JPH0595790A - Modified upstream activated sequence of yeast glycerol-3-phosphate dehydrogenase 3 gene - Google Patents

Abstract

PURPOSE:To provide the subject new sequence capable of preparing a manifestation vector having various manifestation powers such as a manifestation vector having strong manifestation power to be used for the production of protein or a vector for the determination of promoter activity. CONSTITUTION:A modified upstream activation sequence (UAS) having the structure of yeast glycerol-3-phosphate dehydrogenase 3 (TDH3) gene containing partially depleted natural UAS. The modified UAS can be produced e.g. by producing a plasmid pXX from a TDH3 gene separated from the chromosome of a yeast AH22, digesting the plasmid with Xbal and SphI, subjecting to depleting treatment with ExoIII nuclease, cyclizing the plasmid with T4 ligase, transforming an E.coli with the cyclized plasmid, selecting a cell containing a plasmid having depletion mutation from the colony of the E.coli and preparing a single stranded DNA from the colony.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a modified form of the upstream activating sequence (UAS) of the gene for glycerol-3-phosphate dehydrogenase, a fusion promoter containing the modified UAS, and the promoter. The present invention relates to the contained expression vector.

[0002]

2. Description of the Related Art Glycerol-3-phosphate dehydrogenase (or triose dehydrogenase, abbreviated as TDH), which is one of the yeast glycolytic enzymes, is most highly expressed in yeast cells. It is one of the existing enzymes, and its expression level reaches 6% to 20% of the total protein.

This enzyme is encoded by three loci. The loci are TDH1, TDH2, TDH3. The enzyme activity ratio of the enzyme proteins produced by these loci is approximately TDH1: TDH2: TDH3 = 10 to 15:25 to 3 when the cells are grown with glucose as the carbon source.
It is known that it is 0: 50-60 (McAlister, L. & Ho
Lland, MJ, J. Biol. Chem., 260, 15013-15018 (1985) and 15019-15027 (1985)).

Paying attention to the fact that the TDH gene is efficiently expressed, many attempts have been made to express a foreign gene product using yeast using this promoter sequence. For example, examples of using the TDH3 promoter include bovine chymosin, human lysozyme, and hepatitis B virus core antigen, and examples of using the TDH2 promoter include somatoin, which is a plant sweet protein.
(Edens et al., Cell, 37 , 629-633, 1984).

The TDH promoter sequence has already been utilized in various systems as described above, but the knowledge of its mechanism of action, the upstream regulatory sequence controlling the action of the promoter, etc. is scarce. Bitter etc. is TD
The promoter activity of H3 is -26 to -6 upstream of the start codon.
74 is sufficient, and further upstream sequences are not mentioned in the report (Gene, 32, 163-274, 1984), which does not affect the strength of expression. On the other hand, in the case of TDH2 as well, the upstream −844 to −279
Eden et al. Have reported that the sequence present in E. coli affects promoter strength (Cell, 37, 629-633, 1984).
..

[0006] In an attempt to further improve the efficiency by modifying the TDH3 promoter sequence, a report was made to introduce a control sequence of GAL1-GAL10 into −410 of the TDH3 promoter sequence and to add a galactose to form a controllable promoter.
(Gene 69 , 193-207) has been made. Also ADH2 UAS
We also reported an attempt to construct a regulatable promoter by glucose deficiency by combining Escherichia coli with a partial promoter sequence of TDH3 (Yeast Genetic Engineering, Butt
erworth, ISBN 0-409-90117-2,1989, pp83-108). However, there is no report on the analysis of UAS of TDH3 and the use of the element sequences constituting it.

[0007]

Therefore, the present invention is based on the TDH3
Modified UAS having different activities by removing different regions of different UASs to different extents, said UAS
It is intended to provide a fusion promoter containing the above and an expression plasmid containing the fusion promoter.

[0008]

In order to solve the above problems, the present inventor shortened (removed) the UAS region of TDH3 to various degrees and measured its activity to determine the U of TDH3.
AS region has -1 at A immediately upstream of the translation initiation codon ATG
The presence of a UAS region at −409 to −583, and the UAS region is a three element unit sequence having a predetermined function, that is, A (−520 to −499), B (−583 to −540)
And C (-481 to -447), A in these elemental unit sequences contains a sequence highly homologous to the RAP1 binding sequence, and B contains the binding sequence of the yeast GRF2 protein. The present invention has been completed based on these findings and found that there is a sequence having high homology to the existing sequences.

Accordingly, the present invention provides a modified UA in which the natural upstream activation sequence (UAS) of the TDH3 gene is partially deleted.
Provide S.

More specifically, according to the present invention, in the base sequence of UAS shown in Sequence Listing 1, A immediately upstream of the translation initiation codon ATG is set to -1, and element unit sequences B of -583 to -540, -520. ~ -499 element unit array A, and -481
Of the -447 element unit arrays C, (1) element unit array A
Fragments not containing C and C, (2) Elemental unit sequences B and C
Not containing, (3) a fragment not containing the element unit sequence B, (4) a fragment not containing the element unit sequence A, (5)
Provided is a modified UAS comprising either a fragment not containing the elemental unit sequence C or (6) a fragment containing the elemental unit sequences A, B and C.

[0011]

[Detailed Description] The present inventors focused on TDH3, which is the most frequently expressed among TDH, and found that the promoter sequence contained an upstream activating sequence (UAS), which is a sequence capable of efficiently functioning the promoter. It was examined whether it exists. As a result, in the case of TDH3, it was revealed that UAS exists in −409 to −583. Further analysis of this UAS revealed that the UDH of TDH3 is composed of several elemental units that influence activity.

The elemental unit sequence A is a sequence of -520 to -499, which is a sequence which plays the most important role in the activity of the TDH3 promoter. The lack of this sequence significantly reduces activity. Fragments with −583 to −540 were found to have UAS activity. This area is called an element unit array B. UAS activity of element unit B is −
When the region 505 to -539 is present, it appears only weakly, and when this region is deleted, it becomes strong. Moreover, even if only the element unit B is deleted, the UAS of TDH3
There was no effect on activity.

Element unit C was found by using the promoter of ADH1 for activity measurement, -447 to -481.
It is an array that exists in the vicinity. The array around this, that is, −
Since the DNA fragment having 434 to -484 did not show UAS activity, the element unit C was itself a UAS.
It was found to show no activity. The element unit C is AD
It was also clarified that it has a function of increasing the activity of the element unit A when activating the H1 promoter.

By combining an artificially constructed UAS in which elemental unit sequences affecting these activities are combined with a yeast promoter sequence, it is possible to create various promoters having different expression powers. It was revealed.

RAP1 was used by Shore et al. To identify the silencer region of the yeast zygotic locus and the MAT at the same locus.
A purified protein as a protein that binds to the UAS region of the α gene, and the encoding gene was isolated from a yeast chromosome library by using an antibody (Cell, 51,
721-723, 1987).

Subsequent studies revealed that the binding sequence of RAP1 was
In the promoter sequences of glycolytic enzyme genes such as PGK, ADH1, ENO1, and PYK, as well as in yeast telomere sequences, AR
A highly homologous sequence was also found in the S sequence. In addition, the binding sequence of a factor called TUF, which commonly exists in the UAS region of genes of ribosomal constituent proteins, is RAP1.
It is considered that TUF1 and RAP1 are the same protein because they show high homology to the binding sequence of T.

From the analysis of PGK UAS, Stanway et al. Showed that the RAP1 binding sequence in PGK UAS was PGK
It has been shown that it activates transcription from an artificially created promoter sequence, that is, it has UAS activity based on the promoter sequence of PGK.
It is not strong as compared with the original activity of AS, and it has been proposed that the repeated sequence of CTTCC is required to show strong activity (Nucl. Acid. Res. 17 , 9,9205-9218,198).
2).

Buchman et al. Also artificially synthesized the RAP1 binding sequence found in ADH1, PYK1, ENO1 and the like, and investigated its UAS activity using the CYC1 promoter. In order to show, it was shown that the sequence of GCTTCCA existing near the RAP1 binding sequence is necessary (Mol. Cell. Biol. 8 , 5086-5099, 1988).

The element unit A contains a sequence highly homologous to the binding sequence of RAP1. When the fragment containing the elemental unit A activates the ADH1 promoter, it exhibits an activity equivalent to the original activity, so that the elemental sequence C
Need.

However, the element unit C includes the above RA
The sequence itself which has been shown to affect the UAS activity of the P1 binding sequence cannot be found. Instead, in element unit C, a repetitive sequence having GCATCCA as a core sequence can be found. This sequence may correspond to the sequence found in PGK, ADC1 and ENO1.

On the other hand, when the element unit A is combined with the promoter of TDH3, it shows UAS activity which is as strong as the original activity regardless of the presence of the element unit C.

By comparing the sequence of the element unit B with the previously published recognition sequence of the DNA binding protein, a high homology to the sequence proposed as the binding sequence of the yeast GRF2 protein was found in the element unit B. It was revealed that there is a sequence exhibiting sex. Also, overlapping with this array,
It was possible to find a sequence having homology with the binding sequence of the yeast RAP1 protein.

The yeast GRF2 protein is the U of yeast GAL1-GAL10.
It is a protein that binds to AS _G and was found as a factor different from the GAL4 protein. The consensus binding sequence is
5'-YNNYYACCCG-3 'is proposed (Gene
sI Dev., 4 , 503-514, 1990).

Although this binding sequence has a weak UAS activity per se, it is known that when it is combined with a thymidine-rich region existing upstream of the DED1 gene, the activity is synergistically increased. .. This synergistic effect is
It is not detected when combined with the binding site of the L4 protein. In addition, the distance between two sequences is considered to be important for the synergistic effect to appear (Genes and Deve
lopment, 4,503-514,1990).

Determination of upstream activating sequence (UAS) In the present invention, the TDH3 gene isolated from the chromosomal DNA of yeast AH22 was used. In order to investigate the upstream regulatory sequence contained in the TDH3 promoter sequence, the plasmid pXX containing the promoter sequence and containing no structural gene was used. The isolation of the TDH3 gene, and the construction of plasmid and pXX are described in Japanese Patent Application No. 1-328264 (1989, 12, 20 application). The plasmid pXX contains the translation initiation codon of the TDH3 gene and the positions -2 to -1060, where A immediately upstream of the ATG is the first position.

In order to detect the upstream activating sequence, pXX was subjected to a deletion mutation treatment and 3'from the HindIII site of -1059.
Various plasmids containing deletion promoter fragments having deletion mutations in the 5'to 3'direction were obtained. Details of this deletion mutation treatment are described in Example 1.

In order to test the transcription initiation ability of the obtained deleted promoter fragment, it is desirable to insert the fragment to be assayed upstream of the marker gene whose expression level can be assayed relatively easily. Further, in order to introduce the fragment to be assayed using the gene recombination technique, it is desirable that the marker gene is introduced into a shuttle vector that can be replicated in both yeast and E. coli.

As a vector satisfying the above conditions,
In the present invention, an assay system using the neo gene described in Japanese Patent Application No. 1-41604 and a newly created assay vector are used. The assay vector pJDB-NeoC-ATE (Japanese Patent Application No. 1-41604) having the neo gene as a marker gene is
Since it has only the original start codon of the neo gene, it is a vector capable of assaying a weakly active promoter fragment.

This vector is used for replication in yeast.
It is a shuttle vector that has the replication origin of the micron plasmid and has the LEU2 gene as a gene for selecting transformed yeast. Also, since it has HindIII and XhoI sites as restriction enzyme sites for inserting a fragment for assaying promoter activity on the 5'side of the marker gene, the fragment to be assayed can be easily inserted. The method for inserting the deleted promoter fragment to be assayed into this vector is specifically described in Example 2.

On the other hand, the plasmid pL containing the LacZ gene
acZC-37 (see Japanese Patent Application No. 2-226566) and plasmid
From pJDB-NeoC-ATE, promoter assay vectors pRS-LacZC and pRG-LacZC using β-galactosidase as an expression marker are prepared as described in Example 3.

These newly created assay plasmids pR
S-LacZC and pRG-LacZC have an ARS sequence existing near the TRP1 gene as an origin of replication in yeast cells, and may be distributed to daughter cells during meiosis. It is a vector having a CEN region so that it can stably exist in the body with a low copy number.

Like pJDB-NeoC-ATE, it has the LEU2 gene as a gene for selecting transformed yeast. It has the β-galactosidase (LacZ) gene of Escherichia coli as a marker gene for assaying the transcription ability. In addition, HindIII and Xh were used as restriction enzyme sites for inserting a fragment to be assayed on the 5'side of the marker gene.
Since it has an oI site, it is easy to insert the fragment to be assayed.

The expression level in the yeast cells of the assay vector into which the deletion mutant promoter fragment prepared above was inserted was assayed by the method specifically described in Examples 5 and 6. As is clear from Table 1 showing the results obtained from Examples 5 and 6 and FIG. 11, it was found that the expression power was remarkably reduced when the sequence at positions -409 to -589 was deleted.
From this, it was clarified that the UAS of TDH3 is present at -409 to -583. Also in this area, RA
The nucleotide sequence containing the P1 recognition sequence is different from that already reported by Egan et al., And the sequence reported by Egan et al. Is homologous to the consensus RAP1 binding sequence found in the promoter regions of many genes. It became clear that the sex becomes low.

Determination of element unit sequence It was shown above that the UAS of TDH3 is a sequence of -409 to -583. The following shows what kind of functional unit this array is made up of (herein referred to as element unit).

By the method specifically described in Example 7, pUAS1 was obtained from the deletion promoter fragment vector pXX05 having -2 to -583. pUAS1 is -589 to -4
It has an array of 07. The fact that this fragment has UAS activity is specifically described in Examples 7-11.

UAS activity was measured using the TDH3 promoter-
It was carried out by examining the effect of promoting the transcriptional activity of a promoter containing a fragment having 2 to −360.
By introducing this fragment into the promoter assay vector pRG-LacZC, the UAS activity measurement vector pRG-N7-LacZC
Got It is clear from Table 2 that the transformant produces a small amount of β-galactosidase when yeast is transformed with this vector.

When yeast having the HindIII-BglII fragment of pUAS1 inserted into the 5'side of the promoter fragment of this vector was used to transform yeast, as shown in Table 2, the transformant had about 38-fold. The β-galactosidase having the activity value of is produced. Therefore, pUAS1 has −
It is clear that the fragment of 407-583 has UAS activity.

The UAS activity of the fragments contained in pUAS2, pUAS3, pUAS4 and pUAS5 obtained by treating the deletion mutation of pXX05 was assayed using pRG-N7-LacZC.
For the construction of these UAS cassette plasmids,
This is specifically described in Example 7. The insertion into the assay vector is specifically described in Example 10, and the activity measurement is specifically described in Example 11.

Table 2 showing the results obtained from Example 11
As is clear from the above, the deletion of −481 to −505 significantly reduced the UAS activity (about 1/4).
Furthermore, based on pUAS4 deleted up to −481,
In the case of pUAS4-10 obtained by deleting up to 499, the decrease in activity as seen above did not occur.
In addition, based on pUAS4-10, the 5 ′ side was further deleted, and −5
The fragment of pUAS4-11 having 20 to −499 showed the same activity as the fragment of pUAS1 which was not deleted at all.

The results are specifically described in Example 11. The construction of these UAS cassette plasmids is specifically described in Example 7. On the other hand, the insertion into the assay vector was specifically described in Example 10, and the activity measurement was specifically described in Example 11. From these facts, the sequence that brings about the strong activity of pUAS1 is pUAS1.
It was revealed that it exists in S4-11. This pUAS4
The array of -520 to -499 that -11 has is called an element unit array A.

By using the HindIII-BglII fragment of the plasmid pUAS2 having the elemental unit sequence A, a gel mobility shift assay was performed to obtain the elemental unit sequence A.
The existence of a factor that binds to was suggested. Further, the binding sequence of the factor that binds to the element unit sequence A was revealed by the DNA footprinting method using the same fragment and using the inhibition of methylation binding. These operations are specifically described in Reference Example 1.

The revealed binding sequence is shown in -513 -503 GGTGTCTGGGT. By comparing the complementary sequence of the region containing this sequence with the previously published recognition sequence of the DNA binding protein, the complementary sequence of this region shows high homology of the yeast RAP1 protein proposed for the binding sequence. It became clear.

The UAS activity of the fragment contained in pUAS7 obtained by treating the deletion mutation of pXX05 was determined by pRG-N7-L
Assayed using acZC. Experimental details, examples
It is specifically described in 10 and 11. As is clear from Table 2 showing the results obtained from Example 11, the activity which was reduced to about ¼ by deleting up to −516 was increased about 3 times by further deleting up to −540. It turned out to rise.

This UAS activity brings about −583 to −540.
Is called an element unit array B. UA of element unit array B
The S activity is about 1/2 of that of the elemental unit sequence A, and the activity when the elemental unit sequences A and B are combined shows the activity of the elemental unit sequence A, and the activities of both are added. And the fact that there is no effect of being synergized,
It is clear from the results in Table 2.

Generally, UAS has been shown to have the ability to activate transcription regardless of the type of promoter. We investigated whether the UAS of TDH3 and its elemental unit sequence determined in this case can activate transcription from a heterologous promoter. Any promoter may be used as long as it is a yeast promoter transcribed by polymerase II. In this case, for example, the promoter of ADH1 is used for activity measurement.

Part of the promoter sequence of ADH1, -11 to-
The AX promoter fragment having up to 253 was designated as pRG-N7-LacZC
PRG-AX-L introduced in place of the TDH3 promoter fragment
acZC was used. Details of the construction of this assay vector are described in Examples 8 and 9.

This assay vector was constructed from pXX05.
pUAS1, pUAS2, pUAS3, pUAS4, pUAS5, pUAS6, pU
Inserted fragments of AS7, pUAS4-10 and pUAS4-11. Yeast was transformed with the resulting plasmid, and β-galactosidase activity in the cell extract was measured. These details are described in Examples 10 and 11. Example 11
As is clear from Table 3 showing the results obtained in pUAS1
The enzyme activity is increased by about 52 times by inserting the fragment.

From this, the fragment of pUAS1 is AD
It is clear that it activates the transcription of the C1 promoter. This UAS activity did not change even if it was deleted up to -407 to -447, but when it was further deleted up to -481, the activity was reduced to about 1/3. Furthermore, no further decrease in activity was observed in pUAS4-10 in which elemental unit sequence A remained.

As is clear from Table 3, the element unit array A
When a fragment of pUAS4-11 having only the above is inserted, it has the same activity as pUAS4-10 containing element unit sequence B.
Even when activating a heterologous promoter, the effects exerted by both element unit sequences are not added or synergized.

As described above, when -447 to -481 was deleted, UAS activity was decreased, so the sequence of this region is called elemental unit sequence C. pUAS2- was obtained by subjecting pUAS2 to deletion mutation treatment from the 3'side.
When the activity was examined using the D4 and pUAS2-D6 fragments, the activity change due to the deletion of the elemental unit sequence B was not observed, and the fragment having only the elemental unit sequence C showed almost no activity. Table 3 shows that no increase was seen.
Is clear from. From these things, element unit array C
Can be said to be a sequence having a function of increasing the activity of the element unit sequence A. It is also clear that this function is exerted only when combined with a heterologous promoter sequence.

From the above, TATA with different transcription initiation ability
By combining a promoter containing a sequence and a transcription start point with the upstream activation element unit sequence having different activation ability as described above found in TDH3, it is possible to create a promoter having different expression power. is there.

[0052]

EXAMPLES Next, the present invention will be described more specifically by way of examples. The methods commonly used in the examples described below are as follows.

1) ExoIII nuclease and mung bean
5 μg of the deletion mutation-treated plasmid using nuclease was digested with an appropriate restriction enzyme, extracted with phenol-chloroform, and precipitated with ethanol to give DN.
A was collected. Add 50 μl of DNA to ExoIII buffer [50 mM T
ris-HCl (pH 8.0), 100 mM NaCl, 5 mM MgCl2, 10 mM 2-mercaptoethanol]. 50 in another tube
μl of MB buffer [40 mM sodium acetate (pH 4.5), 100 mM
NaCl, 2 mM ZnCl2, 10% glycerol] was added and placed in ice.

180 units of ExoIII nuclease was added to the DNA solution, which was kept warm at 37 ° C. and 5 μm every 30 seconds after the enzyme was added.
1 was sampled and transferred to a tube containing MB buffer. After sampling, put the tube on ice at 65 ℃ for 5
The mixture was kept warm, then cooled to 37 ° C, 50 units of mung bean nuclease was added, and the mixture was kept warm at 37 ° C for 30 minutes. After the reaction, this solution was extracted with phenol saturated with TE, and DNA was recovered by ethanol precipitation.

2) Restriction enzyme treatment Restriction enzyme treatment was carried out under the reaction conditions recommended by the manufacturer.

3) T4 DNA ligase treatment For example, 100 ng of the fragment and 20 ng of the fragment containing the replication origin of E. coli were added to 10 μl of the reaction solution [50 mM Tris.HCl (pH7.5), 10 m
M MgCl2,10mM DTT, 1mM ATP]
A unit of T4 DNA ligase was added, and the mixture was incubated at 16 ° C for 1 hour to overnight.

4) Medium composition E. coli is L medium (1% tryptone, 0.5% yeast extract,
Culture was performed using 0.5% sodium chloride). When ampicillin was added, it was added at a concentration of 50 μg / ml. Further, in the case of an agar medium, agar was added so as to be 1.5%.

The composition of the YPD medium for culturing yeast was 2% polypeptone, 1% yeast extract and 2% butter sugar, and in the case of the agar medium, agar was added so as to be 2%. The composition of the SD medium containing no leucine is as follows. SD medium: 20 μg / ml adenine sulfate,
20 μg / ml arginine hydrochloride, 20 μg / ml methionine,
20 μg / ml histidine hydrochloride, 20 μg / ml tryptophan, 20 μg / ml uracil, 30 μg / ml isoleucine, 30
μg / ml hydrochloride lysine, 30 μg / ml tyrosine, 50 μg /
ml Phenylalanine, 150 μg / ml valine, 0.67% amino acid-free yeast nitrogen base, 2% glucose. Further, in the case of the agar medium, one supplemented with 2% agar was used.

Example 1. Yeast TDH3 gene promoter
Construction of Deletion Mutation Sequence of Region The plasmid pXX was digested with XbaI and SphI and subjected to a deletion mutation treatment by a deletion mutation preparation method using ExoIII nuclease or mung bean nuclease. After circularization with T4 ligase, E. coli XL1-Blue strain was transformed,
The DNA of the obtained ampicillin-resistant colonies was examined, and colonies having the plasmid in which the deletion mutation had occurred were selected.

Single-stranded DNA was prepared from these colonies and sequenced using a Sequenase Sequencing Kit according to the method recommended by the manufacturer.
The deleted region was identified. By these operations, pXXN1
3, pXX08, pXXN12, pXX05, pXXN7, pXXN10, pXXN16
And pXX10 were obtained. The deleted regions of these vectors are shown in Table 1. The state of the remaining upstream area is shown in FIG.

Example 2. Promoter with deletion mutant sequence
Of NeoC expression plasmid for measurement of protein activity (Fig.
1) Digest plasmid pAH-NeoC-ATE with HindIII and XhoI,
The Kb fragment was separated by agarose electrophoresis. This fragment was recovered and purified with the GeneClean kit (BIO101). On the other hand, a plasmid having a deletion mutant promoter sequence was also digested with HindIII and XhoI, the smaller fragment was separated by agarose gel electrophoresis, and purified and recovered with GeneClean kit (BIO101).

20 ng of pAH-NeoC-ATE-derived fragment and 100 ng of promoter-derived fragment were circularized with T4 ligase, and Escherichia coli SCS1 was transformed with the circularized DNA. The DNA of the obtained ampicillin-resistant colonies was examined, and those in which the ADH promoter region of pAH-NeoC-ATE had been replaced with the deletion mutant promoter sequence were selected.

Example 3 Promoter Assay Vector pRS-
Construction of LacZC and pRG-LacZC (Fig. 2) (Fig. 3) Plasmid pLacZC-37 containing LacZ gene (Japanese Patent Application No. 2-2265)
(Described in 66) and a 3.2 Kb fragment obtained by cutting with DraI, XhoI and BamHI, and pJDB-NeoC-ATE (Japanese Patent Application No. 1-441604) with XhoI and BamHI. The 7 Kb fragment was ligated with T4 DNA ligase and circularized to obtain pJDB-LacZC-ATE.

A 3.2 kb fragment was obtained by cutting pJDB-LacZC-ATE with HindIII and SalI. This fragment and pRS315 (Sikorski,
RS and P. Hierter, Genetics 122, p19-27, 1989), T4
The assay vector pRS-LacZC was obtained by ligating with DNA ligase and circularizing.

The plasmid pGAPF1 containing the TDH3 gene (Japanese Patent Application No. 1-328264) was digested with HindIII and XbaI to obtain a 1.05 kb fragment containing the coding region of the TDH3 gene. This fragment was ligated to pUC119 digested with HindIII and XbaI and circularized to obtain plasmid pXH1.05. A 1.05 kb fragment obtained by digesting this plasmid with XbaI and HindIII, as well as HindIII and XbaI.
Ligated with a fragment of about 10 kb from pRS-LacZC cleaved with
The assay vector pRG-LacZC was obtained by circularization.

Example 4 Assay for Deletion Promoter Fragments
(Fig. 4) (Fig. 5) pXXN10, pXXN7, pXXN16, pXX10 and pXX05 are pRG-LacZ
CXX, pXX05 and other promoter fragments are pR
The assay was performed by inserting into S-LacZC. Deletion promoter fragments were obtained by cleaving each promoter cassette vector with HindIII and XhoI and isolating the smaller fragment.

On the other hand, assay vector, pRG-LacZC and pRS-La
Similarly, cZC was cleaved with HindIII and XhoI, and
The 11 kb and 10 kb fragments were isolated. The isolated test vector fragment and the isolated promoter fragment were ligated with T4 DNA ligase for circularization, and Escherichia coli XLl-Blue was transformed to obtain a test vector having the deleted promoter fragment inserted therein.

Example 5. Using NeoC expression plasmid
Assay of deleted promoter fragments Yeast AH using plasmids with deleted promoter fragments
Twenty-two strains were transformed. The transformation method was a conventional method using LiSCN. Selection of transformants was carried out on leucine-free synthetic agar medium. The resulting colony is treated with 5 ml S
The medium D was inoculated and cultured at 30 ° C. for 2 days with shaking. A portion of this culture (2X10 ⁷ cells) was inoculated into 50 ml of SD medium,
The cells were cultivated with shaking at ℃ for 24 hours. Take 20 ml of this culture
The cells were collected by centrifugation at 3000 rpm for 5 minutes.

The cells were resuspended in 1 ml of 1M sorbitol and again collected by centrifugation at 3000 rpm for 5 minutes. 400 μg / ml zymolyase / 1M sorbitol 1 ml
The cells were resuspended in 30 minutes, left at 30 ° C. for 30 minutes, and then centrifuged at 7,000 rpm for 5 minutes to collect the cells. 7 spheroplasts obtained
50 μl of TLES solution [Tris-HCl (pH 7.5), 0.1 M LiCl, 10 mM EDT
A, 1% SDS], 300 μl of TLE saturated phenol was added, and vortex mixing was performed.
After centrifugation at 15000 rpm for 10 minutes, an aqueous layer was obtained. 2 more of this water layer
Water layer 600μ obtained by extraction with TLE saturated phenol
200 μl of 8M LiCl was added to 1 l, mixed well, and allowed to stand at 4 ° C. overnight.

The RNA precipitate was collected by centrifugation at 15000 rpm for 10 minutes. Dissolve this in 300 μl of sterile water and add 100 μl of 8M
LiCl 2 was added, mixed well, and then left standing at 4 ° C. for 2 hours. Centrifuge at 15000 rpm for 10 minutes to collect the RNA precipitate.
It was dissolved in 300 μl of sterilized water, 30 μl of 3M sodium acetate solution (pH 5.2) was added, and 750 μl of ethanol was added, and the mixture was allowed to stand at −80 ° C. for 2 hours. The RNA precipitate was collected by centrifugation at 15000 rpm for 10 minutes, washed with 70% ethanol, and dried under reduced pressure. It was dissolved in an appropriate amount of water and used as an RNA sample.

2 μg of RNA was added to 50% formamide, 2M
Formaldehyde, 1XMOPS buffer (10XMOPS buffer;
0.2M MOPS, 0.05M sodium acetate, 0.01M EDTA,
Adjusted to pH 7.0 with NaOH)
After treatment at 65 ° C for 5 minutes, add 1/10 amount of 0.01% bromophenol blue solution (adjusted with 50% glycerol),
This was separated by electrophoresis on a 1.1% agarose gel (prepared with 1X MOPS buffer and 2M formaldehyde). After electrophoresis, follow the method recommended by the manufacturer to
Transferred to bond-N ^TM filter and irradiated with RNA for RNA
Was fixed to the filter.

This filter was placed in a hybridization solution containing 50% of formamide and incubated at 42 ° C. for 2 hours, and then the probe was transferred to the same solution containing 1 × 10 ⁶ cpm / ml,
Incubated at ℃ overnight. As the probe, a part of the structural gene of yeast phosphoglycerate kinase (abbreviated as PGK) was labeled with α- ³² P-dCTP by the random prime method using DNA polymerase. After washing the filters with 0.1X SSC, 0.1% SDS at 60 ° C, residual radioactivity was detected by exposure to X-AR film.

After the detection, the filter was used as a probe removing solution (50 mM Tris-HCl (pH 7.5), 5X Denhardt's solution, 0.5.
% SDS, 100 μg / ml salmon testis heat-denatured DNA, 50% formamide) and incubated at 80 ° C. for 15 minutes. After repeating this operation, the filter was washed with 0.1XSSC and transferred to the hybridization solution. After incubating at 42 ° C for 2 hours, the probe was transferred to the same solution containing lX10 ⁶ cpm / ml and incubated at 42 ° C overnight.

The probe is a sequence (5'-TAGCCTCTGCACCCAAGC) which forms a complementary chain with the mRNA of the neomycin resistance gene.
GGC) was chemically synthesized, and its 5'end was γ- ³² P-ATP and T4.
The one labeled with a polynucleotide kinase was used. After washing the filter with 2X SSC, 0.1% SDS at 60 ° C,
Residual radioactivity was detected by exposure to X-AR film.

A film obtained by using the PGK gene as a probe and a film obtained by using a sequence that forms a complementary chain with the mRNA of the neomycin resistance gene as a probe were analyzed by a TIAS100 type image processing device manufactured by TEFCO, and the radiation was analyzed. The strength of Noh was quantified. The numerical values obtained respectively, Dpgk, Dneo
And the value of Dneo / Dpgk was calculated. These values were shown relative to each other, with the value obtained using the RNA prepared from yeast obtained by transformation with pXX05-NeoC-ATE being 100. NeoC expression levels were compared using this relative value.
The results are shown in Table 1.

Example 6. Transcriptional ability of deleted promoter fragments
Using the inserted test vectors assay deletion promoter fragment force, by transformation method using LiSCN yeast DC5 strain,
Transformed. Transformed yeast was selected on leucine-free agar synthetic medium. The formed colonies were inoculated into a synthetic liquid medium containing no leucine, and cultured with shaking at 30 ° C for 24 hours.

After culturing, the cells were collected by centrifugation, and 100 μl of Z buffer (disodium phosphate heptahydrate 8.55 g,
Monosodium phosphate monohydrate 2.75 g, potassium chloride
0.375 g, magnesium sulfate heptahydrate 0.123 g, and 2-mercaptoethanol 1.35 ml were dissolved in water and the total amount was made 11). 100 μl of glass beads (diameter 0.4 mm) was added thereto, and vortex mixing was performed for 2 minutes. 100 μl of Z buffer was added thereto, mixed gently, and then centrifuged. After centrifugation, the supernatant was taken to obtain a cell extract.

Cell extract was added to 1 ml of Z buffer,
100 μl of 4 mg / ml o-nitrophenyl-β-
After adding D-galactopyranoside solution and mixing, 28 ℃
To let the reaction take place. The reaction is stopped by adding 1M sodium carbonate solution at an appropriate time.
OD420 was measured. In addition, the protein concentration of the cell extract was
The measurement was performed using a protein assay solution from Rad. The activity value of beta-galactosidase was calculated by the following formula.

[0079]

[Equation 1]

The calculated activity values are shown in Table 1.

[Table 1]

Example 7. UAS Cases Containing Upstream Activating Sequences
Construction of set plasmid (FIG. 6) (FIG. 7) The plasmid pXX 05 was digested with XhoI and subjected to a deletion mutation treatment by a deletion mutation preparation method using ExoIII nuclease and mung bean nuclease. BglII linker (5 '
p-CAGATCTG) and circularized with T4 ligase, E. coli XLl-Blue strain was transformed, and the DNA of the obtained ampicillin resistant colonies was examined to select a colony having a plasmid in which a deletion mutation occurred.

From these colonies, single-stranded DNA was prepared and sequenced using a Sequenase Sequencing Kit according to the method recommended by the manufacturer.
The deleted region was identified, and plasmid pUAS1 having a nucleotide sequence of −583 to −407, pUAS2 having a nucleotide sequence of −583 to −434, pUAS3 having a nucleotide sequence of −583 to −447, −583
PUAS4 with from −481 to −583 with −505
pUAS5, pUAS6 with −583 to −516, and −58
From 3 we obtained pUAS7 with −540.

After cleaving pUAS2 with HindIII, a deletion mutation treatment was carried out by a deletion mutation preparation method using ExoIII nuclease and mung bean nuclease. After circularization with T4 ligase together with HindIII linker (5'p-CCAAGCTTGG), Escherichia coli XLl-blue strain was transformed, and the DNA of the obtained ampicillin-resistant colony was examined. I chose.

Plasmids pUAS2-D4 and pU having deletion mutations
AS2-D6 was digested with HindIII and BglII, the smaller fragment was isolated, and pU was digested with HindIII and BamHI.
Recircularize with C119 to transform E. coli XLl-Blue,
The nucleotide sequence of the introduced fragment was determined.
As a result, it was found that pUAS2-D4 and pUAS2-D6 have the sequences −554 to −434 and −484 to −434, respectively.

PUAS4-10 with -583 to -499 is pUAS4
Was obtained by the following method. pUAS4 to BglI
After cleaving with I, a deletion mutation treatment was performed by a deletion mutation creating method using ExoIII nuclease and mung bean nuclease. The deleted fragment was cleaved with HindIII to give approximately 1
A fragment with a size around 00 bp was separated. PBluescriptI obtained by cutting these fragments with HindIII and SmaI
E. coli XLl-Blue was transformed by ligating with IKS (+) and recircularizing. PUAS4-10 was obtained by examining the DNA of the obtained ampicillin-resistant colonies, selecting a colony having a plasmid having a deletion mutation, and examining the nucleotide sequence thereof.

After cutting pUAS4-10 with HindIII, ExoIII
Deletion mutation treatment was performed by a deletion mutation generation method using nuclease and mung bean nuclease. After circularization with T4 ligase together with HindIII linker (5'p-CCAAGCTTGG), Escherichia coli XLl-Blue strain was transformed, and the DNA of the obtained ampicillin-resistant colony was examined. I chose. From these colonies, single-stranded DNA was prepared and sequenced according to the manufacturer's recommended method using a Sequenase Sequencing Kit to identify the deleted region and have a sequence of -520 to -499. I got pUAS4-11.
The status of these various UAS cassette fragments is shown in FIG.

Example 8. Vector for assaying upstream activation sequences
-Creation of pRG-N7L-LacZC and pRG-AXL-LacZC (Fig. 8) The test vector pXXN7L-NeoD-ATE (Japanese Patent Application No. 2-226566; Microtechnology Research Institute No. 11681) was cut with HindIII and XhoI, About 360
A fragment of b was obtained. This fragment was cloned into pRG-LacZC using HindIII and X.
A fragment of about 11 kb obtained by cutting with hoI,
After ligation and circularization, Escherichia coli XLl-Blue was transformed, the DNA of the obtained ampicillin-resistant colony was examined, and a plasmid in which the fragment had been inserted was selected to obtain the vector pRG-N7L- for upstream activation sequence assay. I got LacZC.

On the other hand, plasmid pAXL-37 (Japanese Patent Application No. 2-2265)
66) was digested with HindIII and XhoI to obtain a fragment of about 260b. This fragment was ligated and circularized with a fragment of about 11 kb obtained by cutting pRG-LacZC with HindIII and XhoI, and transformed into Escherichia coli XLl-Blue, and the DNA of the obtained ampicillin-resistant colony was examined. By selecting a plasmid into which the fragment had been inserted, an upstream activation sequence assay vector pRG-AXL-LacZC was obtained.

Example 9. Upstream activating sequence assay vector
Insertion of a UAS cassette fragment into (Fig. 9) (Fig. 10) pUAS1, pUAS2, pUAS3, pUAS4, pUAS5, pUAS6, pUAS7, p
The fragment contained in UAS2-D4 or pUAS2-D6 was used as the test plasmid pR.
To insert into G-N7L-LacZC or pRG-AXL-LacZC,
The following method was adopted. The UAS cassette plasmid was cut with HindIII and BglII and the smaller fragment isolated. These fragments were ligated with assay plasmid pRG-N7L-LacZC or pRG-AXL-LacZC cleaved with HindIII and BglII, cyclized, and transformed into E. coli XLl-Blue, and the DNA of the obtained ampicillin-resistant colonies was transformed. The plasmid in which the fragment was inserted was selected.

On the other hand, in pUAS4-10 and pUAS4-11, HindIII
It was cut with BamHI and the smaller fragment was isolated. Assay plasmids obtained by cutting these fragments with HindIII and BglII
Escherichia coli XLl-Blue was transformed by binding to pRG-N7L-LacZC or pRG-AXL-LacZC for circularization, and the DNA of the obtained ampicillin-resistant colonies was examined to select the plasmid in which the fragment had been inserted.

Example 10. Transcriptional activation of inserted fragments
Assay of capacity Using the assay plasmid in which the fragment of UAS cassette was inserted, yeast DC5 strain was transformed by the transformation method using LiSCN. Transformed yeast was selected on leucine-free agar synthetic medium. The formed colonies were inoculated into a synthetic liquid medium containing no leucine and incubated at 30 ° C for 24
Culture was performed with shaking for an hour.

After culturing, the cells were collected by centrifugation, and 100 μl of Z buffer (disodium phosphate heptahydrate 8.55 g,
Monosodium phosphate monohydrate 2.75 g, potassium chloride
0.375 g, magnesium sulfate heptahydrate 0.123 g, and 2-mercaptoethanol 1.35 ml were dissolved in water and the total amount was made 11). 100 μl of glass beads (diameter 0.4 mm) was added thereto, and vortex mixing was performed for 2 minutes. 100 μl of Z buffer was added thereto, mixed gently, and then centrifuged. After centrifugation, the supernatant was taken to obtain a cell extract.

Cell extract was added to 1 ml of Z buffer,
100 μl of 4 mg / ml o-nitrophenyl-β-
After adding D-galactopyranoside solution and mixing, 28 ℃
To let the reaction take place. The reaction is stopped by adding 1M sodium carbonate solution at an appropriate time.
OD420 was measured. In addition, the protein concentration of the cell extract was
The measurement was performed using a protein assay solution from Rad. The activity value of beta-galactosidase was calculated by the following formula.

[0094]

[Equation 2]

The calculated activity values are shown in Tables 2 and 3.

[Table 2]

[0096]

[Table 3] Reference example. Demonstration of the existence of factors binding to elemental unit sequences 1) Extraction of nuclear protein from yeast Yeast strain AH22 was shake-cultured in 100 ml of YPD medium at 30 ° C until the OD600 value reached 6. After culturing, the bacterial cells were collected by centrifugation, and the bacterial cells were washed with a 1M sorbitol / 50 mM potassium phosphate (pH 7.0) solution. 400 ml of 10 ml of cells
The cell wall was digested by resuspending it in ml of zymolyase, 1 M sorbitol, 50 mM potassium phosphate (pH 7.0), 1 mM phenylmethylsulfonylfluoride (PMSF) solution and incubating at 30 ° C. for 30 minutes. The following operations are all 4
C. or on ice.

The obtained spheroplasts were collected by centrifugation, and added to 20 mM HEPES (pH 7.4), 15 mM NaCl, 5 mM MgCl 2.
2, 0.5 mM dithiothreitol (DTT), 0.1 mM EDTA, 1
Cells were disrupted by resuspension in mM PMSF. The cell disruption solution was centrifuged at 14700 rpm for 15 minutes to obtain a nuclear fraction. 20mM HEPES (pH7.4), 500mM NaC
l, 5 mM MgCl2, 0.5 mM dithiothreitol (DTT),
Resuspending in 0.1 mM EDTA, 1 mM PMSF, 25% glycerin and placing on ice for 1 hour with vortex mixing every 10 minutes.

Again, it was centrifuged at 14700 rpm for 15 minutes to obtain a supernatant. This supernatant was added to 20 mM HEPES (pH 7.4), 50 mM NaCl,
5 mM MgCl2, 0.5 mM dithiothreitol (DTT), 0.1 mM
Nuclear protein was obtained by dialysis of EDTA, 1 mM PMSF and 5% glycerin against the external solution overnight. The protein concentration was measured using a Bio-Rad protein assay solution, and then stored at -80 ° C.

2) Gel mobility shift assay pUAS2 was cleaved with HindIII, extracted with phenol / chloroform, and recovered by ethanol precipitation. This was dissolved in 50 mM Tris-Cl (pH 8.0), 1 unit of E. coli alkaline phosphatase was added, and the temperature was adjusted to 65 ° C.
The 5'end was dephosphorylated by reacting for 1 hour. The DNA was recovered by phenol extraction and ethanol precipitation and digested with BglII. A 159 base pair fragment was isolated and labeled at the 5'end of the HindIII site with γ- ³² P-ATP and T4 polynucleotidyl kinase. There was thus obtained the labeled fragments of 8.8X10 ⁷ / 2μg DNA.

This labeled fragment 2 × 10 ⁴ cpm, 5 μg of nuclear protein and 1 μg of poly (dI)-(dC) were added to 10 μl of a reaction solution (20 mM HEPES (pH 7.4), 50 mM NaCl, 5 mM MgCl 2,
0.5 mM dithiothreitol (DTT), 0.1 mM EDTA, 1 mM
The reaction was carried out for 15 minutes at room temperature in PMSF, 5% glycerin). When competing with DNA, the UAS cassette vector cleaved with HindIII was added to the above reaction solution in 2
μg was added.

After adding 1 μl of 0.25% bromophenol blue, 0.25% xylene cyanol and 50% glycerin solution to this reaction solution, 5 μl of the solution was applied to a 5% polyacrylamide gel and electrophoresed at 0.5 × TBE. After the electrophoresis was completed, the gel was dried and the radiation was detected using X-AR film. The results are shown in Fig. 11.

Due to the binding of the factors, two bands with altered mobility were detected. These bands are unlabeled pUAS2
It disappeared by the addition of, indicating that it was specifically bound to the pUAS2 fragment. Also
Since the bands were thinned by pUAS3, pUAS4, and pUAS2-D4, and there was almost no change except for these, the binding sequence of the factor was present in the region contained in pUAS4 but not in pUAS5. Was expected.

3) DNA fragment utilizing inhibition of methylation bond
Identification of the binding sequence by the imprint method Labeled fragment obtained as described above,
2 × 10 ⁶ cpm of 400 μl of DMS reaction solution (50 mM sodium cacodylate (pH 7), 1 mM EDTA) was added with 2 μl of dimethyl sulfate, and reacted at room temperature for 5 minutes. 50 μl of stop solution (1.
After adding 5M sodium acetate (pH 7.0) and 1M β-mercaptoethanol, it was quickly applied to a column (NAP-5 column; Pharmacia) equilibrated with sterilized water, and the methylated fragment was separated from the solvent. did. The resulting solution was concentrated by freeze drying.

Methylated fragment, 2X10 ⁴ cpm, and nuclear protein
50 μg was reacted under the same conditions as in the gel mobility assay (50 μl reaction solution), and by 5% polyacrylamide electrophoresis, a band with a factor binding and mobility change and a band with no factor binding were separated. did. These bands were detected by exposing the gel to X-AR film without drying. Cut out the band, 1% agarose, 1X TAE (40 mM Tris, 20 mM sodium acetate, 1 mM EDT
A, which was adjusted to pH 8 with acetic acid).

[0105] The DEAE membrane (NA
45, Schreicher & Schuell) and electrophoresed to transfer the DNA contained in the band to the membrane. DNA from membrane 1M NaCl, 10mM Tris-
Elution was performed with HCl (pH 7.5), 1 mM EDTA. Extract with phenol / chloroform and precipitate with ethanol to obtain D
NA was recovered. 1308cpm from band A, from band B
A DNA of 21336 cpm was detected in the band of 3706 cpm and the factor was not bound. (Count was measured by Cherenkov count.)

1308 cpm was added to each of 6% polyacrylamide, 7 urea, 1XTBE (90 mM Tris, 90 mM boric acid, 2.5 mMEDT).
It was applied to the denaturing gel of A) and electrophoresed. The same labeled fragment was used as a marker for Maxam and Gilb.
What was cut by the ert method was electrophoresed. After completion of the electrophoresis, the gel was dried and exposed to X-AR film to detect radioactivity. The results are shown in Fig. 11. It was found that the factor binding was inhibited by methylation of the sequence shown in the figure. In other words, it can be said that the factor is bound to this sequence.

[Sequence Listing] SEQ ID NO: 1 Sequence length: Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Sequence: AAGCTTACCA GTTCTCACAC GGAACACCAC TAATGGACAC AAATTCGAAA TACTTTGACC -999 CTATTTTCGA GGACCTTGTC ACCTTGAGCC CAAGAGAGCC AAGATTTAAA TTTTCCTATG -939 ACTTGATGCA AATTCCCAAA GCTAATAACA TGCAAGACAC GTACGGTCAA GAAGACATAT -879 TTGACCTCTT AACAGGTTCA GACGCGACTG CCTCATCAGT AAGACCCGTT GAAAAGAACT -819 TACCTGAAAA AAACGAATAT ATACTAGCGT TGAATGTTAG CGTCAACAAC AAGAAGTTTA -759 ATGACGCGGA GGCCAAGGCA AAAAGATTCC TTGATTACGT AAGGGAGTTA GAATCATTTT -699 GAATAAAAAA CACGCTTTTT CAGTTCGAGT TTATCATTAT CAATACTGCC ATTTCAAAGA -639 ATACGTAAAT AATTAATAGT AGTGATTTTC CTAACTTTAT TTAGTCAAAA AATTAGCCTT -579 TTAATTCTGC TGTAACCCGT ACATGCCCAA AATAGGGGGC GGGTTACACA GAATATATAA -519 CATCGTAGGT GTCTGGGTGA ACAGTTTATT CCTGGCATCC ACTAAATATA ATGGAGCCCG -459 CTTTTTAATC TGGCATCCAGATCAACACATCAACAAACAAACATAAAGTAA GGCACAAAC -339 AGGCAAAAAA CGGGCACAAC CTCAATGGAG TGATGCAACC TGCCTGGAGT AAATGATGAC -279 ACAAGGCAAT TGACCCACGC ATGTATCTAT CTCATTTTCT TACACCTTCT ATTACCTTCT -219 GCTCTCTCTG ATTTGGAAAA AGCTGAAAAA AAAGGTTGAA ACCAGTTCCC TGAAATTATT -159 CCCCTACTTG ACTAATAAGT ATATAAAGAC GGTAGGTATT GATTGTAATT CTGTAAATCT -99 ATTTCTTAAA CTTCTTAAAT TCTACTTTTA TAGTTAGTCT TTTTTTTAGT TTTAAAACAC -39 CAAGAACTTA GTTTCGAATA AACACACATA AACAAACAAA -1

[Brief description of drawings]

FIG. 1 shows a process of constructing a NeoC expression plasmid containing a deletion mutant promoter of TDH3 gene.

FIG. 2 is a plasmid pRS-LacZ for assaying UAS activity.
The manufacturing process of C is shown.

FIG. 3 is a plasmid pRG-LacZ for assaying UAS activity.
The manufacturing process of C is shown.

FIG. 4 shows a process of preparing an assay vector in which a deletion mutant promoter of TDH3 gene is inserted.

FIG. 5 shows a process of preparing an assay vector in which a deletion mutant promoter of TDH3 gene is inserted.

FIG. 6 shows a process for preparing a plasmid in which UAS of TDH3 gene is partially removed to various extents by exonuclease.

FIG. 7 shows a process for preparing a plasmid in which UAS of TDH3 gene is partially removed to various extents by exonuclease.

FIG. 8 shows the production process of plasmids pRG-AXL-LacZC and pRG-N7L-LacZC for assaying the activity of modified UAS.

FIG. 9 shows a process of preparing an assay vector in which a modified UAS cassette fragment was inserted.

[Fig. 10] Fig. 10 shows a process for producing an assay vector in which a modified UAS cassette fragment is inserted.

FIG. 11 is an electrophoretogram showing that the binding sequence is included in the UAS of the TDH3 gene, and is a photograph as a drawing.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical indication location C12Q 1/68 A 8114-4B (C12N 15/81 C12R 1:19) (72) Inventor Masanori Suzuki 1-3-1 Nishitsurugaoka, Oi-cho, Iruma-gun, Saitama Prefecture Tonen Corporation Research Institute

Claims (7)

[Claims] 1. A modified UAS in which the natural upstream activating sequence (UAS) of the yeast glycerol-3-phosphate dehydrogenase 3 (TDH3) gene is partially deleted. 2. In the nucleotide sequence of UAS shown in Sequence Listing 1, A immediately upstream of the translation initiation codon ATG is set to -1,
-583 to -540 element unit sequence B, -520 to -499 element unit array A, and -481 to -447 element unit array C, (1) a fragment not containing the element unit sequences A and C,
(2) a fragment not containing the element unit sequences B and C, (3)
A fragment not containing the element unit sequence B, (4) a fragment not containing the element unit sequence A, (5) a fragment not containing the element unit sequence C, or (6) a fragment containing the element unit sequences A, B and C, The modified UAS of claim 1, which comprises any of: 3. The modified UA according to claim 1 or 2.
A fusion promoter in which S and a promoter are linked. 4. The promoter has the UAS removed.
The promoter according to claim 3, which is the TDH3 promoter or the ADH1 promoter with UAS removed. 5. A fusion promoter in which a transcriptional activator recognition sequence and a promoter from which UAS has been removed are linked. 6. The promoter according to claim 5, which comprises the elemental unit sequence C. 7. An expression vector comprising the promoter according to any one of claims 3 to 5.