Mechanism of incomplete cytokinesis during germ cell development
Novel means of generating conditional mutations
The functions of genes predicted from genome sequencing projects must be identified. This relies greatly on the analysis of loss-of-function phenotypes. Conventional gene-targeting techniques generate loss-of-function mutations by permanently deleting the specific gene of interest or by rendering it nonfunctional. However, this strategy falls short for two groups of genes: essential genes and pleiotropic genes. The former are required for viability and the latter function at multiple times and/or different places in the life cycle of an organism. In contrast to conventional gene knock-outs, heat-sensitive mutations (traditionally known as temperature-sensitive (TS) mutations) are powerful tools to study the functions of all genes. This includes both essential and pleiotropic genes, because TS mutations are reversible and easy to use. However, TS alleles are rare and difficult to generate and identify, and this has limited their use in most multicellular organisms. To address this deficiency, one of the goals of my lab is to generate a collection of widely applicable genetic/molecular tools to conditionally ablate protein function(s), including the following:
1. Generating Conditional Mutations Using Temperature-sensitive Intein Alleles .
2. One Step Assembling of Multi-DNA Segments Using Split-marker-mediated Cloning
3. Directly Introducing TS Intein into a Gene of Interest
4. Insect Control with TS Inteins
5. Generating Destabilizing Inteins or Cold-Sensitive Inteins
Mechanism of incomplete cytokinesis during gametogenesis
Our lab is also interested in studying the mechanism of incomplete cytokinesis during gametogenesis. Incomplete cytokinesis is a special kind of cytokinesis and a hallmark of germline stem cell differentiation. Incomplete cytokinesis is a nearly universal process during spermatogenesis; and plays an essential role in oogenesis in Drosophila, humans and other species. Incomplete cytokinesis occurs when the cleavage furrow fails to close completely during cell division. Resulting daughter cells remain interconnected via the cleavage furrow that is subsequently modified and becomes a ring canal. The mechanism mediating arrest of cleavage furrow constriction is unknown. Our studies of IC in wild type flies (Ong and Tan, 2010) has set up a foundation for studying IC on the molecular level in this organism. Furthermore, we have identified the first gene involved in cytokinesis arrest (Ong et al. 2010). By elucidating the mechanism of incomplete cytokinesis in Drosophila, we hope to further our understanding of gametogenesis and stem cell differentiation, as well as to gain insight into infertility pathologies.
Tan C and Tomkins J. 2015. Information processing differences between bacteria and eukarya – implications for the myth of eukaryogenesis, Answers Research Journal. 143–162.
Tan C and Tomkins J. 2015. Information processing differences between archaea and eukaryotes – implications for the myth of homologs and eukaryogenesis. Answers Research Journal. 121–141.
Yamamoto S, Bayat, V, Bellen H, and Tan C. 2013. Protein Phosphatase 1ß Limits Ring Canal Constriction During Drosophila Germline Cyst Formation. PLoS One. 8
Ong S, Foote C, and Tan C. Mutations of DMYPT cause over constriction of contractile rings and ring canals during drosophila germline cyst formation. Dev. Biol. 346: 161-9.
Tan G and Tan C. 2010. SMC, a simple method to rapidly assemble multiple fragments into one construct. Frontiers in Bioscience. E2: 1105-1114.
Ong S and Tan C. 2009. Germline cyst formation and incomplete cytokinesis during Drosophila melanogaster oogenesis. Dev. Biol. 337: 84-98.
Tan G, Chen M, Foote C, and Tan C. 2009. Temperature-sensitive mutations made easy – generating conditional mutations by using TS-inteins that function within different temperature ranges. Genetics. 183(1): 13-22.