Worm Breeder's Gazette 10(3): 78

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.

Trans-Splicing in Ascaris

Tim Nilsen, Christine Faser and Karen Bennett

It has been reported that Ascaris RNAs contain a splice leader 
sequence similar to that of Caenorhabditis 
tesh et al., WBG 10-1:67).  When we first 
sent Ascaris des RNA samples to the laboratory of D.
Hirsh, we also tested them with an oligonucleotide corresponding to 
the C.  elegans spliced leader (SL) and found, as his group did, that 
a similar splice leader is present in Ascaris.  Recently one of us (T.
N.) has shown that the same 22 nt leader is present in the parasitic 
nematode causing human lymphatic filariasis, Brugia malayi (P.N.A.S., 
in press).  Because Ascaris offers potential advantages to do 
biochemistry not available in Brugia, we have recently collaborated in 
further characterizing trans-splicing in Ascaris and in cloning the 
Ascaris leader sequence.  As in C.  elegans, we have found by Northern 
blot analysis that a small (~100nt) polyA-RNA species is recognized by 
the spliced leader, and multiple mRNAs are detected when polyA+ RNAs 
are used.  All RNAs tested by Northern analysis including early 
embryonic (<30 cell), larval, oocyte and gut were positive, implying, 
but certainly not proving, no tissue or stage-specificity.  By 
Southern blot at least two enzymes, ScaI and HaeIII, cut genomic DNA 
to an ~1 kb repeat.  The same pattern of digestion was seen using the 
5S ribosomal gene probe, indicating the trans-spliced leader is 
located in the 5S repeat, as found in C.  elegans and in Brugia.  An 
Ascaris genomic library constructed in lambda EMBL4 (Bennett and Ward, 
Dev.  Bio.  (1986) 118:141) was screened with the 22 mer SL 
oligonucleotide and with an oligonucleotide from the Brugia 5S genes.  
Positive lambda clones which contained the 5S gene and the sequence 
leader have been plaque purified; one has been subcloned and sequenced 
in part.  We have found the Ascaris 22nt sequence leader is exactly 
the same as the C.  elegans and Brugia one, with the rest of the small 
RNA sequence divergent.  To determine the percentage of messages that 
contain the spliced leader, we have used polyA+ RNA from a mixed C.  
elegans population and from various Ascaris tissues and developmental 
stages.  With 0.5  g of C.  elegans polyA+ RNA, the SL oligonucleotide 
hybridizes to 10.6% as much RNA as does a labeled oligo dT probe.  In 
the same assay using Ascaris RNAs, only 1.1-2.0% of the messages 
appear to contain the spliced leader in gut, oocyte and larval RNAs.  
While this one experiment implies that fewer messages are trans-
spliced in Ascaris, the results could also be due to Ascaris messages 
having much longer polyA+ tails than C.  elegans.  However, our 
Northern blots also imply fewer Ascaris messages are trans-spliced 
when Ascaris and C.  elegans polyA+ RNAs are compared.