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The development of multiple DNA methylation analysis techniques, including higher-throughput assays, has resulted in data structures of increasing complexity and diversity. Here, we discuss the general principles of DNA methylation analysis and propose a nomenclature for the various types of methylation analysis. We briefly outline several DNA methylation analysis techniques and discuss how these...
The lack of simple methods to identify relevant T-cell epitopes, the high mutation rate of many pathogens, and restriction of T-cell response to epitopes due to human lymphocyte antigen (HLA) polymorphism have significantly hindered the development of cytotoxic T-lymphocyte (CTL) epitope-based or ''epitope-driven'' vaccines. Previously, CTL epitopes were mapped using large arrays of overlapping synthetic...
Many gene superfamilies have hundreds or thousands of members and hence pose a significant challenge when performing a large-scale phylogenetic analysis. Derivation of the most accurate alignment possible and inference of evolutionary relationships (with an appropriate measure of confidence) are significant ''bottlenecks'' in the process. A generally applicable strategy is outlined for identifying...
TEPITOPE is a prediction model that has been successfully applied to the in silico identification of T cell epitopes in the context of oncology, allergy, infectious diseases, and autoimmune diseases. Like most epitope prediction models, TEPITOPE’s underlying algorithm is based on the prediction of HLA-II peptide binding, which constitutes a major bottleneck in the natural selection of epitopes. An...
High throughput protein analysis (i.e., proteomics) first became possible when sensitive peptide mass mapping techniques were developed, thereby allowing for the possibility of identifying and cataloging most 2D gel electrophoresis spots. Shortly thereafter a few groups pioneered the idea of identifying proteins by using peptide tandem mass spectra to search protein sequence databases. Hence, it became...
MicroRNAs are natural, single-stranded, small RNA molecules that regulate gene expression by binding to target mRNAs and suppress its translation or initiate its degradation. In contrast to the identification and validation of many miRNA genes is the lack of experimental evidence identifying their corresponding mRNA targets. The most fundamental challenge in miRNA biology is to define the rules of...
In bacteria, the expression of transcription units is controlled by regulatory regions, that contain one or more promoters and binding sites for regulatory proteins that activate or repress expression in response to different signals. In this chapter, we explain the diverse approaches that can be used to understand the mechanisms by which the different factors intervene, and how the effects are integrated...
Thirty years of research on gene transcription has uncovered a myriad of factors that regulate, directly or indirectly, the activity of RNA polymerase II (RNAPII) during mRNA synthesis. Yet many regulatory factors remain to be discovered. Using protein affinity purification coupled to mass spectrometry (AP-MS), we recently unraveled a high-density interaction network formed by RNAPII and its accessory...
Selection of protein targets for study is central to structural biology and may be influenced by numerous factors. A key aim is to maximise returns for effort invested by identifying proteins with the balance of biophysical properties that are conducive to success at all stages (e.g. solubility, crystallisation) in the route towards a high resolution structural model. Selected targets can be optimised...
Bioinformatic approaches to the identification of genomic sequences having G-quadruplex forming potential (QFP) has enabled important tests of the structure of these sequences in vitro and of their behavior under conditions where the formation or function of G-quadruplexes is modulated in vivo. Several similar approaches to identifying intramolecular QFP (i.e. forming among G-runs on one strand of...
miRNAs are short (20–23nt) RNAs that are loaded into proteins of the Argonaute (AGO) family and guide them to partially complementary target sites on mRNAs, resulting in mRNA destabilization and/or translational repression. It is estimated that about 60% of the mammalian genes are potentially regulated by miRNAs, and therefore methods for experimental miRNA target determination have become valuable...
Molecular biomarkers found their way into many research fields, especially in molecular medicine, medical diagnostics, disease prognosis, risk assessment but also in other areas like food safety. Different definitions for the term biomarker exist, but on the whole biomarkers are measureable biological molecules that are characteristic for a specific physiological status including drug intervention,...
Selective or Multiple Reaction monitoring (SRM/MRM) is a liquid-chromatography (LC)/tandem-mass spectrometry (MS/MS) method that enables the quantitation of specific proteins in a sample by analyzing precursor ions and the fragment ions of their selected tryptic peptides. Instrumentation software has advanced to the point that thousands of transitions (pairs of primary and secondary m/z values) can...
An emergent strategy for the transcriptome-wide study of protein–RNA interactions is CLIP-seq (crosslinking and immunoprecipitation followed by high-throughput sequencing). We combined CLIP-seq and mRNA-seq to identify direct RNA binding sites of eIF4AIII in human cells. This RNA helicase is a core constituant of the Exon Junction Complex (EJC), a multifunctional protein complex associated with spliced...
Associations between proteins and RNA–RNA duplexes are important in post-transcriptional regulation of gene expression. The CLASH (Cross-linking, Ligation and Sequencing of Hybrids) technique captures RNA–RNA interactions by physically joining two RNA molecules associated with a protein complex into a single chimeric RNA molecule. These events are relatively rare and considerable effort is needed...
Highly parallel sequencing of cDNA derived from endogenous small RNAs (small RNA-seq) is a key method that has accelerated understanding of regulatory small RNAs in eukaryotes. Eukaryotic regulatory small RNAs, which include microRNAs (miRNAs), short interfering RNAs (siRNAs), and Piwi-associated RNAs (piRNAs), typically derive from the processing of longer precursor RNAs. Alignment of small RNA-seq...
The discovery of the small regulatory RNAs has changed our vision of cellular regulations. Indeed, when loaded on Argonaute proteins they form ribonucleoprotein complexes (RNPs) that target complementary sequences to achieve widespread silencing mechanisms conserved in most eukaryotes. The recent development of deep sequencing approaches highly contributed to their detection. Small RNA isolation from...
TAL effectors are transcription factors injected into plant cells by pathogenic bacteria during infection. They find their specific DNA targets via a string of contiguous, structural repeats that individually recognize single nucleotides (with some degeneracy) by virtue of polymorphisms at residue 13. The number of repeats and sequence of the amino acids at position 13 determine the nucleotide sequence...
Over the last decade, development and application of a set of molecular genomic approaches based on the chromosome conformation capture method (3C), combined with increasingly powerful imaging approaches, have enabled high resolution and genome-wide analysis of the spatial organization of chromosomes. The aim of this paper is to provide guidelines for analyzing and interpreting data obtained with...
Studies of the brain’s transcriptome have become prominent in recent years, resulting in an accumulation of datasets with somewhat distinct attributes. These datasets, which are often analyzed only in isolation, also are often collected with divergent goals, which are reflected in their sampling properties. While many researchers have been interested in sampling gene expression in one or a few brain...
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