Worm Breeder's Gazette 10(1): 55

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.

Sequencing ama-1 IV

D.McK. Bird and D.L. Riddle

Figure 1

We have sequenced approximately 8.7 kb of genomic DNA, implicated by 
transcript mapping experiments as spanning ama-1.  Removal of 
sequences resembling introns leaves a sequence which translates to a 
continuous open reading frame of 1795 amino acids.  Alignment of this 
deduced sequence with that encoding the large subunit of yeast RNA 
polymerase II (RP02 1 ) indicates that ama-1.  encodes the analogous 
protein in C.  elegans, and that our sequence begins approximately at 
the codon for amino acid 20.  Currently, we are cloning adjacent 
fragments so as to complete the ama-1 sequence.  We have, however, 
made some preliminary observations.  
The ama-1 gene is broken into 11 exons, ranging in size from 109 bp 
to 1589 bp.  The introns generally are large by worm standards.  Most 
are around 300 bp; the largest is 462 bp and the smallest is 49 bp.  
The splice junctions have the consensus: (with underlined bases 
[See Figure 1]
The length of the 3' untranslated region may be extremely short.  
The only appropriate AAUAAA motif is immediately adjacent to the stop 
codon, with a TGTGT box 34 bp further 3'.  We plan to confirm the 
location of the 3' terminus experimentally.  
The homology of the deduced amino acid sequence of ama-1 with that 
of yeast RP021 extends along the whole length of both peptides.  The 
identity (Wilbur and Lipman algorithm) is approximately 50% overall, 
but includes several regions of more than 30 amino acids common to 
both polypeptides.  
The carboxy terminus of ama-1 is composed of a 'tail' of 31 tandem 
repeats of a heptapeptide with consensus: tyr, ser, pro, thr, ser, pro,
ser.  A similar tail is present in the yeast (26 repeats) and mouse (
52 repeats) polypeptides.  
In addition to sequencing the wild-type gene, we have sequenced a 
portion of m118, an EMS-induced alpha-amanitin resistance allele of 
ama-1.  We have detected a single GC->AT change, resulting in a cys -> 
tyr substitution.  When the amino acid sequences are aligned to 
maximize homology, this change falls at a position only 14 bp from the 
site of the base which is altered in a mouse X-amanitin resistant 
mutant cell line (Bartolomei and Corden, Mol.  Cell.  Biol.  7: 586-
594, 1987).  This position is consistent with the genetic fine-
structure map position of the m118 mutation.  We are planning a 
functional test to confirm that this sequence change indeed conveys 
resistance to alpha-amanitin.

Figure 1