Training Plan: The goal of this research program is to identify genes involved in somatic hypermutation of Ig genes. The approach is to use subtractive hybridization to identify genes that are specifically expressed in the dark zone of the germinal center, a region touted to be specifically and uniquely involved in the somatic hypermutation process. A scheme is presented to isolate germinal center B cells from human tonsils. The first selection is between sIg +B cells versus sIg- B cells, based on a speculation that hypermutation might be more active in cells which had temporarily down-regulated surface Ig gene expression. The second selection is on the basis of binding to peanut agglutinin, a marker for germinal centers. Dr. Kuo notes that the original separation yielded cells with appropriate morphologies in each population of between 60-70%. Both the Ig+ and the Ig-populations were subjected to subtractive hybridization against B cell lymphomas that do not hypermutate. There was an initial characterization of 50 clones from the Ig-subtracted library and 10 clones from the Ig+ subtracted library. Included in these clones were apparently non-specific abundant RNA, some previously identified B cell sequences and 22 novel sequences. Of the 22, 12 had been established through expressed sequence tags in Genbank and 10 appeared to be completely novel. Northern blots showed that several of the clones appeared to be B cell specific, and others appeared to be preferentially expressed by follicular lymphoma, which contains cells that can undergo hypermutation. In situ hybridization on tonsils showed different patterns of hybridization. Four clones showed a distinct germinal center pattern of expression. These preliminary data are the foundation for this proposal.
The aims i nclude identification of more clones that are specific to PNA+ cells. These will be characterized by Northern blots and in situ hybridization. In particular, Dr. Kuo hopes to identify clones that are more abundantly expressed in follicular lymphoma rather than in intermediate lymphocytic lymphoma. In addition, he hopes to find Dark Zone specific expression in germinal centers. So far, he has identified two clones of 60 initial ones that appear to have this property. Full length cDNA sequences will be obtained and determined. An assay for somatic mutation will be applied to Burkitt's lymphoma cell lines into which candidate genes have been transfected. Antibodies to candidate proteins will be raised.
| Kuo, F C; Sklar, J (1997) Augmented expression of a human gene for 8-oxoguanine DNA glycosylase (MutM) in B lymphocytes of the dark zone in lymph node germinal centers. J Exp Med 186:1547-56 |
