STUDIES OF THE CONTROL OF IMMUNOGLOBULIN GENE EXPRESSION

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Specialized nucleic acid elements are involved in the expression of antibody 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. Specially 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 approaches for identifying and isolating the transcriptional factors involved in the expression of immunoglobulin genes. These factors and the control of their synthesis during differentiation may serve as prototypes for intracellular regulatory factors involved in other aspects of development. The first proposed approach is biochemical and involves trying to isolate the enhancer recognition protein directly, 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 asked to be stimulated by the addition of a protein fraction 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 being able to construct and introduce into cells a drug resistance gene whose expression is dependent on an active immunoglobulin promoter and enhancer. 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 with the same depth that has been possible with, for example, the lactose and tryptophan operons in bacteria. A deeper goal is to understand how the irreversible regulatory switch is thrown at the start of the differentiation of B lymphocytes, and how this switch is related to the initiation of transformation in B cell myelomas. 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 provide such a basis molecular description of immunoglobulin regulation and provide key insights into the processes of development and differentiation in general.