Gene expression is precisely regulated by networks of transcription factors that modulate rates of transcriptional by binding enhancers, which integrate spatial and temporal patterning information to specify the tissue fate of a group of cells. Although changes in developmental gene expression play an important role in morphological evolution, the molecular basis for such changes is poorly understood. To characterize the evolutionary dynamics of regulatory sequences and constraints on their function, I constructed and screened fosmid libraries from 12 Acalyptrate fly species and sequenced regions containing important developmental genes. My analyses and functional tests of regulatory regions from these loci are beginning to reveal the constraints on enhancers during evolution.;I analyzed a set of enhancers from the eve locus in six sepsid species, in which I showed that the trans-regulatory network governing eve expression was conserved. RNA expression patterns driven by the sepsid enhancers I identified largely overlap the endogenous D. melanogaster eve pattern despite the nearly complete absence of primary sequence or transcription factor binding site conservation, demonstrating that the constraints on enhancer organization are fairly loose. Strikingly, each eve enhancer contains a small number of highly-conserved elements that overlap known functional sites in D. melanogaster and are enriched for pairs of overlapping or adjacent binding sites, suggesting that functional constraint acts on a larger unit than a single binding site.;The identification of regulatory sequences in animal genomes is still a significant challenge for computational and experimental genomics. Comparative genomic methods that have worked well in vertebrates -- using conservation to predict regulatory function -- have been less successful in Drosophila and other invertebrates. I applied these methods to three species of true fruit flies (Tephritidae) with genomes four to five times larger than D. melanogaster. I showed that there are many discrete non-coding sequences conserved between species and that most have regulatory activity. The success of these methods in tephritids is due to the presence of additional rapidly evolving sequence between functional elements in the larger tephritid genomes. Mapping these conserved elements to the D. melanogaster genome clearly and accurately predicts known regulatory elements using only primary sequence data.
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