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.
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- 134