Worm Breeder's Gazette 10(2): 100
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 previously reported on three stable transgenic lines carrying a novel gene in which an 8 kb fragment of vit-2, including 4 kb of 5' flanking DNA, is fused to a 1.5 kb fragment from the 3' end of vit-6. This gene encodes a 155 kd polypeptide that is produced in parallel with the endogenous vitellogenins exclusively in the adult hermaphrodite intestine. In order to functionally define the vit-2 promoter, we have been injecting the above fusion gene, but with less 5' flanking DNA. We now report on the isolation of several lines containing less 5' flanking DNA, as well as additional lines carrying the original fusion gene. Altogether we have obtained 7 strains with 4 kb of 5' flanking DNA, 1 strain with 1.3 kb, and 7 strains with only 248 bp of upstream DNA. Eight of these strains are viable as homozygotes. All strains contain either a single copy or low copy number tandem arrays of the injected plasmid, each integrated into a single chromosomal location. All strains except two express the 155 kd polypeptide. The two non-expressing strains contain only rearranged copies of the fusion gene. Expression levels roughly correlate with copy number, and the fusion genes are expressed at levels similar to those of the endogenous genes. Thus we see no evidence for gross position effects in any of our 15 lines. Sequence analysis of the vit genes 5' flanking DNA revealed the presence of two repeated heptameric sequences (Box 1 and Box 2) within the first 200 bp upstream of each of the genes. Sequences further from the genes were found to be very A+T rich, but otherwise unrelated to each other. The 5' flanking DNA sequences of the C. briggsae vit genes were quite similar to those of C. elegans in that the Box 1 and Box 2 elements were highly conserved while surrounding DNA was not. Thus we were especially interested in the mode of regulation of the fusion gene containing only 248 bp of 5' flanking DNA. We have now analyzed in detail a line carrying a single copy of the fusion gene integrated by a crossover within the plasmid (pAST 18a) sequences such that the sup-7 gene was upstream of the fusion gene and oriented in the same direction. In this strain we found expression of the fusion gene to be restricted to adult hermaphrodites. In addition, we detected fusion protein synthesis in dissected intestines, but not in heads or gonads. Thus, assuming that the control region is contained within the 5' flanking DNA, these experiments demonstrate that only 248 bp of DNA, containing the Box 1 and Box 2 elements, is required for stage, tissue and sex-specific regulation.