Comparative Analysis of Cancer-Associated Genes and Deve

1）。遗传连锁研究将遗传性前列腺癌的基因座HPCX定位于X染色体长臂的Xq 27。候选区域包含两个编码癌症-睾丸特异性抗原的SPAN基因簇，一个SPAN-A/D亚家族包括五个成员，SPAN-A1、SPAN-A2、SPAN-B、SPAN-C和SPAN-D，和一个SPAN-N亚家族包括四个成员，SPAN-N1、SPAN-N2、SPAN-N3和SPAN-N4。我们先前对SPAN X-B和SPAN X-D基因座的分析鉴定了由基因转换产生的几种新的DNA序列变体，尽管它们都不是X连锁家族特异性的（Kouprina等人，2005年）。在上一个财政年度，我们对X连锁前列腺癌患者候选区域内的其他SPAN X基因（SPAN X-A1、SPAN X-A2、SPAN X-C、SPAN X-N1、SPAN X-N2、SPAN X-N3和SPAN X-N4）进行了突变分析。基因分离物的序列分析没有发现任何遗传性前列腺癌患者的特异性改变。因此，这些和先前的结果表明，在X连锁家族中，要么是SPAN X等位基因的特定组合，要么是SPAN X基因间区域的基因组重排导致前列腺癌的易感性。人类人工染色体（HACs）为研究动粒的形成和开发具有基因治疗潜力的新一代载体提供了一个独特的机会。我们构建了第一个人类人工染色体与条件性着丝粒，可以通过靶向修饰其表观遗传构型在体内失活。使用基于合成α-卫星（α）重复序列的DNA阵列构建HAC，所述合成α-卫星（α）重复序列含有一个天然单体，具有CENP-B（CENP-B盒）的结合位点，和一个完全合成的单体，其中对应于CENP-B盒的区域被四环素操纵子（tetO）替换。tTA转录反式激活因子的结合显著地使HAC不稳定，而其他几种四环素抑制融合蛋白的表达对HAC稳定性没有显著影响。选择性地将不同蛋白质靶向到活性动粒中并由此调节着丝粒功能的机会为人类动粒的前所未有的机制和结构分析以及新的基于HAC的条件基因表达系统的开发开辟了道路。

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.