Functional Comparative Genomics at the Vertebrate Extremes.

SABINA DOMENE; FLAVIO JUNQUEIRA; VIVIANA BUMASCHNY; SOFIA NASIF; MARCELO RUBINSTEIN

San Miguel de Tucuman, Argentina

Congreso; Sociedad Argentina de Investigacion Bioquimica y Biologia Molecular (SAIB); 2009

SAIB

Until recently, the identification of transcriptional enhancers has relied on tedious deletional studies of non coding flanking regions systematically tested in expression assays. The near completion of several vertebrate genomes has dynamized this task by revealing a myriad of conserved potential cis-acting elements such as the neuron-specific enhancers of the proopiomelanocortin gene (Pomc), nPE1 and nPE2, that we discovered to be conserved across mammals. To study the molecular evolution of Pomc expression we analyzed the ability of putative enhancer sequences to drive reporter gene expression to transgenic animals of distant vertebrate Orders: the mammal Mus musculus (mouse) and the teleost fish Danio rerio (zebrafish). To this end, we set up a transgenic zebrafish facility and determined that mouse nPE1 and nPE2 were able to drive EGFP expression to the hypothalamus of transgenic zebrafish embryos. Using the tol2 transposon system, we injected zebrafish eggs with ptol2-nPE1-nPE2-zpomca-egfp and ptol2-deltanPE1-nPE2-zpomca-egfp. Interestingly, both constructs drove EGFP expression to the pituitary and hypothalamus of zebrafish embryos and EGFP colocalized within Pomc neurons as determined by immunofluorescence against ACTH. These results provide a clear example of functional conservation at the vertebrate extremes despite great sequence divergence.