Dr. Tommy C. Douglas

Our defenses against parasitic and infectious diseases rely heavily upon the ability to make specific antibodies. These protective proteins may kill the infectious organisms, physically prevent them from invading tissues, inactivate their toxic products, or kill host cells that have become infected.

The ability to make antibodies is limited to the specific cell lineage known as B lymphocytes. Only these cells can assemble the hundreds of inherited antibody gene segments into the millions of different patterns needed for defense. At a later stage of B lymphocyte differentiation, the assembled genes may be modified through a process of somatic hypermutation which ''fine tunes'' the specificity of the antibody protein for antigen.

The long-term objective of our research is to understand the processes through which the cells of the immune system, and the antibody-forming cells in particular, develop from their progenitors.

We are studying the VH5 family of human antibody heavy chain gene segments, which was originally discovered in cases of familial chronic lymphocytic leukemia. Despite its small size, which makes it favorable for analysis, this gene family can participate in the formation of antibodies against both infectious organisms (HIV-1) and self components (insulin). To permit the study of antibody gene assembly in a highly-simplified genetic context, we utilize ''minilocus'' DNA constructs that contain only small numbers of human V, D, and J antibody heavy chain gene segments. These are stably incorporated into the genome of a recombination-competent B lymphocyte precursor cell line. By manipulating the transcriptional control elements (promoters and enhancers) present on these constructs, we are exploring the influences of transcriptional initiation and elongation on the process of antibody gene assembly. We are also studying the highly unusual patterns of nucleotide insertion that can occur in the VDJ junctions formed from the ''minilocus'' constructs. The sequences of these insertions indicate that the process of antibody gene assembly not only creates diversity, but that it also generates products which are efficient targets for somatic hypermutation.

Our principal experimental methods are ribonuclease protection assay for transcription, polymerase chain reaction for VDJ joining, and DNA sequencing.

Editorial Reviewer:
Biochemical Genetics, Genomics
Journal of Immunology
Journal of Molecular Biology and Evolution

Grant Reviewer:
National Science Foundation, Biological Research Resources Program.

Review Committees:
National Institutes of Health, MBRS Thematic Grant Program Site Visitor.