The Immunoglobulin Variable-Region Gene Repertoire and Its Analysis
Cliburn Chan, Michael G. Hudgens, Shein-Chung Chow in Quantitative Methods for HIV/AIDS Research, 2017
In addition to combinatorial diversity, IgVRG exhibits junctional diversity. The enzymatic complex consisting of the recombination activating genes RAG1 and RAG2 binds to a recombination signal (RS) adjacent to each of the V, D, and J genes, bringing the genes into close proximity. Proper ordering of the genes is enforced by the so-called 12/23 rule. There are two different types of RS, one with a 12-nucleotide spacer and one with a 23-base spacer. Synapsis occurs only between heterogeneous pairs. The recombinase nicks the DNA at the start of each RS, resulting in a DNA hairpin on each gene, which is then cleaved stochastically by the Artemis complex, resulting in variable recombination points. Because of the hairpin structure, this cleavage may occur beyond the end of the coding region into the noncoding strand, resulting in the appearance of p-nucleotides (p for palindromic). Terminal deoxynucleotidyl transferase may then add several n-nucleotides (n for nontemplated), which are yet another source of stochasticity. The strands then pair in complementary regions, and unpaired nucleotides are removed and filled in to form the final junction region [19,25,26]. The site of RAG-mediated cleavage and n-nucleotides together supply the junctional diversity of the antibody repertoire.
Untangling Appetite Circuits with Optogenetics and Chemogenetics
Ruth B.S. Harris in Appetite and Food Intake, 2017
A common way to achieve such explicit specificity is through a two-pronged approach using genetically-engineered mice that express Cre-recombinase in defined neurons, often driven off the endogenous gene promoter, in combination with Cre-dependent viral vectors expressing activity modulators to stimulate or silence neuronal activity using particular wavelengths of light or pharmacologically inert, chemical ligands (Figure 5.1) (Luo, Callaway, and Svoboda 2008). These adeno-associated viruses are designed in such a way that the open reading frame of the coding sequence is inverted, in the antisense orientation, flanked by two pairs of heterotypic, antiparallel loxP-type recombination sites. In the presence of Cre-recombinase, the recombination sites first undergo an inversion of the coding sequence, followed by excision of two sites, leading to one of each orthogonal recombination site oppositely oriented and incapable of further recombination (Atasoy et al. 2008, Schnutgen et al. 2003). This Cre-dependent viral approach is commonly referred to as a FLEX switch or double-floxed inverse open reading frame (DIO) (Atasoy et al. 2008, Witten et al. 2010) and is used to target specific cell types with spatial precision via intracranial surgeries in distinct Cre driver mouse lines.
Immunoglobulins
Constantin A. Bona, Francisco A. Bonilla in Textbook of Immunology, 2019
Many of the molecules participating in the rearrangement process have yet to be identified. Two candidates for components of a “recombinase complex” are the products of the recombinase activation genes: RAG-1 and RAG-2. Recombinase activity is an integral part of the regulation of B cell development. RAG products are prominent in young lymphocytes which are actively rearranging their Ig genes. RAG activity disappears in mature lymphocytes. Fibroblasts do not normally rearrange Ig gene segments. However, when RAG genes are introduced into these cells, they acquire the capacity for Ig gene rearrangement. It is not clear whether these genes encode recombinase components with enzymatic and/or DNA-binding activity, or if they are transcriptional regulators of genes encoding the actual recombinases.
Phase variation of Clostridium difficile virulence factors
Published in Gut Microbes, 2018
Brandon R. Anjuwon-Foster, Rita Tamayo
Phase variation occurs by multiple mechanisms in diverse mucosal bacterial pathogens, including site-specific recombination, general homologous recombination, slip strand mispairing, and DNA methylation.20 For site-specific recombination, a recombinase recognizes a specific DNA sequence and catalyzes strand exchange and DNA inversion. The orientation of the DNA sequence dictates whether or not a nearby gene is expressed. Classically, the invertible DNA element contains a promoter to transcriptionally control an adjacent gene or operon. The best-studied example of this mechanism is the Type I fimbrial biosynthetic operon in E. coli. Here, the invertible element fimS lies upstream of fimA, which encodes the fimbrial subunit. Within fimS is a promoter that drives transcription of fimA when properly oriented.21 Using a series of transcriptional reporters and growth conditions known to be permissive for flagellar gene expression, we found that the flagellar switch in C. difficile does not contain a promoter, and expression of the flgB operon relies on the upstream σA-dependent promoter.15
An insight into clinical and laboratory detections for screening and diagnosis of cervical cancer
Published in Expert Review of Molecular Diagnostics, 2023
Shruthi Padavu, Pooja Aichpure, Ballamoole Krishna Kumar, Anoop Kumar, RadhaKanta Ratho, Shipra Sonkusare, Indrani Karunasagar, Iddya Karunasagar, Praveen Rai
The recombinase polymerase amplification (RPA) has evolved as a novel isothermal amplification technique for the molecular diagnosis of HR-HPV. The RPA, unlike many other isothermal methods, does not require high temperatures and proceeds in the range of 37–42°C within 20–30 minutes [75]. This technique employs the recombinase activity and strand-displacing activity of the enzyme to unwind dsDNA and amplify the target DNA, respectively. The RPA technique is quite helpful in detecting HR-HPV because of its rapidity and near-normal reaction temperature. The end-point of the isothermal amplification product can be interpreted using fluorescent-based nucleic acid detection or gel electrophoresis. End-point detection can also be achieved by chemically labeling the RPA reaction and using lateral flow strips that enable instrument-free signal readouts. TwistAmp® (TwistDx™, UK) offers a variety of RPA kits with varying end-point detection for research and commercial applications [76]. The RPA achieves exponential amplification without the necessity for the pretreatment of the DNA sample. The RPA assay was shown to be a powerful approach for detecting HPV and may be a beneficial tool for the early diagnosis of HPV infection in prognostic applications [77].
Effects of the COVID-19 pandemic: new approaches for accelerated delivery of gene to first-in-human CMC data for recombinant proteins
Published in mAbs, 2023
Hervé Broly, Jonathan Souquet, Alain Beck
Another factor affecting cell line generation timelines is the mode of insertion of the gene(s) of interest into the host cells. Historically, transfection with a non-homologous recombination-based random integration (RI) was the common method for inserting foreign genes into a mammalian cell host to produce recombinant proteins.65 Site-specific recombinase-mediated integration (SSI) is another method.66–68 SSI has the advantage over random integration to reduce the selection and single-cell cloning stages from several weeks to a few days through negative selection (e.g., by supplementing cell culture medium with ganciclovir) to eliminate cells with unwanted recombination events, thus leading to the generation of more consistent and reliable clones.69
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