Genetic analysis of Amanitin resistance in the Nematode Caenorhabditis Elegans
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Mutants resistant to oc-amanitin, a fungal toxin that interferes with messenger RNA production by binding to RNA polymerase II, make possible genetic analyses of RNA polymerase II function. The work reported here involves studies of amanitin resistance in the nematode, Caenorhabditis elegans. The frequency of the amanitin resistance mutation was 3 x 10"6 following EMS mutagenesis. An allele of ama-1, an essential gene encoding the amanitin-binding subunit of RNA polymerase II, was isolated, and the gene was mapped. A recessive-lethal mutation that confers dominant resistance to amanitin defined another gene, ama-2. The ama-2 gene has been mapped to linkage group (LG) V, and the mutant phenotypes have been characterized. Resistance to various levels of amanitin has been tested for several mutant strains carrying mutations in either ama-1 or ama-2. Growth of strains carrying one or two copies of m l 18m252, the embryonic-lethal allele of ama-1, has been characterized, and the data suggest that this allele encodes a deleterious product that interferes with normal RNA polymerase II function. A fine-structure genetic map has been constructed for ama-1. Sixteen EMS-induced recessive-lethal mutations have been positioned in the gene by determining their intragenic recombination distances from m l 18, the resistance mutation. 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 closely linked flanking markers, tine-17 and unc-5, revealed whether the lethal mutation was to the left or right of m ll8, To order the lethal mutations with respect to each other, viable heteroallelic strains were constructed using the free duplication mDpl [unc-17(el 13) dpy-13(+) ama-l(+) ]. 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 order of the lethal mutations. By adding the distances between the extreme left and right mutations, the ama-1 gene was estimated to be 0.011 map unit long. The position of the mutations within the gene is non-random. Functional domains of the ama-1 gene indicated by the various lethal phenotypes are discussed.
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