Levin H L, Boeke J D
Department of Molecular Biology and Genetics, Johns Hopkins School of Medicine, Baltimore, MD 21205.
EMBO J. 1992 Mar;11(3):1145-53. doi: 10.1002/j.1460-2075.1992.tb05155.x.
Tf1, a retrotransposon from fission yeast, has LTRs and coding sequences resembling the protease, reverse transcriptase and integrase domains of retroviral pol genes. A unique aspect of Tf1 is that it contains a single open reading frame whereas other retroviruses and retrotransposons usually possess two or more open reading frames. To determine whether Tf1 can transpose, we overproduced Tf1 transcripts encoded by a plasmid copy of the element marked with a neo gene. Approximately 0.1-4.0% of the cell population acquired chromosomally inherited resistance to G418. DNA blot analysis demonstrated that such strains had acquired both Tf1 and neo specific sequences within a restriction fragment of the same size; the size of this restriction fragment varied between different isolates. Structural analysis of the cloned DNA flanking the Tf1-neo element of two transposition candidates with the same regions in the parent strain showed that the ability to grow on G418 was due to transposition of Tf1-neo and not other types of recombination events.
Tf1是来自裂殖酵母的一种反转录转座子,具有长末端重复序列(LTRs)和编码序列,类似于逆转录病毒pol基因的蛋白酶、逆转录酶和整合酶结构域。Tf1的一个独特之处在于它含有一个单一的开放阅读框,而其他逆转录病毒和反转录转座子通常拥有两个或更多的开放阅读框。为了确定Tf1是否能够转座,我们过量表达了由带有新霉素基因标记的元件的质粒拷贝所编码的Tf1转录本。大约0.1 - 4.0%的细胞群体获得了对G418的染色体遗传性抗性。DNA印迹分析表明,此类菌株在相同大小的限制性片段内同时获得了Tf1和新霉素特异性序列;该限制性片段的大小在不同分离株之间有所不同。对两个转座候选菌株中Tf1 - 新霉素元件两侧克隆DNA的结构分析,以及与亲本菌株相同区域的比较表明,在G418上生长的能力是由于Tf1 - 新霉素的转座,而非其他类型的重组事件。