GENOMIC PLASTICITY IN THE HUMAN U2 SNRNA GENE CLUSTER

人类 U2 SNRNA 基因簇的基因组可塑性

• 批准号: 3299896
• 负责人: ALAN M WEINER
• 金额: $ 15.42万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-04-01 至 1994-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3299896
• 关键词: Adenoviridae; blood donor; cell sorting; chromosome aberrations; chromosome disorders; dihydrofolate reductase; drug resistance; fusion gene; gene conversion; gene mutation; genetic enhancer element; genetic promoter element; genetic strain; genetic transcription; genome; human genetic material tag; human population genetics; in situ hybridization; methotrexate; microorganism genetics; molecular cloning; natural gene amplification; nucleic acid repetitive sequence; nucleic acid sequence; oncogenic virus; site directed mutagenesis; small nuclear RNA; thymidylate kinase; transfection; transposon /insertion element; viral carcinogenesis

Gene amplification and chromosome fragility play important roles in human genetics but the molecular mechanisms responsible for such genomic plasticity are not well understood. Two remarkable features of the multigene family encoding human U2 small nuclear RNA (snRNA) make it ideal for studying both amplification and fragility at the molecular level. First, in contrast to most multigene families, the 10 to 20 human U2 genes are organized in an apparently perfect tandem array; each copy of the 6 kb repeat unit is identical in sequence, and RFLPs are rare or nonexistent. This implies that the homogeneity of the repeats is maintained by a rectification process, or that the cluster itself is young and was originally generated by a mechanism capable of producing perfect tandem repeats from only one or a few initial gene copies. In either case, the repeat unit itself and/or the sequences flanking the tandem array are likely to contain recombinogenic elements that promote rectification, generation, or regeneration of the cluster. Our first major goal is to identify such recombinogenic sequences. The second remarkable feature of the U2 tandem array is that the cluster maps to a site of chromosomal fragility induced by oncogenic strains of adenovirus. In fact, Durnam et al. (1988) have recently shown that the fragile site is highly localized and that breakage often occurs within the U2 gene cluster itself (i.e. within a region of only 60 to 120 kb). Thus the U2 cluster is likely to contain elements that cause chromosomal fragility during adenovirus infection. Our second major goal is to identify these fragility inducing elements. We intend to identify both the recombinogenic elements and the fragility inducing elements by screening the U2 array for sequences which promote gene amplification and/or chromosome fragility when integrated into new chromosomal sites. Cell lines containing all or part of the U2 6kb repeat unit adjacent to a dihydrofolate reductase (DHFR) minigene will be constructed by cotransfection. Recombinogenic elements will be identified by scoring the frequency of spontaneous or induced DHFR amplification by cell sorting or by methotrexate selection. In cell lines having chromosomally amplified DHFR genes, the ability of coamplified U2 sequence elements to confer fragility on the integration site will be assayed after superinfection with adenovirus or transfection with the El region of the virus.

基因扩增和染色体脆弱性起着重要作用 在人类遗传学中，但分子机制负责 基因组可塑性尚不清楚。 两个了不起的 编码人U2小的多基因家族的功能 核RNA（SNRNA）使其非常适合研究两种扩增 和分子水平的脆弱性。 首先，与大多数 多基因家族，10至20个人U2基因在 一个显然是完美的串联阵列； 6 kb重复的每个副本 单位的顺序相同，RFLP罕见或不存在。 这意味着重复的同质性由 纠正过程，或者集群本身很年轻，并且 最初是由能够生产的机制生成的 完美的串联仅重复一个或几个初始 基因副本。 无论哪种情况，重复单元本身和/或 串联阵列两侧的序列可能包含 重组元素促进纠正，产生或 集群的再生。 我们的第一个主要目标是 确定这种重组序列。 第二名 U2串联阵列的特征是群集映射到站点 由致癌菌株诱导的染色体脆性 腺病毒。 实际上，Durnam等人。 （1988）最近表明 脆弱的站点是高度局部的，并且断裂经常发生 在U2基因簇本身中（即仅在60个区域内 至120 kb）。 因此，U2群集可能包含 在腺病毒感染期间引起染色体脆弱性。 我们的 第二个主要目标是识别这些脆弱性诱导元素。 我们打算确定重组元素和 通过筛选U2阵列的序列来诱导元素 促进基因扩增和/或染色体脆弱性 集成到新的染色体位点。 包含所有细胞系 或与二氢叶酸相邻的U2 6KB重复单元的一部分 还原酶（DHFR）小基因将通过共转染构建。 重组元素将通过评分频率来识别 通过细胞分选或通过 甲氨蝶呤选择。 在具有染色体的细胞系中 扩增的DHFR基因，共汇入U2序列的能力 在集成站点上赋予脆弱性的元素将是 用腺病毒或转染过转染后测定 病毒的EL区域。