DENSELY METHYLATED DNA AND MAMMALIAN REPLICATION ORIGINS

密集甲基化 DNA 和哺乳动物复制起源

• 批准号: 2734767
• 负责人: DONALD J ROUFA
• 金额: --
• 依托单位: KANSAS STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-07-01 至 1999-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2734767
• 关键词: 5 methylcytosine; CHO cells; DNA binding protein; DNA footprinting; DNA methylation; DNA replication; DNA replication origin; cell cycle; cell growth regulation; chromosomes; extrachromosomal DNA; flow cytometry; gel mobility shift assay; gene deletion mutation; molecular cloning; nonhistone nucleoprotein; nucleic acid sequence; point mutation; site directed mutagenesis; synchronous cell division

DESCRIPTION (Adapted from Applicant's Abstract): Mammalian chromosomal replication origins contain unusual densely methylated DNA sequence islands in which all cytosine residues, including those in CpC, CpA, CpG, and CpT dinucleotides, are methylated as 5-methylcytosine [Tasheva, E.S. and Roufa, D.J., Mol. Cell Biol., in press (1994)]. Because three islands are fully methylated only in active growing cells, densely methylated islands (DMIs) appear either to regulate or be regulated by chromosomal replication origin activity. A plan to characterize the relationship between DMIs and active mammalian chromosomal replication origins is proposed. Three specific aims are detailed: 1) To investigate the cell cycle regulation of DMI methylation within the replication origin located at the 3' end of the Chinese hamster ribosomal protein S14 gene on chromosome 2q (ori S14). These studies will involve cells synchronized to specific stages of the mitotic cycle as well as G1S and G2 phase cells resolved by FACS. 2) To use in vitro mutagenesis to modify the DMI in ori S14 by deletion and point mutations and then to assess the mutations effect on the origins replication program. The latter will be accomplished using a novel tissue culture episomal DNA replication assay and/or homologous recombinational at the hemizygous RPS14 locus on CHO cell chromosome 2q. and 3) To identify, clone, and characterize nuclear proteins that differentially bind to bilaterally versus hemi-methylated and demethylated DMIs and thereby are likely to mediate interactions between the DMI and the cell's replication machinery.

描述（改编自申请人的摘要）：哺乳动物染色体 复制起源包含异常的密集甲基化的DNA序列 所有胞质残留物，包括CPC，CPA的岛屿， CpG和CPT二核苷酸被甲基化为5-甲基环肽[Tasheva， E.S.和D.J. Roufa，Mol。细胞生物。，在印刷中（1994）]。 因为三个 岛屿仅在活跃的生长细胞中完全甲基化，密集 甲基化岛（DMI）似乎调节或受到调节 染色体复制起源活性。 表征的计划 DMI与活性哺乳动物染色体复制之间的关系 提出了起源。 三个具体目标是详细的：1） 研究DMI甲基化的细胞周期调节 复制起源位于中国仓鼠的3'末端 核糖体蛋白S14基因在2q染色体上（ORI S14）。 这些研究 将涉及与有丝分裂循环的特定阶段同步的细胞 以及由FACS解析的G1和G2相细胞。 2）在体外使用 通过缺失和点突变修改ORI S14中DMI的诱变 然后评估突变对起源复制的影响 程序。 后者将使用新型的组织培养 在 CHO细胞染色体2Q上的Hemizygous RPS14基因座。 3）确定， 克隆并表征差异结合的核蛋白 双侧对半甲基化和脱甲基化的DMI，从而是 可能介导DMI与细胞之间的相互作用 复制机制。