Fine-structure genetics of ama-1, an essential gene encoding the amanitin-binding subunit of RNA polymerase II in Caenorhabditis elegans.

Abstract

A fine-structure genetic map has been constructed for ama-1 IV, an essential gene in Caenorhabditis elegans encoding the amanitin-binding subunit of RNA polymerase II. Sixteen EMS-induced recessive-lethal mutations have been positioned in the gene by determining their intragenic recombination frequencies with m118, a mutation that confers dominant resistance to alpha-amanitin. The 16 mutants, all isolated in the ama-1(m118) background, include 13 that are early larval lethals, and three that are mid-larval lethals, at 25 degrees. Six of the mutants exhibit temperature-dependence in the severity of their phenotype. Intragenic recombination between the lethal site and the parental resistance mutation was detected by means of resistance to amanitin. Recombinants were detected at frequencies as low as 2 X 10(-6). The segregation of the closely linked flanking markers, unc-17 and unc-5, revealed whether the lethal mutation was to the left or the right of m118. By adding the distances between the extreme left and right mutations, the ama-1 gene is estimated to be 0.011 map unit long, with m118 positioned 0.004 map unit from the left-most lethal mutation. To order the lethal mutations with respect to each other, viable heteroallelic strains were constructed using the free duplication, mDp1[unc-17(e113) dpy-13(+) ama-1(+)]. The heteroallelic strains were sensitive to amanitin, and recombination events between the lethal mutations were specifically selected by means of the dominant amanitin resistance encoded on the recombinant chromosome. The segregation of outside markers revealed the left-right order of the lethal mutations. The position of mutations within the gene is nonrandom. Functional domains of the ama-1 gene indicated by the various lethal phenotypes are discussed.