NAME: Mallory Douthitt
 

PROJECT TITLE: Electrophoretic Karyotype Comparisons for Acanthamoeba spp

Acanthamoeba are free-living, eukaryotic amoebae that are most frequently isolated from various types of soil and aquatic environments. Certain strains have been shown to be opportunistically pathogenic, able to cause amoebic keratitis in contact wearers, and other diseases in immunocompromised individuals. Hundreds of isolates and more than 20 named species have been described in the literature, many with unique physiological characteristics (Marciano-Cabral F, Cabral G. 2003). Attempts to classify strains within the Acanthamoeba genus on the basis of cyst morphology have been shown to be inadequate, due to the fact that variation in cyst morphology can arise within the same isolate as a result of different growth media, temperatures, as well as other variables (Khan 2006) Sequencing of small segments of ribosomal genes has revealed the presence of 15 distinctive genotypes (T1-T15) (Stothard et al.1998). However, no previous studies have investigated the variability of the whole genome. The proposed study will utilize whole genome characterization to examine the extent of genome variability and/or conservation between different environmental isolates of Acanthamoeba. Electrophoretic karyotypes will be generated for a wide array of isolates. Banding patterns between different isolates will be examined with regards to phenotypic characters and bands of interest will be isolated from gels for further analysis. In these studies, the initial focus will be on culture methods, sample preparation and optimization of electrophoresis conditions. Techniques will be refined for the isolation of chromosomal DNA fragments and the generation of electrophoretic karyotypes. Procedures for efficient extraction of high molecular weight DNA from gels will be developed. Refinement of these methods will ultimately contribute to development of chromosome specific clone libraries from individual Acanthamoeba chromosomes.