Process for amplifying DNA in cells
The present invention relates to a process for efficiently amplifying a giant DNA. More particularly, the present invention relates to a process for amplifying DNA in a cell, comprising amplifying the DNA as the target of amplification in the presence of DNAs selected from the following (i), (ii) and (iii): (i) DNA encoding a protein selected from the following 1), 2) and 3): 1) a protein consisting of the amino acid sequence of SEQ ID NO: 1, 2) a protein comprising an amino acid sequence which has a deletion, substitution, insertion or addition of one or more amino acids in the amino acid sequence of SEQ ID NO: 1, and 3) a protein comprising an amino acid sequence which has an identity of 90% or more to the amino acid sequence of SEQ ID NO: 1, (ii) DNA consisting of the nucleotide sequence of SEQ ID NO: 2, and (iii) DNA hybridizing to the nucleotide sequence of SEQ ID NO: 2 under stringent conditions.
1. A process for amplifying DNA, comprising:
preparing a recombinant cell comprising:
any one polynucleotide selected from the group consisting of the following (A) to (D), and
a DNA unit disposed in a genome of the recombinant cell, wherein said DNA unit comprises at least first DNA fragment selected from the group consisting of the following (E) to (G), a target gene, and a second DNA fragment selected from the group consisting of (H) to (J),
and wherein said target gene or polynucleotide is exogenous to said recombinant cell;
(A) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 1,
(B) a polynucleotide encoding a protein which consists of an amino acid sequence having an identity of 90% or more to the amino acid sequence of SEQ ID NO: 1,
(C) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2, and
(D) a polynucleotide which hybridizes to the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2 under stringent conditions,
(E) DNA consisting of the nucleotide sequence of SEQ ID NO: 3,
(F) DNA hybridizing to the DNA consisting of the nucleotide sequence of SEQ ID NO: 3 under stringent conditions, and
(G) DNA having an identity of 90% of more to the nucleotide sequence of SEQ ID NO: 3,
(H) DNA represented by the nucleotide of SEQ ID NO: 4,
(I) DNA hybridizing to DNA consisting of the nucleotide sequence of SEQ ID NO: 4 under stringent conditions, and
(J) DNA having an identity of 90% of more to the nucleotide sequence of SEQ ID NO: 4; and
culturing said recombinant cell under conditions for causing gene amplification to amplify said DNA unit.
2. The process according to claim 1 , wherein said DNA unit has a size of 22 to 154 kb.
3. The process according to claim 1 , wherein said DNA unit comprises, in order from the 5′-terminal, the first DNA fragment, the target gene, and the second DNA fragment.
4. The process according to claim 1 , wherein said target gene consists of an antibiotic biosynthetic gene cluster.
5. The process according to claim 1 , wherein said DNA unit further comprises a drug-resistance gene.
6. The process according to claim 1 , wherein said polynucleotide is DNA and is disposed in the genome of said recombinant cell.
7. The process according to claim 1 , wherein said host is an antibiotic-producing strain.
8. A recombinant cell produced by the method of claim 1 , having multiple copies of said DNA unit introduced into the genome of the recombinant cell.
9. The recombinant cell according to claim 8 , wherein said target gene is exogenous to the host.
10. The recombinant cell according to claim 8 , wherein said polynucleotide is exogenous to the host, and said target gene is endogenous or exogenous to the host.
11. The recombinant cell according to claim 10 , wherein said first DNA fragment and said second DNA fragment are exogenous to the host.