2005 Summer
Identification of Transcription Factors That Recognize the A-box, a DNA Sequence Required for Dorsolateral Patterning in Drosophila
Mentor: Angelike Stathopoulos (co-mentor Phoebe Tzou), Caltech (California Institute of Technology), USA
Patterning in the early Drosophila embryo is regulated by transcription factors. These transcription factors act in a sequence specific manner by binding to sites within enhancers or silencers, control regions of genomic DNA that regulate the expression of genes. We have focused on one such motif, ATTCATTCATGA, a conserved sequence which we call the A-box. Our aim was to identify the transcription factor(s) which bind this sequence. The A-box is a novel dorsolateral repression element found within an enhancer for a gene expressed along the dorsoventral axis, the intermediate neuroblast defective (ind) gene. It is necessary to mediate repression of ind expression within dorsal regions. We used a one-hybrid approach to isolate the DNA-binding proteins that recognize this site. At the beginning of this study, cDNA from 0-4 hour’s early (wild type) embryo of Drosophila was prepared and it was tagged with a transcriptional activation domain by insertion into a specific vector (pGADT7-Rec2). By constructing a library from properly staged embryos we hoped to eliminate background and maximize the probability of obtaining true positives. This library was then transformed into yeast strain together with an A-box reporter vector allowing selection based on Histidine auxotrophy (pHIS2 vector with the A-box sequence driving HIS3 expression). We also added an additional screen by creating a second reporter based on lacZ expression. We used the pJL638 vector with A-box sites inserted upstream of lacZ; this vector was integrated into wild-type yeast strain (W303). We screened for library plasmids that bind to the reporter and activate HIS3 as well as lacZ expression. We isolated plasmids from library transformants that passed these two conditions, in order to identify the cDNA insert. We expected to find 1 or 2 transcription factors that bind ATTCATTCATGA with high affinity. This work was important because no other repressor functioning in dorsal regions of the Drosophila early embryo has been characterized to date.
DROSOPHILA PROJECT REPORTS
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