COMPOSITIONS AND METHODS FOR CLONES CONTAINING DNA SEQUENCES ASSOCIATED WITH MULTIDRUG RESISTANCE IN HUMAN CELLS

Abstract: Genomic and cDNA clones of human genes which are selectively amplified or overexpressed in multidrugresistant human tumor cells were isolated. Such clones may be used as probes in diagnostic tests to detect chemotherapy-resistant tumor cells and to predict tumor response to chemotherapy. The complete nucleotide sequence of the coding region of the human mdr1 gene and the complete corresponding amino acid sequence are disclosed.

Description: COMPOSITIONS AND METHODS FOR CLONES CONTAINING DNA SEQUENCES ASSOCIATED WITH MULTIDRUG RESISTANCE IN HUMAN CELLS Roninson et al. This is a Continuation-in-Part of Application Serial No. 845,610, filed March 28, 1986. Background The present invention pertains in general to diagnostic materials and methods and in particular to materials and methods for the detection of multidrug- resistant tumor cells. Selection of mammalian cells for resistance to plant alkaloids or antitu or antibiotics frequently results in the development of cross-resistance to other drugs unrelated in their structure and mode of action to the original selective agent. Biedler et al.. Cancer Res. , 30, 1174 (1970). The phenomenon of multidrug resistance constitutes a major problem in cancer chemo¬ therapy since it involves resistance to some of the most ' commonly used anticancer drugs. Multidrug resistance in most cases appears to result from decreased intracellular drug accumulation, probably as a result of alterations in the plasma mem¬ brane. Biedler et al.. Cancer Treat. Rep., 67, 859 (1983); Ling et al.. Cancer Treat. Rep. , 67, 869 (1983); Ramu et al.. Cancer Treat. Rep., 67, 895 (1983); and Beck et al.. Cancer Res. , 39, 2070 (1979). In some hamster, mouse and human multidrug- resistant cell lines, resistance correlates with over expression of a 170,000 m.w. membrane glycoprotein (P- glycoprotein) or a 19,000 m.w. cytosolic protein. Kart- ner et al.. Science, 221, 1285-1288 (1983); Biedler et al.. Cancer Treat. Rep., 67, 859 (1983). Immunoblotting techniques applied to cells from human cancer patients reveal high levels of P-glycoprotein in some cases of advanced, nonresponsive ovarian cancer. Bell et al., J- Clin. Oncol., _, 311-315 (1985). P-glycoprotein-specific, monoclonal antibodies raised against multidrug-resistant Chinese hamster ovary (CHO) cell lines and cross reactive with human cell lines apparently bind to multidrug-resistant mammalian cells to a degree correlated with the degree of their drug resistance. Kartner et al.. Nature, 316, 820-823 (1985). These monoclonals may all bind to a C-terminal intracellular region of a proposed P-glycoprotein polypeptide. Kartner et al., Nature, 316, 820-823 (1985). P-glycoprotein specific cDNA clones have been isolated from Chinese hamster ovary cells, and these clones revealed amplification of the P-glycoprotein gene in multidrug resistant hamster, mouse and human cells when employed in a Southern blotting procedure. Riordan et al.. Nature, 316, 817-819 (1985). However, Riordan et al. provides no indication whether the hamster P- glycoprotein cDNA clones may be used to detect the expression of human P-glycoprotein genes at the level of ' RNA. In a different approach to the examination of multidrug-resistance, a common region of DNA is found to be amplified in two different multidrug-resistant Chinese hamster cell lines selected for resistance to either colchicine or Adriamycin. Roninson et al., Nature, 309, 626 (1984). This region was found to con¬ tain a transcription unit, presently designated mdr. Expression of the mdr mRNA correlates with multidrug resistance in the hamster cells. Gros et al., J. Cell. Biochem. , 9C (suppl.), 16, A1167 (1985); and Gros et al., Proc. Natl. Acad. Sci. (USA), 8_3, 337 (1986). However, probes derived from the hamster mdr. gene are not useful probes for human cells inasmuch as, even though these probes hybridize to human DNA (as illustrated in Example 2, inf a) , they do not hybridize efficiently with human mdr RNA, despite the impression given in a report on a workshop dealing with multidrug resistance [Kolata, Science, 231, 220-221 (1986)]. Therefore, in the absence of a probe for human mdr gene expression, there is a need for a reliable method for detecting the presence of multidrug-resistant cells in a human tumor either prior to or during chemo¬ therapy. Summary of the Invention The present invention provides an isolated nucleic acid sequence for a human mdr gene associated with multidrug resistance in human cells. A presently-preferred embodiment of the pre¬ sent invention provides an isolated and purified nucleic acid selected from the group consisting of: (a) a nucleic acid comprising a member of the group consisting of a continuous sequence of nucleotides as set forth in Table 4, in Table 5, in pHDR4.4 (ATCC 40227), in pHDR4.5 ' (ATCC 40228), in pHDR5A (ATCC 67040), in pHDR5B (ATCC 67041), in pHDRlO (ATCC 67042) and in pHDR104 (ATCC 67156); (b) a nucleic acid comprising a nucleotide sequence which hybridizes with at least one of the con¬ tinuous sequences of nucleotides as set forth in (a) or which is contained within the same mRNA molecule of human origin or cDNA molecule of human origin as at least one of the continuous sequences of nucleotides as set forth in (a); (c) nucleic acids comprising a nucleotide sequence which hybridizes with any nucleotide sequences described in (b); and (d) nucleic acids comprising a sequence of nucleotides which, but for the degeneracy of the genetic code, would hybridize with at least one of the continuous sequences of nucleotides as set forth in (a), (b), or (c). Standard conditions for identifying the presence or absence of "hybridization" herein are reactions conducted in 4 X SSC and 0.5% SDS at a temperature of 65 degrees C. in the last wash. A nucleic acid probe according to the preferred embodiment may also include a label associated with one of these nucleic acids. Polypeptides encoded by these nucleic acids may be expressed or synthesized chemically, and used, in conjunction with diluents, adjuvants, or carriers of the sort well known to those skilled in the art, to raise monoclonal or polyclonal antibodies or to elicit immune response in patients. Such antibodies may be utilized as a diagnostic reagent using various presently available immunodiagnostic techniques, or employed as immunotherapeutic agents. Brief Description of the Drawings Fig. 1 is a partial restriction map of the cosmid clone cosDR3A which contains a 5' portion of the transcribed mdr region isolated from Chinese hamster cells; Fig. 2 illustrates partial restriction maps of the plasmid clones pHDR4.4 and pHDR4.5, respectively containing mdrl and mdr2 sequences; and Fig. 3 illustrates partial restriction maps of phage cDNA clones λHDR5, λHDRlO, XHDR62, XHDR28, XHDR69A, XHDR103 XHDR104 and XHDR105 containing mdrl sequences. Detailed Descrio —•tion- Preliminary announcements of the obtaining of mdrl clones according to the present invention and of uses therefor have been made by the inventors at the UCLA Symposia on Molecular and Cellular Biology, January 20 - February 15, 1986. Roninson et al., J. Cell. Biochem. , 29 (suppl. 10A) , 12, A18 (1986); Pastan et al., J. Cell. Biochem., 29 (suppl. 10A) , 9, A13 (1986); Clark et al., J. Cell. Biochem., 29 (suppl. 10A) , 49, A130 (1986); and Cornwell et al., J. Cell. Biochem., 29 (suppl. 10A), 50, A131 (1986). A recently published European Patent Application No. 174,180 by John R. Riordan, entitled "Multidrug Resistance in Mammalian Cell Lines And Isolation Of Determinant Glycoprotein DNA," describes isolation of Chinese hamster cDNA clones specific for P- glycoprotein, and it suggests using P-glycoprotein- specific cDNA as a probe in determining multidrug resistance in cells. Although only Southern blot hybridization between hamster cDNA and human genomic DNA is described, claim 18 of Riordan, EPA 174,810, relates to a P-glycoprotein-specific DNA molecule "derived from a source selected from the group consisting of Chinese Hamster Ovary cells, mouse cells and human cells." In the event that the mdr clones described herein represent the human P-glycoprotein gene sequences, which is likely to be the case as discussed in Example 10 below, it should be noted that Riordan, EPA 174,810, does not disclose a human mdr gene or any portion thereof. In face, Riordan, EPA 174,810, post-dates the publication of Roninson et al.. Nature, 309, 626 (1984) which described cloning of a segment of the Chinese hamster mdr region. The work describe in Roninson et al., Nature, 309, was followed by isolation of the entire Chinese hamster mdr gene [Gros et al., J. Cell. Bioche and Proc. Nat' . Acad. Sci. (USA), supra] as opposed to only partial cDNA clones of the Chinese hamster P-glycoprotein genes, as described in Riordan, EPA 174,810. Riordan, EPA 174,810, provides no evidence for the ability of Chinese hamster clones to detect the expression of human P-glycoprotein mRNA. Furthermore, the use of P-glycoprotein cDNA as a probe for detection of multidrug resistance in tumor cells is described in Riordan, EPA 174,180, only in terms of detection of amplified P-glycoprotein genes but not in terms of detection of increased P-glycoprotein mRNA expression. Increased mRNA expression, as described in Example 7 below, provides a much more useful diagnostic marker for multidrug resistance than does gene amplification. In addition, although claiming P-glycoprotein cDNA sequences of human origin, Riordan, EPA 174,810, contains no indication as to how such sequences would be obtained, e.g. the source of human DNA or RNA, or stringency conditions for screening of human cDNA or genomic libraries with a Chinese hamster probe. As shown in Example 2 below, there is a low level of homology between the hamster and human mdr genes, at least within the 5' half of the gene, which presents a considerable technical problem in the isolation of human mdr DNA sequences. In the following exa.αples, nucleic acid clones for human mdr genes and uses for the nucleotide ' sequences of mdr clones are described. In Example 1 a Chinese hamster mdr clone is used to identify sequences hybridizing with human DNA. Example 2 describes the identification and isolation of DNA sequences comprising human mdr genes. In Example 3, amplification of mdr genes in human drug-resistant cells is demonstrated. A characterization of clones containing mdr sequences is presented in Example 4. In Example 5, DNA rearrangement involving mdr genes is examined. In Example 6, transcription of the mdrl gene in human cells is demonstrated. Example 7 describes an investigation into expression levels of the mdrl sequence during the course of development of multidrug resistance in human cells. In Example 8, expression of mdr genes out of proportion to gene amplification is demonstrated. Example 9 provides a description of a genomic clone containing a segment of the mdrl gene. In Example 10, cDNA clones of the mdrl gene and the cDNA sequence of the human mdrl gene is disclosed are described. In Example 11, diagnostic and therapeutic procedures using probes according to the present invention are described. Examole 1 Derivation and characterization of multidrug- resistant sublines of human KB cells are described elsewhere. Akiyama et al., So at. Cell Mol. Genet., 11, 117 (1985); Fojo et al., Cancer Res. , _5 , 3002 (1985); and Richert et al., Proc. Natl. Acad. Sci. (USA), 82, 2330 (1985). The multi-drug resistant phenotype is unstable in the most highly resistant lines, with a decrease in resistance when grown in the absence of the drugs. Using the in-gel DNA renaturation technique [according to Roninson, Nucleic Acids Res., 11, 5413 (1983)], several of the multidrug-resistant sublines of KB cells are known to contain amplified DNA sequences, and karyotypic analysis reveal double minute chromosomes in these cells. Fojo et al., Proc. Natl. Acad. Sci. (USA) , 8_2, 7661 (1985). Sublines of the human KB carcinoma cells, selected for resistance to colchicine, vinblastine or Adriamycin [Akiyama et al., supra; Fojo et al.. Cancer Res. supra; Richert et al., supra and Shen et al.. Science, 232, 643-645 (1986)], demonstrate the multidrug-resistant phenotype. Several of these sublines are described in Table 1. Fojo et al., Proc. Natl. Acad. Sci. USA, 8_2, 7661 (1985). In Table 1, "n/d" means not determined. KB-8-5-11, KB-8-5-11-24, KB-C3 and KB-C4 cell lines are subclones selected in 100 ng/ml, lyg/ml, 3μg/ml and 4 μg/ml Adriamycin, respectively. Relative resistance is expressed as the D T _ Q of the resistant cell line divided by the D-J_ Q of the parental KB-3-1 cells. Akiyama et al., supra. TABLE 1 Relative Resistance To: Cell Line Colchicine Adriamycin Vinblastine KB-3-1 1 1 1 KB-8-5-11 40 23 51 KB-8-5-11-24 128 26 20 KB-C3 487 141 206 KB-C4 1750 254 159 KB-C1-R1 6 3 4 KB-V1 171 422 213 KB-A1 19 97 43 KB-A2 n/d 140 n/d These multidrug-resistant human KB cell lines were used to determine whether DNA sequences homologous to the hamster mdr gene are present in the human genome. The Chinese hamster m r DNA sequences used in this study were derived from the cos id clone cosDR3A, containing a 5' segment of the hamster mdr gene. After digestion with the restriction enzymes Xbal and Kpnl, individual 1.5 - 6 kilobase (kb) restriction fragments from this cosmid were either subcloned into pSP65 plas- mid vector commercially available from Pro ega Biotec, Madison, Wisconsin, or gel-purified prior to labeling with *32 P. A vector including a 4.7 kb Xbal fragment, designated pDR4.7, contained DNA sequences hybridizing to human DNA. In Fig. 1, a partial restriction endonuclease map of the cosmid clone cosDR3A, containing a 5' portion of the transcribed mdr region amplified in multidrug- resistant Chinese hamster cells, is presented along with a dashed line aligned to indicate the portion of ROS DR3A which hybridizes to pDR4.7. In Fig. 1, X denotes an Xbal site and K identifies a Kpnl site. Cloning and characterization of this region are described in Gros et al., Proc. Natl. Acad. Sci. USA, 83, 337 (1986). Example 2 In order to identify and isolate segments of DNA comprising the human mdr genes, individual 1.5 - 6 kilobase (kb) size fragments of the cloned hamster mdr gene were isolated as a series of reco binant subclones in a pSP64 plasmid vector commercially available from Promega Biotec and described in Pro ega Biotec Technical Bulletin No. 13 as well as in Melton, Nucleic Acids Res. , 12, 7055-7056 (1984). Individual subclones were then labeled with 32 P and were used as probes for Southern blot hybridization with human DNA digested with restriction enzymes. The subclones were then used as probes for hybridization with restriction digests of human genomic DNA. Most probes, when used under conditions of low hybridization stringency, produced either no hybridiza¬ tion signal or a continuous smear suggesting cross- hybridization with human repetitive DNA sequences. However, one of the subclones, designated pDR4.7 and illustrated in Fig. 1, gave rise to distinct bands when hybridized to human DNA under low stringency conditions. Inasmuch as subclone pDR4.7, produced a dis¬ tinct hybridizacion signal, this subclone contained hamster DNA sequences homologous to the human mdr genes. pDR4.7 hybridized to two major different EcoRI restriction fragments in human DNA, although in some experiments as many as nine additional EcoRI fragments could be detected. Example 3 In order to determine whether an mdr gene is amplified in multidrug-resistant human cells, DNA extracted from the parental KB-3-1 cells and various. multidrug-resistant sublines described in Table 1 by the procedure of Gros-Bellard et al., Eur. J. Biochem. , 36, 32 (1978) was digested with EcoRI or HindiII, electro- phoresed on agarose gels and hybridized to the pDR4.7 probe by the procedure of Southern [Southern, J. Mol. Biol., 9_8, 503, (1975)]. In the Southern hybridization of pDR4.7 with EcoRI-digested DNA from multidrug-resistant KB cells, DNA was extracted as previously described [Gros-3ellard et al., Eur. J. Biochem. , 36, 32 (1978)]. The concen- tration of EcoRI-digested DNA was determined by the diphenylamine reaction [Giles et al., Nature, 206, 93 (1965)] and 5 ug of DNA were loaded onto each lane. After electrophoresis, DNA was transferred onto a nylon ' (Biodyne) membrane [Southern, supra] . Plasmid pDR4.7 was digested with_XbaI, the insert was gel-purified and labeled with 32 ? to a specific activity of 3 x 10 9 dp /μg by oligolabeling [Feinberg et al. , Analyt. Biochem. , 132, 6 (1983)]. Hybridization was done at 65 β C in 5 x SSPE, 5 x Denhardt's, 0.2% SDS, 500 ug/rtil denatured salmon sperm DNA. After hybridization, the membranes were washed with 4 x SSC, 0.5% SDS at 65°C and autoradiograp ed. The subclone pDR4.7 hybridizes co two EcoRI fragments of 13.5 and 4.5 kb size and co two Hindlll fragments of 10.5 and 4.4 kb size in KB-3-1 DNA when the filters are washed under low stringency conditions (4 x SSC; 65°C). Only the 13.5 kb EcoRI and 4.4 kb Hindlll fragments were detectable under conditions of interme¬ diate stringency (1 x SSC; 65°C). All the fragments were amplified in colchicine-resistant sublines KB-8-5-11, KB-8-5-11-24, KB-C3 and KB-C4. No amplification of either the band corres¬ ponding to the 13.5 kb fragment or the band correspond¬ ing to the 4.4 kb fragment was detected in the revertant subline KB-C1-R1. Unlike the colchicine-selected sub- lines, the subline KB-Vl, selected in vinblastine, shows amplification of only the 13.5 kb EcoRI and the 4.4 kb Hindlll bands. These two bands were also amplified in Adria ycin-resistant cells KB-A1 and KB-A2. KB-A1, in addition, contained a new amplified band of a 7 kb size in the EcoRI digest and of a 6.5 kb size in the Hindlll digest. The same bands were present in KB-Vl DNA, but their intensity suggested that these bands were not amplified. No bands of this size were detected in the parental KB-3-1 DNA, suggesting that they apparently arose as a result of a DNA rearrangement. The different patterns of amplification of the two types of bands hybridizing to the hamster mdr probe in different sublines suggested that they might corres¬ pond to two different related DNA sequences, possibly different members of a multigene family, rather than to two different parts of one contiguous hybridizing region. DNA sequences corresponding to the 13.5 kb EcoRI and 4.4 kb Hindlll fragments were designated mdrl and the sequences corresponding to the 4.5 kb EcoRI and the 10.5 kb Hindlll fragments were designated mdr2. The degree of amplification of mdr sequences in different multidrug-resistant sublines was estimated by comparing the intensity of hybridization signals from serially diluted EcoRI digests of different cellular DNAs. The estimates of the copy number of mdr sequences in different sublines are given in Table 2. In Table 2 , a star indicates the rearrangement of mdr2 DNA sequences. TABLE 2 Degree of Amplification Cell Line mdrl mdr2 KB-3-1 1 1 KB-8-5-11 7-8 7-8 KB-8-5-11-24 9 9 KB-C3 20 20 KB-C4 30 30 KB-C1-R1 1 1 KB-Vl 100 1* KB-A1 70 30* KB-A2 80 1 By comparison of Table 1 with Table 2, it may be observed that in the sublines selected for a 40-700 fold degree of resistance to colchicine, there is a general, but not precise, correlation between increases in drug resistance and in the copy number of mdr sequences. The degree of resistance may correlate more precisely with the expression of md RNA than with the degree of mdr gene amplification. The mdrl and mdr2 sequences appear to be amplified to a similar degree in these cells. The loss of amplified mdr sequences in a revertant of a colchicine-resistant cell line provides strong additional evidence that mdr gene amplification underlies multidrug resistance in the highly resistant cells. The degree of amplification of mdrl in the cells selected for resistance to vinblastine or Adriamycin appears to be higher than in the cells with a similar degree of resistance chat have been selected with col¬ chicine. Example 4 To investigate the nature of the human mdr genes, clones containing mdrl and mdr2 sequences were isolated from the DNA of the colchicine-resistant sub¬ line KB-C3. For this purpose, two phage libraries con¬ taining complete EcoRI or Hindlll digests of KB-C3 DNA were prepared. The EcoRI library was constructed by insertion into the EcoRI site of the λgtll phage vector, and the Hindlll library was made by insertion into the Hindlll site of Charon 28 [Young et al., Proc. Natl. Acad. Sci. (USA), 80, 1194 (1983); Rimm et al.. Gene, 12, 301 (1980)]. Both libraries were screened by plaque hybridization with the Chinese hamster pDR4.7 probe according to the procedure of Benton et al.. Science, 196, 180 (1977). A clone containing the 4.4 kb Hindlll fragment (mdr1) was isolated from the Hindlll library, and a clone containing the 4.5 kb EcoRI fragment (mdr2) was isolated from the EcoRI library. Both inserts were subsequently recloned into the plasmid vector pSP64 [Melton et al.. Nucleic Acids Res. , _2 , 7035 (1984)], giving rise to plasmid clones designated pHDR4.4 and pHDR4.5, respectively. Plasmid clone pHDR4.4 was deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland, as Deposit No. 40227 on March 21, 1986. Likewise, plasmid clone pHDR4.5 was deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MarylanG, as Deposit No. 40228 on March 21, 1986. Partial restriction maps of these clones are shown in Fig. 2 . In Fig. 2, sites for digestion by corresponding restriction endonucleases are identified as follows: "A", Aval/ • M B * ' , 3amHI ' , ΞcoRI; "G" , §2^11; ' Η", Hindlll; I1J.I Haell; "P", PstI; "V", PvulI; and "X" Xbal. In Fig. 2, solid bars indicate the fragments containing highly repeated sequences. These fragments were identified by hybridization of Southern blots con¬ taining restriction digests of cloned DNA with 0.35 x 10 5 dpm/cm 2 of 32 P-labeled total human genomic DNA. Dashed lines indicate the DNA sequences hybridizing to the pDR4.7 clone, as determined by Southern hybridiza¬ tion with the gel-purified pDR4.7 insert. Because the pDR4.7 hamster probe was known to contain transcriptionally active sequences expressed in multidrug-resistant hamster cells [Gros et al., Proc. Nat'l. Acad. Sci. (USA), supra] it seemed likely that the conserved human mdr sequences would provide convenient probes for transcription studies. The hamster pDR4.7 probe hybridized very poorly, if at all, to mRNA from multidrug-resistant human cells, and therefore could not be used as a probe for detection of mdr genes in human cells. Consequently, repeat-free fragments of both clones which hybridized to pDR4.7 were subcloned into the plasmid vector pSP64. The clone containing a 0.75 kb PvuII fragment of pHDR4.4, inserted into the Smal site of the vector, was designated pMDRl. The clone containing a 1.0 kb PstI fragment of pHDR4.5, inserted into the PstI site of the vector, was designated pMDR2. These two clones were found to cross- hybridize with each other under conditions of low hybridization stringency providing additional evidence that mdrl and mdr2 represent related DNA sequences. Example 5 To determine whether the rearranged bands in K3-V1 and XB-A1 correspond to mdrl or mdr2, DNA from different sublines was digested with Hindlll and hybri¬ dized to either hamster pDR4.7 probe or to the human pMDRl or pMDR2 probes. Hybridization with the gel-puri¬ fied insert of the plasmid pDR4.7 was done under condi¬ tions of low stringency (4 X SSC, 0.5% SDS at 65°C) . The same blot wa≤ then rehybridized with gel-purified inserts of the plasmids pMDRl and pMDR2 under high stringency conditions (0.1 X SSC, 0.5% SDS at 65°C) so that the signal resulting from cross-hybridization of mdrl and mdr2 sequences was minimized. This experiment demonstrated that rearranged bands in both KB-Al and KB-Vl sublines correspond to mdr2. The mobility of the new bands appears to be iden¬ tical in several different restriction digests of KB-Vl and KB-Al DNA, indicating that a similar rearrangement may have occurred in both independently selected sub- lines. However, while the rearranged bands are ampli¬ fied in KB-Al, they do not appear amplified in KB-Vl cells. In addition, both types of cells contain bands corresponding to the unrearranged allele of mdr2, which is not amplified. Amplification of the rearranged but not the parental mdr2 band in KB-Al cells suggests that DNA rearrangement either preceded or occurred simul¬ taneously with the onset of gene ampli ication in these cells. In the case of KB-Vl, it is unclear whether mdr2 rearrangement is related to amplification of mdrl. Example 6 To determine whether the evolutionarily con¬ served regions of mdrl and mdr2 contained transcribed sequences, pMDRl and pMDR2 were used as probes for Northern hybridization, performed according to the pro¬ cedure of Thomas, Proc. Natl. Acad. Sci. (USA), 77, 5201-5205 (1980) with poly (A) + RNA extracted from the parental KB-3-1 and multidrug-resistant KB-C2.5 cells [Akiyama et al., supra; Fojo et al.. Cancer Res. , supra; Richert et al., supra and Shen et al., supra] under the conditions of high hybridization stringency as recited in Example 5. Poly (A) + RNA was extracted from the parental drug-sensitive KB-3-1 cells and from the colchicine-resistant KB-C2.5 subline as described in Chirgwin et al., Biochem. , 18, 5294 (1979). One micro- gram of each RNA preparation was electrophoresed in a 1.5% glyoxal agarose gel [McMaster et al., Proc. Natl. Acad. Sci. (USA) , 74_<- 4835 (1977)] and transferred onto Gene Screen Plus™ membrane as available from New England Nuclear, Boston, MA. The membranes were hybridized with 3 x 10 5 dpm/cm 2 of pMDRl or pMDR2 probes. Hybridization was done in 1M NaCl, 10% dextran sulphate, 1% SDS, 50% formamide, 100 ug/ml denatured salmon sperm DNA at 42°C. The membranes were washed with 0.1 x SSC, 0.5% SDS at 65°C and autoradiographed. The size of the RNA band was determined relative to the positions of 28S and 18S ribosomal RNA. The probe pMDRl hybridizes to an mRNA band of a 4.5 kb size which is highly expressed in the drug- resistant cells. This mRNA is not detectable in the parental KB-3-1 cells, indicating little or no expres¬ sion when the probes were labelled either by nick trans¬ lation or oliogolabelling. No distinct bands, however, could be detected when pMDR2 was used as a probe. In addition, no bands were revealed by using other repeat- free subfragments of pHDR4.5 as probes in addition to pMDR2. While the existence of transcriptionally active sequences in other regions of md 2 or transcription of mdr2 sequences at a very low level cannot be excluded by these results, transcription of the amplified region of mdr2 homologous to the Chinese hamster mdr gene is not detected by Northern hybridization. Amplification and over expression of DNA se¬ quences homologous to the Chinese hamster mdr gene in multidrug-resistant human KB carcinoma cells suggests that a similar mechanism may be responsible for multi¬ drug resistance in both human and rodent cells. The nature of the proteins encoded by mdr genes is still unknown. The size of mdrl mRNA is consistent with the possibility that it may code for a 170 kd glycoprotein overexpressed in various multidrug-resistant cell lines [Biedler et al., supra; Ling et al., supra; Ramu et al., supra; Beck et al., supra; Kartner et al.. Science, 221, 1285 (1983); Debenham et al., Mol. Cell. Biol., 2 , 881 (1982); Robertson et al., Mol. Cell. Biol., 4_, 500 (1984)]. It is also unknown whether the same mechanism is utilized in the development of multidrug resistance by human tumor cells iri vitro and in the course of chemotherapy. The availability of cloned probes which detect transcription of mdr DNA in human cells makes it possible now to investigate expression of these sequences in clinical samples of multidrug-resistant tumors . Example 7 In order to examine levels of expression of mdrl sequences during the development of multidrug resistance, multidrug-resistant sublines of human KB carcinoma cells and two other human multidrug resistant cell lines of different origin were studied. Agents used in selecting different sublines in multiple steps were colchicine, Adriamycin and vinblas- tine. In the first two steps of colchicine selection, clones were only obtained if the cell populations were first mutagenized with ethyl ethane sulfonate (EMS). Similarly, KB cell lines selected independently for resistance to Adriamycin or vinblastine [Akiyama et al., supra; Fojo et al.. Cancer Res. , supra; Richert et al. , supra; and Shen et al., supra] were obtained only after mutagenesis with EMS in the first step. Subsequent selection, up to very high levels of resistance, was possible without mutagenesis, and occurred at high frequency. The isolation and some properties of the human multidrug resistant KB carcinoma cell lines has been previously described in Akiyama et al., supra; Fojo et al., Cancer Res. , supra; and Richert et al., supra. The KB cell lines used in this study, the manner of their selection, and their relative resistance to various drugs, are shown in Table 3. CEM is a cell line described in Beck in Advances in Enzyme Regulation, 22, G. Weber, ed. (Pergamon Press, Oxford, 1984), 207, and 2780 is a cell line described in Rogan et al.. Science, 224, 994 (1984). To determine the extent to which mdrl sequences were expressed in these cell lines and the size of the corresponding RNAs, a Northern hybridization was performed with total RNA and poly (A) + -RNA from these cells. A 4.5 kilobase RNA, which migrates just below the 28S ribosomal RNA marker, was clearly visible in all the lanes containing either total or poly (A) + RNA from the resistant lines but was not seen in any of the sensitive cell lines. Slot blot hybridization of total RNA was used to quantitate the expression of md l in various sensi¬ tive and resistant cell lines. RNA prepared as pre- viously described above was applied to filters using a Schleicher and Schuell slot blot apparatus or by blot¬ ting after electrophoresis in 1% agarose containing 13.4% formaldehyde. A gel-purified insert from the pMDRl clone was 32 P-labeled for use as a probe. Nitro- cellulose filters were baked and preincubated for 4-6 hours at 42°C in 50% formamide, 5 x SSC, 10X Denhardt's solution, 0.1% SDS and 100 ug/ml salmon sperm DNA. Filters were hybridized overnight in the above solution containing 32 ?-iabeied probe. Filters were wasned 3 times for 10 minutes at room temperature in 2 x SSC , 0.1% SDS and 3 times for 20 minutes at 50°C in 0.1 x SSC, 0.1% SDS. Levels of mdrl expression were deter¬ mined by densitometry of the autoradiograms. Tracings of peaks were cut out and weighed and compared to the KB-8 peak which was arbitrarily assigned a value of 1. The results are presented in Table 3 along with the relative drug resistances of the human leukemic lympho- blast cell lines, and the human ovarian cancer cell lines used in the study. In Table 3, ND is an abbrevia¬ tion for "none detected". TABLE 3 Relative Resistance to mdrl mRN Cel] Line Selecting Agent C Cooll A Addrr V Vbbll Expressi KB-3-1 parental KB 1 1 1 1 1 1 ND KB-8 colchicine, 5 ng/ml 2 2..1 1 1 1..1 1 1 1..22 1 KB-8-5 colchicine, 10 ng/ml 3 3..8 8 3 3..2 2 6 6..33 3 KB-8-5-11 colchicine, 100 ng/ml 4 400 2 233 5 511 80 KB-C1 colchicine, 1 μg/ml 2 26600 1 16600 9 966 270 KB-C1-R1 revertant of KB-C1 6 6 3 3 4 4 1 KB-C1.5 colchicine, 1.5 μg/inl 3 32200 — — 1 14400 340 KB-C6 colchicine, 6 μg/ml 2 2,,110000 3 32200 3 37700 820 KB-Al Adriamycin, 1 μg/ml 1 199 9 977 4 433 270 KB-Vl vinblastine, 1 μg/ml 1 17700 4 42200 2 21100 320 CEM parental leukemic 1 1 1 1 1 1 ND CEM-Vlb 1Q0 vinblastine 4 455 1 12200 4 42200 250 2780 parental , ova r ian 1 1 1 ND 2780-Ad Adr iamyc i n — 170 15 260 As shown in Table 3, there was a good correla¬ tion between extent of multidrug resistance and the level of mdrl-specific mRNA. As can also be seen in Table 3, there is little or no expression of the mdrl sequences in parental, drug-sensitive cell lines, but increasing expression occurs as the cell lines become more resistant to drugs. A revertant cell line, KB-C1- Rl, subcloned in the absence of colchicine from the colchicine-resistant cell line KB-C1, still expresses mdrl sequences at reduced levels consistent with its low level of multidrug resistance. Tt is not possible to calculate the exact extent of increased expression in the resistant cell lines relative to the parental line, since the hybridi- zation signal from the parental RNA was too weak. How¬ ever, the extent of expression relative to the KB-8 cell line has been calculated and these data are shown in Table 3. Expression appears to correlate well with increasing drug-resistance for every step of selection in KB cells and reaches very high levels in our most resistant KB cell lines. The data summarized in Table 3 indicate that two other human cell lines of different origin, selected for multidrug resistance, also express high levels of the 4.5 kb mRNA. Very little or no expression of this RNA was detected in the parental cell lines. The human leukemic lymphoblast cell line CEM (A.T.C.C. CCL119) and its resistant derivatives CEM-VLB 10Q , selected for resistance to vinblastine (gift of W. Beck, St. Jude's Hospital) (Beck, supra. ) and the ovarian cell line 2780 and its resistant derivative 2780-Ad, selected for resistance to Adriamycin (gift of T. Hamilton and R. Ozols, National Institutes of Health) (Rogan et al., supra) both showed high levels of expression of the 4.5 kb mRNA. Because even low levels of cellular multidrug- resistance may result in clinically refractory tumors, expression of mdrl mRNA in sublines having a low level (2-6 fold) of relative drug resistance but not in the parental drug-sensitive cell lines is of particular interest. In this regard the results presented in Table 3 indicate that quantitation of mdrl mRNA expression may potentially be used for diagnosis of multidrug resis¬ tance in clinical tumor specimens. Example 8 To compare the levels of mdrl mRNA expression with the extent of amplification, of the genomic mdrl sequences genomic DNA was isolated from all of the cell lines described in Example 6. Following digestion with Hindlll, amplification of mdrl was examined by Southern blot analysis. DNA, prepared as previously described in Example 3, was digested with Hindlll and electrophoresed in 0.8% agarose gels before Southern transfer to Gene Screen Plus™ (New England Nuclear). The blots were hybridized with the pMDRl probe for 18 hours at 42°C in 50% formamide, 5 x SSC, 1% SDS with 100 ug/ml salmon sperm DNA. The blots were then washed with 2 x SSC at ' room temperature for 10 minutes, 2 x SSC, 1% SDS at 42°C for 60 minutes and 0.1 x SSC at room temperature for 60 minutes prior to autoradiography. No amplification of mdrl was found in the KB cell lines with low levels of resistance (KB-8, KB-8-5 and the revertant subline, KB-C1-R1), even though these cell lines expressed increased levels of mdrl mRNA. Increased expression of mdrl sequences in human cells may therefore occur prior to gene amplification. Ampli¬ fication of the mdrl gene was detected in highly resis¬ tant sublines of KB cells selected in colchicine, vin- blastine or Adriamycin, as well as in CEM-VLB-. QQ and 2780-Ad cell lines. In the latter two sublines, the degree of gene amplification was estimated by densi- tometry to be approximately 5-10 fold for 2780-Ad and 10-15 fold for CEM-VLB 100 . In all cases, the increase in mRNA expression was clearly greater than the extent of amplification. These results suggest that the evolution of these lines involved a step or steps in which expression was increased out of proportion to gene amplification. A similar dissociation of amplification and expression of the dhfr gene has been reported for human cancer cells selected for resistance to methotrexate i_ vitro. [Frei et al-, Proc. Natl. Acad. Sci. (USA), 81., 2873 (1984); Wolman et al., Proc. Natl. Acad. Sci. (USA), 80, 807 (1983).] The development of multidrug resistance in human KB cells differs in this respect from Chinese hamster V79 cells where a low (5-7 fold) degree of rela¬ tive drug resistance is accompanied by 5-10 fold ampli¬ fication of mdr DNA [Roninson et al., supra; and Gros et al. , supra] . These studies demonstrate a correlation between expression of the mdrl gene and the development of resistance to multiple agents in five independently- derived human cell lines of different origins selected ' for resistance to different cytotoxic drugs. Expression of mdrl may therefore represent a common mechanism of multidrug resistance in human cell lines. Increased expression of mdrl in at least some cases occurs ini¬ tially without gene amplification and may be a prerequi¬ site for the development of multidrug resistance. This observation may be especially relevant for the analysis of the role of the mdr_l gene in the development of multidrug resistance by human tumors in the course of chemotherapy and may have diagnostic potential. Since the tumor cells are expected to have a relatively low degree of resistance, such an analysis may involve quantitation of mdrl RNA expression rather than gene amplification in tumor samples. Example 9 The segment of the mdrl gene cloned into pMDRl was sequenced by the chemical degradation procedure [Maxam et al., Meth. Enzymol., 65, 499, (1980)] and the enzymatic chain-termination sequencing technique [Sanger et al., Proc. Natl. Acad. Sci. USA, 7_4, 5463, (1977)] using supercoiled plasmid DNA as a template [Zagursky et al., Gene Anal. Techn. , 2 , 89, (1985)]. To facilitate sequencing, pMDRl was mapped with Haelll and Rsal and individual 220-400 bp fragments of pMDRl were subcloned into a pUC18 plasmid vector (Bethesda Research Laboratories, Rockville, MD) . The sequence of pMDRl was confirmed by sequencing both strands. The complete sequence of pMDRl is presented in Table 4. Comparison with the sequence of the corresponding cDNA clones in Example 10 below indicated that pMDRl includes segments of two protein-codi g sequences (exons), comprising nucleotides 1-111 and 653-307, and an intervening sequence (intron) which is not expressed as mRNA and which comprises nucleotides 112-652. Table 4 shows that amino acid sequence corresponding to the exons within ' pMDRl. This amino acid sequence therefore defines a segment of the mdrl protein product. TABLE 4 SEQUENCE OF THE pMDRl CLONE 1 T GGA AGA CAA ATA CAC AAA ATT AGA AAA CAG TTT TTT CAT GCT Gly Arg Gin He His Lys He Arg Lys Gin Phe Phe His Ala 44 ATA ATG CGA CAG GAG ATA GGC TGG TTT GAT GTG CAC GAT GTT He MET Arg Gin Glu He Gly Trp Phe Asp Val His Asp Val 86 GGG GAG CTT AAC ACC CGA CTT ACA GA Gly Glu Leu Asn Thr Arg Leu Thr As INJ LΠ 11 2 GTAAGTATT TAGTTTTATG TTGAACTTGG GTGTCGTTCT 151 TATCCTTAGT AAAATGAAAT AGATGTCATC ACATCTGTTA GGAGGTGTTA 201 ATGTATCATT CAAAGGTACT TATGAGACAA AATTCCTTCT AAGCAGCAAC 251 AATGTCGTGT GCATCCTTTT GTTCCCAGTG CCTTGACAGG GTATGGGGGG 301 ACCTGCATGA CTAGCATTAA ATGAAGGACT GGGCTTTCCA GAATGAAGAA 351 ATCCTCTGAG AATGTGCAGT AGAGCAAAAC AAGATACTTT CTGAGGAAAT 401 TTCTGAGCAA TTTGAAATTC CTAGGTTGAA TACTTCTTGT GTACACGATG t > l TCCATTTCCT GGGGCCATGT GGCTATGGΛT TTTTGTTGTT AATGACAAAT 501 ATCCTAGTAC AAACTTCTAC CCTGCTAAAT AAAACAAAGC ATAGGCACAA 551 AATACTCTAG CCATAAACTA CCCTACACTC AAAACAGGCT TCACGAGAAA 601 AGTTGATGTT TACAATTCTG ACAATTATTT CTAACACTAT CTGTTCTTTC 651 AG 653 T GAT GTC TCT AAG A T AAT GAA GTT ATT GGT GAC AAA ATT GGA p Asp Val Ser Lys He Asn Glu Val He Gly Asp Lys He Gly 696 ATG TTC TTT CAG TCA ATG GCA ACA TTT TTC ACT GGG TTT ATA MET Phe Phe Gin Ser MET Ala Thr Phe Phe Thr Gly Phe He TABLE 4 (cont'd. ) 738 GTA GGA TTT ACA CGT GGT TGG AAG CTA ACC CTT GTG ATT TTG Val Gly Phe Thr Arg Gly Trp Lys Leu Thr Leu Val ' He Leu 780 GCC ATC AGT CCT GTT CTT GGA CTG TCA G 807 Ala He Ser Pro Val Leu Gly Leu Ser Example 10 In order to isolate cDNA clones of the mdrl gene, poly(A)+ RNA was isolated as described in Chirgwin et al.. Biochemistry, 18, 5294 (1979) and Aviv et al., Proc. Natl. Acad. Sci. (USA). 69, 1408 (1972) from the subline KB-C2.5, selected with colchicine. A cDNA li¬ brary was constructed using the steps of synthesizing double-stranded cDNA, blunt ending, attachment of EcoRI linkers and insertion into the phage vector λgtll [Young and Davis, supra; Huynh et al., in: DNA Cloning Techni¬ ques: A Practical Approach, D. Glover, ed., IRL Press, Oxford, (1985)]. The cDNA library was screened by pla¬ que hybridization (Benton et al., supra) with the pMDRl probe. Approximately 120 positive clones were iso¬ lated. The inserts from five of these clones (XHDR5, λHDRlO, XKDR28, XHDR62 and λHDR69) were re-cloned into plasmid vectors pGEMl and pGEM4 (Promega Biotec). The partial restriction maps of these clones are shown in Fig. 3. DNA from λHDR5 was treated with EcoRI which generated two fragments, designated 5A and 53. The fragments were subcloned into pGEMl at its EcoRI site to give plasmids pHDR5A and pHDR5B which were deposited ' with the American Type Culture Collection, 12301 Park¬ lawn Drive, Rockville, Maryland, on March 18, 1986, and which received the respective accession numbers ATCC 67040 and ATCC 67041. Similarly, λHDRlO was treated with EcoRI and cloned into the EcoRI site of pGEMl to produce pHDRlO which was deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rock¬ ville, Maryland, on March 18, 1986, as Deposit No. 67042. To isolate the remaining portion of mdrl cDNA, a fragment of the clone ,\HDR5, indicated with a striped bar in Fig. 3, was used to screen the same cDNA library. The inserts from three of the positive clones, designated XHDR103, XHDR104 and λHDRIOϊ, were re-cloned into the EcoRI sites of plasmid vectors pGEMl and pGEM4, giving rise to plasmids designated pHDR103, pHDR104 and pHDR105, respectively. The plasmid pHDR104 was deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland, on July 16, 1986, as Deposit No. 67156. A comparison of the restriction maps of individual clones indicates divergence in the cDNA structure among, for example, clones λHDRlO, λHDR28 and λHDR69. The most highly conserved region among these clones is represented by a 270 bp PvuII fragment, which corresponds to the exon regions of pMDRl and is indicated with a solid bar above the lines in Fig. 3. The variant sequences specific to clones λHDR62 and λHDR105 were detected by DNA sequencing, and they are shown as solid bars underneath the corresponding lines in Fig. 3. In Fig. 3, sites for digestion by corresponding restriction endonucleases are identified as follows: "A", AccI; "E", EcoRI; "H", Hindlll; "N", XmnI; "P", PvuII; "S", StuI; "T", SstI; "V", Aval; and "X", Xbal. The cDNA clones XHDR10, λHDR5 and λHDR104 were sequenced in their entirety using the methods of ' subcloning the inserts into an M13 phage vector [Messing, Meth. Enyzmol. , 101, 20, 1983], generating a series of overlapping deletion subclon.-s [Henikoff, Gene, 28, 351, 1984] and determining their DNA sequence by the enzymatic chain-termination sequencing techniques [Sanger et al., supra] . A part of the cDNA sequence was determined by specific-primer-directed DNA sequencing [Strauss et al., Anal. Biochem., 154, 353 ' 1986] using supercoiled plasmid DNA as a template [Zagursky et al., supra] . The overlapping regions of clones λHDRlO, λHDR5 and λHDR104 were found to be identical, and therefore, these clones are assumed to represent different parts of the same cDNA. The combined cDNA sequence of clones λHDRlO, ..HDR5 and IHDRI04 is shown in Table 5. This table also shows the amino acid sequence of mdrl gene product, derived from the same cDNA sequence. TABLE 5 MDR1 cDNA SEQUENCE (CLONES λHDRlO; XHDR5; XHDR104) 1 CCTACTCTAT TCAGATATTC TCCAGATTCC TAAAGATTAG AGATCATTTC 51 TCATTCTCCT AGGAGTACTC ACTTCAGGAA GCAACCAGAT AAAAGAGAGG 101 TGCAACGGAA GCCAGAACAT TCCTCCTGGA AATTCAACCT GTTTCGCAGT 151 TTCTCGAGGA ATCAGCATTC AGTCAATCCG GGCCGGGAGC AGTCATCTGT 201 GGTGAGGCTG ATTGGCTGGG CAGGAACAGC GCCGGGGCGT GGGCTGAGCA 251 CAGCGCTTCG CTCTCTTTGC CACAGGAAGC CTGAGCTCAT TCGAGTAGCG 301 GCTCTTCCAA GCTCAAAGAA GCAGAGGCCG CTGTTCGTTT CCTTTAGGTC 351 TTTCCACTAA AGTCGGAGTA TCTTCTTCCA AGATTTCACG TCTTGGTGGC 401 CGTTCCAAGG AGCGCGAGGT CGGG ' 425 ATG GAT CTT GAA GGG GAC CGC AAT GGA GGA GCA AAG AAG AAG 466 MET Asp Leu Glu Gly Asp Arg Asn Gly Gly Ala Lys Lys Lys ' 467 AAC TTT TTT AAA CTG AAC AAT AAA AGT GAA AAA GAT AAG AAG 508 Asn Phe Phe Lys Leu Asn Asn Lys Ser Glu Lys Asp Lys Lys 509 GAA AAG AAA CCA ACT GTC AGT GTA TTT TCA ATG TTT CGC TAT 550 Glu Lys Lys Pro Thr \ .._ Ser Vul Phe Ser MET Phe Arg Tyr 551 TCA AAT TGG CTT GAC AAG TTG TAT ATG GTG GTG GGA ACT TTG 592 Ser Asn Trp Leu Asp Lys Leu Tyr MET Val Val Gly Thr Leu 593 GCT GCC ATC ATC CAT GGG GCT GGA CTT CCT CTC ATG ATG CTG 634 Ala Ala He He His Gly Ala Gly Leu Pro Leu MET MET Leu 635 GTG TTT GGA GAA ATG ACA GAT ATC TTT GCA AAT GCA GGA AAT 676 Val Phe Gly Glu MET Thr Asp He Phe Ala Asn Ala Gly Asn TABLE 5 (cont'd. ) 677 TTA GAA GAT CTG ATG TCA AAC ATC ACT AAT AGA AGT GAT ATC 718 Leu Glu Asp Leu MET Ser Asn He Thr Asn Arg Ser Asp He 719 AAT GAT ACA GGG TTC TTC ATG AAT CTG GAG GAA GAC ATG ACC 760 Asn Asp Thr Gly Phe Phe MET Asn Leu Glu Glu Asp MET Thr 761 AGG TAT GCC TAT TAT TAC AGT GGA ATT GGT GCT GGG GTG CTG 802 Arg Tyr Ala Tyr Tyr Tyr Ser Gly He Gly Ala Gly Val Leu 803 GTT GCT GCT TAC ATT CAG GTT TCA TTT TGG TGC CTG GCA GCT 844 Val Ala Ala Tyr He Gin Val Ser Phe Trp Cys Leu Ala Ala 845 GGA AGA CAA ATA CAC AAA ATT AGA AAA CAG TTT TTT CAT GCT 886 Gly Arg Gin He His Lys He Arg Lys Gin Phe Phe His Ala 887 ATA ATG CGA CAG GAG ATA GGC TGG TTT GAT GTG CAC GAT GTT 928 He MET Arg Gin Glu He Gly Trp Phe Asp Val His Asp Val 929 GGG CAG CTT AAC ACC CGA CTT ACA GAT GAT GTC TCT AAG ATT 970 Gly Glu Leu Asn Thr Arg Leu Thr Asp Asp Val Ser Lys He 971 AAT CAA GTT ATT GGT GAC AAA ATT GGA ATG TTC TTT CAG TCA 1012 Asn Glu Val He Gly Asp Lys He Gly MET Phe Phe Gin Ser 1013 ATG GCA ACA TTT TTC ACT GGG TTT ATA GTA GGA TTT ACA CGT 1054 MET Ala Thr Phe Phe Thr Gly Phe He Val Gly Phe Thr Arg 1055 GGT TGG AAG CTA ACC CTT GTG ATT TTG GCC ATC AGT CCT GTT 1096 Gly Trp Lys Leu Thr Leu Val He Leu Ala He Ser Pro Val TABLE 5 (cont'd. ) 1097 CTT GGA CTG TCA GCT GCT GTC TGG GCA AAG ATA CTA TCT TCA 1138 Leu Gly Leu Ser Ala Ala Val Trp Ala Lys He Leu Ser Ser 1139 TTT ACT GAT AAA GAA CTC TTA GCG TAT GCA AAA GCT GGA GCA 1180 Phe Thr Asp Lys Glu Leu Leu Ala Tyr Ala Lys Ala Gly Ala 1181 GTA GCT GAA GAG GTC TTG GCA GCA ATT AGA ACT GTG ATT GCA 1222 Val Ala Glu Glu Val Leu Ala Ala He Arg Thr Val He Ala 1223 TTT GGA GGA CAA AAG AAA GAA CTT GAA AGG TAC AAC AAA AAT 1264 Phe Gly Gly Gin Lys Lys Glu Leu Glu Arg Tyr Asn Lys Asn 1265 TTA GAA GAA GCT AAA AGA ATT GGG ATA AAG AAA GCT ATT ACA 1306 Leu Glu Glu Ala Lys Arg He Gly He Lys Lys Ala He Thr 1 07 GCC AAT ATT TCT ATA GGT GCT GC TTC CTG CTG ATC TAT GCA 1348 Ala Asn He Ser He Cly Ala Ala Phe Leu Leu He Tyr Ala 1349 TCT TAT GCT CTG GCC TTC TGG TAT GGG ACC ACC TTG GTC CTC 1390 Ser Tyi Ala Leu Ala Phe Trp Tyr Gly Thr Thr Leu Val Leu 1391 TCA GGG GAA TAT TCT ATT GGA CAA GTA CTC ACT GTA TTC TTT 1432 Ser Gly Glu Tyr Ser He Gly Gin Val Leu Thr Val Phe Phe 1433 TCT GTA TTA ATT GGG GCT TTT AGT GTT GGA CAG GCA TCT CCA 1474 Ser Val Leu He Gly Ala Phe Ser Val Gly Gin Ala Ser Pro 1475 AGC ATT GAA GCA TTT GCA AAT GCA AGA GGA GCA GCT TAT GAA 1516 Ser He Glu Ala Phe Ala Asn Ala Arg Gly Ala Ala Tyr Glu TABLE 5 (cont'd. ) 1517 ATC TTC AAG ATA ATT GAT AAT AAG CCA AGT ATT GAC AGC TAT 1558 He Phe Lys He He Asp Asn Lys Pro Ser He Asp Ser Tyr 1559 TCG AAG AGT GGG CAC AAA CCA GAT AAT ATT AAG GGA AAT TTG 1600 Ser Lys Ser Gly His Lys Pro Asp Asn He Lys Gly Asn Leu 1601 GAA TTC AGA AAT GTT CAC TTC AGT TAC CCA TCT CGA AAA GAA 1642 Glu Phe Arg Asn Val His Phe Ser Tyr Pro Ser Arg Lys Glu 1643 GTT AAG ATC TTG AAG GGC CTG AAC CTG AAG GTG CAG AGT GGG 1684 Val Lys He Leu Lys Gly Leu Asn Leu Lys Val Gin Ser Gly I 1685 CAG ACG GTC GCC CTG GTT GGA AAC AGT GGC TGT GGG AAG AGC 1726 _ Gln Thr Val Ala Leu Val Gly Asn Ser Gly Cys Gly Lys Ser w I 1727 ACA ACA GTC CAG CTG ATG CAG AGG CTC TAT GAC CCC ACA GAG 1768 Thr Thr Val Gin Leu MET Gin Arg Leu Tyr Asp Pro Thr Glu 1769 GGG ATG GTC AGT GTT GAT GGA CAG GAT ATT AGG ACC ATA AAT 1810 Gly MET Val Ser Val Asp Gly Gin Asp He Arg Thr He Asn 1811 GTA AGG TTT CTA CGG GAA ATC ATT GGT GTG GTG AGT CAG GAA 1852 Val Arg Phe Leu Arg Glu He He Gly Val Val Ser Gin Glu 1853 CCT GTA TTG TTT GCC ACC ACG ATA GCT GAA AAC ATT CGC TAT 1894 Pro Val Leu Phe Ala Thr Thr He Ala Glu Asn He Arg Tyr 1895 GGC CGT GAA AAT GTC ACC ATG GAT GAG ATT GAG AAA GCT GTC 1936 Gly Arg Glu Asn Val Thr MET Asp Glu He Glu Lys Ala Val TABLE 5 (cont'd. ) 1937 AAG GAA GCC AAT GCC TAT GAC TTT ATC ATG AAA CTG CCT CAT 1978 Lys Glu Ala Asn Ala Tyr Asp Phe He MET Lys Leu Pro His 1979 AAA TTT GAC ACC CTG GTT GGA GAG AGA GGG GCC CAG TTG AGT 2020 Lys Phe Asp Thr Leu Val Gly Glu Arg Gly Ala Gin Leu Ser 2021 GGT GGG CAG AAG CAG AGG ATC GCC ATT GCA CGT GCC CTG GTT 2062 Gly Gly Gin Lys Gin Arg He Ala He Ala Arg Ala Leu Val 2063 CGC AAC CCC AAG ATC CTC CTG CTG GAT GAG GCC ACG TCA GCC 2104 Arg Asn Pro Lys He Leu Leu Leu Asp Glu Ala Thr Ser Ala 2105 TTG GAC ACA GAA AGC GAA GCA GTG GTT CAG GTG GCT CTG GAT 2146 Leu Asp Thr Glu Ser Glu Ala Val Val Gin Val Ala Leu Asp 2147 AAG GCC AGA AAA GGT CGG ACC ACC ATT GTG ATA GCT CAT CGT 2188 Lys Ala Arg Lys Gly Arg Thr Thr He Val He Ala His Arg 2189 TTG TCT ACA GTT CGT AAT GCT GAC GTC ATC GCT GGT TTC GAT 2230 Leu Ser Thr Val Arg Asn Ala Asp Val He Ala Gly Phe Asp 2231 GAT GGA GTC ATT GTG GAG AAA GGA AAT CAT GAT GAA CTC ATG 2272 Asp Gly Val lie Val Glu Lys Gly Asn His Asp Glu Leu MET 2273 AAA GAG AAA GGC ATT TAC TTC AAA CTT GTC ACA ATG CAG ACA 2314 Lys Glu Lys Gly He Tyr Phe Lys Leu Val Thr MET Gin Thr 2315 GCA GGA AAT GAA GTT GAA TTA GAA AAT GCA GCT GAT GAA TCC 2356 Ala Gly Asn Glu Val Glu Leu Glu Asn Ala Ala Asp Glu Ser TABLE 5 (Jcont'd. ) L 2357 AAA AGT GAA ATT GAT GCC TTG GAA ATG TCT TCA AAT GAT TCA 2398 Lys Ser Glu He Asp Ala Leu Glu MET Ser Ser Asn Asp Ser 2399 AGA TCC AGT CTA ATA AGA AAA AGA TCA ACT CGT AGG AGT GTC 2440 Arg Ser Ser Leu He Arg Lys Arg Ser Thr Arg Arg Ser Val 2441 CGT GGA TCA CAA GCC CAA GAC AGA AAG CTT AGT ACC AAA GAG 2482 Arg Gly Sei * Gin Ala Gin Asp Arg Lys Leu Ser Thr Lys Glu 2483 GCT CTG GAT GAA AGT ATA CCT CCA GTT TCC TTT TGG AGG ATT 2524 Ala Leu Asp Glu Ser He Pro Pro Val Ser Phe Trp Arg He 2525 ATG AAG CTA AAT TTA ACT GAA TGG CCT TAT TTT GTT GTT GGT 2566 j MET Ly: Leu Asn Leu Thr Glu Trp Pro Tyr Phe Val Val Gly 2567 GTA TTT TGT GCC ATT ATA AAT GGA GGC CTG CAA CCA GCA TTT 2608 Val Phe Cys Ala Tie He Asn Gly Gly Leu Gin Pro Ala Phe 2609 GCA ATA ATA TTT TCA AAG ATT ATA GGG GTT TTT ACA AGA ATT 2650 Ala He He Phe Ser Lys He He Gly Val Phe Thr Arg He 2651 GAT GAT CCT GAA ACA AAA CGA CAG AAT AGT AAC TTG TTT TCA 2692 Asp Asp Pro Glu Thr Lys Arg Gin Asn Ser Asn Leu Phe Ser 2693 CTA TTG TTT CTA GCC CTT GGA ATT ATT TCT TTT ATT ACA TTT 2734 Leu Leu Phe Leu Ala Leu Gly He He Ser Phe He Thr Phe 2735 TTC CTT CAG GGT TTC ACA TTT GGC AAA GCT GGA GAG ATC CTC 2776 Phe Leu Gin Gly Phe Thr Phe Gly Lys Ala Gly Glu He Leu TABLE 5 (cont'd. )_. 2777 ACC AAG CGG CTC CGA TAC ATG GTT TTC CGA TCC ATG CTC AGA 2818 Thr Lys Arg Leu Arg Tyr MET Val Phe Arg Ser MET Leu Arg 2b19 CAG GAT GTG AGT TGG TTT GAT GAC CCT AAA AAC ACC ACT GGA 2860 Gin Asp Val Ser Trp Phe Asp Asp Pro Lys Asn Thr Thr Gly 2861 GCA TTG ACT ACC AGG CTC GCC AAT GAT GCT GCT CAA GTT AAA 2902 Ala Leu Thr Thr Arg Leu Ala Asn Asp Ala Ala Gin Val Lys 2903 GGG GCT ATA GGT TCC AGG CTT GCT GTA ATT ACC CAG AAT ATA 2944 Gly Ala He Gly Ser Arg Leu Ala Val He Thr Gin Asn He I 2945 GCA AAT CTT GGG ACA GGA ATA ATT ATA TCC TTC ATC TAT GGT 2986 «__ Ala Asn Leu Gly Thr Gly He He He Ser Phe He Tyr Gly °- I 2987 TGG CAA CTA ACA CTG TTA CTC TTA GCA ATT GTA CCC ATC ATT 3028 Trp Gin Leu Thr Leu Leu Leu Leu Ala He Val Pro He He 3029 GCA ATA GCA GGA GTT GTT GAA ATG AAA ATG TTG TCT GGA CAA 3070 Ala He Ala Gly Val Val Glu MET Lys MET Leu Ser Gly Gin 3071 GCA CTG AAA GAT AAG AAA GAA CTA GAA GGT GCT GGG AAG ATC 3112 Ala Leu Lys Asp Lys Lys Glu Leu Glu Gly Ala Gly Lys He 3113 GCT ACT GAA GCA ATA GAA AAC TTC CGA ACC GTT GTT TCT TTG 3154 Al«a Thr Glu Ala He Glu Asn Phe Arg Thr Val Val Ser Leu 3155 ACT CAG GAG CAG AAG TTT GAA CAT ATG TAT GCT CAG AGT TTG 3196 Thr Gin Glu Gin Lys Phe Glu His MET Tyr Ala Gin Ser Leu TABLE 5 (Jcont'd. )__ 3197 CAG GTA CCA TAC AGA AAC TCT TTG AGG AAA GCA CAC ATC TTT 3238 Gin Val Pro Tyr Arg Asn Ser Leu Arg Lys Ala His He Phe 3239 GGA ATT ACA TTT TCC TTC ACC CAG GCA ATG ATG TAT TTT TCC 3280 Gly He Thr Phe Ser Phe Thr Gin Ala MET MET Tyr Phe Ser 3281 TAT GCT GGA TGT TTC CGG TTT. GGA GCC TAC TTG GTG GCA CAT 3322 Tyr Ala Gly Cys Phe Arg Phe ' Gly Ala Tyr Leu Val Ala His 3323 AAA C'lϋ ATG AGC TTT GAG GAT GTT CTG TTA GTA TTT TCA GCT 3364 Lys Leu MET Ser Phe Glu Asp Val Leu Leu Val Phe Ser Ala 3365 GTT GTC TTT GGT GCC ATG GCC GTG GGG CAA GTC AGT TCA TTT 3406 ^ Val Val Phe Gly Ala MET Ala Val Gly Gin Val Ser Ser Phe 3407 GCT CCT GAC TAT GCC AAA GCC AAA ATA TCA GCA GCC CAC ATC 3448 Ala Pro Asp Tyr Ala Lys Ala Lys He Ser Ala Ala His He 3449 ATC ATG ATC ATT GAA AAA ACC CCT TTG ATT GAC AGC TAC AGC 3490 He MET He He Glu Lys Thr Pro Leu He Asp Ser Tyr Ser 3491 ACG GAA GGC CTA ATG CCG AAC ACA TTG GAA GGA AAT GTC ACA 3532 Thr Glu Gly Leu MET Pro Asn Thr Leu Glu Gly Asn Val Thr 3533 TTT GGT GAA GTT GTA TTC AAC TAT CCC ACC CGA CCG GAC ATC 3574 Phe Gly Glu Val Val Phe Asn Tyr Pro Thr Arg Pro Asp He 3575 CCA GTG CTT CAG GGA CTG AGC CTG GAG GTG AAG AAG GGC CAG 3616 Pro Val Leu Gin Gly Leu Ser Leu Glu Val Lys Lys Gly Gin TABLE 5 (Scont'd. )_. 3617 ACG CTG GCT CTG GTG GGC AGC AGT GGC TGT GGG AAG AGC ACA 3658 Thr Leu Ala Leu Val Gly Ser Ser Gly Cys Gly Lys Ser Thr 3659 GTG GTC CAG CTC CTG GAG CGG TTC TAC GAC CCC TTG GCA GGG 3700 Val Val Gin Leu Leu Glu Arg Phe Tyr Asp Pro Leu Ala Gly 3701 AAA GTG CTG CTT GAT GGC AAA GAA ATA AAG CGA CTG AAT GTT 3742 Lys Val Leu Leu Asp Gly Lys Glu He Lys Arg Leu Asn Val 3743 CAG TGG CTC CGA GCA CAC CTG GGC ATC GTG TCC CAG GAG CCC 3784 Gin Trp Leu Arg Ala His Leu Gly He Val Ser Gin Glu Pro 3785 ATC CTG TTT GAC TGC AGC ATT GCT GAG AAC ATT GCC TAT GGA 3826 He Leu Phe Asp Cys Ser He Ala Glu Asn He Ala Tyr Gly 3827 GAC AAC AGC CGG GTG GTG TCA CAG GAA GAG ATC GTG AGG GCA 3868 Asp Asn Ser Arg Val Val Ser Gin Glu Glu He Val Arg Ala 3869 GCA AAG GAG GCC AAC ATA CAT GCC TTC ATC GAG TCA CTG CCT 3910 Ala Lys Glu Ala Asn He His Ala Phe He Glu Ser Leu Pro 3911 AAT AAA TAT AGC ACT AAA GTA GGA GAC AAA GGA ACT CAG CTC 3952 Asn Lys Tyr Ser Thr Lys Val Gly Asp Lys Gly Thr Gin Leu 3953 TCT GGT GGC CAG AAA CAA CGC ATT GCC ATA GCT CGT GCC CTT 3994 Ser Gly Gly Gin Lys Gin Arg He Ala He Ala Arg Ala Leu 3995 GTT AGA CAG CCT CAT ATT TTG CTT TTG GAT GAA GCC ACG TCA 4036 Val Arg Gin Pro His He Leu Leu Leu Asp Glu Air* Thq Ser TABLE 5 (Jcont'd.)_. 4037 GCT CTG GAT ACA GAA AGT GAA AAG GTT GTC CAA GAA GCC CTG 4078 Ala Leu Asp Thr Glu Ser Glu Lys Val Val Gin Glu Ala Leu 4079 GAC AAA GCC AGA GAA GGC CGC ACC TGC ATT GTG ATT GCT CAC 4120 Asp Lys Ala Arg Glu Gly Arg Thr Cys He Val He Ala His 121 CGC CTG TCC ACC ATC CAG AAT GCA GAC TTA ATA GTG GTG TTT 4162 Arg Leu Ser Thr He G n Asn Ala Asp Leu He Val Val Phe 1 3 CAG AAT GGC AGA GTC AAG GAG CAT GGC ACG CAT CAG CAG CTG 4204 Gin Asn Gly Arg Val Lys Glu His Gly Thr His Gin Gin Leu 4205 CTG GCA CAG AAA GGC ATC TAT TTT TCA ATG GTC AGT GTC CAG 4246 Leu Ala Gin Lys Gly He Tyr Phe Ser MET Val Ser Val Gin 4247 GCT GGA ACA AAG CGC CAG TGA 4267 Ala Gly Thr Lys Arg Gin TER 4268 ACTCTGACTG TATGAGATGT TAAATACTTT TTAATATTTG TTTAGATATG 4318 ACATTTATTC AAAGTTAAAA GCAAACACTT ACAGAATTAT GAAGAGGTAT 4368 CTGTTTAACA TTTCCTCAGT CAACTTCAGA GTCTTCAGAG ACTTCGTAAT 4 18 TAAAGCAACA GAGTGAGAGA CATCATCAAG TGGAGAGAAA TCATAGTTTA 4468 AACTGCATTA TAAATTTTAT AACAGAATTA AAGTAGATTT TAAAAGATAA 4518 AATGTGTAAT TTTGTTTATA TTTTCCCATT TGGACTGTAA CTGACTGCCT 4568 TGCTAAAAGA TTATAGAAGT AGCAAAAAGT ATTGAAATGT TTGCATAAAG 4618 TGTCTATAAT AAAACTAAAC TTTCATGTGA AAAAAAAAAA AAAAAAAAAA 4668 AA Analysis of the amino acid sequence presented in Table 5 indicates that the mdrl gene product is likely to be a transmembrane protein. This protein may consist of two approximately equal parts, with a considerable sequence homology to each other, indicating that the mdrl gene has likely evolved as a result of an internal duplication. Each half of the protein consists of a hydrophobic and a hydrophilic portion. Each of the hydrophobic portions includes six transmembrane domains, as determined by the algorithm of Eisenberg et al. [J. Mol. Biol., 179, 125-142 (1984)]. Both hydrophilic portions contain two regions that share a high level of amino acic homology with the ATP-binding sites of several known enzymes. The best homology has been observed with the ATP-binding sites of peripheral membrane components of bacterial periplasmic binding protein-dependent transport systems [Higgins et al. , EMBO J. , 4, 1033-1040, (1984)]. The presence of the transmembrane domains and potential glycosylation sites within the protein sequence is consistent with the mdjrl protein being related to the P-glycoprotein, which is described above. Analysis of the DNA and protein sequence ' information presented in Table 5 by the algorithm of Eisenberg et al. , supra, may be used to predict the protein regions that are located on the outside of the cell membrane. These protein regions may be produced either by chemical synthesis or by expression in the appropriate vector systems, and may be used to raise antibodies against cells that express the mdrl gene product, as described in Example 11. Example 11 The recombinant plas ids pMDRl and pMDR2, as well as different individual fragments of recombinant plasmids pHDR4.4 and pHDR4.5, or the latter plasmids as a whole, or cDNA clones λHDR5, λHDRlO, λHDR62, λHDR28 and λHDR69, or other sequences according to the present invention, may be used as diagnostic tools for detection of human tumor cells resistant to chemotherapeutic drugs. These plasmids may be labeled directly with a radioactive isotope, according to the procedures of Rigby et al., Mol. Biol., 113, 237-251 (1977) or Feinberg et al., Anal. Biochem., 132, 6-13 (1983), for example. Alternatively, the plasmids may be labelled with a non-radioactive chemical tag, for example, according to the procedure in Leary et al., Proc. Natl. Acad. Sci. (USA), 8_0, 4045-4049 (1983). The plasmids may also be used to direct synthesis of labeled RNA probes [e.g., according to the procedure in Melton et al., Nucleic Acids Res. , 12, 7035-7055 (1984)]. The labeled probes may then be used to detect the presence of homologous RNA sequences in tumor cells either by the Northern hybridization procedure [according to Thomas, Proc. Natl. Acad. Sci. (USA), 7_7, 5201-5205 (1980)] or by dot blot or slot blot hydridization [according to Kafatos et al.. Nucleic Acids Res. , 7, 1541-1552 (1979) ' and Brown et al., Mol. Cell. Biol., 3, 1097-1107 (1983)], or by in situ hybridization techniques [e.g., those according to the procedures of Brahic et al., Proc. Natl. Acad. Sci. (USA), 7J5, 6125-6129 (1978)]. It is anticipated that in situ hybridization will provide a particularly sensitive method for detection of a small number (1 in 1000 or fewer) of multidrug-resistant cells within a biopsy. The mdr clones may be used to obtain poly- clonαl or monoclonal [Yelton et al. , Ann. Rev. 3iochem. , 50, 657-580 (1981)] antibodies against mdr gene products using either of two strategies. The first strategy involves determination of the cDNA sequences of mdr genes, as described in Example 10. The cDNA sequence may be used to deduce the corresponding protein sequence. Peptides corresponding to different parts of mdr proteins, and preferably comprising at least 15-20 amino acid residues, may be chemically synthesized by solid-phase methods [Marglin et al., Ann. Rev. Biochem., 39, 841-866 (1970)]. Such peptides may then be used to elicit specific polyclonal and monoclonal antibodies [Lerner, Nature, 299, 592-596 (1982); Niman et al., Proc. Natl. Acad. Sci. (USA), 80, 4949-4953 (1983)]. The availability of the full-length mdrl cDNA sequence, as shown in Table 5, greatly facilitates the design of potentially immunogenic peptides, corresponding to different regions of the mdrl protein, including the potential extracytoplasmiσ domains. The second strategy involves expression of either complete or partial mdr gene products in bacteria, yeast or mammalian expression systems using plasmid, phage or viral expression vectors [Vieira et al.. Gene, 19, 259-268 (1982); Zoung et al. , Proc. Natl. Acad. Sci. (USA), 8_0, 1194-1198 (1983); Bitter et al.. Gene, 3_2, 263-274 (1984); Cepko et al.. Cell, 2_, 1053- 62 (1984); and Gorman et al., Mol. Cell. Biol. 2_, 1044- 1051 (1982)]. The expressed proteins may be purified and used in a vaccine or to raise specific antibodies. Antibodies against the mdr gene products may be used as the alternative diagnostic tools for detection of drug- resistant cells. Finally, such antibodies may potentially be used as a basis for a new strategy of cancer immunotherapy. This strategy may involve, for example, conjugation of anti- dr antibodies with radio¬ active isotopes or chemical toxins in order to speci¬ fically eliminate multidrug-resistant tumor cells. This approach may be particularly efficient if used in combi¬ nation with chemotherapy. Alternatively, the binding of anti-mdr antibodies to cells expressing mdr gene products, even in the absence of antibody-mediated cytotoxicity, may be sufficient to reverse the multidrug-resistant phenotype and may therefore render tumor cells susceptible to the cytocidal action of the chemotherapeutic drugs. In addition, complete or partial mdr gene products may be used as a vaccine to elicit an immune reaction in a patient against multidrug resistant tumor cells. Although the present invention has been des¬ cribed in terms of preferred embodiments, it is under¬ stood that variations and modifications will occur to those skilled in the art upon consideration of the pre- sent invention. Therefore, it is intended that all such equivalent variations and modifications should come within the scope of the invention as claimed.

Claims: WHAT IS CLAIMED IS: 1. An isolated nucleic acid sequence for a human mdr gene associated with multidrug resistance in human cells. 2. A nucleic acid selected from the group consisting of: (a) a nucleic acid comprising a member of the group consisting of: a continuous sequence of nucleotides as set forth in Table 4; a continuous sequence of nucleotides as set forth in Table 5; a continuous sequence of nucleotides as set forth in pHDR4.4 (ATCC 40227); a continuous sequence of nucleotides as set forth in pHDR4.5 (ATCC 40228); a continuous sequence of nucleotides as set forth in pHDR5A (ATCC 67040); a continuous sequence of nucleotides as set forth in pHDR5B (ATCC 67041); a continuous sequence of nucleotides as set forth in pHDRlO (ATCC 67042); and a continuous sequence of nucleotides as set forth in pHDR104 (ATTC 67156); (b) a nucleic acid comprising a nucleotide sequence which hybridizes with at least one of the con¬ tinuous sequences of nucleotides as set forth in (a) or ' which is contained within the same mRNA molecule of human origin or cDNA molecule of human origin as at least one of the continuous sequences of nucleotides as set forth in (a) ; (c) a nucleic acid comprising a nucleotide sequence which hybridizes with any nucleotide sequences described in (b); and (d) nucleic acids comprising a sequence of nucleotides which, but for the degeneracy of the genetic code, would hybridize with at least one of the contin¬ uous sequences of nucleotides as set forth in (a), (b), or (c) . 3. A nucleic acid probe comprising: (a) a nucleic acid comprising a member of the group consisting of: a continuous sequence of nucleotides as set forth in Table 4; a continuous sequence of nucleotides as set forth in Table 5; a continuous sequence of nucleotides as set forth in pHDR4.4 (ATCC 40227); a continuous sequence of nucleotides as set forth in pHDR4.5 (ATCC 40228); a continuous sequence of nucleotides as set forth in pHDR5A (ATCC 67040); a continuous sequence of nucleotides as set forth in pHDRSB (ATCC 67041); a continuous sequence of nucleotides as set forth in pHDRlO (ATCC 67042); and a continuous sequence of nucleotides as set forth in pHDR104 (ATTC 67156); (b) a nucleic acid comprising a nucleotide sequence which hybridizes with at least one of the con¬ tinuous sequences of nucleotides as set forth in (a) or which is contained withi" the same mRNA molecule of ' human origin or cDNA molecule of human origin as at least one of the continuous sequences of nucleotides as set forth in (a) ; (c) a nucleic acid comprising a nucleotide sequence which hybridizes with any nucleotide sequences described in (b); and (d) nucleic acids comprising a sequence of nucleotides which, but for the degeneracy of the genetic code, would hybridize with at least one of the contin¬ uous sequences of nucleotides as set forth in (a), (b), or (c) ; and a label associated with said polynucleotide. 4. A polypeptide comprising a continuous sequence of amino acids encoded by a nucleic acid selected from the group consisting of: (a) a nucleic acid comprising a member of the group consisting of: a continuous sequence of nucleotides as set forth in Table 4; a continuous sequence of nucleotides as set forth in Table 5; a continuous sequence of nucleotides as set forth in pHDR4.4 (ATCC 40227); a continuous sequence of nucleotides as set forth in pHDR4.5 (ATCC 40228); a continuous sequence of nucleotides as set forth in pHDR5A (ATCC 67040); a continuous sequence of nucleotides as set forth in pHDR5B (ATCC 67041); a continuous sequence of nucleotides as set forth in pHDRlO (ATCC 67042); and a continuous sequence of nucleotides as set forth in pHDR104 (ATTC 67156); (b) a nucleic acid comprising a nucleotide sequence which hybridizes with at least one of the con¬ tinuous sequences of nucleotides as set forth in (a) or which is contained within the same mRNA molecule of human origin or cDNA molecule of human origin as at least one of the continuous sequences of nucleotides as set forth in (a) ; (c) nucleic acids comprising a nucleotide sequence which hybridizes with any nucleotide sequences described in (b); and (d) nucleic acids comprising a sequence of nucleotides which, but for the degeneracy of the genetic code, would hybridize with at least one of the contin- uous sequences of nucleotides as set forth in (a), (b), or (c) . 5. A composition effective as a vaccine or as an antigen for induction of specific antibodies, com¬ prising a polypeptide as recited in claim 4 and a com¬ patible diluent, adjuvant, or carrier. 6. An antibody to a polypeptide comprising a continuous sequence of amino acids encoded by a nucleic acid selected from the group consisting of: (a) a nucleic acid comprising a member of the group consisting of: a continuous sequence of nucleotides as set forth in Table 4; a continuous sequence of nucleotides as set forth in Table 5; a continuous sequence of nucleotides as set forth in pHDR4.4 (ATCC 40227); a continuous sequence of nucleotides as set forth in pHDR4.5 (ATCC 40228); a continuous sequence of nucleotides as set forth in pHDR5A (ATCC 67040); a continuous sequence of nucleotides as set forth in pHDR5B (ATCC 67041); a continuous sequence of nucleotides as set forth in pHDRlO (ATCC 67042); and a continuous sequence of nucleotides as set forth in pHDR104 (ATTC 67156); (b) a nucleic acid comprising a nucleotide sequence which hybridizes with at least one of the con¬ tinuous sequences of nucleotides as set forth in (a) or which is contained within the same mRNA molecule of human origin or cDNA molecule of human origin as at least one of the continuous sequences of nucleotides as set forth in (a) ; ( ) a nucleic acid comprising a nucleotide sequence which hybridizes with any nucleotide sequences described in (b); and (d) nucleic acids comprising a sequence of nucleotides which, but for the degeneracy of the genetic code, would hybridize with at least one of the con in- uous sequences of nucleotides as set forth in (a), (b), or (c) . 7. A diagnostic reagent comprising: an antibody to a polypeptide comprising a continuous sequence of amino acids encoded by a nucleic acid selected from the group consisting of: (a) a nucleic acid comprising a member of the group consisting of: a continuous sequence of nucleotides as set forth in Table 4; a continuous sequence of nucleotides as set forth in Table 5; a continuous sequence of nucleotides as set forth in pHDR4.4 (ATCC 40227); a continuous sequence of nucleotides as set forth in pHDR4.5 (ATCC 40228); a continuous sequence of nucleotides as set forth in pHDR5A (ATCC 67040); a continuous sequence of nucleotides as set forth in pHDRSB (ATCC 67041); a continuous sequence of nucleotides as set forth in pHDRlO (ATCC 67042); and a continuous sequence of nucleotides as set forth in pHDR104 (ATTC 67516); (b) a nucleic acid comprising a nucleotide ' sequence which hybridizes with at least one of the con- tinuous sequences of nucleotides as set forth in (a) or which is contained within the same mRNA molecule of human origin or cDNA molecule of human origin as at least one of the continuous sequences of nucleotides as set forth in (a) ; (c) a nucleic acid comprising a nucleotide sequence which hybridizes with any nucleotide sequences described in (b); and (d) nucleic acids comprising a sequence of nucleotides which, but for the degeneracy of the genetic code, would hybridize with at least one of the contin¬ uous sequences of nucleotides as set forth in (a), (b), or (c); and a label associated with said monoclonal anti¬ body. 8. An immunotherapeutic preparation compris¬ ing: an antibody to a polypeptide comprising a continuous sequence of amino acids encoded by a nucleic acid selected from the group consisting of: (a) a nucleic acid comprising a member of the group consisting of: a continuous sequence of nucleotides as set forth in Table 4; a continuous sequence of nucleotides as set forth in Table 5; a continuous sequence of nucleotides as set forth in pHDR4.4 (ATCC 40227); a continuous sequence of nucleotides as set forth in pHDR4.5 (ATCC 40228); a continuous sequence of nucleotides as set forth in pHDRSA (ATCC 67040); a continuous sequence of nucleotides as set forth in pHDR5B (ATCC 67041); a continuous sequence of nucleotides as set forth in pHDRlO (ATCC 67042); and a continuous sequence of nucleotides as set forth in pHDR104 (ATTC 67156); (b) a nucleic acid comprising a nucleotide sequence which hybridizes with at least one of the con¬ tinuous sequences of nucleotides as set forth in (a) or which is contained within the same mRNA molecule of human origin or cDNA molecule of human origin as at least one of the continuous sequences of nucleotides as set forth in (a) ; (c) a nucleic acid comprising a nucleotide sequence which hybridizes with any nucleotide sequences described in (b); and (d) nucleic acids comprising a sequence of nucleotides which, but for the degeneracy of the genetic code, would hybridize with at least one of the contin¬ uous sequences of nucleotides as set forth in (a), (b), or (c). 9. The immunotherapeutic preparation as recited in Claim 8 further comprising a cytocidal agent conjugated with said antibody.

Inventors: GOTTESMAN MICHAEL M, PASTAN IRA H, RONINSON IGOR B, GOTTESMAN, MICHAEL, M., PASTAN, IRA, H., RONINSON, IGOR, B.
Applicants: UNIV ILLINOIS, BOARD OF TRUSTEES OF UNIVERSITY OF ILLINOIS
Application year: 1987
Main classification: C12Q1
IPC codes: A61K38, A61K39, A61K47, C07H21, C07K14, C12N15, C12P21, C12Q1, G01N33