Generating Conditional Mutations by Random Insertion of an Intein Switch

University Of Missouri-Columbia, Columbia MO

Investigators

Abstract

Generating Conditional Mutations by Random Insertion of an Intein Switch Reversible and easy to use, temperature-sensitive (TS) mutations are powerful tools to study the functions of all genes, including essential and pleiotropic ones. However, the rarity of TS alleles and the difficulty of generating and identifying them have limited their use. Working with Drosophila, the PI has developed a novel approach to generate TS mutations efficiently using a conditionally-splicing intein called an intein switch. An intein is an exon that splices itself out of its "host" protein, and an intein switch is an intein in which the splicing activity is temperature-sensitive and thus can be "turned off." The presence of the extra exon in the host protein often inactivates that host protein. The PI's intein switch can be inserted into almost any gene. At the permissive temperature (18 degrees C) it excises itself to generate a wild-type host protein. At the non-permissive temperature (30 degrees C), it fails to splice and remains within the host protein, often leading to the loss of the function of the host protein. This project will generate a multi-purpose construct to facilitate isolation of TS mutations created by insertions of intein switches and will determine the efficacy of using the intein switch to generate conditional mutations. The long-term goal is to make the intein switch a universal tool and to generate a genome-wide collection of TS mutations for systematically assigning function(s) to each gene. This project will develop tools to facilitate generating a genome-wide collection of TS mutations and to perform a pilot screen for providing proof of principle for generating such a collection. This project will broaden diversity by providing support for participation of undergraduate students in research. In addition, the results will be disseminated via a community-outreach program, Saturday Morning Science, to high school students as well as other interested non-scientists.