Cloned Human and Mouse Genes Directing Adipogenesis

一个致力于脂肪细胞途径的细胞开始一个多步骤的过程，通过未知的步骤进行可识别的脂肪形成，并以细胞最终分化为充满脂肪的脂肪细胞结束。我们已经克隆了一个1200bp的基因组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，其动力学模拟了3t3来源的前脂肪细胞系3T3-F442A对汇合和血清的反应。克隆A在缺乏外源启动子和任何其他外源调控序列的情况下具有功能。我们准备对克隆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。从第一条链的测序中，我们发现在95%的确定性水平上，1200 bp克隆A不存在于Genbank中。ı3 !初步序列数据显示一个开放的阅读框，两侧是一个TATA盒和一个终止信号，并包含一对一致剪接结。如果克隆A的完整序列包含一个真正的开放阅读框，并具有适当的共识启动信号，我们将在克隆A中创建一个停止密码子突变，并确定它是否阻断克隆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1200bp片段是一个完整的基因，在转染后具有赋予新表型的能力，但在培养的前脂肪细胞中，以1200bp序列为探针的Northern印迹并未显示ac特异性信使RNA。我们将在诱导脂肪形成后的不同时间，在转染细胞、胚胎前脂肪细胞和培养前脂肪细胞中，使用灵敏度更高的RNA-PCR方法寻找AC DNA的转录产物。ı5 !如果最终序列缺乏编码序列，或者根据Aim 3测试的突变体证明与未突变的克隆a DNA具有同等活性，则我们将确定克隆a的活性是由特定编码的新RNA产生的，还是克隆a直接作为转染细胞内的DNA。可能是作为负调节蛋白的结合位点。我们将通过凝胶迁移转移试验来确定来自承诺成纤维细胞的特定蛋白质是否与克隆A DNA和蛋白质复合物。我们将在凝胶移位实验中使用突变的Clone- a DNA来获得克隆a中DNA a结合反应元件的更精确的图谱，我们将使用标准色谱技术纯化proteinıs！具体绑定到克隆A响应元素。脂肪细胞途径尚未在生理或分子水平上得到充分的表征。这项工作将在分子水平上开启这样的研究

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.***//