Recovery And Study Of Yeast Artificial Chromosomes

在此之前，我们已经证明线性酵母人工染色体（YACs）可以携带数百个碱基的DNA，并且可以使用载体pNKG418循环化大的完整基因。高达300 Kb的分子可以成功地转移到细菌中，同时保持其完整性。这些分子可以在细菌中复制，从而制备出大量的DNA。这消除了直接研究基因的一个限制因素。圆形分子可用于进一步的研究，例如转染到哺乳动物细胞中以研究其调控，并专门剖析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YACs，其中一个包含PLAC1基因，该基因在胎盘中特异性表达，另一个基因FOXL2来自3号染色体，在一些女性中负责卵巢早衰（POF）（见AG000647-05）。结合这种方法，我们正在探索在细菌中使用基于重组的方法将绿色荧光蛋白（GFP）作为报告基因和哺乳动物选择性药物标记物整合到细菌人工染色体克隆（BACs）中。在初步研究中，含有PLAC1基因的BAC在3'UTR区至少有60kb的上游序列被IRES-GFP磁带改造，表明BAC包含表达所需的所有调控元件。这与构建10 Kb uspstream序列不足以驱动基因表达形成对比。进一步的实验正在进行中，用FLAG表位标记转染的PLAC1，并切除启动子区域，以确定DNA和染色质中的上游调控区域。另外针对兔抗体的实验表明，PLAC1蛋白以二聚体的形式存在，并在其天然状态下被修饰。实验正在进行中，以进一步分析蛋白质，并通过免疫沉淀寻找相互作用的伙伴。类似的研究将扩展到FOXL2基因。在两篇特邀评论中讨论了这些技术。

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.