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Methods of Evaluation in Orthopaedic Animal Research
Published in Yuehuei H. An, Richard J. Friedman, Animal Models in Orthopaedic Research, 2020
The basic terminology given here is adapted from the reviews by Shore and Kaplan.251,252 A gene is a unit of heredity, consisting of a segment of chromosomal DNA that is required for production of a functional protein or RNA. The gene contains both coding and regulatory regions. A transgene is a foreign gene which has been spliced into an animals original genomic DNA. mRNA is a type of RNA that contains protein coding information. Nucleotide sequence refers to the order of nucleotides in a given segment of DNA or RNA. Translocation is the transfer of a portion of DNA from one chromosome to another. A probe is a DNA or RNA molecule that is labeled, or tagged, and can then be used to locate a complementary DNA or RNA strand through hybridization. Vectors are DNA molecules that are used as carrier molecules for cloned DNA sequences. They contain information which allows recombinant molecules to be replicated in host bacterial cells. A plasmid is a small circular double-stranded DNA molecule which is found in bacteria and replicates independently of the host chromosome. They are commonly used as vectors in molecular cloning. A recombinant DNA molecule is a DNA molecule containing segments of DNA from different origins, such as a piece of human DNA that has been joined to a plasmid DNA. A clone is a term used to describe identical segmental DNA molecules produced by recombinant DNA technique. Molecular cloning is a process by which a specific segment of DNA is isolated and then numerous identical copies, or clones, of that segment of DNA are generated.
Molecular Genetic Analyses of Functional Melanoma-Associated Antigens
Published in Henry T. Lynch, Ramon M. Fusaro, Hereditary Malignant Melanoma, 2019
Lloyd H. Graf, Soldano Ferrone
The cloning approaches described previously can be applied with varying degrees of generality as means of addressing the molecular cloning of immunologically defined molecules. For example, successful use of expression-based cDNA cloning strategies relies on the ability of antibodies to recognize unglycosylated microbial translation products; animal cell cDNA expression cloning relies on the nonexpression by host COS cells, of primate antigen homologs immunologically cross-reactive with the human antigen of interest; and transfection-based approaches can succeed only if the epitope(s) that react with the available antibodies are products of single genes of transfectable (⩽50 kb) size. While the 96 kDa MAA/ICAM-1 molecule may be unusual in meeting all of these criteria, it is probable that the gene(s) encoding any given antigen will be subject to isolation by one or more of the approaches.
Neurotransmission at Parasympathetic Nerve Endings
Published in Kenneth J. Broadley, Autonomic Pharmacology, 2017
Choline acetyltransferase (EC 2.3.1.6) is a globular protein of relative molecular mass in the region of 73 kDa. It is located in the axonal cytoplasm in predominantly soluble form but also ionically bound to membrane lipids. It is transported down the axon after synthesis in the ribosomes of the cell body (perikaryon) and is concentrated at the nerve terminal. There are probably multiple forms of this enzyme and the primary structure has now been identified from molecular cloning (Berrard et al. 1989). The reaction sequence probably occurs with AcCoA binding initially to the enzyme, before choline, and then Ach dissociating first. The binding of AcCoA to the enzyme is probably by the 3′-phosphate to an arginine residue at the active site. The choline appears to bind with its cationic head to an anionic site. The hydroxyl group of choline attacks the thiol-ester groups of AcCoA to release the acetyl group; this is facilitated by the imidazole group of a histidine residue within the active centre (Figure 7.2).
Immunogenicity and safety of seasonal influenza vaccines in children under 3 years of age
Published in Expert Review of Vaccines, 2023
Tia Aoun, Ray Borrow, Peter D. Arkwright
IIVs differ depending on the steps taken after purification of the viral load from the culture. The first-generation IIVs were whole-virus/virion vaccines that used ß-propiolactone or formalin to chemically inactive the virus, attenuating its virulence but retaining its antigenicity [2,12]. Split-virion IIVs were developed to reduce the potential side effects caused by unnecessary viral particles (present in the whole-virus vaccines). After chemical inactivation, the virus is subjected to further disruption of its envelope and the non-antigenic products – nucleic acids, proteins of higher molecular weight, and the splitting agents – are discarded leaving only the Hemagglutinin, Neuraminidase, M, and NP particles [2]. Furthermore, subunit IIVs are derived from split IIVs, where the hemagglutinin and neuraminidase proteins are further concentrated. This construction process has been made easier due to the advancement of molecular cloning techniques; genes encoding the antigenic proteins are inserted into an expression plasmid, which is transferred into a cell (prokaryotic or eukaryotic) to clone and produce the Hemagglutinin and Neuraminidase proteins at larger yields [2]. Virosome IIVs were developed to retain the cell-binding ability of the virus and induce cytotoxic T-cell responses by including the viral phospholipids in addition to the antigenic Hemagglutinin/Neuraminidase proteins [2].
Dietary S. maltophilia induces supersized lipid droplets by enhancing lipogenesis and ER-LD contacts in C. elegans
Published in Gut Microbes, 2022
Kang Xie, Yangli Liu, Xixia Li, Hong Zhang, Shuyan Zhang, Ho Yi Mak, Pingsheng Liu
A forward genetic screen was conducted to identify C. elegans host factors that mediate the S. maltophilia effect on LDs. We used DHS-3::GFP or MDT-28::mCherry as LD markers (Fig. 4a and 4b) and screened 7,000 haploid genomes after chemical mutagenesis with ethyl-methane sulfonate (EMS), and isolated 7 mutant strains (Figure 4a). Genetic mapping based on single nucleotide polymorphisms (SNPs) eventually led to the molecular cloning of two genes: acs-13 and dpy-9 (Figure 4c-Figure 4h). The acs-13 gene encodes an ortholog of human ACSL1, 5, 6 (acyl-CoA synthetase long-chain family member 1, 5, 6). The dpy-9 gene encodes a cuticular collagen family member with similarity to human collagen alpha 5, type IV. Using complementation tests and RNAi, we confirmed that the loss of acs-13 and dpy-9 function blocked the ability of S. maltophilia to induce LD expansion in C. elegans (Figure 4a-4h).
Next-generation sequencing for the diagnosis of hepatitis B: current status and future prospects
Published in Expert Review of Molecular Diagnostics, 2021
Selene Garcia-Garcia, Maria Francesca Cortese, Francisco Rodríguez-Algarra, David Tabernero, Ariadna Rando-Segura, Josep Quer, Maria Buti, Francisco Rodríguez-Frías
Despite its usefulness, Sanger sequencing is limited to detecting variants at relatively high frequencies (>15-20%) [53], without quantifying variant abundance in viral quasispecies. It is often used to obtain a consensus sequence through overall population sequencing. Alternatively, Sanger sequencing may be performed after molecular cloning. In this case, however, it is extremely difficult to process hundreds of clones, and the number of sequences does not represent the magnitude and complexity of the quasispecies population. Sanger sequencing is thus unsuitable for detecting minor circulating variants in viral quasispecies, as well as low-frequency novel mutations selected throughout the infection [54,55,56], which affect disease progression and/or antiviral response. Although this technology remains useful for applications where high throughput is not required [41], the study of quasispecies variability requires sequencing methods that can analyze a higher number of individual sequences.