Patnaik P K, Fang X, Cross G A
Laboratory of Molecular Parasitology, Rockefeller University, New York, NY 10021-6399.
Nucleic Acids Res. 1994 Oct 11;22(20):4111-8. doi: 10.1093/nar/22.20.4111.
We have previously reported the construction and characterization of an autonomously replicating plasmid in Trypanosoma brucei. In this plasmid the procyclic acidic repetitive protein (PARP) gene promoter drives the transcription of a selectable marker. Deletion of this promoter incapacitates the plasmid, suggesting its utilization as a promoter-trap. Three independent libraries were created by inserting variously digested T.brucei genomic DNA into this promoterless construct. Transfection of these libraries into procyclic T.brucei and the subsequent isolation of episomes led only to the reisolation of the PARP promoter. Additionally, a ribosomal RNA promoter failed to keep the construct as an episome, although it can sustain mRNA transcription in T.brucei and was shown to be an efficient promoter in this construct. Finally, by using a transient replication assay involving the methylation-sensitive restriction endonuclease DpnI to distinguish between input and replicated DNA, we showed that the PARP promoter-bearing construct could replicate autonomously in procyclic T.brucei, but the corresponding construct with the rRNA promoter could not. The close association between elements that sustain transcription and DNA replication in T.brucei mirrors results observed in several higher eukaryotes and their viruses and suggests an ancient origin of this feature.
我们之前报道过布氏锥虫中自主复制质粒的构建与特性研究。在该质粒中，前循环酸性重复蛋白（PARP）基因启动子驱动一个选择标记的转录。该启动子的缺失使质粒失去功能，表明其可作为启动子陷阱。通过将经不同酶切的布氏锥虫基因组DNA插入该无启动子构建体，创建了三个独立文库。将这些文库转染到前循环布氏锥虫中，随后分离附加体，结果仅重新分离到了PARP启动子。此外，核糖体RNA启动子虽能在布氏锥虫中维持mRNA转录且在该构建体中显示为高效启动子，但未能使构建体保持为附加体。最后，通过使用涉及甲基化敏感限制性内切酶DpnI的瞬时复制测定法来区分输入DNA和复制DNA，我们表明携带PARP启动子的构建体能够在前循环布氏锥虫中自主复制，而带有rRNA启动子的相应构建体则不能。布氏锥虫中维持转录和DNA复制的元件之间的紧密关联反映了在几种高等真核生物及其病毒中观察到的结果，并表明这一特征具有古老的起源。
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