Worm Breeder's Gazette 10(1): 96

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.

Fine-Structure Map of ama-1 IV

A.M.E. Bullerjahn and D.L. Riddle

Figure 1

A fine-structure genetic map has been constructed for ama-1, an 
essential gene 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 
distances from 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 C.  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 2x10+E-6.  The 
segregation of closely linked flanking markers, unc17 and unc-5, 
revealed whether the 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 units long, with m118 
positioned 0.004 map units from the left-most mutation.  To order the 
lethal mutations with respect to each other, viable heteroallelic 
strains were constructed using the free duplication, mDp1[unc17(e113)
dpy-13(+)ama-1(f)(+).  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.  The ama-1 fine-
structure map is linear, and additive, based on data obtained from 
mapping lethal alleles relative to m118, and on data obtained from 
mapping lethal alleles relative to each other (Figure 1).
The distribution of mutations within the gene is non-random.  The 
early larval lethal mutations map throughout the gene.  Arrested 
development and death in the L1 stage is the ama-1 null phenotype.  
Evidence for this comes from the mDf10 deficiency, which conveys an L1-
lethal phenotype in deficiency homozygotes (Rogalski and Riddle, 
Genetics, in press).  Five mutations are clustered very close to the 
resistance mutation, m118, which maps approximately 1/3 of the way 
through the gene from the dpy-13 (left) end.  Molecular analysis has 
shown that the dpy-13 end is the 5' end of the gene (D.  Bird and D.  
Riddle, unpublished).  Six lethal mutations are positioned to the left 
of m118, and six are to the right of the m118 cluster.  Hence, the 
density of mutations is twice as great near the left end of the gene (
encoding the N-terminus of the protein) than it is on the right side.
The amanitin-binding site is highly conserved among divergent 
species, and this region might be essential to the structural 
integrity of the large subunit.  Attempts to purify RNA polymerase II 
from strains heterozygous for the sterile mutations m236 and m235 
revealed amanitin-resistant species that were degraded rapidly during 
the first steps of purification (M.  Golomb, personal communication).  
It is interesting to note that four of the five sterile mutations (
m235, m368, m369, m236, and m396) map very close to m118, also 
suggesting that this region of the large subunit is important in 
enzyme stability.  These mutants all exhibit temperature-sensitive 
phenotypes (larval lethal at 25 C).
[See Figure 1]

Figure 1