Patchy interspecific sequence similarities efficiently identify positive cis-regulatory elements in the sea urchin
Patchy interspecific sequence similarities efficiently identify positive cis-regulatory elements in the sea urchin
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DOI：
10.1006/dbio.2002.0618
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发表时间：
2002-06-01
影响因子：
2.7
通讯作者：
Davidson, EH
中科院分区：
文献类型：
--
作者：
Yuh, CH;Brown, CT;Davidson, EH
我们证明，种间序列保守可以为鉴定大范围基因组DNA中的功能顺式调控元件提供系统的指导。本试验对紫球藻的otx基因进行了检测。该基因在构成胚胎内胚层规范的基因调控网络中起着重要作用。OTX基因的顺式调控结构被认为是复杂的，因为该基因有三个不同的起始点(X.Li，C.-K.Chuang，C.-A.Mao，L.M.Angerer，和W.H.Klein，1997，Dev.比奥尔。187,253-266)，它在胚胎的许多不同的空间域中表达。从紫球虫和杂色金龟文库中分离出含有OTX基因的BAC重组体，并对其进行了序列测定和注释。对该基因两侧的60个碱基的DNA进行了计算扫描，寻找较短的保守序列元件。为此，我们使用了一个在我们实验室组装的新构建的软件包“家庭关系”。该工具允许在设置为20-50个碱基对的滑动窗口内检测高于所选标准的序列相似性。用聚合酶链式反应(PCR)从紫花链霉菌BAC DNA中扩增出17个部分保守的区域，大部分为几百个碱基对，插入到驱动CAT报告基因的表达载体中，并通过注射到紫毛链霉菌受精卵中检测顺式调节活性。通过使用针对CAT mRNA的探针进行整装原位杂交(WMISH)来评估这些构建物的调节活性。在17个结构中，11个结构显示出空间受限的调控活性，6个在本试验中不活跃。表达顺式调控结构的结构域与WMISH对OTX在胚胎中表达的研究结果大致一致，在该研究中，我们使用了针对三个转录起始点中每一个产生的mRNAs的探针。确定了四个单独的顺式调控元件，它们专门产生内胚层表达，以及普遍存在的活性元件和外胚层特异性元件。我们证实了其他工作中关于在内胚层表达的元素中特定转录因子的靶点的预测。(C)2002年埃尔塞维尔科学公司(美国)。
We demonstrate that interspecific sequence conservation can provide a systematic guide to the identification of functional cis-regulatory elements within a large expanse of genomic DNA. The test was carried out on the otx gene of Strongylocentrotus purpuratus. This gene plays a major role in the gene regulatory network that underlies endomesoderm specification in the embryo. The cis-regulatory organization of the otx gene is expected to be complex, because the gene has three different start sites (X. Li, C.-K. Chuang, C.-A. Mao, L. M. Angerer, and W. H. Klein, 1997, Dev. Biol. 187, 253-266), and it is expressed in many different spatial domains of the embryo. BAC recombinants containing the otx gene were isolated from Strongylocentrotus purpuratus and Lytechinus variegatus libraries, and the ordered sequence of these BACs was obtained and annotated. Sixty kilobases of DNA flanking the gene, and included in the BAC sequence from both species, were scanned computationally for short conserved sequence elements. For this purpose, we used a newly constructed software package assembled in our laboratory, "Family Relations." This tool allows detection of sequence similarities above a chosen criterion within sliding windows set at 20-50 bp. Seventeen partially conserved regions, most a few hundred base pairs long, were amplified from the S. purpuratus BAC DNA by PCR, inserted in an expression vector driving a CAT reporter, and tested for cis-regulatory activity by injection into fertilized S. purpuratus eggs. The regulatory activity of these constructs was assessed by whole-mount in situ hybridization (WMISH) using a probe against CAT mRNA. Of the 17 constructs, 11 constructs displayed spatially restricted regulatory activity, and 6 were inactive in this test. The domains within which the cis-regulatory constructs were expressed are approximately consistent with results from a WMISH study on otx expression in the embryo, in which we used probes specific for the mRNAs generated from each of the three transcription start sites. Four separate cis-regulatory elements that specifically produce endomesodermal expression were identified, as well as ubiquitously active elements, and ectoderm-specific elements. We confirm predictions from other work with respect to target sites for specific transcription factors within the elements that express in the endoderm. (C) 2002 Elsevier Science (USA).