Multidrug resistance in mammalian cell lines and isolation of determinant glycoprotein DNA

Claim 2

An essentially pure DNA molecule CHARACTERIZED IN THAT SAID DNAwhich codes for a polypeptide moiety of a P-glycoprotein having a molecular weight of approximately 170,000 daltons, said P-glycoprotein being a major determinant in multidrug resistance of living cells.

Claim 3

An essentially pure DNA molecule according to claim 1, characterized in that said DNA molecule comprises a gene of a gene family which codes for said polypeptide moiety of corresponding P-glycoproteins.

Claim 4

An essentially pure DNA gene family according to claim 2, characterized in that said gene family comprises a plurality of genes.

Claim 5

An essentially pure DNA gene family according to claim 3, characterized in that said plurality of genes are arranged in tandem array in an amplicon.

Claim 6

An essentially pure nucleotide sequence of said DNA molecule according to claim 1, characterized in that said nucleotide sequence codes for a corresponding polypeptide sequence of said P-glycoprotein.

Claim 7

. An essentially pure nucleotide sequence according to claim 5, characterized in that said DNA comprises cDNA.

Claim 8

An essentially pure nucleotide sequence according to claim 5 characterized in that said DNA comprises mRNA.

Claim 9

An essentially pure nucleotide sequence according to claim 5 characterized in that said DNA comprises a fragment of DNA of said sequence which codes for a corresponding portion of said polypeptide sequence of said P-glycoprotein.

Claim 10

An essentially pure nucleotide sequence according to claim 8, characterized in that said DNA is in recombinant form with a bacterial phage.

Claim 11

An essentially pure nucleotide sequence according to claim 8, charagerized in that said DNA is in recombinant form with a plasmid.

Claim 12

An essentially pure nucleotide sequence according to claim 8, 9 or 10, characterized in that said DNA fragment is approximately 600 base pairs.

Claim 13

An essentially pure nucloetide sequence according to claim 8, 9 or 10, characterized in that said DNA fragement is approximately 2,500 base pairs.

Claim 14

An essentially pure nucleotide sequence according to claims 8 through 12, characterized in that said DNA fragment is cDNA.

Claim 15

An essentially pure nucleotide sequence according to claim 13, characterized in that said cDNA codes for a polypeptide sequence having three different epitopes specific to said P-glycoprotein as indicated by three different antibodies raised to said P-glycoprotein.

Claim 16

An essentially pure nucleotide sequence according to claim 7, characterized in that said mRNA is of approximately 5 kb and has a coding region for said P-glycoprotein of approximately 4 kb which codes for a protein moiety of approximately 140,000 daltons for said P-glycoprotein. ­

Claim 17

An essentially pure nucleotide sequence according to claim 9, characterized in that said bacterial phage is adapted for use as a DNA probe.

Claim 18

An essentially pure nucleotide sequence according to claim 10, characterized in that said plasmid is adapted for use as a DNA probe.

Claim 19

An essentially pure DNA molecule according to claims 1 through 17, characterized in that said molecule is derived from a source selected from the group consisting of Chinese Hamster Ovary cells, mouse cells ahd human cells.

Claim 20

An essentially pure nucleotide sequence according to claim 17, characterized in thatn said DNA probe is labelled with a detectable marker.

Claim 21

A cloning vehicle comprising a bacterial phage according to claim 9.

Claim 22

A cloning vehicle comprising a plasmid according to claim 10.

Claim 23

A host cell comprising a cloning vehicle according to claim 21.

Claim 24

A microorganism comprising a cloning vehicle according to claim 21.

Claim 25

An E. coli microorganism according to claim 23 characterized in having ATCC accession no. 39839 and comprising said cloning vehicle of claim 21.

Claim 26

A method of producing a polypeptide sequence which is a fragment of a P-glycoprotein having a molecular weight of approximately 170,000 daltons characterized in culturing a microorganism according to claim 222 or 31 in an aqueous nutrient medium and isolating said polypeptide.

Claim 28

A method according to claim 26, characterized in that said phage is a bacterial phage.

Claim 29

A method according to claim 27, characterized in that said phage is λgtll.

Claim 30

A method according to claim 28, characterized in that said plasmid is pUC9.

Claim 31

B -galactosidase has a size of about 140,000 daltons. Subtraction of the a-galactosidase molecular weight of 116,600 daltons-yields 23,400 daltons as the molecular weight of the peptide coded for by the insert sequence. This represents about 190 amino acid residues or 570 bp. Therefore, all but about 80 b p of the insert are required to code for the peptide. These 80 bp must represent a portion of the 3' untranslated sequence.

Claim 32

B -galactosidase has a size of about 140,000 daltons. Subtraction of the a-galactosidase molecular weight of 116,600 daltons-yields 23,400 daltons as the molecular weight of the peptide coded for by the insert sequence. This represents about 190 amino acid residues or 570 bp. Therefore, all but about 80 b p of the insert are required to code for the peptide. These 80 bp must represent a portion of the 3' untranslated sequence.

Claim 33

pCHP-1 was used to re-screen the same lamda library and five additional cDNA clones were obtained. These had sizes of 1.2; 1.3; 1.65; 2.0 and 2.5 kb. The longest which codes for approximately one half of complete P-glycoprotein polypeptide was designated

Claim 34

pCHP-1 was used to re-screen the same lamda library and five additional cDNA clones were obtained. These had sizes of 1.2; 1.3; 1.65; 2.0 and 2.5 kb. The longest which codes for approximately one half of complete P-glycoprotein polypeptide was designated

Claim 35

pCHP-2 and deposited with ATCC on July 30th, 1985 and given accession No. 53234.

Claim 36

pCHP-2 and deposited with ATCC on July 30th, 1985 and given accession No. 53234.

Claim 37

It is appreciated, however, that with extensive exacting procedures the nucleic acid sequence for the polypeptide portion of the P-glycoprotein is isolated by selecting.mammalian cell lines which exhibit resistance to drugs, such as adriamycin, actinomycin D, colchicine, daunorubicin, emetine, podophyllotoxin, puromycin, taxol, vinblastine or vincristine, all of which frequently exhibit a pleiotrophic phenotype of multidrug resistance. This complex phenotype involves cross-resistance to structurally and functionally unrelated compounds. Although the plasma membrane of the cell is implicated in this complex phenotype of drug resistance by the cell, the actual mechanisms involved are not fully understood. According to an embodiment of the invention, the extensive procedures for isolating the nucleic acid sequence of the P-glycoprotein gene include selection of multidrug resistant mutant cells; their genetic characterization; drug transport studies'; plasma membrane characterization including isolation of a P-glycoprotein to which antobidies are prepared. The antibodies are then used to screen a bacteria phage cDNA expression vector library prepared from the mRNA isolated from highly multidrug resistant mutants and the positive recombinant bacteria phage clones identified

Claim 38

It is appreciated, however, that with extensive exacting procedures the nucleic acid sequence for the polypeptide portion of the P-glycoprotein is isolated by selecting.mammalian cell lines which exhibit resistance to drugs, such as adriamycin, actinomycin D, colchicine, daunorubicin, emetine, podophyllotoxin, puromycin, taxol, vinblastine or vincristine, all of which frequently exhibit a pleiotrophic phenotype of multidrug resistance. This complex phenotype involves cross-resistance to structurally and functionally unrelated compounds. Although the plasma membrane of the cell is implicated in this complex phenotype of drug resistance by the cell, the actual mechanisms involved are not fully understood. According to an embodiment of the invention, the extensive procedures for isolating the nucleic acid sequence of the P-glycoprotein gene include selection of multidrug resistant mutant cells; their genetic characterization; drug transport studies'; plasma membrane characterization including isolation of a P-glycoprotein to which antobidies are prepared. The antibodies are then used to screen a bacteria phage cDNA expression vector library prepared from the mRNA isolated from highly multidrug resistant mutants and the positive recombinant bacteria phage clones identified