Cloned Human and Mouse Genes Directing Adipogenesis

一个细胞致力于脂肪细胞途径开始一个多步骤 通过未知数量的步骤进行的过程， 可识别的脂肪形成，并以细胞成为 终末分化的充满脂肪的脂肪细胞 我们克隆了一个 1200bp基因组DNA序列（克隆A），其具有以下能力： 提交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刺激下进入脂肪形成， 胰岛素和汇合，遵循模拟反应的动力学 3T3-F442A的3T3-衍生的前脂肪细胞系至汇合 和血清。 克隆A在不存在外源性 启动子和任何其它外源调节序列。 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：1!我们将确定DNA的最小尺寸 通过克隆克隆A的亚片段并测试 这是AC活动。 !我们将完成 通过从第二条链获得完整序列克隆A。 从第一条链的测序中，我们发现， 95%确定1200 bp克隆A不存在于Genbank中。 三!初步序列数据显示开放阅读框，侧翼为 TATA盒和终止信号，并包含一对 共有剪接点 如果克隆A的完整序列 包含一个真正的开放阅读框架，具有适当的共识起点 信号，我们将在克隆A中创建终止密码子突变， 确定其是否阻断克隆A在小鼠中的活性 皮肤成纤维细胞 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。 当1200 bp片段是一个完整的基因，因为它具有 转染后赋予新表型的能力，北方 以1200 bp序列为探针的印迹未显示AC特异性 信使RNA在培养的前脂肪细胞。 我们将使用一种 更高的灵敏度（RNA-PCR）搜索转录产物 AC DNA在转染细胞、胚胎前脂肪细胞和 在诱导后的不同时间培养的前脂肪细胞 脂肪生成 五!万一最后的序列 缺乏编码序列，或者根据目标3测试的突变体证明 同样活跃的未突变的克隆A DNA，我们将承诺， 确定克隆A的活动是否是 特异性编码的新RNA，或者克隆A是否 直接作为转染细胞内的DNA，也许通过充当 负调节蛋白的结合位点。 我们将 确定来自定向成纤维细胞的特定蛋白质 与克隆A DNA和蛋白质的复合物，通过凝胶迁移率变动测定。 我们将在凝胶迁移试验中使用突变的克隆A DNA，以获得 克隆A内DNA A结合反应元件的更精确图谱， 我们会用标准的色谱技术 蛋白质!特异性结合克隆A应答元件。 脂肪细胞途径还没有得到充分的特点， 生理或分子水平。 这项工作将启动 这样的分子水平研究。*//

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.***//