Class switch recombination signals induce lymphocyte-derived Spo11 expression and Spo11 antisense oligonucleotide inhibits class switching.
2001
Abstract Recently, we showed that mouse Spo11 is induced in normal μ(+) B cells by class switch recombination (CSR) stimuli, by RT–PCR using primers based on the reported cDNA sequence of testis-derived Spo11 (test-Spo11) cDNA. In the present study, we first determined the cDNA sequence of lymphocyte-derived Spo11 (lym-Spo11). The 5′ upstream portion had an as yet unreported sequence but the remaining part from exons 2 to 12 and the subsequent 3′UTR was completely identical to that of test-Spo11. RT–PCR analysis indicated that lymphocytes express lym-Spo11 but not test-Spo11. Second, we showed that lym-Spo11 is strongly induced (above eightfold) in the IgA CSR system of LPS-stimulated μ(+)B cells in the presence of all- trans retinoic acid and IL-4. Finally, we examined whether lym-Spo11 antisense S-oligonucleotide (AS) can inhibit CSR reactions in three in vitro CSR systems, IgA ,IgG1, and IgE. Lym-Spo11 AS or the sense oligonucleotide was added to the cultures at the start, and total RNA was extracted after 4 days. IgA, IgG1, and IgE mRNAs (J H C H ) and mature germline C H transcripts (I H C H ) were quantitatively assayed by RT–PCR. AS inhibited J H C H expression dose-dependently. In all three systems, the maximum inhibition by 20 μM AS was in the range of 60 to 90%. Interestingly, I H C H was also inhibited by AS to a similar extent as J H C H . These results suggested that lym-Spo11 plays an important role in the initiation step of CSR.
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