Worm Breeder's Gazette 10(1): 95

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Reversion Analysis of Lethal ama-1 Alleles

T.M. Rogalski and D.L. Riddle

The ama-1 IV gene encodes the largest subunit of RNA polymerase II, 
and is defined by dominant, amanitin-resistance mutations (Sanford, 
Golomb and Riddle, J.  Biol.  Chem.  258:12804-12809, 1983) as well as 
recessive-lethal mutations (Rogalski and Riddle, Genetics, in press).  
The region of chromosome IV surrounding ama-1 has been genetically 
characterized, and deficiencies and duplications that include this 
gene have been identified.  We have determined that the null phenotype 
of ama-1 is developmental arrest after hatching from the egg.  Thus, 
there is sufficient maternal RNA polymerase II in C.  elegans to 
complete embryonic development, and de novo RNA polymerase II 
synthesis is not required until the L1 stage.  Nine of the 21 lethal 
ama-1 alleles obtained thus far exhibit the null phenotype at both 20 
C and 25 C.  One unique allele exhibits an embryonic-arrest phenotype 
at 20 C, but an L1-lethal phenotype at 25 C.  This mutant may encode a 
product that interferes with the wildtype maternal RNA polymerase II 
during embryogenesis.  The mutant function appears to be thermolabile, 
since the null phenotype is observed at 25 C.  The eleven remaining 
ama-1 alleles result in partial loss of RNA polymerase II function.  
One of these results in developmental arrest during the L2 stage, 
whereas the others affect fertility in adult hermaphrodites and/or 
embryonic development in their progeny.  All but one of the ama-1 
hypomorphs exhibit a more severe phenotype at 25 C than they do at 20 
C.  Sixteen of the 21 lethal alleles have been positioned in the ama-1 
fine-structure map (see Bullerjahn and Riddle, this issue).
Reversion experiments were performed with 17 of the ama-1 lethal 
alleles described above to identify intragenic revertants and unlinked 
suppressors of these mutations.  Revertants were detected as rare 
amanitin-resistant progeny of mutagenized dpy-13(e184) 
8mx) / nT1[ama-1(+) (IV); +/nT1(V) hermaphrodites, 
where mx represents the lethal mutation.  Normally hermaphrodites of 
the above genotype do not produce amanitin-resistant progeny, since 
they carry one nonfunctional ama-1 allele and one amanitin-sensitive 
allele.  However, a mutation that restored the dominant, amanitin-
resistance phenotype of ama-1(m118) would result in a resistant 
individual.  Also, resistant animals would be obtained if a new 
amanitin-resistance ama-1 mutation were induced on the nT1(IV) 
chromosome.  The nT1 translocation was used in these experiments to 
prevent intragenic recombination between the lethal and m118 mutations.
Fifteen amanitin-resistant revertants were obtained in these 
experiments after screening a total of 2.5x10+E7 animals.  Eight 
carried new amanitin-resistance mutations linked to the nT1 
translocation, whereas the other seven were apparent intragenic 
revertants.  Five of the intragenic revertants were isolated in the 
m118m236 allele, which exhibits a sterile phenotype at 20 C.  The 
m118m367 and m118m370 null mutations were the other two alleles that 
reverted.  Not all of the ama-1 revertants are wild-type in phenotype, 
suggesting that RNA polymerase II function may not be completely 
restored.  Some of these strains should provide altered enzymes 
suitable for biochemical analysis.