Cloned Human and Mouse Genes Directing Adipogenesis

克隆的人类和小鼠基因指导脂肪生成

• 批准号: 9107166
• 负责人: Robert Pollack
• 金额: $ 19.5万
• 依托单位: Columbia University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-08-01 至 1993-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9107166&HistoricalAwards=false
• 关键词: Cloned; Human; Mouse; Genes; Directing

A cell committed to the adipocytic pathway begins a multi-step process that proceeds through an unknown number of steps to recognizable adipogenesis and ends with the cell becoming a terminally differentiated, fat-filled adipocyte. We have cloned a 1200bp genomic DNA sequence (clone A) that has the capacity to commit 3T3-C2 cells, precrisis mouse skin fibroblasts and 10T1/1 mouse cells to enter adipogenesis upon later stimulation by serum, insulin and confluence, following kinetics that mimic the response of the 3T3-derived preadipocyte cell line 3T3-F442A to confluence and serum. Clone A is functional in the absence of an exogenous promoter and any other exogenous regulatory sequence. We plant to subject Clone A to rigorous molecular analysis. Our specific aims are as follows: ı1! We will determine the minimal size of the DNA with AC activity by cloning subfragments of Clone A and testing these for AC activity. ı2! We will complete the sequencing of Clone A by obtaining complete sequences from the second strand. From sequencing of the first strand we have found that at the level of 95% certainty the 1200 bp Clone A is not present in Genbank. ı3! Preliminary sequence data show an open reading frame flanked by a TATA box and a termination signal, and containing one pair of consensus splice junctions. If the complete sequence of Clone A contains a true open reading frame with proper consensus start signals, we will create a stop-codon mutation in Clone A and determine whether or not it blocks the Clone A's activity in mouse skin fibroblasts. ı4! At this point we cannot exclude the possibility that Clone A does not encode a protein. While the 1200 bp fragment is a complete gene in the sense that it has the capacity to confer a novel phenotype upon transfection, Northern blots with 1200 bp sequence as probe do not reveal an AC-specific messenger RNA in cultured preadipocytes. We will use an assay of greater sensitivity (RNA-PCR) to search for transcription products of AC DNA in transfected cells, embryonic preadipocytes and in cultured preadipocytes at different times after induction of adipogenesis. ı5! In the unlikely event that the final sequence lacks a coding sequence, or if mutants tested under Aim 3 prove equally active to unmutated Clone A DNA, we will undertake to determine whether Clone A's activity is the result of a specifically encoded and novel RNA, or whether Clone A serves directly as a DNA within the transfected cell, perhaps by acting as a binding site for a negative-regulatory protein. We will determine whether specific proteins from committed fibroblasts complex with clone A DNA and protein, by gel mobility shift assay. We will use mutated Clone-A DNA in the gel shift assay to gain a more precise map of DNA A-binding response elements within Clone A, and we will use standard chromatography techniques to purify proteinıs! specifically binding to Clone A response elements. The adipocytic pathway has not been fully characterized at either the physiological or the molecular level. This work will initiate such a study at the molecular level.***//

致力于脂肪细胞途径的细胞开始一个多步骤过程，该过程通过未知数量的步骤进行可识别的脂肪生成，并以细胞成为终末分化的、充满脂肪的脂肪细胞结束。 我们克隆了一个 1200bp 基因组 DNA 序列（克隆 A），该序列能够使 3T3-C2 细胞、危机前小鼠皮肤成纤维细胞和 10T1/1 小鼠细胞在随后的血清、胰岛素和汇合刺激下进入脂肪形成，遵循模拟 3T3 衍生的前脂肪细胞系 3T3-F442A 对汇合和汇合反应的动力学。 血清。 克隆A在缺乏外源启动子和任何其他外源调节序列的情况下具有功能。 我们计划对克隆 A 进行严格的分子分析。 我们的具体目标如下：ı1！我们将通过克隆克隆 A 的子片段并测试它们的 AC 活性来确定具有 AC 活性的 DNA 的最小大小。 2！我们将通过获得第二链的完整序列来完成克隆 A 的测序。 通过第一条链的测序，我们发现 Genbank 中不存在 1200 bp 的克隆 A，其确定性为 95%。 3！初步序列数据显示侧翼有 TATA 盒和终止信号的开放阅读框，并包含一对共有剪接点。 如果克隆 A 的完整序列包含真正的开放阅读框和正确的共有起始信号，我们将在克隆 A 中创建终止密码子突变，并确定它是否会阻断克隆 A 在小鼠皮肤成纤维细胞中的活性。 4！此时我们不能排除克隆 A 不编码蛋白质的可能性。 虽然 1200 bp 片段是一个完整的基因，因为它有能力在转染后赋予新的表型，但以 1200 bp 序列作为探针的 Northern 印迹并未揭示培养的前脂肪细胞中的 AC 特异性信使 RNA。 我们将使用更高灵敏度的测定（RNA-PCR）来寻找诱导脂肪生成后不同时间的转染细胞、胚胎前脂肪细胞和培养的前脂肪细胞中AC DNA的转录产物。 5！万一最终序列缺少编码序列，或者如果在 Aim 3 下测试的突变体证明与未突变的克隆 A DNA 具有同样的活性，我们将确定克隆 A 的活性是否是特定编码的新型 RNA 的结果，或者克隆 A 是否直接作为转染细胞内的 DNA（可能通过充当负调节蛋白的结合位点）。 我们将通过凝胶迁移率变化测定来确定来自定向成纤维细胞的特定蛋白质是否与克隆 A DNA 和蛋白质复合。 我们将在凝胶位移测定中使用突变的克隆 A DNA，以获得克隆 A 内 DNA A 结合反应元件的更精确图谱，并且我们将使用标准层析技术来纯化蛋白质！与克隆 A 响应元件特异性结合。 脂肪细胞途径在生理学或分子水平上尚未得到充分表征。 这项工作将在分子水平上启动这样的研究。***//