FIGURE 3:
mak-1 expression pattern, mutations, and MAK-1 antibodies. (A) The
mak-1 gene is expressed in the intestine and body wall muscle. To detect
mak-1-expressing cells, we created transgenic worms expressing GFP from a 6.4-kb segment of genomic DNA upstream of the
mak-1 predicted translational start. GFP was detected in the intestine (left) and body wall muscle cells (right) at the adult stage. Scale bar, 25 um. (B, C)
mak-1 mutation sites and effects on MAK-1 protein expression. (B) Schematic representation of the
mak-1 gene, with boxes denoting exons, and introns denoting introns, the approximate spans of each deletion represented, and the sequence alterations shown below.
mak-1(
ok2987) is a 754-base pair deletion, essentially as depicted on WormBase. We determined that
mak-1(
tm3455) has both a 7-base pair insertion and a 267-base pair deletion extending from the 3' end of exon 3 until the 5' end of intron 4. (C) Location of immunogens used to generate antibodies to MAK-1 and Western blot analysis of wild-type and
mak-1 mutants. Antibodies raised to either an N-terminal or a C-terminal region detect an 60-kDa protein, the size expected for MAK-1. For the anti-N-terminal antibody, note that on short exposure, MAK-1 cannot be detected from the two mutant alleles. On longer exposure, two novel bands are detected from
tm3455, likely representing truncated MAK-1 polypeptides (indicated by arrows). On longer exposure, no protein is detected from
ok2987, other than bands found from all three strains likely arising from cross-reactivity to bacterial products. For the anti-C-terminal antibody, MAK-1 proteins cannot be detected even after long exposure time.