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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI018790-08
Application #
3128214
Study Section
Immunobiology Study Section (IMB)
Project Start
1982-08-01
Project End
1993-08-31
Budget Start
1989-09-01
Budget End
1990-08-31
Support Year
8
Fiscal Year
1989
Total Cost
Indirect Cost
Name
University of California Berkeley
Department
Type
Schools of Arts and Sciences
DUNS #
094878337
City
Berkeley
State
CA
Country
United States
Zip Code
94704
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Matsuoka, M; Nagawa, F; Okazaki, K et al. (1991) Detection of somatic DNA recombination in the transgenic mouse brain. Science 254:81-6
Shirakata, M; Huppi, K; Usuda, S et al. (1991) HMG1-related DNA-binding protein isolated with V-(D)-J recombination signal probes. Mol Cell Biol 11:4528-36
Yoshida, K; Matsuoka, M; Usuda, S et al. (1990) Immunoglobulin switch circular DNA in the mouse infected with Nippostrongylus brasiliensis: evidence for successive class switching from mu to epsilon via gamma 1. Proc Natl Acad Sci U S A 87:7829-33
Matsuoka, M; Yoshida, K; Maeda, T et al. (1990) Switch circular DNA formed in cytokine-treated mouse splenocytes: evidence for intramolecular DNA deletion in immunoglobulin class switching. Cell 62:135-42
Okazaki, K; Sakano, H (1988) Thymocyte circular DNA excised from T cell receptor alpha-delta gene complex. EMBO J 7:1669-74
Matsuoka, M; Hagiya, M; Hattori, T et al. (1988) Gene rearrangements of T cell receptor beta and gamma chains in HTLV-I infected primary neoplastic T cells. Leukemia 2:84-90
Okazaki, K; Nishikawa, S; Sakano, H (1988) Aberrant immunoglobulin gene rearrangement in scid mouse bone marrow cells. J Immunol 141:1348-52
Okazaki, K; Davis, D D; Sakano, H (1987) T cell receptor beta gene sequences in the circular DNA of thymocyte nuclei: direct evidence for intramolecular DNA deletion in V-D-J joining. Cell 49:477-85

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