We are interested in
lin-15 because it appears to be necessary for 3 cell fates in vulval development and may act antagonistically to let- 23. Previously isolated alleles of
lin-15 fall into two major classes--those that give a Multivulva (Muv) phenotype (e.g.,
n309,
n765 at 20 C) and those that are either class A or class B 'synthetic' (syn) Multivulva alleles, which only give a Muv phenotype in combination with another syn Muv allele of the opposite class ( Ferguson & Horvitz, 1989). Even though there are at least nine known
lin-15 alleles, none of these appear to be true nulls. In an attempt to isolate null alleles of
lin-15, males carrying
mec-5(
e1340) (a closely linked marker) were mutagenized with EMS and mated to
unc-3(
e151)
lin-15(
n765) hermaphrodites to screen for new mutations that failed to complement
n765 for the Muv phenotype at 15 C. At 15 C,
lin-15(
n765)/mnDf4 (a chromosomal deficiency that removes
lin-15) are 60-80% Muv. The rest of the progeny are phenotypically wild type and no apparent lethality is involved. Since
lin-15(
n765) exhibits maternal rescue such that
n765/+ hermaphrodites have
n765/n765 progeny that are not Muv, males were mutagenized and mated to homozygous
n765 hermaphrodites so that there was no maternal contribution of wild type
lin-15 to rescue the Muv phenotype. Although 35,000 F1 worms were screened, no new
lin-15 alleles were found. This was surprising as we should have been able to detect a null allele, which are normally obtained at a frequency of 1/2000-1/5000 worms, and, from our reconstruction experiment, at least 60% of the
lin-15 null alleles should have been recovered in this screen. Possible explanations for not obtaining the null allele include the null being unobtainable by the point mutations usually caused by EMS, the frequency of
lin-15 mutating to a null being very low, or because
n765/Df is not equivalent to
n765/unc-15(null) because the Df might delete a linked dominant suppressor locus or because the Df deletes sequences involved in dosage compensation since
lin-15(
n765) has been shown to be dosage compensated (Meneely & Wood, 1987). We also have reason to believe that
lin-15 syn muvs may not be exclusively defective in only A and B function. Chip Ferguson demonstrated that
n744(class B)/Df are 40% Muv. We've found that
n309( visible Muv)/n767(class A) and
n765(visible Muv at >20 C)/n767 worms can be Muv at 25 C, suggesting that
n767 at 25 C is not a true A and may retain some B function, consistent with Chip's anecdotal observations. At least three more
lin-15B alleles have been found in mutageneses by various lab members and will be further characterized as to the extent of their B and possibly A defect. One approach we have taken towards cloning
lin-15 has been to use three
lin-15 mutants generated in TR679 that were kindly given to us by Stuart Kim. By using 10 different enzymes, we have identified 8 different Tc1 insertions in the three strains. Five were cloned using inverse PCR and one by construction of a size selected library. We are now mapping these bands by probing YAC grids generously provided by Alan Coulson, and by using the strategy of picking Unc non-Sup recombinants from the
unc-3(
e151) +
sup-10(
n983)/+
lin-15 + heterozygote. This mapping should tell us if any of these Tc1 insertions are tightly linked to
lin-15 and if not, should indicate how close they are to
lin-15 and will provide us with molecular markers correlating the genetic and physical maps in this region.