Regulatory sequences for the amplification and replication of the ribosomal DNA minichromosome in Tetrahymena thermophila
Regulatory sequences for the amplification and replication of the ribosomal DNA minichromosome in Tetrahymena thermophila
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DOI：
10.1128/mcb.17.12.7237
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发表时间：
1997-12-01
影响因子：
5.3
通讯作者：
Yao, MC
中科院分区：
文献类型：
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作者：
Blomberg, P;Randolph, C;Yao, MC
我们分析了调控嗜热四膜虫rRNA基因(RDNA)复制的顺式作用序列。这种有纤毛的原生动物的大核含有9000个拷贝的21kbp的小染色体，以回文的形式存在，其中包括两个rDNA拷贝，这些拷贝来自接合过程中通过选择性扩增的单个染色体整合拷贝，并在营养生长期间通过每个细胞周期复制一次来维持。我们已经开发了一个转化载体并进行了缺失分析，以确定复制、扩增和/或稳定维持rDNA分子所需的最小序列。使用包含逐渐延长的缺失的构建体，我们表明只需要一小部分rDNA(类似于900bp)来进行染色体外复制和该分子的稳定维持。这个核心区非常近，但不包括rRNA转录起始点或其假定的启动子，表明复制不依赖于正常的rRNA转录，它包括两个几乎相同的核酸酶敏感结构域(D1和D2)，其中一个(D1)对应于先前确定的复制的物理来源，两个结构域的缺失都会取消复制，而任一个结构域的缺失允许分子复制，表明只需要一个结构域，除了这个核心区外，我们还发现了几个DNA片段，包括21个核苷酸重复(类型II重复)的串联阵列和rRNA编码区内的序列它们在维持rDNA的高拷贝数方面发挥着独特而重要的作用。
We have analyzed the cis-acting sequences that regulate rRNA gene (rDNA) replication in Tetrahymena thermophila. The macronucleus of this ciliated protozoan contains 9,000 copies of a 21-kbp minichromosome in the form of a palindrome comprising two copies of the rDNA, These are derived from a single chromosomally integrated copy during conjugation through selective amplification and are maintained by replicating once per cell cycle during vegetative growth. We have developed a transformation vector and carried out a deletion analysis to determine the minimal sequences required for replication, amplification, and/or stable maintenance of the rDNA molecule. Using constructs containing progressively longer deletions, we show that only a small portion (similar to 900 bp) of the rDNA is needed for extrachromosomal replication and stable maintenance of this molecule. This core region is very near but does not include the rRNA transcription initiation site or its putative promoter, indicating that replication is not dependent on normal rRNA transcription, It includes two nearly identical nuclease-sensitive domains (D1 and D2), one of which (D1) corresponds to the physical origin of replication determined previously, Deletion of both domains abolishes replication, whereas deletion of either domain allows the molecules to replicate, indicating that only one domain is required, In addition to this core region, we have found several DNA segments, including a tandem array of a 21-nucleotide repeat (type II repeats) and sequences within the rRNA coding region, that play distinctive and important roles in maintaining the rDNA at a high copy number.