Cullin C, Minvielle-Sebastia L
Centre de Génétique Moléculaire du C.N.R.S., Laboratoire Propre Associé à l'Université Pierre-et-Marie-Curie, Gif-sur-Yvette, France.
Yeast. 1994 Jan;10(1):105-12. doi: 10.1002/yea.320100110.
In this paper are described a set of new high-copy-number yeast vectors, which are specially designed for the conditional expression of epitope-tagged proteins in vivo. One of the major advantages of these plasmids is that they allow polymerase chain reaction-amplified open reading frames to be automatically fused in frame with the epitope-coding sequence, avoiding longer procedures such as site-directed mutagenesis. This heterologous construction can be realized either at the 5'-end of the coding sequence, in the pYeF1 vector, or at its 3'-end, in pYeF2, generating N- or C-terminal tagged proteins, respectively. Moreover, to increase the usefulness of the method, derivatives of the two basic URA3-borne pYeF1 and pYeF2 were constructed, carrying either the HIS3 or TRP1 gene as a marker of selection. These vectors could be of use for the purpose of functional analysis of the newly discovered genes resulting from the systematic sequencing of the yeast genome. Here, we present results showing the functional expression and the efficient immunoprecipitation of the epitope-tagged Rna15 protein, which is involved in Saccharomyces cerevisiae mRNA stability.
本文描述了一组新的高拷贝数酵母载体，这些载体是专门为体内表位标记蛋白的条件表达而设计的。这些质粒的一个主要优点是，它们允许聚合酶链反应扩增的开放阅读框与表位编码序列自动框内融合，避免了诸如定点诱变等较长的程序。这种异源构建可以在编码序列的5′端（在pYeF1载体中）或其3′端（在pYeF2中）实现，分别产生N端或C端标记的蛋白。此外，为了增加该方法的实用性，构建了两个基本的携带URA3的pYeF1和pYeF2的衍生物，携带HIS3或TRP1基因作为选择标记。这些载体可用于对酵母基因组系统测序产生的新发现基因进行功能分析。在此，我们展示了涉及酿酒酵母mRNA稳定性的表位标记的Rna15蛋白的功能表达和高效免疫沉淀的结果。
