U.S. patent application number 10/263788 was filed with the patent office on 2003-02-27 for human bmp-7 promoter and method for exploring bone-related substance by using the same.
This patent application is currently assigned to Aventis Pharma S.A.. Invention is credited to Kawai, Shinji, Sugiura, Takeyuki.
Application Number | 20030040007 10/263788 |
Document ID | / |
Family ID | 14779761 |
Filed Date | 2003-02-27 |
United States Patent
Application |
20030040007 |
Kind Code |
A1 |
Kawai, Shinji ; et
al. |
February 27, 2003 |
Human BMP-7 promoter and method for exploring bone-related
substance by using the same
Abstract
The present invention provides a method for exploring low
molecular weight compounds which regulate positively or negatively
the expression of human BMP-7 with reference to a reporter activity
by using 5' upstream region gene containing the human BMP-7
promoter and an animal cell introduced with the vector that has
been connected to an appropriate reporter gene. The low molecular
weight compounds and their derivatives obtained by the present
method have morphogenetic activity or inhibiting activity for bone
and cartilage through the expression of the human BMP-7 and are
useful as preventive or therapeutic agents for cartilage and bone
diseases. Furthermore, the low molecular weight compounds and their
derivatives are useful as therapeutic agents for kidney
diseases.
Inventors: |
Kawai, Shinji; (Paris,
FR) ; Sugiura, Takeyuki; (Tokyo, JP) |
Correspondence
Address: |
Charles A. Muserlian
c/o Bierman, Muserlian and Lucas
600 Third Avenue
New York
NY
10016
US
|
Assignee: |
Aventis Pharma S.A.
|
Family ID: |
14779761 |
Appl. No.: |
10/263788 |
Filed: |
October 3, 2002 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10263788 |
Oct 3, 2002 |
|
|
|
09674311 |
Nov 30, 2000 |
|
|
|
09674311 |
Nov 30, 2000 |
|
|
|
PCT/IB99/00733 |
Apr 22, 1999 |
|
|
|
Current U.S.
Class: |
435/6.13 ;
435/91.2; 536/23.2 |
Current CPC
Class: |
A61P 19/00 20180101;
A61P 13/02 20180101; C07K 14/51 20130101; A61P 15/00 20180101 |
Class at
Publication: |
435/6 ; 435/91.2;
536/23.2 |
International
Class: |
C12Q 001/68; C07H
021/04; C12P 019/34 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 30, 1998 |
JP |
10/120174 |
Claims
What is claimed is:
1. A DNA whose nucleotide sequence is represented by the base
sequence No. from 1 to 10877 shown in SEQ ID NO. 1 of the Sequence
Listing that encodes a human bone morphogenetic protein-7 promoter
region, or a fragment thereof.
2. A method for preparing the DNA shown in SEQ ID NO. 1 of the
Sequence Listing comprising the steps of: (1) digestion of a human
placenta genomic DNA with a HindIII restriction enzyme, (2)
isolation by agarose gel electrophoresis, (3) cloning of the
isolated DNA fragment digested with HindIII into a lambda phage
vector .lambda.DASH II treated with the same enzyme, (4) packaging
of said vector into the phage, (5) establishment of genomic DNA
library by infecting Escherichia coli with the phage, (6) screening
by PCR, and (7) subcloning into a plasmid vector.
3. A recombinant expression vector characterized by intearation of
the full length or a part of DNA shown in SEQ ID NO. 1 of the
Sequence Listing into a reporter gene.
4. The method for exploring a bone-related substance characterized
by using the recombinant expression vector according to claim
3.
5. The method for exploring a bone-related substance according to
claim 4, wherein the bone-related substance is osteogenesis
inducing substance.
6. The method for exploring a bone-related substance according to
claim 4, wherein the bone-related substance is osteogenesis
inhibiting substance.
7. The method for exploring a substance effective at the treatment
of a kidney disorder by using a recombinant expression vector
according to claim 3.
Description
BACKGROUND OF THE INVENTION
[0001] (1) Field of the Invention
[0002] The present invention relates to a 5' upstream region DNA
containing a promoter of a human bone morphogenetic protein
(hereafter referred to as BMP-7). Further, the present invention
relates to a method for exploring a low molecular weight compound
positively or negatively which regulates the expression of human
BMP-7 by using a mass of animal or yeast cells that are introduced
with a recombinant expression vector harboring a 5' upstream region
DNA containing the human BMP-7 promoter integrated into a suitable
reporter gene, and by using a reporter activity as an
indicator.
[0003] (2) Description of the Related Art
[0004] At present, a bone morphogenetic activity has been reported
for a bone morphogenetic factor, BMP, belonging to TGF
(transforming growth factor)-.beta. superfamily (Science 150,
893-897, 1965; Science 242, 1528-1534, 1988). Known species of BMP
are BMP-1 to BMP-14. Among them, the members from BMP-2 to BMP-14
have been known as showing the bone morphogenetic activity. BMPs
ranging from BMP-2 to BMP-14 are considered as effective to
therapeutic and preventive treatment for various bone dysfunction
and bone diseases, however, they exist in very small amount in
nature. Therefore, an available large quantity from BMP-2 to BMP-14
used for these treatments requires production of recombinant
protein. The production of the recombinant protein generally is
very expensive compared with a low molecular weight compound.
Furthermore, there are many restrictions as a medical drug in terms
of physical properties and administration methods due to proteinic
characteristics. Considering these points, a small molecular
organic compound having the activity equal to that of the BMP
protein, if any, should be a highly promising medical drug. The
substance obtainable with the exploring method provided by the
present invention has the activity to induce the expression of
human BMP-7, a bone osteogenesis factor, and also has the efficacy
equal to that of human BMP-7, representing very high usefulness. On
the contrary, if human BMP-7 is concerned with bone and cartilage
hyperplasia, inhibiting the expression may prevent
osteohyperplasia. The present invention is able to detect the
inhibition of the human BMP-7 expression and provides a method for
exploring a substance to prevent hyperplasia. In addition, it is
known that human BMP-7 has the ability to enhance the
differentiation of kidney cells (Proc. Natnl. Acad. Sci., U.S.A.,
Vol. 93, p. 9021-9026, 1996). Thus, the experimental system
provided by the present invention can be applied to a method for
exploring the agent for the treatment of the kidney disorder.
[0005] For such an exploring method, an example has been so far
only reported using a murine BMP-2 promoter (WO97/15308), and there
is no example of using the human BMP-7 promoter. In addition, since
the materials of the exploring method provided by the present
invention are all derived from human sources, it can be expected
that discovered substances should show the effects at clinic
practically.
SUMMARY OF THE INVENTION
[0006] The present invention provides a 5' upstream region DNA
containing a promoter of human BMP-7. By using 5' upstream region
gene containing the human BMP-7 promoter and an animal cell
introduced with a recombinant expression vector that has been
connected to an appropriate reporter gene, the low molecular weight
compounds which regulate positively or negatively the expression of
human BMP-7 can be explored with reference to a reporter activity.
The low molecular weight compounds and their derivatives have
morphogenetic activity and inhibiting activity for bone and
cartilage through the expression of human BMP-7 and are effective
as preventive or therapeutic agents for cartilage and bone
diseases, remedies for osteometastasis, or therapeutic and
preventive agents for excess osteogenesis. Furthermore, these low
molecular weight compounds and their derivatives are useful as
preventive or therapeutic agents for kidney disorders.
BRIEF DESCRIPTION OF THE DRAWINGS
[0007] FIG. 1 is an exon-intron structure of 10.8 kb 5' upstream
region of human BMP-7 gene and a restriction enzyme map. A net
shape shows an exon region and an open square shows an intron
region.
[0008] FIG. 2 is a recombinant expression vector (pMSS115)
containing a 5' upstream region of human BMP-7 gene. A promoter
region (4.4 kb) (base No. from 3813 to 8222 shown in SEQ ID NO. 1
of the Sequence Listing, referring from the 2nd XbaI to the 3rd
XbaI from 5' terminal in FIG. 1) was inserted to NheI restriction
enzyme site of pGL3-basic.
[0009] FIG. 3 is a result of measuring human BMP-7 promoter
activity (transiently expression).
DESCRIPTION OF THE PREFERRED EMBODIMENT
[0010] The present invention relates to a DNA whose nucleotide
sequence is represented by the base sequence No. from 1 to 10877
shown in SEQ ID NO. 1 of the Sequence Listing that encodes a human
bone morphogenetic protein-7 promoter region, or a fragment
thereof. SEQ ID NO. 1 of the Sequence Listing shows the 5' upstream
region sequence of the human BMP-7 gene.
[0011] The present invention relates to a method for preparing the
DNA shown in SEQ ID NO. 1 of the Sequence Listing by conducting the
steps of:
[0012] (1) digestion of a human placenta genomic DNA with a HindIII
restriction enzyme,
[0013] (2) isolation by agarose gel electrophoresis,
[0014] (3) cloning of the isolated DNA fragment digested with
HindIII into a lambda phage vector .lambda.DASH II treated with the
same enzyme,
[0015] (4) packaging of said vector into the phage,
[0016] (5) establishment of genomic DNA library by infecting
Escherichia coli with the phage,
[0017] (6) screening by PCR, and
[0018] (7) subcloning into a plasmid vector.
[0019] The plasmid vector used herewith is not restricted and can
be used among ones commercialized. A pUC18 vector can be a
preferable example.
[0020] The present invention relates to a recombinant expression
vector characterized by integration of the full length or a part of
DNA shown in SEQ ID NO. 1 of the Sequence Listing into a reporter
gene. In detail, the recombinant expression vector is constructed
to locate a suitable region of 5' upstream region of the human
BMP-7 gene, that is represented by SEQ ID NO. 1 of the Sequence
Listing, in front of a reporter gene. The reporter gene such as
luciferase or .beta.-galactosidase gene shows an expressing status
on behalf an original product. The vector as the original for the
recombination expression vector is not specially restricted to
allow to use a plasmid vector commercialized. The present invention
used pGL3-basic as a preferable example. The use of pGL3-basic
yielded pMSS115 (9.2 kb) that is a recombination expression vector
containing the human BMP-7 promoter and a luciferase gene. The
present invention assigned it to the recombination expression
vector. It is necessary to introduce the vector to mammalian cells,
preferably a human osteoblast-like cells, such as SaOS-2 cells,
with a liposome. The animal cells stably transfected with the
recombinant expression vector are selected by using a resistance
marker.
[0021] The present invention relates to a method for exploring a
bone-related substance, characterized by using the recombinant
expression vector characterized by integration of the full length
or a part of DNA shown in SEQ ID NO. 1 of the Sequence Listing into
a reporter gene. It relates to the method for exploring a
bone-related substance wherein the bone-related substance is
osteogenesis inducing substance or a bone-related substance wherein
a bone-related substance is osteogenesis inhibiting substance. A
low molecular weight compound which induces or inhibits the
expression of human BMP-7 can be obtained by isolating the promoter
which regulates the expression of the gene, by connecting it to a
suitable reporter gene and by introducing the gene structure to a
suitable mammal cell to make an exploring system. The substance
which regulates the expression of human BMP-7 in the exploring
system works on the promoter to increase or decrease the expression
level of the reporter gene. Therefore, a simple and easy
measurement of the reporter activity makes an exploration of the
aimed substance possible.
[0022] The animal cell transfected with said vector can be used for
a method for screening a chemical compound library by high
throughput screening (Nature, Vol. 384, Suppl., p. 14-16, (1996)
and exploring an active-substance from natural substances. The
substance which increases or decreases an activity is searched by
treating the cell with a substance for an appropriate time period
and thereafter measuring the reporter activity. The substance
obtained hereby can regulate the expression by working directly on
a transcription factor or indirectly on the promoter of human BNP-7
through regulating a signal transduction system. Therefore, these
compounds are effective as a therapeutic agent for
osteocartilaginous diseases, cancer metastasis to bone, or
osteohyperplasia.
[0023] Furthermore, these compounds are useful as a therapeutic
agent for kidney disorders.
[0024] The substance obtained by the present invention has bone or
cartilage morphogenetic activity and is effective as an agent for
therapeutic and preventive treatment in the fields of orthopedic
surgery (fracture, osteoarthritis such as joint osteoarthritis and
hip joint osteoarthritis, arthrosteitis, damage of cartilage such
as damage of meniscus, regeneration of bone and cartilage deficit
caused by injury and tumor dissection, bone reconstruction such as
spinal fusion and vertebral canal enlargement, and congenital
cartilage and bone diseases such as dysoteogenesis and
achondroplasia), or dental fields (bone reconstruction such as
palatoschisis, mandible reconstruction, and residual ridge
construction), and osteoporosis. Moreover, the substance of the
present invention can be used for bone graft in aesthetic surgery.
These therapeutic treatments are effective to therapies in the
fields of veterinary surgery. On the other hand, the present
invention can provide a substance to inhibit bone or cartilage
morphogenesis. In this case, the substance is applied as an agent
for prevention and therapy of bone and cartilarge hyperplasia.
[0025] In addition, the present invention can provide a substance
with ability to enhance the differentiation of kidney cells and it
can be applied to an agent for the treatment and prevention of the
kidney disorder.
EXAMPLES
[0026] This invention shall be more illustratively explained by way
of the following Examples. The following Examples are to be
considered in all respects as illustrative and not restrictive.
Example 1
Isolation of 5' Upstream Region of Human BMP-7 Gene
[0027] A human placenta genomic DNA (a product of CloneTech) was
digested by using various kinds of restriction enzymes (BamHI,
BglII, EcoRI, HindIII, PstI, SacI, SalI, SmaI, SphI, and XbaI),
separated by agarose gel electrophoresis, transferred to a nylon
membrane, and subjected to the Southern hybridization using BMP-7
cDNA (EMBO J. 9: 2085-2093, 1990) as a probe. As the result, it was
found that digestion by the restriction enzyme HindIII among
restriction enzymes used yielded a DNA fragment of ca. 11 kb
containing the longest human BMP-7 gene. Then, a human placenta
genomic DNA was digested by the restriction enzyme HindIII and
separated by agarose gel electrophoresis to extract a DNA fragment
of ca. 11 kb from the agarose gel. The DNA fragment obtained was
cloned to lambda phage vector .lambda.DASH II (Stratagene Ltd.
made) digested by the restriction enzyme HindIII. The vector was in
vitro packaged by Gigapack III XL Extract (Stratagene Ltd. made),
infected to Escherichia coli XL1-Blue MRA (Stratagene Ltd. made) to
make a genomic DNA library. The library was divided into pools.
Each pool was amplified by a screening (Nucleic Acids Res. 21:
2627-2631, 1993) using PCR; namely, the PCR method by using PCR
primers (SEQ ID NO. 2 and SEQ ID NO.3 of the Sequence Listing)
corresponding to translation region to select the objective pool,
to obtain finally 5' upstream region (10.8 kb) of human BMP-7 gene.
In addition, the 5' upstream 10.8 kb fragment was subcloned to a
pUC18 vector (a product of Amersham Pharmacia Biotech). The vector
was named E. coli pKOT 314. The E. coli pKOT 314 was deposited in
National Institute of Bioscience and Human-Technology, Agency of
Industrial Science and Technology, Ministry of International Trade
and Industry 1-3, Higashi 1-chome, Tsukuba-shi Ibaraki-ken 305-8566
Japan, in Mar. 30, 1998 with depository number FERM P-16737 and
transferred to the International Depository Authority under
Budapest Treaty on Feb. 17, 1999 (Deposit No. FERM BP-6651).
Example 2
Determination of DNA Sequence of 5' Upstream Region of Human BMP-7
gene
[0028] The sequence of 5' upstream region of human BMP-7 gene
obtained was determined by Amersham Pharmacia Biotech's ALF DNA
Sequencer according to the method of Sanger et al. (Proc. Natl
Acad. Sci. USA 74: 5463-5467. 1977). The sequence thus analyzed is
described in SEQ ID NO. 1 of the Sequence Listing. The base
sequence No. from 5557 to 10780 of SEQ ID NO. 1 was already been
reported (EMBO J. 9: 2085-2093, 1990). However, there are many
differences from the sequence of this invention.
Example 3
Construction of a Recombinant Expression Vector Containing the
Human BMP-7 Promoter and a Luciferase Reporter Gene
[0029] As shown in FIG. 1, the promoter of human BMP-7 exists in
the upstream of exon 1. Then, a region of 4.4 kb containing a
promoter from XbaI of the second position to XbaI of the third
position from the 5' terminal described in FIG. 1 were--to align in
the upstream of the reporter gene--inserted in the restriction
enzyme site, NheI, of a luciferase reporter vector pGL3-basic (a
product of Pro Mega Ltd.) to construct a recombinant expression
vector pMSS115 (9.2 kb). This is presented in FIG. 2.
Example 4
Measurement of the Activity of the Human BMP-7 Promoter
(Introduction of a Recombinant Expression Vector to a Human Cell
and Transient Expression)
[0030] In order to express transiently the human BMP-7 recombinant
expression vector, said recombinant expression vector (pMSS115) was
mixed with a vector, pRL-SV40 (a product by Pro Mega Co.)
containing sea pansy-luciferase gene as an internal control for
measurement of efficiency of gene introduction in equal quantity.
Then, cationic liposome lipofectamine (a product of Lifetech
Oriental Co.) was added to the mixture solution to add to human
osteosarcoma cells HOS, MG63, and SaOS-2 for transfection. Fire fly
luciferase activity and sea pansy luciferase activity were measured
by Pikka Gene Dual Kit (a product of Toyo Ink Co.). The result is
presented in FIG. 3. The promoter activity was expressed as a ratio
of fire fly luciferase activity to sea pansy luciferase activity.
From the result, it has been known that the DNA of SEQ ID NO. 1 of
the Sequence Listing has a promoter activity.
Example 5
Introduction of the Recombinant Expression Vector to a Human Cell
and Stabilized Expression
[0031] In order to express the human BMP-7 recombinant expression
vector stably, said vector was mixed with a vector pPUR (a product
of CloneTech Ltd.) containing puromycin resistant gene in the
proportion of 10:1 and also mixed with cationic liposome
lipofectamine (a product of Lifetech Oriental Co.) to add to a
human osteosarcoma cell HOS for transfection. The cells to which
the aimed gene had been introduced were selected from a culture
medium containing puromycin (a product of Sigma Ltd.).
Example 6
Screening of Active Low Molecular Weight Compound
[0032] The established cells selected were inoculated in a 96-well
plate, treated with substances of various chemical compound
libraries for 1-3 days, dissolved with a cytolytic agent (a product
of Pro Mega Ltd.), and measured for enzyme activity by employing a
luciferase assay kit (a product of Pro Mega Ltd.). By such
processes, various substances inducing or inhibiting the expression
of human BMP-7 are obtained.
Sequence Listing Free Text
[0033] <210>1
[0034] <223>Human BMP-7 5' upstream gene sequence including
the exon 1 regions.
[0035] <210>2
[0036] <223>Sense PCR primer for cloning 5' upstream human
BMP-7 gene sequence corresponding to the exon 1 region.
[0037] <210>3
[0038] <223>Reverse PCR primer for cloning 5' upstream human
BMP-7 gene sequence corresponding to the exon 1 region.
Sequence CWU 1
1
3 1 10877 DNA HUMAN misc_feature (1)..(10877) Human BMP-7 5'
upstream gene sequence including the exon 1 regions. 1 aagcttggac
atcagatagc tgtgtcattc atgctaactc ttattagttg ttgcatcata 60
tgtaagatga gctcaaatct gttaatcttc ccatacgatc ttttcacatg agttgggact
120 aacctgttat atttagagta atggtttacc aatggggagg gaataaagat
aaaactcgaa 180 gtaaaagaaa gaatacaatt ttttaaatga ggagaaacac
agtgaaggag aggagagaag 240 gaagaaaaca acagagatgg agagaagcac
gacgtggggt ggtgaggagt tcccaggact 300 ctgggagcga ggagcccagt
ctaatccctg attcactgta agactttggg cccagcagtt 360 tctctttggg
tgtcaatttc tctatcttca agatgaagga attgaaccac gcttctgcag 420
gacttttgtt gtaactgact ggaaactccg tacacaatgg cttaaactga acagtcctgg
480 gtggggctgg cttcagggat agtgggctcc aggggctcac tgatgctgtc
agtctcctgc 540 cttttcttgt tttactgtct tgtggcttct ctcacagggc
aggctctccc tgaagggagg 600 gaagatgaca gccagcagca gcccctgacc
tagcaacccc agtgggagga gagacccctt 660 taccggtagt tctagcaaaa
gttctaaaag ttgaatctca ctggcctgga ttaggtcaca 720 tgactgtcac
tgaacccatc gcatgggcat ctctggttga ccaggctggg tcatgtgccc 780
agtctgaatt ttagagaaga ggatagttca acctgaaata cagggactga tggtgaagaa
840 ggggcatttc tactgaggaa aacagaggaa atcttggcca ggcacagtgg
cttatgcctg 900 taatcccagc actttgagag gccaagatgg atggattgtt
tgaggcccgg agttcaagac 960 cagtctggcc agcgtggtgt aaccctgtct
tctactaaaa atacaaaaat tagccaggca 1020 tggtggtgca cacctgtaat
tccagctact cgggggggct gaggagggag aattgcttga 1080 aaccgggagg
cagaggttac agtgagctga gattgtgcca ttgcactcca gcctgggtga 1140
cagagggaga ctctgtctaa aaaaaaaaag aaaagaaaag aaaacagagg aaatatttgc
1200 agaagaagga gaaaaggcag gggtctcaaa ttaggtgagc ttccaaagtg
ttttctgtgg 1260 aaccctggga gcagagaaaa tggtagtgaa aagaaacagg
cctggggact ctgggcccct 1320 gctgggatca cctgataaca ggcagggtgt
ccagtccgat ttgaacttca gataaacaac 1380 gagtaaattt cagtatgagt
atatataata catatactcc aatataatgg gatatagtca 1440 tactaaaatt
cattcattgt ttatctgaaa ttcaaattga actggacacc tatttttttt 1500
tttgcaaaat gtggtaaccc tagtccctcc cctgctctat gtaaccaaag aagctctgtt
1560 tctgttttct gtgtcgagca tgtgtgaaaa actccttgac caaaagtttt
agcgctgcac 1620 aaaagagaag gcctgtatcc ttttaactga catgattgcc
atgccctctc cagctcccca 1680 gtgctgtatc cagcagggct tctggctgca
ggcaacagaa accagcttgg gcaggctggt 1740 gcggaaaagg agaccactag
aggatcttgg gggctcacag aacccctggg aaggccagag 1800 aaccaggctt
gggggctctc tagttgggac acggccccag atcactctgc agaatagtct 1860
gggcctgcac tgatagcttc tggggtgggt gcctctgact ggctgaaact tggtcacctg
1920 tcagggctct ggctgcaaag caggctagac agtgcctctc tggatattca
ccagatgggg 1980 catcctcaac cataagagag ggctcagatc tagctgggag
gagggagatg ctgaaaagca 2040 ggagcaatga acactcactc ctgctcttgc
agatggaatt ctggtttttg atgtttctcg 2100 ggagaggggg agtttatcag
atcagtcacc agggccaaac agcagagttt tgctgggaag 2160 ggactctggg
gaaggttagg gtcagatttt gcctaaaggg gcagacagtt ccagcctttt 2220
caaccactgg ctcagtagcc ctcgaatcat gaattcaatg gagctgtctt gaaatgcacc
2280 atccttggtg gtcctttggt ttgggtgatg ggttggggga ggctgggcac
atcctccaga 2340 cacagcagag ccctttatga ggccaggggc aaaccagatt
catcgaatct gactggtaga 2400 tgtgtcttct ttggtctaat aaccaaaacg
agttgaatta gttgccaaca gataaacatt 2460 gagagatgtc acattcaaac
tcaagtttct agcttctttg gaaaatcaga actttctggt 2520 gatgttggaa
tcaaatttct ggatggcatc acccatgggc atgtggtttc cagttcaccc 2580
cagtctgcac cctccttatt tatccaggcc agtgtctcct gccacctgtc accatgtctg
2640 tgctcatgat tgccttccat taaattaaga ggaagagaaa tattccttga
gcttcatgtt 2700 gcatctctgt atttatgtta cctggttggt gcccgttggc
ttttgagtgt atgacttctg 2760 caataaagga ggaattgaag aagggctcag
aggtatgttc ctcaaacact gatagggcat 2820 ctactgtgtg caaggcctcc
atgggtactg ggaggtccct acaggactaa gacaaaccct 2880 gtgttttggt
gtacctcaat gtttaaggac ttaagtcctg gaacaagata gccggcgtca 2940
gaattctgat tttaccattc actcaccatt ggtgtggctt tgggcaggtt ccctcaagta
3000 tttgagcctc aatttcctca tctgtaaaat ggggataata ataataaaaa
tcatacaagg 3060 tagttgggag aattaatgag ttaattttaa aagacacctg
gaatcataga tattgaagtt 3120 ttaatttgaa gccccaattt gtcagctata
acttcagaaa tctagagcct cgatcgtcaa 3180 catacagcct gaagacgaca
gcctccgagc cccccaggag ctagttagaa gtgcagaatc 3240 tcagctcact
cagactgact gtgggaatct atgttctaac agacccccag gggattcaca 3300
cgcacagttg ggttggagaa gtgttgcttt aggttaaagc aaggaaggaa ggggctgctc
3360 ccaggtcact gaacgtgcac tgatgcttaa aggcctgggc tgtgtagaca
gatgtggctt 3420 tggatcctgg ctccattaca gattagattg gctgtctgac
tctggacaag gtgctctcgg 3480 gatgttggtc ctcctctgca atctgaggag
cagaactagc ctctccatgc agtggggagt 3540 tgcgggtgat gtgggcttgg
gccgtgcagg cctagcaggt gcccagcaga agcaagtgct 3600 cccaacaggt
gacaggtcgg ccgctcccct gtcacgtttt ggaaggagga aagggctacc 3660
tctagtgctg aaccgaggat agttagtgct caaaaccata ccagatttcc tccaatcaag
3720 gaagaaataa cagccctgat aataataaac aaaaccactg cttcccccta
gctggcttcc 3780 agatcctaga aatcctgcat ggtatcagct tctctagagt
gtgtgtgtcg tctgtatttt 3840 ccatttggaa ctgtggccac tgccatgtgt
acttaagact gatggaagac gtttaatctc 3900 ttattctgct gcttttaaag
ttgtgcaaag aaaatctcag agtgggcaag cgtgaggagt 3960 ccaagcctcc
cgtgcaatga gagatctgcg tgggaaacaa aatttcacca caggtgtgct 4020
ctaataattt cctctgcagg gtccacctct gcacacaacg attttcaagc ctgtgcagga
4080 ggcagctctt ggccctctat tccttgttgg ggttggaggt ccaggtcatc
tggcctttgc 4140 caggtggtgt ggacagagag agagcagccc actggcttcc
ttggctgtgc tccccaggga 4200 gcttccaggc cagctgagcc tcctctaggg
cagatggatg aagatgaggt ccccacctcg 4260 ctgaacaatg tgtgtgtcag
cagaattctt ttcttctttt tactgcctta gagagacatg 4320 ctgacttggt
caaaatcact tcagcaagat gtgactgatg atgtaggaaa tgaaaactcc 4380
tggcccagca ctgggaggcc agcaggcgag gggcgcggac actgggaccg ggccacccga
4440 cacaataaca ccccttacaa ttccaggccg tctttcatcc aggcaaaggc
ggccccaggt 4500 gtggagcaga ctggggcaga tcagattacg gtcctggagc
ccagtgctgg gtgccctggc 4560 caggggaacc acaactgggg gtgtgtgggt
tgggggctag ctgctgggga aatgagatag 4620 gacccaggga agtccctggg
ccccagctgg cctgcagggg gcctaggcac aatgtgagga 4680 ttggaatgca
gtgatgacct tttcatctct catgttccct tccacttcac tctctctttt 4740
gctctggtac ttttgctctt gctctcagag tcaggataca tctgcttttc tctgcttggc
4800 aaaaagcaga ccatcatgaa aagttttgtt gtaatttgaa actcaggagg
ctttcctgtt 4860 accatatttt ccttttcatc cagatggtaa cacaataaca
tttatcaagg gttttctctg 4920 tgttccaggc actgtgcaaa gccctttcca
tgaattaagt tctgcaacct cacagcgatc 4980 ccagaagaca ggcgctatca
gtccccccat tttacagatg ggggaactga ggtgtagaga 5040 ggttaagtcc
cttgcccatg gtgcacagct ggaagagaca gagctggagt gtgaatgcgg 5100
ctgggcaggc tccagtgccc aggctacctc ctccacacaa gacttgccct cggcaatctc
5160 aaagcctttt ctggtggtgg gctcagctcc caaactggca tcggatgcac
tcccagccag 5220 atatttcttg cttgccggtt ttcattcatt cattcattca
ttcattcatt cattcattcg 5280 gcattcactg agggcctggt gcagtcttgg
ggatgcctct cggggaggaa acagggaaaa 5340 gaaagacccc cccaccaagc
atggatcaca gaaaagataa ggctaaatgg gggtttgtgg 5400 gacttcagag
gaaaccttat ctcttgaggt cttggatatg aagagcatgt tgtctcttcc 5460
tcttgcatga gaaaagatgg cgtctcagag gaagggttgc tggggtgagg gatctgggag
5520 atgccttagc ttggcgcctg cacagtcagc cctcagtcaa ccggtctctt
taggttttgg 5580 ctgtgcttat tactattcat tcaacaggta ctaattgagc
acctgctgtg tgccaggctc 5640 agaataggct caggtgagat gcacaaagaa
gggtaaacta gaatccttgc ttagacactg 5700 acggatcagt tgtttcatat
gtaaattgta gcaccaagac ctgctgcccc tgcccccagc 5760 ctcacctgct
tgtgaagatc cctccaaaag atttgagagt agataaaaag cagagactac 5820
tactgaagaa cagggctgct ttggctcctt attatttcag actttggaag aaaatgacct
5880 cctttttctc tactggcact ggaggtggca tagctgtccc tagcaagcca
gcgctggagg 5940 gcgtgtgcag ggctggggac cgagcctggt ttctgttccc
tgctctgcag gctcaagcac 6000 ttgctgttcc tccacctggg atgcctttcc
ctggaaaagc ctgtctcttt cttgtctttc 6060 aggactcagg tcagtggcat
ctcctccaaa aactcccctt cccaccctcc atcacctcac 6120 cctgtttatc
tgcgcccccg cccccactgc ctgtcactta ttgcaggctg aagtgaccca 6180
ggctctccag ttgtacactc tcagatggac cctggacgac tgtggcactc ctgcaatttc
6240 cccagtctcc ctggggtagg attcctgctt gccaggatgc ccacctttcc
ttctccctcc 6300 tgcatgtcct cctctgcctg gcttctgaat tgttcccaga
gagagtgata gacaagatct 6360 gcctctcctt cagtccctga atcttattta
aggctcttgc tttgcttccc tggcctggag 6420 gcggctcctt gatggagtct
gccatgtggg ttcgctcatg gccatgtctt cctgcccagc 6480 atggtgcttg
gccctgggac tggccacata atatctgggc caggtgcaaa attagtacgg 6540
ggcagggggt actttgttca taggtgattc agaaccacat atggtgacct cagagtagga
6600 aaccaagtgt ggggccctta agagctgggg ggccctgtac gactgtccag
gttgcaggcc 6660 ccacagctcg cctcctgata tcctgtgctc catgcttgtc
tgttgaagga aggagtgaat 6720 ggatgaagag caggtggtgg gggtggtttg
agggccttgc tggtgggtgg gtagaggccc 6780 ctccctggca tggggctcaa
gacctgttcc atcccacagc ctggggcctg tgtgtaaatg 6840 gccaggacct
gcaggctggc atttttctgc tccttgcctg cctctggcct cccctttctc 6900
cacccatgtg gcccctcagg ttgccatcta gtccaaaagt ccccaaggga gacccagagg
6960 gccacttggc caaactactt ctgctccaga aaactgtaga agaccataat
tctcttcccc 7020 agctctcctg ctccaggaag gacagcccca aagtgaggct
tagccagagc ccctcccaga 7080 caagcgcccc cgcttcccca acctcagccc
ttcccagttc atcccaaagg ccctctgggg 7140 acccactctc tcacccagcc
ccaggagggg aaggagacag gatgaacttt taccccgctg 7200 ccctcactgc
cactctgggt gcagtaattc ccttgagatc ccacaccggc agagggaccg 7260
gtgggttctg agtggtctgg ggactccctg tgacagcgtg catggctcgg tattgattga
7320 gggatgaatg gatgaggaga gacaggagag gaggccgatg gggaggtctc
aggcacagac 7380 ccttggaggg gaagaggatg tgaagaccag cggctggctc
cccaggcact gccacgagga 7440 gggctgatgg gaagccctag tggtggggct
ggggtgtctg gtctcaggct gaggggtggc 7500 tggaaagata cagggccccg
aagaggagga ggtgggaaga acccccccag ctcacacgca 7560 gttcacttat
tcactcaaca aatcgtgact gcgcacgtac agtggctacc aggcgctggg 7620
ttcaaggcac tgcgggtacc agaggtgcgg agaagatcgc tgatccgggc cccagtgctc
7680 tgggtgtcta gcgggggtaa gaaggcaata aagaaggcac ggagtaactc
aaacagcaat 7740 tccagacagc aagagaaact acaggaaaga aaacaaacgt
gcgaggggcg aggcgaggaa 7800 acaacctcag cttggcaggt cttggaggtc
tctgggagga gaaagcagcg tctgatgggg 7860 gcgggaggtg gtgagtgggg
agaggtccag gcggagggaa tggcgagcgc agagacaggc 7920 tggcaacggc
ttcagggagg cgcggagggg tcagcgtggc tggcttaaaa ggatacatgg 7980
gactgagggg caagaccggc tcaagggtca ccgcttccag gaagccttct atttccgcgc
8040 caacctcggc gctcccccaa cttttcccac cgcggtccgc agcccacccg
tcctgctcgg 8100 gccgccttcc tggtccggac cgcgagtgcc gagagggcag
ggccggctcc gattcctcca 8160 gccgcatccc cgcgacgtcc cgccaggctc
taggcacccc gtgggcactc agtaaacatt 8220 tgtcgagcgc tctagaggga
atgaatgaac ccactgggca cagctggggg gagggcgggg 8280 ccgagggcag
gtgggaggcc gccggcgcgg gaggggcccc tcgaagcccg tcctcctcct 8340
cctcctcctc cgcccaggcc ccagcgcgta ccactctggc gctcccgagg cggcctcttg
8400 tgcgatccag ggcgcacaag gctgggagag cgccccgggg cccctgctat
ccgcgccgga 8460 gttggaagag ggtgggttgc cgccgcccga gggcgagagc
gccagaggag cgggaagaag 8520 gagcgctcgc ccgcccgcct gcctcctcgc
tgcctccccg gcgttggctc tctggactcc 8580 taggcttgct ggctgctcct
cccacccgcg cccgcctcct cactcgcctt ttcgttcgcc 8640 ggggctgctt
tccaagccct gcggtgcgcc cgggcgagtg cggggcgagg ggcccggggc 8700
cagcaccgag cagggggcgg gggtccgggc agagcgcggc cggccgggga ggggccatgt
8760 ctggcgcggg cgcagcgggg cccgtctgca gcaagtgacc gacggccggg
acggccgcct 8820 gccccctctg ccacctgggg cggtgcgggc ccggagcccg
gagcccgggt agcgcgtaga 8880 gccggcgcga tgcacgtgcg ctcactgcga
gctgcggcgc cgcacagctt cgtggcgctc 8940 tgggcacccc tgttcctgct
gcgctccgcc ctggccgact tcagcctgga caacgaggtg 9000 cactcgagct
tcatccaccg gcgcctccgc agccaggagc ggcgggagat gcagcgcgag 9060
atcctctcca ttttgggctt gccccaccgc ccgcgcccgc acctccaggg caagcacaac
9120 tcggcaccca tgttcatgct ggacctgtac aacgccatgg cggtggagga
gggcggcggg 9180 cccggcggcc agggcttctc ctacccctac aaggccgtct
tcagtaccca gggcccccct 9240 ctggccagcc tgcaagatag ccatttcctc
accgacgccg acatggtcat gagcttcgtc 9300 aacctcggtg agtaagggca
ggcgagggta cgcgtctcct ttcgggggca ctttgagact 9360 gggagggagg
gagccgcttc ttctatgcag cccgcccagc tttccgctcc tggctgaaat 9420
cgcagtgcct gcccgagggt ctcccaccca cagccctatg actcccaagc tgtgtgcgcc
9480 cccaggtcgg gcgcgctggg ttcggtgagc ctgtaggggt tactgggaag
gagggatcct 9540 ccgaagtccc ctccatgtta cgccgccggc cgcatctctg
gggctggagg caagggccgt 9600 tcaaagcgcg gggctcggtc atgtgagctg
tcccgggccg gcgcggctcg cgtaacctgg 9660 atgtaaaggg cccttcccgg
cgaggctgcc ttgccgccct tcctgggccc ctctcagccc 9720 tgcctggccc
tggcatcgcg gccgtcgcac ccccttaccc tccctgtcaa gccctacctg 9780
tcccctcgtg gtgcgcccgc cttagcgtac cgcgcgctcc gagcgcttgg ggcccctctc
9840 cgggccgccg gatgccccat tctctcttgg ctggagctgg ggaagaaacg
gtgccattgc 9900 taattttctt tgttttcttt ctttgtttat tttttttctt
ttttcttttt ttttcttttc 9960 ttttcttttc tttttttttt ttttttgaga
cggagttcac tcttgtcgcc cagtctggag 10020 tgcaatggcg cgatctctgc
tcaccgcaac ctctgcctcc cgggttcaag cgattctcgt 10080 gcctcagcct
cccgagtagc tgggattaca gcatgcgcca ccatgcctgg ctaattttgt 10140
atttttagta gagacagggt ttctccatgt taggcaggct ggtctcgaac tcccgatctc
10200 aggtgatcct cccgcctcag cctcccaaag tggtgctggg attacaggcg
tgagccactg 10260 tgccctgccg ctagtcttct attttaagta tttagtggta
ggtcccgggc cggcagaatc 10320 tattttcagc atttaccacg tgtggcgcgc
aaaccacagg ttttggcgat tgggttgcgc 10380 gggatctcag actgacgcgc
gggggcggct gggggtcccg gtttccgact ggagccgcga 10440 cgaccccggc
gacgcgagcc tggggctgca gcgagggccg gggagctccc cctccatatg 10500
tgcgcgcaca ttctccagac ttgctcaaac taaccccccg cggcgccagc gcgctgcggg
10560 actgatgatc aaatatttgg tttccgagat aacacacccc gatagcgctg
tttcctgagc 10620 cgctttcatt ctacttgtgt aacttgctgc gaaaacccga
accaagtcaa gacagcaaac 10680 tcaccccacg ggcgctgtgt caacatggaa
ataatgatac tgaagcccca cgctgggcac 10740 ctggggcgtg gactgggggc
gcgggggaag cgcagatccg ccttcatgct tcccccctcc 10800 tgataaggtc
cctggagttc ccgggaggcc attgtctgta cttaataata actaaatcca 10860
actagtgaac caagctt 10877 2 30 DNA HUMAN misc_feature (1)..(30)
Sense PCR primer for cloning 5' upstream human BMP-7 gene sequence
corresponding to the exon 1 region. 2 gggcgcagcg gggcccgtct
gcagcaagtg 30 3 30 DNA HUMAN misc_feature Complement((1)..(30))
Reverse PCR primer for cloning 5' upstream human BMP-7 gene
sequence corresponding to the exon 1 region. 3 agaggatctc
gcgctgcatc tcccgccgct 30
* * * * *