Note: Descriptions are shown in the official language in which they were submitted.
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Production of AVGI-~ ~tjC Seed
The present invention relates to the production of genelically ~,dnsfo""ed plants. In particular
the invention relates interalia to a prucess for inducing apo",i~i~ to the apomictic seeds which
result from the prucess, and to the plants and progeny thereof which result from the gerrnination
of such seeds.
Apomixis which is vegetative (non-sexual) reproduction through seeds is a genetically
Cbr,t~ ed reproductive mechanism found in some polyploid non~ultivated species. The process
is 11assHiecl as gametophytic or non-ga",etophytic. In gametophytic apo",i~is - of which there
are two types (apospory and diplospory) multiple embryo sacs which typically lack anti~odal
nuclei are fomled or else megas~orugenesis in the embryo sac takes place. In adventitious
embryony (non-ga",et~,hytic apomixis) a so",atic embryo develops directly from the cells of the
embryo sac ovary wall or integuments. In advenmious embryony somatic embryos from
surrounding cells invade the sexual ovary one of the somatic embryos out~o",~etes the other
somatic embryos and the sexual embryo and utilizes the produced endo~pemm.
Were apomixis to be a controllable and reproduri~le pheno",enon it would provide many
advantages in plant improvement and cultivar dcvelûp",ent in the case that sexual plants are
available as crosses with the apom: ~ic plant.
For example apG""xis would provide for true-breeding seed prop~g~çd hybrids. Moreover,
apor,~ could shorten and simplify the breeding process so that selfing and progeny testing to
produce and/or stabilize a desirable gene combination could be eliminated. Apomixis would
provide for the use as cultivars of genotypes with unique gene comb ,al;ons since apomictic
genotypes breed tnue irrespective of heterozygosity. Genes or groups of genes could thus be
"pyramided and ~xed~ in super genotypes. Every superior apomictic genotype from a ssxual-
apomictic cross would have the potenlial to be a cultivar. Apomixis would allow plant b,eed~,r:, to
develop cultivars with specHic stable traits for such characters as height seed and forage quality
and maturity. E~reeders would not be limited in their co"""er ial production ot hybrids by (i) a
cytuplaamic-nuclear i,~t~ Aion to produce male sterile female parents or (ii) the fertility ~ t~lilly
.. . . .. ... . . ... . .. . .. . ... . .. . .. . .
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capacity of a pollinator. Almost all cross~r"patil)le ge""pla~", could be a potential parent to
produce apomictic hybrids.
Finally, apo"-ixis would simplify coi-""er~idl hybrid seed production. In particular, (i) the need for
physical isolation of cG"""e.dal hybrid production fields would be eliminated; (ii) all available
land could be used to increase hybrid seed instead of dividing space between pollinators and
male sterile lines; and (iii) the need to maintain parental line seed stocks would be eliminated.
The pote.,lial benefits to accrue from the production of seed v7a apor"i,~is are presel,~ly
unrealized, to a large extent ber-suse of the pr~t'e " of engineering apomictic capacity into
plants of interest. The present invention provides a solution to that problem in that it provides
the means for obtaining plants which exhibit the adventitious embryony type of apu",ixii.
According to the present invention there is provided a method of producing apomictic seeds
co" ,~risi, ,9 the steps of:
(i) trd";,~o",ling plant material with a nucleotide sequence encoding a protein the
presence of which in an active fomm in a cell, or ,-,e"lt-,dne thereof, renders said cell
embryogenic,
(ii) regenerating the thus l,dn:,~u"ned ",ateiial into plants, or carpel-containing parts
thereof, and
(iii) ex~,ressi"y the se~uence in the vicinity of the embryo sac.
By Uvicinity of the embryo sac" is meant in one or more of the following: carpel, integuments,
ovule, ovule pre",G,.lium, ovary wall, chal~7~, nucellus, funicle and placellk~. The skilled man
will recognize that the term Uinteguments~ also includes those tissues, such as endothelium,
which are derived therefrom. By "embryogenic" is meant the capability of cells to develop into
an embryo under pe"";~ive conditions. It will be appreciated that the temm "in an active fomm~
indudes proteins which are truncated or otherwise mutated with the proviso that they initiate or
amplify embryugene:,i, v.h~tl,el or not in doing this they interact with the signal transduction
co""~onents that they ~: ,en,~;se would in the tissues in which they are normally present.
The temm ~plant ~"aterial" includes protoplasts, icolst~qcl plant cells (such as st.-n,a~al guard cells)
possçss;-,y a cell wall, pollen, whole tissues such as ellleryed radicle, stem, leaf, petal,
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hypocotyl section, apical ",er,;il~"l, ovaries, zygotic embryo per se, roots, vascular bundle,
pericycle, anther filament, somatic emblyos and the like.
A further embodiment of the invention relates to a DNA molecule col"pris;"g a nucleotide
sequence encoding a protein the pr~sence of which in an active fomm in a cell, or ",e",brdne
thereof, renders said cell embryogenic.
The said nucleotide sequence may be introduced into the plant material, inter a~ia, via a bacterial
or viral vector, by micro-injection, by co-inc~h~tion of the plant material and sequence in the
presence of a high ",o'ec~ r weight glycol or by coating of the sequence onto the surface of a
biologically inert particle which is then introduced into the material.
Ex~.ression of the sequence may yield a protein kinase c,-~-'le of spanning a plant cell
membrane. Typically the kinase may be a leucine rich repeat ,~ce,ulur like kinase which has the
capacity to auto-phosphorylate. The skilled man will recognize what is meant by the temm
"leucine rich repeat receptor like kinase". Examples of such prute."s include Ar~ ~',pcis RLK5
(Walker, 1993), Ar~h~dop~is RPS2 (Bent et al. 1994), Tomato CF-9 gene product tJones et al.
1994), Tomato N (VVhitham et al. 1994), Petunia PRK1 (Mu et a/. 1994), the product of the
DrosophiJa Toll gene (I lashi",ùlo et al. 1988), the protein kinase encocJed by the rice OsPK10
gene (Zhao et al. 1994), the lldnslalion product of the rice EST clone ric2976 and the product of
the Drosophila Pelle gene (Shelton and Wasse""an, 1993). Still further examples of such
pr~t~ s include the TMK1, Clavatal, Erecta, and TMKL1 gene products from Arabidopsis, the
Flightless-1 gene product from DrDsophila, the TrkC gene product from pig, the rat LhCG
,eceplur and FSH receplor, the dog TSH receptor, and the human Trk ,eceptor kinase. The
protein may cG",~iise a ligand binding domain. a proline box, a trans",e"lb,dne domain, a
kinase domain and a protein binding domain. In many ,~ceptur kinases the extracellular (ligand
binding) domain serves as an inhibitor of the kinase domain in the ligand-free state. This arrest is
removed after binding of the ligand. Accordingly, in one embodiment of the invention the protein
either lacks a ligand binding domain or the domain is functionally inactivated so that the kinase
domain can be constitùtively active in the absence of an activating signal (ligand). Whebher or
not the protein posse~ses a ligand binding domain - full~,hulldl or ~I,en,~ e, once e~lessecl and
GIdted into the plant cell ",er"b,dne the protein binding domain is p~e~t:rdbly located
intra-cellularly.
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In a p,~ "ecJ embodiment of the method, the said sequence further encodes a cell m~"lb,.lne
targeting sequence. The sequence may be that which is depicted in SEQ ID Nos. 1 2 20 or
32 or it may be similar in that it is complementary to a sequence which hyLridi~es under
st~i.,gen~ conditions with the said sequences and which enc~des a ",e"lL,dne bound protein
having kinase act~vity. ~y "similar~ is meant a sequence which is comp ~."ent~ry to a test
sequence which is capable of hybrdi~il,g to the inventive sequence. When the test and
inventive sequences are double stranded the nucleic acid constituting the test sequence
preferably has a TM within 20~C of that of the inventive sequence. In the case that the test and
inventive sequences are mixed togt:ll,er and denatured simultaneously, the TM values of the
sequences are prefe,~Lly within 10~C of each other. More pr~faldbly the h~,iJi~tion is
pe,lo""ed under sl,i"yenl con-litions with either the test or inventive DNA pref~r bly being
slJ~JpGIled. Thus either a denatured test or inventive sequence is preferably first bound to a
support and hyl,ridi~atior, is e~ 1ed for a specified period of time at a temperature of between
50 and 70~C in double ~t~r,ytl, citrate buffered saline (SSC) containing 0.1%SDS followed by
rinsing of the support at the same temperature but with a buffer having a reduced SSC
concent,alion. Depending upon the degree of ~t,i"gel,cy required and thus the degree of
similarity of the sequences at a particular lel"~,erdlure - such as 60~C for example - such
reduced concenl,dlion buffers are typically single sl,t:r,yth SSC containing 0.1%SDS half
~tlt:nglll SSC containing 0.1%SDS and one tenth st~ehytl, SSC containing 0.1%SDS.
Sequences having the y,eale t degree of similarity are those the hyb,idk~tion of which is least
affected by washing in buffers of reduced conceiltldtion. It is most pl~lerl~d that the test and
inventive sequences are so similar that the h~riWi~tion between them is substantially
unaffected by washing or incllh~tion in one tenth ~I,tr,gtl, sodium citrate buffer containing
0.1 %SDS.
Accordingly further cG"",rised by the present invention is a DNA sequence as depicted in SEQ
ID NOS: 22 24 26 28 and 30 or a sequence which is complementary to one which hybrh~kes
under sl,i"yeot conditions with the said sequences and which encodes a ",e"lbrc~ne bound
protein having Wnase activity.
The sequence may be modHied in that known mRNA instability motifs or polyadenylation signals
may be removed and/or codons which are p,efe"~:.l by the plant into which the sequence is to
be i, .se, led may be used so that e~r~ssion of the thus modHied sequence in the said plant may
yield suLs~rlti .I:y similar protein to that obtained by e~-~:ssiot) of the unmodified sequence in
the o,yani_." in which the protein is endogenous.
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ln order to obtain ex~ 3s,0n of the sequence in the regenerated plant (and in particular the
carpel thereof) in a tissue specific manner the sequence is pr~fe,dbly under e~iession control
of an inducible or devel~p",entally regulated pr.""oter, typically one of the fc' ~ J~i- Iy. a pru,),oter
which regulates e,.~u,~ss;on of SERK genes in planta, the AP~cJorc;c ANT gene p,u",oter the
pru",oter of the 0126 gene from rl,alaenopsis the carrot chitinase DcEP3-1 gene pru,,~oler the
Arabidopsis AtChitlV gene pru",oter the Arabidopsis LTP-1 gene ,cru,l,oter the Al . 5i~op-~is bel-
1 gene prv"loter the petunia fbp-7 gene pru",oler, the AQhi~op- is AtDMC1 prul"ote" the
pTA7001 inducible prullloten The DcEP3-1 gene is e~ressed lldns,antly during inner
integument degradation and later in cells that line the inner part of the dcv~ F: ,g encJospel"l.
The AtChilV gene is lldl~5i~:"Uy e~r~ssed in the micropylar endospemm up to cellularisation. The
LTP-1 prulllolt:r is active in the epide,lll;s of the dcv~F.I9 nucel~ both integuments seed
coat and early embryo. The bel-1 gene is e,~ressed in the dc~elop;. I9 inner integument and the
fbb-7 promoter is active during embryo sac dc-.relopment. The Ar~h;dop~ ANT gene is
expressed during integument dcveloprl,en~ and the 0126 gene from Phalaenopsis is e~r~:ssed
in the mature ovule.
It is most p.efe"t:d that the sequence is ex~uressecl in the somatic cells of the embryo sac ovary
wall nucellus,orinteguments.
The endospemm within the apomictic seed results from fusion of polar nuclei within the embryo
sac with a pollen-derived male gamete nucleus. It is preferred that the sequence encoding the
protein is e~r~secl prior to fusion of the polar nuclei with the male gamete nucleus.
The invention further includes a DNA but prt:lerably a recombinant DNA co-."~rs;"g a
sequence encoding a protein the presence of which in an active form in a cell or ~e~lb~ane
thereof, renders said cell embryogenic. Preferred is a DNA encoding a protein which is a leucine
rich repeat r~ tùr like kinase and co",p,ises a ligand binding domain a proline box, a
~dns",e"ll,ra"e domain a kinase domain and a protein binding domain, the ligand binding
domain optionally being absent or h" ,~ionally inactive.
In particular the invention embodies a DNA co"".risil ,9 a DNA sequence encoding a N-terminal
protein l,ayll,erl~ having the f~' ~wi.,g amino acid sequence: ~n SerT~pAsp P~ThrLeuValAsn P~
CysThrTrp PheHisValThrCysAsn.
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A specific embodiment of the invention relates to a DNA co",~iis,"g a DNA sequence encoding
a protein having the sequence depicted in SEQ ID Nos. 3 or 21, or a protein subsk~Antially similar
thereto which is capable of being nle"lb,dne bound and which has kinase activity. By
s~ Antially similar is meant a pure protein having an amino acid sequence which is at least
90% similar to the sequence of the prut~ i. ,s depicted in SEO ID No 3 below. In the context of
the present invention two amino acid sequences with at least 90% similarity to each other have
at least 90% identical or conservatively replnced amino acid resi~ues in a like position when
aligned optimally allowing for up to 8 gaps with the prDWso that in respect of each gap a total not
more than 4 amino acid residues is affected. For the purpose of the present invention
conservative replace",er,~ may be made between amino acids within the following groups:
(i) Serine and Threonine;
(ii) Glutamic acid and Aspartic acid;
(iii) Arginine and Lysine;
(iv) Asparagine and Glutamine;
(v) so ~uc~ne, Leucine Valine and Methionine;
(vi) Phenylalanine Tyrosine and Tryptophan
(vii) Alanine and Glycine
In addition non-conservative replace",ent~ may also occur at a low frequency. Accordingly the
invention futher embodies a DNA co",pris,"g a DNA sequence encoding a N-temminal protein
fragment having the following amino acid sequence: ValXaaGlnSerTrpAspP~ThrLeuValAsnP~
Cys Thr T~p Phe His Val Thr Cys Asn, wi h Xaa being a variable amino a~d but prefelabiy Leu or VaL
Fcpeci~lly preferred within the scope of the invention is a DNA co",pr~ ,g a DNA sequence
encoding a N-tellll;. ,al protein fragment having the fcl A J~i, 19 amino acid sequence: Val Xaa Gln
SerT~ Asp P~ Thr Leu Val Asn P~ Cys ThrTIp Phe Hs Val Thr Cys Asn Xab Xac Xad Xae Val Xaf Arg Val
Asp Leu G~y Asn Xag Xah Leu Ser G~ Hs Leu Xai P~ Glu Leu ~;Jy Xaj Leu Xak Xal Leu Gh with Xaa to
Xak ~epresenti"g variable amino acids, but preIerdbly
Xaa = Leu aVal
Xab=AsnaGln
Xac = Glu orAsp aHs
Xad= Asn aHs
Xae=Ser~Arga~n
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Xaf = lleorThr
Xag=AlaorSer
Xah=GluorAsn
Xai=ValaAla
- Xaj=ValorLys
Xak= Lys orGlu
Xal=AsnaHis
It is pl~fe~ d that the DNA further encodes a cell ",e"lb,dne ~ryetillg sequence and that the
protein encoding region is under exur~s~ion control of a dcv .lop",enldlly rerg~' ted or inducible
prumoter such as, for example a pru",oter which regu'~tr~s e~-~.ression of SERK genes in
planta, the carrot chitinase DcEP3-1 gene plu",uler the Arabidopsis AtChitlV gene p,umoter
the A~l~.idop~is LTP-1 gene pru",olr3r the Ar~h,c~ops;s bel-1 gene pru",oler the petunia fbp-7
gene promoter the Ar~h~ onsic ANT gene pru,l,oter, or the pru",ùt~l of the 0126 gene from
Phalaenopsis; the AP~ :'CF~;S AtDMC1 pru"loler, or the pTA7001 inducible pru",oter.
Particularly preferred embodiments of the said DNA include those depicted in SEQ ID Nos. 1 2
20 or 32 or those which are complementary to one which hyl,r,di~t:s under il~il ,gent conditions
with the said sequences and which encode a ",~"lbldne bound protein having kinase activity.
As indicated above the DNA may be modffied in that known mRNA instability motffs or
polyadenylation signals may be removed and/or codons which are pl~fe~ d by the plant into
which the DNA is to be inserted may be used so that e~,ur~ssion of the thus ",odffied DNA in the
said plant may yield substantially similar protein to that ob~i"ed by e,~,urt:ssion of the unmodffied
DNA in the oryan,s", in which the protein is endogenous.
The invention still further includes a vector which contdi"s DNA as indicated in the three
i"""ediat ly preceding paragraphs plants t~dn~u,,,,ed with the recombinant DNA or vector, and
the progeny of such plants which contain the DNA stably il~ Joldte:d, and/or the apomictic
sesds of such plants or such progeny.
The recombinant DNA i"~'ecules of the invention can be introduced into the plant cell in
a number of art-rec~y"i2ed ways. Those skilled in the art will app,~ciate that the choice
of method might clepend on the type of plant i.e. ",onocot or dicot targeted fortransfo""ation. Suitable methods of transforrning plant cells include m;_~c "~ction
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(Crossway et al., BioTechniques 4:320-334 (1986)), electroporation (Riggs et al, Proc.
Natl. Acad. Sci. USA 83:5602-~606 (1986), Agrobacterium mediated trans~G""alion
(Hinchee et al., Biot~,,hne.~ J~/ 6:9~ 5-921 (1988)), direct gene l,ar,a~er (Paszl~o.vski et al.,
EMBO J. 3:2717-2722 (1984)), ballistic particle acceleration using devices available from
Agracetus, Inc., Madison, Wisconsin and Dupont, Inc., Wilmington, Delaware (see, for
example, Sanford et al., U.S. Patent 4,945,050; and McCabe et al., Bio~chno~Jy
6:923-926 (1988)), and protoplast tranalo""ation/regeneration methods (see U.S. Patent
No. 5,350,689 issued Sept. 27, 1994 to Ciba-Geigy Corp.). Also see, Weissinger et al.,
Annual Rev. Genet. 22:421~77 (1988); Sanford et al., Particvlate Sc~ence and
TechnolD~y 5:27-37 (1 987)(onion); Christou et al., Plant Physiol. 87:671-674
(1988)(soybean); McCabe et al., Bio/Techn~'~J~ 6:923-926 (1988)(soybean); Datta et
al., Bio/Technotogy 8:736-740 (1990)(rice); Klein et al., Proc. Natl. Acad. Sci. USA,
~5:4305-4309 (1 988)(maize); Klein et al., BioJlechnology 6:559-563 (1 988)(maize); Klein
et al., Plant Physiol. 91:440-444 (1 988)(maize); Fromm et al., Bio~Technology~ 8:833-839
(1990); and Gordon-Kamm et al., Plant Cell 2:603-618 (1990)(maize).
Comprised within the scope of the present invention are transgenic plants, in particular
transgenic fertile plants transformed by means of the aforedescribed processes and their
~ceyu~l and/or sexual progeny, which still contain the DNA stably incorporated, and/or the
apomictic seeds of such plants or such progeny.
The transgenic plant according to the invention may be a dicotyledonous or a
monocotyledonous plant. Such plants include field crops, vegetables and fruits including
tomato, pepper, melon, lettuce, cauliflower, ~,oc~, ', r~hb~ge, bnussels sprout, sugar beet,
com, swt,.,tc~"" onion, carrot, leek, cucumber, ~b~r~, aHaHa, aubergine, beet, broad bean,
celery, chicory, cow pea, endive, gourd, groundnut, papaya, pea, peanut, pineapple, potato,
salfloJ~er, snap bean, soybean, spinach, squashes, sl",llo.~er, sorghum, water-melon, and
the like; and or"a",ental crops including Impatiens, Begonia, Petunia, Pelaryonium, Viola,
Cyclamen, Verbena, Vinca, Tagetes, Primula, Saint Paulia, Ageratum, Allla.dllUI.ls,
Anthirrhinum, Aquilegia, Chrysanthemum, Cineraria, Clover, Cosmo, Cowpea, Dahlia, Datura,
Delphinium, Gerbera, Gladiolus, Gloxinia, l~ pe~,.Jm, 1~1e3~ 1,lyarltl,e",um, Salpiglossis,
annia, and the like. In a preferred embodiment, the DNA is e~l~:aaed in nseed crops~ such
as corn, sweet com and peas etc. in such a way that the apomictic seed which results from
such e~.ression is not physically mutated or otl~en~ise damaged in cc,r"parison with seed
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g
from u"l.dr,a~o".,ed like crops. Preferred are monocotyledonous plants of the
Gra""naceae family involving Lolium. Zea. Tnticum. Triticale, Sor~hum. Saccharum,
Bromus. Oryzae. Avena. Ho~eum, Secale and Setaria plants.
More preferred are transgenic maize, wheat, barley, sorghum, rye, oats, turf and forage
grasses, millet and rice. Especially preferred are maize, wheat, sorghum, rye, oats, turf
grasses and rice.
Among the dicotyledonous plants Arabidopsi~, soybean, cotton, sugar beet, sugar cane,
oilseed rape, tobAcco and sunflower are more preferred herein. Especially preferred are
soybean, cotton, tob~cco, sugar beet and oilseed rape.
The expression 'progeny' is understood to embrace both, "asexually" and "sexually"
generated progeny of transgenic plants. This definition is also meant to include all
mutants and variants obtainable by means of known processes, such as for example cell
fusion or mutant selection and which still exhibit the characteristic properties of the initial
transformed plant, together with all crossing and fusion products of the transformed plant
material. This also includes progeny plants that result from a backcrossing, as long as
the said prugeny plants still contain the DNA according to the invention
Another object of the invention concerns the proliferation material of transgenic
plants.
The proliferation material of transgenic plants is defined relative to the invention as any
plant material that may be propagated sexually or asexually in vivo or in vitro. Particularly
preferred within the scope of the present invention are protoplasts, cells, calli, tissues,
organs, seeds, embryos, pollen, egg cells, zygotes, together with any other propAgAting
material obtained from l,dnsgenic plants.
Parts of plants, such as for example flowers, stems, fnuits, leaves, roots oriy;lldlillg in
transgenic plants or their progeny previously transfommed by means of the process of the
invention and therefore consiali"g at least in part of transgenic cells, are also an object
of the present invention. ~speically pr~ft,r,~d a apomictic seeds.
A further object of the invention is a ",ell,ocl of producing apomictic seeds, but pl~ b~
seeds that are of the adventitious embryony type, co, I,pr,~i, ,g the steps of:
(i) l,d":,lo",ling plant ",at~,ial with a nu~leotide sequence encoding a protein the
sence of which in an active form in a cell, or n,e"lL.dne thereof, renders said cell
,. , ., .. , .. , . . , .. . ~ , ., .. _ ~ . .... . . . ..
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embryogenic, but preferdbly a protein which is a protein kinase capable of spanning a
plant cell ",e"ll,,dne and -~F~ Of autophospho~lation.
(ii) regeneraffng the thus l,d"slo",-ed ",alerial into plants, or carpel-conl..i.,;.,y parts
thereof, and
(iii) ex~,es~i"g the sequence in the vicinity of the embryo sac.
The kinase protein being expressed by the DNA accordi.,g to the invention is preferably a
leucine rich repeat receptor like kinase and co"~iises a ligand binding domain, a proline box, a
t,cln~",e",brdne domain, a kinase domain and a protein binding domain. In a specHic
embodiment of the invention, the said kinase protein may lack a functional ligand binding
domain but comprises a proline box, a t,~n~",t:"lL,tdne domain, a kinase domain and a protein
binding domain.
The genetic properties engineered into the transgenic seeds and plants described above
are passed on by sexual reproduction or vegetative growth and can thus be maintained and
prop~g~ted in progeny plants. Generally said maintenance and prop~g~tion make use of
known agricultural methods developed to fit specific purposes such as tilling, sowing or
harvesting. Specialized processes such as hydroponics or greenhouse technologies can
also be applied. As the growing crop is vulnerable to attack and damages caused by insects
or infections as well as to competition by weed plants, measures are undertaken to control
weeds, plant diseases, insects, nematodes, and other adverse conditions to improve yield.
These include mechanical measures such a tillage of the soil or removal of weeds and
infected plants, as well as the application of agrochemicals such as he,L.~ ~es, fungicides,
gametocides, ne".~li.;des, growth regulants, ripening agents and ir secticides
Use of the advantageous genetic properties of the transgenic plants and seeds according to
the invention can further be made in plant breeding which aims at the develop",6,lt of
plants with improved pr~pe,lies such as t~'Erdnce of pests, herbicides, or stress, improved
nutritional value, increased yield, or improved structure causing less loss from lodging or
shall~F" ,g. The various breeding steps are chardl,t.,F,~:d by well-de~i"ed human
intervention such as selecting the lines to be c,ussed, directing pollination of the parental
lines, or selecting apprupriate progeny plants. Dependi"g on the desired properties difl~
breeding measures are taken. The relevant techniques are well known in the art and include
but are not limited to hyL"idi~dtion, inbreeding, backcross breeding, multiline breeding,
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variety blend, interspecific hybridization, aneuploid techn :,ues, etc. Hybticii~a~ion
techniques also include the :,le~ tion of plants to yield male or female sterile plants by
mechanical, chemical or biochemical means. Cross pollination of a male sterile plant with
pollen of a different line assures that the genGi"e of the male sterile but female fertile plant
will uniformly obtain properties of both parental lines. Thus, the transgenic seeds and plants
according to the invention can be used for the breeding of improved plant lines which for
example increase the effectiveness of conventional methods such as herbicide or pestidice
treatment or allow to dispense with said methods due to their modified genetic properties.
Alternatively new crops with improved stress tolerance can be obtained which, due to their
Gpli",i~ed genetic "equi~,",enl~, yield harvested product of better quality than products which
were not able to tolerate comparable adverse develop",ental condilions.
In seeds production germination quality and uniformity of seeds are essential product
chara~terislics, whereas germination quality and unilGIlllity of seeds harvested and sold by
the farmer is not important. As it is difficult to keep a crop free from other crop and weed
seeds, to control seedborne diseases, and to produce seed with good gerrnination, fairiy
extensive and well-defined seed production prclctices have been developed by seed
producers, who are experienced in the art of growing, conditioning and marketing ot pure
seed. Thus, it is cG"""on practice for the farmer to buy certified seed meeting specific
quality standards instead of using seed harvested from his own crop. Prop~tion material
to be used as seeds is customarily treated with a protectant coating co",pris,ng he,l,i~ ides,
insecticides, fungicides, bactericides, nematicides, molluscicicles or mixtures thereof.
Customarily used protectant coatings comprise compounds such as captan, calL,o~-i",
thiram (TMTD~), methalaxyl (Apron6), and pirimiphos-methyl (Actellic-). If desired these
compounds are forrnulated together with further carriers, surfactants or application-
prul"oti"9 adjuvants customarily employed in the art of formulation to provide prulection
against damage caused by bacterial, fungal or animal pests. The p~le~ l coatings may
be applied by i""~fey"ati"g propq.~qtion material with a liquid formulation or by coating with
a combined wet or dry formulation. Other ",ell,ods of application are also possible such as
treatment directed at the buds or the fruit.
It is thus a further obiect of the present invention to provide plant prop~g~qtion ",dl~ial for
cultivated plants, but e~peciqliy plant seed that is treated with an seed proteclant coating
customarily used in seed treatment.
.
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lt is a further aspect of the present invention to provide new agricultural methods such as
the methods examplified above which are characterized by the use of l~dnsgenic plants,
~,dnsgeni~ plant material, or l~,-sgenic seed according to the present invention.
To breed progeny from plants transformed according to the method of the preserltinvention, a method such as that which follows may be used: plants produced as des~;,ibecl
in the examples set forth below are grown in pots in a greenhouse or in soil, as is known in
the art, and permitted to flower. Pollen is obtained from the mature stamens and used to
pollinate the pistils of the same plant, sibling plants, or any desirable plant. Similarly, the
pistils developing on the transforrned plant may be pollinated by pollen obtained from the
same plant, sibling plants, or any desirable plant. Trdns~ur,,,ed progeny obtained by this
method may be distinguished from non-transformed progeny by the presence of the
introduced gene(s) and/or acco",panying DNA (genotype), or the phenotype conferred.
The transformed progeny may similarly be selfed or crossed to other plants, as is nommally
done with any plant carrying a desirable trait. Similarly, tob~cco or other transformed plants
produced by this method may be selfed or crossed as is known in the art in order to
produce progeny with desired characteristics. Similarly, other transgenic o.yariis",s
produced by a combination of the methods known in the art and this invention may be bred
as is known in the art in order to produce progeny with desired chara~:terixtics.
Further co",pr,aed by the invention is a method of obl~i";"g embryogenic cells in plant material,
colllpri:.;"g b~rls~GI"ling the ",c-terial with a recombinant DNA sequence or a vector according to
the invention, ex~rt:s~"~g the sequence in the material or derivatives thereof and subjecting the
said mate,ial or derivatives to a compound which acts as a ligand for the gene product of the
said sequence.
The invention further relates to a method of generating somatic embryos under in vitro
cor,diticins wherein the SERK protein is overexpressed ~ - - F .c ~y.
The invention still fur~er indudes the use of the said DNA in the manufacture of apomictic
seeds, in which use the sequence is e~r~ssed in the vicinity of the embryo sac.
In a specific embodiment of the invention the SERK gene may be e~ressed in l,dr,sgenic plants
such as, for example, an Arab~dopsis plant, under the control of plant e~r~sa;~,n signals,
particula~y a pru",~ter which regulates e~rt:ssion of SERK genes ~n plan~a, but prt:fe,cb~f a
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developr"e"L"y regl~l?tecl or inducible pru",ote, such as for example. the carrot chitinase
DcEP3-1 gene p,u",ùter, the Ara~ rsis AtChitlV gene prc ",~ter the Arah_lop~is LTP-1 gene
p,u.l,citer the Arabidopsis bel-1 gene pru,,,ùler the petunia fbp-7 gene prc".,-~ter the
Arabidopsis ANT gene pru",oter or the pru,,,ù~er of the 0126 gene from rl~ldenopsis, the
Arabidopsis AtDMC1 p,o,..~ter, or the pTA7001 inducible p~u~ut~r.
The pru",ot~,~ of the DcEP3-1 and the AtChit IV genes may be cloned and characterized by
standard procedures. The DcSERK coding sequence (SEQ ID No. 2) is cloned behind the
DcEP3-1 the AtChit IV or the AtLTP-1 prw.,oters and tran~to~",ed into Arahic~l~pc;s The ligation
is pe,lo""ed in such a way that the p,c""oter is operably linked to the sequence to be
l.dnsc,iiJed. This construct which also contains known marker genes providing for selection of
transformed material is inserted into the T-DNA region of a binary vector such as pBlN19 and
transformed into Ara~ 10p-~;s Agr~b2cteriutr1mediated translo-",ation into Ar~J~Or~jS jS
performed bythe vacuum i"~iltl~tion or root l,dr,:,~o""ation procedures known to the skilled man.
T-dr~ o"-,ed seeds are selected and harvested and (where possible) I,~ns~u""ed lines are
established by nomnal selfing. Parallel l-~r~ u""alions with 35S prù",oter-SERK constructs and
the entire SERK gene itseH are used as controls to evaluate over-ex,Jression in many cells or
only in the few cells that naturally express the SERK gene. The 35S p,u",uler-SERK construct
may give embryo forrnation wherever the signal that activates the SERK-",edialed transduction
chain is present in the plant. A testing system based on em~sc~ tion and the generation of
donor plant lines for pollen carrying LTP1 pru",oter-GUS and SERK pru",oler-bamase is
established.
The same constructs (35S, EP3-1, AtChitlV AtLTP-1 and SERK ~pru",ote,~ fused to the SERK
coding sequence) are employed for l,."~:,lo""ation into several A~ 'cp.~is backgrounds. These
backgrounds are wild type, male sterile fis (allelic to emb 173) and pri",o,dia timing (pt)-1 lines
or a combination of two or several of these backgrounds. The wt lines are used as a control to
evaluate possible effects on normal zygobc embryogenesis and to score for seed set u~out
fertilization after errasc~ Qn. The ms lines are used to score directly for seed set without
fertilizabon. The fis lines exhibit a certain degree of seed and embryo dcv.Jlopr.,6rlt without
fertilization so may be expected to have a natural l~nden~y for apomicbc embr~,ugenes,-, which
may be enl)anced by the p,esence of the SERK constructs. The pt-1 line has superior
,tz~ene~ e capabilibes and has been used to initiate the first stably embryogenic Ara~idûpsis
cell suspension cuttures. Comb:.,ations of several of the above backgrounds are obta;.,ed by
..... , . , , ~
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crossing with each other and with lines containing ectopic SERK ex~.essi"g constnucts. Except
for the ms lines, prop~g~tion can proceed by nommal selfing, and analysis of apomictic traits
;ng em~sculetiQn. A similar strategy is followed in which the ATChilV, AtLTP-1 and SERK
p,u",~,le,~ are replece~l by the bel-1 and fbp-7 pru."~te,~ as well by other plulllotela specHic for
cGIllpol)êllls of the female gal"eto~,l,yte.
Additional constructs are generated that have constitutive rèce~.lur kinase activity. Most of the
~eceplor kinases of the SERK type act as homodimeric receptors, requiring aulo~hoaphorylation
before being able to activate do.~.,sbeam signal transduction c~sc~les In many ,ecep~,
kinases the extracellular domain serves as an inhibitor ot the kinase domain in the ligand-free
stage. This arrest is removed after binding of the ligand (Cadena and Gill, 1992). By introduction
of a SERK construct, from which the extracellular ligand-binding domain has been removed,
mutant homodimeric (in cells that do not have a natural population of SERK pr~t~,i"s) or
heterodimeric (in cells that also express the u"",odified forms) pr~t~,i.,~ can be generdted with a
constitutively activated kinase domain. This approach, when coupled to one of the prul,,olera
active in the nucellar region, results in activation of the embryogenic pathway in the absence of
the activating signal. This may be an i",~)o,lanl altemative in cases where it is necess~ry or
desirable to have activation of the SERK pathway only dependant on specific pn.",oter activity
and independent of telll~Joldl re~ulation of an activating signal. Introduction of SERK constructs
that result in fertilization-independent-embryugene~;s (fie) are tested in other species for their
effect. In order to recognize the fie phenotype, the skilled man will use appr~,prial~ male sterile
backgrounds. 1 lo.~e~/cr, pollination is often necess~ry for apo" ,i~is of the adventitious embryony
type, in order to ensure the production of endosperrn.
Whilst the present invention has been particulariy des~"iL,ed by way of the production of
apomictic seed by hel~rc'clgous ex~,r~ssion of the SERK gene in the nucellar region of the
carpel, the skilled man will recognize that other genes, the products of which have a similar
structure/function to the SERK gene product, may likewise be eA~ur~ssed with similar results.
Moreover, aithough the example illustrates apomictic seed production in AQbrdopsis, the
invention is, of course, not limited to the eA~ ssion of apomictic seed-inducing genes solely in
this plant. Moreover, the present r~icclQsure also includes the possibiiity of eA~ s;"g the SERK
(or related) gene sequences in the l,d"~u""ed plant material in a constitutive - tissue non-
specific ",anner (for example under l,dns~ onal control of a CaMV35S or NOS pru,,,oter). In
this case, tissue spe~,ifi.,ity is as-cured by the loc~ d p,~sence within the vicinity of the embryo
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sac of the ligand of the product of the said gene. Furtherrnore the SERK (or related) gene
products may interact with prutei.,s such as trans..,i~,ti~n factors which are involved in regulating
embryogenes;a. This i~,lerdction within tissue which has been l,ar,~u,,,,ed according to the
present ~iicclosure is atso part of the present invenbon.
The skilled man who has the benefit of the present ~isclos~ ~re will also recog"i~e that the SERK
gene (and others as indicated in the preceding paragraph) may be l,an~lo"l,ed into plant
material which may be propagat~d and/or dff,e,~,ltialed and used as an explant from which
somatic embryos can be obtained. Ex~re:ssion of such sequences in the translu,,,,ed tissue
(which is subjected to a ligand of the kinase gene products) suL,~lantially i~l~ reases the
percentage of the cells in the tissue which are competent to form SGI I latic embryos, in
co"lparison with the number present in non-l,dnsfui-"ed like tissue.
The invention will be further apparent from the fc 'ow:.,g descli~JIion and the ~csori~ted dld~ ys
and sequence listings.
SEQ ID NO. 1 depicts the Daucus car~ta yenolll ~ clone of the putative rt:ceplur kinase (SERK)
~sori~trd with the transition of competent to embryogenic cells;
SEQ ID NO. 2 depicts the cDNA of the said putative kinase;
SEQ ID NOs. 3 depicts the the predicted protein sequence of the SERK protein encoded by
the DNA of SEQ ID NO:1.
SEQ ID NOs: 4-16 depict the sequences of various PCR pril"e,~ and
SEQ ID NOs. 17-19 depict specific peptides co,lt~i"ed within the gene product of SEQ ID NO. 2.
SEQ lD NO: 20 depitcts the Ar~ 'Lp~iS thaliana partial genomic clone of the putative receptor
kinase (SERK) Ac5O~ tecl with the l,a,-~ition of cù--,~,att,rlt to embryogenic cells.
SEQ ID NO: 21 depicts the predicted protein sequence of the SERK protein encoded by the
DNA of SEQ ID NO:20.
SEQ ID NOs: 22, 24, 26, 28 and 30 depict the partial DNA sequences of S EST clones with
high homology to the SERK LRR sequences .
SEQ ID NOs. 23 25 27 29 and 31 depict the predicted protein sequence of the partial DNA
sequences of the 5 EST clones of SEQ ID Nos: 22 24 26 28 and 30.
SEQ ID NO: 32 depicts the n~clotide sequence of the SERK cDNA from Arabldopsis thaliana.
SEQ ID NO: 33 depicts the predicted amino acid sequence of the SERK protein fromArabidopsis thaliana enco.led by the DNA of SEQ ID NO: 32.
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Figure 1 shows the results of an RT-PCR experiment pe,~u,,,,ed on RNA extracted from the
indicated tissues. 40 cycles f~ l~wsd by Southem blotting of the resulting bands is necess~y to
visualize SERK e,~ ssion. Lanes include e~la,lt~ at day 7, treated for less (lane 1) or more
(lane 2) then 3 days with 2,4-D. In the original a very faint signal is visible in lane 2 but not in
lane 1. Established embryogenic cultures (lanes 4 6) but not a non-embryogenic control (lane 3)
eA~,r~ss the SERK gene. In carrot plants no ex~ ssion is dete - ~ except for developing
seeds after pollination (lane 7). Up to day 7 after pollination, the carrot zygote ,t:" ,ai"s u, IJiii;led,
sugse~ ~;ng that the obsen/ed signal is coming only from the zygote. At day 10 the early globular
and at day 20 the heart stage is rt:acl,ed in carrot zygotic embryogenesis. No signals are seen
on Northem blots.
Figure 2A shows the results of a whole-mount in sffu hybridization with the SERK cDNA on 7
day e~ lanls treated for 3 days with 2 4 D. Few cells on the surface of the explant express the
SERK gene, and those cells that do are the ones that become embryogenic. Figure 2B shows a
whole mount in situ hybriJi~tion on a partially di~sect~d seed containing a globular zygotic
embryo. HyL,riJi~cltion is visualized by DIG staining.
Figure 3 shows SERK ex~,r~ssion in embryogenic hypocotyl cells during ho""one-induced
activation detemlined by whole mount in situ hyL,iJi~ation . Bar 50 mm
(A-E) Cell population generated by ",ecl,an ~' fragmentation of the activated hypocotyls. Only
few of a certain type of cell defined enlarged cell show SERK e)(~,r~ssion (asterisks). Small
cytuplas"lic cells (c) enlarging cells (eg) and large cells (I) never show SERK ex~ ssion.
(F) Hypocotyl longitudinal section before ho""one-induced activation. It is not possible to detect
any SERK ex~.r~ssion in any tyoe of cell.
(G-l) Proliferating mass coming from the inner hypocotyl tissues 10 days after the beginning of
the hormonal ~,eal",er,l (longitudinal section). In G a single enlarged cells sho.~.,g SERK
expression is clete~ le within a row of negative cells showing the same morphology. In H a
single enlc.,yed cell showing serk e~r~ssion is detaching from the surface of the proliferating
mass. In I a cluster of enlaryed cells sl)o.~;.,g SERK e~rt,ssioo is ~Jet~ le at the surface of
proliferating tissue.
(J) Proliferating mass coming from the inner tissues of the hypocotyl 10 days after the beginning
of the rooting l,ea~",t:,lt (24 hours with 2,4-D followed by l,G""one removal). Both the root
p,i,.,~,clia and the er,lcllyecJ cells detaching from the surface do not show any SERK e~,~aaion.
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Figure 4 shows the phenotype of Arabidopsis WS plants tran~lo,-"ed with the 2200 bp SERK-
lucif~rdse consturct at the seedling level. Pictures were taken at 28 days after germination of T2
seeds. In plant 11 and 111 no clear shoot ",e(ialt:r" is visible at the seedling stage 7 days after ger-
mination. The first two leaves if they develop at all, are needleshaped as hown on the pictures
taken 28 days after germination. At this time plant 1, which shows no clear phenotype already
starts flowering. Secondary shoot IlleliaL~llls are already de\f.'cr:.lg in plant no 11 and will also
develop later from no 111. Shoot ",erisle",s influor~scences and normal flowers eventually
develop on all plants.
Figure 5 shows how the 2200 bpSERKlucife~dse construct affects the number of dcve'r~p:.lg
ovules in the siliques of transfomled plants.
Figure 6 shows aulophosphorylation of purified SERK fusion protein in vitro. Lane 1: purified
SERK fusion protein; Lane 2: serine phosphate; Lane 3: threonine phoa,uhale; Lane 4: thyrosine
phoa~hale.
The following des~ lion illustrates the isolation and cloning of the SERK gene and the
production of apomictic seed by hete,.'-g-us expression of the said gene in the nucellar region
of the carpel so that somatic embryos forrn which penetrate the embryo sac and are
enc~rslJI~ted by the seed as it develops.
. . .
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ISOLATION AND CLONING OF THE SERK GENE FROM DAUCUS CAROTA
Isolation of cDNA clones that are ,u,~F~,.a-~rially O~ eSSli~ in embryogenic cell cultures of
carr~t
In order to increase the chance of success for obtaining genes eA~ ssed in carrot suspension
cells competent to fomm embryos, the number of embryo-forming celis as present in a series of
established cell cultures was determined. A sub-poru'-tiQn of cells that passed through a 30 mm
nylon sieve was isolated from eight di~e,t:rll cultures that ranged in age between 2 months and
4 years. In these sub 30 mm pop~ tions, the number of embryos fommed from the single cells
and small cell clusters was detemmined and eA~,ressed as a pe,.;entage of the total number of
cells present at the start of embryogenesis. Sieved ~30 mm cultures able to fomm somatic
embryos with a frequency of more than 1% were then used as a source for competent cells, and
cultures that produced less than 0.01% embryos were used as non-embryogenic controls. As
main cloning strategies, cold plaque screening (Hodge et al. 1992) and di~e,~r,lial display (dd)
RT-PCR (Liang and Pardee, 1992) were used besides conventional differential screening of
cDNA libraries.
I ~heled probes for di~le,~:nlial screening were obtained from RNA out of a c30 mm sieved
sub-pop~ tion of cells from either embryogenic or non-embryogenic cell cultures. Employing
these probes in a library screen of al~pruA,, ,,ct~,ly 2000 plaques yielded 26 ~ ues that failed to
show any hyL li, li~dtion to either probe. These so-called cold plaques were purified and used for
further analysis. From the total number of p!-ques that did hybridize, about 30 did so only with
the probe from embryogenic cells. ddP~T-PCR reactions using a combination of one anchor
primer and one cleca",er primer were pel~ lled on mRNA isolated from three embryogenic, and
three non-embryogenic su~ ensiol1 cultures. About 50 d~le,~nt ddRT-PCR fragments were
obtained from each reaction. Using co"d,;.,dl,ons of three di~lert:nt anchor and six .li~ .)t
clec~-,-er primers, a total of a~ ~,ruAi."dt.,ly 1000 different cDNA lldylllents was ~cl~ 7ed Six of
these PCR l,.ly",e"~ were only found in lanes made with mRNA from ~30 mm popu'-tiQns of
cells from embryogenic cultures (Table 1 ) and with oligo comb: ,aUons of the anchor primer (5'-
11111111 1 l IGC-3') and the deca",el primers (5'-GGGATCTMG-3'), (5'-ACACGTGGTC-3~,
(5'-TCAGCACAGG-3~. Rec~use differential PCR fragments often consist of several ~ulr~soh~0d
cDNA fragments (Li et a/. 1994), cloning proved to be esse"~idl prior to undertaking further
char~-,le,i~tion of the PCR fragments obtained.
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All clones obtained were subjected to a second screen that consi~lad of spot-dot Northem
hyl,F"~ lion pe,~o""ed under conditions of high ~I,i"gen~. This method, that used RNA from
entire unsieved embryogenic and non-emb~ogenic sus~ansion cultures, proved to be a fast and
reiiable additional scl~tion method. Only one clone (22-28) of the 30 clones obt~i"ed after
differential screening proved to be ,~-~I,i~ad to embryogenic cell cultures while the majonty was
constitutively eA,u,~ssed. The 26 clones obtained from the cold plaque sc,t:en..,g required long
exposure times in the spot-dot Northem analysis. Six of these clones failed to show any
hyl,r,Ji~ation signal and 19 proved to be e~ressed in both embryogenic and non-embryogenic
cell cultures. One clone (31-50) showed low e~r~Kion in all embryogenic cultures and in one
non-embryogenic culture but not in the others. Of the six cloned fragments obtained by ddRT-
PCR display four showed hyl,ridi~a~ion more or less ,~sl,i-Aad to bdns~ ,t~ present in
embryogenic cultures. All clones that passed through the second screening were sequenced.
Two of the ddRT-PCR clones (6-8 and 7-13) were identical to the carrot Lipid Transfer Protein
(LTP) gene previously identffied as a marker for embryogenic carrot cell cultures. LTP
e~r~s,on is rt:~l,ic~t:d to embryogenic cell clusters and the p,ulude"" of somatic and zygoffc
embryos from the early globular stage onwards (Sterk et al. 1991). Therefore while the LTP
gene is not a marker for competent cells its appearance in the screening corlIi""s the validity of
our methods with respect to the cloning of genes exl.ressed early during somaticembryogenesis.
cDNA clone 31-50 e..codes a leucine-rich repeat containing ,~;cep~or-like kinaseThe mRNA co"~spGIlding to the isolated clone 31-50 had an open reading frame of 1659
nucleotides encoding a protein with a c~ ted Mw of 55 kDa. Rec~l-se clone 31-50 is mainly
e~,essed in embryogenic cell cultures it was .t:na",ecJ Somatic Embryogenesis Receptor
Kinase (SERK). The SERK protein co"tai"5 a N-temninal domain with a five-times l~peS~t~CI
leucine-rich motif that is pruposed to act as a protein-binding region in LRR ~t:ceptor kinases
(Kobe and Dcisenbofer, 1994). Between the extracellular LRR domain of SERK and the
~e~llb~dne-spanning region is a 33 amino acid region rich in prolines (13), that is unique for the
SERK protein. Of particular interest is the sequence SPPPP, that is conserved in extensi"s, a
cla~ of universal plant ceU wall ~,rut~ ;.,s (Vamer and Lin, 1989). The p,uposed intracellular
domain of the protein c~lltaills the 11 subdomains cl,a,a- I~F,~tic of the catalytic core of protein
kinases. The core sequences HRDVKAAN and GTLGYIAPE in ~*cpec,i~/ely the kinase
subdomains VB and Vlll sugge.st a function as a serine / threonine kinase (I tanks et al. 1988).
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- 20 -
Another i"lere~ g feature of the intracellular part of the SERK protein is that the C-terrninal 24
amino-acids resembles a single LRR. The serine and threonine r~sidues present within the
intracellular LRR sequence are surrounded by acidic residues and might be targets for the
autophosphorylaffon of SERK, thereby regulating the ability of other ~,,ote;.,s to interact with this
receptor-kinase in a similar fashion as desc,il,ed for the SH2 domain of the EGF family of
tyrosine receptor kinases.
Hybridi~ation of the SERK cDNA clone to the carrot geno",e revealed the pr~:sence of only a
single main hy~ndi~i- ,9 band after digestion with EcoR1, ,crubaLly ~~cfle~til ,9 a single SERK gene
in the carrot genome. This was confirrned after digestion with Ddel an enzyme that cuts three
times within the SERK gene. No signal was observed after N~.lhelll blotting of mRNA from
embryogenic cell cultures and hyl,ridi~clion with labeled SERK probes ,~:fle~ti"g the low levels
of l,dnsc,i~JI present in these cultures. Detection of the SERK l,dns~ l on the original spot-dot
Northems was only possible after long exposure times compared with other probes.
The ability of the SERK protein to autophosphorylate was inve~ ti-J~d in vitro using a previously
desc,iL,ed au~ophospholylation assay (Mu et al. 1994), with a l~a~ nal fusion protein that
contained the con,pete intracellular region of the SERK protein. The bacterially eA~.r~sse.l
SERK fusion protein was able to autopho- ~.horylate, indicating that the SERK protein is able to
fulfill a role as a protein kinase in vivo (Heldin 1995).
C~,r~:ssiG" of the SERK gene cG--~;ponJs with the first ~pez.~.lce of CG~ t~.~l cells
during hypocotyl activation
When carrot hypocotyls are induced with 2,4-D, only the cells of the provascular tissue
proliferate. Cells of epidemmal and cortical origin merely expand suggesffng that the provascular
tissue derived cells fomm the newly initiated suspension culture. After removal of 2 4-D, the
fo""dlion of SGIlldlic embryos occurs after 2-3 weeks. Somaffc embryos are prt:cedPd by
embryogenic cells that are dcveloped in tum from co.ll~ctenl cells. While col..~.ete~lt and
embryogenic cell fo""alion take place in the p,.,sence of 2,4-D, it was not clear when this
occurred and which cells acquired co",petence. Since previous e;~t:,i",ents (Toonen et al.
1994) revealed that cell ll,o"ul,r~yy is not a good criterion, the first appeard,)ce of single
competent cells was detemmined e~er""erltdlly by semi-automaffc cell hdcl~illg pe,f~""ed on
large pop~ tions of immobilized cells. Hypocotyl e.~l,hnt~ activated with 2,4-D for seven days
were mecl,an ~lly f-dy",ent~d and samples of the resulffng popu'~ion of mainly single
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-21-
suspension cells were immobilized to aliow recording of their dcv~lopment by cell tracking. In the
immobiiized cell pop~tions obtained in this way all the morphologically discemible cell types
were present that were also seen in the un-l,ay",enled activated hypocotyls. Rec~l ce the
different cell ~ vh~cgies observed during hypocotyl activation were known (Guzzo et al.
1995), it was possible to trace back the original posibon of each type of cell in the activated
explant. Small cytoplas",-rich cells (16x16 mm) are the proliferating cells that surround the
vascular ele",erlt~. Enlarging v~cuol~ted cells (16x40 mm) are encountered on the surface of
the mass of prolHerating cells and these can detach from the surface when fully enlarged (35x90
mm). Large v~cuoQted cells (more than 60x140 mm) are the non-proliferating ~"",ar,ls of the
hypocotyl epidemmis and cortical parenchyma. The shape of the enlarging and fully enhr~Jed
cells could change from oval to elongate or triangular. Cell tracking on a total of 24 722 cells
,~le~P from seven days activated hypocotyls showed that only 20 single cells fommed a
somatic embryo. Bec~se of their dependance on continued 24-D treatment the embryo-
fomming single cells are still in the competent cell stage. All of the embryo-fomming single cells
belonyecl to the category of 3 511 enlarged cells that contained therefore competent cells in a
frequency of 0.56%. The single cell tracking ex~.e,i",ent~ clea~y reveal that the ability of explant
cells to reinitiate cell division under the influence of 2 4-D resulting in a popu ation of highly
cytuplasn,ic and rapidly proliferating cells does have a causal relation with the ability to become
embryogenic. It is also clear that only a very limited number of the cells that make up the newly
initiated embryogenic suspension culture are actually competent to forrn embryogenic cells.
E3 ~J,ession of the SERK gene detemmined by whole mount in situ h~,L,ridi~lion on a similar
population of cells as used for the cell tldcl~illg experi",ent~ was found to be ~e:S~ ed to only
0.44% of the enla.yed cells. There~urt:, the ex~rt:ss,on of the SEP~K gene appears closely
cc",elalt:d both qualitatively and quantitatively with the p,t:sence of col-")ete.)l single cells.
To obtain insight into the teillpoldl reg~l~tion of SERK ex~ression in the course of explant
activation whole mount 'n s~u hybridization was pe.fu....ed on enbre intact or hand-sectioned
eA~la"t~ treated for different periods with 2 4-D. n_"r~sen~ti.re samples were ~ E~ted at daily
intervals from eA~Jlants u, ~ ated and treated for three days six days, seven days or ten days
with 2,4-D before retuming to B5~. No SERK-ex~ ssi"g cells were ever found in e~larlts
treated for less then three days with 2,4-D. While enla.yed cells became present after the first
five days of culture the first few SERK-eA~rt:ss,.,y enldryecJ cells were found after six-seven
days of culture in the p-t,sence of 2 4-D l,.adt~-,enl. These few cells were present at the surface
-- .. .. . . .
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of the mass of pr~';.erdli"g cells originating from the provascular tissue. In the hypocotyls treated
for ten days with 2.4-D the number of SERK-positive cells had i".;,~ased to 3.04% and included
at this stage also cells present in small clusters. No SERK t~ans~ t was ever cJet~ct~d in small
cytoplas" ,-rich cells or large v~cuol-'ed cells. Hypocotyls were also treated for only one day with
2 4-D and s~ Ihse~uently cultured in ho" "one-free medium for a total of seven or ten days. Under
these conditions explant cells proliferated and gave rise to roots and non-embryogenic cell
cultures while SERK e~ r~ssion could never be clel~d The in situ hy~"idi~lion results
des~,iL,ed above were obldi"ed from a relatively small number of ex~la"t~ and a few hundred
cells so RT-PCR followed by Southern hyb,idi~tion was pe~i~"",ed to obtain more quar,lit~iJc
results. These are shown in Figure 7 and confimm the close l~ oldl co"~lation between the first
a~eardnce of competent cells in e~,ta"l; treated for three days with 2 4-D and the eA~ sa;on
of the SERK gene. Northem hyL"idi~tion never gave any signal after hyL,ricli~tion with SERK
cDNA probes, not even after prolonged exposure in a Phosphorlmager in line with the
extremely re~ ;ted ex~.ression pattem of the SERK gene.
~.ress,~n of the SERK gene cGI~es~Gl~Js with the occumnce of CGI~I~t~.~t cells in
established embryogenic cell cu~tures
While the results described so far indicate that co",pelent and embryogenic cell fG""alion is
le:slli~1ed to a particular class of enlarged cells during explant activation ~he situation in an
established embryogenic cell culture is more complex. Colll~ etcnt single cells in such cultures
do not appear to belong to one cell type in particular but have been shown to originate from all
morphologically different cell types. In cell tracking experi",ent~ embryogenic cells that do not
require exogenous auxin l,t:al,.,enl were never observed to be single but consiilad of clusters
of at least 34 cells (Toonen et al. 1994). SERK e)~ s:,ion was found in all ,,,o,~uh-'ogically
discemible single cell types that were present in an embryogenic cell culture at a frequency
between 0.1 and 0.5% depending on the cell type. In non-embryogenic cultures SERK
e3~ s~i.,g cells were never encountered. As was observed in the activated e~Jh.lt~, SERK
ex~ression was not It:atli-,ted to single cells, but also occurred in small clusters of 2 to 16 cells.
Since clusters of this size are known to consist of embryogenic cells, these data show that
SEP~K e,~ ss;on is not l~tli~ed to co""~elent single cells but may persist in small clusters of
embryogenic cells. No SERK ~ ,r~:,sion was encountered during the late globular, heart and
to" ed~t-ges of somatic embryogenesis.
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The SERK gene is t.~,.si~,ltly eA~,-essed in zygotic embryoge.)esis
The eApre,s,ion of the SERK gene in carrot plants was detemnined by RT-PCR. The results
indicate that no SERK mRNA accumulates in any of the adult plant organs nor in flowers prior to
pollination. The first oc~Acion when SERK eA~ression can be d~t~ . ~d is in flowers at three days
after pollination (DAP), at which stage fertilization has taken place and endosperm develo~," ,ent
has co"""enced. SERK mRNA remains present in flowers up to twenty DAP CO"t,S~ onding
with the early globular stage of the zygotic embryo (Yeung et al. 1996~. Whole mount in sitv
hybridi~ation on partially ~;~e~;ted carrot seeds cc"fi""ed that the SERK gene was only
eA~ ssed in early embryos up to the globular stage. ~ulêssion was observed in the entire
embryo including the sus~ensor. No eA~,ress;ol- was seen in seedlings roots stems leaves
dev~hr: lg and mature flower organs pollen grains and stigma's before and after fertilization.
rlssues in the dcv~leF: Ig seed such as seed coat integuments all embryo sac constituents
before fertilization as well as the endosperm at all stages of dc~/_lop",e"l inves~i-3~Qd did not
show any SERK eAurt:ssion. Later stages of carrot zygotic embryos were also cGIll~!ut~.ly devoid
of SERK mRNA. Given this pattem of eA~,ression that is re~l,i.~ed to the zygotic embryo, the
signal as d~le.1ed by RT-PCR in flowers at 3 and 7 DAP must come from SERK mRNA as
present in zygotes because in carrot the zygote It~ ls undivided up to one week after
pollination (Yeung et al. 1996). Although SERK eA~ ssion persists to slightly later stages in
zygotic globular embryos when compared to the somatic ones these results confimm the
l,dns,anl pattern of expression as observed for the SERK gene during somatic embryugene~
and also imply that there is a correspondence between the fo""ation of competent cells in vitro
and the fo""ation of the zygote in vivo.
METHODS
Cell culture, hypocotyl explant induction and cell llacki~ly
Cell cultures were derived from Daucus car~ta cv. FlaWcese and maintained as previously
described (De Vries et al. 1 988a). Cell su:" ension cultures were maintained at high cell density
in B5 medium (Gamborg et al. 1968) s~ leh,erlted with 2 mM 2 4-D (B5-2 medium). Embryo
cultures with globular heart and torpedo-stage somatic embryos were derived from <30 mm
sieved cell cultures cultured at low cell density (100 000 cells / ml) in B5 medium without 2,4-D
(B5-0). For hypocotyl explant induction e~eff",-:nt~ phr,tle~ were obtained from seed of
, .
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- 24 -
Daucus car~ta cv. S Valery as cles~ ed previously (Guzo et al., 1994). The hypocotyls of one
week old plantlets were divided in seylllellLa of 3-5 mm inc~hA~ed for various periods of time in
B5-2 medium and retumed to B5-0 medium. Seven days after ex~lantdIion and exposure to 2,4-
D the hypocotyl seylll~lltà were Ildy,l,er,l~d on a 170 mm sieve and the resulting cells ~ "e~ted
to fomm a fine cell suspens;on. Immobilization of these cells in B5~.2 medium was pelfu,,,,ed in
a thin layer of phytagel (Toonen et al. 1994). After one week of further culture 24-D was
removed by washing the plates with B5~ medium. This allowed embryos to develop beyond the
globular stage. Recording the dovelou",ent of the immobilized cells was pe,~t,""ecl with a
procedure modified from the previously described by Toonen et al. (1994). The main change
involved a new MicroScan program for automatic 3-axis movement to scan all cells in the
phytagel (Toonen et al. 1996).
Nucleic acid isol~iG,- and ~
RNA was isolated from cultured cells and plant tissues as described by De Vries et al. (1988b).
Poly(A)~-RNA was obtained by pu,ification by oligo (dT) cellulose (Biolabs). For RNA gel blot
analysis samples of 10 mg total RNA were elecl,uphGresed on fo,lllall. ~8 gel, and l,~"a~e"~d to
nytran-plus membranes. For RNA spot-blot analysis 5 mg of total RNA was denatured and
spotted onto nytran-plus filters using a hybridot manifold (BRL).
Genomic DNA was isolated according to Sterk et al. (1991). Samples of 10 mg genomic DNA
were digested with d~ren~ ~sl,i~;tion enzymes and separated on agarose gel and l,~naIt u~d
to nytran-plus ",en,~,dne (Schleicher & Schuell). Hybri-Ji~tion of RNA blots took place at 42~C
in hybr,di~tion buffer containing 50% formamide, 6xSSC, 5xDel-l-al-Jl 0.5% SDS and 0.1
mglml salm sperrn DNA. HyL.r,Ji~d~ion of DNA blots was pe,Iol",ed as previously desc,ibed
(Sterk et al. 1991). F~"~wi.lg hybriJi~lion filters were washed under sl,i"gerit conditions (3x20
min in 0.1% SSC 1% SDS at 65~C). Filters were e~ll-osed to Kodak X~mat AR film. The
integrity and the amount of RNA on the blots was co"~i""ed by hyb,iJi~a~.on with an 18S
,il,oso",al RNA probe. Nucleotide sequence analysis was pe,k"",ed on an ABI 373A au~c",~tt,d
DNA se~uencer (Applied Biosystem).
Sc~ening ~ du~s
Two i,.dependehl cDNA libraries were constructed with equal amounts of poly~A)~-RNA from
total established cell cultures grown for six days in B5-2 medium sieved <125 mm cell cultures
grown for six days in B5~ medium and sieved ~30 mm cell cultures grown for six days in B5
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- 25 -
medium. cDNA synthesis and cloning into the Uni-ZAPTM XR vector was performed according to
the manufacturers protocol (Stratagene).
D~ler~-ltial screening of the cDNA libraries was p~-fo",-ed essential.~r as clesc-il,6d by Scott et
al. (1991). RNA was isolated from either three embryogenic or three non-embryogenic cell
cultures that were grown for seven days in B5-2 after sieving through 30 mm mesh. hrst strand
cDNA synthesis was pell~"lled on 4 mg total RNA using AMV reverse trans~;,i,ulase (Gibco
BRL). [32P}dATP labeled probes were pr~:pared using random prime labeling on first strand
cDNA. Pooled probes from embryogenic and non-embryogenic cell pop~ tions were hyl,ridi~ed
to two pairs of nitrocellulose filters each containing 1000 p'-ques from one cDNA library. After
washing for 3x20 min in 0.1% SSC, 1% SDS at 65~C hybriJi~ation was visu~ d by
autoradiography for two days on Kodak X-omatic film. Plaques that only showed signal with the
embryogenic transcript probe were purified by two further rounds of screening.
In order to identify cDNA clones which are e~,essed at low levels in the c30 mm sieved cell
pop~'otion, cold plaque screening was performed as described by Hodge et al. (1992). Plaques
from the diller~"lial screening that did not show any signal after seven days of autoradiography
were purified by two further rounds of screening. The resulting clones were used as probes for
characterization of the ex~,r~ssion pattern of the corresponding genes.
D~ lial Display RT-PCR
C:'~erential display of mRNA was pe,l~""ed esse,ltially as described by Liang and Pardee
(1992). cDNA synthesis took place by annealing 1 mg of total RNA in 10 ml buffer containing
200 mM KCI, 10 mM Tris-HCI (pH 8.3) and 1 mM EDTA with 100 ng of one ot the following
anchor pr""eia. (5'~ &C-3') (5'-11111111111~ 3') (5'-l l l l l l l l l l l~A-3').
Annealing took place by heating the mix for 3 min. at 83~C followed by in~ h~tion for 30 min at
42~C. Annealing was ~. ~ued by the aJdi~ion of 15 ml pre-wammed cDNA buffer containing 16
mM MgCI2, 24 mM Tris-HCI (pH 8.3), 8 mM DTT, 400 mM dNrP, and 4 Units AMV reverse
h~ns~ Jt~se (Gibco BRL). cDNA synthesis took place at 42~C for 90 min. hrst sband cDNA was
phen~ ;hlo,uphG,,,, extracted and ple~ ecl with ethanol using glycogen as a carrier. The
PCR reaction was pe,lu,,,)ed in a reaction volume of 20 ml containing 10% of the synthesized
cDNA 100 ng of anchor primer, 20 ng of one of the following 10-mer prl",e,a. (5'-
GGGATCTAAG-3'), (5'-TCAGCACAGG-3'), (5'-GACATCGTCG3'), (5'-CCCTACTGGT-3'), (5'-
ACACGTGGTC-3'), (5'-GGTGACTGTC-3'),2 mM dNTP 0.5 UnitTaq enzyme in PCR buffer (10
.
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mM Tris-HCI (pH 9.0), 1.5 mM MgCI2, 50 mM KCI, 0.01% gelatin and 0.1% Triton X100) and 6
nM la-~2P] dATP (Allle~alldl,,)~ PCR pa,d",el~ia were 94~C for 30 sec, 40~C for 1 min, and 72~C
for 30 sec for 40 cycles using a Cetus 9600 (Perkin-Elmer). AmplHied and labeled cDNAs were
separdled on a 6% denaturing DNA sequencing gel. Gels were dried without fixation and bands
were visualized by 16 hours of autoradiography using Kodak X~matic film. Bands containing
dHferentially ex~r~ssed cDNA fragments of 150-450 nu~4Oticles were cut out of the gel and
DNA was extracted from the gel slices by electroelution onto DE~1 paper (VYhdtmdnn). After
washing of the paper in low salt buffer (100 mM LiC12 in 10 mM TE buffer), and elution of the
cDNA in high salt buffer (1 M LiCIz in 10 mM TE buffer with 20% ethanol) the cDNA was
concent,dted by precipitation in ethanol using glycogen as carrier. ReamplHication of the cDNA
~dylllenl~ using the same PCR cycling pa,a",ele,~ as cles~ ed above but PCR buffer
containing 2.5 mM of both the 10-mer and the anchor oligo and 100 mM dNTP. DE~1 paper
allowed an efficient recovery of the DNA fragments and reamplification generated an average of
500 ng DNA after 40 cycles. Amplified PCR products were blunt-ended using the Klenow
Irdylllelll of E.coli DNA Polymerase I (Pharmacia), purHied on Sephacryl-S200 columns
(Pharmacia), ligated into a Smal linearized F''luescript vector ll SK (Stratagene) and
l,dr,~o""ed into E.coli using ele~1,upo,at;ûn.
RT-PCR
Adult plant tissues from Daucus carota were obtained from S8G Seeds (En~huizen). Cont,~
pollination was performed by hand. Flower tissue RNA was obtained from three compete umbels
for each time-point and contained all flower organs including pollen grains. 2 mg of total RNA
from adult plant tissue or cell cultures was annealed at 42~C vnth 50 ng oligo (5'-
TCTTGGACCAGATMTTC-3') in 10 ml annealing buffer (250 mM KCI, 10 mM Tris-HCI pH 8.3,
1 mM EDTA). After 30 min. annealing, 1 unit AMV-reverse l,dns.;,i~.ldse was added in a volume
of 15 ml cDNA buffer (24 mM Tris-HCI pH 8.3, 16 mM MgC12, 8 mM DTT, 0.4 mM dNTP). The
reverse L,d"s.;.i~tion ,ea.,Lion took place for 90 min. at 42~C. PCR amplHicabon of SERK-cDNA
was carried out with two specific oligos for the SERK kinase domain, (5'-
CTCTGATGAC; I I I C~ ;AGT~3') and (5'-AATGGCA I I I GCATGG-3'). Amplification was carried
out with 30 cycles of 30 sec. at 94~C, annealing at 54~C for 30 sec. and e~ter,:,;on at 72~C for 1
min., followed by a final e~-terlsion for 10 min.at 72~C.
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Whole mount in situ hyL.i~ ;G~
Whole mount in situ hyl,rkli~ations were pe-lu""ecl essentially as previously described (Engler et
al. 1994). Cell cultures and somatic embryos were immobilized on poly-L-lysine coated glasses
during fixation to improve handling. Whole mount in situ hybridization on e~lar,t~ took place by
embedding hypocotyls from seven-days old planti -~ in 3% ~~eap'?que agarose (Duchefa) and
p,~cessi"g them in Cppendo,t tubes. Transverse as well as longitudinal sections were made
with a vibrotome (Biorad Microcut). Sections of 50-170 mm thick were inc~ Ih:~tRd in B5-2 medium
for a minimum of three days to induce fo""alion of emb~o-fomming cells. Optimal induction was
achieved with longitudinal hypocotyl se- lions with a thictcness of at least 90 mm. To obtain
proliferating non-embryogenic cell cultures hypocotyl se~;tions were e~l~osed to 2 4-D for only 1
day, and subsequently lldllslerl~d to B5-0 medium (Guzzo et al. 1994). Whole mount in situ
hytJ,i.li~alion on developing seeds was pello""ed by removing the chalazal end of the seeds to
allow easier probe penetration. After hy~,ridi~lion the env~ ~p;.,g layers of integuments and
endosperm were carefully removed to expose the dcv~ ~p:.,g embryos. In situ hyt.,idi~alion on
se~lions was performed as described previously (Sterk et al. 1991) except for the use of non-
radioactive probes.
All samples were fixed for 60 min. ir, PBS containing 70 mM EGTA 4% paraformaldehyde
0.25% glutaraldehyde 0.1% Tween 20 and 10% DMSO. Samples were then washed, treated
with proteinase K for 10 min again washed and fixed a second time. Hybridization solution
consiated of PBS co"~i"ii)g 0.1% Tween 20 330 mM NaCI 50 mg/ml heparin and 50%
deoni~ed fommamide. HyL,ridi~ation took place for 16 hours at 42~C using digoxigenin-labeled
sense or antisense ,il.oprubes (Boet"i"ger Mannheim). After washing the cells were treated with
RNaseA, and inc~h~ted with anti-digoxigenin-alkaline phG:"~ha~se cor, :g~te (Boet"i"yer
Mannheim) which had been pr~absG,L,ed with a plant protein extract. Excess anffbody was
removed by washing followed by rinsing in staining buffer (100 mM Tris-HCI pH 9.~, 100 mM
NaCI 5 mM MgCI2 1 mM levd", s-'~) and the staining reaction was pe.lu,,,,ed for 16 hours in a
buffer containing NBT and BCIP. Observations were pelhJ""ed using a Nikon Optiphot
mic,uscope equipped with Nomarski optics.
AulOpl)Oa~ ilG~lation asaay
A 1.4 kB Sspl cDNA l,dy",en~ of the SERK cDNA encoding most of the open reading frame
apart from the N-temminal three LRRs was cloned into the pGEX e~,t:ss;on vector (Pl,a""ac;a).
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- 28 -
A fusion protein cons-ati,lg of SERK and the glutathione S-t,dn~erdse gene product was
sy"ll,esi~ed by a three hours induction of l-~h~u""ed E.coli with 2 mM IPTG. Fusion protein
was isolated and purified as des.;,il,ed previously (Hom and Walker 1994). Purified fusion
protein was coupled to glutathione agarose beads (Sigma) and inc~ ~h~tsd for 20 min. at 20~C in
a volume of 10 ml buffer 50 mM Hepes (pH 7.6), 10 mM MgCI2, 10 mM MnCI2, 1 mM DTT, 1
mCi [y 32p] (3 000 CUmmol) . Excess label was removed by washing the fusion
protein/glutahone agarose beads three times for 5 min. in 50 mM Tris-HCI (pH 7.3) 10 mM
MgCI2 at 4~C. Protein was removed from the beads by cooking in SDS-PAGE loading buffer.
Equal amounts of protein were sepa,~lecl by SDS-PAGE and protein autophos~horylation was
vicl 1- ' ~ed by autoradiography.
SERK fusion proteins produced with the Baculovirus exl,ression system.
Further fusion pro~3ins containing the intracellular part of the l~ o(ls carota SERK protein
(1.0 kB Hindlll / Sspl fragment of the carrot SERK cDNA clone 31-50) were made using the
baculorvirus vector pAcHLT.
In VitrD phosphorylation studies with this purified protein showed that most if not all of the
autophosphorylation of this SERK fusion protein was at threonine residues (Figure 6)
Construction of viral l,dnsIer vectors
The pAcHLT-B and pAcHLT-C baculovirus transfer vectors were used for the cioning of two
cDNA fragments of the carrot SERK gene. The Sspl 1.41 kB fragment of carrot DcSERK
cDNA was cloned into the Smal site of pAcHLT-B and the Sspl / Pvull 1.07 kB fragment of
carrot DcSERK cDNA was cloned into the Smal site of pAcHLT-C. The first construct
conldins the complete C-terminal part of the DcSERK protein and from the putative
extPcell~ region the proline-rich region and three of the lecuine-rich repeats. The second
construct conldins only the putative intracellular region of the DcSERK gene product.
Nucleotide sequence analysis was pe,(ur",ed in order to confirm the presence and the
G,ienl~liGn of the DcSERK cDNA within the vector.
T.dhs~u,,,,ation of insect cells
The resulting l,dnsfer vectors were used to l-ansfect (lipofect) insect cell culture Sf21 from
S~odoplerd frugiperda in co",~;.)dlion with lineari~ed AcMNPV b~cu'G-inus DNA.
1\1~nolayers of SF21 cells were transfected in 35 mm pe~-id;shes cor,l~ g 2 ml of Hink's
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-29-
medium. One microgram of linearized AcMNPV baculovirus DNA (Baculogold, Invitrogen)
was added to 5 microgram of pAcHLT I SERK vector construct in 25 ")ic,~,liler of water.
Fifteen mi~;r.l;ts~ of Li~ofeclin (BRL) was mixed with 10 microliter of water, after which the
DNA solution was added. After mixing 200 mtcroliter of Hink's medium was added to the mix
and the solution was l,dr,~le"~ecl to the cell monolayer, from which the medium was
removed. After one hour, 500 microliter of Hink's medium was added and the cells were
incubated for anotehr 3 hours. Finally, 1 ml of Hink's medium with 20% foetal bovine serum
(FBS) was added and the cells were incuh~ted for 4 days. After transfection, the viral
infection could be identified by the reduced growth of cells, the swollen shape and the
enlarged nucleus. After four days, infected cells were harvested and the medium containing
infectious budded virus was collected and used for plaque assays and a",plificalion of
recombinant virus stocks.
Iss'-tion of single recombinant viruses
Single recombinant virus plaques were isol~ted from monolayers of cells infected with a
titration range of the primairy virus stock. Infections was performed in 35 mm petridishes
with monolayers of cells. Virus stocks were diluted in 600 microiieter of Graces medium and
added to the cell monolayer, followed by a 90 minutes incubation period at in Graces
medium with 20% FBS. Afterwards, 3% Sea Plaque agarose was autoclaved, mixed with an
equal amount of 2x Graces medium with 20% FBS and from the resulting agarose overlay
solution 2 ml. was spread over the cell monolayers after removal of the viral inoculum. After
4 days of incubation single plaques could be vis~ 7ed and purified for further analysis.
Fusion protein production.
After dehr",i,ling the titer of purified recor"~ ar,l viruses, monolayers of Sf21 cells in 7~
cm2 flasks were infection with a multiplicity of infection (MOI) of 10. Incubation of cells with
the vinus inoculum was performed for 90 min. after which 8 ml. of Hink's medium with
10%FBS was added. After 3 days of inc~h~tion, cells were harvested and washed twice
with PBS. Cells were Iysed for 45 min on ice in twenty volumes of 1 x insect cell Iysis buffer
(10 mM Tris pH 7.5, 130 mM NaCI, 1% Triton, 100 mM NaF, 10 mM NaPi, 10 mM NaPPi,with protei.,ase inhibitors: 16 mgA benzamidine, 10 mg/l phenantl"oline, 10 mg/l aprotinin,
10 mg/l leupeptin, 10 mg/l ~.epsla~in A, 1 mM PMSF).
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The Iysate was cleared by centrifugation at 10.000 9 for 30 min and the supernatant was
bat~ ise inc~h~ted in TALON resin (with high affinity for the 6xHlS tag of the reco",binanl
fusion protein). Binding was pe,~ur,,,ed by gentle ~git~tion for 20 min. at room temp. The
rssin was washed three times with Iysis buffer, followed by an elution step with Iysis buffer
with 200 mM imidazole. Purified fusion protein was ~el eeted and purifty and integrity was
tested by SDS-PAGE.
Autophosphorylation assays
Protein kinase activity was deternined by incubating 1 microgram of purified fusion protein
for 30 min. at room temp. in a buffer containing 10 mM MgCI2 10 mM MnCI2 1 mM DTT
and 10 ~M [gamma-32]ATP (105 pm/pmol ATP). The autophosphorylated fusion proteinwas purified after SDS-PAGE from the gel in a buffer containing 50 mM NH4CO3 0.1%
SDS, 0.25% beta-mercaptoethanol. Protein was prec;~ te~l with 20 ~g/ml BSA and 20%
(w/v) solid l,ichloruacetic acid. The precipitate was collected after centrifugation hydrolysed
in 50 1~l 6N HCI for 1 hour at 120 degrees Celcius. HCI was s~hsequently removed by
Iyophilization and the pellet was resuspended in a buffer consiti"g of 2.2% formic acid and
7.8% acetic acid. Hydrolysed protein was loaded onto cellulose thin layer cl"o",atography
plates together with control amino acid samples (phosphoserine, phosphotl"~onine,
phosphotyrosine). Chromatography was performed in a buffer containing pr.Fionic acid: 1M
ammonium hydroxide: iso~rupyl alcohol (90:35:35 v/v/v). After separation and drying of the
plates the separated amino-acids were visualized by spraying with 0.25% ninhydrin in
aceton followed by heating for 5 min. at 65 degrees Celcius. Plates were afterwards
exrosed to Phospho Imager casettes in order to detect the ~.hos~ ho-labeled aminoacids.
SERKa"t; _: ~s
Purified fusion proteins (10 1~9) were mixed in complete Freund adjuvant and injected IP
into BALBc mice. After 4 weeks booster antigen was i"iec~ed (10 ug purified fusion protein
in i",c~",,,~lete Freund adjuvant). Two weeks later a final boo~l~r was in,r~cted One week
after the final boosler serum was ce"e~tcd from these mice. The sp6cifi~ily and the titer of
the resulting sera was tested on Westem blots using total insect cell extracts with or without
the SERK fusion protei.,s.
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-31-
INTRODUCTION OF THE SERK GENE INTO PLANTA AND THE PRODUCTION OF
APOMICTICSEED
Carrot transformation with a SERK Dromoter fraament/lucileldse aene fusion
The binary vector pMT500 is based on the pBlNt9 vector (Bevan, 1984) and c~ ins the
firefly luciferase gene doJ~r"al,~am of a polylinker containing 5 unique resl.i~:tion sites was
created by uni-di,e.;tional ligation of the firefiy luciferase coding region folloNed by the
polyadenylation sequence from the pea r~cS::E9 gene in the Hindlll-Xbal site of the binary
vector pMOG800 (kindly provided by Mogen N.V. Leiden The Netherlands). The binary
vector pMOG800 is based upon pBlN19 (Bevan 1984) but while in pBlN19 the polylinker is
flanked by the left border and the neomycin phosphol,dr,~lerase (NPT ll) expression
casselle the poiylinker in pMOG800 is flanked by the right border and the NPT llexpression cassette. From a geno,l,ic lambda clone ll~nsc,iplion regulating sequences
from the carrot SERK gene were isolated by digestion with Hindlll and Dral (SEQ ID No. 1),
and cloned into the Hindlll / Smal sites of pBluescript SK+. From the resulting vector a Kpnl
/ Sstl fragment containing the SEF~K genomic DNA was isol~ted and cloned into the Kpnl /
Sstl sites of the binary vector pMT500. The resulting DNA construct, pMT531 contained the
2200 bp genomic SERK DNA fragment as ~ro",ott:r sequence the luciferase gene as vital
reporter and the E9 t~ansc~ tion terminator sequence.
The binary vector pMT531 was transformed by elecl,oporation into Agrobacterium
tumefaciences strains MOG101 and MOG301 (for transformation into carrot cells) and into
Agrobacterium tumefaciences strain C58C1 (for transformation into Arab~dipsis thaliana
plants). Transfonned colonies were selected on LB plates wlth 100mg/l kanamycin.
T,dn~l."",ation of carrot cells
The firefly luciferase coding sequence under control of the geno",ic carrot Hindlll / Dral
2200 bp DNA fragment was introduced into carrot cells by Asi,ubal,teri~lm tumefaciens
mer~iated l,dr,s~ur,,,~lion of hypocotyl segments. T,ansfo""ation of nau~us carota cv.
'Amsterdamse bak' was performed by slicing one week old dark grown seedlings into
segments of 10 to 20 mm. Sey-"ents were inc~h~ted for 20 minutes in a freshly pr~pared
10 fold diluted overnight culture of Agrobacterium.. The segments were dried andtldllsIer,~d to a modified Gamborgs B5 medium (P1 medium; S&G seeds Enkhuizen, The
Netherlands) su~p'e "ented with 2 IJM 2 4-D (P1-2) and solidified with agar (Difco, Detroit
Mi USA ). After two days of culture in the dark at 25 + 0.5 _C seg",snl:, were l,tlnsle,.-~d to
CA 022~4839 l998-ll-l3
W 097143427 PCT~EP97/02443
-32-
sGtidified P1-2 medium supplemented with kanamycin (100 mg ~ ), carbenicillin (500 mg-l~
1; Duchefa) and vancomycin (100 mg-1~1; Duchefa). Atter three weeks segments were
transferred to fresh plates and transfommed calli were selected after an additional three
weeks. Transformed calli were grown on P1-2 plates with antibiotics for 3 weeks at a 16
hour light/8 hour darkness regime. Trdnalull,led embryogenic suspension cultures were
initiated as described by transferring 0.2 g callus to 10 ml liquid P1-2 medium
supplemented with 200 mg-l~1 kanamycin, 250 mg-l~1 carbenicillin and 50 mg-l~1
\,ai1cG",ycin. During the first weeks 1 to 3 volumes of fresh medium were added to the
culture at weekly intervals. After 5 to 7 weeks cultures were s~ Ihcl ~tured to a packed cell
volume of 2 ml per 50 ml medium every two weeks and incuh~t~d at a 16 hour light / 8 hour
darkness regime at 25 + 0.5 ~C.
One week after transfer to kanamycin selection medium, hypocotyl segments were sprayed
with luciferin to test whether luciferase expression could be detecte~l in transformed callus
shortly after tranafor",ation. A large number ot hypocotyl segments sholled luciferase
activity at the cut edges, but did not develop calli. Instead, growth of bacteria occurred,
su~ge~l;ng that the luciferase activity was of bacterial origin. Six to ten weeks after
l,ansfor",ation, calli were obtained that showed luciferase activity in variable amounts, while
no bacterial growth could be observed anymore. After 12 weeks, calli measuring 5 to 10
mm in diameter were used to start suspensi~n cultures. At this time no bacterialconta",i"ation was observed. A control transformation experiment in which luciferase
ex~.ression under influence of the CaMV 35S promoter was observed in single cells and cell
clusters in the suspension culture del"onbl,dli"g that the luciferase protein is active in
Daucus cat~ta suspension cultured cells.
Cell immobilisation
Onc weelc old high-density (1 o6 107 cells-ml~1 ) suspension cultures were sieved through
nylon sieves with successive 300, 125, 50 and 30 ,um pore sizes (Monodur-PES; Versei~ag
Techfab, Walbeck, Germany). Single cells and cell clusters pa55;.~9 the last sieve are
designated as ~ 30 ~m popu'stiQns. Control expe,i",enla with utltlallafolllled cells were
performed with naucus cat~ota cv. 'Trophy' (S&G seeds) suspension cultures grown in P1-2
medium. Size fraL:~ionatetJ cell popu~tiQns smaller then 30 llm were immobi'ised in phytagel
(P81g6; Sigma, St Louis, Mo, USA) in petripemm dishes (Heraeus, Hanau, Germany). The
bottom layer cor,sisled of 1 ml P1-0 medium with 5 mM Ca 2+ and 0.2 % phytagel. Two
CA 022~4839 l998-ll-l3
W 097/43427 PCTAEP97/02443
-33-
hundred thousand cells (~ 30 IJm and c 50 ,um populations) in B5-0 medium without Ca 2+
supplemented with 0.1 % phytagel were poured on top of the bottom layer. For this layer B5
was applied since, at room temperature, phytagel solidified in P1 medium without Ca 2+.
After 2 hours of solidification an additional P1-0 layer with 0.2 % phytagel was poured onto
the cell layer preventing the B5 layer to move. To prevent dehydration of the phytagel layers
and to supply luciferin to the cells, 0.5 ml P1-0 medium containing 0.05 I~M luciferin
(Promega, Madison, Wi, USA) was added after solid;lication. The final luciferin
concentration in the culture was 0.02 ~M. Luciferin detection on single cells was determined
with a CCD camera for a period of 5 times one hour (Schmidt et al. (1997) Development
124:2049-2062). After 7 days of culture, luciferin was removed from the cultures by
extensive washing with P1-0 medium.
ArabidoPsis l.d..~ro. ,..a~ion with a SERK l.ro~ er fra4rne,lUlu~ire-c.se qene fusion
Wildtype WS plants were grown under standard long day cor,Jilions: 16 hours light and 8
hours dark.
The first emerging influorescense was removed in order to increase the
number of influorescenses. Five days later, plants were ready for vacuum infiltration.
Agrobacterium strain C58C1 containing the l-anslur,,,ation plasmid was grown on a LB
plate with 50 mg/l kanamycin, 50 mg/l rifampicin and 25 mg/l gentamycin. A single colony
was used to inoculate 500 ml of LB medium containing 50 mg/l kanamycin, 50 mg/l
rifampicin and 25 mg/l gentamycin. The cultures were grown O/N at 28 degrees Celcius and
the resulting log phase culture (OD600 0.8) was centrifuged to pellet the cells and
resuspended in 150 ml of infiltration medium (0.5x MS medium (pH 5.7) with 5% sucrose
and 10 1~l/l benzyla",inopurine). The i.,llGrescenses of 6 Arabidopsis plants are sub",eryed
in the infiltration suspension while he remaining parts of the plants (which are still potted)
are placed upside down on meshed wire to avoid contact with the i"lill,dtion suspension.
Vacuum is applied to the whole set-up for 10 min. at 50 kPa. Plants are directly al~er~ rJ~
placed under sta.,dard long day cor,ditions. Atter completed seed setting the seeds were
surface sterilized by a 1% sodium hypochlorite soak, then thoroughly washed with sterile
water and plated onto petridishes with 0.5xMS medium and 80 mg/l kanamycin in order to
select for trar,-~;lG,..,ed seeds. After 5 days gemmination under long day conditions (10.000
lux), the t,dr,sl~....ec~ seedlings could be icler,tilied by their green color of their cotyledons
CA 022~4839 1998-11-13
WO 97143427 PCT/EP97/02443
- 34 -
(the untransformed seedlings turn yellow) and were further grown in soil under C1 lab
conditions under long day condilions. This vacuum i"~ ,alion method resulted in
approhi",ately 0.1% transformed seeds.
T,dn~i~u""alion of a construct containing both a gene encoding kanamycin resistance and
the 2200 bp (Hindlll / Dral) SERK genomic DNA fused to the firefly luciferase gene into
Ar~b '~psis thaliana (WS) by vacuum infiltration resulted in six different kanamycin-resistent
primary transforrrlants (I Il Ill IV V and Vl). Plants IV and Vl died at the seedling stage
although they were kanamycin resistant. A T2 generation could be obtained from the four
plants l ll 111 and V (Figure 4). Within the siliques of the T2 generation of plants no. Ill and
V an early inhil,ition in development could be observed in appo~i",alelely 25-50 % of the
seeds. The plants I and ll did not show a reduction in the number of developing
seeds.(Figure 5). Similar results were observed in a T3 generation in which again
approximately 25-50% of the seeds shoY.ed an early inhibition of normal seed dcvelop",ent.
Ar~ Psis transrc" ".dlion with a AtSERK qene
Isolation of the AtSERK genomic and cDNA clones
Using the DcSERK cDNA sequence (seq ID no. 2) as a probe a lambda ZipLox genomiclibrary made form Ar~ 1cp s s Landsberg erecta total genomic DNA is screened for the
presence of homologous sequences. Three different lambda clones with inserts of 14 18
and 20 kb respectively are obtained. The 14 kb clone is digested by EcoRI and the resulting
fragments subcloned into pPlLIescript vectors. Fragments spann;ng the entire coding
sequence of the AtSERK gene are jSQI~terI~ sequenced and colllpared with the Daucus
home'cgues. The resulting sequence is shown as SEQ ID NO: 20.
Using the DcSERK cDNA sequence (SEQ ID NO: 2) as a probe a lambda ZAPII cDNA
library is screened for the presence of holllQ'cgous sequences. Four lambda clones are
obtained and their inserts subcloned into pe'uescript vectors using the helper phage
e,c~;;,ion procedure. Fragments spanning the entire AtSERK cDNA coding sequence of the
AtSERK gene are iso!ate~l sequenced and compared with the Daucus hG",c'~gues. The
resulting sequence is shown as SEQ ID NO: 32.
CA 02254839 1998-11-13
WO 97/43427 PCT~EP97/02443 -35-
rlas",ids containing promoter sequences
Ar~b ..'opcis thaliana LTP1 promoterfragment is obtdi"ed from the binary plasmid pUH1000
(Thoma, S., Hecht, U., Kipper, A., Borella, J., De Vries, S.C., Sommerville, C. (1994) Plant
Physiol. t05, 35-45) by dige-~lion with BamH1 and Hindlll and cloning into pBluescript SK-
(pMT1 21 ).
- The CaMV 35S promoter enhanced by duplication of the -343 to -90 region (Kay et al.,
(1987) Science 236: 1299-1302) is isol ted from the pMON999 vector by digestion with
Hindlll and Sstl and cloned into the p''luescript SK- vector (pMTt20).
- The promoter AtDMC1 (Klimyuk and Jones (1997) Plant Journal 11: 1-14).
Plasmid SLJ 9691 is a construct consisting of pBluescript SK+ in which the Arah~;~op~~s
thaliana DMC1 genomic clone (accession number U76670) is cloned into the EcoRV site.
SW 9691 carries EcoRV fragments of the 5' end of the AtDMC1 gene with the following
modific~tion: a Bglll site instead of the second Hpal site, two ATG codons in the first exon
and an Xhol site at the ATG codon of the second exon.
- The FBP7 pru",oter from Petunia (Angenent et al. (1995) Plant Cell 7: 1569-1582).
The promoter of the FBP7 gene is cloned by subcloning the 0.6 kb Hindlll - Xbal genomic
DNA fragment of FBP7 into the Hindlll - Xbal site of pBluescript KS-, resulting in the vector
FBP201 .
The pAtSERK binary vector constructs.
Based on the pBlN 19 vector, a binary vector pAtSERK is constructed for l,c~nsfo,,,,ation of
the Ara~idopsis thaliana SERK cDNA under the control of different pru",ote-~.
The full length Ar~ls~;c~p-;s thaliana cDNA clone of SERK (Seq ID No. NEW) is obtained
from a pRluescript SK- plasmid. A Smal - Kpnl 2.1 kb Il~y..,ent containing the AtSERK
cDNA is cloned into pBlN19 Smal - Kpnl. The polyadenylation sequence from the pea
rbcS::E9 gene (Millar et al., 1992), Plant Cell 4: 1075-1087) is placed dow..~ am from the
AtSERK cDNA by cloning a Kleno~/ f.~ed EcoRI - Hindlll E9 DNA fragment into the Klenow-
filled Xmal site of the p~lN1 9:AtSERK vector in order to gener~te the binary vector
pAtSERK.
CA 022~4839 1998-11-13
WO 97/43427 PCT/I~:P97102443
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Construction of plant e~,r~ss,on vectors
The pAtSERK binary vector is used to generate the following prc,l"oter-AtSERK constructs.
- The AtLTP1 pru,,,oler is cloned in the Smal site of the pAtSERK binary vector as a
Klenow-filled Kpnl-Sstl DNA fragment to give the pAtLTP1AtSERK vector.
- The CaMV 35S promoter is cloned in the Smal site of the pAtSERK binary vector as a
Klenow-filled Kpnl-Ss~l fragment to give the p35SAtSERK vector.
- The AtDMC1 promoter consisling of the Bglll - Xhol 3.3kB fragment from the clone SLJ
9691 is filled in with Klenow and cloned into the Smal site of the pAtSERK binary vector to
give the pAtDMC1AtSERK vector.
- A Sacl-Kpnl fragment of FBP2101 is filled in with Klenow and cloned into the Smal site of
the pAtSERK binary vector to give the pFBP2101AtSERK vector.
Introduction of plant expression vectors into Ar3h;~0psis thaliana plant cells
The above described vector constructs (pAtLTP1AtSERK p35SAtSERK pAtDMC1AtSERK
pFBP2101AtSERK) have been electrotransformed into Agrobacterium tumifacienses strain
C58C1 as known in the art.
Wild type Ar7~1;L~ S thaliana WS plants are grown under standard long day conditions: 16
hours light and 8 hours dark.
The first emerging inflorescence is removed in order to increase the
number of influorescences. Five days later plants are ready for vacuum i"filtlation.
Agrobacterium strain C58C1 contai,-i.,y the tran~ur,,,alion plas",id (the pAtLTPtAtSERK
vector or the p35SAtSERK or the pAtDMC1 AtSERK vector or the pFBP2101 AtSERK
vector) is grown on a LB plate with 50 mg/l kanamycin, 50 mg/l rifampicin and 25 mg/l
ger,l~",ycin. A single colony is used to inocu'~t.s 500 ml of LB medium containing 50 mgA
kanamycin, 50 mg/l li~dlllp.Ci~l and 25 mg/l genta",ycin. The cultures are grown O/N at 28
degrees Celsius and the resulting log phase culture (OD600 0.8) is centrifuged to pellet the
cells and resusl)6"J6d in 150 ml of infiltration medium (0.5x MS medium (pH 5.7) with 5%
sucrose and 1 mg/l benzylaminopurine). The i,lflGresceoces of 6 Arabidopsis plants are
submerged in the infiltration suspension while the remaining parts ot the plants (which are
still potted) are placed upside down on meshed wire to avoid contact with the infilbdtion
sus~,ension.
CA 02254839 1998-11-13
W O 97/43427 PCT/EP97/02443
-37-
Vacuum is applied to the whole set-up for 10 min. at 50 kPa. Plants are directly afterwards
placed under standard long day condilions. After completed seed setting the seeds are
surface sterilized by a 1% sodium hypochlo,ile soak then thoroughly ished with sterile
water and plated onto pet.idi~hes with 0.5xMS medium and 80 mg/l kanamycin in order to
select for l,dnalul,,,ed seeds. After 5 days germination under long day conditions (10.000
lux), the transforrned seedlings could be itJenlified by their green colour of their cotyledons
(the untransformed seedlings turn yellow) and are further grown in soil under long day
conditions. This vacuum infiltration method resulted in apprûxi,nately 0.1% trar,slor",ed
seeds.
E)~ression of SERK sequences in Ar~' 'cpsis thaliana plant cells
The inflorescences from transgenic and not transgenic Arabidopsis thaliana plants are
analysed by Whole mount ~n sifL hybridisation analysis with AtSERK cDNA as probe. The
i"llor~:scences in different stages of development are fixed for 60 min. in PBS containing 70
mM EGTA 4% pa,~fo""aldehyde, 0.25% glutaraldehyde 0.1% Tween 20, and 10% DMSO.
Samples are then washed treated with p,u~ei.,ase K for 10 min again v:~ ~hed and fixed a
second time. Hybridi~,ation solution cons;sled of PBS containing 0.1% Tween 20 330 mM NaCI
50 mg/ml heparin, and 50% deioni~ecl fommamide. HyL,ridi~alion took place for 16 hours at 42~C
using digoxigenin-labeled sense or antisense ,iboprubes (Boehnnger Mannheim). After washing,
the cells are treated with RNaseA and inc~lh~ted with anti-digoxigenin-alkaline phos~hatase
con ug~te (Boehringer Mannheim) which had been preabsorbed with a plant protein extract.
Excess antibody is removed by washing f~' ~w~ d by rinsing in staining buffer (100 mM Tris-HCI
pH 9.5, 100 mM NaCI, 5 mM MgCI2 1 mM le~,a",: e 'e) and the staining reaction is pe-Ium.ed for
16 hours in a buffer containing NBT and BCIP. Observations are performed using a Nikon
Optiphot m,~;,uscope equipped with Nomarski optics.
The l,dn:,lGr."ed plants show ectopic expression of SERK in the vicinity of the emb~yo sac.
... , . . ~ . , ~,
CA 02254839 lsss-ll-l3
W O 97/43427 PCTAEP97/02443
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.~FQr~r~ LISTING
(1) GENERAL INFORMATIoN:
(i) APPLICANT:
(A) NAME: NCVARTIS AG
(B) STREET: S~A~ l ee 215
(C) CITY: Basel
(E) COUNTRY: Switzerland
(F) POSTAL CODE ~ZIP): 4058
(G) TELEPHCNE: +41 61 69 11 11
(H) TELEFAX: + 41 61 696 79 76
(I) TELEX: 962 991
(ii) TITLE OF ~ N11UN: h~ v~ s in or r~lAtin~ to organic
c~ounds
(iii) NUMBER OF ~ : 33
(iv) COeEUrEa READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) CCMPUTER: IBM PC c~m~At;hle
(C) OPERATIN~ SYSTEM: PC-DOS~MS-DOS
(D) SOFTW~RE: PatentIn Rel~Ace #1.0, Version #1.25 (EPO)
(2) INFORMATICN FOR SEQ ID NO: 1:
C~RaCTER~ CS:
(A) LENGTH: 6695 base p2irs
(B) TYPE: nucleic acid
(C) STRA~ : double
(D) TOPOLDGY: unknown
CA 02254839 l998-ll-l3
W O97143427 PCT/EP97/02443
-39-
(ii) ~nT-T~rTTT-T~' TYPE: ~NA (genomic)
(iii) ANTI-SENSE: NO
~vi) ORIGIN~L SOUR OE:
(A) ORGANISM: Daucus c~ota
~ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATloN: 3696..6617
(ix) FEATURE:
(A) NAME/K~Y: intron
(B) LOCATIoN: 3731..3802
( iX ) ~ ~;A~URE:
(A) NAME/KEY: intron
(B) LOCATICN: 3851..3979
(ix) FEATURE:
(A) NAME/KEY: intron
(B) LOCATICN: 4124..4211
(ix) E~E:
(A) NWME/XEY: intron
(B) LOCATIoN: 4284..4357
(ix) EEWRE:
~A) NAME/KEY: intron
(B) LOCA51oN: 4430..4528
(ix) FEATURE:
(A) NAME/KEY: intran
(B) LOCATION: 4642..4757
CA 02254839 l998-ll-l3
W O 97/43427 PCTAEP97/02443
-40-
(~x) ~3A~E:
~A) N~ME/KEY: intron
(B) LOCATIoN: 4890..4967
(ix) ~E:
(A) N~ME/KEY: intron
(B) LOCATIoN: 5295..5803
(ix) FEATURE:
(A) N~ME/KEY: intron
(B) LOCATIoN: 6197..6339
TCTAGATGAC GAAATCGCGC TAC ~ ~AT TTNGAAATAC TA~ AG TATCTTGATT 60
A~-llll~ll~G ATAl~-ll~l GTAATTTCIT TAG&AGAT~C AAAL~b-l~-ll CATr~AATAT 120
GAGCC~-ll~-l GP~lTt3~aAA AAGTATCTAG CAl~-rll~AT CAoGA3GTAG CTAAAAAGTA 180
~C~1~1-1-1~A TTA~GCACAT AATATTGTAT l~-lATT GGCTATCAAT GaAGTTDGAT 240
GC'AAGTATAT A~-ll~-lATT AT~CATGTGA T&AGGGTATA TAAAAEAALT AAAGAACATT 300
~l~l~lAGc ATrCAT m T ~l~ll~C~lA TAGTTAA~GA ~l-lrl~l~AC ACATCACGIT 360
GAA~CTGGAT ~'l~'L~'l~'l-l~' TTCCA~CTAA ~-l-rlw ATTA CCT&ATAG~T GCT Q A~TTC 420
rl~-l~AGCC rl-ll~lll~ GArllll~ AAGACAAGAT TCTTTAGTTA ATAGTTATTG 480
w ~l~l w C rl~-l~-l~AT m AGGAATC TTAL-~ ~-l-l TTTTAAT~GA GAAACGAAAC 540
CTAC~lllll rl~l~l~ll~ ~-lrllATGA TATCAOCTGC Ile~GGCGT TTAG'AfITTA 600
TCCACCTAAA CTATTCATGT TTAOa~aACA AGCTATACGT ~l-l-L~ jj 660
AC~1~N~AC AAAAGAAG_G CTGATC~ACT &~TTTAATCC ~l~l-lllATT ATATTACACA 720
CA 02254839 1998-11-13
W O 97/43427 PCTAEP97/02443
-41-
TTGAT ~ AT~GAGCTAA TAl~l~ -l TAAATTTCAT GTATATATAT ACC~ll~l 780
TGG CA~l~ lAATT AGCGTACITA ATTATCT&AT GGATACTGTA 840
~ ~LAG ATGAT~-l~AT CAGATTATAC CAl-ll~-ll~-l GCICTACAAA ATAP;AA2CC 900
TCTATTTATG TTCATCTTTT T>A~CAAG TAUC~AATTG Al~C~-lATG TIaA~AGGCG 960
AT~CATTACA CAACTTACGA ACTAGCTTGC AA~ATCCCAA CAAlw ~1~ CAGAaCTCGG 1020
ATCC~ACCCT TGTGoACCCT TGCACATGGT TTCAT&TGAC ATGTAA~AAT GAAAA~AGTG 1080
TTATAA~AGT GTALGTCACT l~llATTA A'lll-ll-llAG CAA~TTAC~A ATA m ACTC 1140
AAT~aAGCAG Al~-l~-l~lll AAATATTTTT ~ lAATTTC TTAGCTAAGC GGAGCATCTA 1200
TCTTAA~TAT CICTACTGAA TTTAACACAT AATACATTTT TTTAAAAAAT CTATTAGAGT 1260
~l-l-l-l-l-l~O~ CACAGCGCAC ATATATCTTT ll-l~-l~-lAA TICAGA~AAC ~lll~l~i 1320
AC~ATAAAAT AATATAAGAT TA~Ll~-ll~ AACTAATTTT TTAlllll~l 'l-l-l~'l'l-l'l-l'A 1380
'l~'1l~l-l-l~ A~AAAGTITC TTAlWl~l-l TIGTGAAAAG TACATTCTAT GATAATTTTT 1440
TGGCAACTCA TATAAATTTA TATATATTCC ATGTAGTTAT AA~TTAAAAA AA~ll~-lA 1500
TTMT~AA GATAGAGGTT CATTTTTATA b-l-l-~ AT OCATGAGT'TT TTGAAAATGT 1560
CAGAAATTTT GllGAaTTM TITTACTTAC CAaCTlTTAT ~-l~AT&C AGrGATCTlG 1620
G~.aADGCAGC ATTATCTGGT CAA~ A GIIGAAAA~T TTACAATACT 1680
T&TMGACCA TATCACTTGG Ml~ l-ll~G TTTTTATACA GCACAAT&CT TTCMTATCT 1740
GIIAAAAGTG TGAAAAUETT GA~-lll~-l~G CTTCAGCAGT ~l~-l~ATA ATATCTAT&A 1800
CA 02254839 1998-11-13
W O 97/43427 PCTAEP97/02443
-42-
AGCACITAAA A~GL1~G~A A1~ 1~1 TATTATTTCA AATATTGITA ATIGTTACTA1860
C11AATATVA TAAACIGATT TAAL1~1~A TG~ 1~1 CAG~CC~A~G 1~G~L1~ATT1920
ALTCAC~IWa TAAAATTG3N GJ~ll~ACA AATATAACTT ~1111~11AA GGICC~GAAA 1980
GAGCACITAT CAAC~'1'1~1~ TAGCGCATA~ CGnCA~A3~3 GGTCAGTCAC GGGCTATCCA 2040
~-111~GGAG ~'1-1-11'AATGA GCAflTA m A~11~1~11 TTAAACGTCT GAGGAT~TTA 2100
TTAAAGTCIa CATCATTCAG AGITTAAATT AGCACrTICA GITGTATTAT GAAT~TACA 2160
T&~AAGATAC ATATCTTAAT ~-11~1AT&C ~'1~11'1~AAC A1~1~1~1AA TA11~-1~-11A 2220
~1~111~1~AT CTTA~AAATG GCACT~uATTA AAAT~TGAGA AAQGTAGTCT TCCAATACCA 2280
TTTC~TGTAT ALCAGAGAAT ATCATAA m TTTTAAATCA TAA~ C CCTAGAÇTTT 2340
TCTCAGTATT ~l'LlA m A TAl'l'l'l'L~AC CA m AGAAC '1~1~1'1~1~A GATGAAAATC 2400
TTGGA~-11~ ACPGAAGATC TTATAGTAAA AGTATrCTTT AGATC'1~AT& ATGA~5rrG 2460
TCA1~b-1~-1~ GcL'l~'l~'A GAA m AAAT CAATC~CATG TCACATGTTT GTTGATCTGA 2520
CTACTCACrG TTAATC~AG AGTAACTATT TGTGAATTAA A'1~'1'11'1'1'1''1-1'1'1~'1-1'L'1'1' 2580
CAT~CTTAGC GTTATAA-AQG ~1~-1ACGTCTC ACTAT~TTT TTAACATGTT ATAb-111-~-1 2640
ACTGAC~AGr TTAAAG m C '1~11~111AC GAATTAAGAA TATATAATAT AAaA~GCTTr 2700
AAL-~-1-1.-~-1 GTGGAAG5rG TTCTTACCTT TTIA~ATATA TATATAGATA CTCa3ACTCr 2760
bIL~ AATT ATATCTTACG AACTTACGAG TATACAGAAC TT~-lATATTA GGTTCAGAT& 2820
A~1W L~ A GTAGAACACC TTAAGC~AGA ACTTAA~rCAT GA~ AA C1L~ 1-~AACT 2880
~ 1-1-1-11AG A1-1-11-1-1~A GTTTAT~GAA AA5TGTAC?CT CAT&AIOGTG ~'1'1'1~'111-~' 2940
CA 02254839 l998-ll-l3
W O97/43427 PCT~EPg7/02443
-43-
ATAAACTTTC C'ATATAA~TC C~111~11~A C~ AT& TA~-l~-l-l~ ALGaGn;ATT 3000
ATTAGCGGIT C~TTCAATAA TCATAAT~TG TCTCAC5TTG ATGaGGCCIa TAC'TTATTAT 3060
TGCA~CTTGC A~IIAACCTT GATCCTCAT& TC'A~-ll~T TGTCATA~TC TACI~ACOGa 3120
GTTGAACATG GTTTATCAT& l~l-l-l-l~AGG TAACAAT&TA G~ l~A~CT ~'l~'l~'l-l~A 3180
TATAG511TA AGG~-l-l~AC C~CACTAG C~-ll-l~-ll~ TTTTATTCAC AGTICACACA 3240
C~TACTAG&A ~ ~A~CT CTA~l~l-l-ll ~l~AAAT AGTA~GAAGT 'l-l~'l'l'l~'A 3300
TAATAGTGGA T&ATCATTTA A&AAATAGTG AATCAAATTA l~-l~-l-lATT ~ AC 3360
TTIGGAATTA AATGAGTraC T&AACATT~T l~l~ll-lAT C~-ll~-l~AAG G~ CCAA 3420
GGAAGaoGAT TAGTAAGAGT GGGCATCC'AA GC~-lll~T CTTGAAGGGG C~ ~C 3480
~-ll~lWATT ~ l~l~l ATTAGAGGAC ATTATCTATA TATACTGATT ATTTATTAGA 3540
ATATAAATCA ACTACTATAT l-l-l-l~l-l-l~l AAT~TTTATA TAGAAATClCC AC~CGTAAAC 3600
TTCACAAATA C~ATTGAAAT ATrTGAACC'T AATTAATTA& TAGn~lrAGG TTTAAATrCA 3660
AACTCATTTA ATTTTAC'TTT AAAAAATAAT TCTATATvAA T~ AACAGT ATAAATATAT 3720
TAAATTACAT GTAl~-l~ CTATATATAG CT&AAT~TCT AATAGACT~C AAGACGGCTG 3780
CTCT~A TGC Cq~GW-l~ AGGCAGTTCA Cn~ADGC5TA O-T5GACAAA TAil~W~ ~ 3840
GTAT&ACATT ~~ G~ArC C~TATCACTG GA~ l~AoOC l~-l~-l-l~AAT 3900
T&ATTTT~AT T&Al~-l~AGTA TTACTAGTTT TATAAATATT ~l-l-l~l~A ATAA m AAC 3960
TaGAa m AA CAATGACAGG GAGCIITACA GCAATAACAT AAGTGGACCA ATTCCTAGTG 4020
. . . .. . . . ~, .
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A'1~'1-1~AA ICT~AChAAT ~ l~AGCT TGGACCTATA CATGAATAGC ~ GAC 4080
CTATACCGGA CACATTAG&A AAGCTTACAA GGCTA~&ATT CTTGTATEAC TACAAATCTT 4140
CACTAGTTTT TAACTTAATG CAATTTGATT A'1~'1-1-1~AA GleA5lG~IT ATATCACAAA 4200
TTACTGGATA G~C~-l~l AA CAACA~TGC ~'1~'1~'1~b'1~ CAATTCCAAT GTCPCTG~Cr 4260
AATATTACAA ~'1~'1-1~AAGT C~TGTA~GTA ITCCG;CCIr TCCA&ATAGT '1-1-1~'11~'1-1~ 4320
T~ATb-11-1~ AATTTTAATA CTAAATATGT TCATCAGGGA TTTATCA~AC AA'1~'1'AT 4380
C;aGa~C;ar ACCGGATAAT GGCTCATTTT ~'1-1-1~'1-1-1'AC ACCTATCAGG TTTAATGCTA 4440
GTAATATCTT TAATATTATG ~l-l~-l-lACTT CTACTGCGAA AGCTAT&ATA ATA1-1-1-1-1-1-1~ 4500
'1~'1~1-1~'AT ATATTATCAC '1-1-1~AGTT TTGGCAATAA m ~AA m A TGTGGaCCIG 4560
TAAClGGaAG G-~1~CC~1~ GGA'l~'l~ CA'1-1-1-1~'1~C ACCAQCTCCG TTCATCCCAC 4620
CATCAAC;aT ACA~'1~A GGTGA m AG TTTTTATATT AA~-1~'1'A ATTAATTTTA 4680
TEACTGTAAA AA~1-1~1~1-1~ AA m ~PCCA ~'1-1~AATA AAGTATTTTC ~'1-1~'1-1-1~'1~' 4740
TTCTTATTAT TATCAAGG;C AAAATGGTCC CACTGGAGCT A'1-1~'1W ~ GAGTAGCTGC 4800
1~1~1~1 TTA~1~1-1-1~ C¦GCACCIGC AATGGCATTT GCA1~ C GGAGAAGAAA 4860
AQ~GC~FGaA CA1-1-1~1-1-1~ A~ G TTA~-1~1~1~ AAATAGATAT CTATTGAAGC 4920
GCTTACTGTC TGT=GaCTTT ~ ACTG TCATTAGTTA ACTr~AGCTG AAGAGGACCC 4980
AGaAGTGcAc ~ ~l~AAC TGAaGAGGTT l~ l~-W~A GAATTGCAAG TQG AACCGA 5040
TACTTTTAGT ACCATC~TTG C~UY3AaaTGG A'11'1W1'AAG GTGTATAAGG GAC~G~11~C 5100
TE~ -l~A CTrGTAGCAG TTAAAaGGCT TAAaGAaGAA CGAAC;CCAG ~'1~AGCT 5160
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GC~l~ Al~ AAATvATTAG CA~ ,'1~ CATCG~AATC '1-l~'l~,'l~'l' 5220
AC~-lwlll~ TGCAT&ACAC CTA~:GCG ~'1'1--'l'l~.'lA TATCCATACA T~CTAAT~ 5280
AAL,-l~ TCAT&~TTAA GiU GTATCI'C AGTTACA~TT ACCATAA~T G~;~Grr 5340
l~,-llliATTA AAAAT&AAAT ATA~ ACACTAT~T A~-~ -lAT AA~l-l-l~lw~G 5400
CAGATCTTAT T~CATIGC A~ATACCAG TTATTAll~l' 'l'l-l'l-l~'l~'l'A ATIGATACC& 5460
GTTATAm~ lAT l-l~l-lATAT GC~AGGATTT CGAGTCTAAT AA~TATCAA 5520
ACIGGAT&CT ATGTITArl~ T&CAATT&AA '1-l~'l-l~'l'l~' Al~-l~l AAA ATATATAT&A 5580
TTCMCT~G MTCAIY l-lA TMTATACT& TGTAAA~A G~-l~J-l-l~iACT TTCATCATTA 5640
ATTAGICTTC ATAMTCAGA Al---l~ AG ~ AGCmAC CGACATACTC TAAAC(~ITC 5700
TTAlw~Y~l GTATATMTC GI~I~A ClllATTCAG '1-l'l~'l~'l~'l' CI~I~AAm 5760
TI~ATCI~A CATTGTGAT& '1~'1-l~,'l'l-l'l~ ATCAAAl~-lA G~ C~TCA~A~C 5820
iAT I~A~A GGGAG~G~T T&CACTAGGA l--'1-l--lAGGG GXTATCTM 5880
ATTGCAT&~C CATT~ iATC C,CAAGATTAT C'CATCGCGAT GT M AAGCI~ CAA,~TATATT 5940
AIT~Y~UA GAATTTGAGG ~l~-l-l~-lAGG T&Allll~ TTAGCTAGGC TCATGGATTA 6000
CA~GGATACC CA~C~aCa~ 'l~'l'AAG G,5GTACCATT GG5CACATAG ~l~AGTA 6060
~-l~-l~ACT GX;U~Y~n~aT CNGaGAAG~C CGA W'~lll GGTTAT&GGA TAhl~-l~-l 6120
Aka~;~DCaTT A¢l~la~hGA ~ 'l-l'l'l~A 'l-'l-l~ iC ~'l'l~P~G ATGATGATGT 6180
TAl~-l-l~-ll~ GAl-l~-lAT ~'l~'l~ L~G 'l~'l'l~'ll'l~ GTT M T-TA~T TCACATATTA 6240
.. . . . . . ~
CA 02254839 l998-ll-l3
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~ ACTA ~'ll-l~l-l~l~ GC~lll~l-l TTTATTTCCT GC~l~-lATTT GATT~TTAGT 6300
CATGTTATGC ATATTGACCT G~ ~LAAT ~~ AGG ITA~AAGCCT ITTGAAAGAG 6360
AAaAAGTTaG AGA~ Wl CGATCCTGAG CTCCAG~hCA ATTACATTGA CACAGAAGTT 6420
GAGCAGCTTA TTCAAGTA~C ATTACTCTGT ACCCPGGGTr CCCCAAlaGA G~ ~'l'AAG 6480
AqGTCAGAGa TAGTCCGAAT GClqGAAGGT GAl~GC~-ll~ CAGAAAAGTG GGACGAGT~ 6540
CPAAAAGTTa A~GTCATCCA TCAAGACGTA GAATTAGCTC CACATCGAAC TTCTGAA1~ 6600
ATCCTAGACT CGACAGATA~ CTTGCATGCT TTTGAATTAT ~ -l~AAG ATAAACAGCA 6660
TATAAAATGT AATGAAATTA ATArlllllA T~T 6695
(2) INFOKMATICN FOR SEQ ID N~: 2:
(i) SEQUENCE CNARALTEBISTICS:
(A) LENGTH: 1815 kase pairs
~B) TYPE: nucleic acid
(C) STRA~LN~S: single
(D) TOPOLOGY: unkncwn
~ ii ) ~nBFr~1T ~F TYPE: c~NA
(iii) ANII-SENSE: NO
Ivi) U~ilNA~ sa~
(A) ~NI5M: Daucus carota
(ix) FE~Tt~
(A) NAMEJXEY: CSS
(B) LOCAIIoN: 94..1752
CA 02254839 l998-ll-l3
WO 97/43427 PCT~EP97/02443
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(xi) SEQUEN~E ~l~llON: SEQ ID NO: 2:
GACAAATACC ATT~AATAT I~ACCrAA TTMTTAGTA Gl~l~ TA~ATT~AA 60
CTCATTTAAT mACTTTM AAAATAATTC TAT AT~; AAT CGT AAC AGT ATA AAT 114
Met Asn Arg Asn Ser Ile Asn
ATA TTA A~T TAC ATG CAG TTC ACT GAT GCT TAC CTT GAC AAA TAT GGG 162
Ile Leu Asn Tyr Met Gln Phe Thr Asp Ala l~r Leu Asp Lys Tyr Gly
10 15 20
GTT CTT AT, ACA TTG GAG CTT TAC AGC AAT MC ATA AGT GGA CCA ATT 210
Val Leu Met Thr Leu Glu Leu Tyr Ser Asn Asn Ile Ser Gly Pro Ile
25 30 35
cCr AGT GAT CTT GGG MT C~ ACA MT TTG GTG AGC Tl'G GAC CTA TAC 258
Pro Ser Asp Leu Gly Asn Leu Thr Asn Leu Val Ser Leu Asp Leu Tyr
40 45 50 55
ATG MT AGC TTC TCT GC;A CCT ATA CCG GAC ACA TTA GGA MG CTT ACA 306
Met Asn Ser Phe Ser Gly Pro Ile Pro Asp Thr Leu Gly Lys Leu Thr
60 65 70
A(;G CTA AGA TTC TI~; CGT CT~ AAC MC MC AGC c~ TCT GGT CCA ATT 354
Arg Leu Arg Phe Leu Arg Leu Asn Asn Asn Ser Leu Ser Gly Pro Ile
75 80 85
CCA ATG TCA CIG ACT MT ATT ACA ACT ~ C~A GTC Cl~ GAT TTA TCA 402
Pro Met Ser Leu Thr Asn Ile Thr Thr Leu Gln Val Leu Asp Leu Ser
90 95 100
AAC AAT C~G CTA T; A GGA CCA GTA CCG GAT MT OE;C TCA m TCT TTG 450
Asrl Asn Arg Leu Ser Gly Pro Val Pro Asp Asn Gly Ser Phe Ser Leu
105 110 115
TTT ACA CCT ATC AGT m GCC AAT AAT T~ AAT TTA TGT GGA CCC GTA 498
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Phe Thr Pro Ile Ser Phe Ala Asn Asn Leu Asn Leu Cys Gly Pro Val
120 125 130 135
ACT GGG AGG CCC T&C CCT GGA TCT CCC CCA m ~1~ CCA CCA CCT CCG 546
Thr Gly Ary Pro Cys Pro Gly Ser Pro Pro Phe Ser Pro Pro Pro Pro
140 145 150
TTC ATC CCA CCA TCA ACA GTA CAG CCT CCA GGA CAA AAT GGT CCC ACT 594
Phe Ile Pro Pro Ser Thr Val Gln Pro Pro Gly Gln Asn Gly Pro Thr
155 160 165
GGA GCT ATT GCT GGG GGA GTA GCT GCT GGT GCT GCT TTA CTG TTT GCT 642
Gly Ala Ile Ala Gly Gly Val Ala Ala Gly Ala Ala Leu Leu Phe Ala
170 175 180
GCA CCT GCA ATG GCA m GCA TCG TGG CGG AGA AGA AAA CCG CGA GAA 690
Ala Pro Ala Met Ala Phe Ala Trp Trp Arg Ary Arg Lys Pro Arg Glu
185 190 195
CAT ~TC m GAT ~l~ CCA GCT GAA GAG GAC CCA GAA GT& CAC CTT GGT 738
His Phe Phe Asp Val Pro Ala Glu Glu Asp Pro Glu Val His Leu Gly
200 205 210 215
CAA CTG AAG AGG m Tc-~ CTG CGA GAA TTG CAA GTC GCA ACG GAT ACT 786
Gln Leu Lys Arg Phe Ser Leu Ary Glu Leu Gln Val Ala Thr Asp Thr
220 225 230
m AGT ACC ATA CIT GGA AGA G-T GGA m GGT AAG GTG TAT AAG G5A 834
Phe Ser Thr Ile Leu Gly Arg Gly Gly Phe Gly Lys Val Tyr Lys Gly
235 240 245
CGC CTT GCT GAT GGC TCA CTT GTA GCA GTT A~A AGG CTI AAA GAA GAA 882
Arg Leu Ala Asp Gly Ser Leu Val Ala Val Lys Arg Leu Lys Glu Glu
250 255 260
CGA ACA CCA GGT GGT GAG CTG CAG m CAA ACA GA~ GTG GAA ATG ATT 930
Arg Thr Pro Gly Gly Glu Leu Gln Phe Gln Thr Glu Val Glu Met Ile
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265 270 275
AGC ATG GCT GTG CAT CGA AAT CTT CIG CGT CTA CGT GGT TTC TGC ATG 978
Ser Met Ala Val His Arg Asn Leu Leu Arg Leu Arg Gly Phe Cys Met
280 285 290 295
ACA CCA ACA GAG CGG CTT CIT GTA TAT CCA TAC ATG GCT AAT GGA AGT 1026
Thr Pro Thr Glu Arg Leu Leu Val Tyr Pro Tyr Met Ala Asn Gly Ser
300 305 310
GTT GCG TCG TGT TTA AGA GAG CGT CAG CCA TCA GAA CCT CCC CTT GAT 1074
Val Ala Ser Cys Leu Arg Glu Arg Gln Pro Ser Glu Pro Pro Leu Asp
315 320 325
TGG CCA ACT AGG AAG ABG ATT GCA CTA GGA TCT GCT AGG GGG CTT TCT 1122
Trp Pro Thr Arg Lys Arg Ile Ala Leu Gly Ser Ala Arg Gly Leu Ser
330 335 340
TAT TTG CAT GAC CAT TGT GAT CCC A~G ATT ATC CAT C~l GAT GTA AAA 1170
Tyr Leu His Asp His Cys Asp Pro Lys Ile Ile His Arg Asp Val Lys
345 350 355
GCT GCA AAT ATA TTA TTG GAC GAA GAA TTT GAG GCT ~-l-l' GTA GGT GAT 1218Ala Ala Asn Ile Leu Leu Asp Glu Glu Phe Glu Ala Val V~l Gly Asp
360 365 370 375
TTT GGG TTA GCT AGG CTC ATG GAT TAC AAG GAT ACC CAT GTT ACA ACT 1266
Phe Gly Leu Ala Arg Leu Met Asp Tyr Lys Asp Thr His Val Thr Thr
380 385 390
GCT GTA AGG Gb-l ADC TIG GGC TAC ATA G~T CCC GAG TAC CTC TCG ACT 1314
Ala Val Arg Gly Thr Leu Gly Tyr Ile Ala Pro Glu Tyr Leu Ser Thr
395 400 405
G5A AAG TCA TCA GAG AAG ACC GAT GT~ TTT GGT TAT GGG ATT ATG CTC 1362
Gly Lys Ser Ser Glu Lys Thr Asp Val Phe Gly Tyr Gly Ile Met Leu
410 415 420
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TTA GAG CTC ATT ACT GGA C'AG AGA GCT TTT GAT CTT GCT CGC CTT GCG 1410
Leu Glu Leu Ile Thr Gly Gln Arg Ala Phe Asp Leu Ala Arg Leu Ala
425 430 435
AAC GAT GAT GAT GTT ATG Tl~ TTG GAT TGG GTT A~A AGC CTT ll~i AAA 1458
Asn Asp Asp Asp Val Met Leu Leu Asp Trp Val Lys Ser Leu Leu Lys
440 445 450 455
GAG AAA AAG TTI~ GAG ATG CTG GTC GAT C'CT GAC CTG GAG AAC AAT TAC 1506
Glu Lys Lys Leu Glu Met Leu Val Asp Pro Asp Leu Glu Asn Asn Tyr
460 465 470
ATT GAC ACA GAA GTT GAG CAG CTT ATT CAA GTA GCA TTA CTC TGT ACC 1554
Ile Asp Thr Glu Val Glu G~n Leu Ile Gln Val Ala Leu Leu Cys Thr
475 480 485
C'AG G5T TCG CCA ATG GAG C~5 CCT AAG ATG TCA GAG GTA GTC CGA ATG 1602
Gln Gly Ser Pro Met Glu Arg Pro Lys Met Ser Glu Val Val Arg Met
490 495 500
CTT GAA G5T GAT GGC Cll GCA GAA AA5 TG5 GAC GAG TGG CAA AAA GTA 1650
Leu Glu Gly Asp Gly Leu Ala Glu Lys Trp Asp Glu Trp Gln Lys Val
505 510 515
GAA GTC ATC CAT C'AA GAC GTA GAA TTA GCT C~A CAT CGA ACT TCT GAA 1698
Glu Val Ile His Gln Asp Val Glu Leu Ala Pro His Arg Thr Ser Glu
520 525 530 535
TGG AT~ CTA GAC TC~G ACA GAT AAC TTG CAT GCT l'IT GAA TTA TCT G5T 1746
Trp Ile Leu Asp Ser Thr Asp Asn Leu His Ala Phe Glu Leu Ser Gly
540 545 550
CCA AGA TAMCAGC~T ATAAAA~-l~ AAT&AAATTA ATA~ r lA IGGrrA~AA 1802
Pro Arg
CA 02254839 1998-11-13
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AAA~AAAAAA AAA 1815
~2) rNFORM~TIoN FOR SEQ ID N~: 3:
a~sllcs
~A) LEWGTH: 553 am~no acids
(B) TYPE: ar~no acid
(D) TOPOLOGY: linear
(ii) MnT.T~Y~.T~. TYPE: protein
~Xi) ~U~N~ ~xI~l~lu~: SEQ ID N~: 3:
Met Asn Arg Asn Ser Ile Asn Ile Leu Asn Tyr Met Gln Phe Thr Asp
1 5 10 15
~la Tyr Leu Asp Lys Tyr Gly Val Leu Met Thr Leu Glu Leu Tyr Ser
Asn Asn Ile Ser Gly Pro Ile Pro Ser Asp Leu Gly Asn Leu Thr Asn
Leu Val Ser Leu Asp Leu Tyr Met Asn Ser Phe Ser Gly Pro Ile Pro
Asp Thr Leu Gly Lys Leu Thr Arg Leu Arg Phe Leu Arg Leu Asn Asn
Asn Ser Leu Ser Gly Pro Ile Pro Met Ser Leu Thr Asn Ile Thr m r
~eu Gln Val Leu Asp Leu Ser Asn Asn Arg Leu Ser Gly Pro Val Pro
100 105 110
Asp Asn Gly Ser Phe Ser Leu Phe Thr Pro Ile Ser Phe Ala Asn Asn
115 120 125
.. _ .. .. .
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Leu ~n Leu Cys Gly Pro Val Thr Gly Arg Pro Cys Pro Gly Ser Pro
130 135 140
Pro Phe Ser Pro Pro Pro Pro Phe Ile Pro Pro Ser Thr Val Gln Pro
145 150 155 160
Pro Gly Gln As~ Gly Pro Thr Gly Ala Ile Ala Gly Gly Val Ala Ala
165 170 175
Gly Ala Ala Leu Leu Phe Ala Ala Pro Ala Met Ala Phe Ala Trp Trp
180 185 190
Arg Arg Arg Lys Pro ~g Glu His Phe Phe Asp Val Pro Ala Glu Glu
195 200 205
Asp Pro Glu Val His Leu Gly Gln Leu Lys Arg Phe Ser Leu Arg Glu
210 215 220
Leu Gln Val Ala Thr Asp m r Phe Ser m r Ile Leu Gly Arg Gly Gly
225 230 235 240
Phe Gly Lys Val Tyr Lys Gly Arg Leu Ala Asp Gly Ser Leu Val Ala
245 250 255
Val Lys Arg Leu Lys Glu Glu Arg Thr Pro Gly Gly Glu Leu Gln Phe
260 265 270
Gln m r Glu Val Glu Met Ile Ser Met Ala Val His Arg Asn Leu Leu
275 280 285
Arg Leu Ary Gly Phe Cys Met Thr Pro ~hr Glu Arg 1~ Leu Val Tyr
290 295 300
Pro Tyr Met Ala Asn Gly Ser Val Ala Ser Cys Leu Arg Glu Ary Gln
305 310 315 320
PrO SOE G1U Pro Pro Leu Asp Trp Pro Thr Arg Lys Ary Ile Ala Leu
,
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325 330 335
Gly Ser Ala Arg Gly Leu Ser Tyr Leu His Asp His Cys Asp Pro Lys
340 345 350
Ile Ile His Arg Asp Val Lys Ala Ala Asn Ile Leu Leu Asp Glu Glu
355 360 365
Phe Glu Ala Val Val Gly Asp Phe Gly Leu Ala Arg Leu Met Asp Tyr
370 375 380
Lys Asp Thr His Val Thr Thr Ala Val Arg Gly Thr Leu Gly Tyr Ile
385 3gO 395 400
Ala Pro Glu Tyr Leu Ser Thr Gly Lys Ser Ser Glu Lys Thr Asp Val
405 410 415
Phe Gly Tyr Gly Ile Met Leu Leu Glu Leu Ile Thr Gly Gln Arg Ala
420 425 430
Phe Asp Leu Ala Arg Leu Ala Asn Asp Asp Asp Val Met Leu Leu Asp
435 440 445
Trp Val Lys Ser Leu Leu Lys Glu Lys Lys Leu Glu Met Leu Val Asp
450 455 460
Pro Asp Leu Glu Asn Asn Tyr Ile Asp Thr Glu Val Glu Gln Leu Ile
465 470 475 480
Gln Val Ala Leu Leu Cys Thr Gln Gly Ser Pro Met Glu Arg Pro Lys
485 490 495
Met Ser Glu Val Val Arg Met Leu Glu Gly Asp Gly Leu Ala Glu Lys
500 505 510
Trp Asp Glu Trp Gln Lys Val Glu Val Ile His Gln Asp Val Glu Leu
515 520 525
CA 02254839 l998-ll-l3
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Ala Pro His Arg Thr Ser Glu Trp Ile Leu Asp Ser Thr Asp Asn Leu
530 535 540
His Ala Phe Glu Leu Ser Gly Pro Ary
545 550
(2) INFORM2112N F0R S~Q ID NO: 4:
CHAR~cqERI~ CS:
(A) LENGTH: 13 kase pairs
~B) TYPE: nucleic acid
(C) STRANDECNESS: single
~D~ TOPOLOGY: unknown
~iii) H~YJln~llLAL: ND
(iii) ANTI-SEWSE: NO
(vi) ORIGIN~L SW ROE:
(A) ORG~NISM: priner
(xi) SEQUENCE D~xl~lluN: SEQ ID NO: 4:
-111'1-1'11-1' TGC 13
(2) INFORMATICN FOR SEQ ID N0: 5:
CH?RA~STICS:
(A) LENGTH: 10 kase pairs
(B) TYPE: ~ ic acid
(C) STRA~ x~: single
(D) T0POLOGY llnl_--
(iii) ANnI-S~NSE: N0
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(vi) u~I~LNAL SOURCE:
~A) ORGANISM: primer
(xi) ~ r~ V~K~ U~ ID N~: 5:
GGGATCTAAG l0
(2) INFORMATIoN FOR SEQ ID N~: 6:
CHARACTERISTICS:
(A) LENGrH: l0 h~e pairs
(B) TYPE: nucleic acid
(C) STRANDEnNESS: single
(D) TOPOLOGY: unknown
(iii) ANTI-SENSE: N~
(vi) ORIGIN~L SOURCE:
(A) ORGANISM: primer
(xi) ~ DESCRlPTIoN: SEQ ID NO: 6:
ACA~ wl~ l0
(2) rNFORMATICN FOR SEy ID N~: 7:
: CH~STICS:
(A) LENGTH: l0 base pairs
~B) TYPE: nucleic acid
~C) sTR~NnFn~ single
(D) TOPOLOGY: unknown
CA 02254839 l998-ll-l3
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(iii) ANTI-SENSE: N~
(vi) ORIOEN~L SCURCE:
(A) ORGANISM: primer
(Xi) ~ ~LK~ U~: SEQ ID N~: 7:
~G 10
(2) INFORMATICN FOR 5Ey ID NO: 8:
(i) ~U~N~ CHARALI~RISTICS:
(A) LENGTH: 14 kase pairs
(B) TYPE: mtrl~ic acid
(C) STRA~ x~: sinyle
(D) TOPOLOGY: unknown
(iii) ANII-SENSE: NO
(vi) ORIGIN~L SOURCE:
(A) ORGANI9M: pr1mer
(xi) ~U~N~ DESCRIPTqoN: SEQ ID NO: 8:
rlllllllll TCTG 14
(2) INFORM~TICN FOR SEQ ID NO: 9:
~A) LENGTH: 13 base pairs
~B) TYPE: n~le;c acid
(C) STRA~ x~: sinyle
~D) TOPOLO~Y:
( iii ) ~ly~~ AL: NO
CA 02254839 1998-11-13
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(iii) ANTI-SENSE: ND
(Vi) ~CilNAL SC~ROE:
(A) ORGANISM: primer
(xi) SEQUENCE v~Lx~ uN: SEQ ID N0: 9:
-1-1-~-1-1-1~1-~-1 TCA 13
(2) INFORMA~IoN FOR SEQ rD NO: 10:
ChiUU~neRI~l~lCS:
(A) LE~X~rH: 10 base pairs
(B) TYPE: nucleic acid
(C) STRANv~LN~SS: single
~D) TOPOLOGY: lmk~ .
(iii) A~TI-SENSE: NO
(vi) ORIGIN~L SOURCE:
(A) ORGANISM: primer
(Xi) ~U~NL~ V~S~Xl~l'l~: SEQ ID N~: 10:
GACAI~X~C 10
(2) INFOR ~ TqCN FOR SEQ ID ND: 11:
CHARALl~h~ S:
(A) LE~:rH: 10 base pairs
(B) TYPE: nllcle;c acid
(C) ~l~Ah~ S: single
(D) TOPOL0GY: un~ wn
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(iii) ~Y~U~ AL: N~
(iii) ANTI-SENSE: NO
(vi) QKl~lNAL SCUR OE :
(A) ORGANISM: priner
(xi) ~U~NL~ v~KI~l~loN: SEQ ID NO: 11:
COCTACTGGT 10
(2) INFORM~TIaN FOR SEQ m NO 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 base pairs
(B) TYPE: nucleic acid
(C) SlRA~vkLN~SS: single
(D) TOPOLOGY: unknown
(iii) ANTI-SENSE: NO
(vi) ORIGIN~L SOUR OE :
(A) ORGANISM: primer
(xi) .~ r~ ~: L~Hl~llU~: SEQ ID NO: 12:
ACAL~ 10
(2) lN~U.~ ~ aN FOR SEQ m No 13:
(i) ~yu~ CN;RALIERISTICS:
(A) LENGTH: 10 kase palrs
(B) TYPE: nucleic acid
(C) STRA~ s single
(D) TOPOLOGY: unknown
CA 02254839 l998-ll-l3
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(iii) ~Y~Ul'~'l'l~AL: NO
(iii~ ANTI-SENSE: N~
(vi) u~l~lNAL SCURCE:
(A) ORGANISM: primer
(xi) S~QUEW~E DESCRIPTIoN: SEQ ID N~: 13:
(2) INFO~MATI5N FOR SEQ ID N~: 14:
(i) ~yu~ CHARA~T$RISTICS:
(A) LENGqH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDE~NESS: single
(D) TOPOLOGY: unknown
(iii) ANTI-SENSE: NO
(vi) ORIGIN~L SCURCE:
(A) ORGPNISM: primer
i) ~CF~ ~Xl~ll~: SEQ ID N~: 14:
CCA G~IAATTC 18
(2) INF3RM~lloN FOR SEQ ID N~: 15:
CH~RACTE~ISTICS:
(A) LENGTH: 19 base paIrs
tB) TYPE: nucleic acid
(C) SIRA~ : single
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~D) T0POLOGY: u ~ own
(iii) ~YHJ~ ~AL: N~
~iii) ANTI-SENSE: NO
~vi) uKI~lNAL SCURCE:
(A) ORGANISM: primer
(Xl) X~ ~K~ CN: SEQ ID N~: 15:
CTCT~ATGAC T~TCC~GTC 19
(2) INFORMATIoN FOR SEQ ID NO: 16:
i ) x~u~; cH~rr~s~cs
(A) LENGTH: 16 base pairs
(B) TYPE: nucleic acid
(C) STRA~w~LNkSS: single
(D) TOPOLOGY: unknown
(iii) H~HJL~llLAL: NO
(iii) ANTI-SENSE: NO
(vi) u~I~LNAL SCUROE:
(A) ORG~NISM: primer
(xi) X~u~N~ ~-x~nl~llu~ SEQ ID NO: 16:
Aa~GCCA~TT GCATGG 16
(2) INFORMATION FOR SEO ID NO: 17:
: C~ARA~TE~ISTICS:
(A) LENGrH: 5 am~no acids
(B) TYPE: amino acid
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(C) STRANDEnNESS: single
(D) TOPOLOGY: unknown
(ii3 ~nT ~T~TT ~ TYPE: peptide
( iii ) ANTI-SENSE: NO
(~i ) ORIG~L SWRCE:
(A) ORGPNISM: Daucus carota
(xi) SE~ICE L~ N: SEQ ID NO: 17:
Ser Pro Pro Pro Pro
l 5
(2) INFORMATICN FOR SEQ ID NO: 18:
(i) SE~UEN~E CHARACTERISTICS:
~A) LENGTH: 8 amlno acids
~B) TYPE: amIno acid
~C) ST~A~rEDNEsS: single
~D) TOPOLOGY: unknown
~ ii ) ~nT-T~TT~T~' TYPE: peptide
(iii) ANTI-SENSE: N~
(vi ) uKl~lNAL SoURCE:
(A) ORGANISM: Daucus c rota
(Xi ) Xl'~,Jh:NI t- I "- 4 ~ ~ ~ SEQ ID N~): 18:
His Arg Asp Val Lys Ala Ala Asn
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(2) INF~MATICN FOR SEQ ID N~: 19:
CHA~STICS:
(A) LE~rH: 9 amlno acids
(B) I~E: amlno acid
(C) STRA~nFrNF-~s: single
(D) TOPOLOGY: unknown
(ii) MnTT~lT.T~' TYPE: peptide
(iii) H~u~ LAL: NO
(iii) ANTI-SENSE: N~
(vi) ORIGIN~L SWROE:
(A) ORGANISM: Daucus carota
(xi) SE~lE~CE ~L~~ uN: SEQ ID N~: 19:
Gly Thr Leu Gly Tyr Ile Ala ~ o Glu
(2) DNFORM~TICN FOR SEQ ID N~: 20:
( i ) ~u~; ~I~STICS:
(A) LE~X~H: 4081 hA~e pairs
(B) TYPE: mlrlPic acid
(C) STR~ S: double
(D) TOPOLOGY: lin~Ar
( ii ) ~)T ~T rrTT ~ TYPE: ~ ( g~nn~i C )
(iii) ~uln~l~AL: N~
(vi) ORIGIN~L SCURCE:
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(A) ORGP.NISM: ArAh~ $ic thAl; An~
(Vii ) T~IM~T~.TE SOURCE:
(B) CLoNE: ArAhi~p~cic SERK gene
(ix) FEATURE:
(A) NAME/XEY: exon
(B) LOCATICN: 1280..1367
(ix) FEATURE:
(A) NAMEtKEY: exon
(B) LOCATICN: 1796..1928
(ix) FEATURE:
(A) N~DE/KEY: exon
~B) LOCATIoN: 2014..2085
(ix) FEATURE:
(A) N~ME/KEY: exon
(B) LOCATION: 2203..2346
(lx) F~:Aq~E:
(A) NEU~E/KEY: exon
(B) LOCATICN: 2450..2521
(ix) FEATURE:
(A) NAME/KEY: eKon
(B) LOCATION: 2617..2688
(ix) FEATURE:
(A) N~MEtKEY: exan
tB) LOCATICN: 2772..2884
(ix) FEATURE:
(A) N~MEtKEY: exon
~B) LOCATIoN: 3015..3146
, _ .
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(ix) FEATURE:
(A) NAME/KEY: exon
(B) LOC~TICN: 3305..3646
(:uc) ~URE:
(A) NAME/KEY: excn
(B) LOCATICN: 3760..4081
(Xi) SEÇUEN~E ~S~K1Y1'1U~: SEQ ID N~: 20:
T -lA ~ TTTTGATCAT AAT&AAAATA AAGAG~C~T CCA~CACATG GGGTAA~CAT 60
AA~ AT ATTTAAAGGG TAAGAAAl~l~ AA~ lll TTATITTACT TTTTACCTCT 120
ACICAAATTG TATGGGCAGT ~lllll~lllll ~llllAAATGA TAAGACAAGT A~l~l~-lllAA 180
TGGTATT~TG ATGAAACAGT AGTAAA~TCA TA~ G~AC GCCATACTAC TTCCACAarG 240
GAAL-~ GCC' AA~llll~-l~ lll~l~l~ CTACAG m C TTCCACCAAA l-l-l-l-l-l~l-l~ 300
ACAAAACTCA AAl~lll~AA TCTCATCr~T GCCAAA~TTG GGITTAGAAA GAATATCAGC 360
AAACACTAAT ATCTTTATTG TT~CATGGTT TATCAATCAC AAhATTCACA ACCA'll~-lAA 420
AAAAA~TTC ACA~lllllw TATGA~ATTG CTCACATGAT AGIGAACCTC mAA~Am 480
TAA~TTTACT TT~ATAAATA CGGGATTACG AATCTTACTT GCATTA~AAA mAGA~AAG 540
-l-ll-l~lAC TTAAAGAAAA AAGGGACCCA ACAGAGaGAG GTTTGACCAG GAGAAACGGG 600
TGCATAGCCT TAAGA~C m CAACrAC m ACCCCAAACC CAAAG33ATG TCACITTCAA 660
C'CA'l--1~1-l~ 1~1~l;~ AU,~,-11-1-1-1 TTGAI~CGGTC AL;1-1U~LA GCAGCACCGT 720
TACGGGCAGC TTATA5TCCT C~l~l-l--~1~ C~CTACACCA CT~T~CCC ATAAATAAAG 780
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C~ A '1~'1'11'AAAAA TATTMATM TATATCAACG AAaAAG~AT mATl~ATA 840
A~aAGa~ GaG~GG~ ACAAC~ ACTAAT~TA ~ G C'AL;t~~ l 900
~;1-1~W~'1'1' AATAAA~ '1~'1'1'1-1~'1'1'A TTATTACT~ ACGTAGAm ~A 960
~1~11~m ~ '1-1-1AA A~Gr TrCAT~rTTA T~m GmTAc~T 1020
W~'1~'1~'1~A GAGAGAaAGT ~1~-1-1-1~AT TGAGaAAAoA CGACOA~GAC A~r~;~G 1080
AATTAGGATT mATTTTAT T1T~TAC~rCT '1'1~'1-1'1~'1'1'1' TM~CTAAT G~'l'l'l'l-lAA 1140
A~TA~ C~AAATGA G~r Gr~ '1~'1~'1~;1AAA ~TMTGG 1200
T~GT~ATTTT CGGAACTTAG ~ l--~ GA~CTaAAGA GAT~AAATCA AGATrCGAAA 1260
mAG,CATTV ~ 1~AAA TGGAGTCGaG TTA~ 1~ m ATC~TAC' m CACDGAT 1320
CITAC ~ AATC~TTCAC ~ 1~C ll~-l~lA~T TTGGPAGGTT C~v-l~-l-lACT 1380
C'AATTAC'TCA GCTTTACTC~ lll~-l~AATT AL1-1'1~1~A 'l-l~ll-l-l-l-lA maGA3GTG 1440
AATCrvCTATC m AGIGTCT GCATTTTGAT TTAT~AAAAT lrv-l-l~v-ll~-l-~ ~lA m 1500
GTA~GA m A ~l~W AGTA cTTTrvAATAc AL1~1111~C l-l-l-l~l-l~vl-l' CAGATC'AACT 1560
TTGTATA~l~ TAAAGGCATG llW ll~vl' DaAAAAGCTG ~llATTTGA TATCTTM GA 1620
TTvAT&T~5T DGATCCAAAC All~l~l~vAA AGACTTCATT l~l-l-l-l-lWl TTTGTAAAGA 1680
All-l~-l-l-lAA TTATTAoCCT Cq~ATCTCAG AG~vL~'l~l' TTGAATAGTT Cl~-~l-l~AA 1740
ATTAGACqTT TCAC~ MTTG ATGCTAATTrv TvTAGATTTG l-l~vll~ll~vl TATAGGTGAT 1800
-l-l~ATA C m GAGGGT TACTCTAGTT GAD C~AACA A1~1~1-1~LA GA~L-l~AT 1860
C~TAOE CTArv T&AATC~TTG C'ACAT>TC CATGrCA~TT GCAACAACGA GAACAGTGTC 1920
CA 02254839 l998-ll-l3
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ATAAG~aT3T AAA~ W TCTACTAATC CCALl-l-l-l-lA AA~STIGA~C TCAGC~ l 1980
TACCGACATT l-l-l~-l-ll~ll TlGT~AAATA CAGTGATrTG GGGAATGCAG AGTTATCTGG 2040
CCATTTAGTT CCAo~GC~TG ~-l~ ~A GAATTTGCAG TATTTGTAAG lTCC~CTr~T 2100
GCATCATGCT TTAASAAAAC AAATCCAAGA TTrG~CAGAA GAAGCPCTGG AGTTACCTTT 2160
~ AATTGAA Al~ll-l-l-lAA CAA~-l-ll~-l-l A1-111~11AC A~GGA3CTTT ACAGTAA~AA 2220
CATAACTG~C CCGATTCCTA GTAAl--ll~C AAATCT&ACA AA~TTAGTGA ~ll-lW ATCT 2280
TTACTTAAAC A~l-l~l~ G~CCTATTCC G&AATCATTG GGAAA XTTT CAAAGCTGAG 2340
A'11-1~1~1~A GTATACATAT GCTTTACCGG CTCA~TTACA w ~l-l-l~lll' AATCTTAGGT 2400
l-l-l~l-l-~A l-l-l-ll~ACTC 'l-l-l~l~AAA ATTTTACATG CAAGAATAGC ~ ACA 2460
A~PACAGICT CAL-l~l~A ATT~-lATGT CACT~A~CAA TATTACTACC CTT~AA{TGT 2520
TGnGAGT~T CTCATTA~CT TICATITATG TCTACTTCAT l~l~l~AG TIGATTTGTT 2580
GAGTTAATGC ACTTAACCTT GATGGATGCA ACACAGAGAT CTATCAAATA ACAGACTCTC 2640
1W11~AGTT CCTaACAATG ~.l..l-l.l~ AL~ 1~ACA CCCATCAGGT TCTATCAIIT 2700
A~ A GTTATITCAG 'll~ll~l~-lC A~-l~ ~AA CITATTCIGA AACTTTCATT 2760
'1~'1'1~'1~'A ~'1-1'1-1~'1'AA TAALTTAGAC CTATGn3G~C CTGTTACAAG TCACCCATGT 2820
C~TGGATCTC ~111-1~ 1~1~ACCA ~111-1~TI~ AAL~-1~.~. A~-111~LACC 2880
CCGAGTAAGC ~'1~'1~'111-1' TAGTTTACAT TATAGGAAAC AG~AGAT&AA A1-1-11~ 1-1 2940
~1~1~1~AAT C~'1'1'11'1~'1~ ATATAACTCA ~1~1-1~AAT AAGGCAATAA CCAAAT~ArC 3000
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TAATTTvATT TCA Wl~Wl AT~TATA~C TGGhGCAATA GCl~l~AG ~ ~AGG 3060
l~l~l-l-l~ ~l~l-l-l~l~ ~l~l~AAT A~lll~-l 'l~'l~C~AC GPAGAAAGCC 3120
A~TAGATATT ~ -l~ATG l~C~l~l~A GTTTATTAIT CGCATTAGTT 'l~'l~'l'l~'l'l'A 3180
GCCPGCAATT ll~l-l-l-l~A GAAAAGTATT GGaAcAAcTG TTAAT&AAAA TCAATACATA 3240
AGTCATrGTT TTTTA~GTTA CAAA~TCT~T T~AGTAAAAT C~GATTGCA AAATCTCTAT 3300
GCAGCCGAA~ AAGATCCAGA AGTTCATCTG GGACAGCnCA AGA Wll-l-l~ ~AG 3360
CTACA~GTGG CGA~T~AT~ GTTTAGTA~C AAGAACATTT TGGGCAGA3G 1~-1-l-l~ 3420
AAAGTCTACA AGGGACGCTT GGCAGAOGGA ALl~-l-l~-l-l~ CTGICAAGAG A~T~AAG~AA 3480
GAGCGAACTC CAGGTGGAGA GCICCAGTTT CAAACAGAAG TAGAGATGAT AAGTAT~GCA 3540
GTT~ATCGAA AC~l~-l-l~AG ATTAC~A~GT l-l~l~-lAT&A CA~CGACCGA GAGATT~CTT 3600
GTGTATCCTT ACAT&GCC~A TGGAAb~C~T GL1-1~1~1~ T~AGAEGTAA AAACTAAACA 3660
ATTAAACATC l-l~l~l~l~ TCTCAATTAC Tr~GAOGrGA A~-l~-l-l-l-l-l-l CAil~l-l-l-l~ 3720
TTTAT&GGTT CATAATTGTT ~-l-lACACTA ATGACACAGA GA~GCCACCG T~ACAAOCrC 3780
~~'l-l~ATT~ GO~AACGCGG AAGAGAATCG CGCTAGGCTC AG~3~GaGGT l-l~l~llACC 3840
TACATGATCA CT~CGATCCG AAGATCATT AO~GTGAOGT AAAAGCAGCA A~CATCCTCT 3900
TAGACGAAGA ATTCGAAGCG ~-ll~-l-l~AG A~-ll~3i~ll GGChA~GCTA ATaGACTATA 3960
AAGPCP~ICA CCn~-ACAACA GCA~l~'G~-l~ GCACCATCGG TCACAT~GCT CCAEAATATC 4020
TCTCAh~C3G AAAATCTTCA CAGAAAACCG AL~-1'11-1~W ATACGGAATC Al~-ll~-lAG 4080
A 4081
, . .. . ..
CA 02254839 l998-ll-l3
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(2) IN~ORM~TION FOR SEQ ID ND: 21:
u~ CHP~A~q~RISTICS
~A) LENGrH: 494 amlno acids
(B) TYPE: amino acid
tc) STRA~-s~: u~
(D) TOPOLOGY: linear
~ii) ~T.FrllT.~ TYPE: protein
(iii) HY~ul~kll~AL: NO
(v) FRAGMENT TYPE: N-t~nmin~l
(Xl) SEQUENCE ~KI~llU~: S~Q ID NO: 21:
Met Glu Ser Ser Tyr Val Val Phe Ile Leu Leu Ser Leu Ile Leu Leu
1 5 10 15
Pro Asn His Ser Leu Trp Leu Ala Ser Ala Asn Leu Glu Gly Asp Ala
Leu His m r Leu Arg Val m r Leu Val Asp Pro Asn Asn Val Leu Gln
Ser Trp Asp Pro Thr Leu Val Asn Pro Cys Thr Trp Phe His Val Thr
Cys Asn Asn Glu Asn Ser Val Ile Arg Val Asp Leu Gly Asn Ala Glu
Leu Ser Gly His Leu Val Pro Glu Leu Gly Val Leu Lys Asn Leu Gln
CA 02254839 1998-11-13
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Glu Leu Tyr Ser Asn Asn Ile Thr Gly Pro Ile Pro Ser Asn Leu Gly
100 105 110
Asn Leu Thr Asn Leu Val Ser Leu Asp Leu Tyr Leu Asn Ser ~he Ser
115 120 125
Gly Pro Ile Pro Glu Ser Leu Gly Lys Leu Ser Lys Leu Arg Phe Leu
130 135 140
Arg Leu Asn Asn Asn Ser Leu Thr Gly Ser Ile Pro Met Ser Leu Thr
145 150 155 160
Asn Ile Thr Thr Leu Gln Val Leu Asp Leu Ser Asn Asn Arg Leu Ser
165 170 175
Gly Ser Val Pro Asp Asn Gly Ser Phe Ser Leu Phe Thr Pro Ile Ser
180 185 190
Phe Ala Asn Asn Leu Asp Leu Cys Gly Pro Val Thr SOE HiS Pro Cys
195 200 205
Pro Gly Ser Pro Pro Phe Ser Pro Pro Pro Pro Phe Ile Gln Pro Pro
210 215 220
Pro Val Ser Thr Pro Ser Gly Tyr Gly Ile Thr Gly Ala Ile Ala Gly
225 230 235 240
Gly Val Ala Ala Gly Ala Ala Leu Leu Phe Ala Ala Pro Ala Ile Ala
245 250 255
Phe Ala Trp Trp Ary Arg Ary Lys Pro Leu Asp Ile Phe Phe Asp Val
260 265 270
Pro Ala Glu Glu Asp Pro Glu Val His Leu Gly Gln Leu Lys Arg Phe
275 280 285
Ser Leu Arg Glu Leu Gln Val Ala SOE Asp Gly Phe SOE Asn Lys Asn
, . . .
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290 295 300
Ile Leu Gly Arg Gly Gly Phe Gly Lys Val Tyr Lys Gly Arg Leu Ala
305 310 315 320
Asp Gly Thr Leu Val Ala Val Lys Arg Leu Lys Glu Glu Arg Thr Pro
325 330 335
Gly Gly Glu Leu Gln Phe Gln Thr Glu Val Glu Met Ile Ser Met Ala
340 345 350
Val His Arg Asn Leu Leu Arg Leu Ary Gly Phe Cys Met Thr Pro Thr
355 360 365
Glu Arg Leu Leu Val Tyr Pro Tyr Met Ala Asn Gly Ser Val Ala Ser
370 375 380
Cys Leu Arg Glu Arg Pro Pro Ser Gln Pro Pro Leu Asp ffl Pro Thr
385 390 395 400
Arg Lys Arg Ile Ala Leu Gly Ser Ala Arg Gly Leu Ser Tyr Leu His
405 410 415
Asp His Cys Asp Pro Lys Ile Ile His Arg Asp Val Lys Ala Ala Asn
420 425 430
Ile Leu Leu Asp Glu Glu Phe Glu Ala Val Val Gly Asp Phe Gly Leu
435 440 445
Ala Lys Leu Met Asp ~yr Lys Asp Thr His Val Thr Thr Ala Val Arg
450 455 460
Gly Thr Ile Gly His Ile Ala Pro Glu Tyr Leu SOE Thr Gly Lys SOE
465 470 475 480
SOE Glu Lys Thr Asp Val Phe Gly Tyr Gly Ile Met Leu Leu
485 490
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~2) INFORMATICN FOR SEQ ID NO: 22:
CHa~A~TE~ISTICS
~A) LENGTH: 1106 base pairs
(B) TYPE: ml~]~i~ acid
(C~ STRA~ single
(D) TOPOLOGY: linear
~ ii ) ~T ,T~ T .T~ 'YPE cl~ to ~RNA
(iii) H~u~ CAL: N~
(ix) FEATURE:
(A) NAME/REY: CDS
(B) LOCATICN: 142..795
(xi) .~ DESCRIPTIoN: SEQ ID NO: 22:
TCGACCCAC~ C~-l~l~A AC~I~AATAA PGGGGAAA{C AACGTAACCC TAAllll~-l 60
'1-1~'1~1~11 I~F~;AAA Allll~'_ll TAcqX3~aAAA 'l-l~l-l~-l~ Alll~'C~-l~-l 120
CTTAA~CTC CGPJU4~nGA C ATG G~G TCT CGA AAC TAT CGC TGG GAG CTC 171
Met Ala Ser Arg Asn Tyr Arg Trp Glu Leu
TTC ~CA GCT TCG TTA AC'C CTA ACC TTA GCT TrG ATT CAC C~G GTC GAA 219
Phe Ala Ala Ser Leu Thr Leu Thr Leu Ala Leu Ile His Leu Val Glu
GCA AAC TCC GAA GGA GAT GCT CTC TAC GCT CTT CGC CGG AGT TTG ACA 267
Ala Asn Ser Glu Gly Asp Ala Leu Tyr Ala Leu Ary Ar~ Ser Leu Thr
.
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GAT C~A GAC CAT GTC crc CAG AGC TGG GAT CCA ACT CTT GTT AAT CCT 315
Asp Pro Asp His Val Leu Gln Ser Trp Asp Pro Thr Leu Val Asn Pro
TGT A~C TGG TrC CAT GTC ACC TGT AAC CAA GAC AAC CGC GTC ACT CGT 363
Cys Thr Trp Phe His Val Thr Cys Asn Gln Asp Asn Arg Val Thr Arg
GTG GAT TTG GGA AAT TCA AAC CTC TCT G5A CAT CTT GCG CCT GAG CTT 411
Val Asp Leu Gly Asn Ser Asn Leu Ser Gly His Leu Ala Pro Glu Leu
GGG AAG rl GAA CAT TTA CAG TAT CTA GAG CTC TAC A~A AAC AAC ATC 459
Gly Lys Leu Glu His Leu Gln Tyr Leu Glu Leu Tyr Lys Asn Asn Ile
100 105
CAA GGA ACT ATA CCT TCC GAA CTT GGA AAT CTG AAG AAT ~'l~ ATC AGC 507
Gln Gly Thr Ile Pro Ser Glu Leu Gly Asn Leu Lys Asn Leu Ile Ser
110 115 120
TTG GAT CTG TAC AAC AAC AAT CTT ACA GGG ATA GTT CCC ACT TrC TrG 555
Leu Asp Leu Tyr Asn Asn Asn Leu Thr Gly Ile Val Pro Thr Phe Leu
125 130 135
GGA AAA TTG AAG TCT CTG GTC m TTA CGG CTT AAT GAC AAC CGA TTG 603
Gly Lys Leu Lys Ser Leu Val Phe Leu Arg Leu Asn Asp Asn Arg Leu
140 145 150
ACC GGT CCA ATC CTA GAG CAC TCA CGG CAA TCC CAA GCC TIT AAA GTT 651
Thr Gly Pro Ile Leu Glu His Ser Arg Gln Ser Gln Ala Phe Lys Val
155 160 165 170
GTT G~C GTC TCA AGC AAT GAT TTG TGT GGG ACA ATC CCA ACA AAC GGA 699
Val Asp Val Ser Ser Asn Asp Leu Cys Gly Thr Ile Pro Thr Asn Gly
175 180 185
ccc m GCT CAC ATT CCT TTA CAG AAC m GAG AAC AAC CCG AGA l-l~ 747
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Pro Phe Ala His Ile Pro Leu Gln Asn Phe Glu Asn Asn Pro Arg Leu
190 195 200
GAG GGA CCG GAA TTA CTC G-T CTT GCA AGC TAC GAC ACT AAC T~C ACC 795
Glu Gly Pro Glu Leu Leu Gly Leu Ala Ser Tyr Asp Thr Asn Cys Thr
205 210 215
TGAA~AA~T GGC;AAACCT GAAAAT~AAG AAll~G~G TGAL~al~-lA A~A~¢ACTTC 855
A~CACTTTAT CAA~TATCAC ATCIATTATC TAATAAGTAT ATATATGTAG TAAAAACAAA 915
AAAAAT&AAG AAT~GAATCG GTAATATCAT ~ AA IlGAoAACTT CGAG~-l~l~-l 975
ATGTA~AATT TCTAAAT~CG A~ -ll ACl~lAATGT l~l-l~l~G GATT~T3A3A 1035
AGTAACATTT GTATTGGTAT GGTATCAAGT ~l~ll~l~l l~-l~l~LAAA AAAAAAAAAA 1095
AaAA~AAAAA A 1106
(2) INFORMATIoN FOR SEQ ID NO: 23:
(i) ~U~NU~ CHARASTERISTICS:
(A) LENGTH: 21~ amino acids
~B~ TYPE: amino acid
(D) TOPOI.OGY: l~ r
(ii) ~nr-r~lr~ TYPE: protein
~Xi) ~U~ ~k~LX~ U~: SEQ ID N~: 23:
Met Ala Ser Ary Asn Tyr Arg Trp Glu Leu Phe Ala Ala Ser Leu Thr
1 5 10 15
Leu Thr Leu Ala Leu Ile His Leu Val Glu Ala Asn Ser Glu Gly Asp
CA 02254839 l998-ll-l3
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Ala Leu Tyr Ala Leu Arg Arg Ser Leu Thr Asp Pro Asp His Val Leu
35 40 45
Gln Ser Trp Asp Pro Thr Leu Val Asn Pro Cys Thr Trp Phe His Val
50 55 60
Thr Cys Asn Gln Asp Asn Arg Val Thr Arg Val Asp Leu Gly Asn Ser
65 70 75 80
Asn Leu Ser Gly His Leu Ala Pro Glu Leu Gly Lys Leu Glu His Leu
85 90 95
Gln Tyr Leu Glu Leu Tyr Lys Asn Asn Ile Gln Gly Thr Ile Pro Ser
100 105 110
Glu Leu Gly Asn Leu Lys Asn Leu Ile Ser Leu Asp Leu Tyr Asn Asn
115 120 125
Asn Leu Thr Gly Ile Val Pro Thr Phe Leu Gly Lys Leu Lys Ser Leu
130 135 140
Val Phe Leu Arg Leu Asn Asp Asn Arg Leu Thr Gly Pro Ile Leu Glu
145 150 155 160
His Ser Arg Gln Ser Gln Ala Phe Lys Val Val Asp Val Ser Ser Asn
165 170 175
Asp Leu Cys Gly Thr Ile Pro Thr Asn Gly Pro Phe Ala His Ile Pro
180 185 190
Leu Gln Asn Phe Glu Asn Asn Pro Arg Leu Glu Gly Pro Glu Leu Leu
195 200 205
Gly Leu Ala Ser Tyr Asp Thr Asn Cys Thr
210 215
~2) INFORM~TICN FO~ SEQ ID N~: 24:
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(i) SEQVENCE CN~A~TE~ISTICS:
(A) LENGTH: 981 ~ase palrs
(B) TYPE: nucleic acid
(C) sTRA~nr~nNFs~s: single
(D) TOPOLOGY: linear
(ii) Mnr.FCUr.~ TYPE: cDN~ to mRNA
(iii) H~HJ~ AL: N~
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATIoN: 104..757
(xi) ~U~NK~: DESCRIPTICN: SEQ ID N~: 24:
AGn~nGAaTA ATTTAGTTTG ~ ll~-lluAGA AAAllll~ m AC ~ 60
AAll~-llll CGArll~'~ lAAACC TCCGAAAGC~ CAC A~G GCG TCT CGA 115
Met Ala Ser Arg
AAC TAT CGG TGG GAG CTC TTC GCA GCT TCG TTA ACC CTA ACC TTA GCT 163
Asn Tyr Arg Trp Glu Leu Phe Ala Ala Ser Leu Thr Leu Thr Leu Ala
5 10 15 20
1-1~ ATT CAC C~G GTC GAA GCA AAC TCC GAA G5A GAT GCT CTC TAC GCT 211
Leu Ile His Leu Val Glu Ala Asn SOE Glu Gly Asp Ala Leu Tyr Ala
25 30 35
CTT CGC CGG AGT TTG ACA GAT CCA GAC CAT GTC CTC CAG AGC T~ GAT 259
Leu Arg Arg Ser Leu Thr Asp Pro Asp His Val Leu Gln Ser ffl Asp
40 45 50
.
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CCA ACT ~ GTT AAT CCT TGT ACC T~G TTC CAT GTC ACC TGT AAC CAA 307
Pro Thr Leu Val Asn Pro Cys Thr Trp Phe His Val Thr Cys Asn Gln
55 60 65
GAC AAC CGC GTC ACT CGT GTG GAT TTG GGA AAT TCA AAC CTC TCT G A 355
Asp Asn Ary Val Thr Ary Val Asp Leu Gly Asn Ser Asn Leu Ser Gly
70 75 80
CAT CTT GCG CCT GAG CTT GGG AAG CTT GAA CAT TTA CAG TAT CTA GAG 403
His Leu Ala Pro Glu Leu Gly Lys Leu Glu His Leu Gln Tyr Leu Glu
85 90 95 100
CTC TAC AAA AAC AAC ATC CAA GGA ACT ATA CCT TC~ GAA CTT GGA AAT 451
Leu Tyr Lys Asn Asn Ile Gln Gly Thr Ile Pro Ser Glu Leu Gly Asn
105 110 115
cIG AAG AAT CTC ATC AGC TTG GAT CTG TAC AAC AAC AAT CTT ACA GGG 499
Leu Lys Asn Leu Ile Ser Leu Asp Leu Tyr Asn Asn Asn Leu Thr Gly
120 125 130
ATA GTT CCC ACT TCT TTG GGA AAA TTG AAG TCT CTG GTC TIT TTA CGG 547
Ile Val Pro Thr Ser Leu Gly Lys Leu Lys Ser Leu Val Phe Leu Ary
135 140 145
CTT AAT GAC AA CGA TTG ACC GGT CCA ATC CCT AGA GCA CTC ACG GCA 595
Leu Asn Asp Asn Arg Leu Thr Gly Pro Ile Pro Arg Ala Leu Thr Ala
150 155 160
ATC CCA AGC CTT AAA GTT ~rl GAC GTC TCA AGC AAT GAT TTG TGT GGA 643
Ile Pro Ser Leu Lys Val Val Asp Val Ser Ser Asn Asp Leu Cys Gly
165 170 175 180
ACA ATC CCA ACA AAC G~A CCC m GCT CAC ATT CCT TTA CAG AAC m 691
Thr Ile Pro Thr Asn Gly Pro Phe Ala His Ile Pro Leu Gln Asn Phe
185 190 195
GAG AAC AAC CCG AGA 1l~ GAG GGA CCG GAA TTA CTC ~GT CTT GCA AGC 739
_
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Glu Asn Asn Pro Arg Leu Glu Gly Pro Glu Leu Leu Gly Leu Ala Ser
200 205 210
TAC GAC ACT A~C TGC ACC TGAAACAACT GC~3UUY4~CT GAAAATGAAG 787
Tyr Asp Thr Asn Cys Thr
215
AArl~x~xiG TGAC~ll~-l~ A42ACACTTC ACX~rrTAT CA~ATATCAC ATCTATTATG 847
TAATA~GTAT ATATATGTAG TAUUUUK~LAA A~AAATGAAG AATCGAATCG GTAATATCAT 907
~ AA IT~F~:U~rr C~A~ AT~-rAAAATT TCTA~ATGCG Allll~l 967
AAATTACTCA CACT 981
~2) INFO~MATIoN FOR SEQ ID NO: 25:
(i) ~Qu~u~ CHARACTERISTICS:
(A) LENGTH: 218 amlno acids
(B) TYPE: amino acid
~D) TOPOLOGY: linear
l~ ~ TYPE: protein
~xi) ~u~ v~x~ oN: SEQ ID NO: 25:
Met Ala Ser Arg Asn Tyr Arg Trp Glu Leu Phe Ala Ala Ser Leu Thr
1 5 10 15
~eu Thr Leu Ala Leu Ile His Leu Val Glu Ala Asn Ser Glu Gly Asp
Ala Leu Tyr Ala Leu Arg Arg Ser Leu Thr Asp Pro Asp His Val Leu
Gln Ser Trp Asp Pro Thr Leu Val Asn Pro Cys Thr Trp Phe His Val
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Thr Cys A~n Gln Asp ~n Arg Val Thr Arg Val Asp Leu Gly Asn SOE
~sn Leu Ser Gly His Leu Ala Pro Glu Leu G~y Lys Leu Glu His Leu
~ln Tyr Leu Glu Leu Tyr Lys Asn Asn Ile Gln Gly T~r Ile Pro Ser
100 105 110
Glu Leu Gly }~3n Leu Lys Asn Leu Ile Ser Leu Asp Leu Tyr Asn Asn
115 120 125
Asn Leu Thr Gly Ile Val Pro Thr Ser Leu Gly Lys Leu Lys Ser Leu
130 135 140
Val Phe Leu Ary Leu Asn Asp Asn Arg Leu Thr Gly Pro Ile Pro Arg
145 150 155 160
~la Leu Thr Ala Ile Pro Ser Leu Lys Val Val Asp Val Ser Ser Asn
165 170 175
~sp Leu Cys Gly Thr Ile Pro Thr Asn Gly Pro Phe Ala His Ile Pro
180 185 190
~eu Gln P~n Phe Glu Asn Asn Pro Ary Leu Glu Gly Pro Glu Leu Leu
195 200 205
Gly Leu Ala Ser Tyr Asp Thr Asn Cys Thr
210 215
(2) INFOR ~ TICN FOR SEy ID N~: 26:
r~ ~: C~ Ll~KI~ S:
(A) LE~X:rH: 789 kase FxLLrs
(8) TYPE: m~ ;c acid
.
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(C) STRA~LN~SS: single
(D) TOPOLOGY: linear
(ii) ~nT.FflTT.~ TYPE: c~NA to mRN~
(iii) HY~U~ LAL: N~
~ix) FEATURE:
~A) N~ME/KEY: CDS
~B) LOCATIoN: 2..661
(xi) SEQWEN~E DESCRIPTIQN: SEQ lD N~: 26:
T CGA CCC ACG ~l CCG CGA AAC TAT CGG TGG GAG ~l~ TT~ GCA GCT 46
Arg Pro Thr Ary Pro Arg Asn Tyr Arg Trp Glu Leu Phe Ala Ala
1 5 10 15
TCG TTA ATC CTA ACC TTA GCT TTG ATT CAC CTG GTC GAA GCA AAC TCC 94
Ser Leu Ile Leu Thr Leu Ala Leu Ile His Leu Val Glu Ala Asn Ser
20 25 30
GAA GGA GAT GCT CTT TAC GCT CTT CGC CGG AGT TTA ACA GAT CCG GAC 142
Glu Gly Asp Ala Leu Tyr Ala Leu Arg Arg Ser Leu Thr Asp Pro Asp
35 40 45
CAT GTT CTC CAG AGC TGG GAT CCA ACT CTT GIT M T CCT TGT ACC TGG 190
His Val Leu Gln Ser ffl Asp Pro Thr Leu Val Asn Pro Cys Thr Trp
50 55 60
TTC CAT Gl~ ACC TGT AAC C M OE AAC CGC Gl~ ACT CGT ~1~ GAT TTG 238
Phe His Val Thr Cy~ Asn Gln Asp Asn Arg Val Thr Ary Val Asp Leu
65 70 75
G ~ AAT TCA AAC CTC TCT GGA CAT CTT GCG CCT GAG CTT GGG A~G CTT 286
Gly Asn Ser Asn Leu Ser Gly His Leu Ala Pro Glu Leu Gly Lys Leu
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80 85 90 95
GAA CAT TTA CAG TAT CTA GAG ~_-l~ TAC AAA AAC A~C ATC CAA GGA ACT 334
Glu His Leu Gln Tyr Leu Glu Leu Tyr Lys Asn Asn Ile Gln Gly Thr
100 105 110
ATA CCT TCC GAA CTT G A AAT CTG AAG AAT CTC ATC AGC TTG GAT CTG 382
Ile Pro Ser Glu Leu Gly Asn Leu Lys Asn Leu Ile Ser Leu Asp Leu
115 120 125
TAC A~C APC AAT CIT ACA GGG ATA ~ CCC ACT TCT TIG GGA AAA T5~ 430
Tyr Asn Asn Asn Leu Thr Gly Ile Val Pro Thr Ser Leu Gly Lys Leu
130 135 lg0
AAG TCT CTG GTC TTT TTA CGG CTT AAT GAC AAC CGA TTG ACG GGG CCA 478
Lys Ser Leu Val Phe Leu Arg Leu Asn Asp Asn Arg Leu Thr Gly Pro
145 150 155
ATC CCT AGA GCA CTC ACT GCA ATC CCA AGC CTT AAA (il-l GTT GAT GTC 526
Ile Pro Arg Ala Leu Thr Ala Ile Pro Ser Leu Lys Val Val Asp Val
160 165 170 175
TCA AGC AAT GAT TTG TGT GGA ACA ATC CCA ACA AAC GGA CCT TTT GCT 574
Ser Ser Asn Asp Leu Cys Gly Thr Ile Pro Thr Asn Gly Pro Phe Ala
180 185 190
CAC ATT CCT TTA CAG AAC m GAG AAC A~C CCG AGG TI~ GAG GGA CCG 622
His Ile Pro Leu Gln Asn Phe Glu Asn Asn Pro Arg Leu Glu Gly Pro
195 200 205
GAA TTA CTC G~;T CTT GCA AGC TAC GAC ACT AAC TGC ACC IWU~TT 671
Glu Leu Leu Gly Leu Ala Ser Tyr A~p Thr Asn Cys Thr
210 215 220
a3C~a;A~ G~AAT~AG AArl~i TGACt-rl~,-lA AGAi:~l~ A~mAT 731
CAAATATCAC ATCTACTATG TAATAA~TAT ATATATGTAG ~AAA~ A~a~ 789
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(2) INFORM~TICN FOR SEQ m N~ 27:
~r~ CHARALl~K~ L~:
(A) LENGT~: 220 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
~ nT~T~TT T~' TYPE: protein
(Xi) ~U~:;M_I~; V~;SU~ rJ: SE~Q ID NO: 27:
Asg Pro Thr Arg Pro Arg Asn Tyr Arg ffl Glu Leu Phe Ala Ala Ser
1 5 10 15
Leu Iie Leu Thr Leu Ala Leu Ile His Leu Val Glu Ala Asn Ser Glu
Gly Asp Ala Leu Tyr Ala Leu Arg Arg Ser Leu Thr Asp Pro Asp His
Val Leu Gln Ser Trp Asp Pro Thr Leu Val Asn Pro Cys Thr Trp Phe
His Val Thr Cys Asn Gln Asp Asn Arg Val Thr Ary Val Asp Leu Gly
Asn Ser Asn Leu Ser Gly His Leu Ala Pro Glu Leu Gly Lys Leu Glu
g5
His Leu Gln Tyr Leu Glu Leu Tyr Lys Asn Asn Ile Gln Gly Thr Ile
100 105 110
Pro Ser Glu Leu Gly Asn Leu Lys Asn Leu Ile Ser Leu Asp Leu Tyr
115 120 125
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Asn Asn Asn Leu Thr Gly Ile Val Pro Thr Ser Leu Gly Lys Leu Lys
130 135 140
Ser Leu Val Phe Leu Arg Leu Asn Asp Asn Arg Leu Thr Gly Pro Ile
145 150 155 160
Pro Arg Ala Leu Thr Ala Ile Pro Ser Leu Lys Val Val Asp Val Ser
165 170 175
Ser Asn Asp Leu Cys Gly Thr Ile Pro Thr Asn Gly Pro Phe Ala His
180 185 190
Ile Pro Leu Gln Asn Phe Glu Asn Asn Pro Arg Leu Glu Gly Pro Glu
195 200 205
Leu Leu Gly Leu Ala Ser Tyr Asp Thr Asn Cys Thr
210 215 220
~2) INFORM~TICN FOR SEQ ID NO: 28:
(i) ~u~: C}~CS:
(A~ LENGTH: 894 base pairs
(B) TYPE: nucleic acid
(C) STRA~u~LN~SS: single
(D) T~PO~OGY: lin~Ar
(ii) ~nT.T~T.T~ TYPE: cDNA to m~NA
(iii) H~u~ ~AL: NO
(ix) E~I~E:
(A) N~ME/KEY: CDS
(B) LOCATIoN: 1..675
~xi) ~yU~NL~ ~Kl~l'l~N: SEQ ID NO: 28:
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GGA CCG ATT CAA GCC TCC GAA GGG GAC GCT CTT CAC GCG CTT CGC CGG 48
Gly Pro Ile Gln Ala Ser Glu Gly Asp Ala Leu His Ala Leu Ary Arg
1 S 10 15
AGC TTA TCA GAT CCA GAC AAT GTT GTT CAG AGT TGG GAT CCA ACT CTT 96
Ser Leu Ser Asp Pro Asp Asn Val Val Gln Ser Trp Asp Pro Thr Leu
GTT AAT CCT T~T ACT TGG TTT CAT GTC ACT TGT AAT CAA CAC CAT CAA 144
Val Asn Pro Cys Thr Trp Phe His Val Thr Cys Asn Gln His His Gln
GTC ACT CGT CTG GAT TT~ GGG AAT TCA AAC TTA TCT GGA C'AT CTA GTA 192
Val Thr Arg Leu Asp Leu Gly Asn Ser Asn Leu Ser Gly His Leu Val
CCT GAA CTT GGG A~G CTT GAA CAT TTA CAA TAT CTG TAT GGA ATC ATC 240
Pro Glu Leu Gly Lys Leu Glu His Leu Gln Tyr Leu Tyr Gly Ile Ile
ACT CTT TTG CCT TTT GAT TAT CTG AAA ACA TTT ACA TTA TCA ~-l~ ACA 288
Thr Leu Leu Pro Phe Asp Tyr Leu Lys Thr Phe Thr Leu Ser Val Thr
CAT ATA ACA TTT T~C TTT GAG TCA TAT AGT GAA CTC TAC A~A AAC GAG 336
His Ile Thr Phe Cys Phe Glu SOE Tyr Ser Glu Leu Tyr Lys Asn Glu
100 105 110
ATT CAA GGA ACT ATA CCT T~T GAG CTT GGA AAT cm A~G AGT CTA ATC 384
Ile Gln Gly Thr Ile Pro Ser Glu Leu Gly Asn Leu Lys Ser Leu Ile
llS 120 125
AGT TTG GAT cm TAC AAC AA~ AAT CT~C ACC GGG AAA ATC C~A TCT TCT 432
Ser Leu Asp Leu Tyr Asn Asn Asn Leu Thr Gly Lys Ile Pro Ser Ser
130 135 140
., , . . ~
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GEA AAA TI~; AAG TCA CrT GTT TIT TI~i CGG ~ AAC GAA. AAC CGA 480
Leu Gly Lys Leu Lys Ser Leu Val Phe Leu Arg Leu Asn Glu Asn Arg
145 150 155 160
TIG ACC G5T CCT ATT CCT AGA GAA CTC ACA GIT ATT TCA AGC t~ AAA 528
Leu Thr Gly Pro Ile Pro Arg Glu Leu Thr Val Ile Ser Ser Leu Lys
165 170 175
ETT G$T GAT GTC TCA GGG AAT GAT Tl~j TGT GGA ACA ATT CCA GTA GAA 576
Val V~ p Val Ser Gly Asn Asp Leu Cys Gly Thr Ile Pro Val Glu
180 185 190
GGA CCT TTT EAA CAC ATT CCT ATG CAA AAC TTT GAG A~C AAC CTG AEA 624
Gly Pro Phe Glu His Ile Pro Met Gln Asn Phe Glu Asn Asn Leu Arg
195 200 205
rl~i GAG GGA CCA GAA CTA CTA E~T ~ GCG AGC TAT EAC ACC AAT TGC 672
Leu Glu Gly Pro Glu Leu Leu Gly Leu Ala Ser Tyr A~ $hr Asn Cys
210 215 220
ACT TMAMGA~G ~ TATAAAGAAG AATE$TAGGT GA~ ~A 725
Thr
225
GAAL~ ,-lA CC~AGla~ GTAAATCTAT ATAG:GCC~ GTllCA$t~TT ATATAT~AA 785
iAGAG ACAGTAA~T GCAAT(~TATT G5TATIGGTA G~A~I~ AAATEAGAAT 845
'l~'l'l'l~'lAA TTGGATl'rGT ~ ll~-rlATG TAaCII~:AT TI~TATTA 894
~2) ~ORMATIaN ~OR SEQ ID ND: 29:
( i ) ~F CHaR~r~STICS:
(A) ~H: 225 am~no acids
~B) TYPE: amino acid
(D) TOPOI~DGY: l~ear
CA 02254839 1998-11-13
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(ii) MnT~T~TT~ TYPE: protein
~Xi) 5E2UEN~E ~K1~1'1U~: SEQ ID N3: 29:
Gly Pro Ile Gln Ala Ser Glu Gly Asp Ala Leu His Ala Leu Arg Arg
1 5 10 15
Ser Leu S OE Asp Pro Asp Asn Val Val Gln S OE Trp Asp Pro Thr Leu
20 25 30
Val Asn Pro Cys Thr Trp Phe His Val Thr Cys Asn Gln His His Gln
35 40 45
Val Thr Arg Leu Asp Leu Gly Asn Ser Asn Leu S OE Gly His Leu Val
50 55 60
Pro Glu Leu Gly Lys Leu Glu His Leu Gln Tyr Leu Tyr Gly Ile Ile
65 70 75 80
Thr Leu Leu Pro Phe Asp Tyr Leu Lys Thr Phe Thr Leu S OE Val Thr
85 90 95
His Ile Thr Phe Cys Phe Glu Ser Tyr S OE Glu Leu Tyr Lys Asn Glu
100 105 110
Ile Gln Gly Thr Ile Pro Ser Glu Leu Gly Asn Leu Lys Ser Leu Ile
115 120 125
Ser Leu Asp Leu Tyr Asn Asn Asn Leu Thr Gly Lys Ile Pro SOE S OE
130 135 140
Leu Gly Lys Leu Lys Ser Leu Val Phe Leu Arg Leu Asn Glu Asn Arg
145 lS0 lSS 160
Leu m r Gly Pro Ile Pro Arg Glu Leu Thr Val Ile Ser Ser Leu Lys
165 170 175
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Val Val Asp Val Ser Gly Asn Asp Leu Cys Gly Thr Ile Pro Val Glu
180 185 190
Gly Pro Phe Glu His Ile Pro Met Gln Asn Phe Glu Asn Asn Leu Arg
195 200 205
Leu Glu Gly Pro Glu Leu Leu Gly Leu Ala Ser Tyr Asp Thr Asn Cys
210 215 220
Thr
225
(2) INFORMATICN FOR SEQ ID ND: 30:
(i) SEQUENCE C~PRA~q~RISTICS:
(A) LENGTH: 1063 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: ~inear
(ii) ~r.r~Y~Irr~ TYPE: cnN~ to mRN~
(ix) ~ E:
(A) N~ME/KEY: CDS
(B) LOCATIoN: 106..759
(Xi) ~ Kl~llU~: SEQ ID N~: 30:
IXX~YXX:i~G C~l~AoGA AACCCTAATT ~ l~l~ Al~-ll~ll~A GAAAAITACT 60
CAAATT~-lA TrA~AT~T ~l~l~ll~A C~l~ATAG CTCAC ATG GCG TCT 114
Met Ala Ser
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CGA AAC TAT CGG TGG GAG CTC TTC GCA GCT TCG TTA ATC CTA ACC TTA 162
Arg Asn Ty.r Arg Trp Glu Leu Phe Ala Ala Ser Leu Ile Leu Thr Leu
GCT TTG ATT CAC CTG GTC GAA GCA AAC TCC GAA GGA GAT GCT CTT TAC 210
Ala Leu Ile His Leu Val Glu Ala Asn Ser Glu Gly Asp Ala Leu Ty.r
GCT CTT CGC CGG AGT TTA ACA GAT C~ GAC CAT GTT ~-~ CAG AGC TGG 258
Ala Leu Arg Arg Ser Leu Thr Asp Pro Asp His Val Leu Gln Ser T~p
gO g5 50
GAT CCA ACT CTT GTT AAT CCT TGT ACC TGG T~C CAT GTC ACC TGT A~C 306
Asp Pro Thr Leu Val Asn Pro Cys Thr Trp Phe His Val Thr Cys Asn
CAA GAC A~C CGC GTC ACT CGT GTG GAT TTG GGG AAT TCA AAC CTC TCT 354
Gln Asp Asn Arg Val Thr Arg Val Asp Leu Gly Asn Ser Asn Leu Ser
GGA CAT CTT GCG CCT GAG Cll GGG AAG CTT GAA CAT TTA CA~ TAT CTA 402
Gly His Leu Ala Pro Glu Leu Gly Lys Leu Glu His Leu Gln Tyr Leu
GAG CTC TAC A~A A~C AAC ATC CAA GGA ACT ATA CCT TCC GAA CTT GGA 450
Glu Leu Tyr LYS Asn Asn Ile Gln Gly Thr Ile Pro Ser Glu Leu Gly
100 105 110 115
AAT ~1~ AAG A~r crc ATC AGC T~ GAT CTG TAC AAC AAC AAT CTT ACA 498
Asn Leu Lys Asn Leu Ile SOE Leu Asp Leu Ty.r Asn Asn Asn Leu Thr
120 125 130
GGG ATA GTT CCC ACT TCT TTG GGA AAA TTG AAG TCT CTG GTC TTT TTA 546
Gly Ile Val P.ro Thr SOE Leu Gly Lys Leu Lys SOE Leu Val Phe Leu
135 140 145
,.
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CGG CTT AAT GAC AAC CGA TTG ACG GGG CCA ATC CCT AGA GCA CTC ACT 5g4
Arg Leu Asn Asp Asn Arg Leu Thr Gly Pro Ile Pro Arg Ala Leu Thr
150 155 160
GCA ATC CCA AGC CTT AAA ~~ GTT GAT GTC TCA AGC AAT GAT TTG l~l 642
Ala Ile Pro Ser Leu Lys Val Val Asp Val SOE Ser Asn Asp Leu Cys
165 170 175
GGA ACA ATC CCA ACA AAC GGA CCT m GCT CAAC ATT CCT TTA CAG AAC 690
Gly Thr Ile Pro Thr Asn Gly Pro Phe Ala His Ile Pro Leu Gln Asn
180 185 190 195
TTT GAG A~C AAC CCG AGG TTG GAG GGA CCG GAA TTA C'TC GGT CTT GCA 738
Phe Glu Asn Asn Pro Arg Leu Glu Gly Pro Glu Leu Leu Gly Leu Ala
200 205 210
AGC TAC GAC ACT AAC TGC ACC lC~;AAAATT GGCPAAACCT GAAAATGAAG 789
Ser Tyr Asp Thr Asn Cys Thr
215
A~ G TGAC~-ll~-lA AGAACACTT~ A~CACTTTAT CAAATATCAC ATCTACTATG 849
TAATAAGTAT ATATAT&TAG TCC~AAAAAA AAAT&AAGAA TCGAATCAGT AATATCATCT 909
G~ AATT GAaAA~TTTa A~l~l~l~l AT~-lAAAATT TCTAAATGCG A~ C~-l 969
AC~ TAAT~T ~l~ wll~lw GATTeTGaa~ AGTA~CATTT GTATTGGTAT GGTATCAAGT 1029
AA AAAAAAAAAA A~AA 1063
(2) INFORM~TICN FOR SEQ ID NO: 31:
CH~ ~RT~CS
~A) LENGIH: 218 amuno acids
(B) TYPE: amino acid
... . ... . .
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(D) TOPOLOGY: }inear
(ii) ~nT-T~TT-~ TYPE: protein
(Xi) .CF~ l~J:;~L~1'1~: SE~ ID ND: 31:
Met Ala Ser Arg Asn Tyr Arg Trp Glu Leu Phe Ala Ala Ser Leu Ile
1 5 10 15
Leu m r Leu Ala Leu Ile His Leu Val Glu Ala Asn Ser Glu Gly Asp
20 25 30
Ala Leu Iyr Ala Leu Arg Arg Ser Leu Thr Asp Pro Asp His Val Leu
35 40 45
Gln Ser Trp Asp Pro Thr Leu Val Asn Pro Cys Thr Trp Phe His Val
50 55 60
Thr Cys Asn Gln Asp Asn Arg Val Thr Arg Val Asp Leu Gly Asn Ser
65 70 75 80
Asn Leu Ser Gly His Leu Ala Pro Glu Leu Gly Lys Leu Glu His Leu
85 90 95
Gln Tyr Leu Glu Leu Tyr Lys Asn Asn Ile Gln Gly Thr Ile Pro Ser
100 105 110
Glu Leu Gly Asn Leu Lys Asn Leu Ile Ser Leu Asp Leu Tyr Asn Asn
115 120 125
Asn Leu m r Gly Ile Val Pro Thr Ser Leu Gly Lys Leu Lys Ser Leu
130 13S 140
Val Phe Leu Arg Leu Asn Asp Asn Arg Leu Thr Gly Pro Ile Pro Arg
145 150 lSS 160
Ala Leu Thr Ala Ile Pro Ser Leu Lys Val Val Asp Val Ser Ser Asn
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165 170 175
Asp Leu Cys Gly Thr Ile Pro Thr Asn Gly Pro Phe Ala His Ile Pro
180 185 190
~eu Gln Aqn Phe Glu Asn Asn Pro Arg Leu Glu Gly Pro Glu Leu Leu
195 200 205
Gly Leu Ala Ser Tyr Asp Thr Asn Cys Thr
210 215
(2) INFORMATICN FOR SEQ ID N~: 32:
(i) ~FQUFNr~ CHaRAOTE~ISTICS:
~AJ LENGTX: 2089 base pairs
(B) TYPE: n~ p;c acid
(C) STR~ : double
(D) TOPOLOGY: linear
(ii) ~nT.Frl~T.T~'. TYPE: c~NA to mRN~
(iii) H~u~ ~AL: N~
(iii) ANTI-SENSE: N~
(vi) OhI~lNAL SOURCE:
(A) ORGANISM: ArAhi~psl~ th~l;An~
(Vii ) TMMF nT~I E SCURCE:
~B) CLCNE: SERK gene cDNA
(ix) FE~TURE:
(A) N~ME/KEY: CDS
(B) LOCATIoN: 195..2069
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(xi) ~v~ DESCRIPTICN: SEQ ID N~: 32:
GGATmTAT TTTATTTTTT AL~ ,-rlllAATG CTAA~l~ mA~GG 60
TTATCG~A MT&~t rGAG ~ l~l~l GTAAAGrGTT M~ 120
Allll~AA GTTAGGGTTT l~-l~ATCT GAA~ATCA AATCA~GATT CG~AATTTAC 180
CArl~,-ll~-ll I~AA ATG &~G TCG AGT TAT GTt GTG TTT ATC TTA CTT qCA 230
Met Glu Ser Ser Tyr Val Val Phe Ile Leu Leu Ser
5 10
CTG ATC TTA C'TT CCG MT CAT TCA CTG TGG CTT GCT TCT GCT AAT TTG 278
Leu Ile Leu Leu Pro Asn His Ser Leu Trp Leu Ala Ser Ala Asn Leu
15 20 25
GAA GGT GAT GCT TTG CAT ACT TTC; AG;G GTT ACT CTA GTT GAT CCA AAC 326
Glu Gly Asp Ala Leu His Thr Leu Arg Val Thr Leu Val Asp Pro Asn
30 35 40
AAT GTC TIG CAG AGC TGG GAT CCT ACG CTA GTG MT CCT TGC ACA TGG 374
Asn Val Leu Gln Ser Trp Asp Pro Thr Leu Val Asn Pro Cys Thr Trp
45 50 55 60
TTC CAT GTC ACT TGC AAC MC GAG AAC AGT GTC ATA AGA GTT GAT T~; 422
Phe His Val Thr Cys Asn Asn Glu Asn Ser Val Ile Ary Val Asp Leu
65 70 75
GGG MT GCA GAG TTA TCT GGC CAT TTA GTT CCA GAG CTT (;GT GTC~ CTC 470
Gly Asn Ala Glu Leu Ser Gly His Leu Val Pro Glu Leu Gly Val T~-
80 85 90
AAG MT TTG CAG TAT TTG GAG CTT TAC AGT AAC AAC ATA ACT GGC CCG 518
Lys Asn Leu Gln Tyr Leu Glu Leu Tyr Ser Asn Asn Ile Thr Gly Pro
95 100 105
ATT CCT AGT AAT CTT GGA MT Clt; ACA AAC TTA GTG AGT TIG GAT CTT 566
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Ile Pro Ser Asn Leu Gly Asn Leu Thr Asn Leu Val Ser Leu Asp Leu
110 115 120
TAC TTA AAC AGC TT~ TCC G&T CCT ATT CCG GAA TCA TTG GGA AAG CTT 614
Tyr Leu Asn Ser Phe Ser Gly Pro Ile Pro Glu Ser Leu Gly Lys Leu
125 130 135 140
TCA AAG CTG AGA TTT CTC CGG CTT AAC AAC AAC AGT CTC ACT G~G TCA 662
Ser Lys Leu Arg Phe Leu Arg Leu Asn Asn Asn Ser Leu Thr Gly Ser
145 150 155
ATT CCT ATG TCA CTG ACC AAT ATT ACT ACC C~T CAA GTG TTA GAT CTA 710
Ile Pro Met Ser Leu Thr Asn Ile Thr Ihr Leu Gln Val Leu Asp Leu
160 165 170
TCA A~T AAC AGA CTC TCT G5T TCA GTT C~T GAC AAT G&C TCC TTC TCA 758
Ser Asn Asn Arg Leu Ser Gly Ser Val Pro Asp Asn Gly Ser Phe Ser
175 180 185
CTC TTC ACA CCC ATC AGT TTT GCT AAT AAC TTA GAC CTA TGT G&A CCT 806
Leu Phe Thr Pro Ile Ser Phe Ala Asn Asn Leu Asp Leu Cys Gly Pro
190 195 200
GTT ACA AGT CAC CCA T~l C~T GGA TCT CCC CCG m TCT CCT CCA CCA 854
Val Thr Ser His Pro Cys Pro Gly Ser Pro Pro Phe Ser Pro Pro Pro
205 210 215 220
CCT m ATT CAA CCT CCC CCA GTT TCC ACC CCG AGT GGG TAT GGT ATA 902
Pro Phe Ile Gln Pro Pro Pro Val SOE Thr Pro Ser Gly Tyr Gly Ile
225 230 235
ACT G&A GCA ATA GCT ~l G~A ~ GCT GCA GGT GCT GrT TT& CCC TTT 950
Thr Gly Ala Ile Ala Gly Gly Val Ala Ala Gly Ala Ala T~l Pro Phe
240 245 250
~l GCT CCT GCA ATA GCC m GCT TGG T&G CGA CGA AGA AGC CCA CTA 998
~la Ala Pro Ala Ile Ala Phe Ala Trp Trp Arg Arg Ary Ser Pro Leu
. .
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255 260 265
GAT ATT TTC TTc GAT GTC CCT GCC GAA &AA &AT CCA &AA GTT CAT CTG 1046
Asp Ile Phe Phe Asp Val Pro Ala Glu Glu Asp Pro Glu Val His Leu
270 275 280
&GA CAG CTC AAG AGG TTT TCT TTG CGG &AG CTA CAA &T& GCG AGT GAT 1094
Gly Gln Leu Lys Ary Phe Ser Leu Arg Glu Leu Gln Val Ala Ser Asp
285 290 295 300
GGG m A&T AAC AAG AAC ATT TT& GGC AGA GGT GG& TTT GGG AAA GTC 1142
Gly Phe Ser Asn Lys Asn Ile Leu Gly Ary Gly Gly Phe Gly Lys Val
305 310 315
TAC AAG GGA CGC TT& GCA GAC GGA ACT CTT GTT GCT GTC AAG AGA CT& 1190
Tyr Lys Gly Arg Leu Ala Asp Gly Thr Leu Val Ala Val Lys Arg Leu
320 325 330
AA& &AA GAG CGA ACT CCA > &GA &A& CTC CA& TTT CAA ACA GAA GTA 1238
Lys Glu &lu Ary Thr Pro Gly Gly Glu Leu Gln Phe Gln Thr Glu Val
335 340 345
GA& AT& ATA A&T AT& GCA GTT CAT CGA AAC CT& TTG AGA TTA CGA > 1286
Glu Met Ile Ser Met Ala Val His Arg Asn Leu Leu Ary Leu Arg Gly
350 355 360
TTC T&T AT& ACA CCG ACC GAG AGA TT~ CTT GT& TAT CCT TAC ATG GCC 1334
Phe Cys Met Thr Pro Thr Glu Arg Leu Leu Val Tyr Pro Tyr Met Ala
365 370 375 380
AAT GGA AGT GTT GCT TCG T~T ~-~ ABA GAG AGG CCA CCG TCA CAA CCT 1382
Asn Gly Ser Val Al~ Ser Cys Leu Arg Glu Arg Pro Pro Ser Gln Pro
385 390 3g5
CCG C~ GAT TGG CCA AC& CGG AAG AGA ATC GCG CTA GGC TCA G~T CGA 1430
Pro Leu Asp Trp Pro Thr Arg Lys Arg Ile Ala Leu Gly Ser Ala Ary
400 405 410
CA 02254839 l998-ll-l3
W 097/43427 PCT~EP97102443
-94-
GGT TTG TCT TAC CTA CAT GAT CAC TGC GAT CCG AAG ATC ATT CAC CGT 1478
Gly Leu Ser Tyr Leu His Asp His Cys Asp Pro Lys Ile Ile His Arg
415 420 425
GAC GTA AAA GCA GCA AAC ATC CTC TTA GAC GAA GAA TTC GAA GCG GTT 1526
Asp Val Lys Ala Ala Asn Ile Leu Leu Asp Glu Glu Phe Glu Ala Val
430 435 440
GTT GGA GAT TTC GGG TTG GCA AAG CTT ATG GAC TAT AAA GAC ACT CAC 1574
Val Gly Asp Phe Gly Leu Ala Lys Leu Met Asp Tyr Lys Asp Thr His
445 450 455 460
GTG ACA ACA GCA GTC CGT GGC AX ATC GGT CAC ATC GCT CCA GAA TAT 1622
Val Thr Thr Ala Val Arg Gly Thr Ile Gly His Ile Ala Pro Glu Tyr
465 470 475
CTC TCA ACC GGA AAA TCT TCA GAG AAA ACC GAC G~T TTC GGA TAC GGA 1670
Leu Ser Thr Gly Lys Ser Ser Glu Lys Thr Asp Val Phe Gly Tyr Gly
480 485 490
ATC ATG CTT CTA GAA CTA ATC ACA GGA CAA AGA GCT TTC GAT CTC GCT 1718
Ile Met Leu Leu Glu Leu Ile Thr Gly Gln Arg Ala Phe Asp Leu Ala
495 500 505
CGG CTA GCT AAC GAC GAC GAG GTC ATG TTA CTT GAC TGG GIG AAA GGA 1766
Arg Leu Ala Asn Asp Asp Asp Val Met Leu Leu Asp Trp Val Lys Gly
510 515 520
TTG TTG AAG GAG AAG AAG CTA GAG ATG TTA w ~ GAT CCA ~AT CTT CAA 1814
Leu Leu Lys Glu Lys Lys Leu Glu Met Leu Val Asp Pro i~sp Leu Gln
525 530 535 540
ACA AAC TAC GAG GAG AGA GAA Cl~ GAA CAA GTG ATA CAA GTG GCG TT~ 1862
Thr Asn Tyr Glu Glu Arg Glu Leu Glu Gln Val Ile Gln ~al Ala Leu
545 550 555
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CTA TGC ACG C M GGA TCA CCA ATG GAA AGA C~A AAG ATG TCT GAA GTT 1910
Leu Cys Thr Gln Gly Ser Pro Met Glu Arg Pro Lys Met Ser Glu Val
560 565 570
GTA AGG ATG CTG GAA GGA GAT GGG CTT GCG GAG AAA TGG GAC GAA TGG 1958
Val Arg Met Leu Glu Gly Asp Gly Leu Ala Glu Lys Trp Asp Glu Trp
575 580 585
CAA AAA GTT GAG ATT TT~ AGG GAA GAG ATT GAT TT~ AGT CCT AAT CCT 2006
Gln Lys Val Glu Ile Leu Arg Glu Glu Ile Asp Leu Ser Pro Asn Pro
590 595 600
AAC TCT GAT TGG ATT CTT GAT TCT ACT TAC AAT Il~ CAC GCC GTT GAG 2054
Asn Ser Asp Trp Ile Leu Asp Ser Thr Tyr Asn Leu His Ala Val Glu
605 610 615 620
TTA TCT GGT CCA AGG TAi~A~AAhA A MUYNV~A~ 2089
Leu Ser Gly Pro Arg
625
(2) INFORMATICW FOR SEQ ID N~: 33:
u~ CH2~ACTERISTICS:
(A) LENGTH: 625 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) ~nTFrl~ TYPE: protein
m N~ 33:
Met Glu Ser Ser Tyr Val Val Phe Ile Leu Leu Ser Leu Ile Leu Leu
1 5 10 15
Pro Asn His Ser Leu Trp Leu Ala Ser Ala Asn Leu Glu Gly Asp Ala
CA 02254839 l998-ll-l3
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-96-
Leu His Thr Leu Ary Val Thr Leu Val Asp Pro Asn Asn Val Leu Gln
35 40 45
Ser Trp Asp Pro Thr Leu Val Asn Pro Cys Thr Trp Phe His Val Thr
50 55 60
Cys Asn Asn Glu Asn Ser Val Ile Arg Val Asp Leu Gly Asn Ala Glu
65 70 75 80
Leu Ser Gly His Leu Val Pro Glu Leu Gly Val Leu Lys Asn Leu Gln
85 90 95
Tyr Leu Glu Leu Tyr Ser Asn Asn Ile Thr Gly Pro Ile Pro Ser Asn
100 105 110
Leu Gly Asn Leu Thr Asn Leu Val Ser Leu Asp Leu Tyr Leu Asn Ser
115 120 125
Phe Ser Gly Pro Ile Pro Glu Ser Leu Gly Lys Leu Ser Lys Leu Arg
130 135 140
Phe Leu Arg Leu Asn Asn Asn Ser Leu Thr Gly Ser Ile Pro ~et Ser
145 150 155 160
Leu Thr Asn Ile Thr Thr Leu Gln Val Leu Asp Leu Ser Asn Asn Arg
165 170 175
Leu Ser Gly Ser Val Pro Asp Asn Gly Ser Phe Ser Leu Phe Thr Pro
180 185 190
Ile Ser Phe Ala Asn Asn Leu Asp Leu Cys Gly Pro Val Thr Ser His
195 200 205
Pro Cys Pro Gly Ser Pro Pro Phe Ser Pro Pro Pro Pro Phe Ile Gln
210 215 220
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Pro Pro Pro Val Ser Thr Pro Ser Gly Tyr Gly Ile Thr Gly Ala Ile
225 230 235 240
Ala Gly Gly Val Ala Ala Gly Ala Ala Leu Pro Phe Ala Ala Pro Ala
245 250 255
Ile Ala Phe Ala Trp Trp Arg Arg Arg Ser Pro Leu Asp Ile Phe Phe
260 265 270
Asp Val Pro Ala Glu Glu Asp Pro Glu Val His Leu Gly Gln Leu Lys
275 280 285
Arg Phe Ser Leu Ary Glu Leu Gln Val Ala Ser Asp Gly Phe Ser Asn
290 295 300
Lys Asn Ile Leu Gly Arg Gly Gly Phe Gly Lys Val Tyr Lys Gly Arg
305 310 315 320
Leu Ala Asp Gly Thr Leu Val Ala Val Lys Arg Leu Lys Glu Glu Arg
325 330 335
Thr Pro Gly Gly Glu Leu Gln Phe Gln Thr Glu Val Glu Met Ile Ser
340 345 350
Met Ala Val His Arg Asn Leu Leu Arg Leu Arg Gly Phe Cys Met Thr
355 360 365
Pro Thr Glu Arg Leu Leu Val Tyr Pro Tyr Met Ala Asn Gly Ser Val
370 375 380
Ala Ser Cys Leu Arg Glu Arg Pro Pro Ser Gln Pro Pro Leu Asp Trp
385 390 395 400
Pro Thr Arg Lys Ary Ile Ala Leu Gly Ser Ala Arg Gly Leu Ser Tyr
405 410 415
Leu His Asp His Cys Asp Pro Lys Ile Ile His Arg Asp Val Lys Ala
CA 02254839 l998-ll-l3
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420 425 430
Ala Asn Ile Leu Leu Asp Glu Glu Phe Glu Ala Val Val Gly Asp Phe
435 440 445
Gly Leu Ala Lys Leu Met Asp Tyr Lys Asp Thr His Val Thr Thr Ala
450 455 460
Val Arg Gly Thr Ile Gly H~s Ile Ala Pro Glu Tyr Leu Ser Thr Gly
465 470 475 480
Lys Ser Ser Glu Lys Thr Asp Val Phe Gly Tyr Gly Ile Met Leu Leu
485 490 495
Glu Leu Ile m r Gly Gln Arg Ala Phe Asp Leu Ala Arg Leu Ala Asn
500 505 510
Asp Asp Asp Val Met Leu Leu Asp Trp Val Lys Gly Leu Leu Lys Glu
515 520 525
Lys Lys Leu Glu Met Leu Val Asp Pro Asp Leu Gln Thr Asn Tyr Glu
530 535 540
Glu Arg Glu Leu Glu Gln Val Ile G~n Val Ala Leu Leu Cys Thr Gln
545 550 555 560
Gly Ser Pro Met Glu Arg Pro Lys Met Ser Glu Val Val Arg Met Leu
565 570 575
Glu Gly Asp Gly Leu Ala Glu Lys Trp Asp Glu Trp Gln Lys Val Glu
580 585 590
Ile Leu Arg Glu Glu Ile Asp Leu Ser Pro Asn Pro Asn Ser Asp Trp
595 600 605
Ile Leu Asp Ser Thr Tyr Asn Leu His Ala Val Glu Leu Ser Gly Pro
610 615 620
CA 02254839 1998-11-13
PCT~EP97/02443
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99
Arg
625
, . . . . .
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