Worm Breeder's Gazette 9(2): 85
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
The amanitin-binding subunit of RNA polymerase II in C. elegans is encoded by the ama-1 gene, located approximately 0.05 map units to the right of dpy-13 IV. ama-1 was initially defined by the dominant mutation, m118, which conveys resistance to amanitin but has very little effect on RNA polymerase II function in vivo. Using an amanitin-resistant strain as a parent, we have isolated several amanitin-sensitive mutants that carry recessive-lethal ama-1 alleles ( WBG: Vol. 8, No. 3). Of 17 EMS-induced mutants examined thus far, one is arrested late in embryogenesis, seven are early larval lethals, two are mid-larval lethals and six are adult steriles, one of which is temperature-sensitive. One ama-1 mutant is non-lethal, but grows very slowly and has reduced fertility. The most severe ama-1 allele obtained (m252) has an embryonic arrest phenotype. The low frequency of this type of mutation suggests that it is a rare event, and not a simple null allele. It may be a small deficiency. Another EMS-induced mutation, which was originally identified as an embryonic lethal allelic of ama-1, has recently been shown to be a deficiency (mDf9) similar to mDf5 and mDf6 by complementation analysis with other genes in the region. The high frequency of early larval lethals in our collection of ama-1 alleles suggests that these may be null mutations. Supporting this hypothesis is the fact that mutants heteroallelic for an early larval lethal ( m328, m329 or m332) and mDf9 still arrest as first stage larvae. Of five early larval lethal alleles tested thus far, none appear to be suppressed by the amber suppressor sup-7(st5)X.Another common type of ama-1 mutation is the adult sterile. The phenotype of mutants carrying these alleles indicates that they are hypomorphs, producing an altered RNA polymerase II with some residual function. Three alleles have been examined at both 20 C and 25 C and were found to exhibit a more severe phenotype at the higher temperature. At 25 C all of the m235 and m335 mutants arrest as L3 or L4 larvae, whereas m236 mutants are early larval lethals. The best temperature-sensitive ama-1 allele is a leaky adult sterile. Temperature-shift experiments have revealed a temperature-critical period that begins during gonadogenesis and is centered at the initiation of egg laying. The major affect of this mutation at the restrictive temperature is to reduce the number of fertilized eggs produced by mutant hermaphrodites from approximately 66 at 20 C to 13 at 25 C. Approximately 50% of the eggs that are laid at 25 C do not hatch, compared to 10% non-hatching at 20 C. With one exception, the lethal alleles of ama-1 described above were isolated as second-site mutations in a gene carrying the m118 mutation. A fine structure map of ama-1 is being constructed by positioning the various lethal mutations relative to m118. An intragenic recombination event occurring between the lethal (mx) and resistant ( m118) mutations in the germline of a + dpy-13(e184) 8mx) + / unc-17(e113) + + + unc-5(e53) heterozygote will result in an F1 individual that is resistant to amanitin. If the lethal is located to the left of m118 then the recombinant chromosome will carry the unc-17 marker (i.e. unc-17 + + m118 +). Conversely, if the lethal lies to the right of m118 a + dpy-13 m118 + unc-5 recombinant chromosome will be obtained. The strategy for the fine- structure mapping experiments, therefore, is to identify intragenic ama-1 recombinants by selecting for rare hermaphrodites that are resistant to amanitin. Three ama-1 alleles have been positioned in this manner (see the accompanying map). The embryonic lethal allele ( m252) lies to the left of m118, whereas two of the early larval lethals are located to the right. The current size of the ama-1 gene is approximately 0.017 map units. We are now hunting for an unlinked duplication of the ama-1 region. Any donations would be gratefully accepted. [See Figure 1]