Regulation of DNA Elimination in Paramecium

发育控制的DNA重排通常发生在真核细胞中，包括哺乳动物中免疫球蛋白基因的体细胞重组和酵母中的交配型转换。 纤毛虫原生动物提供了一个独特的和丰富的来源发育控制的DNA重排。 它们在有性生殖的每一轮中大规模发生，其中大核基因组由生殖细胞微核DNA形成。 整个基因组被包括位点特异性染色体断裂、从头端粒添加和称为内部消除序列（IES）的DNA的精确去除的事件重塑。 在形成新的大核所需的几个小时内，从草履虫基因组中切除了超过10，000个IEs。 这些相对小的DNA序列（27-882 bp）位于开放阅读框以及非编码区中。 最近的研究草履虫IES已经确定了一个共识的反向末端重复序列，具有相似性的Tc 1/水手家族中的转座子的末端。 这是所有II类（DNA）转座子家族中最广泛的。 已在线虫、昆虫、真菌和哺乳动物中发现成员。 草履虫IESs的精确高效切除可能是转座子进化而来的。 这个建议的主要目的是确定顺式作用的DNA元件，调节IES切除。 这将通过两种方法来实现。 1)IES切除缺陷的突变细胞系的分离和分析，以及2）使用体内测定来分析28 bp IES内部和侧翼的突变。 将通过用抗A表面蛋白的抗血清处理诱变细胞来分离突变体。 A基因在编码区内含有7个IES，如果不能消除任何IES，A蛋白的表达就会受到阻碍。 将分离抗血清处理后存活的A突变体，并鉴定IES切除缺陷的亚类。 该程序已被证明成功的IES切除含有缺陷的突变体的分离。 体内测定将允许分析任何核苷酸在DNA切除中的功能。 定点突变将检验IES切除所需的共有反向末端重复序列的假设。 侧翼DNA序列的缺失将决定是否需要切除28 bp IES以外的序列。草履虫大核发育过程中广泛的DNA消除事件的研究可能会发现新的分子机制，或提供类似的，但不太常见的DNA重排在其他生物体的洞察。 草履虫IES和Tc 1/mariner转座子之间的关系可以揭示最初由转座子编码的活性进化转移到宿主细胞使用的功能。

Forney9808285Developmentally controlled DNA rearrangements commonly occur in eukaryotic cells and include examples such as the somatic recombination of immunoglobulin genes in mammals and mating type switching in yeast. Ciliated protozoa provide a unique and rich source of developmentally controlled DNA rearrangements. They occur on a massive scale during each round of sexual reproduction in which the macronuclear genome is formed from the germline micronuclear DNA. The entire genome is remodeled by events that include site specific chromosome breakage, de novo telomere addition and the precise removal of DNAs called internal eliminated sequences (IESs). More than 10,000 IESs are excised from the Paramecium genome during the few hours required to form a new macronucleus. These relatively small DNA sequences (27-882 bp) are located in open reading frames as well as in non-coding regions. Recent studies of Paramecium IESs have identified a consensus inverted terminal repeat that has similarity to the ends of transposons in the Tc1/mariner family. This is the most widespread of all class II (DNA) transposon families. Members have been found in nematodes, insects, fungi, and mammals. It is possible that the precise and highly efficient excision of IESs in Paramecium evolved from transposons. The primary objective of this proposal is to identify the cis acting DNA elements that regulate IES excision. This will be accomplished with two methods. 1) The isolation and analysis of mutant cell lines that are defective for IES excision, and 2) the use of an in vivo assay to analyze mutations inside and flanking a 28 bp IES. Mutants will be isolated by treating mutagenized cells with anti-serum against the A surface protein. The A gene contains seven IESs within the coding region and failure to eliminate any IES prevents A protein expression. A-mutants surviving the treatment with anti-serum will be isolated and the subclass with defects in IES excision will be identified. This procedure has proven successful for the isolation of mutants containing defects in IES excision. The in vivo assay will allow any nucleotide to be analyzed for its function in DNA excision. Site-directed mutations will test the hypothesis that the consensus inverted terminal repeat is required for IES excision. Deletions of the flanking DNA sequence will determine whether sequences outside the 28 bp IES are required for excision.Studies of the extensive DNA elimination events during Paramecium macronuclear development may uncover novel molecular mechanisms or provide insight into similar but less common DNA rearrangements in other organisms. The relationship between Paramecium IESs and the Tc1/mariner transposons could reveal the evolutionary transfer of an activity originally encoded by a transposon into a function used by the host cell.