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.

pgp-4, A New Member of the C. elegans P-Glycoprotein Gene Family.

A. Broeks, D.P.E. Satijn, C. Bontekoe, R.H.A. Plasterk

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
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.
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