Worm Breeder's Gazette 10(2): 134

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.

Analysis of the Polyubiquitin Gene of C. elegans

Roger W. Graham and E. Peter M. Candido

We have cloned the polyubiquitin gene of C.  elegans, which we call 
UbiA, using a yeast ubiquitin probe and recently completed the gene 
sequence.  A number of features are notable about the gene 
organization.  Firstly, there are eleven 228 bp repeat units which are 
tandemly repeated and which each encode monomeric ubiquitin.  All 
eleven repeats of the nematode polyubiquitin gene encode an identical 
amino acid sequence.  This sequence is highly conserved, differing 
from human ubiquitin at only 1 of 76 positions and from yeast and 
plant ubiquitin at only two positions.  A feature of UbiA which so far 
is unique to C.  elegans is that four of the coding repeats contain a 
small intron which interrupts the coding region at an analogous 
position, i.e.  splitting Gly-47 G/GA in each of the first, fourth, 
seventh, and tenth ubiquitin repeats.  The introns are quite similar 
in sequence throughout their length of 50 bp, which suggests that the 
polyubiquitin gene arose by amplification of a tri-ubiquitin cassette. 
The UbiA transcript is trans spliced, much like three of the actin 
transcripts.  There is a 3' splice acceptor sequence six nucleotides 
upstream of the methionine of the first ubiquitin repeat and we have 
been unable to detect any signal on Northern blots with sequences 
further upstream.  S1 analyses with probes spanning the 5' flanking 
region protect only to the 3' splice site and don't reveal a cis 
leader exon.  Primer extension sequencing confirmed the presence of 
the trans splice leader RNA on the mature UbiA mRNA.  The splice 
leader RNA comes from chromosome 5 and UbiA has been mapped to 
chromosome 3 by Donna Albertson, demonstrating that trans splicing can 
occur between loci on different chromosomes (the trans spliced actin 
is also on chromosome 5).  The existence of trans splicing means that 
the start of the UbiA primary transcript lies within the intron 
sequence and as such must be mapped on the unspliced gene product.  
Using S1 mapping and large amounts of RNA (10  g polyA+ RNA) we were 
able to detect the putative start site using long autoradiographic 
exposures.  It maps 455 bp upstream of the 3' splice site.  The 
closest thing to a TATA box is a GAATAA sequence 32 bp upstream of the 
start site.  In the adjacent 500 bp we can find (a) two potential heat 
shock element (HSE) sequences (b) a stretch of cytosine rich sequence (
C15TCC) and (c) a palindrome resembling the binding site for the 
mammalian steroid hormone receptor.  We have also looked at the 
expression of UbiA by Northern blot analysis.  UbiA is constitutive in 
all life cycle stages and is expressed at essentially the same levels 
throughout.  Surprisingly, UbiA doesn't appear to be inducible by heat 
shock (acute or chronic), despite the HSE's in the 5' flanking region. 
We also followed the expression of UbiA from L1 through 
lethargus/moulting and into L2 and saw no apparent regulation of 
transcription during the moulting process.  In the next while we will 
be chasing down the other ubiquitin gene(s) in the worm, as well as 
exploring the chromatin structure of the 5' end of UbiA-