Showing papers by "Nancy A. Jenkins published in 2002"

Human α-synuclein-harboring familial Parkinson's disease-linked Ala-53 → Thr mutation causes neurodegenerative disease with α-synuclein aggregation in transgenic mice

Abstract: Mutations in alpha-synuclein (alpha-Syn) cause Parkinson's disease (PD) in a small number of pedigrees with familial PD. Moreover, alpha-Syn accumulates as a major component of Lewy bodies and Lewy neurites, intraneuronal inclusions that are neuropathological hallmarks of PD. To better understand the pathogenic relationship between alterations in the biology of alpha-Syn and PD-associated neurodegeneration, we generated multiple lines of transgenic mice expressing high levels of either wild-type or familial PD-linked Ala-30 --> Pro (A30P) or Ala-53 --> Thr (A53T) human alpha-Syns. The mice expressing the A53T human alpha-Syn, but not wild-type or the A30P variants, develop adult-onset neurodegenerative disease with a progressive motoric dysfunction leading to death. Pathologically, affected mice exhibit neuronal abnormalities (in perikarya and neurites) including pathological accumulations of alpha-Syn and ubiquitin. Consistent with abnormal neuronal accumulation of alpha-Syn, brain regions with pathology exhibit increases in detergent-insoluble alpha-Syn and alpha-Syn aggregates. Our results demonstrate that the A53T mutant alpha-Syn causes significantly greater in vivo neurotoxicity as compared with other alpha-Syn variants. Further, alpha-Syn-dependent neurodegeneration is associated with abnormal accumulation of detergent-insoluble alpha-Syn.

Identification of an organelle receptor for myosin-Va

Abstract: Little is known about how molecular motors bind to their vesicular cargo. Here we show that myosin-Va, an actin-based vesicle motor, binds to one of its cargoes, the melanosome, by interacting with a receptor-protein complex containing Rab27a and melanophilin, a postulated Rab27a effector. Rab27a binds to the melanosome first and then recruits melanophilin, which in turn recruits myosin-Va. Melanophilin creates this link by binding to Rab27a in a GTP-dependent fashion through its amino terminus, and to myosin-Va through its carboxy terminus. Moreover, this latter interaction, similar to the ability of myosin-Va to colocalize with melanosomes and influence their distribution in vivo, is absolutely dependent on the presence of exon-F, an alternatively spliced exon in the myosin-Va tail. These results provide the first molecular description of an organelle receptor for an actin-based motor, illustrate how alternate exon usage can be used to specify cargo, and further expand the functional repertoire of Rab GTPases and their effectors.

New genes involved in cancer identified by retroviral tagging

Abstract: Retroviral insertional mutagenesis in BXH2 and AKXD mice induces a high incidence of myeloid leukemia and B- and T-cell lymphoma, respectively. The retroviral integration sites (RISs) in these tumors thus provide powerful genetic tags for the discovery of genes involved in cancer. Here we report the first large-scale use of retroviral tagging for cancer gene discovery in the post-genome era. Using high throughput inverse PCR, we cloned and analyzed the sequences of 884 RISs from a tumor panel composed primarily of B-cell lymphomas. We then compared these sequences, and another 415 RIS sequences previously cloned from BXH2 myeloid leukemias and from a few AKXD lymphomas, against the recently assembled mouse genome sequence. These studies identified 152 loci that are targets of retroviral integration in more than one tumor (common retroviral integration sites, CISs) and therefore likely to encode a cancer gene. Thirty-six CISs encode genes that are known or predicted to be genes involved in human cancer or their homologs, whereas others encode candidate genes that have not yet been examined for a role in human cancer. Our studies demonstrate the power of retroviral tagging for cancer gene discovery in the post-genome era and indicate a largely unrecognized complexity in mouse and presumably human cancer.

Dysregulation of T lymphocyte function in itchy mice: a role for Itch in TH2 differentiation.

Abstract: Itch is an E3 ubiquitin ligase that is disrupted in nonagouti-lethal or itchy mice. Itch deficiency leads to severe immune and inflammatory disorders and constant itching of the skin. Here we show that Itch−/− T cells show an activated phenotype and enhanced proliferation. Production of the type 2 T helper (TH2) cell cytokines interleukin 4 (IL-4) and IL-5 by Itch−/− T cells was augmented upon stimulation, and the TH2-dependent serum concentrations of immunoglobulin G1 (IgG1) and IgE in itchy mice were also increased. Molecularly, Itch associated with and induced ubiquitination of JunB, a transcription factor that is involved in TH2 differentiation. These results provide a molecular link between Itch deficiency and the aberrant activation of immune responses in itchy mice.

Synaptic defects in ataxia mice result from a mutation in Usp14 , encoding a ubiquitin-specific protease

Abstract: Mice that are homozygous with respect to a mutation (ax(J)) in the ataxia (ax) gene develop severe tremors by 2-3 weeks of age followed by hindlimb paralysis and death by 6-10 weeks of age. Here we show that ax encodes ubiquitin-specific protease 14 (Usp14). Ubiquitin proteases are a large family of cysteine proteases that specifically cleave ubiquitin conjugates. Although Usp14 can cleave a ubiquitin-tagged protein in vitro, it is unable to process polyubiquitin, which is believed to be associated with the protein aggregates seen in Parkinson disease, spinocerebellar ataxia type 1 (SCA1; ref. 4) and gracile axonal dystrophy (GAD). The physiological substrate of Usp14 may therefore contain a mono-ubiquitin side chain, the removal of which would regulate processes such as protein localization and protein activity. Expression of Usp14 is significantly altered in ax(J)/ax(J) mice as a result of the insertion of an intracisternal-A particle (IAP) into intron 5 of Usp14. In contrast to other neurodegenerative disorders such as Parkinson disease and SCA1 in humans and GAD in mice, neither ubiquitin-positive protein aggregates nor neuronal cell loss is detectable in the central nervous system (CNS) of ax(J) mice. Instead, ax(J) mice have defects in synaptic transmission in both the central and peripheral nervous systems. These results suggest that ubiquitin proteases are important in regulating synaptic activity in mammals.

Mitf and Tfe3, two members of the Mitf-Tfe family of bHLH-Zip transcription factors, have important but functionally redundant roles in osteoclast development

Abstract: The Mitf-Tfe family of basic helix–loop–helix-leucine zipper (bHLH-Zip) transcription factors encodes four family members: Mitf, Tfe3, Tfeb, and Tfec. In vitro, each protein in the family can bind DNA as a homo- or heterodimer with other family members. Mutational studies in mice have shown that Mitf is essential for melanocyte and eye development, whereas Tfeb is required for placental vascularization. Here, we uncover a role for Tfe3 in osteoclast development, a role that is functionally redundant with Mitf. Although osteoclasts seem normal in Mitf or Tfe3 null mice, the combined loss of the two genes results in severe osteopetrosis. We also show that Tfec mutant mice are phenotypically normal, and that the Tfec mutation does not alter the phenotype of Mitf, Tfeb, or Tfe3 mutant mice. Surprisingly, our studies failed to identify any phenotypic overlap between the different Mitf–Tfe mutations. These results suggest that heterodimeric interactions are not essential for Mitf-Tfe function in contrast to other bHLH-Zip families like Myc/Max/Mad, where heterodimeric interactions seem to be essential.

Efficient Cre-loxP-induced mitotic recombination in mouse embryonic stem cells.

Abstract: FLP/FRT-induced mitotic recombination provides a powerful method for creating genetic mosaics in Drosophila and for discerning the function of recessive genes in a heterozygous individual. Here we show that mitotic recombination can be reproducibly induced in mouse embryonic stem (ES) cells, by Cre/loxP technology, at frequencies ranging from 4.2 x 10(-5) (Snrpn) to 7.0 x 10(-3) (D7Mit178) for single allelic loxP sites, and to 5.0 x 10(-2) (D7Mit178) for multiple allelic lox sites, after transient Cre expression. Notably, much of the recombination occurs in G2 and is followed by X segregation, where the recombinant chromatids segregate away from each other during mitosis. It is X segregation that is useful for genetic mosaic analysis because it produces clones of homozygous mutant daughter cells from heterozygous mothers. Our studies confirm the predictions made from studies in Drosophila that suggest that X segregation will not be limited to organisms with strong mitotic pairing, because the forces (sister-chromatid cohesion) responsible for X segregation are an elemental feature of mitosis in all eukaryotes. Our studies also show that genetic mosaic analysis in mice is feasible, at least for certain chromosomal regions.

Differential expression of the seven‐pass transmembrane cadherin genes Celsr1‐3 and distribution of the Celsr2 protein during mouse development

Abstract: Drosophila Flamingo (Fmi) is an evolutionally conserved seven-pass transmembrane receptor of the cadherin superfamily. Fmi plays multiple roles in patterning neuronal processes and epithelial planar cell polarity. To explore the in vivo roles of Fmi homologs in mammals, we previously cloned one of the mouse homologs, mouse flamingo1/Celsr2. Here, we report the results of our study of its embryonic and postnatal expression patterns together with those of two other paralogs, Celsr1 and Celsr3. Celsr1-3 expression was initiated broadly in the nervous system at early developmental stages, and each paralog showed characteristic expression patterns in the developing CNS. These genes were also expressed in several other organs, including the cochlea, where hair cells develop planar polarity, the kidney, and the whisker. The Celsr2 protein was distributed at intercellular boundaries in the whisker and on processes of neuronal cells such as hippocampal pyramidal cells, Purkinje cells, and olfactory neurons. Celsr2 is mapped to a distal region of the mouse chromosome 3. We discussed possible functions of seven-pass transmembrane cadherins in mouse development.

Comparative genetics and evolution of annexin A13 as the founder gene of vertebrate annexins

Abstract: Annexin A13 (ANXA13) is believed to be the original founder gene of the 12-member vertebrate annexin A family, and it has acquired an intestine-specific expression associated with a highly differentiated intracellular transport function. Molecular characterization of this subfamily in a range of vertebrate species was undertaken to assess coding region conservation, gene organization, chromosomal linkage, and phylogenetic relationships relevant to its progenitor role in the structure-function evolution of the annexin gene superfamily. Protein diagnostic features peculiar to this subfamily include an alternate isoform containing a KGD motif, an elevated basic amino acid content with polyhistidine expansion in the 5'-translated region, and the conservation of 15% core tetrad residues specific to annexin A13 members. The 12 coding exons comprising the 58-kb human ANXA13 gene were deduced from BAC clone sequencing, whereas internal repetitive elements and neighboring genes in chromosome 8q24.12 were identified by contig analysis of the draft sequence from the human genome project. A unique exon splicing pattern in the annexin A13 gene was corroborated by coanalysis of mouse, rat, zebrafish, and pufferfish genomic DNA and determined to be the most distinct of all vertebrate annexins. The putative promoter region was identified by phylogenetic footprinting of potential binding sites for intestine-specific transcription factors. Mouse annexin A13 cDNA was used to map the gene to an orthologous linkage group in mouse chromosome 15'(between Sdc2 and Myc by backcross analysis), and the zebrafish cDNA permitted its localization to linkage group 24. Comparative analysis of annexin A13 from nine species traced this gene's speciation history and assessed coding region variation, whereas phylogenetic analysis showed it to be the deepest-branching vertebrate annexin, and computational analysis estimated the gene age and divergence rate. The unique, conserved aspects of annexin A13 primary structure, gene organization, and genetic maps identify it as the probable common ancestor of all vertebrate annexins, beginning with the sequential duplication to annexins' A7 and All 1 approximately 700 MYA, before the emergence of chordates.

Significant Differences Between Mouse and Human Trophinins Are Revealed by Their Expression Patterns and Targeted Disruption of Mouse Trophinin Gene

Abstract: Trophinin has been identified as a membrane protein mediating apical cell adhesion between two human cell lines: trophoblastic HT-H cells, and endometrial epithelial SNG-M cells. Expression patterns of trophinin in humans suggested its involvement in embryo implantation and early placental development. The mouse trophinin gene maps to the distal part of the X chromosome and corresponds to human chromosome Xp11.21-22, the locus where the human trophinin gene maps. Western blot analysis indicates that the molecular weight of mouse trophinin is 110 kDa, which is consistent with the calculated value of 107 kDa. Positive signals for trophinin proteins were detected in preimplantation mouse embryos at the morula and blastocyst stages. Implanting blastocysts do not show detectable levels of trophinin protein, demonstrating that trophinin is not involved in blastocyst adhesion to the uterus in the mouse. Mouse embryo strongly expressed trophinin in the epiblast 1 day after implantation. Trophinin protein was not found in the mouse uteri and placenta after 5.5 days postcoitus (dpc). Targeted disruption of the trophinin gene in the mouse showed a partial embryonic lethality in a 129/SvJ background, but the cause of this lethality remains undetermined. The present study indicates significant differences between mouse and human trophinins in their expression patterns, and it suggests that trophinin is not involved in embryo implantation and placental development in the mouse.

Mmu 16 - Comparative genomic highlights

Abstract: Excitement over the completed human genome sequence may have waned, but the mighty mouse is ready to take center stage. In a lively Perspective, Copeland and colleagues take a look at the mysteries of mouse chromosome 16 ( Mural et al.) and comment on the similarities and differences in the organization of the mouse and human genome.

Molecular cloning of mouse collectin liver 1.

Abstract: Collectins are members of the superfamily of vertebrate C-type lectins that contain a collagen-like region, and are involved in first-line host defense. We earlier cloned and characterized a new kind of collectin, collectin liver 1 (CL-L1). In this study, we isolated the mouse homologue of CL-L1 encoding 277 amino acid residues; its deduced protein sequence was 88% identical with human CL-L1. Mouse CL-L1 mRNA was expressed mainly in the liver and stomach, but was found also in muscles, testes, intestines, and embryos. In mouse embryos, the level of CL-L1 mRNA gradually increased with embryonic age. In 16-day-old mouse embryos, CL-L1 mRNA was expressed in the liver, amnion, and visceral yolk sac. The mouse CL-L1 gene, Cll1 was found on chromosome 15 in a region syntenic with human chromosome 8q. CL-L1 was a highly conserved protein in mammals, birds, and fish.

Chromosomal Localization of the Myelin‐Associated Oligodendrocytic Basic Protein and Expression in the Genetically Linked Neurological Mouse Mutants Ducky and Tippy

Abstract: The alternatively spliced cDNAs encoding the myelin-associated/oligodendrocytic basic proteins (MOBPs) have recently been identified in rat. The Mobp gene maps to the distal part of mouse chromosome 9 at a region syntenic with the human chromosome 3p22-p21.3. Two nonallelic mouse mutants, tippy and ducky, with severe neurological phenotypes map to the vicinity of the Mobp locus. We therefore tested whether MOBP malfunction could explain the tippy and ducky defects. In tippy mutant animals, MOBP expression and that of other myelin markers were indistinguishable from wild type. The ultrastructure of tippy myelin was shown to be normal. Ducky animals showed a slight reduction of the brain size, most evident in the spinal cord, but normal progress of myelination. Both MOBP and myelin basic protein expression were lowered only regionally in the CNS, but were mostly normal in the anterior parts of the brain. Ultrastructurally, ducky myelin appeared normal. MOBP transcript sizes and the molecular weights of the encoded proteins were shown to be normal in both mutants. Finally, the nucleotide sequence of the abundant MOBP-81 cDNA was determined and compared with tippy and ducky MOBP-81. Wild-type mouse MOBP-81 protein was 99% identical to the rat homologue, and tippy and ducky MOBP-81 were identical to the wild-type sequence. Our results suggest that alterations in the Mobp gene are not the cause for the severe neurological phenotypes of ducky and tippy mice.

Murine NFX.1: isolation and characterization of its messenger RNA, mapping of its chromosomal location and assessment of its developmental expression.

Abstract: We have previously isolated (by expression cloning) a human cDNA, termed NFX.1, encoding a nucleic acid-binding protein that interacts with the conserved X1 box cis-element first discovered in class II major histocompatibility complex (MHC) genes. Functional studies involving expression of NFX.1 and assessment of expression from class II reporter constructs and endogenous class II MHC genes indicated that the factor could repress transcription of class II MHC genes. Subsequent studies have extended the biological significance of the factor, indicating that it plays an important role in neuronal development. Indeed, the reiterated RING finger motifs in the central domain of the polypeptide strongly suggest that NF-XI is a probable E3 ubiquitin protein ligase, indicating that the protein may have multiple activities. Here we report the cloning of the mouse homologue of the human NfX.1 cDNA: m-Nfx.1. Comparison of the deduced primary sequence of mouse and human NFX.1 proteins shows very high homology and confirms that m-NFX.1 contains the conserved cysteine-rich DNA-binding motif first described in human NFX.1 (95% homology). Expression of MHC class II genes is substantially reduced following expression of m-NFX.1, which confirms that we have isolated the functional murine homologue of human NfX.1 cDNA. Further evidence comes from the mapping of m-Nfx.1 gene to the proximal region of mouse chromosome 4, a region syntenic to the location of human Nfx.1 (short arm of chromosome 9). Expression profiling shows that m-NFX.1 is expressed ubiquitously in both adult tissues and during development, supporting the hypothesis that it may have yet-undescribed roles in distinct biological processes.

F-MuLV acceleration of myelomonocytic tumorigenesis in SV40 large T antigen transgenic mice is accompanied by retroviral insertion at Fli1 and a novel locus, Fim4

Abstract: We describe here the development of a murine system for the identification of genes involved in myelomonocytic neoplasms. Transgenic C57BL/6J mice expressing SV40 early region under a myelomonocytic promoter develop histiocytic sarcomas with a latency of 167 days. We used retroviral proviral tagging to accelerate tumorigenesis and to uncover genetic changes that contribute to tumor development. Infection of transgenic mice with Friend murine leukemia virus (F-MuLV) shortened the latency of morbidity to 103 days (P< 0.001); this was associated with clonal proviral integrations in tumor DNA. As expected for F-MuLV, proviral insertions occurred at Fli1 in both transgenic and nontransgenic tumors. Four insertions were found at a novel locus, termed Fim4, on chromosome 6. This region is syntenic to human 7q32, a region that is commonly deleted in human myelodysplastic syndrome and acute myeloid leukemia. A murine BAC containing Fim4 was sequenced and analyzed, and while there was significant human-mouse homology in the area of the insertions, no candidate gene has been identified. Thus we have established a system to identify genes involved in myelomonocytic tumors, and have used it to identify Fim4, a new common site of proviral insertion. Study of this locus may provide insight into genes involved in AML-associated 7q32 deletions in humans.

Erratum: New genes involved in cancer identified by retroviral tagging

Abstract: Nature Genet. 32, 166–174 (2002). By error, the subpanel labels for Fig. 1 on page 172 were offset to the right of the corresponding subpanels.