JPH1075793A - Transformation of conjugation transfer of organelle - Google Patents

Abstract

PROBLEM TO BE SOLVED: To directly transduce a required foreign gene to organelle (mitochondrion, chloroplast) in a cell of eukaryote without protoplastization of a cell even in a mild condition. SOLUTION: A vector is transduced into organelle (mitochondrion, chloroplast) in a cell of eukaryote by performing conjugational transfer between prokaryotic and eukaryotic cells of donor bacteria having a replication point acting on both the organelle and donor bacteria, a selected marker gene acting on both the organelle and the donor bacteria, a required foreign gene acting on the organelle, and an expression vector expressing in conjugational transfer organelle and containing oriT sequence required for the conjugational transfer between prokaryotic and eukaryotic cells and a cell of eukaryote which is a host cell by utilizing tra gene and mob gene, for example, in a form of helper plasmid.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention uses an expression vector for conjugative transfer in an organelle, and comprises
The present invention relates to a method for transforming an organelle by introducing the vector into an organelle present in a eukaryotic host cell by conjugative transfer between a prokaryote and a eukaryote using the b gene.

[0002]

BACKGROUND ART Organelles such as mitochondria and chloroplasts present in eukaryotic cells have a structure tightly isolated by a double membrane, have a unique circular DNA genome, and carry genetic information. Express and transmit. In the organelle, the genetic information encoded by the organelle DNA is expressed by the organelle's unique transcription system and translation system, and several proteins required in the organelle are supplied. Organelle DNA is replicated in the DNA synthesis system in the organelle and self-proliferates. Organelles themselves can grow by division, but by organelle DNA self-proliferating, a certain level of organelles is maintained in cells without being diluted or lost by cell division. Among organelles, mitochondria are mainly involved in the electron transport system of respiration, and chloroplasts are involved in the electron transport system of photosynthesis. In these electron transport systems, ATP is generated by oxidative phosphorylation, and the energy required in cells Production. Organelles are involved not only in energy production but also in the production of physiologically important substances, and can be said to be important intracellular organelles for cells. However, since organelle DNA is constantly exposed to reactive oxygen radicals generated in the electron transport system of respiration, it is more susceptible to mutations such as point mutation and deletion due to reactive oxygen radicals than chromosomal DNA in the nucleus. Diseases caused by such mitochondrial DNA mutations (mitochondrial diseases) include Parkinson's disease, mitochondrial diabetes, mitochondrial encephalomyopathy, mitochondrial myopathy, and mitochondrial cardiomyopathy (Takamasa Ozawa, Science, 1994, 45 : 698). If a method for directly transforming an organelle present in a eukaryotic cell could be developed, a desired foreign gene could be introduced into the organelle to restore the reduced function of the organelle by complementing the function of the damaged organelle DNA. It is industrially useful because it is possible to enhance the function or to add a new function to an organelle.

[0003] Gene transfer targeting the nucleus of eukaryotes such as animals, plants, or fungi including yeast has been conventionally performed. However, transfer of a gene to an organelle existing in a cell is performed in the case of the nucleus. Difficult and unlike.
Unlike nuclei that have a nuclear hole in the nuclear envelope, organelles have a strict double membrane, so they were developed for gene transfer into conventional nuclei.
For example, the calcium phosphate method (Graham, FLa)
nd Van der Eb, A. et al. J. , 1973, V
irology, 52 , 456), an electroporation method, a DEAE dextran method, or a lipofection method (Malone, RW et al., 198).
9, Proc. Natl, Acad. Sci. USA,
86 , 6077), there has been no example in which a foreign gene was directly introduced into an organelle under mild conditions and without protoplasting the cells. Fox and others use a powerful particle gun for the purpose of overcoming this problem, and use baker's yeast Saccharomyc.
es cerevisiae mitochondria (Fox, TD et al., 198).
8, Proc. Natl. Acad. Sci. USA,
85 : 7288). However, in this method, a DNA-coated tungsten particle having a diameter of about 1 μm is injected into the cell at a speed close to the speed of sound, so that the intracellular structure of the target cell or the DNA itself to be introduced is physically damaged. There is. Due to the drawback, the introduction efficiency is very poor, and DNA having a large molecular weight cannot be used because of a high probability of causing damage. Since then, the method has not been greatly improved against these drawbacks. As described above, in the prior art of a method called a particle gun or a gene gun, the frequency of obtaining a transformant of an organelle existing in a host cell is low due to physical damage, the reproducibility is poor, and the organelle Not completed as a transformation method. In addition, a method of mixing a protoplast suspension of a plant with a plasmid DNA solution and performing a combination of heat treatment, polyethylene glycol treatment, or electroporation to introduce foreign DNA into plastids and mitochondria has been reported (Potricix, etc.). 1987
However, even if this method is used, in addition to the complexity of the protoplast formation process, physical damage is large, and the cell wall is regenerated from the protoplast and returned to the original cells. There are many problems such as necessity, and the method of organelle transformation has not been completed.

[0004]

DISCLOSURE OF THE INVENTION The present invention uses a novel conjugative transferable organelle expression vector capable of replication and propagation in an organelle, which cannot be achieved by the prior art.
It is intended to provide a highly reproducible method of transforming an organelle by directly introducing a desired foreign gene into the organelle under mild conditions without causing protoplasts and damaging the cells by eukaryotic conjugation transfer. is there.

[0005]

As a method for introducing a desired foreign gene into the nucleus of a eukaryote with high efficiency under mild conditions that do not damage cells, a method called prokaryotic-eukaryotic conjugation transfer has been developed. (Nishikawa, M. et a
l. , 1990, Jpn. J. Genet. , 65 : 3
23, Nishikawa, M .; et al. , 199
2, Curr. Genet. 21 : 101). Although it is known that conjugative transmission across species by high host range plasmids is established in nature among bacteria, Nish
According to the method of conjugation transfer between prokaryotes and eukaryotes shown by Ikawa, M. et al., Escherichia coli having a conjugative transfer shuttle vector containing a sequence (oriT sequence) required for transfer by conjugative transfer, Necessary mob gene, tr
E. coli carrying a helper plasmid containing the a gene,
When yeast and yeast are multiplied under certain conditions, prokaryotic / eukaryotic conjugation occurs, and the conjugative shuttle vector is transmitted to the yeast nucleus by the action of a helper plasmid. Research to date suggests that prokaryotic / eukaryotic junctional transmission occurs in approximately the following steps: That is, E. coli having a prokaryotic / eukaryotic conjugative transfer shuttle vector, E. coli having a helper plasmid, and yeast are mixed under certain artificial conditions to form a coagulum of E. coli and yeast, Next, by the action of the tra gene product, a conjugation tube is formed between the mating partners in the aggregate, and one of the strands of the plasmid DNA, which is the transferable shuttle vector, is oriT by the action of the mob gene product encoded by the helper plasmid. The DNA is cleaved at the site, and the single-stranded DNA enters the yeast cell from the 5 'end and passes through the nuclear membrane to the nucleus. Synthesis of the complementary strand occurs in the nucleus, recircularizes and becomes the structure of the original double-stranded plasmid, and conjugation transfer is completed. Prokaryotic / eukaryotic conjugative transmission has been performed by E. coli and Kleberii yeast, E. coli belonging to E. coli belonging to prokaryotes such as E. coli minicell and baker's yeast, and several yeasts belonging to eukaryotes. Report: Kazuo Yoshida, Masanobu Nishikawa, 1993, Protein nucleic acid enzyme, 3
Eight sixty), also Agrobacterium tum
efaciens and baker's yeast have been shown to occur in donors belonging to prokaryotes other than E. coli (Sawa).
Saki, Y, et al. , 1996, Plant
Cell Physiol. , 37 : 103). Also,
It is known that a very long plasmid (F factor) having a length of 4 Mbp is transmitted in conjugative transfer between Escherichia coli. Therefore, it is expected that the same action can be used in the conjugation transfer between the prokaryotic and eukaryotic kingdoms of the present invention to introduce a plasmid DNA as long as factor F under mild conditions.

The present inventors have proposed the above-mentioned oriT sequence, mo
The mechanism of prokaryotic / eukaryotic conjugation transfer in which the plasmid DNA, which is the main body of the conjugative transfer shuttle vector, is single-stranded and introduced into the yeast nucleus by the action of the b gene and the tra gene is extremely powerful, We thought that prokaryotic / eukaryotic conjugation could occur in the same way as for nuclear. In addition, the function of the organelle causes the synthesis of the complementary strand, and the single-stranded DNA introduced into the organelle from the 5 ′ end will be recircularized and repaired to the original double-stranded plasmid. If the origin of replication that functions in step (1) is included in the conjugative transfer shuttle vector, DNA replication occurs by the organelle's unique DNA synthesis system, and the conjugative transfer shuttle vector self-proliferates in the organelle. It was expected that a higher copy number of the shuttle vector per organelle would be maintained. Furthermore, if a selectable marker gene that functions in an organelle and a desired foreign gene are included in the vector, prokaryotic and
He concluded that eukaryotic organelles could be transformed by eukaryotic conjugation.

That is, the above-mentioned conjugative transfer shuttle vector contains a replication origin that functions in the organelle of the host cell, a selectable marker gene that functions in the organelle, and a desired foreign gene modified to function in the organelle. By preparing a transmissible intracellular expression vector and introducing the vector into an organelle present in eukaryotic cells by a method of conjugation between a prokaryote and a eukaryote, a replication origin that functions in the organelle, a function in the organelle It has been concluded that the vector may be stably maintained in organelles by the function of the selectable marker gene.

Therefore, the present inventors have developed a promoter, terminator, enhancer, and the like capable of expressing an exogenous gene which functions as both a selectable marker gene that functions in the organelle and a desired exogenous gene that can be expressed in the organelle. Functionally linked to, and so as to function in the organelle, to replace the sequence of the foreign gene for organelle, to create a conjugative transfer organelle expression vector,
The vector was introduced into eukaryotic cells by a method of conjugation between prokaryotic and eukaryotic organisms using the tra gene and the mob gene. Then, surprisingly, they found that the vector was transferred into an organelle, and the modified foreign gene was expressed in a eukaryotic organelle by the action of the organella's own transcription system and translation system. It was completed. That is, the present invention provides (1) an origin of replication that functions in an organelle, (2) an origin of replication that functions in a donor, (3) a selectable marker gene that functions in an organelle, (4) a selectable marker gene that functions in a donor, 5) a desired foreign gene that functions in an organelle, and (6) an expression vector in a conjugative organelle containing an oriT sequence necessary for conjugative transfer between prokaryote and eukaryote, a donor bacterium holding the vector, and a host cell A eukaryotic cell and the tra gene,
And a method of performing prokaryotic / eukaryotic conjugative transfer using a mob gene and introducing the vector into an organelle of a host cell.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION The term "organelles present in eukaryotic host cells" of the present invention refers to mitochondria present in eukaryotic cells such as animals, plants or fungi including yeast used as hosts. Alternatively, it means an organelle such as chloroplast. Since chloroplasts and intracellular organelles including their chloroplasts are sometimes collectively referred to as plastids, a part of plastids is included in the organelle of the present invention in the sense that chloroplasts are included.

The "conjugative transfer organelle expression vector" of the present invention is a vector constructed to express a desired gene, and its main body usually takes the form of a plasmid DNA consisting of double-stranded DNA. Further, the vector is `` replication origin that functions in organelles '', `` replication origin that functions in donor bacteria '', `` selection marker gene that functions in organelles '', `` selection marker gene that functions in donor bacteria '',
The “desired foreign gene that functions in the organelle” and the “oriT sequence required for prokaryotic / eukaryotic conjugation transfer” are operatively linked to form a so-called shuttle vector structure.

The “conjugative transfer organelle expression vector” of the present invention is intended to replicate and grow in the organelle of the host cell independently of the organelle DNA.
Including an origin of replication that functions in organelles, while the "conjugative transfer organelle expression vector" of the present invention
Although the probability is low, it may be a state in which the DNA is integrated into the organelle DNA and replicates and grows together with the organelle DNA. In fact, according to the above-mentioned methods such as Fox or Potrix, the introduced foreign DNA does not contain the replication origin for organelles, so it is considered that the introduced foreign DNA was incorporated into the organelle DNA. Therefore, as for the expression vector for conjugative transfer organelle used in the present invention, as in the case of the conventional method, the introduced expression vector for conjugative transfer organelle is composed of organelle DNA.
Is possible, albeit at a lower frequency.

[0012] The "origin of replication functioning in the organelle" contained in the vector of the present invention is defined as the origin of replication in the DNA genome of the organelle itself which functions in the same organelle as the appropriate size within the range having the function. It is obtained by cutting out as a DNA sequence, extracting it, and including it in an expression vector in the conjugative transfer organelle. For example, when the eukaryote is baker's yeast and an expression vector for conjugative mitochondria targeting baker's yeast mitochondria is constructed, the origin of replication encoded by baker's yeast mitochondrial DNA may be used. When the present inventors used a replication origin ( 2 μm-ori ) derived from yeast 2 μm DNA known as an extrachromosomal replication origin in the nucleus of baker's yeast, unexpectedly, replication occurred in yeast mitochondria and “ Since 2 μm-ori was shown to be effective as a “functional replication origin”, this may be used in the case of baker's yeast.

In the case where an eukaryote is an animal and an expression vector for conjugative mitochondria targeting the mitochondria of the animal is constructed, an origin of replication encoded by the mitochondrial DNA of the animal may be used. From the finding that 2 μm-ori is effective in the baker's yeast mitochondria, the origin of extrachromosomal replication in animals, for example, Sim
ian Virus 40 (SV40), bovine papillomavirus (BPV), or Epstein-Barr
Virus (EBV) is also expected to work effectively on mitochondria present in animal cells.

In the present invention, "DNA functioning in donor bacterium"
The “origin of replication” may be a replication origin contained in the genome of the donor bacterium itself, or may be a replication origin of a plasmid present as an episome in the donor bacterium. For example, when the donor bacterium is Escherichia coli, ColE1-type pMB1, pBR32
2, or a sequence of any length including the replication origin of the pUC-based plasmid may be used. In addition, for example,
Agrobacterium tumefaciens
In this case, a sequence of any length including the origin of replication of the Ti plasmid may be used.

The vector of the present invention includes a “selectable marker gene that functions in organelle”, which is composed of a DNA sequence that can be expressed using the transcription and translation systems of the organelle itself. Any gene may be used as long as it is expressed in an organelle and the expression product functions in a host cell to enable selection of a transformant. Organelle translation codons used in the organelle's own translation system include prokaryotic E. coli,
Alternatively, it differs from the universal translation codon commonly used in eukaryotic nuclei. For example, when the organelle is mitochondria, TGA is tryptophan in human, bovine, or yeast translation codons for mitochondria, but is a stop codon in universal translation codons. It is known that ATA is methionine in mitochondrial translation codons, but isoleucine in universal translation codons (Barr).
ell, B .; G, et al. , Proc, Nat
l. Acad. Sci. USA. , 1980, 77 : 3
164, Bonitz, S .; G, et al. , Pr
oc, Natl. Acad. Sci. USA. , 198
0, 77 : 3167).

Therefore, when the organelle is mitochondria, a selectable marker gene prepared by artificially designing and producing a DNA sequence that functions in mitochondria in correspondence with the mitochondrial translation codon may be used.
When the organelle is chloroplast, the heterogeneity of chloroplast tRNA compared to the abundance ratio of nuclear tRNA (Hiroaki Shimada, 19
92, protein nucleic acid enzyme, 37 : 1067), and may be a selectable marker gene artificially designed and prepared for a DNA sequence corresponding to a codon choice resulting from the above. In the case of eukaryotic baker's yeast, codons of baker's yeast mitochondria (Koji Okamoto et al., 1994, protein nucleic acid enzyme, 3
9 : 1638) may be used.

Usually, it is known that a single eukaryotic cell as a host contains a plurality of, for example, several tens, organelles. Is not always introduced into the organelle. Therefore, the transformed cells are cultured for a while in a selection medium under conditions of selective pressure, and the vector plasmid D
Better results are expected if NA cells are grown in an organelle and a transformed cell in which the organelle holding the plasmid DNA is predominant in the cell is selected.

Examples of the selectable marker gene include a chloramphenicol acetyltransferase gene (CAT gene) derived from Escherichia coli, a neomycin (Neo) resistance gene, a hygromycin resistance gene, and a bacterial xanthine guanine phosphoribosyl transferase (XGPRT) gene. , Thymidine kinase (TK) gene, or dihydrofolate reductase (DHFR) gene. These gene sequences are artificially substituted for organelles, and functionally added to promoters, terminators, enhancers, etc. that can be expressed in organelles. By ligating, it can be used as a selectable marker that functions in organelles.

When a thymidine kinase (TK) gene or a dihydrofolate reductase (DHFR) gene is used as a selectable marker gene, these genes are not dominant selectable markers, and the host cells lack each gene. Use of the mutant enables selection of a transformant.

For example, when the eukaryote used as a host is baker's yeast and the CAT gene is used as an example of a selectable marker gene, a baker's yeast mitochondrial CAT gene ( CATM) artificially designed and prepared with a baker's yeast mitochondrial translation codon is used. ) May be used.

Taking the translation codon for baker's yeast mitochondria as an example, the universal translation codon is CTN.
(N is any of A, G, T, C) is translated into leucine, but baker's yeast S. The translation codon for cerevisiae mitochondria is translated into threonine. Similarly, ATA is translated into isoleucine at the universal translation codon for nuclear DNA, but to methionine at the translation codon for baker's yeast mitochondria. Also, TG
A is a stop codon in the universal translation codon for nuclear DNA, but is translated into tryptophan in the translation codon for baker's yeast mitochondria. Therefore, when targeting baker's yeast mitochondria, it is preferable to artificially design a gene sequence using the above-described basal yeast mitochondrial translation codon and perform base substitution. That is, the DNA sequence of a naturally occurring selectable marker gene is designed with reference to the mitochondrial translation codon described above, and the complementary strand of the DNA sequence of the gene is divided into DNA sequences of an appropriate length. Then, a synthetic DNA corresponding to each of the divided DNA sequences is prepared using a fully automatic DNA synthesizer, the 5 ′ end thereof is phosphorylated, each synthetic DNA is annealed, and T4
Total synthesis can be performed by ligation with ligase. Alternatively, site-directed mutagenesis (site-direct
A point mutation may be specifically introduced only at a site requiring conversion using d Mutagenesis method). In order to increase the efficiency of experiments for obtaining DNA sequences mutated by site-directed mutagenesis, a site-directed mutagenesis primer and a selection primer that anneals to the same strand as the mutation-introduced primer, for example, restriction enzyme cleavage Site mutation introducing primers may be used simultaneously.

The “selectable marker gene that functions in a donor bacterium” according to the present invention has a plasmid DNA containing the gene that can be expressed in the donor bacterium under specific culture conditions under which selective pressure is applied. Any bacteria can be used as long as the donor bacteria to be grown can grow. For example, if the donor bacterium is Escherichia coli or Agrobacterium tun
In the case of efaciens , examples of selectable marker genes that function in these prokaryotes include an ampicillin resistance gene, a tetracycline resistance gene, a chloramphenicol resistance gene, and a spectinomycin resistance gene. These are used in the vector of the present invention. It is possible.

The “desired foreign gene that functions in an organelle” in the present invention is a gene that is involved in improving the function of an organelle or in adding a useful function to an organelle, and is a gene derived from nature. Any gene may be used as long as it is a gene whose sequence is modified to function in an organelle, or a gene that functions in an artificially produced organelle. These desired foreign genes are contained in an expression vector for conjugative transfer in an organelle and introduced into the organelle by conjugative transfer between prokaryote and eukaryote. Improvement of organelle function or addition of a useful function to organelles means, for example, when targeting chloroplasts of plants, introduction of a foreign gene that improves the growth rate of plants, or targeting of mitochondria in animals. In this case, it means the introduction of a foreign gene for improving mitochondrial functions such as the respiratory function of an animal. In the case of humans, it refers to gene transfer into mitochondria related to gene therapy aimed at treating diseases caused by mutations in mitochondrial DNA (mitochondrial diseases).

The above-mentioned "selectable marker gene functioning in organelle" and "desired foreign gene functioning in organelle" are respectively transcribed in the organelle, translated by the translation codon for organelle, and the protein is synthesized in the organelle. There is a need. Therefore, a selectable marker gene that functions in an organelle, or a desired foreign gene that functions in an organelle, is not only modified to the aforementioned translation codon for organelles, but also a promoter that operates in an organelle,
It is required that a terminator, an enhancer sequence and the like be functionally linked to form a so-called expression unit. As for the promoter, terminator, enhancer sequence, etc. that operate in this organelle, a promoter, terminator, enhancer sequence, etc. attached to a gene derived from organelle may be used, and the transcription mechanism in the organelle is a prokaryotic type. Because of that,
Prokaryotic, for example, E. coli-derived promoters, terminators, enhancer sequences and the like may be used.

In particular, the cell used as a host is baker's yeast S. cerevisiae. Expression unit of a selectable marker gene that functions in cerevisiae mitochondria
S. The desired exogenous gene expression unit that functions in cerevisiae mitochondria may be prepared using the promoter, terminator sequence, etc. of the E. coli-derived lacZ gene. In the present invention, the baker's yeast S. cerevisiae mitochondria and a selectable marker gene that functions in the baker's yeast S. cerevi
The CAT gene for mitochondria ( CATM ) was used as an exogenous gene that also functions as a desired gene that functions in siae mitochondria. It is not limited to this. Baker's yeast S. c
erevisiae mitochondria and a selectable marker gene that functions in the baker's yeast S. cerevisiae CAT gene for mitochondria ( CATM ) as a foreign gene that also functions as a desired gene that functions in mitochondria
Escherichia coli containing the conjugative transfer organelle expression vector using E. coli was
It has been deposited as MP-15726.

The "oriT sequence necessary for conjugative transfer between prokaryotic and eukaryotic organisms" contained in the conjugative transfer organelle expression vector is recognized by the mob gene product which is an oriT-specific nick enzyme in the donor bacterium. A sequence having a function of initiating transfer of a conjugative transfer organelle expression vector into a host cell into an organelle, which becomes a single-stranded DNA. From the already known mechanism of conjugation transmission, the following tra gene,
And one strand of the plasmid DNA of the expression vector in the conjugative transfer organelle is cleaved at the oriT site in the vector by the action of the mob gene product expressed in the helper plasmid containing the mob gene, and the single-stranded DNA is reduced to 5 '.
It is presumed that they enter the yeast cells from the terminal, pass through the organelle double membrane, and migrate to the organelle. The oriT sequence required for conjugation transfer is, for example, when the donor bacterium is Escherichia coli,
The oriT sequence encoded by Escherichia coli F factor is preferably used. Also, for example, if the donor bacterium is Agrobacter
In the case of ium tumefaciens , an oriT sequence encoded by E. coli F factor or an oriT sequence encoded by a Ti plasmid may be used. When the oriT sequence is contained in the expression vector in the conjugative transfer organelle, the mot encoded in the vicinity of the oriT sequence is expressed.
The b gene may also be included in the vector, but it is unlikely that this will have a bad effect. Therefore, the vector may include the mob gene at the same time as the oriT sequence.

In the present invention, the “tra gene and mo
The “helper plasmid containing the b gene” refers to a helper plasmid having a sequence containing a tra gene and a mob gene required for conjugation transfer between prokaryotes and eukaryotes.

Tra necessary for conjugation transmission between prokaryotic and eukaryotic organisms
The gene is a generic term for a large number of genes consisting of genes involved in cilia formation, junctional tube formation, and junctional control. For example, the tra gene contained in the E. coli F plasmid involved in E. coli It can be used for eukaryotic conjugation transmission.

The tra gene does not need to be present on the conjugative organelle expression vector because it functions as a transelement for prokaryotic / eukaryotic conjugative transmission of the vector. As described above, since the base sequence is composed of a large number of genes and the nucleotide sequence is long, it is preferable to use the tra gene in another helper plasmid. The mob gene also functions as a transelement for prokaryotic / eukaryotic conjugation transfer of the “expression vector for conjugative organelles” as in the case of the tra gene.
The mob gene is not a single gene but mobA, mob
It is considered that it is composed of a group of genes such as B and mobC and that they may act synergistically. As the sequences of the tra gene and the mob gene, for example, a sequence encoded by Escherichia coli F factor may be used.

It is necessary that the helper plasmid contains an origin of replication for autonomous growth in the donor bacterium and a selection marker. For example, when the donor bacterium is Escherichia coli, any replication origin or selection marker that functions in Escherichia coli may be used. Also, for example, if the donor bacterium is Agrobacter
In the case of ium tumefaciens , any origin of replication and selection marker that functions in the cells may be used.

The tra gene derived from Escherichia coli F factor and mo
The recombinant plasmids containing the b gene include, for example, plasmid pRK2013 (Figurski et al., Proc, N
atl. Acad. Sci. USA, 1979, 76 :
1648, available from the ATCC; ATCC 3715
9), and tr prepared by excision from the plasmid
A plasmid containing a replication origin and a selectable marker that functions in Escherichia coli into which a DNA fragment containing the a gene and the mob gene is inserted, and constructing the tra gene and the mob of the present invention
It can be used as a helper plasmid containing a gene.

In the present invention, "tra gene and mo
"Prokaryotic / eukaryotic conjugation transmission using the b gene"
Under appropriate artificial conditions, a donor bacterium holding the “conjugative transfer organelle expression vector” and a donor bacterium holding the “helper plasmid containing the tra gene and the mob gene” and a host eukaryotic cell under appropriate artificial conditions Multiplication refers to a method of prokaryotic / eukaryotic conjugative transfer in which the vector is introduced under mild conditions into an organelle of a eukaryotic cell as a host. Prokaryotic / eukaryotic conjugative transfer includes a donor bacterium holding an "expression vector for conjugative organelle", a donor bacterium holding a helper plasmid containing a tra gene and a mob gene, and a host eukaryote. Tripartal mating (Triparental matin) is performed using the three cells and under conditions of appropriate artificial prokaryotic / eukaryotic conjugation transmission.
By performing g), an expression vector in a conjugative transfer organelle containing a desired foreign gene is introduced into an organelle in a eukaryotic cell as a host.

The coexistence of plasmid DNA in the donor is determined by the incompatibility gene inc, and the same inc species cannot coexist in the same donor for a long time (Kazuo Yoshida, Masanobu Nishikawa,
1993, protein nucleic acid enzyme, 38:60 ). However, even in the case of two plasmids of the same inc species, a helper plasmid carried by one donor is transferred to another donor by conjugation, and then the same ind.
The present inventors have conducted intensive studies and demonstrated that conjugative transfer of a plasmid, which is a c-type conjugative shuttle vector, to an organelle of a eukaryotic cell for the purpose of prokaryotic / eukaryotic conjugative transfer was carried out. This is called a three-parent junction (Tripa
Rental matching). Also, for example, i
It is possible for the donor to simultaneously hold two plasmids, an ncP type conjugative transfer organelle expression vector and an inc type different helper plasmid, for example, the incQ type helper plasmid. The eukaryotic cell, which is the host by prokaryotic-eukaryotic conjugation, under the conditions of appropriate artificial prokaryotic-eukaryotic conjugation transmission, using both fungi and eukaryotic cells as the host Conjugation (Biparan) in which the desired conjugative transfer organelle expression vector is introduced into an organelle
tal mating) is also possible.

For example, baker's yeast S. cerevisia
If the e mitochondrial donor bacteria as a target of E. coli,
For prokaryotic / eukaryotic conjugative transfer, an E. coli suspension carrying a conjugative transmissible mitochondrial expression vector prepared for baker's yeast, an E. coli suspension carrying a helper plasmid containing a tra gene and a mob gene, and bread. The yeast suspension is mixed in an appropriate ratio to form a cell clump, and the cell mixture having formed the cell clump is then dropped on a sterilized filter membrane placed on a culture dish, and the pH is adjusted to a preferable pH, for example, pH 6 to pH 8 , Preferably from pH 7 to pH 7.
5 near neutral and at a preferred temperature, for example 25 ° C to 35 ° C, desirably 28 ° C to 30 ° C, overnight (12 ° C).
Incubate for about an hour). The incubation time during the prokaryotic / eukaryotic conjugation transfer was increased from 10 hours to 16 hours, and the frequency of the conjugation transfer was improved, and a better result was obtained when the incubation time was about 12 hours. Next, after recovering the cell mixture on the membrane with an appropriate medium and washing with the medium, the cells are cultured on a selection agar plate medium for a required number of days, for example, several days, and expressed by a marker gene that can be expressed in mitochondria. It is advisable to examine the degree of overcoming the selective pressure in the selection medium and select a transformed baker's yeast in which the expression vector for conjugative transfer in mitochondria has been introduced into mitochondria. In this case, the mixing ratio of each Escherichia coli and baker's yeast used for prokaryotic / eukaryotic conjugative transfer is not particularly limited as long as any one of them is so small that conjugative transfer is not established.
For example, the ratio of each E. coli to baker's yeast is 1: 1: 10
And good results were obtained. The medium for conjugation transfer was used for E. coli suspensions and baker's yeast suspensions, but for the purpose of avoiding excessive growth of Escherichia coli during conjugation transfer, a yeast medium containing glycerol as a carbon source, for example, a YPG medium ( A medium in which the assimilable carbon source glucose of Escherichia coli contained in the YPD medium for yeast is replaced with glycerol which is more difficult to assimilate in Escherichia coli), or a synthetic medium for yeast in which the growth of Escherichia coli is even worse, such as a synthetic dextrose (SD) medium Use gives good results. Selection of the transformed yeast after the prokaryotic / eukaryotic conjugative transfer is performed is performed using the above-described YPG medium or SD medium, and adding an antibiotic corresponding to the selectable marker, for example, chloramphenicol (Cm). At the same time, for the purpose of suppressing the growth of donor E. coli, nalidixic acid (Nalidic acid: Nal)
At a concentration higher than 1 μg / ml, for example 20 μg / ml
Good results can be obtained by adding at a concentration of from 25 μg / ml to 25 μg / ml. Nalidixic acid
d: Nal) is an antibiotic that inhibits DNA replication by inhibiting the activity of subunit A of prokaryotic DNA gyrase, which may affect the DNA synthesis of yeast mitochondria. At these concentrations, the growth of the yeast was slightly delayed and had no effect on the purpose of the present invention.

Biological experiments on yeast such as a medium for yeast and a yeast culture method are described in, for example, Sherman, F. et al. (Laboratory Course Men)
ual for Methods in Yeast
Genetics, 1986, Cold Spring
The method may be performed in accordance with a known method described in “Harbor Laboratory”.

For example, when the “selectable marker gene that functions in organelles” is a chloramphenicol acetyltransferase ( CATM ) gene having a base replaced by a mitochondrial translation codon, chloramphenicol (Cm) added to the selection medium The amount of Cm is an antibiotic that binds to prokaryotic liposomes and inhibits protein synthesis. It has no protein synthesis inhibitory activity in the nucleus of eukaryotic yeast, so it is usually used for E. coli. Concentration 1
500 μg / m, which is a higher concentration than 70 μg / ml
1 to 1,000 μg / ml, preferably 750 μg / μ
Since the presence of Cm at a concentration near m indicates sensitivity, it is preferable to use Cm at that concentration in a selective medium. CA
It is expected that the TM gene is expressed in a translation system using prokaryotic ribosomes in mitochondria and exhibits chloramphenicol acetyltransferase activity. Ramphenicol acetyltransferase activity was determined by CAT assay (Gorman, CM, et al.).
al. , 1982, Mol. Cell. Biol. ,
2 : 1044), and the same activity was detected in the actually obtained transformed cells.

Prokaryotic / eukaryotic conjugation transmission targeting an animal or plant organelle is carried out under appropriate artificial conditions by using a donor bacterium containing a conjugative transferable organelle expression vector and each cell as a host under appropriate artificial conditions. It is good to do it by multiplying. At that time, strong cell walls exist in prokaryotes such as plant cells, yeast, and Escherichia coli.However, in the method of conjugation between prokaryotes and eukaryotes according to the present invention, it is necessary to devise a method of digesting cell walls and multiplying them as protoplasts. Instead, it is only necessary to perform a direct cross-over process, and then culture in a selection medium to select transformed cells. Separation of the donor cells and the host cells from the transformed organelles can be carried out by a combination of auxotrophy, drug resistance, enzyme treatment with lysozyme or the like. When the host cell is derived from an animal, generally, the selection medium is rich in nutrients, so that the donor bacterium holding the expression vector in the conjugative organelle and the donor bacterium holding the helper plasmid containing the tra gene and the mob gene are used. Since the growth power in the selection medium becomes strong, it may be difficult to remove the donor bacteria after the crossing treatment. Thus, for example, when the donor bacterium is Escherichia coli, it is known that an Escherichia coli minicell having no chromosome can transfer conjugation between yeast and prokaryotic / eukaryotic organisms. May be used for prokaryotic / eukaryotic conjugative transmission targeting organelles.

When the host is an animal cell, the organelle transformed cell produced by the method of the present invention may be subjected to cell fusion with an animal cell having other useful properties, or transplanted into an animal tissue. The animal cell used as a host is Embryon
If it is an ic stem cell, it may be transplanted into an embryo at an early stage of development and differentiated in an individual. A cell may be obtained by cell fusion with a plant cell having, or cultured and grown as a callus and transferred to a plant tissue, or grown under conditions that differentiate the callus.

"Conjugative transfer organelle expression vector"
Construction of plasmid DNA necessary for the preparation of DNA, detection of specific DNA fragment by Southern hybridization,
A series of molecular biology experiments, such as DNA sequencing, are described, for example, in Maniatis, T .; (Molecular Cloning, A Labo)
rattro Manual, 2nd. Ed. , Sam
Brook, J. et al. Fritsch, E .; F, and
T. Maniatis, Cold Spring
Harbor Lab. Press, 1989). Unless otherwise indicated, known enzymes can be used, and examples thereof include those manufactured by Takara Shuzo or Toyobo, but are not limited thereto. DNA fragment length is base pairs (bp)
, But the values set forth in this specification should be read as approximate, in accordance with common knowledge in the art.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

[0041]

【Example】

Example 1 C by site-directed mutagenesis
Conversion of AT gene into mitochondrial translation codon The nucleotide sequence of the E. coli-derived CAT gene coding region represented by SEQ ID NO: 1 in the sequence listing was converted to the baker's yeast S. A desired exogenous gene / selection marker gene was prepared by substituting a base sequence converted to a translation codon for cerevisiae mitochondria and including the expression vector in a conjugative transfer organelle targeting baker's yeast mitochondria. That is, based on the base sequence of the Escherichia coli-derived CAT gene, bases which need to be replaced with baker's yeast mitochondrial translation codons in the 660 base length sequence from the start codon to the stop codon of the Escherichia coli-derived CAT gene. Are 39 (A → T), 115 (C → T), 187 represented by the numbers of the nucleotide sequence of SEQ ID NO: 1 in the sequence listing.
(C → T), 189 (T → A), 196 (C → T), 2
44 (C → T), 252 (A → T), 313 (C →
T), 315 (C → A), 349 (C → T), 357
(A → T), 391 (C → T), 550 (C → T), 5
59 (C → T), 613 (C → T), and 615 (T →
In A), there were a total of 16 locations. Here, the parentheses indicate base substitutions for conversion to mitochondrial translation codons. In site-directed mutagenesis, synthetic DNA
Uses primers to perform base substitution on the target DNA fragment. If the bases to be replaced are close to each other, the base sequence of the synthetic DNA primer for mutagenesis can be designed to be included in the same primer. It is. That is, by using a total of 9 synthetic DNA primers for mutagenesis shown in SEQ ID NO: 2 to SEQ ID NO: 10 in order for 16 base substitutions, all the necessary base substitutions were made to correspond, and SEQ ID NO: 11 The sequence of the coding region of the CATM gene in which the base was substituted corresponding to the translation codon for baker's yeast mitochondria shown was completed. When the positions of the above nine synthetic DNA primers for mutagenesis are indicated using the nucleotide sequence numbers of SEQ ID NO: 1, SEQ ID NO: 2 (27
-49), SEQ ID NO: 3 (105-127), SEQ ID NO: 4
(174-208), SEQ ID NO: 5 (234-262),
SEQ ID NO: 6 (302-327), SEQ ID NO: 7 (339-
370), SEQ ID NO: 8 (381-403) SEQ ID NO: 9
(540-570), and SEQ ID NO: 10 (603-62)
8). The procedure for specifically replacing the CAT gene with a nucleotide sequence converted into a mitochondrial translation codon is shown below.

A 792-base Hin containing the chloramphenicol acetyltransferase (CAT) gene sequence derived from Escherichia coli supplied by Pharmacia Biotech
The dIII DNA fragment was converted to ATCC (American).
Type CultureCollection; Ro
ckville, MD, USA) and the plasmid pUC19 (ATCC 37254) Hin
cloned into the dIII site, the plasmid pUC-C
AT was prepared.

SEQ ID NO: 2 to 9 represented by SEQ ID NO: 10
The synthetic DNA primers for mutagenesis were prepared according to a standard method of oligo DNA synthesis, and the prepared DNA primers were phosphorylated at their 5 'ends using T4 polynucleotide kinase and ATP, respectively. Next, TransformerSi supplied by CloneTech
te-Directed Mutagenesis K
It was used to convert one of the nine synthetic oligo DNAs for mutagenesis and one of the NdeI / NcoI trans-oligo DNAs used as selection primers or the switch oligo NcoI / NdeI into the plasmid p.
Mutagenesis was attempted to be introduced into the E. coli CAT gene sequence by using as primers that anneal to the same strand of UC-CAT. That is, CAT for the purpose of mutation introduction
PUC-CAT plasmid DNA containing the gene sequence, one of nine synthetic oligo DNAs for mutation introduction out of 9 phosphorylated at the 5 'end used as the first primer, and supplied by Clontech as the second primer Of the 5'-terminal phosphorylated NdeI / NcoI trans-oligo DNA was added to 20 μl of 20 mM Tris.
-HCl (pH7.5) -10mM MgCl ₂ -50
The mixture was heated in an mM NaCl buffer at 95 ° C. for 3 minutes, immediately cooled on ice water for 5 minutes, and a double strand was formed between the two primers and the CAT gene sequence by an annealing reaction. For the solution after the annealing reaction,
In vitro DN supplied with Clonetech kit
A synthesis buffer, T4 DNA polymerase, and T4D
A required amount of NA ligase was added, and the mixture was incubated at 37 ° C. for 2 hours. Based on the two annealed primer DNAs, a complementary strand DNA elongation reaction and a ligation reaction were performed to prepare a complete double-stranded plasmid DNA. Heat at 70 ° C for 5 minutes,
The enzymatic reaction was stopped. The obtained reaction solution was used for Escherichia coli BMH71-18 mut S supplied with a kit of Clontech.
(Repair-deficient) competent cells, and the transformed Escherichia coli was cultured overnight at 37 ° C. in an LB medium containing 5 ml of ampicillin.
Plasmid DN extracted from cultured E. coli by a standard method
A 0.1 μg was digested with restriction enzyme NdeI, and
After heating for 10 minutes to stop the enzymatic reaction,
Transformation was performed with 71-18 mut S competent cells, and the cells were cultured overnight at 37 ° C. on an agar plate LB medium containing ampicillin. Several of the obtained ampicillin-resistant transformant colonies were cultured in several clones, plasmid DNA was extracted and purified by a standard method using an alkaline method, the DNA base sequence was determined by the method described below, and site-specific mutation was performed as designed. A recombinant plasmid in which the sequence containing the mutation was confirmed was selected. For DNA sequencing, DNA sequence kit (Dye prime) supplied by PerkinElmer
r cycle sequencing kit; 21
M13primer, AmpliTaq DNA po
lymerase) in each of four tubes A, C, G, or T, DNA (template), dye primer, d / dd mix of A, C, G, or T (each deoxynucleotide / A mixture of dideoxynucleotides serving as terminators), Ta
qDNA polymerase and buffer were added and mixed, and the enzymatic reaction was performed by PCR according to the protocol shown by PerkinElmer.
Reactor (Ato Co., Zymoreactor, AB-
1800) was repeated 25 times. The nucleotide sequence was read from the reaction product using Perkin Elmer's Applied Biosystems 373S DNA sequencer according to the protocol indicated by the company.

The procedure for obtaining the mutant CAT sequence shown above was carried out by sequentially using the nine mutation-introducing oligo DNAs shown in SEQ ID NO: 2 to SEQ ID NO: 10 as first primers and supplied by Clontech. Transoligo N
deI / NcoI and switch oligo NcoI / Nd
The above mutation introduction operation was repeated nine times using eI in turn as the second primer, and alternately using restriction enzymes NdeI and NcoI according to the manual supplied by Clontech. As a result, a total of 16 base substitutions were completed, and a CAT ( CATM ) gene converted to a translation codon for baker's yeast mitochondria was obtained. For the final confirmation of the DNA sequence of the completed CATM gene, the nucleotide sequence was determined from both directions by the aforementioned DNA nucleotide sequence determination method. As a result, the prepared plasmid pUC-C
In the CATM gene sequence contained in the ATM, base substitution was confirmed at a total of 16 positions as designed, and the desired CATM gene sequence represented by SEQ ID NO: 11 was obtained (FIG. 1, p
UC-CATM).

Example 2 Construction of a conjugative transfer mitochondrial expression vector, pAY-CATM A plasmid YEp352 (available from ATCC; ATCC 37675) containing a 2 μm replication origin, which is an extrachromosomal replication origin of yeast, was replaced with a restriction enzyme HpaI. To obtain a blunt-ended linear DNA. Next, plasmid pKT230 (available from ATCC; AT
CC37294) was digested with restriction enzyme EcoRV, fractionated by agarose gel electrophoresis, extracted and purified to obtain a 2.7 kb DNA fragment having blunt ends. The obtained linear YEp352 and the 2.7 kb EcoRV fragment were ligated using T4 ligase (a ligation kit manufactured by Takara Shuzo Co., Ltd.) under the reaction conditions of binding the blunt end shown by the supplier.
The configuration shown in plasmid pAYC7 (oriV -
C, oriT- Q, mob- Q, lacZ , URA3 ,
2 μm-ori , Ap r ). After terminating the reaction with T4 ligase, the TA ligase reaction mixture was used to remove E. coli HB101 ( F- , recAl3 , hsdS20).
can be obtained from proA2 , Sm r , ATCC;
ATCC 33694) by the rubidium chloride method (Hana).
han, D.C. , Techniques of tran
formation of E.S. coli. , In:
DNA Cloning, Ed. Glover, D .;
M. 1985, vol. 1,109-135, IRL
Press, Oxford). Several Ap-resistant transformed Escherichia coli colonies were selected, and transformed Escherichia coli was subjected to liquid culture from the transformed colonies.
The plasmid DNA was extracted and purified, cut with a restriction enzyme, and analyzed by agarose gel electrophoresis. Transformed Escherichia coli harboring the plasmid DNA having the restriction pattern of the target plasmid pAYC7 was selected.

The plasmid DNA prepared in Example 1 (FIG. 1, pUC-CATM) was digested with the control enzyme HindIII, fractionated by agarose gel electrophoresis, cut out, and then subjected to the CATM gene corresponding to the mitochondrial translation codon. Was extracted and purified. 792 bases of H containing the obtained CATM gene
The indIII fragment and the plasmid pAY prepared above
C7 was digested with HindIII to obtain a linear product, which was ligated using T4 ligase to give plasmid pAYC
The plasmid pAY-CATM having the configuration shown in FIG. 1 was constructed in which the promoter and terminator of the LacZ gene contained in No. 7 and the direction of the CATM gene inserted therebetween became forward. After termination of the reaction with T4 ligase, Escherichia coli HB101 was transformed by a rubidium chloride method using a T4 ligase reaction solution containing pAY-CATM. Several Ap-resistant transformed Escherichia coli cells were selected, plasmid DNA was extracted and purified from each, cut with restriction enzymes, and analyzed by agarose gel electrophoresis.
Plasmid pAY-CAT having a control enzyme map shown in FIG.
A transformed Escherichia coli carrying M was selected (Accession No. FERM P-157, Institute of Biotechnology and Industrial Technology, Institute of Industrial Science and Technology)
No. 26). The constructed pAY-CATM ( oriV- C,
oriT -Q, mob -Q, CATM, URA3, 2μ
m-ori, the Ap r), to the position shown in FIG. 1, baker's yeast S. 2 μm replication origin ( 2 μm-ori ) as an origin of replication functioning in cerevisiae mitochondria;
CAT that combines both selectable marker gene and desired foreign gene
M ( CATM in which the LacZ promoter and terminator of Escherichia coli are operatively linked), and a ColE1-type replication origin (ColElori =
oriV -C), the ampicillin resistance gene (Amp = AP r as a selective marker gene in E. coli), oriT sequence of bonding a transfer start point (oriT = oriT -Q), and mob gene (mobB, mobA, mobC = mo
b- Q), which satisfies the requirements for a conjugative transferable mitochondrial expression vector.

Example 3 Helper plasmid pRH22
Construction of plasmid pRK2013 (available from ATCC; ATCC 37159) with EcoRI and Sal
A DNA fragment having a length of 36.3 kb obtained by digestion with I;
Plasmid pHSG576 (available from the Japan Cancer Research Foundation, JCRB Gene Bank; De)
post Number VE037; Takesh
ita, S .; et al. , 1987, Gene, 6
One sixty-three) to EcoRI, and a DNA fragment of about 3.6kb long comprising a pSC101-derived origin of replication and chloramphenicol resistance gene obtained by cutting with SalI, fractionated in each agarose electrophoresis, extracted and purified Prepared. Each of the obtained DNA fragments was ligated with T4 ligase, and a helper plasmid pRH22 of about 39.9 kb in length was used.
0 (oriV -pSC101, oriT -P, tra -
P, mob- P, Cm r ). After terminating the reaction with T4 ligase, Escherichia coli HB101 was transformed by a rubidium chloride method using a T4 ligase reaction solution containing pRH220. Several obtained Ap-resistant transformed Escherichia coli were selected, plasmid DNA was extracted and purified, cut with restriction enzymes, analyzed by agarose gel electrophoresis, and plasmid DNA having a restriction enzyme map of the desired plasmid
The transformed E. coli which retained the E. coli was selected. OriT and mob contained in pAY-CATM constructed in Example 2 are i
Since the tra gene contained in the helper plasmid pRH220 is of the ncQ type and is not of the same inc species, the two plasmids can coexist stably in the same E. coli.

Example 4 Transfer of pAY-CATM from E. coli to Yeast Mitochondria by Prokaryotic / Eukaryotic Conjugative Transfer Escherichia coli HB transformed with plasmid pAY-CATM
101 (pAY-CATM) and plasmid pRH2
E. coli HB101 (pRH220) transformed with
Using baker's yeast S. cerevisiae YNN
281 strains ( MATa , trp1-Δ , ura3-52 ,
ade2-1 , lys2-801 , his3- Δ20
0 , rho + , Yeast Genetic Sto
CK Center, Berkeley, CA, USA) or mitochondrial DNA-deficient baker's yeast S.C. cerevisiae YNN281 rho o strain (mutant strain rho o is subjected to shaking at 28 ° C. for 24 hours while shading at 28 ° C. for 24 hours in the presence of sterilized water containing ethidium bromide at a concentration of 20 μg / ml.
It was obtained by culturing on an agar plate at 28 ° C. for a few days and selecting from colonies that appeared. Mitochondrial DNA deficiency, i.e. the rho strain, is a DAP from Sigma.
I, that is, 4 ', 6-diamin-2--2-ph
Confirmation was performed by staining with nylindol and a genetic method. ) And the following procedure was used to perform prokaryotic / eukaryotic conjugation by the three-parent transmission method.

The baker's yeast S. cerevisiae YNN
281 strains in YPD medium (2% glucose, 1%
Shake culture was performed at 28 ° C. using yeast extract (containing 2% peptone), and turbidity was sometimes measured with a turbidity meter (Fuji ADS-D densitometer, manufactured by Fuji Kogyo Co., Ltd., Tokyo). A yeast culture grown up to a turbidity of 200 was obtained. E. coli carrying pAY-CATM, p
Escherichia coli holding RH220 was isolated by Ap (50 μg).
/ Ml) in LB medium (1% Bacto tryp)
tone, 0.5% yeast extract, 1
% NaCl, containing 5 mM NaOH), shaking culture at 37 ° C., turbidity is sometimes measured with a turbidity meter (Fuji ADS-D densitometer, manufactured by Fuji Kogyo Co., Ltd., Tokyo), and turbidity is measured. An E. coli suspension with a degree of 50 was obtained. Baker's yeast S turbidity has grown to 200. cerevisiae
500 μl of a yeast culture solution of YNN281 strain, pAY-C
50 μl of an Escherichia coli culture grown to a turbidity of 50 retaining ATM and 50 μl of an Escherichia coli culture grown to a turbidity of 50 retaining plasmid pRH220
Was placed in a 1.5 cc Eppendorf tube and mixed.
Next, using a microcentrifuge, centrifugation was performed at 6,000 rpm for 5 minutes to precipitate the cell mixture. The precipitated cell mixture was mixed with 500 μl of TNB buffer (50 mM Tris-HC).
, pH 7.5, containing 0.05% NaCl), and centrifuged again at 6,000 rpm for 5 minutes using a microcentrifuge to precipitate the collected cell mixture in 40 μl of TNB buffer. The mixture was mixed and suspended at room temperature for several minutes. The suspended cell mixture was added to 1.5% agar (Difco,
Filter membrane (typeHA; nitr) manufactured by Millipore on YPD agar medium containing Bacto-agar)
ocellulose / cellulose acet
ate, pore size 0.45 μm;
eter, 25 mm) and dried in the air for 10 minutes, followed by incubation in an incubator at 28 ° C. for 12 hours to carry out prokaryotic / eukaryotic conjugative transfer. After incubation, the cell mixture on the filter was washed with a small amount of TNB buffer, recovered, and resuspended in 400 μl of the same buffer. Then chloramphenicol (Cm) was added to a final concentration of 7
Nalidixic acid (Nal) at a final concentration of 20 μg so as to be 50 μg / ml and for the purpose of inhibiting the growth of Escherichia coli.
/1.5% agar-containing YPG
Medium (3% glycerol, 1% yeast e)
xtract, containing 2% peptone) agar plate 1
After spreading 150 μl of the above-described cell suspension per spreader with a spreader, the cells were cultured in an incubator set at a temperature of 28 ° C. for 3 to 4 days. As a result, about 50 chloramphenicol-resistant yeast colonies were obtained. . Similarly, baker's yeast YN
When only the N281 strain was cultured in the selection medium, some chloramphenicol-resistant yeast colonies appeared. The baker's yeast S. p. The results of introduction into cerevisiae mitochondria are shown in Table 16 below as Table 1.

[0051]

[Table 16]

As shown in the results of Experiment No. 1 in Table 1, the baker's yeast S. cerevisiae YNN281
Frequency of chloramphenicol resistant colonies by tripartite junctions with strain whereas was 3.3 × 10- ^5, the experiment number 2 mitochondrial DNA deficiency baker's yeast S. cer
When Evisiae YNN281 rho o strain was used, mitochondria were deleted, mitochondrial function was insufficient, and mitochondrial transformation did not occur. Therefore, the frequency of appearance of chloramphenicol-resistant colonies was 1 × 10 −. ^No more than ⁸ colonies were substantially obtained. In the experiment lacking the helper plasmid pRH220 of Experiment No. 3 and the prokaryotic-eukaryotic conjugation experiment lacking the expression vector pAY-CATM for mating transfer mitochondria for baker's yeast of Experiment No. 4, the appearance frequency of chloramphenicol-resistant colonies there was also 10- ⁶ or less in the case of only the baker's yeast negative control experiment number 5. Baker's yeast S. cerevisiae is mitochondrial DN
A is a type of mutation that is susceptible to mutation of A, and is therefore called a petit positive yeast, and a mutation that has lost respiratory function, that is, a respiratory-deficient strain (RD) is easily obtained. Table-1
Of Experiment No. 5 of the (negative control) in the experiment a few chloramphenicol resistance baker's yeast only bread yeast appeared (frequency 10 ⁶ or less), the same as in Experiment No. 2 1 × 10- ⁸
The reason that the following did not occur was that chloramphenicol resistance was acquired due to mutations in mitochondrial DNA of genes that act on chloramphenicol encoded by mitochondrial DNA, that is, mitochondrial rRNA and mitochondrial ribosome-related genes. This was attributed to the emergence of developmental mutants.

Example 5 Detection of Plasmid pAY-CATM from Chloramphenicol-Resistant Yeast Mitochondria by Southern Hybridization Method A few of about 50 chloramphenicol-resistant yeast colonies obtained in Example 4 Colonies and yeast Y not transfected to serve as a negative control
Using colonies derived from the NN281 strain as a starting material, yeast mitochondria grown by liquid culture were isolated, DNA was extracted from the isolated mitochondria, and a 792-base HindII encoding CATM obtained in Example 1 was obtained.
An experiment was performed to confirm that the introduced plasmid pAY-CATM was present in mitochondria using the Southern hybridization method using the I fragment as a probe.

The selected yeast colonies were each pre-cultured in 20 ml of YPD medium, and then 1 μl of YPD medium containing 200 ml of chloramphenicol at a concentration of 750 μg / ml.
% Of the mixture, and shaken at 28 ° C. for 2 days to perform main culture. The obtained culture solution was centrifuged at 5,000 rpm for 15 minutes using a low-speed centrifuge to collect the cells, and 200 ml of 10 mM EDT was collected.
A, The cells were suspended in a 0.1% 2-mercaptoethanol solution and collected by centrifugation once again to wash the cells. The washed cells were mixed with 38 ml of 1.2 M Sorbitol,
20 mM EDTA, 7.5 mM NaH ₂ PO ₄ , 4
The suspension was suspended in a 2.5 mM Na ₂ HPO ₄ solution, transferred to a polypropylene (PP) tube (manufactured by Falcon) containing 50 ml, and 0.8 ml of 2-Mercaptoethanol was added.
l and 10 mg / ml concentration of Zymolyase-10
0.8 ml of 0T (manufactured by Seikagaku Corporation) was added, and the mixture was shaken slowly at 37 ° C. for 1 hour to form protoplasts. After confirming that the baker's yeast had been protoplasted by using a microscope, the yeast was centrifuged at 5,000 rpm for 10 minutes using a low-speed centrifuge, and the precipitated protoplast-yeast was added to 40 ml of 0.1 ml.
7M Sorbitol, 10mM EDTA, 50m
M Tris-HCl (pH 7.5) solution.
Transfer to a PP tube containing 0 ml, immerse the tube in ice water and use an ultrasonic crusher manufactured by Tommy Seiko while cooling with ice.
For the purpose of disrupting the yeast cell membrane, several sonications were performed for 10 seconds each. At that time, the degree of destruction of the yeast cell membrane was examined using a microscope every 10 seconds of ultrasonic treatment, and care was taken to prevent excessive destruction of mitochondria. The sonicated cell suspension was centrifuged at 5,000 rpm for 10 minutes with a low-speed centrifuge to precipitate nuclei and cell debris. After centrifugation, the supernatant was added to another 50 ml
Transfer to a P tube, and 5,000 rpm again with a low-speed centrifuge.
centrifugation for 10 minutes to repeatedly precipitate nuclei and cell debris. Next, the supernatant containing mitochondria was transferred to a tube for high-speed centrifugation, and a high-speed centrifuge was used at 16,000 rpm for 10 minutes.
After centrifugation for minutes, mitochondria were precipitated. Precipitated mitochondria were added to 350 μl of 0.7 M Sorbito
1, 2 mM EDTA, 10 mM Tris-HCl
(PH 7.5) and suspended in a 1.5 cc Eppendorf tube. Next, 1.25 μl of 1M MgSO ₄ and 1 μl of DNase I (10 units / μl) were added to the obtained mitochondrial suspension, and the tube was left standing on ice for 30 minutes to perform a DNA degradation reaction. The DNA derived from the nucleus which was considered to be contaminated by adhering to the outside was decomposed and removed by a DNAase enzyme treatment.
This mitochondrial suspension was added at 16,000 rpm, 5
After centrifugation for minutes, the mitochondria were precipitated. Next, mitochondria precipitated by centrifugation were removed by 350 μl of lysis
buffer (10 mM Tris-HCl, pH
8.0, 10 mM EDTA, containing 1% SDS), and proteinase K was added to a final concentration of 100 μg /
ml and incubated at 37 ° C. for 1 hour. After incubation for 1 hour, phenol extraction is performed once using an equal amount of phenol, and phenol / chloroform extraction is performed several times using an equal amount of phenol / chloroform (1: 1), and nucleic acids are precipitated by an ethanol precipitation method. I let it. After collecting and drying the nucleic acid by centrifugation, 100
μl of TE buffer (10 mM Tris-HCl, pH
7.5, containing 1 mM EDTA) and heat treated DNAase-free Pancreatic R
Nase A was added at a final concentration of 10 μg / ml and
For 30 minutes to decompose RNA. Next, phenol extraction was performed once using an equal amount of phenol, and phenol / chloroform extraction was performed once using an equal amount of phenol / chloroform (1: 1), and purified DNA was precipitated by an ethanol precipitation method. . After drying the purified DNA collected by centrifugation, it was dissolved in 20 μl of TE buffer,
Mitochondrial total DNA was used.

The obtained mitochondrial total DNA was cut with restriction enzymes EcoRI, ScaI, SspI or HindIII, respectively, and fractionated by 0.8% agarose gel electrophoresis. Next, the agarose gel was denatured with alkali and neutralized to convert the DNA in the agarose gel into single strands.
The solution was transferred to a nitrocellulose membrane filter (manufactured by Schleicher & Schuell, BA85) by the Southern method using a NaCl, 0.3 M Na-citrate) solution, and the membrane filter was heated in a vacuum oven at 80 ° C for 2 hours. Next, the membrane filter was mixed with 30 ml of a hybridization solution (5 × SSC, 0.1% Sodi).
um N-lauroyl sarcosinate,
0.02% SDS, 0.5% Boehriger M
Blocking reagent made by annheim
Incubation was carried out at 60 ° C. for 3 hours in a plastic bag containing (t. CAT prepared from plasmid pAY-CATM
Using a random primer DNA labeling kit manufactured by Takara Shuzo, a 792-base HindIII fragment encoding the M gene was obtained using [α- ³² P] dCTP (Amers
ham Co., performs a specific activity of about 110TBq / mmol), random primers in labeling kit manufactured by Takara Shuzo Co., Ltd., a reaction using a DNA PolymeraseKlenow fragment (2unites / μl), CA
A TM sequence-specific labeled probe was prepared. After completion of the prehybridization, the prehybridization solution is discarded, and 30 ml of a solution having the same composition as the above prehybridization solution and a CATM sequence-specific labeled probe (about 3 × 10 ⁷ counts / min) are added. The filter was again incubated at 60 ° C. for 19 hours to perform a hybridization reaction. After completion of the hybridization reaction, the membrane filter was gently shaken once at room temperature for 15 minutes using a washing solution (2 × SSC, containing 0.1% SDS), and further washed after washing with a low salt concentration (0.5%). × SSC, containing 0.1% SDS), washed gently twice at room temperature for 15 minutes, and air-dried. The air-dried membrane filter was exposed to an imaging plate (manufactured by Fuji Film) instead of an X-ray film for about 6 hours, and the hybridization pattern was examined using an imaging plate reader manufactured by Fuji Film (FIG. 2). . As a result, no hybridization signal was detected by the CATM probe from the total mitochondrial DNA of the yeast YNN281 used as the host, and a hybridization signal was detected from the total mitochondrial DNA derived from the chloramphenicol-resistant transformed yeast. It was confirmed that the pAY-CATM plasmid DNA was present in mitochondria. That is, as shown in FIG. 2, the signals detected by the CATM probe from the mitochondrial total DNA derived from the chloramphenicol resistant transformed yeast were the restriction enzymes EcoRI, ScaI, and S
In the case of the lane cut with spI, the same hybridization image was obtained by the pattern of the original pAY-CATM used for the introduction and the pattern of the mitochondrial total DNA derived from the chloramphenicol-resistant transformed yeast. This result indicates that the plasmid pAY-CAT
M was shown to be stably present in mitochondria of chloramphenicol resistant transformed baker's yeast. However, in the lane cut with the restriction enzyme HindIII, Hi was used.
Due to the small size of the ndIII fragment, it was observed in the pAY-CATM plasmid DNA of the positive control, but in the mitochondrial total DNA derived from the chloramphenicol-resistant transformed yeast, the hybridization image was weak and the detection sensitivity was lower. .

Example 6 Detection of CATM Gene Expression from Chloramphenicol-Resistant Transformed Baker's Yeast Colony of chloramphenicol-resistant transformed baker's yeast obtained in Example 4 (Example 4, Experiment No. 1 in Table 1) ), The plasmid pAY was added to the yeast mitochondria in Example 5.
CAT assay (Gorman, CM, et. Al.) Was performed on transformants confirmed to contain -CATM.
al. , 1982, Mol. Cell, Biol. ,
2 : 1044) from FAS supplied by Japan Gene
T CAT Green (deoxy) Chloramp
henicol Acetyltransferase
Chloramphenicol acetyltransferase (CAT) activity was detected by a method using a fluorescent substrate using Assay Kit. Saw the above C
Fluorescent substrate (1-deox) included in AT assay kit
y-chloramphenicol) and acety
l CoA and chloramphenicol-resistant transformed baker's yeast cells protoplasted by the method of Example 5 were mixed, incubated at 37 ° C. for 1 hour under the reaction conditions indicated by the supplier, and allowed to react. With a low-speed centrifuge
The mixture was centrifuged at 00 rpm for 10 minutes and centrifuged to remove. The resulting supernatant was separated by thin-layer chromatography (TLC), irradiated with ultraviolet rays (366 nm), and the fluorescent spot was detected. 3-ace
tyl-1-deoxy-chloramphenico
1 spots were examined and CAT activity was measured. As a negative control, baker's yeast S. cerevisia
e YNN281 strain, Example 4
× 10- ⁶ was found at the following frequencies chloramphenicol resistance baker's yeast S. A supernatant obtained by reacting and centrifuging a protoplast prepared from S. cerevisiae YNN281 in the same manner is used, and a cell extract of Escherichia coli HB101 containing plasmid pAY-CATM (including EDTA indicated by the supplier) is used as an anode control. Lysis
prepared using a buffer). Escherichia coli HB101 carrying plasmid pAY-CATM is
Because it exhibited Cm resistance itself, the CATM gene was determined to be expressed as an active protein in E. coli. FIG.
As shown, the prokaryotic-eukaryotic between chloramphenicol resistant transformants plasmid pAY-CATM is obtained by introducing the baker's yeast mitochondria by conjugal transfer, C
Enzyme activity measurements showed that the ATM gene was expressed in mitochondria. On the other hand, Example 4 and Table-1
CAT is in Experiment No. 5 of 1 × 10- ⁶ below were found at the frequency chloramphenicol resistance baker's yeast YNN281 strain
No activity was detected, confirming that the mutant was a naturally occurring mutant that had acquired chloramphenicol resistance.

Example 7 Restriction Enzyme Cleavage Pattern of Plasmids Recovered after Transformation into Escherichia coli from Chloramphenicol-Resistant Yeast Mitochondria Mitochondrial DNA Recovered from Chloramphenicol-Resistant Transformed Yeast Mitochondria by the Method of Example 5
(1 μg) and Escherichia coli HB1 by the rubidium chloride method.
01 and transformation (back transformation), about 20 ampicillin-resistant colonies appeared. Five of the colonies were selected and plasmid DNA was extracted from the transformed E. coli. These plasmid DNAs were cut with the restriction enzyme ScaI, and
% Agarose gel electrophoresis, and compared with the control plasmid pAY-CATM. As shown in FIG.
The plasmid obtained by back-transformation has the same structural pattern as that of the DNA of the M control plasmid DNA. Not shown.

Example 8 Stability of mitochondrial expression plasmid pAY-CATM in conjugative transfer mitochondria Plasmid pAY-CA introduced into yeast mitochondria
To determine whether TM is stably present in mitochondria, chloramphenicol-resistant transformed yeast was transformed into YPD, a non-selective medium containing no chloramphenicol.
Culture was attempted in the medium. For control experiments, CATM
Gene-free plasmid pAYC7 (FIG. 1, URA
Transformed yeast obtained by introducing 3) into the yeast nucleus by the conjugation transfer method was used.

One clone was selected from the chloramphenicol resistant transformed yeast colony obtained in Example 4 and
And shaking culture at 28 ° C. for 20 hours in 5 ml of a YPD medium containing no. At the start of the culture (0 hour), 5 hours, 10 hours, or 20 hours during the culture, take a small amount of the yeast culture, dilute and count, and take the culture containing a certain number of yeasts. On a 2% agar plate prepared in a YPD medium containing no Cm or a 2% agar plate prepared in a YPD medium containing Cm
The cells were spread on an agar plate with a spreader and cultured in an incubator at 28 ° C. for 2 to 4 days. The number of colonies that appeared on the agar plate medium containing no Cm and the number of colonies that appeared on the agar plate medium containing Cm were counted, and the ratio was calculated to determine the stability (%). As shown in FIG.
Time), the number of Cm-resistant yeasts is high even at 5 hours, 10 hours, or 20 hours during the cultivation in the non-selective medium. Even when the number of mitochondria in the mitochondria was increased, it was shown that the plasmid pAY-CATM present in the mitochondria and conferring chloramphenicol resistance was stably maintained.

As a control experiment, a transformed yeast in which a plasmid pAYC7 containing no CATM gene (FIG. 1, encoding URA3) was introduced into the yeast nucleus by the conjugative transfer method was used for 5 ml of yeast containing no uracil (URA). In a synthetic dextrose (SD) medium (SD medium: 2%
glucose, 0.67% Difco Bacto-
yeast nitrogen base witho
ut amino acid, 4% of 25 × am
ino acids and bases solution
ion; 25 × amino acids and ba
ses solution = 0.05% histid
ine, 0.075% tryptophan, 0.1
% Leucine, 0.1% lysine, 0.1
% Adenine, 0.1% uracil), 28
The cells were shake-cultured at 20 ° C for 20 hours. At the start of culture (0 hours)
At 5 hours, 10 hours, or 20 hours during the cultivation, a small amount of yeast culture solution was taken, diluted, counted, and a culture solution containing a certain number of yeasts was taken, and prepared in an SD medium without uracil. S on 2% agar plate or containing uracil
The cells were spread with a spreader on a 2% agar plate prepared in D medium and cultured at 28 ° C. for 4 or 5 days in an incubator. By counting the number of colonies that appeared on the agar plate medium without uracil and the number of colonies that appeared on the agar plate medium with uracil, calculating the ratio and culturing it on a non-selective medium without uracil, the plasmid in yeast was counted. The stability of pAYC7 was determined. As shown in FIG. 5, at the start of culture (0 hour), 5 hours and 10 hours during the culture in the non-selective medium,
Alternatively, the plasmid pAYC7 present in the yeast nucleus by culturing in a non-selective medium at the 20th hour,
Plasmid pAY- which grows in the mitochondria of yeast
It was shown that the protein was stably retained as in the case of CATM.

[0061]

According to the present invention, a conjugative transferable organelle expression vector containing a desired foreign gene can be transformed into a tra gene,
And prokaryotic / eukaryotic conjugative transfer using a helper plasmid containing the mob gene and mob gene, directly into organelles present in eukaryotic cells under mild conditions and without protoplasting cells It became possible to do. In addition, the introduced vector was propagated in an organelle of a eukaryotic cell as a host, and it was shown that the propagated vector was stably present without any functional or structural change. That is, according to the present invention, it is possible to improve the function of an organelle existing in a host cell, or to add a useful function to an organelle, to improve the growth rate by targeting chloroplasts of a plant, or to improve the mitochondrial activity of an animal. To improve the mitochondrial function such as the respiratory function by targeting to humans, it is possible to apply it to the field of creating biologically useful organisms, or in the case of humans, to the field of gene therapy.

[0062]

[Sequence list]

Tables 1 to 15 below show the respective sequences represented by SEQ ID NOS: 1 to 11.

[0063]

[Table 1]

[0064]

[Table 2]

[0065]

[Table 3]

[0066]

[Table 4]

[0067]

[Table 5]

[0068]

[Table 6]

[0069]

[Table 7]

[0070]

[Table 8]

[0071]

[Table 9]

[0072]

[Table 10]

[0073]

[Table 11]

[0074]

[Table 12]

[0075]

[Table 13]

[0076]

[Table 14]

[0077]

[Table 15]

[Brief description of the drawings]

FIG. 1 shows the construction of plasmid pAY-CATM. FIG. 1 shows that the HindIII site in the LacZ gene of the plasmid pAYC7 has a CATM gene coding region 6 designed to correspond to a translation codon for baker's yeast mitochondria.
A 792 base pair long DNA fragment containing 60 base pairs long was
By inserting the promoter and terminator of acZ gene in the forward direction,
1 shows the construction of CATM. The arrow indicates the direction of mRNA transcription.

FIG. 2 shows detection of plasmid pAY-CATM from total mitochondrial DNA isolated from chloramphenicol-resistant baker's yeast by Southern hybridization. A on the left is a photograph of ethidium bromide staining of 0.8% agarose electrophoresis, and B on the right is CAT.
It is a photograph of Southern hybridization by M probe. Lanes 1 to 4 of A show plasmid pAY-CA
TM (positive control), lanes 5 to 8 show mitochondrial total D isolated from chloramphenicol resistant yeast
NA, lane 9 is yeast YNN281 mitochondrial total D
NA (negative control) was used for EcoRI (lanes 1 and 5), ScaI (lanes 2 and 6), and SspI, respectively.
(Lanes 3, 7) or a photograph cut with HindIII (lanes 4, 8), fractionated by 0.8% agarose electrophoresis, and stained with ethidium bromide. Lane M at the left end is a size marker (lambda DNA fragment cut with HindIII). B on the right shows a DNA fragment fractionated by 0.8% agarose electrophoresis (A on the left), which was denatured with an alkali, blotted on a filter membrane, and labeled with ³² P using a CATM probe prepared by Southern hybridization. Shows the result of CATM fragment detection. The numbers on the right end show the position and length of the CATM fragment cut with each restriction enzyme detected by the Southern method.

FIG. 3. CA from chloramphenicol resistant transformed baker's yeast harboring pAY-CATM in mitochondria
FIG. 4 shows detection of TM gene expression by a CAT assay. After completion of the reaction, the product was separated by TLC, and irradiated with ultraviolet light (366 nm) to detect a fluorescent spot. Arrow A
Is the substrate (1-deoxy-chloramphenico)
l), the arrow B indicates the reaction product (3-acetyl-1).
-deoxy-chlorophenicol), and arrow 0 indicates the sample origin. Thick arrows indicate the direction in which the sample is developed by the solvent. Lane 1: substrate (1-deoxy-chloramphe)
Nicol) lane 2; baker's yeast S. cerevisiae YNN2
81 shares lane 3; baker's yeast S. cerevisiae YNN2
Black separated from the 81 strains chloramphenicol resistant spontaneous mutants lane 4; pAY-CATM was obtained by introducing the mitochondrial chloramphenicol resistance baker's yeast S. cere
visiae YNN281 strain lane 5; Escherichia coli HB10 carrying pAY-CATM
1 share

FIG. 4 is an electrophoresis photograph showing a restriction enzyme cleavage pattern of a plasmid DNA obtained by transforming total mitochondrial DNA isolated from chloramphenicol-resistant baker's yeast back into Escherichia coli. Lanes 1 and 8: lambda DNA fragment cut with size marker, HindIII Lanes 2 to 6; ScaI digested fragment of plasmid DNA recovered by back transformation Lane 7; Sca of pAY-CATM plasmid DNA
I-cleaved fragment (control) The numerical values at the left end indicate the length and position of the DNA fragment.

FIG. 5 is a graph showing changes over time in the stability of a conjugative transfer mitochondrial vector introduced by culturing chloramphenicol-resistant baker's yeast in a non-selective medium. Plasmid p
A chloramphenicol-resistant baker's yeast obtained by introducing AY-CATM into baker's yeast mitochondria by a prokaryotic / eukaryotic conjugative transfer method was cultured in a non-selective medium containing no chloramphenicol, and the vector was cultivated over time. The stability was investigated. In a control experiment, a transformed yeast having a plasmid pAYC7 (URA3) containing a URA3 gene introduced into a yeast nucleus by conjugative transfer was used. Perform shaking culture in a non-selective medium at 28 ° C, and at the start of culture (0 hour),
At 5 hours, 10 hours, or 20 hours during the culturing, a small amount of the culture solution is taken and diluted, and a certain number of yeasts are cultured on a selective agar plate medium or a non-selective agar plate medium at 28 ° C. for 2 days to 5 days. Cultured. The stability (%) was calculated by calculating the ratio of colonies that appeared in the selective medium and the non-selective medium after the indicated culture time. The stability of pAY-CATM is indicated by a white circle, and the stability of pAYC7 used as a control is indicated by a black square.

[Explanation of symbols]

Among the codes shown in FIG. 1, ColE1ori is ColE1.
Type origin of replication (also denoted as oriV -C), Amp is (also denoted as Ap r) ampicillin resistance gene is a selection marker gene that functions in E. coli, 2 [mu] m-ori
Is a 2 μm replication origin that is a replication origin that functions in baker's yeast,
In addition, mobB, mobA, and mobC are mob gene groups (also represented as mob- Q), and oriT is an oriT sequence at the initiation point of conjugation transfer (also represented as oriT- Q) URA
3 is a gene involved in the synthesis of uracil derived from plasmid YEp352 ( URA3 : URA gene itself has no functional involvement for the purpose of the present invention, but a restriction enzyme cleavage site ScaI
Was useful in determining the direction of insertion of CATM . )
CATM indicates a chloramphenicol acetyltransferase ( CATM ) gene which also functions as a selectable marker gene and a desired foreign gene prepared by base substitution so as to function in yeast mitochondria. LacZ promoter and terminator are operably linked. Other abbreviations indicate restriction enzyme cleavage sites.

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[Procedure amendment]

[Submission date] May 13, 1997

[Procedure amendment 1]

[Document name to be amended] Drawing

[Correction target item name] Figure 2

[Correction method] Change

[Correction contents]

FIG. 2

[Procedure amendment 2]

[Document name to be amended] Drawing

[Correction target item name] Figure 3

[Correction method] Change

[Correction contents]

FIG. 3

[Procedure amendment 3]

[Document name to be amended] Drawing

[Correction target item name] Fig. 4

[Correction method] Change

[Correction contents]

FIG. 4

Claims (14)

[Claims] (1) an origin of replication that functions in an organelle;
(2) an origin of replication that functions in the donor, (3) a selectable marker gene that functions in the organelle, (4) a selectable marker gene that functions in the donor, (5) a desired foreign gene that functions in the organelle, and (6) A prokaryotic cell using a tra gene and a mob gene is used to transform a donor bacterium having an expression vector in a conjugative transfer organelle containing an oriT sequence necessary for conjugative transfer between a prokaryote and a eukaryote, and a eukaryotic cell as a host cell using a tra gene and a mob gene. A method for performing conjugation transfer between eukaryotes and introducing the vector into an organelle present in a host cell which is a eukaryote. 2. The method according to claim 1, wherein a donor bacterium having a helper plasmid containing a tra gene and a mob gene is used together with a donor bacterium containing an expression vector in a conjugative transfer organelle. 3. The method according to claim 1, wherein the organelle is mitochondria. 4. The method according to claim 1, wherein the eukaryote is yeast. 5. The method according to claim 1, wherein the donor bacterium is Escherichia coli.
Or the method of 4. 6. A tra gene derived from Escherichia coli F factor, and m
The method according to claim 1, 2, 3, 4, or 5, wherein Escherichia coli carrying a helper plasmid containing an ob gene is used. 7. The method according to claim 5, wherein the conjugative transfer organelle expression vector is a plasmid pAY-CATM. 8. An origin of replication functioning in an organelle,
(2) an origin of replication that functions in the donor, (3) a selectable marker gene that functions in the organelle, (4) a selectable marker gene that functions in the donor, (5) a desired foreign gene that functions in the organelle, and (6) Conjugative transferable organelle expression vector containing an oriT sequence required for prokaryotic / eukaryotic conjugative transfer 9. The vector according to claim 8, wherein the donor bacterium is Escherichia coli. 10. The vector according to claim 9, which is pAY-CATM. An Escherichia coli comprising the vector according to claim 9. An Escherichia coli comprising the vector according to claim 10. 13. The Escherichia coli according to claim 12, which has an accession number of FERM P-15726. 14. The method of claim 1, 2, 3, 4, 5, 6, or 7.
A transformant obtained by transforming an organelle by the method described in (1).