Acquisition of DNA Analysis Instrumentation for Molecular Studies

我们的目标是为DNA序列分析建立一个通用的核心设施。我们建议购买：自动DNA测序仪；分子生物学催化剂实验室；寡聚合成器；磷成像仪工作站；Fluorimager;计算机系统；Biomek II机器人；一个热循环仪，一个自动显影剂，一个真空离心机，一个摄影工作站；两个超低温冰柜（零下80度和零下30度）。参与的教师讨论了与生物多样性和生物技术有关的广泛研究。他们的研究都以DNA序列分析为主题，并分为两个主要领域：1)研究基因表达、RNA加工和分子系统发育的分子遗传学方法；2)研究群体遗传学、生态学和进化的分子遗传学方法。在分子遗传学领域研究基因表达的方法。RNA加工。和分子系统发育，教师研究和研究培训包括：Whalen的细菌病原体和植物之间相互作用的分子遗传分析；Marquez-Magana对枯草芽孢杆菌中鞭毛蛋白基因表达调控的分子机制的研究；复生动物剪接前导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。Ramirez的研究重点是克隆RECI基因，并进一步表征酿酒酵母中与受损DNA修复和有丝分裂重组有关的RPD3（又名REC3）基因。Goldman专注于真核生物染色体的一个结构单元，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基因表达调控中的潜在功能单元作用。Perara研究了蛋白质跨细胞膜运输问题的分子方法，主要关注分泌性蛋白质跨内质网（ER）膜和细菌质周膜运输的分子机制的系统比较。在群体遗传学研究中的分子方法领域。生态学和进化，教师研究和研究培训包括：帕克/Cullings对植物群落和进化生态学的研究，植物招募动力学，生活史进化和群落周转率；Smith/Bayliss对进化生态学和保护生物学的研究，研究重点是热带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热带雨林物种保护相关的作用；泥滩无脊椎动物对环境的生理适应、硫化物耐受与解毒、热液喷口生物的生态生理研究；兰德尔对沙漠啮齿类动物交流和社会组织进化的研究以及Routman使用现代分子遗传学来研究种群结构、生物地理学和种内系统发育，以及复杂表型的遗传结构。Larson的研究方向是近岸硬骨鱼的种群生物学，包括它们的行为、摄食生态学、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Agaricales，使用传统和现代系统学的方面，包括分支学和形态计量学分析，利用形态学，生态学，生理学，遗传学和分子数据。Hollibaugh在地球化学循环和食物网中研究细菌群落的生理生态学。Orrego采用分子遗传学技术研究自然种群的遗传变异，通过长距离PCR方法获得的序列显示。我们提出的仪器将增加我们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的DNA/RNA分析，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），以及各种其他DNA/RNA检测和分析方法。随着大量样品的处理能力的提高，可以快速有效地处理更长的序列读取（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个通道/运行，可以在8小时（或过夜）运行和300-500 bp运行在2.25小时内完成，允许在8小时内运行3次。几位教师（Davis， Hollibaugh和Parker）将共同测序超过10,000个样本/年，这代表了一年每天36个样本的一次运行。虽然仅凭这一点就清楚地证明了我们的需要，但收购拟议的仪器也将使我们的能力增加到这一数字的三倍。我们也会利用这些设备开办教师进修短期课程；自1991年以来，我们已经向来自35个州的240多名教师开设了10门这样的课程。NSF表格1358 (1/94

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