Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1996-11-27
1998-09-01
Ketter, James
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
4353201, 536 241, C12P 2102
Patent
active
058010165
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
The present invention relates to a novel DNA fragment having function to promote expression of genes, a vector containing the same and a method for expressing foreign genes using the same.
BACKGROUND ART
Promotion of expression of foreign genes is one of the most important techniques in applying genetic engineering processes to plants. One of the methods therefor is utilization of a DNA having a nucleotide sequence which promotes expression of a gene.
Known nucleotide sequences which promote expression of foreign genes include the intron of the catalase gene of castor bean (Japanese Laid-open Patent Application (Kokai) No. 3-103182; Tanaka et al., Nucleic Acids Res. 18, 6767-6770 (1990)). However, since there are wide varieties of plants to be manipulated and since promotion of expression of genes is required in each of the desired growth stages or tissues of organs, it is desired that wide varieties of DNAs which promote expression of genes can be utilized.
DISCLOSURE OF THE INVENTION
Accordingly, an object of the present invention is to provide a novel DNA, which can promote expression of foreign genes and which has a nucleotide sequence different from those of known DNAs that promote expression of foreign genes.
The present inventors intensively studied to discover introns of rice phospholipase D (hereinafter also referred to as "PLD") gene by comparing a rice cDNA and a rice genomic DNA, and discovered that one of the introns has a function to prominently promote expression of the gene downstream thereof, thereby completing the present invention.
That is, the present invention provides an isolated DNA fragment having a nucleotide sequence shown in SEQ ID NO. 1 in Sequence Listing or having a nucleotide sequence which is the same as the nucleotide sequence shown in SEQ ID NO. 1 in Sequence Listing except that one or a plurality of nucleotides are added, inserted, deleted or substituted, the latter nucleotide sequence having a function to promote expression of a gene downstream thereof. The present invention also provides a recombinant vector comprising the above-mentioned DNA fragment according to the present invention and a foreign gene to be expressed, which is operably linked to the DNA fragment at a downstream region of the DNA fragment. The present invention further provides a method for expressing a foreign gene comprising introducing the recombinant vector according to the present invention into host cells and expressing the foreign gene.
As experimentally confirmed in the Example described below, the DNA fragment according to the present invention largely promotes expression of the gene downstream of the DNA fragment. Therefore, it is expected that the present invention will largely contribute to expression of foreign genes by genetic engineering processes.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the important part of a genetic map of pBI221 into which the DNA fragment according to the present invention is inserted, which was prepared in the Example of the present invention.
BEST MODE FOR CARRING OUT THE INVENTION
As mentioned above, the DNA fragment according to the present invention has a nucleotide sequence shown in SEQ ID NO. 1 in the Sequence Listing. As will be described in detail in the Example below, introns located upstream of rice PLD gene were identified by comparing the nucleotide sequence of the cDNA of rice PLD gene and that of the rice genomic DNA. A fragment containing one of these intron sequences having a size of 173 bp located at the 5'-flanking region was prepared by PCR and the DNA fragment was inserted into an upstream site of a reporter gene of an expression vector containing the reporter gene. By checking the expression activity of the reporter gene, it was confirmed that the DNA fragment has a function to promote expression of the gene downstream thereof. The nucleotide sequence of the DNA fragment according to the present invention corresponds to 1661nt to 1843nt of the nucleotide sequence of the rice genomic PLD gene, which nucleotid
REFERENCES:
Tanaka et al, Nucleic Acids Research, vol. 18, No. 23, pp. 6767-6770 (1990) .
Morioka Shinji
Ueki Jun
Japan Tobacco Inc.
Ketter James
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