Regulation of DNA Elimination in Paramecium

发育控制的DNA重排通常发生在真核细胞中，包括哺乳动物免疫球蛋白基因的体细胞重组和酵母的交配类型转换等例子。纤毛原生动物为发育控制的DNA重排提供了独特而丰富的来源。它们在每一轮有性繁殖中大规模发生，其中大核基因组是由种系微核DNA形成的。整个基因组是通过特定位点的染色体断裂、重新添加端粒和精确去除被称为内部消除序列（IESs）的dna等事件来重塑的。在形成新的巨核所需的几个小时内，草履虫基因组中切除了超过10,000个ie。这些相对较小的DNA序列（27-882 bp）位于开放阅读框和非编码区。最近对草履虫ies的研究已经确定了一个一致的倒置末端重复序列，与Tc1/mariner家族转座子的末端相似。这是所有II类（DNA）转座子家族中分布最广的。在线虫、昆虫、真菌和哺乳动物中都发现了它们的成员。草履虫体内精确高效的ess切除可能是从转座子进化而来的。本提案的主要目的是鉴定调节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突变细胞系进行IES切除，2)使用体内实验分析28bp IES内部和侧翼的突变。用抗A表面蛋白的血清处理诱变细胞，可以分离出突变体。A基因在编码区含有7个IES，不能消除任何IES就会阻止A蛋白的表达。在抗血清治疗下存活下来的a突变体将被分离出来，在IES切除中存在缺陷的亚类将被确定。这一过程已被证明是成功的分离突变体含有缺陷的IES切除。体内试验将允许分析任何核苷酸在DNA切除中的功能。位点定向突变将验证一致的反向末端重复是IES切除所必需的假设。侧翼DNA序列的缺失将决定是否需要切除28bp IES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其他生物中类似但不常见的DNA重排提供见解。草皮虫IESs与Tc1/mariner转座子之间的关系可能揭示了转座子最初编码的活性转化为宿主细胞使用的功能的进化转移。

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.