Construction of a S. cerevisiae Ty1-less strain.

我们最初的观察，反转录转座是自我限制的元件拷贝数，并随后发现，这种控制是由Ty 1反义RNA介导的，在很大程度上依赖于使用一种不寻常的酵母菌株，缺乏任何Ty 1元素。酵母菌的天然（野生）分离株在Ty 1拷贝数上各不相同，尽管它们通常含有比常见的实验室菌株少的元件。啤酒。我们使用的天然无Ty 1应变最初被发现具有异常低的Ty 1元件载荷（一个元件），并且删除该单个元件以获得无Ty 1元件的应变。一项基因组分析，以寻找残留的长末端重复序列（LTR），指示由于LTR-LTR重组而丢失元件的位点，结果显示，至少有11个元件在祖先菌株中存在，并且该菌株不是严格意义上的S。cerevisiae，而是似乎是S. cerevisiae和S.矛盾的。这妨碍了我们对S.酿酒酵母的基因组序列。paradoxus是不完整的，注释也很差，如果不对整个Ty 1-less酵母基因组进行测序，我们就无法辨别杂交的物种或程度。为了解决这一差异，我们已经在实验室菌株S中系统地删除了所有32个Ty 1元件。啤酒。通过用含有侧翼为Ty 1序列的URA 3基因的PCR片段转化。通过在5-氟乳清酸（FOA）存在下的细胞生长，可以监测URA 3基因对Ty 1元件的替换以及随后通过重组的损失，5-氟乳清酸（FOA）是一种当URA 3表达时对细胞有毒的药物。通过脉冲场凝胶电泳检测由该过程产生的不需要的总体染色体异常或重排。使用这种方法，已经构建了具有少至一个元件的菌株。

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.