Fig. 1. eat-4 cloning. This figure summarizes our strategy for and the results from cloningeat-4, starting with the cosmid ZK512. A, The abilities of ZK512 and its derivatives to rescueeat-4 mutants were tested by germline transformation rescue experiments (right column; for details, see Materials and Methods). A 2.4 kb region of ZK512 in which a restriction-fragment length polymorphism (RFLP) was found in eat-4(ad572) (seeC) is marked by ↔. Circles are used to mark the sites of restriction enzymes that did not interfere with the ability of ZK512 to rescue, whereas crosses are used to mark the sites of enzymes that did appear to interfere. pRE4-4 and pRE4-8 are two plasmid subclones of ZK512. The deduced extent (∼6.9 kb) and location of the minimal eat-4 rescuing activity are indicated. At the bottom, drawn to an expanded scale, the relationships among the cDNA, the minigene construct, theeat-4:: reporter constructs, and the cosmid are illustrated. The GenBank accession number for eat-4 cDNA is AF095787. B, Examples of M3 neurotransmission phenotypes, assayed by electropharyngeogram (EPG) recordings, observed in wild-type, eat-4 mutant, and transgene-carrying eat-4 mutant animals. Examples of M3 transients [one in each EPG record, except that of the eat-4(ky5) animal] are marked by asterisks. C, A Southern blot analysis of genomic DNA isolated from animals of three different genotypes: wild type, eat-4(ad572), and a spontaneous intragenic revertant eat-4(ad572 ad613), digested with the restriction enzymesPstI or HindIII and probed with labeled ZK512 cosmid. In the set of lanes for each restriction enzyme a band (marked by >), apparent in both the wild-type and the revertant lanes, disappears in the eat-4(ad572) lane where a novel, apparently 1.3 kb larger, band (marked by anasterisk) is visible. This apparent insertion found ineat-4(ad572) was deduced by restriction patterns to be in a 2.4 kb region of ZK512, as indicated by ↔ inA.