In vitro recombination cloning of entire cDNA libraries in Arabidopsis thaliana and its application to the yeast two-hybrid system

Abstract

In the postgenomic era many experiments rely on the availability of transcript sequence for cloning. As these clones usually originate from cDNA libraries, the quality of these libraries is crucial. If a good library is generated it is desirable to use a versatile cloning system suitable for many different kinds of applications. The cloning systems based on in vitro recombination proves fitting for this task. However, the use of this method for shuttling entire cDNA libraries between different vectors has not yet been studied in great detail. Here we describe the construction of four cDNA libraries from different tissues of Arabidopsis thaliana, the shuttling of the libraries into expression vectors, and evaluation of this method as well as its suitability for downstream applications. Libraries were constructed from seedlings, hormone treated seedlings, flowers, developing seeds and primary leaves in the "entry vector" of the Gateway™ cloning system. After initial characterization of the libraries, they were shuttled into an expression vector (a yeast two-hybrid prey vector). To monitor for a size bias generally assumed to be inherent to in vitro recombination methods, the libraries were characterized before and after the transfer into the expression vector. However no significant difference could be detected. The functionality of the in vitro recombination system for the shuttling of entire libraries was then further tested by protein-protein interaction screens. The results of the library characterization and of the yeast two-hybrid screens and their implications for large-scale proteomic approaches are discussed.

CRFs form protein–protein interactions with each other and with members of the cytokinin signalling pathway in Arabidopsis via the CRF domain

“…The respective cDNAs were shuttled into these vectors via in vitro recombination. Based on the LiAc method, yeast transformations were conducted as described earlier (Bürkle et al , 2005). Weak autoactivation of some hybrid proteins was suppressed by supplementing the interaction medium with 5 mM 3-amino-1,2,4-triazole (3AT), whereas strong autoactivating hybrid proteins were tested in the presence of up to 40 mM 3AT.…”

The ArabidopsisTUMOR PRONE5Gene Encodes an Acetylornithine Aminotransferase Required for Arginine Biosynthesis and Root Meristem Maintenance in Blue Light

“…The PCR products were inserted into the yeast vector p423TEF by directional cloning using the BamHI and SalI sites (Mumberg et al, 1995). The resulting plasmid was transformed into Y37711 by the lithium acetate method (Gietz and Schiestl, 1995;Bürkle et al, 2005). Yeast transformants were first selected for His autotrophy and ampicillin resistance.…”

An Arabidopsis GluTR Binding Protein Mediates Spatial Separation of 5-Aminolevulinic Acid Synthesis in Chloroplasts

“…An Arabidopsis thaliana cDNA library was cloned into the plasmids pGAD10-GW-A, pGAD10-GW-B, and pGAD10-GW-C1 as described by Bü rkle et al (2005). Yeast strain L40ccua (Goehler et al, 2004) was used for protein interaction assays.…”