Worm Breeder's Gazette 11(1): 25

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 from Extrachromosomal Arrays

M. Halligan, X.-Y. Huang, D. Stinchcomb and D. Hirsh

Figure 1

We are microinjecting altered SL1 sequences into worms in order to 
investigate the sequences required for splicing the C.  elegans 
spliced leader (SL1) RNA onto the 5' end of endogenous mRNAs.  The 1 
kilobase pair BamHI-generated fragment encoding both the SL1 and 5S 
RNAs was mutagenized in vitro to produce the SL1-6G7G sequence shown 

[See Figure 1]
A plasmid bearing the 1 kb fragment containing this altered SL1 
sequence was coinjected with phage DNA containing the unc-31 gene (
generously provided by R.  Hoskins) into unc-31(e928) animals.  A Non-
Unc line was established that segregated approximately 60% Non-Unc and 
40% Unc animals per generation.  Results of hybridization analysis of 
DNA purified from this line were consistent with the presence of 
lambda, plasmid and SL1-6G7G sequences.  An oligonucleotide 
complementary to SL1-6G7G was used to probe RNA isolated from the 
transformed line, from Unc segregants of the transformant, and from 
wild-type animals.  The oligonucleotide hybridized to a heterogeneous 
smear of RNA from the transformed line, but failed to hybridize to RNA 
from Unc segregants or wild type worms.  This heterogeneous smear of 
RNA from the transformants did not hybridize to vector sequence probes 
or to 5S RNA probes, so it does not represent run-off transcription 
from the extrachromosomal DNA.  These data suggest that the mutated 
SL1 DNA is being faithfully transcribed and spliced onto a 
heterogeneous collection of endogenous mRNAs.  We are presently 
attempting to clone some of the mRNAs containing SL1-6G7G to determine 
if the normal SL1 specificity has been retained.  We are mutagenizing 
SL1 further to determine the sequence and secondary structure 
requirements for trans-splicing in vivo.

Figure 1