Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
2000-10-18
2004-08-03
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S005000, C435S006120, C435S069100, C435S471000, C435S476000, C435S243000, C435S252300
Reexamination Certificate
active
06770481
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a plasmid vector usable in genetic recombinant technology and to a method for expressing a gene by using the plasmid vector. The present invention also relates to a method for isolating a desired gene by using such a plasmid vector. In addition, the present invention relates to a restriction enzyme and a gene thereof available as a genetic engineering reagent, in more detail to the AccIII restriction endonuclease and a DNA coding therefor.
BACKGROUND ART
In constructing an expression system for a desired gene by genetic recombinant technology, expression of the gene is controlled by bringing it under control of a promoter recognized by the RNA polymerase of the host used. In the case of a gene encoding a protein harmful to the host, however, plasmid construction itself is sometimes hampered by expression of the product of the gene due to the inability to stringently control the expression of the promoter used.
As an expression system resolving that problem, the pET system (produced by Novagen) has been developed, which uses the RNA polymerase of the bacteriophage T7, which infects
Escherichia coli
, with
Escherichia coli
as a host [journal of Molecular Biology, Vol. 189, pp. 113-130 (1986); Gene, Vol. 56, pp. 125-135 (1987)]. The pET system is a system that allows T7 RNA polymerase, which has high promoter recognition specificity and high transcription activity, to be expressed in
Escherichia coli
, which T7 RNA polymerase transcribes a desired gene placed downstream of the T7 promoter on an expression vector and causes high expression of the gene. Because transcription of the desired gene occurs in the presence of T7 RNA polymerase, plasmid construction in the host is possible without expressing the desired gene, provided that the host does not produce the polymerase; plasmid construction itself is never hampered, as in cases where the expression system is constructed, while the desired gene is kept under control of a promoter recognized by the RNA polymerase of the host.
However, because the T7 RNA polymerase gene has been cloned onto the &lgr;-phage vector and lysogenized into the expression host, there is no freedom of host choice; painstaking procedures are needed if the host is changed. In addition, because the expression of T7 RNA polymerase in the host is not stringently controlled, T7 RNA polymerase is expressed even when the host is in a non-inductive condition, resulting in expression of the desired gene placed downstream of the T7 promoter on the expression vector even in a non-inductive condition. To suppress such expression of the desired gene in a non-inductive condition, T7 RNA polymerase activity is inhibited using T7 lysozyme, a T7 RNA polymerase inhibitor [Journal of Molecular Biology, Vol. 219, pp. 37-44 (1991)], or T7 RNA polymerase is prevented from getting access to the T7 promoter by placing a lactose operator downstream of the T7 promoter [Journal of Molecular Biology, Vol. 219, pp. 45-59 (1991)].
However, even these countermeasures are unsatisfactory in terms of effect against T7 RNA polymerase of high transcription activity so that the activity of T7 RNA polymerase in a non-inductive condition cannot be completely inhibited. For this reason, if the desired gene product is lethal to the host, it is impossible in some cases to prepare a transformant for expression of the gene, even when plasmid construction is possible. In other words, the pET system involves two problems to be resolved: one of the inability to freely change the host, and the other of inaccurate control of T7 RNA polymerase expression.
On the other hand, there is a bacteriophage having characteristics similar to those of the bacteriophage T7, known as the bacteriophage SP6 [Science, Vol. 133, pp. 2069-2070 (1961)], which infects
Salmonella typhimurium
. The RNA polymerase produced by the bacteriophage SP6, a single peptide having-a molecular weight of about 100,000, is commonly used for in vitro RNA synthesis since it possesses high promoter recognition specificity and high transcription activity [Journal of Biological Chemistry, Vol. 257, pp. 5772-5778 (1982); Journal of Biological Chemistry, Vol. 257, pp. 5779-5788 (1982)]. In addition, the SP6 RNA polymerase gene has already been cloned and expressed in large amounts in
Escherichia coli
[Nucleic Acids Research, Vol. 15, pp. 2653-2664 (1987)].
Genes whose expression product acts lethally on hosts are exemplified by restriction endonuclease genes. Essentially, restriction endonucleases are utilized for self-defense by cleaving phages and other exogenous DNA entering the cells of microorganisms that produce the restriction endonucleases. On the other hand, microorganisms that produce restriction endonucleases mostly produce modification enzymes that recognize the same base sequences as those of the restriction endonucleases, to protect their own DNA against cleavage by the restriction endonucleases. Specifically, a modification enzyme modifies DNA by adding a methyl group to one or more bases in the base sequence recognized thereby, to make it impossible for the restriction endonuclease that recognizes the same sequence as that of the modification enzyme to bind thereto or to cleave the DNA. This mechanism is called restriction modification system, and the pair of genes of the restriction endonuclease and modification enzyme that constitute the restriction modification system called restriction modification system gene. Therefore, when the restriction endonuclease gene is expressed in a microorganism lacking a modification enzyme gene from the restriction modification system gene, the microorganism's DNA is cleaved, resulting in cell death. In fact, there are two modification enzyme genes in the MboI restriction modification system gene; it has been reported that cloning of restriction endonuclease genes is impossible due to incomplete modification of the host DNA in the case of incomplete methylation in the co-presence of either modification enzyme gene alone [Nucleic Acids Research, Vol. 21, pp. 2309-2313 (1993)].
Also, it has been demonstrated that if a restriction modification system gene is lost from a cell retaining the restriction modification system gene, a lack of modification activity in the cell results in incomplete methylation of genomic DNA, which in turn causes lethal cleavage of its own genomic DNA by a very small amount of restriction endonuclease remaining therein [Science, Vol. 267, pp. 897-899 (1995)]. In summary, in the absence of modification enzymes that constitute a restriction modification system, restriction endonucleases behave as proteins very harmful to cells; separate cloning and expression of their genes have been impossible by prior art technologies.
Concerning restriction endonucleases, restriction endonucleases can be classified by their enzymatic properties into three types: I, II and III. Type II restriction endonucleases, in particular, each of which recognizes a particular DNA base sequence and cleaves it at a particular site in or near the sequence, are extensively used in the field of genetic engineering, and restriction endonucleases of this type with various specificities have been isolated from a variety of microorganisms [Nucleic Acids Research, Vol. 24, pp. 223-235 (1996)]. In the present specification, a type II restriction endonuclease is hereinafter referred to as “restriction endonuclease”. It should be noted, however, that some microorganisms produce only small amounts of restriction endonuclease, and others produce a plurality of restriction endonucleases. For example, the restriction endonuclease AccIII is produced by
Acinetobacter calcoaceticus
(hereinafter referred to as Acc bacterium), which has been deposited under accession number FERM BP-935 at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry [address: 1-3, Higashi 1-chome,
Kato Ikunoshin
Oshima Atsushi
Sagawa Hiroaki
Ueno Harumi
Birch Stewart Kolasch & Birch, LLP.
Guzo David
Takara Shuzo Co. Ltd.
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