Construction of a S. cerevisiae Ty1-less strain.

我们最初观察到逆转录是自我限制的，这取决于元素拷贝数，随后发现这种控制是由Ty1反义RNA介导的，这在很大程度上依赖于使用一种不寻常的缺乏任何Ty1元素的酵母菌菌株。天然（野生）酵母菌的Ty1拷贝数各不相同，尽管它们通常比常见的酿酒酵母实验室菌株含有更少的元素。我们使用的自然Ty1-less菌株最初被发现具有异常低的Ty1元素负载（一个元素），并且该单个元素被删除以获得不含Ty1元素的菌株。一项寻找残余长末端重复序列（LTR）的基因组分析显示，至少有11个元素存在于一个祖先菌株中，并且该菌株不是严格意义上的酿酒葡萄球菌，而是酿酒葡萄球菌和悖论葡萄球菌之间的杂交菌株。这阻碍了我们与酿酒酵母的分析和比较，因为悖论酵母的基因组序列不完整且注释不足，而且没有对整个不含ty1的酵母基因组进行测序，我们无法辨别物种形成或杂交程度。为了解决这一差异，我们系统地删除了酿酒葡萄球菌实验室菌株中所有32个Ty1元素。用含有URA3基因和Ty1序列的PCR片段转化。Ty1元素被URA3基因取代以及随后的重组缺失可以通过在5-氟-羟基乙酸（FOA）存在下的细胞生长来监测，FOA是一种当URA3表达时对细胞有毒的药物。由该过程产生的不需要的总体染色体异常或重排通过脉冲场凝胶电泳检测。使用这种方法，可以构建只有一个元素的应变。

Our initial observation that retrotransposition is self-limiting depending on element copy number, and subsequent discovery that this control is mediated by Ty1 antisense RNA, relied heavily on the use of an unusual Saccharomyces strain that lacked any Ty1 elements. Natural (wild) isolates of Saccharomyces vary in Ty1 copy number, although they usually contain fewer elements than the common laboratory strains of S. cerevisiae. The natural Ty1-less strain we used was initially found to have an unusually low Ty1 element load (one element) and that single element was deleted to derive a strain free of Ty1 elements. A genomic analysis to search for residual long terminal repeats (LTR), indicative of sites where elements had been lost due to LTR-LTR recombination, revealed that at least 11 elements had populated an ancestral strain, and that this strain is not strictly S. cerevisiae , but rather, appeared to be a hybrid between S. cerevisiae and S. paradoxus. This has hindered our analyses and comparisons to S. cerevisiae as the genomic sequence of S. paradoxus is incomplete and poorly annotated, and without sequencing the entire Ty1-less yeast genome, we cannot discern the speciation or extent of hybridization. To address this disparity, we have undertaken the systematic deletion of all 32 Ty1 elements in the laboratory strain of S. cerevisiae. by transformation with a PCR fragment containing the URA3 gene flanked by Ty1 sequence. The replacement of a Ty1 element by the URA3 gene and subsequent loss through recombination can be monitored by growth of the cells in the presence of 5-fluoro-orotic acid (FOA), a drug that is toxic to cells when URA3 is expressed. Unwanted gross chromosomal abnormalities or rearrangements generated by this process are detected by pulse field gel electrophoresis. Using this method, strains with as few as one element have been constructed.