Note: Descriptions are shown in the official language in which they were submitted.
WO 95131564 2 1 8 8 5 6 2 . ~ s 6 1075
' 1
MET~IOD OF L~CUlIUCll~L~ PAT~IOGEN RESISTANCE IN PIANTS
The present invention relates to a method of
introducins pathogen resistance in plants, particularly
broad spectrum pathogen resistance, and plants which
5 may be obtained by said method and which show
resistance to at least one but pref erably more than one
pathogen .
Plants are constantly ~-hRll~nged by pot~nt;711y
pathogenic mi~:Luuly~.-isms. Crop plants are
lC particularly vulnerable, because they are usually grown
as genetically uniform monocultures; when disease
strikes, losses can be severe. ~owever, most plants
are resistant to most plant p7thr-g~nc. To defend
themselves, plants have evolved an array of both
15 preexisting and; n~llri h] e defences which include
barriers to pathogen entry such as thickened or
chemically crosslinked cell wall c c or toxic
chemicals derived from complex plant biosynthetic
pathways. P7thf~.onc must specialize to CiluullLvt:~lt the
20 defence -h7n; 1 of the host, especially those
biotrophic pathogens that derive their nutrition from
an intimate association with living plant cells. If
the pathogen can cause disease, the interaction is said
to be compatible, but if the plant is resistant, the
25 interaction i8 said to be ;n~o~n.pAtihle~
Induced resistance is strongly correlated with
the hypersensitive response (HR), an induced response
Wo9S/31564 2 1 88562 PCIIGs95/0l075
assoc~ated with localized cell death at sites o~ =
attempted pathogen ingress. It is llypothPRi ~P~ that by
HR the plant deprives the pathogen of living host cells
but there is no certai~ty about whether localised cell
5 death results from or induces plant defence ~h~n; om~
Many plant defence -~-h~n; ~ are strongly
induced in response to a challenge by an llnRllrrpRRful
pathogen. Such an induction of enhanced resistance can
be systemic (hereiIIafter referred to as systemic
10 acquired resistance (SAR) ~ (Ross, 1961; Ryals et al.,
992). Acquired resistance can also be local
(hereinafter referred to as LAR) (Ryals et al., 1992).
Acquired resistance has been extensively researched and
various facts have been est~hl i RhPd. For example,
15 biotic stimuli are required to provoke the HR resulting
in areas of dead plant cells on the leaf. Cell death
resulting from wounding or other abiotic stresses wil
not suffice. (Ryals et al., 1992; Enyedi et al.,
1992). In addition, SAR is correlated with the
20 induction of a large array of pa~hn ~PnPR;R-related (PR)
proteins, some of which have demonstrated anti-fungal
activity (Ward et al., 1991).
A variety of examples of SAR have been studied
and include rh~ l l Pn~ing of tobacco carrying the N gene
25 for resistance to tobacco mosaic virus (TMV) with TMV
(Ross, 1961) and challenging cucumber seP~l ;n~s with .-
tobacco recrosis virus or ColletotrichzLm largenarium..
~ WO95/31564 21 88562 r~ 5 1075
Results show that a challenge with one pathogen leads
to Pnh~nr~l resistance to a wide variety of other
pathogens ( Ryal s e t al ., 1 9 9 2 ) .
SAR has also been r rrrPl ~te~i with increased
5 levels of salicylic acid in plants which have been
rh~llPn~ed by pathogens (Malamy et al., l990; Metraux
et al., 1990) which has been rnnfi 1 by studies that
show that a supply of exogenous salicylic acid to
unchallenged plants Gan result in SAR (nard et al.,
1991; Hennig et al ., 1993 ) . Transgenic plants designed
so that salicylic acid ~ t ir,n is prevented by
expression of a salicylate hydroxylase gene show
reduced SAR compared to non-transgenic plants . where
salicylic acid ~ tion is not prevented (Gaffney
15 et al ., 1993 ) . SAR can also be induced by many
chemicals m-nllfAct~red by Ciba-Geigy such as 2,6-
dichloroisonicotinic acid (INA) (Uknes et al., 1992).
SAR is an attractive method by which broad
spectrum disease control can be achieved. However, two
20 major drawbacks hinder its ~ .ial exploitation: SAR
is not a heritable trait and so the rhP- ~n has to
be successfully induced into every plant in the crop
stand; to be effective throughout the crop' s life, the
SAR phenotype has to be re-boosted at regular
25 intervals.
,, Although the - - -h~n i omq causing SAR are not fully
understood, it is believed that when a plant is
~V095131564 21 88562
challenged by a pathogen to which it is reslstant, it
undergoes an HR at the site of attempted ingress of ~he
;n~ -t;hle pathogen. The induced HR leads to a
systemic enhancement and acquisition of plant
5 resistance to virulent pathogens that would normally
cause disease in the unchallenged plant.
It has long been known that HR-associated disease
resistance is often (though not exclusively) cre~
by 1~ n~nt genes (R genes)~ Flor showed that when
lO pathogens mutate to UV~:L~ - such R genes, these
' ::lt; nnq are recessive. Flor c~ncl 1~ d that for ~a R
gene to function, there must also be a ~uLLe~ uu~l~ing
gene in the pathogen, an "avirulence gene" (Avr gene).
To become virulent, p~th~ nc must thus s~op making a
15 product that activates R gene-dependent defence
mech~n; ~ (Flor, 1971) . A broadly accepted working
hypothesis, often termed the elicitor/receptor model,
is that R genes encode products that enable plants to
detect the presence of pathogens, provided said
20 pathogens carry the corr~p~n~ii n~ AVR gene (Gabriel and
Rolfe, l990). This recognition is then t~n~ red into
the activation of a defence response.
The mlo allele of the Mlo gene of barley is the
one example of a recessive disease resistance gene
25 currently widely used in plant breeding. Lines that
are homozygous for the recessive allele of this gene .,
actlvate the defence response (comprising formation of
~ WO g5/3l564 2 1 8 8 5 6 2
cell wall appositions ~ even in the absence of the
pathogen (Wolter et al, 1993) . Thus the mlo - lt~tinn
causes a defence mimic phenotype, also known as a
necrotic or disease lesion mimic phenotype, and appears
5 to deregulate the def ence response, so that it is
activated precociously, or is regulated on more of a
~hair trigger". A number of examples of such disease
lesion mimic mutants exist in maize (Johal et Al, 1994,
Pryor, 1987, Walbot, 1983~. Recently, such mutants
10 have been sought in ArAh; ~npSi c . The characterization
of one such mutant, acdl, has been re~orted (Greenberg
and Ausubel, 1993 ) . Further mutants of this type have
been reported at scientific ~t;n~q (the Arabidopsis
acd2 ~tinn by F.M. Ausubel at a meeting at Rutgers
15 University, New Jersey, IJSA, April 1993; Arabidopsis
~ t;nnq now known as lsd (for lesions si l~t;n5
defence response) mutations by R. Dietrich and J. Dangl
at the ARaPANET ( (Ar~hi~npsic Pathology Network)
workshop in Wye College, ~Cent, UK in April 1993).
20 Manuscripts describing the acd2 and lsd I ~tinnc are
Dietrich et al. and Greenberg et al. (1994). It is
highly likely that the recessive, ~tinnc identified
in such mutant screens that leave the defence response
more constitutively on, or more rapidly activated, or
25 less easily inactivated, are in genes that normally
,, dampen down the defence response to prevent it bc~ n~
so severe that it is deleterious to the plant.
WO9S~31564 2 1 8 8 ,62 ~ ;S.~1075
Conceivably, such gene could be cloned, expressed in an
antisen5e or sen5e configuration to reduce expression
of the corresponding gene (Hamilton, 1990, Napoli et
al, 1989).
Pathogen avirulence genes are still poorly
understood. Several bacterial Avr genes encode
hydrophilic proteins with no homology to other classes
of protein, while others carry repeating units whose
number can be modified to change the range of plants on
which they exhibit avirulence (Keen, 1992; Long and
Sta8kawicz, 1993 ) . Additional bacterial genes (hrp
genes) are required for b~rtPr;ill Avr genes to induce
~, and also for r~thn~pn;city (Keen, 1992; Long and
Staskawicz, 1993 ) . It is not clear why r~thnganq make
products that enable the plant to detect them. It is
widely believed that certain easily discarded Avr genes
contribute to but are not required for p~thn~pnl~it
whereas other Avr genes are less rl;qpPnq~hle (Keen,
1992; ~ong and Staskawicz, 1993). The characterization
of two fungal avirulence genes, Avr 4 and Avr 9 (De Wit
et al_, 1992; Joosten et al., 1994), has also been
reported. Research is also being undertaken to clone
rice blast avirulence genes from the causal organism
~agnaporthe grisea and the avirulence genes (NIP
proteins ) of the barley pathogen Rhynrhnspnrim
secalis. Two viral avirulence genes have also
previously been cloned. Culver and Dawson, 1991, have
~ W095131564 2 1 8 8 5 62
shown that tobacco mo3aic virus coat protein is the
avirulence detPrm; n;: nt for the N' gene product . In
addition, the potato virus X coat protein appears to be
the avirulence det~ n~nt for the Rx and Nx genes
(Kavanagh et al., 1992; Santa-Cruz et al., 1993; Etohm
et al ., 1993 ; Goulden et al ., 1993 ) .
Recently the map based cloning of the tomato Pto
gene that confers "gene-for-gene" resistance to the
bacterial speck pathogen PsPI~ .c syringae pv tomato
(Pst) has been reported (Martin et al., 1993). It has
al60 been recently reported that the Ari~hirlnr~iq Rps2
gene (which confers P~ syri~gae resistance)
and the tobacco N gene (which confers virus resistance)
have been cloned (Key8tone Sympo8ium, January 1994).
Even more recently, the Rps2 and features of the Cf-9
gene Re ~ have been revealed at the 13th An~ual
Symposium in Columbia, Missouri, April 13th-16th 1994,
on the Biology of Communication in Plants.
TntPrnslt;onal Patent Application No: PCT/GB94/02812
describes a method for generally identifying and
cloning plant resistance genes.
The technology for gene isolation based primarily
on genetic criteria has improved dr~r t;c~lly in recent
years, and many workers are currently attempting to
clone a variety of R genes. Targets include (amongst
others) rust resiRtance genes in maize, An~irrhin~ and
flax (by transposon tagging); downy mildew resistance
W0 95/31564 2 1 8 8 5 6 2 ~ 075
genes in lettuce an~ Arabidopsis (by map based cloning
and T-DNA tagging); Cladosporium fulvum (Cf) resistance
genes in tomato (by tagging, map based cloning and
affinity labelling with avirulence gene products);
5 virus resistance genes in tomato and tobacco (by map
based cloning and tagging~; nematode resistance genes
in tomato (by map based cloning); and genes for
resistance to bacterial p~thr~-.nl:~ in Arabidopsis and
tomato (by map based cloning).
Tomato (~ycopersicon esculentum) is susceptible
to disease caused by the leaf mould fungal pathogen
~7;1r7n~rnrium fulvum. According to De Wit, 1992, the
Avr9 gene of C. fulvum, which confers avirulence on C.
fulvum races that attempt to attack tomato varieties
15 that carry the Cf-9 gene, encodes a secreted cysteine-
rich peptide with a final processed size of 28 amino
acids. However, its role in compatible interactions is
not clear. The R genes ~Cf-genes) that act against C.
fulvum have been irl~nt;f;f~rl and bred into cultivated
20 varieties, often from related species of tomato
(Dickinson et al., 1993; Jones et al., 1993).
It has been shown that C. fulvum rfmt::; n~l Avr
genes that confer recognition by plants which contain
the Cf-genes, leading to activation of host defence
25 me~h~n;~ ~ to attack the disea9e (;nl , t;hil;ty).
The Avr4 and Avr9 genes encode amall peptides that are
secreted by the pathogen into the inter~ r spaces
W095131564 .~,1,~,'. 1075
21 88562
of infected leaves, from which they can be extracted.
This has enabled the purification and sequencing of
these peptides and the isolation of the genes that
encode them (De Wit, 1992; Joosten et al., 1994).
Experiments have shown that when the Avr9 gene is
transformed into a race of pathogen that lacks Avr9,
then the race of pathogen becomes avirulent on plants
which are carrying the Cf-9 gene. In addition, it has
been shown that disruption of the Avr9 gene in a
' 10 r~thn~Pn race which is avirulent on plants carrying Cf-
9 gene confers cl ,-t;h;l;ty on the Cf-9 -nnt;l;nin~
plants (Van Den Ackerveken et al., 1992, Marmeisse et
al ., 1993 ) .
In addition, De Wit and colleagues have further
shown that the secreted peptide encoded by the Avr9
gene can be injected into Cf-9 rnnt~;n;n~ tomato leaves
to elicit a necrotic response in the inj ected area .
The necrotic response is consistent with local and
vigorous activation of a defence response (De Wit,
1992; W3 91/15585) . TntPrn~tinn~l Patent Application
No. PCT/GB94/02812 describes the tr InF~gPn; n expression
of the Avr9 gene using the strong n~lll i fl ower mosaic
virus 355 plant promoter to cause lethality in Cf-9
plants. This transgenic Pxpression has been used to
select mutants in which the Cf-9 gene has been
inactivated by transposon insertion in order to isolate
the Cf-9 gene and perform DNA sequence analysis of this
Wo 95/31564 2 1 8 8 5 6 2 PCr/GB95/~l075
'10
gene .
Various pathogen races that overcome these Cf-
genes have emerged and are named af ter the Cf-gene
which they can overcome. For example, C. fulvum race 4
5 can overcome Cf-4; C. fulv~m race 5 can .,v~ Cf-5
and C. fulvuzn race 2.4.5.9 can uvc:~, ~ Cf-2, Cf-4, Cf-
5 and Cf- 9 .
Wû 91/15585 describes a hypothetical method
whereby i f a Cf - 9 gene and/or an Avr9 gene were
0 expressed under the control of a promoter that is
induced by a broad range ûf pathogens, then a general
defence response could be induced. IIowever, there i8 a
lack of Pn~hl; nrJ ~i ~rl r~l~re regarding which
polynucleotide seguences could be used either as the
15 resistance gene or as an actual promoter which would be
suitably affected by a broad range of pathogens. A
further problem with this proposed method is that
necrosis induced by the Cf-9 and Avr9 gene combination
could lead to further i n~lllrt i n of Avr9 and/or Cf -9
20 leading to spreading of the necrosis and severe
reduction in the yield of the plant. This problem may
arise since promoters such as promoters for plant
defence genes and other genes involved in the defence
response such a5 PR genes (pathogenesis related genes ),
25 are induced in both a, ,~t;hle and an in~ ,-t;hle
interaction . Theref ore, even if a promoter exists
which is effectively induced by a broad range of
~ wo95131564 21 88562 r~ 075
pathogens, the method would not be viable unless the
promoter is only induced by the appearance of a
compatible pathogen. If t~e defence response provides
further I n~ r~ i ~An of the promoter the plant might
5 experience spreading necrosis.
The present invention has reeulted from
experiments involving tr~nRrosrn tagging of resistance
genes, the first one being Cf-9. Numerous alleles of
the Cf-9 gene (Cf-9*D8) were isolated that had been
10 inactivated by the maize element Dissociation (Ds).
These ~active Cf-9*Ds genes did not give rise to a
constitutive and lethal activation of def ence
Ah~n;~ in response to constitutively ~.~ ssed Avr9
transgene (35S:SP:Avr9). On backcrossing plants that
carried the Cf-9*Ds and 355:SP:Avr9 genes to tomato
plants carrying an Activator (Ac) transposase gene
(sAc) in the h~ _yy~us state but lacking Cf-9, a
quarter of the resultant progeny carried sAc,
355:5P:Avr9 and Cf-9*D6. These plants showed somatic
20 ~Yr; R; r,n of Dg from the Cf-9*Ds gene, 80matically
restoring Cf-9 function and giving rise to localised
activation in cells of plant defence responses due to
recognition of the constitutively expressed Avr-9
peptide. These cells died and gave rise to small
25 necrotic sectors, the plants phenotypically showing
variegation for a defence-related necrosis, similar to
somatic flecks of necrosis that are associated with the
WO9S/31S64 21 88562 Pcr/Gs9sl0l07s
induction oE SAR in plants rhAl 1 rngc~l with necrotising
pathogens. Further work showed that plants that
variegate for somatic sectors of plant defence response
in this way have increased resistance to a range of
5 pathogens. -
Thus, a f irst aspect of the pre~ent inventionrelates to a method of providing rAthnJ~n resistance,
in particular broad spectrum pathogen resistance, in
plants by induction of variegation in which genes are
10 expressed or suppressed resulting in the activation of
necrosis. A method according to the present invention
comprises: (i) inactivating a nucleotide sequence which
contributes to plant cell necrosis or inactivating one
or more nucleotide sequences forming part o a
15 ' ~ ;nAr;nn of nucleoti~Le sequences which contribute to
plant cell necrosis; (ii) introducing said nucleotide
sequence or serluences into the genome of a plant; and
(iii) restoring said nucleotide sequence or sequences
to a functional form to yield a level of necrosis
20 resulting in pathogen resistance. The plant cell
necrosis is preferably defence-related plant cell
necrosis .
A second aspect of the present invention relates
to a method of providing pathogen resistance in plants
25 by induction of variegation in which genes are
~ ssed or suppressed resulting in the activation of
a plant defence response which comprises: (i)
W0 95~31564 2 1 8 8 5 6 2 i ~ 07s
inactivating a nucleotide sequence which contribute3 to
the plant defence response or ~nactivating one or more
nucleotide sequences forming part of a combination of
nucleotide sequences which contribute to the plant
defence response; (ii) introducing said nucleotide
sequence or sequences into the genome of a plant; and
(iii) restoring said inactivated nucleotide sequence
or sequences to a functional form to result in pathogen
resistance .
0 The variegation will generally be for somatic
sectors. Pathogen re8istance will generally be
increased compared with wild-type.
The nucleotide sequence or sequences comprise one
or more genes . The plant def ence response and/or plant
cell necrosis occurs on expression of the gene or
genes. The defence response and/or plant cell necrosis
can be conditional or unconditional on the expression
of one or more interacting genes. A substance or a
combination of substances nay result in increased
pathogen resistance . Examples are ~li Rr'llR,RPd further
below .
For example, the nucleotide sequence or sequences
may comprise a gene encoding either a substance which
leads to necrosis, e.g. through activation of the plant
defence response, or a substance which leads to a plant
defence response with no sign of necrosis. For
example, the sequence or sequences may comprise a plant
Wo 95/3156~ 2 1 8 8 ~ 6 2 PCT/GB95/0107~
pathogen resistance gene (R), an avirulence gene ~Avr)
or other elicitor or ligand gene (L) of an R gene, or
both and R gene and an L gene.
The inactivation of the nucleotide sequence or
5 sequences which contribute to the plant defence
response and/or plant cell necrosis is preferably
effected by insertion of a tr~ncpoqAhl e genetic element
into the nucleotide sequence or one or more of the
nucleotide sequences forming-a, ' in;~tinn of
lO nucleotide seguences. The transposable genetic element
is preferably a tr~nC~nsnn or a nucleotide sequence
flanked by specific nucleotide sequences 90 that
tr~nRrosnn excision gives rise to activation of the
plant defence response and/or necrosis. Thus,
15 insertion of a genetic lesion into the nllclect;~lP
sequence disrupts the gene to prevent expression of a
product able to function in contributing to the plant
defence response and/or plant cell necrosis. In the
absence of the lesion, e.g. following Pl~n;c1nn of a
20 transposable element such as a transposon, the gene may
be expressed to produce a functional product, i . e . gene
function is restored. The lesion may be inserted into
the part of the gene coding for the expression product,
or may be in a regulatory sequence such as a promoter
25 required for expression of the product.
In this form of the invention, re-activation
within the plant is preferably carried out by
~ Wo 95131564 2 1 8 8 5 6 2 ~ 1075
restoraration o~ the inactivated nucleotide sequence or
sequences resulting in activation of a plant defence
response and/or necrosis. Such restoration may be
caused or allowed by culturing of the plant. Where the
5 nucleotide sequence is inactivated by virtue of
insertion of a transposable element therein, the plant
genome should contain at least one nucleotide sequence
coding for a corr~cpnn~; n~ transposon activation system
~for example, comprising a tranEposase).
0 Alternatively, the inactive form could be flanked by
n:~ce recognition sequences that are acted on by
a site specific re~ ' ;n~ti~n system (comprising a
specific rP ' ;n~ce) 80 that L~_ ` ;nzlt;nn activates
the inactive form of the gene. Hence, when the
15 inactivated nucleotide SP~lPnre or sPs~lpn-pc are
introduced into the plant genome somatic P~ri c j
the tr~nRposnn or recombination of the nucleotide
sequence occurs in some cells leading to ~ctivation of
the plant defence response and/or necrosis in specific
2 0 clones of cells .
The number of cells in which restoration o~
function occurs may vary. As discussed further below,
certain measures are available for opt;m;C;n~ the
system, e.g. by controlling the frequency of
25 crnnt:lnPnu5 excision of a transposable element which is
caused or allowed upon cultivation of a plant with the
requisite ~ucleotide sequence or SPql~Pn~~Pc within its
W095/31S64 2 ~ 8 ~ 56~ //a
genome. ~ ~
The present invention further provides transgenic
plants having increased pathogen resistance obtainable
by the method of the present invention, and any clone
of such a plant, seed, selfed or hybrid progeny and
descendants, and any part of any of these, such as
cuttings, seed. The invention provides any plant
propagule, that is any part which may be used in
reproduction or propagation, sexual or asexual,
1~ including cuttings, seed and so on. Derivatives of
plants are also provided by the present invention. A
derivative is any fl~nct;nn~l unit derived therefrom
h~ ver achieved (e.g. flln~ti nnAl allele of gene
made by a~nf~R;R, re~ ` in~nt DNA, synthesis, or
15 plant which could not have been y ~ ~ -lu-_~d without the
use or manufacture of the plant from which it is
derived. )
Transgenic plants in accordance with the present
invention may demonstrate increased p~thng~n rl~i Rt~n~e
20 since the induced plant defence response and/or
necrosis of plant cells may cause other cells, such as
adj acent cells, to acquire pathogen resistance . The
activation of, for example, a plant resistance gene in
a plant cell is inherited by the progeny and
25 dest f-nrl~nt R of that cell. The expression of this plant
resistance gene leads to initiation of the defence
response in cells which may eventually lead to the
o95/31'64 l,7 '~''' ~a
death of the participating plant cells resulting in an
area of plant cell necrosis. So, plants may haYe
variegation for small somatic sectors in which defence-
related plant cell necrosis is activated. This
response may induce pathogen resistance in other cells.
In an alternative, operating on the same general
principle, the expre6sion of one or more plant pathogen
resistance gene may either lead to initiation of the
defence response only resulting in variegation for
10 small somatic sectors in which the plant defence
response is activated or of plant cell necrosis which
is not related to the plant defence response resulting
in variegation for small somatic sectors in which plant
cell necrosis is activated.
1~ ~Ience, the plant may acquire resistance to a
broad range of path~g~nT~ and not only to the pathogen
associated with the gene or genes contributing to
necrosis, for example, C. fulvu17 in the case of the Cf-
9/Avr gene ~ ;n~t;on ~or example, a transgenic
20 tomato plant according to the present invention may
demonstrate resistance against a broad range of
pathogens such as one or more bacterial plant pathogens
(for example, Xanf7- f,T campegtris, Pse7T' C7
syringae), fungal plant pathogens (for example,
2T7 Phytophthora infestans, Fusariu27 U~y~l~OLIllll, Botrytis
cinerea, Verticilliu~7 dahliae, Altenaria solani,
Rhizoctonia solani) and viral pathogens (for example,
WO95131564 2 1 88 5 ~2 i~ s/01075
18
TMV, PVX, PVY, TSWV). Similarly, other transgenic
plants such as transgenic tobacco, Araoidopsi~ and
potato plants may display resistance to a large number
of ma~or diseases of important crop species such as,
5 Peronospora, Phytophthnra, Puccinia, Erysiphe and
Botrytis .
Thus, according to a further aspect of the
invention there is provided a plant, or any part
thereof, which is phenotypically variegated, with
10 clones of cells expressing a first phenotype and other
cell5 expressing a second phenotype which is increased
pathogen resistance compared with wild-type. The first
phenotype is preferably necrosis and/or a plant defence
r e~ se phenotype . As ~ r-llq~d~ plants variegated by
15 somatic sector for such a phelluLy~e may have f~nhAnred
pathogen resistance as a result of a second phenotype
in cells, which may be adjacent to the cells with the
first phenotype which are necrotic and/or in which a
plant def ence response is activated . The phenotypic
20 variegation is likely to result from expression in
cells with the f irst phenotype of a gene or gene, or
nucleic acid comprising a gene or genes, which
contributes to such phenotype, whereas other cells
without such phenotype lack such gene expression. A8
25 discussed herein, this may result from reactivation of
a previously inactivated gene, such as a resistance
gene, for example by random ~l-r; qinn of a transposable
~ wo95131564 2 1 88562 .~. . 1075
'19
element such as a transposon.
In a further aspect, the present invention
provides a host cell, such as a plant or microbial
cell, or a plant comprising at least one such cell,
5 ~ nt~in1n~ (i) nucleic acid encoding one or more
nucleotide sequences which cause or contribute to the
plant def ence response and/or cell necrosis, at least
one of the nucleotide sequences being reversibly
ina~tivated, for example by insertion of a transposable
10 element such as a trAnqrnsr~n, and (ii) nucleic acid
f~nrf~l;n~ a molecule able to reverse the inactivation,
such as, in the case of a transposon, a tr~nqp~R~qe.
Thus, the cell may comprise a plant resistance gene or
other gene involved in the plant defence response or
lS able to kill a cell when expressed therein (either
alone or ;n~ ' ;n~r;~n with one or more sequences, for
example in the case of an R gene the corrF-qr~nrl;n~
elicitor), the gene being inactivated by insertion
therein of a tr~nqp~snn, and the cell further
20 comprising a gene encoding a tr~nqrss~qe.
In an ~ ry: ' ~ '; ', the genome of the
cell comprises the gene Cf-9, or a mutant, derivative,
variant or allele thereof which retains Cf-9 function,
inactivated by insertion therein of a transposon, the
25 genome also comprising the Avr-9 gene, or a mutant,
derivative, variant or allele thereof which retains
Avr-9 function, and a gene ~n~o~l; n~ a tr~nqros~qe able
Wo 95l3l564 2 1 8 8 5 6 2 PCT/GB95/01075
to exci~e the transposon ~rom the Cf-9 gene or
functional equivalent. Other resistance genes may be
employed, as may genes which do not require the
presence of an elicitor molecule to cau6e cell
5 necrosis, as discu9sed further elsewhere herein.
The cell may comprise the nucleic acid Pnco~i n~
the various genes by virtue of i~troduction into the
cell or an ancestor thereof - of the nucleic acid, e.g.
by transformation, using any suitable technique
10 available to tho9e 9killed in the art. Furt~-~ e,
plants which comprise such cells, and seed therefore,
may be produced by crossing suitable parents to create
a hybrid whose genome (-nntW;nR the required nucleic
acid, in accordance with any available plant breeding
15 technique. For example, a parent strain comprising
within its genome a plant resistance gene rnntw;n;n~ a
trwnqposon or other inactivating lesion may be crossed
with a second strain comprising within its genome a
gene encoding the elicitor molecule for the plant
20 resistance gene a~d a suitable transposase for excision
of the trwnRrnsnn. At least a proportion of the hybrid
progeny of the parents, i . e . seed or plants grown
therefrom, will comprise the required nucleic acid for
activation in the plant of, in this example, the plant
25 resistance gene and, following interaction with the
elicitor, the plant defence response a~d/or plant cell
necrosis .
~ Wo95/31564 21 885~2 r~. 1075
21
,
Plants according to this aspect of the pre6ent
- invention will be variegated genetically. Clones of
cells will have one or more nucleotide sequences which
cause or contribute to the plant defence response
and/or cell necrosis reactivated by removal of the
inactivating lesion such as a transposon, 80 that a
first phenotype such as necrosis is shown, while in
other cells the sequence or sequences will remain
inactivated 80 these cells will not show the first
phenotype.
Within the cell or cells, the nucleic acid may be
incorporated within the ~ - . A gene stably
incorporated into the genome of a plant is pa8sed from
generation to generation to descPnf~nt~ of the plant,
80 such ~lPcpnrl~nt~ should show the desired phenotypic
variegation and so may have Pnl~nt~p~l p~thn~l~n
resistance .
In addition to a plant, the present invention
provides any clone of such a plant, seed, selfed or
hy~rid progeny and clP~rpnr~nt~ and any part of any of
these, such as cuttings, seed. The invention provides
any plant propagule, that is any part which may be used
in reproduction or prora~atinn, sexual or asexual,
including cuttings, seed and so on.
A further aspect of the present invention
provides a method of making such a cell involving
introduction of nucleic acid (e.g. a vector) comprising
.
WO 95/31564 2 1 8 8 5 S 2 P~ 7s
( i ) nucleic acid e~coding one or more nucleotide
sequences which cause or contribute to the plant
defence response and/or cell necrosis, at least one of
the nucleotide sequences being reversibly inactivated,
5 for example by insertion of a transposable element such
as a tr~nApo~t~n~ and/or (ii) nucleic acid encoding a
molecule able to reverse the inactivation, such as, in
the case of a transposon, a transposase into a plant -
cell. Introduction of nucleic acid (i) may be
0 A~ ; ed, preceded or followed by introduction of
nucleic acid (ii). Such introduction may be followed
by ret ' ;n~t;~n between the nucleic acid and the plant
cell genome to introduce the seguence of nucleotides
into the genome. Desc~n~AntA of cells into which
15 nucleic acid has been introduced are ; nrl ~ t~ within
the scope of the present invention.
The level of the plant def ence response and/or
plant cell necrosis in the small somatic sectors should
be sufficient to result in the ;n~ tion of acquired
0 resistance or the ;nt~ tir~n of other defence
n; I ~ . Since this method leads to activation of
acquired resistance but is inherited it is referred to
as Genetic Acquired Resistance (GAR) . Hence, any
system which gives rise to a variegation leading to GAR
5 is applicable to the present invention
Methods and plants etc. according to the present
invention are particularly beneficial since the
WO 95/3 1S64 1 ~ , '. 1 07S
2~ 62
23
nucleotide sequence or seque~ces which contribute to
the plant defence response and/or plant cell necrosis,
for example the avirulence and plant resistance genes,
may be under co~trol of any suitable promoter, such as
5 a constitutive promoter or, in the case of R genes,
their own endogenous promoter, or a cell type specific
promoter Furth,~ e, the restoration of the
nucleotide sequence or sequences, for example by the
somatic excision of a transposon, gives rise to
10 LeuuLLellL and widespread induction of the plant defence
response in many small clones of cells tllLUlyllULlt the
plant, irrespective of whether or not there has been a
challenge by pathogen. The resistance conferred on the
plant is therefore constitutive and broad.
The present invention may be used for many
appl i~t;nnc and is suitable for deployment in Fl
hybrid seed pro~-]ct; ~In system. In such a system, one
of the parents should be 1- 7-yyuus, for example, for
the tr~ncpocAce or recombinase gene. In addition, in a
20 system where two ~ ~ ~ntc are required for ;n~ r;n~
the necrosis such as in the Avr9/Cf-9 gene, ;ni~t;r)n
for example, this parent should also be _yyu~s for
the constitutively expressed genes. The other parent
should be homozygous for the gene that encodes the non-
25 autonomous inactivation system, such as the transposonor recombinase-recognition sequences. After making a
cross between parents of this genetic constitution, on
Wo 95UI564 2 ~ 8 8 5 6 2 PCT/GB9S/01075
somatic excision or recombination, the function of the
gene or genes which give rise to the defence response
and/or plant cell necrosis i8 restored in somatic
sectors in the resulting progeny.
It will be clear to the person skilled in the art
that any gene or ~ ' ini9t;nn of gene8 which contributes
to variegation for the plant defence response and/or
plant cell necrosis may be uGed in the method of the ~
present invention. Furthermore, any system which gives
rise to inactivation of the nucleotide sequence or
sequences and subsequent rest~-r~ti~ n of functional
sequence or SP~lPnC'eR may be used.
The present invention also provides in further
aspects various compositions of matter comprising
, ' in~tir~nR of nucleotide sPquPnre~ encoding various
substances employed herein. Such c ~ ;n~t;~nR of
nucleotide sequences which may be introduced into cells
in accordance with the present invention follow:
(X): represents a nucleotide sequence with one
or more genes of type X
(XY): represents a nucleotide sequence with one
or more genes of type X and one ore more
genes of type Y etc.
R: receptor gene
L: ligand gene (capable of interacting with the R
gene )
W(1 95131S64
21 885~2
I: genetic insert
A: activator of transposition o~ genetic insert.
R may encode a substance whose presence in a
plant results in a plant defence response, necrosis
5 and/or increased pathogen resistance, with I being a
genetic insert able to inactivate R and A .-nrQ~l;nrJ a
substance able to reactivate R inactivated by I:
(l) Any, ' ;n~ti~n of:
l . (R), ( I ) and (A);
2. (R) and (IA);
3 . ( I ) and ~AR); or
4. (A) and (RI);
5. (RIA) .
Alternatively, R and L may encode substances
lS whose presence together in a plant results in a plant
defence response, necrosis and/or increased pathogen
resistance, I being a genetic insert able to inactivate
R and/or L and A ~nro(l; ng a substance able to
reactivate R and/or L inactivated by I:
20 (2) Any combination of:
l. (R), (L), (I) and (A);
2. (R), (LI) and (A)
3. (R), (LA) and (I)
~ . (R), ( IA) and (L)
5 . (L), ( IR) and (A)
Woss~v1s64 2 ~ 6 2 . ~ iv/a
6. (~), (AR) and (I)
7. (I), (LR~ and (A~
8. (R) and (LIA)
9. (L) and (IAR)
lO. (I), and (ARL); or
ll. (A) and (RLI);
12. (RLIA)
If genetic insert (I) is coupled with either the
R or the L gene, the number of possible combinations
lO will then be
(l): (RI~ and ~A); or
(RIA)
(2): (RI) (L) and (A)
(R), (LI) and (A)
(RI) and (LA)
(RA) . and (LI)
(RLIA)
also provided by the present invention is a
method of producing a plant, or a part, propagule,
20 derivative or descendant thereof, cnn~;ninr, nucleic
acid comprising a nucleotide sequence or nucleotide
sequences onrr,~inJ R, I and A, wherein R encodes a
substance whose presence in a-plant results in a plant
~ WO 95/31564 2 1 8 8 5 6 2 ~ 075
2~
defence response, necrosis and/or increased pathogen
resistance, I is a genetic insert able to inactivate R
and A encodes a substance able to reactivate R
inactivated by I, comprising crossing plant lines whose
genomes comprise any of R, I, A and ~ ' ;n~tions
thereof, to produce the plant or an ancestor thereof.
A further aspect provides a method of producing a
plant, or a part, propagule, derivative or ri~cr,on~Ant
thereof, ~ nt~in;n~ nucleic acid comprising a
nucleotide sequence or nucleotide se~uences ~ncoriin~ R,
L, I and A, wherein R and L encode substances whose
presence together in a plant results in a plant def ence
response, necrosis and/or increaged r~qthr~n
resi8tance, I is a genetic insert able to inactivate R
and/or L and A encodes a substance able to reactivate R
and/or L inactivated by I, comprising crossing plant
lines whose genomes comprise any of R, L, I, A and
combinations thereof, to produce the plant or an
ancestor thereof.
Said plant lines may contain nucleic acid
comprising any of R, L, I, A and combinations thereof
as a result of transformation of cells of the plant or
an ancestor thereof
Herein, unless context demands otherwise, a
~receptor" is a product encoded by a gene capable of
interacting with another product, the ligand.
Various ~ i - c of the present invention are
WO95/31564 2 1 88562 .~1 ~ ~lu/a
28
now described in more detail below, by way of example
and not limitation.
Nucleotide Se~uence or ~e~PnrP,q contri~uting to the
Plant Defence Response and/or lVecrosis
The nucleotide sequence or combination of
nucleotide sequences in which at least one of the
sequences is inactivated are rLumerous and may include
an ~ng-; nppred allele of a ubiquitin conjugating enzyme
(Becker et al., 1993), the CaMV gene VI protein
~TAk~hA~-hi et al., 1989), a viral coat protein in the
presence of the appropriate viral resistance gene, for
example Tobacco Mosaic Virus Elicitor Coat Protein and
the gene N' (Culver and DawsonL 1991), a bacterial
harpin protein (Wei et al ., 1992; He et al ., 1993 ), the
gene N (see e.g. Whitham et al (1994) and a ToMV-Ob
gene cloned by Padgett and Beachy (1993), the potato
virus X coat protein and its avirulence determinant,
(Kavanagh et al., 1992; Santa-Cruz et al., 1993; Kohm
et al., 1993; Goulden et al., 1993), Pto and avrPto
~see e.g. Rommens et al., 1995), ~PS2 of ArAhi~r~pqiq
thA7 iAnA and the avirulence gene avr~Pt2 (Bent et al.,
Mindrinos et al. ), and genes of Arahidopsis such as
those identified by Greenberg et al. (1994), Dietrich
et al., (1994) and Bowling et al., (1994).
Genes coding for substances leading to rapid cell
death, such as BA~SE (Mariani et al., 1990) or
WO95i31564 2 1 8 8 5 6 2 PCT~GBg5~01075
29
~iphthPria toxin (Thorsness et al., 1993) may be usable
to induce the changes that lead to GAR even though cell
death in these latter examples is not caused by
activation of the def ence response . It is widely
5 believed amongst researchers in this field that cell
death arises from local induction of the defence
response and that this cell death can activate adj acent
cells to give rise to the defence response. However,
the precise cause and effect rela~ n~hi~ between these
10 events is not clear at the present time. It is also
not clear whether the defence response in plants is
necessarily coupled to necrosis. Hence, cells may
respond to for example the BARNASE-induced death of
adjacent cells by activating a wound-;nf~llr;hle defence
15 L~ullse, such as that leading to the activation of
protease inhibitors or alkaloid biosynthesis (Ryan
lg90). Other genes which may be employed in this way
include a proton pump such as a bacterial proton pump
like the one expressed by Mittler et al (19g5) in
20 transgenic tobacco plants.
A preferred example of the present invention is
the use of the Cf-9/Avr9 gene system. This can involve
the matching of a transposon inactivated allele of the
Cf- 9 gene to constitutive expression of the Avr9 gene .
25 This system can be replaced by similar ~ '-;n~t;rn~ Of
related genes for example the Avr4 and Cf-4 gene,
sequence provided herein (cloning of Cf-4 ifi described
WO9513156~ 2 ~ ~85~ PCJ/GBgS/0107~ ~
in a co-pending GB application ~iled simult;~n~ouRl y
with the present application); the Avr2 and the Cf-2
gene, se~auence provided herein ~ cloning of Cf -2 is
described in GB 9506658,5, priority from which is
5 claimed herein); the Avr5 and the Cf-5 gene, or by
cloning resistance genes and corr~ocrnn-l;n~ avirulence
genes from other systems, such as RPP5, sequence
provided herein (cloning of RPP5 is described in GB
9507232 . 8, priority from which is claimed herein) . It
lO certain cases it may be possible to provoke a suitable
response in plant cells expressing an R gene in the
absence of corresponding Avr, for instance by
~v~le~ 55iOn .
It should also be noted that complete Avr or
15 other ~1; f~; tnr gene may not be required. Instead a
L ~ may be employed, representing a part of the
elicitor molecule which interacts to provoke a plant
defence response and/or plant cell necrosis.
It is possible that the nucleotide sequence
20 comprises the inactivated R gene, the inactivated Avr
gene or both, or comprises both the R and Avr gene
wherein one of the genes is inactivated. Depending of
the genes used, the plant defence response and/or plant
cell necrosis may be ~r~n~nt on the expression of
z5 both genes and so one example would be that the R gene
could be constitutively expressed and the Avr gene
could exhibit somatic variegation for expres~ion due to
~ro gsr3lsG4 2 1 8 8 ~ 6 2 F~
somatic exci9ion and restoration of Avr9 gene
expression, or vice versa.
Nucleotide sequences employed in the present
invention may encode a wild-type sequence (e.g. gene)
5 selected from those available, or a mutant, derivative,
variant or allele, by way of insertion, addition,
deletion or substitution of one or more nucleotides, of
such a se~Iuence. An alteration to or difference in a
nucleotide sequence may or may not be reflected in a
10 change in encoded amino acid sequence, rl~pon(1;n~ on the
~lp~Pn~r;q~y of the genetic code Preferred mutants,
derivatives and alleles are those which retain a
functional characteristic of the protein encoded by the
wild-type gene, in the present context the ability to
15 contribute to a plant defence response and/or plant
cell necrosis. Of course, changes to the nucleic acid
which make no dif f erence to the encoded amino acid
sequence are included.
Similarly, homologues of the various genes whose
2 0 use is disclosed herein from other species or races may
be employed, as may mutants, variants and derivatives
o~ such homologues.
Inactivation and Reactivation of the ~ucleotide
Sequence or Sequences Contributin~ to the Pla~t Defence
25 Response and/or Necrosis
A m~thod according to the present invention may
W095/31564 2 1 8~ ~62 P . . 1 /~
employ any o~ a variety o~ transposon systems known to
the skilled person, including the maize :~
Activator/Dissociation (hereinafter referred to Ac/Ds
system) (Fedoroff, 1989); the maize ~nh~nrPr/suppressor
5 mutator (En/Spm) system (Fedoroff, 1989); and the
~nt;rrhin-lm Taml and Tam3 systems (Coen et al., 1989) .
In addition, any modified recombination systems which
are engineered to yield the appropriate results may be
employed, such as, the bacterial Cre-Loxp (Odell et 21,
1990) or the "FLP/FRT" system (Lloyd and Davis, 199~) .
It will be d~clLe~ to the skilled person that
the particular choice of transposon, re ' ;n~t;nn or
other system used to inactivate the nucleotide sequence
or se~uences which encode substances leading to the
15 plant defence response and/or plant cell necrosis is
not P~R~nt;~l to or a limitation of the present
invention .
In some systems, a tr~n~rnsnn or rP~ ; nAt i nn
system might be so active that an unacceptable level of
20 necrosis is seen. If encountered, this may be overcome
by Pn~;nPPring alleles of the tr2nsposon or rP ' ;n~e
recognition seS~uence in which the frequency at which
activated nucleotide sequences arise is reduced, such
as with Ac(Cla) (Keller et al., 1993) . Alternatively,
25 e hPm; c~l or site-directed mutagenesis may be used to
recover alleles of the necrosis-; n~ ; ng genes which
ar~ less active and therefore result in less severe
Wo 9S/31S64 2 1 8 8 5 6 2
levels of plant cell necrosis (Hammond-Kosack et al.,
19 94 ) .
In other systems, transposition or re~: ' n;
may be inefficient resulting in too few activated
5 nucleotide se~uences leading to an insufficient level
of plant cell necrosis. This may be uv~u",e by
constructing suitable promoter fusions to the
tr~n~ro~Re or re~_ ' ;n~e gene in the plant gene
(Swinburne et al., 1992) to increase the frequency of
10 excision or re~ ' in~t;o~n to efficient levels. The
most suitable promoter might give rise ,Ul~'~ ;n~ntly to
late small sectors of necrosis during organ development
rather than early large sectors.
Many other variations are possible as ;r~
15 for activating the defence response and/or necrosis
after tr~n~pn~nn excision or re~ ` in~inn. A form of
the Cf-9 gene may be constructed so that it activates
the defence response even in the absence of its ligand.
- For example, the Drosophila receptor sevenless
20 (involved in eye development) can be mutated so that it
is activated in the absence of its ligand (Basler et
al, 1991). For example, high level expression of a
disease resistance gene, or expression of a disease
resistance gene in another specie3, may lead to
25 activation of the defence response and/or necrosis even
in the absence of an avirulence product. Bonneus, et
al (1995). In an alternative, the original disease
-
WO 95131S64 2 1 8 8 5 6 2 PCT/GB95/01075
resistance gene may be mutated so that it binds to a
defined chemical such as an agrichemical and this
chemical activates Cf-9 to initiate the defence
response and/or necrosis. Hence, genotypic variegation
5 for excision activating the gene may occur, without
initiation of the somatic necrotic reaction due to the
defence response. The defence response would be
initiated when the agrichemical iæ applied and
recognised by the resistance gene triggering the same
10 reaction as if the avirulence gene product were
present .
rntroducing the Nucleotide Sequence or Seguences which
Contribute to Variegation for the Plant Defence
~esponse an~/or Necrosis into the Plant Genome
The inactivated nucleotide sequence, or
combination of nucleotide sequences at least one of
which is inactivated, codes for a substance or
substances which when expressed in the plant activates
the def ence response and/or leads to plant cell
20 necrosis resulting in broad 8pectrum rath~ n
resistance .
The nucleic acid may be in the form of a
r~, ' ;n~n~ vector, for example a plasmid or
agrobacterium binary vector (Van den Elzen et al.,
25 1985). The nucleic acid may be under the control of an
appropriate promoter and regulatory elements for
WO 9S/3IS64 2 1 8 8 5 6 2 r~, ~ ; /,
expression in a plant cell. In the case of genomic
DNA, this may contain its own promoter and regulatory
elements and in the case of cDNA this may be under the
control of an d~ L iate promoter and regulatory
elements for expression in the host cell.
Those skilled in the art are well able to
construct vectors and design protocols for recombinant
gene expression. Suitable vectors can be chosen or
constructed, c~n~;nin~ cLL,~,v~Liate regulatory
sequences, including promoter sequences, tPrmir~t~r
fr~,~ ' q, polyadenylation sP~ PnrPq~ Pnh~nmPr
sequences, marker genes and other SPqllPn~pc as
Liate. For further details see, for example,
Molecular Cloning: a La~oratory ~anual: 2nd edition,
Sambrook et al, 1989, Cold Spring Harbor Laboratory
Press. Many known techniques and protocols for
~-n;~lllAtion of nucleic acid, for example in
preparation of nucleic acid constructs, mutagenesis,
sequencing, introduction of DN~ into cells and gene
expression, and analysis of proteins, are described in
detail in Short Protocols in ~olecula~ Biology, Second
Edition, Ausubel et al. eds., John Wiley & Sons, 1992.
The disclosures of Sambrook et al. and Ausubel et al.
are incorporated herein by ref erence .
When introducing a chosen gene or gene construct
into a cell, certain considerations must be taken into
account, well known co those skilled in the art. The
Wo 95/31564 2 1 g 8 5 6 2 PCT/GB9S/01075
36
nucleic acid to be inserted may be assembled within a
construct which rnnt~in~ effective rer~ulatory elements
which will drive transcription. There must be available
a method of transporting the construct into the cell.
5 Once the construct is within the cell membrane,
integration into the endogenous chromosomal material
may or may not occur according to different: a; '.C
of ~the invention. In a preferred ~ t, the
nucleic acid of the invention is integrated into the
genome (e.g. chromosome) of the host cell. I~tegration
may be promoted by inclusion of sequences which promote
rel ;n~tion with the genome, in accordance with
atandard technir~ues. Finally, as far as plants are
rnnr~rn~ the target cell type should be such that
cells can be regenerated into whole plants.
Plants transformed with a DNA segment rnntil;n;nrJ
pre-sequence may be produced by standard techni~ues
which are already known f or the genetic manipulation of
plants. DNA can be transformed into plant cells using
any suitable technology, such as a disarmed ~i-plasmid
vector carried by Agrobacterium exploiting its natural
gene transfer ability ~EP-A-270355, EP-A-0116718, NAR
12 ~22) 8711 - 87215 1984), particle or microprojectile
,~ t ~US 5100792, EP-A-444882, EP-A-434616)
microinjection ~WO 92/09696, WO 94/00583, EP 331083, EP
175966), electroporation ~EP 290395, WO 87066~4) or
other ~orms of direct DNA uptake ~DE 4005152, WO
-
~ WO95/31564 21 88562 r~ JUJ~
9012096, US 4684611). Agrnh~rt,orium transformation is
widely used by tho3e skilled in the art to transform
dicotyledonous species. Although Agrobacterium has been
reported to be able to transform foreign DNA into some
monocotyledonous species (WO 92/14828), microprojectile
bombardment, electroporation and direct D~A uptake are
pref erred where Agrobacterium is inef f icient or
ineffective. Alternatively, a combination of different
techniS~ues may be employed to enhance the f~f f i ri ~n. y Of
the transformation process, eg. bombardment with
Agrobacterium coated microparticles (EP-A-486234) or
microproj ectile bombardment to induce wounding followed
by co-cultivation with Agrobacterium (EP-A-486233).
The particular choice of a transformation
technology will be ~ t~rm;n~d by its efficiency to
` transform certain plant species as well as the
experience and preference of the person practising the
invention with a particular ~ hr~r~lo~y of choice. It
will be apparent to the skilled person that the
20 particular choice of a transformation system to
introduce nucleic acid into plant cells is not
essential to or a limitation of the invention.
Selectable genetic markers may be used consisting
of chimaeric genes that conf er selectable phenotypes
25 such as resi3tance to antibiotics such as kanamycin,
hygromycin, phosphinotricin, chlorsulfuron,
methotrexate, gentamycin, srectin~ ycin, ;mi~1~7~1;n~nf~F~
WO 9513156~ . 2 ~ 8 8 5 6 2 1 .I~ l075
38
i~nd glyphosate (~errera-Est~ella et al, 1983; van den
Elzen et al, 1985) .
The present invention is particularly b~n~f i c i
f or use in crop and amenity plants . Examples of
5 suitable plants include tobacco, potato, pepper,
cucurbits, carrot, vegetable brassicas, lettuce,
strawberry, oil seed brassicas, sugar beet, wheat,
barley, maize, rice, soybeans, peas, sunflower,
carnation, cl~ , other orn~ 1 plants, turf
10 grass, poplar, eucalyptus and pine.
Still further details of embodiments of the
present invention are described in the following non-
limiting examples, with reference to the ~ ing
drawings. In the drawings:
Figure 1 srh t;~ lly depicts the Cf-9 gene,
showing tagged alleles. X marks a probable promoter.
Figllre 2 illustrates genetic acquired resistance
to C. fulv~n induced following necrotic sector
formation caused by the excision of a Ds element from
20 ~he Cf-9 resistance gene in an Avr9 expressing tomato
plant. The number of C. fulvu~n pustules per leaf is
indicated, 14 days after inoculation.
Figure 3 illustrates genetic acquired resistance
to Phytophthora lnfestans (late blight of tomato and
25 potato~ . GAR+ and GAR- plants from Cf-9~Ds, mutant
WO 95/3156~ 2 1 8 8 5 6 2 P.~ ' 1075
lines M31 and M50 and cfo plants 3pray inoculated with
10, oOO sporangiospores/mL. In panel A the appearance
of leaves from the muta~t 50 experiment 7 days after
inoculation is shown. In panel B the rate of leaf
5 abscission (in days after ;nnC-llAt;nn) in the various
genotypes inoculated is given.
Fi~ure 4 illustrates genetic acquired resistance
to Phyt~Fhthnra infestans (late blight of tomato and
potato) . GAR+ and GAR- plants from Cf-9*Ds, mutant
lines M31 and M50 and CfO plants were spray innt~ tet~1
with 100 sporangiospores/mL. In panel A the appearance
of leaves from the mutant 50 (GAR+ - right-hand)
experiment 7 days after inoculation is shown, compared
with GAR- (left-hand). In panel B the rate of
15 sporulating lesion formation on the various plant
y~ uLy~es ;nnr~ t~1 is given, with the mean number of
sporulating lesions/leaflet given at 5, 7, 10, 13 and
16 dayg af ter inonl - l ~ t i nn .
Figure 5 shows genetic acquired resistance to
20 Oidium lycopersici (powdery mildew disease). GAR+ and
GAR- plants from Cf-9*Ds, mutant lines M31 and M50 and
Cf O plants were painted with equivalent numbers of
spores. In panel A the appearance of leaves 14 days
after inoculation is shown, GAR- on the left, GAR+ on
25 the right. In B, the rate of chlorotic lesion (upper
panel) and sporulating lesion ~lower panel) formation
on the various plant genotype5 is given for Mutant 31:
WO9SMl564 21 88562 1~1~ . . ,~ ~
mean number of lesions given at 7, 10, 14, 21, 24 and
30 days after inoculation. C shows equivalent results
for Mutant 50.
Figure 6 shows the appearance of tomato fruits on
5 GAR+ (sAc, Cf-9*Ds - right-hand) and GAR- ~6Ac, Cf-9*D6,
Avr-9 - left-hand) plants from mutant line M23 at 2, 3,
4, 5, 6 and 7 weeks after flower poll ;nAt;on Dark
green sectors formed on the GAR+ but not GAR- fruits by
5 weeks. These dark green sectors were not visible on
10 the red fruit.
Figure 7 shows levels of defence-related gene
expression in GAR+ and GAR- plants from Cf-9*Ds mutant
lines M23, M31 and M50 just prior to the pathogen
inoculation experiments. Northern analysis shows in
15 panel A the levels of a basic ,~-1,3 glllr~n~ gene
transcript and in panel B the levels of an anionic
peroxidase gene transcript.
Figure 8 illustrates functional expression of the
Cf-9 gene under the control of its own promoter in
20 tobacco and potato. In panel A is shown a tobacco leaf
that has been injected with intercellular fluid (IF)
either cnnt~;n;ng the Avr9 peptide or lacking the Avr9
peptide. Avr9+ IF~was obtained from transgenic tobacco
or a compatible C. fulvl~n - tomato interaction
25 involving race 5. Avr9- IF was obtained from
untransformed tobacco or a compatible C. fulvum -
tomato interaction involving race 2, 4, 5, 9 . Grey
~ W09~131564 2 1 88562 P~ 075
41
necrosis was vi6ible 3-4 h after injection only in the
leaf panels that had received the Avr+ IF. In panel B
four separate potato leaves are shown that have each
been injected with a single type of IF. Only the two
5 leaves that received the Avr9+IF developed grey
necrosis by 24 h.
Fitgllre 9 shows development of the necrotic lethal
phenotype in seedlings f rom the tobacco cross cv .
Petite Xavana 6201A (355:5P:Avr9)~t 7yyuLe x cos 34.1
10 (genomic Cf-9) heterozygote. A time course for the
period 5-12 days after seed pl~nt;nt3 (dsp) is shown.
50~ of the 5t-t~tll; n~q become chlorotic and die within 2
days of seed germination.
Figure 10 shows development of the necrotic
15 lethal phenotype in st~tll ;n~c from the ~r;~hi~nrsis
cross 6201B4 (355:5P:Avr9) hete- uzyyuLe x cos 138
(genomic Cf-9) heterozygote . Appearance of 5t~t~.11; n~q
19 days after the majority of st~t~t~1 ;n~q had ~t~rm;n~tt?d.
One st-e~l; n5 has died and another has necrotic
20 cotyledons.
Figure 11 shows a single T-DNA construct systems
to apply GAR to potato plants . The T-DNA rt~nr~; nc a
Cf-9 gene seS[uence under the control of its own
promoter which has been inactivated by an ~-ltt nl us Ac
25 element that is only capable of a low level of
- excision, the Ac (Cla) element (Keller et al. 1993;
Schofield et al. 1994) and the 35S:SP:Avr9 transgene.
Wo 95/31564 2 1 ~ ~ ~ 6 2 PCr/GB95101075
42
Figure 12 shows a photograph Df three leaves, two
of which are diseased with C. fulvum and one which is
expressing GAR and is resistant tD the same inoculum of
C. fulvum.
Figure 13 illustrates how GAR+ plants may be made
by crossing stable lines (1) compriaing a Cf-9 gene,
inactivated by insertion of a Ds trAncpnsnn, and an
~vr-9 gene and (2) an Ac transposase gene, as described
in Example 1.
Figure 14 illustrates basic simplified haploid
crossing schemes to produce plants with increased
disease resistance.
T: transgenic line
P: offspring of trAna~n; e line
T1/Pl: line comprising in its genome at
least one of each of the four genes,
R, L,I or A
Tl,2/Pl,2 line comprising in its genome at
least one of each of two of the f our
genes R, L, I or A :
T3~P3: line comprising in its genome at
least one of each of the four genes
R, L, I or A not present in T1 2
T3,4/P3,~,: line comprising in its genome at
least one of two of the four genes R,
L, I or A not present in Tl 2
Tl,2,3/P1,2,3 line comprising in its genome at
w095~ 64 2 ~ 8 8 5 6 2 ~ 1~ cTIu~a
least one of each of three of the
four genes R,L, I or A
T4/P4 line comprising in its genome at
least one of each of the four genes
R,L, I or A not present in T1 2 3
SEQ ID NO. 1 shows the genomic DNA sequence of
the Cf-9 gene. Features: Nucleic acid sequence -
Translation start at nucleotide 898; translation stop
at nucleotide 3487; polyadenylation signal (AATA~A) at
nucleotide 3703-3708; polyadenylation site at
nucleotide 3823; a 115 ~p intron in the 3' non-coding
sequence from nucleotide 3507/9 to nucleotide 3622/4.
Predicted Protein Sequence - primary tr~nRl~ti~-n
product 863 amino acids; signal peptide sequence amino
acids 1-23; mature peptide amino acids 24-863.
SEQ ID NO. 2 shows Cf-9 protein amino acid
sequence .
SEQ ID N0. 3 shows the sequence of one of the Cf-
9 cDNA clones. Tr~nRl~t;nn initiates at the ATG at
20 position +58 . Cf -9 genomic sequence
SEQ ID NO. 4 shows the amino acid sequence and
DNA sequence of the preferred form of the chimaeric
Avr9 gene used as descri~ed herein
SEQ ID NO. 5 shows the genomic DNA sequence of
25 the Cf-2. 1 gene Features: Nucleic acid sequence -
Translation start at nucleotide 1677; translation stop
WO95131564 21 8 ~ 5 62 ~ a
at nucleotide 5012; no consensus polyadenylation
signal ~AATA~A) exists in the characterised sequence
downstream of the translation stop. Predicted Protein
Seriuence - primary translation product 1112 amlno
5 acids; signal peptide ser~uence amino acids 1-26;
mature peptide amino acids 27-1112_
SEQ ID NO. 6 shows Cf-2 protein amino acid
sequence, designated Cf-2.1.
SEQ ID NO. 7 shows the amino acid sequence
10 encoded by the Cf-2.2 gene. Amino acids which dif~er
between the two cf-2 genes are underlined.
SEQ ID NO. 8 shows the sequence of an almost full
length cDNA clone which corresponds to the Cf2-2 gene.
SEQ ID NO. 9 shows the genomic DNA sequenee of
15 the RPP5 gene. ~ntiri~t~ introns are shown in non-
capitalised letters. Features: Nucleic acid sequenee -
Translation start at nueleotide 966; translation stop
at nueleotide 5512.
SEQ ID NO. 10 shows predieted RPP5 protein amino
20 aeid sequenee.
SEQ ID NO. 11 shows genomic DNA sequence of Cf-4.
Features of this sequence include: tr~nR~t;rn start
site at nucleotide 201, translation stop beginning at
nueleotide Z619, rrnR~nRuR polyadenylation seriuenee
25 beginning at nueleotide 2835, spliee donor sequenee in
3 ~ untranslated sequenee at 2641, splice acceptor
ser~uence ending at nucleoti e 2755, proposed site o~
wo 95/31561 2 1 ~ 8 5 6 2 P~ I, ...,, '; . 075
polyadenylation at nucleotide 2955.
SEQ ID NO. 12 shows the predicted Cf-4 amino acid
sequence. The predicted protein sequence is composed
of a primary translation product of 806 amino acids,
signal peptide sequence amino acids 1-23, mature
peptide amino acids 24-806.
SEQ ID NO. 13 shows double-stranded nucleic acid
and deduced amino acid sequence of a ClaI/SalI DNA
f~s ~ encoding the PRla signal peptide sequence
fused to a sequence proposed to encode the mature
processed form of C. fulvum AVR4. Tr;ln~lati~n
initiation codon at nucleotide 5, t-~rrn;n:lt jr-n codon
be~; nn; ng at nucleotide 413 . Amino acids 1-30
represent the signal peptide and amino acids 31-136 the
mature AVR4 peptide.
l~PLE 1
GENETIC ACQrJIR 3D RESISTANCE (G~R) Z7SING Cf-9
(i) B8t~h7 i~2hin~ a stock from w~ich gametes carrying a
mutagenised Cf-9 gene may be obtained and identified
During experiments to isolate the Cf-9 gene by
transposon tagging, alleles of the Cf-9 gene (Cf-9*Ds)
were isolated that had been inactivated by insertion of
the transposon Ds (See Tnt~rn~tion~ Patent Application
No. PCT/GB94/02812 for further details). This
inactivated Cf-9~Ds gene did not give rise to a
WO95/31564 21 885b2 .~1~ 1075
46
constitutive and lethal activation of defence~
.h~n; ! in response to the constitutively expressed
3 5S: SP: Avr9 gene
We have est~h~ hP-l the capacity to carry out
transposon tagging in tomato using the maize transposon
Activator (Ac) and its Dissociation (Ds) derivatives
(Scofield et al 1992; Thomas et al 1993; Carroll et al
1993). The strategy is founded on the fact that these
transposons preferentially transpose to linked sites.
Various lines that carry Dss at positions are useful,
including FT33 (Rommens et al 1992), carrying a Ds
linked to Cf- 9, and lines that carry a construct
SLJ10512 (Scofield et al 1992) which ~ntsl;n~ (a) a
beta-glucuronidase ~GUS) gene (Jefferson et al 1987) to
monitor T-DNA segregation and (b) stable Ac (sAc) that
expresses tr~n~pn~e and can trans-activate a Ds, but
which will not transpose (Scofield et al 1992).
The line FT33 did not carry a cf-g gene. We had
to obtain r~: ` in~nt'~ that placed Cf-9 in cis with the
T-DNA in FT33 in order to carry out linked targeted
tagging. Two strategies were pursued simultaneously:
(a) FT33 was crossed to Cf 9, a stock that carries
the Cf- 9 gene . The resulting F1 was then back crossed
to Co (a stock that carries no Cf- genes). Progeny
that carry the FT33 T-DNA are kanamycin resistant.
Kanamycin resistant progeny were tested for the
wo ss/31564 2 1 8 8 ~ 6 2 ~ J
47
presence of Cf-9; 5 C. fulvll~n resistant individuals
were obtained among 180. We alsogenerated progeny that
were 1- _yy"us for Cf-9 and carried that sAc T-DNA of
SLJ10512. These were crossed to the rPI~ ' ;nAn~S in
5 which Cf-9 and FT33 were ill cis. In the FT33 T-DNA, a
tr~n~rr s~hle Ds element is cloned into a I~YYL' y~:in
resistance gene, preventing its function. The somatic
transactivation of this Ds element, which only occurs
in the presence of trAn~p~ ce gene expression, results
10 in activation of the l1YYLL y~in resistance. Thus
from crossing the recombinants between Cf-9 and FT33,
to the sAc-carrying Cf-9 1- _yyuLe8, l1YYL~ yuin
resistant individuals could be ~htA; r~Prl which carry sAc
and FT33, and are likely to be ~ --yyuus for Cf-9.
15 140 individuals of this ye:~oLy~e were thus obtained.
(b) To accelerate ~hrA;n;ns individuals that
carried sAc, FT33, and were Cf-9 hl -~yu~es, the
FT33/Cf-9 F1 was crossed to a line that was
heterozygous for Cf-9 and sAc. 2596 of the resulting
20 progeny carried both T-DNAs and were LyyL, y-:in
resistant, and of those, slightly more than 5Q~ were
disease resistant because they carried at least one
copy o~ the Cf- 9 gene An RFLP marker was available,
designated CP46, that enabled us to dist;n~liAh between
25 h~ yyutes and heterozygotes for the Cf-9 gene
(3alint-kurti et al 1993). In this manner two
individuals that were Cf-9 h~ yyutes~ and that
W0 9Sl31564 2 1 8 8 5 6 2
48
carried both the FT33 T-DNA and sAc, were obtained.
These two individuals were multiplied by taking
cutting6 80 that more crosses could be made onto this
genotype .
5 (iiJ Establishing a tomato stock that expresse3
functional mature AV~9 protein
A likely freguency for obtaining any desired
mutation in a gene tagging experiment is less than 1 in
lO00, and often less than 1 in 10, 000 (Doring, 1989) .
10 To avoid screening many thousands of plants for
mutations to disease sensitivity, we est~hl; ahf~d a
~election for such mutations based on expressing the
fungal Avr9 gene in plant8.
The sequence of the 28 amino acids of the mature
Avr9 protein is known (van Kan et al 1991). It is a
secreted protein and can be extracted from
intercellular f luid of leaves inf ected with Avr9-
carrying races of C. fulvum. For secretion from plant
cells, we de8igned oligonucleotides to assemble a gene
20 that carried a 30 amino acid plant signal peptide, from
the Prla gene (~r~rn~ sen et al 1987) preceding the
first amino acid of the mature Avr9 protein (see SEQ ID
N0. 4). The preferred Avr9 gene sequence depicted in
SEQ ID N0. 4 shows a r~;r~A~c gene ~n~;n~ed irom the
25 Pr-la signal peptide sequence (Corn~ s~n et al, 1987)
and the Avr9 gene se~uence (van Kan et al, 1991). This
.
-
~ WO 95131564 2 1 8 8 5 6 2 r~ '01075
49
reading frame was fused to the 355 promoter of
cauliflower mosaic viru8 (Odell et al 1984), and the 3
terminator sequences of the octopine synthase gene
(DeGreve et al 1983), and introduced into binary
5 plasmid vectors for plant transformation, using
techniquee well known to those skilled in the art, and
readily available plasmids (Jones et al 1992). We
obtained transformed CfO tomato lines that expressed
this gene.
10 (iii) Crossing AV~Z9 expressiI~g stock with Cf-9
expressing stock
The transformed lines obtained in (ii) were
crossed to plants that carried the Cf-9 gene. When the
resulting progeny were germinated, 50~ l~h;h;tf~ a
15 necrotic phenotype, that mllm;n~ted in 9.o~l ;n~ death.
This outcome was only observed in soorll ;n~q that
cnnt~;nPd the Avr9 gene. When the same tran~Lo,l..d~ts
were crossed to Cf 0 plants, the resulting progeny were
all fully viable.
From selfing the primary transformants,
individuals were ;tl--nt;~;efl that were homozygous for
the Avr9 transgene. When Avr9 homozygotes were crossed
to Cf-9, all progeny died. This system thus provides a
powerful selection for individuals that carry ~tinnc
ln :he Cf.9 gene.
Wo 95/31564 2 l 8 8 5 ~ ~ r ~ /a
(iv) Tagging and inactivati~ Cf-9
Individuals that were 1- - yy~,us f or the Avr9
gene (section (iv) ) were used as male parents to
pollinate individuals that were hc~u,J~yy~us for Cf-9,
5 and carried both sAc and the Ds in the FT33 T-DNA
(sectio~L (iiia) and (iiib) ) Many th~ n~l- of progeny
resulting from such a cross were germinated. Most
died, but some survived.
DNA was obtained f rom survivors and subj ected to
10 Southern blot analysis using a Ds probe. It was
observed that several ; n~ t; nnq were
correlated with insertions of the Ds into a BglII
rl _ - of a consistent size. This suggested that
several independent _tinn~ were a consec~uence of
15 insertion of the Ds into the same DNA Ll _ ~ .
Using primers to the Ds se~Euence, DNA adjacent to the
Ds in tL ~ sed Ds-carrying mutant #18 was amplified
using inverse PCR (Triglia et al 1988) . This DNA was
used as a probe to other mutants, and proved that in
20 independent t~tinn_, the Ds had inserted i~to the
same 6 . 7 kb ~glII ~
The Ds in FT33 contains a bacterial replicon and
a chlc~l , ~; col resistance gene aa a bacterial
selectable marker (Rommens et al 1992). This means
25 that plant DNA carrying this transposed Ds can be
digested with a restriction enzyme that does not cut
within the Ds (such as ~glII), the digestion products
WO95/31564 2 l 8 8 5 6 2 1 ~1 . '0107~
can be r~ri rr~ rized, and then used to transform E~.
coli . ChluL , hrn i col resistant clones can be obtained
that carry the Dr and adjacent plant DNA. This
procedure was used to obtain a clone that carried 1. 8
5 kb of plant DNA on the 3 ' side of the Ds, and 4 . 9 kb of
plant DNA on the 5 ' side of the Ds .
Our present understanding of the Cf-9 gene is
depicted schematically in Figure 1. The Cf-9 gene
sequence and the deduced amino acid sequence are shown
10 in the sequence listing .
A series of primers (Fl, 2, 3, 4, 5, 6, 7, 12,
13, 10, 26, 27 and 25, indicated in Figure 1~ was used
to char~rtf~ri ~e a large number of i n~ "t mutations
by PCR analysis in combination with primers based on
15 the sequence of D8. Therefore, these primers were used
in polymerase chain reactions with primers based on the
maize Ac/Ds tr~nRpo~nn sequence, to characterise the
locations of other ~ lt~tirn~ of Cf-9 that were caused
by transposon insertion. Eighteen i~dependent
20 insertions have been characterized and are located as
shown. Mutants E, #55, #74 and #100 gave in~ _ ,le~te
survival and showed a necrotic phenotype, and based on
the available sequence information, they are 5 ' to the
actual reading frame and might permit enough C~9
25 protein expression to activate an incomplete defence
response.
Using the sequence obtained of the gene,
WO95/~1564 2 1 ~ 5~2 P~
oli~r,nl-r1 ~ntide primers were designed that could be
used in polymerase chain reactions in combination with
primers based on the sequence~of the Ds element, to
characterize both the location and the ori rnt~t; rn Of
5 other transposon insertions in the gene. These are
shown on Figure l Based on the results of such
experiments, the map positions of 17 other DS
insertions have been reliably assigned (as shown in
Figure l ) .
10 (v) Production of ~AF~ plants
On backcrossing plants that carried the Cf-9*Ds
and 35S:SP:Avr9 gene to tomato plant6 that carried an
Ac transposase gene (sAc that lacked the GUS gene) in
the homozygous state, but lacked Cf-9, one quarter of
the resulting progeny carried sAc, 355:5P:Avr9 and Cf-
9*Ds (see Figure 13) plants showed somatic ~Tr;~r,n Of
Ds from the Cf-9*Ds gene, somatically restoring Cf-9
function, and giving rise to necrotic somatic sectors
in which the def ence response was activated .
Phenotypically, these plants thus showed a variegation
for a defence-related necrosis, in the same manner that
plants challenged with necrotizing pathogens show
somatic flecks of ~R that are associated with the
induction of SAR.
Necrotic secto~s were visible on cotyledons,
leaves, stems, petioies, sepals, and green fruits
throughout plant development. Also, the necrotic
95--1564 53
sectors formed in both the lower and upper epidermis,
in all mesophyll layers and in the cells surrounding
the vascular tissue. The size of the necrotic sector
and the frequency of their formation was determined by
5 both the position of the Ds element in the Cf-9
se~uence and the orien~t1c~n of the Ds.
The plants that variegated for necrosis were
tested to assess if they were more resistant to C.
fulvum than their unvariegated ~ihl;n~q that either
10 carried Cf-9*Ds or carried no Cf-9 gene. Plants from
five ;nf~PrPn~Pn~ Cf-g*Ds pedigrees were tested in which
the Ds had ;n~PrPn~Pn~y inserted into five different
locations in the Cf-9 gene. These five ;n~PrPn~Pnt
insertions were between Cf-9 amino acids, 7 and 8
(<M23), 28 and 29 (~M18), 47 and 48 (>M50), 56 and 57
(~M31) and 789 and 790 (~M30) The arrows (~ or :.)
indicates the direction of transcrlption of the Ds
element. Fl plants that developed somatic necrotic
sectors were more resistant to C. fulvum than sibling
20 offspring that did not develop necrotic sectors. On
the plants with necrotic sectors an average of 1-2
small pustules per leaf developed, 14 days after
inoculation with 5 x 105 spores/ml. The plants lacking
a Cf gene and the non variegating individuals all
25 showed on average 3 8 large sporulating pustules per
- leaf. A example of this is shown in Figure 2.
Nine variegated Cf-9*Ds #20 plants, fifteen
WO95131564 21 88562 1~11. 1 ,~
54
variegated Cf-9*Ds #23 plants, eighteen variegated Cf-
9*D8 #30 plants and twenty-eight variegated Cf-9*Ds #31
plants were tested, and compared to one hundred and
ninety eight plants in total that did not variegate for
5 necrosis. Plants were inoculated with C. fulvum (5 x
105 spores/ml) when they were four weeks old and
carried 2 ~An~l,od leaves. A similar result was
obtained when variegated Cf-9*Ds #50 plants and non-
variegated plants were; nnrlll A~ed with C. fulvuzn. On
18 variegated GARt #50 plants 1-3 pustules per lea~
formed, whereas on 42 non-variegated GAR- #50 plants
over 35 pustules per leaf developed by 14 days after
;nnClllAt;rn.
Sensitivity to the pathogen was measured by
rclln~;n~ the number of sporulating pus~ules that were
visible on each genotype 14 days and 21 days after
inoculation. Samples were also taken for microscopic
analysis. The results of the assay after 14 days are
shown in Figure 2, and typical infections on each
genotype after 21 days are shown in Figure 12.
Figure 2 shows a histogram in which the
sensitivity of different individual tomato plants is
ex~oressed on the y axis as the number of sporulating
pustules per leaf. The Ds carried a GUS gene. M20,
M23, M30 and M31 show C. fulvum growth on plants
resulting from crosses between Cf-9*Ds and sAc, and
derive from Cf-9*Ds #20, Cf-9*Ds #23, Cf-9*Ds #30 and
Wo9s/3ls64 2 1 88562 ,~ L~ "
Cf-9*Ds #31, respectively. These individuals segregate
from the Cf-9*Ds and for sAc. CfO carries no R genes
- and M20, M23, M30 and M31 GUS- plants have lost by
segregation both Cf-9*Ds and sAc and are thus
5 disea6e sensitive sibs, providing a good control for
disease symptoms in sensitive individualæ. If plants
receive Ds without sAc they may be GUS+ without
expressing the variegation for necrosis which requires
both Cf-9*Ds and sAc. As can be seen, the necrotic
10 individuals (which all carry the 35S:~vr9 gene) show
distinctly fewer pustules per leaf than their disease
sensitive sibs.
Figure 2 shows that in these experiments, CfO
plants (lacking the Cf-9 gene) exhibited about 38
15 pustules per leaf and non-vari~t; n~ individuals
derived from Cf-9*Ds #20, Cf-9*Ds #23 or cf-g*Ds #3
also showed about 3 8 pustules per leaf . The non-
variegated individuals that carried Cf-9*Ds #30 showed
about 17 pustules per lea~ i~dicating some residual
20 action of the tagged Cf-9 allele. ~lowever, variegated
individuals that carried Cf-9~Ds #20, Cf-9*Ds #23, Cf-
g*D8 #30 or Cf-9*Ds #31 showed 1-3 pustules per leaf.
In total seventy variegated individuals were assessed.
These results demonstrate a very si~n i f i l-~nt level of
25 disease control by this method.
- Figure 12 shows three leaves. Leaf 1 and Leaf 2
are infected with C. fulvum which confers the white
Wo 95/31564 2 ~ 8 8 5 6 2 PCT1Gsg5/01075
fluffy appearance. Leaf l is CfO and Leaf 2 is a
disease sensitive sib from Cf-9*Ds #23. Leaf ~3 showing
minimal sporulation is a necrotic individual (small
sectors of necrosis are discernible) that carried Cf-
9~Ds #23, sAc and 355:Avr9. Leaf 3 is therefore
expressing GAR.
It is important to recosnize that in this example
regions of variegating plants that resist the C fulvurn
pathogen do not contain a functional Cf-9 gene. Indeed
all the cells that do carry a fllnrt j r~n~l Cf-9 gene
(whose function was restored somatically by transposon
f.Yr; R; nn) are killed as they turn on the defence
response after recognition of the ~nrlrg~nrl1cly
expressed Avr9 peptide. Thus, non resistant cells are
being induced to resistance by necrosis being
manifested in adjacent cells.
E:XA~PLE: 2
Pathogen reBi6t~nce of v~riegated pl ntB employlng Cf-9
In addition to demonstrating that variegated
plants produced in Example l have ~nh~nr~d resistance
to C. fulvum, we have est~hl; F~h~ that the plants are
also more resistant to three unrelated fungal
pathogens, Phytophthora infestan~ (the causal agent of
late blight disease of tomato and potato) and Oidium
lycopersici (a powdery mildew) and Colletotrichum
largenarium (which causes leaf and fruit spot).
WO 9S/31564 2 1 ~ ~ ~ 6 2 PcT/Gs95/01075
57
For the P. infestans experiments, sibling
backcross progeny from the mutatnt Cf-g* Ds li~es M31
and M50 that were either variegating for necrosis or
not and control plants lacking a Cf-gene (CfO) were
5 challenged by a spray application of sporangiosspores
(lO,OOO or 100 spores/ml) of the highly virulent
isolate DSSI (Al mating type). After ;nrr~ t;on, the
plants ~qere kept in diffuse light conditions at a
constant 1009~ RH and 16 C and a 12h photoperiod.
Seven days after application of the high spore
dose the leaves of the unvariegated plants and those of
the Cf O plants were completely destroyed by the spread
of P.infestans lesions which had i~hlln~n~
sporangiospores at their margins. In contrast, the
15 variegated plants were infected with P. infestans but
the lesions were 3-5 mm in ~;: ~t~r and non-sporulating
(Figure 3 A, B) . An additional 5-6 days were re~uired
before the enti~e green leaf tissue of the variegated
plants was destroyed and fungal sporulation ~ r~d.
20 At the lower spore dose, by 7 days after inoculation,
an average of 8-10 large sporulating lesions were
present on each leaf of the unvariegated and Cf O plants
whereas on the plants variegating for necrosis there
were 1-2 small non-sporulating lesions per 10 leaves
25 (Figure 4 A, B) . A minimum of 18 plants were used for
- each genotype/spore.
For the Oidium lycope~sici experiments the
WO95/31564 21 88562 1~1~ 5 .~75
.
58
i~nt i ~ l plant genotypes werë used . Each leaf was
inoculated by brushing with an artist paintbrush the
spores from a single 14 day old sporulating pustule
over an entire upper surf ace . The inoculated plants
were then kept under diffuse ~ight conditions at 20 C
during the 16 h photoperiod and at 18 C during the dark
period. The RH was ~n~int;~;n~tl at 7096.
By day 10 post inor~ ti~n 8-10 chlorotic lesions
were evident on the leaves of the unvariegated and Cf 0
plants and in 1-2 of these sporulation had ~ e~l.
By contrast on the variegated plants 1-2 smaller
chlorotic non- sporulating lesions were present on each
leaf (Figure 5). By day 14 post inor~ t;~n more than
20 sporulating lesions per leaf were present on the
unvariegated plants and these were ~: _ i ed by
severe chlorotic symptoms on the l. ; n~-~ of the leaf .
On the variegated plants 2-4 small sporulating lesions
were present per leaf (Figure 5A). An additional 7-10
days were reSEuired before a similar level of
sporulation and chlorosis formed on the variegated
leaves to that f olmd on the unYariegated and Cf 0 leaves
at day 14 post-inoculation. (16 plants each).
E~PLE 3
Variegation in fruit
Dark green sectors formed on green tomato fruits
of GAR plants, 5 weeks after flower po11 in~ti~n (Figure
.
~ Wo 95/31564 2 1 8 8 5 6 2 ~ 075
59
6~ . These sectors were not visible once the tomato
fruit had turned red, which is encouraging for
potential commercial exploitation. When mature red
fruit taken from GAR+ and GAR- plants were injected with
lO0f~1 of spores of Colletotrichum laginariu~ (104
spores/ml) only the GAR- fruit exhibited the typical
soft rot disease symptoms seven day6 later. Repeated
ino~ t;~nc of the GAR+ fruit failed to cau3e disease.
Collectively, the above results attest to a very
significant level of disease control that can be
achieved in the plants variegating for restoration of
Cf-9 gene f-lnct;on whilst constitutively expressing
the Avr9 gene. The data also indicate that the disease
control achievable by this method is pot~nt ~ y broad
spectrum because the four fungal pathogens controlled
have very dissimilar modes of parasitism: C. fulvu~r~ is
a biotroph that does not form haustoria and grows
exclusively in the extr~ r spaces of the leaf
mesophyll layers; 0. Lycopersici is also a biotroph but
colonises only the upper leaf epidermis and forms
complex intracellular haustoria; P. infe6tans and
C.largenarium are hemibiotroph that initially forms
simple haustoria but later on kills host cells in both
the epidermal and mesophyll layers.
- 25 ~1~ y~cJus Cf-9*Ds, 35S:SPAvr9 lines have been
established for the tomato lines M31 and M50. The
W095/3l564 2 1 8 ~ 5 62 .~. ~ 5 ~1075
backcros3 progeny derived from crosses to a h _yyuus
sAc source, may be assessed for their resistance to
various pathogens, including:
Potato virus X, Ps~ syringae pv. tomato,
Necrotrophic fungi - Botrytis spp, Colletotrichum spp,
N: to~f~c - Meloidogyne incognata, Aphids - Green Peach
Aphid, and fruit, pod, root or tuber attacking
pathogens. Also, the effect of GAR on the
est~hl;~h~ of mycorrhizal assor;~ti~n~ may be
tested.
The ~nh~n--cl resistance exhibited in the plants
variegating for necrosis has been termed Genetic
Acquired Resistance (GAR~. It is distinct from SAR
because it is a heritable trait and is active
throughout the entire plants lif e .
When the expression of several defence-related
genes were compared in the GAR- and GAR~ plants,
significantly higher levels of expression of each gene
were found in the GARt plants. Examples of this are
shown in Figure 7 for C~-9*Ds lines from M23, M31 and
M50 pedigrees using a basic tomato ~-1,3 glucanase
probe and a tomato anionic peroxidase probe (pTAP ~ . 5 ) .
The ef f ectiveness of GAR in suppressing plant
disease appears to be inversely related to sector size.
The two; n-l~r~n~nt Cf -9*Ds pedigrees that have the
highest frequency of small necrotic sectors (lines M31
wo ss/31s64 r~ 07~
21 88562
61
and M50) give the best GAR. This indicates that by
carefully manipulating the frequency of somatic
restoration of Cf-9 function even higher levels of
plant protection be developed.
Currently, there are two possible hypotheses to
explain GAR. Either the initially activated host cells
generate local and systemic signals whilst still alive,
and the necrotic lesions are a by-product of the Cf-9-
Avr9 mediated responses. Alternatively, the actual
death and necrotic reactions, the final response of the
activated host cells, generates specif ic local and
systemic signals in a manner analogous to SAR. Exactly
how GAR works does not need to be known for the present
invention to be ~p~^ated. Provided the re~uired
genetic, _ ^-lt~ are present, GAR plants have
~nl~n~ed pathogen resistance ,~ ed with wild-type.
E~A~PI,E: 4
Expre/33ion of Cf-9 in Heterologous Pl~nt~ Species and
Induction of Cell Necro~i~
We have shown that following the transfer of
different genomic clones rnnt~;nin~ the Cf-9 gene into
tobacco and potato, these sequences render the
~Ld~S~ ic plants responsive to Avr9 elicitor (Figure
8) .
Also when transgenic tobacco expression Cf-9 is
crossed to transgenic tobacco plants engineered to
WO 95/31564 2 1 ~ 8 5 6 2
62
express Avr9 peptide constitutively, the Fl seedlings
die within 2 days of seed g~rm;n~t;rm (Figure 9).
When transgenic Ar~hi~ p~is expressing Cf-9 is
crossed to Avr9 expressing transgenic Ar~hi~f~rs;~ the
Fl seedlings die lO days after seed germination (Figure
10) .
Thus we have shown that in a variety of species,
genes required for activation of plant defence,
mediated by the Cf-9 protein, are present and
functional.
EXA~P~E 5
Genetic Ac$~uired liesist~nce Using Cf-9 in Potato
To apply GAR to potato plants a single T-DNA
construct systems is used.
The system i8 based around a single T-DNA
construct (Figure ll) rnnt~;n;ng~ a Cf-g gene seqn~nr~
under the control of its own promoter which has been
inactivated by an ~ltnr ~ Ac element that is only
capable of a low level of ~ n (the Ac (Cla)
element (Keller et al. 1993), and the 355:SP:Avr9
transgene) . The Ac element is inserted at various
positions in the Cf-9 sequence and in both or;!~nt~ti-~n~:
in order to determine the best conf iguration to produce
a high fre~uency of small somatic sectors where Cf-9
function has been restored.
Placing the C~-9 sequence or other R gene
~ W0 95~31564 2 1 8 ~ 5 6 2
63
sequence under the control of a cell-type specific
promoter may enhance the GAR phenotype. Potential
target cellular sites include the epidermis and the
vascular parenchyma cells.
5 E~PLE 6
Expres~ion o Cf-g in trancgenic plants and
demonstration of increased pathogen res~tance
The Cf-4 gene has been tested in transgenic
plants in a number of ways: firstly by inocl~lAtinn with
10 a race of C. .fulvum -,mt:-;n;n~ the corr~qp~n~l;n~
avirulence gene Avr4 to test if that race gives an
; n. t; hle response on the transgenic plant; secondly
by injecting leaves of a transformed plant with
intercellular fluid; Aol~ted from a compatible
15 interaction r~nt~;n;n~ AVR4; thirdly, by delivering
AVR4 in the form of r~__ ` ;n~nt potato virus X as
described previously in studies of the Cf-9/AVR9
interaction (TT i-Kosack et al., 1995).
The DNA sequence of the C. fulvurn gene ,~nr~n~; n~
20 AVR4 has been reported and the amino acid sequence of
the mature processed polypeptide (Joosten et al.,
1994). We amplified by PCR the Avr4 gene from C.
fulvum race 2, 5 using primers to the pllhl i Ch~-:l sequence
and fused a sequence encoding the proposed mature
25 polypeptide to a DNA sequence encoaing the N-t,~nini,
Wo95/31564 21 8 8 5 6 2 PCT1GB95101075
64
signal peptide of the tobacco PRla protein. This would
f acilitate targeting of AVR4 ~o the intercellular space
in transgenic plants where it is expressed. This
chimeric gene (SPAvr4~ was inserted into a cDNA copy of
5 potato virus X, as a ClaI/Sa~I DNA fragment ~SEQ ID NO.
13) as described previously (Hammond-Kosack et
al.,l995) to generate PVX:SPAvr4. Infectious
transcripts of the rerr-~i n~nt virus were generated by
in vitro tran8cription. All nucleic acid r~~n;r~ tions
lO were performed using standard techniques well known to
those skilled in the art.
Toma to
Experiments were designed to test the r/~c ' ;n:lnt
virus in 3 week old tomato 8eedlings. In Cf-4
15 rnnt:~;n;n~ plants inoculated cotyledons appeared
desiccated and eventually Ahsr; ~ed at 3 days
post-inoculation Id.p.i.), in contrast to CfO controls
which only showed signs of slight mechanical damage at
the site of virus innC1~l~tir~n. CfO plants developed
20 visible symptoms of virus infection at 7-lO d.p. i .
comparable to symptoms observed with the wild type
virus i.e. chlorotic mosaic symptoms. At 4-5 d.p.i. in
plants rnnt;3;n;ng Cf-4 necrotic lesions were observed
in the younger leaves, presumably due to systemic
25 spread of the virus as described previously in similar
experiments with PVX -nnt~;n;ng Avr9 on Cf-9 rnnt~;nin~
WO 95131564 6 ~ /a
plants ~Hammond-Kosack et al., 1995). Other features
included necrotic sectors on petioles and the stem.
The necrotic phenotype was seen to spread systemically
and at 14 d p.i. the majority of Cf-4 ~ntA;n1n~
5 seedlings had died . Cf O control plants did not die but
did show symptoms of chlorosis and vein-clearing.
These results confirm that Cf-4 is functional in
transgenic tomato plants, resulting in a necrotic
defence response in the presence of f~ i tor AVR4 .
10 To~acco
Using binary vector cosmids comprising cf-s,
transgenic tobacco plants have also been produced
(Fillatti et al . ,1987; Horsch et al., 1985) using
techni5Iues well known to those skilled in the art.
Transgenic tobacco ~-~nt~;n;n~ cosmids comprising
C-4 were inoculated with PVX:SPAvr4. In most
transformants necrotic lesions were observed at the
site of viru8 inO~1l7At;c n 3-4 d.p.i. similar in
appearance to lesions which appear in response to virus
20 inocll1At;C~n in some virus resistant varieties. In
these individuals the necrosis was not strictly
nf;n~d to local lesions which eventually coalesced
and at 7-10 d.p. i . leaf necrosis was apparent over the
entire region of virus inoclllAt;~n. In several
25 transformants the reaction to PVX:SPAvr4 was more acute
and the necrotic leaf sectors could be observed at 3-4
WO95131564 21 ~ 8 5 ~ i /a
66
d.p . i . Neither o~ these phenotypes were observed in
transgenic tobacco rnnt~;n;nr~ cosmids lacking Cf-4 or
in non-transformed control plants rh~llPnrJP~ with
PVX: SPAvr4 .
F-1nrt;rn~1 expression of cf-s in transgenic
tobacco has thus also been shown, with activation of a
necrotic defence response in the presence of elicitor
AVR4 .
Pa thogen Resi s tan ce
Transgenic plants were propagated by cuttings so
that Cf-4 activity could be detected by inoculation
with PVX:SPAvr4 on 12 tomato transformants. Transgenic
tomato plants rnnt~;n;nJ Cf-4 exhibited leaf necrosis
on ;nn~ tPd leaves 3-4 d.p.i. This necrosis
eventually spread systemioally as previously obsc:L v~d
in Cf-4 rnnt~;n;n~ plants in the expP~i c described
above. Transgenic plants exhibiting necrotic leaf
sectors eventually died.
Cuttings of a number of transgenic plants
obtained in the first round of transformation
experiments were further assayed for Cf-9 function by
inoculation with C. fulvum race 5. In 5 transgenic
plants tested, a positive correlation was observed
between plants exhibiting PVX:SPAvr4 flPren~lPnt necrosis
and resistance to the pathogen. In this P~Pri~^^nt
pathogen growth was .bseLve-l on c -tihle control
~ Wo9Sl31564 2 1 ~ 8 5 6 2 ~ J
67
plants (CfO) but not on incompatible control plants
(Cf2) .
All documents - ; ~n~-cl in the text are
incorporated herein by reference.
5 ~r
Balint-Kurti, et al. (1994~ Theor. App. Genet.
88: 691-700 .
Basler, et al. (1991) Cell, 64, 1069-1081.
Becker,' et al. (1993) Plant Journal 3, 875-881.
Bent, A.F., et al. (1994) Science 265, 1856.
Biffen, R.H. (1905) J. Ag. Sci. 1, 4-48.
Carroll B.J. et al. (1993). Genetics (In Press).
Chang, C., et al. (1992) The Plant Cell 4:1263-1271.
ChristoU, P. (1992) The Plant Journal, 2 (3), 275-281.
Coen, et al. (1989) In Mobile DNA. D.E. Berg and M.M.
Ho~e, eds. (Washington: ASM Pres),
~'r.rn~ s~n, et al. (1987) Nucl .Acids.Res 15 :6799-6811.
Culver, et al. (1991) Molecular Plant Microbe
Interactions 4, 458-463.
De Wit, P.J.G.M. (1992) . Ann. Rev. Phytopathol. 30,
391-418 .
DeGreve, et al. (1983) J.Mol.Appl.Genet. 1:499-511.
Dickinson, et al. (1993) . Mol. Plant Mic. Int. 6, 341-
- 347.
25 Dietrich,~ R.A., et al. (1994) Cell 77, 565-577.
WO 95131564 2 1 8 8 5 6 2 PCT/GB9S/01075
Doring X-P ~1989) . An overview. Maydica 34: 73-88 .
Enyedi, et al. (1992) Cell 70, 879-886.
Fedoroff, N.V. (1989) In Mobile DNA. M. Xowe and D.
Berg, eds. (Washington: ASM Press), pp. 375-411.
Fillatti JJ, et al. (1987). Bio/technol. 5:726-730.
Flor, X.H. (1971) Ann. Rev. Phytopathol. 9, 275-296.
Gabriel, et al. (1990) Ann.Rev.Phytopathol. 28:365-391.
Gaffney, et al. (1993) Science 261, 754-756.
Goulden, et al. (1993) The Plant Cell 5, 921-930.
Greenberg, J.T., et al. (1994) Cell 77, 551-563.
Greenberg, et al . (1993 ) Plant Journal 4, 327-341.
Hamilton, et al. (1990) Nature 346, 284-287.
Hammond-Kosack, K.E., et al. (1994) Proc. Natl. Acad.
Sci. USA 91, 10445-10449.
Xammond-Kosack, et al. (1994) The Plant Cell 6, 361-
374 .
Xammond-Kosack et al. (1995) Mol. Plant-Microbe
Interact . 8 :181-185
Xe, et al. (1993) Cell 73, 1255-1266.
Hennig, et al. (1993) Plant Journal 4, 481-493.
Herrera - Estrella, et al. (1983) EM;30 J. 2, 987-995.
Xorsch RB, et al . (1985) . Science (Wash. )
227: 1229-1231 .
Jefferson, et al. (1987) EM~30.J. 6 :3901-3907.
Johal, et al. (1992) Science (Wash.). 258:985-987.
Johal, et al. (1994) Maydica 39, (in press).
Jones, et al. (1992) Transgen.Res. 1:285-297.
~ WO95131564 21 88562 .~1~ .s~07~
69
Jones, D.A., et al. (1994) Science 266, 789.
Jones, et al. (1993) Mol. Plant Mic. Int. 6, 348-357.
Jones, et al. (1994) Curr. Biol. 4, 67-69.
Joosten, et al. (1994). Nature 367, 384-386.
Kavanagh, et al. (1992) Virology 189, 609-617.
Keen, N.T. (1992) Ann. Rev. Gen. 24, 447-463.
Keller, et al. (1993) Molec. Biol. 21, 159-170.
Kohm, et al. (1993) The Plant Cell 5, 913-920.
I,loyd, et al. (1994) Mol Gen Genet 242, 653-657.
I.ong, et al. (1993) Cell 73, 921-935.
Malamy, et al . (1990) Science (Wash. ) . 250, 1002-1004 .
Mariani, et al. (1990) Nature 347, 737-741.
M~ e, et al. (1993) MPMI 6, 412-417.
Martin, et al. (1993) Science 262, 1432-1436.
Metraux, et al . (1990) Science (Wash. ) . 250, 1004-1006 .
Mindrinos, M., et al. (1994) Cell 78, 1089-1099.
Mittler, R., et al. (1995) The Plant Cell 7, 29-42.
Napoli, et al. (1990). The Plant Cell 2,279-289.
Odell, et al. ~1984) Nature 313:810-812.
Odell, et al. (1990) Mol. Gen. Genet. 223, 369-378.
Padgett, H.S., et al. (1993) The Plant Cell 5, 577-586.
Pryor, et al. (1983) Advanceæ in Plant Pathology 10,
281-305 .
Pryor, T. (1987). Trends. Genet. 3,157-161.
Rommens C.M.T., et al. (1992) Pl.Molec.Biol. 20:61-70.
Rommens, C.M.T., et al. ~1995) The Plant Cell 7, 249-
257 .
WO 95l31564 21 ~ 2 ~ ~ i /a
Ross, A.F. (1961) Virology 14, 340-358.
Ryals, et al. (1992) SEB Symposium 49, 205-229.
Ryan, C.A. (1990) Ann. Rev. Phytopathol. 28, 425-449.
Santa Cruz, et al. (1993~ Molecular Plant-microbe
Interactions 6, 707-714.
Scofield, S., et al. (1992) The Plant Cell 4:573-582.
Scott, A., et al. (1994) The Plant Cell 6, 1845-1857.
S t ein , J . C ., et al . ( 1 9 9 1 ) Pro c . Nat l . Acad . S c i . USA
88: 8816-8820 .
Swinburne, et al (1992) The Plant Cell 4, 583-592.
T~kAh~h;, et al (1989) Molecular & General Genetics
216, 188-194.
Thomas, C.M., et al. (1993) Molecular and General
Genetics (In Pre8s) .
Thorsness, et al (1983) The Plant Cell 5, 253-261.
Triglia, T., et al. (1988) Nucleic Acids Res. 16:8186.
Uknes, et al. (1992) The Plant Cell 4, 645-656.
Valon, C., et al. (1993) Pl.Molec.Biol. 23:415-421.
van den Ackerveken, et al. (1993) Plant Physiol.
van der Beek, et al. (1992) Theor.App.Genet. 84:106-
112 .
van den Elzen, et al. (1985) Plant. Mol. ~3iol. 5, 149-
154 .
van Kan J.A.L., et al. (1991) MPMI 4 :52-59 .
van den Elzen, et al. (1985) Plant. Mol. Biol. 5, 299-
302 .
Van Den Ackerveken, et al. (1992) Plant Journal 2, 359-
~ ~O 95l~5~4 2 1 8 8 5 6 2 P~r/GB9S/01075
366 .
Walbot, V., et al. (1983) T. Kosuge et al, eds. (Plenum
Press), pp. 431-430.
Walker, J.C., (1993) Plant Journal 3 :451-456.
Ward, et al (1991) The Plant Cell 3, 1085-1094.
Nei, et al. (1992) Science (Wash. ) . 257, 85-88.
Whitham, S., et al. (1994) Cell 78, 1101-1115.
Wolter, M., et al. (1983) Molecular & &eneral &enetics
23 9, 122 - 128 .
WO 95131564 2 1 8 8 5 6 2 PCrlGB95/0107~
~2
BEQ ID NO. 1:
CATAGTCTTT r,r r T~TTTRr ATTAAACAGG GGCATTATTG r r rr~ 7` r rTr TTAGATGTAT 6 0
GAaAATTTTG G~rr7~ Tr~ TTGACAACAC GAACATTTTT rr'`''rrD7`rT ATTAACTCAG 120
AATATTTTCC GTTGAATGAA Tr~ TrrrT rrTrrTrD7~T TTTTAGACCA AACTATGAAG lB0
AACATGCCAT GTCTGGACTC CTGCACTATC TTCCATCAAC AGGTCAATTC TCTCAACTCT . 240
ATTGGTGGAA r~rTrr~rr~T ACAaATTGAA TTATATTAD~A AGACAAGCTC rrrTrr~ rT 300
CACTGTTATA rADrDDrrrr r7~DrTrrrrT TCAGCCCCAA ArrrTrrTr~A CCCGAATCAT 360
ATATTGTCAC GAGTTTTTTT TAGAGTATGT TGCATATATT ATACTCAACT TAGGGTTTGT 420
CATTCTGATG CTTCGTACAA ATTTATTGAA TTTTcAacTT TAAAGGTTTA TGAACCAAAT 4 a o
ATTACGCTTA rTrTrATrrr ~ L ' ' ' L GATTAATCAA ACTTATTGAA TTTTCAACTT 540
TADAGGTTTT "~L~7L'il ATrrrrDr7~r TA~Dr~.T TTAaATTATA TAGTCTTTGG 600
ATGGTGACCT ATTTGGATGG TAACATTATT C~ ~ rA7~rrT ATTGATAACG rr"~ ATTrT .660
Tr~ rr r Dr TGAGAAGGAC ATGTCTGGAC L~l ~l L~ ~ ~i L_ LLil_ L~ r,rrr~r~TrrDT 720
TCTTGTGGAD AATTAGCTCG rrr~T~r,rrrr CTATGTGAGG TDrrTr~rTrr TAaATTTTTC 780
TTTGCTTAAT LL~L~ L~TA TDTArrTrDT cTA~D~ATTATT GAATAGTCAC DrD-~'"DDD 840
CATTTCTTGA LLL~LL~L`'L ATCAACATAA CAAGTTTTGA l~I L .L~G TGCDGAA 897
ATG GAT TGT GTA AAD. CTT GTA TTC CTT ATG CTA TAT ACC TTT CTC TGT 945
Met Asp Cy5 Val Ly8 Leu Val Phe Leu Met Leu Tyr Thr Phe Leu Cys
-23 -20 -15 -10
CAA CTT GCT TTA TCC TCA TCC TTG CCT CAT TTG TGC CCC GAA GAT CAA 993
Oln Leu Ala Leu Ser Ser Ser Leu Prr~ }~is Leu Cys Pro Glu Asp Gln
_5 1 5
GCT CTT TCT CTT CTA CAA TTC AAG AAC ATG TTT ACC ATT AAT CCT AAT 1041
Ala Leu Ser Leu Leu Gln Phe Lys Asn Met Phe Thr Ile ABn Pro ABn
10 15 20 25
GCT TCT GAT TAT TGT TAC GAC AATA AGA ACA TAC GTA GAC ATT CAG TCA 1089
Ala Ser Asp Tyr Cys Tyr Asp Ile Arg Thr Tyr Val Asp Ile Gln Ser
30 35 40
TAT CCA AGA A~:T CTT TCT TGG AAC ADA AGC ACA AGT TGC TGC TCA TGG 1137
Tyr Pro Arg Thr Leu Ser Trp Asn Lys Ser Thr Ser Cys Cys Ser Trp
45 50 55
GAT GGC GTT CAT TGT GAC GAG ACG ACA GGA CAA GTG aTT GCG CTT GAC 1185
Asp Gly Val E~i3 Cys Asp Glu Thr Thr Gly Gln Val Ile Ala Leu Asp
60 65 70
CTC CGT TGC AGC CAA CTT C~ GGC AaG TTT CAT TCC AAT AGT AGC CTC 1233
Le~ Arg Cys Ser Gln Leu Gln Gly Lys Phe Elis Ser Asn Ser Ser Leu
75 80 85
TTT CAA CTC TCC AAT CTC AAA AGG CTT GAT TTG TCT TTT AAT AAT TTC 1281
Phe Gln Leu Ser Asn Leu LYR Arg Leu Asp Leu Ser Phe Asn Asn Phe
go 95 100 105
ACT GGA TCA CTC ATT TCA CCA ADA TTT GGT GAG TTT TCA AAT TTG ACG 1329
Thr Gly Ser Leu Ile Ser Pro Lys Phe Gly Glu Phe Ser Asn Leu Thr
110 115 120
_ _ _ _ _ _
W0 95/31564 73 1 ~ 107S
CAT CTC GAT TTG TCG CAT TCT AGT TTT ACA GGT CTA ATT CCT TCT GAA 1377
Xis Leu Asp Leu Ser His Ser Ser Phe Thr Gly Leu Ile Pro Ser Glu
125 130 135
ATC TGT CAC CTT TCT A~ CTA CAC GTT CTT CGT ATA TGT GAT CAA TAT
Ile Cys E~is Leu Ser Lys Leu l~is Val Leu Arg Ile Cys Asp Gln Tyr 1425
140 145 150
GGG CTT AGT CTT GTA CCT TAC AAT TTT GAA CTG CTC CTT A~G AAC TTG 47
Gly Leu Ser Leu Val Pro Tyr A5n Phe Glu Leu Leu LeU Lys Asn LeU 1 3
155 160 165
ACC CAA TTA AGA GAG CTC AAC CTT GAA TCT GTA AAC ATC TCT TCC ACT 521
Thr Gln Leu Arg Glu Leu Asn Leu Glu Ser Val Asn Ile Ser Ser Thr
1~0 175 180 185
ATT CCT TCA AAT TTC TCT TCT CAT TTA ACA ACT CTA CAA CTT TCA GGC 1569
Ile Pro Ser Asn Phe Ser Ser E~is Leu Thr Thr Leu Gln Leu Ser Gly
190 195 200
ACA GAG TTA CAT GGG ATA TTG CCC GAA AGA GTT TTT CAC CTT TCC AAC 61
Thr Glu Leu E~is Gly Ile Leu Pro Glu Arg Val Phe EIis Leu Ser Asn
205 210 215
TTA CAA TCC CTT CAT TTA TCA GTC AAT AC AGG
CCC CAG CTC G GTT TTT 1665
220 225 230
CCC ACA ACC A~A TGG AAT AGC AGT GCA TC~ CTC.ATG ACG TTA TAC GTC 1713
Pro Thr Thr Lys Trp Asn Ser Ser Ala Ser Leu Met Thr Leu Tyr Val
235 240 24s
GAT AGT GTG AAT ATT GCT GAT AGG ATA CCT A~A TCA TTT AGC C~T CTA 1761
Asp ser Val Asn Ile Ala Asp Arg Ile Pro Lys Ser Phe Ser Elis Leu
250 255 260 265
ACT TCA CTT CAT GAG TTG TAC ATG GGT CGT TGT AAT CTG TCA GGG CCT 1809
Thr Ser Leu llis Glu Leu Tyr Met Gly Arg Cys Asn Leu Ser Gly Pro
270 275 280
ATT CCT AaA CCT CTA TGG AAT CTC ACC APC ATA GTG T~T TTG CAC CTT 1857
Ile Pro Ly3 Pro Leu Trp A5n Leu Thr Asn Ile Val Phe Leu Elis Leu
285 290 295
GGT GAT AaC CAT CTT GAA GGA CCA ATT TCC CAT TTC ~CG ATA TTT GAA 1905
Gly Asp Asn His Leu Glu Gly Pro Ile Ser Ili5 Phe Thr Ile Phe Glu
300 305 310
AaG CTC AaG AGG TTA TCA CTT GTA AAT AAC AAC TTT GAT GGC GGA CTT 1953
Lys Leu Lys Arg Leu Ser Leu Val Asn Asn Asn Phe Asp Gly Gly Leu
GAG TTC TTA TCC T~T AAC ACC CAA CTT GAA CGG CTA GAT TTA TCA TCC 2001
Glu Phe Leu Ser Phe Asn Thr Gln Leu Glu Arg Leu Asp Leu Ser Ser
330 335 340 345
AAT TCC CTA ACT GGT CCA ATT CCA TCC AAC ATA AGC GGA CTT CAA AAC 2049
Asn Ser Leu Thr Gly Pro Ile Pro ser Asn Ile Ser Gly Leu Gln Asn
3so 355 360
CTA GAA TGT CTC TAC TTG TCA TCA AAC CAC TTG AAT GGG AGT ATA CCT 2097
Leu Glu Cys Leu Tyr Leu Ser Ser Asn E~is Leu Asn Gly Ser Ile Pro
365 370 37s
TCC TGG ATA TTC TCC CTT CCT TCA CTG GTT GAG TTA GAC TTG AGC AAT 2145
Ser Trp Ile Phe Ser Leu Pro Ser Leu Val Glu Leu Asp Leu Ser Asn
380 38s 390
WO9a/31564 21 8~62 l~vl .v/a
AAC ACT TTC AGT GGA AAA ATT CAA GAG TTC AAG TCC Aa~ ACA TTA AGT 2193
Asn Thr Phe Ser Gly Lys Ile Gln Glu Phe Lys Ser Lys Thr Leu Ser
3gS 400 405
GCC GTT ACT CTA AaA CAA AAT AaG CTG AAA GGT CGT ATT CCG AAT TCA 2241
Ala Val Thr Leu Lys Gln Asn Lys Leu Lys Gly Arg Ile Pro Asn Ser
410 415 420 425
CTC CTA AAC CAG AAG AAC CTA CAA TTA CTT CTC CTT TCA CAC AAT AAT 2289
Leu Leu Asn Gln Lys AGn Leu Gln Leu Leu Leu Leu Ser HiD Asn Asn
430 435 440
ATC AGT GGA CAT ATT TCT TCA GCT ATC TGC AAT CTG AAA ACA TTG ATA 2337
Ile Ser Gly His Ile Ser Ser Ala Ile Cys Asn Leu Lys Thr Leu Ile
445 450 455
TTG TTA GAC TTG GGA AGT AAT AAT TTG GAG GGA ACA ATC CCA CAA TGC 2385
Leu Leu Asp Leu Gly Ser Asn Asn Leu Glu Gly Thr Ile Pro Gln Cys
460 465 470
GTG GTT GAG AGG AaC GAA TAC CTT TCG CAT TTG GAT TTG AGC AaA AAC 2433
Val Val Glu Arg Asn Glu Tyr Leu Ser His Leu Asp Leu Ser Lys Asn
475 480 485
AGA CTT AGT GGG ACA ATC AAT ACA ACT TTT AGT GTT GGA AAC ATT TTA 2481
Arg Leu Ser Gly Thr Ile Asn Thr Thr Phe Ser Val Gly Asn Ile Leu
490 495 500 505
AGG GTC ATT AGC TTG CAC GGG AAT AaG CTA ACG GGG AaA GTC CCA CGA 2529
Arg Val Ile Ser Leu His Gly Asn Ly9 Leu Thr Gly Lys Val Pro Arg
~10 515 520
TCT ATG ATC AAT TGC AaG TAT TTG ACA CTA CTT GAT CTA GGT AaC Aa~T 2577
8er Met Ile Asn Cys Lys Tyr Leu Thr Leu Leu Asp Leu Gly Asn Asn
525 530 535
ATG TTG AAT GAC ACA TTT CCA AAC TGG TTG GGA TAC CTA TTT CAA TTG 2625
Met Leu Asn Asp Thr Phe Pro Asn Trp Leu Gly Tyr Leu Phe Gln Leu
540 545 550
AAG ATT TTA AGC TTG AGA TCA AAT AAG TTG CAT GGT CCC ATC AaA TCT 2673
Lys Ile Leu Ser Leu Arg Ser Asn Lys Leu His Gly Pro Ile Lys Ser
TCA GGG AAT ACA AAC TTG TTT ATG GGT CTT CAA ATT CTT GAT CTA TCA 2721
5er Gly Asn Thr Asn Leu Phe Met Gly Leu Gln Ile Leu Asp Leu Ser
570 575 580 585
TCT AAT GGA TTT AGT GGG AaT TTA CCC GAA AGA ATT TTG GGG AAT TTG 2769
Ser Asn Gly Phe Ser Gly Asn Leu Pro Glu Arg Ile Leu Gly Asn Leu
590 595 600
CAA ACC ATG AaG GAA ATT GAT GAG AGT ACA GGA TTC CCA GAG TAT ATT 2817
Gln Thr Met Lys Glu Ile Asp Glu Ser Thr Gly Phe Pro Glu Tyr Ile
605 610 615
TCT GAT CCA TAT GAT ATT TAT TAC AAT TAT TTG ACG ACA ATT TCT ACA 2865
Ser Asp Pro Tyr A3p Ile Tyr Tyr Asn Tyr Leu Thr Thr Ile Ser Thr
620 625 630
AAG GGA CAA GAT TAT GAT TCT GTT CGA ATT TTG GAT TCT AAC ATG ATT 2913
Lys Gly Gln Asp Tyr Asp Ser Val Arg Ile Leu Asp Ser Asn Met Ile
635 640 645
ATC AAT CTC TCA AAG AAC AGA TTT GAA GGT CAT ATT CCA AGC ATT ATT 2961
Ile Asn Leu Ser Lys Asn Arg Phe Glu Gly His Ile Pro Ser Ile Ile
650 655 660 665
~ ~0 95131564 2 1 8 8 5 6 2 P l ~ C . ,07~
GGA GAT CTT GTT GGA CTT CGT ACG TTG AAC TTG TCT CAC AaT GTC TTG 3009
Gly Asp Leu val Gly Leu Arg Thr Leu Asn Leu Ser His Asn Val Leu
670 67s 680
GAA GGT CAT ATA CCG GCA TCA TTT CAA AAT TTA TCA GTA CTC GAA TCT 3057
Glu Gly His Ile Pro Ala Ser Phe Gl n Asn Leu Ser Val Leu Glu Ser
685 690 69s
TTG GAT CTC TCA TCT AAT AaA ATC AGC GGA GAA ATT CCG CAG CAG CTT 3105
Leu Asp Leu Ser Ser Asn Lys Ile Ser Gly Glu Ile Pro Gln Gln Leu
700 705 710
GCA TCC CTC ACA TTC CTT GAA GTC TTA AAT CTC TCT CAC AAT CAT CTT 3153
Ala Ser Leu Thr Phe Leu Glu Val Leu Asn Leu Ser His Asn His Leu
715 720 725
GTT GGA TGC ATC CCC AAA GGA AaA CAA TTT GAT TCG TTC GGG AAC ACT 3201
Val Gly Cys Ile Pro Lys Gly Lys Gln Phe Asp Ser Phe Gly Asn Thr
730 735 740 745
TCG TAC CAA GGG AAT GAT GGG TTA CGC GGA TTT CCA CTC TCA AaA CTT 3249
Ser Tyr Glr. Gly Asn Asp Gly Leu Arg Gly Phe Pro Leu Ser Lys Leu
750 755 760
TGT GGT GGT GAA GAT CAA GTG ACA ACT CCA GCT GAG CTA GAT CAA GAA 3297
Cys Gly Gly Glu Asp Gln Val Thr Thr Pro Ala Glu Leu Asp Gln Glu
765 770 775
GAG GAG GAA GAA GAT TCA CCA ATG ATC AGT TGG CAG GGG GTT CTC GTG 3345
Glu Glu Glu Glu Asp Ser Pro Met Ile Ser Trp Gln Gly Val Leu Val
780 785 790
GGT TAC GGT TGT GGA CTT GTT ATT GGA CTG TCC GTA ATA TAC ATA ATG 3393
Gly Tyr Gly Cy8 Gly Leu Val Ile Gly Leu Ser Val Ile Tyr Ile Met
795 800 805
TGG TCA ACT CAA TAT CCA GCA TGG TTT TCG AGG ATG GAT TTA AAG TTG 3441
Trp S Thr Gln Tyr Pro Ala Trp Phe Ser Arg Met A~p Leu Lya Leu
810 815 820 825
GAA CAC ATA ATT ACT ACG AAA ATG AAA AAG CAC AAG AaA AGA TAT TAGTGAGTAG 3496
Glu His Ile Ile Thr Thr Lys Met Lys Lys His Lys Lys Arg Tyr
830 835 840
CTATACCTCC AGGTATTCCA ~LL~I~Ll ATCTTTCAGA AGATTAmT I1~1~L~ 3556
ATGA~ATTAT CGACCTCCTT CATCCTCAAA GCTCTTA~CT TTCACTCTTC ATmTGAAA 3616
ATTTCAGGAT TcAaAGATTT CCGAGTTCCC AGTTGCTTGG GATGCAGATA AaAGccmT 3676
TATCTTTCAT AGTTTCTTAT CCTATGAATA AaGATTTTAT TTTCATTTGT CTATGGCACG 3736
TAGATATGTT CCGTCACTAA AaAcATTGTA TTTCTCTCAA ~L~LLO~L~ ACATGATATC 3796
r~r~rT TGACTTCAAT TAAGTTACTG TAGTCTGCTA TTTTAATTTT TTcCATTGaA 3856
ACACAACTGA CGTATCTTGA (''~ "T ATGATCCCCC GGGCTGCAG 3905
8EQ ID N0 . 2:
,
Met Asp Cys Val Lys Leu Val Phe Leu Met Leu Tyr Thr Phe Leu Cys
-23 -20 -15 -10
Gln Leu Ala Leu Ser Ser ser Leu Pro His Leu Cys Pro Glu Asp Gln
_5 1 5
_ _ _ _ . .. .. . .. , . . _ .. _ .. _ . . _ . _ _ . . . . , . .. , .. _ _ . , . _
WO95/31564 2 1 8 8 5 6 2 ~ 5~. /a
Ala Leu Ser Leu Leu Gln Phe Lys Asn Met Phe Thr Ile As Asn
10 lS 20 n Pro
la Ser Asp Tyr Cys Tyr Asp Ile Arg Thr Tyr Val Asp Ile Gln Ser
30 35 40
yr Pro Arg Thr Leu Ser Trp Asn Lys Ser Thr Ser Cys C s Ser T
45 50 y rp
Asp Gly Val Elis Cys Asp Glu Thr Thr Gly Gln Val Ile Ala As
60 65 70 Leu p
Leu Arg Cys Ser Gln Leu Gln Gly Lys Phe His Ser Asn Ser Ser Leu
75 S0 85
Phe Gln Leu Ser Asn Leu Lys Arg Leu Asp Leu Scr Phe Asn Asn Phe
90 95 100 105
hr Gly Ser Leu Ile Ser Pro Lys Phe Gly Glu Phe Ser Asn Leu Thr
110 llS 120
li5 Leu Asp Leu Ser His Ser Ser Phe Thr Gly Leu Ile Pro Ser Glu
125 130 135
Ile Cys Elis Leu Ser Lys I,eu Hi8 Val Leu Arg Ile Cys Asp Gln Tyr
140 145 lS0
Gly Leu Ser Leu Val Pro Tyr Asn Phe Glu Leu Leu Leu Lys Asn Leu
155 160 165
Thr Gln Leu Arg Glu Leu Asn Leu Glu Ser Val Asn Ile
170 - 175 180 Ser Ser Thr
le Pro Ser Asn Phe Ser ser Hi5 Leu Thr Thr Leu Gln Leu Ser Gly
19~ 195 200
hr Glu Leu Hi8 Gly Ile Leu Pro Glu Arg Val Phe His Leu As
205 210 Ser n
Leu Gln S~r Leu l~is Leu Ser Val ~sn Pro Gln Leu Thr Val Arg Phe
220 225 230
Pro Thr Thr Lys Trp Asn Ser Ser Ala Ser Leu Met Thr Leu Val
235 240 245 Tyr
Asp Ser Val Asn Ile Ala ASp Arg Ile Pro Lys Ser Phe Ser P~is Lcu
250 255 260 265
hr Ser Leu ~}is Glu Leu Tyr Met Gly Arg Cys Asn Leu Ser Gly Pro
270 275 280
le Pro Lys Pro Leu Trp Asn Leu Thr Asn Ile Val PhOE Leu His Leu
285 290 295
Gly Asp Asn ~Iis Leu Glu Gly Pro Ile Ser ~Tis Phe Thr Ile Phe Glu
300 305 310
Lys Leu Lys Arg Leu Ser Lcu Val Asn Asn Asn Phe Asp Gly Gly Leu
315 320 325
Glu Phe Leu Ser Phe Asn Thr Gln Leu Glu Arg Leu Asp Leu Ser Ser
330 335 340 34s
Asn Ser Leu Thr Gly Pro Ile Pro Ser Asn Ile Ser Gly Leu Gln Asn
350 355 360
WO95131564 2 1 8 8 5 6 2 F~ J075
eu Glu Cys I.eu Tyr Leu ser Ser Asn llis Leu Asn Gly Ser Ile Pro
365 370 37s
Ser Trp Ile Phe Ser Leu Pro Ser Leu Val Glu Leu Asp Leu Ser Asn
380 3b5 390
Asn Thr Phe Ser Gly Ly6 Ile Gln Glu Phe Lys Ser Lys Thr Leu Ser
395 400 405
Ala Val Thr Leu Lys Gln Asn Lys Leu Lys Gly Arg Ile Pro Asn Ser
410 415 420 425
eu Leu Asn Gln Lys Asn Leu Gln Leu Leu Leu Leu Ser Elis Asn Asn
430 435 440
le Ser Gly ~is Ile Ser Ser Ala Ile Cys Asn Leu Lys Thr Leu Ile
445 450 455
Leu Leu Asp Leu Gly Ser Asn Asn Leu Glu Gly Thr Ile Pro Gln Cys
460 465 470
Val Val Glu Arg Asn Glu Tyr Leu Ser ~lis Leu Asp Leu Ser Lys Asn
Arg Leu Ser Gly Thr Ile Asn Thr Thr Phe Ser Val Gly Asn Ile Leu
490 495 500 505
Arg Val Ile Ser Leu Lis Gly Asn Lys Leu Thr Gly Lys Val Pro Arg
er Met Ile Asn Cys Lys Tyr Leu Thr Leu Leu Asp Leu Gly Asn Asn
525 530 535
Met Leu Asn Asp Thr Phe Pro Asn Trp Leu Gly Tyr Leu Phe Gln Leu
540 545 550
Lys le I.eu Ser Leu Arg 5er Asn Lys Leu His Gly Pro Ile Lys Ser
Ser Gly Asn Thr Asn Leu Phe Met Gly Leu Gln- Ile Leu Asp Leu Ser
570 575 580 585
er Asn Gly Phe ser Gly Asn Leu Pro Glu Arg Ile Leu Gly Asn Leu
590 595 600
ln Thr ~et Lys Glu Ile Asp Glu Ser Thr Gly Phe Pro Glu Tyr Ile
605 610 615
Ser Asp Pro Tyr Asp Ile Tyr Tyr Asn Tyr Leu Thr Thr Ile Ser Thr
620 625 630
Lys Gly Gln Asp Tyr Asp Ser Val Arg Ile Leu Asp Ser Asn Met Ile
63s - 640 645
Ile Asn Leu Ser Lys Asn Arg Phe Glu Gly Lis Ile Pro ser Ile Ile
650 655 660 665
ly Asp Leu val Gly Leu Arg Thr Leu Asn Leu Ser 3~is Asn Val Leu
670 675 680
lu Gly Elis Ile Pro Ala Ser Phe Gln Asn Leu Ser Val Leu Glu Ser
685 690 695
eu Asp Leu Ser Ser Asn Lys Ile Ser Gly Glu Ile Pro Gln Gln Leu
700 705 710
WO95/31564 2 1 ~ /. CiO75
Ala ~er Leu Thr Phe Leu Glu Val Leu Asn Leu Ser His Asn iIis Leu
715 72n ~ 725
Val Gly Cys Ile Pro Ly6 Gly Lys qln Phe Asp Ser Phe Gly Asn Thr
730 735 740 745
er Tyr Gln Gly Asn Asp Gly Leu Arg Gly Phe Pro Leu Ser Lys Leu
750 755 760
ys Gly Gly Glu Asp Gln Val Thr Thr Pro Ala Glu Leu Asp Gln Glu
765 770 775
Glu Glu Glu Glu Asp Ser Pro Met Ile Ser Trp Gln Gly Val Leu Val
780 785 790
Gly Tyr Gly Cys Gly Leu Val Ile Gly Leu Ser Val Ile Ile Met
795 800 805 Tyr
Trp Ser Thr Gln Tyr Pro Ala Trp Phe Ser Arg Met Asp Leu LyD Leu
810 815 820 825
Glu Elis Ile Ile Thr Thr Lys Met Lys Lys E~is Lys Lys Arg Tyr
830 835 840
5BQ ID N0. 3:
ALLL~LL~iA lLL`'lL~L~L DTr~ 'pTP7l CAAGTTTTGA L~L~L~LL~G TGCAqAaATG 60
GATTGTGTAA PprTTr~TpTT rrTTPTGrTP TATACCTTTC TCTGTCPaCT l~i~LLLAi~ 120
TCATCCTTGC ~L~ALLL~L~i rrrrrrn--rT CAAGCTCTTT CTCTTCTACA ATTCDAGAAC 180
ATqTTTACCA TTAATCCTAA TGCTTCTqAT TATTGTTACG PrPTDn--r7~r ATPrr-TP~n-- 240
ATTCPqTCPT ATCCAAqAAC L~LLL~ LL ii~ p7~ p CAAGTTGCTG CTCATGGGAT 300
qqCGTTCATT GTGACGAGAC ~7~--Pr~r-Dn GTGATTGCGC TTGACCTcCq TTrrPrrr7 ?. 360
CTTCDAGGCA AGTTTCATTC rr nTDr.TPr~r CTCTTTCPAC TCTCCDATCT CPAAAqGCTT 420
~AILL~iL~LL TTAATAATTT CACTqGATCP CTCATTTCPC CaaDATTTGq TGAGTTTTCA 480
AaTTTGACGC liTCTCqATTT GTCGCATTCT AGTTTTAcAq GTCTAATTCC TTCTqAaaTC .540
TGTCACCTTT rTP7~n~'TPrA ~ -, ATATGTGATC ~nTATr7nr7rT TPr.TrTTr.T~ 600
CCTTACAATT TTqAACTGCT rrTT~n--rn" TTrArrr7'7`T TAArArAnrT C-AAccTTqAA 660
TCTGTAAACA TCTCTTCCAC ~ AaTTTCTCTT rTrDTTTP7`r AaCTCTACAA . 720
CTTTCAGGCA CAqAGTTA13A ~iW~ALL~i rrrrn~ n-- TTTTTCACCT TTccaAcTTA 780
C,AATCCCTTC ATTTATCAGT CAATCCCCAq CTCACGGTTA GGTTTCCCAC A7.~rA-~-.Tnn 840
AATAGCAqTG CATCACTCAT GACGTTATAC GTCqATAGTG TqAATATTGc Tr~TpnnpTA 900
CCTAAATCAT TTAGCCATCT AACTTCACTT CATqAGTTGT AACATGGGTCG TTGTA~TCTG 960
TCAwGCCTA TTCCTAAACC TCT~TGGAAT CTCaCCAACA TAGTGTTTTT GCACCTTGGT 1020
GATAACCATC TTqAAGGACC AATTTCCCAT TTCACGATAT TTGAaAAGCT CaaGAGGTTA 1080
TCACTTGTAA ATAACAACTT TGATGGCGGA CTTGAqTTCT TATCCTTTAA CACCCAACTT 114 0
rrn ~rrrT~n AL1LA~ e~.I. CAATTCCCTA ACTwTCCAA TTCCATCCAA rPT~ rnD 1200
~ WO95/31564 21 88~62 r~
79
CTTCAAAACC TAGAATGTCT CTACTTGTCA TCABACCACT TrAATGGGAG TATACCTTCC 1 '60
~llO; ll- ACTGGTTGAG TTAGACTTGA Gr7`-TPnADr TTTCAGTGGA 1320
ADAATTCBAG AGTTCDAGTC CAAAACATTA AGTGCCGTTA CTCTABBACA AAATAAGCTG 1380
AaAGGTCGTA TTCCGAATTC ACTCCTAaAC rr~ 7~rr TACAATTACT ~ ~ 1440
rDrD~T~TD TCAGTGGACA TATTTCTTCA r~rT~TrTr-rA ATCTGAABAC ATTGATATTG 1500
TTAGACTTGG r71r~TDrlT~ TTTGGAGGGA ACAATCCCAC AATGCGTGGT Tr~ 1560
GAATACCTTT CGCATTTGGA TTTGAGCABA AACAGACTTA GTGGGACAAT rD~ TDrDDrT 1620
TTTAGTGTTG GA~ACATTTT AAGGGTCATT AGCTTGCACG C~ T7~7~r~rT 7~r~ 1680
GTCCCACGAT CTATGATCAA TTGCAAGTAT TTGACACTAC TTGATCTAGG TAACAATATG 1740
TTGAATGACA CATTTCCBBA ~1~ 7~i~7~ TACCTATTTC D7~TT"'`~'"'T TTTAAGCTTG 1800
AGATCAaATA AGTTGCATGG TCCCATCABA TCTTCBGGGA ATACABACTT 7111~ W71 1860
CTTCAAATTC ll~ ATCTAATGGA TTTAGTGGGA ATTTACCCGA AAGAATTTTG 1920
GGGAATTTGC ~7~7~rrDTr7~ GGAAATTGAT r~ TDrDn GATTCCCAGA ~ -~l 1980
a~TrrDTATr. A~..lll~ll~ CBATTATTTG ACGACAATTT r~TDr~Dr""" ACBAGATTAT 2040
GATTCTGTTC GA~DTTTTGGA TTCTAACATG ATTATCAATC TCTCAAAGAA cAr~ATTTGAA 2100
GGTCATATTC rAAGCATTAT TGGAGATCTT GTTGGACTTC GTACGTTGBA CTTGTCTCAC 2160
AATGTCTTGG AAGGTCATAT ACCGGCATCA TTTCAaAATT T~TrDrTDrT CGAATCTTTG 2220
.l.l~:~T rT~7~T~ T rDrr~ ATTCCGCAGC AGCTTGCATC CCTCACATTC 2280
CTTGAAGTCT TABATCTCTC TCACBATCAT CTTGTTGGAT GCATCCCCBA 7~ 7 r7 r 2340
TTTGATTCGT TCGGGAACAC TTCGTACCAA GGGAATGATG GGTTACGCGG ATTTCCACTC 2400
TCAABACTTT ~lu~ d7l~A AGATCAAGTG ACAACTCCAG rTrDrrTDrD TCAAGAAGAG 2460
r~ 7r. ATTCACCAAT GATCAGTTGG CAGGGGGTTC 1~ .l~ 2520
7 GACTGTCCGT 7`7'TDTDrDTA ATGTGGTCAA rTr~ TDTrr AGCATGGTTT 2580
TCGAGGATGG ATTTAAAGTT rr~ DrDTA ATTACTACGA D"7`T"''7.~ rrDrD~ 2640
AGATATTAGT r'"TDnrTDT ACCTCCAGGA TTCAAAGATT TCCGAGTTCC CAGTTGCTTG Z700
GGATGCAGAT ABAAGCCTTT 1~ lll~ TAGTTTCTTA TCCTATGAAT AAAGATTTTA 2760
TTTTCATTTG TCTATGGCAC rTD--`TDTr~T TCCGTCACTA AAAACATTGT A111.1~ A 2820
ACTCTTTCGT CACATGATAT rD~ DrDr TTGACTTCAA TTAaGTTAAA D7~ D7~7~ 2880
8EQ ID ~o . 4:
ATG GGA TTT GTT CTC TTT TCA CAA TTG CCT TCA TTT CTT CTT GTC TCT 48
Met Gly Phe Val Leu Phe Ser Gln Leu Pro Ser Phe Leu Leu Val Ser
5 10 15
ACA CTT CTC TTA TTC CTA GTA ATA TCC CAC TCT TGC CGT GCC TAC TGT 36
Thr Leu Leu Leu Phe Leu Val Ile Ser Hia Ser cy8 Arg Ala Tyr Cys
20 25 30
_ .. . _ .. .. _, _ .. _ .. _ .. .. _ . . . _ _ _, _ . . _ _ _ , _ .. _ _ ,, . _ .. _ . ~
WO 95/31564 2 ~ 8 3 5 6 2 ~ 1075
AAC AGT TCT TGT ACA AGA GCT TTT GAC TGT ~CTT GGA CAA TGT GGA AGA 144
A~n Ser Ser Cys Thr Arg Ala Phe Asp Cys Leu Gly Gln Cys Gly Arg
35 40 45
TGC GAC TTT CAT ADG CTT CAA TGT GTA CAT TGA 177
Cys Asp Phe His Lys Leu Gln Cys Val ~is
SEQ ID N0 5
CTCGAGTTCG r~rrTDD~7~ rrTDT7\~DDT ATTAZ~TA~AA ATTTTAAaAT
51 GGTATATCAA l l l . l~--.A'L 1 D D rrD 7~ rr. TCAMATCGC TGMACAACA
101 GCGATTTCCT TrDrrrr`'`n AAGCAAaATc GCTACTACTG CAGCGATTTT
151 GCAMATGTA ACTTTTTTT~ ~MAMATGC Ai ~ L~L l ATD~r~rTDTD
2 01 TATTTGAATT TrD D 7~ 7. D D ~ D TATTTGAAAA TCAATAMAT l l ~i l l l l l ~--
251 TACGATTTTC TTTTTAaAAT L~llLL~i GAMATCCCT DrrTDr~r-rDr~
301 w~IlL.~T TTTTAATTTT TTTTADATAA l~rrrrDr~rr~D TTTTCGAMA
351 ~DATTTT AAAAAMATT GADAAAGTCG CTGCCTAGGT AGCGATTTGA
401 ATTTTTTTAA AaAATGTTAT ATTTTGCAZLA ATCGTTGCAG Tarr~DrrDT
4 51 l L l~i~ 1111 .L TTGGAGGA~A l ~i~ l v L 1~1 TCCAGCGATT l L ~ , L L l l
501 rr-TTDDTATD ADATTTTATA TAaCGTTTTG AaATTTTTGT TAATATTTTA
551 TAACTTTTAG GCTCCGGACT CAAGATTACT ~l~ i~L TDrTTTDTD~
601 TGCATAGTCT GAATTTTGAA r`rrrD lDTD GTTTAATTTT rr~rrDTD~T
651 TCAGACATGA AATCTTTADA AaAGTTTAAA TAAaATTTGT ATATGTTGAA
701 ~rTDr~r'`DD A~DGTATTATA ATTCACGATA ATTTATTCAC MGCCATCGT
751 CGGAGTGATC GCGAGTGAAG TGMAGAATT GGAGTTTTTG ATATCCAGAA
801 l~i:~l~ll~i~ GAGGTTGAGA TATCTTAATC TATCTCCA~DT P~D```D``r
851 TATTAATATC CAATTTTCTT GAAGGCCATT ACCTATTCCG ACAAATTCCA
901 CAAGATACTT CATCATATAA AaADATAATC TCCGTGMGA AATTCTTTTA
951 TT~GGAMAT CGATTTTAGA GTCATTGCAA TTTAATTTTA TCA~aATATT
1001 TGAGCATGAA AaATTTGMA TGGAGGTGTC ATDDD~'~TDD AATACCCTTT
1051 ~ DrDrr:r TTTATTGAGT TGACGATAGT TCADGTAGGG ~DDTD7~DTD
1101 ACTTATTAAT Tr``TDT7-7\D ACTTGCAAGA A~MAGTGAT ATTCMATTT
1151 AATTCTGACC ATTATCTCTT GATATTCTTT ~ l TATTTGAATA
1201 TTCATTTTTC A~D~AGTTcr-A CGTcATAaGA CATCADATAT CMGTAGGTC
1251 rrDTD7 ~\D7\T ~DDDTArrrT TCTCAACATG Dr~nDrDD`-- ATTGAaMAT
1301 GACTADCATT TTCTCADAGA rDDDDDrDD7~ ACATGTGAGA GMGACATTA
1351 wAATCATCA TAATCTCTGA GACTGAGMT TGTTAGATAT GGTCCACTAC
WO 9S/3 1~i64 8 P ~, I /~ S _ A075
1401 TrTr~Tr~ AGADTTTTGA rrr~r~TrTA TTATACACTA AGAGTGGTCA
1451 TGATCATTGT GTGATAACAA AACTATTTTG GCDATTGA CTCDGTCCTT
1501 GGCTAAATTA GACCTCTAAC PrrrrnrD7~T CCAD~ArTTG ACTTGAGAAT
1551 GACAA~ATTT I~LL~:1UA TArrrrrrr7` ATTAGCAAAT TTGGAAAAAA
1601 L~ L~i~ll GTTGATCTTT AATTAGTATA Ar~TTACGTAC AATATCCTAT
1651 TGAATTGGAA rrr~TD71rrT rDrrrTDTr~ TGATaGTTTC Trr`'`r`"TD
1701 GTCTCTTCAC TTCAGTTTTT CACTCTTTTC TACCTCTTTA CAGTTGCATT
1751 TGCTTCGACT r~r~ rD7~ ~ LU--~L~LL r~TnrDDr GCAACTTTCA
1801 ~ rrr~ TAATTCCTTT TTGGCTTCAT GGATTCCAAG TTCTAATGCA
1851 TGCAAGGACT GGTATGGAGT ~LU~ LL1 rnTr7rTrr~r, TAAACACGTT
1901 GA~TATTACA AATGCTAGTG TCATTGGTAC ACTCTATGCT LLL.~LLL1
1951 CATCCCTCCC TTCTCTTGA~ AATCTTGATC TTDrrDr''`~ CAATATCTAT
2001 GGTACCATTC CACCTGAGAT TGGTAATCTC ACAAATCTTG L~L~I~LLUA
2051 CTTGAACAAC AATCAGATTT C~ \rr~IT DrrrrrDrr~ ATCGGTTTAC
2101 TArrr7`''rrT TCAGATCATC ~LLL~ ACAATCAATT AAATGr~ATTT
2151 ATTCCTAAAG AAATAGGTTA CCTAaGGTCT rTTDrT~ T~1~LLLU~
2201 TATCAACTTT CTTAGTGGTT CCATTCCTGC TTCAGTGGGG AATCTGAaCA
2251 ACTTGTCT~T LLLUL~I~L TACAATAATC AGCTTTCTGG .L~L~LLLL
2301 r``'""~7.TPD GTTACCTAAG ATCTCTTACT GAGCTAGATT TGAGTGATAA
2351 TGCTCTTAAT ~ LL~L~IL~ L LU~LL~LL rr~r~rTDTr, AACAACTTGT
24 01 ~L L L L L LUL L L~ L L L~L~;A AATCAGCTTT ~LU~7~ Li1~T TCCTGAA~AA
2451 ATATGTTACC TAAGATCTCT TACTTACCTA GATTTGAGTG AGAATGCTCT
2501 TAATGGCTCT AiL~LU~LL CATTGGGGAA TTTGAACAAC LLUL~LLLLL
2551 1ULLL~LLL~ Tr rTrPr ~L LL~ LU;~, ~L~LLi~LU~ r~`7~TDrrT
2601 TPrrTrr~PT CTCTTAATGT CCTAGGTTTG AGTGAGAATG CTCTTAATGG
2651 ~L~L~LL~ 1 GCTTCATTGG GGAATCTGAA AAACTTGTCT AGGTTGAATC
2 7 01 TTGTTAATAA TCAGCTTTCT ~. L .L~I L . CTGCTTCATT GGGGAATCTG
2751 AACAACTTGT ~1~1UL1U1~ TCTTTACAAT AACCAGCTTT 1U~ L~T
2801 1~1U~1L A TTGGGGAATC TGAACAACTT ~ 1.1~1U11U TATCTTTACA
2851 ATAATCAGCT 11~1U~1~L A1L~LU~1L CATTG~3GGAA TCTGAACAAC
2901 TTGTCTAGGT TGTATCTCTA CAATAATCAG ~LL1~1U~1 ~L~L1~1UA
2951 Ar`7`rTpr~GT TACTTGAGTT CTCTTACTTA TCTAGATTTG ArTrrTPrrT
3 0 01 CCATTAATGG A1 L LAL L .. 1 ~i. 11~1 L L U GCAATATGAG CAACTTGGCT
3051 1L111U111~ TTTATGAAAA TCAGCTTGCT AGCTCTGTTC rT~rr~r1~T
WO9S/31564 2 1 8~562 F~,l,. _. 1075
82
3101 AGGTTACCTA AGGTCTCTTA A''~l~L1L~ TTTGAGTGAG ~Tr,rTrTT~
3151 l~ ~ TTCGGGAATT TGAACAaCTT GTCTAGGTTG
3201 AATCTTGTTA ATAaTcAGcT I l~,b~l~ L ATTCCTGAAG 7~-~7`T~rr~TTI~
3251 CCTAAGGTCT CTTAATGTCC TTGATTTGAG TGAGAATGCT CTTAATGGCT
3301 ~ i~ TTCATTCGGG AATTTGA/~CA ACTTGTCTAG GTTGAATCTT
3351 GTTA~AATC ~r~rTTTrTan ~ ~1~ r~7~r7~ T~r~ GTTACCTAAG
3401 ATCTCTTAAT GACCTAGGTT TGAGTGAGAA TGCTCTTAAT ~i~.~l.l~l~
3451 CTGCTTCATT r~--r~ TrTr. ~7~r7~rTTrT ~ ~ Ll~l~ TCTTTACAAT
3501 AATCAGCTTT ~l~,w_l~fLI TCCTGAaGAA ATAGGTTACT TGAGTTCTCT
3551 TACTTATCTA ~ ATAACTCTCT TAATGGACTT }.I ~
3601 CATTTGGCAA TATGAGAAAT CTGCAAGCTC TGATTCTCAA Tr~T7~-~r~-~T
3651 CTCATTGGGG AaATTCCTTC Al.l~ i~ AATTTGACAT rz-rT--~-~rT
3701 ~ , rrr~ r~ ATTTGAaGGG AZU~GTTCCG CAATGTTTGG
3751 GTAATATCAG T7~7~rrTTrz~r- ~Lll~l~W~ TrTr~TrT~7` TAGTTTCAGT
3801 rA~r~rrTrr ~l~ ~I TTCCAATTTA ACATCACTAC AAATACTTGA
3851 TTTTGGCAGA AACAATCTGG Drrrrrr7-7T Z~rr1~r7`'`TGT TTTGGCA7~TA
3901 TTAGTAGCCT CGAGGTTTTT GATATGCAGA ~rr~\r~ rT TTCTGGGACT
3951 rTTrr~7 r7~7~ ATTTTAGCAT TGGATGTTCA CTGATAAGTC TCAACTTGCA
4001 TGGCAATGAA rT~r~ Tr AAATCCCTCG GTCTTTGGAC A1 TTGCAAAA
4051 AGCTGCAAGT I.1.L~1 ~- nr-''r''r~TC ~7`rTr~`rr-~ CACATTTCCC
4101 ~ GAACTTTGCC ~r'`--rTr7'r~ GTTTTAaGGT Tr7~r2~Trr7~7~
4151 Tr~ TT~r~r1~T rr''rrTATal~ r~Tr1~Tr~7~r GGCrGAAATC ~I~511L' ~1~7
4201 ATCTTCGAaT CATAGATCTC TCTCGCAATG CATTCTCGCA AGACTTACCA
4251 ACGAGTCTAT TTGAaCATTT r~ r~ Tr~ AGGAcAGTTG DT~ r2~7,T
4301 GC-~C--'`"rrA l~rTT~Tr''l\7\ arTI~TTDra~ TGACTCGGTG r-T~r-TTaTa~
4351 C~AAGGGATT GGAGCTTGAA ATTGTGAGAA '.L.l~=.l~.L~ GTACACAGTT
4401 ATCGATCm r7\7~rrr71r~7~ ATTTr''~'----'` ~.A'l~,Il.~ll ~l-~l~:~l~i
4451 AGATCTCATT GCGATCCGTA TACTTAATGT ATCTCATAAT GCATTGCAAG
4501 r,rT~T21Tl~rC ATCATCACTT GGAAGTTTAT CTATACTGGA ATCACTArAc
4551 ~l~ \rr~ rTTTc zlr_7~ _7.T~ rr~r~
4601 TACGTTTCTT GAATTCTTAA 1~ ~ rr-TT~TrTr r~ Trr~
4651 TCCCTCAAGG ACCTCAATTC CGTACCTTTG AGAGCAATTC ATATGAAGGT
4701 AATGATGGAT TDrr,Tral~TA TCCAGTTTCA AaAGGTTGTG GCAAAGATCC
4751 TGTGTCAGAG ~ 7.7lrT~T;~ CAGTGTCTGC GrT~r7~ T r~
~ ~O 9S131564 2 1 8 8 5 6 2 ~ ~ ' ~ ~ 1 A J j
83
4801 ATTCTGAATT TTTCAATGAT TTTTGGADAG CAGCTCTGAT (il .i., 113.L~
4851 AGTGGACTGT GTATTGGCAT ATCCATGATA TATATCTTGA TCTCGACTGG
4901 ADATCTAAGA TGGCTTGCAA GAATCATTGA D~ rTC~~D CACAAAATTA
4951 TCATGCAAAG r'-~ - CAGCGAGGTC ADAGA~DATTA rD-~ T~~
S001 AATAATCACT TCTAGACAAG TTACCAATAC AGA~DAGATTT GATTTCAGAA
SOSl CTTCAGGTAT TCACGCTAaG CTCTAACACT TA;~ AGTTTATTCT
5101 A~rDD~-T~7~T ATATGGTTTT TTTTTATCAA r7\D~TDrTTD TTAAGGCTTG
5151 ATACAAATTG CTATAATCAC TTGGAAGCTG TrDT~TDT~D r~ ---rT7~D
5201 A~DATTTATAG TTGTGTGACT CACTTTCTTA TTTTTCAGAT TTTCAGGAGC
5251 CAaGAATTAG AAGACGCTGG TGTADAGGAT 1 l ~ L1~ ~ L~
5301 GCTTATGATT GTTGGATTTG A~ l TTATAAGGTT TTCTTCAGTT
5351 rrrrDD~TrT AATATTTTGA ATTTTGATGA TDTDT7`DT7~D Al~
5 4 01 TTGAATGATG l ~ ~I . L ~ L ~:bW~l ~ ATAATACTCA CCTCAaaGAA
5451 TCTAD~GAG TTAGCGCACG DT7~ r~TD r~D~'DTDrP~I D--~D~ ~DTDr
5501 ATTACAACCT l~ ll~l TATCTTACAC CCCAAAGCTT 41L~' l~
5551 Dr~--`D7`--- CAAGTTTTAT TTTTAGATAT GGGGAGCCTT lil l'ljlli~'l`;'i
5 6 01 TAAGGTTGTA GTGGATAD GG TAACTTCTCC TGTTAATGAA TTGAATGATC
5651 DTDrrD--''-r TGTGTTTADA ~lL'~:L~ 7 TATTAGTTTG TAATATTTGr,
5701 AGGTCTTAAA TTGAACAGAT GCACATCTGT TCGTGAD~GA r,rDTr~ -TDT
5751 TCTTATAAGT CAACTCTCAA GTTCTATAD~A TDTD~---r--T rrTDDD''TDr
5801 rATD~ DDD AACTGCAGTA TDrT~D''----a TTGTTGGATC rTrr~ D
5851 TTGCTGGTAA Cr~rrrT~D~r AaCATAcGTT AL~LL~j~L~; rrrrT~`--7'`--
5901 r.TDrrrDrTr ADATAATCTA GGTTTGCATA ~ 7 r7~ \rD7~rDD
5951 TTATTADACA AAATCCACAC Pr7-rTDrirDr DTr-~-r-TJ~D ADAATTTAAT
6001 GACGAGATGA AAGAAACTCA rrrr~7-DTG GACTTTATCA P71r~7`"7`7`DT
6051 ACATTGTTTG TPrrTTTTr~ ACAACCATTT ATCACTCAAA GAAGATCAAG
6101 ~ ~ ~ TTACATCGTT CTTGGAACAA AATTATGTAC ATA~AACTTA
6151 CAGGAATCAT ~.5 ' 111~7 ' ~71~7 Trr,T7~-~ D7\~ T rrDTr 7~ TAGTCCADriA
6201 TACTGAGATC AAGGATTTCT AAGTGCAGCC AATCTCTTCT CCAGTTCATC
6251 GATCCCCGAA CTGCCAGCAC r7`D7`----Dr7~D r~D~`pDD~Tr TDrpTrr----r,
6301 AGTTACTGAG ATCAAAGAGC ATr~7`DDDD'' GCACTTCATA CTAATATGAT
6351 AACTTCATAC TAATATGATA CAATTATTTA r~\r~rD-~D-~ r-.`D~'.`DTDr
6401 r7~Drrr~7~r rr,rD~rPTPr TTTATCTATT PD''r~""PrT GCACTCAaGA
64S1 TAACTAGTAT lll~ A G
WO95/3l564 ~ 1 88~6~ PCTIGP95/0l075
84
SEQ ID N0. 6:
ohKVV~i SLQFFTLFYL FTVAFASTEE PTPT.T.TrwT~PT FKNQNw'SFLA
51 SWIPSSNACK IJwY~VV~rw~i RVwTLNITNA SVIGTLYAFP FSSLPSLEwL
101 nT.' ~,1 IPPEIGNLTN LVYLDLN~WNQ l~-ill~Ul~i T.T.~T~T.QTTT~T
151 F_NQLNGFIP T~T Tr.VT.PqT.T KLSLGINFLS GSIPASVGNL w'NLSFLYLYw'
201 NQLSGSIPEE TCYT.PqT.TT.'T. nT.qn~ T~T.~ TPPqT.I LSFLFLYGNQ
251 LSGSIPEELC YLRSLTYLDL qF~.TT.T,~..Tp PqT "~TT. .C FLFLYGNQLS
301 ~.~lVh~ l. RSLNELGLSE rTT~T,~rTp::~q Tr.T~T.~T.qTiT. NLVwNQLSGS
351 TP~CT.aNT LqMT.YT, _ LSGSIPASLG NLNNLSMLYL YNrTQT.qnqTP
401 2~qTr.NT. .q T~T.YT, _ .q ~ibl~l~ Yh SSLTYLDLSN NSINGFIPAS
451 FGNMSNLAFL FLYENQLASS VPEEIGYLRS LNELDLSENA LNGSIPASFG
501 NT. .qT T.rTT. ~ _ .qr.CTP Rr'Tr.YT.PqT.rT VLDLSENALN GSIPASFGNL
551 Nw'LSRLNLT~N NQLSGSIPEE TaYT.PqT.`--lT. r.T.q~rT~T.~ rPI~qTr.NT.
601 T.qr~T.YT. _ LSGSIPEEIG YLSSLTYLSL .~-.rP PqT~ Q
651 PT,TT, .T ~ V Wh TSLEVLYMPR Nw'LKGKVPQC LGNISNLQTtL
701 ' .~ i LPSSISNLTS LQILDFGRrwN LEGAIPQCFG NISSLEVFDM
751 1 .qr7TT.P TNFSIGCSLI qT.T~T.~-''.r! DEIPRSLDNC KRLQVLDLGD
801 NQL`wDTFPMW LGTLPELRVL T~T. .TTaP TT~qCPZ-T~rMF PnT.rTTnT.Cr~
851 NAFSQDLPTS LFEELKGMRT V~ ..,Y ~YYL~ IVVV VTKGLELEIV
901 RILSLYTVID L! rr.~rnl PSVhGDLIAI RILNVSENAL QGYIPSSLGS
951 T..qTT.T.'.CT.nT.C FNQLSGEIPQ QLASLTFLEF T.NT.CT~YT.Qr. I.:l~y~5~yrh~
1001 1~ GLRGYPVSKG ~iKu~v~irw YTVSALEDQE ~l~rrl~w
1051 K~ahMGYGSG T.rTnTCMTYT LISTGNhRWL PT~TTT~'T~T.E~TR ~ v
1101 ~ _ EF~
8EQ ID N0. 7:
KKVV~i SLQFFTLFYL FTVAFASTEE PTI~T T-T~WlrPT FRNQNNSFLA
51 SWIPSSNACK ~WI~iVV~l'W~ RVWTLNITNA SVIGTLYAFP FSSLPSLENL
101 nT. ~l IPPEIGNLTN LVY'LDLN.NNQ l~ll~yl~i T.T.7-~TQTTT~T
151 FENQLNGFIP T~T~TrYT.T qT.T KLSLGINFLS GSIPASVGNL NwLSFLYLYN
201 NQLSGSIPEE ISYLRSLTEL nT qn~TrT.~q IPASLGNMNN LSFLFLYGNQ
251 LSGSIPEEIC YLRSLTYLDL SEN~LNGSIP PqTnNT. .C FLFLYGNQLS
~ WO95/31564 21 88562 I~l. 5.'01075
301 ~ lr~:r;l-i~h RSLNVLGLSE ~T~T''r'lTV~q T.r.NT.T~NT.CTT. NLVNNQLSGS
351 Tpl~cT~r~NT LSMLYLYNNQ T..CrCTP~qT.r. NhNNLSMLYL YNNQLSGSIP
401 ~CTrNT c ~TYTYNNQTC ~ Lrrr;1~.5Yh SSLTYLDLSN NSINGFIPAS
451 FGNMSNLAFL FLYENQLASS VPEEIGYLKS LNv~DLSENA LNGSIPASFG
501 NT. .CTT.~T. v _ cacTP RRTr.~T.VcT.N VLDLSENALN GSIPASFGNL
551 NNLSRLNLVN NQT.Cr.CIPRR TrYT.T~CT.~"lT. r~T~cRNi~T~r-c Tp~cT~r~T~
601 T.CMT.YT. _ LSGSIPEEIG YLSSLTYLSL ! ,~TrT,Tp ~ 'Q
651 Z~T,TT, .T ~Ll~ V~ h TSLEVLYMPR NNLRGRvPQC LGNISNLQVL
701 '. 2~ri LPSSISNLTS LQILDFGRNN LEGAIPQCFG NISSLEVFDM
751 _ ~Cr:TT~p TNFSIGCSLI cT.NT.~Tr~TT.~T.R DEIPRSLDNC RRLQVLDLGD
801 NQLNDTFPMW LGTLPELRVL T~T.T~'7,~Tr.p TRCCT~T TMF pnT~T~TTnT~qT~
851 NAFSQDLPTS LFEHLKGMRT VL~ Y ~ YLILJ:.VVV VTKGLELEIV
901 RILSLYTvID T' r~ll PSVLGDLIAI RILNVSHNAL QGYIPSSLGS
951 TCTTWCTnTC FNQLSGEIPQ QLASLTFLEF LNLSI~YLQG l~Q~ yrKl
1001 rLs~.,Yr.~) GLRGYPVSRG ~ }'V~LSJ'.N YTVSALEDQE ~ rrl~rW
1051 RAA~MGYGSG T.rTaTCTTYT LISTGNLRWL ~vTTIPET.T.~nr llrlyKK~yK
1101 ~ _ _F~
8~eQ ~D ~O. 8:
GGTTTCTAGA AAAGTAGTCT CTTrACTTCA GTTTTTCACT CTTTTCTACC
51 TCTTTACAGT ~ l TCGACTGAGG AGGCAACTGC CCTCTTGAAA
101 TGGAAAGCAA CTTTCAAGAA rr~r~T~DT ~ " T
151 TCCAAGTTCT AATGCATGCA ~rr~rTr,GT~ TGGAGTTGTA TGCTTTAaTG
201 r.TDnr~nT~ CACGTTGAAT ATTACAAATG CTAGTGTCAT TGGTACACTC
251 TATGCTT~TC CATTTTCATC ~ ~l CTTGAA~ATC TTGATCTTAG
301 r~-~r~1~r~T ~1~1J~1~ CCATTCCACC TGAGATTGGT AATCTCACAA
351 ~~ .l.lA TCTTGACTTG AACAACAATC AGATTTCAGG 71Tr~TDrr~
401 CCACAAATCG GTTTACTAGC CAAGCTTCAG A1~ TATTTCACAA
451 TCAP,TTAAAT GGATTTATTC rT~ r~ T AGGTTACCTA AGGTCTCTTA
5~1 CTAAGCTATC ~ il~l-- AACTTTCTTA GTGGTTCCAT l~l~i~llr:~
551 GTGGGGAATC TGAACAACTT ~l~l ~ TATCTTTACA ATAATCAGCT
601 1 l~l~ ATTCCTGAAG AAATAAGTTA CCTAAGATCT CTTACTGAGC
651 TAGATTTGAG TGATAATGCT CTTAATGGCT ~ 8ll~ -- TTCATTGGGG
701 AATATGAPCA ACTTGTCTTT ~ lll~ll TATGGAAATC AGCTTTCTGG
WO95/3l564 7 1 ~ a
86
751 ~L~L~1L~1 r`~r`7'7`T~T r.TT~rrT~-~r ATCTCTTAC T TrrrTrrrTT
801 TGAGTGAGAA TGCTCTTAAT iJI ~ , LL~ L1L rrrr`rTrTr
Bsi AACAACTTGT ~LL~L-L~L 1~LLL~L~ AATCAGCTTT,,, ~ T
901 Tr,rTr~r~r AT~rrTTDrr TAAGATCTCT TAATGTCCTA GGTTTGAr~TG
951 AGAATGCTCT TA~TGGCTCT A', ~l .. rr~TTGr-r-r~rr TCTGAAAAAC
1001 TTGTCTAGGT TGAATCTTGT TAATAATCAG ~,, ~ ~l I T l ~1
1051 L ~ W~7 rrTrTr`~rr~ ACTTGTCTAT GTTGTATCTT TDrrrTrrrr
1101 AGCTTTCTGG ~L~_L~L~1 ~rTTrr~TTriri GGAATCTGAA rrrrTTr.TrT
1151 ATGTTGTATC TTTACaATAA TCAGCTTTCT ~ ~L~l~ILi ~L`~ ~I.
1201 GGGGAATCTG AacAAcTTGT CTAGGTTGTA TCTCTACAAT AaTCAGCTTT
1251 ~ -L~L--T T~rrTr`rr`r ATAGGTTACT TGAGTTCTCT TACTTATCTA
1301 GATTTGAGTA ATAaCTCCAT TAATGGATTT A1L~L~ L C~TTTwCAA
1351 Tr~Tr~--rrrr '1L~j~LLLLL 1 7~LL~ LLL~ TGAAAATCAG CTTGCTAGCT
1401 ~L~1L~--L~ AGAAATAGGT TrrrT~rrT CTCTTAATGT ~L~ L L~j
1451 i~GTGAGAATG CTCTTAATGG ~L~LL ~1 ~ BIL~L; GGAATTTGAA
1501 r7~r rTTr~TrT Anr.TTGAATC TTGTTAATAa TCaGCTTTCT ~ L~
1551 rTr~r~ T AwTTACCTA rr-r~TrTrTTA LT~ I ~ TTTGAGTGAG
1601 AATGCTCTTA l TI ~ _T L~ ~ L~ IL TTCr~rr` rTT TGAACAACTT
1651 GTCTAGGTTG liATCTTGTTA DTrrTrArrT ~L~L~j~ L~ ATTCCTGAAG
1701 rrrTAnrTTA CCTAAGATCT CTTAATGACC TAGGTTTGAG TGAGAATGCT
1751 rTTrrTrnrT ~ L~LL~ AATCTGAACA ACTTGTCTAT
1801 J1L~ ~L~ TACAi~AATC rnrTTTrTrr ~:~L~i~L r'~rr'~ ~TAn
1851 GTTACTTGAG TTCTCTTACT TA~ ~ ~ TGGwTaATAA rTrTrTTrl`T
1901 wACTTATTC ~L~i~L~ TGwcaATATG rr~ TrTrr AAGCTCTGAT
1951 TcTcaATG~T AACa~TCTCA TTGGGGAAAT L~1 l ~i~l GTGTGCAATT
2001 TGACATCACT rr~_TrTTn ~L~ A GAAAcaATTT r7lDrrr~7 7 ~
2051 GTTCCGCAAT r.TTTGr,r,TA" TATrDr.Tr"r CTTCaGwTTT TGTCGATGTC
2101 ATCTAATAGT TTCAGTGGAG AGCTCCCTTC ~I,.~.I,~ AATTTAACAT
2151 r~rT~rrrrT ACTTGATTTT rr.rrr~- rrr ATCTGGAGw Ar.r~TArrA
2201 CaATGTTTTG GcaATATTAG TAGCCTCGAG GTTTTTGATA Tr'rA--~r~r~7~
2251 CAAACTTTCT GwACTCTTC CAACAAATTT TAr.rrTTrnA TGTTCACTGA
2301 TAAGTCTCaA CTTGCATGGC 7 rTr"rrTrn AGGATGAAAT ~l~
2351 TTwACaATT r7rA7,r7~-rT GCAAGTTCTT GATTTAwAG ACAATCAACT
2401 r~rr`~ArA ~ l GGTTwGAAC TTTGccaGAG CTGAGAGTTT
~ WO95131564 ~ 1 8 D 5 62
2451 TAAGGTTGAC ATCGAATAAA TTGCATGGAC CTATAAGATC DTrDDrr~rrT
2501 GADATCATGT L~ L'CI TCGAATCATA ~ GCAATGCATT
2551 rTrrr-.rr~r TTACCAACGA ~'C'~ ~ ACATTTGAAA ,rRr.DTr~r--~
2601 CAGTTGAIAA ~Drr7.Trr~ GAACCAAGTT ATGAAAGCTA TTDrC`T~r`r
2651 ,~.. ~:.,,~R TTGTGACaAA GGGATTGGAG CTTGAAATTG TGAGAATTTT
2701 GTCTTTGTAC ~CAGTTATCG ATCTTTCAAG CAACAhATTT r~rr~--DTD
2751 l~ l rrTRGrrr`T CTCATTGCGA TCCGTATACT TAATGTATCT
2801 rhT!\DTr~rAT TerD1`rrrTA TDTDrrDTrD TCACTTGGAA GmATCTAT
2851 ACTGGAATCA CTAGACCTTT CGTTTAACCA ACTTTCAGGA c~r~TDrrAr
2901 AAr--A-AcTTGc TTCTCTTACG mCTTGAAT TCTTAAATCT rTrr,rDrDDT
2951 TATCTCCAAG GATGCATCCC l'rADrr`rrT ChATTCCGTA CCTTTGAGAG
3001 CAATTCATAT GAAGGTAATG ATGGATTACG TGGATATCCA GTTTC~AAAG
3051 GTTGTGGCAA AGATCCTGTG Tr7~r~r~D~D DrTATDrDRT ~~
3101 GAaGATCAAG AAAGCAATTC TGAATTTTTC AATGATTTTT Cr~D~r-Dnr
3151 l.~ihI~ TATRr~rTr~ c~rTGTr~TAT TGGCATATCC AT7`DTDTDTD
3201 TCTTGATCTC r~rTr.r:~D7~T rT7~r~TrRr TTGCAAGAAT CATTGAaGAA
3251 CTGGAACACA AAATTATCAT r~rDD~r--`r` DDr`~'r--DRr, GAGGTcaAAG
3301 AAATTACAGA Ar~r~D7~TD AiCb~l~.~ GACAAGTTAC rD-~TDrrr~D
3351 AGATTTGATT TCAGAACTTC AGACTTTCaG c~rrr7\Dr7~D TD`r``r`rr
3401 CTGGTGTAAA W/~ 1 TGCAGCTTAT ~ ii,hT
3451 TAGAmTTA GTTTTATAAG l~:I.L.Ll~.L~:A GTTGGGAaAA Tr.TD7`TDTTD
3501 TGAATTTGAT GDTDTDrDDT AAATGTTGTG TTTATTGAAA ~D~DDD~ D
3551 DDDDDDDD7~ D~"DD~`D7'D AAA
gBQ ID N0 . 9:
1 tAtAtAtrtt ~I~La.lLyL~ AttrJ~t~-rA aagtgattaa At~t~t~r
51 y~y~y~y~Ly aaatcaggta y~yLLLLyLy ttgttgtttc
101 ~y~yL~ yy t~rttr-_~Jr ~dY~Lyy~yLL ~ -~tr~ ~ J ~ J-- y~
151 Arnr~ ~y~yc~yLLL ctagacgcaa ttrr~rrJ~r~r gcttcttgaa
201 ~L~,yLLyJti~ y~LyLLLy~L ~iyLL~a~ _y ~:.N' :1 Y tr~Jrr~ rJA
251 ~rJrAtJ~rtrtr aagcacgagt trttrtAtrr agtacatgaa arr,rtt~r~
301 Arr~--tJrt rrttr----~Jr . ~~ - ~ Jl --trrJrArJcr tacagttgtt
351 Jrrtr---Jrr~r t:..rJr~-~--- tttr~-~rt:.~ ttatacaatt ctt-------t
401 aaaagagtaa L~ - tagagagtta AttttrArtt ~ A
451 gagagttaat tt~ttt~r _~-t--ttA tAttttrArt ttagtataca
501 attcttagtg LL~aLLLclyL attttr--tt atattatttg --tt~.--.tr
551 rtrAt-~tr~ ~tAtArttAt trtrr-tr~tc catgtgcatg L~LyLe~LLyy
601 r~ ~ tttrJ~tAttA -~ ~-t, --l r. agtacattct tA,rrJ~t-...
651 tgtcttgtac ~ rt rJ~-~rrrr-A aaatatgtgt gtttcaaaat
701 atctgtgtag ily~ y - - LyL~dyL~LC tgtctaattg rrt,r,.rtt
751 r~-'tAttAt ttctgtcttg tArp~-~rt ~gArtt~tr ataattaagt
801 rJ~r~rrArA r---ttr-~t rtrt~---t atctttgtat gtagtgtaaa
WO 9~1~1564 2 ~ 8 8 5 ~ 2 PCTIG1395101075
88
851 aaagctttcg aggaaagtaa gacgaagttt ctrrtrtrtt trtr.~^ArtA
901 LyL.LLy.Ly atttacttct rti~ ir ttcgtctctt ctctgagttc
951 r~rtrtA~rAt ~rtrrrDTnrr ~L~1L~L~ L T,, 11 1.~ r~rrr~r~Tr.
1001 CGACGTTm CCa~AGCTTCA ~ W~ ~ TGTTCGCaAG ACGTTCC~CA
1051 GCCA~CTTCT C~AGGCTCTC r~r~rr-GrrnDT rDPTrrDTrr ~TTrr~TnaT
1101 CATwAATCG rrPrr~ rr rrrr-PTrrrr CCTGAGCTTA TA'~ ,L~
1151 Trrar`DrrT AGGATCTCAA TrrTrr~TrTT CTCTAAGAAC TA~,L~ L
1"01 rPrrr~TGr~Tr~ CTTAaATGAA TTGGTTGAGA TrrrrrPrTQ CTTTAATGAT
1~51 TTAGGTCAAA TGGTGATTCC AGTTTTCTAC GACGTTGATC rTTrrr~a--T
1~01 Tr~~D~arr-r- r-rrr,Qrr~DT TTGGAAAGGT rTTTraDD~r ACATGCGAGG
1~51 TcaGcAAGGA rrPPrrDrrr- GwGATCAGA rprr-a~~~TG GGTGCAAGCT
1401 cTca - rAGATA Tr~Qrr DnTrT ,Drrrrr~~~r GATCTTCTGA ACGGgtacgt
1451 LyLL.It!tc.LL rr--tAtAtr Ly~LLy~yLL ttcaattgtc tr,~--~t,t
1501 ~ I l L Ll ~. ~l ~y~LL~:yyL tcttctttta ggggtgcttc tt~--ttJr----A
1551 -''tt~'rtt LLyLL.lLLd~ GCCTAATGAA GCGCATATGG TTGAAMGAT
1601 ATCCAATGAT GTTTCGAATA AACTTATCAC TCGGTCMAG TGTTTTGATG
1651 ACTTCGTCGG AATTGAAGCT CATATTGAGG. cAaTAAAATc AGTATTGTGC
1701 TTGGAATCCA rrr~rrTPQ AATGGTCGGG ATTTGGGGAC arTrDrrr.DT
1751 TGGTADGAGT ACCATCGGAA GAGCTCTTTT Cr~ GTCAACTC TCTAGCCAGT
1801 TCCACCATCG L~L1LL~L~ DrTTDTDP`` GrDrrDQTr~r~ TDQTr.r~r~.Tr
1851 TCTGGCATGA AGTTGAGTTG QrrDnD`r`r LL I ~ ~ i ~ ~ AAATCTr GG
1901 TCAAD~AGGAC rTD~Dr`TDQ AGCATTTTGG Tr~TQQTrr~~ CaAAGG~ AA
1951 ATCACAAGAA AGTTCTTATC CTTCTTGATG ATGIGGATAA TCTAGAGTTT
2001 rTTr~r~~rT TGGTGGGA~A DQrT~~nTrr. TTTGGATCTG r~rrrDr, IDT
2051 AATTGTGATC ACTCAAGATA GGCAACTTCT CAAGGCTCAT GAGATTG~CC
2101 TTGTATATGA GGTGAAGCTG rrDTrTrPPr ~Pl LL~ LL~1 TrPr~TQDTD
2151 TCCCAA~TG CTTTTGGGAD AGACTCTCCA CCTGATGATT TTr~ D~T
2Z01 AGCATTTGAA GTTGCCGAGC TTrTrQrTDQ 'L~LL~L1 1 ~} QQTrTrDrTQ
2251 ~~ bLLL~ ATCTTTAaAA rr~Prrr~rD A,DGATGAGTG rQTr~
2301 ATGCCTAGGC TTCGAAATGA TTCaGATGAT A~AATTGAGG DDDrDrTD~~
2351 AGTCGGCTAC GATAGGTTAA DTP`DDPP-D TDr'~`~TTD TTTAAGTGCA
2401 TTGCATCTTT TTTCAA~GGT TTT~AAGTCA GTDDrr.TrPP Dr`PTTACTT
2451 r``r`TI rTQ L'~iWL L~C AATGTTGGCT GAGA~GTCCC TrDTDrrTDT
2501 TprDrrrr~rT Cr`TDT~TDQ DrDTrrDrDD TTTGCTAGAG AAATTGGGTA
2551 GAGAAA'`TGA TCGTGCAAAG Trr~'`r~~TD DTCCTr~GaAA ACGTCAATTT
2601 CTGACGAATT TTGAGG~TAT TCG~GAAGTA TTr`rrr`r` AaAcTgtaag
2651 1 1 1 ~ I ~ . J, ~ I , ~,, ~ I . _ .rl yLL L,l~- Ly~: At~rtttAt Atr''tAt''
2701 tr~t.~~tt~ r~tt,-tA --~ tPO~rA ,.l L~ -J. catgcgtaat
2751 taaaacgtag ~LLLy~LyLy tr~ --t aaaaagggtt y~y~LLyLLtl
2801 P~-ttAtAtt agttttcttc Jy- I Ll ~ tr-~r~rr~rr GAAACTCTTC
2851 TTGGAATACG TTTGCCACDC LLL~ ~I~ TTACGACAAG ~L~iL~l L~L
2901 ATAGATGAaA AATCATTCAA DQrrDTr,~rrT r~TrTrrr7~T DTrTD~~--.T
2951 1~jLLL~'LL~j~j TCAGATGGGG TTCTACCTCA GAGCCTCGTT L~L~L~L~L~
3001 GTAaAcTcAA AaGGcTATGG TGGGATAATT GTCCATTGAA ~il l_. I 1~71 ~ .
3051 TCTAATTTTA DQQrTr`~TD TrTrQT~r`' CTCaGAATGG Tr~'TDrTD-
3101 rrTTr~r~r ~ G~CTCAGGT Art~-ttttt ttagtgatca
3151 Attt,rt~ r Ar~ ~tA ~__t~ tgtttaaaat gttcattaac
3201 gtgtgtgctc trttttrrrr L,~LLLL.~LLL tcagCCCCTT GGAAGTCTCA
3251 AGAAGATGGA TTTGTATAAT TrrTDrD7~7.T Tr`'`r'7~DT TCCDGATCTT
3301 TCTTTAGCCA TAaAccTcGA GGAATTADAT CTTGMAGAAT GCGAATCTTT
3351 GGAGACACTT ~ TTrrr~Trc CATTAAACTG AGGGAGT~rAA
3401 I~LLI~LL~; ~ ~L~1L~ ATAGATTTAA D~Trr~TTArr~ Drr,rATrTrT
3451 AATCTCGAAT ATCTATCAGT TCCTAGTTGG Trr~rTpr-r~r7 AATGCACTCA
3501 W~L/ 1~LL LJLILL~L~LL GTAAACTCAA AAGTGTATTG TGGACTAATT
3551 GTCCATTGaA ~--hLLL~LLL TCTAATTTTA Drr.rTr~rTr, TrTrrTTr.-
3601 CTCATAATGG Dr.TrrDQTQA GCTTGAGAAG CTGTGGGATG GTACTCAGgt
3651 Artl~-ttrtA ttagtgataa taaatatgtt æ~'-'-~rtA ---,tr--_
3701 tgtttaa;~at gttcattaac gtgtgtgctc trttttrrrr tattttgtta
3751 tcagTCACTT GGAAGTCTCA AGGAGATGAA TTTGAGGTAT TccAac~DrATT
3801 T~ 'r'-7~T TCCAGATCTT TrTTTrQrrD TAA~ACCTCGA GGAATTAGAT
3851 LL_LLL~jj~L GCGTATCTTT GGTGACAC--T ~LL~L~h~ TTrD"~DTrr
3901 rDrTD~rTr ATCTATTTAG DTDTrDQTrD rTQrr~D~7~T AGAGAGTT
3951 TTCCAACCGT TTTCrACT~G AAATCTCTCG AGTACCTCGA TCTCAGGA
4001 TGCCCGAATT .TGAGAaa~TT rrrDrrD-,--r r~-TrQQDT rTGrrTrr.~
4051 TAGATTATCT rQDDrD'--'T I~LLL~1L-~ Drrrar~ T GAGATCGTGG
4101 TAGAAGATTG TTTGGAAC AAGAATCTCC CTGCTGGDCT r~QrTTDTrTr
4151 GACTGCCTTA TGAGATGTAT GCCTTGTGAA TTTrQrTrDQ r-rr-rTrDr
... .. , . .. ... .. . . . . .... . .. ...... . . . : . . _ . _ . _ . _ .. .
WO95131564 2 1 ~3~562 r
89
, ~
4201 TTTTCTCAAT GTGAGCGGCT GCAAGCTTGA GAAGCTAT~G GAAGGCATCC
4251 AGgtacattg tt~tr,rtAt r,rt~-ttttt gtttaccttc tgttatataa
4301 ctaattaagt AtArrr7~-t ttgtttttat yy~.LLyLyyL rJr ~trrArX
4351 ttatgtctta r~t=rAtArA taataatgtt tP-ttAt~At tttA -~-r51tA
4401 tataggtata --tt~ t ~r?ttAtrAtr gataatgatt ~_ ~J ~
4451 .I~LyLLLLLL tcagTCGCTT GGAAGTCTCG AAGAGATGGA TCTGTCaGAA
4501 TCTGAAAACC TGAAAGAACT TCCAGATCTT TCAAAGGCCA CCAATCTGAA -
4551 ~ 71 CTCAGCGGGT GCAA~AGTTT GGTGACACTT CCTTCTACAA
4601 TTGGGAATCT TCAAAATTTG AGACGTTTGT ACATGAACAG DTGrDrDrnr.
4651 CTGGAGGTTC TTCCGACCGA TGTCAACTTG TCATCTCTCG AaACCCTCGA
4~01 TCTCAGTGGT TGCTCAAGTT TGAGAACTTT TCCTCTGATT TCAACTAATA
4751 ~ CTATCTGGAA 7`rrDrrr-rrD TTGAAGAAAT TCCAGATCTT
4801 TCA~AGGCCA CCAAGCTCGA GTCTTTGATA CTCAACAACT GCAD~GTTT
4851 GGTGACACTT CCTTCTACAA TTGGGAATCT TCAAAATTTG AGACGTT~GT
4901 DrDTr`7~rDr ATGCACAGGG CTGGAGCTTC TTCCGACCGA TGTCAACTTG
4951 TCATCTCTCG AaACCCTCGA TCTCA~TGGT TGCTCAAGTT TGAGAACTTT
5001 TCCTCTGATT TCAACTAGAA TCGAATGTCT CTATCTAGAA D~rDrrr7rrD
5051 TTGAAGAAGT l~ ATTGAGGATT TcAcrArGcT rDrTrTDrTl~
5101 ~ il~il GTTGCCAGAG GTTGAAD~AC ATCTCCCCDA ArATTTTCAG
5151 ACTGACTAGT CTTACGCTCG CCGACTTTAC AGAC''GTAGA (~
5201 AGGCGTTGAG TGATGCAACT GTGGTAGCGA CAATGGAAGA TCACGTTTCT
5251 TGTGTACCAT TATCTGAAAA CATTGAATAT ACATGTGAAC
5301 l~il.:i71~711~L GATTATTACT CTGATGACTT TGAGGTAAAT rrrr~rrrDr
5351 TTAGATTGTC DDrr`TrDrT GTCAACGATG TGGAGTTTAA 7llll~ill~
5401 TCCATTACGA TCAAAGAATG rr~r-Tr~T7~rrD ~lC,~ , TCTATCDJ~QA
5451 ~rrDrr--rDr 7~DrrDrrDrr rTDrrrr~r-- rDrrrrrrrr DTr,rrr~:TDD
5 5 01 ~ D t ~ ~ t t ~ r ~ t--~ _ ~r~ r~ ~ t r l
5551 atttgtttta t~rJ~tr~-~ at~rr~t~gr r~--~rJ---tAr I ~.J_I ~J `
5601 cgatcgtttg AtArAt~-t~ r~r~tr~- - At r,rrJ~rJ---~tr ~.J ~ .J_~
5651 Atr-~~tt~~, . I -I IJ . - ~dLLyL~y~ rJ~r~r~rJ~-r ~ J .~
5701 ~-tAr~rJ ctttcgttag ~J~ J -~_ ~rJrArrAtrA tr-~~Atrtr
5751 t-r-~rJJrrJrJ- a~,_LLL ~LyL ~LLy.-L~ L ---AtrJ~t~-~ tr-~
5801 rJ~trJ~---r-~ t~rrtAtrtt gtatcCtgtt tatggtaact
5851 ts~trAttrt~ ttttr~ctrtt ~l I I _,JJ~I _ ArttrJJrJrAtr J_, _l I _l
5901 At~--tttAt ~-t,J_I ~ r~t LLc ~L~ yLt ~
5g51 ~rAr--grrA ttttttrt~c ~g~tAt~r-~ gatgatatgt ,r,~r~trAtt
81!:Q ID N0 10:
~'`rq.CCCaVT~ uv~r~r~i VDVRKTFLS. TTRDTnrrCT c~lrlu~
51 SRTIAPELIS AIREARISIV IFSKNYAS5T WCLNEL-vEI~. KCFNDLGQMV
101 l~VI'YJVU~S ~k:v~Kyl~ yi KVrr.~ V~ ~UI~U~LUl~Y RWVQALTDIA
151 NTDrFnT.T~7r ~ Kl SNDVSNKLIT ~rUUL~V~i TT~roTFDT~Tc
201 vT rT T~CTr;~Dv ~ L GKSTIGRALF SQLSSQ~R AFLTYKSTSG
251 SDVSGMKLSW QRFTT..CFTTr. yKUlKl~:~r~ VvEQRLNERK VLILLDDVDN
301 LEFL-KTLVGK AEWFGSGSRI IVITQDRQLL KA~3EIDLVYE VRLPSQGLAL
351 KMISQYAFGR Da~uurK ~ AF_VAELVGS LPLGLSVLGS ,CT.Rr-r)RnFw
401 VRMMPRLRND ~nnRrFFTT.T~ vGyDRLr~KRN RELFRCIACF ~n~ Y ~K
451 ELLEDDVGLT MT-DFRCT~TT~T l~l~Yl~ ~ T.T.FRT~rVFTn RARSKGNPGK
501 RQFLTNFEDI REVLT_KTGT FTTTrTVT.P PGYLTTRSFL LUDi~ }~
551 NLQYLEIGYW SDGVLPQSLV YFPRKLRRLW WDNCPLKRLP SNFKAEYLVE
601 LRMVNSKLEK LWDGTQPLGS LKKMDLYNSY KLREIPDLSL DTNT.FFT.NT.T.~
651 EOESLETLPS CTQr DTT~T T~T.` T.~-rWrrT.T.Tn T RCT FrMr~T EYLSVPSWSS
WO 95131564 2 1 8 8 5 6 2 PCTIGB9510107S
701 ~ ilVY~' PRRLKSVLwT NCPLKRLPSN FRAEYLVELI ~TYcr.~T.RRT.W
751 Dr-TQcT~r~qT~R , . KEIPDLSLAI NLEELDLFGC VSLVTLPSSI
801 ~T~TTYTn MSEOENLESF PTVFNLKSLE YLDLTGCPNL RNFPbI~MGC
851 pWT17T,CT~Tr~T, 1"~i7 ~N~lVV ~IJ~rl .P AGLDYLDCLM RCMPCEFRSE
901 QLTFLNVSGC ~T.TCT T.W~rTQ CT.r~CT. . CT.~CT.~NT.RFT,P nT.CRDTNT.RT.
951 LCLSGC~SLV TLPSTIGNLQ NT.rJT T. ~ TGLEVLPTDV l~rr.qCT,T.'TT,nT,
1001 SGCSSLRTFP LISTNIVCLY T.T.~NTDTr~T TP nT.cRDTRT.r c r.TT: ,
1051 TLPSTIGNLQ NT.T TiT, - TGLELLPTDV NT.C.qT.T'TT.nT. SGCSSLRTFP
1101 LISTRIECLY LENTAIEEVP CCIEDFTRLT VLRMYCCQRL KNl:jJ'Nlr~
1151 TSLTLDDFTD CRGVIKDLSD ATVVATMEDH VSCVPLSENI l~ iK~WL~A
lZ01 ~OIJYI~ VNRNPIRLST rll~ V~;rK~ ~l'l'lK~ li VRLLYvYQET
1251 _~llr~JK KRMRVSLLP
82Q ID No. 11:
GACCADACTG GACTCCTGCT ~l.~ A TCbGCAGGTC 7~TTrTrr~Tr~
51 GbD~ATTAGC Trr~--rTrrr r~rDrTDTrTr. Prr.TDrrTDr. TDrT~DTr.T
101 1l~lll~ ,. bATTTGTGCT DT~TPTDrrT rDTrTPDDTT DTTr~DT~r~
151 rDrDrr~ rr A~DACATCTCT TbATTbGTTT TGATCATTTT TDr.Tr,r~r--
~
201 bI~ T7~7~D~rTTr~T GTTTTTCATG rTDTDTr.TrT Ll~' ll '~251 ACTTGTTTCC ~ T~ ~ I ~ACCTrDTTT r.Trrrr,rr~7l GATCAAGCTC
301 ll~ AGAATTCDAG AACATGTTTA CCGTTAATCC TAPTGCTTCT
351 l l...,., .~., . Drr~rDr~Dr ADCTCTTTCT Trr`~r~7`D GCACAAGTTG
401 ~l~=~.l~l~ GATGGCGTTC DTTr.Tr`"nD 7~-~rr~rDr.nD CA~GTGATTG
451 AGCTTGACCT CCGTTGCATC CAACTTCAAG GCAAGTTTCA TTCCAATAGT
501 AGCCTCTTTC AACTCTCCAA ~CTCDAAAGG ~ ., ..-..., ... rTTDT7~~~TaD
551 TTTCACTGGA TCGCCCATTT CACCTADATT TGGTGAGTTT TrDnDTT~nD
601 CGCATCTCGA TTTGTCGCAT TCAAGTTTTA a-r~a-r~Ta-TDnT CCCTTCTGAA
651 ATCTCTCA~C TTTCTADACT ATACGTTCTT CGTATTAGTC TADATGAGCT
701 TACTTTTGGT CCTCACA~TT TTGAATTGCT TCTTAAGAAC TTGACCCAAT
751 TA~DAGTGCT rr~rrTTC~ TCTATCAACA TCTCTTCCAC Ll~
801 AATTTCTCTT CTCATTTADC AAATCTATGG CTTCCATacA CAGAGTTACG
851 l~ Al~ 7 rrrr~D~r~ TTTTCCACCT TTCCGACTTA GAATTTCTCG
901 ATTTATCAAG CAATCCCC~G CTCACGGTTA GGTTTCCCAC ~DrrP~rTr~r~
951 PDTpnrDnTn CATCACTCAT GAAGTTATAT CTCTATAATG TGAATATTGA
WO 95/31564 1~~ . .075
2~ 88562
1001 TGATAGGATA CCTGAATCAT TTAGCCATCT AacTTcAcTT CATAAGTTGT
1051 ACATGAGTCG TTCTAATCTG TCAGGGCCTA TTCCTAAACC TrTATrA~7.T
1101 CTCACCAACA TAGTGTTTTT GGACCTTAaT AATAACCATC TTr7~7~rA~Ar
1151 AATTCCATCC Prrr~TA~rrr, ~ ATAr~ATr7~ rrT~rr~TA
1201 CATCAAACAA CTTAAATGCG ArTATDrrPT CCTGGATATT ~
1251 TCACTGATAG GGTTAGACTT ~A''~r7`rTr~r ACTTTCAGTG GAAAAATTCA
1301 AGAGTTCAAG TCCAAAACAT TA~r.TPrrnT TACTCTAAAA rA~ .TA-~rr
1351 TAAAAGGTCC TATTCCGAAT TCACTCCTAa Arrrr~"r~'` CCTACAATTC
1401 L~ .lll. rArPrr~Tr'~ TATCAGTGGA CATATTTCTT CAGCTATCTC,
1451 CAATCTGAAA ACATTGATAT TGTTAGACTT Arr.Aar.T~ T AATTTGGAGG
1501 A---`rA7'TAr~A GCAATGCGTG GTTGAGAGGA 7\rr.~.~TrrrT TTCGCATTTG
1551 GATTTGAGCA ArA7~rrA~rT TPr.TrrA~Ap ATCAATACAA CTTTTAGT~T
1601 TGGAAACATT TTAAGGGTCA TTAGCTTGCA rcrr~7~Trrr rTArrr.rrr.P
1651 AaGTCCCACG ~I~ AL~: P71TTrrr'"'T A~TTGACACT ACTTGATCTA
1701 Gr~Tr'`rDrT~ TGTTGAATGA QCATTTCCA AACTGGTTGG r~TPrrTATT
1751 TCAATTGAAG ATTTTAaGCT TGAGATcAaA TAAGTTGCAT ~ ~L~_A
1801 AATCTTCAGG r~ TrrA~7lr ~ ."i~ . GTCTTCAaAT ..~ AI-
~1851 TCATCTAATG GATTTAGTGG GAATTTACCC GAAAGAATTT TGGGGAAm
1901 GrA7~ArrpTr~ AAGGAAATTG DTr~r~--Trr AGGATTCCCA GAGTATATTT
1951 rTr7~T~rATP l~I~1.l~I Trr~TTATT TGACGACAAT TTCTACAAAG
2001 GGACAAGATT ~uAl~ l TCGAATTTTG GATTCTAACA TGATTATCAA
2051 TCTCTCAAAG AACAGAmG AAGGTCATAT TCCAAGCATT ATTGGAGATC
2101 TTGTTGGACT TCGTACGTTG AACTTGTCTC ACAATGTCTT GGAAGGTCAT
2151 ATPrrr~:rPT CATTTCAaAA TTTATCAGTA CTCGAATCAT l~
2201 ATCTAATAaA ATCAGCGGAG AaATTCCGCA GCAGCTTGCA TCCCTCACAT
2251 TCCTTGAAGT CTTAAATCTC TCTCACAATC A~ A~
Z301 ~ 'r'`2`'~rr AATTTGATTC GTTCGGGAAC ACTTCGTACC AAGGGAATGA
2351 TGGGTTACGC GGATTTCCAC TCTCAAAACT ll~~ GAAGATCAAG
2401 TGACAACTCC Pr~rTr~rrT~ GATCAAGAAG 7~ r~7~r~ AGATTCACCA
2451 ATGATCAGTT nrrAr~r~r-T l., ,~,~, TACGGTTGTG GACTTGTTAT
2501 TGGACTGTCC rTDATAThrA TAATGTGGTC AACTCAATAT CCaGCATGGT
2551 TTTCGAGGAT GGATTTAAAG TTGGAACACA TAATTACTAC GAaAATGAAA
2601 r~rrPr~r~A AAAGATATTA GTGAGTAGCT ATACCTCCAG GTATTCCACT
2651 TGATCATTAT cTTTcAGAaG ATTATTTTTT GTATATCGAT GAAATTATCG
5/01075
WO 95/31564 21 ~ ~ ~ 6 2 PCT/GB9
92
2701 ACCTCCTTCA TCCTCA~AGC TCTTAACTTT CACTCTTCAT TTTTGAI~AAT
2751 TTCAGGATTC AaAGATTTCC GAGTTCCCAG ~ .~ TGCAGATAAA
2801 AGCCTTTTTA TCTTTCATAG ~ TATr'~`T~ GATTTTATTT
2851 ~ TrGrDraTA GATATGTTCC GTCACTAAAA ACATTGTATT
2901 TCTCTCAACT CTTTCGTCAC ATGATATCAA AGAACACTTG ACTTCAATTA
2951 AGTTACTGTA GTCTGCTATT TTAATTTCTT CCATT.GAAAC ACAACTGACG
3001 TATCTTGAGA ~r`"`"T7~T GATCTCAGAA ATGGGAATCT CCCAATCCAA
S2Q ID No. 12:
MGCvRLVFFM LYVFLFQLVS SSSLPHLCPE DQALALLEFK l....~lvl.. ~c
51 DYCYDRRTLS ~ ~bW CI~iVSl~:lJr l l~i QVIELDLRCI QLQGKFHSNS
101 SLFQLSNLKR LDLSYNDFTG bflb~l~rl~r;l~ CnT.TUT.nT.C~T 5br~1ivl~:,r
151 Tc~TT.cRT.Y~7T. RISLNELTFG PHNFELLL~N LTQLKvLDLE slNl5b~
201 NFSSHLTNLW LPYTELRGIL PERVFHLSDL EFLDLSSNPQ LT'vRFPTTKw
2Sl Naa`CT.MRT.Y LYNVNIDDRI PESFSHLTSL HKLYMSRSNL SGP}PXPLw`N
301 LTNIVFLDLN NNHLEGPIPS NVSGLRNLQI T.WT. ~ SIPSwIFSLP
351 CT.TaT,nT, lr~ lyrir~ SKTLSTVTLK QNKLKGPIPN CT.T~TQR7~T~T7
401 T.T.T. ~TTgC~TrWT.R TT.TT.T.TlT.~`' NLEGTIPQCV VERNEYLSHL
451 nT~c ~CaT ll llrbV~ l LRvIsL~u~GNK LTGKVPRSMI NrR~7T,TT,T,nT,
501 GN~MLNDTFP NWLGYLFQLK TT.CT17~` 'TU ~ LFMGLQILDL
551 SSNGFSGNLP ERILGNLQTM KrilLIri7,~-ir~ riYl::lJrlLllY YNYLTTISTK
601 ~iy~ vr~ll, DSNMIINLSK ~rrr~ bl IGDLVGLRTL NLS~vLEGU
651 IPASFQNLSV T.T.'.CT.nT.! ISGEIPQQLA SLTFLEVLNL SHNHLVGCIP
701 r~yrJ~r-,r TCYQ''`~r.T.T~ GFPLSKLCGG EDQVTTPAEL L~ .v
751 MISWQGVLVG YGCGLVIGLS v ~ PAWFSRMDLK LEHIITTKMX
~01 =~
WO 95131564 2 1 8 8 5 6 2 ~ ~ i /a
~Trr-~G~r~ _~.,___~.____~CAATTGCC'I"'`CI~LL_,___~L ,.... ,., ArAr~r~rCT
1 60
T~rrr~TlD~ r~a~ Ar~TGT~rAArr~AAGTAAAr~AAr~AArAr~AcATc-TGA~GA
3Y G F V L F S Q L P S F L L V S T L L
CTIllAT~l~'rT~r~T~AT~ rrrAl L~LL~ AArrrrrrAAAArTrAA~--rATArAA
cAA~ rr.~r,~ TAArr,n~r G Arrr.rA~ ~7L L L ~ ~ 120
L F L V I S }~ S C R A 3C A. P K T Q P Y N
rrr-q~rr-~-~rrrA-r-AAr.TrATrrArArrA-rTr~rATrr,r,TCCCAACGA~VL-~
a~ ac
=A~_~7L L~7L 1~ ,~A-GTA~.vlv~7L r~ Ar~ArrrAr.{~rjTT~r~A~rAr,~_.,~
P C R P Q E V I D ~r K C ~ G } 1~ D C L Y
r~rrr~Arrrr,~-Ar.~PTr.~r7.Arr~r~r~Lr.Tr~r.T~rrnrTrr~--r~AAGTTrr,rAA
lal I I I I I 1 240
~L-iL~ ~ACATGTTGGAi~ I.,~ Ar-Ar~pcc~rr~ Tr~rlrTcA~ccGTT
P N P D S C T T Y I Q C V P L D E V G N
rr~ LAAGccATGTrrAAAAr~`~Ttjr~r-Tr~`rArc~ rc~TcGcA}~
~CTTcrr-ArArrAAATTrcr-~ rA~r~l LL .~ rrr--- - . L~-l . _, .~.I'AArrnTT
~ R P V V ~ P C P R G L Q W N D ~ V G R
r~n~nn~nrr~ lrr~7.rrT~ ,AAAnArrrrnrA~r~r~~"r
301 1 . I I I 1 360.
cTTr~rr~rr~rTGATAGGT~TGGAcTrArrr~rAr~Ar-r~rr~ ' L ~ L~ 7
X W C D Y P ~ L S T C P V ~C '[! P Q P R P
r~lr~rr--~ r-Ar~ ;Ar~ ArTCG~j
36~ 1 1 1 1 1 1 420
Ll~_L~ ACAGb:~rl-~LL~ nrr~rrll~r~lpAr~r~rr~ r~r~nrr
K R G G V G G R ~ A S V G L P G Y
ArAAr~ r~ Allr~r-TTrlrG~r~rr~r~ , v~ . rrc~
421 1 1 1 ~ ~ 1 4~0
L~7L.L._LL'I I I .'~rrr~-~TTGTr-~r~-rr~Trnll~rTrn~ rr~rr~,.'l 1'1 ,_rnrA
C~ AC
481 ~
GCTG
