Note: Descriptions are shown in the official language in which they were submitted.
CA 02254889 1998-11-16
1
SPECIFICATION
A PROMOTER GENE FORA PORCINECOMPLEMENTINHIBITOR
TECHNICAL FIELD
This invention provides DNA consisting of a specific base sequence.
More particularly, the invention provides a promoter gene for a porcine
complement inhibitor.
BACKGROUND OF THE INVENTION
Recently, organ transplantation has widely been carried out in many
countries. Development of highly effective immunosuppressants (e.g. ,
Cyclosporin and FK506) has solved the problem of rejection of organs
transplanted from man to man, however, lack of donors has become a serious
problem. Such a problem has prompted studies on animal-to-man organ
transplantation, namely xenotransplantation. Although approximately 3,500
instances of heart transplantation have been performed annually in European
countries and the United States, they cover only 20 to 30% of patients who
need heart transplantation. Use of animals closely related to human beings as
donors (for example, such primates as baboons and chimpanzees) involves a
great deal of difficulty due to shortage of these animals and their high
intelligence, but use of domestic animals as donors involves less problems.
Particularly, pigs have advantages of easy supply due to mass rearing, their
organ sizes similar to those of man, and established basic technology
including maintenance of the strains. Consequently, organ transplantation
CA 02254889 1998-11-16
2
from pig to man has been studied.
Of rejections occurring in pig-to-man organ transplantation, acute
rejection by major histocompatibility complex (MHC)-related cellular immunity
may not occur, since evolutional relatedness between pigs and man is so
scarce that there is no similarity between their MHCs. Moreover, application
of
the effective immunosuppressants may avoid such rejection, if ever occurs.
Human blood, however, contains endogenous antibodies against pigs
(namely, natural antibodies). Consequently, if a porcine organ is transplanted
to man, the natural antibodies recognize the organ (antigen) resulting in
formation of antigen-antibody complexes, which activate human complements. .
The activated human complements cause necrosis of the transplanted organ
(rejection). Such a phenomenon occurs immediately (within an hour) after
transplantation, so it is termed hyperacute rejection.
No drug preventing hyperacute rejection caused by complement
activation has ever been developed. No human organ is injured by human
complements, since factors preventing complement activation are expressed
in human organs. Such factors are named complement inhibitors (or
complement-inhibiting factors). Of the complement inhibitors, three factors,
DAF (decay accelerating factor, CD55), MCP (membrane cofactor protein,
CD46) and CD59, are important. It is believed that DAF and MCP inhibit
activation of complements by accelerating the destruction of C3b and C3/C5
convertase, and CD59 does so by inhibiting the C9 step.
The complement inhibitors are species specific. Porcine complement
inhibitors can inhibit the complement activity of pigs but not that of man.
The
porcine complement inhibitors cannot inhibit human complements activated
CA 02254889 1998-11-16
3
by the porcine organ transplanted to man. Therefore, the porcine organ
transplanted to man undergoes necrosis.
Pig-to-man organ transplantation triggers not only hyperacute rejection
but also thrombin formation and platelet coagulation, resulting in thrombosis
in
the host's vascular system as well as the transplanted organ. Components of
the blood coagulation pathway, such as thrombin, fibrin and fibrin degradation
products may also amplify tissue damage, modify immune responses and
augment inflammatory responses (Xenotransplantation, vol. 3(1). 24-34, 1996).
Such problems arising when a porcine organ is transplanted to man will
be solved, if human complement inhibitors and/or such thrombosis-inhibiting
factors as thrombomodulin (collectively termed as complement inhibitors in the
following) are expressed in the porcine organs by genetic engineering. In
transplantation of the porcine heart, there will be no problem if the human
complement inhibitors are being expressed by porcine vascular endothelial
cells.
From such a viewpoint, studies on recombinant pigs (transgenic pigs)
integrated with human complement-inhibitor genes have widely been carried
out.
It has also been considered to be useful to express such factors that are
capable of suppressing thrombosis (e.g., thrombomodulin, Proceedings of the
XVth World Congress of the Transplantation Society, pp. 77-79, 1995) with or
without the human complement inhibitors in the porcine organs by genetic
engineering.
As described above, xenotransplantation by using transgenic pigs
integrated with the human complement-inhibitor genes have been studied. Up
CA 02254889 1998-11-16
4
to the present, promoters derived from the human complement-inhibitor gene
or viruses have been used to prepare such transgenic pigs. For the
complement inhibitors to be expressed in pigs, however, the promoters
originating from pigs may be more efficient. To obtain such promoters, cDNA
of the porcine complement inhibitors is needed. Therefore, the present
inventors carried out studies to isolate and purify cDNA encoding a porcine
complement inhibitor (termed pMCP in the following) and succeeded in
isolating and sequencing its cDNA (see Japanese Pat. Appln. No.178254/1995) .
The present inventors further studied and succeeded in identifying and
sequencing the promoter region of pMCP by preparing porcine genomic
libraries and then screening them with pMCP's cDNA as a probe.
As described above, pMCP's promoter of the invention was derived from
pMCP's genomic DNA, which was isolated by using cDNA of pMCP. pMCP's
cDNA was derived from RNA transcribed in porcine vascular endothelial cells.
Namely, pMCP's promoter of the invention regulates expression of pMCP in
the porcine vascular endothelial cells. Consequently, by using pMCP's
promoter of the invention, genes of human complement inhibitors and those of
such thrombosis-inhibiting factors as thrombomodulin can be expressed in the
porcine organs, particularly the porcine vascular endothelial cells.
Furthermore,
by using pMCP's promoter of the invention, various structural genes can
effectively, selectively and specifically be expressed in the porcine organs,
particularly the porcine vascular endothelial cells.
On the other hand, the promoters derived from the human complement-
inhibitor gene or viral genome, all of which had been employed in the previous
studies, could neither selectively, specifically nor effectively express the
CA 02254889 2004-12-16
human complement inhibitor in the porcine endothelial cells.
This invention was accomplished on the basis of such findings. The
purpose of the invention was to provide DNA possessing an activity of pMCP's
promoter.
5
DISCLOSURE OF THE INVENTION
This invention provides the base sequence defined by Sequence No. 1,
DNA comprising a part of the base sequence, and DNA containing the
sequence.
Another invention provides DNA with an approximately4.1-, 2.4-, 1.7-,
0.9-, 0.5-, 0.07- or 0.05-kb upstream sequence from the 3' end of the base
sequence defined by Sequence No. 1; and DNA with the base sequence
defined by Sequence No. 2.
These DNAs possess pMCP-promoter activities.
In another aspect, the present invention provides an isolated DNA
comprising SEQ ID N0:1 or an isolated DNA comprising a part of the sequence,
wherein said part has a size of at least 0.07 kb from the 3' end of the base
sequence defined as SEQ ID N0:1.
BRIEF DESCRIPTION OF THE FIGURES
The promoter activity of each promoter region is shown in Fig. 1.
The promoter activities of the invention and hDAF are compared in Fig. 2.
THE BEST MODE FOR APPLYING THE INVENTION
The present invention is explained in detail in the following:
The base sequence of this invention defined by Sequence No. 1 is DNA
with the promoter activity of pMCP. Such DNA can be obtained by preparing
genomic DNA of the structural gene of pMCP from a commercially available
porcine genomic library, identifying the promoter region existing on upstream
CA 02254889 1998-11-16
6
region of genomic DNA, and digesting it with appropriate restriction enzymes.
With cDNA of pMCP or its parts as probes, genomic DNA of pMCP's
structural gene can be cloned from porcine genomic library by the
conventional plaque- or colony-hybridization method. By the plaque-
hybridization method, plaques formed by phages containing the pMCP's
genomic DNA are identified as follows: 1~ phages containing the porcine
genomic library and E. coli are co-cultured on agar plates to make plaques; C
the plaques are transferred to nitrocellulose membrane filters, and then phage
DNA in the plaques are fixed on the filters; and ~3 the phage DNAs are
hybridized with pMCP's probes labeled with radioisotope, and then
autoradiographes of the filters are taken. By repeating such procedures,
phages containing the pMCP's genomic DNA can be cloned.
To select the positive phage clones containing the promoter region of the
genomic DNA, a region corresponding to the known first exon of human MCP
cDNA can be excised from pMCP's cDNA and used as a probe to perform the
plaque hybridization similarly as mentioned above, since the promoter gene of
pMCP is believed to possess the first exon.
Thus, pMCP's genomic DNA containing the promoter region can be
prepared from the cloned phage DNA.
The promoter region's DNA of pMCP can be prepared by digesting the
upstream region of pMCP's genomic DNA with proper restriction enzymes,
examining promoter activities of digested fragments, and collecting the
fragments possessing the promoter activities.
The promoter activity can conventionally be examined: e.g., integrating
the restriction enzyme-digested fragment into a vector for luciferase-activity
CA 02254889 1998-11-16
7
determination, transfecting the vector into proper host cells, incubating the
cells for a certain period, lysing the cells and then determining fluorescence
of
luciferase in the cell lysate.
The promoter region of pMCP's genomic DNA was thus obtained from the
porcine genomic library. As shown in Examples described in the following, a
promoter region comprising approximately5,400 by was obtained. By
sequencing the region by a conventional method, the base sequence defined
by Sequence No. 1 (5,418 bases) was identified.
By digesting the promoter region with proper restriction enzymes and
examining the promoter activities of the resulting fragments by the above-
described method, the approximately 1,700-by upstream region proved to
possess a high promoter activity. By sequencing the 1,700-by region, the base
sequence as defined by Sequence No. 2 (1,622 bp) was identified.
As shown in Examples, regions shorter and longer as well than the
promoter defined by Sequence No. 2 were also found to possess the promoter
activities. Consequently, so far as the activity of pMCP's promoter is
retained,
DNA of the present invention may consist of a part of the base sequence
defined by Sequence No. 1 or DNA containing the base sequence defined by
Sequence No. 1.
DNA of the present invention may be used as a promoter to express not
only pMCP but also various other structural genes. In such cases, appropriate
regions can be selected from the entire sequence, depending on the structural
genes to express, host cell, and host animal.
The present invention's DNA thus obtained possesses the promoter
activity to express the structural genes including pMCP and can be used for
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various purposes. As described above, DNA of the invention can favorably be
integrated into the upstream part of human complement-inhibitor genes and
used to generate a transgenic pig transformed with the human complement-
inhibitor genes. Particularly, DNA of the invention can express the complement
inhibitor on the porcine endothelial cells. Moreover, DNA of the invention can
be used as the promoter to express other various structural genes than pMCP
gene.
DNA of the present invention is not restricted to that prepared by the
above-described method, but includes those prepared by other methods. As
far as possessing substantially the same promoter activity, it will be
validated if
parts of the base sequence are deleted or replaced or inserted with one or
more than two bases.
INDUSTRIAL APPLICABILITY
DNA of the present invention is the promoter gene of pMCP. In
generating the transgenic pigs transformed with the human complement-
inhibitor gene, the promoter gene can be integrated into the upstream part of
the human complement-inhibitor genes. Since tissues and organs of such
transgenic pigs do not cause hyperacute rejection, they can be transplanted
to man. Consequently, the problem of the lack of donors for transplantation
can effectively be solved.
EXAMPLES
The present invention will specifically be explained in detail with
examples, but the scope of the invention is not restricted to these examples.
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Example 1
A. Obtaining the promoter region of pMCP
~ Cloning of a phage clone by hybridization
To obtain the genomic clones of pMCP, ,t FIXII (Stratagene) and full-
length pMCPcDNA (prepared by expression cloning and digestion of the
expression vector with Xhol and Nofl) were used for hybridization as the
porcine genomic library and a probe, respectively. The plaque hybridization
was carried out as follows:
First, two million clones of the above-described phage and an E. coli
suspension were spread on 50 plates (totally 100 million clones). The phage
clones formed plaques after overnight incubation at 37 °C. To absorb
the
phages in the plaques, a sheet of nitrocellulose membrane filter for
hybridization was placed on each plate for approximately 1 min.
After that, each filter was turned upside down and immersed in an
alkaline denaturation solution (0.2 M NaOH, 1.5 M NaCI) to brake nuclei for 20
sec and then in a neutralizing solution [x2 SSC, 0.4 M Tris-HCI (pH 7.5)] for
20
sec. After drying, they were cross-linked under UV irradiation (GS Gene
Linker,
Bio Rad).
Second, these filters were blocked in a prehybridization buffer [50
deionized formamide, x4 SSC, 50 mM HEPES (pH 7), x10 Denhardt solution,
100 mg/ml denatured salmon sperm DNA] for 4 h at 37 °C.
The probe was labeled by treating pMCPcDNA with dNTP and Klenow
(DNA polymerase I) in the presence of a radioisotope [«-32P] ATP. A
radioisotope-labeled reverse chain was thus prepared with the cDNA as a
template. The reverse chain can hybridize with the phage clone having the
CA 02254889 1998-11-16
identical DNA sequences.
Then, the radio-labeled probe was placed on a gel-filtration column, spun
down to remove the nonreacted and nonlabeled nucleotides, incubated for 3
min at 90 °C , chilled on ice to maintain a single-chain form of DNA,
and
5 subjected to hybridization.
The prehybridized filters were placed in HybriPack and hybridized
overnight at 37 °C with the above-described probe in the hybridization
buffer.
Next, the filters were washed twice with SSC and SDS, allowed to react
with the image-analyzer plates for 2 h at room temperature, and then analyzed
10 with an image analyzer (Fuji Film Co.).
Finally, 30 positive clones were obtained. Gels of the positive clones were
sucked with Pasteur pipettes and diluted with SM buffer. The clone-containing
solution was thus prepared.
~2 The secondary and further screening
The clone-containing solution was appropriately diluted and spread on
plates (totally 30 plates). After that, hybridization was carried out again.
Many
positive plaques were observed on 20 plates. Twenty positive clones were thus
obtained.
Such procedures were repeated three times. Finally, all of the clones
were purified.
03 Classification of the clones on the basis of the restriction-enzyme
digestion
profiles
To confirm whether the clones were independent, they were digested
with a restriction enzyme EcoRl and then electrophoresed. The identical
clones exhibit the same digestion profile. From various digestion profiles, 10
CA 02254889 1998-11-16
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distinct clones were thus obtained.
~ Selection of the phage clones containing the promoter region by using the
cDNA's leading region
The phage clones containing the promoter region are believed to have
the first exon. Consequently, a region supposedly corresponding to the first
exon of human MCP was excised (Sall-Sphl) from pMCPcDNA and subjected
to hybridization as a probe. Four clones gave positive results.
~5 Selection of the promoter-region containing phage clones by digestion with
a rare restriction-enzyme site
Four clones were digested with Fspl, since its restriction-enzyme site
scarcely exists in the first exon of pMCPcDNA. Only one clone was digested
with the enzyme.
Consequently, it was found highly possible that this phage clone
contained the promoter region.
~ Confirmation of the promoter region by sequencing
The phage clone was digested with a combination of Fspl and another
restriction enzyme (EcoRl ) to localize the Fspl site of the first exon at the
terminal of the digested fragment, integrated into a sequencing vector
pBSIIKS+ (Stratagene) and sequenced (373 DNA Sequencer, Applied
Biosystems, Inc.).
The result showed that the phage clone certainly contained the first exon.
From the restriction-enzyme digestion profile, it was proved that this
phage clone contained the pMCP's promoter region, of which the full length
was approximately 13 kb upstream the genome.
B. Determination of the activity of pMCP's promoter region
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1~ Preparation of a construct for assaying promoter activity
The promoter activity was determined with a system utilizing luciferase
cDNA.
A 5.4-kb promoter region of pMCP bearing a T7 primer recognition site for
sequencing (as the 13-kb DNA of the phage clone was too large to integrate, it
was digested at the EcoRl sites) was digested with restriction enzymes BstEll
and BssHll from the above-described sequencing plasmid. DNA sequence of
the 5.4-kb promoter gene was conventionally determined as shown by
Sequence No. 1 (5,418 bp).
The terminal of the above-described promoter region was blunted with T4
DNA polymerase and subcloned in the pGL-3 basic vector (Promega) for
luciferase-activity determination at the Smal sites. The vector for luciferase
activity determination itself lacks a promoter gene, so it is useful to
evaluate
the promoter activity by integrating a to-be-tested promoter into the upstream
part of the luciferase gene and determining luciferase activity. The
luciferase
activity can be determined by lysing transformed cells to prepare enzyme-
containing lysate, allowing the lysate to react with a substrate of the enzyme
and reading emitting fluorescence intensity.
~2 Preparation of deletion mutants
To obtain the promoter regions of various sizes, deletion mutants were
prepared with a Kilosequence kit (Takara).
A series of pGL-3 vectors bearing promoters of 4.1-, 2.4-, 1.7-, 0.9-, 0.5-,
0.07- and 0.05-kb lengths were thus prepared.
Similarly, a pGL-3 vector bearing an SV40 promoter was prepared for
control.
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Furthermore, a vector bearing a reversed sequence of the above-
described 5.4-kb promoter and that bearing human DAF (hDAF) promoter
(approximately 4 kb) were also prepared for comparison.
~3 Electroporation of the plasmids into porcine aorta endothelial cells
Luciferase activities were determined by using a porcine aorta
endothelial cell line.
Cultured cells were detached with trypsin/EDTA solution, washed and
suspended in HeBS buffer at a concentration of 3x106 cells/800 ~.I.
A 800-ul portion of the cell suspension and a 15-~,g portion of the above-
described plasmids were transferred to a cuvette with 0.4-cm electrode
clearance (a cuvette for cell electroporation) and electroporated with a Gene
Pulser (Bio Rad) at 500 ~,F and 300 V. The plasmids were integrated into the
cells.
~ Determination of the luciferase activity
The above-described cells were cultured for 48 hours, collected, washed
with PBS and treated with the cell-lysing solution for 10 min. Ten ~,I of the
cell
lysate was mixed with 50 ~,I of the substrate of luciferase. Fluorescence of
the
reaction mixture was read with a Luminescence Reader (Aloka).
The results are shown in Fig. 1. The activities per 1 x105 viable cells were
calculated. Each column shows a relative activity, where the activity of a
control pGL-3 vector bearing SV40 promoter is defined as 100. In Fig.l, a
symbol of -5.4 kb means a vector bearing a reversed fragment of about 5.4-kb
promoter region. Similarly, that of hDAF means a vector bearing the hDAF
promoter.
From the luciferase activities, the activities of the promoters ranging from
CA 02254889 1998-11-16
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approximately 0.07 to 1.7 kb were higher than that of the positive control,
which was higher than hDAF promoter's activity (Fig. 1 ). Activities of the
promoters of approximately5.4-, 4.1-, 2.4- and 0.05-kb lengths were also
noticed. The vector bearing the reversed fragment, however, showed no
activity. From these results, it was evident that the above-described regions
certainly possessed promoter activities.
Example 2
Sequencing the promoter region
DNA of an approximately 1.7-kb promoter region possessing a high
activity as shown in Fig. 1 was sequenced.
With the T7 primer-recognition site which had been integrated into the
upstream site of each deletion mutant, DNA was sequenced by the dye primer
method. Sequencing was repeated twice. With regions difficult to identify,
they
were subcloned into pBSIIKS+ and sequenced by using the T3 primer
recognition sites.
As a result, it was evident that the above-described approximately 1.7-kb
promoter region possessed the DNA sequence as defined in Sequence No. 2
(1,622 bases).
Example 3
Determination of the promoter activity (Expression of the human complement
inhibitor
~ Transfection
hDAFcDNA was connected to each of 5.4-kb (Sequence No. 1 ), 1.7-kb
CA 02254889 1998-11-16
(Sequence No. 2) and 0.9-kb pMCP promoters or hDAF promoter. These genes
were linearized, additionally ligated with a neomycin resistance gene as a
selection marker and transfected into to 3x1 O6 porcine cells by
electroporation
at 500 /~F and 300 V with a Gene Pulser (Bio Rad). The cells were cultured in
a
5 culture medium containing 250 ~ug/ml neomycin sulfate (Gibco). Colony-
forming cells were collected. Expression of hDAF on the cell surface was
analyzed with an FACScan (Becton Dickinson) after staining the cells with
biotinylated anti-hDAF monoclonal antibodies (IA10,IIH6 and VIIIA7; Kinoshita,
T., et a1.(1985) J. Exp. Med. 162: 75) and PE-conjugated streptavidin.
10 C Assay for complement resistance
Some clones obtained by the above-described method were selected.
Fifteen-thousand cells of each clone were allowed to react with antibodies
and complement in a 96-well plate. As the antibodies, 100 u1 of inactivated
human serum was added to each well and allowed to stand for 30 min at 4
°C.
15 After washing with PBS, appropriately diluted normal human serum was added
to each well as a complement source and allowed to stand for 3 h at 37
°C.
Surviving cells were determined with a WST-1 (Behringer Mannheim).
03 Results
1. Comparison of expression with various promoters:
A part of the results indicating relationship between each promoter and
hDAF expression is shown in Fig. 2.
The horizontal axis of Fig. 2 , FL2-H, represents the amount of hDAF
expressed; it increases as the axis shifts rightwards. The vertical axis,
count,
represents cell number; it increases as the axis shifts upwards. Each peak
shows the activity of a promoter indicated by the label. Black and blank peaks
i , i 1 i 1 . i i
CA 02254889 2004-12-16
16
indicate FACScan responses obtained by cells reacted without and with anti-
hDAF antibodies, respectively. As a result, no hDAF was expressed without the
promoter. Comparing with hDAF promoter, any of pMCP promoters expressed
hDAF effectively more than 20 times. Namely, it was confirmed that
approximately 5.4-, 1.7- and 0.9-kb promoters effectively expressed the
human complement inhibitor in the porcine endothelial cells.
2. Comparison of resistance against complement by various promoters:
No resistance was noticed in 50% human serum, if hDAF was expressed
by the hDAF promoter; whereas, resistance of more than 80% was noticed
even in 100% human serum, if hDAF was expressed by the pMCP promoters.
Therefore, it was confirmed that hDAF exhibited its original function and
resisted against the human complement, if hDAF was expressed on the
porcine endothelial cells by the pMCP promoters.
20
CA 02254889 1999-OS-17
GENERAL INFORMATION
APPLICANT:
17
NAME: NIPPON MEAT PACKERS, INC.
ADDRESS; 6-14, MINAMIHONMACHI 3-CHOME, CHUG-KU,
O SaIFC.A- S H I
CITY: OSAKA
COL1NTRY ; JAPAN
POSTAL (;ODE/ZII?: 541-0054
TITLE OF INVEN~L'ION: :~ Promoter Gene For A Porcine Complement
Inhibitor
NUMBER OF SEQUI~NCES : 2
CORRESPONDENCE ADDRESS:
NAME: RICHES, McKENZIE & HERBERT
STREET ~~DDRESS: 2 Bloor Street East, Suite 2900
CITY: ~Coront~~
STATE/PROVINCI~: Ontario
POSTAL (:ODE/ZIP: M4W 3J5
TELEPHOPJE: 0416) 961-5000
TELEFAX;; (41n) 961-5081
COMPUTER-READABLE FOIW
COMPUTER: IBM PC Compatible
OPERATILdG SYS'rE;M: MS . DOS Version 6. 21
SOFTWARI~~ : ASCI I Text
CURRENT APPLIC~~TION DATA
APPLICA~.'ION NIJI~ZBER: 2, 254, 889
FILING DATE : 1 ~~ May 1997
C LAS S I F .. CAT I 011:
PRIOR APPLICAT~=ON DATA
APPLICA".'ION NUMBER: JP 148335/1996
FILING DATE: 7.7 May 1996
CLAS S I F7. CAT I ON
PATENT AGENT ITdFORMA'rI:ON
NAME: RICHES,, McKENZIE & HERBERT
REFERENCE NUMBER: P170398
INFORMATION FOR SEQ ID NO.: 1
SEQUENCE CHARAC:TERIS'CI:CS
LENGTH: 5,418
TYPE: Nucleic Acid
STRANDEDNESS: Double Strant
CA 02254889 1999-OS-17
TYPOLOG't : Linear
MOLECULE TYPE: Genomic DNC
HYPOTHETICAL:
ANTI-SENSE:
FRAGMENT TYPE:
18
ORIGINAL SOURCE : 71 F_LXI I porcine genome phage library
IMMEDIATE SOUR(~E
DOS I T I ON IN GElJOME
CHROMOSOME/SEGMENT
MAP DOS=CTION:
UNITS;
FEATURE
NAME / KE '.t
LOCAT I OrJ
IDENTIF=~CATIOIV METHOD:
OTHER IPJFORMA'rION:
PUBLICATION IN3~ORMATION
AUTHORS..
TITLE:
JOURNAL..
VOLUME:
ISSUE:
PAGES:
DATE:
DOCUMENT.' NUMBED, : W097 / 4 4 4 4 9
FILING DATE: 19 May 1997
PUBLICA"'ION DA7.'E: 27 November 1997
RELEVAN".' RESIhLIES IN SEQ ID NO.:
SEQUENCE DESCRIPTION: SEQ ID NO.:
SEQUENCE DESCRIPTIOTd: SEQ I:D NO.: 1
gaattctgcg tacacgggclc cccc~gtggct ttacatcatc gctacagcga 50
catgggatcc gagccgtgt:c tacaacctac acaacaacgc cagatcctta 100
acccaatgca tgaggacaclg gctc~aaacct gcggcctcat agatgctagt 150
cagattcgtt tctgctgaclc cacaatggga actcctaatt ctagatcgat 200
ctagaattag gagttccc~~t tgtc~gctcag cagaaacgaa tctgactagc 250
atctatgagg ccgcagttt:g agcc~c:tgtcc tcatgcattg ggttaaggat 300
ctggcgttgt tgtgtaggt:t gtaclacacgg ctcggatccc atgtcgctgt 350
CA 02254889 2004-12-16
19
agcgatgatg taaagccacc ggggccccgt gctacgcaga attcntgcag 400
cccgggggat ccactagttc tagcnagaga gttgaaaatt taaagaacat 450
ttctccccta atctcccaaa atatgggcaa aggacaggta cccgtggcac 500
tggaaaaata caggcaagca acccatgagt acatgaaaag atgctccagg 550
gttcggccta atggaagcct gaacaatgcc tatcacatcg tgggtttctg 600
aagaagtaac ttaaagaaac tagaaattaa atggctttct tagaatgaaa 650
attctctatc acaaggaaaa atgttgtatg ttgtttttcc cataatggag 700
gtcagtgggc gctatgatta acaaatatct gatgcctgtg actttttaat 750
tgcaagaaat ctgtgnagtt tttttattat ctatgggaaa tattgcatat 800
attaatgata tcacctaact tgtattattg agcaattctg tccacatctg 850
gcctttcatc tttcatctaa aaagcagggg ctggaccaac tgaccttcag 900
tgccattctt actgctaaca ttctaatttt gtttttattg cctttttgta 950
caaaagtgtg agagaagtca ttttaagtct gtgacattaa atgtaatttt 1000
ctgtctccag cattataata agaatcaaag atttaatcta atacaccgat 1050
ggaatattgt ttataacgta tttactgttt caagccttca aaaccaagag 1100
aaaacaaaat gagtacctgt tccttctgag aaatgccctt cttcctgttc 1150
agaatccctg tgtataacag gaatgctctc gagttaacag ccaagtaaga 1200
ggcccatcgg ctggcaggtg cccacctagc taggtgcaag cagaggtggc 1250
agtgctccca ggaccaacag cagaaacatg gcttaactat cctgtgttta 1300
gcagttctct tacgggtttt cacaacacct aaaaagcgcc ctgatggggt 1350
aaagcctctg ccttcatgct gctgccccgt ctctgaaaag caggacgtaa 1400
atatacaatt taggaggtaa gagggacatc tgccattgtt ttctttaaca 1450
cagtcagcct ctgtttaatg aatcccagcc acctccctcc acctaccatc 1500
attcctaagg tttgcagagg agctgccata gagctcaaaa cacggwntac 1550
agacaagcat nttctccatc cctcctcatc ttctcacagg ccgcttgaca 1600
acatctctag gagggggtgg aggcgccacc agtgtttgag cccctcgttc 1650
acgcaaagcc ttgactctgg agttctagtc ctcgcgggac cttaggaagt 1700
tcacggtcaa tactccgccc ttgggctcag acactaagag gatctccggg 1750
taaagagata gacagtagct ccatgcctga tttaggaaaa ctgtccgtac 1800
agacagttgt aattcattcc tttcagagac aaatcctgct ctcttcctag 1850
ttcctgaagt cattaaaatc aaaagctctc agaaacgtcc cagcatttgc 1900
taagtccacg ctgggggagg atgggcagag ccgtgttcag cgcgtttgac 1950
agcaacaccc acttatttca ttyagtatcc ataggcatat atcatgcacc 2000
tggtataggc ctctctctca gcactggaga tacagcaaga aaacgctatt 2050
cctgccccat ggagcttgtw maraaaaata gannnaaaaa ccctttanaa 2100
anggaagctr ccngmtgggn cmaagtnaaa attaagtaaa aagaaawccg 2150
tgarraaacc cttcagtnat attaagaaag aaantagctt gatgaaaccc 2200
caggtgtana aattnncact aaaacaatgs tcccaattaa aacccccmaa 2250
ttcatggaat ttactcnagt ancctgnaac taggraaacc aaattctagc 2300
cnatagtttc tcccttctaa atnttctcat gagaaaacaa yttatttcca 2350
aaganatttt ccatgatggg gaaagttttt ttcaactttg ctcaggtata 2400
aactgaanat acagcattaa agtaaagata gttgcagaga ccaccaaata 2450
gatacccgtt ttcanaaaaa gtgccaacat ggagccagag aacatttccg 2500
ttacatcacg cttttacggc tttgaaaatt aacagagatg ataatccccc 2550
mccttgggtt tccnactccn tccctcctna attttacctc ctttaattgt
catcatgtct ggagattata atccaagata ctaagatgtt tatntcatac 2650
atcgcctcca cacagtgtgt ctnanaagct cttgcaagaa tccaaacatt 2700
gtgctggtct gggtagaaaa ggaaattcca tggtttgttg aacccaggaa 2750
CA 02254889 1999-OS-17
ctcttcagta catctccgag gtaaaactgt ttaaatacaa ttaaagttct 2800
acagttaaag ggtaccctc:c tccac:tgttg gtgggaatgt aaactggtac 2850
aatcactatg aaaaacagc~a tggac~gtact tcagaaaatg aagtatagaa 2900
ctaccacagg atccagcac:t ctcactcctg ggcacctatc aggacaaaaa 2950
attcgctgca aaagatgcat gcacccatag ctatgttcac tgcagcagca 3000
ttcacaatag ccaagacai~g gaaac:gacct aaatgtccat caacagctga 3050
atgcattaag aagacgtgc~t atatacacac aatggaatac tactcaagtc 3100
atgaaaaaga acaaaagaat gccat=ttgca gcaacatggc atggctggaa 3150
ctagagactc atgctaaai=g aagtcagtga gaaagagaaa gacaaatacc 3200
acatgatatc acttatatc:t ggaat:ctaat atacgacaca catgaaactt 3250
tccacagaaa agaaaacci=n cat~ggacttt ggagaacaga cttgtggttt 3300
csccaagggg ggarggggc~g aagac:cgtgg gaggactggg gagctttggg 3350
gttaatagat gcaaaact<it tgcct=ttnga atggataagc caatgggatc 3400
ctgctgtacc agaaccrgc~g aactatanct agtcacttgc kntagaacat 3450
gatggaggat natntgagan aaagaatatn tgtgtgtgtk agagagagag 3500
agactggctc cactttgci~g tatagtagaa aactgacaga acaccgtaaa 3550
ccattaaata aaaatccac~t aaaaatttaa aaataaaaac acacattggt 3600
tccaatgtgt ttaaaagcaa taaagttcta taattgcagc agatgcatct 3650
gaggtttaca cggagagci=t ccatt:cctta ccatcctctc attccttaac 3700
tctaatgtga tacaggttct att~~t=cacca ttctatgaac aaaagagcag 3750
ctgatttaca ggttggatl=t ttc~~aaaaaa aaaatttctt taccaggatc 3800
ccaaatgtaa caaagggtca atat~~gaaaa cttaaaaagc ar_agccaaag 3850
agaaatatac ataagccti=t caa~~t:attaa ttttgattaa tatccaacga 3900
atctcttttt aagtgtatc:a atat~ittatt cattttaata aaagaaattg 3950
caagaggcac ttgctttti:c tgctt:acaaa tacggtttct caaatcgatt 4000
ttttttatat actgtttgca tag,~atttca atccataaag ctacctattg 4050
aaaattcctt atatttct<~c taaac:actta agggcttata ttttctccaa 4100
atttatacat ccttgctcac agttc:tgacg atgtctttgg gataaactct 4150
aaatggaact agaggtttaa aagtt:atgtc catttaaaac ttttaacaca 4200
aaaaaaggta agttaaaaag taaaagtttg gggaggctgc tggtcgcccc 4250
cccaacattg gctgacati:t ttatt:ctttg acaacaaata ggaagaaaat 4300
gtcaatgtct ttttttact=g ctt;~atactg gtcatgttac ttttctttcc 4350
ttttgctaat catacaggc:t tactc:acaac tctacaaaaa aatcttactc 4400
attcctaatg ttccttcat:t gagagattgg tttgccggaa acgttctcac 4450
tctcaccaag tcccaaca<~t ccc;~actcta acgacggtcg ctgcttccag 4500
aaatacggca cttaaggcac cctcc~tcctt acctttttca tgcatgtgta 4550
tttcattttc aataaaacat tgagt:tgttc caaggccaga ccatagagtt 4600
gagccccaac atgctagtc~g ccc;~c~tgtga tgtaataatt taccttccca 4650
ggggtcctct ccggggggc~t aca~~qcgaga ctaagtgact ttaagctgtt 4700
gggagaacaa tggccaaac:c tttcc~tgatt ttgaaatcta tcaggccacg 4750
agacacttcg gtagcggac:g ctcaaccctg ggaatcccaa ctattgtccc 4800
aaattttgcc tgactcgtctc caa<3gattga gccagggccc gggtgtccag 4850
gcagtctgca gtgccccac~t ccccaccaga gccctgaagg gtgtcgggcc 4900
ccacgaaacc gctgcccgctg ctc~r_agggtt tctgttttca ggtcgctgcg 4950
ctttattctc taattcagc:g ttcc~c:gaaag agaccatgag gacccgccca 5000
gtgtccttta caccttccc:g tgtc~c~ggtgg cgacagctgt ttacgaagaa 5050
gagtgcacca ccctttccc:g caac~ccgcag cggttagttc cgcagaagga 5100
ggagccaggg cgtcgggcc:g cagc~t:gggag agaggcccgg cagcgggcgc 5150
cgcggagcag caagggcgt:c cctc~t:ctcgg ccggagcccc gccccgcccc 5200
CA 02254889 1999-OS-17
21
gcccccacgg ccccgccti~g cggcc:cgccc attggctccg ccgggccctg 5250
gagtcactcc ctagagcc<ic ttccclcccag ggcggggccc aggccacgcc 5300
cactggcctg accgcgcgclg aggct:cccgg agaccgtgga ttcttactcc 5350
tgctgtcgga actcgaagag gtctc:cgcta ggctggtgtc gggttacctg 5400
ctcatcttcc cgaaaatg 5418
INFORMATION FOR SEQ ID NO.: 2
SEQUENCE CHARACTERIS'rTCS
LENGTH: 1,622
TYPE: Nucleic Acid
STRANDEDNESS: Double Strant
TYPOLOG'.C: Linear
MOLECULE TYPE: Genomic: DNC
HYPOTHETICAL:
ANTI-SENSE:
FRAGMENT TYPE:
ORIGINAL SOURCE: a F=~X.II porcine genome phage library
IMMEDIATE SOURCE:
POSITION IN GENOME
CHROMOSOME/SEGMENT:
MAP POS7=TION:
UNITS;
FEATURE
NAME/KET:
LOCAT I O1'd
IDENTIF7_CATION METHOD:
OTHER ITIFORMA'C I: ON
PUBLICATION INhORMAT.LC)N
AUTHORS:
TITLE:
JOURNAL:
VOLUME:
ISSUE:
PAGES:
DATE:
DOCUMENT NUMBER,: W097/44449
FILING DATE: 19 May 1997
PUBLICATION DATE: 27 November 1997
RELEVANT RESIDUES IN SEQ ID NO.:
CA 02254889 1999-OS-17
22
SEQUENCE DESC:ftIPTION: SEQ ID NO.:
SEQUENCE DESCRIPTIOZJ: SEQ 7.D NO.: 2
gatcccaaat gtaacaaa<~g gtcaatatag aaaacttaaa aagcacagcc 50
aaagagaaat atacataa<~c ctttc:aacta ttaattttga ttaatatcca 100
acgaatctct ttttaagt<~t atc,~atatat tattcatttt aataaaagaa 150
attgcaagag gcac:ttgci=t ttt~~t:gctta caaatacggt ttctcaaatc 200
gatttttttt atatactgi=t tgc,~t:agaat ttcaatccat aaagctacct 250
attgaaaatt ccttatatt=t ctg~~t:aaaca cttaagggct tatattttct 300
ccaaatttat acatccttc~c tcac~igttct gacgatgtct ttgggataaa 350
ctctaaatgg aactagagc~t ttaaaagtta tgtccattta aaacttttaa 400
cacaaaaaaa ggtaagttaa aaa~~t:aaaag tttggggagg ctgctggtcg 450
cccccccaac attggctgac atttt:tatty tttgacaaca aataggaaga 500
aaatgtcaat gtctttttt=t act~~cttaat actggtcatg ttacttttct 550
ttccttttgc taatcataca ggctt:actca caactctaca aaaaaatctt 600
actcattcct aatgttcct;t cattciagaga ttggtttgcc ggaaacgttc 650
tcactctcac caagtcccaa cagtc:ccaac tctaacgacg gtcgctgctt 700
ccagaaatac ggcactta<ig gcac~c:ctcgt ccttaccttt ttcatgcatg 750
tgtatttcat tttcaata<~a acatt:gagtt gttccaaggc cagaccatag 800
agttgagccc caacatgct:a gtggc:ccagt gtgatgtaat aatttacctt 850
cccaggggtc ctctccgg<ig gggvtacaggc gagactaagt gactttaagc 900
tgttgggaga acaatggcc:a aaccJt:ttcgt gattttgaaa tctatcaggc 950
cacgagacac ttcggtagc:g gac~~c:tcaac cctgggaatc ccaactattg 1000
tcccaaattt tgcctgact:c gtgc~c:aaaga ttgagccagg gcccgggtgt 1050
ccaggcagtc tgcagtgcc:c cagr~c:cccac cagagccctg aagggtgtcg 1100
ggccccacga aaccgctgc:c cggclc:tctag ggtttctgtt ttcaggtcgc 1150
tgcgctttat tctctaatt:c agcgt:tcccg aaagagacca tgaggacccg 1200
cccagtgtcc tttacacct:t cccclt:gtcgg gtggcgacag ctgtttacga 1250
agaagagtgc accaccctt:t cccclc:aagcc gcagcggtta gttccgcaga 1300
aggaggagcc agggcgtcctg gccc~c:agctg ggagagaggc ccggcagcgg 1350
gcgccgcgga gcagcaagctg cgtc~c:ctctc tcggccggag ccccgccccg 1400
ccccgccccc acggcccccic cttc~c:ggccc gcccattggc tccgccgggc 1450
cctggagtca ctccctagag ccac~t:tccgc ccagggcggg gcccaggcca 1500
cgcccactgg cctgaccgc:g cggc~aggctc ccggagaccg tggattctta 1550
ctcctgctgt cggaactccta agac~c~tctcc gctaggctgg tgtcgggtta 1600
cctgctcatc ttcccgaa~~a tg 1622
