The goal of the research is to understand the genetics and evolution of the mariner transposable element and to investigate mariner as a potential tool for genetic manipulation of insect disease vectors and agricultural pests. At least five major subfamilies of mariner (40-56% identical at the nucleotide level) are widespread in invertebrate species. Most of the described elements are genetically defective (nonautonomous), except for certain elements in the mauritiana subfamily. These elements can support germline transformation with exogenous DNA in D. melanogaster. However, the efficiency of germline transformation is low (0.7%), and the transformants are unexpectedly stable in both germline and soma.
Specific Aim 1 seeks to increase the rate of germline transformation by constructing and testing mariner vectors with exogenous DNA fragments of several size classes inserted in at least two unique restriction sites (SacI and SalI) within the element. The vectors will be introduced into D. melanogaster by P-element transformation in order to study germline transposition, as well as germline and somatic excision. This approach allows the genetics of altered mariner elements to be studied, independently of their ability to support mariner-dependent germline transformation. A subset of the vectors, along with various helper elements, will be tested for mariner-dependent germline transformation in D. melanogaster and in progressively more distantly related alternative hosts (D. ananassae, D. pseudoobscura, and D. virilis). A specialized G418-resistance vector will also be constructed for use in other insects.
Specific Aim 2 will determine whether certain naturally occurring deletions of mariner repress mariner activity, which might potentially hinder germline transformation. Deletions account for almost the entire mariner complement in D. teissieri and D. ercepeae. We hypothesize that some deletions function as repressors analogous to the KP elements in the P system. Putative repressor elements will be introduced into D. melanogaster by P-element transformation, and their effects on mariner excision, both somatic and germinal, will be determined.
Specific Aim 3 will characterize additional subfamilies of mariner elements present in the melanogaster species subgroup. Two different subfamilies have been identified in D. erecta and D. melanogaster (the first example of a mariner subfamily in D. melanogaster). We will sequence these elements, determine their distribution among related species, and determine whether the divergent elements were introduced into the genome by vertical or horizontal transmission. Putative functional elements will be tested for autonomous function, as well as interaction with the mauritiana subfamily, using P-element germline transformation.
Showing the most recent 10 out of 23 publications
