David Burke - US grants

Incorrect regulation of gene expression is observed during mammalian aging and may contribute to the pathology of senescence. One class of genetic misregulation is the reactivation of previously repressed genes. The proposed experiments will examine the aging-related re-expression of genes from inactive Xchromosomes. Inactivated X chromosomes of female mammals are transcriptionally repressed and maintained as highly-condensed, late-replicating heterochromatic DNA. The mechanism for maintenance of X-inactivation is uncharacterized, as are the DNA sequences which contribute to the inactive state. In addition, X-inactivation may share common features with the maintenance of other forms of heterochromatin and the programmed inactivation of autosomal genes. Two genetic loci from the mouse X chromosome, sparsefur and Mottled, have previously been shown to undergo aging-dependent reactivation. However, it has not been demonstrated that reactivation is a general phenomenon of X-linked genes. For this proposal, transcriptional reactivation will be examined from a defined set of X-chromosome genes during normal aging. In the female mouse, alleles from each X-chromosome homologue can be assayed independently. Allele-specific expression assays are based on naturally-occurring RNA sequence polymorphisms between interbreeding mouse species. Interspecific hybrid (F1) females maintain one X-chromosome from each parental species. Consequently, these animals are heterozygous for alleles at virtually every genetic locus. For each locus tested, RNA polymorphism assays will be used to detect the relative contribution from each allele in mRNA samples prepared from aged F1 female mice. Aging-dependent reactivation will be detected as increased mRNA expression from the initially repressed allele. The proposed experiments will focus on the reactivation time-course of genes within the X-chromosomal region adjacent to the centromere. The X-pericentromere is a unique genomic location where inactive-X heterochromatin and centromeric heterochromatin may contribute overlapping effects to expression. In addition, the approximate 3 cM centromere-proximal region contains several characterized genetic and molecular loci, including the known reactivated gene ornithine transcarbamylase (Otc=sparsefur). The high density of genomic mapping resources for the region will expedite assembly of a comprehensive physical map. Such a map will be essential for assessing relationships between gene location and aging-dependent reactivation.

This Program Project is the continuation of collaboration between three research groups that have been working together since 1993. The successful interdisciplinary team represents knowledge in chemical collaborative group has focused on the development of microfabricated DNA analysis devices with complete process integration. The continuation proposal will develop a fully integrated DNA sample handling and analysis technology for genotyping and sequencing. All the steps for extracting genetic information will occur within a single self-contained system having minimal operator interaction. The microfluidic, optical, and electronic components of the system are fabricated on silicon, silicon/glass, or silicon/polymer substrates, yielding a micromechanical integrated DNA analysis technology. To ensure inexpensive design, testing, and production, the devices will be made using photolithographic construction techniques. A genomic sequencing device capable of generating raw DNA sequence at less than $0.01 total costs per base is targeted for completion in five years. The proposal has four Program Goals: 1) Design, construct, and evaluate integrated DNA analysis systems. A system-level approach will be used to evaluate all components and fabrication technologies developed by the Program Project. Success will be measured at the level of complete genotyping and sequencing system. (Project 1) 2) Design, construct, and evaluate improved microfluidics, microreactions, and microseparations. Multistep handling and reactions will be tested on microfabricated devices. Advanced gel electrophoresis systems for sequencing t read-length >500 bp will be designed and tested. (Project 2) 3) Develop improved fabrication methods and optical sensors for integrated microsystems. Thin film deposited polymer and micromolded polymer will be examined for use as substrates and water-tight channels. Optical sensors will be designed and tested for improved sensitivity and stability. (Project 3) 4) Provide facilities and support for construction and testing of complex, integrated microfluidic devices. A central assembly and testing facility will be maintained that can rapidly build and test prototypes and incremental improvements for each of the projects (Core Unit).

gene induction /repression; gene expression; developmental genetics; sex chromosomes; aging; animal genetic material tag; female; laboratory mouse;

The Core Unit research engineers will be a central resource for devise assembly and testing expertise for all Projects. The research engineers have over 8 years of combined expertise in silicon, glass, and polymer photolithographic fabrication at the Solid State Engineering Laboratory (SSEL). Personnel also have experience in instrumentation design, circuit design, and chemical processing. The research engineers will assures that components are tested and judged consistently, both in isolation and within integrated devices. As a consequence, component testing between projects will references a common benchmark. The Core facility will also allow a pooling of expensive capital resources that are currently held in separate laboratories or are expensive to replicate at several locations. The Core Unit has four Specific Aims: 1) Provide photolithographic fabrication support for all Projects. support will include training and assistance in SSEL procedures as well as large-scale production runs of silicon wafers. Fabrication processes provided by the Core Unit personnel will be backed upon quality control assessment of each fabrication run. 2) Provide deice assembly support for all Projects. The Core will assist in solving assembly and post-fabrication problems for all projects. This will include flow problems, heat and/or mass transport problems, and macro-to-micro scale interfacing. Production level assembly will be available, including wafer dicing, wire bonding, package gluing, and quality assessment. 3) Provide standardized reagents, testing methods, and operation support for all Projects. Core personnel are available for consultation in the design phase of each Project. The Core will assist in the operation of the devices using biological samples, including provision of quality tested common reagents. Computer software support for laboratory instrumentation is also provided. 4) Provide a common platform for testing of devices. The Core will allow students and research engineers from all Projects to use a common set of testing and analysis equipment. Core personnel will develop instrumentation electronics and the uniform test platform for device testing. Test platforms will be constantly improved over the course of the project.

The Genotyping Core will provide genetic information on the UM-HET3 female mice used for all aspects of the Program Project. The 600 Population 1 animals produced in Animal ore will be genotyped at 150 loci, with each locus able to distinguish the four grandparental alleles. For each animal the genotype scan will provide approximately 15 to 20 cM coverage across the genome. Genotyping will be performed on additional UM-HET3 females to screen for a small number of candidate lifespan-associated loci. Animals identified as having the desired combination of alleles at the loci will be retained as Population 2 (180 animals final total). The Populations will be examined for phenotypes in the individual Projects and assessed for genotype by-phenotype associations by Data Analysis Core. The Specific Aims: Aim 1. Establish a high-throughput genotyping service capable of handling approximately 30,000 PCR-based genotype assays per year. Aim 2. Prepare and quantify genomic DNA from all project animals. Aim 3. Identify, develop, and quality assess 150 polymorphic marker reactions. Additional marker reactions will be identified in candidate regions of the gnome in the final two years of the Project. Aim 4. Generate high-quality genotype data from 150 marker on 600 Population 1 animals. The final dataset will include approximately 90,000 genotypes with less than 3% missing data and less than 1% error. The data will be transferred to Data Animal Core for analysis. Aim 5. Generate high-quality genotype data at six polymorphic loci on approximately 1500 UM-HET3 candidate animals for Population 2. Genotypes will be determined at 8 weeks of age and 180 animals will be selected based on the genotypes. The loci used for the screen will bracket three quantitative trait loci (QTL) proposed as candidates for lifespan. For this screen, approximately 9000 genotype values will be obtained over two years, with percent error and missing data levels comparable to Population I. Aim 6. Develop a laboratory information management system (LIMS) for efficient genotype data handling, resource management, and cost control.

Analysis methods for DNA genotyping and sequencing are linked into multiple-step processing system that begin with DNA source material and extract genetic information. In conventional methods, the processing system operates on batches of microliter-volume reaction samples, together with matched-liquid handling devices and electrophoretic analysis equipment. Project 1 will provide a systems-level approach to evaluate the integrated nano-liter volume components and fabrication technologies developed in the Program Project. The success of microfabricated components will be measured by their ability to work as an autonomous multi-step processing system. Project 1 has four Specific Aims: 1) Develop the microfluidics necessary for integrated nanoliter-scale DNA analysis. Microfluidic components will be used to construct an integrated system that can perform and monitor multiple parallel reactions. An interface with microliter-volume source samples will also be addressed. 2) Demonstrate sequencing and genotyping reactions in an integrated system. Microfluidic devices will be optimized for use with Sanger sequencing or genomic amplification reactions and denaturing gel electrophoresis. The system-level device will perform and monitor polymerase reactions, post reaction treatment, and electrophoretic band migration, with minimal operator intervention. 3) Demonstrate high-quality genomic DNA sequencing in an integrated systems. The resolution of the integrated nanoliter-volume system will be improved to match the level of existing microliter-scale integrated nanoliter-volume system will be improved to match the level of existing microliter-scale technologies. This will include improved fluorescence detectors (with Project 3) and higher resolution gels (with Project 2). Successful completion of this aim will result in a device that can obtain 500-1000 bp of sequence from a sample of template DNA. 4) Demonstrate complex sequencing strategies in an integrated device. Complex sequencing strategies that obtain maximal information from a single DNA sample, such as bi-directional sequencing or simultaneous restriction digestion, will be added to the integrated sequencing device. Issues of quality control, accuracy, and data reproducibility will also be examined.

0304316
Larson
This proposal was received in response to Nanoscale Science and Engineering initiative, NSF 02-148, category NIRT. The objective of this proposal is to design and build a microfabricated platform for study of DNA/protein interactions at the nano-scale. These interactions are at the heart of the cellular machinery for maintaining and transcribing DNA, and include transcription factors, as well as histones and other chromatin proteins. These DNA/protein interactions are under active study at the cellular level, but the next steps towards a detailed understanding of them require their study at smaller length scales, including the nanoscale. Therefore, a microfabricated devise will be built, which contains a main channel of length 20 to 30 microns and of width ranging from 20 nm to 2 microns. Individual DNA molecules will be stretched in the channel and positioned using high frequency electric fields, and anchored at gold electrodes using thiol groups chemically attached to the ends of the DNA molecules. This long, very thin, channel will be joined to thin side channels for addition of DNA-interacting proteins locally to specific regions of the DNA molecule. The proteins will be further localized by use of capture electrodes as well as hydrodynamic focusing using flow through other side streams, and other confinement methods. The channels will be etched in silicon, and bonded to a thin glass substrate, to permit examination by optical microscopy. The broader impacts of the proposal include the integration of the proposed research with the introduction of a new Nanoscale Science and Engineering (NSE) curriculum for graduate students. The new course will focus on nano- fabrication issues related to integration of electronic function with microfluidics and transport issues related to the processing of biological samples.

The Genomic Analysis Core will provide access to genetic resources for University of Michigan Pepper Center scientists, and will enhance the linking of genetic information with studies of the aging process and health of older adults. The Core will assist Center investigators studying the basic science of aging, as well as provide a foundation for human genetic studies. The Genomic Analysis Core has four Specific Aims: Aim 1. Provide genotyping service support to Center investigators. The Core will perform simple sequence length polymorphism and single nucleotide polymorphism detection. The Core will have available pre-tested mouse and human genotyping loci, and will develop custom genotype procedures. Aim 2. Provide genomie analysis, biostatistical, and data handling infrastructure and support. The Core will develop and use genetic analysis tools for quantitative trait locus genome searches, as well as linkage and association studies. The Core will provide a central location for data management of genetic and phenotypic information. Core personnel will provide expert advice on genetic data handling, statistical analysis, and experimental design. Aim 3. Provide an infrastructure for evaluation and development of human genetics projects. The Core will develop a genetic research infrastructure for use with older adults. Consultation services will be provided on population structures, clinical data management, analysis, and subject communication. Personnel will provide expertise on regulatory, legal, and ethical standards for genetic studies. Aim 4. Provide a development structure for novel genetics projects in aging. The Core will solicit, evaluate, and integrate Core Development Projects designed to improve the genomics capabilities of Center investigators. Emphasis will be placed on development projects that co-ordinate, supplement, or enhance existing research activities of the Center.

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).

Soil pH (acidity) has a major influence on soil fertility, and therefore the structure and function of ecosystems. The availability of phosphorous (P), in particular, is highly controlled by soil pH. Both natural processes and human-induced disturbance, such as acid rain, have resulted in acidification of some ecosystems. Soil acidification can lead subsequently to a decrease in phosphorous (P) availability to eastern hardwood forests. However, to date there has not been evidence of P limitation in these forests, whether as a result of natural or anthropogenic acidification. This research is focused on testing the hypothesis that while available P may be reduced by soil acidification, P limitation to plants is ameliorated by microorganisms that are able to liberate P that is normally unavailable to plants, thereby masking P limitation to trees. To test this hypothesis, lime, P, or both will be added to two forests in eastern Ohio to determine if soil acidity or P availability can actually change soil microbial communities and their ability to produce plant-available P.

This research addresses the potential consequences of human activities and the way forest and soil ecosystems may respond to acidification. This project will be used as a teaching tool for undergraduate and high school students in the Appalachian region of southeast Ohio, and a series of collaborative workshops will facilitate the training of K-12 teachers on the importance of soil processes in relation to human prosperity.