Characterization of Triosephosphate Isomerase Mutants with Reduced Enzyme Activity in Mus Musculus

Four heterozygous triosephosphate isomerase (TPI) mutants with approximately 50% reduced activity in blood compared to wild type were detected in offspring of 1-ethyl-1-nitrosourea treated male mice. Breeding experiments displayed an autosomal, dominant mode of inheritance for the mutations. All mutations were found to be homozygous lethal at an early postimplantation stage of embryonic development, probably due to a total lack of TPI activity and consequently to the inability to utilize glucose as a source of metabolic energy. Although activity alteration was also found in liver, lung, kidney, spleen, heart, brain and muscle the TPI deficiency in heterozygotes has no influence on the following physiological traits: hematological parameters, plasma glucose, glucose consumption of blood cells, body weight and organo-somatic indices of liver, spleen, heart, kidney and lung. Biochemical investigations of TPI in the four mutant lines indicated no difference of physicochemical properties compared to the wild type. Results from immunoinactivation assays indicate that the decrease of enzyme activity corresponds to a decrease in the level of an immunologically active moiety. It is suggested that the mutations have affected the Tpi-1 structural locus and resulted in alleles which produce no detectable enzyme activity and no immunologically cross-reacting material. The study furthermore suggests one functional TPI gene per haploid genome in the erythrocyte and seven other tested organs of the mouse.     

Hereditary Lactate Dehydrogenase a-Subunit Deficiency as Cause of Early Postimplantation Death of Homozygotes in Mus Musculus

Two ethylnitrosourea-induced heterozygous mouse mutants with approximately 58 and 50% of wild-type lactate dehydrogenase (LDH) activity and a gamma-ray-induced heterozygous mutant with 50% of wild-type LDH activity in blood, liver and spleen (expressing predominantly the Ldh-1 gene) were recovered in mutagenicity experiments following spermatogonial treatment. Physiological and genetic studies revealed no indications for differences in fertility as well as hematological or other physiological traits between heterozygotes of each mutant line and wild types. This suggests that neither the mutations in the heterozygous state per se nor the resulting approximate 42 to 50% LDH deficiency affect metabolism and fitness. Physicochemical and immunological studies clearly demonstrated that the two mutations with 50% deficiency in heterozygotes result from null alleles of the Ldh-1 structural locus, generating neither enzyme activity nor immunological cross-reacting material. In contrast, the heterozygous mutant with approximately 58% of normal blood LDH activity was shown to be due to a Ldh-1 allele creating protein subunits, which in random assortment with wild-type subunits in vivo exhibit a reduced specific activity and further alterations of kinetic and physicochemical characteristics. All the mutations in the homozygous state were found to be lethal at an early postimplantation stage of embryonic development, probably due to a block of glycolysis with the corresponding loss of the main source of metabolic energy during this ontogenetic stage. The distinct physiological consequences of the total absence of a functioning LDH-A subunit in mice and humans are discussed.(ABSTRACT TRUNCATED AT 250 WORDS)     

A mutation affecting the lactate dehydrogenase locus Ldh-1 in the mouse—I. Genetical and electrophoretical characterization

(101/El × C3H/El)F₁ male mice were injected intraperitoneally with the mutagen procarbazine hydrochloride and immediately caged with untreated test-stock females. Crude liver extracts from the offspring were subjected to polyacrylamide gel isoelectric focusing, and the gels were stained for six enzymes. In the experimental group (mutagen treated spermatogonial germ-cell stage), a dominant inherited banding alteration of the lactate dehydrogenase (LDH) pattern was detected. By crossing the heterozygous mutants, homozygotes were obtained that showed much less gel staining intensity. The mutation is codominantly expressed with 100% penetrance. The banding alteration was also observed in muscle, kidney, heart, blood, brain, testis, spleen, and lung. Polyacrylamide gel electrophoresis was performed with all the tissues examined. The mutation causes the intensity of the band corresponding to LDH-A (primary molecular form in muscle) to decrease from that of the wild type, while the intensity of the bands corresponding to LDH-B (primary molecular form in heart) remains constant. It is concluded that the mutation affects the locus coding for LDH of the muscle type. Ldh-1 ^c is proposed as the allele symbol.     

Linear dose-response relationship of erythrocyte enzyme-activity mutations in offspring of ethylnitrosourea-treated mice

The specific activity of 10 erythrocyte enzymes was measured to detect gene mutations in F1 offspring of male mice treated with 3 different doses of ethylnitrosourea (ENU). After administration of ENU or of the solvent (controls), the (101/El X C3H/El)F1 hybrid males were mated to untreated T-stock females. No enzyme-activity mutant was found in 3610 F1 offspring of the control group. After treatment of postspermatogonial germ-cell stages, 1 mutant in 1125 F1 offspring of males treated with 160 mg ENU/kg body weight, and 2 mutants in 1319 F1 offspring of a 250-mg/kg group were observed. After treatment of spermatogonia, 9 enzyme-activity mutants in 4247 F1 offspring of males treated with 80 mg ENU/kg body weight, 15 mutants in 3396 F1 offspring of a 160-mg/kg group, and 9 mutants in 1402 F1 offspring of a 250-mg/kg group were detected. The mutation frequencies in spermatogonia were significantly different from that of the controls (P less than 0.01). The dose-response curve was found to be linear. The frequencies of enzyme-activity mutations are comparable to those of recessive specific-locus mutations determined in the same experiments. Enzyme-activity mutants with reduced activity as well as mutants with enhanced activity were found. Genetic and biochemical characterization of enzyme-activity mutants was routinely performed. In inter se crossings of heterozygotes, no offspring expressing a third phenotype other than the wild type and the heterozygote were found in approximately half of the mutation studies. The recovered mouse mutants might be used as animal models to study corresponding genetic diseases in humans.     

The mouse Cat4 locus maps to Chromosome 8 and mutants express lens-corneal adhesion

Cat4 is the second largest allelism group in the collection of mouse dominant eye mutations recovered in Neuherberg and carriers express anterior polar cataract, central corneal opacity, and lens-corneal adhesions. We have mapped the Cat4 locus of the mouse to central Chromosome (Chr) 8 at position cM 31. Histological characterization of Cat4 ^a heterozygotes and homozygotes indicates failure of separation of the lens vesicle from the surface ectoderm. Human anterior segment ocular dysgenesis (ASOD) is autosomal dominant, carriers express an eye phenotype similar to that of Cat4 ^a carriers, and it has been mapped to a region of 4q homologous to mouse central Chr 8. Thus, on the basis of phenotype and map position, Cat4 may be a mouse model of human ASOD. The genes Junb, Jund1, Mel, and Zfp42 are discussed as possible candidates for Cat4. Received: 31 October 1996 / Accepted: 20 January 1997     

Characterization of two electrophoretic lactate dehydrogenase-A mutants inMus musculus

Two lactate dehydrogenase (LDH) mutations were recovered independently among offspring of ethylnitrosourea-treated male mice by screening for alterations of isoelectric focusing pattern in liver homogenates. Investigations of physicochemical and kinetic properties of the mutant enzymes indicated that the mutant traits resulted from point mutations at theLdh-1 structural locus. Therefore, the new alleles were designatedLdh-1 ^a-m5Neu andLdh-1 ^a-m6Neu, respectively. Both mutant alleles code for proteins which exhibit an altered stability to heat, in addition to changes in isolectric focusing pattern and a reduction in anodal electrophoretic mobility. While LDH-A^a-m5Neu proteins are markedly less heat stable, LDH-A^a-m6Neu proteins are more heat stable than the wild-type enzyme. Furthermore, a small elevation ofK _m for pyruvate, a slightly reduced inhibition by high pyruvate concentrations, and a slight acidic shift of the pH activity profile distinguish LDH-A^a-m6Neu from both wild-type and LDH-A^a-m5Neu enzymes. Significant alterations of LDH activity were detected in some tissues from LDH-A^a-m5Neu individuals but not in those from LDH-A^a-m6Neu animals. Erythrocytes and blood of LDH-A^a-m5Neu mutants revealed activity levels which were reduced by approximately 6 and 13% compared with those of wild types in heterozygous and homozygous individuals, respectively. In addition, an elevation of approximately 6% in LDH activity was found in skeletal muscle in homozygous mutants. Consistent with the unaltered or only slightly altered LDH activity in tissues, the genetic as well as the physiological characterization yielded no easily detectable effects from either mutation on metabolism or fitness of the affected individuals.This research was supported in part by Contract BI6-156-D from the Commission of the European Communities.     

The liver/erythrocyte pyruvate kinase gene complex [Pk-1] in the mouse: regulatory gene mutations.

Nine enzyme activity variants and one charge variant of liver/erythrocyte pyruvate kinase have been found amongst laboratory and wild mice. Four of the enzyme activity variants were previously reported to be caused by allelic differences in the structural gene, Pk-1s. Analysis of two putative regulatory gene mutations is now reported, both of which map at, or close to, the structural gene on chromosome 3. One of these mutations, in the inbred strain SWR, is tissue specific, affecting enzyme concentration in the liver but not the erythrocyte the other, which arose in a mutation experiment, doubles the enzyme concentration in both tissues. The organization and the nomenclature in the [Pk-1] gene complex are discussed and are compared with the organization of other comprehensively analysed gene complexes in the mouse.     

A mutation resulting in increased triosephosphate isomerase activity in Mus musculus

A mutation resulting in increased triosephosphate isomerase (TPI) activity in blood was recovered in offspring of procarbazine hydrochloride-treated male mice. Breeding experiments indicated a codominant mode of expression. Compared to the wild type, heterozygous and homozygous mutants have mean erythrocyte TPI activities of approximately 140 and 190%, respectively. Besides blood and erythrocytes the increased activity is expressed to a similar degree in spleen, and to a lesser degree in liver, lung, kidney, muscle and brain. Enhanced activity was absent in the heart. Heterozygous and homozygous mutants are viable, fully fertile and exhibit no significant differences in haematological or other physiological traits studied. Biochemical investigations of TPI in both mutant genotypes revealed neither physicochemical nor kinetic differences compared to the wild type. Moreover, immunoinactivation studies showed no difference in the amount of antiplasma required to inactivate a constant amount of TPI activity in all three genotypes, strongly suggesting that the differences in enzyme activity are attributable to differing amounts of enzyme protein expressed per cell. Mapping studies indicated that the mutation is closely linked to the Gapd locus and consequently is located either adjacent to or within the Tpi-1 structural locus. It is hypothesized that the mutation affected a regulatory element contiguous to the Tpi-1 structural locus which acts by increasing the amount of TPI expressed.     

Molecular, genetic and biochemical characterization of lactate dehydrogenase-A enzyme activity mutations in Mus musculus

Four independent heterozygous lactate dehydrogenase (LDH) mutations with approximately 60% of wild-type enzyme activity in whole blood have been recovered. The mutant line Ldh1 ^a2Neu proved to be homozygous lethal, whereas for the three lines Ldh1 ^a7Neu, Ldh1 ^a11Neu, and Ldh1 ^a12Neu homozygous mutants with about 20% residual activity occurred in the progeny of heterozygous inter se matings. However, the number of homozygous mutants was less than expected, suggesting an increased lethality of these animals. Various physicochemical and kinetic properties of LDH are altered. Exons of the Ldh1 gene were PCR amplified and sequenced to determine the molecular lesion in the mutant alleles. Ldh1 ^a2Neu carried an A/T → G/C transition in codon 112 (in exon 3), resulting in an Asn → Asp substitution; Asn112 is part of the helix αD, which is involved in the coenzyme-binding domain. Ldh1 ^a7Neu contained an A/T → C/G transversion within the codon for residue 194 in exon 4, causing an Asp → Ala substitution, which may affect the arrangement of the substrate-binding site. Three base substituions were discovered for the mutation Ldh1 ^a11Neu in exon 7: the transition C/G → T/A, a silent mutation, and two transversions C/G → A/T and C/G → G/C, both missense mutations, which led to the amino acid replacements Ala319 → Glu and Thr321 → Ser, respectively, located in the αH helix structure of the COOH tail of LDHA. We suggest that the mutation is the result of a gene conversion event between Ldh1 ^a wild-type gene and the pseudogene Ldh1-ps. The alteration Ile → Thr of codon 241 in exon 6 caused by the base pair change T/A → C/G was identified in the mutation Ldh1 ^a12Neu; IIe241 is included in the helix α2G, a structure that is indirectly involved in coenzyme binding. Each of the sequence alterations has a potential impact on the structure of the LDHA protein, which is consistent with the decreased LDH activity and biochemical and physiological alterations. Received: 3 July 1997 / Accepted: 30 September 1997     

Glutathione reductase activity deficiency in homozygous Gr1a1Neu mice does not cause haemolytic anaemia

A glutathione reductase (GR) mutant with approximately 50% residual enzyme activity in blood compared with wild-type was detected amongst offspring of isopropyl methanesulphonate-treated male mice. Homozygous mutants with only 2% residual enzyme activity were recovered in progeny of inter se matings of heterozygotes. Results of linkage studies indicate a mutation at the Gr1 structural locus on chromosome 8. The loss of GR activity was evident both in blood and in other tissue extracts. Erythrocyte and organo-somatic indices did not show differences between wild-types and homozygous mutants, indicating no association between the GR deficiency and haemolytic anaemia in this potential animal model.