1 Variant Mutant

In order to show up that everywhere

Someone or something that seems strange, abnormal, or bizarre in the area

Something which has mutated, which has one or more new characteristics from a mutation

Strange, abnormal, or bizarre

That has undergone mutation

Although resulting from mutation; experiencing mutation

The sygnal buzzering buzzering

They wan to entry in the place

Organism that developed as a result of mutation in the dark (with inheritable traits that differ from those of the parent)

Any heritable variation from the wild type that is the result of a mutation Often (but not always) recessive

An organism or cell carrying a mutation An alternative phenotype to the wild-type; the phenotype produced by a non-wildtype allele

(biology) an organism that has characteristics resulting from chromosomal alteration

1 A term applied to a gene or phenotype altered by mutation 2 An individual carrying a mutation

A mutant is an animal or plant that is physically different from others of the same species because of a change in its genes. = mutation

an animal that has undergone mutation (biology) an organism that has characteristics resulting from chromosomal alteration tending to undergo or resulting from mutation; "a mutant gene

Variant organism that differs from its parent because of mutation

Organism, population, gene, or chromosome that differs from the corresponding wild type by one or more base pairs

A cell microorganism that manifests new characteristics due to a change in its genetic material

Effects of Mutations

Bacterial mutants are typically described by comparison to a standard, well-characterized, reference strain called the 'wild-type' strain. Bacterial mutants have often lost some growth property (e.g., failure to utilize a particular carbon or nitrogen source or failure to grow without a particular nutrient), or acquisition of some new growth property (e.g., ability to grow in the presence of some toxic substance). Genes can be divided into two categories based on the phenotypes of the corresponding mutants: nonessential gene products are only required under specific growth conditions, whereas essential gene products are required under all conditions. The genes of lactose catabolism are nonessential because they are only required for growth on medium with lactose as the sole carbon source. In contrast, the genes encoding RNA polymerase are essential because they are required for growth on all media. Null mutations in a nonessential gene will prevent growth on a medium that requires that gene product but such mutants will still grow on other media. In contrast, null mutations in an essential gene are lethal. Consequently, such mutants cannot be recovered from haploid bacteria. Nevertheless, it is possible to isolate more subtle mutations in essential genes. For example, it is possible to isolate mutations that alter a subunit of RNA polymerase that make the organism resistant to the antibiotic rifampicin. It is also possible to isolate mutations where some phenotype is observed under certain 'nonpermissive' conditions but not under other 'permissive' conditions (Table 1).

Because not all mutations have an observable effect, it is important to distinguish the genotype from the resulting phenotype. In bacterial genetic nomenclature a three-letter mnemonic refers to a pathway or discrete cluster of physiologically connected systems. A fourth, capitalized letter represents a particular gene of that set. The genotype is written in lower case letters and italicized (e.g., purB), with a plus superscript indicating the wild-type genotype. (The purB gene encodes one of the enzymes required for purine biosynthesis.) The phenotype is indicated by the same mnemonic but the first letter is upper case and it not italicized (e.g., PurB), with a plus superscript indicating the functional phenotype and a minus indicating a mutant phenotype. The genotype of a cell is usually inferred from its phenotype but may also be determined indirectly by recombination experiments or directly by DNA sequencing

Long-Evans Cinnamon Rats

Long-Evans Cinnamon (LEC) mutant rats have a maturational arrest in the development of CD4+ T-cells, but not CD8+ T-cells, from CD4+CD8+ T-cells (Agui et al., 1990, 1991). This results in a significant decrease in the number of peripheral CD4+ T-cells; however, these cells are present in peripheral lymphoid organs and are most likely generated by an alternative pathway. The deficiency of CD4+ T-cells is owing a single recessive gene system, thid (T-helper immunodeficiency) (Yamada et al., 1991). This mutation is found in bone marrow-derived cells but not in thymic stromal cells (Agui et al., 1991). CD4+ T-cells that are present show functional abnormalities (Sakai et al., 1995). For example, there is no IL-2 production after Con A stimulation. Interestingly, the presence of the allele thid does not prevent the development of CD4+ intraepithelial lymphocytes since LEC rats possess normal numbers of both CD4+CD8+ and CD4+8− intra-epithelial lymphocytes (Sakai et al., 1994).

Transcription Activator-Like Effector Nucleases

TALENs can be used for in vivo genetic engineering of mutant rat models. TALENs are artificial restriction enzymes and can cut DNA strands at any desired sequence, which makes them an attractive tool for genetic engineering. TALENs are generated by fusing DNA binding domains of transcription activator-like (TAL) effectors to DNA cleavage domains. TAL effectors are secreted by Xanthomonas bacteria and can bind DNA sequences (via repetitive amino acid residues in the central domain) and activate gene expression. The simple relationship between amino acids in the TAL effector and the DNA bases in its target provides the possibility of engineering TAL effector proteins with an affinity for a predetermined DNA sequence (Tong et al., 2012). Several researchers have fused the FokI nuclease domain to TAL effector proteins to create TALENs (e.g., Miller et al., 2011). When TALENs are introduced into cells they can be used for genome editing in situ. For example, TALENs were used to disrupt the IgM locus in the rat and to create a heritable mutation that eliminates IgM function. For this, titrations of specifically designed TALENs were microinjected either as DNA or mRNA into one-cell rat embryos (similar to what has been described for ZFN technology), of which a proportion (DNA, 9.5%; mRNA, 58%) showed subsequent alterations to the IgM locus. IgM mutation frequency was a function of TALEN dose as was the rate of biallelically modified rats, which were generated by mRNA (but not DNA) injections only. Genetically modified rats were then bred with wild-type-like control rats and the resulting F1 generation was checked for mutant alleles using PCRs. This procedure established TALEN technology as a valid tool for the generation of in vivo gene knockouts in rats (Tesson et al., 2011).

However, the technique to generate TALEN-mediated DNA double-strand breaks described previously can be technically challenging using the regular cloning methods and is relatively expensive. Tong and coworkers recently developed TALEN-targeting vectors using Golden Gate cloning technique thereby providing a more time-efficient tool to generate gene-targeted rat ES cells (i.e., construction of a pair of TALENs targeting any sequence of interest can be completed in just 5 days) (Tong et al., 2012). Furthermore, TALEN-mediated homologous recombination has been utilized to generate a knock-in rat model using oocyte microinjections of TALENs mRNA with a linear donor (instead of a supercoiled donor, which was ineffective in producing knock-in rats) (Ponce de Leon, Merillat, Tesson, Anegon, & Hummler, 2014).

In conclusion, efficient gene targeting in rat ES cells can be achieved quickly using either TALEN-mediated DNA double-strand breaks (Tong et al., 2012) or integration of TALENs by homologous recombination (Ponce de Leon et al., 2014). Thus, TALENs are an affordable and highly efficient option for the generation of targeted and specific mutagenesis of the rat and will reduce significantly time expenditure.

To give an example, Ferguson and coworkers selected the gene for the toll-like receptor 4 (TLR4) for TALEN-mediated gene inactivation (Ferguson, McKay, Harris, & Homanics, 2013). The team developed a pair of TALEN constructs that specifically target exon 1 immediately downstream of the start of translation. TALEN mRNAs were microinjected into the cytoplasm of one-cell Wistar rat embryos and heterozygous F1 offspring were interbred to produce homozygous F2 animals. The homozygous knockout rats had a markedly attenuated increase in plasma tumor necrosis factor alpha in response to a lipopolysaccharide challenge compared to control rats. TLR4 knockout rats will also be valuable for studies of ethanol action and of inflammatory conditions including septic shock, as TLR4 appears to play a role in ethanol-induced neuroinflammation and neurodegeneration.

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Preventive Role of Renal Kallikrein–Kinin System in the Early Phase of Hypertension and Development of New Antihypertensive Drugs

Makoto Katori, Masataka Majima, in Advances in Pharmacology, 1998

1 Mutant BN-Ka Rats

Damas and Adams, at the Katholiek University of Leuven, Belgium, reported mutant rats of the BN strain (Rattus norvegicus, BN/fMai), which are devoid of plasma kallikrein-like activity and show low levels of kininogen in plasma (Damas and Adams, 1980). This was also studied by another group (Oh-ishi et al., 1982). These BN-strain rats show a prolonged kaolin-activated partial thromboplastin time (APTT) because of the lack of HMW kininogen and the low level of plasma prekallikrein (Oh-ishi et al., 1984). They were designated BN-Ka rats (Oh-ishi et al., 1982), since the original report was published by the Katholiek University of Leuven. Further studies revealed that both HMW and LMW kininogens were almost absent from the plasma (Fig. 6) (Oh-ishi et al., 1986; Majima et al., 1991) and that BN-Ka rats are practically incapable of excreting kinin in the urine (Fig. 6) (Yamasu et al., 1989; Majima et al., 1991). Normal rats of the same strain were kept at the Kitasato University animal facilities and were designated BN-Kitasato (BN-Ki) rats (Oh-ishi et al., 1982). Normal BN-Ki rats show the same levels of kininogens as rats of other strains, such as the SD strain (Majima et al., 1991). The mutant BN-Ka rats, although capable of producing kininogens in the liver, cannot release kininogens into the bloodstream because of the point mutation of Ala163 to threonine in the common heavy chain of the structures of both kininogens (Hayashi et al., 1993). The HMW and LMW kininogens and prekallikrein mRNAs that are present in the liver of BN-Ka rats are of a similar size and abundance to those in BN-Orl rats (Lattion et al., 1988).

The mutant

Results

To identify mutant p53 reactivating drug candidates, we took advantage of recent advances in computational structure-based drug design to perform a virtual screening of small molecule compound library against allosteric site within the R273H mutant p53 protein. A total of >800,000 compounds with drug-like properties were virtually screened against mutant p53 using an iDock tool. Purchasable compounds with the highest predicted negative free binding energies (iDock scores) were selected for further characterization from the top 1000 compounds identified in the virtual screen. The selected compounds were validated in molecular docking studies against mutant p53 structure using a protein ligand docking web service SwissDock. This approach resulted in identification of a total of 12 lead compounds with distinct chemical scaffolds that were selected for further experimental validation in radio-sensitization assays in both wild-type and mutant p53 expressing head and neck cancer cell lines. One of the identified small molecule leads is Compound 1, a quinazoline derivative with structural similarities to known mutant p53-reactivating drugs. The functional characterization of Compound 1, as well as other previously unknown classes of p53 reactivating candidate molecules in radio-sensitization assays will be presented.

A Computational Approach to Discovery of Novel Mutant p53 Reactivating Molecules As Targeted Radio-Sensitizing Agents for Head and Neck Cancer

A. Nikolaev

L. Zeng

S.A. Spencer

J.A. Bonner

E.S. Yang

PlumX Metrics

Purpose/Objective(s)

p53 tumor suppressor plays a central role in the suppression of tumorigenesis by triggering cell growth arrest, apoptosis, and senescence in response to DNA damage, oxidative stress, and dysregulation of tumor oncogenes. Inactivation of p53 function by a gene mutation is present in the majority of human tumors and had been linked to genetic instability and malignant tumor progression. The goal of this project was to identify novel mutant p53 reactivating drug candidates that target the allosteric site of mutant p53, and to validate these candidate compounds in radio-sensitization assays in human head and neck cancer cells expressing both wild-type and mutant p53.

Materials/Methods

A publicly available database ZINC was used as a source of small molecule compounds in ready-to-dock 3D format for virtual screening. Mutant p53 R273H crystal structure obtained from Protein Data Bank. A multithreaded virtual screening tool for flexible ligand docking iDock was used for virtual screens. A protein ligand docking web service SwissDock was used for compound validation in docking studies. Apoptotic cell death following exposure to radiation was quantified by using the Annexin V-FITC apoptosis detection kit (Mountain View, CA) as previously described.

Results

To identify mutant p53 reactivating drug candidates, we took advantage of recent advances in computational structure-based drug design to perform a virtual screening of small molecule compound library against allosteric site within the R273H mutant p53 protein. A total of >800,000 compounds with drug-like properties were virtually screened against mutant p53 using an iDock tool. Purchasable compounds with the highest predicted negative free binding energies (iDock scores) were selected for further characterization from the top 1000 compounds identified in the virtual screen. The selected compounds were validated in molecular docking studies against mutant p53 structure using a protein ligand docking web service SwissDock. This approach resulted in identification of a total of 12 lead compounds with distinct chemical scaffolds that were selected for further experimental validation in radio-sensitization assays in both wild-type and mutant p53 expressing head and neck cancer cell lines. One of the identified small molecule leads is Compound 1, a quinazoline derivative with structural similarities to known mutant p53-reactivating drugs. The functional characterization of Compound 1, as well as other previously unknown classes of p53 reactivating candidate molecules in radio-sensitization assays will be presented.

Conclusion

Computational approach to discovery of mutant p53-reactivating agents yielded distinct classes of small molecule compounds with similarities to the known p53 reactivating molecules, as well as novel previously uncharacterized mutant p53 reactivating drug candidates. Radio-sensitizing properties of these compounds are evaluated in mutant p53 expressing head and neck cancer cells.

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