Taking advantage of recently developed techniques for the transfection of DNA into E. histolytica by her mentor, Dr. Singh proposes to conduct fine sequence analyses of the three promoter elements by using mutational and positional approaches.
The candidate, Dr. Upinder Singh, obtained her B.S. in biochemistry from Ohio State University in 1987 and M.D. in 1992, also from Ohio State-Columbus. Dr. Singh has completed a three year intern/residency program at the University of Virginia and is now a first year fellow in the university's competitive infectious diseases program. Dr. Singh's career development plan is largely focused on her becoming an independent research scientist in the field of clinical infectious diseases. To this end, she will devote greater than 75% of her time and effort to supervised experimentation and classwork; the balance of her efforts will be devoted to continuing her clinical experiences and to teaching in an effort to round out her academic foundation in preparation for an independent career. The mentor, William A. Petri, Jr., is Professor of Medicine, Microbiology and Pathology at the University of Virginia. Dr. Petri obtained his Ph.D. in microbiology and M.D. from the University of Virginia in 1980 and 1982, respectively.
The centerpiece of the candidates experimental training is research aimed at elucidating the role of three conserved and novel cis-acting regulatory sequences within the core promoter region of Entamoeba histolytica protein encoding genes. These three elements are GTATTTMA(G/C) at position -30 and MAGMCT between -30 and -14 from the start codon, and ATAGAC+1M within the transcription initiation site. The sequence regions at -30 and +1 differ significantly from consensus TATA and initiator sequences identified in metazoans, and the presence of a third conserved regulatory sequence is unique and may be the target for interaction with novel transcriptional regulatory proteins previously unidentified in other eukaryotes and higher species. Taking advantage of recently developed techniques for the transfection of DNA into E. histolytica by her mentor, Dr. Singh proposes to conduct fine sequence analyses of the three promoter elements by using mutational and positional approaches. Mutations that alter the sequence, position and orientation of the three elements will be produced and stably transfected into amoeba. The effect of the mutation on the level of gene expression as well as on the site of transcription initiation will be determined. The relative dominance and the required consensus sequences of the conserved elements will be identified. Further, analysis of DNA-protein interactions and identification of protein binding sites within the core promoter will be achieved and identification and cloning of proteins which interact with the unique conserved GAAC element will be accomplished.
It is envisioned that successful completion of these studies will provide fundamental information about the role of the core promoter and novel regulatory elements necessary for gene expression in E. histolytica. The research will be conducted under the supervision of Dr. Petri and a mentoring committee.