Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
~ 72813-38
ISOLATED SILVER FOX GROWTH HORMONE GENE,
AND A RECOMBINANT DNA THEREOF
FIELD OF THE INVENTION
The present invention relates to an isolated gene
encoding silver fox growth hormone and a recombinant DNA thereof.
BACKGROUND OF THE INVENTION
So far, no structure has been known for the growth
hormone gene derived from a silver fox belonging to the super-
family Canoidea and even the gene has never been isolated. It
is known that animal growth hormone is a growth-promoting protein
which is present in the animal pituitary and a purified growth
hormone exhibits the growth promotion of a relevant animal when
administered.
SUMMARY OF THE INVENTION
A first aspect of the present invention provides an
isolated silver fox growth hormone gene encoding the polypeptide
set forth in SEQ ID No. 2~ The isolated silver fox growth
hormone gene is preferably that set forth in SEQ ID No. 1.
Another aspect of the present invention provides a recombinant
DNA comprising the silver fox growth hormone gene inserted into -~
a vector DNA.
BRIEF DESCRIPTION OF THE DRAWING
FIG. l is a restriction map of recombinant plasmid
pFGH300.
DETAILED DESCRIPTION OF THE INVENTION
An object of the present invention is to provide a
silver fox growth hormone gene for producing silver fox growth
2 LO~$34 72813-38
hormone by the techniques of genetic engineering.
As a result of extensive research on silver fox-derived
growth hormone, the present inventors have succeeded in isolation
of the growth hormone gene and subsequent determination of its
structure, to complete the present invention.
Hereinafter, the present invention is described in
detail.
The gene donor used in the invention includes e.g. the
pituitary derived from a silver fox belonging to the super-
family Canoidea. .
From the silver fox pituitary tissue, m-RNA is prepared
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dccording to a method as described e.g. in Proc. Natl. Acad. Sci.
U.S.A., vol. 70, p. 3646 (1973) or "Lab Manual Idenshi Kogaku"
(Laboratory Manual for Genetic Engineering), M. Muramatsu, page
70 (1988).
From the resulting m-RNA, cDNA can be synthesized according
to a method as described e.g. in Mol. Cell. Biol., vol. 2, p. 161
(1982) or Gene, vol. 25, p. 263 (1983).
A wide variety of recombinant phage DNAs are obtained by
inserting the above cDNA into a vector DNA, e.g. phage ~gtlO
(manufactured by Amersham). Then, ln vitro packaging of the
recombinant DNAs into phage particles is carried out using cDNA
cloning system ~gtlO (manufactured by Amersham) by the adapter
method instructed by the manufacture, and the phage particles
were then transformed into organisms such as E. coli L87 (ob-
tained from Amersham).
A cDNA coding for silver fox growth hormone (hereinafter
referred to as "growth hormone cDNA") is obtained from the cDNA
library (vector: ~gtlO) by plaque hybridization with a mink
growth hormone cDNA as a probe (see B.B.~.C., 173, No. 3, pp.
1200-1204 (1990)) on a Hybond blotting membrane (manufactured by
Amersham) according to the manufacture's instruction.
The recombinant phage DNA thus obtained is treated with a
restriction enzyme such as BamHI at 30-40 C, preferably 37 C, for
1-24 hours, preferably 2 hours, and then the reaction solution is
subjected to agarose gel electrophoresis (Molecular Cloning, p.
150, Cold Spring Harbor Laboratory (1982)), whereby a DNA
containing a gene encoding the growth hormone derived from a
silver fox (hereinafter referred to as "growth hormone gene") is
obtained.
In the invention, any vector DNA can be used which includes
plasmid vectorDNA, bacteriophagevectorDNA, etc. Specifically,
DNAs such as plasmid pUC118, pUC119 and pBLUESCRIPTII (manufac-
tured by Stratagene) are preferable. A cleaved vector DNA is
obtained by digesting the above vector DNA with a restriction
enzyme such as BamHI etc. (manufactured by Takara Shuzo Co.,
Ltd.).
The cleaved vector DNA is then mixed with the growth hormone
gene-containing DNA prepared above, followed by treatment e.g.
with T4DNA ligase (manufactured by Boehringer Mannheim GmbH),
thus giving rise to a recombinant DNA.
The recombinant DNA is transformed into e.g. E. coli K-12,
preferably E. coli JM109 (obtained from Takara Shuzo Co., Ltd.),
XLl-Blue (obtained from Funakoshi K.K.), etc., to give a transfo-
rmant originating in each bacterial strain. Transformation can
be effected according to the method described by D. M. Morrison
(Methods in Enzymology, vol. 68, pp. 326-331 (1979)).
The growth hormone gene is determined for its base sequence
according to the experiment under item (6) below. The base
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sequence thus determined is set forth in SEQ ID No. 1, and the
amino acid sequence encoded by this base sequence is set forth in
SEQ ID No. 2.
The present invention provides a silver fox growth hormone
gene and a recombinant DNA containing the same, and is industri-
ally extremely useful because the silver fox growth hormone can
be efficiently produced in a very short time by culturing an ~ .
organism (e.g., a microorganism) carrying said recombinant DNA.
EXAMPLES
Hereinafter, the present invention is further illustrated indetail with reference to the Examples which, however, are not
intended to limit the scope of the present invention. ~-
Cloning of silver fox growth hormone cDNA ~
(1) Preparation of pituitary tissue ;
30 silver foxes (obtained from Zao Mink Ranch) were sacri-
ficed and the pituitary was removed immediately. 0.6 g of -~
pituitary tissue was obtained. It was immediately immersed and
frozen in liquid nitrogen, and then stored in a deep-freezer at
80 C (manufactured by Ebara Co., Ltd.).
(2) Preparation of RNA
50 mg of the pituitary obtained in item (1) above was treated
according to the method described in Molecular Cloning, pp.
,. . ..
p ~ ,3
719-722, Cold Spring Harbor Laboratory(1989), to yield 150 ~g of
RNAs in total.
(3) Synthesis of cDNA
Synthesis of cDNA was carried out using a kit manufactured by
Boehringer Mannheim GmbH. According to the manufacture's
instruction, 60 ~g of the above RNA was treated in accordance
with the method described in Gene, vol. 25, p. 263 (1983), whereby
5 ~g of double-stranded cDNA was obtained.
(4) Preparation of cDNA library
Using a kit manufactured by Amersham (cDNA cloning system
~gtlO, adapter method), 1 ~g of the above cDNA was subjected to
the method indicated by the manufacture, thus giving rise to a
cDNA library (about 2,000,000 strains).
(5) Screening of growth hormone cDNA fragments
For screening of growth hormone cDNA, plaque hybridization
was carried out on a Hybond blotting membrane (manufactured by
Amersham) according to the manufacture's instruction. The
details of the screening are as follows:
The probe used in plaque hybridization was prepared using a
mink cDNA by Random Rrimer Extension Labeling System, NEP-103,
manufactured by DuPont.
For screening of the silver fox growth hormone cDNA, plaque
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~88~ 728l3-38
hybridization was carried out using the above prove on a Hybond
blotting membrane (manufactured by Amersham) according to the
manufacture's instruction. One positive clone strain was
obtained as a result of the screening of 10,000 strains from the
cDNA library obtained under item (4) above.
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(6) Analysis of the silver fox growth hormone gene
From the isolated positive clone, a phage DNA was obtained
according to a conventional method (Molecular Cloning, ed.
t Maniatis et al., pp. 77-85, Cold Spring Harbor Laboratory, USA,
1982), then cleaved with restriction enzymes such as BamHI,
~' EcoRI, etc., and analyzed by the aforementioned electrophoresis.
As a result, the silver fox growth hormone gene was estimated to
i be present on a ca. 800 bp BamHI-cleaved DNA fragment. This
fragment was subjected to agarose electrophoresis and purified
with GENECLEAN II (purchased from Funakoshi K.K.). The resulting
fragment was inserted into BamHI-cleaved pUCll9, thereby giving
recombinant plasmid pFGH300 (see FIG. 1). According to a
conventional method, this recombinant plasmid was transformed
into XL1-~lue, to give a transformant E. col1 XLl-Blue(pFGH300).
This transformant, E. coli XL1-Blue(pFGH300), has beendeposited
t with the accession No. FERM BP-4063 with Fermentation Research
Institute, Agency of Industrial Science and Technology, Japan.
The base sequence of the obtained plasmid was determlned with a
deletion kit for Kilo-Sequence (purchased from Takara Shuzo Co.,
Ltd.) using a 370 DNA Sequencing System (purchased from Applied
siOSystems Inc.). The determined base sequence and the amino
acid sequence encoded by the base sequence are set forth in SEQ ID
Nos. 1 and 2, respectively. The silver fox growth hormone gene
possesses a coding region for 648 bases encoding 216 amino acids.
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~ ,EQUENCE LISTING
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SEQ ID NO~
~, SEQUENCE LENGTH: 648
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~ SEQUENCE TYPE: nucleic acid --
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, STRANDEDNESS: double ~ .
,i~ TOPOLOGY: linear
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:~7 MOLECULAR TYPE: mRNA to cDNA
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' ORIGINAL SOURCE
i'i, ORGANISM: silver fox (Vulpes vulpes)
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~ SEQUENCE:
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,i~ ATG GCT GCA AGC CCT CGG AAC TCT GTG CTC CTG GCC TTC GCC TTG 4S
CTC TGC CTG CCC TGG CCT CAG GAG GTG GGC GCC TTC CCG GCC ATG 90
CCC TTG TCC AGC CTG TTT GCC AAC GCC GTG CTC CGG GCC CAG CAC 135
CTG CAC CAA CTG GCT GCC GAC ACC TAC AAA GAG TTT GAG CGG GCG 180
TAC ATC CCC GAG GGA CAG AGG TAC TCC ATC CAG AAC GCG ChG GCC 225
GCC TTC TGC TTC TCG GAG ACC ATC CCG GCC CCC ACG GGC AAG GAC 270
GhG GCC CAG CAG CGA TCC GAC GTG GAG CTG CTC CGC TTC TCC CTG 315
i~ CTG CTC ATC CAG TCG TGG CTC GGG CCC GTG CAG TTT CTC AGC AGG 360
GTC TTC ACC AAC AGC CTG GTG TTC GGC ACC TCA GAC CGA GTC TAC 405
GAG AAG CTC AAG GAC CTG GAG GAA GGC ATC CAA GCC CTG ATG CGG 450
GAG CTG GAA GAT GGC AGT CCC CGG GCC GGG CAG ATC CTG AAC CAG 495
ACC TAC GAC AAG TTT GAC ACG AAC CTG CGC AGT GAC GAT GCG CTG 540
CTT AAG AAC TAC GGG CTG CTC TCC TGC TTC AAG AAA GAC CTG CAT 585
AAG GCC GAG ACG TAC CTG CGG GTC ATG AhG TGT CGC CGC TTC GTG 630
GAA AGC AGC TGT GCC TTC
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~EQ ID NO: 2
SEQUENCE LENGTH: 216
SEQUENCE TYPE: amino acid
TOPOLOGY: not known
MOLECULAR TYPE: protein
ORIGINAL SOURCE
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ORGANISM: silver fox (Vulpes vulpes)
SEQUENCE:
Met Ala Ala Ser Pro Arg Asn Ser Val leu Leu Ala Phe Ala Leu
Leu Cys Leu Pro Trp Pro Gln Glu Val l,ly Ala Phe Pro Ala Met
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Pro Leu Ser Ser Leu Phe Ala Asn Ala Val Leu Arg Ala Gln His
I() 45
Leu His Gln Leu Ala Ala Asp Thr TYr l~s Glu Phe Glu Arg Ala
S0 .)5 60
Tyr lle Pro Glu Gly Gln Ar~ Tyr Sel lle Gln Asn Ala Gln Ala
, 0 75
Ala Phe CYS Phe Ser Glu Thr lle Pro Ala l'ro Thr GIY Lys Asp
~35 90
Glu Ala Gln Gln Arg Ser Asp Ual Clu leu Leu Arg Phe Ser Leu
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; 5~ 100 10~
.Leu Leu lle Gln Ser Trp Leu GIY Pro Val Gln Phe Leu Ser Arg
110 115 120
Val Phe Thr Asn Ser Leu Val Phe GiY Thr Ser Asp Arg Val TYr
` 125 130 135
Glu Lys Leu Lys Asp Leu Glu Glu G]y lle Gln Ala Leu Met Arg
1~0 14~ 150
Glu Leu Glu Asp Gly Ser Pro Arg Ala Gly Gln lle Leu LYS Gln
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,; 155 160 165
Thr Tyr Asp LYS Phe Asp Thr Asn Leu Arg Ser ASP Asp Ala Leu
... .
~;1 170 175 180
Leu Lys Asn Tyr Gly Leu Leu Ser Cys Phe LYS Lys Asp Leu His
185 190 195
Lys Ala Glu Thr TYr Leu Arg Val Met LYS CYS Arg Arg Phe Val
200 205 210
G I u Ser Ser Cys A I a Phe
215
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