npt gene as part of a chimeric gene construct for plant transformation.

US 6174724 US 5034322 EP 131623 B2
Title Chimeric genes suitable for expression in plant cells
Application No. & date No. 08/435,951
May 4, 1995
No. 07/ 333,802
April 5, 1989
EP 84900782.8
January 16, 1984
Issue date January 16, 2001* July 23, 1991** July 28, 1999*
Language English English (Claims in English, German and French)
Remarks The two United States patents are related to the European patent only through the earliest priority document (the first patent application ever filed on the inventions), which corresponds to the United States application 458,414 filed on January 17, 1983.
*Patent term would be 17 years from the date of issuance, but because of a ” terminal disclaimer”, the term does not extend beyond the expiration date of patent US 5 034 322.
** Patent term is 17 years from the date of issuance.
*The European patent was initially granted on March 6, 1991. As a result of an opposition filed against it by several entities, the scope of the claimed invention was modified. The amended claims and description were published eight years later on the date given. Patent term is 20 years from the date of filing the application.

Monsanto has two granted United States patents and one granted European patent claiming chimeric constructs for plant transformation containing a gene encoding a neomycin phosphotransferase enzyme, which confers antibiotic resistance to the transformed plant. The chimeric constructs of the inventions also comprise a promoter and a polyadenylation signal sequence. The United States patents 6174724 and 5034322 are also analysed in Antibiotic resistance genes in general.

One of the limitations of the inventions lies in the sort of promoter used in the chimeric construct to control the npt gene:

Promoter type
US 6174724 anypromoter naturally expressed in plants
US 5034322 promoter from A. tumefaciens opine synthase gene and
from ribulose-1,5-bis-phosphate carboxylase small subunit (rbcS) gene
EP 131623 B2 promoter from ribulose-1,5-bis-phosphate carboxylase small subunit (rbcS) gene

As discussed in the section Antibiotic resistance genes in general, a promoter “naturally expressed” in plants is not explicitly defined by the inventors. Yet the meaning of the term can be deduced from the description of the invention and the file history of the patent. Both sources tell us that the inventors may have envisioned any promoter from a gene of plant origin and promoters from genes of other organisms such as the nopaline synthase (nos) gene of A. tumefaciens, which is expressed only in a plant cell under natural conditions.

Opine genes are present in the Ti (tumor-inducing) plasmids and Ri (root-inducing) plasmids of Agrobacterium species. These genes are inactive while in the bacterial cells and are expressed only after they enter the plant cells. They code for enzymes that metabolize substances called “opines,” such as octopine, nopaline, and agropine. Opines are utilized by the bacteria as a source of carbon, nitrogen, and energy. The promoter claimed in the US patent 5034322 can be derived from any of the opine genes present in the Ti plasmids of the species A. tumefaciens.

Ribulose-1,5-bis-phosphate carboxylase (Rbc) catalyzes the reduction of atmospheric CO2 during photosynthesis. In higher plants, Rbc is a protein composed of eight copies of chloroplast-encoded large subunits and eight copies of nuclear-encoded small subunits (ss). The promoter claimed in the United States patent 5034322 and the European patent is isolated from a gene encoding a small subunit. There is no limitation on the plant source of the gene; it can be derived from any plant.

The antibiotic resistance gene coding for neomycin phosphotransferase is not restricted to a particular gene sequence. The protected gene in the European patent codes for either a neomycin phosphotransferase I or neomycin phosphotransferase II. According to the invent ors, these are distinct enzymes with major differences in their amino acid sequences and substrate specificity. Thus, in the United States and in Europe, chimeric constructs designed for plant cells having any DNA sequence encoding a neomycin phosphotransferase could be encompassed by the claims.