Family 1. Recombinant DNA cloning vectors having the hpt gene for eukaryotic and prokaryotic cell transformation
Eli Lilly‘s portfolio of patents on this matter starts with this group, which discloses recombinant DNA cloning vectors that confer resistance to hygromycin (hyg B) and G418 antibiotics in both prokaryotic and eukaryotic cells. The patents encompass the initially isolated gene sequences for these antibiotic resistance genes.
The gene coding for an enzyme conferring resistance to G418 does not correspond to the nptI nor the nptII gene from the transposons Tn603 and Tn5, respectively. In this case it is a gene encoding an aminoglycoside acetyltransferase enzyme.
A cloning vector of the invention comprises:
- a eukaryotic promoter;
- structural gene(s) and its associated control sequence(s) for either hyg B or G418 resistance or both; and
- a prokaryotic replicon.
When the host cell is a prokaryote, the antibiotic resistance genes and their control sequences are adjacent to the prokaryote replicon. In the case of a eukaryotic host cell, the eukaryotic promoter drives a single gene, either hpt or G418 resistance gene, but not both.
The plasmid pKC203 from the Escherichia coli JR225 strain is the parent plasmid harboring both antibiotic conferring-resistance genes used for the construction of a series of plasmids for use in transformation of prokaryotic and eukaryotic cells.
| Patents members of Family 1 | |||
| Country | Granted Patent No. | Filing date | Issue date |
|---|---|---|---|
| Australia | AU 555574 B2 AU 582653 B2 |
June 17, 1982 May 27, 1986 |
October 2, 1986 April 6, 1989 |
| Canada | CA 1195626 A1 | June 16, 1982 | October 22, 1985* |
| Denmark | DK 172716 B1 | June 16, 1982 | June 14, 1999 |
| Europe** | EP 68740 B1 | June 17, 1982 | March 22, 1989 |
| Great Britain | GB 2146031 | September 27, 1984 | October 23, 1985 |
| Hungary | HU 195248 B | June 17, 1982 | June 28, 1990 |
| Ireland | IE 8853521 B | June 17, 1982 | December 7, 1988 |
| Former USSR | SU 1250174 A3 | June 16, 1982 | August 7, 1986 |
| United States | US 4727028 | September 30, 1983 | February 23, 1988* |
| *Patent term of the Canadian and United States patents is 17 years from the date of issuance. **The European patent was converted to a national patent in Belgium (BE), France (FR), Great Britain (GB), Germany (DE), Italy (IT); Lichtenstein (LI); Luxemburg (LU); Netherlands (NL), Sweden (SE) and Switzerland (CH). There are related patent applications pending in Greece, Israel and Japan. To view or download the patents as PDF files, click on EP 68740 B1 (2.0 kb) and US 4727028 (2.4 kb). |
|||
The plasmid pKC2O3 is the source of restriction fragments containing both or either one of the antibiotic resistance genes, which are then inserted into different plasmids. This series of plasmids are part of the claimed invention. The claimed fragments are:
- a 7.5 kb Bgl II fragment containing both resistance genes;
- a 2.5 kb Bgl II/Sal I fragment containing both resistance genes;
- a 1.51 kb Sac I/Bgl II fragment containing the hpt gene; and
- a 1.61 kb EcoR I/Sal I fragment containing the G418 resistance gene.
The antibiotic resistance genes of one of the recombinant cloning vectors are claimed in general terms without defining a specific DNA sequence (Claim 1 of the United States andCanadian patents, and Australian patent 555574 B). In these countries, the invention is likely to cover any DNA sequence encoding the enzymes against these antibiotics.
Transformed prokaryotic and eukaryotic host cells are also the subject of independent claims. Eukaryotic host cells include mouse, Escherichia coli, Saccharomyces cerevisiae and human. It does not mean, however, that the recombinant vectors of the invention are only limited to this group of hosts. Other independent claims encompass eukaryotes and prokaryotes in general. Thus, virtually any organism could be covered by the invention.
All the above mentioned features are the subject matter of the Australian, Canadian, European and United States patents. In addition, the United States patent claims the amino acid sequence of the hygromycin phosphotransferase (HPT) enzyme. Patents granted in other countries were not analysed.