Transcription from Recombinationally Activated Immunoglobulin Genes. Specialized nucleic acid elements are involved in the expression of immunoglobulin genes. These elements are 'enhancers' which act positively to promote transcription. The DNA rearrangements that occur during the construction of an antibody gene bring the enhancer near an otherwise weak promoter, thus activating the promoter. Particularly significant is the fact that unlike other enhancers, which are carried on viruses, the immunoglobulin enhancers are tissue specific--they only function in B lymphocytes. This suggests that B cells contain, as a result of their differentiation, proteins which are designed to function as specific transcriptional factors at this site.

This application proposes two general lines of experimentation aimed at identifying and isolating the transcriptional factors involved in the expression of immunoglobulin genes. The first proposed approach is biochemical and involves trying to isolate the enhancer recognition protein directly, for example by assuming that it will specifically bind to enhancer DNA. In addition, an in vitro transcription system will be developed in which messenger RNA synthesis from DNA fragments--containing immunoglobulin genes with or without the enhancer--is tested for stimulation by protein fractions derived from B lymphocytes.

The second general strategy is to seek mutants in the gene which codes for the enhancer recognition protein, and then use the mutants to isolate the gene itself. The fact that the immunoglobulin enhancer works only in B cells has allowed the design of three specific experimental strategies. Each is based on the idea of placing a drug resistance gene (neo) under immunoglobulin enhancer control and introducing the construction into various cells. For example, in B cells which are also caused to contain extra copies of the cloned enhancer DNA sequence, only a mutation to overproduction of the enhancer recognition protein will allow the neo gene to be expressed. This mutation would aid the biochemical studies described above, and potentially also allow the regulatory gene which produces the enhancer recognition protein to be cloned.

With both the protein and nucleic acid components involved in immunoglobulin gene expression in hand, it should be possible to study this example of a regulated eukaryotic gene to the same depth that has been possible with, for example, the lactose and tryptophan operons in bacteria. Ultimately, purification of the enhancer recognition protein and analysis of mutations in the enhancer site that affect the proteins' ability to bind to the DNA will begin to allow a basic molecular description of immunoglobulin regulation and may provide key insights into the processes of development and differentiation in general.

Malaria, DNA Rearrangements and Burkitt Lymphoma. Another experiment related to the recombinational activation of antibody genes attempts to probe the relationship between malaria infection and Burkitt lymphoma, which show a geographic coincidence. By co-cultivation of malaria-infected erythrocytes in vitro with tester mammalian cells, we will test the idea that infected erythrocytes release a clastagenic (chromosome-breaking) compound which could trigger the chromosome translocation associated with Burkitt lymphoma.

R01GM035967

1986-01-01

1991-12-31