Regulation of DNA Elimination in Paramecium

草履虫 DNA 消除的调控

• 批准号: 9808285
• 负责人: James Forney
• 金额: $ 12万
• 依托单位: Purdue Research Foundation
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-07-01 至 2001-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9808285&HistoricalAwards=false
• 关键词: Regulation; DNA; Elimination; Paramecium

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.

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交配类型的转换。纤毛虫为发育控制的DNA重排提供了独特而丰富的来源。它们在每一轮有性生殖过程中大规模发生，在有性繁殖过程中，大核基因组由生殖系微核DNA形成。整个基因组被包括定点染色体断裂、从头添加端粒和被称为内部消除序列(IESS)的DNA的精确移除的事件所重塑。在形成新的大核所需的几个小时内，超过10,000个片段从草履虫基因组中被切除。这些相对较小的DNA序列(27-882bp)位于开放阅读框和非编码区。最近对草履虫Iess的研究发现了一个与Tc1/mariner家族转座子末端相似的共识反向末端重复序列。这是所有第二类(DNA)转座子家族中分布最广泛的。在线虫、昆虫、真菌和哺乳动物中都发现了这种成员。草履虫中精确而高效的Iess切除可能是从转座子进化而来的。这项建议的主要目的是确定调控IES切除的顺式作用DNA元件。这将通过两种方法实现。1)分离和分析有缺陷的IES切除突变细胞系，以及2)使用体内试验来分析28个碱基IES内部和侧翼的突变。用抗A表面蛋白的抗血清处理诱变细胞，可分离出突变体。A基因在编码区有7个缺失，如果不能消除任何IES，就会阻止A蛋白的表达。在抗血清处理后存活下来的A-突变体将被分离出来，并将确定IES切除中存在缺陷的亚类。这一程序已被证明成功地分离出含有IES切除缺陷的突变体。体内试验将允许对任何核苷酸进行分析，以确定其在DNA切除中的功能。定点突变将检验IES切除所需的共识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28bpIES以外的序列。对草履虫大核发育过程中广泛的DNA消除事件的研究可能会揭示新的分子机制，或为深入了解其他生物中类似但不太常见的DNA重排提供线索。草履虫Iess和Tc1/mariner转座子之间的关系可以揭示最初由转座子编码的活动向宿主细胞所使用的功能的进化转移。