Cloned Human and Mouse Genes Directing Adipogenesis

致力于脂肪细胞途径的细胞开始一个多步骤的过程，通过未知数量的步骤进行可识别的脂肪生成，最终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的基因组DNA序列(克隆A)，该序列能够在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、胰岛素和融合刺激下进入成脂状态，其动力学模拟了3T3来源的前脂肪细胞系3T3-F442a对融合和血清的反应。克隆A在没有外源启动子和任何其他外源调控序列的情况下起作用。我们计划对克隆A进行严格的分子分析。我们的具体目标是：ı1！我们将通过克隆克隆A的亚片段并测试它们的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获得完整的序列来完成克隆A的测序。通过对第一链的测序，我们发现在95%的确定性水平上，1200bp的克隆A不存在于Genbank中。ı3！初步测序数据显示，开放阅读框两侧有TATA盒和终止信号，并包含一对一致的剪接连接。如果克隆A的完整序列包含一个真正的开放阅读框架，具有正确的共识起始信号，我们将在克隆A中创建一个终止密码子突变，并确定它是否阻止克隆A在小鼠皮肤成纤维细胞中的活动。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1200bp的片段是一个完整的基因，因为它具有在转染后赋予新的表型的能力，但以1200bP序列为探针的Northern blotting在培养的前脂肪细胞中没有发现AC特异的信使RNA。我们将使用一种更高敏感性的实验(RNA-PCR)来寻找在转基因细胞、胚胎前脂肪细胞和诱导成脂后不同时间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，或者如果在Aim 3下测试的突变体被证明对未突变的克隆A DNA具有同样的活性，我们将致力于确定克隆A的活性是否是特定编码的新RNA的结果，或者克隆A是否直接作为DNA在转基因细胞中发挥作用，也许是作为负调控蛋白的结合位点。我们将通过凝胶迁移率变化分析来确定承诺的成纤维细胞中的特定蛋白是否与克隆A DNA和蛋白质形成复合体。我们将在凝胶移位实验中使用突变的Clone-ADNA，以获得更精确的Clone A内DNAA结合反应元件的图谱，并将使用标准的层析技术来纯化蛋白质ıS！与Clone A反应元件特别结合。无论是在生理水平上还是在分子水平上，脂肪细胞的途径都还没有被完全描述。这项工作将在分子水平上启动这样的研究。

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.***//