Worm Breeder's Gazette 9(2): 85

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Mutations in the ama-1 Gene

T.M. Rogalski, A.M.E. Bullerjahn, and C.L. Ritter

Figure 1

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 
[See Figure 1]

Figure 1