Isolation and characterization of genes specifically expressed at discrete stages of B cell development

Isolation and characterization of genes specifically expressed at discrete stages of B cell development Degree of Doctor of Philosophy, 1998 Y u Zhang Graduate Department of Medical Biophysics in the University of Toronto B cell differentiation involves the coordinate activation and suppression of various genes. Identification of those B lineageand/or developmental stage-specific genes has greatly increased our understanding of this process. However, the genetic program controlling B lymphopoiesis largely remains unresolved. In this thesis, I describe the isolation and characterization of the genes differentially expressed during the transition from pro-B to pre-B cell stages. I used mRNA differential display to isolate genes uniquely present in either IIB4 or 70a3 cells which represent the two stages of interest. In total, nine genes have been identified: three are IIB4-specific and six are 702/3-specific. DNA sequence analysis reveals that all but one of these genes are previously undescribed. Studies of expression pattern demonstrate stage-specificity for all of them. Based on these criteria, three genes were selected for further characterization. 7A3 is specifically expressed by B lineage cells in late developmental stages, including pre-B cells, immature B cells and plasma cells. It encodes a sialic acid-specific 90-acetylesterase. which catalyzes the removal of 9-0-acetyl ester groups from sialic acids present on many glycoconjugates. This enzyme may influence B cell differentiation by regulating the interaction of CD22 with its iigands. 7T9 has an expression pattern similar to that of 7A3 in B lineage cells. It encodes the mouse homologue of human pleckstrin. a major substrate of protein kinase C. This protein may be involved in B cell receptor-mediated signal transduction as suggested by its increased phosphorylation following sIgM aggregation. In addition, it may play a role in further differentiation of pre-B cells since transfection of 702/3 cells with an antisense construct induces NF-KB activity and I ~ K light chain expression. 7G9 is primarily expressed by B cell progenitors. It encodes a precursor protein for a novel tachykinin peptide. The putative peptide resembles substance P both structurally and functionally, raising the possibility that this lymphocyte-specific tachykinin may be the physiological mediator for some immune regulation functions previously ascribed to substance P. Its function in B cell differentiation is not clear. Presumably, it may act as an autocrine growth factor. In summary, I have isolated nine genes differentially expressed at discrete stages of B cell development. Characterization of a selected set of these genes have revealed some novel mechanisms regulating B lymphopoiesis.