Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1998-02-12
2001-04-10
Brusca, John S. (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S468000, C435S320100, C536S023100, C536S024100
Reexamination Certificate
active
06214578
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a method for expressing a foreign gene and a vector therefor. More particularly, the present invention relates to a method for expressing a foreign gene in genetic engineering processes, by which expression of the foreign gene is more strongly promoted than the conventional methods, and to a vector therefor.
BACKGROUND ART
Promotion of expression of foreign genes is one of the most required techniques in genetic engineering processes, especially when the genetic engineering processes are applied to plants.
As one of such methods, it is known to insert an intron-originated DNA fragment into a site upstream of the foreign gene. For example, Japanese Laid-open Patent Application (Kokai) No. 3-103182 discloses that expression of a foreign gene is promoted by inserting an intron-originated DNA fragment of castor-oil plant catalase gene (CAT-1) into a site upstream of the foreign gene, and expressing the foreign gene. Similar phenomena have been reported for various intron-originated DNA fragments.
Although introns have been utilized for the purpose of promoting expression of foreign genes, use of a plurality of introns is not popular, and advantageous effect thereof has not been recognized. For example, although the first intron and the 6th intron of maize alcohol dehydrogenase gene individually promote gene expression, if these introns are ligated, the effect is less than in the case where the 6th intron alone is used (Mascarenhas et al. Plant Mol. Biol., 15, 913-920(1990)). Similarly, in cases where two maize actin third introns are ligated, the effect is less than the case where only one intron is used (Luehrsen et al., Mol. Gen. Genet., 225, 81-93 (1991)).
Although the known methods in which an intron-originated DNA fragment is inserted are effective, the expression-promoting effects are often insufficient. Thus, a method by which gene expression is more strongly promoted is desired.
DISCLOSURE OF THE INVENTION
Accordingly, an object of the present invention is to provide a method for expressing foreign genes by which the foreign genes are more strongly expressed than by the known methods, and to provide recombinant vectors therefor.
The present inventors intensively studied to discover that expression of foreign genes is much more promoted by inserting into one or more sites upstream of the foreign gene a plurality of intron-originated DNA fragments which are the same or different and are capable of promoting expression of foreign genes when inserted into a site upstream of the foreign gene, than in the known methods in which a single intron-originated DNA fragment is inserted, thereby completing the present invention.
That is, the present invention provides a method for expressing a foreign gene comprising inserting said foreign gene into a site downstream of a promoter and expressing said foreign gene in a cell, characterized in that a plurality of intron-originated DNA fragments which are the same or different and are capable of promoting expression of foreign genes are inserted into one or more sites upstream of said foreign gene and said foreign gene is expressed.
The present invention also provides a recombinant vector comprising a promoter, a foreign gene inserted into a site downstream of said promoter, and a plurality of intron-originated DNA fragments which are the same or different and are capable of promoting expression of foreign genes, which are inserted into one or more sites upstream of said foreign gene.
The present inventors discovered that even if a single intron sequence of maize ubiquitin gene, which has a nucleotide sequence shown in SEQ ID NO:3 in the SEQUENCE LISTING, is inserted into a site upstream of a foreign gene, expression of the foreign gene is promoted, thereby completing the second invention of the present application.
That is, the present invention also provides a method for expressing a foreign gene comprising inserting said foreign gene into a site downstream of a promoter, and expressing said foreign gene in a cell, characterized in that the sequence shown in SEQ ID NO: 3 in the SEQUENCE LISTING or a functional variant thereof is inserted into a site upstream of said foreign gene and said foreign gene is expressed.
By the present invention, methods for expressing foreign genes by which expression of the foreign genes are much more strongly promoted than in the known methods, as well as recombinant vectors therefor, were provided. By the present invention, since expression of foreign genes introduced by genetic engineering processes is promoted, it is expected that the present invention will greatly contribute to the field of genetic engineering.
BEST MODE FOR CARRYING OUT THE INVENTION
The method of the present invention is characterized by inserting two or more intron-originated DNA fragments capable of promoting expression of foreign genes into one or more sites upstream of a foreign gene to be expressed, so as to promote expression of the foreign gene.
Here, the term “intron-originated DNA fragment having an effect to promote expression of foreign genes” means a DNA fragment originated from an intron, which is capable of promoting expression of foreign genes to a detectable degree when compared with the case wherein the foreign gene is expressed without inserting the intron-originated DNA fragment. Various such intron-originated DNA fragments per se are known. Examples of such intron-originated DNA fragments include the first intron of catalase gene (CAT-1) of castor-oil plant (Japanese Laid-open Patent Application (Kokai) No. 3-103182; Tanaka et al. Nucleic Acids Res. 18, 6767-6770(1990)); the intron of maize UDP-glucose:flavonol glycosyltransferase (Callis et al., Genes & Develop. 1, 1183-1200 (1987)); the first intron of maize alcohol dehydrogenase-1 (Callis et al., Genes & Develop. 1, 1183-1200 (1987)); the second and the sixth intron of maize alcohol dehydrogenase-1 (Mascarenhas et al., Plant Mol. Biol. 15, 913-920(1990)); the first intron of maize shrunken-1 (Vasil et al., Plant Physiol. 91, 1575-1579(1989)); the first intron of translation elongation factor EF-1&agr; of
Arabidopsis thaliana
); and the first intron of rice actin (McElroy et al. Plant Cell 2, 163-171(1990)). It should be noted, however, the intron-originated DNA fragments which may be employed in the present invention are not restricted thereto and any intron-originated DNA fragments which can promote expression of foreign genes downstream thereof may be employed.
The present inventors previously discovered introns of rice PLD gene by comparing the nucleotide sequences of the cDNA and the genomic DNA of rice phospholipase D (PLD) gene, discovered that one of these introns prominently promotes expression of genes downstream thereof, and filed a patent application directed thereto (PCT/JP96/00812). The nucleotide sequence of this intron is shown in SEQ ID NO:1 in the SEQUENCE LISTING. In the present invention, this intron-originated DNA fragment shown in SEQ ID NO:1 may be employed. Further, the intron sequence of castor-oil plant catalase gene, shown in SEQ ID NO:2 in the SEQUENCE LISTING and the intron sequence shown in maize ubiquitin gene, which has a nucleotide sequence shown in SEQ ID NO:3, may also preferably be employed.
It is well-known in the art that there are cases wherein the physiological activity of a physiologically active DNA sequence is retained even if one or more nucleotides are added, inserted, deleted or substituted. In the present invention, DNA fragments resulting from such a modification of the above-described known intron-originated DNA fragments or the sequence shown in SEQ ID NO:1, which promote expression of the gene downstream thereof, are included in the term “intron-originated DNA fragments” as used herein. That is, DNA fragments which have the same nucleotide sequences as the above-mentioned known intron-originated DNA fragments or the intron-originated DNA fragment having the nucleotide sequence shown in SEQ ID NO:1 except that one or more nucleotides are added, deleted or substitute
Kuraya Yoshiki
Morioka Shinji
Ohta Shozo
Ueki Jun
Birch & Stewart Kolasch & Birch, LLP
Brusca John S.
Japan Tobacco Inc.
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