Worm Breeder's Gazette 8(3): 18

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.

A Genetic Analysis of RNA Polymerase II in C. elegans

T.M. Rogalski, C.E. Haase, R.A. Reenan, D.L. Riddle

Figure 1

Sanford, Golomb and Riddle (J.  Biol.  Chem.  258:12804-12809, 1983) 
have identified a gene, ama-1 IV which appears to code for a subunit 
of RNA polymerase II.  This gene is defined by the dominant mutation 
m118 and has been positioned near dpy-13 on LG IV.  Strains carrying 
ama-1(m118) are resistant to alpha-amanitin and produce an altered RNA 
polymerase II enzyme.  To continue the analysis of RNA polymerase in C.
elegans we have (1) positioned ama-1 relative to dpy-13, (2) 
isolated lethal alleles of ama-1 and (3) identified several essential 
genes near ama-1.  
ama-1 was positioned relative to dpy-13 by testing Dpy recombinants 
from dpy-13(e184) + unc-5(e53)/+ 8)+ hermaphrodites 
for resistance to alpha-amanitin.  The results obtained (2/154 Dpy's 
carried m118) place ama-1 approximately 0.03% to the right of dpy-13.  
The dpy-13(e184)ama-1(m118) double mutant constructed above was used 
in a screen to isolate lethal alleles of ama-1.  Our strategy for 
isolating lethal ama-1 alleles was to first isolate lethal mutations 
on a chromosome carrying the dominant alpha-amanitin resistant allele 
m118 linked to dpy-13, and then screen each lethal strain for loss of 
resistance.  This screen assumes that null alleles of ama-1 are lethal 
and that a second mutation in ama-1(m118) eliminating gene function 
would also eliminate resistance to alpha-amanitin in ama-1(m118mx)/+ 
heterozygotes.  Briefly, homozygous dpy-13(e184)ama-1(m118) 
hermaphrodites were mutagenized (0.025M EMS) and then mated to N2 
males.  Strains carrying a lethal mutation on the dpy-13 
me were identified by the absence of fertile, 
Dpy F2 progeny.  A total of 35 lethal strains were isolated after 
screening the progeny of 3243 F1's.  Each lethal strain (genotype: dpy-
13(e184)ama-1(m118)let-?(mx)/+++) was then tested in alpha-amanitin 
wells and six of the strains were found to be sensitive to alpha-
amanitin.  All six mutations define one complementation group and are 
presumably second site mutations in ama-1(m118).  Two-factor 
recombination mapping of one of these mutations placed it 0.05% to the 
right of dpy-13.  These six ama-1 alleles arrest development of the 
dpy-13(e184)ama-1(m118mx) homozygotes at various stages.  Two are 
apparent embryonic lethals, two are late larval lethals, one is a 
temperature-sensitive sterile and the remaining mutation is not lethal 
but grows slowly and produces very few progeny.  We are planning to do 
reversion experiments with these lethal ama-1 alleles in an attempt to 
identify genes coding for other RNA polymerase II subunits, or other 
genes affecting transcription efficiency.  
The remaining 29 lethal mutations that we have isolated define other 
essential genes in this region of LGIV.  Left-right positioning and 
complementation data for 15 of these mutations have identified 6 
essential genes in addition to ama-1.  The positions of these genes (
based on two-factor mapping of one representative allele) are shown on 
the accompanying genetic map.
We have recently isolated a set of gamma-ray-induced lethal 
mutations linked to dpy-13(e184)ama-1(m118), one of which affects ama-
1 as determined by loss of resistance to alpha-amanitin and failure to 
complement one of the ama-1 lethal alleles.  It also fails to 
complement let-276 which is located approximately 0.5-map unit to the 
right of ama-1.[See Figure 1]

Figure 1