JPH0549271B2 - - Google Patents

Description

[Detailed Description of the Invention] Technical Field of the Invention The present invention relates to the Bean Golden Mosaic Virus (hereinafter sometimes abbreviated as "BGMV").
Concerning DNA and hybrid DNA incorporating it. To explain in more detail, the single strand of BGMV
DNA obtained by double-stranding DNA, this DNA
Hybrid DNA obtained by inserting BGMV into a vector for other organisms, Hybrid DNA obtained by propagating this hybrid DNA, and Hybrid DNA obtained by extracting only the BGMV-derived DNA fragment from this hybrid DNA and circularly ligating it. was given
Concerning DNA and replicative DNA of BGMV. Prior Art DNA genes from viruses have recently been widely developed and used as vectors in genetic recombination technology. An example of a virus that provides such a vector is the simian virus (SV).
40 and papovaviruses such as polyomavirus,
Papillomaviruses and adenoviruses are known. However, these known vectors were discovered as animal-based vectors, and
It does 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 serve as a vector for plant genetic recombination. Recently, Robert M. Goodman and colleagues at the University of Illinois in the United States have discovered that BGMV, a tropical plant virus, has a diameter of about 18 nm.
A single virion is formed by pairing two particles of m, and as a result of analyzing its nucleic acid,
They discovered and reported that the gene of this virus is a circular single-stranded DNA with a size of approximately 2500 bases [Virology 83 171 (1977), Virology 97 388
(1979)]. Subsequently, several types of plant viruses were discovered in which two particles with a diameter of approximately 18 nm are paired to form a pillion, and the gene is a circular single strand. The viruses are called 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 be used not only for dicotyledonous plants, but also for dicotyledonous plants. Monocots such as wheat and corn, which are important grains for humans, can also serve as hosts, so 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 means that when the dieminivirus is vectorized,
This indicates that it is highly possible to modify the nuclear genes of plants themselves. Therefore, it is clear that if the DNA of the dieminivirus could be turned into a vector, it would be of industrial value in itself. Structure of the present invention Therefore, the present inventors have developed a single-stranded DNA of Beer Golden Mosaic Virus (BGMV), 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, the present invention is a DNA obtained by at least partially double-stranding (i) a single-stranded DNA with a length of about 2.57 kilobases from BGMV, and which shows the restriction enzyme map shown in Figure Ia. (hereinafter abbreviated as DNA-a) and (ii) single-stranded DNA with a length of approximately 2.61 kilobases.
DNA obtained by at least partially double-stranded
Among the DNAs (hereinafter abbreviated as DNA-b) shown in the restriction enzyme map of Figure Ib, DNA-b is the main part. That is, according to the present invention, the single-stranded DNA of the bean golden mosaic virus is at least partially double-stranded DNA, and the DNA represented by the genetic map in Figure Ib' (hereinafter referred to as DNA-Ib) (sometimes abbreviated as ) is provided. Furthermore, the above-mentioned DNA, which is characterized in that at least a portion thereof has been methylated in other organisms,
The above DNA, where the other organism is E. coli, in vitro
This includes the above-mentioned DNA double-stranded in vivo, the above-mentioned DAN double-stranded in vivo, the above-mentioned DNA characterized by being a monomer, and the like. Further, according to the present invention, the single-stranded DNA of the bean golden mosaic virus is at least partially double-stranded DNA, and the DNA represented by the genetic map in Figure Ib' is cleaved with a restriction enzyme, Hybrid DNA combined with other biological vector DNA or third DNA is provided. In addition, the above-mentioned hybrid DNA characterized by being at least partially methylated in another organism, the above-mentioned hybrid DNA whose other organism is Escherichia coli, and the above-mentioned hybrid DNA double-stranded in vitro. hybrid DNA etc. are provided. Additionally, the single-stranded DNA of the bean golden mosaic virus was at least partially double-stranded.
DNA, represented by the genetic map in Figure Ia′
A mixture of DNA mono-trimers and DNA mono-trimers represented by the genetic map of Figure Ib' is provided. In the restriction enzyme maps of Figure Ia and Figure Ib, DNA
The target DNA for each restriction enzyme to accurately represent itself.
The corresponding DNA base sequences are shown in Figure Ia' and Figure Ib'. In Figure Ia′, the base sequence at P ₁ to _{P 7} is
The base sequence of one of the double strands is as follows. P ₁ (0.0/2.57): 5′−AAGCTT−3′ P ₂ (0.82): 5′−ATCGAT−3′ P ₃ (0.97): 5′−AGATCT−3′ P ₄ (1.18): 5′− GTCGAC−3′ P ₅ (1.38): 5′−CPyCGPuG−3′ P ₆ (2.08): 5′−ATCGAT−3′ P ₇ (2.21): 5′−GTCGAC−3′ In Figure Ib′, P ₁ The base sequence at ~ _P6 is
The base sequence of one of the double strands is as follows. P ₁ (0.0/2.61): 5′−AAGCTT−3′ P ₂ (0.02): 5′−ATCGAT−3′ P ₃ (0.29): 5′−GTCGAC−3′ P ₄ (1.01): 5′− GTCGAC−3′ P ₅ (1.64): 5′−AAGCTT−3′ P ₆ (1.65): 5′−AGATCT−3′ Bean Golden Mosaic Virus (BGMV)
is a virus that causes golden mosaic-like disease in kidney beans in Central and South America.
Phytopathology 67 (No. 1) 37 (1977) was used to purify the following single strand from infected plants.
DNA can be isolated and purified. (1) Isolation of single-stranded DNA from BGMV Virus was purified from infected plants by the method described in the above literature, and then the purified virus
500μg in 30mM Tris HC1 (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 BGMV The single-stranded DNA obtained as described above is then injected into
Double-stranded in vitro. In this double-stranded formation, 0.002 ~
Preferably, it is used at a rate of 2000 μg. As a primer, G.M. Taylor (J.
Biochemica et Biophysica by M.Tayler et al.
An oligonucleotide obtained by degrading calf thymus DNA with DAaseI by modifying the method described in Acta. 442 325 (1976) can be used. The present inventors used primers obtained by this modified method as described in the Examples below. When using this oligonucleotide as a primer, the amount of primer is 1μ of the single-stranded DNA.
50-400 μg per g, more preferably 100-300 μg
The ratio of g is appropriate. In addition, when using purified DNA containing at least 10 bases and having a completely complementary sequence to the single-stranded DNA of BGMV as a primer,
The usage rate of the primer may be 0.002 to 1 μg, or even 0.004 to 0.02 μg, per 1 μg of single-stranded DNA. 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-32p-decoxycytidine triphosphate (d-32p-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.) is used in a concentration range of 1 to 50 mM, preferably 5 to 21 mM, to promote the enzyme reaction. In this specification, "M" means a concentration unit of "mol/" unless otherwise specified. A buffer such as Tris HC1 is used to maintain the pH within the reaction system in the range of 7.2 to 9, preferably 7.6 to 8.4.
(PH7.6-8.4) at 30-300mM, preferably 70-300mM
Advantageously, a concentration of 200mM is used. Thus, the above mixture is preferably 50~
Maintain at a temperature of 80°C, especially 60-70°C, for 1-10 minutes, preferably 3-7 minutes, then if necessary approx.
Rapid cooling to ℃ or lower. The total amount of the mixed solution is based on the amount of single-stranded virus.
1 to 1000μ, preferably 10 per 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, and examples of the enzyme for double-stranding include Avian myeloblastosis virus (AMV).
reverse transcriptase, T4-DNA polymerase, E. coli DNA polymerase I, DNA polymerase
Examples include large fragment (Klenow enzyme). 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, when using AMV reverse transcriptase (code 120248 manufactured by Seikagaku Corporation), it is effective to use 1 to 20 units, preferably 5 to 10 units, per 1 μg of single-stranded DNA. If the number is less than 1 unit, it may be difficult to form double strands sufficiently. After adding the double-stranded enzyme, firstly at a temperature of 10-30℃, preferably 15-25℃ for 2-30 minutes, preferably 5 minutes.
react for ~20 minutes, then at a temperature of 30-45 °C, preferably 35-40 °C for 30-180 minutes, preferably
It is advantageous to react for 50 to 120 minutes. To stop the reaction, add water-saturated phenol or EDTA aqueous solution (PH = 8.0) to the reaction mixture.
You can add a stopper such as At that time, in the case of water-saturated phenol, 1/10 to 2
It is only necessary to separate and fractionate only the aqueous layer by doubling the volume, shaking, and centrifuging, and in the case of an EDTA aqueous solution, it is sufficient to use an amount sufficient to trap Mg ⁺⁺ present in the mixed solution. The 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 this case, a double-stranded DNA-containing aqueous solution is treated with Sephadex G-10 [manufactured by Pharmacia Huain Chemical Co., Ltd.] or Biogel p30 [Bio-Rad Chemical Co., Ltd.].
The 32p intensity of each fraction is measured by fractionation using a gel-filtration agent, such as the one manufactured by Co., Ltd., and the high molecular weight DNA-containing fraction with high 32p intensity that appears first in each fraction is collected. If this is precipitated in ethanol or isopropanol, double-stranded DNA can be obtained. Double-stranded DNA thus separated
is a double-stranded version based on the single-stranded DNA of BGMV.
It may contain a considerable amount of primer DNA other than DNA. This tendency is remarkable when oligonucleotides from the calf thymus DNA are used as primers. Therefore, in such cases double-stranded DNA
It is desirable to purify it again to make it even more pure. This purification can be carried out using the gel filtration method described above. To give a specific example, when starting from 20g of single-stranded DNA, the double-stranded DNA is diluted with 100 to 500μ of water.
Dissolved in 10mM Tris HC1 (PH=7.4), 100mM
Sephadex G- equilibrated with M NaCl aqueous solution
Gel filtration is performed using a column packed with 50 or Biogel p30 to improve separation performance. 10mM as eluent
Using Tris HC1 (PH = 7.4) and 100mM NaCl, the fraction with a high 32p count in the void volume was collected and precipitated in ethanol or isopropanol to obtain highly purified double-stranded BGMV DNA.
can be obtained. (3) Double-stranded DNA There are two types of DNA in the dieminivirus in BGMV described above, and there are also two types of DNA obtained by double-stranded DNA as described above. This 2
The double-stranded DNA of a species can be separated into individual DNAs, or can be cloned and then separated. Also, before making double strands (in other words, in the state of single stranded DNA), separate each
Double stranding can also be performed in the same way for each. DNA-a mixture of single-stranded DNA of BGMV
In order to separate DNA-b and DNA-b, a method called “strand separation” is used [for example, in the book “Molecular
Cloning-A Laboratory Manual” No. 180~
[method described on page 185] In other words, a method using gel electrophoresis, or a DNA fragment complementary to only either Ia or Ib DNA is added to a cesium chloride solution of a DNA mixture and single-stranded DNA is isolated by equilibrium density gradient centrifugation. It is possible to employ a separation method that utilizes the density difference between the DNA that remains intact and the partially double-stranded DNA. However, the separation of DNA-Ia and DNA-Ib is a single strand.
Separation is easier if the DNA mixture is made into double strands and then cloned into plasmid pBR322, for example, and then separated. This separation method will be explained in detail later. In this way, the DNA obtained by double-stranding the DNA from BGMV has a size of 2.57 ± 0.1 kilobase pairs (hereinafter "kilo base pair" may be abbreviated as "Kbp"). The restriction enzyme cleavage map of Figure Ia is shown. The other one has a size of 2.61±0.1 kilobase pairs (Kbp) and shows the restriction enzyme cleavage map in Figure Ib. The corresponding genetic maps are shown in Figure Ia' and Figure Ib', respectively. In Figure Ia, the Haid cleavage site is set to [0.0/
2.57], each restriction site is shown as a positional coordinate in kilobase pairs (Kbp).
Also, in Figure Ib, one position of Hind is set to [0.0/
2.61], each restriction site is shown as the positional coordinates. Next, the fragments generated by digestion with each restriction enzyme are shown in Table 1 below. In the table, the fragments that appeared densely compared to the size of the fragments were divided into Group A, and the others were classified into Group B. [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. It should be understood that the values for the size of fragment DNA in the table above may vary by ±0.1 Kbp, especially ±0.05 Kbp due to the accuracy of experimental analysis. For restriction enzyme digestion, use a concentration of 10mM Tris.
The test was carried out using HC1 (PH=7.9), 7mM MgCl ₂ , 7mM β-mercaptoethanol, and 0.01% bovine serum albumin as the base solution. Decomposition with Cla was performed using the above basic solution, and Hind. Ava, Bgl, Kpn,
For decomposition by Pst and Pvu, add NaCl to the base solution.
Add to 50mM and also add Sal, BamH
, Xba, and Xho are
NaCl was added to give a concentration of 150mM. In the present invention, restriction enzymes include Hind, Sal,
Bgl, BamH, Kpn, Pst, Pvu and
For Xho, we use products manufactured by Takara Shuzo Co., Ltd., for Cla, we use products manufactured by Boehringer Mannheim, and for Ava and Xba, we use products manufactured by Bethesda Research Laboratories. did. In the present invention, all restriction enzymes for cutting DNA were used at a ratio of at least 4 units/1 μg of DNA, and the digestion was carried out at 37° C. for 4 hours or more. When using two types of restriction enzymes, first digest at 37℃ for 2 hours or more with an enzyme for low salt concentration, then adjust the concentration to a high temperature, and then further digest with an enzyme for high salt concentration.
Decomposed at 37°C for over 2 hours. Thus, the DNA after enzymatic degradation was analyzed by electrophoresis on a 1.5% agarose gel containing 0.5 μg/ml of ethidium bromide to identify the fragments generated by each enzymatic degradation.
We conducted DNA analysis. In addition, during this electrophoresis,
EcoR of λ-DNA as a DNA size marker
/Hind digested product and 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 compared to the single-stranded DNA, and is not necessarily double-stranded over the entire region. Not 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 desirably at least 80% of the base pairs of single-stranded DNA, preferably at least
It is sufficient that 90% of the chain is double-stranded. Ia and Ib
Different values of the DNA ratio can be obtained depending on the growth process of BGMV, but generally the Ia/Ib (mol) ratio is 3 or more. Therefore, as shown in Table 1 above, it is possible to group the fragments into A group fragments and B group fragments. The double-stranded DNA-Ia and Ib of the present invention can be used alone or in combination as a vector for plant genetic recombination. Also DNAIa
Alternatively, Ib can be cleaved at a specific restriction site with a restriction enzyme to form linear DNA. This DNA is mixed with a third useful DNA cut out with the same restriction enzyme in approximately the same molar ratio, and then processed using a conventionally known method.
A third DNA is attached to a specific site of Ia or Ib by a DNA-binding enzyme (e.g. T4-DNA ligase).
It can be a circular DNA containing In this case, at least one kind of restriction enzyme may be used for the cleavage, and although the size of the fragment varies depending on the type of enzyme used, a fragment within a range that does not impair the function as a vector can be used. For example, if the third DNA encodes a gene that confers resistance to the antibiotic Kanamaishi, plant cells can be transformed by using DNA that incorporates this third DNA into DNAIa or Ib. The cells can be transformed into kanamycin resistant cells. In this case, the transformation method is to transform the plant cells into protoplasts.
Ca ⁺⁺ , in the presence of polyethylene glycol or Ca ⁺⁺
A method of causing the above DNA to act in a high PH state in the presence of
Furthermore, there is a method of mechanically introducing the above-mentioned DNA into plant cells (microinjection). Further, the double-stranded DNA Ia or Ib of the present invention can be preferably cut at a single site by an enzyme such as
It can be digested with Hind, Cla or Bgl and then inserted into other biological vectors cleaved with the same restriction enzymes as shown below. In this case, other biological vectors include conventionally known vectors such as plasmid vectors for E. coli (e.g. pBR322),
cosmid vectors (e.g. pKY2662), phage vectors (e.g. Syaron 10), plasmid vectors for Bacillus subtilis (e.g. pUB110, pSA0501),
Vectors for yeast (eg, YRp7) can be used. Hybrids with these other biological vectors
The method for preparing DNA is to prepare double-stranded DNA-Ia
A mixture of DNA-Ib and DNA-Ib is completely digested using, for example, Hind, and this is mixed with a Hind-digested product of another biological vector, such as E. coli plasmid pBR322, and then injected into a DNA binding enzyme (such as T4-DNA ligase). DNA-Ia by bond cyclization
and pBR322 hybrid DNA can be obtained. Similarly, double-stranded DNA-Ia and DNA
The mixture with -Ib is completely digested with Cla, this is mixed with the Cla-digested product of pBR322, and the mixture is bonded and cyclized with a DNA-binding enzyme to form DNA-Ib.
Hybrid DNA with pBR322 can be obtained. 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, for other biological vectors, after cleavage with restriction enzymes, treatment with alkaline phosphatase to dephosphorylate the 5' end of the DNA is recommended.
This is preferable since self-circularization can be largely prevented by using vectors for other organisms alone when preparing vectors. The hybrid DNA thus obtained is also
Like the DNA in Ia or Figure Ib, it is at least partially double-stranded and can be used as a vector for plant genetic recombination. This hybrid DNA is transferred to the original host of other biological vectors, such as E. coli in the case of a hybrid DNA with a vector for E. coli, and Bacillus subtilis in the case of a hybrid DNA with a vector for Bacillus subtilis.
Furthermore, hybrid DNA with yeast vector
In the case of yeast, for example, Escherichia coli and Bacillus subtilis are transformed into competent cells according to known methods, and in the case of yeast, they are treated with Zymolys to form spheroplasts. Transformants can be selected by transformation and appropriate drug resistance or auxotrophy, or by colony hybridization. To explain in more detail one specific example of this method, for example, the hybrid DNA is the Hind digested product of Figure Ia (Cla digested product in the case of Ib) and the Hind/digested product of pBR322 of the E. coli plasmid (in the case of Ib).
If the hybrid DNA is a hybrid DNA obtained by ligating a Cla-digested product) with T4-DNA ligase, this hybrid DNA is prepared by the method of Mandel and Higa [J.Mol.Biol.
53 159 (1970)], and colonies grown on L-Agar plates containing 50 μg/ml ampicillin were transformed using the method of Grunschütstein and Hovnes [Proc.
Natl.Acad.Sci. 72 , 3961] to perform colony hybridization (in this case, 32p-labeled BGMV DNA is used as a probe) to identify DNA that hybridizes with 32p-labeled BGMV DNA. Select a colony. In this way, the DNA and plasmid in Figure Ia (or Ib)
Escherichia coli HB101 can be obtained transformed with a hybrid DNA in which pBR322 is linked at the Hind (Cla in the case of Ib) site. Hybrid DNA can be isolated from the transformant thus obtained by a known method. The isolated hybrid DNA is almost completely double-stranded throughout, and can transform plant cells by incorporating a third DNA into a specific site, and can also grow in bacteria such as Escherichia coli. 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 grow only the hybrid DNA to lyse the bacterial cells, and the DNA is extracted from the aqueous solution containing the hybrid DNA. Just separate it. When the hybrid DNA is, for example, a hybrid DNA with plasmid pBR322 for E. coli, an E. coli transformant containing the hybrid DNA can be used, for example, in the experimental book "Molecular Cloning-A Laboratory" by Maniatis et al.
Manual” [Cold spring Harbor Laboratory
(1982)] using the methods described on pages 88 to 94 to obtain DNA using amplification of hybrid DNA and various lysis methods, and using a covalently closed cycle (hereinafter referred to as
This makes it possible to separate only hybrid DNA of the type (abbreviated as “ccc”). In addition, hybrids separated in this way
By digesting the DNA with the same restriction enzymes used for cloning, it can be separated into the DNA fragment shown in Figure Ia (or Ib) and the vector DNA fragment.
Preferably, the DNA fragment of Figure Ia (or Ib) is isolated,
Next, cyclization is performed using T4-DNA ligase or the like. To separate only the DNA fragment shown in Figure Ia (or Ib), agarose gel electrophoresis is generally performed, and the band portion of the DNA fragment shown in Ia (or Ib) is cut out from the gel, as described on pages 164 to 172 of the above-mentioned experimental book by Maniathes et al. DNA fragments can be separated and purified from gels according to various methods. The DNA fragments thus isolated are processed by known methods, for example, T4-
Combined cyclization using DNA ligase, in which a
(or b) In addition to self-cyclization of only one DNA, some forms are formed by bonding and cyclizing two or more DNAs. Only one of these self-cyclizes, resulting in an apparent 3.3 Kbp band in agarose 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 BGMV can be detected from infected leaves where BGMV is infected and proliferating, such as infected leaves of top crop 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 total DNA described in "Introduction to Plant Cell Breeding (Gakkai Publishing Center)" by Atsushi Hirai et al., pp. 86-88. This can be done using an 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 then the DNA is transferred from this gel to a nitrocellulose filter using Southern's method [see E.M. Southern J.Mol.Biol 98 503-517 (1975)]
of heat-denatured BGMV transferred and labeled with 32p.
Using DNA as a probe, Maniaiatis et al.'s ``Molecular Cloning-A Laboratory''
DNA-DNA hybridization is carried out in the same manner as described on pages 387-389 of the same book, and then autoradiography is carried out in the same manner as described on pages 470-471 of the same book. DNA that hybridizes with 32p labeled BGMV DNA
This part appears on the X-ray film as a black band during autoradiography, and this part is the BGMV.
Indicates that the DNA is derived from Approximately 10 types of such bands are recognized, and their apparent sizes are >20Kbp, 9.8Kbp, 6.8Kbp, 5.2Kbp,
3.8Kbp, 3.1Kbp, 2.5Kbp, 1.9Kbp, 1.5Kbp,
It is 0.87Kbp. DNA that gives these bands
is present in various forms [e.g. CCC, open circle (OC), linear monomer and various forms of dimer,
It is inferred that the formation of many different types of bands is due to the presence of trimers, etc.). Among these bands, the 0.87Kbp band is the single-stranded DNA of BGMV.
, but all others are double-stranded DNA.
This is the replicative DNA of BGMV. These replicative intermediate DNA bands are removed from the agarose gel, and only the DNA is separated from the gel using the same procedure as above, and when digested with Cla, Hind, and Sal, the same DNA as in Figures a and b is obtained. You can get fragments.
By doing this, 9 other than DNA equivalent to 0.87Kbp
The DNA that gives each type of band is shown in figure a or b.
It takes various forms based on DNA.
This is the replicative intermediate DNA of BGMV. A portion of the DNA that gives these bands, equivalent to 1.5 Kbp,
DNA equivalent to 3.1Kbp, DNA equivalent to 9.8Kbp
Since DNA is present in relatively large amounts, it is easy to use. Thus, a portion of DNA corresponding to 1.5 Kbp, 3.1 Kbp or 9.8 Kbp is degraded with Hind and cloned into the Hind site of plasmid pBR322 for E. coli using the above-mentioned method to obtain a hybrid DNA containing the entire DNA-a as described above. Also 1.5Kbp
(or 3.1Kbp or 9.8Kbp) of DNA
By degrading the b-DNA and cloning it into the Cla site of pBR322, a hybrid DNA containing the entire b-DNA can be obtained as described above. When DNA obtained by cloning is replicated in E. coli that has a dan methylase gene, certain base sequences, for example, the N at position 6 of A in GATC, are methylated, and in the DNA obtained by cloning, the restriction enzyme of the original DNA is methylated. Although it may be observed that the site appears to have disappeared, the base sequence itself is the same. Furthermore, DNA obtained through cloning has the same infectivity as a virus, just like DNA extracted directly from a virus. The present invention will be described in detail below with reference to Examples, but the present invention is not limited thereto. Example 1 Isolation and purification of a virus 250ml of 57gr of BGMV-infected top crop leaves
Grind in 0.1M sodium phosphate-10m MEDTA buffer (PH = 7.8) (containing 1.3gr of cysteine), pass through double gauze, and strain the liquid at 10000G-
Centrifugation was performed for 40 minutes at 4°C to obtain 205 ml of supernatant. To this supernatant, 2.4 g of salt and then 8.2 gr of polyethylene glycol (w7800-9000) were added, stirred at 4°C for 1 hour, and centrifuged at 10000G for 25 minutes at 4°C. After removing the supernatant, add 0.1M sodium phosphate buffer (PH=
7.8) Add 10 ml to homogenize, centrifuge at 10,000G for 30 minutes at 4℃, remove the supernatant, and centrifuge the supernatant at 4℃ for 4 hours at 30,000 rpm using a Beckman ultracentrifuge SW40.1 rotor. The mixture was centrifuged to obtain crude virus as a precipitate. 0.5ml of this crude virus
After homogenization in 0.1 M sodium phosphate buffer (PH = 7.8), continuous 10-40% sucrose density gradient centrifugation (using the same SW40.1 rotor as above)
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. These fractions were collected and diluted with double the amount.
Add 0.1M sodium phosphate buffer (PH = 7.8) and homogenize it, then use SW40.1 rotor for 35,000 yen.
Obtain the virus pellet again at 4°C for 3 hours at rpm.
This was homogenized in 0.5ml of 0.1M sodium phosphate buffer (PH7.8) and again 10-40% sucrose continuous density gradient centrifugation (29,000 rpm, 3 hours, 4℃ SW40.1 rotor)
Then, 0.6 ml fractions were collected from the bottom of the centrifuge tube. Only virus peaks were obtained in fractions No. 13 to 18, and these fractions were collected, and after homogenization by adding twice the amount of 0.1M sodium phosphate buffer (PH = 7.8), they were heated at 36,000 rpm for 3 and a half hours at 4℃ SW40. 1 rotor to precipitate the virus to obtain purified BGMV. Isolation of single-stranded DNA genes from b viruses
Purification Add 750μ of sterile water and 15μ of the purified BGMV above.
1M Tris HCl (PH=7.6), 75μ 10% Sodium Dodecyl Sulfate (SDS) 7.5μ Proteinase K
(1 μg/μ solution) was added, shaken at room temperature for 2 minutes, and then diluted with 10 mM Tris HCl (PH=7.6)-1 mM 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 the aqueous layer to extract phenol. This extraction operation was performed three times. The resulting aqueous layer
Add 100μ of 3M sodium acetate buffer (PH = 4.8) and 2.5ml of ethanol to 1000μ and mix at -20°C.
The mixture was kept overnight and centrifuged for 20 minutes at 35,000 rpm in a Beckman ultracentrifuge with an SW50.1 rotor to precipitate DNA. Transfer this DNA to 10mM Tris HCl 0.1mM
BGMV single strand dissolved in 600μ EDTA aqueous solution
A DNA 0.5 μg/μ aqueous solution was prepared. In vitro duplexing of cBGMV single-stranded DNA; BGMV single-stranded DNA (0.5μg/μ) 2μ, sterile water 155μ, 1M Tris HCl (PH = 8.0) 30μ, calf thymus DNA prepared by the following method. Mix 12μ of oligonucleotide (16.6μg/μ) at 70
℃ for 3 minutes, then rapidly cooled to 0℃, and while maintaining at 0℃, 80mM MgCl ₂ 30μ, 10% β
- 30μ of mercaptoethanol aqueous solution, 30μ each of 0.8mM deoxyadenosine triphosphate, 0.8mM deoxyguanosine triphosphate and 0.8mM deoxythymidine triphosphate, 6μ of 0.8mM deoxycytidine triphosphate and deoxycytidine 5′-
[α-32p] Triphosphoric acid (about 3000Ci/mmol, 10mCi/ml code number manufactured by Amashiyam Japan Co., Ltd.)
pB10205) 2.4 μ and AMV reverse transcriptase (Seikagaku Code 120248, 5 units/μ) were added and reacted for 10 minutes at 20°C and then for 1.5 hours at 37°C.
After adding 50μ of phenol and shaking, the mixture was centrifuged for 5 minutes using an Etzpendorf centrifuge, and the aqueous layer was filtered through a gel. Gel filtration is 10mM Tris HCl (PH=7.4) - 0.1M
Sephadex G-75 pre-equilibrated with NaCl aqueous solution
(manufactured by Pharmacia Huain Chemicals) with a diameter of 6
The sample was passed through a column packed in a 5 ml tube. After adding the reaction solution, collect 4 drops (approximately 700μ) each.
The 32p intensity of each fraction was measured. Fraction No.5-12
The first peak appears at No. 15, and unreacted α
A peak due to -32p-dke oxycytidine triphosphate was observed. Collect fractions No. 5 to 12 and collect 60μ
Add 3M NaCl and 1.8ml of ethanol, hold at -20°C for 3 hours, and then run at 25,000 rpm for 20 minutes (Beckman ultracentrifuge).
SW50.1 rotor), centrifuge, remove the supernatant, and add a new
Add 2 ml of 70% ethanol aqueous solution cooled to 20°C.
The mixture was centrifuged at 10,000 rpm for 2 minutes and the supernatant was removed. Precipitate DNA 20
Dry under reduced pressure of mmHg for 1.5 minutes. The obtained DNA is 1
It was dissolved in 100μ of mM Tris HCl (PH=7.4)-0.1mM EDTA aqueous solution and double-stranded in vitro.
Get DNA. (The intensity of 32p was 46 x 10 ⁴ cpm) Preparation of calf thymus DNA oligonucleotide 66 mg of calf thymus DNA was mixed with 0.1M NaCl 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°C for 3 hours, and add 0.66 ml of 0.2 M EDTA to stop the reaction. Ta. This reaction solution was subjected to protein extraction three times with 7 ml of water-saturated phenol, and then 7 ml of ether was added to remove the phenol from the aqueous layer.
Extraction was performed three times with ml. Add 20ml of ethanol to the resulting aqueous layer, leave it at -20℃ for 2 hours, and then shake at 4000G for 4 hours.
Centrifugation was performed for 1 minute 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 42cm, column diameter
Approximately 1.2 ml of the solution was collected by gel filtration using a filter (0.5 cm). A large peak was observed in the crackles No. 10 to 32, and the fractions No. 16 to 26 were collected (total volume 12.5 ml), 31 ml of ethanol was added, left at -20°C for 30 minutes, and then centrifuged (4000G for 8 minutes). 1 ml of DNA pellet
dissolved in distilled water (DNA concentration 16.6 mg/ml)
was used as the primer DNA. Comparative example 1 Calf thymus DNA oligonucleotide (16.6μg/
A double-stranded experiment was performed under the same conditions as in experiment c, except that 1.5 μ of μ) was used instead of 12 μ, and the count of 32p in the polymeric DNA portion was 8×10 ⁴ cpm. It can be seen that less than 1/5 of the double stranding has occurred compared to the case of . 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)
40μ, c experimental oligonucleotide (16.6μ
g/μ) Used instead of 12μ, sterile water 155μ
Double stranding was carried out in the same manner as in experiment C except that 127μ was used instead of . High molecular
The intensity of 32p in the DNA portion was 15×10 ⁴ cpm. The double-stranded DNA obtained in Comparative Example 2 was treated with restriction enzyme Cla and
Decompose with Hind, Sal, Bgl etc.
When we performed autoradiography in exactly the same way as in the previous experiment, we found that in the Hind-digested DNA, the DNA had not moved from the slot position of the agarose gel at all. Also, those digested with other restriction enzymes,
Some of the DNA remained in the slot position, and the DNA that migrated through the gel also became quite smeared, and the band was not as beautiful as in the case of d. Decomposition and analysis of double-stranded DNA using d restriction enzymes,
Restriction enzyme cleavage map 32p-labeled double-stranded DNA obtained in c (32p intensity 46×10 ⁴ cpm/100μ) 3μ (32p intensity
7300cpm), buffer for restriction enzyme digestion [100mM Tris HCl (PH=7.9), 70mM
_MgCl2 , 70mM β-mercaptoethanol, 0.1%
Bovine serum albumin is used as the basic solution. In Cla digestion, this basic solution is called a buffer for restriction enzyme digestion, and it is used to digest Ava, Hind, Bgl, Kpn, Pst,
For Pvu digestion, add 500mM NaCl to the above base solution.
The solution added until Sal, BamH, Xba, Xho
For digestion, a solution in which NaCl is further added to the base solution to a concentration of 1500mM is called a buffer for restriction enzyme digestion] 4μ, distilled water 32μ, and the restriction enzymes listed in Table 1.
Ava (2 units/μ), BamH (6 units/μ), Bal (6 units/μ), Pst
(5 units/μ), Xba (6 units/μ),
μ), Hind (5 units/μ), Kpn
(6 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 Ava manufactured by Boehringer Mannheim, Xba is manufactured by Bethesda Research Laboratory, Others are manufactured by Takara Shuzo Co., Ltd. ]
4 units of DNA were added and DNA was degraded at 37°C for over 4 hours. When decomposing with two types of restriction enzymes, first add an enzyme for low salt concentration, hydrolyze for 4 hours at 37℃ at a salt concentration suitable for the enzyme, and then add an enzyme suitable for high salt concentration. The salt concentration was adjusted, an enzyme for high salt concentration was added, and the mixture was further hydrolyzed at 37°C for 4 hours. 8μ 0.25% Bromophenol Blue after hydrolysis, 50
Add a solution of % glycerol and 10% SDS and incubate at 65℃5.
Heat treatment was performed for 1 minute, followed by 1.5% agarose gel electrophoresis. The agarose used was for type electrophoresis manufactured by Sigma. Electrophoresis buffer: 40mM Tris acetate, 2mM
An EDTA (PH=8.0) aqueous solution 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, 0.5 μg of λ-DNA was added to EcoR as a size marker for DNA fragments.
and Hind completely disassembled and
0.2 μg of pBR322 DNA was completely digested with Taq and used. After electrophoresis, remove the agarose gel, dry it on a gel drying plate, and dry it on page 470 of Maniathes et al.'s experimental book described in the main text.
Autoradiography was performed as described in ~472. The sizes of the DNA fragments degraded and observed using various restriction enzymes are listed in Table 1 in the main text.
It became like that. Furthermore, after autoradiography, it was observed that
The DNA fragments were classified into Group A, which is a group of fragments that appear more densely compared to the size of the fragment, and Group B, which is a group of fragments that appear lighter compared to the size of the fragment. Furthermore, 3.3 in parentheses indicates that the DNA is in an open circle (OC) state and the apparent molecular weight is 3.3 Kbp relative to the linear DNA size marker. From this table, fragments of group A are generated.
Before being digested with restriction enzymes, the DNA is thought to be in the form of an open circle (OC) and approximately 2.57 Kbp in size.
The exact size can ultimately be determined 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 group A fragments can be described as approximately (2.57±0.1 Kbp).From the same consideration, the DNA that produces group B fragments is OC-shaped and has a size of (2.61±0.1) before being digested with restriction enzymes. Kbp.Next, by analyzing the degradation patterns of various restriction enzymes alone and in combination of the two, the relative positional relationship of the various restriction enzyme cleavage points was determined, and the DNA of group A was determined. Figure a is obtained as a restriction enzyme cleavage map for intact DNA that generates fragments, and intact DNA for group B DNA.
Figure b is obtained as a restriction enzyme cleavage map for . e Hybrid DNA of Hind-digested product of double-stranded DNA and plasmid pBR322 for E. coli;
7.4), Biogel equilibrated with 100mM NaCl aqueous solution
In a column packed with P30 to a diameter of 6 mm and a length of 30 cm,
I did a gel filtration. 10mM Tris as eluent
Using HCl (PH=7.4) and 100mM NaCl aqueous solution,
Four drops (approximately 400μ) were collected. A high count peak of 32p was seen in the 17th to 21st fractions, and when the optical density OD ₂₆₀ of the fraction was measured, the OD was found in the 28th fraction.
The value began to increase and showed large values in subsequent fractions. The 17th to 21st fractions, which are double-stranded DNA, were collected (2.1ml), and 200μ of a 3M aqueous sodium acetate solution (PH = 4.8) and 4ml of ethanol were added to -20
After being maintained at °C for 2 hours, it 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.
Centrifugation was performed at 20,000 rpm for 2 minutes, and the supernatant was discarded again to obtain a DNA pellet. After vacuum drying at 2 mmHg for 2 minutes, this
Dissolve the DNA pellet in 50μ of distilled water and collect the DNA.
For 50μ of the solution, add 6μ of the restriction enzyme Hind decomposition buffer (described in Example C) and the restriction enzyme.
Hind3μ was added and reacted at 37°C for 4 hours. next
10μ yeast tRNA (2μg/μ) solution and distilled water
After adding 70μ of water and further adding 100μ of water-saturated phenol and shaking, centrifugation was performed to take out only the aqueous layer, and the phenol was removed from this aqueous layer by ether extraction three times. 10μ of 3M sodium acetate (PH=4.8) and 350μ of ethanol were added and left to stand at -20°C for 4 hours, followed by high speed centrifugation. The resulting DNA pellet was dried and dissolved in 17μ of distilled water. pBR322 shown below for this DNA 17μ solution
1μ of Hind/alkaline phosphatase treatment (DNA 1μg/μ) and buffer for DNA binding enzyme [650mM Tris HCl (PH=7.4), 65
mM _MgCl2 , 10mM DTT, 40mM ATP,
Add 2μ of 40mM Spermide aqueous solution and 0.5μ of T4 DNA ligase (code No. 2010B1.2 units/μ manufactured by Takara Shuzo Co., Ltd.) and react at 14°C for 22 hours, then treat at 65°C for 5 minutes to obtain hybrid DNA. Next, we used this hybrid DNA to develop E. coli bacteria.
Transform HB101 competent cells. Hind/alkaline phosphatase treatment solution of pBR322 (DNA 1μg/μ); 50μg of plasmid pBR322 DNA was added to μ aqueous solution with 40μ of the Hind restriction enzyme buffer and
Add Hind20μ and react for 10 hours at 37℃, then 1M Tris.
44μ of HCl (PH=8.0) was added, and 10μ of bacterial alkaline phosphatase (BAP) (0.4 units/μ, manufactured by Worthington) were added and treated at 65°C for 7 hours. This reaction dephosphorylates the 5' end of DNA. Water-saturated phenol was added to this reaction solution.
Protein removal was performed at 400μ. Then, a mixture of phenol/chloroform (4/1 volume ratio)
Protein was removed twice at 400μ, and finally
Phenol components were extracted from the aqueous solution three times using 600μ ether. 30μ of 3M sodium acetate and 110μ of ethanol were added to the 350μ of the aqueous layer, allowed to stand at −20°C for 2 hours, and centrifuged at high speed. The resulting DNApellet was dried and dissolved in 50μ of distilled water. This is used as a pBR322 hind/alkaline phosphatase treatment (DNA 1 μg/μ) solution. Preparation of competent cells of E. coli HB101 and their transformation; A single colony of E. coli HB101 was grown in culture medium L.
The cells were transferred to 5 ml of -broth and cultured with shaking at 37°C for 11 hours. This 2 ml was inoculated into 200 ml of new L-broth, and cultured with shaking at 37°C for 2 hours and 20 minutes. When the OD ₆₀₀ reached 0.40,
Cool the culture solution to 0°C and use Tommy's refrigerated high-speed centrifuge.
Centrifugation was performed at 5000 rpm for 5 minutes in a No. 9 rotor. Discard the supernatant and add the precipitated E. coli to 50ml of 10mM NaCl.
The bacteria were homogeneously dispersed in water and centrifuged again at high speed (No. 4 rotor, 5000 rpm for 5 minutes) to precipitate the bacteria. To this bacterial pellet, 60 ml of 30mM CaCl ₂ water was added, and the whole was homogenized and kept at 0°C for 20 minutes. High-speed centrifugation again (No. 4 rotor, 4000 rpm for 5 minutes)
Discard the supernatant and add 10ml of 30mM 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, return the competent cells to 0°C, and after about 10 minutes,
Perform the binding reaction with T4 DNA ligase and then 65℃5
The hybrid DNA aqueous solution treated for 1 minute was added, held at 0°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 (diameter 9 cm) containing 50 μg/ml ampicillin at a rate of about 200 μl per plate. Hold at 37℃ for 16 hours
41 transformed HB101 colonies were obtained.
These 41 colonies were plasmidized using the Boiling Lysis method described in the experimental book by Maniathes et al., page 368.
Perform a mini-prep of DNA, then plasmid DNA
Hind disassembled. As a result, 2.58Kbp, 1.62Kbp,
A plasmid containing 102Kbp was obtained. 2.58 Kbp was obtained by Hind cleavage of the DNA in figure a, and 1.62 Kbp and 1.02 Kbp were two types of fragments generated by Hind decomposition of the DNA in figure b. 2.58Kbp fragment and
pBGH1 hybrid DN with pBR322,
Hybrid DNA of 1.62Kbp fragment and pBR322
Hybrid of pBGH29 1.02Kbp and pBR322
The DNA was designated as pBGH4. The DNA of pBGH1 is
pBR322 is replicated in the host E. coli.
The entire DNA was completely double-stranded. Hybrid DNApBGH1 Hind/Bgl,
Hind/Ava, HIND/Sal/Cla,
Hind/Bgl/Ava, Hind/Cla/Bgl
Completely digested with 1.2% agarose gel electrophoresis (at the same time, λDNA was
Ecoe/Hind digest and Taq of pBR322
The degradation product was also electrophoresed), and the DNA fragment size was analyzed and Table 2
The results were obtained. [Table] Values in parentheses are DNA derived from pBR322. The fragments generated by digestion of pBGH1 with each restriction enzyme in Table 2 have a maximum of ±0.05Kbp for each DNA size.
Although there is a measurement error of about 0.1 Kbp, the restriction enzyme cleavage map in Figure a agrees well with the expected one, except that the Cla site 0.82 Kbp away from the Hind site is no longer cleaved by Cla. Ta. The Cla site 0.82Kbp away from the Hind site is
E. coli is no longer cleaved by Cla.
This is thought to be because position 6 N of GAT in ATCGAT TAGCTA was methylated when pBGH ₁ was replicated in HB101. hybrid
Since the infectivity test using DNApBGH1 (Example i) shows completeness, pBGH1 is
One Cla site appears to have disappeared, but the base sequence itself is thought to be the same as aDNA. f Hybrid DNA of Cla-digested double-stranded DNA and E. coli plasmid pBR322;
Perform almost the same procedure as in e, changing the Hind digestion of pBR322 to Cla digestion (change the salt concentration during digestion to a low concentration for Cla digestion),
17μ of sterile water containing double-stranded DNA degraded by Cla, 1μ of pBR322 Cla/alkaline phosphatase treatment (DNA 1μg/μ) solution, 2μ of the buffer for the DNA binding enzyme, and T4 DNA ligase.
Hybrid DNA was obtained in the same manner as in the experiment in e. Using this hybrid DNA, E. coli HB101 competent cells were transformed in the same manner as in e. 41 transformants were obtained. Mini-preparation of plasmid DNA was performed from these 41 colonies by the boiling lysis method as in case e, and the plasmid DNA was digested with Cla. The result is 2.61Kbp,
Three hybrid plasmids containing fragments of 1.33 Kbp and 1.24 Kbp were obtained. This hybrid plasmid DNA was named pBGC1, pBGC2, and pBGC3.
pBGC1 is a hybrid DNA containing the entire DNA of b, and pBGC2 and pBGC3 are hybrid DNA containing two Cla fragments of bDNA.
It was hot. The hybrid DNA pBGC1 was replicated in E. coli and was completely double-stranded throughout the DNA. Hybrid DNApBGC1
was digested with Cla, Cla/Hind, and Cla/Sal, and the degraded fragments were analyzed in the same manner as in e. The sizes of the obtained fragments were as shown in Table 3. [Table] The fragments generated by digestion of pBGC1 with each restriction enzyme in Table 3 are ±0.05 maximum ± the size of each DNA.
Although it included a measurement error of about 0.1 Kbp, it matched well with that shown in the restriction enzyme cleavage map in Figure b. Hybrids obtained in Examples e and f
DNApBGH1 and pBGC1 are shown in Figures a and b, respectively.
It is a hybrid DNA of DNA and pBR322,
These can also be used as vectors for plant recombination. g from hybrid DNA pBGH1 and pBGC1
Separation of aDNA and bDNA and their combined circularization; 20μ each of hybrid DNA pBGH1 and pBGC1
Using 20 units of Hind and 20 units of Cla, respectively, the salt concentration was set using the respective restriction enzyme buffers, and hydrolysis was carried out at 37°C for 10 hours at a total of 400μ. Next, 0.8% agarose gel (slot width
After electrophoresis at 1V/cm for 10 hours using a gel (1.5mm, length 6cm, depth 7mm, gel length 13cm), DNA-a,
Cut out the DNA-b 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. Both are 50μ sterile water (DNA
Approximately 0.1 μg/μ). This linear DNA
-a and DNA-b were added to 20μ of each of the linear DNA-a and DNA-b, respectively, with 5μ of the DNA ligating enzyme buffer, 25μ of sterile water, and T4-
After adding 0.5 μ of DNA ligase and reacting for 19 hours at 12°C, perform 0.8% agarose gel electrophoresis again as above, cut out the DNA band portion with an apparent size of 3.3 Kbp, and perform the same procedure as above.
Only DNA was extracted and purified. This DNA is considered to be circular DNA-a and DNA-b, respectively. Next, 10μ of sterile water was added to each of these DNAs, and 2μ of the DNA was partially degraded with Hind and Cla. Partial degradation of 2μ of DNA
Add 5μ of the restriction enzyme buffer and 43μ of sterile water to the solution, and add Hind for DNA-a.
Add 0.5 units of DNA-b and 0.5 units of Cla for DNA-b, and react at 37°C for 5, 10, and 30 minutes. Each reaction product was subjected to 0.8% agarose gel electrophoresis to observe changes in size. . The 3.3Kbp band gradually decreased as the reaction was carried out for 5, 10, and 30 minutes.
Linear DNA-a and DNA-b
Bands of DNA 2.57Kkbp and 2.61Kbp increased, and no other bands were observed. From this, the above circular DNA-a and DNA-b are both monomeric and circular, and are respectively shown in Figures a and
This is the DNA of b. This DNA is completely double-stranded throughout and can be used as a vector for plant recombination. Extraction and analysis of BGMV replicative DNA from hBGMV-infected plants; method of Halmiton et al. [Nucl Acids Res. 10
4902 (1982)], add 40ml of 0.5M to 21g of BGMV-infected leaves of Topkurotsupin bean.
Add KH ₂ PO ₄ and 0.75% Ha ₂ SO ₃ (PH=7.0), homogenize in a mortar, add 1.2 ml of TritonX-100, and heat at 4°C.
Stirred for 25 hours. Next, wrap it in double gauze,
Centrifuge the liquid portion for 10 minutes at 9000 rpm using a No. 4 rotor in a Tomy high-speed centrifuge, remove the supernatant, and transfer this supernatant to a Beckman ultracentrifuge type SW40.1 rotor.
Centrifugation was performed at 37,000 rpm for 3.5 hours. Discard the supernatant and add 1 ml of 40mM Tris HCl, 5mM acetic acid,
Add 10mM EDTA (PH=8.2) and homogenize to 20μ
10% SDS was added, and proteins etc. were removed from the aqueous layer three times with 1 ml of water-saturated phenol and then three times with 1 ml of phenol/chloroform (4/1).
Next, perform ether extraction four times with 1 ml of ether to remove phenol, and add 100μ of 3M sodium acetate to the aqueous layer.
Then, 3.5 ml of ethanol was added, and the mixture was left at -20°C overnight, followed by high-speed centrifugation to obtain a DNA pellet. this DNA
Add 500μ of sterile water to the pellet (DNA concentration
0.6μg/μ) 100μ of it was subjected to 0.8% agarose gel electrophoresis using the same method as above.
DNA corresponding to 1.5 Kbp of the band that hybridizes with the 32p labeled DNA probe of BGMV, 3.1 Kbp
A corresponding portion of DNA5.2Kbp a corresponding portion of DNA,
The 9.8 Kbp portion of DNA was extracted from the gel and purified in the same manner as in g. (portion equivalent to 5.2Kbp,
The portion corresponding to 9.8Kbp is EcoR/Hind of λ-DNA
Using a size marker, a corresponding portion of each gel was cut out). These four types of DNA are respectively Hind, Cla, and Sal.
The decomposition pattern was analyzed by performing agarose gel electrophoresis. 5.2Kbp equivalent portion,
After agarose gel electrophoresis, the DNA corresponding to 9.8 Kbp was transferred to a nitrocellulose filter, and DNA-DNA hybridization and autoradiography were performed using 32p-labeled BGMV DNA to determine the DNA degradation pattern. Analyzed. The decomposition pattern is 2.58, 1.60, 1.00Kbp by Hind decomposition, 2.57, 1.33, 1.24Kbp by Cla decomposition, 1.90 by Sal decomposition,
It showed degradation patterns of 1.54, 1.03, and 0.71Kbp.
This is the same pattern of decomposition by restriction enzymes a and b, that is, this replicative DNA is DNA
-a or DNA-b, and can be used as a vector for plant recombination. Hybrid of i-replicative DNA and pBR322
Preparation of DNA: The DNA extracted and purified from agarose gel (9 μg DNA/10 μ) of the band portion with an apparent size equivalent to 1.5 Kbp of the replicated DNA of h was added with the Cla
Restriction enzyme buffer 4μ, sterile water 24μ, Cla
3μ was added and hydrolyzed at 37℃ for 2 hours, 60μ of sterile water was added to this, protein was removed twice with 60μ of water-saturated phenol, and then 130μ of ether was added.
Phenol extraction was performed from the aqueous layer four times. next
After adding 7μ of 3M sodium acetate (PH=4.8) and 300μ of ethanol, the mixture was left at -80°C for 30 minutes and then 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. This precipitate is dissolved in 20μ of sterile water, and this is used as a Cla degradation product of replicative DNA. The same as used in f for 4μ of this Cla digested product (approx. 2μg of DNA).
pBR322 Cla degradation/alkaline phosphatase treatment solution (DNA 1μg/μ) 6μ, buffer for the DNA binding enzyme 2μ and T4 DNA ligase
Add 0.5μ and 7.5μ of sterilized water, react at 14℃ for 12 hours, then treat at 65℃ for 5 minutes, transform E. coli HB101 competent cells using the same procedure as in If, and about 500 transformants were obtained. Obtained. L-Agar with colonies of transformants
Place a nitrocellulose filter (BA85 manufactured by Schleicher and Schiill, same as above) on the plate, then remove the nitrocellulose filter, and add 0.5M sodium hydroxide,
Soak in 1.5M saline for 1 minute, then 1M Tris HCl
(PH=7.0) immersed in liquid. This nitrocellulose filter was heat treated at 80° C. for 2 hours under reduced pressure to 1 mmHg. This heat-treated filter was mixed with a prehybridization solution [0.9M NaCl, 0.09M sodium citrate, 0.02% polyvinylpyrrolidone (Wako Pure Chemical Industries, Ltd. Plasdon NP K-30), 0.02% Ficoll (manufactured by Pharmacia Fine Chemicals Co., Ltd.).
Ficoll400) 0.02% bovine serum albumin aqueous solution] and pretreated at 63℃ for 30 minutes, then remove the prehybridization solution and add 10% SDS and yeast to the prehybridization solution.
A hybridization solution containing 40 μg/ml of tRNA and 200×10 ⁴ cpm of the BGMV 32p probe described below were added, and a nitrocellulose filter treated as described above was placed in the solution and DNA-DNA was incubated at 63°C for 24 hours. Hybridization was performed. Next, take out this filter and remove a large amount of
in 0.45M NaCl, 0.045M Sodium Citrate
The filter was washed by gentle shaking at 55°C for 20 minutes. This washing was carried out three times, and after drying, this filter was used to filter the
Autoradiography was performed using the same method as P470-471. Approximately 360 colonies were observed that hybridized with 32p-labeled BGMV DNA. Preparation of BGMV 32p probe; In experiment c, use 0.8mM deoxycytidine triphosphate instead of 10μ. The rest was carried out in the same manner as in c.
The first 32p peak in fractions No. 6 to 11 (total 800
×10 ⁴ cpm), and these fractions were collected (total 900μ), heated at 95°C for 5 minutes, and then rapidly cooled to 0°C. This liquid is 32p labeled BGMV.
Use as a DNA probe. Plasmid DNA was mini-prepared for 30 of the 360 colonies by the Boiling Lysis method described above, and the plasmid DNA was digested with Cla. As a result, a hybrid plasmid containing a 2.61 Kbp fragment was obtained from 7 colonies. One of these hybrid DNAs was named pBGRFC1. Cla/Hind pBGRFC1
When digested with Cla/Sal, the same DNA fragment as in Table 3 of f was shown. j Hybrid of replicative DNA and pBR322
Preparation of DNA: Extract and purify the DNA extracted and purified from the band portion of the replicative DNA of h that has an apparent size equivalent to 3.1 Kbp.
Decompose it with Hind and add it to pBR322 in the same way as e.
Of the 161 transformants obtained by obtaining hybrid DNA with Hind/alkaline phosphatase treatment solution and further transforming E. coli in the same manner as e.
Extract and analyze plasmids from 30 samples in the same way as e.
2.58Kbp, 1.59Kbp,
Hybrid DNA containing 0.99Kbp Hind fragment
pBGRFH1, pBGRFH2, and pBGRFH3 were obtained, respectively. pBGRFH1 is Hind/Bgl, Hind/
Ava, Hind/Cla/Sal's underwear
DNA fragments similar to those in Table 2 were generated. i
From the results of and j, this replicative DNA is shown in Figure aDNA.
and bDNA was found to be included.
Using the method of this experiment, aDNA and bDNA can be separated and propagated. k Infectivity with cloned DNA; hybrid DNA pBGH1 (2.3μ) obtained in e.
g/μ) 25μ to 10μ of 2mM _MgCl2 , as above.
Hind restriction enzyme buffer 13μ, Hind5μ
, add 77μ of sterile water, treat at 37℃ for 7 hours, and then
After treatment at 65°C for 5 minutes, NaCl and sodium citrate were added until the concentrations were 0.15M and 0.015M, respectively. This solution was designated as cloning DNA-a solution. f
Hybrid DNA pBGC1 (0.68μg/μ
) _10μ of 2mM MgCl2 in 100μ, the above Cla
Add 13μ of restriction enzyme buffer, 5μ of Cla, and 2μ of sterilized water, treat at 37℃ for 7 hours, and then incubate at 65℃ for 5 hours.
After treatment for 1 minute, NaCl and sodium citrate were added to 0.15M and 0.015M, respectively. This solution was designated as cloning DNA-b solution. Cloning DNA-a solution and DNA-b solution, respectively
Mix 80μ and store this mixture for 14 hours and 30 hours in daytime conditions.
℃, Biotron (Koito Seisakusho Co., Ltd.) set at 27℃ and humidity of 80% or higher for 10 hours at night.
Using sterile gloves, inoculation was inoculated onto the surface of young leaves of top-cropped kidney beans on the 6th day after germination by rubbing the mixture with celite (mixture 40μ/individual top-crop kidney bean). A total of 5 individuals were inoculated. Thereafter, the conditions in the biotron were maintained as described above, and the onset of bean golden mosaic disease was observed on the true leaves of top-cropped kidney beans. 10 after vaccination
On day 1, bean golden mosaic disease was clearly observed in all four individuals. From the above, cloning DNA-a and
During cloning, DNA-b is methylated at a special base sequence, for example, the 6-N of A in GATC, so that a specific restriction site (for example, one of the Cla sites) in the restriction enzyme cleavage map shown in Figures a and b is methylated. ) has disappeared and appears to be different DNA, but the base sequence itself is the same, and since it exhibits the same infectivity as DNA, it is recognized that it has the same base sequence as the virus DNA. Hybrid DNApMYB4 (0.435μ
g/μ) 460μ with 10μ of 2mM _MgCl2 , as above.
BamH restriction enzyme buffer 55μ, BamH
Add 10μ of sterilized water and 15μ of sterilized water and enzyme treatment at 37℃ for 4 hours. After further treatment at 65℃ for 5 minutes, add 1.5M NaCl and 0.15M.
55μ of sodium citrate aqueous solution was added. This solution was designated as cloning DNA-a solution. f
Hybrid DNA pMYH3 (0.87μg/μ
) _10μ of 2mM MgCl2 in 231μ, Hind
Add 30μ of control enzyme buffer, 10μ of Hind, and 19μ of sterilized water, perform enzyme treatment at 37℃ for 4 hours, and then
After treatment at 65°C for 5 minutes, 30μ of 1.5M NaCl and 0.15M sodium citrate aqueous solution was added. This solution was designated as cloning DNA-b solution. Cloning DNA-a solution 330μ and DNA-
Mix 160 μ of solution B and store this mixture at 30℃ for 14 hours during the daytime and at 27℃ for 10 hours at night with humidity.
In a Biotron (Koitoron manufactured by Koito Seisakusho) set at 80% or higher, inoculation was inoculated by rubbing it together with Celite on the surface of young leaves of Topcropped kidney beans on the 6th day after germination using sterile gloves.
46 μ/1 individual top-cropped green bean). A total of 10 individuals were inoculated. The conditions inside the biotron are
Thereafter, the plants were maintained in the same manner as described above, and the appearance of yellow mosaic lesions was observed on the true leaves of top-cropped kidney beans. On the 18th day after inoculation, yellow mosaic lesions were clearly observed in 9 out of 10 individuals. From the above, the cloning obtained by the present invention
Even if DNA-a and DNA-b have a special base sequence, such as methylation of N at position 6 of A of GATC, due to cloning, virus
Shows the same infectivity as DNA. From this, it can be considered that they have substantially the same base sequence.

[Brief explanation of drawings]

FIG. 1 and FIG. 2 are each according to the present invention.
This shows a restriction enzyme cleavage map of DNA. Figures 3 and 4 correspond to the target DNA in the restriction enzyme maps of Figures 1 and 2, respectively.
This is to show the DNA base sequence.

Claims (1)

[Claims] 1. Single strand of bean golden mosaic virus
The DNA is at least partially double-stranded DNA, and is a mono- to trimeric DNA of the DNA represented by the genetic map of Ib' below. [Here, the base sequence of one strand of the double strands of P ₁ to _{P 6} is as follows. P ₁ (0.0/2.61): 5′−AAGCTT−3′ P ₂ (0.02): 5′−ATCGAT−3′ P ₃ (0.29): 5′−GTCGAC−3′ P ₄ (1.01): 5′− GTCGAC−3′ P ₅ (1.64):5′−AAGCTT−3′ P ₆ (1.65):5′−AGATCT−3′] 2 Characterized by being at least partially methylated in other organisms Claim No. 1
DNA described in section. 3 Claim 2 in which the other organism is E. coli
DNA 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 Bean golden mosaic virus single strand
The DNA is at least partially double-stranded DNA, and the DNA represented by the genetic map Ib′ below is cleaved with a restriction enzyme, and the vector DNA for other organisms is
or a hybrid combined with a third DNA
DNA. [Here, the base sequence of one strand of the double strands of P ₁ to _{P 6} is as follows. P ₁ (0.0/2.61): 5′−AAGCTT−3′ P ₂ (0.02): 5′−ATCGAT−3′ P ₃ (0.29): 5′−GTCGAC−3′ P ₄ (1.01): 5′− GTCGAC−3′ P ₅ (1.64):5′−AAGCTT−3′ P ₆ (1.65):5′−AGATCT−3′] 8 In other organisms, at least a portion of it has been methylated. The hybrid DNA according to claim 7 characterized by: 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.