The Neurological Mouse Mutations Jittery and Hesitant Are Allelic and Map to the Region of Mouse Chromosome 10 Homologous to 19p13.3

Jittery (ji) is a recessive mouse mutation on Chromosome 10 characterized by progressive ataxic gait, dystonic movements, spontaneus seizures, and death by dehydration/starvation before fertility. Recently, a viable neurological recessive mutation, hesitant, was discovered. It is characterized by hesitant, uncoordinated movements, exaggerated stepping of the hind limbs, and reduced fertility in males. In a complementation test and by genetic mapping we have shown here that hesitant and jittery are allelic. Using several large intersubspecific backcrosses and intercrosses we have genetically mappedjinear the markerAmhand microsatellite markersD10Mit7, D10Mit21,andD10Mit23.The linked region of mouse Chromosome 10 is homologous to human 19p13.3, to which several human ataxia loci have recently been mapped. By excluding genes that map to human 21q22.3 (Pfkl) and 12q23 (Nfyb), we conclude that jittery is not likely to be a genetic mouse model for human Unverricht–Lundborg progressive myoclonus epilepsy (EPM1) on 21q22.3 nor for spinocerebellar ataxia II (SCA2) on 12q22–q24. The closely linked markers presented here will facilitate positional cloning of thejigene.     

Phospholipase A2 and arachidonic acid in Alzheimer's disease

Essential fatty acids (EFA) play a critical role in the brain and regulate many of the processes altered in Alzheimer's disease (AD). Technical advances are allowing for the dissection of complex lipid pathways in normal and diseased states. Arachidonic acid (AA) and specific isoforms of phospholipase A₂ (PLA₂) appear to be critical mediators in amyloid-β (Aβ)-induced pathogenesis, leading to learning, memory, and behavioral impairments in mouse models of AD. These findings and ongoing research into lipid biology in AD and related disorders promise to reveal new pharmacological targets that may lead to better treatments for these devastating conditions.     

Detailed Comparative Map of Human Chromosome 19q and Related Regions of the Mouse Genome

One of the larger contiguous blocks of mouse–human genomic homology includes the proximal portion of mouse chromosome 7 and the long arm of human chromosome 19. Previous studies have demonstrated the close relationship between the two regions, but have also indicated significant rearrangements in the relative orders of homologous mouse and human genes. Here we present the genetic locations of the homologs of 42 human chromosome 19q markers in the mouse, with an emphasis on genes also included in the human chromosome 19 physical map. Our results demonstrate that despite an overall inversion of sequences relative to the centromere, apparent “transpositions” of three gene-rich segments, and a local inversion of markers mapping near the 19q telomere, gene content, order, and spacing are remarkably well conserved throughout the lengths of these related mouse and human regions. Although most human 19q markers have remained genetically linked in mouse, one small human segment forms a separate region of homology between human chromosome 19q and mouse chromosome 17. Three of the four rearrangements of mouse versus human 19q sequences involve segments that are located directly adjacent to each other in 19q13.3–q13.4, suggesting either the coincident occurrence of these events or their common association with unstable DNA sequences. These data permit an unusually in-depth examination of this large region of mouse–human genomic homology and provide an important new tool to aid in the mapping of genes and associated phenotypes in both species.     

Phospholipase A2 from sheep erythrocyte membrane CA dependence and localization

The calcium dependence and the time course of phosphatidylethanolamine and phosphatidylcholine degradation by sheep erythrocyte membrane suspensions in presence of Triton X-100 were investigated. One enzyme with phospholipase A₂ specificity was found to be responsible for both phosphatidylethanolamine and phosphatidylcholine degradation.The localization of this enzyme in the membrane of the sheep erythrocyte was investigated by proteolytic treatment of sealed erythrocyte ghosts from the outside and of ghosts which had both sides of the membrane exposed to chymotrypsin. The inability of sealed ghosts to take up chymotrypsin was followed by flux measurements of [¹⁴C]dextran carboxyl previously trapped in the ghosts. No efflux of the marker was found during the proteolytic treatment. By comparing the residual phospholipase activities in the membranes from both ghost preparations, we concluded that the phospholipase is oriented to the exterior of the sheep erythrocyte.     

Phospholipase A2 activity in pancreatic islets is calcium-dependent and stimulated by glucose

Phospholipase A₂ activity in islet cell homogenates and dispersed islet cells of the rat was determined using an exogenous radiolabeled phospholipid substrate from $\text{E.}$$\text{coli}$ membranes. Phospholipase A₂ activity in islet homogenates was found to have two pH optima in acid or neutral/alkaline pH ranges. The enzyme activity at pH 7.5 was calcium dependent and responded to increasing calcium concentrations with graded increases in phospholipid hydrolysis. Preincubation of islets with a concentration of glucose known to elicit maximum rates of insulin secretion resulted in a stable activation of phospholipase A₂ activity which was assayable in islet homogenates. Glucose stimulated phospholipase A₂ in these preparations by as much as 220% above control. 2-Deoxy-D-glucose, a nonsecretory analogue of glucose, did not elicit a significant increase in islet phospholipase A₂ activity. The glucose sensitive enzyme was associated with a membrane-enriched subcellular fraction in which the glucose-stimulated activity was greater than 2-fold higher than control activity. Glucose stimulation potentiated the phospholipase A₂ activity measured in the presence of high calcium concentrations. Phospholipase A₂ activity was also found in dispersed islet cell preparations where glucose stimulation of what may be a partly externalized membrane enzyme was most apparent at low calcium concentrations. These data indicate that islet cells possess phospholipase A₂ activity which may be in part localized to the plasma membrane as well as other membrane systems, and which exhibits the characteristic properties of pH and calcium dependency, and sensitivity to secretagogue stimulation reported for the enzyme in other secretory systems.     

Phospholipase A2-derived lysophosphatidylcholine triggers Ca entry in dystrophic skeletal muscle fibers

Duchenne muscular dystrophy is an inherited disease caused by the absence of dystrophin, a structural protein normally located under the sarcolemma of skeletal muscle fibers. Muscle degeneration occurring in this disease is thought to be partly caused by increased Ca²⁺ entry through sarcolemmal cationic channels. Using the Mn²⁺ quench method, we show here that Mn²⁺ entry triggered by Ca²⁺ store depletion but not basal Mn²⁺ entry relies on Ca²⁺-independent PLA₂ (iPLA₂) activity in dystrophic fibers isolated from a murine model of Duchenne muscular dystrophy, the mdx^5cv mouse. iPLA₂ was found to be localized in the vicinity of the sarcolemma and consistently, the iPLA₂ lipid product lysophosphatidylcholine was found to trigger Ca²⁺ entry through sarcolemmal channels, suggesting that it acts as an intracellular messenger responsible for store-operated channels opening in dystrophic fibers. Our results suggest that inhibition of iPLA₂ and lysophospholipid production may be of interest to reduce Ca²⁺ entry and subsequent degeneration of dystrophic muscle.     

YAC Contigs of theRab1andwobbler(wr) Spinal Muscular Atrophy Gene Region on Proximal Mouse Chromosome 11 and of the Homologous Region on Human Chromosome 2p

Despite rapid progress in the physical characterization of murine and human genomes, little molecular information is available on certain regions, e.g., proximal mouse chromosome 11 (Chr 11) and human chromosome 2p (Chr 2p). We have localized thewobblerspinal atrophy genewrto proximal mouse Chr 11, tightly linked toRab1,a gene coding for a small GTP-binding protein, andGlns-ps1,an intronless pseudogene of the glutamine synthetase gene. We have now used these markers to construct a 1.3-Mb yeast artificial chromosome (YAC) contig of theRab1region on mouse Chr 11. Four YAC clones isolated from two independent YAC libraries were characterized by rare-cutting analysis, fluorescencein situhybridization (FISH), and sequence-tagged site (STS) isolation and mapping.Rab1andGlns-ps1were found to be only 200 kb apart. A potential CpG island near a methylatedNarI site and a trapped exon,ETG1.1,were found between these loci, and a new STS,AHY1.1,was found over 250 kb fromRab1.Two overlapping YACs were identified that contained a 150-kb region of human Chr 2p, comprising theRAB1locus,AHY1.1,and the human homologue ofETG1.1,indicating a high degree of conservation of this region in the two species. We mappedAHY1.1and thus humanRAB1on Chr 2p13.4–p14 using somatic cell hybrids and a radiation hybrid panel, thus extending a known region of conserved synteny between mouse Chr 11 and human Chr 2p. Recently, the geneLMGMD2Bfor a human recessive neuromuscular disease, limb girdle muscular dystrophy type 2B, has been mapped to 2p13–p16. The conservation between the mouseRab1and humanRAB1regions will be helpful in identifying candidate genes for thewobblerspinal muscular atrophy and in clarifying a possible relationship betweenwrandLMGMD2B.     

The Mouse Glutathione PeroxidaseGpx2Gene Maps to Chromosome 12; Its PseudogeneGpx2-psMaps to Chromosome 7

TheGPX2gene codes for GSHPx-GI, a glutathione peroxidase whose mRNA is readily detectable in the gastrointestinal tract. AlthoughGPX2is a single gene in humans, there are two genes in the mouse genome with homology toGPX2.By analyzing a panel of mouse interspecies DNA from the Jackson Laboratory's backcross resource, we have chromosomally mapped these two genes. One was mapped to the central region of mouse chromosome 12 betweenD12Mit4andD12Mit5,nearfosandTgfb3.This region is homologous to human 14q24.1, where humanGPX2has been mapped, and most likely represents the functional mouseGpx2gene. The otherGpx2-like gene was mapped to mouse chromosome 7 betweenPcsk3andHbb.We have isolated the latter gene from a P1 phage library. Its pseudogene nature is revealed by the sequence analysis: (a) it is intronless; (b) it has a single nucleotide deletion in the coding region; and (c) it has a poly(A) tail at its 3′-untranslated region.     

Two Distinct P2 Purinergic Receptors, P2Y and P2U, Are Coupled to Phospholipase C in Mouse Pineal Gland Tumor Cells

Abstract: We found that extracellular ATP can increase the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in mouse pineal gland tumor (PGT-β) cells. Studies of the [Ca²⁺]_i rise using nucleotides and ATP analogues established the following potency order: ATP, adenosine 5′-O-(3-thiotriphosphate) ≥ UTP > 2-chloro-ATP > 3′-O-(4-benzoyl)benzoyl ATP, GTP ≥ 2-methylthio ATP, adenosine 5′-O-(2-thiodiphosphate) (ADPβS) > CTP. AMP, adenosine, α,β-methyleneadenosine 5′-triphosphate, β,γ-methyleneadenosine 5′-triphosphate, and UMP had little or no effect on the [Ca²⁺]_i rise. Raising the extracellular Mg²⁺ concentration to 10 mM decreases the ATP-and UTP-induced [Ca²⁺]_i rise, because the responses depend on the ATP^4? and UTP^4? concentrations, respectively. The P_2U purinoceptor-selective agonist UTP and the P_2Y purinoceptor-selective agonist ADPβS induce inositol 1,4,5-trisphosphate generation in a concentration-dependent manner with maximal effective concentrations of ～100 µM. In sequential stimulation, UTP and ADPβS do not interfere with each other in raising the [Ca²⁺]_i. Costimulation with UTP and ADPβS results in additive inositol 1,4,5-trisphosphate generation to a similar extent as is achieved with ATP alone. Pretreatment with pertussis toxin inhibits the action of UTP and ATP by maximally 45–55%, whereas it has no effect on the ADPβS response. Treatment with 1 µM phorbol 12-myristate 13-acetate inhibits the ADPβS-induced [Ca²⁺]_i rise more effectively than the ATP- and UTP-induced responses. These results suggest that P_2U and P_2Y purinoceptors coexist on PGT-β cells and that both receptors are linked to phospholipase C.     

Mice Deficient in Group IV Cytosolic Phospholipase A2 Are Resistant to MPTP Neurotoxicity

Abstract: Phospholipase A₂ (PLA₂) enzymes are critical regulators of prostaglandin and leukotriene synthesis, and they may also play an important role in the generation of intracellular free radicals. The group IV cytosolic form of phospholipase A₂ (cPLA₂) is regulated by changes in intracellular calcium concentration, and the enzyme preferentially acts to release arachidonic acid esterified at the sn -2 position of phospholipids. We examined the susceptibility of mice carrying a targeted mutation of the cPLA₂ gene to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity. Mutant mice have no functional cPLA₂ activity. Mice that were homozygous for the mutation (cPLA₂^−/−) were significantly resistant to MPTP-induced dopamine depletion as compared with littermate control (cPLA₂^+/+) and heterozygous mice (cPLA₂^+/−). These findings provide evidence that cPLA₂ plays a role in MPTP neurotoxicity and suggest that cPLA₂ may play a role in the development of Parkinson's disease in humans.     

The Tight Skin (Tsk) Mutation in the Mouse, a Model for Human Fibrotic Diseases, Is Tightly Linked to the β2-Microglobulin (B2m) Gene on Chromosome 2

The Tsk mutation in the mouse is characterized by the excessive accumulation of collagen in skin and various internal organs, including the heart and lungs. These connective tissue abnormalities are similar to those present in human systemic sclerosis or scleroderma. The Tsk mutation provides an opportunity to investigate, at the molecular level, the pathogenesis of tissue fibrosis. As a first step to cloning the Tsk gene, we report the localization of the Tsk mutation with respect to known molecular markers on mouse chromosome 2. N2 progeny carrying the Tsk mutation were obtained from an intersubspecific backcross of [(C57BL/6-pa +/+ Tsk × Mus castaneus)F1 × M. castaneus ] mice. Genomic DNA from each N2 mouse was subjected to Southern and PCR analyses to identify restriction fragment length polymorphisms and simple sequence length polymorphisms, respectively. Our results refine the location of Tsk to a 3-cM region, eliminate several genes from consideration as the Tsk mutation, identify molecular probes tightly linked with Tsk, and suggest candidate genes responsible for the Tsk phenotype.     

Assignment of the Y4Receptor Gene (PPYR1) to Human Chromosome 10q11.2 and Mouse Chromosome 14

The human and mouse genes for the neuropeptide Y₄receptor have been isolated, sequenced, and shown to contain no introns within the coding region of the gene. Nonisotopicin situhybridization and interspecific mouse backcross mapping have localized the genes to human chromosome 10q11.2 and mouse chromosome 14. Five nucleotide variants, which do not alter the protein sequence, have been identified within the coding region of the human receptor gene. The human Y₄subtype is most closely related to the Y₁-receptor subtype (42%), suggesting that it evolved from an ancestral Y₁-like receptor via an RNA-mediated transpositional event.     

Assignment of the βB1 Crystallin Gene (CRYBB1) to Human Chromosome 22 and Mouse Chromosome 5

By using primers complementary to the rat βB1 crystallin gene sequence, we amplified exons 5 and 6 of the orthologous human gene (CRYBB1). The amplified human segments displayed greater than 88% sequence homology to the corresponding rat and bovine sequences.CRYBB1was assigned to the group 5 region in 22q11.2–q12.1 by hybridizing the exon 6 PCR product to somatic cell hybrids containing defined portions of human chromosome 22. The exon 5 and exon 6 PCR products ofCRYBB1were used to localize, by interspecific backcross mapping, the mouse gene (Crybb1) to the central portion of chromosome 5. Three other β crystallin genes (βB2(−1), βB3, and βA4) have previously been mapped to the same regions in human and mouse. We demonstrate that the βB1 and βA4 crystallin genes are very closely linked in the two species. These assignments complete the mapping and identification of the human and mouse homologues of the major β crystallins genes that are expressed in the bovine lens.