ROOT-SPECIFIC PROMOTER

PROMOTEUR SPECIFIQUE DES RACINES

English Abstract

A DNA which has the sequence shown in Figure 5 and which defines a gene
promoter region has been isolated from maize roots. The promoter may be used
for driving expression of foreign genes in the roots of plants. This is
particularly useful for expressing an insecticidal toxin, such as a delta-
endotoxin of Bacillus thuringiensis, to impart resistance to insect attack on
the roots of plants by Coleopteran insects.

French Abstract

Un ADN dont la séquence est illustrée dans la figure 5, et qui définit une région promotrice d'un gène, a été isolé des racines du maïs. Ce promoteur peut être utilisé pour diriger l'expression de gènes étrangers dans les racines de végétaux, ce qui est particulièrement utile pour exprimer une toxine insecticide, par exemple la delta-endotoxine de Bacillus thurigiensis, laquelle est capable de rendre les racines de ces végétaux résistantes aux dégâts causés par les coléoptères.

Claims

1. An isolated DNA sequence which codes for a promoter of a root-expressed
plant gene, said gene
having the sequence of FIGS. 5A-F (SEQ ID NO:3).
2. The isolated DNA sequence of claim 1 wherein said promoter has the sequence
of a 1.9 Kb XbaI
fragment of the sequence of FIGS. 5A-F (SEQ ID NO:3).
3. The isolated DNA sequence of claim 1 wherein said sequence is isolated from
the root tissue of a
target plant species.
4. The isolated DNA sequence of claim 3, wherein the target plant species is
Zea mays.
5. An isolated DNA construct comprising, in sequence, the DNA sequence of
claim 1 which comprises a
promoter, a coding region located downstream and controlled by said promoter
and a 3'-untranslated
region including a polyadenylation signal.
6. An isolated DNA construct comprising, in sequence, the DNA sequence of
claim 2 which comprises a
promoter, a coding region located downstream and controlled by said promoter
and a 3'-untranslated
region including a polyadenylation signal.
7. The construct of claim 5, wherein the coding region encodes a protein which
is toxic to root-attacking
organisms.
8. The construct of claim 6, wherein the coding region encodes a protein which
is toxic to root-attacking
organisms.
9. The construct of claim 7, wherein the protein is an insecticidal endotoxin
of Bacillus thuringiensis.
10. The construct of claim 8, wherein the protein is an insecticidal endotoxin
of Bacillus thuringiensis.
11. A plant genome into which the construct of claim 5, has been inserted such
that said promoter is
transcriptionally active.
12. A plant genome into which the construct of claim 6 has been inserted such
that said promoter is
transcriptionally active.
13. A plant genome into which the construct of claim 7 has been inserted such
that said promoter is
transcriptionally active.
14. A plant genome into which the construct of claim 8 has been inserted such
that said promoter is
transcriptionally active.
15. A plant genome into which the construct of claim 9 has been inserted such
that said promoter is
transcriptionally active.
16. A plant genome into which the construct of claim 10 has been inserted such
that said promoter is
transcriptionally active.

Description

CA 02317723 2000-07-11
wo aon~4 Pcrns9siozooo
R04T-SPECIFIC PROMOTER
This invention relates to a gene promoter
sequence which directs expression of a gene to the
root tissue of plants.
In the genetic improvement of plants by
molecular techniques,, it is desirable that
expression of inserted foreign genes be restricted
to tissue where that expression will have
significant effect. There are two principal
reasons for this. First, restricted expression,
rather than total (constitutive) is likely to be
less demanding on the metabolism of the plant.
Secondly, it would be good practice to direct
expression of the foreign gene to those parts of
the plant which are not used for human or animal
food when the expressed protein has no effect on
such food parts. This second reason may be
important when the effect which ingestion of the
expressed protein may have is not be fully known.
One widespread target for genetic improvement
of crop plants is the introduction of resistance to
insect attack. Certain insect species attack green
leaf tissue, whereas other, for example Coleoptera,
attack the roots. Similarly there are certain
disease-inducing microorganisms which attack the
below-ground plant tissue and any genetic
modification to impart resistance to such organisms
will require expression of the resistance-imparting
gene in the roots.

CA 02317723 2000-07-11
_ WO OOI29594 PCT/IB98l02000
An object of the present invention is to
provide a root-specific gene promoter sequence and
means for isolating same of root DNA.
According to the present invention there is
provided a DNA sequence, defining a promoter of a
root-expressed plant gene, having the sequence set
forth in Figure 5 herewith.
The said DNA may be isolated from the root
tissue of a particular target plant species of
interest. The preferred species is Zea mays.
The invention a1'~o provides a gene construct
comprising, in sequence, the aforesaid gene
promoter of the invention, a coding region located
downstream and controlled by the said promoter and
a 3'-untranslated region including a
polyadenylation signal.
Preferably the coding region encodes a protein
which is toxic to root-attacking organisms and more
preferably the protein is an insecticidal endotoxin
of Bacillus thurinaiensis.
Further according to the invention there is
provided a plant genome into which the gene
construct of the invention has been inserted by
transformation.
The promoter sequence of the invention may be
isolated from the genomic sequence to which a cDNA
derived from a root-expressed gene hybridises. A
genomic library is screened using the said cDNA as
a probe. Those geraomia fragments which hybridise
to the cDNA probe carry not~only the structural
gene but the promoter sequence associated
therewith. The promoter may then be isolated by
cleavage of the sequence around the location of the
translation start point of the structural gene
_ 2 _

CA 02317723 2000-07-11
_ WO OOIZ9594 PCTIIB98/02000
sequence. The sequences of suitable such cDNAs are
shown in Figures 1 and 2 were isolated from maize.
These cDNAs have been deposited (1) in a
plasmid designated pMR7 in an E.coli DHSa host and
(2) in a plasmid designated pMR7/10.1 in an oli
DHSa host, at the National Collection of Industrial
and Marine Bacteria, Aberdeen, United Kingdom, on
15th March 1990, under the Accession Number 40267.
These deposits were made under the terms of the
Budapest Treaty on the deposit of microorganisms
for patent purposes.'°'
Many genes specifying insecticidal proteins,
particularly delta-endotoxin genes of Bacillus
~,hurinaiensis have been reported in the
literature.
The invention will now be described, by way of
illustration, by the following Examples.
EXAMPLE 1
Total RNA was extracted from root tissue of
five-day old and fourteen-day old maize plants.
For use in certain comparative tests which will be
described later, total RNA was also isolated from
maize leaf and immature cob.
The RNA samples were purified using the
guanidinium thiocyanate/caesium chloride method and
poly(a)+mRNA purified on an oligo(dT) column. The
corresponding cDNAs were synthesised using the
oligo dT priming method and the cDNA cloned into
plasmid pUCl3 afte~e=Tinkering.
The success of each of-these stages was
monitored by incorporation of a label. Digests of
randomly picked clones from the cDNA library showed
a size distribution for inserts of between 300 and
1300 base pairs.
-3-

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Recombinants were individually transferred to
microtitre wells, in total the library consisted of
about 7,000 clones.
Clones representing genes with root enhanced
expression were identified by differential
screening. Identical filters were prepared from
the microtitre plates and hybridised separately
with probes prepared by first strand synthesis of
root mRNA and four week old leaf mRNA. The
autoradiographs were superimposed and recombinants
showing root enhanced expression were selected as
showing a more intense signal with the root probe
than with the leaf probe. Interestingly, none of
the selected clones showing differential
hybridisation fell into the highly expressed
category; all examples of this type showed equally
intense signals to both probes.
By this procedure, 235 clones were selected as
potentially showing a degree of differential
hybridisation after the first screen. This number
was reduced to thirteen after further screens.
The cDNA inserts of these thirteen clones
ranged from 300 to 1100 base pairs as judged by
restriction digestion or PCR. The inserts of each
of the thirteen candidate inserts were then used in
Northern hybridisations to confirm their tissue
specificity.
RNAs from the five-day and fourteen day old
root tissue and, fer comparison, from leaf and cob
tissue were probed to identify any which were
expressed in root tissue but not in leaf or cob.
By these procedures, the clone designated pMR7
showed enhanced expression in both the five and the
fourteen day old root and only insignificant
- 4 -

CA 02317723 2000-07-11
WO.00/29594 PCT/IB98I02000
expression in leaf and cob.
Figure 3 herewith shows the autoradiograph of
a Northern blot probed with pMR7. Fox comparison
purposes, Figure 4 shows the auto- radiograph of a
Northern blot probed with pMRl2 which was typical
of those clones which do not show root enhanced
expression. Comparison of Figures 3 and 4 shows
that whereas pMR7 hybridised to both five- and
fourteen-day old root RNA with little hybridisation
to either leaf-or cob RNA,~pMR2 gave strong signals
on five-day old root~RRA, much reduced signal on
fourteen day old root but strong signals to both
leaf and cob RNA.
Thus pMR7 has been selected for further
analysis. The insert of pMR7 is 700 base pairs in
length and has been fully sequenced by walking
through its length by synthesising oligonucleotides
at approximately 200 base pair intervals and
performing direct plasmid sequencing. There is a
poly(A)+ tail. The sequence of the pMR7 insert is
given as Figure 1 herewith.
Maize genomic DNA digests have been probed
using pMR7 as a probe. Southern blots have
indicated that the corresponding gene is of low
copy number, that is, only a small number of
hybridising bands are detectable at the level of
stringency used.
From the screen of a partial M~gl genomic
library a number of-gutative positives have been
identified and from these the-upstream promoter
sequence which directs expression to root tissue
can be isolated and sequenced.
The pMR7 insert was used to screen a second
maize seedling root cDNA library constructed in the
-5-

CA 02317723 2000-07-11
WO 00/29594 PGTI1B98/02000
cloning vector 1ZAP II. From a number of
positively hybridising clones, one, pMR7/10.1, was
selected for further analysis. DNA sequencing
indicated that pMR7/10.1 was completely homologous
with pMR7 but was of longer length, perhaps
representing the full length cDNA clone. The
sequence of pMR7/10.1 is given as Figure 2
herewith.
EXAMPLE 2
A 'genespecific' probe, representing the
entire 3' untranslated region of the MR7 gene, was
radioactively labelled and used to screen a
commercial corn genomic library obtained from
Clontech, USA (line W22). The probe, obtained by
PCR using the cDNA as a template, was 350bp in
length and of lower G+C content than the. entire
cDNA, thereby reducing the chances of non-specific
hybridisation.
Five clones were selected for further analysis
after three rounds of plaque purification. Each.
hybridised strongly to oligonucleotide probes
designed throughout the length of the pMR7 cDNA,
confirming that they were closely related to the
original cDNA. Restriction analysis of purified
DNA obtained from these lambda clones indicated
that 4 of them (numbers 7, 11, 14 and 15) were
clearly related on the basis of similarity of
restriction profiles. The other clone, number 10
had a different pr'bfile. Hybridisation of the MR7
gene-specific probe confirmed this relationship.
Single or few hybridisation bands resulted from
probing digests of each of the 5 lambda isolates,
number 10 having a different profile than the other
f our .
- fi -

CA 02317723 2000-07-11
_ WO OOIZ9594 PCT/IB98/02000
Of the four more. closely related lambda
clones, number 7 was chosen for further analysis on
the basis of ita larger insert size of
approximately l6kb,~estimated from restriction
analysis (the other inserts ranging in size from
9.0 to 13.5kb).
In order to identify a genomic fragment
containing the MR7 promoter, the insert from lambda
clone 7 was subcloned into pUClB vectors. pMRPl
represents a--lbkb EcoRI fragment subcloned from
lambda clone number 7:r Partial sequencing with an
internal primer confirmed that this fragment
contained DNA related to that of pMR7/10.1 cDNA, as
opposed to any related but distinguishably
different classes of the MR7 gene.
Utilising restriction sites identified at the
5' end of the pMR7 cDNA, the upstream region of the
MR7 gene contained within lambda clone 7 was
identified and subsequently isolated on the basis
of hybridisation to specific oligonucleotide probes
designed against sequence in the cDNA upstream of
the aforementioned sites. A 4.2kb NcoI fragment
was subcloned into pUClB (pMRP2) which represents
the region of the gene immediatly upstream of the
ATG translation startpoint (the ATG being a part of
the 3' Ncol restriction site).
From within the insert of pMRP2, a l.9kb XbaI
fragment Was also identified which represented a
region expected to-contain an active gene promoter.
The entire 4.2kb region of pMRP2 was
sequenced. The sequence is given in Figure 5
herewith. Short sequences sharing homology with a
number of promoter 'sequence motifs' described in
the literature ca be recognised.
_7_

CA 02317723 2000-07-11
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The technique of primer extension was utilised
to identify the transcription start point within
the promoter region. A possible transcription
start point was identified 25 nucleotides
downstream, of the A+T region thought to represent
the 'TATA' box of the MR7 promoter.
To confirm the activity of the putative
promoter regions, both the 4.2kb NcoI fragment and
the l.9kb XbaI fragment Were cloned into a
'promoter assay construct', in which they were
fused to a the easily'assayable B-glucuronidase
(GUS) gene. In the former case (pMRP3), there was
precise fusion through the ATG of the NcoI site.
In the latter case (pMRP4), the fusion was a
transcriptional one, the resulting expression
construct also containing the 'enhancing' maize
AdhI Intron I sequence within the transcribed
region.
Plasmid DNA of both pMRP3 and pMR.P4 were used
in transient expression experiments in maize
protoplasts derived from several sources, including
root, leaf and endosperm tissue. In each case,
expression of GUS from the constructs was classifed
as 'high', being greater than control plasmids in
which GUS expression was driven by 'standard'
promoters such as 35S and maize Adh. High level
GUS expression from these two constructs was also
demonstrated by bombardment of root, leaf and
coleoptile tissues of. maize seedlings.
-8-

CA 02317723 2000-07-11
WO OOI29594 PCT/IB98/02000
SE(~UENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: ADVANTA B.V.
(ii) TITLE OF INVENTION: ROOT-SPECIFIC PROMOTER
(iii) NUMBER OF SEQUENCES: 3
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSe.,E: PILLSBURY MADISON & SUTRO, L.L.P.
(B) STREET: 1100 New York Avenue, N.W.
(C) CITY: Washington
(D) STATE: D.C.
(E) COUNTRY: U.S.A.
(F} ZIP: 20005-3918
(v) COMPGTER REi-.DABLE FORM:
(A) MEDICii~' TYPE: Diskette
(B) COMPUTER: IBM PC compatible
(C) OPERATI\G SYSTEM: PC-DOS/MS-DOS
(D) SOFTh~R: PatentIn Release #1.0, Version #1.25
(vi) CURRE\T AP?T_.T_CATION DATA:
(A) ~PPL~:,~iION NUMBER: PCT/IB98/02000
(B) =ILING SATE: November 16, 1998
(C) ;.LASSI:ICATION:
(viii} ATTORNEY/~~ENT INFORMATION:
(A) \AME: AUL N. KOKULIS
(3) REGISTRt:T ION NUMBER: 16, 773
(C) REFEn=.\:.~/DOCKET NUMBER: SEE35669 PCT
(ix) TELECOMMUN=C=.TION INFORMATION:
(A) TELEF_=0\E: 1 (202) 861-3000
(B) TELEF=.1: 1 (202) 822-0944
(C) TELEX: c714627 CUSH
(2) INFORMATIC_~: FOR ~=Q ID NO:1:
(i) SEQU=::CE
CH_--_~~CTERISTICS:
(A) TENGTH: 1333
Lase pairs
(B) TYPE: -:u~leic
acid
(C) STRA\l:=CHESS:
single
i~) TOPOT_~~': linear
( ii
) MOLEC::~LE
T'_'FC
: cDNE
(xi)
SEQUEtdCE
i.E~CRIPTION:
SEQ
ID N0:1:
ACTGAAGCCAGTC~ATAGC~ :GTTCTAGAACTAGTAGATAGCCTGCTGAT CTGT=CTGTT60
GTTiAGTTCGCAzaGCC'=T~. 'GTTTCGGCGACCATGGAGGATGAGAGGAA CACCCAGCAG120
CACCAGGGCGGTVAGGCCvR uCAGGACGCTGCCGGTCAGGTGGAGGTGAA GGATiGGGGG180
CTCCTGGACAGCCTTCTC:::: CAGGAAGAAGCACGACGACGACCAGGAGAA GAAG.3AGCAG240
ACGGAGGAGCTGGCGACCG:: CATGG~:GAAGGTCACGGTGTCCGAGCCCGA GAAGCACGGG300
CACP.AGGAGGAGG-GCACA GGTCGTCGGCGAGAAGAAGGAGGGCCTTTT CGCCAzIGCTG360
CACCGCACCAGTTCCAGCTC CAGCT~GTCGAGCGACGAGGAAGAGGAGGC GATCGATGAG420
AACGGCGAGATTATCAAG=G GAAGAAGAAGAAGGTGGGCCTCAAGGAGAA GATC:jAGGAG480
AAGCTGCCGGGCGCACGP.AG GACGGCCACCACACGGCCGCACCGTCCCCG GCGCCCGCGC540
1
SUBSTtTttrE SHEET (RULE 26)

CA 02317723 2000-07-11
WO 00/29594 PCTIIB98/02000
CCGCGCCCGTGGAGACGCATGCCCACCACCAGGAGGAAGCGNATCACNGG~CCGCACGTCG 600
TCCCGGCCCCGGCGCCTCCACCGCACGTGGAGACGCACGTCCACCAGCACGACCACGGCG 660
TCGTCGTCCAGr..AGGTCGAC:GACGACGTGAAGACCGAGACCCCGCCGCATGCACCGGGGG 720
AGGAGAAGAAAG~CCTGCT~GACAAGATCAAGGAGAAGCTCCCC~GTGGCCACAACAAGA 780
AGCCTGAAGCC~CTGCCGC=CCGGCTCCGCCCGTCCACGCGCCGGCGCCAGCGCCGCACG 840
CCGAGt~ACGTG=::CAGCCCGGATGGCAAGGAGAAGAAGGGTTTGCTGGGCAAGATCATGG 900
ACAAGATAGCCGGCTACCA:.AAGAGCTCAGGTGAGGAAGCAGACCACAAGGCGGACGCCT 960
GCCGGCGAGCAC=:GGACCA~CTCCTCTAATTAAGGTCGCAGTCC~AGCGTGTCCTGGCCG 1020
TGGGCGGCGGAT~'AGAAGCTAGCTAGCGTTGGCATGTGTGTTGGuTTCTGGTTTGCTTTT 1080
ACCAA_~AGTTTGTTTCAAGGTGGATCGCCTGGTCAAGGTCCGTGTGCTCTATTAAGGTGG 1140
ATCGCGTGAC TCTGGCAGzC-. AGTGT ,GCTG CTTGTGTAGG ACGT~GTACG TACGGGCTTT 1200
ATTTTGGTCC C=~.AGTCAA~.~=i GTCACGGTCG GTCTGGATGT TGTGTACTTG GGTTTGTTGA 1260
ATTATGAGCA GCTGCGTGrT GTAAT_CGGC TGGGCTACCT GGA~'GCGGTT AATAATTGCT 1320
TTGGTTTCTG CCC 1333
(2) INFORMAT=,"'.N FOR SEQ ID _~0:2:
Vii) SEQWNCE
CHARACTERISTICS:
(A; LENGTH: 758
base pairs
(B! TYPE: nucleic
acid
(C; STRANCEDNESS:
double
(D? TOPOLOGY: llncar
(ii) MOLECiILE
T'_'~E:
cDN
(xi) SEQ:IE~~CE
DE~CRIPTi~;t:
SEQ ID
N0:2:
CGCCP~ GCGCTGCACC TCGAC'_'CTAGAGGATCCCCGGGCG!GCTCG AATTCCTTTT60
GCTT
TTTTT=TTTTTIT?P.TGATA ATTGC:.ATATATATATACACGCTAACACGC TCGCGCGCTG120
GGCAG F_L~:GCAATTH TTAACGCATCCAGGTAGCCCAGCCGAATTA CAACACGCAG180
~~ACC
CTGCTCATAATTC?ACAAAC CCAAG=ACACAACATCCAGACCGACCGTGA CTTTTGACTT240
GGGACCAAAATAAHGCCCC:I ACGTr~CACGTCCTACACAAGCrCCAACAC TCACTGCCAG300
AGTCACGCGATCCACCTTLA TAGA6;.ACACGGACCTTGACCAGCCGATCC ACCTTGAAAC360
AAACT~TTGGTAP_~AGCAAH CAGAtCC:CAACACACATGCC=.AC6CTAGCT AGCTTCTAAT420
CCGCCGCCNACCCCCAGG .C ACGCT~-CGACGCGACCTTAA~T~~AGGAGC TGGTCCTGTG980
CTCGCa?7NGNC:.GCCTTGTC GTCTGCTTCCTCACCTGAGT~TTGTGGTAG CCGGCTATCT540
TGTCC~'TGATCTTGCCCAGC AAACC~'TTCTTCTCCTTGCCATCCGGGCGC TCACGTTCTC600
GGCGTGCGGCGCTGGCGCCC GCGCGTGGACGGCGGAGCGGTGCGGCAGCG GCTTCAGCTT660
CTTGTTGTGG CCACCGGGGA GCTTCTCCTT GATCTTGTCC AGCAGGCCTT TCTTGGAATT 720
CACTGGCCGT CGTTTTCAAC GTCGTGACTG GGAAAACC 75g
(2) I14FORMAT=0~9 FOR SJQ ID Iv0:3:
2
SUB~nTUTE SHEET (RULE 26)

CA 02317723 2000-07-11
WO 00/29594 PCT/IB98/02000
(i) SEQUENCE
CHARACTERISTICS:
(A) LENGTH:9203 base
pairs
(B) TYPE:
nucleic
acid
(C) STRANDEDNESS:
single
(D) TOPOLOGY:
linear
(ii) OLECULE
M TYPE:
cDNA
(xi) EQUENCE
S DESCRIPTION:
SEQ ID
N0:3:
CCATGGCTGCACACAATGTGAGGACTCTCATCTACTCCAGCACGTGTGTGACCTATGGAG60
AGCCTGACAAGATGCCCATCGCTGAAGGAACTCCCCAGGTCAGCAGTTCAGGTCTGATTT120
CTGA~ACCATTAGTTCCTTTCTAACATAGCATGTTCCTAGGTTGCTTTCTTATTTGTCTG180
TGTTGTCTCCCACATGTTCTTATATCTGCATCTTTAGAAAGCTTGGATATTGATGACATC240
TATTATTAGGTCCATGCTCTCAGGCGTTGTTTGGACGGTGGCTAACAGGCTCCCAAGCAA300
ACCCACCTAGGCTCATGTTTATTATCTATCTCTTTTTGAAAAGTTACACATTTACTTTGT360
TGCCTGTGAGCAGGGAATACGTTTGGAGAAAATGTATCACATTTGGTGCCAGGTTCAATT420
TGGTTTCTGCAAAGTTTATCACTCCTACATTTTCGCAATTAGTTTCTACAAAGTATATCA480
CTCCATTCCACTCCTATGAaATTACTATGACTTAATTTCAATCGAGGTCATCTTCTTGCT540
CCTTCGCTTGCTTAGCAGTAAGACATAACTTCCTTTACCTTGCTCAATAGTTTGCCTiTT600
AATTTGAACAAAAATCTAATCACCTGACATTGCATGGGAGGTAAGCTCCTGTTTTTCF.CA660
AACTTTATCGGTGGACAGATCACAGTCCTGACAGACCCATTAGTCCGATAGAACAGTTAG720
CATTAGGTAAATATTTTGCCAATTGGCAATTTTGATCTACTCCTATTTTAAAATGCCATC780
ATAGGGGTGCTTGCATTTCTTGTTCATGATTTTATTACTCAAGTCAAAAGTCTGCTTTTT840
ATATTACCTATTACATATGCATGGAAAAGCATGTAGAAGGTAACACCAATAAAGTTTGGA900
TCATATGTTTCCATCTATAi-~TGGTTGTCTTGGTATTCTCAATCAGTGGACTTGTGCP.FCT,960
ATGTAATTTGCAGTCTCCArAAGGATGCTAATGATAGGTCCTCAACACAAGCCTTATTGG1020
TAAGCTGAAA:~P.CAACTTChCACCTTCATTTCATTTCAATAATCGTCTACAAGACTP.PAC1080
CACTTATCTTaTCCTTCCCTTCCTGTTGTCTTTGATGCAGGACCATCCATTCTTGAGCGT1140
GTATGATGACCTACATGTAGGACGGGATCTCCCCTCGCCACCTTCAAGTAATGACAGTGT1200
ACTTGTTCTT~TAATCTTT~CTTTTTATGTATCCATCGTTGTGCACATAAGTG=TAC~.TT1260
TTATTTTACGTTTCAGGCA%:CTCTAATATTTATCCTCCTTATTAAGCAAAGAGTGTGGTG1320
ACACATTTCCCTTTTGGGC~:AGGGTTGGGTTGTGTACTGAGCTGTAATGATTCrCAAiTC1380
ACCTGATATC:~TGATTTi~GATGGTTTTCTGAAAGTGCATTGAG.~.CATTAGGAAt~CACP.AG1440
TGGGF.iGTAGT.GATAACAP=~TCTT'fT'!'AGTCACAAAGATTTTTTTTCTTGGAACCATiAA1500
TAGTTGGCTAACAGCTACAuTGATACAAGCGTTTGTTTTAATATGTTGTGAATTGCAATG1560
GTTACAATTGCCTTGTTTTGTTTGCAAACAGACTACCTTATCTGGTTCTCAAGGTTCCTT1620
CATGGCCTTGACTCTCAAGATCAGAGTATTCTTGTCAATGGGATATCAATGAAGGTAAAG1680
GTTTCCACCCCTATCTTTTCTCAACCTACCCATTTTCTCTAAAATACAATAAAP.AGCTTT1790
TGAATTATTGAGTTTGGAAACATGCAATTCACAAAAAAATGGAATTTCTCCTAAATTGAA1800
3
SUBSTITUTE SHEET (RULE 26)

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WO 00/29594 PCT/IB98IOZ000
GAATTTGTAATCTTCTCTTGTAGGCTCGTTCGCCTTGTCACAAGCTCCATCGTTTAAGGA 1860
AAGAATATGCATACACATTTGAAAGCACAAGCTTATCTTATCAGTGTTCTGAGGTTACCA 1920
GGATCTTGCAAGGTAGCGCACCATAGTAATTGCAGCCATAATGAAGACAGGCTGAGCTTA 1980
GATCAGAGCCCAGCAAGAAGGTATGGATCTTTACTTGTTGCTATTCTTGTCCATTTGGTT 2040
CAGGGGGTGGTTTATGAATATTCAATCTGTTTATTACACCATTCACAATTCGGCAGTATC 2100
GCATGGTTAAATTCAGAAATCAAAGATCTGATATAATGGTGTATTGGAATCATGAGTAGT 2160
TTGAGAAGATTCCTGGTTACATGGAATGAAAGGCTGACTTACACTTCAAGTTTCATCAAG 2220
TCATCACTATTAGTGATGTCGTCCTCATTGATGTCACTTGCTGTGTGCCTGTGCTCATGT 2280
TCTAGAATTTAATTACTGATTACCATTGGTGGGCATTTTATATGTAATATGCTGCTCCTG 2340
TTTTCTGGAGGCAGGGGTGTTAAAGTAGTGTCATCAATTATACAAAGTCAAATTTCTTGC 2400
AGGGAACCATGTGATCTGTTGTGTTTAAAGCTTGTTTATTAGTTTTCTATAAGCTGAACA 2460
AGTTCCTTCGCATTTGTTTTGGATTGCAGAATGAATACTTTTTCAAGTACAGGGACGCCC 2520
GAAATGCAGCAGAAACTTTCAGAGCCAATGGAGATATAA~.TAGACTTATATCACACTGTA 2580
ATAGTCAGGTAAATCGCACAGCCTGTCTTCATTATGGCTGCAATTACTATATCAGCATTT 2640
AATCTGGTTTGGTTTCTGTTGATTAAGCTGGTTTGATATTCCATATGCCTTTTGCTAATT 2700
AAGTAACGGTACAAGTTCATACCATTAATGTTTGCAAGTGCTTCTGCTCATTATATGTAT 2760
TCCAGTACTACAACTAAGCATTGTATCTGAAGTCCTACCCTTCGAATAACTACCAGCGTT 2820
TTGGAGACTCCTACATTTTTTTGTCCATTGGACTGTTATTAAAGTTCAACTCTCATGTGT 2880
ACTGGTTCTAAAATACA~TGCTCTCTTGTCTCATCATTTTGTGACGTGCCAGCCAATATT 2990
TTTTTCCTCTTTTAGATTGAGAGAGTTATGGAAATATGGAACP.AAAACGAGGACTTCCGC 3000
AAGCAGTATGTTGAATCGAACAAGGTTAGCACACTAAAGAGATTAGGGACCCATGACGGA 3060
CGAAAACTTGGCCCTGGTGAGGATCCTCCGGTCATTCCAAGCCGAAGACCAAGCAACATT 3120
TATCCATTGTCTGCCTCAAGCCCGGAAGTGATCACCTTAGCTTCAACACCAGCACCTGTA 3180
TTGGCTGCTGCAGCTGCAGTCCCTTCCAAAGAGAACTCTTTTCCTGCTTTGGACGCCCCT 3240
CACATTGTTTCGATTCTCTGTGGTCTGTACTTAATAAGT.3GTCTATTTATTTCGTGTGAT 3300
TGATCAGACACCGTTCTCTGCATGCCAACATC~AGCTGA!GP.~:GCACCCTCCTGAAGTTA 3360
TTTGATATTGTATACTGATAAGTAATAAACTAGATTATGTAGiTCCTATAATTTTTATCA 3420
TATTGATTCCGTAGCAACGCACGAGCATATACCTATAACAATATAAGACATATTTTGTAT 3480
ATATAACACA=GTGCATATATAAGTTATCGAGATATTATCGTCTCTCGTTGCAACGCACG 3540
TGCACTGACCTATAAAAGTATAACACJiCATTTGTACATAGTT'_'ATCGTGGTTTTATACGT 3600
TTCGTTGCAACGCACGGGCACTCTCCTAGTATATATTTATTGATGGTTTCATCTCTACCC 3660
GTATGTAAATATTCTTATATTATTTGTGAATGATTCATCTCTAACCGTCTGTGAATGGTT 3720
TATATATATGCGTTATTCTTCACTAGGCAAAACAAAAACCACGCCGACCGCCGACCGACC 3780
CGGCCTGTCCACATGGCGCCGACCTCTCCCGACGCCGTCCACCGCAGTGATACCGCACCT 3840
CGACTCCTCGAGCATCGCCACATGCCCACGTCCGATCCGGGCGCCCCACGCGCGGGGTAC 3900
4
SUBSTITUTE SHEET (RULE 26)

- CA 02317723 2000-07-11
- wo oon9s9a PcTnB9aroZOOo
GACAGCGTCGTGGCGCGACTGGCCACCAGACATGTCCTCGTCGGCCAACCGACAGTCCGT3960
TTCCGCCGCGGCTGATCTGTCCCCTCCTGCGTGCGTAGCCTACGCGTACACGAAAACGAA4020
CGTGACTTTCGGTGGCCTAGCTTGCTGATGCTCTATATAAGGACTGCCGGCCTCGATACC4080
TCTCCATCCCTAAGCCAAAAGGCACTGAAGAAGCCAGTCAATAGCGAGTTCTAGAACTAG4140
TAGCTAGCCTGCTGATCTGTTCTGTTGTTTAGTTCGCAAAGCCTTCTGTTTCGGCGACCA9200
TGG
4203
SUBSTITUTE SHEET (RULE 26~
ATAGTCAGGTAAATC

Representative Drawing