Isolation of proteins involved in posttranscriptional gene silencing and methods of use - Patents

Isolation of proteins involved in posttranscriptional gene silencing and methods of use
United States Patent 7,001,739
Mirkov , et al.
February 21, 2006
Inventors: Mirkov; T. Erik (Harlingen, TX); Ingelbrecht; Ivan L. (Jabbeke, BE)
Assignee: The Texas A&M University System (College Station, TX)
Appl. No.: 226715
Filed: August 23, 2002
Abstract
The present invention includes a method for detecting and isolating sugarcane proteins that interact with the HC-Pro and P1 proteins of SrMV and other proteins involved in gene silencing, particularly in sugarcane. The method uses a two hybrid assay with an HC-Pro, P1, or other silencing-related protein-containing bait protein and a prey protein containing a polypeptide encoded by a DNA molecule in a cDNA library. The method also includes identification of false positives through reverse two-hybrid assays and using in vitro techniques such as farwestern blots or pull down assays where plant physiological conditions may be replicated. Finally, interactions may be confirmed in planta. Some novel proteins used in and discovered using the these methods are also identified. Methods of using viral and plant proteins to regulate silencing in plants such as sugarcane are also discussed.