Worm Breeder's Gazette 9(3): 61

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.

Molecular Analysis of ama-1 IV, the Gene Encoding the alpha-amanitin Sensitive Subunit of C. elegans RNA Polymerase II

D.McK. Bird and D.L. Riddle

Figure 1

Figure 2

We have been continuing our analysis of ama-1 IV and envirous, the 
cloning of which has been reported in a previous edition (WBG, 9(1)). 
Using a conventional walking approach, we have expanded the area 
around the region of Drosophila homology to about 29 kb [Sulston and 
Coulson et al. were unable to find a cosmid contig, perhaps as a 
result of the paucity of HindIII sites]. About 20 kb of the ama-1 
region is shown in Figure 1. 
Using restriction fragments spanning this region (bottom line of 
Figure I ), we have, barring any very large introns, delineated the 
approximate extent of the region encoding the 5.9 kb ama-1 transcript. 
Based on hybridization signal strength, ama-1 transcripts are about 
150 times less abundant than unc-54 transcripts in total RNA. 
While doing northerns, we observed that a probe approximately 5 kb 
rightward of ama- I detected an abundant smear of transcripts at 1.2 - 
1.4 kb. Since this resembled the pattern for a collagen probe, we sent 
a flanking sequence to Jim Kramer who very kindly screened the 
collagen gene bank (Cox et al., MCB, 4, 2389, 1984), identifying this 
as the collagen gene in  CG41.  CG41 extends an additional 2 kb more 
rightward than shown in Figure 1. This collagen gene has been assigned 
the gene name col-33.{Figure 
1}
The analogues of ama- I in yeast (RP02 1 ) and mouse have been 
sequenced (Allison et aL, Cell, 42, 599, 1985; M. Bartolemei, personal 
communication). Both were found to encode a C-terminal domain 
comprised of an heptapeptide, with consensus [tyr-ser-pro-thr-ser-pro-
ser], tandemly repeated (26 times in yeast and 52 times in mouse). 
Loss of this domain during preparation of the pol II enzyme from yeast 
cells results in a characteristic 190 k degradation product, which is 
also observed for C. M. Golomb, personal 
communication), suggesting that worms may also have this strange tail. 

To test this, we have begun DNA sequencing the region that we 
suspect encodes the C-terminus of the ama- I product. Although 
preliminary, we have detected regions of strong homology between the 
ama- I sequence and the heptapeptide tail of RP02 1 (Figure 2), 
thereby orienting the ama- I transcription unit S' - 3' as shown left 
to right in Figure 1. We haven't yet observed the tandem array seen in 
yeast or mouse, although this motif may be present a little further 5',
where hybridization to the Drosophila probe is much stronger (solid 
box in Figure 1). Sequencing in this area is currently in progress. 
{Figure 
2}
We are continuing to walk leftward from ama-1 so as to locate both 
the mDf4 breakpoint and dpy-13 (see Albert and Riddle, this issue).

Figure 1

Figure 2