Does Spontanious Mutation Tcg Continue to Go Up

Strains, Stocks and Mutant Mice

Cathleen M. Lutz , ... Muriel T. Davisson , in The Laboratory Mouse (Second Edition), 2012

A brief word on nomenclature

Spontaneous mutations are alleles of initially unknown genes and are given allele names and symbols based on their phenotype (e.g. diabetes, db). Recessive mutations (i.e. requiring two copies of the mutated allele to manifest the phenotype) are represented by all lower-case letters while dominant (i.e. one or two copies of the mutated allele produces the phenotype) and semidominant (i.e. one mutant allele produces an intermediate phenotype) spontaneous mutations are represented by an upper-case first letter, followed by lower-case letters. Once the gene responsible for the mutant phenotype has been identified, the allele symbol is superscripted to an approved gene symbol (e.g. the diabetes mutation is a point mutation in the leptin receptor gene, Lepr^db ). The Mouse Genomic Database Nomenclature Committee approves and assigns gene names and symbols, which may be registered online (http://www.informatics.jax.org) or requested by email ([email protected]). Gene names and symbols may change as the function of a gene is better understood or to better correspond with gene symbols of other species (primarily human).

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Hunger and Satiation

Nori Geary , in Encyclopedia of Endocrine Diseases, 2004

Disorders of CCK Satiation

Spontaneous mutations have been identified in the CCK-1 receptor. Rats without CCK-1 receptors overeat at every meal, gain weight, and become diabetic ( Fig. 3). Humans without CCK-1 receptors apparently do the same. This syndrome indicates not only that CCK is an important part of the natural process of satiation but also that the physiological system controlling food intake, however complicated, is not completely redundant. Rather, the lack of a single basic control of meal size can produce uncompensated hyperphagia and obesity.

Figure 3. Null mutation of the CCK-1 receptor leading to increased meal size and obesity. Top graph shows weights of normal male rats (LETO strain) and the same rats lacking the CCK-1 receptor (OLETF). Lower graphs show daily food intakes and spontaneous meal sizes and meal frequencies (means ± SEMs) in the same rats at approximately 30 weeks of age. Modified with permission from Moran, T. H., Katz, L. F., Plata-Salaman, C. R., and Schwartz, G. J. (1998). Disordered food intake and obesity in rats lacking cholecystokinin A receptors. Am. J. Physiol. 274, R618–R625.

Disorders in CCK satiation occur in eating disorders. Patients with bulimia nervosa display reduced prandial CCK secretion and reduced subjective experience of satiation during meals. Although these abnormalities improve together with improvement in binge eating, it is possible that they both facilitate the onset of the disorder and retard recovery from it.

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Genome Composition, Organization, and Expression

Roger Hull , in Plant Virology (Fifth Edition), 2014

viii Functional Regions Within a Gene

Spontaneous mutations and deletions can be used to identify important functional regions within a gene. However, mutants obtained by site-directed mutagenesis, and deletions constructed in vitro can give similar information in a more systematic and controlled manner. For example, the construction and transcription of cDNA representing various portions of the TEV genome and translation in vitro and testing of the polypeptide products showed that the proteolytic activity of the 49-kDa viral proteinase lies in the 3′-terminal region. The amino acid sequence in this region suggested that it is a thiol protease related in mechanism to papain (Carrington and Dougherty, 1987). Viral proteinases are described in Box 6.2.

However, care must be taken with this approach. Many functions depend upon the three-dimensional (3D) structure of the protein and mutations, not at the active site, and may have a secondary effect on the protein structure.

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Mouse Models: Approaches to Generate In Vivo Models for Hereditary Disorders of Mineral and Skeletal Homeostasis

Siân E. Piret , Rajesh V. Thakker , in Genetics of Bone Biology and Skeletal Disease (Second Edition), 2018

2.1 Nontargeted Strategies

Spontaneous mutations in mice may result in benign phenotypes, such as variable coat colors, or in disorders that have similarities to diseases in man, for example, the hyperphosphatemia ( Hyp) mouse, which is representative of X-linked hyperphosphatemia in man. ⁵⁹ Such spontaneous mutations occur at very-low frequencies, thus several techniques that increase the rate of mutation induction in the mouse genome by either nontargeted (random) or targeted strategies have been developed (Tables 7.1 and 7.2). An early example of nontargeted mutagenesis is provided by irradiation, which generated the Gy mouse, a second model for X-linked hypophosphatemia. ⁵⁹ More recently, chemical mutagens have been used in large-scale mutagenesis programs. Successful agents include isopropyl methane sulfonate (iPMS) used to generate the Nuf mouse model with an activating calcium-sensing receptor (CaSR) mutation, and N-ethyl-N-nitrosourea (ENU) used to generate a mouse model for osteogenesis imperfecta with a collagen 1 alpha 1 (COL1A1) mutation. ENU, which is an alkylating agent that primarily introduces point mutations via transfer of the ENU alkyl group to the DNA base followed by mispairing and subsequent basepair substitution during the next round of DNA replication (Fig. 7.1A ), is the most potent mutagen in mice. ¹⁴ Intraperitoneal injections of ENU to male mice generate approximately one mutation per 1–1.5 Mbp of sperm DNA, ¹⁴ which allows the mutations to be inherited (Fig. 7.1B). ENU mutagenesis programs utilize two complementary approaches, which are phenotype-driven and genotype-driven screens. In phenotype-driven screens, the offspring of mutagenized mice are assessed for phenotypic variances, using a panel of morphological, biochemical, or behavioral tests, in a "hypothesis-generating" strategy, which aims to elucidate new genes, pathways, and mechanisms for a disease phenotype ¹⁴ (Fig. 7.1B). By establishing appropriate matings, phenotype-driven screens can be used to identify dominant or recessive phenotypes. Genotype-driven screens, in which mutations in a gene of interest are sought, are "hypothesis driven" and are feasible by using available parallel archives of DNA and sperm samples from mutagenized male mice (Fig. 7.1B). Archived DNA samples from the mutagenized male mice are used to search for mutations in the gene of interest, and once mutations are identified in the mouse DNA, then the corresponding sperm sample for the male mouse harboring the mutation is used to establish progeny carrying the mutation by in vitro fertilization. ¹⁴ It is estimated that the probability of finding three or more mutant alleles in an archive of >5000 DNA samples is >90%. ⁶⁰ Thus, the gene-driven approach can be used to generate an "allelic series" of mutations within one gene, which may yield insights into genotype–phenotype correlations in the gene and disease of interest. ⁶¹

Figure 7.1. Methods for nontargeted (random) mutagenesis.

(A) Chemical mutagenesis using N-ethyl-N-nitrosourea (ENU). ENU is an alkylating agent that transfers its ethyl group to one of a number of reactive sites on DNA nucleotides, including the O6 of guanine as shown. Modification of guanine with the ethyl group to produce O⁶-ethylGuanine (O-eG), causes mispairing during DNA replication, for example, at spermatogenesis, and during subsequent replication a mutation is introduced. (B) ENU-mutagenized G0 male mice, harboring induced mutations in their sperm DNA, are mated with wild-type females of the same strain to generate G1 mice. G1 males are examined for phenotypic abnormalities (i.e., the phenotype-driven screens). Males with phenotypic abnormalities of interest are then mated with wild-type females to facilitate inheritance testing and genetic mapping in affected offspring (G2) to identify the mutation causing the phenotypic abnormality. DNA and sperm from all the G1 males are also archived to facilitate genotype-driven screens. m, Mutant allele; +, wild-type allele. (C) Insertion mutagenesis using gene trap vectors. Gene trap vectors consist of a strong splice acceptor (SA), a reporter gene, such as β-galactosidase (β-gal) and a polyadenine tract (pA). The gene trap randomly inserts into the host genome (dashed lines), and during splicing, the SA is used in preference to the normal genomic splice sites (splicing pattern shown by dotted lines). Filled/striped boxes denote coding sequences and open boxes denote noncoding sequences.

Source: Reproduced from Piret SE, Thakker RV. Mouse models for inherited endocrine and metabolic disorders. J Endocrinol 2011;211:211–30. ¹

ENU mutations most frequently result in missense mutations (>80%) that may generate hypo- and hypermorphs, although occasionally nonsense or frame-shift mutations (<10%) generating knock-out models may be obtained. ⁶² However, a more recent and reliable method for generating nontargeted knock-out models on a large scale is by the use of insertional mutagenesis, utilizing gene-trap strategies. ^63,64 Gene-trap vectors usually consist of a reporter gene, either with or without a promoter, and a strong splice acceptor (SA) site, which causes any upstream exons to splice directly to the gene trap ¹⁵ (Fig. 7.1C). The vector is either electroporated or retrovirally infected into embryonic stem (ES) cells, after which it randomly inserts into the genome. Mutagenized ES cells are then reintroduced into developing blastocysts to generate chimeric mice, from which germline mutant mice can be bred (Fig. 7.2). A recent refinement of the gene-trap strategy is targeted trapping, in which the vector also contains regions homologous to the targeted gene, thereby facilitating the deletion of a specific gene. ^16,63

Figure 7.2. Gene targeting by modification of embryonic stem (ES) cells.

Totipotent ES cells are isolated from the inner cell mass of a blastocyst [e.g., from a 129Sv embryo (shown)] and cultured. The targeting vector is transferred to the ES cells, and those in which homologous recombination or integration has been successful are selected. These are injected into the inner cell mass of a blastocyst from a different mouse strain [e.g., C57BL/6 (shown)], which is transferred to the uterus of a pseudopregnant female. The resulting chimeric offspring (usually males are selected) are bred with wild type, for example, C57BL/6 mice (usually females are selected) to achieve germline transmission. m, Mutant allele; +, wild-type allele.

Source: Reproduced from Piret SE, Thakker RV. Mouse models for inherited endocrine and metabolic disorders. J Endocrinol 2011;211:211–30. ¹

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Biology of Gap Junctions

Richard D. Veenstra , in Cell Physiology Source Book (Fourth Edition), 2012

XIVB Demyelinating Neuropathies of Cx32 and Cx47 Mutations

Spontaneous mutations of Cx32 result in a common peripheral neuropathy in humans, Charcot-Marie-Tooth disease, which affects one of every 2500 individuals. While this demyelinating disease may arise as a consequence of mutations of a peripheral myelin protein PMP22 (on chromosome 17) or of the myelin constituent Po (on chromosome 1), numerous families have been identified with an X-linked dominant form of the disease characterized by as many as 150 different mutations of Cx32 (CMTX). Mutations may involve single-base substitutions, formation of a premature stop codon, frame-shift or elimination of an amino acid residue. They may occur in amino or carboxyl termini, cytoplasmic or extracellular loops, or in transmembrane regions of the connexin. The causal relationship between these diverse mutations of Cx32 and demyelination of peripheral nerves is obviously varied since the mutations are quite diverse and may or may not affect channel properties. In some cases, it seems possible that mutations may directly affect the function of Cx32 gap junctions in peripheral myelin by interfering with their possible function as ATP-sensitive hemichannels, their function in the exchange of nutrients between the perinuclear region of the Schwann cell and the Schmidt–Lantermann incisures and paranodal processes at the node of Ranvier, or their possible function in signaling between internodes. In other cases, it is thought that the mutations may indirectly affect Cx32 function through effects on Cx32 synthesis and trafficking within the cell. Targeted knockout of connexin 32 in mice also results in changes in liver enzyme activities and in glucose mobilization but, most interestingly, these Cx32-deficient mice have an increased susceptibility to hepatocarcinogenesis. Otherwise, they are vital and fertile. There are >300 GJB1 mutations thus far identified. A list of these CMTX mutations can be found at http://www.molgen.ua.ac.be/CMTMutations/Mutations/MutByGene.cfm. Oligodendrocytes in the central nervous system also develop a demyelinating neuropathy, Pelizaeus–Merzbacher–like disease (PLMD1) owing to mutations in Cx47 (GJC2, formerly GJA12). For additional information go to http://www.ncbi.nlm.nih.gov/omim/608804.

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Dwarfism, in Mice

K. Douglas , S.A. Camper , in Encyclopedia of Genetics, 2001

Mouse Mutants

Spontaneous mutations have been identified in genes acting at various levels of growth regulation. The little (lit) mouse mutation is an autosomal recessive mutation resulting in proportionate dwarfism visible at 2 weeks of age. Adult lit mice have a body weight two-thirds the size of control littermates. Female mice are fertile but often fail to nurse their first litters, whereas male mice have reduced fertility. Dwarfism in little mice is due to a missense mutation in the GHRH receptor (Ghrhr) gene (Godfrey et al., 1993). The mutation substitutes a glycine residue for a conserved aspartic acid residue within the ligand-binding domain of the receptor. This amino acid substitution greatly reduces the sensitivity of the receptor to GHRH. Therefore, little mice do not receive the hypothalamic GHRH signal to secrete GH. Serum levels of GH and IGF-I are low and the mice are dwarfed. In addition, lit/lit pituitaries have fewer somatotropes (GH-producing cells), because GHRH normally stimulates proliferation of these cells.

The Ames dwarf (df) mouse mutation is an autosomal recessive mutation resulting in more severe growth defect than lit. df/df mice are proportionately dwarfed by 3 weeks of age and are half the size of control littermates as adults. The mice are infertile and hypothyroid as well. At the cellular level, Ames dwarf mice are almost completely lacking in pituitary somatotropes, lactotropes (prolactin-producing cells), and thyrotropes (TSH-producing cells). The lack of these three cell types causes the lack of GH, prolactin (PRL), and TSH in the mice. A missense mutation in the Prophet of Pit1 (Prop1) gene is responsible for Ames dwarfism. The Prop1 gene encodes a transcription factor which contains a paired-like homeodomain. Ames dwarf mice have a serine to proline amino acid substitution within the DNA-binding domain of PROP1 (Sornson et al., 1996). Mutant PROP1 does not bind DNA effectively to regulate transcription of downstream genes, resulting in the failure of three pituitary cell types to differentiate and proliferate during development.

Snell dwarf (dw) mice have a phenotype nearly indistinguishable from Ames dwarf mice. There are two noncomplementing alleles of the Snell dwarf mutation that are autosomal recessive and cause dwarfism, infertility, and hypothyroidism. dw/dw pituitaries completely lack GH-, PRL-, and TSH-producing cells. The Snell dwarf phenotype is due to mutations in the pituitary specific transcription factor1 (Pit1) gene (Camper et al., 1990; Li et al., 1990). One of these mutations is a gene rearrangement and the other is a point mutation in the DNA-binding domain of this transcription factor. PIT1 is necessary for expression of the GH, TSH, and PRL genes and for proliferation of the cells that produce these hormones. Both Ames and Snell dwarfs are unable to respond to hypothalamic signals to secrete GH and TSH due to pituitary defects.

The hypothyroid (hyt) mouse mutation is an autosomal recessive mutation resulting in growth retardation, infertility, elevated TSH levels, undetectable TH, and extreme hypothyroidism. hyt/hyt mice have a mutation in the TSH receptor (Tshr) gene (Stein et al., 1994; Gu et al., 1995). The mutation is a leucine amino acid substitution at a conserved proline within the transmembrane domain of the TSHR. The mutant TSHR does not bind TSH, therefore the thyroid gland does not receive the pituitary signal to secrete TH. The end result is an unresponsive thyroid gland and reduced TH levels.

The congenital goiter (cog) mouse mutation is an example of target organ failure. The cog mutation is an autosomal recessive mutation resulting in hypothyroidism, goiter, and small size early in life. As cog/cog mice age, serum TH levels increase, and they are able to overcome their growth retardation (Adkison et al., 1990). A point mutation within the thyroglobulin (Tgn) gene causes the cog phenotype (Kim et al., 1998). Thyroglobulin is converted to TH within the thyroid gland. Therefore, cog mice receive the pituitary TSH signal to produce and secrete TH, but are unable to produce TH efficiently due to a defect in the Tgn gene.

These examples of spontaneous mouse mutations involve the endocrine axis regulating growth. lit, df, and dw are pituitary defects; hyt and cog are thyroid defects. Each produces a similar phenotype of proportionate dwarfism. Many spontaneous mouse mutants not discussed here have skeletal defects resulting in nonproportionate dwarfism.

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Spontaneous, Surgically and Chemically Induced Models of Disease

Dwight R. Owens , in The Laboratory Rat (Second Edition), 2006

2. Polycystic Kidney Rat

A spontaneous mutation was observed in an inbred, albino rat that developed polycystic lesions in the liver as well as the kidneys and that has an autosomal recessive mode of inheritance ( Lager et al., 2001). The mutation occurred in a colony of CRJ: CD (SD) BR rats in Japan, and the rats develop congenital intrahepatic biliary dilatation associated with congenital hepatic fibrosis. The lesions found in this model are similar to those seen in humans with Caroli's disease (Sanzen et al., 2001). Genetic analysis has shown the polycystic kidney rat model to be orthologous to the autosomal recessive polycystic kidney disease that occurs in man (Harris, 2002).

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Toxicology Testing and Evaluation

J.E. Klaunig , L.M. Kamendulis , in Comprehensive Toxicology, 2010

3.09.4.1.4(i) Mismatch repair

Many spontaneous mutations are point mutations, a change in a single base pair in the DNA sequence. An inherent problem with the mismatch repair process is determining the normal from the mutant DNA strand, such that the correct base pairs are restored on the mutated strand. Depurination occurs commonly in mammals and results in the formation of AP sites. If these lesions are left unrepaired, they can generate mutations during DNA replication as DNA synthetic machinery is unable to determine the appropriate base with which to pair. All mammalian cells possess AP endonucleases that function to cut DNA near AP sites. The scission is then extended by exonucleases, and the resulting gap repaired by DNA polymerases and ligases.

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Forward and Reverse Genetics to Model Human Diseases in the Mouse

Yoichi Gondo , ... Ryutaro Fukumura , in Animal Models for the Study of Human Disease (Second Edition), 2017

7 Mutant Mice as Disease Models

The spontaneous mutation rate is very low; for instance, roughly three orders of magnitude lower than that induced by the most potent mutagen, ethylnitrosourea (ENU) in the mouse ( Russell et al., 1981). Thus, the development and establishment of mutant strains are often laborious and take time. Once mutant strains are established and maintained, however, any researchers may access to the strains as a research resource.

Mutations usually occur randomly in the genome. Most of the mutations are a loss-of-function type due to the disturbance or disruption of the functional DNA sequences (Figs. 28.1 and 28.2). Thereby, a biological effect(s) of the most of the mutations is a detrimental to the organisms. Namely, mutations are likely to induce some disorders modeling many diseases and deficiencies in human.

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Pathology of Peripheral Neuron Cell Bodies

MICHAEL J. GROVES , FRANCESCO SCARAVILLI , in Peripheral Neuropathy (Fourth Edition), 2005

Mouse Motor Neuron Degeneration

This spontaneous mutation in C57Bl/6 mice follows an autosomal dominant inheritance pattern, and produces a disorder that begins by 6 months of age with limping of the hind limbs and progresses to spastic paralysis of the hind and, later, forelimbs. ²⁹³ The mutation produces a progressively larger loss of spinal and cranial motor neurons, particularly those in the lumbosacral region, which undergo chromatolytic changes and accumulation of ubiquitinated and autofluorescent lipofuscinoid inclusions that contain various lysosomal enzymes. ^{289, 294} The apoptosis-associated enzyme transglutaminase type 2 is "superactivated" in the spinal cord at the same time that the motor neuron disease begins to manifest itself. ¹⁹⁶ It is now apparent that this mouse is a homologue ¹⁹⁶ of a human neuronal ceroid lipofuscinosis (progressive epilepsy with mental retardation) caused by a mutation in the CLN8 gene, which encodes for a membrane protein with as yet unknown functions. ³⁶⁴

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