Hybrid plasmid and process of making same
AssigneeThe Upjohn Company
InventorFritz Reusser
Life of this patent
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Abstract
A novel chemical compound, plasmid pUC1060, which was constructed from Streptomyces sp. 3022a chromosomal DNA and plasmid pBR322. Hybrid plasmid pUC1060 contains a functional tet gene promoter composed of streptomycete and E. coli DNA, and, thus, is useful as a cloning vehicle in recombinant DNA work. For example, using well known DNA methodology, a desired gene, for example, the insulin gene, can be inserted into pUC1060 and the resulting plasmid can then be transformed into a suitable host microbe which, upon culturing, produces the desired insulin.
Claims6
E. coli HB101 (pUC1060) having the deposit accession number NRRL B-12376.
Hybrid plasmid pUC1060 characterized as follows:
- (a) it is a hybrid plasmid which has the entire nucleotide sequence of plasmid pBR322 and a foreign DNA insert between the Eco R.sub.1 and HindIII sites of pBR322;
- (b) said DNA insert is located upstream from the HindIII site of pBR322;
- (c) said DNA insert restriction enzyme map is shown in the drawing;
- and
- (d) said DNA insert nucleotide sequence and restriction enzyme sites are as follows:
- ##STR2##
A process for preparing hybrid plasmid pUC1060 which comprises:
- (a) linearizing plasmid pBR322 with EcoR, and HindIII to obtain linear plasmid DNA;
- (b) obtaining chromosomal DNA from Streptomyces sp. 3022a NRRL B-11441.
- (c) digesting said chromosomal DNA;
- with Eco R.sub.1 and HindIII and
- (d) ligating said digested linear plasmid DNA from pBR322 and said digested chromosomal DNA from Streptomyces sp. 3022a NRRL B-11441, to obtain hybrid plasmid pUC1060.
A process for cloning plasmid pUC1060 into a suitable host by transformation wherein said suitable host is grown in an aqueous nutrient medium, isolated from the medium and suspended in a CaCl.sub.2 solution, and mixed with ligase mixture containing plasmid pUC1060.
Dependencies are inferred from the claim text.