| Volvox carteri is a multicellular green alga comprised of two distinct cell types: ∼16 large reproductive cells called gonidia and ∼ 2000 small motile somatic cells. The cells of a V. carteri individual are arranged spherically, with the somatic cells distributed at the surface of the spheroid, and the gonidia positioned slightly below. Since V. carteri possesses a basic 3-dimensional morphology and exhibits the simplest type of complete division of labor, it is an ideal system to study fundamental developmental mechanisms. Since V. carteri is haploid, it is relatively easy to find developmental mutants which disrupt asymmetric division, spheroid morphology, and somatic cell fate. Previously Jordan and Idaten, two cold-inducible transposons, were used to tag and clone several genes from these developmental mutant classes. Unfortunately, extensive efforts to tag and clone certain types of developmental genes with these transposons have failed. Though it is relatively easy to obtain developmental mutants in V. carteri, it is particularly difficult to conduct antisense or gene knockdown studies because of low transformation efficiency. Other knockdown approaches such as antisense technology have also been used in V. carteri, but are cumbersome and sometimes cause off targets effects. Knockdown studies are valuable for reverse genetic analyses on candidate genes suspected of playing important developmental roles. Presently, gene knockdown approaches are more routinely achieved by highly specific artificial microRNA technologies. The goal of this dissertation has been two fold, first, to identify and characterize additional class II transposons for gene tagging and second, to develop a more robust gene knockdown system in V. carteri. Here, we report the characterization and classification of 14 novel V. carteri class II transposon families and have identified members with the potential for transposon tagging experiments to identify and clone other important genes in V. carteri. Additionally, we have demonstrated that V. carteri possesses an endogenous miRNA system and present the first V. carteri amiRNA vectors that facilitate stable and specific knockdowns of target genes. |
