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Three purposes are listed for this paper: (1) to share the results of a national survey of vocational education exemplary projects; (2) to emphasize the importance of a project director's communication with five clientele audiences; and (3) to suggest specific actions likely to result in institutionalization of a demonstration project. A brief procedural overview is presented of the national survey which investigated the activities of demonstration project directors through questionnaire/interview contacts with two groups: a primary population consisting of the project directors of the projects funded between 1970 and 1973; and a secondary population consisting of the sites using results from the projects. The remainder of the paper is an outline listing suggested activities of project directors which, based on the study findings, can lead to effective implementation and institutionalization. The activities are listed as they relate to five key clientele audiences: host site decision makers, project staff, community representatives, students, and potential replication site representatives. Because timing is a critical factor in the success of a project, the suggested activities with each audience are discussed in relation to three phases of a project: pre-grant period, grant period, and post-grant period. In summary, a list is provided of eight suggested guidelines for project directors as they implement a demonstration project. (VB)

Analyzing the three-dimensional (3D) structure of specimens has become increasingly important in biomedical research fields, and microcomputed tomography (micro-CT) has emerged as an effective method for rapidly and non-destructively quantifying the geometry and morphology of tissues. Though micro-CT was originally designed to assess cortical and trabecular bone microarchitecture, applications for micro-CT now extend to the imaging of soft tissues. In particular, vascular anatomy and articular cartilage degradation are of major interest to investigators in the orthopedic research areas. This chapter outlines processes and results involved in utilizing X-ray contrast agents to enhance micro-CT analyses of vasculature and articular cartilage. [ABSTRACT FROM AUTHOR]

Efficient tagging methodologies are an integral aspect of protein complex characterization by proteomic approaches. Owing to the very high affinity of biotin for avidin and streptavidin, biotinylation tagging offers an attractive approach for the efficient purification of protein complexes. The very high affinity of the biotin/(strept)avidin system also offers the potential for the single-step capture of lower abundance protein complexes, such as transcription factor complexes. The identification of short peptide tags that are efficiently biotinylated by the bacterial BirA biotin ligase led to an approach for the singlestep purification of transcription factor complexes by specific in vivo biotinylation tagging. A short sequence tag fused N-terminally to the transcription factor of interest is very efficiently biotinylated by BirA coexpressed in the same cells, as was demonstrated by the tagging of the essential hematopoietic transcription factor GATA-1. The direct binding to streptavidin of biotinylated GATA-1 in nuclear extracts resulted in the single-step capture of the tagged factor and associated proteins, which were eluted and identified by mass spectrometry. This led to the characterization of several distinct GATA-1 complexes with other transcription factors and chromatin remodeling cofactors, which are involved in activation and repression of gene targets. Thus, BirA-mediated tagging is an efficient approach for the direct capture and characterization of transcription factor complexes. [ABSTRACT FROM AUTHOR]

A subset of the proteome that binds to specific DNA sequences is at the center of genome function, integrity, and dynamics. We present a detailed protocol that allows the isolation of any specific DNA binding protein and its subsequent identification by mass spectrometry. The procedure involves prefractionation of crude nuclear extract by phosphocellulose (P11) chromatography, followed by a series of positive/negative selections on wild-type and site-mutated ligand DNA in a magnetic microparticulate format. DNAaffinity capture requires concatamerized and biotinylated ligand, selective salt conditions, and improved competitor DNAs. The amount of protein(s) captured on DNA-magnetic beads is generally sufficient for successful MALDI-TOF and TOF/TOF MS-based protein identification. As an example, we describe the procedures used to isolate and identify four specific transcription factors from 2 ↔ 109 promyelocytic NB4 cells. [ABSTRACT FROM AUTHOR]

Binding of nuclear transactivators to sequence-specific regulatory elements on the promoter regions is of fundamental importance in gene expression and regulation. DNAbound transactivators recruit transcription coactivators or repressors and an array of associated proteins that interact with the basal transcription factors, thereby activating the transcription machinery. Analysis of the large complex of proteins that bind to DNA is an important step in elucidating the mechanisms by which gene expressions are regulated. Commonly used techniques to determine DNA-protein binding such as the electrophoretic mobility shift assay (EMSA) have limited value for analyzing simultaneously a large number of proteins in the complex. We describe here a streptavidin-agarose pulldown assay that is capable of analyzing quantitatively binding of an array of proteins to DNA probes. The assay is easy to perform and does not require radiolabeled probes. It involves incubation of nuclear extract proteins with 5'biotinylated double-stranded DNA probes and streptavidin-agarose beads. The complex is pulled down, and proteins in the complex are dissociated and analyzed by Western blotting. This method has been shown to be useful in determining the regulation of binding of transactivators, p300/CBP, and associated proteins to the cyclooxygenase-2 (COX-2) promoter. [ABSTRACT FROM AUTHOR]

Recent studies on genome-wide localization of transcription factor (TF) binding to DNA have shown that a large proportion of identified sequences do not contain consensus motifs predicted by databases of transcription factor binding sites, such as TRANSFAC. The main limitation of these databases is that they are based on a literature search of published examples of binding; consequently the data are not from a systematic survey and may be subject to sampling biases if investigators focused on particular motifs. Thus, there is an urgent need for systematic profiling of vertebrate transcription factor binding to DNA. We have developed a high-throughput platform for the quantitative analysis of protein-DNA interactions based on microarray technology. [ABSTRACT FROM AUTHOR]

DNA binding proteins play a number of key roles in cells,in processes including transcriptional regulation,recombination,genome rearrangements,and DNA replication, repair,and modification.Of particular interest are the interactions between transcription factors and their DNA binding sites,as they are an integral part of the transcriptional regulatory networks that control gene expression.Despite their importance,the DNA binding specificities of most DNA binding proteins remain unknown,as earlier technologies aimed at characterizing DNA-protein interactions have been time consuming and not highly scalable.We have developed a new DNA microarray-based technology,termed protein binding microarrays (PBMs),that allows rapid,high-throughput characterization of the in vitro DNA binding site sequence specificities of transcription factors in a single day. The resulting DNA binding site data can be used in a number of ways,including for the prediction of the genes regulated by a given transcription factor,annotation of transcription factor function,and functional annotation of the predicted target genes. [ABSTRACT FROM AUTHOR]

Effective methods of probing chromatin structure without disrupting DNA-protein interactions and associations are necessary for creating an accurate picture of chromatin and its processes in vivo. Expression of cytidine-5 DNA methyltransferases (C5 DMTases) in Saccharomyces cerevisiae provides a powerful noninvasive method of assaying relative DNA accessibility in chromatin. DNA MTases are occluded from protein-associated DNA based on the strength and span of the DNA-protein interaction. Ectopic regulation of C5 DMTase expression systems allows for minimal disruption of yeast physiology. Methylated sites are detected by bisulfite genomic sequencing, which leads to a positive signal corresponding to modified cytidine residues. High-resolution C5 DMTases with dinucleotide recognition specificity are shown to provide sufficient coverage to map interactions spanning a relatively short distance. [ABSTRACT FROM AUTHOR]

Nucleosome arrangements and possibly chromatin higher order structures differ in different regions of genomic DNA, and these differences could be functionally important. Nucleosome arrangements are reflected by the nucleosome ladders they give rise to upon micrococcal nuclease digestion of the chromatin. Here we describe how Southern hybridization can be used to detect the nucleosome ladders arising from different regions of DNA in mouse liver nuclei. [ABSTRACT FROM AUTHOR]

The Stanford Microarray Database (SMD) is a DNA microarray research database that provides a large amount of data for public use. This chapter describes the use of the primary tools for searching, browsing, retrieving, and analyzing data available for SMD. With this introduction, researchers and students will be able to examine and analyze a large body of gene expression and other experiments. Additional tools for depositing, annotating, sharing, and analyzing data, available only to registered users, are also described. SMD is available for installation as a local database. [ABSTRACT FROM AUTHOR]

The Gene Expression Omnibus (GEO) at the National Center for Biotechnology Information (NCBI) has emerged as the leading fully public repository for gene expression data. This chapter describes how to use Web-based interfaces, applications, and graphics to effectively explore, visualize, and interpret the hundreds of microarray studies and millions of gene expression patterns stored in GEO. Data can be examined from both experimentcentric and gene-centric perspectives using user-friendly tools that do not require specialized expertise in microarray analysis or time-consuming download of massive data sets. The GEO database is publicly accessible through the World Wide Web at http://www. ncbi.nlm.nih.gov/geo. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are a class of noncoding RNAs that regulate gene expression by single-stranded RNAs of 18 to 25 nucleotides in length. Hundreds of miRNAs have been found in animals and plants, some of which play important roles in development or differentiation. Increasing attention has thus been paid to their biogenesis and regulation mechanisms and the identification of target genes. We are constructing a comprehensive expression vector library containing predicted human miRNAs. miRNA expression vectors containing human RNA polymerase II or III promoters, and utilizing a flexible vector system, can be useful for functional analysis. [ABSTRACT FROM AUTHOR]

The analysis of allele-specific gene expression has been of long-standing interest in the study of genomic imprinting, but there is growing awareness that differences in allelic expression are widespread among autosomal nonimprinted genes. Recent research into cis-acting regulatory polymorphisms has utilized the analysis of allele-specific gene expression to identify functionally important regulatory haplotypes and specific genetic polymorphisms. Allele-specific effects are typically of modest magnitude, requiring techniques for analysis of high sensitivity and specificity. Here, strategic approaches to the analysis of allele-specific gene expression are reviewed with protocols for in vivo analysis. These include analysis of the relative allelic abundance of transcribed RNA and of transcription factor recruitment and Pol II loading by chromatin immunoprecipitation. [ABSTRACT FROM AUTHOR]

A major challenge in systems biology is to discover and reconstruct the cis-regulatory networks through which the expression of genes is controlled. Even though a variety of sequences have been shown to interact with the transcription factors that bind DNA, extensive work is needed to discover and classify regulatory "codes" and to elucidate the role played by the sequence context of genomic DNA in the regulation of genes. Databases of sequence elements extracted from regulatory regions may facilitate this process. This report provides a Toolkit and instructions for creating a database for collecting and analyzing 9-base elements (9-mers) from a large collection of DNA sequences. A reference set consisting of all possible 9-mers is included for extracting potential control elements, irrespective of their orientation and order in DNA. [ABSTRACT FROM AUTHOR]

Discovery of lexical characteristics of specific sequence motifs in human genomic DNA can help with predicting and classifying regulatory cis elements according to the genes they control. In lexical models, some "words" may serve as downstream targets of signaling systems, whereas other "words" may specify sequences that selectively control the expression of a subset of genes to produce the various cell types and tissues. To discover lexical features of potential regulatory "words," we have created a database of 9-mers derived from the promoter regions of a subset of human protein-coding genes. This report describes the procedure for extracting information from that database through the web. [ABSTRACT FROM AUTHOR]

Genome-wide studies are fast becoming the norm, partly fueled by the availability of genome sequences and the feasibility of high-throughput experimental platforms, e.g., microarrays. An important aspect in any genome-wide studies is determination of regulatory relationships, believed to be primarily transacted through transcription factor binding to DNA. Identification of specific transcription factor binding sites in the cis-regulatory regions of genes makes it possible to list direct targets of transcription factors, model transcriptional regulatory networks, and mine other associated datasets for relevant targets for experimental and clinical manipulation. We have developed a web-based tool to assist biologists in efficiently carrying out the analysis of genes from studies of specific transcription factors or otherwise. The batch extraction and analysis of cis-regulatory regions (BEARR) facilitates identification, extraction, and analysis of regulatory regions from the large amount of data that is typically generated in genome-wide studies. This chapter highlights features and serves as a tutorial for using this publicly available software. The URL is http://giscompute.gis.a-star.edu.sg/R2vega/BEARR1.0/. [ABSTRACT FROM AUTHOR]

CORG is a versatile web-based workbench for comparative promoter analysis in vertebrate model organisms. Two kinds of information are explicitly considered in the automated annotation process. First, local conservation patterns in upstream regions of homologous genes: These phylogenetic footprints are likely to stem from sequence elements that are under selective pressure. The CORG pipeline detects and exploits patterns of local similarity to annotate promoter regions. Second, experimental data on transcription start sites: exon positions and DNA binding site descriptions complete the promoter annotation. These data are made available via an interactive web portal. Individual promoter studies are supported by a JAVA applet that supplies all data down to the nucleotide level. [ABSTRACT FROM AUTHOR]

One major goal of genomics is to identify all the functional sequences in genomes, including sequences that regulate the expression of genes. Sequence conservation is a good, albeit imperfect, guide to these functional elements. We describe how to use publicly available servers (Galaxy, the UCSC Table Browser, and GALA) to find genomic sequences whose alignments (from blastZ and multiZ) show properties associated with cis-regulatory modules, such as high conservation score, high regulatory potential score, and conserved transcription factor binding sites. Links to these servers can be accessed at http:// www.bx.psu.edu/ and http://genome.ucsc.edu/. [ABSTRACT FROM AUTHOR]

Comparative analysis of DNA sequences is becoming one of the major methods for discovery of functionally important genomic intervals. Presented here the VISTA family of computational tools was built to help researchers in this undertaking. These tools allow the researcher to align DNA sequences, quickly visualize conservation levels between them, identify highly conserved regions, and analyze sequences of interest through one of the following approaches: • Browse precomputed whole-genome alignments of vertebrates and other groups of organisms.• Submit sequences to GenomeVISTA to align them to whole genomes.• Submit two or more sequences to mVISTA to align them with each other (a variety of alignment programs with several distinct capabilities are made available).• Submit sequences to Regulatory VISTA (rVISTA) to perform transcription factor binding site predictions based on conservation within sequence alignments.• Use stand-alone alignment and visualization programs to run comparative sequence analysis locally. All VISTA tools use standard algorithms for visualization and conservation analysis to make comparison of results from different programs more straightforward. The web page http://genome.lbl.gov/vista/ serves as a portal for access to all VISTA tools. Our support group can be reached by email at vista@lbl.gov. [ABSTRACT FROM AUTHOR]

Primed in situ labeling (PRINS) is a sensitive and specific method that can be used for the localization of single copy genes and sequences too small for detection by conventional fluorescence in situ hybridization. By the use of PRINS, the human SRY gene was localized to Yp11.31-p11.32 and the SOX3 gene to Xq26-q27. In other studies, we localized specific deletions of RBM and DAZ, candidate genes for AZF (azoospermia factor) to proximal Yq11.2, the AZF region, in an infertile male. Locus-specific oligonucleotide probes (PRINS primers) were annealed to chromosomal DNA in situ and extended on preparations fixed on glass slides in the presence of dATP, dCTP, dGTP, dTTP, biotin-16-dUTP, and Taq DNA polymerase. After addition of avidin-conjugated fluorophore, signals were visualized by fluorescence microscopy in metaphase spreads from patients and controls. With further development, the PRINS method may prove useful for localization of single-copy genes, in general, and for the detection of gene deletions. [ABSTRACT FROM AUTHOR]

Efforts to prepare a first draft of the human DNA genomic sequence forced multidisciplinary teams of researchers to face unique challenges.At the same time,these unprecedented obstacles stimulated the development of many highly innovative approaches to biomedical problem solving,robotics,and bioinformatics.High-resolution physical maps are required for ordering individual segments of information for the construction of a comprehensive map of the entire genome.This chapter describes a novel way to identify,delineate,and characterize selected,often small DNA sequences along a larger piece of the human genome.The technology is based on immobilization of high molecular weight DNA molecules on a solid substrate (such as a glass slide)followed by uniform stretching of the DNA molecule by the force of a receding meniscus.The hydrodynamic force stretches the DNA molecules homogeneously to approximately 2.3 kb/μm,so that distances measured after probe binding in μm can be converted directly into kb distances.Out of a large number of applications,this article focuses on mapping of genomic sequences relative to one another,the assembly of physical maps with near kb resolution,and,finally,quality control during physical map assembly and sequencing. [ABSTRACT FROM AUTHOR]

The completion of whole genome sequencing projects offers the opportunity to exam-ine the organization of genes and the discovery of evolutionarily related genes in a given species. For the beginners in the field, through a specific example, this chapter provides a step-by-step procedure for identifying paralogous genes, using the genome browser at UCSC (http://genome.ucsc.edu/). The example describes identification and mapping in the human genome, the paralogs of TCF12/HTF4. The example identifies TCF3 and TCF4 as paralogs of the TCF12/HTF4 gene. The example also identifies a related sequence, cor-responding to a pseudogene, in one of the introns of the JAK2 gene. The procedure described should be applicable to the discovery and creation of maps of paralogous genes in the gen-omic DNA sequences that are available at the genome browser at UCSC. [ABSTRACT FROM AUTHOR]

The aim of this chapter is to provide instruction for analyzing and mapping recent segmental and gene duplications in eukaryotic genomes. We describe a bioinformatics-based approach utilizing computational tools to manage eukaryotic genome sequences to characterize and understand the evolutionary fates and trajectories of duplicated genes. An introduction to bioinformatics tools and programs such as BLAST, Perl, BioPerl, and the GFF specification provides the necessary background to complete this analysis for any eukaryotic genome of interest. [ABSTRACT FROM AUTHOR]

The completion of whole-genome sequencing projects offers the opportunity of creating high-resolution maps of specific segments in a known genomic DNA sequence. For this purpose, several genome browsers have been created. They include the map-view (http:// www.ncbi.nlm.nih.gov/mapview/), the Ensembl genome browser (http://www.ensembl. org/), and the genome browser at UCSC (http://genome.ucsc.edu/). For the beginners in the field, through a specific example, this chapter provides a step-by-step procedure for creating a map using the genome browser at UCSC. The example describes mapping, in the human genome, the promoter region of the NF-IL6 gene. The procedure is applicable to creating maps of the desired regions in genomes of other species available at the genome browser at UCSC. [ABSTRACT FROM AUTHOR]

Chapter 2.02 of the book "Treatise on Geochemistry: The Mantle and the Core" is presented. The chapter focuses on the seismological constraints upon the composition of the mantle of the Earth. It also explores seismic wave velocities, the mineralogy of the upper mantle and hydrothermal alteration of basalts.

Changes in blood pressure (BP) from low to high, are genetically controlled by many genes. Identifying genes that regulate BP is important for two major reasons: (1) for delineating the biochemical/physiological mechanisms for BP control; and (2) for utilizing this knowledge to improve clinical management strategies for maintenance of normal physiological BP, which is an important factor in reducing the risk for susceptibility to cardiovascular diseases. Genetic approaches to identify BP regulatory genes involve the use of rat models with elevated BP (hypertension). Typically, genetic linkage analyses and substitution mapping using congenic strains are performed. These studies are designed to enable the identification of BP regulatory genes primarily by virtue of their location on the rat genome. This chapter captures the current status of studies aimed at identifying BP causative genes using various congenic rat strains. [ABSTRACT FROM AUTHOR]

A foreword to the book "Food Sovereignty and Uncultivated Biodiversity in South Asia: Essays on the Poverty of Food Policy and the Wealth of the Social Landscape" is presented.