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 candidate sexperimental 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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Clinical Investigator Award (CIA) (K08)
Project #
5K08AI001453-04
Application #
2886052
Study Section
Microbiology and Infectious Diseases B Subcommittee (MID)
Program Officer
Fairfield, Alexandra
Project Start
1997-09-01
Project End
2002-08-31
Budget Start
1999-09-01
Budget End
2000-08-31
Support Year
4
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Stanford University
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
800771545
City
Stanford
State
CA
Country
United States
Zip Code
94305
Boothroyd, John C; Blader, Ira; Cleary, Michael et al. (2003) DNA microarrays in parasitology: strengths and limitations. Trends Parasitol 19:470-6
Cleary, Michael D; Singh, Upinder; Blader, Ira J et al. (2002) Toxoplasma gondii asexual development: identification of developmentally regulated genes and distinct patterns of gene expression. Eukaryot Cell 1:329-40
Singh, Upinder; Gilchrist, Carol A; Schaenman, Joanna M et al. (2002) Context-dependent roles of the Entamoeba histolytica core promoter element GAAC in transcriptional activation and protein complex assembly. Mol Biochem Parasitol 120:107-16
Matrajt, Mariana; Donald, Robert G K; Singh, Upinder et al. (2002) Identification and characterization of differentiation mutants in the protozoan parasite Toxoplasma gondii. Mol Microbiol 44:735-47
Singh, Upinder; Brewer, Jeremy L; Boothroyd, John C (2002) Genetic analysis of tachyzoite to bradyzoite differentiation mutants in Toxoplasma gondii reveals a hierarchy of gene induction. Mol Microbiol 44:721-33
Singh, U; Rogers, J B (1998) The novel core promoter element GAAC in the hgl5 gene of Entamoeba histolytica is able to direct a transcription start site independent of TATA or initiator regions. J Biol Chem 273:21663-8