Studies on thyrotropin releasing hormone in cerebellar ataxia mutant mouse brain

The effects of cathecholamine on the regional TRH distribution in the brain was studied in rolling mouse Nagoya (RMN) and non-affected C3H mice. TRH was extracted from the hypothalamus, brain stem, cerebellum, and cerebrum one hour after i.p. injection of the precursor or inhibitors of cathecholamine. TRH was distributed throughout the brain of both affected and non-affected mice; however, in RMN, TRH levels were lower in the hypothalamus and higher in other areas. 1-Dopa caused a decrease of TRH in the brain stem but no change in other regions in the RMN brain, whereas it caused an increase in TRH levels in all areas of the C3H brain. Fusaric acid increased TRH in the hypothalamus of RMN and decreased it in the cerebellum; alpha-MPT also caused a decrease in the TRH level in the cerebellum. Reserpine increased the TRH level in the hypothalamus and decreased it in the cerebrum. From these results, it appears that cerebellar ataxia in RMN does not result from a decrease in the TRH, which is actually increased in the cerebellum. Catecholamine had different effects on TRH levels in RMN and the controls; this might be due to the excess accumulation of noradrenaline in the RMN brain.     

A new mouse mutant, skijumper

Low blood sugar levels are a well-known cause of severe illness and often death in newborn humans, especially those that are small for age. Few of the causes of neonatal hypoglycemia are known, and many remain to be found. We describe a novel mouse mutant, skijumper (skimp), in which pups, despite feeding well, have low levels of glucose and develop opisthotonos, followed by death typically within a few days after birth. Genetic mapping studies have localized the lesion to a approximately 1 cM interval on mouse Chromosome (Chr) 7 between D7Mit318 and D7Mit93. We have carried out extensive analysis to define the phenotype and its likely cause. In addition to low blood glucose, affected skijumper mice have lowglycogen and ketone levels. Mass spectrometric analysis of blood samples has excluded major defects in amino acid metabolism. Initial biochemical analyses suggested a defect in ketogenesis as one possible cause of this phenotype. However, measurements of levels and activities of carnitine, carnitine palmitoyl transferases, and other enzymes involved in ketogenesis, along with studies of mitochondrial structure and function, did not demonstrate significant differences between skijumper, unaffected littermates, and control wild-type mice. These results indicate that abnormal enzyme activity in known pathways does not appear to be the primary biochemical lesion in skijumper. The skijumper may be a new valuable model for studying and understanding one type of neonatal morbidity and death.     

Patients with an inherited syndrome characterized by immunodeficiency, microcephaly, and chromosomal instability: genetic relationship to ataxia telangiectasia.

Fibroblast cultures from six unrelated patients having a familial type of immunodeficiency combined with microcephaly, developmental delay, and chromosomal instability were studied with respect to their response to ionizing radiation. The cells from five of them resembled those from individuals with ataxia telangiectasia (AT) in that they were two to three times more radiosensitive on the basis of clonogenic cell survival. In addition, after exposure to either X-rays or bleomycin, they showed an inhibition of DNA replication that was less pronounced than that in normal cells and characteristic of AT fibroblasts. However, the patients are clinically very different from AT patients, not showing any signs of neurocutaneous symptoms. Genetic complementation studies in fused cells, with the radioresistant DNA synthesis used as a marker, showed that the patients' cells could complement representatives of all presently known AT complementation groups. Furthermore, they were shown to constitute a genetically heterogeneous group as well. It is concluded that these patients are similar to AT patients with respect to cytological parameters. The clinical differences between these patients and AT patients are a reflection of genetic heterogeneity. The data indicate that the patients suffer from a chromosome-instability syndrome that is distinct from AT.     

A novel mutant of mouse lymphoma cells sensitive to alkylating agents and caffeine

Two methyl-methanesulfonate-sensitive strains have been isolated, one of which, M10, was cross-sensitive to X-rays as reported before. Sensitivities of parental L5178Y, M10, and newly isolated MS-1 cells to various mutagens were examined. Mutgans tested were UV, X-rays, 4-nitroquinoline 1-oxide (4NQO), caffeine and alkylating agents; methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS) and mitomycin C (MMC).In terms of D₃₇ values, M10 cells were 2.5–7 times more sensitive to EMS, MMC and 4NQO as well as to MMS and X-rays than were parental L5178Y cells, while the new mutant MS-1 was about 3 times more sensitive to MMS, EMS, MMC and caffeine than were parental cells. The characteristics in sensitivities of M10 cells to X-rays, alkylating agents and 4NQO resemble some ataxia telangiectasia cells; and MS-1 cells to alkylating agents and caffeine are novel among mammalian cell mutants so far reported. Sensitivity of M10 cells to mutagens has so far been stable for one year, and that of MS-1 cells was stable for 6 months in continuous culture.     

Sprague Dawley rat mutant with tremor, ataxia, tonic immobility episodes, epilepsy and paralysis

A spontaneous neurological mutation was detected in a colony of Sprague Dawley rats. The animals developed a progressive neurological syndrome characterized by tremor (which appeared at the age of 1 month), ataxia (at 4 months), immobility episodes (after 5-6 months), audiogenic seizures and hindlimb paralysis (after 10 months). Cross breeding experiments indicate that this is an autosomal recessive mutation, which we have named taiep subline.     

A cytoplasmically inherited mutant controlling early chloroplast development in barley seedlings

Cytoplasmic line 2 (CL2) has been previously reported as a cytoplasmically inherited chlorophyll-deficient mutant selected from a chloroplast-mutator genotype of barley. It was characterized by a localized effect on the upper part of the first-leaf blade. At emergence the CL2 seedlings-phenotype varied from a grainy light green to an albino color. They gradually greened during the following days, starting from the base of the blade and extending to cover most of its surface when it was fully grown. The present results, from both light microscopy and transmission electron microscopy (TEM), confirmed the previously described positional and time-dependent expression of the CL2 syndrome along the first-leaf blade. During the first days after emergence, light microscopy showed a normally developed chloroplast at the middle part of the CL2 first-leaf blade, meanwhile at the tip only small plastids were observed. TEM showed that the shapes and the internal structure of the small plastids were abnormal, presenting features of proplastids, amyloplasts and/or senescent gerontoplasts. Besides, they lack plastid ribosomes, contrasting with what was observed inside chloroplasts from normal tips, which presented abundant ribosomes. Phenotypic observations and spectrophotometric analysis of seedlings produced by mother plants that had been grown under different temperatures indicated that higher temperatures during seed formation were negatively associated with pigment content in CL2 seedlings. In contrast, higher temperatures during the growth of CL2 seedlings have been associated with increased pigment content. Aqueous solution with kanamycin and streptomycin, which are antibiotics known to interfere with plastid gene translation, were used for imbibition of wild-type and CL2 seeds. Antibiotic treatments differentially reduced the chlorophyll content in the upper part of the first-leaf blade in CL2, but not in wild-type seedlings. These results suggest that in the wild-type, plastid-gene proteins which are necessary for chloroplast development and chlorophyll synthesis in the upper part of the first-leaf blade are usually synthesized during embryogenesis. However, under certain circumstances, in CL2 seedlings, they would be synthesized after germination. In addition, a shortening of the sheath has been observed in association with pigment decrease suggesting the existence of plastid factors affecting the expression of some nuclear genes. We consider the CL2 mutant a unique experimental material useful to study biological phenomena and external factors regulating plastid, and nuclear gene expression during embryogenesis and early seedling development.Communicated by R. Hagemann     

EMMA--the European mouse mutant archive.

The European Mouse Mutant Archive (EMMA) offers the worldwide scientific community a free archiving service for its mutant mouse lines and access to a wide range of disease models and other research tools. EMMA is currently comprised of seven partners who operate as the primary mouse repository in Europe. EMMA' s primary objectives are to establish and manage a unified repository for maintaining mouse mutations and to make them available to the scientific community. In addition to these core services, the consortium can generate germ-free (axenic) mice for its customers and also hosts courses in cryopreservation. EMMA is a founder member of the Federation of International Mouse Resources (FIMRe). The EMMA network is funded by the participating institutes, national research programmes and the European Commission Research Infrastructures Programme.     

A point mutation in a cadherin gene, Cdh23, causes deafness in a novel mutant, Waltzer mouse niigata

A novel mouse model for human nonsyndromic hearing loss, Waltzer niigata (v(ngt)), is found and subjected to positional cloning analysis. Genome-wide scan of 1648 backcross mice maps v(ngt) to the D10Mit258 locus near Waltzer (v). Recombination breakpoints are positioned on a physical map consisting of 13 BACs relative to the flanking markers in the vicinity of v(ngt). Allelism test done in parallel shows that v(ngt) and v are allelic. Sequence analysis reveals one-base deletion in the cDNA encoding a cadherin-related protein, Cdh23, mutation of which is recently reported in v mutants. The frame-shift change, producing a truncated protein of 51 amino acids, is ascribed to a base-substitution of G to A in the acceptor site of splicing junction which is predicted to cause one-base shift of the splicing position.     

Evaluation of the mouse mutant "wasted" as an animal model for ataxia telangiectasia. I. Age-dependent and tissue-specific effects

The wasted mouse, an animal model proposed for the genetically transmitted human disease ataxia telangiectasia (AT), was examined for its biological, cytogenetic and biochemical properties. In affected homozygotes, a marked age-dependent decrease in the ratio of spleen and thymus to body weight, and a slight but significant decrease in the liver to body weight ratio were observed while no such change was found in the kidney. An age-dependent increase was observed in the frequency of both spontaneous and gamma-ray-induced chromosomal aberrations in bone marrow cells of wasted mice. In littermate control mice, neither of these alterations was observed in an age-dependent manner. The activity of a primer activating enzyme, which has been reported to be deficient in AT cells, also decreased with age in spleen cells, but not in liver cells of affected mice. However, alterations in apurinic DNA endonuclease activity were not detected in the developmental stages examined. These data indicate that this mouse mutant may serve as a useful animal model for studying the relationships between DNA repair and lymphoid tissue differentiation.     

A YAC mouse model for Huntington's disease with full-length mutant huntingtin, cytoplasmic toxicity, and selective striatal neurodegeneration.

We have produced yeast artificial chromosome (YAC) transgenic mice expressing normal (YAC18) and mutant (YAC46 and YAC72) huntingtin (htt) in a developmental and tissue-specific manner identical to that observed in Huntington's disease (HD). YAC46 and YAC72 mice show early electrophysiological abnormalities, indicating cytoplasmic dysfunction prior to observed nuclear inclusions or neurodegeneration. By 12 months of age, YAC72 mice have a selective degeneration of medium spiny neurons in the lateral striatum associated with the translocation of N-terminal htt fragments to the nucleus. Neurodegeneration can be present in the absence of macro- or microaggregates, clearly showing that aggregates are not essential to initiation of neuronal death. These mice demonstrate that initial neuronal cytoplasmic toxicity is followed by cleavage of htt, nuclear translocation of htt N-terminal fragments, and selective neurodegeneration.     

entla, a novel epileptic and ataxic Cacna2d2 mutant of the mouse

entla (ent) is a novel recessive phenotype of mice. The underlying mutation was mapped to chromosome 9 (60.1 centimorgans) and identified as an allele of the Cacna2d2 gene encoding the alpha2delta-2 subunit of voltage-gated calcium channels. The Cacna2d2entla allele harbors a 38-kb duplication comprising the 117 nucleotides of exon 3. The predicted duplication of 39 amino acid residues near the subunit's N terminus results in the expression of a full-length, membrane-associated protein. Western blot data were consistent with correct cleavage of the alpha2delta-2entla precursor into alpha2entla and delta2 proteins but indicated loss of the disulfide linkage between the two proteins. ent/ent mice develop ataxia by postnatal day 13-15, followed by paroxysmal dyskinesia a few days later. Two distinct types of cortical and hippocampal epileptic activity at 2 and 4 Hz were recorded, indicative of absence epilepsy. Homozygotes display reduced size and weight, increased mortality before weaning, and female infertility. No overt neuroanatomical abnormalities were detected. Ca2+ current densities recorded from acutely dissociated Purkinje cells of homozygous entla animals were reduced by 50% compared with wild type. Ligand binding assays using the antiepileptic drug [3H]gabapentin, a specific ligand of the alpha2delta-1 and alpha2delta-2 subunits, revealed a >60% reduced maximum binding to cerebellar membranes of ent/ent compared with unaffected littermates. entla is allelic to ducky and ducky2J, representing the third murine Cacna2d2 allele identified and so far the only one encoding an untruncated protein that is incorporated into membranes.     

A novel ethanol-hypersensitive mutant of Arabidopsis

A novel ethanol-hypersensitive mutant, geko1 (gek1), was isolated from Arabidopsis thaliana. The gek1 mutant displays an enhanced sensitivity (10-100 times greater than the wild type) to ethanol in growth medium, while it grows normally in the absence of ethanol, and responds normally to other alcohols and to environmental stresses such as heat shock and high salinity. The ethanol-hypersensitive phenotype of gek1 requires alcohol dehydrogenase activity, indicating that gek1 is sensitive not to ethanol itself but to the metabolites of ethanol. Consistent with this, gek1 shows enhanced sensitivity to acetaldehyde in the medium. The endogenous acetaldehyde levels were not different between gek1-2 and wild-type seedlings treated with ethanol. These results indicate that the ethanol hypersensitivity of gek1 is due to an enhanced sensitivity to acetaldehyde toxicity, instead of abnormally elevated accumulation of toxic acetaldehyde, which has been thought to be the major cause of ethanol toxicity in mammal cells.     

A novel retroviruslike family in mouse DNA.

In the course of structural analysis of VL30 DNA elements, a recombinant retroviruslike element was encountered that contained non-VL30 long terminal repeats (LTRs) flanking internal VL30 sequences. With the aid of this novel LTR sequence probe, we cloned several DNA elements that were apparently members of a new retroviruslike family. A particular DNA element representative of this family (designated GLN) was characterized. It was approximately 8 kilobase pairs long and contained LTRs that are 430 base pairs long. It possessed an unusual primer-binding site sequence that corresponds to tRNAGln and a polypurine tract primer that is adjacent to the 3' LTR. The nucleotide sequences of the LTRs and their adjacent regions (which together housed all cis-acting retroviral functions) were different from those of known retroviruses and retroviruslike families. The comparison of three different GLN LTR sequences revealed a marked heterogeneity of U3 sequences relative to the homogeneity of R and U5 sequences. We estimate that approximately 20 to 50 copies of GLN elements are dispersed in all species of mice. GLN-related LTRs, however, are present in a much higher copy number (1,000 to 1,500 per genome). Nucleotide sequences that are more distantly related to GLN DNA are present in multiple copies in DNAs of other rodents but not in nonrodent genomes.     

Defect of a fiber cell-specific 94-kDa protein in the lens of inherited microphthalmic mutant mouse Elo

Deficiency in a 94,000-dalton protein in the non-crystallin fraction from the Elo mouse lens was shown. To perform further investigations, we raised an antibody against the 94,000-dalton protein isolated from normal mouse lens. Western blot analysis with the antibody indicated that the protein was only present in the lens and not in the brain, lung, heart, liver, and kidney. In the lens, it was unique to the cortex and nucleus fractions, not being present in the epithelial cells. Furthermore, it was observed in the water-soluble fraction as well as in the urea-soluble fraction. The antibody weakly but clearly reacted with the chick CP97 lens peptide, a fiber cell-specific protein, and anti-CP97 antibody also reacted with the 94,000-dalton protein. From these results, we concluded that the protein corresponds to CP97 cytoskeletal protein in the mouse lens. The protein was deficient in the lenses from Elo mice, but microphthalmic lenses from CTA mice contained a normal level.     

A novel mitochondrial mutation m.8989G>C associated with neuropathy,ataxia, retinitis pigmentosa — The NARP syndrome

The archetypal NARP syndrome is almost exclusively associated with the m.8993T>C/G mutation in the sixth subunit of the mitochondrial ATP synthase, whereas other mutations in the MT-ATP6 gene primarily associate with Leigh syndrome or Leber's hereditary optic neuropathy (LHON). We report a novel mitochondrial point mutation, m.8989G>C, in a patient presenting with neuropathy, ataxia and retinitis pigmentosa constituting the classical NARP phenotype. This mutation alters the amino acid right next to canonical NARP mutation. We suggest that classic NARP syndrome relates to a defined dysfunction of p.MT-ATP6.     

Circling mouse, a spontaneous mutant in the inner ear.

A spontaneous mutant was established in the ICR mouse strain. The affected mice became hyperactive at about 7 days of age, and then showed circling behavior. The body weight decreased significantly 2 weeks after birth, and developmental defects were revealed in the middle ear, cochlea, cochlear nerve, and semicircular canal areas. The mutation was inherited by an autosomal single recessive gene and is referred to as cir.