Tschunko A H, Loechel R H, McLaren N C, Allen S L
Department of Biology, University of Michigan, Ann Arbor 48109.
Genetics. 1987 Nov;117(3):451-66. doi: 10.1093/genetics/117.3.451.
DNA sequence elimination and rearrangement occurs during the development of somatic cell lineages of eukaryotes and was first discovered over a century ago. However, the significance and mechanism of chromatin elimination are not understood. DNA elimination also occurs during the development of the somatic macronucleus from the germinal micronucleus in unicellular ciliated protozoa such as Tetrahymena thermophila. In this study foldback DNA from the micronucleus was used as a probe to isolate ten clones. All of those tested (4/4) contained sequences that were repetitive in the micronucleus and rearranged in the macronucleus. The presence of inverted repeated sequences was clearly demonstrated in one of them by electron microscopy. DNA sequence analysis showed that the left portion of this clone contains three tandem, directly repeated copies of a 340-bp sequence, a 120-bp portion of which appears in inverted orientation at a 1.6-kb distance. This clone, pTtFB1, was subjected to a detailed analysis of its developmental fate. Subregions were subcloned and used as probes against Southern blots of micronuclear and macronuclear DNA. We found that all subregions defined repeated sequence families in the micronuclear genome. A minimum of four different families was defined, two of which are retained in the macronucleus and two of which are completely eliminated. The inverted repeat family is retained with little rearrangement. Two of the families, defined by subregions that do not contain parts of the inverted repeat, one in the "loop" and one in the "right flanking region," are totally eliminated during macronuclear development--and contain open reading frames. A fourth family occurs in the "loop" region and is rearranged extensively during development. The two gene families that are eliminated are stable in the micronuclear genome but are not clustered together as evidenced by experiments in which DNAs from nullisomic strains are used to map family members to specific micronuclear chromosomes. The inverted repeat family is also stable in the micronuclear genome and is dispersed among several chromosomes. The significance of retained inverted repeats to the process of elimination is discussed.
DNA序列消除和重排在真核生物体细胞谱系发育过程中发生，这在一个多世纪前就首次被发现。然而，染色质消除的意义和机制尚不清楚。在单细胞纤毛原生动物（如嗜热四膜虫）中，从生殖微核发育成体大核的过程中也会发生DNA消除。在本研究中，来自微核的回文DNA被用作探针来分离十个克隆。所有测试的克隆（4/4）都包含在微核中重复且在大核中重排的序列。通过电子显微镜在其中一个克隆中清楚地证明了反向重复序列的存在。DNA序列分析表明，该克隆的左侧部分包含一个340bp序列的三个串联、直接重复拷贝，其中120bp的部分以反向方向出现在1.6kb的距离处。这个克隆，pTtFB1，对其发育命运进行了详细分析。将亚区域亚克隆，并用作针对微核和大核DNA的Southern杂交印迹的探针。我们发现所有亚区域在微核基因组中定义了重复序列家族。至少定义了四个不同的家族，其中两个保留在大核中，两个完全被消除。反向重复家族保留下来，几乎没有重排。由不包含反向重复部分的亚区域定义的两个家族，一个在“环”中，一个在“右侧侧翼区域”，在大核发育过程中完全被消除，并且包含开放阅读框。第四个家族出现在“环”区域，在发育过程中广泛重排。被消除的两个基因家族在微核基因组中是稳定的，但不是聚集在一起的，这通过使用来自缺体菌株的DNA将家族成员定位到特定微核染色体的实验得到证明。反向重复家族在微核基因组中也是稳定的，并且分散在几条染色体中。讨论了保留的反向重复对消除过程的意义。
