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Sample records for mouse meiotic genes

  1. Analysis of the gene expression profile of mouse male meiotic germ cells.

    PubMed

    Rossi, Pellegrino; Dolci, Susanna; Sette, Claudio; Capolunghi, Federica; Pellegrini, Manuela; Loiarro, Maria; Di Agostino, Silvia; Paronetto, Maria Paola; Grimaldi, Paola; Merico, Daniele; Martegani, Enzo; Geremia, Raffaele

    2004-05-01

    Wide genome analysis of difference in gene expression between spermatogonial populations from 7-day-old mice and pachytene spermatocytes from 18-day-old mice was performed using Affymetrix gene chips representing approximately 12,500 mouse known genes or EST sequences, spanning approximately 1/3rd of the mouse genome. To delineate differences in the profile of gene expression between mitotic and meiotic stages of male germ cell differentiation, expressed genes were grouped in functional clusters. The analysis confirmed the previously described pre-meiotic or meiotic expression for several genes, in particular for those involved in the regulation of the mitotic and meiotic cell cycle, and for those whose transcripts are accumulated during the meiotic stages to be translated later in post-meiotic stages. Differential expression of several additional genes was discovered. In few cases (pro-apoptotic factors Bak, Bad and Bax), data were in conflict with the previously published stage-dependent expression of genes already known to be expressed in male germ cells. Northern blot analysis of selected genes confirmed the results obtained with the microarray chips. Six of these were novel genes specifically expressed in pachytene spermatocytes: a chromatin remodeling factor (chrac1/YCL1), a homeobox gene (hmx1), a novel G-coupled receptor for an unknown ligand (Gpr19), a glycoprotein of the intestinal epithelium (mucin 3), a novel RAS activator (Ranbp9), and the A630056B21Rik gene (predicted to encode a novel zinc finger protein). These studies will help to delineate the global patterns of gene expression characterizing male germ cell differentiation for a better understanding of regulation of spermatogenesis in mammals.

  2. The SMAGE gene family is expressed in post-meiotic spermatids during mouse germ cell differentiation

    SciTech Connect

    Chomez, P.; Williams, R.; Vennstroem, B.

    1996-06-01

    The human melanoma cell line MZ2-MEL expresses several tumor antigens defined in vitro by autologous cytolytic T lymphocytes. One of these antigens, MZ2-E, has been identified as a nonapeptide encoded by the MAGE1 gene and presented at the tumor cell surface by the HLA-Al molecule. Gene MAGE1 belongs to a family of closely related genes. The MAGE genes are clustered within two distinct regions on chromosome X. MAGE1 to -12 are located in the q terminal region of the chromosome (Xq26-qter), while an additional member (MAGE-Xp) has been identified in the Xp21.3 locus. The MAGE gene family is silent in healthy adult tissues, with two important exceptions: testis, where all members but MAGE7 are expressed, and placenta, where transcripts for MAGE3 and -4 have been detected. In contrast, MAGE1, -2, -3, -4, -6, and -12 are frequently expressed at high levels in a significant proportion of tumors of various histological types, including melanomas, colon carcinomas, leukemias, lung cancers, sarcomas, and breast cancers. In addition to the peptide corresponding to antigen MZ2-E, an additional peptide derived from MAGE1 and a peptide derived from MAGE3 have recently been identified as tumor antigens recognized by autologous cytolytic T cells. The MAGE proteins are therefore considered to be attractive targets for cancer immunotherapy. However, it remains to be demonstrated that such a therapy will not affect tissues, like testis, where the corresponding genes are expressed. 15 refs., 3 figs.

  3. The Microtubule-Associated Protein ASPM Regulates Spindle Assembly and Meiotic Progression in Mouse Oocytes

    PubMed Central

    Xu, Xiao-Ling; Ma, Wei; Zhu, Yu-Bo; Wang, Chao; Wang, Bing-Yuan; An, Na; An, Lei; Liu, Yan; Wu, Zhong-Hong; Tian, Jian-Hui

    2012-01-01

    The microtubule-associated protein ASPM (abnormal spindle-like microcephaly-associated) plays an important role in spindle organization and cell division in mitosis and meiosis in lower animals, but its function in mouse oocyte meiosis has not been investigated. In this study, we characterized the localization and expression dynamics of ASPM during mouse oocyte meiotic maturation and analyzed the effects of the downregulation of ASPM expression on meiotic spindle assembly and meiotic progression. Immunofluorescence analysis showed that ASPM localized to the entire spindle at metaphase I (MI) and metaphase II (MII), colocalizing with the spindle microtubule protein acetylated tubulin (Ac-tubulin). In taxol-treated oocytes, ASPM colocalized with Ac-tubulin on the excessively polymerized microtubule fibers of enlarged spindles and the numerous asters in the cytoplasm. Nocodazole treatment induced the gradual disassembly of microtubule fibers, during which ASPM remained colocalized with the dynamic Ac-tubulin. The downregulation of ASPM expression by a gene-specific morpholino resulted in an abnormal meiotic spindle and inhibited meiotic progression; most of the treated oocytes were blocked in the MI stage with elongated meiotic spindles. Furthermore, coimmunoprecipitation combined with mass spectrometry and western blot analysis revealed that ASPM interacted with calmodulin in MI oocytes and that these proteins colocalized at the spindle. Our results provide strong evidence that ASPM plays a critical role in meiotic spindle assembly and meiotic progression in mouse oocytes. PMID:23152892

  4. Axin-1 Regulates Meiotic Spindle Organization in Mouse Oocytes

    PubMed Central

    Liu, Rui; Liu, Yu; Zhang, Fei; Zhang, Zhen; Shen, Yu-Ting; Xu, Lin; Chen, Ming-Huang; Wang, Ya-Long; Xu, Bai-Hui; Yang, Xiang-Jun; Wang, Hai-Long

    2016-01-01

    Axin-1, a negative regulator of Wnt signaling, is a versatile scaffold protein involved in centrosome separation and spindle assembly in mitosis, but its function in mammalian oogenesis remains unknown. Here we examined the localization and function of Axin-1 during meiotic maturation in mouse oocytes. Immunofluorescence analysis showed that Axin-1 was localized around the spindle. Knockdown of the Axin1 gene by microinjection of specific short interfering (si)RNA into the oocyte cytoplasm resulted in severely defective spindles, misaligned chromosomes, failure of first polar body (PB1) extrusion, and impaired pronuclear formation. However, supplementing the culture medium with the Wnt pathway activator LiCl improved spindle morphology and pronuclear formation. Downregulation of Axin1 gene expression also impaired the spindle pole localization of γ-tubulin/Nek9 and resulted in retention of the spindle assembly checkpoint protein BubR1 at kinetochores after 8.5 h of culture. Our results suggest that Axin-1 is critical for spindle organization and cell cycle progression during meiotic maturation in mouse oocytes. PMID:27284927

  5. Hybrid Sterility Locus on Chromosome X Controls Meiotic Recombination Rate in Mouse

    PubMed Central

    Balcova, Maria; Faltusova, Barbora; Gergelits, Vaclav; Bhattacharyya, Tanmoy; Mihola, Ondrej; Trachtulec, Zdenek; Knopf, Corinna; Fotopulosova, Vladana; Chvatalova, Irena; Gregorova, Sona; Forejt, Jiri

    2016-01-01

    Meiotic recombination safeguards proper segregation of homologous chromosomes into gametes, affects genetic variation within species, and contributes to meiotic chromosome recognition, pairing and synapsis. The Prdm9 gene has a dual role, it controls meiotic recombination by determining the genomic position of crossover hotspots and, in infertile hybrids of house mouse subspecies Mus m. musculus (Mmm) and Mus m. domesticus (Mmd), it further functions as the major hybrid sterility gene. In the latter role Prdm9 interacts with the hybrid sterility X 2 (Hstx2) genomic locus on Chromosome X (Chr X) by a still unknown mechanism. Here we investigated the meiotic recombination rate at the genome-wide level and its possible relation to hybrid sterility. Using immunofluorescence microscopy we quantified the foci of MLH1 DNA mismatch repair protein, the cytological counterparts of reciprocal crossovers, in a panel of inter-subspecific chromosome substitution strains. Two autosomes, Chr 7 and Chr 11, significantly modified the meiotic recombination rate, yet the strongest modifier, designated meiotic recombination 1, Meir1, emerged in the 4.7 Mb Hstx2 genomic locus on Chr X. The male-limited transgressive effect of Meir1 on recombination rate parallels the male-limited transgressive role of Hstx2 in hybrid male sterility. Thus, both genetic factors, the Prdm9 gene and the Hstx2/Meir1 genomic locus, indicate a link between meiotic recombination and hybrid sterility. A strong female-specific modifier of meiotic recombination rate with the effect opposite to Meir1 was localized on Chr X, distally to Meir1. Mapping Meir1 to a narrow candidate interval on Chr X is an important first step towards positional cloning of the respective gene(s) responsible for variation in the global recombination rate between closely related mouse subspecies. PMID:27104744

  6. Hybrid Sterility Locus on Chromosome X Controls Meiotic Recombination Rate in Mouse.

    PubMed

    Balcova, Maria; Faltusova, Barbora; Gergelits, Vaclav; Bhattacharyya, Tanmoy; Mihola, Ondrej; Trachtulec, Zdenek; Knopf, Corinna; Fotopulosova, Vladana; Chvatalova, Irena; Gregorova, Sona; Forejt, Jiri

    2016-04-01

    Meiotic recombination safeguards proper segregation of homologous chromosomes into gametes, affects genetic variation within species, and contributes to meiotic chromosome recognition, pairing and synapsis. The Prdm9 gene has a dual role, it controls meiotic recombination by determining the genomic position of crossover hotspots and, in infertile hybrids of house mouse subspecies Mus m. musculus (Mmm) and Mus m. domesticus (Mmd), it further functions as the major hybrid sterility gene. In the latter role Prdm9 interacts with the hybrid sterility X 2 (Hstx2) genomic locus on Chromosome X (Chr X) by a still unknown mechanism. Here we investigated the meiotic recombination rate at the genome-wide level and its possible relation to hybrid sterility. Using immunofluorescence microscopy we quantified the foci of MLH1 DNA mismatch repair protein, the cytological counterparts of reciprocal crossovers, in a panel of inter-subspecific chromosome substitution strains. Two autosomes, Chr 7 and Chr 11, significantly modified the meiotic recombination rate, yet the strongest modifier, designated meiotic recombination 1, Meir1, emerged in the 4.7 Mb Hstx2 genomic locus on Chr X. The male-limited transgressive effect of Meir1 on recombination rate parallels the male-limited transgressive role of Hstx2 in hybrid male sterility. Thus, both genetic factors, the Prdm9 gene and the Hstx2/Meir1 genomic locus, indicate a link between meiotic recombination and hybrid sterility. A strong female-specific modifier of meiotic recombination rate with the effect opposite to Meir1 was localized on Chr X, distally to Meir1. Mapping Meir1 to a narrow candidate interval on Chr X is an important first step towards positional cloning of the respective gene(s) responsible for variation in the global recombination rate between closely related mouse subspecies.

  7. Silencing of unsynapsed meiotic chromosomes in the mouse.

    PubMed

    Turner, James M A; Mahadevaiah, Shantha K; Fernandez-Capetillo, Oscar; Nussenzweig, André; Xu, Xiaoling; Deng, Chu-Xia; Burgoyne, Paul S

    2005-01-01

    In Neurospora, DNA unpaired in meiosis both is silenced and induces silencing of all DNA homologous to it. This process, called meiotic silencing by unpaired DNA, is thought to protect the host genome from invasion by transposable elements. We now show that silencing of unpaired (unsynapsed) chromosome regions also takes place in the mouse during both male and female meiosis. The tumor suppressor protein BRCA1 is implicated in this silencing, mirroring its role in the meiotic silencing of the X and Y chromosomes in normal male meiosis. These findings impact on the interpretation of the relationship between synaptic errors and sterility in mammals and extend our understanding of the biology of Brca1.

  8. Effects of clinostat rotation on mouse meiotic maturation in vitro

    NASA Technical Reports Server (NTRS)

    Wolgemuth, D. J.; Grills, G. S.

    1984-01-01

    The effects of microgravity on meiosis, fertilization, and early embryonic development in mammals are being examined by using a clinostat to reorient the cells with respect to the gravity vector. A clinostat capable of supporting mammalian cells in tissue culture has been developed. Initial studies have focused on examining the effects of clinostat rotation on meiotic maturation in mouse oocytes. Oocytes recovered from ovarian follicles were subjected to clinostat rotation on a horizontal or vertical axis or to static conditions for a 16 hr period. No gross morphological changes and no effects on germinal vesicle breakdown were observed under any rotation conditions (1/4, 1, 10, 30, 100 RPM). Success of meiotic progression to Metaphase II was comparable among experimental and control groups except at 100 RPM, where a slight inhibition was observed.

  9. The role of chromatin modifications in progression through mouse meiotic prophase.

    PubMed

    Crichton, James H; Playfoot, Christopher J; Adams, Ian R

    2014-03-20

    Meiosis is a key event in gametogenesis that generates new combinations of genetic information and is required to reduce the chromosome content of the gametes. Meiotic chromosomes undergo a number of specialised events during prophase to allow meiotic recombination, homologous chromosome synapsis and reductional chromosome segregation to occur. In mammalian cells, DNA physically associates with histones to form chromatin, which can be modified by methylation, phosphorylation, ubiquitination and acetylation to help regulate higher order chromatin structure, gene expression, and chromosome organisation. Recent studies have identified some of the enzymes responsible for generating chromatin modifications in meiotic mammalian cells, and shown that these chromatin modifying enzymes are required for key meiosis-specific events that occur during meiotic prophase. This review will discuss the role of chromatin modifications in meiotic recombination, homologous chromosome synapsis and regulation of meiotic gene expression in mammals. PMID:24656230

  10. Depletion of the LINC complex disrupts cytoskeleton dynamics and meiotic resumption in mouse oocytes

    PubMed Central

    Luo, Yibo; Lee, In-Won; Jo, Yu-Jin; Namgoong, Suk; Kim, Nam-Hyung

    2016-01-01

    The SUN (Sad-1/UNC-84) and KASH (Klarsicht/ANC-1/Syne/homology) proteins constitute the linker of nucleoskeleton and cytoskeleton (LINC) complex on the nuclear envelope. To date, the SUN1/KASH5 complex is known to function as meiotic-specific factors. In this study, gene-silencing methods were used to explore the roles of SUN1 and KASH5 in mouse oocytes after prophase. SUN1 was detected throughout the nucleus; however, KASH5 was dispersed through the cell. After germinal vesicle breakdown (GVBD), SUN1 and KASH5 migrated during spindle formation and localized to the spindle poles at the MII stage. Most oocytes were arrested at the germinal vesicle (GV) stage after depletion of either SUN1 or KASH5. The DNA damage response was triggered in SUN1-depleted oocytes and thus gave rise to the G2/M checkpoint protein, p-CHK1. Oocytes that underwent GVBD had relatively small and abnormal spindles and lower levels of cytoplasm F-actin mesh. Immunofluorescence results also indicated the dislocation of pericentrin and P150Glued after SUN1 or KASH5 depletion. Furthermore, KASH5 localized exclusively near the oocyte cortex after SUN1 depletion, but SUN1 localization was unaffected in KASH5-depleted oocytes. Taken together, the results suggest that SUN1 and KASH5 are essential factors in the regulation of meiotic resumption and spindle formation. PMID:26842404

  11. Maternal Setdb1 Is Required for Meiotic Progression and Preimplantation Development in Mouse.

    PubMed

    Kim, Jeesun; Zhao, Hongbo; Dan, Jiameng; Kim, Soojin; Hardikar, Swanand; Hollowell, Debra; Lin, Kevin; Lu, Yue; Takata, Yoko; Shen, Jianjun; Chen, Taiping

    2016-04-01

    Oocyte meiotic progression and maternal-to-zygote transition are accompanied by dynamic epigenetic changes. The functional significance of these changes and the key epigenetic regulators involved are largely unknown. Here we show that Setdb1, a lysine methyltransferase, controls the global level of histone H3 lysine 9 di-methyl (H3K9me2) mark in growing oocytes. Conditional deletion of Setdb1 in developing oocytes leads to meiotic arrest at the germinal vesicle and meiosis I stages, resulting in substantially fewer mature eggs. Embryos derived from these eggs exhibit severe defects in cell cycle progression, progressive delays in preimplantation development, and degeneration before reaching the blastocyst stage. Rescue experiments by expressing wild-type or inactive Setdb1 in Setdb1-deficient oocytes suggest that the catalytic activity of Setdb1 is essential for meiotic progression and early embryogenesis. Mechanistically, up-regulation of Cdc14b, a dual-specificity phosphatase that inhibits meiotic progression, greatly contributes to the meiotic arrest phenotype. Setdb1 deficiency also leads to derepression of transposons and increased DNA damage in oocytes, which likely also contribute to meiotic defects. Thus, Setdb1 is a maternal-effect gene that controls meiotic progression and is essential for early embryogenesis. Our results uncover an important link between the epigenetic machinery and the major signaling pathway governing meiotic progression. PMID:27070551

  12. Maternal Setdb1 Is Required for Meiotic Progression and Preimplantation Development in Mouse

    PubMed Central

    Dan, Jiameng; Kim, Soojin; Hardikar, Swanand; Hollowell, Debra; Lin, Kevin; Lu, Yue; Takata, Yoko; Shen, Jianjun; Chen, Taiping

    2016-01-01

    Oocyte meiotic progression and maternal-to-zygote transition are accompanied by dynamic epigenetic changes. The functional significance of these changes and the key epigenetic regulators involved are largely unknown. Here we show that Setdb1, a lysine methyltransferase, controls the global level of histone H3 lysine 9 di-methyl (H3K9me2) mark in growing oocytes. Conditional deletion of Setdb1 in developing oocytes leads to meiotic arrest at the germinal vesicle and meiosis I stages, resulting in substantially fewer mature eggs. Embryos derived from these eggs exhibit severe defects in cell cycle progression, progressive delays in preimplantation development, and degeneration before reaching the blastocyst stage. Rescue experiments by expressing wild-type or inactive Setdb1 in Setdb1-deficient oocytes suggest that the catalytic activity of Setdb1 is essential for meiotic progression and early embryogenesis. Mechanistically, up-regulation of Cdc14b, a dual-specificity phosphatase that inhibits meiotic progression, greatly contributes to the meiotic arrest phenotype. Setdb1 deficiency also leads to derepression of transposons and increased DNA damage in oocytes, which likely also contribute to meiotic defects. Thus, Setdb1 is a maternal-effect gene that controls meiotic progression and is essential for early embryogenesis. Our results uncover an important link between the epigenetic machinery and the major signaling pathway governing meiotic progression. PMID:27070551

  13. Oocyte heterogeneity with respect to the meiotic silencing of unsynapsed X chromosomes in the XY female mouse.

    PubMed

    Taketo, Teruko; Naumova, Anna K

    2013-10-01

    In the XY pachytene spermatocyte, the sex chromosomes do not synapse except for the pseudoautosomal region and become transcriptionally silenced. It has been suggested that the meiotic silencing of unsynapsed chromatin (MSUC) also occurs in oocytes. In the XY sex-reversed female mouse, the sex chromosomes fail to pair in the majority of oocytes and a greater number of oocytes are eliminated during the meiotic prophase compared to the XX female. Yet, many XY oocytes survive to reach the second meiotic metaphase. The goal of our current study was to determine whether the single X chromosome shows the characteristics of asynapsis and meiotic silencing in a proportion of XY oocytes, which can explain the survival of the remaining oocytes. We first examined the accumulation of markers associated with asynapsis or transcriptional silencing, i.e., BRCA1, γH2AX, H3K9me3, and H3K27me3, at the single X chromosome in the XY oocyte. We found that γH2AX and BRCA1 were enriched on the single X chromosome whereas H3K9me3 was not, and H3K27me3 was enriched at all chromosomes in the majority of XY oocytes. We next examined the meiotic silencing of the single X chromosome using enrichment of the X-encoded ATRX protein. On average, ATRX enrichment was lower in XY oocytes than in XX oocytes as expected from its half gene dosage. However, the intensity of ATRX staining in XY oocytes harboring γH2AX domains showed a remarkable heterogeneity. We conclude that MSUC occurs with varying consequences, resulting in a heterogeneous population of oocytes with respect to protein enrichment in the XY female mouse. PMID:23760560

  14. Inventory and Phylogenetic Analysis of Meiotic Genes in Monogonont Rotifers

    PubMed Central

    2013-01-01

    A long-standing question in evolutionary biology is how sexual reproduction has persisted in eukaryotic lineages. As cyclical parthenogens, monogonont rotifers are a powerful model for examining this question, yet the molecular nature of sexual reproduction in this lineage is currently understudied. To examine genes involved in meiosis, we generated partial genome assemblies for 2 distantly related monogonont species, Brachionus calyciflorus and B. manjavacas. Here we present an inventory of 89 meiotic genes, of which 80 homologs were identified and annotated from these assemblies. Using phylogenetic analysis, we show that several meiotic genes have undergone relatively recent duplication events that appear to be specific to the monogonont lineage. Further, we compare the expression of “meiosis-specific” genes involved in recombination and all annotated copies of the cell cycle regulatory gene CDC20 between obligate parthenogenetic (OP) and cyclical parthenogenetic (CP) strains of B. calyciflorus. We show that “meiosis-specific” genes are expressed in both CP and OP strains, whereas the expression of one of the CDC20 genes is specific to cyclical parthenogenesis. The data presented here provide insights into mechanisms of cyclical parthenogenesis and establish expectations for studies of obligate asexual relatives of monogononts, the bdelloid rotifer lineage. PMID:23487324

  15. The meiotic recombination checkpoint is regulated by checkpoint rad+ genes in fission yeast

    PubMed Central

    Shimada, Midori; Nabeshima, Kentaro; Tougan, Takahiro; Nojima, Hiroshi

    2002-01-01

    During the course of meiotic prophase, intrinsic double-strand breaks (DSBs) must be repaired before the cell can engage in meiotic nuclear division. Here we investigate the mechanism that controls the meiotic progression in Schizosaccharomyces pombe that have accumulated excess meiotic DSBs. A meiotic recombination-defective mutant, meu13Δ, shows a delay in meiotic progression. This delay is dependent on rec12+, namely on DSB formation. Pulsed-field gel electrophoresis analysis revealed that meiotic DSB repair in meu13Δ was retarded. We also found that the delay in entering nuclear division was dependent on the checkpoint rad+, cds1+ and mek1+ (the meiotic paralog of Cds1/Chk2). This implies that these genes are involved in a checkpoint that provides time to repair DSBs. Consistently, the induction of an excess of extrinsic DSBs by ionizing radiation delayed meiotic progression in a rad17+-dependent manner. dmc1Δ also shows meiotic delay, however, this delay is independent of rec12+ and checkpoint rad+. We propose that checkpoint monitoring of the status of meiotic DSB repair exists in fission yeast and that defects other than DSB accumulation can cause delays in meiotic progression. PMID:12032093

  16. The meiotic recombination checkpoint is regulated by checkpoint rad+ genes in fission yeast.

    PubMed

    Shimada, Midori; Nabeshima, Kentaro; Tougan, Takahiro; Nojima, Hiroshi

    2002-06-01

    During the course of meiotic prophase, intrinsic double-strand breaks (DSBs) must be repaired before the cell can engage in meiotic nuclear division. Here we investigate the mechanism that controls the meiotic progression in Schizosaccharomyces pombe that have accumulated excess meiotic DSBs. A meiotic recombination-defective mutant, meu13Delta, shows a delay in meiotic progression. This delay is dependent on rec12+, namely on DSB formation. Pulsed-field gel electrophoresis analysis revealed that meiotic DSB repair in meu13Delta was retarded. We also found that the delay in entering nuclear division was dependent on the checkpoint rad+, cds1+ and mek1+ (the meiotic paralog of Cds1/Chk2). This implies that these genes are involved in a checkpoint that provides time to repair DSBs. Consistently, the induction of an excess of extrinsic DSBs by ionizing radiation delayed meiotic progression in a rad17(+)-dependent manner. dmc1Delta also shows meiotic delay, however, this delay is independent of rec12+ and checkpoint rad+. We propose that checkpoint monitoring of the status of meiotic DSB repair exists in fission yeast and that defects other than DSB accumulation can cause delays in meiotic progression. PMID:12032093

  17. Kinetochore Fibers Are Not Involved in the Formation of the First Meiotic Spindle in Mouse Oocytes, but Control the Exit from the First Meiotic M Phase

    PubMed Central

    Brunet, Stéphane; Maria, Angélica Santa; Guillaud, Philippe; Dujardin, Denis; Kubiak, Jacek Z.; Maro, Bernard

    1999-01-01

    During meiosis, two successive divisions occur without any intermediate S phase to produce haploid gametes. The first meiotic division is unique in that homologous chromosomes are segregated while the cohesion between sister chromatids is maintained, resulting in a reductional division. Moreover, the duration of the first meiotic M phase is usually prolonged when compared with mitotic M phases lasting 8 h in mouse oocytes. We investigated the spindle assembly pathway and its role in the progression of the first meiotic M phase in mouse oocytes. During the first 4 h, a bipolar spindle forms and the chromosomes congress near the equatorial plane of the spindle without stable kinetochore– microtubule end interactions. This late prometaphase spindle is then maintained for 4 h with chromosomes oscillating in the central region of the spindle. The kinetochore–microtubule end interactions are set up at the end of the first meiotic M phase (8 h after entry into M phase). This event allows the final alignment of the chromosomes and exit from metaphase. The continuous presence of the prometaphase spindle is not required for progression of the first meiotic M phase. Finally, the ability of kinetochores to interact with microtubules is acquired at the end of the first meiotic M phase and determines the timing of polar body extrusion. PMID:10402455

  18. Live Imaging of Intracellular Dynamics During Meiotic Maturation in Mouse Oocytes.

    PubMed

    Yoshida, Shuhei; Sakakibara, Yogo; Kitajima, Tomoya S

    2016-01-01

    Fluorescence live imaging is a powerful approach to study intracellular dynamics during cellular events such as cell division. By applying automated confocal live imaging to mouse oocytes, in which meiotic maturation can be induced in vitro after the introduction of fluorescent proteins through microinjection, the meiotic dynamics of intracellular structures, such as chromosomes, can be monitored at high resolution. A combination of this method with approaches for the perturbation of specific proteins opens up opportunities for understanding the molecular and intracellular basis of mammalian meiosis. PMID:27557586

  19. An expanded inventory of conserved meiotic genes provides evidence for sex in Trichomonas vaginalis.

    PubMed

    Malik, Shehre-Banoo; Pightling, Arthur W; Stefaniak, Lauren M; Schurko, Andrew M; Logsdon, John M

    2008-01-01

    Meiosis is a defining feature of eukaryotes but its phylogenetic distribution has not been broadly determined, especially among eukaryotic microorganisms (i.e. protists)-which represent the majority of eukaryotic 'supergroups'. We surveyed genomes of animals, fungi, plants and protists for meiotic genes, focusing on the evolutionarily divergent parasitic protist Trichomonas vaginalis. We identified homologs of 29 components of the meiotic recombination machinery, as well as the synaptonemal and meiotic sister chromatid cohesion complexes. T. vaginalis has orthologs of 27 of 29 meiotic genes, including eight of nine genes that encode meiosis-specific proteins in model organisms. Although meiosis has not been observed in T. vaginalis, our findings suggest it is either currently sexual or a recent asexual, consistent with observed, albeit unusual, sexual cycles in their distant parabasalid relatives, the hypermastigotes. T. vaginalis may use meiotic gene homologs to mediate homologous recombination and genetic exchange. Overall, this expanded inventory of meiotic genes forms a useful "meiosis detection toolkit". Our analyses indicate that these meiotic genes arose, or were already present, early in eukaryotic evolution; thus, the eukaryotic cenancestor contained most or all components of this set and was likely capable of performing meiotic recombination using near-universal meiotic machinery. PMID:18663385

  20. An expanded inventory of conserved meiotic genes provides evidence for sex in Trichomonas vaginalis.

    PubMed

    Malik, Shehre-Banoo; Pightling, Arthur W; Stefaniak, Lauren M; Schurko, Andrew M; Logsdon, John M

    2008-01-01

    Meiosis is a defining feature of eukaryotes but its phylogenetic distribution has not been broadly determined, especially among eukaryotic microorganisms (i.e. protists)-which represent the majority of eukaryotic 'supergroups'. We surveyed genomes of animals, fungi, plants and protists for meiotic genes, focusing on the evolutionarily divergent parasitic protist Trichomonas vaginalis. We identified homologs of 29 components of the meiotic recombination machinery, as well as the synaptonemal and meiotic sister chromatid cohesion complexes. T. vaginalis has orthologs of 27 of 29 meiotic genes, including eight of nine genes that encode meiosis-specific proteins in model organisms. Although meiosis has not been observed in T. vaginalis, our findings suggest it is either currently sexual or a recent asexual, consistent with observed, albeit unusual, sexual cycles in their distant parabasalid relatives, the hypermastigotes. T. vaginalis may use meiotic gene homologs to mediate homologous recombination and genetic exchange. Overall, this expanded inventory of meiotic genes forms a useful "meiosis detection toolkit". Our analyses indicate that these meiotic genes arose, or were already present, early in eukaryotic evolution; thus, the eukaryotic cenancestor contained most or all components of this set and was likely capable of performing meiotic recombination using near-universal meiotic machinery.

  1. Preaching about the converted: how meiotic gene conversion influences genomic diversity.

    PubMed

    Cole, Francesca; Keeney, Scott; Jasin, Maria

    2012-09-01

    Meiotic crossover (CO) recombination involves a reciprocal exchange between homologous chromosomes. COs are often associated with gene conversion at the exchange site where genetic information is unidirectionally transferred from one chromosome to the other. COs and independent assortment of homologous chromosomes contribute significantly to the promotion of genomic diversity. What has not been appreciated is the contribution of another product of meiotic recombination, noncrossovers (NCOs), which result in gene conversion without exchange of flanking markers. Here, we review our comprehensive analysis of recombination at a highly polymorphic mouse hotspot. We found that NCOs make up ∼90% of recombination events. Preferential recombination initiation on one chromosome allowed us to estimate the contribution of CO and NCO gene conversion to transmission distortion, a deviation from Mendelian inheritance in the population. While NCO gene conversion tracts are shorter, and thus have a more punctate effect, their higher frequency translates into an approximately two-fold greater contribution than COs to gene conversion-based allelic shuffling and transmission distortion. We discuss the potential impact of mammalian NCO characteristics on evolution and genomic diversity. PMID:22954222

  2. Mechanism and regulation of rapid telomere prophase movements in mouse meiotic chromosomes

    PubMed Central

    Lee, Chih-Ying; Horn, Henning F.; Stewart, Colin L.; Burke, Brian; Bolcun-Filas, Ewelina; Schimenti, John C.; Dresser, Michael E.; Pezza, Roberto J.

    2015-01-01

    SUMMARY Telomere-led rapid prophase movements (RPMs) in meiotic prophase have been observed in diverse eukaryote species. A shared feature of RPMs is that the force that drives the chromosomal movements is transmitted from the cytoskeleton, through the nuclear envelope, to the telomeres. Studies in mice suggested that dynein movement along microtubules is transmitted to telomeres through SUN1/KASH5 nuclear envelope bridges to generate RPMs. We monitored RPMs in mouse seminiferous tubules using four-dimensional fluorescence imaging and quantitative motion analysis to characterize patterns of movement in the RPM process. We find that RPMs reflect a combination of nuclear rotation and individual chromosome movements. The telomeres move along microtubule tracks which are apparently continuous with the cytoskeletal network, and exhibit characteristic arrangements at different stages of prophase. Quantitative measurements confirmed that SUN1/KASH5, microtubules, and dynein but not actin were necessary for RPMs and that defects in meiotic recombination and synapsis resulted in altered RPMs. PMID:25892231

  3. Conserved meiotic genes point to sex in the choanoflagellates.

    PubMed

    Carr, Martin; Leadbeater, Barry S C; Baldauf, Sandra L

    2010-01-01

    The choanoflagellates are a widespread group of heterotrophic aquatic nanoflagellates, which have recently been confirmed as the sister-group to Metazoa. Asexual reproduction is the only mode of cell division that has been observed within the group; at present the range of reproductive modes, as well as the ploidy level, within choanoflagellates are unknown. The recent discovery of long terminal repeat retrotransposons within the genome of Monosiga brevicollis suggests that this species also has sexual stages in its life cycle because asexual organisms cannot tolerate retrotransposons due to the rapid accumulation of deleterious mutations caused by their transposition. We screened the M. brevicollis genome for known eukaryotic meiotic genes, using a recently established "meiosis detection toolkit" of 19 genes. Eighteen of these genes were identified, none of which appears to be a pseudogene. Four of the genes were also identified in expressed sequence tag data from the distantly related Monosiga ovata. The presence of these meiosis-specific genes provides evidence for meiosis, and by implication sex, within this important group of protists.

  4. X Chromosome Control of Meiotic Chromosome Synapsis in Mouse Inter-Subspecific Hybrids

    PubMed Central

    Bhattacharyya, Tanmoy; Reifova, Radka; Gregorova, Sona; Simecek, Petr; Gergelits, Vaclav; Mistrik, Martin; Martincova, Iva; Pialek, Jaroslav; Forejt, Jiri

    2014-01-01

    Hybrid sterility (HS) belongs to reproductive isolation barriers that safeguard the integrity of species in statu nascendi. Although hybrid sterility occurs almost universally among animal and plant species, most of our current knowledge comes from the classical genetic studies on Drosophila interspecific crosses or introgressions. With the house mouse subspecies Mus m. musculus and Mus m. domesticus as a model, new research tools have become available for studies of the molecular mechanisms and genetic networks underlying HS. Here we used QTL analysis and intersubspecific chromosome substitution strains to identify a 4.7 Mb critical region on Chromosome X (Chr X) harboring the Hstx2 HS locus, which causes asymmetrical spermatogenic arrest in reciprocal intersubspecific F1 hybrids. Subsequently, we mapped autosomal loci on Chrs 3, 9 and 13 that can abolish this asymmetry. Combination of immunofluorescent visualization of the proteins of synaptonemal complexes with whole-chromosome DNA FISH on pachytene spreads revealed that heterosubspecific, unlike consubspecific, homologous chromosomes are predisposed to asynapsis in F1 hybrid male and female meiosis. The asynapsis is under the trans- control of Hstx2 and Hst1/Prdm9 hybrid sterility genes in pachynemas of male but not female hybrids. The finding concurred with the fertility of intersubpecific F1 hybrid females homozygous for the Hstx2Mmm allele and resolved the apparent conflict with the dominance theory of Haldane's rule. We propose that meiotic asynapsis in intersubspecific hybrids is a consequence of cis-acting mismatch between homologous chromosomes modulated by the trans-acting Hstx2 and Prdm9 hybrid male sterility genes. PMID:24516397

  5. X chromosome control of meiotic chromosome synapsis in mouse inter-subspecific hybrids.

    PubMed

    Bhattacharyya, Tanmoy; Reifova, Radka; Gregorova, Sona; Simecek, Petr; Gergelits, Vaclav; Mistrik, Martin; Martincova, Iva; Pialek, Jaroslav; Forejt, Jiri

    2014-02-01

    Hybrid sterility (HS) belongs to reproductive isolation barriers that safeguard the integrity of species in statu nascendi. Although hybrid sterility occurs almost universally among animal and plant species, most of our current knowledge comes from the classical genetic studies on Drosophila interspecific crosses or introgressions. With the house mouse subspecies Mus m. musculus and Mus m. domesticus as a model, new research tools have become available for studies of the molecular mechanisms and genetic networks underlying HS. Here we used QTL analysis and intersubspecific chromosome substitution strains to identify a 4.7 Mb critical region on Chromosome X (Chr X) harboring the Hstx2 HS locus, which causes asymmetrical spermatogenic arrest in reciprocal intersubspecific F1 hybrids. Subsequently, we mapped autosomal loci on Chrs 3, 9 and 13 that can abolish this asymmetry. Combination of immunofluorescent visualization of the proteins of synaptonemal complexes with whole-chromosome DNA FISH on pachytene spreads revealed that heterosubspecific, unlike consubspecific, homologous chromosomes are predisposed to asynapsis in F1 hybrid male and female meiosis. The asynapsis is under the trans- control of Hstx2 and Hst1/Prdm9 hybrid sterility genes in pachynemas of male but not female hybrids. The finding concurred with the fertility of intersubpecific F1 hybrid females homozygous for the Hstx2(Mmm) allele and resolved the apparent conflict with the dominance theory of Haldane's rule. We propose that meiotic asynapsis in intersubspecific hybrids is a consequence of cis-acting mismatch between homologous chromosomes modulated by the trans-acting Hstx2 and Prdm9 hybrid male sterility genes. PMID:24516397

  6. Mitofusin-2 is required for mouse oocyte meiotic maturation

    PubMed Central

    Zhang, Jing-Hua; Zhang, Teng; Gao, Si-Hua; Wang, Ke; Yang, Xiu-Yan; Mo, Fang-Fang; Na Yu; An, Tian; Li, Yu-Feng; Hu, Ji-Wei; Jiang, Guang-Jian

    2016-01-01

    Mitofusin-2 (Mfn2) is essential for embryonic development, anti-apoptotic events, protection against free radical-induced lesions, and mitochondrial fusion in many cells. However, little is known about its mechanism and function during oocyte maturation. In this study, we found that Mfn2 was expressed in the cytoplasm during different stages of mouse oocyte maturation. Mfn2 was mainly associated with α-tubulin during oocyte maturation. Knockdown of Mfn2 by specific siRNA injection into oocytes caused the mitochondrial morphology and quantity to change, resulting in severely defective spindles and misaligned chromosomes. This led to metaphase I arrest and the failure of first polar body extrusion. Furthermore, Mfn2 depletion from GV stage oocytes caused the redistribution of p38 MAPK in oocyte cytoplasm. These findings provide insights into potential mechanisms of Mfn2-mediated cellular alterations, which may have significant implications for oocyte maturation. PMID:27485634

  7. Loss of MAX results in meiotic entry in mouse embryonic and germline stem cells

    PubMed Central

    Suzuki, Ayumu; Hirasaki, Masataka; Hishida, Tomoaki; Wu, Jun; Okamura, Daiji; Ueda, Atsushi; Nishimoto, Masazumi; Nakachi, Yutaka; Mizuno, Yosuke; Okazaki, Yasushi; Matsui, Yasuhisa; Belmonte, Juan Carlos Izpisua; Okuda, Akihiko

    2016-01-01

    Meiosis is a unique process that allows the generation of reproductive cells. It remains largely unknown how meiosis is initiated in germ cells and why non-germline cells do not undergo meiosis. We previously demonstrated that knockdown of Max expression, a gene encoding a partner of MYC family proteins, strongly activates expression of germ cell-related genes in ESCs. Here we find that complete ablation of Max expression in ESCs results in profound cytological changes reminiscent of cells undergoing meiotic cell division. Furthermore, our analyses uncovers that Max expression is transiently attenuated in germ cells undergoing meiosis in vivo and its forced reduction induces meiosis-like cytological changes in cultured germline stem cells. Mechanistically, Max depletion alterations are, in part, due to impairment of the function of an atypical PRC1 complex (PRC1.6), in which MAX is one of the components. Our data highlight MAX as a new regulator of meiotic onset. PMID:27025988

  8. APCFZR1 prevents nondisjunction in mouse oocytes by controlling meiotic spindle assembly timing

    PubMed Central

    Holt, Janet E.; Lane, Simon I. R.; Jennings, Phoebe; García-Higuera, Irene; Moreno, Sergio; Jones, Keith T.

    2012-01-01

    FZR1 is an anaphase-promoting complex (APC) activator best known for its role in the mitotic cell cycle at M-phase exit, in G1, and in maintaining genome integrity. Previous studies also established that it prevents meiotic resumption, equivalent to the G2/M transition. Here we report that mouse oocytes lacking FZR1 undergo passage through meiosis I that is accelerated by ∼1 h, and this is due to an earlier onset of spindle assembly checkpoint (SAC) satisfaction and APCCDC20 activity. However, loss of FZR1 did not compromise SAC functionality; instead, earlier SAC satisfaction was achieved because the bipolar meiotic spindle was assembled more quickly in the absence of FZR1. This novel regulation of spindle assembly by FZR1 led to premature bivalent attachment to microtubules and loss of kinetochore-bound MAD2. Bivalents, however, were observed to congress poorly, leading to nondisjunction rates of 25%. We conclude that in mouse oocytes FZR1 controls the timing of assembly of the bipolar spindle and in so doing the timing of SAC satisfaction and APCCDC20 activity. This study implicates FZR1 as a major regulator of prometaphase whose activity helps to prevent chromosome nondisjunction. PMID:22918942

  9. Neuregulins are essential for spermatogonial proliferation and meiotic initiation in neonatal mouse testis.

    PubMed

    Zhang, JiDong; Eto, Ko; Honmyou, Asuka; Nakao, Kazuki; Kiyonari, Hiroshi; Abé, Shin-ichi

    2011-08-01

    The transition from mitosis to meiosis is unique to germ cells. In murine embryonic ovaries and juvenile testes, retinoic acid (RA) induces meiosis via the stimulated by retinoic acid gene 8 (Stra8), but its molecular pathway requires elucidation. We present genetic evidence in vivo and in vitro that neuregulins (NRGs) are essential for the proliferation of spermatogonia and the initiation of meiosis. Tamoxifen (TAM) was injected into 14-day post-partum (dpp) Sertoli cell-specific conditional Nrg1(Ser-/-) mutant mice. TAM induced testis degeneration, suppressed BrdU incorporation into spermatogonia and pre-leptotene primary spermatocytes, and decreased and increased the number of STRA8-positive and TUNEL-positive cells, respectively. In testicular organ cultures from 5-6 dpp wild-type mice and cultures of their re-aggregated spermatogonia and Sertoli cells, FSH, RA [all-trans-retinoic acid (ATRA), AM580, 9-cis-RA] and NRG1 promoted spermatogonial proliferation and meiotic initiation. However, TAM treatment of testicular organ cultures from the Nrg1(Ser-/-) mutants suppressed spermatogonial proliferation and meiotic initiation that was promoted by FSH or AM580. In re-aggregated cultures of purified spermatogonia, NRG1, NRG3, ATRA and 9-cis-RA promoted their proliferation and meiotic initiation, but neither AM580 nor FSH did. In addition, FSH, RAs and NRG1 promoted Nrg1 and Nrg3 mRNA expression in Sertoli cells. These results indicate that in juvenile testes RA and FSH induced meiosis indirectly through Sertoli cells when NRG1 and NRG3 were upregulated, as NRG1 amplified itself and NRG3. The amplified NRG1 and NRG3 directly induced meiosis in spermatogonia. In addition, ATRA and 9-cis-RA activated spermatogonia directly and promoted their proliferation and eventually meiotic initiation.

  10. Co-Localization of Somatic and Meiotic Double Strand Breaks Near the Myc Oncogene on Mouse Chromosome 15

    PubMed Central

    Ng, Siemon H.; Maas, Sarah A.; Petkov, Petko M.; Mills, Kevin D.; Paigen, Kenneth

    2010-01-01

    Both somatic and meiotic recombinations involve the repair of DNA double strand breaks (DSBs) that occur at preferred locations in the genome. Improper repair of DSBs during either mitosis or meiosis can lead to mutations, chromosomal aberration such as translocations, cancer and/or cell death. Currently, no model exists that explains the locations of either spontaneous somatic DSBs or programmed meiotic DSBs or relates them to each other. One common class of tumorigenic translocations arising from DSBs is chromosomal rearrangements near the Myc oncogene. Myc translocations have been associated with Burkitt lymphoma in humans, plasmacytoma in mice and immunocytoma in rats. Comparing the locations of somatic and meiotic DSBs near the mouse Myc oncogene, we demonstrated that the placement of these DSBs is not random and that both events clustered in the same short discrete region of the genome. Our work shows that both somatic and meiotic DSBs tend to occur in proximity to each other within the Myc region, suggesting that they share common originating features. It is likely that some regions of the genome are more susceptible to both somatic and meiotic DSBs, and the locations of meiotic hotspots may be an indicator of genomic regions more susceptible to DNA damage. PMID:19603522

  11. Factors affecting meiotic and developmental competence of primary spermatocyte nuclei injected into mouse oocytes.

    PubMed

    Kimura, Y; Tateno, H; Handel, M A; Yanagimachi, R

    1998-10-01

    Mature mouse oocytes that have received the nuclei of pachytene primary spermatocytes (or metaphase I chromosomes of primary spermatocytes) can develop into fertile offspring. However, success rate in this study was low. No more than 3.8% of transferred 2-cell embryos arising from spermatocyte-injected oocytes developed to full term. Nevertheless, the birth of normal offspring seems to suggest that at least in some primary spermatocytes the functional genomic imprinting is complete before transfer and/or consolidated after the transfer. Although injected spermatocyte nuclei could undergo two successive meiotic divisions within oocytes, abnormalities of both divisions were commonly observed, and sister chromatids often separated prematurely during the second meiotic division. Chromosome breakage/rearrangements were also frequently seen before the first cleavage. Such abnormalities of chromosome behavior are probably the major causes of the poor preimplantation development of zygotes arising from primary spermatocyte-injected oocytes. Thus, clinical use of primary spermatocytes as substitutes for spermatozoa in assisted fertilization is not advisable until the causes of chromosomal abnormalities are better understood through extensive animal studies. PMID:9746737

  12. ACTIN-RELATED PROTEIN6 Regulates Female Meiosis by Modulating Meiotic Gene Expression in Arabidopsis.

    PubMed

    Qin, Yuan; Zhao, Lihua; Skaggs, Megan I; Andreuzza, Sebastien; Tsukamoto, Tatsuya; Panoli, Aneesh; Wallace, Kirsten N; Smith, Steven; Siddiqi, Imran; Yang, Zhenbiao; Yadegari, Ramin; Palanivelu, Ravishankar

    2014-04-15

    In flowering plants, meiocytes develop from subepidermal cells in anthers and ovules. The mechanisms that integrate gene-regulatory processes with meiotic programs during reproductive development remain poorly characterized. Here, we show that Arabidopsis thaliana plants deficient in ACTIN-RELATED PROTEIN6 (ARP6), a subunit of the SWR1 ATP-dependent chromatin-remodeling complex, exhibit defects in prophase I of female meiosis. We found that this meiotic defect is likely due to dysregulated expression of meiotic genes, particularly those involved in meiotic recombination, including DMC1 (DISRUPTED MEIOTIC cDNA1). Analysis of DMC1 expression in arp6 mutant plants indicated that ARP6 inhibits expression of DMC1 in the megasporocyte and surrounding nonsporogeneous ovule cells before meiosis. After cells enter meiosis, however, ARP6 activates DMC1 expression specifically in the megasporocyte even as it continues to inhibit DMC1 expression in the nonsporogenous ovule cells. We further show that deposition of the histone variant H2A.Z, mediated by the SWR1 chromatin-remodeling complex at the DMC1 gene body, requires ARP6. Therefore, ARP6 regulates female meiosis by determining the spatial and temporal patterns of gene expression required for proper meiosis during ovule development. PMID:24737671

  13. Ten years of gene discovery for meiotic event control in rice.

    PubMed

    Luo, Qiong; Li, Yafei; Shen, Yi; Cheng, Zhukuan

    2014-03-20

    Meiosis is the crucial process by which sexually propagating eukaryotes give rise to haploid gametes from diploid cells. Several key processes, like homologous chromosomes pairing, synapsis, recombination, and segregation, sequentially take place in meiosis. Although these widely conserved events are under both genetic and epigenetic control, the accurate details of molecular mechanisms are continuing to investigate. Rice is a good model organism for exploring the molecular mechanisms of meiosis in higher plants. So far, 28 rice meiotic genes have been characterized. In this review, we give an overview of the discovery of rice meiotic genes in the last ten years, with a particular focus on their functions in meiosis. PMID:24656233

  14. Meiotic genes and sexual reproduction in the green algal class Trebouxiophyceae (Chlorophyta).

    PubMed

    Fučíková, Karolina; Pažoutová, Marie; Rindi, Fabio

    2015-06-01

    Sexual reproduction is widespread in eukaryotes and is well documented in chlorophytan green algae. In this lineage, however, the Trebouxiophyceae represent a striking exception: in contrast to its relatives Chlorophyceae and Ulvophyceae this group appears to be mostly asexual, as fertilization has been rarely observed. Assessments of sexual reproduction in the Trebouxiophyceae have been based on microscopic observation of gametes fusing. New genomic data offer now the opportunity to check for the presence of meiotic genes, which represent an indirect evidence of a sexual life cycle. Using genomic and transcriptomic data for 12 taxa spanning the phylogenetic breadth of the class, we tried to clarify whether genuine asexuality or cryptic sexuality is the most likely case for the numerous putatively asexual trebouxiophytes. On the basis of these data and a bibliographic review, we conclude that the view of trebouxiophytes as primarily asexual is incorrect. In contrast to the limited number of reports of fertilization, meiotic genes were found in all genomes and transcriptomes examined, even in species presumed asexual. In the taxa examined the totality or majority of the genes were present, Helicosporidium and Auxenochlorella being the only partial exceptions (only four genes present). The evidence of sex provided by the meiotic genes is phylogenetically widespread in the class and indicates that sexual reproduction is not associated with any particular morphological or ecological trait. On the basis of the results, we expect that the existence of the meiotic genes will be documented in all trebouxiophycean genomes that will become available in the future. PMID:26986659

  15. Meiotic genes and sexual reproduction in the green algal class Trebouxiophyceae (Chlorophyta).

    PubMed

    Fučíková, Karolina; Pažoutová, Marie; Rindi, Fabio

    2015-06-01

    Sexual reproduction is widespread in eukaryotes and is well documented in chlorophytan green algae. In this lineage, however, the Trebouxiophyceae represent a striking exception: in contrast to its relatives Chlorophyceae and Ulvophyceae this group appears to be mostly asexual, as fertilization has been rarely observed. Assessments of sexual reproduction in the Trebouxiophyceae have been based on microscopic observation of gametes fusing. New genomic data offer now the opportunity to check for the presence of meiotic genes, which represent an indirect evidence of a sexual life cycle. Using genomic and transcriptomic data for 12 taxa spanning the phylogenetic breadth of the class, we tried to clarify whether genuine asexuality or cryptic sexuality is the most likely case for the numerous putatively asexual trebouxiophytes. On the basis of these data and a bibliographic review, we conclude that the view of trebouxiophytes as primarily asexual is incorrect. In contrast to the limited number of reports of fertilization, meiotic genes were found in all genomes and transcriptomes examined, even in species presumed asexual. In the taxa examined the totality or majority of the genes were present, Helicosporidium and Auxenochlorella being the only partial exceptions (only four genes present). The evidence of sex provided by the meiotic genes is phylogenetically widespread in the class and indicates that sexual reproduction is not associated with any particular morphological or ecological trait. On the basis of the results, we expect that the existence of the meiotic genes will be documented in all trebouxiophycean genomes that will become available in the future.

  16. Mouse Y-linked Zfy1 and Zfy2 are expressed during the male-specific interphase between meiosis I and meiosis II and promote the 2nd meiotic division.

    PubMed

    Vernet, Nadège; Mahadevaiah, Shantha K; Yamauchi, Yasuhiro; Decarpentrie, Fanny; Mitchell, Michael J; Ward, Monika A; Burgoyne, Paul S

    2014-06-01

    Mouse Zfy1 and Zfy2 encode zinc finger transcription factors that map to the short arm of the Y chromosome (Yp). They have previously been shown to promote meiotic quality control during pachytene (Zfy1 and Zfy2) and at the first meiotic metaphase (Zfy2). However, from these previous studies additional roles for genes encoded on Yp during meiotic progression were inferred. In order to identify these genes and investigate their function in later stages of meiosis, we created three models with diminishing Yp and Zfy gene complements (but lacking the Y-long-arm). Since the Y-long-arm mediates pairing and exchange with the X via their pseudoautosomal regions (PARs) we added a minute PAR-bearing X chromosome derivative to enable formation of a sex bivalent, thus avoiding Zfy2-mediated meiotic metaphase I (MI) checkpoint responses to the unpaired (univalent) X chromosome. Using these models we obtained definitive evidence that genetic information on Yp promotes meiosis II, and by transgene addition identified Zfy1 and Zfy2 as the genes responsible. Zfy2 was substantially more effective and proved to have a much more potent transactivation domain than Zfy1. We previously established that only Zfy2 is required for the robust apoptotic elimination of MI spermatocytes in response to a univalent X; the finding that both genes potentiate meiosis II led us to ask whether there was de novo Zfy1 and Zfy2 transcription in the interphase between meiosis I and meiosis II, and this proved to be the case. X-encoded Zfx was also expressed at this stage and Zfx over-expression also potentiated meiosis II. An interphase between the meiotic divisions is male-specific and we previously hypothesised that this allows meiosis II critical X and Y gene reactivation following sex chromosome silencing in meiotic prophase. The interphase transcription and meiosis II function of Zfx, Zfy1 and Zfy2 validate this hypothesis. PMID:24967676

  17. Mouse Y-Linked Zfy1 and Zfy2 Are Expressed during the Male-Specific Interphase between Meiosis I and Meiosis II and Promote the 2nd Meiotic Division

    PubMed Central

    Vernet, Nadège; Mahadevaiah, Shantha K.; Yamauchi, Yasuhiro; Decarpentrie, Fanny; Mitchell, Michael J.; Ward, Monika A.; Burgoyne, Paul S.

    2014-01-01

    Mouse Zfy1 and Zfy2 encode zinc finger transcription factors that map to the short arm of the Y chromosome (Yp). They have previously been shown to promote meiotic quality control during pachytene (Zfy1 and Zfy2) and at the first meiotic metaphase (Zfy2). However, from these previous studies additional roles for genes encoded on Yp during meiotic progression were inferred. In order to identify these genes and investigate their function in later stages of meiosis, we created three models with diminishing Yp and Zfy gene complements (but lacking the Y-long-arm). Since the Y-long-arm mediates pairing and exchange with the X via their pseudoautosomal regions (PARs) we added a minute PAR-bearing X chromosome derivative to enable formation of a sex bivalent, thus avoiding Zfy2-mediated meiotic metaphase I (MI) checkpoint responses to the unpaired (univalent) X chromosome. Using these models we obtained definitive evidence that genetic information on Yp promotes meiosis II, and by transgene addition identified Zfy1 and Zfy2 as the genes responsible. Zfy2 was substantially more effective and proved to have a much more potent transactivation domain than Zfy1. We previously established that only Zfy2 is required for the robust apoptotic elimination of MI spermatocytes in response to a univalent X; the finding that both genes potentiate meiosis II led us to ask whether there was de novo Zfy1 and Zfy2 transcription in the interphase between meiosis I and meiosis II, and this proved to be the case. X-encoded Zfx was also expressed at this stage and Zfx over-expression also potentiated meiosis II. An interphase between the meiotic divisions is male-specific and we previously hypothesised that this allows meiosis II critical X and Y gene reactivation following sex chromosome silencing in meiotic prophase. The interphase transcription and meiosis II function of Zfx, Zfy1 and Zfy2 validate this hypothesis. PMID:24967676

  18. Mouse Y-linked Zfy1 and Zfy2 are expressed during the male-specific interphase between meiosis I and meiosis II and promote the 2nd meiotic division.

    PubMed

    Vernet, Nadège; Mahadevaiah, Shantha K; Yamauchi, Yasuhiro; Decarpentrie, Fanny; Mitchell, Michael J; Ward, Monika A; Burgoyne, Paul S

    2014-06-01

    Mouse Zfy1 and Zfy2 encode zinc finger transcription factors that map to the short arm of the Y chromosome (Yp). They have previously been shown to promote meiotic quality control during pachytene (Zfy1 and Zfy2) and at the first meiotic metaphase (Zfy2). However, from these previous studies additional roles for genes encoded on Yp during meiotic progression were inferred. In order to identify these genes and investigate their function in later stages of meiosis, we created three models with diminishing Yp and Zfy gene complements (but lacking the Y-long-arm). Since the Y-long-arm mediates pairing and exchange with the X via their pseudoautosomal regions (PARs) we added a minute PAR-bearing X chromosome derivative to enable formation of a sex bivalent, thus avoiding Zfy2-mediated meiotic metaphase I (MI) checkpoint responses to the unpaired (univalent) X chromosome. Using these models we obtained definitive evidence that genetic information on Yp promotes meiosis II, and by transgene addition identified Zfy1 and Zfy2 as the genes responsible. Zfy2 was substantially more effective and proved to have a much more potent transactivation domain than Zfy1. We previously established that only Zfy2 is required for the robust apoptotic elimination of MI spermatocytes in response to a univalent X; the finding that both genes potentiate meiosis II led us to ask whether there was de novo Zfy1 and Zfy2 transcription in the interphase between meiosis I and meiosis II, and this proved to be the case. X-encoded Zfx was also expressed at this stage and Zfx over-expression also potentiated meiosis II. An interphase between the meiotic divisions is male-specific and we previously hypothesised that this allows meiosis II critical X and Y gene reactivation following sex chromosome silencing in meiotic prophase. The interphase transcription and meiosis II function of Zfx, Zfy1 and Zfy2 validate this hypothesis.

  19. Identification of novel Drosophila meiotic genes recovered in a P-element screen.

    PubMed Central

    Sekelsky, J J; McKim, K S; Messina, L; French, R L; Hurley, W D; Arbel, T; Chin, G M; Deneen, B; Force, S J; Hari, K L; Jang, J K; Laurençon, A C; Madden, L D; Matthies, H J; Milliken, D B; Page, S L; Ring, A D; Wayson, S M; Zimmerman, C C; Hawley, R S

    1999-01-01

    The segregation of homologous chromosomes from one another is the essence of meiosis. In many organisms, accurate segregation is ensured by the formation of chiasmata resulting from crossing over. Drosophila melanogaster females use this type of recombination-based system, but they also have mechanisms for segregating achiasmate chromosomes with high fidelity. We describe a P-element mutagenesis and screen in a sensitized genetic background to detect mutations that impair meiotic chromosome pairing, recombination, or segregation. Our screen identified two new recombination-deficient mutations: mei-P22, which fully eliminates meiotic recombination, and mei-P26, which decreases meiotic exchange by 70% in a polar fashion. We also recovered an unusual allele of the ncd gene, whose wild-type product is required for proper structure and function of the meiotic spindle. However, the screen yielded primarily mutants specifically defective in the segregation of achiasmate chromosomes. Although most of these are alleles of previously undescribed genes, five were in the known genes alphaTubulin67C, CycE, push, and Trl. The five mutations in known genes produce novel phenotypes for those genes. PMID:10353897

  20. The Role of Microfilaments in Early Meiotic Maturation of Mouse Oocytes

    NASA Astrophysics Data System (ADS)

    Calarco, Patricia G.

    2005-04-01

    Mouse oocyte microfilaments (MF) were perturbed by depolymerization (cytochalasin B) or stabilization (jasplakinolide) and correlated meiotic defects examined by confocal microscopy. MF, microtubules, and mitochondria were vitally stained; centrosomes ([gamma]-tubulin), after fixation. MF depolymerization by cytochalasin in culture medium did not affect central migration of centrosomes, mitochondria, or nuclear breakdown (GVBD); some MF signal was localized around the germinal vesicle (GV). In maturation-blocking medium (containing IBMX), central movement was curtailed and cortical MF aggregations made the plasma membrane wavy. Occasional long MF suggested that not all MF were depolymerized. MF stabilization by jasplakinolide led to MF aggregations throughout the cytoplasm. GVBD occurred (unless IBMX was present) but no spindle formed. Over time, most oocytes constricted creating a dumbbell shape with MF concentrated under one-half of the oocyte cortex and on either side of the constriction. In IBMX medium, the MF-containing half of the dumbbell over time sequestered the GV, MF, mitochondria, and one to two large cortical centrosomes; the non-MF half appeared empty. Cumulus processes contacted the oocyte surface (detected by microtubule content) and mirrored MF distribution. Results demonstrated that MF play an essential role in meiosis, primarily through cortically mediated events, including centrosome localization, spindle (or GV) movement to the periphery, activation of (polar body) constriction, and establishment of oocyte polarity. The presence of a cortical “organizing pole” is hypothesized.

  1. A Maternal Screen for Genes Regulating Drosophila Oocyte Polarity Uncovers New Steps in Meiotic Progression

    PubMed Central

    Barbosa, Vitor; Kimm, Naomi; Lehmann, Ruth

    2007-01-01

    Meiotic checkpoints monitor chromosome status to ensure correct homologous recombination, genomic integrity, and chromosome segregation. In Drosophila, the persistent presence of double-strand DNA breaks (DSB) activates the ATR/Mei-41 checkpoint, delays progression through meiosis, and causes defects in DNA condensation of the oocyte nucleus, the karyosome. Checkpoint activation has also been linked to decreased levels of the TGFα-like molecule Gurken, which controls normal eggshell patterning. We used this easy-to-score eggshell phenotype in a germ-line mosaic screen in Drosophila to identify new genes affecting meiotic progression, DNA condensation, and Gurken signaling. One hundred eighteen new ventralizing mutants on the second chromosome fell into 17 complementation groups. Here we describe the analysis of 8 complementation groups, including Kinesin heavy chain, the SR protein kinase cuaba, the cohesin-related gene dPds5/cohiba, and the Tudor-domain gene montecristo. Our findings challenge the hypothesis that checkpoint activation upon persistent DSBs is exclusively mediated by ATR/Mei-41 kinase and instead reveal a more complex network of interactions that link DSB formation, checkpoint activation, meiotic delay, DNA condensation, and Gurken protein synthesis. PMID:17507684

  2. Genetic control of mammalian meiotic recombination. I. Variation in exchange frequencies among males from inbred mouse strains.

    PubMed

    Koehler, Kara E; Cherry, Jonathan P; Lynn, Audrey; Hunt, Patricia A; Hassold, Terry J

    2002-09-01

    Genetic background effects on the frequency of meiotic recombination have long been suspected in mice but never demonstrated in a systematic manner, especially in inbred strains. We used a recently described immunostaining technique to assess meiotic exchange patterns in male mice. We found that among four different inbred strains--CAST/Ei, A/J, C57BL/6, and SPRET/Ei--the mean number of meiotic exchanges per cell and, thus, the recombination rates in these genetic backgrounds were significantly different. These frequencies ranged from a low of 21.5 exchanges in CAST/Ei to a high of 24.9 in SPRET/Ei. We also found that, as expected, these crossover events were nonrandomly distributed and displayed positive interference. However, we found no evidence for significant differences in the patterns of crossover positioning between strains with different exchange frequencies. From our observations of >10,000 autosomal synaptonemal complexes, we conclude that achiasmate bivalents arise in the male mouse at a frequency of 0.1%. Thus, special mechanisms that segregate achiasmate chromosomes are unlikely to be an important component of mammalian male meiosis.

  3. Correlation of meiotic events in testis sections and microspreads of mouse spermatocytes relative to the mid-pachytene checkpoint.

    PubMed

    Ashley, Terry; Gaeth, Ann P; Creemers, Laura B; Hack, Adelle M; de Rooij, Dirk G

    2004-09-01

    In mouse, asynaptic meiotic mutants arrest at Testis Epithelial Stage IV. This arrest is 4.5 days after homologous chromosomes begin to synapse and approximately 2.5 days after synapsis is usually completed. To correlate cytological events with meiotic progression in testis and to determine which meiotic events are normally completed by Stage IV, we induced spermatogenic arrest by placing mice on a vitamin A deficient diet. Subsequent injection of retinoic acid and a return to a normal diet resulted in resumption of spermatogenesis with all spermatocytes proceeding through meiosis in a highly synchronous cohort. Between Days 11 and 16 post-injection we prepared one testis for immunocytological and the other for histological evaluation, then used antibodies to SCP3 and either RPA, or MLH1 to follow quantitative changes in synapsis and recombination. RPA was found at sites along the synaptonemal complex as soon as homologs synapsed, and most, but not all, RPA disappeared by Stage IV. MLH1 foci appeared between Stage II and IV and remained through Stage VII, the end point of the study. The data suggest that the earliest the mid-pachytene checkpoint can be activated is Epithelial Stage IV, but that activities monitored by the checkpoint may not be completed by this time.

  4. Meiotic chromosome dynamics dependent upon the rec8(+), rec10(+) and rec11(+) genes of the fission yeast Schizosaccharomyces pombe.

    PubMed

    Krawchuk, M D; DeVeaux, L C; Wahls, W P

    1999-09-01

    During meiosis homologous chromosomes replicate once, pair, experience recombination, and undergo two rounds of segregation to produce haploid meiotic products. The rec8(+), rec10(+), and rec11(+) genes of the fission yeast Schizosaccharomyces pombe exhibit similar specificities for meiotic recombination and rec8(+) is required for sister chromatid cohesion and homolog pairing. We applied cytological and genetic approaches to identify potential genetic interactions and to gauge the fidelity of meiotic chromosome segregation in the mutants. The rec8(+) gene was epistatic to rec10(+) and to rec11(+), but there was no clear epistatic relationship between rec10(+) and rec11(+). Reciprocal (crossover) recombination in the central regions of all three chromosomes was compromised in the rec mutants, but recombination near the telomeres was nearly normal. Each of the mutants also exhibited a high rate of aberrant segregation for all three chromosomes. The rec8 mutations affected mainly meiosis I segregation. Remarkably, the rec10 and rec11 mutations, which compromised recombination during meiosis I, affected mainly meiosis II segregation. We propose that these genes encode regulators or components of a "meiotic chromatid cohesion" pathway involved in establishing, maintaining, and appropriately releasing meiotic interactions between chromosomes. A model of synergistic interactions between sister chromatid cohesion and crossover position suggests how crossovers and cohesion help ensure the proper segregation of chromosomes in each of the meiotic divisions. PMID:10471700

  5. Meiotic chromosome dynamics dependent upon the rec8(+), rec10(+) and rec11(+) genes of the fission yeast Schizosaccharomyces pombe.

    PubMed Central

    Krawchuk, M D; DeVeaux, L C; Wahls, W P

    1999-01-01

    During meiosis homologous chromosomes replicate once, pair, experience recombination, and undergo two rounds of segregation to produce haploid meiotic products. The rec8(+), rec10(+), and rec11(+) genes of the fission yeast Schizosaccharomyces pombe exhibit similar specificities for meiotic recombination and rec8(+) is required for sister chromatid cohesion and homolog pairing. We applied cytological and genetic approaches to identify potential genetic interactions and to gauge the fidelity of meiotic chromosome segregation in the mutants. The rec8(+) gene was epistatic to rec10(+) and to rec11(+), but there was no clear epistatic relationship between rec10(+) and rec11(+). Reciprocal (crossover) recombination in the central regions of all three chromosomes was compromised in the rec mutants, but recombination near the telomeres was nearly normal. Each of the mutants also exhibited a high rate of aberrant segregation for all three chromosomes. The rec8 mutations affected mainly meiosis I segregation. Remarkably, the rec10 and rec11 mutations, which compromised recombination during meiosis I, affected mainly meiosis II segregation. We propose that these genes encode regulators or components of a "meiotic chromatid cohesion" pathway involved in establishing, maintaining, and appropriately releasing meiotic interactions between chromosomes. A model of synergistic interactions between sister chromatid cohesion and crossover position suggests how crossovers and cohesion help ensure the proper segregation of chromosomes in each of the meiotic divisions. PMID:10471700

  6. Perturbing microtubule integrity blocks AMP-activated protein kinase-induced meiotic resumption in cultured mouse oocytes

    PubMed Central

    Ya, Ru; Downs, Stephen M.

    2014-01-01

    AMP-activated protein kinase (AMPK) is an important cellular energy sensor that is activated by a low AMP-to-ATP ratio. AMPK is involved in meiotic induction of mouse oocytes and also promotes the completion of maturation, but suppress premature activation. This study was conducted to further examine the activity and localization of AMPK in relation to microtubule (MT) integrity. Immunostaining with tubulin revealed that active AMPK localized with gamma tubulin during metaphase I and II, while it localized at the spindle midzone during anaphase. This discrete localization pattern was dependent on MT integrity. Treatment with nocodazole, which depolymerized spindle MTs, led to disruption of proper spindle pole localization of active AMPK. In contrast, active AMPK was localized at each pole and with small asters when treated with paclitaxel, which induced excessive polymerization of spindle MTs. Disturbing spindle integrity not only influenced active AMPK localization, but also blocked AMPK activity, as both hormone- and AMPK activator- induced oocyte maturation were blocked by MT-disrupting agents. In concert with these data, the treatments inhibited active AMPK germinal vesicle staining as well, which appears before germinal vesicle breakdown (GVB) in meiotically induced oocytes. Targeting actin filament polymerization had only a marginal effect on meiotic induction. Although oocytes stimulated by AMPK activator increased the rate of GVB, spindle formation and PB extrusion, blocking AMPK activity did not influence peripheral movement of the spindle. These data suggest that the meiosis-inducing action and localization of AMPK are regulated by MT spindle integrity during mouse oocyte maturation. PMID:23199370

  7. Mitotic and Meiotic Gene Conversion of Ty Elements and Other Insertions in Saccharomyces Cerevisiae

    PubMed Central

    Vincent, A.; Petes, T. D.

    1989-01-01

    We examined meiotic and mitotic gene conversion events involved in deletion of Ty elements and other insertions from the genome of the yeast Saccharomyces cerevisiae. We found that Ty elements and one other insertion were deleted by mitotic gene conversion less frequently than point mutations at the same loci. One non-Ty insertion similar in size to Ty, however, did not show this bias. Mitotic conversion events deleting insertions were more frequently associated with crossing over than those deleting point mutations. In meiosis, conversion events duplicating the element were more common than those that deleted the element for one of the loci (HIS4) examined. PMID:2547693

  8. Region-specific meiotic recombination in Schizosaccharomyces pombe: the rec11 gene.

    PubMed

    Li, Y F; Numata, M; Wahls, W P; Smith, G R

    1997-03-01

    Mutations in the rec11 gene of Schizosaccharomyces pombe reduce meiotic recombinant frequencies by as much as a factor of 300 on chromosome III but less than a factor of 4 in the intervals tested on chromosomes I and II. To gain insight into the function of this region- (or chromosome-) specific activator of recombination, we have cloned and sequenced the rec11 gene. Meiotic crosses with rec11 disruption mutations placed the rec11 gene 6 cM from ade6 on chromosome III. Transcripts of rec11 accumulated transiently at 2-3 h after induction of melosis in a pat1-114 (Ts) mutant. Reverse transcriptase/polymerase chain reaction (RT-PCR) analysis of these transcripts revealed eight introns. The spliced RNA is predicted to encode a polypeptide of 923 amino acids with only very limited homology to reported proteins. The transient accumulation of rec11 transcripts and the phenotype of rec11 mutations suggest that the novel rec11 gene product acts early in meiosis to activate recombination preferentially on chromosome III. PMID:9076725

  9. Meiotic drive impacts expression and evolution of x-linked genes in stalk-eyed flies.

    PubMed

    Reinhardt, Josephine A; Brand, Cara L; Paczolt, Kimberly A; Johns, Philip M; Baker, Richard H; Wilkinson, Gerald S

    2014-01-01

    Although sex chromosome meiotic drive has been observed in a variety of species for over 50 years, the genes causing drive are only known in a few cases, and none of these cases cause distorted sex-ratios in nature. In stalk-eyed flies (Teleopsis dalmanni), driving X chromosomes are commonly found at frequencies approaching 30% in the wild, but the genetic basis of drive has remained elusive due to reduced recombination between driving and non-driving X chromosomes. Here, we used RNAseq to identify transcripts that are differentially expressed between males carrying either a driving X (XSR) or a standard X chromosome (XST), and found hundreds of these, the majority of which are X-linked. Drive-associated transcripts show increased levels of sequence divergence (dN/dS) compared to a control set, and are predominantly expressed either in testes or in the gonads of both sexes. Finally, we confirmed that XSR and XST are highly divergent by estimating sequence differentiation between the RNAseq pools. We found that X-linked transcripts were often strongly differentiated (whereas most autosomal transcripts were not), supporting the presence of a relatively large region of recombination suppression on XSR presumably caused by one or more inversions. We have identified a group of genes that are good candidates for further study into the causes and consequences of sex-chromosome drive, and demonstrated that meiotic drive has had a profound effect on sequence evolution and gene expression of X-linked genes in this species.

  10. Arabidopsis meiotic crossover hotspots overlap with H2A.Z nucleosomes at gene promoters

    PubMed Central

    Choi, Kyuha; Zhao, Xiaohui; Kelly, Krystyna A.; Venn, Oliver; Higgins, James D.; Yelina, Nataliya E.; Hardcastle, Thomas J.; Ziolkowski, Piotr A.; Copenhaver, Gregory P.; Franklin, F. Chris H.; McVean, Gil; Henderson, Ian R.

    2013-01-01

    PRDM9 directs human meiotic crossover hotspots to intergenic sequence motifs, whereas budding yeast hotspots overlap low nucleosome density regions in gene promoters. To investigate hotspots in plants, which lack PRDM9, we used coalescent analysis of Arabidopsis genetic variation. Crossovers increase towards gene promoters and terminators, and hotspots are associated with active chromatin modifications, including H2A.Z, histone H3K4me3, low nucleosome density and low DNA methylation. Hotspot-enriched A-rich and CTT-repeat DNA motifs occur upstream and downstream of transcriptional start respectively. Crossovers are asymmetric around promoters and highest over CTT-motifs and H2A.Z-nucleosomes. Pollen-typing, segregation and cytogenetic analysis show decreased crossovers in the arp6 H2A.Z deposition mutant, at multiple scales. During meiosis H2A.Z and DMC1/RAD51 recombinases form overlapping chromosomal foci. As arp6 reduces DMC1/RAD51 foci, H2A.Z may promote formation or processing of meiotic DNA double-strand breaks. We propose that gene chromatin ancestrally designates hotspots within eukaryotes and PRDM9 is a derived state within vertebrates. PMID:24056716

  11. Correlation between induction of meiotic delay and aneuploidy in male mouse germ cells

    SciTech Connect

    Adler, I.D.; Gassner, P.; Schriever-Schwemmer, G.; Min, Zhou Ru

    1993-12-31

    No aneuploidy assays are prescribed in any international guidelines for chemical safety testing up to now. The CEC-sponsored Aneuploidy Project has the aim to validate test methods for aneuploidy induction which could be used as screening tests. Furthermore, one of the major goals is to develop an understanding of mechanisms by which aneuploidy is induced. The present paper describes the investigation of meiotic delay and aneuploidy induction with the drug diazepam (DZ), the environmentally important mutagen acrylamide (AA) and the spindle poison colchicine (COL), which is used as a positive control. The time course of events was investigated. It is concluded that the assessment of meiotic delay can be used to preselect chemicals which require evaluation of aneuploidy induction during MMI in male germ cells.

  12. SLX2 interacting with BLOS2 is differentially expressed during mouse oocyte meiotic maturation.

    PubMed

    Zhuang, Xin-Jie; Shi, Yu-Qiang; Xu, Bo; Chen, Lei; Tang, Wen-Hao; Huang, Jin; Lian, Ying; Liu, Ping; Qiao, Jie

    2014-01-01

    Gametogenesis is a complex biological process of producing cells for sexual reproduction. Xlr super family members containing a conserved COR1 domain play essential roles in gametogenesis. In the present study, we identified that Slx2, a novel member of Xlr super family, is specifically expressed in the meiotic oocytes, which is demonstrated by western blotting and immunohistochemistry studies. In the first meiotic prophase, SLX2 is unevenly distributed in the nuclei of oocytes, during which phase SLX2 is partly co-localized with SYCP3 in synaptonemal complex and γH2AX in the nucleus of oocytes. Interestingly, the localization of SLX2 was found to be switched into the cytoplasm of oocytes after prometaphase I during oocyte maturation. Furthermore, yeast two-hybrid and coimmunoprecipitation studies demonstrated that SLX2 interacts with BLOS2, which is a novel centrosome-associated protein, and co-localized with γ-Tubulin, which is a protein marker of chromosome segregation in meiosis. These results indicated that SLX2 might get involved in chromosomes segregation during meiosis by interaction with BLOS2. In conclusion, SLX2 might be a novel gametogenesis-related protein that could play multiple roles in regulation of meiotic processes including synaptonemal complex assembly and chromosome segregation. PMID:24870619

  13. The Meiotic Nuclear Lamina Regulates Chromosome Dynamics and Promotes Efficient Homologous Recombination in the Mouse

    PubMed Central

    Schmitt, Johannes; Göb, Eva; Baar, Johannes; Ortega, Sagrario; Benavente, Ricardo; Alsheimer, Manfred

    2013-01-01

    The nuclear lamina is the structural scaffold of the nuclear envelope and is well known for its central role in nuclear organization and maintaining nuclear stability and shape. In the past, a number of severe human disorders have been identified to be associated with mutations in lamins. Extensive research on this topic has provided novel important clues about nuclear lamina function. These studies have contributed to the knowledge that the lamina constitutes a complex multifunctional platform combining both structural and regulatory functions. Here, we report that, in addition to the previously demonstrated significance for somatic cell differentiation and maintenance, the nuclear lamina is also an essential determinant for germ cell development. Both male and female mice lacking the short meiosis-specific A-type lamin C2 have a severely defective meiosis, which at least in the male results in infertility. Detailed analysis revealed that lamin C2 is required for telomere-driven dynamic repositioning of meiotic chromosomes. Loss of lamin C2 affects precise synapsis of the homologs and interferes with meiotic double-strand break repair. Taken together, our data explain how the nuclear lamina contributes to meiotic chromosome behaviour and accurate genome haploidization on a mechanistic level. PMID:23382700

  14. Effects of simulated weightlessness on mammalian development. Part 1: Development of clinostat for mammalian tissue culture and use in studies on meiotic maturation of mouse oocytes

    NASA Technical Reports Server (NTRS)

    Wolegemuth, D. J.; Grills, G. S.

    1984-01-01

    The effects of weightlessness on three aspects of mammalian reproduction: oocyte development, fertilization, and early embryogenesis was studied. Zero-gravity conditions within the laboratory by construction of a clinostat designed to support in vitro tissue culture were simulated and the effects of simulated weightlessness on meiotic maturation in mammalian oocytes using mouse as the model system were studied. The timing and frequency of germinal vesicule breakdown and polar body extrusion, and the structural and numerical properties of meiotic chromosomes at Metaphase and Metaphase of meiosis are assessed.

  15. Effects of Simulated Weightlessness on Mammalian Development. Part 2: Meiotic Maturation of Mouse Oocytes During Clinostat Rotation

    NASA Technical Reports Server (NTRS)

    Wolgemuth, D. J.; Grills, G. S.

    1985-01-01

    In order to understand the role of gravity in basic cellular processes that are important during development, the effects of a simulated microgravity environment on mammalian gametes and early embryos cultured in vitro are examined. A microgravity environment is simulated by use of a clinostat, which essentially reorients cells relative to the gravity vector. Initial studies have focused on assessing the effects of clinostat rotation on the meiotic progression of mouse oocytes. Modifications centered on providing the unique in vitro culture of the clinostat requirements of mammalian oocytes and embryos: 37 C temperature, constant humidity, and a 5% CO2 in air environment. The oocytes are observed under the dissecting microscope for polar body formation and gross morphological appearance. They are then processed for cytogenetic analysis.

  16. The Chromatin Protein DUET/MMD1 Controls Expression of the Meiotic Gene TDM1 during Male Meiosis in Arabidopsis.

    PubMed

    Andreuzza, Sébastien; Nishal, Bindu; Singh, Aparna; Siddiqi, Imran

    2015-09-01

    Meiosis produces haploid cells essential for sexual reproduction. In yeast, entry into meiosis activates transcription factors which trigger a transcriptional cascade that results in sequential co-expression of early, middle and late meiotic genes. However, these factors are not conserved, and the factors and regulatory mechanisms that ensure proper meiotic gene expression in multicellular eukaryotes are poorly understood. Here, we report that DUET/MMD1, a PHD finger protein essential for Arabidopsis male meiosis, functions as a transcriptional regulator in plant meiosis. We find that DUET-PHD binds H3K4me2 in vitro, and show that this interaction is critical for function during meiosis. We also show that DUET is required for proper microtubule organization during meiosis II, independently of its function in meiosis I. Remarkably, DUET protein shows stage-specific expression, confined to diplotene. We identify two genes TDM1 and JAS with critical functions in cell cycle transitions and spindle organization in male meiosis, as DUET targets, with TDM1 being a direct target. Thus, DUET is required to regulate microtubule organization and cell cycle transitions during male meiosis, and functions as a direct transcription activator of the meiotic gene TDM1. Expression profiling showed reduced expression of a subset comprising about 12% of a known set of meiosis preferred genes in the duet mutant. Our results reveal the action of DUET as a transcriptional regulator during male meiosis in plants, and suggest that transcription of meiotic genes is under stagewise control in plants as in yeast.

  17. Meiotic Drive Impacts Expression and Evolution of X-Linked Genes in Stalk-Eyed Flies

    PubMed Central

    Reinhardt, Josephine A.; Brand, Cara L.; Paczolt, Kimberly A.; Johns, Philip M.; Baker, Richard H.; Wilkinson, Gerald S.

    2014-01-01

    Although sex chromosome meiotic drive has been observed in a variety of species for over 50 years, the genes causing drive are only known in a few cases, and none of these cases cause distorted sex-ratios in nature. In stalk-eyed flies (Teleopsis dalmanni), driving X chromosomes are commonly found at frequencies approaching 30% in the wild, but the genetic basis of drive has remained elusive due to reduced recombination between driving and non-driving X chromosomes. Here, we used RNAseq to identify transcripts that are differentially expressed between males carrying either a driving X (XSR) or a standard X chromosome (XST), and found hundreds of these, the majority of which are X-linked. Drive-associated transcripts show increased levels of sequence divergence (dN/dS) compared to a control set, and are predominantly expressed either in testes or in the gonads of both sexes. Finally, we confirmed that XSR and XST are highly divergent by estimating sequence differentiation between the RNAseq pools. We found that X-linked transcripts were often strongly differentiated (whereas most autosomal transcripts were not), supporting the presence of a relatively large region of recombination suppression on XSR presumably caused by one or more inversions. We have identified a group of genes that are good candidates for further study into the causes and consequences of sex-chromosome drive, and demonstrated that meiotic drive has had a profound effect on sequence evolution and gene expression of X-linked genes in this species. PMID:24832132

  18. A Genetic Analysis of the Drosophila mcm5 Gene Defines a Domain Specifically Required for Meiotic Recombination

    PubMed Central

    Lake, Cathleen M.; Teeter, Kathy; Page, Scott L.; Nielsen, Rachel; Hawley, R. Scott

    2007-01-01

    Members of the minichromosome maintenance (MCM) family have pivotal roles in many biological processes. Although originally studied for their role in DNA replication, it is becoming increasingly apparent that certain members of this family are multifunctional and also play roles in transcription, cohesion, condensation, and recombination. Here we provide a genetic dissection of the mcm5 gene in Drosophila that demonstrates an unexpected function for this protein. First, we show that homozygotes for a null allele of mcm5 die as third instar larvae, apparently as a result of blocking those replication events that lead to mitotic divisions without impairing endo-reduplication. However, we have also recovered a viable and fertile allele of mcm5 (denoted mcm5A7) that specifically impairs the meiotic recombination process. We demonstrate that the decrease in recombination observed in females homozygous for mcm5A7 is not due to a failure to create or repair meiotically induced double strand breaks (DSBs), but rather to a failure to resolve those DSBs into meiotic crossovers. Consistent with their ability to repair meiotically induced DSBs, flies homozygous for mcm5A7 are fully proficient in somatic DNA repair. These results strengthen the observation that members of the prereplicative complex have multiple functions and provide evidence that mcm5 plays a critical role in the meiotic recombination pathway. PMID:17565942

  19. EZH2 is required for mouse oocyte meiotic maturation by interacting with and stabilizing spindle assembly checkpoint protein BubRI

    PubMed Central

    Qu, Yi; Lu, Danyu; Jiang, Hao; Chi, Xiaochun; Zhang, Hongquan

    2016-01-01

    Enhancer of zeste homolog 2 (EZH2) trimethylates histone H3 Lys 27 and plays key roles in a variety of biological processes. Stability of spindle assembly checkpoint protein BubR1 is essential for mitosis in somatic cells and for meiosis in oocytes. However, the role of EZH2 in oocyte meiotic maturation was unknown. Here, we presented a mechanism underlying EZH2 control of BubR1 stability in the meiosis of mouse oocytes. We identified a methyltransferase activity-independent function of EZH2 by demonstrating that EZH2 regulates spindle assembly and the polar body I extrusion. EZH2 was increased with the oocyte progression from GVBD to MII, while EZH2 was concentrated on the chromosomes. Interestingly, inhibition of EZH2 methyltranferase activity by DZNep or GSK343 did not affect oocyte meiotic maturation. However, depletion of EZH2 by morpholino led to chromosome misalignment and abnormal spindle assembly. Furthermore, ectopic expression of EZH2 led to oocyte meiotic maturation arrested at the MI stage followed by chromosome misalignment and aneuploidy. Mechanistically, EZH2 directly interacted with and stabilized BubR1, an effect driving EZH2 into the concert of meiosis regulation. Collectively, we provided a paradigm that EZH2 is required for mouse oocyte meiotic maturation. PMID:27226494

  20. [The effect of in-situ nerve growth factor from different biological sources on the reinitiation of mouse oocyte meiotic maturation in culture and on parthenogenetic activation].

    PubMed

    Fedorushchenko, A N; Koval', T Iu; Khamidov, D Kh

    1999-01-01

    We studied the capacity of mouse oocytes to complete meiotic maturation in vitro and form the female pronucleus upon parthenogenetic activation by cycloheximide, in response to a single injection into the mouse ovaries in situ of a purified fraction of 2.5 S NGF from mouse submaxillary glands and beta-NGF from bovine sperm. Injection of NGF from both sources at 10 ng/ml with subsequent incubation of the ovaries for 1 h increased the capacity of matured oocytes for parthenogenetic formation of the pronucleus. The frequency of pronucleus formation in both "naked oocyte" and oocytes surrounded by the cumulus cells was four times that in the control. PMID:10624718

  1. High Fat Diet Induced Developmental Defects in the Mouse: Oocyte Meiotic Aneuploidy and Fetal Growth Retardation/Brain Defects

    PubMed Central

    Purcell, Scott H.; Chi, Maggie; Jimenez, Patricia T.; Grindler, Natalia; Schedl, Tim; Moley, Kelle H.

    2012-01-01

    Background Maternal obesity is associated with poor outcomes across the reproductive spectrum including infertility, increased time to pregnancy, early pregnancy loss, fetal loss, congenital abnormalities and neonatal conditions. Furthermore, the proportion of reproductive-aged woman that are obese in the population is increasing sharply. From current studies it is not clear if the origin of the reproductive complications is attributable to problems that arise in the oocyte or the uterine environment. Methodology/Principal Findings We examined the developmental basis of the reproductive phenotypes in obese animals by employing a high fat diet mouse model of obesity. We analyzed very early embryonic and fetal phenotypes, which can be parsed into three abnormal developmental processes that occur in obese mothers. The first is oocyte meiotic aneuploidy that then leads to early embryonic loss. The second is an abnormal process distinct from meiotic aneuploidy that also leads to early embryonic loss. The third is fetal growth retardation and brain developmental abnormalities, which based on embryo transfer experiments are not due to the obese uterine environment but instead must be from a defect that arises prior to the blastocyst stage. Conclusions/Significance Our results suggest that reproductive complications in obese females are, at least in part, from oocyte maternal effects. This conclusion is consistent with IVF studies where the increased pregnancy failure rate in obese women returns to the normal rate if donor oocytes are used instead of autologous oocytes. We postulate that preconceptional weight gain adversely affects pregnancy outcomes and fetal development. In light of our findings, preconceptional counseling may be indicated as the preferable, earlier target for intervention in obese women desiring pregnancy and healthy outcomes. PMID:23152876

  2. The Sin3p PAH Domains Provide Separate Functions Repressing Meiotic Gene Transcription in Saccharomyces cerevisiae ▿

    PubMed Central

    Mallory, Michael J.; Law, Michael J.; Buckingham, Lela E.; Strich, Randy

    2010-01-01

    Meiotic genes in budding yeast are repressed during vegetative growth but are transiently induced during specific stages of meiosis. Sin3p represses the early meiotic gene (EMG) by bridging the DNA binding protein Ume6p to the histone deacetylase Rpd3p. Sin3p contains four paired amphipathic helix (PAH) domains, one of which (PAH3) is required for repressing several genes expressed during mitotic cell division. This report examines the roles of the PAH domains in mediating EMG repression during mitotic cell division and following meiotic induction. PAH2 and PAH3 are required for mitotic EMG repression, while electrophoretic mobility shift assays indicate that only PAH2 is required for stable Ume6p-promoter interaction. Unlike mitotic repression, reestablishing EMG repression following transient meiotic induction requires PAH3 and PAH4. In addition, the role of Sin3p in reestablishing repression is expanded to include additional loci that it does not control during vegetative growth. These findings indicate that mitotic and postinduction EMG repressions are mediated by two separate systems that utilize different Sin3p domains. PMID:20971827

  3. DNA damage induces a meiotic arrest in mouse oocytes mediated by the spindle assembly checkpoint

    PubMed Central

    Collins, Josie K.; Lane, Simon I. R.; Merriman, Julie A.; Jones, Keith T.

    2015-01-01

    Extensive damage to maternal DNA during meiosis causes infertility, birth defects and abortions. However, it is unknown if fully grown oocytes have a mechanism to prevent the creation of DNA-damaged embryos. Here we show that DNA damage activates a pathway involving the spindle assembly checkpoint (SAC) in response to chemically induced double strand breaks, UVB and ionizing radiation. DNA damage can occur either before or after nuclear envelope breakdown, and provides an effective block to anaphase-promoting complex activity, and consequently the formation of mature eggs. This contrasts with somatic cells, where DNA damage fails to affect mitotic progression. However, it uncovers a second function for the meiotic SAC, which in the context of detecting microtubule–kinetochore errors has hitherto been labelled as weak or ineffectual in mammalian oocytes. We propose that its essential role in the detection of DNA damage sheds new light on its biological purpose in mammalian female meiosis. PMID:26522232

  4. Meiotic Interactors of a Mitotic Gene TAO3 Revealed by Functional Analysis of its Rare Variant.

    PubMed

    Gupta, Saumya; Radhakrishnan, Aparna; Nitin, Rachana; Raharja-Liu, Pandu; Lin, Gen; Steinmetz, Lars M; Gagneur, Julien; Sinha, Himanshu

    2016-01-01

    Studying the molecular consequences of rare genetic variants has the potential to identify novel and hitherto uncharacterized pathways causally contributing to phenotypic variation. Here, we characterize the functional consequences of a rare coding variant of TAO3, previously reported to contribute significantly to sporulation efficiency variation in Saccharomyces cerevisiae During mitosis, the common TAO3 allele interacts with CBK1-a conserved NDR kinase. Both TAO3 and CBK1 are components of the RAM signaling network that regulates cell separation and polarization during mitosis. We demonstrate that the role of the rare allele TAO3(4477C) in meiosis is distinct from its role in mitosis by being independent of ACE2-a RAM network target gene. By quantitatively measuring cell morphological dynamics, and expressing the TAO3(4477C) allele conditionally during sporulation, we show that TAO3 has an early role in meiosis. This early role of TAO3 coincides with entry of cells into meiotic division. Time-resolved transcriptome analyses during early sporulation identified regulators of carbon and lipid metabolic pathways as candidate mediators. We show experimentally that, during sporulation, the TAO3(4477C) allele interacts genetically with ERT1 and PIP2, regulators of the tricarboxylic acid cycle and gluconeogenesis metabolic pathways, respectively. We thus uncover a meiotic functional role for TAO3, and identify ERT1 and PIP2 as novel regulators of sporulation efficiency. Our results demonstrate that studying the causal effects of genetic variation on the underlying molecular network has the potential to provide a more extensive understanding of the pathways driving a complex trait. PMID:27317780

  5. Meiotic Interactors of a Mitotic Gene TAO3 Revealed by Functional Analysis of its Rare Variant

    PubMed Central

    Gupta, Saumya; Radhakrishnan, Aparna; Nitin, Rachana; Raharja-Liu, Pandu; Lin, Gen; Steinmetz, Lars M.; Gagneur, Julien; Sinha, Himanshu

    2016-01-01

    Studying the molecular consequences of rare genetic variants has the potential to identify novel and hitherto uncharacterized pathways causally contributing to phenotypic variation. Here, we characterize the functional consequences of a rare coding variant of TAO3, previously reported to contribute significantly to sporulation efficiency variation in Saccharomyces cerevisiae. During mitosis, the common TAO3 allele interacts with CBK1—a conserved NDR kinase. Both TAO3 and CBK1 are components of the RAM signaling network that regulates cell separation and polarization during mitosis. We demonstrate that the role of the rare allele TAO3(4477C) in meiosis is distinct from its role in mitosis by being independent of ACE2—a RAM network target gene. By quantitatively measuring cell morphological dynamics, and expressing the TAO3(4477C) allele conditionally during sporulation, we show that TAO3 has an early role in meiosis. This early role of TAO3 coincides with entry of cells into meiotic division. Time-resolved transcriptome analyses during early sporulation identified regulators of carbon and lipid metabolic pathways as candidate mediators. We show experimentally that, during sporulation, the TAO3(4477C) allele interacts genetically with ERT1 and PIP2, regulators of the tricarboxylic acid cycle and gluconeogenesis metabolic pathways, respectively. We thus uncover a meiotic functional role for TAO3, and identify ERT1 and PIP2 as novel regulators of sporulation efficiency. Our results demonstrate that studying the causal effects of genetic variation on the underlying molecular network has the potential to provide a more extensive understanding of the pathways driving a complex trait. PMID:27317780

  6. The Chromatin Protein DUET/MMD1 Controls Expression of the Meiotic Gene TDM1 during Male Meiosis in Arabidopsis

    PubMed Central

    Andreuzza, Sébastien; Nishal, Bindu; Singh, Aparna; Siddiqi, Imran

    2015-01-01

    Meiosis produces haploid cells essential for sexual reproduction. In yeast, entry into meiosis activates transcription factors which trigger a transcriptional cascade that results in sequential co-expression of early, middle and late meiotic genes. However, these factors are not conserved, and the factors and regulatory mechanisms that ensure proper meiotic gene expression in multicellular eukaryotes are poorly understood. Here, we report that DUET/MMD1, a PHD finger protein essential for Arabidopsis male meiosis, functions as a transcriptional regulator in plant meiosis. We find that DUET-PHD binds H3K4me2 in vitro, and show that this interaction is critical for function during meiosis. We also show that DUET is required for proper microtubule organization during meiosis II, independently of its function in meiosis I. Remarkably, DUET protein shows stage-specific expression, confined to diplotene. We identify two genes TDM1 and JAS with critical functions in cell cycle transitions and spindle organization in male meiosis, as DUET targets, with TDM1 being a direct target. Thus, DUET is required to regulate microtubule organization and cell cycle transitions during male meiosis, and functions as a direct transcription activator of the meiotic gene TDM1. Expression profiling showed reduced expression of a subset comprising about 12% of a known set of meiosis preferred genes in the duet mutant. Our results reveal the action of DUET as a transcriptional regulator during male meiosis in plants, and suggest that transcription of meiotic genes is under stagewise control in plants as in yeast. PMID:26348709

  7. MEI4 – a central player in the regulation of meiotic DNA double-strand break formation in the mouse

    PubMed Central

    Kumar, Rajeev; Ghyselinck, Norbert; Ishiguro, Kei-ichiro; Watanabe, Yoshinori; Kouznetsova, Anna; Höög, Christer; Strong, Edward; Schimenti, John; Daniel, Katrin; Toth, Attila; de Massy, Bernard

    2015-01-01

    The formation of programmed DNA double-strand breaks (DSBs) at the beginning of meiotic prophase marks the initiation of meiotic recombination. Meiotic DSB formation is catalyzed by SPO11 and their repair takes place on meiotic chromosome axes. The evolutionarily conserved MEI4 protein is required for meiotic DSB formation and is localized on chromosome axes. Here, we show that HORMAD1, one of the meiotic chromosome axis components, is required for MEI4 localization. Importantly, the quantitative correlation between the level of axis-associated MEI4 and DSB formation suggests that axis-associated MEI4 could be a limiting factor for DSB formation. We also show that MEI1, REC8 and RAD21L are important for proper MEI4 localization. These findings on MEI4 dynamics during meiotic prophase suggest that the association of MEI4 to chromosome axes is required for DSB formation, and that the loss of this association upon DSB repair could contribute to turning off meiotic DSB formation. PMID:25795304

  8. A phylogenomic inventory of meiotic genes; evidence for sex in Giardia and an early eukaryotic origin of meiosis.

    PubMed

    Ramesh, Marilee A; Malik, Shehre-Banoo; Logsdon, John M

    2005-01-26

    Sexual reproduction in eukaryotes is accomplished by meiosis, a complex and specialized process of cell division that results in haploid cells (e.g., gametes). The stereotypical reductive division in meiosis is a major evolutionary innovation in eukaryotic cells, and delineating its history is key to understanding the evolution of sex. Meiosis arose early in eukaryotic evolution, but when and how meiosis arose and whether all eukaryotes have meiosis remain open questions. The known phylogenetic distribution of meiosis comprises plants, animals, fungi, and numerous protists. Diplomonads including Giardia intestinalis (syn. G. lamblia) are not known to have a sexual cycle; these protists may be an early-diverging lineage and could represent a premeiotic stage in eukaryotic evolution. We surveyed the ongoing G. intestinalis genome project data and have identified, verified, and analyzed a core set of putative meiotic genes-including five meiosis-specific genes-that are widely present among sexual eukaryotes. The presence of these genes indicates that: (1) Giardia is capable of meiosis and, thus, sexual reproduction, (2) the evolution of meiosis occurred early in eukaryotic evolution, and (3) the conserved meiotic machinery comprises a large set of genes that encode a variety of component proteins, including those involved in meiotic recombination.

  9. Meta-analysis of clinical data using human meiotic genes identifies a novel cohort of highly restricted cancer-specific marker genes

    PubMed Central

    Feichtinger, Julia; Almutairi, Mikhlid; Almatrafi, Ahmed; Alsiwiehri, Naif; Griffiths, Keith; Stuart, Nicholas; Wakeman, Jane A.; Larcombe, Lee; McFarlane, Ramsay J.

    2012-01-01

    Identifying cancer-specific biomarkers represents an ongoing challenge to the development of novel cancer diagnostic, prognostic and therapeutic strategies. Cancer/testis (CT) genes are an important gene family with expression tightly restricted to the testis in normal individuals but which can also be activated in cancers. Here we develop a pipeline to identify new CT genes. We analysed and validated expression profiles of human meiotic genes in normal and cancerous tissue followed by meta-analyses of clinical data sets from a range of tumour types resulting in the identification of a large cohort of highly specific cancer biomarker genes, including the recombination hot spot activator PRDM9 and the meiotic cohesin genes SMC1beta and RAD21L. These genes not only provide excellent cancer biomarkers for diagnostics and prognostics, but may serve as oncogenes and have excellent drug targeting potential. PMID:22918178

  10. CYS3, a hotspot of meiotic recombination in Saccharomyces cerevisiae. Effects of heterozygosity and mismatch repair functions on gene conversion and recombination intermediates.

    PubMed Central

    Vedel, M; Nicolas, A

    1999-01-01

    We have examined meiotic recombination at the CYS3 locus. Genetic analysis indicates that CYS3 is a hotspot of meiotic gene conversion, with a putative 5'-3' polarity gradient of conversion frequencies. This gradient is relieved in the presence of msh2 and pms1 mutations, indicating an involvement of mismatch repair functions in meiotic recombination. To investigate the role of mismatch repair proteins in meiotic recombination, we performed a physical analysis of meiotic DNA in wild-type and msh2 pms1 strains in the presence or absence of allelic differences at CYS3. Neither the mutations in CYS3 nor the absence of mismatch repair functions affects the frequency and distribution of nearby recombination-initiating DNA double-strand breaks (DSBs). Processing of DSBs is also similar in msh2 pms1 and wild-type strains. We conclude that mismatch repair functions do not control the distribution of meiotic gene conversion events at the initiating steps. In the MSH2 PMS1 background, strains heteroallelic for frameshift mutations in CYS3 exhibit a frequency of gene conversion greater than that observed for either marker alone. Physical analysis revealed no modification in the formation of DSBs, suggesting that this marker effect results from subsequent processing events that are not yet understood. PMID:10101154

  11. Fission yeast Mus81.Eme1 Holliday junction resolvase is required for meiotic crossing over but not for gene conversion.

    PubMed Central

    Smith, Gerald R; Boddy, Michael N; Shanahan, Paul; Russell, Paul

    2003-01-01

    Most models of homologous recombination invoke cleavage of Holliday junctions to explain crossing over. The Mus81.Eme1 endonuclease from fission yeast and humans cleaves Holliday junctions and other branched DNA structures, leaving its physiological substrate uncertain. We report here that Schizosaccharomyces pombe mus81 mutants have normal or elevated frequencies of gene conversion but 20- to 100-fold reduced frequencies of crossing over. Thus, gene conversion and crossing over can be genetically separated, and Mus81 is required for crossing over, supporting the hypothesis that the fission yeast Mus81.Eme1 protein complex resolves Holliday junctions in meiotic cells. PMID:14704204

  12. MBTD1 is associated with Pr-Set7 to stabilize H4K20me1 in mouse oocyte meiotic maturation.

    PubMed

    Luo, Yi-Bo; Ma, Jun-Yu; Zhang, Qing-Hua; Lin, Fei; Wang, Zhong-Wei; Huang, Lin; Schatten, Heide; Sun, Qing-Yuan

    2013-04-01

    H4K20me1 is a critical histone lysine methyl modification in eukaryotes. It is recognized and "read" by various histone lysine methyl modification binding proteins. In this study, the function of MBTD1, a member of the Polycomb protein family containing four MBT domains, was comprehensively studied in mouse oocyte meiotic maturation. The results showed that depletion of MBTD1 caused reduced expression of histone lysine methyl transferase Pr-Set7 and H4K20me1 as well as increased oocyte arrest at the GV stage. Increased γH2AX foci were formed, and DNA damage repair checkpoint protein 53BP1 was downregulated. Furthermore, depletion of MBTD1 activated the cell cycle checkpoint protein Chk1 and downregulated the expression of cyclin B1 and cdc2. MBTD1 knockdown also affected chromosome configuration in GV stage oocytes and chromosome alignment at the MII stage. All these phenotypes were reproduced when the H4K20 methyl transferase Pr-Set7 was depleted. Co-IP demonstrated that MBTD1 was correlated with Pr-Set7 in mouse oocytes. Our results demonstrate that MBTD1 is associated with Pr-Set7 to stabilize H4K20me1 in mouse oocyte meiotic maturation. PMID:23475131

  13. Time-Lapse Dynamics of the Mouse Oocyte Chromatin Organisation during Meiotic Resumption

    PubMed Central

    Redi, Carlo Alberto; Zuccotti, Maurizio

    2014-01-01

    In the mammalian oocyte, distinct patterns of centromeres and pericentromeric heterochromatin localisation correlate with the gamete's developmental competence. Mouse antral oocytes display two main types of chromatin organisation: SN oocytes, with a ring of Hoechst-positive chromatin surrounding the nucleolus, and NSN oocytes lacking this ring. When matured to MII and fertilised, only SN oocytes develop beyond the 2-cell, and reach full term. To give detailed information on the dynamics of the SN or NSN chromatin during meiosis resumption, we performed a 9 hr time-lapse observation. The main significant differences recorded are: (1) reduction of the nuclear area only in SN oocytes; (2) ~17 min delay of GVBD in NSN oocytes; (3) chromatin condensation, after GVBD, in SN oocytes; (4) formation of 4-5 CHCs in SN oocytes; (5) increase of the perivitelline space, ~57 min later in NSN oocytes; (6) formation of a rosette-like disposition of CHCs, ~84 min later in SN oocytes; (7) appearance of the MI plate ~40 min later in NSN oocytes. Overall, we described a pathway of transition from the GV to the MII stage that is punctuated of discrete recordable events showing their specificity and occurring with different time kinetics in the two types of oocytes. PMID:24864231

  14. The rad9 gene of Coprinus cinereus encodes a proline-rich protein required for meiotic chromosome condensation and synapsis

    SciTech Connect

    Seitz, L.C.; Tang, Keliang; Cummings, W.J.; Zolan, M.E.

    1996-04-01

    The rad9 gene of Coprinus cinereus is essential for the normal completion of meiosis. We examined surface-spread preparations of wild-type and rad9-1 nuclei from the meiotic stages of karyogamy through metaphase I, and we determined the primary sequence, structure, and meiotic expression of the rad9 gene. In wild-type C. cinereus, karyogamy is followed by condensation and alignment of homologous chromosomes. Condensation and axial core development largely precede synapsis, which often initiates at telomeres. A diffuse diplotene phase coincides with dissolution of the synaptonemal complex, and subsequently chromosomes further condense as the cells progress into metaphase I. In contrast, although karyogamy and nucleolar fusion are apparently normal in rad9-1 basidia, only short stretches of synaptonemal complex form. These correlate with stretches of condensed chromatin, mostly at apparent chromosome ends, and regions of presumptive triple synapsis are numerous. rad9-1 basidia enter the diffuse stages of early diplotene, and then 50% of these cells enter metaphase I by the criteria of nucleolar elimination and at least some chromatin condensation. rad9 gene expression is induced after gamma irradiation and during meiosis. The gene has 27 exons and encodes a predicted protein of 2157 amino acids, with a proline-rich amino terminus. 62 refs., 10 figs.

  15. FSH Modulates PKAI and GPR3 Activities in Mouse Oocyte of COC in a Gap Junctional Communication (GJC)-Dependent Manner to Initiate Meiotic Resumption

    PubMed Central

    Xia, Guoliang

    2012-01-01

    Many studies have shown that cyclic adenosine-5′-monophosphate (cAMP)-dependent protein kinase A (PKA) and G-protein-coupled receptor 3 (GPR3) are crucial for controlling meiotic arrest in oocytes. However, it is unclear how gonadotropins modulate these factors to regulate oocyte maturation, especially by gap junctional communication (GJC). Using an in vitro meiosis-arrested mouse cumulus-oocyte complex (COC) culture model, we showed that there is a close relationship between follicle-stimulating hormone (FSH) and the PKA type I (PKAI) and GPR3. The effect of FSH on oocyte maturation was biphasic, initially inhibitory and then stimulatory. During FSH-induced maturation, rapid cAMP surges were observed in both cumulus cells and oocyte. Most GJC between cumulus cells and oocyte ceased immediately after FSH stimulation and recommenced after the cAMP surge. FSH-induced maturation was blocked by PKAI activator 8-AHA-cAMP. Levels of PKAI regulatory subunits and GPR3 decreased and increased, respectively, after FSH stimulation. In the presence of the GJC inhibitor carbenoxolone (CBX), FSH failed to induce the meiotic resumption and the changes in PKAI, GPR3 and cAMP surge in oocyte were no longer detected. Furthermore, GPR3 was upregulated by high cAMP levels, but not by PKAI activation. When applied after FSH stimulation, the specific phosphodiesterase 3A (PDE3A) inhibitor cilostamide immediately blocked meiotic induction, regardless of when it was administered. PKAI activation inhibited mitogen-activated protein kinase (MAPK) phosphorylation in the oocytes of COCs, which participated in the initiation of FSH-induced meiotic maturation in vitro. Just before FSH-induced meiotic maturation, cAMP, PKAI, and GPR3 returned to basal levels, and PDE3A activity and MAPK phosphorylation increased markedly. These experiments show that FSH induces a transient increase in cAMP levels and regulates GJC to control PKAI and GPR3 activities, thereby creating an inhibitory phase. After

  16. Efficient Gene Knockdown in Mouse Oocytes through Peptide Nanoparticle-Mediated SiRNA Transfection.

    PubMed

    Jin, Zhen; Li, Ruichao; Zhou, Chunxiang; Shi, Liya; Zhang, Xiaolan; Yang, Zhixia; Zhang, Dong

    2016-01-01

    The use of mouse oocytes as a model for studying female meiosis is very important in reproductive medicine. Gene knockdown by specific small interfering RNA (siRNA) is usually the first step in the study of the function of a target gene in mouse oocytes during in vitro maturation. Traditionally, the only way to introduce siRNA into mouse oocytes is through microinjection, which is certainly less efficient and strenuous than siRNA transfection in somatic cells. Recently, in research using somatic cells, peptide nanoparticle-mediated siRNA transfection has been gaining popularity over liposome nanoparticle-mediated methods because of its high efficiency, low toxicity, good stability, and strong serum compatibility. However, no researchers have yet tried transfecting siRNA into mouse oocytes because of the existence of the protective zona pellucida surrounding the oocyte membrane (vitelline membrane). We therefore tested whether peptide nanoparticles can introduce siRNA into mouse oocytes. In the present study, we showed for the first time that our optimized program can efficiently knock down a target gene with high specificity. Furthermore, we achieved the expected meiotic phenotypes after we knocked down a test unknown target gene TRIM75. We propose that peptide nanoparticles may be superior for preliminary functional studies of unknown genes in mouse oocytes. PMID:26974323

  17. Efficient Gene Knockdown in Mouse Oocytes through Peptide Nanoparticle-Mediated SiRNA Transfection

    PubMed Central

    Jin, Zhen; Li, Ruichao; Zhou, Chunxiang; Shi, Liya; Zhang, Xiaolan; Yang, Zhixia; Zhang, Dong

    2016-01-01

    The use of mouse oocytes as a model for studying female meiosis is very important in reproductive medicine. Gene knockdown by specific small interfering RNA (siRNA) is usually the first step in the study of the function of a target gene in mouse oocytes during in vitro maturation. Traditionally, the only way to introduce siRNA into mouse oocytes is through microinjection, which is certainly less efficient and strenuous than siRNA transfection in somatic cells. Recently, in research using somatic cells, peptide nanoparticle-mediated siRNA transfection has been gaining popularity over liposome nanoparticle-mediated methods because of its high efficiency, low toxicity, good stability, and strong serum compatibility. However, no researchers have yet tried transfecting siRNA into mouse oocytes because of the existence of the protective zona pellucida surrounding the oocyte membrane (vitelline membrane). We therefore tested whether peptide nanoparticles can introduce siRNA into mouse oocytes. In the present study, we showed for the first time that our optimized program can efficiently knock down a target gene with high specificity. Furthermore, we achieved the expected meiotic phenotypes after we knocked down a test unknown target gene TRIM75. We propose that peptide nanoparticles may be superior for preliminary functional studies of unknown genes in mouse oocytes. PMID:26974323

  18. The PHD Finger Protein MMD1/DUET Ensures the Progression of Male Meiotic Chromosome Condensation and Directly Regulates the Expression of the Condensin Gene CAP-D3.

    PubMed

    Wang, Jun; Niu, Baixiao; Huang, Jiyue; Wang, Hongkuan; Yang, Xiaohui; Dong, Aiwu; Makaroff, Christopher; Ma, Hong; Wang, Yingxiang

    2016-08-01

    Chromosome condensation, a process mediated by the condensin complex, is essential for proper chromosome segregation during cell division. Unlike rapid mitotic chromosome condensation, meiotic chromosome condensation occurs over a relatively long prophase I and is unusually complex due to the coordination with chromosome axis formation and homolog interaction. The molecular mechanisms that regulate meiotic chromosome condensation progression from prophase I to metaphase I are unclear. Here, we show that the Arabidopsis thaliana meiotic PHD-finger protein MMD1/DUET is required for progressive compaction of prophase I chromosomes to metaphase I bivalents. The MMD1 PHD domain is required for its function in chromosome condensation and binds to methylated histone tails. Transcriptome analysis and qRT-PCR showed that several condensin genes exhibit significantly reduced expression in mmd1 meiocytes. Furthermore, MMD1 specifically binds to the promoter region of the condensin subunit gene CAP-D3 to enhance its expression. Moreover, cap-d3 mutants exhibit similar chromosome condensation defects, revealing an MMD1-dependent mechanism for regulating meiotic chromosome condensation, which functions in part by promoting condensin gene expression. Together, these discoveries provide strong evidence that the histone reader MMD1/DUET defines an important step for regulating the progression of meiotic prophase I chromosome condensation.

  19. The PHD Finger Protein MMD1/DUET Ensures the Progression of Male Meiotic Chromosome Condensation and Directly Regulates the Expression of the Condensin Gene CAP-D3.

    PubMed

    Wang, Jun; Niu, Baixiao; Huang, Jiyue; Wang, Hongkuan; Yang, Xiaohui; Dong, Aiwu; Makaroff, Christopher; Ma, Hong; Wang, Yingxiang

    2016-08-01

    Chromosome condensation, a process mediated by the condensin complex, is essential for proper chromosome segregation during cell division. Unlike rapid mitotic chromosome condensation, meiotic chromosome condensation occurs over a relatively long prophase I and is unusually complex due to the coordination with chromosome axis formation and homolog interaction. The molecular mechanisms that regulate meiotic chromosome condensation progression from prophase I to metaphase I are unclear. Here, we show that the Arabidopsis thaliana meiotic PHD-finger protein MMD1/DUET is required for progressive compaction of prophase I chromosomes to metaphase I bivalents. The MMD1 PHD domain is required for its function in chromosome condensation and binds to methylated histone tails. Transcriptome analysis and qRT-PCR showed that several condensin genes exhibit significantly reduced expression in mmd1 meiocytes. Furthermore, MMD1 specifically binds to the promoter region of the condensin subunit gene CAP-D3 to enhance its expression. Moreover, cap-d3 mutants exhibit similar chromosome condensation defects, revealing an MMD1-dependent mechanism for regulating meiotic chromosome condensation, which functions in part by promoting condensin gene expression. Together, these discoveries provide strong evidence that the histone reader MMD1/DUET defines an important step for regulating the progression of meiotic prophase I chromosome condensation. PMID:27385818

  20. The pam1 gene is required for meiotic bouquet formation and efficient homologous synapsis in maize (Zea mays L.).

    PubMed Central

    Golubovskaya, Inna N; Harper, Lisa C; Pawlowski, Wojciech P; Schichnes, Denise; Cande, W Zacheus

    2002-01-01

    The clustering of telomeres on the nuclear envelope (NE) during meiotic prophase to form the bouquet arrangement of chromosomes may facilitate homologous chromosome synapsis. The pam1 (plural abnormalities of meiosis 1) gene is the first maize gene that appears to be required for telomere clustering, and homologous synapsis is impaired in pam1. Telomere clustering on the NE is arrested or delayed at an intermediate stage in pam1. Telomeres associate with the NE during the leptotene-zygotene transition but cluster slowly if at all as meiosis proceeds. Intermediate stages in telomere clustering including miniclusters are observed in pam1 but not in wild-type meiocytes. The tight bouquet normally seen at zygotene is a rare event. In contrast, the polarization of centromeres vs. telomeres in the nucleus at the leptotene-zygotene transition is the same in mutant and wild-type cells. Defects in homologous chromosome synapsis include incomplete synapsis, nonhomologous synapsis, and unresolved interlocks. However, the number of RAD51 foci on chromosomes in pam1 is similar to that of wild type. We suggest that the defects in homologous synapsis and the retardation of prophase I arise from the irregularity of telomere clustering and propose that pam1 is involved in the control of bouquet formation and downstream meiotic prophase I events. PMID:12524364

  1. From genes to games: cooperation and cyclic dominance in meiotic drive.

    PubMed

    Traulsen, Arne; Reed, Floyd A

    2012-04-21

    Evolutionary change can be described on a genotypic level or a phenotypic level. Evolutionary game theory is typically thought of as a phenotypic approach, although it is frequently argued that it can also be used to describe population genetic evolution. Interpreting the interaction between alleles in a diploid genome as a two player game leads to interesting alternative perspectives on genetic evolution. Here we focus on the case of meiotic drive and illustrate how meiotic drive can be directly and precisely interpreted as a social dilemma, such as the prisoners dilemma or the snowdrift game, in which the drive allele takes more than its fair share. Resistance to meiotic drive can lead to the well understood cyclic dominance found in the rock-paper-scissors game. This perspective is well established for the replicator dynamics, but there is still considerable ground for mutual inspiration between the two fields. For example, evolutionary game theorists can benefit from considering the stochastic evolutionary dynamics arising from finite population size. Population geneticists can benefit from game theoretic tools and perspectives on genetic evolution.

  2. Meiotic abnormalities

    SciTech Connect

    1993-12-31

    Chapter 19, describes meiotic abnormalities. These include nondisjunction of autosomes and sex chromosomes, genetic and environmental causes of nondisjunction, misdivision of the centromere, chromosomally abnormal human sperm, male infertility, parental age, and origin of diploid gametes. 57 refs., 2 figs., 1 tab.

  3. Whole-Genome Analysis of Individual Meiotic Events in Drosophila melanogaster Reveals That Noncrossover Gene Conversions Are Insensitive to Interference and the Centromere Effect

    PubMed Central

    Miller, Danny E.; Smith, Clarissa B.; Kazemi, Nazanin Yeganeh; Cockrell, Alexandria J.; Arvanitakis, Alexandra V.; Blumenstiel, Justin P.; Jaspersen, Sue L.; Hawley, R. Scott

    2016-01-01

    A century of genetic analysis has revealed that multiple mechanisms control the distribution of meiotic crossover events. In Drosophila melanogaster, two significant positional controls are interference and the strongly polar centromere effect. Here, we assess the factors controlling the distribution of crossovers (COs) and noncrossover gene conversions (NCOs) along all five major chromosome arms in 196 single meiotic divisions to generate a more detailed understanding of these controls on a genome-wide scale. Analyzing the outcomes of single meiotic events allows us to distinguish among different classes of meiotic recombination. In so doing, we identified 291 NCOs spread uniformly among the five major chromosome arms and 541 COs (including 52 double crossovers and one triple crossover). We find that unlike COs, NCOs are insensitive to the centromere effect and do not demonstrate interference. Although the positions of COs appear to be determined predominately by the long-range influences of interference and the centromere effect, each chromosome may display a different pattern of sensitivity to interference, suggesting that interference may not be a uniform global property. In addition, unbiased sequencing of a large number of individuals allows us to describe the formation of de novo copy number variants, the majority of which appear to be mediated by unequal crossing over between transposable elements. This work has multiple implications for our understanding of how meiotic recombination is regulated to ensure proper chromosome segregation and maintain genome stability. PMID:26944917

  4. The Mouse Gene Expression Database (GXD)

    PubMed Central

    Ringwald, Martin; Eppig, Janan T.; Begley, Dale A.; Corradi, John P.; McCright, Ingeborg J.; Hayamizu, Terry F.; Hill, David P.; Kadin, James A.; Richardson, Joel E.

    2001-01-01

    The Gene Expression Database (GXD) is a community resource of gene expression information for the laboratory mouse. By combining the different types of expression data, GXD aims to provide increasingly complete information about the expression profiles of genes in different mouse strains and mutants, thus enabling valuable insights into the molecular networks that underlie normal development and disease. GXD is integrated with the Mouse Genome Database (MGD). Extensive interconnections with sequence databases and with databases from other species, and the development and use of shared controlled vocabularies extend GXD’s utility for the analysis of gene expression information. GXD is accessible through the Mouse Genome Informatics web site at http://www.informatic s.jax.org/ or directly at http://www.informatics.jax.org/me nus/expression_menu.shtml. PMID:11125060

  5. Boule gene expression underpins the meiotic arrest in spermatogenesis in male rainbow trout (Oncorhynchus mykiss) exposed to DEHP and butachlor.

    PubMed

    Ahmadivand, Sohrab; Farahmand, Hamid; Teimoori-Toolabi, Ladan; Mirvaghefi, Alireza; Eagderi, Soheil; Geerinckx, Tom; Shokrpoor, Sara; Rahmati-Holasoo, Hooman

    2016-01-01

    Boule, the ancestor of the DAZ (Deleted in AZoospermia) gene family, in most organisms is mainly involved in male meiosis. The present study investigates the effects of the plasticizer DEHP (50mg/kg body weight) and herbicide butachlor (0.39mg/L) on male rainbow trout (Oncorhynchus mykiss) for a 10-day period in two independent experiments. The results showed that plasma testosterone (T) concentrations were significantly lower in fish exposed to either DEHP or butachlor compared to the control fish (P<0.05). Fish showed a significantly elevated hepatosomatic index (HSI) in the butachlor treatment (P<0.05). However, no significant difference was observed in HSI values in the DEHP treatment (P>0.05). In addition, no significant differences were found in the gonadosomatic index (GSI) in both DEHP and butachlor treatments (P>0.05). Histologically, testes of male trout in the control groups were well differentiated and filled with large numbers of cystic structures containing spermatozoa. In contrast, the testes of male trout contained mostly spermatocytes with few spermatozoa in both treated group, suggesting that DEHP and butachlor may inhibit the progression of meiosis. Also, boule gene expression was significantly lower in the testes of male trout affected by DEHP and butachlor in comparison with their control groups (P<0.05), which confirmed the meiotic arrest in affected trout. Based on the results, the present study demonstrated that DEHP and butachlor can inhibit the progression of spermatogenesis in male trout, potentially by causing an arrest of meiosis, maybe due to down-regulation of boule gene expression through T and/or IGF1 via ERK1/2 signaling in T-independent pathways. In addition, these results confirmed that boule can be considered as a predictive marker to assess meiotic efficiency.

  6. Boule gene expression underpins the meiotic arrest in spermatogenesis in male rainbow trout (Oncorhynchus mykiss) exposed to DEHP and butachlor.

    PubMed

    Ahmadivand, Sohrab; Farahmand, Hamid; Teimoori-Toolabi, Ladan; Mirvaghefi, Alireza; Eagderi, Soheil; Geerinckx, Tom; Shokrpoor, Sara; Rahmati-Holasoo, Hooman

    2016-01-01

    Boule, the ancestor of the DAZ (Deleted in AZoospermia) gene family, in most organisms is mainly involved in male meiosis. The present study investigates the effects of the plasticizer DEHP (50mg/kg body weight) and herbicide butachlor (0.39mg/L) on male rainbow trout (Oncorhynchus mykiss) for a 10-day period in two independent experiments. The results showed that plasma testosterone (T) concentrations were significantly lower in fish exposed to either DEHP or butachlor compared to the control fish (P<0.05). Fish showed a significantly elevated hepatosomatic index (HSI) in the butachlor treatment (P<0.05). However, no significant difference was observed in HSI values in the DEHP treatment (P>0.05). In addition, no significant differences were found in the gonadosomatic index (GSI) in both DEHP and butachlor treatments (P>0.05). Histologically, testes of male trout in the control groups were well differentiated and filled with large numbers of cystic structures containing spermatozoa. In contrast, the testes of male trout contained mostly spermatocytes with few spermatozoa in both treated group, suggesting that DEHP and butachlor may inhibit the progression of meiosis. Also, boule gene expression was significantly lower in the testes of male trout affected by DEHP and butachlor in comparison with their control groups (P<0.05), which confirmed the meiotic arrest in affected trout. Based on the results, the present study demonstrated that DEHP and butachlor can inhibit the progression of spermatogenesis in male trout, potentially by causing an arrest of meiosis, maybe due to down-regulation of boule gene expression through T and/or IGF1 via ERK1/2 signaling in T-independent pathways. In addition, these results confirmed that boule can be considered as a predictive marker to assess meiotic efficiency. PMID:26027538

  7. Effect of inhibition of sterol delta 14-reductase on accumulation of meiosis-activating sterol and meiotic resumption in cumulus-enclosed mouse oocytes in vitro.

    PubMed

    Leonardsen, L; Strömstedt, M; Jacobsen, D; Kristensen, K S; Baltsen, M; Andersen, C Y; Byskov, A G

    2000-01-01

    Two sterols of the cholesterol biosynthetic pathway induce resumption of meiosis in mouse oocytes in vitro. The sterols, termed meiosis-activating sterols (MAS), have been isolated from human follicular fluid (FF-MAS, 4,4-dimethyl-5 alpha-cholest-8,14,24-triene-3 beta-ol) and from bull testicular tissue (T-MAS, 4,4-dimethyl-5 alpha-cholest-8,24-diene-3 beta-ol). FF-MAS is the first intermediate in the cholesterol biosynthesis from lanosterol and is converted to T-MAS by sterol delta 14-reductase. An inhibitor of delta 7-reductase and delta 14 reductase, AY9944-A-7, causes cells with a constitutive cholesterol biosynthesis to accumulate FF-MAS and possibly other intermediates between lanosterol and cholesterol. The aim of the present study was to evaluate whether AY9944-A-7 added to cultures of cumulus-oocyte complexes (COC) from mice resulted in accumulation of MAS and meiotic maturation. AY9944-A-7 stimulated dose dependently (5-25 mumol l-1) COC to resume meiosis when cultured for 22 h in alpha minimal essential medium (alpha-MEM) containing 4 mmol hypoxanthine l-1, a natural inhibitor of meiotic maturation. In contrast, naked oocytes were not induced to resume meiosis by AY9944-A-7. When cumulus cells were separated from their oocytes and co-cultured, AY9944-A-7 did not affect resumption of meiosis, indicating that intact oocyte-cumulus cell connections are important for AY9944-A-7 to exert its effect on meiosis. Cultures of COC with 10 mumol AY9944-A-7 l-1 in the presence of [3H]mevalonic acid, a natural precursor for steroid synthesis, resulted in accumulation of labelled FF-MAS, which had an 11-fold greater amount of radioactivity incorporated per COC compared with the control culture without AY9944-A-7. In contrast, incorporation of radioactivity into the cholesterol fraction was reduced 30-fold in extracts from the same oocytes. The present findings demonstrate for the first time that COC can synthesize cholesterol from mevalonate and accumulate FF-MAS in

  8. Dis1: A Yeast Gene Required for Proper Meiotic Chromosome Disjunction

    PubMed Central

    Rockmill, B.; Fogel, S.

    1988-01-01

    Mutants at a newly identified locus, DIS1 (disjunction), were detected by screening for mutants that generate aneuploid spores (chromosome VIII disomes) at an increased frequency. Strains carrying the partially dominant alleles, DIS1-1 or DIS1-2, generate disomes at rates up to 100 times the background level. Mitotic nondisjunction is also increased 10- to 50-fold over background. Half-tetrad analysis of disomes for a marked interval on chromosome VIII yields wild-type map distances, indicating that a general recombination deficiency is not the cause of nondisjuction. Meiotic nondisjunction in DIS1 mutants is not chromosome specific; 5% of the spores disomic for chromosome VIII are also disomic for chromosome III. Although only one disomic spore is found per exceptional ascus most of the disomes appear to be generated in the first meiotic division because recovered chromosome VIII disomes contain mostly nonsister chromosomes. We propose that disome generation in the DIS1 mutants results from precocious separation of sister centromeres. PMID:3294101

  9. The mouse fidgetin gene defines a new role for AAA family proteins in mammalian development.

    PubMed

    Cox, G A; Mahaffey, C L; Nystuen, A; Letts, V A; Frankel, W N

    2000-10-01

    The mouse mutation fidget arose spontaneously in a heterogeneous albino stock. This mutant mouse is characterized by a side-to-side head-shaking and circling behaviour, due to reduced or absent semicircular canals. Fidget mice also have small eyes, associated with cell-cycle delay and insufficient growth of the retinal neural epithelium, and lower penetrance skeletal abnormalities, including pelvic girdle dysgenesis, skull bone fusions and polydactyly. By positional cloning, we found the gene mutated in fidget mice, fidgetin (Fign), which encodes a new member of the 'meiotic' or subfamily-7 (SF7; ref. 7) group of ATPases associated with diverse cellular activities (AAA proteins). We also discovered two closely related mammalian genes. AAA proteins are molecular chaperones that facilitate a variety of functions, including membrane fusion, proteolysis, peroxisome biogenesis, endosome sorting and meiotic spindle formation, but functions for the SF7 AAA proteins are largely unknown. Fidgetin is the first mutant AAA protein found in a mammalian developmental mutant, thus defining a new role for these proteins in embryonic development.

  10. The polyubiquitin gene Ubi-p63E is essential for male meiotic cell cycle progression and germ cell differentiation in Drosophila.

    PubMed

    Lu, Chenggang; Kim, Jongmin; Fuller, Margaret T

    2013-09-01

    The ubiquitin proteasome system (UPS) regulates many biological pathways by post-translationally ubiquitylating proteins for degradation. Although maintaining a dynamic balance between free ubiquitin and ubiquitylated proteins is key to UPS function, the mechanisms that regulate ubiquitin homeostasis in different tissues through development are not clear. Here we show, via analysis of the magellan (magn) complementation group, that loss of function of the Drosophila polyubiquitin Ubi-p63E results specifically in meiotic arrest sterility in males. Ubi-p63E contributes predominantly to maintaining the free ubiquitin pool in testes. The function of Ubi-p63E is required cell-autonomously for proper meiotic chromatin condensation, cell cycle progression and spermatid differentiation. magn mutant germ cells develop normally to the spermatocyte stage but arrest at the G2/M transition of meiosis I, with lack of protein expression of the key meiotic cell cycle regulators Boule and Cyclin B. Loss of Ubi-p63E function did not strongly affect the spermatocyte transcription program regulated by the testis TBP-associated factor (tTAF) or meiosis arrest complex (tMAC) genes. Knocking down proteasome function specifically in spermatocytes caused a different meiotic arrest phenotype, suggesting that the magn phenotype might not result from general defects in protein degradation. Our results suggest a conserved role of polyubiquitin genes in male meiosis and a potential mechanism leading to meiosis I maturation arrest.

  11. The behavior of the X- and Y-chromosomes in the oocyte during meiotic prophase in the B6.Y(TIR)sex-reversed mouse ovary.

    PubMed

    Alton, Michelle; Lau, Mau Pan; Villemure, Michele; Taketo, Teruko

    2008-02-01

    Sexual differentiation of the germ cells follows gonadal differentiation, which is determined by the presence or the absence of the Y-chromosome. Consequently, oogenesis and spermatogenesis take place in the germ cells with XX and XY sex chromosomal compositions respectively. It is unclear how sexual dimorphic regulation of meiosis is associated with the sex-chromosomal composition. In the present study, we examined the behavior of the sex chromosomes in the oocytes of the B6.Y(TIR) sex-reversed female mouse, in comparison with XO and XX females. As the sex chromosomes fail to pair in both XY and XO oocytes during meiotic prophase, we anticipated that the pairing failure may lead to excessive oocyte loss. However, the total number of germ cells, identified by immunolabeling of germ cell nuclear antigen 1 (GCNA1), did not differ between XY and XX ovaries or XO and XX ovaries up to the day of delivery. The progression of meiotic prophase, assessed by immunolabeling of synaptonemal complex components, was also similar between the two genotypes of ovaries. These observations suggest that the failure in sex-chromosome pairing is not sufficient to cause oocyte loss. On the other hand, labeling of phosphorylated histone gammaH2AX, known to be associated with asynapsis and transcriptional repression, was seen over the X-chromosome but not over the Y-chromosome in the majority of XY oocytes at the pachytene stage. For comparison, gammaH2AX labeling was seen only in the minority of XX oocytes at the same stage. We speculate that the transcriptional activity of sex chromosomes in the XY oocyte may be incompatible with ooplasmic maturation. PMID:18239052

  12. In situ localization of mRNAs coding for mouse testicular structural genes

    SciTech Connect

    Hecht, N.B. ); Penshow, J.D. )

    1987-11-01

    In situ hybridization histochemistry has been used to localize mRNA transcripts of five nuclear and cytoplasmic structural genes in the mouse testis. The mRNAs for three nuclear structural proteins involved in chromatin transformation during spermatogenesis (the two protamine variants of the mouse and one of the testis-specific proteins) are restricted solely to postmeiotic germ cells. In contrast, mRNAs for two other structural proteins, actin and {alpha} tubulin, are detected throughout spermatogenesis. Although present in premeiotic, meiotic, and postmeiotic cell types, the mRNA levels of actin and {alpha} tubulin differ considerably during spermiogenesis, the haploid phase of spermatogenesis. Actin mRNA levels decrease markedly as the male gamete differentiates during spermiogenesis whereas {alpha}-tubulin mRNAs are equally abundant in the haploid round and elongating spermatids.

  13. Unique subcellular distribution of phosphorylated Plk1 (Ser137 and Thr210) in mouse oocytes during meiotic division and pPlk1(Ser137) involvement in spindle formation and REC8 cleavage.

    PubMed

    Du, Juan; Cao, Yan; Wang, Qian; Zhang, Nana; Liu, Xiaoyu; Chen, Dandan; Liu, Xiaoyun; Xu, Qunyuan; Ma, Wei

    2015-01-01

    Polo-like kinase 1 (Plk1) is pivotal for proper mitotic progression, its targeting activity is regulated by precise subcellular positioning and phosphorylation. Here we assessed the protein expression, subcellular localization and possible functions of phosphorylated Plk1 (pPlk1(Ser137) and pPlk1(Thr210)) in mouse oocytes during meiotic division. Western blot analysis revealed a peptide of pPlk1(Ser137) with high and stable expression from germinal vesicle (GV) until metaphase II (MII), while pPlk1(Thr210) was detected as one large single band at GV stage and 2 small bands after germinal vesicle breakdown (GVBD), which maintained stable up to MII. Immunofluorescence analysis showed pPlk1(Ser137) was colocalized with microtubule organizing center (MTOC) proteins, γ-tubulin and pericentrin, on spindle poles, concomitantly with persistent concentration at centromeres and dynamic aggregation between chromosome arms. Differently, pPlk1(Thr210) was persistently distributed across the whole body of chromosomes after meiotic resumption. The specific Plk1 inhibitor, BI2536, repressed pPlk1(Ser137) accumulation at MTOCs and between chromosome arms, consequently disturbed γ-tubulin and pericentrin recruiting to MTOCs, destroyed meiotic spindle formation, and delayed REC8 cleavage, therefore arresting oocytes at metaphase I (MI) with chromosome misalignment. BI2536 completely reversed the premature degradation of REC8 and precocious segregation of chromosomes induced with okadaic acid (OA), an inhibitor to protein phosphatase 2A. Additionally, the protein levels of pPlk1(Ser137) and pPlk1(Thr210), as well as the subcellular distribution of pPlk1(Thr210), were not affected by BI2536. Taken together, our results demonstrate that Plk1 activity is required for meiotic spindle assembly and REC8 cleavage, with pPlk1(Ser137) is the action executor, in mouse oocytes during meiotic division. PMID:26654596

  14. Mei-1, a Gene Required for Meiotic Spindle Formation in Caenorhabditis Elegans, Is a Member of a Family of ATPases

    PubMed Central

    Clark-Maguire, S.; Mains, P. E.

    1994-01-01

    Meiotic spindle formation in the female germline of Caenorhabditis elegans requires expression of the gene mei-1. We have cloned mei-1 by transformation rescue and found that it resides near a hot spot for recombination, in an area of high gene density. The highest levels of mei-1 mRNA accumulate in the female germline of adult hermaphrodites as well as in fertilized embryos. The message persists for several hours after the protein functions in embryos, implying the need for post-transcriptional regulation. Two alternatively spliced messages are made that would result in proteins that differ internally by three amino acids; the larger of the two mRNAs is preferentially enriched in the female germline. The sequence of mei-1 shows that it is a member of a newly described family of ATPases that share a highly conserved nucleotide-binding site; four dominant-negative mutations of mei-1 are found at or near this region. Divergent roles ascribed to this family include membrane function, proteolysis, transcription and cell cycle regulation. PMID:8150281

  15. Protist homologs of the meiotic Spo11 gene and topoisomerase VI reveal an evolutionary history of gene duplication and lineage-specific loss.

    PubMed

    Malik, Shehre-Banoo; Ramesh, Marilee A; Hulstrand, Alissa M; Logsdon, John M

    2007-12-01

    Spo11 is a meiotic protein of fundamental importance as it is a conserved meiosis-specific transesterase required for meiotic recombination initiation in fungi, animals, and plants. Spo11 is homologous to the archaebacterial topoisomerase VIA (Top6A) gene, and its homologs are broadly distributed among eukaryotes, with some eukaryotes having more than one homolog. However, the evolutionary relationships among these genes are unclear, with some debate as to whether eukaryotic homologs originated by lateral gene transfer. We have identified and characterized protist Spo11 homologs by degenerate polymerase chain reaction (PCR) and sequencing and by analyses of sequences from public databases. Our phylogenetic analyses show that Spo11 homologs evolved by two ancient eukaryotic gene duplication events prior to the last common ancestor of extant eukaryotes, resulting in three eukaryotic paralogs: Spo11-1, Spo11-2, and Spo11-3. Spo11-1 orthologs encode meiosis-specific proteins and are distributed broadly among eukaryotic lineages, though Spo11-1 is absent from some protists. This absence coincides with the presence of Spo11-2 orthologs, which are meiosis-specific in Arabidopsis and are found in plants, red algae, and some protists but absent in animals and fungi. Spo11-3 encodes a Top6A subunit that interacts with topoisomerase VIB (Top6B) subunits, which together play a role in vegetative growth in Arabidopsis. We identified Spo11-3 (Top6A) and Top6B homologs in plants, red algae, and a few protists, establishing a broader distribution of these genes among eukaryotes, indicating their likely vertical descent followed by lineage-specific loss.

  16. Cuf2 Is a Transcriptional Co-Regulator that Interacts with Mei4 for Timely Expression of Middle-Phase Meiotic Genes

    PubMed Central

    Ioannoni, Raphaël; Brault, Ariane; Labbé, Simon

    2016-01-01

    The Schizosaccharomyces pombe cuf2+ gene encodes a nuclear regulator that is required for timely activation and repression of several middle-phase genes during meiotic differentiation. In this study, we sought to gain insight into the mechanism by which Cuf2 regulates meiotic gene expression. Using a chromatin immunoprecipitation approach, we demonstrate that Cuf2 is specifically associated with promoters of both activated and repressed target genes, in a time-dependent manner. In case of the fzr1+ gene whose transcription is positively affected by Cuf2, promoter occupancy by Cuf2 results in a concomitant increased association of RNA polymerase II along its coding region. In marked contrast, association of RNA polymerase II with chromatin decreases when Cuf2 negatively regulates target gene expression such as wtf13+. Although Cuf2 operates through a transcriptional mechanism, it is unable to perform its function in the absence of the Mei4 transcription factor, which is a member of the conserved forkhead protein family. Using coimmunoprecipitation experiments, results showed that Cuf2 is a binding partner of Mei4. Bimolecular fluorescence complementation experiments brought further evidence that an association between Cuf2 and Mei4 occurs in the nucleus. Analysis of fzr1+ promoter regions revealed that two FLEX-like elements, which are bound by the transcription factor Mei4, are required for chromatin occupancy by Cuf2. Together, results reported here revealed that Cuf2 and Mei4 co-regulate the timely expression of middle-phase genes during meiosis. PMID:26986212

  17. Regulation of Meiotic Recombination

    SciTech Connect

    Gregory p. Copenhaver

    2011-11-09

    Meiotic recombination results in the heritable rearrangement of DNA, primarily through reciprocal exchange between homologous chromosome or gene conversion. In plants these events are critical for ensuring proper chromosome segregation, facilitating DNA repair and providing a basis for genetic diversity. Understanding this fundamental biological mechanism will directly facilitate trait mapping, conventional plant breeding, and development of genetic engineering techniques that will help support the responsible production and conversion of renewable resources for fuels, chemicals, and the conservation of energy (1-3). Substantial progress has been made in understanding the basal recombination machinery, much of which is conserved in organisms as diverse as yeast, plants and mammals (4, 5). Significantly less is known about the factors that regulate how often and where that basal machinery acts on higher eukaryotic chromosomes. One important mechanism for regulating the frequency and distribution of meiotic recombination is crossover interference - or the ability of one recombination event to influence nearby events. The MUS81 gene is thought to play an important role in regulating the influence of interference on crossing over. The immediate goals of this project are to use reverse genetics to identify mutants in two putative MUS81 homologs in the model plant Arabidopsis thaliana, characterize those mutants and initiate a novel forward genetic screen for additional regulators of meiotic recombination. The long-term goal of the project is to understand how meiotic recombination is regulated in higher eukaryotes with an emphasis on the molecular basis of crossover interference. The ability to monitor recombination in all four meiotic products (tetrad analysis) has been a powerful tool in the arsenal of yeast geneticists. Previously, the qrt mutant of Arabidopsis, which causes the four pollen products of male meiosis to remain attached, was developed as a facile system

  18. Meiotic recombination at the Lmp2 hotspot tolerates minor sequence divergence between homologous chromosomes

    SciTech Connect

    Yoshino, Masayasu; Sagai, Tomoko; Shiroishi, Toshihiko

    1996-06-01

    Recombination is widely considered to linearly depend on the length of the homologous sequences. An 11% mismatch decreases the rate of phage-plasmid recombination 240-fold. Two single nucleotide mismatches, which reduce the longest uninterrupted stretch of similarity from 232 base pairs (bp) to 134 bp, reduce gene conversion in mouse L cells 20-fold. The efficiency of gene targeting through homologous recombination in mouse embryonic stem cells can be increased by using an isogenic, rather than a non-isogenic, DNA construct. In this study we asked whether a high degree of sequence identity between homologous mouse chromosomes enhances meiotic recombination at a hotspot. Sites of meiotic recombination in the mouse major histocompatibility complex (MHC) class II region are not randomly distributed but are almost all clustered within short segments known as recombinational hotspots. The wm7 MHC haplotype, derived from Japanese wild mice Mus musculus molossinus, enhances meiotic recombination at a hotspot near the Lmp2 gene. Heterozygotes between the wm7 haplotype and the b or k haplotypes have yielded a high frequency of recombination (2.1%) in 1.3 kilobase kb segment of this hotspot. 20 refs., 2 figs.

  19. Swi6, a Gene Required for Mating-Type Switching, Prohibits Meiotic Recombination in the Mat2-Mat3 ``cold Spot'' of Fission Yeast

    PubMed Central

    Klar, AJS.; Bonaduce, M. J.

    1991-01-01

    Mitotic interconversion of the mating-type locus (mat1) of the fission yeast Schizosaccharomyces pombe is initiated by a double-strand break at mat1. The mat2 and mat3 loci act as nonrandom donors of genetic information for mat1 switching such that switches occur primarily (or only) to the opposite mat1 allele. Location of the mat1 ``hot spot'' for transposition should be contrasted with the ``cold spot'' of meiotic recombination located within the adjoining mat2-mat3 interval. That is, meiotic interchromosomal recombination in mat2, mat3 and the intervening 15-kilobase region does not occur at all. swi2 and swi6 switching-deficient mutants possess the normal level of double-strand break at mat1, yet they fail to switch efficiently. By testing for meiotic recombination in the cold spot, we found the usual lack of recombination in a swi2 mutant but a significant level of recombination in a swi6 mutant. Therefore, the swi6 gene function is required to keep the donor loci inert for interchromosomal recombination. This finding, combined with the additional result that switching primarily occurs intrachromosomally, suggests that the donor loci are made accessible for switching by folding them onto mat1, thus causing the cold spot of recombination. PMID:1783290

  20. The human and mouse homologs of the yeat RAD52 gene: cDNA cloning, sequence analysis, assignment to human chromosome 12p12.2-p13, and mRNA expression in mouse tissues

    SciTech Connect

    Shen, Z.; Chen, D.J.; Denison, K.

    1995-01-01

    The yeast Saccharomyces cerevisiae RAD52 gene is involved in DNA double-strand break repair and mitotic/meiotic recombination. The N-terminal amino acid sequence of yeast S. cerevisiae, Schizosaccharomyces pombe, and Kluyveromyces lactis and chicken is highly conserved. Using the technology of mixed oligonucleotide primed amplification of cDNA (MOPAC), two mouse RAD52 homologous cDNA fragments were amplified and sequenced. Subsequently, we have cloned the cDNA of the human and mouse homologs of yeast RAD52 gene by screening cDNA libraries using the identified mouse cDNA fragments. Sequence analysis of cDNA derived amino acid revealed a highly conserved N-terminus among human, mouse, chicken, and yeast RAD52 genes. The human RAD52 gene was assigned to chromosome 12p12.2-p13 by fluorescence in situ hybridization, R-banding, and DNA analysis of somatic cell hybrids. Unlike chicken RAD52 and mouse RAD51, no significant difference in mouse RAD52 mRNA level was found among mouse heart, brain, spleen, lung, liver, skeletal muscle, kidney, and testis. In addition to an {approximately}1.9-kb RAD52 mRNA band that is present in all of the tested tissues, an extra mRNA species of {approximately}0.85 kb was detectable in mouse testis. 40 refs., 7 figs., 1 tab.

  1. Physical mapping of male fertility and meiotic drive quantitative trait loci in the mouse t complex using chromosome deficiencies.

    PubMed Central

    Planchart, A; You, Y; Schimenti, J C

    2000-01-01

    The t complex spans 20 cM of the proximal region of mouse chromosome 17. A variant form, the t haplotype (t), exists at significant frequencies in wild mouse populations and is characterized by the presence of inversions that suppress recombination with wild-type (+) chromosomes. Transmission ratio distortion and sterility are associated with t and affect males only. It is hypothesized that these phenomena are caused by trans-acting distorter/sterility factors that interact with a responder locus (Tcr(t)) and that the distorter and sterility factors are the same because homozygosity of the distorters causes male sterility. One factor, Tcd1, was previously shown to be amorphic using a chromosome deletion. To overcome limitations imposed by recombination suppression, we used a series of deletions within the t complex in trans to t chromosomes to characterize the Tcd1 region. We find that the distorter activity of Tcd1 is distinct from a linked sterility factor, originally called tcs1. YACs mapped with respect to deletion breakpoints localize tcs1 to a 1.1-Mb interval flanked by D17Aus9 and Tctex1. We present evidence for the existence of multiple proximal t complex regions that exhibit distorter activity. These studies demonstrate the utility of chromosome deletions for complex trait analysis. PMID:10835401

  2. Three genes for metabolism of the phytoalexin maackiain in the plant pathogen Nectria haematococca: Meiotic instability and relationship to a new gene for pisatin demethylase

    SciTech Connect

    Miao, V.P.W.; Vanetten, H.D. )

    1992-03-01

    Some isolates of the plant-pathogenic fungus Nectria haematococca mating population (MP) VI metabolize maackiain and medicarpin, two antimicrobial compounds (phytoalexins) synthesized by chickpea (Cicer arietinum L.). The enzymatic modifications by the fungus convert the phytoalexins to less toxic derivatives, and this detoxification has been proposed to be important for pathogenesis on chickpea. In the present study, loci controlling maackiain metabolism (Mak genes) were identified by crosses among isolates of N. haematococca MP VI that differed in their ability to metabolize the phytoalexin. Strains carrying Mak1 or Mak2 converted maackiain to 1a-hydroxymaackiain, while those with Mak3 converted it to 6a-hydroxymaackiain. Mak1 and Mak2 were unusual in that they often failed to be inherited by progeny. Mak1 was closely linked to Pda6, a new member in a family of genes in N. haematococca MP VI that encode enzymes for detoxification of pisatin, the phytoalexin synthesized by garden pea. Like Mak1, Pda6 was also transmitted irregularly to progeny. Although the unusual meiotic behaviors of some Mak genes complicate genetic analysis, identification of these genes should afford a more thorough evaluation of the role of phytoalexin detoxification in the pathogenesis of N. haematococca MP VI on chickpea.

  3. The PHD Finger Protein MMD1/DUET Ensures the Progression of Male Meiotic Chromosome Condensation and Directly Regulates the Expression of the Condensin Gene CAP-D3[OPEN

    PubMed Central

    Wang, Jun; Niu, Baixiao; Huang, Jiyue; Wang, Hongkuan; Yang, Xiaohui; Dong, Aiwu

    2016-01-01

    Chromosome condensation, a process mediated by the condensin complex, is essential for proper chromosome segregation during cell division. Unlike rapid mitotic chromosome condensation, meiotic chromosome condensation occurs over a relatively long prophase I and is unusually complex due to the coordination with chromosome axis formation and homolog interaction. The molecular mechanisms that regulate meiotic chromosome condensation progression from prophase I to metaphase I are unclear. Here, we show that the Arabidopsis thaliana meiotic PHD-finger protein MMD1/DUET is required for progressive compaction of prophase I chromosomes to metaphase I bivalents. The MMD1 PHD domain is required for its function in chromosome condensation and binds to methylated histone tails. Transcriptome analysis and qRT-PCR showed that several condensin genes exhibit significantly reduced expression in mmd1 meiocytes. Furthermore, MMD1 specifically binds to the promoter region of the condensin subunit gene CAP-D3 to enhance its expression. Moreover, cap-d3 mutants exhibit similar chromosome condensation defects, revealing an MMD1-dependent mechanism for regulating meiotic chromosome condensation, which functions in part by promoting condensin gene expression. Together, these discoveries provide strong evidence that the histone reader MMD1/DUET defines an important step for regulating the progression of meiotic prophase I chromosome condensation. PMID:27385818

  4. Genomic organization of mouse gene zfp162.

    PubMed

    Wrehlke, C; Wiedemeyer, W R; Schmitt-Wrede, H P; Mincheva, A; Lichter, P; Wunderlich, F

    1999-05-01

    We report the cloning and characterization of the alternatively spliced mouse gene zfp162, formerly termed mzfm, the homolog of the human ZFM1 gene encoding the splicing factor SF1 and a putative signal transduction and activation of RNA (STAR) protein. The zfp162 gene is about 14 kb long and consists of 14 exons and 13 introns. Comparison of zfp162 with the genomic sequences of ZFM1/SF1 revealed that the exon-intron structure and exon sequences are well conserved between the genes, whereas the introns differ in length and sequence composition. Using fluorescent in situ hybridization, the zfp162 gene was assigned to chromosome 19, region B. Screening of a genomic library integrated in lambda DASH II resulted in the identification of the 5'-flanking region of zfp162. Sequence analysis of this region showed that zfp162 is a TATA-less gene containing an initiator control element and two CCAAT boxes. The promoter exhibits the following motifs: AP-2, CRE, Ets, GRE, HNF5, MRE, SP-1, TRE, TCF1, and PU.1. The core promoter, from position -331 to -157, contains the motifs CRE, SP-1, MRE, and AP-2, as determined in transfected CHO-K1 cells and IC-21 cells by reporter gene assay using a secreted form of human placental alkaline phosphatase. The occurrence of PU.1/GRE supports the view that the zfp162 gene encodes a protein involved not only in nuclear RNA metabolism, as the human ZFM1/SF1, but also in as yet unknown macrophage-inherent functions. PMID:10360842

  5. Expression of mouse metallothionein genes in tobacco

    SciTech Connect

    Maiti, I.B.; Yeargan, R.; Wagner, G.J.; Hunt, A.G. )

    1990-05-01

    We have expressed a mouse metallothionein (NT) gene in tobacco under control of the cauliflower mosaic virus (CaMV) 35S promoter and a pea ribulose-1,5-bisphosphate carboxylase small subunit (rbcS) gene promoter. Seedlings in which MT gene expression is driven by the 35S promoter are resistant to toxic levels of cadmium. Mature plants carrying the 35S-MT gene accumulate less Cd in their leaves when exposed to low levels of Cd in laboratory growth conditions. Plants with the rbcS-MT construction express this gene in a light-regulated and tissue-specific manner, as expected. Moreover, the MT levels in leaves in these plants are about 20% of those seen in 35S-MT plants. These plants are currently being tested for Cd resistance. In addition, a small field evaluation of 35S-MT lines for Cd levels is being evaluated. These experiments will address the possibility of using MTs to alter Cd levels in crop species.

  6. [Mapping of meiotic genes in rye (Secale cereale L.): localization of sy19 mutation, impairing homologous synapsis, by means of isozyme and microsatellite markers].

    PubMed

    Dolmatovich, T V; Malyshev, S V; Sosnikhina, S P; Tsvetkova, N V; Kartel', N A; Voiĭlokov, A V

    2013-05-01

    The sy19 mutation, which impairs the homology of meiotic chromosome synapsis in rye, were mapped using a specially created F2 population by means of isozyme Acph 1 locus and microsatellite (SSR) markers. The sy19gene was localized in the chromosome 7R in the pericentromeric region of long armbased on the linked inheritance with the Acph 1 locus. The locus was linked with five rye SSR markers, with the Xrems 1234 locus being located closest to the sy19 gene (6.4 cM). The genetic map of the analyzed chromosome 7R region includes ten markers and the sy19 locus. A possible function of the Sy1 and Sy19 genes based on the data on comparative genomics is discussed. PMID:24159800

  7. The Mouse Genome Database (MGD): from genes to mice--a community resource for mouse biology.

    PubMed

    Eppig, Janan T; Bult, Carol J; Kadin, James A; Richardson, Joel E; Blake, Judith A; Anagnostopoulos, A; Baldarelli, R M; Baya, M; Beal, J S; Bello, S M; Boddy, W J; Bradt, D W; Burkart, D L; Butler, N E; Campbell, J; Cassell, M A; Corbani, L E; Cousins, S L; Dahmen, D J; Dene, H; Diehl, A D; Drabkin, H J; Frazer, K S; Frost, P; Glass, L H; Goldsmith, C W; Grant, P L; Lennon-Pierce, M; Lewis, J; Lu, I; Maltais, L J; McAndrews-Hill, M; McClellan, L; Miers, D B; Miller, L A; Ni, L; Ormsby, J E; Qi, D; Reddy, T B K; Reed, D J; Richards-Smith, B; Shaw, D R; Sinclair, R; Smith, C L; Szauter, P; Walker, M B; Walton, D O; Washburn, L L; Witham, I T; Zhu, Y

    2005-01-01

    The Mouse Genome Database (MGD) forms the core of the Mouse Genome Informatics (MGI) system (http://www.informatics.jax.org), a model organism database resource for the laboratory mouse. MGD provides essential integration of experimental knowledge for the mouse system with information annotated from both literature and online sources. MGD curates and presents consensus and experimental data representations of genotype (sequence) through phenotype information, including highly detailed reports about genes and gene products. Primary foci of integration are through representations of relationships among genes, sequences and phenotypes. MGD collaborates with other bioinformatics groups to curate a definitive set of information about the laboratory mouse and to build and implement the data and semantic standards that are essential for comparative genome analysis. Recent improvements in MGD discussed here include the enhancement of phenotype resources, the re-development of the International Mouse Strain Resource, IMSR, the update of mammalian orthology datasets and the electronic publication of classic books in mouse genetics.

  8. The biology of novel animal genes: Mouse APEX gene knockout

    SciTech Connect

    MacInnes, M.; Altherr, M.R.; Ludwig, D.; Pedersen, R.; Mold, C.

    1997-07-01

    This is the final report of a one-year, Laboratory Directed Research and Development (LDRD) project at the Los Alamos National Laboratory (LANL). The controlled breeding of novel genes into mice, including the gene knockout (KO), or conversely by adding back transgenes provide powerful genetic technologies that together suffice to determine in large part the biological role(s) of novel genes. Inbred mouse remains the best understood and most useful mammalian experimental system available for tackling the biology of novel genes. The major mammalian apurinic/apyrimidinic (AP) endonuclease (APE), is involved in a key step in the repair of spontaneous and induced AP sites in DNA. Efficient repair of these lesions is imperative to prevent the stable incorporation of mutations into the cellular genome which may lead to cell death or transformation. Loss or modulation of base excison repair activity in vivo may elevate the spontaneous mutation rate in cells, and may lead to a substantial increase in the incidence of cancer. Despite extensive biochemical analysis, however, the significance of these individual APE functions in vivo has not been elucidated. Mouse embryonic stem (ES) cells heterozygous for a deletion mutation in APE have been generated and whole animals containing the APE mutation have been derived from these ES cells. Animals homozygous for the APE null mutation die early in gestation, underscoring the biological significance of this DNA repair gene.

  9. Gene Expression Profile Analysis of Type 2 Diabetic Mouse Liver

    PubMed Central

    Zhang, Fang; Xu, Xiang; Zhang, Yi; Zhou, Ben; He, Zhishui; Zhai, Qiwei

    2013-01-01

    Liver plays a key role in glucose metabolism and homeostasis, and impaired hepatic glucose metabolism contributes to the development of type 2 diabetes. However, the precise gene expression profile of diabetic liver and its association with diabetes and related diseases are yet to be further elucidated. In this study, we detected the gene expression profile by high-throughput sequencing in 9-week-old normal and type 2 diabetic db/db mouse liver. Totally 12132 genes were detected, and 2627 genes were significantly changed in diabetic mouse liver. Biological process analysis showed that the upregulated genes in diabetic mouse liver were mainly enriched in metabolic processes. Surprisingly, the downregulated genes in diabetic mouse liver were mainly enriched in immune-related processes, although all the altered genes were still mainly enriched in metabolic processes. Similarly, KEGG pathway analysis showed that metabolic pathways were the major pathways altered in diabetic mouse liver, and downregulated genes were enriched in immune and cancer pathways. Analysis of the key enzyme genes in fatty acid and glucose metabolism showed that some key enzyme genes were significantly increased and none of the detected key enzyme genes were decreased. In addition, FunDo analysis showed that liver cancer and hepatitis were most likely to be associated with diabetes. Taken together, this study provides the digital gene expression profile of diabetic mouse liver, and demonstrates the main diabetes-associated hepatic biological processes, pathways, key enzyme genes in fatty acid and glucose metabolism and potential hepatic diseases. PMID:23469233

  10. Pathophysiology of gene-targeted mouse models for cystic fibrosis.

    PubMed

    Grubb, B R; Boucher, R C

    1999-01-01

    Pathophysiology of Gene-Targeted Mouse Models for Cystic Fibrosis. Physiol. Rev. 79, Suppl.: S193-S214, 1999. - Mutations in the gene causing the fatal disease cystic fibrosis (CF) result in abnormal transport of several ions across a number of epithelial tissues. In just 3 years after this gene was cloned, the first CF mouse models were generated. The CF mouse models generated to date have provided a wealth of information on the pathophysiology of the disease in a variety of organs. Heterogeneity of disease in the mouse models is due to the variety of gene-targeting strategies used in the generation of the CF mouse models as well as the diversity of the murine genetic background. This paper reviews the pathophysiology in the tissues and organs (gastrointestinal, airway, hepatobiliary, pancreas, reproductive, and salivary tissue) involved in the disease in the various CF mouse models. Marked similarities to and differences from the human disease have been observed in the various murine models. Some of the CF mouse models accurately reflect the ion-transport abnormalities and disease phenotype seen in human CF patients, especially in gastrointestinal tissue. However, alterations in airway ion transport, which lead to the devastating lung disease in CF patients, appear to be largely absent in the CF mouse models. Reasons for these unexpected findings are discussed. This paper also reviews pharmacotherapeutic and gene therapeutic studies in the various mouse models. PMID:9922382

  11. Chromosomal localization of the human and mouse hyaluronan synthase genes

    SciTech Connect

    Spicer, A.P.; McDonald, J.A.; Seldin, M.F.

    1997-05-01

    We have recently identified a new vertebrate gene family encoding putative hyaluronan (HA) synthases. Three highly conserved related genes have been identified, designated HAS1, HAS2, and HAS3 in humans and Has1, Has2, and Has3 in the mouse. All three genes encode predicted plasma membrane proteins with multiple transmembrane domains and approximately 25% amino acid sequence identity to the Streptococcus pyogenes HA synthase, HasA. Furthermore, expression of any one HAS gene in transfected mammalian cells leads to high levels of HA biosynthesis. We now report the chromosomal localization of the three HAS genes in human and in mouse. The genes localized to three different positions within both the human and the mouse genomes. HAS1 was localized to the human chromosome 19q13.3-q13.4 boundary and Has1 to mouse Chr 17. HAS2 was localized to human chromosome 8q24.12 and Has2 to mouse Chr 15. HAS3 was localized to human chromosome 16q22.1 and Has3 to mouse Chr 8. The map position for HAS1 reinforces the recently reported relationship between a small region of human chromosome 19q and proximal mouse chromosome 17. HAS2 mapped outside the predicted critical region delineated for the Langer-Giedion syndrome and can thus be excluded as a candidate gene for this genetic syndrome. 33 refs., 2 figs.

  12. Comprehensive comparative homeobox gene annotation in human and mouse

    PubMed Central

    Wilming, Laurens G.; Boychenko, Veronika; Harrow, Jennifer L.

    2015-01-01

    Homeobox genes are a group of genes coding for transcription factors with a DNA-binding helix-turn-helix structure called a homeodomain and which play a crucial role in pattern formation during embryogenesis. Many homeobox genes are located in clusters and some of these, most notably the HOX genes, are known to have antisense or opposite strand long non-coding RNA (lncRNA) genes that play a regulatory role. Because automated annotation of both gene clusters and non-coding genes is fraught with difficulty (over-prediction, under-prediction, inaccurate transcript structures), we set out to manually annotate all homeobox genes in the mouse and human genomes. This includes all supported splice variants, pseudogenes and both antisense and flanking lncRNAs. One of the areas where manual annotation has a significant advantage is the annotation of duplicated gene clusters. After comprehensive annotation of all homeobox genes and their antisense genes in human and in mouse, we found some discrepancies with the current gene set in RefSeq regarding exact gene structures and coding versus pseudogene locus biotype. We also identified previously un-annotated pseudogenes in the DUX, Rhox and Obox gene clusters, which helped us re-evaluate and update the gene nomenclature in these regions. We found that human homeobox genes are enriched in antisense lncRNA loci, some of which are known to play a role in gene or gene cluster regulation, compared to their mouse orthologues. Of the annotated set of 241 human protein-coding homeobox genes, 98 have an antisense locus (41%) while of the 277 orthologous mouse genes, only 62 protein coding gene have an antisense locus (22%), based on publicly available transcriptional evidence. PMID:26412852

  13. Female meiotic sex chromosome inactivation in chicken.

    PubMed

    Schoenmakers, Sam; Wassenaar, Evelyne; Hoogerbrugge, Jos W; Laven, Joop S E; Grootegoed, J Anton; Baarends, Willy M

    2009-05-01

    During meiotic prophase in male mammals, the heterologous X and Y chromosomes remain largely unsynapsed, and meiotic sex chromosome inactivation (MSCI) leads to formation of the transcriptionally silenced XY body. In birds, the heterogametic sex is female, carrying Z and W chromosomes (ZW), whereas males have the homogametic ZZ constitution. During chicken oogenesis, the heterologous ZW pair reaches a state of complete heterologous synapsis, and this might enable maintenance of transcription of Z- and W chromosomal genes during meiotic prophase. Herein, we show that the ZW pair is transiently silenced, from early pachytene to early diplotene using immunocytochemistry and gene expression analyses. We propose that ZW inactivation is most likely achieved via spreading of heterochromatin from the W on the Z chromosome. Also, persistent meiotic DNA double-strand breaks (DSBs) may contribute to silencing of Z. Surprisingly, gammaH2AX, a marker of DSBs, and also the earliest histone modification that is associated with XY body formation in mammalian and marsupial spermatocytes, does not cover the ZW during the synapsed stage. However, when the ZW pair starts to desynapse, a second wave of gammaH2AX accumulates on the unsynapsed regions of Z, which also show a reappearance of the DSB repair protein RAD51. This indicates that repair of meiotic DSBs on the heterologous part of Z is postponed until late pachytene/diplotene, possibly to avoid recombination with regions on the heterologously synapsed W chromosome. Two days after entering diplotene, the Z looses gammaH2AX and shows reactivation. This is the first report of meiotic sex chromosome inactivation in a species with female heterogamety, providing evidence that this mechanism is not specific to spermatogenesis. It also indicates the presence of an evolutionary force that drives meiotic sex chromosome inactivation independent of the final achievement of synapsis. PMID:19461881

  14. Female Meiotic Sex Chromosome Inactivation in Chicken

    PubMed Central

    Schoenmakers, Sam; Wassenaar, Evelyne; Hoogerbrugge, Jos W.; Laven, Joop S. E.; Grootegoed, J. Anton; Baarends, Willy M.

    2009-01-01

    During meiotic prophase in male mammals, the heterologous X and Y chromosomes remain largely unsynapsed, and meiotic sex chromosome inactivation (MSCI) leads to formation of the transcriptionally silenced XY body. In birds, the heterogametic sex is female, carrying Z and W chromosomes (ZW), whereas males have the homogametic ZZ constitution. During chicken oogenesis, the heterologous ZW pair reaches a state of complete heterologous synapsis, and this might enable maintenance of transcription of Z- and W chromosomal genes during meiotic prophase. Herein, we show that the ZW pair is transiently silenced, from early pachytene to early diplotene using immunocytochemistry and gene expression analyses. We propose that ZW inactivation is most likely achieved via spreading of heterochromatin from the W on the Z chromosome. Also, persistent meiotic DNA double-strand breaks (DSBs) may contribute to silencing of Z. Surprisingly, γH2AX, a marker of DSBs, and also the earliest histone modification that is associated with XY body formation in mammalian and marsupial spermatocytes, does not cover the ZW during the synapsed stage. However, when the ZW pair starts to desynapse, a second wave of γH2AX accumulates on the unsynapsed regions of Z, which also show a reappearance of the DSB repair protein RAD51. This indicates that repair of meiotic DSBs on the heterologous part of Z is postponed until late pachytene/diplotene, possibly to avoid recombination with regions on the heterologously synapsed W chromosome. Two days after entering diplotene, the Z looses γH2AX and shows reactivation. This is the first report of meiotic sex chromosome inactivation in a species with female heterogamety, providing evidence that this mechanism is not specific to spermatogenesis. It also indicates the presence of an evolutionary force that drives meiotic sex chromosome inactivation independent of the final achievement of synapsis. PMID:19461881

  15. Gene Expression Analysis of Parthenogenetic Embryonic Development of the Pea Aphid, Acyrthosiphon pisum, Suggests That Aphid Parthenogenesis Evolved from Meiotic Oogenesis

    PubMed Central

    Srinivasan, Dayalan G.; Abdelhady, Ahmed; Stern, David L.

    2014-01-01

    Aphids exhibit a form of phenotypic plasticity, called polyphenism, in which genetically identical females reproduce sexually during one part of the life cycle and asexually (via parthenogenesis) during the remainder of the life cycle. The molecular basis for aphid parthenogenesis is unknown. Cytological observations of aphid parthenogenesis suggest that asexual oogenesis evolved either through a modification of meiosis or from a mitotic process. As a test of these alternatives, we assessed the expression levels and expression patterns of canonical meiotic recombination and germline genes in the sexual and asexual ovaries of the pea aphid, Acyrthosiphon pisum. We observed expression of all meiosis genes in similar patterns in asexual and sexual ovaries, with the exception that some genes encoding Argonaute-family members were not expressed in sexual ovaries. In addition, we observed that asexual aphid tissues accumulated unspliced transcripts of Spo11, whereas sexual aphid tissues accumulated primarily spliced transcripts. In situ hybridization revealed Spo11 transcript in sexual germ cells and undetectable levels of Spo11 transcript in asexual germ cells. We also found that an obligately asexual strain of pea aphid produced little spliced Spo11 transcript. Together, these results suggest that parthenogenetic oogenesis evolved from a meiosis-like, and not a mitosis-like, process and that the aphid reproductive polyphenism may involve a modification of Spo11 gene activity. PMID:25501006

  16. Meiotic drive of chromosomal knobs reshaped the maize genome.

    PubMed Central

    Buckler, E S; Phelps-Durr, T L; Buckler, C S; Dawe, R K; Doebley, J F; Holtsford, T P

    1999-01-01

    Meiotic drive is the subversion of meiosis so that particular genes are preferentially transmitted to the progeny. Meiotic drive generally causes the preferential segregation of small regions of the genome; however, in maize we propose that meiotic drive is responsible for the evolution of large repetitive DNA arrays on all chromosomes. A maize meiotic drive locus found on an uncommon form of chromosome 10 [abnormal 10 (Ab10)] may be largely responsible for the evolution of heterochromatic chromosomal knobs, which can confer meiotic drive potential to every maize chromosome. Simulations were used to illustrate the dynamics of this meiotic drive model and suggest knobs might be deleterious in the absence of Ab10. Chromosomal knob data from maize's wild relatives (Zea mays ssp. parviglumis and mexicana) and phylogenetic comparisons demonstrated that the evolution of knob size, frequency, and chromosomal position agreed with the meiotic drive hypothesis. Knob chromosomal position was incompatible with the hypothesis that knob repetitive DNA is neutral or slightly deleterious to the genome. We also show that environmental factors and transposition may play a role in the evolution of knobs. Because knobs occur at multiple locations on all maize chromosomes, the combined effects of meiotic drive and genetic linkage may have reshaped genetic diversity throughout the maize genome in response to the presence of Ab10. Meiotic drive may be a major force of genome evolution, allowing revolutionary changes in genome structure and diversity over short evolutionary periods. PMID:10471723

  17. Mouse T-cell receptor variable gene segment families

    SciTech Connect

    Arden, B.; Kabelitz, D.; Clark, S.P.; Mak, T.W.

    1995-10-01

    All mouse T-cell receptor {alpha}/{delta}, {beta}, and {gamma} variable (Tcra/d-, b-, and g-V) gene segments were aligned to compare the sequences with one another, to group them into subfamilies, and to derive a name which complies with the standard nomenclature. it was necessary to change the names of some V gene segments because they conflicted with those of other segments. The traditional classification into subfamilies was re-evaluated using a much larger pool of sequences. In the mouse, most V gene segments can be grouped into subfamilies of closely related genes with significantly less similarity between different subfamilies. 118 refs., 11 figs., 4 tabs.

  18. Recommended nomenclature for five mammalian carboxylesterase gene families: human, mouse, and rat genes and proteins.

    PubMed

    Holmes, Roger S; Wright, Matthew W; Laulederkind, Stanley J F; Cox, Laura A; Hosokawa, Masakiyo; Imai, Teruko; Ishibashi, Shun; Lehner, Richard; Miyazaki, Masao; Perkins, Everett J; Potter, Phillip M; Redinbo, Matthew R; Robert, Jacques; Satoh, Tetsuo; Yamashita, Tetsuro; Yan, Bingfan; Yokoi, Tsuyoshi; Zechner, Rudolf; Maltais, Lois J

    2010-10-01

    Mammalian carboxylesterase (CES or Ces) genes encode enzymes that participate in xenobiotic, drug, and lipid metabolism in the body and are members of at least five gene families. Tandem duplications have added more genes for some families, particularly for mouse and rat genomes, which has caused confusion in naming rodent Ces genes. This article describes a new nomenclature system for human, mouse, and rat carboxylesterase genes that identifies homolog gene families and allocates a unique name for each gene. The guidelines of human, mouse, and rat gene nomenclature committees were followed and "CES" (human) and "Ces" (mouse and rat) root symbols were used followed by the family number (e.g., human CES1). Where multiple genes were identified for a family or where a clash occurred with an existing gene name, a letter was added (e.g., human CES4A; mouse and rat Ces1a) that reflected gene relatedness among rodent species (e.g., mouse and rat Ces1a). Pseudogenes were named by adding "P" and a number to the human gene name (e.g., human CES1P1) or by using a new letter followed by ps for mouse and rat Ces pseudogenes (e.g., Ces2d-ps). Gene transcript isoforms were named by adding the GenBank accession ID to the gene symbol (e.g., human CES1_AB119995 or mouse Ces1e_BC019208). This nomenclature improves our understanding of human, mouse, and rat CES/Ces gene families and facilitates research into the structure, function, and evolution of these gene families. It also serves as a model for naming CES genes from other mammalian species.

  19. Immunologic Applications of Conditional Gene Modification Technology in the Mouse

    PubMed Central

    Sharma, Suveena; Zhu, Jinfang

    2014-01-01

    Since the success of homologous recombination in altering mouse genome and the discovery of Cre-loxP system, the combination of these two breakthroughs has created important applications for studying the immune system in the mouse. Here, we briefly summarize the general principles of this technology and its applications in studying immune cell development and responses; such implications include conditional gene knockout and inducible and/or tissue-specific gene over-expression, as well as lineage fate mapping. We then discuss the pros and cons of a few commonly used Cre-expressing mouse lines for studying lymphocyte development and functions. We also raise several general issues, such as efficiency of gene deletion, leaky activity of Cre, and Cre toxicity, all of which may have profound impacts on data interpretation. Finally, we selectively list some useful links to the Web sites as valuable mouse resources. PMID:24700321

  20. Cloning, characterization and targeting of the mouse HEXA gene

    SciTech Connect

    Wakamatsu, N.; Trasler, J.M.; Gravel, R.A.

    1994-09-01

    The HEXA gene, encoding the {alpha} subunit of {beta}-hexosaminidase A, is essential for the metabolism of ganglioside G{sub M2}, and defects in this gene cause Tay-Sachs disease in humans. To elucidate the role of the gene in the nervous system of the mouse and to establish a mouse model of Tay-Sachs disease, we have cloned and characterized the HEXA gene and targeted a disruption of the gene in mouse ES cells. The mouse HEXA gene spans {approximately}26 kb and consists of 14 exons, similar to the human gene. A heterogeneous transcription initiation site was identified 21-42 bp 5{prime} of the initiator ATG, with two of the sites fitting the consensus CTCA (A = start) as seen for some weak initiator systems. Promoter analysis showed that the first 150 bp 5{prime} of the ATG contained 85% of promoter activity observed in constructs containing up to 1050 bp of 5{prime} sequence. The active region contained a sequence matching that of the adenovirus major late promoter upstream element factor. A survey of mouse tissues showed that the highest mRNA levels were in (max to min): testis (5.5 x brain cortex), adrenal, epididymis, heart, brain, lung, kidney, and liver (0.3 x brain cortex). A 12 kb BstI/SalI fragment containing nine exons was disrupted with the insertion of the bacterial neo{sup r} gene in exon 11 and was targeted into 129/Sv ES cells by homologous recombination. Nine of 153 G418 resistant clones were correctly targeted as confirmed by Southern blotting. The heterozygous ES cells were microinjected into mouse blastocysts and implanted into pseudo-pregnant mice. Nine male chimeric mice, showing that 40-95% chimerism for the 129/Sv agouti coat color marker, are being bred in an effort to generate germline transmission of the disrupted HEXA gene.

  1. Genetic and molecular characterization of sting, a gene involved in crystal formation and meiotic drive in the male germ line of Drosophila melanogaster.

    PubMed Central

    Schmidt, A; Palumbo, G; Bozzetti, M P; Tritto, P; Pimpinelli, S; Schäfer, U

    1999-01-01

    The sting mutation, caused by a P element inserted into polytene region 32D, was isolated by a screen for male sterile insertions in Drosophila melanogaster. This sterility is correlated with the presence of crystals in spermatocytes and spermatids that are structurally indistinguishable from those produced in males carrying a deficiency of the Y-linked crystal (cry) locus. In addition, their morphology is needle-like in Ste+ flies and star-shaped in Ste flies, once again as observed in cry- males. The sti mutation leads to meiotic drive of the sex chromosomes, and the strength of the phenomenon is correlated with the copy number of the repetitive Ste locus. The same correlation is also true for the penetrance of the male sterile mutation. A presumptive sti null allele results in male sterility and lethal maternal effect. The gene was cloned and shown to code for a putative protein that is 866 amino acids long. A C-terminal domain of 82 amino acids is identified that is well conserved in proteins from different organisms. The gene is expressed only in the germline of both sexes. The interaction of sting with the Ste locus can also be demonstrated at the molecular level. While an unprocessed 8-kb Ste primary transcript is expressed in wild-type males, in X/Y homozygous sti males, as in X/Y cry- males, a 0.7-kb mRNA is produced. PMID:9927466

  2. The plant-specific protein FEHLSTART controls male meiotic entry, initializing meiotic synchronization in Arabidopsis.

    PubMed

    Li, Junhua; Dukowic-Schulze, Stefanie; Lindquist, Ingrid E; Farmer, Andrew D; Kelly, Bridget; Li, Tao; Smith, Alan G; Retzel, Ernest F; Mudge, Joann; Chen, Changbin

    2015-11-01

    Meiosis marks the transition from the sporophyte to the gametophyte generation in the life cycle of flowering plants, and creates genetic variations through homologous recombination. In most flowering plants, meiosis is highly synchronized within each anther, which is significant for efficient fertilization. To date, little is known about the molecular mechanisms of entry into meiosis and exit from it, and only a few genes in Arabidopsis have been characterized with a role in regulating meiotic progression. In this study, we report the functional characterization of a plant-specific basic helix-loop-helix (bHLH) protein, FEHLSTART (FST), a defect in which leads to premature meiotic entry and asynchronous meiosis, and results in decreased seed yield. Investigation of the time course of meiosis showed that the onset of leptotene, the first stage of prophase I, frequently occurred earlier in fst-1 than in the wild type. Asynchronous meiosis followed, which could manifest in the disruption of regular spindle structures and symmetric cell divisions in fst-1 mutants during the meiosis I/II transition. In accordance with frequently accelerated meiotic entry, whole-transcriptome analysis of fst-1 anthers undergoing meiosis revealed that 19 circadian rhythm genes were affected and 47 pollen-related genes were prematurely expressed at a higher level. Taken together, we propose that FST is required for normal meiotic entry and the establishment of meiotic synchrony. PMID:26382719

  3. Functional dynamics of H3K9 methylation during meiotic prophase progression.

    PubMed

    Tachibana, Makoto; Nozaki, Masami; Takeda, Naoki; Shinkai, Yoichi

    2007-07-25

    Histone H3 lysine 9 (H3K9) methylation is a crucial epigenetic mark of heterochromatin formation and transcriptional silencing. G9a is a major mammalian H3K9 methyltransferase at euchromatin and is essential for mouse embryogenesis. Here we describe the roles of G9a in germ cell development. Mutant mice in which G9a is specifically inactivated in the germ-lineage displayed sterility due to a drastic loss of mature gametes. G9a-deficient germ cells exhibited perturbation of synchronous synapsis in meiotic prophase. Importantly, mono- and di-methylation of H3K9 (H3K9me1 and 2) in G9a-deficient germ cells were significantly reduced and G9a-regulated genes were overexpressed during meiosis, suggesting that G9a-mediated epigenetic gene silencing is crucial for proper meiotic prophase progression. Finally, we show that H3K9me1 and 2 are dynamically and sex-differentially regulated during the meiotic prophase. This genetic and biochemical evidence strongly suggests that a specific set of H3K9 methyltransferase(s) and demethylase(s) coordinately regulate gametogenesis. PMID:17599069

  4. Inducible and combinatorial gene manipulation in mouse brain

    PubMed Central

    Dogbevia, Godwin K.; Marticorena-Alvarez, Ricardo; Bausen, Melanie; Sprengel, Rolf; Hasan, Mazahir T.

    2015-01-01

    We have deployed recombinant adeno-associated viruses equipped with tetracycline-controlled genetic switches to manipulate gene expression in mouse brain. Here, we show a combinatorial genetic approach for inducible, cell type-specific gene expression and Cre/loxP mediated gene recombination in different brain regions. Our chemical-genetic approach will help to investigate ‘when’, ‘where’, and ‘how’ gene(s) control neuronal circuit dynamics, and organize, for example, sensory signal processing, learning and memory, and behavior. PMID:25954155

  5. Recent segmental and gene duplications in the mouse genome

    PubMed Central

    Cheung, Joseph; Wilson, Michael D; Zhang, Junjun; Khaja, Razi; MacDonald, Jeffrey R; Heng, Henry HQ; Koop, Ben F; Scherer, Stephen W

    2003-01-01

    Background The high quality of the mouse genome draft sequence and its associated annotations are an invaluable biological resource. Identifying recent duplications in the mouse genome, especially in regions containing genes, may highlight important events in recent murine evolution. In addition, detecting recent sequence duplications can reveal potentially problematic regions of the genome assembly. We use BLAST-based computational heuristics to identify large (≥ 5 kb) and recent (≥ 90% sequence identity) segmental duplications in the mouse genome sequence. Here we present a database of recently duplicated regions of the mouse genome found in the mouse genome sequencing consortium (MGSC) February 2002 and February 2003 assemblies. Results We determined that 33.6 Mb of 2,695 Mb (1.2%) of sequence from the February 2003 mouse genome sequence assembly is involved in recent segmental duplications, which is less than that observed in the human genome (around 3.5-5%). From this dataset, 8.9 Mb (26%) of the duplication content consisted of 'unmapped' chromosome sequence. Moreover, we suspect that an additional 18.5 Mb of sequence is involved in duplication artifacts arising from sequence misassignment errors in this genome assembly. By searching for genes that are located within these regions, we identified 675 genes that mapped to duplicated regions of the mouse genome. Sixteen of these genes appear to have been duplicated independently in the human genome. From our dataset we further characterized a 42 kb recent segmental duplication of Mater, a maternal-effect gene essential for embryogenesis in mice. Conclusion Our results provide an initial analysis of the recently duplicated sequence and gene content of the mouse genome. Many of these duplicated loci, as well as regions identified to be involved in potential sequence misassignment errors, will require further mapping and sequencing to achieve accuracy. A Genome Browser database was set up to display the

  6. Sequence conservation of the rad21 Schizosaccharomyces pombe DNA double-strand break repair gene in human and mouse

    SciTech Connect

    McKay, M.J.; Troelstra, C.; Kanaar, R.

    1996-09-01

    The rad21 gene of Schizosaccharomyces pombe is involved in the repair of ionizing radiation-induced DNA double-strand breaks. The isolation of mouse and human putative homologs of rad21 is reported here. Alignment of the predicted amino acid sequence of Rad21 with the mammalian proteins showed that the similarity was distributed across the length of the proteins, with more highly conserved regions at both termini. The mHR21{sup sp} (mouse homolog of Rad21, S. pombe) and hHR21{sup sp} (human homolog of Rad21, S. pombe) predicted proteins were 96% identical, whereas the human and S. pombe proteins were 25% identical and 47% similar. RNA blot analysis showed that mHR21{sup sp} mRNA was abundant in all adult mouse tissues examined, with highest expression in testis and thymus. In addition to a 3.1-kb constitutive mRNA transcript, a 2.2-kb transcript was present at a high level in postmeiotic spermatids, while expression of the 3.1-kb mRNA in testis was confined to the meiotic compartment. hHR21{sup sp} mRNA was cell-cycle regulated in human cells, increasing in late S phase to a peak in G2 phase. The level of hHR21{sup sp} transcripts was not altered by exposure of normal diploid fibroblasts to 10 Gy ionizing radiation. In situ hybridization showed that mHR21{sup sp} resided on chromosome 15D3, whereas hHR21{sup sp} localized to the syntenic 8q24 region. Elevated expression of mHR21{sup sp} in testis and thymus supports a possible role for the rad21 mammalian homologs in V(D)J and meiotic recombination, respectively. Cell cycle regulation of rad21, retained from S. pombe to human, is consistent with a conservation of function between S. pombe and human rad21 genes. 62 refs., 8 figs., 1 tab.

  7. A unified gene catalog for the laboratory mouse reference genome.

    PubMed

    Zhu, Y; Richardson, J E; Hale, P; Baldarelli, R M; Reed, D J; Recla, J M; Sinclair, R; Reddy, T B K; Bult, C J

    2015-08-01

    We report here a semi-automated process by which mouse genome feature predictions and curated annotations (i.e., genes, pseudogenes, functional RNAs, etc.) from Ensembl, NCBI and Vertebrate Genome Annotation database (Vega) are reconciled with the genome features in the Mouse Genome Informatics (MGI) database (http://www.informatics.jax.org) into a comprehensive and non-redundant catalog. Our gene unification method employs an algorithm (fjoin--feature join) for efficient detection of genome coordinate overlaps among features represented in two annotation data sets. Following the analysis with fjoin, genome features are binned into six possible categories (1:1, 1:0, 0:1, 1:n, n:1, n:m) based on coordinate overlaps. These categories are subsequently prioritized for assessment of annotation equivalencies and differences. The version of the unified catalog reported here contains more than 59,000 entries, including 22,599 protein-coding coding genes, 12,455 pseudogenes, and 24,007 other feature types (e.g., microRNAs, lincRNAs, etc.). More than 23,000 of the entries in the MGI gene catalog have equivalent gene models in the annotation files obtained from NCBI, Vega, and Ensembl. 12,719 of the features are unique to NCBI relative to Ensembl/Vega; 11,957 are unique to Ensembl/Vega relative to NCBI, and 3095 are unique to MGI. More than 4000 genome features fall into categories that require manual inspection to resolve structural differences in the gene models from different annotation sources. Using the MGI unified gene catalog, researchers can easily generate a comprehensive report of mouse genome features from a single source and compare the details of gene and transcript structure using MGI's mouse genome browser.

  8. Overlapping genes in the human and mouse genomes

    PubMed Central

    Sanna, Chaitanya R; Li, Wen-Hsiung; Zhang, Liqing

    2008-01-01

    Background Increasing evidence suggests that overlapping genes are much more common in eukaryotic genomes than previously thought. In this study we identified and characterized the overlapping genes in a set of 13,484 pairs of human-mouse orthologous genes. Results About 10% of the genes under study are overlapping genes, the majority of which are different-strand overlaps. The majority of the same-strand overlaps are embedded forms, whereas most different-strand overlaps are not embedded and in the convergent transcription orientation. Most of the same-strand overlapping gene pairs show at least a tenfold difference in length, much larger than the length difference between non-overlapping neighboring gene pairs. The length difference between the two different-strand overlapping genes is less dramatic. Over 27% of the different-strand-overlap relationships are shared between human and mouse, compared to only ~8% conservation for same-strand-overlap relationships. More than 96% of the same-strand and different-strand overlaps that are not shared between human and mouse have both genes located on the same chromosomes in the species that does not show the overlap. We examined the causes of transition between the overlapping and non-overlapping states in the two species and found that 3' UTR change plays an important role in the transition. Conclusion Our study contributes to the understanding of the evolutionary transition between overlapping genes and non-overlapping genes and demonstrates the high rates of evolutionary changes in the un-translated regions. PMID:18410680

  9. Initiation of meiotic recombination in Ustilago maydis.

    PubMed

    Kojic, Milorad; Sutherland, Jeanette H; Pérez-Martín, José; Holloman, William K

    2013-12-01

    A central feature of meiosis is the pairing and recombination of homologous chromosomes. Ustilago maydis, a biotrophic fungus that parasitizes maize, has long been utilized as an experimental system for studying recombination, but it has not been clear when in the life cycle meiotic recombination initiates. U. maydis forms dormant diploid teliospores as the end product of the infection process. Upon germination, teliospores complete meiosis to produce four haploid basidiospores. Here we asked whether the meiotic process begins when teliospores germinate or at an earlier stage in development. When teliospores homozygous for a cdc45 mutation temperature sensitive for DNA synthesis were germinated at the restrictive temperature, four nuclei became visible. This implies that teliospores have already undergone premeiotic DNA synthesis and suggests that meiotic recombination initiates at a stage of infection before teliospores mature. Determination of homologous recombination in plant tissue infected with U. maydis strains heteroallelic for the nar1 gene revealed that Nar(+) recombinants were produced at a stage before teliospore maturation. Teliospores obtained from a spo11Δ cross were still able to germinate but the process was highly disturbed and the meiotic products were imbalanced in chromosomal complement. These results show that in U. maydis, homologous recombination initiates during the infection process and that meiosis can proceed even in the absence of Spo11, but with loss of genomic integrity.

  10. Cellular functions of genetically imprinted genes in human and mouse as annotated in the gene ontology.

    PubMed

    Hamed, Mohamed; Ismael, Siba; Paulsen, Martina; Helms, Volkhard

    2012-01-01

    By analyzing the cellular functions of genetically imprinted genes as annotated in the Gene Ontology for human and mouse, we found that imprinted genes are often involved in developmental, transport and regulatory processes. In the human, paternally expressed genes are enriched in GO terms related to the development of organs and of anatomical structures. In the mouse, maternally expressed genes regulate cation transport as well as G-protein signaling processes. Furthermore, we investigated if imprinted genes are regulated by common transcription factors. We identified 25 TF families that showed an enrichment of binding sites in the set of imprinted genes in human and 40 TF families in mouse. In general, maternally and paternally expressed genes are not regulated by different transcription factors. The genes Nnat, Klf14, Blcap, Gnas and Ube3a contribute most to the enrichment of TF families. In the mouse, genes that are maternally expressed in placenta are enriched for AP1 binding sites. In the human, we found that these genes possessed binding sites for both, AP1 and SP1. PMID:23226257

  11. Identification and characterization of a second mouse Nramp gene

    SciTech Connect

    Gruenheid, S.; Cellier, M.; Vidal, S.

    1995-01-20

    Nramp gene was isolated as a candidate for the host resistance locus Bcg/Ity/Lsh, which controls natural resistance of mice to several types of infections. We have isolated by cross-hybridization cDNA clones corresponding to a second mouse Nramp gene, which we designate Nramp2. Nucleotide and predicted amino acid sequence analyses of full-length cDNA clones for Nramp2 indicate that this novel Nramp protein is closely homologous to the previously described Nramp and that the two genes form part of a small gene family. The two Nramp proteins encode integral membrane proteins that share 63% identical residues and an overall homology of 78%. They share very similar secondary structure, including identical hydropathy profiles and predicted membrane organization, with a minimum of 10 and most probably 12 transmembrane domains, a cluster of predicted N-linked glycosylation sites, and a consensus transport motif. Analysis of the distribution of Nramp2 mRNA transcripts in normal mouse tissues by Northern blotting revealed that the Nramp2 gene produces several mRNAs, including prominent 3.3- and 2.3-kb species generated by the use of alternative polyadenylation signals. In contrast to the previously described macrophage-specific Nramp gene, Nramp2 mRNAs were found to be expressed at low levels in all tissues tested. Using a polymorphic (GT)26 dinucleotide repeat identified in the 3{prime} untranslated region of the mRNA, we have mapped the Nramp2 gene to the distal part of mouse chromosome 15 between markers D15Mit41 and D15Mit15, with the gene order and intergene distance (in cM): centromere-56.1-D15Mit41-(1{+-}1)-Nramp2-(5{+-}2)-D15Mit15. 59 refs., 5 figs., 1 tab.

  12. Cdk2 catalytic activity is essential for meiotic cell division in vivo.

    PubMed

    Chauhan, Sangeeta; Diril, M Kasim; Lee, Joanna H S; Bisteau, Xavier; Manoharan, Vanessa; Adhikari, Deepak; Ratnacaram, Chandrahas Koumar; Janela, Baptiste; Noffke, Juliane; Ginhoux, Florent; Coppola, Vincenzo; Liu, Kui; Tessarollo, Lino; Kaldis, Philipp

    2016-09-15

    Cyclin-dependent kinases (Cdks) control the eukaryotic cell cycle by phosphorylating serine and threonine residues in key regulatory proteins, but some Cdk family members may exert kinase-independent functions that cannot easily be assessed using gene knockout approaches. While Cdk2-deficient mice display near-normal mitotic cell proliferation due to the compensatory activities of Cdk1 and Cdk4, they are unable to undergo meiotic generation of gametes and are consequently sterile. To investigate whether Cdk2 regulates meiosis via protein phosphorylation or by alternative kinase-independent mechanisms, we generated two different knockin mouse strains in which Cdk2 point mutations ablated enzyme activity without altering protein expression levels. Mice homozygous for the mutations Cdk2(D145N/D145N) or Cdk2(T160A/T160A) expressed only 'kinase-dead' variants of Cdk2 under the control of the endogenous promoter, and despite exhibiting normal expression of cell cycle regulatory proteins and complexes, both mutations rendered mice sterile. Mouse cells that expressed only 'kinase-dead' variants of Cdk2 displayed normal mitotic cell cycle progression and proliferation both in vitro and in vivo, indicating that loss of Cdk2 kinase activity exerted little effect on this mode of cell division. In contrast, the reproductive organs of Cdk2 mutant mice exhibited abnormal morphology and impaired function associated with defective meiotic cell division and inability to produce gametes. Cdk2 mutant animals were therefore comparable to gene knockout mice, which completely lack the Cdk2 protein. Together, our data indicate that the essential meiotic functions of Cdk2 depend on its kinase activity, without which the generation of haploid cells is disrupted, resulting in sterility of otherwise healthy animals. PMID:27371320

  13. Spatial gene expression in the T-stage mouse metanephros.

    PubMed

    Caruana, Georgina; Cullen-McEwen, Luise; Nelson, Amy L; Kostoulias, Xenia; Woods, Kyra; Gardiner, Brooke; Davis, Melissa J; Taylor, Darrin F; Teasdale, Rohan D; Grimmond, Sean M; Little, Melissa H; Bertram, John F

    2006-10-01

    The E11.5 mouse metanephros is comprised of a T-stage ureteric epithelial tubule sub-divided into tip and trunk cells surrounded by metanephric mesenchyme (MM). Tip cells are induced to undergo branching morphogenesis by the MM. In contrast, signals within the mesenchyme surrounding the trunk prevent ectopic branching of this region. In order to identify novel genes involved in the molecular regulation of branching morphogenesis we compared the gene expression profiles of isolated tip, trunk and MM cells using Compugen mouse long oligo microarrays. We identified genes enriched in the tip epithelium, sim-1, Arg2, Tacstd1, Crlf-1 and BMP7; genes enriched in the trunk epithelium, Innp1, Itm2b, Mkrn1, SPARC, Emu2 and Gsta3 and genes spatially restricted to the mesenchyme surrounding the trunk, CSPG2 and CV-2, with overlapping and complimentary expression to BMP4, respectively. This study has identified genes spatially expressed in regions of the developing kidney involved in branching morphogenesis, nephrogenesis and the development of the collecting duct system, calyces, renal pelvis and ureter.

  14. Analysis of Meiotic Recombination Pathways in the Yeast Saccharomyces Cerevisiae

    PubMed Central

    Mao-Draayer, Y.; Galbraith, A. M.; Pittman, D. L.; Cool, M.; Malone, R. E.

    1996-01-01

    In the yeast, Saccharomyces cerevisiae, several genes appear to act early in meiotic recombination. HOP1 and RED1 have been classified as such early genes. The data in this paper demonstrate that neither a red1 nor a hop1 mutation can rescue the inviable spores produced by a rad52 spo13 strain; this phenotype helps to distinguish these two genes from other early meiotic recombination genes such as SPO11, REC104, or MEI4. In contrast, either a red1 or a hop1 mutation can rescue a rad50S spo13 strain; this phenotype is similar to that conferred by mutations in the other early recombination genes (e.g., REC104). These two different results can be explained because the data presented here indicate that a rad50S mutation does not diminish meiotic intrachromosomal recombination, similar to the mutant phenotypes conferred by red1 or hop1. Of course, RED1 and HOP1 do act in the normal meiotic interchromosomal recombination pathway; they reduce interchromosomal recombination to ~10% of normal levels. We demonstrate that a mutation in a gene (REC104) required for initiation of exchange is completely epistatic to a mutation in RED1. Finally, mutations in either HOP1 or RED1 reduce the number of double-strand breaks observed at the HIS2 meiotic recombination hotspot. PMID:8878674

  15. The mouse Engrailed genes: a window into Autism

    PubMed Central

    Kuemerle, Barbara; Gulden, Forrest; Cherosky, Natalie; Williams, Elizabeth; Herrup, Karl

    2009-01-01

    The complex behavioral symptoms and neuroanatomical abnormalities observed in autistic individuals strongly suggest a multi-factorial basis for this perplexing disease. Although not the perfect model, we believe the Engrailed genes provide an invaluable “window” into the elusive etiology of Autism Spectrum Disorder. The Engrailed-2 gene has been associated with autism in genetic linkage studies. The En2 knock-out mouse harbors cerebellar abnormalities that are similar to those found in autistic individuals and, as we report here, has a distinct anterior shift in the position of the amygdala in the cerebral cortex. Our initial analysis of background effects in the En1 mouse knock-out provides insight as to possible molecular mechanisms and gender differences associated with autism. These findings further the connection between Engrailed and autism and provide new avenues to explore in the ongoing study of the biological basis of this multifaceted disease. PMID:17055592

  16. Cloning and chromosome localization of the mouse Ews gene

    SciTech Connect

    Plougastel, B.; Thomas, G.; Delattre, O.; Mattei, M.G.

    1994-09-01

    The human EWS gene encodes a putative RNA binding protein. As a result of acquired chromosome rearrangement, the N-terminal portion of the EWS protein is fused to the DNA binding domain of either FLI1-or ERG in the Ewing family of tumors and to the DNA binding domain of ATF1 in malignant melanoma of soft parts. We have determined the cDNA sequence of the mouse Ews gene. Its nucleotide sequence and its translation product demonstrate 93 and 98% homology with the human EWS cDNA and protein, respectively. The murine Ews locus lies within a conserved synteny segment between human chromosome 22q12 and mouse chromosome 11A1-A3.

  17. The meiotic transcriptome architecture of plants

    PubMed Central

    Dukowic-Schulze, Stefanie; Chen, Changbin

    2014-01-01

    Although a number of genes that play key roles during the meiotic process have been characterized in great detail, the whole process of meiosis is still not completely unraveled. To gain insight into the bigger picture, large-scale approaches like RNA-seq and microarray can help to elucidate the transcriptome landscape during plant meiosis, discover co-regulated genes, enriched processes, and highly expressed known and unknown genes which might be important for meiosis. These high-throughput studies are gaining more and more popularity, but their beginnings in plant systems reach back as far as the 1960's. Frequently, whole anthers or post-meiotic pollen were investigated, while less data is available on isolated cells during meiosis, and only few studies addressed the transcriptome of female meiosis. For this review, we compiled meiotic transcriptome studies covering different plant species, and summarized and compared their key findings. Besides pointing to consistent as well as unique discoveries, we finally draw conclusions what can be learned from these studies so far and what should be addressed next. PMID:24926296

  18. Overexpression of mouse TTF-2 gene causes cleft palate

    PubMed Central

    Meng, Tian; Shi, Jia-Yu; Wu, Min; Wang, Yan; Li, Ling; Liu, Yan; Zheng, Qian; Huang, Lei; Shi, Bing

    2012-01-01

    In humans, mutations of the gene encoding for thyroid transcription factor-2 (TTF-2 or FOXE1) result in Bamforth syndrome. Bamforth syndrome is characterized by agenesis, cleft palate, spiky hair and choanal atresia. TTF-2 null mice (TTF-2−/−) also exhibit cleft palate, suggesting its involvement in the palatogenesis. However, the molecular pathology and genetic regulation by TTF2 remain largely unknown. In the present study, the recombinant expression vector pBROAD3-TTF-2 containing the promoter of the mouse ROSA26 gene was created to form the structural gene of mouse TTF-2 and was microinjected into the male pronuclei of fertilized ova. Sequence analysis confirmed that the TTF-2 transgenic mouse model was established successfully. The transgenic mice displayed a phenotype of cleft palate. In addition, we found that TTF-2 was highly expressed in the medial edge epithelium (MEE) from the embryonic day 12.5 (E12.5) to E14.5 in TTF-2 transgenic mice. These observations suggest that overexpression of TTF-2 during palatogenesis may contribute to formation of cleft palate. PMID:22304410

  19. DEVELOPMENT OF A 950-GENE DNA ARRAY FOR EXAMINING GENE EXPRESSION PATTERNS IN MOUSE TESTIS

    EPA Science Inventory

    Development of a 950-gene DNA array for examining gene expression patterns in mouse testis.

    Rockett JC, Christopher Luft J, Brian Garges J, Krawetz SA, Hughes MR, Hee Kirn K, Oudes AJ, Dix DJ.

    Reproductive Toxicology Division, National Health and Environmental Effec...

  20. MOLECULAR ANALYSIS OF MUTATIONS INDUCED BY MUTAGENS IN THE TK GENE OF MOUSE LYMPHOMA CELLS

    EPA Science Inventory

    MOLECULAR ANALYSIS OF MUTATIONS INDUCED BY BROMATE AND N- ETHYL-N-NITROSOUREA IN THE TK GENE OF MOUSE L YMPHOMA CELLS

    The mouse lymphoma assay is widely used to identify chemical mutagens The Tk +1- gene located on an autosome in mouse lymphoma cells may recover a wide ra...

  1. An anatomic gene expression atlas of the adult mouse brain.

    PubMed

    Ng, Lydia; Bernard, Amy; Lau, Chris; Overly, Caroline C; Dong, Hong-Wei; Kuan, Chihchau; Pathak, Sayan; Sunkin, Susan M; Dang, Chinh; Bohland, Jason W; Bokil, Hemant; Mitra, Partha P; Puelles, Luis; Hohmann, John; Anderson, David J; Lein, Ed S; Jones, Allan R; Hawrylycz, Michael

    2009-03-01

    Studying gene expression provides a powerful means of understanding structure-function relationships in the nervous system. The availability of genome-scale in situ hybridization datasets enables new possibilities for understanding brain organization based on gene expression patterns. The Anatomic Gene Expression Atlas (AGEA) is a new relational atlas revealing the genetic architecture of the adult C57Bl/6J mouse brain based on spatial correlations across expression data for thousands of genes in the Allen Brain Atlas (ABA). The AGEA includes three discovery tools for examining neuroanatomical relationships and boundaries: (1) three-dimensional expression-based correlation maps, (2) a hierarchical transcriptome-based parcellation of the brain and (3) a facility to retrieve from the ABA specific genes showing enriched expression in local correlated domains. The utility of this atlas is illustrated by analysis of genetic organization in the thalamus, striatum and cerebral cortex. The AGEA is a publicly accessible online computational tool integrated with the ABA (http://mouse.brain-map.org/agea). PMID:19219037

  2. Sleeping Beauty mouse models identify candidate genes involved in gliomagenesis.

    PubMed

    Vyazunova, Irina; Maklakova, Vilena I; Berman, Samuel; De, Ishani; Steffen, Megan D; Hong, Won; Lincoln, Hayley; Morrissy, A Sorana; Taylor, Michael D; Akagi, Keiko; Brennan, Cameron W; Rodriguez, Fausto J; Collier, Lara S

    2014-01-01

    Genomic studies of human high-grade gliomas have discovered known and candidate tumor drivers. Studies in both cell culture and mouse models have complemented these approaches and have identified additional genes and processes important for gliomagenesis. Previously, we found that mobilization of Sleeping Beauty transposons in mice ubiquitously throughout the body from the Rosa26 locus led to gliomagenesis with low penetrance. Here we report the characterization of mice in which transposons are mobilized in the Glial Fibrillary Acidic Protein (GFAP) compartment. Glioma formation in these mice did not occur on an otherwise wild-type genetic background, but rare gliomas were observed when mobilization occurred in a p19Arf heterozygous background. Through cloning insertions from additional gliomas generated by transposon mobilization in the Rosa26 compartment, several candidate glioma genes were identified. Comparisons to genetic, epigenetic and mRNA expression data from human gliomas implicates several of these genes as tumor suppressor genes and oncogenes in human glioblastoma.

  3. Mouse lysozyme M gene: isolation, characterization, and expression studies.

    PubMed Central

    Cross, M; Mangelsdorf, I; Wedel, A; Renkawitz, R

    1988-01-01

    We have isolated and characterized both cDNA and genomic DNA of the mouse lysozyme M gene. Derivation of the amino acid sequence from the nucleotide sequences revealed six positions in the carboxyl terminus that differ from partial sequences previously published. The differential detection of specific mRNAs from the closely related lysozyme M and P genes has revealed different but overlapping tissue specificities of expression. The M gene is expressed weakly in myeloblasts, moderately in immature macrophages, and strongly in both mature macrophages and macrophage-rich tissues, while high levels of P transcripts are present only in small intestine. Sites of protein accumulation, rather than gene expression, have been identified by comparative quantitation of mRNA and enzyme levels. Images PMID:3413093

  4. Meiotic recombination mechanisms.

    PubMed

    Grelon, Mathilde

    2016-01-01

    Meiosis is a specialized cell division at the origin of the haploid cells that eventually develop into the gametes. It therefore lies at the heart of Mendelian heredity. Recombination and redistribution of the homologous chromosomes arising during meiosis constitute an important source of genetic diversity, conferring to meiosis a particularly important place in the evolution and the diversification of the species. Our understanding of the molecular mechanisms governing meiotic recombination has considerably progressed these last decades, benefiting from complementary approaches led on various model species. An overview of these mechanisms will be provided as well as a discussion on the implications of these recent discoveries. PMID:27180110

  5. Identification and sequence analysis of a new member of the mouse HSP70 gene family and characterization of its unique cellular and developmental pattern of expression in the male germ line.

    PubMed Central

    Zakeri, Z F; Wolgemuth, D J; Hunt, C R

    1988-01-01

    A unique member of the mouse HSP70 gene family has been isolated and characterized with respect to its DNA sequence organization and expression. The gene contains extensive similarity to a heat shock-inducible HSP70 gene within the coding region but diverges in both 3' and 5' nontranslated regions. The gene does not yield transcripts in response to heat shock in mouse L cells. Rather, the gene appears to be activated uniquely in the male germ line. Analysis of RNA from different developmental stages and from enriched populations of spermatogenic cells revealed that this gene is expressed during the prophase stage of meiosis. A transcript different in size from the major heat-inducible mouse transcripts is most abundant in meiotic prophase spermatocytes and decreases in abundance in postmeiotic stages of spermatogenesis. This pattern of expression is distinct from that observed for another member of this gene family, which was previously shown to be expressed abundantly in postmeiotic germ cells. These observations suggest that specific HSP70 gene family members play distinct roles in the differentiation of the germ cell lineage in mammals. Images PMID:3405224

  6. A Mouse Model for Imprinting of the Human Retinoblastoma Gene

    PubMed Central

    Tasiou, Vasiliki; Hiber, Michaela; Steenpass, Laura

    2015-01-01

    The human RB1 gene is imprinted due to integration of the PPP1R26P1 pseudogene into intron 2. PPP1R26P1 harbors the gametic differentially methylated region of the RB1 gene, CpG85, which is methylated in the female germ line. The paternally unmethylated CpG85 acts as promoter for the alternative transcript 2B of RB1, which interferes with expression of full-length RB1 in cis. In mice, PPP1R26P1 is not present in the Rb1 gene and Rb1 is not imprinted. Assuming that the mechanisms responsible for genomic imprinting are conserved, we investigated if imprinting of mouse Rb1 can be induced by transferring human PPP1R26P1 into mouse Rb1. We generated humanized Rb1_PPP1R26P1 knock-in mice that pass human PPP1R26P1 through the mouse germ line. We found that the function of unmethylated CpG85 as promoter for an alternative Rb1 transcript and as cis-repressor of the main Rb1 transcript is maintained in mouse tissues. However, CpG85 is not recognized as a gametic differentially methylated region in the mouse germ line. DNA methylation at CpG85 is acquired only in tissues of neuroectodermal origin, independent of parental transmission of PPP1R26P1. Absence of CpG85 methylation in oocytes and sperm implies a failure of imprint methylation establishment in the germ line. Our results indicate that site-specific integration of a proven human gametic differentially methylated region is not sufficient for acquisition of DNA methylation in the mouse germ line, even if promoter function of the element is maintained. This suggests a considerable dependency of DNA methylation induction on the surrounding sequence. However, our model is suited to determine the cellular function of the alternative Rb1 transcript. PMID:26275142

  7. Genetic mapping of tumor susceptibility genes involved in mouse plasmacytomagenesis

    SciTech Connect

    Mock, B.A.; Krall, M.M.; Dosik, J.K. )

    1993-10-15

    Plasmacytomas (PCTs) were induced in 47% of BALB/cAnPt mice by the intraperitoneal injection of pristane, in 2% of (BALB/c [times] DBA/2N)F[sub 1], and in 11% of 773 BALB/cAnPt [times] (BALB/cAnPt [times] DBA/2N)F[sub 1]N[sub 2] backcross mice. This result indicates a multigenic mode of inheritance for PCT susceptibility. To locate genes controlling this complex genetic trait, tumor susceptibility in backcross progeny generated from BALB/c and DBA/2N (resistant) mice was correlated with alleles of 83 marker loci. The genotypes of the PCT-susceptible progeny displayed an excess homozygosity for BALB/c alleles with a 32-centimorgan stretch of mouse chromosome 4 (>95% probability of linkage) with minimal recombination (12%) near Gt10. Another susceptibility gene on mouse chromosome 1 may be linked to Fcgr2 (90% probability of linkage); there were excess heterozygotes for Fcgr2 among the susceptible progeny and excess homozygotes among the resistant progeny. Regions of mouse chromosomes 4 and 1 that are correlated with PCT susceptibility share extensive linkage homology with regions of human chromosome 1 that have been associated with cytogenetic abnormalities in multiple myeloma and lymphoid, breast, and endocrine tumors. 68 refs., 2 figs., 1 tab.

  8. Structure and genetic polymorphism of the mouse KCC1 gene.

    PubMed

    Shmukler, B E; Brugnara, C; Alper, S L

    2000-07-24

    The KCC1 K-Cl cotransporter is a major regulator of erythroid and non-erythroid cell volume, and the KCC1 gene is a candidate modifier gene for sickle cell disease and other hemoglobinopathies. We have cloned and sequenced the mouse KCC1 (mKCC1) gene, defined its intron-exon junctions, and analyzed (AC)/(TG) intragenic polymorphisms. A highly polymorphic (AC) repeat of mKCC1 intron 1 was characterized in musculus strains, and used to prove lack of linkage between the mKCC1 gene and the rol (resistant to osmotic lysis) locus. The intron 1 (AC) repeat in CAST/Ei and SPRET/Ei was not only more divergent in length but also underwent additional sequence variation. A dimorphic (TG) repeat in intron 2 distinguished CAST/Ei from other strains, and an intron 17 B1 Alu-like SINE present in all musculus strains was found to be absent from intron 17 in SPRET/Ei. These and additional described strain-specific polymorphisms will be useful mapping and genetic tools in the study of mouse models of sickle cell disease.

  9. Complex elaboration: making sense of meiotic cohesin dynamics

    PubMed Central

    Rankin, Susannah

    2015-01-01

    In mitotically dividing cells, the cohesin complex tethers sister chromatids, the products of DNA replication, together from the time they are generated during S phase until anaphase. Cohesion between sister chromatids ensures accurate chromosome segregation, and promotes normal gene regulation and certain kinds of DNA repair. In somatic cells, the core cohesin complex is composed of four subunits: Smc1, Smc3, Rad21 and an SA subunit. During meiotic cell divisions meiosis-specific isoforms of several of the cohesin subunits are also expressed and incorporated into distinct meiotic cohesin complexes. The relative contributions of these meiosis-specific forms of cohesin to chromosome dynamics during meiotic progression have not been fully worked out. However, the localization of these proteins during chromosome pairing and synapsis, and their unique loss-of-function phenotypes, suggest non-overlapping roles in controlling meiotic chromosome behavior. Many of the proteins that regulate cohesin function during mitosis also appear to regulate cohesin during meiosis. Here we review how cohesin contributes to meiotic chromosome dynamics, and explore similarities and differences between cohesin regulation during the mitotic cell cycle and meiotic progression. A deeper understanding of the regulation and function of cohesin in meiosis will provide important new insights into how the cohesin complex is able to promote distinct kinds of chromosome interactions under diverse conditions. PMID:25895170

  10. Control of Oocyte Growth and Meiotic Maturation in C. elegans

    PubMed Central

    Kim, Seongseop; Spike, Caroline; Greenstein, David

    2013-01-01

    In sexually reproducing animals, oocytes arrest at diplotene or diakinesis and resume meiosis (meiotic maturation) in response to hormones. Chromosome segregation errors in female meiosis I are the leading cause of human birth defects, and age-related changes in the hormonal environment of the ovary are a suggested cause. C. elegans is emerging as a genetic paradigm for studying hormonal control of meiotic maturation. The meiotic maturation processes in C. elegans and mammals share a number of biological and molecular similarities. Major sperm protein (MSP) and luteinizing hormone (LH), though unrelated in sequence, both trigger meiotic resumption using somatic Gαs-adenylate cyclase pathways and soma-germline gap-junctional communication. At a molecular level, the oocyte responses apparently involve the control of conserved protein kinase pathways and post-transcriptional gene regulation in the oocyte. At a cellular level, the responses include cortical cytoskeletal rearrangement, nuclear envelope breakdown, assembly of the acentriolar meiotic spindle, chromosome segregation, and likely changes important for fertilization and the oocyte-to-embryo transition. This chapter focuses on signaling mechanisms required for oocyte growth and meiotic maturation in C. elegans and discusses how these mechanisms coordinate the completion of meiosis and the oocyte-to-embryo transition. PMID:22872481

  11. Mutations in TUBB8 cause human oocyte meiotic arrest

    PubMed Central

    Feng, Ruizhi; Sang, Qing; Kuang, Yanping; Sun, Xiaoxi; Yan, Zheng; Zhang, Shaozhen; Shi, Juanzi; Tian, Guoling; Luchniak, Anna; Fukuda, Yusuke; Li, Bin; Yu, Min; Chen, Junling; Xu, Yao; Guo, Luo; Qu, Ronggui; Wang, Xueqian; Sun, Zhaogui; Liu, Miao; Shi, Huijuan; Wang, Hongyan; Feng, Yi; Shao, Ruijin; Chai, Renjie; Li, Qiaoli; Xing, Qinghe; Zhang, Rui; Nogales, Eva; Jin, Li; He, Lin; Gupta, Mohan L.; Cowan, Nicholas J.; Wang, Lei

    2016-01-01

    Background Successful human reproduction depends on the fusion of a mature oocyte with a sperm cell to form a fertilized egg. The genetic events that lead to human oocyte maturation arrest are unknown. Methods We recruited a rare four-generation family with female infertility as a consequence of oocyte meiosis I arrest. We applied whole-exome and direct Sanger sequencing to an additional 23 patients following identification of mutations in a candidate gene, TUBB8. Expression of TUBB8 and all other β-tubulin isotypes was measured in human oocytes, early embryos, sperm cells and several somatic tissues by qRT-PCR. The effect of the TUBB8 mutations was assessed on α/β tubulin heterodimer assembly in vitro, on microtubule architecture in HeLa cells, on microtubule dynamics in yeast cells, and on spindle assembly in mouse and human oocytes via microinjection of the corresponding cRNAs. Results We identified seven mutations in the primate-specific gene TUBB8 that are responsible for human oocyte meiosis I arrest in seven families. TUBB8 expression is unique to oocytes and the early embryo, where this gene accounts for almost all of the expressed β-tubulin. The mutations affect the chaperone-dependent folding and assembly of the α/β-tubulin heterodimer, induce microtubule chaos upon expression in cultured cells, alter microtubule dynamics in vivo, and cause catastrophic spindle assembly defects and maturation arrest upon expression in mouse and human oocytes. Conclusions TUBB8 mutations function via dominant negative effects that massively disrupt proper microtubule behavior. TUBB8 is a key gene involved in human oocyte meiotic spindle assembly and maturation. PMID:26789871

  12. Gene transfer techniques in whole embryo cultured post-implantation mouse embryos.

    PubMed

    Sakai, Daisuke; Trainor, Paul A

    2014-01-01

    Gene transfer techniques such as electroporation and lipofection are powerful systems for investigating gene function. In this chapter we focus on the methods and applications of gene transfer into specific cells and tissues of post-implantation mouse embryos.

  13. Rapid cloning of any rearranged mouse immunoglobulin variable genes

    SciTech Connect

    Dattamajumdar, A.K.; Jacobson, D.P.; Hood, L.E.; Osman, G.E.

    1996-12-31

    Immunoglobulins (Ig) have been the focus of extensive study for several decades and have become an important research area for immunologists and molecular biologists. The use of polymerase chain reaction (PCR) technology has accelerated the cloning, sequencing, and characterization of genes of the immune system. However, cloning and sequencing the Ig variable (V) genes using the PCR technology has been a challenging task, primarily due to the very diverse nature of Ig V region genes. We have developed a simple, rapid, and reproducible PCR-based technique to clone any rearranged mouse Ig heavy or light chain genes. A close examination of all Ig heavy and light chain V gene families has resulted in the design of 5{prime} and 3{prime} universal primers from regions that are highly conserved across all heavy or light chain V gene families, and the joining or constant regions, respectively. We present our strategy for designing universal primers for Ig V gene families. These primers were able to rapidly amplify the rearranged Ig V genes, belonging to diverse Ig V gene families from very different cell lines, i.e., J558, MOPC-21, 36-60, and a chicken ovalbumin specific B-cell hybridoma. In addition, the present study provides the complete alignment of nucleotide sequences of all heavy and light chain variable gene families. This powerful method of cloning Ig V genes, therefore, allows rapid and precise analysis of B-cell hybridomas, B-cell repertoire, and B-cell ontogeny. 55 refs., 5 figs., 2 tabs.

  14. Gene Expression by Mouse Inner Ear Hair Cells during Development

    PubMed Central

    Scheffer, Déborah I.; Shen, Jun

    2015-01-01

    Hair cells of the inner ear are essential for hearing and balance. As a consequence, pathogenic variants in genes specifically expressed in hair cells often cause hereditary deafness. Hair cells are few in number and not easily isolated from the adjacent supporting cells, so the biochemistry and molecular biology of hair cells can be difficult to study. To study gene expression in hair cells, we developed a protocol for hair cell isolation by FACS. With nearly pure hair cells and surrounding cells, from cochlea and utricle and from E16 to P7, we performed a comprehensive cell type-specific RNA-Seq study of gene expression during mouse inner ear development. Expression profiling revealed new hair cell genes with distinct expression patterns: some are specific for vestibular hair cells, others for cochlear hair cells, and some are expressed just before or after maturation of mechanosensitivity. We found that many of the known hereditary deafness genes are much more highly expressed in hair cells than surrounding cells, suggesting that genes preferentially expressed in hair cells are good candidates for unknown deafness genes. PMID:25904789

  15. Adult mouse brain gene expression patterns bear an embryologic imprint.

    PubMed

    Zapala, Matthew A; Hovatta, Iiris; Ellison, Julie A; Wodicka, Lisa; Del Rio, Jo A; Tennant, Richard; Tynan, Wendy; Broide, Ron S; Helton, Rob; Stoveken, Barbara S; Winrow, Christopher; Lockhart, Daniel J; Reilly, John F; Young, Warren G; Bloom, Floyd E; Lockhart, David J; Barlow, Carrolee

    2005-07-19

    The current model to explain the organization of the mammalian nervous system is based on studies of anatomy, embryology, and evolution. To further investigate the molecular organization of the adult mammalian brain, we have built a gene expression-based brain map. We measured gene expression patterns for 24 neural tissues covering the mouse central nervous system and found, surprisingly, that the adult brain bears a transcriptional "imprint" consistent with both embryological origins and classic evolutionary relationships. Embryonic cellular position along the anterior-posterior axis of the neural tube was shown to be closely associated with, and possibly a determinant of, the gene expression patterns in adult structures. We also observed a significant number of embryonic patterning and homeobox genes with region-specific expression in the adult nervous system. The relationships between global expression patterns for different anatomical regions and the nature of the observed region-specific genes suggest that the adult brain retains a degree of overall gene expression established during embryogenesis that is important for regional specificity and the functional relationships between regions in the adult. The complete collection of extensively annotated gene expression data along with data mining and visualization tools have been made available on a publicly accessible web site (www.barlow-lockhart-brainmapnimhgrant.org).

  16. Expression of a mouse metallothionein-Escherichia coli. beta. -galactosidase fusion gene (MT-. beta. gal) in early mouse embryos

    SciTech Connect

    Stevens, M.E.; Meneses, J.J.; Pedersen, R.A. )

    1989-08-01

    The authors have microinjected DNA containing the inducible mouse metallothionein-I (MT-I) promoter, coupled to the structural gene for Escherichia coli {beta}-galactosidase (lacZ), into the pronuclei of one-cell mouse embryos. A qualitative histochemical assay, with 5-bromo-4-chloro-3-indolyl {beta}-D-galactopyranoside (X-Gal) as a substrate, was used to detect expression of lacZ at several preimplantation stages. They observed staining indicative of exogenous {beta}-galactosidase activity in 5-17% of DNA-injected embryos assayed at preimplantation stages after 16-24 h treatment with ZnSO{sub 4}. Thus, lacZ can be used as an indicator gene for promoter function during early mouse embryogenesis, and the incorporation of the MT-I promoter into fusion genes can be a useful means of controlling the expression of exogenous genes in preimplantation mouse embryos.

  17. ATM localization and gene expression in the adult mouse eye

    PubMed Central

    Leemput, Julia; Masson, Christel; Bigot, Karine; Errachid, Abdelmounaim; Dansault, Anouk; Provost, Alexandra; Gadin, Stéphanie; Aoufouchi, Said; Menasche, Maurice

    2009-01-01

    Purpose High levels of metabolism and oxygen consumption in most adult murine ocular compartments, combined with exposure to light and ultraviolet (UV) radiation, are major sources of oxidative stress, causing DNA damage in ocular cells. Of all mammalian body cells, photoreceptor cells consume the largest amount of oxygen and generate the highest levels of oxidative damage. The accumulation of such damage throughout life is a major factor of aging tissues. Several multiprotein complexes have recently been identified as the major sensors and mediators involved in the maintenance of DNA integrity. The activity of these complexes initially seemed to be restricted to dividing cells, given their ultimate role in major cell cycle checkpoints. However, it was later established that they are also active in post-mitotic cells. Recent findings demonstrate that the DNA damage response (DDR) is essential for the development, maintenance, and normal functioning of the adult central nervous system. One major molecular factor in the DDR is the protein, ataxia telangiectasia mutated (ATM). It is required for the rapid induction of cellular responses to DNA double-strand breaks. These cytotoxic DNA lesions may be caused by oxidative damage. To understand how ATM prevents oxidative stress and participates in the maintenance of genomic integrity and cell viability of the adult retina, we determined the ATM expression patterns and studied its localization in the adult mouse eye. Methods Atm gene expression was analyzed by RT–PCR experiments and its localization by in situ hybridization on adult mouse ocular and cerebellar tissue sections. ATM protein expression was determined by western blot analysis of proteins homogenates extracted from several mouse tissues and its localization by immunohistochemistry experiments performed on adult mouse ocular and cerebellar tissue sections. In addition, subcellular localization was realized by confocal microscopy imaging of ocular tissue

  18. Gene expression and dental enamel structure in developing mouse incisor.

    PubMed

    Sehic, Amer; Risnes, Steinar; Khan, Qalb-E-Saleem; Khuu, Cuong; Osmundsen, Harald

    2010-04-01

    At the mouse incisor tip the initially differentiated ameloblasts produce a thin, prism-free enamel, while further apically, in the immediate adjacent segment, the enamel thickness increases and the four-layered enamel of mouse incisor is formed. Comparative gene-expression profiling was carried out on RNA isolated from these two segments of incisor tooth germs at embryonic day (E)17.5 and at postnatal days (P)0, 1, 2, and 10 using microarrays to measure messenger RNA (mRNA) and microRNA (miRNA) species present in the segments. Validation of expression data was achieved using real-time reverse transcription-polymerase chain reaction (RT-PCR) and western blotting. Bioinformatic data suggested enhanced cellular apoptosis in the incisal tip segment, which, together with diminished expression of the Amelx and Enam genes, may contribute to the production of the thin enamel seen in this tooth segment. For genes exhibiting higher levels of expression in the adjacent segment where complex enamel is being formed, bioinformatic analysis suggested significant associations with cellular functions involving the actin cytoskeleton, cellular development, morphology, and movement. This is suggested to reflect that ameloblasts with Tomes' process are being organized in transverse rows, facilitating the transverse movement that results in prism decussation in the inner enamel of the adjacent segment. Bioinformatic analysis of miRNA expression data lends support to these suggestions.

  19. EMAGE mouse embryo spatial gene expression database: 2010 update

    PubMed Central

    Richardson, Lorna; Venkataraman, Shanmugasundaram; Stevenson, Peter; Yang, Yiya; Burton, Nicholas; Rao, Jianguo; Fisher, Malcolm; Baldock, Richard A.; Davidson, Duncan R.; Christiansen, Jeffrey H.

    2010-01-01

    EMAGE (http://www.emouseatlas.org/emage) is a freely available online database of in situ gene expression patterns in the developing mouse embryo. Gene expression domains from raw images are extracted and integrated spatially into a set of standard 3D virtual mouse embryos at different stages of development, which allows data interrogation by spatial methods. An anatomy ontology is also used to describe sites of expression, which allows data to be queried using text-based methods. Here, we describe recent enhancements to EMAGE including: the release of a completely re-designed website, which offers integration of many different search functions in HTML web pages, improved user feedback and the ability to find similar expression patterns at the click of a button; back-end refactoring from an object oriented to relational architecture, allowing associated SQL access; and the provision of further access by standard formatted URLs and a Java API. We have also increased data coverage by sourcing from a greater selection of journals and developed automated methods for spatial data annotation that are being applied to spatially incorporate the genome-wide (∼19 000 gene) ‘EURExpress’ dataset into EMAGE. PMID:19767607

  20. EMAGE mouse embryo spatial gene expression database: 2010 update.

    PubMed

    Richardson, Lorna; Venkataraman, Shanmugasundaram; Stevenson, Peter; Yang, Yiya; Burton, Nicholas; Rao, Jianguo; Fisher, Malcolm; Baldock, Richard A; Davidson, Duncan R; Christiansen, Jeffrey H

    2010-01-01

    EMAGE (http://www.emouseatlas.org/emage) is a freely available online database of in situ gene expression patterns in the developing mouse embryo. Gene expression domains from raw images are extracted and integrated spatially into a set of standard 3D virtual mouse embryos at different stages of development, which allows data interrogation by spatial methods. An anatomy ontology is also used to describe sites of expression, which allows data to be queried using text-based methods. Here, we describe recent enhancements to EMAGE including: the release of a completely re-designed website, which offers integration of many different search functions in HTML web pages, improved user feedback and the ability to find similar expression patterns at the click of a button; back-end refactoring from an object oriented to relational architecture, allowing associated SQL access; and the provision of further access by standard formatted URLs and a Java API. We have also increased data coverage by sourcing from a greater selection of journals and developed automated methods for spatial data annotation that are being applied to spatially incorporate the genome-wide (approximately 19,000 gene) 'EURExpress' dataset into EMAGE. PMID:19767607

  1. The mouse Gene Expression Database (GXD): 2014 update.

    PubMed

    Smith, Constance M; Finger, Jacqueline H; Hayamizu, Terry F; McCright, Ingeborg J; Xu, Jingxia; Berghout, Joanne; Campbell, Jeff; Corbani, Lori E; Forthofer, Kim L; Frost, Pete J; Miers, Dave; Shaw, David R; Stone, Kevin R; Eppig, Janan T; Kadin, James A; Richardson, Joel E; Ringwald, Martin

    2014-01-01

    The Gene Expression Database (GXD; http://www.informatics.jax.org/expression.shtml) is an extensive and well-curated community resource of mouse developmental expression information. GXD collects different types of expression data from studies of wild-type and mutant mice, covering all developmental stages and including data from RNA in situ hybridization, immunohistochemistry, RT-PCR, northern blot and western blot experiments. The data are acquired from the scientific literature and from researchers, including groups doing large-scale expression studies. Integration with the other data in Mouse Genome Informatics (MGI) and interconnections with other databases places GXD's gene expression information in the larger biological and biomedical context. Since the last report, the utility of GXD has been greatly enhanced by the addition of new data and by the implementation of more powerful and versatile search and display features. Web interface enhancements include the capability to search for expression data for genes associated with specific phenotypes and/or human diseases; new, more interactive data summaries; easy downloading of data; direct searches of expression images via associated metadata; and new displays that combine image data and their associated annotations. At present, GXD includes >1.4 million expression results and 250,000 images that are accessible to our search tools. PMID:24163257

  2. The mouse Gene Expression Database (GXD): 2007 update

    PubMed Central

    Smith, Constance M.; Finger, Jacqueline H.; Hayamizu, Terry F.; McCright, Ingeborg J.; Eppig, Janan T.; Kadin, James A.; Richardson, Joel E.; Ringwald, Martin

    2007-01-01

    The Gene Expression Database (GXD) provides the scientific community with an extensive and easily searchable database of gene expression information about the mouse. Its primary emphasis is on developmental studies. By integrating different types of expression data, GXD aims to provide comprehensive information about expression patterns of transcripts and proteins in wild-type and mutant mice. Integration with the other Mouse Genome Informatics (MGI) databases places the gene expression information in the context of genetic, sequence, functional and phenotypic information, enabling valuable insights into the molecular biology that underlies developmental and disease processes. In recent years the utility of GXD has been greatly enhanced by a large increase in data content, obtained from the literature and provided by researchers doing large-scale in situ and cDNA screens. In addition, we have continued to refine our query and display features to make it easier for users to interrogate the data. GXD is available through the MGI web site at or directly at . PMID:17130151

  3. BodyMap: a human and mouse gene expression database.

    PubMed

    Hishiki, T; Kawamoto, S; Morishita, S; Okubo, K

    2000-01-01

    BodyMap is a human and mouse gene expression database that has been maintained since 1993. It is based on site-directed 3'-ESTs collected from non-biased cDNA libraries constructed at Osaka University and contains >270 000 sequences from 60 human and 38 mouse tissues. The site-directed nature of the sequence tags allows unequivocal grouping of tags representing the same transcript and provides abundance information for each transcript in different parts of the body. Our collection of ESTs was compared periodically with other public databases for cross referencing. The histological resolution of source tissues and unique cloning strategy that minimized cloning bias enabled BodyMap to support three unique mRNA based experiments in silico. First, the recurrence information for clones in each library provides a rough estimate of the mRNA composition of each source tissue. Second, a user can search the entire data set with nucleotide sequences or keywords to assess expression patterns of particular genes. Third, and most important, BodyMap allows a user to select genes that have a desired expression pattern in humans and mice. BodyMap is accessible through the WWW at http://bodymap.ims.u-tokyo.ac.jp PMID:10592203

  4. Simultaneous synthesis of human-, mouse- and chimeric epidermal growth factor genes via 'hybrid gene synthesis' approach.

    PubMed Central

    Sung, W L; Zahab, D M; Yao, F L; Wu, R; Narang, S A

    1986-01-01

    Simultaneous synthesis of two DNA duplexes encoding human and mouse epidermal growth factors (EGF) was accomplished in a single step. A 174 b.p. DNA heteroduplex, with 16 single and double base pair mismatches, was designed. One strand encoded the human EGF, and the opposite strand indirectly encoded the mouse EGF. The heteroduplex DNA was synthesized by ligation of seven overlapping oligodeoxyribonucleotides with a linearized plasmid. After transformation in E. coli HB101 (recA 13), the resulting heteroduplex plasmid served as the template in plasmid replication. Two different plasmid progenies bearing either the human or mouse EGF-coding sequence were identified by colony hybridization using the appropriate probes. However, in E. coli JM103, the same process yielded plasmid progenies encoding different chimeric EGF molecules, presumably due to crossover of human and mouse EGF gene sequences. Images PMID:3529034

  5. The mouse Gene Expression Database (GXD): 2011 update

    PubMed Central

    Finger, Jacqueline H.; Smith, Constance M.; Hayamizu, Terry F.; McCright, Ingeborg J.; Eppig, Janan T.; Kadin, James A.; Richardson, Joel E.; Ringwald, Martin

    2011-01-01

    The Gene Expression Database (GXD) is a community resource of mouse developmental expression information. GXD integrates different types of expression data at the transcript and protein level and captures expression information from many different mouse strains and mutants. GXD places these data in the larger biological context through integration with other Mouse Genome Informatics (MGI) resources and interconnections with many other databases. Web-based query forms support simple or complex searches that take advantage of all these integrated data. The data in GXD are obtained from the literature, from individual laboratories, and from large-scale data providers. All data are annotated and reviewed by GXD curators. Since the last report, the GXD data content has increased significantly, the interface and data displays have been improved, new querying capabilities were implemented, and links to other expression resources were added. GXD is available through the MGI web site (www.informatics.jax.org), or directly at www.informatics.jax.org/expression.shtml. PMID:21062809

  6. ATM controls meiotic double-strand break formation

    PubMed Central

    Lange, Julian; Pan, Jing; Cole, Francesca; Thelen, Michael P.; Jasin, Maria; Keeney, Scott

    2011-01-01

    In many organisms, developmentally programmed double-strand breaks (DSBs) formed by the SPO11 transesterase initiate meiotic recombination, which promotes pairing and segregation of homologous chromosomes1. Because every chromosome must receive a minimum number of DSBs, attention has focused on factors that support DSB formation2. However, improperly repaired DSBs can cause meiotic arrest or mutation3,4, thus having too many DSBs is likely as deleterious as having too few. Only a small fraction of SPO11 protein ever makes a DSB in yeast or mouse5, and SPO11 and its accessory factors remain abundant long after most DSB formation ceases1, implying the existence of mechanisms that restrain SPO11 activity to limit DSB numbers. Here we report that the number of meiotic DSBs in mouse is controlled by ATM, a kinase activated by DNA damage to trigger checkpoint signaling and promote DSB repair. Levels of SPO11-oligonucleotide complexes, by-products of meiotic DSB formation, are elevated at least ten-fold in spermatocytes lacking ATM. Moreover, Atm mutation renders SPO11-oligonucleotide levels sensitive to genetic manipulations that modulate SPO11 protein levels. We propose that ATM restrains SPO11 via a negative feedback loop in which kinase activation by DSBs suppresses further DSB formation. Our findings explain previously puzzling phenotypes of Atm-null mice and provide a molecular basis for the gonadal dysgenesis observed in ataxia telangiectasia, the human syndrome caused by ATM deficiency. PMID:22002603

  7. Mutations in TUBB8 and Human Oocyte Meiotic Arrest.

    PubMed

    Feng, Ruizhi; Sang, Qing; Kuang, Yanping; Sun, Xiaoxi; Yan, Zheng; Zhang, Shaozhen; Shi, Juanzi; Tian, Guoling; Luchniak, Anna; Fukuda, Yusuke; Li, Bin; Yu, Min; Chen, Junling; Xu, Yao; Guo, Luo; Qu, Ronggui; Wang, Xueqian; Sun, Zhaogui; Liu, Miao; Shi, Huijuan; Wang, Hongyan; Feng, Yi; Shao, Ruijin; Chai, Renjie; Li, Qiaoli; Xing, Qinghe; Zhang, Rui; Nogales, Eva; Jin, Li; He, Lin; Gupta, Mohan L; Cowan, Nicholas J; Wang, Lei

    2016-01-21

    Background Human reproduction depends on the fusion of a mature oocyte with a sperm cell to form a fertilized egg. The genetic events that lead to the arrest of human oocyte maturation are unknown. Methods We sequenced the exomes of five members of a four-generation family, three of whom had infertility due to oocyte meiosis I arrest. We performed Sanger sequencing of a candidate gene, TUBB8, in DNA samples from these members, additional family members, and members of 23 other affected families. The expression of TUBB8 and all other β-tubulin isotypes was assessed in human oocytes, early embryos, sperm cells, and several somatic tissues by means of a quantitative reverse-transcriptase-polymerase-chain-reaction assay. We evaluated the effect of the TUBB8 mutations on the assembly of the heterodimer consisting of one α-tubulin polypeptide and one β-tubulin polypeptide (α/β-tubulin heterodimer) in vitro, on microtubule architecture in HeLa cells, on microtubule dynamics in yeast cells, and on spindle assembly in mouse and human oocytes. Results We identified seven mutations in the primate-specific gene TUBB8 that were responsible for oocyte meiosis I arrest in 7 of the 24 families. TUBB8 expression is unique to oocytes and the early embryo, in which this gene accounts for almost all the expressed β-tubulin. The mutations affect chaperone-dependent folding and assembly of the α/β-tubulin heterodimer, disrupt microtubule behavior on expression in cultured cells, alter microtubule dynamics in vivo, and cause catastrophic spindle-assembly defects and maturation arrest on expression in mouse and human oocytes. Conclusions TUBB8 mutations have dominant-negative effects that disrupt microtubule behavior and oocyte meiotic spindle assembly and maturation, causing female infertility. (Funded by the National Basic Research Program of China and others.). PMID:26789871

  8. Characterization of the p16 gene in the mouse: Evidence for a large gene family

    SciTech Connect

    Fountain, J.W.; Giendening, J.M.; Flores, J.F.

    1994-09-01

    The p16 gene product is an inhibitor of the cyclin-dependent kinase 4 (CDK4)/cyclin D complex. When uninhibited, the CDK4/cyclin D complex participates in the phosphorylation of the retinoblastoma (RB) protein and renders it inactive. Upon inactivation of the RB protein, transition from the G{sub 1} to the S phase of mitosis occurs and results in cellular proliferation. Thus, p16 is presumed to act as a negative regulator of cell growth by preventing the phosphorylation, and thereby subsequent inactivation, of RB by CDK4/cyclin D. Recently, the p16 gene (also known as the multiple tumor suppressor 1 (MTS1) gene) has been mapped to chromosome 9p21 and found to be deleted or mutated in a number of tumor cell lines. These findings support the role of p16 as a growth inhibitor or tumor suppressor gene and suggest that the mutation of this gene may have global implications in carcinogenesis. We have chosen to test the functional significance of p16 mutations in vivo through the generation of a mouse mutant for p16. In preparation for this undertaking, eight apparently independent (as judged by restriction enzyme digestion and differential hybridization) mouse genomic embryonic stem cell clones have been identified using exon 2 from the human p16 gene as a probe. The identification of these multiple nonoverlapping clones was not entirely surprising since the reduced stringency hybridization of a zoo blot with the same probe also revealed 10-15 positive EcoRI fragments in all species tested, including human, monkey, cow, dog, cat, rabbit, hamster, mouse, chicken and D. melanogaster. Taken together, these findings suggest that the p16 gene is a member of a large gene family. The location of these genomic clones, as well as their potential expression in the mouse, is currently under investigation.

  9. Mouse models for genes involved in impaired spermatogenesis.

    PubMed

    O'Bryan, M K; de Kretser, D

    2006-02-01

    Since the introduction of molecular biology and gene ablation technologies there have been substantial advances in our understanding of how sperm are made and fertilization occurs. There have been at least 150 different models of specifically altered gene function produced that have resulted in male infertility spanning virtually all aspects of the spermatogenic, sperm maturation and fertilization processes. While each has, or potentially will reveal, novel aspects of these processes, there is still much of which we have little knowledge. The current review is by no means a comprehensive list of these mouse models, rather it gives an overview of the potential for such models which up to this point have generally been 'knockouts'; it presents alternative strategies for the production of new models and emphasizes the importance of thorough phenotypic analysis in order to extract a maximum amount of information from each model.

  10. Inheritance of a functional mouse metallothionein gene in tobacco

    SciTech Connect

    Maiti, I.B.; Wagner, G.J.; Yeargan, R.; Hunt, A.G. )

    1989-04-01

    Morphologically normal plants were obtained from progeny (Ro, R1 and R2) originating from tobacco leaf tissue transformed with Agrobacterium tumefaciens containing a chimeric gene for kanamycin resistance an the mouse metallothionein cDNA gene directed by the constitutive promote 35S from CaMV. Integration and expression in R1 progeny was demonstrated by Southern, Northern blot analysis and metallothionein assay. Kanamycin resistance analysis of R1 and R2 progeny showed inheritance to be as a dominant Mendelian trait. Seedlings obtained from self-fertilized transgenic tobacco are more tolerant to cadmium stress than nontransformed controls. Cadmium accumulation in leaves of transgenic seedlings exposed to a low, field-like Cd concentration was about 20% lower than that in nontransformed controls.

  11. Comparative importance of fatty acid beta-oxidation to nuclear maturation, gene expression, and glucose metabolism in mouse, bovine, and porcine cumulus oocyte complexes.

    PubMed

    Paczkowski, Melissa; Silva, Elena; Schoolcraft, William B; Krisher, Rebecca L

    2013-05-01

    The objective of these experiments was to evaluate the importance of fatty acid beta-oxidation (FAO) in the cumulus oocyte complex (COC) during in vitro maturation (IVM) to oocyte nuclear maturation and gene expression in both the oocyte and cumulus cells in three species with differing amounts of oocyte intracellular lipids (mouse, low; bovine, moderate; porcine, high). We inhibited FAO using etomoxir at 0, 10, 25, 100, or 250 μM. Completion of oocyte nuclear maturation was inhibited after COC exposure to 250 μM etomoxir in mouse oocytes, 100 μM etomoxir in bovine oocytes, and as little as 10 μM etomoxir in porcine oocytes (P < 0.05). When FAO was inhibited in mouse and porcine COCs resulting in inhibition of meiosis, the abundance of FAO, glycolytic, and oxidative stress gene transcripts were decreased in oocytes and cumulus cells (P < 0.05), although to a much greater extent in the pig. In bovine oocytes and cumulus cells, FAO gene transcripts were increased and glycolytic gene expression altered following meiotic inhibition due to etomoxir. Etomoxir, at doses that did not inhibit nuclear maturation in bovine and murine COCs, increased glucose consumption (P < 0.05), suggesting glucose metabolism is increased to meet the metabolic demands of the COCs when fatty acid metabolism is compromised. Our data demonstrates that FAO is essential to oocyte nuclear maturation in all three species. Sensitivity of nuclear maturation to FAO inhibition reflects the amount of lipid present in the ooplasm and may suggest a relative reliance on this metabolic pathway.

  12. Localization of dystrophin gene transcripts during mouse embryogenesis

    PubMed Central

    1992-01-01

    The spatial and temporal expression of the dystrophin gene has been examined during mouse embryogenesis, using in situ hybridization on tissue sections with a probe from the 5' end of the dystrophin coding sequence. In striated muscle, dystrophin transcripts are detectable from about 9 d in the heart and slightly later in skeletal muscle. However, there is an important difference between the two types of muscle: the heart is already functional as a contractile organ before the appearance of dystrophin transcripts, whereas this is not the case in skeletal muscle, where dystrophin and myosin heavy chain transcripts are first detectable at the same time. In the heart, dystrophin transcripts accumulate initially in the outflow tract and, at later stages, in both the atria and ventricles. In skeletal muscle, the gene is expressed in all myocytes irrespective of fiber type. In smooth muscle dystrophin transcripts are first detectable from 11 d post coitum in blood vessels, and subsequently in lung bronchi and in the digestive tract. The other major tissue where the dystrophin gene is expressed is the brain, where transcripts are clearly detectable in the cerebellum from 13 d. High-level expression of the gene is also seen in particular regions of the forebrain involved in the regulation of circadian rhythms, the endocrine system, and olfactory function, not previously identified in this context. The findings are discussed in the context of the pathology of Duchenne muscular dystrophy. PMID:1429837

  13. Structure and polymorphism of the mouse prion protein gene.

    PubMed Central

    Westaway, D; Cooper, C; Turner, S; Da Costa, M; Carlson, G A; Prusiner, S B

    1994-01-01

    Missense mutations in the prion protein (PrP) gene, overexpression of the cellular isoform of PrP (PrPC), and infection with prions containing the scrapie isoform of PrP (PrPSc) all cause neurodegenerative disease. To understand better the physiology and expression of PrPC, we retrieved mouse PrP gene (Prn-p) yeast artificial chromosome (YAC), cosmid, phage, and cDNA clones. Physical mapping positions Prn-p approximately 300 kb from ecotropic virus integration site number 4 (Evi-4), compatible with failure to detect recombination between Prn-p and Evi-4 in genetic crosses. The Prn-pa allele encompasses three exons, with exons 1 and 2 encoding the mRNA 5' untranslated region. Exon 2 has no equivalent in the Syrian hamster and human PrP genes. The Prn-pb gene shares this intron/exon structure but harbors an approximately 6-kb deletion within intron 2. While the Prn-pb open reading frame encodes two amino acid substitutions linked to prolonged scrapie incubation periods, a deletion of intron 2 sequences also characterizes inbred strains such as RIII/S and MOLF/Ei with shorter incubation periods, making a relationship between intron 2 size and scrapie pathogenesis unlikely. The promoter regions of a and b Prn-p alleles include consensus Sp1 and AP-1 sites, as well as other conserved motifs which may represent binding sites for as yet unidentified transcription factors. Images PMID:7912827

  14. Sleeping Beauty Mouse Models Identify Candidate Genes Involved in Gliomagenesis

    PubMed Central

    Vyazunova, Irina; Maklakova, Vilena I.; Berman, Samuel; De, Ishani; Steffen, Megan D.; Hong, Won; Lincoln, Hayley; Morrissy, A. Sorana; Taylor, Michael D.; Akagi, Keiko; Brennan, Cameron W.; Rodriguez, Fausto J.; Collier, Lara S.

    2014-01-01

    Genomic studies of human high-grade gliomas have discovered known and candidate tumor drivers. Studies in both cell culture and mouse models have complemented these approaches and have identified additional genes and processes important for gliomagenesis. Previously, we found that mobilization of Sleeping Beauty transposons in mice ubiquitously throughout the body from the Rosa26 locus led to gliomagenesis with low penetrance. Here we report the characterization of mice in which transposons are mobilized in the Glial Fibrillary Acidic Protein (GFAP) compartment. Glioma formation in these mice did not occur on an otherwise wild-type genetic background, but rare gliomas were observed when mobilization occurred in a p19Arf heterozygous background. Through cloning insertions from additional gliomas generated by transposon mobilization in the Rosa26 compartment, several candidate glioma genes were identified. Comparisons to genetic, epigenetic and mRNA expression data from human gliomas implicates several of these genes as tumor suppressor genes and oncogenes in human glioblastoma. PMID:25423036

  15. A mouse embryonic stem cell bank for inducible overexpression of human chromosome 21 genes

    PubMed Central

    2010-01-01

    Background Dosage imbalance is responsible for several genetic diseases, among which Down syndrome is caused by the trisomy of human chromosome 21. Results To elucidate the extent to which the dosage imbalance of specific human chromosome 21 genes perturb distinct molecular pathways, we developed the first mouse embryonic stem (ES) cell bank of human chromosome 21 genes. The human chromosome 21-mouse ES cell bank includes, in triplicate clones, 32 human chromosome 21 genes, which can be overexpressed in an inducible manner. Each clone was transcriptionally profiled in inducing versus non-inducing conditions. Analysis of the transcriptional response yielded results that were consistent with the perturbed gene's known function. Comparison between mouse ES cells containing the whole human chromosome 21 (trisomic mouse ES cells) and mouse ES cells overexpressing single human chromosome 21 genes allowed us to evaluate the contribution of single genes to the trisomic mouse ES cell transcriptome. In addition, for the clones overexpressing the Runx1 gene, we compared the transcriptome changes with the corresponding protein changes by mass spectroscopy analysis. Conclusions We determined that only a subset of genes produces a strong transcriptional response when overexpressed in mouse ES cells and that this effect can be predicted taking into account the basal gene expression level and the protein secondary structure. We showed that the human chromosome 21-mouse ES cell bank is an important resource, which may be instrumental towards a better understanding of Down syndrome and other human aneuploidy disorders. PMID:20569505

  16. Lysosomal Dysfunctions Associated with Mutations at Mouse Pigment Genes

    PubMed Central

    Novak, Edward K.; Swank, Richard T.

    1979-01-01

    Melanosomes and lysosomes share several structural and biosynthetic properties. Therefore, a large number of mouse pigment mutants were tested to determine whether genes affecting melanosome structure or function might also affect the lysosome. Among 31 mouse pigment mutants, six had 1.5- to 2.5- fold increased concentrations of kidney β-glucuronidase. Three mutants, pale ear, pearl and pallid, had a generalized effect on lysosomal enzymes since there were coordinate increases in kidney β-galactosidase and α-mannosidase. The effects of these three mutations are lysosome specific since rates of kidney protein synthesis and activities of three nonlysosomal kidney enzymes were normal. Also, the mutants are relatively tissue specific in that all had normal liver lysomal enzyme concentrations.—A common dysfunction in all three mutants was a lowered rate of lysosomal enzyme secretion from kidney into urine. While normal C57BL/6J mice daily secreted 27 to 30% of total kidney β-glucuronidase and β-galactosidase, secretion of these two enzymes was coordinately depressed to 1 to 2%, 8 to 9% and 4 to 5% of total kidney enzyme in the pale-ear, pearl and pallid mutants, respectively. Although depressed lysosomal enzyme secretion is the major pigment mutant alteration, the higher lysomal enzyme concentrations in pearl and pallid may be partly due to an increase in lysosomal enzyme synthesis. In these mutants kidney glucuronidase synthetic rate was increased 1.4- to 1.5-fold.—These results suggest that there are several critical genes in mammals that control the biogenesis, processing and/or function of related classes of subcellular organelles. The mechanism of action of these genes is amenable to further analysis since they have been incorporated into congenic inbred strains of mice. PMID:115747

  17. Gene Expression and Functional Annotation of the Human and Mouse Choroid Plexus Epithelium

    PubMed Central

    Janssen, Sarah F.; van der Spek, Sophie J. F.; ten Brink, Jacoline B.; Essing, Anke H. W.; Gorgels, Theo G. M. F.; van der Spek, Peter J.; Jansonius, Nomdo M.; Bergen, Arthur A. B.

    2013-01-01

    Background The choroid plexus epithelium (CPE) is a lobed neuro-epithelial structure that forms the outer blood-brain barrier. The CPE protrudes into the brain ventricles and produces the cerebrospinal fluid (CSF), which is crucial for brain homeostasis. Malfunction of the CPE is possibly implicated in disorders like Alzheimer disease, hydrocephalus or glaucoma. To study human genetic diseases and potential new therapies, mouse models are widely used. This requires a detailed knowledge of similarities and differences in gene expression and functional annotation between the species. The aim of this study is to analyze and compare gene expression and functional annotation of healthy human and mouse CPE. Methods We performed 44k Agilent microarray hybridizations with RNA derived from laser dissected healthy human and mouse CPE cells. We functionally annotated and compared the gene expression data of human and mouse CPE using the knowledge database Ingenuity. We searched for common and species specific gene expression patterns and function between human and mouse CPE. We also made a comparison with previously published CPE human and mouse gene expression data. Results Overall, the human and mouse CPE transcriptomes are very similar. Their major functionalities included epithelial junctions, transport, energy production, neuro-endocrine signaling, as well as immunological, neurological and hematological functions and disorders. The mouse CPE presented two additional functions not found in the human CPE: carbohydrate metabolism and a more extensive list of (neural) developmental functions. We found three genes specifically expressed in the mouse CPE compared to human CPE, being ACE, PON1 and TRIM3 and no human specifically expressed CPE genes compared to mouse CPE. Conclusion Human and mouse CPE transcriptomes are very similar, and display many common functionalities. Nonetheless, we also identified a few genes and pathways which suggest that the CPE between mouse and

  18. Meiotic process and aneuploidy

    SciTech Connect

    Grell, R.F.

    1985-01-01

    The process of meiosis is analyzed by dissecting it into its component parts using the early oocyte of Drosophila as a model. Entrance of the oocytes into premeiotic interphase signals initiation of DNA replication which continues for 30 h. Coincidentally, extensive synaptonemal complexes appear, averaging 50 ..mu..m (132 h), peaking at 75 ..mu..m (144 h) and continuing into early vitellarial stages. Recombinational response to heat, evidenced by enhancement or induction of exchange, is limited to the S-phase with a peak at 144 h coinciding with maximal extension of the SC. Coincidence of synapsis and recombination response with S at premeiotic interphase is contrary to their conventional localization at meiotic prophase. The interrelationship between exchange and nondisjunction has been clarified by the Distributive Pairing Model of meiosis. Originally revealed through high frequencies of nonrandom assortment of nonhomologous chromosomes, distributive pairing has been shown to follow and to be noncompetitive with exchange, to be based on size-recognition, not homology, and as a raison d'etre, to provide a segregational mechanism for noncrossover homologues. Rearrangements, recombination mutants and aneuploids may contribute noncrossover chromosomes to the distributive pool and so promote the nonhomologous associations responsible for nondisjunction of homologues and regular segregation of nonhomologues. 38 references, 15 figures. (ACR)

  19. Meiotic recombination initiated by a double-strand break in rad50{Delta} yeast cells otherwise unable to initiate meiotic recombination

    SciTech Connect

    Malkova, A.; Haber, J.E.; Dawson, D.

    1996-06-01

    Meiotic recombination in Saccharomyces cerevisiae is initiated by double-strand breaks (DSBs). We have developed a system to compare the properties of meiotic DSBs with those created by the site-specific HO endonuclease. HO endonuclease was expressed under the control of the meiotic-specific SPO13 promoter, creating a DSB at a single site on one of yeast`s 16 chromosomes. In Rad{sup +} strains the times of appearance of the HO-induced DSBs and of subsequent recombinants are coincident with those induced by normal meiotic DSBs. Physical monitoring of DNA showed that SPO13::HO induced gene conversions both in Rad{sup +} and in rad50{Delta} cells that cannot initiate normal meiotic DSBs. We find that the RAD50 gene is important, but not essential, for recombination even after a DSB has been created in a meiotic cell. In rad50{Delta} cells, some DSBs are not repaired until a broken chromosome has been packaged into a spore and is subsequently germinated. This suggests that a broken chromosome does not signal an arrest of progression through meiosis. The recombination defect in rad50{Delta} diploids is not, however, meiotic specific, as mitotic rad50 diploids, experiencing an HO-induced DSB, exhibit similar departures from wild-type recombination. 57 refs., 5 figs., 3 tabs.

  20. Genomic organization of the mouse dystrobrevin gene: Comparative analysis with the dystrophin gene

    SciTech Connect

    Ambrose, H.J.; Blake, D.J.; Nawrotzki, R.A.; Davies, K.E.

    1997-02-01

    Dystrobrevin, the mammalian orthologue of the Torpedo 87-kDa postsynaptic protein, is a member of the dystrophin gene family with homology to the cysteine-rich carboxy-terminal domain of dystrophin. Torpedo dystrobrevin copurifies with the acetylcholine receptors and is thought to form a complex with dystrophin and syntrophin. This complex is also found at the sarcolemma in vertebrates and defines the cytoplasmic component of the dystrophin-associated protein complex. Previously we have cloned several dystrobrevin isoforms from mouse brain and muscle. Here we show that these transcripts are the products of a single gene located on proximal mouse chromosome 18. To investigate the diversity of dystrobrevin transcripts we have determined that the mouse dystrobrevin gene is organized into 24 coding exons that span between 130 and 170 kb at the genomic level. The gene encodes at least three distinct protein isoforms that are expressed in a tissue-specific manner. Interestingly, although there is only 27% amino acid identity between the homologous regions of dystrobrevin and dystrophin, the positions of 8 of the 15 exon-intron junctions are identical. 47 refs., 4 figs., 2 tabs.

  1. Mouse histone H2A and H2B genes: four functional genes and a pseudogene undergoing gene conversion with a closely linked functional gene.

    PubMed Central

    Liu, T J; Liu, L; Marzluff, W F

    1987-01-01

    The sequence of five mouse histone genes, two H2a and three H2b genes on chromosome 13 has been determined. The three H2b genes all code for different proteins, each differing in two amino acids from the others. The H2b specific elements present 5' to H2b genes from other species are present in all three mouse H2b genes. All three H2b genes are expressed in the same relative amounts in three different mouse cell lines and fetal mice. The H2b gene with the H2b specific sequence closest to the TATAA sequence is expressed in the highest amount. One of the H2a genes lacks the first 9 amino acids, the promoter region, the last 3 amino acids and contains an altered 3' end sequence. Despite these multiple defects, there is only one nucleotide change between the two H2a genes from codon 9 to 126. This indicates that a recent gene conversion has occurred between these two genes. The similarity of the nucleotide sequences in the coding regions of mouse histone genes is probably due to gene conversion events targeted precisely at the coding region. Images PMID:3562244

  2. Targeted disruption of the mouse Lipoma Preferred Partner gene

    SciTech Connect

    Vervenne, Hilke B.V.K.; Crombez, Koen R.M.O.; Delvaux, Els L.; Janssens, Veerle; Ven, Wim J.M. van de Petit, Marleen M.R.

    2009-02-06

    LPP (Lipoma Preferred Partner) is a zyxin-related cell adhesion protein that is involved in the regulation of cell migration. We generated mice with a targeted disruption of the Lpp gene and analysed the importance of Lpp for embryonic development and adult functions. Aberrant Mendelian inheritance in heterozygous crosses suggested partial embryonic lethality of Lpp{sup -/-} females. Fertility of Lpp{sup -/-} males was proven to be normal, however, females from Lpp{sup -/-} x Lpp{sup -/-} crosses produced a strongly reduced number of offspring, probably due to a combination of female embryonic lethality and aberrant pregnancies. Apart from these developmental and reproductive abnormalities, Lpp{sup -/-} mice that were born reached adulthood without displaying any additional macroscopic defects. On the other hand, Lpp{sup -/-} mouse embryonic fibroblasts exhibited reduced migration capacity, reduced viability, and reduced expression of some Lpp interaction partners. Finally, we discovered a short nuclear form of Lpp, expressed mainly in testis via an alternative promoter.

  3. Assignment of the lactotransferrin gene to human chromosome 3 and to mouse chromosome 9.

    PubMed

    Teng, C T; Pentecost, B T; Marshall, A; Solomon, A; Bowman, B H; Lalley, P A; Naylor, S L

    1987-11-01

    Lactotransferrin (LTF), a member of the transferrin family of genes, is the major iron-binding protein in milk and body secretions. The amino acid sequence of LTF consists of two homologous domains homologous to proteins in the transferrin family. Recent isolation of cDNA encoding mouse LTF has expedited the mapping of both mouse and human LTF genes. Southern blot analysis of DNA from mouse-Chinese hamster and human-mouse somatic cell hybrids maps the LTF gene to mouse chromosome 9 and to human chromosome 3, respectively. Furthermore, analysis of cell hybrids containing defined segments of human chromosome 3 demonstrates that the gene is located in the 3q21-qter region. These results suggest that LTF and associated genes of the transferrin family have existed together on the same chromosomal region for 300-500 million years. PMID:3478818

  4. Identification of a set of genes showing regionally enriched expression in the mouse brain

    PubMed Central

    D'Souza, Cletus A; Chopra, Vikramjit; Varhol, Richard; Xie, Yuan-Yun; Bohacec, Slavita; Zhao, Yongjun; Lee, Lisa LC; Bilenky, Mikhail; Portales-Casamar, Elodie; He, An; Wasserman, Wyeth W; Goldowitz, Daniel; Marra, Marco A; Holt, Robert A; Simpson, Elizabeth M; Jones, Steven JM

    2008-01-01

    Background The Pleiades Promoter Project aims to improve gene therapy by designing human mini-promoters (< 4 kb) that drive gene expression in specific brain regions or cell-types of therapeutic interest. Our goal was to first identify genes displaying regionally enriched expression in the mouse brain so that promoters designed from orthologous human genes can then be tested to drive reporter expression in a similar pattern in the mouse brain. Results We have utilized LongSAGE to identify regionally enriched transcripts in the adult mouse brain. As supplemental strategies, we also performed a meta-analysis of published literature and inspected the Allen Brain Atlas in situ hybridization data. From a set of approximately 30,000 mouse genes, 237 were identified as showing specific or enriched expression in 30 target regions of the mouse brain. GO term over-representation among these genes revealed co-involvement in various aspects of central nervous system development and physiology. Conclusion Using a multi-faceted expression validation approach, we have identified mouse genes whose human orthologs are good candidates for design of mini-promoters. These mouse genes represent molecular markers in several discrete brain regions/cell-types, which could potentially provide a mechanistic explanation of unique functions performed by each region. This set of markers may also serve as a resource for further studies of gene regulatory elements influencing brain expression. PMID:18625066

  5. Analysis of Yeast Sporulation Efficiency, Spore Viability, and Meiotic Recombination on Solid Medium.

    PubMed

    Börner, G Valentin; Cha, Rita S

    2015-11-01

    Under conditions of nutrient deprivation, yeast cells initiate a differentiation program in which meiosis is induced and spores are formed. During meiosis, one round of genome duplication is followed by two rounds of chromosome segregation (meiosis I and meiosis II) to generate four haploid nuclei. Meiotic recombination occurs during prophase I. During sporogenesis, each nucleus becomes surrounded by an individual spore wall, and all four haploid spores become contained as a tetrad within an ascus. Important insights into the meiotic function(s) of a gene of interest can be gained by observing the effects of gene mutations on spore viability and viability patterns among tetrads. Moreover, recombination frequencies among viable spores can reveal potential involvement of the gene during meiotic exchange between homologous chromosomes. Here, we describe methods for inducing spore formation on solid medium, determining spore viability, and measuring, via tetrad analysis, frequencies of crossing over and gene conversion as indicators of meiotic chromosome exchange. PMID:26527763

  6. Chromosome choreography: the meiotic ballet.

    PubMed

    Page, Scott L; Hawley, R Scott

    2003-08-01

    The separation of homologous chromosomes during meiosis in eukaryotes is the physical basis of Mendelian inheritance. The core of the meiotic process is a specialized nuclear division (meiosis I) in which homologs pair with each other, recombine, and then segregate from each other. The processes of chromosome alignment and pairing allow for homolog recognition. Reciprocal meiotic recombination ensures meiotic chromosome segregation by converting sister chromatid cohesion into mechanisms that hold homologous chromosomes together. Finally, the ability of sister kinetochores to orient to a single pole at metaphase I allows the separation of homologs to two different daughter cells. Failures to properly accomplish this elegant chromosome dance result in aneuploidy, a major cause of miscarriage and birth defects in human beings. PMID:12907787

  7. The mouse homologue of the polycystic kidney disease gene (Pkd1) is a single-copy gene

    SciTech Connect

    Olsson, P.G.; Loehning, C.; Frischauf, A.M.

    1996-06-01

    The mouse homologue of the polycystic kidney disease 1 gene (PKD1) was mapped to chromosome 17 using somatic cell hybrid, BXD recombinant inbred strains, and FISH. The gene is located within a previously defined conserved synteny group that includes the mouse homologue of tuberous sclerosis 2 (TSC2) and is linked to the {alpha} globin pseudogene Hba-ps4. Although the human genome contains multiple copies of genes related to PKD1, there is no evidence for more than one copy in the mouse genome. Like their human counterparts, the mouse Tsc2 and Pkd1 genes are arranged in a tail-to-tail orientation with a distance of only 63 bp between the polyadenylation signals of the two genes. 17 refs., 3 figs.

  8. DNA sequence of a gene encoding a BALB/c mouse Ld transplantation antigen.

    PubMed

    Moore, K W; Sher, B T; Sun, Y H; Eakle, K A; Hood, L

    1982-02-01

    The sequence of a gene, denoted 27.5, encoding a transplantation antigen for the BALB/c mouse has been determined. Gene transfer studies and comparison of the translated sequence with the partial amino acid sequence of the Ld transplantation antigen establish that gene 27.5 encodes an Ld polypeptide. A comparison of the gene 27.5 sequence with several complementary DNA sequences suggests that the BALB/c mouse may contain a number of closely related L-like genes. Gene 27.5 has eight exons that correlate with the structural domains of the transplantation antigen. PMID:7058332

  9. Organization and evolution of D region class I genes in the mouse major histocompatibility complex

    PubMed Central

    1986-01-01

    Chromosome walking has been used to study the organization of the class I genes in the D and Qa regions of the MHC of the BALB/c mouse and in the D region of the AKR mouse. Five and eight class I genes are found in the D and Qa regions of the BALB/c mouse, respectively, while the AKR mouse contains only a single class I D region gene that has been identified by transfection as the Dk gene. Restriction map homologies and crosshybridization experiments suggest that the multiple class I genes in the D region of the BALB/c mouse have been generated by unequal crossing-over involving class I genes from the Qa region. The expanded D region of BALB/c and other H-2d haplotype mouse strains appears to be metastable, since evidence for gene contraction in the Dd region has been found in two mutant strains. Thus the D region and also the Qa region class I genes are in a dynamic state, evolving by gene expansion and contraction. PMID:3701254

  10. Control of mouse U1 snRNA gene expression during in vitro differentiation of mouse embryonic stem cells.

    PubMed Central

    Cheng, Y; Lund, E; Kahan, B W; Dahlberg, J E

    1997-01-01

    Early in mouse development, two classes of U1 RNAs, mU1a and mU1b, are synthesized, but as development proceeds, transcription of the embryo-specific mU1b genes is selectively down-regulated to a barely detectable level. We show here that during in vitro differentiation of mouse embryonic stem (ES) cells, both exogenously introduced and endogenous U1b genes are subject to normal developmental regulation. Thus, ES cells represent a convenient isogenic system for studying the control of expression of developmentally regulated snRNA genes. Using this system, we have identified a region in the proximal 5'flanking region, located outside the PSE element, that is responsible for differential transcription of the mU1a and mU1b genes in both developing cells and transiently transfected NIH 3T3 cells. PMID:9153321

  11. A Study of Differential Expression of Testicular Genes in Various Reproductive Phases of Hemidactylus flaviviridis (Wall Lizard) to Derive Their Association with Onset of Spermatogenesis and Its Relevance to Mammals

    PubMed Central

    Sarkar, Hironmoy; Arya, Satyapal; Rai, Umesh; Majumdar, Subeer S.

    2016-01-01

    Testis of Hemidactylus flaviviridis, commonly known as Indian wall lizard, displays a lack of cellular and metabolic activity in regressed phase of testis during non-breeding season of the year. Retracted Sertoli cells (Sc), fibroid myoid cells and pre-meiotic resting spermatogonia are observed in such testis. This situation is akin to certain forms of infertility in men where hormone supplementation fails to generate sperm despite the presence of Sc and germ cells (Gc) in testis. In testis of lizard, spermatogenesis is reinitiated upon increased level of hormones during appropriate season (phase of recrudescence). Study of genes associated with generation of sperm, from regressed adult testis in lizard, may provide valuable information for understanding certain forms of male idiopathic infertility. Subtractive hybridization using testicular RNA obtained from the regressed and active phases of lizard reproductive cycle led to identify eight partial mRNA sequences that showed sequence homology with mice genes. We further evaluated the gene expression prolife by real-time PCR in three different reproductive phases of H. flaviviridis: regressed (pre-meiotic), recrudescent (meiotic) and active (post meiotic), for comparison with the corresponding testicular phases found in testis of 5 days (pre-meiotic), 20 days (meiotic) and 60 days (post-meiotic) old mouse. This is the first report where genes associated with progression of spermatogenesis during active phase, which follows a regressed state of adult testis, were identified in lizard and found to be conserved in mouse. Six important genes, Hk1, Nme5, Akap4, Arih1, Rassf7 and Tubb4b were found to be strictly associated with active spermatogenesis in both mouse and lizard. Factors interfering with the expression of any of these genes may potentially abrogate the process of spermatogenesis leading to infertility. Such information may shed light on unknown causes of idiopathic male infertility. PMID:26963275

  12. A Study of Differential Expression of Testicular Genes in Various Reproductive Phases of Hemidactylus flaviviridis (Wall Lizard) to Derive Their Association with Onset of Spermatogenesis and Its Relevance to Mammals.

    PubMed

    Sarkar, Hironmoy; Arya, Satyapal; Rai, Umesh; Majumdar, Subeer S

    2016-01-01

    Testis of Hemidactylus flaviviridis, commonly known as Indian wall lizard, displays a lack of cellular and metabolic activity in regressed phase of testis during non-breeding season of the year. Retracted Sertoli cells (Sc), fibroid myoid cells and pre-meiotic resting spermatogonia are observed in such testis. This situation is akin to certain forms of infertility in men where hormone supplementation fails to generate sperm despite the presence of Sc and germ cells (Gc) in testis. In testis of lizard, spermatogenesis is reinitiated upon increased level of hormones during appropriate season (phase of recrudescence). Study of genes associated with generation of sperm, from regressed adult testis in lizard, may provide valuable information for understanding certain forms of male idiopathic infertility. Subtractive hybridization using testicular RNA obtained from the regressed and active phases of lizard reproductive cycle led to identify eight partial mRNA sequences that showed sequence homology with mice genes. We further evaluated the gene expression prolife by real-time PCR in three different reproductive phases of H. flaviviridis: regressed (pre-meiotic), recrudescent (meiotic) and active (post meiotic), for comparison with the corresponding testicular phases found in testis of 5 days (pre-meiotic), 20 days (meiotic) and 60 days (post-meiotic) old mouse. This is the first report where genes associated with progression of spermatogenesis during active phase, which follows a regressed state of adult testis, were identified in lizard and found to be conserved in mouse. Six important genes, Hk1, Nme5, Akap4, Arih1, Rassf7 and Tubb4b were found to be strictly associated with active spermatogenesis in both mouse and lizard. Factors interfering with the expression of any of these genes may potentially abrogate the process of spermatogenesis leading to infertility. Such information may shed light on unknown causes of idiopathic male infertility.

  13. The Maize Divergent spindle-1 (dv1) Gene Encodes a Kinesin-14A Motor Protein Required for Meiotic Spindle Pole Organization.

    PubMed

    Higgins, David M; Nannas, Natalie J; Dawe, R Kelly

    2016-01-01

    The classic maize mutant divergent spindle-1 (dv1) causes failures in meiotic spindle assembly and a decrease in pollen viability. By analyzing two independent dv1 alleles we demonstrate that this phenotype is caused by mutations in a member of the kinesin-14A subfamily, a class of C-terminal, minus-end directed microtubule motors. Further analysis demonstrates that defects in early spindle assembly are rare, but that later stages of spindle organization promoting the formation of finely focused spindle poles are strongly dependent on Dv1. Anaphase is error-prone in dv1 lines but not severely so, and the majority of cells show normal chromosome segregation. Live-cell imaging of wild type and mutant plants carrying CFP-tagged β-tubulin confirm that meiosis in dv1 lines fails primarily at the pole-sharpening phase of spindle assembly. These data indicate that plant kinesin-14A proteins help to enforce bipolarity by focusing spindle poles and that this stage of spindle assembly is not required for transition through the spindle checkpoint but improves the accuracy of chromosome segregation. PMID:27610117

  14. The Maize Divergent spindle-1 (dv1) Gene Encodes a Kinesin-14A Motor Protein Required for Meiotic Spindle Pole Organization

    PubMed Central

    Higgins, David M.; Nannas, Natalie J.; Dawe, R. Kelly

    2016-01-01

    The classic maize mutant divergent spindle-1 (dv1) causes failures in meiotic spindle assembly and a decrease in pollen viability. By analyzing two independent dv1 alleles we demonstrate that this phenotype is caused by mutations in a member of the kinesin-14A subfamily, a class of C-terminal, minus-end directed microtubule motors. Further analysis demonstrates that defects in early spindle assembly are rare, but that later stages of spindle organization promoting the formation of finely focused spindle poles are strongly dependent on Dv1. Anaphase is error-prone in dv1 lines but not severely so, and the majority of cells show normal chromosome segregation. Live-cell imaging of wild type and mutant plants carrying CFP-tagged β-tubulin confirm that meiosis in dv1 lines fails primarily at the pole-sharpening phase of spindle assembly. These data indicate that plant kinesin-14A proteins help to enforce bipolarity by focusing spindle poles and that this stage of spindle assembly is not required for transition through the spindle checkpoint but improves the accuracy of chromosome segregation. PMID:27610117

  15. The Maize Divergent spindle-1 (dv1) Gene Encodes a Kinesin-14A Motor Protein Required for Meiotic Spindle Pole Organization

    PubMed Central

    Higgins, David M.; Nannas, Natalie J.; Dawe, R. Kelly

    2016-01-01

    The classic maize mutant divergent spindle-1 (dv1) causes failures in meiotic spindle assembly and a decrease in pollen viability. By analyzing two independent dv1 alleles we demonstrate that this phenotype is caused by mutations in a member of the kinesin-14A subfamily, a class of C-terminal, minus-end directed microtubule motors. Further analysis demonstrates that defects in early spindle assembly are rare, but that later stages of spindle organization promoting the formation of finely focused spindle poles are strongly dependent on Dv1. Anaphase is error-prone in dv1 lines but not severely so, and the majority of cells show normal chromosome segregation. Live-cell imaging of wild type and mutant plants carrying CFP-tagged β-tubulin confirm that meiosis in dv1 lines fails primarily at the pole-sharpening phase of spindle assembly. These data indicate that plant kinesin-14A proteins help to enforce bipolarity by focusing spindle poles and that this stage of spindle assembly is not required for transition through the spindle checkpoint but improves the accuracy of chromosome segregation.

  16. Evidence for meiotic sex in bdelloid rotifers.

    PubMed

    Signorovitch, Ana; Hur, Jae; Gladyshev, Eugene; Meselson, Matthew

    2016-08-22

    In their study of genetic exchange in the bdelloid rotifer Adineta vaga, Debortoli et al. [1] conclude that the patchwork pattern of allele sharing among three individuals in the genomic regions they examined is "…unlikely to arise in cases of PTH (Oenothera-like) meiosis since haplotypes are transferred as entire blocks…" and therefore that "Genetic exchange among bdelloid rotifers is more likely due to horizontal gene transfer than to meiotic sex." This assumes without justification that horizontal gene transfer (HGT) in bdelloids precludes the sexual transmission of entire haplotypes, for which we have reported evidence in the bdelloid Macrotrachela quadricornifera[2]. And it does not consider the contribution to such a patchwork pattern that would result from conversion and subsequent outcrossing, even in Oenothera-like systems. PMID:27554650

  17. Meiotic drive and evolution of female choice.

    PubMed

    Reinhold, K; Engqvist, L; Misof, B; Kurtz, J

    1999-07-01

    As a special version of the good-genes hypothesis, it was recently proposed that females could benefit from choosing drive-resistant males in a meiotic drive system. Here, we examine with a three-locus, six-allele population genetic model whether female choice for drive resistance can evolve. An allele leading to female preference for drive-resistant males was introduced at low frequency into a population polymorphic for meiotic drive and drive resistance. Our simulations show that female choice of drive-resistant males is disadvantageous when resistance is Y-linked. This disadvantage occurs because, at equilibrium, drive-resistant males have lower reproductive success than drive-susceptible males. Thus, female choice of drive-susceptible males can evolve when resistance is Y-linked. When resistance is autosomal, selection on female choice for drive resistance is less strong and depends on the frequency of choice: female preference of resistant males is favoured when choice is rare and disadvantageous when choice is frequent, leading to a stable equilibrium at a low frequency of the choice allele. Independent of the location of drive resistance alleles, males with the non-driving allele always have above average reproductive success. Female choice is therefore beneficial when choosy females prefer males with the non-driving allele.

  18. Meiotic drive and evolution of female choice.

    PubMed Central

    Reinhold, K; Engqvist, L; Misof, B; Kurtz, J

    1999-01-01

    As a special version of the good-genes hypothesis, it was recently proposed that females could benefit from choosing drive-resistant males in a meiotic drive system. Here, we examine with a three-locus, six-allele population genetic model whether female choice for drive resistance can evolve. An allele leading to female preference for drive-resistant males was introduced at low frequency into a population polymorphic for meiotic drive and drive resistance. Our simulations show that female choice of drive-resistant males is disadvantageous when resistance is Y-linked. This disadvantage occurs because, at equilibrium, drive-resistant males have lower reproductive success than drive-susceptible males. Thus, female choice of drive-susceptible males can evolve when resistance is Y-linked. When resistance is autosomal, selection on female choice for drive resistance is less strong and depends on the frequency of choice: female preference of resistant males is favoured when choice is rare and disadvantageous when choice is frequent, leading to a stable equilibrium at a low frequency of the choice allele. Independent of the location of drive resistance alleles, males with the non-driving allele always have above average reproductive success. Female choice is therefore beneficial when choosy females prefer males with the non-driving allele. PMID:10445289

  19. Cloning the mouse homologue of the human lysosomal acid {alpha}-glucosidase gene

    SciTech Connect

    Ding, J.H.; Yang, B.Z.; Liu, H.M.

    1994-09-01

    Pompe disease (GSD II) is an autosomal recessive disorder caused by a deficiency of lysosomal acid {alpha}-glucosidase (GAA). In an attempt to create a mouse model for Pompe disease, we isolated and characterized the gene encoding the mouse homologue of the human GAA. Twenty clones that extend from exon 2 to the poly(A) tail were isolated from a mouse liver cDNA library, but the remainder of the mRNA proved difficult to obtain by conventional cDNA library screening. Sequences spanning exons 1-2 were cloned by RACE from mouse liver RNA. The full-length liver GAA cDNA contains 3365 nucleotides with a coding region of 2859 nucleotides and a 394 base pair 3{prime}-nontranslated region. The deduced amino acid sequence of the mouse GAA shows 84% identity to the human GAA. Southern blot analysis demonstrated that the mouse GAA was encoded by a single copy gene. Then six bacteriophages containing DNA from the GAA gene were isolated by screening 10{sup 6} phage plaques of a mouse 129 genomic library using a mouse GAA cDNA as a probe. From one of these bacteriophages, an 11-kilobase EcoRI fragment containing exons 3 to 15 was subcloned and sequenced. Work is in progress using this genomic clone to disrupt the GAA gene in murine embryonic stem cells in order to create GSD II mice.

  20. REC-1 and HIM-5 distribute meiotic crossovers and function redundantly in meiotic double-strand break formation in Caenorhabditis elegans.

    PubMed

    Chung, George; Rose, Ann M; Petalcorin, Mark I R; Martin, Julie S; Kessler, Zebulin; Sanchez-Pulido, Luis; Ponting, Chris P; Yanowitz, Judith L; Boulton, Simon J

    2015-09-15

    The Caenorhabditis elegans gene rec-1 was the first genetic locus identified in metazoa to affect the distribution of meiotic crossovers along the chromosome. We report that rec-1 encodes a distant paralog of HIM-5, which was discovered by whole-genome sequencing and confirmed by multiple genome-edited alleles. REC-1 is phosphorylated by cyclin-dependent kinase (CDK) in vitro, and mutation of the CDK consensus sites in REC-1 compromises meiotic crossover distribution in vivo. Unexpectedly, rec-1; him-5 double mutants are synthetic-lethal due to a defect in meiotic double-strand break formation. Thus, we uncovered an unexpected robustness to meiotic DSB formation and crossover positioning that is executed by HIM-5 and REC-1 and regulated by phosphorylation.

  1. REC-1 and HIM-5 distribute meiotic crossovers and function redundantly in meiotic double-strand break formation in Caenorhabditis elegans

    PubMed Central

    Chung, George; Rose, Ann M.; Petalcorin, Mark I.R.; Martin, Julie S.; Kessler, Zebulin; Sanchez-Pulido, Luis; Ponting, Chris P.; Yanowitz, Judith L.; Boulton, Simon J.

    2015-01-01

    The Caenorhabditis elegans gene rec-1 was the first genetic locus identified in metazoa to affect the distribution of meiotic crossovers along the chromosome. We report that rec-1 encodes a distant paralog of HIM-5, which was discovered by whole-genome sequencing and confirmed by multiple genome-edited alleles. REC-1 is phosphorylated by cyclin-dependent kinase (CDK) in vitro, and mutation of the CDK consensus sites in REC-1 compromises meiotic crossover distribution in vivo. Unexpectedly, rec-1; him-5 double mutants are synthetic-lethal due to a defect in meiotic double-strand break formation. Thus, we uncovered an unexpected robustness to meiotic DSB formation and crossover positioning that is executed by HIM-5 and REC-1 and regulated by phosphorylation. PMID:26385965

  2. Structure of the chromosomal gene for granulocyte-macrophage colony stimulating factor: comparison of the mouse and human genes.

    PubMed Central

    Miyatake, S; Otsuka, T; Yokota, T; Lee, F; Arai, K

    1985-01-01

    A cDNA clone that expresses granulocyte-macrophage colony stimulating factor (GM-CSF) activity in COS-7 cells has been isolated from a pcD library prepared from mRNA derived from concanavalin A-activated mouse helper T cell clones. Based on homology with the mouse GM-CSF cDNA sequence, the mouse GM-CSF gene was isolated. The human GM-CSF gene was also isolated based on homology with the human GM-CSF cDNA sequence. The nucleotide sequences determined for the genes and their flanking regions revealed that both the mouse and human GM-CSF genes are composed of three introns and four exons. The organization of the mouse and human GM-CSF genes are highly homologous and strong sequence homology between the two genes is found both in the coding and non-coding regions. A 'TATA'-like sequence was found 20-25 bp upstream from the transcription initiation site. In the 5'-flanking region, there is a highly homologous region extending 330 bp upstream of the putative TATA box. This sequence may play a role in regulation of expression of the GM-CSF gene. These structures are compared with those of different lymphokine genes and their regulatory regions. Images Fig. 2. Fig. 6. PMID:3876930

  3. Genomic structure and chromosomal assignment of the mouse Ku70 gene

    SciTech Connect

    Takiguchi, Yuichi |; Kurimasa, Akihiro; Chen, Fanqing

    1996-07-01

    DNA-dependent protein kinase (DNA-PK) consists of three polypeptide subunits: Ku70, Ku80, and the DNA-PK catalytic subunit (DNA-PKcs). Mammalian mutants deficient in either Ku80 or DNA-PKcs function have been shown to be lacking in DNA double-strand break repair and V(D)J recombination, respectively. The precise role of the Ku70 gene in this process has not yet been determined, in part because no cell lines, animals, or human diseases involved with deficiencies in this gene have yet been identified. Both the human and the mouse Ku70 cDNAs have been cloned, and the human gene has been mapped to chromosome 22q13. The original mouse cDNA clones, however, lacked a complete 5{prime}-region, and none of the mammalian Ku70 genomic sequences have been characterized. This report contains an analysis of the 5{prime}-region of the mouse cDNA sequence, a characterization of the mouse Ku70 genomic structure, and fluorescence in situ hybridization data that map the mouse gene to chromosome 15. The deduced amino acid sequence of the mouse gene consists of 608 amino acids compared to 609 for the human gene. The genomic sequence is 24 kb and consists of 13 exons, including an untranslated first exon. Sequences form the upstream region of exon 1 revealed four consensus GC box sequences and a strong transcription initiation site at a reasonable location. The assignment of the mouse Ku70 gene to chromosome 15 is consistent with the syntenic relationship of this gene in human (chromosome 22q13) and mouse and adds to the comparative mapping data for the genes involved in the SCID phenotype. 39 refs., 3 figs.

  4. Identification and Applications of Repetitive Probes for Gene Mapping in the Mouse

    PubMed Central

    Siracusa, L. D.; Jenkins, N. A.; Copeland, N. G.

    1991-01-01

    Interspecific mouse hybrids that are viable and fertile provide a wealth of genetic variation that is useful for gene mapping. We are using this genetic variation to develop multilocus linkage maps of the mouse genome. As an outgrowth of this work, we have identified three repetitive probes that collectively identify 28 loci dispersed on 16 of the 19 mouse autosomes and the X chromosome. These loci establish a skeleton linkage map that can be used to detect linkage over much of the mouse genome. The molecular probes are derived from the mouse mammary tumor virus envelope gene, the ornithine decarboxylase gene, and the triose phosphate isomerase gene. The ability to scan the mouse genome quickly and efficiently in an interspecific cross using these three repetitive probes makes this system a powerful tool for identifying the chromosomal location of mutations that have yet to be cloned, mapping multigenic traits, and identifying recessive protooncogene loci associated with murine neoplastic disease. Ultimately, interspecific hybrids in conjunction with repetitive and single-copy probes will provide a rapid means to access virtually any gene of interest in the mouse genome at the molecular level. PMID:1673105

  5. Interallelic and intergenic incompatibilities of the Prdm9 (Hst1) gene in mouse hybrid sterility.

    PubMed

    Flachs, Petr; Mihola, Ondřej; Simeček, Petr; Gregorová, Soňa; Schimenti, John C; Matsui, Yasuhisa; Baudat, Frédéric; de Massy, Bernard; Piálek, Jaroslav; Forejt, Jiří; Trachtulec, Zdenek

    2012-01-01

    The Dobzhansky-Muller model of incompatibilities explains reproductive isolation between species by incorrect epistatic interactions. Although the mechanisms of speciation are of great interest, no incompatibility has been characterized at the gene level in mammals. The Hybrid sterility 1 gene (Hst1) participates in the arrest of meiosis in F(1) males of certain strains from two Mus musculus subspecies, e.g., PWD from M. m. musculus and C57BL/6J (henceforth B6) from M. m. domesticus. Hst1 has been identified as a meiotic PR-domain gene (Prdm9) encoding histone 3 methyltransferase in the male offspring of PWD females and B6 males, (PWD×B6)F(1). To characterize the incompatibilities underlying hybrid sterility, we phenotyped reproductive and meiotic markers in males with altered copy numbers of Prdm9. A partial rescue of fertility was observed upon removal of the B6 allele of Prdm9 from the azoospermic (PWD×B6)F(1) hybrids, whereas removing one of the two Prdm9 copies in PWD or B6 background had no effect on male reproduction. Incompatibility(ies) not involving Prdm9(B6) also acts in the (PWD×B6)F(1) hybrids, since the correction of hybrid sterility by Prdm9(B6) deletion was not complete. Additions and subtractions of Prdm9 copies, as well as allelic replacements, improved meiotic progression and fecundity also in the progeny-producing reciprocal (B6×PWD)F(1) males. Moreover, an increased dosage of Prdm9 and reciprocal cross enhanced fertility of other sperm-carrying male hybrids, (PWD×B6-C3H.Prdm9)F(1), harboring another Prdm9 allele of M. m. domesticus origin. The levels of Prdm9 mRNA isoforms were similar in the prepubertal testes of all types of F(1) hybrids of PWD with B6 and B6-C3H.Prdm9 despite their different prospective fertility, but decreased to 53% after removal of Prdm9(B6). Therefore, the Prdm9(B6) allele probably takes part in posttranscriptional dominant-negative hybrid interaction(s) absent in the parental strains.

  6. Effects of gene regulatory reprogramming on gene expression in human and mouse developing hearts.

    PubMed

    Hsu, Chih-Hao; Ovcharenko, Ivan

    2013-01-01

    Lineage-specific regulatory elements underlie adaptation of species and play a role in disease susceptibility. We compared functionally conserved and lineage-specific enhancers by cross-mapping 5042 human and 6564 mouse heart enhancers. Of these, 79 per cent are lineage-specific, lacking a functional orthologue. Heart enhancers tend to cluster and, commonly, there are multiple heart enhancers in a heart locus providing a regulatory stability to the locus. We observed little cross-clustering, however, between lineage-specific and functionally conserved heart enhancers suggesting regulatory function acquisition and development in loci previously lacking heart activity. We also identified 862 human-specific heart enhancers: 417 featuring sequence conservation with mouse (class II) and 445 with neither sequence nor function conservation (class III). Ninety-eight per cent of class III enhancers were deleted from the mouse genome, and we estimated a similar-sized enhancer gain in the human lineage. Human-specific enhancers display no detectable decrease in the negative selection pressure and are strongly associated with genes partaking in the heart regulatory programmes. The loss of a heart enhancer could be compensated by activity of a redundant heart enhancer; however, we observed redundancy in only 15 per cent of class II and III enhancer loci indicating a large-scale reprogramming of the heart regulatory programme in mammals.

  7. Meiotic Crossing over between Nonhomologous Chromosomes Affects Chromosome Segregation in Yeast

    PubMed Central

    Jinks-Robertson, S.; Sayeed, S.; Murphy, T.

    1997-01-01

    Meiotic recombination between artificial repeats positioned on nonhomologous chromosomes occurs efficiently in the yeast Saccharomyces cerevisiae. Both gene conversion and crossover events have been observed, with crossovers yielding reciprocal translocations. In the current study, 5.5-kb ura3 repeats positioned on chromosomes V and XV were used to examine the effect of ectopic recombination on meiotic chromosome segregation. Ura(+) random spores were selected and gene conversion vs. crossover events were distinguished by Southern blot analysis. Approximately 15% of the crossover events between chromosomes V and XV were associated with missegregation of one of these chromosomes. The missegregation was manifest as hyperploid spores containing either both translocations plus a normal chromosome, or both normal chromosomes plus one of the translocations. In those cases where it could be analyzed, missegregation occurred at the first meiotic division. These data are discussed in terms of a model in which ectopic crossovers compete efficiently with normal allelic crossovers in directing meiotic chromosome segregation. PMID:9136001

  8. Cloning, analysis, and chromosomal localization of myoxin (MYH12), the human homologue to the mouse dilute gene

    SciTech Connect

    Engle, L.J.; Kennett, R.H. )

    1994-02-01

    The mouse dilute gene encodes a novel type of non-muscle myosin that structurally combines elements from both nonmuscle myosin type I and nonmuscle myosin type II. Phenotypically, mutations in the mouse dilute gene result not only in the lightening of coat color, but also in the onset of severe neurological defects shortly after birth. This may indicate that the mouse dilute gene is important in maintaining the normal neuronal function in the mouse. The authors report the isolation and sequencing of [open quotes]myoxin[close quotes] (MYH12), the human homologue of the mouse dilute gene, and its assignment to human chromosome 15. 35 refs., 6 figs.

  9. Mouse Palmitoyl Protein Thioesterase: Gene Structure and Expression of cDNA

    PubMed Central

    Salonen, Tarja; Hellsten, Elina; Horelli-Kuitunen, Nina; Peltonen, Leena; Jalanko, Anu

    1998-01-01

    Palmitoyl protein thioesterase (PPT) is the defective enzyme in infantile neuronal ceroid lipofuscinosis (INCL), which is a recessively inherited, progressive neurodegenerative disorder. We present here the cloning, chromosomal mapping, genomic structure, and the expression of the cDNA of mouse PPT. The mouse PPT gene spans >21 kb of genomic DNA and contains nine exons with a coding sequence of 918 bp. Fluorescence in situ hybridization to metaphase chromosomes localized the mouse PPT gene to the chromosome 4 conserved syntenic region with human chromosome 1p32 where the human PPT is located. PPT is expressed widely in a variety of mouse tissues. The mouse PPT cDNA is conserved highly with the human and rat PPT both at the nucleotide and amino acid sequence level. Transient expression of mouse PPT in COS-1 cells yielded a 38/36-kD differentially glycosylated polypeptide that was also secreted into culture media. Immunofluorescence analysis of transiently transfected HeLa cells indicated lysosomal localization of mouse PPT. Based on the high conservation of the gene and polypeptide structure as well as similar processing and intracellular localization, the function of PPT in mouse and human are likely to be very similar. [The sequence data described in this paper have been submitted to GenBank under accession no. AF071O25.] PMID:9685319

  10. Aup1, a novel gene on mouse Chromosome 6 and human Chromosome 2p13

    SciTech Connect

    Jang, Wonhee; Weber, J.S.; Meisler, M.H.

    1996-09-01

    We have cloned a novel mouse cDNA, Aup1, encoding a predicted protein of 410 amino acid residues. The 1.5-kb Aup1 transcript is ubiquitously expressed in mouse tissues. An evolutionary relationship to the Caenorhabditis elegans predicted protein F44b9.5 is indicated by the 35% identity and 53% conservation of the amino acid sequences. Nineteen related human ESTs spanning 80% of the protein have also been identified, with a predicted amino acid sequence identity of 86% between the human and the mouse proteins. The gene has been mapped to a conserved linkage group on human chromosome 2p13 and mouse Chromosome 6. Aup1 was eliminated as a candidate gene for two closely linked disorders, human LGMD2B and mouse mnd2. 15 refs., 2 figs.

  11. Synteny conservation of the Huntington's disease gene and surrounding loci on mouse Chromosome 5.

    PubMed

    Grosson, C L; MacDonald, M E; Duyao, M P; Ambrose, C M; Roffler-Tarlov, S; Gusella, J F

    1994-07-01

    The mouse homologs of the Huntington's disease (HD) gene and 17 other human Chromosome (Chr) 4 loci (including six previously unmapped) were localized by use of an interspecific cross. All loci mapped in a continuous linkage group on mouse Chr 5, distal to En2 and I16, whose human counterparts are located on Chr 7. The relative order of the loci on human Chr 4 and mouse Chr 5 was maintained, except for a break between D5H4S115E and Idua/rd, with relocation of the latter to the opposite end of the map. The mouse HD homolog (Hdh) mapped within a cluster of seven genes that were completely linked in our data set. In human these loci span a approximately 1.8 Mb stretch of human 4p16.3 that has been entirely cloned. To date, there is no phenotypic correspondence between human and mouse mutations mapping to this region of synteny conservation.

  12. Lgn1, a gene that determines susceptibility to Legionella pneumophila, maps to mouse chromosome 13

    SciTech Connect

    Dietrich, W.F.; Damron, D.M.; Lander, E.S.

    1995-04-10

    The intracellular pathogen Legionella pneumophila is unable to replicate in macrophages derived from most inbred mouse strains. Here, we report the mapping of a gene, called Lgn1, that determines whether mouse macrophages are permissive for the intracellular replication of L. pneumophila. Although Lgn1 has been previously reported to map to mouse chromosome 15, we show here that it actually maps to chromosome 13, between D13Mit128 and D13Mit70. In the absence of any regional candidates for Lgn1, this map position will facilitate positional cloning attempts directed at this gene. 22 refs., 2 figs., 2 tabs.

  13. Sisters unbound is required for meiotic centromeric cohesion in Drosophila melanogaster.

    PubMed

    Krishnan, Badri; Thomas, Sharon E; Yan, Rihui; Yamada, Hirotsugu; Zhulin, Igor B; McKee, Bruce D

    2014-11-01

    Regular meiotic chromosome segregation requires sister centromeres to mono-orient (orient to the same pole) during the first meiotic division (meiosis I) when homologous chromosomes segregate, and to bi-orient (orient to opposite poles) during the second meiotic division (meiosis II) when sister chromatids segregate. Both orientation patterns require cohesion between sister centromeres, which is established during meiotic DNA replication and persists until anaphase of meiosis II. Meiotic cohesion is mediated by a conserved four-protein complex called cohesin that includes two structural maintenance of chromosomes (SMC) subunits (SMC1 and SMC3) and two non-SMC subunits. In Drosophila melanogaster, however, the meiotic cohesion apparatus has not been fully characterized and the non-SMC subunits have not been identified. We have identified a novel Drosophila gene called sisters unbound (sunn), which is required for stable sister chromatid cohesion throughout meiosis. sunn mutations disrupt centromere cohesion during prophase I and cause high frequencies of non-disjunction (NDJ) at both meiotic divisions in both sexes. SUNN co-localizes at centromeres with the cohesion proteins SMC1 and SOLO in both sexes and is necessary for the recruitment of both proteins to centromeres. Although SUNN lacks sequence homology to cohesins, bioinformatic analysis indicates that SUNN may be a structural homolog of the non-SMC cohesin subunit stromalin (SA), suggesting that SUNN may serve as a meiosis-specific cohesin subunit. In conclusion, our data show that SUNN is an essential meiosis-specific Drosophila cohesion protein. PMID:25194162

  14. Sisters Unbound Is Required for Meiotic Centromeric Cohesion in Drosophila melanogaster

    PubMed Central

    Krishnan, Badri; Thomas, Sharon E.; Yan, Rihui; Yamada, Hirotsugu; Zhulin, Igor B.; McKee, Bruce D.

    2014-01-01

    Regular meiotic chromosome segregation requires sister centromeres to mono-orient (orient to the same pole) during the first meiotic division (meiosis I) when homologous chromosomes segregate, and to bi-orient (orient to opposite poles) during the second meiotic division (meiosis II) when sister chromatids segregate. Both orientation patterns require cohesion between sister centromeres, which is established during meiotic DNA replication and persists until anaphase of meiosis II. Meiotic cohesion is mediated by a conserved four-protein complex called cohesin that includes two structural maintenance of chromosomes (SMC) subunits (SMC1 and SMC3) and two non-SMC subunits. In Drosophila melanogaster, however, the meiotic cohesion apparatus has not been fully characterized and the non-SMC subunits have not been identified. We have identified a novel Drosophila gene called sisters unbound (sunn), which is required for stable sister chromatid cohesion throughout meiosis. sunn mutations disrupt centromere cohesion during prophase I and cause high frequencies of non-disjunction (NDJ) at both meiotic divisions in both sexes. SUNN co-localizes at centromeres with the cohesion proteins SMC1 and SOLO in both sexes and is necessary for the recruitment of both proteins to centromeres. Although SUNN lacks sequence homology to cohesins, bioinformatic analysis indicates that SUNN may be a structural homolog of the non-SMC cohesin subunit stromalin (SA), suggesting that SUNN may serve as a meiosis-specific cohesin subunit. In conclusion, our data show that SUNN is an essential meiosis-specific Drosophila cohesion protein. PMID:25194162

  15. The fission yeast MTREC and EJC orthologs ensure the maturation of meiotic transcripts during meiosis.

    PubMed

    Marayati, Bahjat Fadi; Hoskins, Victoria; Boger, Robert W; Tucker, James F; Fishman, Emily S; Bray, Andrew S; Zhang, Ke

    2016-09-01

    Meiosis is a highly regulated process by which genetic information is transmitted through sexual reproduction. It encompasses unique mechanisms that do not occur in vegetative cells, producing a distinct, well-regulated meiotic transcriptome. During vegetative growth, many meiotic genes are constitutively transcribed, but most of the resulting mRNAs are rapidly eliminated by the Mmi1-MTREC (Mtl1-Red1 core) complex. While Mmi1-MTREC targets premature meiotic RNAs for degradation by the nuclear 3'-5' exoribonuclease exosome during mitotic growth, its role in meiotic gene expression during meiosis is not known. Here, we report that Red5, an essential MTREC component, interacts with pFal1, an ortholog of eukaryotic translation initiation factor eIF4aIII in the fission yeast Schizosaccharomyces pombe In mammals, together with MAGO (Mnh1), Rnps1, and Y14, elF4AIII (pFal1) forms the core of the exon junction complex (EJC), which is essential for transcriptional surveillance and localization of mature mRNAs. In fission yeast, two EJC orthologs, pFal1 and Mnh1, are functionally connected with MTREC, specifically in the process of meiotic gene expression during meiosis. Although pFal1 interacts with Mnh1, Y14, and Rnps1, its association with Mnh1 is not disrupted upon loss of Y14 or Rnps1. Mutations of Red1, Red5, pFal1, or Mnh1 produce severe meiotic defects; the abundance of meiotic transcripts during meiosis decreases; and mRNA maturation processes such as splicing are impaired. Since studying meiosis in mammalian germline cells is difficult, our findings in fission yeast may help to define the general mechanisms involved in accurate meiotic gene expression in higher eukaryotes.

  16. The fission yeast MTREC and EJC orthologs ensure the maturation of meiotic transcripts during meiosis

    PubMed Central

    Marayati, Bahjat Fadi; Hoskins, Victoria; Boger, Robert W.; Tucker, James F.; Fishman, Emily S.; Bray, Andrew S.; Zhang, Ke

    2016-01-01

    Meiosis is a highly regulated process by which genetic information is transmitted through sexual reproduction. It encompasses unique mechanisms that do not occur in vegetative cells, producing a distinct, well-regulated meiotic transcriptome. During vegetative growth, many meiotic genes are constitutively transcribed, but most of the resulting mRNAs are rapidly eliminated by the Mmi1-MTREC (Mtl1-Red1 core) complex. While Mmi1-MTREC targets premature meiotic RNAs for degradation by the nuclear 3′–5′ exoribonuclease exosome during mitotic growth, its role in meiotic gene expression during meiosis is not known. Here, we report that Red5, an essential MTREC component, interacts with pFal1, an ortholog of eukaryotic translation initiation factor eIF4aIII in the fission yeast Schizosaccharomyces pombe. In mammals, together with MAGO (Mnh1), Rnps1, and Y14, elF4AIII (pFal1) forms the core of the exon junction complex (EJC), which is essential for transcriptional surveillance and localization of mature mRNAs. In fission yeast, two EJC orthologs, pFal1 and Mnh1, are functionally connected with MTREC, specifically in the process of meiotic gene expression during meiosis. Although pFal1 interacts with Mnh1, Y14, and Rnps1, its association with Mnh1 is not disrupted upon loss of Y14 or Rnps1. Mutations of Red1, Red5, pFal1, or Mnh1 produce severe meiotic defects; the abundance of meiotic transcripts during meiosis decreases; and mRNA maturation processes such as splicing are impaired. Since studying meiosis in mammalian germline cells is difficult, our findings in fission yeast may help to define the general mechanisms involved in accurate meiotic gene expression in higher eukaryotes. PMID:27365210

  17. The fission yeast MTREC and EJC orthologs ensure the maturation of meiotic transcripts during meiosis.

    PubMed

    Marayati, Bahjat Fadi; Hoskins, Victoria; Boger, Robert W; Tucker, James F; Fishman, Emily S; Bray, Andrew S; Zhang, Ke

    2016-09-01

    Meiosis is a highly regulated process by which genetic information is transmitted through sexual reproduction. It encompasses unique mechanisms that do not occur in vegetative cells, producing a distinct, well-regulated meiotic transcriptome. During vegetative growth, many meiotic genes are constitutively transcribed, but most of the resulting mRNAs are rapidly eliminated by the Mmi1-MTREC (Mtl1-Red1 core) complex. While Mmi1-MTREC targets premature meiotic RNAs for degradation by the nuclear 3'-5' exoribonuclease exosome during mitotic growth, its role in meiotic gene expression during meiosis is not known. Here, we report that Red5, an essential MTREC component, interacts with pFal1, an ortholog of eukaryotic translation initiation factor eIF4aIII in the fission yeast Schizosaccharomyces pombe In mammals, together with MAGO (Mnh1), Rnps1, and Y14, elF4AIII (pFal1) forms the core of the exon junction complex (EJC), which is essential for transcriptional surveillance and localization of mature mRNAs. In fission yeast, two EJC orthologs, pFal1 and Mnh1, are functionally connected with MTREC, specifically in the process of meiotic gene expression during meiosis. Although pFal1 interacts with Mnh1, Y14, and Rnps1, its association with Mnh1 is not disrupted upon loss of Y14 or Rnps1. Mutations of Red1, Red5, pFal1, or Mnh1 produce severe meiotic defects; the abundance of meiotic transcripts during meiosis decreases; and mRNA maturation processes such as splicing are impaired. Since studying meiosis in mammalian germline cells is difficult, our findings in fission yeast may help to define the general mechanisms involved in accurate meiotic gene expression in higher eukaryotes. PMID:27365210

  18. c-Rel Regulates Inscuteable Gene Expression during Mouse Embryonic Stem Cell Differentiation.

    PubMed

    Ishibashi, Riki; Kozuki, Satoshi; Kamakura, Sachiko; Sumimoto, Hideki; Toyoshima, Fumiko

    2016-02-12

    Inscuteable (Insc) regulates cell fate decisions in several types of stem cells. Although it is recognized that the expression levels of mouse INSC govern the balance between symmetric and asymmetric stem cell division, regulation of mouse Insc gene expression remains poorly understood. Here, we showed that mouse Insc expression transiently increases at an early stage of differentiation, when mouse embryonic stem (mES) cells differentiate into bipotent mesendoderm capable of producing both endoderm and mesoderm in defined culture conditions. We identified the minimum transcriptional regulatory element (354 bases) that drives mouse Insc transcription in mES cells within a region >5 kb upstream of the mouse Insc transcription start site. We found that the transcription factor reticuloendotheliosis oncogene (c-Rel) bound to the minimum element and promoted mouse Insc expression in mES cells. In addition, short interfering RNA-mediated knockdown of either mouse INSC or c-Rel protein decreased mesodermal cell populations without affecting differentiation into the mesendoderm or endoderm. Furthermore, overexpression of mouse INSC rescued the mesoderm-reduced phenotype induced by knockdown of c-Rel. We propose that regulation of mouse Insc expression by c-Rel modulates cell fate decisions during mES cell differentiation.

  19. Characterization, expression, and evolution of the mouse embryonic zeta-globin gene.

    PubMed Central

    Leder, A; Weir, L; Leder, P

    1985-01-01

    We have determined the complete sequence of the embryonic alpha-like, zeta (zeta)-globin gene of the BALB/c mouse. The structure of this gene establishes the amino acid sequence of the mouse embryonic zeta-globin polypeptide chain and allows us to identify sequences within the gene that may be important for its expression. One of these is a 300-base segment that is tightly conserved between mice and humans and is located at the 5' end of the zeta-globin gene. By introducing the cloned gene into permanently transfected mouse erythroleukemic cell lines and comparing its transcript with that of zeta-globin mRNA derived from embryonic yolk sac erythrocytes, we are able to show that the cloned gene is transcriptionally active and that its transcript is correctly initiated and processed. Interestingly, the zeta-globin gene is also active when permanently transfected into an immunoglobulin-producing B-cell, a cell that presumably has tissue-specific requirements for gene expression. Further, a comparison of the amino acid coding sequence of the mouse zeta-globin gene to that of zeta-like globin genes of other species supports a revised evolutionary lineage in which goats and humans are closely related, whereas mice are further removed. Images PMID:4000117

  20. Distribution of the mammalian Stat gene family in mouse chromosomes

    SciTech Connect

    Copeland, N.G.; Gilbert, D.J.; Jenkins, N.A.

    1995-09-01

    Studies of transcriptional activation by interferons and a variety of cytokines have led to the identification of a family of proteins that serve as signal transducers and activators of transcription, Stats. Here, we report that the seven mouse Stat loci map in three clusters, with each cluster located on a different mouse autosome. The data suggest that the family has arisen via a tandem duplication of the ancestral locus, followed by dispersion of the linked loci to different mouse chromosomes. 28 refs., 1 fig., 1 tab.

  1. MEIOTIC F-BOX Is Essential for Male Meiotic DNA Double-Strand Break Repair in Rice[OPEN

    PubMed Central

    Wang, Chong; Yu, Junping; Zong, Jie; Lu, Pingli

    2016-01-01

    F-box proteins constitute a large superfamily in plants and play important roles in controlling many biological processes, but the roles of F-box proteins in male meiosis in plants remain unclear. Here, we identify the rice (Oryza sativa) F-box gene MEIOTIC F-BOX (MOF), which is essential for male meiotic progression. MOF belongs to the FBX subfamily and is predominantly active during leptotene to pachytene of prophase I. mof meiocytes display disrupted telomere bouquet formation, impaired pairing and synapsis of homologous chromosomes, and arrested meiocytes at late prophase I, followed by apoptosis. Although normal, programmed double-stranded DNA breaks (DSBs) form in mof mutants, foci of the phosphorylated histone variant γH2AX, a marker for DSBs, persist in the mutant, indicating that many of the DSBs remained unrepaired. The recruitment of Completion of meiosis I (COM1) and Radiation sensitive51C (RAD51C) to DSBs is severely compromised in mutant meiocytes, indicating that MOF is crucial for DSB end-processing and repair. Further analyses showed that MOF could physically interact with the rice SKP1-like Protein1 (OSK1), indicating that MOF functions as a component of the SCF E3 ligase to regulate meiotic progression in rice. Thus, this study reveals the essential role of an F-box protein in plant meiosis and provides helpful information for elucidating the roles of the ubiquitin proteasome system in plant meiotic progression. PMID:27436711

  2. MEIOTIC F-BOX Is Essential for Male Meiotic DNA Double-Strand Break Repair in Rice.

    PubMed

    He, Yi; Wang, Chong; Higgins, James D; Yu, Junping; Zong, Jie; Lu, Pingli; Zhang, Dabing; Liang, Wanqi

    2016-08-01

    F-box proteins constitute a large superfamily in plants and play important roles in controlling many biological processes, but the roles of F-box proteins in male meiosis in plants remain unclear. Here, we identify the rice (Oryza sativa) F-box gene MEIOTIC F-BOX (MOF), which is essential for male meiotic progression. MOF belongs to the FBX subfamily and is predominantly active during leptotene to pachytene of prophase I. mof meiocytes display disrupted telomere bouquet formation, impaired pairing and synapsis of homologous chromosomes, and arrested meiocytes at late prophase I, followed by apoptosis. Although normal, programmed double-stranded DNA breaks (DSBs) form in mof mutants, foci of the phosphorylated histone variant γH2AX, a marker for DSBs, persist in the mutant, indicating that many of the DSBs remained unrepaired. The recruitment of Completion of meiosis I (COM1) and Radiation sensitive51C (RAD51C) to DSBs is severely compromised in mutant meiocytes, indicating that MOF is crucial for DSB end-processing and repair. Further analyses showed that MOF could physically interact with the rice SKP1-like Protein1 (OSK1), indicating that MOF functions as a component of the SCF E3 ligase to regulate meiotic progression in rice. Thus, this study reveals the essential role of an F-box protein in plant meiosis and provides helpful information for elucidating the roles of the ubiquitin proteasome system in plant meiotic progression. PMID:27436711

  3. Comparison of human and mouse T-cell receptor variable gene segment subfamilies

    SciTech Connect

    Clark, S.P.; Arden, B.; Kabelitz, D.; Mak, T.W.

    1995-10-01

    Like the immunoglobulin Igh-V and Igk-V gene families, the human or mouse TCRV gene families may be grouped into subfamilies displaying {ge} 75% nucleic acid sequence similarity among their members. Systematic interspecies sequence comparisons reveal that most mouse Tcr-V subfamilies exhibit clear homology to human TCRV subfamilies ({ge}60% amino acid sequence similarity). Homologous paris of TCRV genes in mice and humans show higher sequence similarity than TCRV genes from different subfamilies within either species, indicating trans-species evolution of TCRV genes. Mouse and human homologues show conservation of their relative map order, particularly in the 3{prime} region and a similar sequential and developmentally programmed expression. When the V regions from both species were analyzed together, local length differences and conserved residues in the loop regions were revealed, characteristic of each of the four TCRV families. 31 refs., 4 figs.

  4. Precise and in situ genetic humanization of 6 Mb of mouse immunoglobulin genes.

    PubMed

    Macdonald, Lynn E; Karow, Margaret; Stevens, Sean; Auerbach, Wojtek; Poueymirou, William T; Yasenchak, Jason; Frendewey, David; Valenzuela, David M; Giallourakis, Cosmas C; Alt, Frederick W; Yancopoulos, George D; Murphy, Andrew J

    2014-04-01

    Genetic humanization, which involves replacing mouse genes with their human counterparts, can create powerful animal models for the study of human genes and diseases. One important example of genetic humanization involves mice humanized for their Ig genes, allowing for human antibody responses within a mouse background (HumAb mice) and also providing a valuable platform for the generation of fully human antibodies as therapeutics. However, existing HumAb mice do not have fully functional immune systems, perhaps because of the manner in which they were genetically humanized. Heretofore, most genetic humanizations have involved disruption of the endogenous mouse gene with simultaneous introduction of a human transgene at a new and random location (so-called KO-plus-transgenic humanization). More recent efforts have attempted to replace mouse genes with their human counterparts at the same genetic location (in situ humanization), but such efforts involved laborious procedures and were limited in size and precision. We describe a general and efficient method for very large, in situ, and precise genetic humanization using large compound bacterial artificial chromosome-based targeting vectors introduced into mouse ES cells. We applied this method to genetically humanize 3-Mb segments of both the mouse heavy and κ light chain Ig loci, by far the largest genetic humanizations ever described. This paper provides a detailed description of our genetic humanization approach, and the companion paper reports that the humoral immune systems of mice bearing these genetically humanized loci function as efficiently as those of WT mice.

  5. A Survey for Novel Imprinted Genes in the Mouse Placenta by mRNA-seq

    PubMed Central

    Wang, Xu; Soloway, Paul D.; Clark, Andrew G.

    2011-01-01

    Many questions about the regulation, functional specialization, computational prediction, and evolution of genomic imprinting would be better addressed by having an exhaustive genome-wide catalog of genes that display parent-of-origin differential expression. As a first-pass scan for novel imprinted genes, we performed mRNA-seq experiments on embryonic day 17.5 (E17.5) mouse placenta cDNA samples from reciprocal cross F1 progeny of AKR and PWD mouse strains and quantified the allele-specific expression and the degree of parent-of-origin allelic imbalance. We confirmed the imprinting status of 23 known imprinted genes in the placenta and found that 12 genes reported previously to be imprinted in other tissues are also imprinted in mouse placenta. Through a well-replicated design using an orthogonal allelic-expression technology, we verified 5 novel imprinted genes that were not previously known to be imprinted in mouse (Pde10, Phf17, Phactr2, Zfp64, and Htra3). Our data suggest that most of the strongly imprinted genes have already been identified, at least in the placenta, and that evidence supports perhaps 100 additional weakly imprinted genes. Despite previous appearance that the placenta tends to display an excess of maternally expressed imprinted genes, with the addition of our validated set of placenta-imprinted genes, this maternal bias has disappeared. PMID:21705755

  6. Identification of the class I genes of the mouse major histocompatibility complex by DNA-mediated gene transfer.

    PubMed

    Goodenow, R S; McMillan, M; Nicolson, M; Sher, B T; Eakle, K; Davidson, N; Hood, L

    1982-11-18

    DNA-mediated gene transfer was used to identify cloned class I genes from the major histocompatibility complex of the BALB/c mouse. Three genes encoding the transplantation antigens H-2 Kd, Dd and Ld were identified as well as genes encoding the Qa-2,3 and two TL differentiation antigens. As many as 10 putative novel class I genes were detected by the association of their gene products with beta 2-microglobulin. Alloantiserum prepared to one of the novel antigens was used to demonstrate the expression of the previously undetected antigen on spleen cells of various inbred, congeneic, and recombinant congeneic strains of mice. PMID:6815535

  7. Discovery of novel genes and gene isoforms by integrating transcriptomic and proteomic profiling from mouse liver.

    PubMed

    Wu, Peng; Zhang, Hongyu; Lin, Weiran; Hao, Yunwei; Ren, Liangliang; Zhang, Chengpu; Li, Ning; Wei, Handong; Jiang, Ying; He, Fuchu

    2014-05-01

    Comprehensively identifying gene expression in both transcriptomic and proteomic levels of one tissue is a prerequisite for a deeper understanding of its biological functions. Alternative splicing and RNA editing, two main forms of transcriptional processing, play important roles in transcriptome and proteome diversity and result in multiple isoforms for one gene, which are hard to identify by mass spectrometry (MS)-based proteomics approach due to the relative lack of isoform information in standard protein databases. In our study, we employed MS and RNA-Seq in parallel into mouse liver tissue and captured a considerable catalogue of both transcripts and proteins that, respectively, covered 60 and 34% of protein-coding genes in Ensembl. We then developed a bioinformatics workflow for building a customized protein database that for the first time included new splicing-derived peptides and RNA-editing-caused peptide variants, allowing us to more completely identify protein isoforms. Using this experimentally determined database, we totally identified 150 peptides not present in standard biological databases at false discovery rate of <1%, corresponding to 72 novel splicing isoforms, 43 new genetic regions, and 15 RNA-editing sites. Of these, 11 randomly selected novel events passed experimental verification by PCR and Sanger sequencing. New discoveries of gene products with high confidence in two omics levels demonstrated the robustness and effectiveness of our approach and its potential application into improve genome annotation. All the MS data have been deposited to the iProx ( http://ww.iprox.org ) with the identifier IPX00003601.

  8. A High Incidence of Meiotic Silencing of Unsynapsed Chromatin Is Not Associated with Substantial Pachytene Loss in Heterozygous Male Mice Carrying Multiple Simple Robertsonian Translocations

    PubMed Central

    Vasco, Chiara; Berríos, Soledad; Parra, María Teresa; Viera, Alberto; Rufas, Julio S.; Zuccotti, Maurizio; Garagna, Silvia; Fernández-Donoso, Raúl

    2009-01-01

    Meiosis is a complex type of cell division that involves homologous chromosome pairing, synapsis, recombination, and segregation. When any of these processes is altered, cellular checkpoints arrest meiosis progression and induce cell elimination. Meiotic impairment is particularly frequent in organisms bearing chromosomal translocations. When chromosomal translocations appear in heterozygosis, the chromosomes involved may not correctly complete synapsis, recombination, and/or segregation, thus promoting the activation of checkpoints that lead to the death of the meiocytes. In mammals and other organisms, the unsynapsed chromosomal regions are subject to a process called meiotic silencing of unsynapsed chromatin (MSUC). Different degrees of asynapsis could contribute to disturb the normal loading of MSUC proteins, interfering with autosome and sex chromosome gene expression and triggering a massive pachytene cell death. We report that in mice that are heterozygous for eight multiple simple Robertsonian translocations, most pachytene spermatocytes bear trivalents with unsynapsed regions that incorporate, in a stage-dependent manner, proteins involved in MSUC (e.g., γH2AX, ATR, ubiquitinated-H2A, SUMO-1, and XMR). These spermatocytes have a correct MSUC response and are not eliminated during pachytene and most of them proceed into diplotene. However, we found a high incidence of apoptotic spermatocytes at the metaphase stage. These results suggest that in Robertsonian heterozygous mice synapsis defects on most pachytene cells do not trigger a prophase-I checkpoint. Instead, meiotic impairment seems to mainly rely on the action of a checkpoint acting at the metaphase stage. We propose that a low stringency of the pachytene checkpoint could help to increase the chances that spermatocytes with synaptic defects will complete meiotic divisions and differentiate into viable gametes. This scenario, despite a reduction of fertility, allows the spreading of Robertsonian

  9. Mapping of the Tuple1 gene to mouse chromosome 16A-B1

    SciTech Connect

    Mattei, M.G.; Halford, S.; Scambler, P.J.

    1994-10-01

    The human TUPLE1 gene encodes a putative transcriptional regulator and maps to chromosome 22, and therefore may play a role in Di-George syndrome (DGS), relo-cardio-facial syndrome (VCFS), or a related pathology. The murine TUPLE1 gene has also been cloned and shows strong sequence similarity to TUPLE1. Comparative mapping is useful in the study of chromosome evolution and is sometimes able to indicate possible mouse mutations that are potential models of human genetic disorders. As TIPLE1 is a candidate gene for the haploinsufficient phenotype in DGS, we mapped TUPLE1 to mouse chromosome 16A-B1. 6 refs., 1 fig.

  10. Mapping of 195 genes in cattle and updated comparative map with man, mouse, rat and pig.

    PubMed

    Hayes, H; Elduque, C; Gautier, M; Schibler, L; Cribiu, E; Eggen, A

    2003-01-01

    Our on-going goal is to improve and update the comparative genome organization between cattle and man but also among the most detailed mammalian species genomes i.e. cattle, mouse, rat and pig. In this work, we localized 195 genes in cattle and checked all human/bovine non-concordant localizations found in the literature. Next, we compiled all the genes mapped in cattle, goat, sheep and pig (2,166) for which the human ortholog with its chromosomal position is known, added corresponding data in mouse and rat, and ordered the genes relatively to the human genome sequence. We estimate that our compilation provides bovine mapping information for about 89% of the human autosomes. Thus, a near complete, overall and detailed picture of the number, distribution and extent of bovine conserved syntenies (regardless of gene order) on human R-banded autosomes is proposed as well as a comparison with mouse, rat and pig genomes. PMID:14970673

  11. Meiotic behavior and chromosome number of Urochloa adspersa (Trin.) R. D. Webster from the Brazilian Chaco.

    PubMed

    Felismino, M F; Maior, R L S; Damasceno, G A; Pott, A; Pagliarini, M S

    2015-01-01

    This is the first report of meiotic division in Uro-chloa adspersa (Trin.) collected from the Brazilian Chaco. Meiotic analyses were performed on three specimens of U. adspersa named G10, G15, and G16. Inflorescences were collected and fixed in a mixture of ethanol and acetic acid (3:1, v/v) for 24 h and then stored in 70% alcohol. Diakinesis revealed different chromosome numbers and ploidy levels. All three plants were polyploids: G10 and G15 exhibited 2n = 6x = 54 chromosomes (arranged in 27 bivalents), while G16 exhibited 2n = 4x = 36 chromosomes (18 bivalents). Meiotic behavior was mainly normal in the hexaploid G15 and the tetraploid G16 (5.3 and 6.2% of the cells were abnormal, respective-ly), revealing only a few meiotic abnormalities that are common to polyploids, i.e., those related to irregular chromosome segregation. G10 exhibited other meiotic abnormalities during meiosis II, such as chromosome stickiness, irregular spindle orientation, and irregular cytokinesis, which led to the formation of a few triads, resulting in 16.9% of the cells being abnormal. The origin of these abnormalities is discussed, and we suggest that the genes that control meiotic steps may be present in the Urochloa gene pool. PMID:26214424

  12. Phylogenetic conservation of immunoglobulin heavy chains: direct comparison of hamster and mouse Cmu genes.

    PubMed Central

    McGuire, K L; Duncan, W R; Tucker, P W

    1985-01-01

    We have analyzed the JH-Cmu locus of the Syrian hamster by DNA cloning and sequencing. The single Cmu gene is highly homologous to that of the mouse. The hamster equivalents of the JH and switch (S) recombination regions are arranged as in the mouse, but surprisingly are not highly conserved. Also unlike its close murine relative, the Smu regions among inbred hamster strains are not polymorphic. The complete nucleotide sequence of hamster and mouse Cmu genes have been compared to partial Cmu sequences of other species. Conservation within a portion of the 3' untranslated region may signify functional requirements for 3' end processing. Mutational frequencies within exons and introns of hamster and mouse do not support the theory that the rate of DNA transitions to transversions decreases with evolutionary distance. Images PMID:2994005

  13. Sequence divergence and chromosomal rearrangements during the evolution of human pseudoautosomal genes and their mouse homologs

    SciTech Connect

    Ellison, J.; Li, X.; Francke, U.

    1994-09-01

    The pseudoautosomal region (PAR) is an area of sequence identity between the X and Y chromosomes and is important for mediating X-Y pairing during male meiosis. Of the seven genes assigned to the human PAR, none of the mouse homologs have been isolated by a cross-hybridization strategy. Two of these homologs, Csfgmra and II3ra, have been isolated using a functional assay for the gene products. These genes are quite different in sequence from their human homologs, showing only 60-70% sequence similarity. The Csfgmra gene has been found to further differ from its human homolog in being isolated not on the sex chromosomes, but on a mouse autosome (chromosome 19). Using a mouse-hamster somatic cell hybrid mapping panel, we have mapped the II3ra gene to yet another mouse autosome, chromosome 14. Attempts to clone the mouse homolog of the ANT3 locus resulted in the isolation of two related genes, Ant1 and Ant2, but failed to yield the Ant3 gene. Southern blot analysis of the ANT/Ant genes showed the Ant1 and Ant2 sequences to be well-conserved among all of a dozen mammals tested. In contrast, the ANT3 gene only showed hybridization to non-rodent mammals, suggesting it is either greatly divergent or has been deleted in the rodent lineage. Similar experiments with other human pseudoautosomal probes likewise showed a lack of hybridization to rodent sequences. The results show a definite trend of extensive divergence of pseudoautosomal sequences in addition to chromosomal rearrangements involving X;autosome translocations and perhaps gene deletions. Such observations have interesting implications regarding the evolution of this important region of the sex chromosomes.

  14. Sequencing the mouse Y chromosome reveals convergent gene acquisition and amplification on both sex chromosomes.

    PubMed

    Soh, Y Q Shirleen; Alföldi, Jessica; Pyntikova, Tatyana; Brown, Laura G; Graves, Tina; Minx, Patrick J; Fulton, Robert S; Kremitzki, Colin; Koutseva, Natalia; Mueller, Jacob L; Rozen, Steve; Hughes, Jennifer F; Owens, Elaine; Womack, James E; Murphy, William J; Cao, Qing; de Jong, Pieter; Warren, Wesley C; Wilson, Richard K; Skaletsky, Helen; Page, David C

    2014-11-01

    We sequenced the MSY (male-specific region of the Y chromosome) of the C57BL/6J strain of the laboratory mouse Mus musculus. In contrast to theories that Y chromosomes are heterochromatic and gene poor, the mouse MSY is 99.9% euchromatic and contains about 700 protein-coding genes. Only 2% of the MSY derives from the ancestral autosomes that gave rise to the mammalian sex chromosomes. Instead, all but 45 of the MSY's genes belong to three acquired, massively amplified gene families that have no homologs on primate MSYs but do have acquired, amplified homologs on the mouse X chromosome. The complete mouse MSY sequence brings to light dramatic forces in sex chromosome evolution: lineage-specific convergent acquisition and amplification of X-Y gene families, possibly fueled by antagonism between acquired X-Y homologs. The mouse MSY sequence presents opportunities for experimental studies of a sex-specific chromosome in its entirety, in a genetically tractable model organism.

  15. Surface-based mapping of gene expression and probabilistic expression maps in the mouse cortex.

    PubMed

    Ng, Lydia; Lau, Chris; Sunkin, Susan M; Bernard, Amy; Chakravarty, M Mallar; Lein, Ed S; Jones, Allan R; Hawrylycz, Michael

    2010-02-01

    The Allen Brain Atlas (ABA, www.brain-map.org) is a genome wide, spatially registered collection of cellular resolution in situ hybridization gene expression image data of the C57Bl/6J mouse brain. Derived from the ABA, the Anatomic Gene Expression Atlas (AGEA, http://mouse.brain-map.org/agea) has demonstrated both laminar and areal spatial gene expression correlations in the mouse cortex. While the mouse cortex is lissencephalic, its curvature and substantial bending in boundary areas renders it difficult to visualize and analyze laminar versus areal effects in a rectilinear coordinate framework. In context of human and non-human primate cortex, surface-based representation has proven useful for understanding relative locations of laminar, columnar, and areal features. In this paper, we describe a methodology for constructing surface-based flatmaps of the mouse cortex that enables mapping of gene expression data from individual genes in the ABA, or probabilistic expression maps from the AGEA, to identify and visualize genetic relationships between layers and areas. PMID:19818854

  16. The neuronal nicotinic acetylcholine receptor {alpha}7 subunit gene: Cloning, mapping, structure, and targeting in mouse

    SciTech Connect

    Orr-Urtreger, A.; Baldini, A.; Beaudet, A.L.

    1994-09-01

    The neuronal nicotinic acetylcholine receptor {alpha}7 subunit is a member of a family of ligand-gated ion channels, and is the only subunit know to bind {alpha}-bungarotoxin in mammalian brain. {alpha}-Bungarotoxin binding sites are known to be more abundant in the hippocampus of mouse strains that are particularly sensitive to nicotine-induced seizures. The {alpha}7 receptor is highly permeable to calcium, which could suggest a role in synaptic plasticity in the nervous system. Auditory gating deficiency, an abnormal response to a second auditory stimulus, is characteristic of schizophrenia. Mouse strains that exhibit a similar gating deficit have reduced hippocampal expression of the {alpha}7 subunit. We have cloned and sequenced the full length cDNA for the mouse {alpha}7 gene (Acra-7) and characterized its gene structure. The murine {alpha}7 shares amino acid identity of 99% and 93% with the rat and human {alpha}7 subunits, respectively. Using an interspecies backcross panel, the murine gene was mapped to chromosome 7 near the p locus, a region syntenic with human chromosome 15; the human gene (CHRNA7) was confirmed to map to 15q13-q14 by FISH. To generate a mouse {alpha}7 mutant by homologous recombination, we have constructed a replacement vector which will delete transmembrane domains II-IV and the cytoplasmic domain from the gene product. Recombinant embryonic stem (ES) cell clones were selected and used to develop mouse chimeras that are currently being bred to obtain germline transmission.

  17. Sequencing the mouse Y chromosome reveals convergent gene acquisition and amplification on both sex chromosomes

    PubMed Central

    Soh, Y.Q. Shirleen; Alföldi, Jessica; Pyntikova, Tatyana; Brown, Laura G.; Graves, Tina; Minx, Patrick J.; Fulton, Robert S.; Kremitzki, Colin; Koutseva, Natalia; Mueller, Jacob L.; Rozen, Steve; Hughes, Jennifer F.; Owens, Elaine; Womack, James E.; Murphy, William J.; Cao, Qing; de Jong, Pieter; Warren, Wesley C.; Wilson, Richard K.; Skaletsky, Helen; Page, David C.

    2014-01-01

    Summary We sequenced the MSY (Male-Specific region of the Y chromosome) of the C57BL/6J strain of the laboratory mouse Mus musculus. In contrast to theories that Y chromosomes are heterochromatic and gene poor, the mouse MSY is 99.9% euchromatic and contains about 700 protein-coding genes. Only two percent of the MSY derives from the ancestral autosomes that gave rise to the mammalian sex chromosomes. Instead, all but 50 of the MSY's genes belong to three acquired, massively amplified gene families that have no homologs on primate MSYs, but do have acquired, amplified homologs on the mouse X chromosome. The complete mouse MSY sequence brings to light dramatic forces in sex chromosome evolution: lineage-specific convergent acquisition and amplification of X-Y gene families, possibly fueled by antagonism between acquired X-Y homologs. The mouse MSY sequence presents opportunities for experimental studies of a sex-specific chromosome in its entirety, in a genetically tractable model organism. PMID:25417157

  18. Molecular dissection of Neurospora Spore killer meiotic drive elements

    PubMed Central

    Hammond, Thomas M.; Rehard, David G.; Xiao, Hua; Shiu, Patrick K. T.

    2012-01-01

    Meiotic drive is a non-Mendelian inheritance phenomenon in which certain selfish genetic elements skew sexual transmission in their own favor. In some cases, progeny or gametes carrying a meiotic drive element can survive preferentially because it causes the death or malfunctioning of those that do not carry it. In Neurospora, meiotic drive can be observed in fungal spore killing. In a cross of Spore killer (Sk) × WT (Sk-sensitive), the ascospores containing the Spore killer allele survive, whereas the ones with the sensitive allele degenerate. Sk-2 and Sk-3 are the most studied meiotic drive elements in Neurospora, and they each theoretically contain two essential components: a killer element and a resistance gene. Here we report the identification and characterization of the Sk resistance gene, rsk (resistant to Spore killer). rsk seems to be a fungal-specific gene, and its deletion in a killer strain leads to self-killing. Sk-2, Sk-3, and naturally resistant isolates all use rsk for resistance. In each killer system, rsk sequences from an Sk strain and a resistant isolate are highly similar, suggesting that they share the same origin. Sk-2, Sk-3, and sensitive rsk alleles differ from each other by their unique indel patterns. Contrary to long-held belief, the killer targets not only late but also early ascospore development. The WT RSK protein is dispensable for ascospore production and is not a target of the spore-killing mechanism. Rather, a resistant version of RSK likely neutralizes the killer element and prevents it from interfering with ascospore development. PMID:22753473

  19. Gene for the catalytic subunit of mouse DNA-dependent protein kinase maps to the scid locus.

    PubMed Central

    Miller, R D; Hogg, J; Ozaki, J H; Gell, D; Jackson, S P; Riblet, R

    1995-01-01

    The gene encoding the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) has been proposed recently as a candidate gene for the mouse severe combined immune deficiency (scid) locus. We have used a partial cDNA clone for human DNA-PKcs to map the mouse homologue using a large interspecific backcross panel. We found that the mouse gene for DNA-PKcs does not recombine with scid, consistent with the hypothesis that scid is a mutation in the mouse gene for DNA-PKcs. Images Fig. 3 PMID:7479885

  20. A Survey of Imprinted Gene Expression in Mouse Trophoblast Stem Cells

    PubMed Central

    Calabrese, J. Mauro; Starmer, Joshua; Schertzer, Megan D.; Yee, Della; Magnuson, Terry

    2015-01-01

    Several hundred mammalian genes are expressed preferentially from one parental allele as the result of a process called genomic imprinting. Genomic imprinting is prevalent in extra-embryonic tissue, where it plays an essential role during development. Here, we profiled imprinted gene expression via RNA-Seq in a panel of six mouse trophoblast stem lines, which are ex vivo derivatives of a progenitor population that gives rise to the placental tissue of the mouse. We found evidence of imprinted expression for 48 genes, 31 of which had been described previously as imprinted and 17 of which we suggest as candidate imprinted genes. An equal number of maternally and paternally biased genes were detected. On average, candidate imprinted genes were more lowly expressed and had weaker parent-of-origin biases than known imprinted genes. Several known and candidate imprinted genes showed variability in parent-of-origin expression bias between the six trophoblast stem cell lines. Sixteen of the 48 known and candidate imprinted genes were previously or newly annotated noncoding RNAs and six encoded for a total of 60 annotated microRNAs. Pyrosequencing across our panel of trophoblast stem cell lines returned levels of imprinted expression that were concordant with RNA-Seq measurements for all eight genes examined. Our results solidify trophoblast stem cells as a cell culture-based experimental model to study genomic imprinting, and provide a quantitative foundation upon which to delineate mechanisms by which the process is maintained in the mouse. PMID:25711832

  1. Chromosomal localization of genes encoding guanine nucleotide-binding protein subunits in mouse and human.

    PubMed

    Blatt, C; Eversole-Cire, P; Cohn, V H; Zollman, S; Fournier, R E; Mohandas, L T; Nesbitt, M; Lugo, T; Jones, D T; Reed, R R

    1988-10-01

    A variety of genes have been identified that specify the synthesis of the components of guanine nucleotide-binding proteins (G proteins). Eight different guanine nucleotide-binding alpha-subunit proteins, two different beta subunits, and one gamma subunit have been described. Hybridization of cDNA clones with DNA from human-mouse somatic cell hybrids was used to assign many of these genes to human chromosomes. The retinal-specific transducin subunit genes GNAT1 and GNAT2 were on chromosomes 3 and 1; GNAI1, GNAI2, and GNAI3 were assigned to chromosomes 7, 3, and 1, respectively; GNAZ and GNAS were found on chromosomes 22 and 20. The beta subunits were also assigned--GNB1 to chromosome 1 and GNB2 to chromosome 7. Restriction fragment length polymorphisms were used to map the homologues of some of these genes in the mouse. GNAT1 and GNAI2 were found to map adjacent to each other on mouse chromosome 9 and GNAT2 was mapped on chromosome 17. The mouse GNB1 gene was assigned to chromosome 19. These mapping assignments will be useful in defining the extent of the G alpha gene family and may help in attempts to correlate specific genetic diseases with genes corresponding to G proteins. PMID:2902634

  2. Structural organization and transcription of the mouse gastric H+, K(+)-ATPase beta subunit gene.

    PubMed Central

    Canfield, V A; Levenson, R

    1991-01-01

    We have cloned and characterized the mouse gene encoding the beta subunit of H+, K(+)-ATPase (EC 3.6.1.36). The entire 10.5-kilobase transcription unit of the H+,K(+)-ATPase beta subunit gene was cloned in three overlapping cosmids encompassing approximately 46 kilobases of genomic DNA. A tight cluster of transcription initiation sites has been localized 24-25 nucleotides upstream of the translation start site and 28-29 nucleotides downstream of a TATA-like sequence. The H+, K(+)-ATPase beta subunit gene is split into seven exons encoding predicted structural domains of the beta subunit protein. The intracellular amino-terminal and putative transmembrane domains are encoded by individual exons, and the extracellular carboxyl-terminal domain is encoded by five exons. The exon/intron organization of the mouse H+,K(+)-ATPase beta subunit gene is identical to that of the mouse Na+,K(+)-ATPase beta 2 subunit gene. The conservation of genomic organization, together with the high sequence homology, indicates that the mouse H+,K(+)-ATPase beta and Na+,K(+)-ATPase beta 2 subunit genes originated from a common ancestral gene. Images PMID:1654563

  3. Generation and gene expression profiling of 48 transcription-factor-inducible mouse embryonic stem cell lines

    PubMed Central

    Yamamizu, Kohei; Sharov, Alexei A.; Piao, Yulan; Amano, Misa; Yu, Hong; Nishiyama, Akira; Dudekula, Dawood B.; Schlessinger, David; Ko, Minoru S. H.

    2016-01-01

    Mouse embryonic stem cells (ESCs) can differentiate into a wide range – and possibly all cell types in vitro, and thus provide an ideal platform to study systematically the action of transcription factors (TFs) in cell differentiation. Previously, we have generated and analyzed 137 TF-inducible mouse ESC lines. As an extension of this “NIA Mouse ESC Bank,” we generated and characterized 48 additional mouse ESC lines, in which single TFs in each line could be induced in a doxycycline-controllable manner. Together, with the previous ESC lines, the bank now comprises 185 TF-manipulable ESC lines (>10% of all mouse TFs). Global gene expression (transcriptome) profiling revealed that the induction of individual TFs in mouse ESCs for 48 hours shifts their transcriptomes toward specific differentiation fates (e.g., neural lineages by Myt1 Isl1, and St18; mesodermal lineages by Pitx1, Pitx2, Barhl2, and Lmx1a; white blood cells by Myb, Etv2, and Tbx6, and ovary by Pitx1, Pitx2, and Dmrtc2). These data also provide and lists of inferred target genes of each TF and possible functions of these TFs. The results demonstrate the utility of mouse ESC lines and their transcriptome data for understanding the mechanism of cell differentiation and the function of TFs. PMID:27150017

  4. Backcrossing to increase meiotic stability in triticale.

    PubMed

    Giacomin, R M; Assis, R; Brammer, S P; Nascimento Junior, A; Da-Silva, P R

    2015-01-01

    Triticale (X Triticosecale Wittmack) is an intergeneric hybrid derived from a cross between wheat and rye. As a newly created allopolyploid, the plant shows instabilities during the meiotic process, which may result in the loss of fertility. This genomic instability has hindered the success of triticale-breeding programs. Therefore, strategies should be developed to obtain stable triticale lines for use in breeding. In some species, backcrossing has been effective in increasing the meiotic stability of lineages. To assess whether backcrossing has the same effect in triticale, indices of meiotic abnormalities, meiotic index, and pollen viability were determined in genotypes from multiple generations of triticale (P1, P2, F1, F2, BC1a, and BC1b). All analyzed genotypes exhibited instability during meiosis, and their meiotic index values were all lower than normal. However, the backcrosses BC1a and BC1b showed the lowest mean meiotic abnormalities and the highest meiotic indices, demonstrating higher stability. All genotypes showed a high rate of pollen viability, with the backcrosses BC1a and BC1b again exhibiting the best values. Statistical analyses confirmed that backcrossing positively affects the meiotic stability of triticale. Our results show that backcrossing should be considered by breeders aiming to obtain triticale lines with improved genomic stability. PMID:26400358

  5. Comparative gene expression profiling in two congenic mouse strains following Bordetella pertussis infection

    PubMed Central

    Banus, Sander; Vandebriel, Rob J; Pennings, Jeroen LA; Gremmer, Eric R; Wester, Piet W; van Kranen, Henk J; Breit, Timo M; Demant, Peter; Mooi, Frits R; Hoebee, Barbara; Kimman, Tjeerd G

    2007-01-01

    Background Susceptibility to Bordetella pertussis infection varies widely. These differences can partly be explained by genetic host factors. HcB-28 mice are more resistant to B. pertussis infection than C3H mice, which could partially be ascribed to the B. pertussis susceptibility locus-1 (Bps1) on chromosome 12. The presence of C57BL/10 genome on this locus instead of C3H genome resulted in a decreased number of bacteria in the lung. To further elucidate the role of host genetic factors, in particular in the Bps1 locus, in B. pertussis infection, and to identify candidate genes within in this region, we compared expression profiles in the lungs of the C3H and HcB-28 mouse strains following B. pertussis inoculation. Twelve and a half percent of the genomes of these mice are from a different genetic background. Results Upon B. pertussis inoculation 2,353 genes were differentially expressed in the lungs of both mouse strains. Two hundred and six genes were differentially expressed between the two mouse strains, but, remarkably, none of these were up- or down-regulated upon B. pertussis infection. Of these 206 genes, 17 were located in the Bps1 region. Eight of these genes, which showed a strong difference in gene expression between the two mouse strains, map to the immunoglobulin heavy chain complex (Igh). Conclusion Gene expression changes upon B. pertussis infection are highly identical between the two mouse strains despite the differences in the course of B. pertussis infection. Because the genes that were differentially regulated between the mouse strains only showed differences in expression before infection, it appears likely that such intrinsic differences in gene regulation are involved in determining differences in susceptibility to B. pertussis infection. Alternatively, such genetic differences in susceptibility may be explained by genes that are not differentially regulated between these two mouse strains. Genes in the Igh complex, among which Igh-1a

  6. Assessment of orthologous splicing isoforms in human and mouse orthologous genes

    PubMed Central

    2010-01-01

    Background Recent discoveries have highlighted the fact that alternative splicing and alternative transcripts are the rule, rather than the exception, in metazoan genes. Since multiple transcript and protein variants expressed by the same gene are, by definition, structurally distinct and need not to be functionally equivalent, the concept of gene orthology should be extended to the transcript level in order to describe evolutionary relationships between structurally similar transcript variants. In other words, the identification of true orthology relationships between gene products now should progress beyond primary sequence and "splicing orthology", consisting in ancestrally shared exon-intron structures, is required to define orthologous isoforms at transcript level. Results As a starting step in this direction, in this work we performed a large scale human- mouse gene comparison with a twofold goal: first, to assess if and to which extent traditional gene annotations such as RefSeq capture genuine splicing orthology; second, to provide a more detailed annotation and quantification of true human-mouse orthologous transcripts defined as transcripts of orthologous genes exhibiting the same splicing patterns. Conclusions We observed an identical exon/intron structure for 32% of human and mouse orthologous genes. This figure increases to 87% using less stringent criteria for gene structure similarity, thus implying that for about 13% of the human RefSeq annotated genes (and about 25% of the corresponding transcripts) we could not identify any mouse transcript showing sufficient similarity to be confidently assigned as a splicing ortholog. Our data suggest that current gene and transcript data may still be rather incomplete - with several splicing variants still unknown. The observation that alternative splicing produces large numbers of alternative transcripts and proteins, some of them conserved across species and others truly species-specific, suggests that, still

  7. Introduction of human gamma 1 immunoglobulin genes into fertilized mouse eggs.

    PubMed

    Yamamura, K; Kikutani, H; Takahashi, N; Taga, T; Akira, S; Kawai, K; Fukuchi, K; Kumahara, Y; Honjo, T; Kishimoto, T

    1984-08-01

    A rearranged human gamma 1 immunoglobulin gene was introduced into fertilized mouse eggs. The phage Ch4A-VCE-gamma 1 was constructed by ligating an EcoRI and BglII fragment of pBR322-CESSV(CE-1) containing the VDJ region with an EcoRI and BamHI fragment of Ch4A-HIg gamma 1-10 containing the gamma 1 constant region. About 200 copies of Ch4A-VCE-gamma 1 genes were introduced into fertilized mouse eggs. Of 489 eggs injected with these genes, 319 survived and were transferred to oviducts of foster mothers. Thirtyeight mice were born and were screened for the presence of human gamma 1 immunoglobulin genes by Southern blot hybridization. Five of these 38 mice had integrated human gamma 1 immunoglobulin genes. None of the human gamma 1 copies in each mouse had undergone deletions or rearrangements as judged by the Southern blotting patterns for several restriction enzymes. Human gamma 1 gene was present in several different tissues. All the mice tested so far transmit the human gamma 1 gene to a fraction of their offspring in an autosomal dominant manner. Spleen cells from transgenic mice were analyzed for immunoglobulin production by reverse plaque assay or immunofluorescence staining of cytoplasmic immunoglobulin, but synthesis and secretion of human gamma 1 chains could not be detected. No human gamma 1 immunoglobulin mRNA was detected in the liver and spleen of a transgenic mouse. The presence of the human gamma 1 immunoglobulin gene appeared to have no effect on the expression of endogenous mouse immunoglobulin genes.

  8. Meiotic Recombination: The Essence of Heredity.

    PubMed

    Hunter, Neil

    2015-10-28

    The study of homologous recombination has its historical roots in meiosis. In this context, recombination occurs as a programmed event that culminates in the formation of crossovers, which are essential for accurate chromosome segregation and create new combinations of parental alleles. Thus, meiotic recombination underlies both the independent assortment of parental chromosomes and genetic linkage. This review highlights the features of meiotic recombination that distinguish it from recombinational repair in somatic cells, and how the molecular processes of meiotic recombination are embedded and interdependent with the chromosome structures that characterize meiotic prophase. A more in-depth review presents our understanding of how crossover and noncrossover pathways of meiotic recombination are differentiated and regulated. The final section of this review summarizes the studies that have defined defective recombination as a leading cause of pregnancy loss and congenital disease in humans.

  9. Structure and polymorphism of the mouse myelin/oligodendrocyte glycoprotein gene

    SciTech Connect

    Daubas, P.; Pham-Dinh, D.; Dautigny, A.

    1994-09-01

    The authors have isolated and characterized genomic clones containing the mouse myelin/oligodendrocyte glycoprotein (MOG) gene. It spans a region of 12.5 kb and consists of eight exons. Its exon-intron structure differs from that of classical MHC-class I genes, with which it is linked in the mouse genome. Nucleotide sequencing of the 5{prime} flanking region revelas that it contains several putative protein-binding sites, some of them in common with other myelin gene promoters. One intragenic polymorphism has been identified: it consists of a GA repeat, defining at least three alleles in mouse inbred strains, and is easily detectable using the polymerase chain reaction method.

  10. Areal and laminar differentiation in the mouse neocortex using large scale gene expression data.

    PubMed

    Hawrylycz, Mike; Bernard, Amy; Lau, Chris; Sunkin, Susan M; Chakravarty, M Mallar; Lein, Ed S; Jones, Allan R; Ng, Lydia

    2010-02-01

    Although cytoarchitectonic organization of the mammalian cortex into different lamina has been well-studied, identifying the architectural differences that distinguish cortical areas from one another is more challenging. Localization of large anatomical structures is possible using magnetic resonance imaging or invasive techniques (such as anterograde or retrograde tracing), but identifying patterns in gene expression architecture is limited as gene products do not necessarily identify an immediate functional consequence of a specialized area. Expression of specific genes in the mouse and human cortex is most often identified across entire lamina, and areal patterning of expression (when it exists) is most easily differentiated on a layer-by-layer basis. Since cortical organization is defined by the expression of large sets of genes, the task of identifying individual (or groups of structures) cannot be done using individual areal markers. In this manuscript we describe a methodology for clustering gene expression correlation profiles in the C57Bl/6J mouse cortex to identify large-scale genetic relationships between layers and areas. By using the Anatomic Gene Expression Atlas (http://mouse.brain-map.org/agea/) derived from in situ hybridization data in the Allen Brain Atlas, we show that a consistent expression based organization of areal patterning in the mouse cortex exists when clustered on a laminar basis. Surface-based mapping and visualization techniques are used as a representation to clarify these relationships. PMID:19800006

  11. Construction of a mouse model of factor VIII deficiency by gene targeting

    SciTech Connect

    Bi, L.; Lawler, A.; Gearhart, J.

    1994-09-01

    To develop a small animal model of hemophilia A for gene therapy experiments, we set out to construct a mouse model for factor VIII deficiency by gene targeting. First, we screened a mouse liver cDNA library using a human FVIII cDNA probe. We cloned a 2.6 Kb partial mouse factor VIII cDNA which extends from 800 base pairs of the 3{prime} end of exon 14 to the 5{prime} end of exon 26. A mouse genomic library made from strain 129 was then screened to obtain genomic fragments covering the exons desired for homologous recombination. Two genomic clones were obtained, and one covering exon 15 through 22 was used for gene targeting. To make gene targeting constructs, a 5.8 Kb genomic DNA fragment covering exons 15 to 19 of the mouse FVIII gene was subcloned, and the neo expression cassette was inserted into exons 16 and 17 separately by different strategies. These two constructs were named MFVIIIC-16 and MFVIIIC-17. The constructs were linearized and transfected into strain 129 mouse ES cells by electroporation. Factor VIII gene-knockout ES cell lines were selected by G-418 and screened by genomic Southern blots. Eight exon 16 targeted cell lines and five exon 17 targeted cell lines were obtained. Three cell lines from each construct were injected into blastocysts and surgically transferred into foster mothers. Multiple chimeric mice with 70-90% hair color derived from the ES-cell genotype were seen with both constructs. Germ line transmission of the ES-cell genotype has been obtained for the MFVIIIC-16 construct, and multiple hemophilia A carrier females have been identified. Factor VIII-deficient males will be conceived soon.

  12. High-resolution gene expression atlases for adult and developing mouse brain and spinal cord.

    PubMed

    Henry, Alex M; Hohmann, John G

    2012-10-01

    Knowledge of the structure, genetics, circuits, and physiological properties of the mammalian brain in both normal and pathological states is ever increasing as research labs worldwide probe the various aspects of brain function. Until recently, however, comprehensive cataloging of gene expression across the central nervous system has been lacking. The Allen Institute for Brain Science, as part of its mission to propel neuroscience research, has completed several large gene-mapping projects in mouse, nonhuman primate, and human brain, producing informative online public resources and tools. Here we present the Allen Mouse Brain Atlas, covering ~20,000 genes throughout the adult mouse brain; the Allen Developing Mouse Brain Atlas, detailing expression of approximately 2,000 important developmental genes across seven embryonic and postnatal stages of brain growth; and the Allen Spinal Cord Atlas, revealing expression for ~20,000 genes in the adult and neonatal mouse spinal cords. Integrated data-mining tools, including reference atlases, informatics analyses, and 3-D viewers, are described. For these massive-scale projects, high-throughput industrial techniques were developed to standardize and reliably repeat experimental goals. To verify consistency and accuracy, a detailed analysis of the 1,000 most viewed genes for the adult mouse brain (according to website page views) was performed by comparing our data with peer-reviewed literature and other databases. We show that our data are highly consistent with independent sources and provide a comprehensive compendium of information and tools used by thousands of researchers each month. All data and tools are freely available via the Allen Brain Atlas portal (www.brain-map.org).

  13. High-resolution gene expression atlases for adult and developing mouse brain and spinal cord.

    PubMed

    Henry, Alex M; Hohmann, John G

    2012-10-01

    Knowledge of the structure, genetics, circuits, and physiological properties of the mammalian brain in both normal and pathological states is ever increasing as research labs worldwide probe the various aspects of brain function. Until recently, however, comprehensive cataloging of gene expression across the central nervous system has been lacking. The Allen Institute for Brain Science, as part of its mission to propel neuroscience research, has completed several large gene-mapping projects in mouse, nonhuman primate, and human brain, producing informative online public resources and tools. Here we present the Allen Mouse Brain Atlas, covering ~20,000 genes throughout the adult mouse brain; the Allen Developing Mouse Brain Atlas, detailing expression of approximately 2,000 important developmental genes across seven embryonic and postnatal stages of brain growth; and the Allen Spinal Cord Atlas, revealing expression for ~20,000 genes in the adult and neonatal mouse spinal cords. Integrated data-mining tools, including reference atlases, informatics analyses, and 3-D viewers, are described. For these massive-scale projects, high-throughput industrial techniques were developed to standardize and reliably repeat experimental goals. To verify consistency and accuracy, a detailed analysis of the 1,000 most viewed genes for the adult mouse brain (according to website page views) was performed by comparing our data with peer-reviewed literature and other databases. We show that our data are highly consistent with independent sources and provide a comprehensive compendium of information and tools used by thousands of researchers each month. All data and tools are freely available via the Allen Brain Atlas portal (www.brain-map.org). PMID:22832508

  14. Intervening sequence of DNA identified in the structural portion of a mouse beta-globin gene.

    PubMed Central

    Tilghman, S M; Tiemeier, D C; Seidman, J G; Peterlin, B M; Sullivan, M; Maizel, J V; Leder, P

    1978-01-01

    The unusual electron microscopic appearance of a hybrid formed between 9S mouse beta-globin mRNA and its corresponding cloned gene segment is caused by at least one, and possibly two, intervening sequences of DNA that interrupt the mouse beta-globin gene. Such an interpretation is consistent with a paradoxical restriction site pattern previously noted in this gene and with the nucleotide sequence of that portion of the gene that spans both structural and intervening sequences. The large intervening sequence, approximately 550 base pairs in length, occurs in the structural globin sequence and immediately follows the beta-globin codon corresponding to amino acid 104. A smaller, putative intervening sequence is located close to the 5' end of the beta-globin-coding sequence but may reside beyond its initiation codon. The beta-globin gene thus appears to be encoded in two, and possibly three, discontinuous segments. Images PMID:273235

  15. Systematic identification and integrative analysis of novel genes expressed specifically or predominantly in mouse epididymis

    PubMed Central

    Oh, Jungsu; Lee, Jiae; Woo, Jong-Min; Choi, Eunyoung; Park, Inju; Han, Cecil; Baek, Namhoe; Lee, Hoyong; Kim, Do Han; Cho, Chunghee

    2006-01-01

    Background Maturation of spermatozoa, including development of motility and the ability to fertilize the oocyte, occurs during transit through the microenvironment of the epididymis. Comprehensive understanding of sperm maturation requires identification and characterization of unique genes expressed in the epididymis. Results We systematically identified 32 novel genes with epididymis-specific or -predominant expression in the mouse epididymis UniGene library, containing 1505 gene-oriented transcript clusters, by in silico and in vitro analyses. The Northern blot analysis revealed various characteristics of the genes at the transcript level, such as expression level, size and the presence of isoform. We found that expression of the half of the genes is regulated by androgens. Further expression analyses demonstrated that the novel genes are region-specific and developmentally regulated. Computational analysis showed that 15 of the genes lack human orthologues, suggesting their implication in male reproduction unique to the mouse. A number of the novel genes are putative epididymal protease inhibitors or β-defensins. We also found that six of the genes have secretory activity, indicating that they may interact with sperm and have functional roles in sperm maturation. Conclusion We identified and characterized 32 novel epididymis-specific or -predominant genes by an integrative approach. Our study is unique in the aspect of systematic identification of novel epididymal genes and should be a firm basis for future investigation into molecular mechanisms underlying sperm maturation in the epididymis. PMID:17166261

  16. Identification and chromosomal localization of Atm, the mouse homolog of the ataxia-telangiectasia gene

    SciTech Connect

    Pecker, I.; Savitsky, K.; Rotman, G.

    1996-07-01

    Atm, the mouse homolog of the human ATM gene defective in ataxia-telangiectasia (A-T), has been identified. The entire coding sequence of the Atm transcript was cloned and found to contain an open reading frame encoding a protein of 3066 amino acids with 84% overall identity and 91% similarity to the human ATM protein. Variable levels of expression of Atm were observed in different tissues. Fluorescence in situ hybridization and linkage analysis located the Atm gene on mouse chromosome 9, band 9C, in a region homologous to the ATM region on human chromosome 11q22-q23. 32 refs., 6 figs.

  17. Male eyespan size is associated with meiotic drive in wild stalk-eyed flies (Teleopsis dalmanni)

    PubMed Central

    Cotton, A J; Földvári, M; Cotton, S; Pomiankowski, A

    2014-01-01

    This study provides the first direct evidence from wild populations of stalk-eyed flies to support the hypothesis that male eyespan is a signal of meiotic drive. Several stalk-eyed fly species are known to exhibit X-linked meiotic drive. A recent quantitative trait locus analysis in Teleopsis dalmanni found a potential link between variation in male eyespan, a sexually selected ornamental trait, and the presence of meiotic drive. This was based on laboratory populations subject to artificial selection for male eyespan. In this study, we examined the association between microsatellite markers and levels of sex ratio bias (meiotic drive) in 12 wild T. dalmanni populations. We collected two data sets: (a) brood sex ratios of wild-caught males mated to standard laboratory females and (b) variation in a range of phenotypic traits associated with reproductive success of wild-caught males and females. In each case, we typed individuals for eight X-linked microsatellite markers, including several that previously were shown to be associated with male eyespan and meiotic drive. We found that one microsatellite marker was very strongly associated with meiotic drive, whereas a second showed a weaker association. We also found that, using both independent data sets, meiotic drive was strongly associated with male eyespan, with smaller eyespan males being associated with more female-biased broods. These results suggest that mate preference for exaggerated male eyespan allows females to avoid mating with males carrying the meiotic drive gene and is thus a potential mechanism for the maintenance and evolution of female mate preference. PMID:24398884

  18. Male eyespan size is associated with meiotic drive in wild stalk-eyed flies (Teleopsis dalmanni).

    PubMed

    Cotton, A J; Földvári, M; Cotton, S; Pomiankowski, A

    2014-04-01

    This study provides the first direct evidence from wild populations of stalk-eyed flies to support the hypothesis that male eyespan is a signal of meiotic drive. Several stalk-eyed fly species are known to exhibit X-linked meiotic drive. A recent quantitative trait locus analysis in Teleopsis dalmanni found a potential link between variation in male eyespan, a sexually selected ornamental trait, and the presence of meiotic drive. This was based on laboratory populations subject to artificial selection for male eyespan. In this study, we examined the association between microsatellite markers and levels of sex ratio bias (meiotic drive) in 12 wild T. dalmanni populations. We collected two data sets: (a) brood sex ratios of wild-caught males mated to standard laboratory females and (b) variation in a range of phenotypic traits associated with reproductive success of wild-caught males and females. In each case, we typed individuals for eight X-linked microsatellite markers, including several that previously were shown to be associated with male eyespan and meiotic drive. We found that one microsatellite marker was very strongly associated with meiotic drive, whereas a second showed a weaker association. We also found that, using both independent data sets, meiotic drive was strongly associated with male eyespan, with smaller eyespan males being associated with more female-biased broods. These results suggest that mate preference for exaggerated male eyespan allows females to avoid mating with males carrying the meiotic drive gene and is thus a potential mechanism for the maintenance and evolution of female mate preference.

  19. Potential translational targets revealed by linking mouse grooming behavioral phenotypes to gene expression using public databases.

    PubMed

    Roth, Andrew; Kyzar, Evan J; Cachat, Jonathan; Stewart, Adam Michael; Green, Jeremy; Gaikwad, Siddharth; O'Leary, Timothy P; Tabakoff, Boris; Brown, Richard E; Kalueff, Allan V

    2013-01-10

    Rodent self-grooming is an important, evolutionarily conserved behavior, highly sensitive to pharmacological and genetic manipulations. Mice with aberrant grooming phenotypes are currently used to model various human disorders. Therefore, it is critical to understand the biology of grooming behavior, and to assess its translational validity to humans. The present in-silico study used publicly available gene expression and behavioral data obtained from several inbred mouse strains in the open-field, light-dark box, elevated plus- and elevated zero-maze tests. As grooming duration differed between strains, our analysis revealed several candidate genes with significant correlations between gene expression in the brain and grooming duration. The Allen Brain Atlas, STRING, GoMiner and Mouse Genome Informatics databases were used to functionally map and analyze these candidate mouse genes against their human orthologs, assessing the strain ranking of their expression and the regional distribution of expression in the mouse brain. This allowed us to identify an interconnected network of candidate genes (which have expression levels that correlate with grooming behavior), display altered patterns of expression in key brain areas related to grooming, and underlie important functions in the brain. Collectively, our results demonstrate the utility of large-scale, high-throughput data-mining and in-silico modeling for linking genomic and behavioral data, as well as their potential to identify novel neural targets for complex neurobehavioral phenotypes, including grooming.

  20. Manual Gene Ontology annotation workflow at the Mouse Genome Informatics Database.

    PubMed

    Drabkin, Harold J; Blake, Judith A

    2012-01-01

    The Mouse Genome Database, the Gene Expression Database and the Mouse Tumor Biology database are integrated components of the Mouse Genome Informatics (MGI) resource (http://www.informatics.jax.org). The MGI system presents both a consensus view and an experimental view of the knowledge concerning the genetics and genomics of the laboratory mouse. From genotype to phenotype, this information resource integrates information about genes, sequences, maps, expression analyses, alleles, strains and mutant phenotypes. Comparative mammalian data are also presented particularly in regards to the use of the mouse as a model for the investigation of molecular and genetic components of human diseases. These data are collected from literature curation as well as downloads of large datasets (SwissProt, LocusLink, etc.). MGI is one of the founding members of the Gene Ontology (GO) and uses the GO for functional annotation of genes. Here, we discuss the workflow associated with manual GO annotation at MGI, from literature collection to display of the annotations. Peer-reviewed literature is collected mostly from a set of journals available electronically. Selected articles are entered into a master bibliography and indexed to one of eight areas of interest such as 'GO' or 'homology' or 'phenotype'. Each article is then either indexed to a gene already contained in the database or funneled through a separate nomenclature database to add genes. The master bibliography and associated indexing provide information for various curator-reports such as 'papers selected for GO that refer to genes with NO GO annotation'. Once indexed, curators who have expertise in appropriate disciplines enter pertinent information. MGI makes use of several controlled vocabularies that ensure uniform data encoding, enable robust analysis and support the construction of complex queries. These vocabularies range from pick-lists to structured vocabularies such as the GO. All data associations are supported

  1. A cross-species genetic analysis identifies candidate genes for mouse anxiety and human bipolar disorder

    PubMed Central

    Ashbrook, David G.; Williams, Robert W.; Lu, Lu; Hager, Reinmar

    2015-01-01

    Bipolar disorder (BD) is a significant neuropsychiatric disorder with a lifetime prevalence of ~1%. To identify genetic variants underlying BD genome-wide association studies (GWAS) have been carried out. While many variants of small effect associated with BD have been identified few have yet been confirmed, partly because of the low power of GWAS due to multiple comparisons being made. Complementary mapping studies using murine models have identified genetic variants for behavioral traits linked to BD, often with high power, but these identified regions often contain too many genes for clear identification of candidate genes. In the current study we have aligned human BD GWAS results and mouse linkage studies to help define and evaluate candidate genes linked to BD, seeking to use the power of the mouse mapping with the precision of GWAS. We use quantitative trait mapping for open field test and elevated zero maze data in the largest mammalian model system, the BXD recombinant inbred mouse population, to identify genomic regions associated with these BD-like phenotypes. We then investigate these regions in whole genome data from the Psychiatric Genomics Consortium's bipolar disorder GWAS to identify candidate genes associated with BD. Finally we establish the biological relevance and pathways of these genes in a comprehensive systems genetics analysis. We identify four genes associated with both mouse anxiety and human BD. While TNR is a novel candidate for BD, we can confirm previously suggested associations with CMYA5, MCTP1, and RXRG. A cross-species, systems genetics analysis shows that MCTP1, RXRG, and TNR coexpress with genes linked to psychiatric disorders and identify the striatum as a potential site of action. CMYA5, MCTP1, RXRG, and TNR are associated with mouse anxiety and human BD. We hypothesize that MCTP1, RXRG, and TNR influence intercellular signaling in the striatum. PMID:26190982

  2. Assessing fluctuating evolutionary pressure in yeast and mammal evolutionary rate covariation using bioinformatics of meiotic protein genetic sequences

    NASA Astrophysics Data System (ADS)

    Dehipawala, Sunil; Nguyen, A.; Tremberger, G.; Cheung, E.; Holden, T.; Lieberman, D.; Cheung, T.

    2013-09-01

    The evolutionary rate co-variation in meiotic proteins has been reported for yeast and mammal using phylogenic branch lengths which assess retention, duplication and mutation. The bioinformatics of the corresponding DNA sequences could be classified as a diagram of fractal dimension and Shannon entropy. Results from biomedical gene research provide examples on the diagram methodology. The identification of adaptive selection using entropy marker and functional-structural diversity using fractal dimension would support a regression analysis where the coefficient of determination would serve as evolutionary pathway marker for DNA sequences and be an important component in the astrobiology community. Comparisons between biomedical genes such as EEF2 (elongation factor 2 human, mouse, etc), WDR85 in epigenetics, HAR1 in human specificity, clinical trial targeted cancer gene CD47, SIRT6 in spermatogenesis, and HLA-C in mosquito bite immunology demonstrate the diagram classification methodology. Comparisons to the SEPT4-XIAP pair in stem cell apoptosis, testesexpressed taste genes TAS1R3-GNAT3 pair, and amyloid beta APLP1-APLP2 pair with the yeast-mammal DNA sequences for meiotic proteins RAD50-MRE11 pair and NCAPD2-ICK pair have accounted for the observed fluctuating evolutionary pressure systematically. Regression with high R-sq values or a triangular-like cluster pattern for concordant pairs in co-variation among the studied species could serve as evidences for the possible location of common ancestors in the entropy-fractal dimension diagram, consistent with an example of the human-chimp common ancestor study using the FOXP2 regulated genes reported in human fetal brain study. The Deinococcus radiodurans R1 Rad-A could be viewed as an outlier in the RAD50 diagram and also in the free energy versus fractal dimension regression Cook's distance, consistent with a non-Earth source for this radiation resistant bacterium. Convergent and divergent fluctuating evolutionary

  3. Transposon Tagging of a Male-Sterility, Female-Sterility Gene, St8, Revealed that the Meiotic MER3 DNA Helicase Activity Is Essential for Fertility in Soybean

    PubMed Central

    Baumbach, Jordan; Pudake, Ramesh N.; Johnson, Callie; Kleinhans, Kaylin; Ollhoff, Alexandrea; Palmer, Reid G.; Bhattacharyya, Madan K.; Sandhu, Devinder

    2016-01-01

    The W4 locus in soybean encodes a dihydroflavonol-4-reductase (DFR2) that regulates pigmentation patterns in flowers and hypocotyl. The mutable w4-m allele that governs variegated flowers has arisen through insertion of a CACTA-type transposable element, Tgm9, in DFR2. In the w4-m line, reversion from variegated to purple flower indicates excision of Tgm9, and its insertion at a new locus. Previously, we have identified a male-sterile, female-sterile mutant among the selfed progenies of a revertant plant carrying only purple flowers. Co-segregation between Tgm9 and the sterility phenotype suggested that the mutant was generated by insertion of Tgm9 at the St8 locus. The transposon was localized to exon 10 of Glyma.16G072300 that shows high identity to the MER3 DNA helicase involved in crossing over. Molecular analysis of fertile branches from two independent revertant plants confirmed precise excision of Tgm9 from the st8 allele, which restored fertility. In soybean, the gene is expressed in flower-buds, trifoliate leaves and stem. Phylogenetic analysis placed St8 in a clade with the Arabidopsis and rice MER3 suggesting that St8 is most likely the orthologous MER3 soybean gene. This study established the utility of Tgm9 in gene identification as well as in forward and reverse genetics studies. PMID:26930200

  4. Transposon Tagging of a Male-Sterility, Female-Sterility Gene, St8, Revealed that the Meiotic MER3 DNA Helicase Activity Is Essential for Fertility in Soybean.

    PubMed

    Baumbach, Jordan; Pudake, Ramesh N; Johnson, Callie; Kleinhans, Kaylin; Ollhoff, Alexandrea; Palmer, Reid G; Bhattacharyya, Madan K; Sandhu, Devinder

    2016-01-01

    The W4 locus in soybean encodes a dihydroflavonol-4-reductase (DFR2) that regulates pigmentation patterns in flowers and hypocotyl. The mutable w4-m allele that governs variegated flowers has arisen through insertion of a CACTA-type transposable element, Tgm9, in DFR2. In the w4-m line, reversion from variegated to purple flower indicates excision of Tgm9, and its insertion at a new locus. Previously, we have identified a male-sterile, female-sterile mutant among the selfed progenies of a revertant plant carrying only purple flowers. Co-segregation between Tgm9 and the sterility phenotype suggested that the mutant was generated by insertion of Tgm9 at the St8 locus. The transposon was localized to exon 10 of Glyma.16G072300 that shows high identity to the MER3 DNA helicase involved in crossing over. Molecular analysis of fertile branches from two independent revertant plants confirmed precise excision of Tgm9 from the st8 allele, which restored fertility. In soybean, the gene is expressed in flower-buds, trifoliate leaves and stem. Phylogenetic analysis placed St8 in a clade with the Arabidopsis and rice MER3 suggesting that St8 is most likely the orthologous MER3 soybean gene. This study established the utility of Tgm9 in gene identification as well as in forward and reverse genetics studies. PMID:26930200

  5. Exploring novel candidate genes from the Mouse Genome Informatics database: Potential implications for avian migration research.

    PubMed

    Contina, Andrea; Bridge, Eli S; Kelly, Jeffrey F

    2016-07-01

    To search for genes associated with migratory phenotypes in songbirds, we selected candidate genes through annotations from the Mouse Genome Informatics database and assembled an extensive candidate-gene library. Then, we implemented a next-generation sequencing approach to obtain DNA sequences from the Painted Bunting genome. We focused on those sequences that were conserved across avian species and that aligned with candidate genes in our mouse library. We genotyped short sequence repeats from the following candidate genes: ADRA1d, ANKRD17, CISH and MYH7. We studied the possible correlations between allelic variations occurring in these novel candidate migration genes and avian migratory phenotypes available from the published literature. We found that allele variation at MYH7 correlated with a calculated index of speed of migration (km/day) across 11 species of songbirds. We highlight the potential of the Mouse Genome Informatics database in providing new candidate genes that might play a crucial role in regulating migration in birds and possibly in other taxa. Our research effort shows the benefits and limitations of working with extensive genomic datasets and offers a snapshot of the challenges related to cross-species validation in behavioral and molecular ecology studies.

  6. [Cloning and identification of a mouse zinc finger protein gene ZF-12-related pseudogene].

    PubMed

    Li, Jian Zhong; Zhang, Ya Zhou; Wang, Shui Liang; Yang, Hua; Li, Jian; Yu, Long; Fu, Ji Liang

    2002-06-01

    The mouse zinc finger protein ZF-12 gene is homologous to human gene and encodes a protein of 368 amino acids, which contains four tandem C2H2-type zinc finger motifs in the N-terminal and one SCAN domain in the C-terminal. Some recent studies suggest that ZNF191 might be a hepatocarcinogenesis-associated gene. We screened a mouse lambda genomic library with a human ZNF191 cDNA probe and isolated a ZF-12-like gene, named ZF12p (GenBank AY040222). This intronless gene closely resembles ZF-12 but displays several mutations, suggesting that ZF12p represents a ZF-12-related pseudogene. RT-PCR analysis on total RNA from mouse tissue and bioinformatis analysis on promoter region of ZF12p gene, suggest the transcripts of ZF12p may be not synthesized. BLAST on the data of the human genome in the GenBank with ZNF191 cDNA and Southern blotting show there is no any psedogene related to ZNF191 gene in the human genome. The high similarity of ZF12p to ZF-12 might be of considerable importance for mutation and evolution analysis of ZF-12.

  7. Exploring novel candidate genes from the Mouse Genome Informatics database: Potential implications for avian migration research.

    PubMed

    Contina, Andrea; Bridge, Eli S; Kelly, Jeffrey F

    2016-07-01

    To search for genes associated with migratory phenotypes in songbirds, we selected candidate genes through annotations from the Mouse Genome Informatics database and assembled an extensive candidate-gene library. Then, we implemented a next-generation sequencing approach to obtain DNA sequences from the Painted Bunting genome. We focused on those sequences that were conserved across avian species and that aligned with candidate genes in our mouse library. We genotyped short sequence repeats from the following candidate genes: ADRA1d, ANKRD17, CISH and MYH7. We studied the possible correlations between allelic variations occurring in these novel candidate migration genes and avian migratory phenotypes available from the published literature. We found that allele variation at MYH7 correlated with a calculated index of speed of migration (km/day) across 11 species of songbirds. We highlight the potential of the Mouse Genome Informatics database in providing new candidate genes that might play a crucial role in regulating migration in birds and possibly in other taxa. Our research effort shows the benefits and limitations of working with extensive genomic datasets and offers a snapshot of the challenges related to cross-species validation in behavioral and molecular ecology studies. PMID:27061206

  8. Five RecA-like proteins of Schizosaccharomyces pombe are involved in meiotic recombination.

    PubMed Central

    Grishchuk, A L; Kohli, J

    2003-01-01

    The genome of Schizosaccharomyces pombe contains five genes that code for proteins with sequence similarity to the Escherichia coli recombination protein RecA: rad51+, rhp55+, rhp57+, rlp1+, and dmc1+. We analyzed the effect of deletion of each of these genes on meiotic recombination and viability of spores. Meiotic recombination levels were different from wild type in all recA-related mutants in several genetic intervals, suggesting that all five RecA homologs of S. pombe are required for normal levels of meiotic recombination. Spore viability was reduced in rad51, rhp55, and rhp57 mutants, but not in rlp1 and dmc1. It is argued that reduction of crossover is not the only cause for the observed reduction of spore viability. Analysis of double and triple mutants revealed that Rad51 and Dmc1 play major and partially overlapping roles in meiotic recombination, while Rhp55, Rhp57, and Rlp1 play accessory roles. Remarkably, deletion of Rlp1 decreases the frequency of intergenic recombination (crossovers), but increases intragenic recombination (gene conversion). On the basis of our results, we present a model for the involvement of five RecA-like proteins of S. pombe in meiotic recombination and discuss their respective roles. PMID:14668362

  9. Characterization and chromosomal mapping of a mouse ortholog of the late-infantile ceroid-lipofuscinosis gene CLN2.

    PubMed

    Katz, M L; Liu, P C; Grob-Nunn, S E; Shibuya, H; Johnson, G S

    1999-11-01

    Late-infantile ceroid-lipofuscinosis (CLN2) is an autosomal recessively inherited, neurodegenerative disease in humans. The CLN2 locus has been mapped to Chromosome (Chr) 11p15, and its sequence and genomic organization have recently been reported. In the present study, the cDNA sequence, exon/intron organization, and chromosomal localization of a mouse ortholog of the CLN2 gene are described. The mouse cDNA contains an open reading frame that predicts a protein product of 562 amino acids. The mouse and human coding regions are 86% and 88% identical at the nucleic acid and amino acid levels, respectively. One less codon appears in the mouse cDNA when compared with the human ortholog. The mouse gene (Cln2) spans more than 6 kb and consists of 13 exons separated by introns ranging in size from 111 to 1259 bp. Length polymorphism in an (AC)(n) microsatellite in intron 3 of the mouse Cln2 gene was used to perform segregation analysis with The Jackson Laboratory DNA Panel Mapping Resource. On the basis of this analysis, the Cln2 gene was localized to a region of mouse Chr 7 that corresponds to human Chr 11p15. Characterization of the mouse Cln2 gene will facilitate generation of a mouse model for late-infantile ceroid-lipofuscinosis by gene targeting and identification of functionally important regions of the Cln2 protein.

  10. Unresolved issues in pre-meiotic anther development.

    PubMed

    Kelliher, Timothy; Egger, Rachel L; Zhang, Han; Walbot, Virginia

    2014-01-01

    Compared to the diversity of other floral organs, the steps in anther ontogeny, final cell types, and overall organ shape are remarkably conserved among Angiosperms. Defects in pre-meiotic anthers that alter cellular composition or function typically result in male-sterility. Given the ease of identifying male-sterile mutants, dozens of genes with key roles in early anther development have been identified and cloned in model species, ordered by time of action and spatiotemporal expression, and used to propose explanatory models for critical steps in cell fate specification. Despite rapid progress, fundamental issues in anther development remain unresolved, and it is unclear if insights from one species can be applied to others. Here we construct a comparison of Arabidopsis, rice, and maize immature anthers to pinpoint distinctions in developmental pace. We analyze the mechanisms by which archesporial (pre-meiotic) cells are specified distinct from the soma, discuss what constitutes meiotic preparation, and review what is known about the secondary parietal layer and its terminal periclinal division that generates the tapetal and middle layers. Finally, roles for small RNAs are examined, focusing on the grass-specific phasiRNAs. PMID:25101101

  11. Analysis of mammalian gene function through broad based phenotypic screens across a consortium of mouse clinics

    PubMed Central

    Adams, David J; Adams, Niels C; Adler, Thure; Aguilar-Pimentel, Antonio; Ali-Hadji, Dalila; Amann, Gregory; André, Philippe; Atkins, Sarah; Auburtin, Aurelie; Ayadi, Abdel; Becker, Julien; Becker, Lore; Bedu, Elodie; Bekeredjian, Raffi; Birling, Marie-Christine; Blake, Andrew; Bottomley, Joanna; Bowl, Mike; Brault, Véronique; Busch, Dirk H; Bussell, James N; Calzada-Wack, Julia; Cater, Heather; Champy, Marie-France; Charles, Philippe; Chevalier, Claire; Chiani, Francesco; Codner, Gemma F; Combe, Roy; Cox, Roger; Dalloneau, Emilie; Dierich, André; Di Fenza, Armida; Doe, Brendan; Duchon, Arnaud; Eickelberg, Oliver; Esapa, Chris T; El Fertak, Lahcen; Feigel, Tanja; Emelyanova, Irina; Estabel, Jeanne; Favor, Jack; Flenniken, Ann; Gambadoro, Alessia; Garrett, Lilian; Gates, Hilary; Gerdin, Anna-Karin; Gkoutos, George; Greenaway, Simon; Glasl, Lisa; Goetz, Patrice; Da Cruz, Isabelle Goncalves; Götz, Alexander; Graw, Jochen; Guimond, Alain; Hans, Wolfgang; Hicks, Geoff; Hölter, Sabine M; Höfler, Heinz; Hancock, John M; Hoehndorf, Robert; Hough, Tertius; Houghton, Richard; Hurt, Anja; Ivandic, Boris; Jacobs, Hughes; Jacquot, Sylvie; Jones, Nora; Karp, Natasha A; Katus, Hugo A; Kitchen, Sharon; Klein-Rodewald, Tanja; Klingenspor, Martin; Klopstock, Thomas; Lalanne, Valerie; Leblanc, Sophie; Lengger, Christoph; le Marchand, Elise; Ludwig, Tonia; Lux, Aline; McKerlie, Colin; Maier, Holger; Mandel, Jean-Louis; Marschall, Susan; Mark, Manuel; Melvin, David G; Meziane, Hamid; Micklich, Kateryna; Mittelhauser, Christophe; Monassier, Laurent; Moulaert, David; Muller, Stéphanie; Naton, Beatrix; Neff, Frauke; Nolan, Patrick M; Nutter, Lauryl MJ; Ollert, Markus; Pavlovic, Guillaume; Pellegata, Natalia S; Peter, Emilie; Petit-Demoulière, Benoit; Pickard, Amanda; Podrini, Christine; Potter, Paul; Pouilly, Laurent; Puk, Oliver; Richardson, David; Rousseau, Stephane; Quintanilla-Fend, Leticia; Quwailid, Mohamed M; Racz, Ildiko; Rathkolb, Birgit; Riet, Fabrice; Rossant, Janet; Roux, Michel; Rozman, Jan; Ryder, Ed; Salisbury, Jennifer; Santos, Luis; Schäble, Karl-Heinz; Schiller, Evelyn; Schrewe, Anja; Schulz, Holger; Steinkamp, Ralf; Simon, Michelle; Stewart, Michelle; Stöger, Claudia; Stöger, Tobias; Sun, Minxuan; Sunter, David; Teboul, Lydia; Tilly, Isabelle; Tocchini-Valentini, Glauco P; Tost, Monica; Treise, Irina; Vasseur, Laurent; Velot, Emilie; Vogt-Weisenhorn, Daniela; Wagner, Christelle; Walling, Alison; Weber, Bruno; Wendling, Olivia; Westerberg, Henrik; Willershäuser, Monja; Wolf, Eckhard; Wolter, Anne; Wood, Joe; Wurst, Wolfgang; Yildirim, Ali Önder; Zeh, Ramona; Zimmer, Andreas; Zimprich, Annemarie

    2015-01-01

    The function of the majority of genes in the mouse and human genomes remains unknown. The mouse ES cell knockout resource provides a basis for characterisation of relationships between gene and phenotype. The EUMODIC consortium developed and validated robust methodologies for broad-based phenotyping of knockouts through a pipeline comprising 20 disease-orientated platforms. We developed novel statistical methods for pipeline design and data analysis aimed at detecting reproducible phenotypes with high power. We acquired phenotype data from 449 mutant alleles, representing 320 unique genes, of which half had no prior functional annotation. We captured data from over 27,000 mice finding that 83% of the mutant lines are phenodeviant, with 65% demonstrating pleiotropy. Surprisingly, we found significant differences in phenotype annotation according to zygosity. Novel phenotypes were uncovered for many genes with unknown function providing a powerful basis for hypothesis generation and further investigation in diverse systems. PMID:26214591

  12. EMAGE: a spatial database of gene expression patterns during mouse embryo development

    PubMed Central

    Christiansen, Jeffrey H.; Yang, Yiya; Venkataraman, Shanmugasundaram; Richardson, Lorna; Stevenson, Peter; Burton, Nicholas; Baldock, Richard A.; Davidson, Duncan R.

    2006-01-01

    EMAGE () is a freely available, curated database of gene expression patterns generated by in situ techniques in the developing mouse embryo. It is unique in that it contains standardized spatial representations of the sites of gene expression for each gene, denoted against a set of virtual reference embryo models. As such, the data can be interrogated in a novel and abstract manner by using space to define a query. Accompanying the spatial representations of gene expression patterns are text descriptions of the sites of expression, which also allows searching of the data by more conventional text-based methods. PMID:16381949

  13. A novel non-mouse mammary tumor virus activation of the Int-3 gene in a spontaneous mouse mammary tumor.

    PubMed Central

    Kordon, E C; Smith, G H; Callahan, R; Gallahan, D

    1995-01-01

    In a mouse mammary tumor model system in which carcinogenic progression can be investigated, we have found a unique mutation of Int-3 associated with progression from premalignant lobular hyperplasia to tumor. Sequence analysis of the rearranged fragment revealed an insertion of an intracisternal type A particle (IAP) within the Int-3 gene. Int-3 is mutated frequently in mouse mammary tumor virus (MMTV)-induced mammary tumors by insertion of MMTV proviral DNA into this intragenic region. In these mutations, the insertion produces a chimeric Int-3 transcript encoding the cytoplasmic portion of the Int-3 protein driven by the MMTV long terminal repeat promoter. In this case, the IAP DNA was inserted in the opposite transcriptional orientation relative to Int-3; nevertheless, a similar chimeric RNA transcript driven by a cryptic promoter in the oppositely oriented 5' IAP long terminal repeat was generated. This is the first demonstration that an insertional mutation unrelated to MMTV activates an Int gene commonly associated with mammary tumorigenesis. PMID:7494323

  14. Evidence for meiotic drive at the myotonic dystrophy locus

    SciTech Connect

    Shaw, A.M.; Barnetson, R.A.; Phillips, M.F.

    1994-09-01

    Myotonic dystrophy (DM), an autosomal dominant disorder, is the most common form of adult muscular dystrophy, affecting at least 1 in 8000 of the population. It is a multisystemic disorder, primarily characterized by myotonia, muscle wasting and cataract. The molecular basis of DM is an expanded CTG repeat located within the 3{prime} untranslated region of a putative serine-threonine protein kinase on chromosome 19q13.3. DM exhibits anticipation, that is, with successive generations there is increasing disease severity and earlier age of onset. This mechanism and the fact that the origin of the disease has been attributed to one or a small number of founder chromosomes suggests that, in time, DM should die out. Meiotic drive has been described as a way in which certain alleles are transmitted to succeeding generations in preference to others: preferential transmission of large CTG alleles may account for their continued existence in the gene pool. There is evidence that a CTG allele with > 19 repeats may gradually increase in repeat number over many generations until it is sufficiently large to give a DM phenotype. We report a study of 495 transmissions from individuals heterozygous for the CTG repeat and with repeat numbers within the normal range (5-30). Alleles were simply classified as large or small relative to the other allele in an individual. Of 242 male meioses, 126 transmissions from parent to child were of the larger allele to their offspring (57.7%, p=0.014). This shows that there is strong evidence for meiotic drive favoring the transmission of the larger DM allele in unaffected individuals. Contrary to a previous report of meiotic drive in the male, we have shown that females preferentially transmit the larger DM allele. Taken together, the data suggest the occurrence of meiotic drive in both males and females in this locus.

  15. Characterization of the genomic structure of the mouse APLP1 gene

    SciTech Connect

    Zhong, Sue; Wu, Kuo; Black, I.B.; Schaar, D.G.

    1996-02-15

    This article reports on the organization of the mouse APLP1 gene, an evolutionarily conserved amyloid precursor-like protein. The amyloid beta protein, important in Alzheimer diseases, is derived from these precursor proteins. By investigating the expression and structure of this murine gene, it is hoped that more will be learned about the function and regulation of the human homologue. 27 refs., 2 figs.

  16. Molecular cloning of the mouse CCK gene: expression in different brain regions and during cortical development.

    PubMed Central

    Vitale, M; Vashishtha, A; Linzer, E; Powell, D J; Friedman, J M

    1991-01-01

    In this paper we describe experiments that address specific issues concerning the regulation of the mouse cholecystokinin gene in brain and intestine. The mouse cholecystokinin gene was cloned and sequenced. Extensive homology among the mouse, man and rat genes was noted particularly in the three exons and the regions upstream of the RNA start site. RNAse protection assays for each of the three exons were used to demonstrate that CCK is expressed in only a subset of tissues and that the same cap site and splice choices are used in brain, intestine as well as in cerebellum, cortex, midbrain, hypothalamus and hippocampus. CCK RNA was also noted to be detectable in kidney. Thus the same gene using the same promoter is expressed in subsets of cells that differ in their biochemical, morphologic and functional characteristics. The level of expression of CCK was also monitored during mouse cortical development and the appearance of CCK RNA was compared to glutamate decarboxylase (GAD), enkephalin and somatostatin. It was noted that each of these cortical markers was first expressed at different times during cortical development. The appearance of CCK RNA during intestinal development was also measured and found to precede appearance in cortex by several days. Images PMID:2011497

  17. Chromosomal localization and genomic characterization of the mouse melastatin gene (Mlsn1).

    PubMed

    Hunter, J J; Shao, J; Smutko, J S; Dussault, B J; Nagle, D L; Woolf, E A; Holmgren, L M; Moore, K J; Shyjan, A W

    1998-11-15

    We recently described a novel gene, melastatin, whose expression is inversely correlated with melanoma aggressiveness. Chromosomal localization of this gene places it on mouse chromosome 7 and in the 15q13-q14 region of the human genome. Although expression patterns and chromosomal localization in the mouse are consistent with involvement of melastatin mutations in the mouse ruby-eye-2 defect, congenic analysis showed genetic segregation of the two loci. Cloning of the full-length human cDNA revealed a much larger transcript than we had previously identified, corresponding to a 1533-amino-acid protein product with homology to members of the transient receptor potential (Trp) family of calcium channels. The mouse melastatin gene contains 27 exons and spans at least 58 kb of genomic DNA. The promoter region of Mlsn1 contains four potential microphthalmia binding sites including an M box, a transcriptional regulatory element unique to genes with a restricted melanocytic expression pattern. A 1-kb PvuII fragment from this region was capable of driving high levels of luciferase expression in B16 melanoma cells. PMID:9806836

  18. Forging Links between Human Mental Retardation–Associated CNVs and Mouse Gene Knockout Models

    PubMed Central

    Webber, Caleb; Hehir-Kwa, Jayne Y.; Nguyen, Duc-Quang; de Vries, Bert B. A.; Veltman, Joris A.; Ponting, Chris P.

    2009-01-01

    Rare copy number variants (CNVs) are frequently associated with common neurological disorders such as mental retardation (MR; learning disability), autism, and schizophrenia. CNV screening in clinical practice is limited because pathological CNVs cannot be distinguished routinely from benign CNVs, and because genes underlying patients' phenotypes remain largely unknown. Here, we present a novel, statistically robust approach that forges links between 148 MR–associated CNVs and phenotypes from ∼5,000 mouse gene knockout experiments. These CNVs were found to be significantly enriched in two classes of genes, those whose mouse orthologues, when disrupted, result in either abnormal axon or dopaminergic neuron morphologies. Additional enrichments highlighted correspondences between relevant mouse phenotypes and secondary presentations such as brain abnormality, cleft palate, and seizures. The strength of these phenotype enrichments (>100% increases) greatly exceeded molecular annotations (<30% increases) and allowed the identification of 78 genes that may contribute to MR and associated phenotypes. This study is the first to demonstrate how the power of mouse knockout data can be systematically exploited to better understand genetically heterogeneous neurological disorders. PMID:19557186

  19. Characterization of the mouse TFF1 (pS2) gene promoter region.

    PubMed

    Terada, T; Sakagami, R; Tabuchi, Y; Maeda, M

    2001-02-01

    Trefoil peptides (TFFs) with a unique trefoil domain(s) are presumed to function in protection and repair of the gastrointestinal epithelial layer. Three peptide family members are differently distributed in the mouse gastrointestinal tract: TFF1/pS2 specifically in stomach, TFF2/SP mainly in stomach, pancreas and duodenum, and TFF3/ITF in intestine. We cloned and sequenced the mouse TFF1 gene 5'-upstream region by means of the genomic walking procedure. The cloned region was ligated to the luciferase reporter gene and then introduced into mouse gastric surface mucous GSM10 cells which express TFF1 and TFF2. The minimum promoter was located in the region containing the TATA-box between -39 and the transcriptional start site. Further upstream regions stimulated (-2192-- -1630bp, -641-- -243bp, -137-- -39bp) and inhibited (-1630-- -641bp, -243-- -137 bp) luciferase gene expression. These regions as well as short segments conserved in the mouse and human 5'-upstream sequences may be important for modulation of the mRNA level of the TFF1 gene.

  20. The mouse Gene Expression Database (GXD): new features and how to use them effectively

    PubMed Central

    Finger, Jacqueline H.; Smith, Constance M.; Hayamizu, Terry F.; McCright, Ingeborg J.; Xu, Jingxia; Eppig, Janan T.; Kadin, James A.; Richardson, Joel E.; Ringwald, Martin

    2015-01-01

    The Gene Expression Database (GXD) is an extensive and freely available community resource of mouse developmental expression data. GXD curates and integrates expression data from the literature, via electronic data submissions, and by collaborations with large-scale projects. As an integral component of the Mouse Genome Informatics (MGI) Resource, GXD combines expression data with genetic, functional, phenotypic and disease-related data, and provides tools for the research community to search for and analyze expression data in this larger context. Recent enhancements include: an interactive browser to navigate the mouse developmental anatomy and find expression data for specific anatomical structures; the capability to search for expression data of genes located in specific genomic regions, supporting the identification of disease candidate genes; a summary displaying all the expression images that meet specified search criteria; interactive matrix views that provide overviews of spatio-temporal expression patterns (Tissue × Stage Matrix) and enable the comparison of expression patterns between genes (Tissue × Gene Matrix); data zoom and filter utilities to iteratively refine summary displays and data sets; and gene-based links to expression data from other model organisms, such as chicken, Xenopus and zebrafish, fostering comparative expression analysis for species that are highly relevant for developmental research. PMID:26045019

  1. Chromosome localizations of genes for five cAMP-specific phosphodiesterases in man and mouse

    SciTech Connect

    Milatovich, A.; Francke, U. ); Bolger, G.; Michaeli, T. )

    1994-03-01

    Cyclic nucleotides are important second messengers that mediate a number of cellular responses to external signals. Cyclic nucleotide phosphodiesterases play a role in signal transduction by regulating the cellular concentrations of these messengers. Here, the authors have applied Southern analyses of somatic cell hybrid lines and of recombinant inbred (RI) mouse strains as well as fluorescence chromosomal in situ hybridization (FISH) to chromosomally localize five cAMP-specific nucleotide phosphodiesterase genes in human and mouse. Genes DPDE1, DPDE2, DPDE3, and DPDE4 that share sequence homology with the Drosophila dunce gene were assigned to human chromosomes 19 (DPDE1 and DPDE2), ga12 (DPDE3), and 1p31 (DPDE4) and to mouse chromosomes 8, 9, 13, and 4, respectively. The high-affinity cAMP-specific phosphodiesterase gene (HCP1) was mapped to human chromosome 8q13-q22. Since these genes are potential candidates for involvement in psychiatric or behavioral disorders, knowledge of their chromosomal localizations will facilitate the discovery of their association with disease genes as they are being mapped by linkage studies.

  2. Automatic Summarization of Mouse Gene Information by Clustering and Sentence Extraction from MEDLINE Abstracts

    PubMed Central

    Yang, Jianji; Cohen, Aaron M.; Hersh, William

    2007-01-01

    Tools to automatically summarize gene information from the literature have the potential to help genomics researchers better interpret gene expression data and investigate biological pathways. The task of finding information on sets of genes is common for genomic researchers, and PubMed is still the first choice because the most recent and original information can only be found in the unstructured, free text biomedical literature. However, finding information on a set of genes by manually searching and scanning the literature is a time-consuming and daunting task for scientists. We built and evaluated a query-based automatic summarizer of information on mouse genes studied in microarray experiments. The system clusters a set of genes by MeSH, GO and free text features and presents summaries for each gene by ranked sentences extracted from MEDLINE abstracts. Evaluation showed that the system seems to provide meaningful clusters and informative sentences are ranked higher by the algorithm. PMID:18693953

  3. Induction of cell cycle progression by hepatitis B virus HBx gene expression in quiescent mouse fibroblasts.

    PubMed Central

    Koike, K; Moriya, K; Yotsuyanagi, H; Iino, S; Kurokawa, K

    1994-01-01

    The HBx gene of hepatitis B virus has been shown to induce hepatic tumors in transgenic mice and is implicated in hepatocarcinogenesis in human hepatitis B virus infection. To further characterize the role of HBx gene in carcinogenesis, we established mouse fibroblast cell lines in which the expression of HBx gene could be controlled by glucocorticoid hormone and examined the effect of HBx gene expression on cell growth in vitro. Along with the expression of HBx gene, most cells in the G0/G1 phase moved into the S phase in 24 h, and the cell cycle progressed further toward 48 h. Induction of DNA synthesis was also demonstrated by bromo-deoxyuridine labeling analysis. These results indicate that HBx gene has a function to trigger the synthesis of cellular DNA and suggest that HBx gene may play a role in hepatocarcinogenesis in human infection by driving deregulated cell cycle progression. Images PMID:8040286

  4. Apoptosis-related genes change their expression with age and hearing loss in the mouse cochlea.

    PubMed

    Tadros, Sherif F; D'Souza, Mary; Zhu, Xiaoxia; Frisina, Robert D

    2008-11-01

    To understand possible causative roles of apoptosis gene regulation in age-related hearing loss (presbycusis), apoptotic gene expression patterns in the CBA mouse cochlea of four different age and hearing loss groups were compared, using GeneChip and real-time (qPCR) microarrays. GeneChip transcriptional expression patterns of 318 apoptosis-related genes were analyzed. Thirty eight probes (35 genes) showed significant differences in expression. The significant gene families include Caspases, B-cell leukemia/lymphoma2 family, P53, Calpains, Mitogen activated protein kinase family, Jun oncogene, Nuclear factor of kappa light chain gene enhancer in B-cells inhibitor-related and tumor necrosis factor-related genes. The GeneChip results of 31 genes were validated using the new TaqMan Low Density Array (TLDA). Eight genes showed highly correlated results with the GeneChip data. These genes are: activating transcription factor3, B-cell leukemia/lymphoma2, Bcl2-like1, caspase4 apoptosis-related cysteine protease 4, Calpain2, dual specificity phosphatase9, tumor necrosis factor receptor superfamily member12a, and Tumor necrosis factor superfamily member13b, suggesting they may play critical roles in inner ear aging.

  5. Structural characterization and chromosomal location of the mouse macrophage migration inhibitory factor gene and pseudogenes

    SciTech Connect

    Bozza, M.; Gerard, C.; Kolakowski, L.F. Jr.

    1995-06-10

    Macrophage migration inhibitory factor, MIF, is a cytokine released by T-lymphocytes, macrophages, and the pituitary gland that serves to integrate peripheral and central inflammatory responses. Ubiquitous expression and developmental regulation suggest that MIF may have additional roles outside of the immune system. Here we report the structure and chromosomal location of the mouse Mif gene and the partial characterization of five Mif pseudogenes. The mouse Mif gene spans less than 0.7 kb of chromosomal DNA and is composed of three exons. A comparison between the mouse and the human genes shows a similar gene structure and common regulatory elements in both promoter regions. The mouse Mif gene maps to the middle region of chromosome 10, between Bcr and S100b, which have been mapped to human chromosomes 22q11 and 21q22.3, respectively. The entire sequence of two pseudogenes demonstrates the absence of introns, the presence of the 5{prime} untranslated region of the cDNA, a 3{prime} poly(A) tail, and the lack of sequence similarity with untranscribed regions of the gene. The five pseudogenes are highly homologous to the cDNA, but contain a variable number of mutations that would produce mutated or truncated MIF-like proteins. Phylogenetic analyses of MIF genes and pseudogenes indicate several independent genetic events that can account for multiple genomic integrations. Three of the Mif pseudogenes were also mapped by interspecific backcross to chromosomes 1, 9, and 17. These results suggest that Mif pseudogenes originated by retrotransposition. 46 refs., 5 figs., 1 tab.

  6. Combination of mouse models and genomewide association studies highlights novel genes associated with human kidney function.

    PubMed

    Jing, Jiaojiao; Pattaro, Cristian; Hoppmann, Anselm; Okada, Yukinori; Fox, Caroline S; Köttgen, Anna

    2016-10-01

    Genomewide association studies have identified numerous chronic kidney disease-associated genetic variants, but often do not pinpoint causal genes. This limitation was addressed by combining Mouse Genome Informatics with human genomewide association studies of kidney function. Genes for which mouse models showed abnormal renal physiology, morphology, glomerular filtration rate (GFR), or urinary albumin-to-creatinine ratio were identified from Mouse Genome Informatics. The corresponding human orthologs were then evaluated for GFR-associated single-nucleotide polymorphisms in 133,814 individuals and urinary albumin-to-creatinine ratio-associated SNPs in 54,451 individuals in genome-wide association studies meta-analysis of the CKDGen Consortium. After multiple testing corrections, significant associations with estimated GFR in humans were identified for single-nucleotide polymorphisms in 2, 7, and 17 genes causing abnormal GFR, abnormal physiology, and abnormal morphology in mice, respectively. Genes identified for abnormal kidney morphology showed significant enrichment for estimated GFR-associated single-nucleotide polymorphisms. In total, 19 genes contained variants associated with estimated GFR or the urinary albumin-to-creatinine ratio of which 16 mapped into previously reported genomewide significant loci. CYP26A1 and BMP4 emerged as novel signals subsequently validated in a large, independent study. An additional gene, CYP24A1, was discovered after conditioning on a published nearby association signal. Thus, our novel approach to combine comprehensive mouse phenotype information with human genomewide association studies data resulted in the identification of candidate genes for kidney disease pathogenesis.

  7. Combination of mouse models and genomewide association studies highlights novel genes associated with human kidney function.

    PubMed

    Jing, Jiaojiao; Pattaro, Cristian; Hoppmann, Anselm; Okada, Yukinori; Fox, Caroline S; Köttgen, Anna

    2016-10-01

    Genomewide association studies have identified numerous chronic kidney disease-associated genetic variants, but often do not pinpoint causal genes. This limitation was addressed by combining Mouse Genome Informatics with human genomewide association studies of kidney function. Genes for which mouse models showed abnormal renal physiology, morphology, glomerular filtration rate (GFR), or urinary albumin-to-creatinine ratio were identified from Mouse Genome Informatics. The corresponding human orthologs were then evaluated for GFR-associated single-nucleotide polymorphisms in 133,814 individuals and urinary albumin-to-creatinine ratio-associated SNPs in 54,451 individuals in genome-wide association studies meta-analysis of the CKDGen Consortium. After multiple testing corrections, significant associations with estimated GFR in humans were identified for single-nucleotide polymorphisms in 2, 7, and 17 genes causing abnormal GFR, abnormal physiology, and abnormal morphology in mice, respectively. Genes identified for abnormal kidney morphology showed significant enrichment for estimated GFR-associated single-nucleotide polymorphisms. In total, 19 genes contained variants associated with estimated GFR or the urinary albumin-to-creatinine ratio of which 16 mapped into previously reported genomewide significant loci. CYP26A1 and BMP4 emerged as novel signals subsequently validated in a large, independent study. An additional gene, CYP24A1, was discovered after conditioning on a published nearby association signal. Thus, our novel approach to combine comprehensive mouse phenotype information with human genomewide association studies data resulted in the identification of candidate genes for kidney disease pathogenesis. PMID:27263491

  8. The Evolutionary Fates of a Large Segmental Duplication in Mouse.

    PubMed

    Morgan, Andrew P; Holt, J Matthew; McMullan, Rachel C; Bell, Timothy A; Clayshulte, Amelia M-F; Didion, John P; Yadgary, Liran; Thybert, David; Odom, Duncan T; Flicek, Paul; McMillan, Leonard; de Villena, Fernando Pardo-Manuel

    2016-09-01

    Gene duplication and loss are major sources of genetic polymorphism in populations, and are important forces shaping the evolution of genome content and organization. We have reconstructed the origin and history of a 127-kbp segmental duplication, R2d, in the house mouse (Mus musculus). R2d contains a single protein-coding gene, Cwc22 De novo assembly of both the ancestral (R2d1) and the derived (R2d2) copies reveals that they have been subject to nonallelic gene conversion events spanning tens of kilobases. R2d2 is also a hotspot for structural variation: its diploid copy number ranges from zero in the mouse reference genome to >80 in wild mice sampled from around the globe. Hemizygosity for high copy-number alleles of R2d2 is associated in cis with meiotic drive; suppression of meiotic crossovers; and copy-number instability, with a mutation rate in excess of 1 per 100 transmissions in some laboratory populations. Our results provide a striking example of allelic diversity generated by duplication and demonstrate the value of de novo assembly in a phylogenetic context for understanding the mutational processes affecting duplicate genes. PMID:27371833

  9. Characterization of the mouse DAX-1 gene reveals evolutionary conservation of a unique amino-terminal motif and widespread expression in mouse tissue.

    PubMed

    Bae, D S; Schaefer, M L; Partan, B W; Muglia, L

    1996-09-01

    The human genetic disorder adrenal hypoplasia congenita with hypogonadotropic hypogonadism results from mutations in the recently isolated DAX-1 gene, a member of the nuclear hormone receptor superfamily. To study the role of DAX-1 in adrenal development and activation of the hypothalamic pituitary-gonadal axis, animal model systems will be essential. Here, we report the isolation and characterization of the mouse DAX-1 gene and its tissue-specific pattern of expression. The mouse DAX-1 gene codes for a 472-amino acid protein, with 75% overall nucleotide sequence homology to its human homolog. The 3.5 amino-terminal repeats of a unique motif with probable DNA-binding activity have been conserved between mouse and human, although highest conservation in the DAX-1 peptide exists in the carboxy-terminal ligand-binding domain. The DAX-1 gene remains X-linked in the mouse, consistent with its potential role in sex determination. We have developed a sensitive reverse transcription-PCR assay that detects DAX-1 messenger RNA in the central nervous system, pituitary, lung, heart, spleen, kidney, and thymus in addition to the adrenal and testis DAX-1 expression noted for the human DAX-1 gene. Future studies using mouse models of altered DAX-1 expression will be critical in defining the role of this factor in tissue- and development-specific gene regulation.

  10. Comparative Gene Expression Analysis of Mouse and Human Cardiac Maturation.

    PubMed

    Uosaki, Hideki; Taguchi, Y-H

    2016-08-01

    Understanding how human cardiomyocytes mature is crucial to realizing stem cell-based heart regeneration, modeling adult heart diseases, and facilitating drug discovery. However, it is not feasible to analyze human samples for maturation due to inaccessibility to samples while cardiomyocytes mature during fetal development and childhood, as well as difficulty in avoiding variations among individuals. Using model animals such as mice can be a useful strategy; nonetheless, it is not well-understood whether and to what degree gene expression profiles during maturation are shared between humans and mice. Therefore, we performed a comparative gene expression analysis of mice and human samples. First, we examined two distinct mice microarray platforms for shared gene expression profiles, aiming to increase reliability of the analysis. We identified a set of genes displaying progressive changes during maturation based on principal component analysis. Second, we demonstrated that the genes identified had a differential expression pattern between adult and earlier stages (e.g., fetus) common in mice and humans. Our findings provide a foundation for further genetic studies of cardiomyocyte maturation. PMID:27431744

  11. Mechanism and regulation of meiotic recombination initiation

    PubMed Central

    Lam, Isabel; Keeney, Scott

    2015-01-01

    Meiotic recombination involves the formation and repair of programmed DNA double-strand breaks (DSBs) catalyzed by the conserved Spo11 protein. This review summarizes recent studies pertaining to the formation of meiotic DSBs, including the mechanism of DNA cleavage by Spo11, proteins required for break formation, and mechanisms that control the location, timing, and number of DSBs. Where appropriate, findings in different organisms are discussed to highlight evolutionary conservation or divergence. PMID:25324213

  12. Cytological analysis of Arabidopsis thaliana meiotic chromosomes.

    PubMed

    Armstrong, Susan J; Sanchez-Moran, Eugenio; Franklin, F Chris H

    2009-01-01

    Advances in molecular biology and in the genetics of Arabidopsis thaliana have led to this organism becoming an important model for the analysis of meiosis in plants. Cytogenetic investigations are pivotal to meiotic studies and a number of technological improvements for Arabidopsis cytology have provided a range of tools to investigate chromosome behaviour during meiosis. This chapter includes protocols on basic cytology, FISH analysis, immunocytology, a procedure for a meiotic time course and electron microscopy.

  13. The mouse and human genes encoding the recognition component of the N-end rule pathway

    PubMed Central

    Kwon, Yong Tae; Reiss, Yuval; Fried, Victor A.; Hershko, Avram; Yoon, Jeong Kyo; Gonda, David K.; Sangan, Pitchai; Copeland, Neal G.; Jenkins, Nancy A.; Varshavsky, Alexander

    1998-01-01

    The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. The N-end rule pathway is one proteolytic pathway of the ubiquitin system. The recognition component of this pathway, called N-recognin or E3, binds to a destabilizing N-terminal residue of a substrate protein and participates in the formation of a substrate-linked multiubiquitin chain. We report the cloning of the mouse and human Ubr1 cDNAs and genes that encode a mammalian N-recognin called E3α. Mouse UBR1p (E3α) is a 1,757-residue (200-kDa) protein that contains regions of sequence similarity to the 225-kDa Ubr1p of the yeast Saccharomyces cerevisiae. Mouse and human UBR1p have apparent homologs in other eukaryotes as well, thus defining a distinct family of proteins, the UBR family. The residues essential for substrate recognition by the yeast Ubr1p are conserved in the mouse UBR1p. The regions of similarity among the UBR family members include a putative zinc finger and RING-H2 finger, another zinc-binding domain. Ubr1 is located in the middle of mouse chromosome 2 and in the syntenic 15q15-q21.1 region of human chromosome 15. Mouse Ubr1 spans ≈120 kilobases of genomic DNA and contains ≈50 exons. Ubr1 is ubiquitously expressed in adults, with skeletal muscle and heart being the sites of highest expression. In mouse embryos, the Ubr1 expression is highest in the branchial arches and in the tail and limb buds. The cloning of Ubr1 makes possible the construction of Ubr1-lacking mouse strains, a prerequisite for the functional understanding of the mammalian N-end rule pathway. PMID:9653112

  14. Arabidopsis Cell Division Cycle 20.1 Is Required for Normal Meiotic Spindle Assembly and Chromosome Segregation[OPEN

    PubMed Central

    Niu, Baixiao; Wang, Liudan; Ren, Ding; Ren, Ren

    2015-01-01

    Cell division requires proper spindle assembly; a surveillance pathway, the spindle assembly checkpoint (SAC), monitors whether the spindle is normal and correctly attached to kinetochores. The SAC proteins regulate mitotic chromosome segregation by affecting CDC20 (Cell Division Cycle 20) function. However, it is unclear whether CDC20 regulates meiotic spindle assembly and proper homolog segregation. Here, we show that the Arabidopsis thaliana CDC20.1 gene is indispensable for meiosis and male fertility. We demonstrate that cdc20.1 meiotic chromosomes align asynchronously and segregate unequally and the metaphase I spindle has aberrant morphology. Comparison of the distribution of meiotic stages at different time points between the wild type and cdc20.1 reveals a delay of meiotic progression from diakinesis to anaphase I. Furthermore, cdc20.1 meiocytes exhibit an abnormal distribution of a histone H3 phosphorylation mark mediated by the Aurora kinase, providing evidence that CDC20.1 regulates Aurora localization for meiotic chromosome segregation. Further evidence that CDC20.1 and Aurora are functionally related was provided by meiosis-specific knockdown of At-Aurora1 expression, resulting in meiotic chromosome segregation defects similar to those of cdc20.1. Taken together, these results suggest a critical role for CDC20.1 in SAC-dependent meiotic chromosome segregation. PMID:26672070

  15. Meiotic segregation of a homeologous chromosome pair.

    PubMed

    Maxfield Boumil, R; Kemp, B; Angelichio, M; Nilsson-Tillgren, T; Dawson, D S

    2003-03-01

    During meiosis, the alignment of homologous chromosomes facilitates their subsequent migration away from one another to opposite spindle poles at anaphase I. Recombination is part of the mechanism by which chromosomes identify their homologous partners, and serves to link the homologs in a way that, in some organisms, has been shown to promote proper attachment to the meiotic spindle. We have built a diploid strain that contains a pair of homeologous chromosomes V': one is derived from Saccharomyces cerevisiae and one originates from S. carlsbergensis. Sequence analysis reveals that these chromosomes share 71% sequence identity. The homeologs experience high levels of meiotic double-stranded breaks. Despite their relatedness and their competence to initiate recombination, the meiotic segregation behavior of the homeologous chromosomes suggests that, in most meioses, they are partitioned by a meiotic segregation system that has been shown previously to partition non-exchange chromosomes and pairs with no homology. Though the homeologous chromosomes show a degree of meiotic segregation fidelity similar to that of other non-exchange pairs, our data provide evidence that their limited sequence homology may provide some bias in meiotic partner choice. PMID:12655401

  16. Changes in gene expression associated with retinal degeneration in the rd3 mouse

    PubMed Central

    Cheng, Christiana L.

    2013-01-01

    Purpose To identify and characterize changes in gene expression associated with photoreceptor degeneration in the rd3 mouse model of Leber congenital amaurosis (LCA) type 12. Methods Global genome expression profiling using microarray technology was performed on total RNA extracts from rd3 and wild-type control mouse retinas at postnatal day 21. Quantitative PCR analysis of selected transcripts was performed to validate the microarray results. Results Functional annotation of differentially regulated genes in the rd3 mouse defined key canonical pathways, including phototransduction, glycerophospholipid metabolism, tumor necrosis factor receptor 1 signaling, and endothelin signaling. Overall, 1,140 of approximately 55,800 transcripts were differentially represented. In particular, a large percentage of the upregulated transcripts encode proteins involved in the immune response; whereas the downregulated transcripts encode proteins involved in phototransduction and lipid metabolism. Conclusions This analysis has elucidated several candidate genes and pathways, thus providing insight into the pathogenic mechanisms underlying the photoreceptor degeneration in the rd3 mouse retina and indicating directions for future studies. PMID:23687432

  17. In vivo analysis of mouse gastrin gene regulation in enhanced GFP-BAC transgenic mice.

    PubMed

    Takaishi, Shigeo; Shibata, Wataru; Tomita, Hiroyuki; Jin, Guangchun; Yang, Xiangdong; Ericksen, Russell; Dubeykovskaya, Zinaida; Asfaha, Samuel; Quante, Michael; Betz, Kelly S; Shulkes, Arthur; Wang, Timothy C

    2011-02-01

    Gastrin is secreted from a subset of neuroendocrine cells residing in the gastric antrum known as G cells, but low levels are also expressed in fetal pancreas and intestine and in many solid malignancies. Although past studies have suggested that antral gastrin is transcriptionally regulated by inflammation, gastric pH, somatostatin, and neoplastic transformation, the transcriptional regulation of gastrin has not previously been demonstrated in vivo. Here, we describe the creation of an enhanced green fluorescent protein reporter (mGAS-EGFP) mouse using a bacterial artificial chromosome that contains the entire mouse gastrin gene. Three founder lines expressed GFP signals in the gastric antrum and the transitional zone to the corpus. In addition, GFP(+) cells could be detected in the fetal pancreatic islets and small intestinal villi, but not in these organs of the adult mice. The administration of acid-suppressive reagents such as proton pump inhibitor omeprazole and gastrin/CCK-2 receptor antagonist YF476 significantly increased GFP signal intensity and GFP(+) cell numbers in the antrum, whereas these parameters were decreased by overnight fasting, octreotide (long-lasting somatostatin ortholog) infusion, and Helicobacter felis infection. GFP(+) cells were also detected in the anterior lobe of the pituitary gland and importantly in the colonic tumor cells induced by administration with azoxymethane and dextran sulfate sodium salt. This transgenic mouse provides a useful tool to study the regulation of mouse gastrin gene in vivo, thus contributing to our understanding of the mechanisms involved in transcriptional control of the gastrin gene.

  18. Editing of mouse and human immunoglobulin genes by CRISPR-Cas9 system

    PubMed Central

    Cheong, Taek-Chin; Compagno, Mara; Chiarle, Roberto

    2016-01-01

    Applications of the CRISPR-Cas9 system to edit the genome have widely expanded to include DNA gene knock-out, deletions, chromosomal rearrangements, RNA editing and genome-wide screenings. Here we show the application of CRISPR-Cas9 technology to edit the mouse and human immunoglobulin (Ig) genes. By delivering Cas9 and guide-RNA (gRNA) with retro- or lenti-virus to IgM+ mouse B cells and hybridomas, we induce class-switch recombination (CSR) of the IgH chain to the desired subclass. Similarly, we induce CSR in all human B cell lines tested with high efficiency to targeted IgH subclass. Finally, we engineer mouse hybridomas to secrete Fab′ fragments instead of the whole Ig. Our results indicate that Ig genes in mouse and human cells can be edited to obtain any desired IgH switching helpful to study the biology of normal and lymphoma B cells. We also propose applications that could transform the technology of antibody production. PMID:26956543

  19. Editing of mouse and human immunoglobulin genes by CRISPR-Cas9 system.

    PubMed

    Cheong, Taek-Chin; Compagno, Mara; Chiarle, Roberto

    2016-03-09

    Applications of the CRISPR-Cas9 system to edit the genome have widely expanded to include DNA gene knock-out, deletions, chromosomal rearrangements, RNA editing and genome-wide screenings. Here we show the application of CRISPR-Cas9 technology to edit the mouse and human immunoglobulin (Ig) genes. By delivering Cas9 and guide-RNA (gRNA) with retro- or lenti-virus to IgM(+) mouse B cells and hybridomas, we induce class-switch recombination (CSR) of the IgH chain to the desired subclass. Similarly, we induce CSR in all human B cell lines tested with high efficiency to targeted IgH subclass. Finally, we engineer mouse hybridomas to secrete Fab' fragments instead of the whole Ig. Our results indicate that Ig genes in mouse and human cells can be edited to obtain any desired IgH switching helpful to study the biology of normal and lymphoma B cells. We also propose applications that could transform the technology of antibody production.

  20. A bipartite operator interacts with a heat shock element to mediate early meiotic induction of Saccharomyces cerevisiae HSP82

    SciTech Connect

    Szent-Gyorgyi, C.

    1995-12-01

    This report seeks to characterize the activation of meiotic gene in terms of cis-acting DNA elements and their associated factors in Saccharomyces cerevisiae. It was found that vegetative repression and meiotic induction depend on interactions of the promoter-proximal heat shock element with a nearby bipartite repression element. The experiments described explore how two different regulatory pathways induce transcription by stimulating a single classical activation element, a nonspecific heat shock element. 81 refs., 10 figs., 1 tab.

  1. Chromosomal localization of a new mouse lens opacity gene (lop18)

    SciTech Connect

    Chang, Bo; Hawes, N.L.; Smith, R.S.

    1996-08-15

    Examination of mouse strains with a slit lamp and indirect ophthalmoscopy revealed that strain CBA/CaGnLe has a white cataract obvious at weaning age. It soon progresses to a large white nuclear cataract with mild cortical changes. Crosses with C57BL/GJ showed that this is inherited as a single recessive fully penetrant gene, which we have designated lop18 (lens opacity 18). Linkage analysis using visible marker T (brachyury), histocompatibility marker H2, and microsatellite markers D17MU21, D17MU28, D17MU38, and D17MU46 shows that the 1op18 gene is located, {approximately}16 cM from the centromere on mouse Chromosome 17. It is a likely candidate mutation for the {alpha}-crystallin (Cryal) gene. 14 refs., 1 fig., 1 tab.

  2. Perinatal Gjb2 gene transfer rescues hearing in a mouse model of hereditary deafness.

    PubMed

    Iizuka, Takashi; Kamiya, Kazusaku; Gotoh, Satoru; Sugitani, Yoshinobu; Suzuki, Masaaki; Noda, Tetsuo; Minowa, Osamu; Ikeda, Katsuhisa

    2015-07-01

    Hearing loss is the most widespread sensory disorder, with an incidence of congenital genetic deafness of 1 in 1600 children. For many ethnic populations, the most prevalent form of genetic deafness is caused by recessive mutations in the gene gap junction protein, beta 2, 26 kDa (GJB2), which is also known as connexin 26 (Cx26). Despite this knowledge, existing treatment strategies do not completely recover speech perception. Here we used a gene delivery system to rescue hearing in a mouse model of Gjb2 deletion. Mice lacking Cx26 are characterized by profound deafness from birth and improper development of cochlear cells. Cochlear delivery of Gjb2 using an adeno-associated virus significantly improved the auditory responses and development of the cochlear structure. Using gene replacement to restore hearing in a new mouse model of Gjb2-related deafness may lead to the development of therapies for human hereditary deafness.

  3. Extraordinary sequence divergence at Tsga8, an X-linked gene involved in mouse spermiogenesis.

    PubMed

    Good, Jeffrey M; Vanderpool, Dan; Smith, Kimberly L; Nachman, Michael W

    2011-05-01

    The X chromosome plays an important role in both adaptive evolution and speciation. We used a molecular evolutionary screen of X-linked genes potentially involved in reproductive isolation in mice to identify putative targets of recurrent positive selection. We then sequenced five very rapidly evolving genes within and between several closely related species of mice in the genus Mus. All five genes were involved in male reproduction and four of the genes showed evidence of recurrent positive selection. The most remarkable evolutionary patterns were found at Testis-specific gene a8 (Tsga8), a spermatogenesis-specific gene expressed during postmeiotic chromatin condensation and nuclear transformation. Tsga8 was characterized by extremely high levels of insertion-deletion variation of an alanine-rich repetitive motif in natural populations of Mus domesticus and M. musculus, differing in length from the reference mouse genome by up to 89 amino acids (27% of the total protein length). This population-level variation was coupled with striking divergence in protein sequence and length between closely related mouse species. Although no clear orthologs had previously been described for Tsga8 in other mammalian species, we have identified a highly divergent hypothetical gene on the rat X chromosome that shares clear orthology with the 5' and 3' ends of Tsga8. Further inspection of this ortholog verified that it is expressed in rat testis and shares remarkable similarity with mouse Tsga8 across several general features of the protein sequence despite no conservation of nucleotide sequence across over 60% of the rat-coding domain. Overall, Tsga8 appears to be one of the most rapidly evolving genes to have been described in rodents. We discuss the potential evolutionary causes and functional implications of this extraordinary divergence and the possible contribution of Tsga8 and the other four genes we examined to reproductive isolation in mice. PMID:21186189

  4. Gene expression profiling in primary mouse hepatocytes discriminates true from false-positive genotoxic compounds.

    PubMed

    Mathijs, K; Brauers, K J J; Jennen, D G J; Lizarraga, D; Kleinjans, J C S; van Delft, J H M

    2010-11-01

    Well-established in vitro methods for testing the genotoxic potency of chemicals--such as the Ames/Salmonella test, the mouse lymphoma assay, the micronucleus test and the chromosomal aberration test--show a high false-positive rate for predicting in vivo genotoxicity and carcinogenicity. Thus, there is a need for more reliable in vitro assays. We investigated whether gene expression profiling in metabolically competent primary mouse hepatocytes is capable of discriminating true genotoxic (GTX) compounds from false-positive genotoxic (FP-GTX) compounds. Sandwich-cultured primary hepatocytes from male C57Bl6 mice were treated for 24 and 48 h with five true GTX and five FP-GTX compounds. Whole genome gene expression modifications were analysed by means of Affymetrix mouse genome 430 2.0 microarrays. Filtered genes were used for hierarchical clustering and class prediction methods. Classifiers were generated by prediction analysis of microarray using a leave-one-compound-out method and selecting the genes that were common to the 10 training sets. For the training compounds, all but one were correctly classified. Validation of the classification model with five new compounds resulted in a 100% correct classification at 24 h and 80% at 48 h. The generated classifiers were mostly involved in metabolic and biosynthetic processes, immune responses and apoptosis. Applying genes whose expression change correlates with γH2AX foci, a measure for DNA damage, did not improve the classification. The present study shows that gene expression profiling in primary mouse hepatocytes is capable of discriminating between true GTX and FP-GTX compounds.

  5. Gene transfer to the developing mouse inner ear by in vivo electroporation.

    PubMed

    Wang, Lingyan; Jiang, Han; Brigande, John V

    2012-06-30

    The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and the organ of Corti in the coiled cochlea. The cristae and maculae contain vestibular hair cells that transduce mechanical stimuli to subserve the special sense of balance, while auditory hair cells in the organ of Corti are the primary transducers for hearing. Cell fate specification in these sensory epithelia and morphogenesis of the semicircular canals and cochlea take place during the second week of gestation in the mouse and are largely completed before birth. Developmental studies of the mouse inner ear are routinely conducted by harvesting transgenic embryos at different embryonic or postnatal stages to gain insight into the molecular basis of cellular and/or morphological phenotypes. We hypothesize that gene transfer to the developing mouse inner ear in utero in the context of gain- and loss-of-function studies represents a complimentary approach to traditional mouse transgenesis for the interrogation of the genetic mechanisms underlying mammalian inner ear development(6). The experimental paradigm to conduct gene misexpression studies in the developing mouse inner ear demonstrated here resolves into three general steps: 1) ventral laparotomy; 2) transuterine microinjection; and 3) in vivo electroporation. Ventral laparotomy is a mouse survival surgical technique that permits externalization of the uterus to gain experimental access to the implanted embryos. Transuterine microinjection is the use of beveled, glass capillary micropipettes to introduce expression plasmid into the lumen of the otic vesicle or otocyst. In vivo electroporation is the application of square wave, direct current pulses to drive expression plasmid into progenitor cells. We previously described this electroporation-based gene transfer technique and included detailed notes on each step of the protocol(11). Mouse experimental

  6. Two genes substitute for the mouse Y chromosome for spermatogenesis and reproduction.

    PubMed

    Yamauchi, Yasuhiro; Riel, Jonathan M; Ruthig, Victor A; Ortega, Eglė A; Mitchell, Michael J; Ward, Monika A

    2016-01-29

    The mammalian Y chromosome is considered a symbol of maleness, as it encodes a gene driving male sex determination, Sry, as well as a battery of other genes important for male reproduction. We previously demonstrated in the mouse that successful assisted reproduction can be achieved when the Y gene contribution is limited to only two genes, Sry and spermatogonial proliferation factor Eif2s3y. Here, we replaced Sry by transgenic activation of its downstream target Sox9, and Eif2s3y, by transgenic overexpression of its X chromosome-encoded homolog Eif2s3x. The resulting males with no Y chromosome genes produced haploid male gametes and sired offspring after assisted reproduction. Our findings support the existence of functional redundancy between the Y chromosome genes and their homologs encoded on other chromosomes.

  7. Silencing of X-Linked MicroRNAs by Meiotic Sex Chromosome Inactivation.

    PubMed

    Royo, Hélène; Seitz, Hervé; ElInati, Elias; Peters, Antoine H F M; Stadler, Michael B; Turner, James M A

    2015-10-01

    During the pachytene stage of meiosis in male mammals, the X and Y chromosomes are transcriptionally silenced by Meiotic Sex Chromosome Inactivation (MSCI). MSCI is conserved in therian mammals and is essential for normal male fertility. Transcriptomics approaches have demonstrated that in mice, most or all protein-coding genes on the X chromosome are subject to MSCI. However, it is unclear whether X-linked non-coding RNAs behave in a similar manner. The X chromosome is enriched in microRNA (miRNA) genes, with many exhibiting testis-biased expression. Importantly, high expression levels of X-linked miRNAs (X-miRNAs) have been reported in pachytene spermatocytes, indicating that these genes may escape MSCI, and perhaps play a role in the XY-silencing process. Here we use RNA FISH to examine X-miRNA expression in the male germ line. We find that, like protein-coding X-genes, X-miRNAs are expressed prior to prophase I and are thereafter silenced during pachynema. X-miRNA silencing does not occur in mouse models with defective MSCI. Furthermore, X-miRNAs are expressed at pachynema when present as autosomally integrated transgenes. Thus, we conclude that silencing of X-miRNAs during pachynema in wild type males is MSCI-dependent. Importantly, misexpression of X-miRNAs during pachynema causes spermatogenic defects. We propose that MSCI represents a chromosomal mechanism by which X-miRNAs, and other potential X-encoded repressors, can be silenced, thereby regulating genes with critical late spermatogenic functions.

  8. REC, Drosophila MCM8, drives formation of meiotic crossovers.

    PubMed

    Blanton, Hunter L; Radford, Sarah J; McMahan, Susan; Kearney, Hutton M; Ibrahim, Joseph G; Sekelsky, Jeff

    2005-09-01

    Crossovers ensure the accurate segregation of homologous chromosomes from one another during meiosis. Here, we describe the identity and function of the Drosophila melanogaster gene recombination defective (rec), which is required for most meiotic crossing over. We show that rec encodes a member of the mini-chromosome maintenance (MCM) protein family. Six MCM proteins (MCM2-7) are essential for DNA replication and are found in all eukaryotes. REC is the Drosophila ortholog of the recently identified seventh member of this family, MCM8. Our phylogenetic analysis reveals the existence of yet another family member, MCM9, and shows that MCM8 and MCM9 arose early in eukaryotic evolution, though one or both have been lost in multiple eukaryotic lineages. Drosophila has lost MCM9 but retained MCM8, represented by REC. We used genetic and molecular methods to study the function of REC in meiotic recombination. Epistasis experiments suggest that REC acts after the Rad51 ortholog SPN-A but before the endonuclease MEI-9. Although crossovers are reduced by 95% in rec mutants, the frequency of noncrossover gene conversion is significantly increased. Interestingly, gene conversion tracts in rec mutants are about half the length of tracts in wild-type flies. To account for these phenotypes, we propose that REC facilitates repair synthesis during meiotic recombination. In the absence of REC, synthesis does not proceed far enough to allow formation of an intermediate that can give rise to crossovers, and recombination proceeds via synthesis-dependent strand annealing to generate only noncrossover products.

  9. Stable Somatic Gene Expression in Mouse Lungs Following Electroporation-mediated Tol2 Transposon Delivery.

    PubMed

    Muliawan, Hary Sakti; Nakayama, Kazuhiko; Yagi, Keiko; Ikeda, Koji; Yagita, Kazuhiro; Hirata, Ken-ichi; Emoto, Noriaki

    2015-10-07

    Gene delivery to the lung has rapidly progressed as an important method for studying various chronic lung diseases. Viral vectors, albeit highly efficient, are limited by the host immune response. Electroporation, a well-known non-viral method, can efficiently deliver genes to the lung, but is unable to induce stable gene expression. The Tol2 transposon is another non-viral method that can induce stable gene expression by reinserting its genes into the host genome. In this study, we combined electroporation and Tol2 transposons to obtain stable, high-level gene expression in the mouse lung. Tol2 transposon plasmids (pT2A-EGFP; Tol2, pCAGGS-TP; transposase) were optimized in vitro, and the electroporation procedure (pCAG-EGFP) was optimized in mouse lungs. After optimization, a combination of electroporation plus the Tol2 transposon was used in a comparative analysis with electroporation plus pCAG-EGFP. GFP expression levels were quantified and visualized on days 4 and 7 post-electroporation. We successfully reproduced the Tol2 transposon system in vitro and the electroporation procedure in vivo. We observed sustainable GFP expression using electroporation plus the Tol2 transposon on days 4 and 7, while electroporation plus pCAG-EGFP resulted in decreased GFP expression on day 7. We were able to induce high-level, stable gene expression in mouse lungs using a combination of electroporation and the Tol2 transposon. This represents a safer method for lung gene delivery that can be used as an alternative to viral vectors.

  10. A gene expression resource generated by genome-wide lacZ profiling in the mouse

    PubMed Central

    Tuck, Elizabeth; Estabel, Jeanne; Oellrich, Anika; Maguire, Anna Karin; Adissu, Hibret A.; Souter, Luke; Siragher, Emma; Lillistone, Charlotte; Green, Angela L.; Wardle-Jones, Hannah; Carragher, Damian M.; Karp, Natasha A.; Smedley, Damian; Adams, Niels C.; Bussell, James N.; Adams, David J.; Ramírez-Solis, Ramiro; Steel, Karen P.; Galli, Antonella; White, Jacqueline K.

    2015-01-01

    ABSTRACT Knowledge of the expression profile of a gene is a critical piece of information required to build an understanding of the normal and essential functions of that gene and any role it may play in the development or progression of disease. High-throughput, large-scale efforts are on-going internationally to characterise reporter-tagged knockout mouse lines. As part of that effort, we report an open access adult mouse expression resource, in which the expression profile of 424 genes has been assessed in up to 47 different organs, tissues and sub-structures using a lacZ reporter gene. Many specific and informative expression patterns were noted. Expression was most commonly observed in the testis and brain and was most restricted in white adipose tissue and mammary gland. Over half of the assessed genes presented with an absent or localised expression pattern (categorised as 0-10 positive structures). A link between complexity of expression profile and viability of homozygous null animals was observed; inactivation of genes expressed in ≥21 structures was more likely to result in reduced viability by postnatal day 14 compared with more restricted expression profiles. For validation purposes, this mouse expression resource was compared with Bgee, a federated composite of RNA-based expression data sets. Strong agreement was observed, indicating a high degree of specificity in our data. Furthermore, there were 1207 observations of expression of a particular gene in an anatomical structure where Bgee had no data, indicating a large amount of novelty in our data set. Examples of expression data corroborating and extending genotype-phenotype associations and supporting disease gene candidacy are presented to demonstrate the potential of this powerful resource. PMID:26398943

  11. Introducing desirable transgenes into insect populations using Y-linked meiotic drive - a theoretical assessment.

    PubMed

    Huang, Yunxin; Magori, Krisztian; Lloyd, Alun L; Gould, Fred

    2007-04-01

    The use of genetic drive mechanisms to replace native mosquito genotypes with individuals bearing antipathogen transgenes is a potential strategy for repressing insect transmission of human diseases such as malaria and dengue. Antipathogen transgenes have been developed and tested, but efficient gene drive mechanisms are lacking. Here we theoretically assess the feasibility of introducing antipathogen genes into wild Aedes aegypti populations by using a naturally occurring meiotic drive system. We consider the release of males having both a Y-linked meiotic drive gene and an X-linked drive-insensitive response allele to which an antipathogen gene is linked. We use mathematical models and computer simulations to determine how the post-introduction dynamics of the antipathogen gene are affected by specific genetic characteristics of the system. The results show that when the natural population is uniformly sensitive to the meiotic drive gene, the antipathogen gene may be driven close to fixation if the fitness costs of the drive gene, the insensitive response allele, and the antipathogen gene are low. However, when the natural population has a small proportion of an X-linked insensitive response allele or an autosomal gene that strongly reduces the effect of the drive gene, the antipathogen gene does not spread if it has an associated fitness cost. Our modeling results provide a theoretical foundation for further experimental tests.

  12. Gene structure and chromosomal localization of the mouse homologue of rat OX40 protein.

    PubMed

    Birkeland, M L; Copeland, N G; Gilbert, D J; Jenkins, N A; Barclay, A N

    1995-04-01

    The OX40 protein is expressed only on activated rat CD4+ T blasts and is a member of a superfamily of cell surface molecules which includes CD40, CD30, CD95 (Fas), CD27, 4-1BB antigens and the receptors for tumor necrosis factor (TNF) and nerve growth factor (NGF). The proteins of this group are related to each other by having three to six repeats of a cysteine-rich sequence in their extracellular domains. Members of this family of receptors have also been shown to bind to ligands which are structurally related to TNF. The mouse homologue of the rat OX40 protein was cloned at the cDNA and genomic levels. The gene structure shows that there are several intron/exon borders shared between OX40 and CD27, CD40, TNF receptor type I, CD95 and 4-1BB genes. This group of genes is less closely related structurally to the gene structure of the NGF receptor. The gene encoding murine OX40 has been placed on mouse chromosome 4, in an area which contains the genes for TNF receptor type II and 4-1BB, and is syntenic with a region of human chromosome 1 which contains human TNF receptor type II, OX40, and CD30 genes. PMID:7737295

  13. Effect of light on global gene expression in the neuroglobin-deficient mouse retina

    PubMed Central

    ILMJÄRV, STEN; REIMETS, RIIN; HUNDAHL, CHRISTIAN ANSGAR; LUUK, HENDRIK

    2014-01-01

    Several previous studies have raised controversy over the functional role of neuroglobin (Ngb) in the retina. Certain studies indicate a significant impact of Ngb on retinal physiology, whereas others are conflicting. The present is an observational study that tested the effect of Ngb deficiency on gene expression in dark- and light-adapted mouse retinas. Large-scale gene expression profiling was performed using GeneChip® Mouse Exon 1.0 ST arrays and the results were compared to publicly available data sets. The lack of Ngb was found to have a minor effect on the light-induced retinal gene expression response. In addition, there was no increase in the expression of marker genes associated with hypoxia, endoplasmic reticulum-stress and oxidative stress in the Ngb-deficient retina. By contrast, several genes were identified that appeared to be differentially expressed between the genotypes when the effect of light was ignored. The present study indicates that Ngb deficiency does not lead to major alternations in light-dependent gene expression response, but leads to subtle systemic differences of a currently unknown functional significance. PMID:25279145

  14. Analysis of spatial-temporal gene expression patterns reveals dynamics and regionalization in developing mouse brain

    PubMed Central

    Chou, Shen-Ju; Wang, Chindi; Sintupisut, Nardnisa; Niou, Zhen-Xian; Lin, Chih-Hsu; Li, Ker-Chau; Yeang, Chen-Hsiang

    2016-01-01

    Allen Brain Atlas (ABA) provides a valuable resource of spatial/temporal gene expressions in mammalian brains. Despite rich information extracted from this database, current analyses suffer from several limitations. First, most studies are either gene-centric or region-centric, thus are inadequate to capture the superposition of multiple spatial-temporal patterns. Second, standard tools of expression analysis such as matrix factorization can capture those patterns but do not explicitly incorporate spatial dependency. To overcome those limitations, we proposed a computational method to detect recurrent patterns in the spatial-temporal gene expression data of developing mouse brains. We demonstrated that regional distinction in brain development could be revealed by localized gene expression patterns. The patterns expressed in the forebrain, medullary and pontomedullary, and basal ganglia are enriched with genes involved in forebrain development, locomotory behavior, and dopamine metabolism respectively. In addition, the timing of global gene expression patterns reflects the general trends of molecular events in mouse brain development. Furthermore, we validated functional implications of the inferred patterns by showing genes sharing similar spatial-temporal expression patterns with Lhx2 exhibited differential expression in the embryonic forebrains of Lhx2 mutant mice. These analysis outcomes confirm the utility of recurrent expression patterns in studying brain development. PMID:26786896

  15. Gene expression profiles in liver of mouse after chronic exposure to drinking water.

    PubMed

    Wu, Bing; Zhang, Yan; Zhao, Dayong; Zhang, Xuxiang; Kong, Zhiming; Cheng, Shupei

    2009-10-01

    cDNA micorarray approach was applied to hepatic transcriptional profile analysis in male mouse (Mus musculus, ICR) to assess the potential health effects of drinking water in Nanjing, China. Mice were treated with continuous exposure to drinking water for 90 days. Hepatic gene expression was analyzed with Affymetrix Mouse Genome 430A 2.0 arrays, and pathway analysis was carried out by Molecule Annotation System 2.0 and KEGG pathway database. A total of 836 genes were found to be significantly altered (1.5-fold, P < or = 0.05), including 294 up-regulated genes and 542 down-regulated genes. According to biological pathway analysis, drinking water exposure resulted in aberration of gene expression and biological pathways linked to xenobiotic metabolism, signal transduction, cell cycle and oxidative stress response. Further, deregulation of several genes associated with carcinogenesis or tumor progression including Ccnd1, Egfr, Map2k3, Mcm2, Orc2l and Smad2 was observed. Although transcription changes in identified genes are unlikely to be used as a sole indicator of adverse health effects, the results of this study could enhance our understanding of early toxic effects of drinking water exposure and support future studies on drinking water safety.

  16. Reporter Gene Silencing in Targeted Mouse Mutants Is Associated with Promoter CpG Island Methylation

    PubMed Central

    Kirov, Julia V.; Adkisson, Michael; Nava, A. J.; Cipollone, Andreana; Willis, Brandon; Engelhard, Eric K.; Lloyd, K. C. Kent; de Jong, Pieter; West, David B.

    2015-01-01

    Targeted mutations in mouse disrupt local chromatin structure and may lead to unanticipated local effects. We evaluated targeted gene promoter silencing in a group of six mutants carrying the tm1a Knockout Mouse Project allele containing both a LacZ reporter gene driven by the native promoter and a neo selection cassette. Messenger RNA levels of the reporter gene and targeted gene were assessed by qRT-PCR, and methylation of the promoter CpG islands and LacZ coding sequence were evaluated by sequencing of bisulfite-treated DNA. Mutants were stratified by LacZ staining into presumed Silenced and Expressed reporter genes. Silenced mutants had reduced relative quantities LacZ mRNA and greater CpG Island methylation compared with the Expressed mutant group. Within the silenced group, LacZ coding sequence methylation was significantly and positively correlated with CpG Island methylation, while promoter CpG methylation was only weakly correlated with LacZ gene mRNA. The results support the conclusion that there is promoter silencing in a subset of mutants carrying the tm1a allele. The features of targeted genes which promote local silencing when targeted remain unknown. PMID:26275310

  17. Adaptive Evolution of the Insulin Two-Gene System in Mouse

    PubMed Central

    Shiao, Meng-Shin; Liao, Ben-Yang; Long, Manyuan; Yu, Hon-Tsen

    2008-01-01

    Insulin genes in mouse and rat compose a two-gene system in which Ins1 was retroposed from the partially processed mRNA of Ins2. When Ins1 originated and how it was retained in genomes still remain interesting problems. In this study, we used genomic approaches to detect insulin gene copy number variation in rodent species and investigated evolutionary forces acting on both Ins1 and Ins2. We characterized the phylogenetic distribution of the new insulin gene (Ins1) by Southern analyses and confirmed by sequencing insulin genes in the rodent genomes. The results demonstrate that Ins1 originated right before the mouse–rat split (∼20 MYA), and both Ins1 and Ins2 are under strong functional constraints in these murine species. Interestingly, by examining a range of nucleotide polymorphisms, we detected positive selection acting on both Ins2 and Ins1 gene regions in the Mus musculus domesticus populations. Furthermore, three amino acid sites were also identified as having evolved under positive selection in two insulin peptides: two are in the signal peptide and one is in the C-peptide. Our data suggest an adaptive divergence in the mouse insulin two-gene system, which may result from the response to environmental change caused by the rise of agricultural civilization, as proposed by the thrifty-genotype hypothesis. PMID:18245324

  18. Gene structure and chromosomal localization of the mouse homologue of rat OX40 protein.

    PubMed

    Birkeland, M L; Copeland, N G; Gilbert, D J; Jenkins, N A; Barclay, A N

    1995-04-01

    The OX40 protein is expressed only on activated rat CD4+ T blasts and is a member of a superfamily of cell surface molecules which includes CD40, CD30, CD95 (Fas), CD27, 4-1BB antigens and the receptors for tumor necrosis factor (TNF) and nerve growth factor (NGF). The proteins of this group are related to each other by having three to six repeats of a cysteine-rich sequence in their extracellular domains. Members of this family of receptors have also been shown to bind to ligands which are structurally related to TNF. The mouse homologue of the rat OX40 protein was cloned at the cDNA and genomic levels. The gene structure shows that there are several intron/exon borders shared between OX40 and CD27, CD40, TNF receptor type I, CD95 and 4-1BB genes. This group of genes is less closely related structurally to the gene structure of the NGF receptor. The gene encoding murine OX40 has been placed on mouse chromosome 4, in an area which contains the genes for TNF receptor type II and 4-1BB, and is syntenic with a region of human chromosome 1 which contains human TNF receptor type II, OX40, and CD30 genes.

  19. Mouse in utero electroporation: controlled spatiotemporal gene transfection.

    PubMed

    Matsui, Asuka; Yoshida, Aya C; Kubota, Mayumi; Ogawa, Masaharu; Shimogori, Tomomi

    2011-01-01

    In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance molecules, local gene transfer or knock- out is required. Gene targeting knock-in or knock-out into local regions is possible to perform with combination with a specific CRE line, which is laborious, costly, and time consuming. Therefore, a simple transfection method, an in utero electroporation technique, which can be performed with short time, will be handy to test the possible function of candidate genes prior to the generation of transgenic animals. In addition to this, in utero electroporation targets areas of the brain where no specific CRE line exists, and will limit embryonic lethality. Here, we present a method of in utero electroporation combining two different types of electrodes for simple and convenient gene transfer into target areas of the developing brain. First, a unique holding method of embryos using an optic fiber optic light cable will make small embryos (from E9.5) visible for targeted DNA solution injection into ventricles and needle type electrodes insertion to the targeted brain area. The patterning of the brain such as cortical area occur at early embryonic stage, therefore, these early electroporation from E9.5 make a big contribution to understand entire area patterning event. Second, the precise shape of a capillary prevents uterine damage by making holes by insertion of the capillary. Furthermore, the precise shape of the needle electrodes are created with tungsten and platinum wire and sharpened using sand paper and insulated with nail polish, a method which is described in great detail in this protocol. This unique technique allows transfection of plasmid DNA into restricted areas of the brain and will enable small embryos to be electroporated. This will help to, open a new window for many scientists who are working on cell differentiation, cell migration, axon guidance in very early

  20. Mapping of the Sca1 and pcd genes on mouse chromosome 13 provides evidence that they are different genes

    SciTech Connect

    Servadio, A.; McCall, A.; Zoghbi, H.; Eicher, E.M.

    1995-10-10

    It is well established that large chromosomal segments have remained intact during the evolution of different mammalian species. Thus, mapping information for a gene in mammalian species facilitates mapping the same gene in another mammalian species. In addition, phenotypically similar diseases that map to linkage conserved regions in two species may be caused by mutations in the same gene. Spinocerebellar ataxia type 1 (SCA1) is a dominantly inherited human disorder characterized by progressive ataxia, dysarthria, and dysmetria. SCA1 maps to the short arm of human chromosome (Chr) 6 in the 6p23-p22 region. SCA1 is caused by the expansion of an unstable CAG repeat located within the coding region of a novel protein, ataxin-1, Purkinje cell degeneration (pcd) is a recessively inherited mouse disorder characterized by a moderate ataxia, usually noted by 3-4 weeks of age. Progressive degeneration of Purkinje cells is the underlying pathogenesis in this disorder. The pcd gene was assigned to mouse Chr 13 because it showed linkage to extra toes (Xt) and pearl (pe). Some doubt about this assignment existed, however, because the calculated genetic distance between pcd and Xt was 32 cM and that between pcd and pe was 18 cM. If pcd is located in Chr 13, its placement relative to Xt and pe suggests that it would be located in the region that shares linkage homology with the region that shares linkage homology with the region of human Chr 6 that contains SCA1. Here, we present data that confirm the assignment of pcd to Chr 13, map the mouse Sca1 gene to Chr 13, and eliminate Sca1 as a candidate gene for pcd. 11 refs., 1 tab.

  1. [Cloning of mouse adam10 gene promoter and construction and identification of dual luciferase reporter system].

    PubMed

    Chen, Wei; Chen, Chong; Zhang, Huan-Xin; Cao, Jiang; Sang, Wei; Wu, Qing-Yun; Zhao, Kai; Zang, Yu; Zeng, Ling-Yu; Xu, Kai-Lin

    2012-06-01

    This study was aimed to clone mouse adam10 gene promoter and construct its dual luciferase report vector, and to investigate its transcriptional activity. Total DNA was extracted from mouse brain and used for amplifying the fragment containing adam10 gene promoter by PCR. The amplified product was inserted into pGL-4.10 vector to construct pGL4.10-adam10. The pGL4.10-adam10 and control plasmid pGL4.74 were co-transfected into HEK293 FT cells by lipofectamine 2000. The activity of adam10 gene promoter was assayed by luciferase system. The results showed that the recombinant plasmid pGL4.10-adam10 containing promoter of mouse adam10 was correctly constructed. The method was optimized by changing ratio of two plasmids. Moreover, the transcriptional activity of pGL4.10-adam10 stimulated by ionomycin increased. It is concluded that the dual luciferase reporter system is successfully established, which is useful in bioluminescence imaging technology in vitro. The effect of ionomycin can enhance the transcriptional activity of adam10 gene promoter.

  2. Regulation of X-linked gene expression during early mouse development by Rlim

    PubMed Central

    Wang, Feng; Shin, JongDae; Shea, Jeremy M; Yu, Jun; Bošković, Ana; Byron, Meg; Zhu, Xiaochun; Shalek, Alex K; Regev, Aviv; Lawrence, Jeanne B; Torres, Eduardo M; Zhu, Lihua J; Rando, Oliver J; Bach, Ingolf

    2016-01-01

    Mammalian X-linked gene expression is highly regulated as female cells contain two and male one X chromosome (X). To adjust the X gene dosage between genders, female mouse preimplantation embryos undergo an imprinted form of X chromosome inactivation (iXCI) that requires both Rlim (also known as Rnf12) and the long non-coding RNA Xist. Moreover, it is thought that gene expression from the single active X is upregulated to correct for bi-allelic autosomal (A) gene expression. We have combined mouse genetics with RNA-seq on single mouse embryos to investigate functions of Rlim on the temporal regulation of iXCI and Xist. Our results reveal crucial roles of Rlim for the maintenance of high Xist RNA levels, Xist clouds and X-silencing in female embryos at blastocyst stages, while initial Xist expression appears Rlim-independent. We find further that X/A upregulation is initiated in early male and female preimplantation embryos. DOI: http://dx.doi.org/10.7554/eLife.19127.001 PMID:27642011

  3. Thalidomide induced early gene expression perturbations indicative of human embryopathy in mouse embryonic stem cells

    SciTech Connect

    Gao, Xiugong Sprando, Robert L.; Yourick, Jeffrey J.

    2015-08-15

    Developmental toxicity testing has traditionally relied on animal models which are costly, time consuming, and require the sacrifice of large numbers of animals. In addition, there are significant disparities between human beings and animals in their responses to chemicals. Thalidomide is a species-specific developmental toxicant that causes severe limb malformations in humans but not in mice. Here, we used microarrays to study transcriptomic changes induced by thalidomide in an in vitro model based on differentiation of mouse embryonic stem cells (mESCs). C57BL/6 mESCs were allowed to differentiate spontaneously and RNA was collected at 24, 48, and 72 h after exposure to 0.25 mM thalidomide. Global gene expression analysis using microarrays revealed hundreds of differentially expressed genes upon thalidomide exposure that were enriched in gene ontology (GO) terms and canonical pathways associated with embryonic development and differentiation. In addition, many genes were found to be involved in small GTPases-mediated signal transduction, heart development, and inflammatory responses, which coincide with clinical evidences and may represent critical embryotoxicities of thalidomide. These results demonstrate that transcriptomics in combination with mouse embryonic stem cell differentiation is a promising alternative model for developmental toxicity assessment. - Highlights: • Studied genomic changes in mouse embryonic stem cells upon thalidomide exposure • Identified gene expression changes that may represent thalidomide embryotoxicity • The toxicogenomic changes coincide well with known thalidomide clinical outcomes. • The mouse embryonic stem cell model is suitable for developmental toxicity testing. • The model has the potential for high-throughput screening of a multitude of compounds.

  4. Linkage analysis of the whirler deafness gene on mouse chromosome 4

    SciTech Connect

    Fleming, J.; Rogers, M.J.C.; Steel, K.P. ); Brown, S.D.M. )

    1994-05-01

    The whirler mouse harbors an autosomal recessive mutation on mouse chromosome 4 that causes deafness and vestibular dysfunction in the adult that is manifested as head-bobbing and circling behavior. Although there is no obvious human homologue for this mutation as yet, whirler is a potential mouse model for human autosomal recessive deafness. Many genetic markers for this region of mouse chromosome 4 are now available, and the authors have used these to construct genetic linkage maps in both inter- and intraspecific backcrosses as the first step toward the cloning of the whirler gene. A total of 19 loci were analyzed in these crosses, giving the following gene orders: Interspecific cross, centromere-(D4Mit5, D4Mit38)-D4Mit6-(Lv,Tzn,D4Mit44)-wi-Hxb-(D4Mit25, D4Nds9)-(D4Mit7, D4Ler2)-b-D4Mit45-(D4Wsm1, D4Mit27b)-(D4Rck65, D4Mit15), and intraspecific cross, centromere-(Mup-1, wi, Hxb)-b-D4Wsm1. This analysis has positioned the wi locus in the interval between the genes for [delta]-aminolevulinate dehydratase (Lv) and hexabrachion (Hxb). The human homologues of these genes, ALAD and HXB, both lie on human chromosome 9q32-q34. They therefore predict that a human homologue of the wi gene, involved in autosomal recessive deafness, lies in this region of conserved homology on 9q32-q34. 36 refs., 2 figs., 4 tabs.

  5. Glucocorticoid modulation of casein gene transcription in mouse mammary gland.

    PubMed Central

    Ganguly, R; Mehta, N M; Ganguly, N; Banerjee, M R

    1979-01-01

    The influence of cortisol and prolactin on casein gene expression in the mammary gland of lactating BALB/c mice was measured by using a specific cDNA probe to 15S casein mRNA (cDNAcsn). Casein mRNA (mRNAcsn) level in the mammary gland was decreased by 85% 5 days after adrenal ablation, but then was increased 4.4-fold 12 hr after a single injection of hydrocortisone-21-acetate. An 80% decrease in serum prolactin level, induced by the prolactin inhibitor 2-bromo-alpha-ergocryptin (CB-154), did not alter the level of mRNAcsn in the gland. Specific transcription of the casein gene in nuclei isolated from lactating mammary glands was measured by cDNAcsn hybridization to the in vitro synthesized Hg-CTP-containing RNA (Hg-RNA), which was purified by SH-agarose chromatography. The level of the mRNAcsn in Hg-RNA synthesized in the isolated nuclei was 0.09% and this was decreased 85% by alpha-amanitin, indicating that the mRNAcsn sequences in the Hg-RNA were the products of RNA polymerase II-directed DNA-dependent RNA synthesis. Transcription of the mRNAcsn in isolated nuclei was decreased by 70% 5 days after adrenalectomy and a single injection of the glucocorticoid then increased the transcription level 2-fold at 6 hr. Essentially no alteration of the level of transcription was detectable in mammary nuclei isolated from lactating mice with 80% decreased serum prolactin level, induced by CB-154 treatment. The results thus demonstrate a glucocorticoid involvement on the modulation of casein gene expression at the transcriptional level of control. PMID:293734

  6. Php4 Is a Key Player for Iron Economy in Meiotic and Sporulating Cells

    PubMed Central

    Brault, Ariane; Rallis, Charalampos; Normant, Vincent; Garant, Jean-Michel; Bähler, Jürg; Labbé, Simon

    2016-01-01

    Meiosis is essential for sexually reproducing organisms, including the fission yeast Schizosaccharomyces pombe. In meiosis, chromosomes replicate once in a diploid precursor cell (zygote), and then segregate twice to generate four haploid meiotic products, named spores in yeast. In S. pombe, Php4 is responsible for the transcriptional repression capability of the heteromeric CCAAT-binding factor to negatively regulate genes encoding iron-using proteins under low-iron conditions. Here, we show that the CCAAT-regulatory subunit Php4 is required for normal progression of meiosis under iron-limiting conditions. Cells lacking Php4 exhibit a meiotic arrest at metaphase I. Microscopic analyses of cells expressing functional GFP-Php4 show that it colocalizes with chromosomal material at every stage of meiosis under low concentrations of iron. In contrast, GFP-Php4 fluorescence signal is lost when cells undergo meiosis under iron-replete conditions. Global gene expression analysis of meiotic cells using DNA microarrays identified 137 genes that are regulated in an iron- and Php4-dependent manner. Among them, 18 genes are expressed exclusively during meiosis and constitute new putative Php4 target genes, which include hry1+ and mug14+. Further analysis validates that Php4 is required for maximal and timely repression of hry1+ and mug14+ genes. Using a chromatin immunoprecipitation approach, we show that Php4 specifically associates with hry1+ and mug14+ promoters in vivo. Taken together, the results reveal that in iron-starved meiotic cells, Php4 is essential for completion of the meiotic program since it participates in global gene expression reprogramming to optimize the use of limited available iron. PMID:27466270

  7. The Multi-Copy Mouse Gene Sycp3-Like Y-Linked (Sly) Encodes an Abundant Spermatid Protein That Interacts with a Histone Acetyltransferase and an Acrosomal Protein1

    PubMed Central

    Reynard, Louise N.; Cocquet, Julie; Burgoyne, Paul S.

    2009-01-01

    Deletion analysis has established that genes on the Y chromosome are essential for normal sperm production in humans, mice, and Drosophila. In mice, long-arm deletions have an impact on spermiogenesis, with the most extensive deletions resulting in severe sperm head malformations and infertility. Intriguingly, smaller deletions are compatible with fertility but result in a distorted sex ratio in favor of females, and recently it was found that Y long-arm deletions are also associated with a marked upregulation of several X-encoded and Y-encoded spermatid-expressed genes. The mouse Y long arm encodes a number of distinct transcripts, each of which derives from multiple gene copies. Of these multicopy genes, the recently described Sly has been favored as the gene underlying the spermiogenic defects associated with Y long-arm deletions. To assess the candidacy of Sly, the expression of this gene was examined in the testis at the transcript and protein levels. Sly is transcribed after the first meiotic division in secondary spermatocytes and round spermatids and encodes two transcript variants, Sly_v1 and Sly_v2 (proteins referred to as SLY1 and SLY2). We raised an antibody against SLY1 which detected the protein in round and early elongating spermatids, where it is predominantly cytoplasmic. Yeast two-hybrid and coimmunoprecipitation studies demonstrated that SLY1 interacts with the acrosomal protein DKKL1, the histone acetyltransferase KAT5 (also known as TIP60), and the microtubule-associated protein APPBP2. Together, these data suggest SLY1 may be involved in multiple processes during spermiogenesis, including the control of gene expression and the development or function of the acrosome. PMID:19176879

  8. Gene order is conserved within the human chromosome 21 linkage group on mouse chromosome 10

    SciTech Connect

    Irving, N.G.; Cabin, D.E.; Swanson, D.A.; Reeves, R.H. )

    1994-05-01

    One hundred progeny from each of two intersubspecific mouse backcrosses were used to construct a comparative genetic map of a region of mouse chromosome 10 (MMU10) that is homologous to the distal tip of the long arm of human chromosome 21 (HSA21). The analysis included five genes and three simple sequence repeat markers, two of which flanked the HSA21-homologous cluster on either side. Analysis of 200 backcross progeny detected at least one crossover between each pair of adjacent genes and demonstrated that the proximal to distal orientation of the cluster was reversed between human and mouse. The order was determined to be Fyn-1-D10Mit20-S100b-Col6a1-Itgb2-Pfk1/D10Mit7-D10Mit11. Comparative mapping supports the order of corresponding markers on HSA21 determined using pulsed-field gel electrophoresis and radiation hybrid line data. However, sequence tagged site content mapping of human yeast artificial chromosomes (YACs) yielded conflicting data on the relative positions of human COL6A1 and S100B on HSA21. This discrepancy was resolved here by demonstrating that several key YACs used in the human contig analysis were mistyped for S100B. The murine map reported here provides a scaffold for construction of physical maps and yeast artificial chromosome contigs that will be useful in the development of mouse models for the study of Down syndrome. 28 refs., 4 figs., 2 tabs.

  9. The mouse collagen X gene: complete nucleotide sequence, exon structure and expression pattern.

    PubMed Central

    Elima, K; Eerola, I; Rosati, R; Metsäranta, M; Garofalo, S; Perälä, M; De Crombrugghe, B; Vuorio, E

    1993-01-01

    Overlapping genomic clones covering the 7.2 kb mouse alpha 1(X) collagen gene, 0.86 kb of promoter and 1.25 kb of 3'-flanking sequences were isolated from two genomic libraries and characterized by nucleotide sequencing. Typical features of the gene include a unique three-exon structure, similar to that in the chick gene, with the entire triple-helical domain of 463 amino acids coded by a single large exon. The highest degree of amino acid and nucleotide sequence conservation was seen in the coding region for the collagenous and C-terminal non-collagenous domains between the mouse and known chick, bovine and human collagen type X sequences. More divergence between the sequences occurred in the N-terminal non-collagenous domain. Similarity between the mammalian collagen X sequences extended into the 3'-untranslated sequence, particularly near the polyadenylation site. The promoter of the mouse collagen X gene was found to contain two TATAA boxes 159 bp apart; primer extension analyses of the transcription start site revealed that both were functional. The promoter has an unusual structure with a very low G + C content of 28% between positions -220 and -1 of the upstream transcription start site. Northern and in situ hybridization analyses confirmed that the expression of the alpha 1(X) collagen gene is restricted to hypertrophic chondrocytes in tissues undergoing endochondral calcification. The detailed sequence information of the gene is useful for studies on the promoter activity of the gene and for generation of transgenic mice. Images Figure 3 Figure 5 Figure 6 PMID:8424763

  10. Alternative Induction of Meiotic Recombination From Single-Base Lesions of DNA Deaminases

    PubMed Central

    Pauklin, Siim; Burkert, Julia S.; Martin, Julie; Osman, Fekret; Weller, Sandra; Boulton, Simon J.; Whitby, Matthew C.; Petersen-Mahrt, Svend K.

    2009-01-01

    Meiotic recombination enhances genetic diversity as well as ensures proper segregation of homologous chromosomes, requiring Spo11-initiated double-strand breaks (DSBs). DNA deaminases act on regions of single-stranded DNA and deaminate cytosine to uracil (dU). In the immunoglobulin locus, this lesion will initiate point mutations, gene conversion, and DNA recombination. To begin to delineate the effect of induced base lesions on meiosis, we analyzed the effect of expressing DNA deaminases (activation-induced deaminase, AID, and APOBEC3C) in germ cells. We show that meiotic dU:dG lesions can partially rescue a spo11Δ phenotype in yeast and worm. In rec12 Schizosaccharomyces pombe, AID expression increased proper chromosome segregation, thereby enhancing spore viability, and induced low-frequency meiotic crossovers. Expression of AID in the germ cells of Caenorhabditis elegans spo-11 induced meiotic RAD-51 foci formation and chromosomal bivalency and segregation, as well as an increase in viability. RNAi experiments showed that this rescue was dependent on uracil DNA-glycosylase (Ung). Furthermore, unlike ionizing radiation-induced spo-11 rescue, AID expression did not induce large numbers of DSBs during the rescue. This suggests that the products of DNA deamination and base excision repair, such as uracil, an abasic site, or a single-stranded nick, are sufficient to initiate and alter meiotic recombination in uni- and multicellular organisms. PMID:19237686

  11. A sex-ratio Meiotic Drive System in Drosophila simulans. II: An X-linked Distorter

    PubMed Central

    Tao, Yun; Araripe, Luciana; Kingan, Sarah B; Ke, Yeyan; Xiao, Hailian; Hartl, Daniel L

    2007-01-01

    The evolution of heteromorphic sex chromosomes creates a genetic condition favoring the invasion of sex-ratio meiotic drive elements, resulting in the biased transmission of one sex chromosome over the other, in violation of Mendel's first law. The molecular mechanisms of sex-ratio meiotic drive may therefore help us to understand the evolutionary forces shaping the meiotic behavior of the sex chromosomes. Here we characterize a sex-ratio distorter on the X chromosome (Dox) in Drosophila simulans by genetic and molecular means. Intriguingly, Dox has very limited coding capacity. It evolved from another X-linked gene, which also evolved de nova. Through retrotransposition, Dox also gave rise to an autosomal suppressor, not much yang (Nmy). An RNA interference mechanism seems to be involved in the suppression of the Dox distorter by the Nmy suppressor. Double mutant males of the genotype dox; nmy are normal for both sex-ratio and spermatogenesis. We postulate that recurrent bouts of sex-ratio meiotic drive and its subsequent suppression might underlie several common features observed in the heterogametic sex, including meiotic sex chromosome inactivation and achiasmy. PMID:17988173

  12. Meiotic failure in male mice lacking an X-linked factor

    PubMed Central

    Yang, Fang; Gell, Katarina; van der Heijden, Godfried W.; Eckardt, Sigrid; Leu, N. Adrian; Page, David C.; Benavente, Ricardo; Her, Chengtao; Höög, Christer; McLaughlin, K. John; Wang, Peijing Jeremy

    2008-01-01

    Meiotic silencing of sex chromosomes may cause their depletion of meiosis-specific genes during evolution. Here, we challenge this hypothesis by reporting the identification of TEX11 as the first X-encoded meiosis-specific factor in mice. TEX11 forms discrete foci on synapsed regions of meiotic chromosomes and appears to be a novel constituent of meiotic nodules involved in recombination. Loss of TEX11 function causes chromosomal asynapsis and reduced crossover formation, leading to elimination of spermatocytes, respectively, at the pachytene and anaphase I stages. Specifically, TEX11-deficient spermatocytes with asynapsed autosomes undergo apoptosis at the pachytene stage, while those with only asynapsed sex chromosomes progress. However, cells that survive the pachytene stage display chromosome nondisjunction at the first meiotic division, resulting in cell death and male infertility. TEX11 interacts with SYCP2, which is an integral component of the synaptonemal complex lateral elements. Thus, TEX11 promotes initiation and/or maintenance of synapsis and formation of crossovers, and may provide a physical link between these two meiotic processes. PMID:18316482

  13. Meiotic failure in male mice lacking an X-linked factor.

    PubMed

    Yang, Fang; Gell, Katarina; van der Heijden, Godfried W; Eckardt, Sigrid; Leu, N Adrian; Page, David C; Benavente, Ricardo; Her, Chengtao; Höög, Christer; McLaughlin, K John; Wang, Peijing Jeremy

    2008-03-01

    Meiotic silencing of sex chromosomes may cause their depletion of meiosis-specific genes during evolution. Here, we challenge this hypothesis by reporting the identification of TEX11 as the first X-encoded meiosis-specific factor in mice. TEX11 forms discrete foci on synapsed regions of meiotic chromosomes and appears to be a novel constituent of meiotic nodules involved in recombination. Loss of TEX11 function causes chromosomal asynapsis and reduced crossover formation, leading to elimination of spermatocytes, respectively, at the pachytene and anaphase I stages. Specifically, TEX11-deficient spermatocytes with asynapsed autosomes undergo apoptosis at the pachytene stage, while those with only asynapsed sex chromosomes progress. However, cells that survive the pachytene stage display chromosome nondisjunction at the first meiotic division, resulting in cell death and male infertility. TEX11 interacts with SYCP2, which is an integral component of the synaptonemal complex lateral elements. Thus, TEX11 promotes initiation and/or maintenance of synapsis and formation of crossovers, and may provide a physical link between these two meiotic processes.

  14. Disruption of Chtf18 Causes Defective Meiotic Recombination in Male Mice

    PubMed Central

    Berkowitz, Karen M.; Sowash, Aislinn R.; Koenig, Lydia R.; Urcuyo, Dawnette; Khan, Fahmida; Yang, Fang; Wang, P. Jeremy; Jongens, Thomas A.; Kaestner, Klaus H.

    2012-01-01

    CHTF18 (chromosome transmission fidelity factor 18) is an evolutionarily conserved subunit of the Replication Factor C-like complex, CTF18-RLC. CHTF18 is necessary for the faithful passage of chromosomes from one daughter cell to the next during mitosis in yeast, and it is crucial for germline development in the fruitfly. Previously, we showed that mouse Chtf18 is expressed throughout the germline, suggesting a role for CHTF18 in mammalian gametogenesis. To determine the role of CHTF18 in mammalian germ cell development, we derived mice carrying null and conditional mutations in the Chtf18 gene. Chtf18-null males exhibit 5-fold decreased sperm concentrations compared to wild-type controls, resulting in subfertility. Loss of Chtf18 results in impaired spermatogenesis; spermatogenic cells display abnormal morphology, and the stereotypical arrangement of cells within seminiferous tubules is perturbed. Meiotic recombination is defective and homologous chromosomes separate prematurely during prophase I. Repair of DNA double-strand breaks is delayed and incomplete; both RAD51 and γH2AX persist in prophase I. In addition, MLH1 foci are decreased in pachynema. These findings demonstrate essential roles for CHTF18 in mammalian spermatogenesis and meiosis, and suggest that CHTF18 may function during the double-strand break repair pathway to promote the formation of crossovers. PMID:23133398

  15. Rapid and efficient gene delivery into the adult mouse brain via focal electroporation

    PubMed Central

    Nomura, Tadashi; Nishimura, Yusuke; Gotoh, Hitoshi; Ono, Katsuhiko

    2016-01-01

    In vivo gene delivery is required for studying the cellular and molecular mechanisms of various biological events. Virus-mediated gene transfer or generation of transgenic animals is widely used; however, these methods are time-consuming and expensive. Here we show an improved electroporation technique for acute gene delivery into the adult mouse brain. Using a syringe-based microelectrode, local DNA injection and the application of electric current can be performed simultaneously; this allows rapid and efficient gene transduction of adult non-neuronal cells. Combining this technique with various expression vectors that carry specific promoters resulted in targeted gene expression in astrocytic cells. Our results constitute a powerful strategy for the genetic manipulation of adult brains in a spatio-temporally controlled manner. PMID:27430903

  16. A meta-analysis study of gene expression datasets in mouse liver under PPARα knockout.

    PubMed

    He, Kan; Wang, Zhen; Wang, Qishan; Pan, Yuchun

    2013-06-01

    Gene expression profiling of peroxisome-proliferator-activated receptor α (PPARα) has been used in several studies, but there were no consistent results on gene expression patterns involved in PPARα activation in genome-wide due to different sample sizes or platforms. Here, we employed two published microarray datasets both PPARα dependent in mouse liver and applied meta-analysis on them to increase the power of the identification of differentially expressed genes and significantly enriched pathways. As a result, we have improved the concordance in identifying many biological mechanisms involved in PPARα activation. We suggest that our analysis not only leads to more identified genes by combining datasets from different resources together, but also provides some novel hepatic tissue-specific marker genes related to PPARα according to our re-analysis. PMID:23938112

  17. Rapid and efficient gene delivery into the adult mouse brain via focal electroporation.

    PubMed

    Nomura, Tadashi; Nishimura, Yusuke; Gotoh, Hitoshi; Ono, Katsuhiko

    2016-01-01

    In vivo gene delivery is required for studying the cellular and molecular mechanisms of various biological events. Virus-mediated gene transfer or generation of transgenic animals is widely used; however, these methods are time-consuming and expensive. Here we show an improved electroporation technique for acute gene delivery into the adult mouse brain. Using a syringe-based microelectrode, local DNA injection and the application of electric current can be performed simultaneously; this allows rapid and efficient gene transduction of adult non-neuronal cells. Combining this technique with various expression vectors that carry specific promoters resulted in targeted gene expression in astrocytic cells. Our results constitute a powerful strategy for the genetic manipulation of adult brains in a spatio-temporally controlled manner. PMID:27430903

  18. The mouse formin (Fmn) gene: Genomic structure, novel exons, and genetic mapping

    SciTech Connect

    Wang, C.C.; Chan, D.C.; Leder, P.

    1997-02-01

    Mutations in the mouse formin (Fmn) gene, formerly known as the limb deformity (ld) gene, give rise to recessively inherited limb deformities and renal malformations or aplasia. The Fmn gene encodes many differentially processed transcripts that are expressed in both adult and embryonic tissues. To study the genomic organization of the Fmn locus, we have used Fmn probes to isolate and characterize genomic clones spanning 500 kb. Our analysis of these clones shows that the Fmn gene is composed of at least 24 exons and spans 400 kb. We have identified two novel exons that are expressed in the developing embryonic limb bud as well as adult tissues such as brain and kidney. We have also used a microsatellite polymorphism from within the Fmn gene to map it genetically to a 2.2-cM interval between D2Mit58 and D2Mit103. 36 refs., 6 figs., 1 tab.

  19. Random Monoallelic Expression of Three Genes Clustered within 60 kb of Mouse t Complex Genomic DNA

    PubMed Central

    Sano, Yuri; Shimada, Tokihiko; Nakashima, Hiroshi; Nicholson, Rhonda H.; Eliason, James F.; Kocarek, Thomas A.; Ko, Minoru S.H.

    2001-01-01

    Mammals achieve gene dosage control by (1) random X-chromosome inactivation in females, (2) parental origin-specific imprinting of selected autosomal genes, and (3) random autosomal inactivation. Genes belonging to the third category of epigenetic phenomenon are just now emerging, with only six identified so far. Here we report three additional genes, Nubp2, Igfals, and Jsap1, that show 50%-methylated CpG sites by Southern blot analyses and primarily monoallelic expression in single-cell allele-specific RT-PCR analysis of bone marrow stromal cells and hepatocytes. Furthermore, we show that, in contrast to X inactivation, alleles can switch between active and inactive states during the formation of daughter cells. These three genes are the first in their category to exist as a tight cluster, in the proximal region of mouse chromosome 17, providing a thus far unique example of a region of autosomal random monoallelic expression. PMID:11691847

  20. Meiotic silencing by unpaired DNA: properties, regulation and suppression.

    PubMed

    Shiu, Patrick K T; Metzenberg, Robert L

    2002-08-01

    In Neurospora, a gene not paired with a homolog in prophase I of meiosis generates a signal that transiently silences all sequences homologous to it by a process called meiotic silencing by unpaired DNA (MSUD). Thus a deletion mutation in a heterozygous cross is formally "ascus-dominant" because its unpaired wild-type partner silences itself. We describe in detail the isolation of a mutation, Sad-1(UV), that suppresses the dominance of various ascus-dominant mutations. Additional dominant, semidominant, and recessive Sad-1 alleles have been generated by RIP; the DNA of the dominant RIP alleles becomes methylated, but dim-2-dependent methylation is not necessary for silencing. The barrenness of homozygous Sad-1 crosses is not due to the failure to silence unpaired mating-type mat A-2 mat A-3 genes. Transcripts of sad-1(+) can be detected during the sexual phase in a homozygous wild-type cross, indicating that the gene is expressed even if all DNA can pair normally. Meiotic silencing is confined to the ascus in which DNA is unpaired, and silencing does not spread to neighboring asci in a fruiting body of mixed genetic constitution.

  1. [Site of fidget gene action and its interaction with the ocular retardation gene in cultured mouse embryo retinas].

    PubMed

    Koniukhov, B V; Ugol'kova, T S

    1978-01-01

    The eye rudiments of 10 and 11 days old mouse embryos of the genotypes +/+ +/+, fi/fi +/+, +/+ or/or, fi/fi or/or and 11 days old embryos of the genotype fi/+ or/+ were cultivated in vitro during 24, 48 and 72 hrs. The expression of the fi gene was shown in the cells of the cultivated fi/fi +/+ retina: its proliferative activity was inhibited. The fi gene was not active in the cells of the developing lens and the anomalies of the latter in homozygotes arose secondarily, due to the inhibition of growth of the retinal rudiment. The fi and or genes interacted synergically in the cultivated fi/fi or/or retina, thus resulting in the marked inhibition of its mitotic activity. This suggests that both the genes act in the retinal cells and, apparently, affect different links of the biochemical chain of events in the preparation of DNA replication.

  2. Double replacement gene targeting for the production of a series of mouse strains with different prion protein gene alterations

    SciTech Connect

    Moore, R.C.; Redhead, N.J.; Selfridge, J.

    1995-09-01

    We have developed a double replacement gene targeting strategy which enables the production of a series of mouse strains bearing different subtle alterations to endogenous genes. This is a two-step process in which a region of the gene of interest is first replaced with a selectable marker to produce an inactivated allele, which is then re-targeted with a second vector to reconstruct the inactivated allele, concomitantly introducing an engineered mutation. Five independent embryonic stem cell lines have been produced bearing different targeted alterations to the prion protein gene, including one which raises the level of expression. We have constructed mice bearing the codon 101 proline to leucine substitution linked to the human familial prion disease, Gerstmann-Straussler-Scheinker syndrome. We anticipate that this procedure will have applications to the study of human inherited diseases and the development of therapies. 43 refs., 6 figs., 1 tab.

  3. AAV-mediated gene therapy in mouse models of recessive retinal degeneration

    PubMed Central

    Pang, Ji-jing; Lei, Lei; Dai, Xufeng; Shi, Wei; Liu, Xuan; Dinculescu, Astra; McDowell, J. Hugh

    2013-01-01

    In recent years, more and more mutant genes that cause retinal diseases have been detected. At the same time, many naturally occurring mouse models of retinal degeneration have also been found, which show similar changes to human retinal diseases. These, together with improved viral vector quality allow more and more traditionally incurable inherited retinal disorders to become potential candidates for gene therapy. Currently, the most common vehicle to deliver the therapeutic gene into target retinal cells is the adeno-associated viral vector (AAV). Following delivery to the immuno-priviledged subretinal space, AAV-vectors can efficiently target both retinal pigment epithelium and photoreceptor cells, the origin of most retinal degenerations. This review focuses on the AAV-based gene therapy in mouse models of recessive retinal degenerations, especially those in which delivery of the correct copy of the wild-type gene has led to significant beneficial effects on visual function, as determined by morphological, biochemical, electroretinographic and behavioral analysis. The past studies in animal models and ongoing successful LCA2 clinical trials, predict a bright future for AAV gene replacement treatment for inherited recessive retinal diseases. PMID:22300136

  4. Systems Biology-Based Identification of Mycobacterium tuberculosis Persistence Genes in Mouse Lungs

    PubMed Central

    Dutta, Noton K.; Bandyopadhyay, Nirmalya; Veeramani, Balaji; Lamichhane, Gyanu; Karakousis, Petros C.; Bader, Joel S.

    2014-01-01

    ABSTRACT Identifying Mycobacterium tuberculosis persistence genes is important for developing novel drugs to shorten the duration of tuberculosis (TB) treatment. We developed computational algorithms that predict M. tuberculosis genes required for long-term survival in mouse lungs. As the input, we used high-throughput M. tuberculosis mutant library screen data, mycobacterial global transcriptional profiles in mice and macrophages, and functional interaction networks. We selected 57 unique, genetically defined mutants (18 previously tested and 39 untested) to assess the predictive power of this approach in the murine model of TB infection. We observed a 6-fold enrichment in the predicted set of M. tuberculosis genes required for persistence in mouse lungs relative to randomly selected mutant pools. Our results also allowed us to reclassify several genes as required for M. tuberculosis persistence in vivo. Finally, the new results implicated additional high-priority candidate genes for testing. Experimental validation of computational predictions demonstrates the power of this systems biology approach for elucidating M. tuberculosis persistence genes. PMID:24549847

  5. Expression Profile of DNA Damage Signaling Genes in Proton Exposed Mouse Brain

    NASA Astrophysics Data System (ADS)

    Ramesh, Govindarajan; Wu, Honglu

    Exposure of living systems to radiation results in a wide assortment of lesions, the most signif-icant of is damage to genomic DNA which induce several cellular functions such as cell cycle arrest, repair, apoptosis etc. The radiation induced DNA damage investigation is one of the im-portant area in biology, but still the information available regarding the effects of proton is very limited. In this report, we investigated the differential gene expression pattern of DNA damage signaling genes particularly, damaged DNA binding, repair, cell cycle arrest, checkpoints and apoptosis using quantitative real-time RT-PCR array in proton exposed mouse brain tissues. The expression profiles showed significant changes in DNA damage related genes in 2Gy proton exposed mouse brain tissues as compared with control brain tissues. Furthermore, we also show that significantly increased levels of apoptotic related genes, caspase-3 and 8 activities in these cells, suggesting that in addition to differential expression of DNA damage genes, the alteration of apoptosis related genes may also contribute to the radiation induced DNA damage followed by programmed cell death. In summary, our findings suggest that proton exposed brain tissue undergo severe DNA damage which in turn destabilize the chromatin stability.

  6. Glutamate-related gene expression changes with age in the mouse auditory midbrain.

    PubMed

    Tadros, Sherif F; D'Souza, Mary; Zettel, Martha L; Zhu, Xiaoxia; Waxmonsky, Nicole C; Frisina, Robert D

    2007-01-01

    Glutamate is the main excitatory neurotransmitter in both the peripheral and central auditory systems. Changes of glutamate and glutamate-related genes with age may be an important factor in the pathogenesis of age-related hearing loss-presbycusis. In this study, changes in glutamate-related mRNA gene expression in the CBA mouse inferior colliculus with age and hearing loss were examined and correlations were sought between these changes and functional hearing measures, such as the auditory brainstem response (ABR) and distortion product otoacoustic emissions (DPOAEs). Gene expression of 68 glutamate-related genes was investigated using both genechip microarray and real-time PCR (qPCR) molecular techniques for four different age/hearing loss CBA mouse subject groups. Two genes showed consistent differences between groups for both the genechip and qPCR. Pyrroline-5-carboxylate synthetase enzyme (Pycs) showed down-regulation with age and a high-affinity glutamate transporter (Slc1a3) showed up-regulation with age and hearing loss. Since Pycs plays a role in converting glutamate to proline, its deficiency in old age may lead to both glutamate increases and proline deficiencies in the auditory midbrain, playing a role in the subsequent inducement of glutamate toxicity and loss of proline neuroprotective effects. The up-regulation of Slc1a3 gene expression may reflect a cellular compensatory mechanism to protect against age-related glutamate or calcium excitoxicity.

  7. A brain-specific gene cluster isolated from the region of the mouse obesity locus is expressed in the adult hypothalamus and during mouse development

    SciTech Connect

    Laig-Webster, M.; Lim, M.E.; Chehab, F.F.

    1994-09-01

    The molecular defect underlying an autosomal recessive form of genetic obesity in a classical mouse model C57 BL/6J-ob/ob has not yet been elucidated. Whereas metabolic and physiological disturbances such as diabetes and hypertension are associated with obesity, the site of expression and the nature of the primary lesion responsible for this cascade of events remains elusive. Our efforts aimed at the positional cloning of the ob gene by YAC contig mapping and gene identification have resulted in the cloning of a brain-specific gene cluster from the ob critical region. The expression of this gene cluster is remarkably complex owing to the multitude of brain-specific mRNA transcripts detected on Northern blots. cDNA cloning of these transcripts suggests that they are expressed from different genes as well as by alternate splicing mechanisms. Furthermore, the genomic organization of the cluster appears to consist of at least two identical promoters displaying CpG islands characteristic of housekeeping genes, yet clearly involving tissue-specific expression. Sense and anti-sense synthetic RNA probes were derived from a common DNA sequence on 3 cDNA clones and hybridized to 8-16 days mouse embryonic stages and mouse adult brain sections. Expression in development was noticeable as of the 11th day of gestation and confined to the central nervous system mainly in the telencephalon and spinal cord. Coronal and sagittal sections of the adult mouse brain showed expression only in 3 different regions of the brain stem. In situ hybridization to mouse hypothalamus sections revealed the presence of a localized and specialized group of cells expressing high levels of mRNA, suggesting that this gene cluster may also be involved in the regulation of hypothalamic activities. The hypothalamus has long been hypothesized as a primary candidate tissue for the expression of the obesity gene mainly because of its well-established role in the regulation of energy metabolism and food intake.

  8. In vivo gene editing in dystrophic mouse muscle and muscle stem cells.

    PubMed

    Tabebordbar, Mohammadsharif; Zhu, Kexian; Cheng, Jason K W; Chew, Wei Leong; Widrick, Jeffrey J; Yan, Winston X; Maesner, Claire; Wu, Elizabeth Y; Xiao, Ru; Ran, F Ann; Cong, Le; Zhang, Feng; Vandenberghe, Luk H; Church, George M; Wagers, Amy J

    2016-01-22

    Frame-disrupting mutations in the DMD gene, encoding dystrophin, compromise myofiber integrity and drive muscle deterioration in Duchenne muscular dystrophy (DMD). Removing one or more exons from the mutated transcript can produce an in-frame mRNA and a truncated, but still functional, protein. In this study, we developed and tested a direct gene-editing approach to induce exon deletion and recover dystrophin expression in the mdx mouse model of DMD. Delivery by adeno-associated virus (AAV) of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 endonucleases coupled with paired guide RNAs flanking the mutated Dmd exon23 resulted in excision of intervening DNA and restored the Dmd reading frame in myofibers, cardiomyocytes, and muscle stem cells after local or systemic delivery. AAV-Dmd CRISPR treatment partially recovered muscle functional deficiencies and generated a pool of endogenously corrected myogenic precursors in mdx mouse muscle.

  9. Endocrine parameters and phenotypes of the growth hormone receptor gene disrupted (GHR-/-) mouse.

    PubMed

    List, Edward O; Sackmann-Sala, Lucila; Berryman, Darlene E; Funk, Kevin; Kelder, Bruce; Gosney, Elahu S; Okada, Shigeru; Ding, Juan; Cruz-Topete, Diana; Kopchick, John J

    2011-06-01

    Disruption of the GH receptor (GHR) gene eliminates GH-induced intracellular signaling and, thus, its biological actions. Therefore, the GHR gene disrupted mouse (GHR-/-) has been and is a valuable tool for helping to define various parameters of GH physiology. Since its creation in 1995, this mouse strain has been used by our laboratory and others for numerous studies ranging from growth to aging. Some of the most notable discoveries are their extreme insulin sensitivity in the presence of obesity. Also, the animals have an extended lifespan, which has generated a large number of investigations into the roles of GH and IGF-I in the aging process. This review summarizes the many results derived from the GHR-/- mice. We have attempted to present the findings in the context of current knowledge regarding GH action and, where applicable, to discuss how these mice compare to GH insensitivity syndrome in humans. PMID:21123740

  10. In vivo gene editing in dystrophic mouse muscle and muscle stem cells#

    PubMed Central

    Cheng, Jason K.W.; Chew, Wei Leong; Widrick, Jeffrey J.; Yan, Winston X.; Maesner, Claire; Wu, Elizabeth Y.; Xiao, Ru; Ran, F. Ann; Cong, Le; Zhang, Feng; Vandenberghe, Luk H.; Church, George M.; Wagers, Amy J.

    2016-01-01

    Frame-disrupting mutations in the DMD gene, encoding dystrophin, compromise myofiber integrity and drive muscle deterioration in Duchenne muscular dystrophy (DMD). Removing one or more exons from the mutated transcript can produce an in-frame mRNA and a truncated but still functional protein. In this study, we develop and test a direct gene editing approach to induce exon deletion and recover dystrophin expression in the mdx mouse model of DMD. Delivery by adeno-associated virus (AAV) of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 endonucleases coupled with paired guide RNAs flanking the mutated Dmd exon23 resulted in excision of intervening DNA and restored Dystrophin reading frame in myofibers, cardiomyocytes and muscle stem cells following local or systemic delivery. AAV-Dmd CRISPR-treatment partially recovered muscle functional deficiencies and generated a pool of endogenously corrected myogenic precursors in mdx mouse muscle. PMID:26721686

  11. Endocrine Parameters and Phenotypes of the Growth Hormone Receptor Gene Disrupted (GHR−/−) Mouse

    PubMed Central

    List, Edward O.; Sackmann-Sala, Lucila; Berryman, Darlene E.; Funk, Kevin; Kelder, Bruce; Gosney, Elahu S.; Okada, Shigeru; Ding, Juan; Cruz-Topete, Diana

    2011-01-01

    Disruption of the GH receptor (GHR) gene eliminates GH-induced intracellular signaling and, thus, its biological actions. Therefore, the GHR gene disrupted mouse (GHR−/−) has been and is a valuable tool for helping to define various parameters of GH physiology. Since its creation in 1995, this mouse strain has been used by our laboratory and others for numerous studies ranging from growth to aging. Some of the most notable discoveries are their extreme insulin sensitivity in the presence of obesity. Also, the animals have an extended lifespan, which has generated a large number of investigations into the roles of GH and IGF-I in the aging process. This review summarizes the many results derived from the GHR−/− mice. We have attempted to present the findings in the context of current knowledge regarding GH action and, where applicable, to discuss how these mice compare to GH insensitivity syndrome in humans. PMID:21123740

  12. Extensive compensatory cis-trans regulation in the evolution of mouse gene expression

    PubMed Central

    Goncalves, Angela; Leigh-Brown, Sarah; Thybert, David; Stefflova, Klara; Turro, Ernest; Flicek, Paul; Brazma, Alvis; Odom, Duncan T.; Marioni, John C.

    2012-01-01

    Gene expression levels are thought to diverge primarily via regulatory mutations in trans within species, and in cis between species. To test this hypothesis in mammals we used RNA-sequencing to measure gene expression divergence between C57BL/6J and CAST/EiJ mouse strains and allele-specific expression in their F1 progeny. We identified 535 genes with parent-of-origin specific expression patterns, although few of these showed full allelic silencing. This suggests that the number of imprinted genes in a typical mouse somatic tissue is relatively small. In the set of nonimprinted genes, 32% showed evidence of divergent expression between the two strains. Of these, 2% could be attributed purely to variants acting in trans, while 43% were attributable only to variants acting in cis. The genes with expression divergence driven by changes in trans showed significantly higher sequence constraint than genes where the divergence was explained by variants acting in cis. The remaining genes with divergent patterns of expression (55%) were regulated by a combination of variants acting in cis and variants acting in trans. Intriguingly, the changes in expression induced by the cis and trans variants were in opposite directions more frequently than expected by chance, implying that compensatory regulation to stabilize gene expression levels is widespread. We propose that expression levels of genes regulated by this mechanism are fine-tuned by cis variants that arise following regulatory changes in trans, suggesting that many cis variants are not the primary targets of natural selection. PMID:22919075

  13. From zebrafish heart jogging genes to mouse and human orthologs: using Gene Ontology to investigate mammalian heart development.

    PubMed

    Khodiyar, Varsha K; Howe, Doug; Talmud, Philippa J; Breckenridge, Ross; Lovering, Ruth C

    2013-01-01

    For the majority of organs in developing vertebrate embryos, left-right asymmetry is controlled by a ciliated region; the left-right organizer node in the mouse and human, and the Kuppfer's vesicle in the zebrafish. In the zebrafish, laterality cues from the Kuppfer's vesicle determine asymmetry in the developing heart, the direction of 'heart jogging' and the direction of 'heart looping'.  'Heart jogging' is the term given to the process by which the symmetrical zebrafish heart tube is displaced relative to the dorsal midline, with a leftward 'jog'. Heart jogging is not considered to occur in mammals, although a leftward shift of the developing mouse caudal heart does occur prior to looping, which may be analogous to zebrafish heart jogging. Previous studies have characterized 30 genes involved in zebrafish heart jogging, the majority of which have well defined orthologs in mouse and human and many of these orthologs have been associated with early mammalian heart development.    We undertook manual curation of a specific set of genes associated with heart development and we describe the use of Gene Ontology term enrichment analyses to examine the cellular processes associated with heart jogging.  We found that the human, mouse and zebrafish 'heart jogging orthologs' are involved in similar organ developmental processes across the three species, such as heart, kidney and nervous system development, as well as more specific cellular processes such as cilium development and function. The results of these analyses are consistent with a role for cilia in the determination of left-right asymmetry of many internal organs, in addition to their known role in zebrafish heart jogging.    This study highlights the importance of model organisms in the study of human heart development, and emphasises both the conservation and divergence of developmental processes across vertebrates, as well as the limitations of this approach.

  14. HIM-8 Binds to the X Chromosome Pairing Center and Mediates Chromosome-Specific Meiotic Synapsis

    PubMed Central

    Phillips, Carolyn M.; Wong, Chihunt; Bhalla, Needhi; Carlton, Peter M.; Weiser, Pinky; Meneely, Philip M.; Dernburg, Abby F.

    2015-01-01

    SUMMARY The him-8 gene is essential for proper meiotic segregation of the X chromosomes in C. elegans. Here we show that loss of him-8 function causes profound X chromosome-specific defects in homolog pairing and synapsis. him-8 encodes a C2H2 zinc-finger protein that is expressed during meiosis and concentrates at a site on the X chromosome known as the meiotic pairing center (PC). A role for HIM-8 in PC function is supported by genetic interactions between PC lesions and him-8 mutations. HIM-8 bound chromosome sites associate with the nuclear envelope (NE) throughout meiotic prophase. Surprisingly, a point mutation in him-8 that retains both chromosome binding and NE localization fails to stabilize pairing or promote synapsis. These observations indicate that stabilization of homolog pairing is an active process in which the tethering of chromosome sites to the NE may be necessary but is not sufficient. PMID:16360035

  15. Mnd1p: an evolutionarily conserved protein required for meiotic recombination.

    PubMed

    Gerton, Jennifer L; DeRisi, Joseph L

    2002-05-14

    We used a functional genomics approach to identify a gene required for meiotic recombination, YGL183c or MND1. MND1 was spliced in meiotic cells, extending the annotated YGL183c ORF N terminus by 45 aa. Saccharomyces cerevisiae mnd1-1 mutants, in which the majority of the MND1 coding sequence was removed, arrested before the first meiotic division with a phenotype reminiscent of dmc1 mutants. Physical and genetic analysis showed that these cells initiated recombination, but did not form heteroduplex DNA or double Holliday junctions, suggesting that Mnd1p is involved in strand invasion. Orthologs of MND1 were identified in protists, several yeasts, plants, and mammals, suggesting that its function has been conserved throughout evolution.

  16. Searching for a Spore killer: A meiotic drive element in Neurospora fungi

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mendelian inheritance predicts that different alleles of the same gene will have an equal chance of being transmitted to the next generation. However, meiotic drive is a phenomenon where certain alleles evolve the ability to bias transmission in their own favor. In this study we are investigating a ...

  17. Meta-analysis of differentiating mouse embryonic stem cell gene expression kinetics reveals early change of a small gene set.

    PubMed

    Glover, Clive H; Marin, Michael; Eaves, Connie J; Helgason, Cheryl D; Piret, James M; Bryan, Jennifer

    2006-11-24

    Stem cell differentiation involves critical changes in gene expression. Identification of these should provide endpoints useful for optimizing stem cell propagation as well as potential clues about mechanisms governing stem cell maintenance. Here we describe the results of a new meta-analysis methodology applied to multiple gene expression datasets from three mouse embryonic stem cell (ESC) lines obtained at specific time points during the course of their differentiation into various lineages. We developed methods to identify genes with expression changes that correlated with the altered frequency of functionally defined, undifferentiated ESC in culture. In each dataset, we computed a novel statistical confidence measure for every gene which captured the certainty that a particular gene exhibited an expression pattern of interest within that dataset. This permitted a joint analysis of the datasets, despite the different experimental designs. Using a ranking scheme that favored genes exhibiting patterns of interest, we focused on the top 88 genes whose expression was consistently changed when ESC were induced to differentiate. Seven of these (103728_at, 8430410A17Rik, Klf2, Nr0b1, Sox2, Tcl1, and Zfp42) showed a rapid decrease in expression concurrent with a decrease in frequency of undifferentiated cells and remained predictive when evaluated in additional maintenance and differentiating protocols. Through a novel meta-analysis, this study identifies a small set of genes whose expression is useful for identifying changes in stem cell frequencies in cultures of mouse ESC. The methods and findings have broader applicability to understanding the regulation of self-renewal of other stem cell types.

  18. Left-Right Function of dmrt2 Genes Is Not Conserved between Zebrafish and Mouse

    PubMed Central

    Lourenço, Raquel; Lopes, Susana S.; Saúde, Leonor

    2010-01-01

    Background Members of the Dmrt family, generally associated with sex determination, were shown to be involved in several other functions during embryonic development. Dmrt2 has been studied in the context of zebrafish development where, due to a duplication event, two paralog genes dmrt2a and dmrt2b are present. Both zebrafish dmrt2a/terra and dmrt2b are important to regulate left-right patterning in the lateral plate mesoderm. In addition, dmrt2a/terra is necessary for symmetric somite formation while dmrt2b regulates somite differentiation impacting on slow muscle development. One dmrt2 gene is also expressed in the mouse embryo, where it is necessary for somite differentiation but with an impact on axial skeleton development. However, nothing was known about its role during left-right patterning in the lateral plate mesoderm or in the symmetric synchronization of somite formation. Methodology/Principal Findings Using a dmrt2 mutant mouse line, we show that this gene is not involved in symmetric somite formation and does not regulate the laterality pathway that controls left-right asymmetric organ positioning. We reveal that dmrt2a/terra is present in the zebrafish laterality organ, the Kupffer's vesicle, while its homologue is excluded from the mouse equivalent structure, the node. On the basis of evolutionary sub-functionalization and neo-functionalization theories we discuss this absence of functional conservation. Conclusions/Significance Our results show that the role of dmrt2 gene is not conserved during zebrafish and mouse embryonic development. PMID:21203428

  19. Transcriptome-Wide Identification of Novel Imprinted Genes in Neonatal Mouse Brain

    PubMed Central

    Wang, Xu; Sun, Qi; McGrath, Sean D.; Mardis, Elaine R.; Soloway, Paul D.; Clark, Andrew G.

    2008-01-01

    Imprinted genes display differential allelic expression in a manner that depends on the sex of the transmitting parent. The degree of imprinting is often tissue-specific and/or developmental stage-specific, and may be altered in some diseases including cancer. Here we applied Illumina/Solexa sequencing of the transcriptomes of reciprocal F1 mouse neonatal brains and identified 26 genes with parent-of-origin dependent differential allelic expression. Allele-specific Pyrosequencing verified 17 of them, including three novel imprinted genes. The known and novel imprinted genes all are found in proximity to previously reported differentially methylated regions (DMRs). Ten genes known to be imprinted in placenta had sufficient expression levels to attain a read depth that provided statistical power to detect imprinting, and yet all were consistent with non-imprinting in our transcript count data for neonatal brain. Three closely linked and reciprocally imprinted gene pairs were also discovered, and their pattern of expression suggests transcriptional interference. Despite the coverage of more than 5000 genes, this scan only identified three novel imprinted refseq genes in neonatal brain, suggesting that this tissue is nearly exhaustively characterized. This approach has the potential to yield an complete catalog of imprinted genes after application to multiple tissues and developmental stages, shedding light on the mechanism, bioinformatic prediction, and evolution of imprinted genes and diseases associated with genomic imprinting. PMID:19052635

  20. Evaluation of OPEN Zinc Finger Nucleases for Direct Gene Targeting of the ROSA26 Locus in Mouse Embryos

    PubMed Central

    Hermann, Mario; Maeder, Morgan L.; Rector, Kyle; Ruiz, Joseph; Becher, Burkhard; Bürki, Kurt; Khayter, Cyd; Aguzzi, Adriano; Joung, J. Keith

    2012-01-01

    Zinc finger nucleases (ZFNs) enable precise genome modification in a variety of organisms and cell types. Commercial ZFNs were reported to enhance gene targeting directly in mouse zygotes, whereas similar approaches using publicly available resources have not yet been described. Here we report precise targeted mutagenesis of the mouse genome using Oligomerized Pool Engineering (OPEN) ZFNs. OPEN ZFN can be constructed using publicly available resources and therefore provide an attractive alternative for academic researchers. Two ZFN pairs specific to the mouse genomic locus gt(ROSA26)Sor were generated by OPEN selections and used for gene disruption and homology-mediated gene replacement in single cell mouse embryos. One specific ZFN pair facilitated non-homologous end joining (NHEJ)-mediated gene disruption when expressed in mouse zygotes. We also observed a single homologous recombination (HR)-driven gene replacement event when this ZFN pair was co-injected with a targeting vector. Our experiments demonstrate the feasibility of achieving both gene ablation through NHEJ and gene replacement by HR by using the OPEN ZFN technology directly in mouse zygotes. PMID:22970113

  1. Meiotic chromosome structure and function in plants.

    PubMed

    Mainiero, Samantha; Pawlowski, Wojciech P

    2014-01-01

    Chromosome structure is important for many meiotic processes. Here, we outline 3 main determinants of chromosome structure and their effects on meiotic processes in plants. Cohesins are necessary to hold sister chromatids together until the first meiotic division, ensuring that homologous chromosomes and not sister chromatids separate during anaphase I. During meiosis in maize, Arabidopsis, and rice, cohesins are needed for establishing early prophase chromosome structure and recombination and for aligning bivalents at the metaphase plate. Condensin complexes play pivotal roles in controlling the packaging of chromatin into chromosomes through chromatin compaction and chromosome individualization. In animals and fungi, these complexes establish a meiotic chromosome structure that allows for proper recombination, pairing, and synapsis of homologous chromosomes. In plants, information on the role of condensins in meiosis is limited, but they are known to be required for successful completion of reproductive development. Therefore, we speculate that they play roles similar to animal and fungal condensins during meiosis. Plants generally have large and complex genomes due to frequent polyploidy events, and likely, condensins and cohesins organize chromosomes in such a way as to ensure genome stability. Hexaploid wheat has evolved a unique mechanism using a Ph1 locus-controlled chromosome organization to ensure proper chromosome pairing in meiosis. Altogether, studies on meiotic chromosome structure indicate that chromosome organization is not only important for chromatin packaging but also fulfills specific functions in facilitating chromosome interactions during meiosis, including pairing and recombination. PMID:25096046

  2. A characterization of the elements comprising the promoter of the mouse ribosomal protein gene RPS16.

    PubMed

    Hariharan, N; Perry, R P

    1989-07-11

    The elements comprising the mouse rpS16 promoter were characterized by transfection experiments with mutant genes in which various portions of the 5' flanking region and exon I were removed or substituted with extraneous DNA sequence. These experiments were carried out with otherwise intact rpS16 genes transfected into monkey kidney (COS) cells and also with chimeric rpS16-CAT gene constructs transfected into mouse plasmacytoma cells and COS cells. The locations of the functionally important elements were generally correlated with the locations of binding sites for specific nuclear factors, which were identified by gel-mobility shift analyses and methylation interference footprints. The most upstream element, which is located approximately 165 bp from the cap site, binds the Sp1 transcription factor and augments the promoter activity by 2 to 2.5-fold. In addition, there is a complex bipartite element in the -83 to -59 region, an element in the -37 to -12 region and an element in the +9 to +29 region of exon I, all of which are essential for rpS16 expression. The rpS16 promoter has a general architecture that resembles other mouse rp promoters; however, it also possesses some distinctive characteristics. PMID:2762128

  3. The construction of transgenic and gene knockout/knockin mouse models of human disease.

    PubMed

    Doyle, Alfred; McGarry, Michael P; Lee, Nancy A; Lee, James J

    2012-04-01

    The genetic and physiological similarities between mice and humans have focused considerable attention on rodents as potential models of human health and disease. Together with the wealth of resources, knowledge, and technologies surrounding the mouse as a model system, these similarities have propelled this species to the forefront of biomedical research. The advent of genomic manipulation has quickly led to the creation and use of genetically engineered mice as powerful tools for cutting edge studies of human disease research including the discovery, refinement, and utility of many currently available therapeutic regimes. In particular, the creation of genetically modified mice as models of human disease has remarkably changed our ability to understand the molecular mechanisms and cellular pathways underlying disease states. Moreover, the mouse models resulting from gene transfer technologies have been important components correlating an individual's gene expression profile to the development of disease pathologies. The objective of this review is to provide physician-scientists with an expansive historical and logistical overview of the creation of mouse models of human disease through gene transfer technologies. Our expectation is that this will facilitate on-going disease research studies and may initiate new areas of translational research leading to enhanced patient care. PMID:21800101

  4. The Construction of Transgenic and Gene Knockout/Knockin Mouse Models of Human Disease

    PubMed Central

    Doyle, Alfred; McGarry, Michael P.; Lee, Nancy A.; Lee, James J.

    2012-01-01

    The genetic and physiological similarities between mice and humans have focused considerable attention on rodents as potential models of human health and disease. Together with the wealth of resources, knowledge, and technologies surrounding the mouse as a model system, these similarities have propelled this species to the forefront of biomedical research. The advent of genomic manipulation has quickly led to the creation and use of genetically engineered mice as powerful tools for cutting edge studies of human disease research, including the discovery, refinement, and utility of many currently available therapeutic regimes. In particular, the creation of genetically modified mice as models of human disease has remarkably changed our ability to understand the molecular mechanisms and cellular pathways underlying disease states. Moreover, the mouse models resulting from gene transfer technologies have been important components correlating an individual’s gene expression profile to the development of disease pathologies. The objective of this review is to provide physician-scientists with an expansive historical and logistical overview of the creation of mouse models of human disease through gene transfer technologies. Our expectation is that this will facilitate on-going disease research studies and may initiate new areas of translational research leading to enhanced patient care. PMID:21800101

  5. Sequence analysis of the ERCC2 gene regions in human, mouse, and hamster reveals three linked genes

    SciTech Connect

    Lamerdin, J.E.; Stilwagen, S.A.; Ramirez, M.H.

    1996-06-15

    The ERCC2 (excision repair cross-complementing rodent repair group 2) gene product is involved in transcription-coupled repair as an integral member of the basal transcription factor BTF2/TFIIH complex. Defects in this gene can result in three distinct human disorders, namely the cancer-prone syndrome xeroderma pigmentosum complementation group D, trichothiodystrophy, and Cockayne syndrome. We report the comparative analysis of 91.6 kb of new sequence including 54.3 kb encompassing the human ERCC2 locus, the syntenic region in the mouse (32.6 kb), and a further 4.7 kb of sequence 3{prime} of the previously reported ERCC2 region in the hamster. In addition to ERCC2, our analysis revealed the presence of two previously undescribed genes in all three species. The first is centromeric (in the human) to ERCC2 and is most similar to the kinesin light chain gene in sea urchin. The second gene is telomeric (in the human) to ERCC2 and contains a motif found in ankyrins, some cell proteins, and transcription factors. Multiple EST matches to this putative new gene indicate that it is expressed in several human tissues, including breast. The identification and description of two new genes provides potential candidate genes for disorders mapping to this region of 19q13.2. 42 refs., 6 figs., 3 tabs.

  6. LSD1 is essential for oocyte meiotic progression by regulating CDC25B expression in mice

    PubMed Central

    Kim, Jeesun; Singh, Anup Kumar; Takata, Yoko; Lin, Kevin; Shen, Jianjun; Lu, Yue; Kerenyi, Marc A.; Orkin, Stuart H.; Chen, Taiping

    2015-01-01

    Mammalian oocytes are arrested at prophase I until puberty when hormonal signals induce the resumption of meiosis I and progression to meiosis II. Meiotic progression is controlled by CDK1 activity and is accompanied by dynamic epigenetic changes. Although the signalling pathways regulating CDK1 activity are well defined, the functional significance of epigenetic changes remains largely unknown. Here we show that LSD1, a lysine demethylase, regulates histone H3 lysine 4 di-methylation (H3K4me2) in mouse oocytes and is essential for meiotic progression. Conditional deletion of Lsd1 in growing oocytes results in precocious resumption of meiosis and spindle and chromosomal abnormalities. Consequently, most Lsd1-null oocytes fail to complete meiosis I and undergo apoptosis. Mechanistically, upregulation of CDC25B, a phosphatase that activates CDK1, is responsible for precocious meiotic resumption and also contributes to subsequent spindle and chromosomal defects. Our findings uncover a functional link between LSD1 and the major signalling pathway governing meiotic progression. PMID:26626423

  7. The Mouse Solitary Odorant Receptor Gene Promoters as Models for the Study of Odorant Receptor Gene Choice

    PubMed Central

    Degl'Innocenti, Andrea

    2016-01-01

    Background In vertebrates, several anatomical regions located within the nasal cavity mediate olfaction. Among these, the main olfactory epithelium detects most conventional odorants. Olfactory sensory neurons, provided with cilia exposed to the air, detect volatile chemicals via an extremely large family of seven-transmembrane chemoreceptors named odorant receptors. Their genes are expressed in a monogenic and monoallelic fashion: a single allele of a single odorant receptor gene is transcribed in a given mature neuron, through a still uncharacterized molecular mechanism known as odorant receptor gene choice. Aim Odorant receptor genes are typically arranged in genomic clusters, but a few are isolated (we call them solitary) from the others within a region broader than 1 Mb upstream and downstream with respect to their transcript's coordinates. The study of clustered genes is problematic, because of redundancy and ambiguities in their regulatory elements: we propose to use the solitary genes as simplified models to understand odorant receptor gene choice. Procedures Here we define number and identity of the solitary genes in the mouse genome (C57BL/6J), and assess the conservation of the solitary status in some mammalian orthologs. Furthermore, we locate their putative promoters, predict their homeodomain binding sites (commonly present in the promoters of odorant receptor genes) and compare candidate promoter sequences with those of wild-caught mice. We also provide expression data from histological sections. Results In the mouse genome there are eight intact solitary genes: Olfr19 (M12), Olfr49, Olfr266, Olfr267, Olfr370, Olfr371, Olfr466, Olfr1402; five are conserved as solitary in rat. These genes are all expressed in the main olfactory epithelium of three-day-old mice. The C57BL/6J candidate promoter of Olfr370 has considerably varied compared to its wild-type counterpart. Within the putative promoter for Olfr266 a homeodomain binding site is predicted. As a

  8. Transposon mutagenesis identifies genes driving hepatocellular carcinoma in a chronic hepatitis B mouse model

    PubMed Central

    Bard-Chapeau, Emilie A.; Nguyen, Anh-Tuan; Rust, Alistair G.; Sayadi, Ahmed; Lee, Philip; Chua, Belinda Q; New, Lee-Sun; de Jong, Johann; Ward, Jerrold M.; Chin, Christopher KY.; Chew, Valerie; Toh, Han Chong; Abastado, Jean-Pierre; Benoukraf, Touati; Soong, Richie; Bard, Frederic A.; Dupuy, Adam J.; Johnson, Randy L.; Radda, George K.; Chan, Eric CY.; Wessels, Lodewyk FA.; Adams, David J.

    2014-01-01

    The most common risk factor for developing hepatocellular carcinoma (HCC) is chronic infection with hepatitis B virus (HBV). To better understand the evolutionary forces driving HCC we performed a near saturating transposon mutagenesis screen in a mouse HBV model of HCC. This screen identified 21 candidate early stage drivers, and a bewildering number (2860) of candidate later stage drivers, that were enriched for genes mutated, deregulated, or that function in signaling pathways important for human HCC, with a striking 1199 genes linked to cellular metabolic processes. Our study provides a comprehensive overview of the genetic landscape of HCC. PMID:24316982

  9. Genetically diverse CC-founder mouse strains replicate the human influenza gene expression signature.

    PubMed

    Elbahesh, Husni; Schughart, Klaus

    2016-05-19

    Influenza A viruses (IAV) are zoonotic pathogens that pose a major threat to human and animal health. Influenza virus disease severity is influenced by viral virulence factors as well as individual differences in host response. We analyzed gene expression changes in the blood of infected mice using a previously defined set of signature genes that was derived from changes in the blood transcriptome of IAV-infected human volunteers. We found that the human signature was reproduced well in the founder strains of the Collaborative Cross (CC) mice, thus demonstrating the relevance and importance of mouse experimental model systems for studying human influenza disease.

  10. Localization of complement factor H gene expression and protein distribution in the mouse outer retina

    PubMed Central

    Smit-McBride, Zeljka; Oltjen, Sharon L.; Radu, Roxana A.; Estep, Jason; Nguyen, Anthony T.; Gong, Qizhi

    2015-01-01

    Purpose To determine the localization of complement factor H (Cfh) mRNA and its protein in the mouse outer retina. Methods Quantitative real-time PCR (qPCR) was used to determine the expression of Cfh and Cfh-related (Cfhr) transcripts in the RPE/choroid. In situ hybridization (ISH) was performed using the novel RNAscope 2.0 FFPE assay to localize the expression of Cfh mRNA in the mouse outer retina. Immunohistochemistry (IHC) was used to localize Cfh protein expression, and western blots were used to characterize CFH antibodies used for IHC. Results Cfh and Cfhr2 transcripts were detected in the mouse RPE/choroid using qPCR, while Cfhr1, Cfhr3, and Cfhrc (Gm4788) were not detected. ISH showed abundant Cfh mRNA in the RPE of all mouse strains (C57BL/6, BALB/c, 129/Sv) tested, with the exception of the Cfh−/− eye. Surprisingly, the Cfh protein was detected by immunohistochemistry in photoreceptors rather than in RPE cells. The specificity of the CFH antibodies was tested by western blotting. Our CFH antibodies recognized purified mouse Cfh protein, serum Cfh protein in wild-type C57BL/6, BALB/c, and 129/Sv, and showed an absence of the Cfh protein in the serum of Cfh−/− mice. Greatly reduced Cfh protein immunohistological signals in the Cfh−/− eyes also supported the specificity of the Cfh protein distribution results. Conclusions Only Cfh and Cfhr2 genes are expressed in the mouse outer retina. Only Cfh mRNA was detected in the RPE, but no protein. We hypothesize that the steady-state concentration of Cfh protein is low in the cells due to secretion, and therefore is below the detection level for IHC. PMID:25684976

  11. The Immunoglobulin-like Gene spe-45 Acts during Fertilization in Caenorhabditis elegans like the Mouse Izumo1 Gene.

    PubMed

    Nishimura, Hitoshi; Tajima, Tatsuya; Comstra, Heather Skye; Gleason, Elizabeth J; L'Hernault, Steven W

    2015-12-21

    The Caenorhabditis elegans spe-9 class genes, which show specific or predominant expression in the male germline, are indispensable for fertilization [1, 2]. However, due to the rapid evolution of genes involved in reproduction, we do not currently know if there are spe-9 class genes in mammals that play similar roles during fertilization to those found in C. elegans. In mice, the Izumo1 gene encodes a sperm-specific transmembrane (TM) protein with a single immunoglobulin (Ig)-like domain that is absolutely required for gamete fusion [3, 4]. In this study, we hypothesized that C. elegans has a new member of the spe-9 class genes coding for an IZUMO1-like protein. We screened C. elegans microarray data [5, 6] to identify male germline-enriched genes that encode membrane proteins with Ig-like domains. A deletion (tm3715) in one such gene (F28D1.8) caused hermaphrodites to show a male germline-dependent self-sterility, so we have named it spe-45. Mutant spe-45 worms seemed to normally undergo spermatogenesis (spermatid production by meiosis) and spermiogenesis (spermatid activation into actively motile spermatozoa). spe-45 mutant spermatozoa, however, could not complete gamete fusion, which is a characteristic of all spe-9 class mutants [1, 2]. Moreover, spe-45 self-sterile worms were rescued by a transgene expressing chimeric SPE-45 protein in which its Ig-like domain was replaced by the Ig-like domain from mouse IZUMO1. Hence, C. elegans SPE-45 and mouse IZUMO1 appear to have retained a common function(s) that is required during fertilization.

  12. Adeno-associated virus-mediated gene transfer targeting normal and traumatized mouse utricle.

    PubMed

    Wang, G-P; Guo, J-Y; Peng, Z; Liu, Y-Y; Xie, J; Gong, S-S

    2014-11-01

    Balance dysfunction is closely associated with loss of vestibular hair cells (HCs). Gene therapy shows promise when used to protect or regenerate vestibular HCs to preserve or restore adequate vestibular function. Adeno-associated virus (AAV) vectors allow long-term gene expression in the absence of toxicity. To noninvasively define an AAV serotype exhibiting favorable tropism toward the vestibular sensory epithelium, we characterized the transgene expression potential of AAV vectors (serotypes 1, 2, 5, 6 and 8) inoculated into adult mouse utricle via canalostomy. We found that AAV8 was the most effective AAV vector in utricular gene transfer. Swim tests and measurements of auditory brainstem response revealed minimal loss of vestibular function and hearing after canalostomy. In the normal utricle after AAV8 infusion, transduction efficiency peaked at 7 days, and was maintained thereafter, in vestibular HCs, and at 3 days in supporting cells (SCs). In the streptomycin-lesioned utricle, the SC transduction efficiency peaked at 7 days and decreased at 30 days. In conclusion, AAV8-mediated gene transfer via canalostomy facilitates efficient and safe transduction in mouse vestibular sensory epithelium, and may in the future become clinically relevant for human vestibular gene therapy. PMID:25119376

  13. Isolation and characterization of two mouse Pi-class glutathione S-transferase genes.

    PubMed Central

    Bammler, T K; Smith, C A; Wolf, C R

    1994-01-01

    Pi-class glutathione S-transferases (GSTs) play an important role in the detoxification of chemical toxins and mutagens and are implicated in neoplastic development and drug resistance. In all species characterized to date, only one functional Pi-class GST gene has been described. In this report we have identified two actively transcribed murine Pi-class GST genes, Gst p-1 and Gst p-2. The coding regions of Gst p-1 and the mouse Pi-class GST cDNA (GST-II) reported by Hatayama, Satoh and Satoh (1990) (Nucleic Acids Res. 18, 4606) are identical, whereas Gst p-2 encodes a protein that has not been described previously. The two genes are approximately 3 kb long and contain seven exons interrupted by six introns. In addition to a TATA box and a sequence motif matching the phorbol-ester-responsive element, the promoters of Gst p-1 and Gst p-2 exhibit one and two G+C boxes (GGGCGG) respectively. The cDNAs of the two genes were isolated from total liver RNA using reverse PCR. The peptide sequence deduced from the cDNAs share 97% identity and differ in six amino acids. Both genes are transcribed at significantly higher levels in male mouse liver than in female, and Gst p-1 mRNA is more abundant in both sexes than Gst p-2. Images Figure 4 Figure 5 PMID:8135745

  14. Integrated proteomic and transcriptomic profiling of mouse lung development and Nmyc target genes

    PubMed Central

    Cox, Brian; Kislinger, Thomas; Wigle, Dennis A; Kannan, Anitha; Brown, Kevin; Okubo, Tadashi; Hogan, Brigid; Jurisica, Igor; Frey, Brendan; Rossant, Janet; Emili, Andrew

    2007-01-01

    Although microarray analysis has provided information regarding the dynamics of gene expression during development of the mouse lung, no extensive correlations have been made to the levels of corresponding protein products. Here, we present a global survey of protein expression during mouse lung organogenesis from embryonic day E13.5 until adulthood using gel-free two-dimensional liquid chromatography coupled to shotgun tandem mass spectrometry (MudPIT). Mathematical modeling of the proteomic profiles with parallel DNA microarray data identified large groups of gene products with statistically significant correlation or divergence in coregulation of protein and transcript levels during lung development. We also present an integrative analysis of mRNA and protein expression in Nmyc loss- and gain-of-function mutants. This revealed a set of 90 positively and negatively regulated putative target genes. These targets are evidence that Nmyc is a regulator of genes involved in mRNA processing and a repressor of the imprinted gene Igf2r in the developing lung. PMID:17486137

  15. Cloning and partial characterization of the mouse glutamine:fructose-6-phosphate amidotransferase (GFAT) gene promoter.

    PubMed Central

    Sayeski, P P; Wang, D; Su, K; Han, I O; Kudlow, J E

    1997-01-01

    Glutamine:fructose-6-phosphate amidotransferase (GFAT) is the enzyme that is rate limiting in the synthesis of glucosamine and hexosamines. Glucosamine has been proposed to contribute to the glucotoxicity of diabetes. Evidence that the gene encoding GFAT is transcriptionally regulated prompted us to clone and characterize its promoter. The position of the mouse GFAT promoter relative to the translational start site was located by primer extension and found to be 149 bp upstream of the translational start site. A 1.9 kb SacI fragment of the GFAT gene was found to contain the promoter and 88 bp of sequence downstream of the transcriptional start site. This promoter segment could drive expression of a luciferase reporter gene, could confer correct transcriptional initiation to the reporter and could confer the EGF-responsiveness previously observed in the native gene. The mouse GFAT promoter lacks a canonical TATA box and has several GC boxes within a highly GC-rich region. Deletional analysis of the promoter indicated that a proximal element extending to -120 relative to the transcriptional start site could confer reporter expression at a level of 57% of the 1.9 kb construct. Detailed analysis of this proximal region by DNase I footprinting, electrophoretic mobility shift assays and site-directed mutagenesis indicated that Sp1 binds to three elements in this proximal promoter segment and plays a vital role in regulation of transcription from this gene. PMID:9060444

  16. Retroviral-mediated gene transfer and expression of human phenylalanine hydroxylase in primary mouse hepatocytes

    SciTech Connect

    Peng, H.; Armentano, D.; Mackenzie-Graham, L.; Shen, R.F.; Darlington, G.; Ledley, F.D.; Woo, S.L.C. )

    1988-11-01

    Genetic therapy for phenylketonuria (severe phenylalanine hydroxylase deficiency) may require introduction of a normal phenylalanine hydroxylase gene into hepatic cells of patients. The authors report development of a recombinant retrovirus based on the N2 vector for gene transfer and expression of human phenylalanine hydroxylase cDNA in primary mouse hepatocytes. This construct contains an internal promoter of the human {alpha}{sub 1}-antitrypsin gene driving transcription of the phenylalanine hydroxylase cDNA. Primary mouse hepatocytes were isolated from newborn mice, infected with the recombinant virus, and selected for expression of the neomycin-resistance gene. Hepatocytes transformed with the recombinant virus contained high levels of human phenylalanine hydroxylase mRNA transcripts originating from the retroviral and internal promoters. These results demonstrate that the transcriptional regulatory elements of the {alpha}{sub 1} antitrypsin gene retain their tissue-specific function in the recombinant provirus and establish a method for efficient transfer and high-level expression of human phenylalanine hydroxylase in primary hepatocytes.

  17. Genetic and Molecular Basis of QTL of Diabetes in Mouse: Genes and Polymorphisms

    PubMed Central

    Gao, Peng; Jiao, Yan; Xiong, Qing; Wang, Cong-Yi; Gerling, Ivan; Gu, Weikuan

    2008-01-01

    A systematic study has been conducted of all available reports in PubMed and OMIM (Online Mendelian Inheritance in Man) to examine the genetic and molecular basis of quantitative genetic loci (QTL) of diabetes with the main focus on genes and polymorphisms. The major question is, What can the QTL tell us? Specifically, we want to know whether those genome regions differ from other regions in terms of genes relevant to diabetes. Which genes are within those QTL regions, and, among them, which genes have already been linked to diabetes? whether more polymorphisms have been associated with diabetes in the QTL regions than in the non-QTL regions. Our search revealed a total of 9038 genes from 26 type 1 diabetes QTL, which cover 667,096,006 bp of the mouse genomic sequence. On one hand, a large number of candidate genes are in each of these QTL; on the other hand, we found that some obvious candidate genes of QTL have not yet been investigated. Thus, the comprehensive search of candidate genes for known QTL may provide unexpected benefit for identifying QTL genes for diabetes. PMID:19471607

  18. High-throughput screening of mouse gene knockouts identifies established and novel skeletal phenotypes

    PubMed Central

    Brommage, Robert; Liu, Jeff; Hansen, Gwenn M; Kirkpatrick, Laura L; Potter, David G; Sands, Arthur T; Zambrowicz, Brian; Powell, David R; Vogel, Peter

    2014-01-01

    Screening gene function in vivo is a powerful approach to discover novel drug targets. We present high-throughput screening (HTS) data for 3 762 distinct global gene knockout (KO) mouse lines with viable adult homozygous mice generated using either gene-trap or homologous recombination technologies. Bone mass was determined from DEXA scans of male and female mice at 14 weeks of age and by microCT analyses of bones from male mice at 16 weeks of age. Wild-type (WT) cagemates/littermates were examined for each gene KO. Lethality was observed in an additional 850 KO lines. Since primary HTS are susceptible to false positive findings, additional cohorts of mice from KO lines with intriguing HTS bone data were examined. Aging, ovariectomy, histomorphometry and bone strength studies were performed and possible non-skeletal phenotypes were explored. Together, these screens identified multiple genes affecting bone mass: 23 previously reported genes (Calcr, Cebpb, Crtap, Dcstamp, Dkk1, Duoxa2, Enpp1, Fgf23, Kiss1/Kiss1r, Kl (Klotho), Lrp5, Mstn, Neo1, Npr2, Ostm1, Postn, Sfrp4, Slc30a5, Slc39a13, Sost, Sumf1, Src, Wnt10b), five novel genes extensively characterized (Cldn18, Fam20c, Lrrk1, Sgpl1, Wnt16), five novel genes with preliminary characterization (Agpat2, Rassf5, Slc10a7, Slc26a7, Slc30a10) and three novel undisclosed genes coding for potential osteoporosis drug targets. PMID:26273529

  19. An in situ hybridization-based screen for heterogeneously expressed genes in mouse ES cells.

    PubMed

    Carter, Mark G; Stagg, Carole A; Falco, Geppino; Yoshikawa, Toshiyuki; Bassey, Uwem C; Aiba, Kazuhiro; Sharova, Lioudmila V; Shaik, Nabeebi; Ko, Minoru S H

    2008-02-01

    We previously reported that Zscan4 showed heterogeneous expression patterns in mouse embryonic stem (ES) cells. To identify genes that show similar expression patterns, we carried out high-throughput in situ hybridization assays on ES cell cultures for 244 genes. Most of the genes are involved in transcriptional regulation, and were selected using microarray-based comparisons of gene expression profiles in ES and embryonal carcinoma (EC) cells versus differentiated cell types. Pou5f1 (Oct4, Oct3/4) and Krt8 (EndoA) were used as controls. Hybridization signals were detected on ES cell colonies for 147 genes (60%). The majority (136 genes) of them showed relatively homogeneous expression in ES cell colonies. However, we found that two genes unequivocally showed Zscan4-like spotted expression pattern (spot-in-colony pattern; Whsc2 and Rhox9). We also found that nine genes showed relatively heterogeneous expression pattern (mosaic-in-colony pattern: Zfp42/Rex1, Rest, Atf4, Pa2g4, E2f2, Nanog, Dppa3/Pgc7/Stella, Esrrb, and Fscn1). Among these genes, Zfp42/Rex1 showed unequivocally heterogeneous expression in individual ES cells prepared by the CytoSpin. These results show the presence of different types or states of cells within ES cell cultures otherwise thought to be undifferentiated and homogeneous, suggesting a previously unappreciated complexity in ES cell cultures.

  20. Retinoic acid homeostasis through aldh1a2 and cyp26a1 mediates meiotic entry in Nile tilapia (Oreochromis niloticus).

    PubMed

    Feng, Ruijuan; Fang, Lingling; Cheng, Yunying; He, Xue; Jiang, Wentao; Dong, Ranran; Shi, Hongjuan; Jiang, Dongneng; Sun, Lina; Wang, Deshou

    2015-01-01

    Meiosis is a process unique to the differentiation of germ cells. Retinoic acid (RA) is the key factor controlling the sex-specific timing of meiotic initiation in tetrapods; however, the role of RA in meiotic initiation in teleosts has remained unclear. In this study, the genes encoding RA synthase aldh1a2, and catabolic enzyme cyp26a1 were isolated from Nile tilapia (Oreochromis niloticus), a species without stra8. The expression of aldh1a2 was up-regulated and expression of cyp26a1 was down-regulated before the meiotic initiation in ovaries and in testes. Treatment with RA synthase inhibitor or disruption of Aldh1a2 by CRISPR/Cas9 resulted in delayed meiotic initiation, with simultaneous down-regulation of cyp26a1 and up-regulation of sycp3. By contrast, treatment with an inhibitor of RA catabolic enzyme and disruption of cyp26a1 resulted in earlier meiotic initiation, with increased expression of aldh1a2 and sycp3. Additionally, treatment of XY fish with estrogen (E2) and XX fish with fadrozole led to sex reversal and reversion of meiotic initiation. These results indicate that RA is indispensable for meiotic initiation in teleosts via a stra8 independent signaling pathway where both aldh1a2 and cyp26a1 are critical. In contrast to mammals, E2 is a major regulator of sex determination and meiotic initiation in teleosts. PMID:25976364

  1. Retinoic acid homeostasis through aldh1a2 and cyp26a1 mediates meiotic entry in Nile tilapia (Oreochromis niloticus)

    PubMed Central

    Feng, Ruijuan; Fang, Lingling; Cheng, Yunying; He, Xue; Jiang, Wentao; Dong, Ranran; Shi, Hongjuan; Jiang, Dongneng; Sun, Lina; Wang, Deshou

    2015-01-01

    Meiosis is a process unique to the differentiation of germ cells. Retinoic acid (RA) is the key factor controlling the sex-specific timing of meiotic initiation in tetrapods; however, the role of RA in meiotic initiation in teleosts has remained unclear. In this study, the genes encoding RA synthase aldh1a2, and catabolic enzyme cyp26a1 were isolated from Nile tilapia (Oreochromis niloticus), a species without stra8. The expression of aldh1a2 was up-regulated and expression of cyp26a1 was down-regulated before the meiotic initiation in ovaries and in testes. Treatment with RA synthase inhibitor or disruption of Aldh1a2 by CRISPR/Cas9 resulted in delayed meiotic initiation, with simultaneous down-regulation of cyp26a1 and up-regulation of sycp3. By contrast, treatment with an inhibitor of RA catabolic enzyme and disruption of cyp26a1 resulted in earlier meiotic initiation, with increased expression of aldh1a2 and sycp3. Additionally, treatment of XY fish with estrogen (E2) and XX fish with fadrozole led to sex reversal and reversion of meiotic initiation. These results indicate that RA is indispensable for meiotic initiation in teleosts via a stra8 independent signaling pathway where both aldh1a2 and cyp26a1 are critical. In contrast to mammals, E2 is a major regulator of sex determination and meiotic initiation in teleosts. PMID:25976364

  2. Comparison of Mouse and Human Retinal Pigment Epithelium Gene Expression Profiles: Potential Implications for Age-Related Macular Degeneration

    PubMed Central

    Bennis, Anna; Gorgels, Theo G. M. F.; ten Brink, Jacoline B.; van der Spek, Peter J.; Bossers, Koen; Heine, Vivi M.; Bergen, Arthur A.

    2015-01-01

    Background The human retinal pigment epithelium (RPE) plays an important role in the pathogenesis of age related macular degeneration (AMD). AMD is the leading cause of blindness worldwide. There is currently no effective treatment available. Preclinical studies in AMD mouse models are essential to develop new therapeutics. This requires further in-depth knowledge of the similarities and differences between mouse and human RPE. Methods We performed a microarray study to identify and functionally annotate RPE specific gene expression in mouse and human RPE. We used a meticulous method to determine C57BL/6J mouse RPE signature genes, correcting for possible RNA contamination from its adjacent layers: the choroid and the photoreceptors. We compared the signature genes, gene expression profiles and functional annotations of the mouse and human RPE. Results We defined sets of mouse (64), human (171) and mouse–human interspecies (22) RPE signature genes. Not unexpectedly, our gene expression analysis and comparative functional annotation suggested that, in general, the mouse and human RPE are very similar. For example, we found similarities for general features, like “organ development” and “disorders related to neurological tissue”. However, detailed analysis of the molecular pathways and networks associated with RPE functions, suggested also multiple species-specific differences, some of which may be relevant for the development of AMD. For example, CFHR1, most likely the main complement regulator in AMD pathogenesis was highly expressed in human RPE, but almost absent in mouse RPE. Furthermore, functions assigned to mouse and human RPE expression profiles indicate (patho-) biological differences related to AMD, such as oxidative stress, Bruch’s membrane, immune-regulation and outer blood retina barrier. Conclusion These differences may be important for the development of new therapeutic strategies and translational studies in age-related macular

  3. Expression of the Bgp gene and characterization of mouse colon biliary glycoprotein isoforms.

    PubMed

    McCuaig, K; Rosenberg, M; Nédellec, P; Turbide, C; Beauchemin, N

    1993-05-30

    The biliary glycoprotein (BGP)-encoding gene is a member of the human carcinoembryonic antigen (CEA) gene family. We have now cloned several mouse Bgp cDNAs from an outbred CDR-1 mouse colon cDNA library, as well as by reverse transcription-PCR amplification of colon RNA. The distinguishing features of the deduced Bgp protein isoforms are found in the two divergent N-terminal domains, the highly conserved internal C2-set immunoglobulin domains, and an intracytoplasmic domain of either 10 or 73 amino acids (aa). The cDNA structures suggest that these mRNAs are produced through alternative splicing of a Bgp gene and the usage of multiple transcriptional terminators. The Bgp deduced aa sequences are highly homologous to several well characterized rat hepatocyte proteins such as the cell CAM105/ecto-ATPase/pp120/HA4 proteins. Oligodeoxyribonucleotide probes representing the various cDNA isoform domains revealed predominant transcripts of 1.8, 3.1 and 4.0 kb on Northern analyses of mouse colon RNA; some of these bands are actually composed of several co-migrating transcripts. The transcripts encoding the long intracytoplasmic-tailed Bgp proteins are expressed at one-tenth the relative abundance of the shorter-tailed species. We have previously demonstrated that several mouse Bgp cDNAs, when transfected into eukaryotic cells, express BGP proteins at the cell surface and function in vitro as cell adhesion molecules, much like their human and rat counterparts. The expression of the many Bgp isoforms at the surface of epithelial cells, such as colon, suggests that these proteins play a determinant role, through self- or heterologous contact, in renewal and/or differentiation of their epithelia.

  4. Genetic‐Genomic Replication to Identify Candidate Mouse Atherosclerosis Modifier Genes

    PubMed Central

    Hsu, Jeffrey; Smith, Jonathan D.

    2013-01-01

    Objective Genetics plays a large role in atherosclerosis susceptibility in humans and mice. We attempted to confirm previously determined mouse atherosclerosis‐associated loci and use bioinformatics and transcriptomics to create a catalog of candidate atherosclerosis modifier genes at these loci. Methods and Results A strain intercross was performed between AKR and DBA/2 mice on the apoE−/− background generating 166 F2 progeny. Using the phenotype log10 of the aortic root lesion area, we identified 3 suggestive atherosclerosis quantitative trait loci (Ath QTLs). When combined with our prior strain intercross, we confirmed 3 significant Ath QTLs on chromosomes 2, 15, and 17, with combined logarithm of odds scores of 5.9, 5.3, and 5.6, respectively, which each met the genome‐wide 5% false discovery rate threshold. We identified all of the protein coding differences between these 2 mouse strains within the Ath QTL intervals. Microarray gene expression profiling was performed on macrophages and endothelial cells from this intercross to identify expression QTLs (eQTLs), the loci that are associated with variation in the expression levels of specific transcripts. Cross tissue eQTLs and macrophage eQTLs that replicated from a prior strain intercross were identified. These bioinformatic and eQTL analyses produced a comprehensive list of candidate genes that may be responsible for the Ath QTLs. Conclusions Replication studies for clinical traits as well as gene expression traits are worthwhile in identifying true versus false genetic associations. We have replicated 3 loci on mouse chromosomes 2, 15, and 17 that are associated with atherosclerosis. We have also identified protein coding differences and multiple replicated eQTLs, which may be useful in the identification of atherosclerosis modifier genes. PMID:23525445

  5. Senataxin controls meiotic silencing through ATR activation and chromatin remodeling.

    PubMed

    Yeo, Abrey J; Becherel, Olivier J; Luff, John E; Graham, Mark E; Richard, Derek; Lavin, Martin F

    2015-01-01

    Senataxin, defective in ataxia oculomotor apraxia type 2, protects the genome by facilitating the resolution of RNA-DNA hybrids (R-loops) and other aspects of RNA processing. Disruption of this gene in mice causes failure of meiotic recombination and defective meiotic sex chromosome inactivation, leading to male infertility. Here we provide evidence that the disruption of Setx leads to reduced SUMOylation and disruption of protein localization across the XY body during meiosis. We demonstrate that senataxin and other DNA damage repair proteins, including ataxia telangiectasia and Rad3-related protein-interacting partner, are SUMOylated, and a marked downregulation of both ataxia telangiectasia and Rad3-related protein-interacting partner and TopBP1 leading to defective activation and signaling through ataxia telangiectasia and Rad3-related protein occurs in the absence of senataxin. Furthermore, chromodomain helicase DNA-binding protein 4, a component of the nucleosome remodeling and deacetylase chromatin remodeler that interacts with both ataxia telangiectasia and Rad3-related protein and senataxin was not recruited efficiently to the XY body, triggering altered histone acetylation and chromatin conformation in Setx (-/-) pachytene-staged spermatocytes. These results demonstrate that senataxin has a critical role in ataxia telangiectasia and Rad3-related protein- and chromodomain helicase DNA-binding protein 4-mediated transcriptional silencing and chromatin remodeling during meiosis providing greater insight into its critical role in gene regulation to protect against neurodegeneration. PMID:27462424

  6. Senataxin controls meiotic silencing through ATR activation and chromatin remodeling

    PubMed Central

    Yeo, Abrey J; Becherel, Olivier J; Luff, John E; Graham, Mark E; Richard, Derek; Lavin, Martin F

    2015-01-01

    Senataxin, defective in ataxia oculomotor apraxia type 2, protects the genome by facilitating the resolution of RNA–DNA hybrids (R-loops) and other aspects of RNA processing. Disruption of this gene in mice causes failure of meiotic recombination and defective meiotic sex chromosome inactivation, leading to male infertility. Here we provide evidence that the disruption of Setx leads to reduced SUMOylation and disruption of protein localization across the XY body during meiosis. We demonstrate that senataxin and other DNA damage repair proteins, including ataxia telangiectasia and Rad3-related protein-interacting partner, are SUMOylated, and a marked downregulation of both ataxia telangiectasia and Rad3-related protein-interacting partner and TopBP1 leading to defective activation and signaling through ataxia telangiectasia and Rad3-related protein occurs in the absence of senataxin. Furthermore, chromodomain helicase DNA-binding protein 4, a component of the nucleosome remodeling and deacetylase chromatin remodeler that interacts with both ataxia telangiectasia and Rad3-related protein and senataxin was not recruited efficiently to the XY body, triggering altered histone acetylation and chromatin conformation in Setx−/− pachytene-staged spermatocytes. These results demonstrate that senataxin has a critical role in ataxia telangiectasia and Rad3-related protein- and chromodomain helicase DNA-binding protein 4-mediated transcriptional silencing and chromatin remodeling during meiosis providing greater insight into its critical role in gene regulation to protect against neurodegeneration. PMID:27462424

  7. Mouse Y-Encoded Transcription Factor Zfy2 Is Essential for Sperm Head Remodelling and Sperm Tail Development

    PubMed Central

    Vernet, Nadege; Mahadevaiah, Shantha K.; Decarpentrie, Fanny; Longepied, Guy; de Rooij, Dirk G.; Burgoyne, Paul S.; Mitchell, Michael J.

    2016-01-01

    A previous study indicated that genetic information encoded on the mouse Y chromosome short arm (Yp) is required for efficient completion of the second meiotic division (that generates haploid round spermatids), restructuring of the sperm head, and development of the sperm tail. Using mouse models lacking a Y chromosome but with varying Yp gene complements provided by Yp chromosomal derivatives or transgenes, we recently identified the Y-encoded zinc finger transcription factors Zfy1 and Zfy2 as the Yp genes promoting the second meiotic division. Using the same mouse models we here show that Zfy2 (but not Zfy1) contributes to the restructuring of the sperm head and is required for the development of the sperm tail. The preferential involvement of Zfy2 is consistent with the presence of an additional strong spermatid-specific promotor that has been acquired by this gene. This is further supported by the fact that promotion of sperm morphogenesis is also seen in one of the two markedly Yp gene deficient models in which a Yp deletion has created a Zfy2/1 fusion gene that is driven by the strong Zfy2 spermatid-specific promotor, but encodes a protein almost identical to that encoded by Zfy1. Our results point to there being further genetic information on Yp that also has a role in restructuring the sperm head. PMID:26765744

  8. Mouse Y-Encoded Transcription Factor Zfy2 Is Essential for Sperm Head Remodelling and Sperm Tail Development.

    PubMed

    Vernet, Nadege; Mahadevaiah, Shantha K; Decarpentrie, Fanny; Longepied, Guy; de Rooij, Dirk G; Burgoyne, Paul S; Mitchell, Michael J

    2016-01-01

    A previous study indicated that genetic information encoded on the mouse Y chromosome short arm (Yp) is required for efficient completion of the second meiotic division (that generates haploid round spermatids), restructuring of the sperm head, and development of the sperm tail. Using mouse models lacking a Y chromosome but with varying Yp gene complements provided by Yp chromosomal derivatives or transgenes, we recently identified the Y-encoded zinc finger transcription factors Zfy1 and Zfy2 as the Yp genes promoting the second meiotic division. Using the same mouse models we here show that Zfy2 (but not Zfy1) contributes to the restructuring of the sperm head and is required for the development of the sperm tail. The preferential involvement of Zfy2 is consistent with the presence of an additional strong spermatid-specific promotor that has been acquired by this gene. This is further supported by the fact that promotion of sperm morphogenesis is also seen in one of the two markedly Yp gene deficient models in which a Yp deletion has created a Zfy2/1 fusion gene that is driven by the strong Zfy2 spermatid-specific promotor, but encodes a protein almost identical to that encoded by Zfy1. Our results point to there being further genetic information on Yp that also has a role in restructuring the sperm head. PMID:26765744

  9. Selection-Independent Generation of Gene Knockout Mouse Embryonic Stem Cells Using Zinc-Finger Nucleases

    PubMed Central

    Osiak, Anna; Radecke, Frank; Guhl, Eva; Radecke, Sarah; Dannemann, Nadine; Lütge, Fabienne; Glage, Silke; Rudolph, Cornelia; Cantz, Tobias; Schwarz, Klaus; Heilbronn, Regine; Cathomen, Toni

    2011-01-01

    Gene knockout in murine embryonic stem cells (ESCs) has been an invaluable tool to study gene function in vitro or to generate animal models with altered phenotypes. Gene targeting using standard techniques, however, is rather inefficient and typically does not exceed frequencies of 10−6. In consequence, the usage of complex positive/negative selection strategies to isolate targeted clones has been necessary. Here, we present a rapid single-step approach to generate a gene knockout in mouse ESCs using engineered zinc-finger nucleases (ZFNs). Upon transient expression of ZFNs, the target gene is cleaved by the designer nucleases and then repaired by non-homologous end-joining, an error-prone DNA repair process that introduces insertions/deletions at the break site and therefore leads to functional null mutations. To explore and quantify the potential of ZFNs to generate a gene knockout in pluripotent stem cells, we generated a mouse ESC line containing an X-chromosomally integrated EGFP marker gene. Applying optimized conditions, the EGFP locus was disrupted in up to 8% of ESCs after transfection of the ZFN expression vectors, thus obviating the need of selection markers to identify targeted cells, which may impede or complicate downstream applications. Both activity and ZFN-associated cytotoxicity was dependent on vector dose and the architecture of the nuclease domain. Importantly, teratoma formation assays of selected ESC clones confirmed that ZFN-treated ESCs maintained pluripotency. In conclusion, the described ZFN-based approach represents a fast strategy for generating gene knockouts in ESCs in a selection-independent fashion that should be easily transferrable to other pluripotent stem cells. PMID:22194948

  10. Early response of gene clusters is associated with mouse lung resistance or sensitivity to cigarette smoke.

    PubMed

    Cavarra, Eleonora; Fardin, Paolo; Fineschi, Silvia; Ricciardi, Annamaria; De Cunto, Giovanna; Sallustio, Fabio; Zorzetto, Michele; Luisetti, Maurizio; Pfeffer, Ulrich; Lungarella, Giuseppe; Varesio, Luigi

    2009-03-01

    We have investigated the effects of cigarette smoke exposure in three different strains of mice. DBA/2 and C57BL/6J are susceptible to smoke and develop different lung changes in response to chronic exposure, whereas ICR mice are resistant to smoke and do not develop emphysema. The present study was carried out to determine early changes in the gene expression profile of mice exposed to cigarette smoke with either a susceptible or resistant phenotype. The three strains of mice were exposed to smoke from three cigarettes per day, 5 days/wk, for 4 wk. Microarray analysis was carried out on total RNA extracted from the lung using the Affymetrix platform. Cigarette smoke modulates several clusters of genes (i.e., proemphysematous, acute phase response, and cell adhesion) in smoke-sensitive DBA/2 or C57BL/6J strains, but the same genes are not altered by smoke in ICR resistant mice. Only a few genes were commonly modulated by smoke in the three strains of mice. This pattern of gene expression suggests that the response to smoke is strain-dependent and may involve different molecular signaling pathways. Real-time quantitative PCR was used to verify the pattern of modulation of selected genes and their potential biological relevance. We conclude that gene expression response to smoke is highly dependent on the mouse genetic background. We speculate that the definition of gene clusters associated, to various degrees, with mouse susceptibility or resistance to smoke may be instrumental in defining the molecular basis of the individual response to smoke-induced lung injury in humans.

  11. The chromosomal integration site determines the tissue-specific methylation of mouse mammary tumour virus proviral genes.

    PubMed Central

    Günzburg, W H; Groner, B

    1984-01-01

    Multiple endogenous mouse mammary tumour virus (MMTV) proviral genes are present at different chromosomal locations in inbred mouse strains. Proviral DNA methylation is location and tissue specific. The methylation patterns are stably inherited and appear to be conferred upon the viral DNA by the flanking mouse genomic DNA. In transformed cells, either mammary carcinoma cells, or cells immortalized by SV40 in vitro, the stable pattern of methylation is lost. Although hypomethylation of proviral genes, both in normal and in transformed tissue, accompanies MMTV-specific RNA expression, it is also observed in non-expressing tissues. Images Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:6329738

  12. The fibulin-1 gene (FBLN1) is located on human chromosome 22 and on mouse chromosome 15

    SciTech Connect

    Mattei, M.G.; Pan, T.C.; Zhang, R.Z.

    1994-07-15

    Fibulin-1 is a calcium-binding glycoprotein present in the extracellular matrix and in the serum. The gene coding for fibulin-1 (FBLN1) was located by in situ hybridization of {sup 3}H-labeled cDNA probes to human and mouse metaphase chromosomes. The gene was assigned to the q13.2-q13.3 region of human chromosome 22 and to the E-F band of mouse chromosome 15. The finding extends the evolutionary conservation between human chromosome 22 and mouse chromosome 15. 11 refs., 2 figs.

  13. Mouse muscle nicotinic acetylcholine receptor gamma subunit: cDNA sequence and gene expression.

    PubMed Central

    Yu, L; LaPolla, R J; Davidson, N

    1986-01-01

    Clones coding for the mouse nicotinic acetylcholine receptor (AChR) gamma subunit precursor have been selected from a cDNA library derived from a mouse myogenic cell line and sequenced. The deduced protein sequence consists of a signal peptide of 22 amino acid residues and a mature gamma subunit of 497 amino acid residues. There is a high degree of sequence conservation between this mouse sequence and published human and calf AChR gamma subunits and, after allowing for functional amino acid substitutions, also to the more distantly related chicken and Torpedo AChR gamma subunits. The degree of sequence conservation is especially high in the four putative hydrophobic membrane spanning regions, supporting the assignment of these domains. RNA blot hybridization showed that the mRNA level of the gamma subunit increases by 30 fold or more upon differentiation of the two mouse myogenic cell lines, BC3H-1 and C2C12, suggesting that the primary controls for changes in gene expression during differentiation are at the level of transcription. One cDNA clone was found to correspond to a partially processed nuclear transcript containing two as yet unspliced intervening sequences. Images PMID:3010242

  14. Chromosomal localization of three mouse diacylglycerol kinase (DAGK) genes: Genes sharing sequence homology to the Drosophila retinal degeneration A (rdgA) gene

    SciTech Connect

    Pilz, A.; Hunt, D.; Fitzgibbon, J.

    1995-04-10

    There is growing evidence to support some form of light-activated phosphoinositide signal transduction pathway in the mammalian retina. Although this pathway plays no obvious role in mammalian phototransduction, mutations in this pathway cause retinal degenerations in Drosophila. These include the retinal-degeneration A mutant, which is caused by an alteration in an eye-specific diacylglycerol kinase (DAGK) gene. In our efforts to consider genes mutated in Drosophila as candidates for mammalian eye disease, we have initially determined the map position of three DAGK genes in the mouse. 21 refs., 2 figs.

  15. Identifying aging-related genes in mouse hippocampus using gateway nodes

    PubMed Central

    2014-01-01

    Background High-throughput studies continue to produce volumes of metadata representing valuable sources of information to better guide biological research. With a stronger focus on data generation, analysis models that can readily identify actual signals have not received the same level of attention. This is due in part to high levels of noise and data heterogeneity, along with a lack of sophisticated algorithms for mining useful information. Networks have emerged as a powerful tool for modeling high-throughput data because they are capable of representing not only individual biological elements but also different types of relationships en masse. Moreover, well-established graph theoretic methodology can be applied to network models to increase efficiency and speed of analysis. In this project, we propose a network model that examines temporal data from mouse hippocampus at the transcriptional level via correlation of gene expression. Using this model, we formally define the concept of “gateway” nodes, loosely defined as nodes representing genes co-expressed in multiple states. We show that the proposed network model allows us to identify target genes implicated in hippocampal aging-related processes. Results By mining gateway genes related to hippocampal aging from networks made from gene expression in young and middle-aged mice, we provide a proof-of-concept of existence and importance of gateway nodes. Additionally, these results highlight how network analysis can act as a supplement to traditional statistical analysis of differentially expressed genes. Finally, we use the gateway nodes identified by our method as well as functional databases and literature to propose new targets for study of aging in the mouse hippocampus. Conclusions This research highlights the need for methods of temporal comparison using network models and provides a systems biology approach to extract information from correlation networks of gene expression. Our results identify a

  16. Using mouse models of autism spectrum disorders to study the neurotoxicology of gene-environment interactions

    PubMed Central

    Schwartzer, Jared J.; Koenig, Claire M.; Berman, Robert F

    2012-01-01

    To better study the role of genetics in autism, mouse models have been developed which mimic the genetics of specific autism spectrum and related disorders. These models have facilitated research on the role genetic susceptibility factors in the pathogenesis of autism in the absence of environmental factors. Inbred mouse strains have been similarly studied to assess the role of environmental agents on neurodevelopment, typically without the complications of genetic heterogeneity of the human population. What has not been as actively pursued, however, is the methodical study of the interaction between these factors (e.g., gene and environmental interactions in neurodevelopment). This review suggests that a genetic predisposition paired with exposure to environmental toxicants play an important role in the etiology of neurodevelopmental disorders including autism, and may contribute to the largely unexplained rise in the number of children diagnosed with autism worldwide. Specifically, descriptions of the major mouse models of autism and toxic mechanisms of prevalent environmental chemicals are provided followed by a discussion of current and future research strategies to evaluate the role of gene and environment interactions in neurodevelopmental disorders. PMID:23010509

  17. NFAT5 regulates transcription of the mouse telomerase reverse transcriptase gene

    SciTech Connect

    Fujiki, Tsukasa; Udono, Miyako; Kotake, Yojiro; Yamashita, Makiko; Shirahata, Sanetaka; Katakura, Yoshinori

    2010-12-10

    We aimed to clarify the transcription-regulation mechanisms of the mouse telomerase reverse transcriptase gene (mTERT). First, we searched for the promoter region required for transcriptional activation of mTERT and identified an enhancer cis-element (named mTERT-EE) located between - 200 and - 179 bp of the mouse TERT gene (mTERT). EMSA results suggested that nuclear factor of activated T cells (NFAT) member proteins bind to mTERT-EE. We then identified NFAT5 as the factor binding to mTERT-EE and found that it activates the transcription of the mTERT core promoter. The results that siRNA directed against NFAT5 significantly reduced mTERT expression and mTERT core promoter activity and that the expressions of NFAT5 and mTERT were well correlated in various mouse tissues except liver suggest that NFAT5 dominantly and directly regulates mTERT expression. To clarify their functionality further, we investigated the effect of hypertonic stress, a known stimulus affecting the expression and transcriptional activity of NFAT5, on mTERT expression. The result indicated that hypertonic stress activates mTERT transcription via the activation and recruitment of NFAT5 to the mTERT promoter. These results provide useful information about the transcription-regulation mechanisms of mTERT.

  18. Methylation-Associated Gene Silencing of RARB in Areca Carcinogens Induced Mouse Oral Squamous Cell Carcinoma

    PubMed Central

    Tsou, Yung-An; Fan, Shin-Ru; Tsai, Ming-Hsui; Chen, Hsiao-Ling; Chang, Nai-Wen; Cheng, Ju-Chien

    2014-01-01

    Regarding oral squamous cell carcinoma (OSCC) development, chewing areca is known to be a strong risk factor in many Asian cultures. Therefore, we established an OSCC induced mouse model by 4-nitroquinoline-1-oxide (4-NQO), or arecoline, or both treatments, respectively. These are the main two components of the areca nut that could increase the occurrence of OSCC. We examined the effects with the noncommercial MCGI (mouse CpG islands) microarray for genome-wide screening the DNA methylation aberrant in induced OSCC mice. The microarray results showed 34 hypermethylated genes in 4-NQO plus arecoline induced OSCC mice tongue tissues. The examinations also used methylation-specific polymerase chain reaction (MS-PCR) and bisulfite sequencing to realize the methylation pattern in collected mouse tongue tissues and human OSCC cell lines of different grades, respectively. These results showed that retinoic acid receptor β (RARB) was indicated in hypermethylation at the promoter region and the loss of expression during cancer development. According to the results of real-time PCR, it was shown that de novo DNA methyltransferases were involved in gene epigenetic alternations of OSCC. Collectively, our results showed that RARB hypermethylation was involved in the areca-associated oral carcinogenesis. PMID:25197641

  19. Quadruple zebrafish mutant reveals different roles of Mesp genes in somite segmentation between mouse and zebrafish.

    PubMed

    Yabe, Taijiro; Hoshijima, Kazuyuki; Yamamoto, Takashi; Takada, Shinji

    2016-08-01

    The segmental pattern of somites is generated by sequential conversion of the temporal periodicity provided by the molecular clock. Whereas the basic structure of this clock is conserved among different species, diversity also exists, especially in terms of the molecular network. The temporal periodicity is subsequently converted into the spatial pattern of somites, and Mesp2 plays crucial roles in this conversion in the mouse. However, it remains unclear whether Mesp genes play similar roles in other vertebrates. In this study, we generated zebrafish mutants lacking all four zebrafish Mesp genes by using TALEN-mediated genome editing. Contrary to the situation in the mouse Mesp2 mutant, in the zebrafish Mesp quadruple mutant embryos the positions of somite boundaries were clearly determined and morphological boundaries were formed, although their formation was not completely normal. However, each somite was caudalized in a similar manner to the mouse Mesp2 mutant, and the superficial horizontal myoseptum and lateral line primordia were not properly formed in the quadruple mutants. These results clarify the conserved and species-specific roles of Mesp in the link between the molecular clock and somite morphogenesis. PMID:27385009

  20. Identification of a conserved set of upregulated genes in mouse skeletal muscle hypertrophy and regrowth

    PubMed Central

    Chaillou, Thomas; Jackson, Janna R.; England, Jonathan H.; Kirby, Tyler J.; Richards-White, Jena; Esser, Karyn A.; Dupont-Versteegden, Esther E.

    2014-01-01

    The purpose of this study was to compare the gene expression profile of mouse skeletal muscle undergoing two forms of growth (hypertrophy and regrowth) with the goal of identifying a conserved set of differentially expressed genes. Expression profiling by microarray was performed on the plantaris muscle subjected to 1, 3, 5, 7, 10, and 14 days of hypertrophy or regrowth following 2 wk of hind-limb suspension. We identified 97 differentially expressed genes (≥2-fold increase or ≥50% decrease compared with control muscle) that were conserved during the two forms of muscle growth. The vast majority (∼90%) of the differentially expressed genes was upregulated and occurred at a single time point (64 out of 86 genes), which most often was on the first day of the time course. Microarray analysis from the conserved upregulated genes showed a set of genes related to contractile apparatus and stress response at day 1, including three genes involved in mechanotransduction and four genes encoding heat shock proteins. Our analysis further identified three cell cycle-related genes at day and several genes associated with extracellular matrix (ECM) at both days 3 and 10. In conclusion, we have identified a core set of genes commonly upregulated in two forms of muscle growth that could play a role in the maintenance of sarcomere stability, ECM remodeling, cell proliferation, fast-to-slow fiber type transition, and the regulation of skeletal muscle growth. These findings suggest conserved regulatory mechanisms involved in the adaptation of skeletal muscle to increased mechanical loading. PMID:25554798

  1. PCR detection of retinoblastoma gene deletions in radiation-induced mouse lung adenocarcinomas

    SciTech Connect

    Churchill, M.E.; Gemmell, M.A.; Woloschak, G.E.

    1994-05-01

    From 1971--1986, Argonne National Laboratory conducted a series of large-scale studies of tumor incidence in 40,000 BCF{sub 1} mice irradiated with {sup 60}Co {gamma}-rays or JANUS fission-spectrum neutrons. Polymerase chain reaction (PCR) technique was used to detect deletions in the mouse retinoblastoma (mRb) gene. Six mRb gene exon fragments were amplified in a 40-cycle, 3-temperature PCR protocol. Absence of any of these fragments on a Southern blot indicated a deletion of that portion of the mRb gene. Tumors chosen for analysis were lung adenocarcinomas that were judged to be the cause of death in post-mortem analyses. Spontaneous tumors as well as those from irradiated mice were analyzed for mRb deletions. In all normal mouse tissues studies all six mRb exon fragments were present on Southern blots. Tumors in six neutron-irradiated mice also had no mRb deletions. However, 1 of 6 tumors from {gamma}-irradiated mice and 6 of 18 spontaneous tumors from unirradiated mice showed a deletion in one or both mRb alleles. All deletions detected were in the 5{prime} region of the mRb gene.

  2. EHMT2 directs DNA methylation for efficient gene silencing in mouse embryos

    PubMed Central

    Auclair, Ghislain; Borgel, Julie; Sanz, Lionel A.; Vallet, Judith; Guibert, Sylvain; Dumas, Michael; Cavelier, Patricia; Girardot, Michael; Forné, Thierry; Feil, Robert; Weber, Michael

    2016-01-01

    The extent to which histone modifying enzymes contribute to DNA methylation in mammals remains unclear. Previous studies suggested a link between the lysine methyltransferase EHMT2 (also known as G9A and KMT1C) and DNA methylation in the mouse. Here, we used a model of knockout mice to explore the role of EHMT2 in DNA methylation during mouse embryogenesis. The Ehmt2 gene is expressed in epiblast cells but is dispensable for global DNA methylation in embryogenesis. In contrast, EHMT2 regulates DNA methylation at specific sequences that include CpG-rich promoters of germline-specific genes. These loci are bound by EHMT2 in embryonic cells, are marked by H3K9 dimethylation, and have strongly reduced DNA methylation in Ehmt2−/− embryos. EHMT2 also plays a role in the maintenance of germline-derived DNA methylation at one imprinted locus, the Slc38a4 gene. Finally, we show that DNA methylation is instrumental for EHMT2-mediated gene silencing in embryogenesis. Our findings identify EHMT2 as a critical factor that facilitates repressive DNA methylation at specific genomic loci during mammalian development. PMID:26576615

  3. Thalidomide induced early gene expression perturbations indicative of human embryopathy in mouse embryonic stem cells.

    PubMed

    Gao, Xiugong; Sprando, Robert L; Yourick, Jeffrey J

    2015-08-15

    Developmental toxicity testing has traditionally relied on animal models which are costly, time consuming, and require the sacrifice of large numbers of animals. In addition, there are significant disparities between human beings and animals in their responses to chemicals. Thalidomide is a species-specific developmental toxicant that causes severe limb malformations in humans but not in mice. Here, we used microarrays to study transcriptomic changes induced by thalidomide in an in vitro model based on differentiation of mouse embryonic stem cells (mESCs). C57BL/6 mESCs were allowed to differentiate spontaneously and RNA was collected at 24, 48, and 72h after exposure to 0.25mM thalidomide. Global gene expression analysis using microarrays revealed hundreds of differentially expressed genes upon thalidomide exposure that were enriched in gene ontology (GO) terms and canonical pathways associated with embryonic development and differentiation. In addition, many genes were found to be involved in small GTPases-mediated signal transduction, heart development, and inflammatory responses, which coincide with clinical evidences and may represent critical embryotoxicities of thalidomide. These results demonstrate that transcriptomics in combination with mouse embryonic stem cell differentiation is a promising alternative model for developmental toxicity assessment.

  4. Survival benefit and phenotypic improvement by hamartin gene therapy in a tuberous sclerosis mouse brain model

    PubMed Central

    Prabhakar, Shilpa; Zhang, Xuan; Goto, June; Han, Sangyeul; Lai, Charles; Bronson, Roderick; Sena-Esteves, Miguel; Ramesh, Vijaya; Stemmer-Rachamimov, Anat; Kwiatkowski, David J.; Breakefield, Xandra O.

    2016-01-01

    We examined the potential benefit of gene therapy in a mouse model of tuberous sclerosis complex (TSC) in which there is embryonic loss of Tsc1 (hamartin) in brain neurons. An adeno-associated virus (AAV) vector (serotype rh8) expressing a tagged form of hamartin was injected into the cerebral ventricles of newborn pups with the genotype Tsc1cc (homozygous for a conditional floxed Tsc1 allele) SynI-cre+, in which Tsc1 is lost selectively in neurons starting at embryonic day 12. Vector-treated Tsc1ccSynIcre+ mice showed a marked improvement in survival from a mean of 22 days in non-injected mice to 52 days in AAV hamartin vector-injected mice, with improved weight gain and motor behavior in the latter. Pathologic studies showed normalization of neuron size and a decrease in markers of mTOR activation in treated as compared to untreated mutant littermates. Hence, we show that gene replacement in the brain is an effective therapeutic approach in this mouse model of TSC1. Our strategy for gene therapy has the advantages that therapy can be achieved from a single application, as compared to repeated treatment with drugs, and that AAV vectors have been found to have minimal to no toxicity in clinical trials for other neurologic conditions. Although there are many additional issues to be addressed, our studies support gene therapy as a useful approach in TSC patients. PMID:26019056

  5. Cell-type-specific neuroanatomy of cliques of autism-related genes in the mouse brain

    PubMed Central

    Grange, Pascal; Menashe, Idan; Hawrylycz, Michael

    2015-01-01

    Two cliques of genes identified computationally for their high co-expression in the mouse brain according to the Allen Brain Atlas, and for their enrichment in genes related to autism spectrum disorder (ASD), have recently been shown to be highly co-expressed in the cerebellar cortex, compared to what could be expected by chance. Moreover, the expression of these cliques of genes is not homogeneous across the cerebellar cortex, and it has been noted that their expression pattern seems to highlight the granular layer. However, this observation was only made by eye, and recent advances in computational neuroanatomy allow to rank cell types in the mouse brain (characterized by their transcriptome profiles) according to the similarity between their spatial density profiles and the spatial expression profiles of the cliques. We establish by Monte Carlo simulation that with probability at least 99%, the expression profiles of the two cliques are more similar to the density profile of granule cells than 99% of the expression of cliques containing the same number of genes (Purkinje cells also score above 99% in one of the cliques). Thresholding the expression profiles shows that the signal is more intense in the granular layer. Finally, we work out pairs of cell types whose combined expression profiles are more similar to the expression profiles of the cliques than any single cell type. These pairs predominantly consist of one cortical pyramidal cell and one cerebellar cell (which can be either a granule cell or a Purkinje cell). PMID:26074809

  6. A short upstream promoter region mediates transcriptional regulation of the mouse doublecortin gene in differentiating neurons

    PubMed Central

    2010-01-01

    Background Doublecortin (Dcx), a MAP (Microtubule-Associated Protein), is transiently expressed in migrating and differentiating neurons and thereby characterizes neuronal precursors and neurogenesis in developing and adult neurogenesis. In addition, reduced Dcx expression during development has been related to appearance of brain pathologies. Here, we attempt to unveil the molecular mechanisms controlling Dcx gene expression by studying its transcriptional regulation during neuronal differentiation. Results To determine and analyze important regulatory sequences of the Dcx promoter, we studied a putative regulatory region upstream from the mouse Dcx coding region (pdcx2kb) and several deletions thereof. These different fragments were used in vitro and in vivo to drive reporter gene expression. We demonstrated, using transient expression experiments, that pdcx2kb is sufficient to control specific reporter gene expression in cerebellar cells and in the developing brain (E14.5). We determined the temporal profile of Dcx promoter activity during neuronal differentiation of mouse embryonic stem cells (mESC) and found that transcriptional activation of the Dcx gene varies along with neuronal differentiation of mESC. Deletion experiments and sequence comparison of Dcx promoters across rodents, human and chicken revealed the importance of a highly conserved sequence in the proximal region of the promoter required for specific and strong expression in neuronal precursors and young neuronal cells. Further analyses revealed the presence in this short sequence of several conserved, putative transcription factor binding sites: LEF/TCF (Lymphoid Enhancer Factor/T-Cell Factor) which are effectors of the canonical Wnt pathway; HNF6/OC2 (Hepatocyte Nuclear Factor-6/Oncecut-2) members of the ONECUT family and NF-Y/CAAT (Nuclear Factor-Y). Conclusions Studies of Dcx gene regulatory sequences using native, deleted and mutated constructs suggest that fragments located upstream of the

  7. The human and mouse receptors of hyaluronan-mediated motility, RHAMM, genes (HMMR) map to human chromosome 5q33.2-qter and mouse chromosome 11

    SciTech Connect

    Spicer, A.P.; McDonald, J.A.; Roller, M.L.; Camper, S.A.

    1995-11-01

    The gene for the receptor for hyaluronan-mediated motility, RHAAM (designated hyaluronan-mediated motility receptor, HMMR (human) and Hmmr (mouse), for mapping purposes), was localized to human chromosome 5q33.2-qter by somatic cell and radiation hybrid analyses. Investigation of two interspecific back-crosses localized the mouse RHAMM (Hmmr) locus 18 cM from the centromere of mouse chromosome 11 within a region of synteny homology with human chromosome 5q23-q35 genes. The map position of the human RHAMM gene places it in a region comparatively rich in disease-associated genes, including those for low-frequency hearing loss, dominant limb-girdle muscular dystrophy, diastrophic dysplasia, Treacher Collins syndrome, and myeloid disorders associated with the 5q-syndrome. The RHAMM gene location and its ability to transform cells when overexpressed implicate RHAMM as a possible candidate gene in the pathogenesis of the recently described t(5;14)(q33-q34;q11) acute lymphoblastic leukemias. 18 refs., 1 fig.

  8. Activation of an imprinted Igf 2 gene in mouse somatic cell cultures.

    PubMed Central

    Eversole-Cire, P; Ferguson-Smith, A C; Sasaki, H; Brown, K D; Cattanach, B M; Gonzales, F A; Surani, M A; Jones, P A

    1993-01-01

    The mouse insulin-like growth factor II gene (Igf 2), located on distal chromosome 7, is parentally imprinted such that the paternal allele is expressed while the maternal allele is transcriptionally silent. We derived a cell line from a mouse embryo maternally disomic and paternally deficient for distal chromosome 7 (MatDi7) to determine the stability of gene repression in culture. MatDi7 cells maintained Igf2 in a repressed state even after immortalization, except for one randomly picked clone which spontaneously expressed the gene. Igf 2 was expressed in a cell culture derived from a normal littermate; this expression was growth regulated, with Igf 2 mRNA levels increasing in the stationary phase of growth. Analysis of the methylation status of 28 sites distributed over 10 kb of the gene did not show consistent differences associated with expression level in the normal and MatDi7 cell lines, and the CpG island in the Igf 2 promoter remained unmethylated in all of the cell lines. Only with an oncogenically transformed cell line did the promoter become extensively methylated. We attempted to derepress the imprinted gene in MatDi7 cells by treatments known to alter gene expression. Expression of the Igf 2 allele in MatDi7 cells was increased in a dose-dependent manner by treatment with 5-aza-2'-deoxycytidine or bromodeoxyuridine, agents known to change DNA methylation patterns or chromatin conformation. Treatment of the cells with 1-beta-D-arabinofuranosylcytosine, 2'-deoxycytidine, calcium ionophore, heat shock, cold shock, or sodium butyrate did not result in increases in the levels of Igf 2 expression. It seems likely that the mechanism of the Igf 2 imprint involves subtle changes in the methylation or chromatin conformation of the gene which are affected by 5-aza-2'-deoxycytidine and bromodeoxyuridine. Images PMID:8336727

  9. A lacZ reporter gene expression atlas for 313 adult KOMP mutant mouse lines

    PubMed Central

    Pasumarthi, Ravi K.; Baridon, Brian; Djan, Esi; Trainor, Amanda; Griffey, Stephen M.; Engelhard, Eric K.; Rapp, Jared; Li, Bowen; de Jong, Pieter J.; Lloyd, K.C. Kent

    2015-01-01

    Expression of the bacterial beta-galactosidase reporter gene (lacZ) in the vector used for the Knockout Mouse Project (KOMP) is driven by the endogenous promoter of the target gene. In tissues from KOMP mice, histochemical staining for LacZ enzyme activity can be used to determine gene expression patterns. With this technique, we have produced a comprehensive resource of gene expression using both whole mount (WM) and frozen section (FS) LacZ staining in 313 unique KOMP mutant mouse lines. Of these, ∼80% of mutants showed specific staining in one or more tissues, while ∼20% showed no specific staining, ∼13% had staining in only one tissue, and ∼25% had staining in >6 tissues. The highest frequency of specific staining occurred in the brain (∼50%), male gonads (42%), and kidney (39%). The WM method was useful for rapidly identifying whole organ and some substructure staining, while the FS method often revealed substructure and cellular staining specificity. Both staining methods had >90% repeatability in biological replicates. Nonspecific LacZ staining occurs in some tissues due to the presence of bacteria or endogenous enzyme activity. However, this can be effectively distinguished from reporter gene activity by the combination of the WM and FS methods. After careful annotation, LacZ staining patterns in a high percentage of mutants revealed a unique structure-function not previously reported for many of these genes. The validation of methods for LacZ staining, annotation, and expression analysis reported here provides unique insights into the function of genes for which little is currently known. PMID:25591789

  10. PCR detection of retinoblastoma gene deletions in radiation-induced mouse lung adenocarcinomas

    SciTech Connect

    Churchill, M.E.; Gemmell, M.A.; Woloschak, G.E.

    1993-04-01

    From 1971 to 1986, Argonne National Laboratory conducted a series of large-scale studies of tumor incidence in 40,000 BCF{sub 1} mice irradiated with {sup 60}Co {gamma} rays or JANUS fission-spectrum neutrons; normal and tumor tissues from mice in these studies were preserved in paraffin blocks. A polymerase chain reaction (PCR) technique has been developed to detect deletions in the mouse retinoblastoma (mRb) gene in the paraffin-embedded tissues. Microtomed sections were used as the DNA source in PCR reaction mixtures. Six mRb gene exon fragments were amplified in a 40-cycle, 3-temperature PCR protocol. The absence of any of these fragments (relative to control PCR products) on a Southern blot indicated a deletion of that portion of the mRb gene. The tumors chosen for analysis were lung adenocarcinomas that were judged to be the cause of death in post-mortem analyses. Spontaneous tumors as well as those from irradiated mice (569 cGy of {sup 60}Co {gamma} rays or 60 cGy of JANUS neutrons, doses that have been found to have approximately equal biological effectiveness in the BCF, mouse) were analyzed for mRb deletions. In all normal mouse tissues studies, all six mRb exon fragments were present on Southem blots. Tumors in six neutron-irradiated mice also had no mRb deletions. However, I of 6 tumors from {gamma}-irradiated mice and 6 of 18 spontaneous tumors from unirradiated mice had a deletion in one or both mRb alleles. All deletions detected were in the 5{prime} region of the mRb gene.

  11. PCR detection of retinoblastoma gene deletions in radiation-induced mouse lung adenocarcinomas

    SciTech Connect

    Churchill, M.E.; Gemmell, M.A.; Woloschak, G.E.

    1993-01-01

    From 1971 to 1986, Argonne National Laboratory conducted a series of large-scale studies of tumor incidence in 40,000 BCF[sub 1] mice irradiated with [sup 60]Co [gamma] rays or JANUS fission-spectrum neutrons; normal and tumor tissues from mice in these studies were preserved in paraffin blocks. A polymerase chain reaction (PCR) technique has been developed to detect deletions in the mouse retinoblastoma (mRb) gene in the paraffin-embedded tissues. Microtomed sections were used as the DNA source in PCR reaction mixtures. Six mRb gene exon fragments were amplified in a 40-cycle, 3-temperature PCR protocol. The absence of any of these fragments (relative to control PCR products) on a Southern blot indicated a deletion of that portion of the mRb gene. The tumors chosen for analysis were lung adenocarcinomas that were judged to be the cause of death in post-mortem analyses. Spontaneous tumors as well as those from irradiated mice (569 cGy of [sup 60]Co [gamma] rays or 60 cGy of JANUS neutrons, doses that have been found to have approximately equal biological effectiveness in the BCF, mouse) were analyzed for mRb deletions. In all normal mouse tissues studies, all six mRb exon fragments were present on Southem blots. Tumors in six neutron-irradiated mice also had no mRb deletions. However, I of 6 tumors from [gamma]-irradiated mice and 6 of 18 spontaneous tumors from unirradiated mice had a deletion in one or both mRb alleles. All deletions detected were in the 5[prime] region of the mRb gene.

  12. Meiotic Recombination in Drosophila Females Depends on Chromosome Continuity Between Genetically Defined Boundaries

    PubMed Central

    Sherizen, Dalia; Jang, Janet K.; Bhagat, Rajal; Kato, Naohiro; McKim, Kim S.

    2005-01-01

    In the pairing-site model, specialized regions on each chromosome function to establish meiotic homolog pairing. Analysis of these sites could provide insights into the mechanism used by Drosophila females to form a synaptonemal complex (SC) in the absence of meiotic recombination. These specialized sites were first established on the X chromosome by noting that there were barriers to crossover suppression caused by translocation heterozygotes. These sites were genetically mapped and proposed to be pairing sites. By comparing the cytological breakpoints of third chromosome translocations to their patterns of crossover suppression, we have mapped two sites on chromosome 3R. We have performed experiments to determine if these sites have a role in meiotic homolog pairing and the initiation of recombination. Translocation heterozygotes exhibit reduced gene conversion within the crossover-suppressed region, consistent with an effect on the initiation of meiotic recombination. To determine if homolog pairing is disrupted in translocation heterozygotes, we used fluorescent in situ hybridization to measure the extent of homolog pairing. In wild-type oocytes, homologs are paired along their entire lengths prior to accumulation of the SC protein C(3)G. Surprisingly, translocation heterozygotes exhibited homolog pairing similar to wild type within the crossover-suppressed regions. This result contrasted with our observations of c(3)G mutant females, which were found to be defective in pairing. We propose that each Drosophila chromosome is divided into several domains by specialized sites. These sites are not required for homolog pairing. Instead, the initiation of meiotic recombination requires continuity of the meiotic chromosome structure within each of these domains. PMID:15545646

  13. A high throughput genetic screen identifies new early meiotic recombination functions in Arabidopsis thaliana.

    PubMed

    De Muyt, Arnaud; Pereira, Lucie; Vezon, Daniel; Chelysheva, Liudmila; Gendrot, Ghislaine; Chambon, Aurélie; Lainé-Choinard, Sandrine; Pelletier, Georges; Mercier, Raphaël; Nogué, Fabien; Grelon, Mathilde

    2009-09-01

    Meiotic recombination is initiated by the formation of numerous DNA double-strand breaks (DSBs) catalysed by the widely conserved Spo11 protein. In Saccharomyces cerevisiae, Spo11 requires nine other proteins for meiotic DSB formation; however, unlike Spo11, few of these are conserved across kingdoms. In order to investigate this recombination step in higher eukaryotes, we took advantage of a high-throughput meiotic mutant screen carried out in the model plant Arabidopsis thaliana. A collection of 55,000 mutant lines was screened, and spo11-like mutations, characterised by a drastic decrease in chiasma formation at metaphase I associated with an absence of synapsis at prophase, were selected. This screen led to the identification of two populations of mutants classified according to their recombination defects: mutants that repair meiotic DSBs using the sister chromatid such as Atdmc1 or mutants that are unable to make DSBs like Atspo11-1. We found that in Arabidopsis thaliana at least four proteins are necessary for driving meiotic DSB repair via the homologous chromosomes. These include the previously characterised DMC1 and the Hop1-related ASY1 proteins, but also the meiotic specific cyclin SDS as well as the Hop2 Arabidopsis homologue AHP2. Analysing the mutants defective in DSB formation, we identified the previously characterised AtSPO11-1, AtSPO11-2, and AtPRD1 as well as two new genes, AtPRD2 and AtPRD3. Our data thus increase the number of proteins necessary for DSB formation in Arabidopsis thaliana to five. Unlike SPO11 and (to a minor extent) PRD1, these two new proteins are poorly conserved among species, suggesting that the DSB formation mechanism, but not its regulation, is conserved among eukaryotes.

  14. Cardiac gene expression data and in silico analysis provide novel insights into human and mouse taste receptor gene regulation.

    PubMed

    Foster, Simon R; Porrello, Enzo R; Stefani, Maurizio; Smith, Nicola J; Molenaar, Peter; dos Remedios, Cristobal G; Thomas, Walter G; Ramialison, Mirana

    2015-10-01

    G protein-coupled receptors are the principal mediators of the sweet, umami, bitter, and fat taste qualities in mammals. Intriguingly, the taste receptors are also expressed outside of the oral cavity, including in the gut, airways, brain, and heart, where they have additional functions and contribute to disease. However, there is little known about the mechanisms governing the transcriptional regulation of taste receptor genes. Following our recent delineation of taste receptors in the heart, we investigated the genomic loci encoding for taste receptors to gain insight into the regulatory mechanisms that drive their expression in the heart. Gene expression analyses of healthy and diseased human and mouse hearts showed coordinated expression for a subset of chromosomally clustered taste receptors. This chromosomal clustering mirrored the cardiac expression profile, suggesting that a common gene regulatory block may control the taste receptor locus. We identified unique domains with strong regulatory potential in the vicinity of taste receptor genes. We also performed de novo motif enrichment in the proximal promoter regions and found several overrepresented DNA motifs in cardiac taste receptor gene promoters corresponding to ubiquitous and cardiac-specific transcription factor binding sites. Thus, combining cardiac gene expression data with bioinformatic analyses, this study has provided insights into the noncoding regulatory landscape for taste GPCRs. These findings also have broader relevance for the study of taste GPCRs outside of the classical gustatory system, where understanding the mechanisms controlling the expression of these receptors may have implications for future therapeutic development.

  15. Cardiac gene expression data and in silico analysis provide novel insights into human and mouse taste receptor gene regulation.

    PubMed

    Foster, Simon R; Porrello, Enzo R; Stefani, Maurizio; Smith, Nicola J; Molenaar, Peter; dos Remedios, Cristobal G; Thomas, Walter G; Ramialison, Mirana

    2015-10-01

    G protein-coupled receptors are the principal mediators of the sweet, umami, bitter, and fat taste qualities in mammals. Intriguingly, the taste receptors are also expressed outside of the oral cavity, including in the gut, airways, brain, and heart, where they have additional functions and contribute to disease. However, there is little known about the mechanisms governing the transcriptional regulation of taste receptor genes. Following our recent delineation of taste receptors in the heart, we investigated the genomic loci encoding for taste receptors to gain insight into the regulatory mechanisms that drive their expression in the heart. Gene expression analyses of healthy and diseased human and mouse hearts showed coordinated expression for a subset of chromosomally clustered taste receptors. This chromosomal clustering mirrored the cardiac expression profile, suggesting that a common gene regulatory block may control the taste receptor locus. We identified unique domains with strong regulatory potential in the vicinity of taste receptor genes. We also performed de novo motif enrichment in the proximal promoter regions and found several overrepresented DNA motifs in cardiac taste receptor gene promoters corresponding to ubiquitous and cardiac-specific transcription factor binding sites. Thus, combining cardiac gene expression data with bioinformatic analyses, this study has provided insights into the noncoding regulatory landscape for taste GPCRs. These findings also have broader relevance for the study of taste GPCRs outside of the classical gustatory system, where understanding the mechanisms controlling the expression of these receptors may have implications for future therapeutic development. PMID:25986534

  16. Restricted Immunoglobulin Variable Region (Ig V) Gene Expression Accompanies Secondary Rearrangements of Light Chain Ig V Genes in Mouse Plasmacytomas

    PubMed Central

    Diaw, Lena; Siwarski, David; Coleman, Allen; Kim, Jennifer; Jones, Gary M.; Dighiero, Guillaume; Huppi, Konrad

    1999-01-01

    The many binding studies of monoclonal immunoglobulin (Ig) produced by plasmacytomas have found no universally common binding properties, but instead, groups of plasmacytomas with specific antigen-binding activities to haptens such as phosphorylcholine, dextrans, fructofuranans, or dinitrophenyl. Subsequently, it was found that plasmacytomas with similar binding chain specificities not only expressed the same idiotype, but rearranged the same light (VL) and heavy (VH) variable region genes to express a characteristic monoclonal antibody. In this study, we have examined by enzyme-linked immunosorbent assay five antibodies secreted by silicone-induced mouse plasmacytomas using a broader panel of antigens including actin, myosin, tubulin, single-stranded DNA, and double-stranded DNA. We have determined the Ig heavy and light chain V gene usage in these same plasmacytomas at the DNA and RNA level. Our studies reveal: (a) antibodies secreted by plasmacytomas bind to different antigens in a manner similar to that observed for natural autoantibodies; (b) the expressed Ig heavy genes are restricted in V gene usage to the VH-J558 family; and (c) secondary rearrangements occur at the light chain level with at least three plasmacytomas expressing both κ and λ light chain genes. These results suggest that plasmacytomas use a restricted population of B cells that may still be undergoing rearrangement, thereby bypassing the allelic exclusion normally associated with expression of antibody genes. PMID:10562316

  17. cDNA cloning and chromosomal mapping of the mouse type VII collagen gene (Col7a1): Evidence for rapid evolutionary divergence of the gene

    SciTech Connect

    Li, Kehua; Christiano, A.M.; Chu, Mon Li; Uitto, J. Thomas Jefferson Univ., Philadelphia, PA ); Copeland, N.G.; Gilbert, D.J. )

    1993-06-01

    Type VII collagen is the major component of anchoring fibrils, critical attachment structures at the dermal-epidermal basement membrane zone. Genetic linkage analyses with recently cloned human type VII collagen cDNAs have indicated that the corresponding gene, COL7A1, is the candidate gene in the dystrophic forms of epidermolysis bullosa. To gain insight into the evolutionary conservation of COL7A1, in this study the authors have isolated mouse type VII collagen cDNAs by screening a mouse epidermal keratinocyte cDNA library with a human COL7A1 cDNA. Two overlapping mouse cDNAs were isolated, and Northern hybridization of mouse epidermal keratinocyte RNA with one of them revealed the presence of a mRNA transcript of [approximately]9.5 kb, the approximate size of the human COL7A1 mRNA. Nucleotide sequencing of the mouse cDNAs revealed a 2760-bp open reading frame that encodes the 5[prime] half of the collagenous domain and a segment of the NC-1, the noncollagenous amino-terminal domain of type VII collagen. Comparison of the mouse amino acid sequences with the corresponding human sequences deduced from cDNAs revealed 82.5% identity. The evolutionary divergence of the gene was relatively rapid in comparison to other collagen genes. Despite the high degree of sequence variation, several sequences, including the size and the position of noncollagenous imperfections and interruptions within the Gly-X-Y repeat sequence, were precisely conserved. Finally, the mouse Col7a1 gene was located by interspecific backcross mapping to mouse Chromosome 9, a region that corresponds to human chromosome 3p21, the position of human COL7Al. This assignment confirms and extends the relationship between the mouse and the human chromosomes in this region of the genome. 33 refs., 5 figs., 1 tab.

  18. Specific amplification by PCR of rearranged genomic variable regions of immunoglobulin genes from mouse hybridoma cells.

    PubMed

    Berdoz, J; Monath, T P; Kraehenbuhl, J P

    1995-04-01

    We have designed a novel strategy for the isolation of the rearranged genomic fragments encoding the L-VH-D-JH and L-V kappa/lambda-J kappa/lambda regions of mouse immunoglobulin genes. This strategy is based on the PCR amplification of genomic DNA from mouse hybridomas using multiple specific primers chosen in the 5'-untranslated region and in the intron downstream of the rearranged JH/J kappa/lambda sequences. Variable regions with intact coding sequences, including full-length leader peptides (L) can be obtained without previous DNA sequencing. Our strategy is based on a genomic template that produces fragments that do not need to be adapted for recombinant antibody expression, thus facilitating the generation of chimeric and isotype-switched immunoglobulins.

  19. The Invalidation of HspB1 Gene in Mouse Alters the Ultrastructural Phenotype of Muscles.

    PubMed

    Kammoun, Malek; Picard, Brigitte; Astruc, Thierry; Gagaoua, Mohammed; Aubert, Denise; Bonnet, Muriel; Blanquet, Véronique; Cassar-Malek, Isabelle

    2016-01-01

    Even though abundance of Hsp27 is the highest in skeletal muscle, the relationships between the expression of HspB1 (encoding Hsp27) and muscle characteristics are not fully understood. In this study, we have analysed the effect of Hsp27 inactivation on mouse development and phenotype. We generated a mouse strain devoid of Hsp27 protein by homologous recombination of the HspB1 gene. The HspB1-/- mouse was viable and fertile, showing neither apparent morphological nor anatomical alterations. We detected a gender dimorphism with marked effects in males, a lower body weight (P < 0.05) with no obvious changes in the growth rate, and a lower plasma lipids profile (cholesterol, HDL and triglycerides, 0.001 < P< 0.05). The muscle structure of the animals was examined by optical microscopy and transmission electron microscopy. Not any differences in the characteristics of muscle fibres (contractile and metabolic type, shape, perimeter, cross-sectional area) were detected except a trend for a higher proportion of small fibres. Different myosin heavy chains electrophoretic profiles were observed in the HspB1-/- mouse especially the presence of an additional isoform. Electron microscopy revealed ultrastructural abnormalities in the myofibrillar structure of the HspB1-/- mouse mutant mice (e.g. destructured myofibrils and higher gaps between myofibrils) especially in the m. Soleus. Combined with our previous data, these findings suggest that Hsp27 could directly impact the organization of muscle cytoskeleton at the molecular and ultrastructural levels. PMID:27512988

  20. The Invalidation of HspB1 Gene in Mouse Alters the Ultrastructural Phenotype of Muscles

    PubMed Central

    Kammoun, Malek; Picard, Brigitte; Astruc, Thierry; Gagaoua, Mohammed; Aubert, Denise; Bonnet, Muriel; Blanquet, Véronique; Cassar-Malek, Isabelle

    2016-01-01

    Even though abundance of Hsp27 is the highest in skeletal muscle, the relationships between the expression of HspB1 (encoding Hsp27) and muscle characteristics are not fully understood. In this study, we have analysed the effect of Hsp27 inactivation on mouse development and phenotype. We generated a mouse strain devoid of Hsp27 protein by homologous recombination of the HspB1 gene. The HspB1-/- mouse was viable and fertile, showing neither apparent morphological nor anatomical alterations. We detected a gender dimorphism with marked effects in males, a lower body weight (P < 0.05) with no obvious changes in the growth rate, and a lower plasma lipids profile (cholesterol, HDL and triglycerides, 0.001 < P< 0.05). The muscle structure of the animals was examined by optical microscopy and transmission electron microscopy. Not any differences in the characteristics of muscle fibres (contractile and metabolic type, shape, perimeter, cross-sectional area) were detected except a trend for a higher proportion of small fibres. Different myosin heavy chains electrophoretic profiles were observed in the HspB1-/- mouse especially the presence of an additional isoform. Electron microscopy revealed ultrastructural abnormalities in the myofibrillar structure of the HspB1-/- mouse mutant mice (e.g. destructured myofibrils and higher gaps between myofibrils) especially in the m. Soleus. Combined with our previous data, these findings suggest that Hsp27 could directly impact the organization of muscle cytoskeleton at the molecular and ultrastructural levels. PMID:27512988

  1. Isolation of the mouse (MFH-1) and human (FKHL14) mesenchyme fork head-1 genes reveals conservation of their gene and protein structures

    SciTech Connect

    Miura, Naoyuki; Iida, Kiyoshi; Yang, Xiao-Li

    1997-05-01

    The very recently found evolutionarily conserved DNA-binding domain of 100 amino acids, termed the fork head domain, emerged from a sequence comparison of the rat hepatocyte transcription factor HNF-3{alpha} and the homeotic gene fork head of Drosophila. We previously isolated a new member of this family, the mesenchyme fork head-1 (MFH-1) gene, which is expressed in developing mesenchyme. Here we describe the isolation of the mouse (MFH-1) and human (FKHL14) chromosomal MFH-1 genes and the determination of the gene and protein structures of MFH-1. We found that the MFH-1 gene has no introns and that the identity of the amino acid sequences of mouse and human MFH-1 proteins is 94%. We also investigated the transcriptional activity of the mouse and human MFH-1 proteins and found that both proteins act as positive transactivators. 31 refs., 3 figs.

  2. Transcriptome Analysis of Targeted Mouse Mutations Reveals the Topography of Local Changes in Gene Expression

    PubMed Central

    Adkisson, Michael; Nava, A. J.; Kirov, Julia V.; Cipollone, Andreanna; Willis, Brandon; Rapp, Jared; de Jong, Pieter J.; Lloyd, Kent C.

    2016-01-01

    The unintended consequences of gene targeting in mouse models have not been thoroughly studied and a more systematic analysis is needed to understand the frequency and characteristics of off-target effects. Using RNA-seq, we evaluated targeted and neighboring gene expression in tissues from 44 homozygous mutants compared with C57BL/6N control mice. Two allele types were evaluated: 15 targeted trap mutations (TRAP); and 29 deletion alleles (DEL), usually a deletion between the translational start and the 3’ UTR. Both targeting strategies insert a bacterial beta-galactosidase reporter (LacZ) and a neomycin resistance selection cassette. Evaluating transcription of genes in +/- 500 kb of flanking DNA around the targeted gene, we found up-regulated genes more frequently around DEL compared with TRAP alleles, however the frequency of alleles with local down-regulated genes flanking DEL and TRAP targets was similar. Down-regulated genes around both DEL and TRAP targets were found at a higher frequency than expected from a genome-wide survey. However, only around DEL targets were up-regulated genes found with a significantly higher frequency compared with genome-wide sampling. Transcriptome analysis confirms targeting in 97% of DEL alleles, but in only 47% of TRAP alleles probably due to non-functional splice variants, and some splicing around the gene trap. Local effects on gene expression are likely due to a number of factors including compensatory regulation, loss or disruption of intragenic regulatory elements, the exogenous promoter in the neo selection cassette, removal of insulating DNA in the DEL mutants, and local silencing due to disruption of normal chromatin organization or presence of exogenous DNA. An understanding of local position effects is important for understanding and interpreting any phenotype attributed to targeted gene mutations, or to spontaneous indels. PMID:26839965

  3. Quantitative Evaluation and Selection of Reference Genes for Quantitative RT-PCR in Mouse Acute Pancreatitis

    PubMed Central

    Yan, Zhaoping; Gao, Jinhang; Lv, Xiuhe; Yang, Wenjuan; Wen, Shilei; Tong, Huan; Tang, Chengwei

    2016-01-01

    The analysis of differences in gene expression is dependent on normalization using reference genes. However, the expression of many of these reference genes, as evaluated by quantitative RT-PCR, is upregulated in acute pancreatitis, so they cannot be used as the standard for gene expression in this condition. For this reason, we sought to identify a stable reference gene, or a suitable combination, for expression analysis in acute pancreatitis. The expression stability of 10 reference genes (ACTB, GAPDH, 18sRNA, TUBB, B2M, HPRT1, UBC, YWHAZ, EF-1α, and RPL-13A) was analyzed using geNorm, NormFinder, and BestKeeper software and evaluated according to variations in the raw Ct values. These reference genes were evaluated using a comprehensive method, which ranked the expression stability of these genes as follows (from most stable to least stable): RPL-13A, YWHAZ > HPRT1 > GAPDH > UBC > EF-1α > 18sRNA > B2M > TUBB > ACTB. RPL-13A was the most suitable reference gene, and the combination of RPL-13A and YWHAZ was the most stable group of reference genes in our experiments. The expression levels of ACTB, TUBB, and B2M were found to be significantly upregulated during acute pancreatitis, whereas the expression level of 18sRNA was downregulated. Thus, we recommend the use of RPL-13A or a combination of RPL-13A and YWHAZ for normalization in qRT-PCR analyses of gene expression in mouse models of acute pancreatitis. PMID:27069927

  4. Cep55 regulates spindle organization and cell cycle progression in meiotic oocyte

    PubMed Central

    Xu, Zhao-Yang; Ma, Xue-Shan; Qi, Shu-Tao; Wang, Zhen-Bo; Guo, Lei; Schatten, Heide; Sun, Qing-Yuan; Sun, Ying-Pu

    2015-01-01

    Cep55 is a relatively novel member of the centrosomal protein family. Here, we show that Cep55 is expressed in mouse oocytes from the germinal vesicle (GV) to metaphase II (MII) stages. Immuostaining and confocal microscopy as well as time lapse live imaging after injection of mRNA encoding fusion protein of Cep55 and GFP identified that Cep55 was localized to the meiotic spindle, especially to the spindle poles at metaphase, while it was concentrated at the midbody in telophase in meiotic oocytes. Knockdown of Cep55 by specific siRNA injection caused the dissociation of γ-tubulin from the spindle poles, resulting in severely defective spindles and misaligned chromosomes, leading to metaphase I arrest and failure of first polar body (PB1) extrusion. Correspondingly, cyclin B accumulation and spindle assembly checkpoint (SAC) activation were observed in Cep55 knockdown oocytes. Our results suggest that Cep55 may act as an MTOC-associated protein regulating spindle organization, and thus cell cycle progression during mouse oocyte meiotic maturation. PMID:26582107

  5. DNA context represents transcription regulation of the gene in mouse embryonic stem cells

    PubMed Central

    Ha, Misook; Hong, Soondo

    2016-01-01

    Understanding gene regulatory information in DNA remains a significant challenge in biomedical research. This study presents a computational approach to infer gene regulatory programs from primary DNA sequences. Using DNA around transcription start sites as attributes, our model predicts gene regulation in the gene. We find that H3K27ac around TSS is an informative descriptor of the transcription program in mouse embryonic stem cells. We build a computational model inferring the cell-type-specific H3K27ac signatures in the DNA around TSS. A comparison of embryonic stem cell and liver cell-specific H3K27ac signatures in DNA shows that the H3K27ac signatures in DNA around TSS efficiently distinguish the cell-type specific H3K27ac peaks and the gene regulation. The arrangement of the H3K27ac signatures inferred from the DNA represents the transcription regulation of the gene in mESC. We show that the DNA around transcription start sites is associated with the gene regulatory program by specific interaction with H3K27ac. PMID:27075878

  6. Differential gene expression in mouse liver associated with the hepatoprotective effect of clofibrate

    SciTech Connect

    Moffit, Jeffrey S.; Koza-Taylor, Petra H.; Holland, Ricky D.; Thibodeau, Michael S.; Beger, Richard D.; Lawton, Michael P.; Manautou, Jose E. . E-mail: jose.manautou@uconn.edu

    2007-07-15

    Pretreatment of mice with the peroxisome proliferator clofibrate (CFB) protects against acetaminophen (APAP)-induced hepatotoxicity. Previous studies have shown that activation of the nuclear peroxisome proliferator activated receptor-alpha (PPAR{alpha}) is required for this effect. The present study utilizes gene expression profile analysis to identify potential pathways contributing to PPAR{alpha}-mediated hepatoprotection. Gene expression profiles were compared between wild type and PPAR{alpha}-null mice pretreated with vehicle or CFB (500 mg/kg, i.p., daily for 10 days) and then challenged with APAP (400 mg/kg, p.o.). Total hepatic RNA was isolated 4 h after APAP treatment and hybridized to Affymetrix Mouse Genome MGU74 v2.0 GeneChips. Gene expression analysis was performed utilizing GeneSpring (registered) software. Our analysis identified 53 genes of interest including vanin-1, cell cycle regulators, lipid-metabolizing enzymes, and aldehyde dehydrogenase 2, an acetaminophen binding protein. Vanin-1 could be important for CFB-mediated hepatoprotection because this protein is involved in the synthesis of cysteamine and cystamine. These are potent antioxidants capable of ameliorating APAP toxicity in rodents and humans. HPLC-ESI/MS/MS analysis of liver extracts indicates that enhanced vanin-1 gene expression results in elevated cystamine levels, which could be mechanistically associated with CFB-mediated hepatoprotection.

  7. Preliminary evidence of phenytoin-induced alterations in embryonic gene expression in a mouse model.

    PubMed

    Musselman, A C; Bennett, G D; Greer, K A; Eberwine, J H; Finnell, R H

    1994-01-01

    SWV mouse embryos collected on gestational days (GD) 9:12 and 10:00 following chronic in utero exposure to teratogenic concentrations of phenytoin were utilized for in situ transcription studies of gene expression. The substrate cDNA obtained from the frozen embryo sections was amplified into radiolabelled antisense RNA (RT/aRNA) and used as a probe to screen a panel of 20 cDNA clones representing genes that are important regulators of craniofacial and neural development. The magnitude of alteration in gene expression following phenytoin treatment was determined densitometrically by changes in the hybridization intensity of the aRNA probes to the cDNA clones immobilized to the slot blots. We found that both Wnt-1 and the calcium channel gene were developmentally regulated, as their level of expression decreased significantly between the two collection times. Phenytoin treatment produced a significant downregulation in the level of expression for 25% of the genes examined in the GD 9:12 embryos, including the growth factors TGF-beta and NT3, the proto-oncogene Wnt-1, the nicotinic receptor, and the voltage sensitive calcium channel gene. Additional changes in the coordinate expression of several of the growth and transcription factors were observed at both gestational timepoints. The application of RT/aRNA technology has extended our appreciation of the normal patterns of gene expression during craniofacial and neural development, and provided the first demonstration of multiple coordinate changes in transcription patterns following teratogenic insult.

  8. DNA context represents transcription regulation of the gene in mouse embryonic stem cells

    NASA Astrophysics Data System (ADS)

    Ha, Misook; Hong, Soondo

    2016-04-01

    Understanding gene regulatory information in DNA remains a significant challenge in biomedical research. This study presents a computational approach to infer gene regulatory programs from primary DNA sequences. Using DNA around transcription start sites as attributes, our model predicts gene regulation in the gene. We find that H3K27ac around TSS is an informative descriptor of the transcription program in mouse embryonic stem cells. We build a computational model inferring the cell-type-specific H3K27ac signatures in the DNA around TSS. A comparison of embryonic stem cell and liver cell-specific H3K27ac signatures in DNA shows that the H3K27ac signatures in DNA around TSS efficiently distinguish the cell-type specific H3K27ac peaks and the gene regulation. The arrangement of the H3K27ac signatures inferred from the DNA represents the transcription regulation of the gene in mESC. We show that the DNA around transcription start sites is associated with the gene regulatory program by specific interaction with H3K27ac.

  9. DNA demethylation reactivates a subset of imprinted genes in uniparental mouse embryonic fibroblasts.

    PubMed

    El Kharroubi, A; Piras, G; Stewart, C L

    2001-03-23

    Although most imprinted genes show allelic differences in DNA methylation, it is not clear whether methylation regulates the expression of some or all imprinted genes in somatic cells. To examine the mechanisms of silencing of imprinted alleles, we generated novel uniparental mouse embryonic fibroblasts exclusively containing either the paternal or the maternal genome. These fibroblasts retain parent-of-origin allele-specific expression of 12 imprinted genes examined for more than 30 cell generations. We show that p57(Kip2) (cyclin-dependent kinase inhibitor protein 2) and Igf2 (insulin-like growth factor 2) are induced by inhibiting histone deacetylases; however, their activated state is reversed quickly by withdrawal of trichostatin A. In contrast, DNA demethylation results in the heritable expression of a subset of imprinted genes including H19 (H19 fetal liver mRNA), p57(Kip2), Peg3/Pw1 (paternally expressed gene 3), and Zac1 (zinc finger-binding protein regulating apoptosis and cell cycle arrest). Other imprinted genes such as Grb10 (growth factor receptor-bound protein 10), Peg1/Mest (paternally expressed gene 1/mesoderm-specific transcript), Sgce (epsilon-sarcoglycan), Snrpn (small nuclear ribonucleoprotein polypeptide N), and U2af1 (U2 small nuclear ribonucleoprotein auxiliary factor), remain inactive, despite their exposure to inhibitors of histone deacetylases and DNA methylation. These results demonstrate that changes in DNA methylation but not histone acetylation create a heritable epigenetic state at some imprinted loci in somatic cells. PMID:11124954

  10. High-resolution prediction of mouse brain connectivity using gene expression patterns.

    PubMed

    Fakhry, Ahmed; Ji, Shuiwang

    2015-02-01

    The brain is a multi-level system in which the high-level functions are generated by low-level genetic mechanisms. Thus, elucidating the relationship among multiple brain levels via correlative and predictive analytics is an important area in brain research. Currently, studies in multiple species have indicated that the spatiotemporal gene expression patterns are predictive of brain wiring. Specifically, results on the worm Caenorhabditis elegans have shown that the prediction of neuronal connectivity using gene expression signatures yielded statistically significant results. Recent studies on the mammalian brain produced similar results at the coarse regional level. In this study, we provide the first high-resolution, large-scale integrative analysis of the transcriptome and connectome in a single mammalian brain at a fine voxel level. By using the Allen Brain Atlas data, we predict voxel-level brain connectivity based on the gene expressions in the adult mouse brain. We employ regularized models to show that gene expression is predictive of connectivity at the voxel-level with an accuracy of 93%. We also identify a set of genes playing the most important role in connectivity prediction. We use only this small number of genes to predict the brain wiring with an accuracy over 80%. We discover that these important genes are enriched in neurons as compared to glia, and they perform connectivity-related functions. We perform several interesting correlative studies to further elucidate the transcriptome-connectome relationship.

  11. Identification of peroxisome-proliferator responsive element in the mouse HSL gene

    SciTech Connect

    Yajima, Hiroaki . E-mail: hyajima@kirin.co.jp; Kobayashi, Yumie; Kanaya, Tomoka; Horino, Yoko

    2007-01-12

    Hormone-sensitive lipase (HSL) catalyzes the rate-limiting step of lipolysis in adipose tissue. Several studies suggest that protein phosphorylation regulates the HSL enzymatic activity. On the other hand, the precise mechanism of the transcriptional regulation of the HSL gene remains to be elucidated. Here, we identified a functional peroxisome-proliferator responsive element (PPRE) in the mouse HSL promoter by reporter assay in CV-1 cells using serial deletion and point mutants of the 5'-flanking region. Chromatin immunoprecipitation (ChIP) analysis revealed that both peroxisome-proliferator activated receptor (PPAR{gamma}) and retinoid X receptor (RXR{alpha}) interacted with the region. Binding of the PPAR{gamma}/RXR{alpha} heterodimer to the PPRE sequence was also confirmed by electrophoretic mobility shift assay. These results indicate that the HSL gene is transcriptionally regulated by PPAR{gamma}/RXR{alpha} heterodimer, and suggest that a cis-acting element regulates the HSL gene expression.

  12. Gene expression analysis of mouse embryonic stem cells following levitation in an ultrasound standing wave trap.

    PubMed

    Bazou, Despina; Kearney, Roisin; Mansergh, Fiona; Bourdon, Celine; Farrar, Jane; Wride, Michael

    2011-02-01

    In the present paper, gene expression analysis of mouse embryonic stem (ES) cells levitated in a novel ultrasound standing wave trap (USWT) (Bazou et al. 2005a) at variable acoustic pressures (0.08-0.85 MPa) and times (5-60 min) was performed. Our results showed that levitation of ES cells at the highest employed acoustic pressure for 60 min does not modify gene expression and cells maintain their pluripotency. Embryoid bodies (EBs) also expressed the early and late neural differentiation markers, which were also unaffected by the acoustic field. Our results suggest that the ultrasound trap microenvironment is minimally invasive as the biologic consequences of ES cell replication and EB differentiation proceed without significantly affecting gene expression. The technique holds great promise in safe cell manipulation techniques for a variety of applications including tissue engineering and regenerative medicine. PMID:21208732

  13. Gene Expression Analysis of Mouse Embryonic Stem Cells Following Levitation in an Ultrasound Standing Wave Trap

    PubMed Central

    Bazou, Despina; Kearney, Roisin; Mansergh, Fiona; Bourdon, Celine; Farrar, Jane; Wride, Michael

    2011-01-01

    In the present paper, gene expression analysis of mouse embryonic stem (ES) cells levitated in a novel ultrasound standing wave trap (USWT) (Bazou et al. 2005a) at variable acoustic pressures (0.08–0.85 MPa) and times (5–60 min) was performed. Our results showed that levitation of ES cells at the highest employed acoustic pressure for 60 min does not modify gene expression and cells maintain their pluripotency. Embryoid bodies (EBs) also expressed the early and late neural differentiation markers, which were also unaffected by the acoustic field. Our results suggest that the ultrasound trap microenvironment is minimally invasive as the biologic consequences of ES cell replication and EB differentiation proceed without significantly affecting gene expression. The technique holds great promise in safe cell manipulation techniques for a variety of applications including tissue engineering and regenerative medicine. (E-mail: Bazoud@tcd.ie) PMID:21208732

  14. Identification of peroxisome-proliferator responsive element in the mouse HSL gene.

    PubMed

    Yajima, Hiroaki; Kobayashi, Yumie; Kanaya, Tomoka; Horino, Yoko

    2007-01-12

    Hormone-sensitive lipase (HSL) catalyzes the rate-limiting step of lipolysis in adipose tissue. Several studies suggest that protein phosphorylation regulates the HSL enzymatic activity. On the other hand, the precise mechanism of the transcriptional regulation of the HSL gene remains to be elucidated. Here, we identified a functional peroxisome-proliferator responsive element (PPRE) in the mouse HSL promoter by reporter assay in CV-1 cells using serial deletion and point mutants of the 5'-flanking region. Chromatin immunoprecipitation (ChIP) analysis revealed that both peroxisome-proliferator activated receptor (PPARgamma) and retinoid X receptor (RXRalpha) interacted with the region. Binding of the PPARgamma/RXRalpha heterodimer to the PPRE sequence was also confirmed by electrophoretic mobility shift assay. These results indicate that the HSL gene is transcriptionally regulated by PPARgamma/RXRalpha heterodimer, and suggest that a cis-acting element regulates the HSL gene expression.

  15. Identification of the meiotic life cycle stage of Trypanosoma brucei in the tsetse fly

    PubMed Central

    Peacock, Lori; Ferris, Vanessa; Sharma, Reuben; Sunter, Jack; Bailey, Mick; Carrington, Mark; Gibson, Wendy

    2011-01-01

    Elucidating the mechanism of genetic exchange is fundamental for understanding how genes for such traits as virulence, disease phenotype, and drug resistance are transferred between pathogen strains. Genetic exchange occurs in the parasitic protists Trypanosoma brucei, T. cruzi, and Leishmania major, but the precise cellular mechanisms are unknown, because the process has not been observed directly. Here we exploit the identification of homologs of meiotic genes in the T. brucei genome and demonstrate that three functionally distinct, meiosis-specific proteins are expressed in the nucleus of a single specific cell type, defining a previously undescribed developmental stage occurring within the tsetse fly salivary gland. Expression occurs in clonal and mixed infections, indicating that the meiotic program is an intrinsic but hitherto cryptic part of the developmental cycle of trypanosomes. In experimental crosses, expression of meiosis-specific proteins usually occurred before cell fusion. This is evidence of conventional meiotic division in an excavate protist, and the functional conservation of the meiotic machinery in these divergent organisms underlines the ubiquity and basal evolution of meiosis in eukaryotes. PMID:21321215

  16. A Germline Clone Screen on the X Chromosome Reveals Novel Meiotic Mutants in Drosophila melanogaster

    PubMed Central

    Collins, Kimberly A.; Callicoat, Jonathon G.; Lake, Cathleen M.; McClurken, Cailey M.; Kohl, Kathryn P.; Hawley, R. Scott

    2012-01-01

    In an effort to isolate novel meiotic mutants that are severely defective in chromosome segregation and/or exchange, we employed a germline clone screen of the X chromosome of Drosophila melanogaster. We screened over 120,000 EMS-mutagenized chromosomes and isolated 19 mutants, which comprised nine complementation groups. Four of these complementation groups mapped to known meiotic genes, including mei-217, mei-218, mei-9, and nod. Importantly, we have identified two novel complementation groups with strong meiotic phenotypes, as assayed by X chromosome nondisjunction. One complementation group is defined by three alleles, and the second novel complementation group is defined by a single allele. All 19 mutants are homozygous viable, fertile, and fully recessive. Of the 9 mutants that have been molecularly characterized, 5 are canonical EMS-induced transitions, and the remaining 4 are transversions. In sum, we have identified two new genes that are defined by novel meiotic mutants, in addition to isolating new alleles of mei-217, mei-218, mei-9, and nod. PMID:23173088

  17. Dbl2 Regulates Rad51 and DNA Joint Molecule Metabolism to Ensure Proper Meiotic Chromosome Segregation

    PubMed Central

    Hyppa, Randy W.; Benko, Zsigmond; Misova, Ivana; Schleiffer, Alexander; Smith, Gerald R.; Gregan, Juraj

    2016-01-01

    To identify new proteins required for faithful meiotic chromosome segregation, we screened a Schizosaccharomyces pombe deletion mutant library and found that deletion of the dbl2 gene led to missegregation of chromosomes during meiosis. Analyses of both live and fixed cells showed that dbl2Δ mutant cells frequently failed to segregate homologous chromosomes to opposite poles during meiosis I. Removing Rec12 (Spo11 homolog) to eliminate meiotic DNA double-strand breaks (DSBs) suppressed the segregation defect in dbl2Δ cells, indicating that Dbl2 acts after the initiation of meiotic recombination. Analyses of DSBs and Holliday junctions revealed no significant defect in their formation or processing in dbl2Δ mutant cells, although some Rec12-dependent DNA joint molecules persisted late in meiosis. Failure to segregate chromosomes in the absence of Dbl2 correlated with persistent Rad51 foci, and deletion of rad51 or genes encoding Rad51 mediators also suppressed the segregation defect of dbl2Δ. Formation of foci of Fbh1, an F-box helicase that efficiently dismantles Rad51-DNA filaments, was impaired in dbl2Δ cells. Our results suggest that Dbl2 is a novel regulator of Fbh1 and thereby Rad51-dependent DSB repair required for proper meiotic chromosome segregation and viable sex cell formation. The wide conservation of these proteins suggests that our results apply to many species. PMID:27304859

  18. Identification of the meiotic life cycle stage of Trypanosoma brucei in the tsetse fly.

    PubMed

    Peacock, Lori; Ferris, Vanessa; Sharma, Reuben; Sunter, Jack; Bailey, Mick; Carrington, Mark; Gibson, Wendy

    2011-03-01

    Elucidating the mechanism of genetic exchange is fundamental for understanding how genes for such traits as virulence, disease phenotype, and drug resistance are transferred between pathogen strains. Genetic exchange occurs in the parasitic protists Trypanosoma brucei, T. cruzi, and Leishmania major, but the precise cellular mechanisms are unknown, because the process has not been observed directly. Here we exploit the identification of homologs of meiotic genes in the T. brucei genome and demonstrate that three functionally distinct, meiosis-specific proteins are expressed in the nucleus of a single specific cell type, defining a previously undescribed developmental stage occurring within the tsetse fly salivary gland. Expression occurs in clonal and mixed infections, indicating that the meiotic program is an intrinsic but hitherto cryptic part of the developmental cycle of trypanosomes. In experimental crosses, expression of meiosis-specific proteins usually occurred before cell fusion. This is evidence of conventional meiotic division in an excavate protist, and the functional conservation of the meiotic machinery in these divergent organisms underlines the ubiquity and basal evolution of meiosis in eukaryotes.

  19. Using standard nomenclature to adequately name transgenes, knockout gene alleles and any mutation associated to a genetically modified mouse strain.

    PubMed

    Montoliu, Lluís; Whitelaw, C Bruce A

    2011-04-01

    Mice provide an unlimited source of animal models to study mammalian gene function and human diseases. The powerful genetic modification toolbox existing for the mouse genome enables the creation of, literally, thousands of genetically modified mouse strains, carrying spontaneous or induced mutations, transgenes or knock-out/knock-in alleles which, in addition, can exist in hundreds of different genetic backgrounds. Such an immense diversity of individuals needs to be adequately annotated, to ensure that the most relevant information is kept associated with the name of each mouse line, and hence, the scientific community can correctly interpret and benefit from the reported animal model. Therefore, rules and guidelines for correctly naming genes, alleles and mouse strains are required. The Mouse Genome Informatics Database is the authoritative source of official names for mouse genes, alleles, and strains. Nomenclature follows the rules and guidelines established by the International Committee on Standardized Genetic Nomenclature for Mice. Herewith, both from the International Society for Transgenic Technologies (ISTT) and from the scientific journal Transgenic Research, we would like to encourage all our colleagues to adhere and follow adequately the standard nomenclature rules when describing mouse models. The entire scientific community using genetically modified mice in experiments will benefit.

  20. Effects of Subretinal Gene Transfer at Different Time Points in a Mouse Model of Retinal Degeneration

    PubMed Central

    Dai, Xufeng; Zhang, Hua; Han, Juanjuan; He, Ying; Zhang, Yangyang; Qi, Yan; Pang, Ji-jing

    2016-01-01

    Lysophosphatidylcholine acyltransferase 1 (LPCAT1) is necessary for photoreceptors to generate an important lipid component of their membranes. The absence of LPCAT1 results in early and rapid rod and cone degeneration. Retinal degeneration 11 (rd11) mice carry a mutation in the Lpcat1 gene, and are an excellent model of early-onset rapid retinal degeneration (RD). To date, no reports have documented gene therapy administration in the rd11 mouse model at different ages. In this study, the AAV8 (Y733F)-smCBA-Lpcat1 vector was subretinally injected at postnatal day (P) 10, 14, 18, or 22. Four months after injection, immunohistochemistry and analysis of retinal morphology showed that treatment at P10 rescued about 82% of the wild-type retinal thickness. However, the diffusion of the vector and the resulting rescue were limited to an area around the injection site that was only 31% of the total retinal area. Injection at P14 resulted in vector diffusion that covered approximately 84% of the retina, and we found that gene therapy was more effective against RD when exposure to light was limited before and after treatment. We observed long-term preservation of electroretinogram (ERG) responses, and preservation of retinal structure, indicating that early treatment followed by limited light exposure can improve gene therapy effectiveness for the eyes of rd11 mice. Importantly, delayed treatment still partially preserved M-cones, but not S-cones, and M-cones in the rd11 retina appeared to have a longer window of opportunity for effective preservation with gene therapy. These results provide important information regarding the effects of subretinal gene therapy in the mouse model of LPCAT1-deficiency. PMID:27228218

  1. Sexual differentiation in the developing mouse brain: contributions of sex chromosome genes.

    PubMed

    Wolstenholme, J T; Rissman, E F; Bekiranov, S

    2013-03-01

    Neural sexual differentiation begins during embryogenesis and continues after birth for a variable amount of time depending on the species and brain region. Because gonadal hormones were the first factors identified in neural sexual differentiation, their role in this process has eclipsed investigation of other factors. Here, we use a mouse with a spontaneous translocation that produces four different unique sets of sex chromosomes. Each genotype has one normal X-chromosome and a unique second sex chromosome creating the following genotypes: XY(*x) , XX, XY(*) , XX(Y) (*) . This Y(*) mouse line is used by several laboratories to study two human aneuploid conditions: Turner and Klinefelter syndromes. As sex chromosome number affects behavior and brain morphology, we surveyed brain gene expression at embryonic days 11.5 and 18.5 to isolate X-chromosome dose effects in the developing brain as possible mechanistic changes underlying the phenotypes. We compared gene expression differences between gonadal males and females as well as individuals with one vs. two X-chromosomes. We present data showing, in addition to genes reported to escape X-inactivation, a number of autosomal genes are differentially expressed between the sexes and in mice with different numbers of X-chromosomes. Based on our results, we can now identify the genes present in the region around the chromosomal break point that produces the Y(*) model. Our results also indicate an interaction between gonadal development and sex chromosome number that could further elucidate the role of sex chromosome genes and hormones in the sexual differentiation of behavior.

  2. Identification of novel SHOX target genes in the developing limb using a transgenic mouse model.

    PubMed

    Beiser, Katja U; Glaser, Anne; Kleinschmidt, Kerstin; Scholl, Isabell; Röth, Ralph; Li, Li; Gretz, Norbert; Mechtersheimer, Gunhild; Karperien, Marcel; Marchini, Antonio; Richter, Wiltrud; Rappold, Gudrun A

    2014-01-01

    Deficiency of the human short stature homeobox-containing gene (SHOX) has been identified in several disorders characterized by reduced height and skeletal anomalies such as Turner syndrome, Léri-Weill dyschondrosteosis and Langer mesomelic dysplasia as well as isolated short stature. SHOX acts as a transcription factor during limb development and is expressed in chondrocytes of the growth plates. Although highly conserved in vertebrates, rodents lack a SHOX orthologue. This offers the unique opportunity to analyze the effects of human SHOX expression in transgenic mice. We have generated a mouse expressing the human SHOXa cDNA under the control of a murine Col2a1 promoter and enhancer (Tg(Col2a1-SHOX)). SHOX and marker gene expression as well as skeletal phenotypes were characterized in two transgenic lines. No significant skeletal anomalies were found in transgenic compared to wildtype mice. Quantitative and in situ hybridization analyses revealed that Tg(Col2a1-SHOX), however, affected extracellular matrix gene expression during early limb development, suggesting a role for SHOX in growth plate assembly and extracellular matrix composition during long bone development. For instance, we could show that the connective tissue growth factor gene Ctgf, a gene involved in chondrogenic and angiogenic differentiation, is transcriptionally regulated by SHOX in transgenic mice. This finding was confirmed in human NHDF and U2OS cells and chicken micromass culture, demonstrating the value of the SHOX-transgenic mouse for the characterization of SHOX-dependent genes and pathways in early limb development. PMID:24887312

  3. Premeiotic events and meiotic chromosome pairing.

    PubMed

    Bennett, M D

    1984-01-01

    There is practical difficulty in identifying when meiosis begins. Moreover, because of contradictory definitions there is ambiguity and some confusion as to when, in terms of the cell cycle, premeiosis ends and meiosis begins. Nevertheless, results for several organisms show clearly that meiotic chromosome behaviour is affected by premeiotic events and especially by events during the final premeiotic mitosis and/or premeiotic interphase. This review considers only premeiotic events which do (or might) affect meiotic chromosome pairing by their effect on genomic characters, such as: chromosome number, homology, condition and position, with particular emphasis on the last. Interpreted in its widest sense 'premeiotic events affecting meiotic chromosome pairing' must include karyogamy. Moreover, while karyogamy is the normal means of achieving the diploid chromosome number and pairs of homologues essential for normal chromosome pairing, it is not the only way, as illustrated by the remarkable premeiotic adaptations seen in the apogamous ferns and the frog Rana esculenta. Little is known about the condition (including the molecular organization) of chromosomes during their approach and switch to meiosis. However, completion during premeiosis of some DNA synthesis may be essential for normal meiotic chromosome pairing. Various results (including different effects of colchicine given first at different premeiotic stages) have been claimed as evidence of one or other type of premeiotic spatial ordering of chromosomes which might favour, or be essential for, meiotic chromosome pairing. Chromosome placement has been studied recently using the electron microscope, serial thin-section, reconstruction technique. This has revealed clear evidence of non-random spatial placement of chromosomes in non-meiotic and premeiotic cells. For example, in root-tip cells of barley, Hordeum vulgare L. cv. Tuleen 346 (2n = 2x = 14), it showed: a significant spatial separation of two haploid

  4. Chromosomal assignment of the genes for proprotein convertases PC4, PC5, and PACE 4 in mouse and human

    SciTech Connect

    Mbikay, M.; Seidah, N.G.; Chretien, M.

    1995-03-01

    The genes for three subtilisin/kexin-like proprotein convertases, PC4, PC5, and PACE4, were mapped in the mouse by RFLP analysis of a DNA panel from a (C57BL/6JEi x SPRET/Ei) F{sub 1} x SPRET/Ei backcross. The chromosomal locations of the human homologs were determined by Southern blot analysis of a DNA panel from human-rodent somatic cell hybrids, most of which contained a single human chromosome each. The gene for PC4 (Pcsk4 locus) mapped to mouse chromosome 10, close to the Adn (adipsin, a serine protease) locus and near the Amh (anti-Mullerian hormone) locus; in a human, the gene was localized to chromosome 19. The gene for PC5 (Pcsk5 locus) mapped to mouse chromosome 19 close to the Lpc1 (lipoacortin-1) locus and, in human, was localized to chromosome 9. The gene for PACE4 (Pcsk6 locus) mapped to mouse chromosome 7, at a distance of 13 cM from the Pcsk3 locus, which specifies furin, another member of this family of enzymes previoulsy mapped to this chromosome. This is in concordance with the known close proximity of these two loci in the homologous region on human chromosome 15q25-qter. Pcsk3 and Pcsk6 mapped to a region of mouse chromosome 7 that has been associated cytogenetically with postnatal lethality in maternal disomy, suggesting that these genes might be candidates for imprinting. 43 refs., 3 figs., 2 tabs.

  5. Gene organization and transcription of duplicated MBP genes of myelin deficient (shi(mld)) mutant mouse.

    PubMed Central

    Okano, H; Tamura, T; Miura, M; Aoyama, A; Ikenaka, K; Oshimura, M; Mikoshiba, K

    1988-01-01

    A hereditary dysmyelinating mutation, named myelin deficient (shi(mld)), is characterized by reduced expression of myelin basic protein (MBP). In shi(mld), the MBP gene is duplicated and its reduced expression is mainly determined by the level of mRNA. We have characterized the structure and function of the promoter regions of the duplicated MBP genes in shi(mld). Among the lambda clones containing promoter regions of the duplicated MBP genes in shi(mld), one (gene 1) had the same restriction enzyme pattern as that in control mice, but another (gene 2) had a rearrangement on a distal part of the promoter. A 712-bp nucleotide sequence upstream of the first exons of both of the duplicated MBP genes of shi(mld) was completely consistent with that of the control. Promoter activities of 1.3-kb 5'-flanking regions from respective genes of shi(mld) measured by in vitro run-off assay using HeLa whole-cell extracts were indistinguishable from that of the control MPB gene. Chromosomal mapping by in situ hybridization suggested that the duplicated MBP genes were located closely to each other at the distal part of chromosome 18. A recombinational event including the inversion seemed to have occurred within gene 1 and its possible relationship to the reduced expression of MBP is discussed. Images PMID:2452084

  6. Identification of mouse retinal genes differentially regulated by dim and bright cyclic light rearing.

    PubMed

    Huang, Hu; Frank, Mark Barton; Dozmorov, Igor; Cao, Wei; Cadwell, Craig; Knowlton, Nick; Centola, Michael; Anderson, Robert E

    2005-05-01

    Bright cyclic light rearing protects BALB/c mice from light-induced photoreceptor apoptosis compared to dim cyclic light rearing. We used a microarray approach to search for putative neuroprotection genes that were up- or down-regulated under these environmental conditions. Retinal protection by bright cyclic rearing was determined by quantitative histology and DNA fragmentation analysis. Total RNA was isolated from 5-week-old mice raised in bright (400 lux) or dim (5 lux) cyclic light and prepared for analysis on microarrays produced using a 70-mer oligonucleotide library that represented 16,463 mouse genes. Genes of interest were identified using statistically robust bioinformatics analysis methods that were developed in-house. Changes in some genes were confirmed with quantitative real time PCR. We found that 952 genes were up- or down-regulated by bright cyclic light rearing compared to dim cyclic light rearing. One hundred and eighty-four of them, having >/=2-fold differences, were grouped into 13 categories, and selected for further consideration. Eleven up-regulated and two down-regulated genes were confirmed by semi-quantitative PCR. Five neuroprotection-associated genes were up-regulated by bright cyclic light rearing as confirmed by real-time PCR. The human orthologue chromosomal location of 22 differentially expressed genes map to known retinal degeneration loci. Using PathwayAssist software, we modeled the pathway networks of up- and down-regulated genes that are functionally related to the retina. We identified retinal genes that are differentially regulated by environmental light history. Those that directly affect cell processes such as survival, apoptosis, and transcription are likely play a pivotal role in the regulation of retinal neuroprotection against light-induced photoreceptor apoptosis.

  7. Gene expression in mouse ovarian follicle development in vivo versus an ex vivo alginate culture system.

    PubMed

    Parrish, Elizabeth M; Siletz, Anaar; Xu, Min; Woodruff, Teresa K; Shea, Lonnie D

    2011-08-01

    Ovarian follicle maturation results from a complex interplay of endocrine, paracrine, and direct cell-cell interactions. This study compared the dynamic expression of key developmental genes during folliculogenesis in vivo and during in vitro culture in a 3D alginate hydrogel system. Candidate gene expression profiles were measured within mouse two-layered secondary follicles, multi-layered secondary follicles, and cumulus-oocyte complexes (COCs). The expression of 20 genes involved in endocrine communication, growth signaling, and oocyte development was investigated by real-time PCR. Gene product levels were compared between i) follicles of similar stage and ii) COCs derived either in vivo or by in vitro culture. For follicles cultured for 4 days, the expression pattern and the expression level of 12 genes were the same in vivo and in vitro. Some endocrine (cytochrome P450, family 19, subfamily A, polypeptide 1 (Cyp19a1) and inhibin βA subunit (Inhba)) and growth-related genes (bone morphogenetic protein 15 (Bmp15), kit ligand (Kitl), and transforming growth factor β receptor 2 (Tgfbr2)) were downregulated relative to in vivo follicles. For COCs obtained from cultured follicles, endocrine-related genes (inhibin α-subunit (Inha) and Inhba) had increased expression relative to in vivo counterparts, whereas growth-related genes (Bmp15, growth differentiation factor 9, and kit oncogene (Kit)) and zona pellucida genes were decreased. However, most of the oocyte-specific genes (e.g. factor in the germline α (Figla), jagged 1 (Jag1), and Nlrp5 (Mater)) were expressed in vitro at the same level and with the same pattern as in vivo-derived follicles. These studies establish the similarities and differences between in vivo and in vitro cultured follicles, guiding the creation of environments that maximize follicle development and oocyte quality. PMID:21610168

  8. Gene expression based mouse brain parcellation using Markov random field regularized non-negative matrix factorization

    NASA Astrophysics Data System (ADS)

    Pathak, Sayan D.; Haynor, David R.; Thompson, Carol L.; Lein, Ed; Hawrylycz, Michael

    2009-02-01

    Understanding the geography of genetic expression in the mouse brain has opened previously unexplored avenues in neuroinformatics. The Allen Brain Atlas (www.brain-map.org) (ABA) provides genome-wide colorimetric in situ hybridization (ISH) gene expression images at high spatial resolution, all mapped to a common three-dimensional 200μm3 spatial framework defined by the Allen Reference Atlas (ARA) and is a unique data set for studying expression based structural and functional organization of the brain. The goal of this study was to facilitate an unbiased data-driven structural partitioning of the major structures in the mouse brain. We have developed an algorithm that uses nonnegative matrix factorization (NMF) to perform parts based analysis of ISH gene expression images. The standard NMF approach and its variants are limited in their ability to flexibly integrate prior knowledge, in the context of spatial data. In this paper, we introduce spatial connectivity as an additional regularization in NMF decomposition via the use of Markov Random Fields (mNMF). The mNMF algorithm alternates neighborhood updates with iterations of the standard NMF algorithm to exploit spatial correlations in the data. We present the algorithm and show the sub-divisions of hippocampus and somatosensory-cortex obtained via this approach. The results are compared with established neuroanatomic knowledge. We also highlight novel gene expression based sub divisions of the hippocampus identified by using the mNMF algorithm.

  9. Mapping the mouse dactylaplasia mutation, Dac, and a gene that controls its expression, mdac

    SciTech Connect

    Johnson, K.R.; Lane, P.W.; Ward-Bailey, P.; Davisson, M.T.

    1995-09-20

    Dactylaplasia is an inherited mouse limb malformation whose manifestation is clearly dependent on the interaction of two genes and thus represents an excellent model system for studying such gene interactions in vivo. The Dac mutation is inherited as a semidominant trait and may be a model for some forms of human ectrodactyly. Heterozygotes show absence of digits on each foot; the long bones are normal. On the SM/Ckc background on which the mutation occurred, Dac homozygotes die around birth. We mapped Dac to the distal end of Chr 19 by backcross segregation analysis. A closely linked marker was then used to distinguish -/+, Dac/+, and Dac/Dac genotypes of embryos and adults. When intercrossed with the NZB/BINJ strain, DAC homozygotes were shown to be viable and fertile, but had a more severe limb malformation (only a single remaining digit) than heterozygotes. Expression of the abnormal limb phenotypes of Dac/+ and Dac/Dac mice also depends on homozygosity for a recessive allele of another unlinked gene, mdac, that is polymorphic among inbred mouse strains. We mapped mdac to the middle Chr 13 by segregation analysis of both recombinant inbred strains and backcross progeny. 52 refs., 3 figs., 2 tabs.

  10. DNA methylation map of mouse and human brain identifies target genes in Alzheimer's disease.

    PubMed

    Sanchez-Mut, Jose V; Aso, Ester; Panayotis, Nicolas; Lott, Ira; Dierssen, Mara; Rabano, Alberto; Urdinguio, Rocio G; Fernandez, Agustin F; Astudillo, Aurora; Martin-Subero, Jose I; Balint, Balazs; Fraga, Mario F; Gomez, Antonio; Gurnot, Cecile; Roux, Jean-Christophe; Avila, Jesus; Hensch, Takao K; Ferrer, Isidre; Esteller, Manel

    2013-10-01

    The central nervous system has a pattern of gene expression that is closely regulated with respect to functional and anatomical regions. DNA methylation is a major regulator of transcriptional activity, and aberrations in the distribution of this epigenetic mark may be involved in many neurological disorders, such as Alzheimer's disease. Herein, we have analysed 12 distinct mouse brain regions according to their CpG 5'-end gene methylation patterns and observed their unique epigenetic landscapes. The DNA methylomes obtained from the cerebral cortex were used to identify aberrant DNA methylation changes that occurred in two mouse models of Alzheimer's disease. We were able to translate these findings to patients with Alzheimer's disease, identifying DNA methylation-associated silencing of three targets genes: thromboxane A2 receptor (TBXA2R), sorbin and SH3 domain containing 3 (SORBS3) and spectrin beta 4 (SPTBN4). These hypermethylation targets indicate that the cyclic AMP response element-binding protein (CREB) activation pathway and the axon initial segment could contribute to the disease.

  11. Differential gene expression in mouse retina related to regional differences in vulnerability to hyperoxia

    PubMed Central

    Natoli, Riccardo; Valter, Krisztina; Stone, Jonathan

    2010-01-01

    Purpose In the C57BL/6J mouse retina, hyperoxia-induced degeneration of photoreceptors shows strong regional variation, beginning at a locus ~0.5 mm inferior to the optic disc. To identify gene expression differences that might underlie this variability in vulnerability, we have used microarray techniques to describe regional (superior-inferior) variations in gene expression in the retina. Methods Young adult C57BL/6J mice raised in dim cyclic illumination (12 h at 5 lx and 12 h in darkness) were exposed to hyperoxia (75% oxygen for two weeks). Retinas were collected from hyperoxia-exposed and control animals without fixation and divided into superior and inferior halves. RNA was extracted from each sample, purified, and hybridized to Mouse Gene 1.0 ST arrays (Affymetrix). The consistency of the microarray results was assessed using quantitative PCR for selected genes. Expression data were analyzed to identify genes and ncRNAs whose differential expression between the superior and inferior retina could be associated with relative vulnerability to hyperoxia. Results In control retinas, only two genes showed a fold difference in expression >2 between the superior and inferior retina; another 25 showed a fold difference of 1.5–2.0. Of these 27, the functions of six genes, including ventral anterior homeobox containing gene 2 (Vax2) and T-box 5 (Tbox5), are related to parameters of anatomic development and the functions of five are related to sensory perception. Among the latter, short-wave-sensitive cone opsin (Opn1sw) was more strongly expressed in the inferior retina and medium-wave-sensitive cone opsin (Opn1mw) in the superior retina. This is consistent with known differences in S- and M-cone distribution, confirming our separation of retinal regions. The highest fold difference was reported for membrane metalloendopeptidase (Mme), a member from the metallothionein group of cytoprotective proteins. To identify genes whose regulation by hyperoxia was

  12. Chromosomal locations of the human and mouse genes for precursors of epidermal growth factor and the. beta. subunit of nerve growth factor

    SciTech Connect

    Zabel, B.U.; Eddy, R.L.; Lalley, P.A.; Scott, J.; Bell, G.I.; Shows, T.B.

    1985-01-01

    DNA probes for pre-pro-epidermal growth factor (EGF) and the precursor of the ..beta.. subunit of nerve growth factor (NGF) were used to chromosomally map human and mouse EGF and NGF genes in panels of human-mouse and mouse-Chinese hamster somatic cell hybrids. The EGF and NGF genes were mapped to human chromosomes 4 and 1, respectively, by using human-mouse cell hybrids. A combination of regional mapping using a chromosome 1 translocation and comparative gene mapping suggests that the human NGF gene is in the p21-p22.1 region of chromosome 1. In mouse-Chinese hamster cell hybrids, both genes were assigned to mouse chromosome 3. A knowledge of the chromosomal assignment of these genes should help in our understanding of their regulation and role in development and disease.

  13. Microarray analysis of gene expression in mouse (strain 129) embryonic stem cells after typical synthetic musk exposure.

    PubMed

    Shi, Jiachen; Li, Ming; Jiao, Zhihao; Zhang, Jing; Feng, Yixing; Shao, Bing

    2013-01-01

    Synthetic musks are widely used in personal-care products and can readily accumulate in the adipose tissue, breast milk, and blood of humans. In this study, the Affymetrix Mouse Genome GeneChip was used to identify alterations in gene expression of embryonic stem cells from the 129 strain of the laboratory mouse after treatment with the synthetic musk tonalide (AHTN). Among the 45,037 transcripts in the microarray, 2,879 genes were differentially expressed. According to the microarray analysis, the potential influence of AHTN on the development to embryo should be of concern, and the toxicological effects of it and related musk compounds should be studied further.

  14. The pronatriodilatin gene is located on the distal short arm of human chromosome 1 and on mouse chromosome 4.

    PubMed

    Yang-Feng, T L; Floyd-Smith, G; Nemer, M; Drouin, J; Francke, U

    1985-11-01

    Atrial natriuretic factors (ANF) are polypeptides having natriuretic, diuretic, and smooth muscle-relaxing activities that are synthesized from a single larger precursor: pronatriodilatin. Chromosomal assignment of the gene coding for human pronatriodilatin was accomplished by in situ hybridization of a [3H]-labeled pronatriodilatin probe to human chromosome preparations and by Southern blot analysis of somatic cell hybrid DNAs with normal and rearranged chromosomes 1. The human pronatriodilatin gene was mapped to the distal short arm of chromosome 1, in band 1p36. Southern blot analysis of mouse X Chinese hamster somatic cell hybrids was used to assign the mouse pronatriodilatin gene to chromosome 4. This assignment adds another locus to the conserved syntenic group of homologous genes located on the distal half of the short arm of human chromosome 1 and on mouse chromosome 4.

  15. The HOX-5 and surfeit gene clusters are linked in the proximal portion of mouse chromosome 2.

    PubMed

    Stubbs, L; Huxley, C; Hogan, B; Evans, T; Fried, M; Duboule, D; Lehrach, H

    1990-04-01

    Using an interspecies backcross, we have mapped the HOX-5 and surfeit (surf) gene clusters within the proximal portion of mouse chromosome 2. While the HOX-5 cluster of homeobox-containing genes has been localized to chromosome 2, bands C3-E1, by in situ hybridization, its more precise position relative to the genes and cloned markers of chromosome 2 was not known. Surfeit, a tight cluster of at least six highly conserved "housekeeping" genes, has not been previously mapped in mouse, but has been localized to human chromosome 9q, a region of the human genome with strong homology to proximal mouse chromosome 2. The data presented here place HOX-5 in the vicinity of the closely linked set of developmental mutations rachiterata, lethargic, and fidget and place surf close to the proto-oncogene Abl, near the centromere of chromosome 2.

  16. Neuroinformatics for genome-wide 3D gene expression mapping in the mouse brain.

    PubMed

    Ng, Lydia; Pathak, Sayan D; Kuan, Chihchau; Lau, Chris; Dong, Hongwei; Sodt, Andrew; Dang, Chinh; Avants, Brian; Yushkevich, Paul; Gee, James C; Haynor, David; Lein, Ed; Jones, Allan; Hawrylycz, Mike

    2007-01-01

    Large scale gene expression studies in the mammalian brain offer the promise of understanding the topology, networks and ultimately the function of its complex anatomy, opening previously unexplored avenues in neuroscience. High-throughput methods permit genome-wide searches to discover genes that are uniquely expressed in brain circuits and regions that control behavior. Previous gene expression mapping studies in model organisms have employed situ hybridization (ISH), a technique that uses labeled nucleic acid probes to bind to specific mRNA transcripts in tissue sections. A key requirement for this effort is the development of fast and robust algorithms for anatomically mapping and quantifying gene expression for ISH. We describe a neuroinformatics pipeline for automatically mapping expression profiles of ISH data and its use to produce the first genomic scale 3-D mapping of gene expression in a mammalian brain. The pipeline is fully automated and adaptable to other organisms and tissues. Our automated study of over 20,000 genes indicates that at least 78.8 percent are expressed at some level in the adult C56BL/6J mouse brain. In addition to providing a platform for genomic scale search, high-resolution images and visualization tools for expression analysis are available at the Allen Brain Atlas web site (http://www.brain-map.org).

  17. Functional expression of a mouse H-2Kb gene isolated from non-expressing teratocarcinoma cells.

    PubMed Central

    Daniel-Vedele, F; Morello, D; Benicourt, C; Transy, C; Le Bail, O; Plata, F; Kourilsky, P

    1984-01-01

    Embryonal carcinoma cells do not express H-2 antigens or beta 2-microglobulin. Recent studies have suggested that the expression of these antigens is likely to be controlled at the level of transcription. To study the precise organization of the corresponding genes and their possible expression in adult mouse cells, we have isolated H-2-related genes from a genomic cosmid library constructed with PCC4-aza-RI from DNA of EC cells. Clones isolated from the library after stringent hybridization with an H-2 cDNA probe were tested for their ability to direct H-2 antigen synthesis after DNA-mediated gene transfer in a fibroblastic L cell. Four clones have been found to code for the major transplantation antigen H-2Kb. Structural analysis showed that these clones contained the same entire H-2Kb gene, identical to the corresponding gene isolated from differentiated C57Bl/10 cells. Furthermore, the present studies showed that this embryonal carcinoma gene was expressed and was functional when transfected into a differentiated cell. PMID:6714227

  18. Identification of candidate lung cancer susceptibility genes in mouse using oligonucleotide arrays

    PubMed Central

    Lemon, W; Bernert, H; Sun, H; Wang, Y; You, M

    2002-01-01

    We applied microarray gene expression profiling to lungs from mouse strains having variable susceptibility to lung tumour development as a means to identify, within known quantitative trait loci (QTLs), candidate genes responsible for susceptibility or resistance to lung cancer. At least eight chromosomal regions of mice have been mapped and verified to be linked with lung tumour susceptibility or resistance. In this study, high density oligonucleotide arrays were used to measure the relative expression levels of >36 000 genes and ESTs in lung tissues of A/J, BALB/cJ, SM/J, C3H/HeJ, and C57BL/6J mice. A number of differentially expressed genes were found in each of the lung cancer susceptibility QTLs. Bioinformatic analysis of the differentially expressed genes located within QTLs produced 28 susceptibility candidates and 22 resistance candidates. These candidates may be extremely helpful in the ultimate identification of the precise genes responsible for lung tumour susceptibility or resistance in mice and, through follow up, humans. Complete data sets are available at http://thinker.med.ohio-state.edu. PMID:12205107

  19. Cloning of immunoglobulin kappa light chain genes from mouse liver and myeloma MOPC 173.

    PubMed

    Steinmetz, M; Zachau, H G; Mach, B

    1979-07-25

    The organization of the kappa chain constant region gene was compared in DNA from an immunoglobulin-producing mouse myeloma (MOPC 173) and from liver. In situ hybridization using the Southern blotting technique revealed constant region gene-containing EcoRI-DNA fragments of 14 and 20 kb in the myeloma tissue whereas one EcoRI-DNA fragment with a length of 15 kb was found in liver DNA. After enrichment by RPC-5 chromatography and preparative electrophoresis the 14 kb fragment from MOPC 173 DNA and the 15 kb fragment from liver DNA were cloned in the bacteriophage lambda vector Charon 4A using in vitro packaging. Extensive characterization of the two fragments by restriction endonuclease mapping, in situ hybridization, and electron microscopy (R-loop and heteroduplex) showed that both fragments contain the constant region but no MOPC 173 variable region gene. Both fragments are homologous over a length of 12.5 kb including the constant region but differ from one another starting about 2.7 kb from the 5' end of the constant region gene. This indicates that the 14 kb EcoRI-DNA fragment from the myeloma tissue clearly resulted from somatic DNA rearrangement although it does not seem to carry the MOPC 173 variable region gene. These observations