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TCP-seq
Translation complex profile sequencing (TCP-seq) is a molecular biology method for obtaining snapshots of momentary distribution of protein synthesis complexes along messenger RNA (mRNA) chains. Application Expression of genetic code in all life forms consists of two major processes, synthesis of copies of the genetic code recorded in DNA into the form of mRNA (transcription), and protein synthesis itself (translation), whereby the code copies in mRNA are decoded into amino acid sequences of the respective proteins. Both transcription and translation are highly regulated processes essentially controlling everything of what happens in live cells (and multicellular organisms, consequently). Control of translation is especially important in eukaryotic cells where it forms part of post-transcriptional regulatory networks of genes expression. This additional functionality is reflected in the increased complexity of the translation process, making it a hard object to investigate. Yet ...
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ChIP-sequencing
ChIP-sequencing, also known as ChIP-seq, is a method used to analyze protein interactions with DNA. ChIP-seq combines chromatin immunoprecipitation (ChIP) with massively parallel DNA sequencing to identify the binding sites of DNA-associated proteins. It can be used to map global binding sites precisely for any protein of interest. Previously, ChIP-on-chip was the most common technique utilized to study these protein–DNA relations. Uses ChIP-seq is primarily used to determine how transcription factors and other chromatin-associated proteins influence phenotype-affecting mechanisms. Determining how proteins interact with DNA to regulate gene expression is essential for fully understanding many biological processes and disease states. This epigenetic information is complementary to genotype and expression analysis. ChIP-seq technology is currently seen primarily as an alternative to ChIP-chip which requires a hybridization array. This introduces some bias, as an array is restr ...
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Molecular Biology
Molecular biology is the branch of biology that seeks to understand the molecular basis of biological activity in and between cells, including biomolecular synthesis, modification, mechanisms, and interactions. The study of chemical and physical structure of biological macromolecules is known as molecular biology. Molecular biology was first described as an approach focused on the underpinnings of biological phenomena - uncovering the structures of biological molecules as well as their interactions, and how these interactions explain observations of classical biology. In 1945 the term molecular biology was used by physicist William Astbury. In 1953 Francis Crick, James Watson, Rosalind Franklin, and colleagues, working at Medical Research Council unit, Cavendish laboratory, Cambridge (now the MRC Laboratory of Molecular Biology), made a double helix model of DNA which changed the entire research scenario. They proposed the DNA structure based on previous research done by ...
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Ribonuclease
Ribonuclease (commonly abbreviated RNase) is a type of nuclease that catalyzes the degradation of RNA into smaller components. Ribonucleases can be divided into endoribonucleases and exoribonucleases, and comprise several sub-classes within the EC 2.7 (for the phosphorolytic enzymes) and 3.1 (for the hydrolytic enzymes) classes of enzymes. Function All organisms studied contain many RNases of two different classes, showing that RNA degradation is a very ancient and important process. As well as clearing of cellular RNA that is no longer required, RNases play key roles in the maturation of all RNA molecules, both messenger RNAs that carry genetic material for making proteins and non-coding RNAs that function in varied cellular processes. In addition, active RNA degradation systems are the first defense against RNA viruses and provide the underlying machinery for more advanced cellular immune strategies such as RNAi. Some cells also secrete copious quantities of non-specific ...
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Molecular Biology Techniques
A molecule is a group of two or more atoms held together by attractive forces known as chemical bonds; depending on context, the term may or may not include ions which satisfy this criterion. In quantum physics, organic chemistry, and biochemistry, the distinction from ions is dropped and ''molecule'' is often used when referring to polyatomic ions. A molecule may be homonuclear, that is, it consists of atoms of one chemical element, e.g. two atoms in the oxygen molecule (O2); or it may be heteronuclear, a chemical compound composed of more than one element, e.g. water (molecule), water (two hydrogen atoms and one oxygen atom; H2O). In the kinetic theory of gases, the term ''molecule'' is often used for any gaseous particle regardless of its composition. This relaxes the requirement that a molecule contains two or more atoms, since the noble gases are individual atoms. Atoms and complexes connected by non-covalent interactions, such as hydrogen bonds or ionic bonds, are typic ...
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Yeast
Yeasts are eukaryotic, single-celled microorganisms classified as members of the fungus kingdom. The first yeast originated hundreds of millions of years ago, and at least 1,500 species are currently recognized. They are estimated to constitute 1% of all described fungal species. Yeasts are unicellular organisms that evolved from multicellular ancestors, with some species having the ability to develop multicellular characteristics by forming strings of connected budding cells known as pseudohyphae or false hyphae. Yeast sizes vary greatly, depending on species and environment, typically measuring 3–4 µm in diameter, although some yeasts can grow to 40 µm in size. Most yeasts reproduce asexually by mitosis, and many do so by the asymmetric division process known as budding. With their single-celled growth habit, yeasts can be contrasted with molds, which grow hyphae. Fungal species that can take both forms (depending on temperature or other conditions) are ...
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Polysome
A polyribosome (or polysome or ergosome) is a group of ribosomes bound to an mRNA molecule like “beads” on a “thread”. It consists of a complex of an mRNA molecule and two or more ribosomes that act to translate mRNA instructions into polypeptides. Originally coined "ergosomes" in 1963, they were further characterized by Jonathan Warner, Paul M. Knopf, and Alex Rich Alexander Rich (15 November 1924 – 27 April 2015) was an American biologist and biophysicist. He was the William Thompson Sedgwick Professor of Biophysics at MIT (since 1958) and Harvard Medical School. Rich earned an A.B. (''magna cum .... Polysomes are formed during the elongation phase when ribosomes and elongation factors synthesize the encoded polypeptide. Multiple ribosomes move along the coding region of mRNA, creating a polysome. The ability of multiple ribosomes to function on an mRNA molecule explains the limited abundance of mRNA in the cell. Polyribosome structure differs between pro ...
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Ribosome Profiling
Ribosome profiling, or Ribo-Seq (also named ribosome footprinting), is an adaptation of a technique developed by Joan Steitz and Marilyn Kozak almost 50 years ago that Nicholas Ingolia and Jonathan Weissman adapted to work with next generation sequencing that uses specialized messenger RNA (mRNA) sequencing to determine which mRNAs are being actively translated. A related technique that can also be used to determine which mRNAs are being actively translated is the Translating Ribosome Affinity Purification (TRAP) methodology, which was developed by Nathaniel Heintz at Rockefeller University (in collaboration with Paul Greengard and Myriam Heiman). TRAP does not involve ribosome footprinting but provides cell type-specific information. Description It produces a “global snapshot” of all the ribosomes actively translating in a cell at a particular moment, known as a translatome. Consequently, this enables researchers to identify the location of translation start sites, the co ...
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Polysome Profiling
Polysome profiling is a technique in molecular biology that is used to study the association of mRNAs with ribosomes. It is important to note that this technique is different from ribosome profiling. Both techniques have been reviewed and both are used in analysis of the translatome, but the data they generate are at very different levels of specificity. When employed by experts, the technique is remarkably reproducible: the 3 profiles in the first image are from 3 different experiments. The procedure The procedure begins by making a cell lysate of the cells of interest. This lysate contains polysomes, monosomes (composed of one ribosome residing on an mRNA), the small (40S in eukaryotes) and large (60S in eukaryotes) ribosomal subunits, "free" mRNA and a host of other soluble cellular components. The procedure continues by making a continuous sucrose gradient of continuously variable density in a centrifuge tube. At the concentrations used (15-45% in the example), sucrose does ...
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Transcriptomics Technologies
Transcriptomics technologies are the techniques used to study an organism's transcriptome, the sum of all of its RNA transcripts. The information content of an organism is recorded in the DNA of its genome and expressed through transcription. Here, mRNA serves as a transient intermediary molecule in the information network, whilst non-coding RNAs perform additional diverse functions. A transcriptome captures a snapshot in time of the total transcripts present in a cell. Transcriptomics technologies provide a broad account of which cellular processes are active and which are dormant. A major challenge in molecular biology is to understand how a single genome gives rise to a variety of cells. Another is how gene expression is regulated. The first attempts to study whole transcriptomes began in the early 1990s. Subsequent technological advances since the late 1990s have repeatedly transformed the field and made transcriptomics a widespread discipline in biological sciences. There ar ...
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Transcriptome
The transcriptome is the set of all RNA transcripts, including coding and non-coding, in an individual or a population of cells. The term can also sometimes be used to refer to all RNAs, or just mRNA, depending on the particular experiment. The term ''transcriptome'' is a portmanteau of the words ''transcript'' and ''genome''; it is associated with the process of transcript production during the biological process of transcription. The early stages of transcriptome annotations began with cDNA libraries published in the 1980s. Subsequently, the advent of high-throughput technology led to faster and more efficient ways of obtaining data about the transcriptome. Two biological techniques are used to study the transcriptome, namely DNA microarray, a hybridization-based technique and RNA-seq, a sequence-based approach. RNA-seq is the preferred method and has been the dominant transcriptomics technique since the 2010s. Single-cell transcriptomics allows tracking of transcript chang ...
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Sedimentation Coefficient
The sedimentation coefficient () of a particle characterizes its sedimentation during centrifugation. It is defined as the ratio of a particle's sedimentation velocity to the applied acceleration causing the sedimentation. : s = \frac The sedimentation speed is also the terminal velocity. It is constant because the force applied to a particle by gravity or by a centrifuge (typically in multiples of tens of thousands of gravities in an ultracentrifuge) is balanced by the viscous resistance (or "drag") of the fluid (normally water) through which the particle is moving. The applied acceleration can be either the gravitational acceleration , or more commonly the centrifugal acceleration . In the latter case, is the angular velocity of the rotor and is the distance of a particle to the rotor axis ( radius). The viscous resistance for a spherical particle is given by Stokes' law: , where is the viscosity of the medium, is the radius of the particle and is the velocity ...
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Cross-link
In chemistry and biology a cross-link is a bond or a short sequence of bonds that links one polymer chain to another. These links may take the form of covalent bonds or ionic bonds and the polymers can be either synthetic polymers or natural polymers (such as proteins). In polymer chemistry "cross-linking" usually refers to the use of cross-links to promote a change in the polymers' physical properties. When "crosslinking" is used in the biological field, it refers to the use of a probe to link proteins together to check for protein–protein interactions, as well as other creative cross-linking methodologies. Although the term is used to refer to the "linking of polymer chains" for both sciences, the extent of crosslinking and specificities of the crosslinking agents vary greatly. As with all science, there are overlaps, and the following delineations are a starting point to understanding the subtleties. Polymer chemistry Crosslinking is the general term for the process of ...
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