Identification of functional centromeric sequences in barley through analysis of structurally modified chromosomes.

为了鉴定大麦染色体上的功能着丝粒序列，对小麦中经过结构修饰的大麦染色体进行了分析。首先，我们从大麦染色体7H短臂同工染色体中鉴定出两条结构修饰的染色体7HS^*和7HS^<**>。我们的细胞学分析表明，7HS^*和7HS^**染色体不具有着丝粒c带，也不具有着丝粒特异性重复序列（cereba和（GAAAGA）n卫星序列）。这些截短的染色体正常地传递给下一代，表明有丝分裂和减数分裂中着丝点的正常形成。通过对着丝粒特异性蛋白的免疫染色证实有丝分裂中功能性着丝粒的形成（Nasuda et al. 2005a）。虽然我们尝试用各种方法从7HS^*和7HS^<**>染色体中分离出着丝粒序列，但均未成功。原因之一是六倍体的ChIP分析困难。此外，小麦染色体在流动分选中的污染也是一个问题。然而，7HS^*和7HS^<**>中的断点映射正在进行中（Nasuda et al. 2005b）。到目前为止，我们在短臂的非常近的区域获得了一个标记，最终在修饰的染色体中缺失。我计划利用大麦和水稻的基因组信息来进一步澄清断点。在正常大麦中成功鉴定了功能性着丝粒序列（Houben et al. 2007）。通过对叶组织核的ChIP（染色质免疫沉淀）分析，我们可以清楚地表明，大脑反转录转座子和（AGGGAG）n卫星序列与着丝粒特异性组蛋白H3 （CENH3）相互作用，该蛋白定位于迄今所分析的所有真核生物物种的功能着丝粒上。在大麦中，cereba和（AGGGAG）n序列的互作水平无显著差异，表明两者都形成着丝粒染色质。

In order to identify functional centromeric sequences on barley chromosomes, we analyzed the structurally-modified barley chromosomes in wheat. First, we identified two structurally-modified barley chromosomes (7HS^* and 7HS^<**>)that were originated from an isochromosome of short arm of barley chromosome 7H. Our cytological analyses indicated that both 7HS^* and 7HS^** chromosomes did not possess centromeric C-bands as well as centromere-specific repetitive sequences (cereba and (GAAAGA)n satellite sequences). These truncated chromosome normally transmit to next generation, indicating the normal formation of kinetochores in both mitotic and meiotic division. Formation of functional centromeres in mitosis was confirmed by immunostaining of centromere-specific proteins (Nasuda et al. 2005a). Although we tried to isolated centromeric sequences from 7HS^* and 7HS^<**> chromosomes with various method, our attempt was not successful. One reason was difficulty of ChIP analysis in hexaploid. And also the contamination of wheat chromosomes in flow-sorting was problematic. However, mapping the breakpoints in 7HS^* and 7HS^<**> is ongoing (Nasuda et al. 2005b). So far, we obtained a marker at very proximal region of the short arm that is eventually missing in the modified chromosomes. I am planning to use genomic information of barley and rice to further clarify the breakpoints.Identification of functional centromeric sequences in normal barley was successful (Houben et al. 2007). By ChIP (chromatin immunoprecipitation) analyses of nuclei isolated from leaf tissue, we could clearly indicate that the cereba retrotransposons and (AGGGAG)n satellite sequences have interaction with the centromere specific histone H3 (CENH3), which localize to functional centromeres in all eukaryotic species so far analyzed. The levels of interaction were not significantly different between cereba and (AGGGAG)n sequences, indicating that both form the centromeric chromatin in barley.