Genetic, biochemical, and characterization of neurological mutant 3, a new mouse model for Parkinson's disease

We have identified a new mutant mouse that we have named new mouse neurological mutant 3 (NM3); it may be a useful model to understand the underlying molecular and genetic basis of Parkinson's disease (PD). A mouse carrying the NM3 mutation arose spontaneously in an RIIIS/J breeding colony and was identified as having a movement disorder. Upon neurological examination of these mice, their movement was found to be slow and abnormal, with characteristic choreaform and bradykinetic-type movements, typical of PD. The importance of the gene mutation in NM3 in the molecular pathway involved in this pathology is underscored by the fact that these mice do not survive past weaning age if they are homozygous for the genetic mutation. We localized the gene mutation by positional cloning and genetic mapping to mouse chromosome 2 in an area that corresponds to human chromosome 2q24-31, which does not contain any known genes associated with PD. However, there was a significant decrease of 15-20% in the levels of dopamine, and its principal metabolite, 3,4-dihydroxyphenylacetic acid, in the midbrain of affected mice. Low concentrations of these substances are associated with PD in human patients, making these mutant mice candidates for studies of this disease.     

Genetic variation in alkaline phosphatase of the house mouse (Mus musculus) with emphasis on a manganese-requiring isozyme

Genetic variation among inbred strains is described for electrophoretic migration of alkaline phosphatase from intestine, kidney, blood plasma, and three isozymes of liver. A manganese-requiring isozyme of liver and kidney unaffected by neuraminidase is described, and the locus controlling variation in this isozyme is designated Akp-1. Data from recombinant inbred strains place the locus on chromosome 1 at a distance of 3.6 +/- 2.9 cM from the M1s locus on the side distal to the centromere. Test-cross data show the following gene order and recombination percentages: Dip-1 19.0 +/- 3.8% Lp 7.4 +/- 2.2% Akp-1.     

Genetic characterization of esterase 28 (ES-28) of the house mouse

The genetics of esterase-28, the major esterase of cauda epididymidis of the house mouse, has been studied after separation by polyacrylamide gel electrophoresis and isoelectric focusing. Four phenotypes are distinguished. Segregation ofEs-28 in two backcross series indicated linkage to Es-1, Es-9, and Es-22. The Es-28 locus was placed into esterase cluster 1 on chromosome 8.This work was supported by the Deutsche Forschungsgemeinschaft.This is communication No. 69 of a research program devoted to the cellular distribution, genetics, and regulation of nonspecific esterases.     

Genetic and biochemical characterization of a new extracellular lipase from Streptomyces cinnamomeus.

Streptomyces cinnamomeus Tü89 secretes a 30-kDa esterase and a 50-kDa lipase. The lipase-encoding gene, lipA, was cloned from genomic DNA into Streptomyces lividans TK23 with plasmid vector pIJ702. Two lipase-positive clones were identified; each recombinant plasmid had a 5.2-kb MboI insert that contained the complete lipA gene. The two plasmids differed in the orientation of the insert and the degree of lipolytic activity produced. The lipA gene was sequenced; lipA encodes a proprotein of 275 amino acids (29,213 Da) with a pI of 5.35. The LipA signal peptide is 30 amino acids long, and the mature lipase sequence is 245 amino acids long (26.2 kDa) and contains six cysteine residues. The conserved catalytic serine residue of LipA is in position 125. Sequence similarity of the mature lipases (29% identity, 60% similarity) was observed mainly in the N-terminal 104 amino acids with the group II Pseudomonas lipases; no similarity to the two Streptomyces lipase sequences was found. lipA was also expressed in Escherichia coli under the control of lacZ promoter. In the presence of the inducer isopropyl-beta-D-thiogalactopyranoside (IPTG), growth of the E. coli clone was severely affected, and the cells lysed in liquid medium. Lipase activity in the E. coli clone was found mainly in the pellet fraction. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, three additional protein bands of 50, 29, and 27 kDa were visible. The 27-kDa protein showed lipolytic activity and represents the mature lipase; the 29- and 50-kDa forms showed no activity and very probably represent the unprocessed form and a dimeric misfolded form, respectively. For higher expression of lipA in S. lividans, the gene was cloned next to the strong aphII promoter. In contrast to the lipA-expressing E. coli clone, S. cinnamomeus and the corresponding S. lividans clone secreted only an active protein of 50 kDa. The lipase showed highest activity with C6 and C18 triglycerides; no activity was observed with phospholipids, Tween 20, or p-nitrophenylesters. Upstream of lipA and in the same orientation, an open reading frame, orfA, is found whose deduced protein sequence (519 amino acids) shows similarity to various membrane-localized transporters. Downstream of lipA and in the opposite orientation, an open reading frame, orfB (encoding a 199-amino-acid protein) is found, which shows no conspicuous sequence similarity to known proteins, other than an NAD and flavin adenine dinucleotide binding-site sequence.     

A new variant of chromosome 1 in the house mouse

In wild mouse populations of Siberia, animals with a new variant of chromosome 1 were found. The total length of this chromosome was 1.3 times as great as the normal homologue. The G-banding technique revealed two additional insertions Is(HSR; 1C5)1Icg and Is(HSR; 1E3)2Icg located between bands 1C5 and 1D, and 1E3 and 1E4, resp. The C-banding of both the insertions was positive and lighter than that of the centromeric heterochromatin. The size of each insertion was approximately 15% of new variant of chromosome 1. No meiotic disturbances were found in heterozygous male mice. Chromosome 1 with insertions has been introduced into the laboratory mouse stock.     

Esterase-30 (ES-30) of the house mouse: Biochemical characterization and genetics of a new carboxylesterase isozyme linked to cluster-2 loci on chromosome 8

A new carboxylesterase isozyme (EC 3.1.1.1), designated ES-30, is described in mouse liver. Two phenotypes were distinguished, ES-30A, a possible null type, was found in SPE/Pas and in other lines derived fromMus spretus, and ES-30B was found in BALB/cJ and other laboratory inbred strains. ES-30B is characterized by a distinct electrophoretic band when stained using 5-bromoindoxyl acetate as the substrate. After isolation and purification from other esterases by ion-exchange chromatography and molecular sieving, the molecular mass was estimated by two independent methods to be 62 and 64 kDa, respectively. The activity of ES-30B is higher in adult males than in females and can be stimulatedin vivo by testosterone. The distribution of phenotypes on the progeny of a backcross series suggests a separate locus,Es-30, with the allele a for absence andb for presence of the isozyme. LocusEs-30 is shown to be closely linked toEs-2 and toEs-7 of cluster-2 on chromosome 8. The gene orderEs-9—Got-2—(Es-2, Es-7, Es-30) is suggested. This work was supported by the Deutsche Forschungsgemeinschaft. This is communication No. 72 of a research program devoted to the cellular distribution, genetics, and regulation of nonspecific esterases.     

Genetic and molecular characteristics of a new natural haplotype twMP1 of the house mouse (Mus domesticus R.) from Peru

A new natural haplotype, twWP1, found in a population of house mouse Mus domesticus from Peru, was subjected to genetic and molecular analyses. Experiments were performed to study the complementation of the new haplotype, fertility of twMP1/tx heterozygotes, and transmission ratio distortion (TRD) of the t-carrying chromosome in the progeny of heterozygous males. Molecular analysis included blot hybridization with t-specific probes Tu48, Tu66, and Tu119. The results were collated with the structure and properties of the t complex, and the new haplotype was identified as a complete lethal one.     

Isolation and characterization of a cDNA and a pseudogene for mouse lactate dehydrogenase-A isozyme

A mouse lactate dehydrogenase-A cDNA was isolated and it was shown to contain the 393bp of the protein-coding sequence and 488bp of the 3' untranslated region. The amino acid sequence deduced from its open reading frame provided independent evidence for the sequence of residues 201-331 of mouse LDH-A subunit (muscle). This cDNA clone was used as a probe to isolate a mouse genomic clone containing a truncated, processed LDH-A pseudogene. This pseudogene showed 81.6% homology at 713 positions compared with the LDH-A cDNA sequence. The divergence of this pseudogene was estimated to have occurred 39 million years ago.     

Genetic and behavioral characterization of a Kmt2d mouse mutant,a new model for Kabuki Syndrome

The recessive mutant mice bate palmas (bapa) ‐ claps in Portuguese arose from N‐ethyl‐N‐nitrosourea mutagenesis. A single nucleotide, T > C, change in exon 13, leading to a Thr₁₂₈₉Ala substitution, was identified in the lysine (K)‐specific methyltransferase 2D gene (Kmt2d) located on chromosome 15. Mutations with a loss‐of‐function in the KMT2D gene on chromosome 12 in humans are responsible for Kabuki syndrome (KS). Phenotypic characterization of the bapa mutant was performed using a behavioral test battery to evaluate the parameters related to general activity, the sensory nervous system, the psychomotor system, and the autonomous nervous system, as well as to measure motor function and spatial memory. Relative to BALB/cJ mice, the bapa mutant showed sensory and psychomotor impairments, such as hypotonia denoted by a surface righting reflex impairment and hindquarter fall, and a reduction in the auricular reflex, suggesting hearing impairment. Additionally, the enhanced general activity showed by the increased rearing and grooming frequency, distance traveled and average speed possibly presupposes the presence of hyperactivity of bapa mice compared with the control group. A slight motor coordination dysfunction was showed in bapa mice, which had a longer crossing time on the balance beam compared with BALB/cJ controls. Male bapa mice also showed spatial gait pattern changes, such as a shorter stride length and shorter step length. In conclusion, the bapa mouse may be a valuable animal model to study the mechanisms involved in psychomotor and behavior impairments, such as hypotonia, fine motor coordination and hyperactivity linked to the Kmt2d mutation.     

Jimpy, a new totally sex-linked gene in the house mouse

Summary A new recessive mutant jimpy, jp, is described. It is situated on the differential segment of the sex chromosome and is lethal in the hemizygous condition. The jimpy mice show a behaviour disorder distinguished by an intention tremor first visible at about 11 days; after about three weeks they are liable to go into a convulsion if disturbed; death occurs usually between twenty-five and thirty-two days. jp shows about 21% recombination with Ta and 36% with Bn. It has not been tested directly against Br and Mo, but they both have been shown to be about four units beyond Ta with respect to Bn (Falconer 1954). The presumed order is therefore Bn, Ta, Br and Mo, jp.Member of the Scientific Staff, Medical Research Council.     

Immunocytochemical and biochemical characterization of FMRP, FXR1P, and FXR2P in the mouse

Fragile X syndrome is caused by the absence of expression of the FMR1 gene. Both FXR1 and FXR2 are autosomal gene homologues of FMR1. The products of the three genes are belonging to a family of RNA-binding proteins, called FMRP, FXR1P, and FXR2P, respectively, and are associated with polyribosomes as cytoplasmic mRNP particles. The aim of the present study is to obtain more knowledge about the cellular function of the three proteins (Fxr proteins) and their interrelationships in vivo. We have utilized monospecific antibodies raised against each of these proteins and performed Western blotting and immunolabeling at the light-microscopic level on tissues of wild-type and Fmr1 knockout adult mice. In addition, we have performed immunoelectron microscopy on hippocampal neurons of wild-type mice to study the subcellular distribution of the Fxr proteins. A high expression was found in brain and gonads for all three proteins. Skeletal muscle tissue showed only a high expression for Fxr1p. In the brain the three proteins were colocalized in the cytoplasm of the neurons; however, in specific neurons Fxr1p was also found in the nucleolus. Immunoelectronmicrsocopy on hippocampal neurons demonstrated the majority of the three proteins in association with ribosomes and a minority in the nucleus. The colocalization of the Fxr proteins in neurons is consistent with similar cellular functions in those specific cells. The presence of the three proteins in the nucleus of hippocampal neurons suggests a nucleocytoplasmic shuttling for the Fxr proteins. In maturing and adult testis a differential expression was observed for the three proteins in the spermatogenic cells. The similarities and differences between the distribution of the Fxr proteins have implications with respect to their normal function and the pathogenesis of the fragile X syndrome.     

Genetic and biochemical characterization of MbeA,the relaxase involved in plasmid ColE1 conjugative mobilization

MbeA is a 60 kDa protein encoded by plasmid ColE1. It plays a key role in conjugative mobilization. MbeA*, a slightly truncated version of MbeA, was purified for in vitro analysis. MbeA* catalysed DNA cleavage and strand-transfer reactions using oligonucleotides embracing the ColE1 nic site, which was mapped to 5'-(1469)CTGG/CTTA(1462)-3'. Thus MbeA is the relaxase for ColE1 conjugal mobilization, in spite of the fact that it lacks a three histidine motif considered the invariant signature of conjugative relaxases. Amino acid sequence comparisons suggest MbeA is nevertheless related to the common relaxase protein family. For instance, MbeA residue Y19 could correspond to the invariant tyrosine in Motif I, whereas H97, E104 and N106 may constitute the equivalent residues to the histidine triad in Motif III. This hypothesis was tested by site-directed mutagenesis. MbeA amino acid residues Y19, H97, E104 and N106 were changed to alanine. MbeA mutant N106A showed reduced oligonucleotide cleavage and strand-transfer activities, whereas mutation in the other three residues resulted in proteins without detectable activity, suggesting they are directly implicated in catalysis of DNA-cleavage and strand-transfer reactions. A double substitution of E104 and N106 by histidines, therefore reconstituting the canonical histidine triad, restored relaxase activities to 1% of wild type. Thus, MbeA is a variant of the common relaxase theme with a HEN signature motif, which has to be added to the canonical three histidine motif of previously reported relaxases.     

Purification and characterization of esterase 1F, the albumin esterase of the house mouse (Mus musculus)

Esterase 1F was isolated from mouse serum and purified by ion-exchange chromatography, isoelectrofocusing, and molecular sieve chromatography. It is considered to be a glycoprotein with an apparent molecular weight of 75 000. The equivalent weight (approximately equal to 77 000 X g/mol) was estimated by titration of the catalytic site with diethyl p-nitrophenyl phosphate. The Michaelis constant Km and the catalytic constant kcat of the enzyme for 4-nitrophenyl hexanoate were determined. Esterase 1F is characterized by its ability to split a wide spectrum of substrates and its relatively low turnover rates towards the substrates tested. It belongs to the isozyme system of carboxylesterase (EC 3.1.1.1) coded for by chromosome 8. Esterase 1F was compared with three other genetically related isozymes, esterase 2, esterase 7 and esterase 9, with respect to some physical and catalytic properties.     

Cloning and biochemical characterization of a novel mouse ADP-dependent glucokinase

Glycolysis, the catabolism of glucose to pyruvate, is an iconic central metabolic pathway and often used as a paradigm for explaining the general principles of the regulation/control of cellular metabolism. The ubiquitous mammalian ATP-dependent hexokinases I-III and hexokinase IV, also termed glucokinase, initiate the process by phosphorylating glucose to glucose-6-phosphate. Despite glycolysis having been studied extensively for over 70 years and the last new mammalian ATP-dependent hexokinase isotype having been described in the 1960s, we report here the biochemical characterization of a recombinant ADP-dependent glucokinase cloned from a full-length Mus musculus cDNA, identified by sequence analysis. The recombinant enzyme is quite specific for glucose, is monomeric, has an apparent Km for glucose and ADP of 96 and 280 microM, respectively, and is inhibited by both high concentrations of glucose and AMP. The metabolic role of this enzyme in cells would be dependent on the relative level of its activity to those of the ATP-dependent hexokinases. The greatest advantage of an ADP-GK would clearly be during ischemia/hypoxia, clinically relevant conditions in multiple major disease states, by decreasing the priming cost for the phosphorylation of glucose, saving ATP.     

A molecular characterization of the charismatic Faroe house mouse

Faroe house mice are a ‘classic’ system of rapid and dramatic morphological divergence highlighted by J. S. Huxley during the development of the Modern Synthesis. In the present study, we characterize these charismatic mice using modern molecular techniques, examining specimens from all Faroe islands occupied by mice. The aims were to classify the mice within the modern house mouse taxonomy (i.e. as either Mus musculus domesticus or Mus musculus musculus) using four molecular markers and a morphological feature, and to examine the genetic diversity and possible routes of colonization using mitochondrial (mt) control region DNA sequences and microsatellite data (15 loci). Mice on the most remote islands were characterized as M. m. domesticus and exhibited exceptionally low genetic diversity, whereas those on better connected islands were more genetically diverse and had both M. m. musculus and M. m. domesticus genetic elements, including one population which was morphologically M. m. musculus‐like. The mtDNA data indicate that the majority of the mice had their origins in south‐western Norway (or possibly southern Denmark/northern Germany), and probably arrived with the Vikings, earlier than suggested by Huxley. The M. m. musculus genetic component appears to derive from recent mouse immigration from Denmark. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 102 , 471–482.     

Genetic differences in alteration of albumin in the house mouse,Mus musculus

Two strains of mice were shown to differ in relative proportion of two major protein bands of liver under acid conditions of electrophoresis. Genetic control was autosomal and by a pair of dominant and recessive genes. The difference was observed only if liver homogenates were extracted by treating with dilute acid and alkali. The two protein bands were identified as albumin on the basis of electrophoretic migration, molecular weight, and immunological response. No differences were found between strains in relative amounts of comparable bands of plasma. However, in studies with extraction of mixtures, liver homogenates of one strain but not another were capable of converting a large proportion of albumin from several sources to a faster-migrating form. It was concluded that the strains differ in a factor in liver capable of causing a secondary modification of the albumin molecule.