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

摘要9970256 Funds将用于购买一台应用生物系统公司的Prism 377自动DNA测序仪和一台转基因组学波型高效液相色谱仪，供宾夕法尼亚州立大学分子进化遗传学研究所的发育和分子进化遗传学家共享。这些仪器将被用来建立一个灵活、高效和具有成本效益的测序和基因分型设施。该战略将采用分层测序方法，使用dHPLC来识别片段差异，并对不同的片段进行自动测序。分层方法是在种群大小的样本中评估DNA多态的一种快速而熟练的方法。四名主要研究人员将在一系列重叠的项目中使用这些工具，这些项目整合了广泛生物的发育和进化遗传学。这些项目包括：与适应性进化相关的基因的分子变异(安德鲁·克拉克)。序列分析将被用来探索果蝇代谢酶编码基因调控的变异。此外，还将进行功能变异和DNA序列多态性的联合分析，以表征和量化果蝇抗菌防御的分子进化和母体合子基因在早期发育中的表达；第三染色体倒位中核苷酸多样性的组织(Stephen Schaeffer)。比较DNA序列分析将被用来确定选择、漂移和连锁的相互作用，以确定在经典的假暗纹夜蛾第三染色体倒位中观察到的遗传变异模式；早期发育的遗传调节以及与物种间体型差异的关联(Billie Swalla)。这个项目是通过确定导致近缘海鞘物种之间形态差异的基因来研究身体平面的进化。分子系统发生将被确定，可能负责形态进化的候选发育基因将在半角类和尾索类中被检查；毒力实验进化的遗传基础(托马斯·惠特姆)分子方法使得毒力的进化可以直接在实验室进行研究。在微生物宿主-寄生虫系统的实验进化制度之后，该项目将比较军团菌的祖先和衍生菌株，并确定用于利用原生动物宿主的已知毒力相关基因上的特定核苷酸替换。除了这些主要项目外，IMEG的活跃成员还有另外八个小用户概述了dHPLC和DNA序列和片段分析的创造性使用。

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.