Terminal deoxynucleotidyl transferase is an unusual DNA polymerase that appears to be important in development of the immune system and has a highly conserved protein structure. The enzyme is highly sensitive to proteolytic degradation during isolation and several molecular weight species as well as phosphorylated forms have been discovered. This project will examine primary protein structure and the modifications that occur during cell differentiation. A mouse monoclonal antibody column has been developed, allowing single step isolation of terminal deoxynucleotidyl transferase as the 58,000 dalton or 32,000 dalton form calf thymus gland. The isolation provides material for studies on several important aspects of terminal transferase structure. The enzyme peptides will be used directly for protein sequencing. They will also be used as antigens for development of new monoclonal and polyclonal antibodies of different specificities. The monoclonal antibody columns can be used to study detailed structural aspects of this protein, such as phosphorylation, and other consequences of differentiation that lead to protein degradation. Antibody technology will also be used to isolate TdT mRNA. The mRNA will be reverse transcribed and cloned into plasmids for DNA sequencing and gene regulation studies.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM031393-05
Application #
3279380
Study Section
Biochemistry Study Section (BIO)
Project Start
1983-03-01
Project End
1989-02-28
Budget Start
1987-03-01
Budget End
1989-02-28
Support Year
5
Fiscal Year
1987
Total Cost
Indirect Cost
Name
U.S. Uniformed Services University of Health Science
Department
Type
Schools of Medicine
DUNS #
City
Bethesda
State
MD
Country
United States
Zip Code
20814
Chang, L M; Rafter, E; Rusquet-Valerius, R et al. (1988) Expression and processing of recombinant human terminal transferase in the baculovirus system. J Biol Chem 263:12509-13
Chang, L M; Bollum, F J (1986) Molecular biology of terminal transferase. CRC Crit Rev Biochem 21:27-52
Bollum, F J; Chang, L M (1986) Terminal transferase in normal and leukemic cells. Adv Cancer Res 47:37-61
Peterson, R C; Cheung, L C; Mattaliano, R J et al. (1985) Expression of human terminal deoxynucleotidyl transferase in Escherichia coli. J Biol Chem 260:10495-502