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了一个多步骤过程，该过程通过未知数量的步骤进行识别脂肪形成，并以细胞成为末端分化的，充满脂肪的脂肪细胞而结束。我们已经克隆了一个1200BP基因组DNA序列（克隆A），该序列具有3T3-C2细胞，小鼠皮肤纤维成纤维细胞和10T1/1小鼠细胞的能力，在通过血清，胰岛素和融合的后来刺激后进入脂肪形成，随后在模拟3T3-T3-T3-declive declience to的响应中刺激脂肪形成。血清。克隆A在没有外源启动子和任何其他外源调节序列的情况下起作用。我们种植对克隆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的子纹理并测试AC活性来确定具有AC活性的DNA的最小尺寸。 ı2！我们将通过从第二链中获得完整的序列来完成克隆A的测序。从第一线的测序中，我们发现在95％的确定性水平上，1200 bp克隆A在GenBank中不存在。 ı3！初步序列数据显示了一个开放的阅读框架，该框架是塔塔框和一个终止信号，并包含一对共识剪接连接。如果克隆A的完整序列包含具有适当共识启动信号的真正开放阅读框架，我们将在克隆A中创建一个停止构造的突变，并确定它是否阻止了克隆A在小鼠皮肤成纤维细胞中的活性。 ı4！在这一点上，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，因为它具有在翻译时赋予新型表型的能力，但具有1200 bp序列的北部印迹，因为探针在培养的前脂肪细胞中没有揭示AC特异性的Messenger RNA。我们将使用对更高敏感性（RNA-PCR）的评估来搜索翻译细胞，胚胎前脂肪细胞和培养的前脂肪细胞中AC DNA的转录产物。 ı5！在不太可能的情况下，最终序列缺乏编码序列，或者在AIM 3下测试的突变体证明对未灭绝的克隆A DNA同样活跃，我们将承诺确定克隆A的活性是否是专门编码和新颖的RNA的结果，还是克隆A在翻译单元中直接用作翻译单元中的DNA，也许是通过粘合的位置，也可能是由粘合的位置，也可能是粘合的位置。我们将通过凝胶迁移率转移测定法确定是否与克隆A DNA和蛋白质的成纤维细胞复合物中的特定蛋白质。我们将在凝胶移位评估中使用突变的A DNA，以获得克隆A内的DNA A结合响应元件的更精确图，我们将使用标准色谱技术来纯化蛋白质！特异性结合到克隆响应元素。脂肪细胞途径尚未在生理或分子水平上充分表征。这项工作将在分子水平上启动此类研究。*** //