Worm Breeder's Gazette 13(4): 29 (October 1, 1994)
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.
Carnegie Institution of Washington, Baltimore, Md 21210
The E. coli gene lacZ (encoding 337-galactosidase) has been used extensively as a reporter to assay gene expression in C. elegans transgenics. Advantages of 337-gal include highly reliable histochemical assays and a lack of endogenous enzyme activity. As we have worked with lacZ vectors, several disadvantages have also become apparent. In particular, patterns of lacZ expression, although generally similar to the endogenous promoter, are often highly mosaic: even in integrated lines, fusion transgenes tend to express stochastically in varying fractions of cells from the target tissue. In addition, no lacZ fusion construct to date produces 337-gal in the germline or in pre l2 -cellembryos (e.g., Hope, Dev. 113,399; Seydoux & Fire wbg 12#4, 20). In seeking to improve the utility of lacZ vectors, we were guided by results from Geraldine Seydoux and Pete Okkema: they found that several different lacZ fusion transcripts had a strong tendency to accumulate in the nucleus (often at the putative sites of transcription) while the corresponding endogenous messages were efficiently transported to the cytoplasm (e.g., Seydoux & Fire wbg 13#3, 33). One potential difference between lacZ transcripts and equivalent endogenous messages was the long intron-less coding region of lacZ . Consistent with this possibility, we had found several years ago (Okkema et al., Genetics 135, 385) that a single intron near the 5' end of lacZ could stimulate expression from some promoters. We therefore attempted to construct a vector that was interrupted within the lacZ coding region by multiple introns. In doing so, it was important that the final spliced transcript retain the original lacZ coding sequence. To achieve this, we synthesized a degenerate double stranded "transposon" population with consensus splice junctions abutting the blunt ends: GTAAGTTTAAAC*******TACTAACTAAC*******ATTTAAATTTTCAG CATTCAAATTTG*******ATGATTGATTG*******TAAATTTAAAAGTC where *s are degenerate bases (34%GC+67%AT) This was cloned sequentially into blunt-end restriction sites in the lacZ coding region. Surprisingly every insertion site so far used in lacZ (also in gfp) appears to allow splicing out of the transposon oligonucleotide (i.e., the resulting transcript produces active protein). Although we've constructed functional lacZ genes with one to eight introns interspersed through the transcript, most of our results to date have been with a five intron-lacZ driven by a variety of different promoters (these are compared to constructs with just a single intron upstream of the lacZ coding region). With weak and moderately strong promoters ( skn-1 glp-1 , cey-2 , hlh-1 ), and with minimal segments from unc-54 , myo-3 ,and myo-2 ,the additional introns cause a dramatic increase in both the frequency and intensity of somatic expression. Stimulation is seen in both F1 assays (1-2 orders of magnitude) and transgenic lines (not quantitated by probably comparable). Intron-stimulation was somewhat less dramatic starting with already-strong promoters; these may already be saturating enzyme assays or ability of cells to make product. For both strong and weak promoters, we saw no changes in overall tissue specificity following intron insertion [but note that all aspects of expression are stimulated, including putative low level 'background' (e.g., glp-1 in gut)]. We hypothesize that the ability of inserted sequences to stimulate expression reflects post-transcriptional effects, perhaps advantages in transport or utilization of mRNAs formed by multiple splicing events. [Note that the inserted intron sequences are unlikely to be working as transcriptional enhancers, since an "inactive promoter segment" (which can activate in response to many enhancers) was still inactive when driving the five-intron-containing lacZ .] We are currently addressing several questions using these vectors: 1) Do added introns decrease the tendency of lacZ transcripts to be retained in the nucleus? 2) Do intron-containing vectors allow reliable expression in the germline or very early embryonic lineages? At this point, several conclusions can be drawn from these adventures. First, the intron containing lacZ constructs will be useful as increased efficiency reporter-fusion vectors for assaying gene expression. Second, expression of heterologous (and of homologous) coding regions in C. elegans may be dramatically improved by inclusion or insertion of introns. Third, the ability of almost any sequence within a gene to act as a site for intron residence suggests that evolutionary insertion of introns could have occurred by dispersion of a blunt-end-splice-junction transposon similar to the one that we have constructed by design.