Worm Breeder's Gazette 8(3): 93
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 have constructed fusions of the col-1 gene from the worm with the E. coli -glucuronidase gene, introduced them into the germline of the worm, and studied their expression. The fusions consist of the promoter region and the first several nucleotides of the col-1 coding sequence, the entire coding sequence of the bacterial -glucuronidase gene, col-1 sequences encoding its 3' intron and the terminator region. The fusions were either in frame or out of frame with the col-1 initiator codon. These constructions were injected into the gonad of gus-1(DH408) worms and detected in the progeny by dot blot hybridization. DH408 is a strain with no detectable -glucuronidase activity, and is used to increase the sensitivity with which we can measure the chimeric activity (Bazzicallupo, Jefferson and Hirsh, unpubl.). -glucuronidase activity was measured in extracts of the transformed worms. DH408 transformed with the in-frame fusion showed glucuronidase activity, while two independent transformed lines carrying the out-of-frame fusion showed no detectable activity. As the DNA was in the long extrachromosomal tandem array, we could use its characteristic instability to perform a cosegregation analysis. There was always a strict correlation between the presence of the transforming DNA and the glucuronidase activity. We verified that the glucuronidase activity in extracts from the transformed line was due to the bacterial enzyme by using affinity purified antibodies directed against the purified bacterial glucuronidase. col-1 has been shown to be transcribed primarily in the embryo ( presumably during the synthesis of the L1 cuticle) but also at lower levels in later stages (Kramer, Cox and Hirsh, in press). To see if the col-1: glucuronidase fusion was being regulated temporally, we prepared extracts from staged populations of transformed worms and measured glucuronidase specific activity. Specific activity is highest in eggs, and decreases markedly in later stages. These experiments are being repeated, but although they are consistent with the wild-type col-1 transcription profiles, they are not sufficient to indicate temporal regulation. To analyze spatial distribution of the glucuronidase activity, we have used enzyme histochemistry and immuno- cytochemical techniques. Although we can visualize activity in eggs using the histochemical stains, we have not yet achieved the resolution to say anything about localization. These experiments are in progress.