Mutational analysis of a vitellogenin promoter in vivo.

摘要

Gene regulation involves the recognition of specific DNA sequences by cell specific factors which affect gene expression. This thesis contains a study of vitellogenin (vit) gene regulation in the worm, Caenorhabditis elegans. The vit genes are expressed only in the adult, hermaphrodite intestinal cells. Two highly conserved sequences, vit promoter elements 1 and 2 (VPE1 and VPE2), were identified in the 5;I created transgenic worms which contained modifications in the VPEs or deletions of vit DNA linked to a marker gene and determined how these alterations affected regulation of the marker gene in vivo.;By using transgenic worms, I demonstrated experimentally that these sequences are sites for activator function, since removal or mutation of VPE1 and VPE2 resulted in a loss or reduction of marker gene expression. VPE1 is repeated five times in the vit-2 regulatory DNA, but only one VPE1 was found to be required for gene activity. The other VPE1's could be individually dispensed with. One VPE2 was absolutely required for gene activity, while the other one was necessary for high level gene expression. When I added a synthetic VPE2 in either orientation to the vit-2 regulatory DNA, only small effects were produced on the regulation of the marker gene when analyzed in transgenic worms. I concluded that there may be additional important regulatory sequences not yet identified. When I added a synthetic VPE1 in one orientation to the vit-2 regulatory DNA, a small reduction or no change in marker gene expression was seen. When VPE1 was added in the opposite orientation, marker gene expression was barely detectable, which suggested that this somehow poisoned the transcription complex.