Aminoglycoside phosphotransferase gene conferring resistance to G418

There are several aminoglycoside phosphotransferases conferring resistance to aminoglycoside antibiotics. The aminoglycoside phosphotransferase I (aph-I) enzyme and the aminoglycoside (or neomycin) phosphotransferase II (APH-II or NPTII) are unrelated except for their ability to inactivate the antibiotic G418. The aph-I gene was originally found on transposon Tn601, also known as Tn903. According to some reports, aph-I is approximately four times more effective than aph-II in inactivating G418.

Cetus Corporation (acquired by Hoffman-La Roche in the early 90’s) has two granted patents, one in the United States and one in Canada, on the DNA sequence of a modified aph-I enzyme. Modified truncated aph-I gene can be used as a selectable marker for both prokaryotic and eukaryotic organisms.

US 4784949 CA 1337716 A1
Title Universal dominant selectable marker cassette
Application No. & Filing date US 602118
April 19, 1984
CA 475153 A
February 26, 1985
Issue date November 15, 1988* December 12, 1995*
Remarks The related granted U.S. patent 5116750 directed to a fusion protein containing aph-I enzyme expired due to lack of payment of fees on September 26, 2000.
*The patent term of both the United States and the Canadian patents is 17 years from the date of issuance.

Both patents claim a truncated DNA sequence of an aph-I gene, which contains restriction sites immediately upstream of the start codon (Claim 1). This set-up ensures a precise reproducible translation of the gene and permits the construction of fusion proteins that contain the aph-I sequences at the C-terminal end. The aph-I gene of the invention is further modified by removing the codons for amino acids in positions 2-10, inclusive, and a couple of restriction sites within the codifying region. This modified truncated aph-I (mt aph -I) gene is particularly effective against G418. It also inactivates the antibiotic neomycin effectively, but it is less effective against kanamycin than the nptII (or aph-II) gene.

The Canadian patent further claims:

  • a recombinant expression vector having the mt aph-I gene capable of conferring resistance to G418 on prokaryotes and eukaryotes (Claim 4);
  • an expression vector with the mt aph-I gene as described under the control of promoter for eukaryotic cells (Claim 17);
  • an expression vector for eukaryotes having a sequence for a fusion protein that has a mt aph-I gene as described in its C-terminal (Claim 19);
  • a fusion protein that contains a C-terminal mt aph-I sequence and a N-terminal sequence of:
    • any desired protein (Claim 30);
    • ß-isopropylmalate dehydrogenase (Claim 31); and
    • yeast enolase (Claim 35).
  • a method of purifying a fusion protein that comprises the mt aph-I gene as described (Claim 37).

There may be some marginal overlap between the United States patents filed by Monsanto and the United States patent granted to Cetus Corporation. Although Monsanto‘s patents are directed to chimeric genes, the DNA sequence coding for a neomycin phosphotransferase is not limited to any in particular. So, it might well include a neomycin phosphotransferase I (aph-I) gene or a neomycin phosphotransferase II (nptII or aph-II) gene.

The sequence claimed by Cetus Corporation has some limitations. It is a modified and truncated version of aph -I. Thus, it is likely that an aph-I gene without the modifications of the claimed version of the gene would not be encompassed by the claims.