Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1998-12-22
2001-11-20
Brusca, John S. (Department: 1631)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C536S023100, C435S320100, C435S006120, C435S471000, C435S091400, C435S091420, C435S235100
Reexamination Certificate
active
06319692
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a method for transferring a foreign gene into a host chromosome, and more specifically, to a method for transferring a foreign gene into a chromosome by using a lysogenic vector, wherein genes originating in the vector and being necessary at the time of transfer but unnecessary for the expression and conservation of the foreign gene are finally deleted. Additionally, the present invention relates to a vector capable of integrating a foreign gene into the chromosome of for example
Lactobacillus casei
(referred to as
L. casei
hereinafter).
BACKGROUND ART
By using plasmids, foreign genes have been transferred into host cells of various microorganisms including
Escherichia coli
(referred to as
E. coli
hereinafter). So as to allow the characteristic properties of a foreign gene to be exerted in host cells in a more stable manner, attempts have been made to integrate a foreign gene into a host chromosome by using a lysogenic phage.
Meanwhile,
L. casei
Shirota strains, YIT9018 and YIT9029, have been utilized widely in for example lactic acid beverages and fermented milk, and it is revealed that these strains have excellent physiological actions, particularly antitumor activity, blood cholesterol reducing action, hypotensive action and anti-ulcer effect (Japanese Patent Laid-open Nos. 113718/1980, 5236/1992, 25055/1993, and 116155/1994).
A number of reports have been issued about shuttle vectors in hosts such as these lactic acid bacteria, particularly
L. casei
(see Japanese Patent Laid-open Nos. 128692/1990, 259086/1991, and 58890/1992). Most of these shuttle vectors in reports have been prepared via transformation as plasmids, so not any of these shuttle vectors has been satisfactory in terms of stability.
According to a conventional method for transferring a foreign gene into a host cell by utilizing a lysogenic phage, not only the objective foreign gene but also DNA sequences unnecessary after the transfer are simultaneously transferred. For example, a site-specific recombination enzyme gene region, a chemically resistant gene region necessary for recombinant selection, and a replication origin functioning in
E. coli
are simultaneously integrated into the chromosome of a host, but functionally, these regions are not necessary after the transfer of the gene.
From the standpoint of the subsequent utilization of a transformant, these regions functionally unnecessary after the transfer of the gene may sometimes be disadvantageous.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide a method for transferring a foreign gene into a chromosome, wherein unnecessary genes originating in a vector, etc., except for the foreign gene, can be deleted finally. It is the other object of the present invention to provide a recombination-type vector preferable for integration into the chromosome of
L. casei
strain YIT9029, in particular.
According to the present invention, more specifically, unnecessary genes derived from a vector, etc., except for the foreign gene, can be deleted finally by contriving procedures to integrate the foreign gene in the vector by utilizing a lysogenic phage just when the foreign gene is to be transferred into a host cell.
In one embodiment of the present invention, a method for integrating a foreign gene into a host chromosome by using a vector with the properties of lysogenic phage, comprises:
a first step of modifying the vector so that a lysogenic phage integration site (attP site) might be arranged between a first partial sequence being composed of the foreign gene to be transferred but lacking one terminal region thereof and a second partial sequence being composed of the gene but lacking the other terminal region thereof and having an overlapping region with a portion of the first partial sequence;
a second step of integrating the vector obtained in the first step into the host chromosome; and
a third step of screening from among the recombinants obtained in the second step a recombinant from which unnecessary genes originating in the vector have been deleted owing to the homologous recombination mechanism functioning in the overlapping region between the first and second partial sequences.
In accordance with the present invention, the term “vector with the characteristic properties of lysogenic phage” means a vector to be functionally integrated into a host chromosome when transferred in a host cell. More specifically, any vector having an integration site (for example, attP site) triggering the recombination of a different recombination site (for example, attB site) into the chromosome of a host cell may be satisfactory. A recombinant prepared by integrating a foreign gene into a host chromosome by using such vector carries integrated genes derived from the vector, together with the foreign gene. In accordance with the present invention, therefore, it should be prepared, prior to integration, a vector with a lysogenic phage integration site (attP site) arranged between a first partial sequence lacking one terminal region of the objective foreign gene for transfer and a second partial sequence lacking the other terminal region thereof and having an overlapping region with a portion of the first partial sequence. At the integration step, the vector is integrated into the chromosome of a host for promoting homologous recombination to recover a recombinant.
These first partial sequence and second partial sequence have an overlapping region to each other. As the length of the sequence in the overlapping region is shorter, the frequency of the homologous recombination after integration is decreased, so it is difficult to delete the unnecessary genes via such homologous recombination. Even by alternatively elongating the length of the sequence of the overlapping region, for example by allowing the first partial sequence and the second partial sequence to have absolutely the same sequence, with no deletion of any terminal region of the foreign gene, such homologous recombination occurs. When intending to use the expression of the foreign gene as a marker for final selection, potentially, there will be no chance that a transformation recombinant with the deletion of unnecessary vector-derived genes is selected among the resulting recombinants. Therefore, the lower limit of the length of the sequence of the overlapping region is satisfactorily a length at which the homologous recombination mechanism functions at a sufficient frequency. The length of the sequence of the overlapping region has not upper limit from the standpoint of homologous recombination, but when intending to use the expression of the foreign gene as a marker, the length may satisfactorily be a length at which no foreign gene expression occurs in the partial sequences with the deletion of one terminal region.
The first partial sequence and the second partial sequence are arranged in a direction along which the homologous recombination occurs in the overlapping region, so that the resulting sequence from such homologous recombination serves as the essential foreign gene. More specifically, the first and second partial sequences should be arranged toward the same direction, to align the individual termini N and C of the foreign gene so as to interpose the integration site (attP site) of the lysogenic phage between the termini.
In accordance with the present invention, the term “foreign genes” does not necessarily mean those derived from bacteria other than a host bacterium into which the gene is to be integrated, but means those from the same species of bacteria and those from the same bacterial strain, for example a gene harbored on a plasmid to be transferred into the chromosome. Furthermore, the foreign gene may satisfactorily be a set of genes as a combination of plural types of protein information (for example, the gene of an enzyme composed of plural subunits, in its entirety).
So as to select a recombinant where transformation has occurred after the deletion of the unnecessary genes derived from a vector, the l
Kadota Mariko
Kiwaki Mayumi
Sako Tomoyuki
Sawaki Saeko
Shirasawa Yukio
Brusca John S.
Frishauf, Holtz Goodman, Langer & Chick, P.C.
Kabushiki Kaisha Yakul't Honsha
Lundgren Jeffrey S.
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