Acquisition of DNA Analysis Instrumentation for Molecular Studies

收购用于分子研究的DNA分析仪器我们的目标是建立一个用于DNA序列分析的共同核心设施。我们建议购买：一台自动DNA测序仪；一台分子生物催化剂实验室站；一台寡聚合成器；一台磷光成像仪工作站；一台荧光成像仪；一台计算机系统；一台Biomek II机器人；一台热循环仪：AL自动胶片显影仪：一台真空离心机：一台摄影文档站；以及两台超冷冷冻机(-80和-30)。参与研究的教师从事与生物多样性和生物技术相关的广泛研究。他们的努力有一个共同的DNA序列分析主题，并分为两个主要领域：1)研究基因表达、RNA加工和分子系统学的分子遗传学方法；2)研究种群遗传学、生态学和进化论的分子方法。在研究基因表达的分子遗传学方法领域。RNA处理。Whalen对细菌病原体与植物之间相互作用的分子遗传学分析；Marquez-Magana对调节枯草芽孢杆菌鞭毛基因表达的分子机制的研究；Davis对后生动物剪接前导RNA反式剪接的功能意义和调控；扁虫寄生的进化；以及扁虫和早期后生动物的分子系统发育。克隆RECI基因并进一步鉴定RPD3(又名RPD3)是拉米雷斯·S研究的重点。酿酒酵母REC3)基因与DNA损伤修复和有丝分裂重组有关。戈德曼专注于真核染色体的一个结构单位，即染色质结构域或环，以及它在哺乳动物X染色体失活和基因组印记中作为功能单位调节基因表达的潜在作用。Perara研究了蛋白质跨细胞膜运输问题的分子方法，主要集中在系统比较分泌蛋白质跨越内质网(ER)膜和细菌周质膜运输的分子机制。在群体遗传学研究中的分子方法领域。生态学和进化论、教师研究和研究培训包括：Parker/Culling对植物群落和进化生态学、植物招募动态、生活史进化和群落周转率的研究；Smith/Bayliss对进化生态学和保护生物学的研究，重点是雨林和稀树草原之间的过渡带在物种形成中的作用及其与雨林物种保护有关的作用；Arp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、热液生物的生态学和生理学的研究；Randall对沙漠啮齿动物交流和社会组织进化的研究；鲁特曼的S利用现代分子遗传学研究了种群结构、生物地理学和种内系统发育，以及复杂表型的遗传结构。拉森的研究方向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和系统发育，主要是蘑菇目，使用传统和现代的系统学方面，包括支序和形态计量分析，利用形态、生态、生理、遗传和分子数据。霍利堡研究了地化循环和食物网中细菌群落的生理生态。Orrego利用分子遗传学技术研究自然种群的遗传变异，如通过长距离聚合酶链式反应方法获得的序列所显示的那样。我们建议的仪器将增加我们的研究和研究工作的数量和效率，并使我们能够培养更多的学生。所要求的设备将提供现有最高分辨率的DNA/RNA分析，使用的是聚合酶链式反应扩增、DNA测序、变性梯度凝胶电泳(DGGE)、单链构象多态(SSCP)以及各种其他DNA/RNA检测和分析方法。随着大量样品的吞吐能力的增加，这些样品可以通过更长的序列读数(700 Bp)快速有效地处理，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，允许一个人读取500个碱基/运行和36个通道/运行，可以在8小时(或一夜)运行完成，300-500个核苷酸运行在2.25小时，允许在8小时内运行3个。几个教员(Davis、Hollibaugh和Parker)将每年对超过10,000个样本进行集体测序，这相当于一年中每天36个样本的一次排序。虽然单凭这一点就清楚地证明了我们的需要，但购置拟议的仪器也将使我们有能力将这一数字增加两倍。我们还将使用这些设备举办教师强化短期课程；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