JPS59140884A - Cloned dna fragment coding corticotropin releasing factor - Google Patents

Abstract

NEW MATERIAL:The cloned DNA fragment originated from the gene coding the corticotropin releasing factor (CRF) manifested in the hypothalamus, and containing a base sequence. USE:Useful as a probe and primer for cloning the large CRF precursor cDNA and the CRF cDNA of heterologous animals including man. PREPARATION:The mRNA of the objective gene is separated, purified and concentrated in high concentration as far as possible, from the total DNA extracted from the hypothalamus of sheep. The mRNA is used as the template to synthesize cDNA with a reverse transcriptase using oligodeoxythymidylate as a primer. The synthesized cDNA is integrated in a proper site of a proper vector to obtain a recombinant vector DNA, which is subjected to the hybridization using a proper marker probe. The product is screened to separate the clone containing the objective cDNA.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to corticotropin-releasing factor, a peptide expressed in the hypothalamus.
The present invention relates to cloned DNA useful for the genetic engineering production of in-releasing fac'Lor (hereinafter abbreviated as CRF), vector DNA incorporating the same, and transformed microorganisms.

More specifically, from the pituitary gland adrenofluticotropin (ACTH), β-endorphin (β-EP), β
- A cloned DNA 7 fragment derived from the gene encoding CRF expressed in the hypothalamus that promotes the release of lipotropin (β-LPH), etc., and containing the base sequence shown in Figure 1. The present invention relates to an integrated vector DNA, a microorganism transformed with this recombinant vector, and a method for producing the same.

・Togami's fields and conventional techniques'j The existence of CRF has been confirmed as a hypothalamic factor that promotes the release of ACTH from the pituitary gland [qui
llemin, R. et al., Endocrino
599 (1955) and 5affra
n, M., eLal, Can, J., Biocbe.
+n, Pbysiol, 3 3+408 (195
5)]. Isolation of CRF has been unsuccessful for a long time, and only recently has the group of Peil et al.
A peptide consisting of 41 amino acids thought to be F was isolated and its amino acid sequence was determined [Vale W,
et 'al, 5science 2↓3,1394
(1981) and 5piess, J.

et aLProc, natl, Acad,, S
ci, U, S, A.

I↑Takashi, 6517 (1981)]. According to the knowledge obtained to date, the CRF of the hypothalamus is A C', PH
ACTH1β made from a common precursor called -β-L P H precursor or preproobiomela/frutin
- Promote the synthesis and release of peptide hormones such as EP, β-L, PH in the pituitary gland [Yasuda,
N+et al, E++docr, Rev, 3.
] 23 (1982) and Numa, S, et
al,Trendsbiocbe+o,Sci,
6 r 274 (1981)]. Also, the sheep CRF is i
It is known to release ACTH in rats and humans in vitro and in vivo [Vale, W, et'al, 5cien.
ce 213.139.4 (1981), Turke
lsor+, G,, M, etat, PepLide
s 2, 425 (1981), R1vier.

C, eL al, Endocrinology
1 1.0.272 (1982), and Gross+
aan+A, at aLLanceLi, 921(
1982), CI+an, J., ,S., D., ,eL.
at.

Endo-crinology 1↓↓, 1388 (
1982), White+M, C, et aLLan
cet, May 29°1251 (1982)].

Since CRF mediates the innervation of the pituitary gland-adrenal cortex system, it is thought to play an important role in the endocrine response to Stotos, and is therefore a peptide that is thought to be useful as a drug for diseases related to these. be.

However, it has been difficult to produce such a high molecular weight peptide using conventional techniques.

3rd term 9 and fishing ・The present inventor has completed the present invention as a result of repeated research to find a method for efficiently producing eel CRF by applying genetic engineering technology, which has made remarkable progress in recent years. This is what I did.

To produce CRF by applying genetic engineering technology, CR
It is critical to clone cDNA complementary to the mRNA of F or its precursor. The present inventor obtained +nRNA from the total RNA extracted from the hypothalamus of Nanatsuji.
was isolated, cDNA was synthesized using this as a template, and cloning was performed using this, thereby successfully creating a cloned DNA 7 fragment containing part or all of CRF cDNA.

Therefore, the present invention provides ACTH1β-EP from the pituitary gland.
It is derived from the gene that feeds CRF, which is expressed in the hypothalamus and promotes the release of peptides such as , β-LP, and H.
It provides a cloned DNA 7 fragment containing the nucleotide sequence shown in Figure 1, and furthermore, a recombinant vector 1)''NA in which this cloned DNA 7 fragment is integrated into an appropriate vector, and its vector DNA.
The purpose of the present invention is to provide a transformed microorganism obtained by transformation using A.

2. A DNA fragment containing part or all of the cloned CRF cDNA can be used as a larger CRF precursor cDNA or a CRF cDNA of a different species (including humans).
cDNA containing the entire translated region of CRF or its precursor is itself a material for creating CRI'' producing microorganisms.

Structure and effect of the invention In order to prepare a cloned DNA 7 fragment, the +nRNA of the target gene is purified and concentrated to the highest possible concentration from the total DNA usually extracted from the sheep hypothalamus, and this +nRNA is used as a template to create an oligomer. c using reverse transcriptase with deoxythymicylate as a primer.
DNA is synthesized, this cDNA is inserted into an appropriate position in an appropriate vector to obtain a recombinant vector DNA, which is subjected to hybridization using an appropriate labeled probe, and subjected to screening to determine whether it contains the desired cDNA. A method of searching for clones is adopted.

Synthetic oligodeoxynucleotides are often used as probes when the amino acid sequence of the target substance is known; however, in the case of hypothalamic peptide hormones for which mRNA exists only in extremely small amounts, such methods can be applied. It's not clear what. In fact, sheep CR
In the case of F, in such a commonly used method, 100
Despite screening over 10,000 transformants, no clones containing CRF cDNA could be detected.

In order to increase the concentration of CRF cRNA, the present inventor synthesized a cDNA fragment by reverse transcription from +nRNA using a synthetic oligodeoxynucleotide mixture corresponding to a part of the amino acid sequence of CRF as a primer, and incorporated this into a vector.
The RF cDNA library I was constructed by screening this library using a different synthetic oligodeoxynucleotide mixture corresponding to part of the amino acid coding region of CRF as a probe. The corresponding cDNA fragment was cloned.

Since the cloned cDNA 'A fragment lacks the nucleotide sequence of the part corresponding to the downstream of the nucleotide sequence used as a primer, the method of Okayama et al. [0kaya+n ,i eLal
, Mo1. Ce11. Bic+l, 2.161
(4982)] was cleaved with restriction enzymes from the aforementioned cloned CRF cDNA. 1) Various clones containing the base sequence encoding the desired CRF amino acid sequence were screened using the NA fragment as a probe. Chemical formula 1) NA7 fragment was obtained. Cloning of the three 1) By repeating the screening using restriction enzyme-cleaved fragments of NA as primers or probes, we obtained a large number of DNA fragment clones of the CRF precursor and examined their nucleotide sequences. , Gila:) The primary structure of CRF precursor mRNA and CD'NA was determined.

In addition, as a vector for integrating the synthetic cDNA in the above method, any vector that proliferates in microorganisms when microorganisms are transformed after integrating the cDNA can be used, such as plasmids (E. coli plasmid pBR322, etc.). ), 7age (such as Ramug7age derivatives), viruses (such as SV40), which can be used alone or in combination, such as +)BR322
It may also be used in the form of -8V40 hybrid plasmid, etc. ().

Cloning I) of the present invention, NA fragment, is derived from the gene encoding CRF, which is expressed in the hypothalamus and promotes the release of peptides such as ACT H1β-EP and β-LPH from the pituitary gland. It contains the base sequence shown in FIG. The base sequence shown in Figure 1 is based on the primary structure of RNA (in the human CRF precursor book described later).
(see Figure 2), only the part corresponding to CRF is shown (however, U in Figure 2 is replaced with T), and -X- and -Y- can be arbitrary base sequences. Through research by the present inventors, the primary structure of sheep CRF precursor mRNA was clarified for the first time based on the base sequences of a large number of cloned DNA fragments obtained by the present invention. This is shown in Figure 2 (in the figure, if U is set to 'l', it indicates the primary structure of cDNA).

In Figure 2, the numbers at the bottom indicate the translation initiation codon A U
(The first base of i is numbered 1, and the numbers are given to the bases as + for the 3' end and - for the 5' end.

'' The numbers in two rows are the numbers assigned to the amino acids from the N-terminus to the C-terminus, with 1 being the first amino acid methionine in the translated region. AA LI AA in the 3' untranslated region
A (underlined) is the P1B A) A1J addition signal,
Arrows are polyadenylation sites. The translated region has base sequences 1 to 570, which corresponds to a 190 amino acid sequence. 1
No. 72 is considered to be a sequence corresponding to a signal peptide often found at the N-terminus of secreted proteins. 442-56
No. 4 (boxed part) is a sequence encoding the amino acid sequence of CRF, which is preceded by a sequence that covers four consecutive basic amino acids: Arg-Lys-Arg-Arg, and followed by a sequence that encodes Gly- There is a sequence encoding Lys followed by a stop codon (UGA). In addition, there are basic amino acid pairs at two positions upstream of the CRF code (amino acid sequence 1
Arg-Arg of numbers 16-117 and 127-128
Lys-Argo at No. 127 (Lys at No. 127 is changed to Glu depending on the clone).

The CRF precursor mRNA nucleotide sequence shows polymorphism, with three locations in the translated region (nucleotide sequences 369, 377 and 480).
The bases differ from clone to clone at two positions (base sequence number 578 and 923) in the untranslated region. That is, by the method described above, 7 fragments of phosphorylated DNA that share the same CRF amino acid sequence shown in FIG. 1) CRF8, pCRF25
, pcRF30 and pcRF31, the above five bases were as shown in F.

369th: l) G for CRF 31 and p'CRF25, U for pcRF30.

No. 379 1'l: pcRF31, pcRF25 and 1
) for CRF8 is A, 1) for CRF30 is C;.

480th: I) CRF 31, pCRF25 and I
) U in CRF 8, 1) C0923 in CRF30: 1 Sat U in pCRF 31, pCRF 3 t)
C0 578 No. 1'':l] G (overline) in CRF 31 and prison + CRF25, deleted in pcRF30.

This may be because the human hypothalamus that served as the raw material for mRNA extraction was pooled from many individuals. Base sequence 379th base 1 is from A to G
The amino acid at position 127 changes from lysine to glutamic acid, and I, ys-A at positions 127 and -128
It is thought that the basic amino sequence pair of rB disappears, causing a slight change in the processing of the CRF precursor. base sequence 369
．． Since the changes at bases 480.578 and 923 do not change the amino acid sequence, they are considered functionally synonymous.

As far as we have investigated, the amino acid sequences of CRF are exactly the same and no polymorphisms were observed.

The basic amino acid site deduced from the primary structure of to RNA A is considered to be the cleavage site during proteolysis [5teiner, D, F, eL al, A
++n, N.

Y, Acad, Sci, 3 above 3.1 (1980)]
Therefore, when the CRF precursor is processed, in addition to CRF, 2
It appears that ~3 new peptides are generated. That is, the structure of the sheep CRF precursor is schematically shown in FIG. 3. In FIG. 3, the CRF portion is indicated by a black frame, and the portion presumed to be a signal peptide is indicated by a dotted bar. Sites with basic amino acid pairs are marked with two vertical lines at the top. The basic amino acid pair portion is considered to be a processing site by proteolytic enzymes. ”
The second part of the numbers is the amino acid sequence number, starting with the N-terminal amino acid as number 1 and sequentially numbering toward the C-terminus.

A CRF production vector can be obtained by incorporating the phosphorylated DNA 7 fragment of the present invention into an appropriate expression vector. Any of the vectors mentioned above can be used, and the DNA 7 fragment integration site can be selected arbitrarily. That is, an appropriate operon of an appropriate expression vector can be cleaved using an appropriate restriction enzyme in a conventional manner, and the phantom cloned DNA 7 fragment can be processed to an appropriate length and integrated into the cleavage site.

A microorganism expressing a trait can be obtained by transforming a suitable microorganism by a conventional method with a vector incorporating the cloned DNA fragment.

The operon for gene expression is β-lactosidase,
Examples include operons such as tryptophan, β-lactamase, and alkaline phosphatase. Furthermore, examples of microorganisms used for transformation include Escherichia coli, Bacillus subtilis, and yeast.

Next, an example of the process from synthesis of a cloned L)NA fragment to production of a transformed microorganism according to the present invention will be described.

From the ovine hypothalamus, for example, the method 1cI of Chertsin et al.
+irgu+in, J1M, eL at, Biocl
+e+ois5y-1, 5294 (1979)], all RN and A were extracted, and this was added to oligo (d
T) Cellulose column chromatography [Aviv, [
(, eL al.

Proc, natl, Acad, Sci, U,
S, A, 69° 1408 (1972)] to separate poly(A)mR and NA. This lllRNA
as a template, amino acid residues 176 to 17 of the CRF precursor
cDNA using reverse transcriptase as a synthetic oligonucleotide mer corresponding to GlrGln-Ala-His at position 9
Synthesize A, convert it into double-stranded cDNA, l]BR
322 Pst1 sites using 7 methods [Noda+M,
eL at.

Nature 295, 202 (1982)], by the poly(dG)-poly(dc) homopolymer extension method. The obtained recombinant plasmid was subjected to the method of Cohen et al.
, Nacl.

Acad, Sci, 69.2110 (1'97
2)], transform E. coli to obtain a tetracycline-resistant strain. Tetracycline-resistant transformants were treated with 5′-”P-labeled oligonucleotides (C,
Amino acid residues 167-1°71 of the RF precursor (;
lu-MeL (corresponding to Tbr-Lys-Ala), and colonies containing a plasmid containing a nucleotide sequence complementary to this probe are screened. In this way, a strain containing a cloned DNA plasmid l+cRF 8 ) containing a base sequence that covers the CRF amino acid sequence portion is obtained.

The above-mentioned bacterial strain has a plasmid containing a nucleotide sequence corresponding to a portion of the blind RNA for CRF, but the nucleotide sequence lacks sequences downstream of the portion used as a primer. Therefore, as detailed below, this cloned cDNA fragment was used as a probe.
A more complete cDNA7 was created by screening the DNA library according to the method of Okayama et al.
It comes off by getting the ragment.

That is, a hybrid plasmid of 1]BR322 and SV40 is digested with the restriction enzyme KpnI, and the DNA is recovered by a conventional method. To this, an oligo (<IT) tail (=1) is added to the cleavage site of Kpnl using terminal dioxynucleotidyl trans7-erase.

After cutting the oligo(dT) tail DNA thus obtained with restriction enzyme) al endonuclease, the longer D
Separate and purify the N and A fragments and prepare plasmid primers.

Separately, the pBf (322 and s V40) 1 ifrid plasmid was cut with the restriction enzyme PsL l endonuclease, and the terminal deoxynucleotinol trans7
Oligo(d(i) tail is added by erase.

This is further digested with the restriction enzyme H1ndl Ifl endonuclease, and the shorter DNA fragment is separated and purified to obtain oligo(d(i) tail linker-DNA).

The thus obtained mRNA raw materials and plasmid primers were subjected to dAi''P, d'FTP in the presence of reverse transcriptase.
, dGTP, and dc'r'P to prepare a plasmid-CDNA:111 RNA complex. The resulting plasmid-cDNA:+nRNA complex was reacted with dCTP in the presence of terminal deoxynucleotidyl transferase to transform the oligo (clc) tail (=1
add Plasmid-cDNA:+0RNA complex with oligo(dC) tail added with restriction enzyme Hindll
After digestion with l, the DNA is reacted with the previously prepared oligo(dG) tail linker DNA in the presence of DNA ligase to cyclize it. Circular cDNA thus obtained: +.

The RNA complex plasmid was injected into four types of deoxyribonucleotide phosphates, namely clA""P, dTTF),
dGTP and dCTP and DNA ligase, 1) N
React in the presence of A polymerase and ribonucleotide l-1 to replace the RNA strand with a DNA strand, producing cDNA
Prepare fragment-containing plasmids.

Using the thus obtained plasmid, Escherichia coli is transformed to obtain an ampicillin-resistant strain, for example, according to the method of Cohen et al.

The thus transformed microorganism is cloned as described above.I) Using the NA fragment as a probe, a colony having a plasmid containing a sequence complementary to this probe is picked up among the colonies.

The E. coli strain thus obtained is a plasmid (pCRF3) containing the cloned DNA fragment of the present invention.
1.1) CRF30.1) CRF2S etc.). The plasmid can then be replicated by its multiplication. This strain also exhibits properties derived from this plasmid, such as ampicillin resistance due to the pBR322 origin of this plasmid, but its properties are common to the parent strain.

The recombinant DNA plasmid of the present invention can be easily obtained from the thus obtained strain of E. coli or the like by a conventional method. In addition, this plasmid was used in a conventional manner.
The cloned DNA 7-? of the present invention can be obtained by decomposing it with a restriction enzyme or the like. can be easily obtained.

The above clone had the entire translated region of CRF,
Since a part of the translated region of the CRF precursor was deleted, in order to re-clon the deleted site, we added an oligodeoxyribonucleotide 5'-GGGTCTCCATCGAGGT- complementary to the 224th to 238th base sequence of the mRNA.
3' by the triester method [Ito, H, et al.
Nucleic Ac1dsRes, I O,1
75'5 (1982)], and using this as a primer, cl)NA was synthesized from poly(A) mRNA, inserted into the ++BR322 vector according to the method described above, and pcR
The DdeI(131)-Xn+a1(237) fragment of F31 is 32p-labeled and hybridized using this as a probe at 60°C to obtain a recombinant DN'A plasmid (+)CRF38).

14aell(88)-Dde of this pCRF38
Further hybridization was performed at 50'C using the (131) fragment as a probe □ to generate a DNA containing a cD'NA fragment encoding the N-terminal region of the CRF precursor.
A plasmid (+)CRF43) was obtained, and this I]C
RF 43 Pst I(-26)-XmaI(94
) fragment as a probe, hybridization was performed at 60°C, and Ike's DNA plasmid (pCRF
90 and pcRF95).

The types of cloned CRF precursor cDNA fragments obtained as described above and the outline of the method for determining their base sequences are shown in FIG. 4.

In FIG. 4, the thick line at the bottom is the cloned cDNA7 fragment, and the thin line with an arrow indicates the size and direction of the restriction enzyme-digested DNA7 fragment used for base sequencing. The 5' end label is shown as a short vertical line, and the 3' end label is shown as a white circle. The diagonal line at the end of the arrow indicates that the 5' end label is on the vector DNA. At the top, the relevant restriction enzyme cleavage sites are indicated along with the number of the 5' terminal base resulting from the cleavage [indicated in parentheses]. The black frame represents the base sequence site used as a primer, and the diagonal line represents the base sequence site of the probe used for hybridization.

The numbers on the top row are the first base of the methionine fudon at the N-terminus of the CRF precursor as number 1, and the numbers are +, 5' towards the 3' end.
These are base sequence numbers assigned sequentially as - towards the end.

As is clear from FIG. 4, the cloned DNA plasmid prepared according to the present invention 1) CRF3.

1, pcRF30 and pCRF25 are both CRF
The cloned DNA 7 fragment is suitably treated and incorporated into a vector, and a microorganism expressing the expression can be obtained by transforming the microorganism.

The nucleotide sequence analysis of the cloned DN'A 7 fragment of the present invention revealed that CRF is produced by processing from a larger precursor protein.

In general, short-chain peptides such as insulin are often rapidly degraded within bacterial cells, so a method of synthesizing them as a high-molecular-weight fusion protein is used for expression. However, such fusion proteins often have lost their activity, and in order to obtain biologically active proteins, the fusion proteins must be treated with appropriate methods. The aforementioned CR
In the case of the F precursor protein, there is no need to process such a fusion protein; simply ligate the CRF cDNA precursor fragment with a vector that has an appropriate promoter upstream of the cDNA followed by a protein synthesis initiation signal. is achieved. This is because it is thought that in the hypothalamus, where the CRF precursor gene is expressed, the CRF precursor is appropriately processed by the action of an appropriate enzyme, and CRF is excised. This is an advantage when using a CRF precursor protein or a portion of a CRF precursor protein that includes CRF.

Ohsheno: Next, the present invention will be explained in more detail with reference to Examples.

[I] Isolation of pCR'F 8 [Preparation of +n RNA fraction from sheep hypothalamus] 100 ml of an aqueous guaninorthiocyanate solution (4M) was added to 5 sheep hypothalamus (30 g) and homogenized. This homogenate was mixed with a cesium chloride aqueous solution (5.9M
) and centrifuged at 30,000 x 20 hours to obtain a fraction containing a+ RNA in the form of a pellet. This fraction was treated with Tris-HCI and EDTA at 10% each.
Aqueous solution (pH 7,4) containing concentrations of mM and 1 mM (
Below, 10mM Tris-HCI-1mMEDTA (p
It is abbreviated as H7,4). )2I111, add KCI solution to this to make it 0.5M, and then purify by oligo(dT) cellulose column chromatography [eluent: 10+oMTris-HCI-1u+MEDTA
(pH 7, 4)] to obtain Po1A) mRNA fraction.

[Synthesis of cDNA 1 50+nM'rris-HCI (pH 8,3), L++
MMgCL, 31)mMKcI, 0,3u+M dithiothreitol, 2+nMdATP', 2mMdTTP,
A) mRNA obtained on Saturday and Sunday was added to 40 μm of an aqueous solution containing 2+J4dGTP and 2+oMdCTP.
Add 200 μ8 of the fraction and synthetic oligodeoxynucleotides and add 100 μ8 of atrimyeloid leukemia virus reverse transcriptase.
c'DNA was synthesized by reacting at 42° C. for 90 minutes in the presence of U. The reaction was stopped by adding 10% sodium dodecyl sulfate aqueous solution to the reaction solution, extracted with a water-saturated phenol-chloroform mixed solvent, and ethanol was added to the aqueous layer to extract cDN.
A: +nRNA complex was precipitated, washed with ethanol, and then recovered. The obtained μ-pellet was dissolved in 10'0 μm water, and 1,8N NaOH150+nMED was added to this.
20 μm of an aqueous solution containing TA was added and reacted at 70° C. for 30 minutes to decompose and remove +oRNA. 2NHC1 for this
After neutralizing by adding 18 μm of an aqueous solution, ethanol was added to precipitate single-stranded cDNA, and the single-stranded cDNA was recovered.

This -heavy chain cDNA'A-containing precipitate was treated with 0.5+oM each of dATP, dTTP, dGTP and dcTF' in 5 m
b'1Mgc'12,'70+oMKC1,15+oM
- 100 InM containing β-mercaptoethanol□nol and 8 U of DNA polymerase (large subunit)
Hepes buffer (pH 6,9) dissolved in 40μm
Double-stranded cDNA was synthesized by reacting at ℃ for 4 hours. The reaction was stopped by adding an aqueous solution of sodium dodecyl sulfate to the reaction solution, and after extraction with a water-saturated phenol-chloroform mixed solvent, ethanol was added to the aqueous layer to precipitate double-stranded CDNA, which was washed with ethanol and collected. . The resulting pellet was mixed with 50mM sodium acetate, 1mM ZnO,,
250mM NaCl and 0.25U S1 nuclease
was dissolved in 50 μm of an aqueous solution containing the following, and reacted at 115° C. for 30 minutes. The reaction was stopped by adding an aqueous solution of sodium dodecyl sulfate to the reaction solution, extracted with a water-saturated phenol-chloroform mixed solvent, and the aqueous layer was separated by ethanol precipitation.
DNA was collected.

Preparation of r oligo(dC) tail cDNA 1. The cDNA-containing pellet was mixed with 1+nMCoCI2.0.1mM dithiothreitol, 0.2μg poly(A), 0.1mM"H
-dCTP (1200 cpm/pmol) and terminal deoxynucleotinol trans7 errorase 2
130IoM sodium cacodylate containing 0(1)-30
+oMTris-HCI mild Tii solution (It) 16.8)
25 μm. In order to add 10 to 15 residues of dcMP per terminal, the reaction was carried out at 37° C. for 20 minutes, and the reaction was stopped by adding an aqueous solution of sodium dodecyl sulfate. After extraction with a water-saturated phenol-chloroform mixed solvent, ethanol was added to the aqueous layer to precipitate the oligo(dC) tail cDNA, and the resulting pellet was washed with ethanol and collected. This was mixed with 100mM Tris-HCI (pH 7,
4) Dissolved in an aqueous solution containing 10mM EDTA and 200mM NaCl to contain 0.2 μB of oligo(dC) tail cDNA per 1101.

[Oligo(dG) tail plasmid pBR322DNA
Preparation 1 20+aMTris-HCI (pH 7,4), 10mM
MgC12,50+nM(NH,), So, and 11
Dissolve pBR322 in an aqueous solution containing 0.1 TII8 bovine serum albumin per o1,
Add 15 U of restriction enzyme PstI endonuclease,
The reaction was carried out at 7°C for 1 hour. After the reaction is complete, pour the reaction solution into 7 parts.
- extract and ethanol precipitation from the aqueous layer.
A was collected. The obtained DNA was combined with the aforementioned oligo (dC).
Aqueous solution used for tail addition (contains 0.1+cM3H-dGTP instead of 3H-dCTP) 200 μm
and reacted at 37°C for 20 minutes using 80 U of terminal deoxynucleotidyl trans7-erase.
Approximately 10-15 dG residues were incorporated into this. The reaction solution was diluted with water-saturated 7-7-chloroform mixed solvent (1:1
), and oligo(
dG) Tail plasmid pBR322 DNA was recovered. , buffer this as in the case of oligo(dc) tail cDNA? In the evening, the oligo'(dG) tail plasmid pBR322 DNA was lysed to contain 92μ8 per 1+nl.

1 Preparation of recombinant plasmid 1 Add 50 μm of oligo(dC) tail cDNA solution to oligo(dC) tail cDNA solution.
dG) Tail 1) Water-soluble i night 5 containing BR322DN'A
()μm for 10 minutes at 65°C, then at 46°C.
for 120 minutes at 37℃, 60 minutes at 37℃ and
The recombinant tree plasmid solution was prepared by incubating for a minute to perform annealing.

Using the plasmid solution obtained in [Transformant Selection 1], E. coli X1776 strain was transformed according to the method of Cohen et al. with some modifications.

That is, E. coli
Culture in 20+al of L-broth supplemented with /ml at 37°C until the absorbance (600++11+) reaches 0.5, collect the cells using a centrifuge with a condenser, and add 5'OInM.
10+aMTris-1-1 containing CaCl2
cI buffer (+)I-17,3) Disperse 101 and 0
It was centrifuged again at ℃. The collected bacterial cells were added to the same buffer solution 21.
o1 and left at 0°C for 5 minutes. This dispersion 0.
Add the above recombinant plasmid solution 0.11111 to 2n+1.
Add and mix, leave to stand at () °C for 15 minutes, and then incubate at 42
After being kept at 0.degree. C. for 2 minutes, 0.51 o1 of L-broth having the same composition as that used in the previous culture was added and cultured with shaking for 1 hour. A portion of this culture solution was taken and added to the above-mentioned component pond containing L-1 containing 15 μg/+ol of tetracycline.
It was spread on a broth agar plate and cultured at 37°C for about 12 hours.

[Hybridization test J In order to screen for strains carrying a plasmid containing cDNA encoding CRF, a 32Fl-labeled synthetic oligonucleotide corresponding to part of the amino acid sequence of CRF (amino acid sequence No. 167-171 in Figure 2) was used. G Ic+
Using a mixed aqueous solution of the base sequence corresponding to -Met-Thr-Lys-Ala, the third base of the Ala codon is missing), using Hanahan and Meselson's method I.
Hanaban, D., & Meselson+N.
+C; elle + Yumaru, 63 (1980)],
Screening was carried out using these labeled synthetic nucleotides as probes, and strains containing plasmids containing sequences complementary to this probe were picked up.

Four colonies out of approximately 7.5 x 10 5 colonies were positive.

[Preparation of cloned DNA 7 fragment] The bacterial strain thus selected was cultured in the aforementioned L-broth supplemented with diaminopimelic acid and thymidine to obtain bacterial cells.

This bacterial cell was collected using the method of Schleif et al. (Scl+1eif,
R.

et al, “Practical Method
s in Molecular Biology” (
1981) tspri118er Verlag+
101) to obtain plasmid DNA (yield: 200 μg/culture solution 11). The plasmid DNA thus obtained was digested with the restriction enzyme Pst I and purified and separated to obtain a cloned DNA fragment. The nucleotide sequence of this cloned DNA fragment was determined by the method described below, and it was found that the cloned DNA 7 fragment of plasmid pCRF8 contained part of the amino acid coding region of CRF.

[111ρCRF25.1) CRF30 and CRF
The isolated pCRF8 of 31 lacks the nucleotide sequence downstream of the CRF nucleotide sequence used as a primer, so it contains a more complete CR.
To clone the FcDNA, the pCRF8 l
-1palI(346)-H,aeIII(512)
Using the fragment as Pro7, cDNA for sheep hypothalamic mRNA was prepared by the method of Okayama et al.
Performed library screening.

[Preparation of plasmid primers 1 60ug of a hybrid plasmid (map unit 0.71 to 0.86) of pBR322 and S V jO was mixed with 6n+M' ris-HCI (pH 7,5), 6mM
Dissolved in 1.5 n+l of an aqueous solution containing gC12,6+aM NaCl, 6mM 2-mercaptoethanol and 0.1 mg of bovine serum albumin per in+l, restriction enzyme K
Add 1.2000 pn I endonuclease, 37
After reacting at °C for 4 hours, a 10% aqueous sodium dodecyl sulfate solution was added to stop the reaction, and DNA was recovered from the resulting digestive fluid by ethanol precipitation according to a conventional method. This was mixed with 140 mM sodium cacodylate, 30
mM'l"ris-HCI (pi-16,8), 100
0.3 m of an aqueous solution containing MCoC12,0,1+aM dithiothreitol and 0,25n+MdTTP
10% sodium dodecyl sulfate. After terminating the reaction by adding an aqueous solution, ethanol precipitation was performed again to recover 1) NA.

The collected l)NA was added to 10+ oMTris-HCI (
1 mountain 7°4), 10mM MgCI2.20+aMKC1
, 1+J4 dithiothreitol and 0.1 per ml
The mixture was dissolved in an aqueous solution containing mg of bovine serum albumin, added with the restriction enzyme HpaI endonuclease 101) U, and reacted at 37°C for 5 hours to cleave the DNA.

The reaction solution was subjected to 1% agarose gel electrophoresis using the glass powder method (VolgelsLein, B+eL aL).
Proc, Na1l, Acad, Sci, Yu6,61
5 (1979) 1 with some modifications, the larger DNA was recovered. Add this DNA to 10+nMTr
is-HCI(l]H7,3), dissolved in 1 part 1 of an aqueous solution containing 1+nMEDTA and IM'Na'CI and
Oligo(dT) chromatography was carried out at
) Plasmid primer with tail approximately 14 ()μ8
I got it.

Preparation of E oligo (clG) tail linker-DNA 1 Oligo dG) tail linker-D according to the method of Okayama et al.
NA was prepared. 1) 100 μg of hybridoplasmid containing BR322 and SV 40 (map unit 0.19-0.32) was mixed with 6+nM Tris-HCI (pH
7,4), 6mM MgCl2, 6mM 2-mercaptoethanol, 50+nM NaCl and 1. An aqueous solution containing 0.1 mg of bovine serum albumin per ml. Restriction enzyme Pst I endonuclease 12
After the reaction, the reaction solution was extracted with phenol, and the aqueous layer was precipitated with ethanol to recover the DNA. The aqueous solution used for JT detail addition (however, 0.25+nM dTTP was used instead of 0.25+nM dTTP).
About 10 to about 15 dG residues were incorporated by dissolving in 50 μM (containing 1 mM dGTP) and reacting with 60 U of terminal deoxynucleotidyl trans7-erase at 37° C. for 200 minutes. The reaction solution was extracted with a water-saturated 7×7×9× roform mixed solvent (1:1), and DNA was recovered from the aqueous layer by ethanol precipitation.

This I) N A is 20. mMTris-HCI (pH
7,4),? m'M MgCl 2.60+IIMN
Dissolved in 50 μm of an aqueous solution containing aCI and 0.1 mg of bovine serum albumin per ml, and added the restriction enzyme Hi.
Add 50 U of nd III endonuclease and incubate at 37°C.
The reaction solution was subjected to 1.8% arose gel electrophoresis, and the small oligo (dG) tail linker DNA generated by restriction enzyme Hincl III endonuclease digestion was collected in the same manner as in the recovery of plasmid primers described above. It was collected using the method.

[Preparation of cDNA library I SOmMTris-HCI (pH 8,3), 8+n
MMgCI2.30mM KCI, 0.3+nM dithiothreitol, 2+nMdATP, 2mMd
l'TP, 2+oMdGTP and 32PdCTP
Aqueous solution 1 containing (1600, cln++/ pmol)
At 0 μm, the [11[16RN from the human hypothalamus]
A) mRNA fraction 2μ obtained in Preparation 1 of A fraction
8 and 1.4 μg of the plasmid primer obtained in [11 and Preparation of Plasmid Primer 1] were added and reacted at 37°C for 200 minutes in the presence of 5 U of avian myeloid leukemia virus reverse transcriptase to convert plasmid cDNA:llRNA.
The complex was synthesized.

The reaction was stopped by adding an aqueous solution of sodium dodecyl sulfate to the reaction solution, and the plasmid cDN was isolated by ethanol precipitation.
A: The n+R'NA complex was collected in the form of a pellet.

The pellet containing the plasmid cDNA:+oR'NA complex was mixed with 11M CoCl2.0, 1mM dithiothreitol, 0.2μM 3', 66μM 3'
130n+M sodium cacodylate-30 containing P-dCTP (1+200 cpm/pmol) and 18 U of terminal deoxynucleotidyl trans7 errorase
mMTris-HCI buffer (IIHO, 8) 15/7
It was dissolved in 1. The reaction was carried out at 37°C for 5 minutes to add 10 to 15 residues of dcMP per terminal, and the reaction was stopped by adding an aqueous solution of sodium dodecyl sulfate. After extraction with a water-saturated phenol-chloroform mixed solvent, the aqueous layer was
- Add oligo (dC) tail plasmid cD N
A: +nRNA A complex was precipitated, and the resulting pellet was collected after washing with ethanol.

The obtained pellet was treated with 20"IIMTris-HCI (p
H7゜4), 7mM MHCl, 2.60mM NaCl, and 0.1n+H bovine serum albumin per 1+nl were dissolved in a buffer solution, digested with 2.5U of restriction enzyme Hind III endonuclease at 37°C for 1 hour, and precipitated with ethanol and collected. . This is 10+OMTris
-Dissolve in 10 μm of HCIII^MEDTA<pH 7,3), add 3 μl of ethanol, and dissolve the oligo(dC).
A tail cDNA-mRNA plasmid solution was obtained.

1 μm of this solution was added to 10 mM'r'ris-H, CI (p
H7゜5), 1+nMEDTA, 0.1M NaCl, and the linker-DNAO obtained in the above [Preparation of oligo (dG) tail linker-DNA], '041111101 was dissolved in 10 μm of an aqueous solution, and after incubation at 65°C for 2 minutes, Annealing was performed at 42°C for 30 minutes. This was cooled to 0° C., 0.6 μg of E. coli DNA ligase was added, and the mixture was incubated overnight.

Add 40 μM each of d'ATP, dTTP, and dGT to this solution.
P and clcTP, 0.15mM β-NAD, 0,4
#g E, coli DNA ligase, 0.3μ
gE, E. coli DNA polymerase, 1 μg E, and E. coli ribonuclease H were added to bring the total volume to 100 μm, and the mixture was incubated first at 12° C. and then at room temperature for 1 hour each. Add cold 10+oMT to the reaction solution.
A plasmid solution was prepared by adding ris-HCI (pH 7,3) 0.91111 to stop the reaction.

Using this plasmid solution, transformation was carried out by the method described in [I] [Transformant Selection 1].
- A cDNA library was prepared by spreading on broth agar plates and selecting resistant bacteria.

1cRIF”c, Cloning of DNA7 fragment I
In order to screen for strains having a plasmid containing cDNA for +aRNA encoding CRF, preparation of 7 cloned DNA fragments of [I] Cloning of pCRF8 as described in Section 1 I) NA
7 fragments of Hpal1Flaell fragment
cD labeled with 2F+ and prepared in the previous section according to the method of Hanahan and Meselson using this as a probe.
A seven-NA library was screened to select strains that hybridized with this probe. Approximately 1.5X1
Five of the 0' colonies were positive.

From the positive strain obtained above, plasmid I) NA was extracted by the same method as in [11. Purification and separation were performed to obtain 7 cloned DNA fragments. As a result of determining the base sequence as shown below, three clones 1)
DNs of CRF25, pCRF3() and pcRF31
All A7 fragments were CRF cDNA7 fragments containing all base sequences corresponding to the CRF amino acid sequence.

[Determination of base sequence of cloned DNA 1 The plasmid DNA thus obtained was digested with various restriction enzymes, and the base sequence of appropriate restriction enzyme fragments was determined.

That is, the plasmid DNA (for example, pCRF31-
)50/'H to 6+aMTris-1-ICI (pH 7
,4), 6+nMMgCl□, 50+aMNaCl,6
200 μm of aqueous solution containing mM 2-mercaptoethanol
Digested with 50 U of Haell for 1 hour at 37°C, and purified by 5% polyacrylamide del electrophoresis at 200 U.
1 μB of bp Haell17 fragment was recovered. This was mixed with 20+oM Tris-1-I CI buffer (
Lll-17, 9) in 50 μm, E. coli alkaline 7 male 7 atase 0. Dephosphorylation was performed using IU at 55° C. for 30 minutes. Tsuide, 501oM
Tris-HCI (pi-17,6), 10IIIMM
Polynucleotide kinase 0.5 μm in an aqueous solution containing gC12 and 10n+M2-mercaptoethyl.
IU and 7-32P-ATP (>5000 Ci/
+o+ool) for 1 hour at 37°C.
' end was labeled with 32p. 6+aM of labeled DNA
Tris-HCI (pH 7,4), 5 (Ill
5au3A in 40 μM aqueous solution containing M NaCl, 5 mM MgCl□, and 6 mM 2-mercaptoethanol.
125b was reacted for 1 hour at 37°C using
A 1+ HaellLSau3 A I 7 fragment was recovered.

This DNA fragment is subjected to a base-specific chemical reaction [
Maxan++A0M, et al, “Metl+
od orEnzy+nology"65 + 49
9<1980); Rubin, C0M, et
al, Nucleic Ac1ds Re5
earthb, 8.4613 (1980) 1, D
A partial decomposition product of NA was obtained. This was electrophoresed on an 8% polyacrylamide, 8M urea gel and a 20% polyacrylamide, 8M urea gel. After the electrophoresis was completed, the gel was subjected to autoradiography to determine the base sequence.

As a result, the base sequence is the 312th GGCCG in the base sequence shown in Figure 2 (however, U can be read as r).
It was up to the 413th 12 CGCCA from CC...

13 Furthermore, 0 μg of the same plasmid DNA was added to 6+OMTri.
s-HCI(pi-(8,0), 10+nM MgC
l 2.60o + MNaC1 and 6mM 2-mercaptoethanol dissolved in an aqueous solution containing Aval, I1
After reacting for 1 hour at 37° C. using 0 U, 4 μ8 of Aval17 fragment of 2300+)l) was recovered by 5% polyacrylamide gel electrophoresis. This was subjected to dephosphorylation and end labeling with 32p as described above. Labeled DNA was treated with 6mM Tris-HCI (pH 8,0).
, 12+oMMgCl2.50mM NaCl and 6m
Dissolved in an aqueous solution containing M2-mercaptoethanol, R
Digested with saI4U to obtain 350b1+ Avail
-Rsa I 7 fragments were recovered. Haell
A base-specific chemical reaction and polyacrylamide gel electrophoresis were carried out in the same manner as in the case of the base sequence of the -8au3 A I 7 fragment, and the base sequence was determined by 1-autotrapography. The determined base sequence is 3 in Figure 2.
577th 7 from 7()th GGTCC...
0...A A It was up to CG.

77 From these results, it was determined that there was an overlap of I (the aellisau3 A I fragment and the Avall-Rsa I 7 fragment were at least 43 bl), and that this was a continuous base sequence. These results and the results of determining the base sequence of DNA 7 fragments obtained by cutting this plasmid DNA with various restriction enzymes using a similar method revealed that CRF precursor +n RN
It was determined that A had the base sequence shown in Figure 2.

[Brief explanation of the drawing]

Figure 1 is the base sequence corresponding to CRF, Figure 2 is sheep C
Primary structure of RF precursor + oRNA, Figure 3 shows sheep CR
FIG. 4, a drawing schematically showing the structure of the F precursor, shows the types of cloned CRF precursor cDNA fragments and an outline of the method for determining their base sequences. Patent applicant: Dainippon Pharmaceutical Co., Ltd. (1 agent) Patent attorney Seihaku Ao Haka (procedural amendment form) Commissioner of the Japan Patent Office (1) Indication of case 1 Patent application No. 013511 of 1988 2, Title of invention case Relationship with Patent applicant address 3-25 Doshomachi, Higashi-ku, Osaka Name (291
) Dainippon Pharmaceutical Co., Ltd. (and 1 other person) 4. Agent 7, Contents of amendment (1) Amend "Figure 2" in Mei IIB, page 11, line 5, to include all "Figure 2 (I) and ■" do. (2) Insert the following phrase between "Fig. 2" and "in" on page 1-1, line 7 of the same book. "(Figure 2 (2) is a continuation of Figure 2 (I))" (
3) "Figure 2" on page 44, line 3 of the same book is corrected to read "Figure 2 + IJ and tapping." (4) In the drawings, “Figure 2” and “Figure 2 (continued)”
8. List of attached documents. 8. List of attached documents.

Claims (6)

[Claims] (1) Cloned D derived from the gene encoding fluticotropin releasing factor (hereinafter abbreviated as CRF) expressed in the hypothalamus and containing the base sequence shown in Figure 1.
NA7 Ragment. (2) Vector DNA incorporating a cloned DNA 7 fragment derived from the gene encoding CRF expressed in the hypothalamus and containing the base sequence shown in Figure 1
0 (3) Vector D N that incorporates a cloned DNA 7 fragment derived from the gene encoding CRF expressed in the hypothalamus and containing the base sequence shown in Figure 1.
Microorganisms transformed by A. (4) The microorganism according to claim (3), wherein the transformed bacterial strain is Escherichia coli. (5) Extract total RNA from the sheep hypothalamus with guanidyl thiocyanate, separate the m RNA fraction by affinity column chromatography, and use this as a template for C
, a cDNA fragment is synthesized using reverse transcriptase using a synthetic oligodeoxynucleotide corresponding to a part of the RF amino acid sequence as a primer, and this is incorporated into a vector. Next, microorganisms are transformed with this vector DNA, and then this transgenic A clone containing a CRF cDNA fragment is obtained by screening the formant using a labeled synthetic oligodeoxynucleotide corresponding to a part of the CRF amino acid sequence as a probe, and then a part of the CRF cDNA fragment in this clone is screened. was used as a probe, and a cDNA library created using vector primers and linkers was screened and CR
DN having the base sequence of the entire amino acid coding region of F
CRF, characterized by taking a clone containing the A fragment
A method for producing a microorganism having a vector DNA incorporating a CDN derived from a gene and an A'7 fragment. (6) The production method according to claim (5), wherein the microorganism carrying the vector DNA incorporating cDNA 7 ragmen F derived from the CRF gene is Escherichia coli.