BIOLOGY OF HUMAN RETROTRANSPOSITION

人类逆转录转座生物学

• 批准号: 3748215
• 负责人: G SWERGOLD
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3748215
• 关键词: DNA binding protein; DNA footprinting; chromosome aberrations; computer assisted sequence analysis; developmental genetics; gene expression; genetic enhancer element; germ cell neoplasms; human genetic material tag; hybrid cells; molecular cloning; nonhistone nucleoprotein; nucleic acid sequence; tissue /cell culture; transcription factor; transposon /insertion element

The human LINE-1 (L1Hs) element is a transposon that causes insertional mutagenesis upon transposition into sensitive genomic loci. Examples of both somatic and presumed germ-line insertional mutagenesis events have been reported and new examples are being discovered regularly. About 5% of the human genome is composed of the estimated 4000 full length and 100,000 truncated L1Hs elements that it contains.Expression of L1Hs is highly regulated and occurs mostly in cells of germ line origin. The sequences necessary and sufficient for the appropriate expression of L1Hs are found within the elements 5~ UTR. We have continued our investigation of a 140 bp fragment of the L1Hs 5~ UTR that we previously showed acts as an enhancer in NTera2D1 human teratocarcinoma cells. Partial deletions of this fragment results in partial decrease in activity and partial fragments contain enhancer activity when cloned into heterologous promoters suggesting that the intact fragment contains more than one enhancer sequence. Mutations of a motif within the fragment that we previously showed is footprinted by nuclear proteins from NTera2D1 cells in DNAse I protection experiments results in increased enhancer activity suggesting that this sequence may be acting as a transcriptional silencer. We are continuing to narrow down the regions of DNA that contain the enhancers by deletion analysis. A recent search of the Genbank revealed a strong similarity between a motif in the fragment and a previously unstudied region of the JC virus regulatory region. A related sequence in other genes is known to bind to members of the HMG family of DNA binding proteins. We are testing the hypothesis that an HMG protein family member is acting as a transcriptional regulating factor of L1Hs.During the past year we have continued our efforts to investigate the expression of L1Hs in M28 cells, a somatic cell hybrid line containing a human isochromosome 12p. This abnormal chromosome is a feature of both testicular germ cell tumors and the Pallister-Killian Syndrome. Our studies indicate that the similar but not identical regions of the L1Hs 5~ UTR govern expression of L1Hs in NTera2D1 and M28 cells. Experiments designed to clone the gene responsible for the enhanced expression of L1Hs in M28 cells are under way.

人类 LINE-1 (L1Hs) 元件是一种转座子，可导致插入 转座到敏感基因组位点后发生诱变。的例子 体细胞和推测的种系插入突变事件均已发生 已被报道，并且定期发现新的例子。 关于 人类基因组的 5% 由估计的 4000 个全长和 它包含 100,000 个截断的 L1Hs 元素。L1Hs 的表达式为 受到高度调控，主要发生在种系来源的细胞中。 这 L1Hs 适当表达所必需且充分的序列 存在于元素 5~ UTR 内。 我们继续我们的 对我们之前的 L1Hs 5~ UTR 的 140 bp 片段的研究 显示在 NTera2D1 人畸胎癌细胞中充当增强子。 该片段的部分删除导致部分减少 活性和部分片段克隆时含有增强子活性 异源启动子表明完整片段包含更多 多于一个增强子序列。 片段内基序的突变 我们之前展示的核蛋白足迹 NTera2D1 细胞在 DNAse I 保护实验中结果增加 增强子活性表明该序列可能充当 转录沉默子。 我们正在继续缩小区域范围 通过缺失分析分析含有增强子的 DNA。 最近的搜索 Genbank 的结果显示， JC 病毒调控片段和之前未研究的区域 地区。已知其他基因中的相关序列与以下成员结合 DNA 结合蛋白的 HMG 家族。 我们正在测试假设 HMG 蛋白家族成员充当转录因子 L1Hs的调节因素。在过去的一年里，我们继续致力于 努力研究 L1Hs 在 M28 细胞中的表达，M28 细胞是一种体细胞 含有人等染色体 12p 的细胞杂交系。 这个不正常的 染色体是睾丸生殖细胞肿瘤和睾丸生殖细胞肿瘤的一个特征 帕利斯特-基利安综合症。 我们的研究表明，相似但 L1Hs 5~ UTR 的不同区域控制 L1Hs 的表达 NTera2D1 和 M28 细胞。 旨在克隆基因的实验 负责 M28 细胞中 L1Hs 表达增强的研究 方式。