Acquisition of DNA Analysis Instrumentation for Molecular Studies

购买用于分子研究的DNA分析仪器 我们的目标是建立一个共同的核心设施的DNA序列分析。我们建议购买：自动DNA测序仪;分子生物学CATALYST实验室工作站;寡核苷酸合成仪;磷光成像仪工作站;荧光成像仪;计算机系统; Biomek II机器人;热循环仪;自动胶片显影仪;真空显影仪;照片记录站;和两个超低温冷冻机（-80和-30）。 参与的教师涉及与生物多样性和生物技术有关的广泛研究。他们的工作有一个共同的主题，DNA序列分析，并分为两个主要领域：1）分子遗传学方法研究基因表达，RNA加工，和分子生物学，和2）分子方法在种群遗传学，生态学和进化的研究。在分子遗传学方法研究基因表达领域。RNA加工和分子生物学，教师的研究和研究培训包括：惠伦的细菌病原体和植物之间的相互作用的分子遗传学分析;马奎斯-马格纳的调查，调节鞭毛蛋白基因表达的枯草芽孢杆菌的分子机制;以及Davis的多细胞动物中剪接前导RNA反式剪接的功能意义和调控，扁虫寄生的进化，以及扁形虫和早期后生动物的分子进化。 拉米雷斯研究的重点是克隆RECI基因，并进一步表征RPD 3（又名RPD 3）。REC 3）基因，其涉及受损DNA的修复和有丝分裂重组。戈德曼专注于真核染色体的一个结构单元，染色质结构域或环，及其作为哺乳动物X染色体失活和基因组印记中基因表达调控的功能单元的潜在作用。Perara研究了跨细胞膜蛋白质运输问题的分子方法，主要关注于跨内质网（ER）膜和细菌周质膜分泌蛋白质运输的分子机制的系统比较。在群体遗传学研究的分子方法领域。生态与进化，教师研究和研究培训包括：帕克/Cullings的植物群落和进化生态学，植物补充，生活史进化和社区周转率的动态研究;史密斯/贝利斯的进化生态学和保护生物学的调查，研究重点是热带雨林和萨凡纳之间的过渡区在物种形成中的作用，以及它们与保护环境有关的作用。雨林物种;阿普对环境的生理适应、泥滩无脊椎动物的硫化物耐受性和解毒作用、热液喷口生物的生态学和生理学的研究;兰德尔对沙漠啮齿动物的交流和社会组织进化的研究;科尔曼利用现代分子遗传学研究种群结构、地理学和种内遗传学，以及复杂表型的遗传结构。拉森的研究领域是近岸硬骨鱼的种群生物学，包括它们的行为、摄食生态、生活史和种群结构。 Desjardin专注于肉质真菌的分类学和系统发生学，主要是伞菌目，使用传统和现代的系统学方面，包括分支和形态测量分析，利用形态，生态，生理，遗传和分子数据。Hollibaugh致力于地球化学循环和食物网中细菌群落的生理生态学。Orrego采用分子遗传学技术研究自然群体中的遗传变异，这些遗传变异是通过长距离PCR方法获得的序列显示的。 我们提出的仪器将增加我们的研究和培训工作的数量和效率，使我们能够培训更多的学生。所要求的设备将提供最高分辨率，用于使用PCR扩增、DNA测序、变性梯度凝胶电泳（DGGE）、单链构象多态性（SSCP）和各种其他DNA/RNA检测和分析方法进行DNA/RNA分析。随着大量样品的吞吐量增加，可以快速有效地处理更长的序列阅读（700 bp），我们将能够比过去更快地对更多数量的样品进行新的高分辨率分析。例如，ABI 377测序仪提供了新的更长的凝胶格式，允许读取500个碱基/运行和36个泳道/运行，可以在8小时（或过夜）运行中完成，300-500 bp运行在2.25小时内完成，允许在8小时内运行3次。几名教师（Davis、Hollibaugh和帕克）将每年对超过10，000个样本进行集体测序，这代表一年内每天运行36个样本。虽然这一点本身就证明了我们的需要，但购买拟议的仪器也将使我们能够将这一数字增加两倍。 我们还将使用这些设备进行教师短期培训;自1991年以来，我们已经向来自35个州的240多名教师提供了10门这样的课程。 NSF表格1358（1/94）4

Acquisition of DNA Analysis Instrumentation for Molecular Studies Our goal is to establish a common core facility for DNA sequence analysis. We propose to purchase: an Automated DNA Sequencer; a molecular biology CATALYST Labstation; an Oligo Synthesizer; a PhosphorImager workstation; a Fluorimager; a Computer System; a Biomek II robot; a Thermocycler: al Automated Film Developer: a Vacuum Centrifuge: a Photodocumentation Station; and two Ultra Cold Freezers (-80 and -30). The participating faculty address a wide range of research related to biodiversity and biotechnology. Their efforts share a common theme of DNA sequence analysis and fall into two major areas: 1) Molecular genetic approaches to study gene expression, RNA processing, and molecular phylogeny, and 2) Molecular approaches in the study of Population Genetics, Ecology, and Evolution. In the area of Molecular genetic approaches to study gene expression. RNA processing. and molecular phylogeny, faculty research and research training include: Whalen's molecular genetic analyses of interactions between bacterial pathogens and plants; Marquez-Magana's investigations of the molecular mechanisms that regulate flagellin gene expression in Bacillus subtilis; and Davis's functional significance and regulation of spliced leader RNA trans-splicing in metazoa, the evolution of parasitism in flatworms, and the molecular phylogeny of flatworms and early metazoa. The focus of Ramirez' s research is to clone the RECI gene and further characterize the RPD3 (a.k.a. REC3) genes of Saccharomyces cerevisiae which are implicated in the repair of damaged DNA and in mitotic recombination. Goldman focuses on a structural unit of the eukaryotic chromosome, the chromatin domain or loop, and its potential role as a functional unit in the regulation of gene expression in mammalian X- chromosome inactivation and genomic imprinting. Perara investigates molecular approaches to the problem of protein transport across cell ular membranes, with the primary focus on a systematic comparison of the molecular mechanisms of secretory protein transport across the endoplasmic reticulum (ER) membrane and the bacterial periplasmic membrane. In the area of Molecular approaches in the study of Population Genetics. Ecology and Evolution, faculty research and research training include: Parker/Cullings' studies of plant community and evolutionary ecology, dynamics of plant recruitment, life history evolution and community turnover rates; Smith/Bayliss' investigations of evolutionary ecology and conservation biology with research focus on the role of transition zones between rain forest and savannah in speciation and their role as related to conservation of rain forest species; Arp's studies of physiological adaptation to environment, sulfide tolerance and detoxification in mudflat invertebrates, ecology and physiology of hydrothermal vent organisms; Randall's investigations of evolution of communication and social organization in desert rodents; and Routman' s use of modem molecular genetics to examine population structure, biogeography and intraspecific phylogeny, and the genetic architecture of complex phenotypes. Larson's research is in population biology of nearshore teleost fishes, including their behavior, feeding ecology, life histories, and population structure. Desjardin focuses on taxonomy and phylogeny of fleshy fungi, primarily Agaricales, using traditional and modern aspects of systematics, including cladistic and morphometric analysis, utilizing morphological, ecological, physiological, genetic and molecular data. Hollibaugh addresses physiological ecology of bacterial communities in geochemical cycles and foodwebs. Orrego employs the techniques of molecular genetics for the study of genetic variation in natural populations as displayed by sequences obtained via long distance PCR methods. Our proposed instrumentation will increase the amount and efficiency of our research and tr aining efforts and enable us to train a greater number of students. The requested equipment will provide the highest resolution available for the analysis of DNA/RNA using PCR amplification, DNA sequencing, Denaturing Gradient Gel Electrophoresis (DGGE), Single Strand Conformation Polymorphisms (SSCP), and a variety of other DNA/RNA detection and analytical methods. With the increased throughput capacity for large numbers of samples that can be processed rapidly and efficiently with longer sequence reading (700 bp), we will be able to do new high resolution analysis on much greater numbers of samples more rapidly than in the past. For example, the ABI 377 Sequencer provides new longer gel formats that allow one to read 500 bases/run and 36 lanes/run that can be completed in 8 hour (or overnight) runs and 300-500 bp runs in 2.25 hours, allowing 3 runs in an eight hour period. Several faculty (Davis, Hollibaugh, and Parker) will collectively sequence in excess of 10,000 samples/year which represents one run of 36 samples each day for a year. While this alone clearly justifies our need, the acquisition of the proposed instrumentation would also allow us the capacity to triple this number. We will also use the equipment for faculty enhancement short courses; since 1991 we have presented 10 such courses-to over 240 faculty from 35 states. NSF FORM 1358 (1/94) 4