JPH02142482A - Synthetic intron dna, recominant dna containing the same and transformant transformed therewith - Google Patents

Abstract

NEW MATERIAL:A synthetic intron DNA, having a region containing the 5' and 3'-terminal consesus sequences and a region containing a sequence of a branching part, either of at least one set of recurring base sequences in the opposite directions being a sequence of region containing a consesus sequence and/or a region containing the branching part and at least one of the above- mentioned one set of the base sequences in the opposite directions, present in base sequences other than the above-mentioned regions and sandwiching regions containing the sequence of the branching part. USE:A genetic expression controlling agent and reagent for elucidating a splicing mechanism. PREPARATION:For example, a synthetic intron is linked to a plasmid pUC118 and cloned to provided a plasmid pUC-INTRON, which is then cleaved with restriction enzymes SnaBI and PstI to afford DNA fragments. The resultant DNA fragments are then linked to a DNA fragment cleaved with restriction enzymes XbaI and PstI using a T4DNA ligase to provided a synthetic intron DNA.

Description

DETAILED DESCRIPTION OF THE INVENTION [Background of the Invention] Technical Field The present invention relates to synthetic intron DNA, recombinant DNA containing the same, and transformants transformed with this recombinant DNA.

Prior art Currently, the genes used in genetic engineering are mainly (1
) genes consisting of cDNA obtained by reverse transcription from mRNA; (2) genes consisting of genomic DNA isolated from a living body; and (3) genes consisting of chemically synthesized DNA.

Of these, (2) usually contains an intron in eukaryotes. On the other hand, (1) and (3) usually do not contain introns. Also, even if (2),
In lower eukaryotes such as yeast and prokaryotes, genes that do not have introns are common.

A gene with intron sequences removed can be obtained relatively easily by isolating the cDNA of the gene.

In addition, recently, a technology has been established in which only the intron sequence part is synthesized or isolated and inserted into any gene (see Japanese Patent Application No. 62-276944 for details). Therefore, applications in the field of genetic engineering are expected.

However, at present, there have been no reports of cases in which introns themselves have been artificially synthesized and modified to add new functions.

[Summary of the Invention] Summary The present invention aims to further develop the technical possibilities of gene manipulation, and the present invention aims to further develop the technical possibilities of genetic manipulation. Synthetic intron D with
NA, recombinant DNA containing it, and this recombinant I)
The purpose is to achieve the above object by providing a transformant transformed with NA.

That is, the synthetic intron DNA according to the present invention has a 5'
Intron D with a region containing the end and 3' end consensus sequences and a region containing the branching site sequence
NA is characterized in that the intron DNA has at least one set of inverted repeat base sequence regions defined by (1) and (2) below.

(1) One of the at least one set of inverted repeat base sequences is a sequence of a region including a consensus sequence and/or a region including a branch forming site sequence; (2) the at least one set of inverted repeats. The base sequence is present in a base sequence region other than the region containing the consensus sequence and the region containing the branch forming site sequence, and at least one of the set of reverse base sequences is present in the region containing the branch forming site sequence. To exist in between.

Furthermore, the recombinant DNA according to the present invention is characterized in that this synthetic intron DNA or any gene containing this DNA is inserted or ligated into an expression vector.

Furthermore, the transformant according to the present invention is characterized in that it has been transformed with a recombinant DNA in which this synthetic intron DNA or any gene containing this DNA is inserted or ligated into an expression vector.

Effects By using the synthetic intron DNA according to the present invention, a recombinant DNA containing the same, and a transformant transformed with this recombinant DNA, synthetic intron DNA can be obtained.
The transcript RNA of any gene desired to be expressed, including
Depending on the temperature conditions, that is, the lower the temperature, the more intramolecular hybridization occurs between the sites corresponding to the base sequence sites in the synthetic intron DNA that are in the relationship of mutually inverted repeat base sequences, and the splicing reaction is inhibited. Therefore, by appropriately adjusting the length of this hybridizing region, etc., gene expression can be regulated at the culture temperature of the host.

By utilizing this fact, the following effects can be expected.

(1) It is possible to control the expression of any gene whose base sequence can be expressed constitutively. Therefore, even when a foreign gene is expressed using a strong but unregulatable promoter (for example, the yeast PGK promoter shown in the experimental example below), the expression of the foreign gene can be regulated. , the expression of the gene can be induced at the most appropriate time.

(2) Expression of a gene linked to a regulatable promoter can be regulated using the present invention as a second regulation mechanism in addition to regulation by the promoter. Therefore, when regulation of the promoter alone is incomplete, expression of the gene can be more completely controlled.

(3) Temperature-sensitive mutant strains of isolated unknown or known genes can be easily created. That is, since the expression of any gene that can be expressed into which the synthetic intron DNA of the present invention has been inserted can be controlled by temperature, it is easy to convert the gene to be temperature sensitive. Therefore, it can be used for genetic analysis to elucidate gene function.

(4) In any of the above (1), (2), and (3), subtle regulation of gene expression is possible. That is, by controlling the culture temperature of the host, the expression level can be subtly changed. Thereby, it is easy to adjust the expression level of the foreign gene to the most convenient level for the host, and it is also possible to examine the effects on the host due to subtle differences in the expression level.

(5) It can be used to elucidate the mechanism of splicing.

For example, by using a temperature-sensitive strain of the gene described in (3) and isolating a complementary mutant strain thereof, it is possible to identify genes involved in splicing.

[Detailed description of the invention]

Synthetic intron DNA An intron in the present invention is a special base sequence site that exists in a gene and is not involved in translation, and is transcribed by RNA polymerase, but is removed from the transcript RNA by a reaction called post-transcriptional splicing. A DNA sequence that encodes a sequence. There are various types of these introns, including those that exist in DNA that encodes tRNA, those that exist in DNA that encodes rRNA, and those that exist in DNA that encodes polypeptides (Ann, LEV, Biochem,
4L1035-69 (1979), Ann, Re
v, Biochem, 50, 349-83 <
1981), Ann.

Rev. Biochem, 52.441-88 (1
983), ^nn, Rev, Bioch-etn, ,
55, 597-1329 (198B), An
n, Rev, Biochem, , 55°1119-
1.150 (198B), Ann, Rev, Genet
, 20.671-708 (1986)).

By the way, introns of genes encoding polypeptides have at least three special base sequences, namely (1
) 5' end consensus sequence, (2) branch forming site sequence, and (3) 3' end consensus sequence, and these sequences are said to be necessary for splicing. Therefore, when using an intron derived from a gene encoding the above polypeptide, this intron must contain at least these sequences.

The synthetic intron DNA according to the present invention is an intron DNA having a region containing the 5' end and 3' end consensus sequences as described above and a region containing the branch forming site sequence, and the above intron DNA has the following (1
) and (or) (2), and is characterized by having at least one set of inverted repeat base sequence regions defined by (2), and the temperature at which the expression of a desired gene can be made temperature dependent. As mentioned above, the DNA has a function as a sensitive intron.

(1) One of the at least one set of inverted repeat base sequences is a sequence of a region including a consensus sequence and/or a region including a branch forming site sequence; (2) the at least one set of inverted repeats. The nucleotide sequence is present in a nucleotide sequence region other than the region containing the consensus sequence and the region containing the branch formation site sequence (hereinafter referred to as an arbitrary nucleotide sequence region), and at least one of the set of inverted repeat nucleotide sequences be present between the region containing the sequence of the branch formation site.

Therefore, the synthetic intron DNA according to the present invention includes those defined by (1) above, those defined by (2) above, and those defined by both (1) and (2). Become.

The synthetic intron DNA defined in (1) above is 5'
In a base sequence region other than both the region containing the end or 3' end consensus sequence and the region containing the branch formation site sequence (i.e., arbitrary base sequence region), an inverted repeat base sequence is added to at least one of the regions. It is made up of areas that have the following relationship.

This synthetic intron DNA preferably has a region consisting of an inverted repeat base sequence or a branch forming site sequence in an arbitrary base sequence region on the 3' end of the region containing the 5' consensus sequence. It has a region consisting of an inverted repeat base sequence with respect to the region in an arbitrary base sequence region on the 5' end side of the region.

Being in a relationship of inverted repeat base sequences means that one base sequence has a base sequence that is completely or almost complementary to the other base sequence but is in the opposite direction. Here, "complementary" means that the bases at a small number of positions, for example, one or two, are not complementary to each other, but the base sequences at all other positions are complementary. do.

By having these portions in the relationship of mutually inverted repeat base sequences, the transcription product RNA can be kept at low temperatures, for example (30
)'C, these parts hybridize with each other, masking the consensus sequence in the intron or the sequence part of the branch formation site, and inhibiting the splicing reaction.

The synthetic intron DNA defined in (2) above is the fourth
As shown in the figure and FIG. 5, in at least one set of reverse base sequences, the base sequence region (R and R'1) of the set
The name sequence region is the region containing the sequence of the branch formation site (B
) are present in arbitrary base sequence regions on each side of the region (see each figure (a)), but as shown in Figure 5, other pairs of inverted repeat base sequences (R2 and R2 '), this pair (R2 and R2') may exist only in an arbitrary base sequence region on one side of region (B) without sandwiching it.

When the RNA portion of the transcript produced by this intron DNA portion is bent due to hybridization and exhibits a double structure, the reverse base sequence R1 to R R′ and R″′ corresponding to the ′ site hybridize with each other to form a loop structure, and as a result, the region B′ portion corresponding to region B containing the sequence of the branch forming site is confined within this loop structure. As a result, the region B' portion becomes in a masked state.

In the case of Fig. 5, another set of inverted repeat base sequences (R
and R') are also hybridized with each other, and the masking state of the region B' becomes stronger.

In addition, C and C2 in figure (a) are the 5' end or ■ indicates the region containing the 3' end consensus sequence.

Furthermore, the synthetic intron DNA according to the present invention is described in (1) above.
As mentioned above, it also includes what is defined by both the requirements (1) and (2), but in this case, when the RNA portion of the transcript exhibits a folded structure, the consensus sequence and/or branch The region corresponding to the region containing the formation site (C', C2' or B') also becomes hybridized.

Specific examples of these synthetic intron DNAs include the following formula (I
) and (n).

[In the formula, N represents one or more arbitrary base sequences selected from ASC, G and T,
Any combination is possible. A plurality of N or N' bases in each formula may be the same or different. N' may be any sequence complementary to N.

In addition, the number of bases of N or N' is preferably 0 to 1000,
0 to 100 is more preferable. ] As shown in these two formulas, a set of base sequences that are mutually inverted repeat base sequences, in this case, the sequence of each region including the above sequence of an intron and the reverse direction to the region It may be directly bonded to a region consisting of a repetitive base sequence, or may be bonded via an arbitrary base sequence.

Furthermore, the region containing the above-mentioned sequence of an intron as used in the present invention includes each sequence, that is, a consensus sequence or a branch forming site sequence, which is composed of itself.

Preparation of synthetic intron DNA Base sequences that are mutually inverted repeat base sequences, that is, the 5' end or 3' end of the synthetic intron DNA, or the 5' end of the region containing the consensus sequence or the branch forming site sequence. a region consisting of an inverted repeat base sequence with respect to the region, located on the side or 3'end;
Alternatively, if the length of the base sequences that are mutually inverted repeat base sequences in the arbitrary base sequence region is made sufficiently long, the splicing reaction will be inhibited at any temperature at which the host for transformation can grow. , and conversely, when shortened, the correct splicing reaction function is maintained at any temperature at which the host can grow.

Therefore, by appropriately adjusting the length of this inverted repeat base sequence, the degree of complementarity of the inverted repeat base sequences, or the spacing between both base sequences that are in the relationship of mutually inverted repeat base sequences, it is possible to A tunable temperature-sensitive intron, ie, a synthetic intronic DNA according to the present invention, can be created at which temperature the splicing reaction can occur. Such a synthetic intron DN
In A, the preparation of both base sequences or other parts of the base sequences that are mutually inverted repeat base sequences is D.
Any method can be used, such as chemical synthesis using an NA synthesizer or use of the necessary intron portion of a known gene.

Preferred specific examples of synthetic intron DNA can be prepared into sequences as shown in formulas (I) and (II) above.

Temperature-sensitive synthetic intron DN thus prepared
A can be prepared in large quantities by cloning it into an appropriate cloning vector, such as an E. coli plasmid. Furthermore, if this synthetic intron DNA is cloned in such a way that it cannot be excised using an appropriate restriction enzyme, it can be excised when necessary. In this case, it is preferable to clone in such a way that it can be excised with a restriction enzyme that produces blunt ends. A specific example of this is to set the base sequence of the synthetic intron DNA to have restriction enzyme recognition sites of 5naBI and PvuI, as shown in the experimental examples below.

Recombinant DNA containing synthetic intron DNA The recombinant DNA containing synthetic intron DNA according to the present invention is obtained by inserting or ligating the above synthetic intron DNA or any gene containing the DNA into an expression vector. The above expression vector is a synthetic intron DNA or
It refers to a carrier DNA that plays the role of transferring any gene containing A into a host cell for transformation.

Therefore, the expression vector may be one that grows autonomously within the host cell, one that is integrated into the host chromosome, or one that replaces a part of the host chromosome, and , it may be circular or linear that behaves like a host chromosome. Such vectors include plasmids, phages, cosmids, etc., with plasmids being more preferred. Also,
These vectors preferably have a gene that serves as a marker indicating that any gene into which the synthetic intron DNA has been inserted has been transferred into the host by the vector.

Specific examples of these include 1) multicopy plasmid YEp13.11, which has the yeast LEU2 gene as a marker and grows autonomously in yeast, as shown in the Examples below;
) A circular CEN-type plasmid pFN that has the yeast URA3 gene as a marker and behaves like a chromosome in yeast.
There are 8 etc.

Any gene into which synthetic intron DNA is inserted and transferred into the host cell is a gene that is desired to be expressed in the host cell, and may be derived from the host cell or may be a foreign gene, and may be a gene that is desired to be expressed in the host cell, and may be derived from the host cell or may be a foreign gene. RNA, such as tRNA, rRNA,
It may also be coded. A specific example of this is the yeast URA3 gene shown in the experimental example below.

Details regarding the insertion of an intron into a gene are described in the specification of Japanese Patent Application No. 62-276944, and the outline thereof will be explained below in relation to the present invention.

introns (i.e. synthetic intronic DNs according to the invention)
If the end of A) is blunt, the gene can be cut to create a blunt end with just the right number of bases, and the synthetic intron DNA can be inserted there in the correct direction (the correct direction means post-transcriptional splicing). refers to the direction in which the synthetic intron DNA is removed from the transcript by a reaction called Although various methods are possible for this, for example, synthetic intron DNA can be inserted into a gene as follows.

1) Cut with a restriction enzyme that produces blunt ends, and insert synthetic intron DNA therein in the correct orientation.

2) After cutting with a restriction enzyme that produces a sticky end, one end is made blunt with DNA polymerase, and the other end is made blunt with single-strand specific DNAase. Insert the synthetic intron DNA there in the correct direction.

If it is difficult to cut the gene so as to produce a blunt end with no excess or deficiency of bases, insert the necessary base pairs at one or both ends of the synthetic intron DNA so that the insertion does not result in excess or deficiency of bases in the gene. After adding or removing extra base pairs from the ends, the synthetic intron DNA must be inserted in the correct orientation (
Please refer to the above specification).

The insertion position of synthetic intron DNA into any gene is
It may be located anywhere within the transcribed region, and specific examples include a polypeptide-encoding region (exon portion) included in the transcription product of a gene, or the above-mentioned RNA-encoding region. Preferably, it is a region encoding the above polypeptide, more preferably,
This is near the 5' end.

Transformants A transformant according to the present invention can be obtained by transfecting into an appropriate host cell the above-mentioned recombinant DNA into which a synthetic intron DNA or any gene containing the DNA has been inserted or ligated into an expression vector. When the expression vector used has a marker gene, for example, a gene related to auxotrophy, drug resistance, etc., transformants can be easily identified using this marker gene as an indicator.

The host serving as a transformant may be any microorganism or cultured cell (plant or animal cell, etc.) as long as it has the following properties.

1) Having a mechanism for splicing synthetic intron DNA.

ii) Ability to grow at various temperatures to regulate splicing of synthetic intronic DNA.

Preferably, it is a yeast strain, and a specific example thereof is Saccaromyces cerevisiae (Sacc), which is shown in the experimental example below.
haromyces cerevis Jae).

[Experiment example]

Preparation of temperature-sensitive synthetic intron DNA (1) Preparation of synthetic intron DNA having base sequences that are mutually inverted repeat base sequences (a) Plasmid pUC
-lNTR0N (Synthesized introns A and B were each ligated to plasmid pUc118 and cloned, then restriction enzyme DNA fragments containing each intron were purified from the two plasmids, and both fragments were ligated and cloned. The nucleotide sequence of the intron portion of this clone is shown in Figure 3.
276944) (50 μg) was treated with restriction enzyme 5
After cutting with naBI and PstI, polyacrylamide gel electrophoresis revealed that the approximately 33 base pair intron 5
A fragment containing the 'end consensus sequence was purified. this,
Plasmid pUC-1NTR0N (3 μg) was cut with restriction enzyme Xbal, blunt-ended with Escherichia coli DNA polymerase Klenow fragment, and then ligated with the restriction enzyme PstI using T4 DNA ligase.

(b) Plasmid ptrc-lNTR0N (5
0 μg) was digested with restriction enzymes RvuII and Sac I, and then subjected to polyacrylamide gel electrophoresis.
A fragment containing the base-paired intron branching site and the 3' consensus sequence was purified. This was combined with plasmid pUC-lNTR0N (3 μg) using restriction enzyme Xb.
After cutting with Al, the ends were made blunt with Escherichia coli DNA polymerase Klenow fragment, which was further cut with restriction enzyme 5acl and ligated with T4 DNA ligase.

Using these DNAs, Escherichia coli E was produced according to a conventional method.

Clones were obtained by transforming E. coli H8101 strain and selecting ampicillin-resistant strains.
INT・A%pUC-INT・B).

After cutting the plasmid pUC-INT-A (10, czg) with the restriction enzyme Pstl, the DNA was excised to various lengths using Ba131 nuclease. Further D
Make blunt ends with NA polymerase Klenow fragment and
aI linker is ligated using T4 DNA ligase,
DNA fragments of various lengths obtained by digestion with restriction enzymes Xbal and Sca I were purified by agarose electrophoresis.

This was ligated to ptrc-INT-A (2 μg) cut with restriction enzymes Xbal and Sca I using DNA ligase.

After cutting plasmid pUC-INT-B (10 μg) with restriction enzyme Sac I, the DNA was excised to various lengths using BAL-31 nuclease. Furthermore, the ends were made blunt with DNA polymerase Klenow fragment,
Xbal linkers were ligated using T4 DNA ligase, and DNA fragments of various lengths obtained by cutting with restriction enzymes Xbal and 5caI were purified by agarose electrophoresis. This was combined with pUC-INT-B (2 μg) cut with restriction enzymes Xbal and 5caI and DNA
It was ligated using A ligase.

Using these DNAs, Escherichia coli E was produced according to conventional methods.

By transforming E. coli H8101 strain and selecting ampicillin-resistant strains, introns with inverted repeat base sequences of various lengths for the 5'-end consensus sequence or the region containing the branch-forming site consensus sequence were created. A clone was obtained (pUC-INT-
A' 1-40, pUC-INT-B' 1-40
).

(2) Screening of temperature-sensitive synthetic intron DNA Among introns with inverted repeat base sequences of various lengths for the region containing the above-mentioned 5'-end consensus sequence or branch formation site consensus sequence, the following introns are temperature-sensitive. It was screened as follows.

Plasmid pUC-PGKp (3 μg) containing the promoter region of the yeast phosphoglycerate kinase gene
) was cut with the restriction enzyme Sph-D, made blunt-ended with T4 DNA polymerase, and further digested with the restriction enzyme Hi, n d
After cutting with m, agarose electrophoresis revealed approximately 16 cells containing the promoter region of the phosphoglycerate kinase gene.
A 00 base pair fragment was purified. This is plasmid pUC
-I NT-A' 1-40 or plasmid p
UC-INT-B'1 to 40 (1 μg each) were digested with restriction enzymes 5naBI and Hind]II and ligated using T4 DNA ligase. These DNA
E. coli using conventional methods.

The desired clone was obtained by transforming E.coli H8101 strain and selecting an ampicillin-resistant strain (
pUC-PGKp-INT-A' 1-40, pUC
-PGKp-INT-B' 1-40) The above plasmid pUC-PGKp-INT-A' 1-40, p
UC-PGKp-INT-B'1-40 (3 μg each
) was cut with the restriction enzyme PvuII, a BglU linker was ligated with T4 DNA ligase, and then the restriction enzyme H
After digestion with indIII and BglII, a DNA fragment of about 1700 base pairs was purified by agarose electrophoresis. This was transformed into plasmid pFN8 (F, Naga
wa,et,al,,Proc,Na-tl,Acad
, Sc1. USA, 828557-8561 (1985
)) was digested with the restriction enzymes Hindm and BamHI and ligated using T4 DNA ligase. Using these DNAs, extract Escherichia coli E, coliHBIO according to the conventional method.
The target clone was obtained by transforming the I strain and selecting an ampicillin-resistant strain (pFN-PGKp-
INT-A' 1-40, pFN-PGKpiNT-
B' 1-40).

The above plasmid pFN-PGKp-TNT-A' 1~
40 and pFN-PGKp-INT-B'1-40
yeast Saccharom-yces according to a conventional method using
ccrevlsjac NY6 to eight stocks (MATa
ura3-52.1eu2-3.1eu2-1
12, his4-519) to obtain a URA+ transformant.

These transformants were cultured at 16°C or 36°C,
The activity of β-galactosidase was examined, and clones with decreased activity when cultured at 16°C compared to when cultured at 36°C were obtained (pFN-PGKp-INT-A' co-, pFN-PGKp-INT-B '1).

When the mRNA synthesized from the β-galactosidase gene of these transformants was analyzed by the Brimer extension method, a band with the same length as the wild type detected when cultured at 36°C was detected when cultured at 16°C. No cases were detected. This clearly shows that splicing of this intron is temperature sensitive.

The intron base sequences of these clones are as shown in FIGS. 1 and 2.

In addition, the symbols 1 =----) at two positions in each figure
indicates that the base sequences within both ranges are in an inverted repeat sequence relationship.

Gene expression regulation using temperature-sensitive synthetic intron DNA Temperature plasmid pUC-INT-A'1 or pUC
-INT-B' IC (50 μg each) was added with restriction enzyme 5
After digestion with naBI and PvuU, a DNA Ir piece containing an intron of about 123 base pairs or about 104 base pairs was purified by acrylamide gel electrophoresis.

This is converted into plasmid Y I p5 [1,) 5tr
u1. ,etat proc,NaL,Acad,S
c1. USA, 76.1035-+039 (1979
), 2) Dotstein, D. eL al., Gen.
e, 8.17-24 (+979), 3) Rose, e
tal, Gene, 29, 113-124 (198
4)) (2 μg) was digested with restriction enzyme 5tuI and ligated with T4 DNA ligase.

Using these DNAs, Escherichia coli E, col
i By transforming the H8101 strain and selecting ampicillin-resistant strains, the intron was transformed into yeast S, cerev
A clone inserted into the URA3 gene of Y. isiae in the correct orientation was obtained (Ylp-INT-A'
1, Ylp-INT-B' 1).

The above plasmid YIp-INT-A' 1 or
After cutting YIp-INT-B' 1 (3 μg each) with restriction enzymes sph I and Pvu II, a DNA fragment of about 2500 base pairs containing the URA3 gene into which an intron sequence had been inserted was purified by agarose electrophoresis.

This was transformed into plasmid Y E p 13 (Broach
j,R,,Methods in Enzymolo-
gy, lot, 307 (1983)) (2 μg
) was digested with restriction enzymes sph I and Pvu II and ligated using T4 DNA ligase.

Using these DNAs, Escherichia coli E was produced according to conventional methods.

The desired clone was obtained by transforming the colt H8101 strain and selecting an ampicillin-resistant strain (Y
Ep-INT-A' 1 and YEp-INT-B'
1).

The above plasmids YEp-INT-A' 1 and YE
Using p-INT-B', yeast S, ee
revisiae NYBA strain (MataSura3-
52, Ieu2-3, 1eu2-112, his4-
519) to obtain a LEU+ transformant.

These transformants were cultured at 36°C and 23°C and uracil auxotrophy was examined. Three days after culture, it was URA when cultured at 36°C, but URA when cultured at 23°C.

From these results, plasmid pUC-INT-A'1
Alternatively, it is clear that by inserting an intron derived from pUC-INT-B'1 into any gene, the expression of that gene can be regulated by temperature.

[Brief Description of the Drawings] Figure 1 shows the base sequence of the intron region of plasmid pUC-INT-A'1, and Figure 2 shows the base sequence of the intron region of plasmid pUC-INT-A'1.
Figure 3 shows the base sequence of the intron region of plasmid pUC-INT-B'1.
This shows the base sequence of the intron region of UC-1NTR0N. FIG. 4 and FIG. 5 are explanatory diagrams showing the positional relationship of inverted repeat base sequences in intron DNA according to the present invention and the secondary structure during hybridization of transcript RNA, respectively.

Claims (1)

[Scope of Claims] In an intronic DNA having a region containing consensus sequences at the 1, 5' and 3' ends, and a region containing a branch formation site sequence, the above intronic DNA contains the following (1) and/or (2). ) A synthetic intron DNA characterized by having at least one set of inverted repeat base sequence regions defined by: (1) One of the at least one set of inverted repeat base sequences is a sequence of a region containing a consensus sequence and/or a region containing a branch forming site sequence; (2) the at least one set of inverted repeat base sequences; The base sequence is present in a base sequence region other than the region containing the consensus sequence and the region containing the branch forming site sequence, and at least one of the set of reverse base sequences is present in the region containing the branch forming site sequence. To exist in between. 2. The synthetic intron DNA according to claim 1 or the DNA
A recombinant DNA obtained by inserting or ligating any gene containing A into an expression vector. 3. The synthetic intron DNA according to claim 1 or the DNA
A transformant transformed with a recombinant DNA in which any gene containing A is inserted or ligated into an expression vector.