In vitro recombination cloning of entire cDNA libraries in Arabidopsis thaliana and its application to the yeast two-hybrid system.
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.
Bürkle
Lukas
L
Max Planck Institute for Molecular Genetics, Berlin, Germany.
Meyer
Svenja
S
Dortay
Hakan
H
Lehrach
Hans
H
Heyl
Alexander
A
D017360:Arabidopsis / Q000235:genetics*; D018076:DNA, Complementary; D015723:Gene Library; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D020798:Two-Hybrid System Techniques
D018076:DNA, Complementary