Efficient Approaches for Generating GFP Fusion and Epitope-Tagging Constructs in Filamentous Fungi
For functional characterization of predicted genes encoding hypothetical proteins in fungal genomes, it is complementary to genetic studies to determine their expression and subcellular localization patterns in different developmental or infection stages. It is also important to identify and characterize other proteins that are physically associated with or functionally related to these genes in vivo by co-immunoprecipitation or affinity purification analyses. In this chapter, we described a set of yeast shuttle vectors and protocols to generate fusion constructs by the yeast gap repair approach. Because of the simplicity and efficiency of yeast gap repair, these vectors and the general methods described in this chapter are suitable for functional genomics studies in filamentous fungi.
- Construction of a Bacterial Artificial Chromosome Library
- Printing DNA Microarrays Using the Biomek 2000 Laboratory Automation Workstation
- High-Throughput Analysis of Protein-DNA Binding Affinity
- PCR-SSCP Analysis of Polymorphism: A Simple and Sensitive Method for Detecting Differences Between Short Segments of DNA
- Discontinuous Polyacrylamide Gel Electrophoresis of DNA Fragments
- Tapping Genomics to Unravel Ectomycorrhizal Symbiosis
- BAC Transgenes, DNA Purification, and Transgenic Mouse Production
- Molecular Assays to Investigate Chromatin Changes During DNA Double-Strand Break Repair in Yeast
- Genetic Distance and Markers Used in Linkage Mapping
- Ribosome Display for Rapid Protein Evolution by Consecutive Rounds of Mutation and Selection