REGULATION OF IMMUNOGLOBULIN GENE EXPRESSION

点击翻译按钮获取中文摘要

When we injected a cloned kappa immunoglobulin gene into Xenopus oocytes, no specific transcription initiating at the kappa promoter was observed. When we coinjected nuclei or nuclear extracts from antibody-producing myeloma cells together with the cloned gene, high level transcription from the kappa promoter occurred. Hence, the cell-type specific factors that activate immunoglobulin transcription in lymphoid cells must be able to function in oocytes, providing an in vivo assay for these factors. We wish to use this oocyte complementation assay to purify the cellular initiation factors for transcription of immunoglobulin genes. Experiments with the in vivo system using suitable plasmid substrates, and later DNA binding studies with the purified activating factors, will allow us to determine whether the factors act on the kappa promotor itself or on the downstream enhancing element or both. Cells of B-lymphocyte type can produce either a membrane-bound or secreted form of IgM antibody, with a program to switch from the first to the second form during cellular development. The heavy chains of the membrane and secreted form are synthesized from different mRNAs transcribed off the same gene, with the two mRNAs utilizing different polyadenylation (poly A) sites. We wish to isolate and study the cellular factors that cause the switch from membrane-bound to secreted antibody during development, evidently by determining which poly A signal is used. For this purpose, nuclei from lymphoid cells at various stages of development will be injected into oocytes together with a cloned Mu heavy chain gene. If the nuclei can be made to alter the polyadenylation pattern of transcripts extending through the gene, we will use this assay to purify the factors involved in selecting the poly A site.