Identification of Xenopus CENP-A and an associated centromeric DNA repeat
Identification of Xenopus CENP-A and an associated centromeric DNA repeat
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DOI：
10.1091/mbc.e04-09-0788
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发表时间：
2005-04-01
影响因子：
3.3
通讯作者：
Murray, AW
中科院分区：
文献类型：
--
作者：
Edwards, NS;Murray, AW
着丝粒是聚集在着丝粒DNA上并指导真核生物染色体分离的蛋白质复合物。高等真核细胞定义着丝粒的机制知之甚少。可能的分子贡献着丝粒规范包括潜在的DNA序列和表观遗传因素，如着丝粒组蛋白着丝粒蛋白A（CENP-A）的结合。蛙卵提取物是研究着丝粒定义和动粒组装的一个有吸引力的系统。为了促进此类研究，我们克隆了CENP-A的非洲爪蟾同源物（XCENP-A）。我们通过染色质免疫沉淀法克隆与XCENP-A共纯化的DNA片段来鉴定着丝粒相关DNA序列。XCENP-A与青蛙着丝粒重复序列1（Fcr 1）相关，Fcr 1是一个174个碱基对的重复序列，含有一个可能的CENP-B盒。部分消化的基因组DNA的Southern印迹显示基因组中Fcr 1的大的有序阵列。Fcr 1探针荧光原位杂交染色大多数着丝粒在培养的细胞。通过染色灯刷染色体，我们特别确定了11（18）染色体染色与Fcr 1探针一致。
Kinetochores are the proteinaceous complexes that assemble on centromeric DNA and direct eukaryotic chromosome segregation. The mechanisms by which higher eukaryotic cells define centromeres are poorly understood. Possible molecular contributors to centromere specification include the underlying DNA sequences and epigenetic factors such as binding of the centromeric histone centromere protein A (CENP-A). Frog egg extracts are an attractive system for studying centromere definition and kinetochore assembly. To facilitate such studies, we cloned a Xenopus laevis homologue of CENP-A (XCENP-A). We identified centromere-associated DNA sequences by cloning fragments of DNA that copurified with XCENP-A by chromatin immunoprecipitation. XCENP-A associates with frog centromeric repeat 1 (Fcr1), a 174-base pair repeat containing a possible CENP-B box. Southern blots of partially digested genomic DNA revealed large ordered arrays of Fcr1 in the genome. Fluorescent in situ hybridization with Fcr1 probes stained most centromeres in cultured cells. By staining lampbrush chromosomes, we specifically identified the 11 (of 18) chromosomes that stain consistently with Fcr1 probes.