Automated DNA Sequencer and dHPLC Heteroduplex Analysis for Molecular Evolution and Development

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ABSTRACT 9970256Funds will be used to purchase an Applied Biosystems Prism 377 automated DNA sequencer and a Transgenomics WAVE high performance liquid chromatograph (dHPLC) to be shared by developmental and molecular evolutionary geneticists in the Institute of Molecular Evolutionary Genetics (IMEG) at Penn State University. These instruments will be used to establish a flexible, efficient, and cost-effective sequencing and genotyping facility. The strategy will employ a stratified sequencing approach, using dHPLC to identify fragment differences and automated sequencing of distinct fragments. The stratified approach is a rapid and proficient method for assessing DNA polymorphism in population-sized samples.The four principal investigators will use these instruments in a set of overlapping projects that integrate developmental and evolutionary genetics in a wide range of organisms. The projects include: Molecular variation in genes associated with adaptive evolution (Andrew Clark). Sequence analysis will be used to explore variation in the regulation of genes encoding metabolic enzymes in Drosophila. In addition, combined analysis of functional variation and DNA sequence polymorphism will be performed to characterize and quantify the molecular evolution of antibacterial defense in Drosophila and maternal-zygotic gene expression in early development; Organization of nucleotide diversity in the third chromosomal inversions (Stephen Schaeffer). Comparative DNA sequence analysis will be used to determine the interplay of selection, drift, and linkage in determining observed patterns of genetic variation in the classical third chromosome inversions of D. pseudoobscura; Genetic regulation of early development and associations with interspecific differences in body plans (Billie Swalla). This project is investigating the evolution of body plans by identifying the genes responsible for morphological differences between closely related species of ascidians in the genus Molgula. Molecular phylogenies will be determined and candidate developmental genes that may be responsible for morphological evolution will be examined in hemichordates and urochordates and; Genetic basis of experimental evolution of virulence (Thomas Whittam). Molecular methods render the evolution of virulence amenable to direct study in the laboratory. Following a regime of experimental evolution in a microbial host-parasite system, this project will compare ancestral and derived strains of Legionella and identify specific nucleotide substitutions at known virulence-associated loci used to exploit protozoan hosts. In addition to these major projects, creative use of dHPLC and DNA sequence and fragment analysis is outlined by an additional eight minor users who are active members of IMEG.