Worm Breeder's Gazette 13(2): 99 (February 1, 1994)
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.
gum-1 I: gum-1 ( q373 ),cropped up spontaneously in a Bristol stock. q373 is recessive and causes large (³40 µm), transparent vesicles to accumulate in the intestine of both males and hermaphrodites. Otherwise, the animals appear quite happy (although they are slightly dumpy). Three factor mapping puts q373 just to the right of bli-4 (0.1-0.2 cM).
In order to identify additional alleles of gum-1 ,we did a noncomplementation screen: Mutagenized N2 males were crossed with gum-1 ( q373 ) unc-13 ( e51 )hermaphrodites. F1 progeny were screened for Gum-non-Uncs. By screening ³1 x10 +E4 F1 s,we recovered an additional allele, dx2 .At 25°, dx2 homozygotes derived from a heterozygous mother are viable and Gum; however, the progeny of these animals succumb during the early larval stages. At 20°, dx2 homozygotes are Gum, viable and fertile, but not extremely healthy.
It is possible that gum-1 is equivalent to one of let loci previously identified by Ann Rose's lab. dx2 / hDf8 has a sterile Gum phenotype at 20°. We are in the process of testing candidate sterile let mutations for allelism. Thank you Ann Rose, Mark Edgley and Theresa Stiernagle for providing hDf8 and other things in the region.
gum( dx4 )V: The phenotype of dx4 is very similar to that of gum-1 ( q373 ).Initial mapping has P1 aced dx4 on the left arm of V, with good linkage to dpy-1 .We are screening for new alleles by noncomplementation. Thanks to Susan Mango for dx4 .
gum( oz124 )X?: oz124 has a Gum phenotype similar to that of gum-1 ( q373 ).However, oz140 is dominant, and it is not expressed in males. We have tentatively mapped oz124 to the X. Thank you Tim Schedl for providing oz124 .
As a first step toward figuring out which compartment is represented by the large vesicles in these gum mutants, we tried soaking live worms in two different vital dyes:
1) CDCFDA (Molecular Probes). This dye is a lysosome/vacuole-specific marker in yeast. N2 hermaphrodites that have been soaked in 50 µM CDCFDA for a few hours exhibit bright staining of variously sized vesicles in the gut. The staining pattern is very similar to that of the endogenous gut "granules". However, l) the fluorescence is much brighter than in untreated animals, 2) intense blue-light (fluorescein filter) illumination initiates lysis of the vesicles within about 5 seconds, 3) the staining is bright in the most anterior intestinal cells, which have only a low level of background gut granule staining, and 4) in some animals, embryos within the uterus exhibit fluorescent vesicles in all blastomeres. We still aren't certain that CDCFDA identifies lysosome in C. elegans; however, the dye does not localize to the large vesicles in the gum mutants.
2) NBD-ceramide (Molecular Probes). This dye becomes localized to the Golgi in many types of cultured cells. Overnight soaking of worms in 12 µM NBD-ceramide resulted in moderate staining of a heterogeneously-sized population of vesicles. Again, the pattern is similar to that of the gut granules, but somewhat brighter and the vesicles are light-sensitive. No staining is seen in embryos. It is possible that the Golgi is actually stained by this dye, since (in some animals) the vesicles stained in the anterior-most intestinal cells are loosely clustered in the vicinity of the nucleus. NBD-ceramide does not accumulate in the large vesicles of any of the gum mutants.
We are currently testing other staining methods etc. to determine vesicle identity.