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，我们对小麦的结构修饰大麦染色体进行了分析。首先，我们鉴定了两条结构修饰的大麦染色体(7hs^*和7hs^&lt；**&gt；)，它们起源于大麦7H染色体短臂的一条等位染色体。我们的细胞学分析表明，7HS^*和7HS^**染色体都没有着丝粒C带和着丝粒特异重复序列(Cereba和(GAAAGA)n卫星序列)。这些被截断的染色体通常传递给下一代，表明有丝分裂和减数分裂中动粒的正常形成。着丝粒特异蛋白的免疫染色证实了有丝分裂中功能着丝粒的形成(Nasuda等人。2005a)。虽然我们尝试用各种方法从7HS^*和7HS^&lt；**&gt；染色体中分离着丝粒序列，但没有成功。其中一个原因是六倍体的芯片分析困难。此外，小麦染色体在流动分选过程中的污染也是个问题。但是，7hs^*和7hs^&lt；**&gt；中的断点映射仍在进行中(Nasuda等人)。2005B)。到目前为止，我们在短臂的非常近端区域获得了一个标记，该标记最终在修饰后的染色体中缺失。我计划使用大麦和水稻的基因组信息来进一步澄清断裂点。在正常大麦中成功地鉴定了功能着丝粒序列(Houben等人。2007)。通过对叶片组织中分离的细胞核进行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)存在相互作用，CENH3定位于所有已分析的真核物种中的功能着丝粒。大麦中的(AGGGAG)n和Cereba序列之间的相互作用水平没有显著差异，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.