ggt tca cta ttt att 3117
aag acg tta cgc cat
Phe Glu Leu Asn Glu Asp Gly Ser Leu Phe Ile
Lys Thr Leu Arg His
780 785 790
get ctt ggt aaa tat gtt get att aat cct tca 3165
act acg caa ttt atc
Ala Leu Gly Lys 'Iyr Val Ala Ile Asn Pro Ser
'Ihr Thr Gln Phe Ile
795 800 805
ttc ttt gca caa gga aag tac agt gaa ttt atc 3213
atg aat gcc tta aag
Phe Phe Ala Gln Gly Lys 'I~r Ser Glu Phe Ile
Met Asn Ala Leu Lys
810 815 820
aca gtt gaa gac gaa tta tca aaa cgt tat cga 3261
gtc aga att att cct
Thr Val Glu Asp Glu Leu Ser Lys Arg Tyr Arg
Val Arg Ile Ile Pro
825 830 835 840
gaa ttg caa ggg ccg tat tat ggc ttt gaa ctt 3309
gat att ctt tct att
Glu Leu Gln Gly Pro Zyr 'Iyr Gly Phe Glu Leu
Asp Tle Leu Ser Ile
845 850 855
aca get taa ttcacaatat tatggagagt gtt atg gaa 3362
aag aaa ata aca aca
Met Glu Lys Lys Ile ~hr Thr
860 865
ttt acc att gag aaa act gat gac aat ttt tat 3410
get aat ggg cgt cat
Phe Thr Ile Glu Lys Thr Asp Asp Asn Phe err
Ala Asn Gly Arg His
870 875 880
caa tgt atg gta aaa atc tct gta ctt aaa caa 3458
gaa tat agg aat ggt
Gln Cps Met Val Lys Ile Ser Val Leu Lys Gln
Glu 'I~rr Arg Asn Gly
885 890 895
gat tgg ata aaa tta gca ctt agt gag get gaa 3506
aaa aga tcg att cag
Asp Trp Ile Lys I~eu Ala I~u Ser Glu Ala Glu
Lys Arg Ser Ile Gln
900 905 910
gtg gcg gca tta agt gat agc ctc ata tat gac 3554
caa tta aaa atg cct
Val Ala Ala Leu Ser Asp Ser Leu Ile err Asp
Gln Leu Lys Met Pro
915 920 925 930
tca ggt tgg aca acg aca gat gca aga aat aaa 3602
ttt gat ctt ggg tta
Ser Gly Trp Thr Thr Thr Asp Ala Ang Asn Lys
Phe Asp Leu Gly Leu
935 940 945
tta aat ggt gtt tat cat get gat get ttt att 3650
gac gaa cag gta aca
Leu Asn Gly Val 'iyr His Ala Asp Ala Phe Ile
Asp Glu Gln Val ~Ihr
950 955 960
gat cgt gcg gga gat tgc tgc aca aat gaa aac 3698
tat cag aac agt gtg
Asp Arg Ala Gly Asp Cars Cys ~'hr Asn Glu Asn
Tyr Gln Asn Ser Val
965 970 975
aaa agt gtt cct gaa att atc tat cgt tat gtc 3746
agt agt aat aga aca
Lys Sex Val Pro Glu Ile Ile 'I~rr Arg Tyr Val
Ser Ser Asn Arg Thr
980 985 990
CA 02320801 2000-08-14
WO 99/42589 PCT/EP99/01015
-6-
agc aca gaa tac cta atg gca aaa atg aca ttt gaa gat acg gat ggg 3794
Ser Thr Glu 'iyr Leu Met Ala Lys Met Thr Phe Glu Asp Thr Asp Gly
995 1000 1005 1010
aaa cgc aca tta aca acg aat atg tca gtt ggt gat gaa gtt ttt gac 3842
Lys Arg 'Ilzr Leu 'Ilzr Thr Asn Met Ser Val Gly Asp Glu Val Phe Asp
1015 1020 1025
agc aag gtt tta tta aaa gcc att get cct tat gca att aat aca aat 3890
Ser Lys Val Leu Leu Lys Ala Ile Ala Pro 'Iyr Ala Ile Asn 2lzr Asn
1030 1035 1040
caa ttg cat gaa aac atc aat aca ttg ttt gat aaa aca gaa gag ccg 3938
Gln Leu His Glu Asn Ile Asn Thr Leu Phe Asp Lys Thr Glu Glu Pro
1045 1050 1055
aca aaa tcc gat act cat cat caa ata att aat ctt tat cgc tgg aca 3986
'Ihr Lys Ser Asp Zfir His His Gln Ile Ile Asn Leu 'Iyr Arg Trp Thr
1060 1065 1070
ttg cca tat cat ttg agg att ctt gaa ggg aat gac agt act gtt aat 4034
Leu Pro ~r His Leu Arg Ile Leu Glu Gly Asn Asp Ser 'ilzr Val Asn
1075 1080 1085 1090
aga ata tat gtc ctt ggt aaa gag cca tca aat gat aga ttc ctg aca 4082
Arg Ile 'Iyr Val Leu Gly Lys Glu Pro Ser Asn Asp Arg Phe Leu Thr
1095 1100 1105
aga gga agg gGa ttt aaa cga gga act cat atg tga atgcacgtga 4128
Arg Gly Arg Val Phe Lys Arg Gly Thr His Met
1110 1115
taatgtqagt ggaggatgtg ttatggacta tgcttatacc gtaactattc cggacacgca 4188
gcttgctgct gaagtgcttc atgtgacagg gtgttcgtgg acgagtggtt attatgatgg 4248
atatcatgat gtcacaatca ttgataacta cggttgtcag cataaattta gaatttcttc 4308
ggttaatatt ggacgtgcgc taagcatagc gagaataagt tgattttcct tagtaaaaaa 4368
cctttgttta tgctggtaaa cgcatgtgcg tttgccagca attaatatat tccattattg 4428
aaataggaat atagccatat ctgtaattat acataaacga atttttactc gaatataatt 4488
ttaattgatc aaacaggaaa tttaaa atg aaa get acc gat ata tat tcc aat 4541
Met Lys Ala Thr Asp Ile Tyr Ser Asn
1120 1125
get ttt aat ttc ggt tct tat att aat act ggt gtc gat ccc aga aca 4589
Ala Phe Asn Phe Gly Ser 'Iyr Ile Asn Thr Gly Val Asp Pro Arg Thr
1130 1135 1140
ggt caa tat agt gca aat att aat att atc acg tta aga cct aat aat 4637
Gly Gln Tar Ser Ala Asn Ile Asn Ile Ile Thr Leu Arg Pro Asn Asn
1145 1150 1155
gtg ggt aat tcg gaa caa aca ttg agc cta tca ttc tcg cca tta aca 4685
Val Gly Asn Ser Glu Gln Thr Leu Ser Txu Ser Phe Ser pro Leu ~~.
1160 1165 1170 1175
acg tta aac aat ggc ttt ggt att ggc tgg cgc ttt tca tta aca aca 4733
'I2~r Leu Asn Asn Gly Phe Gly Ile Gly Trp Arg Phe Ser Leu Thr Thr
1180 1185 1190
CA 02320801 2000-08-14
WO 99/42589 PCT/EP99/01015
_7_
tta gat ata aaa aca ctt aca ttt agc cga gca 4781
aat ggg gag caa ttt
Leu Asp Ile Lys Thr Leu 'Itzr Phe Ser Arg
Ala Asn Gly Glu Gln Phe
1195 1200 1205
aaa tgt aag cca ttg ccg cct aat aat aat gat 4829
ctt agt ttt aaa gat
Lys Cars Lys Pro Lieu Pro Pro Asn Asn Asn
Asp Leu Ser Phe Lys Asp
1210 1215 1220
aaa aaa cta aaa gat ttg cgc gta tat aag ctc 4877
gat agc aat act ttt
Lys Lys Leu Lys Asp Leu Arg Val Tyr Lys Leu
Asp Ser Asn ~'hr Phe
1225 1230 1235
tat gtt tat aac aaa aac ggc att ata gag ata 4925
ctt aaa cga att ggg
Tyr Val Tyr Asn Lys Asn Gly Ile Ile Glu Ile
Leu Lys Arg Ile Gly
1240 1245 1250 1255
tcg agt gat att gca aaa aca gtt gca ctt gaa 4973
ttt cct gat ggt gaa
Ser Ser Asp Ile Ala Lys '1'hr Val Ala Leu
Glu Phe Pro Asp Gly Glu
1260 1265 1270
gca ttt gat tta att tat aat tca aga ttt gca 5021
ttg tcc gaa ata aaa
Ala Phe Asp Leu Ile Tyr Asn Ser Arg phe Ala
Leu Ser Glu Ile Lys
1275 1280 1285
tac cgt gtg aca ggt aaa act tat ctt aaa ctc 5069
aat tac tct gga aat
'Iyr Arg Val Thr Gly Lys Thr Tyr Leu Lys Leu
Asn err Ser Gly Asn
1290 1295 1300
aac tgt aca tca gtg gaa tac cct gat gat aat 5117
aat att tct gcg aaa
Asn Cys Thr Ser Val Glu Zyr Pro Asp Asp Asn
Asn Ile Ser Ala Lys
1305 1310 1315
ata gca ttc gat tat cgt aac gat tac ctt att 5165
acg gtg act gta cct
Ile Ala Phe Asp Zyr Arg Asn Asp Tyr Leu Ile
Thr Val Thr Val Pro
1320 1325 1330 1335
tac gat get tct ggt cct att gat tct gcc cga 5213
ttt aag atg acc tat
Zyr Asp Ala Ser Gly Pro Ile Asp Ser Ala Arg
Phe Lys Met Zhr 2~rr
1340 1345 1350
cag aca tta aaa ggc gta ttt cca gtt atc agc 5261
acc ttc cgt aca cca
Gln Thr Leu Lys Gly Val Phe Pro Val Ile Ser
Thr Ptye Arg 'rhr pro
1355 1360 1365
acc ggt tat gtt gag ctg gtg agt tat aaa gag 5309
aat ggg cat aaa gtg
Thr Gly Tyr Val Glu Leu Val Ser Zyr Lys Glu
Asn Gly His Lys Val
1370 1375 1380
acg gac acg gaa tat att cct tat gcg get gca 5357
ctc act att caa ccc
Thr Asp Thr Glu 'Iyr Ile Pro Tyr Ala Ala Ala
Leu Thr Ile Gln Pro
1385 1390 1395
ggc aat gga caa cct gcg gtc agc aaa tcc tat 5405
gaa tat agt tca gta
Gly Asn Gly Gln Pro Ala Val Ser Lys Ser 2~rr
Glu 'lyr Ser Ser Val
1400 1405 1410 1415
cat aac ttc ttg ggc tat tct tct ggc cgg aca 5453
agc ttt gat tcc agt
His Asn Phe Leu Gly 'Iyr Ser Ser Gly Arg ~l~r
Ser Phe Asp Ser Ser
1420 1425 1430
caa gat aat ttg tat ttg gtc aca ggg aaa tac 5501
act tat tca tcc att
Gln Asp Asn Leu err Leu Val Thr Gly Lys 2~rr
'Ilzr 'i~rr Ser Ser Ile
1435 1440 1445
gaa cgg gtt tta gat ggt caa agt gtg gtt tca gta ata gaa cga gta 5549
CA 02320801 2000-08-14
WO 99/42589 PCT/EP99101015
_g_
Glu Arg Val Leu Asp Gly Gln Ser Val Val Ser
Val Ile Glu Arg Val
1450 1455 1460
ttt aat aaa ttc cat tta atg acc aaa gaa gca 5597
aaa aca caa gat aat
Phe Asn Lys Phe His Leu Met Thr Lys Glu Ala
Lys Zl~r Gln Asp Asn
1465 1470 1475
aag aga att aca aca gaa att act tac aat gag 5645
gat cta tca aaa agt
Lys Arg Ile Thr 'Ihr Glu Ile ~Ilzr err Asn
Glu Asp Leu Ser Lys Ser
1480 1485 1490 1495
ttc tca gag caa cca gaa aat tta caa caa cct 5693
tct cgc gtg tta acc
Phe Ser Glu Gln Pro Glu Asn Leu Gln Gln Pro
Ser Arg Val Leu Z'hr
1500 1505 1510
cgt tat acg gat ata caa aca aat act tca cga 5741
gaa gag act gtc aat
Arg Tyr Thr Asp Ile Gln Thr Asn Thr Ser And
Glu Glu Thr Val Asn
1515 1520 1525
att aaa agt gat gat tgg gga aat act cta ctt 5789
att act gag acc agt
Ile Lys Ser Asp Asp Trp Gly Asn Thr Leu Leu
Ile 'rhr Glu Zhr Ser
1530 1535 1540
ggg ata cag aaa gaa tac gtt tat tat ccg gtc 5837
aat ggc gaa ggt aat
Gly Ile Gln Lys Glu Zyr Val Tyrr err Pro Val
Asn Gly Glu Gly Asn
1545 1550 1555
agt tgc cct gcc gat ccc ttg ggt ttt tct cgg 5885
ttc tta aaa tca gtt
Ser Cys Pro Ala Asp Pro Leu Gly Phe Ser Arg
Phe Leu Lys Ser Val
1560 1565 1570 1575
acg caa aaa gga tcg cct gat get get caa agt 5933
gtc gca aat aaa gtg
2lzr Gln Lys Gly Ser Pro Asp Ala Ala Gln Ser
Val Ala Asn Lys Val
1580 1585 1590
att cat tat aca tat caa aaa ttt cct act ttt 5981
acc ggc get tat gtt
Ile His 2yr Thr 'Iyr Gln Lys Phe Pro Thr Phe
Thr Gly Ala 'Iyr Val
1595 1600 1605
aag gaa tat gtc agt aaa gtc tca gag acg ata 6029
gac aat aaa ata gcg
Lys GlWyr Val Ser Lys Val Ser Glu 'Ihr Ile
Asp Asn Lys Ile Ala
1610 1615 1620
aga acc ttt agc tat gtt aac tca ccg acg agt 6077
aaa tct cat ggt tcg
Arg Zlzr phe Ser Z~r Val Asn Ser Pro 'I3zr
Ser Lys Ser His Gly Ser
1625 1630 1635
tta gca aaa ata acg tca gtg atg aat aac cag 6125
caa acg gtc acc aca
Leu Ala Lys Ile Thr Ser Val Met Asn Asn Gln
Gln Thr Val I'hr Thr
1640 1645 1650 1655
ttt aaa tat gaa tat tca gaa agt gag atg acc 6173
aca aat get acg gtg
Phe Lys 'Iyr Glu 'Iyr Ser Glu Ser Glu Met
Thr Thr Asn Ala 'rhr Val
1660 1665 1670
acc ggt ttt gat ggc gca cat atg gaa tcg aaa 6221
aat gtg acg tct att
Thr Gly Phe Asp Gly Ala His Met Glu Ser Lys
Asn Val Thr Ser Ile
1675 1680 1685
tat acc cat cg~g caa ctt cgt aaa gtt gat gta 6269
aac cac gtg att acc
Zyr Thr His Arg Gln Leu Arg Lys Val Asp Val
Asn His Val Ile 2'hr
1690 1695 1700
gat cag tct tat gat ctt ttg ggt cgc att aca 631?
ggg ca,a att att gat
Asp Gln Ser Ayr Asp Leu Leu Gly Arg Ile Thr
Gly Gln Ile Ile Asp
CA 02320801 2000-08-14
WO 99/42589 PCT/EP99/01015
-9-
1705 1710 1715
ccc ggc acg gca aga gaa att aaa cgt aat tac 6365
gtt tat caa tat ccc
Pro Gly 'ihr Ala Arg Glu Ile Lys Arg Asn Tyr
Val Tyr Gln Tyr Pro
1720 1725 1730 1735
ggc ggt gac gaa aat gat ttt tgg ccg gtg atg 6413
ata gaa gtt gat tct
Gly Gly Asp Glu Asn Asp Phe Tzp Pro Val Met
Ile Glu Val Asp Ser
1740 1745 1750
caa ggc gtc aga cgt aaa acc cat tac gat gga 6461
atg gga cgt att tgt
Gln Gly Val Arg Arg Lys Thr His Tyr Asp Gly
Met Gly Arg Ile Gys
1755 1760 1765
tcg att gaa gaa caa gat gat gat ggc gcc tgg 6509
~c aca tcg ggg att
Ser Ile Glu Glu Gln Asp Asp Asp Gly Ala Trp
Gly Thr Ser Gly Ile
1770 1775 1780
tat caa ggc aca tat cga aaa gtt ctt gcc aga 6557
caa tat gat gtt ttg
Tyr G3n Gly Thr Tyr Arg Lys Val Leu Ala Arg
Gln Tyr Asp Val Leu
1785 1790 1795
ggg cag ttg agc aag gaa att tca aat gat tgg 6605
tta tgg aat ttz tct
Gly Gln Leu Ser Lys Glu Ile Ser Asn Asp Tip
Leu Txp Asn Leu Ser
1800 1805 1810 1815
gcc aat cct ttg gtt ~t ctt get acc ccg ttg 6653
gtt aca acg aaa acc
Ala Asn Pro Leu Val Arg Leu Ala Thr Pro Leu
Val Thr Thr Lys Thr
1820 1825 1830
tat aaa tat gat ggt tgg gga aat ctt tac agc 6701
acg gaa tac agt gat
Tyr Lys Tyr Asp Gly Tip Gly Asn Leu Tyr Ser
Thr Glu Tyr Ser Asp
1835 1840 1845
ggt cgg ata gag ctg gaa atc cat gat cct att 6749
acg agg aca att act
Gly Arg Ile Glu L~eu Glu Ile His Asp Pro Ile
T1~ Arg 2~r Ile Thrr
1850 1855 1860
caa ggg gtc aaa gga tta ggg atg tta aat att 6797
cag caa aat aat ttt
Gln Gly Val Lys Gly L~ Gly Met Leu Asn Ile
Gln Gln Asn Asn Phe
1865 1870 1875
gag caa ccg get tcg atc aaa get gtg tat cct 6845
gat ggt acg ata tat
Glu Gln Pro Ala Ser Ile Lys Ala Val Tyr Pro
Asp Gly Thr Ile Tyr
1880 1885 1890 1895
agc acc cgt act tat cgt tat gat gga ttt ggt 6893
cgt aca gtg acg gaa
Ser Thr Arg Thr Tyr Arg Tyr Asp Gly Ptbe Gly
Anyl~r Val 'Il~r Glu
1900 1905 1910
aca gat gca gaa ggt cat get acc caa att gga 6941
tat gat gtg ttt gat
Thr Asp Ala Glu Gly His Ala Thr Gln Ile Gly
Tyr Asp Val Phe Asp
1915 1920 1925
cgt ata gtg aaa aaa acg ttg cca gac gga aca 6989
ata tta gaa tcc get
Arg Ile Val Lys Lys Thr Leu Pro Asp Gly Thr
Ile Leu Glu Ser Ala
1930 1935 1940
tat gca agc ttt agc cat gaa gaa tta att tcg 7037
gca ctg aac gtg aat
Tyr Ala Ser Phe Ser His Glu Glu Leu Ile Ser
Ala Leu Asn Val Asn
1945 1950 1955
ggc aca cag ttg ggg gca tta gtt tat gat ggt 7085
ctt ggg cgg gta ata
Gly 'ilzr Gln Leu Gly Ala Leu Val Tyr Asp
Gly Leu Gly Arg Val Ile
1960 1965 1970 1975
CA 02320801 2000-08-14
WO 99/42589 PCT/EP99/01015
-10-
agt gat acg gtg ggt ggt cgc aaa acg gaa tat 7133
tta tat ggg cct caa
Ser Asp 'I2~r Val Gly Gly Arg Lys 'Ilzr Glu
2yr LeWyr Gly Pro Gln
1980 1985 1990
ggt gac aaa ccg att cag tca att act cct tcg 7181
cat aat aag caa aat
Gly Asp Lys Pro Ile Gln Ser Ile T'hr Pro Ser
His Asn Lys Gln Asn
1995 2000 2005
atg gat tac ctc tac tat ctt ggt agt gtg atg 7229
tcc aaa ttt acc acg
Met Asp Zyr Leu 'Iyr 'Iyr Leu Gly Ser Val
Met Ser Lys Phe 'Ihr Thr
2010 2015 2020
ggg aca gac caa caa aac ttt cgt tat cat tcg 7277
aaa acg gga aca tta
Gly Thx~ Asp Gln Gln Asn Phe Arg err His Ser
Lys Thr Gly 'Ihr L~eu
2025 2030 2035
tta tct gcg tca gaa ggc gta tct cag act aat 7325
tac agt tat ttc cca
Leu Ser Ala Ser Glu Gly Val Ser Gln Thr Asn
Tyr Ser Ayr Phe pro
2040 2045 2050 2055
tcg ggt gta tta cag cga gaa tca ttt tta cgg 7373
gat aat aaa ccg att
Ser Gly Val Leu Gln Arg Glu Ser Phe Leu Arg
Asp Asn Lys Pro Ile
2060 2065 2070
tca tcg ggc gag tac ctt tat acg atg tcc ggt 7421
ttg att caa cgt cat
Ser Ser Gly Glu Tyr Leu Tyr Thr Met Ser Gly
Leu Ile Gln Ang His
2075 2080 2085
aaa gat agt ttt ggt cat aat cat gtt tat agt 7469
tac gat get cag gga
Lys Asp Ser Phe Gly His Asn His Val Zyr Ser
T~rr Asp Ala Gln Gly
2090 2095 2100
aga ttg gtc aaa aca gaa cag gat gca caa tac 7517
get aca ttt gaa tat
Arg Leu Val Lys Thr Glu Gln Asp Ala Gln 'I~rr
Ala Thr Phe Glu Zyr
2105 2110 2115
gac aat gtt ggg cga ttg ata aca acg acg acc 7565
aaa gac acg acg tca
Asp Asn Val Gly Arg Leu Ile Thr Zhr ~'hr Thr
Lys Asp 'Ihr Thr Ser
2120 2125 2130 2135
tta tcc caa tta gtg aca aaa atc gaa tat gat 7613
get ttt gat cga gaa
Leu Ser Gln Leu Val 'Ii~r Lys Ile Glu Zyr
Asp Ala Phe Asp Arg Glu
2140 2145 2150
ata aaa cgc tcg cta att agt gac ttc tca ata 7661
caa gtt att acc tta
Ile Lys Arg Ser heu Ile Ser Asp Phe Ser Ile
Gln Val Ile Thr Leu
2155 2160 2165
agc tat acg aag aat aat caa atc agt caa cgt 7709
atc acc tcc atc gat
Ser Zyr Thr Lys Asn Asn Gln Ile Ser Gln Arg
Ile Thr Ser Ile Asp
2170 2175 2180
ggg gtg gtt atg aaa aat gaa cgt tat caa tat 7757
gat aat aat caa cgc
Gly Val Val Met Lys Asn Glu Arg 'I~r GlWyr
Asp Asn Asn Gln Arg
2185 2190 2195
tta agc caa tac caa tgt gag gga gaa caa tct 7805
ccg att gat cat acg
1xu Ser Gln 'I~r Gln Cars Glu Gly Glu Gln
Ser Pro Ile Asp His 'Ilzr
2200 2205 2210 2215
ggt cgt gta tta aat cag cag att tac cat tat 7853
gac caa tgg gga aat
Gly Arg Val Leu Asn Gln Gln Ile Zyr His Tyr
Asp Gln Trp Gly Asn
2220 2225 2230
CA 02320801 2000-08-14
WO 99/42589 PC'T/EP99/01015
-11-
att aag cgg ctc gat aat aca tat cga gat ggt 7901
aag gaa acg gtg gat
Ile Lys Arg Leu Asp Asn ~'hr Zyr Arg Asp Gly
Lys Glu 'Ilzr Val Asp
2235 2240 2245
tat cat ttc agt caa gcc gat cca act caa ctt 7949
att cgt att acc agc
'Iyr Isis type Ser Gln Ala Asp Pro 'Ihr Gln
Leu Ile Arg Ile Thr Ser
2250 2255 2260
gac aaa cag cag ata gag tta agt tat gat get 7997
aat ggc aac cta aca
Asp Lys Gln Gln Ile Glu Leu Ser Zyx~ Asp Ala
Asn Gly Asn Leu Thr
2265 2270 2275
cgt gac gaa aaa ggg caa acg ctc att tac gat 8045
cag aat aat cgc ttg
Arg Asp Glu Lys Gly Gln Zhr Leu Ile Zyr Asp
Gln Asn Asn Arg Leu
2280 2285 2290 2295
gta cag gtc aaa gac cgg ttg ggc aat ctg gtg 8093
tgc agc tac cag tat
Val Gln Val Lys Asp Arg Leu Gly Asn Leu Val
C]rs Ser Tyr Gln Tyr
2300 2305 2310
gat gca ttg aac aaa tta acc gca cag gtt ttg 8141
gcg aat ggt acc gtt
Asp Ala Leu Asn Lys Leu Thr Ala Gln Val Leu
Ala A~ Gly Thr Val
2315 2320 2325
aat cga cag cat tat get tcc ggt aaa gtg acg 8189
aat att caa ttg ggt
Asn Arg Gln His Tyr Ala Ser Gly Lys Val Thr
Asn Ile Gln Leu Gly
2330 2335 2340
gat gaa gcg att act tgg ttg agc agt gat aag 8237
caa cga att gga cat
Asp Glu Ala Ile Zlzr Tzp Leu Ser Ser Asp Lys
Gln Arg Ile Gly His
2345 2350 2355
caa agc gcc aag aat ggt caa tca gtc tac tat 8285
caa tat ggt att gac
Gln Ser Ala Lys Asn Gly Gln Ser Val Tyr Tyr
Gln Tyr Gly Ile Asp
2360 2365 2370 2375
cat aac agt acg gtt atc gcc agt cag aac gaa 8333
aac gag ttg atg get
His Asn Ser Thr Val Ile Ala Ser Gln Asn Glu
Asn Glu Leu Met Ala
2380 2385 2390
tta tcc tat aca cct tat ggc ttt agg agt tta 8381
att tcc tca tta ccg
Leu Ser Zyr Thr Pro Zyr Gly Phe Arg Ser Leu
Ile Ser Ser Leu Pro
2395 2400 2405
ggt ttg aat ggc gca cag gtt gat cca gta aca 8429
ggc tgg tac ttc tta
Gly Leu Asn Gly Ala Gln Val Asp Pro Val Thr
Gly Trp Tyr pne r_en,
2410 2415 2420
Gy 8477
g
~
t
t
Asn
Gl
y 'Iyr Arg Val
Phe Asn Pro al Leu Met Arg
Phe His ~-
2425 2430 2435
ccc gat agt tgg agt cct ttt ggt cgg gga ggg 8525
att aac cct tat acc
Pro Asp Ser Txp Ser Pro Phe Gly Arg Gly Gly
Ile Asn Pro Tyr ~'hr
2440 2445 2450 2455
tat tgc caa ggc gat ccc ata aac cgg att gat 8573
ctg aac ggt cat ctt
Tyr Cars Gln Gly Asp Pro Ile Asn Arg Ile Asp
Leu Asn Gly His Leu
2460 2465 2470
agt gcc ggc ggg ata tta ggc att gtg cta ggg 8621
gca att ggc atc att
Ser Ala Gly Gly ile Leu Gly Ile Val Leu Gly
Ala Ile Gly Ile Ile
2475 2480 2485
gtc ggg att gta tca ctg gga gcc gga gcg gcg att agc gcg ggt ctc 8669
CA 02320801 2000-08-14
WO 99/42589 PCT/EP99/01015
-12-
Val Gly Ile Val Ser Leu Gly Ala Gly Ala Ala
Ile Ser Ala Gly Leu
2490 2495 2500
att get gcg ggg ggc get ttg ggg gcg att get 8717
tct acc agc gcg ctt
Ile Ala Ala Gly Gly Ala Leu Gly Ala Ile Ala
Ser Thr Ser Ala L~eu
2505 2510 2515
gca gtt act gcg act gtc att gga ttg get gcc 8765
gat tcg ata ggg att
Ala Val ~hr Ala 2'hr Val Ile Gly Leu Ala Ala
Asp Ser Ile Gly Ile
2520 2525 2530 2535
gcg tca gca gca tta tcg gaa aaa gat ccg aaa 8813
aca tct ggg ata tta
Ala Ser Ala Ala Leu Ser Glu Lys Asp Pro Lys
Zhr Ser Gly Ile Leu
2540 2545 2550
aat tgg att agt gcg gga ttg ggg gtt tta agc 8861
ttt ggt atc agc gca
Asn Txp Ile Ser Ala Gly Leu Gly Val Leu Ser
Phe Gly Ile Ser Ala
2555 2560 2565
ata acc ttt acc tct tcg ctg gta aaa tcg gca 8909
cgg agt ggt tct cag
Ile 'Ihr I?he Thr Ser Ser Leu Val Lys Ser
Ala Arg Ser Gly Ser Gln
2570 2575 2580
gca gtc agc gcg ggt gtt atc ggg tca gtg cct 8957
ctt gaa ttt ggt gaa
Ala Val Ser Ala Gly Val Ile Gly Ser Val Pro
Leu Glu Plze Gly Glu
2585 2590 2595
gtt get agc cgt tcc agc aga cga tgg gat att 9005
gcg tta tct tcg ata
Val Ala Ser Arg Ser Ser Arg Arg Trp Asp Ile
Ala Leu Ser Ser Ile
2600 2605 2610 2615
tcg ttg ggc gca aat gcg gcg tct ctc tct acg 9053
ggg ata gcg gcg gog
Ser Leu Gly Ala Asn Ala Ala Ser Leu Ser Thr
Gly Ile Ala Ala Ala
2620 2625 2630
gcg gtt gca gac agt aat gcg aat gca get aat 9101
att ctg gga tgg gta
Ala Val Ala Asp Ser Asn Ala Asn Ala Ala Asn
Ile Leu Gly Trp Val
2635 2640 2645
tcc ttt ggt ttt ggt gca gta tcg aca acc tca 9149
gga ata att gag ctt
Ser Pte Gly Phe Gly Ala Val Ser Thr Thr Ser
Gly Ile Ile Glu Leu
2650 2655 2660
acg cgt aca get tat gca gtg aat cat cag act 9197
tgg gaa ctg agt tca
Thr Arg Thr Ala Zyr Ala Val Asn His Gln Thr
Trp Glu Leu Ser Ser
2665 2670 2675
tca gca ggt act tcg gag gaa gtg aag cct ata 9245
cgt tgt ctc gtt tca
Ser Ala Gly ?!zr Ser Glu Glu Val Lys Pro Ile
Arg Cys Leu Val Ser
2680 2685 2690 2695
cac cgc tgg aat cag aag cag tga atgttaaccc 9299
tcct~ca gttgagttaa
His An3 Trp Asn Gln Lys Gln
2700
tcaaacgttt cgaaatagta ccacta tttagccaat cgtccattga aacccgtaat 9359
gtgttgcgac gtcgtttgac aatataaaga ttctgcgaac cgattggtta agtctcacga 9419
aaaataacta ttag~gcgaca tttgcgtcgc cttttttaag gaactttatc aggttacatt 9479
tataagaagc tattttgttt tcgacggatg ttggtttctc tgagataaaa aatagaggga 9539
aatgatgtca agggtgataa tggttaattg taaaatatgt gatattattc gcatttatat 9599
CA 02320801 2000-08-14
WO 99/42589 PCT/EP99/01015
-13-
gtcaatgtaa ttcctcttat tatttaattt tattgcattt gctacgcgaa atcgccttat 9659
aattttattt ttaataaatt attatttcat cattaaacta aaataaatta tttctaga 9717
<210> 2
<211> 417
<212> PftT
<213> Photorhabdus lmninesc~s
<400> 2
Met Gln Arg Ala Gln Arg Val Val Ile Thr Gly Met Gly Ala Val Thr
1 5 10 15
Pro Ile Gly Glu Asp Val Glu Ser Gys Trp Gln Ser Ile Ile Glu Lys
20 25 30
Gln His Arg Phe His Arg Ile Glu Phe Pro Asp Ser Phe Ile Asn Ser
35 40 45
Arg Phe Phe Ser Phe Leu Ala Pro Asn Pro Ser Ar~g Zyr Gln Leu Leu
50 55 60
Pro Lys Lys Leu Thr His 'Ihr L~eu Ser Asp Cys Gly Lys Ala Ala Leu
65 70 75 8p
Lys Ala Thr Tyr Gln Ala Phe Thr Gln Ala Phe Gly Val Asn Ile Ser
85 90 95
Pro Val Glu 'Iyr 'Ijrr Asp Lys Tyr Glu Cps Gly Val Ile Leu Gly Ser
100 105 110
Gly Trp Gly Ala Ile Asp Asn Ala Gly Asp His Ala Cys G1n 2yr Lys
115 120 125
Gln Ala Lys Leu Ala His Pro Met Ser Asn Leu Ile Thr Met Pro Ser
130 135 140
Ser Niet I'hr Ala Ala Cys Ser Ile l~t 'Iyr Gly Leu Arg Gly 'Iyr Gln
145 150 155 160
Asn 'rhr Val Met Ala Ala Cps Ala Thr Gly Thr Met Ala Ile Gly Asp
165 170 175
Ala Phe Glu Ile Ile Arg Ser Gly Arg Ala Lys Gars Met Ile Ala Gly
180 185 190
Ala Ala Glu Ser L~ Thr Ar,g Glu Cys Asn Ile Trp Ser Ile Asp Val
195 200 205
Leu Asn Ala Leu Ser Lys Glu Gln Ala Asp Pro Asn Leu Ala Cys Cars
210 215 220
Pro Phe Ser Leu Asp Arg Ser Gly Phe Val Leu Ala Glu Gly Ala Ala
225 230 235 240
Val Val Cys Leu Glu Asn Tyr Asp Ser Ala Ile Ala And Gly Ala Thr
245 250 255
Ile Leu Ala Glu Ile Lys Gly 2yr Ala Gln 25rr Ser Asp Ala Val Asn
260 265 270
Leu Thr Arg Pro 'I'hr Glu Asp Ile Glu Pro Lys Ile Ireu Ala Ile 'Ihr
275 280 285
CA 02320801 2000-08-14
WO 99/42589 PCT/EP99/01015
-14-
Lys Ala Ile Glu Gln Ala Gln Ile Ser Pro Lys Asp Ile Asp Zyr Ile
290 295 300
Asn Ala His Gly Thr Ser Thr Pro Leu Asn Asp Leu Tyr Glu Thr Gln
305 310 315 320
Ala Ile Lys Ala Ala Leu Gly Gln Tyr Ala 'Iyr Gln Val Pro Ile Ser
325 330 335
Ser Zhr Lys Ser 'I'yr 'Irir Gly His Leu Ile Ala Ala Ala Gly Ser Phe
340 345 350
Glu 'I'hr Ile Val Cps Val Lys Ala Leu Ala Glu Asn Cys Leu pro Ala
355 360 365
'I2zr Leu Asn Leu His Arg Ala Asp Pro Asp Cars Asp Leu Asn 2yr Leu
370 375 380
Pro Asn Gln His Cps 'Iyr Z2~r Ala Gln Pro Glu Val Thr Leu Asn Ile
385 390 395 400
Ser Ala Gly Phe Gly Gly His Asn Ala Ala Leu Val Ile Ala Lys Val
405 410 415
<210> 3
<211> 253
<212> PItT
<213> Photorhabc~us haninescens
<400> 3
Met Glu Asp Ile Glu His Tip Ser Asn Phe Ser Gly Asp Phe Asn Pro
1 5 10 15
Ile His Z]rr Ser Ala Lys Ser Glu Ser Leu Arg Asn Ile Gln Gln His
20 25 30
Pro Val Gln Gly Met Leu Sex Leu Leu 2~rr Val Arg Gln Gln Phe Ser
35 40 45
Gln Leu Thr Ser Ala Phe ~lir Thr Gly Ile Leu Asn Ile Asp Ala Ser
50 55 60
Pl~e Arg Gln err Val Zyr Thr Ala Leu Pro His Gln Leu Arg Ile Asn
65 70 75 80
Thr Lys Asn Lys Thr Phe Lys Leu Glu Asn Pro Ser Lys Glu Asn Thr
85 90 95
Leu Phe Gly Asn Thr Ser Val Glu Asn Thr Met Glu Ser Ile Glu Asp
100 105 110
Trp Ile Val Gln Asp Asn Cars Gln Lys Leu Zhr Ile Thr Gly Glu Glu
115 120 125
Val Cps Glu Lys 'I~rr Ala Val Phe Arg Tyr 2~rr Phe pro Ser Val Thr
130 135 140
Ser Ile Gly Trp Phe Leu Asp Ala Leu Ala Phe His L~eu Ile Ile Asn
145 150 155 160
Ser 'Ihr Gly Phe Leu Asn Phe Glu His 'Tyr His Phe Asn Gln Leu Gln
165 170 175
CA 02320801 2000-08-14
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-15-
Asp Tyr Leu Ser Gln Ser Phe Thr Leu His 'Ihr Gly Gln Ala Ile Lys
180 185 190
Ile Arg Lys Glu Ile Val Asn Ser Thr Val Leu Leu Ser Ser Pro Asp
195 200 205
Ile Cps Val Glu Leu Asn Pro Pro Leu Leu Ile Lys Asn Gly Asp Lys
210 215 220
Asp 'Iyr Ile Arg Ile Phe 'I~rr Tyr Arg Cars Le~ta TSrr Asp Lys Lys Pro
225 230 235 240
Ile Phe Val Ser Lys Thr Ser Ile Ile Ser Lys Met Lys
245 250
<210> 4
<211> 186
<212> PRT
<213> Photorhabdus lLm~escens
<400> 4
Met Asn Val Leu Glu Gln Gly Lys Val Ala Ala Leu ~r Ser Ala err
1 5 10 15
Ser Glu Thr Glu Gly Ser Ser Trp Val Gly Asn Leu Gys Cys Phe Ser
20 25 30
Ser Asp Arg Glu His Leu Pro Ile Ile Val Asn Gly Arg Arg Phe Leu
35 40 45
Ile Glu Phe Val Ile Pro Asp His Lieu Leu Asp Lys Thr Val Lys Pro
50 55 60
Arg Val Phe Asp Leu Asp Ile Asn Lys Gln Phe Leu Leu Arg Arg Asp
65 70 75 80
His Arg Glu Ile Asn Ile Zyr I~eu Leu Gly Glu Gly Asn Phe Met Asp
85 90 95
Arg 29~r Thr 'I~r Asp Lys Asn Leu Phe Glu Leu Asn Glu Asp Gly Ser
100 105 T10
Leu hhe Ile Lys ~hr Leu Arg His Ala Leu Gly Lys 'Iyr Val Ala Ile
115 120 125
Asn Pro Ser Thr Thr Gln Phe Ile Phe Phe Ala Gln Gly Lys 'Iyr Ser
130 135 140
Glu Phe Ile Met Asn Ala Leu Lys Thr Val Glu Asp Glu Leu Ser Lys
145 150 155 160
Arg 'Iyr Arg Val Arg Ile Ile Pro Glu Ieu Gln Gly Pro 'I]rr 'Iyr Gly
165 170 175
Phe Glu Leu Asp Ile Leu Ser Ile Thr Ala
180 185
<210> 5
<211> 258
<212> PRT
<213> Photorhabdus lum:in~escens
CA 02320801 2000-08-14
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-16-
<400> 5
Met Glu Lys Lys Ile Thr Thr Phe ~'hr Ile Glu Lys Trbr Asp
1 5 10
Asp Asn Phe Zyr Ala Asn Gly Arg His Gln Cys Met Val Lys Ile Ser
15 20 25 30
Val Leu Lys Gln Glu Tyr Arg Asn Gly Asp Trp Ile Lys Leu Ala Leu
35 40 45
Ser Glu Ala Glu Lys Arg Ser Ile Gln Val Ala Ala I~eu Ser Asp Ser
50 55 60
Leu Ile Tyr Asp Gln Leu Lys Met Pro Ser Gly 2~p ~iizr Thr Thr Asp
65 70 75
Ala Arg Asn Lys Phe Asp Leu Gly L~ Leu Asn Gly Val Tyr His Ala
80 85 90
Asp Ala Phe Ile Asp Glu Gln Val Thr Asp Arg Ala Gly Asp Cys Cys
95 100 105 110
Thr Asn Glu Asn 'Iyr Gln Asn Ser Val Lys Ser Val Pro Glu Ile Ile
115 120 125
'Iyr Arg Zyr Val Ser Ser Asn Arg 'Ilzx~ Ser Thr Glu err Leu Met Ala
130 135 140
Lys Met T'hr Phe Glu Asp ~'hr Asp Gly Lys Arg 'Ilzr Leu Thr 2~r Asn
145 150 155
Met Ser Val Gly Asp Glu Val Phe Asp Ser Lys Val Leu Leu Lys Ala
160 165 170
Ile Ala Pro 'Iyr Ala Ile Asn '1'tbr Asn Gln Leu His Glu Asw Ile As~n
175 180 185 190
'Ihr Leu Plebe Asp Lys 'I~r Glu Glu Pro 2fbr Lys Ser Asp 'Ihr His His
195 200 205
Gln Ile Ile Asn Leu 'Iyr Arg Trp Thr Leu Pro Tyr His Leu Arg Ile
210 215 220
Leu Glu Gly Asn Asp Ser Z'hr Val Asn Arg Ile Zyr Val Leu Gly Lys
225 230 235
Glu Pro Ser Asn Asp Axg Plebe Leu Thr Arg Gly Arg Val Phe Lys Arg
240 245 250
Gly 'Ihr His Met
255
<210> 6
<211> 1584
<212> PRT
<213> Photorhabdus lscer~s
<400> 6
Met Lys Ala Zhr Asp Ile err Ser Asn Ala Phe Asn Phe Gly Ser Tyr
1 5 10 15
Ile Asn Thr Gly Val Asp Pro Arg Ttbr Gly Gln 'Iyr Ser Ala Asn Ile
20 25 30
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_17_
Asn Ile Ile Thr Leu Arg Pro Asn Asn Val Gly Asn Ser Glu Gln 'I~r
35 40 45
Leu Ser Leu Ser Phe Ser Pro Leu Thr Thr Leu Asn Asn Gly Phe Gly
50 55 60
Ile Gly Trp Arg Phe Ser Leu 'Ihr Thr Leu Asp Ile Lys Thr Leu Thr
65 70 75 g0
Phe Ser Arg Ala Asn Gly Glu Gln Phe Lys Cys Lys Pro Leu Pro Pro
85 90 95
Asn Asn Asn Asp Leu Ser Phe Lys Asp Lys Lys Leu Lys Asp Leu Arg
100 105 110
Val Tyr Lys Leu Asp Ser Asn Thr Phe err Val err Asn Lys Asn Gly
115 120 125
Ile Ile Glu Ile Leu Lys Arg Ile Gly Ser Sex Asp Ile Ala Lys 'Ihr
130 135 140
Val Ala Leu Glu Phe Pro Asp Gly Glu Ala Phe Asp L~eu Ile err Asn
145 150 155 160
Ser Arg Phe Ala 1xu Ser Glu Ile Lys ~Iyr Arg Val Thr Gly Lys Thr
165 170 175
2~rr I~ Lys Leu Asn Tyr Ser Gly Asn Asn-Cys Thr Ser Val Glu err
180 185 190
Pro Asp Asp Asn Asn Ile Ser Ala Lys Ile Ala Phe Asp Tyr Arg Asn
195 200 205
Asp Tyr Leu Ile Thr Val Thr Val Pro Zyr Asp Ala Ser Gly Pro Ile
210 215 220
Asp Ser Ala Arg Phe Lys Met 2hr Tyr Gln Thr Leu Lys Gly Val Phe
225 230 235 240
Pro Val Ile Ser 'I~r Phe Arg 'i'hr Pro Thr Gly 2yr Val Glu Leu Val
245 250 255
Ser ~I)rr Lys Glu Asn Gly His Lys Val 'I~r Asp Thr Glu Tyr Ile Pro
260 265 27p
Zyr Ala Ala Ala Leu Thr Ile Gln Pro Gly Asn Gly Gln Pro Ala Val
275 280 285
Ser Lys Ser err Glu err Ser Ser Val His Asn Phe Leu Gly err Ser
290 295 300
Ser Gly Arg Thr Ser Phe Asp Ser Ser Gln Asp Asn I~ ~Iyr Leu Val
305 310 315 320
Thr Gly Lys 'l~rr 'rhr ~Iyr Ser Ser Ile Glu Arg Val Leu Asp Gly Gln
325 330 335
Ser Val Val Ser Val Ile Glu Arg Val Phe Asn Lys Phe His Leu Met
340 345 350
Thr Lys Glu Ala Lys 'Ihr Gln Asp Asn Lys Arg Ile Thr Thr Glu Ile
355 360 365
'I~r Tyr Asn Glu Asp L~ Ser Lys Ser Phe Ser Glu Gln Pro Glu Asn
370 375 380
CA 02320801 2000-08-14
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_18_
Leu Gln Gln Pro Ser Arg Val Leu Thr An3 Tyr Thr Asp Ile Gln Thr
385 390 395 400
Asn Thr Ser Arg Glu Glu 2'hr Val Asn Ile Lys Ser Asp Asp Trp Gly
405 410 415
Asn Thr Leu Leu Ile Thr Glu Thr Ser Gly Ile Gln Lys Glu Tyr Val
420 425 430
Tyr Tyr Pro Val Asn Gly Glu Gly Asn Ser Cys Pro Ala Asp Pro Leu
435 440 445
Gly Phe Ser Arg Phe Leu Lys Ser Val Thr Gln Lys Gly Ser Pro Asp
450 455 460
Ala Ala Gln Ser Val Ala Asn Lys Val Ile His Tyr 'Ihr Tyr Gln Lys
465 470 475 480
Phe Pro Thr Phe Thr Gly Ala Tyr Val Lys Glu Tyr Val Ser Lys Val
485 490 495
Ser Glu Thr Ile Asp Asn Lys Ile Ala Arg ~Ihr Phe Ser Tyr Val Asn
500 505 510
Ser Pro Thr Ser Lys Ser His Gly Ser Leu Ala Lys Ile Thr Sex Val
515 520 525
Met Asn Asn Gln Gln Thr Val Thr Thr Phe Lys Tyr Glu Tyr Ser Glu
530 535 540
Ser Glu Met Thr Thr Asn Ala Zlzr Val Thr Gly Phe Asp Gly Ala His
545 550 555 560
Met Glu Ser Lys Asn Val Thr Ser Ile Tyr Thr His Arg Gln Leu Arg
565 570 575
Lys Val Asp Val Asn His Val Ile Thr Asp Gln Ser Tyr Asp Leu Leu
580 585 590
Gly Arg Ile Thr Gly Gln Ile Ile Asp Pro Gly Thr Ala Arg Glu Ile
595 600 605
Lys Arg Asn Tyr Val Tyr Gln Tyr Pro Gly Gly Asp Glu Asn Asp Phe
610 615 620
Tip Pro Val Met Ile Glu Val Asp Ser Gln Gly Val Arg Arg Lys 'Ilzr
625 630 635 640
His Tyr Asp Gly Met Gly Arg Ile Cys Ser Ile Glu Glu Gln Asp Asp
645 650 655
Asp Gly Ala Trp Gly Thr Ser Gly Ile Tyr Gln Gly Thr Tyr Arg Lys
660 665 670
Val Leu Ala Arg Gln Tyr Asp Val Liz Gly Gln Leu Ser Lys Glu Ile
675 680 685
Ser Asn Asp Trp Leu Trp Asn Leu Ser Ala Asn Pro Leu Val Arg Leu
690 695 700
Ala Thr Pro Lau Val Thr Thr Lys Thr Tyr Lys Tyr Asp Gly Txp Gly
705 710 715 720
Asn Leu Tyr Ser Thr Glu Tyr Ser Asp Gly Arg Ile Glu Leu Glu Ile
CA 02320801 2000-08-14
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-i9-
725 730 735
His Asp Pro Ile Thr Arg Thr Ile Thr Gln Gly Val Lys Gly Leu Gly
740 745 750
Met Leu Asn Ile Gln Gln Asn Asn Phe Glu Gln Pro Ala Ser Ile Lys
755 760 765
Ala Val err Pro Asp Gly 2'hr Ile Tyr Ser Thr Arg ~'hr err Ang Tyr
770 775 780
Asp Gly Phe Gly Arg Thr Val Thr Glu Thr Asp Ala Glu Gly His Ala
785 790 795 800
'rhr Gln Ile Gly Tyx- Asp Val Phe Asp Arg Ile Val Lys Lys Thr Leu
805 810 815
Pro Asp Gly 'Ihr Ile Leu Glu Ser Ala ~Iyr Ala Ser Phe Ser His Glu
820 825 830
Glu Leu Ile Ser Ala I~ Asn Val Asn Gly Thr Gln Leu Gly Ala Leu
835 840 g45
Val Tyr Asp Gly Leu Gly Arg Val Ile Ser Asp 'Thr Val Gly Gly And
850 855 860
Lys 2hr Glu Tyr Leu 'I~r Gly Pro Gln Gly Asp Lys Pro Ile Gln Ser
865 870 875 880
Ile Ztir Pro Ser His Asn Lys Gln Asn Met Asp Tyr Leu err 'Iyr Leu
885 890 895
Gly Ser Val Met Ser Lys Phe Thr I'hr Gly Thr Asp Gln Gln Asn Phe
900 905 910
Arg Zyr His Ser Lys Thr Gly Thr Leu Leu Ser Ala Ser Glu Gly Val
915 920 925
Sex' Gln Thr Asn err Ser 'iyr Phe Pro Ser Gly Val Leu Gln Arg Glu
930 935 940
Ser Phe Leu Arg Asp Asn Lys Pro Ile Ser Ser Gly Glu Tyr Leu Zyr
945 950 955 960
Thr Met Ser Gly Leu Ile Gln Arg His Lys Asp Ser Phe Gly His Asn
965 970 975
His Val 2yr Ser Zyr Asp Ala Gln Gly Ang Leu Val Lys Thr Glu Gln
980 985 990
Asp Ala Gln Zyr Ala 'ihr Phe Glu Tyr Asp Asn Val Gly Arg Leu Ile
995 1000 1005
'Ihr Thr Thr Thr Lys Asp 'rhr Thr Ser Leu Ser Gln Leu Val 'Ihr Lys
1010 1015 1020
Ile Glu Tyr Asp Ala Phe Asp Arg Glu Ile Lys Arg Ser Leu Ile Ser
025 1030 1035 1040
Asp Phe Ser Ile Gln Val Ile Thr Leu Ser Zyr Thr Lys Asn Asn Gln
1045 1050 1055
Ile Ser Gln Arg Ile Thr Ser Ile Asp Gly Val Val Met Lys Asn Glu
1060 1065 1070
CA 02320801 2000-08-14
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-20-
Arg Tyr Gln Zyr Asp Asa Asn Gln Arg L~u Ser Gln Zyr Gln Cys Glu
1075 1080 1085
Gly Glu Gln Ser Pro Ile Asp His Thr Gly Arg Val Leu Asn Gln Gln
1090 1095 1100
Ile Tyr His Zyr Asp Gln Trp Gly Asn Ile Lys Arg Leu Asp Asn Thr
105 1110 1115 1120
?yr Arg Asp Gly Lys Glu 'Ihr Val Asp 'Iyr His Phe Ser Gin Ala Asp
1125 1130 1135
Pro 'Ilzr Gln Leu Ile Arg Ile Thr Ser Asp Lys Gln Gln Ile Glu Leu
1140 1145 1150
Ser err Asp Ala Asn Gly Asn Leu '1"hr Arg Asp Glu Lys Gly Gln Thr
1155 1160 1165
Leu Ile 'lyr Asp Gln Asn Asn Arg Leu Val Gln Val Lys Asp Axg Leu
1170 1175 1180
Gly Asn Leu Val Cys Ser ~I~rr Gln Tyr Asp Ala Leu Asn Lys Leu Thr
185 1190 1195 1200
Ala Gln Val Leu Ala Asn Gly Thr Val Asn Arg Gln His 2yr Ala Ser
1205 1210 1215
Gly Lys Val Thr Asn Ile Gln Leu Gly Asp Glu Ala Ile Thr Trp Leu
1220 1225 1230
Ser Ser Asp Lys Gln Arg Ile Gly His Gln Ser Ala Lys Asn Gly Gln
1235 1240 1245
Ser Val Tyr Ayr Gln err Gly Ile Asp His Asn Ser Thr Val Ile Ala
1250 1255 1260
Ser G1n Asn Glu Asn Glu Leu hZet Ala Leu Ser Tyr ~"hr pro 'I~rr Gly
265 1270 1275 1280
Phe Arg Ser Leu Ile Ser Ser Leu Pro Gly Leu Asn Gly Ala Gln Val
1285 1290 1295
Asp Pro Val Thr Gly Trp err Phe Leu Gly Asn Gly Tyr Arg Val Phe
1300 1305 1310
Asn Pro Val Leu Nlet Arg Phe His Ser Pro Asp Ser Trp Ser Pro Phe
1315 1320 1325
Gly Arg Gly Gly Ile Asn Pro Zyr Z2zr Tyr Cps Gln Gly Asp Pro Ile
1330 1335 1340
Asn Arg Ile Asp Leu Asn Gly His Leu Ser Ala Gly Gly Ile Leu Gly
345 1350 1355 1360
Ile Val Leu Gly Ala Ile Gly Ile Ile Val Gly Ile Val Ser Leu Gly
1365 1370 1375
Ala Gly Ala Ala Ile Ser Ala Gly Leu Ile Ala Ala Gly Gly Ala Leu
1380 1385 1390
Gly Ala Ile Ala Ser Thr Ser Ala Leu Ala Val Thr Ala Thr Val Ile
1395 1400 1405
Gly Lieu Ala Ala Asp Ser Ile Gly Ile Ala Ser Ala Ala Lieu Ser Glu
1410 1415 1420
CA 02320801 2000-08-14
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-21 -
Lys Asp Pro Lys Thr Ser Gly Ile Leu Asn Trp Ile Ser Ala Gly Leu
425 1430 1435 1440
Gly Val Leu Ser Phe Gly Ile Ser Ala Ile 2hr Phe Thr Ser Ser Leu
1445 1450 1455
Val Lys Ser Ala Arg Ser Gly Ser Gln Ala Val Ser Ala Gly Val Ile
1460 1465 1470
Gly Ser Val Pro Leu Glu Phe Gly Glu Val Ala Ser Arg Ser Ser Arg
1475 1480 1485
Arg Tip Asp Ile Ala Leu Ser Ser Ile Ser Lieu Gly Ala Asn Ala Ala
1490 1495 1500
Ser Leu Ser Thr Gly Ile Ala Ala Ala Ala Val Ala Asp Ser Asn Ala
505 1510 1515 1520
Asn Ala Ala Asn Ile I~ Gly Trp Val Ser Phe Gly Phe Gly Ala Val
1525 1530 1535
Ser Thr Thr Ser Gly Ile Ile Glu Leu Thr Arg Thr Ala Tyr Ala Val
1540 1545 1550
Asn His Gln 2'hr Tip Glu Leu Ser Ser Ser Ala Gly 2hr Ser Glu Glu
1555 1560 1565
Val Lys Pro Ile Arg Cars Leu Val Ser His Arg Trp Asn Gln Lys Gln
1570 1575 1580
<210> 7
<211> 18
<212> LIB
<213> Artificial Sequ~ce
<220>
<223> Description of Artificial Sequ~ce:oligonucleotide
<400> 7
acacagcagg ttcgtcag 18
<210> 8
<211> 18
<212> 1NA
<213> Artificial Sequ~ce
<220>
<223> Description of Artificial Sequence:oligariucleotide
<400> 8
ggcagaagca ctcaactc 18
<210> 9
<211> 20
<212> Lid
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:oligonucleotide
<400> 9
Gly Lieu Ala Ala Asp Ser Ile Gly Ile Ala
CA 02320801 2000-08-14
WO 99/42589 PCT/EP99/01015
-22-
attgatagca cgoggcgacc 20
<210> 10
<211> 22
<212> LIB
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:oligonucleotide
<400> 10
tag 9ag~9~ct gg 22
<210> 11
<211> 37948
<212> L1~.
<213> Photorhabdus lscens
<220>
<221> C~S
<222> (15171)..(18035)
<223> orf5
<220>
<221> C~5
<222> (23768)..(31336)
<223> hph2
<220>
<221> C~6
<222> (31393)..(35838)
<223> orf2
<400> 11
tgttgctgga ccgtggagat tatgcctatc gtcagttaga ac.~gagacacg ctcaatgaag 60
ccaagatgtg gtatatgcaa gcactgcatc tgttaggcga taaacctcat ctatcgttca 120
gttcagagtg gagcaaaccg agtttaggcg acgctgccgg ~~aga ~agcaac 180
acgcccaagc aatggccgct ctgcgacaag g~~tag tcgg~caac aaaxgacag 240
atcttttctt gccacaggtc aatgaagtga tgcaaaacta tt~gcaaaaa ttggaa~caac 300
ggctgtataa cctgcgtcat aacctcacta ttgacggcca accgctacat ctgcctattt 360
acgctacacc ggcagatcca aaagcattac ttagcgccgc tgtcgctagc tccgaaggtg 420
gggtagctct ctcacagcca tttatgtcac tgtggcgttt cccacacatg ctggaaaacg 480
cgcgtggtat ggtcagtcag ctcactcaat tcggctctac gctacaaaat attatcgaac 540
gtcaggatgc ggaagcttta aacacgctct tgcagaatca agcagcagaa ctgatattga 600
ctcatctcag catacaggac aaaaccatcg cagagctgga tgcggaaaaa atcgtactgg 660
aaaaatccaa agccggggcg caatcacgct ttgacagcta caaaaagtta tacgacgaaa 720
atatcaatgc gggtgaaaac cgggctatag cattgcatgc ctccgttgct ggcctcagca 780
ctgccctgca agcatcacgt ctggcgggcg ctgcgcttga tctggcgccc aacattttcg 840
gtctcgctga tggcggtagc cgttggggag cgattgccga agcgacaggt aatgttatgg 900
CA 02320801 2000-08-14
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aattctccgc cagtgtgatg aacaccgaag cggataaaat cagccagtca gaagcctatc 960
gccgtcgccg tcaggaatgg gaaatccagc gtaatcatgc cgaagcagag ataaaacaga 1020
tcgatgctca acttcaatca ctggcagtac gccgtgaagc cgcggtattg cagaaagcca 1080
gcctaaaaac ccaacaggaa cagactcatg ctcaattgac tttcctgcaa cgtaaattca 1140
gtaatcaagc gttgtactac tggctacgcg gtcggctagc tgctatttac ttccaatttt 1200
acgatttggc cgtagcgcgt tgtctgatgg ctgaaatggc ttatcgttgg gagactaatg 1260
agaccgcggc aagctttatc aaacccggcg cctggcaggg aacccatgcg ggtttactgg 1320
ctggtgdaac cttgatgctg aatctggcgc aaatggaaga tgcccatttg aggtgggatc 2380
aacgcgctct ggaagtggaa cggaccattt cattgacgca acactatgga gcactgccag 1440
aaaaatcgtt taatttagcc acacggattt ctaccctgct agcaggtggt acaactgact 1500
ccattgatga tcatcccgtt acattagaaa acgaccaact tagtgccaaa atctctctgt 1560
caggtctgtc attagataat gactacccag atggcaacgg cgtaggcaac attcgacgca 1620
ttaaacaaat cagtgtcacc ttgccagccc tgttaggacc atatcaggat gtacaagcta 1680
ttctgtccta cggaggaagt gaaatcggat tagctgaaag ctgtaaatca ctggcgatct 1740
ctcatgggat caatgacagt ggtcaattcc agttggattt taacaatggt aagttcctgc 1800
cgtttgaagg gattgogatt aacgatactg gcacattgac actcagtttc ccccaatgcg 1860
actgtcaaac aagaaaacat gttgcagact ttgagtgata ttattctgca tattcgctat 1920
accatccgcc aataaccacc tcaattaaat accaaaaaca ggctcctaaa cggggcctga 1980
acttttcacg aatatatacc actcacagtc tgctctcttt acctgtctga cgctcgttat 2040
aacagagata tttccttttc tcgtgagtcc catcacctac tataaaatat caaccctctt 2100
ctttttcata atatgcaata tgtaacaaat gcaattattt catttagtta ttgttaacta 2160
gttatattac ttatgatgta attataaatt ttgttattgc atcacaatag ccatttaaat 2220
aaataataac gttgtgaaat agttgatagt taaatggtgt ttttatttag ccgttatttt 2280
caacccaatt tcagaccgct atcagacgtt acctgtgttg cctttgtttt gatagatata 2340
aataacctta tttatatcca cggtactcag accagcataa atgttttatt tacctaacat 2400
ttaaaaggaa taaacatgaa cacactcaaa tccgaatatg aaaacgcgtt agtagcaggt 2460
tttaataatc taaccgatat ttgtcatctc tcttttgacg aacttcgcaa aaaagtgaag 2520
gacaaactct catggtcaca gacccaaagc ttatatcttg aagcacagca agtgcaaaag 2580
gacaatctcc tgcatgaagc ccatattctg aaacgcgcca atcctcattt acaaagtgcg 2640
gtccatcttg ccctgacaac acctcatgct gaccagcaag gttataatag cagatttggc 2700
aatcgcgcca gcaaatatac agccccaggc gcaatttctt ccatgttttc tcctgcggct 2760
tatttagctg aactttatcg tcaggcacgg aatttacatg atgaaaattc tatttatcat 2820
CA 02320801 2000-08-14
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ttggatacac ggcgtccgga tctaaaatca ttggtgctca gccagaaaaa tatggatacg 2880
gagatttcca cactttctct gtctaatgac atgttgctag agggtattaa aactctgttc 2940
aaggacaagc tgctggaggc tctgaagaat attaaatctc tgtccaagga cgagctgctg 3000
gaggctttga agaatattaa acctctgtcc aaggacgatc tgctggaggc tttgaagaat 3060
attaaacctc tgtccaagga cgatctgctg gaggctttga agaatattaa acctctgtcc 3120
aaggacgatc tgctggaggc tttgaagaat attaaacctc tgtccaagga cgatctgctg 3180
gaggctttga agaatattaa acctctgtcc aaggacgatc tgctggaggc tttgaagaat 3240
attaaacctc tgtccaagga cgatctgctg gaggctttga agaatattaa acctctgtcc 3300
aaggacgatc tacaggaatg tattgaaatt ctattcaatc tggacagcca cactaaagta 3360
atgaaagcgt tatccaattt ccgcgtttct ggcatgatgc catatcacga tgcttatgaa 3420
agcgtgcgta aggttgttca attacaggct ccggtgtttg aacacgttgt tagtacatca 3480
ctagaaacga ctatcgatga actaaaatat caagcttctt tgttggaaat taattcttct 3540
gtctcgccta aattatttac tatcttgact gaagaaatta ctacaatcaa tgcaagaagt 3600
ctctatgagg aaaattttgg taatattaaa ccttctctaa taggaaaacc c~aatatctg 3660
aaaagttatt acaatctgag tgatgaagag tttagcgatt tcattaaaat aagaactata 3720
cttcttccag aagaagaaat agcaattact gatcttgcat cgcgtactac tagtacacaa 3780
cagactatcg aaaatcctga ttatcgtgct ctattgaaaa ttaataagtt tattcgtcta 3840
ttcaaagcta taaacttatc accgacggta ttaagtggaa tcctccgcag catcagcaca 3900
gaattcaata tcaataaaga aatattacaa aaaatctttc gtgttaaata ctatatgcaa 3960
cgttatggta ttgacactga gactgcatta atactatgca aggtaccaat ttcacaatat 4020
atcaatgacg gacatctaag tcagtttgat cgtttattta attcccccaa actgaatggt 4080
caagattttt ccgtcaatgg tactcagaat attgatttaa ccctaagcag taccaacaac 4140
tggaataaaa cagtacttaa acgtgctttt aacctcgatg atatctcatt aaatcgacta 4200
ctaaaaatta ccaatccggt caatactacc gaaat~taa ctaa
tgatat agagaatctt 4260
tctcatctct ataggacaaa attactggca gatatccatc agttaactat tgatgaactg 4320
gggttactgt tggaagccat aggtaaagga acaaccaatt tatctgagat tactcctgac 4380
aatctggtta ctctaattaa caaactctat gctgtcacta gctggctacg tacacaaaag 4440
tggagtgtct atcagttgtt tatgatgact actgataaat ataacaaaac cctaaccccg 4500
gaaatacgga atttactgga taccgtctac aatggcttgc aagattttaa taagaagatg 4560
ttgaaagctg aagaagatct agagaaaacc aaaaagaaat tgcagagcgc caaggaaaat 4620
ctggaaaaat tcccggaaaa ccagccacaa ctccaagaag acaggaaaaa agcccagaga 4680
agactgaata aagctgaaga gacccacgaa aaagccgaga aaaacctaga tgaggtcagg 4740
aaaaatctgc caaaagccat atctccttat atcgccgccg ctctgcaatt accatctgaa 4800
CA 02320801 2000-08-14
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-25-
catgcggcat attccatact catctgggca gataatctgg aacccggcat aggaaaaatg 4860
acagcggaaa aattatggaa ctggttgcgg aaaaatcccg ttacggctca acctgaattc 4920
caaaaacaag ctgaacctgt ggtccagtat tgccagcgcc tggcacaact agcgttgatt 4980
taccgttcta ccggccttaa cgaaaacacc ttaagtctgt ttgtgacaaa gccgcaacac 5040
tttgttatta aaaccaaagc acccgaaaca actgaaacaa caccagcaca tgacgtatca 5100
acactaatgt cactaacgcg ttttactgac tgggttaact cactaggtga aaacgcctct 5160
tctgtactaa ccgaatttaa aaaaggaaca ttaacagcag aactattggc taaggctatg 5220
aatcttgata aaaatctact ggagcaagcc aagattcagg caaaaactga tttgtccaac 5280
tggccatcta tcgacaacct attgcagtgg attaacatct cacgtcaatt gaacatctct 5340
ccacaaggca tttccacact gactcaagta ttgaccgcag aacctcccgc taactatacc 5400
caatgggaaa acgccgctgc gatattaacc gccgggctgg acacccaaaa gactaaogcc 5460
ctacatgcgt ttctggatga gtctcgcagt gctgcgttaa gcacatacta tatttattct 5520
cataaccaaa aagatcgaga agcaagaaaa catacagtaa ttaaaaacog tgatgatcta 5580
tatcaatacc tattgatcga taaccaagtt tccgccgaca ttaaaactac agagatcgct 5640
gaagctatcg ctagtatcca actgtatatt aaccgcgcgt tgaaaaatat acacaaaaat 5700
actgtcacaa gtgtcaccag ccgctcattc ttcaccaact gggataaata caataaacgc 5760
tacag~cactt gggccggtat ggctaaactc ctttactatc cagagaatta catcgatccg 5820
acgctacgta ttgggcagac aaaaatgatg gatacgttgc tgcaatccat cagccaaagc 5880
caattaaata tcgataccgt agaagatgcc tttaaatctt acctaacatc attcgaacag 5940
gtggctaatc tggaaatcct cagcgcctac catgacaaca ttaataatga tcaaggatta 6000
acttacttta tcggacgtag taaaacagaa gtgaatcaat attattggcg cagtgtggat 6060
cacaataaat ccagcgaagg taaattcccc gctaatgcct ggagtgagtg gtacaaaatt 6120
gattgtccaa ttaaccccta caaagatact attcacccgg taattttcca atctcgcctg 6180
tatcttatct ggctggaaca aaaaaaggcg actaaacagg aaggtgataa aaccgcctcg 6240
ggttattatt atgaactgaa attagcgcat atccgttatg acggcacctg gaatacacca 6300
gtcacctttg atgtaaacca aaaaatatcc gatttaaatc tgggaaataa aacacctgga 6360
ctttactgct caagctttca aggcagagat gaattgctgg tgatgtttta taaaaaacaa 6420
gatcaattaa atcaatacac aaacacagta ecaataaaag gactatatat cacttccaat 6480
atgtcttcta aggaaatgac acctgaaaat cacaaaccta acgcttataa acagtttgat 6540
actaatagta ttattggtgt caataatcgc tatgcagaaa gctacgaaat cccttcatca 6600
gtaaatagta ataacggtta tgattgggga gatggctatc tgagtatggt gtatggcgga 6660
aatatttcag ccatcaaact ggagtcctca tcagataagt taaaactctc accaaggtta 6720
CA 02320801 2000-08-14
WO 99/42589 ~ PCT/EP99/01015
- 26 -
agaattattc ataatggact tgtaggccga caacgcaacc aatgcaacct gatgaagaaa 6780
tacggtcagc ttggtgataa atttattatt tatactactc taggtattaa ccccaataat 6840
ttgtcgaata aaaaattcat ttaccctgtt tatcagtata gtgggaacac taccaataat 6900
gagaaaggac gtctgctgtt ttatcgagaa agtactacta actttgtaag agcctggttc 6960
cctaaccttc cctctggctc tcaagaaatg tccacaacca ctggcggtga catragtggt 7020
aactatggtt atattgataa caaacatagt gacgatgttc catttaaaca atatttctat 7080
atggatgacc acggtggtat tgacactgat gtttcaggga tattatctat taatacgaac 7140
attaatcatt caaaagttaa agtaatagtg aaagccgaag gtatcacaga gcaaactttt 7200
gtagcgagcg aaaacagtaa tgtccccacc aatccgtccc gcttcgaaga aatgaattat 7260
cagtttaaag agcttgaaat agatatctcc acactgacat ttcataataa tgaagcaagt 7320
attgatatca cctttatcgc atttgctgag aaatttgacg ataatagtaa tgatcgtaac 7380
ttaggcgaag aacatttcag tattcgtatt atcaaaaaag cggaaactga taatgccctg 7440
accctgcacc ataatgcaaa cqgggcgcaa tatatgcagt ggggaaactc ttgtattcgc 7500
cttaatacgc tatttgcccg tcaattaatt agccgagcca acgcaaggat agatactatt 7560
ttgagcatgg acactcagaa tattcaggaa cctaaattag gagaagattc tcctgatgct 7620
atggaaccaa tggacttcaa cgg~CCaac agcctctatt tctgggaact gttctactac 7680
accccgatgc tgattgctca acgtttgctg cacgaacaaa acttcgatga ggctaaccgt 7740
tggctgaaat atgtctggaa cccatccggt tatattgtca atggtcaaat gcaacattac 7800
cgctggaatg ttcgcccatt acaagaagac actagttgga acgatgatcc gttggattca 7860
tttgatcctg ataccatagc tcaacatgat ccaatgcact acaaagtcgc cacctttatg 7920
cgcaccctag atctgttgat cgaacgggga gattacgcct atcgccaatt ggagcgggac 7980
acactcgctg aagccaaaat gtggtatatg caggcactgc atctattggg tgataaacct 8040
catctaccac tcagttcagc atggaatgat ccagagctag aagaggccgc agctcttgaa 8100
aaacaacagg cacatgccaa agaaatagca gatttacgac aaggacttcc tacatccaca 8160
gggtctaaag atgaaatcaa aacagatctt ttcctgccgc aagtcaacga agtgatgctg 8220
agctactggc agaaactaga acaacggttg tataacctgc gccataacct ctctattgat 8280
ggtcaacctt tacatttgcc tattttcgca acaccagcag atccaaaagc gctgctcagc 8340
gccgctgtcg ccagttcaca aggtggaagt aatcttccat cagaatttat atcagtgtgg 8400
cgtttccctc atatgctgga aaacgcccgt agtatggtca gtcagctaac ccaattcggc 8460
tccacattgc aaaatattat cgaacgtcaa gatgcggagg cattaaacac gctgttgcaa 8520
aatcaggcgg cagaactgat attgaccaat ctcagcatac aggacaaaac catccaagag 8580
ctggatgctg aaaaaactgt gctagaaaaa aaccgcgccg gaacccagtc gcgttttgat 8640
agctacagca aattctacga tgaagacatc aacgcgggtg aaaaacaggc aatggcgttg 8700
CA 02320801 2000-08-14
WO 99/42589 PCT/EP99/01015
-27-
cgtgcttccg tcgctggcat ctctacagcc cttcaagcat cacatctggc gggcgcagca 8760
cttgatctgg cgcccaacat cttcggcttc gctgatggtg gcagccgttg gggggcaatc 8820
gcccaagcca caggtaatgt catggagttc tccgccagtg ttatgaacac cgaagcggat 8880
aaaatcagcc aatctgaagc ctaccgtcgg cgtcgtcagg aatgggaaat tcagcgtaat 8940
aacgccgagg cagagctgaa acaaatcgat gctcaacttg gttcgctggc agtgcgccgt 9000
gaagccgcag tattgcagaa aaccagccta aaaacccaac aagagcagac tcatgcacaa 9060
ctgaccttcc tgcaacataa gttcagtaat caggcgctgt acaactggct gcgtggtcga 9120
ttgtccgcca tttacttcca gttctatgat ttaacggtag ctcgctgttt gatggcggaa 9180
atggcctatc gctgggagac taacgatacc gcatcacgct ttatcaaacc cggcgcctgg 9240
cagggaaccc atgccggttt gctcgcgggt gaaaccttaa tgctgaatct ggcacagatg 9300
gaagatgccc acctgaaaca ggataaacgc gtactggagg tagaacgtac cgtttcgctg 9360
gccgaagtct atgccaaatt accgcaagat aaatttatcc tgactcagga aatagagaag 9420
ttggtgagta aaggttcagg cagggccggc aaggacaata ataagctggc gtttagtacc 9480
aataccaata cctctctaga agcgtccatt tcgttatcta ccttgaacat tagcagcgat 9540
tatcctgatt ctattggtaa aacccgtcgt attaaacaga tcagcgttac cctgccagca 9600
ctgctaggac cctatcagga tgtgcaagca attctgtctt acagcggaaa agcctctgaa 9660
ttggctgaaa gttgcaaatc attagcggtt tctcatggga tgaatgacag cggtcagttc 9720
caactggatt tcaacgatgg caaattcctg ccgttcgaag gaatcaaaat cgatgaaggt 9780
acgctgacat tgagcttccc aaatgcaatt agtaaagaag acaaaaaaga cgaaaaaggc 9840
aaacaacaag ccatgctgga gagtctgaac gacatcattc tgcatattcg ctacaccatt 9900
cgccaataac gattttaatt aagtgctaaa acaggcccct aagcggggcc tgcaaggagt 9960
ctttcatgca aaattcacaa gatttcagta ttacagaact atcattgccc aaaggaggag 10020
gcgctatcac ~atgggg gaagctttaa cccccaccgg gccggatggg atggcc~cgc 10080
tgtctctgcc gttgcctatc tctgccgggc gcggttatgc tccgtcactc gccttaaact 10140
acaacagcgg cgccggtaac agcccatttg gtctgggctg ggattgcaac gttatgacca 10200
tccgccgccg cacccatttt ggcgttccac attatgatga aaccgatacc tttctggggc 10260
cagatggcga ggtactggtg gtagc~ggatc aatcccgcga cgaatcgaca ttacagggta 10320
tcaacttagg caccgccttt accgttaccg gataccgttc ccgtctggag agtcatttca 10380
gccgattgga atattggcaa cccaaggcaa cacccaagac aactggcaaa acagattttt 10440
ggctgatata tagcccagat ggacaagtac atttactggg taaatcacca caagcccgga 10500
tcagcaaccc gtcagacatc actcaaacag cacaatggtt gctagaagcc tctgtgtcac 10560
cacatggtga acaaatttat tatcaatatc gggccgagga taacaccggt tgcgaagctg 10620
CA 02320801 2000-08-14
WO 99/42589 PCT/EP99/01015
-28-
atgaaattac tctccatcca caggccgCaacgtta tctacacaca gtgtattacg 10680
gcaaccggac agccagcaaa acgttacccg gtctggatgg cac,~cgcccca ccacaagcag 10740
actggttatt ctatctggta tttgattacg gcgaacgcag taacaacctg agaacgccgc 10800
cagcattttc gactacaggt agctggcttt gtcgccagga ccgtttttcc cgttatgaat 10860
atggttttga gattcgtacc cgccgcttat gccgtcaggt attgatgtat caccacctgc 10920
aagctctgga tagcgagata aaagaacaca acggaccaac gctg~gtttca cgcctgatac 10980
tcaattatga cgaaagcgca atcgccagca cgctggtatt cgttcgtcga gtaggccacg 11040
agcaagacgg tactgccgtc accctgccgc cattagaatt ggcgtatcag gatttttcac 11100
cgcaacataa cgctcgctgg caatcgatga atgtgctggc aaacttcaat gccattcagc 11160
gctggcaact agttgatcta aaaggcgaag gattccccgg tctgctatat caagataaag 11220
gcgcct~ gta~ctcc gcac:aacgtt ttggcaaaat tggctcagat gccgtcactt 11280
gqgaaaaaat gcaacctttg tcggttatcc cttccttgca aagtaatgcc tcgctggtgg 11340
atatcaatgg agacggCCaa cttgactggg ttatcaccgg ac~ggatta cggggatatc 11400
atagtcagca tccagatggc agttggacac gttttacccc gctcaacgct ctgccagtgg 11460
aatatactca tccacgcgcg caactcgccg atttaatggg agctggactt tctgatttag 11520
tactgatcgg ccctaagagt gtacgtttat atgccaatac ccgcgacggc tttgccaaag 11580
gaaaagatgt agtgcaatcc ggtgatatca cactgccagt accgggcgcc gatccgtgta 11640
agttggtggc atttagtgat gtattgggtt ccggtc:aggc acatctggtt gaagtgagcg 11700
cgactaaagt cacctgctgg cctaatctgg ggcacggacg ttttggtcaa ccaattactc 11760
ttccgggatt tagccaacca gaagcgacgt ttaatcctgc tcaagtttat ctggccgatc 11820
tagatggcag cggcccgact gatctgattt atgttcacac agatcgtctg gatatcttcc 11880
tgaataaaag cggCaacggc ttcgccgcac cagtaactct ccccttccca gccggagtgc 11940
gttttgatca tacctgtcag ttacaagtgg ccgatgtaca agggttaggc gtc~gccagcc 12000
tgatattaag tgtgccgcat atgactcccc atcactggcg ttgcgatctg accaacacaa 12060
aaccgtggtt actcagtgaa atgaacaaca atatgggggc tcatcacacc ctgcgttacc 12120
gtagttccgc ccagttctgg ctggatgaaa aagccacggc actggatgcc ggacaaatac 12180
cagtttgtta tctacccttc ccggtacaca ccctatggca aacggaaata gaggatgaaa 12240
tcagcggcaa caaattagtc acaatactac gttatgcaca tggcgcctgg gatggacgtg 12300
agcgagaatt tcgcggattt ggttatgttg aacagaaaga cagccatcaa ctggcccaag 12360
gcagtgcgcc agaatgcaca ccacctgcac tgacccaagg caacgcgcct gaactcacat 12420
cacccgcgct gacccaaggc aacgctccag aactcacacc acctgcgatg acccaaagca 12480
acgcgcctga actcacatca cccgcgctga cccaaggcaa cgcgccagaa ttcacatcac 12540
ccgcgctggc ccaaggcaat gcgccagaac tcacaccacc tgcgatgacc aaaaactggt 12600
CA 02320801 2000-08-14
WO 99/42589 PCT/EP99/01015
_29_
atgccaccgg aatacccatg atagataaca cattatcgac agagtattgg catggtgatc 12660
accaagcttt tgccggtttt tcaccacgct ttacgacctg gcaagatggt caagatattc 12720
tgctcacacc ggaaaatgat aacagtcagt actggctaaa ccgggcactg aaaggtcaac 12780
tgctacgcag tgaactgtac ggcgaggatg gcagtacaca ggaaaaaatt ccctacacag 12840
tcactgaatt tcgcccacag gtacgtcggt tacagcatac cgatagccga taccttgtgc 12900
tttggtcatc tgtagttgaa agccgcaact atcattacga acgtatcgcc agcgatcctc 12960
aatgcagcca aaagattacg ctatccagcg atctatttgg tcaaccgcta aaacaggttt 13020
cggtacagta tccacgccgc cagcaaccgg caagcagtcc gtatcctgat acgttgcctg 13080
ataagttatt tgctaacagc tatgatgacc agcaacacaa attacggctc acctatcaac 13140
agttcagttg gcatcatctg accgacaata ccattctgat gttaggatta ccggatagta 13200
cccgcagcga tatctttgct tatagcgctg aacatgtccc tactggtggt ctaaatctgg 13260
aaatcctaaa tgataaaaat agtctgattg cggagaataa acctcgtgaa tacctcggcc 13320
agcaaaaaac cgtttatacc gacgggcaaa atgcaacgcc atcgcaaacg ccaacacgac 13380
aagcgctgat tgccttcacc gagacaacag tatttaatca atccacacta tcagcgtttg 13440
atgggagtat ctcatctgct caattgtcaa cgacgctgga acaagccgga taccagcaaa 13500
cagattatct attcccgcgc actggagaag ataaagtctg ggcagctcgt cgtggctata 13560
ctgattacgg cacagccgaa cagttctggc ggccgcaaaa acagacgCaac actcaactca 13620
cgggcaaaat cacgctcact tgggatgcaa actattgcgt cgtcacacaa acrcgggatg 13680
cggctggact gacaacctca gccagatatg attggcgttt tctgaccccc gttcaactca 13740
cggatatcaa cgacaatcag caccttacca cgctggatgc actgggccga ccaatcacac 13800
tgcgcttttg gggaaccgaa aacggtaaga tgactggtta ttcttcaccg gaaaaaatat 13860
cgttttctcc aayatctgat gttgacgccg cgattaagtt aacaacgcca atccctgtag 13920
cacagtgtca ggtctacgca cccgaaagct g~tgcccat attaaagaaa accctcaata 13980
acctggcaga gcaagagcgg aaagagttat ataacacccg aatcatcacc gaagacggac 14040
gcatctgtac cctagctcac egccgctggg taaaaagcca aagtgcagtc acccagccaa 14100
tcaatctgtc aaacggcagt ccccgtttac cccctcatag cctcacattg actacggatc 14160
gttatgaccg cgatcttaag caacagattc gtcaacaagt agtattcagt gatggctttg 14220
gccgtttact gcaagcatct gtacgacatg aagcaggcga agcctggcaa cgtaaccaag 14280
acggcgctct ggtgacaaaa atggaagata ccaaaacgcg ctgggcggtt ac~cgca 14340
ctgaatatga caataaggga caaccgatac gcacctatca accctatttc ctcaacgact 14400
ggcaatacgt cagtaatgac agtgcccggc ggacagaaga agcctatgca gatacccatg 14460
tctatgatcc cattggtcga gaaatcaagg tcactaccgc aaaaggctgg ttccgtcgaa 14520
CA 02320801 2000-08-14
WO 99/42589 PCT/EP99/01015
-30-
ccttgttcac tccttggttt actgtcaatg aagatgaaaa tgacacagct actgaggtga 14580
aggtaaagaa gaaagaatgt aaagaaggta aagaaggtaa agatgtaatt tgatcaatcc 14640
cgcccggttg aagggcggga aacataacat aatatagagg tgaaacgtgt cattcataat 14700
gccgtcagat actcaactta tgagttggtt gatcattggt tttattgcgg cctggggcgg 14760
attagtaagg tacctcattg atatacaaaa caaacaatgt aaatggaatt ggatcaacgt 14820
actctgtcaa ctcattatct cctgttttac cggtatattg actgc tgagttttga 14880
aagcggcggc agcccctata tgacttttgc gattgccggg ctatttggca ccacgggaag 14940
ttctggattg aactggatct ggcgtcgcct ttttatgcat tatcgcgatg atggaggaaa 15000
gcaataaggc attccractg ccgcaaaaac catctgtctc cggcagttaa accgggaaat 15060
tacctactac aactattgta agaaaacgaa tatatagaaa aactaacatg cagataaaaa 15120
ctgcgattgc agaacagatg acacacaacg ccccaacaac gaggtaaatc atg aaa 15176
Met Lys
1
aac atc gat cct aaa ctt tat caa aag acc cct gtc gtc aac atc tac 15224
Asn Ile Asp Pro Lys Leu Tyr Gln Lys Thr Pro Val Val Asn Ile Tyr
10 15
gat aac cga ggt cta acg atc cgt aac atc gac ttt cac cgt acc acc 15272
Asp Asn Arg Gly Leu Thr Ile Arg Asn Ile Asp Phe His A~ ~'hr Thr
20 25 30
gca aac ggc gat acc gat atc cgt att act cgc cat caa tat gac tcc 15320
Ala Asn Gly Asp Thr Asp Ile Arg Ile Thr Arg His Gln err Asp Ser
35 40 45 50
ctt ggg cac cta agc caa agc acc gat ccg cgt cta tat gaa gcc aaa 15368
Leu Gly His Leu Ser Gln Ser Ztir Asp Pro Arg Leu Zyr Glu Ala Lys
55 60 65
caa aaa tct aac ttt ctc tgg cag tat gat ttg acc ggt aat att ttg 15416
Gln Lys Ser Asn Phe Leu Trp Gln Tyr Asp Leu Thr Gly Asn Ile Leu
70 75 80
tgt aca gaa agc gtc gat get ggt cgc act gtc acc ttg aat gat att 15464
Cys Thr Glu Ser Val Asp Ala Gly Arg Thr Val Thr Leu Asn Asp Ile
85 90 95
gaa ggc cgt ccg cta ctg aca gta act gca aca ggt gtc ata caa acc 15512
Glu Gly Arg Pro Leu Leu Thr Val Thr Ala Thr Gly Val Ile Gln Thr
100 105 110
cga caa tat gaa acg tct tcc cta ccc ggt cgt ctg ttg tct gtt acc 15560
And Gln Z]rr Glu 'lfir Ser Ser Leu Pro Gly An3 Leu Leu Ser Val mhr
115 120 125 130
gaa caa ata cca gaa aaa aca tcc cgt atc acc gaa cgc ctg att tgg 15608
Glu Gln Ile Pro Glu Lys 'i'hr Ser Arg Ile Thr Glu Arg Leu Ile Tzp
135 140 145
get ggc aat agc gaa gca gag aaa aac cat aat ctt gcc agc cag tgc 15656
Ala Gly Asn Ser Glu Ala Glu Lys Asn His Asn Leu Ala Ser Gln Cys
150 155 160
gtg cgc cac tat gac acg gcg gga gtc acc cga tta gag agt ttg tca 15704
CA 02320801 2000-08-14
WO 99/42589 PCT/EP99/01015
-31 -
Val Arg His Zyz~ Asp 'Ihr Ala Gly Val 'Ihr
Arg Leu Glu Ser Leu Ser
165 170 175
ctg acc ggt act gtt tta tct caa tcc agc caa 15752
cta ttg agc gac act
Leu 'iizr Gly Thr Val Leu Sex Gln Ser Ser
Gln Leu Leu Ser Asp Thr
180 185 190
caa gaa get agc tgg aca ggt gat aat gaa acc 15800
gtc tgg caa aac atg
Gln Glu Ala Ser Trp I'hr Gly Asp Asn Glu Thr
Val Trp Gln Asn Met
195 200 205 210
ctg get gat gac atc tac aca acc ctg agc gcc 15848
ttt gat gcc acc ggc
Leu Ala Asp Asp Ile Tyr 'rhr Zhr Leu Ser Ala
Phe Asp Ala Thr Gly
215 220 225
get tta ctc act cag acc gat gcg aaa ggg aac 15896
att cag agg cta acc
Ala Leu Leu 'Phr Gln Thr Asp Ala Lys Gly Asn
Ile Gln Arg Leu Thr
230 235 240
tat gat gtg gcc ggg cag cta aac ggg agc tgg 15944
tta acc tta aaa gac
'Iyr Asp Val Ala Gly Gln Leu Asn Gly Ser Trp
Leu Thr Leu Lys Asp
245 250 255
caa ccg gaa caa gtg att atc aga tcc ctg acc 15992
tat tcc gcc gcc gga
Gln Pro Glu Gln Val Ile Ile Arg Ser Leu 'I2~r
Zyr Ser Ala Ala Gly
260 265 270
caa aaa tta cgc gag gaa cac ggc aat ggt gtt 16040
atc acc gaa tac agt
Gln Lys Leu Arg Glu Glu His Gly Asn Gly Val
Ile Thr Glu Tyr Ser
275 280 285 290
tat gaa ccg gaa acc caa cag ctt atc ggt acc 16088
aaa acc cac cgt ccg
'I~rr Glu Pro Glu 'I~x~ Gln Gln Leu Ile Gly
Thr Lys Thr His Arg Pro
295 300 305
tca gat gcc aaa gtg ttg caa gat cta cgt tat 16136
gag tat gac ccg gta
Ser Asp Ala Lys Val Leu Gln Asp Leu Arg Tyr
Glu 'Iyr Asp Pro Val
310 315 320
ggc aat gtc atc agt atc cgt aat gac gca gaa 16184
gcc acc cgc ttc tgg
Gly Asn Val Ile Ser Ile Arg Asn Asp Ala Glu
Ala Thr Arg Phe Txp
325 330 335
cac aat cag aaa gtg gcg ccg gaa aac act tat 16232
acc tac gac tcc ttg
His Asn Gln Lys Val Ala Pro Glu Asn Thr ~Iyr
Thr ~Iyr Asp Ser Leu
340 345 350
tat cag ctt atc agc gca acc ggg cgc gag atg 16280
gcg aat ata ggt cag
Zyr Gln Leu Ile Ser Ala Thr Gly Arg Glu Met
Ala Asn Ile Gly Gln
355 360 365 370
caa agt aac caa ctt ccc tcc ctc acc cta cct 16328
tct gat aac aac acc
Gln Ser Asn Gln Leu Pro Ser Leu Thr Leu Pro
Ser Asp Asn Asn Thr
375 380 385
tac acc aac tat acc cgt act tat act tat gac 16376
cgt ggc ggc aat ttg
Z'Yz' '~ ~ '~' 'I'hr Arg 'rhr 'Iyr 'IZzr 'I~'r
Asp Arg Gly Gly Asn Leu
390 395 400
act aaa atc cag cac agt tca ccg gcg acg caa 16424
aac aac tac acc aca
Thr Lys Ile Gln His Ser Ser Pro Ala Thr Gln
Asn Asn 'Iyr I'hr Thr
405 410 415
aac atc acg gtt tct aac cgg agc aat cgc gca 16472
gta ctc agc act ctg
Asn Ile Thr Val Ser Asn Ar~g Ser Asn Arg Ala
Val Leu Ser 'rhr Leu
CA 02320801 2000-08-14
WO 99/42589 PCT/EP99/OlOi 5
-32-
420 425 430
acc gaa gat ccg gcg caa gta gat get tta ttt 16520
gat gca ggc gga cat
Thr Glu Asp Pro Ala Gln Val Asp Ala Leu Phe
Asp Ala Gly Gly His
435 440 445 450
cag aac acg ttg ata tca gga caa aac ctg aac 16568
tgg aat aca cgc ggt
Gln Asn Thr Leu Ile Ser Gly Gln Asn Leu Asn
Trp Asn Thr Arg Gly
455 460 465
gaa cta caa cat gtg aca ttg gtg aaa cgg gac 16616
aag ggc gcc aat gat
Glu Leu Gln His Val ~lzr Leu Val Lys Arg Asp
Lys Gly Ala Asn Asp
470 475 480
gat cgg gaa tgg tat cgc tat agt agt gac ggg 16664
aga agg ata tta aaa
Asp Arg Glu Trp Tyr Arg Tyr Ser Ser Asp Gly
Arg Arg Ile Leu Lys
485 490 495
atc aat gaa cag cag acc agc agc aac tct caa 16712
aca cag aga ata act
Ile Asn Glu Gln Gln Thr Ser Ser Asn Ser Gln
Thr Gln Arg Ile Thr
500 505 510
tat ttg ccg agc tta gaa ctt cgt cta aca caa 16760
aac agc acg atc aca
Zyr Leu Pro Ser Leu Glu Leu Arg Leu Zlir Gln
Asn Ser 'Ihr Ile Thr
515 520 525 530
acc gaa gat ttg caa gtt atc aca gta gga gaa 16808
gcg ggt cgg gca cag
'Ihr Glu Asp Leu Gln Val Ile Zhr Val Gly Glu
Ala Gly Arg Ala Gln
535 540 545
gta cga gta tta cat tgg gat agc ggt caa ccg 16856
gaa gat atc gac aat
Val Arg Val Leu His Tzp Asp Ser Gly Gln Pro
Glu Asp Ile Asp Asn
550 555 560
aat cag cta cgt tat agc tac gat aat ctt atc 16904
ggt tcc agt caa ctt
Asn Gln Leu Arg 2yr Ser 'I~rr Asp Asn 1xu
Ile Gly Ser Ser Gln Leu
565 570 575
gaa tta gac agc aaa gga gaa att att agt gag 16952
gaa gag tac tat ccc
Glu Txu Asp Ser Lys Gly Glu Ile Ile Ser Glu
Glu Glu Zyr 2yr Pro
580 585 590
tat ggc ggc acg gca tta tgg gca aca agg aag 17000
cgg aca gaa gcc agt
2yr Gly Gly Thr Ala Leu Trp Ala 'IYZr Arg
Lys Arg Thr Glu Ala Ser
595 600 605 610
tat aaa acc atc cgt tat tca ggt aaa gag cgg 17048
gat gcc acc gga cta
err Lys 'Ihr Ile Arg Tyr Ser Gly Lys Glu Arg
Asp Ala 22~r Gly Leu
615 620 625
tat tat tac ggt tac cga tat tat cag cct tgg 17096
gta gga cga tgg tta
2yr 'Iyr Tyr Glyyr Arg Zyr 'Iyr Gln Pro Trp
Val Gly Arg Trp Leu
630 635 640
agt gcc gat ccg gca gga aca gta gat ggg ttg 17144
aat tta tat cgg atg
Ser Ala Asp Pro Ala Gly Thr Val Asp Gly Liz
Asn I~ ~Iyr Arg Met
645 650 655
gta agg aat aat ccg gtt act ctg ctt gat cct 17192
gat gga tta atg cca
Val Arg Asn Asn Pro Val Thr Leu Leu Asp Pro
Asp Gly Leu Met Pro
660 665 670
aca att gca gaa cgc ata gca gca ctg caa aaa 17240
aat aaa gta gca gat
'Iizr Ile Ala Glu Arg Ile Ala Ala Lieu Gln
Lys Asn Lys Val Ala Asp
675 680 685 690
CA 02320801 2000-08-14
WO 99/42589 PCT/EP99/01015
-33-
tca gcg cct tcg cca aca aat gcc aca aac gta 17288
gcg ata aac atc cgc
Ser Ala Pro Ser Pro 'Ihr Asn Ala Thr Asn Val
Ala Ile Asn Ile Arg
695 700 705
ccg ccc gta gca cca aaa cct acc tta ccc aaa 17336
gca tca acg agt agc
Pro Pro Val Ala Pro Lys Pro Thr Leu Pro Lys
Ala Ser 'Ihr Ser Ser
710 715 720
caa tca act aca tac ccc atc aaa tct gca agc 17384
ata aaa cca acg acg
Gln Ser Zlir Thr Tyr Pro Ile Lys Ser Ala Ser
Ile Lys Pro Thr Thr
725 730 735
tcg gga tca tcc att act get cca ctg agt cca 17432
gta gga aat aaa tct
Ser Gly Ser Ser Ile Thr Ala Pro Leu Ser Pro
Val Gly Asn Lys Ser
740 745 750
act cct gaa ata tct ctt cca gaa agc act caa 17480
agc aat tct tca agc
Thr Pro Glu Ile Ser Leu Pro Glu Ser 'rhr Gln
Ser Asn Ser Ser Ser
755 760 765 770
get att tca aca aat cta cag aaa aag tca ttt 17528
act tta tat aga gcg
Ala Ile Ser Zhr Asn Leu Gln Lys Lys Ser Phe
Thr Leu ~Iyr Arg Ala
775 780 785
gat aat aga tcc ttt gaa gac atg cag agt aaa 17576
ttc cct gaa gga ttt
Asp Asn Arg Ser Phe Glu Asp Met Gln Ser Lys
Phe Pro Glu Gly Phe
790 795 800
aaa gcc tgg act cct cta gat act aag atg gca 17624
agg cag ttt get agt
Lys Ala Txp Thr Pro I~u Asp 'Ilzr Lys Met
Ala Arg Gln Phe Ala Ser
805 810 815
gtc ttt att ggt cag aaa gat act tct aat tta 17672
cct aaa gaa aca gtc
Val Phe Ile Gly Gln Lys Asp Thr Ser Asn Leu
Pro Lys Glu Thr Val
820 825 830
aag aat ata aac aca tgg gga aca aaa cca aaa 17720
tta aat gat ctc tca
Lys Asn Ile Asn 'llzr Trp Gly 'W r Lys Pro
Lys Leu Asn Asp Leu Ser
835 840 845 850
act tac ata aaa tat acc aag gac aaa tct aca 17768
gta tgg gtc tct act
Thr ~I~r Ile Lys Zyr 2hr Lys Asp Lys Ser Thr
Val '1'rp Val Ser Thr
855 860 865
gca att aat act gaa gca ggt gga caa agt tca 17816
ggg get cca ctc cat
Ala Ile Asn Thr Glu Ala Gly Gly Gln Ser Ser
Gly Ala Pro Leu His
870 875 880
gaa att aat atg gat ctt tat gag ttt acc att 17864
gac gga caa aag cta
Glu Ile Asn lit Asp Leu 'I~r Glu Phe Thr Ile
Asp Gly Gln Lys Leu
885 890 895
aat cca cta cca agg gga aga tct aaa gac agg 17912
gtg cct tca cta tta
Asn Pro Leu Pro Arg Gly Arg Ser Lys Asp Arg
Val Pro Ser Leu Leu
900 905 910
ctt gac aca cca gaa ata gaa aca gca tcc ata 17960
att gca ctt aat cat
Leu Asp Thr Pro Glu Ile Glu Thr Ala Ser Ile
Ile Ala Leu Asn His
915 920 925 930
gga ccg gta aat gat gca gaa gtt tca ttc cta 18008
aca aca att ccg ctt
Gly Pro Val Asn Asp Ala Glu Val Ser Pl~e Leu
Thr Zhr Ile Pro Leu
935 940 945
CA 02320801 2000-08-14
WO 99/42589 PCT/EP99/OI015
-34-
aaa aat gta aaa cct tat aag aga taa cgaaaaatta atattcttta 18055
Lys Asn Val Lys Pro Ayr Lys Arg
950 955
tctactttta atagccctct tgaacttaca ctcaarg ggaaaccaaa taagaaacca 18115
tctttaataa caagccatga aagaatattt atttcatggc ttgattactt ttaacattca 18175
atattaaata attaaaacaa tatctaacca attaaaataa caatacctta tttatcatat 18235
taaaatatca aatcagaaat taatgaattt aagggttctt tatatttatt tctgagagca 18295
taggcacaat accttaccga tggcgctgga cgtgattcaa aatccagaaa tgctatattt 18355
tcatcaatat gggcagaata gcgcatttca ttgggagtca ttaaacttat cgcgacaccc 18415
gcttttacca gatccaatct attagtaaaa tcagggaccg tcaataacgc taaattttgg 18475
tattcaggga gataattcaa tggcataaaa ttattgcatt gttttaaaaa agcactatta 18535
tgctgaacaa aaggaaaact agatattatt tcatcagcgt gactttctgg ttctaaaata 18595
tcatgggata cagcaagaga cagcatttga taagcaccat ctatcctgat gatatcatca 18655
ttatctggat aacattcagt cgtcacataa actgttatat cccctttcat taaggaagaa 18715
aataccgcat cttgccttat taaatcatca attagaaaat tgttgattat acaaatatcg 18775
cgataatgat aacgttgcac cgctcttttt acgaccgtag atattttatt aacatattct 18835
ccacttgtgc caataaccag tttgtctctg tttgataatt tataatttct acgacaattc 18895
caattattct caactttcag gatcctttca taacacggca gcaactcttg ata
tagtgcc 18955
tttccctctt ctgtgagctt ggtttttccc ggtagtcgct caaacaattg acaccccaca 19015
cgctgttcca gttgatatac gagcctgcta agtggagaag gggtaataca aagcgtatcc 19075
gccgctaacg tgaatgactc tttcttagct gattccataa aatactttag ttgctttgaa 19135
caaaatatca tcacataccc tcttgttttc attccagaaa tagaatatta accatagaac 19195
atgacaacga tgtttctact ttgcattctt ttacattagg acatgcgtta atggacattg 19255
aatttcacta catcaattgt taatatttat ttaatacttg cacaataatt ataaaataaa 19315
tataacttag ttaattattt cttgatattg atcatggtaa gttttcctca atacctacag 19375
aagtagatat tattttatct tccagtaatc tatcgtttgg cgacggaggt cgattcttcc 19435
attgggatat tcaacccatt cgccgccttt cttattaatt acagtgattt ttggcatttt 19495
ggtttcatcc aacttaggtt tataggtgat tttccattta gcacccggtg ttaacttcaa 19555
cctaaaggga tacataccaa cttcaccttg taagaatatt ctgtttggtc taccttcaac 19615
gactttcaaa atggggtaaa taaccgggct aaaatcaatc gtatccaatg catcaatttc 19675
gctgatattt gtccgggctg catcattgat aaatgcgatt aaatcggttg ctgaatacgg 19735
aatagcatct ttcactagat gacggacatc ggtataactc actgacacaa aggctcggtc 19795
aatcttccac ttacatcgac cgccaccatt aaaaggtagt tttgcctgaa agtaaccggt 19855
tttcggatca gcttttacat ccagacgtaa tccgttataa gttggtacct taaaaggcga 19915
CA 02320801 2000-08-14
WO 99/42589 PCT/EP99/01015
-35-
catattggaa tctaaaegat atttaaggca atcttttgag atatacacag cggatacatg 19975
cggctgtgtg tatttaggtg cgactccttc tacagtaatc cactgattct ctttgggagg 20035
agagagcggc tcatttgggt cagcacagcc tgatattaaa atcacggata agacagataa 20095
gtatttcttg atatttatca tggtaagttt tcctcaactc ctacagcgtt atctgcatgt 20155
gtgtccaatt ccagatcttc ctgtttatct atttagaaat aaataagcta cgctgatagc 20215
attacttcat atttccatac atgaatcgaa aatcgacttc ttgagtgccg ttatcaattt 20275
tgccgcccgg atattcaacc cactcgccgc ctttcttatt agtcaccgtg accttcgcca 20335
ttttggtttc atccagctta ggcttaaaaa taattttcca tttagctcct ggagttaacg 20395
tgagttgaaa aggacgcatt tttaatactt caccttgtaa gaatattctg ttcgggcgac 20455
cttcaacgac tttcaaaaca gggtaaataa ccgggctaaa atcaatcgta ttcaatgtcg 20515
agattttgct aatattcatc tggactatgc cattgataga tgcgattaaa ccggttgctg 20575
aatacggaat agcatctttc accagatggc tgacatcagt ataactcacc gatacaaagg 20635
cccggttaat tttccattta catcgtcccc ctccattaaa aggtagtttt gcttgaaaat 20695
aaccggtttg tggatcggcc ttcactttca gacgaagccc attataggtc ggcactttaa 20755
aaggcgacat attggaatcc agacgatact caaggcaatc ctttgatatg tattctgcgg 20815
atacatgtgg ttcggtatat ttcggcgcta ccccttctac cgtgatccat tgattttctt 20875
taggaa!~rga aagcggctca tttgggtcag cacagcctga tattaaaatc actgacaaga 20935
caaataagta ttttttaaca tttatcatgg taagttttcc tcaattccta cagcattatc 20995
cgcataaata tcctgtcaag aatagcgttc attgatttcg tcacaaaaga aacaagatag 21055
taaaaatcct attaccacag ataaaaaaca ccgcttatgc cgtgagtaat agtgagttga 21115
gcgacaggga tacagcagtg catccccatc aattagtccc tttgaataaa gggaacagaa 21175
tttgaaattt ccgtcatacc gtccatatta cggaacttag attatgatta ttaaatcacc 21235
accaaatggc aagaaaaatt ttcatttttt aatttacgaa gaatgaattt gtaagaaagt 21295
gttacaaact taatagaaat taatttactg ttaatctaat gaaggatgaa attataaaaa 21355
taacccattt ctcagggaca acaatccaca atatatagaa ccactggtcc tcacttaatt 21415
tcctgtcagg agtagaaata tcctgatgac tcagtcgatg acatacagca atgtcattgg 21475
tattgagact accgactgtt taataaattt cttttgtctt taatggcgag atacaagtga 21535
ttcactattt aagcactatc gataaataag attccaaaat agcgccatat cttacaccac 21595
tcataattct atgtataaca attggttaaa taggatcatg tgtaacagga ttatgaaacg 21655
ttatttatat caaatctatc aattatttta tatatagttt cacagtcaca ctcgctatct 21715
ggtaccttca taaccaactg ccctccctgc gctaccttct gataacaaca gctacactaa 21775
ctatacecgc gcctataatt atgaccgtgt gaaaattcag cgtagttcac cggccacgca 21835
ttggtacct taaaaggcga 19
CA 02320801 2000-08-14
WO 99/42589 PCT/EP99/01015
-36-
aaataactac acgaaatatt gctccccaga gaaacaccgt tcgaggttgt ttcaatgaaa 21895
catcaaggta gagacaccta tgtattatta caagatatta aaccctctgc gattactcat 21955
aggaatgtac gtaatactta tacaggcaac ttcacgtcat ccagagaaaa ttaagttgta 22015
caaaatagac atcaactaat atagtaatag aaaatcccct gaaaatagat tcaggggatt 22075
taataaatta accaaaaatc ataataaaaa tttatttcat tattttagga taaatattta 22135
attagcctaa taatgaatta ttacttaaag taattcctaa acaatcaaat cggaaattaa 22195
taaattcaat ggttcttgat atttatgcct gagagtataa gcacaatatt tcactgaggg 22255
tgtcggatgc gatttaaaat tcaaaaaggt aatgccttta tgaaggtcag cagaacagag 22315
cacttcattc ggtgtcatta aacttatcgc gatacctgat ttcactaagt ccaaccgatt 22375
agtaaaatca ggcacagtta gcaaagttaa attctggtat tcaggtagac aattcagtga 22435
aataaaattg ccgcactgtt taaagaaagc actattatgc tgagccaaag gcaatgtata 22495
tataatatta tcagcgttac tttccgatcc taaaatatca ttagctacag caaggcgcaa 22555
agtctgataa gttccatcca ctctgataat atcatcgttg tcaggataat gttcagtcgt 22615
cacatagacg gttatatctt ccttcattaa agaagaaaat attgtctctt ttcttgccga 22675
atcatcaacc agaaaattgt tttttatgta aatatcacga taatgataac gttgtaccgc 22735
tctttttatc actgccgaaa ttttattaat atattctcca cttgtcccga tgaccagttt 22795
gccggtgttt gctaattttc cgcttctacg ataatgccaa ttatcctcaa ctcgctgaat 22855
tctctcataa cacggcaata gctcctgata taatgccttc ccctcttcag tgagtttggt 22915
ccttcccggt agtcgttcaa atagttgaca gcccacacgt tgttccagtt gatatatgat 22975
cctacttaga ggagagggag taatacaaag cgtatccgcg gctaaagtaa atgactcttt 23035
tttcgctgac tccataaaat atttcaagct ctttgaacaa aatagcatca tatatccttc 23095
ttattttaat tcattgttcc atccgaaata gaatggaatg ttaacaagaa aacattacaa 23155
ctacttttct tctttgcatt atttaacatc aaagtatgca ttaactgaga ttgagtttta 23215
tcatctttat tcttaacagt tatcaaacaa ttttcattat tattgcaaaa taaatacaac 23275
cccttcttat gttacaataa tgattataaa gaaatttcac atattatcat taagtaataa 23335
tgggcacaat taaccattta attaaacatt tcaattggtt gacaaagact cattatgttc 23395
aacatgtaat gagcgcaatt ttaacattaa ataaattaca tagttcatat tcattatcac 23455
tgagatcagc ttttttcgta tagtacatca tgtgaacaat accgtgccat ttcctgccaa 23515
atcttattaa aaagtcagtt gcaaattttg catctgcttt ttttgcaaca gctatttaaa 23575
gaaaacagtg agatagtgat tatccgagag atcaagatat gtctgctctt tacgcacaaa 23635
ctgcaaacca tttctatgca tatctcagct atttctcaaa acctgtattt aatcatctct 23695
tattccgatg gaacggaatc attctctgat tgattcatga tgtaaagaca atatggatgt 23?55
ttcatttact tt atg att tta aaa gga ata aat atg aat tcg cct gta aaa 23806
CA 02320801 2000-08-14
WO 99/42589 PCT/EP99/01015
-37-
Met Ile Leu Lys Gly Ile Asn Met Asn Ser Pro
Val Lys
960 965
gag ata cct gat gta tta aaa atc cag tgt ggt 23854
ttt cag tgt ctg aca
Glu Ile Pro Asp Val Leu Lys Ile Gln Cps Gly
Plebe Gln Cys Leu Thr
970 975 980
gat att agc cac agc tct ttt aac gaa ttt cac 23902
cag caa gta tcc gaa
Asp Ile Ser His Ser Ser Phe Asn Glu Phe His
Gln Gln Val Ser Glu
985 990 995 1000
cac ctc tcc tgg tcc gaa gca cac gac tta tat 23950
cat gat gca caa cag
His Leu Ser Trp Ser Glu Ala His Asp Leu Tyr
His Asp Ala Gln Gln
1005 1010 1015
gcc caa aag gat aat cgg ctg tat gaa gcg cgt 23998
att ctt aaa cgc acg
Ala Gln Lys Asp Asn Arg Leu 'Iyr Glu Ala Arg
Ile Leu Lys Arg 'Il~r
1020 1025 1030
aat cct caa tta caa aat get gta cat ctt gcc 24046
atc gta gcg cct aat
Asn Pro Gln Leu Gln Asn Ala Val His Leu Ala
Ile Val Ala Pro Asn
1035 1040 1045
get gaa ctg ata ggc tat aac aac caa ttt agc 24094
ggc agg gcc agt caa
Ala Glu Leu Ile Gly 'Iyr Asn Asn Gln Phe Ser
Gly Arg Ala Ser Gln
1050 1055 1060
tat gtc gcg ccg ggt acc gtt tcc tcc atg ttc 24142
tcc ccc gcc get tat
'i~r Val Ala Pro Gly ~'hr Val Ser Ser Met Phe
Ser Pro Ala Ala 2~rr
1065 1070 1075 1080
ttg act gag ctt tat cgt gaa gca cgc aat tta 24190
cac gcc agc gat tcc
Leu Thr Glu Leu 2yr Arg Glu Ala Arg Asn Leu
His Ala Ser Asp Ser
1085 1090 1095
gtt tat cgc ctg gat act cgc cgc cca gat ctc 24238
aaa tca atg gcg ctc
Val Zyr Arg Leu Asp 'I2br Arg Arg Pro Asp Leu
Lys Ser Met Ala Leu
1100 1105 1110
agt caa caa aat atg gat acg gaa ctt tcc act 24286
ctc tct tta tcc aat
Ser Gln Gln Asn Met Asp Thr Glu Lieu Ser Thr
Leu Ser Leu Ser Asn
1115 1120 1125
gag cta tta ttg gaa agc att aaa act gag tct 24334
aag ctg gat aat tat
Glu Leu L~ Leu Glu Ser Ile Lys Thr Glu Ser
Lys Leu Asp Asn 'Iyr
1130 1135 1140
act caa gtg atg gaa atg ctc tcc get ttc cgt 24382
cct tcc ggc gcg acg
Thr Gln Val Met Glu Met Leu Ser Ala Phe Arg
Pro Ser Gly Ala Thr
1145 1150 1155 1160
cct tat cac gat get tac gaa aat gtg cgt aaa 24430
gtt atc cag cta caa
Pro 'Iyr His Asp Ala err Glu Asn Val Arg Lys
Val Ile Gln Leu Gln
1165 1170 1175
gat cct ggg ctt gag caa tta aat get tca cca 24478
gcc att gcc ggg ctg
Asp Pro Gly Leu Glu Gln Leu Asn Ala Ser Pro
Ala Ile Ala Gly Leu
1180 1185 1190
atg cat caa get tcc cta tta ggt att aac get 24526
tca atc tca cct gag
Met His Gln Ala Ser Leu Leu Gly Ile Asn Ala
Ser Ile Ser Pro Glu
1195 1200 1205
ttg ttt aat att ctg acg gag gag att act gaa 245?4
ggt aat get gag gaa
Leu Phe Asn Ile Leu Zhr Glu Glu Ile Thr Glu
Gly Asn Ala Glu Glu
CA 02320801 2000-08-14
WO 99/42589 PCT/EP99/01015
-38-
1210 1215 1220
ctt tat aag aaa aat ttt ggt aat atc gaa ccg 24622
get tca ctg get atg
Leu 'Iyr Lys Lys Asn Phe Gly Asn Ile Glu Pro
Ala Ser Leu Ala Met
1225 1230 1235 1240
ccg gaa Cac ctt aga cgt tat tac aat tta agt 24670
gat gaa gaa ctc agc
Pro Glu 'Iyr Leu Arg Arg 'Iyr Zyr Asn Leu
Ser Asp Glu Glu Leu Ser
1245 1250 1255
cag ttt att ggt aaa gcc agc aat ttc ggc caa 24718
caa gaa tat agt aat
Gln Phe Ile Gly Lys Ala Ser Asn Phe Gly Gln
Gln Glu 'Iyrr Ser Asn
1260 1265 1270
aac caa ctc att act ccg ata gtc aac agc aat 24766
gat ggc aca gtc aag
Asn Gln Leu Ile 'Inr Pro Ile Val Asn Ser Asn
Asp Gly Thr Val Lys
1275 1280 1285
gta tat cga att acc cgc gaa tat aca aca aat 24814
gcc aat caa gta gac
Val err Arg Ile 'I~r Arg Glu Tyr Thr Thr Asn
Ala Asn Gln Val Asp
1290 1295 1300
gtg gag ctg ttt ccc tac ggt gga gaa aat tat 24862
cag tta aat tac aaa
Val Glu Leu Pk~e Pro Tyr Gly Gly Glu Asn 'I~r
Gln Leu Asn Tyr Lys
1305 1310 1315 1320
ttc aaa gat tct cgt cag gat gtc tcc tat tta 24910
tcc atc aaa tta aat
Phe Lys Asp Ser Arg Gln Asp Val Ser Zyr Leu
Ser Ile Lys Leu Asn
1325 1330 1335
gac aaa aga gaa ctt atc cga att gaa gga gcg 24958
cct cag gtc aac atc
Asp Lys Arg Glu Leu Ile Arg Ile Glu Gly Ala
Pro Gln Val A~ Ile
1340 1345 1350
gaa tat tca gaa cat atc aca tta agt aca act 25006
gat atc agt caa cct
Glu Zyr Ser Glu His Ile 'Ihr Leu Ser Thr Thr
Asp Ile Ser Gln Pro
1355 1360 1365
ttt gaa atc ggc cta aca cga gta tat cct tct 25054
agt tct tgg gca tat
Phe Glu Ile Gly Leu 'rhr Arg Val 'Iyr Pro
Ser Ser Ser Trp Ala Tyr
1370 1375 1380
gca gcc gca aaa ttt acc att gag gaa tat aac 25102
caa tac tct ttc ctg
Ala Ala Ala Lys Pl~ Thz- Ile Glu Glu 2yr Asn
Gln ~Iyr Ser Phe Leu
1385 1390 1395 1400
tta aaa ctc aat aaa get att cgt cta tct cgt 25150
gcg aca gaa tta tca
Leu Lys Leu Asn Lys Ala Ile Arg Leu Ser Arg
Ala 'I'hr Glu Leu Ser
1405 1410 1415
ccc acc att ctg gaa agt att gtg cgt agt gtt 25198
aat cag caa ctg gat
Pro 'It~r Ile Leu Glu Ser Ile Val Arg Ser
Val Asn Gln Gln Leu Asp
1420 1425 1430
atc aac gca gaa gta tta ggt aaa gtt ttt ctg 25246
act aaa tat tat atg
Ile Asn Ala Glu Val Leu Gly Lys Val Phe Leu
'rhr Lys ~Iyr 'Iyr Met
1435 1440 1445
caa cgt tat get att aat get gaa act gcc cta 25294
ata cta tgc aat gca
Gln Arg 'ryr Ala Ile Asn Ala Glu Thr Ala Leu
Ile Lieu Cys Asn Ala
1450 1455 1960
ctt att tca caa cgt tca tat gat aat caa cct 25342
agc caa ttt gat cgc
Leu Ile Ser Gln Arg Ser 'Iyr Asp Asn Gln Pro
Ser Gln Phe Asp Arg
1465 1970 1475 1480
CA 02320801 2000-08-14
WO 99/42589 PCT/EP99/01015
-39-
ctg ttt aat acg cca tta ctg aac ggc caa tat 25390
ttt tct acc gga gat
Leu Phe Asn Thr Pro Leu Leu Asn Gly Gln 'Iyr
Phe Ser Thr Gly Asp
1485 1490 1495
gaa gag att gat tta aat cca ggt agt act ggc 25438
gat tgg cgt aaa tcc
Glu Glu Ile Asp Leu Asn Pro Gly Ser Thr Gly
Asp Trp Arg Lys Ser
1500 1505 1510
gtg ctt aaa cgt gca ttt aat atc gat gat att 25486
tcc ctc tac cgc ctg
Val Leu Lys Arg Ala Phe Asn Ile Asp Asp Ile
Ser Leu 'Iyr Arg Leu
1515 1520 1525
ctt aaa att acc aac cat aat aat caa gat gga 25534
aag att aaa aat aac
Leu Lys Ile Thr Asn His Asn Asn Gln Asp Gly
Lys Ile Lys Asn Asn
1530 1535 1540
tta aat aat ctt tct gat tta tat att ggg aaa 25582
tta ctg gca gaa att
Leu Asn Asn Leu Ser Asp Leu err Ile Gly Lys
Leu Leu Ala Glu Ile
1545 1550 1555 1560
cat caa tta acc att gat gaa ttg gat tta ttg 25630
ctg gtt gcc gtg ggt
His Gln Leu Thr Ile Asp Glu Leu Asp L~ L~
Leu Val Ala Val Gly
1565 1570 1575
gaa gga gaa act aat tta tcc get atc agt gat 25678
aaa caa ctg gcg gca
Glu Gly Glu Thr Asn Leu Ser Ala Ile Ser Asp
Lys Gln Lieu Ala Ala
1580 1585 1590
ctg atc aga aaa ctc aat acc att acc gtc tgg 25726
cta cag aca cag aag
Leu Ile Arg Lys Leu Asn Thr Ile 'Ihr Val Trp
Leu Gln Thr Gln Lys
1595 1600 1605
tgg agt gcg ttc caa tta ttt gtt atg act tcc 25774
acc agc tat aac aaa
Trp Ser Ala Phe Gln Leu Phe Val Met I'hr Ser
Thr Ser 'Iyr Asn Lys
1610 1615 1620
acg ctg acg cct gaa att aag aat ctg ctg gat 25822
acc gtc tac cac ggt
Thr L~eu 'Ilzr Pro Glu Ile Lys Asn Lieu Leu
Asp Thr Val Tyr His Gly
1625 1630 1635 1640
tta caa ggc ttt gat aaa gac aag gca aat tta 25870
ctg cat gtt atg gcg
Leu Gln Gly Phe Asp Lys Asp Lys Ala Asn Leu
Leu His Val Met Ala
1645 1650 1655
ccc tat att gcg gcc acc tta caa tta tca tcg 25918
gaa aat gtc gcc cat
Pro 'Iyr Ile Ala Ala Thr Leu Gln Leu Ser Ser
Glu Asn Val Ala His
1660 1665 1670
tct gtg ctg ctt tgg gca gac aag tta aag ccc 25966
ggc gac ggc gca atg
Ser Val Leu Leu Tzp Ala Asp Lys Leu Lys Pro
Gly Asp Gly Ala Met
1675 1680 1685
aca gcc gaa aaa ttc tgg gac tgg ttg aat act 26014
caa tat acg cca gat
'i'hr Ala Glu Lys Phe Trp Asp Trp Leu Asn
Thr Gln 'Iyr Thr Pro Asp
1690 1695 1700
tca tcg gaa gta tta gca aca cag gaa cat att 26062
gtt cag tat tgt cag
Ser Ser Glu Val Leu Ala Thr Gln Glu His Ile
Val Gln 'Iyr Cys Gln
1705 1710 1715 1720
gcg ttg gcg caa tta gaa atg gtt tac cat tcc 26110
acc ggt atc aat gaa
Ala Leu Ala Gln Leu Glu Met Val 'Iyr' His
Ser Thr Gly Ile Asn Glu
1725 1730 1735
CA 02320801 2000-08-14
WO 99/42589 PCT/EP99/01015
-40
aac gcc ttc cgc ctg ttt gtg aca aaa cca gag 26158
atg ttt ggc tcg tca
Asn Ala Phe Arg Leu Phe Val Thr Lys Pro Glu
Met Phe Gly Ser Ser
1740 1745 1750
act gag gca gta cct gcg cat gat gca ctt tca 26206
ctg atc atg ctg acg
Thr Glu Ala Val Pro Ala His Asp Ala Leu Ser
Leu Ile Met Leu 'rhr
1755 1760 1765
cgt ttt gca gat tgg gtt aat gcg tta ggc gaa 26254
aaa gcc tct tcc gta
Arg Phe Ala Asp Trp Val Asn Ala Leu Gly Glu
Lys Ala Ser Ser Val
1770 1775 1780
cta gcg gca ttt gaa get aac agt tta acg gca 26302
gaa caa ttg get gat
Leu Ala Ala Phe Glu Ala Asn Ser Leu Thr Ala
Glu Gln Leu Ala Asp
1785 1790 1795 1800
gcc atg aat ctt gat get aat ttg cta ttg caa 26350
gcc agt act caa gca
Ala Met Asn Leu Asp Ala Asn Leu Leu Leu Gln
Ala Ser 'Ihr Gln Ala
1805 1810 1815
caa aac cat caa cat ctt ccc cca gtg acg caa 26398
aaa aat get ttc tcc
Gln Asn His Gln His Leu Pro Pro Val ~hr Gln
Lys Asn Ala Phe Ser
1820 1825 1830
tgt tgg aca tct atc gac act atc ctg caa tgg 26446
gtt aat gtt gca caa
C.ys Tzp Zhr Ser Ile Asp Thr Ile Leu Gln Trp
Val Asn Val Ala Gln
1835 1840 1845
caa ttg aat gtc gcc cca cag gga gtt tcc get 26494
ttg gtc ggg ctg gat
Gln Leu Asn Val Ala Pro Gln Gly Val Ser Ala
Leu Val Gly Leu Asp
1850 1855 1860
tat att caa tta aat caa aaa atc ccc acc tat 26542
gcc cag tgg gaa agt
~Iyr Ile Gln Leu Asn Gln Lys Ile Pro Thr Tyr
Ala G7n Trp Glu Ser
1865 1870 1875 1880
get ggg gaa ata ttg act gcc gga ttg aat tca 26590
caa cag get gat ata
Ala Gly Glu Ile Leu 'I~r Ala Gly Leu Asn Ser
Gln Gln Ala Asp Ile
1885 1890 1895
tta cac get ttt ttg gac gaa tct cgc agt gcc 26638
gca tta agc acc tac
Leu His Ala Phe Leu Asp Glu Ser Arg Ser Ala
Ala Leu Ser Thr Tyr
1900 1905 1910
tat atc cgt caa gtc gcc aag cca gcg gca gcc 26686
ata aaa agc cgt gat
Zyr Ile Arg Gln Val Ala Lys Pro Ala Ala Ala
Ile Lys Ser Arg Asp
1915 1920 1925
gac ttg tac caa tac tta cta att gat aat cag 26734
gtt tcc get gca atc
Asp Leu 'Iyr Gln 'Iyr Leu Leu Ile Asp Asn
Gln Val Ser Ala Ala Ile
1930 1935 1940
aaa act acc cgg att gcc gaa gcc att gcc agc 26782
att caa ctg tac gtc
Lys Thr Thr Arg Ile Ala Glu Ala Ile Ala Ser
Ile Gln Leu Zyr Val
1945 1950 1955 1960
aac cgc acg ctg gaa aat gta gaa gaa aat gcc 26830
cat tca ggg gtt atc
Asn Arg Thr Leu Glu Asn Val Glu Glu Asn Ala
His Ser Gly Val Ile
1965 19?0 1975
agc cgt cag ttc ttt atc gac tgg gac aaa tat 26878
aac aaa cgc tac agc
Ser Arg Gln Phe Phe Ile Asp Trp Asp Lys 2yr
Asn Lys Arg 'Iyr Ser
1980 1985 1990
acc tgg gcg ggt gtt tct caa tta gtt tac tac 26926
ccg gaa aac tat att
CA 02320801 2000-08-14
WO 99/42589 PCT/EP99/01015
-41 -
'Ilir Trp Ala Gly Val Ser Gln Leu Val 'Iyr
'Iyr Pro Glu Asn 'Iyr Ile
1995 2000 2005
gat ccc acc atg cgt atc gga caa acc aaa atg 26974
atg gac gca tta ttg
Asp Pro 'Ihr Met Arg Ile Gly Gln I'hr Lys Met
Met Asp Ala Leu Leu
2010 2015 2020
caa tcc gtc agc caa agc caa tta aat gcc gat 27022
act gtc gaa gac gcc
Gln Ser Val Ser Gln Ser Gln Leu Asn Ala Asp
Thr Val Glu Asp Ala
2025 2030 2035 2040
ttt atg tct tat ctg aca tcg ttt gag caa gtg 27070
get aat ctt aaa gtt
Phe Met Ser 'Iyr Leu Thr Ser Phe Glu Gln Val
Ala Asn Leu Lys Val
2045 2050 2055
att agc gcg tat cac gat aat att aac aac gat 27118
caa ggg ctg acc tat
Ile Ser Ala 'Iyr His Asp Asn Ile Asn Asn Asp
Gln Gly Leu 'Ihr 'Iyr
2060 2065 2070
ttt atc ggc ctc agt gaa act gat acc ggt gaa 27166
tac tat tgg cgc agt
Phe Ile Gly Leu Ser Glu Thr Asp 'Ihr Gly Glu
2yr ~r Trp Arg Ser
2075 2080 2085
gtc gat cac agt aaa ttc age gac ggt aaa ttc 27214
gce get aat gcc tgg
Val Asp His Ser Lys Phe Ser Asp Gly Lys Phe
Ala Ala Asn Ala Trp
2090 2095 2100
agt gaa tgg cac aaa att gat tgt cca att aat 27262
cct tac cga agc act
Ser Glu Trp His Lys Ile Asp Cys Pro Ile Asn
Pro Tyr Arg Ser Thr
2105 2110 2115 2120
atc cgt cct gtg atg tac aaa tcc cgc ttg tat 27310
ctg ctc tgg ttg gaa
Ile Arg Pro Val Met 'Iyr Lys Ser Arg Leu 'IyzW~eu
I~u Trp Leu Glu
2125 2130 2135
caa aag gag atc act aaa caa aca gga aat agc 27358
aaa gat ggc tat caa
Gln Lys Glu Ile 'Ilzr Lys Gln Thr Gly Asn Ser
Lys Asp Gly Tyr Gln
2140 2145 2150
acc gag aca gat tat cgt tat gag cta aaa ttg 27406
gcg cat atc cgt tat
Thr Glu '1'hr Asp 'I'yr Arg 'i~rr Glu Leu Lys
I~u Ala His Ile Arg 'I~r
2155 2160 2165
gac ggt acc tgg aat acg cca atc act ttt gat 27454
gtc aat gaa aaa ata
Asp Gly ~'hr Trp Asn Thr Pro Ile Thr Phe Asp
Val Asn Glu Lys Ile
2170 2175 2180
tcc aag cta gaa ctg gca aaa aat aaa gcg eet 27502
ggg ctc tat tgt get
Ser Lys Leu Glu Leu Ala Lys Asn Lys Ala Pro
Gly Leu Tyr Cys Ala
2185 2190 2195 2200
ggt tat caa ggt gaa gat acg ttg ctg gtt atg 27550
ttt tat aac caa caa
Gly 'Iyr Gln Gly Glu Asp Zhr Leu Leu Val Met
Phe 'I~r Asn Gln Gln
2205 2210 2215
gat aca ctc gat agt tat aaa ace get tca atg 27598
caa ggg eta tat ate
Asp 'I'hr Leu Asp Ser 'Iyr Lys Thr Ala Ser
Met Gln Gly Leu 'Iyr Ile
2220 2225 2230
ttt gcc gat atg gaa tat aaa gat atg acc gat 27646
gga caa tac aaa tct
Phe Ala Asp Met Glu 'Iyr Lys Asp Met Thr Asp
Gly Gln 'Iyr Lys Ser
2235 2240 2245
tat cgg gac aac agc tat aaa caa ttc gat act 27694
aat agt gtc aga aga
'Iyr Arg Asp Asn Ser 'Iyr Lys Gln Phe Asp Thr
Asn Ser Val Arg Arg
CA 02320801 2000-08-14
WO 99/42589 PCT1EP99/01015
-42-
2250 2255 2260
gtg aat aac cgc tat gca gag gat tat gaa att 27742
ccc tca tcg gta aat
Val Asn Asn Arg 'Iyr Ala Glu Asp 'Iyr Glu
Ile Pro Ser Ser Val A~
2265 2270 2275 2280
agc cgt aaa ggc tat gat tgg gga gat tat tat 27790
ctc agt atg gta tat
Ser Arg Lys Gly 'Iyr Asp Trp Gly Asp 'iyr
Tyr Leu Ser Met Val 2yr
2285 2290 2295
aac gga gat att cca act att agt tac aaa gcc 27838
aca tca agt gat tta
Asn Gly Asp Ile Pro Thr Ile Ser 'i~r Lys Ala
'Ihr Ser Ser Asp Leu
2300 2305 2310
aaa atc tat atc tcg cca aaa tta aga att att 27886
cat aat gga tat gaa
Lys Ile Tyr Ile Ser Pro Lys Leu Arg Ile Ile
His Asn Gly 'Iyr Glu
2315 2320 2325
ggg cag caa cgc aat caa tgc aat cta atg aat 27934
aaa tat ggc aaa cta
Gly Gln Gln Arg Asn Gln Cps Asn Leu Met Asn
Lys 'iyr Gly Lys heu
2330 2335 2340
ggt gat aaa ttt att gtt tat act agc ttg gga 27982
gtt aat cca aat aat
Gly Asp Lys Phe Ile Val 'I~r 'Ihr Ser Leu
Gly Val Asn Pro Asn Asn
2345 2350 2355 2360
tcg tca aat aag ctg atg ttt tac ccc gtt tat 28030
caa tat aac gga aat
Ser Ser Asn Lys Leu Met Phe Tyr Pro Val 'Iyr
Gln Tyr Asn Gly Asn
2365 2370 2375
gtc agt ggg ctt agt caa ggg aga tta cta ttc 28078
cac cgt gac acc aat
Val Ser Gly Leu Ser Gln Gly Arg Leu Leu Phe
His Arg Asp Thr Asn
2380 2385 2390
tat tca tct aaa gta gaa get tgg att cct gga 28126
gca gga cgt tct cta
~Iyr Ser Ser Lys Val Glu Ala Trp Ile Pro Gly
Ala Gly Arg Sex Leu
2395 2400 2405
acc aat ccg aat get gcc att ggt gat gat tat 28174
get aca gac tcg tta
'1'hr Asn Pro Asn Ala Ala Ile Gly Asp Asp
'Iyr Ala 'rhr Asp Ser Leu
2410 2415 2420
aac aaa ccg aat gat ctt aag caa tac gtc tat 28222
atg act gac agt aaa
Asn Lys Pro Asn Asp heu Lys Gln Tyr Val Z'yr
Met Thr Asp Ser Lys
2425 2430 2435 2440
ggt act get acc gat gtc tca gga cca gta gat 28270
atc aat act gca att
Gly 'Ihr Ala Thr Asp Val Ser Gly Pro Val Asp
Ile Asn Thr Ala Ile
2445 2450 2455
tcc ccg gca aaa gtt cag gta aca gta aaa gcc 28318
ggt agc aaa gaa caa
Ser Pro Ala Lys Val Gln Val 'Itzr Val Lys
Ala Gly Ser Lys Glu Gln
2460 2465 2470
acg ttt acc gcg gat aaa aat gtc tcc att cag 28366
cca tcc cct agc ttt
'I2zr Phe Thr Ala Asp Lys Asn Val Ser Ile
Gln Pro Ser Pro Ser Phe
2475 2480 2485
gat gaa atg aat tat caa ttt aat get ctc gaa 28414
ata gat ggc tca agt
Asp Glu Met Asn 'Iyr Gln Phe Asn Ala Leu Glu
Ile Asp Gly Ser Ser
2490 2995 2500
ctg aat ttt act aac aat tca gcc agt att gat 28462
att acc ttt acc gca
Leu Asn Phe Thr Asn Asn Ser Ala Ser Ile Asp
Ile 'I'hr Phe Thr Ala
2505 2510 2515 2520
CA 02320801 2000-08-14
WO 99/42589 PCT/EP99/01015
-43-
ttt gca gag gat gga cgt aaa ctg ggt tat gaa 28510
agt ttc agt att cct
Phe Ala Glu Asp Gly Arg Lys Leu Gly Tyr Glu
Ser Phe Ser Ile Pro
2525 2530 2535
att acc cgc aag gtg agt act gat aat tcc ctg 28558
acc ctg cgc cat aat
Ile 'I97r Arg Lys Val Ser 'Ihr Asp Asn Ser
Leu 22zr Lieu Arg His Asn
2540 2545 2550
gaa aat ggt gcg caa tat atg caa tgg gga gtc 28606
tat cgc att cgt ctt
Glu Asn Gly Ala Gln Tyr Met Gln Trp Gly Val
Tyr Arg Ile Arg 1xu
2555 2560 2565
aat act tta ttt get cgc caa tta gtt gcg cga 28654
gcc act acc ggt att
Asn Thr Leu Phe Ala Arg Gln Leu Val Ala Arg
Ala ~'hr Thr Gly Ile
2570 2575 2580
gat acg att ctg agt atg gaa act cag aat att 28702
cag gaa cca cag tta
Asp 'rhr Ile Leu Ser Met Glu 'i~ir Gln Asn
Ile Gln Glu Pro Gln Leu
2585 2590 2595 2600
ggc aaa ggt ttc tac get acg ttc gtg ata cct 28750
ccg tat aac cca tca
Gly Lys Gly Phe Tyr Ala 'I9zr Phe Val Ile
Pm Pro Tyr Asn Pro Ser
2605 2610 2615
act cat ggt gat gaa cgt tgg ttt aag ctt tat 28798
atc aaa cat gtt gtt
Thr His Gly Asp Glu Arg Tip Phe Lys Leu Tyr
Ile Lys His Val Val
2620 2625 2630
gat aat aat tca cat att atc tat tca ggt cag 28846
cta aaa gat aca aat
Asp Asn Asn Ser His Ile Ile Tyr Ser Gly Gln
Leu Lys Asp 'I~ir Asn
2635 2640 2645
ata agc acc acg tta ttt atc cct ctt gat gat 28894
gtt cca ttg aac caa
Ile Ser Thr 'I~r Leu Phe Ile Pro Leu Asp Asp
Val Pro Leu Asn Gln
2650 2655 2660
gat tac agc gcc aag gtt tac atg acc ttc aag 28942
aaa tca cca tca gat
Asp Zyr Ser Ala Lys Val Tyr Met 'I3~r Phe
Lys Lys Ser Pro Ser Asp
2665 2670 2675 2680
ggt acc tgg tgg ggc cct cac ttt gtt aga gat 28990
gat aaa gga ata gta
Gly ~hr Trp Tip Gly Pro His Phe Val Arg Asp
Asp Lys Gly Ile Val
2685 2690 2695
aca ata aac cct aaa tcc att ttg acc cac ttt 29038
gag agc gtc aat gtc
Thr Ile Asn Pro Lys Ser Ile Leu Thr His Phe
Glu Ser Val Asn Val
2700 2705 2710
ctg aat aat att agt agc gaa cca atg gat ttc 29086
agc ggc get aac agc
Leu Asn Asn Ile Ser Ser Glu Pro Met Asp Phe
Ser Gly Ala Asn Ser
2715 2720 2725
ctc tat ttt tgg gaa ctg ttc tac tat acc ccg 29134
atg ctg gtt gcc caa
Leu Tyr Phe Trp Glu Leu Phe Tyr 'Iyr Thr Pro
Met Leu Val Ala Gln
2730 2735 2740
cgt ttg ttg cat gag caa aac ttt gat gaa gcg 29182
aac cgc tgg ctg aaa
Arg Leu Leu His Glu Gln Asn Phe Asp Glu Ala
Asn Arg Trp Leu Lys
2745 2750 2755 2760
tat gtc tgg agc cca tcc ggg tat att gtt cac 29230
ggc cag att cag aat
Tyr Val Trp Ser Pro Ser Gly Tyr Ile Val His
Gly Gln Ile Gln Asn
2765 2770 2775
CA 02320801 2000-08-14
WO 99/42589 PCT/EP99/01015
-44-
tat caa tgg aac gtc cgc ccg tta ttg gaa gat 29278
acc agt tgg aac agt
'Iyr Gln Trp Asn Val Arg Pro Leu Leu Glu Asp
Thr Ser Trp Asn Ser
2780 2785 2790
gat cct ttg gat tcc gtc gat cct gac gcg gta 29326
gcg cag cac gat ccg
Asp Pro Leu Asp Ser Val Asp Pro Asp Ala Val
Ala Gln His Asp Pro
2795 2800 2805
atg cac tat aaa gtt tca acc ttt atg cgc acc 29374
ctt gat ctg ttg atc
Met His 'Iyr Lys Val Ser Thr Phe Met Arg 'rhr
1xu Asp Leu Leu Ile
2810 2815 2820
gcg cgc ggc gac cat get tac cgc caa ttg gag 29422
cgc gat acg ctt aac
Ala Arg Gly Asp His Ala 'Iyr Arg Gln Leu Glu
Arg Asp '1'hr Leu Asn
2825 2830 2835 2840
gaa gcg aag atg tgg tat atg caa gcg ctg cat 29470
ctg tta ggc gat aaa
Glu Ala Lys Met Trp 'Iyr Met Gln Ala Leu His
Leu Leu Gly Asp Lys
2845 2850 2855
cct tat ctg ccg ctg agt acc aca tgg aat gat 29518
cca cga ctg gac aaa
Pro Zyr L~ Pro Leu Ser Thr Thr Trp Asn Asp
Pro Arg Leu Asp Lys
2860 2865 2870
gcc gcg gat att act acc caa agt get cat tcc 29566
agc tca ata gtc get
Ala Ala Asp Ile Thr Thr Gln Ser Ala His Ser
Ser Ser Ile Val Ala
2875 2880 2885
ttg cgg cag agt aca ccg gcg ctt tta tca ttg 29614
cgc agc gcc aat acc
r_p!~ Arg Gln Ser ~I~r Pro Ala Leu Leu Ser
Leu Arg Ser Ala Asn Thr
2890 2895 2900
ctg acc gat ctc ttc ctg ccg caa atc aat gaa 29662
gtg atg atg aat tac
Leu Thr Asp Leu Phe Lsu Pro Gln Ile Asn Glu
Val Met Met Asn 'I~r
2905 2910 2915 2920
tgg caa aca tta get cag aga gta tac aac ctg 29710
cgc cac aac ctc tct
Trp Gln Ttir Leu Ala Gln Arg Val Zyr Asn Leu
Arg His Asn Leu Ser
2925 2930 2935
atc gac ggt cag ccg tta tat ctg cca atc tat 29758
gcc aca ccg gcg gac
Ile Asp Gly Gln Pro LeWyr Leu Pro Ile 'lyr
Ala Zhr Pro Ala Asp
2940 2945 2950
ccg aaa gcg tta ctc agc gcc get gtt gcc act 29806
tct caa ggt gga ggc
Pro Lys Ala L~ Leu Ser Ala Ala Val Ala 'Ihx~
Ser Gln Gly Gly Gly
2955 2960 2965
aag ctg ccg gag tca ttt atg tcc ctg tgg cgt 29854
ttc ccg cac atg ctg
Lys Leu Pro Glu Ser Phe Met Ser Leu Trp Arg
Phe Pro His Met Leu
2970 2975 2980
gaa aat get cgc agc atg gtt agc cag ctc acc 29902
caa ttc ggc tcc acg
Glu Asn Ala Arg Ser Met Val Ser Gln Leu Thr
Gln Phe Gly Ser Z'hr
2985 2990 2995 3000
tta caa aat att atc gaa cgt cag gac gca gaa 29950
gcg ctc aat gcg tta
Leu Gln Asn Ile Ile Glu Arg Gln Asp Ala Glu
Ala Leu Asn Ala Leu
3005 3010 3015
tta caa aat cag gcc gca gag ctg ata ttg act 29998
aac ctg agt att caa
Leu Gln Asn Gln Ala Ala Glu Leu Ile Leu 'I'hr
Asn Leu Ser Ile Gln
3020 3025 3030
gac aaa acc att gaa gaa ctg gat gcc gag aaa 30046
acc gtg ctg gaa aaa
CA 02320801 2000-08-14
WO 99/42589 PCT/EP99/01015
-45-
Asp Lys ~'hr Ile Glu Glu Leu Asp Ala Glu Lys
'Ilzr Val Leu Glu Lys
3035 3040 3045
tcc aaa gcg gga gca caa tcg cgc ttt gat agc 30094
tat agc aaa ctg cat
Ser Lys Ala Gly Ala Gln Ser Arg Phe Asp Ser
'Iyr Ser Lys Leu His
3050 3055 3060
gat gaa aac atc aac gcc ggt gaa aac caa get 30142
atg acg cta cga gcg
Asp Glu Asn Ile Asn Ala Gly Glu Asn Gln Ala
Met Thr Leu Arg Ala
3065 3070 3075 3080
tcc gca gcc ggg ctt acc acg gcg gtt cag gca 30190
tcc cgt ctg gcc ggc
Ser Ala Ala Gly Leu 'I2~r Thr Ala Val Gln
Ala Ser Arg Leu Ala Gly
3085 3090 3095
gca gcg get gat ctg gtg cct aac atc ttc ggc 30238
ttc gcc ggt ggt ggt
Ala Ala Ala Asp Leu Val Pro Asn Ile Phe Gly
Phe Ala Gly Gly Gly
3100 3105 3110
agc cgt tgg ggg get atc get gag gcg acc ggc 30286
tat gta atg gaa ttt
Ser Arg Txp Gly Ala Ile Ala Glu Ala Thr G1y
Zyr Val Met Glu Phe
3115 3120 3125
tcc get aat gtt atg aat acc gaa gcg gat aaa 30334
att agc caa tct gaa
Ser Ala Asn Val Met Asn 'rhr Glu Ala Asp Lys
Ile Ser Gln Ser Glu
3130 3135 3140
acc tac cgt cgt cgc cgt cag gag tgg gaa att 30382
cag cgt aat aat gcc
'Ihr 'Iyr Arg Arg Arg Arg Gln Glu Trp Glu
Ile Gln Arg Asn Asn Ala
3145 3150 3155 3160
gaa gcg gag ctg aaa caa ctc gat gcc caa ctt 30430
aaa tcg ctg gca gta
Glu Ala Glu Leu Lys Gln Leu Asp Ala G7n Leu
Lys Ser Leu Ala Val
3165 3170 3175
cgc cgt gaa gcc gcc gta ttg caa aaa acc agc 30478
ctg aaa acc caa caa
Arg Arg Glu Ala Ala Val Leu Gln Lys Thr Ser
Leu Lys 'rhr Gln Gln
3180 3185 3190
gag cag acc caa gcc caa ttg gcc ttc ctg caa 30526
cgt aag ttc agc aat
Glu Gln Thr Gln Ala Gln Leu Ala Phe Leu G7n
Arg Lys Phe Ser Asn
3195 3200 3205
caa gcg ttg tac aac tgg cta cgt ggc cga ctg 30574
gca gca att tac ttc
Gln Ala Leu ~F'yr Asn Trp Leu Arg Gly Arg
Leu Ala Ala Ile ~Iyr Phe
3210 3215 3220
caa ttc tac gac ttg get atc gcg cgt tgt tta 30622
atg gca gag cag get
Gln Phe ~r Asp Leu Ala Ile Ala Arg Cys Leu
Met Ala Glu Gln Ala
3225 3230 3235 3240
tac cgt tgg gaa att agc gat gac tct get cgc 30670
ttt att aaa ccg ggc
Tyr Arg Trp Glu Ile Ser Asp Asp Ser Ala Arg
Phe Ile Lys Pro Gly
3245 3250 3255
gcc tgg caa gga acc tat gca ggt ctg ctg gca 30718
ggt gaa acc ttg atg
Ala Trp Gln Gly 'I9zr 'Iyr Ala Gly Leu Leu
Ala Gly Glu Thr Leu Met
3260 3265 3270
cta agt ttg gca caa atg gaa gac gcc cat tta 30766
aga cgc gat aaa cgc
Leu Ser Leu Ala Gln Met Glu Asp Ala His Leu
Arg Arg Asp Lys Arg
3275 3280 3285
gca tta gag gtc gaa cgt aca gta tcg ctg gcc 30814
gaa att tat get ggt
Ala Leu Glu Val Glu Arg Thr Val Ser Leu Ala
Glu Ile 'Iyr Ala Gly
CA 02320801 2000-08-14
WO 99/42589 PCT/EP99/01015
-46-
3290 3295 3300
tta ccg caa gat aaa ggc cca ttc tcc ctg acg 30862
caa gaa atc gag aag
Leu Pro Gln Asp Lys Gly Pro Phe Ser Leu Thr
Gln Glu Ile Glu Lys
3305 3310 3315 3320
ctg gtg aat gca ggt tca ggc agc gcc ggc agt 30910
ggt aat aat aat ttg
Leu Val Asn Ala Gly Ser Gly Ser Ala Gly Ser
Gly Asn Asn Asn Leu
3325 3330 3335
gca ttt ggc gcc ggc acg gac act aaa act tct 30958
ttg cag gca tcc att
Ala Phe Gly Ala Gly Thr Asp Thr Lys Thr Ser
Leu Gln Ala Ser Ile
3340 ~ 3345 3350
tca tta get gat tta aaa att cgt gag gat tac 31006
ccg gaa tct att ggc
Ser Leu Ala Asp Leu Lys Ile Arg Glu Asp Zyr
Pro Glu Ser Ile Gly
3355 3360 3365
aaa atc cga cgc atc aaa cag atc agc gtt acc 31054
ctg ccg gcg cta ttg
Lys Ile Arg Arg Ile Lys Gln Ile Ser Val Thr
L~eu Pro Ala Leu I~eu
3370 3375 3380
gga cct tat cag gat gtg cag gca ata tta tct 31102
tac ggc gat aaa gcc
Gly Pro 'I~r Gln Asp Val Gln Ala Ile Leu Ser
'Iyr Gly Asp Lys Ala
3385 3390 3395 3400
gga tta gcg aac ggc tgt gca gcg ctg gcc gtt 31150
tcc cac ggt acg aat
Gly Leu Ala Asn Gly Cys Ala Ala Leu Ala Val
Ser His Gly Thr Asn
3405 3410 3415
gac agc ggt caa ttc cag ctc gat ttc aac gat 31198
ggc aaa ttc ctg ccg
Asp Ser Gly Gln Phe Gln Leu Asp Phe Asn Asp
Gly Lys Phe Leu Pm
3420 3425 3430
ttt gaa ggt atc gcc att gat caa ggt acg cta 31246
aca ctg agt ttt cct
Phe Glu Gly Ile Ala Ile Asp Gln Gly 'I~r Leu
Thr Leu Ser Phe Pro
3435 3440 3445
aat gca tca acg cca gcc aaa ggt aaa caa gcc 31294
act atg tta aaa acc
Asn Ala Ser Thr Pro Ala Lys Gly Lys Gln Ala
Thr Met Leu Lys Thr
3450 3455 3460
ctg aac gat atc att ttg cat att cgc tac acc 31336
att aag taa
L~eu Asn Asp Ile Ile Leu His Ile Arg Tyr 'I~r
Ile Lys
3465 3470 3475
ccatcccaac acagaactaa gacaggcccc gaatcggggt 31395
ctggtaagga gtttct atg
Met
cag aat tca cag aca ttc agc atg acc gag ctg 31443
tca tta cct aag ggc
Gln Asn Ser Gln 'Ibr Phe Ser Met Thr Glu Leu
Ser Leu Pro Lys Gly
3480 3485 3490 3495
ggc ggc gcc att acc ggt atg ggt gaa gca tta 31491
acg ccg gcc ggg ccg
Gly Gly Ala Ile 'lfir Gly Met Gly Glu Ala Leu
Thr Pro Ala Gly Pro
3500 3505 3510
gat ggt atg gca gcc tta tcg ctg cca ttg ccc 31539
att tct gcc gga cgt
Asp Gly Met Ala Ala Leu Ser Leu Pro Leu Pro
Ile Ser Ala Gly Arg
3515 3520 3525
ggt tat gcc ccc tcg ctc acg ctg aac tac aac 31587
agc gga acc ggt aac
Gly 'Iyr Ala Pro Ser Leu 'Ihr Leu Asn 'Tyr
Asn Ser Gly Thr Gly Asn
3530 3535 3540
CA 02320801 2000-08-14
WO 99/42589 PCT/EP99/01015
-47-
agc ccg ttc ggt ctc ggt tgg gac tgt aac gtc 31635
atg aca att cgt cgt
Ser Pro Phe Gly Leu Gly Trp Asp Cys Asn Val
Met Thr Ile Arg Arg
3545 3550 3555
cgc acc agt acc ggc gtg ccg aat tat gat gaa 31683
acc gat act ttt ctg
Arg Thr Ser Thr Gly Val Pro Asn 'Iyr Asp Glu
Thr Asp Thr Phe Leu
3560 3565 3570 3575
ggg ccg gaa ggt gaa gtg ttg gtc gta gca tta 31731
aat gag gca ggt caa
Gly Pro Glu Gly Glu Val Leu Val Val Ala Leu
Asn Glu Ala Gly Gln
3580 3585 3590
get gat atc cgc agt gaa tcc tca tta cag ggc 31779
atc aat ttg ggg atg
Ala Asp Ile Arg Ser Glu Ser Ser Leu Gln Gly
Ile Asn Leu Gly Met
3595 3600 3605
acc ttc acc gtt acc ggt tat cgc tcc cgt ttg 31827
gaa agc cac ttt agc
Thr Phe Thr Val 2hr Gly 'iyr Arg Ser Arg Leu
Glu Ser His Phe Ser
3610 3615 3620
cgg ttg gaa tac tgg caa ccc caa aca aca ggc 31875
gca acc gat ttc tgg
Arg Leu Glu 'Iyr Trp Gln Pro Gln Thr 'Ilzr
Gly Ala Thr Asp Phe Txp
3625 3630 3635
ctg ata tac agc ccc gac gga caa gcc cat tta 31923
ctg ggc aaa aat cct
Leu Ile Tyr Ser Pro Asp Gly Gln Ala His Leu
Leu Gly Lys Asn Pro
3640 3645 3650 3655
caa gca cgc atc agc aat cca cta aat gtt aac 31971
caa aca gcg caa tgg
Gln Ala Arg Ile Ser Asn Pro Leu Asn Val Asn
Gln 'Itir Ala Gln T'rp
3660 3665 3670
cta ttg gaa gcc tcg gta tca tcc cac ggc gag 32019
cag att tat tat cag
Lieu Leu Glu Ala Ser Val Ser Ser His Gly Glu
Gln Ile Tyr err Gln
3675 3680 3685
tat cga gcc gaa gat gaa act gat tgc gaa act 32067
gac gaa ctc aca gcc
'Iyr Arg Ala Glu Asp Glu Thr Asp Cys Glu 'Ihr
Asp Glu Txu Thr Ala
3690 3695 3700
cac ccg aac aca acc gtc cag cgc tac ctg caa 32115
gta gta cat tac ggt
His Pro Asn Thr Thr Val Gln Arg Ayr Leu Gln
Val Val His Tyr Giy
3705 3710 3715
aat cta acc gcc agc gaa gta ttt ccc acg cta 32163
aat gga gat gat cca
Asn Leu Thr Ala Ser Glu Val Phe Pro Thr Leu
Asn Gly Asp Asp Pro
3720 3725 3730 3735
ctc aaa tct ggc tgg ttg ttc tgt tta gta ttt 32211
gat tac ggt gag cgc
Leu Lys Ser Gly Trp Leu Phe Cys Leu Val Phe
Asp 'Iyr Gly Glu Arg
3740 3745 3750
aaa aac agc tta tct gaa atg ccg cca ttt aaa 32259
gcc aca agt aac tgg
Lys Asn Ser Leu Ser Glu Met Pro Pro Phe Lys
Ala 'I'hr Ser Asn Trp
3755 3760 3765
ctt tgc cgc aaa gac cgt ttt tcc cgt tat gaa 32307
tac ggt ttt gca ttg
Leu Cys Arg Lys Asp Arg Phe Ser Arg 'Iyr Glu
'Iyr Gly Pl~ Ala Leu
3770 3775 3780
cgc acc cgg cgc tta tgt cgc caa ata ctg atg 32355
ttt cac cgt ctg caa
Arg 'Ihr Arg Arg Leu Cys Arg Gln Ile Leu Met
Phe His Arg Leu Gln
3785 3790 3795
acc ctg tct ggt cag gca aaa ggc gac gat gaa 32903
ccc gca tta gtt tca
CA 02320801 2000-08-14
WO 99/42589 PCT/EP99/01015
-48-
'Ilzr Leu Ser Gly Gln Ala Lys Gly Asp Asp
Glu Pro Ala Leu Val Ser
3800 3805 3810 3815
cgt ctg ata ctg gat tat gac gaa aac gcg gtg 32451
gtc agt acg ctc gtt
Arg Leu Ile Leu Asp 'Iyr Asp Glu Asn Ala Val
Val Ser Thr Leu Val
3820 3825 3830
tct gtc cgc cga gtg gga cat gag caa gat ggc 32499
aca acg gcg gtc gcc
Ser Val Arg Arg Val Gly His Glu Gln Asp Gly
'Ilzr Thr Ala Val Ala
3835 3840 3845
ctg ccg cca ttg gaa ctg get tat cag cct ttt 32547
gaa cca gaa caa aaa
Leu Pro Pro Leu Glu Leu Ala 'Iyr Gln Pro Phe
Glu Pro Glu Gln Lys
3850 3855 3860
gca ctc tgg cga cca atg gat gta ctg gcg aat 32595
ttc aac acc atc caa
Ala Leu Trp Arg Pro Met Asp Val Leu Ala Asn
Phe Asn Thr Ile Gln
3865 3870 3875
cgc tgg caa ctg ctt gat ctg caa ggc gaa ggc 32643
gta ccc ggt att ctg
Arg Trp Gln Leu Lieu Asp Leu G7n Gly Glu Gly
Val Pro Gly Ile Leu
3880 3885 3890 3895
tat cag gat aaa aat ggc tgg tgg tat cga tct 32691
get caa cgt cag aca
'Iyr Gln Asp Lys Asn Gly Trp Trp 'i~rr Arg
Ser Ala Gln Arg Gln Thr
3900 3905 3910
gg~g gaa gag atg aat gcg gtc acc tgg ggc aaa 32739
atg caa ctc ctt cct
Gly Glu Glu Met A.sn Ala Val Thr Trp Gly Lys
Met Gln Leu Leu Pro
3915 3920 3925
atc acg ccc get att cag gat aac gcc tca ctg 32787
atg gat att aat ggt
Ile 29zr Pro Ala Ile Gln Asp Asn Ala Ser Leu
Met Asp Ile Asn Gly
3930 3935 3940
gat ggg caa ctg gat tgg gtt atc acc ggt ccg 32835
ggg cta agg ggt tat
Asp Gly Gln Leu Asp Trp Val Ile Thr Gly Pro
Gly Leu Arg Gly Tyr
3945 3950 3955
cac agc cag cat cca gat ggc agt tgg aca cgt 32883
ttt acg ccg ttg cac
His Ser Gln His Pro Asp Gly Ser Trp Thr Arg
Phe Thr Pro Leu His
3960 3965 3970 3975
gcc tta ccg ata gaa tat acc cat ccc cgc gcc 32931
caa ctt gcg gat tta
Ala Leu Pro Ile Glu Zyr 'Ifir His Pro Arg
Ala Gln Leu Ala Asp Leu
3980 3985 3990
atg ggg gcc ggg ctg tcc gat tta gtg ctg att 32979
ggt ccc aaa agc gtg
Met Gly Ala Gly Leu Ser Asp Leu Val Leu Ile
Gly Pro Lys Ser Val
3995 4000 4005
cgt ttg tat gcc aat aac cgt gat ggt ttt acc 33027
gaa gga cgg gat gtg
Arg Leu 'Iyr Ala Asn Asn Arg Asp Gly Phe 'Ihr
Glu Gly Arg Asp Val
4010 4015 4020
gtg caa tcc ggt ggt atc acc ctg ccg tta ccg 33075
ggc gcc gat gcg cgt
Val Gln Ser Gly Gly Ile Thr Leu Pro Lsu Pro
Gly Ala Asp Ala Arg
4025 4030 4035
aag tta gtg gcc ttt agc gac gta ctc ggt tca 33123
ggc caa gca cat ttg
Lys Leu Val Ala Phe Ser Asp Val Leu Gly Ser
Gly Gln Ala His Leu
4040 4045 4050 4055
gtt gaa gtt agt gcg acg aaa gtc acc tgc tgg 33171
cca aat ctg gga cat
Val Glu Val Ser Ala Thr Lys Val Thr Cys 'I'rp
Pro Asn Leu Gly His
CA 02320801 2000-08-14
WO 99/42589 PC'f/EP99/01015
- 49 -
4060 4065 4070
ggc cgt ttt ggt cag cca atc aca ttg ccg gga 33219
ttt agc caa tcc gcc
Gly Arg Phe Gly Gln Pro Ile 'Ilzr L~eu Pro
Gly Phe Ser Gln Ser Ala
4075 4080 4085
gcc aat ttt aat cct gat cga gtt cat ctg gcc 33267
gat ctg gac ggt agt
Ala Asn Phe Asn Pro Asp Arg Val His Leu Ala
Asp Leu Asp Gly Ser
4090 4095 4100
ggt cct gcc gat ctg att tat gtt cat get gac 33315
cat ctg gat att ttc
Gly Pro Ala Asp Leu Ile 'Iyr Val His Ala Asp
His Leu Asp Ile Phe
4105 4110 4115
agc aat gaa agt ggt aac ggt ttt gca caa cca 33363
ttc aca ctc cgt ttt
Ser Asn Glu Ser Gly Asn Gly Phe Ala Gln Pro
Phe Thr Leu Arg Phe
4120 4125 4130 4135
cct gac ggc ctg cgt ttt gat gat act tgc cag 33411
cta caa gtg get gat
Pro Asp Gly Leu Arg Phe Asp Asp ~'hr Cys Gln
Leu Gln Val Ala Asp
4140 4145 4150
gta cag gga tta ggg gtt gtc agc ctg atc ctg 33459
agc gta ccg cat atg
Val Gln Gly Leu Gly Val Val Ser Leu Ile Leu
Ser Val Pro His Met
4155 4160 4165
gcg cca cac cat tgg cgc tgc gat ctg acc aac 33507
gcg aaa ccg tgg tta
Ala Pro His His 'I~p Arg Cys Asp Leu 'rhr Asn
Ala Lys Pro Trp rte,
4170 4175 4180
ctc agt gaa atg aac aac aac atg gga gcc cat 33555
cac acc ctg cat tac
L~eu Ser Glu Met Asn Asn A~ Met Gly Ala His
His 'rhr Leu His 'Iyr
4185 4190 4195
cgt agc tcc gtc cag ttt tgg ctg gat gaa aaa 33603
gcc gca gcc tta get
Arg Ser Ser Val Gln Phe Trp Leu Asp Glu Lys
Ala Ala Ala L~ Ala
4200 4205 4210 4215
acc gga caa aca ccg gtc tgt tac ctg ccc ttc 33651
ccg gtc cat acc ctg
Thr Gly Gln 'rhr Pro Val Cps 2yr heu Pro Phe
Pro Val His Thr heu
4220 4225 4230
tgg caa aca gaa acc gag gat gaa atc agc ggc 33699
aat aaa tta gtg acc
Trp Gln Thr Glu Thr Glu Asp Glu Ile Ser Gly
Asn Lys Leu Val 'I~r
4235 4240 4245
act tta cgt tac get cac ggc gcc tgg gat gga 33747
cgt gag cgg gaa ttt
'Ihr L~eu Arg 'Iyr Ala His Gly Ala Trp Asp
Gly Arg Glu Arg Glu Phe
4250 4255 4260
cgc ggc ttt ggc tat gtt gag cag aca gac agc 33795
cat caa ctg get caa
Arg Gly Phe Gly 'Iyr Val Glu Gln 'I'hr Asp
Ser His Gln Leu Ala Gln
4265 4270 4275
ggc aat gcg ccg gaa cgt aca tca ccg gca ctt 33893
acc aaa aac tgg tat
Gly Asn Ala Pro Glu Arg 'Inr Ser Pro Ala Leu
'Ilzr Lys Asn Trp 'Iyr
4280 4285 4290 4295
gcc acc gga atc cct gag gta gac aat acg cta 33891
tct gcc ggg tat tgg
Ala 'I"hr Gly Ile Pro Glu Val Asp Asn Thr Leu
Ser Ala Gly 'Iyr 'I'rp
4300 9305 4310
cgc ggt gat acg cag get ttc act ggt ttt acg 33939
cca cac ttt act ctc
Arg Gly Asp Thr Gln Ala Phe Thr Gly Phe Thr
Pro His Phe Thr Leu
4315 4320 4325
CA 02320801 2000-08-14
WO 99/42589 PCT/EP99/01015
-50-
tgg aaa gag ggc aaa gat gtt cca ctg aca ccg 33987
gaa gat gac cac aat
'IYp Lys Glu Gly Lys Asp Val Pro Leu Thr Pro
Glu Asp Asp His Asn
4330 4335 4340
ctg tac tgg tta aac cqg gca cta aaa ggt caa 34035
cca ctg cgt agt gaa
Leu 'i'yr Trp Leu Asn Arg Ala Leu Lys Gly
Gln Pro Leu Arg Ser Glu
4345 4350 4355
ctc tac ggg cta gat ggc agc gca cag cag aag 34083
atc ccc tat aca gtg
Leu 'Iyr Gly L~eu Asp Gly Ser Ala Gln Gln
Lys Ile Pro Zyr Thr Val
4360 4365 4370 4375
act gaa tcc cgc cca caa gtg cgc caa tta caa 34131
gat aac act acc ctt
Thr Glu Ser Arg Pro Gln Val Arg Gln Leu Gln
Asp Asn Thr Thr Leu
4380 4385 4390
tcc ccg gtg ctc tgg gcc tca gtg gtg gaa agt 34179
cgt agt tat cac tat
Ser Pro Val Leu Trp Ala Ser Val Val Glu Ser
Arg Ser 'Iyr His Tyr
4395 4400 4405
gaa cgt atc atc agc gat ccc caa tgc aat cag 34227
gat atc act ctg tcc
Glu Arg Ile Ile Ser Asp Pro Gln Cys Asn Gln
Asp Ile Thr Leu Ser
4410 4415 4420
agt gac cta ttc ggg caa ccg ctg aaa cag gtt 34275
tca gtg caa tat ccc
Ser Asp Leu Phe Gly Gln Pro Leu Lys Gln Val
Ser Val Gln Tyr Pro
4425 4430 4435
cgc cgc aat aaa cca aca acc aat ccg tat ccc 34323
gat aca cta cca gat
Arg Arg Asn Lys Pro 2'hr Thr Asn Pro Tyr Pro
Asp 1'hr Leu Pro Asp
4440 4445 4450 4455
act ctg ttt gcc agc agt tat gac gac caa caa 34371
caa cta ttg cgg tta
'I2zr Leu f~ Ala Ser Ser Tyr Asp Asp Gln Gln
Gln Leu Leu Arg Leu
4460 4465 4470
acc tac cag caa tcc agt tgg cat cat cta att 34419
get aat gaa ctc aga
Thr 'Iyr Gln Gln Ser Ser Trp His His Leu Ile
Ala Asn Glu Leu Arg
4475 4480 4485
gtg tta gga tta ccg gat ggt aca cgc agt gat 34467
get ttc act tac gat
Val Leu Gly Leu Pro Asp Gly Thr Arg Ser Asp
Ala Phe Thr 2yr Asp
4490 4495 4500
get aaa cac gtg cct gtt gat ggt tta aat ctg 34515
gaa get cta tgt get
Ala Lys His Val Pro Val Asp Gly Leu Asn Leu
Glu Ala Leu Cps Ala
4505 4510 4515
gaa aat agc ctg att gcc gat gat aaa cct cgc 34563
gaa tac ctc aac cag
Glu Asn Ser Leu Ile Ala Asp Asp Lys Pro Arg
Glu 'Iyr Leu Asn Gln
4520 4525 4530 4535
caa cga acg ttc tat acc gat ggg aaa acc gat 34611
gga aaa aat cca acg
Gln Arg 'Inr Phe 'Iyr 'Ilzr Asp Gly Lys Thr
Asp Gly Lys Asn Pro Thr
4540 4545 4550
cca ctg aaa aca ccg aca cga cag get tta atc 34659
gcc ttt acc gaa aeg
Pro Leu Lys Thr Pro Thr Arg Gln Ala Leu Ile
Ala Phe Thr Glu Thr
4555 4560 4565
gcg gta tta acg gaa tct ctg tta tcc gca ttt 34707
gat ggc ggt atc acg
Ala Val Leu 'Ihr Glu Ser Leu Leu Ser Ala Phe
Asp Gly Gly Ile 'Il~r
4570 4575 4580
CA 02320801 2000-08-14
WO 99/42589 PC'T/EP99/01015
-51 -
cca ~t gaa tta ccc ggc ctt ctg aca caa gca 34755
gga tac caa caa gaa
Pro Asp Glu Leu Pro Gly Leu Leu Thr G7n Ala
Gly Tyr Gln Gln Glu
4585 4590 4595
cct tat ctg ttc cca ctc agt ggc gaa aac caa 34803
gtc tgg gta gca cgc
Pro Tyr Leu Phe Pro Leu Ser Gly Glu Asn Gln
Val Trp Val Ala Arg
4600 4605 4610 4615
aaa ggc tat acc gat tac gga act gag gta caa 34851
ttt tgg cgt cct gtc
Lys Gly Tyr Thr Asp Tyr Gly 'I~r Glu Val Gln
Plebe Trp Azg Pro Val
4620 4625 4630
gca caa cgt aac acc cag tta acc ggg aaa acg 34899
act cta aaa tgg gat
Ala Gln Ax~g Asn Thr Gln Leu Ttbr Gly Lys
Thr Thr Leu Lys Trp Asp
4635 4640 4645
acc cac tac tgt gtc atc act caa acc caa gac 34947
gcg get ggt ttg act
'Itbr His Tyr Cps Val Ile Thr Gln Thr Gln
Asp Ala Ala Gly Leu 'rhr
4650 4655 4660
gtc tca gcc aat tat gac tgg cgt ttt ctc aca 34995
cct atg caa ctg act
Val Ser Ala Asn Tyr Asp Txp Arg Phe Leu Thr
Pm Met Gln Leu Thr
4665 4670 4675
gat atc aac gat aat gtg cat atc ata acc ttg 35043
gat gcg cta gga cgc
Asp Ile Asn Asp Asn Val His Ile Ile 'rhr Leu
Asp Ala Leu Gly Arg
4680 4685 4690 4695
cct gtc act caa cgt ttc tgg gga atc gaa aat 35091
ggt gtg gca aca ggt
Pro Val Thr Gln Arg Phe Trp Gly Ile Glu Asn
Gly Val Ala Thr Gly
4700 4705 4710
tac tct tca as gaa gca aaa cca ttc act cca 35139
cca gtc gat gtc aat
Tyr Ser Ser Pro Glu Ala Lys Pro Phe Thr Pro
Pro Val Asp Val Asn
4715 4720 4725
get gcc att get ctg acc gga cca ctc cct gtc 35187
gcg cag tgt ctg gtc
Ala Ala Ile Ala L~eu Thr Gly Pro Leu Pro Val
Ala Gln C~s I~eu Val
4730 4735 4740
tat gcg ccg gac agt tgg atg ccg cta ttc ggt 35235
cag gaa acc ttc aac
Tyr Ala Pro Asp Ser Trp Met Pro L~ Phe Gly
Gln GlWhr Phe Asn
4745 4750 4755
aca tta acg cag gaa gag caa aag aca ctg cgt 35283
gat tta cgg att atc
Thr L~eu Thr Gln Glu Glu Gln Lys Thr Leu Arg
Asp Leu Arg Ile Ile
4760 4765 4770 4775
aca gaa gat tgg cgt att tgc gca ctg get cgc 35331
cgc cgt tgg cta caa
Ttbr Glu Asp Tip Arg Ile Cars Ala Leu Ala
An3 Arg Arg Trg Leu Gln
4780 4785 4790
agt caa aaa gcc ggc aca cca ttg gtt aag ctg 35379
tta acc aac agc atc
Ser Gln Lys Ala Gly 'Ihr Pro Leu Val Lys Leu
Leu Ttbr Asn Ser Ile
4795 4800 4805
ggt tta cct ccc cac aac ctc atg ctg get acg 35427
gac cgt tat gac cgt
Gly Leu Pro Pro His Asn L~ Met Leu Ala Thr
Asp Arg Tyr Asp Arg
4810 4815 4820
gat tct gaa cag caa att cgt caa caa gtc gca 35475
ttc agt gat ggt ttt
Asp Ser Glu Gln Gln Ile Arg Gln Gln Val Ala
Phe Ser Asp Gly Phe
4825 4830 4835
ggc cgt ttg ttg caa gcg get gtg cgg cat gag gca ggc gaa gcc tgg 35523
CA 02320801 2000-08-14
WO 99/42589 PCT/EP99/01015
-52-
Gly Arg Leu I~ Gln Ala Ala Val Arg His Glu Ala Gly Glu Ala Trp
4840 4845 4850 4855
caa cgt aac caa gac ggt tct ctg gtg aca aaa atg gaa gat acc aaa 35571
Gln Ang Asn Gln Asp Gly Ser Leu Val 'lhr Lys Met Glu Asp Thr Lys
4860 4865 4870
acg cgc tgg gcg att acg gga cgc act gaa tat gac aat aag ggg cag 35619
'mr Arg Trp Ala Ile 'Ilzr Gly Arg Thr Glu Tyr Asp Asn Lys Gly Gin
4875 4880 4885
gcg ata cga act tat cag ccc tat ttc ctc aat gac tgg cga tat gtg 35667
Ala Ile Arg Thr Tyr Gln Pro 'Iyr Phe Leu Asn Asp Tip Arg Tyr Val
4890 4895 4900
agt gat gac agc gcc aga aaa gag gcc tat gcc gat act cat atc tat 35715
Ser Asp Asp Ser Ala Arg Lys Glu Ala 'Iyr Ala Asp Tnr His Ile Tyr
4905 4910 4915
gat ccg att ggg cgg gaa atc caa gtt atc acg gca aaa ggc tgg ctg 35763
Asp Pro Ile Giy Arg Glu Ile Gin Val Ile 'I~r Ala Lys Gly Trp Leu
4920 4925 4930 4935
cg~g cag aac caa tat ttc ccg tgg ttt acc gtg agt gaa gat gaa aat 35811
Arg Gln Asn Gln Tyr Phe Pro Trp Phe ~IY~r Val Ser Glu Asp Glu Asn
4940 4945 4950
gat ttg tcc get gac gcg ctc gtg taa ttgaatcaag attcgctcgt 35858
Asp Leu Ser Ala Asp Ala Leu Val
4955 4960
ttaatgttaa cga~gaata taatatacct aatagatttc gagttgcagc gcggcggcaa 35918
gtgaacgaat ccccaggagc atagataact atgtgactgg ggtgagtgaa agcagccaac 35978
aaagcagcag cttgaaagat gaagggtata aataagaaac tgcattgtga gttctaaata 36038
gagtagcagc atattttatt gccttttatt tcataggtaa taaaattcaa ttgctgr_aaa 36098
aatctgtcat catgagaact aaaaataaca actttctctt ctgcaagaga aatcaataat 36158
tcaattaaaa atgttataga atctgaatca agaccatttg ttggctcatc aaaaatataa 36218
acatccgcat cggtaataaa agctgatgtc aatagaaatt tcttttttat cccaagtgac 36278
atatgtccat actcaatacc agaataatta gatataccaa aaccatttaa atagtaatct 3633 8
aattgatatt ttaaattact tttcctataa cgctgactta aattaatcac atccattccc 36398
gtgatgaaat tataaaagtt aacattatcc gatagataaa aaccatgctg ttgcaaatta 36458
aatcggctct tttctccctt ttttataaaa ttaaccattc cttttttaac cttatttaca 36518
ccagcaatac ttgaaagaaa agtcgtttta cccgccccat taactcccgc aatacggttt 36578
aatccaaccc gaaaatcaca attgactcct gaaaaaatag tcttaccatt aataacaacc 36638
tctaacccaa taacttcaag cataaataac ccctaaaaat aacgtaaaaa agaaaataac 36698
accaacaata ataattttcg tgtattgcgt tctcaacaga gaaatagaag aaacaataat 36758
agaaagaaaa gcataagata aaaatataat cacaggaaaa gatttaacaa caagaaagca 36818
aaaaataaaa aaacaaagca aataaaaaaa caaagaaata ccataattaa aaaagaatat 36878
CA 02320801 2000-08-14
WO 99/42589 PCT/EP99/01015
-53-
tttccgcaca gataaaaagt tggacaaata tgaaagataa tttatttcaa tatatgatag 36938
attataaaat aacaacatgc atatatataa aacaacactg gcatatatta atgatatata 36998
atcagccttg tttgggattt gagaaaaggc actttcacat aatagatata aaagcagaac 37058
agataatgcc ataataagca cagacatttt atttttatta aaacaataac gaagattcat 37118
tatataaggc aatgaaaaaa aacctgatga aaataatttt ttatttctat taattatata 37178
acatggtgtg aaattcaaat ataatatcaa tgctactaat ggaataacta atgtaaaaat 37238
caaatcatat aatattccac tcctgaatga tgccgccaga agaaagaaca cagcaacaat 37298
aaaaaaatgc aaaaaactta attcaaataa gcaaaatcca attacagcaa aagaaactat 37358
caaaaaaaac acagatgaaa ggtaatgcaa ataattaaca ttttcgtaaa aaaaacctat 37418
aaagaagaaa ataactatcg gaaaagcact ataaataaaa aaaacgatac gactaaaaaa 37478
caacgttttt ttacctacca aagaaacgat gattgaattc tcctttgcag aaggaaaaaa 37538
ccttatgtta atcaaataaa ataccatata taccattaaa gatatggcag taaaataaaa 37598
tgattttatg tagccatctg gaataataat attggaagat aaagttatta aaacctcaaa 37658
gataccactg aactttgccg gaagtaataa aagaaaaagg aatataatga catttttatt 37718
cccagacgca aatttcttta tcctaccttt atattccaag gcatcagcga ttattaaatt 37778
catactgcct ctctaaaacc aaaatctaaa taatgtcctt ggtgaatctt tagggaattt 37838
cgtcctggaa tgcaaatata aatagttact gaaaacaata cattgatttt taattaaata 37898
ctggcgatat gaccttaatg atgctacttt attttccagt attcaattcg 37948
<210> 12
<211> 954
<212> PRT
<213> Photorhabdus luminescens
<400> 12
Met Lys Asn Ile Asp Pro Lys 1xu 'I'yr Gln Lys Zlzr Pro Val Val Asn
1 5 10 15
Ile 'Iyr Asp Asn Arg Gly Leu Thr Ile Arg Asn Ile Asp Phe His Arg
20 25 30
Thr Thr Ala Asn Gly Asp Thr Asp Ile Arg Ile 'rhr Arg His Gln Tyr
35 40 45
Asp Ser Leu Gly His Leu Ser Gln Ser 'rhr Asp Pro Arg Leu 'iyr Glu
50 55 60
Ala Lys Gln Lys Ser Asn Phe Leu Trp Gln 'Iyr Asp Leu Thr Gly Asn
65 70 75 80
Ile Leu Cys 'Ihr Glu Ser Val Asp Ala Gly Arg 'Itir Val 'I7zr Leu Asn
85 90 95
Asp Ile Glu Gly Arg Pro Leu Leu 'rhr Val 'I'hr Rla 'I'hr Gly Val Ile
100 105 110
Gln Thr Arg Gln 'Iyr Glu Thr Ser Ser Leu Pro Gly Arg Leu Leu Ser
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115 120 125
Val 'Ihr Glu Gln Ile Pro Glu Lys Thr Ser Arg Ile Thr Glu Arg Leu
130 135 140
Ile Trp Ala Gly Asn Ser Glu Ala Glu Lys Asn His Asn Leu Ala Ser
145 150 155 160
Gln Cys Val Arg His 'Iyr Asp 'I~' Ala Gly Val 22zr Arg Leu Glu Ser
165 170 175
Leu Ser Leu 2hr Gly 'I4zr Val Leu Ser G1n Ser Ser Gln Leu Leu Ser
180 185 190
Asp Thr Gln Glu Ala Ser 'I~p Thr Gly Asp Asn Glu Thr Val Trp Gln
195 200 205
Asn Met Leu Ala Asp Asp Ile 'Iyr 'Ihr Thr Leu Ser Ala Phe Asp Ala
210 215 220
Thr Gly Ala Ieu 1xu 'Ihr Gln 'Ilzr Asp Ala Lys Gly Asn Ile Gln Arg
225 230 235 240
Leu Thr 'iyr Asp Val Ala Gly Gln Leu Asn Gly Ser Trp Leu 'Ihr Leu
245 250 255
Lys Asp Gln Pro Glu Gln Val Ile Ile Arg Ser Leu Thr Tyr Ser Ala
260 265 270
Ala Gly Gln Lys Leu Arg Glu Glu His Gly Asn Gly Val Ile Zhr Glu
275 280 285
'Iyr Ser Tyr Glu Pro Glu 'rhr Gln Gln Leu Ile Gly'hr Lys 'I9zr His
290 295 300
Arg Pro Ser Asp Ala Lys Val Leu Gln Asp Lieu Arg 'Iyr Glu Tyr Asp
305 310 315 320
Pro Val Gly Asn Val Ile Ser Ile Arg Asn Asp Ala Glu Ala 'l~r Arg
325 330 335
Pl~e Trp His Asn Gln Lys Val Ala Pro Glu Asn Thr Tyr 'ilzr 2yr Asp
340 345 350
Ser Leu 'Iyr Gln Leu Ile Ser Ala Thr Gly Arg Glu Met Ala Asn Ile
355 360 365 '
Gly Gln Gln Ser Asn Gln Leu Pro Ser Leu 'Inr Leu Pro Ser Asp Asn
370 375 380
Asn 'Ifir 'Iyr '13~r Asn Zyr Thr Arg Thr 'Iyr 'I9~r 'tyr Asp Arg Gly Gly
385 390 395 400
Asn Leu 'Ihr Lys Ile Gln His Ser Ser Pro Ala 'Ihr Gln Asn Asn 'Iyr
405 410 415
'I'hr 'Ihr Asn Ile Thr Val Ser Asn Arg Ser Asn Arg Ala Val Leu Ser
420 425 430
Thr Leu Thr Glu Asp Pro Ala Gln Val Asp Ala Leu Phe Asp Ala Gly
435 440 445
Gly His Gln Asn Thr Leu Ile Ser Gly Gln Asn Leu Asn Trp Asn Thr
450 455 460
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Arg Gly Glu Leu Gln His Val 'Ilzr I~u Val Lys Arg Asp Lys Gly Ala
465 470 475 480
Asn Asp Asp Arg Glu Trp 'Iyr Arg Tyr Ser Ser Asp Gly Arg Arg Ile
485 490 495
Leu Lys Ile Asn Glu G7n G1n Zhr Ser Ser Asn Ser Gln Thr Gln Arg
500 505 510
Ile Zlzr 'Iyr Leu Pro Ser 1xu Glu Leu Arg Leu 'Inr Gln Asn Ser Thr
515 520 525
Ile Thr Thr Glu Asp Leu Gln Val Ile Thr Val Gly Glu Ala Gly Arg
530 535 540
Ala Gln Val Arg Val Leu His Trp Asp Ser Gly Gln Pro Glu Asp Ile
545 550 555 560
Asp Asn Asn Gln Leu Arg 'I~rr Ser ~I~r Asp Asn Leu Ile Gly Ser Ser
565 570 575
Gln Leu Glu Leu Asp Ser Lys Gly Glu Ile Ile Ser Glu Glu Glu Zyr
580 585 590
'tyr Pro Zyr Gly Gly Thr Ala Leu Txp Ala 'rhr Arg Lys Arg Thr Glu
595 600 605
Ala Ser ~tyr Lys 'Ihr Ile Arg Tyr Ser Gly Lys Glu Arg Asp Ala Thr
610 615 620
Gly Leu err Tyr 'Iyrr Gly 'Iyz~ Arg Tyr Zyr Gln Pro Trp Val Gly Arg
625 630 635 640
Ttp Leu Ser Ala Asp Pro Ala Gly Zhr Val Asp Gly Leu Asn Leu Tyr
645 650 655
Arg Met Val Arg Asn Asn Pro Val 'Ilzr Leu Leu Asp Pro Asp Gly Leu
660 665 670
Met Pro 'Ihr Ile Ala Glu Arg Ile Ala Ala Leu Gln Lys Asn Lys Val
675 680 685
Ala Asp Ser Ala Pro Ser Pro Thr Asn Ala Thr Asn Val Ala Ile Asn
690 695 700
Ile Arg Pro Pro Val Ala Pro Lys Pro Thr Leu Pro Lys Ala Ser Thr
705 710 715 720
Ser Ser Gln Ser 'Ihr Thr 'I~rr Pro Ile Lys Ser Ala Ser Ile Lys Pro
725 730 735
'lfir Thr Ser Gly Ser Ser Ile 'Ihr Ala Pro Leu Ser Pro Val Gly Asn
740 745 750
Lys Ser Thr Pro Glu Ile Ser Leu Pro Glu Ser 'Ifir Gln Ser Asn Ser
755 760 765
Ser Ser Ala Ile Ser 'Inr Asn Leu Gln Lys Lys Ser Phe Thr Leu 'Iyr
770 775 780
Arg Ala Asp Asn Arg Ser Phe Glu Asp Met Gln Ser Lys Phe Pro Glu
785 790 795 800
Gly Phe Lys Ala Trp Thr Pro Leu Asp Thr Lys Met Ala Arg Gln Phe
805 810 815
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Ala Ser Val Phe Ile Gly Gln Lys Asp Thr Ser Asn Leu Pro Lys Glu
820 825 830
Thr Val Lys Asn Ile Asn Thr Trp Gly Thr Lys Pro Lys L~u Asn Asp
835 840 845
Leu Ser 'Ihr 'Iyr Ile Lys 2~r Thr Lys Asp Lys Ser 'Ilzr Val Trp Val
850 855 860
Ser 'Ilzr Ala Ile Asn Thr Glu Ala Gly Gly Gln Ser Ser Gly Ala Pro
865 870 875 880
Leu His Glu Ile Asn Met Asp Leu 'Iyr Glu Phe 'Ihr Ile Asp Gly Gln
885 890 895
Lys Leu Asn Pro Lsu Pro Arg Gly Arg Ser Lys Asp Arg Val Pro Ser
900 905 910
Leu Leu Leu Asp Thr Pro Glu Ile Glu 'I'hr Ala Ser Ile Ile Ala Leu
915 920 925
Asn His Gly Pro Val Asn Asp Ala Glu Val Ser Phe Leu Thr Zhr Ile
930 935 940
Pro Lsu Lys Asn Val Lys Pro 'Iyr Lys And
945 950
<210> 13
<211> 2522
<212> PRT
<213> Photorhabdus luminescens
<400> 13
Met Ile Leu Lys Gly Ile Asn Met Asn Ser Pro Val Lys Glu Ile Pro
1 5 10 15
Asp Val Leu Lys Ile Gln Cys Gly Phe Gln Cys Leu Thr Asp Ile Ser
20 25 30
His Ser Ser Phe Asn Glu Phe His Gln Glri Val Ser Glu His Leu Ser
35 40 45
Trp Ser Glu Ala His Asp Leu 'I~~r His Asp Ala Gln G7n Ala Gln Lys
50 55 60
Asp Asn Arg I~eu 'Iyr Glu Ala Arg Ile 1xu Lys Arg Thr Asn Pro Gln
65 70 75 80
Leu Gln Asn Ala Val His Leu Ala Ile Val Ala Pro Asn Ala Glu Leu
85 90 95
Ile Gly 'Iyr Asn Asn Gln Phe Ser Gly Arg Ala Ser G7n 'Iyr Val Ala
100 105 110
Pro Giy 'ihr Val Ser Ser Met Phe Ser Pro Ala Ala 'Iyx~ Leu 'rhr Glu
115 120 125
Leu 'Iyr Arg Glu Ala Arg Asn Leu His Ala Ser Asp Ser Val ~Iyr Arg
130 135 140
Leu Asp Thr Arg Arg Pro Asp Leu Lys Ser Met Ala L~ Ser Gln Gln
145 150 155 160
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Asn Met Asp Ttir Glu Leu Ser Thr Leu Ser Leu Ser Asn Glu Leu Leu
165 170 175
Leu Glu Ser Ile Lys Thr Glu Ser Lys Leu Asp Asn Tyr 'I'hr Gln Val
180 185 190
Met Glu Met Leu Ser Ala Phe Arg Pro Ser Gly Ala Zhr Pro 'Iyr His
195 200 205
Asp Ala 'Iyr Glu Asn Val Arg Lys Val Ile Gln Leu Gln Asp Pro Gly
210 215 220
Leu Glu Gln Leu Asn Ala Ser Pro Ala Ile Ala Gly Leu Met His Gln
225 230 235 240
Ala Ser re" Leu Gly Ile Asn Ala Ser Ile Ser Pro Glu Leu Phe Asn
245 250 255
Ile Leu Thr Glu Glu Ile Thr Glu Gly Asn Ala Glu Glu Leu 'Iyr Lys
260 265 270
Lys Asn Phe Gly Asn Ile Glu Pro Ala Ser L~ Ala Met Pro Glu Tyr
275 280 285
Leu Arg Arg 'Iyr 'Iyr Asn Leu Ser Asp Glu Glu Leu Ser Gln Phe Ile
290 295 300
Gly Lys Ala Ser Asn Ph,e Gly Gln Gln Glu Tyr Ser Asn Asn Gln Leu
305 310 315 320
Ile Thr Pro Ile Val Asn Ser Asn Asp Gly Thr Val Lys Val Tyr Arg
325 330 335
Ile 'Ihr Arg Glu 2yr 'It~r 'I'hr Asn Ala Asn Gln Val Asp Val Glu Leu
340 345 350
Phe Pro Tyr Gly Gly Glu Asn Tyr Gln Leu Asn Tyr Lys Phe Lys Asp
355 360 365
Ser Arg Gln Asp Val Ser Tyr Lieu Ser Ile Lys L~eu Asn Asp Lys Arg
370 375 380
Glu Leu Ile Arg Ile Glu Gly Ala Pro Gln Val Asn Ile Glu Tyr Ser
385 390 395 400
Glu His Ile Zizr Leu Ser Thr Thr Asp Ile Ser Gln Pro Phe Glu Ile
405 410 415
Gly Leu Thr Arg Val Tyr Pro Ser Ser Ser Trp Ala Zyr Ala Ala Ala
420 425 430
Lys Phe 'I'hr Ile Glu Glu 'Iyr Asn Gln 'Iyr Ser Phe Leu Leu Lys Leu
435 440 445
Asn Lys Ala Ile Arg Leu Ser Arg Ala 'I~r Glu Leu Ser Pro 'Ihr Ile
450 455 460
Leu Glu Ser Ile Val Arg Ser Val Asn Gln Gln Leu Asp Ile Asn Ala
465 470 475 480
Glu Val Leu Gly Lys Val Phe Leu 'Tlzr Lys 'iyr Zyr Met Gln Arg Tyr
485 490 495
Ala Ile Asn Ala Glu 'Ihr Ala Leu Ile Leu Cys Asn Ala Ixu Ile Ser
500 505 510
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Gln Arg Ser 'Iyr Asp Asn Gln Pro Ser Gln Phe Asp Arg Leu Phe Asn
515 520 525
Thr Pro Leu Leu Asn Gly Gln ~lyr Pk~e Ser Thr Gly Asp Glu Glu Ile
530 535 540
Asp Leu Asn Pro Gly Ser Thr Gly Asp Trp Arg Lys Ser Val 1xu Lys
545 550 555 560
Arg Ala Phe Asn Ile Asp Asp Ile Ser Leu Zyr Arg Leu Leu Lys Ile
565 570 575
'Ihr Asn His Asn Asn Gln Asp Gly Lys Ile Lys Asn Asn Leu Asn Asn
580 585 590
L~eu Ser Asp Leu Tyr Ile Gly Lys 1xu Leu Ala Glu Ile His Gln Leu
595 600 605
'Ihr Ile Asp Glu Lieu Asp Lieu I~eu Leu Val Ala Val Gly Glu Gly Glu
610 615 620
Thr Asn Leu Ser Ala Ile Ser Asp Lys Gln Leu Ala Ala Leu Ile Arg
625 630 635 640
Lys Leu Asn Thr Ile I'hr Val Trp Leu Gln Thr Gln Lys Trp Ser Ala
645 650 655
Phe Gln Leu Phe Val Met Thr Ser 'Ilzr Ser Tyr Asn Lys Thr Leu 'Ilzr
660 665 670
Pro Glu Ile Lys Asn Leu Leu Asp 'Ihr Val 'lyr His Gly Leu Gln Gly
675 680 685
Phe Asp Lys Asp Lys Ala Asn Leu Leu His Val Met Ala Pro 'I~rr Ile
690 695 700
Ala Ala Thr Leu Gln Leu Ser Ser Glu Asn Val Ala His Ser Val Leu
705 710 715 720
Leu Trp Ala Asp Lys Leu Lys Pro Gly Asp Gly Ala Met Thr Ala Glu
725 730 735
Lys Phe Trp Asp Trp Leu Asn 'Itir Gln 2yr Thr Pro Asp Ser Ser Glu
740 745 750
Val Leu Ala Thr Gln Glu His Ile Val Gln 'Iyr Cys Gln Ala Leu Ala
755 760 765
Gln Leu Glu Met Val 'Iyr His Ser Thr Gly Ile Asn Glu Asn Ala Phe
770 775 780
Arg Leu Phe Val T'hr Lys Pro Glu Met Phe Gly Ser Ser Thr Glu Ala
785 790 795 800
Val Pro Ala His Asp Ala Leu Ser Leu Ile Met Leu Thr Arg Phe Ala
805 810 815
Asp Trp Val Asn Ala Leu Gly Glu Lys Ala Ser Ser Val Leu Ala Ala
820 825 830
Phe Glu Ala Asn Ser Leu Thr Ala Glu Gln Leu Ala Asp Ala Met Asn
835 840 845
Leu Asp Ala Asn Leu Leu Leu Gln Ala Ser Thr Gln Ala Gln Asn His
CA 02320801 2000-08-14
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850 855 860
Gln His Leu Pro Pro Val Thr Gln Lys Asn Ala Phe Ser Cps Trp '1'hr
865 870 875 880
Ser Ile Asp 'i'hr Ile Leu Gln Trp Val Asn Val Ala Gln Gln Leu Asn
885 890 895
Val Ala Pro Gln Gly Val Ser Ala Ieu Val Gly Leu Asp 'Iyr Ile Gln
900 905 910
L~ Asn Gln Lys Ile Pro Thr 'I~r Ala Gln Trp Glu Ser Ala Gly Glu
915 920 925
Ile Leu Thr A,la Gly Leu Asn Ser Gln Gln Ala Asp Ile Leu His Ala
930 935 940
Phe Leu Asp Glu Ser Arg Ser Ala Ala Leu Ser Thr Tyr Zyr Ile Arg
945 950 955 960
Gln Val Ala Lys Pro Ala Ala Ala Ile Lys Ser Arg Asp Asp Leu 'Iyr
965 970 975
Gln Tyr Leu Leu Ile Asp Asn Gln Val Ser Ala Ala Ile Lys ~Ilzr Thr
980 985 990
Arg Ile Ala Glu Ala Ile Ala Ser Ile Gln Leu 'I~r Val Asn Arg Thr
995 1000 1005
Leu Glu Asn Val Glu Glu Asn Aia His Ser Gly Val Ile Ser Arg Gln
1010 1015 1020
Phe Pie Ile Asp Trp Asp Lys 'Iyr Asn Lys Arg 'Iyr Ser Thr Trp Ala
025 1030 1035 1040
Gly Val Ser Gln Leu Val Tyr err Pro Glu Asn 'Iyr Ile Asp Pro ~'hr
1045 1050 1055
Met Arg Ile Gly Gln Thr Lys Met Met Asp Ala Leu Leu Gln Ser Val
1060 1065 1070
Ser Gln Ser Gln Leu Asn Ala Asp Thr Val Glu Asp Ala Phe Met Ser
1075 1080 1085
err Leu Thr Ser Phe Glu Gln Val Ala Asn Leu Lys Val Ile Ser Ala
1090 1095 1100
'Iyr I-Iis Asp Asn Ile Asn Asn Asp Gln Gly Leu Thr 'Iyr Phe Ile Gly
105 1110 1115 1120
Leu Ser Glu Zlzr Asp Thr Gly Glu 'Iyr 'Iyr Trp Arg Ser Val Asp His
1125 1130 1135
Ser Lys Plve Ser Asp Gly Lys Ptie Ala Ala Asn Ala Trp Ser Glu Trp
1140 1145 1150
His Lys Ile Asp Cys Pro Ile Asn Pro 'Iyr Arg Ser 'Ilzr Ile Arg Pro
1155 1160 1165
Val Met 'Iyr Lys Ser Arg L~eWyr Leu Leu Trp Leu Glu Gln Lys Glu
1170 1175 1180
Ile 'Ifir Lys Gln Ztir Gly Asn Ser Lys Asp Gly 'Iyr Gln Thr Glu T'hr
185 1190 1195 1200
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Asp Tyr Arg Tyr Glu Leu Lys Leu Ala His Ile Arg Tyr Asp Gly Thr
1205 1210 1215
Tip Asn Thr Pro Ile Thr Phe Asp Val Asn Glu Lys Ile Ser Lys Leu
1220 1225 1230
Glu Leu Ala Lys Asn Lys Ala Pro Gly I~ Tyr Cys Ala Gly Tyr Gln
1235 1240 1245
Gly Glu Asp Thr Lsu Leu Val Met Phe Tyr Asn Gln Gln Asp Thr Lieu
1250 1255 1260
Asp Ser Tyr Lys Thr Ala Ser Met Gln Gly Ixu Tyr Ile Phe Ala Asp
265 1270 1275 1280
Met Glu Tyr Lys Asp Met Thr Asp Gly Gln Tyr Lys Ser Tyr Arg Asp
1285 1290 1295
Asn Ser Tyr Lys Gln Plve Asp Thr Asn Ser Val Arg Arg Val Asn Asn
1300 1305 1310
Arg Tyr Ala Glu Asp Tyr Glu Ile Pro Ser Ser Val Asn Ser Arg Lys
1315 1320 1325
Gly Tyr Asp Trp Gly Asp Tyr Tyr Leu Ser Met Val Tyr Asn Gly Asp
1330 1335 1340
Ile Pro '1'hr Ile Ser Tyr Lys Ala Thr Ser Ser Asp Leu Lys Ile Tyr
345 1350 1355 1360
Ile Ser Pro Lys Leu Arg Ile Ile Isis Asn Gly Tyr Glu Gly Gln Gln
1365 1370 1375
Arg Asn Gln Cys Asn Leu Met Asn Lys Tyr Gly Lys Leu Gly Asp Lys
1380 1385 1390
Phe Ile Val Tyr Thr Ser Leu Gly Val Asn Pro Asn Asn Ser Ser Asn
1395 1400 1405
Lys Leu Met Phe Tyr Pro Val Tyr Gln Tyr Asn Gly Asn Val Ser Gly
1410 1415 1420
Leu Ser Gln Gly Arg i~eu I~eu Phe His Arg Asp Thr Asn Tyr Ser Ser
425 1430 1435 1440
Lys Val Glu Ala Trp Ile Pro Gly Ala Gly Arg Ser Leu Thr Asn Pro
1445 1450 1455
Asn Ala Ala Ile Gly Asp Asp Tyr Ala Thr Asp Ser Leu Asn Lys Pro
1460 1465 1470
Asn Asp Leu Lys Gln Tyr Val Tyr Met Thr Asp Ser Lys Gly Thr Ala
1475 1480 1485
Thr Asp Val Ser Gly Pro Val Asp Ile Asn Thr Ala Ile Ser Pro Ala
1490 1495 1500
Lys Val Gln Val Thr Val Lys Ala Gly Ser Lys Glu Gln Thr Phe Thr
505 1510 1515 1520
Ala Asp Lys Asn Val Ser Ile Gln Pro Ser Pro Ser Phe Asp Glu Met
1525 1530 1535
Asn Tyr Gln Phe Asn Ala Leu Glu Ile Asp Gly Ser Ser Leu Asn Phe
1540 1545 1550
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Thr Asn Asn Ser Ala Ser Ile Asp Ile 'Ihr Phe 't'hr Ala Phe Ala Glu
1555 1560 1565
Asp Gly Arg Lys lxu Gly Tyr Glu Ser Phe Ser Ile Pro Ile Thr Arg
1570 1575 1580
Lys Val Ser Thr Asp Asn Ser Lieu Thr Ixu Arg His Asn Glu Asn Gly
585 1590 1595 1600
Ala Gln Tyr Met Gln Trp Gly Val Tyr Arg Ile Arg Leu Asn Thr Leu
1605 1610 1615
Phe Ala Arg Gln Leu Val Ala Arg Ala 'rhr Thr Gly Ile Asp Thr Ile
1620 1625 1630
Leu Ser Met Glu 'I~r Gln Asn Ile Gln Glu Pro Gln Leu Gly Lys Gly
1635 1640 1645
Phe Tyr Ala Zlzr Phe Val Ile Pro Pro Tyr Asn Pro Ser Thr His Gly
1650 1655 1660
Asp Glu Arg Trp Phe Lys Leu Tyr Ile Lys His Val Val Asp Asn Asn
665 1670 1675 1680
Ser His Ile Ile Tyr Ser Gly Gln Leu Lys Asp Thr Asn Ile Ser Thr
1685 1690 1695
Thr Leu Phe Ile Pro Leu Asp Asp Val Pro Leu Asn Gln Asp Tyr Ser
1700 1705 1710
Ala Lys Val Tyr Met Thr Phe Lys Lys Ser Pro Ser Asp Gly Thr Trp
1715 1720 1725
Tip Gly Pro His Phe Val Arg Asp Asp Lys Gly Ile Val 'Inr Ile Asn
1730 1735 1740
Pro Lys Ser Ile Leu Thr His Phe Glu Ser Val Asn Val Leu Asn Asn
745 1750 1755 1760
Ile Ser Sex Glu Pro Met Asp Phe Ser Gly Ala Asn Ser Leu Tyr Phe
1765 1770 1775
Txp Glu Leu Phe Tyr Tyr 'Ihr Pro Met Leu Val Ala Gln Arg Leu Leu
1780 1785 1790
His Glu Gln Asn Phe Asp Glu Ala Asn Arg Trp Leu Lys Tyr Val Trp
1795 1800 1805
Ser Pro Ser Gly Tyr Ile Val His Gly Gln Ile Gln Asn Tyr Gln Trp
1810 1815 1820
Asn Val Arg Pro Leu Leu Glu Asp Thr Ser Trp Asn Ser Asp Pro Leu
825 1830 1835 1840
Asp Ser Val Asp Pro Asp Ala Val Ala Gln His Asp Pro Met His Tyr
1845 1850 1855
Lys Val Ser Thr Phe Met Arg Thr Lsu Asp Leu Leu Ile Ala Arg Gly
1860 1865 1870
Asp His Ala Tyr Arg Gln L~ Glu Arg Asp 'Ihr Leu As-n Glu Ala Lys
1875 1880 1885
Met Trp Tyr Met Gln Ala 1xu His Leu Leu Gly Asp Lys Pro Tyr Leu
CA 02320801 2000-08-14
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1890 1895 1900
Pro Leu Ser Thr 'l~r Trp As-n Asp Pro Arg Leu Asp Lys Ala Ala Asp
905 1910 1915 1920
Ile 'Ilzr Thr Gln Ser Ala His Ser Ser Ser Ile Val Ala Leu Arg Gln
1925 1930 1935
Ser Thr Pro Ala Leu Leu Ser Leu Arg Ser Ala Asn 'rhr Leu 'Itir Asp
1940 1945 1950
Leu Phe Leu Pro Gln Ile Asn Glu Val Met Met Asn 'Iyr Trp Gln 2hr
1955 1960 1965
Leu Ala Gln Arg Val 2~rr Asn Leu Arg His Asn Leu Ser Ile Asp Gly
1970 1975 1980
Gln Pro Leu 'Iyr Leu Pro Ile 'Iyr Ala Thr Pro Ala Asp Pro Lys Ala
985 1990 1995 2000
Leu Leu Ser Ala Ala Val Ala Thr Ser Gln Gly Gly Gly Lys Leu Pro
2005 2010 2015
Glu Ser Phe Met Ser Leu Trp Arg Phe Pro His Met Leu Glu Asn Ala
2020 2025 2030
Axg Ser Met Val Ser Gln Leu 'itir Gln Phe Gly Ser Thr Leu Gln Asn
2035 2040 2045
Ile Ile Glu Arg Gln Asp Ala Glu Ala Leu Asn Ala Leu Leu Gln Asn
2050 2055 2060
Gln Ala Ala Glu Leu Ile Leu Zhr Asn Lieu Ser Ile Gln Asp Lys 'lhr
065 2070 2075 2080
Ile Glu Glu Leu Asp Ala Glu Lys Thr Val Isu Glu Lys Ser Lys Ala
2085 2090 2095
Gly Ala Gln.Ser Arg Phe Asp Ser 'Iyr Ser Lys Leu His Asp Glu Asn
2100 2105 2110
Ile Asn Ala Gly Glu Asn Gln Ala Met 'Ihr I~eu Arg Ala Ser Ala Ala
2115 2120 2125
Gly Leu '1'hr Zl~r Ala Val Gln Ala Ser Arg Leu Ala Gly Ala Ala Ala
2130 2135 2140
Asp I~eu Val Pro Asn Ile Phe Gly Phe Ala Gly Gly Gly Ser Arg Trp
145 2150 2155 2160
Gly Ala Ile Ala Glu Ala Thr Gly Tyr Val Met Glu Phe Ser Ala Asn
2165 2170 2175
Val Met Asn ~I9zr Glu Ala Asp Lys Ile Ser Gln Ser Glu Thr Tyr Arg
2180 2185 2190
Arg Arg Arg Gln Glu Trp Glu Ile Gln Arg Asn Asn Ala Glu Ala Glu
2195 2200 2205
Leu Lys Gln Leu Asp Ala Gln Leu Lys Ser Leu Ala Val Arg Arg Glu
2210 2215 2220
Ala Ala Val L~eu Gln Lys 'Itir Ser Leu Lys Thr Gln Gln Glu Gln Thr
225 2230 2235 2240
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Gln Ala Gln Leu Ala Phe Leu Gln Arg Lys Phe Ser Asn Gln Ala Leu
2245 2250 2255
Tyr Asn Trp Leu Arg Gly Arg Leu Ala Ala Ile Tyr Phe Gln Phe Zyr
2260 2265 2270
Asp Tati Ala Ile Ala Arg Cys Leu Met Ala Glu Gln Ala '1)rr Arg Trp
2275 2280 2285
Glu Ile Ser Asp Asp Ser Ala Arg Phe Ile Lys Pro Gly Ala Trp Gln
2290 2295 2300
Gly 'rhr 'Iyr Ala Gly Leu I~eu Ala Gly Glu Thr Leu Met Leu Ser Leu
305 2310 2315 2320
Ala Gln Met Glu Asp Ala His re,i Arg Arg Asp Lys Arg Ala Leu Glu
2325 2330 2335
Val Glu An3 Thr Val Ser Lieu Ala Glu Ile Zyr Ala Gly I~eu Pro Gln
2340 2345 2350
Asp Lys Gly Pro PYie Ser Ixu 'Ilzr Gln Glu Ile Glu Lys Lieu Val Asn
2355 2360 2365
Ala Gly Ser Gly Ser Ala Gly Ser Gly Asn Asn Asn Leu Ala PY~e Gly
2370 2375 2380
Ala Gly Thr Asp Thr Lys T3Zr Ser Leu Gln Ala Ser Ile Ser Leu Ala
385 2390 2395 2400
Asp Leu Lys Ile Arg Glu Asp Ayr Pro Glu Ser Ile Gly Lys Ile Arg
2405 2410 2415
Arg Ile Lys Gln Ile Ser Val Thr Leu Pro Ala Leu Leu Gly Pro Tyr
2420 2425 2430
Gln Asp Val Gln Ala Ile Leu Ser Tyr Gly Asp Lys Ala Gly Leu Ala
2435 2440 2445
Asn Gly Cys Ala Ala Leu Ala Val Ser His Gly 'rhr Asn Asp Ser Gly
2950 2455 2460
Gln Phe Gln Leu Asp Phe Asn Asp Gly Lys Phe Leu Pro Phe Glu Gly
465 2470 2475 2480
Ile Ala Ile Asp Gln Gly 'Itir Leu '1lzr Leu Ser Phe Pro Asn Ala Ser
2485 2490 2495
Thr Pro Ala Lys Gly Lys Gln Ala Thr Met Leu Lys Thr Leu Asn Asp
2500 2505 2510
Ile Ile Leu His Ile Arg 'I~r Thr Ile Lys
2515 2520
<210> 14
<211> 1481
<212> PRT
<213> Photorhabdus luminesc~s
<400> 14
Met Gln Asn Ser Gln Thr Phe Ser Met 'I'~r Glu Leu Ser Leu Pro Lys
1 5 10 15
Gly Gly Gly Ala Ile 2hr Gly Met Gly Glu Ala Leu Thr Pro Ala Gly
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20 25 30
Pro Asp Gly Met Ala Ala Leu Ser I~u Pro Leu Pro Ile Ser Ala Gly
35 40 45
Arg Gly err Ala Pro Ser Leu Thr Leu Asn 'Iyr Asn Ser Gly 2lzr Gly
50 55 60
Asn Ser Pro Phe Gly Leu Gly Trp Asp Cys Asn Val Met Thr Tle Arg
65 70 75 80
Arg Arg 'Ihr Ser 'Ihr Gly Val Pro Asn ~Iyr Asp Glu 'Ihr Asp Thr Phe
85 90 95
Leu Gly Pro Glu Gly Glu Val Leu Val Val Ala Leu Asn Glu Ala Gly
100 105 110
Gln Ala Asp Ile Arg Ser Glu Ser Ser Leu Gln Gly Ile Asn Leu Gly
115 120 125
Met Thr Phe 'rhr Val 'Ihr Gly Tyr Arg Ser Arg Leu Glu Ser His Phe
130 135 140
Ser Arg Leu Glu Z]rr Txp Gln Pro Gln I'hr Thr Gly Ala Thr Asp Phe
145 150 155 160
Trp Leu Ile Ayr Ser Pro Asp Gly Gln Ala His L~ Leu Gly Lys Asn
165 170 175
Pro Gln Ala Arg Ile Ser Asn Pro Leu Asn Val Asn Gln Z'hr Ala Gln
180 185 190
Trp Leu Leu Glu Ala Ser Val Ser Ser His Gly Glu Gln Ile err Tyr
195 200 205
Gln err Arg Ala Glu Asp Glu Zl~r Asp Cps Glu Thr Asp Glu Lieu Zhr
210 215 220
Ala His Pro Asn 'Ihr I'hr Val Gln Arg Zyr Leu Gln Val Val His 'I~r
225 230 235 240
Gly Asn Leu Thr Ala Ser Glu Val Phe Pro Thr L~eu Asn Gly Asp Asp
245 250 255
Pro Leu Lys Ser Gly Trp Leu Pie Cys Leu Val Phe Asp Tyr Gly Glu
260 265 270
Arg Lys Asn Ser Leu Ser Glu Met Pro Pro Phe Lys Ala Thr Sex Asn
275 280 285
Trp Leu Cars Arg Lys Asp Arg 1~ Sex' Arg Zyr Glu 'Iyr Gly Phe Ala
290 295 300
Leu Arg 'Ihr Arg Arg Leu Cys Arg Gln Ile Leu Met Phe His Arg Leu
305 310 315 320
Gln 'Ihr Leu Ser Gly Gln Ala Lys Gly Asp Asp Glu Pro Ala Leu Val
325 330 335
Ser Arg Leu Ile Leu Asp 'Iyr Asp Glu Asn Ala Val Val Ser 'Itm Leu
340 345 350
Val Ser Val Arg Arg Val Gly His Glu Gln Asp Gly 'Phr I'hr Ala Val
355 360 365
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Ala Leu Pro Pro Leu Glu Leu Ala Tyr Gln Pro Phe Glu Pro Glu Gln
370 375 380
Lys Ala Leu Trp Arg Pro Met Asp Val Leu Ala Asn Phe Asn Thr Ile
385 390 395 400
Gln Arg Tip Gln Leu Lieu Asp Leu Gln Gly Glu Gly Val Pro Gly Ile
405 410 415
Leu Tyr Gln Asp Lys Asn Gly Txp Trp Tyr Arg Ser Ala Gln Arg Gln
420 425 430
Thr Gly Glu Glu Met Asn Ala Val 'Ihr Trp Gly Lys Met Gln Leu Leu
435 440 445
Pro Ile Thr Pro Ala Ile Gln Asp Asn Ala Ser Leu Met Asp Ile Asn
450 455 460
Gly Asp Gly Gln Leu Asp Trp Val Ile Z'hr Gly Pro Gly Leu And Gly
465 470 475 480
Tyr His Ser Gln His Pro Asp Gly Ser Txp Thr Arg Phe Thr Pro Leu
485 490 495
His Ala Leu Pro Ile Glu Tyr Thr His Pro Arg Ala Gln Leu Ala Asp
500 505 510
Leu Met Gly Ala Gly Lieu Ser Asp Leu Val Leu Ile Gly Pro Lys Ser
515 520 525
Val Arg Leu Tyr Ala Asn Asn Arg Asp Gly Phe Z'hr Glu Gly Arg Asp
530 535 540
Val Val Gln Ser Gly Gly Ile Thr Leu Pro Leu Pro Gly Ala Asp Ala
545 550 555 560
Arg Lys Leu Val Ala Phe Ser Asp Val Leu Gly Ser Gly Gln Ala His
565 570 575
Leu Val Glu Val Ser Ala Thr Lys Val Thr Cys Trp Pro Asn Leu Gly
580 585 590
His Gly Arg Phe Gly Gln Pro Ile Thr Leu Pro Gly Pile Ser Gln Ser
595 600 605
Ala Ala Asn Phe Asn Pro Asp Arg Val His Leu Ala Asp Leu Asp Gly
610 615 620
Ser Gly Pro Ala Asp Leu Ile Tyr Val His Ala Asp His Leu Asp Ile
625 630 635 640
Phe Ser Asn Glu Ser Gly Asn Gly Phe Ala Gln Pro Phe Thr Leu Arg
645 650 655
Phe Pro Asp Gly Leu Arg Phe Asp Asp Thr Cys Gln Leu Gln Val Ala
660 665 670
Asp Val Gln Gly Leu Gly Val Val Ser Leu Ile Leu Ser Val Pro His
675 680 685
Met Ala Pro His His Trp Arg Cys Asp Leu ~'hr Asn Ala Lys Pro Trp
690 695 700
Leu Leu Ser Glu Met Asn Asn Asn Met Gly Ala His His Ihr Leu His
705 710 715 720
CA 02320801 2000-08-14
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Tyr Arg Ser Ser Val Gln Phe Trp Leu Asp Glu Lys Ala Ala Ala Leu
725 730 735
Ala 1'hr Gly Gln ~hr Pro Val Cys 'Iyr Leu Pro Phe Pro Val His Thr
740 745 750
Leu Trp Gln Zhr Glu Thr Glu Asp Glu Ile Ser Gly Asn Lys Leu Val
755 760 765
1'hr ~r Leu Arg 'Iyr Ala His Gly Ala Trp Asp Gly Arg Glu Arg Glu
770 775 780
Fyhe Arg Gly Phe Gly Zyr Val Glu Gln 'It~r Asp Ser His Gln Leu Ala
785 790 795 800
Gln Gly Asn Ala Pro Glu Arg Thr Ser Pro Ala Leu Thr Lys Asn Trp
805 810 815
'Iyr Ala Thr Gly Ile Pro Glu Val Asp Asn 'I'hr Leu Ser Ala Gly 'i~r
820 825 830
Trp Arg Gly Asp Thr Gln Ala Phe Thr Gly Pte Thr Pro His Phe 'rhr
835 840 845
Leu Trp Lys Glu Gly Lys Asp Val Pro Leu Thr Pro Glu Asp Asp His
850 855 860
Asn Leu 'Iyr Trp Leu Asn Arg Ala Leu Lys Gly Gln Pro Leu Arg Ser
865 870 875 880
Glu Leu Tyr Gly Leu Asp Gly Sex Ala Gln Gln Lys Ile Pro Tyr Thr
885 890 895
Val Thr Glu Ser Arg Pro Gln Val Arg Gln Leu Gln Asp Asn xhr 2~r
900 905 910
Leu Ser Pro Val Leu Txp Ala Ser Val Val Glu Ser Arg Ser Tyr His
915 920 925
Tyr Glu Arg Ile Ile Ser Asp Pro Gln (.ys Asn Gln Asp Ile Thr 1xu
930 935 940
Ser Ser Asp Leu Phe Gly Gln Pro Leu Lys Gln Val Ser Val Gln Tyr
945 9S0 955 960
Pro Arg Arg Asn Lys Pro Thr Zhr Asn Pro Tyr Pro Asp Thr Leu Pro
965 970 975
Asp Thr Leu Phe Ala Ser Ser Tyr Asp Asp Gln Gln Gln Leu Leu Arg
980 985 990
Leu 'Ihr Zyr Gln Gln Ser Ser Trp His His Leu Ile Ala Asn Glu Leu
995 1000 1005
Arg Val Leu Gly L~eu Pro Asp Gly Thr Arg Ser Asp Ala Phe 'rhr 'Iyr
1010 1015 1020
Asp Ala Lys His Val Pro Val Asp Gly Leu Asn Leu Glu Ala Leu Cys
025 1030 1035 1040
Ala Glu Asn Ser Leu Ile Ala Asp Asp Lys Pro Arg Glu 'Iyr Leu Asn
1045 1050 1055
Gln Gln Arg Thr Phe Tyr 'Iizr Asp Gly Lys Thr Asp Gly Lys Asn Pro
CA 02320801 2000-08-14
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1060 1065 1070
'I~r Pro Leu Lys 'Ihr Pro Thr Arg Gln Ala Leu Ile Ala Phe ~r Glu
1075 1080 1085
Thr Ala Val Leu Thr Glu Ser Leu Leu Ser Ala Phe Asp Gly Gly Ile
1090 1095 1100
Thr Pro Asp Glu Leu Pro Gly Leu Leu ~'hr Gln Ala Gly 'Iyr Gln Gln
105 1110 1115 1120
Glu Pro Tyr Leu Phe Pro Leu Ser Gly Glu Asn Gln Val Trp Val Ala
1125 1130 1135
Arg Lys Gly 'Iyr 'rhr Asp 'I~r Gly 'Ihr Glu Val Gln Phe Tzp Arg Pro
1140 1145 1150
Val Ala Gln Arg Asn Thr Gln Leu 'Ihr Gly Lys Thr 'Ihr Leu Lys Trp
1155 1160 1165
Asp 'I9zr His 'ISrr Cars Val Ile 'T9zr Gln 'Il~r Gln Asp Ala Ala Gly Jxu
1170 1175 1180
Thr Val Ser Ala Asn err Asp Trp Arg ~e Leu 'rhr Pro Met Gln r ~ ~
185 1190 1195 1200
2'hr Asp Ile Asn Asp Asn Val His Ile Ile Ztir Leu Asp Ala Leu Gly
1205 1210 1215
Arg Pm Val 'rhr Gln Arg Phe Txp Gly Ile Glu Asn Gly Val Ala ~'hr
1220 1225 1230
Gly Tyr Ser Ser Pro Glu Ala Lys Pro Phe Thr Pro Pro Val Asp Val
1235 1240 1245
Asn Ala Ala Ile Ala Leu Thr Gly Pro Leu Pro Val Ala Gln Cys Leu
1250 1255 1260
Val ~Iyr Ala Pro Asp Sex Trp Met Pro Leu Phe Gly Gln Glu 'Il~r Phe
265 1270 1275 1280
Asn Thr Leu Thr Gln Glu Glu Gln Lys Ztir Leu Arg Asp L~ Arg Ile
1285 1290 1295
Ile Thr Glu Asp Trp Arg Ile Cars Ala Leu Ala Arg Arg Arg 'I'rp Leu
1300 1305 1310
Gln Ser Gln Lys Ala Gly ~'hr Pro Leu Val Lys Leu Lieu ~'hr Asn Ser
1315 1320 1325
Ile Gly Leu Pro Pro His Asn Leu Met Leu Ala 'Ilir Asp Arg Tyr Asp
1330 1335 1340
Arg Asp Ser Glu Gln Gln Ile Arg Gln Gln Val Ala Phe Ser Asp Gly
345 1350 1355 1360
Phe Gly Arg Leu Leu Gln Ala Ala Val Arg His Glu Ala Gly Glu Ala
1365 1370 1375
Trp Gln Arg Asn GJ.n Asp Gly Ser Leu Val 'Ihr Lys Met Glu Asp Zfir
1380 1385 1390
Lys 'Itir Arg Trp Ala Ile Thr Gly Arg 'I'hr Glu 'Iyr Asp Asn Lys Gly
1395 1400 1405
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Gln Ala Ile Arg Thr 'Iyr Gln Pro 'iyr P'h~e Leu Asn Asp Trp Arg Zyr
1410 1415 1420
Val Ser Asp Asp Ser Ala Arg Lys Glu Ala Zyr Ala Asp Thr His Ile
425 1430 1435 1440
Tyr Asp Pro Ile Gly Arg Glu Ile Gln Val Ile Thr Ala Lys Gly Trp
1445 1450 1455
Leu Arg Gln Asn Gln err Phe Pro Trp Phe Thr Val Ser Glu Asp Glu
1460 1465 1470
Asn Asp Leu Ser Ala Asp Ala Leu Val
1475 1480
<210> 15
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequ~ce:oligo~ucleotide
<400> 15
cg~tccga tgattttaaa agg 23
<210> 16
<211> 16
<212> INF1
<213> Artificial Sequ~ce
<220>
<223> Description of Artificial Sequence:oligariucleotide
<400> 16
gcgccattga tttgag 16
<210> 17
<211> 19
<212> INA
<213> Artificial Sequsxe
<220>
<223> Description of Artificial Sequence:oligonucleotide
<400> 17
cattagaggt cgaacgtac 19
<210> 18
<211> 26
<212> DNA
<213> Artificial Sequence
<220>
<223> Descripti~ of Artificial Sequence:oligoarucleotide
<400> 18
gagcgagctc ttacttaatg gtgtag 26
<210> 19
CA 02320801 2000-08-14
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<211> 28
<212> n~m
<213> Artificial Sequence
<220>
<223> Description of Artificial Segue~u;e:oliga~ucleotide
<400> 19
~~~ ~t ~ 28
<210> 20
<211> 18
<212> Il~r
<213> Artificial S
<220>
<223> Description of Artificial Segu~ce:oligodmcleotide
<400> 20
99~t9'~ ~a~g 18
<210> 21
<211> 18
<212> I1~1
<213a Artificial Sequence
<220>
<223> Descxiptian of Artificial Sequc~ce:oligrxatcleotide
<400> 21
cattaaogca ggaagagc 18
<210> 22
<211> 26
<Z12> Its
<213> Artificial Sequence
420>
<223> Description of Artificial Se:oligan~ccleotide
<400> 22
9~~~gcg 26
