Intragenic deletion in the gene encoding ubiquitin carboxy-terminal hydrolase in gad mice.

The gracile axonal dystrophy (gad) mouse is an autosomal recessive mutant that shows sensory ataxia at an early stage, followed by motor ataxia at a later stage. Pathologically, the mutant is characterized by 'dying-back' type axonal degeneration and formation of spheroid bodies in nerve terminals. Recent pathological observations have associated brain ageing and neurodegenerative diseases with progressive accumulation of ubiquitinated protein conjugates. In gad mice, accumulation of amyloid beta-protein and ubiquitin-positive deposits occur retrogradely along the sensory and motor nervous systems. We previously reported that the gad mutation was transmitted by a gene on chromosome 5 (refs 10,11). Here we find that the gad mutation is caused by an in-frame deletion including exons 7 and 8 of Uchl1, encoding the ubiquitin carboxy-terminal hydrolase (UCH) isozyme (Uch-l1) selectively expressed in the nervous system and testis. The gad allele encodes a truncated Uch-l1 lacking a segment of 42 amino acids containing a catalytic residue. As Uch-l1 is thought to stimulate protein degradation by generating free monomeric ubiquitin, the gad mutation appears to affect protein turnover. Our data suggest that altered function of the ubiquitin system directly causes neurodegeneration. The gad mouse provides a useful model for investigating human neurodegenerative disorders.

Peg3 imprinted gene on proximal chromosome 7 encodes for a zinc finger protein.

Genetic and embryological studies in the mouse demonstrated functional differences between parental chromosomes during development. This is due to imprinted genes whose expression is dependent on their parental origin. In a recent systematic screen for imprinted genes, we detected Peg3 (paternally expressed gene 3). Peg3 is not expressed in parthenogenones. In interspecific hybrids, only the paternal copy of the gene is expressed in the embryos, individual tissues examined in d9.5-13.5 embryos, neonates and adults. Peg3 mRNA is a 9 kb transcript encoding an unusual zinc finger protein with eleven widely spaced C2H2 type motifs and two groups of amino acid repeats. Peg3 is expressed in early somites, branchial arches and other mesodermal tissues, as well as in the hypothalamus. Peg3 maps to the proximal region of chromosome 7. Consistent with our findings, maternal duplication of the proximal chromosome 7 causes neonatal lethality. This region is syntenic with human chromosome 19q13.1-13.3 (refs 10,11), where the genes for myotonic dystrophy and a putative tumour suppressor gene are located.

Diffusion tensor imaging in children with periventricular leukomalacia: variability of injuries to white matter tracts.

BACKGROUND AND PURPOSE: Conventional MR imaging shows evidence of brain injury and/or maldevelopment in 70%-90% of children with cerebral palsy (CP), though its capability to identify specific white matter tract injury is limited. The great variability of white matter lesions in CP already demonstrated by postmortem studies is thought to be one of the reasons why response to treatment is so variable. Our hypothesis is that diffusion tensor imaging (DTI) is a suitable technique to provide in vivo characterization of specific white matter tract lesions in children with CP associated with periventricular leukomalacia (PVL). MATERIALS AND METHODS: In this study, 24 children with CP associated with PVL and 35 healthy controls were evaluated with DTI. Criteria for identification of 26 white matter tracts on the basis of 2D DTI color-coded maps were established, and a qualitative scoring system, based on visual inspection of the tracts in comparison with age-matched controls, was used to grade the severity of abnormalities. An ordinal grading system (0=normal, 1=abnormal, 2=severely abnormal or absent) was used to score each white matter tract. RESULTS: There was marked variability in white matter injury pattern in patients with PVL, with the most frequent injury to the retrolenticular part of the internal capsule, posterior thalamic radiation, superior corona radiata, and commissural fibers. CONCLUSION: DTI is a suitable technique for in vivo assessment of specific white matter lesions in patients with PVL and, thus, a potentially valuable diagnostic tool. The tract-specific evaluation revealed a family of tracts that are highly susceptible in PVL, important information that can potentially be used to tailor treatment options in the future.

Genetic loci controlling susceptibility to gamma-ray-induced thymic lymphoma.

BALB/c is a susceptible strain for the development of gamma-ray induced mouse thymic lymphoma whereas MSM shows resistance. Association analysis of 220 backcross mice between the two strains using 67 markers was carried out to identify loci involved in the control of susceptibility. The genotype of mice with lymphoma showed excess heterozygosity relative to MSM homozygosity at D2Mit15 and D4Mit12 and was skewed toward MSM-derived alleles at D5Mit5. The P values in Mantel-Cox test were 0.0048 (D2Mit15), 0.0034 (D4Mit12) and 0.0048 (D5Mit5), suggesting association at the three loci in the susceptibility. Cooperative effect on lymphomagenesis was also observed among the three loci. To obtain independent evidence for linkage at D4Mit12, we made partially congenic mice in which a D4Mit12 region in BALB/c was replaced by MSM-derived homolog. Examination for the lymphoma susceptibility in 78 progeny of the congenic mice confirmed the effect of the locus near D4Mit12 (P=0.0037). The result, together with the linkage analysis, shows that the locus near D4Mit12 is regarded as a confirmed linkage but the other two loci as marginally suggestive.

Novel mouse microsatellites: primer sequences and chromosomal location.

Sixty-nine sequences containing microsatellites were determined by analysis of clones from a pUC118 library of total genomic mouse DNA. These sequences were examined for size variation using polymerase chain reaction and gel electrophoresis. Fifty-one of them showed allelic variations between C57BL/6 and MSM, the two strains used for genetic mapping. Hence, their chromosomal location was determined using a panel consisting of 131 backcross mice that had been typed with 85 anchor loci. The microsatellites were distributed to most chromosomes except for chromosomes 16 and 19. These novel markers with defined locations are useful in linkage and genome mapping studies.

Validation of transgenic mice carrying the human prototype c-Ha-ras gene as a bioassay model for rapid carcinogenicity testing.

Carcinogenicity testing is indispensable for identifying environmental carcinogens and for evaluating the safety of drugs in the process of development. Conventional 2-year rodent bioassays are one of the most resource-consuming tests in terms of animals, time, and costs. Development of rapid carcinogenicity testing systems that can assess carcinogenicity within a short period has become a social demand and is essential to improve efficacy in the identification of environmental carcinogens as well as in the development of new drugs. In this review we introduce the rapid carcinogenicity testing system using transgenic (Tg) mice carrying the human prototype c-Ha-ras gene, namely rasH2 mouse (CB6F1-TgHras2 mouse is the same mouse). The studies have been conducted to validate the rasH2 mouse as a model for the rapid carcinogenicity testing system. Our current validation studies revealed that rasH2 mice are able to detect various types of mutagenic carcinogens within 6 months. The rasH2 mice may also be able to detect various nonmutagenic carcinogens. The validation studies also revealed that rasH2 mice are generally much more susceptible to both mutagenic and nonmutagenic carcinogens than control non-Tg mice. No significant tumor induction has been observed in rasH2 mice with either mutagenic or nonmutagenic noncarcinogens. More rapid onset and higher incidence of more malignant tumors can be expected with a high probability after treatment with various carcinogens in the rasH2 mice than in control non-Tg mice. The rasH2 mouse appears to be a promising candidate as an animal model for development of a rapid carcinogenicity testing system.

Radiation-associated loss of heterozygosity at the Znfn1a1 (Ikaros) locus on chromosome 11 in murine thymic lymphomas.

Although information on the molecular pathways in radiation carcinogenesis is accumulating, the data are still relatively scanty. To find the tumor suppressor locus associated with radiation carcinogenesis, we determined the frequency and distribution of loss of heterozygosity (LOH) of X-ray-induced thymic lymphomas of B6C3F(1) mice using 58 microsatellite markers and compared the results with those for spontaneous lymphomas and N-ethylnitrosourea (ENU)-induced lymphomas. Based on the results, we describe a unique locus with frequent LOH in the centromeric region of chromosome 11 of X-ray-induced lymphomas. This locus has never been observed to be altered similarly in either ENU-induced or spontaneous lymphomas, suggesting radiation-specific molecular alteration. The LOH patterns of individual thymic lymphomas indicated that the common region of LOH was located within 1.6 cM between D11Mit62 and D11Mit204, a region syntenic to human chromosome 7p13. Linkage analysis revealed that the markers of the common LOH region were genetically linked to Ikaros (now known as Znfn1a1), a master gene of lymphopoiesis. Although the presence of radiation-associated LOH in other loci cannot be ruled out, these results suggest a novel molecular pathway in induction of thymic lymphomas by ionizing radiation.

Low frequency of Ras gene mutation in spontaneous and gamma-ray-induced thymic lymphomas of scid mice.

Scid mice, which have a defect in the capacity to repair DNA double-strand breaks, were highly prone to the induction of thymic lymphomas after exposure to ionizing radiation; approximately 70% of mice developed lymphomas within 1 year after exposure to 1-3 Gy, whereas approximately 20% of unirradiated control mice developed lymphomas. To gain information on the possible role of Ras activation in development of thymic lymphomas in scid mice, we have examined both the frequency and the spectrum of Kras and Nras mutations in spontaneous and radiation-induced lymphomas. Neither activated Kras nor Nras genes were detected in spontaneous lymphomas, while Kras mutations increased in a dose-dependent manner in radiation-induced lymphomas. However, Kras mutations were infrequent (6% in lymphomas in mice exposed to 1 Gy, 12.5% in those exposed to 2 Gy, 16.7% in those exposed to 3 Gy), and no mutations were detected in Nras genes, suggesting that Ras mutation was not significantly involved in the development of thymic lymphomas in scid mice. Analysis of the spectrum of Kras mutations demonstrated unique mutations in both codons 13 (GGC to GAC) and 61 (CAA to CTA) in addition to the commonly identified substitution of GAT for GGT in codon 12 of Kras.

Gene mapping of SEZ group genes and determination of pentylenetetrazol susceptible quantitative trait loci in the mouse chromosome.

Gene mapping of the newly discovered SEZ genes (seizure-related genes) in the mouse was performed by linkage analysis. SEZ6 was on chromosome 11, SEZ12 on chromosome 16, SEZ15 on chromosome 3 and SEZ17 (PTZ17) on chromosome 18. The mouse chromosomal locus related to high susceptibility to pentylenetetrazol (PTZ) was also determined by linkage analysis using the recombinant inbred mouse, BXD (C57BLxDBA). A significant level of PTZ susceptibility was found on chromosome 2. Chromosomal loci of the newly discovered SEZ genes were not coincident with the significant chromosomal loci to PTZ susceptibility. Since epilepsy is assumed to be a disease syndrome which is probably manifested by abnormal expression of multifocal genes, determination of the role of each chromosomal locus in the provocation of seizure activity is important.

Molecular phylogeny of East Asian moles inferred from the sequence variation of the mitochondrial cytochrome b gene.

Taxonomic analysis has previously revealed that the species of moles that inhabit Japan are characterized by exceptional species richness and a high level of endemism. Here, we focused on the evolutionary history of the four Japanese mole species of the genera Euroscapter and Mogera, examining mitochondrial cytochrome b (cyt b) gene sequences and comparing them with those of continental Mogera wogura (Korean and Russian populations), M. insularis from Taiwan, and Talpa europaea and T. altaica from the western and central Eurasian continent, respectively. Our data support the idea that in a radiation center somewhere on the Eurasian continent, a parental stock evolved to modern mole-like morph and radiated several times intermittently during the course of the evolution, spreading its branches to other peripheral geographic domains at each stage of the radiation. Under this hypothesis, the four lineages of Japanese mole species, E. mizura, M. tokudae, M. imaizumii, and M. wogura, could be explained to have immigrated to Japan in this order. Mogera wogura and M. imaizumii showed substantial amounts of geographic variation and somewhat complicated distributions of the cyt b gene types. These intraspecific variations are likely to be associated with the expansion processes of moles in the Japanese Islands during the Pleistocene glacial ages.

Overexpression of human H-ras transgene is responsible for tumors induced by chemical carcinogens in mice.

Level of human prototype H-ras transgene expression in tumors induced by chemical carcinogens (N-ethyl-N-nitrosourea and N-methyl-N-nitrosourea) was analyzed in human H-ras transgenic mice (CB6F1-TgrasH2 Jic mice). All forestomach tumors examined revealed about 2-fold overexpression of the human H-ras transgene with or without point mutation at codon 12 or codon 61. However, endogenous mouse H- and K-ras genes exhibited neither point mutation nor overexpression. These results suggested that increased levels of ras gene products in the cell played an important role in facilitating chemical carcinogenesis in transgenic mice.

Reduced sensitivity to and ras mutation spectrum of N-ethyl-N-nitrosourea-induced thymic lymphomas in adult C.B-17 scid mice.

Scid mice are defective in the ability to repair DNA double strand breaks and, as a consequence, their cells are radiosensitive. Further, they have been shown to be prone to develop thymic lymphomas (TLs) after small doses of ionizing radiation. Little is known, however, on the role of scid mutation in chemical carcinogenesis. To determine if scid mutation increased predisposition to chemical carcinogenesis, we examined both the susceptibility of scid mice to N-ethyl-N-nitrosourea (ENU)-induced lymphomagenesis and the involvement of ras gene activation. Adult female mice at 8 weeks of age were given ENU in their drinking water at 400 ppm for 2-10 weeks. Contrary to expectations, we observed a two to three-fold reduction in TL development in the scid mice. The highest incidence was achieved by ENU treatment for 8 weeks for scid and wild-type C.B-17 mice, of 42 and 85%, respectively (P<0.05). We investigated whether this was attributable to the usage of the ras mutation pathway. There was, however, no significant difference in the frequency and spectrum of K-ras mutation between the scid and wild-type C.B-17 mice. Most of the K-ras mutations were either GGT to GAT transition in codon 12 (11/23: 48%) or CAA to CCA transversion in codon 61 (8/23: 35%) that was independent of scid background. The incidence of N-ras mutation was very low. These results indicate that scid mice are less susceptible to ENU-induced lymphomagenesis and ras gene mutation frequently occurs in both scid and wild-type C.B-17 mice.

A new variant of the Es-4 locus in the rat.

A new variant of kidney esterase in the DK/Nac rat strain is reported. The new esterase was tentatively named ES-4C determined by a third allele of the Es-4 locus of Linkage Group V (LGV). Strain distribution was surveyed using 17 inbred strains, but no strain except for the DK/Nac strain possessed the ES-4C type. Although we surveyed outbred stocks (Jcl: Wistar and Jcl: SD) we could not find rats carrying the ES-4C type. Genetic analysis of the ES-4C type was carried out using mating experiments between DK/Nac and BUF/Nac (ES-4B). The results indicated that the new variant was controlled by the Es-4 locus and it was named the Es-4c allele.

A variant in the restriction endonuclease cleavage pattern of mitochondrial DNA in the domestic fowl, Gallus gallus domesticus.

The cleavage patterns of mitochondrial DNAs (mtDNAs) were investigated from 15 lines of domestic fowls, Gallus gallus domesticus, using 11 restriction endonucleases. The cleavage patterns with 10 restriction endonucleases were identical in all the lines. A variant was found in a line of White Leghorn in the pattern with MspI digestions. Cleavage patterns of the red jungle fowl, Gallus gallus gallus, were identical to the common patterns shown by the 14 lines of domestic fowls.

Mapping of the Es-4 locus and linear order of esterase genes (linkage group V; LGV) in the rat.

There are three different linear orders of esterase loci of linkage group V (LGV) in the rat (Rattus norvegicus). The first is Es-2-Es-3-Es-1, the second Es-3-(Es-2,Es-4)-Es-1, and the third Es-3-Es-2-Es-1-Es-4. We carried out mating experiments to define the order clearly. Linkage analyses of the four esterase loci, Es-1, Es-2, Es-3, and Es-4, were carried out using two inbred strains carrying different alleles at the four loci. Six locus combinations examined in this study were as follows: Es-1-Es-2, Es-1-Es-3, Es-1-Es-4, Es-2-Es-3, Es-2-Es-4, and Es-3-Es-4. The recombination frequencies of each combination were 6.3, 6.3, 6.3, 5.2, 1.8, and 3.4%, respectively. The first recombination between Es-2 and Es-4 was observed. We propose that the esterase loci of LGV be classified into three clusters according to distances between the loci. The linear order of the four loci is shown to be as follows: [Es-3] (cluster II)-3.4 +/- 2.4%-[Es-4-1.8 +/- 1.7%-Es-2] (cluster III)-6.3 +/- 6.1%-[Es-1] (cluster I).

Mitochondrial DNA polymorphism in native Philippine cattle based on restriction endonuclease cleavage patterns.

An analysis of patterns of cleavage of mtDNA by restriction endonucleases was performed for nine individuals from the Philippine population of native cattle. MtDNA polymorphisms were detected in the restriction patterns generated by the following six enzymes, BamHI, BglII, EcoRV, HindIII, PstI, and ScaI. The restriction patterns showing polymorphisms were distributed nonrandomly among the nine individuals examined from the Philippine population of native cattle, indicating the existence of two separate types of mtDNA. These two types of mtDNA are very different from each other, at the level of subspecies. Since the native Philippine cattle are considered to represent an admixture of European and Indian cattle, the two types of mtDNA must be derived from the mtDNAs of both varieties. The polymorphic sites in mtDNA have been located on a restriction map, and the nucleotide substitutions at some of the sites have also been estimated.

A duplicated zone of polarizing activity in polydactylous mouse mutants.

The positional signaling along the anteroposterior axis of the developing vertebrate limb is provided by the zone of polarizing activity (ZPA) located at the posterior margin. Recently, it was established that the Sonic hedgehog (Shh) mediates ZPA activity. Here we report that a new mouse mutant, Recombination induced mutant 4 (Rim4), and two old mutants, Hemimelic extra toes (Hx) and Extra toes (Xt), exhibit mirror-image duplications of the skeletal pattern of the digits. In situ hybridization of the embryos of these mutants revealed ectopic expression of Shh and fibroblast growth factor-4 (Fgf-4) genes at the anterior margin of limb buds. The new mutation, Rim4, was mapped to chromosome 6 with linkage to HoxAbut segregated from HoxA. No linkage to other known polydactylous mutations was detected. In this mutant, ectopic expression of the Hoxd-11 gene, thought to be downstream of ZPA, was also observed at the anterior margin of the limb buds. All results indicate the presence of an additional ZPA at the anterior margin of limb buds in these mutants. Thus, it appears that multiple endogenous genes regulate the spatial localization of the ZPA in the developing mouse limb bud.