Recovery And Study Of Yeast Artificial Chromosomes

此前，我们已经证明了线性酵母人工染色体(YAC)可以携带数百个千碱基的DNA，并且可以使用载体pNKG418来循环大的完整基因。高达300kb的分子可以成功地转移到细菌中，同时保持它们的完整性。然后，这些分子可以在细菌中复制，并可以制备大量的DNA。这消除了直接研究基因的一个限制因素。这些环状分子可以用于进一步的研究，例如，将其导入哺乳动物细胞以研究其调控，并具体分析负责其组织特异性表达模式的控制元件。我们现在将这种方法扩展到两个YAC循环，其中一个包含PLAC1基因，它在胎盘中特异表达，第二个基因FOXL2，来自第三染色体，负责一些妇女的卵巢早衰(POF)(见AG000647-05)。结合这一方法，我们正在探索将绿色荧光蛋白(GFP)作为报告基因和哺乳动物可选药物标记纳入细菌人工染色体克隆(BAC)中，使用基于细菌重组的方法。在初步研究中，含有PLAC1基因的BAC在3‘UTR区用IRES-GFP盒改造的至少60kb的上游序列获得了表达，这表明BAC包含了表达所需的所有调控元件。这与用10kb的uspstream序列构建的情况相反，后者不足以驱动基因表达。进一步的实验正在进行中，用标志表位标记转基因的PLAC1，并切除启动子区域，以定义DNA和染色质中的上游调节区。用针对选定表位的兔抗体进行的其他实验表明，PLAC1蛋白以二聚体的形式存在，并在其天然状态的基础上进行了修饰。进一步分析蛋白质并通过免疫沉淀寻找相互作用伙伴的实验正在进行中。类似的研究将扩展到FOXL2基因。这些技术在两篇特邀评论中进行了讨论。

Previously we have shown that linear Yeast Artificial chromosomes (YACs) can carry hundreds of kilobases of DNA, and large complete genes, can be circularized using the vector, pNKG418. Molecules of up to 300 Kb can be transferred successfully into bacteria while maintaining their integrity. These molecules can then be replicated in bacteria and large amounts of DNA can be prepared. This eliminates a limiting factor in studying the genes directly. The circular molecules can be used for further studies, such as transfection into mammalian cells to study their regulation, and to specifically dissect the control elements responsible their tissue-specific expression patterns. We are now extending this method to circularize two YACs, one of which contains the PLAC1 gene, which is expressed specifically in placenta, and a second gene, FOXL2, from chromosome 3, responsible for Premature Ovarian Failure (POF) in some women (see AG000647-05). In conjunction with this approach we are exploring the incorporation into bacterial artificial chromosome clones (BACs) of GFP (green fluorescence protein) as a reporter gene and a mammalian selectable drug marker, using recombination-based methods in bacteria. In preliminary studies a BAC containing PLAC1 gene with at least 60 Kb of upstream sequences retrofitted with IRES-GFP cassette in 3'UTR region gives expression, suggesting that the BAC contains all regulatory elements necessary for expression. This is in contrast to construct with 10 Kb uspstream sequence which was not sufficient to drive gene expression. Further experiments are in progress to tag the transfected PLAC1 with a FLAG epitope and resect the promoter region to define the upstream regulatory regions in DNA and chromatin. Additional experiments with rabbit antibodies raised against selected epitopes suggest that the PLAC1 protein exists as a dimer and is modified from in its native state. Experiments are in progress to analyze the protein further and look for interacting partners by immunoprecipitation. Similar studies will be extended to the FOXL2 gene. The techniques are discussed in two invited reviews.