The purpose of this proposed research is to analyze the molecular mechanisms of somatic DNA recombination in immunoglobulin (Ig) and T-cell receptor (TCR) genes. We will define each individual step of variable (V) region gene rearrangement, and characterize the enzymatic machinary for the recombination. We have identified a pre-B cell nuclear protein which specifically interacts with the recombination signal sequences (RSS's). This protein is assumed to be a DNA binding component of a putative recombinase. In the proposed research, we will purify the RSS binding protein with various chromatography columns including a DNA cellulose (affinity) column. The peptide sequence will be determine for the binding protein, and oligonucleotide probes will be snythesized for the cDNA cloning. A cDNA library from the pre-B cell line, 38B9, will be made with the expression vector, phage lambda gtll. The library will be screened with the oligonucleotide probes to isolate the gene coding for the RSS binding protein. We have identified the TG-endonuclease which introduces a double-strand cut adjacent to the recombination signal, CACTGTG. This nuclease is found in the recombination- competent pre-B cell line, 38B9, and appears to be a good candidate for the cleavage activity of the recombinase. We will purify the TG-endonuclease and isolate the cDNA clone in order to study the structure and expression of the nuclease. We have demonstrated that two sets of recombination signal sequences, CACTGTG and GGTTTTTGT, were sufficient to cause the V-J type of recombination when they were introduced into the recombination competent pre-B cells. With various mutant RSS substrates, we will determine the structural requirements of substrate DNA for the V-J type of recombination. We have characterized the extrachromosomal circular DNA which is an excision product of the gene rearrangement. We will extend our study of the circular DNA to the developmental regulation of the Ig and TCR gene rearrangement. It is hoped that the proposed research will clarify not only the basic process of DNA recombination, but the molecular mechanisms of immuno-deficiencies and leukemogenesis caused by DNA rearrangement.
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