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.
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. One of our long-term goals is to make our intein switch a universal tool and to generate a genome-wide collection of TS mutations for systematically assigning function(s) to each gene. The intein switch is a TS-intein that can be inserted into any gene. At the permissive temperature it is excised to generate a wild-type host protein. At the non-permissive temperature, it fails to splice and remains within the host protein, leading to loss-of-function of the host protein. This pilot project aims at developing tools to facilitate generating a genome-wide collection of TS mutations. The NSF funds have enabled us to generate widely applicable tools for the science community. We have generated and characterized 41 intein switches, temperature-sensitive Sce VMA mutations, each of which splices at its own unique permissive temperatures to generate intact host proteins (Tan et al., 2009). Our collection of TS-intein switches constitutes a new toolbox for the generation of TS mutations tailored to specific experimental conditions or growth requirements of a particular organism. We have also developed a simple method, named SMC (split-marker-mediated cloning), to rapidly assemble complex constructs in yeast (Tan and Tan, 2010). In this approach, a selectable marker is split into two non-functional, overlapping halves, of which one is located on the plasmid backbone. Homologous recombination reconstitutes the marker gene and assembles all DNA fragments in the desired order. This method allows rapid one-step fusion of various DNA fragments that contain ~30 base pair overlaps in yeast. In addition, the NSF funding has allowed us to develop methods to directly target specific genes in Drosophila, as well as in haploid yeast cells, to control the activities of endogenous genes in a temperature-dependent manner. With the support of the NSF funding, six people, four undergraduate students, a graduate student, and a postdoc have been trained. These four undergraduates, of whom three are women, had never worked in a laboratory before. The reagents generated in this project have been distributed to 31 laboratories in ten countries to generate conditional mutations in bacteria, yeast, worms, flies, zebrafish, and plants. Reference: Tan, G., Chen, M., Foote, C., and Tan, C. (2009). Temperature-sensitive mutations made easy: generating conditional mutations by using temperature-sensitive inteins that function within different temperature ranges. Genetics 183, 13-22. Tan, G., and Tan, C. (2010). SMC, a simple method to rapidly assemble multiple fragments into one construct. Front Biosci (Elite Ed) 2, 1105-1114.
