JPH064030B2 - DNA - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a DNA fragment useful for the production of peptides, and more particularly to a DNA fragment containing a base sequence encoding a precursor of cardionatrin.

Cardionatrin is a peptide consisting of 28 amino acids [Ser-Leu-Arg It is known that it has Na ⁺ diuretic action and vasorelaxant action with the precursor as the mother substance.

Although various studies have been conducted on the precursor itself at the peptide level, the present inventor has elucidated the structure of the precursor at the DNA level and contains a nucleotide sequence encoding a precursor of cardiononatrin. As a result of studies to obtain a DNA fragment, the present invention has been completed.

(Structure of the Invention) That is, the gist of the present invention resides in a DNA containing a base sequence encoding a precursor of cardionatrin shown by the amino acid sequence in FIG.

Hereinafter, the present invention will be described in detail.

First, an example of a method for preparing a DNA fragment according to the present invention will be shown.

Human heart fragments were homogenized with guadinyl thiocyanate and total RNA was analyzed by CsCl equilibrium density gradient ultracentrifugation.
(Chirgwin et al., Biochemistry 18 , 5294-5299, 1979). Then, this is purified by oligo (dt) cellulose column chromatography by a conventional method to isolate poly (A) -containing RNA (mRNA raw material).

From this mRNA raw material, the method of Okayama and Berg (Moleculer and Cellular Biology (Molecule)
ar and Cellular Biology) 2 , 161-170, 1982),
Obtain a cDNA library. That is, pBR322 and SV40
Using the hybrid plasmid of, a vector primer and an oligo (dG) tail linker are obtained. Reverse transcriptase is allowed to act in the presence of this vector primer and the above-mentioned mRNA to synthesize cDNA, which is then digested with the restriction enzyme Hind III, and then cyclized using the above-mentioned linker. afterwards,
By replacing the mRNA portion with DNA, a plasmid containing the cDNA fragment is obtained.

Then, this was subjected to Escherichia coli (Escherichia coli
i) etc. to obtain ampicillin resistant strain.

Next, a nucleotide complementary to the nucleotide encoding Met-Asp-Arg-Ile-Gly of cardionatrin was synthesized as a probe.

This probe is used to screen the above-mentioned cDNA library to select clones having complementarity. The cDNA fragment obtained from the clone was obtained by the method of Maxam and Gilbert (Methods).
Methods in Enzymology 65 , 49
9-560, 1980) to determine the nucleotide sequence.

The DNA fragment according to the present invention is a DNA fragment containing a base sequence encoding a precursor of cardionatrin, and the same sequence contains a base sequence encoding the amino acid sequence of cardionatrin. First example
As shown in the figure, the DNA fragment according to the present invention is not necessarily required to have a certain number of nucleotide residues.

Further, the DNA fragment according to the present invention is not limited to the above human origin, and other higher animals such as bovine,
It may be derived from pig, horse, mouse, rat and the like.

(Effects of the Invention) The DNA fragment according to the present invention can be introduced into the cloning site of an expression vector and expressed by a conventional method to produce a peptide. The resulting peptides (including cardionatrin) are useful as diuretics and vasodilators.

Hereinafter, the present invention will be described in more detail with reference to Examples.

Example 1 (1) Human heart fragments were disrupted in liquid nitrogen, and then an aqueous guanidinium thiocyanate solution was added to homogenize.
The obtained homogenate was subjected to the method of Chirgwin et al. (Biochemistry 18 , 5,294-52).
99, 1979), and total RNA was isolated by cesium chloride equilibrium density gradient ultracentrifugation. Then, this was purified by oligo (dT) cellulose column chromatography by a conventional method, and poly (A) -containing RNA was isolated and used as an mRNA raw material.

(2) On the other hand, the method of Okayama and Berg (Molecular and Cellular Biology)
lular Biology) 2 , 161-170, 1982), pBR322 and SV40
Using the hybrid plasmid of, a vector primer and an oligo (dG) tail linker were obtained.

That is, pBR322 and SV40 (map unit 0.71−0.8
400 μg of the hybrid plasmid of 6) was incubated with a restriction enzyme Kpn I at 37 ° C. for 4 times in a buffer containing bovine serum albumin.
Digested for hours.

Then, DNA was recovered by ethanol precipitation by a conventional method, dissolved in a buffer solution containing dTTP, terminal deoxynucleotidyl transferase was added, and the mixture was reacted at 37 ° C. for 30 minutes to generate a restriction enzyme Kpn. After adding about 60 dT tails to the digestion site of I, DNA was recovered by ethanol precipitation. Then, the restriction enzyme Hpa I was added to the DNA in a buffer containing bovine serum albumin.
(37 ° C., 5 hours). Larger DNA
The fragment was purified by agarose gel electrophoresis and subjected to the glass powder method (Vogelstein).
Proc.Natl.Acad.Sc, Proceedings of the National Academy of Sciences of the NA
iUSA) 76, have been conducted under the recovery in 615-619,1979). Then, this DNA was applied to an oligo (dA) cellulose column at 0 ° C., eluted with water, and then recovered with ethanol to obtain a vector primer having an oligo (dT) tail.

On the other hand, 100 μg of a hybrid plasmid of pBR322 and SV40 (map unit 0.19 to 0.32) was digested with the restriction enzyme Pet I in a buffer containing bovine serum albumin (37 ° C., 1 hour and a half). Then collect the DNA and dG
Dissolve in a buffer solution containing TP, add terminal deoxynucleotidyl transferase to 20 at 37 ° C.
The reaction was carried out for 1 minute, and about 10 to 15 dG tails were added.
The recovered DNA was digested with a restriction enzyme Hind III in a buffer containing bovine serum albumin (37 ° C, 1 hour), and then subjected to agarose gel (1.8%) electrophoresis to give a small oligo (dG) tail linker DNA. Extract the
Recovered.

(3) Okayama and Berg (Molecular and Cellular Biology)
Biology) 2 , 161-170, 1982) to obtain a cDNA library.

That, Tris-HCl (pH8.3), MgCl 2, KCl, dithiothreitol, dATP, dTTP, in dGTP and ^[32 P] mRNA30μg obtained above (1) in an aqueous solution containing dCTP and above (2) 10 μg of the obtained vector primer was added, and the mixture was allowed to react at 37 ° C. for 20 minutes in the presence of reverse transcriptase, and plasmid-
cDNA: mRNA was synthesized, and this was ethanol-precipitated and recovered in pellet form. This pellet was dissolved in a buffer solution containing CoCl ₂ , dithiothreitol, poly (A), [ ³² P] dCTP and terminal deoxynucleotidyl transferase and allowed to react at 37 ° C. for 10 minutes to give 10 dCMP per terminal. ~ 15 residues were added.

Then, the recovered oligo (dC) tail plasmid-cD
NA: mRNA-containing pellets were dissolved in a buffer containing bovine serum albumin, and the restriction enzyme Hind III was added thereto at 37 ° C.
After digesting for 1 hour, HindIII-digested oligo (dC) tail cDNA: mRNA plasmid was recovered by ethanol precipitation.

This was dissolved in the buffer solution containing the oligo (dG) tail linker DNA obtained in (2) above, incubated at 65 ° C for 2 minutes, kept at 42 ° C for 30 minutes, and cooled to 0 ° C. Then, in the presence of β-NAD (nicotine adenine dinucleotide), E. coli DNA ligase was added and incubated overnight. After that, dATP, dT
TP, dGTP, dCTP, β-NAD, E. coli DNA
Ligase, E. coli DNA polymerase and E. coli R Nas
After eH was added, the mixture was incubated at 12 ° C. for 1 hour and then at room temperature for 1 hour, and then cooled to stop the reaction to obtain the desired cDNA fragment-containing plasmid.

Then, using this plasmid, E. coli HB101 was transformed by a conventional method.

(4) On the other hand, Met-Asp-Arg-Ile-Gly of cardioonatrine
Nucleotides that were complementary to the nucleotides that code for were synthesized in two groups.

Then, a cDNA library was screened using the oligos I and II as probes, and 12 complementary clones were selected from 40,000 transformants. A cDNA fragment was extracted from these 12 clones and a restriction enzyme map was prepared.
It was discovered that the A fragment was present. Its common fragment is Maxam and Gilber.
t) (Methods in Enzymology (Methods
in Enzymology) 65 , 499-560, 1980). FIG. 1 shows the nucleotide sequence of this DNA fragment and the amino acid sequence assumed from it. Shows the sequence part of cardiotonatrine (in addition, shows the restriction enzyme cleavage site).

[Brief description of drawings]

FIG. 1 shows the nucleotide sequence of the DNA fragment according to the present invention and the amino acid sequence expected therefrom.

Continuation of front page (51) Int.Cl. ⁵ Identification number Office reference number FI technical display area C07K 99:00

Claims (1)

[Claims] [Claim 1] the following amino acid sequence Ser Ala Leu Leu Lys Ser Lys Leu Arg Ala Leu Leu Thr Ala Pro Arg Ser Leu Arg Arg Ser Ser Cys Phe Gly Gly Arg Met Asp Arg Ile Gly Ala Gln Ser Gly Leu Gly Cys Asn Ser A DNA containing a nucleotide sequence encoding a precursor of cardionatrin represented by Phe Arg Tyr-term.