Worm Breeder's Gazette 12(2): 65b (January 1, 1992)

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.

An expressing vit/lacZ fusion

John Spieth, Kris Lea, Tom Blumenthal

Department of Biology, Indiana University, Bloomington IN 47405

For years we have used an unusual reporter gene composed of a fusion between the 5' end of vit-2 and the 3' end of vit-6 to test vit-2 promoter function in transgenic worms. This construct has enabled us to determine that VPE1 and VPE2 serve as activation sites and that 174 bp of 5' flanking vit-2 DNA is sufficient upstream DNA for high level, correctly regulated expression. In order to determine whether sequences within the gene may also play a role in vit expression, we fused 274 bp of vit-2 5' flanking DNA to one of Andy Fire's lacZ vectors containing a nuclear localization signal and a synthetic intron (Fire et al. 1990. Gene 93:189). Transformants containing this construct in co-arrays with rol-6 did not stain for ß-galactosidase activity and did not produce detectable fusion gene mRNA, as assayed by Northern blots and PCR. Thus it appeared that sequences downstream of the transcription initiation site were required for expression of vit-2 .

In order to localize such sequences we made a shortened vit-2 /6fusion gene by deleting most of the vit-2 portion of the original fusion gene, such that the product is only 31 kD (compared with 155 kD for the original gene). We found that this gene was regulated correctly, so we fused it in-frame to lacZ containing the synthetic intron and nuclear localization signal. We made two versions: one with unmodified vit-2 sequence and one in which the signal sequence was mutagenized. We found that strains carrying either of these genes in extrachromosomal coarrays with rol-6 made abundant fusion mRNA, but that ß-galactosidase activity or protein could be detected only if the signal sequence was eliminated. As expected, the activity was restricted to intestinal nuclei. We are now working on localizing the sequences which permit this gene to be expressed.