JPH0322986A - Novel isolated dna - Google Patents

Abstract

NEW MATERIAL:A DNA coding amino acid sequence of cerebral sodium diuretic peptide derived from rat expressed by formula I (X1 expresses existence or absence of Met). USE:Used to produce various peptides having precursor of rat cerebral sodium diuretic peptide and biological activity similar to the precursor. PREPARATION:Whole RNA is separated from rat tissue considered as containing cerebral sodium diuretic peptide (rBNP) and mRNA is purified to construct cDNA library. The cDNA library is subjected to screening by hybridization using DNA fragment coding precursor of swine BNP as a probe to afford rBNP clone.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to novel DNA isolates and peptides, and in particular to novel DNA isolates and peptides encoding brain natriuretic bebutide derived from rats. .

C
de Bold, ^l. et. al. , Life S
cionce 28, 89.1981: Keele
r, R. , Can. J. Phyciol. Pharselfa
col. , 60, 1078, (1982)]
The entity that causes natriuresis has been studied, and natriuretic bebutide has already been isolated from rat and human atria, and its structure has been elucidated [FIynn, T.
G. et. at. , Biochem. Bio
phys. Res. Commun,, 117,
859-865 (1983); Kangawa,
K. et. al. , Biochem. Bio
phys. Res. Commun. 118, 13
1-139 (1984)]. Atrial Natriuretic Pep
These bebutides, named Tide, AN It has become.

On the other hand, a peptide of 26 amino acid residues and a peptide with an extended N-terminus were recently identified from pig brain extracts [Sudo, T. at. al. , Na
ture, 332. 78-81 (19811);
Sudo, T. et. al. + Bioch
em. Biophys, Res. Commun.
155, 726-732 (i9gg)]. these are,
It is very similar in structure and pharmacological activity to ANP, and 2
The six amino acid residue peptide is a porcine brain natriuretic peptide.
etic PeptideSpBNP]. Furthermore, the structure of the cDNA encoding the pBNP precursor has been established by the present inventors [S
udo, T. et. a1. , Biochem
．． Biophys. Res. Commun. 1
57. 4 to-4 16 (1988)].

Problems of the Invention As mentioned above, pBNP and the DNA sequence encoding it have become known, but
P is not even known to exist.

Even when the antiserum against the 26 amino acid pBNP was used, no immunoreactivity could be found in rat tissues.

The present inventors have demonstrated that not only ANP but also BNP in pig species.
In view of the discovery that these two pezutids may be widely present in mammalian species and that they play a role in maintaining body fluid homeostasis, we hypothesized that BNP of L.
We have conducted extensive research to find out.

The present inventors used the cDNA fragment of the pBNP precursor as a probe to screen a rat tissue cDNA library.
The present invention was completed by isolating a cDNA encoding NP (hereinafter abbreviated as rBNP), cloning it, and determining the amino acid sequence of the corresponding peptide.

The structure and effects of the invention, namely, the present invention provides rat-derived brain natriuretic bebutide represented by the sequence formula (I): (wherein, X, X, X3 represents the presence or absence of Met) or its I) NA isolate encoding the amino acid sequence of the active region fragment. Furthermore, the present invention provides, as a preferred embodiment, the sequence formula (bi) 1 Y, GAT CTC CAG AAG GT, which encodes Lanoto cerebral natriuretic bebutide.
G CTG CCC CAG ATG31 ATT
CTC CTC CTG CTT TTC CTT A
AT CTG TCG61 CCC CTG GGA
CGT CAC TCC CAT CCC CTG
GGA91 AGT CCT ACC CAG TC
T CCA GAA CAA TCC ACG121
ATG CAG AAG CTG CTG GAG
CTC ATA AGA GAA151 ^^G T
CA GAG GAA ATG GCT CAG AG
A CAIE CTC+81 TC^^^G GAC
CAA GGC CCT ACA AAA GAA
CTT2+1 CTA ^^A AGA GTC C
TT AGO TCT CAA GAC ^6C241
GCC TTC CGG ATC CAG GAG
AGA CTT CGA AAT271 TCC
AAG ATG GCA CAT AGT
TCA AGC TGC TTT301
GGσCAG AAG ATA GAC CGG AT
C GGC GCA GTCLeu Gly Ser
Pro Ser Gin Ser Pro Glu G
in Ser Thr Met GinGin Arg
Gin Leu Ser Lys Asp Gin
Gly Pro Thr Lys Glu LeuLe
u Lys Arg Val Leu Arg Ser
GI++ Asp Ser Ala Phe Arg
lieCys Phe Gly Gin Lys l
le Asp Arg lle Gly Ala Va
l Ser ArgLeu Gly Cys Asp
Gly Leu Arg Leu Phe, array formula (■
); Thr Met Gin Lys Leu Leu G
Fu Leu lie Arg Glu Lys Se
r GluLys GluLeuLeu
Lys Arg l/al Leu Arg
Ser Gin Asp Ser AlaP
he Arg lie Gln Glu Arg Le
u Arg Asn Ser Lys Met Ala
HisVat Ser Arg Leu Gly C
ys Asp Gly Leu Arg Leu Ph
e, or array formula (Ill): 331 AGT CGC TTG GGC TGT
GAC GGG CTG AGG TTG 3603
61 TTT, Sequence formula (II'): Y. CAT CCC C Ding G CCA GAA CAA TCC GAG CTG AT^^G^ GCT CAG AGA CAG CC Ding ^CA A^^ G^^ ^GG TCT CAA GAC GAG AGA CTT CG^^GT TCA
AGC TGC CGG ATC GGC GC^ GAC GGG CTG AGG or array 2〇F): GGA AGT CCT AGC AGO ATG CAG AAG GAA AAG TCA GAG CTC TCA AAG GAC CTT CTA ^^A AG^ ^GC GCC TTC CGG AAT TCC ^^ G ATG TTT GGG CAG ＾＾G GTC AGT CGC TTG TTG TTT 363, CAG TCT 105 CT[E ＣＧ １３５ GAA ATC 165 C＾＾GGC 195 GTC CTT 225 ^TC CAG 255 GCA CAT 285 ^TA GAC 315 GGC TGT 345 Y3 AAT TCC 295 TGC TTT GGG 325 GCA GTC AGT 355 ^GG TTG TTT ^^G ATG GCA CAT AGT TCA A
GC 294CAG AAG ATA GAC CGG
ATC GGC 324CGC TTG GGC T
GT GAC GGG CTG 354 (wherein, Y,
Y,,Y,represents the presence or absence of ATG. )"' is provided.

Furthermore, the present invention provides a DNA containing the amino acid sequence represented by the above sequence formula (r), (U) or (1).
An isolate, preferably a DNA isolate having the sequence formula (■゛), (■゛) or (■゜), is introduced into an expression plasmid, and a host cell transformed with the expression plasmid is cultured. The resulting rat brain natriuretic bebutide is also provided.

The amino acid sequence represented by the sequence formula (1) consists of the amino acids from position -26 or -25 to position 95 in the amino acid sequence of the brain natriuretic peptide derived from Lanoto shown in Figure 2, and is represented by the sequence formula (II). The amino acid sequence shown consists of the amino acids from position 1 to position 95 in the amino acid sequence shown in FIG. The amino acid sequence is the amino acid from position 64 to position 95 in the amino acid sequence shown in Figure 2, or further 6
Consists of amino acids with Met at the N-terminus at position 4.

Among the above amino acid sequences, those that do not have Met at the N-terminus are rBNP containing a signal peptide.
, rBNP lacking signal conjugate, and rBN
It represents an active region fragment of rBNP that retains the activity of P. In addition, when the above-mentioned bebutide is expressed in the large intestine by genetic recombination technology, Met at the N-terminus corresponding to the start codon may not be removed and may remain at the N-terminus. is a fact known to those skilled in the art, and such bebutides are also included in the present invention.

In this specification and drawings, abbreviations representing amino acids are commonly used in the art and have the following meanings.

Asp: L-aspartic acid, Ser: L-serine, Gly+glycine, Cys: L-cysteine, Phe: L-phenylalanine, Arg: L-arginine, Lue: L-leucine, 11e: L-inleucine, Asn: L-asparagine, Vat: L-valine, Tyr: L-tyrosine.

The DNA isolate of the present invention is prepared, for example, as follows.

First, total RNA is isolated from Lanoto silk which is thought to contain rBNP, RNA III is purified from this, and a cDNA library is constructed by a conventional method. Next, this cDNA library is screened by hybridization using a DNA fragment encoding the pBNP precursor as a probe to obtain an rBNP clone. These operations will be specifically explained below.

(1) Construction of cDNAΔ library Total RNA was isolated by grinding rat atrial tissue with guanidine thiocyanate and then subjecting it to ultracentrifugation. Purification of poly(A) RNA (mRNA) was performed by oligo-dT column chromatography [Chirgwin, etal. , Bi
oche+*istry, 18. 5294-529
9 (1979)]. Construction of cDNA libraries from mRNA can be carried out using the Gubler and
Hoffman. Gene, 25, 263-269 (
1983)].

(2) Screening of rat cDNA clone pBN
The established DNA sequence of the cDNA encoding P was derived from the known rat ANP [Kangawa, at
a1. , Nature, 312, 152-15
5 (1984)], pBNP (nucleotides 298-393) consisting of 32 amino acids located at the 3' end of the PBNP reading frame [hereinafter referred to as pB
Relatively high homology (approximately 61.1%) was observed only in the region corresponding to NP-32, and less than 45% homology was observed in the remaining 5' side. In order to clearly distinguish between rANP and rBNP that exist in rat atrial tissue (rANP is more rBNP in rat atrial tissue),
(considered to be more abundant than BNP), first, r
l of the pBNP precursor showing low homology to the ANP precursor.
9 5bp Hhal (9 2)/ Bcnl (2
87) The fragment was used to screen a cDNA library to clone the rBNP precursor. However, even when the stringency of hybridization was lowered, no positive blur was obtained. This suggests that pBNP and rBNP have extremely low sequence homology in the region corresponding to this probe. Then, the above pBN
The probe corresponding to the I”32 area (hereinafter referred to as the probe [p
Screening was carried out using BNP (referred to as BNP). When hybridization was performed at a formamide concentration of 30% or higher, no positive plaques were obtained. However, under lower stringency conditions, running the hybridization at a 20% formamide concentration, more positive blanks were obtained than expected.

This suggested that undesired ANP clones were also detected by cross-hybridization with this probe. Among these positive clones, 625b, which corresponds to almost the entire region of rat ANP-cDNA, was used to exclude ANP clones.
A second hybridization was performed using a long p probe (referred to as probe [rANP]) under high stringency conditions of 50% formamide. In this way, from among the 250,000 silk exchange brak,
Eighteen clones showing positive signals only by probe [pBNP] were successfully cloned.

(3) Sequencing of cDNA The clone designated λrBNP5I7 having the longest cDNA insert was sequenced by the chain terminator method based on the restriction site map shown in FIG.

The complete nucleotide sequence of 628 bp thus established (excluding the (A) tail) is shown in the accompanying Figure 2. Two other clones, λrBNP107 and λr
BNP518 was found to have a relatively short cDNA insert identical to nucleotides '129-57 11 and [6-562] of λrBNP517. The nucleotide sequence of λrBNP107 was found to have nucleotides 321, 336 and Although C was substituted with A at position 390, no amino acid substitution occurred.

The longest rBNP contained in clone λrBNP517
- The cDNA runs from the ATG start codon (nucleotides 1-3) to the TAG stop codon (nucleotides 364-3).
It has a single-stranded reading frame up to 366) (Figure 2). The 3°-untranslated region contains the hexanucleotide sequence AATAAA (nucleotide 543,548), which is known to precede the polyadenylation site in many eukaryotic mRNAs. This reading frame encodes the entire molecule of the 121 amino acid residue predicted rat BNP precursor, which is similar to pBNP-3.
It has a C-terminal sequence homologous to 2.

The predicted amino acid sequence of the la7}BNP precursor is shown in the accompanying Figure 2. As previously mentioned, this precursor is clearly different from the rat ANP precursor, but structurally similar to the porcine BNP precursor. The rBNP precursor is significantly different from the rANP precursor in that it contains an AT-rich region (nucleotides 453-48) in the 3' untranslated region of rBNP-cDNA.
5) is recognized (Figure 2), which indicates that ANP-
It cannot be found in cDNA. This AT- is it rich? The columns are present in the PBNP precursor as well.

Such AT-rich nucleotide sequences are known to destabilize III RNA in cells, as seen in lymphokines and broto-oncogenics, indicating that BNP expression is ANP
This suggests that it may be regulated in a manner different from that of .

Introducing the DNA fragment of the present invention into an expression vector,
By the conventional means in the art of transforming host cells with this and culturing the transformants,
It is possible to produce P precursors as well as various peptides having similar biological activities. The obtained rat BNP has a smooth muscle relaxing effect as well as lid BNP.
It is thought to have diuretic and natriuretic effects and hypotensive effects, and may be useful as a therapeutic agent for, for example, cardiac edema, hypertension, congestive heart failure, acute and chronic renal failure, and the like.

Figure 1: Restriction site map for sequencing the cDNA insert in clone λrBNP517. This map includes
Only restriction endonuclease sites relevant to the invention are shown. Numbers in parentheses are 5 generated by cleavage
' indicates the number of the terminal nucleotide. Each arrow indicates the direction of sequencing and its length. The predicted γ-rBNP (95 amino acid residues considered to be the active expression part) and the corresponding part of the signal peptide are indicated by white bands and diagonal lines, respectively.

FIG. 2; Nucleotide sequence and predicted amino acid residue sequence of the cDNA insert in λrBNP517. Nucleotide residue sequence numbering begins with rAJ, the ATG codon encoding the predicted initiation methionine, and nucleotide residue sequences 5' to nucleotide 1 are numbered negatively. The stop codon is indicated by three consecutive stars. Arrows indicate sites likely to be processed by signal bebutidase. The hexanucleotide preceding the polyadenylation site is underlined. 3' untranslated region (nucleotide 453i8
The dotted line portion in 5) represents a 3° AT-rich sequence.

Examples are given below to help understand the present invention.
The preparation of DNA isolates according to the present invention is not limited thereto.

Rat atrium was used as the rat tissue for preparing aRNA. As mentioned above, rBNP did not immunoreact with the pBNP-specific antiserum, so it was not possible to determine in which tissue of the rat rBNP was present in large amounts. Therefore, in pigs, rat atrium was used in this study because pBNP was detected most frequently in the heart, particularly in the atrium.

Total RNA was extracted from the atria of 22 rats by the guanidine thiocyanate method. Grabler and Hoffman [
Gubler and [Ioffan, Gene,
25. Double-stranded cDNA was synthesized from 3 μg of rat atrium "Aori(A)" RNA by the method of 263269 (19g: {)].

After ligating EcoRI linkers, the cDNA was isolated from phage λgt 10 [Bethesda Rese
arch Laboratory] and in vitro packaging was performed. This yielded approximately 3.8XlO' independent clones per ng of BoIvaA) RNA.

B. rBNP clones were isolated by a two-step method of cDNA library screening and sequencing.

After labeling with 3″P by multi-priming method, the resulting cDNA library was screened using probe [pBNPJ and probe [rANP]. Probe [pBNP] was pBNP (96-13
] 0 5bp of pBNP-cDNA corresponding to 0)
Bcnl (2 8 7) / Rsal (3 9 2
) fragment, and the blope [rANP] is rA
It was a 625 bp fragment (nucleotides -57 to 568 (Pst1 site)) of NP-cDNA. Hybridization was performed at 37°C for 16 hours in a solution of 3xSSC, 501IM Tris-HCl (pH 7.5), 1mM EDTA 1xDenhardt's, and 20μg/xQ of denatured salmon sperm DNA. Two different concentrations of formamide were carried out to adjust the hybridization stringency. First, approximately 250,000 recombinant phages were transferred to a nitrocellulose membrane and screened using a probe [pBNP] in a 20% formamide solution (low stringency). This film is 3XSSC,0
．． Washed twice with 1% SDS (37°C, 1 hour) and then twice with 0.1xSSC, 0.1% SDS (42°C, 1 hour). After autoradiography, the membranes were boiled in water to remove the radiolabeled probe. The washed membrane was then probed [rANP] in a 50% formamide solution (high stringency).
hybridized with. Membrane 3xSSC, 0.1% SD
Wash twice at 0.S (68°C for 140 minutes) and then at 0.5°C for 140 minutes.
1×SSC, 0. 2 in 1% SDS (68°C, 1 hour)
Washed twice. Clones showing a positive signal only with probe [pBNP] were selected and analyzed. the largest c
Clones λrBNP517 with a DNA insert, and λrBNP107 and λrBNP5 with relatively short inserts]. 8 was subcloned into the M13 vector using the chain terminator method [Sanger, et.
al. , Proc. Natl. Acad.
Sci. u. s. ^． , 74. 5463
-5489 (1977)].

[Brief explanation of drawings]

FIG. 1 is a restriction site map for sequencing the cDNA insert in clone λrBNP517, and FIG. 2 is a schematic showing the nucleotide sequence and predicted amino acid residue sequence of the cDNA insert in λrBNP517. It is a diagram.

Claims (1)

[Claims] 1. Array formula (I): X_1 Asp Leu Gln Lys Val L
eu Pro Gln Met Ile Leu Le
u LeuLeu Phe Leu Asn Leu
Ser Pro Leu Gly Gly His S
er His ProLeu Gly Ser Pro
Ser Gln Ser Pro Glu Gln
Ser Thr Met GlnLys Leu Le
u Glu Leu Ile Arg Glu Lys
Ser Glu Glu Met AlaGln A
rg Gln Leu Ser Lys Asp Gl
n Gly Pro Thr Lys Glu Leu
Leu Lys Arg Val Leu Arg S
er Gln Asp Ser Ala Phe Ar
g IleGln Glu Arg Leu Arg
Asn Ser Lys Met Ala His S
er Ser Ser Ser Cys Phe Gly Gln
Lys Ile Asp Arg Ile Gly
Ala Val Ser ArgLeu Gly Cy
s Asp Gly Leu Arg Leu Phe
(In the formula, X_1 represents the presence or absence of Met.)
A DNA isolate encoding the amino acid sequence of rat-derived brain natriuretic peptide shown in 2. Array formula (I'): Y_1 GAT CTC CAG AAG GTG C
TG CCC CAG ATGATT CTG CTC
CTG CTT TTC CTT AAT CTG
TCGCCG CTG GGA GGT CAC TC
C CAT CCC CTG GGAAGT CCT
AGC CAG TCT CCA GAA CAA T
CC ACGATG CAG AAG CTG CTG
GAG CTG ATA AGA GAAAAG T
CA GAG GAA ATG GCT CAG AG
A CAG CTCTCA AAG GAC CAA
GGC CCT ACA AAA GAA CTTCT
A AAA AGA GTC CTT AGG TCT
CAA GAC AGCGCC TTC CGG A
TC CAG GAG AGA CTT CGA AA
TTCC AAG ATG GCA CAT AGT
TCA AGC TGC TTTGGG CAG AA
G ATA GAC CGG ATC GGC GCA
GTCAGT CGC TTG GGC TGT G
AC GGG CTG AGG TTGTTT (In the formula, Y_1 represents the presence or absence of ATG.)
The DNA isolate according to claim 1, which is represented by: 3. Sequence formula (II): X_2 His Pro Leu Gly Ser P
ro Ser Gln Ser Pro Glu Gl
n SerThr Met Gln Lys Leu
Leu Glu Leu Ile Arg Glu L
ys Ser GluGlu Met Ala Gln
Arg Gln Leu Ser Lys Asp
Gln Gly Pro ThrLys Glu Le
u Leu Lys Arg Val Leu Arg
Ser Gln Asp Ser AlaPhe A
rg Ile Gln Glu Arg Leu Ar
g Asn Ser Lys Met Ala His
Ser Ser Ser Cys Phe Gly G
ln Lys Ile Asp Arg Ile Gl
y AlaVal Ser Arg Leu Gly
Cys Asp Gly Leu Arg Leu P
A DNA isolate encoding the amino acid sequence of a rat-derived brain natriuretic peptide represented by he (in the formula, X_2 is synonymous with X_1). 4. Array formula (II'): Y_2 CAT CCC CTG GGA AGT C
CT AGC CAG TCTCCA GAA CAA
TCC ACG ATG CAG AAG CTG
CTGGAG CTG ATA AGA GAA AA
G TCA GAG GAA ATGGCT CAG
AGA CAG CTC TCA AAG CAC C
AA GGCCCT ACA AAA GAA CTT
CTA AAA AGA GTC CTTAGG T
CT CAA GAC AGC GCC TTC CG
G ATC CAGGAG AGA CTT CGA
AAT TCC AAG ATG GCA CATAG
T TCA AGC TGC TTT GGG CAG
AAG ATA GACCGG ATC GGC G
CA GTC AGT CGC TTC GGC TG
TGAC GGG CTG AGG TTG TTT (
In the formula, Y_2 has the same meaning as Y_1. ) The DNA isolate according to claim 3. 5. Sequence formula (III): X_3 Asn Ser Lys Met Ala H
is Ser Ser Ser Cys Phe Gl
y GlnLys Ile Asp Arg Ile
Gly Ala Val Ser Arg Leu G
ly Cys AspGly Leu Arg Leu
Phe (wherein, X_3 is synonymous with X_1)
NA isolate. 6. Array formula (III'): Y_3 AAT TCC AAG ATG GCA C
AT AGT TCA AGCTGC TTT GGG
CAG AAG ATA GAC CGG ATC
GGCGCA GTC AGT CGC TTG GG
C TGT GAC GGG CTGAGG TTG
The DNA isolate according to claim 5, represented by TTT (wherein, Y_3 is synonymous with Y_1). 7. DN according to any one of claims 1 to 6
Rat brain natriuretic peptide obtained by culturing recombinant host cells transformed with an expression plasmid into which isolate A was introduced.