Worm Breeder's Gazette 10(2): 69
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.
In our initial attempts to isolate mutants that express stage specific surface antigenic determinants at inappropriate stages, we've turned up some mutants with a novel, unexpected phenotype. In immunofluorescence tests, these mutants stain uniformly with a rabbit antiserum that does not stain the surface of wild-type animals. This antiserum was prepared by treating an anti-wild-type cuticle serum (by adsorption with wild-type adults) to remove antibodies capable of binding to the wild-type surface. Thus, antigenic determinants that are not available on the wild-type surface are exposed in the mutants. We describe here isolation and preliminary genetic characterization of the mutants. Mutants were isolated by screening synchronous populations of F2 progeny of animals mutagenized with EMS. Populations were synchronized by refeeding F2 dauer larvae, or by gently washing plates to remove egglaying F1 adult hermaphrodites while leaving F2 eggs stuck in the E. coli lawn. In the latter case, eggs were allowed to hatch for 1-2 hours and then L1's were washed off and transferred to fresh plates for growth. The egg-hatching and transferring steps were usually repeated to obtain a second 'wave' of synchronous F2's. Populations were harvested at the L4 stage or younger and screened with L4-adsorbed antiadult serum (Genetics 117:467 1987) by indirect FITC immunofluorescence. Samples in 100-200 l PBS were spread in thin layers onto Pyrex Petri dishes; up to 10,000 animals per sample were screened at a time. Samples were screened using an epifluorescent stereomicroscope (Leitz-Wild). Immunofluorescent larvae were observed at low frequency; these were picked using a mouth tube and a drawn-out 100 l capillary micropipet. Clones were established and cloned stocks were rescreened by immunofluorescence to eliminate false positives. Viable mutants were obtained at a frequency of about 5x10+E-4 per F1. Of 12 mutants that we have analyzed, 11 fit the pattern described in the first paragraph above; the remaining one produces an incompletely penetrant 'small adult' phenotype that may be of interest, but this is not its story. Initially, we looked at which stages besides adult stained with the L4-adsorbed antibody. Eight of the 11 mutants stained at all stages, while the remaining three stained only at late larval stages. We looked at several of the mutants to see if they expressed adult lateral alae at earlier stages, but surprisingly, found no larvae that carried alae. yj10 is the only mutant in the set that has a readily apparent visible phenotype; it is somewhat scrawny and has a cold-sensitive weak Left Roller phenotype. These results led us to consider that the mutant phenotypes might result from cuticle structural defects rather than, or in addition to, precocious expression of an adult-specific cuticle type. The 11 mutants were then tested for staining with the same parent antiserum after it had been adsorbed with wild-type adults to remove all antibodies capable of reacting with the wild-type surface. All of the mutants were antigen-positive; wild-type was antigen-negative (this is the result summarized in the first paragraph). The 8 mutants that light up at all stages with this serum are termed 'super-bright' in our lab jargon and the other three that exhibit apparent stage-specificity stain much more weakly. We considered the possibility that known apparent cuticle phenotypes like Sqt and Rol might share this phenotype with our new mutants. We obtained a set of ten sqt and rol mutants from Bob Edgar and tested them with adult-adsorbed antibodies. None were antigen positive. Thus our mutants have a new phenotype that is not shared with the classical morphological cuticle mutants we have tested. We have made some progress in analyzing the 'super-bright' mutants genetically. It has been possible to use the difference between wildtype and mutant antigen phenotypes to do linkage, complementation, and mapping using methods similar to those described for mapping of the srf-1 antigenic polymorphism (Genetics paper again). All mutants were backcrossed to wild-type twice and an antigen-positive (mutant) segregant picked after each backcross. Penetrance of the surface antigen phenotype is complete; it is very unusual to find a non- staining individual in a stained population of one of the homozygous mutant stocks. This is in contrast to the srf-1 phenotype which we found to be incompletely penetrant both in wild-type and srf-1 mutant strains. All of the mutant antigen phenotypes are recessive, the best evidence for this is that in crosses with unlinked unc markers, 25% or less of Unc segregants issuing from an unc +/+ srf parent are antigen- positive. Linkage was determined by using unc markers on the autosomes as selected markers and staining for antigen-positive Unc segregants of the double heterozygote. Absence of the double homozygous recombinant in a small population was taken as evidence for linkage. For complementation testing, srf-a/+ males were mated with srf-b hermaphrodites. F1 male progeny were collected and stained. Failure to complement was indicated by presence of antigen-positive heterozygous males. So far there are two complementation groups. At least 5 non-complementing mutations are linked to unc-13(e51)I, and yj10 shows linkage to unc-24(e138)IV. We are presently designating the chromosome I mutations as srf-2 and yj10(IV) as srf-3. We have mapped srf-2(yj262) by 2 and 3 factor crosses; it is on IR in the vicinity of lin-11 and unc-75. A 3 factor cross with these two markers is in progress. Mutant phenotypes were compared serologically by the adsorption method. Antiserum adsorbed with srf-2(yj262) adults binds to yj10 adults but not to yj262 or any of the other srf-2 mutants. The same parent antiserum adsorbed with yj10 adults doesn't bind yj10 or any of the srf-2 mutants. Thus the two complementation groups correspond to two distinct serological phenotypes, and the results fit a model in which the antigens exposed in srf-2 mutants are a subset of those exposed in srf-3(yj10).To summarize, all mutants in this set share the general characteristic of expressing surface antigenic determinants that are not on the wild-type surface. The fact that the antisera were raised against a wild-type cuticle immunogen indicates that the antigens exposed in the mutants are probably wild-type antigens and not novel structures. The fact that the mutant phenotypes are recessive, even though they constitute an apparent gain of antigenicity, suggests that the mutant lesions are defects in cuticle structure that uncover normally hidden antigenic determinants. Our hope is that these mutants and the genes that they identify will contribute to understanding genetic control of the layered organization of the cuticle in a way that is complementary to what can be learned from morphological mutants.