Family 2. Modified hygromycin B resistance-conferring gene

This family of patents is directed to modified DNA sequences of the hpt gene. The modified hpt gene is useful for cloning, isolating and characterizing promoters and also for constructing gene fusions that act as dominant selectable markers in appropriate host cells.

Differences to the wild-type sequence of the gene lie in the removal of the first, first and second, or first, second and third codons in the amino (N-) terminus of the HPT enzyme.

Plasmids bearing such truncated versions of the hpt gene are also part of the claimed invention. The plasmids also comprise non-native prokaryotic and eukaryotic transcriptional and translational activator sequences.

In addition, the Canadian and the United States patents claim the amino acid sequence of a modified HPT enzyme.

E. coli and Saccharomyces cerevisiae transformants with the vectors of the invention are also claimed in the Australian patent.

The disclosed modified sequence corresponds to the structural sequence of the hpt gene contained in the 1.45 kb restriction fragment of the pKC222 plasmid, except for the first N-terminal amino acids and the stop codon at the C-terminus, which are variable.

Patents members of Family 2
Country Granted Patent No. Filing date Issue date
Australia AU 565625 B2 July 20, 1984 September 24, 1987
Canada CA 1278540 A1 July 16, 1984 January 2, 1991*
Europe** EP 135291 B1 July 17, 1984 March 14, 1990
Hungary HU 200366 B July 20, 1984 May 28, 1990
Ireland IE 9357776 B July 17, 1984 April 7, 1993
United States US 4960704 May 31, 1988 October 2, 1990**
* 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 Austria (AT), Belgium (BE), France (FR), Germany (DE), Italy (IT); Liechtenstein (LI); Luxemburg (LU); Netherlands (NL), Sweden (SE) and Switzerland (CH). There are related patent applications pending in Denmark, Finland, Greece, Israel, and Japan.

The modified hygromycin phosphotransferase sequence is inserted into a plasmid either alone or with a transcriptional and translational activator sequence. In the examples given, the inventors used bacterial sequences such as the coding sequence for the first 12 amino acids of the E. coli lacZ gene and eukaryotic sequences such as the yeast heat shock genes YG101 and YG100 and the phosphoglycerate kinase gene (PGK) as transcriptional and translational activator sequences.

The inventors state that other synthesized sequences encoding the same amino acids as those encoded by the disclosed sequence are within the scope of the invention.