GENOMIC PLASTICITY IN THE HUMAN U2 SNRNA GENE CLUSTER

基因扩增和染色体脆性起着重要作用。 在人类遗传学中，但导致这种现象的分子机制 基因组的可塑性还没有被很好地理解。两个不同寻常的 编码人类U2小分子的多基因家族的特征 核糖核酸(SnRNA)使其成为研究这两种扩增的理想工具 以及分子水平上的脆弱性。首先，与大多数人不同的是 多基因家族，人类的10到20个U2基因被组织成 一个明显完美的串联阵列；6kb重复序列的每个拷贝 单位在序列上是相同的，RFLP很少或根本不存在。 这意味着重复序列的同质性是由 整顿过程，或者集群本身是年轻的和 最初是由一种能够产生 完美串联仅从一个或几个首字母重复 基因拷贝。在任一情况下，重复单元本身和/或 串联阵列两侧的序列可能包含 促进整流、生成或重组的重组元件 集群的再生。我们的第一个主要目标是 以鉴定这样的重组序列。第二个非凡之处 U2串联阵列的特点是集群映射到一个站点 致癌菌株诱发的染色体脆性 腺病毒。事实上，Durnam等人。(1988)最近表明 易碎性部位高度局部化，经常发生破损 在U2基因簇本身内(即在仅60个区域内 到120kb)。因此，U2星团可能包含以下元素 在腺病毒感染过程中导致染色体脆性。我们的 第二个主要目标是确定这些导致脆弱性的因素。 我们打算鉴定重组元件和 通过筛选U2序列的脆性诱导元件 在以下情况下促进基因扩增和/或染色体脆性 整合到新的染色体位置。包含所有细胞的细胞系 或与二氢叶酸相邻的U2 6kb重复单位的一部分 通过共转染法构建还原酶(DHFR)微基因。 将通过对频率的评分来识别重组元件 通过细胞分选或通过细胞分类或通过 甲氨蝶呤选择。在具有染色体的细胞系中 扩增dhfr基因，共扩增U2序列的能力 使集成站点变得脆弱的要素将是 重叠感染或转染腺病毒后的检测 病毒的EL区。

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.