Molecular analysis of four ENU induced triosephosphate isomerase null mutants in Mus musculus

doi:10.1016/0027-5107(95)00004-3

Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis

Volume 328, Issue 2, May 1995, Pages 163-173

https://doi.org/10.1016/0027-5107(95)00004-3 Get rights and content

Abstract

Four ENU-induced mutations were previously identified at the triosephosphate isomerase (TPI) locus in mouse germinal mutation experiments. Each of the mutants is associated with a 50% loss of enzymatic activity in the F₁ (heterozygous) animals. Exons of the TPI gene from control mice and heterozygous mutant mice were PCR amplified and sequenced as necessary to determine the molecular lesion in the mutant alleles.

Mutants Tpi^∗M-1NEU and Tpi^∗M-2NEU carried the same T:A to A:T transversion in exon 6, resulting in a Leu to Gln substitution at residue 192. Amino acid residue 192 is located in α-helix H6 of the protein. Tpi^∗M-4NEU contained a T:A to A:T transversion within the codon for residue 162 in exon 5, also causing a Leu to Gln substitution. This mutation is located at the beginning of β-strand B6, within a highly conserved sequence region surrounding the active site residue Glu 165. Sequence analysis of Tpi^∗M-3NEU revealed an A:T to C:G transversion, changing the stop codon to a codon for Cys, with the resulting addition of 19 predominantly hydrophobic amino acids to the protein. All four mutations occurred at an A:T base pair. In each case, the mutation site was flanked on both sides by G:C base pairs.

Each of the sequence alterations has a potential impact on the structure of the TPI protein that is consistent with the existence of a null allele. In addition to providing insight into the molecular basis of ENU induced germ cell mutations and the differences in mutation spectra among organisms, these mutants represent models for structure-function studies of this highly conserved enzyme.

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The triosephosphate isomerase (TPI) functions at a metabolic cross-road ensuring the rapid equilibration of the triosephosphates produced by aldolase in glycolysis, which is interconnected to lipid metabolism, to glycerol-3-phosphate shuttle and to the pentose phosphate pathway. The enzyme is a stable homodimer, which is catalytically active only in its dimeric form. TPI deficiency is an autosomal recessive multisystem genetic disease coupled with hemolytic anemia and neurological disorder frequently leading to death in early childhood. Various genetic mutations of this enzyme have been identified; the mutations result in decrease in the catalytic activity and/or the dissociation of the dimers into inactive monomers. The impairment of TPI activity apparently does not affect the energy metabolism at system level; however, it results in accumulation of dihydroxyacetone phosphate followed by its chemical conversion into the toxic methylglyoxal, leading to the formation of advanced glycation end products. By now, the research on this disease seems to enter a progressive stage by adapting new model systems such as Drosophila, yeast strains and TPI-deficient mouse, which have complemented the results obtained by prediction and experiments with recombinant proteins or erythrocytes, and added novel data concerning the complexity of the intracellular behavior of mutant TPIs. This paper reviews the recent studies on the structural and catalytic changes caused by mutation and/or nitrotyrosination of the isomerase leading to the formation of an aggregation-prone protein, a characteristic of conformational disorders.
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Citation Excerpt :
An animal model of TPI deficiency caused by missense and nonsense mutations has been developed in ethylnitrosourea-treated mice. Heterozygous animals display a 50% loss of enzymatic activity, whereas homozygosity for these TPI null alleles is lethal at an early stage of development.62 Phosphoglycerate kinase generates one molecule of ATP by catalyzing the reversible conversion of 1,3-bisphosphoglycerate to 3-phosphoglycerate (Figure 1).
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In 424 African-American and 75 white subjects, we found that the −5 (TPI 592 A→G), −8 (TPI 589 G→A), and −24 (TPI 573 T→G) variants in the triosephosphate isomerase (TPI) gene occurred frequently (41.0%) in the African-American subjects but did not occur in the whites. These data suggest that this set of polymorphisms may turn out to be one of the higher-incidence molecular markers of African lineage, a surprising finding because others had reported that these nucleotide substitutions were restricted to a small subset of African Americans who had been characterized as TPI-deficiency heterozygotes. Additionally, we investigated the relationship of these variants to TPI-enzyme activity. Although the variant substitutions (occurring in three haplotypes: −5 alone, −5 −8, and −5 −8 −24) were associated with moderate reduction in enzyme activity, severe-deficiency heterozygotes could not be identified with certainty, and none of the haplotypes were restricted to subjects with marked reduction of enzyme activity. Three subjects were homozygous for the −5 −8 haplotype, a finding inconsistent with the putative role of this haplotype as the cause of a null variant incompatible with life in homozygotes. Despite these findings, the possibility remains that the −5 −8 or −5 −8 −24 haplotypes may in some instances contribute to compound heterozygosity and clinical TPI deficiency.
© 1998 by The American Society of Hematology.
The mutagenic activity of ethylnitrosourea at low doses in spermatogonia of the mouse as assessed by the specific-locus test
1998, Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis
Ethylnitrosourea is the most efficient chemical mutagen in spermatogonial stem cells of the mouse and its mutagenic activity has been intensively studied. The pertinent specific-locus mutation test results for a discussion of low dose–effect studies have been summarized and indicate: (1) A threshold dose response best characterizes the relationship between dose and mutation rate. (2) The reduced effectiveness of ethylnitrosourea in the low dose range is likely due to a saturable repair process. (3) The recovery of the saturable repair process as assessed in fractionated dose experiments is long (ca. 168 h). The dynamics of stem cell spermatogonia suggests a long time interval before the cell population passes through at least one cell division and this may be relevant to an interpretation of the fractionation effects. (4) There is a slight but important discrepancy between the predicted and observed mutagenic activity of ethylnitrosourea in the low dose range. This is interpreted to be due to the differences between a mathematical abstraction and the biological realities of the system being studied.
The effect of the interval between dose applications on the observed specific-locus mutation rate in the mouse following fractionated treatments of spermatogonia with ethylnitrosourea
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Our earlier analyses have suggested an apparent threshold dose-response for ethylnitrosourea-induced specific-locus mutations in treated spermatogonia of the mouse to be due to a saturable repair process. In the current study a series of fractionated-treatment experiments was carried out in which male (102×C3H)F₁ mice were exposed to 4×10, 2×40, 4×20 or 4×40 mg ethylnitrosourea per kg body weight with 24 h between applications; 4×40 mg ethylnitrosourea per kg body weight with 72 h between dose applications; and 2×40, 4×20 and 4×40 mg ethylnitrosourea per kg body weight with 168 h between dose applications. For all experiments with 24-h intervals between dose applications, there was no effect due to dose fractionation on the observed mutation rates, indicating the time interval between dose applications to be shorter than the recovery time of the repair processes acting on ethylnitrosourea-induced DNA adducts. In contrast, a fractionation interval of 168 h was associated with a significant reduction in the observed mutation rate due to recovery of the repair process. However, although reduced, the observed mutation rates for fractionation intervals of 168 h were higher than the spontaneous specific-locus mutation rate. These observations contradict the expectation for a true threshold dose response. We interpret this discrepancy to be due to the differences in the predictions of a mathematical abstraction of experimental data and the complexities of the biological system being studied. Biologically plausible explanations of the discrepancy are presented.
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A glucose-6-phosphate dehydrogenase (G6PD) deficient strain of mouse (GPDX) which was developed using the ethylating agent ethylnitrosourea (ENU) has been used to study clonality in epithelial tissues. While the biochemical defect has been quantified, the genetic basis of the deficiency is unknown. The G6PD gene is composed of 13 exons. Exon 1 is not translated, and the ATG start site is near the 5′ end of exon 2. Direct sequencing of the exonic regions of the gene from GPDX, C3H, 101, C57BL/6 and BALB/c mice was carried out. The coding region, in which (with a single exception) all mutations found to cause G6PD deficiency in man are situated, showed identical sequences in three of the four strains studied (101 coding region sequence was not examined). However, the G6PD gene in the GPDX mouse showed a single base difference from the other four strains and from the published mouse G6PD sequence (BALB/c) in the 5′ splice site consensus sequence at the 3′ end of exon 1, part of the untranslated region. The difference was confirmed in four different GPDX mice. This mutation was of the type (A to T transversion) that is known to be induced by ENU; its effect is likely to be exerted through a defect in transcription, splicing or translation, leading to a reduction in protein levels. By Western blot we have found a marked decrease in the G6PD protein levels in the GPDX mouse, with the C3H×GPDX heterozygote showing a lesser decrease. Recently, an increasing number of mutations in the untranslated regions of genes have been found which have effects on protein levels. We believe that the reduced enzyme activity in the GPDX mouse is due to the mutation in the 5′ untranslated region (UTR), and that similar mutations may be relevant in other inherited conditions.

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Research paperMolecular analysis of four ENU induced triosephosphate isomerase null mutants in Mus musculus

Abstract

Mutation Res.

Mutation Res.

Mutation Res.

J. Mol. Biol.

Mutation Res.

Mutation Res.

Mutation Res.

J. Mol. Biol.

FEBS Lett.

Structure of chicken muscle triose phosphate isomerase determined crystallographically at 2.5 angstrom resolution using amino acid sequence data

Nature

Efficient repair of O6-ethylguanine, but not O4-ethylthymine or O2-ethylthymine, is dependent upon O6-alkylguanine-DNA alkyltransferase and nucleotide excision repair activities in human cells

Cancer Res.

Characterization of the functional gene and several processed pseudogenes in the human triosephosphate isomerase gene family

Mol. Cell. Biol.

Mutagenesis and transgenic systems: perspective from the mutagen, N-ethyl-N-nitrosourea

Environ. Mol. Mutagen.

Influence of neighboring base sequence on the distribution and repair of N-ethyl-N-nitrosourea-induced lesions in Escherichia coli

Cancer Res.

Human triosephosphate isomerase deficiency resulting from mutation of Phe-240

Am. J. Hum. Genet.

Nucleotide sequence of murine triosephosphate isomerase cDNA

Nucleic Acids Res.

Human triose-phosphate isomerase deficiency: a single amino acid substitution results in a thermolabile enzyme

Molecular analysis of mutations induced in human cells by N-ethyl-N-nitrosourea

Mol. Carcinogen.

Toward an understanding of the mechanisms of mutation induction by ethylnitrosourea in germ cells of the mouse

Banbury Report

Catalog of mutant genes and polymorphic loci

DNA sequence analysis of spontaneous and N-ethyl-N-nitrosourea-induced hprt mutations arising in vivo in cynomolgus monkey T-lymphocytes

Environ. Mol. Mutagen.

The incidence of rare alleles determining electrophoretic variants: data on 43 enzyme loci in man

Ann. Hum. Genet.

Research paper
Molecular analysis of four ENU induced triosephosphate isomerase null mutants in Mus musculus