Genetic studies have shown that
glp-1 is necessary for at least two regulatory cell-cell interactions during C. elegans development ( Austin and Kimble, 1987; Priess, Schnabel and Schnabel, 1987). As a first step towards understanding the molecular mechanism by which glp- 1 affects these interactions, we have cloned the gene. Our strategy relied on the fact that
glp-1 maps 0.02% to the right of
lin-12. Since the ratio of physical and recombinational distance in this region is about 830 kb/map unit (Greenwald et al. 1987), we estimated that the
glp-1-
lin-12 intergenic distance would be approximately 17kb. Therefore, we screened DNAs from 8 EMS and 2 gamma-ray induced
glp-1 alleles with two hybridization probes: T26E12, a cosmid that carries
lin-12, and ZK506, a cosmid that overlaps and is to the right of T26E12 (both cosmids kindly provided by J. Priess). None of the glp- 1 alleles showed altered restriction fragment patterns when probed with T26E12 but 1/8 EMS and 2/2 gamma-ray induced alleles showed alterations in the same 1.5 kb R1 fragment when probed with ZK506.
q172 has a 300bp deletion within the 1.5 kb R1 fragment, qDf2 has a large deletion with one endpoint in this fragment, and
q339 has a DNA rearrangment where this fragment is replaced by novel fragments of 1.1 and 1.9 kb. At the same time, John Yochem, in Iva Greenwald's lab, had identified a sequence with strong similarity to the
lin-12 sequence (J. Yochem and I. Greenwald, personal communication; see also Yochem and Greenwald, this issue) that was then localized to ZK506 (J. Sulston and A. Coulson, personal communication). Using a phage containing this
lin-12 homologue as a probe for Southern blots of
q172 and qDf2, we were able to determine that the DNA alterations associated with these two mutations lay within the region of similarity to
lin-12. In particular, the 1.5 kb R1 fragment is in a region that corresponds to the extracellular domain of
lin-12 (J. Yochem and I. Greenwald, personal communication). On northerns,
glp-1 probes hybridize to a single abundant transcript of 4.5 kb in the RNA of adult hermaphrodites. This is the same size as the major
lin-12 transcript (J. Yochem and I. Greenwald, personal communication). Which raises the issue of whether crosshybridization is occurring. We believe that we presently have gene specific hybridization but plan to test this using
glp-1 and
lin-12 alleles that produce transcripts of altered size. Comparison of the level of
glp-1 transcript in wildtype hermaphrodites and hermaphrodites where the germ line is absent suggests that the
glp-1 message is located primarily in the germ line. The presence of
glp-1 message in the germ line is consistent with earlier results that indicated that
glp-1 acts in the germ line to control the switch of germ cells from mitosis to meiosis (Austin and Kimble, 1987).