Haney Steven A, Keeney David, Chen Lei, Moghazeh Soraya, Projan Steve, Rasmussen Beth
Department of Infectious Disease, Wyeth Research, 401 N, Middletown Rd, Pearl River, NY 10965, USA.
BMC Genomics. 2003 Sep 10;4(1):36. doi: 10.1186/1471-2164-4-36.
Cloning of genes in expression libraries, such as the yeast two-hybrid system (Y2H), is based on the assumption that the loss of target genes is minimal, or at worst, managable. However, the expression of genes or gene fragments that are capable of interacting with E. coli or yeast gene products in these systems has been shown to be growth inhibitory, and therefore these clones are underrepresented (or completely lost) in the amplified library.
Analysis of candidate genes as Y2H fusion constructs has shown that, while stable in E. coli and yeast for genetic studies, they are rapidly lost in growth conditions for genomic libraries. This includes the rapid loss of a fragment of the E. coli cell division gene ftsZ which encodes the binding site for ZipA and FtsA. Expression of this clone causes slower growth in E. coli. This clone is also rapidly lost in yeast, when expressed from a GAL1 promoter, relative to a vector control, but is stable when the promoter is repressed. We have demonstrated in this report that the construction of libraries for the E. coli and B. subtilis genomes without passaging through E. coli is practical, but the number of transformants is less than for libraries cloned using E. coli as a host. Analysis of several clones in the libraries that are strongly growth inhibitory in E. coli include genes for many essential cellular processes, such as transcription, translation, cell division, and transport.
Expression of Y2H clones capable of interacting with E. coli and yeast targets are rapidly lost, causing a loss of complexity. The strategy for preparing Y2H libraries described here allows the retention of genes that are toxic when inappropriately expressed in E. coli, or yeast, including many genes that represent potential antibacterial targets. While these methods are generally applicable to the generation of Y2H libraries from any source, including mammalian and plant genomes, the potential of functional clones interacting with host proteins to inhibit growth would make this approach most relevant for the study of prokaryotic genomes.
在诸如酵母双杂交系统（Y2H）等表达文库中克隆基因，是基于目标基因损失最小这一假设，或者说，即便损失最大，也是可控的。然而，在这些系统中，能够与大肠杆菌或酵母基因产物相互作用的基因或基因片段的表达已被证明具有生长抑制作用，因此这些克隆在扩增文库中的代表性不足（或完全丢失）。
将候选基因作为Y2H融合构建体进行分析表明，虽然它们在大肠杆菌和酵母中用于遗传研究时是稳定的，但在基因组文库的生长条件下会迅速丢失。这包括大肠杆菌细胞分裂基因ftsZ的一个片段的快速丢失，该片段编码ZipA和FtsA的结合位点。该克隆的表达导致大肠杆菌生长缓慢。当从GAL1启动子表达时，该克隆在酵母中相对于载体对照也会迅速丢失，但当启动子被抑制时则是稳定的。我们在本报告中证明，不经过大肠杆菌传代构建大肠杆菌和枯草芽孢杆菌基因组文库是可行的，但转化子数量比以大肠杆菌为宿主克隆的文库要少。对文库中几个在大肠杆菌中具有强烈生长抑制作用的克隆进行分析，发现其中包括许多参与细胞基本过程的基因，如转录、翻译、细胞分裂和运输。
能够与大肠杆菌和酵母靶标相互作用的Y2H克隆的表达会迅速丢失，导致复杂性降低。本文所述的制备Y2H文库的策略能够保留那些在大肠杆菌或酵母中不适当表达时具有毒性的基因，包括许多代表潜在抗菌靶点的基因。虽然这些方法通常适用于从任何来源（包括哺乳动物和植物基因组）生成Y2H文库，但与宿主蛋白相互作用的功能克隆抑制生长的可能性使得这种方法在原核基因组研究中最为适用。
