Worm Breeder's Gazette 12(1): 28 (September 1, 1991)

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

Cloning and characterization of P-glycoprotein genes of C. elegans

Carsten R. Lincke, Inge The, Marjon van Groenigen, Piet Borst

Division of Molecular Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands (phone +31-20-5122880, FAX +31-20-6172625)

P-glycoproteins (P-gp's) were first identified in mammalian tumor cells as causing multi-drug resistance (MDR) by active drug export (generally large hydrophobic cytotoxic drugs of plant or fungal origin) when overproduced. These evolutionarily well conserved proteins are encoded by small gene families (2 members in human, 3 in mouse and hamster). They are localized in the plasma membrane and belong to a superfamily of ATP-binding proteins (review: Endicott & Ling. Annu. Rev. Biochem. 1989;58:137-171). Their physiological function is unknown; they may play a role in eliminating ubiquitous toxins from the body. Some data suggest that endogenous substances (like small peptide or steroid hormones) might be substrates for P-gp's. We cloned three genes encoding proteins highly similar to mammalian P-gp's from C. elegans by cross-hybridization with human MDR cDNA. These genes, named cepgpA, cepgpB and cepgpC, were physically mapped by A. Coulson to respectively chromosome IV (near unc-31 ),I (near dpy-5 )and X (near lin-14 ),and are transcribed into relatively low abundant mRNAs of 5, 4.5 and 4 kb. Mutant phenotypes have not yet been described. Sequence analysis of genomic and cDNA clones showed that cepgpA covers 8kb and consists of 14 exons. cepgpC is 5kb long, and it is composed of 13 exons. cepgpB has not yet been analyzed in detail. The gene structure of the nematode genes does not resemble that of mammalian P-gp genes, which is highly conserved (27 coding exons). A few intron positions are, however, absolutely conserved between nematodes and mammals (4 in cepgpA; 5 in cepgpC). In contrast, cepgpA and cepgpC share only a single conserved intron position. cepgpA and cepgpB pre-mRNA are trans-spliced to a trans-spliced leader (SL1) at splice acceptor sites only a few nucleotides upstream of the translation initiation codons. The lengths of the predicted proteins are 1320 aa for cepgpA, and 1254 aa for cepgpC, similar to mammalian P-gp's (about 1280 aa). Their hydrophobicity profiles are virtually superimposable on that of mammalian P-gp's, predicting two similar halves, each with 6 transmembrane domains, followed by ATP-binding consensus motifs. The overall similarity/identity with human and mouse P-gp's is about 65/45%, but approaches 100% in the regions around the nucleotide binding domains. Surprisingly, the identity values for the C. elegans P-gp genes compared with each other are only about 50% (~75% in human and mouse). Thus, the intra-species differences in the nematode P-gp genes are much more pronounced than in mammals, possibly indicating stronger divergence in function. We used RNase protection assays to quantitatively examine the expression of cepgp genes (relative to actin-IV mRNA) upon exposure of nematodes to various kinds of chemical and physical stress: sodium arsenite (reported to induce human MDR1 transcription) at sublethal concentrations had no effect on the mRNA levels of any of the P-gp genes. Heat shock led to an about five-fold increase in cepgpA mRNA levels which remained elevated for at least 6 hours after recovery at room temperature and was normal again after 18 hours recovery. cepgpB mRNA levels also increased slightly under these conditions, whereas cepgpC mRNA levels were not clearly affected. No induction of cepgp gene transcription was found upon exposure to actinomycin D or emetine (substrates of mammalian P-gp's). To investigate whether overexpression of cepgp genes can confer drug resistance to nematodes we cultured EMS-mutagenized worms in the presence of emetine or actinomycin D for several months (initial studies with several MDR-related drugs showed that C. elegans is extremely resistant to most of them, generally 1000 times more than mammalian tumor cells) . Non-mutagenized animals exposed to either of the drugs did not respond with detectable changes in expression levels. Prolonged selection of mutagenized worms with emetine, however, yielded lines that had elevated cepgpA and/or cepgpC mRNA levels (2 to 10-fold). These lines had a low level (2 to 4-fold) of resistance to emetine compared to wild-type animals. These results suggest that C. elegans P-gp's share both structural and functional properties with their mammalian counterparts . (We thank Ronald Plasterk for advice and support)