ANALYSIS OF HUMAN CHROMOSOME 13Q NEOCENTROMERE FORMATION

本提案的目的是检查人类着丝粒的形成，它确保在细胞分裂过程中适当的染色体分离。染色体分离错误导致非整倍体和嵌合体，导致出生缺陷和肿瘤。正常的人类着丝粒含有大量重复的α卫星DNA，这对分析它们的形成有一定的限制。然而，从正常着丝粒分离的有丝分裂稳定的重排染色体片段上发现的新着丝粒不包含重复序列，并且可以定位于低拷贝或单拷贝基因组DNA。因此，新着丝粒提供了一种新的方法来研究当前的模型，即着丝粒的形成需要不同的初级DNA序列，例如α卫星或新着丝粒DNA，或很大程度上独立于序列的表观遗传修饰，例如不同的染色质结构或DNA复制中DNA复制的时间差异。因此，以下三个特定目的研究了7个独立细胞系的独特集合，每个细胞系都包含一个额外的倒置/重复13q染色体和一个新着丝粒。1)分子细胞遗传学FISH定位将确定13q染色体上新中心粒的位置和单个或重叠的粒体的反转断点。在这个集合中至少有四个新中心粒在细胞遗传学上定位于染色体带13q32。这种FISH映射将潜在地定义13q中具有新中心粒形成和/或与反转断点关系的高倾向的区域。2)原DNA序列在着丝粒形成中的作用将通过新着丝粒DNA的分离和序列分析来解决，使用两种互补的方法。保留着丝粒蛋白CENP-C的改良扩展染色质技术将用于高分辨率定位基因组克隆到新着丝粒。新着丝粒序列将通过使用针对着丝粒特异性组蛋白CENP-1的抗体免疫沉淀着丝粒染色质来分离。3) DNA复制时间在着丝粒形成中的作用将通过检查新着丝粒DNA的复制时间和与同源染色体中相应DNA序列的比较来解决，使用两种方法。BrdU结合将允许评估在新着丝粒和非着丝粒状态下特定染色体区域的复制。原位复制时间将允许评估特定基因组序列在新中心粒和正常染色体上的相对复制时间。

The objective of this proposal is to examine the formation of human centromeres, which ensure proper chromosome segregation during cell division. Errors in chromosome segregation lead to aneuploidy and mosaicism, resulting in birth defects and neoplasias. Normal human centromeres contain large amounts of repetitive alpha satellite DNA, which presents certain limitations to the analysis of their formation. However, neocentromeres, found on mitotically stable rearranged chromosomal fragments separated from normal centromeres, do not contain repetitive sequences and can be found localized to low or single copy genomic DNA. Thus, neocentromeres provide a novel approach to investigate current models that centromere formation requires either a distinct primary DNA sequence, e.g. alpha satellite or neocentromere DNA, or largely sequence independent epigenetic modifications, e.g. a distinct chromatin structure or temporal differences in DNA replication in DNA replication. Thus, the following three Specific Aims examine a unique collection of seven independent cell lines that each contain a supernumerary inversion/duplication 13q chromosome with a neocentromere. 1) Molecular cytogenetic FISH mapping will determine the positions of the neocentromeres and the inversion breakpoints to individual or overlapping cosmids from chromosome 13q. At least four neocentromeres in this collection have been cytogenetically localized to chromosome band 13q32. This FISH mapping will potentially define regions in 13q with a high propensity for neocentromere formation and/or a relationship to inversion breakpoints. 2) The role of primary DNA sequence in centromere formation will be addressed by isolation and sequence analysis of neocentromere DNA, using two complementary approaches. Modified extended chromatin techniques that retain the kinetochore protein CENP-C will be used to localize genomic clones to neocentromeres to high resolution. Neocentromere sequences will be isolated by immunoprecipitation of centromeric chromatin using antibodies to the centromere-specific histone CENP-1. 3) The role of DNA replication timing in centromere formation will be addressed by examination of the replication timing of neocentromere DNA and comparison to corresponding DNA sequences in homologous chromosomes, using two approaches. BrdU incorporation will permit assessment of replication of specific chromosomal regions in neocentromeric and non-centromeric states. In situ replication timing will permit assessment of the relative replication timing of specific genomic sequences at neocentromeres and on normal chromosomes.