A Positive Control for the Green Fluorescent Protein-Based One-Hybrid System

✍ Scribed by Thomas Barz; Karin Ackermann; Walter Pyerin

• Publisher: Elsevier Science
• Year: 2002
• Tongue: English
• Weight: 127 KB
• Volume: 304
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.2002.5618

✦ Synopsis

The yeast one-hybrid system (1H-system) is applied to studies of transcription-linked DNA-protein interactions under in vivo conditions detected by reporter gene expression. By use of a variant of green fluorescent protein (GFP) as a reporter, the new 1H-system generation represents a time-and work-saving alternative to the established HIS3/lacZ-based form. However, hitherto, a positive control proving the functionality of the system has been missing. We designed a corresponding control vector combination by subcloning mouse p53 cDNA and its binding sequence and, to get the system working, modified the distance between cloning site and reporter gene promoter in the reporter vector by site-directed mutagenesis. This provides, for the first time, a positive control vector combination for the GFP 1H-system, crucial for its employment in any DNA-protein interaction studies.