JPH1084970A - New transcription control factor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a DNA comprising a DNA coding a polypeptide having such transcription control activity as to enable the mass secretive expression of useful extraneous protein. SOLUTION: This DNA comprises a DNA consisting of part or the whole of an amino acid sequence of the formula and coding a polypeptide having transcription control activity (i.e., a DNA coding a polypeptide having activity to promote or inhibit transcription from a DNA to mRNA), e.g. transcription control factor gene ERN4 of Saccharomyces cerevisiae. This DNA is capable of cloning the gene ERN4 by One-Hybrid method.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel transcription factor. Specifically, the present invention relates to a method for controlling a stress response of eukaryotic cells using the transcriptional regulator and a method for expressing a foreign protein.

[0002]

BACKGROUND OF THE INVENTION Heat shock proteins (HSPs) are at the center of stress proteins, but a group of other proteins collectively referred to as glucose regulatory proteins (GRPs) has long been known. In eukaryotic cells from yeast to mammals, glucose regulatory proteins are present in the lumen of the endoplasmic reticulum in a well-preserved form, and folding of secretory proteins in the endoplasmic reticulum as molecular chaperones and folding enzymes ) Plays a very important role. For example,
Yeast Kar2p (also called GRP78 or BiP, hereinafter GRP78) is essential for vital activity in yeast, and GRP78 and GRP94 in mammalian cells are deeply involved in the folding process of antibodies and cell membrane receptors. In the experiment, GSP was used together with HSP70 mRNA when subjected to stress such as ischemia.
It has been reported that RP78 mRNA is induced.
Although it is considered to be a clinically important molecule in this way, stress proteins in the endoplasmic reticulum (molecular chaperones and folding enzymes) are compared with cytoplasmic stress proteins represented by heat shock proteins (HSPs).
The current situation is that research is far behind.

While HSP is induced by heat shock, GRP is not induced by heat shock, and cells are exposed to various stresses, such as glucose starvation, tunicamycin (T), a protein glycosylation inhibitor.
M), the GRP stress response is induced at the transcriptional level by accumulation of a protein (unfolded protein) that cannot take a normal three-dimensional structure in the endoplasmic reticulum by the calcium ionophore A23187, a calcium chelator or a reducing agent, etc. It is considered that the process and the HSP response process are different systems. However, GRP
The analysis of the expression induction mechanism of Escherichia coli is greatly delayed as compared with HSP, and little is known about the pathway of the transmission of stress signals from the endoplasmic reticulum to the nucleus and the transcription control sequences and transcription factors of the GRP gene.

[0004] Saccharomy (Saccharomy)
ces cerevisiae), when unfolded protein accumulates in the endoplasmic reticulum due to stress, information is transmitted from the endoplasmic reticulum to the cytoplasm and further to the nucleus via Ern1p, which activates the transcriptional regulator (UPRF). It has been clarified that the transcription of the endoplasmic reticulum stress protein gene such as GRP78 is finally induced (Mori, K. et al., Cell 74, 743-756 (1993)). Also,
A cis sequence (UPRE) that responds to endoplasmic reticulum stress on the promoter of the yeast GRP78 gene was also identified (Mo
ri, K. et al., EMBO J. 11, 2583-2593 (1992): Kohno, K. et a
l., Mol. Cell. Biol. 13, 877-890 (1993)).

[0005]

The newly synthesized secreted proteins and membrane proteins become functional proteins by folding of peptide chains and association of subunits to form oligomeric proteins and disulfide bonds in the endoplasmic reticulum. Is transported to the Golgi. The formation of higher-order structures of such proteins in the endoplasmic reticulum is believed to proceed rapidly and accurately through the intervention of molecular chaperones and folding enzymes in the lumen of the endoplasmic reticulum. On the other hand, when a useful foreign protein is secreted and expressed in a host by recombinant DNA, the molecular chaperone or folding enzyme in the endoplasmic reticulum is insufficient to cope with a large amount of the foreign protein, and a small high-order structure cannot be formed. Degradation or accumulation in the endoplasmic reticulum often prevented the desired protein from being expressed at high levels as expected. Techniques for quantitatively controlling the molecular chaperones and folding enzymes that constitute GRP are important techniques that enable large-scale secretory expression of useful foreign proteins.

[0006] Therefore, it is necessary to clarify the molecular mechanism of GRP induction by so-called endoplasmic reticulum stress, which causes accumulation of unfolded proteins in the endoplasmic reticulum, and to determine a technique enabling large-scale secretory expression of useful foreign proteins. Is required. A first object of the present invention is to provide a DNA containing the amino acid sequence set forth in SEQ ID NO: 2 or 3 in the sequence listing, or a part thereof, and containing a DNA encoding a polypeptide having a transcription regulatory activity. To provide. A second object of the present invention is to provide a Saccharomyces cerevisiae mutant strain in which the DNA has been disrupted.
is to provide n4Δ. Furthermore, a third object of the present invention is to provide a method for controlling a molecular chaperone and a folding enzyme in a yeast endoplasmic reticulum by transforming a yeast with an expression vector containing the DNA,
An object of the present invention is to provide a method for expressing a foreign protein by transforming yeast with an expression vector containing the expression vector and a foreign protein expression vector.

[0007]

Means for Solving the Problems In view of the above, the present inventors have made intensive studies in the analysis using Saccharomyces cerevisiae, taking advantage of the fact that genetic techniques can be used.
First, a cis-sequence (UPRE) that responds to endoplasmic reticulum stress on the GRP gene promoter was analyzed in detail, and based on this finding, a novel transcriptional regulator (UPRF) gene (designated ERN4) involved in the induction of endoplasmic reticulum stress proteins was obtained. Was successfully cloned, and the present invention was completed.

That is, the gist of the present invention is to provide (1) a DNA encoding the polypeptide having the amino acid sequence of SEQ ID NO: 2 or a part thereof and having a transcription regulatory activity. (2) a DNA containing a DNA encoding a polypeptide having the amino acid sequence of SEQ ID NO: 3 in the sequence listing or a part thereof and having a transcriptional regulatory activity, (3) the sequence listing SEQ ID NO: 1 or a part thereof, and D which encodes a polypeptide having a transcription regulatory activity
The DNA according to the above (1) or (2), which comprises NA,
(4) the amino acid sequence of SEQ ID NO: 2 in the sequence listing,
Or an amino acid sequence consisting of 1 or 2
DNA encoding a polypeptide having at least one of the deletion, addition, insertion or substitution of the above amino acid residues and having a transcription regulatory activity, (5) the amino acid set forth in SEQ ID NO: 3 in the sequence listing, DNA encoding a polypeptide having at least one of deletion, addition, insertion or substitution of one or more amino acid residues in a sequence or an amino acid sequence consisting of a part thereof and having a transcriptional regulatory activity (6) a DNA that is hybridizable to the DNA according to any one of (1) to (5) and encodes a polypeptide having a transcription regulatory activity or a functionally equivalent activity thereof; 7) DNA is Saccharomyces cerevisia
e) the DNA according to any one of the above (1) to (6), which is the transcription regulatory factor gene ERN4 or a mutant gene thereof;
(8) a polypeptide encoded by the DNA according to any one of (1) to (7), (9) a polypeptide comprising the transcription activation domain of a transcriptional regulator that functions in yeast and the polypeptide according to (8). Fusion protein, (10) the transcription activation domain is the transcription activation domain Gal4p (a) of the transcription regulator Gal4p of Saccharomyces cerevisiae.
(11) the fusion protein according to the above (9), which is d);
DNA encoding the fusion protein according to (9) or (10), (12) (1) to (7) or (1).
1) a recombinant DNA containing the DNA of any one of the above,
(13) an expression vector containing the recombinant DNA according to (12), (14) an antibody that specifically binds to the polypeptide according to (8) or the fusion protein according to (9) or (10), or The fragment, (15) E
Ern4Δ, a Saccharomyces cerevisiae mutant strain in which the RN4 gene has been disrupted, (16) A method for controlling a molecular chaperone and a folding enzyme in the lumen of a yeast vesicle, comprising transforming a yeast with the expression vector according to (13). (17) the control method according to (16), wherein the yeast is a mutant strain ern4Δ in which the ERN4 gene has been disrupted; (18) the yeast is transformed with the expression vector according to (13) and a foreign protein expression vector. (19) A method for expressing a foreign protein according to (18), wherein the yeast is a mutant strain ern4Δ in which the ERN4 gene has been disrupted.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION The DNA encoding the polypeptide having the transcription regulating activity of the present invention is defined as a DNA derived from mRN.
The DNA is not particularly limited as long as it encodes a polypeptide having the activity of activating or suppressing the transcription into A. For example, the amino acid sequence described in SEQ ID NO: 2 or 3 in the sequence listing, or one of them. And a DNA having the base sequence of SEQ ID NO: 1 or a part thereof. In addition, at least one of deletion, addition, insertion or substitution of one or more amino acid residues occurs in the amino acid sequence of SEQ ID NO: 2 or 3 in the sequence listing or an amino acid sequence consisting of a part thereof. It may be a DNA encoding the polypeptide to be made.
Furthermore, DNAs capable of hybridizing to these DNAs
Are also included in the DNA of the present invention. Specific examples include the transcription regulatory factor gene ERN4 of Saccharomyces cerevisiae or a mutant gene thereof.

[0010] The polypeptide of the present invention comprises the above-described DN.
The polypeptide is not particularly limited as long as it is a polypeptide encoded by A and has a transcription regulatory activity, and also includes a fusion protein of the transcription activation domain (ad) of a transcription regulatory factor that functions in yeast and the polypeptide. . As the transcription activation domain, Gal4p (ad) derived from the transcription regulatory factor Gal4p of Saccharomyces cerevisiae and the like are preferable.

[0011] The recombinant DNA of the present invention contains a DNA encoding the polypeptide having the transcription regulating activity of the present invention, including a DNA encoding the fusion protein described above.

The expression vector of the present invention is not particularly limited as long as it contains the recombinant DNA of the present invention and is introduced into a target host and expresses a desired protein.

Antibodies that specifically bind to the polypeptide of the present invention can be prepared by, for example, a conventional method (Current Protocols in Immunolog).
y (Coligan, JEet al.eds) 2.4.1-2.4.7, John Wiley &
After immunizing rabbits or the like with the polypeptide as an antigen according to Sons, New York (1991)), blood can be collected to obtain an antiserum. Further, using the polypeptide as an antigen,
Ordinary method (Cell & Tissue Culture; Laboratory Procedure (D
oyle, A. et al., eds) 25A: 1-25C: 4, John Wiley & Sons, Ne
w York (1994)). Fragments generated by protease digestion after purification of these antibodies are also included in the antibodies of the present invention.

In the present invention, the molecular chaperone in the lumen of the endoplasmic reticulum refers to a protein that temporarily stabilizes an unfolded or partially folded region of a target protein and causes inappropriate intramolecular or intermolecular interaction. Saccharomyces cerevisiae, GRP78
(DnaK homolog of Escherichia coli) and Scjlp (DnaJ homolog of Escherichia coli) and the like.

[0015] Folding enzymes are a group of accessory proteins that catalyze specific isomerization that limit the rate of folding of certain proteins. Examples of Saccharomyces cerevisiae include Pdilp (promotion of disulfide bond formation), Euglp (a multicopy suppressor of PDI [protein disulfide isomerase] 1-disrupted strain), and Fkb2p (proline cis-trans isomerase).

The following three points have been elucidated so far regarding the mechanism of induction of stress proteins in the endoplasmic reticulum of Saccharomyces cerevisiae. i) The proteins induced include GRP78 and Scjlp, which are thought to function as molecular chaperones, and the folding enzymes Pdilp, Euglp, Fk
b2p must be a total of 5 types. ii) When unfolded protein accumulates in the endoplasmic reticulum due to stress, Ernlp, a transmembrane glycoprotein present in the endoplasmic reticulum, is activated and information is transmitted to the cytoplasmic side (changes in the endoplasmic reticulum change Promotes dimerization, and consequently activates a phosphorylase region present on the cytoplasmic side of Ernlp to phosphorylate a substrate present in the cytoplasm). iii) The information transmitted to the cytoplasm side is transmitted to the nucleus by some mechanism, the transcriptional regulator UPRF is activated, and the cis sequence UP present on the promoter of the induced gene.
Induction of transcription through RE. UPRE is GRP
It has been identified as a 22 bp sequence that exists on the promoter of 78 genes and functions as necessary and sufficient conditions for induction (FIG. 1).

(1) Cloning of transcriptional regulator UPRF First, a detailed structure-activity relationship concerning UPRE was clarified, and based on this information, a DNA-binding protein such as a transcriptional regulator was genetically cloned using yeast. One-Hybrid
d) Cloning the UPRF by the method.

Analysis of cis sequence UPRE The reporter plasmid is CYC, which is often used in yeast.
1-lacZ (beta-galactosidase gene linked to cytochrome C isoform 1 promoter) expression plasmid pLGΔ-178 (Guarente, L. and Maso
n, T. Cell 32, 1279-1286 (1983)).
A wild-type (designated Y) UPRE is inserted upstream of CYC1-lacZ and introduced into yeast cells by transformation. Tunicamycin (TM) is added as a stress at a concentration of 5 μg / ml, and the cells are cultured for 3 hours. Tunicamycin is known to inhibit the process of adding sugar chains to nascent proteins in the endoplasmic reticulum, thereby accumulating proteins with an abnormally folded structure and inducing endoplasmic reticulum stress. The transformant into which the reporter plasmid has been introduced is cultured in the presence or absence of stress, and the β-galactosidase activity expressed in the cells is measured.

Next, for the purpose of knowing the detailed structure-activity relationship of UPRE, point mutants were prepared for 19 bases of the 22 bp UPRE sequence, and their activities were measured in the same manner as in the case of wild-type Y ( (Fig. 1). As a result,
When mutations are introduced into the 12th CAG and the 14th to 16th GTG, even if stress is applied, β-galactosidase activity is hardly increased, and almost no UPRE activity is lost. Since the sequence of CAG-GTG is the same as the so-called E box (CANNTG) consensus sequence, the region rich in basic amino acids is
It is considered that a transcription regulatory factor having an NA binding domain is likely to bind to this portion. Substitution of the 13th C between CAG and GTG with G, A, and T, respectively, decreases β-gactosidase activity. Also, CAG
When the spacing between GTG and GTG is reduced to 0, the activity becomes about 7%. Conversely, when the spacing is increased to 2 or more by inserting C, the activity completely disappears (FIG. 2). Therefore, the presence of only one C between CAG and GTG is extremely important for UPRE activity.

This region partially has a palindromic structure. As shown in Table 1, in wild-type Y, 4
This is a palindrome consisting of bp. 12th G is C
(12C), the G at position 19 is replaced with C (19C), and both are replaced to a complete palindrome consisting of 7 bp (UY). These mutations and β
-The correlation of galactosidase activity is examined in the presence and absence of stress. UY shows about twice the activity of wild type Y, and UPRF is presumed to recognize palindromic.

Furthermore, the GAA portion of the second to fourth GAA of UPRE also plays an important role in activity, and when a mutation is introduced into this portion, the inducing activity is considerably reduced (FIG. 1). From the above results, UPRE contains the second to fourth GAA,
Three transcriptional regulators recognizing mainly the 12th CAG and the 14th to 16th GTG bind, of which 10
It is presumed that the transcriptional regulator having a region rich in basic amino acids as a DNA binding domain is likely to bind as a homo- or heterodimer to the CGCCGTG portion at the 16th position.

Cloning of UPRF Based on UPRE information, a one-hybrid method is first selected as a technique for cloning a gene encoding a protein that binds to CAGCCGTG. The one-hybrid method refers to the yeast transcriptional regulator Gal4p.
This is a method for cloning a DNA-binding protein using a fusion protein with a transcription activation domain (ad) and using the activation of a reporter gene in yeast as an index. That is, if the sequence UPRE recognized by UPRF is inserted upstream of the reporter, UPRF becomes Gal4p
When a fusion protein is formed with the transcription activation domain of E. coli, this fusion protein is considered to constitutively activate the transcription of the reporter gene regardless of stress. If yeast HIS3 is used as a reporter gene,
Cells expressing the fusion protein of interest can grow without histidine in the medium and, if the expression level of HIS3 is high, is resistant to 3-aminotriazole (an antagonist of the HIS3 gene product) Can be acquired. However, it appears that some of the clones obtained are not specific for UPRE and more generally promote transcription. To eliminate these at an early stage of screening, lacZ is used as another negative reporter, and a cis sequence UPRE of a mutant not recognized by UPRF is inserted upstream thereof. The clone of interest promotes the transcription of HIS3 and confers drug resistance to the cells, but does not promote the transcription of lacZ.
-Expect to show a phenotype that galactosidase activity remains low.

[0023] As shown in FIG. 3, as positive reporter _{UPRE (UY) -P ERN1 -HIS3,} UPRE (Tv10) as negative reporter -P _ERNI
-LacZ is constructed in one plasmid (pRCE4
NR3), and integrated into the chromosome of the ERN1-disrupted host cell strain (ern1Δ) by homologous recombination using the URA3 gene. In the ERN1-disrupted strain, the signal transduction pathway from the endoplasmic reticulum to the nucleus is blocked, so that even when the fusion protein moves into the endoplasmic reticulum and becomes an endoplasmic reticulum stress, it does not activate endogenous UPRF, The background can be kept low. UPRF is UPRE (U
Y) binds more strongly than wild-type Y,
Hardly binds to (Tv10). As a promoter, about 300 bp upstream of ERN1 having low basal transcription activity
(P _ERNI ) is used. The resulting transformants were screened for pRC integrated at the ura3-52 locus.
YHCE4 strain having one copy of E4 is obtained.

Dr. Eridge of Houston, USA (Dr.
A cDNA library of yeast prepared by E. Ledge (Elledge) was obtained, and Gal of the pACT vector in FIG. 4 was obtained.
YHCE4 strain was transformed with a plasmid having the cDNA inserted downstream of the DNA encoding 4p (ad), and Hi
The cDNA of interest is isolated by double screening and sequencing of s ⁺ and β-galactosidase activity.

Next, it is examined whether the DNA binding specificity of the protein encoded by this gene matches the specificity of the endogenous UPRF shown in FIGS. 1 and 2 and Table 1.
PACT vector expressing a fusion protein of Gal4p (ad) and this gene product and various UPREs
A reporter gene upstream of YC1-lacZ is introduced into yeast cells together, and the constitutively expressed β-galactosidase activity is measured (FIG. 5). U of this gene product
PRE-specific binding is in good agreement with what is expected for UPRF.

Ern4p, which is a gene product exhibiting UPRE binding, is composed of 230 amino acids shown in SEQ ID NO: 2, and as shown in FIG.
This gene is termed ERN4 because it is considered to be a transcriptional regulator having a leucine zipper structure and its activity and structure are in good agreement with those expected as UPRF.

ERN isolated from pACT library
Yeast genomic library (ATCC 37323; Nasmyth, KA and Reed, SI, P) constructed on YEp13, a multicopy yeast vector, using 4 cDNAs.
Acad. Sci. USA 77, 2119-2123 (1980)) to obtain genomic ERN4 DNA.
The sequence of the protein coding region of the obtained clone is determined using a synthetic oligonucleotide primer with a DNA sequencer. ER described in SEQ ID NO: 1
The N4 gene starts translation from the ATG at nucleotide position 1037, and terminates translation at the stop codon of TGA at position 1727. A 2.5 kb BamHI-EcoNI fragment containing the entire ERN4 gene described in SEQ ID NO: 1 was converted to YCp-L
2 to make YCp-ERN4. YCp-L
2 is CEN4-ARS1 containing the LEU2 selection marker
Base single copy yeast vector, pSB
32 (Rose, MDand Broach, JR, Meth. Enzymol. 194, 19)
5-230 (1991)) 0.53 kb HindIII-Sph
It is constructed by replacing the I fragment with the multiple cloning site of pUC119. YCp-ERN1 is identical to known pERN1BC (Mori, K. et al, Cell 74,7).
43-756 (1993)).

(2) Preparation of ERN4 Disrupted Saccharomyces cerevisiae Mutant (ern4 △) The ERN1 and ERN4 genes on the chromosome are disrupted by a known method (Rothstein, R., Meth. Enzymol. 194, 281-30).
1 (1991)). Central SalI-SacI fragment of ERN1 (corresponding to amino acid sequence 1-747) or central SpeI-MfeI fragment of ERN4 (amino acid sequence 11-215)
By the yeast TRP1 gene,
Prepare pKOE1TRP or pKOE4TRP, respectively. The plasmid was linearized, SEC ⁺ and sec
After transformation of 53 haploid cells, Trp ⁺ transformants are isolated and disruption is confirmed by Southern analysis using an appropriate probe. ERN4 by one-step homologous recombination
When an attempt was made to produce a disrupted strain, it was found that a disrupted strain could be obtained even in haploid cells, indicating that ERN4 is not an essential gene.

Next, ERN4 was prepared using haploid cells.
To investigate whether stress-induced reporter gene activation occurs in the disrupted strain. As shown in FIG.
RN ⁺ ), UPN (Y)
The β-galactosidase activity expressed from the -CYC1-lacZ reporter gene is induced about 10-fold, whereas the ERN1-disrupted strain (ernl △),
No induction was observed even in the RN4 disrupted strain (ern4 △). ERN1 is the same as IRE1 isolated as a gene complementary to the inositol-requiring mutant, and the ERN1-disrupted strain exhibits inositol-requirement for optimal growth. Therefore, when the cell suspension diluted with water was spotted on a medium containing no inositol so that the absorbance at 600 nm (A ₆₀₀ ) became 0.1 and examined, the ERN4-disrupted strain also showed a requirement for synositol ( (Fig. 6).

Further, in order to confirm whether the β-galactosidase-inducing activity lost in the ERN4-disrupted strain was really due to ERN4, the plasmid YCp-ERN4 containing ERN4 in a single copy vector was replaced with ERN4.
Introduce to 4 disrupted strains. When a wild-type strain (ERN ⁺ ) into which YCp-V containing only a single copy vector was introduced as a control, no change was observed when YCp-V was introduced into the ERN4 disrupted strain (ern4Δ), but the ERN4 disrupted strain was not found. When YCp-ERN4 was introduced into E. coli, the inducible activity was completely restored to the level of the wild-type strain (FIG. 7). The inositol requirement of ERN4 disrupted strains is also completely complemented by YCp-ERN4. Therefore, the product of ERN4 (Ern4p) is a transcriptional regulator constituting UPRF. Also, ERN
Even if ERN1 is introduced into 4 disrupted strains by multicopy (YE
p-ERN1), neither inducible activity nor inositol requirement is restored (FIG. 7).

On the other hand, the inducing activity lost in the ERN1-disrupted strain (ernl △) was determined by the
It is completely recovered by the RN1 gene YCp-ERN1 (FIG. 7). When ERN4 was introduced into ERN1-disrupted strains by multicopy (YEp-ERN4), the inducible activity was not recovered at all (FIG. 7), but the inositol auxotrophy was recovered although not completely, and the inositol auxotrophy was recovered. As for ERN4, it can be seen that ERN4 functions as a multicopy suppressor for ERN1-disrupted strains. Loss of the inducing activity is always associated with inositol requirement, but the mechanism at the finest level seems to be quite different.

(3) Constitutive activation of unfolded protein response process by GAL4 (ad) -ERN4 gene SEC ⁺ ERN ⁺ , ern1Δ and ern4Δ strains
pACT vector alone or ERN4 with GAL4 (a
The pACT expression vector containing the gene in frame with d) and the pSCZ reporter plasmid into which various UASs (upstream activation sequences) have been inserted are introduced together, and the resulting transformants are subjected to CYC1 without stress treatment. −la
The activity of β-galactosidase expressed from the cZ gene is measured (FIG. 5). In addition, Kar2p (GRP78) and Pd in total protein (50 μg) extracted from the culture of each transformant by the immunoblotting method
Determine the amount of i1p (FIG. 5). GAL4 (ad) -E
By expressing the RN4 gene, UPRE (Y)
Β-galactosidase having UPR and UPRE (UY) is activated even in the absence of stress, and Kar2p (G
RP78) and Pdi1p expression are also induced.

(4) Necessity of ERN4 gene product for signal transduction from endoplasmic reticulum to nucleus Single copy UPRE (Y) -CYC1-lacZ
ERN ⁺ strain of SEC ⁺ with reporter gene, er
tunicamycin (5 μg)
/ Ml) for 3 hours in the absence and presence of β-galactosidase activity (FIG. 6). SC containing 2 μl of culture with or without inositol
When spotted on (-Ura) plates and incubated at 30 ° C. for 2 days, two mutants (ern1Δ and ern4Δ) show inositol auxotrophy (FIG. 6).

Next, ERN ⁺ , ern1Δ in sec53
And ern4Δ strain were cultured at 23 ° C. (permissive temperature), and part of the logarithmic growth phase was 1 hour at 23 ° C. (no accumulation of unfolded protein in the endoplasmic reticulum (-)) or unfolded protein accumulated in the endoplasmic reticulum Incubate (+) at 30 ° C (semi-non-permissive temperature) to extract total RNA. KAR2
(GRP78), SCJ1, PDI1, EUG1, FK
Using a DNA probe specific for B2, lacZ and ACT1 (yeast actin), analysis is performed by Northern hybridization (FIG. 8). In the ERN ⁺ strain, the accumulation of unfolded proteins in the endoplasmic reticulum
2 (GRP78), SCJ1, PDI1, EUG1 and FKB2 RNA transcription were induced, compared to ern
The 1Δ and ern4Δ strains do not induce transcription of these RNAs.

(5) Effect of ERN4 gene deletion on protection against endoplasmic reticulum stress Single copy UPRE (Y) -CYC1-lacZ
A sec53 ERN ⁺ strain with a reporter gene and a single copy (YCp) expression plasmid, ern1
The Δ strain and the ern4Δ strain were cultured overnight at 23 ° C. in SC (-Ura, Leu) medium, and the culture was cultured at an OD ₆₀₀ of about 0.2.
And further incubate at 30 ° C. The ratio of β-galactosidase activity to viable cells is measured every 2 hours from 0 to 10 hours (FIG. 9). Next, Kar2p and Pdi1p are detected by immunoblotting using 10 μg of the total protein derived from the sample collected every two hours (FIG. 10). In the ern4Δ strain,
By expressing exogenous Ern4p, cells can be protected from endoplasmic reticulum stress.

(6) Expression, purification and specific binding to the UPRE of the Ern4p-fusion protein 0.8 kb SpeI-HindIII fragment of ERN4 (
Amino acid sequence 10-230)
Glutathione S on EX-2TK (Pharmacia)
-A nucleotide sequence encoding a transferase or a nucleotide sequence encoding a maltose binding protein on plasmid pMAL-c2 (New England Biolabs) was fused in frame with the 3 ^' end, and GST-Ern4p and MBP-, respectively. Ern4p is expressed. Both fusion proteins were 1 mM isopropyl β-
When induced by D-thiogalactopyranoside, it is efficiently expressed in Escherichia coli and purified by affinity chromatography.

UPRE (Y, Tv10, UY and Tv2
34 (UPRE triple mutants 2-4)) and C
5 of the double-stranded synthetic oligonucleotide encoding RE
^'And 3 ^{' at} both ends, Klen of DNA polymerase I
radiolabeled with the ow fragment and [α- ³² P] dATP,
Purify by electrophoresis on an 8% polyacrylamide gel. 0.25-0.3 ng of radiolabeled probe (8.0
00-10,000 cpm), 1 μg of poly (dI-d
C): Poly (dI-dC) (Pharmacia) and 1
MBP-Ern4p (0.1 μg) or GST-Er purified in a binding buffer containing μg of denatured salmon sperm DNA
The binding reaction is started by adding n4p (0.5 μg). Five times the amount of GST-Ern4p was used because GST-Ern4p tends to aggregate and stay at the top of the gel. 1 at 4 ° C. in a final volume of 20 μl
After a 0 minute incubation, the sample is electrophoresed on a 5% polyacrylamide gel containing 0.5% ficoll under non-denaturing conditions, the gel is dried and exposed to X-ray film (FIG. 11). Purified MBP-Ern4p and GST
-Ern4p is both UPRE (Y) and UPR
Specific binding to E (UY).

(7) Method for Controlling Yeast Endoplasmic Reticulum Molecular Chaperone and Folding Enzyme by Transformation with Ern4p Expression Vector Expression of Ern4p or a fusion protein of Ern4p with the transcriptional activation domain of a transcriptional regulator that functions in yeast. Thus, an expression plasmid is constructed in which DNAs encoding these are ligated downstream of the promoter of the Ern4p gene itself or another suitable promoter. As a vector for a yeast host, YIp integrated into the chromosome in one copy is used.
-Type plasmid, YCp-type plasmid maintained at one copy as a plasmid in yeast, Y maintained at multiple copies
Ep-type plasmids and YRp-type plasmids can be used.
Examples of the promoter include the promoter of the ERN4 gene itself, ADH1, ENO1, PGK1, GA
It can be used by selecting from promoters such as PDH, GAL1, GAL10, GAL7, and PHO5. AD
By using the H1, ENO1, PGK1 and GAPDH promoters, constitutive expression is enhanced by GAL1, GAL1
By using 10 and GAL7 promoters, it is possible to induce the expression by galactose, and to suppress the expression by the PHO5 promoter by inorganic phosphate. By transforming a yeast, preferably an ern4Δ strain, with such an Ern4p expression vector, Ern4
The amount of p can be adjusted artificially. In this case, the expressed Ern4p responds to endoplasmic reticulum stress to induce transcription of genes encoding molecular chaperones and folding enzymes in the endoplasmic reticulum. On the other hand, a fusion protein of the transcription activation domain of another transcription factor and Ern4p has a constitutive transcription activity, and in a yeast transformed with an expression vector of the fusion protein, the amount of the expressed fusion protein is reduced. Independently, the genes of endoplasmic reticulum molecular chaperones and folding enzymes are transcribed. At this time, the host yeast does not need to be the ern4Δ strain.

(8) Method for expressing foreign protein using Ern4p expression vector The foreign protein expression vector is a YIp type, YCp type,
ADH using YEp type or YRp type plasmid
1, ENO1, PGK1, GAPDH, GAL1, GA
A gene encoding a foreign protein of interest is linked downstream of a promoter such as L10, GAL7, PHO5 or the like to construct. SUC for secretory expression of foreign proteins
2. Use a foreign protein gene prepared so as to add a signal peptide derived from PHO5, MFα1 or a heterologous gene to the N-terminus. The thus-produced foreign protein expression vector can be used as an Ern4p expression vector or a transcriptional activation domain of a transcriptional regulator that functions in yeast.
The expression vector of the fusion protein with rn4p is introduced into yeast together to obtain a co-transformant. Next, the co-transformant is cultured by a commonly used method, and expression is induced as necessary to produce a foreign protein.

(9) Analysis of Ern4p activation mechanism Ern4p of the present invention is a polypeptide consisting of 230 amino acid residues shown in SEQ ID NO: 2,
Although there is a region (basic region, see FIG. 4) that specifically binds to PRE, there is no region considered to be a transcriptional regulatory activation region. Therefore, the activation mechanism of Ern4p itself will be analyzed.

First, the transcript of the ERN4 gene is analyzed by Northern blotting. Total RNA was extracted from the cell line of Saccharomyces cerevisiae treated with tunicamycin at regular intervals, subjected to electrophoresis, blotted on a nitrocellulose membrane, and hybridized using probes specific to ERN4, KAR2 and PDI1. Thus, the transcript of the ERN4 gene is detected. As an internal standard, A
A probe specific to CT1 (yeast actin) is used.
The 1.4 kb band, which is regularly seen before tunicamycin treatment, decreases rapidly after tunicamycin treatment, producing two new short bands (1.2 kb and 0.7 kb) (FIG. 12). Furthermore, since this phenomenon starts before the induction of the expression of the KAR2 and PDI1 genes (FIG. 13) and is not observed in the ern1Δ strain (FIG. 12), the existence of a stress-specific ERN4 gene post-transcriptional regulatory mechanism exists. It is suggested.

Second, the short-chain transcript is analyzed by the RT-PCR method. Using the cDNA synthesized from the total RNA using an oligo-dT primer as a template, ERN
Various primers are designed from the regions inside the four genes (FIG. 14), and RT-PCR is performed (FIG. 15). When a primer for the 5 ^′ untranslated region and a primer for the 3 ^′ untranslated region were used, a PC derived from tunicamycin-treated cDNA was used.
Only in the R amplification product, in addition to the expected length band,
A band about 250 bp shorter than that is seen. This difference in length is considered to be due to a change from 1.4 kb to 1.2 kb in the analysis result by the Northern blotting. The band is cloned and its nucleotide sequence is determined. As a result, the primers f and o in FIG.
A deletion is found during Saccharomyces cerevisiae, but splicing suggests that the deletion has occurred. As a result of splicing, a new open reading frame (ORF) appears,
Ern4p consisting of 230 amino acid residues is 221
A polypeptide consisting of 238 amino acid residues (SEQ ID NO: 3) having different amino acid sequences after the position (FIG. 1)
6).

Third, five types of oligonucleotide probes that specifically hybridize to the region where the deletion was found (corresponding to an intron) or the region not deleted (corresponding to an exon) were synthesized ( FIG. 16) and Northern blotting analysis is performed (FIG. 17). Since the probe hybridizing with the intron region does not hybridize with the 1.2 kb band induced by the stress caused by the tunicamycin treatment, the probe hybridizes from 1.4 kb to 1.2 kb.
It is concluded that the change of band to b is in fact a consequence of about 250 bases being removed by splicing. The 0.7 kb band that does not hybridize with the 3 ^′ probe in FIG. 16 is expected to be a 5 ^′ exon.

Fourth, the polypeptide (SEQ ID NO: 3) derived from the novel ORF is examined for its transcriptional regulatory activity. ERN4
Using the original promoter of the gene, a DNA was constructed on the single copy vector so that the polypeptide derived from the novel ORF excluding the intron was expressed, and the reporter gene (UPRE (Y) -CYC1-lacZ) was used together with the ern4Δ strain. And the β-galactosidase-inducing activity is examined as described above (FIG. 18). New OR
The F-derived polypeptide can be expressed in β-
Because of the galactosidase-inducing activity, this polypeptide is Ern4p, which has the ability to activate transcriptional regulation, and is hereinafter referred to as activated Ern4p. It is suggested that the activity of Ern4p is regulated by stress-specific splicing.

[0045]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, which should not be construed as limiting the present invention.

Strains and Microbiological Techniques The strains of Saccharomyces cerevisiae used herein are as follows: KMY1005 (MATαl)
eu2-3,112 ura3-52 his3-Δ2
00 trp1-Δ901 lys2-801); KM
Y1015 (KMY1005 ern1Δ :: TRP
1); KMY1045 (KMY1005 ern4
Δ :: TRP1); KMY2005 (MATα leu)
2-3, 112ura3-52 his3-Δ200
trp1-Δ901 lys2-801sec53-
6); KMY2015 (KMY2005 ern1
Δ :: TRP1); KMY2045 (KMY2005
ern4Δ :: TRP1). Genetic techniques and rich broth medium (YPD) and SC (-Ura) or SC
The composition of the selective medium for transformants such as (-Ura, Leu) is described in Sherman, F. et al.
s in Yeast Genetics, 1986 Cold Spring Harbor Labora
tory Press ". Inositol-free medium is available from Culbertson and Henry (Culbertson, M .;
R. and Henry, SA) "Genetics 80, 23-40 (1975)". Transformation of yeast was performed by the lithium acetate method (Ito, H. et al., J. Bacteriol. 1).
53,163-168 (1983)).

Construction of Reporter Plasmids Recombinant DNA techniques are described in Sambrook, J. et al.
l.) “Molecular Cloning: A Laboratory Manual, 2nd.
Edition, 1989 Cold Spring Harbor Laboratory Press ". Reporter plasmid pMCZ2
The construction of (2 μm DNA-based multicopy plasmid containing the URA3 selectable marker) and pSCZ2 (CEN4-ARS1-based single copy plasmid containing the URA3 selectable marker) are reported separately (Mori,
K.et al., Posting). Both pMCZ2 and pSCZ2 contain a CYC1-lacZ fusion gene with EcoRI and XhoI sites located upstream of the CYC1 promoter for UPRE cloning. EcoRI
And 5 ^{'at the} protruding ends generated by XhoI digestion, respectively.
And various double-stranded, synthetic oligonucleotides with complementary 3 ^' ends were inserted between the EcoRI and XhoI sites.

Assays, Probes and Antibodies Assays for β-galactosidase activity in yeast cell extracts and Northern hybridization assays for yeast RNA were performed as previously described (Mori, K. et al., Cell.
74, 743-756 (1993)). The yeast PDI1 probe (middle 1.47 kb XhoI-SalI fragment) was constructed using pMTY
17 (Tachikawa, H. et al., J. Biochem. 110, 306-313 (19
91)). Probes for the yeast SCJ1 and FKB2 genes were prepared by the PCR method. The immunoblotting was performed basically according to the previous report (Kohno, K. e.
etal., Mol. Cell. Biol. 13, 877-890 (1993)). Rabbit polyclonal antibodies specific to plasmids pMTY17 and Pdi1p were obtained from Dr. Tachikawa (Tachikawa, H., Tokyo University of Agriculture and Technology).
Provided by

Example 1 Analysis of the Cis Sequence UPRE The EcoRI and XhoI sites located upstream of the CYC1 promoter of the reporter plasmid pMCZ2 were replaced with wild-type UPRE (named Y) or the positions shown on the horizontal axis of FIG. Point mutant (G,
A, T, and C are replaced with T, C, G, and A, respectively.
19) and insert it with the yeast wild strain KMY10.
05, and the resulting Ura ⁺ transformant was transformed into SC
The cells were cultured in (-Ura) medium at 30 ° C., and the cells in the logarithmic growth phase were incubated for 3 hours in the presence or absence of tunicamycin (TM, 5 μg / ml) to obtain CYC1-lac.
Β-galactosidase activity expressed from the Z fusion gene was measured (FIG. 1). In the case of the wild type UPRE, the β-galactosidase activity was increased about 50-fold by TM treatment. 10th to 12th CAG and 14th to 16th GT
In the case of the point mutant of G, it was revealed that β-galactosidase activity hardly increased even after TM treatment, and UPRE activity was almost lost. Also, 2
The fourth point mutant of GAA also reduced β-galactosidase activity. Next, the 13th C between CAG and GTG
In order to investigate the importance of, the 13th C is replaced with another base G,
Mutants were prepared by substituting A or T, removing the 13th C (spacing 0), and inserting 1 to 4 Cs (spacing 2 to 5), and similarly β-galactosidase The activity was measured (FIG. 2). When the spacing was set to 0, the activity was about 7% of that of the wild type, and when the spacing was set to 2 or more, the activity almost disappeared. Therefore, it was shown that the presence of only one C between CAG and GTG is extremely important for UPRE activity. Further, the region containing CAG-GTG has a palindromic structure partially. As shown in Table 1, wild-type Y is a palindrome consisting of 4 bp, and β-galactosidase activity was increased about 50-fold by stress as described above. Replace the 12th G with C (12C),
When the 19th G was replaced with C (19C), β-galactosidase activity increased in both the presence and absence of stress compared to wild-type Y. Furthermore, when both were substituted to form a complete palindromic structure consisting of 7 bp (UY), the activity increased about 4 times before stress and about 2 times after stress compared to wild type Y. . Therefore, yeast U
PRF has been shown to recognize palindromic structures.

[0050]

[Table 1]

Example 2 One-hybrid of UPRF
d) Screening method A SalI site was created immediately upstream of the translation start site of the yeast HIS3 gene cloned in pUC119 (Takara Shuzo). 0.3 including two TATAA boxes
kb of the DraI-SalI fragment was inserted into the ERN1 gene (Mo
ri, K. et al., Cell 74, 743-756 (1993)) and isolated from pSZWT (Mori, K. et al., EMBO J. 11,
2583-2593 (1992)) or upstream of the HIS3 gene. The ERN1 promoter is the smallest GAL1 promoter previously used (Wang, MM and Reed, RR, Nature 364, 121-126 (199).
3); Chong, JA et al., Cell 80, 949-957 (1995)) because it has a very low basal transcription activity. Mutant UPREs (Tv10 and UY) were inserted upstream of the ERN1 promoter (P _ERN1 ), which controls lacZ and HIS3 expression, respectively. UPRE (T
v10) -P _ERN1 -lacZ construct was converted to UPRE (UY)
This was fused with a plasmid having the -P _ERN1 -HIS3 gene. The yeast URA3 gene was further inserted and pRCE4
(FIG. 3). pRCE4 is digested with NcoI,
ern1Δ strain (KMY1015).
The use of ern1Δ cells as a host was for the reasons described above. The GAL4 and GAL80 genes on the chromosome did not interfere with the screen. Southern
rn) YHC with one copy of pRCE4 integrated at the ura3-52 locus after screening by analytical method
E4 strain was obtained. The YHCE4 strain has very low levels of β-
It expressed galactosidase and did not grow on plates without histidine. The yeast cDNA library was provided by Dr. Eledge (S., Baylor College of Medicine) through Dr. Hayashi (N., Kyushu University). The pACT vector (Durfee, T. et al., Gene
7, 555-569 (1993)), a plasmid was constructed in which the cDNA was inserted into the XhoI site downstream of the DNA encoding Gal4p (ad). 100 μg of YHCE4 cells
And the His ⁺ colonies were selected. 100 H from about 5 × 10 ⁶ transformants
Of the is ⁺ colonies, 23 were discarded because β-galactosidase levels were significantly increased (more than 2-fold). Forty-seven clones out of the 77 candidates contained 1 mM 3
-Could grow in the presence of aminotriazole. This indicated that UPRE-dependent transcription of HIS3 was strongly activated.

When a plasmid derived from the library was recovered from these colonies and the nucleotide sequence was examined, it was found that all the inserts differing in origin but were derived from one gene.

Example 3 Construction of Plasmid for ERN4 Gene Expression Using the ERN4 cDNA isolated from the pACT library, the multicopy yeast vector YEp13 was used.
The yeast genomic library constructed above (ATCC3
7323; Nasmyth, KA and Reed, SI, Proc. Natl. Aca
d.Sci.USA 77,2119-2123 (1980)) to obtain ERN4 genomic DNA. One clone obtained contained all genes having a flanking region, and the sequence of the region encoding the protein was determined using a synthetic oligonucleotide primer with a DNA sequencer manufactured by Applied Biosystems. The obtained nucleotide sequence was different from the previously reported sequence (Nojima, H. et a
l., Nucleic Acids Res. 22, 5279-5288 (1994); Murakami,
Y. et al., Nature Genet. 10, 261-268 (1995)). All ERN
A 2.5 kb BamHI-EcoNI fragment containing the four genes was ligated to YCp-L2 to produce YCp-ERN4. YCp-L2 is a CE containing LEU2 selectable marker.
N4-ARS1-based single copy yeast vector, pSB32 (Rose, MD and Broach, JR, Meth.
Enzymol. 194, 195-230 (1991)) 0.53 kb Hin
It was constructed by replacing the dIII-SphI fragment with the multiple cloning site of pUC119. YCp-ER
N1 is the same as known pERN1BC (Mori, Ke
tal., Cell 74, 743-756 (1993)).

Example 4 Disruption of the ERN4 Locus The ERN1 and ERN4 genes on the chromosome were disrupted by a known method (Rothstein, R., Meth. Enzymol. 194, 281-30).
1 (1991)). Central SalI-SacI fragment of ERN1 (corresponding to amino acid sequence 1-747) or central SpeI-MfeI fragment of ERN4 (amino acid sequence 11-215)
By the yeast TRP1 gene,
PKOE1TRP or pKOE4TRP was prepared, respectively. The plasmid was linearized and SEC ⁺ and
c53 haploid cells were transformed. Transformants of Trp ⁺ were isolated and disruption was confirmed by Southern analysis using an appropriate probe.

Example 5 Unfall by GAL4 (ad) -ERN4 gene
Constitutively activated SEC ^{+ in} the ERN ⁺ , ern1Δ and ern4Δ strains
pACT vector alone or ERN4 with GAL4 (a
A transformant was obtained by introducing together a pACT expression vector containing the gene whose frame was matched to d) and a pSCZ reporter plasmid into which various UASs (upstream activation sequences) were inserted. The transformant was transformed with SC (-Ura, Le
u) The cells were cultured at 30 ° C. in a medium, and the cells in the logarithmic growth phase were subjected to the stress-treated β-galactosidase activity expressed from the CYC1-lacZ gene without stress treatment (FIG. 5). Three separate transformants were measured twice to determine the value. In addition, the amounts of Kar2p (GRP78) and Pdi1p in the total protein (50 μg) extracted from the culture of each transformant were determined by the immunoblotting method (FIG. 5). By expressing the GAL4 (ad) -ERN4 gene, UPRE (Y) and UPRE (UY)
Β-galactosidase upstream of is activated even in the absence of stress, and Kar2p (GRP78) and Pdi1
Expression of p was also induced.

Example 6 ERN4 Gene Product Signaling from ER to Nucleus
Need for single copy UPRE (Y) -CYC1-lacZ
ERN ⁺ strain of SEC ⁺ with reporter gene, er
The n1Δ strain and the ern4Δ strain were cultured in SC (-Ura) medium for 3 hours.
The cells were cultured at 0 ° C. Some of the cells in the logarithmic growth phase were collected, and 3 cells were collected in the presence or absence of tunicamycin (5 μg / ml).
Incubated for hours. β-galactosidase activity was expressed as the average of two measurements (FIG. 6). 2 μl of the culture were spotted on SC (-Ura) plates with or without inositol, incubated at 30 ° C. for 2 days,
Two mutant strains (ern1Δ strain and ern4Δ strain) showed inositol auxotrophy (FIG. 6).

Next, the ERN ⁺ strain of sec53, ern1
The Δ strain and the ern4Δ strain are cultured at 23 ° C. (permissible temperature),
Part of the cells in the logarithmic growth phase are cultured for 1 hour at 23 ° C. (unfolded protein (−) in the endoplasmic reticulum) or at 30 ° C. (semi-nonpermissive temperature) at which unfolded protein accumulates (+) in the endoplasmic reticulum. did. Total RNA is extracted and KAR2
(GRP78), SCJ1, PDI1, EUG1, FK
Using DNA probes specific for B2, lacZ and ACT1 (yeast actin), analysis was performed by Northern hybridization (FIG. 8). In the ERN ⁺ strain, KAR2 (G
RP78), SCJ1, PDI1, EUG1, and FKB
The transcription of these RNAs was not induced in the ern1Δ strain and the ern4Δ strain, whereas the transcription of 2 RNAs was induced.

Example 7 ERN4 gene protection in endoplasmic reticulum stress
Effect of deletion Single copy UPRE (Y) -CYC1-lacZ
Sec53 ERN ⁺ , ern1Δ with reporter gene and single copy (YCp) expression plasmid
And the ern4Δ strain were cultured overnight at 23 ° C. in SC (-Ura, Leu) medium. The culture was diluted to an OD ₆₀₀ of about 0.2 and further incubated at 30 ° C. The ratio of β-galactosidase activity to viable cells was measured every 2 hours from 0 to 10 hours. The percentage of viable cells is
It was determined by appropriate dilution and seeding on a YPD plate. Values are expressed as% of control (0 hours) (FIG. 9). next,
10μ of total protein from samples taken every 2 hours
g and Kar2p by immunoblotting
And Pdi1p were detected (FIG. 10). In the ern4Δ strain, it was found that by expressing exogenous Ern4p, cells could be protected from endoplasmic reticulum stress.

Example 8 Expression and purification of Ern4p-fusion protein 0.8 kb SpeI-HindIII fragment of ERN4 (
Amino acid sequence 10-230)
Glutathione S on EX-2TK (Pharmacia)
-A nucleotide sequence encoding a transferase or a nucleotide sequence encoding a maltose binding protein on plasmid pMAL-c2 (New England Biolabs) was fused in frame with the 3 ^' end, and GST-Ern4p and MBP-, respectively. Ern4p was expressed. Both fusion proteins were 1 mM isopropyl β-
When induced with D-thiogalactopyranoside, it was efficiently expressed in Escherichia coli and purified by affinity chromatography according to the manufacturer's instructions.

Example 9 Specific Binding of ERN4 Gene Product to UPRE The DNA polymerase was used to bind both 5 ^′ and 3 ^′ ends of double-stranded synthetic oligonucleotides encoding UPRE (Y, Tv10, UY and Tv234) and CRE. I's Kle
Now fragment and [α- ³² P] dATP (3000 Ci / m
mol) and purified by electrophoresis on an 8% polyacrylamide gel. The composition of the binding buffer was 20 mM HEPES (pH 7.9), 50 mM
KCl, 10 mM MgCl ₂ , 0.25 mM EDT
A, 0.5 mM dithiothreitol, 2% ficoll and 5% glycerol. 0.25-0.3
ng of radiolabeled probe (8,000-10,000 c
pm), 1 μg of poly (dI-dC): poly (dI-d
C) (Pharmacia) and 1 μg of denatured salmon sperm D
MBP-Ern4p purified in binding buffer containing NA
(0.1 μg) or GST-Ern4p (0.5 μg)
Was added to initiate the binding reaction. GST-Ern4p was used in an amount of 5 times the amount of GST-Ern4p.
Is likely to aggregate and stay at the top of the gel.
After incubation for 10 minutes at 4 ° C. in a final volume of 20 μl, the samples were electrophoresed on a 5% polyacrylamide gel containing 0.5% ficoll under non-denaturing conditions (the ratio of acrylamide to bisacrylamide is 30:
0.8). The gel pre-runs for 2-3 hours,
The sample was run in 0.5 × TBE at 4 ° C. and 150 V for 3 hours. The gel was then dried and exposed to X-ray film (FIG. 11). Purified MBP-Ern4p and GST-
Ern4p are both UPRE (Y) and UPRE
(UY) was found to specifically bind.

Example 10 Analysis of Ern4p Activation Mechanism 10-1) ERN4 Residue by Northern Blotting
Analysis of gene transcripts From the wild-type strain of Saccharomyces cerevisiae and the ern1Δ strain treated with tunicamycin in the same manner as in Example 1, all RNs were taken at regular intervals (0, 10, 20, 30, 45, 60 minutes).
A was extracted and subjected to electrophoresis on a 1.5% agarose formamide gel, and then a transcription product of the ERN4 gene was detected by Northern blotting using a probe specific to ERN4, KAR2 and PDI1. In addition, a probe specific to ACT1 (yeast actin) was used as an internal standard (FIG. 12). The radiation dose of each band of the autoradiography in FIG. 12 was quantified using a bioimaging analyzer BAS2000 (manufactured by Fuji Film Co., Ltd.) and graphed (FIG. 13). FIG.
Thus, 1.4 which is regularly observed before tunicamycin treatment was obtained.
The kb band (lanes 1 and 3) decreased rapidly after tunicamycin treatment and two new short bands (1.
2 kb and 0.7 kb) (lanes 4-8). Furthermore, this phenomenon starts before the induction of the expression of the KAR2 and PDI1 genes (lane 4 and FIG. 13).
Not being observed in the Δ strain (lane 2), suggesting the presence of a post-transcriptional regulatory mechanism of the stress-specific ERN4 gene.

10-2) Short Transcription by RT-PCR
Analysis of product From total RNA of the strain treated with tunicamycin for 45 minutes, oligo-dT primer and reverse transcriptase
DNA was synthesized. Using the cDNA as a template, various primers designed from the region inside the ERN4 gene (FIG. 1)
4) and using Takara ExTaq (Takara Shuzo)
R was performed (FIG. 15). As shown in FIG. 15, when primer b of the 5 ^′ untranslated region and primer o or m of the 3 ^′ untranslated region were used, cDNA treated with tunicamycin was used.
Only in the derived PCR amplification product, in addition to the expected length band, a band approximately 250 bp shorter was seen (lanes 9, 11). The difference between the lengths was from 1.4 kb to 1.2 k in the analysis result by the Northern blotting.
b.

10-3) Amplification Product by RT-PCR Method
The nucleotide sequence of the PCR amplification product was determined in the same manner as in Example 3. As a result, a deletion was found between the primers f and o in FIG. 14, suggesting that the deletion occurred due to splicing, although the frequency was low in Saccharomyces cerevisiae. As a result of splicing,
A new open reading frame (ORF) appears, which differs from Ern4p (SEQ ID NO: 2) consisting of 230 amino acid residues in the amino acid sequence after position 221.
A polypeptide consisting of 38 amino acid residues (SEQ ID NO:
3) occurred (FIG. 16).

10-4) By Northern Blotting
Analysis of splicing Five types of oligonucleotide probes that specifically hybridize to the region where the deletion was found (corresponding to an intron) or the region where the deletion was not found (corresponding to an exon) were synthesized (FIG. 16). And Northern blotting analysis (FIG. 17). As shown in FIG. 17, the probes (af, jo and lm) hybridizing to the region outside the intron were induced by the stress caused by the tunicamycin treatment.
Hybridized with the kb band (lanes 2, 8 and 10). On the other hand, the probes (gd and hi) that hybridize with the intron region do not hybridize with the 1.2 kb band induced by the stress caused by tunicamycin treatment (lanes 4 and 6).
The band change from kb to 1.2 kb is actually about 25
This was a phenomenon resulting from the removal of 0 bases by splicing. The 0.7 kb band that does not hybridize with the probe recognizing these 3 ^' regions is 5
^'The exon was expected.

10-5) Novel ORF-derived polypeptide
Measurement of transcriptional regulatory activity of E. coli using the original promoter of the ERN4 gene so that a polypeptide derived from the novel ORF excluding introns is expressed on the single copy vector YCp-L2.
A was constructed and a reporter plasmid (UPRE (Y)-
CYC1-lacZ) was introduced into the ern4Δ strain, and β-galactosidase-inducing activity was measured as described above (FIG. 18). From FIG. 18, since the polypeptide derived from the novel ORF has β-galactosidase-inducing activity even in a stress-free state, it was found that this polypeptide is Ern4p, which has the ability to activate transcriptional regulation. Designated as type Ern4p. Ern4p
Activity was regulated by stress-specific splicing.

[0066]

EFFECT OF THE INVENTION Using the transcription regulatory factor Ern4p or activated Ern4p of the present invention, G
The transcription induction mechanism of RP78 has been analyzed, and it has become possible to express useful proteins in large amounts in yeast.

[0067]

[Sequence list]

SEQ ID NO: 1 Sequence length: 2535 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: Genomic DNA Origin Organism name: Saccharomyces (Saccharomyces)
cerevisiae) strain name: AB320 sequence GGATCCGAGA ATAAGGTCTG CAATGTACGT CGGAGAAGAG CCACTATCAT CGGCGACTCC 60 TGTGATCGTG TTCCACTGTG GAGAGCCATA TCGCACCTTG CCGTCCAGTT CCAGCTCCAA 120 GATCATACAC GGATTCTTCT CTGTGGCCAG CTGCAGATAT TTTTCATAAT TGGAGGAAGT 180 CAATGAGCTT GGGTTCGAAT TCGATGATAT CGGGGAGAGC TTGTTTATGC CAAGTCCCTC 240 TTTCATAGCC TGAGATCCGC CTGCGGTGTT ACTTCTATTG AACATCTGTC TTCCTCTACT 300 GGGCTTATCT ATTCTATCAA ATGAGGCAAG AGAAAGGAAA GGGAAAAAAA AAAAGGGAAA 360 ATGCTTGATG AGTTAGGAAT TCAGTAAAAG CACCCACCAG AACCTCTACA GCAGGCCTTC 420 CAGGAGTGGT TAAGAGGATA GAGAATTCTG TATATATATA TACGTCTATA TCTATACAAA 480 AAACGGCAGA CAATGCAGAA GTTGAAAATG CTGTGATACG AACAACAACT TATTTTTACA 540 ATGAAAAACA ACACCAAGCT GCCAAAAGCT GTATATTAAA GTTAAAGCTC CCTCATTTTT 600 AGTAAGGACG CTTTCTTTTC CCACAAAAGG GACGAGGAAA AAAAGAAAAA CCTGGAAATA 660 TAGTCACGTG ACATGAATAA ACAAGACCTA GGGCAATATG GAAAAAGAAG CTAGGATAAA 720 AATACATTTA TGAGGGTTGT AAGGCAAAGT GGCTCAGCAT TAAGTAGGGC TAAAAAGAGT 780 CGCAAATATA CATCGTACCG AGTGATGAAC GTGGTTTTGA A CACCTTGTT CTCTTTTGTT 840 CTCGCTCCCT ACATTCACTA TATATTAGAA GAAATTTCAC CTCAATGGAC AACTCGAGAA 900 ATGAATACAG AAATATGTTT TTTAGCGAAA TTTTCCTTTC TTCTTGTCTT CTTGTTTTAT 960 TTAAACTTCC AAGGCTTTAA CTCAGTGTCA AACATAACAA CCTCCTCCTC CCCCACCTAC 1020 GACAACAACC GCCACTATGG AAATGACTGA TTTTGAACTA ACTAGTAATT CGCAATCGAA 1080 CTTGGCTATC CCTACCAACT TCAAGTCGAC TCTGCCTCCA AGGAAAAGAG CCAAGACAAA 1140 AGAGGAAAAG GAACAGCGAA GGATCGAGCG TATTTTGAGA AACAGAAGAG CTGCTCACCA 1200 GAGCAGAGAG AAAAAAAGAC TACATCTGCA GTATCTCGAG AGAAAATGTT CTCTTTTGGA 1260 AAATTTACTG AACAGCGTCA ACCTTGAAAA ACTGGCTGAC CACGAAGACG CGTTGACTTG 1320 CAGCCACGAC GCTTTTGTTG CTTCTCTTGA CGAGTACAGG GATTTCCAGA GCACGAGGGG 1380 CGCTTCACTG GACACCAGGG CCAGTTCGCA CTCGTCGTCT GATACGTTCA CACCTTCACC 1440 TCTGAACTGT ACAATGGAGC CTGCGACTTT GTCGCCCAAG AGTATGCGCG ATTCCGCGTC 1500 GGACCAAGAG ACTTCATGGG AGCTGCAGAT GTTTAAGACG GAAAATGTAC CAGAGTCGAC 1560 GACGCTACCT GCCGTAGACA ACAACAATTT GTTTGATGCG GTGGCCTCGC CGTTGGCAGA 1620 CCCACTCTGC GACGATATAG CGGGAAACAG TCTACCCTTT GACAATTCAA TTGATCTTGA 1680 CAATTGGCGT AATCCAGCCG TGATTACGAT GACCAGGAAA CTACAGTGAA CAAGAACACT 1740 AGCCCCAGCT TTTGCTTTCT GCTTTTTTTC TTTTTTTTTT TTTTTAGTCG TGGTTCTCTG 1800 ATGGGGGAGG AGCCGGTTAA AGTACCTTCA AAAGCAGAAT GCAGGGTTAT TGGAAGCTTT 1860 CTTTTTTTCT TTTATGCTAG TTTTTCCTGA ACAAATAGAG CCATTCTTTT CTTATTACTA 1920 AGAAATGGAC GGCTTGCTTG TACTGTCCGA AGCGCAGTCA GGTTTGAATT CATTTGAATT 1980 GAATGATTTC TTCATCACTT CATGAAGACA ATCGCAAGAG GGTATAATTT TTTTTTTCTC 2040 GTTATTATCG CTGTTGGTGG GTTTTTTCTT TTCATATATT TCTTTTTCGC TTAGTGGTTT 2100 CTACTGTTCT GTCTCCGGTT AGTGTGTGCT ACTTCAACCG AAGAAGAAGA GGCTTTTCAA 2160 GAATGCAAAC GTGAGGTTGG CGCGCCCTCC TACAATTATT TGTGGCGACT GGGCAGCGAC 2220 ACTGAACATA GCTCTTGAAC AAGACCCTTT TTTGGCTGCA AGGAGCAAGA CTGGCTGGGG 2280 TTCCACCTCA AAGAGCCACG CTCTGCTTTT TTTCTATCTG TTTGTGTCAT ATCTATCTGT 2340 CTATTTATCT ATATATATAT TTTTTTATAT AAAACTATAA AGAATTCTTG ATGTATGCCC 2400 TTAGGTTGGG CAGCTTTTCA ACCTTAGACT TGATGCTAAC GCCGCTCTGT TCCTTCTCCC 2460 GTGCTCCCGC AAGCGAACAT CTCCCCCTAA CTCCGGGCCA ATGAGTGATA ACTAT ATCAA 2520 ATACCTTCGA AAGGA 2535

SEQ ID NO: 2 Sequence length: 230 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Met Glu Met Thr Asp Phe Glu Leu Thr Ser Asn Ser Gln Ser Asn Leu 1 5 10 15 Ala Ile Pro Thr Asn Phe Lys Ser Thr Leu Pro Pro Arg Lys Arg Ala 20 25 30 Lys Thr Lys Glu Glu Lys Glu Gln Arg Arg Ile Glu Arg Ile Leu Arg 35 40 45 Asn Arg Arg Ala Ala His Gln Ser Arg Glu Lys Lys Arg Leu His Leu 50 55 60 Gln Tyr Leu Glu Arg Lys Cys Ser Leu Leu Glu Asn Leu Leu Asn Ser 65 70 75 80 Val Asn Leu Glu Lys Leu Ala Asp His Glu Asp Ala Leu Thr Cys Ser 85 90 95 His Asp Ala Phe Val Ala Ser Leu Asp Glu Tyr Arg Asp Phe Gln Ser 100 105 110 Thr Arg Gly Ala Ser Leu Asp Thr Arg Ala Ser Ser His Ser Ser Ser 115 120 125 Asp Thr Phe Thr Pro Ser Pro Leu Asn Cys Thr Met Glu Pro Ala Thr 130 135 140 Leu Ser Pro Lys Ser Met Arg Asp Ser Ala Ser Asp Gln Glu Thr Ser 145 150 155 160 Trp Glu Leu Gln Met Phe Lys Thr Glu Asn Val Pro Glu Ser Thr Thr 165 170 175 Leu Pro Ala Va l Asp Asn Asn Asn Leu Phe Asp Ala Val Ala Ser Pro 180 185 190 Leu Ala Asp Pro Leu Cys Asp Asp Ile Ala Gly Asn Ser Leu Pro Phe 195 200 205 Asp Asn Ser Ile Asp Leu Asp Asn Trp Arg Asn Pro Ala Val Ile Thr 210 215 220 Met Thr Arg Lys Leu Gln 225 230

SEQ ID NO: 3 Sequence length: 238 Sequence type: amino acid Topology: Linear Sequence type: Peptide sequence Met Glu Met Thr Asp Phe Glu Leu Thr Ser Asn Ser Gln Ser Asn Leu 1 5 10 15 Ala Ile Pro Thr Asn Phe Lys Ser Thr Leu Pro Pro Arg Lys Arg Ala 20 25 30 Lys Thr Lys Glu Glu Lys Glu Gln Arg Arg Ile Glu Arg Ile Leu Arg 35 40 45 Asn Arg Arg Ala Ala His Gln Ser Arg Glu Lys Lys Arg Leu His Leu 50 55 60 Gln Tyr Leu Glu Arg Lys Cys Ser Leu Leu Glu Asn Leu Leu Asn Ser 65 70 75 80 Val Asn Leu Glu Lys Leu Ala Asp His Glu Asp Ala Leu Thr Cys Ser 85 90 95 His Asp Ala Phe Val Ala Ser Leu Asp Glu Tyr Arg Asp Phe Gln Ser 100 105 110 Thr Arg Gly Ala Ser Leu Asp Thr Arg Ala Ser Ser His Ser Ser Ser 115 120 125 Asp Thr Phe Thr Pro Ser Pro Leu Asn Cys Thr Met Glu Pro Ala Thr 130 135 140 Leu Ser Pro Lys Ser Met Arg Asp Ser Ala Ser Asp Gln Glu Thr Ser 145 150 155 160 Trp Glu Leu Gln Met Phe Lys Thr Glu Asn Val Pro Glu Ser Thr Thr 165 170 175 Leu Pro Ala Val Asp Asn Asn Asn Leu Phe Asp Ala Val Ala Ser Pro 180 185 190 Leu Ala Asp Pro Leu Cys Asp Asp Ile Ala Gly Asn Ser Leu Pro Phe 195 200 205 Asp Asn Ser Ile Asp Leu Asp Asn Trp Arg Asn Pro Glu Ala Gln Ser 210 215 220 Gly Leu Asn Ser Phe Glu Leu Asn Asp Phe Phe Ile Thr Ser 225 230 235

[Brief description of the drawings]

FIG. 1 shows that yeast wild strain KMY1005 is 1-19.
FIG. 4 is a diagram showing β-galactosidase activity when transformed with a reporter plasmid containing a second UPRE sequence point mutation and treated with tunicamycin (TM). In the figure,
Y indicates the sequence of the parent UPRE.

FIG. 2 is a diagram showing β-galactosidase activity when the 13th C of UPRE is substituted, deleted or inserted, and transformed by the same method as in FIG. 1.

FIG. 3 is a schematic diagram of the reporter plasmid pRCE4.

FIG. 4 is a schematic diagram of a plasmid vector pACT. CDNA (ERN) downstream of GAL4 (ad)
4) Insert The structure of the protein encoded by ERN4 is shown below.

FIG. 5 shows that SEC ⁺ ERN ⁺ , ern1Δ and ern4Δ strains were isolated from Gal4p (ad) -Ern4p
FIG. 4 shows the results of measuring β-galactosidase without stress treatment by transforming with a vector expressing a fusion protein and a reporter plasmid. The inserted part in the figure is the Kar2p in the protein (50 μg) extracted from each transformant by the immunoblotting method.
FIG. 4 is a diagram showing the amount of Pdi1p.

FIG. 6 is a single copy UPRE (Y) -C.
It is a figure which shows (beta) -galactosidase activity when the ERN ⁺ strain of SEC ⁺ which has a YC1-lacZ reporter gene, the ern1 (DELTA) strain, and the ern4 (DELTA) strain are untreated or treated with tunicamycin (TM). The insert in the figure shows the result of culturing a part (2 μl) of the aforementioned culture on an (+) or (-) SC (-Ura) plate containing inositol.

FIG. 7 shows a single copy UPRE (Y) -C.
ERN having YC1-lacZ reporter gene
⁺ , Ern1Δ and ern4Δ strains with single copy (YCp) or multicopy (YEp) ERN
FIG. 2 is a diagram showing β-galactosidase activity when one expression plasmid or a single copy (YCp) or multicopy (YEp) ERN4 expression plasmid was introduced.

FIG. 8 is a diagram showing single copy UPRE (Y) -C;
Sec53 having YC1-lacZ reporter gene
The ERN ⁺ , ern1Δ and ern4Δ strains of 23
After culturing at 30 ° C. (unfolded protein in the endoplasmic reticulum (−)) or 30 ° C. (unfolded protein in the endoplasmic reticulum (+)), the extracted total RNA was analyzed by Northern hybridization. It is a photograph of the electrophoresis shown.

FIG. 9 shows a single copy UPR (Y) -C.
ERN of sec53 with YC1-lacZ reporter gene and single copy (YCp) expression plasmid
⁺ , Ern1Δ and ern4Δ strains were cultured overnight at 23 ° C., diluted and further cultured at 30 ° C.
-It is a figure which shows galactosidase activity and cell viability. The symbols in the figure indicate the same contents as the symbols shown at the top of the panel in FIG.

FIG. 10 shows Kar2p detected by immunoblotting using 10 μg of total protein prepared from each strain of FIG. 9 cultured at 30 ° C. every 2 hours.
5 is an electrophoretic photograph showing the amounts of Pdi1p and Pdi1p.

FIG. 11 is a photograph of electrophoresis showing an autoradiography result of a gel shift assay using a purified Ern4p fusion protein and a ³² P-labeled synthetic double-stranded oligonucleotide.

FIG. 12 shows that total RNA was extracted at regular intervals from wild-type and ern1Δ strains of Saccharomyces cerevisiae treated with tunicamycin, and ERN4, KAR2, PDI1, and ACT1 were isolated by Northern blotting.
It is a photograph of the electrophoresis which shows the result of the autoradiography which detected the mRNA of (internal standard). Lanes 1 and 2 show RNAs of 0 and 60 minutes after tunicamycin treatment of the ern1Δ strain, and lanes 3 to 8 show 0, 10, 20, and 3 respectively after treatment of tunicamycin of the wild strain.
Represents RNA at 0, 45 and 60 minutes.

FIG. 13 is a graph in which the radiation dose of each band of autoradiography in lanes 3 to 8 in FIG. 12 is quantified using a bioimaging analyzer BAS2000 (manufactured by Fuji Film Co., Ltd.) and is a graph.

FIG. 14 is a schematic diagram showing positions of an ERN4 gene and a primer.

FIG. 15 is a photograph of electrophoresis of RT-PCR using total primers of FIG. 14 from total RNA of a wild-type strain treated with tunicamycin for 45 minutes. Lanes 1 and 12 show molecular weight markers. Lanes 2, 3
Are primer b and primer f, lanes 4 and 5 are primer b and primer d, lanes 6 and 7 are primer b and primer i, lanes 8 and 9 are primer b and primer o, and lanes 10 and 11 are The results using primer b and primer m are shown, where even numbers indicate no tunicamycin treatment and odd numbers indicate tunicamycin treatment.

FIG. 16 is a schematic diagram showing the arrangement of a new ORF and the position of a probe used in the Northern blotting method of FIG. 17;

FIG. 17 is a photograph of electrophoresis by Northern blotting using the probe of FIG. Lanes 1 and 2 are probe af, lanes 3 and 4 are probe gd, lanes 5 and 6 are probe hi, lanes 7 and 8,
Indicates the results of using the probe jo and lanes 9 and 10 using the probe lm, and the odd numbers indicate no tunicamycin treatment and the even numbers indicate the tunicamycin treatment.

FIG. 18 is a diagram showing β-galactosidase-inducing activity by expression of a polypeptide derived from a novel ORF excluding introns.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI // C12P 21/08 C12P 21/08 (C12N 15/09 ZNA C12R 1: 865) (C12N 1/19 C12R 1: 865) ( C12P 21/02 C12R 1: 865)

Claims (19)

[Claims] 1. A DNA comprising a DNA encoding the polypeptide having the amino acid sequence of SEQ ID NO: 2 or a part thereof and having a transcriptional regulatory activity.
A. 2. A DNA comprising a DNA encoding the amino acid sequence of SEQ ID NO: 3 or a part thereof and encoding a polypeptide having a transcription regulatory activity.
A. 3. The method according to claim 1, wherein the DNA comprises a base sequence as set forth in SEQ ID NO: 1 or a part thereof and which encodes a polypeptide having a transcription regulating activity.
Or the DNA of 2. 4. An amino acid sequence represented by SEQ ID NO: 2 in the sequence listing or an amino acid sequence comprising a part thereof
Alternatively, DNA encoding a polypeptide having at least one of deletion, addition, insertion or substitution of two or more amino acid residues and having a transcription regulatory activity. 5. An amino acid sequence represented by SEQ ID NO: 3 in the sequence listing or an amino acid sequence comprising a part thereof
Alternatively, DNA encoding a polypeptide having at least one of deletion, addition, insertion or substitution of two or more amino acid residues and having a transcription regulatory activity. 6. A DNA capable of hybridizing to the DNA according to any one of claims 1 to 5 and encoding a polypeptide having a transcription regulatory activity or a functionally equivalent activity thereof. 7. The method according to claim 7, wherein the DNA is a transcription regulatory gene E of Saccharomyces cerevisiae.
The DNA according to any one of claims 1 to 6, which is RN4 or a mutant gene thereof. A polypeptide encoded by the DNA according to any one of claims 1 to 7. 9. A fusion protein comprising the transcriptional activation domain of a transcriptional regulator that functions in yeast and the polypeptide according to claim 8. 10. The fusion protein according to claim 9, wherein the transcription activation domain is a transcription activation domain Gal4p (ad) of the transcriptional regulator Gal4p of Saccharomyces cerevisiae. 11. A DNA encoding the fusion protein according to claim 9 or 10. A recombinant DNA containing the DNA according to any one of claims 1 to 7 or 11. 13. An expression vector containing the recombinant DNA according to claim 12. 14. An antibody or a fragment thereof that specifically binds to the polypeptide according to claim 8 or the fusion protein according to claim 9 or 10. 15. ern4Δ which is a Saccharomyces cerevisiae mutant strain in which the ERN4 gene has been disrupted. 16. A method for controlling a molecular chaperone and a folding enzyme in the lumen of a yeast vesicle, which comprises transforming a yeast with the expression vector according to claim 13. 17. The control method according to claim 16, wherein the yeast is a mutant strain ern4Δ in which the ERN4 gene has been disrupted. 18. A method for expressing a foreign protein, comprising transforming a yeast with the expression vector according to claim 13 and a foreign protein expression vector. 19. The method for expressing a foreign protein according to claim 18, wherein the yeast is a mutant strain ern4Δ in which the ERN4 gene has been disrupted.