This revised proposal stems from work begun during a FIRST Award to the Applicant. The Investigator will further characterize the cis- and trans-acting factors that are involved in directing splicing of beta-spectrin pre-mRNA along erythroid- specific or non-erythroid pathways. The Investigator has identified the splice acceptor and donor sites for both pathways of pre-mRNA processing. He has established a minigene/transfection assay system that allows him to examine both tissue and developmental specificity of RNA processing. His plan of attack calls for deletional analysis as well as site-directed mutagenesis to approach the problem. He will use the information gained from examination of the cis-acting elements in the splicing process to construct RNA that contains the minimum required amount of sequence to direct splicing correctly. This construct will be used as a probe in the search for trans-acting factors in the system. The methodology will involve gel retardation studies with nuclear extracts from non-erythroid cells and erythroid cells at various stages of maturation. Other studies will involve UV cross-linking of the RNA probe to the transacting factors. The trans-acting factors will be isolated and identified. Previously undescribed factors will then be cloned.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL050462-03
Application #
2415602
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1995-05-01
Project End
1999-04-30
Budget Start
1997-05-01
Budget End
1998-04-30
Support Year
3
Fiscal Year
1997
Total Cost
Indirect Cost
Name
University of Cincinnati
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
City
Cincinnati
State
OH
Country
United States
Zip Code
45221