TTLL1 and TTLL4 polyglutamylases are required for the neurodegenerative phenotypes in pcd mice

Polyglutamylation is a dynamic posttranslational modification where glutamate residues are added to substrate proteins by 8 tubulin tyrosine ligase-like (TTLL) family members (writers) and removed by the 6 member Nna1/CCP family of carboxypeptidases (erasers). Genetic disruption of polyglutamylation leading to hyperglutamylation causes neurodegenerative phenotypes in humans and animal models; the best characterized being the Purkinje cell degeneration (pcd) mouse, a mutant of the gene encoding Nna1/CCP1, the prototypic eraser. Emphasizing the functional importance of the balance between glutamate addition and elimination, loss of TTLL1 prevents Purkinje cell degeneration in pcd. However, whether Ttll1 loss protects other vulnerable neurons in pcd, or if elimination of other TTLLs provides protection is largely unknown. Here using a mouse genetic rescue strategy, we characterized the contribution of Ttll1, 4, 5, 7, or 11 to the degenerative phenotypes in cerebellum, olfactory bulb and retinae of pcd mutants. Ttll1 deficiency attenuates Purkinje cell loss and function and reduces olfactory bulb mitral cell death and retinal photoreceptor degeneration. Moreover, degeneration of photoreceptors in pcd is preceded by impaired rhodopsin trafficking to the rod outer segment and likely represents the causal defect leading to degeneration as this too is rescued by elimination of TTLL1. Although TTLLs have similar catalytic properties on model substrates and several are highly expressed in Purkinje cells (e.g. TTLL5 and 7), besides TTLL1 only TTLL4 deficiency attenuated degeneration of Purkinje and mitral cells in pcd. Additionally, TTLL4 loss partially rescued photoreceptor degeneration and impaired rhodopsin trafficking. Despite their common properties, the polyglutamylation profile changes promoted by TTLL1 and TTLL4 deficiencies in pcd mice are very different. We also report that loss of anabolic TTLL5 synergizes with loss of catabolic Nna1/CCP1 to promote photoreceptor degeneration. Finally, male infertility in pcd is not rescued by loss of any Ttll. These data provide insight into the complexity of polyglutamate homeostasis and function in vivo and potential routes to ameliorate disorders caused by disrupted polyglutamylation.     

Comparison of the Enzymatic and Functional Properties of Three Cytosolic Carboxypeptidase Family Members

Nna1 (CCP1) defines a subfamily of M14 metallocarboxypeptidases (CCP1–6) and is mutated in pcd (Purkinje cell degeneration) mice. Nna1, CCP4, and CCP6 are involved in the post-translational process of polyglutamylation, where they catalyze the removal of polyglutamate side chains. However, it is unknown whether these three cytosolic carboxypeptidases share identical enzymatic properties and redundant biological functions. We show that like Nna1, purified recombinant CCP4 and CCP6 deglutamylate tubulin, but unlike Nna1, neither rescues Purkinje cell degeneration in pcd mice, indicating that they do not have identical functions. Using biotin-based synthetic substrates, we established that the three enzymes are distinguishable based upon individual preferences for glutamate chain length, the amino acid immediately adjacent to the glutamate chain, and whether their activity is enhanced by nearby acidic amino acids. Nna1 and CCP4 remove the C-terminal glutamate from substrates with two or more glutamates, whereas CCP6 requires four or more glutamates. CCP4 behaves as a promiscuous glutamase, with little preference for chain length or neighboring amino acid composition. Besides glutamate chain length dependence, Nna1 and CCP6 exhibit higher k_cat/K_m when substrates contain nearby acidic amino acids. All cytosolic carboxypeptidases exhibit a monoglutamase activity when aspartic acid precedes a single glutamate, which, together with their other individual preferences for flanking amino acids, greatly increases the potential substrates for these enzymes and the biological processes in which they act. Additionally, Nna1 metabolized substrates mimicking the C terminus of tubulin in a way suggesting that the tyrosinated form of tubulin will accumulate in pcd mice.     

Serotonin fiber innervation of cerebellar cell suspensions intraparenchymally grafted to the cerebellum ofpcd mutant mice

One aspect of integration of implanted neurons into the neuronal circuitry of a defective host brain is the re-establishment of a host-to-graft afferent innervation. We addressed this issue by using the adult cerebellum of Purkinje cell degeneration (pcd) mutant mice, which lack virtually all Purkinje cells after postnatal day (P) 45. Purkinje cells constitute one of the cerebellar cell types being innervated by axons of raphé serotonin (5-HT) neurons. In normal mice, 5-HT-immunoreactive fibers are distributed to all cerebellar folia. Following Purkinje cell loss inpcd mice, cerebellar 5-HT-immunoreactive fibers persist. Cerebellar cell suspensions were prepared from embryonic day (E) 11–13 normal mouse embryos and were intraparenchymally grafted into the cerebellum ofpcd mutants either directly or after pre-treatment with 5, 7-dihydroxytryptamine (5,7-DHT) to selectively remove 5-HT cells of donor origin. The state of Purkinje cells and 5-HT axons was monitored in alternate sections by 28-kDa Ca²⁺-binding protein (CaBP) and 5-HT immunocytochemistry, respectively. Serotonin-immunoreactive axons were seen in the grafts from 5 to 32 days after transplantation. In some of the grafts which had not been pre-treated with 5,7-DHT, a small number of 5-HT-immunoreactive cell bodies was found, indicating that part of the 5-HT fiber innervation of the graft could actually derive from donor cells. On the other hand, in grafts pre-treated with 5,7-DHT, no 5-HT cell bodies were seen in the grafted cerebellum; 5-HT fibre innervation of the grafts occurred, but it appeared to be slightly less robust compared to situations of co-grafted 5-HT cell bodies. These findings suggest that host 5-HT fibers are able to provide afferent innervation to donor cerebellar tissue; the presence of co-grafted 5-HT cells may augment such an innervation.Special issue dedicated to Dr. Morris H. Aprison.     

Stable expression of a single-copy rolA gene in transgenic Arabidopsis thaliana plants allows an exhaustive mutagenic analysis of the transgene-associated phenotype

Several publications have documented the instability of transgene expression in plants. Previous genetic approaches to the study of transgene-associated phenotypes in plants were limited by this phenomenon. Here we show that a transgene can be expressed in plants with sufficient stability to allow an exhaustive mutagenic analysis of the resulting phenotype. We have expressed the morphogenic rolA gene from the TL-DNA of Agrobacterium rhizogenes Ri plasmid in transgenic Arabidopsis thaliana plants. The resulting pleiotropic RolA phenotype allows a visual screen for reversion to detect germinal as well as somatic instability of transgene expression. However no spontaneous reversions of the RolA phenotype were observed in 65 000 progeny of two independent transgenic A. thaliana lines, each carrying a single homozygous rolA locus. In contrast, 12 revertants of the RolA phenotype were isolated from 360000 ethyl methane sulphonate (EMS)-mutagenized M2 progeny. All revertants were shown genetically to carry stable recessive mutations in the rolA locus, thus establishing a series of loss-of-function alleles. Molecular characterization revealed that the loss-of-function alleles were structurally intact and expressed in all rolA mutants. A wild-type rolA locus and two loss-of-function alleles were reisolated and sequenced; base pair substitutions were found in each loss-of-function allele leading to single amino acid substitutions in the rolA open reading frame. Therefore no instability of expression of the rolA locus was detected in any of the 425 000 individuals studied in this analysis. Furthermore even under conditions of saturation mutagenesis, no extragenic suppressor locus was detected.     

Novel mutations affecting the Na,K ATPase alpha model complex neurological diseases and implicate the sodium pump in increased longevity

Mutations affecting the Na⁺, K⁺ ATPase alpha subunit have been implicated in at least two distinct human diseases, rapid-onset dystonia Parkinsonism (RDP), and familial hemiplegic migraine (FHM). Over 40 mutations have been mapped to the human ATP1A2 and ATP1A3 genes and are known to result in RDP, FHM or a variant of FHM with neurological complications. To develop a genetically tractable model system for investigating the role of the Na⁺, K⁺ ATPase in neural pathologies we performed genetic screens in Drosophila melanogaster to isolate loss-of-function alleles affecting the Na⁺, K⁺ ATPase alpha subunit. Flies heterozygous for these mutations all exhibit reduced respiration, consistent with a loss-of-function in the major ATPase. However, these mutations do not affect all functions of the Na⁺, K⁺ ATPase alpha protein since embryos homozygous for these mutations have normal septate junction paracellular barrier function and tracheal morphology. Importantly, all of these mutations cause neurological phenotypes and, akin to the mutations that cause RDP and FHM, these new alleles are missense mutations. All of these alleles exhibit progressive stress-induced locomotor impairment suggesting neuromuscular dysfunction, yet neurodegeneration is observed in an allele-specific manner. Surprisingly, studies of longevity demonstrate that mild hypomorphic mutations in the sodium pump significantly improve longevity, which was verified using the Na⁺, K⁺ ATPase antagonist ouabain. The isolation and characterization of a series of new missense alleles of ATPalpha in Drosophila provides the foundation for further studies of these neurological diseases and the role of sodium pump impairment in animal longevity.     

Lack of Cytosolic Carboxypeptidase 1 Leads to Subfertility due to the Reduced Number of Antral Follicles in pcd3J-/- Females

Females homozygous for the Purkinje cell degeneration mutation (pcd) are fertile, although the success rate is much lower than in the wild type. We performed detailed analysis of reproductive abnormalities of pcd females. The number of oocytes produced following exogenous gonadotropin treatment was much lower in pcd ^3J-/- females than in pcd ^3J+/+ females. Furthermore, the estrous cyclicity of pcd ^3J-/- females according to the appearance of the vagina was almost undetectable comparing to that of the wild type. Histological analyses and follicle counting of 4- and 8-week-old pcd ^3J-/- ovaries showed an increase in the number of secondary follicles and a decrease in the number of antral follicles, indicating that AGTPBP1/ CCP1 plays an important role in the development of secondary follicles into antral follicles. Consistent with a previous analysis of the pcd cerebellum, pcd ^3J-/- ovaries also showed a clear increase in the level of polyglutamylation. Gene expression analysis showed that both oocytes and cumulus cells express CCP1. However, Ccp4 and CCP6, which can compensate the function of CCP1, were not expressed in mouse ovaries. Failure of microtubule deglutamylation did not affect the structure and function of the meiotic spindle in properly aligning chromosomes in the center of the nucleus during meiosis in pcd ^3J-/- females. We also showed that the pituitary-derived growth and reproduction-related endocrine system functions normally in pcd ^3J-/- mice. The results of this study provide insight into additional functions of CCP1, which cannot be fully explained by the side chain deglutamylation of microtubules alone.     

Analysis of a zebrafish behavioral mutant reveals a dominant mutation in atp2a1/SERCA1

Zebrafish embryos demonstrate robust swimming behavior, which consists of smooth, alternating body bends. In contrast, several motility mutants have been identified that perform sustained, bilateral trunk muscle contractions which result in abnormal body shortening. Unlike most of these mutants, accordion (acc)^dta5 demonstrates a semidominant effect: Heterozygotes exhibit a distinct but less severe phenotype than homozygotes. Using molecular‐genetic mapping and candidate gene analysis, we determined that acc^dta5 mutants harbor a novel mutation in atp2a1, which encodes SERCA1, a calcium pump important for muscle relaxation. Previous studies have shown that eight other acc alleles compromise SERCA1 function, but these alleles were all reported to be recessive. Quantitative behavioral assays, complementation testing, and analysis of molecular models all indicate that the acc^dta5 mutation diminishes SERCA1 function to a greater degree than other acc alleles through either haploinsufficient or dominant‐negative molecular mechanisms. Since mutation of human ATP2A1 results in Brody disease, an exercise‐induced impairment of muscle relaxation, acc^dta5 mutants may provide a particularly sensitive model of this disorder. genesis, 48:354–361, 2010. © 2010 Wiley‐Liss, Inc.     

Stable expression of a single-copy rolA gene in transgenic Arabidopsis thaliana plants allows an exhaustive mutagenic analysis of the transgene-associated phenotype

Several publications have documented the instability of transgene expression in plants. Previous genetic approaches to the study of transgene-associated phenotypes in plants were limited by this phenomenon. Here we show that a transgene can be expressed in plants with sufficient stability to allow an exhaustive mutagenic analysis of the resulting phenotype. We have expressed the morphogenic rolA gene from the TL-DNA of Agrobacterium rhizogenes Ri plasmid in transgenic Arabidopsis thaliana plants. The resulting pleiotropic RolA phenotype allows a visual screen for reversion to detect germinal as well as somatic instability of transgene expression. However no spontaneous reversions of the RolA phenotype were observed in 65 000 progeny of two independent transgenic A. thaliana lines, each carrying a single homozygous rolA locus. In contrast, 12 revertants of the RolA phenotype were isolated from 360000 ethyl methane sulphonate (EMS)-mutagenized M2 progeny. All revertants were shown genetically to carry stable recessive mutations in the rolA locus, thus establishing a series of loss-of-function alleles. Molecular characterization revealed that the loss-of-function alleles were structurally intact and expressed in all rolA mutants. A wild-type rolA locus and two loss-of-function alleles were reisolated and sequenced; base pair substitutions were found in each loss-of-function allele leading to single amino acid substitutions in the rolA open reading frame. Therefore no instability of expression of the rolA locus was detected in any of the 425 000 individuals studied in this analysis. Furthermore even under conditions of saturation mutagenesis, no extragenic suppressor locus was detected.     

Absence of BiP Co-chaperone DNAJC3 Causes Diabetes Mellitus and Multisystemic Neurodegeneration

Diabetes mellitus and neurodegeneration are common diseases for which shared genetic factors are still only partly known. Here, we show that loss of the BiP (immunoglobulin heavy-chain binding protein) co-chaperone DNAJC3 leads to diabetes mellitus and widespread neurodegeneration. We investigated three siblings with juvenile-onset diabetes and central and peripheral neurodegeneration, including ataxia, upper-motor-neuron damage, peripheral neuropathy, hearing loss, and cerebral atrophy. Exome sequencing identified a homozygous stop mutation in DNAJC3. Screening of a diabetes database with 226,194 individuals yielded eight phenotypically similar individuals and one family carrying a homozygous DNAJC3 deletion. DNAJC3 was absent in fibroblasts from all affected subjects in both families. To delineate the phenotypic and mutational spectrum and the genetic variability of DNAJC3, we analyzed 8,603 exomes, including 506 from families affected by diabetes, ataxia, upper-motor-neuron damage, peripheral neuropathy, or hearing loss. This analysis revealed only one further loss-of-function allele in DNAJC3 and no further associations in subjects with only a subset of the features of the main phenotype. Our findings demonstrate that loss-of-function DNAJC3 mutations lead to a monogenic, recessive form of diabetes mellitus in humans. Moreover, they present a common denominator for diabetes and widespread neurodegeneration. This complements findings from mice in which knockout of Dnajc3 leads to diabetes and modifies disease in a neurodegenerative model of Marinesco-Sjögren syndrome.     

Scd1 ab-Xyk : a new asebia allele characterized by a CCC trinucleotide insertion in exon 5 of the stearoyl-CoA desaturase 1 gene in mouse

We describe here a spontaneous, autosomal recessive mutant mouse suffering from skin and hair defects, which arose in the outbred Kunming strain. By haplotype analysis and direct sequencing of PCR products, we show that this mutation is a new allele of the asebia locus with a naturally occurring mutation in the Scd1 gene (a CCC insertion at nucleotide position 835 in exon 5), which codes for stearoyl-CoA desaturase 1. This mutation introduces an extra proline residue at position 279 in the Scd1 protein. The mutant mice, originally designated km/km but now assigned the name Scd1 ^ab-Xyk (hereafter abbreviated as ab ^Xyk / ab ^Xyk ), have a similar gross and histological phenotype to that reported for previously characterized allelic asebia mutations ( Scd1 ^ab , Scd1 ^abJ , Scd1 ^ab2J , and Scd1 ^tm1Ntam ). Histological analysis showed they were also characterized by hypoplasic sebaceous glands and abnormal hair follicles. In a cross between Kunming- ab ^Xyk / ab ^Xyk and ABJ/Le- ab ^J / ab ^J mice, all the progeny showed the same phenotype, indicating that the two mutations were non-complementing and therefore allelic. Comparisons with the other four allelic mutants indicate that the Scd1 ^ab-Xyk mutation causes the mildest change in Scd1 function. This new mouse mutant is a good model not only for the study of scarring alopecias in humans, which are characterized by hypoplasic sebaceous glands, but also for studying the structure and function of the Scd1 protein.Communicated by G. ReuterThe first two authors contribute equally to this work     

The mouse mutants <Emphasis Type="Italic">recoil wobbler</Emphasis> and <Emphasis Type="Italic">nmf373</Emphasis> represent a series of <Emphasis Type="Italic">Grm1</Emphasis> mutations

The identification of novel mutant alleles is important for understanding critical functional domains of a protein and establishing genotype:phenotype correlations. The recoil wobbler (rcw) allelic series of spontaneous ataxic mutants and the ENU-induced mutant nmf373 genetically mapped to a shared region of chromosome 10. Their mutant phenotypes are strikingly similar; all have an ataxic phenotype that is recessive, early-onset, and is not associated with neurodegeneration. In this study we used complementation tests to show that these series of mutants are allelic to a knockout mutant of Grm1. Subsequently, a duplication of exon 4 and three missense mutations were identified in Grm1: I160T, E292D, and G337E. All mutations occurred within the ligand-binding region and changed conserved amino acids. In the rcw mutant, the Grm1 gene is expressed and the protein product is properly localized to the molecular layer of the cerebellar cortex. Grm1 is responsible for the generation of inositol 1,4,5-trisphosphate (IP₃). The inositol second messenger system is the central mechanism for calcium release from intracellular stores in cerebellar Purkinje cells. Several of the genes involved in this pathway are mutated in mouse ataxic disorders. The novel rcw mutants represent a resource that will have utility for further studies of inositol second-messenger-system defects in neurogenetic disorders.     

Regional mutagenesis using Dissociation in maize

We describe genetic screens, molecular methods and web resources newly available to utilize Dissociation (Ds) as an insertional mutagen in maize. Over 1700 Ds elements have been distributed throughout the maize genome to serve as donor elements for local or regional mutagenesis. Two genetic screens are described to identify Ds insertions in genes-of-interest (goi). In scheme I, Ds is used to generate insertion alleles when a recessive reference allele is available. A Ds insertion will enable the cloning of the target gene and can be used to create an allelic series. In scheme II, Ds insertions in a goi are identified using a PCR-based screen to identify the rare insertion alleles among a population of testcross progeny. We detail an inverse PCR protocol to rapidly amplify sequences flanking Ds insertion alleles and describe a high-throughput 96-well plate-based DNA extraction method for the recovery of high-quality genomic DNA from seedling tissues. We also describe several web-based tools for browsing, searching and accessing the genetic materials described. The development of these Ds insertion lines promises to greatly accelerate functional genomics studies in maize.     

High‐resolution insight into recombination events at the SD1 locus in rice

Recombination during meiosis plays an important role in genome evolution by reshuffling existing genetic variations into fresh combinations with the possibility of recovery of lost ancestral genotypes. While crossover (CO) events have been well studied, gene conversion events (GCs), which represent non‐reciprocal information transfer between chromosomes, are poorly documented and difficult to detect due to their relatively small converted tract size. Here, we document these GC events and their phenotypic effects at an important locus in rice containing the SD1 gene, where multiple defective alleles contributed to the semi‐dwarf phenotype of rice in the ‘Green Revolution’ of the 1960s. Here, physical separation of two defects allows recombination to generate the wild‐type SD1 gene, for which plant height can then be used as a reporter. By screening 18 000 F₂ progeny from a cross between two semi‐dwarf cultivars that carry these different defective alleles, we detected 24 GC events, indicating a conversion rate of ~3.3 × 10^?4 per marker per generation in a single meiotic cycle in rice. Furthermore, our data show that indels and single‐nucleotide polymorphisms (SNPs) do not differ significantly in GC rates, at least at the SD1 locus. Our results provide strong evidence that GC by itself can regain an ancestral phenotype that was lost through mutation. This GC detection approach is likely to be broadly applicable to natural or artificial alleles of other phenotype‐related functional genes, which are abundant in other plant genomes.     

Molecular Basis for the rhino (hr)Phenotype: A Nonsense Mutation in the MousehairlessGene

The hairless (hr) and rhino (hr^rh) mutations are autosomal recessive allelic mutations that map to mouse Chromosome 14. Both hairless and rhino mice have a number of skin and nail abnormalities and develop a striking form of total alopecia at approximately 3–4 weeks of age. The molecular basis of the hairless mouse phenotype was previously found to be the result of a murine leukemia proviral insertion in intron 6 of thehrgene that resulted in aberrant splicing. In this study, we report a 2-bp substitution in exon 4 of thehrgene in a second allele ofhr,rhino 8J (hr^rh-8J), leading to a nonsense mutation. These findings document the molecular basis of the rhino phenotype for the first time and suggest that rhino is a functional knock-out of thehrgene.     

Drosophila purine auxotrophy: New alleles ofadenosine2 exhibiting a complex visible phenotype

New mutant alleles of theadenosine2 locus (ade2; 2–17.7) have been isolated using the eye-color phenotype exhibited by the prototype auxotrophic alleleade2 ¹ as the screening criterion. The new mutants form a single complementation group, suggesting that they all exhibit purine auxotrophy and defective formylglycineamide ribotide amidotransferase enzyme, likeade2 ¹. Tests carried out on particular new alleles confirm these suggestions. The new mutants all exhibit more extreme physical defects than the prototype. They have wing abnormalities like mutants defective in pyrimidine biosynthesis and reduced bristles like those defective in protein synthesis; thus they exhibit the combined visible phenotype ofrudimentary wings,rosy eyes, andbobbed bristles. Cytogenetic analysis places the locus in the interband proximal to26B1-2.This work was supported by NSERC Operating Grant A3269 to D.N., an Alberta Heritage Foundation for Medical Research Postdoctoral Fellowship to S.Y.K.T., and National Institute on Aging Grant AG00029 to D.P.     

Selection of human cells having two different types of mutations in individual cells (genetic/artificial mutants)

Summary We have previously reported the establishment and characterization of B cell lines from patients and family members with various types of adenine phosphoribosyltransferase (APRT) deficiencies. These cell lines contain, at the APRT locus, three different alleles (APRT ^* 1, APRT ^* Q0, and APRT ^* J) that are clearly distinguishable from each other. From five genetically heterozygous cell lines with two different genotypes (APRT ^* 1/APRTQ0 and APRT ^* 1/APRT ^* J), we have selected 48 clones resistant to 2,6-diaminopurine. Resistance to this adenine analogue is a characteristic of cells having defects in both of the APRT alleles in individual cells. The mutant clones from a cell line from a complete-type heterozygote had APRT activities close to zero (mean=0.04 nmol/min per milligram protein) in the cell extracts, while 15 clones from four cell lines from the four Japanese-type heterozygotes had significant enzyme activities (mean=3.88 nmol/min per milligram protein). Kinetic studies on two of the mutants from two Japancse-type heterozygous cell lines have shown that affinity to substrate 5-phosphoribosyl-1-pyrophosphate was reduced, indicating that APRT in those clones reflected the characteristics of the Japanese-type enzyme. The data presented here indicate that clones we obtained are genetic/artificial mutants, each having a genetic mutation in a single allele (APRT ^* J or APRT ^* Q0) and an artificially produced mutation in the other previously functional allele (APRT ^*1). The present procedure provided the only diagnostic method for Japanese-type APRT heterozygotes (APRT ^* 1/APRT ^* J).     

Suppression of distinct ovo phenotypes in the Drosophila female germline by maleless− and Sex-lethalM

Mutations in ovo result in several different phenotypes, which we show are due to the regulation of distinct developmental pathways. Two X (female) germ cells require ovo⁺ activity for viability, but 1X (male) germ cells do not. In our study, we observed suppression of the ovo germline-lethality phenotype in loss-of-function maleless (mle) females indicating that ovo⁺ and mle⁺ have opposing effects in female germ cells; or that they are hierarchically related. Gain-of-function Sex-lethal (Sxl) alleles and male specific lethal-2 alleles did not suppress the ovo germline death phenotype. Many of the surviving germ cells in females mutant for both ovo and mle showed ovarian tumors. In contrast to the germline viability phenotype, we did observe suppression of the tumor phenotype in females heterozygous for gain-of-function alleles of Sxl. Further, females mutant for some hypomorphic ovo alleles were rendered fertile by Sxl gain-of-function alleles. Thus, ovo⁺ is required for at least two distinct functions, one involving mle⁺, and one mediated by Sxl⁺ gene products. The existence of ovo⁺ functions independent of mle⁺ and Sxl⁺ is likely. Dev. Genet. 23:335–346, 1998. © 1998 Wiley-Liss, Inc.     

Quantitative Peptidomics of Purkinje Cell Degeneration Mice

Cytosolic carboxypeptidase 1 (CCP1) is a metallopeptidase that removes C-terminal and side-chain glutamates from tubulin. The Purkinje cell degeneration (pcd) mouse lacks CCP1 due to a mutation. Previously, elevated levels of peptides derived from cytosolic and mitochondrial proteins were found in adult pcd mouse brain, raising the possibility that CCP1 functions in the degradation of intracellular peptides. To test this hypothesis, we used a quantitative peptidomics technique to compare peptide levels in wild-type and pcd mice, examining adult heart, spleen, and brain, and presymptomatic 3 week-old amygdala and cerebellum. Contrary to adult mouse brain, young pcd brain and adult heart and spleen did not show a large increase in levels of intracellular peptides. Unexpectedly, levels of peptides derived from secretory pathway proteins were altered in adult pcd mouse brain. The pattern of changes for the intracellular and secretory pathway peptides in pcd mice was generally similar to the pattern observed in mice lacking primary cilia. Collectively, these results suggest that intracellular peptide accumulation in adult pcd mouse brain is a secondary effect and is not due to a role of CCP1 in peptide turnover.     

Mapping of genetic modifiers affecting the eye phenotype of ocular retardation (Chx10 or-J ) mice

Ocular retardation is a recessive murine mutation whose phenotypic expression is greatly affected by genetic background effects. Mice of the inbred 129/SvJ background that are homozygous for the Chx10^or-J mutation are blind and have a thin, poorly differentiated retina and no optic nerve. A backcross between 129/SvJ and Mus musculus castaneus (CASA/Rk) produced animals that were homozygous for the Chx10^or-J mutation, yet showed a much milder phenotype. Such animals, when brother-sister mated and selected for mild phenotype for several generations, resulted in partial recovery of visual function, including presence of an optic nerve and pupillary response. In this article we report a genome scan of phenotypic extremes of the backcross to identify the genetic loci affecting this phenotype modification. Our scan revealed significant loci on Chromosomes 6 and 14 where the CASA/Rk alleles are maintained selectively. Markers were developed near candidate genes, but no candidate gene could be identified unequivocally. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users.     

Two loss-of-function alleles of the glutathione S-transferase (GST) gene cause anthocyanin deficiency in flower and fruit skin of peach (Prunus persica)

Flower and fruit colors are important agronomic traits. To date, there is no forward genetic evidence that the glutathione S-transferase (GST) gene is responsible for the white flower color in peach (Prunus persica). In this study, genetic analysis indicated that the white-flower trait is monogenetic, is recessive to the non-white allele, and shows pleiotropic effects with non-white-flowered types. The genetic locus underpinning this trait was mapped onto chromosome 3 between 0.421951 and 3.227115 Mb by using bulked segregant analysis in conjunction with whole-genome sequencing, and was further mapped between 0 and 1.178149 Mb by using the backcross 1 (BC₁) population. Finally, the locus was fine-mapped within 535.974- and 552.027-kb intervals by using 151 F₂ individuals and 75 individuals from a BC₁ self-pollinated (BC₁S₁) population, respectively. Pp3G013600, encoding a GST that is known to transport anthocyanin, was identified within the mapping interval. The analysis of genome sequence data showed Pp3G013600 in white flowers has a 2-bp insertion or a 5-bp deletion in the third exon. These variants likely render the GST non-functional because of early stop codons that reduce the protein length from 215 amino acids to 167 and 175 amino acids, respectively. Genetic markers based on these variants validated a complete correlation between the GST loss-of-function alleles and white flower in 128 peach accessions. This correlation was further confirmed by silencing of Pp3G013600 using virus-induced gene silencing technology, which reduced anthocyanin accumulation in peach fruit. The new knowledge from this study is useful for designing peach breeding programs to generate cultivars with white flower and fruit skin.