JP3220845B2 - Chromosomal DNA synthesis method and chromosome production method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for synthesizing chromosomal DNA and a method for producing chromosome, and more specifically, a method in which a certain type of deoxyribonucleotide polymerase (hereinafter, simply referred to as "DNA polymerase") reads the genetic information of a template. "Creative" rather than "replication", that is, synthesizing DNA as new genetic information, and a part of the DNA thus synthesized is a centromere region, a telomere region, and a replication initiation region essential for chromosomal DNA. The present invention relates to a method for synthesizing a chromosomal DNA and a method for producing a chromosome, comprising:

[0002]

2. Description of the Related Art The chromosome of an organism is composed of double-stranded linear DNA (chromosomal DNA) and proteins such as histones.

[0003] The double-stranded DNA has three essential regions for functioning as chromosomes. The three regions are the centromere region (participating in the proper distribution of the replicated chromosomes during mitosis and meiosis),
Telomere region (involved in chromosome stabilization and replication) and replication initiation region (ARS) (having the origin of replication,
Involvement in the initiation of NA replication), and chromosomal DNA becomes unstable and disappears during cell division, regardless of which region is missing.

[0004] Since the total length of the nucleotide sequences of the above-mentioned regions (1) and (2) exceeds 1,000 base pairs, it is extremely difficult to synthesize them from scratch.

[0005] Therefore, heretofore, the following art for producing an artificial chromosome has been proposed. That is, the biological DN
A technique has been proposed in which A is cut out by a technique of a genetic recombination technique to extract only a necessary part, and this part is joined to another DNA to produce an independent artificial chromosome.

[0006] As described above, if the above three regions, which are essential as chromosomal DNA, are joined one by one,
It would be possible to produce artificial chromosomes. However, the work up to that is extremely complicated,
Also, the productivity was poor.

[Object of the Invention] The present invention has been made in view of the above-mentioned circumstances, and has as its object to provide a connection comprising a centromere region, a telomere region, and a replication initiation region, which are necessary elements for a chromosome. Double-stranded linear DNA
(Chromosomal DNA) is easily synthesized, and a chromosome is produced using the synthesized chromosomal DNA.

[0008]

A chromosome D according to claim 1
In order to prevent a gene such as a drug resistance gene to be introduced into the nucleus from disappearing from the nucleus in the process of meiosis, the method of synthesizing NA includes, at the end of the gene, a repetitive base sequence that is a centromere region, a telomere region. , A linked double-stranded linear DN having a guanine-rich repeating base sequence such as [TTGGGG]
A method for synthesizing chromosomal DNA for additionally synthesizing A by additionally synthesizing A, wherein the chromosomal DNA of a bacterium belonging to the genus Sermococcus, Sermas and Pyrococcus in a reaction system in which no template and / or primer is present. A repeating base sequence that is a centromere region by polymerizing deoxyribonucleotides in the presence of at least one thermostable deoxyribonucleotide polymerase selected from the group consisting of deoxyribonucleotide polymerases,
It is characterized by synthesizing a linked double-stranded linear DNA having a guanine-rich repeated base sequence such as a telomeric region [TTGGG] and the replication initiation region (ARS).

The method for synthesizing chromosomal DNA according to the second aspect of the present invention provides a method for synthesizing a gene such as a drug resistance gene to be introduced into the nucleus, so that the gene does not disappear from the nucleus during meiosis. Region, a telomeric region such as [TTGGGG], etc., and a replication starting region (AR
Chromosomal DNA for additionally synthesizing a linked double-stranded linear DNA having
A method for synthesizing a cell, comprising inserting a DNA encoding at least one thermostable deoxyribonucleotide polymerase selected from the group consisting of bacterial deoxyribonucleotide polymerases belonging to the genus Sermococcus, Sermus and Pyrococcus into a cell. To produce the thermostable deoxyribonucleotide polymerase in the cells, and polymerize deoxyribonucleotides in the absence of a template and / or primer in the presence of the thermostable deoxyribonucleotide polymerase to repeat the centromere region. A nucleotide sequence, a guanine-rich repetitive nucleotide sequence such as a telomeric region [TTTGGG], and a replication initiation region (AR
A method for synthesizing chromosomal DNA, comprising synthesizing a continuous double-stranded linear DNA having S).

[0010] According to a third aspect of the present invention, there is provided a method for synthesizing chromosomal DNA according to the first or second aspect, wherein the repetitive base sequence of the centromere region is [CTAGA].
TAT].

According to a fourth aspect of the present invention, there is provided a method for producing a chromosome, wherein the chromosomal DNA obtained by the synthesis method according to any one of the first to third aspects is used when the chromosomal DNA is synthesized extracellularly. Is a production method characterized by introducing into a cell and, when the chromosomal DNA is synthesized in the cell, converting the chromosome into a chromosome in the cell as it is.

[0012]

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION The present inventor paid attention to a DNA (gene) synthesis method using a protein in order to solve the above-mentioned problems.

Certain proteins, especially DNA polymerases, have long been known to synthesize DNA replicatively. Replicative means that, using an existing single-stranded DNA as a template, a DNA having a base sequence complementary thereto is synthesized,
Therefore, it is finally to be double-stranded. Therefore,
In this reaction, DNA as new genetic information cannot be created. This is because the genetic information is already contained in the single-stranded DNA as a template.

[0015] In view of this, as a result we have fully examined the type and reaction conditions of the DNA polymerase, certain DNA polymerase, for example Anselmo litoralis (The
It has been found that an extremely long (about 100 to 50,000 base pairs) double-stranded linear DNA can be produced without using a template at all for a DNA polymerase of R. ococcus litoralis .

Moreover, surprisingly, the DNA synthesized by this reaction has a repetitive sequence of 8 bases or 12 bases in its base sequence, which is recognized as the above three regions necessary to function as chromosomal DNA. Alternatively, a sequence having no repeat is observed, and further, when this chromosomal DNA is introduced into a cell, the chromosomal DNA is a protein (such as another histone) naturally produced in the cell. With two essential elements).

Therefore, DNA is synthesized extracellularly or intracellularly using the DNA polymerase,
Using the synthesized DNA, the chromosome D
NA can be made into a chromosome.

That is, a double-stranded linear DNA having three essential elements, a centromere region, a telomere region, and a replication initiation region on the same molecule, is synthesized in advance outside the cell (in a test tube or the like) by a DNA polymerase, and then By introducing this DNA into the cell, the connected DNA (chromosomal DNA) binds to proteins such as histones in the cell and functions as one chromosome. Then, once functioning as a chromosome, it is replicated only once in synchronization with each cell division and transmitted to each daughter cell one by one. Alternatively, using an expression vector, a DNA polymerase is produced in a cell,
Then, the DNA polymerase further increases the chromosome DN.
The chromosome can be made after A is made.

Those that function as chromosomal DNA are proteins, for example, some molecules in the novel DNA molecule population synthesized by DNA polymerase, and the synthesized novel DNA molecule population includes chromosomal DNA. Some molecules do not function as Such nonfunctional DNA
When is introduced into cells, it disappears (does not remain) in the process of cell division. In other words, if the synthesized novel DNA molecule population is introduced into cells and the cells are cultured, and there is DNA remaining after a certain number of cell divisions, the DNA is required as chromosomal DNA. It can be said that it has sufficient elements and can function sufficiently.

It is preferable that the DNA polymerase does not inactivate even at a temperature of about 20 ° C. or more, preferably about 60 ° C. or more, and more preferably about 70 ° C. or more. For example,
Sermococcus litoralis listed above ( Thermoc
occus litoralis ) and other species of the genus Sermococcus, such as Cellus aquaticus ( Thermus)
aquaticus ), the same as Sermophilus ( T. th .
and DNA polymerases of bacteria belonging to the genus Cellmas, such as E. thermophilus .

One of the above DNA polymerases may be used alone, or two or more (two, three,...) May be used in combination as a mixed system. When deoxyribonucleotides are polymerized by using two or more DNA polymerases in combination, the DNA synthesized at one time is more diversified (that is, a DNA (group) having a more varied base sequence than the case where one type is polymerized alone. ) Is obtained), chromosome DN
The content of DNA that can be A increases.

As deoxyribonucleotides, dAT
P, dTTP, dGTP, and dCTP are used, and the reaction proceeds if four or three of them are present in the reaction system.

The reaction between the deoxyribonucleotide and the DNA polymerase is preferably carried out at a pH of about 7 or a weak alkaline pH of 10 or less. Reaction temperature and time are D
It can be selected as long as NA polymerase is not inactivated,
By allowing the reaction to proceed under a temperature condition in which the polymerase activity is exhibited at about 20 ° C. or higher, the reaction can proceed promptly. For example, the reaction may be carried out at 74 ° C. for several hours, and may be carried out under ordinary PCR reaction conditions, for example, (1)
Hold at 95 ° C for 1 minute, (2) Hold at 45 ° C for 2 minutes,
(3) The cycle of holding at 74 ° C. for 3 minutes may be repeated. The reaction starts after a short delay in the beginning,
Eventually the maximum speed is reached. It is considered that chromosomal DNA synthesized by the method of the present invention is synthesized not depending on DNA or RNA to be used as a template or primer, but on information of DNA polymerase present in the reaction system.

As described above, the synthesized DNA is introduced into cells to form chromosomes. Specific examples of cells that can be used here include human HeLa cells and human KB.
Cells, human CAPAN-1 cells, mouse L1210 cells, mouse 3T3 cells, monkey COS7 cells, human PAN
C-1 cells and the like. In addition, in this reaction, if a desired gene such as a drug resistance gene is put into the reaction system as a double-stranded linear form, new DNA is additionally synthesized at both ends of the DNA, and the desired gene is included. One independent chromosome can be created. That is, the gene of your choice can be “chromosomized”.

The method for producing the thermostable DNA polymerase used in the present invention or the method for purifying the gene encoding the same are known today and can be easily carried out by those skilled in the art. The related publications are listed below. JP-A-2-60585, JP-B-8-24570
(JP-A-2-434), JP-A-5-68747, JP-B-7-59195 (JP-A-5-130871),
JP-A-5-328969, JP-A-7-51061,
JP-A-6-339373, JP-A-6-7160

[0026]

The present invention will be described below with reference to examples.
The present invention is not limited by this.

Example 1 Synthesis of Chromosomal DNA 10 mM KCl, 10 mM (NH ₄ ) ₂ SO ₄ , 2
0 mM Tris-HCl (pH 8.8), 6 mM MgC
l ₂ , 0.1% octoxynol (Triton X
-100, manufactured by Sigma), deoxyribonucleotide triphosphate [0.2 mM dATP, 0.2 mM dTTP,
0.2 mM dGTP, 0.2 mM dCTP], 20
Unit / ml Sermococcus litoralis ( Therm
ococcus litoralis) DNA polymerase (trade name: Vent DNA polymerase, consisting of New England Biolabs, Inc.) reaction solution 1ml
Was reacted at 74 ° C. for 3 hours.

Thereafter, a portion (20 μl) of the reaction solution was taken, and 1% agarose gel (Samrock et al., Molecular Cloning (A Laboratory Manual), Cold Spring Harbor Laboratory Press,
1989), stained with 0.5 μg / ml ethidium bromide, and photographed using an ultraviolet lamp. As a result, it was confirmed that DNA having a size of 100 to 50,000 base pairs was synthesized.

For the remaining 980 μl, phenol treatment, chloroform washing, ethanol precipitation, cesium chloride equilibrium density centrifugation (Molecular Genetics Experiment, written by Haruo Koseki et al.
The DNA was purified by Kyoritsu Shuppan Co., Ltd., 1983). As a result, about 20 μg of DNA was obtained.

Nucleotide Sequence of Chromosomal DNA Half of the obtained DNA was used for DNA base sequence determination (molecular genetics experiment). A part of the base sequence revealed by this is shown in Formulation Tables 1 to 3. The nucleotide sequence of DNA was diverse, and there were repetitive sequences or some non-repeated sequences. Specifically, it was found that the obtained DNA had a multiply repeated portion of an 8-base pair sequence [CTAGATAT] (see Table 1). It is considered that the repetition part performs the function of the centromere. Also, [TTG
GGG]. This part is thought to perform the function of telomere. In addition, [TAGATATC
It was found to have a repeating sequence such as [TATC] and [GGAAT].

Introduction of Chromosomal DNA into Cells The remaining half of the obtained DNA is introduced into cells (human HeLa cells) by a calcium phosphate coprecipitation method (after the introduction, the DNA binds to protein histones). This was subcultured. Therefore, at the time of division about 10 times, DNA was extracted from the cells, and (CTAGA
TAT) ₁₅ was used as a probe to search for DNA by Southern blotting (molecular genetics experiment). As a result, it was found that the DNA remained in the cells.

It is apparent from the above that the obtained DNA contains chromosomal DNA functioning as a chromosome (if the synthesized DNA has a centromere region, telomere region, or replication initiation region). If it is not provided, it will no longer be able to function as chromosomal DNA, and there should be no DNA remaining after cell division, and no search will be performed when a search is performed using (CTAGATAT) ₁₅ as a probe. It should be). In addition, He
The existing DNA in the La cell (that is, the chromosomal DNA of the HeLa cell) and the DNA introduced in this example are:
Since the sizes are different, it can be distinguished, and confusion does not occur.

Next, mitotic chromosomes could be observed by in situ hybridization using a microscope with sufficiently high magnification using a fluorescently labeled probe as a mark.

Example 2 Thermococcus litoralis used in Example 1 described above was used.
s ) Instead of DNA polymerase, Thermus aquaticus DN
Even when A polymerase was used, the same results as in Example 1 were obtained.

[0035]Example 3 Sermococcus lit used in Example 1 above
Laris (Thermococcus litorari
s) Instead of DNA polymerase, Sermas Sermo
Filth (Thermus thermophilu
s) Even when DNA polymerase was used,
Similar results were obtained.

Example 4 In this example, chromosomal DNA was synthesized in cells,
The method for producing a chromosome in a cell as it is is shown.

A plasmid pN897 in which the S. litoralis DNA polymerase gene was inserted into an eukaryotic cell expression vector was prepared (see FIG. 1) (this preparation is easily performed by those skilled in the art. JP-A-6-7160). See the publications, etc. The following can also be used: GenBank accession number
r: M74196, ATCC deposit number 68487, AT
CC deposit number 68447). This plasmid was introduced into monkey cell COS7 by a conventional calcium phosphate method. The introduction into COS7 cells indicates that the neomycin resistance gene of pN897 was
418 was confirmed by growth in a medium containing 400 μg / ml (Dulbecco's medium containing 20% fetal bovine serum).

The LTR promoter of the mouse mammary gland virus in pN897 was activated by culturing the COS7 cells into which pN897 had been introduced in a medium containing 10 μg / ml of dexamethasone for 2 days. Thus, it was confirmed by Western blotting that the production of Sermococcus litoralis DNA polymerase protein in COS7 cells was performed. This protein is
It was confirmed in the same manner as in Example 1 that chromosomal DNA was synthesized in the cell, and that a part thereof functioned as chromosome of the cell.

[0039]

According to the present invention, a continuous double-stranded linear DNA (chromosomal DNA) having a centromere region, a telomere region, and a replication initiation region can be easily synthesized, and the obtained chromosomal DNA can be easily synthesized. The chromosome can be easily produced using

[0040]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 56 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: other nucleic acid (newly synthesized DNA) Antisense: Yes TATCTAGATA TCTAGATATC TAGATATCTA GATATCTAGA TATCTAGATA TCTAGA 56

[0041]

[Sequence list]

 SEQ ID NO: 2 Sequence length: 34 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: other nucleic acid (newly synthesized DNA) Antisense: Yes CTAGATATCT ATCTAGATAT CTATCTAGAT ATCT 34

[0042]

[Sequence list]

 SEQ ID NO: 3 Sequence length: 34 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: other nucleic acid (newly synthesized DNA) Antisense: Yes TAGATATCTA GATCTAGATA TCTATCTAGA TATC 34

[Brief description of the drawings]

FIG. 1 is a diagram showing an expression vector pN897.

──────────────────────────────────────────────────続 き Continuation of the front page (56) References JP-A-9-285289 (JP, A) JP-A-5-317058 (JP, A) International Publication 96/7669 (WO, A1) Mol. Cell. Biol. , Vol. 8, No. 11 (1988), p 4642-4650 (58) Fields investigated (Int. Cl. ⁷ , DB name) C12N 15/00-15/90 BIOSIS (DIALOG) WPI (DIALOG)

Claims (4)

(57) [Claims] 1. A gene such as a drug resistance gene to be introduced into the nucleus.
Should not disappear from the nucleus during meiosis.
At the end of the gene, a repetitive centromere region
Base sequence, telomeric region [TTTGGG], etc.
Guanine-rich repetitive nucleotide sequence and replication initiation region
Double-stranded linear DNA with a single region (ARS)
Staining for additional synthesis and chromosomalization of the gene
A process for the synthesis of body DNA, in a reaction system template and / or primer is not present, CERUMO genus, Serumasu genus and Pirokokka
Deoxyribonucleotide polymer of bacteria belonging to the genus Genus
At least one heat-resistant resin selected from the group consisting of
The deoxyribonucleotide is polymerized in the presence of oxyribonucleotide polymerase to form a centromere region.
[TTGGGG] which is a telomere region
Guanine-rich repetitive nucleotide sequences such as
Connected double-stranded linear DN with start region (ARS)
Method of synthesizing chromosomal DNA characterized that you synthesize A. 2. A gene such as a drug resistance gene to be introduced into the nucleus.
Should not disappear from the nucleus during meiosis.
At the end of the gene, a repetitive centromere region
Base sequence, telomeric region [TTTGGG], etc.
Guanine-rich repetitive nucleotide sequence and replication initiation region
Double-stranded linear DNA with a single region (ARS)
Staining for additional synthesis and chromosomalization of the gene
A method for synthesizing body DNA, comprising the steps of Serumococcus, Sermus and Pyrococcus
A bacterial deoxyribonucleotide polymerase?
At least one heat-resistant deoxy selected from the group consisting of
An expression vector into which a DNA encoding a ribonucleotide polymerase has been inserted is introduced into cells, and the heat-resistant deoxyribonucleotide polymerase is produced in the cells, and the absence of a template and / or a primer
Under the deoxyribonucleotides are polymerized in the presence of the heat-resistant deoxyribonucleotide polymerase <br/> by
Repetitive nucleotide sequence in the telomeric region,
Guanine-rich repetitive salts such as certain [TTGGGG]
Connection with base sequence and replication initiation region (ARS)
Duplex method for the synthesis of linear DNA synthesis to chromosomal DNA characterized by Rukoto of. 3. The repeating base which is the centromere region
The sequence is a repetitive sequence of [CTAGATAT]
The method for synthesizing chromosomal DNA according to claim 1 or 2, characterized in that : 4. The method according to claim 1, wherein the chromosomal DNA obtained by the method according to any one of claims 1 to 3 is introduced into a cell when the chromosomal DNA is synthesized outside the cell. A method for producing a chromosome, characterized in that when the chromosomal DNA is synthesized in a cell, the chromosome is converted into a chromosome in the cell.