Formation of artificial chromosomes in Caenorhabditis elegans and analyses of their segregation in mitosis, DNA sequence composition and holocentromere organization. Formation of artificial chromosomes in Caenorhabditis elegans and analyses of their segregation in mitosis, DNA sequence composition and holocentromere organization

To investigate how exogenous DNA concatemerizes to form episomal artificial chromosomes (ACs), acquire equal segregation ability and maintain stable holocentromeres, we injected DNA sequences with different features, including sequences that are repetitive or complex, and sequences with different AT-contents, into the gonad of Caenorhabditis elegans to form ACs in embryos, and monitored AC mitotic segregation. We demonstrated that AT-poor sequences (26% AT-content) delayed the acquisition of segregation competency of newly formed ACs. We also co-injected fragmented Saccharomyces cerevisiae genomic DNA, differentially expressed fluorescent markers and ubiquitously expressed selectable marker to construct a less repetitive, more complex AC. We sequenced the whole genome of a strain which propagates this AC through multiple generations, and de novo assembled the AC sequences. We discovered CENP-AHCP-3 domains/peaks are distributed along the AC, as in endogenous chromosomes, suggesting a holocentric architecture. We found that CENP-AHCP-3 binds to the unexpressed marker genes and many fragmented yeast sequences, but is excluded in the yeast extremely high-AT-content centromeric and mitochondrial DNA (> 83% AT-content) on the AC. We identified A-rich motifs in CENP-AHCP-3 domains/peaks on the AC and on endogenous chromosomes, which have some similarity with each other and similarity to some non-germline transcription factor binding sites. Overall design: EXPERIMENT TYPE: CHIP-seq. BIOLOGICAL SOURCE: Strain: WYY35; Developmental Stage: Early Embryos; Genotype: wild type; Sex: Hermaphrodite; NUMBER OF REPLICATES: 2; temp (temperature) 22 degree celsius; Antibody: SDQ0804 (Rabbit anti-HCP-3)

为探究外源DNA如何通过串联形成附加体型人工染色体（episomal artificial chromosomes, ACs）、获得均等分离能力并维持稳定的全着丝粒（holocentromeres）结构，我们将具备不同特征的DNA序列——包括重复序列与复杂序列，以及不同AT含量的序列——注入秀丽隐杆线虫（Caenorhabditis elegans）的性腺中，使其在胚胎内形成人工染色体，并监测其有丝分裂分离过程。我们证实，AT含量较低的序列（AT占比26%）会延缓新形成的人工染色体获得分离能力的进程。我们还联合注射了片段化的酿酒酵母（Saccharomyces cerevisiae）基因组DNA、差异表达的荧光标记基因与广谱表达的筛选标记基因，以构建低重复、高复杂度的人工染色体。我们对一株可将该人工染色体稳定传递多代的菌株开展全基因组测序，并从头组装了人工染色体的序列。我们发现，与内源染色体一致，CENP-AHCP-3结构域/峰在人工染色体上呈分布式分布，提示其具备全着丝粒结构。我们进一步观察到，CENP-AHCP-3可结合未表达的标记基因与大量片段化的酵母序列，但会被排除在人工染色体上AT含量极高的酵母着丝粒序列与线粒体DNA序列（AT占比>83%）之外。我们在人工染色体与内源染色体的CENP-AHCP-3结构域/峰中鉴定出了富含A的基序，这类基序之间存在一定相似性，且与部分非生殖系转录因子的结合位点具有相似性。整体实验设计：实验类型：染色质免疫共沉淀测序（CHIP-seq）；生物样本来源：菌株：WYY35；发育阶段：早期胚胎；基因型：野生型；性别：雌雄同体；生物学重复数：2；培养温度：22℃；抗体：SDQ0804（兔抗HCP-3）