A large number of in vitro experiments have confirmed that DNA molecules can form i-motif advanced structure when multiple cytosines exist in the sequence. However, whether these structures are present in vivo environment still lacks sufficient experimental evidence. In this paper, we report the in vivo visualization of i-motif structures in the nuclei and chromosomes of the testis of the invertebrate Bombyx mori using immunofluorescence staining with an antibody specifically recognizing the endogenous transcription factor BmILF, which binds i-motif structure with high specificity. The number of i-motif structures observed in the genome increased when the pH was changed from basic to acidic and decreased under treatment with an i-motif inhibitor, the porphyrin compound TMPyP4. The pH change affected the transcription of genes that contain i-motif sequences. Moreover, there were more i-motif structures observed in the testis cells in interphase than in any other cell cycle stage. In this study, the i-motif structures in invertebrates were detected for the first time at the cell and organ levels. The formation of the structures depended on cell cycle and pH and affected gene expression.

中文翻译：

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家蚕睾丸中i-基序DNA二级结构的体内可视化

大量的体外实验已经证实，当序列中存在多个胞嘧啶时，DNA分子可以形成i-motif高级结构。但是，这些结构是否存在于体内环境仍缺乏足够的实验证据。在本文中，我们报告了使用免疫荧光染色和特异性识别内源性转录因子BmILF的抗体进行的免疫荧光染色，对无脊椎动物Bombyx mori睾丸的核和染色体中i-motif结构进行了体内可视化，特异性。当pH从碱性改变为酸性时，基因组中观察到的i-基序结构数目增加，而在用i-基序抑制剂卟啉化合物TMPyP4处理时，i-基序结构的数目减少。pH值变化会影响包含i-基序序列的基因的转录。此外，在相间的睾丸细胞中观察到的i-基序结构比在任何其他细胞周期阶段都多。在这项研究中，首次在细胞和器官水平检测到无脊椎动物的i-基序结构。结构的形成取决于细胞周期和pH以及受影响的基因表达。