bluuebunny/crossref_metadata_2025 huggingface dataset:
Interaction Trap/Two‐Hybrid System to Identify Interacting Proteins
Authors: Golemis Erica A. , Serebriiskii Ilya , Finley Russell L. , Kolonin Mikhail G. , Gyuris Jeno , Brent Roger
Published: June 2004

This unit presents protocols designed to detect interacting proteins. Using yeast as a “test tube” and transcriptional activation of a reporter system, interacting proteins can be identified. The system can also be used to test complex formation for proteins for which there exists a reason to expect interaction.
Mapping Protein‐Protein Interactions with Phage‐Displayed Combinatorial Peptide Libraries
Authors: Kay Brian K. , Castagnoli Luisa
Published: June 2004

This unit describes the process and analysis of affinity selecting bacteriophage M13 from libraries displaying combinatorial peptides fused to either a minor or major capsid protein. Direct affinity selection uses target protein bound to a microtiter plate followed by purification of selected phage by ELISA. Alternatively, there is a bead‐based affinity selection method. These methods allow one to readily isolate peptide ligands that bind to a protein target of interest and use the consensus sequence to search proteomic databases for putative interacting proteins.
Protein‐Protein Interactions Identified by Pull‐Down Experiments and Mass Spectrometry
Authors: Brymora Adam , Valova Valentina A. , Robinson Phillip J.
Published: June 2004

The aim of this unit is to provide a method for the identification of new protein‐protein interactions. Pull‐down experiments with GST fusion proteins attached to glutathione beads are a screening technique for identification of protein‐protein interactions. When coupled with mass spectrometry, pull‐downs can be considered as the protein‐based equivalent of a yeast two‐hybrid screen. To improve the isolation of specific binding partners, pull‐down methods are described involving the use of cross‐linking, large‐scale tissue lysates, and spin columns. Alternative techniques are detailed for isolating activation state‐dependent protein interactions with small GTPases. Appropriate methods of sample preparation for mass spectrometry‐based identification of interacting proteins are described, including specialized gel staining techniques, band excision, and in‐gel tryptic digestion. Data interpretation and the most commonly encountered problems are discussed.
Measuring Protein Interactions by Optical Biosensors
Authors: Schuck Peter , Boyd Lisa F. , Andersen Peter S.
Published: June 2004

In recent years, optical evanescent wave biosensors have been used to characterize protein‐protein interactions, including determination of equilibrium binding constants and bimolecular rate constants. This surface binding technique can provide information about chemical on‐rate constants, the lifetimes of complexes formed, and the time course of the signal. This unit provides a thorough, well‐illustrated discussion of the principles of optical biosensors, experimental design, ligand immobilization, experimental data analysis, and common obstacles and possible solutions.
Assessment of Apoptosis and Necrosis by DNA Fragmentation and Morphological Criteria
Authors: Zhivotosky Boris , Orrenius Sten
Published: June 2004

Apoptotic cells share a number of common features, such as phosphatidylserine (PS) exposure, cell shrinkage, chromatin cleavage, nuclear condensation, and formation of pyknotic bodies of condensed chromatin. Necrotic cells exhibit nuclear swelling, chromatin flocculation, loss of nuclear basophilia, breakdown of cytoplasmic structure and organelle function, and cytolysis by swelling. This unit describes some of the techniques most commonly used to detect cell death. A number of assays are used for characterizing and distinguishing apoptosis and necrosis. Morphological assays include trypan blue exclusion, differential staining, and Hoechst staining. Methods to detect chromatin cleavage include TUNEL assays for whole cells and paraffin sections, DNA fragmentation assays using whole cells, assays of total genomic DNA, analysis of DNA fragmentation by agarose gel electrophoresis, phenol extraction of DNA for analysis of fragmentation, a quantitative assay for DNA fragmentation, and detection of DNA fragmentation by pulsed‐field gel electrophoresis. A protocol is also provided for Cytospin preparations from cell suspensions.
Quantitative Fluorescence In Situ Hybridization (Q‐FISH)
Authors: Poon Steven S.S. , Lansdorp Peter M.
Published: June 2004

This unit describes a quantitative technique for measuring the lengths of telomere repeat sequences in individual chromosomes from single metaphase cells. The technique is based on fluorescence in situ hybridization (FISH) adapted for use with peptide nucleic acid (PNA) probes. PNA is an example of novel synthetic oligonucleotide “mimetic” which has a higher affinity than regular oligonucleotide (RNA or DNA) probes for complementary single‐strand (ss) DNA sequences. PNA oligonucleotides have excellent penetration properties due to their small size (typically 15 to 18‐mers) and can be directly labeled with fluorochromes. These properties have been exploited to develop quantitative fluorescence in situ hybridization (Q‐FISH) onto denatured single‐stranded chromosomal DNA target sequences. The latter can be present in preparations of fixed metaphase cells on slides (Q‐FISH) or in heat‐treated (interphase) cells in suspension (flow‐FISH).
Analysis of Telomeres and Telomerase
Authors: Herbert Brittney‐Shea , Shay Jerry W. , Wright Woodring E.
Published: June 2004

This unit describes techniques to analyze telomeric length and telomerase activity in human cells. Telomere length can be determined by a modification of Southern blotting in which the analysis of chromosome terminal restriction fragments (TRFs) provides the average lengths of all telomeres in a cell population. Telomerase activity can be measured in vitro by a sensitive and efficient polymerase chain reaction (PCR)‐based detection method, also known as telomeric repeat amplification protocol (TRAP). These assays can be used to study the in vitro cellular effects of aging and cancer treatments on telomere biology and telomerase activity.
Flow Cytometry of Apoptosis
Authors: Pozarowski Piotr , Grabarek Jerzy , Darzynkiewicz Zbigniew
Published: June 2004

Common methods applicable to flow cytometry make it possible to: (1) identify and quantify dead or dying cells, (2) reveal a mode of cell death (apoptosis or necrosis), and (3) study mechanisms involved in cell death. Gross changes in cell morphology and chromatin condensation, which occur during apoptosis, can be detected by analysis with laser light beam scattering. Early events of apoptosis, dissipation of the mitochondrial transmembrane potential and caspase activation, can be detected using either fluorochrome reporter groups or appropriate antibodies. Exposure of phosphatidylserine on the exterior surface of the plasma membrane can be detected by the binding of fluoresceinated annexin V. Another apoptotic event, DNA fragmentation based on DNA content of cells with fractional (“sub‐G1”) or DNA strand‐break labeling, TUNEL; or In Situ End Labeling, ISEL;. Still another hallmark of apoptosis is the activation of tissue transglutaminase (TGase), the enzyme that crosslinks protein and thereby makes them less immunogenic. The major advantage of flow cytometry in these applications is that it provides the possibility of multiparametric measurements of cell attributes.
Overview of Metastasis Assays
Authors: Paweletz Cloud P. , Charboneau Lu , Liotta Lance A.
Published: June 2004

During tumor progression, cells acquire genetic and proteomic changes as they transform from normal to hyperplastic, through dysplasia, to carcinoma in situ, and finally to invasive and metastatic. The time course of progression may extend as far back as 10 years prior to diagnosis. Discerning the mechanism whereby tumor cells execute metastatic dissemination may provide the foundation necessary for successful treatment of the disease. For example, direct genetic evidence has linked in situ breast cancer to invasive carcinoma of the breast supporting the generally accepted assumption that carcinoma in situ of the breast is a clonal expansion of hyperproliferating cells. This in turn may provide a more comprehensive and/or functionally directed target strategy for intervention and prevention of breast cancer. This overview provides a picture of the processes related to metastasis and the experimental approaches used to study these processes.
Microanalysis of Gene Expression in Tissues Using T7‐SAGE: Serial Analysis of Gene Expression After High‐Fidelity T7‐Based RNA Amplification
Authors: Sakai Takayoshi , Larsen Melinda , Yamada Kenneth M.
Published: June 2004

In this unit, the authors describe a new technique, T7‐SAGE, in which a high‐fidelity T7 amplification step is combined with SAGE analysis. In order to avoid extra PCR or other forms of amplification, the authors incorporate only two cycles of T7‐based RNA amplification as the initial step. This T7‐based amplification step has high accuracy. In addition, T7 RNA polymerase has high processivity and functions effectively even when broad stretches of nucleotides are being amplified. Although no protocol that includes an amplification step can claim to permit determination of absolute transcript number, since slight changes in estimated transcript frequency are always possible, T7 procedures appear to be the safest to date. This new T7‐SAGE procedure should facilitate application of SAGE for gene‐expression profiling using minimal quantities of starting material, such as from embryonic tissues and microdissected cells from histological sections of tissues.
The Chick Chorioallantoic Membrane as an In Vivo Angiogenesis Model
Authors: Ponce M. Lourdes , Kleinmann Hynda K.
Published: June 2004

The chick chorioallantoic membrane (CAM) assay for angiogenic activity is a model originally developed to study the angiogenic activity of tumor samples. It is an in vivo assay that can be readily performed in any laboratory setting. The effects of a test compound on angiogenesis are tested by exposing day 10 embryos to the compound and following the patterns of blood vessel development, scoring the appearance of the CAM at day 12 or 13.
Experimental Metastasis Assays in the Chick Embryo
Authors: Wilson Sylvia M. , Chambers Ann F.
Published: June 2004

Experimental metastasis assays are used to measure the ability of cancer cells to grow in secondary organs following injection of the cells into the circulation of an experimental animal. The chicken embryo provides an alternative to the more commonly used assays in mice. Details of the experimental metastasis assay in chick embryo are provided, including protocols for maintenance of fertilized chicken eggs, injection of cells into the circulation of 11‐day‐old chick embryos, recovery and quantification of cancer cells from chick liver using the ouabain plating assay, labeling of cells with fluorescent nanospheres, and monitoring of individual steps in the metastatic process in the chick chorioallantoic membrane using intravital videomicroscopy. These assays provide a cost‐effective, readily accessible, and rapid approach for studying the process of cancer metastasis.
Direct Introduction of Molecules into Cells
Authors: McNeil Paul L.
Published: June 2004

Techniques for introducing normally impermeant macromolecules into the living cell—referred to as “cell‐loading techniques”—are useful in a variety of settings for the cell biologist. Microinjection is probably the most commonly used technique for introducing fluorescent probes, fluorescently tagged proteins, and antibodies into living cells for short‐term studies of cell physiology and protein location and function. It is, however, not the only technique available, nor the easiest or least expensive to implement. Among the alternatives are several closely related techniques that, like microinjection, rely on the cell's ability to reseal a mechanically induced plasma membrane disruption created in order to gain temporary access to cell cytosol. Four such techniques are described in this unit: scrape loading, scratch loading, bead loading, and syringe loading. Unlike microinjection, these techniques allow one to rapidly load (in a matter of minutes) thousands or even many millions of many types of mammalian cells with normally impermeant molecules, and so to facilitate quantitative analyses of the effect of loading.
Protein Transduction: Generation of Full‐Length Transducible Proteins Using the TAT System
Authors: Becker‐Hapak Michelle , Dowdy Steven F.
Published: June 2004

This unit describes the technology that allows an investigator to transduce full‐length proteins by utilizing a minimal, eleven‐amino acid, HIV‐TAT transduction domain that can be fused to a protein of choice using the pTAT or pTAT‐HA protein expression plasmids. Bacterial expression, followed by solubilization of protein aggregates with a denaturing agent, affords high yields of transducible fusion protein. The fusion protein, once added to the culture medium, can cross the cell membrane and then be degraded or refolded by the cellular machinery. Correct targeting and function of the fusion protein can be easily examined by fluorescent microscopy or immunohistochemistry. This strategy was established and improved to its current state by the purification and transduction of a multitude of fusion proteins. Because the pool of fusion proteins spans many different functions, the protocols cover a wide variety of commonly used protein isolation and characterization methods.
Calcium Phosphate Transfection
Authors: Kingston Robert E. , Chen Claudia A. , Okayama Hiroto
Published: June 2004

This unit presents two methods of calcium phosphate‐based eukaryotic cell transfection that can be used for both transient and stable transfections. In these protocols, plasmid DNA is introduced to monolayer cell cultures via a precipitate that adheres to the cell surface. A HEPES‐buffered solution is used to form a calcium phosphate precipitate that is directly layered onto the cells. For some cells, shocking the cells with glycerol or DMSO improves transfection efficiency. In the alternate high‐efficiency method, a BES‐buffered system is used that allows the precipitate to form gradually in the medium and then drop onto the cells. While the alternate method is particularly efficient for stable transformation of cells with circular plasmid DNA, both protocols yield similar results for transformation with linear plasmid or genomic DNA, or for transient expression.
Transfection Using DEAE‐Dextran
Authors: Gulick Tod
Published: June 2004

Transfection of cultured mammalian cells using diethylaminoethyl (DEAE)‐dextran/DNA can be an attractive alternative to other transfection methods in many circumstances. The major advantages of the technique are its relative simplicity and speed, limited expense, and remarkably reproducible interexperimental and intraexperimental transfection efficiency. Disadvantages include inhibition of cell growth and induction of heterogeneous morphological changes in cells. Furthermore, the concentration of serum in the culture medium must be transiently reduced during the transfection. In general, DEAE‐dextran DNA transfection is ideal for transient transfections with promoter/reporter plasmids in analyses of promoter and enhancer functions, and is suitable for overexpression of recombinant protein in transient transfections or for generation of stable cell lines using vectors designed to exist in the cell as episomes. This unit presents a general description of DEAE‐dextran transfection, as well as two more specific protocols for typical experimental applications. The basic protocol is suitable for transfection of anchorage‐dependent (attached) cells. For cells that grow in suspension, electroporation or lipofection is usually preferred, although DEAE‐dextran‐mediated transfection can be used.
Transfection by Electroporation
Authors: Potter Huntington
Published: June 2004

Electroporation–the use of high‐voltage electric shocks to introduce DNA into cells–can be used with most cell types, yields a high frequency of both stable transformation and transient gene expression, and, because it requires fewer steps, can be easier than alternate techniques. In this unit, the basic protocol describes the electroporation of mammalian cells and an alternate protocol outlines modifications for preparation and transfection of plant protoplasts.
Transfection of Cultured Eukaryotic Cells Using Cationic Lipid Reagents
Authors: Hawley‐Nelson Pamela , Ciccarone Valentina
Published: June 2004

The development of high‐efficiency methods for the introduction of functional genetic material into eukaryotic cells using cationic lipids has accelerated biological research in the studies of gene expression, control of cell growth, and cell lineage. Transfection mediated by cationic lipids is commonly used in industrial protein production as well as in some clinical gene therapy protocols. Replacing our previous unit on this topic, this new version describes how to perform transfection of adherent and suspension cells, insect cells, and RNA transfection using the cationic lipid system.
Optimization of Transfection
Authors: Rose John K.
Published: June 2004

When embarking upon any transfection procedure, a critical first step is to optimize conditions. Every mammalian cell type has a characteristic set of requirements for optimal introduction of foreign DNA; there is a tremendous degree of variability in the transfection conditions that work, even among cell types that are very similar to one another. Often, an experimenter must screen a wide variety of cell types for a desired regulatory trait, such as an appropriate response to a particular effector molecule. It is thus helpful to have a straightforward, systematic approach to optimizing transfection efficiency. This unit provides guidelines to optimizing transfection experiments.
Measuring Protein Mobility by Photobleaching GFP Chimeras in Living Cells
Authors: Snapp Erik L. , Altan Nihal , Lippincott‐Schwartz Jennifer
Published: June 2004

This unit describes fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) using commercially available confocal scanning laser microscopy (CSLM). Photobleaching is the photo‐induced change in a fluorphore that abolishes that molecule's fluorescence. The different characteristics of green fluorescent protein (GFP) chimeras in a cell can be studied by FRAP, in which a selected region of the cell is photobleached with intense light. The movement of unbleached molecules into a photobleached region is quantified by imaging with an attenuated light source. The movement of molecules between cellular compartments can be determined by FLIP, in which the same region of a cell expressing a GFP chimera is repeatedly photobleached. The loss of fluorescence from regions outside the photobleached region is monitored to characterize the movement of a protein. Together these two techniques are providing fundamentally new insights into the kinetic properties of proteins in cells.
Identification of Motifs in Protein Sequences
Authors: Sonnhammer Erik L.L. , Wolfsberg Tyra G.
Published: June 2004

This brief appendix serves as a guide for the analysis of functional motifs in proteins. Several database search engines that can be accessed via the World Wide Web are described. Such computerized searches have become the preferred method to scan large sequence and motif databases, as the searches are efficient and the databases are updated frequently. A short list of sorting signals is also included, since these motifs often cannot be predicted reliably by a computer search.
Safe Use of Radioisotopes
Authors: Meisenhelder Jill , Semba Kentaro
Published: June 2004

The use of radioisotopes to label specific molecules in a defined way has greatly advanced the discovery and dissection of biochemical pathways. The development of methods to inexpensively synthesize such tagged biological compounds on an industrial scale has enabled them to be used routinely in laboratory protocols, including many detailed in this manual. Although most of these protocols involve the use of only microcurie (mCi) amounts of radioactivity, some (particularly those describing the metabolic labeling of proteins or nucleic acids within cells) require amounts on the order of millicuries (mCi). In all cases where radioisotopes are used, depending on the quantity and nature of the isotope, certain precautions must be taken to ensure the safety of the investigator. It is essential to use good safety practices and proper protection to handle radioactive substances. This unit discusses storage, handling, and disposal of 35S, 32P, 33P, and 125I.
Absorption and Emission Maxima for Common Fluorophores
Authors: Herman Brian
Published: June 2004

This unit lists absorption and emission maximum wavelengths for common fluorophores used in cell biology. This information can be used in designing experiments and selecting appropriate filters for use with the fluorophores.
Importing Biological Materials
Authors: Bryant Paula Wolf
Published: June 2004

Scientific cooperation is no longer confined to laboratories at the same institution, nor between laboratories within the boundaries of the United States. More than ever, scientists are forming collaborations with their peers outside the U.S., leading not only to the exchange of knowledge and ideas, but also to the exchange of biological reagents. Thus, investigators need to be aware that an import permit may be required to obtain purified proteins, chemically synthesized materials, cell lines, tissues, and other biological materials from abroad. Before requesting any biological material from a laboratory outside the U.S., it is the responsibility of the “receiver” to ascertain whether an import permit is required, to apply for the permit if one is determined to be necessary, and to provide the “shipper” with an official copy of the permit to be included with the shipped material. This unit identifies the appropriate agencies and steps required to obtain a permit.
Internet Basics for Biologists
Authors: Baxevanis Andreas D. , Ouellette B. F. Francis
Published: June 2004

With the explosion of sequence and structural information available to researchers, the field of bioinformatics is playing an increasingly large role in the study of fundamental biomedical problems. This information is generally available through the Internet and access to the World Wide Web. This unit provides an introduction to the internet–how it is organized, how you connect to it, using email and file transfer protocols, and finding your way around the Web.
Spectrophotometric Determination of Protein Concentration
Authors: Simonian Michael H.
Published: June 2004

This unit describes spectrophotometric and colorimetric methods for measuring the concentration of a sample protein in solution. Absorbance measured at 280 nm (A280) is used to calculate protein concentration by comparison with a standard curve or published absorptivity values for that protein (a280). Alternatively, absorbance measured at 205 nm (A205) is used to calculate the protein concentration. The A280 and A205 methods can be used to quantify total protein in crude lysates and purified or partially purified protein. A spectrofluorometer or a filter fluorometer can be used to measure the intrinsic fluorescence emission of a sample solution; this value is compared with the emissions from standard solutions to determine the sample concentration. The fluorescence emission method is used to quantify purified protein. This simple method is useful for dilute protein samples and can be completed in a short amount of time. There are two colorimetric methods: the Bradford colorimetric method, based upon binding of the dye Coomassie brilliant blue to the protein of interest, and the Lowry method, which measures colorimetric reaction of tyrosyl residues in the protein sample.
Dialysis and Concentration of Protein Solutions
Authors: Andrew Sarah. M. , Titus Julie. A. , Zumstein Louis
Published: June 2004

Conventional dialysis separates small molecules from large molecules by allowing diffusion of only the small molecules through selectively permeable membranes. Dialysis is usually used to change the salt (small‐molecule) composition of a macromolecule‐containing solution. This unit describes dialysis of large‐ and small‐volume samples using cellulose membranes with pore sizes designed to exclude molecules above a selected molecular weight. A support protocol describes preparation of membranes for dialysis and discusses issues related to the selection of membranes including commercial kits.
Quantification of DNA and RNA with Absorption and Fluorescence Spectroscopy
Authors: Gallagher Sean R.
Published: June 2004

Reliable quantitation of nanogram and microgram amounts of DNA and RNA in solution is essential to researchers in cell and molecular biology. In addition to the traditional absorbance measurements at 260 nm, two more sensitive fluorescence techniques–Hoescht 33258 dye binding, ethidium bromide binding are described. These three procedures cover a range from 5 to 10 ng/ml DNA to 50m g/ml DNA.
Silanizing Glassware
Authors: Seed Brian
Published: June 2004

This unit provides a protocol for silanizing (siliconizing) glassware to prevent adsorption of solute to the glass surface and to increase the hydrophobicity of the surface. This is particularly important when dealing with low concentrations of particularly “sticky” solutes such as single‐stranded nucleic acids or proteins or cells.
Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization
Authors: Kramer Martha F. , Coen Donald M.
Published: June 2004

This appendix describes a method for amplifying DNA enzymatically by the polymerase chain reaction (PCR), including procedures to quickly determine conditions for successful amplification of the sequence and primer sets of interest, and to optimize for specificity, sensitivity, and yield.
Micro RT‐PCR
Authors: Paweletz Cloud P. , Charboneau Lu , Liotta Lance A.
Published: June 2004

This unit presents a collection of protocols for the microisolation, manipulation, and amplification of the RNA content of microdissected cells. Even though emphasis in these protocols is given for microdissected cells, these protocols have successfully been used for bulk tissue (i.e., less than 10 ug).
The Colorimetric Detection and Quantitation of Total Protein
Authors: Krohn Randall I.
Published: June 2004

Protein quantification is an important step for handling protein samples for isolation and characterization; it is a prerequisite step before submitting proteins for chromatographic, electrophoretic, or immunochemical analysis and separation. Colorimetric methods are fast, simple, and not laborious. This unit describes a number of assays able to detect protein concentrations in the low microgram to milligram per milliliter ranges in a variety of formats.
Searching Online Mendelian Inheritance in Man (OMIM) for Information for Genetic Loci Involved in Human Disease
Authors: Baxevanis Andreas D.
Published: June 2004

Online Mendelian Inheritance in Man (OMIM) is a non‐sequence‐based information resource that can be of tremendous use to genomics researchers, physicians, and patients. OMIM is the electronic version of the catalog of human genes and genetic disorders. It provides concise textual information from the literature on most human conditions having a genetic basis, as well as pictures illustrating the condition or disorder (where appropriate) and full citation information. This unit gives an overview of the OMIM database, the layout of the records, and the information that is available within each entry.
Searching the NCBI Databases Using Entrez
Authors: Murphy Juliane , Baxevanis Andreas D.
Published: June 2004

One of the most widely‐used interfaces for the retrieval of information from biological databases is the NCBI Entrez system. Entrez capitalizes on the fact that there are pre‐existing, logical relationships between the individual entries found in numerous public databases. The existence of such natural connections, mostly biological in nature, argued for the development of a method through which all the information about a particular biological entity could be found without having to sequentially visit and query disparate databases. Two Basic Protocols describe simple, text‐based searches, illustrating the types of information that can be retrieved through the Entrez system. An builds upon the first Basic Protocol, using additional, built‐in features of the Entrez system, as well as providing alternative ways to issue the initial query. The reviews how to save frequently‐issued queries.
The UCSC Genome Browser
Authors: Karolchik Donna , Kent W. James
Published: June 2004

The UCSC Genome Browser is a popular Web‐based tool for quickly displaying a requested portion of a genome at any scale, accompanied by a series of aligned annotation tracks. The annotations ‐ some of which are generated at the University of California, Santa Cruz (UCSC) and some by external collaborators ‐ display gene predictions, mRNA alignments, expressed sequence tag alignments, cross‐species homologies, single nucleotide polymorphisms, and several other types of data. All information relevant to a region is presented in one window, facilitating biological analysis and interpretation. Users can also upload custom annotation tracks for research or educational use. This unit describes how to use and customize the UCSC Genome Browser, download the underlying database tables, and create and display custom annotation tracks.
Using the TIGR Gene Index Databases for Biological Discovery
Authors: Lee Yuandan , Quackenbush John
Published: June 2004

The TIGR Gene Index web pages provide access to analyses of ESTs and gene sequences for nearly 60 species, as well as a number of resources derived from these. Each species‐specific database is presented using a common format with a homepage. A variety of methods exist that allow users to search each species‐specific database. Methods implemented currently include nucleotide or protein sequence queries using WU‐BLAST, text‐based searches using various sequence identifiers, searches by gene, tissue and library name, and searches using functional classes through Gene Ontology assignments. This protocol provides guidance for using the Gene Index Databases to extract information.
Searching the Mouse Genome Informatics (MGI) Resources for Information on Mouse Biology from Genotype to Phenotype
Authors: Shaw David
Published: June 2004

The Mouse Genome Informatics (MGI, http://www.informatics.jax.org/) resource provides the research community with access to information on the genetics, genomics, and biology of the laboratory mouse. Core data in MGI include gene characterization and function, phenotype and disease model descriptions, DNA and protein sequence data, gene expression data, mammalian orthologies, polymorphisms, mapping data, and links to other bioinformatics databases. Semantic integration is supported through the use of standardized nomenclature and controlled vocabularies such as the mouse Anatomical Dictionary and the Gene Ontologies. MouseBLAST offers a fast, sequence‐based method for identifying genes and searching for similarity, and provides links from sequence to MGI biological data. MGI extracts and organizes data from primary literature. MGI data are shared with and widely displayed by other bioinformatics resources. The database is updated nightly with curated annotations and regularly adds new datasets and enhances user interface features. This unit provides a guide to using the MGI bioinformatics resource.
An Introduction to Recognizing Functional Domains
Authors: Stormo Gary
Published: June 2004

This unit provides an overview of issues involved in domain recognition in protein and DNA sequences. It opens with a discussion of the two primary methods of domain representation, namely consensus sequences and alignment matrices (e.g., the log‐odds matrix). The unit continues with a brief overview of some of the resources available for identifying functional domains in nucleotide sequences (e.g., TRANSFAC, TESS, and MEME). In addition, it reviews databases such as Pfam, and Blocks, which are available for protein analysis.
Multiple Sequence Alignment Using ClustalW and ClustalX
Authors: Thompson Julie D. , Gibson Toby. J. , Higgins Des G.
Published: June 2004

The Clustal programs are widely used for carrying out automatic multiple alignment of nucleotide or amino acid sequences. The most familiar version is ClustalW, which uses a simple text menu system that is portable to more or less all computer systems. ClustalX features a graphical user interface and some powerful graphical utilities for aiding the interpretation of alignments and is the preferred version for interactive usage. Users may run Clustal remotely from several sites using the Web or the programs may be downloaded and run locally on PCs, Macintosh, or Unix computers. The protocols in this unit discuss how to use ClustalX and ClustalW to construct an alignment, and create profile alignments by merging existing alignments.
Discovering Novel Sequence Motifs with MEME
Authors: Bailey Timothy L.
Published: June 2004

This unit illustrates how to use MEME to discover motifs in a group of related nucleotide or peptide sequences. A MEME motif is a sequence pattern that occurs repeatedly in one or more sequences in the input group. MEME can be used to discover novel patterns because it bases its discoveries only on the input sequences, not on any prior knowledge (such as databases of known motifs). The input to MEME is a set of unaligned sequences of the same type (peptide or nucleotide). For each motif it discovers, MEME reports the occurrences (sites), consensus sequence, and the level of conservation (information content) at each position in the pattern. MEME also produces block diagrams showing where all of the discovered motifs occur in the training set sequences. MEME's hypertext (HTML) output also contains buttons that allow for the convenient use of the motifs in other searches.
Using TESS to Predict Transcription Factor Binding Sites in DNA Sequence
Authors: Schug Jonathan
Published: June 2004

This unit describes how to use the Transcription Element Search System (TESS). This Web site predicts transcription factor binding sites in DNA sequence using two different kinds of models of sites, strings and positional weight matrices. The binding of transcription factors to DNA is a major part of the control of gene expression. Transcription factors exhibit sequence‐specific binding; they form stronger bonds to some DNA sequences than to others. Identification of a good binding site in the promoter for a gene suggests the possibility that the corresponding factor may play a role in the regulation of that gene. However, the sequences transcription factors recognize are typically short and allow for some amount of mismatch. Because of this, binding sites for a factor can typically be found at random every few hundred to a thousand base pairs. TESS has features to help sort through and evaluate the significance of predicted sites.
The InterPro Database and Tools for Protein Domain Analysis
Authors: Mulder Nicola J. , Apweiler Rolf
Published: June 2004

InterPro provides a one‐stop shop for protein‐sequence classification, freeing the user from having to visit multiple database separately and rationalize the different results in varying formats. This unit describes how to submit a sequence to InterProScan via a Web or E‐mail server. It also provides instructions for installing and running InterProScan locally. In addition, details on browsing InterPro families and domains of interest using the InterPro Web and sequence retrieval system (SRS) are provided.
An Overview of Sequence Similarity (“Homology”) Searching
Authors: Davison Daniel B.
Published: June 2004

Sequence similarity searching is an essential tool for molecular biologists. It is used to support inference of protein function and for phylogenetic analysis. Every searching procedure requires some understanding of the underlying principles, so at the very least the investigator's selection of parameters is correct. The principles underlying the most commonly used procedures are presented in this unit. The discussion is intentionally non‐mathematical, but does contain references for those who desire a mathematical and statistical discussion of these procedures.
Finding Homologs to Nucleotide Sequences Using Network BLAST Searches
Authors: Ladunga Istvan
Published: June 2004

Basic Local Alignment Search Tool (BLAST) can identify possible homologs in nucleotide and protein databases with high sensitivity and selectivity, and at an amazing speed. Those homologs may provide inference for the biochemical function, the exon boundaries, the domain architecture, the secondary and tertiary structure of the protein, and many other features. The purpose of this unit is not restricted to providing optimal ways of applying the BLAST tool. Just running a BLAST search is an easy task and still can be of great service to researchers. The unit details how to fine tune the arguments of the programs, allowing the user to take advantage of frequently overlooked capabilities of the tool. It also addresses the pitfalls in over‐ or under‐interpretation of results.
Constructing and Refining Multiple Sequence Alignments with PileUp, SeqLab, and the GCG Suite
Authors: Thompson Steven M.
Published: June 2004

This unit discusses how the Accelrys GCG Wisconsin Package SeqLab graphical user interface can be used to align, annotate, analyze, and export into alternative formats, multiple biological sequence data. The emphasis is on discovering and recognizing common elements within the dataset. The GCG programs, or implementations of public domain programs thereof, investigated include: LookUp, PileUp, PlotSimilarity, FASTA, Motifs, MEME/MotifSearch, the Profile Package, the HMMER Package, PAUPSearch, and ToFastA. ReadSeq, a non‐GCG, public domain program is also used.
Computing Multiple Sequence/Structure Alignments with theT‐Coffee Package
Authors: Notredame Cedric , Suhre Karsten
Published: June 2004

The FASTA package provides a comprehensive set of similarity searching programs, similar to those provided by the BLAST package, and some additional programs for searching with short peptides and oligonucleotides that are not provided by BLAST. The FASTA programs work with a wide variety of database formats, including mySQL sequence databases. FASTA provides very accurate statistical significance estimates, and is more sensitive than BLASTN when comparing DNA sequences. These protocols describe how to use the FASTA programs to characterize protein and DNA sequences, using protein:protein, protein:DNA, and DNA:DNA comparisons.
Finding Protein and Nucleotide Similarities with FASTA
Authors: Pearson William
Published: June 2004

The FASTA package provides a comprehensive set of similarity searching programs, similar to those provided by the BLAST package, and some additional programs that are not provided by BLAST for searching with short peptides and oligonucleotides. The FASTA programs work with a wide variety of database formats, including mySQL sequence databases. FASTA provides very accurate statistical significance estimates, and is more sensitive than BLASTN when comparing DNA sequences. These protocols describe how to use the FASTA programs to characterize protein and DNA sequences, using protein:protein, protein:DNA, and DNA:DNA comparisons.
Mathematically Complete Nucleotide and Protein Sequence Searching Using Ssearch
Authors: Ropelewski Alexander J. , Nicholas Hugh B. , Deerfield David W.
Published: June 2004

In this unit a protocol is described for predicting the structure of simple transmembrane a‐helical bundles. The protocol is based on a global molecular dynamics search (GMDS) of the configuration space of the helical bundle, yielding several candidate structures. The correct structure among these candidates is selected using information from silent amino acid substitutions, employing the premise that only the correct structure must (by definition) accept all of the silent amino acid substitutions. Thus, the correct structure is found by repeating the GMDS for several close homologs and selecting the structure that persists in all of the trials.
Installing, Maintaining, and Using a Local Copy of BLAST for Intranet and Workstation Use
Authors: Littlejohn Timothy G.
Published: June 2004

The Basic Local Alignment Search Tool (BLAST) is one of the widest used and most useful applications in sequence‐based bioinformatics analysis. Frequently it is not practical or possible to use remote BLAST services through the Internet due to restrictions of a security or technical nature or the need for high‐throughput analysis requiring greater amounts of processing power than are available from remote services. This unit describes the steps involved in obtaining and installing a copy of the BLAST software for use on a local intranet or stand‐alone workstation. Once installed, the BLAST package can be used to create BLAST‐searchable nucleotide and protein sequence databanks. Various popular hardware (PPC, Intel) and operating system (MacOSX, FreeBSD and Linux) options for running and maintaining the software are discussed. Finally, steps for indexing proprietary and third party (publicly available) sequence databanks for use with BLAST and managing these resources are discussed.
An Overview of Gene Identification: Approaches, Strategies, and Considerations
Authors: Baxevanis Andreas D.
Published: June 2004

Modern biology is on the verge of officially ushering in a new era in science with the completion of the sequencing of the human genome in April 2003. While often erroneously called the “post‐genome era”, this will actually truly mark the beginning of the “genome era,” a time in which the availability of sequence data for many genomes will have a significant effect on how science is performed in the 21st century. This unit offers an overview of many of the gene prediction methods that are currently available and offers a general assessment of how well the methods work for various problems.
Using MZEF to Find Internal Coding Exons
Authors: Zhang Micheal Q.
Published: June 2004

MZEF (Michael Zhang's Exon Finder) was designed to help identify one of the most important classes of exons, i.e. the internal coding exons, in human genomic DNA sequences. It is neither for predicting intronless genes, nor for assembling predicted exons into complete gene models. There is also a mouse version (mMZEF) and an Arabidopsis version (aMZEF). This unit presents the Unix and Web versions of MZEF and reviews how to interpret the MZEF results.
Using geneid to Identify Genes
Authors: Blanco Enrique , Parra Genís , Guigó Roderic
Published: June 2004

The unit describes the usage of geneid. geneid is a very efficient gene‐finding program. It allows for the analysis of large genomic sequences, including whole mammalian chromosomes. These sequences can be partially annotated, and geneid can be used to refine this initial annotation. Parameter configurations exist for a number of eukaryotic species. Geneid produces output in a variety of standard formats. The results, thus, can be processed by a variety of software tools, including visualization programs. Geneid software is in the public domain, and it is undergoing constant development. It is easy to install and use. Exhaustive benchmark evaluations show that geneid compares favorably with other existing gene finding tools.
Using GlimmerM to Find Genes in Eukaryotic Genomes
Authors: Pertea Mihaela , Salzberg Steven L.
Published: June 2004

GlimmerM is a eukaryotic gene finder that has been used in the annotation of the genomes of Plasmodium falciparum (the malaria parasite), the model plant Arabidopsis thaliana, Oryza sativa (rice), the parasite Theileria parva, and the fungus Aspergillus fumigatus. A unique feature of the system compared to other eukaryotic gene finders is a module that allows users to provide their own data and train GlimmerM for any organism.
Prokaryotic Gene Prediction Using GeneMark and GeneMark.hmm
Authors: Borodovsky Mark , Mills Ryan , Besemer John , Lomsadze Alex
Published: June 2004

In this unit, the GeneMark and GeneMark.hmm programs are presented as two different methods for the in silico prediction of genes in prokaryotes. GeneMark can be used for whole genome analysis as well as for the local analysis of a particular gene and its surrounding regions. GeneMark.hmm makes use of Hidden Markov models to find the transition points (boundaries) between protein coding states and noncoding states and can be efficiently used for larger genome sequences. These methods can be used in conjunction with each other for a higher sensitivity of gene detection.
Eukaryotic Gene Prediction Using GeneMark.hmm
Authors: Borodovsky Mark , Lomsadze Alex , Ivanov Nikolai , Mills Ryan
Published: June 2004

In this unit, eukaryotic GeneMark.hmm is presented as a method for detecting genes in eukaryotic DNA sequences. The eukaryotic GeneMark.hmm uses Markov models of protein coding and noncoding sequences, as well as positional nucleotide frequency matrices for prediction of the translational start, translational termination and splice sites. All these models along with length distributions of exons, introns and intergenic regions are integrated into one Hidden Markov model. The unit describes running the program over the Internet and locally on a Unix machine. It also discusses GeneMarkS EV, which can be used to detect genes in eukaryotic viruses.
Application of FirstEF to Find Promoters and First Exons in the Human Genome
Authors: Davuluri Ramana V.
Published: June 2004

Predicting first exons and promoters is an important part of gene finding in DNA sequence analysis. This unit presents FirstEF as a method for predicting the first exons and promoters. A combines FirstEF predictions with other information such as cDNA/EST matches.
GrailEXP and Genome Analysis Pipeline for Genome Annotation
Authors: Uberbacher Edward C. , Hyatt Doug , Shah Manesh
Published: June 2004

The Basic Protocol describes the use of GrailEXP, the latest version of the gene finding system from Oak Ridge National Laboratory. GrailEXP provides gene models, by making use of sequence similarity with Expressed Sequence Tags (ESTs) and known genes. GrailEXP also provides alternatively spliced constructs for each gene based on the available EST evidence. The Support Protocol describes the use of the Genome Analysis Pipeline, a web application which allows users to perform comprehensive sequence analysis by offering a selection from a wide choice of supported gene finders, other biological feature finders, and database searches.
Using RepeatMasker to Identify Repetitive Elements in Genomic Sequences
Authors: Chen Nansheng
Published: June 2004

The RepeatMasker program is used for identifying repetitive elements in nucleotide sequences for further detailed analyses. Users can run RepeatMasker remotely via a Web site, or, for larger input sequences, the program and its dependent programs may be downloaded and run locally on Unix/Linux computers. The protocols in this chapter detail how to use RepeatMasker both remotely and locally to extract repetitive elements data and mask these repetitive elements in nucleotide sequences.
Introduction to Inferring Evolutionary Relationships
Authors: Page Roderic D.M.
Published: June 2004

This unit provides a general introduction to phylogeny. It defines common terms and discusses the issue of rooting trees, in addition to comparing gene and species trees. Methods for inferring phylogenies, such as distance methods, parsimony methods, and maximum likelihood are also presented. The unit concludes with discussion of how to assess tree confidence.