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US 4 727 028

Recombinant DNA cloning vectors having the hpt gene for eukaryotic and prokaryotic cell transformation

Patents granted to Eli Lilly Co. Actual granted independent claims

This patent was assigned to Novartis

US 4727028
Claim 1A recombinant DNA cloning vector comprising:
a) a eukaryotic promoter;
b) one or two different structural genes and associated control elements that convey resistance to either or both antibiotics hygromycin B and G418 when transformed into a host cell that is sensitive to either or both antibiotics for which resistance is conveyed, said host cell being susceptible to transformation, cell division and culture; and
c) a prokaryotic replicon, said replicon being functional when said host cell is prokaryotic, subject to the limitations

  • that the one or two structural genes and associated control elements are adjacent to and, in a eukaryotic host cell, transcribed from the eukaryotic promoter,
  • that a single gene and associated control sequence convey resistance to only either one of hygromycin B or G418, and
  • that the gene conveying resistance to G418 does not code for the enzyme phosphotransferase.
Claim 2A recombinant DNA cloning vector comprising:
a) a eukaryotic promoter;
b) the 7.5 kb Bgl II restriction fragment of plasmid pKC203; and
c) a prokaryotic replicon, said replicon being functional when said vector is transformed into a prokaryotic host cell, subject to the limitations

  • that said 7.5 kb Bgl II restriction fragment is adjacent to the eukaryotic promoter, and
  • that any antibiotic resistance-conferring gene contained within said 7.5 kb Bgl II restriction fragment is, in a eukaryotic host cell, transcribed from the eukaryotic promoter.
Claim 20A plasmid selected from the group consisting of plasmid pKC203, pKC222, pKC214, pKC215, pGD10, pGD11, pGD12, pGD13, pGD14 and pGD151.
1 These E. coli plasmids contain either or both resistance genes for hygromycin and G418 in different orientations.
Claim 26A restriction fragment selected from the group consisting of:

a) the 7.5 kb Bgl II restriction fragment of plasmid pKC203;
b) the 2.75 kb Bgl II/Sal I restriction fragment of plasmid pKC203;
c) the 1.5 kb Sac I/Bgl II restriction fragment of plasmid pKC222; and
d) the 1.65 kb EcoR I/Sal I restriction fragment of plasmid pKC222.
Claim 51A transformed host cell selected from the group consisting of:

a) Mouse Ltk–/pKC2142;
b) Mouse Ltk–/pKC2152;
c) E. coli K12/pKC214;
d) E. coli K12/pKC215;
e) E. coli K12 BE827/pKC214;
f) E.coli K12 BE827/pKC215;
g) human/pKC214 cell; and
h) mammalian/ pKC214 cell.

2 Plasmids pKC214 and pKC215 have the 7.5 kb Bgl II restriction fragment containing resistance genes for both hygromycin and G418 in 5′ to 3′ and 3′ to 5′ direction, respectively.
Claim 59A transformed host cell selected from the group consisting of:

a) E. coli K12 BE827/pKC2223;
b) E. coli K12 BE827/pGD104;
c) E. coli K12 BE827/pGD114;
d) E. coli K12 BE827/pGD125;
e)E. coli K12 BE827/pGD135;
f) E. coli K12 BE827/pGD146;
g) E. coli K12 BE827/pGD156;
h) Mouse Ltk–/ pGD10;
i) Mouse Ltk–/ pGD11;
j) Mouse Ltk–/ pGD12;
k) Mouse Ltk–/ pGD13;
l) Mouse Ltk–/ pGD14; and
m) Mouse Ltk–/ pGD15. 3 Plasmid pKC222 contains the 2.5 kb Sal II/Bgl II restriction fragment with resistance genes for both hygromycin and G418.
4 Plasmids pGD10 and pGD11 contain each the 1.51 kb Bgl II/Sac I fragment with a hygromycin B resistance gene in different orientations.
5 Plasmids pGD12 and pGD13 contain each the 1.65 kb EcoR I/Sal I fragment with a G418 resistance gene in different orientations.
6 Plasmids pGD14 and pGD15 contain each the 2.75 kb Sal I/Bgl II fragment with resistance genes for both hygromycin and G418 in different orientations.
Claim 64A plasmid selected from the group consisting of plasmid pSC701, pKC257, pKC259, pKC261, pKC275, pKC264, pLO378, pKC273, pLO314, pLO315, pLO316, pLO317, pLO318, pLO319, pLO320, and pLO3217.
7 The plasmid pSC701 contains the 7.5 Bgl II fragment with resistance genes for both hygromycin and G418. The plasmid pKC259 also confers resistance to both antibiotics. The remaining plasmids all convey resistance to hygromycin B only and are functional in both prokaryotic and eukaryotic host cells.
Claim 97A eukaryotic host cell transformed with a vector selected from the group consisting of plasmids pLO378, pLO314, pLO315, pLO316, pLO317, pLO318, pLO319, pLO320, and pLO3218. 8 All these plasmids confer resistance to hygromycin B only.
Claim 98A constructed hygromycin B phosphotransferase encoding DNA sequence, which comprises recombinant DNA, comprising the sequence:
[Sequence] wherein A is deoxyadenyl, G is deoxyguanidyl, C is deoxycytisyl and T is thymidyl.
Claim 99A constructed hygromycin B phosphotransferase encoding DNA sequence, which comprises recombinant DNA, comprising the sequence:
[Sequence9] wherein A is deoxyadenyl, G is deoxyguanidyl, C is deoxycytisyl and T is thymidyl.
9 DNA sequence corresponds to the coding sequence for the enzyme and associated control sequence.
Claim 100A constructed DNA sequence that comprises recombinant DNA that encodes a polypeptide comprising the amino acid sequence
[polypeptide sequence10] wherein MET is methionine, LYS is lysine, PRO is proline, GLU is glutamic acid, LEU is leucine, THR is threonine, ALA is alanine, SER is serine, VAL is valine, PHE is phenylalanine, ILE is isoleucine, GLY is glycine, ASP is aspartic acid, GLN is glutamine, ARG is arginine, CYS is cysteine, TRP is tryptophan, ASN is asparagine, HIS is histidine and TYR is tyrosine. 10Sequence corresponds to the 341 amino acid residues of hygromycin B phosphotransferase.