Law R, Kuwabara M D, Briskin M, Fasel N, Hermanson G, Sigman D S, Wall R
Molecular Biology Institute, University of California at Los Angeles 90024.
Proc Natl Acad Sci U S A. 1987 Dec;84(24):9160-4. doi: 10.1073/pnas.84.24.9160.
mRNAs specifying immunoglobulin mu and delta heavy chains are encoded by a single large, complex transcription unit (mu + delta gene). The transcriptional activity of delta gene segments in terminally differentiated, IgM-secreting B lymphocytes is 10-20 times lower than in earlier B-lineage cells expressing delta mRNA. We find that transcription of the mu + delta gene in IgM-secreting murine myeloma cells terminates within a region of 500-1000 nucleotides immediately following the mu membrane (mu m) polyadenylylation site. Transcription decreases only minimally through this region in murine cell lines representative of earlier stages in B-cell development. A DNA fragment containing the mu m polyadenylylation signal gives protein-DNA complexes with different mobilities in gel retardation assays with nuclear extracts from myeloma cells than with nuclear extracts from earlier B-lineage cells. However, using a recently developed "footprinting" procedure in which protein-DNA complexes resolved in gel retardation assays are subjected to nucleolytic cleavage while still in the polyacrylamide gel, we find that the DNA sequences protected by factors from the two cell types are indistinguishable. The factor-binding site on the DNA is located 5' of the mu m polyadenylylation signal AATAAA and includes the 15-nucleotide-long A + T-rich palindrome CTGTAAACAAATGTC. This type of palindromic binding site exhibits orientation-dependent activity consistent with the reported properties of polymerase II termination signals. This binding site is followed by two sets of directly repeated DNA sequences with different helical conformation as revealed by their reactivity with the chemical nuclease 1,10-phenanthroline-copper. The close proximity of these features to the signals for mu m mRNA processing may reflect a linkage of the processes of developmentally regulated mu m polyadenylylation and transcription termination.
编码免疫球蛋白μ和δ重链的mRNA由一个单一的大型复杂转录单元（μ+δ基因）编码。在终末分化的分泌IgM的B淋巴细胞中，δ基因片段的转录活性比表达δmRNA的早期B系细胞低10 - 20倍。我们发现，在分泌IgM的鼠骨髓瘤细胞中，μ+δ基因的转录在μ膜（μm）聚腺苷酸化位点之后立即终止于500 - 1000个核苷酸的区域内。在代表B细胞发育早期阶段的鼠细胞系中，转录通过该区域时仅略有下降。含有μm聚腺苷酸化信号的DNA片段在与骨髓瘤细胞核提取物进行凝胶阻滞分析时，与早期B系细胞核提取物相比，会产生具有不同迁移率的蛋白质-DNA复合物。然而，使用最近开发的一种“足迹法”，即在凝胶阻滞分析中解析的蛋白质-DNA复合物仍在聚丙烯酰胺凝胶中时进行核酸酶切割，我们发现两种细胞类型的因子所保护的DNA序列无法区分。DNA上的因子结合位点位于μm聚腺苷酸化信号AATAAA的5'端，包括15个核苷酸长的富含A + T的回文序列CTGTAAACAAATGTC。这种类型的回文结合位点表现出与聚合酶II终止信号的报道特性一致的方向依赖性活性。该结合位点之后是两组具有不同螺旋构象的直接重复DNA序列，这通过它们与化学核酸酶1,10 - 菲咯啉 - 铜的反应性得以揭示。这些特征与μm mRNA加工信号的紧密接近可能反映了发育调控的μm聚腺苷酸化和转录终止过程之间的联系。
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