Bradshaw M S, Shashikant C S, Belting H G, Bollekens J A, Ruddle F H
Department of Biology, Yale University, New Haven, Connecticut 06520, USA.
Proc Natl Acad Sci U S A. 1996 Mar 19;93(6):2426-30. doi: 10.1073/pnas.93.6.2426.
Hox genes are located in highly conserved clusters. The significance of this organization is unclear, but one possibility is that regulatory regions for individual genes are dispersed throughout the cluster and shared with other Hox genes. This hypothesis is supported by studies on several Hox genes in which even large genomic regions immediately surrounding the gene fail to direct the complete expression pattern in transgenic mice. In particular, previous studies have identified proximal regulatory regions that are primarily responsible for early phases of mouse Hoxc8 expression. To locate additional regulatory regions governing expression during the later periods of development, a yeast homologous recombination-based strategy utilizing the pClasper vector was employed. Using homologous recombination into pClasper, we cloned a 27-kb region around the Hoxc8 gene from a yeast artificial chromosome. A reporter gene was introduced into the coding region of the isolated gene by homologous recombination in yeast. This large fragment recapitulates critical aspects of Hoxc8 expression in transgenic mice. We show that the regulatory elements that maintain the anterior boundaries of expression in the neural tube and paraxial mesoderm are located between 11 and 19 kb downstream of the gene.
Hox基因位于高度保守的基因簇中。这种组织形式的意义尚不清楚，但一种可能性是单个基因的调控区域分散在整个基因簇中，并与其他Hox基因共享。这一假设得到了对多个Hox基因的研究支持，在这些研究中，即使是基因周围紧邻的大片基因组区域，在转基因小鼠中也无法指导完整的表达模式。特别是，先前的研究已经确定了主要负责小鼠Hoxc8基因早期表达阶段的近端调控区域。为了定位在发育后期控制表达的其他调控区域，采用了基于酵母同源重组的策略，利用pClasper载体。通过同源重组到pClasper中，我们从酵母人工染色体中克隆了Hoxc8基因周围一个27kb的区域。通过酵母中的同源重组，将一个报告基因引入到分离基因的编码区。这个大片段概括了转基因小鼠中Hoxc8基因表达的关键方面。我们表明，维持神经管和轴旁中胚层表达前界的调控元件位于该基因下游11至19kb之间。
