Genetics as a Tool to Understand Structure and Function
Peter M. Gresshoff in Molecular Biology of Symbiotic Nitrogen Fixation, 2018
The quaternary structure of proteins may lead to complications in the interpretation of genetic analysis, even when subunits are identical and are determined by a single gene. Thus, in complementation tests, the restoration of the wild phenotype when two mutant genomes affecting the same function are introduced into the same cell normally implies that the mutations are in different genes; the heterokaryon has one good gene of each type. However, if the protein is an aggregate of two or more identical subunits, mutations at different locations in the same gene may produce nonoverlapping defects in the subunits which can then aggregate randomly and stabilize one another; thus, a functional protein is produced, but it is usually less active than the wild type. This is called "intraallelic complementation".
An Approach to Inherited Pulmonary Disease
Stephen D. Litwin in Genetic Determinants of Pulmonary Disease, 2020
The concept of complementation has been applied with great success to the analysis of genetic heterogeneity in organisms ranging from viruses to humans. Autosomal recessive deficiencies of enzymes commonly result in the accumulation of substrates proximal to the altered enzyme in the metabolic pathway, and deficiency of products distal to the enzyme. Both accumulation and deficiency of metabolites have been implicated in the pathogenesis of various disease phenotypes. Complementation is the name given to the observation that when cells with one enzyme deficiency are combined with cells with deficiency of another enzyme in the same metabolic pathway, the deficiency in each cell is compensated by normal activity of that enzyme in the other cell; the two deficiencies complement one another to give sufficient joint activity of the metabolic pathway to abolish the abnormal phenotype. If two cells each have a defect in the same metabolic pathway and they do not complement one another the interpretation is that the same enzyme is defective in both cells.
Integrins, Integrin Regulators, and the Extracellular Matrix
Bruce S. Bochner in Adhesion Molecules in Allergic Disease, 2020
A genetic approach has also been employed to elucidate the mechanisms by which activation upregulates β1 integrin activity (91). Mutants of the Jurkat T cell line were isolated by γ-irradiation and selection for cells that were unable to bind to Fn-coated plates following stimulation with PMA or anti-CD3 mAbs. These mutant cell lines expressed normal levels of β1 integrins. Furthermore, the ability of these mutants to bind to Fn and VCAM-1 following direct stimulation of β1 integrins with an activating β1-specific mAb suggested that the mutation induced in these cells did not alter the structural integrity of the ligand-binding domain. Genetic complementation studies indicated that at least three genetically distinct mutant types were isolated. The defect in β1 integrin function in one of these mutant types was also associated with the expression of an altered form of the MAPK isoform ERK-1 and defective production of IL-2 following CD3 stimulation. While the precise nature of the defects in β 1 integrin function in these mutant cells remains to be elucidated, early signaling events that impact integrin functional activity, such as tyrosine kinase activity, PKC activity, and intracellular Ca2+ flux, appear normal.
How to discover new antibiotic resistance genes?
Published in Expert Review of Molecular Diagnostics, 2019
Linda Hadjadj, Sophie Alexandra Baron, Seydina M. Diene, Jean-Marc Rolain
Mutagenesis is a process by which the genetic information (DNA) of an organism is changed, resulting in a mutation. This mutation, caused by a substitution, a deletion or an insertion of nucleotides, can make the bacteria resistant to antibiotics [33,35]. To create a specific mutation in a plasmid, protocols can be used [29,37,38], as well as commercial kits as Site-Directed Mutagenesis kit (NEB, Thermo). To determine the action of a specific mutation, two cloning strategies can be employed: original cloning and cloning by complementation. The first cloning method consists of incorporating a plasmid containing the sequence of a gene with a mutation into a bacteria susceptible to antibiotics and analyzing its effect. For the complementation strategy, a plasmid containing the non-mutated gene is inserted into the resistant bacteria carrying the mutation. Transformants are screened on a medium with and without antibiotics. If the bacterium that was originally resistant to this antibiotic becomes susceptible then the cloned gene is considered to be responsible for this resistance. The wild type gene introduced into resistant bacteria can compensate for the defect caused by the mutated gene as described on mutations in the mgrB gene for colistin resistance [29] (Figure 2A).
Returning Results to Family Members: Professional Duties in Genomics Research in the United States
Published in Journal of Legal Medicine, 2018
Dov Fox, Emily Spencer, Ali Torkamani
No one has expressly proposed mandating disclosure of uncertain genomic results. Yet, there has been no rigorous account to date of why no such duty should attach to return at least those findings of known significance. The key insight is that genomic research poses three distinct layers of uncertainty. First is whether an observed variant alters or destroys gene function. Second is whether altering the function of that gene can lead to the observed phenotype.77See R. Scott Hawley and William D. Gilliland, Sometimes the Result Is Not the Answer: The Truths and the Lies That Come from Using the Complementation Test, 174 Genetics 5 (2006). Third is the distinctive risk for relatives of individuals with particular variants. Their family members are certainly at a higher risk of having a variant than the general population. However, it is not known whether they have it until they are tested themselves. That would require them to either become a research participant themselves or that a test for that variant be clinically available. If no test is available and the presence or absence of the variant is not a research interest of the lab, a mandate would essentially force the lab to become a clinical service provider, performing testing that does not have a direct research benefit.
Regenerating the kidney using human pluripotent stem cells and renal progenitors
Published in Expert Opinion on Biological Therapy, 2018
Francesca Becherucci, Benedetta Mazzinghi, Marco Allinovi, Maria Lucia Angelotti, Paola Romagnani
The generation of transplantable kidneys is among the ultimate goals of regenerative nephrology due to a shortage of donor organs that represents a critical obstacle to the expansion of transplantation programs. Notwithstanding this, the complex 3D cellular and tissue interactions required for organogenesis are quite difficult to recapitulate in vitro. Blastocyst complementation is a method used to overcome these obstacles. Briefly, it consists in generating organs in vivo by injecting pluripotent SCs (either ESCs or iPSCs) into blastocyst-stage embryos (mainly, but not exclusively, rodents). This finally leads to the generation of chimeric embryos in which pluripotent SCs contribute to the generation of host tissues and organs [79]. Interspecies blastocyst complementation is a variation of the classical technique in which the recipient host is genetically manipulated to carry DNA mutations that prevent the development of a target organ [80]. Ideally, the injection of donor-derived pluripotent SCs would developmentally compensate for the defect and form the missing organ. This strategy had been initially used for the reconstitution of bone marrow but was subsequently applied to the generation of entire organs (e.g. pancreas, heart, eye) [81]. The resulting organs are composed almost entirely of cells derived from donor, even if the blastocyst complementation involves different species.