Comparative Analysis of Cancer-Associated Genes and Deve

遗传连锁研究将遗传性前列腺癌HPCX的一个位点定位在Xq27的X染色体长臂上。候选区域包含两个SPANX基因簇，一个是SPANX- a /D亚家族，包括5个成员，分别是spax - a1、spax - a2、spax - b、spax - c和spax -D；一个是SPANX- n亚家族，包括4个成员，分别是spax - n1、spax - n2、spax - n3和spax - n4，编码睾丸癌特异性抗原。我们之前对spax - b和spax - d基因座的分析发现了几个由基因转换产生的新的DNA序列变异，尽管它们都不是x连锁家族所特有的（Kouprina等，2005）。在上一财年，我们对x连锁前列腺癌患者候选区域内的其他SPANX基因（SPANX- a1、SPANX- a2、SPANX- c、SPANX- n1、SPANX- n2、SPANX- n3和SPANX- n4）进行了突变分析。基因分离物的序列分析未发现遗传性前列腺癌患者特有的任何改变。因此，这些和先前的结果表明，在x连锁家族中，SPANX等位基因的特定组合或SPANX基因间区域的基因组重排导致了前列腺癌的易感性。人类人工染色体（Human artificial chromosome, HACs）为研究着丝点形成和开发具有基因治疗潜力的新一代载体提供了独特的机会。我们构建了第一个具有条件着丝粒的人类人工染色体，该着丝粒可以通过靶向修饰其体内表观遗传结构而失活。HAC的构建是基于一个人工合成的α -卫星（alphoid）重复序列的DNA阵列，该重复序列包含一个天然单体，具有CENP-B的结合位点（CENP-B box），以及一个完全人工合成的单体，其中与CENP-B box对应的区域被四环素算子（tetO）取代。tTA转录激活子的结合极大地破坏了HAC的稳定性，而其他几种四环素抑制融合蛋白的表达对HAC的稳定性没有显著影响。有机会选择性地将不同的蛋白质靶向到一个活跃的着丝粒中，从而调节着丝粒的功能，为人类着丝粒的前所未有的机制和结构分析以及开发新的基于hac的条件基因表达系统开辟了道路。

1). Genetic linkage studies mapped a locus for hereditary prostate cancer, HPCX, to the long arm of the X chromosome at Xq27. The candidate region contains two clusters of SPANX genes, a SPANX-A/D subfamily including five members, SPANX-A1, SPANX-A2, SPANX-B, SPANX-C, and SPANX-D, and a SPANX-N subfamily including four members, SPANX-N1, SPANX-N2, SPANX-N3, and SPANX-N4, encoding cancer-testis specific antigens. Our previous analysis of the SPANX-B and SPANX-D loci identified several new DNA sequence variants resulted from gene conversion, though none of them was specific for X-linked families (Kouprina et al., 2005). During last fiscal year, we carried out mutational analysis of other SPANX genes (SPANX-A1, SPANX-A2, SPANX-C, SPANX-N1, SPANX-N2, SPANX-N3, and SPANX-N4) localized within the candidate region in the X-linked prostate cancer patients. Sequence analysis of the gene isolates did not reveal any alterations specific for the patients with hereditary prostate cancer. Thus, these and previous results indicate that either a specific combination of SPANX alleles or genomic rearrangements in the SPANX intergenic regions results in the predisposition to prostate cancer in X-linked families.2). Human artificial chromosomes (HACs) provide a unique opportunity to study kinetochore formation and to develop a new generation of vectors with potential in gene therapy. We constructed the first human artificial chromosome with a conditional centromere that can be inactivated by targeted modification of its epigenetic configuration in vivo. The HAC was built up using a DNA array based upon a synthetic alpha-satellite (alphoid) repeat containing one natural monomer, with a binding site for CENP-B (CENP-B box), and one completely synthetic monomer in which the region corresponding to the CENP-B box was replaced with a tetracycline operator (tetO). Binding of the tTA transcriptional transactivator dramatically destabilized the HAC, whilst expression of several other tetracycline-repression fusion proteins had no significant effect on HAC stability. The opportunity to selectively target different proteins into an active kinetochore and thereby regulate centromere function opens the way for an unprecedented mechanistic and structural analysis of the human kinetochore as well as for development of new HAC-based conditional gene expression systems.