JPH0549272B2 - - Google Patents

Description

[Detailed description of the invention]

Technical Field of the Invention The present invention relates to DNA related to Mungbean Yellow Mosaic Virus (hereinafter sometimes abbreviated as "MYMV") and hybrids incorporating the same.
Regarding DNA. To explain in more detail,
Obtained by double-stranding the single-stranded DNA of MYMV
DNA, hybrid DNA obtained by cleaving this DNA with a restriction enzyme and combining it with another biological vector or a third DNA, hybrid DNA obtained by propagating this hybrid DNA
This invention relates to DNA, DNA obtained by extracting only the MYMV-derived DNA fragment portion from this hybrid DNA, and performing ring-closing ligation, and replicative MYMV DNA. Prior Art Recently, DNA genes from viruses have been widely developed and used as vectors in genetic recombination technology. Examples of viruses that provide such vectors include papovaviruses such as simian virus 40 and polyomavirus, papillomaviruses, and adenoviruses. However, these known vectors were discovered as animal vectors and do not proliferate in plant cells. Therefore, these known vectors cannot be used as vectors for plant genetic recombination. To date, vectors that can be used as plant genetic modification vectors include the extranuclear vector possessed by Agrobacterium tumefaciens, which causes tumors in dicotyledonous plants such as tomatoes and tobacco. The only known genes are the Ti plasmid and the DNA gene from the cauliflower mosaic virus, which causes diseases in cabbage and Chinese cabbage.
Other than these, vectors suitable for plant genetic recombination have not yet been developed. Furthermore, it can be said that no plant virus other than the above-mentioned cauliflower mosaic virus is known to have a DNA gene that could potentially be used as a vector for plant genetic recombination. Recently, Robert M. Goodman and his colleagues at the University of Illinois in the United States analyzed the nucleic acid of a tropical plant virus that forms a single virion, which looks like two paired particles with a diameter of approximately 18 nm. As a result, the genes of this virus were approximately
discovered and reported that it is a circular single-stranded DNA with a size of 2500 bases [Virology 83 , 171
(1977), Virology 97 , 388 (1979)]. Subsequently, several types of plant viruses were discovered that form a single virion in which two particles with a diameter of approximately 18 nm are paired together, and whose genes are circular single-stranded. These viruses have been named the “gieminivirus group.” As mentioned above, only cauliflower mosaic virus and dieminivirus are known as plant DNA viruses, and dieminivirus is extremely promising as a target for the development of vectors for plant genetic modification. Conventionally, the Ti plasmid and cauliflower mosaic virus, which have been proposed as vectors for plant genetic modification, can only infect dicotyledonous plants, that is, host plants, whereas dieminivirus can infect not only dicotyledonous plants. Since monocots such as wheat and corn, which are important grains for humans, can also serve as hosts, this dieminivirus is
Converting DNA into a vector for plant genetic modification is in itself extremely significant. On the other hand, cauliflower mosaic virus multiplies within the cytoplasm of plant cells, whereas dieminivirus multiplies both within the cytoplasm and nucleus. This indicates that when dieminivirus is used as a vector, it is possible to modify the nuclear genes of plants themselves. Therefore, the dieminivirus
It is clear that if DNA could be turned into a vector, it would be of industrial value in itself. Structure of the Invention The present inventors have developed the single-stranded DNA of mung bean yellow mosaic virus (MYMV), which is a type of dieminivirus, into a double-stranded vector that is technically easy to handle in genetic recombination operations. Hybrid incorporating
The present invention was achieved as a result of research into DNA and the like. That is, according to the present invention, the single-stranded DNA of the mung bean yellow mosaic virus is at least partially double-stranded DNA, and the mono- to trimeric DNA of the DNA represented by the genetic map in FIG. is provided. FIG. 4 corresponds to the restriction enzyme cleavage map shown in FIG. 2, and shows the DNA base sequence corresponding to the target DNA of each restriction enzyme shown in FIG. In FIG. 4, the base sequence at P ₁ to _{P 6} is:
The base sequence of one of the double strands is as follows. P ₁ (0.0/2.70): 5′−GGATCC−3′ P ₂ (0.96): 5′−AAGCTT−3′ P ₃ (1.30): 5′−CTGCAG−3′ P ₄ (1.74): 5′− AGATCT-3′ P ₅ (2.03):5′-GGATCC-3′ Further, according to the present invention, the single-stranded DNA of Mung Bean Yellow Mosaic Virus is at least partially double-stranded DNA, The DNA shown in the genetic map in Figure 4 is cleaved with a restriction enzyme,
Hybrid DNA combined with other biological vector DNA or third DNA is provided. Further, a hybrid DNA is provided, which is DNA in which the single-stranded DNA of Mung Bean Yellow Mosaic Virus is at least partially double-stranded, and includes the DNA represented by the genetic map of FIG. 4.
Furthermore, the single-stranded DNA of mung bean yellow mosaic virus was at least partially double-stranded.
A mixture of DNA monomers to trimers shown in FIG. 3 and monomers to trimers of DNA shown in FIG. 4 is provided. The present invention is a DNA obtained by at least partially double-stranding a single-stranded DNA of (i) approximately 2.67 kilobases in length from MYMV, which shows the restriction enzyme cleavage map shown in Figure 1. DNA (hereinafter abbreviated as DNA-a)
(ii) DNA obtained by partially double-stranding single-stranded DNA with a length of about 2.70 kilobases, and the restriction enzyme cleavage map shown in Figure 2 is DNA (hereinafter abbreviated as DNA-b). Among them, DNA-b shown in FIG. 2 is the subject of the invention. Further, according to the present invention, the double-stranded
A hybrid DNA obtained by decomposing DNA-b with a restriction enzyme and combining it with another biological vector or third DNA cleaved with the same restriction enzyme is provided. Hybrids obtained by returning this hybrid DNA to the original vector host and propagating it
DNA, DNA obtained by digesting this hybrid DNA with restriction enzymes and then ring-closing it.
is provided. Furthermore, according to the present invention, a replicative DNA containing 1 to 3 base sequences of DNA-b is provided. Myg bean yellow mosaic virus (MYMV) is a type of dieminivirus discovered in Thailand (this virus is a virus that causes mosaic disease in mung bean, top black bean, etc., and is preserved at the National Institute of Plant Virology). and available there),
Purify the following single strand from infected plants by the method described in Phytopathology 67 (No. 1) 37 (1977).
DNA can be isolated and purified. (1) Isolation of single-stranded DNA from MYMV Virus was purified from infected plants by the method described in the above-mentioned literature. Approximately 500 μg of the purified virus was then added to 30 mM Tris HCl (PH=7.6) 1%.
The mixture was shaken for 2 minutes with SDS and proteinase K at 10 μg/ml, and then protein extraction was performed three times with 700 μg of phenol. Dissolved phenol can be removed from the aqueous layer by ether extraction, and then the single-stranded DNA of the virus can be isolated and purified by ethanol precipitation. (2) Double-stranding the single-stranded DNA of MYMV The single-stranded DNA obtained as above was then injected into
Double-stranded in vitro. In this double-stranded process, 0.002 µg of single-stranded DNA is added to the primer.
Preferably, it is used in a proportion of ~2000 μg. As a primer, Biochimica et al. by JMTayler et al.
By modifying the method described in Biophysica Acta, 442 , 325 (1976), oligonucleotides obtained by digesting calf thymus DNA with DNaseI can be used. The present inventors used primers obtained by this modified method as described in the Examples below. When this oligonucleotide is used as a primer, the appropriate amount of primer is 50 to 400 μg, more preferably 100 to 300 μg, per 1 μg of the single-stranded DNA. In addition, when using purified DNA with a completely complementary sequence of at least 10 bases as a primer to the single-stranded DNA of MYMV, the ratio of primers to be used per 1 μg of single-stranded DNA is
A range of 0.002 to 1 μg, especially 0.004 to 0.02 μg is sufficient. After adding primers to single-stranded DNA in the proportions described above, deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP) and deoxythymidine triphosphate (dTTP) each at a final concentration of 10-300 μM,
It is preferably added in a range of 50 to 150 μM. At this time, for example, a part of dCTP is converted into α- ³² P-deoxycytidine triphosphate (α- ³² P-dCTP).
It is convenient to substitute , as it makes it easy to track the progress of double-stranded DNA and the double-stranded DNA in subsequent reactions. Furthermore, Mg ⁺⁺ (eg, magnesium chloride, magnesium sulfate, etc.) can be used in a concentration range of 1 to 50 mM, preferably 5 to 20 mM, to promote the enzyme reaction. In this specification, "M" means a concentration unit of "mol/" unless otherwise specified. PH in the reaction system is 7.2-9, preferably 7.6-9
Buffers to be maintained in the range of 8.4, e.g.
Advantageously, Tris HCl (PH 7.6-8.4) is used in a concentration of 30-300mM, preferably 70-200mM. Thus, the above mixture is heated to a temperature of preferably 50 to 80°C, especially 60 to 70°C for 1 to 10 minutes.
minutes, preferably 3 to 7 minutes, and then, if necessary, rapidly cooled to a temperature of about 0°C or less. The total amount of the mixed solution is based on the amount of single-stranded virus.
10 to 1000 μg, preferably 50 μg of DNA
It is preferable to set it in the range of ~500μ. It is also effective to use commonly used enzyme stabilizers, such as α-mercaptoethanol and dithiothreitol (DTT). The concentration of the stabilizer in the mixed solution is 0.1 to 5% (by weight), preferably 0.5 to 5% (by weight).
It can be used in a range of 2% (by weight). Next, an enzyme for double-stranding is applied. Examples of enzymes for double-stranding include Avian myeloblastosis virus (AMV) reverse transcriptase, T4-DNA polymerase, and Escherichia coli DNA polymerase. I, DNA polymerase large fragment (Klenow enzyme), etc. Among these, AMV reverse transcriptase or DNA polymerase large fragment is preferred. The proportion of double-stranded enzyme used depends primarily on its type. For example, in the case of AMV reverse transcriptase [when using code 120248 manufactured by Seikagaku Corporation], per 1 μg of single-stranded DNA,
It is effective to use 1 to 20 units, preferably 5 to 10 units. If the number is less than 1 unit, it may be difficult to form double strands sufficiently. After adding the double-stranded enzyme, first heat at 10-30℃.
Preferably react at a temperature of 15-25°C for 2-30 minutes, preferably 5-20 minutes, then 30-45°C,
It is advantageous to react for 30 to 180 minutes, preferably for 50 to 120 minutes, preferably at a temperature of 35 to 40°C. To stop the reaction, add, for example, a water-saturated phenol or EDTA aqueous solution (PH
= 8.0) may be added. At this time, in the case of water-saturated phenol, it is sufficient to add 1/10 to 2 times the volume of the reaction mixture, and after shaking, centrifuge to separate and fractionate only the aqueous layer. Just use enough to capture the Mg ⁺⁺ that you want. Double-stranded DNA may be separated from the DNA-containing aqueous solution that has been subjected to the reaction termination treatment, and a preferred separation operation is a gel filtration method. To explain one specific example of that case, double-stranded
The DNA-containing aqueous solution was prepared using Sephadex G-10 [manufactured by Pharmacia Huain Chemical Co., Ltd.] or Biogel.
Fractionation was performed using a gelatinizing agent such as P30 [manufactured by Bio-Rad Co., Ltd.], and the ^32P intensity of each fraction was measured.
^Double- stranded DNA can be obtained by collecting the high molecular weight DNA-containing fractions that appear first in each fraction and precipitating them in ethanol or isopropanol. The double-stranded DNA thus isolated is the single-stranded DNA of MYMV.
may contain considerable amount of primer DNA other than double-stranded DNA based on This tendency is remarkable when the oligonucleotide from the calf thymus DNA is used as a primer. Therefore, in such cases double-stranded DNA
When it is desirable to purify it again in order to achieve a high degree of purity. This purification can be performed using the gel filtration method described above.
To give a specific example, when starting from 20 μg of single-stranded DNA, the double-stranded DNA is
Dissolved in 500μ, 10mM Tris HCl (PH=
7.4), equilibrated with 100mM NaCl aqueous solution
Gel filtration is performed using a column packed with Sephadex G-50 or Biogel P30 with enhanced separation ability. 10mM Tris HCl (PH=7.4) as eluent, 100
^32P out of the void volume using mM NaCl
Collect the fractions with high counts and precipitate them in ethanol or isopropanol to obtain highly purified
Double-stranded DNA of MYMV can be obtained. (3) Double-stranded DNA In the aforementioned MYMV, two types of DNA
exists, and was obtained by double-stranded as described above.
There are also two types of DNA. These two types of double-stranded DNA can be separated into individual DNAs, and can be separated after cloning. Also, before becoming double-stranded (that is, single-stranded)
It is also possible to separate each (in the state of DNA) and double-strand it in the same way. DNA-a mixture of single-stranded DNA of MYMV
To separate DNA-b and DNA-b, a method called “strand separation” is used [for example, in the book “Molecular Cloning
[method described in "A Laboratory Manual" pages 180 to 185], that is, the method using gel electrophoresis, or the method of adding DNA to a cesium chloride solution of a DNA mixture.
Complementary DNA fragments are added to only either DNA a or DNA-b, and the density difference between the DNA that remains single-stranded and the partially double-stranded DNA is determined by equilibrium density gradient centrifugation. It is possible to adopt a method of separating by using However, the separation of DNA-a and DNA-b is a single strand.
It is easier to double-strand the DNA mixture and then clone it into, for example, plasmid pBR322 and separate it. This separation method will be explained in detail later. In this way, the DNA obtained by double-stranding the DNA from MYMV has a size of 2.67 ± 0.1 kilobase pairs (hereinafter "kilo base pair" may be abbreviated as "Kbp"). The restriction enzyme cleavage map of FIG. 1 is shown. The other one has a size of 2.70±0.1 kilobase pairs (Kbp) and shows the restriction enzyme cleavage map shown in FIG. FIG. 3 shows the DNA corresponding to the target DNA of each restriction enzyme in the restriction enzyme map of FIG. 1. In FIG. 3, the base sequence of one strand of the double strands in P ₁ to _{P 6} is as follows. P ₁ (0.0/2.67): 5′−GGATCC−3′ P ₂ (0.22): 5′−TCTAGA−3′ P ₃ (0.27): 5′−AGATCT−3′ P ₄ (1.24): 5′− ATCGAT-3' P ₅ (1.68): 5'-TCTAGA-3' P ₆ (1.92): 5'-AGATCT-3' The restriction enzyme map in Figure 2 shows the DNA corresponding to the target DNA of each restriction enzyme. Figure 4 shows this. In FIG. 4, the base sequence of one strand of the double strands in P ₁ to _{P 6} is as follows. P ₁ (0.0/2.70): 5′−GGATCC−3′ P ₂ (0.96): 5′−AAGCTT−3′ P ₃ (1.30): 5′−CTGCAG−3′ P ₄ (1.74): 5′− AGATCT-3' P ₅ (2.03):5'-GGATCC-3' In Figure 1, the cleavage site by BamH is (0.0/2.67), and each restriction site is shown as a positional coordinate, and the unit is kilobase pairs ( Kbp). Furthermore, in FIG. 2, each restriction site is shown as a positional coordinate, with one position of BamH being (0.0/2.70). Next, the fragments generated by digestion with each restriction enzyme are shown in Table 1 below. The fragments that appear densely compared to the size of the fragments in the table are grouped into A group, and the other fragments are grouped into B group.
Shown in groups.

[Table] In the above table, (3.3) means that the DNA remains as an open ring without being cut at any point, and the apparent molecular weight is 3.3 Kbp relative to the size marker of linear DNA. It shows. The size values of the fragment DNA in the above table are:
It should be understood that the accuracy of experimental analysis may vary by ±0.1 Kbq, especially ±0.05 Kbq. For restriction enzyme digestion, use a concentration of 10mM Tris.
HCl (PH = 7.9), 7mM MgCl ₂ , 7mM β-mercaptoethanol, 0.01% bovine serum albumin base solution was used. Digestion with Cal was performed with the above base solution, and digestion with Hind, Bgl, Kpn, and Pvu was performed with the above base solution. Add NaCl to 50mM, and also add Sal, BamH, Xba, Xho
The decomposition was carried out by adding NaCl to the base solution at a concentration of 150 mM. In the present invention, restriction enzymes include Hind, SalI,
Bgl, BamH, Kpn, Pst, Pvu and
In the case of Xho, those manufactured by Takara Shuzo Co., Ltd. were used, in the case of Cla, those manufactured by Boehringer Mannheim, and in the case of Xba, those manufactured by Bethesda Research Laboratory were used. In the present invention, each restriction enzyme for cutting DNA has at least 4 units/
1 μg of DNA was used, and the digestion was carried out at 37° C. for 4 hours or more. When digesting with two types of restriction enzymes, first use a low-salt enzyme.
It was decomposed at 37°C for 2 hours or more, then adjusted to a high salt concentration, and further digested at 37°C for 2 hours or more using an enzyme designed for high salt concentrations. The DNA thus enzymatically degraded was subjected to 1.5% agarose gel electrophoresis containing 0.5 μg/ml of ethidium bromide to analyze the DNA fragments produced by each enzymatic degradation. In addition, during this electrophoresis, λ was used as a DNA size marker.
−DNA degraded with EcoR/Hind,
Plasmid pBR322 digested with Taq was used. The double-stranded DNA according to the present invention only needs to be at least partially double-stranded with respect to the single-stranded DNA, and it is not necessary that the double-stranded DNA is completely double-stranded over the entire region. Not necessarily necessary. The double-stranded DNA of the present invention is used, for example, as a vector for plant genetic recombination, and is therefore used after being cut with a certain enzyme. In that case, as long as the site to be cut is at least double-stranded, the non-double-stranded site will be repaired and almost completely double-stranded by subsequent cloning or propagation. Therefore, the double-stranded DNA of the present invention only needs to be at least partially double-stranded, but is preferably single-stranded.
It is sufficient that at least 80%, preferably at least 90% of the DNA is double-stranded. The ratio of DNA between DNA-a and DNA-b is
Different values are obtained depending on the growth process of MYMV, but generally the ratio of DNA-a/DNA-b (mol) is 2 or more. Therefore, as shown in Table 1 above, it is possible to group the fragments into group A fragments and group B fragments. The double-stranded DNA of the present invention is DNA-a and DNA
-b can be used alone or in combination as a vector for plant genetic recombination. Further, DNA-a or DNA-b can be cleaved at a specific restriction site with a restriction enzyme to form linear DNA. This DNA was mixed with a third DNA cut out using the same restriction enzyme at approximately the same molar ratio, and then a DNA-binding enzyme (e.g. T4
- DNA ligase) to form a circular DNA containing a third DNA at a specific site of DNA-a or DNA-b. For example, if the third DNA encodes a gene that confers resistance to the antibiotic kanamycin, by using DNA into which this third DNA has been incorporated into DNA-a or DNA-b, Plant cells can be transformed to become kanamycin resistant. In this case, transformation methods include, for example, a method in which the above DNA is applied to protoplast-formed plant cells in the presence of Ca ⁺⁺ , polyethylene glycol, or in a high pH state in the presence of Ca ⁺⁺ , and further into the plant cells. There is a method of mechanically introducing the above-mentioned DNA (microinjection). Moreover, double-stranded DNA-a or DNA- of the present invention
an enzyme capable of cleaving b, preferably at one location;
For example BamH, Hind, Pst, Cla or
It can be digested with Bgl and then inserted into other biological vectors cleaved with the same restriction enzymes as shown below. In this case, other biological vectors include conventional vectors such as plasmid vectors for Escherichia coli (e.g. pBR322), cosmid vectors (e.g. pKY2662), phage vectors (e.g. Sialon 10), and plasmid vectors for Bacillus subtilis. (eg, pUB110, pSA0501), vectors for yeast (eg, YRp7), etc. can be used. Hybrids with these other biological vectors
The method for preparing DNA consists of double-stranded DNA-a
and DNA-b, for example, Cal (or
BamH) and use it as a vector for other organisms, such as a plasmid for E. coli.
Mix with Cla (or BamH) decomposition product of pBR322 and use DNA binding enzyme (e.g. T4-DNA ligase).
DNA-a and
Hybrid DNA with pBR322 can be obtained. Similarly, double-stranded DNA-a and DNA
The mixture with -b is completely decomposed with Hind (or Pst), and this is mixed with Hind (or Pst) of pBR322.
) A hybrid DNA of DNA-b and pBR322 can be obtained by mixing with a decomposition product and cyclizing the mixture with a DNA-binding enzyme. When obtaining these hybrid DNAs, it is better to mix the two DNAs you want to combine in approximately equimolar proportions to more efficiently produce the desired hybrid DNA.
DNA can be obtained. In addition, when preparing hybrid DNA, it is recommended that vectors for other organisms be cleaved with restriction enzymes and then treated with alkaline phosphatase to dephosphorylate less than 5' of the DNA. This is preferable because self-circularization can be largely prevented by using the vector alone. The hybrid DNA thus obtained is also at least partially double-stranded, like the DNA shown in FIG. 1 or 2, and can be used as a vector for plant genetic recombination. This hybrid DNA is transferred to the original vector host for other organisms; for example, in the case of a hybrid DNA with an E. coli vector, it is transferred to E. coli, and in the case of a hybrid DNA with a Bacillus subtilis vector, it is transferred to Bacillus subtilis and then transferred to a yeast vector. hybrid of
In the case of DNA, transform yeast into yeast according to known methods.
For example, in the case of Escherichia coli and Bacillus subtilis, they are transformed into competent cells by known methods, and in the case of yeast, they are transformed into spheroplasts by treatment with Zymolyses, and then transformed using appropriate drug resistance or auxotrophy. Transformants can be selected or colony hybridization can be performed to select transformants. To explain in more detail one specific example of this method, for example, when hybrid DNA is
Cla-digested product (Hind-digested product in the case of Figure 2) and Cla-digested product of E. coli plasmid pBR322 (second
In the case of hybrid DNA (in the case of the figure, Hind degradation product) linked with T4-DNA ligase,
This hybrid DNA was transformed into E. coli using the method of Mandel and Higa [see J.Mol.Biol. 53 , 159 (1970)].
Colony hybridization was performed using the method of Grunstein and Hogness [Proc. Natl. Acad. Sci. 72 , 3961] after transforming HB101 and generating colonies on agar (L-Agar) plates containing 50 μg/ml ampicillin. zesion (at this time,
³² P-labeled MYMV DNA is used as a probe). ³² Select colonies that have DNA that hybridizes with P-labeled MYMV DNA. In this way, Figure 1 (or Figure 2)
The DNA and plasmid pBR322 are
) E. coli HB101 transformed with the hybrid DNA ligated at the site can be obtained. Hybrid DNA can be isolated from the transformant thus obtained by a known method. The isolated hybrid DNA is completely double-stranded throughout, and a third DNA can be inserted into a specific site to transform plant cells, and it can also be grown in bacteria such as Escherichia coli. It is possible. In order to prepare a large amount of the desired hybrid DNA, a bacterial strain containing the hybrid DNA is grown, and if necessary, an operation is performed to amplify only the hybrid DNA to lyse the bacterial cells, and the hybrid DNA is used as a plasmid for E. coli, for example.
If it is a hybrid DNA with pBR322,
From the E. coli transformant containing the hybrid DNA, for example, "Molecular Cloning-A Laboratory
Manual”［Cold Spring Harbor Laboratory］
(1982)], pages 88 to 94, hybrid DNA amplification and lysis methods were used to obtain DNA, and a covalently closed circuit (hereinafter referred to as "CCC") was obtained using an equilibrium density gradient of cesium chloride. This makes it possible to separate only hybrid DNA of the type (abbreviated). In addition, hybrids separated in this way
By digesting the DNA with the same restriction enzyme, the DNA fragment shown in Figure 1 (or Figure 2) and the vector DNA are obtained.
It can be divided into fragments. Preferably, the DNA fragment of Figure 1 (or Figure 2) is isolated and then T4-
Combine and cyclize using DNA ligase or the like. 1st
To separate only the DNA fragments shown in the figure (or figure 2), agarose gel electrophoresis is generally performed to separate the DNA fragments.
-a (or DNA-b) DNA fragment band portion gel is cut out, and the DNA fragment is separated from the gel and purified according to various methods described on pages 164 to 172 of the experimental book by Maniathes et al. be able to.
The DNA fragments thus separated are ligated and circularized using, for example, T4-DNA ligase by a known method, and at this time, DNA-a (or DNA-b) is
In addition to self-circularized DNA with only one DNA, two
A part of the compound is formed by bonding and cyclizing two or more of them. Only one of these molecules self-cyclizes, resulting in an apparent 3.3 Kbp band in angarose gel electrophoresis.
DNA can be extracted and purified from this banded gel in the same manner as described above. This DNA is completely double-stranded throughout the ring and is useful as a vector for plant genetic recombination. Next, the replicative DNA in the present invention will be explained. The replication form of MYMV can be detected from the infected leaves of infected leaves where MYMV is infected and proliferating, such as the infected leaves of top and black beans (for example, top crop of Taiki Seed Co., Ltd.).
To isolate DNA, use the method of WDO Hamilton et al. [see Nucl. Acids Res. 10 , 4902 (1982)] or the complete text of "Introduction to Plant Cell Breeding (Gakkai Publishing Center)" by Atsushi Hirai et al., pp. 86-88. This can be done using a DNA extraction method. Total DNA extracted according to these methods was subjected to 0.8% agarose gel electrophoresis (at this time, λ- was used as a DNA size marker).
EcoR/Hind digests of DNA are run together), and DNA is then transferred from this gel to a nitrocellulose filter using Southern's method [see E.M. Southern J.Mol.Biol 98 , 503-517 (1975)]. Heat-denatured MYMV labeled with ^32P
Using DNA as a probe, Maniathes et al.'s ``Molecular Cloning-A Laboratory''
DNA-DNA hybridization is carried out in the same manner as described on pages 387 to 389 of the same book, and then autoradiography is carried out in the same manner as described on pages 470 to 471 of the same book. The DNA portion that hybridizes with ^{the 32P-} labeled MYMV DNA appears as a black band on the X-ray film during autoradiography, indicating that this portion is MYMV-derived DNA. Approximately nine types of such bands have been observed, and their apparent sizes are >20Kbp, 10Kbp, 6.8Kbp, 5.4Kbp,
They are 4.0Kbp, 3.2Kbp, 2.7Kbp, 1.6Kbp, and 0.89Kbp. The DNA that gives these bands comes in various forms [e.g. CCC, open circle (OC),
It is inferred that because it contains linear monomers and various forms of dimers, trimers, etc., it forms many types of bands. Of these bands,
The 0.89 Kbp band corresponds to the single-stranded DNA of MYMV, but all the others are double-stranded DNA and are replicative DNA of MYMV. These replicative intermediate DNA bands were removed from the agarose gel and DNA was extracted by the same procedure as above.
Separate only BamH, Bgl, bgl/Pst
When decomposed with
The same DNA fragments can be obtained by decomposing . This shows that the DNA that gives eight types of bands other than the DNA equivalent to 0.89 Kbp is the replicative intermediate DNA of MYMV, which takes various forms based on the DNA shown in Figure 1 or Figure 2. . Of the DNA that gives these bands,
DNA equivalent to 1.6Kbp, DNA equivalent to 3.2Kbp
DNA, a portion equivalent to 10.0 Kbp, is present in relatively large amounts and is easy to use. Thus, by decomposing a portion of DAN corresponding to 1.6 Kbp, 3.2 Kbp, or 10.0 Kbp with Cla and cloning it into the Cla site of plasmid pBR322 for E. coli using the method described above, a hybrid DNA containing the entire DNA-a can be obtained as described above. Also 1.5Kbp
(or 3.2Kbp or 10.0Kbp) equivalent portion of DNA
By digesting with Hind and cloning into the Hind site of pBR322, a hybrid DNA containing the entire DNA-a can be obtained as described above. When the DNA obtained by cloning is replicated in E. coli, which has the dam methylase gene, a specific base sequence, for example, the N at the 6th position of the A position of GATC, is methylated, and in the DNA obtained by cloning, the 6th N of the A position of GATC is often methylated. Although it may be observed that the restriction enzyme site appears to be missing, the base sequence itself is the same. Furthermore, DNA obtained through cloning exhibits the same infectivity as a virus, just like DNA extracted directly from a virus. EXAMPLES The present invention will be described in detail with reference to Examples below, but the present invention is not limited thereto. Example 1 a Isolation and purification of virus 53gr of MYMV-infected Topcrop leaves were
250ml 0.1M Sodium Phosphate - 10mM
EDTA buffer (PH=7.8) (cysteine
(contains 1.3gr) and pass through double gauze.
Centrifuge the solution at 10,000G for 40 minutes at 4℃ to 205ml.
The supernatant was obtained. Add 2.4g of salt to this supernatant, then polyethylene glycol (7800-9000),
After adding 8.2gr and stirring at 4℃ for 1 hour, 10000G, 25
Centrifugation was performed for 4 minutes at 4°C. After removing the supernatant, 10 ml of 0.1 M sodium phosphate buffer (PH = 7.8) was added to the precipitated polyethylene glycol pellet to homogenize it, and the mixture was heated at 10,000 G for 30 minutes at 4°C.
The supernatant was centrifuged at 4° C. for 4 hours at 30,000 rpm using a Beckman ultracentrifuge SW40.1 rotor to obtain a crude virus as a precipitate. 0.5ml of this crude virus
0.1M sodium phosphate buffer (PH=
7.8) After homogenization, perform continuous 10-40% sucrose density gradient centrifugation (using the same SW40.1 rotor as above, 32,000 rpm, 3 hours, 4°C),
After centrifugation, 0.6 ml fractions were collected from the bottom of the centrifuge tube. The absorbance _A260 of each fraction was measured, and virus peaks were observed in fractions No. 9 to 16.
This fraction was collected and homogenized by adding twice the amount of 0.1M sodium phosphate buffer (PH = 7.8).
35,000 rpm with SW40.1 rotor, 3 hours, 4℃
Obtain a virus pellet again and add 0.5ml of this to
0.1M sodium phosphate buffer (PH=
7.8) and centrifuged again with a 10-40% sucrose density gradient (29,000 rpm, 3 hours, 4°C, SW40.1 rotor), and 0.6 ml fractions were collected from the bottom of the centrifuge tube. Only virus peaks were obtained in fractions No. 15 to 19, and these fractions were collected and
Double the amount of 0.1M sodium phosphate buffer (PH
7.8) and after homogenization, 36,000 rpm, 3 and a half hours,
The virus was precipitated in an SW40.1 rotor at 4°C to obtain purified MYMV. Isolation of single-stranded DNA genes from Ib virus
Purification To the above purified MYMV, add 750μ distilled water, 15μ
Add 1M Tris HCl (PH = 7.6), 75μ of 10% sodium dodecyl sulfate (SDS), and 7.5μ of proteinase K (1μg/μ solution) and shake at room temperature for 2 minutes.
Then 10mM Tris HCl (PH=7.6)-1mM EDTA
700μ of phenol saturated with an aqueous solution was added, and the mixture was shaken for 3 minutes, centrifuged for 5 minutes in an Etzpendorf small centrifuge, and the aqueous layer was taken out. This aqueous layer is subjected to the same phenol extraction procedure two more times, resulting in an aqueous layer.
Obtain 950μ, then add 700μ of chloroform to the aqueous layer.
After shaking for 2 minutes, remove the aqueous layer and add 700μ of ether to extract the phenol. This phenol extraction operation was performed three times. Add 100μ of 3M sodium acetate buffer (PH = 4.8) and 2.5ml of ethanol to 1000μ of the resulting aqueous layer, keep at -20℃ overnight, and centrifuge in a Beckman ultracentrifuge.
Centrifuge for 20 minutes at 35,000 rpm in an SW50.1 rotor.
DNA was obtained as a precipitate. 10mM of this DNA
The MYMV single-stranded DNA was dissolved in 400μ of Tris HCl 0.1mM EDTA aqueous solution to give ~0.1μg/μ aqueous solution. Ic In vitro double-stranding of MYMV single-stranded DNA MYMV single-stranded DNA (~0.1μg/μ) 10μ
Sterile water 84μ, 1M Tris HCl (PH=8.0)
30μ, calf thorax adjusted by the method below.
DNA oligonucleotide (16.6μg/μ)
Mix 12μ and hold at 70℃ for 3 minutes, then 0℃
After rapid cooling to 80mM while keeping at 0℃
30μ _{of MgCl2} , 30μ of 10% β-mercaptoethanol aqueous solution, 30μ each of 0.8mM deoxyadenosine triphosphate, 0.8mM deoxyguanomine triphosphate and 0.8mM deoxythymidine triphosphate, 10μ of 0.8mM deoxycytidine triphosphate. and deoxycytidine 5′-[α- ³² P]
Triphosphoric acid (approximately 3000 Ci/manufactured by Amashiyam Japan Co., Ltd.)
mmol, 10m Ci/ml, code number
pB10205) 3μ and AMV reverse transcriptase (Seikagaku Corporation, code 12048, 5 units/μ) 1μ
After the mixture was reacted at 20°C for 10 minutes and then at 37°C for 1.5 hours, 50μ of phenol was added, shaken, and centrifuged for 5 minutes in an Etzpendorf centrifuge, and the aqueous layer was gel-filtered. Gel run in 10mM Tris HCl
(PH=7.4) - 5 ml of Sephadex G-75 (manufactured by Pharmacia Fine Chemical Co., Ltd.) equilibrated with a 0.1 M NaCl aqueous solution was passed through a column packed in a 6 mm diameter tube. After adding the reaction solution, add 4 drops (approx.
The ^32P intensity of each fraction was measured. The first peak appeared in fractions No. 6 to 12, and a peak due to unreacted α- ³² P-deoxycytidine triphosphate was observed after No. 14. Collect fractions No. 6 to 12 and add 60μ of 3M NaCl.
Add 2.2 ml of ethanol and hold at -20℃ for 2 hours, then run at 30,000 rpm for 25 minutes (Beckman ultracentrifuge)
SW50.1 rotor) centrifugation and remove the supernatant, add 3 ml of 70% ethanol aqueous solution cooled to -20°C, centrifuge at 20,000 rpm for 2 minutes, and remove the supernatant. precipitation
The DNA was dried under reduced pressure at 20 mmHg for 1.5 minutes. The obtained DNA was diluted with 1mM Tris HCl (PH=7.4)-0.1
Obtain double-stranded DNA in vitro by dissolving it in 100μ of an mM EDTA aqueous solution (the intensity of ^32P is 32×
¹⁰⁴ cpm). Preparation of calf thoracic DNA oligonucleotide 66 mg of calf thoracic DNA was added to 0.1M NaCl and 10mM.
Dissolve MgCl ₂ in 6.6 ml of 10 mM Tris HCl (PH = 7.4), add 460 μg of DNase (manufactured by Millipore Corporation, 2182 units/g),
Incubate at 37℃ for 3 hours and 0.2M
The reaction was stopped by adding 0.66 ml of EDTA. This reaction solution was subjected to protein extraction three times with 7 ml of water-saturated phenol, and then extracted three times with 7 ml of ether to remove phenol from the aqueous layer. 20 ml of ethanol was added to the resulting aqueous layer, and the mixture was left at -20°C for 2 hours, and then centrifuged at 4000G for 4 minutes to obtain a DNA pellet. This DNA pellet was mixed with 0.5ml of 0.1M NaCl, 10mM Tris HCl (PH
=7.4), 0.1M NaCl, 10mM Tris
Sephadex G pre-equilibrated with HCl (PH=7.4)
-75 (manufactured by Pharmacia) (column length: 42 cm, column diameter: 0.5 cm) and fractionated into approximately 1.2 ml portions. A large peak was observed in fractions No. 10 to 32, and fractions No. 16 to 26 were collected (total amount
12.5ml) Add 31ml of ethanol, leave at -20℃ for 30 minutes, and centrifuge (4000G, 8 minutes).
A DNA pellet dissolved in 1 ml of distilled water (DNA concentration 16 m ³ /ml) was used as primer DNA. Comparative Example 1 Calf thymus DNA oligonucleotide (16.6μ
When a double-stranded experiment was performed under the same conditions as experiment c except that 2 μg/μ) was used instead of 12μ, the count of ^32P in the polymeric DNA part was 2×10 ⁴ cpm. It can be seen that less than 1/10 of the double stranding has occurred compared to the case of Atsuta c. Comparative Example 2 The primer was prepared by G.M. Theiler et al.
Biochimica et Biophysica Acta, 442 , 325
Primer mixture from calf thymus DNA prepared by the method of (1976) (DNA concentration 5 μg/μ
) was used instead of 12μ of the oligonucleotide (16.6μg/μ) in experiment c, and 55μ was used instead of 83μ of sterile water, and double stranding was carried out in the same manner as in experiment c. The strength of ^32P in the polymeric DNA part is 13×
It was 10 ⁴ cpm. The double-stranded DNA obtained in Comparative Example 2 was treated with restriction enzymes under exactly the same conditions as in the experiment d described below.
When the DNA was digested with BamH, Hind, Bgl, Xba, etc. and subjected to autoradiography in exactly the same manner as in experiment d below, the DNA in the Hind-digested DNA did not move from the slot position of the agarose gel at all. Also, some of the DNA digested with other restriction enzymes remained in the slot position, and the DNA that migrated through the gel was quite smeared, and the band was not as beautiful as in the case of d. d Degradation of double-stranded DNA using restriction enzymes
Analysis (restriction enzyme cleavage map) ^32P -labeled double-stranded DNA obtained in c ( ^32P intensity 32× ¹⁰⁴ cpm/100μ) 3μ ( ^32P intensity
7300cpm) and buffer for restriction enzyme digestion (100mM Tris HCl (PH=7.9) 70mM
_MgCl2 , 70mM β-mercaptoethanol, 0.1
% bovine serum albumin as the basic solution, Cla decomposition is performed using this basic solution, Hind, Bgl, Kpn,
For Pst and Pvu decomposition, the above basic solution is further
A solution in which NaCl was added to 500mM, or
For Sal, BamH, Xba, and
(6 units/μ), Pst (5 units/μ)
), Xba (6 units/μ), Hind
(5 units/μ), Kpn (6 units/μ)
), Mlu (5 units/μ), Pvu (6
units/μ), Sal (6 units/μ),
Xho (7.5 units/μ), Cla (5 units/μ) Among the restriction enzymes in Table 1, Cla is manufactured by Boehringer Mannheim, Xba is manufactured by Bethesda Research Laboratory, and the others are manufactured by Takara Shuzo Co., Ltd.). Add 4 units, heat to 37℃,
DNA degradation was performed for 2-4 hours. When decomposing with two types of restriction enzymes, first add an enzyme for low salt concentration, hydrolyze at 37℃ for 4 hours at a salt concentration suitable for the enzyme, and then add the enzyme suitable for high salt concentration. An enzyme for high salt concentrations was added, and the mixture was further hydrolyzed at 37°C for 4 hours. After hydrolysis, 8 μ of a solution of 0.25% bromophenol blue, 50% glycerol, 10% SDS was added, heat treated at 65°C for 5 minutes, and then 1.5%
Agarose gel electrophoresis was performed. The agarose used was for type electrophoresis manufactured by Sigma.
As an electrophoresis buffer, an aqueous solution of 40mM Tris acetate and 2mM EDTA (PH=8.0) was used. Electrophoresis was carried out in a 5 mm thick horizontal gel at a voltage of 1.5 V/cm for 11 to 15 hours. During this electrophoresis, as a size marker for DNA fragments,
0.5μg of λ-DNA was completely digested with EcoR and Hind and 0.2μg of pBR322DNA was digested with Taq.
I used one that had been completely disassembled. After electrophoresis, remove the agarose gel and dry it on a gel drying plate.
P470 of the experimental book by Maniathes et al. mentioned in the main text
Autoradiography was performed as described in 472. As a result, the sizes of DNA fragments observed after being degraded by various restriction enzymes were as shown in Table 1 of the text. Furthermore, the DNA fragments observed after autoradiography are classified and recorded by classifying the fragments that appear densely compared to the size of the fragment into group A, and the group of fragments that appear faintly compared to the size of the fragment as group B. did. Also, 3.3 in parentheses is
When your DNA is in an open circle (OC) state,
The apparent molecular weight of the linear DNA size marker is 3.3 Kbp. From this table, it is thought that the DNA that produces the fragments of group A is open circle (OC) shaped and approximately 2.67 Kbp in size before being digested with restriction enzymes. The exact size can ultimately be known by determining the base sequence, but with the current method, a minimum measurement error of 0.05 Kbp is unavoidable. The size of the DNA that produces the group A fragment is (2.67±0.1) Kbp
It can be described as a degree. Similar observations show that the DNA that produces group B fragments is OC before restriction enzyme digestion.
The size is described as 2.70±0.1Kbp. Next, by analyzing the degradation patterns of various restriction enzymes alone and in combination, the relative positional relationships of the various restriction enzyme cleavage points are determined, and the intact DNA that produces Group A DNA fragments is determined. Figure 1 is obtained as a restriction enzyme cleavage map for group B DNA, and Figure 2 is obtained as a restriction enzyme cleavage map for intact DNA of group B DNA. e Hybrid DNA of BamH-digested double-stranded DNA and plasmid pBR322 for E. coli 50μ of the 100μ solution of double-stranded DNA obtained in Example c was preliminarily mixed with 10mM Tris HCl (PH=
7.4) Bio equilibrated in aqueous solution with 100mM NaCl
Gel filtration was performed using a column packed with GelP30 with a diameter of 10 mm and a length of 10 cm. 10mM as eluent
Using Tris HCl (PH=7.4) 100mM NaCl aqueous solution, 10 portions (approximately 400μ) were collected. 4th to 6th
A high count peak of ^32P was seen in the fraction, and when the optical density OD ₂₆₀ of the fraction was measured, the OD value started to increase from the 4th fraction, increasing to a large value in the 9th fraction, and up to the 16th fraction. showed a large value. double stranded
Collect the 4th to 6th fractions, which are the DNA part (1200μ
), and 3M sodium acetate aqueous solution (PH=4.8)
After adding 120μ and 3 ml of ethanol and keeping at -20°C for 2 hours, the mixture was centrifuged at 30,000 rpm for 30 minutes in a Beckman ultracentrifuge SW50.1 rotor. Discard the supernatant and
Add 5 ml of 75% ethanol at -20°C.
Centrifuge at 20,000 rpm for 2 minutes and discard the supernatant again.
Got pellets. After vacuum drying at 2 mmHg for 2 minutes,
Dissolve this DNA pellet in 50μ of distilled water and
Add the above restriction enzyme BamH to 50μ of DNA solution.
6μ of a digestion buffer (described in Example C) and 3μ of the restriction enzyme BamH were added and reacted at 37°C for 4 hours. Next, 10μ of yeast tRNA (2μg/μ
) solution and 70μ of distilled water were added, and 100μ of water-saturated phenol was added, followed by immersion and centrifugation to take out only the aqueous layer, and the phenol was removed from this aqueous layer by ether extraction three times. Add 10μ of 3M sodium acetate (PH=4.8) and 350μ of ethanol, and keep at -20℃ for 4 hours.
After drying the DNA pellet obtained by high-speed centrifugation, 17μ
of distilled water. pBR322 shown below for this DNA 17μ solution
1μ of BamH/alkaline phosphatase treatment (DNA 1μg/μ) and buffer for DNA binding enzyme [650mM Tris HCl (PH=7.4), 65mM
M MgCl ₂ , 10mM MDTT, 4mM ATP, 40mM
Add 2μ of Spermidine aqueous solution and 0.5μ of T4 DNA ligase (manufactured by Takara Shuzo Co., Ltd., code No. 2010B, 1.2 units/μ), react at 14°C for 22 hours, then at 65°C for 5 minutes to form a hybrid.
Get DNA. Next, we used this hybrid DNA to infect E. coli.
Transform HB101 competent cells. BamH/alkaline phosphatase treatment solution for pBR322: Add 40μ of the BamH restriction enzyme buffer to a 340μ aqueous solution containing 50μg of plasmid pBR322 DNA.
Add 20μ of BamH and react at 37℃ for 10 hours, then add 44μ of 1M Tris HCl (PH=8.0) to react with bacterial alkaine phosphatase (BAP).
(Manufactured by Worthington, 0.4 units/μ) 10μ
The mixture was then treated at 65°C for 7 hours. Due to this reaction
The 5′ end of DNA is dephosphorylated. For this reaction solution, add 400μ of water-saturated phenol.
Protein removal was performed at Next, 400μ of a phenol/chloroform (4/1 volume ratio) mixture was
The protein was removed several times, and finally the phenol component was extracted from the aqueous solution three times using 600 μl of ether. Add 30μ of 3M sodium acetate and 1100μ of ethanol to 350μ of the aqueous layer, and leave at -20℃ for 2 hours.
After drying the DNA pellet obtained by high-speed centrifugation,
Dissolved in 50μ of distilled water. Add this to pBR322
Use BmH/allical phosphatase treatment (DNA 1 μg/μ) solution. Preparation of E. coli HB101 competent cells and their transformation; A single colony of E. coli HB101 was grown in culture medium L.
-brcth (5 ml) and cultured with shaking at 37°C for 11 hours.
Inoculate 200ml of new L-broth with this 2ml, and
C. for 2 hours and 20 minutes, and when the OD ₆₀₀ reached 0.40, the culture solution was cooled to 0.degree. C. and stretched at 5000 rpm for 5 minutes in a Tomy refrigerated high-speed centrifuge No. 9 rotor. Discard the supernatant and add the precipitated E. coli to 50ml of 10mM.
Homogeneously dispersed in NaCl water, centrifuged again at high speed (Tommy No. 4 rotor, 5000 rpm, 5 minutes) to precipitate the bacteria, and add 60 ml of 30mM CaCl ₂ to this bacterial pellet.
Water was added to homogenize the whole, and the mixture was kept at 0°C for 20 minutes. Centrifuge again at high speed (No. 4 rotor 4000 rpm, 5 minutes), discard the supernatant, and add 10 ml of 30 mM to the bacterial pellet.
Add CaCl ₂ and a 15% glycerol aqueous solution to gently homogenize the whole, dispense 200μ into 1.5ml Eppendorf tubes, and store frozen at -80°C.
This CaCl ₂ -treated E. coli HB101 (referred to as competent cells) was transformed. For transformation, the competent cells were returned to 0°C, and after about 10 minutes, a ligation reaction was performed using the T4 DNA ligase described above, and then 65
Add the DNA aqueous solution that has been treated for 5 minutes at 0°C.
The mixture was held at 42°C for 40 minutes and then heated at 42°C for 2 minutes. Next, 1.5 ml of L-broth was added and kept at 37°C for 1 hour, and the culture solution was spread on 8 L-agar plates (9 cm in diameter) containing 50 μg/ml of ampicillin at a rate of about 200 μl per plate. The mixture was kept at 37°C for 16 hours to obtain 86 transformed HB101 colonies. These 86 colonies were treated with ampicillin 50μg/
Transfer the two plates to the positions corresponding to the L-agar plate containing ml and the L-agar plate containing 25 μg/ml tetracycline.
℃ for 8 hours, and 51 ampicillin-resistant and tetracycline-sensitive colonies were selected. Mini-preparation of plasmid DNA was performed on 15 of these 51 colonies by the Boiling Lysis method described in the experimental book by Maniathes et al., page 368, and then the plasmid DNA was digested with BamH. As a result, approx.
Plasmids containing 2.67Kbp, approximately 2.02Kbp, and approximately 0.67Kbp were obtained. The DNA in Figure 1 for 2.67Kbp is BamH
The cleaved fragments, 2.02 Kbp and 0.67 Kbp, were two types of fragments generated when the DNA shown in Figure 2 was degraded with BamH. Hybrid DNA of 2.67Kbp fragment and pBR322
was designated pMYB4, the hybrid DNA of the 2.02 Kbp fragment and pBR322 was designated pMYB5, and the hybrid DNA of the 0.67 Kbp fragment and pBR322 was designated pMYB3. pMYB3
The DNA of ~5 was replicated and propagated in the host E. coli of pBR322, and was completely double-stranded throughout the DNA. BamH hybrid DNApMYB4,
BamH/Bg1, BamH/Bg1/Cla,
After complete digestion with BamH/Xba and BamH/XbaI/Bgl, 1.2% agarose gel electrophoresis was performed (at the same time, EcoR of λDNA was used as a size marker).
/Hind digest and Taq digest of pBR322 were also electrophoresed), and the DNA fragment size was analyzed. Also
pMYB5 to BamH, BamH/Hind/Bgl
, BamH/Hind/pst and similar analysis was performed to obtain the results shown in Table 2.

[Table] The fragments generated by digestion of pMYB4 with each restriction enzyme in Table 2 are shown in the restriction enzyme cleavage map in Figure 1, although each DNA size includes a measurement error of ±0.05 Kbp and a maximum of ±0.1 Kbp. It matches well. Furthermore, the Hind site was determined by analysis of fragments from pMYB5 and pMYB3, and the restriction enzyme cleavage map shown in FIG. 2 was confirmed. If hybrid DNA of Hind-digested double-stranded DNA and E. coli plasmid pBR322; double-stranded DNA and pBR322 in Ie experiment
Perform almost the same procedure as e, replacing BamH digestion with Hind digestion (change the salt concentration during digestion to a low concentration for Hind digestion), and prepare an aqueous solution of Hind-digested double-stranded DNA.
17μ and pBR322 Hind/alkaline phosphatase treatment (DNA 1μg/1μ) solution 1μ and the buffer for DNA binding enzyme 2μ and
A reaction was carried out at 14°C for 22 hours using 0.5μ of T4 DNA ligase, and hybrid DNA was obtained in the same manner as in the experiment in e. Using this hybrid DNA, E. coli HB101 was transformed into competent cells by the same procedure as in e. 37 transformants were obtained.
Plasmids from 15 of these 37 colonies
Perform a mini-prep of DNA and this plasmid DNA
was decomposed into Hind. As a result, a hybrid plasmid containing a fragment of approximately 2.69 Kbp was obtained. This hybrid plasmid DNA was named pMYH3. The hybrid DNA pMYH3 was replicated and propagated in E. coli, and the entire DNA was completely double-stranded. Hind hybrid DNA pMYH3,
Decompose with Hind/BamH, Hind/Pst,
The degraded fragments were analyzed in the same manner as e-1. The sizes of the obtained fragments were as shown in Table 3.

[Table] The fragments generated by digestion of pMYH3 with each restriction enzyme in Table 3 are shown in the restriction enzyme cleavage map in Figure 2, although the size of each DNA includes a measurement error of about ±0.05 maximum ±0.1 Kbp. It matched well. Hybrid obtained in Examples e and If
DNA pMYB4 and pMYH3 are hybrids of the DNA shown in Figures 1 and 2 and pBR322, respectively.
DNA, and these can also be used as vectors for plant recombination. g Hybrid DNA pMYB4 and pMYH3
Separation of aDNA and bDNA from and their combined circularization 20 μg each of hybrid DNA pMYB4 and pMYH3 was mixed with 20 units of BmH,
Using Hind20 units, set the salt concentration with the buffer for each restriction enzyme,
Hydrolysis was carried out at 37°C for 10 hours at a total of 400μ.
Next, 0.8% agarose gel (slot width 1.5 mm,
(length 6 cm, depth 7 mm, gel length 13 cm) was used.
After electrophoresis at 1V/cm for 10 hours, aDNA,
Cut out the bDNA band part along with the gel and use the method described on pages 164 to 165 of the experimental book by Maniathes et al.
DNA was extracted from the gel, and then purified according to the method of P166. This linear
Both aDNA and bDNA are sterilized with 50μ water.
(approximately 0.1 μg/μ DNA). Each of these linear DNA-a and DNA-b
Add 5μ of the buffer for DNA binding enzyme, 25μ of sterile water, and T4 DNA ligase to each 20μ.
After adding 0.5 μ and reacting at 12℃ for 19 hours, perform 0.8% agarose gel electrophoresis again as above, cut out the DNA band portion with an apparent size of 3.3 Kbp along with the gel, and use the same method as above. Only the DNA was extracted and purified. This DNA is considered to be circular DNA-a and DNA-b, respectively. Next, add 10μ of sterile water to each of this DNA, and for each 2μ
Partial decomposition by BamH and Hind was performed.
For partial degradation, add 5μ of the restriction enzyme buffer and 43μ of sterile water to a 2μ solution of DNA, and then add
For DNA-a, 0.5 unit of BamH,
Add Hind0.5 unit to DNA-b,
The reaction mixture was reacted at 37°C for 5 minutes, 10 minutes, and 30 minutes, and each reaction product was subjected to 0.8% agarose gel electrophoresis to observe changes in size. As the reaction was carried out for 5, 10, and 30 minutes, the 3.3Kbp band gradually decreased.
DNA/a and DNA-b linear DNA 2.70Kbp,
The 2.69 Kbp band increased, and no other bands were observed. From this, the above circular DNA-a, DNA-
b are both monomeric and cyclic,
These are the DNAs shown in Figures 1 and 2, respectively. this
DNA is completely double-stranded throughout and can be used as a vector for plant recombination. h MYMV replicative DNA from MYMV-infected plants
extraction analysis; Hamilton et al.'s method [Nucl. Acids, Res,
10, 4902 (1982)], 40 ml of 0.5 M
Add KH ₂ PO ₄ and 0.75% Na 2 SO ₃ (PH=7.0) and homogenize in a mortar, then add 1.2 ml of Triton X-100 4
Stirred at ℃ for 12 hours. Next, pass through double gauze, centrifuge the liquid portion at 7000 rpm for 15 minutes using a Tommy high-speed centrifuge No. 4 rotor, remove the supernatant, and transfer this supernatant to a Beckman ultracentrifuge with a type 32 angle rotor. Centrifugation was performed at 32,000 rpm for 4.5 hours. Discard the supernatant and add 1ml of 40mM to the pellet.
Tris HCl, 5mM acetic acid, 10mM EDTA (PH=
Add 8.2) and homogenize, add 20 μ of 10% SDS, add 1 ml of water-saturated phenol three times, then 1 ml.
Proteins, etc. were poured out from the aqueous layer three times with phenol/chloroform (4/1). Next, ether extraction was performed three times with 1 ml of ether to remove the phenol, and 100μ of 3M sodium acetate and 3 ml of ethanol were added to the aqueous layer at -20℃.
After standing for 5 hours, high speed centrifugation was performed to obtain a DNA pellet. Add 500μ of sterile water to this DNA pellet (DNA concentration 0.47μg/μ), perform 0.8% agarose gel electrophoresis on 100μ using the same method as above, and perform band 1.6 that hybridizes with the ^32P -labeled DNA probe of MYMV. DNA equivalent to Kbp, DNA equivalent to 3.2Kbp,
5.4Kbp portion of DNA, 10Kbp portion of DNA
was removed from the gel and purified in the same manner as in g (the portion corresponding to 5.4 Kbp and the portion corresponding to 10 Kbp were λ-
Using DNA EcoR/Hind size markers, a corresponding portion of each gel was cut out). These four types of DNA are BamH and Bal, respectively.
, and Bal/Pst, agarose gel electrophoresis was performed, and the degradation pattern was analyzed.
For the DNA equivalent to 5.4 Kbp and 10.0 Kbp, the agarose gel electrophoresis DNA was transferred to a nitrocellulose filter, and DNA-DNA hybridization and autoradiography were performed using ^32P -labeled MYMV DNA. The decomposition pattern of was analyzed. The degradation pattern is 2.66, 2.01, and 0.68Kbp for BamH degradation, and 2.66, 2.01, and 0.68Kbp for Bal degradation.
2.71, 1.64, 1.04 Kbp, and Bal/Pst digestion showed a degradation pattern of 2.27, 1.67, 1.02, and 0.44 Kbp, which is the same as the degradation pattern of restriction enzymes a and b, that is, this replication type The DNA contains DNA-a or DNA-b and can be used as a vector for plant recombination. i Hybrid of replicative DNA and pBR322
Preparation of DNA: The above-mentioned DNA (0.9 μg DNA/10 μ) was extracted and purified from agarose gel from a band portion with an apparent size equivalent to 1.6 Kbp of the replicated DNA of h.
Hind restriction enzyme buffer 4μ, sterile water 24μ
, add 3 μ of Hind and hydrolyze at 37℃ for 2 hours.
Add 65 μg of sterile water and 20 μg of yeast tRNA to this.
The protein was removed twice with 100μ of water-saturated phenol, and then phenol was extracted from the aqueous layer three times with 150μ of ether. Then 10μ 1.5M
Sodium acetate (PH=4.8), plus ethanol
After adding 300μ and standing at -20°C for 2 hours, the mixture was centrifuged at high speed to obtain a precipitate. Cold 70% ethanol 500μ
The precipitate was washed with water, centrifuged again, and the supernatant was discarded, and the precipitate was dried at 2 mmHg for 3 minutes. 10μ of this precipitate
Dissolve the DNA in sterile water and use it as Hind of the replicative DNA.
Treat it as a decomposition product. Hind-digested pBR322, the same as used in f, was added to this Hind-digested product (6 μg of DNA ~0.5 μg).
Alkaline phosphatase treatment solution (DNA1μg/
μ) 4 μ, buffer for the DNA-binding enzyme
2μ and T4 DNA ligase 0.5μ, sterile water 7.5μ
In addition, the reaction was carried out at 14°C for 13 hours, then at 65°C for 5 minutes, and E. coli was isolated using the same procedure as in f.
Transform HB101 competent cells,
Approximately 130 transformants were obtained. L- in which colonies of transformants have arisen
Place a nitrocellulose filter (BA85 manufactured by Schleicher and Schiill, same as above) on the Agar plate, then remove the nitrocellulose filter, soak it in 0.5M sodium hydroxide 1.5M saline solution for 1 minute, and then 1M
Immersed in Tris HC1 (PH=7.0) solution. This nitrocellulose filter was heated to 80°C for 2 hours to 1mm
The pressure was reduced to Hg and heat treated. Add this heat-treated filter to the prehybridization solution [0.9M
NaCl 0.09M sodium citrate, 0.02% polyvinylpyrrolidone (Wako Pure Chemical Industries, Ltd. Plasdone NP)
K-30), 0.02% Ficoll (Ficoll 400, manufactured by Pharmacia Fine Chemicals), and 0.02% bovine serum albumin aqueous solution], pretreated at 63°C for 30 minutes, and then removed from the prehybridization solution and freshened. The prehybridization solution contained 10% SDS and 40 μg/ml yeast tRNA.
Hybridization solution and the MYMV ^32P probe below were added 200x.
Add 10 ⁴ cpm, place the treated nitrocellulose filter into this solution, and heat at 63°C, 24°C.
Time DNA-DNA hybridization was performed. Next, take out this filter and remove a large amount of
The filter was washed by gentle shaking in 0.45M NaCl and 0.045M sodium citrate at 55°C for 20 minutes. This washing is carried out three times, dried, and then this filter is used to filter the
Autoradiography was performed using the same method as P470-471. ³² P-labeled MYMV DNA
Eight colonies were found to hybridize with. Preparation of ^32P probe for MYMV; Perform the same procedure as in c except that 0.8mM deoxycytidine triphosphate was added to 0μ instead of 10μ in the experiment in c, and Nos. 6 to 11 were prepared by gel filtration with Sephadex G-75. The first ³² P peak (total 300×10 ⁴ cpm) was found in the fractions, and these fractions were collected (total 800 μ), heated at 95° C. for 5 minutes, and then rapidly cooled to 0° C. This liquid was labeled with ³² P.
Use as a DNA probe for MYMV. From these 8 colonies, the above-mentioned boiling
Mini-preparation of plasmid DNA was performed using the Lysis method, and the plasmid DNA was subjected to Hind degradation. As a result, a hybrid plasmid containing a fragment of approximately 2.69 Kbp was obtained from 6 colonies. One of these hybrid DNAs was named pMYRFH.
pMYRFH Hind/BamH, Hind/
When digested with BamH/Bgl, the same DNA fragments in Table 3 of If were shown. Ii Hybrid between replicative DNA and pBR322
Preparation of DNA; DNA extracted and purified from the band portion of the Ih replicative DNA with an apparent size equivalent to 3.2 Kbp.
Digested with BamH and converted to pBR322, which is similar to e.
Hybrid DNA was obtained with BamH/alkaline phosphatase treatment solution, and further E. coli
Plasmids were extracted and analyzed from the diameter transformants obtained by transforming HB101 in the same manner as Ie, and the Bam site of pBR322 contained approximately 2.68 Kbp, approximately
Hybrid DNAs containing 2.03Kbp and approximately 0.68Kbp BamH fragments, pMYRFB1 and
pMYRFB3 and pMYRFB4 were obtained. pMYRFB1 is BamH/Bgl, BamH/
In the decomposition of Xba, the same as in Table 2 of e-1
Generated DNA fragments. From the results of i and j, this replicative DNA is
It was confirmed that DNA-a and DNA-b were included. Using the method of this experiment, DNA-a,
It is also possible to separate each DNA-b and also to propagate it.

[Brief explanation of the drawing]

FIG. 1 and FIG. 2 are each according to the present invention.
This shows a restriction prime cleavage map of DNA. Third
The diagram corresponds to the target DNA of each restriction enzyme in Figure 1.
This is a genetic map showing the DNA base sequence. Figure 4 corresponds to the target DNA of each restriction enzyme in Figure 2.
This is a genetic map showing the DNA base sequence.

Claims (1)

[Claims] 1. The single-stranded DNA of the mung bean yellow mosaic virus is at least partially double-stranded.
DNA, represented by the genetic map below
Mono-trimeric DNA. [Here, the base sequence of one strand of the double strands of P ₁ to _{P 6} is as follows. P ₁ (0.0/2.70): 5′−GGATCC−3′ P ₂ (0.96): 5′−AAGCTT−3′ P ₃ (1.30): 5′−CTGCAG−3′ P ₄ (1.74): 5′− AGATCT-3′ P ₅ (2.03):5′-GGATCC-3′] 2 Claim 1 characterized in that at least a part of it has been methylated in another organism.
DNA as described in section. 3 Claim 2 in which the other organism is E. coli
DNA as described in section. 4. The DNA according to claim 1, which has been double-stranded in vitro. 5. The DNA according to claim 1, which has been double-stranded in vivo. 6. The DNA according to claim 1, which is a monomer. 7 The single-stranded DNA of Mung Bean Yellow Mosaic Virus has become at least partially double-stranded.
DNA, represented by the genetic map below
Hybrid DNA containing DNA. [Here, the base sequence of one strand of the double strands of P ₁ to _{P 6} is as follows. P ₁ (0.0/2.70): 5′−GGATCC−3′ P ₂ (0.96): 5′−AAGCTT−3′ P ₃ (1.30): 5′−CTGCAG−3′ P ₄ (1.74): 5′− AGATCT-3′ P ₅ (2.03):5′-GGATCC-3′] 8 The hybrid according to claim 7, which is at least partially methylated in another organism. DNA. 9 Claim 8 in which the other organism is E. coli
Hybrid DNA as described in Section. 10. The hybrid DNA according to claim 7, which has been double-stranded in vitro.