Gabriel A, Mules E H
Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, New Jersey 08855, USA.
Ann N Y Acad Sci. 1999 May 18;870:108-18. doi: 10.1111/j.1749-6632.1999.tb08871.x.
Ty1, the genetically tractable retrotransposable element found in the yeast Saccharomyces cerevisiae, closely resembles vertebrate retroviruses both in structure and in mechanism of replication. By direct sequence analysis, we examined the rate and spectrum of new mutations appearing during a single cycle of Ty1 replication. The rate of new mutations was comparable to those seen for replicating retroviruses. All observed changes were base substitutions, and their location suggested that template ends may be hot spots for generating these mutations. To test this, we developed methods to examine, at the nucleotide level, the end structure of the expected Ty1 replication intermediates. Our results demonstrate that Ty1 reverse transcriptase can add terminal non-templated bases in vivo during each step in replication. Furthermore, Ty1 RNAse H creates multiple template ends by imprecisely cleaving RNA. This expands the range of sites of subsequent non-templated base addition. Finally, on reaching template ends, Ty1 reverse transcriptase can strand transfer to inappropriate templates. Taken together, these mutagenic mechanisms may influence the evolution of particular regions of the Ty1 genome and serve as a mechanism to regulate the overall level of Ty1 transposition in its host cell.
Ty1是在酿酒酵母中发现的一种可进行遗传操作的逆转座子元件，在结构和复制机制上都与脊椎动物逆转录病毒极为相似。通过直接序列分析，我们检测了Ty1复制单周期内出现的新突变的速率和谱。新突变的速率与复制型逆转录病毒的情况相当。所有观察到的变化都是碱基替换，其位置表明模板末端可能是产生这些突变的热点。为了验证这一点，我们开发了在核苷酸水平检测预期Ty1复制中间体末端结构的方法。我们的结果表明，Ty1逆转录酶在体内复制的每个步骤中都能添加末端非模板化碱基。此外，Ty1核糖核酸酶H通过不精确切割RNA产生多个模板末端。这扩大了后续非模板化碱基添加的位点范围。最后，当到达模板末端时，Ty1逆转录酶可以链转移到不适当的模板上。综上所述，这些诱变机制可能影响Ty1基因组特定区域的进化，并作为一种调节其在宿主细胞中整体转座水平的机制。
