Worm Breeder's Gazette 13(5): 72 (February 1, 1995)
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.
Division of Molecular Biolo3y, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.
P-glycoproteins belong to the superfamily of ATP binding cassette (ABC) transporters. They are evolutionary well conserved and present in organisms ranging from man to bacteria. P-glycoproteins function as membrane bound ATP dependent extrusion pumps and they can cause Multi Drug Resistance (MDR) in mammalian tumor cells by active export of drugs. Substrates are generally large hydrophobic molecules of plant or microbial origin ( reviewed by Gottesman and Pastan, 1993). In C elegans four P-glycoprotein gene homologs are identified; pgp- 1, -2, -3 and 4 (C.R. Lincke et al., 1992). So far pgp-1 and pgp-3 have been analyzed in detail, expression pattem and function have been identified (C.R. Uncke er al., 1993; A. Broeks et al., 1994). The nucleotide sequence determination of pgp-2 is almost finished, the 5' promoter region has to be identified. The pgp4 nucleotide sequence determination has been finished, the genomic DNA covers 4,756 nucleotides from the 5' ATG to the stopcodon. Sequence data are available from the author and will be made available through the EMBL database. Based on the nucleotide sequence we predict it encodes an ATP-binding membrane spanning protein of 1265 amino acids. Comparing the predicted protein sequence of pgp4 with already identified family members we found that PGP4 and PGP3 are 75 percent identical. The identity with PGP1 and the human homolog MDR1 is about 40 percent. The twelve transmembrane domains can be pointed out in homology with PGP3, two well conserved nucleotide binding domains are also present. A high degree of divergence is found at the N-terminal end and at the linker region. pgp4 is localized on the X-chromosome, 2 kb downstream of pgp-3. Using RNAse protection assay and RT PCR a pgp4 transcript could be detected. The determination of stage specific transcription is in progress. The tissue specific expression of pgp4 has been determined using Lac-Z fusion constructs. Two constructs were made. One construct contains 2 kb of 5' sequences (the region in between pgp-3 and pgp-4) the first exon and part of the first intron cloned in pPD 21.28 (Fire et al., 1990). The second construct is the same, except that this construct contains 8 kb 5' sequences including the pgp-3 promoter and coding region. Both constructs gave the same expression pattern: staining was found in only one nucleus in the head region at the site of the terminal bulb of the pharynx. Based on size and shape this is in all likelihood the nucleus of the excretory cell. Removal of the NLS did not result in staining in the excretory cell, apparently expression was too weak to be detected. Staining in the single nucleus could be detected at all stages. Staining was also found in nuclei of late stage embryos (approximately half of all nuclei), which particular cells are involved has not be defined yet. pgp-1 and pgp-3 are both expressed in the apical membrane of the intestinal cells and pgp-3 is also expressed in the apical membrane of the excretory cell. pgp4 was found not to be expressed in the intestinal cells, but only in the excretory cell, this could indicate that pgp4 and pgp-3 have a similar function in this cell and that pop-3 and pop-l have a more similar function in the intestinal cells. To investigate biological function Tc1 transposon insertions have been isolated; one insertion in exon 12 and one insertion in the exon 7 / intron 7 boundary. Both insertion mutants are apparently viable and fertile and thus far we did not find altered resistance to several drugs. A Tc1 induced deletion mutant will be isolated to obtain a null-allele. We will try to determine the role of pgp4 in drug resistance, in protection of the animal against toxins present in environment. references C.R. Lincke et al., (1992) Mol Cell Biol., 228, 701-71 1 C.R. Lincke et al., (1993) EMBO J., 12, 1615-1620 A. Broeks et al., (1994) WBG, 13, #3, 69 A. Fre etal., (1990) Gene, 93, 189-198