GOALS


The Lecom Developmental Gene Expression Laboratory investigates the control of gene expression in mammalian cells and the consequences of altered gene expression. The effects of ethanol on gene expression in liver cells are of primary interest at the moment. When exposed to ethanol, the liver increases expression of mitochondrial aspartate aminotransferase, and the increase in the amount of this enzymatic activity in serum is a clinical marker for alcoholic liver disease. Interestingly, this protein also serves a second function in the body, facilitating fatty acid uptake into cells. Another hallmark of alcoholic liver disease is that fat accumulates in liver cells, causing hepatic steatosis. As one gene may be involved in both functions, we are concentrating our efforts on this particular gene. The questions of how ethanol increases expression of the gene, and how it functions in fatty acid uptake, are being investigated.

The promoter of the gene is being examined to determine where ethanol responsive elements may reside using human hepatoma cells transfected with reporter constructs. Once specific regions are identified, standard techniques such as DNA Footprinting and electrophoretic mobility shift assays will be employed to confirm the findings. Newer methods for identifying transcription factor binding sites will also be employed as they are becoming available, such as array-based techniques. In related studies, the putative fatty acid binding site has been identified. Mutagenesis of key residues has suggested that fatty acid binding does occur in this region. Transfection of expression constructs into fibroblasts show that wild-type protein expression increases fatty acid uptake, while mutated constructs do not. The contribution of various amino acids to fatty acid binding and uptake will be further assessed. New methods of measuring fatty acid uptake will be employed, such as uptake of fluorescent analogs using our new fluorescent-luminescent plate reader. Further mutants will be prepared and studied, to further refine the parameters of fatty acid binding.

Other areas that will be investigated involve the early development of mammals and development of the germ cells. Observations made in previous studies may allow for new and simple methods of tracing early germ cell development and delineation of specific structures during spermatogenesis. Advanced embryo culture techniques are also under investigation.