Nonredundant Roles of Iron Acquisition Systems in Vibrio cholerae

PubMed Central

Peng, Eric D.; Wyckoff, Elizabeth E.; Mey, Alexandra R.; Fisher, Carolyn R.

2015-01-01

Vibrio cholerae, the causative agent of the severe diarrheal disease cholera, thrives in both marine environments and the human host. To do so, it must encode the tools necessary to acquire essential nutrients, including iron, under these vastly different conditions. A number of V. cholerae iron acquisition systems have been identified; however, the precise role of each system is not fully understood. To test the roles of individual systems, we generated a series of mutants in which only one of the four systems that support iron acquisition on unsupplemented LB agar, Feo, Fbp, Vct, and Vib, remains functional. Analysis of these mutants under different growth conditions showed that these systems are not redundant. The strain carrying only the ferrous iron transporter Feo grew well at acidic, but not alkaline, pH, whereas the ferric iron transporter Fbp promoted better growth at alkaline than at acidic pH. A strain defective in all four systems (null mutant) had a severe growth defect under aerobic conditions but accumulated iron and grew as well as the wild type in the absence of oxygen, suggesting the presence of an additional, unidentified iron transporter in V. cholerae. In support of this, the null mutant was only moderately attenuated in an infant mouse model of infection. While the null mutant used heme as an iron source in vitro, we demonstrate that heme is not available to V. cholerae in the infant mouse intestine. PMID:26644383

Neural Crest Migration and Survival Are Susceptible to Morpholino-Induced Artifacts

PubMed Central

Jette, Cicely A.

2016-01-01

The neural crest (NC) is a stem cell-like embryonic population that is essential for generating and patterning the vertebrate body, including the craniofacial skeleton and peripheral nervous system. Defects in NC development underlie many birth defects and contribute to formation of some of the most malignant cancers in humans, such as melanoma and neuroblastoma. For these reasons, significant research efforts have been expended to identify genes that control NC development, as it is expected to lead to a deeper understanding of the genetic mechanisms controlling vertebrate development and identify new treatments for NC-derived diseases and cancers. However, a number of inconsistencies regarding gene function during NC development have emerged from comparative analyses of gene function between mammalian and non-mammalian systems (chick, frog, zebrafish). This poses a significant barrier to identification of single genes and/or redundant pathways to target in NC diseases. Here, we determine whether technical differences, namely morpholino-based approaches used in non-mammalian systems, could contribute to these discrepancies, by examining the extent to which NC phenotypes in fascin1a (fscn1a) morphant embryos are similar to or different from fscn1a null mutants in zebrafish. Analysis of fscn1a morphants showed that they mimicked early NC phenotypes observed in fscn1a null mutants; however, these embryos also displayed NC migration and derivative phenotypes not observed in null mutants, including accumulation of p53-independent cell death. These data demonstrate that morpholinos can cause seemingly specific NC migration and derivative phenotypes, and thus have likely contributed to the inconsistencies surrounding NC gene function between species. We suggest that comparison of genetic mutants between different species is the most rigorous method for identifying conserved genetic mechanisms controlling NC development and is critical to identify new treatments for NC diseases. PMID:28005909

Difference in C3-C4 metabolism underlies tradeoff between growth rate and biomass yield in Methylobacterium extorquens AM1.

PubMed

Fu, Yanfen; Beck, David A C; Lidstrom, Mary E

2016-07-19

Two variants of Methylobacterium extorquens AM1 demonstrated a trade-off between growth rate and biomass yield. In addition, growth rate and biomass yield were also affected by supplementation of growth medium with different amounts of cobalt. The metabolism changes relating to these growth phenomena as well as the trade-off were investigated in this study. (13)C metabolic flux analysis was used to generate a detailed central carbon metabolic flux map with both absolute and normalized flux values. The major differences between the two variants occurred at the formate node as well as within C3-C4 inter-conversion pathways. Higher relative fluxes through formyltetrahydrofolate ligase, phosphoenolpyruvate carboxylase, and malic enzyme led to higher biomass yield, while higher relative fluxes through pyruvate kinase and pyruvate dehydrogenase led to higher growth rate. These results were then tested by phenotypic studies on three mutants (null pyk, null pck mutant and null dme mutant) in both variants, which agreed with the model prediction. In this study, (13)C metabolic flux analysis for two strain variants of M. extorquens AM1 successfully identified metabolic pathways contributing to the trade-off between cell growth and biomass yield. Phenotypic analysis of mutants deficient in corresponding genes supported the conclusion that C3-C4 inter-conversion strategies were the major response to the trade-off.

Identifying Neurofibromin Specific Regulatory Nodes for Therapeutic Targeting in NF1

DTIC Science & Technology

2017-10-01

neurofibromin depends on the adapter protein SPRED1, to function, and we are utilizing the latest technical innovations including CRISPR technology... CRISPR technology to find genes that regulate neurofibromin SPRED function. Keywords Neurofibromin, Spred1, Spred2, EGFR, mutant EGFR(L858R), Ras...Establish good NF1 and Spred1/2 knockdown protocols for indicated cell lines NF1-Null and Spred1-Null HEK 293T cells have been generated using CRISPR /Cas9

The ftsH gene of Bacillus subtilis is involved in major cellular processes such as sporulation, stress adaptation and secretion.

PubMed

Deuerling, E; Mogk, A; Richter, C; Purucker, M; Schumann, W

1997-03-01

The ftsH gene of Bacillus subtilis has been identified as a general stress gene which is transiently induced after thermal or osmotic upshift. The FtsH protein exhibits 70.1% homology to FtsH of Escherichia coli which constitutes an essential ATP- and Zn(2+)-dependent protease anchored in the cytoplasmic membrane via two N-terminal transmembrane domains. This paper describes the isolation and functional characterization of an ftsH null mutant which was obtained by integration of a cat-cassette near the 5' end of ftsH, thereby preventing the synthesis of FtsH protein. In contrast to the situation in E. coli, ftsH is dispensable in B. subtilis but results in a pleiotropic phenotype. While the mutant cells grew mostly as large filaments under physiological conditions, they turned out to be extremely sensitive to heat and salt stress. Although ftsH is necessary for adaptation to heat, it is not involved in the regulation of the heat-shock response. The induction profiles of representative genes of the CIRCE and sigma-B regulon and class III heat-shock genes ion and clpC were identical in the wild type and the ftsH null mutant. Furthermore, the ftsH knockout strain was unable to sporulate, and this failure was probably due to the absence of Spo0A protein which is essential for entry into the sporulation programme. In addition, secretion of bulk exoproteins was severely impaired in the ftsH null mutant after entry into stationary phase. The alpha-amylase and subtilisin activity in the supernatant was specifically tested. Whereas the activity of alpha-amylase increased after entry into stationary phase in both the wild type and the ftsH mutant strain, that of subtilisin encoded by aprE was prevented at the level of transcription in the mutant. Most of these results can be explained by the failure to synthesize appropriate amounts of Spo0A protein in the ftsH null mutant and point to ftsH as a developmental checkpoint.

Both LOV1 and LOV2 domains of phototropin2 function as the photosensory domain for hypocotyl phototropic responses in Arabidopsis thaliana (Brassicaceae).

PubMed

Suetsugu, Noriyuki; Kong, Sam-Geun; Kasahara, Masahiro; Wada, Masamitsu

2013-01-01

Phototropins (phot) are blue light receptor proteins that mediate phototropism and control photomovement responses, such as chloroplast photorelocation movement and stomatal opening. Arabidopsis thaliana has two phototropins, phot1 and phot2. Although both phot1 and phot2 redundantly mediate photomovement responses, phot2 uniquely regulates phototropism and the chloroplast avoidance response under high-intensity blue light. However, compared to that of phot1, the mechanistic basis of phot2 function is poorly understood, and in particular, the importance of the LOV2 domain in phot2 function has not been clearly demonstrated. Indeed, photocycle-deficient LOV2 transgenic lines expressing phot2 in a phot1phot2 mutant background retained phototropism, although with less sensitivity than wild-type plants. We isolated 11 alleles of phot2 mutants and determined the molecular lesion in each allele. We analyzed hypocotyl phototropism, chloroplast photorelocation movement, and leaf flattening in the phot2 mutant and the respective phot1phot2 double mutant plants. We demonstrated that unlike the phot2 null mutant, the phot2-10 mutant, which has the defective phot2 LOV2 domain, retained the phototropic response and had unusual chloroplast movement. Mutants phot2-2 and phot2-6, which have a missense mutation in the kinase activation loop of phot2, had the phot2-null mutant phenotype. Furthermore, we convincingly demonstrated that the commonly used phot2-1 mutant allele is a phot2-null mutant. The analyses of the multiple phot2 mutant alleles provided strong evidence for the importance of both LOV domains and the kinase activation loop of phot2 in phototropism and other phot-dependent responses and also demonstrated that phot2-1 allele is a null mutant.

High-Throughput Genetic Screens Identify a Large and Diverse Collection of New Sporulation Genes in Bacillus subtilis.

PubMed

Meeske, Alexander J; Rodrigues, Christopher D A; Brady, Jacqueline; Lim, Hoong Chuin; Bernhardt, Thomas G; Rudner, David Z

2016-01-01

The differentiation of the bacterium Bacillus subtilis into a dormant spore is among the most well-characterized developmental pathways in biology. Classical genetic screens performed over the past half century identified scores of factors involved in every step of this morphological process. More recently, transcriptional profiling uncovered additional sporulation-induced genes required for successful spore development. Here, we used transposon-sequencing (Tn-seq) to assess whether there were any sporulation genes left to be discovered. Our screen identified 133 out of the 148 genes with known sporulation defects. Surprisingly, we discovered 24 additional genes that had not been previously implicated in spore formation. To investigate their functions, we used fluorescence microscopy to survey early, middle, and late stages of differentiation of null mutants from the B. subtilis ordered knockout collection. This analysis identified mutants that are delayed in the initiation of sporulation, defective in membrane remodeling, and impaired in spore maturation. Several mutants had novel sporulation phenotypes. We performed in-depth characterization of two new factors that participate in cell-cell signaling pathways during sporulation. One (SpoIIT) functions in the activation of σE in the mother cell; the other (SpoIIIL) is required for σG activity in the forespore. Our analysis also revealed that as many as 36 sporulation-induced genes with no previously reported mutant phenotypes are required for timely spore maturation. Finally, we discovered a large set of transposon insertions that trigger premature initiation of sporulation. Our results highlight the power of Tn-seq for the discovery of new genes and novel pathways in sporulation and, combined with the recently completed null mutant collection, open the door for similar screens in other, less well-characterized processes.

High-Throughput Genetic Screens Identify a Large and Diverse Collection of New Sporulation Genes in Bacillus subtilis

PubMed Central

Brady, Jacqueline; Lim, Hoong Chuin; Bernhardt, Thomas G.; Rudner, David Z.

2016-01-01

The differentiation of the bacterium Bacillus subtilis into a dormant spore is among the most well-characterized developmental pathways in biology. Classical genetic screens performed over the past half century identified scores of factors involved in every step of this morphological process. More recently, transcriptional profiling uncovered additional sporulation-induced genes required for successful spore development. Here, we used transposon-sequencing (Tn-seq) to assess whether there were any sporulation genes left to be discovered. Our screen identified 133 out of the 148 genes with known sporulation defects. Surprisingly, we discovered 24 additional genes that had not been previously implicated in spore formation. To investigate their functions, we used fluorescence microscopy to survey early, middle, and late stages of differentiation of null mutants from the B. subtilis ordered knockout collection. This analysis identified mutants that are delayed in the initiation of sporulation, defective in membrane remodeling, and impaired in spore maturation. Several mutants had novel sporulation phenotypes. We performed in-depth characterization of two new factors that participate in cell–cell signaling pathways during sporulation. One (SpoIIT) functions in the activation of σE in the mother cell; the other (SpoIIIL) is required for σG activity in the forespore. Our analysis also revealed that as many as 36 sporulation-induced genes with no previously reported mutant phenotypes are required for timely spore maturation. Finally, we discovered a large set of transposon insertions that trigger premature initiation of sporulation. Our results highlight the power of Tn-seq for the discovery of new genes and novel pathways in sporulation and, combined with the recently completed null mutant collection, open the door for similar screens in other, less well-characterized processes. PMID:26735940

Role of Cystathionine β-Lyase in Catabolism of Amino Acids to Sulfur Volatiles by Genetic Variants of Lactobacillus helveticus CNRZ 32▿

PubMed Central

Lee, Won-Jae; Banavara, Dattatreya S.; Hughes, Joanne E.; Christiansen, Jason K.; Steele, James L.; Broadbent, Jeffery R.; Rankin, Scott A.

2007-01-01

Catabolism of sulfur-containing amino acids plays an important role in the development of cheese flavor. During ripening, cystathionine β-lyase (CBL) is believed to contribute to the formation of volatile sulfur compounds (VSCs) such as methanethiol and dimethyl disulfide. However, the role of CBL in the generation of VSCs from the catabolism of specific sulfur-containing amino acids is not well characterized. The objective of this study was to investigate the role of CBL in VSC formation by Lactobacillus helveticus CNRZ 32 using genetic variants of L. helveticus CNRZ 32 including the CBL-null mutant, complementation of the CBL-null mutant, and the CBL overexpression mutant. The formation of VSCs from methionine, cystathionine, and cysteine was determined in a model system using gas chromatography-mass spectrometry with solid-phase microextraction. With methionine as a substrate, CBL overexpression resulted in higher VSC production than that of wild-type L. helveticus CNRZ 32 or the CBL-null mutant. However, there were no differences in VSC production between the wild type and the CBL-null mutant. With cystathionine, methanethiol production was detected from the CBL overexpression variant and complementation of the CBL-null mutant, implying that CBL may be involved in the conversion of cystathionine to methanethiol. With cysteine, no differences in VSC formation were observed between the wild type and genetic variants, indicating that CBL does not contribute to the conversion of cysteine. PMID:17337535

Decrease in Leaf Sucrose Synthesis Leads to Increased Leaf Starch Turnover and Decreased RuBP-limited Photosynthesis But Not Rubisco-limited Photosynthesis in Arabidopsis Null Mutants of SPSA1

USDA-ARS?s Scientific Manuscript database

SPS (Sucrose phosphate synthase) isoforms from dicots cluster into families A, B and C. In this study, we investigated the individual effect of null mutations of each of the four SPS genes in Arabidopsis (spsa1, spsa2, spsb and spsc) on photosynthesis and carbon partitioning. Null mutants spsa1 and ...

Six post-implantation lethal knockouts of genes for lipophilic MAPK pathway proteins are expressed in preimplantation mouse embryos and trophoblast stem cells.

PubMed

Xie, Yufen; Wang, Yingchun; Sun, Tong; Wang, Fangfei; Trostinskaia, Anna; Puscheck, Elizabeth; Rappolee, Daniel A

2005-05-01

Mitogen-activated protein kinase (MAPK) signaling pathways play an important role in controlling embryonic proliferation and differentiation. It has been demonstrated that sequential lipophilic signal transduction mediators that participate in the MAPK pathway are null post-implantation lethal. It is not clear why the lethality of these null mutants arises after implantation and not before. One hypothesis is that the gene product of these post-implantation lethal null mutants are not present before implantation in normal embryos and do not have function until after implantation. To test this hypothesis, we selected a set of lipophilic genes mediating MAPK signal transduction pathways whose null mutants result in early peri-implantation or placental lethality. These included FRS2alpha, GAB1, GRB2, SOS1, Raf-B, and Raf1. Products of these selected genes were detected and their locations and functions indicated by indirect immunocytochemistry and Western blotting for proteins and RT-polymerase chain reaction (PCR) for mRNA transcription. We report here that all six signal mediators are detected at the protein level in preimplantation mouse embryo, placental trophoblasts, and in cultured trophoblast stem cells (TSC). Proteins are all detected in E3.5 embryos at a time when the first known mitogenic intercellular communication has been documented. mRNA transcripts of two post-implantation null mutant genes are expressed in mouse preimplantation embryos and unfertilized eggs. These mRNA transcripts were detected as maternal mRNA in unfertilized eggs that could delay the lethality of null mutants. All of the proteins were detected in the cytoplasm or in the cell membrane. This study of spatial and temporal expression revealed that all of these six null mutants post-implantation genes in MAPK pathway are expressed and, where tested, phosphorylated/activated proteins are detected in the blastocyst. Studies on RNA expression using RT-PCR suggest that maternal RNA could play an important role in delaying the presence of the lethal phenotype of null mutations. Copyright (c) 2005 Wiley-Liss, Inc.

An efficient method for generation of bi-allelic null mutant mouse embryonic stem cells and its application for investigating epigenetic modifiers

PubMed Central

Cho, Lily Ting-yin; Andrews, Robert; Carroll, Thomas; Iyer, Vivek; Tate, Peri; Rosen, Barry; Stunnenberg, Hendrik G.; Fisher, Amanda G.; Skarnes, William C.

2017-01-01

Abstract Mouse embryonic stem (ES) cells are a popular model system to study biological processes, though uncovering recessive phenotypes requires inactivating both alleles. Building upon resources from the International Knockout Mouse Consortium (IKMC), we developed a targeting vector for second allele inactivation in conditional-ready IKMC ‘knockout-first’ ES cell lines. We applied our technology to several epigenetic regulators, recovering bi-allelic targeted clones with a high efficiency of 60% and used Flp recombinase to restore expression in two null cell lines to demonstrate how our system confirms causality through mutant phenotype reversion. We designed our strategy to select against re-targeting the ‘knockout-first’ allele and identify essential genes in ES cells, including the histone methyltransferase Setdb1. For confirmation, we exploited the flexibility of our system, enabling tamoxifen inducible conditional gene ablation while controlling for genetic background and tamoxifen effects. Setdb1 ablated ES cells exhibit severe growth inhibition, which is not rescued by exogenous Nanog expression or culturing in naive pluripotency ‘2i’ media, suggesting that the self-renewal defect is mediated through pluripotency network independent pathways. Our strategy to generate null mutant mouse ES cells is applicable to thousands of genes and repurposes existing IKMC Intermediate Vectors. PMID:28981838

A Drosophila SNAP-25 null mutant reveals context-dependent redundancy with SNAP-24 in neurotransmission.

PubMed Central

Vilinsky, Ilya; Stewart, Bryan A; Drummond, James; Robinson, Iain; Deitcher, David L

2002-01-01

The synaptic protein SNAP-25 is an important component of the neurotransmitter release machinery, although its precise function is still unknown. Genetic analysis of other synaptic proteins has yielded valuable information on their role in synaptic transmission. In this study, we performed a mutagenesis screen to identify new SNAP-25 alleles that fail to complement our previously isolated recessive temperature-sensitive allele of SNAP-25, SNAP-25(ts). In a screen of 100,000 flies, 26 F(1) progeny failed to complement SNAP-25(ts) and 21 of these were found to be null alleles of SNAP-25. These null alleles die at the pharate adult stage and electroretinogram recordings of these animals reveal that synaptic transmission is blocked. At the third instar larval stage, SNAP-25 nulls exhibit nearly normal neurotransmitter release at the neuromuscular junction. This is surprising since SNAP-25(ts) larvae exhibit a much stronger synaptic phenotype. Our evidence indicates that a related protein, SNAP-24, can substitute for SNAP-25 at the larval stage in SNAP-25 nulls. However, if a wild-type or mutant form of SNAP-25 is present, then SNAP-24 does not appear to take part in neurotransmitter release at the larval NMJ. These results suggest that the apparent redundancy between SNAP-25 and SNAP-24 is due to inappropriate genetic substitution. PMID:12242238

Difference in C3–C4 metabolism underlies tradeoff between growth rate and biomass yield in Methylobacterium extorquens AM1

DOE PAGES

Fu, Yanfen; Beck, David A. C.; Lidstrom, Mary E.

2016-07-19

In this study, two variants of Methylobacterium extorquens AM1 demonstrated a trade-off between growth rate and biomass yield. In addition, growth rate and biomass yield were also affected by supplementation of growth medium with different amounts of cobalt. The metabolism changes relating to these growth phenomena as well as the trade-off were investigated in this study. 13C metabolic flux analysis was used to generate a detailed central carbon metabolic flux map with both absolute and normalized flux values. As a result, the major differences between the two variants occurred at the formate node as well as within C3-C4 inter-conversion pathways.more » Higher relative fluxes through formyltetrahydrofolate ligase, phosphoenolpyruvate carboxylase, and malic enzyme led to higher biomass yield, while higher relative fluxes through pyruvate kinase and pyruvate dehydrogenase led to higher growth rate. These results were then tested by phenotypic studies on three mutants (null pyk, null pck mutant and null dme mutant) in both variants, which agreed with the model prediction. In this study, 13C metabolic flux analysis for two strain variants of M. extorquens AM1 successfully identified metabolic pathways contributing to the trade-off between cell growth and biomass yield. Phenotypic analysis of mutants deficient in corresponding genes supported the conclusion that C3-C4 inter-conversion strategies were the major response to the trade-off.« less

Difference in C3–C4 metabolism underlies tradeoff between growth rate and biomass yield in Methylobacterium extorquens AM1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fu, Yanfen; Beck, David A. C.; Lidstrom, Mary E.

In this study, two variants of Methylobacterium extorquens AM1 demonstrated a trade-off between growth rate and biomass yield. In addition, growth rate and biomass yield were also affected by supplementation of growth medium with different amounts of cobalt. The metabolism changes relating to these growth phenomena as well as the trade-off were investigated in this study. 13C metabolic flux analysis was used to generate a detailed central carbon metabolic flux map with both absolute and normalized flux values. As a result, the major differences between the two variants occurred at the formate node as well as within C3-C4 inter-conversion pathways.more » Higher relative fluxes through formyltetrahydrofolate ligase, phosphoenolpyruvate carboxylase, and malic enzyme led to higher biomass yield, while higher relative fluxes through pyruvate kinase and pyruvate dehydrogenase led to higher growth rate. These results were then tested by phenotypic studies on three mutants (null pyk, null pck mutant and null dme mutant) in both variants, which agreed with the model prediction. In this study, 13C metabolic flux analysis for two strain variants of M. extorquens AM1 successfully identified metabolic pathways contributing to the trade-off between cell growth and biomass yield. Phenotypic analysis of mutants deficient in corresponding genes supported the conclusion that C3-C4 inter-conversion strategies were the major response to the trade-off.« less

The Aspergillus fumigatus SchASCH9 kinase modulates SakAHOG1 MAP kinase activity and it is essential for virulence

PubMed Central

Alves de Castro, Patrícia; dos Reis, Thaila Fernanda; Dolan, Stephen K.; Manfiolli, Adriana Oliveira; Brown, Neil Andrew; Jones, Gary W.; Doyle, Sean; Riaño-Pachón, Diego M.; Squina, Fábio Márcio; Caldana, Camila; Singh, Ashutosh; Del Poeta, Maurizio; Hagiwara, Daisuke; Silva-Rocha, Rafael; Goldman, Gustavo H.

2016-01-01

Summary The serine-threonine kinase TOR, the Target of Rapamycin, is an important regulator of nutrient, energy and stress signaling in eukaryotes. Sch9, a Ser/Thr kinase of AGC family (the cAMP-dependent PKA, cGMP- dependent protein kinase G and phospholipid-dependent protein kinase C family), is a substrate of TOR. Here, we characterized the fungal opportunistic pathogen Aspergillus fumigatus Sch9 homologue (SchA). The schA null mutant was sensitive to rapamycin, high concentrations of calcium, hyperosmotic stress and SchA was involved in iron metabolism. The ΔschA null mutant showed increased phosphorylation of SakA, the A. fumigatus Hog1 homologue. The schA null mutant has increased and decreased trehalose and glycerol accumulation, respectively, suggesting SchA performs different roles for glycerol and trehalose accumulation during osmotic stress. The schA was transcriptionally regulated by osmotic stress and this response was dependent on SakA and MpkC. The double ΔschA ΔsakA and ΔschA ΔmpkC mutants were more sensitive to osmotic stress than the corresponding parental strains. Transcriptomics and proteomics identified direct and indirect targets of SchA post-exposure to hyperosmotic stress. Finally, ΔschA was avirulent in a low dose murine infection model. Our results suggest there is a complex network of interactions amongst the A. fumigatus TOR, SakA and SchA pathways. PMID:27538790

The Aspergillus fumigatus SchASCH9 kinase modulates SakAHOG1 MAP kinase activity and it is essential for virulence.

PubMed

Alves de Castro, Patrícia; Dos Reis, Thaila Fernanda; Dolan, Stephen K; Oliveira Manfiolli, Adriana; Brown, Neil Andrew; Jones, Gary W; Doyle, Sean; Riaño-Pachón, Diego M; Squina, Fábio Márcio; Caldana, Camila; Singh, Ashutosh; Del Poeta, Maurizio; Hagiwara, Daisuke; Silva-Rocha, Rafael; Goldman, Gustavo H

2016-11-01

The serine-threonine kinase TOR, the Target of Rapamycin, is an important regulator of nutrient, energy and stress signaling in eukaryotes. Sch9, a Ser/Thr kinase of AGC family (the cAMP-dependent PKA, cGMP- dependent protein kinase G and phospholipid-dependent protein kinase C family), is a substrate of TOR. Here, we characterized the fungal opportunistic pathogen Aspergillus fumigatus Sch9 homologue (SchA). The schA null mutant was sensitive to rapamycin, high concentrations of calcium, hyperosmotic stress and SchA was involved in iron metabolism. The ΔschA null mutant showed increased phosphorylation of SakA, the A. fumigatus Hog1 homologue. The schA null mutant has increased and decreased trehalose and glycerol accumulation, respectively, suggesting SchA performs different roles for glycerol and trehalose accumulation during osmotic stress. The schA was transcriptionally regulated by osmotic stress and this response was dependent on SakA and MpkC. The double ΔschA ΔsakA and ΔschA ΔmpkC mutants were more sensitive to osmotic stress than the corresponding parental strains. Transcriptomics and proteomics identified direct and indirect targets of SchA post-exposure to hyperosmotic stress. Finally, ΔschA was avirulent in a low dose murine infection model. Our results suggest there is a complex network of interactions amongst the A. fumigatus TOR, SakA and SchA pathways. © 2016 John Wiley & Sons Ltd.

Cloning, sequencing, disruption and phenotypic analysis of uvsC, an Aspergillus nidulans homologue of yeast RAD51.

PubMed

van Heemst, D; Swart, K; Holub, E F; van Dijk, R; Offenberg, H H; Goosen, T; van den Broek, H W; Heyting, C

1997-05-01

We have cloned the uvsC gene of Aspergillus nidulans by complementation of the A. nidulans uvsC114 mutant. The predicted protein UVSC shows 67.4% sequence identity to the Saccharomyces cerevisiae Rad51 protein and 27.4% sequence identity to the Escherichia coli RecA protein. Transcription of uvsC is induced by methyl-methane sulphonate (MMS), as is transcription of RAD51 of yeast. Similar levels of uvsC transcription were observed after MMS induction in a uvsC+ strain and the uvsC114 mutant. The coding sequence of the uvsC114 allele has a deletion of 6 bp, which results in deletion of two amino acids and replacement of one amino acid in the translation product. In order to gain more insight into the biological function of the uvsC gene, a uvsC null mutant was constructed, in which the entire uvsC coding sequence was replaced by a selectable marker gene. Meiotic and mitotic phenotypes of a uvsC+ strain, the uvsC114 mutant and the uvsC null mutant were compared. The uvsC null mutant was more sensitive to both UV and MMS than the uvsC114 mutant. The uvsC114 mutant arrested in meiotic prophase-I. The uvsC null mutant arrested at an earlier stage, before the onset of meiosis. One possible interpretation of these meiotic phenotypes is that the A. nidulans homologue of Rad51 of yeast has a role both in the specialized processes preceding meiosis and in meiotic prophase I.

Candida albicans Iff11, a secreted protein required for cell wall structure and virulence.

PubMed

Bates, Steven; de la Rosa, José M; MacCallum, Donna M; Brown, Alistair J P; Gow, Neil A R; Odds, Frank C

2007-06-01

The Candida albicans cell wall is the immediate point of contact with the host and is implicated in the host-fungal interaction and virulence. To date, a number of cell wall proteins have been identified and associated with virulence. Analysis of the C. albicans genome has identified the IFF gene family as encoding the largest family of cell wall-related proteins. This family is also conserved in a range of other Candida species. Iff11 differs from other family members in lacking a GPI anchor, and we have demonstrated it to be O glycosylated and secreted in C. albicans. A null mutant lacking IFF11 was hypersensitive to cell wall-damaging agents, suggesting a role in cell wall organization. In a murine model of systemic infection the null mutant was highly attenuated in virulence, and survival-standardized infections suggest it is required to establish an infection. This work provides the first evidence of the importance of this gene family in the host-fungal interaction and virulence.

New Class of Precision Antimicrobials Redefines Role of Clostridium difficile S-layer in Virulence and Viability

PubMed Central

Kirk, Joseph A.; Gebhart, Dana; Buckley, Anthony M.; Lok, Stephen; Scholl, Dean; Douce, Gillian R.; Govoni, Gregory R.; Fagan, Robert P.

2017-01-01

Avidocin-CDs are a new class of precision bactericidal agents that do not damage resident gut microbiota and are unlikely to promote the spread of antibiotic resistance. The precision killing properties result from the fusion of bacteriophage receptor binding proteins (RBPs) to a lethal contractile scaffold from an R-type bacteriocin. We recently described the prototypic Avidocin-CD, Av-CD291.2, that specifically kills C. difficile ribotype 027 strains and prevents colonization of mice. We have since selected two rare Av-CD291.2 resistant mutants of strain R20291 (RT027; S-layer cassette type-4, SCLT-4). These mutants have distinct point mutations in the slpA gene that result in an S-layer null phenotype. Reversion of the mutations to wild-type restored normal SLCT-4 S-layer formation and Av-CD291.2 sensitivity; however, complementation with other SCLT alleles did not restore Av-CD291.2 sensitivity despite restoring S-layer formation. Using newly identified phage RBPs, we constructed a panel of new Avidocin-CDs that kill C. difficile isolates in an SLCT-dependent manner, confirming the S-layer as the receptor in every case. In addition to bacteriophage adsorption, characterization of the S-layer null mutant also uncovered important roles for SlpA in sporulation, resistance to lysozyme and LL-37, and toxin production. Surprisingly, the S-layer-null mutant was found to persist in the hamster gut despite its completely attenuated virulence. Avidocin-CDs have significant therapeutic potential for the treatment and prevention of C. difficile Infection (CDI) given their exquisite specificity for the pathogen. Furthermore, the emergence of resistance forces mutants to trade virulence for continued viability and, therefore, greatly reduce their potential clinical impact. PMID:28878013

A second component of the SltA-dependent cation tolerance pathway in Aspergillus nidulans.

PubMed

Mellado, Laura; Calcagno-Pizarelli, Ana Maria; Lockington, Robin A; Cortese, Marc S; Kelly, Joan M; Arst, Herbert N; Espeso, Eduardo A

2015-09-01

The transcriptional response to alkali metal cation stress is mediated by the zinc finger transcription factor SltA in Aspergillus nidulans and probably in other fungi of the pezizomycotina subphylum. A second component of this pathway has been identified and characterized. SltB is a 1272 amino acid protein with at least two putative functional domains, a pseudo-kinase and a serine-endoprotease, involved in signaling to the transcription factor SltA. Absence of SltB activity results in nearly identical phenotypes to those observed for a null sltA mutant. Hypersensitivity to a variety of monovalent and divalent cations, and to medium alkalinization are among the phenotypes exhibited by a null sltB mutant. Calcium homeostasis is an exception and this cation improves growth of sltΔ mutants. Moreover, loss of kinase HalA in conjunction with loss-of-function sltA or sltB mutations leads to pronounced calcium auxotrophy. sltA sltB double null mutants display a cation stress sensitive phenotype indistinguishable from that of single slt mutants showing the close functional relationship between these two proteins. This functional relationship is reinforced by the fact that numerous mutations in both slt loci can be isolated as suppressors of poor colonial growth resulting from certain null vps (vacuolar protein sorting) mutations. In addition to allowing identification of sltB, our sltB missense mutations enabled prediction of functional regions in the SltB protein. Although the relationship between the Slt and Vps pathways remains enigmatic, absence of SltB, like that of SltA, leads to vacuolar hypertrophy. Importantly, the phenotypes of selected sltA and sltB mutations demonstrate that suppression of null vps mutations is not dependent on the inability to tolerate cation stress. Thus a specific role for both SltA and SltB in the VPS pathway seems likely. Finally, it is noteworthy that SltA and SltB have a similar, limited phylogenetic distribution, being restricted to the pezizomycotina subphylum. The relevance of the Slt regulatory pathway to cell structure, intracellular trafficking and cation homeostasis and its restricted phylogenetic distribution makes this pathway of general interest for future investigation and as a source of targets for antifungal drugs. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Msc1 acts through histone H2A.Z to promote chromosome stability in Schizosaccharomyces pombe.

PubMed

Ahmed, Shakil; Dul, Barbara; Qiu, Xinxing; Walworth, Nancy C

2007-11-01

As a central component of the DNA damage checkpoint pathway, the conserved protein kinase Chk1 mediates cell cycle progression when DNA damage is generated. Msc1 was identified as a multicopy suppressor capable of facilitating survival in response to DNA damage of cells mutant for chk1. We demonstrate that loss of msc1 function results in an increased rate of chromosome loss and that an msc1 null allele exhibits genetic interactions with mutants in key kinetochore components. Multicopy expression of msc1 robustly suppresses a temperature-sensitive mutant (cnp1-1) in the centromere-specific histone H3 variant CENP-A, and localization of CENP-A to the centromere is compromised in msc1 null cells. We present several lines of evidence to suggest that Msc1 carries out its function through the histone H2A variant H2A.Z, encoded by pht1 in fission yeast. Like an msc1 mutant, a pht1 mutant also exhibits chromosome instability and genetic interactions with kinetochore mutants. Suppression of cnp1-1 by multicopy msc1 requires pht1. Likewise, suppression of the DNA damage sensitivity of a chk1 mutant by multicopy msc1 also requires pht1. We present the first genetic evidence that histone H2A.Z may participate in centromere function in fission yeast and propose that Msc1 acts through H2A.Z to promote chromosome stability and cell survival following DNA damage.

Msc1 Acts Through Histone H2A.Z to Promote Chromosome Stability in Schizosaccharomyces pombe

PubMed Central

Ahmed, Shakil; Dul, Barbara; Qiu, Xinxing; Walworth, Nancy C.

2007-01-01

As a central component of the DNA damage checkpoint pathway, the conserved protein kinase Chk1 mediates cell cycle progression when DNA damage is generated. Msc1 was identified as a multicopy suppressor capable of facilitating survival in response to DNA damage of cells mutant for chk1. We demonstrate that loss of msc1 function results in an increased rate of chromosome loss and that an msc1 null allele exhibits genetic interactions with mutants in key kinetochore components. Multicopy expression of msc1 robustly suppresses a temperature-sensitive mutant (cnp1-1) in the centromere-specific histone H3 variant CENP-A, and localization of CENP-A to the centromere is compromised in msc1 null cells. We present several lines of evidence to suggest that Msc1 carries out its function through the histone H2A variant H2A.Z, encoded by pht1 in fission yeast. Like an msc1 mutant, a pht1 mutant also exhibits chromosome instability and genetic interactions with kinetochore mutants. Suppression of cnp1-1 by multicopy msc1 requires pht1. Likewise, suppression of the DNA damage sensitivity of a chk1 mutant by multicopy msc1 also requires pht1. We present the first genetic evidence that histone H2A.Z may participate in centromere function in fission yeast and propose that Msc1 acts through H2A.Z to promote chromosome stability and cell survival following DNA damage. PMID:17947424

Gene disruptions indicate an essential function for the LmmCRK1 cdc2-related kinase of Leishmania mexicana.

PubMed

Mottram, J C; McCready, B P; Brown, K G; Grant, K M

1996-11-01

The generation of homozygous null mutants for the crk1 Cdc2-Related Kinase of Leishmania mexicana was attempted using targeted gene disruption. Promastigote mutants heterozygous for crk1 were readily isolated with a hyg-targeting fragment, but attempts to create null mutants by second-round transfections with a bie-targeting fragment yielded two classes of mutant, neither of which was null. First, the transfected fragment formed an episome; second, the cloned transfectants were found to contain wild-type crk1 alleles as well as hyg and ble integrations. DNA-content analysis revealed that these mutants were triploid or tetraploid. Plasticity in chromosome number following targeting has been proposed as a means by which Leishmania avoids deletion of essential genes. These data support this theory and implicate crk1 as an essential gene, validating CRK1 as a potential drug target. L mexicana transfected with a Trypanosoma brucel homologue, tbcrk1, was shown to be viable in an immcrk1 null background, thus showing complementation of function between these trypanosomatid genes. The expression of crk1 was further manipulated by engineering a six-histidine tag at the C-terminus of the kinase, allowing purification of the active complex by affinity selection on Nl(2+)-nitriloacetic acid (NTA) agarose.

An efficient method for generation of bi-allelic null mutant mouse embryonic stem cells and its application for investigating epigenetic modifiers.

PubMed

Fisher, Cynthia L; Marks, Hendrik; Cho, Lily Ting-Yin; Andrews, Robert; Wormald, Sam; Carroll, Thomas; Iyer, Vivek; Tate, Peri; Rosen, Barry; Stunnenberg, Hendrik G; Fisher, Amanda G; Skarnes, William C

2017-12-01

Mouse embryonic stem (ES) cells are a popular model system to study biological processes, though uncovering recessive phenotypes requires inactivating both alleles. Building upon resources from the International Knockout Mouse Consortium (IKMC), we developed a targeting vector for second allele inactivation in conditional-ready IKMC 'knockout-first' ES cell lines. We applied our technology to several epigenetic regulators, recovering bi-allelic targeted clones with a high efficiency of 60% and used Flp recombinase to restore expression in two null cell lines to demonstrate how our system confirms causality through mutant phenotype reversion. We designed our strategy to select against re-targeting the 'knockout-first' allele and identify essential genes in ES cells, including the histone methyltransferase Setdb1. For confirmation, we exploited the flexibility of our system, enabling tamoxifen inducible conditional gene ablation while controlling for genetic background and tamoxifen effects. Setdb1 ablated ES cells exhibit severe growth inhibition, which is not rescued by exogenous Nanog expression or culturing in naive pluripotency '2i' media, suggesting that the self-renewal defect is mediated through pluripotency network independent pathways. Our strategy to generate null mutant mouse ES cells is applicable to thousands of genes and repurposes existing IKMC Intermediate Vectors. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Evidence for a functional link between Dd-STATa and Dd-PIAS, a Dictyostelium PIAS homologue.

PubMed

Kawata, Takefumi; Hirano, Tatsunori; Ogasawara, Shun; Aoshima, Ryota; Yachi, Ayako

2011-09-01

Several mammalian protein families inhibit the activity of signal transducer and activator of transcription (STAT) proteins. The protein inhibitor of activated STAT (PIAS) was initially identified through its ability to interact with human STAT proteins. We isolated a gene (pisA) encoding a Dictyostelium orthologue of PIAS, Dd-PIAS, which possesses almost all the representative motifs and domains of mammalian PIAS proteins. A Dd-PIAS null mutant strain displays a normal terminal morphology but with accelerated development once cells are aggregated. In contrast, Dd-PIAS overexpressor strains demonstrate delayed aggregation, almost no slug phototaxis, impaired slug motility, and a prolonged slug migration period. This strain is a near phenocopy of the Dd-STATa null mutant, although it eventually forms a fruiting body, albeit inefficiently. The expression of several Dd-STATa-activated genes is upregulated in the Dd-PIAS null mutant and there is ectopic expression of pstAB makers. The concentration of a PIAS-green fluorescent protein (GFP) fusion protein, expressed under the PIAS promoter, is greatest in the pstO cells and gradually decreases with proximity to the tip of the slug and culminant: a pattern diametrically opposite to that of Dd-STATa. Our results suggest a functional interrelationship between Dd-PIAS and Dd-STATa that influences gene expression and development. © 2011 The Authors. Development, Growth & Differentiation © 2011 Japanese Society of Developmental Biologists.

The α Glycerophosphate Cycle in Drosophila melanogaster V. Molecular Analysis of α Glycerophosphate Dehydrogenase and α Glycerophosphate Oxidase Mutants

PubMed Central

Carmon, Amber; Chien, Jeff; Sullivan, David

2010-01-01

Two enzymes, α glycerophosphate dehydrogenase (GPDH-1) in the cytoplasm and α glycerophosphate oxidase (GPO-1) in the mitochondrion cooperate in Drosophila flight muscles to generate the ATP needed for muscle contraction. Null mutants for either enzyme cannot fly. Here, we characterize 15 ethyl methane sulfonate (EMS)-induced mutants in GPDH-1 at the molecular level and assess their effects on structural and evolutionarily conserved domains of this enzyme. In addition, we molecularly characterize 3 EMS-induced GPO-1 mutants and excisions of a P element insertion in the GPO-1 gene. The latter represent the best candidate for null or amorphic mutants in this gene. PMID:19995806

The alpha glycerophosphate cycle in Drosophila melanogaster V. molecular analysis of alpha glycerophosphate dehydrogenase and alpha glycerophosphate oxidase mutants.

PubMed

Carmon, Amber; Chien, Jeff; Sullivan, David; MacIntyre, Ross

2010-01-01

Two enzymes, alpha glycerophosphate dehydrogenase (GPDH-1) in the cytoplasm and alpha glycerophosphate oxidase (GPO-1) in the mitochondrion cooperate in Drosophila flight muscles to generate the ATP needed for muscle contraction. Null mutants for either enzyme cannot fly. Here, we characterize 15 ethyl methane sulfonate (EMS)-induced mutants in GPDH-1 at the molecular level and assess their effects on structural and evolutionarily conserved domains of this enzyme. In addition, we molecularly characterize 3 EMS-induced GPO-1 mutants and excisions of a P element insertion in the GPO-1 gene. The latter represent the best candidate for null or amorphic mutants in this gene.

A Toxoplasma MORN1 Null Mutant Undergoes Repeated Divisions but Is Defective in Basal Assembly, Apicoplast Division and Cytokinesis

PubMed Central

Lorestani, Alexander; Sheiner, Lilach; Yang, Kevin; Robertson, Seth D.; Sahoo, Nivedita; Brooks, Carrie F.; Ferguson, David J. P.; Striepen, Boris; Gubbels, Marc-Jan

2010-01-01

The membrane occupation and recognition nexus protein 1 (MORN1) is highly conserved among apicomplexan parasites and is associated with several structures that have a role in cell division. Here we dissected the role of MORN1 using the relatively simple budding process of Toxoplasma gondii as a model. Ablation of MORN1 in a conditional null mutant resulted in pronounced defects suggesting a central role for MORN1 in apicoplast segregation and in daughter cell budding. Lack of MORN1 resulted in double-headed parasites. These Janus-headed parasites form two complete apical complexes but fail to assemble a basal complex. Moreover, these parasites were capable of undergoing several more budding rounds resulting in the formation of up to 16-headed parasites conjoined at the basal end. Despite this segregation defect, the mother's cytoskeleton was completely disassembled in every budding round. Overall this argues that successful completion of the budding is not required for cell cycle progression. None of the known basal complex components, including a set of recently identified inner membrane complex (IMC) proteins, localized correctly in these multi-headed parasites. These data suggest that MORN1 is essential for assembly of the basal complex, and that lack of the basal complex abolishes the contractile capacity assigned to the basal complex late in daughter formation. Consistent with this hypothesis we observe that MORN1 mutants fail to efficiently constrict and divide the apicoplast. We used the null background provided by the mutant to dissect the function of subdomains of the MORN1 protein. This demonstrated that deletion of a single MORN domain already prevented the function of MORN1 whereas a critical role for the short linker between MORN domains 6 and 7 was identified. In conclusion, MORN1 is required for basal complex assembly and loss of MORN1 results in defects in apicoplast division and daughter segregation. PMID:20808817

Identification of biochemical adaptations in hyper- or hypocontractile hearts from phospholamban mutant mice by expression proteomics.

PubMed

Pan, Yan; Kislinger, Thomas; Gramolini, Anthony O; Zvaritch, Elena; Kranias, Evangelia G; MacLennan, David H; Emili, Andrew

2004-02-24

Phospholamban (PLN) is a critical regulator of cardiac contractility through its binding to and regulation of the activity of the sarco(endo)plasmic reticulum Ca2+ ATPase. To uncover biochemical adaptations associated with extremes of cardiac muscle contractility, we used high-throughput gel-free tandem MS to monitor differences in the relative abundance of membrane proteins in standard microsomal fractions isolated from the hearts of PLN-null mice (PLN-KO) with high contractility and from transgenic mice overexpressing a superinhibitory PLN mutant in a PLN-null background (I40A-KO) with diminished contractility. Significant differential expression was detected for a subset of the 782 proteins identified, including known membrane-associated biomarkers, components of signaling pathways, and previously uninvestigated proteins. Proteins involved in fat and carbohydrate metabolism and proteins linked to G protein-signaling pathways activating protein kinase C were enriched in I40A-KO cardiac muscle, whereas proteins linked to enhanced contractile function were enriched in PLN-KO mutant hearts. These data demonstrate that Ca2+ dysregulation, leading to elevated or depressed cardiac contractility, induces compensatory biochemical responses.

The Agr-Like Quorum Sensing System Is Required for Pathogenesis of Necrotic Enteritis Caused by Clostridium perfringens in Poultry.

PubMed

Yu, Qiang; Lepp, Dion; Mehdizadeh Gohari, Iman; Wu, Tao; Zhou, Hongzhuan; Yin, Xianhua; Yu, Hai; Prescott, John F; Nie, Shao-Ping; Xie, Ming-Yong; Gong, Joshua

2017-06-01

Clostridium perfringens encodes at least two different quorum sensing (QS) systems, the Agr-like and LuxS, and recent studies have highlighted their importance in the regulation of toxin production and virulence. The role of QS in the pathogenesis of necrotic enteritis (NE) in poultry and the regulation of NetB, the key toxin involved, has not yet been investigated. We have generated isogenic agrB -null and complemented strains from parent strain CP1 and demonstrated that the virulence of the agrB -null mutant was strongly attenuated in a chicken NE model system and restored by complementation. The production of NetB, a key NE-associated toxin, was dramatically reduced in the agrB mutant at both the transcriptional and protein levels, though not in a luxS mutant. Transwell assays confirmed that the Agr-like QS system controls NetB production through a diffusible signal. Global gene expression analysis of the agrB mutant identified additional genes modulated by Agr-like QS, including operons related to phospholipid metabolism and adherence, which may also play a role in NE pathogenesis. This study provides the first evidence that the Agr-like QS system is critical for NE pathogenesis and identifies a number of Agr-regulated genes, most notably netB , that are potentially involved in mediating its effects. The Agr-like QS system thus may serve as a target for developing novel interventions to prevent NE in chickens. © Crown copyright 2017.

Expression and function of orphan nuclear receptor TLX in adult neural stem cells.

PubMed

Shi, Yanhong; Chichung Lie, D; Taupin, Philippe; Nakashima, Kinichi; Ray, Jasodhara; Yu, Ruth T; Gage, Fred H; Evans, Ronald M

2004-01-01

The finding of neurogenesis in the adult brain led to the discovery of adult neural stem cells. TLX was initially identified as an orphan nuclear receptor expressed in vertebrate forebrains and is highly expressed in the adult brain. The brains of TLX-null mice have been reported to have no obvious defects during embryogenesis; however, mature mice suffer from retinopathies, severe limbic defects, aggressiveness, reduced copulation and progressively violent behaviour. Here we show that TLX maintains adult neural stem cells in an undifferentiated, proliferative state. We show that TLX-expressing cells isolated by fluorescence-activated cell sorting (FACS) from adult brains can proliferate, self-renew and differentiate into all neural cell types in vitro. By contrast, TLX-null cells isolated from adult mutant brains fail to proliferate. Reintroducing TLX into FACS-sorted TLX-null cells rescues their ability to proliferate and to self-renew. In vivo, TLX mutant mice show a loss of cell proliferation and reduced labelling of nestin in neurogenic areas in the adult brain. TLX can silence glia-specific expression of the astrocyte marker GFAP in neural stem cells, suggesting that transcriptional repression may be crucial in maintaining the undifferentiated state of these cells.

Seven functional classes of Barth syndrome mutation.

PubMed

Whited, Kevin; Baile, Matthew G; Currier, Pamela; Claypool, Steven M

2013-02-01

Patients with Barth syndrome (BTHS), a rare X-linked disease, suffer from skeletal and cardiomyopathy and bouts of cyclic neutropenia. The causative gene encodes tafazzin, a transacylase, which is the major determinant of the final acyl chain composition of the mitochondrial-specific phospholipid, CL. In addition to numerous frame shift and splice-site mutations, 36 missense mutations have been associated with BTHS. Previously, we established a BTHS-mutant panel in the yeast Saccharomyces cerevisiae that successfully models 18/21 conserved pathogenic missense mutations and defined the loss-of-function mechanism associated with a subset of the mutant tafazzins. Here, we report the biochemical and cell biological characterization of the rest of the yeast BTHS-mutant panel and in so doing identify three additional modes of tafazzin dysfunction. The largest group of mutant tafazzins is catalytically null, two mutants encode hypomorphic alleles, and another two mutants are temperature sensitive. Additionally, we have expanded the defects associated with previously characterized matrix-mislocalized-mutant tafazzins to include the rapid degradation of aggregation-prone polypeptides that correctly localize to the mitochondrial IMS. In sum, our in-depth characterization of the yeast BTHS-mutant panel has identified seven functional classes of BTHS mutation.

Zn2+ Uptake in Streptococcus pyogenes: Characterization of adcA and lmb Null Mutants.

PubMed

Tedde, Vittorio; Rosini, Roberto; Galeotti, Cesira L

2016-01-01

An effective regulation of metal ion homeostasis is essential for the growth of microorganisms in any environment and in pathogenic bacteria is strongly associated with their ability to invade and colonise their hosts. To gain a better insight into zinc acquisition in Group A Streptococcus (GAS) we characterized null deletion mutants of the adcA and lmb genes of Streptococcus pyogenes strain MGAS5005 encoding the orthologues of AdcA and AdcAII, the two surface lipoproteins with partly redundant roles in zinc homeostasis in Streptococcus pneumoniae. Null adcA and lmb mutants were analysed for their capability to grow in zinc-depleted conditions and were found to be more susceptible to zinc starvation, a phenotype that could be rescued by the addition of Zn2+ ions to the growth medium. Expression of AdcA, Lmb and HtpA, the polyhistidine triad protein encoded by the gene adjacent to lmb, during growth under conditions of limited zinc availability was examined by Western blot analysis in wild type and null mutant strains. In the wild type strain, AdcA was always present with little variation in expression levels between conditions of excess or limited zinc availability. In contrast, Lmb and HtpA were expressed at detectable levels only during growth in the presence of low zinc concentrations or in the null adcA mutant, when expression of lmb is required to compensate for the lack of adcA expression. In the latter case, Lmb and HtpA were overexpressed by several fold, thus indicating that also in GAS AdcA is a zinc-specific importer and, although it shares this function with Lmb, the two substrate-binding proteins do not show fully overlapping roles in zinc homeostasis.

Protein expression profiling of the drosophila fragile X mutant brain reveals up-regulation of monoamine synthesis.

PubMed

Zhang, Yong Q; Friedman, David B; Wang, Zhe; Woodruff, Elvin; Pan, Luyuan; O'donnell, Janis; Broadie, Kendal

2005-03-01

Fragile X syndrome is the most common form of inherited mental retardation, associated with both cognitive and behavioral anomalies. The disease is caused by silencing of the fragile X mental retardation 1 (fmr1) gene, which encodes the mRNA-binding, translational regulator FMRP. Previously we established a disease model through mutation of Drosophila fmr1 (dfmr1) and showed that loss of dFMRP causes defects in neuronal structure, function, and behavioral output similar to the human disease state. To uncover molecular targets of dFMRP in the brain, we use here a proteomic approach involving two-dimensional difference gel electrophoresis analyses followed by mass spectrometry identification of proteins with significantly altered expression in dfmr1 null mutants. We then focus on two misregulated enzymes, phenylalanine hydroxylase (Henna) and GTP cyclohydrolase (Punch), both of which mediate in concert the synthetic pathways of two key monoamine neuromodulators, dopamine and serotonin. Brain enzymatic assays show a nearly 2-fold elevation of Punch activity in dfmr1 null mutants. Consistently brain neurochemical assays show that both dopamine and serotonin are significantly increased in dfmr1 null mutants. At a cellular level, dfmr1 null mutant neurons display a highly significant elevation of the dense core vesicles that package these monoamine neuromodulators for secretion. Taken together, these data indicate that dFMRP normally down-regulates the monoamine pathway, which is consequently up-regulated in the mutant condition. Elevated brain levels of dopamine and serotonin provide a plausible mechanistic explanation for aspects of cognitive and behavioral deficits in human patients.

Metabotropic glutamate receptor-mediated use-dependent down-regulation of synaptic excitability involves the fragile X mental retardation protein.

PubMed

Repicky, Sarah; Broadie, Kendal

2009-02-01

Loss of the mRNA-binding protein FMRP results in the most common inherited form of both mental retardation and autism spectrum disorders: fragile X syndrome (FXS). The leading FXS hypothesis proposes that metabotropic glutamate receptor (mGluR) signaling at the synapse controls FMRP function in the regulation of local protein translation to modulate synaptic transmission strength. In this study, we use the Drosophila FXS disease model to test the relationship between Drosophila FMRP (dFMRP) and the sole Drosophila mGluR (dmGluRA) in regulation of synaptic function, using two-electrode voltage-clamp recording at the glutamatergic neuromuscular junction (NMJ). Null dmGluRA mutants show minimal changes in basal synapse properties but pronounced defects during sustained high-frequency stimulation (HFS). The double null dfmr1;dmGluRA mutant shows repression of enhanced augmentation and delayed onset of premature long-term facilitation (LTF) and strongly reduces grossly elevated post-tetanic potentiation (PTP) phenotypes present in dmGluRA-null animals. Null dfmr1 mutants show features of synaptic hyperexcitability, including multiple transmission events in response to a single stimulus and cyclic modulation of transmission amplitude during prolonged HFS. The double null dfmr1;dmGluRA mutant shows amelioration of these defects but does not fully restore wildtype properties in dfmr1-null animals. These data suggest that dmGluRA functions in a negative feedback loop in which excess glutamate released during high-frequency transmission binds the glutamate receptor to dampen synaptic excitability, and dFMRP functions to suppress the translation of proteins regulating this synaptic excitability. Removal of the translational regulator partially compensates for loss of the receptor and, similarly, loss of the receptor weakly compensates for loss of the translational regulator.

Role of Tir and Intimin in the Virulence of Rabbit Enteropathogenic Escherichia coli Serotype O103:H2

PubMed Central

Marchès, Olivier; Nougayrède, Jean-Philippe; Boullier, Séverine; Mainil, Jacques; Charlier, Gérard; Raymond, Isabelle; Pohl, Pierre; Boury, Michèle; De Rycke, Jean; Milon, Alain; Oswald, Eric

2000-01-01

Attaching and effacing (A/E) rabbit enteropathogenic Escherichia coli (REPEC) strains belonging to serogroup O103 are an important cause of diarrhea in weaned rabbits. Like human EPEC strains, they possess the locus of enterocyte effacement clustering the genes involved in the formation of the A/E lesions. In addition, pathogenic REPEC O103 strains produce an Esp-dependent but Eae (intimin)-independent alteration of the host cell cytoskeleton characterized by the formation of focal adhesion complexes and the reorganization of the actin cytoskeleton into bundles of stress fibers. To investigate the role of intimin and its translocated coreceptor (Tir) in the pathogenicity of REPEC, we have used a newly constructed isogenic tir null mutant together with a previously described eae null mutant. When human HeLa epithelial cells were infected, the tir mutant was still able to induce the formation of stress fibers as previously reported for the eae null mutant. When the rabbit epithelial cell line RK13 was used, REPEC O103 produced a classical fluorescent actin staining (FAS) effect, whereas both the eae and tir mutants were FAS negative. In a rabbit ligated ileal loop model, neither mutant was able to induce A/E lesions. In contrast to the parental strain, which intimately adhered to the enterocytes and destroyed the brush border microvilli, bacteria of both mutants were clustered in the mucus without reaching and damaging the microvilli. The role of intimin and Tir was then analyzed in vivo by oral inoculation of weaned rabbits. Although both mutants were still present in the intestinal flora of the rabbits 3 weeks after oral inoculation, neither mutant strain induced any clinical signs or significant weight loss in the inoculated rabbits whereas the parental strain caused the death of 90% of the inoculated rabbits. Nevertheless, an inflammatory infiltrate was present in the lamina propria of the rabbits infected with both mutants, with an inflammatory response greater for the eae null mutant. In conclusion, we have confirmed the role of intimin in virulence, and we have shown, for the first time, that Tir is also a key factor in vivo for pathogenicity. PMID:10722617

Role of tir and intimin in the virulence of rabbit enteropathogenic Escherichia coli serotype O103:H2.

PubMed

Marchès, O; Nougayrède, J P; Boullier, S; Mainil, J; Charlier, G; Raymond, I; Pohl, P; Boury, M; De Rycke, J; Milon, A; Oswald, E

2000-04-01

Attaching and effacing (A/E) rabbit enteropathogenic Escherichia coli (REPEC) strains belonging to serogroup O103 are an important cause of diarrhea in weaned rabbits. Like human EPEC strains, they possess the locus of enterocyte effacement clustering the genes involved in the formation of the A/E lesions. In addition, pathogenic REPEC O103 strains produce an Esp-dependent but Eae (intimin)-independent alteration of the host cell cytoskeleton characterized by the formation of focal adhesion complexes and the reorganization of the actin cytoskeleton into bundles of stress fibers. To investigate the role of intimin and its translocated coreceptor (Tir) in the pathogenicity of REPEC, we have used a newly constructed isogenic tir null mutant together with a previously described eae null mutant. When human HeLa epithelial cells were infected, the tir mutant was still able to induce the formation of stress fibers as previously reported for the eae null mutant. When the rabbit epithelial cell line RK13 was used, REPEC O103 produced a classical fluorescent actin staining (FAS) effect, whereas both the eae and tir mutants were FAS negative. In a rabbit ligated ileal loop model, neither mutant was able to induce A/E lesions. In contrast to the parental strain, which intimately adhered to the enterocytes and destroyed the brush border microvilli, bacteria of both mutants were clustered in the mucus without reaching and damaging the microvilli. The role of intimin and Tir was then analyzed in vivo by oral inoculation of weaned rabbits. Although both mutants were still present in the intestinal flora of the rabbits 3 weeks after oral inoculation, neither mutant strain induced any clinical signs or significant weight loss in the inoculated rabbits whereas the parental strain caused the death of 90% of the inoculated rabbits. Nevertheless, an inflammatory infiltrate was present in the lamina propria of the rabbits infected with both mutants, with an inflammatory response greater for the eae null mutant. In conclusion, we have confirmed the role of intimin in virulence, and we have shown, for the first time, that Tir is also a key factor in vivo for pathogenicity.

The functionally conserved nucleoporins Nup124p from fission yeast and the human Nup153 mediate nuclear import and activity of the Tf1 retrotransposon and HIV-1 Vpr.

PubMed

Varadarajan, Padmapriya; Mahalingam, Sundarasamy; Liu, Peiyun; Ng, Sarah Boon Hsi; Gandotra, Sheetal; Dorairajoo, Desmond Suresh Kumar; Balasundaram, David

2005-04-01

We report that the fission yeast nucleoporin Nup124p is required for the nuclear import of both, retrotransposon Tf1-Gag as well as the retroviral HIV-1 Vpr. Failure to import Tf1-Gag into the nucleus in a nup124 null mutant resulted in complete loss of Tf1 transposition. Similarly, nuclear import of HIV-1 Vpr was impaired in nup124 null mutant strains and cells became resistant to Vpr's cell-killing activity. On the basis of protein domain similarity, the human nucleoporin Nup153 was identified as a putative homolog of Nup124p. We demonstrate that in vitro-translated Nup124p and Nup153 coimmunoprecipitate Tf1-Gag or HIV-1 Vpr. Though full-length Nup153 was unable to complement the Tf1 transposition defect in a nup124 null mutant, we provide evidence that both nucleoporins share a unique N-terminal domain, Nup124p(AA264-454) and Nup153(AA448-634) that is absolutely essential for Tf1 transposition. Epigenetic overexpression of this domain in a wild-type (nup124(+)) background blocked Tf1 activity implying that sequences from Nup124p and the human Nup153 challenged the same pathway affecting Tf1 transposition. Our results establish a unique relationship between two analogous nucleoporins Nup124p and Nup153 wherein the function of a common domain in retrotransposition is conserved.

Neural circuit architecture defects in a Drosophila model of Fragile X syndrome are alleviated by minocycline treatment and genetic removal of matrix metalloproteinase

PubMed Central

Siller, Saul S.; Broadie, Kendal

2011-01-01

SUMMARY Fragile X syndrome (FXS), caused by loss of the fragile X mental retardation 1 (FMR1) product (FMRP), is the most common cause of inherited intellectual disability and autism spectrum disorders. FXS patients suffer multiple behavioral symptoms, including hyperactivity, disrupted circadian cycles, and learning and memory deficits. Recently, a study in the mouse FXS model showed that the tetracycline derivative minocycline effectively remediates the disease state via a proposed matrix metalloproteinase (MMP) inhibition mechanism. Here, we use the well-characterized Drosophila FXS model to assess the effects of minocycline treatment on multiple neural circuit morphological defects and to investigate the MMP hypothesis. We first treat Drosophila Fmr1 (dfmr1) null animals with minocycline to assay the effects on mutant synaptic architecture in three disparate locations: the neuromuscular junction (NMJ), clock neurons in the circadian activity circuit and Kenyon cells in the mushroom body learning and memory center. We find that minocycline effectively restores normal synaptic structure in all three circuits, promising therapeutic potential for FXS treatment. We next tested the MMP hypothesis by assaying the effects of overexpressing the sole Drosophila tissue inhibitor of MMP (TIMP) in dfmr1 null mutants. We find that TIMP overexpression effectively prevents defects in the NMJ synaptic architecture in dfmr1 mutants. Moreover, co-removal of dfmr1 similarly rescues TIMP overexpression phenotypes, including cellular tracheal defects and lethality. To further test the MMP hypothesis, we generated dfmr1;mmp1 double null mutants. Null mmp1 mutants are 100% lethal and display cellular tracheal defects, but co-removal of dfmr1 allows adult viability and prevents tracheal defects. Conversely, co-removal of mmp1 ameliorates the NMJ synaptic architecture defects in dfmr1 null mutants, despite the lack of detectable difference in MMP1 expression or gelatinase activity between the single dfmr1 mutants and controls. These results support minocycline as a promising potential FXS treatment and suggest that it might act via MMP inhibition. We conclude that FMRP and TIMP pathways interact in a reciprocal, bidirectional manner. PMID:21669931

Drosophila GPCR Han is a receptor for the circadian clock neuropeptide PDF.

PubMed

Hyun, Seogang; Lee, Youngseok; Hong, Sung-Tae; Bang, Sunhoe; Paik, Donggi; Kang, Jongkyun; Shin, Jinwhan; Lee, Jaejung; Jeon, Keunhye; Hwang, Seungyoon; Bae, Eunkyung; Kim, Jaeseob

2005-10-20

The pigment-dispersing factor (PDF) is a neuropeptide controlling circadian behavioral rhythms in Drosophila, but its receptor is not yet known. From a large-scale temperature preference behavior screen in Drosophila, we isolated a P insertion mutant that preferred different temperatures during the day and night. This mutation, which we named han, reduced the transcript level of CG13758. We found that Han was expressed specifically in 13 pairs of circadian clock neurons in the adult brain. han null flies showed arrhythmic circadian behavior in constant darkness. The behavioral characteristics of han null mutants were similar to those of pdf null mutants. We also found that PDF binds specifically to S2 cells expressing Han, which results in the elevation of cAMP synthesis. Therefore, we herein propose that Han is a PDF receptor regulating circadian behavioral rhythm through coordination of activities of clock neurons.

Compensatory changes in CYP expression in three different toxicology mouse models: CAR-null, Cyp3a-null, and Cyp2b9/10/13-null mice

EPA Science Inventory

Targeted mutant models are common in mechanistic toxicology experiments investigating the absorption, metabolism, distribution, or elimination (ADME) of chemicals from individuals. Key models include those for xenosensing transcription factors and cytochrome P450s (CYP). Here we ...

Drug discrimination and neurochemical studies in alpha7 null mutant mice: tests for the role of nicotinic alpha7 receptors in dopamine release.

PubMed

Quarta, Davide; Naylor, Christopher G; Barik, Jacques; Fernandes, Cathy; Wonnacott, Susan; Stolerman, Ian P

2009-04-01

The nicotine discriminative stimulus has been linked to beta2-containing (beta2*) nicotinic receptors, with little evidence of a role for alpha7 nicotinic receptors, because nicotine discrimination was very weak in beta2 null mutant mice but normal in alpha7 mutants. As both alpha7 and beta2* nicotinic receptors have been implicated in nicotine-stimulated dopamine overflow, this study focused on the dopamine-mediated element in the nicotine stimulus by examining cross-generalisation between amphetamine and nicotine. Male alpha7 nicotinic receptor null mutant mice and wild-type controls were bred in-house and trained to discriminate nicotine (0.8 mg/kg) or (+)-amphetamine (0.6 mg/kg) from saline in a two-lever procedure with a tandem VI-30 FR-10 schedule of food reinforcement. Dopamine release from striatal slices was determined in parallel experiments. An alpha7 nicotinic receptor-mediated component of dopamine release was demonstrated in tissue from wild-type mice using choline as a selective agonist. This response was absent in tissue from null mutant animals. The mutation did not influence acquisition of drug discriminations but subtly affected the results of cross-generalisation tests. In mice trained to discriminate nicotine or amphetamine, there was partial cross-generalisation in wild-type mice and, at certain doses, these effects were attenuated in mutants. Further support for an alpha7 nicotinic receptor-mediated component was provided by the ability of the alpha7 nicotinic receptor antagonist methyllycaconitine to attenuate responses to nicotine and amphetamine in wild-type mice. These findings support the concept of an alpha7 nicotinic receptor-mediated dopaminergic element in nicotine discrimination, warranting further tests with selective dopamine agonists.

MIB-1 Is Required for Spermatogenesis and Facilitates LIN-12 and GLP-1 Activity in Caenorhabditis elegans.

PubMed

Ratliff, Miriam; Hill-Harfe, Katherine L; Gleason, Elizabeth J; Ling, Huiping; Kroft, Tim L; L'Hernault, Steven W

2018-05-01

Covalent attachment of ubiquitin to substrate proteins changes their function or marks them for proteolysis, and the specificity of ubiquitin attachment is mediated by the numerous E3 ligases encoded by animals. Mind Bomb is an essential E3 ligase during Notch pathway signaling in insects and vertebrates. While Caenorhabditis elegans encodes a Mind Bomb homolog ( mib-1 ), it has never been recovered in the extensive Notch suppressor/enhancer screens that have identified numerous pathway components. Here, we show that C. elegans mib-1 null mutants have a spermatogenesis-defective phenotype that results in a heterogeneous mixture of arrested spermatocytes, defective spermatids, and motility-impaired spermatozoa. mib-1 mutants also have chromosome segregation defects during meiosis, molecular null mutants are intrinsically temperature-sensitive, and many mib-1 spermatids contain large amounts of tubulin. These phenotypic features are similar to the endogenous RNA intereference (RNAi) mutants, but mib-1 mutants do not affect RNAi. MIB-1 protein is expressed throughout the germ line with peak expression in spermatocytes followed by segregation into the residual body during spermatid formation. C. elegans mib-1 expression, while upregulated during spermatogenesis, also occurs somatically, including in vulva precursor cells. Here, we show that mib-1 mutants suppress both lin-12 and glp-1 ( C. elegans Notch) gain-of-function mutants, restoring anchor cell formation and a functional vulva to the former and partly restoring oocyte production to the latter. However, suppressed hermaphrodites are only observed when grown at 25°, and they are self-sterile. This probably explains why mib-1 was not previously recovered as a Notch pathway component in suppressor/enhancer selection experiments. Copyright © 2018 by the Genetics Society of America.

Retinoic acid from the meninges regulates cortical neuron generation.

PubMed

Siegenthaler, Julie A; Ashique, Amir M; Zarbalis, Konstantinos; Patterson, Katelin P; Hecht, Jonathan H; Kane, Maureen A; Folias, Alexandra E; Choe, Youngshik; May, Scott R; Kume, Tsutomu; Napoli, Joseph L; Peterson, Andrew S; Pleasure, Samuel J

2009-10-30

Extrinsic signals controlling generation of neocortical neurons during embryonic life have been difficult to identify. In this study we demonstrate that the dorsal forebrain meninges communicate with the adjacent radial glial endfeet and influence cortical development. We took advantage of Foxc1 mutant mice with defects in forebrain meningeal formation. Foxc1 dosage and loss of meninges correlated with a dramatic reduction in both neuron and intermediate progenitor production and elongation of the neuroepithelium. Several types of experiments demonstrate that retinoic acid (RA) is the key component of this secreted activity. In addition, Rdh10- and Raldh2-expressing cells in the dorsal meninges were either reduced or absent in the Foxc1 mutants, and Rdh10 mutants had a cortical phenotype similar to the Foxc1 null mutants. Lastly, in utero RA treatment rescued the cortical phenotype in Foxc1 mutants. These results establish RA as a potent, meningeal-derived cue required for successful corticogenesis.

Dependence of paranodal junctional gap width on transverse bands.

PubMed

Rosenbluth, Jack; Petzold, Chris; Peles, Elior

2012-08-15

Mouse mutants with paranodal junctional (PNJ) defects display variable degrees of neurological impairment. In this study we compare control paranodes with those from three mouse mutants that differ with respect to a conspicuous PNJ component, the transverse bands (TBs). We hypothesize that TBs link the apposed junctional membranes together at a fixed distance and thereby determine the width of the junctional gap, which may in turn determine the extent to which nodal action currents can be short-circuited underneath the myelin sheath. Electron micrographs of aldehyde-fixed control PNJs, in which TBs are abundant, show a consistent junctional gap of ∼3.5 nm. In Caspr-null PNJs, which lack TBs entirely, the gap is wider (∼6-7 nm) and more variable. In CST-null PNJs, which have only occasional TBs, the mean PNJ gap width is comparable to that in Caspr-null mice. In the shaking mutant, in contrast, which has approximately 60% of the normal complement of TBs, mean PNJ gap width is not significantly different from that in controls. Correspondingly, shaking mice are much less impaired neurologically than either Caspr-null or CST-null mice. We conclude that in the absence or gross diminution of TBs, mean PNJ gap width increases significantly and suggest that this difference could underlie some of the neurological impairment seen in those mutants. Surprisingly, even in the absence of TBs, paranodes are to some extent maintained in their usual form, implying that in addition to TBs, other factors govern the formation and maintenance of overall paranodal structure. Copyright © 2012 Wiley Periodicals, Inc.

Alteration of medial-edge epithelium cell adhesion in two Tgf-β3 null mouse strains

PubMed Central

Martínez-Sanz, Elena; Del Río, Aurora; Barrio, Carmen; Murillo, Jorge; Maldonado, Estela; Garcillán, Beatriz; Amorós, María; Fuerte, Tamara; Fernández, Álvaro; Trinidad, Eva; Rabadán, M Ángeles; López, Yamila; Martínez, M Luisa; Martínez-Álvarez, Concepción

2008-01-01

Although palatal shelf adhesion is a crucial event during palate development, little work has been carried out to determine which molecules are responsible for this process. Furthermore, whether altered palatal shelf adhesion causes the cleft palate presented by Tgf-β3 null mutant mice has not yet been clarified. Here, we study the presence/distribution of some extracellular matrix and cell adhesion molecules at the time of the contact of palatal shelves in both wild-type and Tgf-β3 null mutant palates of two strains of mice (C57/BL/6J (C57), and MF1) that develop cleft palates of different severity. We have performed immunohistochemistry with antibodies against collagens IV and IX, laminin, fibronectin, the α5- and β1-integrins, and ICAM-1; in situ hybridization with a Nectin-1 riboprobe; and palatal shelf cultures treated or untreated with TGF-β3 or neutralizing antibodies against fibronectin or the α5-integrin. Our results show the location of these molecules in the wild-type mouse medial edge epithelium (MEE) of both strains at the time of the contact of palatal shelves; the heavier (C57) and milder (MF1) alteration of their presence in the Tgf-β3 null mutants; the importance of TGF-β3 to restore their normal pattern of expression; and the crucial role of fibronectin and the α5-integrin in palatal shelf adhesion. We thus provide insight into the molecular bases of this important process and the cleft palate presented by Tgf-β3 null mutant mice. PMID:18431835

Identification of nucleolus-associated chromatin domains reveals the role of the nucleolus in the 3D organisation of the A. thaliana genome

PubMed Central

Pontvianne, Frédéric; Carpentier, Marie-Christine; Durut, Nathalie; Pavlištová, Veronika; Jaške, Karin; Schořová, Šárka; Parrinello, Hugues; Rohmer, Marine; Pikaard, Craig S; Fojtová, Miloslava; Fajkus, Jiří; Saez-Vasquez, Julio

2017-01-01

The nucleolus is the site of ribosomal RNA (rRNA) gene transcription, rRNA processing and ribosome biogenesis. However, the nucleolus also plays additional roles in the cell. We isolated nucleoli by Fluorescence Activated Cell Sorting (FACS) and identified Nucleolus-Associated Chromatin Domains (NADs) by deep sequencing, comparing wild-type plants and null mutants for the nucleolar protein, NUCLEOLIN 1 (NUC1). NADs are primarily genomic regions with heterochromatic signatures and include transposable elements (TEs), sub-telomeric regions and mostly inactive protein-coding genes. However, NADs also include active ribosomal RNA genes, and the entire short arm of chromosome 4 adjacent to them. In nuc1 null mutants, which alter rRNA gene expression and overall nucleolar structure, NADs are altered, telomere association with the nucleolus is decreased and telomeres become shorter. Collectively, our studies reveal roles for NUC1 and the nucleolus in the spatial organization of chromosomes as well as telomere maintenance. PMID:27477271

Expansion of stem cells counteracts age-related mammary regression in compound Timp1/Timp3 null mice.

PubMed

Jackson, Hartland W; Waterhouse, Paul; Sinha, Ankit; Kislinger, Thomas; Berman, Hal K; Khokha, Rama

2015-03-01

Age is the primary risk factor for breast cancer in women. Bipotent basal stem cells actively maintain the adult mammary ductal tree, but with age tissues atrophy. We show that cell-extrinsic factors maintain the adult stem cell pool during ageing and dictate tissue stoichiometry. Mammary stem cells spontaneously expand more than 11-fold in virgin adult female mice lacking specific genes for TIMPs, the natural metalloproteinase inhibitors. Compound Timp1/Timp3 null glands exhibit Notch activation and accelerated gestational differentiation. Proteomics of mutant basal cells uncover altered cytoskeletal and extracellular protein repertoires, and we identify aberrant mitotic spindle orientation in these glands, a process that instructs asymmetric cell division and fate. We find that progenitor activity normally declines with age, but enriched stem/progenitor pools prevent tissue regression in Timp mutant mammary glands without affecting carcinogen-induced cancer susceptibility. Thus, improved stem cell content can extend mouse mammary tissue lifespan without altering cancer risk in this mouse model.

Hes1 expression is reduced in Tbx1 null cells and is required for the development of structures affected in 22q11 deletion syndrome

PubMed Central

van Bueren, Kelly Lammerts; Papangeli, Irinna; Rochais, Francesca; Pearce, Kerra; Roberts, Catherine; Calmont, Amelie; Szumska, Dorota; Kelly, Robert G.; Bhattacharya, Shoumo; Scambler, Peter J.

2010-01-01

22q11 deletion syndrome (22q11DS) is characterised by aberrant development of the pharyngeal apparatus and the heart with haploinsufficiency of the transcription factor TBX1 being considered the major underlying cause of the disease. Tbx1 mutations in mouse phenocopy the disorder. In order to identify the transcriptional dysregulation in Tbx1-expressing lineages we optimised fluorescent-activated cell sorting of β-galactosidase expressing cells (FACS-Gal) to compare the expression profile of Df1/Tbx1lacZ (effectively Tbx1 null) and Tbx1 heterozygous cells isolated from mouse embryos. Hes1, a major effector of Notch signalling, was identified as downregulated in Tbx1−/− mutants. Hes1 mutant mice exhibited a partially penetrant range of 22q11DS-like defects including pharyngeal arch artery (PAA), outflow tract, craniofacial and thymic abnormalities. Similar to Tbx1 mice, conditional mutagenesis revealed that Hes1 expression in embryonic pharyngeal ectoderm contributes to thymus and pharyngeal arch artery development. These results suggest that Hes1 acts downstream of Tbx1 in the morphogenesis of pharyngeal-derived structures. PMID:20122914

Deletion of vanilloid receptor (TRPV1) in mice alters behavioral effects of ethanol

PubMed Central

Blednov, Y.A.; Harris, R.A.

2009-01-01

The vanilloid receptor TRPV1 is activated by ethanol and this may be important for some of the central and peripheral actions of ethanol. To determine if this receptor has a role in ethanol-mediated behaviors, we studied null mutant mice in which the Trpv1 gene was deleted. Mice lacking this gene showed significantly higher preference for ethanol and consumed more ethanol in a two-bottle choice test as compared with wild type littermates. Null mutant mice showed shorter duration of loss of righting reflex induced by low doses of ethanol (3.2 and 3.4 g/kg) and faster recovery from motor incoordination induced by ethanol (2 g/kg). However, there were no differences between null mutant and wild type mice in severity of ethanol-induced acute withdrawal (4 g/kg) or conditioned taste aversion to ethanol (2.5 g/kg). Two behavioral phenotypes (decreased sensitivity to ethanol-induced sedation and faster recovery from ethanol-induced motor incoordination) seen in null mutant mice were reproduced in wild type mice by injection of a TRPV1 antagonist, capsazepine (10 mg/kg). These two ethanol behaviors were changed in the opposite direction after injection of capsaicin, a selective TRPV1 agonist, in wild type mice. The studies provide the first evidence that TRPV1 is important for specific behavioral actions of ethanol. PMID:19705551

Brassinosteroid-Insensitive Dwarf Mutants of Arabidopsis Accumulate Brassinosteroids1

PubMed Central

Noguchi, Takahiro; Fujioka, Shozo; Choe, Sunghwa; Takatsuto, Suguru; Yoshida, Shigeo; Yuan, Heng; Feldmann, Kenneth A.; Tax, Frans E.

1999-01-01

Seven dwarf mutants resembling brassinosteroid (BR)-biosynthetic dwarfs were isolated that did not respond significantly to the application of exogenous BRs. Genetic and molecular analyses revealed that these were novel alleles of BRI1 (Brassinosteroid-Insensitive 1), which encodes a receptor kinase that may act as a receptor for BRs or be involved in downstream signaling. The results of morphological and molecular analyses indicated that these represent a range of alleles from weak to null. The endogenous BRs were examined from 5-week-old plants of a null allele (bri1-4) and two weak alleles (bri1-5 and bri1-6). Previous analysis of endogenous BRs in several BR-biosynthetic dwarf mutants revealed that active BRs are deficient in these mutants. However, bri1-4 plants accumulated very high levels of brassinolide, castasterone, and typhasterol (57-, 128-, and 33-fold higher, respectively, than those of wild-type plants). Weaker alleles (bri1-5 and bri1-6) also accumulated considerable levels of brassinolide, castasterone, and typhasterol, but less than the null allele (bri1-4). The levels of 6-deoxoBRs in bri1 mutants were comparable to that of wild type. The accumulation of biologically active BRs may result from the inability to utilize these active BRs, the inability to regulate BR biosynthesis in bri1 mutants, or both. Therefore, BRI1 is required for the homeostasis of endogenous BR levels. PMID:10557222

The Functionally Conserved Nucleoporins Nup124p from Fission Yeast and the Human Nup153 Mediate Nuclear Import and Activity of the Tf1 Retrotransposon and HIV-1 VprV⃞

PubMed Central

Varadarajan, Padmapriya; Mahalingam, Sundarasamy; Liu, Peiyun; Ng, Sarah Boon Hsi; Gandotra, Sheetal; Dorairajoo, Desmond Suresh Kumar; Balasundaram, David

2005-01-01

We report that the fission yeast nucleoporin Nup124p is required for the nuclear import of both, retrotransposon Tf1-Gag as well as the retroviral HIV-1 Vpr. Failure to import Tf1-Gag into the nucleus in a nup124 null mutant resulted in complete loss of Tf1 transposition. Similarly, nuclear import of HIV-1 Vpr was impaired in nup124 null mutant strains and cells became resistant to Vpr's cell-killing activity. On the basis of protein domain similarity, the human nucleoporin Nup153 was identified as a putative homolog of Nup124p. We demonstrate that in vitro–translated Nup124p and Nup153 coimmunoprecipitate Tf1-Gag or HIV-1 Vpr. Though full-length Nup153 was unable to complement the Tf1 transposition defect in a nup124 null mutant, we provide evidence that both nucleoporins share a unique N-terminal domain, Nup124pAA264–454 and Nup153AA448–634 that is absolutely essential for Tf1 transposition. Epigenetic overexpression of this domain in a wild-type (nup124+) background blocked Tf1 activity implying that sequences from Nup124p and the human Nup153 challenged the same pathway affecting Tf1 transposition. Our results establish a unique relationship between two analogous nucleoporins Nup124p and Nup153 wherein the function of a common domain in retrotransposition is conserved. PMID:15659641

Identification and characterization of a putative cyclic nucleotide-gated channel, CNG-1, in C. elegans.

PubMed

Cho, Suk-Woo; Cho, Jeong-Hoon; Song, Hyun-Ok; Park, Chul-Seung

2005-02-28

Cyclic nucleotide-gated (CNG) channels encoded by the tax-4 and tax-2 genes are required for chemosensing and thermosensing in the nematode C. elegans. We identified a gene in the C. elegans genome, which we designated cng-1, that is highly homologous to tax-4. Partial CNG-1 protein tagged with green fluorescent protein was expressed in several sensory neurons of the amphid. We created a deletion mutant of cng-1, cng-1 (jh111), to investigate its in vivo function. The mutant worms had no detectable abnormalities in terms of their basic behavior or morphology. Whereas tax-4 and tax-2 mutants failed to respond to water-soluble or volatile chemical attractants, the cng-1 null mutant exhibited normal chemotaxis to such chemicals and a tax-4;cng-1 double mutant had a similar phenotype to tax-4 single mutants. Interestingly, cng-1 and tax-4 had a synergistic effect on brood size.

Characterization of a New Pink-Fruited Tomato Mutant Results in the Identification of a Null Allele of the SlMYB12 Transcription Factor.

PubMed

Fernandez-Moreno, Josefina-Patricia; Tzfadia, Oren; Forment, Javier; Presa, Silvia; Rogachev, Ilana; Meir, Sagit; Orzaez, Diego; Aharoni, Aspah; Granell, Antonio

2016-07-01

The identification and characterization of new tomato (Solanum lycopersicum) mutants affected in fruit pigmentation and nutritional content can provide valuable insights into the underlying biology, as well as a source of new alleles for breeding programs. To date, all characterized pink-pigmented tomato fruit mutants appear to result from low SlMYB12 transcript levels in the fruit skin. Two new mutant lines displaying a pink fruit phenotype (pf1 and pf2) were characterized in this study. In the pf mutants, SlMYB12 transcripts accumulated to wild-type levels but exhibited the same truncation, which resulted in the absence of the essential MYB activation domain coding region. Allelism and complementation tests revealed that both pf mutants were allelic to the y locus and showed the same recessive null allele in homozygosis: Δy A set of molecular and metabolic effects, reminiscent of those observed in the Arabidopsis (Arabidopsis thaliana) myb11 myb12 myb111 triple mutant, were found in the tomato Δy mutants. To our knowledge, these have not been described previously, and our data support the idea of their being null mutants, in contrast to previously described transcriptional hypomorphic pink fruit lines. We detected a reduction in the expression of several flavonol glycosides and some associated glycosyl transferases. Transcriptome analysis further revealed that the effects of the pf mutations extended beyond the flavonoid pathway into the interface between primary and secondary metabolism. Finally, screening for Myb-binding sites in the candidate gene promoter sequences revealed that 141 of the 152 co-down-regulated genes may be direct targets of SlMYB12 regulation. © 2016 American Society of Plant Biologists. All Rights Reserved.

Loss- and Gain-of-Function Mutations in the F1-HAMP Region of the Escherichia coli Aerotaxis Transducer Aer

PubMed Central

del Carmen Burón-Barral, Maria; Gosink, Khoosheh K.; Parkinson, John S.

2006-01-01

The Escherichia coli Aer protein contains an N-terminal PAS domain that binds flavin adenine dinucleotide (FAD), senses aerotactic stimuli, and communicates with the output signaling domain. To explore the roles of the intervening F1 and HAMP segments in Aer signaling, we isolated plasmid-borne aerotaxis-defective mutations in a host strain lacking all chemoreceptors of the methyl-accepting chemotaxis protein (MCP) family. Under these conditions, Aer alone established the cell's run/tumble swimming pattern and modulated that behavior in response to oxygen gradients. We found two classes of Aer mutants: null and clockwise (CW) biased. Most mutant proteins exhibited the null phenotype: failure to elicit CW flagellar rotation, no aerosensing behavior in MCP-containing hosts, and no apparent FAD-binding ability. However, null mutants had low Aer expression levels caused by rapid degradation of apparently nonnative subunits. Their functional defects probably reflect the absence of a protein product. In contrast, CW-biased mutant proteins exhibited normal expression levels, wild-type FAD binding, and robust aerosensing behavior in MCP-containing hosts. The CW lesions evidently shift unstimulated Aer output to the CW signaling state but do not block the Aer input-output pathway. The distribution and properties of null and CW-biased mutations suggest that the Aer PAS domain may engage in two different interactions with HAMP and the HAMP-proximal signaling domain: one needed for Aer maturation and another for promoting CW output from the Aer signaling domain. Most aerotaxis-defective null mutations in these regions seemed to affect maturation only, indicating that these two interactions involve structurally distinct determinants. PMID:16672601

Interleukin-6 is an essential determinant of on-time parturition in the mouse.

PubMed

Robertson, Sarah A; Christiaens, Inge; Dorian, Camilla L; Zaragoza, Dean B; Care, Alison S; Banks, Anke M; Olson, David M

2010-08-01

IL-6 abundance in amniotic fluid and uterine tissues increases in late gestation or with infection-associated preterm labor. A role in regulation of labor onset is suggested by observations that IL-6 increases expression of genes controlling prostaglandin synthesis and signaling in isolated uterine cells, but whether IL-6 is essential for normal parturition is unknown. To evaluate the physiological role of IL-6 in parturition in mice, we investigated the effect of Il6 null mutation on the timing of parturition and expression of genes associated with uterine activation. Il6 null mutant mice delivered 24 h later than wild-type mice, although circulating progesterone fell similarly in both genotypes during the prepartal period. Il6 null mutant mice were also refractory to low doses of lipopolysaccharide sufficient to induce preterm delivery in wild-type mice. The characteristic late-gestation elevation in uterine expression of Oxtr mRNA encoding oxytocin receptor, and peripartal increases in Ptgfr and Ptgs2 mRNAs regulating prostaglandin synthesis and signaling were delayed by 24 h in Il6 null mutant mice. Conversely, Ptger4 mRNA encoding the prostaglandin E receptor-4 was abnormally elevated in late-gestation in Il6 null mutant mice. Administration of recombinant IL-6 from d 11.5 postcoitum until term restored the normal timing of delivery and normalized Ptger4 mRNA expression in late gestation. We conclude that IL-6 has a key role in controlling the progression of events culminating in parturition and that it acts downstream of luteolysis in the uterus to regulate genes involved in the prostaglandin-mediated uterine activation cascade.

Mutations associated with base excision repair deficiency and methylation-induced genotoxic stress

PubMed Central

Sobol, Robert W.; Watson, David E.; Nakamura, Jun; Yakes, F. Michael; Hou, Esther; Horton, Julie K.; Ladapo, Joseph; Van Houten, Bennett; Swenberg, James A.; Tindall, Kenneth R.; Samson, Leona D.; Wilson, Samuel H.

2002-01-01

The long-term effect of exposure to DNA alkylating agents is entwined with the cell's genetic capacity for DNA repair and appropriate DNA damage responses. A unique combination of environmental exposure and deficiency in these responses can lead to genomic instability; this “gene–environment interaction” paradigm is a theme for research on chronic disease etiology. In the present study, we used mouse embryonic fibroblasts with a gene deletion in the base excision repair (BER) enzymes DNA β-polymerase (β-pol) and alkyladenine DNA glycosylase (AAG), along with exposure to methyl methanesulfonate (MMS) to study mutagenesis as a function of a particular gene–environment interaction. The β-pol null cells, defective in BER, exhibit a modest increase in spontaneous mutagenesis compared with wild-type cells. MMS exposure increases mutant frequency in β-pol null cells, but not in isogenic wild-type cells; UV light exposure or N-methyl-N′-nitro-N-nitrosoguanidine exposure increases mutant frequency similarly in both cell lines. The MMS-induced increase in mutant frequency in β-pol null cells appears to be caused by DNA lesions that are AAG substrates, because overexpression of AAG in β-pol null cells eliminates the effect. In contrast, β-pol/AAG double null cells are slightly more mutable than the β-pol null cells after MMS exposure. These results illustrate that BER plays a role in protecting mouse embryonic fibroblast cells against methylation-induced mutations and characterize the effect of a particular combination of BER gene defect and environmental exposure. PMID:11983862

Differential Radiosensitivity Phenotypes of DNA-PKcs Mutations Affecting NHEJ and HRR Systems following Irradiation with Gamma-Rays or Very Low Fluences of Alpha Particles

PubMed Central

Little, John B.; Kato, Takamitsu A.; Shih, Hung-Ying; Xie, Xian-Jin; Wilson Jr., Paul F.; Brogan, John R.; Kurimasa, Akihiro; Chen, David J.; Bedford, Joel S.; Chen, Benjamin P. C.

2014-01-01

We have examined cell-cycle dependence of chromosomal aberration induction and cell killing after high or low dose-rate γ irradiation in cells bearing DNA-PKcs mutations in the S2056 cluster, the T2609 cluster, or the kinase domain. We also compared sister chromatid exchanges (SCE) production by very low fluences of α-particles in DNA-PKcs mutant cells, and in homologous recombination repair (HRR) mutant cells including Rad51C, Rad51D, and Fancg/xrcc9. Generally, chromosomal aberrations and cell killing by γ-rays were similarly affected by mutations in DNA-PKcs, and these mutant cells were more sensitive in G1 than in S/G2 phase. In G1-irradiated DNA-PKcs mutant cells, both chromosome- and chromatid-type breaks and exchanges were in excess than wild-type cells. For cells irradiated in late S/G2 phase, mutant cells showed very high yields of chromatid breaks compared to wild-type cells. Few exchanges were seen in DNA-PKcs-null, Ku80-null, or DNA-PKcs kinase dead mutants, but exchanges in excess were detected in the S2506 or T2609 cluster mutants. SCE induction by very low doses of α-particles is resulted from bystander effects in cells not traversed by α-particles. SCE seen in wild-type cells was completely abolished in Rad51C- or Rad51D-deficient cells, but near normal in Fancg/xrcc9 cells. In marked contrast, very high levels of SCEs were observed in DNA-PKcs-null, DNA-PKcs kinase-dead and Ku80-null mutants. SCE induction was also abolished in T2609 cluster mutant cells, but was only slightly reduced in the S2056 cluster mutant cells. Since both non-homologous end-joining (NHEJ) and HRR systems utilize initial DNA lesions as a substrate, these results suggest the possibility of a competitive interference phenomenon operating between NHEJ and at least the Rad51C/D components of HRR; the level of interaction between damaged DNA and a particular DNA-PK component may determine the level of interaction of such DNA with a relevant HRR component. PMID:24714417

The transcription factor SKN7 regulates conidiation, thermotolerance, apoptotic-like cell death and parasitism in the nematode endoparasitic fungus Hirsutella minnesotensis

PubMed Central

Hussain, Muzammil; Hamid, M. Imran; Wang, Niuniu; Bin, Lin; Xiang, Meichun; Liu, Xingzhong

2016-01-01

The transcription factor SKN7 is a highly conserved protein among fungi and was initially recognized as a response regulator that protects cells from oxidative stress and maintains cell wall integrity in yeast. Orthologs of SKN7 are extensively present in biocontrol agents of plant pathogens, but they had not been functionally characterized. Here, we identified and characterized the transcription factor SKN7 in the nematode endoparasitic fungus Hirsutella minnesotensis. Null mutant lacking HIM-SKN7 (HIM_03620), which was generated by a gene disruption strategy, demonstrated reduced conidiation, increased sensitivity to high temperature, hydrogen peroxide, mannitol and ethanol, and reduced fungal resistance to farnesol. However, over-expression mutant showed increased conidial production, thermotolerance and resistance to farnesol, suggesting that HIM-SKN7 regulates antiapoptotic-like cell death in H. minnesotensis. Moreover, the results showed that in null mutant, H. minnesotensis had decreased endoparasitic ability as compared to wild type and over-expression strain. During the infection process, the relative expression of the HIM-SKN7 gene was significantly induced in the wild type and over-expression strain. The results of the present study advance our understanding of the functions of the SKN7 gene in biocontrol agents, in particular, nematode endoparasitic fungi. PMID:27436205

The mitochondrial SIR2 related protein 2 (SIR2RP2) impacts Leishmania donovani growth and infectivity

PubMed Central

Mittal, Nimisha; Muthuswami, Rohini

2017-01-01

Background Leishmania donovani, a protozoan parasite is the major causative agent of visceral leishmaniasis. Increased toxicity and resistance to the existing repertoire of drugs has been reported. Hence, an urgent need exists for identifying newer drugs and drug targets. Previous reports have shown sirtuins (Silent Information Regulator) from kinetoplastids as promising drug targets. Leishmania species code for three SIR2 (Silent Information Regulator) related proteins. Here, we for the first time report the functional characterization of SIR2 related protein 2 (SIR2RP2) of L. donovani. Methodology Recombinant L. donovani SIR2RP2 was expressed in E. coli and purified. The enzymatic functions of SIR2RP2 were determined. The subcellular localization of LdSIR2RP2 was done by constructing C-terminal GFP-tagged full-length LdSIR2RP2. Deletion mutants of LdSIR2RP2 were generated in Leishmania by double targeted gene replacement methodology. These null mutants were tested for their proliferation, virulence, cell cycle defects, mitochondrial functioning and sensitivity to known SIR2 inhibitors. Conclusion Our data suggests that LdSIR2RP2 possesses NAD+-dependent ADP-ribosyltransferase activity. However, NAD+-dependent deacetylase and desuccinylase activities were not detected. The protein localises to the mitochondrion of the promastigotes. Gene deletion studies showed that ΔLdSIR2RP2 null mutants had restrictive growth phenotype associated with accumulation of cells in the G2/M phase and compromised mitochondrial functioning. The null mutants had attenuated infectivity. Deletion of LdSIR2RP2 resulted in increased sensitivity of the parasites to the known SIR2 inhibitors. The sirtuin inhibitors inhibited the ADP-ribosyltransferase activity of recombinant LdSIR2RP2. In conclusion, sirtuins could be used as potential new drug targets for visceral leishmaniasis. PMID:28493888

The dev Operon Regulates the Timing of Sporulation during Myxococcus xanthus Development.

PubMed

Rajagopalan, Ramya; Kroos, Lee

2017-05-15

Myxococcus xanthus undergoes multicellular development when starved. Thousands of rod-shaped cells coordinate their movements and aggregate into mounds in which cells differentiate into spores. Mutations in the dev operon impair development. The dev operon encompasses a clustered regularly interspaced short palindromic repeat-associated (CRISPR-Cas) system. Null mutations in devI , a small gene at the beginning of the dev operon, suppress the developmental defects caused by null mutations in the downstream devR and devS genes but failed to suppress defects caused by a small in-frame deletion in devT We provide evidence that the original mutant has a second-site mutation. We show that devT null mutants exhibit developmental defects indistinguishable from devR and devS null mutants, and a null mutation in devI suppresses the defects of a devT null mutation. The similarity of DevTRS proteins to components of the CRISPR-associated complex for antiviral defense (Cascade), together with our molecular characterization of dev mutants, support a model in which DevTRS form a Cascade-like subcomplex that negatively autoregulates dev transcript accumulation and prevents DevI overproduction that would strongly inhibit sporulation. Our results also suggest that DevI transiently inhibits sporulation when regulated normally. The mechanism of transient inhibition may involve MrpC, a key transcription factor, whose translation appears to be weakly inhibited by DevI. Finally, our characterization of a devI devS mutant indicates that very little exo transcript is required for sporulation, which is surprising since Exo proteins help form the polysaccharide spore coat. IMPORTANCE CRISPR-Cas systems typically function as adaptive immune systems in bacteria. The dev CRISPR-Cas system of M. xanthus has been proposed to prevent bacteriophage infection during development, but how dev controls sporulation has been elusive. Recent evidence supported a model in which DevR and DevS prevent overproduction of DevI, a predicted 40-residue inhibitor of sporulation. We provide genetic evidence that DevT functions together with DevR and DevS to prevent DevI overproduction. We also show that spores form about 6 h earlier in mutants lacking devI than in the wild type. Only a minority of natural isolates appear to have a functional dev promoter and devI , suggesting that a functional dev CRISPR-Cas system evolved recently in niches where delayed sporulation and/or protection from bacteriophage infection proved advantageous. Copyright © 2017 American Society for Microbiology.

The dev Operon Regulates the Timing of Sporulation during Myxococcus xanthus Development

PubMed Central

Rajagopalan, Ramya

2017-01-01

ABSTRACT Myxococcus xanthus undergoes multicellular development when starved. Thousands of rod-shaped cells coordinate their movements and aggregate into mounds in which cells differentiate into spores. Mutations in the dev operon impair development. The dev operon encompasses a clustered regularly interspaced short palindromic repeat-associated (CRISPR-Cas) system. Null mutations in devI, a small gene at the beginning of the dev operon, suppress the developmental defects caused by null mutations in the downstream devR and devS genes but failed to suppress defects caused by a small in-frame deletion in devT. We provide evidence that the original mutant has a second-site mutation. We show that devT null mutants exhibit developmental defects indistinguishable from devR and devS null mutants, and a null mutation in devI suppresses the defects of a devT null mutation. The similarity of DevTRS proteins to components of the CRISPR-associated complex for antiviral defense (Cascade), together with our molecular characterization of dev mutants, support a model in which DevTRS form a Cascade-like subcomplex that negatively autoregulates dev transcript accumulation and prevents DevI overproduction that would strongly inhibit sporulation. Our results also suggest that DevI transiently inhibits sporulation when regulated normally. The mechanism of transient inhibition may involve MrpC, a key transcription factor, whose translation appears to be weakly inhibited by DevI. Finally, our characterization of a devI devS mutant indicates that very little exo transcript is required for sporulation, which is surprising since Exo proteins help form the polysaccharide spore coat. IMPORTANCE CRISPR-Cas systems typically function as adaptive immune systems in bacteria. The dev CRISPR-Cas system of M. xanthus has been proposed to prevent bacteriophage infection during development, but how dev controls sporulation has been elusive. Recent evidence supported a model in which DevR and DevS prevent overproduction of DevI, a predicted 40-residue inhibitor of sporulation. We provide genetic evidence that DevT functions together with DevR and DevS to prevent DevI overproduction. We also show that spores form about 6 h earlier in mutants lacking devI than in the wild type. Only a minority of natural isolates appear to have a functional dev promoter and devI, suggesting that a functional dev CRISPR-Cas system evolved recently in niches where delayed sporulation and/or protection from bacteriophage infection proved advantageous. PMID:28264995

LKB1 promotes cell survival by modulating TIF-IA-mediated pre-ribosomal RNA synthesis under uridine downregulated conditions

PubMed Central

Liu, Xiuju; Huang, Henry; Wilkinson, Scott C.; Zhong, Diansheng; Khuri, Fadlo R.; Fu, Haian; Marcus, Adam; He, Yulong; Zhou, Wei

2016-01-01

We analyzed the mechanism underlying 5-aminoimidazole-4-carboxamide riboside (AICAR) mediated apoptosis in LKB1-null non-small cell lung cancer (NSCLC) cells. Metabolic profile analysis revealed depletion of the intracellular pyrimidine pool after AICAR treatment, but uridine was the only nucleotide precursor capable of rescuing this apoptosis, suggesting the involvement of RNA metabolism. Because half of RNA transcription in cancer is for pre-ribosomal RNA (rRNA) synthesis, which is suppressed by over 90% after AICAR treatment, we evaluated the role of TIF-IA-mediated rRNA synthesis. While the depletion of TIF-IA by RNAi alone promoted apoptosis in LKB1-null cells, the overexpression of a wild-type or a S636A TIF-IA mutant, but not a S636D mutant, attenuated AICAR-induced apoptosis. In LKB1-null H157 cells, pre-rRNA synthesis was not suppressed by AICAR when wild-type LKB1 was present, and cellular fractionation analysis indicated that TIF-IA quickly accumulated in the nucleus in the presence of a wild-type LKB1 but not a kinase-dead mutant. Furthermore, ectopic expression of LKB1 was capable of attenuating AICAR-induced death in AMPK-null cells. Because LKB1 promotes cell survival by modulating TIF-IA-mediated pre-rRNA synthesis, this discovery suggested that targeted depletion of uridine related metabolites may be exploited in the clinic to eliminate LKB1-null cancer cells. PMID:26506235

LKB1 promotes cell survival by modulating TIF-IA-mediated pre-ribosomal RNA synthesis under uridine downregulated conditions.

PubMed

Liu, Fakeng; Jin, Rui; Liu, Xiuju; Huang, Henry; Wilkinson, Scott C; Zhong, Diansheng; Khuri, Fadlo R; Fu, Haian; Marcus, Adam; He, Yulong; Zhou, Wei

2016-01-19

We analyzed the mechanism underlying 5-aminoimidazole-4-carboxamide riboside (AICAR) mediated apoptosis in LKB1-null non-small cell lung cancer (NSCLC) cells. Metabolic profile analysis revealed depletion of the intracellular pyrimidine pool after AICAR treatment, but uridine was the only nucleotide precursor capable of rescuing this apoptosis, suggesting the involvement of RNA metabolism. Because half of RNA transcription in cancer is for pre-ribosomal RNA (rRNA) synthesis, which is suppressed by over 90% after AICAR treatment, we evaluated the role of TIF-IA-mediated rRNA synthesis. While the depletion of TIF-IA by RNAi alone promoted apoptosis in LKB1-null cells, the overexpression of a wild-type or a S636A TIF-IA mutant, but not a S636D mutant, attenuated AICAR-induced apoptosis. In LKB1-null H157 cells, pre-rRNA synthesis was not suppressed by AICAR when wild-type LKB1 was present, and cellular fractionation analysis indicated that TIF-IA quickly accumulated in the nucleus in the presence of a wild-type LKB1 but not a kinase-dead mutant. Furthermore, ectopic expression of LKB1 was capable of attenuating AICAR-induced death in AMPK-null cells. Because LKB1 promotes cell survival by modulating TIF-IA-mediated pre-rRNA synthesis, this discovery suggested that targeted depletion of uridine related metabolites may be exploited in the clinic to eliminate LKB1-null cancer cells.

Retinoic acid from the meninges regulates cortical neuron generation

PubMed Central

Siegenthaler, Julie A.; Ashique, Amir M.; Zarbalis, Konstantinos; Patterson, Katelin P.; Hecht, Jonathan H.; Kane, Maureen A.; Folias, Alexandra E.; Choe, Youngshik; May, Scott R.; Kume, Tsutomu; Napoli, Joseph L.; Peterson, Andrew S.; Pleasure, Samuel J.

2009-01-01

Summary Extrinsic signals controlling generation of neocortical neurons during embryonic life have been difficult to identify. In this study we demonstrate that the dorsal forebrain meninges communicate with the adjacent radial glial endfeet and influence cortical development. We took advantage of Foxc1 mutant mice with defects in forebrain meningeal formation. Foxc1 dosage and loss of meninges correlated with a dramatic reduction in both neuron and intermediate progenitor production and elongation of the neuroepithelium. Several types of experiments demonstrate that retinoic acid (RA) is the key component of this secreted activity. In addition, Rdh10 and Raldh2 expressing cells in the dorsal meninges were either reduced or absent in the Foxc1 mutants and Rdh10 mutants had a cortical phenotype similar to the Foxc1-null mutants. Lastly, in utero RA treatment rescued the cortical phenotype in Foxc1 mutants. These results establish RA as a potent, meningeal-derived cue required for successful corticogenesis. PMID:19879845

Superoxide dismutases and glutaredoxins have a distinct role in the response of Candida albicans to oxidative stress generated by the chemical compounds menadione and diamide.

PubMed

Chaves, Guilherme Maranhão; da Silva, Walicyranison Plinio

2012-12-01

To cope with oxidative stress, Candida albicans possesses several enzymes involved in a number of biological processes, including superoxide dismutases (Sods) and glutaredoxins (Grxs). The resistance of C. albicans to reactive oxygen species is thought to act as a virulence factor. Genes such as SOD1 and GRX2, which encode for a Sod and Grx, respectively, in C. albicans are widely recognised to be important for pathogenesis. We generated a double mutant, Δgrx2/sod1, for both genes. This strain is very defective in hyphae formation and is susceptible to killing by neutrophils. When exposed to two compounds that generate reactive oxygen species, the double null mutant was susceptible to menadione and resistant to diamide. The reintegration of the SOD1 gene in the null mutant led to recovery in resistance to menadione, whereas reintegration of the GRX2 gene made the null mutant sensitive to diamide. Despite having two different roles in the responses to oxidative stress generated by chemical compounds, GRX2 and SOD1 are important for C. albicans pathogenesis because the double mutant Δgrx2/sod1 was very susceptible to neutrophil killing and was defective in hyphae formation in addition to having a lower virulence in an animal model of systemic infection.

New phenotypes generated by the G57R mutation of BUD23 in Saccharomyces cerevisiae.

PubMed

Lin, Jyun-Liang; Yu, Hui-Chia; Chao, Ju-Lan; Wang, Chung; Cheng, Ming-Yuan

2012-12-01

BUD23 in Saccharomyces cerevisiae encodes for a class I methyltransferase, and deletion of the gene results in slow growth and random budding phenotypes. Herein, two BUD23 mutants defective in methyltransferase activity were generated to investigate whether the phenotypes of the null mutant might be correlated with a loss in enzymatic activity. Expression at the physiological level of both D77A and G57R mutants was able to rescue the phenotypes of the bud23-null mutant. The result implied that the methyltransferase activity of the protein was not necessary for supporting normal growth and bud site selection of the cells. High-level expression of Bud23 (G57R), but not Bud23 or Bud23 (D77A), in BUD23 deletion cells failed to complement these phenotypes. However, just like Bud23, Bud23 (G57R) was localized in a DAPI-poor region in the nucleus. Distinct behaviour in Bud23 (G57R) could not be originated from a mislocalization of the protein. Over-expression of Bud23 (G57R) in null cells also produced changes in actin organization and additional septin mutant-like phenotypes. Therefore, the absence of Bud23, Bud23 (G57R) at a high level might affect the cell division of yeast cells through an as yet unidentified mechanism. Copyright © 2012 John Wiley & Sons, Ltd.

Csf2 null mutation alters placental gene expression and trophoblast glycogen cell and giant cell abundance in mice.

PubMed

Sferruzzi-Perri, Amanda N; Macpherson, Anne M; Roberts, Claire T; Robertson, Sarah A

2009-07-01

Genetic deficiency in granulocyte-macrophage colony-stimulating factor (CSF2, GM-CSF) results in altered placental structure in mice. To investigate the mechanism of action of CSF2 in placental morphogenesis, the placental gene expression and cell composition were examined in Csf2 null mutant and wild-type mice. Microarray and quantitative RT-PCR analyses on Embryonic Day (E) 13 placentae revealed that the Csf2 null mutation caused altered expression of 17 genes not previously known to be associated with placental development, including Mid1, Cd24a, Tnfrsf11b, and Wdfy1. Genes controlling trophoblast differentiation (Ascl2, Tcfeb, Itgav, and Socs3) were also differentially expressed. The CSF2 ligand and the CSF2 receptor alpha subunit were predominantly synthesized in the placental junctional zone. Altered placental structure in Csf2 null mice at E15 was characterized by an expanded junctional zone and by increased Cx31(+) glycogen cells and cyclin-dependent kinase inhibitor 1C (CDKN1C(+), P57(Kip2+)) giant cells, accompanied by elevated junctional zone transcription of genes controlling spongiotrophoblast and giant cell differentiation and secretory function (Ascl2, Hand1, Prl3d1, and Prl2c2). Granzyme genes implicated in tissue remodeling and potentially in trophoblast invasion (Gzmc, Gzme, and Gzmf) were downregulated in the junctional zone of Csf2 null mutant placentae. These data demonstrate aberrant placental gene expression in Csf2 null mutant mice that is associated with altered differentiation and/or functional maturation of junctional zone trophoblast lineages, glycogen cells, and giant cells. We conclude that CSF2 is a regulator of trophoblast differentiation and placental development, which potentially influences the functional capacity of the placenta to support optimal fetal growth in pregnancy.

Identification of Nucleolus-Associated Chromatin Domains Reveals a Role for the Nucleolus in 3D Organization of the A. thaliana Genome.

PubMed

Pontvianne, Frédéric; Carpentier, Marie-Christine; Durut, Nathalie; Pavlištová, Veronika; Jaške, Karin; Schořová, Šárka; Parrinello, Hugues; Rohmer, Marine; Pikaard, Craig S; Fojtová, Miloslava; Fajkus, Jiří; Sáez-Vásquez, Julio

2016-08-09

The nucleolus is the site of rRNA gene transcription, rRNA processing, and ribosome biogenesis. However, the nucleolus also plays additional roles in the cell. We isolated nucleoli using fluorescence-activated cell sorting (FACS) and identified nucleolus-associated chromatin domains (NADs) by deep sequencing, comparing wild-type plants and null mutants for the nucleolar protein NUCLEOLIN 1 (NUC1). NADs are primarily genomic regions with heterochromatic signatures and include transposable elements (TEs), sub-telomeric regions, and mostly inactive protein-coding genes. However, NADs also include active rRNA genes and the entire short arm of chromosome 4 adjacent to them. In nuc1 null mutants, which alter rRNA gene expression and overall nucleolar structure, NADs are altered, telomere association with the nucleolus is decreased, and telomeres become shorter. Collectively, our studies reveal roles for NUC1 and the nucleolus in the spatial organization of chromosomes as well as telomere maintenance. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

NanI Sialidase, CcpA, and CodY Work Together To Regulate Epsilon Toxin Production by Clostridium perfringens Type D Strain CN3718.

PubMed

Li, Jihong; Freedman, John C; McClane, Bruce A

2015-10-01

Clostridium perfringens type D strains are usually associated with diseases of livestock, and their virulence requires the production of epsilon toxin (ETX). We previously showed (J. Li, S. Sayeed, S. Robertson, J. Chen, and B. A. McClane, PLoS Pathog 7:e1002429, 2011, http://dx.doi.org/10.1371/journal.ppat.1002429) that BMC202, a nanI null mutant of type D strain CN3718, produces less ETX than wild-type CN3718 does. The current study proved that the lower ETX production by strain BMC202 is due to nanI gene disruption, since both genetic and physical (NanI or sialic acid) complementation increased ETX production by BMC202. Furthermore, a sialidase inhibitor that interfered with NanI activity also reduced ETX production by wild-type CN3718. The NanI effect on ETX production was shown to involve reductions in codY and ccpA gene transcription levels in BMC202 versus wild-type CN3718. Similar to CodY, CcpA was found to positively control ETX production. A double codY ccpA null mutant produced even less ETX than a codY or ccpA single null mutant. CcpA bound directly to sequences upstream of the etx or codY start codon, and bioinformatics identified putative CcpA-binding cre sites immediately upstream of both the codY and etx start codons, suggesting possible direct CcpA regulatory effects. A ccpA mutation also decreased codY transcription, suggesting that CcpA effects on ETX production can be both direct and indirect, including effects on codY transcription. Collectively, these results suggest that NanI, CcpA, and CodY work together to regulate ETX production, with NanI-generated sialic acid from the intestines possibly signaling type D strains to upregulate their ETX production and induce disease. Clostridium perfringens NanI was previously shown to increase ETX binding to, and cytotoxicity for, MDCK host cells. The current study demonstrates that NanI also regulates ETX production via increased transcription of genes encoding the CodY and CcpA global regulators. Results obtained using single ccpA or codY null mutants and a ccpA codY double null mutant showed that codY and ccpA regulate ETX production independently of one another but that ccpA also affects codY transcription. Electrophoretic mobility shift assays and bioinformatic analyses suggest that both CodY and CcpA may directly regulate etx transcription. Collectively, results of this study suggest that sialic acid generated by NanI from intestinal sources signals ETX-producing C. perfringens strains, via CcpA and CodY, to upregulate ETX production and cause disease. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Mutations reducing replication from R-loops suppress the defects of growth, chromosome segregation and DNA supercoiling in cells lacking topoisomerase I and RNase HI activity.

PubMed

Usongo, Valentine; Martel, Makisha; Balleydier, Aurélien; Drolet, Marc

2016-04-01

R-loop formation occurs when the nascent RNA hybridizes with the template DNA strand behind the RNA polymerase. R-loops affect a wide range of cellular processes and their use as origins of replication was the first function attributed to them. In Escherichia coli, R-loop formation is promoted by the ATP-dependent negative supercoiling activity of gyrase (gyrA and gyrB) and is inhibited by topoisomerase (topo) I (topA) relaxing transcription-induced negative supercoiling. RNase HI (rnhA) degrades the RNA moiety of R-loops. The depletion of RNase HI activity in topA null mutants was previously shown to lead to extensive DNA relaxation, due to DNA gyrase inhibition, and to severe growth and chromosome segregation defects that were partially corrected by overproducing topo III (topB). Here, DNA gyrase assays in crude cell extracts showed that the ATP-dependent activity (supercoiling) of gyrase but not its ATP-independent activity (relaxation) was inhibited in topA null cells lacking RNase HI. To characterize the cellular event(s) triggered by the absence of RNase HI, we performed a genetic screen for suppressors of the growth defect of topA rnhA null cells. Suppressors affecting genes in replication (holC2::aph and dnaT18::aph) nucleotide metabolism (dcd49::aph), RNA degradation (rne59::aph) and fimbriae synthesis (fimD22::aph) were found to reduce replication from R-loops and to restore supercoiling, thus pointing to a correlation between R-loop-dependent replication in topA rnhA mutants and the inhibition of gyrase activity and growth. Interestingly, the position of fimD on the E. coli chromosome corresponds to the site of one of the five main putative origins of replication from R-loops in rnhA null cells recently identified by next-generation sequencing, thus suggesting that the fimD22::aph mutation inactivated one of these origins. Furthermore, we show that topo III overproduction is unable to complement the growth defect of topA rnhA null mutants at low temperatures that stabilizes hyper-negatively supercoiled DNA. Copyright © 2016 Elsevier B.V. All rights reserved.

GRANULOCYTE INFILTRATION AND EXPRESSION OF THE PRO-ANGIOGENIC BV8 PROTEIN IN EXPERIMENTAL EL4 AND LEWIS LUNG CARCINOMA TUMORS.

PubMed

Jiang, Kan; Kwak, Hyeongil; Tosato, Giovanna

2013-01-18

Although Vascular Endothelial Growth Factor (VEGF)-targeted therapies have shown efficacy in the treatment of certain advanced cancers, benefits to patients have been modest, which is attributed to tumor resistance to VEGF neutralization. Recent efforts to identify new targets to inhibit tumor angiogenesis have identified Bv8 (prokineticin 2), a myeloid cell-derived protein that promotes endothelial cell growth and tumor angiogenesis, but many mechanistic aspects of the pro-tumorigenic function of Bv8 are unclear. Here we demonstrate that CD11b+, Ly6C+, Ly6G+ granulocytes are the predominant cell source of Bv8 expression in bone marrow, spleen and in tumor tissues. Using granulocyte-deficient Growth factor independence-1 (Gfi1)-null mutant mice and normal littermates, we found that EL4 lymphoma tumors grow significantly larger in the granulocyte and Bv8-deficient mutant mice in comparison to the normal mice that display abundant tumor-associated granulocytes and Bv8 expression. Conversely, Lewis lung carcinoma (LLC-1) tumors grew to a significantly greater size in the normal mice in comparison to the Gfi1-null mice, but normal granulocyte tumor infiltration was modest. Quantitative analysis of tissue vascularization showed that EL4 and LLC-1 tumors from normal and Gfi1-mutant mice are similarly vascularized. These results confirm the critical contribution of the tumor microenvironment in determining the rate of tumor progression independently of tumor angiogenesis, and reveal some of the complexities of granulocyte and Bv8 functions in modulating tumor growth.

GRANULOCYTE INFILTRATION AND EXPRESSION OF THE PRO-ANGIOGENIC BV8 PROTEIN IN EXPERIMENTAL EL4 AND LEWIS LUNG CARCINOMA TUMORS

PubMed Central

Jiang, Kan; Kwak, Hyeongil; Tosato, Giovanna

2014-01-01

Although Vascular Endothelial Growth Factor (VEGF)-targeted therapies have shown efficacy in the treatment of certain advanced cancers, benefits to patients have been modest, which is attributed to tumor resistance to VEGF neutralization. Recent efforts to identify new targets to inhibit tumor angiogenesis have identified Bv8 (prokineticin 2), a myeloid cell-derived protein that promotes endothelial cell growth and tumor angiogenesis, but many mechanistic aspects of the pro-tumorigenic function of Bv8 are unclear. Here we demonstrate that CD11b+, Ly6C+, Ly6G+ granulocytes are the predominant cell source of Bv8 expression in bone marrow, spleen and in tumor tissues. Using granulocyte-deficient Growth factor independence-1 (Gfi1)-null mutant mice and normal littermates, we found that EL4 lymphoma tumors grow significantly larger in the granulocyte and Bv8-deficient mutant mice in comparison to the normal mice that display abundant tumor-associated granulocytes and Bv8 expression. Conversely, Lewis lung carcinoma (LLC-1) tumors grew to a significantly greater size in the normal mice in comparison to the Gfi1-null mice, but normal granulocyte tumor infiltration was modest. Quantitative analysis of tissue vascularization showed that EL4 and LLC-1 tumors from normal and Gfi1-mutant mice are similarly vascularized. These results confirm the critical contribution of the tumor microenvironment in determining the rate of tumor progression independently of tumor angiogenesis, and reveal some of the complexities of granulocyte and Bv8 functions in modulating tumor growth. PMID:25493215

Increased prevalence of mutant null alleles that cause hereditary fructose intolerance in the American population.

PubMed

Coffee, Erin M; Yerkes, Laura; Ewen, Elizabeth P; Zee, Tiffany; Tolan, Dean R

2010-02-01

Mutations in the aldolase B gene (ALDOB) impairing enzyme activity toward fructose-1-phosphate cleavage cause hereditary fructose intolerance (HFI). Diagnosis of the disease is possible by identifying known mutant ALDOB alleles in suspected patients; however, the frequencies of mutant alleles can differ by population. Here, 153 American HFI patients with 268 independent alleles were analyzed to identify the prevalence of seven known HFI-causing alleles (A149P, A174D, N334K, Delta4E4, R59Op, A337V, and L256P) in this population. Allele-specific oligonucleotide hybridization analysis was performed on polymerase chain reaction (PCR)-amplified genomic DNA from these patients. In the American population, the missense mutations A149P and A174D are the two most common alleles, with frequencies of 44% and 9%, respectively. In addition, the nonsense mutations Delta4E4 and R59Op are the next most common alleles, with each having a frequency of 4%. Together, the frequencies of all seven alleles make up 65% of HFI-causing alleles in this population. Worldwide, these same alleles make up 82% of HFI-causing mutations. This difference indicates that screening for common HFI alleles is more difficult in the American population. Nevertheless, a genetic screen for diagnosing HFI in America can be improved by including all seven alleles studied here. Lastly, identification of HFI patients presenting with classic symptoms and who have homozygous null genotypes indicates that aldolase B is not required for proper development or metabolic maintenance.

Cloning of soluble alkaline phosphatase cDNA and molecular basis of the polymorphic nature in alkaline phosphatase isozymes of Bombyx mori midgut.

PubMed

Itoh, M; Kanamori, Y; Takao, M; Eguchi, M

1999-02-01

A cDNA coding for soluble type alkaline phosphatase (sALP) of Bombyx mori was isolated. Deduced amino acid sequence showed high identities to various ALPs and partial similarities to ATPase of Manduca sexta. Using this cDNA sequence as a probe, the molecular basis of electrophoretic polymorphism in sALP and membrane-bound type ALP (mALP) was studied. As for mALP, the result suggested that post-translational modification was important for the proteins to express activity and to represent their extensive polymorphic nature, whereas the magnitude of activities was mainly regulated by transcription. On the other hand, sALP zymogram showed poor polymorphism, but one exception was the null mutant, in which the sALP gene was largely lost. Interestingly, the sALP gene was shown to be transcribed into two mRNAs of different sizes, 2.0 and 2.4 Kb. In addition to the null mutant of sALP, we found a null mutant for mALP. Both of these mutants seem phenotypically silent, suggesting that the functional differentiation between these isozymes is not perfect, so that they can still work mutually and complement each other as an indispensable enzyme for B. mori.

HPV-18 E6 mutants reveal p53 modulation of viral DNA amplification in organotypic cultures

PubMed Central

Kho, Eun-Young; Wang, Hsu-Kun; Banerjee, N. Sanjib; Broker, Thomas R.; Chow, Louise T.

2013-01-01

Human papillomaviruses (HPVs) amplify in differentiated strata of a squamous epithelium. The HPV E7 protein destabilizes the p130/retinoblastoma susceptibility protein family of tumor suppressors and reactivates S-phase reentry, thereby facilitating viral DNA amplification. The high-risk HPV E6 protein destabilizes the p53 tumor suppressor and many other host proteins. However, the critical E6 targets relevant to viral DNA amplification have not been identified, because functionally significant E6 mutants are not stably maintained in transfected cells. Using Cre-loxP recombination, which efficiently generates HPV genomic plasmids in transfected primary human keratinocytes, we have recapitulated a highly productive infection of HPV-18 in organotypic epithelial cultures. By using this system, we now report the characterization of four HPV-18 E6 mutations. An E6 null mutant accumulated high levels of p53 and amplified very poorly. p53 siRNA or ectopic WT E6 partially restored amplification, whereas three missense E6 mutations that did not effectively destabilize p53 complemented the null mutant poorly. Unexpectedly, in cis, two of the missense mutants amplified, albeit to a lower extent than the WT and only in cells with undetectable p53. These observations and others implicate p53 and additional host proteins in regulating viral DNA amplification and also suggest an inhibitory effect of E6 overexpression. We show that high levels of viral DNA amplification are critical for late protein expression and report several previously undescribed viral RNAs, including bicistronic transcripts predicted to encode E5 and L2 or an alternative form of E1^E4 and L1. PMID:23572574

Analysis of the presence of cell proliferation-related molecules in the Tgf-β3 null mutant mouse palate reveals misexpression of EGF and Msx-1.

PubMed

del Río, A; Barrio, M C; Murillo, J; Maldonado, E; López-Gordillo, Y; Martínez-Sanz, E; Martínez, M L; Martínez-Álvarez, C

2011-01-01

The Tgf-β(3) null mutant mouse palate presents several cellular anomalies that lead to the appearance of cleft palate. One of them concerns the cell proliferation of both the palatal medial edge epithelium and mesenchyme. In this work, our aim was to determine whether there was any variation in the presence/distribution of several cell proliferation-related molecules that could be responsible for the cell proliferation defects observed in these palates. Our results showed no difference in the presence of EGF-R, PDGF-A, TGF-β(2), Bmp-2, and Bmp-4, and differences were minimal for FGF-10 and Shh. However, the expression of EGF and Msx-1 changed substantially. The shift of the EGF protein expression was the one that most correlated with that of cell proliferation. This molecule is regulated by TGF-β(3), and experiments blocking its activity in culture suggest that EGF misexpression in the Tgf-β(3) null mutant mouse palate plays a role in the cell proliferation defect observed. Copyright © 2010 S. Karger AG, Basel.

Inactivation of Mre11 does not affect VSG gene duplication mediated by homologous recombination in Trypanosoma brucei.

PubMed

Robinson, Nicholas P; McCulloch, Richard; Conway, Colin; Browitt, Alison; Barry, J David

2002-07-19

We demonstrate, by gene deletion analysis, that Mre11 has a critical role in maintaining genomic integrity in Trypanosoma brucei. mre11(-/-) null mutant strains exhibited retarded growth but no delay or disruption of cell cycle progression. They showed also a weak hyporecombination phenotype and the accumulation of gross chromosomal rearrangements, which did not involve sequence translocation, telomere loss, or formation of new telomeres. The trypanosome mre11(-/-) strains were hypersensitive to phleomycin, a mutagen causing DNA double strand breaks (DSBs) but, in contrast to mre11(-/-) null mutants in other organisms and T. brucei rad51(-/-) null mutants, displayed no hypersensitivity to methyl methanesulfonate, which causes point mutations and DSBs. Mre11 therefore is important for the repair of chromosomal damage and DSBs in trypanosomes, although in this organism the intersection of repair pathways appears to differ from that in other organisms. Mre11 inactivation appears not to affect VSG gene switching during antigenic variation of a laboratory strain, which is perhaps surprising given the importance of homologous recombination during this process.

Stat1-independent regulation of gene expression in response to IFN-γ

PubMed Central

Ramana, Chilakamarti V.; Gil, M. Pilar; Han, Yulong; Ransohoff, Richard M.; Schreiber, Robert D.; Stark, George R.

2001-01-01

Although Stat1 is essential for cells to respond fully to IFN-γ, there is substantial evidence that, in the absence of Stat1, IFN-γ can still regulate the expression of some genes, induce an antiviral state and affect cell growth. We have now identified many genes that are regulated by IFN-γ in serum-starved Stat1-null mouse fibroblasts. The proteins induced by IFN-γ in Stat1-null cells can account for the substantial biological responses that remain. Some genes are induced in both wild-type and Stat1-null cells and thus are truly Stat1-independent. Others are subject to more complex regulation in response to IFN-γ, repressed by Stat1 in wild-type cells and activated in Stat1-null cells. Many genes induced by IFN-γ in Stat1-null fibroblasts also are induced by platelet-derived growth factor in wild-type cells and thus are likely to be involved in cell proliferation. In mouse cells expressing the docking site mutant Y440F of human IFN-γ receptor subunit 1, the mouse Stat1 is not phosphorylated in response to human IFN-γ, but c-myc and c-jun are still induced, showing that the Stat1 docking site is not required for Stat1-independent signaling. PMID:11390994

Evidence for complete epistasis of null mutations in murine Fanconi anemia genes Fanca and Fancg.

PubMed

van de Vrugt, Henri J; Koomen, Mireille; Bakker, Sietske; Berns, Mariska A D; Cheng, Ngan Ching; van der Valk, Martin A; de Vries, Yne; Rooimans, Martin A; Oostra, Anneke B; Hoatlin, Maureen E; Te Riele, Hein; Joenje, Hans; Arwert, Fré

2011-12-10

Fanconi anemia (FA) is a heritable disease characterized by bone marrow failure, congenital abnormalities, and cancer predisposition. The 15 identified FA genes operate in a molecular pathway to preserve genomic integrity. Within this pathway the FA core complex operates as an ubiquitin ligase that activates the complex of FANCD2 and FANCI to coordinate DNA repair. The FA core complex is formed by at least 12 proteins. However, only the FANCL subunit displays ubiquitin ligase activity. FANCA and FANCG are members of the FA core complex for which no other functions have been described than to participate in protein interactions. In this study we generated mice with combined null alleles for Fanca and Fancg to identify extended functions for these genes by characterizing the double mutant mice and cells. Double mutant a(-/-)/g(-/-) mice were born at near Mendelian frequencies without apparent developmental abnormalities. Histological analysis of a(-/-)/g(-/-) mice revealed a Leydig cell hyperplasia and frequent vacuolization of Sertoli cells in testes, while ovaries were depleted from developing follicles and displayed an interstitial cell hyperplasia. These gonadal aberrations were associated with a compromised fertility of a(-/-)/g(-/-) males and females. During the first year of life a(-/-)/g(-/-) did not develop malignancies or bone marrow failure. At the cellular level a(-/-)/g(-/-), Fanca(-/-), and Fancg(-/-) cells proved equally compromised in DNA crosslink and homology-directed repair. Overall the phenotype of a(-/-)/g(-/-) double knockout mice and cells appeared highly similar to the phenotype of Fanca or Fancg single knockouts. The lack of an augmented phenotype suggest that null mutations in Fanca or Fancg are fully epistatic, making additional important functions outside of the FA core complex highly unlikely. 2011 Elsevier B.V. All rights reserved.

A mutant of the Arabidopsis thaliana Toc159 gene accumulates reduced levels of linolenic acid and monogalactosyldiacylglycerol

USDA-ARS?s Scientific Manuscript database

Previous studies have shown that a null mutant of Arabidopsis that lacks Toc159 receptor is impaired in chloroplast biogenesis and incapable of importing photosynthetic proteins. The mutant is referred to as plastid protein import 2 or ppi2, and has an albino phenotype. In this study, we measured ...

Identification of two new genes, mukE and mukF, involved in chromosome partitioning in Escherichia coli.

PubMed

Yamanaka, K; Ogura, T; Niki, H; Hiraga, S

1996-02-25

We have previously reported that the MukB protein is essential for chromosome partitioning in Escherichia coli and that mukB mutants produce anucleate cells and are temperature-sensitive for colony formation. The mukB gene maps at 21 min on the E. coli chromosome and smtA-mukF-mukE-mukB genes might comprise an operon, which is transcribed in a clockwise direction. Here, we report that mukF and mukE null mutants are both temperature-sensitive for colony formation and produce anucleate cells even at the permissive temperature. These phenotypes are the same as those observed in the mukB null mutant. The primary sequence of MukF includes a leucine zipper structure and an acidic domain. Mutational analysis revealed that both are required for MukF function. When the MukF protein was overproduced in the wild-type strain, anucleate cells were produced. In contrast, overproduction of either MukE or MukB did not cause the defect. In null mutants for the mukF, mukE, and mukB genes, the synchronous initiation of chromosome replication was not affected. The mini-F plasmid was as stably maintained in these mutants as in the wild-type strain. These results indicate that the MukF, MukE, and MukB proteins are involved in the chromosome partitioning steps, but are not required for mini-F plasmid partitioning.

Altered Function of the DnaJ Family Cochaperone DNJ-17 Modulates Locomotor Circuit Activity in a Caenorhabditis elegans Seizure Model.

PubMed

Takayanagi-Kiya, Seika; Jin, Yishi

2016-07-07

The highly conserved cochaperone DnaJ/Hsp40 family proteins are known to interact with molecular chaperone Hsp70, and can regulate many cellular processes including protein folding, translocation, and degradation. In studies of Caenorhabditis elegans locomotion mutants, we identified a gain-of-function (gf) mutation in dnj-17 closely linked to the widely used e156 null allele of C. elegans GAD (glutamic acid decarboxylase) unc-25 dnj-17 encodes a DnaJ protein orthologous to human DNAJA5. In C. elegans DNJ-17 is a cytosolic protein and is broadly expressed in many tissues. dnj-17(gf) causes a single amino acid substitution in a conserved domain, and behaves as a hypermorphic mutation. The effect of this dnj-17(gf) is most prominent in mutants lacking GABA synaptic transmission. In a seizure model caused by a mutation in the ionotropic acetylcholine receptor acr-2(gf), dnj-17(gf) exacerbates the convulsion phenotype in conjunction with absence of GABA. Null mutants of dnj-17 show mild resistance to aldicarb, while dnj-17(gf) is hypersensitive. These results highlight the importance of DnaJ proteins in regulation of C. elegans locomotor circuit, and provide insights into the in vivo roles of DnaJ proteins in humans. Copyright © 2016 Takayanagi-Kiya and Jin.

Expression and function of FGF10 in mammalian inner ear development

NASA Technical Reports Server (NTRS)

Pauley, Sarah; Wright, Tracy J.; Pirvola, Ulla; Ornitz, David; Beisel, Kirk; Fritzsch, Bernd

2003-01-01

We have investigated the expression of FGF10 during ear development and the effect of an FGF10 null mutation on ear development. Our in situ hybridization data reveal expression of FGF10 in all three canal crista sensory epithelia and the cochlea anlage as well as all sensory neurons at embryonic day 11.5 (E11.5). Older embryos (E18.5) displayed strong graded expression in all sensory epithelia. FGF10 null mutants show complete agenesis of the posterior canal crista and the posterior canal. The posterior canal sensory neurons form initially and project rather normally by E11.5, but they disappear within 2 days. FGF10 null mutants have no posterior canal system at E18.5. In addition, these mutants have deformations of the anterior and horizontal cristae, reduced formation of the anterior and horizontal canals, as well as altered position of the remaining sensory epithelia with respect to the utricle. Hair cells form but some have defects in their cilia formation. No defects were detected in the organ of Corti at the cellular level. Together these data suggest that FGF10 plays a major role in ear morphogenesis. Most of these data are consistent with earlier findings on a null mutation in FGFR2b, one of FGF10's main receptors. Copyright 2003 Wiley-Liss, Inc.

Paranodal permeability in `myelin mutants'

PubMed Central

Shroff, S.; Mierzwa, A.; Scherer, S.S.; Peles, E.; Arevalo, J.C.; Chao, M.V.; Rosenbluth, J.

2011-01-01

Fluorescent dextran tracers of varying sizes have been used to assess paranodal permeability in myelinated sciatic nerve fibers from control and three `myelin mutant' mice, Caspr-null, cst-null and shaking. We demonstrate that in all of these the paranode is permeable to small tracers (3kDa, 10kDa), which penetrate most fibers, and to larger tracers (40kDa, 70kDa), which penetrate far fewer fibers and move shorter distances over longer periods of time. Despite gross diminution in transverse bands in the Caspr-null and cst-null mice, the permeability of their paranodal junctions is equivalent to that in controls. Thus, deficiency of transverse bands in these mutants does not increase the permeability of their paranodal junctions to the dextrans we used, moving from the perinodal space through the paranode to the internodal periaxonal space. In addition, we show that the shaking mice, which have thinner myelin and shorter paranodes, show increased permeability to the same tracers despite the presence of transverse bands. We conclude that the extent of penetration of these tracers does not depend on the presence or absence of transverse bands but does depend on the length of the paranode and, in turn, on the length of `pathway 3', the helical extracellular pathway that passes through the paranode parallel to the lateral edge of the myelin sheath. PMID:21618613

Coenzyme Q supplementation or over-expression of the yeast Coq8 putative kinase stabilizes multi-subunit Coq polypeptide complexes in yeast coq null mutants.

PubMed

He, Cuiwen H; Xie, Letian X; Allan, Christopher M; Tran, Uyenphuong C; Clarke, Catherine F

2014-04-04

Coenzyme Q biosynthesis in yeast requires a multi-subunit Coq polypeptide complex. Deletion of any one of the COQ genes leads to respiratory deficiency and decreased levels of the Coq4, Coq6, Coq7, and Coq9 polypeptides, suggesting that their association in a high molecular mass complex is required for stability. Over-expression of the putative Coq8 kinase in certain coq null mutants restores steady-state levels of the sensitive Coq polypeptides and promotes the synthesis of late-stage Q-intermediates. Here we show that over-expression of Coq8 in yeast coq null mutants profoundly affects the association of several of the Coq polypeptides in high molecular mass complexes, as assayed by separation of digitonin extracts of mitochondria by two-dimensional blue-native/SDS PAGE. The Coq4 polypeptide persists at high molecular mass with over-expression of Coq8 in coq3, coq5, coq6, coq7, coq9, and coq10 mutants, indicating that Coq4 is a central organizer of the Coq complex. Supplementation with exogenous Q6 increased the steady-state levels of Coq4, Coq7, and Coq9, and several other mitochondrial polypeptides in select coq null mutants, and also promoted the formation of late-stage Q-intermediates. Q supplementation may stabilize this complex by interacting with one or more of the Coq polypeptides. The stabilizing effects of exogenously added Q6 or over-expression of Coq8 depend on Coq1 and Coq2 production of a polyisoprenyl intermediate. Based on the observed interdependence of the Coq polypeptides, the effect of exogenous Q6, and the requirement for an endogenously produced polyisoprenyl intermediate, we propose a new model for the Q-biosynthetic complex, termed the CoQ-synthome. Copyright © 2014 Elsevier B.V. All rights reserved.

Coenzyme Q supplementation or over-expression of the yeast Coq8 putative kinase stabilizes multi-subunit Coq polypeptide complexes in yeast coq null mutants*

PubMed Central

He, Cuiwen H.; Xie, Letian X.; Allan, Christopher M.; Tran, UyenPhuong C.; Clarke, Catherine F.

2014-01-01

Coenzyme Q biosynthesis in yeast requires a multi-subunit Coq polypeptide complex. Deletion of any one of the COQ genes leads to respiratory deficiency and decreased levels of the Coq4, Coq6, Coq7, and Coq9 polypeptides, suggesting that their association in a high molecular mass complex is required for stability. Over-expression of the putative Coq8 kinase in certain coq null mutants restores steady-state levels of the sensitive Coq polypeptides and promotes the synthesis of late-stage Q-intermediates. Here we show that over-expression of Coq8 in yeast coq null mutants profoundly affects the association of several of the Coq polypeptides in high molecular mass complexes, as assayed by separation of digitonin extracts of mitochondria by two-dimensional blue-native/SDS PAGE. The Coq4 polypeptide persists at high molecular mass with over-expression of Coq8 in coq3, coq5, coq6, coq7, coq9, and coq10 mutants, indicating that Coq4 is a central organizer of the Coq complex. Supplementation with exogenous Q6 increased the steady-state levels of Coq4, Coq7, Coq9, and several other mitochondrial polypeptides in select coq null mutants, and also promoted the formation of late-stage Q-intermediates. Q supplementation may stabilize this complex by interacting with one or more of the Coq polypeptides. The stabilizing effects of exogenously added Q6 or over-expression of Coq8 depend on Coq1 and Coq2 production of a polyisoprenyl intermediate. Based on the observed interdependence of the Coq polypeptides, the effect of exogenous Q6, and the requirement for an endogenously produced polyisoprenyl intermediate, we propose a new model for the Q-biosynthetic complex, termed the CoQ-synthome. PMID:24406904

Autism-Relevant Social Abnormalities and Cognitive Deficits in Engrailed-2 Knockout Mice

PubMed Central

Brielmaier, Jennifer; Matteson, Paul G.; Silverman, Jill L.; Senerth, Julia M.; Kelly, Samantha; Genestine, Matthieu; Millonig, James H.

2012-01-01

ENGRAILED 2 (En2), a homeobox transcription factor, functions as a patterning gene in the early development and connectivity of rodent hindbrain and cerebellum, and regulates neurogenesis and development of monoaminergic pathways. To further understand the neurobiological functions of En2, we conducted neuroanatomical expression profiling of En2 wildtype mice. RTQPCR assays demonstrated that En2 is expressed in adult brain structures including the somatosensory cortex, hippocampus, striatum, thalamus, hypothalamus and brainstem. Human genetic studies indicate that EN2 is associated with autism. To determine the consequences of En2 mutations on mouse behaviors, including outcomes potentially relevant to autism, we conducted comprehensive phenotyping of social, communication, repetitive, and cognitive behaviors. En2 null mutants exhibited robust deficits in reciprocal social interactions as juveniles and adults, and absence of sociability in adults, replicated in two independent cohorts. Fear conditioning and water maze learning were impaired in En2 null mutants. High immobility in the forced swim test, reduced prepulse inhibition, mild motor coordination impairments and reduced grip strength were detected in En2 null mutants. No genotype differences were found on measures of ultrasonic vocalizations in social contexts, and no stereotyped or repetitive behaviors were observed. Developmental milestones, general health, olfactory abilities, exploratory locomotor activity, anxiety-like behaviors and pain responses did not differ across genotypes, indicating that the behavioral abnormalities detected in En2 null mutants were not attributable to physical or procedural confounds. Our findings provide new insight into the role of En2 in complex behaviors and suggest that disturbances in En2 signaling may contribute to neuropsychiatric disorders marked by social and cognitive deficits, including autism spectrum disorders. PMID:22829897

Genetic Screen in Drosophila Larvae Links ird1 Function to Toll Signaling in the Fat Body and Hemocyte Motility

PubMed Central

Schmid, Martin R.; Anderl, Ines; Vo, Hoa T. M.; Valanne, Susanna; Yang, Hairu; Kronhamn, Jesper; Rämet, Mika; Rusten, Tor Erik

2016-01-01

To understand how Toll signaling controls the activation of a cellular immune response in Drosophila blood cells (hemocytes), we carried out a genetic modifier screen, looking for deletions that suppress or enhance the mobilization of sessile hemocytes by the gain-of-function mutation Toll10b (Tl10b). Here we describe the results from chromosome arm 3R, where five regions strongly suppressed this phenotype. We identified the specific genes immune response deficient 1 (ird1), headcase (hdc) and possibly Rab23 as suppressors, and we studied the role of ird1 in more detail. An ird1 null mutant and a mutant that truncates the N-terminal kinase domain of the encoded Ird1 protein affected the Tl10b phenotype, unlike mutations that affect the C-terminal part of the protein. The ird1 null mutant suppressed mobilization of sessile hemocytes, but enhanced other Tl10b hemocyte phenotypes, like the formation of melanotic nodules and the increased number of circulating hemocytes. ird1 mutants also had blood cell phenotypes on their own. They lacked crystal cells and showed aberrant formation of lamellocytes. ird1 mutant plasmatocytes had a reduced ability to spread on an artificial substrate by forming protrusions, which may explain why they did not go into circulation in response to Toll signaling. The effect of the ird1 mutation depended mainly on ird1 expression in hemocytes, but ird1-dependent effects in other tissues may contribute. Specifically, the Toll receptor was translocated from the cell membrane to intracellular vesicles in the fat body of the ird1 mutant, and Toll signaling was activated in that tissue, partially explaining the Tl10b-like phenotype. As ird1 is otherwise known to control vesicular transport, we conclude that the vesicular transport system may be of particular importance during an immune response. PMID:27467079

Mutation of the ptsG Gene Results in Increased Production of Succinate in Fermentation of Glucose by Escherichia coli

PubMed Central

Chatterjee, Ranjini; Millard, Cynthia Sanville; Champion, Kathleen; Clark, David P.; Donnelly, Mark I.

2001-01-01

Escherichia coli NZN111 is blocked in the ability to grow fermentatively on glucose but gave rise spontaneously to a mutant that had this ability. The mutant carries out a balanced fermentation of glucose to give approximately 1 mol of succinate, 0.5 mol of acetate, and 0.5 mol of ethanol per mol of glucose. The causative mutation was mapped to the ptsG gene, which encodes the membrane-bound, glucose-specific permease of the phosphotransferase system, protein EIICBglc. Replacement of the chromosomal ptsG gene with an insertionally inactivated form also restored growth on glucose and resulted in the same distribution of fermentation products. The physiological characteristics of the spontaneous and null mutants were consistent with loss of function of the ptsG gene product; the mutants possessed greatly reduced glucose phosphotransferase activity and lacked normal glucose repression. Introduction of the null mutant into strains not blocked in the ability to ferment glucose also increased succinate production in those strains. This phenomenon was widespread, occurring in different lineages of E. coli, including E. coli B. PMID:11133439

Minimal Phenotype of Mice Homozygous for a Null Mutation in the Forkhead/Winged Helix Gene, Mf2

PubMed Central

Kume, Tsutomu; Deng, Keyu; Hogan, Brigid L. M.

2000-01-01

Mf2 (mesoderm/mesenchyme forkhead 2) encodes a forkhead/winged helix transcription factor expressed in numerous tissues of the mouse embryo, including paraxial mesoderm, somites, branchial arches, vibrissae, developing central nervous system, and developing kidney. We have generated mice homozygous for a null mutation in the Mf2 gene (Mf2lacZ) to examine its role during embryonic development. The lacZ allele also allows monitoring of Mf2 gene expression. Homozygous null mutants are viable and fertile and have no major developmental defects. Some mutants show renal abnormalities, including kidney hypoplasia and hydroureter, but the penetrance of this phenotype is only 40% or lower, depending on the genetic background. These data suggest that Mf2 can play a unique role in kidney development, but there is functional redundancy in this organ and other tissues with other forkhead/winged helix genes. PMID:10648626

Minimal phenotype of mice homozygous for a null mutation in the forkhead/winged helix gene, Mf2.

PubMed

Kume, T; Deng, K; Hogan, B L

2000-02-01

Mf2 (mesoderm/mesenchyme forkhead 2) encodes a forkhead/winged helix transcription factor expressed in numerous tissues of the mouse embryo, including paraxial mesoderm, somites, branchial arches, vibrissae, developing central nervous system, and developing kidney. We have generated mice homozygous for a null mutation in the Mf2 gene (Mf2(lacZ)) to examine its role during embryonic development. The lacZ allele also allows monitoring of Mf2 gene expression. Homozygous null mutants are viable and fertile and have no major developmental defects. Some mutants show renal abnormalities, including kidney hypoplasia and hydroureter, but the penetrance of this phenotype is only 40% or lower, depending on the genetic background. These data suggest that Mf2 can play a unique role in kidney development, but there is functional redundancy in this organ and other tissues with other forkhead/winged helix genes.

Lung tumors with distinct p53 mutations respond similarly to p53 targeted therapy but exhibit genotype-specific statin sensitivity

PubMed Central

Turrell, Frances K.; Kerr, Emma M.; Gao, Meiling; Thorpe, Hannah; Doherty, Gary J.; Cridge, Jake; Shorthouse, David; Speed, Alyson; Samarajiwa, Shamith; Hall, Benjamin A.; Griffiths, Meryl; Martins, Carla P.

2017-01-01

Lung adenocarcinoma accounts for ∼40% of lung cancers, the leading cause of cancer-related death worldwide, and current therapies provide only limited survival benefit. Approximately half of lung adenocarcinomas harbor mutations in TP53 (p53), making these mutants appealing targets for lung cancer therapy. As mutant p53 remains untargetable, mutant p53-dependent phenotypes represent alternative targeting opportunities, but the prevalence and therapeutic relevance of such effects (gain of function and dominant-negative activity) in lung adenocarcinoma are unclear. Through transcriptional and functional analysis of murine KrasG12D-p53null, -p53R172H (conformational), and -p53R270H (contact) mutant lung tumors, we identified genotype-independent and genotype-dependent therapeutic sensitivities. Unexpectedly, we found that wild-type p53 exerts a dominant tumor-suppressive effect on mutant tumors, as all genotypes were similarly sensitive to its restoration in vivo. These data show that the potential of p53 targeted therapies is comparable across all p53-deficient genotypes and may explain the high incidence of p53 loss of heterozygosity in mutant tumors. In contrast, mutant p53 gain of function and their associated vulnerabilities can vary according to mutation type. Notably, we identified a p53R270H-specific sensitivity to simvastatin in lung tumors, and the transcriptional signature that underlies this sensitivity was also present in human lung tumors, indicating that this therapeutic approach may be clinically relevant. PMID:28790158

Reduced alcohol consumption in mice lacking preprodynorphin.

PubMed Central

Blednov, Yuri A.; Walker, Danielle; Martinez, Marni; Harris., R. Adron

2007-01-01

Many studies suggest a role for endogenous opioid peptides and their receptors in regulation of ethanol intake. It is commonly accepted that the κ-opioid receptors and their endogenous ligands, dynorphins, produce a dysphoric state and therefore may be responsible for avoidance of alcohol. We used mutant mice lacking preprodynorphin in a variety of behavioral tests of alcohol actions. Null mutant female, but not male, mice showed significantly lower preference for alcohol and consumed lower amounts of alcohol in a two-bottle choice test as compared with wild-type littermates. In the same test, knockout mice of both sexes showed a strong reduction of preference for saccharin compared to control mice. In contrast, under conditions of limited (4 hours) access (light phase of the light/dark cycle), null mutant mice did not show any differences in consumption of saccharin but they showed significantly reduced intake of sucrose. To determine the possible cause for reduction of ethanol preference and intake, we studied other ethanol-related behaviors in mice lacking the preprodynorphin gene. There were no differences between null mutant and wild type mice in ethanol-induced loss of righting reflex, acute ethanol withdrawal, ethanol-induced conditioned place preference or conditioned taste aversion to ethanol. These results indicate that deletion of preprodynorphin leads to substantial reduction of alcohol intake in female mice, and suggest thath this is caused by decreased orosensory reward of alcohol (sweet taste and/or palatability). PMID:17307643

Reduced alcohol consumption in mice lacking preprodynorphin.

PubMed

Blednov, Yuri A; Walker, Danielle; Martinez, Marni; Harris, R Adron

2006-10-01

Many studies suggest a role for endogenous opioid peptides and their receptors in regulation of ethanol intake. It is commonly accepted that the kappa-opioid receptors and their endogenous ligands, dynorphins, produce a dysphoric state and therefore may be responsible for avoidance of alcohol. We used mutant mice lacking preprodynorphin in a variety of behavioral tests of alcohol actions. Null mutant female, but not male, mice showed significantly lower preference for alcohol and consumed lower amounts of alcohol in a two-bottle choice test as compared with wild-type littermates. In the same test, knockout mice of both sexes showed a strong reduction of preference for saccharin compared to control mice. In contrast, under conditions of limited (4 h) access (light phase of the light/dark cycle), null mutant mice did not show any differences in consumption of saccharin, but they showed significantly reduced intake of sucrose. To determine the possible cause for reduction of ethanol preference and intake, we studied other ethanol-related behaviors in mice lacking the preprodynorphin gene. There were no differences between null mutant and wild-type mice in ethanol-induced loss of righting reflex, acute ethanol withdrawal, ethanol-induced conditioned place preference, or conditioned taste aversion to ethanol. These results indicate that deletion of preprodynorphin leads to substantial reduction of alcohol intake in female mice, and suggest that this is caused by decreased orosensory reward of alcohol (sweet taste and/or palatability).

Generation of Esr1-Knockout Rats Using Zinc Finger Nuclease-Mediated Genome Editing

PubMed Central

Dhakal, Pramod; Kubota, Kaiyu; Chakraborty, Damayanti; Lei, Tianhua; Larson, Melissa A.; Wolfe, Michael W.; Roby, Katherine F.; Vivian, Jay L.

2014-01-01

Estrogens play pivotal roles in development and function of many organ systems, including the reproductive system. We have generated estrogen receptor 1 (Esr1)-knockout rats using zinc finger nuclease (ZFN) genome targeting. mRNAs encoding ZFNs targeted to exon 3 of Esr1 were microinjected into single-cell rat embryos and transferred to pseudopregnant recipients. Of 17 live births, 5 had biallelic and 1 had monoallelic Esr1 mutations. A founder with monoallelic mutations was backcrossed to a wild-type rat. Offspring possessed only wild-type Esr1 alleles or wild-type alleles and Esr1 alleles containing either 482 bp (Δ482) or 223 bp (Δ223) deletions, indicating mosaicism in the founder. These heterozygous mutants were bred for colony expansion, generation of homozygous mutants, and phenotypic characterization. The Δ482 Esr1 allele yielded altered transcript processing, including the absence of exon 3, aberrant splicing of exon 2 and 4, and a frameshift that generated premature stop codons located immediately after the codon for Thr157. ESR1 protein was not detected in homozygous Δ482 mutant uteri. ESR1 disruption affected sexually dimorphic postnatal growth patterns and serum levels of gonadotropins and sex steroid hormones. Both male and female Esr1-null rats were infertile. Esr1-null males had small testes with distended and dysplastic seminiferous tubules, whereas Esr1-null females possessed large polycystic ovaries, thread-like uteri, and poorly developed mammary glands. In addition, uteri of Esr1-null rats did not effectively respond to 17β-estradiol treatment, further demonstrating that the Δ482 Esr1 mutation created a null allele. This rat model provides a new experimental tool for investigating the pathophysiology of estrogen action. PMID:24506075

Generation of Esr1-knockout rats using zinc finger nuclease-mediated genome editing.

PubMed

Rumi, M A Karim; Dhakal, Pramod; Kubota, Kaiyu; Chakraborty, Damayanti; Lei, Tianhua; Larson, Melissa A; Wolfe, Michael W; Roby, Katherine F; Vivian, Jay L; Soares, Michael J

2014-05-01

Estrogens play pivotal roles in development and function of many organ systems, including the reproductive system. We have generated estrogen receptor 1 (Esr1)-knockout rats using zinc finger nuclease (ZFN) genome targeting. mRNAs encoding ZFNs targeted to exon 3 of Esr1 were microinjected into single-cell rat embryos and transferred to pseudopregnant recipients. Of 17 live births, 5 had biallelic and 1 had monoallelic Esr1 mutations. A founder with monoallelic mutations was backcrossed to a wild-type rat. Offspring possessed only wild-type Esr1 alleles or wild-type alleles and Esr1 alleles containing either 482 bp (Δ482) or 223 bp (Δ223) deletions, indicating mosaicism in the founder. These heterozygous mutants were bred for colony expansion, generation of homozygous mutants, and phenotypic characterization. The Δ482 Esr1 allele yielded altered transcript processing, including the absence of exon 3, aberrant splicing of exon 2 and 4, and a frameshift that generated premature stop codons located immediately after the codon for Thr157. ESR1 protein was not detected in homozygous Δ482 mutant uteri. ESR1 disruption affected sexually dimorphic postnatal growth patterns and serum levels of gonadotropins and sex steroid hormones. Both male and female Esr1-null rats were infertile. Esr1-null males had small testes with distended and dysplastic seminiferous tubules, whereas Esr1-null females possessed large polycystic ovaries, thread-like uteri, and poorly developed mammary glands. In addition, uteri of Esr1-null rats did not effectively respond to 17β-estradiol treatment, further demonstrating that the Δ482 Esr1 mutation created a null allele. This rat model provides a new experimental tool for investigating the pathophysiology of estrogen action.

Arabidopsis kinetochore null2 is an upstream component for centromeric histone H3 variant cenH3 deposition at centromeres.

PubMed

Lermontova, Inna; Kuhlmann, Markus; Friedel, Swetlana; Rutten, Twan; Heckmann, Stefan; Sandmann, Michael; Demidov, Dmitri; Schubert, Veit; Schubert, Ingo

2013-09-01

The centromeric histone H3 variant cenH3 is an essential centromeric protein required for assembly, maintenance, and proper function of kinetochores during mitosis and meiosis. We identified a kinetochore null2 (KNL2) homolog in Arabidopsis thaliana and uncovered features of its role in cenH3 loading at centromeres. We show that Arabidopsis KNL2 colocalizes with cenH3 and is associated with centromeres during all stages of the mitotic cell cycle, except from metaphase to mid-anaphase. KNL2 is regulated by the proteasome degradation pathway. The KNL2 promoter is mainly active in meristematic tissues, similar to the cenH3 promoter. A knockout mutant for KNL2 shows a reduced level of cenH3 expression and reduced amount of cenH3 protein at chromocenters of meristematic nuclei, anaphase bridges during mitosis, micronuclei in pollen tetrads, and 30% seed abortion. Moreover, knl2 mutant plants display reduced expression of suppressor of variegation 3-9 homologs2, 4, and 9 and reduced DNA methylation, suggesting an impact of KNL2 on the epigenetic environment for centromere maintenance.

Methionine biosynthesis is essential for infection in the rice blast fungus Magnaporthe oryzae.

PubMed

Saint-Macary, Marie Emmanuelle; Barbisan, Crystel; Gagey, Marie Josèphe; Frelin, Océane; Beffa, Roland; Lebrun, Marc Henri; Droux, Michel

2015-01-01

Methionine is a sulfur amino acid standing at the crossroads of several biosynthetic pathways. In fungi, the last step of methionine biosynthesis is catalyzed by a cobalamine-independent methionine synthase (Met6, EC 2.1.1.14). In the present work, we studied the role of Met6 in the infection process of the rice blast fungus, Magnaporthe oryzae. To this end MET6 null mutants were obtained by targeted gene replacement. On minimum medium, MET6 null mutants were auxotrophic for methionine. Even when grown in presence of excess methionine, these mutants displayed developmental defects, such as reduced mycelium pigmentation, aerial hypha formation and sporulation. They also displayed characteristic metabolic signatures such as increased levels of cysteine, cystathionine, homocysteine, S-adenosylmethionine, S-adenosylhomocysteine while methionine and glutathione levels remained unchanged. These metabolic perturbations were associated with the over-expression of MgCBS1 involved in the reversed transsulfuration pathway that metabolizes homocysteine into cysteine and MgSAM1 and MgSAHH1 involved in the methyl cycle. This suggests a physiological adaptation of M. oryzae to metabolic defects induced by the loss of Met6, in particular an increase in homocysteine levels. Pathogenicity assays showed that MET6 null mutants were non-pathogenic on both barley and rice leaves. These mutants were defective in appressorium-mediated penetration and invasive infectious growth. These pathogenicity defects were rescued by addition of exogenous methionine and S-methylmethionine. These results show that M. oryzae cannot assimilate sufficient methionine from plant tissues and must synthesize this amino acid de novo to fulfill its sulfur amino acid requirement during infection.

An essential role for IGF2 in cartilage development and glucose metabolism during postnatal long bone growth.

PubMed

Uchimura, Tomoya; Hollander, Judith M; Nakamura, Daisy S; Liu, Zhiyi; Rosen, Clifford J; Georgakoudi, Irene; Zeng, Li

2017-10-01

Postnatal bone growth involves a dramatic increase in length and girth. Intriguingly, this period of growth is independent of growth hormone and the underlying mechanism is poorly understood. Recently, an IGF2 mutation was identified in humans with early postnatal growth restriction. Here, we show that IGF2 is essential for longitudinal and appositional murine postnatal bone development, which involves proper timing of chondrocyte maturation and perichondrial cell differentiation and survival. Importantly, the Igf2 null mouse model does not represent a simple delay of growth but instead uncoordinated growth plate development. Furthermore, biochemical and two-photon imaging analyses identified elevated and imbalanced glucose metabolism in the Igf2 null mouse. Attenuation of glycolysis rescued the mutant phenotype of premature cartilage maturation, thereby indicating that IGF2 controls bone growth by regulating glucose metabolism in chondrocytes. This work links glucose metabolism with cartilage development and provides insight into the fundamental understanding of human growth abnormalities. © 2017. Published by The Company of Biologists Ltd.

Impairment of Infectivity and Immunoprotective Effect of a LYT1 Null Mutant of Trypanosoma cruzi▿

PubMed Central

Zago, M. Paola; Barrio, Alejandra B.; Cardozo, Rubén M.; Duffy, Tomás; Schijman, Alejandro G.; Basombrío, Miguel A.

2008-01-01

Trypanosoma cruzi infection of host cells is a complex process in which many proteins participate but only a few of these proteins have been identified experimentally. One parasite factor likely to be involved is the protein product of LYT1, a single-copy gene cloned, sequenced, and characterized by Manning-Cela et al. (Infect. Immun. 69:3916-3923, 2001). This gene was potentially associated with infectivity, since the deletion of both LYT1 alleles in the CL Brenner strain (the wild type [WT]) resulted in a null mutant T. cruzi clone (L16) that shows an attenuated phenotype in cell culture models. The aim of this work was to characterize the infective behavior of L16 in the insect vector and murine models. The infection of adult Swiss mice with 103 trypomastigotes of both clones revealed a significant reduction in infective behavior of L16, as shown by direct parasitemia, spleen index, and quantitation of tissue parasite burden, suggesting the loss of virulence in the null mutant clone. Although L16 blood counts were almost undetectable, blood-based PCRs indicated the presence of latent and persistent infection during all of the study period and epimastigotes were reisolated from hemocultures until 12 months postinfection. Nevertheless, virulence was not restored in L16 by serial passages in mice, and reisolated parasites lacking the LYT1 gene and bearing the antibiotic resistance genes revealed the stability of the genetic manipulation. Histopathological studies showed a strong diminution in the muscle inflammatory response triggered by L16 compared to that triggered by the WT group, consistent with a lower tissue parasite load. A strong protection against a virulent challenge in both L16- and WT-infected mice was observed; however, the immunizing infection by the genetically modified parasite was highly attenuated. PMID:17938222

WhiD and WhiB, homologous proteins required for different stages of sporulation in Streptomyces coelicolor A3(2).

PubMed

Molle, V; Palframan, W J; Findlay, K C; Buttner, M J

2000-03-01

The whiD locus, which is required for the differentiation of Streptomyces coelicolor aerial hyphae into mature spore chains, was localized by map-based cloning to the overlap between cosmids 6G4 and D63 of the minimal ordered library of Redenbach et al. (M. Redenbach et al., Mol. Microbiol. 21:77-96, 1996). Subcloning and sequencing showed that whiD encodes a homologue of WhiB, a protein required for the initiation of sporulation septation in S. coelicolor. WhiD and WhiB belong to a growing family of small (76- to 112-residue) proteins of unknown biochemical function in which four cysteines are absolutely conserved; all known members of this family are found in the actinomycetes. A constructed whiD null mutant showed reduced levels of sporulation, and those spores that did form were heat sensitive, lysed extensively, and were highly irregular in size, arising at least in part from irregularity in septum placement. The whiD null mutant showed extreme variation in spore cell wall deposition; most spores had uniformly thin (20- to 30-nm) walls, but spore chains were frequently observed in which there was irregular but very pronounced (up to 170 nm) cell wall thickening at the junctions between spores. whiD null mutant spores were frequently partitioned into irregular smaller units through the deposition of additional septa, which were often laid down in several different planes, very close to the spore poles. These "minicompartments" appeared to be devoid of chromosomal DNA. Two whiD promoters, whiDp1 and whiDp2, were identified, and their activities were analyzed during development of wild-type S. coelicolor on solid medium. Both promoters were developmentally regulated; whiDp1 and whiDp2 transcripts were detected transiently, approximately at the time when sporulation septa were observed in the aerial hyphae.

ςBldN, an Extracytoplasmic Function RNA Polymerase Sigma Factor Required for Aerial Mycelium Formation in Streptomyces coelicolor A3(2)

PubMed Central

Bibb, Maureen J.; Molle, Virginie; Buttner, Mark J.

2000-01-01

Sporulation mutants of Streptomyces coelicolor appear white because they are defective in the synthesis of the gray polyketide spore pigment, and such white (whi) mutants have been used to define 13 sporulation loci. whiN, one of five new whi loci identified in a recent screen of NTG (N-methyl-N′-nitro-N-nitrosoguanidine)-induced whi strains (N. J. Ryding et al., J. Bacteriol. 181:5419–5425, 1999), was defined by two mutants, R112 and R650. R650 produced frequent spores that were longer than those of the wild type. In contrast, R112 produced long, straight, undifferentiated hyphae, although rare spore chains were observed, sometimes showing highly irregular septum placement. Subcloning and sequencing showed that whiN encodes a member of the extracytoplasmic function subfamily of RNA polymerase sigma factors and that the sigma factor has an unusual N-terminal extension of approximately 86 residues that is not present in other sigma factors. A constructed whiN null mutant failed to form aerial mycelium (the “bald” phenotype) and, as a consequence, whiN was renamed bldN. This observation was not totally unexpected because, on some media, the R112 point mutant produced substantially less aerial mycelium than its parent, M145. The bldN null mutant did not fit simply into the extracellular signaling cascade proposed for S. coelicolor bld mutants. Expression of bldN was analyzed during colony development in wild-type and aerial mycelium-deficient bld strains. bldN was transcribed from a single promoter, bldNp. bldN transcription was developmentally regulated, commencing approximately at the time of aerial mycelium formation, and depended on bldG and bldH, but not on bldA, bldB, bldC, bldF, bldK, or bldJ or on bldN itself. Transcription from the p1 promoter of the response-regulator gene bldM depended on bldN in vivo, and the bldMp1 promoter was shown to be a direct biochemical target for ςBldN holoenzyme in vitro. PMID:10913095

Identification of new modes of Dd-STATa regulation of gene expression in Dictyostelium by in situ hybridisation.

PubMed

Shimada, Nao; Maeda, Mineko; Urushihara, Hideko; Kawata, Takefumi

2004-09-01

Signal Transducers and Activators of Transcription (STATs) are transcription factors which lie at the end of cytokine and growth signal transduction pathways. Dictyostelium Dd-STATa is a functional homologue of metazoan STATs. It is activated by cAMP and, at the slug stage, it translocates into the nuclei of the tip cells, which are a subset of the anterior, prestalk A (pstA) cells. Here we searched for novel Dd-STATa regulated genes by in situ hybridisation. A set of 54 cDNA clones whose gene expression patterns are known to be prestalk-specific (Maeda et al., 2003), were chosen as probes and we compared their expression patterns in parental and Dd-STATa-null strains. We identified 13 genes which are candidates for direct induction by Dd-STATa. In the parental strain, most of these genes are expressed in the cone shaped mass of pstAB cells which is located within the prestalk region. These cDNAs show little or no expression in the Dd-STATa-null strain. This contrasts markedly with the paradigmatic ecmB gene which is expressed in pstAB cells in parental cells, but which is expressed throughout the prestalk zone in the Dd-STATa-null strain. We also identified several genes which are normally expressed in pstA cells, or throughout the prestalk region, but whose expression is markedly down-regulated in the null mutant. Again, this contrasts with markers derived from the paradigmatic, ecmA gene which are expressed normally in the Dd-STATa-null strain. The identification of these novel genes provides valuable tools to investigate the role of Dd-STATa.

Prolonged Stationary-Phase Incubation Selects for lrp Mutations in Escherichia coli K-12

PubMed Central

Zinser, Erik R.; Kolter, Roberto

2000-01-01

Evolution by natural selection occurs in cultures of Escherichia coli maintained under carbon starvation stress. Mutants of increased fitness express a growth advantage in stationary phase (GASP) phenotype, enabling them to grow and displace the parent as the majority population. The first GASP mutation was identified as a loss-of-function allele of rpoS, encoding the stationary-phase global regulator, ςS (M. M. Zambrano, D. A. Siegele, M. A. Almirón, A. Tormo, and R. Kolter, Science 259:1757–1760, 1993). We now report that a second global regulator, Lrp, can also play a role in stationary-phase competition. We found that a mutant that took over an aged culture of an rpoS strain had acquired a GASP mutation in lrp. This GASP allele, lrp-1141, encodes a mutant protein lacking the critical glycine in the turn of the helix-turn-helix DNA-binding domain. The lrp-1141 allele behaves as a null mutation when in single copy and is dominant negative when overexpressed. Hence, the mutant protein appears to retain stability and the ability to dimerize but lacks DNA-binding activity. We also demonstrated that a lrp null allele generated by a transposon insertion has a fitness gain identical to that of the lrp-1141 allele, verifying that cells lacking Lrp activity have a competitive advantage during prolonged starvation. Finally, we tested by genetic analysis the hypothesis that the lrp-1141 GASP mutation confers a fitness gain by enhancing amino acid catabolism during carbon starvation. We found that while amino acid catabolism may play a role, it is not necessary for the lrp GASP phenotype, and hence the lrp GASP phenotype is due to more global physiological changes. PMID:10894750

Mixed Neurodevelopmental and Neurodegenerative Pathology in Nhe6-Null Mouse Model of Christianson Syndrome

PubMed Central

Xu, Meiyu; Ouyang, Qing; Gong, Jingyi; Pescosolido, Matthew F.; Mishra, Sasmita; Schmidt, Michael; Jones, Richard N.; Gamsiz Uzun, Ece D.; Lizarraga, Sofia B.

2017-01-01

Abstract Christianson syndrome (CS) is an X-linked disorder resulting from loss-of-function mutations in SLC9A6, which encodes the endosomal Na+/H+ exchanger 6 (NHE6). Symptoms include early developmental delay, seizures, intellectual disability, nonverbal status, autistic features, postnatal microcephaly, and progressive ataxia. Neuronal development is impaired in CS, involving defects in neuronal arborization and synaptogenesis, likely underlying diminished brain growth postnatally. In addition to neurodevelopmental defects, some reports have supported neurodegenerative pathology in CS with age. The objective of this study was to determine the nature of progressive changes in the postnatal brain in Nhe6-null mice. We examined the trajectories of brain growth and atrophy in mutant mice from birth until very old age (2 yr). We report trajectories of volume changes in the mutant that likely reflect both brain undergrowth as well as tissue loss. Reductions in volume are first apparent at 2 mo, particularly in the cerebellum, which demonstrates progressive loss of Purkinje cells (PCs). We report PC loss in two distinct Nhe6-null mouse models. More widespread reductions in tissue volumes, namely, in the hippocampus, striatum, and cortex, become apparent after 2 mo, largely reflecting delays in growth with more limited tissue losses with aging. Also, we identify pronounced glial responses, particularly in major fiber tracts such as the corpus callosum, where the density of activated astrocytes and microglia are substantially increased. The prominence of the glial response in axonal tracts suggests a primary axonopathy. Importantly, therefore, our data support both neurodevelopmental and degenerative mechanisms in the pathobiology of CS. PMID:29349289

The inability of Bacillus licheniformis perR mutant to grow is mainly due to the lack of PerR-mediated fur repression.

PubMed

Kim, Jung-Hoon; Yang, Yoon-Mo; Ji, Chang-Jun; Ryu, Su-Hyun; Won, Young-Bin; Ju, Shin-Yeong; Kwon, Yumi; Lee, Yeh-Eun; Youn, Hwan; Lee, Jin-Won

2017-06-01

PerR, a member of Fur family protein, is a metal-dependent H 2 O 2 sensing transcription factor that regulates genes involved in peroxide stress response. Industrially important bacterium Bacillus licheniformis contains three PerR-like proteins (PerR BL , PerR2, and PerR3) compared to its close relative Bacillus subtilis. Interestingly, unlike other bacteria including B. subtilis, no authentic perR BL null mutant could be established for B. licheniformis. Thus, we constructed a conditional perR BL mutant using a xylose-inducible promoter, and investigated the genes under the control of PerR BL . PerR BL regulon genes include katA, mrgA, ahpC, pfeT, hemA, fur, and perR as observed for PerR BS . However, there is some variation in the expression levels of fur and hemA genes between B. subtilis and B. licheniformis in the derepressed state. Furthermore, katA, mrgA, and ahpC are strongly induced, whereas the others are only weakly or not induced by H 2 O 2 treatment. In contrast to the B. subtilis perR null mutant which frequently gives rise to large colony phenotype mainly due to the loss of katA, the suppressors of B. licheniformis perR mutant, which can form colonies on LB agar, were all catalase-positive. Instead, many of the suppressors showed increased levels of siderophore production, suggesting that the suppressor mutation is linked to the fur gene. Consistent with this, perR fur double mutant could grow on LB agar without Fe supplementation, whereas perR katA double mutant could only grow on LB agar with Fe supplementation. Taken together, our data suggest that in B. licheniformis, despite the similarity in PerR BL and PerR BS regulon genes, perR is an essential gene required for growth and that the inability of perR null mutant to grow is mainly due to elevated expression of Fur.

PIERCE1 is critical for specification of left-right asymmetry in mice.

PubMed

Sung, Young Hoon; Baek, In-Jeoung; Kim, Yong Hwan; Gho, Yong Song; Oh, S Paul; Lee, Young Jae; Lee, Han-Woong

2016-06-16

The specification of left-right asymmetry of the visceral organs is precisely regulated. The earliest breakage of left-right symmetry occurs as the result of leftward flow generated by asymmetric beating of nodal cilia, which eventually induces asymmetric Nodal/Lefty/Pitx2 expression on the left side of the lateral plate mesoderm. PIERCE1 has been identified as a p53 target gene involved in the DNA damage response. In this study, we found that Pierce1-null mice exhibit severe laterality defects, including situs inversus totalis and heterotaxy with randomized situs and left and right isomerisms. The spectrum of laterality defects was closely correlated with randomized expression of Nodal and its downstream genes, Lefty1/2 and Pitx2. The phenotype of Pierce1-null mice most closely resembled that of mutant mice with impaired ciliogenesis and/or ciliary motility of the node. We also found the loss of asymmetric expression of Cerl2, the earliest flow-responding gene in the node of Pierce1-null embryos. The results suggest that Pierce1-null embryos have defects in generating a symmetry breaking signal including leftward nodal flow. This is the first report implicating a role for PIERCE1 in the symmetry-breaking step of left-right asymmetry specification.

Methionine Biosynthesis is Essential for Infection in the Rice Blast Fungus Magnaporthe oryzae

PubMed Central

Gagey, Marie Josèphe; Frelin, Océane; Beffa, Roland; Lebrun, Marc Henri; Droux, Michel

2015-01-01

Methionine is a sulfur amino acid standing at the crossroads of several biosynthetic pathways. In fungi, the last step of methionine biosynthesis is catalyzed by a cobalamine-independent methionine synthase (Met6, EC 2.1.1.14). In the present work, we studied the role of Met6 in the infection process of the rice blast fungus, Magnaporthe oryzae. To this end MET6 null mutants were obtained by targeted gene replacement. On minimum medium, MET6 null mutants were auxotrophic for methionine. Even when grown in presence of excess methionine, these mutants displayed developmental defects, such as reduced mycelium pigmentation, aerial hypha formation and sporulation. They also displayed characteristic metabolic signatures such as increased levels of cysteine, cystathionine, homocysteine, S-adenosylmethionine, S-adenosylhomocysteine while methionine and glutathione levels remained unchanged. These metabolic perturbations were associated with the over-expression of MgCBS1 involved in the reversed transsulfuration pathway that metabolizes homocysteine into cysteine and MgSAM1 and MgSAHH1 involved in the methyl cycle. This suggests a physiological adaptation of M. oryzae to metabolic defects induced by the loss of Met6, in particular an increase in homocysteine levels. Pathogenicity assays showed that MET6 null mutants were non-pathogenic on both barley and rice leaves. These mutants were defective in appressorium-mediated penetration and invasive infectious growth. These pathogenicity defects were rescued by addition of exogenous methionine and S-methylmethionine. These results show that M. oryzae cannot assimilate sufficient methionine from plant tissues and must synthesize this amino acid de novo to fulfill its sulfur amino acid requirement during infection. PMID:25856162

Impaired proteoglycan glycosylation, elevated TGF-β signaling, and abnormal osteoblast differentiation as the basis for bone fragility in a mouse model for gerodermia osteodysplastica

PubMed Central

Chan, Wing Lee; Steiner, Magdalena; Egerer, Johannes; Mizumoto, Shuji; Pestka, Jan M.; Zhang, Haikuo; Khayal, Layal Abo; Ott, Claus-Eric; Kolanczyk, Mateusz; Schinke, Thorsten; Paganini, Chiara; Rossi, Antonio; Sugahara, Kazuyuki; Amling, Michael; Knaus, Petra; Chan, Danny; Mundlos, Stefan

2018-01-01

Gerodermia osteodysplastica (GO) is characterized by skin laxity and early-onset osteoporosis. GORAB, the responsible disease gene, encodes a small Golgi protein of poorly characterized function. To circumvent neonatal lethality of the GorabNull full knockout, Gorab was conditionally inactivated in mesenchymal progenitor cells (Prx1-cre), pre-osteoblasts (Runx2-cre), and late osteoblasts/osteocytes (Dmp1-cre), respectively. While in all three lines a reduction in trabecular bone density was evident, only GorabPrx1 and GorabRunx2 mutants showed dramatically thinned, porous cortical bone and spontaneous fractures. Collagen fibrils in the skin of GorabNull mutants and in bone of GorabPrx1 mutants were disorganized, which was also seen in a bone biopsy from a GO patient. Measurement of glycosaminoglycan contents revealed a reduction of dermatan sulfate levels in skin and cartilage from GorabNull mutants. In bone from GorabPrx1 mutants total glycosaminoglycan levels and the relative percentage of dermatan sulfate were both strongly diminished. Accordingly, the proteoglycans biglycan and decorin showed reduced glycanation. Also in cultured GORAB-deficient fibroblasts reduced decorin glycanation was evident. The Golgi compartment of these cells showed an accumulation of decorin, but reduced signals for dermatan sulfate. Moreover, we found elevated activation of TGF-β in GorabPrx1 bone tissue leading to enhanced downstream signalling, which was reproduced in GORAB-deficient fibroblasts. Our data suggest that the loss of Gorab primarily perturbs pre-osteoblasts. GO may be regarded as a congenital disorder of glycosylation affecting proteoglycan synthesis due to delayed transport and impaired posttranslational modification in the Golgi compartment. PMID:29561836

Goalpha regulates volatile anesthetic action in Caenorhabditis elegans.

PubMed Central

van Swinderen, B; Metz, L B; Shebester, L D; Mendel, J E; Sternberg, P W; Crowder, C M

2001-01-01

To identify genes controlling volatile anesthetic (VA) action, we have screened through existing Caenorhabditis elegans mutants and found that strains with a reduction in Go signaling are VA resistant. Loss-of-function mutants of the gene goa-1, which codes for the alpha-subunit of Go, have EC(50)s for the VA isoflurane of 1.7- to 2.4-fold that of wild type. Strains overexpressing egl-10, which codes for an RGS protein negatively regulating goa-1, are also isoflurane resistant. However, sensitivity to halothane, a structurally distinct VA, is differentially affected by Go pathway mutants. The RGS overexpressing strains, a goa-1 missense mutant found to carry a novel mutation near the GTP-binding domain, and eat-16(rf) mutants, which suppress goa-1(gf) mutations, are all halothane resistant; goa-1(null) mutants have wild-type sensitivities. Double mutant strains carrying mutations in both goa-1 and unc-64, which codes for a neuronal syntaxin previously found to regulate VA sensitivity, show that the syntaxin mutant phenotypes depend in part on goa-1 expression. Pharmacological assays using the cholinesterase inhibitor aldicarb suggest that VAs and GOA-1 similarly downregulate cholinergic neurotransmitter release in C. elegans. Thus, the mechanism of action of VAs in C. elegans is regulated by Goalpha, and presynaptic Goalpha-effectors are candidate VA molecular targets. PMID:11404329

Heme deficiency in erythroid lineage causes differentiation arrest and cytoplasmic iron overload.

PubMed Central

Nakajima, O; Takahashi, S; Harigae, H; Furuyama, K; Hayashi, N; Sassa, S; Yamamoto, M

1999-01-01

Erythroid 5-aminolevulinate synthase (ALAS-E) catalyzes the first step of heme biosynthesis in erythroid cells. Mutation of human ALAS-E causes the disorder X-linked sideroblastic anemia. To examine the roles of heme during hematopoiesis, we disrupted the mouse ALAS-E gene. ALAS-E-null embryos showed no hemoglobinized cells and died by embryonic day 11.5, indicating that ALAS-E is the principal isozyme contributing to erythroid heme biosynthesis. In the ALAS-E-null mutant embryos, erythroid differentiation was arrested, and an abnormal hematopoietic cell fraction emerged that accumulated a large amount of iron diffusely in the cytoplasm. In contrast, we found typical ring sideroblasts that accumulated iron mostly in mitochondria in adult mice chimeric for ALAS-E-null mutant cells, indicating that the mode of iron accumulation caused by the lack of ALAS-E is different in primitive and definitive erythroid cells. These results demonstrate that ALAS-E, and hence heme supply, is necessary for differentiation and iron metabolism of erythroid cells. PMID:10562540

In vivo replication of an ICP34.5 second-site suppressor mutant following corneal infection correlates with in vitro regulation of eIF2 alpha phosphorylation.

PubMed

Ward, Stephen L; Scheuner, Donalyn; Poppers, Jeremy; Kaufman, Randal J; Mohr, Ian; Leib, David A

2003-04-01

In animal models of herpes simplex virus type 1 (HSV-1) infection, ICP34.5-null viruses are avirulent and also fail to grow in a variety of cultured cells due to their inability to prevent RNA-dependent protein kinase (PKR)-mediated inhibition of protein synthesis. We show here that the inability of ICP34.5 mutants to grow in vitro is due specifically to the accumulation of phosphorylated eIF2 alpha. Mutations suppressing the in vitro phenotype of ICP34.5-null mutants have been described which map to the unique short region of the HSV-1 genome, resulting in dysregulated expression of the US11 gene. Despite the inability of the suppressor mutation to suppress the avirulent phenotype of the ICP34.5-null parental virus following intracranial inoculation, the suppressor mutation enhanced virus growth in the cornea, trigeminal ganglia, and periocular skin following corneal infection compared to that with the ICP34.5-null virus. The phosphorylation state of eIF2 alpha following in vitro infection with the suppressor virus was examined to determine if in vivo differences could be attributed to differential regulation of eIF2 alpha phosphorylation. The suppressor virus prevented accumulation of phosphorylated eIF2 alpha, while the wild-type virus substantially reduced eIF2 alpha phosphorylation levels. These data suggest that US11 functions as a PKR antagonist in vivo, although its activity may be modulated by tissue-specific differences in translation regulation.

In Vivo Replication of an ICP34.5 Second-Site Suppressor Mutant following Corneal Infection Correlates with In Vitro Regulation of eIF2α Phosphorylation

PubMed Central

Ward, Stephen L.; Scheuner, Donalyn; Poppers, Jeremy; Kaufman, Randal J.; Mohr, Ian; Leib, David A.

2003-01-01

In animal models of herpes simplex virus type 1 (HSV-1) infection, ICP34.5-null viruses are avirulent and also fail to grow in a variety of cultured cells due to their inability to prevent RNA-dependent protein kinase (PKR)-mediated inhibition of protein synthesis. We show here that the inability of ICP34.5 mutants to grow in vitro is due specifically to the accumulation of phosphorylated eIF2α. Mutations suppressing the in vitro phenotype of ICP34.5-null mutants have been described which map to the unique short region of the HSV-1 genome, resulting in dysregulated expression of the US11 gene. Despite the inability of the suppressor mutation to suppress the avirulent phenotype of the ICP34.5-null parental virus following intracranial inoculation, the suppressor mutation enhanced virus growth in the cornea, trigeminal ganglia, and periocular skin following corneal infection compared to that with the ICP34.5-null virus. The phosphorylation state of eIF2α following in vitro infection with the suppressor virus was examined to determine if in vivo differences could be attributed to differential regulation of eIF2α phosphorylation. The suppressor virus prevented accumulation of phosphorylated eIF2α, while the wild-type virus substantially reduced eIF2α phosphorylation levels. These data suggest that US11 functions as a PKR antagonist in vivo, although its activity may be modulated by tissue-specific differences in translation regulation. PMID:12663769

Disruption of insulin-like growth factor-II imprinting during embryonic development rescues the dwarf phenotype of mice null for pregnancy-associated plasma protein-A.

PubMed

Bale, Laurie K; Conover, Cheryl A

2005-08-01

Pregnancy-associated plasma protein-A (PAPP-A), an insulin-like growth factor-binding protein (IGFBP) protease, increases insulin-like growth factor (IGF) activity through cleavage of inhibitory IGFBP-4 and the consequent release of IGF peptide for receptor activation. Mice homozygous for targeted disruption of the PAPP-A gene are born as proportional dwarfs and exhibit retarded bone ossification during fetal development. Phenotype and in vitro data support a model in which decreased IGF-II bioavailability during embryogenesis results in growth retardation and reduction in overall body size. To test the hypothesis that an increase in IGF-II during embryogenesis would overcome the growth deficiencies, PAPP-A-null mice were crossed with DeltaH19 mutant mice, which have increased IGF-II expression and fetal overgrowth due to disruption of IgfII imprinting. DeltaH19 mutant mice were 126% and PAPP-A-null mice were 74% the size of controls at birth. These size differences were evident at embryonic day 16.5. Importantly, double mutants were indistinguishable from controls both in terms of size and skeletal development. Body size programmed during embryo development persisted post-natally. Thus, disruption of IgfII imprinting and consequent elevation in IGF-II during fetal development was associated with rescue of the dwarf phenotype and ossification defects of PAPP-A-null mice. These data provide strong genetic evidence that PAPP-A plays an essential role in determining IGF-II bioavailability for optimal fetal growth and development.

CodY Promotes Sporulation and Enterotoxin Production by Clostridium perfringens Type A Strain SM101.

PubMed

Li, Jihong; Freedman, John C; Evans, Daniel R; McClane, Bruce A

2017-03-01

Clostridium perfringens type D strains cause enterotoxemia and enteritis in livestock via epsilon toxin production. In type D strain CN3718, CodY was previously shown to increase the level of epsilon toxin production and repress sporulation. C. perfringens type A strains producing C. perfringens enterotoxin (CPE) cause human food poisoning and antibiotic-associated diarrhea. Sporulation is critical for C. perfringens type A food poisoning since spores contribute to transmission and resistance in the harsh food environment and sporulation is essential for CPE production. Therefore, the current study asked whether CodY also regulates sporulation and CPE production in SM101, a derivative of C. perfringens type A food-poisoning strain NCTC8798. An isogenic codY -null mutant of SM101 showed decreased levels of spore formation, along with lower levels of CPE production. A complemented strain recovered wild-type levels of both sporulation and CPE production. When this result was coupled with the earlier results obtained with CN3718, it became apparent that CodY regulation of sporulation varies among different C. perfringens strains. Results from quantitative reverse transcriptase PCR analysis clearly demonstrated that, during sporulation, codY transcript levels remained high in SM101 but rapidly declined in CN3718. In addition, abrB gene expression patterns varied significantly between codY -null mutants of SM101 and CN3718. Compared to the levels in their wild-type parents, the level of abrB gene expression decreased in the CN3718 codY -null mutant strain but significantly increased in the SM101 codY -null mutant strain, demonstrating CodY-dependent regulation differences in abrB expression between these two strains. This difference appears to be important since overexpression of the abrB gene in SM101 reduced the levels of sporulation and enterotoxin production, supporting the involvement of AbrB repression in regulating C. perfringens sporulation. Copyright © 2017 American Society for Microbiology.

CodY Promotes Sporulation and Enterotoxin Production by Clostridium perfringens Type A Strain SM101

PubMed Central

Li, Jihong; Freedman, John C.; Evans, Daniel R.

2017-01-01

ABSTRACT Clostridium perfringens type D strains cause enterotoxemia and enteritis in livestock via epsilon toxin production. In type D strain CN3718, CodY was previously shown to increase the level of epsilon toxin production and repress sporulation. C. perfringens type A strains producing C. perfringens enterotoxin (CPE) cause human food poisoning and antibiotic-associated diarrhea. Sporulation is critical for C. perfringens type A food poisoning since spores contribute to transmission and resistance in the harsh food environment and sporulation is essential for CPE production. Therefore, the current study asked whether CodY also regulates sporulation and CPE production in SM101, a derivative of C. perfringens type A food-poisoning strain NCTC8798. An isogenic codY-null mutant of SM101 showed decreased levels of spore formation, along with lower levels of CPE production. A complemented strain recovered wild-type levels of both sporulation and CPE production. When this result was coupled with the earlier results obtained with CN3718, it became apparent that CodY regulation of sporulation varies among different C. perfringens strains. Results from quantitative reverse transcriptase PCR analysis clearly demonstrated that, during sporulation, codY transcript levels remained high in SM101 but rapidly declined in CN3718. In addition, abrB gene expression patterns varied significantly between codY-null mutants of SM101 and CN3718. Compared to the levels in their wild-type parents, the level of abrB gene expression decreased in the CN3718 codY-null mutant strain but significantly increased in the SM101 codY-null mutant strain, demonstrating CodY-dependent regulation differences in abrB expression between these two strains. This difference appears to be important since overexpression of the abrB gene in SM101 reduced the levels of sporulation and enterotoxin production, supporting the involvement of AbrB repression in regulating C. perfringens sporulation. PMID:28052992

Drosophila fragile X mental retardation protein and metabotropic glutamate receptor A convergently regulate the synaptic ratio of ionotropic glutamate receptor subclasses.

PubMed

Pan, Luyuan; Broadie, Kendal S

2007-11-07

A current hypothesis proposes that fragile X mental retardation protein (FMRP), an RNA-binding translational regulator, acts downstream of glutamatergic transmission, via metabotropic glutamate receptor (mGluR) G(q)-dependent signaling, to modulate protein synthesis critical for trafficking ionotropic glutamate receptors (iGluRs) at synapses. However, direct evidence linking FMRP and mGluR function with iGluR synaptic expression is limited. In this study, we use the Drosophila fragile X model to test this hypothesis at the well characterized glutamatergic neuromuscular junction (NMJ). Two iGluR classes reside at this synapse, each containing common GluRIIC (III), IID and IIE subunits, and variable GluRIIA (A-class) or GluRIIB (B-class) subunits. In Drosophila fragile X mental retardation 1 (dfmr1) null mutants, A-class GluRs accumulate and B-class GluRs are lost, whereas total GluR levels do not change, resulting in a striking change in GluR subclass ratio at individual synapses. The sole Drosophila mGluR, DmGluRA, is also expressed at the NMJ. In dmGluRA null mutants, both iGluR classes increase, resulting in an increase in total synaptic GluR content at individual synapses. Targeted postsynaptic dmGluRA overexpression causes the exact opposite GluR phenotype to the dfmr1 null, confirming postsynaptic GluR subtype-specific regulation. In dfmr1; dmGluRA double null mutants, there is an additive increase in A-class GluRs, and a similar additive impact on B-class GluRs, toward normal levels in the double mutants. These results show that both dFMRP and DmGluRA differentially regulate the abundance of different GluR subclasses in a convergent mechanism within individual postsynaptic domains.

GABAA Receptors Containing ρ1 Subunits Contribute to In Vivo Effects of Ethanol in Mice

PubMed Central

Blednov, Yuri A.; Benavidez, Jillian M.; Black, Mendy; Leiter, Courtney R.; Osterndorff-Kahanek, Elizabeth; Johnson, David; Borghese, Cecilia M.; Hanrahan, Jane R.; Johnston, Graham A. R.; Chebib, Mary; Harris, R. Adron

2014-01-01

GABAA receptors consisting of ρ1, ρ2, or ρ3 subunits in homo- or hetero-pentamers have been studied mainly in retina but are detected in many brain regions. Receptors formed from ρ1 are inhibited by low ethanol concentrations, and family-based association analyses have linked ρ subunit genes with alcohol dependence. We determined if genetic deletion of ρ1 in mice altered in vivo ethanol effects. Null mutant male mice showed reduced ethanol consumption and preference in a two-bottle choice test with no differences in preference for saccharin or quinine. Null mutant mice of both sexes demonstrated longer duration of ethanol-induced loss of righting reflex (LORR), and males were more sensitive to ethanol-induced motor sedation. In contrast, ρ1 null mice showed faster recovery from acute motor incoordination produced by ethanol. Null mutant females were less sensitive to ethanol-induced development of conditioned taste aversion. Measurement of mRNA levels in cerebellum showed that deletion of ρ1 did not change expression of ρ2, α2, or α6 GABAA receptor subunits. (S)-4-amino-cyclopent-1-enyl butylphosphinic acid (“ρ1” antagonist), when administered to wild type mice, mimicked the changes that ethanol induced in ρ1 null mice (LORR and rotarod tests), but the ρ1 antagonist did not produce these effects in ρ1 null mice. In contrast, (R)-4-amino-cyclopent-1-enyl butylphosphinic acid (“ρ2” antagonist) did not change ethanol actions in wild type but produced effects in mice lacking ρ1 that were opposite of the effects of deleting (or inhibiting) ρ1. These results suggest that ρ1 has a predominant role in two in vivo effects of ethanol, and a role for ρ2 may be revealed when ρ1 is deleted. We also found that ethanol produces similar inhibition of function of recombinant ρ1 and ρ2 receptors. These data indicate that ethanol action on GABAA receptors containing ρ1/ρ2 subunits may be important for specific effects of ethanol in vivo. PMID:24454882

Left cardiac isomerism in the Sonic hedgehog null mouse.

PubMed

Hildreth, Victoria; Webb, Sandra; Chaudhry, Bill; Peat, Jonathan D; Phillips, Helen M; Brown, Nigel; Anderson, Robert H; Henderson, Deborah J

2009-06-01

Sonic hedgehog (Shh) is a secreted morphogen necessary for the production of sidedness in the developing embryo. In this study, we describe the morphology of the atrial chambers and atrioventricular junctions of the Shh null mouse heart. We demonstrate that the essential phenotypic feature is isomerism of the left atrial appendages, in combination with an atrioventricular septal defect and a common atrioventricular junction. These malformations are known to be frequent in humans with left isomerism. To confirm the presence of left isomerism, we show that Pitx2c, a recognized determinant of morphological leftness, is expressed in the Shh null mutants on both the right and left sides of the inflow region, and on both sides of the solitary arterial trunk exiting from the heart. It has been established that derivatives of the second heart field expressing Isl1 are asymmetrically distributed in the developing normal heart. We now show that this population is reduced in the hearts from the Shh null mutants, likely contributing to the defects. To distinguish the consequences of reduced contributions from the second heart field from those of left-right patterning disturbance, we disrupted the movement of second heart field cells into the heart by expressing dominant-negative Rho kinase in the population of cells expressing Isl1. This resulted in absence of the vestibular spine, and presence of atrioventricular septal defects closely resembling those seen in the hearts from the Shh null mutants. The primary atrial septum, however, was well formed, and there was no evidence of isomerism of the atrial appendages, suggesting that these features do not relate to disruption of the contributions made by the second heart field. We demonstrate, therefore, that the Shh null mouse is a model of isomerism of the left atrial appendages, and show that the recognized associated malformations found at the venous pole of the heart in the setting of left isomerism are likely to arise from the loss of the effects of Shh in the establishment of laterality, combined with a reduced contribution made by cells derived from the second heart field.

Maize opaque5 Encodes Monogalactosyldiacylglycerol Synthase and Specifically Affects Galactolipids Necessary for Amyloplast and Chloroplast Function[C][W][OA

PubMed Central

Myers, Alan M.; James, Martha G.; Lin, Qiaohui; Yi, Gibum; Stinard, Philip S.; Hennen-Bierwagen, Tracie A.; Becraft, Philip W.

2011-01-01

The maize (Zea mays) opaque5 (o5) locus was shown to encode the monogalactosyldiacylglycerol synthase MGD1. Null and point mutations of o5 that affect the vitreous nature of mature endosperm engendered an allelic series of lines with stepwise reductions in gene function. C18:3/C18:2 galactolipid abundance in seedling leaves was reduced proportionally, without significant effects on total galactolipid content. This alteration in polar lipid composition disrupted the organization of thylakoid membranes into granal stacks. Total galactolipid abundance in endosperm was strongly reduced in o5- mutants, causing developmental defects and changes in starch production such that the normal simple granules were replaced with compound granules separated by amyloplast membrane. Complete loss of MGD1 function in a null mutant caused kernel lethality owing to failure in both endosperm and embryo development. The data demonstrate that low-abundance galactolipids with five double bonds serve functions in plastid membranes that are not replaced by the predominant species with six double bonds. Furthermore, the data identify a function of amyloplast membranes in the development of starch granules. Finally, the specific changes in lipid composition suggest that MGD1 can distinguish the constituency of acyl groups on its diacylglycerol substrate based upon the degree of desaturation. PMID:21685260

Evidence that the Dictyostelium STAT protein Dd-STATa plays a role in the differentiation of inner basal disc cells and identification of a promoter element essential for expression in these cells.

PubMed

Shimada, Nao; Maruo, Toshinari; Maeda, Mineko; Urushihara, Hideko; Kawata, Takefumi

2005-02-01

Dd-STATa, a Dictyostelium homolog of the metazoan STAT (signal transducers and activators of transcription) proteins, is necessary in the slug for correct entry into culmination. Dd-STATa-null mutant fails to culminate and its phenotype correlates with the loss of a funnel-shaped core region, the pstAB core region, which expresses both the ecmA and ecmB genes. To understand how the differentiation of pstAB core cells is regulated, we identified an EST that is expressed in the core cells of normal slugs but down-regulated in the Dd-STATa-null mutant. This EST, SSK348, encodes a close homolog of the Dictyostelium acetyl-CoA synthetase (ACS). A promoter fragment of the cognate gene, aslA (acetyl-CoA synthetase-like A), was fused to a lacZ reporter and the expression pattern determined. As expected from the behavior of the endogenous aslA gene, the aslA::lacZ fusion gene is not expressed in Dd-STATa-null slugs. In parental cells, the aslA promoter is first activated in the funnel-shaped core cells located at the slug anterior, the "pstAB core." During culmination, the pstAB core cells move down, through the prespore cells, to form the inner part of the basal disc. As the spore mass climbs the stalk, the aslA gene comes to be expressed in cells of the upper and lower cups, structures that cradle the spore head. Deletion and point mutation analyses of the promoter identified an AT-rich sequence that is necessary for expression in the pstAB core. This acts in combination with repressor regions that prevent ectopic aslA expression in the pre-stalk regions of slugs and the stalks of culminants. Thus, this study confirms that Dd-STATa is necessary for the differentiation of pstAB core cells, by showing that it is needed for the activation of the aslA gene. It also identifies aslA promoter elements that are likely to be regulated, directly or indirectly, by Dd-STATa.

Glutamine Deprivation Causes Enhanced Plating Efficiency of a Herpes Simplex Virus Type 1 ICP0-Null Mutant ▿

PubMed Central

Bringhurst, Ryan M.; Dominguez, Antonia A.; Schaffer, Priscilla A.

2008-01-01

Isoleucine deprivation of cellular monolayers prior to infection has been reported to result in partial complementation of a herpes simplex virus type 1 (HSV-1) ICP0 null (ICP0−) mutant. We now report that glutamine deprivation alone is able to enhance the plating efficiency of an ICP0− virus and that isoleucine deprivation has little or no effect. Because a low glutamine level is associated with stress and because stress is known to induce reactivation, low levels of glutamine may be relevant to the reactivation of HSV-1 from latency. Additionally, we demonstrate that arginine and methionine deprivation result in partial complementation of the ICP0− virus. PMID:18768961

AP2 hemicomplexes contribute independently to synaptic vesicle endocytosis

PubMed Central

Gu, Mingyu; Liu, Qiang; Watanabe, Shigeki; Sun, Lin; Hollopeter, Gunther; Grant, Barth D; Jorgensen, Erik M

2013-01-01

The clathrin adaptor complex AP2 is thought to be an obligate heterotetramer. We identify null mutations in the α subunit of AP2 in the nematode Caenorhabditis elegans. α-adaptin mutants are viable and the remaining μ2/β hemicomplex retains some function. Conversely, in μ2 mutants, the alpha/sigma2 hemicomplex is localized and is partially functional. α-μ2 double mutants disrupt both halves of the complex and are lethal. The lethality can be rescued by expression of AP2 components in the skin, which allowed us to evaluate the requirement for AP2 subunits at synapses. Mutations in either α or μ2 subunits alone reduce the number of synaptic vesicles by about 30%; however, simultaneous loss of both α and μ2 subunits leads to a 70% reduction in synaptic vesicles and the presence of large vacuoles. These data suggest that AP2 may function as two partially independent hemicomplexes. DOI: http://dx.doi.org/10.7554/eLife.00190.001 PMID:23482940

Variant-specific quantification of factor H in plasma reveals null alleles associated with atypical hemolytic uremic syndrome

PubMed Central

Hakobyan, Svetlana; Tortajada, Agustín; Harris, Claire L.; de Córdoba, Santiago Rodríguez; Morgan, B. Paul

2011-01-01

Atypical hemolytic uremic syndrome (aHUS) associates with complement alternative pathway defects in over 50% of cases. Mutations in factor H (fH) are most common, usually point mutations affecting complement surface regulation and sometimes null mutations in heterozygosity. The latter are difficult to identify; although consistently low plasma fH concentration is suggestive, definitive proof has required the demonstration that the mutant sequence does not express in vitro. Here, novel reagents and assays that distinguish and individually quantify the common fH-Y402H polymorphic variants were used to identify alleles of the CFH gene resulting in low or no (‘null’) expression of full-length fH, but normal or increased expression of the alternative splice product FHL-1, also detected in these assays. Their use in an aHUS cohort identified three Y402H heterozygotes with low or absent fH-H402 but normal or increased FHL-1 levels. Novel mutations in heterozygosis explained the null phenotype in two cases, confirmed by family studies in one. In the third case, family studies showed that a known mutation was present on the Y allele; the cause of the reduced expression of H allele was not found, although data suggested altered fH/FHL-1 splicing. In each family, inheritance of “low expression” or “null” alleles for fH strongly associated with aHUS. These assays provide a rapid means to identify fH expression defects in aHUS without resorting to gene sequencing or expression analysis. PMID:20703214

The Caenorhabditis elegans mucolipin-like gene cup-5 is essential for viability and regulates lysosomes in multiple cell types.

PubMed

Hersh, Bradley M; Hartwieg, Erika; Horvitz, H Robert

2002-04-02

The misregulation of programmed cell death, or apoptosis, contributes to the pathogenesis of many diseases. We used Nomarski microscopy to screen for mutants containing refractile cell corpses in a C. elegans strain in which all programmed cell death is blocked and such corpses are absent. We isolated a mutant strain that accumulates refractile bodies resembling irregular cell corpses. We rescued this mutant phenotype with the C. elegans mucolipidosis type IV (ML-IV) homolog, the recently identified cup-5 (coelomocyte-uptake defective) gene. ML-IV is a human autosomal recessive lysosomal storage disease characterized by psychomotor retardation and ophthalmological abnormalities. Our null mutations in cup-5 cause maternal-effect lethality. In addition, cup-5 mutants contain excess lysosomes in many and possibly all cell types and contain lamellar structures similar to those observed in ML-IV cell lines. The human ML-IV gene is capable of rescuing both the maternal-effect lethality and the lysosome-accumulation abnormality of cup-5 mutants. cup-5 mutants seem to contain excess apoptotic cells as detected by staining with terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling. We suggest that the increased apoptosis seen in cup-5 mutants is a secondary consequence of the lysosomal defect, and that abnormalities in apoptosis may be associated with human lysosomal storage disorders.

Epidermal growth factor impairs palatal shelf adhesion and fusion in the Tgf-β 3 null mutant.

PubMed

Barrio, M Carmen; Del Río, Aurora; Murillo, Jorge; Maldonado, Estela; López-Gordillo, Yamila; Paradas-Lara, Irene; Hernandes, Luzmarina; Catón, Javier; Martínez-Álvarez, Concepción

2014-01-01

The cleft palate presented by transforming growth factor-β3 (Tgf-β3) null mutant mice is caused by altered palatal shelf adhesion, cell proliferation, epithelial-to-mesenchymal transformation and cell death. The expression of epidermal growth factor (EGF), transforming growth factor-β1 (Tgf-β1) and muscle segment homeobox-1 (Msx-1) is modified in the palates of these knockout mice, and the cell proliferation defect is caused by the change in EGF expression. In this study, we aimed to determine whether this change in EGF expression has any effect on the other mechanisms altered in Tgf-β3 knockout mouse palates. We tested the effect of inhibiting EGF activity in vitro in the knockout palates via the addition of Tyrphostin AG 1478. We also investigated possible interactions between EGF, Tgf-β1 and Msx-1 in Tgf-β3 null mouse palate cultures. The results show that the inhibition of EGF activity in Tgf-β3 null mouse palate cultures improves palatal shelf adhesion and fusion, with a particular effect on cell death, and restores the normal distribution pattern of Msx-1 in the palatal mesenchyme. Inhibition of TGF-β1 does not affect either EGF or Msx-1 expression. © 2014 S. Karger AG, Basel.

Wild-type myoblasts rescue the ability of myogenin-null myoblasts to fuse in vivo.

PubMed

Myer, A; Wagner, D S; Vivian, J L; Olson, E N; Klein, W H

1997-05-15

Skeletal muscle is formed via a complex series of events during embryogenesis. These events include commitment of mesodermal precursor cells, cell migration, cell-cell recognition, fusion of myoblasts, activation of structural genes, and maturation. In mice lacking the bHLH transcription factor myogenin, myoblasts are specified and positioned correctly, but few fuse to form multinucleated fibers. This indicates that myogenin is critical for the fusion process and subsequent differentiation events of myogenesis. To further define the nature of the myogenic defects in myogenin-null mice, we investigated whether myogenin-null myoblasts are capable of fusing with wild-type myoblasts in vivo using chimeric mice containing mixtures of myogenin-null and wild-type cells. Chimeric embryos demonstrated that myogenin-null myoblasts readily fused in the presence of wild-type myoblasts. However, chimeric myofibers did not express wild-type levels of muscle-specific gene products, and myofibers with a high percentage of mutant nuclei appeared abnormal, suggesting that the wild-type nuclei could not fully rescue mutant nuclei in the myofibers. These data demonstrate that myoblast fusion can be uncoupled from complete myogenic differentiation and that myogenin regulates a specific subset of genes with diverse function. Thus, myogenin appears to control not only transcription of muscle structural genes but also the extracellular environment in which myoblast fusion takes place. We propose that myogenin regulates the expression of one or more extracellular or cell surface proteins required to initiate the muscle differentiation program.

Beyond Tryptophan Synthase: Identification of Genes That Contribute to Chlamydia trachomatis Survival during Gamma Interferon-Induced Persistence and Reactivation

PubMed Central

Muramatsu, Matthew K.; Brothwell, Julie A.; Stein, Barry D.; Putman, Timothy E.; Rockey, Daniel D.

2016-01-01

Chlamydia trachomatis can enter a viable but nonculturable state in vitro termed persistence. A common feature of C. trachomatis persistence models is that reticulate bodies fail to divide and make few infectious progeny until the persistence-inducing stressor is removed. One model of persistence that has relevance to human disease involves tryptophan limitation mediated by the host enzyme indoleamine 2,3-dioxygenase, which converts l-tryptophan to N-formylkynurenine. Genital C. trachomatis strains can counter tryptophan limitation because they encode a tryptophan-synthesizing enzyme. Tryptophan synthase is the only enzyme that has been confirmed to play a role in interferon gamma (IFN-γ)-induced persistence, although profound changes in chlamydial physiology and gene expression occur in the presence of persistence-inducing stressors. Thus, we screened a population of mutagenized C. trachomatis strains for mutants that failed to reactivate from IFN-γ-induced persistence. Six mutants were identified, and the mutations linked to the persistence phenotype in three of these were successfully mapped. One mutant had a missense mutation in tryptophan synthase; however, this mutant behaved differently from previously described synthase null mutants. Two hypothetical genes of unknown function, ctl0225 and ctl0694, were also identified and may be involved in amino acid transport and DNA damage repair, respectively. Our results indicate that C. trachomatis utilizes functionally diverse genes to mediate survival during and reactivation from persistence in HeLa cells. PMID:27430273

Disruptions in valine degradation affect seed development and germination in Arabidopsis.

PubMed

Gipson, Andrew B; Morton, Kyla J; Rhee, Rachel J; Simo, Szabolcs; Clayton, Jack A; Perrett, Morgan E; Binkley, Christiana G; Jensen, Erika L; Oakes, Dana L; Rouhier, Matthew F; Rouhier, Kerry A

2017-06-01

We have functionally characterized the role of two putative mitochondrial enzymes in valine degradation using insertional mutants. Prior to this study, the relationship between branched-chain amino acid degradation (named for leucine, valine and isoleucine) and seed development was limited to leucine catabolism. Using a reverse genetics approach, we show that disruptions in the mitochondrial valine degradation pathway affect seed development and germination in Arabidopsis thaliana. A null mutant of 3-hydroxyisobutyryl-CoA hydrolase (CHY4, At4g31810) resulted in an embryo lethal phenotype, while a null mutant of methylmalonate semialdehyde dehydrogenase (MMSD, At2g14170) resulted in seeds with wrinkled coats, decreased storage reserves, elevated valine and leucine, and reduced germination rates. These data highlight the unique contributions CHY4 and MMSD make to the overall growth and viability of plants. It also increases our knowledge of the role branched-chain amino acid catabolism plays in seed development and amino acid homeostasis. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

Functional mapping of yeast genomes by saturated transposition

PubMed Central

Michel, Agnès H; Hatakeyama, Riko; Kimmig, Philipp; Arter, Meret; Peter, Matthias; Matos, Joao; De Virgilio, Claudio; Kornmann, Benoît

2017-01-01

Yeast is a powerful model for systems genetics. We present a versatile, time- and labor-efficient method to functionally explore the Saccharomyces cerevisiae genome using saturated transposon mutagenesis coupled to high-throughput sequencing. SAturated Transposon Analysis in Yeast (SATAY) allows one-step mapping of all genetic loci in which transposons can insert without disrupting essential functions. SATAY is particularly suited to discover loci important for growth under various conditions. SATAY (1) reveals positive and negative genetic interactions in single and multiple mutant strains, (2) can identify drug targets, (3) detects not only essential genes, but also essential protein domains, (4) generates both null and other informative alleles. In a SATAY screen for rapamycin-resistant mutants, we identify Pib2 (PhosphoInositide-Binding 2) as a master regulator of TORC1. We describe two antagonistic TORC1-activating and -inhibiting activities located on opposite ends of Pib2. Thus, SATAY allows to easily explore the yeast genome at unprecedented resolution and throughput. DOI: http://dx.doi.org/10.7554/eLife.23570.001 PMID:28481201

Expression of the voltage-gated potassium channel KCNQ1 in mammalian taste bud cells and the effect of its null-mutation on taste preferences.

PubMed

Wang, Hong; Iguchi, Naoko; Rong, Qi; Zhou, Minliang; Ogunkorode, Martina; Inoue, Masashi; Pribitkin, Edmund A; Bachmanov, Alexander A; Margolskee, Robert F; Pfeifer, Karl; Huang, Liquan

2009-01-20

Vertebrate taste buds undergo continual cell turnover. To understand how the gustatory progenitor cells in the stratified lingual epithelium migrate and differentiate into different types of mature taste cells, we sought to identify genes that were selectively expressed in taste cells at different maturation stages. Here we report the expression of the voltage-gated potassium channel KCNQ1 in mammalian taste buds of mouse, rat, and human. Immunohistochemistry and nuclear staining showed that nearly all rodent and human taste cells express this channel. Double immunostaining with antibodies against type II and III taste cell markers validated the presence of KCNQ1 in these two types of cells. Co-localization studies with cytokeratin 14 indicated that KCNQ1 is also expressed in type IV basal precursor cells. Null mutation of the kcnq1 gene in mouse, however, did not alter the gross structure of taste buds or the expression of taste signaling molecules. Behavioral assays showed that the mutant mice display reduced preference to some umami substances, but not to any other taste compounds tested. Gustatory nerve recordings, however, were unable to detect any significant change in the integrated nerve responses of the mutant mice to umami stimuli. These results suggest that although it is expressed in nearly all taste bud cells, the function of KCNQ1 is not required for gross taste bud development or peripheral taste transduction pathways, and the reduced preference of kcnq1-null mice in the behavioral assays may be attributable to the deficiency in the central nervous system or other organs.

Perception of sweet taste is important for voluntary alcohol consumption in mice.

PubMed

Blednov, Y A; Walker, D; Martinez, M; Levine, M; Damak, S; Margolskee, R F

2008-02-01

To directly evaluate the association between taste perception and alcohol intake, we used three different mutant mice, each lacking a gene expressed in taste buds and critical to taste transduction: alpha-gustducin (Gnat3), Tas1r3 or Trpm5. Null mutant mice lacking any of these three genes showed lower preference score for alcohol and consumed less alcohol in a two-bottle choice test, as compared with wild-type littermates. These null mice also showed lower preference score for saccharin solutions than did wild-type littermates. In contrast, avoidance of quinine solutions was less in Gnat3 or Trpm5 knockout mice than in wild-type mice, whereas Tas1r3 null mice were not different from wild type in their response to quinine solutions. There were no differences in null vs. wild-type mice in their consumption of sodium chloride solutions. To determine the cause for reduction of ethanol intake, we studied other ethanol-induced behaviors known to be related to alcohol consumption. There were no differences between null and wild-type mice in ethanol-induced loss of righting reflex, severity of acute ethanol withdrawal or conditioned place preference for ethanol. Weaker conditioned taste aversion (CTA) to alcohol in null mice may have been caused by weaker rewarding value of the conditioned stimulus (saccharin). When saccharin was replaced by sodium chloride, no differences in CTA to alcohol between knockout and wild-type mice were seen. Thus, deletion of any one of three different genes involved in detection of sweet taste leads to a substantial reduction of alcohol intake without any changes in pharmacological actions of ethanol.

PERCEPTION OF SWEET TASTE IS IMPORTANT FOR VOLUNTARY ALCOHOL CONSUMPTION IN MICE

PubMed Central

Blednov, Y.A.; Walker, D.; Martinez, M.; Levine, M.; Damak, S.; Margolskee, R.F.

2012-01-01

To directly evaluate the association between taste perception and alcohol intake, we used three different mutant mice, each lacking a gene expressed in taste buds and critical to taste transduction: α-gustducin (Gnat3), Tas1r3 or Trpm5. Null mutant mice lacking any of these three genes showed lower preference score for alcohol and consumed less alcohol in a two-bottle choice test, as compared with wild-type littermates. These null mice also showed lower preference score for saccharin solutions than did wild-type littermates. In contrast, avoidance of quinine solutions was less in Gnat3 or Trpm5 knockout mice than in wild type mice, whereas Tas1r3 null mice were not different from wild-type in their response to quinine solutions. There were no differences in null vs. wild-type mice in their consumption of sodium chloride solutions. To determine the cause for reduction of ethanol intake, we studied other ethanol-induced behaviors known to be related to alcohol consumption. There were no differences between null and wild-type mice in ethanol-induced loss of righting reflex, severity of acute ethanol withdrawal or conditioned place preference for ethanol. Weaker conditioned taste aversion to alcohol in null mice may have been caused by weaker rewarding value of the conditioned stimulus (saccharin). When saccharin was replaced by sodium chloride, no differences in conditioned taste aversion to alcohol between knockout and wild-type mice were seen. Thus, deletion of any one of three different genes involved in detection of sweet taste leads to a substantial reduction of alcohol intake without any changes in pharmacological actions of ethanol. PMID:17376151

Maternal nicotine exposure effects on adolescent learning and memory are abolished in alpha(α)2* nicotinic acetylcholine receptor-null mutant mice.

PubMed

Mojica, Celina; Bai, Yu; Lotfipour, Shahrdad

2018-06-01

The objective of the current study is to test the hypothesis that the deletion of alpha(α)2* nicotinic acetylcholine receptors (nAChRs) (encoded by the Chrna2 gene) ablate maternal nicotine-induced learning and memory deficits in adolescent mice. We use a pre-exposure-dependent contextual fear conditioning behavioral paradigm that is highly hippocampus-dependent. Adolescent wild type and α2-null mutant offspring are exposed to vehicle or maternal nicotine exposure (200 μg/ml, expressed as base) in the drinking water throughout pregnancy until weaning. Adolescent male offspring mice are tested for alterations in growth and development characteristics as well as modifications in locomotion, anxiety, shock-reactivity and learning and memory. As expected, maternal nicotine exposure has no effects on pup number, weight gain and only modestly reduces fluid intake by 19%. Behaviorally, maternal nicotine exposure impedes extinction learning in adolescent wild type mice, a consequence that is abolished in α2-null mutant mice. The effects on learning and memory are not confounded by alternations in stereotypy, locomotion, anxiety or sensory shock reactivity. Overall, the findings highlight that the deletion of α2* nAChRs eliminate the effects of maternal nicotine exposure on learning and memory in adolescent mice. Copyright © 2018 Elsevier Ltd. All rights reserved.

Performance deficits of mGluR8 knockout mice in learning tasks: the effects of null mutation and the background genotype.

PubMed

Gerlai, R; Adams, B; Fitch, T; Chaney, S; Baez, M

2002-08-01

mGluR8 is a G-protein coupled metabotropic glutamate receptor expressed in the mammalian brain. Members of the mGluR family have been shown to be modulators of neural plasticity and learning and memory. Here we analyze the consequences of a null mutation at the mGluR8 gene locus generated using homologous recombination in embryonic stem cells by comparing the learning performance of the mutants with that of wild type controls in the Morris water maze (MWM) and the context and cue dependent fear conditioning (CFC). Our results revealed robust performance deficits associated with the genetic background, the ICR outbred strain, in both mGluR8 null mutant and the wild type control mice. Mice of this strain origin suffered from impaired vision as compared to CD1 or C57BL/6 mice, a significant impediment in MWM, a visuo-spatial learning task. The CFC task, being less dependent on visual cues, allowed us to reveal subtle performance deficits in the mGluR8 mutants: novelty induced hyperactivity and temporally delayed and blunted responding to shocks and temporally delayed responding to contextual stimuli were detected. The role of mGluR8 as a presynaptic autoreceptor and its contribution to cognitive processes are hypothesized and the utility of gene targeting as compared to pharmacological methods is discussed.

Chromosomal Translocations in the Parasite Leishmania by a MRE11/RAD50-Independent Microhomology-Mediated End Joining Mechanism

PubMed Central

Laffitte, Marie-Claude N.; Leprohon, Philippe; Hainse, Maripier; Légaré, Danielle; Masson, Jean-Yves; Ouellette, Marc

2016-01-01

The parasite Leishmania often relies on gene rearrangements to survive stressful environments. However, safeguarding a minimum level of genome integrity is important for cell survival. We hypothesized that maintenance of genomic integrity in Leishmania would imply a leading role of the MRE11 and RAD50 proteins considering their role in DNA repair, chromosomal organization and protection of chromosomes ends in other organisms. Attempts to generate RAD50 null mutants in a wild-type background failed and we provide evidence that this gene is essential. Remarkably, inactivation of RAD50 was possible in a MRE11 null mutant that we had previously generated, providing good evidence that RAD50 may be dispensable in the absence of MRE11. Inactivation of the MRE11 and RAD50 genes led to a decreased frequency of homologous recombination and analysis of the null mutants by whole genome sequencing revealed several chromosomal translocations. Sequencing of the junction between translocated chromosomes highlighted microhomology sequences at the level of breakpoint regions. Sequencing data also showed a decreased coverage at subtelomeric locations in many chromosomes in the MRE11-/-RAD50-/- parasites. This study demonstrates an MRE11-independent microhomology-mediated end-joining mechanism and a prominent role for MRE11 and RAD50 in the maintenance of genomic integrity. Moreover, we suggest the possible involvement of RAD50 in subtelomeric regions stability. PMID:27314941

Different CHD chromatin remodelers are required for expression of distinct gene sets and specific stages during development of Dictyostelium discoideum

PubMed Central

Platt, James L.; Rogers, Benjamin J.; Rogers, Kelley C.; Harwood, Adrian J.; Kimmel, Alan R.

2013-01-01

Control of chromatin structure is crucial for multicellular development and regulation of cell differentiation. The CHD (chromodomain-helicase-DNA binding) protein family is one of the major ATP-dependent, chromatin remodeling factors that regulate nucleosome positioning and access of transcription factors and RNA polymerase to the eukaryotic genome. There are three mammalian CHD subfamilies and their impaired functions are associated with several human diseases. Here, we identify three CHD orthologs (ChdA, ChdB and ChdC) in Dictyostelium discoideum. These CHDs are expressed throughout development, but with unique patterns. Null mutants lacking each CHD have distinct phenotypes that reflect their expression patterns and suggest functional specificity. Accordingly, using genome-wide (RNA-seq) transcriptome profiling for each null strain, we show that the different CHDs regulate distinct gene sets during both growth and development. ChdC is an apparent ortholog of the mammalian Class III CHD group that is associated with the human CHARGE syndrome, and GO analyses of aberrant gene expression in chdC nulls suggest defects in both cell-autonomous and non-autonomous signaling, which have been confirmed through analyses of chdC nulls developed in pure populations or with low levels of wild-type cells. This study provides novel insight into the broad function of CHDs in the regulation development and disease, through chromatin-mediated changes in directed gene expression. PMID:24301467

Compound Heterozygosity for Null Mutations and a Common Hypomorphic Risk Haplotype in TBX6 Causes Congenital Scoliosis.

PubMed

Takeda, Kazuki; Kou, Ikuyo; Kawakami, Noriaki; Iida, Aritoshi; Nakajima, Masahiro; Ogura, Yoji; Imagawa, Eri; Miyake, Noriko; Matsumoto, Naomichi; Yasuhiko, Yukuto; Sudo, Hideki; Kotani, Toshiaki; Nakamura, Masaya; Matsumoto, Morio; Watanabe, Kota; Ikegawa, Shiro

2017-03-01

Congenital scoliosis (CS) occurs as a result of vertebral malformations and has an incidence of 0.5-1/1,000 births. Recently, TBX6 on chromosome 16p11.2 was reported as a disease gene for CS; about 10% of Chinese CS patients were compound heterozygotes for rare null mutations and a common haplotype defined by three SNPs in TBX6. All patients had hemivertebrae. We recruited 94 Japanese CS patients, investigated the TBX6 locus for both mutations and the risk haplotype, examined transcriptional activities of mutant TBX6 in vitro, and evaluated clinical and radiographic features. We identified TBX6 null mutations in nine patients, including a missense mutation that had a loss of function in vitro. All had the risk haplotype in the opposite allele. One of the mutations showed dominant negative effect. Although all Chinese patients had one or more hemivertebrae, two Japanese patients did not have hemivertebra. The compound heterozygosity of null mutations and the common risk haplotype in TBX6 also causes CS in Japanese patients with similar incidence. Hemivertebra was not a specific type of spinal malformation in TBX6-associated CS (TACS). A heterozygous TBX6 loss-of-function mutation has been reported in a family with autosomal-dominant spondylocostal dysostosis, but it may represent a spectrum of the same disease with TACS. © 2017 WILEY PERIODICALS, INC.

Irxl1 mutant mice show reduced tendon differentiation and no patterning defects in musculoskeletal system development.

PubMed

Kimura, Wataru; Machii, Masashi; Xue, XiaoDong; Sultana, Nishat; Hikosaka, Keisuke; Sharkar, Mohammad T K; Uezato, Tadayoshi; Matsuda, Masashi; Koseki, Haruhiko; Miura, Naoyuki

2011-01-01

Irxl1 (Iroquois-related homeobox like-1) is a newly identified three amino-acid loop extension (TALE) homeobox gene, which is expressed in various mesoderm-derived tissues, particularly in the progenitors of the musculoskeletal system. To analyze the roles of Irxl1 during embryonic development, we generated mice carrying a null allele of Irxl1. Mice homozygous for the targeted allele were viable, fertile, and showed reduced tendon differentiation. Skeletal morphology and skeletal muscle weight in Irxl1-knockout mice appeared normal. Expression patterns of several marker genes for cartilage, tendon, and muscle progenitors in homozygous mutant embryos were unchanged. These results suggest that Irxl1 is required for the tendon differentiation but dispensable for the patterning of the musculoskeletal system in development. Copyright © 2010 Wiley-Liss, Inc.

A novel Ras-interacting protein required for chemotaxis and cyclic adenosine monophosphate signal relay in Dictyostelium.

PubMed

Lee, S; Parent, C A; Insall, R; Firtel, R A

1999-09-01

We have identified a novel Ras-interacting protein from Dictyostelium, RIP3, whose function is required for both chemotaxis and the synthesis and relay of the cyclic AMP (cAMP) chemoattractant signal. rip3 null cells are unable to aggregate and lack receptor activation of adenylyl cyclase but are able, in response to cAMP, to induce aggregation-stage, postaggregative, and cell-type-specific gene expression in suspension culture. In addition, rip3 null cells are unable to properly polarize in a cAMP gradient and chemotaxis is highly impaired. We demonstrate that cAMP stimulation of guanylyl cyclase, which is required for chemotaxis, is reduced approximately 60% in rip3 null cells. This reduced activation of guanylyl cyclase may account, in part, for the defect in chemotaxis. When cells are pulsed with cAMP for 5 h to mimic the endogenous cAMP oscillations that occur in wild-type strains, the cells will form aggregates, most of which, however, arrest at the mound stage. Unlike the response seen in wild-type strains, the rip3 null cell aggregates that form under these experimental conditions are very small, which is probably due to the rip3 null cell chemotaxis defect. Many of the phenotypes of the rip3 null cell, including the inability to activate adenylyl cyclase in response to cAMP and defects in chemotaxis, are very similar to those of strains carrying a disruption of the gene encoding the putative Ras exchange factor AleA. We demonstrate that aleA null cells also exhibit a defect in cAMP-mediated activation of guanylyl cyclase similar to that of rip3 null cells. A double-knockout mutant (rip3/aleA null cells) exhibits a further reduction in receptor activation of guanylyl cyclase, and these cells display almost no cell polarization or movement in cAMP gradients. As RIP3 preferentially interacts with an activated form of the Dictyostelium Ras protein RasG, which itself is important for cell movement, we propose that RIP3 and AleA are components of a Ras-regulated pathway involved in integrating chemotaxis and signal relay pathways that are essential for aggregation.

Direct and Indirect Regulation of Spinal Cord Ia Afferent Terminal Formation by the γ-Protocadherins

PubMed Central

Prasad, Tuhina; Weiner, Joshua A.

2011-01-01

The Pcdh-γ gene cluster encodes 22 protocadherin adhesion molecules that interact as homophilic multimers and critically regulate synaptogenesis and apoptosis of interneurons in the developing spinal cord. Unlike interneurons, the two primary components of the monosynaptic stretch reflex circuit, dorsal root ganglion sensory neurons and ventral motor neurons (MNs), do not undergo excessive apoptosis in Pcdh-γdel/del null mutants, which die shortly after birth. However, as we show here, mutants exhibit severely disorganized Ia proprioceptive afferent terminals in the ventral horn. In contrast to the fine net-like pattern observed in wild-type mice, central Ia terminals in Pcdh-γ mutants appear clumped, and fill the space between individual MNs; quantitative analysis shows a ~2.5-fold increase in the area of terminals. Concomitant with this, there is a 70% loss of the collaterals that Ia afferents extend to ventral interneurons (vINs), many of which undergo apoptosis in the mutants. The Ia afferent phenotype is ameliorated, though not entirely rescued, when apoptosis is blocked in Pcdh-γ null mice by introduction of a Bax null allele. This indicates that loss of vINs, which act as collateral Ia afferent targets, contributes to the disorganization of terminals on motor pools. Restricted mutation of the Pcdh-γ cluster using conditional mutants and multiple Cre transgenic lines (Wnt1-Cre for sensory neurons; Pax2-Cre for vINs; Hb9-Cre for MNs) also revealed a direct requirement for the γ-Pcdhs in Ia neurons and vINs, but not in MNs themselves. Together, these genetic manipulations indicate that the γ-Pcdhs are required for the formation of the Ia afferent circuit in two ways: First, they control the survival of vINs that act as collateral Ia targets; and second, they provide a homophilic molecular cue between Ia afferents and target vINs. PMID:22275881

Direct and Indirect Regulation of Spinal Cord Ia Afferent Terminal Formation by the γ-Protocadherins.

PubMed

Prasad, Tuhina; Weiner, Joshua A

2011-01-01

The Pcdh-γ gene cluster encodes 22 protocadherin adhesion molecules that interact as homophilic multimers and critically regulate synaptogenesis and apoptosis of interneurons in the developing spinal cord. Unlike interneurons, the two primary components of the monosynaptic stretch reflex circuit, dorsal root ganglion sensory neurons and ventral motor neurons (MNs), do not undergo excessive apoptosis in Pcdh-γ(del/del) null mutants, which die shortly after birth. However, as we show here, mutants exhibit severely disorganized Ia proprioceptive afferent terminals in the ventral horn. In contrast to the fine net-like pattern observed in wild-type mice, central Ia terminals in Pcdh-γ mutants appear clumped, and fill the space between individual MNs; quantitative analysis shows a ~2.5-fold increase in the area of terminals. Concomitant with this, there is a 70% loss of the collaterals that Ia afferents extend to ventral interneurons (vINs), many of which undergo apoptosis in the mutants. The Ia afferent phenotype is ameliorated, though not entirely rescued, when apoptosis is blocked in Pcdh-γ null mice by introduction of a Bax null allele. This indicates that loss of vINs, which act as collateral Ia afferent targets, contributes to the disorganization of terminals on motor pools. Restricted mutation of the Pcdh-γ cluster using conditional mutants and multiple Cre transgenic lines (Wnt1-Cre for sensory neurons; Pax2-Cre for vINs; Hb9-Cre for MNs) also revealed a direct requirement for the γ-Pcdhs in Ia neurons and vINs, but not in MNs themselves. Together, these genetic manipulations indicate that the γ-Pcdhs are required for the formation of the Ia afferent circuit in two ways: First, they control the survival of vINs that act as collateral Ia targets; and second, they provide a homophilic molecular cue between Ia afferents and target vINs.

Nuclear Photosynthetic Gene Expression Is Synergistically Modulated by Rates of Protein Synthesis in Chloroplasts and Mitochondria[W

PubMed Central

Pesaresi, Paolo; Masiero, Simona; Eubel, Holger; Braun, Hans-Peter; Bhushan, Shashi; Glaser, Elzbieta; Salamini, Francesco; Leister, Dario

2006-01-01

Arabidopsis thaliana mutants prors1-1 and -2 were identified on the basis of a decrease in effective photosystem II quantum yield. Mutations were localized to the 5′-untranslated region of the nuclear gene PROLYL-tRNA SYNTHETASE1 (PRORS1), which acts in both plastids and mitochondria. In prors1-1 and -2, PRORS1 expression is reduced, along with protein synthesis in both organelles. PRORS1 null alleles (prors1-3 and -4) result in embryo sac and embryo development arrest. In mutants with the leaky prors1-1 and -2 alleles, transcription of nuclear genes for proteins involved in photosynthetic light reactions is downregulated, whereas genes for other chloroplast proteins are upregulated. Downregulation of nuclear photosynthetic genes is not associated with a marked increase in the level of reactive oxygen species in leaves and persists in the dark, suggesting that the transcriptional response is light and photooxidative stress independent. The mrpl11 and prpl11 mutants are impaired in the mitochondrial and plastid ribosomal L11 proteins, respectively. The prpl11 mrpl11 double mutant, but neither of the single mutants, resulted in strong downregulation of nuclear photosynthetic genes, like that seen in leaky mutants for PRORS1, implying that, when organellar translation is perturbed, signals derived from both types of organelles cooperate in the regulation of nuclear photosynthetic gene expression. PMID:16517761

WhiB5, a Transcriptional Regulator That Contributes to Mycobacterium tuberculosis Virulence and Reactivation

PubMed Central

Casonato, Stefano; Cervantes Sánchez, Axel; Haruki, Hirohito; Rengifo González, Monica; Provvedi, Roberta; Dainese, Elisa; Jaouen, Thomas; Gola, Susanne; Bini, Estela; Vicente, Miguel; Johnsson, Kai; Ghisotti, Daniela; Palù, Giorgio; Hernández-Pando, Rogelio

2012-01-01

The proteins belonging to the WhiB superfamily are small global transcriptional regulators typical of actinomycetes. In this paper, we characterize the role of WhiB5, a Mycobacterium tuberculosis protein belonging to this superfamily. A null mutant was constructed in M. tuberculosis H37Rv and was shown to be attenuated during both progressive and chronic mouse infections. Mice infected with the mutant had smaller bacillary burdens in the lungs but a larger inflammatory response, suggesting a role of WhiB5 in immunomodulation. Most interestingly, the whiB5 mutant was not able to resume growth after reactivation from chronic infection, suggesting that WhiB5 controls the expression of genes involved in this process. The mutant was also more sensitive than the wild-type parental strain to S-nitrosoglutathione (GSNO) and was less metabolically active following prolonged starvation, underscoring the importance of GSNO and starvation in development and maintenance of chronic infection. DNA microarray analysis identified 58 genes whose expression is influenced by WhiB5, including sigM, encoding an alternative sigma factor, and genes encoding the constituents of two type VII secretion systems, namely, ESX-2 and ESX-4. PMID:22733573

Fission yeast dam1-A8 mutant is resistant to and rescued by an anti-microtubule agent

DOE Office of Scientific and Technical Information (OSTI.GOV)

Griffiths, Karen; Masuda, Hirohisa; Dhut, Susheela

2008-04-11

The Dam1/DASH outer kinetochore complex is required for high-fidelity chromosome segregation in budding and fission yeast. Unlike budding yeast, the fission yeast complex is non-essential, however it promotes bipolar microtubule attachment in conjunction with microtubule-depolymerising kinesin-8 Klp5 and Klp6. Here, we screened for dam1 temperature sensitive mutants in a klp5 null background and identified dam1-A8 that contains two amino acid substitutions in the C-terminus (H126R and E149G). dam1-A8klp5 mutant cells display massive chromosome missegregation with lagging chromosomes and monopolar attachment of sister chromatids to one SPB (spindle pole body). Unexpectedly contrary to a deletion mutant that is hypersensitive to microtubule-destabilisingmore » drugs, dam1-A8 is resistant and furthermore the temperature sensitivity of dam1-A8klp5 is rescued by addition of these drugs. This indicates that the hyper-stabilised rigidity of kinetochore-spindle mal-attachments is the primary cause of lethality. Our result shows that fine-tuning of Dam1 activity is essential for chromosome bi-orientation.« less

Region of Herpes Simplex Virus Type 1 Latency-Associated Transcript Sufficient for Wild-Type Spontaneous Reactivation Promotes Cell Survival in Tissue Culture

PubMed Central

Inman, Melissa; Perng, Guey-Chuen; Henderson, Gail; Ghiasi, Homayon; Nesburn, Anthony B.; Wechsler, Steven L.; Jones, Clinton

2001-01-01

The latency-associated transcript (LAT) is the only abundant herpes simplex virus type 1 (HSV-1) transcript expressed during latency. In the rabbit eye model, LAT null mutants do not reactivate efficiently from latency. We recently demonstrated that the LAT null mutant dLAT2903 induces increased levels of apoptosis in trigeminal ganglia of infected rabbits compared to LAT+ strains (G.-C. Perng, C. Jones, J. Ciacci-Zarella, M. Stone, G. Henderson, A. Yokht, S. M. Slanina, F. M. Hoffman, H. Ghiasi, A. B. Nesburn, and C. S. Wechsler, Science 287:1500–1503, 2000).The same study also demonstrated that a plasmid expressing LAT nucleotides 301 to 2659 enhanced cell survival of transfected cells after induction of apoptosis. Consequently, we hypothesized that LAT enhances spontaneous reactivation in part, because it promotes survival of infected neurons. Here we report on the ability of plasmids expressing different portions of the 5′ end of LAT to promote cell survival after induction of apoptosis. A plasmid expressing the first 1.5 kb of LAT (LAT nucleotides 1 to 1499) promoted cell survival in neuro-2A (mouse neuronal) and CV-1 (monkey fibroblast) cells. A plasmid expressing just the first 811 nucleotides of LAT promoted cell survival less efficiently. Plasmids expressing the first 661 nucleotides or less of LAT did not promote cell survival. We previously showed that a mutant expressing just the first 1.5 kb of LAT has wild-type spontaneous reactivation in rabbits, and a mutant expressing just the first 811 nucleotides of LAT has a reactivation frequency higher than that of dLAT2903 but lower than that of wild-type virus. In addition, mutants reported here for the first time, expressing just the first 661 or 76 nucleotides of LAT, had spontaneous reactivation indistinguishable from that of the LAT null mutant dLAT2903. In summary, these studies provide evidence that there is a functional relationship between the ability of LAT to promote cell survival and its ability to enhance spontaneous reactivation. PMID:11264353

SpxA1 Involved in Hydrogen Peroxide Production, Stress Tolerance and Endocarditis Virulence in Streptococcus sanguinis

PubMed Central

Chen, Lei; Ge, Xiuchun; Wang, Xiaojing; Patel, Jenishkumar R.; Xu, Ping

2012-01-01

Streptococcus sanguinis is one of the most common agents of infective endocarditis. Spx proteins are a group of global regulators that negatively or positively control global transcription initiation. In this study, we characterized the spxA1 gene in S. sanguinis SK36. The spxA1 null mutant displayed opaque colony morphology, reduced hydrogen peroxide (H2O2) production, and reduced antagonistic activity against Streptococcus mutans UA159 relative to the wild type strain. The ΔspxA1 mutant also demonstrated decreased tolerance to high temperature, acidic and oxidative stresses. Further analysis revealed that ΔspxA1 also exhibited a ∼5-fold reduction in competitiveness in an animal model of endocarditis. Microarray studies indicated that expression of several oxidative stress genes was downregulated in the ΔspxA1 mutant. The expression of spxB and nox was significantly decreased in the ΔspxA1 mutant compared with the wild type. These results indicate that spxA1 plays a major role in H2O2 production, stress tolerance and endocarditis virulence in S. sanguinis SK36. The second spx gene, spxA2, was also found in S. sanguinis SK36. The spxA2 null mutant was found to be defective for growth under normal conditions and showed sensitivity to high temperature, acidic and oxidative stresses. PMID:22768210

SpxA1 involved in hydrogen peroxide production, stress tolerance and endocarditis virulence in Streptococcus sanguinis.

PubMed

Chen, Lei; Ge, Xiuchun; Wang, Xiaojing; Patel, Jenishkumar R; Xu, Ping

2012-01-01

Streptococcus sanguinis is one of the most common agents of infective endocarditis. Spx proteins are a group of global regulators that negatively or positively control global transcription initiation. In this study, we characterized the spxA1 gene in S. sanguinis SK36. The spxA1 null mutant displayed opaque colony morphology, reduced hydrogen peroxide (H(2)O(2)) production, and reduced antagonistic activity against Streptococcus mutans UA159 relative to the wild type strain. The ΔspxA1 mutant also demonstrated decreased tolerance to high temperature, acidic and oxidative stresses. Further analysis revealed that ΔspxA1 also exhibited a ∼5-fold reduction in competitiveness in an animal model of endocarditis. Microarray studies indicated that expression of several oxidative stress genes was downregulated in the ΔspxA1 mutant. The expression of spxB and nox was significantly decreased in the ΔspxA1 mutant compared with the wild type. These results indicate that spxA1 plays a major role in H(2)O(2) production, stress tolerance and endocarditis virulence in S. sanguinis SK36. The second spx gene, spxA2, was also found in S. sanguinis SK36. The spxA2 null mutant was found to be defective for growth under normal conditions and showed sensitivity to high temperature, acidic and oxidative stresses.

Methylation of Gibberellins by Arabidopsis GAMT1 and GAMT2[W

PubMed Central

Varbanova, Marina; Yamaguchi, Shinjiro; Yang, Yue; McKelvey, Katherine; Hanada, Atsushi; Borochov, Roy; Yu, Fei; Jikumaru, Yusuke; Ross, Jeannine; Cortes, Diego; Ma, Choong Je; Noel, Joseph P.; Mander, Lew; Shulaev, Vladimir; Kamiya, Yuji; Rodermel, Steve; Weiss, David; Pichersky, Eran

2007-01-01

Arabidopsis thaliana GAMT1 and GAMT2 encode enzymes that catalyze formation of the methyl esters of gibberellins (GAs). Ectopic expression of GAMT1 or GAMT2 in Arabidopsis, tobacco (Nicotiana tabacum), and petunia (Petunia hybrida) resulted in plants with GA deficiency and typical GA deficiency phenotypes, such as dwarfism and reduced fertility. GAMT1 and GAMT2 are both expressed mainly in whole siliques (including seeds), with peak transcript levels from the middle until the end of silique development. Within whole siliques, GAMT2 was previously shown to be expressed mostly in developing seeds, and we show here that GAMT1 expression is also localized mostly to seed, suggesting a role in seed development. Siliques of null single GAMT1 and GAMT2 mutants accumulated high levels of various GAs, with particularly high levels of GA1 in the double mutant. Methylated GAs were not detected in wild-type siliques, suggesting that methylation of GAs by GAMT1 and GAMT2 serves to deactivate GAs and initiate their degradation as the seeds mature. Seeds of homozygous GAMT1 and GAMT2 null mutants showed reduced inhibition of germination, compared with the wild type, when placed on plates containing the GA biosynthesis inhibitor ancymidol, with the double mutant showing the least inhibition. These results suggest that the mature mutant seeds contained higher levels of active GAs than wild-type seeds. PMID:17220201

Premature chain termination is a unifying mechanism for COL1A1 null alleles in osteogenesis imperfecta type I cell strains

DOE Office of Scientific and Technical Information (OSTI.GOV)

Willing, M.C.; Deschenes, S.P.; Roberts, E.J.

Nonsense and frameshift mutations, which predict premature termination of translation, often cause a dramatic reduction in the amount of transcript from the mutant allele (nonsense-mediated mRNA decay). In some genes, these mutations also influence RNA splicing and induce skipping of the exon that contains the nonsense codon. To begin to dissect how premature termination alters the metabolism of RNA from the COL1A1 gene, we studied nonsense and frameshift mutations distributed over exons 11-49 of the gene. These mutations were originally identified in 10 unrelated families with osteogenesis imperfecta (OI) type I. We observed marked reduction in steady-state amounts of mRNAmore » from the mutant allele in both total cellular and nuclear RNA extracts of cells from affected individuals, suggesting that nonsense-mediated decay of COL1A1 RNA is a nuclear phenomenon. Position of the mutation within the gene did not influence this observation. None of the mutations induced skipping of either the exon containing the mutation or, for the frameshifts, the downstream exons with the new termination sites. Our data suggest that nonsense and frameshift mutations throughout most of the COL1A1 gene result in a null allele, which is associated with the predictable mild clinical phenotype, OI type I. 42 refs., 6 figs., 1 tab.« less

The signal transducing photoreceptors of plants.

PubMed

Franklin, Keara A; Larner, Victoria S; Whitelam, Garry C

2005-01-01

Light signals are amongst the most important environmental cues regulating plant development. In addition to light quantity, plants measure the quality, direction and periodicity of incident light and use the information to optimise growth and development to the prevailing environmental conditions. Red and far-red wavelengths are perceived by the photoreversible phytochrome family of photoreceptors, whilst the detection of blue and ultraviolet (UV)-A wavelengths is conferred by the cryptochromes and phototropins. Higher plants contain multiple discrete phytochromes, the apoproteins of which are encoded by a small divergent gene family. In Arabidopsis, two cryptochrome and two phototropin family members have been identified and characterized. Photoreceptor action regulates development throughout the lifecycle of plants, from seed germination through to architecture of the mature plant and the onset of reproduction. The roles of individual photoreceptors in mediating plant development have, however, often been confounded by redundant, synergistic and in some cases mutually antagonistic mechanisms of action. The isolation of mutants null for individual photoreceptors and the construction of mutants null for multiple photoreceptors have therefore been paramount in elucidating photoreceptor functions. Photoreceptor action does not, however, operate in isolation from other signalling systems. The integration of light signals with other environmental cues enables plants to adapt their physiology to changing seasonal environments. This paper summarises current understanding of photoreceptor families and their functions throughout the lifecycle of plants. The integration of light signals with other environmental stimuli is also discussed.

MyTH4-FERM myosins have an ancient and conserved role in filopod formation

PubMed Central

Goodson, Holly V.; Arthur, Ashley L.; Luxton, G. W. Gant; Houdusse, Anne; Titus, Margaret A.

2016-01-01

The formation of filopodia in Metazoa and Amoebozoa requires the activity of myosin 10 (Myo10) in mammalian cells and of Dictyostelium unconventional myosin 7 (DdMyo7) in the social amoeba Dictyostelium. However, the exact roles of these MyTH4-FERM myosins (myosin tail homology 4-band 4.1, ezrin, radixin, moesin; MF) in the initiation and elongation of filopodia are not well defined and may reflect conserved functions among phylogenetically diverse MF myosins. Phylogenetic analysis of MF myosin domains suggests that a single ancestral MF myosin existed with a structure similar to DdMyo7, which has two MF domains, and that subsequent duplications in the metazoan lineage produced its functional homolog Myo10. The essential functional features of the DdMyo7 myosin were identified using quantitative live-cell imaging to characterize the ability of various mutants to rescue filopod formation in myo7-null cells. The two MF domains were found to function redundantly in filopod formation with the C-terminal FERM domain regulating both the number of filopodia and their elongation velocity. DdMyo7 mutants consisting solely of the motor plus a single MyTH4 domain were found to be capable of rescuing the formation of filopodia, establishing the minimal elements necessary for the function of this myosin. Interestingly, a chimeric myosin with the Myo10 MF domain fused to the DdMyo7 motor also was capable of rescuing filopod formation in the myo7-null mutant, supporting fundamental functional conservation between these two distant myosins. Together, these findings reveal that MF myosins have an ancient and conserved role in filopod formation. PMID:27911821

Seizure susceptibility of neuropeptide-Y null mutant mice in amygdala kindling and chemical-induced seizure models.

PubMed

Shannon, Harlan E; Yang, Lijuan

2004-01-01

Neuropeptide Y (NPY) administered exogenously is anticonvulsant, and, NPY null mutant mice are more susceptible to kainate-induced seizures. In order to better understand the potential role of NPY in epileptogenesis, the present studies investigated the development of amygdala kindling, post-kindling seizure thresholds, and anticonvulsant effects of carbamazepine and levetiracetam in 129S6/SvEv NPY(+/+) and NPY(-/-) mice. In addition, susceptibility to pilocarpine- and kainate-induced seizures was compared in NPY(+/+) and (-/-) mice. The rate of amygdala kindling development did not differ in the NPY(-/-) and NPY(+/+) mice either when kindling stimuli were presented once daily for at least 20 days, or, 12 times daily for 2 days. However, during kindling development, the NPY(-/-) mice had higher seizure severity scores and longer afterdischarge durations than the NPY(+/+) mice. Post-kindling, the NPY(-/-) mice had markedly lower afterdischarge thresholds and longer afterdischarge durations than NPY (+/+) mice. Carbamazepine and levetiracetam increased the seizure thresholds of both NPY (-/-) and (+/+) mice. In addition, NPY (-/-) mice had lower thresholds for both kainate- and pilocarpine-induced seizures. The present results in amygdala kindling and chemical seizure models suggest that NPY may play a more prominent role in determining seizure thresholds and severity of seizures than in events leading to epileptogenesis. In addition, a lack of NPY does not appear to confer drug-resistance in that carbamazepine and levetiracetam were anticonvulsant in both wild type (WT) and NPY null mutant mice.

The Arabidopsis SRR1 gene mediates phyB signaling and is required for normal circadian clock function

PubMed Central

Staiger, Dorothee; Allenbach, Laure; Salathia, Neeraj; Fiechter, Vincent; Davis, Seth J.; Millar, Andrew J.; Chory, Joanne; Fankhauser, Christian

2003-01-01

Plants possess several photoreceptors to sense the light environment. In Arabidopsis cryptochromes and phytochromes play roles in photomorphogenesis and in the light input pathways that synchronize the circadian clock with the external world. We have identified SRR1 (sensitivity to red light reduced), a gene that plays an important role in phytochrome B (phyB)-mediated light signaling. The recessive srr1 null allele and phyB mutants display a number of similar phenotypes indicating that SRR1 is required for normal phyB signaling. Genetic analysis suggests that SRR1 works both in the phyB pathway but also independently of phyB. srr1 mutants are affected in multiple outputs of the circadian clock in continuous light conditions, including leaf movement and expression of the clock components, CCA1 and TOC1. Clock-regulated gene expression is also impaired during day–night cycles and in constant darkness. The circadian phenotypes of srr1 mutants in all three conditions suggest that SRR1 activity is required for normal oscillator function. The SRR1 gene was identified and shown to code for a protein conserved in numerous eukaryotes including mammals and flies, implicating a conserved role for this protein in both the animal and plant kingdoms. PMID:12533513

Compensation for PKMζ in long-term potentiation and spatial long-term memory in mutant mice.

PubMed

Tsokas, Panayiotis; Hsieh, Changchi; Yao, Yudong; Lesburguères, Edith; Wallace, Emma Jane Claire; Tcherepanov, Andrew; Jothianandan, Desingarao; Hartley, Benjamin Rush; Pan, Ling; Rivard, Bruno; Farese, Robert V; Sajan, Mini P; Bergold, Peter John; Hernández, Alejandro Iván; Cottrell, James E; Shouval, Harel Z; Fenton, André Antonio; Sacktor, Todd Charlton

2016-05-17

PKMζ is a persistently active PKC isoform proposed to maintain late-LTP and long-term memory. But late-LTP and memory are maintained without PKMζ in PKMζ-null mice. Two hypotheses can account for these findings. First, PKMζ is unimportant for LTP or memory. Second, PKMζ is essential for late-LTP and long-term memory in wild-type mice, and PKMζ-null mice recruit compensatory mechanisms. We find that whereas PKMζ persistently increases in LTP maintenance in wild-type mice, PKCι/λ, a gene-product closely related to PKMζ, persistently increases in LTP maintenance in PKMζ-null mice. Using a pharmacogenetic approach, we find PKMζ-antisense in hippocampus blocks late-LTP and spatial long-term memory in wild-type mice, but not in PKMζ-null mice without the target mRNA. Conversely, a PKCι/λ-antagonist disrupts late-LTP and spatial memory in PKMζ-null mice but not in wild-type mice. Thus, whereas PKMζ is essential for wild-type LTP and long-term memory, persistent PKCι/λ activation compensates for PKMζ loss in PKMζ-null mice.

RAP-1 and the RAL-1/exocyst pathway coordinate hypodermal cell organization in Caenorhabditis elegans

PubMed Central

Frische, Ester W; Pellis-van Berkel, Wendy; van Haaften, Gijs; Cuppen, Edwin; Plasterk, Ronald H A; Tijsterman, Marcel; Bos, Johannes L; Zwartkruis, Fried J T

2007-01-01

The small Ras-like GTPase Rap1 has been identified as a regulator of integrin activation and cadherin-mediated cell–cell contacts. Surprisingly, null mutants of RAP-1 in Caenorhabditis elegans are viable and fertile. In a synthetic lethal RNAi screen with C. elegans rap-1 mutants, the Ras-like GTPase ral-1 emerged as one of seven genes specifically required for viability. Depletion of exoc-8 and sec-5, encoding two putative RAL-1 effectors and members of the exocyst complex, also caused lethality of rap-1 mutants, but did not affect wild-type worms. The RAP-1 and the RAL-1/exocyst pathway appear to coordinate hypodermal cell movement and elongation during embryonic development. They mediate their effect in part through targeting the α-catenin homologue HMP-1 to the lateral membrane. Genetic interactions show that the RAP-1 and RAL-1/exocyst pathway also act in parallel during larval stages. Together these data provide in vivo evidence for the exocyst complex as a downstream RAL-1 effector in cell migration. PMID:17989692

RAP-1 and the RAL-1/exocyst pathway coordinate hypodermal cell organization in Caenorhabditis elegans.

PubMed

Frische, Ester W; Pellis-van Berkel, Wendy; van Haaften, Gijs; Cuppen, Edwin; Plasterk, Ronald H A; Tijsterman, Marcel; Bos, Johannes L; Zwartkruis, Fried J T

2007-12-12

The small Ras-like GTPase Rap1 has been identified as a regulator of integrin activation and cadherin-mediated cell-cell contacts. Surprisingly, null mutants of RAP-1 in Caenorhabditis elegans are viable and fertile. In a synthetic lethal RNAi screen with C. elegans rap-1 mutants, the Ras-like GTPase ral-1 emerged as one of seven genes specifically required for viability. Depletion of exoc-8 and sec-5, encoding two putative RAL-1 effectors and members of the exocyst complex, also caused lethality of rap-1 mutants, but did not affect wild-type worms. The RAP-1 and the RAL-1/exocyst pathway appear to coordinate hypodermal cell movement and elongation during embryonic development. They mediate their effect in part through targeting the alpha-catenin homologue HMP-1 to the lateral membrane. Genetic interactions show that the RAP-1 and RAL-1/exocyst pathway also act in parallel during larval stages. Together these data provide in vivo evidence for the exocyst complex as a downstream RAL-1 effector in cell migration.

PiggyBac Transposon-Mediated Mutagenesis in Rats Reveals a Crucial Role of Bbx in Growth and Male Fertility1

PubMed Central

Wang, Chieh-Ying; Tang, Ming-Chu; Chang, Wen-Chi; Furushima, Kenryo; Jang, Chuan-Wei; Behringer, Richard R; Chen, Chun-Ming

2016-01-01

Bobby sox homolog (Bbx) is an evolutionally conserved gene, but its biological function remains elusive. Here, we characterized defects of Bbx mutant rats that were created by PiggyBac-mediated insertional mutagenesis. Smaller body size and male infertility were the two major phenotypes of homozygous Bbx mutants. Bbx expression profile analysis showed that Bbx was more highly expressed in the testis and pituitary gland than in other organs. Histology and hormonal gene expression analysis of control and Bbx-null pituitary glands showed that loss of Bbx appeared to be dispensable for pituitary histogenesis and the expression of major hormones. BBX was localized in the nuclei of postmeiotic spermatids and Sertoli cells in wild-type testes, but absent in mutant testes. An increased presence of aberrant multinuclear giant cells and apoptotic cells was observed in mutant seminiferous tubules. TUNEL-positive cells costained with CREM (round spermatid marker), but not PLZF (spermatogonia marker), gammaH2Ax (meiotic spermatocyte marker), or GATA4 (Sertoli cell marker). Finally, there were drastically reduced numbers and motility of epididymal sperm from Bbx-null rats. These results suggest that loss of BBX induces apoptosis of postmeiotic spermatids and results in spermiogenesis defects and infertility. PMID:27465138

The ADAR RNA editing enzyme controls neuronal excitability in Drosophila melanogaster

PubMed Central

Li, Xianghua; Overton, Ian M.; Baines, Richard A.; Keegan, Liam P.; O’Connell, Mary A.

2014-01-01

RNA editing by deamination of specific adenosine bases to inosines during pre-mRNA processing generates edited isoforms of proteins. Recoding RNA editing is more widespread in Drosophila than in vertebrates. Editing levels rise strongly at metamorphosis, and Adar5G1 null mutant flies lack editing events in hundreds of CNS transcripts; mutant flies have reduced viability, severely defective locomotion and age-dependent neurodegeneration. On the other hand, overexpressing an adult dADAR isoform with high enzymatic activity ubiquitously during larval and pupal stages is lethal. Advantage was taken of this to screen for genetic modifiers; Adar overexpression lethality is rescued by reduced dosage of the Rdl (Resistant to dieldrin), gene encoding a subunit of inhibitory GABA receptors. Reduced dosage of the Gad1 gene encoding the GABA synthetase also rescues Adar overexpression lethality. Drosophila Adar5G1 mutant phenotypes are ameliorated by feeding GABA modulators. We demonstrate that neuronal excitability is linked to dADAR expression levels in individual neurons; Adar-overexpressing larval motor neurons show reduced excitability whereas Adar5G1 null mutant or targeted Adar knockdown motor neurons exhibit increased excitability. GABA inhibitory signalling is impaired in human epileptic and autistic conditions, and vertebrate ADARs may have a relevant evolutionarily conserved control over neuronal excitability. PMID:24137011

Cloning and Sequencing of a Candida albicans Catalase Gene and Effects of Disruption of This Gene†

PubMed Central

Wysong, Deborah R.; Christin, Laurent; Sugar, Alan M.; Robbins, Phillips W.; Diamond, Richard D.

1998-01-01

Catalase plays a key role as an antioxidant, protecting aerobic organisms from the toxic effects of hydrogen peroxide, and in some cases has been postulated to be a virulence factor. To help elucidate the function of catalase in Candida albicans, a single C. albicans-derived catalase gene, designated CAT1, was isolated and cloned. Degenerate PCR primers based on highly conserved areas of other fungal catalase genes were used to amplify a 411-bp product from genomic DNA of C. albicans ATCC 10261. By using this product as a probe, catalase clones were isolated from genomic libraries of C. albicans. Nucleotide sequence analysis revealed an open reading frame encoding a protein of 487 amino acid residues. Construction of a CAT1-deficient mutant was achieved by using the Ura-blaster technique for sequential disruption of multiple alleles by integrative transformation using URA3 as a selectable marker. Resulting mutants exhibited normal morphology and comparable growth rates of both yeast and mycelial forms. Enzymatic analysis revealed an abundance of catalase in the wild-type strain but decreasing catalase activity in heterozygous mutants and no detectable catalase in a homozygous null mutant. In vitro assays showed the mutant strains to be more sensitive to damage by both neutrophils and concentrations of exogenous peroxide that were sublethal for the parental strain. Compared to the parental strain, the homozygous null mutant strain was far less virulent for mice in an intravenous infection model of disseminated candidiasis. Definitive linkage of CAT1 with virulence would require restoration of activity by reintroduction of the gene into mutants. However, initial results in mice, taken together with the enhanced susceptibility of catalase-deficient hyphae to damage by human neutrophils, suggest that catalase may enhance the pathogenicity of C. albicans. PMID:9573075

Role of Iron Uptake Systems in Pseudomonas aeruginosa Virulence and Airway Infection

PubMed Central

Minandri, Fabrizia; Imperi, Francesco; Frangipani, Emanuela; Bonchi, Carlo; Visaggio, Daniela; Facchini, Marcella; Pasquali, Paolo; Bragonzi, Alessandra

2016-01-01

Pseudomonas aeruginosa is a leading cause of hospital-acquired pneumonia and chronic lung infections in cystic fibrosis patients. Iron is essential for bacterial growth, and P. aeruginosa expresses multiple iron uptake systems, whose role in lung infection deserves further investigation. P. aeruginosa Fe3+ uptake systems include the pyoverdine and pyochelin siderophores and two systems for heme uptake, all of which are dependent on the TonB energy transducer. P. aeruginosa also has the FeoB transporter for Fe2+ acquisition. To assess the roles of individual iron uptake systems in P. aeruginosa lung infection, single and double deletion mutants were generated in P. aeruginosa PAO1 and characterized in vitro, using iron-poor media and human serum, and in vivo, using a mouse model of lung infection. The iron uptake-null mutant (tonB1 feoB) and the Fe3+ transport mutant (tonB1) did not grow aerobically under low-iron conditions and were avirulent in the mouse model. Conversely, the wild type and the feoB, hasR phuR (heme uptake), and pchD (pyochelin) mutants grew in vitro and caused 60 to 90% mortality in mice. The pyoverdine mutant (pvdA) and the siderophore-null mutant (pvdA pchD) grew aerobically in iron-poor media but not in human serum, and they caused low mortality in mice (10 to 20%). To differentiate the roles of pyoverdine in iron uptake and virulence regulation, a pvdA fpvR double mutant defective in pyoverdine production but expressing wild-type levels of pyoverdine-regulated virulence factors was generated. Deletion of fpvR in the pvdA background partially restored the lethal phenotype, indicating that pyoverdine contributes to the pathogenesis of P. aeruginosa lung infection by combining iron transport and virulence-inducing capabilities. PMID:27271740

In vivo neuronal function of the fragile X mental retardation protein is regulated by phosphorylation

PubMed Central

Coffee, R. Lane; Williamson, Ashley J.; Adkins, Christopher M.; Gray, Marisa C.; Page, Terry L.; Broadie, Kendal

2012-01-01

Fragile X syndrome (FXS), caused by loss of the Fragile X Mental Retardation 1 (FMR1) gene product (FMRP), is the most common heritable cause of intellectual disability and autism spectrum disorders. It has been long hypothesized that the phosphorylation of serine 500 (S500) in human FMRP controls its function as an RNA-binding translational repressor. To test this hypothesis in vivo, we employed neuronally targeted expression of three human FMR1 transgenes, including wild-type (hFMR1), dephosphomimetic (S500A-hFMR1) and phosphomimetic (S500D-hFMR1), in the Drosophila FXS disease model to investigate phosphorylation requirements. At the molecular level, dfmr1 null mutants exhibit elevated brain protein levels due to loss of translational repressor activity. This defect is rescued for an individual target protein and across the population of brain proteins by the phosphomimetic, whereas the dephosphomimetic phenocopies the null condition. At the cellular level, dfmr1 null synapse architecture exhibits increased area, branching and bouton number. The phosphomimetic fully rescues these synaptogenesis defects, whereas the dephosphomimetic provides no rescue. The presence of Futsch-positive (microtubule-associated protein 1B) supernumerary microtubule loops is elevated in dfmr1 null synapses. The human phosphomimetic restores normal Futsch loops, whereas the dephosphomimetic provides no activity. At the behavioral level, dfmr1 null mutants exhibit strongly impaired olfactory associative learning. The human phosphomimetic targeted only to the brain-learning center restores normal learning ability, whereas the dephosphomimetic provides absolutely no rescue. We conclude that human FMRP S500 phosphorylation is necessary for its in vivo function as a neuronal translational repressor and regulator of synaptic architecture, and for the manifestation of FMRP-dependent learning behavior. PMID:22080836

The Leishmania major BBSome subunit BBS1 is essential for parasite virulence in the mammalian host

PubMed Central

Price, Helen P; Paape, Daniel; Hodgkinson, Michael R; Farrant, Katie; Doehl, Johannes; Stark, Meg; Smith, Deborah F

2013-01-01

Summary Bardet–Biedl syndrome (BBS) is a human genetic disorder with a spectrum of symptoms caused by primary cilium dysfunction. The disease is caused by mutations in one of at least 17 identified genes, of which seven encode subunits of the BBSome, a protein complex required for specific trafficking events to and from the primary cilium. The molecular mechanisms associated with BBSome function remain to be fully elucidated. Here, we generated null and complemented mutants of the BBSome subunit BBS1 in the protozoan parasite, Leishmania. In the absence of BBS1, extracellular parasites have no apparent defects in growth, flagellum assembly, motility or differentiation in vitro but there is accumulation of vacuole-like structures close to the flagellar pocket. Infectivity of these parasites for macrophages in vitro is reduced compared with wild-type controls but the null parasites retain the ability to differentiate to the intracellular amastigote stage. However, infectivity of BBS1 null parasites is severely compromised in a BALB/c mouse footpad model. We hypothesize that the absence of BBS1 in Leishmania leads to defects in specific trafficking events that affect parasite persistence in the host. This is the first report of an association between the BBSome complex and pathogen infectivity. PMID:23998526

Mutation of the Na+/K+-ATPase Atp1a1a.1 causes QT interval prolongation and bradycardia in zebrafish.

PubMed

Pott, Alexander; Bock, Sarah; Berger, Ina M; Frese, Karen; Dahme, Tillman; Keßler, Mirjam; Rinné, Susanne; Decher, Niels; Just, Steffen; Rottbauer, Wolfgang

2018-05-08

The genetic underpinnings that orchestrate the vertebrate heart rate are not fully understood yet, but of high clinical importance, since diseases of cardiac impulse formation and propagation are common and severe human arrhythmias. To identify novel regulators of the vertebrate heart rate, we deciphered the pathogenesis of the bradycardia in the homozygous zebrafish mutant hiphop (hip) and identified a missense-mutation (N851K) in Na + /K + -ATPase α1-subunit (atp1a1a.1). N851K affects zebrafish Na + /K + -ATPase ion transport capacity, as revealed by in vitro pump current measurements. Inhibition of the Na + /K + -ATPase in vivo indicates that hip rather acts as a hypomorph than being a null allele. Consequently, reduced Na + /K + -ATPase function leads to prolonged QT interval and refractoriness in the hip mutant heart, as shown by electrocardiogram and in vivo electrical stimulation experiments. We here demonstrate for the first time that Na + /K + -ATPase plays an essential role in heart rate regulation by prolonging myocardial repolarization. Copyright © 2018. Published by Elsevier Ltd.

THE α-GLYCEROPHOSPHATE CYCLE IN DROSOPHILA MELANOGASTER

PubMed Central

O'Brien, Stephen J.; Shimada, Yoshio

1974-01-01

"Null" mutations previously isolated at the αGpdh-1 locus of Drosophila melanogaster, because of disruption of the energy-producing α-glycerophosphate cycle, severely restrict the flight ability and relative viability of affected individuals. Two "null" alleles, αGpdh-1 BO-1-4, and αGpdh-1 BO-1-5, when made hemizygous with a deficiency of the αGpdh-1 locus, Df(2L)GdhA, were rendered homozygous by recombination with and selective elimination of the Df(2L)GdhA chromosome. After over 25 generations, a homozygous αGpdh-1 BO-1-4 stock regained the ability to fly despite the continued absence of measurable αGPDH activity. Inter se heterozygotes of three noncomplementing αGpdh-1 "null" alleles and the "adapted" αGpdh-1 BO-1-4 homozygotes were examined for metabolic enzymatic activities related to the energy-producing and pyridine nucleotide-regulating functions of the α-glycerophosphate cycle in Drosophila. The enzyme functions tested included glyceraldehyde-3-phosphate dehydrogenase, cytoplasmic and soluble malate dehydrogenase, lactate dehydrogenase, mitochondrial NADH oxidation, oxidative phosphorylation, and respiratory control with the substrates α-glycerophosphate, succinate, and pyruvate. These activities in any of the mutant genotypes in early adult life were indistinguishable from those in the wild type. There was, however, a premature deterioration and atrophy of the ultrastructural integrity of flight muscle sarcosomes observed by electron microscopy in the "null" mutants. These observations were correlated with a decrease in state 3 mitochondrial oxidation with α-glycerophosphate, succinate, and pyruvate, as well as with loss of respiratory control in adults as early as 2 wk after eclosion. Such observations, which normally are seen in aged dipterans, were accompanied by premature mortality of the mutant heterozygotes. The adapted αGpdh-1 BO-1-4 was identical with wild type in each of the aging characters with the single exception of lowered rates of mitochondrial oxidative phosphorylation. PMID:4154945

Characterisation of a DNA sequence element that directs Dictyostelium stalk cell-specific gene expression.

PubMed

Ceccarelli, A; Zhukovskaya, N; Kawata, T; Bozzaro, S; Williams, J

2000-12-01

The ecmB gene of Dictyostelium is expressed at culmination both in the prestalk cells that enter the stalk tube and in ancillary stalk cell structures such as the basal disc. Stalk tube-specific expression is regulated by sequence elements within the cap-site proximal part of the promoter, the stalk tube (ST) promoter region. Dd-STATa, a member of the STAT transcription factor family, binds to elements present in the ST promoter-region and represses transcription prior to entry into the stalk tube. We have characterised an activatory DNA sequence element, that lies distal to the repressor elements and that is both necessary and sufficient for expression within the stalk tube. We have mapped this activator to a 28 nucleotide region (the 28-mer) within which we have identified a GA-containing sequence element that is required for efficient gene transcription. The Dd-STATa protein binds to the 28-mer in an in vitro binding assay, and binding is dependent upon the GA-containing sequence. However, the ecmB gene is expressed in a Dd-STATa null mutant, therefore Dd-STATa cannot be responsible for activating the 28-mer in vivo. Instead, we identified a distinct 28-mer binding activity in nuclear extracts from the Dd-STATa null mutant, the activity of this GA binding activity being largely masked in wild type extracts by the high affinity binding of the Dd-STATa protein. We suggest, that in addition to the long range repression exerted by binding to the two known repressor sites, Dd-STATa inhibits transcription by direct competition with this putative activator for binding to the GA sequence.

Isocitrate dehydrogenase 1 mutations prime the all-trans retinoic acid myeloid differentiation pathway in acute myeloid leukemia

PubMed Central

Boutzen, Héléna; Saland, Estelle; Larrue, Clément; de Toni, Fabienne; Gales, Lara; Castelli, Florence A.; Cathebas, Mathilde; Zaghdoudi, Sonia; Stuani, Lucille; Kaoma, Tony; Riscal, Romain; Yang, Guangli; Hirsch, Pierre; David, Marion; De Mas-Mansat, Véronique; Delabesse, Eric; Vallar, Laurent; Delhommeau, François; Jouanin, Isabelle; Ouerfelli, Ouathek; Le Cam, Laurent; Linares, Laetitia K.; Junot, Christophe; Portais, Jean-Charles; Vergez, François; Récher, Christian

2016-01-01

Acute myeloid leukemia (AML) is characterized by the accumulation of malignant blasts with impaired differentiation programs caused by recurrent mutations, such as the isocitrate dehydrogenase (IDH) mutations found in 15% of AML patients. These mutations result in the production of the oncometabolite (R)-2-hydroxyglutarate (2-HG), leading to a hypermethylation phenotype that dysregulates hematopoietic differentiation. In this study, we identified mutant R132H IDH1-specific gene signatures regulated by key transcription factors, particularly CEBPα, involved in myeloid differentiation and retinoid responsiveness. We show that treatment with all-trans retinoic acid (ATRA) at clinically achievable doses markedly enhanced terminal granulocytic differentiation in AML cell lines, primary patient samples, and a xenograft mouse model carrying mutant IDH1. Moreover, treatment with a cell-permeable form of 2-HG sensitized wild-type IDH1 AML cells to ATRA-induced myeloid differentiation, whereas inhibition of 2-HG production significantly reduced ATRA effects in mutant IDH1 cells. ATRA treatment specifically decreased cell viability and induced apoptosis of mutant IDH1 blasts in vitro. ATRA also reduced tumor burden of mutant IDH1 AML cells xenografted in NOD–Scid–IL2rγnull mice and markedly increased overall survival, revealing a potent antileukemic effect of ATRA in the presence of IDH1 mutation. This therapeutic strategy holds promise for this AML patient subgroup in future clinical studies. PMID:26951332

Drosophila Rolling Blackout Displays Lipase Domain-Dependent and Independent Endocytic Functions Downstream of Dynamin

PubMed Central

Vijayakrishnan, Niranjana; Phillips, Scott E.; Broadie, Kendal

2010-01-01

Drosophila temperature-sensitive rolling blackout (rbots) mutants display a total block of endocytosis in non-neuronal cells and a weaker, partial defect at neuronal synapses. RBO is an integral plasma membrane protein and is predicted to be a serine esterase. To determine if lipase activity is required for RBO function, we mutated the catalytic serine 358 to alanine in the G-X-S-X-G active site, and assayed genomic rescue of rbo mutant non-neuronal and neuronal phenotypes. The rboS358A mutant is unable to rescue rbo null 100% embryonic lethality, indicating that the lipase-domain is critical for RBO essential function. Likewise, the rboS358A mutant cannot provide any rescue of endocytic blockade in rbots Garland cells, demonstrating that the lipase-domain is indispensable for non-neuronal endocytosis. In contrast, rbots conditional paralysis, synaptic transmission block and synapse endocytic defects are all fully rescued by the rboS358A mutant, showing that the RBO lipase-domain is dispensable in neuronal contexts. We identified a synthetic lethal interaction between rbots and the well-characterized dynamin GTPase conditional shibire (shits1) mutant. In both non-neuronal cells and neuronal synapses, shits1;rbots phenocopies shits1 endocytic defects, indicating that dynamin and RBO act in the same pathway, with dynamin functioning upstream of RBO. We conclude that RBO possesses both lipase-domain dependent and scaffolding functions with differential requirements in non-neuronal versus neuronal endocytosis mechanisms downstream of dynamin GTPase activity. PMID:21029287

Over-expression of COQ10 in Saccharomyces cerevisiae inhibits mitochondrial respiration.

PubMed

Zampol, Mariana A; Busso, Cleverson; Gomes, Fernando; Ferreira-Junior, Jose Ribamar; Tzagoloff, Alexander; Barros, Mario H

2010-11-05

COQ10 deletion in Saccharomyces cerevisiae elicits a defect in mitochondrial respiration correctable by addition of coenzyme Q(2). Rescue of respiration by Q(2) is a characteristic of mutants blocked in coenzyme Q(6) synthesis. Unlike Q(6) deficient mutants, mitochondria of the coq10 null mutant have wild-type concentrations of Q(6). The physiological significance of earlier observations that purified Coq10p contains bound Q(6) was examined in the present study by testing the in vivo effect of over-expression of Coq10p on respiration. Mitochondria with elevated levels of Coq10p display reduced respiration in the bc1 span of the electron transport chain, which can be restored with exogenous Q(2). This suggests that in vivo binding of Q(6) by excess Coq10p reduces the pool of this redox carrier available for its normal function in providing electrons to the bc1 complex. This is confirmed by observing that extra Coq8p relieves the inhibitory effect of excess Coq10p. Coq8p is a putative kinase, and a high-copy suppressor of the coq10 null mutant. As shown here, when over-produced in coq mutants, Coq8p counteracts turnover of Coq3p and Coq4p subunits of the Q-biosynthetic complex. This can account for the observed rescue by COQ8 of the respiratory defect in strains over-producing Coq10p. Copyright © 2010 Elsevier Inc. All rights reserved.

Functional Analysis of Human NF1 in Drosophila

DTIC Science & Technology

2008-12-01

also have learning problem. Such learning phenotypes have been recapitulated in animal models, including in mouse and Drosophila mutants. This proposal...by examining the phenotypes of mutated human genes expressed in Drosophila NF1 null mutants. We also propose that Gsα/NF1 activated AC pathway...in both Drosophila and mouse NF1 models. Our previous work has shown that defective cAMP signaling leads to the learning phenotype in Drosophila Nf1

Suppression of gain-of-function mutant p53 with metabolic inhibitors reduces tumor growth in vivo

PubMed Central

Jung, Chae Lim; Mun, Hyemin; Jo, Se-Young; Oh, Ju-Hee; Lee, ChuHee; Choi, Eun-Kyung; Jang, Se Jin; Suh, Young-Ah

2016-01-01

Mutation of p53 occasionally results in a gain of function, which promotes tumor growth. We asked whether destabilizing the gain-of-function protein would kill tumor cells. Downregulation of the gene reduced cell proliferation in p53-mutant cells, but not in p53-null cells, indicating that the former depended on the mutant protein for survival. Moreover, phenformin and 2-deoxyglucose suppressed cell growth and simultaneously destabilized mutant p53. The AMPK pathway, MAPK pathway, chaperone proteins and ubiquitination all contributed to this process. Interestingly, phenformin and 2-deoxyglucose also reduced tumor growth in syngeneic mice harboring the p53 mutation. Thus, destabilizing mutant p53 protein in order to kill cells exhibiting “oncogene addiction” could be a promising strategy for combatting p53 mutant tumors. PMID:27765910

Suppression of gain-of-function mutant p53 with metabolic inhibitors reduces tumor growth in vivo.

PubMed

Jung, Chae Lim; Mun, Hyemin; Jo, Se-Young; Oh, Ju-Hee; Lee, ChuHee; Choi, Eun-Kyung; Jang, Se Jin; Suh, Young-Ah

2016-11-22

Mutation of p53 occasionally results in a gain of function, which promotes tumor growth. We asked whether destabilizing the gain-of-function protein would kill tumor cells. Downregulation of the gene reduced cell proliferation in p53-mutant cells, but not in p53-null cells, indicating that the former depended on the mutant protein for survival. Moreover, phenformin and 2-deoxyglucose suppressed cell growth and simultaneously destabilized mutant p53. The AMPK pathway, MAPK pathway, chaperone proteins and ubiquitination all contributed to this process. Interestingly, phenformin and 2-deoxyglucose also reduced tumor growth in syngeneic mice harboring the p53 mutation. Thus, destabilizing mutant p53 protein in order to kill cells exhibiting "oncogene addiction" could be a promising strategy for combatting p53 mutant tumors.

Trehalose synthesis in Aspergillus niger: characterization of six homologous genes, all with conserved orthologs in related species

PubMed Central

2014-01-01

Background The disaccharide trehalose is a major component of fungal spores and is released upon germination. Moreover, the sugar is well known for is protective functions, e.g. against thermal stress and dehydration. The properties and synthesis of trehalose have been well investigated in the bakers’ yeast Saccharomyces cerevisiae. In filamentous fungi, such knowledge is limited, although several gene products have been identified. Results Using Aspergillus niger as a model fungus, the aim of this study was to provide an overview of all genes involved in trehalose synthesis. This fungus has three potential trehalose-6-phosphate synthase encoding genes, tpsA-C, and three putative trehalose phosphate phosphatase encoding genes, tppA-C, of which two have not previously been identified. Expression of all six genes was confirmed using real-time PCR, and conserved orthologs could be identified in related Aspergilli. Using a two-hybrid approach, there is a strong indication that four of the proteins physically interact, as has previously been shown in S. cerevisiae. When creating null mutants of all the six genes, three of them, ΔtpsA, ΔtppA and ΔtppB, had lower internal trehalose contents. The only mutant with a pronounced morphological difference was ΔtppA, in which sporulation was severely reduced with abnormal conidiophores. This was also the only mutant with accumulated levels of trehalose-6-phosphate, indicating that the encoded protein is the main phosphatase under normal conditions. Besides ΔtppA, the most studied deletion mutant in this work was ΔtppB. This gene encodes a protein conserved in filamentous Ascomycota. The ΔtppB mutant displayed a low, but not depleted, internal trehalose content, and conidia were more susceptible to thermal stress. Conclusion A. niger contains at least 6 genes putatively involved in trehalose synthesis. Gene expressions related to germination have been quantified and deletion mutants characterized: Mutants lacking tpsA, tppA or tppB have reduced internal trehalose contents. Furthermore, tppA, under normal conditions, encodes the functional trehalose-6-phosphate-phosphatase. PMID:24725382

A Novel Ras-interacting Protein Required for Chemotaxis and Cyclic Adenosine Monophosphate Signal Relay in Dictyostelium

PubMed Central

Lee, Susan; Parent, Carole A.; Insall, Robert; Firtel, Richard A.

1999-01-01

We have identified a novel Ras-interacting protein from Dictyostelium, RIP3, whose function is required for both chemotaxis and the synthesis and relay of the cyclic AMP (cAMP) chemoattractant signal. rip3 null cells are unable to aggregate and lack receptor activation of adenylyl cyclase but are able, in response to cAMP, to induce aggregation-stage, postaggregative, and cell-type-specific gene expression in suspension culture. In addition, rip3 null cells are unable to properly polarize in a cAMP gradient and chemotaxis is highly impaired. We demonstrate that cAMP stimulation of guanylyl cyclase, which is required for chemotaxis, is reduced ∼60% in rip3 null cells. This reduced activation of guanylyl cyclase may account, in part, for the defect in chemotaxis. When cells are pulsed with cAMP for 5 h to mimic the endogenous cAMP oscillations that occur in wild-type strains, the cells will form aggregates, most of which, however, arrest at the mound stage. Unlike the response seen in wild-type strains, the rip3 null cell aggregates that form under these experimental conditions are very small, which is probably due to the rip3 null cell chemotaxis defect. Many of the phenotypes of the rip3 null cell, including the inability to activate adenylyl cyclase in response to cAMP and defects in chemotaxis, are very similar to those of strains carrying a disruption of the gene encoding the putative Ras exchange factor AleA. We demonstrate that aleA null cells also exhibit a defect in cAMP-mediated activation of guanylyl cyclase similar to that of rip3 null cells. A double-knockout mutant (rip3/aleA null cells) exhibits a further reduction in receptor activation of guanylyl cyclase, and these cells display almost no cell polarization or movement in cAMP gradients. As RIP3 preferentially interacts with an activated form of the Dictyostelium Ras protein RasG, which itself is important for cell movement, we propose that RIP3 and AleA are components of a Ras-regulated pathway involved in integrating chemotaxis and signal relay pathways that are essential for aggregation. PMID:10473630

A method distinguishing expressed vs. null mutations of the Col1A1 gene in osteogenesis imperfecta

DOE Office of Scientific and Technical Information (OSTI.GOV)

Redford-Badwal, D.A.; Stover, M.L.; McKinstry, M.

Osteogenesis imperfecta (OI) is a heterogeneous group of heritable disorders of bone characterized by increased susceptibility to fracture. Most of the causative mutations were identified in patients with the lethal form of the disease. Attention is now shifting to the milder forms of OI where glycine substitutions and null producing mutations have been found. Single amino acid substitutions can be identified by RT/PCR of total cellular RNA, but this approach does not work well for null mutations since the defective transcript does not accumulate in the cytoplasm. We have altered our RNA extraction method to separate RNA from the nuclearmore » and cytoplasmic compartments of cultured fibroblasts. Standard methods of mutation identification (RT/PCR followed by SSCP) is applied to each RNA fraction. DNA from an abnormal band on the SSCP gel is eluted and amplified by PCR for cloning and sequencing. Using this approach we have identified an Asp to Asn change in exon 50 (type II OI) and a Gly to Arg in exon 11 (type I OI) of the COL1A1 gene. These changes were found in both nuclear and cytoplasmic compartments. These putative mutations are currently being confirmed by protein studies. In contrast, three patients with mild OI associated with reduced {proportional_to}(I)mRNA, had distinguishing SSCP bands present in the nuclear but not the cytoplasmic compartment. In one case a frame shift mutation was observed, while the other two revealed polymorphisms. The compartmentalization of the mutant allele has directed us to look elsewhere in the transcript for the causative mutation. This approach to mutation identification is capable of distinguishing these fundamentally different types of mutations and allows for preferential cloning and sequencing of the abnormal allele.« less

NAD Deficiency, Congenital Malformations, and Niacin Supplementation.

PubMed

Shi, Hongjun; Enriquez, Annabelle; Rapadas, Melissa; Martin, Ella M M A; Wang, Roni; Moreau, Julie; Lim, Chai K; Szot, Justin O; Ip, Eddie; Hughes, James N; Sugimoto, Kotaro; Humphreys, David T; McInerney-Leo, Aideen M; Leo, Paul J; Maghzal, Ghassan J; Halliday, Jake; Smith, Janine; Colley, Alison; Mark, Paul R; Collins, Felicity; Sillence, David O; Winlaw, David S; Ho, Joshua W K; Guillemin, Gilles J; Brown, Matthew A; Kikuchi, Kazu; Thomas, Paul Q; Stocker, Roland; Giannoulatou, Eleni; Chapman, Gavin; Duncan, Emma L; Sparrow, Duncan B; Dunwoodie, Sally L

2017-08-10

Congenital malformations can be manifested as combinations of phenotypes that co-occur more often than expected by chance. In many such cases, it has proved difficult to identify a genetic cause. We sought the genetic cause of cardiac, vertebral, and renal defects, among others, in unrelated patients. We used genomic sequencing to identify potentially pathogenic gene variants in families in which a person had multiple congenital malformations. We tested the function of the variant by using assays of in vitro enzyme activity and by quantifying metabolites in patient plasma. We engineered mouse models with similar variants using the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 system. Variants were identified in two genes that encode enzymes of the kynurenine pathway, 3-hydroxyanthranilic acid 3,4-dioxygenase (HAAO) and kynureninase (KYNU). Three patients carried homozygous variants predicting loss-of-function changes in the HAAO or KYNU proteins (HAAO p.D162*, HAAO p.W186*, or KYNU p.V57Efs*21). Another patient carried heterozygous KYNU variants (p.Y156* and p.F349Kfs*4). The mutant enzymes had greatly reduced activity in vitro. Nicotinamide adenine dinucleotide (NAD) is synthesized de novo from tryptophan through the kynurenine pathway. The patients had reduced levels of circulating NAD. Defects similar to those in the patients developed in the embryos of Haao-null or Kynu-null mice owing to NAD deficiency. In null mice, the prevention of NAD deficiency during gestation averted defects. Disruption of NAD synthesis caused a deficiency of NAD and congenital malformations in humans and mice. Niacin supplementation during gestation prevented the malformations in mice. (Funded by the National Health and Medical Research Council of Australia and others.).

Unique and shared functions of nuclear lamina LEM domain proteins in Drosophila.

PubMed

Barton, Lacy J; Wilmington, Shameika R; Martin, Melinda J; Skopec, Hannah M; Lovander, Kaylee E; Pinto, Belinda S; Geyer, Pamela K

2014-06-01

The nuclear lamina is an extensive protein network that contributes to nuclear structure and function. LEM domain (LAP2, emerin, MAN1 domain, LEM-D) proteins are components of the nuclear lamina, identified by a shared ∼45-amino-acid motif that binds Barrier-to-autointegration factor (BAF), a chromatin-interacting protein. Drosophila melanogaster has three nuclear lamina LEM-D proteins, named Otefin (Ote), Bocksbeutel (Bocks), and dMAN1. Although these LEM-D proteins are globally expressed, loss of either Ote or dMAN1 causes tissue-specific defects in adult flies that differ from each other. The reason for such distinct tissue-restricted defects is unknown. Here, we generated null alleles of bocks, finding that loss of Bocks causes no overt adult phenotypes. Next, we defined phenotypes associated with lem-d double mutants. Although the absence of individual LEM-D proteins does not affect viability, loss of any two proteins causes lethality. Mutant phenotypes displayed by lem-d double mutants differ from baf mutants, suggesting that BAF function is retained in animals with a single nuclear lamina LEM-D protein. Interestingly, lem-d double mutants displayed distinct developmental and cellular mutant phenotypes, suggesting that Drosophila LEM-D proteins have developmental functions that are differentially shared with other LEM-D family members. This conclusion is supported by studies showing that ectopically produced LEM-D proteins have distinct capacities to rescue the tissue-specific phenotypes found in single lem-d mutants. Our findings predict that cell-specific mutant phenotypes caused by loss of LEM-D proteins reflect both the constellation of LEM-D proteins within the nuclear lamina and the capacity of functional compensation of the remaining LEM-D proteins. Copyright © 2014 by the Genetics Society of America.

Unique and Shared Functions of Nuclear Lamina LEM Domain Proteins in Drosophila

PubMed Central

Barton, Lacy J.; Wilmington, Shameika R.; Martin, Melinda J.; Skopec, Hannah M.; Lovander, Kaylee E.; Pinto, Belinda S.; Geyer, Pamela K.

2014-01-01

The nuclear lamina is an extensive protein network that contributes to nuclear structure and function. LEM domain (LAP2, emerin, MAN1 domain, LEM-D) proteins are components of the nuclear lamina, identified by a shared ∼45-amino-acid motif that binds Barrier-to-autointegration factor (BAF), a chromatin-interacting protein. Drosophila melanogaster has three nuclear lamina LEM-D proteins, named Otefin (Ote), Bocksbeutel (Bocks), and dMAN1. Although these LEM-D proteins are globally expressed, loss of either Ote or dMAN1 causes tissue-specific defects in adult flies that differ from each other. The reason for such distinct tissue-restricted defects is unknown. Here, we generated null alleles of bocks, finding that loss of Bocks causes no overt adult phenotypes. Next, we defined phenotypes associated with lem-d double mutants. Although the absence of individual LEM-D proteins does not affect viability, loss of any two proteins causes lethality. Mutant phenotypes displayed by lem-d double mutants differ from baf mutants, suggesting that BAF function is retained in animals with a single nuclear lamina LEM-D protein. Interestingly, lem-d double mutants displayed distinct developmental and cellular mutant phenotypes, suggesting that Drosophila LEM-D proteins have developmental functions that are differentially shared with other LEM-D family members. This conclusion is supported by studies showing that ectopically produced LEM-D proteins have distinct capacities to rescue the tissue-specific phenotypes found in single lem-d mutants. Our findings predict that cell-specific mutant phenotypes caused by loss of LEM-D proteins reflect both the constellation of LEM-D proteins within the nuclear lamina and the capacity of functional compensation of the remaining LEM-D proteins. PMID:24700158

A retrospective: Use of Escherichia coli as a vehicle to study phospholipid synthesis and function

PubMed Central

Dowhan, William

2012-01-01

Although the study of individual phospholipids and their synthesis began in the 1920’s first in plants and then mammals, it was not until the early 1960’s that Eugene Kennedy using Escherichia coli initiated studies of bacterial phospholipid metabolism. With the base of information already available from studies of mammalian tissue, the basic blueprint of phospholipid biosynthesis in E. coli was worked out by the late 1960’s. In 1970’s and 1980’s most of the enzymes responsible for phospholipid biosynthesis were purified and many of the genes encoding these enzymes were identified. By the late 1990’s conditional and null mutants were available along with clones of the genes for every step of phospholipid biosynthesis. Most of these genes had been sequenced before the complete E. coli genome sequence was available. Strains of E. coli were developed in which phospholipid composition could be changed in a systematic manner while maintaining cell viability. Null mutants, strains in which phospholipid metabolism was artificially regulated, and strains synthesizing foreign lipids not found in E. coli have been used to this day to define specific roles for individual phospholipid. This review will trace the findings that have led to the development of E. coli as an excellent model system to study mechanisms underlying the synthesis and function of phospholipids that are widely applicable to other prokaryotic and eukaryotic systems. PMID:22925633

Compensation for PKMζ in long-term potentiation and spatial long-term memory in mutant mice

PubMed Central

Tsokas, Panayiotis; Hsieh, Changchi; Yao, Yudong; Lesburguères, Edith; Wallace, Emma Jane Claire; Tcherepanov, Andrew; Jothianandan, Desingarao; Hartley, Benjamin Rush; Pan, Ling; Rivard, Bruno; Farese, Robert V; Sajan, Mini P; Bergold, Peter John; Hernández, Alejandro Iván; Cottrell, James E; Shouval, Harel Z; Fenton, André Antonio; Sacktor, Todd Charlton

2016-01-01

PKMζ is a persistently active PKC isoform proposed to maintain late-LTP and long-term memory. But late-LTP and memory are maintained without PKMζ in PKMζ-null mice. Two hypotheses can account for these findings. First, PKMζ is unimportant for LTP or memory. Second, PKMζ is essential for late-LTP and long-term memory in wild-type mice, and PKMζ-null mice recruit compensatory mechanisms. We find that whereas PKMζ persistently increases in LTP maintenance in wild-type mice, PKCι/λ, a gene-product closely related to PKMζ, persistently increases in LTP maintenance in PKMζ-null mice. Using a pharmacogenetic approach, we find PKMζ-antisense in hippocampus blocks late-LTP and spatial long-term memory in wild-type mice, but not in PKMζ-null mice without the target mRNA. Conversely, a PKCι/λ-antagonist disrupts late-LTP and spatial memory in PKMζ-null mice but not in wild-type mice. Thus, whereas PKMζ is essential for wild-type LTP and long-term memory, persistent PKCι/λ activation compensates for PKMζ loss in PKMζ-null mice. DOI: http://dx.doi.org/10.7554/eLife.14846.001 PMID:27187150

A magnesium-dependent mreB null mutant: implications for the role of mreB in Bacillus subtilis.

PubMed

Formstone, Alex; Errington, Jeffery

2005-03-01

MreB shares a common prokaryotic ancestor with actin and is present in almost all rod-shaped bacteria. MreB proteins have been implicated in a range of important cell processes, including cell morphogenesis, chromosome segregation and cell polarity. The mreB gene frequently lies at the beginning of a cluster of genes, immediately upstream of the conserved mreC and mreD genes. RNA analysis showed that in Bacillus subtilis mreB is co-transcribed with mreC and that these genes form part of an operon under the control of a promoter(s) upstream of mreB. Construction of an in-frame deletion of mreB and its complementation by mreB(+) only, in trans, established that the gene is important for maintenance of cell width and cell viability under normal growth conditions, independent of polar effects on downstream genes. Remarkably, virtually normal growth was restored to the mreB null mutant in the presence of high concentrations of magnesium, especially when high concentrations of the osmoprotectant, sucrose were also present. Under these conditions, cells could be maintained in the complete absence of an mreB gene, with almost normal morphology. No detectable effect on chromosome segregation was evident in the mutant, nor was there an effect on the topology of nascent peptidoglycan insertion. A GFP-MreB fusion was used to look at the localization of MreB in live cells. The pattern of localization was similar to that previously described, but no tight linkage to nucleoid positioning was evident. Propagation of the mreB null mutant in the absence of magnesium and sucrose led to a progressive increase in cell width, culminating in cell lysis. Cell division was also perturbed but this effect may be secondary to the disturbance in cell width. These results suggest that the major role of MreB in B. subtilis lies in the control of cell diameter.

A BDNF loop-domain mimetic acutely reverses spontaneous apneas and respiratory abnormalities during behavioral arousal in a mouse model of Rett syndrome

PubMed Central

Kron, Miriam; Lang, Min; Adams, Ian T.; Sceniak, Michael; Longo, Frank; Katz, David M.

2014-01-01

Reduced levels of brain-derived neurotrophic factor (BDNF) are thought to contribute to the pathophysiology of Rett syndrome (RTT), a severe neurodevelopmental disorder caused by loss-of-function mutations in the gene encoding methyl-CpG-binding protein 2 (MeCP2). In Mecp2 mutant mice, BDNF deficits have been associated with breathing abnormalities, a core feature of RTT, as well as with synaptic hyperexcitability within the brainstem respiratory network. Application of BDNF can reverse hyperexcitability in acute brainstem slices from Mecp2-null mice, suggesting that therapies targeting BDNF or its receptor, TrkB, could be effective at acute reversal of respiratory abnormalities in RTT. Therefore, we examined the ability of LM22A-4, a small-molecule BDNF loop-domain mimetic and TrkB partial agonist, to modulate synaptic excitability within respiratory cell groups in the brainstem nucleus tractus solitarius (nTS) and to acutely reverse abnormalities in breathing at rest and during behavioral arousal in Mecp2 mutants. Patch-clamp recordings in Mecp2-null brainstem slices demonstrated that LM22A-4 decreases excitability at primary afferent synapses in the nTS by reducing the amplitude of evoked excitatory postsynaptic currents and the frequency of spontaneous and miniature excitatory postsynaptic currents. In vivo, acute treatment of Mecp2-null and -heterozygous mutants with LM22A-4 completely eliminated spontaneous apneas in resting animals, without sedation. Moreover, we demonstrate that respiratory dysregulation during behavioral arousal, a feature of human RTT, is also reversed in Mecp2 mutants by acute treatment with LM22A-4. Together, these data support the hypothesis that reduced BDNF signaling and respiratory dysfunction in RTT are linked, and establish the proof-of-concept that treatment with a small-molecule structural mimetic of a BDNF loop domain and a TrkB partial agonist can acutely reverse abnormal breathing at rest and in response to behavioral arousal in symptomatic RTT mice. PMID:25147297

Drosophila Spastin Regulates Synaptic Microtubule Networks and Is Required for Normal Motor Function

PubMed Central

Sherwood, Nina Tang; Sun, Qi; Xue, Mingshan; Zhang, Bing

2004-01-01

The most common form of human autosomal dominant hereditary spastic paraplegia (AD-HSP) is caused by mutations in the SPG4 (spastin) gene, which encodes an AAA ATPase closely related in sequence to the microtubule-severing protein Katanin. Patients with AD-HSP exhibit degeneration of the distal regions of the longest axons in the spinal cord. Loss-of-function mutations in the Drosophila spastin gene produce larval neuromuscular junction (NMJ) phenotypes. NMJ synaptic boutons in spastin mutants are more numerous and more clustered than in wild-type, and transmitter release is impaired. spastin-null adult flies have severe movement defects. They do not fly or jump, they climb poorly, and they have short lifespans. spastin hypomorphs have weaker behavioral phenotypes. Overexpression of Spastin erases the muscle microtubule network. This gain-of-function phenotype is consistent with the hypothesis that Spastin has microtubule-severing activity, and implies that spastin loss-of-function mutants should have an increased number of microtubules. Surprisingly, however, we observed the opposite phenotype: in spastin-null mutants, there are fewer microtubule bundles within the NMJ, especially in its distal boutons. The Drosophila NMJ is a glutamatergic synapse that resembles excitatory synapses in the mammalian spinal cord, so the reduction of organized presynaptic microtubules that we observe in spastin mutants may be relevant to an understanding of human Spastin's role in maintenance of axon terminals in the spinal cord. PMID:15562320

γ-glutamyl transpeptidase 1 specifically suppresses green-light avoidance via GABAA receptors in Drosophila.

PubMed

Liu, Jiangqu; Gong, Zhefeng; Liu, Li

2014-08-01

Drosophila larvae innately show light avoidance behavior. Compared with robust blue-light avoidance, larvae exhibit relatively weaker green-light responses. In our previous screening for genes involved in larval light avoidance, compared with control w(1118) larvae, larvae with γ-glutamyl transpeptidase 1 (Ggt-1) knockdown or Ggt-1 mutation were found to exhibit higher percentage of green-light avoidance which was mediated by Rhodopsin6 (Rh6) photoreceptors. However, their responses to blue light did not change significantly. By adjusting the expression level of Ggt-1 in different tissues, we found that Ggt-1 in malpighian tubules was both necessary and sufficient for green-light avoidance. Our results showed that glutamate levels were lower in Ggt-1 null mutants compared with controls. Feeding Ggt-1 null mutants glutamate can normalize green-light avoidance, indicating that high glutamate concentrations suppressed larval green-light avoidance. However, rather than directly, glutamate affected green-light avoidance indirectly through GABA, the level of which was also lower in Ggt-1 mutants compared with controls. Mutants in glutamate decarboxylase 1, which encodes GABA synthase, and knockdown lines of the GABAA receptor, both exhibit elevated levels of green-light avoidance. Thus, our results elucidate the neurobiological mechanisms mediating green-light avoidance, which was inhibited in wild-type larvae. © 2014 International Society for Neurochemistry.

Role of urease in megasome formation and Helicobacter pylori survival in macrophages

PubMed Central

Schwartz, Justin T.; Allen, Lee-Ann H.

2007-01-01

Previous studies have demonstrated that Helicobacter pylori (Hp) delays its entry into macrophages and persists inside megasomes, which are poorly acidified and accumulate early endosome autoantigen 1. Herein, we explored the role of Hp urease in bacterial survival in murine peritoneal macrophages and J774 cells. Plasmid-free mutagenesis was used to replace ureA and ureB with cat in Hp Strains 11637 and 11916. ureAB null Hp lacked detectable urease activity and did not express UreA or UreB as judged by immunoblotting. Deletion of ureAB had no effect on Hp binding to macrophages or the rate or extent of phagocytosis. However, intracellular survival of mutant organisms was impaired significantly. Immunofluorescence microscopy demonstrated that (in contrast to parental organisms) mutant Hp resided in single phagosomes, which were acidic and accumulated the lysosome marker lysosome-associated membrane protein-1 but not early endosome autoantigen 1. A similar phenotype was observed for spontaneous urease mutants derived from Hp Strain 60190. Treatment of macrophages with bafilomycin A1, NH4Cl, or chloroquine prevented acidification of phagosomes containing mutant Hp. However, only ammonium chloride enhanced bacterial viability significantly. Rescue of ureAB null organisms was also achieved by surface adsorption of active urease. Altogether, our data indicate a role for urease and urease-derived ammonia in megasome formation and Hp survival. PMID:16543403

Characterization of a Null Allelic Mutant of the Rice NAL1 Gene Reveals Its Role in Regulating Cell Division

PubMed Central

Jiang, Dan; Fang, Jingjing; Lou, Lamei; Zhao, Jinfeng; Yuan, Shoujiang; Yin, Liang; Sun, Wei; Peng, Lixiang; Guo, Baotai; Li, Xueyong

2015-01-01

Leaf morphology is closely associated with cell division. In rice, mutations in Narrow leaf 1 (NAL1) show narrow leaf phenotypes. Previous studies have shown that NAL1 plays a role in regulating vein patterning and increasing grain yield in indica cultivars, but its role in leaf growth and development remains unknown. In this report, we characterized two allelic mutants of NARROW LEAF1 (NAL1), nal1-2 and nal1-3, both of which showed a 50% reduction in leaf width and length, as well as a dwarf culm. Longitudinal and transverse histological analyses of leaves and internodes revealed that cell division was suppressed in the anticlinal orientation but enhanced in the periclinal orientation in the mutants, while cell size remained unaltered. In addition to defects in cell proliferation, the mutants showed abnormal midrib in leaves. Map-based cloning revealed that nal1-2 is a null allelic mutant of NAL1 since both the whole promoter and a 404-bp fragment in the first exon of NAL1 were deleted, and that a 6-bp fragment was deleted in the mutant nal1-3. We demonstrated that NAL1 functions in the regulation of cell division as early as during leaf primordia initiation. The altered transcript level of G1- and S-phase-specific genes suggested that NAL1 affects cell cycle regulation. Heterogenous expression of NAL1 in fission yeast (Schizosaccharomyces pombe) further supported that NAL1 affects cell division. These results suggest that NAL1 controls leaf width and plant height through its effects on cell division. PMID:25658704

Acentriolar mitosis activates a p53-dependent apoptosis pathway in the mouse embryo

PubMed Central

Bazzi, Hisham; Anderson, Kathryn V.

2014-01-01

Centrosomes are the microtubule-organizing centers of animal cells that organize interphase microtubules and mitotic spindles. Centrioles are the microtubule-based structures that organize centrosomes, and a defined set of proteins, including spindle assembly defective-4 (SAS4) (CPAP/CENPJ), is required for centriole biogenesis. The biological functions of centrioles and centrosomes vary among animals, and the functions of mammalian centrosomes have not been genetically defined. Here we use a null mutation in mouse Sas4 to define the cellular and developmental functions of mammalian centrioles in vivo. Sas4-null embryos lack centrosomes but survive until midgestation. As expected, Sas4−/− mutants lack primary cilia and therefore cannot respond to Hedgehog signals, but other developmental signaling pathways are normal in the mutants. Unlike mutants that lack cilia, Sas4−/− embryos show widespread apoptosis associated with global elevated expression of p53. Cell death is rescued in Sas4−/− p53−/− double-mutant embryos, demonstrating that mammalian centrioles prevent activation of a p53-dependent apoptotic pathway. Expression of p53 is not activated by abnormalities in bipolar spindle organization, chromosome segregation, cell-cycle profile, or DNA damage response, which are normal in Sas4−/− mutants. Instead, live imaging shows that the duration of prometaphase is prolonged in the mutants while two acentriolar spindle poles are assembled. Independent experiments show that prolonging spindle assembly is sufficient to trigger p53-dependent apoptosis. We conclude that a short delay in the prometaphase caused by the absence of centrioles activates a previously undescribed p53-dependent cell death pathway in the rapidly dividing cells of the mouse embryo. PMID:24706806

Extracellular matrix family proteins that are potential targets of Dd-STATa in Dictyostelium discoideum.

PubMed

Shimada, Nao; Nishio, Keiko; Maeda, Mineko; Urushihara, Hideko; Kawata, Takefumi

2004-10-01

Dd-STATa is a functional Dictyostelium homologue of metazoan STAT (signal transducers and activators of transcription) proteins, which is activated by cAMP and is thereby translocated into the nuclei of anterior tip cells of the prestalk region of the slug. By using in situ hybridization analyses, we found that the SLF308 cDNA clone, which contains the ecmF gene that encodes a putative extracellular matrix protein and is expressed in the anterior tip cells, was greatly down-regulated in the Dd-STATa-null mutant. Disruption of the ecmF gene, however, resulted in almost no phenotypic change. The absence of any obvious mutant phenotype in the ecmF-null mutant could be due to a redundancy of similar genes. In fact, a search of the Dictyostelium whole genome database demonstrates the existence of an additional 16 homologues, all of which contain a cellulose-binding module. Among these homologues, four genes show Dd-STATa-dependent expression, while the others are Dd-STATa-independent. We discuss the potential role of Dd-STATa in morphogenesis via its effect on the interaction between cellulose and these extracellular matrix family proteins.

Molecular basis for the catalytic inactivity of a naturally occurring near-null variant of human ALOX15.

PubMed

Horn, Thomas; Ivanov, Igor; Di Venere, Almerinda; Kakularam, Kumar Reddy; Reddanna, Pallu; Conrad, Melanie L; Richter, Constanze; Scheerer, Patrick; Kuhn, Hartmut

2013-12-01

Mammalian lipoxygenases belong to a family of lipid-peroxidizing enzymes, which have been implicated in cardiovascular, hyperproliferative and neurodegenerative diseases. Here we report that a naturally occurring mutation in the hALOX15 gene leads to expression of a catalytically near-null enzyme variant (hGly422Glu). The inactivity may be related to severe misfolding of the enzyme protein, which was concluded from CD-spectra as well as from thermal and chemical stability assays. In silico mutagenesis experiments suggest that most mutations at hGly422 have the potential to induce sterical clash, which might be considered a reason for protein misfolding. hGly422 is conserved among ALOX5, ALOX12 and ALOX15 isoforms and corresponding hALOX12 and hALOX5 mutants also exhibited a reduced catalytic activity. Interestingly, in the hALOX5 Gly429Glu mutants the reaction specificity of arachidonic acid oxygenation was shifted from 5S- to 8S- and 12R-H(p)ETE formation. Taken together, our data indicate that the conserved glycine is of functional importance for these enzyme variants and most mutants at this position lose catalytic activity. © 2013.

System-level analysis of genes and functions affecting survival during nutrient starvation in Saccharomyces cerevisiae.

PubMed

Gresham, David; Boer, Viktor M; Caudy, Amy; Ziv, Naomi; Brandt, Nathan J; Storey, John D; Botstein, David

2011-01-01

An essential property of all cells is the ability to exit from active cell division and persist in a quiescent state. For single-celled microbes this primarily occurs in response to nutrient deprivation. We studied the genetic requirements for survival of Saccharomyces cerevisiae when starved for either of two nutrients: phosphate or leucine. We measured the survival of nearly all nonessential haploid null yeast mutants in mixed populations using a quantitative sequencing method that estimates the abundance of each mutant on the basis of frequency of unique molecular barcodes. Starvation for phosphate results in a population half-life of 337 hr whereas starvation for leucine results in a half-life of 27.7 hr. To measure survival of individual mutants in each population we developed a statistical framework that accounts for the multiple sources of experimental variation. From the identities of the genes in which mutations strongly affect survival, we identify genetic evidence for several cellular processes affecting survival during nutrient starvation, including autophagy, chromatin remodeling, mRNA processing, and cytoskeleton function. In addition, we found evidence that mitochondrial and peroxisome function is required for survival. Our experimental and analytical methods represent an efficient and quantitative approach to characterizing genetic functions and networks with unprecedented resolution and identified genotype-by-environment interactions that have important implications for interpretation of studies of aging and quiescence in yeast.

Replicable in vivo physiological and behavioral phenotypes of the Shank3B null mutant mouse model of autism.

PubMed

Dhamne, Sameer C; Silverman, Jill L; Super, Chloe E; Lammers, Stephen H T; Hameed, Mustafa Q; Modi, Meera E; Copping, Nycole A; Pride, Michael C; Smith, Daniel G; Rotenberg, Alexander; Crawley, Jacqueline N; Sahin, Mustafa

2017-01-01

Autism spectrum disorder (ASD) is a clinically and biologically heterogeneous condition characterized by social, repetitive, and sensory behavioral abnormalities. No treatments are approved for the core diagnostic symptoms of ASD. To enable the earliest stages of therapeutic discovery and development for ASD, robust and reproducible behavioral phenotypes and biological markers are essential to establish in preclinical animal models. The goal of this study was to identify electroencephalographic (EEG) and behavioral phenotypes that are replicable between independent cohorts in a mouse model of ASD. The larger goal of our strategy is to empower the preclinical biomedical ASD research field by generating robust and reproducible behavioral and physiological phenotypes in animal models of ASD, for the characterization of mechanistic underpinnings of ASD-relevant phenotypes, and to ensure reliability for the discovery of novel therapeutics. Genetic disruption of the SHANK3 gene, a scaffolding protein involved in the stability of the postsynaptic density in excitatory synapses, is thought to be responsible for a relatively large number of cases of ASD. Therefore, we have thoroughly characterized the robustness of ASD-relevant behavioral phenotypes in two cohorts, and for the first time quantified translational EEG activity in Shank3B null mutant mice. In vivo physiology and behavioral assays were conducted in two independently bred and tested full cohorts of Shank3B null mutant ( Shank3B KO) and wildtype littermate control (WT) mice. EEG was recorded via wireless implanted telemeters for 7 days of baseline followed by 20 min of recording following pentylenetetrazol (PTZ) challenge. Behaviors relevant to the diagnostic and associated symptoms of ASD were tested on a battery of established behavioral tests. Assays were designed to reproduce and expand on the original behavioral characterization of Shank3B KO mice. Two or more corroborative tests were conducted within each behavioral domain, including social, repetitive, cognitive, anxiety-related, sensory, and motor categories of assays. Relative to WT mice, Shank3B KO mice displayed a dramatic resistance to PTZ seizure induction and an enhancement of gamma band oscillatory EEG activity indicative of enhanced inhibitory tone. These findings replicated in two separate cohorts. Behaviorally, Shank3B KO mice exhibited repetitive grooming, deficits in aspects of reciprocal social interactions and vocalizations, and reduced open field activity, as well as variable deficits in sensory responses, anxiety-related behaviors, learning and memory. Robust animal models and quantitative, replicable biomarkers of neural dysfunction are needed to decrease risk and enable successful drug discovery and development for ASD and other neurodevelopmental disorders. Complementary to the replicated behavioral phenotypes of the Shank3B mutant mouse is the new identification of a robust, translational in vivo neurophysiological phenotype. Our findings provide strong evidence for robustness and replicability of key translational phenotypes in Shank3B mutant mice and support the usefulness of this mouse model of ASD for therapeutic discovery.

Fascin1-Dependent Filopodia are Required for Directional Migration of a Subset of Neural Crest Cells

PubMed Central

Boer, Elena F.; Howell, Elizabeth D.; Schilling, Thomas F.; Jette, Cicely A.; Stewart, Rodney A.

2015-01-01

Directional migration of neural crest (NC) cells is essential for patterning the vertebrate embryo, including the craniofacial skeleton. Extensive filopodial protrusions in NC cells are thought to sense chemo-attractive/repulsive signals that provide directionality. To test this hypothesis, we generated null mutations in zebrafish fascin1a (fscn1a), which encodes an actin-bundling protein required for filopodia formation. Homozygous fscn1a zygotic null mutants have normal NC filopodia due to unexpected stability of maternal Fscn1a protein throughout NC development and into juvenile stages. In contrast, maternal/zygotic fscn1a null mutant embryos (fscn1a MZ) have severe loss of NC filopodia. However, only a subset of NC streams display migration defects, associated with selective loss of craniofacial elements and peripheral neurons. We also show that fscn1a-dependent NC migration functions through cxcr4a/cxcl12b chemokine signaling to ensure the fidelity of directional cell migration. These data show that fscn1a-dependent filopodia are required in a subset of NC cells to promote cell migration and NC derivative formation, and that perdurance of long-lived maternal proteins can mask essential zygotic gene functions during NC development. PMID:25607881

Loss of virulence in Ustilago maydis by Umchs6 gene disruption.

PubMed

Garcerá-Teruel, Ana; Xoconostle-Cázares, Beatriz; Rosas-Quijano, Raymundo; Ortiz, Lucila; León-Ramírez, Claudia; Specht, Charles A; Sentandreu, Rafael; Ruiz-Herrera, José

2004-03-01

A gene encoding a sixth chitin synthase (Umchs6, sequence GenBank accession No. ) from the plant pathogenic hemibasidiomycete Ustilago maydis (DC.) Cda. was isolated and characterized. The predicted protein is 1103 amino acids in length with a calculated molecular mass of 123.5 kDa. a2b2 null mutants were obtained by substitution of a central fragment of the Umchs6 gene with the hygromycin resistance cassette, and a1b1 null mutants were obtained by genetic recombination in plants of an a2b2deltach6 and a wild-type a1b1 strain. The mutation had no effect on the dimorphic transition in vitro or on mating, and growth rate of the mutants was only slightly reduced. On the other hand, they displayed important alterations in cell morphology, particularly at the mycelial stage, and in the staining pattern with calcofluor white. Levels of chitin synthase activity in vitro and chitin content were reduced. The most noticeable characteristic of the mutants was their almost complete loss of virulence to maize (Zea mays L.). This was a recessive character. Microscopic observations during the infectious process suggest that chitin synthase 6 activity is very important for growth of the fungus into the plant. Transformation of a2b2deltach6 mutants with an autonomous replicating plasmid carrying the full Umchs6 gene restored their normal morphological phenotype and virulence. These results are evidence that the mutation in the Umchs6 gene was solely responsible for the phenotypic alterations observed.

NCAM deficiency in the mouse forebrain impairs innate and learned avoidance behaviours.

PubMed

Brandewiede, J; Stork, O; Schachner, M

2014-06-01

The neural cell adhesion molecule (NCAM) has been implicated in the development and plasticity of neural circuits and the control of hippocampus- and amygdala-dependent learning and behaviour. Previous studies in constitutive NCAM null mutants identified emotional behaviour deficits related to disturbances of hippocampal and amygdala functions. Here, we studied these behaviours in mice conditionally deficient in NCAM in the postmigratory forebrain neurons. We report deficits in both innate and learned avoidance behaviours, as observed in elevated plus maze and passive avoidance tasks. In contrast, general locomotor activity, trait anxiety or neophobia were unaffected by the mutation. Altered avoidance behaviour of the conditional NCAM mutants was associated with a deficit in serotonergic signalling, as indicated by their reduced responsiveness to (±)-8-hydroxy-2-(dipropylamino)-tetralin-induced hypothermia. Another serotonin-dependent behaviour, namely intermale aggression that is massively increased in constitutively NCAM-deficient mice, was not affected in the forebrain-specific mutants. Our data suggest that genetically or environmentally induced changes of NCAM expression in the late postnatal and mature forebrain determine avoidance behaviour and serotonin (5-HT)1A receptor signalling. © 2014 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.

Suppressor Mutations for Presenilin 1 Familial Alzheimer Disease Mutants Modulate γ-Secretase Activities.

PubMed

Futai, Eugene; Osawa, Satoko; Cai, Tetsuo; Fujisawa, Tomoya; Ishiura, Shoichi; Tomita, Taisuke

2016-01-01

γ-Secretase is a multisubunit membrane protein complex containing presenilin (PS1) as a catalytic subunit. Familial Alzheimer disease (FAD) mutations within PS1 were analyzed in yeast cells artificially expressing membrane-bound substrate, amyloid precursor protein, or Notch fused to Gal4 transcriptional activator. The FAD mutations, L166P and G384A (Leu-166 to Pro and Gly-384 to Ala substitution, respectively), were loss-of-function in yeast. We identified five amino acid substitutions that suppress the FAD mutations. The cleavage of amyloid precursor protein or Notch was recovered by the secondary mutations. We also found that secondary mutations alone activated the γ-secretase activity. FAD mutants with suppressor mutations, L432M or S438P within TMD9 together with a missense mutation in the second or sixth loops, regained γ-secretase activity when introduced into presenilin null mouse fibroblasts. Notably, the cells with suppressor mutants produced a decreased amount of Aβ42, which is responsible for Alzheimer disease. These results indicate that the yeast system is useful to screen for mutations and chemicals that modulate γ-secretase activity. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Presynaptic establishment of the synaptic cleft extracellular matrix is required for post-synaptic differentiation

PubMed Central

Rohrbough, Jeffrey; Rushton, Emma; Woodruff, Elvin; Fergestad, Tim; Vigneswaran, Krishanthan; Broadie, Kendal

2007-01-01

Formation and regulation of excitatory glutamatergic synapses is essential for shaping neural circuits throughout development. In a Drosophila genetic screen for synaptogenesis mutants, we identified mind the gap (mtg), which encodes a secreted, extracellular N-glycosaminoglycan-binding protein. MTG is expressed neuronally and detected in the synaptic cleft, and is required to form the specialized transsynaptic matrix that links the presynaptic active zone with the post-synaptic glutamate receptor (GluR) domain. Null mtg embryonic mutant synapses exhibit greatly reduced GluR function, and a corresponding loss of localized GluR domains. All known post-synaptic signaling/scaffold proteins functioning upstream of GluR localization are also grossly reduced or mislocalized in mtg mutants, including the dPix–dPak–Dock cascade and the Dlg/PSD-95 scaffold. Ubiquitous or neuronally targeted mtg RNA interference (RNAi) similarly reduce post-synaptic assembly, whereas post-synaptically targeted RNAi has no effect, indicating that presynaptic MTG induces and maintains the post-synaptic pathways driving GluR domain formation. These findings suggest that MTG is secreted from the presynaptic terminal to shape the extracellular synaptic cleft domain, and that the cleft domain functions to mediate transsynaptic signals required for post-synaptic development. PMID:17901219

Role of an expansin-like molecule in Dictyostelium morphogenesis and regulation of its gene expression by the signal transducer and activator of transcription protein Dd-STATa.

PubMed

Ogasawara, Shun; Shimada, Nao; Kawata, Takefumi

2009-02-01

Expansins are proteins involved in plant morphogenesis, exerting their effects on cellulose to extend cell walls. Dictyostelium is an organism that possesses expansin-like molecules, but their functions are not known. In this study, we analyzed the expL7 (expansin-like 7) gene, which has been identified as a putative target of Dd-STATa, a Dictyostelium homolog of the metazoan signal transducer and activator of transcription (STAT) proteins. Promoter fragments of the expL7 were fused to a lacZ reporter and the expression patterns determined. As expected from the behavior of the endogenous expL7 gene, the expL7/lacZ fusion gene was downregulated in Dd-STATa null slugs. In the parental strain, the expL7 promoter was activated in the anterior tip region. Mutational analysis of the promoter identified a sequence that was necessary for expression in tip cells. In addition, an activator sequence for pstAB cells was identified. These sequences act in combination with the repressor region to prevent ectopic expL7 expression in the prespore and prestalk regions of the slug and culminant. Although the expL7 null mutant showed no phenotypic change, the expL7 overexpressor showed aberrant stalk formation. These results indicate that the expansin-like molecule is important for morphogenesis in Dictyostelium.

Drosophila rolling blackout displays lipase domain-dependent and -independent endocytic functions downstream of dynamin.

PubMed

Vijayakrishnan, Niranjana; Phillips, Scott E; Broadie, Kendal

2010-12-01

Drosophila temperature-sensitive rolling blackout (rbo(ts) ) mutants display a total block of endocytosis in non-neuronal cells and a weaker, partial defect at neuronal synapses. RBO is an integral plasma membrane protein and is predicted to be a serine esterase. To determine if lipase activity is required for RBO function, we mutated the catalytic serine 358 to alanine in the G-X-S-X-G active site, and assayed genomic rescue of rbo mutant non-neuronal and neuronal phenotypes. The rbo(S358A) mutant is unable to rescue rbo null 100% embryonic lethality, indicating that the lipase domain is critical for RBO essential function. Likewise, the rbo(S358A) mutant cannot provide any rescue of endocytic blockade in rbo(ts) Garland cells, showing that the lipase domain is indispensable for non-neuronal endocytosis. In contrast, rbo(ts) conditional paralysis, synaptic transmission block and synapse endocytic defects are all fully rescued by the rbo(S358A) mutant, showing that the RBO lipase domain is dispensable in neuronal contexts. We identified a synthetic lethal interaction between rbo(ts) and the well-characterized dynamin GTPase conditional shibire (shi(ts1)) mutant. In both non-neuronal cells and neuronal synapses, shi(ts1); rbo(ts) phenocopies shi(ts1) endocytic defects, indicating that dynamin and RBO act in the same pathway, with dynamin functioning upstream of RBO. We conclude that RBO possesses both lipase domain-dependent and scaffolding functions with differential requirements in non-neuronal versus neuronal endocytosis mechanisms downstream of dynamin GTPase activity. © 2010 John Wiley & Sons A/S.

acn-1, a C. elegans homologue of ACE, genetically interacts with the let-7 microRNA and other heterochronic genes.

PubMed

Metheetrairut, Chanatip; Ahuja, Yuri; Slack, Frank J

2017-10-02

The heterochronic pathway in C. elegans controls the relative timing of cell fate decisions during post-embryonic development. It includes a network of microRNAs (miRNAs), such as let-7, and protein-coding genes, such as the stemness factors, LIN-28 and LIN-41. Here we identified the acn-1 gene, a homologue of mammalian angiotensin-converting enzyme (ACE), as a new suppressor of the stem cell developmental defects of let-7 mutants. Since acn-1 null mutants die during early larval development, we used RNAi to characterize the role of acn-1 in C. elegans seam cell development, and determined its interaction with heterochronic factors, including let-7 and its downstream interactors - lin-41, hbl-1, and apl-1. We demonstrate that although RNAi knockdown of acn-1 is insufficient to cause heterochronic defects on its own, loss of acn-1 suppresses the retarded phenotypes of let-7 mutants and enhances the precocious phenotypes of hbl-1, though not lin-41, mutants. Conversely, the pattern of acn-1 expression, which oscillates during larval development, is disrupted by lin-41 mutants but not by hbl-1 mutants. Finally, we show that acn-1(RNAi) enhances the let-7-suppressing phenotypes caused by loss of apl-1, a homologue of the Alzheimer's disease-causing amyloid precursor protein (APP), while significantly disrupting the expression of apl-1 during the L4 larval stage. In conclusion, acn-1 interacts with heterochronic genes and appears to function downstream of let-7 and its target genes, including lin-41 and apl-1.

Integration of Genomic, Biologic, and Chemical Approaches to Target p53 Loss and Gain-of-Function in Triple Negative Breast Cancer

DTIC Science & Technology

2016-09-01

in this progress report: p53 triple-negative breast cancer subtypes gene expression somatic cell genetics CRISPR /Cas 3. ACCOMPLISHMENTS Major...report, we described the creation of an isogenic p53 mutant TNBC cell line panel using CRISPR /Cas-mediated genome editing8 and the resultant...LOF null state. To validate that mutant p53 is directly responsible for this altered transcription, we will use the same CRISPR -mediated genome

Zebrafish pit1 mutants lack three pituitary cell types and develop severe dwarfism.

PubMed

Nica, Gabriela; Herzog, Wiebke; Sonntag, Carmen; Hammerschmidt, Matthias

2004-05-01

The Pou domain transcription factor Pit-1 is required for lineage determination and cellular commitment processes during mammalian adenohypophysis development. Here we report the cloning and mutational analysis of a pit1 homolog from zebrafish. Compared with mouse, zebrafish pit1 starts to be expressed at a much earlier stage of adenohypophysis development. However, as in the mouse, expression is restricted to a subset of pituitary cell types, excluding proopiomelanocortin (pomc)-expressing cells (corticotropes, melanotropes) and possibly gonadotropes. We could identify two N-ethyl-N-nitrosourea-induced zebrafish pit1 null mutants. Most mutants die during larval stages, whereas survivors develop severe dwarfism. Mutant larvae lack lactotropes, somatotropes, and thyrotropes, although the adenohypophysis is of normal size, without any sign of increased apoptosis rates. Instead, mutant embryos initiate ectopic expression of pomc in pit1-positive cells, leading to an expansion of the Pomc lineage. Similarly, the number of gonadotropes seems increased, as indicated by the expression of gsualpha, a marker for thyrotropes and gonadotropes. In pit1 mutants, the total number of gsualpha-positive cells is normal despite the loss of gsualpha and tshbeta coexpressing cells. Together, these data suggest a transfating of the Pit1 lineage to the Pomc and possibly the gonadotroph lineages in the mutant, and a pomc- and gonadotropin-repressive role of Pit1 during normal zebrafish development. This is different from mouse, for which a repressive role of Pit-1 has only been reported for the gonadotropin Lhbeta, but not for Pomc. In sum, our data point to both conserved and class-specific aspects of Pit1 function during pituitary development in different vertebrate species.

The UL21 Tegument Protein of Herpes Simplex Virus 1 Is Differentially Required for the Syncytial Phenotype

PubMed Central

Starkey, Jason; Mellinger, Erica; Zhang, Dan; Chadha, Pooja; Carmichael, Jillian

2017-01-01

ABSTRACT The initial goal of this study was to reexamine the requirement of UL21 for herpes simplex virus 1 (HSV-1) replication. Previous studies suggested that UL21 is dispensable for replication in cell cultures, but a recent report on HSV-2 challenges those findings. As was done for the HSV-2 study, a UL21-null virus was made and propagated on complementing cells to discourage selection of compensating mutations. This HSV-1 mutant was able to replicate in noncomplementing cells, even at a low multiplicity of infection (MOI), though a reduction in titer was observed. Also, increased proportions of empty capsids were observed in the cytoplasm, suggesting a role for UL21 in preventing their exit from the nucleus. Surprisingly, passage of the null mutant resulted in rapid outgrowth of syncytial (Syn) variants. This was unexpected because UL21 has been shown to be required for the Syn phenotype. However, earlier experiments made use of only the A855V syncytial mutant of glycoprotein B (gB), and the Syn phenotype can also be produced by substitutions in glycoprotein K (gK), UL20, and UL24. Sequencing of the syncytial variants revealed mutations in the gK locus, but UL21 was shown to be dispensable for UL20Syn and UL24Syn. To test whether UL21 is needed only for the A855V mutant, additional gBSyn derivatives were examined in the context of the null virus, and all produced lytic rather than syncytial sites of infection. Thus, UL21 is required only for the gBSyn phenotype. This is the first example of a differential requirement for a viral protein across the four syn loci. IMPORTANCE UL21 is conserved among alphaherpesviruses, but its role is poorly understood. This study shows that HSV-1 can replicate without UL21, although the virus titers are greatly reduced. The null virus had greater proportions of empty (DNA-less) capsids in the cytoplasm of infected cells, suggesting that UL21 may play a role in retaining them in the nucleus. This is consistent with reports showing UL21 to be capsid associated and localized to the nuclei of infected cells. UL21 also appears to be needed for viral membrane activities. It was found to be required for virus-mediated cell fusion, but only for mutants that harbor syncytial mutations in gB (not variants of gK, UL20, or UL24). The machinery needed for syncytial formation is similar to that needed for direct spread of the virus through cell junctions, and these studies show that UL21 is required for cell-to-cell spread even in the absence of syncytial mutations. PMID:28794039

Ubiquitin orchestrates proteasome dynamics between proliferation and quiescence in yeast

PubMed Central

Gu, Zhu Chao; Wu, Edwin; Sailer, Carolin; Jando, Julia; Styles, Erin; Eisenkolb, Ina; Kuschel, Maike; Bitschar, Katharina; Wang, Xiaorong; Huang, Lan; Vissa, Adriano; Yip, Christopher M.; Yedidi, Ravikiran S.; Friesen, Helena; Enenkel, Cordula

2017-01-01

Proteasomes are essential for protein degradation in proliferating cells. Little is known about proteasome functions in quiescent cells. In nondividing yeast, a eukaryotic model of quiescence, proteasomes are depleted from the nucleus and accumulate in motile cytosolic granules termed proteasome storage granules (PSGs). PSGs enhance resistance to genotoxic stress and confer fitness during aging. Upon exit from quiescence PSGs dissolve, and proteasomes are rapidly delivered into the nucleus. To identify key players in PSG organization, we performed high-throughput imaging of green fluorescent protein (GFP)-labeled proteasomes in the yeast null-mutant collection. Mutants with reduced levels of ubiquitin are impaired in PSG formation. Colocalization studies of PSGs with proteins of the yeast GFP collection, mass spectrometry, and direct stochastic optical reconstitution microscopy of cross-linked PSGs revealed that PSGs are densely packed with proteasomes and contain ubiquitin but no polyubiquitin chains. Our results provide insight into proteasome dynamics between proliferating and quiescent yeast in response to cellular requirements for ubiquitin-dependent degradation. PMID:28768827

Protein Kinase A Regulatory Subunit Isoforms Regulate Growth and Differentiation in Mucor circinelloides: Essential Role of PKAR4

PubMed Central

Ocampo, J.; McCormack, B.; Navarro, E.; Moreno, S.; Garre, V.

2012-01-01

The protein kinase A (PKA) signaling pathway plays a role in regulating growth and differentiation in the dimorphic fungus Mucor circinelloides. PKA holoenzyme is comprised of two catalytic (C) and two regulatory (R) subunits. In M. circinelloides, four genes encode the PKAR1, PKAR2, PKAR3, and PKAR4 isoforms of R subunits. We have constructed null mutants and demonstrate that each isoform has a different role in growth and differentiation. The most striking finding is that pkaR4 is an essential gene, because only heterokaryons were obtained in knockout experiments. Heterokaryons with low levels of wild-type nuclei showed an impediment in the emission of the germ tube, suggesting a pivotal role of this gene in germ tube emergence. The remaining null strains showed different alterations in germ tube emergence, sporulation, and volume of the mother cell. The pkaR2 null mutant showed an accelerated germ tube emission and was the only mutant that germinated under anaerobic conditions when glycine was used as a nitrogen source, suggesting that pkaR2 participates in germ tube emergence by repressing it. From the measurement of the mRNA and protein levels of each isoform in the wild-type and knockout strains, it can be concluded that the expression of each subunit has its own mechanism of differential regulation. The PKAR1 and PKAR2 isoforms are posttranslationally modified by ubiquitylation, suggesting another regulation point in the specificity of the signal transduction. The results indicate that each R isoform has a different role in M. circinelloides physiology, controlling the dimorphism and contributing to the specificity of cyclic AMP (cAMP)-PKA pathway. PMID:22635921

Regulation of onset of female mating and sex pheromone production by juvenile hormone in Drosophila melanogaster

PubMed Central

Bilen, Julide; Atallah, Jade; Azanchi, Reza; Levine, Joel D.; Riddiford, Lynn M.

2013-01-01

Juvenile hormone (JH) coordinates timing of female reproductive maturation in most insects. In Drosophila melanogaster, JH plays roles in both mating and egg maturation. However, very little is known about the molecular pathways associated with mating. Our behavioral analysis of females genetically lacking the corpora allata, the glands that produce JH, showed that they were courted less by males and mated later than control females. Application of the JH mimic, methoprene, to the allatectomized females just after eclosion rescued both the male courtship and the mating delay. Our studies of the null mutants of the JH receptors, Methoprene tolerant (Met) and germ cell-expressed (gce), showed that lack of Met in Met27 females delayed the onset of mating, whereas lack of Gce had little effect. The Met27 females were shown to be more attractive but less behaviorally receptive to copulation attempts. The behavioral but not the attractiveness phenotype was rescued by the Met genomic transgene. Analysis of the female cuticular hydrocarbon profiles showed that corpora allata ablation caused a delay in production of the major female-specific sex pheromones (the 7,11-C27 and -C29 dienes) and a change in the cuticular hydrocarbon blend. In the Met27 null mutant, by 48 h, the major C27 diene was greatly increased relative to wild type. In contrast, the gce2.5k null mutant females were courted similarly to control females despite changes in certain cuticular hydrocarbons. Our findings indicate that JH acts primarily via Met to modulate the timing of onset of female sex pheromone production and mating. PMID:24145432

AAV-Mediated Administration of Myostatin Pro-Peptide Mutant in Adult Ldlr Null Mice Reduces Diet-Induced Hepatosteatosis and Arteriosclerosis

PubMed Central

Guo, Wen; Wong, Siu; Bhasin, Shalender

2013-01-01

Genetic disruption of myostatin or its related signaling is known to cause strong protection against diet-induced metabolic disorders. The translational value of these prior findings, however, is dependent on whether such metabolically favorable phenotype can be reproduced when myostatin blockade begins at an adult age. Here, we reported that AAV-mediated delivery of a myostatin pro-peptide D76A mutant in adult mice attenuates the development of hepatic steatosis and arteriosclerosis, two common diet-induced metabolic diseases. A single dose of AAV-D76A in adult Ldlr null mice resulted in sustained expression of myostatin pro-peptide in the liver. Compared to vehicle-treated mice, D76A-treated mice gained similar amount of lean and fat mass when fed a high fat diet. However, D76A-treated mice displayed significantly reduced aortic lesions and liver fat, in association with a reduction in hepatic expression of lipogenic genes and improvement in liver insulin sensitivity. This suggests that muscle and fat may not be the primary targets of treatment under our experimental condition. In support to this argument, we show that myostatin directly up-regulated lipogenic genes and increased fat accumulation in cultured liver cells. We also show that both myostatin and its receptor were abundantly expressed in mouse aorta. Cultured aortic endothelial cells responded to myostatin with a reduction in eNOS phosphorylation and an increase in ICAM-1 and VCAM-1 expression. Conclusions: AAV-mediated expression of myostatin pro-peptide D76A mutant in adult Ldlr null mice sustained metabolic protection without remarkable impacts on body lean and fat mass. Further investigations are needed to determine whether direct impact of myostatin on liver and aortic endothelium may contribute to the related metabolic phenotypes. PMID:23936482

Neuron class-specific requirements for Fragile X Mental Retardation Protein in critical period development of calcium signaling in learning and memory circuitry.

PubMed

Doll, Caleb A; Broadie, Kendal

2016-05-01

Neural circuit optimization occurs through sensory activity-dependent mechanisms that refine synaptic connectivity and information processing during early-use developmental critical periods. Fragile X Mental Retardation Protein (FMRP), the gene product lost in Fragile X syndrome (FXS), acts as an activity sensor during critical period development, both as an RNA-binding translation regulator and channel-binding excitability regulator. Here, we employ a Drosophila FXS disease model to assay calcium signaling dynamics with a targeted transgenic GCaMP reporter during critical period development of the mushroom body (MB) learning/memory circuit. We find FMRP regulates depolarization-induced calcium signaling in a neuron-specific manner within this circuit, suppressing activity-dependent calcium transients in excitatory cholinergic MB input projection neurons and enhancing calcium signals in inhibitory GABAergic MB output neurons. Both changes are restricted to the developmental critical period and rectified at maturity. Importantly, conditional genetic (dfmr1) rescue of null mutants during the critical period corrects calcium signaling defects in both neuron classes, indicating a temporally restricted FMRP requirement. Likewise, conditional dfmr1 knockdown (RNAi) during the critical period replicates constitutive null mutant defects in both neuron classes, confirming cell-autonomous requirements for FMRP in developmental regulation of calcium signaling dynamics. Optogenetic stimulation during the critical period enhances depolarization-induced calcium signaling in both neuron classes, but this developmental change is eliminated in dfmr1 null mutants, indicating the activity-dependent regulation requires FMRP. These results show FMRP shapes neuron class-specific calcium signaling in excitatory vs. inhibitory neurons in developing learning/memory circuitry, and that FMRP mediates activity-dependent regulation of calcium signaling specifically during the early-use critical period. Copyright © 2016 Elsevier Inc. All rights reserved.

A novel cytosolic regulator, Pianissimo, is required for chemoattractant receptor and G protein-mediated activation of the 12 transmembrane domain adenylyl cyclase in Dictyostelium

PubMed Central

Chen, Mei-Yu; Long, Yu; Devreotes, Peter N.

1997-01-01

Genetic analysis was applied to identify novel genes involved in G protein-linked pathways controlling development. Using restriction enzyme-mediated integration (REMI), we have identified a new gene, Pianissimo (PiaA), involved in cAMP signaling in Dictyostelium discoideum. PiaA encodes a 130-kD cytosolic protein required for chemoattractant receptor and G protein-mediated activation of the 12 transmembrane domain adenylyl cyclase. In piaA− null mutants, neither chemoattractant stimulation of intact cells nor GTPγS treatment of lysates activates the enzyme; constitutive expression of PiaA reverses these defects. Cytosols of wild-type cells that contain Pia protein reconstitute the GTPγS stimulation of adenylyl cyclase activity in piaA− lysates, indicating that Pia is directly involved in the activation. Pia and CRAC, a previously identified cytosolic regulator, are both essential for activation of the enzyme as lysates of crac− piaA− double mutants require both proteins for reconstitution. Homologs of PiaA are found in Saccharomyces cerevisiae and Schizosaccaromyces pombe; disruption of the S. cerevisiae homolog results in lethality. We propose that homologs of Pia and similar modes of regulation of these ubiquitous G protein-linked pathways are likely to exist in higher eukaryotes. PMID:9389653

Inositol Hexakisphosphate Kinase-3 Regulates the Morphology and Synapse Formation of Cerebellar Purkinje Cells via Spectrin/Adducin

PubMed Central

Fu, Chenglai; Xu, Jing; Li, Ruo-Jing; Crawford, Joshua A.; Khan, A. Basit; Ma, Ting Martin; Cha, Jiyoung Y.; Snowman, Adele M.; Pletnikov, Mikhail V.

2015-01-01

The inositol hexakisphosphate kinases (IP6Ks) are the principal enzymes that generate inositol pyrophosphates. There are three IP6Ks (IP6K1, 2, and 3). Functions of IP6K1 and IP6K2 have been substantially delineated, but little is known of IP6K3's role in normal physiology, especially in the brain. To elucidate functions of IP6K3, we generated mice with targeted deletion of IP6K3. We demonstrate that IP6K3 is highly concentrated in the brain in cerebellar Purkinje cells. IP6K3 physiologically binds to the cytoskeletal proteins adducin and spectrin, whose mutual interactions are perturbed in IP6K3-null mutants. Consequently, IP6K3 knock-out cerebella manifest abnormalities in Purkinje cell structure and synapse number, and the mutant mice display deficits in motor learning and coordination. Thus, IP6K3 is a major determinant of cytoskeletal disposition and function of cerebellar Purkinje cells. SIGNIFICANCE STATEMENT We identified and cloned a family of three inositol hexakisphosphate kinases (IP6Ks) that generate the inositol pyrophosphates, most notably 5-diphosphoinositol pentakisphosphate (IP7). Of these, IP6K3 has been least characterized. In the present study we generated IP6K3 knock-out mice and show that IP6K3 is highly expressed in cerebellar Purkinje cells. IP6K3-deleted mice display defects of motor learning and coordination. IP6K3-null mice manifest aberrations of Purkinje cells with a diminished number of synapses. IP6K3 interacts with the cytoskeletal proteins spectrin and adducin whose altered disposition in IP6K3 knock-out mice may mediate phenotypic features of the mutant mice. These findings afford molecular/cytoskeletal mechanisms by which the inositol polyphosphate system impacts brain function. PMID:26245967

RNA interference can target pre-mRNA: consequences for gene expression in a Caenorhabditis elegans operon.

PubMed Central

Bosher, J M; Dufourcq, P; Sookhareea, S; Labouesse, M

1999-01-01

In nematodes, flies, trypanosomes, and planarians, introduction of double-stranded RNA results in sequence-specific inactivation of gene function, a process termed RNA interference (RNAi). We demonstrate that RNAi against the Caenorhabditis elegans gene lir-1, which is part of the lir-1/lin-26 operon, induced phenotypes very different from a newly isolated lir-1 null mutation. Specifically, lir-1(RNAi) induced embryonic lethality reminiscent of moderately strong lin-26 alleles, whereas the lir-1 null mutant was viable. We show that the lir-1(RNAi) phenotypes resulted from a severe loss of lin-26 gene expression. In addition, we found that RNAi directed against lir-1 or lin-26 introns induced similar phenotypes, so we conclude that lir-1(RNAi) targets the lir-1/lin-26 pre-mRNA. This provides direct evidence that RNA interference can prevent gene expression by targeting nuclear transcripts. Our results highlight that caution may be necessary when interpreting RNA interference without the benefit of mutant alleles. PMID:10545456

Deleterious CHEK2 1100delC and L303X mutants identified among 38 human breast cancer cell lines.

PubMed

Wasielewski, Marijke; Hanifi-Moghaddam, Pejman; Hollestelle, Antoinette; Merajver, Sofia D; van den Ouweland, Ans; Klijn, Jan G M; Ethier, Stephen P; Schutte, Mieke

2009-01-01

The CHEK2 protein plays a major role in the regulation of DNA damage response pathways. Mutations in the CHEK2 gene, in particular 1100delC, have been associated with increased cancer risks, but the precise function of CHEK2 mutations in carcinogenesis is not known. Human cancer cell lines with CHEK2 mutations are therefore of main interest. Here, we have sequenced 38 breast cancer cell lines for mutations in the CHEK2 gene and identified two cell lines with deleterious CHEK2 mutations. Cell line UACC812 has a nonsense truncating mutation in the CHEK2 kinase domain (L303X) and cell line SUM102PT has the well-known oncogenic CHEK2 1100delC founder mutation. Immunohistochemical analysis revealed that the two CHEK2 mutant cell lines expressed neither CHEK2 nor P-Thr(68) CHEK2 proteins, implying abrogation of normal CHEK2 DNA repair functions. Cell lines UACC812 and SUM102PT thus are the first human CHEK2 null cell lines reported and should therefore be a major help in further unraveling the function of CHEK2 mutations in carcinogenesis.

Biofilm formation ability of Salmonella enterica serovar Typhimurium acrAB mutants.

PubMed

Schlisselberg, Dov B; Kler, Edna; Kisluk, Guy; Shachar, Dina; Yaron, Sima

2015-10-01

Recent studies offer contradictory findings about the role of multidrug efflux pumps in bacterial biofilm development. Thus, the aim of this study was to investigate the involvement of the AcrAB efflux pump in biofilm formation by investigating the ability of AcrB and AcrAB null mutants of Salmonella enterica serovar Typhimurium to produce biofilms. Three models were used to compare the ability of S. Typhimurium wild-type and its mutants to form biofilms: formation of biofilm on polystyrene surfaces; production of biofilm (mat model) on the air/liquid interface; and expression of curli and cellulose on Congo red-supplemented agar plates. All three investigated genotypes formed biofilms with similar characteristics. However, upon exposure to chloramphenicol, formation of biofilms on solid surfaces as well as the production of curli were either reduced or were delayed more significantly in both mutants, whilst there was no visible effect on pellicle formation. It can be concluded that when no selective pressure is applied, S. Typhimurium is able to produce biofilms even when the AcrAB efflux pumps are inactivated, implying that the use of efflux pump inhibitors to prevent biofilm formation is not a general solution and that combined treatments might be more efficient. Other factors that affect the ability to produce biofilms depending on efflux pump activity are yet to be identified. Copyright © 2015 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

Multiple phytochromes are involved in red-light-induced enhancement of first-positive phototropism in arabidopsis thaliana

DOE Office of Scientific and Technical Information (OSTI.GOV)

Janoudi, A.K.; Gordon, W.R.; Poff, K.L.

1997-03-01

The amplitude of phototropic curvature to blue light is enhanced by a prior exposure of seedlings to red light. This enhancement is mediated by phytochrome. Fluence-response relationships have been constructed for red-light-induced enhancement in the phytochrome A (phyA) null mutant, the phytochrome B- (phyB) deficient mutant, and in two transgenic lines of Arabidopsis thaliana that overexpress either phyA or phyB. These fluence-response relationships demonstrate the existence of two responses in enhancement, a response in the very-low-to-low-fluence range, and a response in the high-fluence range. Only the response in the high-fluence range is present in the phyA null mutant. In contrast,more » the phyB-deficient mutant is indistinguishable from the wild-type parent in red-light responsiveness. These data indicate that phyA is necessary for the very-low-to-low but not the high-fluence response, and that phyB is not necessary for either response range. Based on these results, the high-fluence response, if controlled by a single phytochrome, must be controlled by a phytochrome other than phyA or phyB. Overexpression of phyA has a negative effect and overexpression of phyB has an enhancing effect in the high fluence range. These results suggest that overexpression of either phytochrome perturbs the function of the endogenous photoreceptor system in unpreditable fashion. 25 refs., 3 figs.« less

A Defect in DNA Ligase4 Enhances the Frequency of TALEN-Mediated Targeted Mutagenesis in Rice1[OPEN

PubMed Central

Cermak, Tomas; Sugimoto, Kazuhiko; Saika, Hiroaki; Mori, Akiko; Osakabe, Keishi; Hamada, Masao; Katayose, Yuichi; Voytas, Daniel F.

2016-01-01

We have established methods for site-directed mutagenesis via transcription activator-like effector nucleases (TALENs) in the endogenous rice (Oryza sativa) waxy gene and demonstrated stable inheritance of TALEN-induced somatic mutations to the progeny. To analyze the role of classical nonhomologous end joining (cNHEJ) and alternative nonhomologous end joining (altNHEJ) pathways in TALEN-induced mutagenesis in plant cells, we investigated whether a lack of DNA Ligase4 (Lig4) affects the kinetics of TALEN-induced double-strand break repair in rice cells. Deep-sequencing analysis revealed that the frequency of all types of mutations, namely deletion, insertion, combination of insertion with deletion, and substitution, in lig4 null mutant calli was higher than that in a lig4 heterozygous mutant or the wild type. In addition, the ratio of large deletions (greater than 10 bp) and deletions repaired by microhomology-mediated end joining (MMEJ) to total deletion mutations in lig4 null mutant calli was higher than that in the lig4 heterozygous mutant or wild type. Furthermore, almost all insertions (2 bp or greater) were shown to be processed via copy and paste of one or more regions around the TALENs cleavage site and rejoined via MMEJ regardless of genetic background. Taken together, our findings indicate that the dysfunction of cNHEJ leads to a shift in the repair pathway from cNHEJ to altNHEJ or synthesis-dependent strand annealing. PMID:26668331

The missing link in plant histidine biosynthesis: Arabidopsis myoinositol monophosphatase-like2 encodes a functional histidinol-phosphate phosphatase.

PubMed

Petersen, Lindsay N; Marineo, Sandra; Mandalà, Salvatore; Davids, Faezah; Sewell, Bryan T; Ingle, Robert A

2010-03-01

Histidine (His) plays a critical role in plant growth and development, both as one of the standard amino acids in proteins, and as a metal-binding ligand. While genes encoding seven of the eight enzymes in the pathway of His biosynthesis have been characterized from a number of plant species, the identity of the enzyme catalyzing the dephosphorylation of histidinol-phosphate to histidinol has remained elusive. Recently, members of a novel family of histidinol-phosphate phosphatase proteins, displaying significant sequence similarity to known myoinositol monophosphatases (IMPs) have been identified from several Actinobacteria. Here we demonstrate that a member of the IMP family from Arabidopsis (Arabidopsis thaliana), myoinositol monophosphatase-like2 (IMPL2; encoded by At4g39120), has histidinol-phosphate phosphatase activity. Heterologous expression of IMPL2, but not the related IMPL1 protein, was sufficient to rescue the His auxotrophy of a Streptomyces coelicolor hisN mutant. Homozygous null impl2 Arabidopsis mutants displayed embryonic lethality, which could be rescued by supplying plants heterozygous for null impl2 alleles with His. In common with the previously characterized HISN genes from Arabidopsis, IMPL2 was expressed in all plant tissues and throughout development, and an IMPL2:green fluorescent protein fusion protein was targeted to the plastid, where His biosynthesis occurs in plants. Our data demonstrate that IMPL2 is the HISN7 gene product, and suggest a lack of genetic redundancy at this metabolic step in Arabidopsis, which is characteristic of the His biosynthetic pathway.

Regulation of ciaXRH Operon Expression and Identification of the CiaR Regulon in Streptococcus mutans▿

PubMed Central

Wu, Chenggang; Ayala, Eduardo A.; Downey, Jennifer S.; Merritt, Justin; Goodman, Steven D.; Qi, Fengxia

2010-01-01

The ciaRH operon in Streptococcus mutans contains 3 contiguous genes, ciaXRH. Unlike the CiaRH system in other streptococci, only the ciaH-null mutant displays defective phenotypes, while the ciaR-null mutant behaves like the wild type. The objective of this study was to determine the mechanism of this unusual property. We demonstrate that the ciaH mutation caused a >20-fold increase in ciaR transcript synthesis. A ciaRH double deletion reversed the ciaH phenotype, suggesting that overexpressed ciaR might be responsible for the observed ciaH phenotypes. When ciaR was forced to be overexpressed by a transcriptional fusion to the ldh promoter in the wild-type background, the same ciaH phenotypes were restored, confirming the involvement of overexpressed ciaR in the ciaH phenotypes. The ciaH mutation and ciaR overexpression also caused transcriptional alterations in 100 genes, with 15 genes upregulated >5-fold. Bioinformatics analysis identified a putative CiaR regulon consisting of 8 genes/operons, including the ciaXRH operon itself, all of which were upregulated. In vitro footprinting on 4 of the 8 promoters revealed a protected region of 26 to 28 bp encompassing two direct repeats, NTTAAG-n5-WTTAAG, 10 bp upstream of the −10 region, indicating direct binding of the CiaR protein to these promoters. Taken together, we conclude that overexpressed CiaR, as a result of either ciaH deletion or forced expression from a constitutive promoter, is a mediator in the CiaH-regulated phenotypes. PMID:20639331

LuxO controls extracellular protease, haemolytic activities and siderophore production in fish pathogen Vibrio alginolyticus.

PubMed

Wang, Q; Liu, Q; Ma, Y; Rui, H; Zhang, Y

2007-11-01

To characterize the luxO gene in fish pathogen Vibrio alginolyticus MVP01 and investigate its roles in regulation of extracellular products (ECP) and siderophore production. The luxO gene was cloned from V. alginolyticus MVP01. Genetic analysis revealed that it encoded a protein with high similarity to other LuxO homologues. The luxO in-frame deletion mutant and rpoN null mutant were constructed with suicide plasmids. We demonstrated that sole deletion in LuxO increased the secretion of extracellular protease and haemolytic products, but decreased siderophore production for V. alginolyticus MVP01. Mutants with null rpoN displayed significantly enhanced protease level and siderophore production while notable reduction in haemolytic activities of ECP. Vibrio alginolyticus harbours functional luxO gene that regulates the secretion of extracellular protease and haemolytic materials as well as siderophore production in either sigma(54) dependent or independent manners. The current study demonstrated that V. alginolyticus MVP01 produces extracellular protease and haemolytic activity material as well as siderophore, which may be characteristics of the virulence of the strain. Revelations that secretion of these products is under the regulation of LuxO and sigma(54) as well as the potential quorum sensing systems in V. alginolyticus MVP01 will expedite the understanding of vibriosis pathogenesis.

Proliferation, differentiation and apoptosis in connexin43-null osteoblasts

NASA Technical Reports Server (NTRS)

Furlan, F.; Lecanda, F.; Screen, J.; Civitelli, R.

2001-01-01

Osteoblasts are highly coupled by gap junctions formed primarily by connexin43 (Cx43). We have shown that interference with Cx43 expression or function disrupts transcriptional regulation of osteoblast genes, and that deletion of Cx43 in the mouse causes skeletal malformations, delayed mineralization, and osteoblast dysfunction. Here, we studied the mechanisms by which genetic deficiency of Cx43 alters osteoblast development. While cell proliferation rates were similar in osteoblastic cells derived from calvaria of Cx43-null and wild type mice, camptothecin-induced apoptosis was 3-fold higher in mutant compared to wild type osteoblasts. When grown in mineralizing medium, Cx43-null cells were able to produce mineralized matrix but it took one week longer to reach the same mineralization levels as in normal cells. Likewise, expression of alkaline phosphatase activity per cell--a marker of osteoblast differentiation--was maximal only 2 weeks later in Cx43-null relative to wild-type cells. These observations suggest that Cx43 is important for a normal and timely development of the osteoblastic phenotype. Delayed differentiation and increase programmed cell death may explain the skeletal phenotype of Cx43-null mice.

Aberrant Muscle Antigen Exposure in Mice Is Sufficient to Cause Myositis in a Treg Cell–Deficient Milieu

PubMed Central

Young, Nicholas A; Sharma, Rahul; Friedman, Alexandra K; Kaffenberger, Benjamin H; Bolon, Brad; Jarjour, Wael N

2013-01-01

Objective Myositis is associated with muscle-targeted inflammation and is observed in some Treg cell–deficient mouse models. Because an autoimmune pathogenesis has been strongly implicated, the aim of this study was to investigate the hypothesis that abnormal exposure to muscle antigens, as observed in muscle injury, can induce autoimmune-mediated myositis in susceptible hosts. Methods FoxP3 mutant (scurfy) mice were mated to synaptotagmin VII (Syt VII) mutant mice, which resulted in a new mouse strain that combines impaired membrane resealing with Treg cell deficiency. Lymphocyte preparations from double-mutant mice were adoptively transferred intraperitoneally, with or without purified Treg cells, into recombination-activating gene 1 (RAG-1)–null recipients. Lymph node cells from mice with the FoxP3 mutation were transferred into RAG-1–null mice either 1) intraperitoneally in conjunction with muscle homogenate or purified myosin protein or 2) intramuscularly with or without cotransfer of purified Treg cells. Results FoxP3-deficient mouse lymph node cells transferred in conjunction with myosin protein or muscle homogenate induced robust skeletal muscle inflammation. The infiltrates consisted predominantly of CD4+ and CD8+ T cells, a limited number of macrophages, and no B cells. Significant inflammation was also seen in similar experiments using lymph node cells from FoxP3/Syt VII double-mutant mice but was absent in experiments using adoptive transfer of FoxP3 mutant mouse cells alone. The cotransfer of Treg cells completely suppressed myositis. Conclusion These data, derived from a new, reproducible model, demonstrate the critical roles of Treg cell deficiency and aberrant muscle antigen exposure in the priming of autoreactive cells to induce myositis. This mouse system has multifaceted potential for examining the interplay in vivo between tissue injury and autoimmunity. PMID:24022275

Genetic separation of phototropism and blue light inhibition of stem elongation

NASA Technical Reports Server (NTRS)

Liscum, E.; Young, J. C.; Poff, K. L.; Hangarter, R. P.

1992-01-01

Blue light-induced regulation of cell elongation is a component of the signal response pathway for both phototropic curvature and inhibition of stem elongation in higher plants. To determine if blue light regulates cell elongation in these responses through shared or discrete pathways, phototropism and hypocotyl elongation were investigated in several blue light response mutants in Arabidopsis thaliana. Specifically, the blu mutants that lack blue light-dependent inhibition of hypocotyl elongation were found to exhibit a normal phototropic response. In contrast, a phototropic null mutant (JK218) and a mutant that has a 20- to 30-fold shift in the fluence dependence for first positive phototropism (JK224) showed normal inhibition of hypocotyl elongation in blue light. F1 progeny of crosses between the blu mutants and JK218 showed normal phototropism and inhibition of hypocotyl elongation, and approximately 1 in 16 F2 progeny were double mutants lacking both responses. Thus, blue light-dependent inhibition of hypocotyl elongation and phototropism operate through at least some genetically distinct components.

Inactivation of retinoblastoma protein does not overcome the requirement for human cytomegalovirus UL97 in lamina disruption and nuclear egress.

PubMed

Reim, Natalia I; Kamil, Jeremy P; Wang, Depeng; Lin, Alison; Sharma, Mayuri; Ericsson, Maria; Pesola, Jean M; Golan, David E; Coen, Donald M

2013-05-01

Human cytomegalovirus (HCMV) encodes one conventional protein kinase, UL97. During infection, UL97 phosphorylates the retinoblastoma tumor suppressor protein (pRb) on sites ordinarily phosphorylated by cyclin-dependent kinases (CDK), inactivating the ability of pRb to repress host genes required for cell cycle progression to S phase. UL97 is important for viral DNA synthesis in quiescent cells, but this function can be replaced by human papillomavirus type 16 E7, which targets pRb for degradation. However, viruses in which E7 replaces UL97 are still defective for virus production. UL97 is also required for efficient nuclear egress of viral nucleocapsids, which is associated with disruption of the nuclear lamina during infection, and phosphorylation of lamin A/C on serine 22, which antagonizes lamin polymerization. We investigated whether inactivation of pRb might overcome the requirement of UL97 for these roles, as pRb inactivation induces CDK1, and CDK1 phosphorylates lamin A/C on serine 22. We found that lamin A/C serine 22 phosphorylation during HCMV infection correlated with expression of UL97 and was considerably delayed in UL97-null mutants, even when E7 was expressed. E7 failed to restore gaps in the nuclear lamina seen in wild-type but not UL97-null virus infections. In electron microscopy analyses, a UL97-null virus expressing E7 was as impaired as a UL97-null mutant in cytoplasmic accumulation of viral nucleocapsids. Our results demonstrate that pRb inactivation is insufficient to restore efficient viral nuclear egress of HCMV in the absence of UL97 and instead argue further for a direct role of UL97 in this stage of the infectious cycle.

The heparan sulphate deficient Hspg2 exon 3 null mouse displays reduced deposition of TGF-β1 in skin compared to C57BL/6 wild type mice.

PubMed

Shu, Cindy; Smith, Susan M; Melrose, James

2016-06-01

This was an observational study where we examined the role of perlecan HS on the deposition of TGF-β1 in C57BL/6 and Hspg2(∆3-/∆3-) perlecan exon 3 null mouse skin. Despite its obvious importance in skin repair and tissue homeostasis no definitive studies have immunolocalised TGF-β1 in skin in WT or Hspg2(∆3-/∆3-) perlecan exon 3 null mice. Vertical parasagittal murine dorsal skin from 3, 6 and 12 week old C57BL/6 and Hspg2(∆3-/∆3-) mice were fixed in neutral buffered formalin, paraffin embedded and 4 μm sections stained with Mayers haematoxylin and eosin (H & E). TGF-β1 was immunolocalised using a rabbit polyclonal antibody, heat retrieval and the Envision NovaRED detection system. Immunolocalisation of TGF-β1 differed markedly in C57BL/6 and Hspg2(∆3-/∆3-) mouse skin, ablation of exon 3 of Hspg2 resulted in a very severe reduction in the deposition of TGF-β1 in skin 3-12 weeks postnatally. The reduced deposition of TGF-β1 observed in the present study would be expected to impact detrimentally on the remodelling and healing capacity of skin in mutant mice compounding on the poor wound-healing properties already reported for perlecan exon 3 null mice due to an inability to signal with FGF-2 and promote angiogenic repair processes. TGF-β1 also has cell mediated effects in tissue homeostasis and matrix stabilisation a reduction in TGF-β1 deposition would therefore be expected to detrimentally impact on skin homeostasis in the perlecan mutant mice.

Long-term improvements in sensory inhibition with gestational choline supplementation linked to α7 nicotinic receptors through studies in Chrna7 null mutation mice.

PubMed

Stevens, Karen E; Choo, Kevin S; Stitzel, Jerry A; Marks, Michael J; Adams, Catherine E

2014-03-13

Perinatal choline supplementation has produced several benefits in rodent models, from improved learning and memory to protection from the behavioral effects of fetal alcohol exposure. We have shown that supplemented choline through gestation and lactation produces long-term improvement in deficient sensory inhibition in DBA/2 mice which models a similar deficit in schizophrenia patients. The present study extends that research by feeding normal or supplemented choline diets to DBA/2 mice carrying the null mutation for the α7 nicotinic receptor gene (Chrna7). DBA/2 mice heterozygotic for Chrna7 were bred together. Dams were placed on supplemented (5 gm/kg diet) or normal (1.1 gm/kg diet) choline at mating and remained on the specific diet until offspring weaning. Thereafter, offspring were fed standard rodent chow. Adult offspring were assessed for sensory inhibition. Brains were obtained to ascertain hippocampal α7 nicotinic receptor levels. Choline-supplemented mice heterozygotic or null-mutant for Chrna7 failed to show improvement in sensory inhibition. Only wildtype choline-supplemented mice showed improvement with the effect solely through a decrease in test amplitude. This supports the hypothesis that gestational-choline supplementation is acting through the α7 nicotinic receptor to improve sensory inhibition. Although there was a significant gene-dose-related change in hippocampal α7 receptor numbers, binding studies did not reveal any choline-dose-related change in binding in any hippocampal region, the interaction being driven by a significant genotype main effect (wildtype>heterozygote>null mutant). These data parallel a human study wherein the offspring of pregnant women receiving choline supplementation during gestation, showed better sensory inhibition than offspring of women on placebo. Published by Elsevier B.V.

Long-term improvements in sensory inhibition with gestational choline supplementation linked to α7 nicotinic receptors through studies in Chrna7 null mutation mice

PubMed Central

Stevens, Karen E.; Choo, Kevin S.; Stitzel, Jerry A.; Marks, Michael J.; Adams, Catherine E.

2014-01-01

Perinatal choline supplementation has produced several benefits in rodent models, from improved learning and memory to protection from the behavioral effects of fetal alcohol exposure. We have shown that supplemented choline through gestation and lactation produces long-term improvement in deficient sensory inhibition in DBA/2 mice which models a similar deficit in schizophrenia patients. The present study extends that research by feeding normal or supplemented choline diets to DBA/2 mice carrying the null mutation for the α7 nicotinic receptor gene (Chrna7). DBA/2 mice heterozygotic for Chrna7 were bred together. Dams were placed on supplemented (5 gm/kg diet) or normal (1.1 gm/kg diet) choline at mating and remained on the specific diet until offspring weaning. Thereafter, offspring were fed standard rodent chow. Adult offspring were assessed for sensory inhibition. Brains were obtained to ascertain hippocampal α7 nicotinic receptor levels. Choline-supplemented mice heterozygotic or null-mutant for Chrna7 failed to show improvement in sensory inhibition. Only wildtype choline-supplemented mice showed improvement with the effect solely through a decrease in test amplitude. This supports the hypothesis that gestational-choline supplementation is acting through the α7 nicotinic receptor to improve sensory inhibition. Although there was a significant gene-dose-related change in hippocampal α7 receptor numbers, binding studies did not reveal any choline-dose-related change in binding in any hippocampal region, the interaction being driven by a significant genotype main effect (wildtype>heterozygote>null mutant). These data parallel a human study wherein the offspring of pregnant women receiving choline supplementation during gestation, showed better sensory inhibition than offspring of women on placebo. PMID:24462939

Usher syndrome: animal models, retinal function of Usher proteins, and prospects for gene therapy

PubMed Central

Williams, David S.

2009-01-01

Usher syndrome is a deafness-blindness disorder. The blindness occurs from a progressive retinal degeneration that begins after deafness and after the retina has developed. Three clinical subtypes of Usher syndrome have been identified, with mutations in any one of six different genes giving rise to type 1, in any one of three different genes to type 2, and in one identified gene causing Usher type 3. Mutant mice for most of the genes have been studied; while they have clear inner ear defects, retinal phenotypes are relatively mild and have been difficult to characterize. The retinal functions of the Usher proteins are still largely unknown. Protein binding studies have suggested many interactions among the proteins, and a model of interaction among all the proteins in the photoreceptor synapse has been proposed. However this model is not supported by localization data from some laboratories, or the indication of any synaptic phenotype in mutant mice. An earlier suggestion, based on patient pathologies, of Usher protein function in the photoreceptor cilium continues to gain support from immunolocalization and mutant mouse studies, which are consistent with Usher protein interaction in the photoreceptor ciliary/periciliary region. So far, the most characterized Usher protein is myosin VIIa. It is present in the apical RPE and photoreceptor ciliary/periciliary region, where it is required for organelle transport and clearance of opsin from the connecting cilium, respectively. Usher syndrome is amenable to gene replacement therapy, but also has some specific challenges. Progress in this treatment approach has been achieved by correction of mutant phenotypes in Myo7a-null mouse retinas, following lentiviral delivery of MYO7A. PMID:17936325

Sf29 Gene of Spodoptera frugiperda Multiple Nucleopolyhedrovirus Is a Viral Factor That Determines the Number of Virions in Occlusion Bodies▿

PubMed Central

Simón, Oihane; Williams, Trevor; Asensio, Aaron C.; Ros, Sarhay; Gaya, Andrea; Caballero, Primitivo; Possee, Robert D.

2008-01-01

The genome of Spodoptera frugiperda multiple nucleopolyhedrovirus (NPV) was inserted into a bacmid (Sfbac) and used to produce a mutant lacking open reading frame 29 (Sf29null). Sf29null bacmid DNA was able to generate an infection in S. frugiperda. Approximately six times less DNA was present in occlusion bodies (OBs) produced by the Sf29null bacmid in comparison to viruses containing this gene. This reduction in DNA content was consistent with fewer virus particles being packaged within Sf29null bacmid OBs, as determined by fractionation of dissolved polyhedra and comparison of occlusion-derived virus (ODV) infectivity in cell culture. DNA from Sfbac, Sf29null, or Sf29null-repair, in which the gene deletion had been repaired, were equally infectious when used to transfect S. frugiperda. All three viruses produced similar numbers of OBs, although those from Sf29null were 10-fold less infectious than viruses with the gene. Insects infected with Sf29null bacmid died ∼24 h later than positive controls, consistent with the reduced virus particle content of Sf29null OBs. Transcripts from Sf29 were detected in infected insects 12 h prior to those from the polyhedrin gene. Homologs to Sf29 were present in other group II NPVs, and similar sequences were present in entomopoxviruses. Analysis of the Sf29 predicted protein sequence revealed signal peptide and transmembrane domains, but the presence of 12 potential N-glycosylation sites suggest that it is not an ODV envelope protein. Other motifs, including zinc-binding and threonine-rich regions, suggest degradation and adhesion functions. We conclude that Sf29 is a viral factor that determines the number of ODVs occluded in each OB. PMID:18550678

The Sterol Methyltransferases SMT1, SMT2, and SMT3 Influence Arabidopsis Development through Nonbrassinosteroid Products1[W][OA

PubMed Central

Carland, Francine; Fujioka, Shozo; Nelson, Timothy

2010-01-01

Plant sterols are structural components of cell membranes that provide rigidity, permeability, and regional identity to membranes. Sterols are also the precursors to the brassinosteroid signaling molecules. Evidence is accumulating that specific sterols have roles in pattern formation during development. COTYLEDON VASCULAR PATTERNING1 (CVP1) encodes C-24 STEROL METHYLTRANSFERASE2 (SMT2), one of three SMTs in Arabidopsis (Arabidopsis thaliana). SMT2 and SMT3, which also encodes a C-24 SMT, catalyze the reaction that distinguishes the synthesis of structural sterols from signaling brassinosteroid derivatives and are highly regulated. The deficiency of SMT2 in the cvp1 mutant results in moderate developmental defects, including aberrant cotyledon vein patterning, serrated floral organs, and reduced stature, but plants are viable, suggesting that SMT3 activity can substitute for the loss of SMT2. To test the distinct developmental roles of SMT2 and SMT3, we identified a transcript null smt3 mutant. Although smt3 single mutants appear wild type, cvp1 smt3 double mutants show enhanced defects relative to cvp1 mutants, such as discontinuous cotyledon vein pattern, and produce novel phenotypes, including defective root growth, loss of apical dominance, sterility, and homeotic floral transformations. These phenotypes are correlated with major alterations in the profiles of specific sterols but without significant alterations to brassinosteroid profiles. The alterations to sterol profiles in cvp1 mutants affect auxin response, demonstrated by weak auxin insensitivity, enhanced axr1 auxin resistance, ectopically expressed DR5:β-glucuronidase in developing embryos, and defective response to auxin-inhibited PIN2-green fluorescent protein endocytosis. We discuss the developmental roles of sterols implied by these results. PMID:20421456

Suppression of calbindin-D28k expression exacerbates SCA1 phenotype in a disease mouse model.

PubMed

Vig, Parminder J S; Wei, Jinrong; Shao, Qingmei; Lopez, Maripar E; Halperin, Rebecca; Gerber, Jill

2012-09-01

Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurological disorder caused by the expansion of a polyglutamine tract in the mutant protein ataxin-1. The cerebellar Purkinje cells (PCs) are the major targets of mutant ataxin-1. The mechanism of PC death in SCA1 is not known; however, previous work indicates that downregulation of specific proteins involved in calcium homeostasis and signaling by mutant ataxin-1 is the probable cause of PC degeneration in SCA1. In this study, we explored if targeted deprivation of PC specific calcium-binding protein calbindin-D28k (CaB) exacerbates ataxin-1 mediated toxicity in SCA1 transgenic (Tg) mice. Using behavioral tests, we found that though both SCA1/+ and SCA1/+: CaB null (-/+) double mutants exhibited progressive impaired performance on the rotating rod, a simultaneous enhancement of exploratory activity, and absence of deficits in coordination, the double mutants were more severely impaired than SCA1/+ mice. With increasing age, SCA1/+ mice showed a progressive loss in the expression and localization of CaB and other PC specific calcium-binding and signaling proteins. In double mutants, these changes were more pronounced and had an earlier onset. Gene expression profiling of young mice exhibiting no behavior or biochemical deficits revealed a differential expression of many genes common to SCA1/+ and CaB-/+ lines, and unique to SCA1/+: CaB-/+ phenotype. Our study provides further evidence for a critical role of CaB in SCA1 pathogenesis, which may help identify new therapeutic targets to treat SCA1 or other cerebellar ataxias.

Increased lipolysis and altered lipid homeostasis protect y-synuclein null mutant mice from diet-induced obesity

USDA-ARS?s Scientific Manuscript database

Synucleins are a family of homologous proteins principally known for their involvement in neurodegeneration. In neurons a-synuclein promotes assembly of SNARE complexes required for fusion of synaptic vesicles with the plasma membrane during neurotransmitter release. Y-synuclein is highly expressed ...

Synapsin Is Selectively Required for Anesthesia-Sensitive Memory

ERIC Educational Resources Information Center

Knapek, Stephan; Gerber, Bertram; Tanimoto, Hiromu

2010-01-01

Odor-shock memory in "Drosophila melanogaster" consists of heterogeneous components each with different dynamics. We report that a null mutant for the evolutionarily conserved synaptic protein Synapsin entails a memory deficit selectively in early memory, leaving later memory as well as sensory motor function unaffected. Notably, a consolidated…

Fragile X mental retardation protein has a unique, evolutionarily conserved neuronal function not shared with FXR1P or FXR2P

PubMed Central

Coffee, R. Lane; Tessier, Charles R.; Woodruff, Elvin A.; Broadie, Kendal

2010-01-01

SUMMARY Fragile X syndrome (FXS), resulting solely from the loss of function of the human fragile X mental retardation 1 (hFMR1) gene, is the most common heritable cause of mental retardation and autism disorders, with syndromic defects also in non-neuronal tissues. In addition, the human genome encodes two closely related hFMR1 paralogs: hFXR1 and hFXR2. The Drosophila genome, by contrast, encodes a single dFMR1 gene with close sequence homology to all three human genes. Drosophila that lack the dFMR1 gene (dfmr1 null mutants) recapitulate FXS-associated molecular, cellular and behavioral phenotypes, suggesting that FMR1 function has been conserved, albeit with specific functions possibly sub-served by the expanded human gene family. To test evolutionary conservation, we used tissue-targeted transgenic expression of all three human genes in the Drosophila disease model to investigate function at (1) molecular, (2) neuronal and (3) non-neuronal levels. In neurons, dfmr1 null mutants exhibit elevated protein levels that alter the central brain and neuromuscular junction (NMJ) synaptic architecture, including an increase in synapse area, branching and bouton numbers. Importantly, hFMR1 can, comparably to dFMR1, fully rescue both the molecular and cellular defects in neurons, whereas hFXR1 and hFXR2 provide absolutely no rescue. For non-neuronal requirements, we assayed male fecundity and testes function. dfmr1 null mutants are effectively sterile owing to disruption of the 9+2 microtubule organization in the sperm tail. Importantly, all three human genes fully and equally rescue mutant fecundity and spermatogenesis defects. These results indicate that FMR1 gene function is evolutionarily conserved in neural mechanisms and cannot be compensated by either FXR1 or FXR2, but that all three proteins can substitute for each other in non-neuronal requirements. We conclude that FMR1 has a neural-specific function that is distinct from its paralogs, and that the unique FMR1 function is responsible for regulating neuronal protein expression and synaptic connectivity. PMID:20442204

Pofut1 point-mutations that disrupt O-fucosyltransferase activity destabilize the protein and abolish Notch1 signaling during mouse somitogenesis

PubMed Central

Suzuki, Emiko; Saga, Yumiko

2017-01-01

The segmental pattern of the vertebrate body is established via the periodic formation of somites from the presomitic mesoderm (PSM). This periodical process is controlled by the cyclic and synchronized activation of Notch signaling in the PSM. Protein O-fucosyltransferase1 (Pofut1), which transfers O-fucose to the EGF domains of the Notch1 receptor, is indispensable for Notch signaling activation. The Drosophila homologue Ofut1 was reported to control Notch localization via two different mechanisms, working as a chaperone for Notch or as a regulator of Notch endocytosis. However, these were found to be independent of O-fucosyltransferase activity because the phenotypes were rescued by Ofut1 mutants lacking O-fucosyltransferase activity. Pofut1 may also be involved in the Notch receptor localization in mice. However, the contribution of enzymatic activity of Pofut1 to the Notch receptor dynamics remains to be elucidated. In order to clarify the importance of the O-fucosyltransferase activity of Pofut1 for Notch signaling activation and the protein localization in the PSM, we established mice carrying point mutations at the 245th a.a. or 370-372th a.a., highly conserved amino-acid sequences whose mutations disrupt the O-fucosyltransferase activity of both Drosophila Ofut1 and mammalian Pofut1, with the CRISPR/Cas9 mediated genome-engineering technique. Both mutants displayed the same severely perturbed somite formation and Notch1 subcellular localization defects as the Pofut1 null mutants. In the mutants, Pofut1 protein, but not RNA, became undetectable by E9.5. Furthermore, both wild-type and mutant Pofut1 proteins were degraded through lysosome dependent machinery. Pofut1 protein loss in the point mutant embryos caused the same phenotypes as those observed in Pofut1 null embryos. PMID:29095923

Tissue-specific roles of Tbx1 in the development of the outer, middle and inner ear, defective in 22q11DS patients

PubMed Central

Arnold, Jelena S.; Braunstein, Evan M.; Ohyama, Takahiro; Groves, Andrew K.; Adams, Joe C.; Brown, M. Christian; Morrow, Bernice E.

2007-01-01

Most 22q11.2 deletion syndrome (22q11DS) patients have middle and outer ear anomalies, whereas some have inner ear malformations. Tbx1, a gene hemizygously deleted in 22q11DS patients and required for ear development, is expressed in multiple tissues during embryogenesis. To determine the role of Tbx1 in the first pharyngeal pouch (PPI) in forming outer and middle ears, we tissue-specifically inactivated the gene using Foxg1-Cre. In the conditional mutants, PPI failed to outgrow, preventing the middle ear bone condensations from forming. Tbx1 was also inactivated in the otic vesicle (OV), resulting in the failure of inner ear sensory organ formation, and in duplication of the cochleovestibular ganglion (CVG). Consistent with the anatomical defects, the sensory genes, Otx1 and Bmp4 were downregulated, whereas the CVG genes, Fgf3 and NeuroD, were upregulated. To delineate Tbx1 cell-autonomous roles, a more selective ablation, exclusively in the OV, was performed using Pax2-Cre. In contrast to the Foxg1-Cre mutants, Pax2-Cre conditional mutant mice survived to adulthood and had normal outer and middle ears but had the same inner ear defects as the Tbx1 null mice, with the same gene expression changes. These results demonstrate that Tbx1 has non-cell autonomous roles in PPI in the formation of outer and middle ears and cell-autonomous roles in the OV. Periotic mesenchymal markers, Prx2 and Brn4 were normal in both conditional mutants, whereas they were diminished in Tbx1−/− embryos. Thus, Tbx1 in the surrounding mesenchyme in both sets of conditional mutants cannot suppress the defects in the OV that occur in the null mutants. PMID:16600992

Identification and partial characterization of the enzyme of omega: one of five putative DPP IV genes in Drosophila melanogaster

PubMed Central

Chihara, Carol J.; Song, Chunyan; LaMonte, Greg; Fetalvero, Kristina; Hinchman, Kristy; Phan, Helen; Pineda, Mario; Robinson, Kelly; Schneider, Gregory P.

2005-01-01

The omega (ome) gene product is a modifier of larval cuticle protein 5 and its alleles (and duplicates) in the third instar of Drosophila melanogaster. Using deletion mapping the locus mapped to 70F-71A on the left arm of chromosome 3. A homozygote null mutant (ome 1) shows a pleiotropic phenotype that affected the size, developmental time of the flies, and the fertility (or perhaps the behavior) of homozygous mutant males. The omega gene was verified as producing a dipeptidyl peptidase IV (DPPIV) by genetic analysis, substrate specificity and pH optimum. The identity of the gene was confirmed as CG32145 (cytology 70F4) in the Celera Database (Berkeley Drosophila Genome Project), which is consistent with its deletion map position. The genomic structure of the gene is described and the decrease in DPPIV activity in the mutant ome1 is shown to be due to the gene CG32145 (omega). The D. melanogaster omega DPPIV enzyme was partially purified and characterized. The exons of the ome1 mutant were sequenced and a base substitution mutation in exon 4 was identified that would yield a truncated protein caused by a stop codon. A preliminary study of the compartmentalization of the omega DPPIV enzyme in several organs is also reported. Abbreviations: DPPIV dipeptidyl peptidase IV LCP5 & LCP6 third instar larval cuticle proteins 5 & 6 ome & ome1 omega locus name (CG32145) and mutant allele in D. melanogaster pNA paranotroanilide PMID:17119608

Alteration of cell wall xylan acetylation triggers defense responses that counterbalance the immune deficiencies of plants impaired in the β-subunit of the heterotrimeric G-protein.

PubMed

Escudero, Viviana; Jordá, Lucía; Sopeña-Torres, Sara; Mélida, Hugo; Miedes, Eva; Muñoz-Barrios, Antonio; Swami, Sanjay; Alexander, Danny; McKee, Lauren S; Sánchez-Vallet, Andrea; Bulone, Vincent; Jones, Alan M; Molina, Antonio

2017-11-01

Arabidopsis heterotrimeric G-protein complex modulates pathogen-associated molecular pattern-triggered immunity (PTI) and disease resistance responses to different types of pathogens. It also plays a role in plant cell wall integrity as mutants impaired in the Gβ- (agb1-2) or Gγ-subunits have an altered wall composition compared with wild-type plants. Here we performed a mutant screen to identify suppressors of agb1-2 (sgb) that restore susceptibility to pathogens to wild-type levels. Out of the four sgb mutants (sgb10-sgb13) identified, sgb11 is a new mutant allele of ESKIMO1 (ESK1), which encodes a plant-specific polysaccharide O-acetyltransferase involved in xylan acetylation. Null alleles (sgb11/esk1-7) of ESK1 restore to wild-type levels the enhanced susceptibility of agb1-2 to the necrotrophic fungus Plectosphaerella cucumerina BMM (PcBMM), but not to the bacterium Pseudomonas syringae pv. tomato DC3000 or to the oomycete Hyaloperonospora arabidopsidis. The enhanced resistance to PcBMM of the agb1-2 esk1-7 double mutant was not the result of the re-activation of deficient PTI responses in agb1-2. Alteration of cell wall xylan acetylation caused by ESK1 impairment was accompanied by an enhanced accumulation of abscisic acid, the constitutive expression of genes encoding antibiotic peptides and enzymes involved in the biosynthesis of tryptophan-derived metabolites, and the accumulation of disease resistance-related secondary metabolites and different osmolites. These esk1-mediated responses counterbalance the defective PTI and PcBMM susceptibility of agb1-2 plants, and explain the enhanced drought resistance of esk1 plants. These results suggest that a deficient PTI-mediated resistance is partially compensated by the activation of specific cell-wall-triggered immune responses. © 2017 The Authors The Plant Journal published by John Wiley & Sons Ltd and Society for Experimental Biology.

Secreted Citrate Serves as Iron Carrier for the Marine Pathogen Photobacterium damselae subsp damselae

PubMed Central

Balado, Miguel; Puentes, Beatriz; Couceiro, Lucía; Fuentes-Monteverde, Juan C.; Rodríguez, Jaime; Osorio, Carlos R.; Jiménez, Carlos; Lemos, Manuel L.

2017-01-01

Photobacterium damselae subsp damselae (Pdd) is a Vibrionaceae that has a wide pathogenic potential against many marine animals and also against humans. Some strains of this bacterium acquire iron through the siderophore vibrioferrin. However, there are virulent strains that do not produce vibrioferrin, but they still give a strong positive reaction in the CAS test for siderophore production. In an in silico search on the genome sequences of this type of strains we could not find any ORF which could be related to a siderophore system. To identify genes that could encode a siderophore-mediated iron acquisition system we used a mini-Tn10 transposon random mutagenesis approach. From more than 1,400 mutants examined, we could isolate a mutant (BP53) that showed a strong CAS reaction independently of the iron levels of the medium. In this mutant the transposon was inserted into the idh gene, which encodes an isocitrate dehydrogenase that participates in the tricarboxylic acid cycle. The mutant did not show any growth impairment in rich or minimal media, but it accumulated a noticeable amount of citrate (around 7 mM) in the culture medium, irrespective of the iron levels. The parental strain accumulated citrate, but in an iron-regulated fashion, being citrate levels 5–6 times higher under iron restricted conditions. In addition, a null mutant deficient in citrate synthase showed an impairment for growth at high concentrations of iron chelators, and showed almost no reaction in the CAS test. Chemical analysis by liquid chromatography of the iron-restricted culture supernatants resulted in a CAS-positive fraction with biological activity as siderophore. HPLC purification of that fraction yielded a pure compound which was identified as citrate from its MS and NMR spectral data. Although the production of another citrate-based compound with siderophore activity cannot be ruled out, our results suggest that Pdd secretes endogenous citrate and use it for iron scavenging from the cell environment. PMID:28848719

Compensatory changes in CYP expression in three different toxicology mouse models: CAR-null, Cyp3a-null, and Cyp2b9/10/13-null mice

PubMed Central

Kumar, Ramiya; Mota, Linda C.; Litoff, Elizabeth J.; Rooney, John P.; Boswell, W. Tyler; Courter, Elliott; Henderson, Charles M.; Hernandez, Juan P.; Corton, J. Christopher; Moore, David D.

2017-01-01

Targeted mutant models are common in mechanistic toxicology experiments investigating the absorption, metabolism, distribution, or elimination (ADME) of chemicals from individuals. Key models include those for xenosensing transcription factors and cytochrome P450s (CYP). Here we investigated changes in transcript levels, protein expression, and steroid hydroxylation of several xenobiotic detoxifying CYPs in constitutive androstane receptor (CAR)-null and two CYP-null mouse models that have subfamily members regulated by CAR; the Cyp3a-null and a newly described Cyp2b9/10/13-null mouse model. Compensatory changes in CYP expression that occur in these models may also occur in polymorphic humans, or may complicate interpretation of ADME studies performed using these models. The loss of CAR causes significant changes in several CYPs probably due to loss of CAR-mediated constitutive regulation of these CYPs. Expression and activity changes include significant repression of Cyp2a and Cyp2b members with corresponding drops in 6α- and 16β-testosterone hydroxylase activity. Further, the ratio of 6α-/15α-hydroxylase activity, a biomarker of sexual dimorphism in the liver, indicates masculinization of female CAR-null mice, suggesting a role for CAR in the regulation of sexually dimorphic liver CYP profiles. The loss of Cyp3a causes fewer changes than CAR. Nevertheless, there are compensatory changes including gender-specific increases in Cyp2a and Cyp2b. Cyp2a and Cyp2b were down-regulated in CAR-null mice, suggesting activation of CAR and potentially PXR following loss of the Cyp3a members. However, the loss of Cyp2b causes few changes in hepatic CYP transcript levels and almost no significant compensatory changes in protein expression or activity with the possible exception of 6α-hydroxylase activity. This lack of a compensatory response in the Cyp2b9/10/13-null mice is probably due to low CYP2B hepatic expression, especially in male mice. Overall, compensatory and regulatory CYP changes followed the order CAR-null > Cyp3a-null > Cyp2b-null mice. PMID:28350814

Compensatory changes in CYP expression in three different toxicology mouse models: CAR-null, Cyp3a-null, and Cyp2b9/10/13-null mice.

PubMed

Kumar, Ramiya; Mota, Linda C; Litoff, Elizabeth J; Rooney, John P; Boswell, W Tyler; Courter, Elliott; Henderson, Charles M; Hernandez, Juan P; Corton, J Christopher; Moore, David D; Baldwin, William S

2017-01-01

Targeted mutant models are common in mechanistic toxicology experiments investigating the absorption, metabolism, distribution, or elimination (ADME) of chemicals from individuals. Key models include those for xenosensing transcription factors and cytochrome P450s (CYP). Here we investigated changes in transcript levels, protein expression, and steroid hydroxylation of several xenobiotic detoxifying CYPs in constitutive androstane receptor (CAR)-null and two CYP-null mouse models that have subfamily members regulated by CAR; the Cyp3a-null and a newly described Cyp2b9/10/13-null mouse model. Compensatory changes in CYP expression that occur in these models may also occur in polymorphic humans, or may complicate interpretation of ADME studies performed using these models. The loss of CAR causes significant changes in several CYPs probably due to loss of CAR-mediated constitutive regulation of these CYPs. Expression and activity changes include significant repression of Cyp2a and Cyp2b members with corresponding drops in 6α- and 16β-testosterone hydroxylase activity. Further, the ratio of 6α-/15α-hydroxylase activity, a biomarker of sexual dimorphism in the liver, indicates masculinization of female CAR-null mice, suggesting a role for CAR in the regulation of sexually dimorphic liver CYP profiles. The loss of Cyp3a causes fewer changes than CAR. Nevertheless, there are compensatory changes including gender-specific increases in Cyp2a and Cyp2b. Cyp2a and Cyp2b were down-regulated in CAR-null mice, suggesting activation of CAR and potentially PXR following loss of the Cyp3a members. However, the loss of Cyp2b causes few changes in hepatic CYP transcript levels and almost no significant compensatory changes in protein expression or activity with the possible exception of 6α-hydroxylase activity. This lack of a compensatory response in the Cyp2b9/10/13-null mice is probably due to low CYP2B hepatic expression, especially in male mice. Overall, compensatory and regulatory CYP changes followed the order CAR-null > Cyp3a-null > Cyp2b-null mice.

Boron Tolerance in Aspergillus nidulans Is Sustained by the SltA Pathway Through the SLC-Family Transporters SbtA and SbtB

PubMed Central

Villarino, María; Mendizabal, Gorka; Garzia, Aitor; Ugalde, Unai

2017-01-01

Microbial cells interact with the environment by adapting to external changes. Signal transduction pathways participate in both sensing and responding in the form of modification of gene expression patterns, enabling cell survival. The filamentous fungal-specific SltA pathway regulates tolerance to alkalinity, elevated cation concentrations and, as shown in this work, also stress conditions induced by borates. Growth of sltA− mutants is inhibited by increasing millimolar concentrations of boric acid or borax (sodium tetraborate). In an attempt to identify genes required for boron-stress response, we determined the boric acid or borax-dependent expression of sbtA and sbtB, orthologs of Saccharomyces cerevisiae bor1, and a reduction in their transcript levels in a ΔsltA mutant. Deletion of sbtA, but mainly that of sbtB, decreased the tolerance to boric acid or borax. In contrast, null mutants of genes coding for additional transporters of the Solute Carrier (SLC) family, sB, sbtD or sbtE, showed an unaltered growth pattern under the same stress conditions. Taken together, our results suggest that the SltA pathway induces, through SbtA and SbtB, the export of toxic concentrations of borates, which have largely recognized antimicrobial properties. PMID:28753996

The sps Gene Products Affect the Germination, Hydrophobicity, and Protein Adsorption of Bacillus subtilis Spores

PubMed Central

Cangiano, Giuseppina; Sirec, Teja; Panarella, Cristina; Isticato, Rachele; Baccigalupi, Loredana; De Felice, Maurilio

2014-01-01

The multilayered surface of the Bacillus subtilis spore is composed of proteins and glycans. While over 70 different proteins have been identified as surface components, carbohydrates associated with the spore surface have not been characterized in detail yet. Bioinformatic data suggest that the 11 products of the sps operon are involved in the synthesis of polysaccharides present on the spore surface, but an experimental validation is available only for the four distal genes of the operon. Here, we report a transcriptional analysis of the sps operon and a functional study performed by constructing and analyzing two null mutants lacking either all or only the promoter-proximal gene of the operon. Our results show that both sps mutant spores apparently have normal coat and crust but have a small germination defect and are more hydrophobic than wild-type spores. We also show that spores lacking all Sps proteins are highly adhesive and form extensive clumps. In addition, sps mutant spores have an increased efficiency in adsorbing a heterologous enzyme, suggesting that hydrophobic force is a major determinant of spore adsorption and indicating that a deep understanding of the surface properties of the spore is essential for its full development as a surface display platform. PMID:25239894

Seizure phenotypes, periodicity, and sleep-wake pattern of seizures in Kcna-1 null mice.

PubMed

Wright, Samantha; Wallace, Eli; Hwang, Youngdeok; Maganti, Rama

2016-02-01

This study was undertaken to describe seizure phenotypes, natural progression, sleep-wake patterns, as well as periodicity of seizures in Kcna-1 null mutant mice. These mice were implanted with epidural electroencephalography (EEG) and electromyography (EMG) electrodes, and simultaneous video-EEG recordings were obtained while animals were individually housed under either diurnal (LD) condition or constant darkness (DD) over ten days of recording. The video-EEG data were analyzed to identify electrographic and behavioral phenotypes and natural progression and to examine the periodicity of seizures. Sleep-wake patterns were analyzed to understand the distribution and onset of seizures across the sleep-wake cycle. Four electrographically and behaviorally distinct seizure types were observed. Regardless of lighting condition that animals were housed in, Kcna-1 null mice initially expressed only a few of the most severe seizure types that progressively increased in frequency and decreased in seizure severity. In addition, a circadian periodicity was noted, with seizures peaking in the first 12h of the Zeitgeber time (ZT) cycle, regardless of lighting conditions. Interestingly, seizure onset differed between lighting conditions where more seizures arose out of sleep in LD conditions, whereas under DD conditions, the majority occurred out of the wakeful state. We suggest that this model be used to understand the circadian pattern of seizures as well as the pathophysiological implications of sleep and circadian disturbances in limbic epilepsies. Copyright © 2015 Elsevier Inc. All rights reserved.

Involvement of MoVMA11, a Putative Vacuolar ATPase c’ Subunit, in Vacuolar Acidification and Infection-Related Morphogenesis of Magnaporthe oryzae

PubMed Central

Chen, Guoqing; Liu, Xiaohong; Zhang, Lilin; Cao, Huijuan; Lu, Jianping; Lin, Fucheng

2013-01-01

Many functions of vacuole depend on the activity of vacuolar ATPase which is essential to maintain an acidic lumen and create the driving forces for massive fluxes of ions and metabolites through vacuolar membrane. In filamentous fungus Magnaporthe oryzae , subcellular colocalization and quinacrine staining suggested that the V1V0 domains of V-ATPase were fully assembled and the vacuoles were kept acidic during infection-related developments. Targeted gene disruption of MoVMA11 gene, encoding the putative c’ subunit of V-ATPase, impaired vacuolar acidification and mimicked the phenotypes of yeast V-ATPase mutants in the poor colony morphology, abolished asexual and sexual reproductions, selective carbon source utilization, and increased calcium and heavy metals sensitivities, however, not in the typical pH conditional lethality. Strikingly, aerial hyphae of the MoVMA11 null mutant intertwined with each other to form extremely thick filamentous structures. The results also implicated that MoVMA11 was involved in cell wall integrity and appressorium formation. Abundant non-melanized swollen structures and rare, small appressoria without penetration ability were produced at the hyphal tips of the ΔMovma11 mutant on onion epidermal cells. Finally, the MoVMA11 null mutant lost pathogenicity on both intact and wounded host leaves. Overall, our data indicated that MoVMA11, like other fungal VMA genes, is associated with numerous cellular functions and highlighted that V-ATPase is essential for infection-related morphogenesis and pathogenesis in M . oryzae . PMID:23826342

Ku must load directly onto the chromosome end in order to mediate its telomeric functions.

PubMed

Lopez, Christopher R; Ribes-Zamora, Albert; Indiviglio, Sandra M; Williams, Christopher L; Haricharan, Svasti; Bertuch, Alison A

2011-08-01

The Ku heterodimer associates with the Saccharomyces cerevisiae telomere, where it impacts several aspects of telomere structure and function. Although Ku avidly binds DNA ends via a preformed channel, its ability to associate with telomeres via this mechanism could be challenged by factors known to bind directly to the chromosome terminus. This has led to uncertainty as to whether Ku itself binds directly to telomeric ends and whether end association is crucial for Ku's telomeric functions. To address these questions, we constructed DNA end binding-defective Ku heterodimers by altering amino acid residues in Ku70 and Ku80 that were predicted to contact DNA. These mutants continued to associate with their known telomere-related partners, such as Sir4, a factor required for telomeric silencing, and TLC1, the RNA component of telomerase. Despite these interactions, we found that the Ku mutants had markedly reduced association with telomeric chromatin and null-like deficiencies for telomere end protection, length regulation, and silencing functions. In contrast to Ku null strains, the DNA end binding defective Ku mutants resulted in increased, rather than markedly decreased, imprecise end-joining proficiency at an induced double-strand break. This result further supports that it was the specific loss of Ku's telomere end binding that resulted in telomeric defects rather than global loss of Ku's functions. The extensive telomere defects observed in these mutants lead us to propose that Ku is an integral component of the terminal telomeric cap, where it promotes a specific architecture that is central to telomere function and maintenance.

Ku Must Load Directly onto the Chromosome End in Order to Mediate Its Telomeric Functions

PubMed Central

Lopez, Christopher R.; Ribes-Zamora, Albert; Indiviglio, Sandra M.; Williams, Christopher L.; Haricharan, Svasti; Bertuch, Alison A.

2011-01-01

The Ku heterodimer associates with the Saccharomyces cerevisiae telomere, where it impacts several aspects of telomere structure and function. Although Ku avidly binds DNA ends via a preformed channel, its ability to associate with telomeres via this mechanism could be challenged by factors known to bind directly to the chromosome terminus. This has led to uncertainty as to whether Ku itself binds directly to telomeric ends and whether end association is crucial for Ku's telomeric functions. To address these questions, we constructed DNA end binding–defective Ku heterodimers by altering amino acid residues in Ku70 and Ku80 that were predicted to contact DNA. These mutants continued to associate with their known telomere-related partners, such as Sir4, a factor required for telomeric silencing, and TLC1, the RNA component of telomerase. Despite these interactions, we found that the Ku mutants had markedly reduced association with telomeric chromatin and null-like deficiencies for telomere end protection, length regulation, and silencing functions. In contrast to Ku null strains, the DNA end binding defective Ku mutants resulted in increased, rather than markedly decreased, imprecise end-joining proficiency at an induced double-strand break. This result further supports that it was the specific loss of Ku's telomere end binding that resulted in telomeric defects rather than global loss of Ku's functions. The extensive telomere defects observed in these mutants lead us to propose that Ku is an integral component of the terminal telomeric cap, where it promotes a specific architecture that is central to telomere function and maintenance. PMID:21852961

Regulation of glycolysis in Kluyveromyces lactis: role of KlGCR1 and KlGCR2 in glucose uptake and catabolism.

PubMed

Neil, H; Lemaire, M; Wésolowski-Louvel, M

2004-03-01

In Kluyveromyces lactis, the casein kinase I (Rag8p) regulates the transcription of glycolytic genes and the expression of the low-affinity glucose transporter gene RAG1. This control involves the transcription factor Sck1p, a homologue of Sgc1p of Saccharomyces cerevisiae. SGC1 is known to interact genetically with ScGCR1 and ScGCR2, which code for regulators of glycolytic gene expression. Therefore, we studied the role of KlGCR1 and KlGCR2 genes in K. lactis. The Klgcr1 null mutant could not grow on glucose when respiration was blocked by antimycin A (Rag(- )phenotype). In contrast, the Klgcr2 null mutant could grow under the same conditions, although at a reduced rate. In both mutants, the transcription of glycolytic genes was affected, while that of ribosomal protein genes was not modified. Furthermore, the transcription of the glucose permease genes was also found to be affected in the two mutants, although dissimilarly. While RAG1 transcription decreased at high glucose concentrations, the expression of the high-affinity glucose permease gene HGT1 was unexpectedly impaired under gluconeogenic conditions, in the absence of glucose. Gel mobility shift assays performed with purified maltose-binding protein-KlGcr1p showed that KlGcr1p could interact directly with the promoters of the glycolytic genes, but not with the promoters of the glucose permease genes. Thus, the control exerted by KlGcr1p and KlGcr2p upon glucose transporter genes is probably indirect.

Nanoridges that characterize the surface morphology of flowers require the synthesis of cutin polyester

PubMed Central

Li-Beisson, Yonghua; Pollard, Mike; Sauveplane, Vincent; Pinot, Franck; Ohlrogge, John; Beisson, Fred

2009-01-01

Distinctive nanoridges on the surface of flowers have puzzled plant biologists ever since their discovery over 75 years ago. Although postulated to help attract insect pollinators, the function, chemical nature, and ontogeny of these surface nanostructures remain uncertain. Studies have been hampered by the fact that no ridgeless mutants have been identified. Here, we describe two mutants lacking nanoridges and define the biosynthetic pathway for 10,16-dihydroxypalmitate, a major cutin monomer in nature. Using gene expression profiling, two candidates for the formation of floral cutin were identified in the model plant Arabidopsis thaliana: the glycerol-3-phosphate acyltransferase 6 (GPAT6) and a member of a cytochrome P450 family with unknown biological function (CYP77A6). Plants carrying null mutations in either gene produced petals with no nanoridges and no cuticle could be observed by either scanning or transmission electron microscopy. A strong reduction in cutin content was found in flowers of both mutants. In planta overexpression suggested GPAT6 preferentially uses palmitate derivatives in cutin synthesis. Comparison of cutin monomer profiles in knockouts for CYP77A6 and the fatty acid ω-hydroxylase CYP86A4 provided genetic evidence that CYP77A6 is an in-chain hydroxylase acting subsequently to CYP86A4 in the synthesis of 10,16-dihydroxypalmitate. Biochemical activity of CYP77A6 was demonstrated by production of dihydroxypalmitates from 16-hydroxypalmitate, using CYP77A6-expressing yeast microsomes. These results define the biosynthetic pathway for an abundant and widespread monomer of the cutin polyester, show that the morphology of floral surfaces depends on the synthesis of cutin, and identify target genes to investigate the function of nanoridges in flower biology. PMID:19959665

Nanoridges that characterize the surface morphology of flowers require the synthesis of cutin polyester.

PubMed

Li-Beisson, Yonghua; Pollard, Mike; Sauveplane, Vincent; Pinot, Franck; Ohlrogge, John; Beisson, Fred

2009-12-22

Distinctive nanoridges on the surface of flowers have puzzled plant biologists ever since their discovery over 75 years ago. Although postulated to help attract insect pollinators, the function, chemical nature, and ontogeny of these surface nanostructures remain uncertain. Studies have been hampered by the fact that no ridgeless mutants have been identified. Here, we describe two mutants lacking nanoridges and define the biosynthetic pathway for 10,16-dihydroxypalmitate, a major cutin monomer in nature. Using gene expression profiling, two candidates for the formation of floral cutin were identified in the model plant Arabidopsis thaliana: the glycerol-3-phosphate acyltransferase 6 (GPAT6) and a member of a cytochrome P450 family with unknown biological function (CYP77A6). Plants carrying null mutations in either gene produced petals with no nanoridges and no cuticle could be observed by either scanning or transmission electron microscopy. A strong reduction in cutin content was found in flowers of both mutants. In planta overexpression suggested GPAT6 preferentially uses palmitate derivatives in cutin synthesis. Comparison of cutin monomer profiles in knockouts for CYP77A6 and the fatty acid omega-hydroxylase CYP86A4 provided genetic evidence that CYP77A6 is an in-chain hydroxylase acting subsequently to CYP86A4 in the synthesis of 10,16-dihydroxypalmitate. Biochemical activity of CYP77A6 was demonstrated by production of dihydroxypalmitates from 16-hydroxypalmitate, using CYP77A6-expressing yeast microsomes. These results define the biosynthetic pathway for an abundant and widespread monomer of the cutin polyester, show that the morphology of floral surfaces depends on the synthesis of cutin, and identify target genes to investigate the function of nanoridges in flower biology.

On the cellular site of two-pore channel TPC1 action in the Poaceae.

PubMed

Dadacz-Narloch, Beata; Kimura, Sachie; Kurusu, Takamitsu; Farmer, Edward E; Becker, Dirk; Kuchitsu, Kazuyuki; Hedrich, Rainer

2013-11-01

The slow vacuolar (SV) channel has been characterized in different dicots by patch-clamp recordings. This channel represents the major cation conductance of the largest organelle in most plant cells. Studies with the tpc1-2 mutant of the model dicot plant Arabidopsis thaliana identified the SV channel as the product of the TPC1 gene. By contrast, research on rice and wheat TPC1 suggested that the monocot gene encodes a plasma membrane calcium-permeable channel. To explore the site of action of grass TPC1 channels, we expressed OsTPC1 from rice (Oryza sativa) and TaTPC1 from wheat (Triticum aestivum) in the background of the Arabidopsis tpc1-2 mutant. Cross-species tpc1 complementation and patch-clamping of vacuoles using Arabidopsis and rice tpc1 null mutants documented that both monocot TPC1 genes were capable of rescuing the SV channel deficit. Vacuoles from wild-type rice but not the tpc1 loss-of-function mutant harbor SV channels exhibiting the hallmark properties of dicot TPC1/SV channels. When expressed in human embryonic kidney (HEK293) cells OsTPC1 was targeted to Lysotracker-Red-positive organelles. The finding that the rice TPC1, just like those from the model plant Arabidopsis and even animal cells, is localized and active in lyso-vacuolar membranes associates this cation channel species with endomembrane function. © 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.

Flies lacking all synapsins are unexpectedly healthy but are impaired in complex behaviour.

PubMed

Godenschwege, Tanja A; Reisch, Dietmar; Diegelmann, Sören; Eberle, Kai; Funk, Natalja; Heisenberg, Martin; Hoppe, Viviane; Hoppe, Jürgen; Klagges, Bert R E; Martin, Jean-René; Nikitina, Ekaterina A; Putz, Gabi; Reifegerste, Rita; Reisch, Natascha; Rister, Jens; Schaupp, Michael; Scholz, Henrike; Schwärzel, Martin; Werner, Ursula; Zars, Troy D; Buchner, Sigrid; Buchner, Erich

2004-08-01

Vertebrate synapsins are abundant synaptic vesicle phosphoproteins that have been proposed to fine-regulate neurotransmitter release by phosphorylation-dependent control of synaptic vesicle motility. However, the consequences of a total lack of all synapsin isoforms due to a knock-out of all three mouse synapsin genes have not yet been investigated. In Drosophila a single synapsin gene encodes several isoforms and is expressed in most synaptic terminals. Thus the targeted deletion of the synapsin gene of Drosophila eliminates the possibility of functional knock-out complementation by other isoforms. Unexpectedly, synapsin null mutant flies show no obvious defects in brain morphology, and no striking qualitative changes in behaviour are observed. Ultrastructural analysis of an identified 'model' synapse of the larval nerve muscle preparation revealed no difference between wild-type and mutant, and spontaneous or evoked excitatory junction potentials at this synapse were normal up to a stimulus frequency of 5 Hz. However, when several behavioural responses were analysed quantitatively, specific differences between mutant and wild-type flies are noted. Adult locomotor activity, optomotor responses at high pattern velocities, wing beat frequency, and visual pattern preference are modified. Synapsin mutant flies show faster habituation of an olfactory jump response, enhanced ethanol tolerance, and significant defects in learning and memory as measured using three different paradigms. Larval behavioural defects are described in a separate paper. We conclude that Drosophila synapsins play a significant role in nervous system function, which is subtle at the cellular level but manifests itself in complex behaviour.

Deconstructing mammalian reproduction: using knockouts to define fertility pathways.

PubMed

Roy, Angshumoy; Matzuk, Martin M

2006-02-01

Reproduction is the sine qua non for the propagation of species and continuation of life. It is a complex biological process that is regulated by multiple factors during the reproductive life of an organism. Over the past decade, the molecular mechanisms regulating reproduction in mammals have been rapidly unraveled by the study of a vast number of mouse gene knockouts with impaired fertility. The use of reverse genetics to generate null mutants in mice through targeted disruption of specific genes has enabled researchers to identify essential regulators of spermatogenesis and oogenesis in vivo and model human disorders affecting reproduction. This review focuses on the merits, utility, and the variations of the knockout technology in studies of reproduction in mammals.

Glucuronylated core 1 glycans are required for precise localization of neuromuscular junctions and normal formation of basement membranes on Drosophila muscles.

PubMed

Itoh, Kazuyoshi; Akimoto, Yoshihiro; Kondo, Shu; Ichimiya, Tomomi; Aoki, Kazuhiro; Tiemeyer, Michael; Nishihara, Shoko

2018-04-15

T antigen (Galβ1-3GalNAcα1-Ser/Thr) is an evolutionary-conserved mucin-type core 1 glycan structure in animals synthesized by core 1 β1,3-galactosyltransferase 1 (C1GalT1). Previous studies showed that T antigen produced by Drosophila C1GalT1 (dC1GalT1) was expressed in various tissues and dC1GalT1 loss in larvae led to various defects, including decreased number of circulating hemocytes, hyper-differentiation of hematopoietic stem cells in lymph glands, malformation of the central nervous system, mislocalization of neuromuscular junction (NMJ) boutons, and ultrastructural abnormalities in NMJs and muscle cells. Although glucuronylated T antigen (GlcAβ1-3Galβ1-3GalNAcα1-Ser/Thr) has been identified in Drosophila, the physiological function of this structure has not yet been clarified. In this study, for the first time, we unraveled biological roles of glucuronylated T antigen. Our data show that in Drosophila, glucuronylation of T antigen is predominantly carried out by Drosophila β1,3-glucuronyltransferase-P (dGlcAT-P). We created dGlcAT-P null mutants and found that mutant larvae showed lower expression of glucuronylated T antigen on the muscles and at NMJs. Furthermore, mislocalization of NMJ boutons and a partial loss of the basement membrane components collagen IV (Col IV) and nidogen (Ndg) at the muscle 6/7 boundary were observed. Those two phenotypes were correlated and identical to previously described phenotypes in dC1GalT1 mutant larvae. In addition, dGlcAT-P null mutants exhibited fewer NMJ branches on muscles 6/7. Moreover, ultrastructural analysis revealed that basement membranes that lacked Col IV and Ndg were significantly deformed. We also found that the loss of dGlcAT-P expression caused ultrastructural defects in NMJ boutons. Finally, we showed a genetic interaction between dGlcAT-P and dC1GalT1. Therefore, these results demonstrate that glucuronylated core 1 glycans synthesized by dGlcAT-P are key modulators of NMJ bouton localization, basement membrane formation, and NMJ arborization on larval muscles. Copyright © 2018 Elsevier Ltd. All rights reserved.

A Drosophila model of spinal muscular atrophy uncouples snRNP biogenesis functions of survival motor neuron from locomotion and viability defects.

PubMed

Praveen, Kavita; Wen, Ying; Matera, A Gregory

2012-06-28

The spinal muscular atrophy (SMA) protein, survival motor neuron (SMN), functions in the biogenesis of small nuclear ribonucleoproteins (snRNPs). SMN has also been implicated in tissue-specific functions; however, it remains unclear which of these is important for the etiology of SMA. Smn null mutants display larval lethality and show significant locomotion defects as well as reductions in minor-class spliceosomal snRNAs. Despite these reductions, we found no appreciable defects in the splicing of mRNAs containing minor-class introns. Transgenic expression of low levels of either wild-type or an SMA patient-derived form of SMN rescued the larval lethality and locomotor defects; however, snRNA levels were not restored. Thus, the snRNP biogenesis function of SMN is not a major contributor to the phenotype of Smn null mutants. These findings have major implications for SMA etiology because they show that SMN's role in snRNP biogenesis can be uncoupled from the organismal viability and locomotor defects. Copyright © 2012 The Authors. Published by Elsevier Inc. All rights reserved.

Eliminating anti-nutritional plant food proteins: the case of seed protease inhibitors in pea.

PubMed

Clemente, Alfonso; Arques, Maria C; Dalmais, Marion; Le Signor, Christine; Chinoy, Catherine; Olias, Raquel; Rayner, Tracey; Isaac, Peter G; Lawson, David M; Bendahmane, Abdelhafid; Domoney, Claire

2015-01-01

Several classes of seed proteins limit the utilisation of plant proteins in human and farm animal diets, while plant foods have much to offer to the sustainable intensification of food/feed production and to human health. Reduction or removal of these proteins could greatly enhance seed protein quality and various strategies have been used to try to achieve this with limited success. We investigated whether seed protease inhibitor mutations could be exploited to enhance seed quality, availing of induced mutant and natural Pisum germplasm collections to identify mutants, whilst acquiring an understanding of the impact of mutations on activity. A mutant (TILLING) resource developed in Pisum sativum L. (pea) and a large germplasm collection representing Pisum diversity were investigated as sources of mutations that reduce or abolish the activity of the major protease inhibitor (Bowman-Birk) class of seed protein. Of three missense mutations, predicted to affect activity of the mature trypsin / chymotrypsin inhibitor TI1 protein, a C77Y substitution in the mature mutant inhibitor abolished inhibitor activity, consistent with an absolute requirement for the disulphide bond C77-C92 for function in the native inhibitor. Two further classes of mutation (S85F, E109K) resulted in less dramatic changes to isoform or overall inhibitory activity. The alternative strategy to reduce anti-nutrients, by targeted screening of Pisum germplasm, successfully identified a single accession (Pisum elatius) as a double null mutant for the two closely linked genes encoding the TI1 and TI2 seed protease inhibitors. The P. elatius mutant has extremely low seed protease inhibitory activity and introgression of the mutation into cultivated germplasm has been achieved. The study provides new insights into structure-function relationships for protease inhibitors which impact on pea seed quality. The induced and natural germplasm variants identified provide immediate potential for either halving or abolishing the corresponding inhibitory activity, along with associated molecular markers for breeding programmes. The potential for making large changes to plant protein profiles for improved and sustainable food production through diversity is illustrated. The strategy employed here to reduce anti-nutritional proteins in seeds may be extended to allergens and other seed proteins with negative nutritional effects. Additionally, the novel variants described for pea will assist future studies of the biological role and health-related properties of so-called anti-nutrients.

Glycosylation of Cblns attenuates their receptor binding.

PubMed

Rong, Yongqi; Bansal, Parmil K; Wei, Peng; Guo, Hong; Correia, Kristen; Parris, Jennifer; Morgan, James I

2018-05-18

Cbln1 is the prototype of a family (Cbln1-Cbln4) of secreted glycoproteins and is essential for normal synapse structure and function in cerebellum by bridging presynaptic Nrxn to postsynaptic Grid2. Here we report the effects of glycosylation on the in vitro receptor binding properties of Cblns. Cbln1, 2 and 4 harbor two N-linked glycosylation sites, one at the N-terminus is in a region implicated in Nrxn binding and the second is in the C1q domain, a region involved in Grid2 binding. Mutation (asparagine to glutamine) of the N-terminal site, increased neurexin binding whereas mutation of the C1q site markedly increased Grid2 binding. These mutations did not influence subunit composition of Cbln trimeric complexes (mediated through the C1q domain) nor their assembly into hexamers (mediated by the N-terminal region). Therefore, glycosylation likely masks the receptor binding interfaces of Cblns. As Cbln4 has undetectable Grid2 binding in vitro we assessed whether transgenic expression of wild type Cbln4 or its glycosylation mutants rescued the Cbln1-null phenotype in vivo. Cbln4 partially rescued and both glycosylation mutants completely rescued ataxia in cbln1-null mice. Thus Cbln4 has intrinsic Grid2 binding that is attenuated by glycosylation, and glycosylation mutants exhibit gain of function in vivo. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Proliferation of smooth muscle cells stimulated by Porphyromonas gingivalis is inhibited by apple polyphenol.

PubMed

Inaba, Hiroaki; Tagashira, Motoyuki; Kanda, Tomomasa; Amano, Atsuo

2011-11-01

Porphyromonas gingivalis (Pg) is thought to be involved in the progression of occlusive arterial lesions, whereas vascular smooth muscle cell (SMC) proliferation is considered to be involved in occlusive arterial disease. We previously showed that bacteremia caused by Pg infection induced proliferation of mouse aortic SMCs. Furthermore, human SMCs stimulated with human plasma incubated with Pg showed a marked transformation from the contractile to proliferative phenotype. In the present study, we examine the involvement of Pg gingipains and fimbriae in induction of the SMC transformation and proliferation, and effective inhibitors. Pg strains including gingipain- and fimbria-null mutants were incubated in human plasma, after which the bacteria were removed and the supernatants were added to cultured SMCs. To evaluate the effects of inhibitors, Pg organisms were incubated in plasma in the presence of apple polyphenol (AP), epigallocatechin gallate, KYT-1 (Arg-gingipain inhibitor), and KYT-36 (Lys-gingipain inhibitor). Plasma supernatants from wild-type and fimbria-mutant cultures markedly stimulated cellular proliferation, whereas those containing gingipain-null mutants showed negligible effects. SMC proliferation was also induced by plasma treated with trypsin. Furthermore, plasma supernatants cultured in the presence of KYT-1/KYT-36 and AP showed significant inhibitory effects on SMC proliferation, whereas cultures with epigallocatechin gallate did not. Our results suggest that Pg gingipains are involved in the induction of SMC transformation and proliferation, whereas this was inhibited by AP.

Comparative Proteomic Analysis of Normal and Collagen IX Null Mouse Cartilage Reveals Altered Extracellular Matrix Composition and Novel Components of the Collagen IX Interactome*

PubMed Central

Brachvogel, Bent; Zaucke, Frank; Dave, Keyur; Norris, Emma L.; Stermann, Jacek; Dayakli, Münire; Koch, Manuel; Gorman, Jeffrey J.; Bateman, John F.; Wilson, Richard

2013-01-01

The cartilage extracellular matrix is essential for endochondral bone development and joint function. In addition to the major aggrecan/collagen II framework, the interacting complex of collagen IX, matrilin-3, and cartilage oligomeric matrix protein (COMP) is essential for cartilage matrix stability, as mutations in Col9a1, Col9a2, Col9a3, Comp, and Matn3 genes cause multiple epiphyseal dysplasia, in which patients develop early onset osteoarthritis. In mice, collagen IX ablation results in severely disturbed growth plate organization, hypocellular regions, and abnormal chondrocyte shape. This abnormal differentiation is likely to involve altered cell-matrix interactions but the mechanism is not known. To investigate the molecular basis of the collagen IX null phenotype we analyzed global differences in protein abundance between wild-type and knock-out femoral head cartilage by capillary HPLC tandem mass spectrometry. We identified 297 proteins in 3-day cartilage and 397 proteins in 21-day cartilage. Components that were differentially abundant between wild-type and collagen IX-deficient cartilage included 15 extracellular matrix proteins. Collagen IX ablation was associated with dramatically reduced COMP and matrilin-3, consistent with known interactions. Matrilin-1, matrilin-4, epiphycan, and thrombospondin-4 levels were reduced in collagen IX null cartilage, providing the first in vivo evidence for these proteins belonging to the collagen IX interactome. Thrombospondin-4 expression was reduced at the mRNA level, whereas matrilin-4 was verified as a novel collagen IX-binding protein. Furthermore, changes in TGFβ-induced protein βig-h3 and fibronectin abundance were found in the collagen IX knock-out but not associated with COMP ablation, indicating specific involvement in the abnormal collagen IX null cartilage. In addition, the more widespread expression of collagen XII in the collagen IX-deficient cartilage suggests an attempted compensatory response to the absence of collagen IX. Our differential proteomic analysis of cartilage is a novel approach to identify candidate matrix protein interactions in vivo, underpinning further analysis of mutant cartilage lacking other matrix components or harboring disease-causing mutations. PMID:23530037

CLP1 founder mutation links tRNA splicing and maturation to cerebellar development and neurodegeneration.

PubMed

Schaffer, Ashleigh E; Eggens, Veerle R C; Caglayan, Ahmet Okay; Reuter, Miriam S; Scott, Eric; Coufal, Nicole G; Silhavy, Jennifer L; Xue, Yuanchao; Kayserili, Hulya; Yasuno, Katsuhito; Rosti, Rasim Ozgur; Abdellateef, Mostafa; Caglar, Caner; Kasher, Paul R; Cazemier, J Leonie; Weterman, Marian A; Cantagrel, Vincent; Cai, Na; Zweier, Christiane; Altunoglu, Umut; Satkin, N Bilge; Aktar, Fesih; Tuysuz, Beyhan; Yalcinkaya, Cengiz; Caksen, Huseyin; Bilguvar, Kaya; Fu, Xiang-Dong; Trotta, Christopher R; Gabriel, Stacey; Reis, André; Gunel, Murat; Baas, Frank; Gleeson, Joseph G

2014-04-24

Neurodegenerative diseases can occur so early as to affect neurodevelopment. From a cohort of more than 2,000 consanguineous families with childhood neurological disease, we identified a founder mutation in four independent pedigrees in cleavage and polyadenylation factor I subunit 1 (CLP1). CLP1 is a multifunctional kinase implicated in tRNA, mRNA, and siRNA maturation. Kinase activity of the CLP1 mutant protein was defective, and the tRNA endonuclease complex (TSEN) was destabilized, resulting in impaired pre-tRNA cleavage. Germline clp1 null zebrafish showed cerebellar neurodegeneration that was rescued by wild-type, but not mutant, human CLP1 expression. Patient-derived induced neurons displayed both depletion of mature tRNAs and accumulation of unspliced pre-tRNAs. Transfection of partially processed tRNA fragments into patient cells exacerbated an oxidative stress-induced reduction in cell survival. Our data link tRNA maturation to neuronal development and neurodegeneration through defective CLP1 function in humans. Copyright © 2014 Elsevier Inc. All rights reserved.

Myocardial-specific R-spondin3 drives proliferation of the coronary stems primarily through the Leucine Rich Repeat G Protein coupled receptor LGR4.

PubMed

Da Silva, Fabio; Massa, Filippo; Motamedi, Fariba Jian; Vidal, Valerie; Rocha, Ana Sofia; Gregoire, Elodie P; Cai, Chen-Leng; Wagner, Kay Dietrich; Schedl, Andreas

2018-05-31

Coronary artery anomalies are common congenital disorders with serious consequences in adult life. Coronary circulation begins when the coronary stems form connections between the aorta and the developing vascular plexus. We recently identified the WNT signaling modulator R-spondin 3 (Rspo3), as a crucial regulator of coronary stem proliferation. Using expression analysis and tissue-specific deletion we now demonstrate that Rspo3 is primarily produced by cardiomyocytes. Moreover, we have employed CRISPR/Cas9 technology to generate novel Lgr4-null alleles that showed a significant decrease in coronary stem proliferation and thus phenocopied the coronary artery defects seen in Rspo3 mutants. Interestingly, Lgr4 mutants displayed slightly hypomorphic right ventricles, an observation also made after myocardial specific deletion of Rspo3. These results shed new light on the role of Rspo3 in heart development and demonstrate that LGR4 is the principal R-spondin 3 receptor in the heart. Copyright © 2018 Elsevier Inc. All rights reserved.

CRISPR/Cas9-mediated targeted mutagenesis of GmFT2a delays flowering time in soya bean.

PubMed

Cai, Yupeng; Chen, Li; Liu, Xiujie; Guo, Chen; Sun, Shi; Wu, Cunxiang; Jiang, Bingjun; Han, Tianfu; Hou, Wensheng

2018-01-01

Flowering is an indication of the transition from vegetative growth to reproductive growth and has considerable effects on the life cycle of soya bean (Glycine max). In this study, we employed the CRISPR/Cas9 system to specifically induce targeted mutagenesis of GmFT2a, an integrator in the photoperiod flowering pathway in soya bean. The soya bean cultivar Jack was transformed with three sgRNA/Cas9 vectors targeting different sites of endogenous GmFT2a via Agrobacterium tumefaciens-mediated transformation. Site-directed mutations were observed at all targeted sites by DNA sequencing analysis. T1-generation soya bean plants homozygous for null alleles of GmFT2a frameshift mutated by a 1-bp insertion or short deletion exhibited late flowering under natural conditions (summer) in Beijing, China (N39°58', E116°20'). We also found that the targeted mutagenesis was stably heritable in the following T2 generation, and the homozygous GmFT2a mutants exhibited late flowering under both long-day and short-day conditions. We identified some 'transgene-clean' soya bean plants that were homozygous for null alleles of endogenous GmFT2a and without any transgenic element from the T1 and T2 generations. These 'transgene-clean' mutants of GmFT2a may provide materials for more in-depth research of GmFT2a functions and the molecular mechanism of photoperiod responses in soya bean. They will also contribute to soya bean breeding and regional introduction. © 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Dmc1 Functions in a Saccharomyces Cerevisiae Meiotic Pathway That Is Largely Independent of the Rad51 Pathway

PubMed Central

Dresser, M. E.; Ewing, D. J.; Conrad, M. N.; Dominguez, A. M.; Barstead, R.; Jiang, H.; Kodadek, T.

1997-01-01

Meiotic recombination in the yeast Saccharomyces cerevisiae requires two similar recA-like proteins, Dmc1p and Rad51p. A screen for dominant meiotic mutants provided DMC1-G126D, a dominant allele mutated in the conserved ATP-binding site (specifically, the A-loop motif) that confers a null phenotype. A recessive null allele, dmc1-K69E, was isolated as an intragenic suppressor of DMC1-G126D. Dmc1-K69Ep, unlike Dmc1p, does not interact homotypically in a two-hybrid assay, although it does interact with other fusion proteins identified by two-hybrid screen with Dmc1p. Dmc1p, unlike Rad51p, does not interact in the two-hybrid assay with Rad52p or Rad54p. However, Dmc1p does interact with Tid1p, a Rad54p homologue, with Tid4p, a Rad16p homologue, and with other fusion proteins that do not interact with Rad51p, suggesting that Dmc1p and Rad51p function in separate, though possibly overlapping, recombinational repair complexes. Epistasis analysis suggests that DMC1 and RAD51 function in separate pathways responsible for meiotic recombination. Taken together, our results are consistent with a requirement for DMC1 for meiosis-specific entry of DNA double-strand break ends into chromatin. Interestingly, the pattern on CHEF gels of chromosome fragments that result from meiotic DNA double-strand break formation is different in DMC1 mutant strains from that seen in rad50S strains. PMID:9335591

PTBP1 Is Required for Embryonic Development before Gastrulation

PubMed Central

Suckale, Jakob; Wendling, Olivia; Masjkur, Jimmy; Jäger, Melanie; Münster, Carla; Anastassiadis, Konstantinos; Stewart, A. Francis; Solimena, Michele

2011-01-01

Polypyrimidine-tract binding protein 1 (PTBP1) is an important cellular regulator of messenger RNAs influencing the alternative splicing profile of a cell as well as its mRNA stability, location and translation. In addition, it is diverted by some viruses to facilitate their replication. Here, we used a novel PTBP1 knockout mouse to analyse the tissue expression pattern of PTBP1 as well as the effect of its complete removal during development. We found evidence of strong PTBP1 expression in embryonic stem cells and throughout embryonic development, especially in the developing brain and spinal cord, the olfactory and auditory systems, the heart, the liver, the kidney, the brown fat and cartilage primordia. This widespread distribution points towards a role of PTBP1 during embryonic development. Homozygous offspring, identified by PCR and immunofluorescence, were able to implant but were arrested or retarded in growth. At day 7.5 of embryonic development (E7.5) the null mutants were about 5x smaller than the control littermates and the gap in body size widened with time. At mid-gestation, all homozygous embryos were resorbed/degraded. No homozygous mice were genotyped at E12 and the age of weaning. Embryos lacking PTBP1 did not display differentiation into the 3 germ layers and cavitation of the epiblast, which are hallmarks of gastrulation. In addition, homozygous mutants displayed malformed ectoplacental cones and yolk sacs, both early supportive structure of the embryo proper. We conclude that PTBP1 is not required for the earliest isovolumetric divisions and differentiation steps of the zygote up to the formation of the blastocyst. However, further post-implantation development requires PTBP1 and stalls in homozygous null animals with a phenotype of dramatically reduced size and aberration in embryonic and extra-embryonic structures. PMID:21423341

PTBP1 is required for embryonic development before gastrulation.

PubMed

Suckale, Jakob; Wendling, Olivia; Masjkur, Jimmy; Jäger, Melanie; Münster, Carla; Anastassiadis, Konstantinos; Stewart, A Francis; Solimena, Michele

2011-02-17

Polypyrimidine-tract binding protein 1 (PTBP1) is an important cellular regulator of messenger RNAs influencing the alternative splicing profile of a cell as well as its mRNA stability, location and translation. In addition, it is diverted by some viruses to facilitate their replication. Here, we used a novel PTBP1 knockout mouse to analyse the tissue expression pattern of PTBP1 as well as the effect of its complete removal during development. We found evidence of strong PTBP1 expression in embryonic stem cells and throughout embryonic development, especially in the developing brain and spinal cord, the olfactory and auditory systems, the heart, the liver, the kidney, the brown fat and cartilage primordia. This widespread distribution points towards a role of PTBP1 during embryonic development. Homozygous offspring, identified by PCR and immunofluorescence, were able to implant but were arrested or retarded in growth. At day 7.5 of embryonic development (E7.5) the null mutants were about 5x smaller than the control littermates and the gap in body size widened with time. At mid-gestation, all homozygous embryos were resorbed/degraded. No homozygous mice were genotyped at E12 and the age of weaning. Embryos lacking PTBP1 did not display differentiation into the 3 germ layers and cavitation of the epiblast, which are hallmarks of gastrulation. In addition, homozygous mutants displayed malformed ectoplacental cones and yolk sacs, both early supportive structure of the embryo proper. We conclude that PTBP1 is not required for the earliest isovolumetric divisions and differentiation steps of the zygote up to the formation of the blastocyst. However, further post-implantation development requires PTBP1 and stalls in homozygous null animals with a phenotype of dramatically reduced size and aberration in embryonic and extra-embryonic structures.

Candida albicans Swi/Snf and Mediator Complexes Differentially Regulate Mrr1-Induced MDR1 Expression and Fluconazole Resistance.

PubMed

Liu, Zhongle; Myers, Lawrence C

2017-11-01

Long-term azole treatment of patients with chronic Candida albicans infections can lead to drug resistance. Gain-of-function (GOF) mutations in the transcription factor Mrr1 and the consequent transcriptional activation of MDR1 , a drug efflux coding gene, is a common pathway by which this human fungal pathogen acquires fluconazole resistance. This work elucidates the previously unknown downstream transcription mechanisms utilized by hyperactive Mrr1. We identified the Swi/Snf chromatin remodeling complex as a key coactivator for Mrr1, which is required to maintain basal and induced open chromatin, and Mrr1 occupancy, at the MDR1 promoter. Deletion of snf2 , the catalytic subunit of Swi/Snf, largely abrogates the increases in MDR1 expression and fluconazole MIC observed in MRR1 GOF mutant strains. Mediator positively and negatively regulates key Mrr1 target promoters. Deletion of the Mediator tail module med3 subunit reduces, but does not eliminate, the increased MDR1 expression and fluconazole MIC conferred by MRR1 GOF mutations. Eliminating the kinase activity of the Mediator Ssn3 subunit suppresses the decreased MDR1 expression and fluconazole MIC of the snf2 null mutation in MRR1 GOF strains. Ssn3 deletion also suppresses MDR1 promoter histone displacement defects in snf2 null mutants. The combination of this work with studies on other hyperactive zinc cluster transcription factors that confer azole resistance in fungal pathogens reveals a complex picture where the induction of drug efflux pump expression requires the coordination of multiple coactivators. The observed variations in transcription factor and target promoter dependence of this process may make the search for azole sensitivity-restoring small molecules more complicated. Copyright © 2017 American Society for Microbiology.

PspG, a New Member of the Yersinia enterocolitica Phage Shock Protein Regulon

PubMed Central

Green, Rebecca C.; Darwin, Andrew J.

2004-01-01

The Yersinia enterocolitica phage shock protein (Psp) system is induced when the Ysc type III secretion system is produced or when only the YscC secretin component is synthesized. Some psp null mutants have a growth defect when YscC is produced and a severe virulence defect in animals. The Y. enterocolitica psp locus is made up of two divergently transcribed cistrons, pspF and pspABCDycjXF. pspA operon expression is dependent on RpoN (σ54) and the enhancer-binding protein PspF. Previous data indicated that PspF also controls at least one gene that is not part of the psp locus. In this study we describe the identification of pspG, a new member of the PspF regulon. Predicted RpoN-binding sites upstream of the pspA genes from different bacteria have a common divergence from the consensus sequence, which may be a signature of PspF-dependent promoters. The Y. enterocolitica pspG gene was identified because its promoter also has this signature. Like the pspA operon, pspG is positively regulated by PspF, negatively regulated by PspA, and induced in response to the production of secretins. Purified His6-PspF protein specifically interacts with the pspA and pspG control regions. A pspA operon deletion mutant has a growth defect when the YscC secretin is produced and a virulence defect in a mouse model of infection. These phenotypes were exacerbated by a pspG null mutation. Therefore, PspG is the missing component of the Y. enterocolitica Psp regulon that was previously predicted to exist. PMID:15262928

Horizontal acquisition of a hypoxia-responsive molybdenum cofactor biosynthesis pathway contributed to Mycobacterium tuberculosis pathoadaptation.

PubMed

Levillain, Florence; Poquet, Yannick; Mallet, Ludovic; Mazères, Serge; Marceau, Michael; Brosch, Roland; Bange, Franz-Christoph; Supply, Philip; Magalon, Axel; Neyrolles, Olivier

2017-11-01

The unique ability of the tuberculosis (TB) bacillus, Mycobacterium tuberculosis, to persist for long periods of time in lung hypoxic lesions chiefly contributes to the global burden of latent TB. We and others previously reported that the M. tuberculosis ancestor underwent massive episodes of horizontal gene transfer (HGT), mostly from environmental species. Here, we sought to explore whether such ancient HGT played a part in M. tuberculosis evolution towards pathogenicity. We were interested by a HGT-acquired M. tuberculosis-specific gene set, namely moaA1-D1, which is involved in the biosynthesis of the molybdenum cofactor. Horizontal acquisition of this gene set was striking because homologues of these moa genes are present all across the Mycobacterium genus, including in M. tuberculosis. Here, we discovered that, unlike their paralogues, the moaA1-D1 genes are strongly induced under hypoxia. In vitro, a M. tuberculosis moaA1-D1-null mutant has an impaired ability to respire nitrate, to enter dormancy and to survive in oxygen-limiting conditions. Conversely, heterologous expression of moaA1-D1 in the phylogenetically closest non-TB mycobacterium, Mycobacterium kansasii, which lacks these genes, improves its capacity to respire nitrate and grants it with a marked ability to survive oxygen depletion. In vivo, the M. tuberculosis moaA1-D1-null mutant shows impaired survival in hypoxic granulomas in C3HeB/FeJ mice, but not in normoxic lesions in C57BL/6 animals. Collectively, our results identify a novel pathway required for M. tuberculosis resistance to host-imposed stress, namely hypoxia, and provide evidence that ancient HGT bolstered M. tuberculosis evolution from an environmental species towards a pervasive human-adapted pathogen.

Icm/Dot-Independent Entry of Legionella pneumophila into Amoeba and Macrophage Hosts

PubMed Central

Bandyopadhyay, Purnima; Xiao, Huifang; Coleman, Hope A.; Price-Whelan, Alexa; Steinman, Howard M.

2004-01-01

Legionella pneumophila, the causative agent of Legionnaires' disease, expresses a type IVB secretion apparatus that translocates bacterial proteins into amoeba and macrophage hosts. When stationary-phase cultures are used to infect hosts, the type IVB apparatus encoded by the icm/dot genes is required for entry, delay of phagosome-lysosome fusion, and intracellular multiplication within host cells. Null mutants with mutations in icm/dot genes are defective in these phenotypes. Here a new model is described in which hosts are infected with stationary-phase cultures that have been incubated overnight in pH 6.5 buffer. This model is called Ers treatment because it enhances the resistance to acid, hydrogen peroxide, and antibiotic stress beyond that of stationary-phase cultures. Following Ers treatment entry into amoeba and macrophage hosts does not require dotA, which is essential for Legionella virulence phenotypes when hosts are infected with stationary-phase cultures, dotB, icmF, icmV, or icmX. Defective host entry is also suppressed for null mutants with mutations in the KatA and KatB catalase-peroxidase enzymes, which are required for proper intracellular growth in amoeba and macrophage hosts. Ers treatment-induced suppression of defective entry is not associated with increased bacterial adhesion to host cells or with morphological changes in the bacterial envelope but is dependent on protein expression during Ers treatment. By using proteomic analysis, Ers treatment was shown to induce a protein predicted to contain eight tetratricopeptide repeats, a motif previously implicated in enhanced entry of L. pneumophila. Characterization of Ers treatment-dependent changes in expression is proposed as an avenue for identifying icm/dot-independent factors that function in the entry of Legionella into amoeba and macrophage hosts. PMID:15271914

Drosophila Fatty Acid Transport Protein Regulates Rhodopsin-1 Metabolism and Is Required for Photoreceptor Neuron Survival

PubMed Central

Dourlen, Pierre; Bertin, Benjamin; Chatelain, Gilles; Robin, Marion; Napoletano, Francesco; Roux, Michel J.; Mollereau, Bertrand

2012-01-01

Tight regulation of the visual response is essential for photoreceptor function and survival. Visual response dysregulation often leads to photoreceptor cell degeneration, but the causes of such cell death are not well understood. In this study, we investigated a fatty acid transport protein (fatp) null mutation that caused adult-onset and progressive photoreceptor cell death. Consistent with fatp having a role in the retina, we showed that fatp is expressed in adult photoreceptors and accessory cells and that its re-expression in photoreceptors rescued photoreceptor viability in fatp mutants. The visual response in young fatp-mutant flies was abnormal with elevated electroretinogram amplitudes associated with high levels of Rhodopsin-1 (Rh1). Reducing Rh1 levels in rh1 mutants or depriving flies of vitamin A rescued photoreceptor cell death in fatp mutant flies. Our results indicate that fatp promotes photoreceptor survival by regulating Rh1 abundance. PMID:22844251

IMP2, a nuclear gene controlling the mitochondrial dependence of galactose, maltose and raffinose utilization in Saccharomyces cerevisiae.

PubMed

Donnini, C; Lodi, T; Ferrero, I; Puglisi, P P

1992-02-01

The IMP2 gene of Saccharomyces cerevisiae is involved in the nucleo-mitochondrial control of maltose, galactose and raffinose utilization as shown by the inability of imp2 mutants to grow on these carbon sources in respiratory-deficient conditions or in the presence of ethidium bromide and erythromycin. The negative phenotype cannot be scored in the presence of inhibitors of respiration and oxidative phosphorylation, indicating that the role of the mitochondria in the utilization of the above-mentioned carbon sources in imp2 mutants is not at the energetical level. Mutations in the IMP2 gene also confer many phenotypic alterations in respiratory-sufficient conditions, e.g. leaky phenotype on oxidizable carbon sources, sensitivity to heat shock and sporulation deficiency. The IMP2 gene has been cloned, sequenced and disrupted. The phenotype of null imp2 mutants is indistinguishable from that of the originally isolated mutant.

The putative RNA helicase Dbp6p functionally interacts with Rpl3p, Nop8p and the novel trans-acting Factor Rsa3p during biogenesis of 60S ribosomal subunits in Saccharomyces cerevisiae.

PubMed Central

de la Cruz, Jesús; Lacombe, Thierry; Deloche, Olivier; Linder, Patrick; Kressler, Dieter

2004-01-01

Ribosome biogenesis requires at least 18 putative ATP-dependent RNA helicases in Saccharomyces cerevisiae. To explore the functional environment of one of these putative RNA helicases, Dbp6p, we have performed a synthetic lethal screen with dbp6 alleles. We have previously characterized the nonessential Rsa1p, whose null allele is synthetically lethal with dbp6 alleles. Here, we report on the characterization of the four remaining synthetic lethal mutants, which reveals that Dbp6p also functionally interacts with Rpl3p, Nop8p, and the so-far-uncharacterized Rsa3p (ribosome assembly 3). The nonessential Rsa3p is a predominantly nucleolar protein required for optimal biogenesis of 60S ribosomal subunits. Both Dbp6p and Rsa3p are associated with complexes that most likely correspond to early pre-60S ribosomal particles. Moreover, Rsa3p is co-immunoprecipitated with protA-tagged Dbp6p under low salt conditions. In addition, we have established a synthetic interaction network among factors involved in different aspects of 60S-ribosomal-subunit biogenesis. This extensive genetic analysis reveals that the rsa3 null mutant displays some specificity by being synthetically lethal with dbp6 alleles and by showing some synthetic enhancement with the nop8-101 and the rsa1 null allele. PMID:15126390

Mesenchyme-specific knockout of ESET histone methyltransferase causes ectopic hypertrophy and terminal differentiation of articular chondrocytes.

PubMed

Lawson, Kevin A; Teteak, Colin J; Zou, Junhui; Hacquebord, Jacques; Ghatan, Andrew; Zielinska-Kwiatkowska, Anna; Fernandes, Russell J; Chansky, Howard A; Yang, Liu

2013-11-08

The exact molecular mechanisms governing articular chondrocytes remain unknown in skeletal biology. In this study, we have found that ESET (an ERG-associated protein with a SET domain, also called SETDB1) histone methyltransferase is expressed in articular cartilage. To test whether ESET regulates articular chondrocytes, we carried out mesenchyme-specific deletion of the ESET gene in mice. ESET knock-out did not affect generation of articular chondrocytes during embryonic development. Two weeks after birth, there was minimal qualitative difference at the knee joints between wild-type and ESET knock-out animals. At 1 month, ectopic hypertrophy, proliferation, and apoptosis of articular chondrocytes were seen in the articular cartilage of ESET-null animals. At 3 months, additional signs of terminal differentiation such as increased alkaline phosphatase activity and an elevated level of matrix metalloproteinase (MMP)-13 were found in ESET-null cartilage. Staining for type II collagen and proteoglycan revealed that cartilage degeneration became progressively worse from 2 weeks to 12 months at the knee joints of ESET knock-out mutants. Analysis of over 14 pairs of age- and sex-matched wild-type and knock-out mice indicated that the articular chondrocyte phenotype in ESET-null mutants is 100% penetrant. Our results demonstrate that expression of ESET plays an essential role in the maintenance of articular cartilage by preventing articular chondrocytes from terminal differentiation and may have implications in joint diseases such as osteoarthritis.

Fe65 is required for Tip60-directed histone H4 acetylation at DNA strand breaks

PubMed Central

Stante, Maria; Minopoli, Giuseppina; Passaro, Fabiana; Raia, Maddalena; Vecchio, Luigi Del; Russo, Tommaso

2009-01-01

Fe65 is a binding partner of the Alzheimer's β-amyloid precursor protein APP. The possible involvement of this protein in the cellular response to DNA damage was suggested by the observation that Fe65 null mice are more sensitive to genotoxic stress than WT counterpart. Fe65 associated with chromatin under basal conditions and its involvement in DNA damage repair requires this association. A known partner of Fe65 is the histone acetyltransferase Tip60. Considering the crucial role of Tip60 in DNA repair, we explored the hypothesis that the phenotype of Fe65 null cells depended on its interaction with Tip60. We demonstrated that Fe65 knockdown impaired recruitment of Tip60-TRRAP complex to DNA double strand breaks and decreased histone H4 acetylation. Accordingly, the efficiency of DNA repair was decreased upon Fe65 suppression. To explore whether APP has a role in this mechanism, we analyzed a Fe65 mutant unable to bind to APP. This mutant failed to rescue the phenotypes of Fe65 null cells; furthermore, APP/APLP2 suppression results in the impairment of recruitment of Tip60-TRRAP complex to DNA double strand breaks, decreased histone H4 acetylation and repair efficiency. On these bases, we propose that Fe65 and its interaction with APP play an important role in the response to DNA damage by assisting the recruitment of Tip60-TRRAP to DNA damage sites. PMID:19282473

Peroxiredoxin 5 confers protection against oxidative stress and apoptosis and also promotes longevity in Drosophila

PubMed Central

Radyuk, Svetlana N.; Michalak, Katarzyna; Klichko, Vladimir I.; Benes, Judith; Rebrin, Igor; Sohal, Rajindar S.; Orr, William C.

2010-01-01

Peroxiredoxin 5 is a distinct isoform of the peroxiredoxin gene family. The antioxidative and anti-apoptotic functions of peroxiredoxin 5 have been extensively demonstrated in cell culture experiments. In the present paper, we provide the first functional analysis of peroxiredoxin 5 in a multicellular organism, Drosophila melanogaster. Similar to its mammalian, yeast or human counterparts, dPrx5 (Drosophila peroxiredoxin 5) is expressed in several cellular compartments, including the cytosol, nucleus and the mitochondrion. Global overexpression of dPrx5 in flies increased resistance to oxidative stress and extended their life span by up to 30% under normal conditions. The dprx5−/− null flies were comparatively more susceptible to oxidative stress, had higher incidence of apoptosis, and a shortened life span. TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling) analysis revealed that the dprx5−/− null mutant had discernible tissue-specific apoptotic patterns, similar to those observed in control flies exposed to paraquat. In addition, apoptosis was particularly notable in oenocytes. During development the dPrx5 levels co-varied with ecdysone pulses, suggesting inter-relationship between ecdystreroids and dPrx5 expression. The importance of dPrx5 for development was further underscored by the embryonic lethal phenotype of progeny derived from the dprx5−/− null mutant. Results from the present study suggest that the antioxidant and anti-apoptotic activities of dPrx5 play a critical role in development and aging of the fly. PMID:19128239

Otoconin-90 deletion leads to imbalance but normal hearing: a comparison with other otoconia mutants.

PubMed

Zhao, X; Jones, S M; Yamoah, E N; Lundberg, Y Wang

2008-04-22

Our sense of gravitation and linear acceleration is mediated by stimulation of vestibular hair cells through displacement of otoconia in the utricle and saccule (the gravity receptor organ). We recently showed that otoconin-90 (Oc90) deletion led to formation of giant otoconia. In the present study, we determined the extent to which the giant otoconia affected balance and gravity receptor sensory input and compared the findings with other otoconia mutants. We employed a wide spectrum of balance behavioral tests, including reaching and air-righting reflexes, gait, swimming, beam-crossing, rotorod latencies, and a direct measure of gravity receptor input, vestibular evoked potentials (VsEPs). All tests on homozygous adult mutants consistently ranked the order of imbalance as (from worst to best) Nox3(het)

The replication defect of ICP0-null mutant herpes simplex virus 1 can be largely complemented by the combined activities of human cytomegalovirus proteins IE1 and pp71.

PubMed

Everett, Roger D; Bell, Adam J; Lu, Yongxu; Orr, Anne

2013-01-01

Herpes simplex virus 1 (HSV-1) immediate-early protein ICP0 is required for efficient lytic infection and productive reactivation from latency and induces derepression of quiescent viral genomes. Despite being unrelated at the sequence level, ICP0 and human cytomegalovirus proteins IE1 and pp71 share some functional similarities in their abilities to counteract antiviral restriction mediated by components of cellular nuclear structures known as ND10. To investigate the extent to which IE1 and pp71 might substitute for ICP0, cell lines were developed that express either IE1 or pp71, or both together, in an inducible manner. We found that pp71 dissociated the hDaxx-ATRX complex and inhibited accumulation of these proteins at sites juxtaposed to HSV-1 genomes but had no effect on the promyelocytic leukemia protein (PML) or Sp100. IE1 caused loss of the small ubiquitin-like modifier (SUMO)-conjugated forms of PML and Sp100 and inhibited the recruitment of these proteins to HSV-1 genome foci but had little effect on hDaxx or ATRX in these assays. Both IE1 and pp71 stimulated ICP0-null mutant plaque formation, but neither to the extent achieved by ICP0. The combination of IE1 and pp71, however, inhibited recruitment of all ND10 proteins to viral genome foci, stimulated ICP0-null mutant HSV-1 plaque formation to near wild-type levels, and efficiently induced derepression of quiescent HSV-1 genomes. These results suggest that ND10-related intrinsic resistance results from the additive effects of several ND10 components and that the effects of IE1 and pp71 on subsets of these components combine to mirror the overall activities of ICP0.

The Replication Defect of ICP0-Null Mutant Herpes Simplex Virus 1 Can Be Largely Complemented by the Combined Activities of Human Cytomegalovirus Proteins IE1 and pp71

PubMed Central

Bell, Adam J.; Lu, Yongxu; Orr, Anne

2013-01-01

Herpes simplex virus 1 (HSV-1) immediate-early protein ICP0 is required for efficient lytic infection and productive reactivation from latency and induces derepression of quiescent viral genomes. Despite being unrelated at the sequence level, ICP0 and human cytomegalovirus proteins IE1 and pp71 share some functional similarities in their abilities to counteract antiviral restriction mediated by components of cellular nuclear structures known as ND10. To investigate the extent to which IE1 and pp71 might substitute for ICP0, cell lines were developed that express either IE1 or pp71, or both together, in an inducible manner. We found that pp71 dissociated the hDaxx-ATRX complex and inhibited accumulation of these proteins at sites juxtaposed to HSV-1 genomes but had no effect on the promyelocytic leukemia protein (PML) or Sp100. IE1 caused loss of the small ubiquitin-like modifier (SUMO)-conjugated forms of PML and Sp100 and inhibited the recruitment of these proteins to HSV-1 genome foci but had little effect on hDaxx or ATRX in these assays. Both IE1 and pp71 stimulated ICP0-null mutant plaque formation, but neither to the extent achieved by ICP0. The combination of IE1 and pp71, however, inhibited recruitment of all ND10 proteins to viral genome foci, stimulated ICP0-null mutant HSV-1 plaque formation to near wild-type levels, and efficiently induced derepression of quiescent HSV-1 genomes. These results suggest that ND10-related intrinsic resistance results from the additive effects of several ND10 components and that the effects of IE1 and pp71 on subsets of these components combine to mirror the overall activities of ICP0. PMID:23135716

The Identification of Zebrafish Mutants Showing Alterations in Senescence-Associated Biomarkers

PubMed Central

Uchiyama, Junzo; Koshimizu, Eriko; Qi, Jie; Nanjappa, Purushothama; Imamura, Shintaro; Islam, Asiful; Neuberg, Donna; Amsterdam, Adam; Roberts, Thomas M.

2008-01-01

There is an interesting overlap of function in a wide range of organisms between genes that modulate the stress responses and those that regulate aging phenotypes and, in some cases, lifespan. We have therefore screened mutagenized zebrafish embryos for the altered expression of a stress biomarker, senescence-associated β-galactosidase (SA-β-gal) in our current study. We validated the use of embryonic SA-β-gal production as a screening tool by analyzing a collection of retrovirus-insertional mutants. From a pool of 306 such mutants, we identified 11 candidates that showed higher embryonic SA-β-gal activity, two of which were selected for further study. One of these mutants is null for a homologue of Drosophila spinster, a gene known to regulate lifespan in flies, whereas the other harbors a mutation in a homologue of the human telomeric repeat binding factor 2 (terf2) gene, which plays roles in telomere protection and telomere-length regulation. Although the homozygous spinster and terf2 mutants are embryonic lethal, heterozygous adult fish are viable and show an accelerated appearance of aging symptoms including lipofuscin accumulation, which is another biomarker, and shorter lifespan. We next used the same SA-β-gal assay to screen chemically mutagenized zebrafish, each of which was heterozygous for lesions in multiple genes, under the sensitizing conditions of oxidative stress. We obtained eight additional mutants from this screen that, when bred to homozygosity, showed enhanced SA-β-gal activity even in the absence of stress, and further displayed embryonic neural and muscular degenerative phenotypes. Adult fish that are heterozygous for these mutations also showed the premature expression of aging biomarkers and the accelerated onset of aging phenotypes. Our current strategy of mutant screening for a senescence-associated biomarker in zebrafish embryos may thus prove to be a useful new tool for the genetic dissection of vertebrate stress response and senescence mechanisms. PMID:18704191

NUA Activities at the Plant Nuclear Pore

PubMed Central

Xu, Xianfeng Morgan; Rose, Annkatrin

2007-01-01

NUA (Nuclear Pore Anchor), the Arabidopsis homolog of Tpr (Translocated Promoter Region), is one of the few nuclear pore proteins conserved between animals, yeast and plants. In the May issue of Plant Cell, we report that null mutants of NUA show a pleiotropic, early flowering phenotype accompanied by changes in SUMo and RNA homeostasis. We have shown that the early flowering phenotype is caused by changed abundances of flowering time regulators involved in several pathways. Arabidopsis nua mutants phenocopy mutants lacking the ESD4 (EARlY IN ShoRT DAYS 4) SUMo protease, similar to mutants of their respective yeast homologs. however, in contrast to the comparable yeast mutants, ESD4 does not appear to be delocalized from the nuclear pore in nua mutants. Taken together, our experimental data suggests a role for NUA in controlling mRNA export from the nucleus as well as SUMo protease activity at the nuclear pore, comparable but not identical to its homologs in other eukaryotes. Furthermore, characterization of NUA illustrates a potential link at the nuclear pore between SUMo modification, RNA homeostasis and plant developmental control. PMID:19704557

Leishmania infantum HSP70-II null mutant as candidate vaccine against leishmaniasis: a preliminary evaluation.

PubMed

Carrión, Javier; Folgueira, Cristina; Soto, Manuel; Fresno, Manuel; Requena, Jose M

2011-07-27

Visceral leishmaniasis is the most severe form of leishmaniasis and no effective vaccine exists. The use of live attenuated vaccines is emerging as a promising vaccination strategy. In this study, we tested the ability of a Leishmania infantum deletion mutant, lacking both HSP70-II alleles (ΔHSP70-II), to provide protection against Leishmania infection in the L. major-BALB/c infection model. Administration of the mutant line by either intraperitoneal, intravenous or subcutaneous route invariably leads to the production of high levels of NO and the development in mice of type 1 immune responses, as determined by analysis of anti-Leishmania IgG subclasses. In addition, we have shown that ΔHSP70-II would be a safe live vaccine as immunodeficient SCID mice, and hamsters (Mesocricetus auratus), infected with mutant parasites did not develop any sign of pathology. The results suggest that the ΔHSP70-II mutant is a promising and safe vaccine, but further studies in more appropriate animal models (hamsters and dogs) are needed to appraise whether this attenuate mutant would be useful as vaccine against visceral leishmaniasis.

The Impact of ExoS on Pseudomonas aeruginosa Internalization by Epithelial Cells Is Independent of fleQ and Correlates with Bistability of Type Three Secretion System Gene Expression

PubMed Central

Kroken, Abby R.; Chen, Camille K.; Evans, David J.; Yahr, Timothy L.

2018-01-01

ABSTRACT Pseudomonas aeruginosa is internalized into multiple types of epithelial cell in vitro and in vivo and yet is often regarded as an exclusively extracellular pathogen. Paradoxically, ExoS, a type three secretion system (T3SS) effector, has antiphagocytic activities but is required for intracellular survival of P. aeruginosa and its occupation of bleb niches in epithelial cells. Here, we addressed mechanisms for this dichotomy using invasive (ExoS-expressing) P. aeruginosa and corresponding effector-null isogenic T3SS mutants, effector-null mutants of cytotoxic P. aeruginosa with and without ExoS transformation, antibiotic exclusion assays, and imaging using a T3SS-GFP reporter. Except for effector-null PA103, all strains were internalized while encoding ExoS. Intracellular bacteria showed T3SS activation that continued in replicating daughter cells. Correcting the fleQ mutation in effector-null PA103 promoted internalization by >10-fold with or without ExoS. Conversely, mutating fleQ in PAO1 reduced internalization by >10-fold, also with or without ExoS. Effector-null PA103 remained less well internalized than PAO1 matched for fleQ status, but only with ExoS expression, suggesting additional differences between these strains. Quantifying T3SS activation using GFP fluorescence and quantitative reverse transcription-PCR (qRT-PCR) showed that T3SS expression was hyperinducible for strain PA103ΔexoUT versus other isolates and was unrelated to fleQ status. These findings support the principle that P. aeruginosa is not exclusively an extracellular pathogen, with internalization influenced by the relative proportions of T3SS-positive and T3SS-negative bacteria in the population during host cell interaction. These data also challenge current thinking about T3SS effector delivery into host cells and suggest that T3SS bistability is an important consideration in studying P. aeruginosa pathogenesis. PMID:29717012

The lateral mobility of cell adhesion molecules is highly restricted at septate junctions in Drosophila.

PubMed

Laval, Monique; Bel, Christophe; Faivre-Sarrailh, Catherine

2008-07-18

A complex of three cell adhesion molecules (CAMs) Neurexin IV(Nrx IV), Contactin (Cont) and Neuroglian (Nrg) is implicated in the formation of septate junctions between epithelial cells in Drosophila. These CAMs are interdependent for their localization at septate junctions and e.g. null mutation of nrx IV or cont induces the mislocalization of Nrg to the baso-lateral membrane. These mutations also result in ultrastructural alteration of the strands of septate junctions and breakdown of the paracellular barrier. Varicose (Vari) and Coracle (Cora), that both interact with the cytoplasmic tail of Nrx IV, are scaffolding molecules required for the formation of septate junctions. We conducted photobleaching experiments on whole living Drosophila embryos to analyze the membrane mobility of CAMs at septate junctions between epithelial cells. We show that GFP-tagged Nrg and Nrx IV molecules exhibit very stable association with septate junctions in wild-type embryos. Nrg-GFP is mislocalized to the baso-lateral membrane in nrx IV or cont null mutant embryos, and displays increased mobile fraction. Similarly, Nrx IV-GFP becomes distributed to the baso-lateral membrane in null mutants of vari and cora, and its mobile fraction is strongly increased. The loss of Vari, a MAGUK protein that interacts with the cytoplasmic tail of Nrx IV, has a stronger effect than the null mutation of nrx IV on the lateral mobility of Nrg-GFP. The strands of septate junctions display a stable behavior in vivo that may be correlated with their role of paracellular barrier. The membrane mobility of CAMs is strongly limited when they take part to the multimolecular complex forming septate junctions. This restricted lateral diffusion of CAMs depends on both adhesive interactions and clustering by scaffolding molecules. The lateral mobility of CAMs is strongly increased in embryos presenting alteration of septate junctions. The stronger effect of vari by comparison with nrx IV null mutation supports the hypothesis that this scaffolding molecule may cross-link different types of CAMs and play a crucial role in stabilizing the strands of septate junctions.

Cysteine shotgun–mass spectrometry (CS-MS) reveals dynamic sequence of protein structure changes within mutant and stressed cells

PubMed Central

Krieger, Christine C.; An, Xiuli; Tang, Hsin-Yao; Mohandas, Narla; Speicher, David W.; Discher, Dennis E.

2011-01-01

Questions of if and when protein structures change within cells pervade biology and include questions of how the cytoskeleton sustains stresses on cells—particularly in mutant versus normal cells. Cysteine shotgun labeling with fluorophores is analyzed here with mass spectrometry of the spectrin–actin membrane skeleton in sheared red blood cell ghosts from normal and diseased mice. Sheared samples are compared to static samples at 37 °C in terms of cell membrane intensity in fluorescence microscopy, separated protein fluorescence, and tryptic peptide modification in liquid chromatography–tandem mass spectrometry (LC-MS/MS). Spectrin labeling proves to be the most sensitive to shear, whereas binding partners ankyrin and actin exhibit shear thresholds in labeling and both the ankyrin-binding membrane protein band 3 and the spectrin–actin stabilizer 4.1R show minimal differential labeling. Cells from 4.1R-null mice differ significantly from normal in the shear-dependent labeling of spectrin, ankyrin, and band 3: Decreased labeling of spectrin reveals less stress on the mutant network as spectrin dissociates from actin. Mapping the stress-dependent labeling kinetics of α- and β-spectrin by LC-MS/MS identifies Cys in these antiparallel chains that are either force-enhanced or force-independent in labeling, with structural analyses indicating the force-enhanced sites are sequestered either in spectrin’s triple-helical domains or in interactions with actin or ankyrin. Shear-sensitive sites identified comprehensively here in both spectrin and ankyrin appear consistent with stress relief through forced unfolding followed by cytoskeletal disruption. PMID:21527722

The Arabidopsis cyclic nucleotide-gated ion channels AtCNGC2 and AtCNGC4 work in the same signaling pathway to regulate pathogen defense and floral transition.

PubMed

Chin, Kimberley; DeFalco, Thomas A; Moeder, Wolfgang; Yoshioka, Keiko

2013-10-01

Arabidopsis (Arabidopsis thaliana) cyclic nucleotide-gated ion channels (CNGCs) form a large family consisting of 20 members and have been implicated in Ca(2+) signaling related to various physiological processes, such as pathogen defense, development, and thermotolerance. The null mutant of AtCNGC2, defense, no death (dnd1), exhibits autoimmune phenotypes, while it is impaired in mounting the hypersensitive response, which is a hallmark of effector-triggered (i.e. RESISTANCE-gene mediated) resistance. It has been suggested that AtCNGC2 is involved in defense responses and likely other aspects of physiology through its role as a Ca(2+)-conducting channel. However, the downstream signaling components and its relation with AtCNGC4, which is the closest paralog of AtCNGC2, remain elusive. Despite the fact that cngc4 mutants display almost identical phenotypes to those seen in cngc2 mutants, not much is known about their relationship. Here, we report the identification and characterization of the Arabidopsis mutant repressor of defense no death1 (rdd1), obtained from a suppressor screen of a transfer DNA insertion knockout mutant of AtCNGC2 in order to identify downstream components of dnd1-mediated signal transduction. rdd1 suppressed the majority of dnd1-mediated phenotypes except Ca(2+) hypersensitivity. In addition, rdd1 also suppressed the dnd1-mediated late-flowering phenotype that was discovered in this study. Our genetic analysis conducted to elucidate the relationship between AtCNGC2 and AtCNGC4 indicates that RDD1 is also involved in AtCNGC4-mediated signal transduction. Lastly, bimolecular fluorescence complementation analysis suggests that AtCNGC2 and AtCNGC4 are likely part of the same channel complex.

Fray, a Drosophila serine/threonine kinase homologous to mammalian PASK, is required for axonal ensheathment

NASA Technical Reports Server (NTRS)

Leiserson, W. M.; Harkins, E. W.; Keshishian, H.

2000-01-01

Fray is a serine/threonine kinase expressed by the peripheral glia of Drosophila, whose function is required for normal axonal ensheathment. Null fray mutants die early in larval development and have nerves with severe swelling and axonal defasciculation. The phenotype is associated with a failure of the ensheathing glia to correctly wrap peripheral axons. When the fray cDNA is expressed in the ensheathing glia of fray mutants, normal nerve morphology is restored. Fray belongs to a novel family of Ser/Thr kinases, the PF kinases, whose closest relatives are the PAK kinases. Rescue of the Drosophila mutant phenotype with PASK, the rat homolog of Fray, demonstrates a functional homology among these proteins and suggests that the Fray signaling pathway is widely conserved.

Intranasal Oxytocin for the Treatment of Pain Associated with Interstitial Cystitis

DTIC Science & Technology

2014-09-01

THIS PAGE U UU 8 19b. TELEPHONE NUMBER (include area code ) Table of Contents...electrical nerve stimulation, changes in diet, cessation in smoking, exercise, bladder training, physical therapy, and surgery . Unfortunately...Matzuk MM, Insel TR (2000) Infant vocalization , adult aggression, and fear behavior of an oxytocin null mutant mouse. Horm Behav 37:145–155.

EvoluZion: A Computer Simulator for Teaching Genetic and Evolutionary Concepts

ERIC Educational Resources Information Center

Zurita, Adolfo R.

2017-01-01

EvoluZion is a forward-in-time genetic simulator developed in Java and designed to perform real time simulations on the evolutionary history of virtual organisms. These model organisms harbour a set of 13 genes that codify an equal number of phenotypic features. These genes change randomly during replication, and mutant genes can have null,…

Hypomorphic alleles reveal FCA-independent roles for FY in the regulation of FLOWERING LOCUS C.

PubMed

Feng, Wei; Jacob, Yannick; Veley, Kira M; Ding, Lei; Yu, Xuhong; Choe, Goh; Michaels, Scott D

2011-03-01

The autonomous floral promotion pathway plays a key role in the regulation of flowering in rapid-cycling Arabidopsis (Arabidopsis thaliana) by providing constitutive repression of the floral inhibitor FLOWERING LOCUS C (FLC). As a result, autonomous pathway mutants contain elevated levels of FLC and are late flowering. Winter annual Arabidopsis, in contrast, contain functional alleles of FRIGIDA (FRI), which acts epistatically to the autonomous pathway to up-regulate FLC and delay flowering. To further explore the relationship between FRI and the autonomous pathway, we placed autonomous pathway mutants in a FRI-containing background. Unexpectedly, we found that a hypomorphic allele of the autonomous pathway gene fy (fy null alleles are embryo lethal) displayed background-specific effects on FLC expression and flowering time; in a rapid-cycling background fy mutants contained elevated levels of FLC and were late flowering, whereas in a winter annual background fy decreased FLC levels and partially suppressed the late-flowering phenotype conferred by FRI. Because FY has been shown to have homology to polyadenylation factors, we examined polyadenylation site selection in FLC transcripts. In wild type, two polyadenylation sites were detected and used at similar levels. In fy mutant backgrounds, however, the ratio of products was shifted to favor the distally polyadenylated form. FY has previously been shown to physically interact with another member of the autonomous pathway, FCA. Interestingly, we found that fy can partially suppress FLC expression in an fca null background and promote proximal polyadenylation site selection usage in the absence of FCA. Taken together, these results indicate novel and FCA-independent roles for FY in the regulation of FLC.

Hypomorphic Alleles Reveal FCA-Independent Roles for FY in the Regulation of FLOWERING LOCUS C1[C][W][OA

PubMed Central

Feng, Wei; Jacob, Yannick; Veley, Kira M.; Ding, Lei; Yu, Xuhong; Choe, Goh; Michaels, Scott D.

2011-01-01

The autonomous floral promotion pathway plays a key role in the regulation of flowering in rapid-cycling Arabidopsis (Arabidopsis thaliana) by providing constitutive repression of the floral inhibitor FLOWERING LOCUS C (FLC). As a result, autonomous pathway mutants contain elevated levels of FLC and are late flowering. Winter annual Arabidopsis, in contrast, contain functional alleles of FRIGIDA (FRI), which acts epistatically to the autonomous pathway to up-regulate FLC and delay flowering. To further explore the relationship between FRI and the autonomous pathway, we placed autonomous pathway mutants in a FRI-containing background. Unexpectedly, we found that a hypomorphic allele of the autonomous pathway gene fy (fy null alleles are embryo lethal) displayed background-specific effects on FLC expression and flowering time; in a rapid-cycling background fy mutants contained elevated levels of FLC and were late flowering, whereas in a winter annual background fy decreased FLC levels and partially suppressed the late-flowering phenotype conferred by FRI. Because FY has been shown to have homology to polyadenylation factors, we examined polyadenylation site selection in FLC transcripts. In wild type, two polyadenylation sites were detected and used at similar levels. In fy mutant backgrounds, however, the ratio of products was shifted to favor the distally polyadenylated form. FY has previously been shown to physically interact with another member of the autonomous pathway, FCA. Interestingly, we found that fy can partially suppress FLC expression in an fca null background and promote proximal polyadenylation site selection usage in the absence of FCA. Taken together, these results indicate novel and FCA-independent roles for FY in the regulation of FLC. PMID:21209277

Aberrant muscle antigen exposure in mice is sufficient to cause myositis in a Treg cell-deficient milieu.

PubMed

Young, Nicholas A; Sharma, Rahul; Friedman, Alexandra K; Kaffenberger, Benjamin H; Bolon, Brad; Jarjour, Wael N

2013-12-01

Myositis is associated with muscle-targeted inflammation and is observed in some Treg cell-deficient mouse models. Because an autoimmune pathogenesis has been strongly implicated, the aim of this study was to investigate the hypothesis that abnormal exposure to muscle antigens, as observed in muscle injury, can induce autoimmune-mediated myositis in susceptible hosts. FoxP3 mutant (scurfy) mice were mated to synaptotagmin VII (Syt VII) mutant mice, which resulted in a new mouse strain that combines impaired membrane resealing with Treg cell deficiency. Lymphocyte preparations from double-mutant mice were adoptively transferred intraperitoneally, with or without purified Treg cells, into recombination-activating gene 1 (RAG-1)-null recipients. Lymph node cells from mice with the FoxP3 mutation were transferred into RAG-1-null mice either 1) intraperitoneally in conjunction with muscle homogenate or purified myosin protein or 2) intramuscularly with or without cotransfer of purified Treg cells. FoxP3-deficient mouse lymph node cells transferred in conjunction with myosin protein or muscle homogenate induced robust skeletal muscle inflammation. The infiltrates consisted predominantly of CD4+ and CD8+ T cells, a limited number of macrophages, and no B cells. Significant inflammation was also seen in similar experiments using lymph node cells from FoxP3/Syt VII double-mutant mice but was absent in experiments using adoptive transfer of FoxP3 mutant mouse cells alone. The cotransfer of Treg cells completely suppressed myositis. These data, derived from a new, reproducible model, demonstrate the critical roles of Treg cell deficiency and aberrant muscle antigen exposure in the priming of autoreactive cells to induce myositis. This mouse system has multifaceted potential for examining the interplay in vivo between tissue injury and autoimmunity. © 2013 The Authors. Arthritis & Rheumatism is published by Wiley Periodicals, Inc. on behalf of the American College of Rheumatology.

The OmpL porin does not modulate redox potential in the periplasmic space of Escherichia coli.

PubMed

Sardesai, Abhijit A; Genevaux, Pierre; Schwager, Françoise; Ang, Debbie; Georgopoulos, Costa

2003-04-01

The Escherichia coli DsbA protein is the major oxidative catalyst in the periplasm. Dartigalongue et al. (EMBO J., 19, 5980-5988, 2000) reported that null mutations in the ompL gene of E.coli fully suppress all phenotypes associated with dsbA mutants, i.e. sensitivity to the reducing agent dithiothreitol (DTT) and the antibiotic benzylpenicillin, lack of motility, reduced alkaline phosphatase activity and mucoidy. They showed that OmpL is a porin and hypothesized that ompL null mutations exert their suppressive effect by preventing efflux of a putative oxidizing-reducing compound into the medium. We have repeated these experiments using two different ompL null alleles in at least three different E.coli K-12 genetic backgrounds and have failed to reproduce any of the ompL suppressive effects noted above. Also, we show that, contrary to earlier results, ompL null mutations alone do not result in partial DTT sensitivity or partial motility, nor do they appreciably affect bacterial growth rates or block propagation of the male-specific bacteriophage M13. Thus, our findings clearly demonstrate that ompL plays no perceptible role in modulating redox potential in the periplasm of E.coli.

The impact of the SSIIa null mutations on grain traits and composition in durum wheat.

PubMed

Botticella, Ermelinda; Sestili, Francesco; Ferrazzano, Gianluca; Mantovani, Paola; Cammerata, Alessandro; D'Egidio, Maria Grazia; Lafiandra, Domenico

2016-09-01

Starch represents a major nutrient in the human diet providing essentially a source of energy. More recently the modification of its composition has been associated with new functionalities both at the nutritional and technological level. Targeting the major starch biosynthetic enzymes has been shown to be a valuable strategy to manipulate the amylose-amylopectin ratio in reserve starch. In the present work a breeding strategy aiming to produce a set of SSIIa (starch synthases IIa) null durum wheat is described. We have characterized major traits such as seed weight, total starch, amylose, protein and β-glucan content in a set of mutant families derived from the introgression of the SSIIa null trait into Svevo, an elite Italian durum wheat cultivar. A large degree of variability was detected and used to select wheat lines with either improved quality traits or agronomic performances. Semolina of a set of two SSIIa null lines showed new rheological behavior and an increased content of all major dietary fiber components, namely arabinoxylans, β-glucans and resistant starch. Furthermore the investigation of gene expression highlighted important differences in some genes involved in starch and β-glucans biosynthesis.

Synergistic roles of bone morphogenetic protein 15 and growth differentiation factor 9 in ovarian function.

PubMed

Yan, C; Wang, P; DeMayo, J; DeMayo, F J; Elvin, J A; Carino, C; Prasad, S V; Skinner, S S; Dunbar, B S; Dube, J L; Celeste, A J; Matzuk, M M

2001-06-01

Knockout mouse technology has been used over the last decade to define the essential roles of ovarian-expressed genes and uncover genetic interactions. In particular, we have used this technology to study the function of multiple members of the transforming growth factor-beta superfamily including inhibins, activins, and growth differentiation factor 9 (GDF-9 or Gdf9). Knockout mice lacking GDF-9 are infertile due to a block in folliculogenesis at the primary follicle stage. In addition, recombinant GDF-9 regulates multiple cumulus granulosa cell functions in the periovulatory period including hyaluronic acid synthesis and cumulus expansion. We have also cloned an oocyte-specific homolog of GDF-9 from mice and humans, which is termed bone morphogenetic protein 15 (BMP-15 or Bmp15). To define the function of BMP-15 in mice, we generated embryonic stem cells and knockout mice, which have a null mutation in this X-linked gene. Male chimeric and Bmp15 null mice are normal and fertile. In contrast to Bmp15 null males and Gdf9 knockout females, Bmp15 null females (Bmp15(-/-)) are subfertile and usually have minimal ovarian histopathological defects, but demonstrate decreased ovulation and fertilization rates. To further decipher possible direct or indirect genetic interactions between GDF-9 and BMP-15, we have generated double mutant mice lacking one or both alleles of these related homologs. Double homozygote females (Bmp15(-/-)Gdf9(-/-)) display oocyte loss and cysts and resemble Gdf9(-/-) mutants. In contrast, Bmp15(-/-)Gdf9(+/-) female mice have more severe fertility defects than Bmp15(-/-) females, which appear to be due to abnormalities in ovarian folliculogenesis, cumulus cell physiology, and fertilization. Thus, the dosage of intact Bmp15 and Gdf9 alleles directly influences the destiny of the oocyte during folliculogenesis and in the periovulatory period. These studies have important implications for human fertility control and the maintenance of fertility and normal ovarian physiology.

Non-muscle myosin IIB (Myh10) is required for epicardial function and coronary vessel formation during mammalian development

PubMed Central

Mitchell, Karen; Al-Anbaki, Ali; Shaikh Qureshi, Wasay Mohiuddin; Tenin, Gennadiy; Lu, Yinhui; Clowes, Christopher; Robertson, Abigail; Barnes, Emma; Wright, Jayne A.; Keavney, Bernard; Lovell, Simon C.

2017-01-01

The coronary vasculature is an essential vessel network providing the blood supply to the heart. Disruptions in coronary blood flow contribute to cardiac disease, a major cause of premature death worldwide. The generation of treatments for cardiovascular disease will be aided by a deeper understanding of the developmental processes that underpin coronary vessel formation. From an ENU mutagenesis screen, we have isolated a mouse mutant displaying embryonic hydrocephalus and cardiac defects (EHC). Positional cloning and candidate gene analysis revealed that the EHC phenotype results from a point mutation in a splice donor site of the Myh10 gene, which encodes NMHC IIB. Complementation testing confirmed that the Myh10 mutation causes the EHC phenotype. Characterisation of the EHC cardiac defects revealed abnormalities in myocardial development, consistent with observations from previously generated NMHC IIB null mouse lines. Analysis of the EHC mutant hearts also identified defects in the formation of the coronary vasculature. We attribute the coronary vessel abnormalities to defective epicardial cell function, as the EHC epicardium displays an abnormal cell morphology, reduced capacity to undergo epithelial-mesenchymal transition (EMT), and impaired migration of epicardial-derived cells (EPDCs) into the myocardium. Our studies on the EHC mutant demonstrate a requirement for NMHC IIB in epicardial function and coronary vessel formation, highlighting the importance of this protein in cardiac development and ultimately, embryonic survival. PMID:29084269

Galactose Metabolism Plays a Crucial Role in Biofilm Formation by Bacillus subtilis

PubMed Central

Chai, Yunrong; Beauregard, Pascale B.; Vlamakis, Hera; Losick, Richard; Kolter, Roberto

2012-01-01

ABSTRACT Galactose is a common monosaccharide that can be utilized by all living organisms via the activities of three main enzymes that make up the Leloir pathway: GalK, GalT, and GalE. In Bacillus subtilis, the absence of GalE causes sensitivity to exogenous galactose, leading to rapid cell lysis. This effect can be attributed to the accumulation of toxic galactose metabolites, since the galE mutant is blocked in the final step of galactose catabolism. In a screen for suppressor mutants restoring viability to a galE null mutant in the presence of galactose, we identified mutations in sinR, which is the major biofilm repressor gene. These mutations caused an increase in the production of the exopolysaccharide (EPS) component of the biofilm matrix. We propose that UDP-galactose is the toxic galactose metabolite and that it is used in the synthesis of EPS. Thus, EPS production can function as a shunt mechanism for this toxic molecule. Additionally, we demonstrated that galactose metabolism genes play an essential role in B. subtilis biofilm formation and that the expressions of both the gal and eps genes are interrelated. Finally, we propose that B. subtilis and other members of the Bacillus genus may have evolved to utilize naturally occurring polymers of galactose, such as galactan, as carbon sources. PMID:22893383

Regulation of c–myc expression by IFN–γ through Stat1-dependent and -independent pathways

PubMed Central

Ramana, Chilakamarti V.; Grammatikakis, Nicholas; Chernov, Mikhail; Nguyen, Hannah; Goh, Kee Chuan; Williams, Bryan R.G.; Stark, George R.

2000-01-01

Interferons (IFNs) inhibit cell growth in a Stat1-dependent fashion that involves regulation of c–myc expression. IFN–γ suppresses c–myc in wild-type mouse embryo fibroblasts, but not in Stat1-null cells, where IFNs induce c–myc mRNA rapidly and transiently, thus revealing a novel signaling pathway. Both tyrosine and serine phosphorylation of Stat1 are required for suppression. Induced expression of c–myc is likely to contribute to the proliferation of Stat1-null cells in response to IFNs. IFNs also suppress platelet-derived growth factor (PDGF)-induced c–myc expression in wild-type but not in Stat1-null cells. A gamma-activated sequence element in the promoter is necessary but not sufficient to suppress c–myc expression in wild-type cells. In PKR-null cells, the phosphorylation of Stat1 on Ser727 and transactivation are both defective, and c–myc mRNA is induced, not suppressed, in response to IFN–γ. A role for Raf–1 in the Stat1-independent pathway is revealed by studies with geldanamycin, an HSP90-specific inhibitor, and by expression of a mutant of p50cdc37 that is unable to recruit HSP90 to the Raf–1 complex. Both agents abrogated the IFN–γ-dependent induction of c–myc expression in Stat1-null cells. PMID:10637230

Reduced wheel running and blunted effects of voluntary exercise in LPA1-null mice: The importance of assessing the amount of running in transgenic mice studies

PubMed Central

Castilla-Ortega, Estela; Rosell-Valle, Cristina; Blanco, Eduardo; Pedraza, Carmen; Chun, Jerold; de Fonseca, Fernando Rodríguez; Estivill-Torrús, Guillermo; Santín, Luis J.

2014-01-01

This work was aimed to assess whether voluntary exercise rescued behavioral and hippocampal alterations in mice lacking the lysophosphatidic acid LPA1 receptor (LPA1-null mice), studying the potential relationship between the amount of exercise performed and its effects. Normal and LPA1-null mice underwent 23 days of free wheel running and were tested for open-field behavior and adult hippocampal neurogenesis (cell proliferation, immature neurons, cell survival). Running decreased anxiety-like behavior in both genotypes but increased exploration only in the normal mice. While running affected all neurogenesis-related measures in normal mice (especially in the suprapyramidal blade of the dentate gyrus), only a moderate increase in cell survival was found in the mutants. Importantly, the LPA1-nulls showed notably reduced running. Analysis suggested that defective running in the LPA1-null mice could contribute to explain the scarce benefit of the voluntary exercise treatment. On the other hand, a literature review revealed that voluntary exercise is frequently used to modulate behavior and the hippocampus in transgenic mice, but half of the studies did not assess the quantity of running, overlooking any potential running impairments. This study adds evidence to the relevance of the quantity of exercise performed, emphasizing the importance of its assessment in transgenic mice research. PMID:24055600

Buffering of protons released by mineral formation during amelogenesis in mice.

PubMed

Bronckers, Antonius L J J; Lyaruu, Don M; Jalali, Rozita; DenBesten, Pamela K

2016-10-01

Regulation of pH by ameloblasts during amelogenesis is critical for enamel mineralization. We examined the effects of reduced bicarbonate secretion and the presence or absence of amelogenins on ameloblast modulation and enamel mineralization. To that end, the composition of fluorotic and non-fluorotic enamel of several different mouse mutants, including enamel of cystic fibrosis transmembrane conductance regulator-deficient (Cftr null), anion exchanger-2-deficient (Ae2a,b null), and amelogenin-deficient (Amelx null) mice, was determined by quantitative X-ray microanalysis. Correlation analysis was carried out to compare the effects of changes in the levels of sulfated-matrix (S) and chlorine (Cl; for bicarbonate secretion) on mineralization and modulation. The chloride (Cl - ) levels in forming enamel determined the ability of ameloblasts to modulate, remove matrix, and mineralize enamel. In general, the lower the Cl - content, the stronger the negative effects. In Amelx-null mice, modulation was essentially normal and the calcium content was reduced least. Retention of amelogenins in enamel of kallikrein-4-deficient (Klk4-null) mice resulted in decreased mineralization and reduced the length of the first acid modulation band without changing the total length of all acidic bands. These data suggest that buffering by bicarbonates is critical for modulation, matrix removal and enamel mineralization. Amelogenins also act as a buffer but are not critical for modulation. © 2016 Eur J Oral Sci.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zampol, Mariana A.; Busso, Cleverson; Gomes, Fernando

Research highlights: {yields} COQ10 deletion elicits a defect in mitochondrial respiration correctable by addition of coenzyme Q{sub 2}, a synthetic diffusible ubiquinone. {yields} The significance that purified Coq10p contains bound Q{sub 6} was examined by testing over-expression of Coq10p on respiration. {yields} Inhibition of CoQ function due to Coq10p excess strength our hypothesis of Coq10p function in CoQ delivery. {yields} Respiratory deficiency caused by more Coq10p was specific and restored by Q{sub 2} in mitochondria or by Coq8p in cells. {yields} Coq8p over-production on other coq mutants revealed a surprisingly higher stability of other Coq proteins. -- Abstract: COQ10 deletionmore » in Saccharomyces cerevisiae elicits a defect in mitochondrial respiration correctable by addition of coenzyme Q{sub 2}. Rescue of respiration by Q{sub 2} is a characteristic of mutants blocked in coenzyme Q{sub 6} synthesis. Unlike Q{sub 6} deficient mutants, mitochondria of the coq10 null mutant have wild-type concentrations of Q{sub 6}. The physiological significance of earlier observations that purified Coq10p contains bound Q{sub 6} was examined in the present study by testing the in vivo effect of over-expression of Coq10p on respiration. Mitochondria with elevated levels of Coq10p display reduced respiration in the bc1 span of the electron transport chain, which can be restored with exogenous Q{sub 2}. This suggests that in vivo binding of Q{sub 6} by excess Coq10p reduces the pool of this redox carrier available for its normal function in providing electrons to the bc1 complex. This is confirmed by observing that extra Coq8p relieves the inhibitory effect of excess Coq10p. Coq8p is a putative kinase, and a high-copy suppressor of the coq10 null mutant. As shown here, when over-produced in coq mutants, Coq8p counteracts turnover of Coq3p and Coq4p subunits of the Q-biosynthetic complex. This can account for the observed rescue by COQ8 of the respiratory defect in strains over-producing Coq10p.« less

Drosophila larvae lacking the bcl-2 gene, buffy, are sensitive to nutrient stress, maintain increased basal target of rapamycin (Tor) signaling and exhibit characteristics of altered basal energy metabolism

PubMed Central

2012-01-01

Background B cell lymphoma 2 (Bcl-2) proteins are the central regulators of apoptosis. The two bcl-2 genes in Drosophila modulate the response to stress-induced cell death, but not developmental cell death. Because null mutants are viable, Drosophila provides an optimum model system to investigate alternate functions of Bcl-2 proteins. In this report, we explore the role of one bcl-2 gene in nutrient stress responses. Results We report that starvation of Drosophila larvae lacking the bcl-2 gene, buffy, decreases survival rate by more than twofold relative to wild-type larvae. The buffy null mutant reacted to starvation with the expected responses such as inhibition of target of rapamycin (Tor) signaling, autophagy initiation and mobilization of stored lipids. However, the autophagic response to starvation initiated faster in larvae lacking buffy and was inhibited by ectopic buffy. We demonstrate that unusually high basal Tor signaling, indicated by more phosphorylated S6K, was detected in the buffy mutant and that removal of a genomic copy of S6K, but not inactivation of Tor by rapamycin, reverted the precocious autophagy phenotype. Instead, Tor inactivation also required loss of a positive nutrient signal to trigger autophagy and loss of both was sufficient to activate autophagy in the buffy mutant even in the presence of enforced phosphoinositide 3-kinase (PI3K) signaling. Prior to starvation, the fed buffy mutant stored less lipid and glycogen, had high lactate levels and maintained a reduced pool of cellular ATP. These observations, together with the inability of buffy mutant larvae to adapt to nutrient restriction, indicate altered energy metabolism in the absence of buffy. Conclusions All animals in their natural habitats are faced with periods of reduced nutrient availability. This study demonstrates that buffy is required for adaptation to both starvation and nutrient restriction. Thus, Buffy is a Bcl-2 protein that plays an important non-apoptotic role to promote survival of the whole organism in a stressful situation. PMID:22824239

DOE Office of Scientific and Technical Information (OSTI.GOV)

Noda, Kazuo, E-mail: knoda@kuhp.kyoto-u.ac.jp; Nakamura, Tomoyuki; Komatsu, Yoshihiro

Craniofacial sutures govern the shape of the craniofacial skeleton during postnatal development. The differentiation of suture mesenchymal cells to osteoblasts is precisely regulated in part by signaling through cell surface receptors that interact with extracellular proteins. Here we report that fibulin-5, a key extracellular matrix protein, is important for craniofacial skeletal development in mice. Fibulin-5 is deposited as a fibrous matrix in cranial neural crest-derived mesenchymal tissues, including craniofacial sutures. Fibulin-5-null mice show decreased premaxillary bone outgrowth during postnatal stages. While premaxillo-maxillary suture mesenchymal cells in fibulin-5-null mice were capable of differentiating into osteoblasts, suture cells in mutant mice weremore » less proliferative. Our study provides the first evidence that fibulin-5 is indispensable for the regulation of facial suture mesenchymal cell proliferation required for craniofacial skeletal morphogenesis. - Highlights: • Fibulin-5 is deposited in cranial neural crest-derived mesenchymal tissues. • Fibulin-5-null mice show decreased premaxillary bone growth during postnatal stage. • Fibulin-5 is indispensable for facial suture mesenchymal cell proliferation.« less

Gα modulates salt-induced cellular senescence and cell division in rice and maize

DOE PAGES

Urano, Daisuke; Colaneri, Alejandro; Jones, Alan M.

2014-09-16

The plant G-protein network, comprising Gα, Gβ, and Gγ core subunits, regulates development, senses sugar, and mediates biotic and abiotic stress responses. Here in this paper, we report G-protein signalling in the salt stress response using two crop models, rice and maize. Loss-of-function mutations in the corresponding genes encoding the Gα subunit attenuate growth inhibition and cellular senescence caused by sodium chloride (NaCl). Gα null mutations conferred reduced leaf senescence, chlorophyll degradation, and cytoplasm electrolyte leakage under NaCl stress. Sodium accumulated in both wild-type and Gα-mutant shoots to the same levels, suggesting that Gα signalling controls cell death in leavesmore » rather than sodium exclusion in roots. Growth inhibition is probably initiated by osmotic change around root cells, because KCl and MgSO 4 also suppressed seedling growth equally as well as NaCl. NaCl lowered rates of cell division and elongation in the wild-type leaf sheath to the level of the Gα-null mutants; however there was no NaCl-induced decrease in cell division in the Gα mutant, implying that the osmotic phase of salt stress suppresses cell proliferation through the inhibition of Gα-coupled signalling. These results reveal two distinct functions of Gα in NaCl stress in these grasses: attenuation of leaf senescence caused by sodium toxicity in leaves, and cell cycle regulation by osmotic/ionic stress.« less

Impairment of enzymatic antioxidant defenses is associated with bilirubin-induced neuronal cell death in the cerebellum of Ugt1 KO mice

PubMed Central

Bortolussi, G; Codarin, E; Antoniali, G; Vascotto, C; Vodret, S; Arena, S; Cesaratto, L; Scaloni, A; Tell, G; Muro, A F

2015-01-01

Severe hyperbilirubinemia is toxic during central nervous system development. Prolonged and uncontrolled high levels of unconjugated bilirubin lead to bilirubin-induced encephalopathy and eventually death by kernicterus. Despite extensive studies, the molecular and cellular mechanisms of bilirubin toxicity are still poorly defined. To fill this gap, we investigated the molecular processes underlying neuronal injury in a mouse model of severe neonatal jaundice, which develops hyperbilirubinemia as a consequence of a null mutation in the Ugt1 gene. These mutant mice show cerebellar abnormalities and hypoplasia, neuronal cell death and die shortly after birth because of bilirubin neurotoxicity. To identify protein changes associated with bilirubin-induced cell death, we performed proteomic analysis of cerebella from Ugt1 mutant and wild-type mice. Proteomic data pointed-out to oxidoreductase activities or antioxidant processes as important intracellular mechanisms altered during bilirubin-induced neurotoxicity. In particular, they revealed that down-representation of DJ-1, superoxide dismutase, peroxiredoxins 2 and 6 was associated with hyperbilirubinemia in the cerebellum of mutant mice. Interestingly, the reduction in protein levels seems to result from post-translational mechanisms because we did not detect significant quantitative differences in the corresponding mRNAs. We also observed an increase in neuro-specific enolase 2 both in the cerebellum and in the serum of mutant mice, supporting its potential use as a biomarker of bilirubin-induced neurological damage. In conclusion, our data show that different protective mechanisms fail to contrast oxidative burst in bilirubin-affected brain regions, ultimately leading to neurodegeneration. PMID:25950469

Distinct roles for RDE-1 and RDE-4 during RNA interference in Caenorhabditis elegans.

PubMed

Parrish, S; Fire, A

2001-10-01

RNA interference (RNAi) is a cellular defense mechanism that uses double-stranded RNA (dsRNA) as a sequence-specific trigger to guide the degradation of homologous single-stranded RNAs. RNAi is a multistep process involving several proteins and at least one type of RNA intermediate, a population of small 21-25 nt RNAs (called siRNAs) that are initially derived from cleavage of the dsRNA trigger. Genetic screens in Caenorhabditis elegans have identified numerous mutations that cause partial or complete loss of RNAi. In this work, we analyzed cleavage of injected dsRNA to produce the initial siRNA population in animals mutant for rde-1 and rde-4, two genes that are essential for RNAi but that are not required for organismal viability or fertility. Our results suggest distinct roles for RDE-1 and RDE-4 in the interference process. Although null mutants lacking rde-1 show no phenotypic response to dsRNA, the amount of siRNAs generated from an injected dsRNA trigger was comparable to that of wild-type. By contrast, mutations in rde-4 substantially reduced the population of siRNAs derived from an injected dsRNA trigger. Injection of chemically synthesized 24- or 25-nt siRNAs could circumvent RNAi resistance in rde-4 mutants, whereas no bypass was observed in rde-1 mutants. These results support a model in which RDE-4 is involved before or during production of siRNAs, whereas RDE-1 acts after the siRNAs have been formed.

Distinct roles for RDE-1 and RDE-4 during RNA interference in Caenorhabditis elegans.

PubMed Central

Parrish, S; Fire, A

2001-01-01

RNA interference (RNAi) is a cellular defense mechanism that uses double-stranded RNA (dsRNA) as a sequence-specific trigger to guide the degradation of homologous single-stranded RNAs. RNAi is a multistep process involving several proteins and at least one type of RNA intermediate, a population of small 21-25 nt RNAs (called siRNAs) that are initially derived from cleavage of the dsRNA trigger. Genetic screens in Caenorhabditis elegans have identified numerous mutations that cause partial or complete loss of RNAi. In this work, we analyzed cleavage of injected dsRNA to produce the initial siRNA population in animals mutant for rde-1 and rde-4, two genes that are essential for RNAi but that are not required for organismal viability or fertility. Our results suggest distinct roles for RDE-1 and RDE-4 in the interference process. Although null mutants lacking rde-1 show no phenotypic response to dsRNA, the amount of siRNAs generated from an injected dsRNA trigger was comparable to that of wild-type. By contrast, mutations in rde-4 substantially reduced the population of siRNAs derived from an injected dsRNA trigger. Injection of chemically synthesized 24- or 25-nt siRNAs could circumvent RNAi resistance in rde-4 mutants, whereas no bypass was observed in rde-1 mutants. These results support a model in which RDE-4 is involved before or during production of siRNAs, whereas RDE-1 acts after the siRNAs have been formed. PMID:11680844

Impairment of enzymatic antioxidant defenses is associated with bilirubin-induced neuronal cell death in the cerebellum of Ugt1 KO mice.

PubMed

Bortolussi, G; Codarin, E; Antoniali, G; Vascotto, C; Vodret, S; Arena, S; Cesaratto, L; Scaloni, A; Tell, G; Muro, A F

2015-05-07

Severe hyperbilirubinemia is toxic during central nervous system development. Prolonged and uncontrolled high levels of unconjugated bilirubin lead to bilirubin-induced encephalopathy and eventually death by kernicterus. Despite extensive studies, the molecular and cellular mechanisms of bilirubin toxicity are still poorly defined. To fill this gap, we investigated the molecular processes underlying neuronal injury in a mouse model of severe neonatal jaundice, which develops hyperbilirubinemia as a consequence of a null mutation in the Ugt1 gene. These mutant mice show cerebellar abnormalities and hypoplasia, neuronal cell death and die shortly after birth because of bilirubin neurotoxicity. To identify protein changes associated with bilirubin-induced cell death, we performed proteomic analysis of cerebella from Ugt1 mutant and wild-type mice. Proteomic data pointed-out to oxidoreductase activities or antioxidant processes as important intracellular mechanisms altered during bilirubin-induced neurotoxicity. In particular, they revealed that down-representation of DJ-1, superoxide dismutase, peroxiredoxins 2 and 6 was associated with hyperbilirubinemia in the cerebellum of mutant mice. Interestingly, the reduction in protein levels seems to result from post-translational mechanisms because we did not detect significant quantitative differences in the corresponding mRNAs. We also observed an increase in neuro-specific enolase 2 both in the cerebellum and in the serum of mutant mice, supporting its potential use as a biomarker of bilirubin-induced neurological damage. In conclusion, our data show that different protective mechanisms fail to contrast oxidative burst in bilirubin-affected brain regions, ultimately leading to neurodegeneration.

Protein kinase FgSch9 serves as a mediator of the target of rapamycin and high osmolarity glycerol pathways and regulates multiple stress responses and secondary metabolism in Fusarium graminearum.

PubMed

Gu, Qin; Zhang, Chengqi; Yu, Fangwei; Yin, Yanni; Shim, Won-Bo; Ma, Zhonghua

2015-08-01

Saccharomyces cerevisiae protein kinase Sch9 is one of the downstream effectors of the target of rapamycin (TOR) complex 1 and plays multiple roles in stress resistance, longevity and nutrient sensing. However, the functions of Sch9 orthologs in filamentous fungi, particularly in pathogenic species, have not been characterized to date. Here, we investigated biological and genetic functions of FgSch9 in Fusarium graminearum. The FgSCH9 deletion mutant (ΔFgSch9) was defective in aerial hyphal growth, hyphal branching and conidial germination. The mutant exhibited increased sensitivity to osmotic and oxidative stresses, cell wall-damaging agents, and to rapamycin, while showing increased thermal tolerance. We identified FgMaf1 as one of the FgSch9-interacting proteins that plays an important role in regulating mycotoxin biosynthesis and virulence of F. graminearum. Co-immunoprecipitation and affinity capture-mass spectrometry assays showed that FgSch9 also interacts with FgTor and FgHog1. More importantly, both ΔFgSch9 and FgHog1 null mutant (ΔFgHog1) exhibited increased sensitivity to osmotic and oxidative stresses. This defect was more severe in the FgSch9/FgHog1 double mutant. Taken together, we propose that FgSch9 serves as a mediator of the TOR and high osmolarity glycerol pathways, and regulates vegetative differentiation, multiple stress responses and secondary metabolism in F. graminearum. © 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.

Comparative proteomic analysis of normal and collagen IX null mouse cartilage reveals altered extracellular matrix composition and novel components of the collagen IX interactome.

PubMed

Brachvogel, Bent; Zaucke, Frank; Dave, Keyur; Norris, Emma L; Stermann, Jacek; Dayakli, Münire; Koch, Manuel; Gorman, Jeffrey J; Bateman, John F; Wilson, Richard

2013-05-10

Collagen IX is an integral cartilage extracellular matrix component important in skeletal development and joint function. Proteomic analysis and validation studies revealed novel alterations in collagen IX null cartilage. Matrilin-4, collagen XII, thrombospondin-4, fibronectin, βig-h3, and epiphycan are components of the in vivo collagen IX interactome. We applied a proteomics approach to advance our understanding of collagen IX ablation in cartilage. The cartilage extracellular matrix is essential for endochondral bone development and joint function. In addition to the major aggrecan/collagen II framework, the interacting complex of collagen IX, matrilin-3, and cartilage oligomeric matrix protein (COMP) is essential for cartilage matrix stability, as mutations in Col9a1, Col9a2, Col9a3, Comp, and Matn3 genes cause multiple epiphyseal dysplasia, in which patients develop early onset osteoarthritis. In mice, collagen IX ablation results in severely disturbed growth plate organization, hypocellular regions, and abnormal chondrocyte shape. This abnormal differentiation is likely to involve altered cell-matrix interactions but the mechanism is not known. To investigate the molecular basis of the collagen IX null phenotype we analyzed global differences in protein abundance between wild-type and knock-out femoral head cartilage by capillary HPLC tandem mass spectrometry. We identified 297 proteins in 3-day cartilage and 397 proteins in 21-day cartilage. Components that were differentially abundant between wild-type and collagen IX-deficient cartilage included 15 extracellular matrix proteins. Collagen IX ablation was associated with dramatically reduced COMP and matrilin-3, consistent with known interactions. Matrilin-1, matrilin-4, epiphycan, and thrombospondin-4 levels were reduced in collagen IX null cartilage, providing the first in vivo evidence for these proteins belonging to the collagen IX interactome. Thrombospondin-4 expression was reduced at the mRNA level, whereas matrilin-4 was verified as a novel collagen IX-binding protein. Furthermore, changes in TGFβ-induced protein βig-h3 and fibronectin abundance were found in the collagen IX knock-out but not associated with COMP ablation, indicating specific involvement in the abnormal collagen IX null cartilage. In addition, the more widespread expression of collagen XII in the collagen IX-deficient cartilage suggests an attempted compensatory response to the absence of collagen IX. Our differential proteomic analysis of cartilage is a novel approach to identify candidate matrix protein interactions in vivo, underpinning further analysis of mutant cartilage lacking other matrix components or harboring disease-causing mutations.

A Mycobacterium tuberculosis Cytochrome bd Oxidase Mutant Is Hypersensitive to Bedaquiline

PubMed Central

Hartman, Travis E.

2014-01-01

ABSTRACT The new medicinal compound bedaquiline (BDQ) kills Mycobacterium tuberculosis by inhibiting F1Fo-ATP synthase. BDQ is bacteriostatic for 4 to 7 days and kills relatively slowly compared to other frontline tuberculosis (TB) drugs. Here we show that killing with BDQ can be improved significantly by inhibiting cytochrome bd oxidase, a non-proton-pumping terminal oxidase. BDQ was instantly bactericidal against a cytochrome bd oxidase null mutant of M. tuberculosis, and the rate of killing was increased by more than 50%. We propose that this exclusively bacterial enzyme should be a high-priority target for new drug discovery. PMID:25028424

Selective reconstitution of liver cholesterol biosynthesis promotes lung maturation but does not prevent neonatal lethality in Dhcr7 null mice.

PubMed

Yu, Hongwei; Li, Man; Tint, G Stephen; Chen, Jianliang; Xu, Guorong; Patel, Shailendra B

2007-04-04

Targeted disruption of the murine 3beta-hydroxysterol-Delta7-reductase gene (Dhcr7), an animal model of Smith-Lemli-Opitz syndrome, leads to loss of cholesterol synthesis and neonatal death that can be partially rescued by transgenic replacement of DHCR7 expression in brain during embryogenesis. To gain further insight into the role of non-brain tissue cholesterol deficiency in the pathophysiology, we tested whether the lethal phenotype could be abrogated by selective transgenic complementation with DHCR7 expression in the liver. We generated mice that carried a liver-specific human DHCR7 transgene whose expression was driven by the human apolipoprotein E (ApoE) promoter and its associated liver-specific enhancer. These mice were then crossed with Dhcr7+/- mutants to generate Dhcr7-/- mice bearing a human DHCR7 transgene. Robust hepatic transgene expression resulted in significant improvement of cholesterol homeostasis with cholesterol concentrations increasing to 80~90 % of normal levels in liver and lung. Significantly, cholesterol deficiency in brain was not altered. Although late gestational lung sacculation defect reported previously was significantly improved, there was no parallel increase in postnatal survival in the transgenic mutant mice. The reconstitution of DHCR7 function selectively in liver induced a significant improvement of cholesterol homeostasis in non-brain tissues, but failed to rescue the neonatal lethality of Dhcr7 null mice. These results provided further evidence that CNS defects caused by Dhcr7 null likely play a major role in the lethal pathogenesis of Dhcr7-/- mice, with the peripheral organs contributing the morbidity.

Calcium Homeostasis and Muscle Energy Metabolism Are Modified in HspB1-Null Mice.

PubMed

Picard, Brigitte; Kammoun, Malek; Gagaoua, Mohammed; Barboiron, Christiane; Meunier, Bruno; Chambon, Christophe; Cassar-Malek, Isabelle

2016-05-04

Hsp27-encoded by HspB1- is a member of the small heat shock proteins (sHsp, 12-43 kDa (kilodalton)) family. This protein is constitutively present in a wide variety of tissues and in many cell lines. The abundance of Hsp27 is highest in skeletal muscle, indicating a crucial role for muscle physiology. The protein identified as a beef tenderness biomarker was found at a crucial hub in a functional network involved in beef tenderness. The aim of this study was to analyze the proteins impacted by the targeted invalidation of HspB1 in the Tibialis anterior muscle of the mouse . Comparative proteomics using two-dimensional gel electrophoresis revealed 22 spots that were differentially abundant between HspB1 -null mice and their controls that could be identified by mass spectrometry. Eighteen spots were more abundant in the muscle of the mutant mice, and four were less abundant. The proteins impacted by the absence of Hsp27 belonged mainly to calcium homeostasis (Srl and Calsq1), contraction (TnnT3), energy metabolism (Tpi1, Mdh1, PdhB, Ckm, Pygm, ApoA1) and the Hsp proteins family (HspA9). These data suggest a crucial role for these proteins in meat tenderization. The information gained by this study could also be helpful to predict the side effects of Hsp27 depletion in muscle development and pathologies linked to small Hsps.

Calcium Homeostasis and Muscle Energy Metabolism Are Modified in HspB1-Null Mice

PubMed Central

Picard, Brigitte; Kammoun, Malek; Gagaoua, Mohammed; Barboiron, Christiane; Meunier, Bruno; Chambon, Christophe; Cassar-Malek, Isabelle

2016-01-01

Hsp27—encoded by HspB1—is a member of the small heat shock proteins (sHsp, 12–43 kDa (kilodalton)) family. This protein is constitutively present in a wide variety of tissues and in many cell lines. The abundance of Hsp27 is highest in skeletal muscle, indicating a crucial role for muscle physiology. The protein identified as a beef tenderness biomarker was found at a crucial hub in a functional network involved in beef tenderness. The aim of this study was to analyze the proteins impacted by the targeted invalidation of HspB1 in the Tibialis anterior muscle of the mouse. Comparative proteomics using two-dimensional gel electrophoresis revealed 22 spots that were differentially abundant between HspB1-null mice and their controls that could be identified by mass spectrometry. Eighteen spots were more abundant in the muscle of the mutant mice, and four were less abundant. The proteins impacted by the absence of Hsp27 belonged mainly to calcium homeostasis (Srl and Calsq1), contraction (TnnT3), energy metabolism (Tpi1, Mdh1, PdhB, Ckm, Pygm, ApoA1) and the Hsp proteins family (HspA9). These data suggest a crucial role for these proteins in meat tenderization. The information gained by this study could also be helpful to predict the side effects of Hsp27 depletion in muscle development and pathologies linked to small Hsps. PMID:28248227

Goa1p of Candida albicans Localizes to the Mitochondria during Stress and Is Required for Mitochondrial Function and Virulence▿ †

PubMed Central

Bambach, Adrienne; Fernandes, Mariana P.; Ghosh, Anup; Kruppa, Michael; Alex, Deepu; Li, Dongmei; Fonzi, William A.; Chauhan, Neeraj; Sun, Nuo; Agrellos, Orlando A.; Vercesi, Anibal E.; Rolfes, Ronda J.; Calderone, Richard

2009-01-01

Using a Tn7 transposon library of Candida albicans, we have identified a mutant that exhibited sensitivity in drop plate assays to oxidants such as menadione and hydrogen peroxide. To verify the role of the mutated gene in stress adaptation, null mutants were constructed and phenotypically characterized. Because of its apparent functions in growth and oxidant adaptation, we have named the gene GOA1. Goa1p appears to be unique to the CTG subclade of the Saccharomycotina, including C. albicans. Mutants of C. albicans lacking goa1 (strain GOA31) were more sensitive to 6 mM H2O2 and 0.125 mM menadione than the wild type (wt) or a gene-reconstituted (GOA32) strain. The sensitivity to oxidants correlated with reduced survival of the GOA31 mutant in human neutrophils and avirulence compared to control strains. Other phenotypes of GOA31 include reduced growth and filamentation in 10% serum, Spider, and SLAD agar media and an inability to form chlamydospores. Since Goa1p has an N-terminal mitochondrion localization site, we also show that green fluorescent protein-tagged Goa1p shows a mitochondrionlike distribution during oxidant or osmotic stress. Further, the inability of GOA31 to grow in medium containing lactate, ethanol, or glycerol as the sole carbon source indicates that the mitochondria are defective in the mutant. To determine how Goa1p contributes to mitochondrial function, we compared the wt, GOA32, and GOA31 strains for mitochondrial electrical membrane potential, respiration, and oxidative phosphorylation. We now show that GOA31, but not the wt or GOA32, had decreased respiration and mitochondrial membrane potential such that mutant cells are unable to drive oxidative phosphorylation. This is the first report in C. albicans of a respiratory defect caused by a loss of mitochondrial membrane potential. PMID:19717740

Root hair-specific disruption of cellulose and xyloglucan in AtCSLD3 mutants, and factors affecting the post-rupture resumption of mutant root hair growth.

PubMed

Galway, Moira E; Eng, Ryan C; Schiefelbein, John W; Wasteneys, Geoffrey O

2011-05-01

The glycosyl transferase encoded by the cellulose synthase-like gene CSLD3/KJK/RHD7 (At3g03050) is required for cell wall integrity during root hair formation in Arabidopsis thaliana but it remains unclear whether it contributes to the synthesis of cellulose or hemicellulose. We identified two new alleles, root hair-defective (rhd) 7-1 and rhd7-4, which affect the C-terminal end of the encoded protein. Like root hairs in the previously characterized kjk-2 putative null mutant, rhd7-1 and rhd7-4 hairs rupture before tip growth but, depending on the growth medium and temperature, hairs are able to survive rupture and initiate tip growth, indicating that these alleles retain some function. At 21°C, the rhd7 tip-growing root hairs continued to rupture but at 5ºC, rupture was inhibited, resulting in long, wild type-like root hairs. At both temperatures, the expression of another root hair-specific CSLD gene, CSLD2, was increased in the rhd7-4 mutant but reduced in the kjk-2 mutant, suggesting that CSLD2 expression is CSLD3-dependent, and that CSLD2 could partially compensate for CSLD3 defects to prevent rupture at 5°C. Using a fluorescent brightener (FB 28) to detect cell wall (1 → 4)-β-glucans (primarily cellulose) and CCRC-M1 antibody to detect fucosylated xyloglucans revealed a patchy distribution of both in the mutant root hair cell walls. Cell wall thickness varied, and immunogold electron microscopy indicated that xyloglucan distribution was altered throughout the root hair cell walls. These cell wall defects indicate that CSLD3 is required for the normal organization of both cellulose and xyloglucan in root hair cell walls.

RML prions act through Mahogunin and Attractin-independent pathways.

PubMed

Gunn, Teresa M; Carlson, George A

2013-01-01

While the conversion of the normal form of prion protein to a conformationally distinct pathogenic form is recognized to be the primary cause of prion disease, it is not clear how this leads to spongiform change, neuronal dysfunction and death. Mahogunin ring finger-1 (Mgrn1) and Attractin (Atrn) null mutant mice accumulate vacuoles throughout the brain that appear very similar to those associated with prion disease, but they do not accumulate the protease-resistant scrapie form of the prion protein or become sick. A study demonstrating an interaction between cytosolically-exposed prion protein and MGRN1 suggested that disruption of MGRN1 function may contribute to prion disease pathogenesis, but we recently showed that neither loss of MGRN1 nor MGRN1 overexpression influences the onset or progression of prion disease following intracerebral inoculation with Rocky Mountain Laboratory prions. Here, we show that loss of ATRN also has no effect on prion disease onset or progression and discuss possible mechanisms that could cause vacuolation of the central nervous system in Mgrn1 and Atrn null mutant mice and whether the same pathways might contribute to this intriguing phenotype in prion disease.

Bacteriophage P2 ogr and P4 delta genes act independently and are essential for P4 multiplication.

PubMed Central

Halling, C; Calendar, R

1990-01-01

Satellite bacteriophage P4 requires the products of the late genes of a helper phage such as P2 for lytic growth. Expression of the P2 late genes is positively regulated by the P2 ogr gene in a process requiring P2 DNA replication. Transactivation of P2 late gene expression by P4 requires the P4 delta gene product and works even in the absence of P2 DNA replication. We have made null mutants of the P2 ogr and P4 delta genes. In the absence of the P4 delta gene product, P4 multiplication required both the P2 ogr protein and P2 DNA replication. In the absence of the P2 ogr gene product, P4 multiplication required the P4 delta protein. In complementation experiments, we found that the P2 ogr protein was made in the absence of P2 DNA replication but could not function unless P2 DNA replicated. We produced P4 delta protein from a plasmid and found that it complemented the null P4 delta and P2 ogr mutants. Images PMID:2193911

The transcription factor MTF-1 is essential for basal and heavy metal-induced metallothionein gene expression.

PubMed

Heuchel, R; Radtke, F; Georgiev, O; Stark, G; Aguet, M; Schaffner, W

1994-06-15

We have described and cloned previously a factor (MTF-1) that binds specifically to heavy metal-responsive DNA sequence elements in the enhancer/promoter region of metallothionein genes. MTF-1 is a protein of 72.5 kDa that contains six zinc fingers and multiple domains for transcriptional activation. Here we report the disruption of both alleles of the MTF-1 gene in mouse embryonic stem cells by homologous recombination. The resulting null mutant cell line fails to produce detectable amounts of MTF-1. Moreover, due to the loss of MTF-1, the endogenous metallothionein I and II genes are silent, indicating that MTF-1 is required for both their basal and zinc-induced transcription. In addition to zinc, other heavy metals, including cadmium, copper, nickel and lead, also fail to activate metal-responsive promoters in null mutant cells. However, cotransfection of an MTF-1 expression vector and metal-responsive reporter genes yields strong basal transcription that can be further boosted by zinc treatment of cells. These results demonstrate that MTF-1 is essential for metallothionein gene regulation. Finally, we present evidence that MTF-1 itself is a zinc sensor, which exhibits increased DNA binding activity upon zinc treatment.

Essential roles for Cdx in murine primitive hematopoiesis.

PubMed

Brooke-Bisschop, Travis; Savory, Joanne G A; Foley, Tanya; Ringuette, Randy; Lohnes, David

2017-02-15

The Cdx transcription factors play essential roles in primitive hematopoiesis in the zebrafish where they exert their effects, in part, through regulation of hox genes. Defects in hematopoiesis have also been reported in Cdx mutant murine embryonic stem cell models, however, to date no mouse model reflecting the zebrafish Cdx mutant hematopoietic phenotype has been described. This is likely due, in part, to functional redundancy among Cdx members and the early lethality of Cdx2 null mutants. To circumvent these limitations, we used Cre-mediated conditional deletion to assess the impact of concomitant loss of Cdx1 and Cdx2 on murine primitive hematopoiesis. We found that Cdx1/Cdx2 double mutants exhibited defects in primitive hematopoiesis and yolk sac vasculature concomitant with reduced expression of several genes encoding hematopoietic transcription factors including Scl/Tal1. Chromatin immunoprecipitation analysis revealed that Scl was occupied by Cdx2 in vivo, and Cdx mutant hematopoietic yolk sac differentiation defects could be rescued by expression of exogenous Scl. These findings demonstrate critical roles for Cdx members in murine primitive hematopoiesis upstream of Scl. Copyright © 2017 Elsevier Inc. All rights reserved.

Mice that lack the angiogenesis inhibitor, thrombospondin 2, mount an altered foreign body reaction characterized by increased vascularity

PubMed Central

Kyriakides, Themis R.; Leach, Kathleen J.; Hoffman, Allan S.; Ratner, Buddy D.; Bornstein, Paul

1999-01-01

Disruption of the thrombospondin 2 gene (Thbs2) in mice results in a complex phenotype characterized chiefly by abnormalities in fibroblasts, connective tissues, and blood vessels. Consideration of this phenotype suggested to us that the foreign body reaction (FBR) might be altered in thrombospondin 2 (TSP2)-null mice. To investigate the participation of TSP2 in the FBR, polydimethylsiloxane (PDMS) and oxidized PDMS (ox-PDMS) disks were implanted in TSP2-null and control mice. Growth of TSP2-null and control skin fibroblasts in vitro also was evaluated on both types of disks. Normal fibroblasts grew as a monolayer on both surfaces, but attachment of the cells to ox-PDMS was weak and sensitive to movement. TSP2-null fibroblasts grew as aggregates on both surfaces, and their attachment was further compromised on ox-PDMS. After a 4-week implantation period, both types of PDMS elicited a similar FBR with a collagenous capsule in both TSP2-null and control mice. However, strikingly, the collagenous capsule that formed in TSP2-null mice was highly vascularized and thicker than that formed in normal mice. In addition, abnormally shaped collagen fibers were observed in capsules from mutant mice. These observations indicate that the presence or absence of an extracellular matrix component, TSP2, can influence the nature of the FBR, in particular its vascularity. The expression of TSP2 therefore could represent a molecular target for local inhibitory measures when vascularization of the tissue surrounding an implanted device is desired. PMID:10200282

Thioredoxin-interacting protein (Txnip) is a critical regulator of hepatic glucose production.

PubMed

Chutkow, William A; Patwari, Parth; Yoshioka, Jun; Lee, Richard T

2008-01-25

Thioredoxin-interacting protein (Txnip) has been recently described as a possible link between cellular redox state and metabolism; Txnip binds thioredoxin and inhibits its disulfide reductase activity in vitro, while a naturally occurring strain of Txnip-deficient mice has hyperlipidemia, hypoglycemia, and ketosis exacerbated by fasting. We generated Txnip-null mice to investigate the role of Txnip in glucose homeostasis. Txnip-null mice were hypoglycemic, hypoinsulinemic, and had blunted glucose production following a glucagon challenge, consistent with a central liver glucose-handling defect. Glucose release from isolated Txnip-null hepatocytes was 2-fold lower than wild-type hepatocytes, whereas beta-hydroxybutyrate release was increased 2-fold, supporting an intrinsic defect in hepatocyte glucose metabolism. While hepatocyte-specific gene deletion of Txnip did not alter glucose clearance compared with littermate controls, Txnip expression in the liver was required for maintaining normal fasting glycemia and glucose production. In addition, hepatic overexpression of a Txnip transgene in wild-type mice resulted in elevated serum glucose levels and decreased ketone levels. Liver homogenates from Txnip-null mice had no significant differences in the glutathione oxidation state or in the amount of available thioredoxin. However, overexpression of wild-type Txnip in Txnip-null hepatocytes rescued cellular glucose production, whereas overexpression of a C247S mutant Txnip, which does not bind thioredoxin, had no effect. These data demonstrate that Txnip is required for normal glucose homeostasis in the liver. While available thioredoxin is not changed in Txnip-null mice, the effects of Txnip on glucose homeostasis are abolished by a single cysteine mutation that inhibits binding to thioredoxin.

The vitamin D receptor functions as a transcription regulator in the absence of 1,25-dihydroxyvitamin D3.

PubMed

Lee, Seong Min; Pike, J Wesley

2016-11-01

The vitamin D receptor (VDR) is a critical mediator of the biological actions of 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ). As a nuclear receptor, ligand activation of the VDR leads to the protein's binding to specific sites on the genome that results in the modulation of target gene expression. The VDR is also known to play a role in the hair cycle, an action that appears to be 1,25(OH) 2 D 3 -independent. Indeed, in the absence of the VDR as in hereditary 1,25-dihydroxyvitamin D resistant rickets (HVDRR) both skin defects and alopecia emerge. Recently, we generated a mouse model of HVDRR without alopecia wherein a mutant human VDR lacking 1,25(OH) 2 D 3 -binding activity was expressed in the absence of endogenous mouse VDR. While 1,25(OH) 2 D 3 failed to induce gene expression in these mice, resulting in an extensive skeletal phenotype, the receptor was capable of restoring normal hair cycling. We also noted a level of secondary hyperparathyroidism that was much higher than that seen in the VDR null mouse and was associated with an exaggerated bone phenotype as well. This suggested that the VDR might play a role in parathyroid hormone (PTH) regulation independent of 1,25(OH) 2 D 3 . To evaluate this hypothesis further, we contrasted PTH levels in the HVDRR mouse model with those seen in Cyp27b1 null mice where the VDR was present but the hormone was absent. The data revealed that PTH was indeed higher in Cyp27b1 null mice compared to VDR null mice. To evaluate the mechanism of action underlying such a hypothesis, we measured the expression levels of a number of VDR target genes in the duodena of wildtype mice and in transgenic mice expressing either normal or hormone-binding deficient mutant VDRs. We also compared expression levels of these genes between VDR null mice and Cyp27b1 null mice. In a subset of cases, the expression of VDR target genes was lower in mice containing the VDR as opposed to mice that did not. We suggest that the VDR may function as a selective suppressor/de-repressor of gene expression in the absence of 1,25(OH) 2 D 3 . Copyright © 2015 Elsevier Ltd. All rights reserved.

Leishmania infantum HSP70-II null mutant as candidate vaccine against leishmaniasis: a preliminary evaluation

PubMed Central

2011-01-01

Background Visceral leishmaniasis is the most severe form of leishmaniasis and no effective vaccine exists. The use of live attenuated vaccines is emerging as a promising vaccination strategy. Results In this study, we tested the ability of a Leishmania infantum deletion mutant, lacking both HSP70-II alleles (ΔHSP70-II), to provide protection against Leishmania infection in the L. major-BALB/c infection model. Administration of the mutant line by either intraperitoneal, intravenous or subcutaneous route invariably leads to the production of high levels of NO and the development in mice of type 1 immune responses, as determined by analysis of anti-Leishmania IgG subclasses. In addition, we have shown that ΔHSP70-II would be a safe live vaccine as immunodeficient SCID mice, and hamsters (Mesocricetus auratus), infected with mutant parasites did not develop any sign of pathology. Conclusions The results suggest that the ΔHSP70-II mutant is a promising and safe vaccine, but further studies in more appropriate animal models (hamsters and dogs) are needed to appraise whether this attenuate mutant would be useful as vaccine against visceral leishmaniasis. PMID:21794145

Drosophila melanogaster White Mutant w1118 Undergo Retinal Degeneration

PubMed Central

Ferreiro, María José; Pérez, Coralia; Marchesano, Mariana; Ruiz, Santiago; Caputi, Angel; Aguilera, Pedro; Barrio, Rosa; Cantera, Rafael

2018-01-01

Key scientific discoveries have resulted from genetic studies of Drosophila melanogaster, using a multitude of transgenic fly strains, the majority of which are constructed in a genetic background containing mutations in the white gene. Here we report that white mutant flies from w1118 strain undergo retinal degeneration. We observed also that w1118 mutants have progressive loss of climbing ability, shortened life span, as well as impaired resistance to various forms of stress. Retinal degeneration was abolished by transgenic expression of mini-white+ in the white null background w1118. We conclude that beyond the classical eye-color phenotype, mutations in Drosophila white gene could impair several biological functions affecting parameters like mobility, life span and stress tolerance. Consequently, we suggest caution and attentiveness during the interpretation of old experiments employing white mutant flies and when planning new ones, especially within the research field of neurodegeneration and neuroprotection. We also encourage that the use of w1118 strain as a wild-type control should be avoided. PMID:29354028

Loss of Atg16 delays the alcohol-induced sedation response via regulation of Corazonin neuropeptide production in Drosophila.

PubMed

Varga, Kata; Nagy, Péter; Arsikin Csordás, Katarina; Kovács, Attila L; Hegedűs, Krisztina; Juhász, Gábor

2016-10-06

Autophagy defects lead to the buildup of damaged proteins and organelles, reduced survival during starvation and infections, hypersensitivity to stress and toxic substances, and progressive neurodegeneration. Here we show that, surprisingly, Drosophila mutants lacking the core autophagy gene Atg16 are not only defective in autophagy but also exhibit increased resistance to the sedative effects of ethanol, unlike Atg7 or Atg3 null mutant flies. This mutant phenotype is rescued by the re-expression of Atg16 in Corazonin (Crz)-producing neurosecretory cells that are known to promote the sedation response during ethanol exposure, and RNAi knockdown of Atg16 specifically in these cells also delays the onset of ethanol-induced coma. We find that Atg16 and Crz colocalize within these neurosecretory cells, and both Crz protein and mRNA levels are decreased in Atg16 mutant flies. Thus, Atg16 promotes Crz production to ensure a proper organismal sedation response to ethanol.

Drosophila melanogaster White Mutant w 1118 Undergo Retinal Degeneration.

PubMed

Ferreiro, María José; Pérez, Coralia; Marchesano, Mariana; Ruiz, Santiago; Caputi, Angel; Aguilera, Pedro; Barrio, Rosa; Cantera, Rafael

2017-01-01

Key scientific discoveries have resulted from genetic studies of Drosophila melanogaster , using a multitude of transgenic fly strains, the majority of which are constructed in a genetic background containing mutations in the white gene. Here we report that white mutant flies from w 1118 strain undergo retinal degeneration. We observed also that w 1118 mutants have progressive loss of climbing ability, shortened life span, as well as impaired resistance to various forms of stress. Retinal degeneration was abolished by transgenic expression of mini-white + in the white null background w 1118 . We conclude that beyond the classical eye-color phenotype, mutations in Drosophila white gene could impair several biological functions affecting parameters like mobility, life span and stress tolerance. Consequently, we suggest caution and attentiveness during the interpretation of old experiments employing white mutant flies and when planning new ones, especially within the research field of neurodegeneration and neuroprotection. We also encourage that the use of w 1118 strain as a wild-type control should be avoided.

A missense mutation in Grm6 reduces but does not eliminate mGluR6 expression or rod depolarizing bipolar cell function.

PubMed

Peachey, Neal S; Hasan, Nazarul; FitzMaurice, Bernard; Burrill, Samantha; Pangeni, Gobinda; Karst, Son Yong; Reinholdt, Laura; Berry, Melissa L; Strobel, Marge; Gregg, Ronald G; McCall, Maureen A; Chang, Bo

2017-08-01

GRM6 encodes the metabotropic glutamate receptor 6 (mGluR6) used by retinal depolarizing bipolar cells (DBCs). Mutations in GRM6 lead to DBC dysfunction and underlie the human condition autosomal recessive complete congenital stationary night blindness. Mouse mutants for Grm6 are important models for this condition. Here we report a new Grm6 mutant, identified in an electroretinogram (ERG) screen of mice maintained at The Jackson Laboratory. The Grm6 nob8 mouse has a reduced-amplitude b-wave component of the ERG, which reflects light-evoked DBC activity. Sequencing identified a missense mutation that converts a highly conserved methionine within the ligand binding domain to leucine (p.Met66Leu). Consistent with prior studies of Grm6 mutant mice, the laminar size and structure in the Grm6 nob8 retina were comparable to control. The Grm6 nob8 phenotype is distinguished from other Grm6 mutants that carry a null allele by a reduced but not absent ERG b-wave, decreased but present expression of mGluR6 at DBC dendritic tips, and mislocalization of mGluR6 to DBC somas. Consistent with a reduced but not absent b-wave, there were a subset of retinal ganglion cells whose responses to light onset have times to peak within the range of those in control retinas. These data indicate that the p.Met66Leu mutant mGluR6 is trafficked less than control. However, the mGluR6 that is localized to the DBC dendritic tips is able to initiate DBC signal transduction. The Grm6 nob8 mouse extends the Grm6 allelic series and will be useful for elucidating the role of mGluR6 in DBC signal transduction and in human disease. NEW & NOTEWORTHY This article describes a mouse model of the human disease complete congenital stationary night blindness in which the mutation reduces but does not eliminate GRM6 expression and bipolar cell function, a distinct phenotype from that seen in other Grm6 mouse models.

Osteogenesis imperfecta type I: Molecular heterogeneity for COL1A1 null alleles of type I collagen

DOE Office of Scientific and Technical Information (OSTI.GOV)

Willing, M.C.; Deschenes, S.P.; Pitts, S.H.

Osteogenesis imperfecta (OI) type I is the mildest form of inherited brittle-bone disease. Dermal fibroblasts from most affected individuals produce about half the usual amount of type I procollagen, as a result of a COL1A1 {open_quotes}null{close_quotes} allele. Using PCR amplification of genomic DNA from affected individuals, followed by denaturing gradient gel electrophoresis (DGGE) and SSCP, we identified seven different COL1A1 gene mutations in eight unrelated families with OI type I. Three families have single nucleotide substitutions that alter 5{prime} donor splice sites; two of these unrelated families have the same mutation. One family has a point mutation, in an exon,more » that creates a premature termination codon, and four have small deletions or insertions, within exons, that create translational frameshifts and new termination codons downstream of the mutation sites. Each mutation leads to both marked reduction in steady-state levels of mRNA from the mutant allele and a quantitative decrease in type I procollagen production. Our data demonstrate that different molecular mechanisms that have the same effect on type I collagen production result in the same clinical phenotype. 58 refs., 4 figs., 1 tab.« less

Clustering of Genetically Defined Allele Classes in the Caenorhabditis elegans DAF-2 Insulin/IGF-1 Receptor

PubMed Central

Patel, Dhaval S.; Garza-Garcia, Acely; Nanji, Manoj; McElwee, Joshua J.; Ackerman, Daniel; Driscoll, Paul C.; Gems, David

2008-01-01

The DAF-2 insulin/IGF-1 receptor regulates development, metabolism, and aging in the nematode Caenorhabditis elegans. However, complex differences among daf-2 alleles complicate analysis of this gene. We have employed epistasis analysis, transcript profile analysis, mutant sequence analysis, and homology modeling of mutant receptors to understand this complexity. We define an allelic series of nonconditional daf-2 mutants, including nonsense and deletion alleles, and a putative null allele, m65. The most severe daf-2 alleles show incomplete suppression by daf-18(0) and daf-16(0) and have a range of effects on early development. Among weaker daf-2 alleles there exist distinct mutant classes that differ in epistatic interactions with mutations in other genes. Mutant sequence analysis (including 11 newly sequenced alleles) reveals that class 1 mutant lesions lie only in certain extracellular regions of the receptor, while class 2 (pleiotropic) and nonconditional missense mutants have lesions only in the ligand-binding pocket of the receptor ectodomain or the tyrosine kinase domain. Effects of equivalent mutations on the human insulin receptor suggest an altered balance of intracellular signaling in class 2 alleles. These studies consolidate and extend our understanding of the complex genetics of daf-2 and its underlying molecular biology. PMID:18245374

The RNA Binding Protein Tudor-SN Is Essential for Stress Tolerance and Stabilizes Levels of Stress-Responsive mRNAs Encoding Secreted Proteins in Arabidopsis[C][W][OA

PubMed Central

dit Frey, Nicolas Frei; Muller, Philippe; Jammes, Fabien; Kizis, Dimosthenis; Leung, Jeffrey; Perrot-Rechenmann, Catherine; Bianchi, Michele Wolfe

2010-01-01

Tudor-SN (TSN) copurifies with the RNA-induced silencing complex in animal cells where, among other functions, it is thought to act on mRNA stability via the degradation of specific dsRNA templates. In plants, TSN has been identified biochemically as a cytoskeleton-associated RNA binding activity. In eukaryotes, it has recently been identified as a conserved primary target of programmed cell death–associated proteolysis. We have investigated the physiological role of TSN by isolating null mutations for two homologous genes in Arabidopsis thaliana. The double mutant tsn1 tsn2 displays only mild growth phenotypes under nonstress conditions, but germination, growth, and survival are severely affected under high salinity stress. Either TSN1 or TSN2 alone can complement the double mutant, indicating their functional redundancy. TSN accumulates heterogeneously in the cytosol and relocates transiently to a diffuse pattern in response to salt stress. Unexpectedly, stress-regulated mRNAs encoding secreted proteins are significantly enriched among the transcripts that are underrepresented in tsn1 tsn2. Our data also reveal that TSN is important for RNA stability of its targets. These findings show that TSN is essential for stress tolerance in plants and implicate TSN in new, potentially conserved mechanisms acting on mRNAs entering the secretory pathway. PMID:20484005

Regulated nuclear trafficking of rpL10A mediated by NIK1 represents a defense strategy of plant cells against virus.

PubMed

Carvalho, Claudine M; Santos, Anésia A; Pires, Silvana R; Rocha, Carolina S; Saraiva, Daniela I; Machado, João Paulo B; Mattos, Eliciane C; Fietto, Luciano G; Fontes, Elizabeth P B

2008-12-01

The NSP-interacting kinase (NIK) receptor-mediated defense pathway has been identified recently as a virulence target of the geminivirus nuclear shuttle protein (NSP). However, the NIK1-NSP interaction does not fit into the elicitor-receptor model of resistance, and hence the molecular mechanism that links this antiviral response to receptor activation remains obscure. Here, we identified a ribosomal protein, rpL10A, as a specific partner and substrate of NIK1 that functions as an immediate downstream effector of NIK1-mediated response. Phosphorylation of cytosolic rpL10A by NIK1 redirects the protein to the nucleus where it may act to modulate viral infection. While ectopic expression of normal NIK1 or a hyperactive NIK1 mutant promotes the accumulation of phosphorylated rpL10A within the nuclei, an inactive NIK1 mutant fails to redirect the protein to the nuclei of co-transfected cells. Likewise, a mutant rpL10A defective for NIK1 phosphorylation is not redirected to the nucleus. Furthermore, loss of rpL10A function enhances susceptibility to geminivirus infection, resembling the phenotype of nik1 null alleles. We also provide evidence that geminivirus infection directly interferes with NIK1-mediated nuclear relocalization of rpL10A as a counterdefensive measure. However, the NIK1-mediated defense signaling neither activates RNA silencing nor promotes a hypersensitive response but inhibits plant growth and development. Although the virulence function of the particular geminivirus NSP studied here overcomes this layer of defense in Arabidopsis, the NIK1-mediated signaling response may be involved in restricting the host range of other viruses.

A novel mutation in the SLC25A15 gene in a Turkish patient with HHH syndrome: Functional analysis of the mutant protein

PubMed Central

Ersoy Tunalı, Nagehan; Marobbio, Carlo M.T.; Tiryakioğlu, N. Ozan; Punzi, Giuseppe; Saygılı, Seha K.; Önal, Hasan; Palmieri, Ferdinando

2014-01-01

The hyperornithinemia–hyperammonemia–homocitrullinuria syndrome is a rare autosomal recessive disorder caused by the functional deficiency of the mitochondrial ornithine transporter 1 (ORC1). ORC1 is encoded by the SLC25A15 gene and catalyzes the transport of cytosolic ornithine into mitochondria in exchange for citrulline. Although the age of onset and the severity of the symptoms vary widely, the disease usually manifests in early infancy. The typical clinical features include protein intolerance, lethargy, episodic confusion, cerebellar ataxia, seizures and mental retardation. In this study, we identified a novel p.Ala15Val (c.44C > T) mutation by genomic DNA sequencing in a Turkish child presenting severe tantrum, confusion, gait disturbances and loss of speech abilities in addition to hyperornithinemia, hyperammonemia and homocitrullinuria. One hundred Turkish control chromosomes did not possess this variant. The functional effect of the novel mutation was assessed by both complementation of the yeast ORT1 null mutant and transport assays. Our study demonstrates that the A15V mutation dramatically interferes with the transport properties of ORC1 since it was shown to inhibit ornithine transport nearly completely. PMID:24721342

Fission yeast cdc24(+) encodes a novel replication factor required for chromosome integrity.

PubMed

Gould, K L; Burns, C G; Feoktistova, A; Hu, C P; Pasion, S G; Forsburg, S L

1998-07-01

A mutation within the Schizosaccharomyces pombe cdc24(+) gene was identified previously in a screen for cell division cycle mutants and the cdc24(+) gene was determined to be essential for S phase in this yeast. We have isolated the cdc24(+) gene by complementation of a new temperature-sensitive allele of the gene, cdc24-G1. The DNA sequence predicts the presence of an open reading frame punctuated by six introns which encodes a pioneer protein of 58 kD. A cdc24 null mutant was generated by homologous recombination. Haploid cells lacking cdc24(+) are inviable, indicating that cdc24(+) is an essential gene. The transcript of cdc24(+) is present at constant levels throughout the cell cycle. Cells lacking cdc24(+) function show a checkpoint-dependent arrest with a 2N DNA content, indicating a block late in S phase. Arrest is accompanied by a rapid loss of viability and chromosome breakage. An S. pombe homolog of the replicative DNA helicase DNA2 of S. cerevisiae suppresses cdc24. These results suggest that Cdc24p plays a role in the progression of normal DNA replication and is required to maintain genomic integrity.

Fission yeast cdc24(+) encodes a novel replication factor required for chromosome integrity.

PubMed Central

Gould, K L; Burns, C G; Feoktistova, A; Hu, C P; Pasion, S G; Forsburg, S L

1998-01-01

A mutation within the Schizosaccharomyces pombe cdc24(+) gene was identified previously in a screen for cell division cycle mutants and the cdc24(+) gene was determined to be essential for S phase in this yeast. We have isolated the cdc24(+) gene by complementation of a new temperature-sensitive allele of the gene, cdc24-G1. The DNA sequence predicts the presence of an open reading frame punctuated by six introns which encodes a pioneer protein of 58 kD. A cdc24 null mutant was generated by homologous recombination. Haploid cells lacking cdc24(+) are inviable, indicating that cdc24(+) is an essential gene. The transcript of cdc24(+) is present at constant levels throughout the cell cycle. Cells lacking cdc24(+) function show a checkpoint-dependent arrest with a 2N DNA content, indicating a block late in S phase. Arrest is accompanied by a rapid loss of viability and chromosome breakage. An S. pombe homolog of the replicative DNA helicase DNA2 of S. cerevisiae suppresses cdc24. These results suggest that Cdc24p plays a role in the progression of normal DNA replication and is required to maintain genomic integrity. PMID:9649516

Comparison of the Gene Expression Profiles from Normal and Fgfrl1 Deficient Mouse Kidneys Reveals Downstream Targets of Fgfrl1 Signaling

PubMed Central

Gerber, Simon D.; Amann, Ruth; Wyder, Stefan; Trueb, Beat

2012-01-01

Fgfrl1 (fibroblast growth factor receptor-like 1) is a transmembrane receptor that is essential for the development of the metanephric kidney. It is expressed in all nascent nephrogenic structures and in the ureteric bud. Fgfrl1 null mice fail to develop the metanephric kidneys. Mutant kidney rudiments show a dramatic reduction of ureteric branching and a lack of mesenchymal-to-epithelial transition. Here, we compared the expression profiles of wildtype and Fgfrl1 mutant kidneys to identify genes that act downstream of Fgfrl1 signaling during the early steps of nephron formation. We detected 56 differentially expressed transcripts with 2-fold or greater reduction, among them many genes involved in Fgf, Wnt, Bmp, Notch, and Six/Eya/Dach signaling. We validated the microarray data by qPCR and whole-mount in situ hybridization and showed the expression pattern of candidate genes in normal kidneys. Some of these genes might play an important role during early nephron formation. Our study should help to define the minimal set of genes that is required to form a functional nephron. PMID:22432025

New tools for targeted disruption of cholinergic synaptic transmission in Drosophila melanogaster.

PubMed

Mejia, Monica; Heghinian, Mari D; Marí, Frank; Godenschwege, Tanja A

2013-01-01

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels. The α7 subtype of nAChRs is involved in neurological pathologies such as Parkinson's disease, Alzheimer's disease, addiction, epilepsy and autism spectrum disorders. The Drosophila melanogaster α7 (Dα7) has the closest sequence homology to the vertebrate α7 subunit and it can form homopentameric receptors just as the vertebrate counterpart. The Dα7 subunits are essential for the function of the Giant Fiber circuit, which mediates the escape response of the fly. To further characterize the receptor function, we generated different missense mutations in the Dα7 nAChR's ligand binding domain. We characterized the effects of targeted expression of two UAS-constructs carrying a single mutation, D197A and Y195T, as well as a UAS-construct carrying a triple D77T, L117Q, I196P mutation in a Dα7 null mutant and in a wild type background. Expression of the triple mutation was able to restore the function of the circuit in Dα7 null mutants and had no disruptive effects when expressed in wild type. In contrast, both single mutations severely disrupted the synaptic transmission of Dα7-dependent but not glutamatergic or gap junction dependent synapses in wild type background, and did not or only partially rescued the synaptic defects of the null mutant. These observations are consistent with the formation of hybrid receptors, consisting of D197A or Y195T subunits and wild type Dα7 subunits, in which the binding of acetylcholine or acetylcholine-induced conformational changes of the Dα7 receptor are altered and causes inhibition of cholinergic responses. Thus targeted expression of D197A or Y195T can be used to selectively disrupt synaptic transmission of Dα7-dependent synapses in neuronal circuits. Hence, these constructs can be used as tools to study learning and memory or addiction associated behaviors by allowing the manipulation of neuronal processing in the circuits without affecting other cellular signaling.

New Tools for Targeted Disruption of Cholinergic Synaptic Transmission in Drosophila melanogaster

PubMed Central

Mejia, Monica; Heghinian, Mari D.; Marí, Frank; Godenschwege, Tanja A.

2013-01-01

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels. The α7 subtype of nAChRs is involved in neurological pathologies such as Parkinson’s disease, Alzheimer’s disease, addiction, epilepsy and autism spectrum disorders. The Drosophila melanogaster α7 (Dα7) has the closest sequence homology to the vertebrate α7 subunit and it can form homopentameric receptors just as the vertebrate counterpart. The Dα7 subunits are essential for the function of the Giant Fiber circuit, which mediates the escape response of the fly. To further characterize the receptor function, we generated different missense mutations in the Dα7 nAChR’s ligand binding domain. We characterized the effects of targeted expression of two UAS-constructs carrying a single mutation, D197A and Y195T, as well as a UAS-construct carrying a triple D77T, L117Q, I196P mutation in a Dα7 null mutant and in a wild type background. Expression of the triple mutation was able to restore the function of the circuit in Dα7 null mutants and had no disruptive effects when expressed in wild type. In contrast, both single mutations severely disrupted the synaptic transmission of Dα7-dependent but not glutamatergic or gap junction dependent synapses in wild type background, and did not or only partially rescued the synaptic defects of the null mutant. These observations are consistent with the formation of hybrid receptors, consisting of D197A or Y195T subunits and wild type Dα7 subunits, in which the binding of acetylcholine or acetylcholine-induced conformational changes of the Dα7 receptor are altered and causes inhibition of cholinergic responses. Thus targeted expression of D197A or Y195T can be used to selectively disrupt synaptic transmission of Dα7-dependent synapses in neuronal circuits. Hence, these constructs can be used as tools to study learning and memory or addiction associated behaviors by allowing the manipulation of neuronal processing in the circuits without affecting other cellular signaling. PMID:23737994

Reduced wheel running and blunted effects of voluntary exercise in LPA1-null mice: the importance of assessing the amount of running in transgenic mice studies.

PubMed

Castilla-Ortega, Estela; Rosell-Valle, Cristina; Blanco, Eduardo; Pedraza, Carmen; Chun, Jerold; Rodríguez de Fonseca, Fernando; Estivill-Torrús, Guillermo; Santín, Luis J

2013-11-01

This work was aimed to assess whether voluntary exercise rescued behavioral and hippocampal alterations in mice lacking the lysophosphatidic acid LPA1 receptor (LPA1-null mice), studying the potential relationship between the amount of exercise performed and its effects. Normal and LPA1-null mice underwent 23 days of free wheel running and were tested for open-field behavior and adult hippocampal neurogenesis (cell proliferation, immature neurons, cell survival). Running decreased anxiety-like behavior in both genotypes but increased exploration only in the normal mice. While running affected all neurogenesis-related measures in normal mice (especially in the suprapyramidal blade of the dentate gyrus), only a moderate increase in cell survival was found in the mutants. Importantly, the LPA1-nulls showed notably reduced running. Analysis suggested that defective running in the LPA1-null mice could contribute to explain the scarce benefit of the voluntary exercise treatment. On the other hand, a literature review revealed that voluntary exercise is frequently used to modulate behavior and the hippocampus in transgenic mice, but half of the studies did not assess the quantity of running, overlooking any potential running impairments. This study adds evidence to the relevance of the quantity of exercise performed, emphasizing the importance of its assessment in transgenic mice research. Copyright © 2013 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

Ubiad1 Is an Antioxidant Enzyme that Regulates eNOS Activity by CoQ10 Synthesis

PubMed Central

Mugoni, Vera; Postel, Ruben; Catanzaro, Valeria; De Luca, Elisa; Turco, Emilia; Digilio, Giuseppe; Silengo, Lorenzo; Murphy, Michael P.; Medana, Claudio; Stainier, Didier Y.R.; Bakkers, Jeroen; Santoro, Massimo M.

2013-01-01

Summary Protection against oxidative damage caused by excessive reactive oxygen species (ROS) by an antioxidant network is essential for the health of tissues, especially in the cardiovascular system. Here, we identified a gene with important antioxidant features by analyzing a null allele of zebrafish ubiad1, called barolo (bar). bar mutants show specific cardiovascular failure due to oxidative stress and ROS-mediated cellular damage. Human UBIAD1 is a nonmitochondrial prenyltransferase that synthesizes CoQ10 in the Golgi membrane compartment. Loss of UBIAD1 reduces the cytosolic pool of the antioxidant CoQ10 and leads to ROS-mediated lipid peroxidation in vascular cells. Surprisingly, inhibition of eNOS prevents Ubiad1-dependent cardiovascular oxidative damage, suggesting a crucial role for this enzyme and nonmitochondrial CoQ10 in NO signaling. These findings identify UBIAD1 as a nonmitochondrial CoQ10-forming enzyme with specific cardiovascular protective function via the modulation of eNOS activity. PMID:23374346

The Dictyostelium MAP kinase kinase DdMEK1 regulates chemotaxis and is essential for chemoattractant-mediated activation of guanylyl cyclase.

PubMed Central

Ma, H; Gamper, M; Parent, C; Firtel, R A

1997-01-01

We have identified a MAP kinase kinase (DdMEK1) that is required for proper aggregation in Dictyostelium. Null mutations produce extremely small aggregate sizes, resulting in the formation of slugs and terminal fruiting bodies that are significantly smaller than those of wild-type cells. Time-lapse video microscopy and in vitro assays indicate that the cells are able to produce cAMP waves that move through the aggregation domains. However, these cells are unable to undergo chemotaxis properly during aggregation in response to the chemoattractant cAMP or activate guanylyl cyclase, a known regulator of chemotaxis in Dictyostelium. The activation of guanylyl cyclase in response to osmotic stress is, however, normal. Expression of putative constitutively active forms of DdMEK1 in a ddmek1 null background is capable, at least partially, of complementing the small aggregate size defect and the ability to activate guanylyl cyclase. However, this does not result in constitutive activation of guanylyl cyclase, suggesting that DdMEK1 activity is necessary, but not sufficient, for cAMP activation of guanylyl cyclase. Analysis of a temperature-sensitive DdMEK1 mutant suggests that DdMEK1 activity is required throughout aggregation at the time of guanylyl cyclase activation, but is not essential for proper morphogenesis during the later multicellular stages. The activation of the MAP kinase ERK2, which is essential for chemoattractant activation of adenylyl cyclase, is not affected in ddmek1 null strains, indicating that DdMEK1 does not regulate ERK2 and suggesting that at least two independent MAP kinase cascades control aggregation in Dictyostelium. PMID:9250676

Combined glutathione S transferase M1/T1 null genotypes is associated with type 2 diabetes mellitus

PubMed Central

POROJAN, MIHAI D.; BALA, CORNELIA; ILIES, ROXANA; CATANA, ANDREEA; POPP, RADU A.; DUMITRASCU, DAN L.

2015-01-01

Background Due to new genetic insights, a considerably large number of genes and polymorphic gene variants are screened and linked with the complex pathogenesis of type 2 diabetes (DM). Our study aimed to investigate the association between the two isoforms of the glutathione S-transferase genes (Glutathione S transferase isoemzyme type M1- GSTM1 and Glutathione S transferase isoemzyme type T1-GSTT1) and the prevalence of DM in the Northern Romanian population. Methods We conducted a cross-sectional, randomized, case-control study evaluating the frequency of GSTM1 and GSTT1 null alleles in patients diagnosed with DM. A total of 106 patients diagnosed with DM and 124 healthy controls were included in the study. GSTM1 and GSTT1 null alleles genotyping was carried out using Multiplex PCR amplification of relevant gene fragments, followed by gel electrophoresis analysis of the resulting amplicons. Results Molecular analysis did not reveal an increased frequency of the null GSTM1 and GSTT1 alleles (mutant genotypes) respectively in the DM group compared to controls (p=0.171, OR=1.444 CI=0.852–2.447; p=0.647, OR=0.854, CI=0.436–1.673). Nevertheless, the combined GSTM1/GSTT1 null genotypes were statistically significantly higher in DM patients compared to control subjects (p=0.0021, OR=0.313, CI=0.149–0.655) Conclusions The main finding of our study is that the combined, double GSTM1/GSTT1 null genotypes are to be considered among the polymorphic genetic risk factors for type 2 DM. PMID:26528065

Annealing of Complementary DNA Sequences During Double-Strand Break Repair in Drosophila Is Mediated by the Ortholog of SMARCAL1.

PubMed

Holsclaw, Julie Korda; Sekelsky, Jeff

2017-05-01

DNA double-strand breaks (DSBs) pose a serious threat to genomic integrity. If unrepaired, they can lead to chromosome fragmentation and cell death. If repaired incorrectly, they can cause mutations and chromosome rearrangements. DSBs are repaired using end-joining or homology-directed repair strategies, with the predominant form of homology-directed repair being synthesis-dependent strand annealing (SDSA). SDSA is the first defense against genomic rearrangements and information loss during DSB repair, making it a vital component of cell health and an attractive target for chemotherapeutic development. SDSA has also been proposed to be the primary mechanism for integration of large insertions during genome editing with CRISPR/Cas9. Despite the central role for SDSA in genome stability, little is known about the defining step: annealing. We hypothesized that annealing during SDSA is performed by the annealing helicase SMARCAL1, which can anneal RPA-coated single DNA strands during replication-associated DNA damage repair. We used unique genetic tools in Drosophila melanogaster to test whether the fly ortholog of SMARCAL1, Marcal1, mediates annealing during SDSA. Repair that requires annealing is significantly reduced in Marcal1 null mutants in both synthesis-dependent and synthesis-independent (single-strand annealing) assays. Elimination of the ATP-binding activity of Marcal1 also reduced annealing-dependent repair, suggesting that the annealing activity requires translocation along DNA. Unlike the null mutant, however, the ATP-binding defect mutant showed reduced end joining, shedding light on the interaction between SDSA and end-joining pathways. Copyright © 2017 by the Genetics Society of America.

Analysis of a homologue of the adducin head gene which is a potential target for the Dictyostelium STAT protein Dd-STATa.

PubMed

Aoshima, Ryota; Hiraoka, Rieko; Shimada, Nao; Kawata, Takefumi

2006-01-01

A Dd-STATa-null mutant, which is defective in expression of a Dictyostelium homologue of the metazoan STAT (signal transducers and activators of transcription) proteins, fails to culminate and this phenotype correlates with the loss of expression of various prestalk (pst) genes. An EST clone, SSK395, encodes a close homologue of the adducin amino-terminal head domain and harbors a putative actin-binding domain. We fused promoter fragments of the cognate gene, ahhA (adducin head homologue A), to a lacZ reporter and determined their expression pattern. The proximal promoter region is necessary for the expression of ahhA at an early (pre-aggregative) stage of development and this expression is Dd-STATa independent. The distal promoter region is necessary for expression at later stages of development in pstA cells, of the slug and in upper cup and pstAB cells during culmination. The distal region is partly Dd-STATa-dependent. The ahhA-null mutant develops almost normally until culmination, but it forms slanting culminants that tend to collapse on to the substratum. The mutant also occasionally forms fruiting bodies with swollen papillae and with constrictions in the prestalk region. The AhhA protein localizes to the stalk tube entrance and also to the upper cup cells and in cells at or near to the constricted region where an F-actin ring is localized. These findings suggest that Dd-STATa regulates culmination and may be necessary for straight downward elongation of the stalk, via the putative actin-binding protein AhhA.

Mutational analysis of RsrA, a zinc-binding anti-sigma factor with a thiol-disulphide redox switch.

PubMed

Paget, M S; Bae, J B; Hahn, M Y; Li, W; Kleanthous, C; Roe, J H; Buttner, M J

2001-02-01

In the Gram-positive bacterium, Streptomyces coelicolor A3(2), expression of the thioredoxin system is modulated by a sigma factor called sigmaR in response to changes in the cytoplasmic thiol-disulphide status, and the activity of sigmaR is controlled post-translationally by an anti-sigma factor, RsrA. In vitro, the anti-sigma factor activity of RsrA, which contains seven cysteines, correlates with its thiol-disulphide redox status. Here, we investigate the function of RsrA in vivo. A constructed rsrA null mutant had very high constitutive levels of disulphide reductase activity and sigmaR-dependent transcription, confirming that RsrA is a negative regulator of sigmaR and a key sensor of thiol-disulphide status. Targeted mutagenesis revealed that three of the seven cysteines in RsrA (C11, C41 and C44) were essential for anti-sigma factor activity and that a mutant RsrA protein containing only these three cysteines was active and still redox sensitive in vivo. We also show that RsrA is a metalloprotein, containing near-stoichiometric amounts of zinc. On the basis of these data, we propose that a thiol-disulphide redox switch is formed between two of C11, C41 and C44, and that all three residues play an essential role in anti-sigma factor activity in their reduced state, perhaps by acting as ligands for zinc. Unexpectedly, rsrA null mutants were blocked in sporulation, probably as a consequence of an increase in the level of free sigmaR.

Alanine scan of core positions in ubiquitin reveals links between dynamics, stability, and function

PubMed Central

Lee, Shirley Y.; Pullen, Lester; Virgil, Daniel J.; Castañeda, Carlos A.; Abeykoon, Dulith; Bolon, Daniel N. A.; Fushman, David

2014-01-01

Mutations at solvent inaccessible core positions in proteins can impact function through many biophysical mechanisms including alterations to thermodynamic stability and protein dynamics. As these properties of proteins are difficult to investigate, the impacts of core mutations on protein function are poorly understood for most systems. Here, we determined the effects of alanine mutations at all 15 core positions in ubiquitin on function in yeast. The majority (13 of 15) of alanine substitutions supported yeast growth as the sole ubiquitin. The two null mutants (I30A and L43A) were both less stable to temperature-induced unfolding in vitro than wild-type, but were well folded at physiological temperatures. Heteronuclear NMR studies indicated that the L43A mutation reduces temperature stability while retaining a ground-state structure similar to wild-type. This structure enables L43A to bind to common ubiquitin receptors in vitro. Many of the core alanine ubiquitin mutants, including one of the null variants (I30A), exhibited an increased accumulation of high molecular weight species, suggesting that these mutants caused a defect in the processing of ubiquitin-substrate conjugates. In contrast, L43A exhibited a unique accumulation pattern with reduced levels of high molecular weight species and undetectable levels of free ubiquitin. When conjugation to other proteins was blocked, L43A ubiquitin accumulated as free ubiquitin in yeast. Based on these findings we speculate that ubiquitin's stability to unfolding may be required for efficient recycling during proteasome-mediated substrate degradation. PMID:24361330

LINKING GABAA RECEPTOR SUBUNITS TO ALCOHOL-INDUCED CONDITIONED TASTE AVERSION AND RECOVERY FROM ACUTE ALCOHOL INTOXICATION

PubMed Central

Blednov, Y.A.; Benavidez, J.M.; Black, M.; Chandra, D.; Homanics, G.E.; Rudolph, U.; Harris, R.A.

2012-01-01

GABA type A receptors (GABAA-R) are important for ethanol actions and it is of interest to link individual subunits with specific ethanol behaviors. We studied null mutant mice for six different GABAA-R subunits (α1, α2, α3, α4, α5 and δ). Only mice lacking the α2 subunit showed reduction of conditioned taste aversion (CTA) to ethanol. These results are in agreement with data from knock-in mice with mutation of the ethanol-sensitive site in the α2-subunit (Blednov et al., 2011) and indicate this aversive property of ethanol is dependent on ethanol action on α2-containing GABAA-R. Deletion of the α2-subunit led to faster recovery whereas absence of the α3-subunit slowed recovery from ethanol-induced incoordination (rotarod). Deletion of the other four subunits did not affect this behavior. Similar changes in this behavior for the α2 and α3 null mutants were found for flurazepam motor-incoordination. However, no differences in recovery were found in motor-incoordinating effects of an α1-selective modulator (zolpidem) or an α4-selective agonist (gaboxadol). Therefore, recovery of rotarod incoordination is under control of two GABAA-R subunits: α2 and α3. For motor activity, α3 null mice demonstrated higher activation by ethanol (1 g/kg) whereas both α2 and α3 (-/-) knockout mice were less sensitive to ethanol-induced reduction of motor activity (1.5 g/kg). These studies demonstrate that the effects of ethanol at GABAergic synapses containing α2 subunit are important for specific behavioral effects of ethanol which may be relevant to the genetic linkage of the α2 subunit with human alcoholism. PMID:23147414

Expression pattern of two paralogs encoding cinnamyl alcohol dehydrogenases in Arabidopsis. Isolation and characterization of the corresponding mutants.

PubMed

Sibout, Richard; Eudes, Aymerick; Pollet, Brigitte; Goujon, Thomas; Mila, Isabelle; Granier, Fabienne; Séguin, Armand; Lapierre, Catherine; Jouanin, Lise

2003-06-01

Studying Arabidopsis mutants of the phenylpropanoid pathway has unraveled several biosynthetic steps of monolignol synthesis. Most of the genes leading to monolignol synthesis have been characterized recently in this herbaceous plant, except those encoding cinnamyl alcohol dehydrogenase (CAD). We have used the complete sequencing of the Arabidopsis genome to highlight a new view of the complete CAD gene family. Among nine AtCAD genes, we have identified the two distinct paralogs AtCAD-C and AtCAD-D, which share 75% identity and are likely to be involved in lignin biosynthesis in other plants. Northern, semiquantitative restriction fragment-length polymorphism-reverse transcriptase-polymerase chain reaction and western analysis revealed that AtCAD-C and AtCAD-D mRNA and protein ratios were organ dependent. Promoter activities of both genes are high in fibers and in xylem bundles. However, AtCAD-C displayed a larger range of sites of expression than AtCAD-D. Arabidopsis null mutants (Atcad-D and Atcad-C) corresponding to both genes were isolated. CAD activities were drastically reduced in both mutants, with a higher impact on sinapyl alcohol dehydrogenase activity (6% and 38% of residual sinapyl alcohol dehydrogenase activities for Atcad-D and Atcad-C, respectively). Only Atcad-D showed a slight reduction in Klason lignin content and displayed modifications of lignin structure with a significant reduced proportion of conventional S lignin units in both stems and roots, together with the incorporation of sinapaldehyde structures ether linked at Cbeta. These results argue for a substantial role of AtCAD-D in lignification, and more specifically in the biosynthesis of sinapyl alcohol, the precursor of S lignin units.

Expression Pattern of Two Paralogs Encoding Cinnamyl Alcohol Dehydrogenases in Arabidopsis. Isolation and Characterization of the Corresponding Mutants1

PubMed Central

Sibout, Richard; Eudes, Aymerick; Pollet, Brigitte; Goujon, Thomas; Mila, Isabelle; Granier, Fabienne; Séguin, Armand; Lapierre, Catherine; Jouanin, Lise

2003-01-01

Studying Arabidopsis mutants of the phenylpropanoid pathway has unraveled several biosynthetic steps of monolignol synthesis. Most of the genes leading to monolignol synthesis have been characterized recently in this herbaceous plant, except those encoding cinnamyl alcohol dehydrogenase (CAD). We have used the complete sequencing of the Arabidopsis genome to highlight a new view of the complete CAD gene family. Among nine AtCAD genes, we have identified the two distinct paralogs AtCAD-C and AtCAD-D, which share 75% identity and are likely to be involved in lignin biosynthesis in other plants. Northern, semiquantitative restriction fragment-length polymorphism-reverse transcriptase-polymerase chain reaction and western analysis revealed that AtCAD-C and AtCAD-D mRNA and protein ratios were organ dependent. Promoter activities of both genes are high in fibers and in xylem bundles. However, AtCAD-C displayed a larger range of sites of expression than AtCAD-D. Arabidopsis null mutants (Atcad-D and Atcad-C) corresponding to both genes were isolated. CAD activities were drastically reduced in both mutants, with a higher impact on sinapyl alcohol dehydrogenase activity (6% and 38% of residual sinapyl alcohol dehydrogenase activities for Atcad-D and Atcad-C, respectively). Only Atcad-D showed a slight reduction in Klason lignin content and displayed modifications of lignin structure with a significant reduced proportion of conventional S lignin units in both stems and roots, together with the incorporation of sinapaldehyde structures ether linked at Cβ. These results argue for a substantial role of AtCAD-D in lignification, and more specifically in the biosynthesis of sinapyl alcohol, the precursor of S lignin units. PMID:12805615

Identification of the gravitropism-related rice gene LAZY1 and elucidation of LAZY1-dependent and -independent gravity signaling pathways.

PubMed

Yoshihara, Takeshi; Iino, Moritoshi

2007-05-01

We identified the gene responsible for three allelic lazy1 mutations of Japonica rice (Oryza sativa L.) by map-based cloning, complementation and RNA interference. Sequence analysis and database searches indicated that the wild-type gene (LAZY1) encodes a novel and unique protein (LAZY1) and that rice has no homologous gene. Two lazy1 mutants were LAZY1 null. Confirming and advancing the previously reported results on lazy1 mutants, we found the following. (i) Gravitropism is impaired, but only partially, in lazy1 coleoptiles. (ii) Circumnutation, observed in dark-grown coleoptiles, is totally absent from lazy1 coleoptiles. (iii) Primary roots of lazy1 mutants show normal gravitropism and circumnutation. (iv) LAZY1 is expressed in a tissue-specific manner in gravity-sensitive shoot tissues (i.e. coleoptiles, leaf sheath pulvini and lamina joints) and is little expressed in roots. (v) The gravitropic response of lazy1 coleoptiles is kinetically separable from that absent from lazy1 coleoptiles. (vi) Gravity-induced lateral translocation of auxin, found in wild-type coleoptiles, does not occur in lazy1 coleoptiles. Based on the genetic and physiological evidence obtained, it is concluded that LAZY1 is specifically involved in shoot gravitropism and that LAZY1-dependent and -independent signaling pathways occur in coleoptiles. It is further concluded that, in coleoptiles, only the LAZY1-dependent gravity signaling involves asymmetric distribution of auxin between the two lateral halves and is required for circumnutation.

Carboxylic acids permeases in yeast: two genes in Kluyveromyces lactis.

PubMed

Lodi, Tiziana; Fontanesi, Flavia; Ferrero, Iliana; Donnini, Claudia

2004-09-15

Two new genes KlJEN1 and KlJEN2 were identified in Kluyveromyces lactis. The deduced structure of their products is typical of membrane-bound carriers and displays high similarity to Jen1p, the monocarboxylate permease of Saccharomyces cerevisiae. Both KlJEN1 and KlJEN2 are under the control of glucose repression mediated by FOG1 and FOG2, corresponding to S. cerevisiae GAL83 and SNF1 respectively, and KlCAT8, proteins involved in glucose signalling cascade in K. lactis. KlJEN1, but not KlJEN2, is induced by lactate. KlJEN2 in contrast is expressed at high level in ethanol and succinate. The physiological characterization of null mutants showed that KlJEN1 is the functional homologue of ScJEN1, whereas KlJEN2 encodes a dicarboxylic acids transporter. In fact, KlJen1p [transporter classification (TC) number: 2.A.1.12.2.] is required for lactate uptake and therefore for growth on lactate. KlJen2p is required for succinate transport, as demonstrated by succinate uptake experiments and by inability of Kljen2 mutant to grow on succinate. This carrier appears to transport also malate and fumarate because the Kljen2 mutant cannot grow on these substrates and the succinate uptake is competed by these carboxylic acids. We conclude that KlJEN2 is the first yeast gene shown to encode a dicarboxylic acids permease.

The Putzig-NURF nucleosome remodeling complex is required for ecdysone receptor signaling and innate immunity in Drosophila melanogaster.

PubMed

Kugler, Sabrina J; Gehring, Eva-Maria; Wallkamm, Veronika; Krüger, Victoria; Nagel, Anja C

2011-05-01

Putzig (Pzg) was originally identified as being an integral component of the TRF2/DREF complex in Drosophila melanogaster, thereby regulating the transcriptional activation of replication-related genes. In a DREF-independent manner, Pzg was shown to mediate Notch target gene activation. This function of Pzg entails an association with the nucleosome remodeling factor complex NURF, which directly binds the ecdysone receptor EcR and coregulates targets of the EcR via the NURF-specific subunit Nurf-301. In contrast, Nurf-301 acts as a negative regulator of JAK/STAT signaling. Here, we provide evidence to show that Pzg is fundamental for these functions of NURF, apart from the regulation of Notch signaling activity. A jump-out mutagenesis provided us with a pzg null mutant displaying early larval lethality, defects in growth, and molting accompanied by aberrant feeding behavior. We show that Pzg is associated with EcR in vivo and required for the transcriptional induction of EcR target genes, whereas reduced ecdysteroid levels imply a NURF-independent function of Pzg. Moreover, pzg interferes with JAK/STAT-signaling activity by acting as a corepressor of Ken. Lamellocyte differentiation was consistently affected in a JAK/STAT mutant background and the expression level of defense response genes was elevated in pzg mutants, leading to the formation of melanotic tumors. Our results suggest that Pzg acts as an important partner of NURF in the regulation of EcR and JAK/STAT signaling.

Multiple histone deacetylases are recruited by corepressor Sin3 and contribute to gene repression mediated by Opi1 regulator of phospholipid biosynthesis in the yeast Saccharomyces cerevisiae.

PubMed

Grigat, Mathias; Jäschke, Yvonne; Kliewe, Felix; Pfeifer, Matthias; Walz, Susanne; Schüller, Hans-Joachim

2012-06-01

Yeast genes of phospholipid biosynthesis are negatively regulated by repressor protein Opi1 when precursor molecules inositol and choline (IC) are available. Opi1-triggered gene repression is mediated by recruitment of the Sin3 corepressor complex. In this study, we systematically investigated the regulatory contribution of subunits of Sin3 complexes and identified Pho23 as important for IC-dependent gene repression. Two non-overlapping regions within Pho23 mediate its direct interaction with Sin3. Previous work has shown that Sin3 recruits the histone deacetylase (HDAC) Rpd3 to execute gene repression. While deletion of SIN3 strongly alleviates gene repression by IC, an rpd3 null mutant shows almost normal regulation. We thus hypothesized that various HDACs may contribute to Sin3-mediated repression of IC-regulated genes. Indeed, a triple mutant lacking HDACs, Rpd3, Hda1 and Hos1, could phenocopy a sin3 single mutant. We show that these proteins are able to contact Sin3 in vitro and in vivo and mapped three distinct HDAC interaction domains, designated HID1, HID2 and HID3. HID3, which is identical to the previously described structural motif PAH4 (paired amphipathic helix), can bind all HDACs tested. Chromatin immunoprecipitation studies finally confirmed that Hda1 and Hos1 are recruited to promoters of phospholipid biosynthetic genes INO1 and CHO2.

High-Throughput Genome Editing and Phenotyping Facilitated by High Resolution Melting Curve Analysis

PubMed Central

Thomas, Holly R.; Percival, Stefanie M.; Yoder, Bradley K.; Parant, John M.

2014-01-01

With the goal to generate and characterize the phenotypes of null alleles in all genes within an organism and the recent advances in custom nucleases, genome editing limitations have moved from mutation generation to mutation detection. We previously demonstrated that High Resolution Melting (HRM) analysis is a rapid and efficient means of genotyping known zebrafish mutants. Here we establish optimized conditions for HRM based detection of novel mutant alleles. Using these conditions, we demonstrate that HRM is highly efficient at mutation detection across multiple genome editing platforms (ZFNs, TALENs, and CRISPRs); we observed nuclease generated HRM positive targeting in 1 of 6 (16%) open pool derived ZFNs, 14 of 23 (60%) TALENs, and 58 of 77 (75%) CRISPR nucleases. Successful targeting, based on HRM of G0 embryos correlates well with successful germline transmission (46 of 47 nucleases); yet, surprisingly mutations in the somatic tail DNA weakly correlate with mutations in the germline F1 progeny DNA. This suggests that analysis of G0 tail DNA is a good indicator of the efficiency of the nuclease, but not necessarily a good indicator of germline alleles that will be present in the F1s. However, we demonstrate that small amplicon HRM curve profiles of F1 progeny DNA can be used to differentiate between specific mutant alleles, facilitating rare allele identification and isolation; and that HRM is a powerful technique for screening possible off-target mutations that may be generated by the nucleases. Our data suggest that micro-homology based alternative NHEJ repair is primarily utilized in the generation of CRISPR mutant alleles and allows us to predict likelihood of generating a null allele. Lastly, we demonstrate that HRM can be used to quickly distinguish genotype-phenotype correlations within F1 embryos derived from G0 intercrosses. Together these data indicate that custom nucleases, in conjunction with the ease and speed of HRM, will facilitate future high-throughput mutation generation and analysis needed to establish mutants in all genes of an organism. PMID:25503746

The role of RNase H2 in processing ribonucleotides incorporated during DNA replication.

PubMed

Williams, Jessica S; Gehle, Daniel B; Kunkel, Thomas A

2017-05-01

Saccharomyces cerevisiae RNase H2 resolves RNA-DNA hybrids formed during transcription and it incises DNA at single ribonucleotides incorporated during nuclear DNA replication. To distinguish between the roles of these two activities in maintenance of genome stability, here we investigate the phenotypes of a mutant of yeast RNase H2 (rnh201-RED; ribonucleotide excision defective) that retains activity on RNA-DNA hybrids but is unable to cleave single ribonucleotides that are stably incorporated into the genome. The rnh201-RED mutant was expressed in wild type yeast or in a strain that also encodes a mutant allele of DNA polymerase ε (pol2-M644G) that enhances ribonucleotide incorporation during DNA replication. Similar to a strain that completely lacks RNase H2 (rnh201Δ), the pol2-M644G rnh201-RED strain exhibits replication stress and checkpoint activation. Moreover, like its null mutant counterpart, the double mutant pol2-M644G rnh201-RED strain and the single mutant rnh201-RED strain delete 2-5 base pairs in repetitive sequences at a high rate that is topoisomerase 1-dependent. The results highlight an important role for RNase H2 in maintaining genome integrity by removing single ribonucleotides incorporated during DNA replication. Published by Elsevier B.V.

Nuclei pulposi formation from the embryonic notochord occurs normally in GDF-5-deficient mice.

PubMed

Maier, Jennifer A; Harfe, Brian D

2011-11-15

The transition of the mouse embryonic notochord into nuclei pulposi was determined ("fate mapped") in vivo in growth and differentiating factor-5 (GDF-5)-null mice using the Shhcre and R26R alleles. To determine whether abnormal nuclei pulposi formation from the embryonic notochord was responsible for defects present in adult nuclei pulposi of Gdf-5-null mice. The development, maintenance, and degeneration of the intervertebral disc are not understood. Previously, we demonstrated that all cells in the adult nucleus pulposus of normal mice are derived from the embryonic notochord. Gdf-5-null mice have been reported to contain intervertebral discs in which the nucleus pulposus is abnormal. It is currently unclear if disc defects in Gdf-5-null mice arise during the formation of nuclei pulposi from the notochord during embryogenesis or result from progressive postnatal degeneration of nuclei pulposi. Gdf-5 messenger RNA expression was examined in the discs of wild-type embryos by RNA in situ hybridization to determine when and where this gene was expressed. To examine nucleus pulposus formation in Gdf-5-null mice, intervertebral discs in which embryonic notochord cells were marked were analyzed in newborn and 24-week-old mice. Our Gdf-5 messenger RNA in situ experiments determined that this gene is localized to the annulus fibrosus and not the nucleus pulposus in mouse embryos. Notochord fate-mapping experiments revealed that notochord cells in Gdf-5-null mice correctly form nuclei pulposi. Our data suggest that the defects reported in the nucleus pulposus of adult Gdf-5-null mice do not result from abnormal patterning of the embryonic notochord. The use of mouse alleles to mark cells that produce all cell types that reside in the adult nucleus pulposus will allow for a detailed examination of disc formation in other mouse mutants that have been reported to contain disc defects.

Nuclei pulposi formation from the embryonic notochord occurs normally in GDF5-deficient mice

PubMed Central

Maier, Jennifer A.; Harfe, Brian D.

2011-01-01

Study Design The transition of the mouse embryonic notochord into nuclei pulposi was determined (“fate mapped”) in vivo in GDF-5 null mice using the Shhcre and R26R alleles. Objective To determine if abnormal nuclei pulposi formation from the embryonic notochord was responsible for defects present in adult nuclei pulposi of Gdf-5 null mice. Summary of Background Data The development, maintenance, and degeneration of the intervertebral disc are not understood. Previously, we demonstrated that all cells in the adult nucleus pulposus of normal mice are derived from the embryonic notochord. Gdf-5 null mice have been reported to contain intervertebral discs in which the nucleus pulposus is abnormal. It is currently unclear if disc defects in Gdf-5 null mice arise during the formation of nuclei pulposi from the notochord during embryogenesis or resulted from progressive postnatal degeneration of nuclei pulposi. Methods Gdf-5 mRNA expression was examined in the discs of wild-type embryos by RNA in situ hybridization to determine when and where this gene was expressed. To examine nucleus pulposus formation in Gdf-5 null mice, intervertebral discs in which embryonic notochord cells were marked were analyzed in newborn and 24 week old mice. Results Our Gdf-5 mRNA in situ experiments determined that this gene is localized to the annulus fibrosus and not the nucleus pulposus in mouse embryos. Notochord fate mapping experiments revealed that notochord cells in Gdf-5 null mice correctly form nuclei pulposi. Conclusion Our data suggest that the defects reported in the nucleus pulposus of adult Gdf-5 null mice do not result from abnormal patterning of the embryonic notochord. The use of mouse alleles to mark cells that produce all cell types that reside in the adult nucleus pulposus will allow for a detailed examination of disc formation in other mouse mutants that have been reported to contain disc defects. PMID:21278629

Opaque-2 is a transcriptional activator that recognizes a specific target site in 22-kD zein genes.

PubMed Central

Schmidt, R J; Ketudat, M; Aukerman, M J; Hoschek, G

1992-01-01

opaque-2 (o2) is a regulatory locus in maize that plays an essential role in controlling the expression of genes encoding the 22-kD zein proteins. Through DNase I footprinting and DNA binding analyses, we have identified the binding site for the O2 protein (O2) in the promoter of 22-kD zein genes. The sequence in the 22-kD zein gene promoter that is recognized by O2 is similar to the target site recognized by other "basic/leucine zipper" (bZIP) proteins in that it contains an ACGT core that is necessary for DNA binding. The site is located in the -300 region relative to the translation start and lies about 20 bp downstream of the highly conserved zein gene sequence motif known as the "prolamin box." Employing gel mobility shift assays, we used O2 antibodies and nuclear extracts from an o2 null mutant to demonstrate that the O2 protein in maize endosperm nuclei recognizes the target site in the zein gene promoter. Mobility shift assays using nuclear proteins from an o2 null mutant indicated that other endosperm proteins in addition to O2 can bind the O2 target site and that O2 may be associated with one of these proteins. We also demonstrated that in yeast cells the O2 protein can activate expression of a lacZ gene containing a multimer of the O2 target sequence as part of its promoter, thus confirming its role as a transcriptional activator. A computer-assisted search indicated that the O2 target site is not present in the promoters of zein genes other than those of the 22-kD class. These data suggest a likely explanation at the molecular level for the differential effect of o2 mutations on expression of certain members of the zein gene family. PMID:1392590

Heterologous Expression in Remodeled C. elegans: A Platform for Monoaminergic Agonist Identification and Anthelmintic Screening.

PubMed

Law, Wenjing; Wuescher, Leah M; Ortega, Amanda; Hapiak, Vera M; Komuniecki, Patricia R; Komuniecki, Richard

2015-04-01

Monoamines, such as 5-HT and tyramine (TA), paralyze both free-living and parasitic nematodes when applied exogenously and serotonergic agonists have been used to clear Haemonchus contortus infections in vivo. Since nematode cell lines are not available and animal screening options are limited, we have developed a screening platform to identify monoamine receptor agonists. Key receptors were expressed heterologously in chimeric, genetically-engineered Caenorhabditis elegans, at sites likely to yield robust phenotypes upon agonist stimulation. This approach potentially preserves the unique pharmacologies of the receptors, while including nematode-specific accessory proteins and the nematode cuticle. Importantly, the sensitivity of monoamine-dependent paralysis could be increased dramatically by hypotonic incubation or the use of bus mutants with increased cuticular permeabilities. We have demonstrated that the monoamine-dependent inhibition of key interneurons, cholinergic motor neurons or body wall muscle inhibited locomotion and caused paralysis. Specifically, 5-HT paralyzed C. elegans 5-HT receptor null animals expressing either nematode, insect or human orthologues of a key Gαo-coupled 5-HT1-like receptor in the cholinergic motor neurons. Importantly, 8-OH-DPAT and PAPP, 5-HT receptor agonists, differentially paralyzed the transgenic animals, with 8-OH-DPAT paralyzing mutant animals expressing the human receptor at concentrations well below those affecting its C. elegans or insect orthologues. Similarly, 5-HT and TA paralyzed C. elegans 5-HT or TA receptor null animals, respectively, expressing either C. elegans or H. contortus 5-HT or TA-gated Cl- channels in either C. elegans cholinergic motor neurons or body wall muscles. Together, these data suggest that this heterologous, ectopic expression screening approach will be useful for the identification of agonists for key monoamine receptors from parasites and could have broad application for the identification of ligands for a host of potential anthelmintic targets.

Heterologous Expression in Remodeled C. elegans: A Platform for Monoaminergic Agonist Identification and Anthelmintic Screening

PubMed Central

Law, Wenjing; Wuescher, Leah M.; Ortega, Amanda; Hapiak, Vera M.; Komuniecki, Patricia R.; Komuniecki, Richard

2015-01-01

Monoamines, such as 5-HT and tyramine (TA), paralyze both free-living and parasitic nematodes when applied exogenously and serotonergic agonists have been used to clear Haemonchus contortus infections in vivo. Since nematode cell lines are not available and animal screening options are limited, we have developed a screening platform to identify monoamine receptor agonists. Key receptors were expressed heterologously in chimeric, genetically-engineered Caenorhabditis elegans, at sites likely to yield robust phenotypes upon agonist stimulation. This approach potentially preserves the unique pharmacologies of the receptors, while including nematode-specific accessory proteins and the nematode cuticle. Importantly, the sensitivity of monoamine-dependent paralysis could be increased dramatically by hypotonic incubation or the use of bus mutants with increased cuticular permeabilities. We have demonstrated that the monoamine-dependent inhibition of key interneurons, cholinergic motor neurons or body wall muscle inhibited locomotion and caused paralysis. Specifically, 5-HT paralyzed C. elegans 5-HT receptor null animals expressing either nematode, insect or human orthologues of a key Gαo-coupled 5-HT1-like receptor in the cholinergic motor neurons. Importantly, 8-OH-DPAT and PAPP, 5-HT receptor agonists, differentially paralyzed the transgenic animals, with 8-OH-DPAT paralyzing mutant animals expressing the human receptor at concentrations well below those affecting its C. elegans or insect orthologues. Similarly, 5-HT and TA paralyzed C. elegans 5-HT or TA receptor null animals, respectively, expressing either C. elegans or H. contortus 5-HT or TA-gated Cl- channels in either C. elegans cholinergic motor neurons or body wall muscles. Together, these data suggest that this heterologous, ectopic expression screening approach will be useful for the identification of agonists for key monoamine receptors from parasites and could have broad application for the identification of ligands for a host of potential anthelmintic targets. PMID:25928899

AsrR Is an Oxidative Stress Sensing Regulator Modulating Enterococcus faecium Opportunistic Traits, Antimicrobial Resistance, and Pathogenicity

PubMed Central

Lebreton, François; van Schaik, Willem; Sanguinetti, Maurizio; Posteraro, Brunella; Torelli, Riccardo; Le Bras, Florian; Verneuil, Nicolas; Zhang, Xinglin; Giard, Jean-Christophe; Dhalluin, Anne; Willems, Rob J. L.; Leclercq, Roland; Cattoir, Vincent

2012-01-01

Oxidative stress serves as an important host/environmental signal that triggers a wide range of responses in microorganisms. Here, we identified an oxidative stress sensor and response regulator in the important multidrug-resistant nosocomial pathogen Enterococcus faecium belonging to the MarR family and called AsrR (antibiotic and stress response regulator). The AsrR regulator used cysteine oxidation to sense the hydrogen peroxide which results in its dissociation to promoter DNA. Transcriptome analysis showed that the AsrR regulon was composed of 181 genes, including representing functionally diverse groups involved in pathogenesis, antibiotic and antimicrobial peptide resistance, oxidative stress, and adaptive responses. Consistent with the upregulated expression of the pbp5 gene, encoding a low-affinity penicillin-binding protein, the asrR null mutant was found to be more resistant to β-lactam antibiotics. Deletion of asrR markedly decreased the bactericidal activity of ampicillin and vancomycin, which are both commonly used to treat infections due to enterococci, and also led to over-expression of two major adhesins, acm and ecbA, which resulted in enhanced in vitro adhesion to human intestinal cells. Additional pathogenic traits were also reinforced in the asrR null mutant including greater capacity than the parental strain to form biofilm in vitro and greater persistance in Galleria mellonella colonization and mouse systemic infection models. Despite overexpression of oxidative stress-response genes, deletion of asrR was associated with a decreased oxidative stress resistance in vitro, which correlated with a reduced resistance to phagocytic killing by murine macrophages. Interestingly, both strains showed similar amounts of intracellular reactive oxygen species. Finally, we observed a mutator phenotype and enhanced DNA transfer frequencies in the asrR deleted strain. These data indicate that AsrR plays a major role in antimicrobial resistance and adaptation for survival within the host, thereby contributes importantly to the opportunistic traits of E. faecium. PMID:22876178

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shiau, Celia E.; Kaufman, Zoe; Meireles, Ana M.

Interferon regulatory factor 8 (Irf8) is critical for mammalian macrophage development and innate immunity, but its role in teleost myelopoiesis remains incompletely understood. Specifically, genetic tools to analyze the role of irf8 in zebrafish macrophage development at larval and adult stages are lacking. In this study, we generated irf8 null mutants in zebrafish using TALEN-mediated targeting. Our analysis defines different requirements for irf8 at different stages. irf8 is required for formation of all macrophages during primitive and transient definitive hematopoiesis, but not during adult-phase definitive hematopoiesis starting at 5-6 days postfertilization. At early stages, irf8 mutants have excess neutrophils andmore » excess cell death in pu.1-expressing myeloid cells. Macrophage fates were recovered in irf8 mutants after wildtype irf8 expression in neutrophil and macrophage lineages, suggesting that irf8 regulates macrophage specification and survival. In juvenile irf8 mutant fish, mature macrophages are present, but at numbers significantly reduced compared to wildtype, indicating an ongoing requirement for irf8 after embryogenesis. As development progresses, tissue macrophages become apparent in zebrafish irf8 mutants, with the possible exception of microglia. Our study defines distinct requirement for irf8 in myelopoiesis before and after transition to the adult hematopoietic system.« less

Association of the cad-n1 allele with increased stem growth and wood density in full-sib families of loblolly pine

Treesearch

Q. Yu; B. Li; C.D. Nelson; S.E. McKeand; T.J. Mullin

2005-01-01

Stem growth and wood density associated with a mutant null (cad-nl) allele were examined in three 15-year old loblolly pine half-diallel tests established on two sites in the southern United States. In each half-diallel test, one or two cad-nl heterozygous parents were crossed with five unrelated wild-type parents to produce five...

Genetic metabolic complementation establishes a requirement for GDP-fucose in Leishmania.

PubMed

Guo, Hongjie; Novozhilova, Natalia M; Bandini, Giulia; Turco, Salvatore J; Ferguson, Michael A J; Beverley, Stephen M

2017-06-23

To survive in its sand fly vector, the trypanosomatid protozoan parasite Leishmania first attaches to the midgut to avoid excretion, but eventually it must detach for transmission by the next bite. In Leishmania major strain Friedlin, this is controlled by modifications of the stage-specific adhesin lipophosphoglycan (LPG). During differentiation to infective metacyclics, d-arabinopyranose (d-Ara p ) caps the LPG side-chain galactose residues, blocking interaction with the midgut lectin PpGalec, thereby leading to parasite detachment and transmission. Previously, we characterized two closely related L. major genes ( FKP40 and AFKP80 ) encoding bifunctional proteins with kinase/pyrophosphorylase activities required for salvage and conversion of l-fucose and/or d-Ara p into the nucleotide-sugar substrates required by glycosyltransferases. Whereas only AFKP80 yielded GDP-d-Ara p from exogenous d-Ara p , both proteins were able to salvage l-fucose to GDP-fucose. We now show that Δ afkp80 - null mutants ablated d-Ara p modifications of LPG as predicted, whereas Δ fkp40 - null mutants resembled wild type (WT). Fucoconjugates had not been reported previously in L. major , but unexpectedly, we were unable to generate fkp40 - / afkp80 - double mutants, unless one of the A/FKPs was expressed ectopically. To test whether GDP-fucose itself was essential for Leishmania viability, we employed "genetic metabolite complementation." First, the trypanosome de novo pathway enzymes GDP-mannose dehydratase (GMD) and GDP-fucose synthetase (GMER) were expressed ectopically; from these cells, the Δ fkp40 - /Δ afkp80 - double mutant was now readily obtained. As expected, the Δ fkp40 - /Δ afkp80 - /+ TbGMD-GMER line lacked the capacity to generate GDP-Ara p , while synthesizing abundant GDP-fucose. These results establish a requirement for GDP-fucose for L. major viability and predict the existence of an essential fucoconjugate(s). © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Inactivation of the alpha C protein antigen gene, bca, by a novel shuttle/suicide vector results in attenuation of virulence and immunity in group B Streptococcus.

PubMed

Li, J; Kasper, D L; Ausubel, F M; Rosner, B; Michel, J L

1997-11-25

The alpha C protein of group B Streptococcus (GBS) is a major surface-associated antigen. Although its role in the biology and virulence of GBS has not been defined, it is opsonic and capable of eliciting protective immunity. The alpha C protein is widely distributed among clinical isolates and is a potential protein carrier and antigen in conjugate vaccines to prevent GBS infections. The structural gene for the alpha C protein, bca, has been cloned and sequenced. The protein encoded by bca is related to a class of surface-associated proteins of gram-positive cocci involved in virulence and immunity. To investigate the potential roles of the alpha C protein, bca null mutants were generated in which the bca gene was replaced with a kanamycin resistance cassette via homologous recombination using a novel shuttle/suicide vector. Studies of lethality in neonatal mice showed that the virulence of the bca null mutants was attenuated 5- to 7-fold when compared with the isogenic wild-type strain A909. Significant differences in mortality occurred in the first 24 h, suggesting that the role of the alpha antigen is important in the initial stages of the infection. In contrast to A909, bca mutants were no longer killed by polymorphonuclear leukocytes in the presence of alpha-specific antibodies in an in vitro opsonophagocytic assay. In contrast to previous studies, alpha antigen expression does not appear to play a role in resistance to opsonophagocytosis in the absence of alpha-specific antibodies. In addition, antibodies to the alpha C protein did not passively protect neonatal mice from lethal challenge with bca mutants, suggesting that these epitopes are uniquely present within the alpha antigen as expressed from the bca gene. Therefore, the alpha C protein is important in the pathogenesis of GBS infection and is a target for protective immunity in the development of GBS vaccines.

Genetic metabolic complementation establishes a requirement for GDP-fucose in Leishmania

PubMed Central

Novozhilova, Natalia M.; Turco, Salvatore J.

2017-01-01

To survive in its sand fly vector, the trypanosomatid protozoan parasite Leishmania first attaches to the midgut to avoid excretion, but eventually it must detach for transmission by the next bite. In Leishmania major strain Friedlin, this is controlled by modifications of the stage-specific adhesin lipophosphoglycan (LPG). During differentiation to infective metacyclics, d-arabinopyranose (d-Arap) caps the LPG side-chain galactose residues, blocking interaction with the midgut lectin PpGalec, thereby leading to parasite detachment and transmission. Previously, we characterized two closely related L. major genes (FKP40 and AFKP80) encoding bifunctional proteins with kinase/pyrophosphorylase activities required for salvage and conversion of l-fucose and/or d-Arap into the nucleotide-sugar substrates required by glycosyltransferases. Whereas only AFKP80 yielded GDP-d-Arap from exogenous d-Arap, both proteins were able to salvage l-fucose to GDP-fucose. We now show that Δafkp80− null mutants ablated d-Arap modifications of LPG as predicted, whereas Δfkp40− null mutants resembled wild type (WT). Fucoconjugates had not been reported previously in L. major, but unexpectedly, we were unable to generate fkp40−/afkp80− double mutants, unless one of the A/FKPs was expressed ectopically. To test whether GDP-fucose itself was essential for Leishmania viability, we employed “genetic metabolite complementation.” First, the trypanosome de novo pathway enzymes GDP-mannose dehydratase (GMD) and GDP-fucose synthetase (GMER) were expressed ectopically; from these cells, the Δfkp40−/Δafkp80− double mutant was now readily obtained. As expected, the Δfkp40−/Δafkp80−/+TbGMD-GMER line lacked the capacity to generate GDP-Arap, while synthesizing abundant GDP-fucose. These results establish a requirement for GDP-fucose for L. major viability and predict the existence of an essential fucoconjugate(s). PMID:28465349

Constitutively active follicle-stimulating hormone receptor enables androgen-independent spermatogenesis.

PubMed

Oduwole, Olayiwola O; Peltoketo, Hellevi; Poliandri, Ariel; Vengadabady, Laura; Chrusciel, Marcin; Doroszko, Milena; Samanta, Luna; Owen, Laura; Keevil, Brian; Rahman, Nafis A; Huhtaniemi, Ilpo T

2018-05-01

Spermatogenesis is regulated by the 2 pituitary gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH). This process is considered impossible without the absolute requirement of LH-stimulated testicular testosterone (T) production. The role of FSH remains unclear because men and mice with inactivating FSH receptor (FSHR) mutations are fertile. We revisited the role of FSH in spermatogenesis using transgenic mice expressing a constitutively strongly active FSHR mutant in a LH receptor-null (LHR-null) background. The mutant FSHR reversed the azoospermia and partially restored fertility of Lhr-/- mice. The finding was initially ascribed to the residual Leydig cell T production. However, when T action was completely blocked with the potent antiandrogen flutamide, spermatogenesis persisted. Hence, completely T-independent spermatogenesis is possible through strong FSHR activation, and the dogma of T being a sine qua non for spermatogenesis may need modification. The mechanism for the finding appeared to be that FSHR activation maintained the expression of Sertoli cell genes considered androgen dependent. The translational message of our findings is the possibility of developing a new strategy of high-dose FSH treatment for spermatogenic failure. Our findings also provide an explanation of molecular pathogenesis for Pasqualini syndrome (fertile eunuchs; LH/T deficiency with persistent spermatogenesis) and explain how the hormonal regulation of spermatogenesis has shifted from FSH to T dominance during evolution.

Stress responses at the endometrial-placental interface regulate labyrinthine placental differentiation from trophoblast stem cells.

PubMed

Rappolee, D A; Zhou, S; Puscheck, E E; Xie, Y

2013-05-01

Development can happen in one of two ways. Cells performing a necessary function can differentiate from stem cells before the need for it arises and stress does not develop. Or need arises before function, stress develops and stress signals are part of the normal stimuli that regulate developmental mechanisms. These mechanisms adjust stem cell differentiation to produce function in a timely and proportional manner. In this review, we will interpret data from studies of null lethal mutants for placental stress genes that suggest the latter possibility. Acknowledged stress pathways participate in stress-induced and -regulated differentiation in two ways. These pathways manage the homeostatic response to maintain stem cells during the stress. Stress pathways also direct stem cell differentiation to increase the first essential lineage and suppress later lineages when stem cell accumulation is diminished. This stress-induced differentiation maintains the conceptus during stress. Pathogenic outcomes arise because population sizes of normal stem cells are first depleted by decreased accumulation. The fraction of stem cells is further decreased by differentiation that is induced to compensate for smaller stem cell populations. Analysis of placental lethal null mutant genes known to mediate stress responses suggests that the labyrinthine placenta develops during, and is regulated by, hypoxic stress.

Trypanosoma brucei (UMP synthase null mutants) are avirulent in mice, but recover virulence upon prolonged culture in vitro while retaining pyrimidine auxotrophy

PubMed Central

Ong, Han B; Sienkiewicz, Natasha; Wyllie, Susan; Patterson, Stephen; Fairlamb, Alan H

2013-01-01

African trypanosomes are capable of both de novo synthesis and salvage of pyrimidines. The last two steps in de novo synthesis are catalysed by UMP synthase (UMPS) – a bifunctional enzyme comprising orotate phosphoribosyl transferase (OPRT) and orotidine monophosphate decarboxylase (OMPDC). To investigate the essentiality of pyrimidine biosynthesis in Trypanosoma brucei, we generated a umps double knockout (DKO) line by gene replacement. The DKO was unable to grow in pyrimidine-depleted medium in vitro, unless supplemented with uracil, uridine, deoxyuridine or UMP. DKO parasites were completely resistant to 5-fluoroorotate and hypersensitive to 5-fluorouracil, consistent with loss of UMPS, but remained sensitive to pyrazofurin indicating that, unlike mammalian cells, the primary target of pyrazofurin is not OMPDC. The null mutant was unable to infect mice indicating that salvage of host pyrimidines is insufficient to support growth. However, following prolonged culture in vitro, parasites regained virulence in mice despite retaining pyrimidine auxotrophy. Unlike the wild-type, both pyrimidine auxotrophs secreted substantial quantities of orotate, significantly higher in the virulent DKO line. We propose that this may be responsible for the recovery of virulence in mice, due to host metabolism converting orotate to uridine, thereby bypassing the loss of UMPS in the parasite. PMID:23980694

Leishmania dihydroxyacetonephosphate acyltransferase LmDAT is important for ether lipid biosynthesis but not for the integrity of detergent resistant membranes.

PubMed

Zufferey, Rachel; Al-Ani, Gada K; Dunlap, Kara

2009-12-01

Glycerolipid biosynthesis in Leishmania initiates with the acylation of glycerol-3-phosphate by a single glycerol-3-phosphate acyltransferase, LmGAT, or of dihydroxyacetonephosphate by a dihydroxyacetonephosphate acyltransferase, LmDAT. We previously reported that acylation of the precursor dihydroxyacetonephosphate rather than glycerol-3-phosphate is the physiologically relevant pathway for Leishmania parasites. We demonstrated that LmDAT is important for normal growth, survival during the stationary phase, and for virulence. Here, we assessed the role of LmDAT in glycerolipid metabolism and metacyclogenesis. LmDAT was found to be implicated in the biosynthesis of ether glycerolipids, including the ether lipid derived virulence factor lipophosphoglycan and glycosylphosphatidylinositol-anchored proteins. The null mutant produced longer lipophosphoglycan molecules that were not released in the medium, and augmented levels of glycosylphosphatidylinositol-anchored proteins. In addition, the integrity of detergent resistant membranes was not affected by the absence of the LmDAT gene. Further, our genetic analyses strongly suggest that LmDAT was synthetic lethal with the glycerol-3-phosphate acyltransferase encoding gene LmGAT, implying that Leishmania expresses only two acyltransferases that initiate the biosynthesis of its cellular glycerolipids. Last, despite the fact that LmDAT is important for virulence the null mutant still exhibited the typical characteristics of metacyclics.

Constitutive role of the Fanconi anemia D2 gene in the replication stress response.

PubMed

Tian, Yanyan; Shen, Xi; Wang, Rui; Klages-Mundt, Naeh L; Lynn, Erica J; Martin, Sara K; Ye, Yin; Gao, Min; Chen, Junjie; Schlacher, Katharina; Li, Lei

2017-12-08

In response to DNA cross-linking damage, the Fanconi anemia (FA) core complex activates the FA pathway by monoubiquitinating Fanconi anemia complementation group D2 (FANCD2) for the initiation of the nucleolytic processing of the DNA cross-links and stabilization of stalled replication forks. Given that all the classic FA proteins coordinately monoubiquitinate FANCD2, it is unclear why losses of individual classic FA genes yield varying cellular sensitivities to cross-linking damage. To address this question, we generated cellular knock-out models of FA core complex components and FANCD2 and found that FANCD2-null mutants display higher levels of spontaneous chromosomal damage and hypersensitivity to replication-blocking lesions than Fanconi anemia complementation group L (FANCL)-null mutants, suggesting that FANCD2 provides a basal level of DNA protection countering endogenous lesions in the absence of monoubiquitination. FANCD2's ubiquitination-independent function is likely involved in optimized recruitment of nucleolytic activities for the processing and protection of stressed replication forks. Our results reveal that FANCD2 has a ubiquitination-independent role in countering endogenous levels of replication stress, a function that is critical for the maintenance of genomic stability. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Cerebellar Ataxia, Seizures, Premature Death, and Cardiac Abnormalities in Mice with Targeted Disruption of the Cacna2d2 Gene

PubMed Central

Ivanov, Sergey V.; Ward, Jerrold M.; Tessarollo, Lino; McAreavey, Dorothea; Sachdev, Vandana; Fananapazir, Lameh; Banks, Melissa K.; Morris, Nicole; Djurickovic, Draginja; Devor-Henneman, Deborah E.; Wei, Ming-Hui; Alvord, Gregory W.; Gao, Boning; Richardson, James A.; Minna, John D.; Rogawski, Michael A.; Lerman, Michael I.

2004-01-01

CACNA2D2 is a putative tumor suppressor gene located in the human chromosome 3p21.3 region that shows frequent allelic imbalances in lung, breast, and other cancers. The α2δ-2 protein encoded by the gene is a regulatory subunit of voltage-dependent calcium channels and is expressed in brain, heart, and other tissues. Here we report that mice homozygous for targeted disruption of the Cacna2d2 gene exhibit growth retardation, reduced life span, ataxic gait with apoptosis of cerebellar granule cells followed by Purkinje cell depletion, enhanced susceptibility to seizures, and cardiac abnormalities. The Cacna2d2tm1NCIF null phenotype has much in common with that of Cacna1a mutants, such as cerebellar neuro-degeneration associated with ataxia, seizures, and premature death. A tendency to bradycardia and limited response of null mutants to isoflurane implicate α2δ-2 in sympathetic regulation of cardiac function. In summary, our findings provide genetic evidence that the α2δ-2 subunit serves in vivo as a component of P/Q-type calcium channels, is indispensable for the central nervous system function, and may be involved in hereditary cerebellar ataxias and epileptic disorders in humans. PMID:15331424

Beadex Function in the Motor Neurons Is Essential for Female Reproduction in Drosophila melanogaster

PubMed Central

Kairamkonda, Subhash; Nongthomba, Upendra

2014-01-01

Drosophila melanogaster has served as an excellent model system for understanding the neuronal circuits and molecular mechanisms regulating complex behaviors. The Drosophila female reproductive circuits, in particular, are well studied and can be used as a tool to understand the role of novel genes in neuronal function in general and female reproduction in particular. In the present study, the role of Beadex, a transcription co-activator, in Drosophila female reproduction was assessed by generation of mutant and knock down studies. Null allele of Beadex was generated by transposase induced excision of P-element present within an intron of Beadex gene. The mutant showed highly compromised reproductive abilities as evaluated by reduced fecundity and fertility, abnormal oviposition and more importantly, the failure of sperm release from storage organs. However, no defect was found in the overall ovariole development. Tissue specific, targeted knock down of Beadex indicated that its function in neurons is important for efficient female reproduction, since its neuronal knock down led to compromised female reproductive abilities, similar to Beadex null females. Further, different neuronal class specific knock down studies revealed that Beadex function is required in motor neurons for normal fecundity and fertility of females. Thus, the present study attributes a novel and essential role for Beadex in female reproduction through neurons. PMID:25396431

Thctf1 transcription factor of Trichoderma harzianum is involved in 6-pentyl-2H-pyran-2-one production and antifungal activity.

PubMed

Rubio, M Belén; Hermosa, Rosa; Reino, José Luis; Collado, Isidro G; Monte, Enrique

2009-01-01

We describe the cloning and characterization of the Trichoderma harzianum Thctf1 gene, which shows high sequence identity with a transcription factor gene of Fusarium solani f. sp. pisi. In T. harzianum, disruption of the Thctf1 gene by homologous recombination gave rise to transformants that in plate experiments did not show the yellow pigmentation observed in the wild-type strain. In several Trichoderma spp. a yellow pigmentation and a coconut aroma have been related to the production of 6-pentyl-2H-pyran-2-one (6PP) compounds. Prompted by this, we explored whether the loss of pigmentation in the Thctf1 null mutants of T. harzianum might be related to the synthesis of 6PP. Chromatographic and spectroscopic analyses revealed that the disruptants did not produce two secondary metabolites, derived from 6PP and not previously described in the Trichoderma genus, that are present in wild-type culture filtrates. Since 6PP is a recognized antifungal compound, this ability was analyzed in both the disruptants and wild-type, observing that the Thctf1 null mutants of T. harzianum had reduced antimicrobial capacity. Our results point to the significant role of THCTF1 in the production of secondary metabolites and in the antifungal activity of T. harzianum.

The ARG1-LIKE2 gene of Arabidopsis functions in a gravity signal transduction pathway that is genetically distinct from the PGM pathway

NASA Technical Reports Server (NTRS)

Guan, Changhui; Rosen, Elizabeth S.; Boonsirichai, Kanokporn; Poff, Kenneth L.; Masson, Patrick H.

2003-01-01

The arl2 mutants of Arabidopsis display altered root and hypocotyl gravitropism, whereas their inflorescence stems are fully gravitropic. Interestingly, mutant roots respond like the wild type to phytohormones and an inhibitor of polar auxin transport. Also, their cap columella cells accumulate starch similarly to wild-type cells, and mutant hypocotyls display strong phototropic responses to lateral light stimulation. The ARL2 gene encodes a DnaJ-like protein similar to ARG1, another protein previously implicated in gravity signal transduction in Arabidopsis seedlings. ARL2 is expressed at low levels in all organs of seedlings and plants. arl2-1 arg1-2 double mutant roots display kinetics of gravitropism similar to those of single mutants. However, double mutants carrying both arl2-1 and pgm-1 (a mutation in the starch-biosynthetic gene PHOSPHOGLUCOMUTASE) at the homozygous state display a more pronounced root gravitropic defect than the single mutants. On the other hand, seedlings with a null mutation in ARL1, a paralog of ARG1 and ARL2, behave similarly to the wild type in gravitropism and other related assays. Taken together, the results suggest that ARG1 and ARL2 function in the same gravity signal transduction pathway in the hypocotyl and root of Arabidopsis seedlings, distinct from the pathway involving PGM.

Targeted inactivation of the mouse locus encoding coagulation factor XIII-A: hemostatic abnormalities in mutant mice and characterization of the coagulation deficit.

PubMed

Lauer, Peter; Metzner, Hubert J; Zettlmeissl, Gerd; Li, Meng; Smith, Austin G; Lathe, Richard; Dickneite, Gerhard

2002-12-01

Blood coagulation factor XIII (FXIII) promotes cross-linking of fibrin during blood coagulation; impaired clot stabilization in human genetic deficiency is associated with marked pathologies of major clinical impact, including bleeding symptoms and deficient wound healing. To investigate the role of FXIII we employed homologous recombination to generate a targeted deletion of the inferred exon 7 of the FXIII-A gene. FXIII transglutaminase activity in plasma was reduced to about 50% in mice heterozygous for the mutant allele, and was abolished in homozygous null mice. Plasma fibrin gamma-dimerization was also indetectable in the homozygous deficient animals, confirming the absence of activatable FXIII. Homozygous mutant mice were fertile, although reproduction was impaired. Bleeding episodes, hematothorax, hematoperitoneum and subcutaneous hemorrhage in mutant mice were associated with reduced survival. Arrest of tail-tip bleeding in FXIII-A deficient mice was markedly and significantly delayed; replacement of mutant mice with human plasma FXIII (Fibrogammin P) restored bleeding time to within the normal range. Thrombelastography (TEG) experiments demonstrated impaired clot stabilization in FXIII-A mutant mice, replacement with human FXIII led to dose-dependent TEG normalization. The mutant mice thus reiterate some key features of the human genetic disorder: they will be valuable in assessing the role of FXIII in other associated pathologies and the development of new therapies.

Nuclear sequestration of COL1A1 mRNA transcript associated with type I osteogenesis imperfecta (OI)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Primorac, D.; Stover, M.L.; McKinstry, M.B.

Previously we identified an OI type I patient with a splice donor mutation that resulted in intron 26 retention instead of exon skipping and sequestration of normal levels of the mutant transcript in the nuclear compartment. Intron retention was consistent with the exon definition hypothesis for splice site selection since the size of the exon-intron-exon unit was less than 300 bp. Furthermore, the retained intron contained in-frame stop codons which is thought to cause the mutant RNA to remain within the nucleus rather than appearing in the cytoplasm. To test these hypotheses, genomic fragments containing the normal sequence or themore » donor mutation were cloned into a collagen minigene and expressed in stably tansfected NIH 3T3 cells. None of the modifications to the normal intron altered the level of RNA that accumulated in the cytoplasm, as expected. However none of the modifications to the mutant intron allowed accumulation of normal levels of mRNA in the cytoplasm. Moreover, in contrast to our findings in the patient`s cells only low levels of mutant transcript were found in the nucleus; a fraction of the transcript did appear in the cytoplasm which had spliced the mutant donor site correctly. Nuclear run-on experiments demonstrated equal levels of transcription from each transgene. Expression of another donor mutation known to cause in-frame exon skipping in OI type IV was accurately reproduced in the minigene in transfected 3T3 cells. Our experience suggests that either mechanism can lead to formation of a null allele possibly related to the type of splicing events surrounding the potential stop codons. Understanding the rules governing inactivation of a collagen RNA transcript may be important in designing a strategy to inactivate a dominate negative mutation associated with the more severe forms of OI.« less

Plant perception and response to the signal in gravity resistance

NASA Astrophysics Data System (ADS)

Hoson, Takayuki; Soga, Kouichi; Wakabayashi, Kazuyuki; Kamisaka, Seiichiro; Zhang, Yan; Otomi, Yasuhiro; Hashimoto, Takashi; Iida, Hidetoshi

2012-07-01

Gravity resistance, mechanical resistance to the gravitational force, is a principal graviresponse in plants, distinct from gravitropism. Plants increase the rigidity of their cell walls in the final step of gravity resistance. We studied cellular events leading to or related to the cell wall changes under hypergravity conditions produced by centrifugation and under microgravity conditions in space. The involvement of mechanosensitive ion channels (mechanoreceptors) in signal perception in gravity resistance has been suggested by experiments with inhibitors. As a candidate for the mechanoreceptor, we identified MCA1 and MCA2 in Arabidopsis. mca-null and MCA-overexpressing seedlings were normal in growth in the dark at 1 g. However, suppression by hypergravity of elongation growth was reduced in hypocotyls of mca-null seedlings. On the contrary, MCA-overexpressing seedlings were hypersensitive to hypergravity. These results suggest that MCAs act as the mechanoreceptor in signal perception of gravity resistance. Cortical microtubules play an essential role in maintenance of normal growth phenotype under hypergravity conditions. In Space Seed experiment in the Kibo Module (PI: S. Kamisaka), we examined the effects of microgravity on growth phenotypes of Arabidopsis tubulin mutant, tua6. Inflorescences of the mutant emerged earlier and elongated rapidly under microgravity conditions than under on-orbit or ground 1 g conditions. Also, the inflorescences grown under microgravity conditions showed higher cell wall extensibilities than the controls. The tubulin mutant thus grew and developed more or less normally under microgravity conditions, supporting the principal role of microtubules also in plant resistance to 1 g gravity. On the other hand, the cellular osmotic properties, as well as the cell wall properties, are important factors determining the rigidity of plant body. Azuki bean epicotyls were capable of maintaining osmoregulation even under hypergravity conditions for a short period. By long-term hypergravity treatment, the increase in level of total osmotic solutes was suppressed, which was accounted by suppression of translocation of organic solutes, such as sugars and amino acids, from seed to epicotyl. Nevertheless, the ATP content per epicotyl or fresh weight was kept constant even under hypergravity conditions for a long period. The maintenance of osmoregulation may contribute to plant resistance to hypergravity. Space experiments on the International Space Station will further clarify the mechanism of gravity resistance.

Molecular identification of rare FY*Null and FY*X alleles in Caucasian thalassemic family from Sardinia.

PubMed

Manfroi, Silvia; Scarcello, Antonio; Pagliaro, Pasqualepaolo

2015-10-01

Molecular genetic studies on Duffy blood group antigens have identified mutations underlying rare FY*Null and FY*X alleles. FY*Null has a high frequency in Blacks, especially from sub-Saharan Africa, while its frequency is not defined in Caucasians. FY*X allele, associated with Fy(a-b+w) phenotype, has a frequency of 2-3.5% in Caucasian people while it is absent in Blacks. During the project of extensive blood group genotyping in patients affected by hemoglobinopathies, we identified FY*X/FY*Null and FY*A/FY*Null genotypes in a Caucasian thalassemic family from Sardinia. We speculate on the frequency of FY*X and FY*Null alleles in Caucasian and Black people; further, we focused on the association of FY*X allele with weak Fyb antigen expression on red blood cells and its identification performing high sensitivity serological typing methods or genotyping. Copyright © 2015 Elsevier Ltd. All rights reserved.

Recruitment of DNA Replication and Damage Response Proteins to Viral Replication Centers during Infection with NS2 Mutants of Minute Virus of Mice (MVM)

PubMed Central

Ruiz, Zandra; Mihaylov, Ivailo S.; Cotmore, Susan F.; Tattersall, Peter

2010-01-01

MVM NS2 is essential for viral DNA amplification, but its mechanism of action is unknown. A classification scheme for autonomous parvovirus-associated replication (APAR) center development, based on NS1 distribution, was used to characterize abnormal APAR body maturation in NS2null mutant infections, and their organization examined for defects in host protein recruitment. Since acquisition of known replication factors appeared normal, we looked for differences in invoked DNA damage responses. We observed widespread association of H2AX/MDC1 damage response foci with viral replication centers, and sequestration and complex hyperphosphorylation of RPA32, which occurred in wildtype and mutant infections. Quantifying these responses by western transfer indicated that both wildtype and NS2 mutant MVM elicited ATM activation, while phosphorylation of ATR, already basally activated in asynchronous A9 cells, was downregulated. We conclude that MVM infection invokes multiple damage responses that influence the APAR environment, but that NS2 does not modify the recruitment of cellular proteins. PMID:21193212

Recruitment of DNA replication and damage response proteins to viral replication centers during infection with NS2 mutants of Minute Virus of Mice (MVM).

PubMed

Ruiz, Zandra; Mihaylov, Ivailo S; Cotmore, Susan F; Tattersall, Peter

2011-02-20

MVM NS2 is essential for viral DNA amplification, but its mechanism of action is unknown. A classification scheme for autonomous parvovirus-associated replication (APAR) center development, based on NS1 distribution, was used to characterize abnormal APAR body maturation in NS2null mutant infections, and their organization examined for defects in host protein recruitment. Since acquisition of known replication factors appeared normal, we looked for differences in invoked DNA damage responses. We observed widespread association of H2AX/MDC1 damage response foci with viral replication centers, and sequestration and complex hyperphosphorylation of RPA(32), which occurred in wildtype and mutant infections. Quantifying these responses by western transfer indicated that both wildtype and NS2 mutant MVM elicited ATM activation, while phosphorylation of ATR, already basally activated in asynchronous A9 cells, was downregulated. We conclude that MVM infection invokes multiple damage responses that influence the APAR environment, but that NS2 does not modify the recruitment of cellular proteins. Copyright © 2010 Elsevier Inc. All rights reserved.

Genes and Alcohol Consumption: Studies with Mutant Mice

PubMed Central

Mayfield, Jody; Arends, Michael A.; Harris, R. Adron; Blednov, Yuri A.

2017-01-01

In this chapter, we review the effects of global null mutant and overexpressing transgenic mouse lines on voluntary self-administration of alcohol. We examine approximately 200 publications pertaining to the effects of 155 mouse genes on alcohol consumption in different drinking models. The targeted genes vary in function and include neurotransmitter, ion channel, neuroimmune, and neuropeptide signaling systems. The alcohol self-administration models include operant conditioning, two- and four-bottle choice continuous and intermittent access, drinking in the dark limited access, chronic intermittent ethanol, and scheduled high alcohol consumption tests. Comparisons of different drinking models using the same mutant mice are potentially the most informative, and we will highlight those examples. More mutants have been tested for continuous two-bottle choice consumption than any other test; of the 137 mouse genes examined using this model, 97 (72%) altered drinking in at least one sex. Overall, the effects of genetic manipulations on alcohol drinking often depend on the sex of the mice, alcohol concentration and time of access, genetic background, as well as the drinking test. PMID:27055617

Brucella abortus Cyclic β-1,2-Glucan Mutants Have Reduced Virulence in Mice and Are Defective in Intracellular Replication in HeLa Cells

PubMed Central

Briones, Gabriel; Iñón de Iannino, Nora; Roset, Mara; Vigliocco, Ana; Paulo, Patricia Silva; Ugalde, Rodolfo A.

2001-01-01

Null cyclic β-1,2-glucan synthetase mutants (cgs mutants) were obtained from Brucella abortus virulent strain 2308 and from B. abortus attenuated vaccinal strain S19. Both mutants show greater sensitivity to surfactants like deoxycholic acid, sodium dodecyl sulfate, and Zwittergent than the parental strains, suggesting cell surface alterations. Although not to the same extent, both mutants display reduced virulence in mice and defective intracellular multiplication in HeLa cells. The B. abortus S19 cgs mutant was completely cleared from the spleens of mice after 4 weeks, while the 2308 mutant showed a 1.5-log reduction of the number of brucellae isolated from the spleens after 12 weeks. These results suggest that cyclic β-1,2-glucan plays an important role in the residual virulence of the attenuated B. abortus S19 strain. Although the cgs mutant was cleared from the spleens earlier than the wild-type parental strain (B. abortus S19) and produced less inflammatory response, its ability to confer protection against the virulent strain B. abortus 2308 was fully retained. Equivalent levels of induction of spleen gamma interferon mRNA and anti-lipopolysaccharide (LPS) of immunoglobulin G2a (IgG2a) subtype antibodies were observed in mice injected with B. abortus S19 or the cgs mutant. However, the titer of anti-LPS antibodies of the IgG1 subtype induced by the cgs mutant was lower than that observed with the parental S19 strain, thus suggesting that the cgs mutant induces a relatively exclusive Th1 response. PMID:11401996

Mammalian Exo1 encodes both structural and catalytic functions that play distinct roles in essential biological processes

PubMed Central

Schaetzlein, Sonja; Chahwan, Richard; Avdievich, Elena; Roa, Sergio; Wei, Kaichun; Eoff, Robert L.; Sellers, Rani S.; Clark, Alan B.; Kunkel, Thomas A.; Scharff, Matthew D.; Edelmann, Winfried

2013-01-01

Mammalian Exonuclease 1 (EXO1) is an evolutionarily conserved, multifunctional exonuclease involved in DNA damage repair, replication, immunoglobulin diversity, meiosis, and telomere maintenance. It has been assumed that EXO1 participates in these processes primarily through its exonuclease activity, but recent studies also suggest that EXO1 has a structural function in the assembly of higher-order protein complexes. To dissect the enzymatic and nonenzymatic roles of EXO1 in the different biological processes in vivo, we generated an EXO1-E109K knockin (Exo1EK) mouse expressing a stable exonuclease-deficient protein and, for comparison, a fully EXO1-deficient (Exo1null) mouse. In contrast to Exo1null/null mice, Exo1EK/EK mice retained mismatch repair activity and displayed normal class switch recombination and meiosis. However, both Exo1-mutant lines showed defects in DNA damage response including DNA double-strand break repair (DSBR) through DNA end resection, chromosomal stability, and tumor suppression, indicating that the enzymatic function is required for those processes. On a transformation-related protein 53 (Trp53)-null background, the DSBR defect caused by the E109K mutation altered the tumor spectrum but did not affect the overall survival as compared with p53-Exo1null mice, whose defects in both DSBR and mismatch repair also compromised survival. The separation of these functions demonstrates the differential requirement for the structural function and nuclease activity of mammalian EXO1 in distinct DNA repair processes and tumorigenesis in vivo. PMID:23754438

The Chlamydomonas Dhc1 gene encodes a dynein heavy chain subunit required for assembly of the I1 inner arm complex.

PubMed Central

Myster, S H; Knott, J A; O'Toole, E; Porter, M E

1997-01-01

Multiple members of the dynein heavy chain (Dhc) gene family have been recovered in several organisms, but the relationships between these sequences and the Dhc isoforms that they encode are largely unknown. To identify Dhc loci and determine the specific functions of the individual Dhc isoforms, we have screened a collection of motility mutants generated by insertional mutagenesis in Chlamydomonas. In this report, we characterize one strain, pf9-3, in which the insertion event was accompanied by a deletion of approximately 13 kb of genomic DNA within the transcription unit of the Dhc1 gene. Northern blot analysis confirms that pf9-3 is a null mutation. Biochemical and structural studies of isolated axonemes demonstrate that the pf9-3 mutant fails to assemble the I1 inner arm complex, a two-headed dynein isoform composed of two Dhcs (1 alpha and 1 beta) and three intermediate chains. To determine if the Dhc1 gene product corresponds to one of the Dhcs of the I1 complex, antibodies were generated against a Dhc1-specific peptide sequence. Immunoblot analysis reveals that the Dhc1 gene encodes the 1 alpha Dhc subunit. These studies thus, identify the first inner arm Dhc locus to be described in any organism and further demonstrate that the 1 alpha Dhc subunit plays an essential role in the assembly of the I1 inner arm complex. Images PMID:9247642

A novel human pain insensitivity disorder caused by a point mutation in ZFHX2

PubMed Central

Habib, Abdella M; Matsuyama, Ayako; Okorokov, Andrei L; Santana-Varela, Sonia; Bras, Jose T; Aloisi, Anna Maria; Emery, Edward C; Bogdanov, Yury D; Follenfant, Maryne; Gossage, Sam J; Gras, Mathilde; Humphrey, Jack; Kolesnikov, Anna; Le Cann, Kim; Li, Shengnan; Minett, Michael S; Pereira, Vanessa; Ponsolles, Clara; Sikandar, Shafaq; Torres, Jesus M; Yamaoka, Kenji; Zhao, Jing; Komine, Yuriko; Yamamori, Tetsuo; Maniatis, Nikolas; Panov, Konstantin I; Houlden, Henry; Ramirez, Juan D; Bennett, David L H; Marsili, Letizia; Bachiocco, Valeria; Wood, John N; Cox, James J

2018-01-01

Abstract Chronic pain is a major global public health issue causing a severe impact on both the quality of life for sufferers and the wider economy. Despite the significant clinical burden, little progress has been made in terms of therapeutic development. A unique approach to identifying new human-validated analgesic drug targets is to study rare families with inherited pain insensitivity. Here we have analysed an otherwise normal family where six affected individuals display a pain insensitive phenotype that is characterized by hyposensitivity to noxious heat and painless bone fractures. This autosomal dominant disorder is found in three generations and is not associated with a peripheral neuropathy. A novel point mutation in ZFHX2, encoding a putative transcription factor expressed in small diameter sensory neurons, was identified by whole exome sequencing that segregates with the pain insensitivity. The mutation is predicted to change an evolutionarily highly conserved arginine residue 1913 to a lysine within a homeodomain. Bacterial artificial chromosome (BAC) transgenic mice bearing the orthologous murine p.R1907K mutation, as well as Zfhx2 null mutant mice, have significant deficits in pain sensitivity. Gene expression analyses in dorsal root ganglia from mutant and wild-type mice show altered expression of genes implicated in peripheral pain mechanisms. The ZFHX2 variant and downstream regulated genes associated with a human pain-insensitive phenotype are therefore potential novel targets for the development of new analgesic drugs. PMID:29253101

MDS1, a dosage suppressor of an mck1 mutant, encodes a putative yeast homolog of glycogen synthase kinase 3.

PubMed Central

Puziss, J W; Hardy, T A; Johnson, R B; Roach, P J; Hieter, P

1994-01-01

The yeast gene MCK1 encodes a serine/threonine protein kinase that is thought to function in regulating kinetochore activity and entry into meiosis. Disruption of MCK1 confers a cold-sensitive phenotype, a temperature-sensitive phenotype, and sensitivity to the microtubule-destabilizing drug benomyl and leads to loss of chromosomes during growth on benomyl. A dosage suppression selection was used to identify genes that, when present at high copy number, could suppress the cold-sensitive phenotype of mck1::HIS3 mutant cells. Several unique classes of clones were identified, and one of these, designated MDS1, has been characterized in some detail. Nucleotide sequence data reveal that MDS1 encodes a serine/threonine protein kinase that is highly homologous to the shaggy/zw3 kinase in Drosophila melanogaster and its functional homolog, glycogen synthase kinase 3, in rats. The presence of MDS1 in high copy number rescues both the cold-sensitive and the temperature-sensitive phenotypes, but not the benomyl-sensitive phenotype, associated with the disruption of MCK1. Analysis of strains harboring an mds1 null mutation demonstrates that MDS1 is not essential during normal vegetative growth but appears to be required for meiosis. Finally, in vitro experiments indicate that the proteins encoded by both MCK1 and MDS1 possess protein kinase activity with substrate specificity similar to that of mammalian glycogen synthase kinase 3. Images PMID:8264650

TET2 mutations predict response to hypomethylating agents in myelodysplastic syndrome patients

PubMed Central

Lord, Allegra; Stevenson, Kristen; Bar-Natan, Michal; Pérez-Ladaga, Albert; Zaneveld, Jacques; Wang, Hui; Caughey, Bennett; Stojanov, Petar; Getz, Gad; Garcia-Manero, Guillermo; Kantarjian, Hagop; Chen, Rui; Stone, Richard M.; Neuberg, Donna; Steensma, David P.; Ebert, Benjamin L.

2014-01-01

Only a minority of myelodysplastic syndrome (MDS) patients respond to hypomethylating agents (HMAs), but strong predictors of response are unknown. We sequenced 40 recurrently mutated myeloid malignancy genes in tumor DNA from 213 MDS patients collected before treatment with azacitidine (AZA) or decitabine (DEC). Mutations were examined for association with response and overall survival. The overall response rate of 47% was not different between agents. Clonal TET2 mutations predicted response (odds ratio [OR] 1.99, P = .036) when subclones unlikely to be detected by Sanger sequencing (allele fraction <10%) were treated as wild-type (WT). Response rates were highest in the subset of TET2 mutant patients without clonal ASXL1 mutations (OR 3.65, P = .009). Mutations of TP53 (hazard ratio [HR] 2.01, P = .002) and PTPN11 (HR 3.26, P = .006) were associated with shorter overall survival but not drug response. Murine-competitive bone marrow transplantation followed by treatment with AZA demonstrated that Tet2-null cells have an engraftment advantage over Tet2-WT cells. AZA significantly decreased this advantage for Tet2-null cells (P = .002) but not Tet2-WT cells (P = .212). Overall, Tet2 loss appears to sensitize cells to treatment with AZA in vivo, and TET2 mutations can identify patients more likely to respond to HMAs. PMID:25224413

TET2 mutations predict response to hypomethylating agents in myelodysplastic syndrome patients.

PubMed

Bejar, Rafael; Lord, Allegra; Stevenson, Kristen; Bar-Natan, Michal; Pérez-Ladaga, Albert; Zaneveld, Jacques; Wang, Hui; Caughey, Bennett; Stojanov, Petar; Getz, Gad; Garcia-Manero, Guillermo; Kantarjian, Hagop; Chen, Rui; Stone, Richard M; Neuberg, Donna; Steensma, David P; Ebert, Benjamin L

2014-10-23

Only a minority of myelodysplastic syndrome (MDS) patients respond to hypomethylating agents (HMAs), but strong predictors of response are unknown. We sequenced 40 recurrently mutated myeloid malignancy genes in tumor DNA from 213 MDS patients collected before treatment with azacitidine (AZA) or decitabine (DEC). Mutations were examined for association with response and overall survival. The overall response rate of 47% was not different between agents. Clonal TET2 mutations predicted response (odds ratio [OR] 1.99, P = .036) when subclones unlikely to be detected by Sanger sequencing (allele fraction <10%) were treated as wild-type (WT). Response rates were highest in the subset of TET2 mutant patients without clonal ASXL1 mutations (OR 3.65, P = .009). Mutations of TP53 (hazard ratio [HR] 2.01, P = .002) and PTPN11 (HR 3.26, P = .006) were associated with shorter overall survival but not drug response. Murine-competitive bone marrow transplantation followed by treatment with AZA demonstrated that Tet2-null cells have an engraftment advantage over Tet2-WT cells. AZA significantly decreased this advantage for Tet2-null cells (P = .002) but not Tet2-WT cells (P = .212). Overall, Tet2 loss appears to sensitize cells to treatment with AZA in vivo, and TET2 mutations can identify patients more likely to respond to HMAs. © 2014 by The American Society of Hematology.

Tissue-specific roles for sonic hedgehog signaling in establishing thymus and parathyroid organ fate

PubMed Central

Bain, Virginia E.; Gordon, Julie; O'Neil, John D.; Ramos, Isaias; Richie, Ellen R.

2016-01-01

The thymus and parathyroids develop from third pharyngeal pouch (3rd pp) endoderm. Our previous studies show that Shh null mice have smaller, aparathyroid primordia in which thymus fate specification extends into the pharynx. SHH signaling is active in both dorsal pouch endoderm and neighboring neural crest (NC) mesenchyme. It is unclear which target tissue of SHH signaling is required for the patterning defects in Shh mutants. Here, we used a genetic approach to ectopically activate or delete the SHH signal transducer Smo in either pp endoderm or NC mesenchyme. Although no manipulation recapitulated the Shh null phenotype, manipulation of SHH signaling in either the endoderm or NC mesenchyme had direct and indirect effects on both cell types during fate specification and organogenesis. SHH pathway activation throughout pouch endoderm activated ectopic Tbx1 expression and partially suppressed the thymus-specific transcription factor Foxn1, identifying Tbx1 as a key target of SHH signaling in the 3rd pp. However, ectopic SHH signaling was insufficient to expand the GCM2-positive parathyroid domain, indicating that multiple inputs, some of which might be independent of SHH signaling, are required for parathyroid fate specification. These data support a model in which SHH signaling plays both positive and negative roles in patterning and organogenesis of the thymus and parathyroids. PMID:27633995

DOE Office of Scientific and Technical Information (OSTI.GOV)

Burles, Kristin; Irwin, Chad R.; Burton, Robyn-Lee

Currently, little is known about the ankyrin/F-box protein B4. Here, we report that B4R-null viruses exhibited reduced plaque size in tissue culture, and decreased ability to spread, as assessed by multiple-step growth analysis. Electron microscopy indicated that B4R-null viruses still formed mature and extracellular virions; however, there was a slight decrease of virions released into the media following deletion of B4R. Deletion of B4R did not affect the ability of the virus to rearrange actin; however, VACV811, a large vaccinia virus deletion mutant missing 55 open reading frames, had decreased ability to produce actin tails. Using ectromelia virus, a naturalmore » mouse pathogen, we demonstrated that virus devoid of EVM154, the B4R homolog, showed decreased spread to organs and was attenuated during infection. This initial characterization suggests that B4 may play a role in virus spread, and that other unidentified mediators of actin tail formation may exist in vaccinia virus. - Highlights: • B4R-null viruses show reduced plaque size, and decreased ability to spread. • B4R-null viruses formed mature and extracellular virions; and rearranged actin. • Virus devoid of EVM154, the B4R homolog, was attenuated during infection. • Initial characterization suggests that B4 may play a role in virus spread. • Unidentified mediators of actin tail formation may exist in vaccinia virus.« less

Mapped Clone and Functional Analysis of Leaf-Color Gene Ygl7 in a Rice Hybrid (Oryza sativa L. ssp. indica)

PubMed Central

Deng, Xiao-juan; Zhang, Hai-qing; Wang, Yue; He, Feng; Liu, Jin-ling; Xiao, Xiao; Shu, Zhi-feng; Li, Wei; Wang, Guo-huai; Wang, Guo-liang

2014-01-01

Leaf-color is an effective marker to identify the hybridization of rice. Leaf-color related genes function in chloroplast development and the photosynthetic pigment biosynthesis of higher plants. The ygl7 (yellow-green leaf 7) is a mutant with spontaneous yellow-green leaf phenotype across the whole lifespan but with no change to its yield traits. We cloned gene Ygl7 (Os03g59640) which encodes a magnesium-chelatase ChlD protein. Expression of ygl7 turns green-leaves to yellow, whereas RNAi-mediated silence of Ygl7 causes a lethal phenotype of the transgenic plants. This indicates the importance of the gene for rice plant. On the other hand, it corroborates that ygl7 is a non-null mutants. The content of photosynthetic pigment is lower in Ygl7 than the wild type, but its light efficiency was comparatively high. All these results indicated that the mutational YGL7 protein does not cause a complete loss of original function but instead acts as a new protein performing a new function. This new function partially includes its preceding function and possesses an additional feature to promote photosynthesis. Chl1, Ygl98, and Ygl3 are three alleles of the OsChlD gene that have been documented previously. However, mutational sites of OsChlD mutant gene and their encoded protein products were different in the three mutants. The three mutants have suppressed grain output. In our experiment, plant materials of three mutants (ygl7, chl1, and ygl98) all exhibited mutational leaf-color during the whole growth period. This result was somewhat different from previous studies. We used ygl7 as female crossed with chl1 and ygl98, respectively. Both the F1 and F2 generation display yellow-green leaf phenotype with their chlorophyll and carotenoid content falling between the values of their parents. Moreover, we noted an important phenomenon: ygl7-NIL's leaf-color is yellow, not yellowy-green, and this is also true of all back-crossed offspring with ygl7. PMID:24932524

Mapped clone and functional analysis of leaf-color gene Ygl7 in a rice hybrid (Oryza sativa L. ssp. indica).

PubMed

Deng, Xiao-juan; Zhang, Hai-qing; Wang, Yue; He, Feng; Liu, Jin-ling; Xiao, Xiao; Shu, Zhi-feng; Li, Wei; Wang, Guo-huai; Wang, Guo-liang

2014-01-01

Leaf-color is an effective marker to identify the hybridization of rice. Leaf-color related genes function in chloroplast development and the photosynthetic pigment biosynthesis of higher plants. The ygl7 (yellow-green leaf 7) is a mutant with spontaneous yellow-green leaf phenotype across the whole lifespan but with no change to its yield traits. We cloned gene Ygl7 (Os03g59640) which encodes a magnesium-chelatase ChlD protein. Expression of ygl7 turns green-leaves to yellow, whereas RNAi-mediated silence of Ygl7 causes a lethal phenotype of the transgenic plants. This indicates the importance of the gene for rice plant. On the other hand, it corroborates that ygl7 is a non-null mutants. The content of photosynthetic pigment is lower in Ygl7 than the wild type, but its light efficiency was comparatively high. All these results indicated that the mutational YGL7 protein does not cause a complete loss of original function but instead acts as a new protein performing a new function. This new function partially includes its preceding function and possesses an additional feature to promote photosynthesis. Chl1, Ygl98, and Ygl3 are three alleles of the OsChlD gene that have been documented previously. However, mutational sites of OsChlD mutant gene and their encoded protein products were different in the three mutants. The three mutants have suppressed grain output. In our experiment, plant materials of three mutants (ygl7, chl1, and ygl98) all exhibited mutational leaf-color during the whole growth period. This result was somewhat different from previous studies. We used ygl7 as female crossed with chl1 and ygl98, respectively. Both the F1 and F2 generation display yellow-green leaf phenotype with their chlorophyll and carotenoid content falling between the values of their parents. Moreover, we noted an important phenomenon: ygl7-NIL's leaf-color is yellow, not yellowy-green, and this is also true of all back-crossed offspring with ygl7.

Evaluation of Multimodal Imaging Biomarkers of Prostate Cancer

DTIC Science & Technology

2015-09-01

and PET images. Figure 2 highlights the dynamic uptake of TSPO as compared to muscle. Across 60 minutes the %ID/cc continues to increase which is...p53 double null mutant mouse model. Towards that end, we have successfully acquired anatomic MRI and PET data in orthotopic tumors within the Pten...castration resistant prostate cancer, MRI, PET , FDHT, image optimization 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18. NUMBER OF PAGES

An extensive allelic series of Drosophila kae1 mutants reveals diverse and tissue-specific requirements for t6A biogenesis

PubMed Central

Lin, Ching-Jung; Smibert, Peter; Zhao, Xiaoyu; Hu, Jennifer F.; Ramroop, Johnny; Kellner, Stefanie M.; Benton, Matthew A.; Govind, Shubha; Dedon, Peter C.; Sternglanz, Rolf; Lai, Eric C.

2015-01-01

N6-threonylcarbamoyl-adenosine (t6A) is one of the few RNA modifications that is universally present in life. This modification occurs at high frequency at position 37 of most tRNAs that decode ANN codons, and stabilizes cognate anticodon–codon interactions. Nearly all genetic studies of the t6A pathway have focused on single-celled organisms. In this study, we report the isolation of an extensive allelic series in the Drosophila ortholog of the core t6A biosynthesis factor Kae1. kae1 hemizygous larvae exhibit decreases in t6A that correlate with allele strength; however, we still detect substantial t6A-modified tRNAs even during the extended larval phase of null alleles. Nevertheless, complementation of Drosophila Kae1 and other t6A factors in corresponding yeast null mutants demonstrates that these metazoan genes execute t6A synthesis. Turning to the biological consequences of t6A loss, we characterize prominent kae1 melanotic masses and show that they are associated with lymph gland overgrowth and ectopic generation of lamellocytes. On the other hand, kae1 mutants exhibit other phenotypes that reflect insufficient tissue growth. Interestingly, whole-tissue and clonal analyses show that strongly mitotic tissues such as imaginal discs are exquisitely sensitive to loss of kae1, whereas nonproliferating tissues are less affected. Indeed, despite overt requirements of t6A for growth of many tissues, certain strong kae1 alleles achieve and sustain enlarged body size during their extended larval phase. Our studies highlight tissue-specific requirements of the t6A pathway in a metazoan context and provide insights into the diverse biological roles of this fundamental RNA modification during animal development and disease. PMID:26516084

Deficiency of Sbds in the mouse pancreas leads to features of Shwachman-Diamond syndrome, with loss of zymogen granules.

PubMed

Tourlakis, Marina E; Zhong, Jian; Gandhi, Rikesh; Zhang, Siyi; Chen, Lingling; Durie, Peter R; Rommens, Johanna M

2012-08-01

Shwachman-Diamond syndrome (SDS) is the second leading cause of hereditary exocrine pancreatic dysfunction. More than 90% of patients with SDS have biallelic loss-of-function mutations in the Shwachman-Bodian Diamond syndrome (SBDS) gene, which encodes a factor involved in ribosome function. We investigated whether mutations in Sbds lead to similar pancreatic defects in mice. Pancreas-specific knock-out mice were generated using a floxed Sbds allele and bred with mice carrying a null or disease-associated missense Sbds allele. Cre recombinase, regulated by the pancreatic transcription factor 1a promoter, was used to disrupt Sbds specifically in the pancreas. Models were assessed for pancreatic dysfunction and growth impairment. Disruption of Sbds in the mouse pancreas was sufficient to recapitulate SDS phenotypes. Pancreata of mice with Sbds mutations had decreased mass, fat infiltration, but general preservation of ductal and endocrine compartments. Pancreatic extracts from mutant mice had defects in formation of the 80S ribosomal complex. The exocrine compartment of mutant mice was hypoplastic and individual acini produced few zymogen granules. The null Sbds allele resulted in an earlier onset of phenotypes as well as endocrine impairment. Mutant mice had reduced serum levels of digestive enzymes and overall growth impairment. We developed a mouse model of SDS with pancreatic phenotypes similar to those of the human disease. This model could be used to investigate organ-specific consequences of Sbds-associated ribosomopathy. Sbds genotypes correlated with phenotypes. Defects developed specifically in the pancreata of mice, reducing growth of mice and production of digestive enzymes. SBDS therefore appears to be required for normal pancreatic development and function. Copyright © 2012 AGA Institute. Published by Elsevier Inc. All rights reserved.

Dishevelled 2 is essential for cardiac outflow tract development, somite segmentation and neural tube closure.

PubMed

Hamblet, Natasha S; Lijam, Nardos; Ruiz-Lozano, Pilar; Wang, Jianbo; Yang, Yasheng; Luo, Zhenge; Mei, Lin; Chien, Kenneth R; Sussman, Daniel J; Wynshaw-Boris, Anthony

2002-12-01

The murine dishevelled 2 (Dvl2) gene is an ortholog of the Drosophila segment polarity gene Dishevelled, a member of the highly conserved Wingless/Wnt developmental pathway. Dvl2-deficient mice were produced to determine the role of Dvl2 in mammalian development. Mice containing null mutations in Dvl2 present with 50% lethality in both inbred 129S6 and in a hybrid 129S6-NIH Black Swiss background because of severe cardiovascular outflow tract defects, including double outlet right ventricle, transposition of the great arteries and persistent truncus arteriosis. The majority of the surviving Dvl2(-/-) mice were female, suggesting that penetrance was influenced by sex. Expression of Pitx2 and plexin A2 was attenuated in Dvl2 null mutants, suggesting a defect in cardiac neural crest development during outflow tract formation. In addition, approximately 90% of Dvl2(-/-) mice have vertebral and rib malformations that affect the proximal as well as the distal parts of the ribs. These skeletal abnormalities were more pronounced in mice deficient for both Dvl1 and Dvl2. Somite differentiation markers used to analyze Dvl2(-/-) and Dvl1(-/-);Dvl2(-/-) mutant embryos revealed mildly aberrant expression of Uncx4.1, delta 1 and myogenin, suggesting defects in somite segmentation. Finally, 2-3% of Dvl2(-/-) embryos displayed thoracic spina bifida, while virtually all Dvl1/2 double mutant embryos displayed craniorachishisis, a completely open neural tube from the midbrain to the tail. Thus, Dvl2 is essential for normal cardiac morphogenesis, somite segmentation and neural tube closure, and there is functional redundancy between Dvl1 and Dvl2 in some phenotypes.

EMK protein kinase-null mice: dwarfism and hypofertility associated with alterations in the somatotrope and prolactin pathways.

PubMed

Bessone, S; Vidal, F; Le Bouc, Y; Epelbaum, J; Bluet-Pajot, M T; Darmon, M

1999-10-01

Gene trapping was used in embryonic stem (ES) cells in an attempt to inactivate genes involved in development. The Emk (ELKL motif kinase) gene has been disrupted and a mutant mouse line derived. Previous work had shown that EMK kinases, called MARK in the rat, exert a major control on microtubule stability by phosphorylating microtubule-associated proteins and that genes homologous to Emk in yeast or Caenorhabditis elegans are essential for cell and embryonic polarity. Although we found the Emk gene to be active in the preimplantation mouse embryo and then to show a widespread expression, Emk-null mice had no embryonic defect and were viable. They show an overall proportionate dwarfism and a peculiar hypofertility: homozygotes are not fertile when intercrossed, but are fertile in other types of crosses. Insulin-like growth factor I (IGF I) and IGF-binding protein 3 (IGFBP3) were reduced in the plasma of homozygotes of both sexes. A direct implication of the EMK kinase in IGF I plasmatic production is unlikely because the Emk gene does not seem to be expressed in hepatocytes. Nevertheless, GH assayed at arbitrary times in plasma did not show differences between genotypes and GH concentrations in pituitary extracts were not found to be altered in homozygotes. Our results, though, do not exclude the possibility that in the mutants the overall quantity of GH secreted daily is reduced. Our observation of a smaller size of the pituitaries of the mutants is in favor of this hypothesis. The prolactin concentration in the pituitaries was much lowered in homozygous females, but it was normal in males. The possible involvement of EMK protein kinase in hormone secretion in the pituitary and/or the hypothalamus, via the microtubule network, is discussed. Copyright 1999 Academic Press.

Glucosylceramide is Critical for Cell-Type Differentiation and Organogenesis, but not for Cell Viability in Arabidopsis

PubMed Central

Msanne, Joseph; Chen, Ming; Luttgeharm, Kyle D.; Bradley, Amanda M.; Mays, Elizabeth S.; Paper, Janet M.; Boyle, Daniel L.; Cahoon, Rebecca E.; Schrick, Kathrin; Cahoon, Edgar B.

2015-01-01

Summary Glucosylceramides (GlcCer), glucose-conjugated sphingolipids, are major components of the endomembrane system and plasma membrane in most eukaryote cells. Yet, the quantitative significance and cellular functions of GlcCer are not well characterized in plants and other multi-organ eukaryotes. To address this, we examined Arabidopsis lines lacking or deficient in GlcCer by insertional disruption or by RNAi suppression of the single gene for GlcCer synthase (GCS, At2g19880), the enzyme that catalyzes GlcCer synthesis. Null mutants for GCS (designated “gcs-1”) were viable as seedlings, albeit strongly reduced in size, and failed to develop beyond the seedling stage. Heterozygous plants harboring the insertion allele exhibited reduced transmission through the male gametophyte. Undifferentiated calli generated from gcs-1 seedlings and lacking GlcCer proliferated in a manner similar to calli from wild-type plants. However, gcs-1 calli, in contrast to wild-type calli, were unable to develop organs on differentiation media. Consistent with a role for GlcCer in organ-specific cell differentiation, calli from gcs-1 mutants formed roots and leaves on media supplemented with the glucosylated sphingosine glucopsychosine, which was readily converted to GlcCer independent of GCS. Underlying these phenotypes, gcs-1 cells had altered Golgi morphology and fewer cisternae per Golgi apparatus relative to wild-type cells, indicative of protein trafficking defects. Despite seedling lethality in the null mutant, GCS RNAi suppression lines with ≤2% of wild-type GlcCer levels were viable and fertile. Collectively, these results indicate that GlcCer are essential for cell-type differentiation and organogenesis, and plant cells produce GlcCer amounts in excess of that required for normal development. PMID:26313010

CASK regulates CaMKII autophosphorylation in neuronal growth, calcium signaling, and learning

PubMed Central

Gillespie, John M.; Hodge, James J. L.

2013-01-01

Calcium (Ca2+)/calmodulin (CaM)-dependent kinase II (CaMKII) activity plays a fundamental role in learning and memory. A key feature of CaMKII in memory formation is its ability to be regulated by autophosphorylation, which switches its activity on and off during synaptic plasticity. The synaptic scaffolding protein CASK (calcium (Ca2+)/calmodulin (CaM) associated serine kinase) is also important for learning and memory, as mutations in CASK result in intellectual disability and neurological defects in humans. We show that in Drosophila larvae, CASK interacts with CaMKII to control neuronal growth and calcium signaling. Furthermore, deletion of the CaMK-like and L27 domains of CASK (CASK β null) or expression of overactive CaMKII (T287D) produced similar effects on synaptic growth and Ca2+ signaling. CASK overexpression rescues the effects of CaMKII overactivity, consistent with the notion that CASK and CaMKII act in a common pathway that controls these neuronal processes. The reduction in Ca2+ signaling observed in the CASK β null mutant caused a decrease in vesicle trafficking at synapses. In addition, the decrease in Ca2+ signaling in CASK mutants was associated with an increase in Ether-à-go-go (EAG) potassium (K+) channel localization to synapses. Reducing EAG restored the decrease in Ca2+ signaling observed in CASK mutants to the level of wildtype, suggesting that CASK regulates Ca2+ signaling via EAG. CASK knockdown reduced both appetitive associative learning and odor evoked Ca2+ responses in Drosophila mushroom bodies, which are the learning centers of Drosophila. Expression of human CASK in Drosophila rescued the effect of CASK deletion on the activity state of CaMKII, suggesting that human CASK may also regulate CaMKII autophosphorylation. PMID:24062638

A modified acetylcholine receptor δ-subunit enables a null mutant to survive beyond sexual maturation

PubMed Central

Epley, Kimberly E.; Urban, Jason M.; Ikenaga, Takanori; Ono, Fumihito

2008-01-01

The contraction of skeletal muscle is dependent upon synaptic transmission through acetylcholine receptors (AChRs) at the neuromuscular junction (NMJ). The lack of an AChR subunit causes a fetal akinesia in humans, leading to death in the first trimester and characteristic features of Fetal Akinesia Deformation Sequences (FADS). A corresponding null mutation of the δ-subunit in zebrafish (sofa potato; sop−/−) leads to the death of embryos around 5 days post-fertilization (dpf). In sop−/− mutants, we expressed modified δ-subunits, with one (δ1YFP) or two yellow fluorescent protein (δ2YFP) molecules fused at the intracellular loop, under the control of an α-actin promoter. AChRs containing these fusion proteins are fluorescent, assemble on the plasma membrane, make clusters under motor neuron endings, and generate synaptic current. We screened for germ-line transmission of the transgene and established a line of sop−/− fish stably expressing the δ2YFP. These δ2YFP/sop−/− embryos can mount escape behavior close to that of their wild type siblings. Synaptic currents in these embryos had a smaller amplitude, slower rise time, and slower decay when compared to wild type fish. Remarkably, these embryos grow to adulthood and display complex behaviors such as feeding and breeding. To the best of our knowledge, this is the first case of a mutant animal corresponding to first trimester lethality in human that has been rescued by a transgene and survived to adulthood. In the rescued fish, a foreign promoter drove the transgene expression and the NMJ had altered synaptic strength. The survival of the transgenic animal delineates requirements for gene therapies of NMJ. PMID:19052214

Mice That Lack Thrombospondin 2 Display Connective Tissue Abnormalities That Are Associated with Disordered Collagen Fibrillogenesis, an Increased Vascular Density, and a Bleeding Diathesis

PubMed Central

Kyriakides, Themis R.; Zhu, Yu-Hong; Smith, Lynne T.; Bain, Steven D.; Yang, Zhantao; Lin, Ming T.; Danielson, Keith G.; Iozzo, Renato V.; LaMarca, Mary; McKinney, Cindy E.; Ginns, Edward I.; Bornstein, Paul

1998-01-01

Thrombospondin (TSP) 2, and its close relative TSP1, are extracellular proteins whose functions are complex, poorly understood, and controversial. In an attempt to determine the function of TSP2, we disrupted the Thbs2 gene by homologous recombination in embryonic stem cells, and generated TSP2-null mice by blastocyst injection and appropriate breeding of mutant animals. Thbs2−/− mice were produced with the expected Mendelian frequency, appeared overtly normal, and were fertile. However, on closer examination, these mice displayed a wide variety of abnormalities. Collagen fiber patterns in skin were disordered, and abnormally large fibrils with irregular contours were observed by electron microscopy in both skin and tendon. As a functional correlate of these findings, the skin was fragile and had reduced tensile strength, and the tail was unusually flexible. Mutant skin fibroblasts were defective in attachment to a substratum. An increase in total density and in cortical thickness of long bones was documented by histology and quantitative computer tomography. Mutant mice also manifested an abnormal bleeding time, and histologic surveys of mouse tissues, stained with an antibody to von Willebrand factor, showed a significant increase in blood vessels. The basis for the unusual phenotype of the TSP2-null mouse could derive from the structural role that TSP2 might play in collagen fibrillogenesis in skin and tendon. However, it seems likely that some of the diverse manifestations of this genetic disorder result from the ability of TSP2 to modulate the cell surface properties of mesenchymal cells, and thus, to affect cell functions such as adhesion and migration. PMID:9442117

Knocking off the suppressors of cytokine signaling (SOCS): their roles in mammalian pregnancy.

PubMed

Fitzgerald, Justine S; Toth, Bettina; Jeschke, Udo; Schleussner, Ekkehard; Markert, Udo R

2009-12-01

This review discusses the possible role of the suppressor of cytokine signaling (SOCS) proteins in mammalian reproduction. SOCS are regulatory proteins that are rapidly transcribed in response to intracellular Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling, a cascade governing biological functions including cytokine-induced immunological responses and reproductive processes. For instance STAT3 appears to mediate trophoblast invasion induced by LIF. The SOCS family includes 8 members (cytokine-inducible SH2 protein [CIS] and SOCS1-7) that orchestrate distinct reactions by antagonizing STAT activation. Emerging evidence points to a role of some family members in synchronizing Th1/Th2 cell profiles, the balance in which is considered vital to pregnancy maintenance. The reproductive phenotypes of mutant mice harboring targeted disruption of SOCS gene isoforms offer insights for reproductive immunology, trophoblast function and human pregnancy. CIS transgenic mice display impaired responses to IL-2 and resemble STAT5 deficient mice, except they are fertile. SOCS1 deficiency leads to an overabundance of IFNgamma signaling, yet SOCS1 null mutant mice are able to reproduce. Lack of SOCS3 is embryonically lethal due to placental insufficiency, while SOCS3 over-expression leads to elevated Th2 responses. SOCS3 seems to be vital for reproduction by regulating LIF-driven trophoblast differentiation. SOCS5 inhibits IL-4 signaling, yet the SOCS5 transgenic mouse has no conspicuous reproductive phenotype. SOCS-6 and SOCS-7 null mutant mice display growth retardation. In summary, SOCS proteins are avidly involved in fine regulation of immunological and other vital cellular responses. Many of the above phenotypes present contradictions to accepted reproductive immunological paradigms.

Novel functions for glycosyltransferases Jhp0562 and GalT in Lewis antigen synthesis and variation in Helicobacter pylori.

PubMed

Pohl, Mary Ann; Kienesberger, Sabine; Blaser, Martin J

2012-04-01

Lewis (Le) antigens are fucosylated oligosaccharides present in the Helicobacter pylori lipopolysaccharide. Expression of these antigens is believed to be important for H. pylori colonization, since Le antigens also are expressed on the gastric epithelia in humans. A galactosyltransferase encoded by β-(1,3)galT is essential for production of type 1 (Le(a) and Le(b)) antigens. The upstream gene jhp0562, which is present in many but not all H. pylori strains, is homologous to β-(1,3)galT but is of unknown function. Because H. pylori demonstrates extensive intragenomic recombination, we hypothesized that these two genes could undergo DNA rearrangement. A PCR screen and subsequent sequence analyses revealed that the two genes can recombine at both the 5' and 3' ends. Chimeric β-(1,3)galT-like alleles can restore function in a β-(1,3)galT null mutant, but neither native nor recombinant jhp0562 can. Mutagenesis of jhp0562 revealed that it is essential for synthesis of both type 1 and type 2 Le antigens. Transcriptional analyses of both loci showed β-(1,3)galT expression in all wild-type (WT) and mutant strains tested, whereas jhp0562 was not expressed in jhp0562 null mutants, as expected. Since jhp0562 unexpectedly displayed functions in both type 1 and type 2 Le synthesis, we asked whether galT, part of the type 2 synthesis pathway, had analogous functions in type 1 synthesis. Mutagenesis and complementation analysis confirmed that galT is essential for Le(b) production. In total, these results demonstrate that galT and jhp0562 have functions that cross the expected Le synthesis pathways and that jhp0562 provides a substrate for intragenomic recombination to generate diverse Le synthesis enzymes.

Novel Functions for Glycosyltransferases Jhp0562 and GalT in Lewis Antigen Synthesis and Variation in Helicobacter pylori

PubMed Central

Kienesberger, Sabine; Blaser, Martin J.

2012-01-01

Lewis (Le) antigens are fucosylated oligosaccharides present in the Helicobacter pylori lipopolysaccharide. Expression of these antigens is believed to be important for H. pylori colonization, since Le antigens also are expressed on the gastric epithelia in humans. A galactosyltransferase encoded by β-(1,3)galT is essential for production of type 1 (Lea and Leb) antigens. The upstream gene jhp0562, which is present in many but not all H. pylori strains, is homologous to β-(1,3)galT but is of unknown function. Because H. pylori demonstrates extensive intragenomic recombination, we hypothesized that these two genes could undergo DNA rearrangement. A PCR screen and subsequent sequence analyses revealed that the two genes can recombine at both the 5′ and 3′ ends. Chimeric β-(1,3)galT-like alleles can restore function in a β-(1,3)galT null mutant, but neither native nor recombinant jhp0562 can. Mutagenesis of jhp0562 revealed that it is essential for synthesis of both type 1 and type 2 Le antigens. Transcriptional analyses of both loci showed β-(1,3)galT expression in all wild-type (WT) and mutant strains tested, whereas jhp0562 was not expressed in jhp0562 null mutants, as expected. Since jhp0562 unexpectedly displayed functions in both type 1 and type 2 Le synthesis, we asked whether galT, part of the type 2 synthesis pathway, had analogous functions in type 1 synthesis. Mutagenesis and complementation analysis confirmed that galT is essential for Leb production. In total, these results demonstrate that galT and jhp0562 have functions that cross the expected Le synthesis pathways and that jhp0562 provides a substrate for intragenomic recombination to generate diverse Le synthesis enzymes. PMID:22290141

Candida albicans ISW2 Regulates Chlamydospore Suspensor Cell Formation and Virulence In Vivo in a Mouse Model of Disseminated Candidiasis

PubMed Central

Lionakis, Michail S.; Nickerson, Kenneth W.

2016-01-01

Formation of chlamydospores by Candida albicans was an established medical diagnostic test to confirm candidiasis before the molecular era. However, the functional role and pathological relevance of this in vitro morphological transition to pathogenesis in vivo remain unclear. We compared the physical properties of in vitro-induced chlamydospores with those of large C. albicans cells purified by density gradient centrifugation from Candida-infected mouse kidneys. The morphological and physical properties of these cells in kidneys of mice infected intravenously with wild type C. albicans confirmed that chlamydospores can form in infected kidneys. A previously reported chlamydospore-null Δisw2/Δisw2 mutant was used to investigate its role in virulence and chlamydospore induction. Virulence of the Δisw2/Δisw2 mutant strain was reduced 3.4-fold compared to wild type C. albicans or the ISW2 reconstituted strain. Altered host inflammatory reactions to the null mutant further indicate that ISW2 is a virulence factor in C. albicans. ISW2 deletion abolished chlamydospore formation within infected mouse kidneys, whereas the reconstituted strain restored chlamydospore formation in kidneys. Under chlamydospore inducing conditions in vitro, deletion of ISW2 significantly delayed chlamydospore formation, and those late induced chlamydospores lacked associated suspensor cells while attaching laterally to hyphae via novel spore-hypha septa. Our findings establish the induction of chlamydospores by C. albicans during mouse kidney colonization. Our results indicate that ISW2 is not strictly required for chlamydospores formation but is necessary for suspensor cell formation. The importance of ISW2 in chlamydospore morphogenesis and virulence may lead to additional insights into morphological differentiation and pathogenesis of C. albicans in the host microenvironment. PMID:27727302

An in silico agent-based model demonstrates Reelin function in directing lamination of neurons during cortical development.

PubMed

Caffrey, James R; Hughes, Barry D; Britto, Joanne M; Landman, Kerry A

2014-01-01

The characteristic six-layered appearance of the neocortex arises from the correct positioning of pyramidal neurons during development and alterations in this process can cause intellectual disabilities and developmental delay. Malformations in cortical development arise when neurons either fail to migrate properly from the germinal zones or fail to cease migration in the correct laminar position within the cortical plate. The Reelin signalling pathway is vital for correct neuronal positioning as loss of Reelin leads to a partially inverted cortex. The precise biological function of Reelin remains controversial and debate surrounds its role as a chemoattractant or stop signal for migrating neurons. To investigate this further we developed an in silico agent-based model of cortical layer formation. Using this model we tested four biologically plausible hypotheses for neuron motility and four biologically plausible hypotheses for the loss of neuron motility (conversion from migration). A matrix of 16 combinations of motility and conversion rules was applied against the known structure of mouse cortical layers in the wild-type cortex, the Reelin-null mutant, the Dab1-null mutant and a conditional Dab1 mutant. Using this approach, many combinations of motility and conversion mechanisms can be rejected. For example, the model does not support Reelin acting as a repelling or as a stopping signal. In contrast, the study lends very strong support to the notion that the glycoprotein Reelin acts as a chemoattractant for neurons. Furthermore, the most viable proposition for the conversion mechanism is one in which conversion is affected by a motile neuron sensing in the near vicinity neurons that have already converted. Therefore, this model helps elucidate the function of Reelin during neuronal migration and cortical development.

Miro GTPase controls mitochondrial behavior affecting stress tolerance and virulence of a fungal insect pathogen.

PubMed

Guan, Yi; Wang, Ding-Yi; Ying, Sheng-Hua; Feng, Ming-Guang

2016-08-01

Miro homologues are small mitochondrial Rho GTPases belonging to the Ras superfamily across organisms and are generally unexplored in filamentous fungi. Here we identified a Miro orthologue (bMiro) in Beauveria bassiana, a filamentous fungal insect pathogen as a classic biological control agent of insect pests. This orthologue was proven to anchor on mitochondrial outer membrane in a manner depending completely upon a short C-terminal transmembrane domain. As a result of bmiro deletion, mitochondria in hyphal cells were largely aggregated, and their mass and mobility were reduced, accompanied with a remarkable decrease in ATP content but little change in mitochondrial morphology. The deletion mutant became 42%, 37%, 19% and 10% more tolerant to Ca(2+), Mn(2+), Zn(2+) and Mg(2+) than wild-type, respectively, during cultivation in a minimal medium under normal conditions. The deletion mutant also showed mild defects in conidial germination, vegetative growth, thermotolerance, UV-B resistance and virulence despite null response to oxidative and osmotic stresses. All these phenotypic changes were restored by targeted gene complementation. Our results indicate that bMiro can control mitochondrial distribution and movement required for the transport of ATP-form energy and metal ions and contributes significantly to the fungal potential against insect pests through the control. Copyright © 2016 Elsevier Inc. All rights reserved.

Transcriptional organization and in vivo role of the Escherichia coli rsd gene, encoding the regulator of RNA polymerase sigma D.

PubMed

Jishage, M; Ishihama, A

1999-06-01

The regulator of sigma D (Rsd) was identified as an RNA polymerase sigma70-associated protein in stationary-phase Escherichia coli with the inhibitory activity of sigma70-dependent transcription in vitro (M. Jishage and A. Ishihama, Proc. Natl. Acad. Sci. USA 95:4953-4958, 1998). Primer extension analysis of rsd mRNA indicated the presence of two promoters, sigmaS-dependent P1 and sigma70-dependent P2 with the gearbox sequence. To get insight into the in vivo role of Rsd, the expression of a reporter gene fused to either the sigma70- or sigmaS-dependent promoter was analyzed in the absence of Rsd or the presence of overexpressed Rsd. In the rsd null mutant, the sigma70- and sigmaS-dependent gene expression was increased or decreased, respectively. On the other hand, the sigma70- or sigmaS-dependent transcription was reduced or enhanced, respectively, after overexpression of Rsd. The repression of the sigmaS-dependent transcription in the rsd mutant is overcome by increased production of the sigmaS subunit. Together these observations support the prediction that Rsd is involved in replacement of the RNA polymerase sigma subunit from sigma70 to sigmaS during the transition from exponential growth to the stationary phase.

Discovery of a Coregulatory Interaction between Kaposi's Sarcoma-Associated Herpesvirus ORF45 and the Viral Protein Kinase ORF36.

PubMed

Avey, Denis; Tepper, Sarah; Pifer, Benjamin; Bahga, Amritpal; Williams, Hunter; Gillen, Joseph; Li, Wenwei; Ogden, Sarah; Zhu, Fanxiu

2016-07-01

Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of three human malignancies. KSHV ORF36 encodes a serine/threonine viral protein kinase, which is conserved throughout all herpesviruses. Although several studies have identified the viral and cellular substrates of conserved herpesvirus protein kinases (CHPKs), the precise functions of KSHV ORF36 during lytic replication remain elusive. Here, we report that ORF36 interacts with another lytic protein, ORF45, in a manner dependent on ORF36 kinase activity. We mapped the regions of ORF36 and ORF45 involved in the binding. Their association appears to be mediated by electrostatic interactions, since deletion of either the highly basic N terminus of ORF36 or an acidic patch of ORF45 abolished the binding. In addition, the dephosphorylation of ORF45 protein dramatically reduced its association with ORF36. Importantly, ORF45 enhances both the stability and kinase activity of ORF36. Consistent with previous studies of CHPK homologs, we detected ORF36 protein in extracellular virions. To investigate the roles of ORF36 in the context of KSHV lytic replication, we used bacterial artificial chromosome mutagenesis to engineer both ORF36-null and kinase-dead mutants. We found that ORF36-null/mutant virions are moderately defective in viral particle production and are further deficient in primary infection. In summary, our results uncover a functionally important interaction between ORF36 and ORF45 and indicate a significant role of ORF36 in the production of infectious progeny virions. Kaposi's sarcoma-associated herpesvirus (KSHV) is a human tumor virus with a significant public health burden. KSHV ORF36 encodes a serine/threonine viral protein kinase, whose functions throughout the viral life cycle have not been elucidated. Here, we report that ORF36 interacts with another KSHV protein, ORF45. We mapped the regions of ORF36 and ORF45 involved in their association and further characterized the consequences of this interaction. We engineered ORF36 mutant viruses in order to investigate the functional roles of ORF36 in the context of KSHV lytic replication, and we confirmed that ORF36 is a component of KSHV virions. Moreover, we found that ORF36 mutants are defective in virion production and primary infection. In summary, we discovered and characterized a functionally important interaction between KSHV ORF36 and ORF45, and our results suggest a significant role of ORF36 in the production of infectious progeny virions, a process critical for KSHV pathogenesis. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Generation and characterization of Kctd15 mutations in zebrafish

PubMed Central

Heffer, Alison; Marquart, Gregory D.; Aquilina-Beck, Allisan; Saleem, Nabil; Burgess, Harold A.

2017-01-01

Potassium channel tetramerization domain containing 15 (Kctd15) was previously found to have a role in early neural crest (NC) patterning, specifically delimiting the region where NC markers are expressed via repression of transcription factor AP-2a and inhibition of Wnt signaling. We used transcription activator-like effector nucleases (TALENs) to generate null mutations in zebrafish kctd15a and kctd15b paralogs to study the in vivo role of Kctd15. We found that while deletions producing frame-shift mutations in each paralog showed no apparent phenotype, kctd15a/b double mutant zebrafish are smaller in size and show several phenotypes including some affecting the NC, such as expansion of the early NC domain, increased pigmentation, and craniofacial defects. Both melanophore and xanthophore pigment cell numbers and early markers are up-regulated in the double mutants. While we find no embryonic craniofacial defects, adult mutants have a deformed maxillary segment and missing barbels. By confocal imaging of mutant larval brains we found that the torus lateralis (TLa), a region implicated in gustatory networks in other fish, is absent. Ablation of this brain tissue in wild type larvae mimics some aspects of the mutant growth phenotype. Thus kctd15 mutants show deficits in the development of both neural crest derivatives, and specific regions within the central nervous system, leading to a strong reduction in normal growth rates. PMID:29216270

Aspergillus flavus GPI-anchored protein-encoding ecm33 has a role in growth, development, aflatoxin biosynthesis, and maize infection.

PubMed

Chang, Perng-Kuang; Zhang, Qi; Scharfenstein, Leslie; Mack, Brian; Yoshimi, Akira; Miyazawa, Ken; Abe, Keietsu

2018-06-01

Many glycosylphosphatidylinositol-anchored proteins (GPI-APs) of fungi are membrane enzymes, organization components, and extracellular matrix adhesins. We analyzed eight Aspergillus flavus transcriptome sets for the GPI-AP gene family and identified AFLA_040110, AFLA_063860, and AFLA_113120 to be among the top 5 highly expressed genes of the 36 family genes analyzed. Disruption of the former two genes did not drastically affect A. flavus growth and development. In contrast, disruption of AFLA_113120, an orthologue of Saccharomyces cerevisiae ECM33, caused a significant decrease in vegetative growth and conidiation, promoted sclerotial production, and altered conidial pigmentation. The A. flavus ecm33 null mutant, compared with the wild type and the complemented strain, produced predominantly aflatoxin B 2 but accumulated comparable amounts of cyclopiazonic acid. It showed decreased sensitivity to Congo red at low concentrations (25-50 μg/mL) but had increased sensitivity to calcofluor white at high concentrations (250-500 μg/mL). Analyses of cell wall carbohydrates indicated that the α-glucan content was decreased significantly (p < 0.05), but the contents of chitin and ß-glucan were increased in the mutant strain. In a maize colonization study, the mutant was shown to be impaired in its infectivity and produced 3- to 4-fold lower amounts of conidia than the wild type and the complemented strain. A. flavus Ecm33 is required for proper cell wall composition and plays an important role in normal fungal growth and development, aflatoxin biosynthesis, and seed colonization.

A cyclic nucleotide-gated channel (CNGC16) in pollen is critical for stress tolerance in pollen reproductive development.

PubMed

Tunc-Ozdemir, Meral; Tang, Chong; Ishka, Maryam Rahmati; Brown, Elizabeth; Groves, Norman R; Myers, Candace T; Rato, Claudia; Poulsen, Lisbeth R; McDowell, Stephen; Miller, Gad; Mittler, Ron; Harper, Jeffrey F

2013-02-01

Cyclic nucleotide-gated channels (CNGCs) have been implicated in diverse aspects of plant growth and development, including responses to biotic and abiotic stress, as well as pollen tube growth and fertility. Here, genetic evidence identifies CNGC16 in Arabidopsis (Arabidopsis thaliana) as critical for pollen fertility under conditions of heat stress and drought. Two independent transfer DNA disruptions of cngc16 resulted in a greater than 10-fold stress-dependent reduction in pollen fitness and seed set. This phenotype was fully rescued through pollen expression of a CNGC16 transgene, indicating that cngc16-1 and 16-2 were both loss-of-function null alleles. The most stress-sensitive period for cngc16 pollen was during germination and the initiation of pollen tube tip growth. Pollen viability assays indicate that mutant pollen are also hypersensitive to external calcium chloride, a phenomenon analogous to calcium chloride hypersensitivities observed in other cngc mutants. A heat stress was found to increase concentrations of 3',5'-cyclic guanyl monophosphate in both pollen and leaves, as detected using an antibody-binding assay. A quantitative PCR analysis indicates that cngc16 mutant pollen have attenuated expression of several heat-stress response genes, including two heat shock transcription factor genes, HsfA2 and HsfB1. Together, these results provide evidence for a heat stress response pathway in pollen that connects a cyclic nucleotide signal, a Ca(2+)-permeable ion channel, and a signaling network that activates a downstream transcriptional heat shock response.

A mathematical model of communication between groups of circadian neurons in Drosophila melanogaster.

PubMed

Risau-Gusman, Sebastián; Gleiser, Pablo M

2014-12-01

In the fruit fly, circadian behavior is controlled by a small number of specialized neurons, whose molecular clocks are relatively well known. However, much less is known about how these neurons communicate among themselves. In particular, only 1 circadian neuropeptide, pigment-dispersing factor (PDF), has been identified, and most aspects of its interaction with the molecular clock remain to be elucidated. Furthermore, it is speculated that many other peptides should contribute to circadian communication. We have developed a relatively detailed model of the 2 main groups of circadian pacemaker neurons (sLNvs and LNds) to investigate these issues. We have proposed many possible mechanisms for the interaction between the synchronization factors and the molecular clock, and we have compared the outputs with the experimental results reported in the literature both for the wild-type and PDF-null mutant. We have studied how different the properties of each neuron should be to account for the observations reported for the sLNvs in the mutant. We have found that only a few mechanisms, mostly related to the slowing down of nuclear entry of a circadian protein, can synchronize neurons that present these differences. Detailed immunofluorescent recordings have suggested that, whereas in the mutant, LNd neurons are synchronized, in the wild-type, a subset of the LNds oscillate faster than the rest. With our model, we find that a more likely explanation for the same observations is that this subset is being driven outside its synchronization range and displays therefore a complex pattern of oscillation.

Synthetic organotelluride compounds induce the reversal of Pdr5p mediated fluconazole resistance in Saccharomyces cerevisiae.

PubMed

Reis de Sá, Leandro Figueira; Toledo, Fabiano Travanca; de Sousa, Bruno Artur; Gonçalves, Augusto César; Tessis, Ana Claudia; Wendler, Edison P; Comasseto, João V; Dos Santos, Alcindo A; Ferreira-Pereira, Antonio

2014-07-26

Resistance to fluconazole, a commonly used azole antifungal, is a challenge for the treatment of fungal infections. Resistance can be mediated by overexpression of ABC transporters, which promote drug efflux that requires ATP hydrolysis. The Pdr5p ABC transporter of Saccharomyces cerevisiae is a well-known model used to study this mechanism of antifungal resistance. The present study investigated the effects of 13 synthetic compounds on Pdr5p. Among the tested compounds, four contained a tellurium-butane group and shared structural similarities that were absent in the other tested compounds: a lateral hydrocarbon chain and an amide group. These four compounds were capable of inhibiting Pdr5p ATPase activity by more than 90%, they demonstrated IC50 values less than 2 μM and had an uncompetitive pattern of Pdr5p ATPase activity inhibition. These organotellurides did not demonstrate cytotoxicity against human erythrocytes or S. cerevisiae mutant strains (a strain that overexpress Pdr5p and a null mutant strain) even in concentrations above 100 μM. When tested at 100 μM, they could reverse the fluconazole resistance expressed by both the S. cerevisiae mutant strain that overexpress Pdr5p and a clinical isolate of Candida albicans. We have identified four organotellurides that are promising candidates for the reversal of drug resistance mediated by drug efflux pumps. These molecules will act as scaffolds for the development of more efficient and effective efflux pump inhibitors that can be used in combination therapy with available antifungals.

Synthetic organotelluride compounds induce the reversal of Pdr5p mediated fluconazole resistance in Saccharomyces cerevisiae

PubMed Central

2014-01-01

Background Resistance to fluconazole, a commonly used azole antifungal, is a challenge for the treatment of fungal infections. Resistance can be mediated by overexpression of ABC transporters, which promote drug efflux that requires ATP hydrolysis. The Pdr5p ABC transporter of Saccharomyces cerevisiae is a well-known model used to study this mechanism of antifungal resistance. The present study investigated the effects of 13 synthetic compounds on Pdr5p. Results Among the tested compounds, four contained a tellurium-butane group and shared structural similarities that were absent in the other tested compounds: a lateral hydrocarbon chain and an amide group. These four compounds were capable of inhibiting Pdr5p ATPase activity by more than 90%, they demonstrated IC50 values less than 2 μM and had an uncompetitive pattern of Pdr5p ATPase activity inhibition. These organotellurides did not demonstrate cytotoxicity against human erythrocytes or S. cerevisiae mutant strains (a strain that overexpress Pdr5p and a null mutant strain) even in concentrations above 100 μM. When tested at 100 μM, they could reverse the fluconazole resistance expressed by both the S. cerevisiae mutant strain that overexpress Pdr5p and a clinical isolate of Candida albicans. Conclusions We have identified four organotellurides that are promising candidates for the reversal of drug resistance mediated by drug efflux pumps. These molecules will act as scaffolds for the development of more efficient and effective efflux pump inhibitors that can be used in combination therapy with available antifungals. PMID:25062749

In Candida parapsilosis the ATC1 Gene Encodes for an Acid Trehalase Involved in Trehalose Hydrolysis, Stress Resistance and Virulence

PubMed Central

Sánchez-Fresneda, Ruth; Martínez-Esparza, María; Maicas, Sergi; Argüelles, Juan-Carlos; Valentín, Eulogio

2014-01-01

An ORF named CPAR2-208980 on contig 005809 was identified by screening a Candida parapsilosis genome data base. Its 67% identity with the acid trehalase sequence from C. albicans (ATC1) led us to designate it CpATC1. Homozygous mutants that lack acid trehalase activity were constructed by gene disruption at the two CpATC1 chromosomal alleles. Phenotypic characterization showed that atc1Δ null cells were unable to grow on exogenous trehalose as carbon source, and also displayed higher resistance to environmental challenges, such as saline exposure (1.2 M NaCl), heat shock (42°C) and both mild and severe oxidative stress (5 and 50 mM H2O2). Significant amounts of intracellular trehalose were specifically stored in response to the thermal upshift in both wild type and mutant strains. Analysis of their antioxidant activities revealed that catalase was only triggered in response to heat shock in atc1Δ cells, whereas glutathione reductase was activated upon mild oxidative stress in wild type and reintegrant strains, and in response to the whole set of stress treatments in the homozygous mutant. Furthermore, yeast cells with double CpATC1 deletion were significantly attenuated in non-mammalian infection models, suggesting that CpATC1 is required for the pathobiology of the fungus. Our results demonstrate the involvement of CpAtc1 protein in the physiological hydrolysis of external trehalose in C. parapsilosis, where it also plays a major role in stress resistance and virulence. PMID:24922533

SOT1, a pentatricopeptide repeat protein with a small MutS-related domain, is required for correct processing of plastid 23S-4.5S rRNA precursors in Arabidopsis thaliana.

PubMed

Wu, Wenjuan; Liu, Sheng; Ruwe, Hannes; Zhang, Delin; Melonek, Joanna; Zhu, Yajuan; Hu, Xupeng; Gusewski, Sandra; Yin, Ping; Small, Ian D; Howell, Katharine A; Huang, Jirong

2016-03-01

Ribosomal RNA processing is essential for plastid ribosome biogenesis, but is still poorly understood in higher plants. Here, we show that SUPPRESSOR OF THYLAKOID FORMATION1 (SOT1), a plastid-localized pentatricopeptide repeat (PPR) protein with a small MutS-related domain, is required for maturation of the 23S-4.5S rRNA dicistron. Loss of SOT1 function leads to slower chloroplast development, suppression of leaf variegation, and abnormal 23S and 4.5S processing. Predictions based on the PPR motif sequences identified the 5' end of the 23S-4.5S rRNA dicistronic precursor as a putative SOT1 binding site. This was confirmed by electrophoretic mobility shift assay, and by loss of the abundant small RNA 'footprint' associated with this site in sot1 mutants. We found that more than half of the 23S-4.5S rRNA dicistrons in sot1 mutants contain eroded and/or unprocessed 5' and 3' ends, and that the endonucleolytic cleavage product normally released from the 5' end of the precursor is absent in a sot1 null mutant. We postulate that SOT1 binding protects the 5' extremity of the 23S-4.5S rRNA dicistron from exonucleolytic attack, and favours formation of the RNA structure that allows endonucleolytic processing of its 5' and 3' ends. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

A Cyclic Nucleotide-Gated Channel (CNGC16) in Pollen Is Critical for Stress Tolerance in Pollen Reproductive Development1[W][OA

PubMed Central

Tunc-Ozdemir, Meral; Tang, Chong; Ishka, Maryam Rahmati; Brown, Elizabeth; Groves, Norman R.; Myers, Candace T.; Rato, Claudia; Poulsen, Lisbeth R.; McDowell, Stephen; Miller, Gad; Mittler, Ron; Harper, Jeffrey F.

2013-01-01

Cyclic nucleotide-gated channels (CNGCs) have been implicated in diverse aspects of plant growth and development, including responses to biotic and abiotic stress, as well as pollen tube growth and fertility. Here, genetic evidence identifies CNGC16 in Arabidopsis (Arabidopsis thaliana) as critical for pollen fertility under conditions of heat stress and drought. Two independent transfer DNA disruptions of cngc16 resulted in a greater than 10-fold stress-dependent reduction in pollen fitness and seed set. This phenotype was fully rescued through pollen expression of a CNGC16 transgene, indicating that cngc16-1 and 16-2 were both loss-of-function null alleles. The most stress-sensitive period for cngc16 pollen was during germination and the initiation of pollen tube tip growth. Pollen viability assays indicate that mutant pollen are also hypersensitive to external calcium chloride, a phenomenon analogous to calcium chloride hypersensitivities observed in other cngc mutants. A heat stress was found to increase concentrations of 3′,5′-cyclic guanyl monophosphate in both pollen and leaves, as detected using an antibody-binding assay. A quantitative PCR analysis indicates that cngc16 mutant pollen have attenuated expression of several heat-stress response genes, including two heat shock transcription factor genes, HsfA2 and HsfB1. Together, these results provide evidence for a heat stress response pathway in pollen that connects a cyclic nucleotide signal, a Ca2+-permeable ion channel, and a signaling network that activates a downstream transcriptional heat shock response. PMID:23370720

Phosphorylation of p53 modifies sensitivity to ionizing radiation.

PubMed

Okaichi, Kumio; Nose, Kanako; Kotake, Takako; Izumi, Nanaka; Kudo, Takashi

2011-06-01

Phosphorylation is an important modification involved in the control of p53 activity. We examined the relationship between p53 phosphorylation and cell radiosensitivity. We prepared H1299 cells (p53-null) with various mutations of p53 at three sites (serine 15, 20 and 46) and examined the radiosensitivity of the cells. In three mutant forms of p53--S15A, S20A and S46A--serine was converted to alanine at these sites to prevent phosphorylation, and in two other mutant forms, S15D and S20D, serine was converted to aspartic acid to mimic phosphorylation. H1299 cells were more radioresistant than cells with wild-type p53. Cells with the S15A and S46A mutant forms of p53 were radiosensitive, whereas those with the S15D, S20A and S20D forms showed medium radiosensitivity. Thus the sensitivity of cells to ionizing radiation varies according to the site of phosphorylation of p53.

Pyruvate decarboxylase provides growing pollen tubes with a competitive advantage in petunia.

PubMed

Gass, Nathalie; Glagotskaia, Tatiana; Mellema, Stefan; Stuurman, Jeroen; Barone, Mario; Mandel, Therese; Roessner-Tunali, Ute; Kuhlemeier, Cris

2005-08-01

Rapid pollen tube growth places unique demands on energy production and biosynthetic capacity. The aim of this work is to understand how primary metabolism meets the demands of such rapid growth. Aerobically grown pollen produce ethanol in large quantities. The ethanolic fermentation pathway consists of two committed enzymes: pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH). Because adh mutations do not affect male gametophyte function, the obvious question is why pollen synthesize an abundant enzyme if they could do just as well without. Using transposon tagging in Petunia hybrida, we isolated a null mutant in pollen-specific Pdc2. Growth of the mutant pollen tubes through the style is reduced, and the mutant allele shows reduced transmission through the male, when in competition with wild-type pollen. We propose that not ADH but rather PDC is the critical enzyme in a novel, pollen-specific pathway. This pathway serves to bypass pyruvate dehydrogenase enzymes and thereby maintain biosynthetic capacity and energy production under the unique conditions prevailing during pollen-pistil interaction.

Alanine–glyoxylate aminotransferase-deficient mice, a model for primary hyperoxaluria that responds to adenoviral gene transfer

PubMed Central

Salido, Eduardo C.; Li, Xiao M.; Lu, Yang; Wang, Xia; Santana, Alfredo; Roy-Chowdhury, Namita; Torres, Armando; Shapiro, Larry J.; Roy-Chowdhury, Jayanta

2006-01-01

Mutations in the alanine–glyoxylate amino transferase gene (AGXT) are responsible for primary hyperoxaluria type I, a rare disease characterized by excessive hepatic oxalate production that leads to renal failure. We generated a null mutant mouse by targeted mutagenesis of the homologous gene, Agxt, in embryonic stem cells. Mutant mice developed normally, and they exhibited hyperoxaluria and crystalluria. Approximately half of the male mice in mixed genetic background developed calcium oxalate urinary stones. Severe nephrocalcinosis and renal failure developed after enhancement of oxalate production by ethylene glycol administration. Hepatic expression of human AGT1, the protein encoded by AGXT, by adenoviral vector-mediated gene transfer in Agxt−/− mice normalized urinary oxalate excretion and prevented oxalate crystalluria. Subcellular fractionation and immunofluorescence studies revealed that, as in the human liver, the expressed wild-type human AGT1 was predominantly localized in mouse hepatocellular peroxisomes, whereas the most common mutant form of AGT1 (G170R) was localized predominantly in the mitochondria. PMID:17110443

c-Type Cytochrome Assembly Is a Key Target of Copper Toxicity within the Bacterial Periplasm

PubMed Central

Durand, Anne; Azzouzi, Asma; Bourbon, Marie-Line; Steunou, Anne-Soisig; Liotenberg, Sylviane; Maeshima, Akinori; Astier, Chantal; Argentini, Manuela; Saito, Shingo

2015-01-01

ABSTRACT In the absence of a tight control of copper entrance into cells, bacteria have evolved different systems to control copper concentration within the cytoplasm and the periplasm. Central to these systems, the Cu+ ATPase CopA plays a major role in copper tolerance and translocates copper from the cytoplasm to the periplasm. The fate of copper in the periplasm varies among species. Copper can be sequestered, oxidized, or released outside the cells. Here we describe the identification of CopI, a periplasmic protein present in many proteobacteria, and show its requirement for copper tolerance in Rubrivivax gelatinosus. The ΔcopI mutant is more susceptible to copper than the Cu+ ATPase copA mutant. CopI is induced by copper, localized in the periplasm and could bind copper. Interestingly, copper affects cytochrome c membrane complexes (cbb3 oxidase and photosystem) in both ΔcopI and copA-null mutants, but the causes are different. In the copA mutant, heme and chlorophyll synthesis are affected, whereas in ΔcopI mutant, the decrease is a consequence of impaired cytochrome c assembly. This impact on c-type cytochromes would contribute also to the copper toxicity in the periplasm of the wild-type cells when they are exposed to high copper concentrations. PMID:26396241

Differential requirement for irf8 in formation of embryonic and adult macrophages in zebrafish

DOE PAGES

Shiau, Celia E.; Kaufman, Zoe; Meireles, Ana M.; ...

2015-01-23

Interferon regulatory factor 8 (Irf8) is critical for mammalian macrophage development and innate immunity, but its role in teleost myelopoiesis remains incompletely understood. Specifically, genetic tools to analyze the role of irf8 in zebrafish macrophage development at larval and adult stages are lacking. In this study, we generated irf8 null mutants in zebrafish using TALEN-mediated targeting. Our analysis defines different requirements for irf8 at different stages. irf8 is required for formation of all macrophages during primitive and transient definitive hematopoiesis, but not during adult-phase definitive hematopoiesis starting at 5-6 days postfertilization. At early stages, irf8 mutants have excess neutrophils andmore » excess cell death in pu.1-expressing myeloid cells. Macrophage fates were recovered in irf8 mutants after wildtype irf8 expression in neutrophil and macrophage lineages, suggesting that irf8 regulates macrophage specification and survival. In juvenile irf8 mutant fish, mature macrophages are present, but at numbers significantly reduced compared to wildtype, indicating an ongoing requirement for irf8 after embryogenesis. As development progresses, tissue macrophages become apparent in zebrafish irf8 mutants, with the possible exception of microglia. Our study defines distinct requirement for irf8 in myelopoiesis before and after transition to the adult hematopoietic system.« less

Engineering an efficient and tight D-amino acid-inducible gene expression system in Rhodosporidium/Rhodotorula species.

PubMed

Liu, Yanbin; Koh, Chong Mei John; Ngoh, Si Te; Ji, Lianghui

2015-10-26

Rhodosporidium and Rhodotorula are two genera of oleaginous red yeast with great potential for industrial biotechnology. To date, there is no effective method for inducible expression of proteins and RNAs in these hosts. We have developed a luciferase gene reporter assay based on a new codon-optimized LUC2 reporter gene (RtLUC2), which is flanked with CAR2 homology arms and can be integrated into the CAR2 locus in the nuclear genome at >90 % efficiency. We characterized the upstream DNA sequence of a D-amino acid oxidase gene (DAO1) from R. toruloides ATCC 10657 by nested deletions. By comparing the upstream DNA sequences of several putative DAO1 homologs of Basidiomycetous fungi, we identified a conserved DNA motif with a consensus sequence of AGGXXGXAGX11GAXGAXGG within a 0.2 kb region from the mRNA translation initiation site. Deletion of this motif led to strong mRNA transcription under non-inducing conditions. Interestingly, DAO1 promoter activity was enhanced about fivefold when the 108 bp intron 1 was included in the reporter construct. We identified a conserved CT-rich motif in the intron with a consensus sequence of TYTCCCYCTCCYCCCCACWYCCGA, deletion or point mutations of which drastically reduced promoter strength under both inducing and non-inducing conditions. Additionally, we created a selection marker-free DAO1-null mutant (∆dao1e) which displayed greatly improved inducible gene expression, particularly when both glucose and nitrogen were present in high levels. To avoid adding unwanted peptide to proteins to be expressed, we converted the original translation initiation codon to ATC and re-created a translation initiation codon at the start of exon 2. This promoter, named P DAO1-in1m1 , showed very similar luciferase activity to the wild-type promoter upon induction with D-alanine. The inducible system was tunable by adjusting the levels of inducers, carbon source and nitrogen source. The intron 1-containing DAO1 promoters coupled with a DAO1 null mutant makes an efficient and tight D-amino acid-inducible gene expression system in Rhodosporidium and Rhodotorula genera. The system will be a valuable tool for metabolic engineering and enzyme expression in these yeast hosts.

Saccharomyces Cerevisiae Cho2 Mutants Are Deficient in Phospholipid Methylation and Cross-Pathway Regulation of Inositol Synthesis

PubMed Central

Summers, E. F.; Letts, V. A.; McGraw, P.; Henry, S. A.

1988-01-01

Five allelic Saccharomyces cerevisiae mutants deficient in the methylation of phosphatidylethanolamine (PE) have been isolated, using two different screening techniques. Biochemical analysis suggested that these mutants define a locus, designated CHO2, that may encode a methyltransferase. Membranes of cho2 mutant cells grown in defined medium contain approximately 10% phosphatidylcholine (PC) and 40-50% PE as compared to wild-type levels of 40-45% PC and 15-20% PE. In spite of this greatly altered phospholipid composition, cho2 mutant cells are viable in defined medium and are not auxotrophic for choline or other phospholipid precursors such as monomethylethanolamine (MME). However, analysis of yeast strains carrying more than one mutation affecting phospholipid biosynthesis indicated that some level of methylated phospholipid is essential for viability. The cho2 locus was shown by tetrad analysis to be unlinked to other loci affecting phospholipid synthesis. Interestingly, cho2 mutants and other mutant strains that produce reduced levels of methylated phospholipids are unable to properly repress synthesis of the cytoplasmic enzyme inositol-1-phosphate synthase. This enzyme was previously shown to be regulated at the level of mRNA abundance in response to inositol and choline in the growth medium. We cloned the CHO2 gene on a 3.6-kb genomic DNA fragment and created a null allele of cho2 by disrupting the CHO2 gene in vivo. The cho2 disruptant, like all other cho2 mutants, is viable, exhibits altered regulation of inositol biosynthesis and is not auxotrophic for choline or MME. PMID:3066687

Drosophila CHIP protects against mitochondrial dysfunction by acting downstream of Pink1 in parallel with Parkin.

PubMed

Chen, Jia; Xue, Jin; Ruan, Jingsong; Zhao, Juan; Tang, Beisha; Duan, Ranhui

2017-12-01

Mitochondrial kinase PTEN-induced putative kinase 1 (PINK1) and E3 ubiquitin ligase Parkin function in a common pathway to regulate mitochondrial homeostasis contributing to the pathogenesis of Parkinson disease. The carboxyl terminus of Hsc70-interacting protein (CHIP) acts as a heat shock protein 70/heat shock protein 90 cochaperone to mediate protein folding or as an E3 ubiquitin ligase to target proteins for degradation. In this study, overexpression of Drosophila CHIP suppressed a range of Pink1 mutant phenotypes in flies, including abnormal wing posture, thoracic indentation, locomotion defects, muscle degeneration, and loss of dopaminergic neurons. Mitochondrial defects of Pink1 mutant, such as excessive fusion, reduced ATP content, and crista disorganization, were rescued by CHIP but not its ligase-dead mutants. Similar phenotypes and mitochondrial impairment were ameliorated in Parkin mutant flies by wild-type CHIP. Inactivation of CHIP with null fly mutants resulted in mitochondrial defects, such as reduced thoracic ATP content at 3 d old, decreased thoracic mitochondrial DNA content, and defective mitochondrial morphology at 60 d old. CHIP mutants did not exacerbate the phenotypes of Pink1 mutant flies but markedly shortened the life span of Parkin mutant flies. These results indicate that CHIP is involved in mitochondrial integrity and may act downstream of Pink1 in parallel with Parkin.-Chen, J., Xue, J., Ruan, J., Zhao, J., Tang, B., Duan, R. Drosophila CHIP protects against mitochondrial dysfunction by acting downstream of Pink1 in parallel with Parkin. © FASEB.

ICBP90 Regulation of DNA Methylation, Histone Ubiquitination, and Tumor Suppressor Gene Expression in Breast Cancer Cells

DTIC Science & Technology

2013-09-01

accomplishments include creation of relevant plant lines, development of in vitro assays, and profiling of mRNA expression in null mutants. 15. SUBJECT TERMS...DNA methylation, UHRF1, VIM1, ubiquitination, epigenetics, chromatin 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18. NUMBER OF...Molecular Basis of Human Disease ,” which covered several weeks’ worth of material specifically related to the molecular and epigenetic basis of cancer

Targeting SRC Family Kinases and HSP90 in Lung Cancer

DTIC Science & Technology

2016-12-01

inhalation of Adeno-Cre, followed by MRI imaging at regular intervals to detect tumor initiation and growth, followed by euthanasia and processing of...experimental endpoint. 10 mice were used per time point Representative MRI data describing tumor volume (TV) are shown in Figure 1. Quantification of data is...dasatinib, we were able to make several conclusions. Figure 1. Representative MRI images from Nedd9wt or Nedd9 null Kras mutant mice, treated with

Investigating the Role of Helicobacter pylori PriA Protein.

PubMed

Singh, Aparna; Blaskovic, Dusan; Joo, Jungsoo; Yang, Zhen; Jackson, Sharon H; Coleman, William G; Yan, Ming

2016-08-01

In bacteria, PriA protein, a conserved DEXH-type DNA helicase, plays a central role in replication restart at stalled replication forks. Its unique DNA binding property allows it to recognize and stabilize stalled forks and the structures derived from them. PriA plays a very critical role in replication fork stabilization and DNA repair in E. coli and N. gonorrhoeae. In our in vivo expression technology screen, priA gene was induced in vivo when Helicobacter pylori infects mouse stomach. We decided to elucidate the role of H. pylori PriA protein in survival in mouse stomach, survival in gastric epithelial cells and macrophage cells, DNA repair, acid stress, and oxidative stress. The priA null mutant strain was unable to colonize mice stomach mucosa after long-term infections. Mouse colonization was observed after 1 week of infection, but the levels were much lower than the wild-type HpSS1 strain. PriA protein was found to be important for intracellular survival of epithelial cell-/macrophage cell-ingested H. pylori. Also, a priA null mutant was more sensitive to DNA-damaging agents and was much more sensitive to acid and oxidative stress as compared to the wild-type strain. These data suggest that the PriA protein is needed for survival and persistence of H. pylori in mice stomach mucosa. © 2016 John Wiley & Sons Ltd.

Trypanosoma brucei (UMP synthase null mutants) are avirulent in mice, but recover virulence upon prolonged culture in vitro while retaining pyrimidine auxotrophy.

PubMed

Ong, Han B; Sienkiewicz, Natasha; Wyllie, Susan; Patterson, Stephen; Fairlamb, Alan H

2013-10-01

African trypanosomes are capable of both de novo synthesis and salvage of pyrimidines. The last two steps in de novo synthesis are catalysed by UMP synthase (UMPS) - a bifunctional enzyme comprising orotate phosphoribosyl transferase (OPRT) and orotidine monophosphate decarboxylase (OMPDC). To investigate the essentiality of pyrimidine biosynthesis in Trypanosoma brucei, we generated a umps double knockout (DKO) line by gene replacement. The DKO was unable to grow in pyrimidine-depleted medium in vitro, unless supplemented with uracil, uridine, deoxyuridine or UMP. DKO parasites were completely resistant to 5-fluoroorotate and hypersensitive to 5-fluorouracil, consistent with loss of UMPS, but remained sensitive to pyrazofurin indicating that, unlike mammalian cells, the primary target of pyrazofurin is not OMPDC. The null mutant was unable to infect mice indicating that salvage of host pyrimidines is insufficient to support growth. However, following prolonged culture in vitro, parasites regained virulence in mice despite retaining pyrimidine auxotrophy. Unlike the wild-type, both pyrimidine auxotrophs secreted substantial quantities of orotate, significantly higher in the virulent DKO line. We propose that this may be responsible for the recovery of virulence in mice, due to host metabolism converting orotate to uridine, thereby bypassing the loss of UMPS in the parasite. © 2013 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.

STRIP1, a core component of STRIPAK complexes, is essential for normal mesoderm migration in the mouse embryo.

PubMed

Bazzi, Hisham; Soroka, Ekaterina; Alcorn, Heather L; Anderson, Kathryn V

2017-12-19

Regulated mesoderm migration is necessary for the proper morphogenesis and organ formation during embryonic development. Cell migration and its dependence on the cytoskeleton and signaling machines have been studied extensively in cultured cells; in contrast, remarkably little is known about the mechanisms that regulate mesoderm cell migration in vivo. Here, we report the identification and characterization of a mouse mutation in striatin-interacting protein 1 ( Strip1 ) that disrupts migration of the mesoderm after the gastrulation epithelial-to-mesenchymal transition (EMT). STRIP1 is a core component of the biochemically defined mammalian striatin-interacting phosphatases and kinase (STRIPAK) complexes that appear to act through regulation of protein phosphatase 2A (PP2A), but their functions in mammals in vivo have not been examined. Strip1 -null mutants arrest development at midgestation with profound disruptions in the organization of the mesoderm and its derivatives, including a complete failure of the anterior extension of axial mesoderm. Analysis of cultured mesoderm explants and mouse embryonic fibroblasts from null mutants shows that the mesoderm migration defect is correlated with decreased cell spreading, abnormal focal adhesions, changes in the organization of the actin cytoskeleton, and decreased velocity of cell migration. The results show that STRIPAK complexes are essential for cell migration and tissue morphogenesis in vivo. Copyright © 2017 the Author(s). Published by PNAS.

Trio’s Rho-specific GEF domain is the missing Gαq effector in C. elegans

PubMed Central

Williams, Stacey L.; Lutz, Susanne; Charlie, Nicole K.; Vettel, Christiane; Ailion, Michael; Coco, Cassandra; Tesmer, John J.G.; Jorgensen, Erik M.; Wieland, Thomas; Miller, Kenneth G.

2007-01-01

The Gαq pathway is essential for animal life and is a central pathway for driving locomotion, egg laying, and growth in Caenorhabditis elegans, where it exerts its effects through EGL-8 (phospholipase Cβ [PLCβ]) and at least one other effector. To find the missing effector, we performed forward genetic screens to suppress the slow growth and hyperactive behaviors of mutants with an overactive Gαq pathway. Four suppressor mutations disrupted the Rho-specific guanine-nucleotide exchange factor (GEF) domain of UNC-73 (Trio). The mutations produce defects in neuronal function, but not neuronal development, that cause sluggish locomotion similar to animals lacking EGL-8 (PLCβ). Strains containing null mutations in both EGL-8 (PLCβ) and UNC-73 (Trio RhoGEF) have strong synthetic phenotypes that phenocopy the arrested growth and near-complete paralysis of Gαq-null mutants. Using cell-based and biochemical assays, we show that activated C. elegans Gαq synergizes with Trio RhoGEF to activate RhoA. Activated Gαq and Trio RhoGEF appear to be part of a signaling complex, because they coimmunoprecipitate when expressed together in cells. Our results show that Trio’s Rho-specific GEF domain is a major Gαq effector that, together with PLCβ, mediates the Gαq signaling that drives the locomotion, egg laying, and growth of the animal. PMID:17942708

Arabidopsis SYT1 maintains stability of cortical endoplasmic reticulum networks and VAP27-1-enriched endoplasmic reticulum-plasma membrane contact sites.

PubMed

Siao, Wei; Wang, Pengwei; Voigt, Boris; Hussey, Patrick J; Baluska, Frantisek

2016-11-01

Arabidopsis synaptotagmin 1 (SYT1) is localized on the endoplasmic reticulum-plasma membrane (ER-PM) contact sites in leaf and root cells. The ER-PM localization of Arabidopsis SYT1 resembles that of the extended synaptotagmins (E-SYTs) in animal cells. In mammals, E-SYTs have been shown to regulate calcium signaling, lipid transfer, and endocytosis. Arabidopsis SYT1 was reported to be essential for maintaining cell integrity and virus movement. This study provides detailed insight into the subcellular localization of SYT1 and VAP27-1, another ER-PM-tethering protein. SYT1 and VAP27-1 were shown to be localized on distinct ER-PM contact sites. The VAP27-1-enriched ER-PM contact sites (V-EPCSs) were always in contact with the SYT1-enriched ER-PM contact sites (S-EPCSs). The V-EPCSs still existed in the leaf epidermal cells of the SYT1 null mutant; however, they were less stable than those in the wild type. The polygonal networks of cortical ER disassembled and the mobility of VAP27-1 protein on the ER-PM contact sites increased in leaf cells of the SYT1 null mutant. These results suggest that SYT1 is responsible for stabilizing the ER network and V-EPCSs. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

The mir-279/996 cluster represses receptor tyrosine kinase signaling to determine cell fates in the Drosophila eye.

PubMed

Duan, Hong; de Navas, Luis F; Hu, Fuqu; Sun, Kailiang; Mavromatakis, Yannis E; Viets, Kayla; Zhou, Cyrus; Kavaler, Joshua; Johnston, Robert J; Tomlinson, Andrew; Lai, Eric C

2018-04-09

Photoreceptors in the crystalline Drosophila eye are recruited by receptor tyrosine kinase (RTK)/Ras signaling mediated by Epidermal growth factor receptor (EGFR) and the Sevenless (Sev) receptor. Analyses of an allelic deletion series of the mir-279/996 locus, along with a panel of modified genomic rescue transgenes, show that Drosophila eye patterning depends on both miRNAs. Transcriptional reporter and activity sensor transgenes reveal expression and function of miR-279/996 in non-neural cells of the developing eye. Moreover, mir-279/996 mutants exhibit substantial numbers of ectopic photoreceptors, particularly of R7, and cone cell loss. These miRNAs restrict RTK signaling in the eye, since mir-279/996 nulls are dominantly suppressed by positive components of the EGFR pathway and enhanced by heterozygosity for an EGFR repressor. miR-279/996 limit photoreceptor recruitment by targeting multiple positive RTK/Ras signaling components that promote photoreceptor/R7 specification. Strikingly, deletion of mir-279/996 sufficiently derepresses RTK/Ras signaling so as to rescue a population of R7 cells in R7-specific RTK null mutants boss and sev , which otherwise completely lack this cell fate. Altogether, we reveal a rare setting of developmental cell specification that involves substantial miRNA control. © 2018. Published by The Company of Biologists Ltd.

Leishmania infantum Asparagine Synthetase A Is Dispensable for Parasites Survival and Infectivity.

PubMed

Faria, Joana; Loureiro, Inês; Santarém, Nuno; Macedo-Ribeiro, Sandra; Tavares, Joana; Cordeiro-da-Silva, Anabela

2016-01-01

A growing interest in asparagine (Asn) metabolism has currently been observed in cancer and infection fields. Asparagine synthetase (AS) is responsible for the conversion of aspartate into Asn in an ATP-dependent manner, using ammonia or glutamine as a nitrogen source. There are two structurally distinct AS: the strictly ammonia dependent, type A, and the type B, which preferably uses glutamine. Absent in humans and present in trypanosomatids, AS-A was worthy of exploring as a potential drug target candidate. Appealingly, it was reported that AS-A was essential in Leishmania donovani, making it a promising drug target. In the work herein we demonstrate that Leishmania infantum AS-A, similarly to Trypanosoma spp. and L. donovani, is able to use both ammonia and glutamine as nitrogen donors. Moreover, we have successfully generated LiASA null mutants by targeted gene replacement in L. infantum, and these parasites do not display any significant growth or infectivity defect. Indeed, a severe impairment of in vitro growth was only observed when null mutants were cultured in asparagine limiting conditions. Altogether our results demonstrate that despite being important under asparagine limitation, LiAS-A is not essential for parasite survival, growth or infectivity in normal in vitro and in vivo conditions. Therefore we exclude AS-A as a suitable drug target against L. infantum parasites.

Deletion analysis of Streptococcus pneumoniae late competence genes distinguishes virulence determinants that are dependent or independent of competence induction.

PubMed

Zhu, Luchang; Lin, Jingjun; Kuang, Zhizhou; Vidal, Jorge E; Lau, Gee W

2015-07-01

The competence regulon of Streptococcus pneumoniae (pneumococcus) is crucial for genetic transformation. During competence development, the alternative sigma factor ComX is activated, which in turn, initiates transcription of 80 'late' competence genes. Interestingly, only 16 late genes are essential for genetic transformation. We hypothesized that these late genes that are dispensable for competence are beneficial to pneumococcal fitness during infection. These late genes were systematically deleted, and the resulting mutants were examined for their fitness during mouse models of bacteremia and acute pneumonia. Among these, 14 late genes were important for fitness in mice. Significantly, deletion of some late genes attenuated pneumococcal fitness to the same level in both wild-type and ComX-null genetic backgrounds, suggesting that the constitutive baseline expression of these genes was important for bacterial fitness. In contrast, some mutants were attenuated only in the wild-type genetic background but not in the ComX-null background, suggesting that specific expression of these genes during competence state contributed to pneumococcal fitness. Increased virulence during competence state was partially caused by the induction of allolytic enzymes that enhanced pneumolysin release. These results distinguish the role of basal expression versus competence induction in virulence functions encoded by ComX-regulated late competence genes. © 2015 John Wiley & Sons Ltd.

Mdm-2 binding and TAF(II)31 recruitment is regulated by hydrogen bond disruption between the p53 residues Thr18 and Asp21.

PubMed

Jabbur, James R; Tabor, Amy D; Cheng, Xiaodong; Wang, Hua; Uesugi, Motonari; Lozano, Guillermina; Zhang, Wei

2002-10-10

Analyses of five wild-type p53 containing cell lines revealed lineage specific differences in phosphorylation of Thr18 after treatment with ionizing (IR) or ultraviolet (UV) radiation. Importantly, Thr18 phosphorylation correlated with induction of the p53 downstream targets p21(Waf1/Cip1) (p21) and Mdm-2, suggesting a transactivation enhancing role. Thr18 phosphorylation has been shown to abolish side-chain hydrogen bonding between Thr18 and Asp21, an interaction necessary for stabilizing alpha-helical conformation within the transactivation domain. Mutagenesis-derived hydrogen bond disruption attenuated the interaction of p53 with the transactivation repressor Mdm-2 but had no direct effect on the interaction of p53 with the basal transcription factor TAF(II)31. However, prior incubation of p53 mutants with Mdm-2 modulated TAF(II)31 interaction with p53, suggesting Mdm-2 blocks the accessibility of p53 to TAF(II)31. Consistently, p53-null cells transfected with hydrogen bond disrupting p53 mutants demonstrated enhanced endogenous p21 expression, whereas p53/Mdm-2-double null cells exhibited no discernible differences in p21 expression. We conclude disruption of intramolecular hydrogen bonding between Thr18 and Asp21 enhances p53 transactivation by modulating Mdm-2 binding, facilitating TAF(II)31 recruitment.

Grhl3 and Lmo4 play coordinate roles in epidermal migration.

PubMed

Hislop, Nikki R; Caddy, Jacinta; Ting, Stephen B; Auden, Alana; Vasudevan, Sumitha; King, Sarah L; Lindeman, Geoffrey J; Visvader, Jane E; Cunningham, John M; Jane, Stephen M

2008-09-01

In addition to its role in formation of the epidermal barrier, the mammalian transcription factor Grainy head-like 3 (Grhl3) is also essential for neural tube closure and wound repair, processes that are dependent in part on epidermal migration. Here, we demonstrate that the LIM-only domain protein, LMO4 serves as a functional partner of GRHL3 in its established roles, and define a new cooperative role for these factors in another developmental epidermal migration event, eyelid fusion. GRHL3 and LMO4 interact biochemically and genetically, with mutant mice exhibiting fully penetrant exencephaly, thoraco-lumbo-sacral spina bifida, defective skin barrier formation, and a co-incident eyes-open-at-birth (EOB) phenotype, which is not observed in the original individual null lines. The two genes are co-expressed in the surface ectoderm of the migrating eyelid root, and electron microscopy of Grhl3/Lmo4-null eyes reveals a failure in epithelial extension and a lack of peridermal clump formation at the eyelid margins. Accumulation of actin fibers is also absent in the circumference of these eyelids, and ERK1/2 phosphorylation is lost in the epidermis and eyelids of Grhl3(-/-)/Lmo4(-/-) embryos. Keratinocytes from mutant mice fail to "heal" in in vitro scratch assays, consistent with a general epidermal migratory defect that is dependent on ERK activation and actin cable formation.

Vaccination with a Leishmania infantum HSP70-II null mutant confers long-term protective immunity against Leishmania major infection in two mice models

PubMed Central

Solana, José Carlos; Ramírez, Laura; Corvo, Laura; de Oliveira, Camila Indiani; Barral-Netto, Manoel; Requena, José María

2017-01-01

Background The immunization with genetically attenuated Leishmania cell lines has been associated to the induction of memory and effector T cell responses against Leishmania able to control subsequent challenges. A Leishmania infantum null mutant for the HSP70-II genes has been described, possessing a non-virulent phenotype. Methodology/Principal findings The L. infantum attenuated parasites (LiΔHSP70-II) were inoculated in BALB/c (intravenously and subcutaneously) and C57BL/6 (subcutaneously) mice. An asymptomatic infection was generated and parasites diminished progressively to become undetectable in most of the analyzed organs. However, inoculation resulted in the long-term induction of parasite specific IFN-γ responses able to control the disease caused by a challenge of L. major infective promastigotes. BALB/c susceptible mice showed very low lesion development and a drastic decrease in parasite burdens in the lymph nodes draining the site of infection and internal organs. C57BL/6 mice did not show clinical manifestation of disease, correlated to the rapid migration of Leishmania specific IFN-γ producing T cells to the site of infection. Conclusion/Significance Inoculation of the LiΔHSP70-II attenuated line activates mammalian immune system for inducing moderate pro-inflammatory responses. These responses are able to confer long-term protection in mice against the infection of L. major virulent parasites. PMID:28558043

Pathogenesis of Lethal Cardiac Arrhythmias in Mecp2 Mutant Mice: Implication for Therapy in Rett Syndrome

PubMed Central

McCauley, Mark D.; Wang, Tiannan; Mike, Elise; Herrera, Jose; Beavers, David L.; Huang, Teng-Wei; Ward, Christopher S.; Skinner, Steven; Percy, Alan K.; Glaze, Daniel G.; Wehrens, Xander H. T.; Neul, Jeffrey L.

2013-01-01

Rett Syndrome is a neurodevelopmental disorder typically caused by mutations in Methyl-CpG-Binding Protein 2 (MECP2) in which 26% of deaths are sudden and of unknown cause. To explore the hypothesis that these deaths may be due to cardiac dysfunction, we characterized the electrocardiograms (ECGs) in 379 people with Rett syndrome and found that 18.5% show prolongation of the corrected QT interval (QTc), indicating a repolarization abnormality that can predispose to the development of an unstable fatal cardiac rhythm. Male mice lacking MeCP2 function, Mecp2Null/Y, also have prolonged QTc and show increased susceptibility to induced ventricular tachycardia. Female heterozygous null mice, Mecp2Null/+, show an age-dependent prolongation of QTc associated with ventricular tachycardia and cardiac-related death. Genetic deletion of MeCP2 function in only the nervous system was sufficient to cause long QTc and ventricular tachycardia, implicating neuronally-mediated changes to cardiac electrical conduction as a potential cause of ventricular tachycardia in Rett syndrome. The standard therapy for prolonged QTc in Rett syndrome, β-adrenergic receptor blockers, did not prevent ventricular tachycardia in Mecp2Null/Y mice. To determine whether an alternative therapy would be more appropriate, we characterized cardiomyocytes from Mecp2Null/Y mice and found increased persistent sodium current, which was normalized when cells were treated with the sodium channel-blocking anti-seizure drug phenytoin. Treatment with phenytoin reduced both QTc and sustained ventricular tachycardia in Mecp2Null/Y mice. These results demonstrate that cardiac abnormalities in Rett syndrome are secondary to abnormal nervous system control, which leads to increased persistent sodium current. Our findings suggest that treatment in people with Rett syndrome would be more effective if it targeted the increased persistent sodium current in order to prevent lethal cardiac arrhythmias. PMID:22174313

The relationship of the local food environment with obesity: A systematic review of methods, study quality, and results.

PubMed

Cobb, Laura K; Appel, Lawrence J; Franco, Manuel; Jones-Smith, Jessica C; Nur, Alana; Anderson, Cheryl A M

2015-07-01

To examine the relationship between local food environments and obesity and assess the quality of studies reviewed. Systematic keyword searches identified studies from US and Canada that assessed the relationship of obesity to local food environments. We applied a quality metric based on design, exposure and outcome measurement, and analysis. We identified 71 studies representing 65 cohorts. Overall, study quality was low; 60 studies were cross-sectional. Associations between food outlet availability and obesity were predominantly null. Among non-null associations, we saw a trend toward inverse associations between supermarket availability and obesity (22 negative, 4 positive, 67 null) and direct associations between fast food and obesity (29 positive, 6 negative, 71 null) in adults. We saw direct associations between fast food availability and obesity in lower income children (12 positive, 7 null). Indices including multiple food outlets were most consistently associated with obesity in adults (18 expected, 1 not expected, 17 null). Limiting to higher quality studies did not affect results. Despite the large number of studies, we found limited evidence for associations between local food environments and obesity. The predominantly null associations should be interpreted cautiously due to the low quality of available studies. © 2015 The Obesity Society.

The relationship of the local food environment with obesity: A systematic review of methods, study quality and results

PubMed Central

Cobb, Laura K; Appel, Lawrence J; Franco, Manuel; Jones-Smith, Jessica C; Nur, Alana; Anderson, Cheryl AM

2015-01-01

Objective To examine the relationship between local food environments and obesity and assess the quality of studies reviewed. Methods Systematic keyword searches identified studies from US and Canada that assessed the relationship of obesity to local food environments. We applied a quality metric based on design, exposure and outcome measurement, and analysis. Results We identified 71 studies representing 65 cohorts. Overall, study quality was low; 60 studies were cross-sectional. Associations between food outlet availability and obesity were predominantly null. Among non-null associations, we saw a trend toward inverse associations between supermarket availability and obesity (22 negative, 4 positive, 67 null) and direct associations between fast food and obesity (29 positive, 6 negative, 71 null) in adults. We saw direct associations between fast food availability and obesity in lower income children (12 positive, 7 null). Indices including multiple food outlets were most consistently associated with obesity in adults (18 expected, 1 not expected, 17 null). Limiting to higher quality studies did not affect results. Conclusions Despite the large number of studies, we found limited evidence for associations between local food environments and obesity. The predominantly null associations should be interpreted cautiously due to the low quality of available studies. PMID:26096983

The unique GGA clathrin adaptor of Drosophila melanogaster is not essential.

PubMed

Luan, Shan; Ilvarsonn, Anne M; Eissenberg, Joel C

2012-01-01

The Golgi-localized, γ-ear-containing, ARF binding proteins (GGAs) are a highly conserved family of monomeric clathrin adaptor proteins implicated in clathrin-mediated protein sorting between the trans-Golgi network and endosomes. GGA RNAi knockdowns in Drosophila have resulted in conflicting data concerning whether the Drosophila GGA (dGGA) is essential. The goal of this study was to define the null phenotype for the unique Drosophila GGA. We describe two independently derived dGGA mutations. Neither allele expresses detectable dGGA protein. Homozygous and hemizygous flies with each allele are viable and fertile. In contrast to a previous report using RNAi knockdown, GGA mutant flies show no evidence of age-dependent retinal degeneration or cathepsin missorting. Our results demonstrate that several of the previous RNAi knockdown phenotypes were the result of off-target effects. However, GGA null flies are hypersensitive to dietary chloroquine and to starvation, implicating GGA in lysosomal function and autophagy.

Mutant Kras copy number defines metabolic reprogramming and therapeutic susceptibilities

PubMed Central

Kerr, Emma; Gaude, Edoardo; Turrell, Frances; Frezza, Christian; Martins, Carla P

2016-01-01

Summary The RAS/MAPK-signalling pathway is frequently deregulated in non-small cell lung cancer (NSCLC), often through KRAS activating mutations1-3. A single endogenous mutant Kras allele is sufficient to promote lung tumour formation in mice but malignant progression requires additional genetic alterations4-7. We recently showed that advanced lung tumours from KrasG12D/+;p53-null mice frequently exhibit KrasG12D allelic enrichment (KrasG12D/Kraswild-type>1)7, implying that mutant Kras copy gains are positively selected during progression. Through a comprehensive analysis of mutant Kras homozygous and heterozygous MEFs and lung cancer cells we now show that these genotypes are phenotypically distinct. In particular, KrasG12D/G12D cells exhibit a glycolytic switch coupled to increased channelling of glucose-derived metabolites into the TCA cycle and glutathione biosynthesis, resulting in enhanced glutathione-mediated detoxification. This metabolic rewiring is recapitulated in mutant KRAS homozygous NSCLC cells and in vivo, in spontaneous advanced murine lung tumours (which display a high frequency of KrasG12D copy gain), but not in the corresponding early tumours (KrasG12D heterozygous). Finally, we demonstrate that mutant Kras copy gain creates unique metabolic dependences that can be exploited to selectively target these aggressive mutant Kras tumours. Our data demonstrate that mutant Kras lung tumours are not a single disease but rather a heterogeneous group comprised of two classes of tumours with distinct metabolic profiles, prognosis and therapeutic susceptibility, which can be discriminated based on their relative mutant allelic content. We also provide the first in vivo evidence of metabolic rewiring during lung cancer malignant progression. PMID:26909577

No Significant Increase in the Δ4- and Δ7-Dafachronic Acid Concentration in the Long-Lived glp-1 Mutant, nor in the Mutants Defective in Dauer Formation.

PubMed

Li, Tie-Mei; Liu, Weilong; Lu, Shan; Zhang, Yan-Ping; Jia, Le-Mei; Chen, Jie; Li, Xiangke; Lei, Xiaoguang; Dong, Meng-Qiu

2015-05-12

The steroid hormone dafachronic acid (DA) regulates dauer formation and lifespan in Caenorhabditis elegans by binding to the nuclear receptor DAF-12. However, little is known about how DA concentrations change under various physiologic conditions and about how DA/DAF-12 signaling interacts with other signaling pathways that also regulate dauer formation and lifespan. Using a sensitive bioanalytical method, we quantified the endogenous DA concentrations in a long-lived germline-less glp-1 mutant and in the Dauer formation-defective (Daf-d) mutants daf-12, daf-16, daf-5, and daf-3. We found that the DA concentration in the glp-1 mutant was similar to that in the wild type (WT). This result is contrary to the long-held belief that germline loss-induced longevity involves increased DA production and suggests instead that this type of longevity involves an enhanced response to DA. We also found evidence suggesting that increased DA sensitivity underlies lifespan extension triggered by exogenous DA. At the L2/L3 stage, the DA concentration in a daf-12 null mutant decreased to 22% of the WT level. This finding is consistent with the previously proposed positive feedback regulation between DAF-12 and DA production. Surprisingly, the DA concentrations in the daf-16, daf-5, and daf-3 mutants were only 19-34% of the WT level at the L2/L3 stage, slightly greater than those in the Dauer formation-constitutive (Daf-c) mutants at the pre-dauer stage (4-15% of the WT L2 control). Our experimental evidence suggested that the positive feedback between DA and DAF-12 was partially induced in the three Daf-d mutants. Copyright © 2015 Li et al.

Arabidopsis G-protein β subunit AGB1 interacts with NPH3 and is involved in phototropism.

PubMed

Kansup, Jeeraporn; Tsugama, Daisuke; Liu, Shenkui; Takano, Tetsuo

2014-02-28

Heterotrimeric G proteins (Gα, Gβ and Gγ) have pleiotropic roles in plants, but molecular mechanisms underlying them remain to be elucidated. Here we show that Arabidopsis Gβ (AGB1) interacts with NPH3, a regulator of phototropism. Yeast two-hybrid assays, in vitro pull-down assays and bimolecular fluorescence complementation assays showed that AGB1 and NPH3 physically interact. NPH3-null mutation (nph3) is known to completely abolish hypocotyl phototropism. Loss-of-function mutants of AGB1 (agb1-1 and agb1-2) showed decreased hypocotyl phototropism, and agb1/nph3 double mutants showed no hypocotyl phototropism. These results suggest that AGB1 is involved in the NPH3-mediated regulation of phototropism. Copyright © 2014 Elsevier Inc. All rights reserved.

Dissecting Arabidopsis Gβ Signal Transduction on the Protein Surface1[W][OA

PubMed Central

Jiang, Kun; Frick-Cheng, Arwen; Trusov, Yuri; Delgado-Cerezo, Magdalena; Rosenthal, David M.; Lorek, Justine; Panstruga, Ralph; Booker, Fitzgerald L.; Botella, José Ramón; Molina, Antonio; Ort, Donald R.; Jones, Alan M.

2012-01-01

The heterotrimeric G-protein complex provides signal amplification and target specificity. The Arabidopsis (Arabidopsis thaliana) Gβ-subunit of this complex (AGB1) interacts with and modulates the activity of target cytoplasmic proteins. This specificity resides in the structure of the interface between AGB1 and its targets. Important surface residues of AGB1, which were deduced from a comparative evolutionary approach, were mutated to dissect AGB1-dependent physiological functions. Analysis of the capacity of these mutants to complement well-established phenotypes of Gβ-null mutants revealed AGB1 residues critical for specific AGB1-mediated biological processes, including growth architecture, pathogen resistance, stomata-mediated leaf-air gas exchange, and possibly photosynthesis. These findings provide promising new avenues to direct the finely tuned engineering of crop yield and traits. PMID:22570469

A G protein alpha null mutation confers prolificacy potential in maize

DOE PAGES

Urano, Daisuke; Jackson, David; Jones, Alan M.

2015-05-06

Plasticity in plant development is controlled by environmental signals through largely unknown signalling networks. Signalling coupled by the heterotrimeric G protein complex underlies various developmental pathways in plants. The morphology of two plastic developmental pathways, root system architecture and female inflorescence formation, was quantitatively assessed in a mutant compact plant 2 (ct2) lacking the alpha subunit of the heterotrimeric G protein complex in maize. The ct2 mutant partially compensated for a reduced shoot height by increased total leaf number, and had far more ears, even in the presence of pollination signals. Lastly, the maize heterotrimeric G protein complex is importantmore » in some plastic developmental traits in maize. In particular, the maize Gα subunit is required to dampen the overproduction of female inflorescences.« less

Characterization of highly efficient heavy-ion mutagenesis in Arabidopsis thaliana.

PubMed

Kazama, Yusuke; Hirano, Tomonari; Saito, Hiroyuki; Liu, Yang; Ohbu, Sumie; Hayashi, Yoriko; Abe, Tomoko

2011-11-15

Heavy-ion mutagenesis is recognised as a powerful technology to generate new mutants, especially in higher plants. Heavy-ion beams show high linear energy transfer (LET) and thus more effectively induce DNA double-strand breaks than other mutagenic techniques. Previously, we determined the most effective heavy-ion LET (LETmax: 30.0 keV μm(-1)) for Arabidopsis mutagenesis by analysing the effect of LET on mutation induction. However, the molecular structure of mutated DNA induced by heavy ions with LETmax remains unclear. Knowledge of the structure of mutated DNA will contribute to the effective exploitation of heavy-ion beam mutagenesis. Dry Arabidopsis thaliana seeds were irradiated with carbon (C) ions with LETmax at a dose of 400 Gy and with LET of 22.5 keV μm(-1) at doses of 250 Gy or 450 Gy. The effects on mutation frequency and alteration of DNA structure were compared. To characterise the structure of mutated DNA, we screened the well-characterised mutants elongated hypocotyls (hy) and glabrous (gl) and identified mutated DNA among the resulting mutants by high-resolution melting curve, PCR and sequencing analyses. The mutation frequency induced by C ions with LETmax was two-fold higher than that with 22.5 keV μm(-1) and similar to the mutation frequency previously induced by ethyl methane sulfonate. We identified the structure of 22 mutated DNAs. Over 80% of the mutations caused by C ions with both LETs were base substitutions or deletions/insertions of less than 100 bp. The other mutations involved large rearrangements. The C ions with LETmax showed high mutation efficiency and predominantly induced base substitutions or small deletions/insertions, most of which were null mutations. These small alterations can be determined by single-nucleotide polymorphism (SNP) detection systems. Therefore, C ions with LETmax might be useful as a highly efficient reverse genetic system in conjunction with SNP detection systems, and will be beneficial for forward genetics and plant breeding.

Survival of glucose phosphate isomerase null somatic cells and germ cells in adult mouse chimaeras

PubMed Central

Keighren, Margaret A.; Flockhart, Jean H.

2016-01-01

ABSTRACT The mouse Gpi1 gene encodes the glycolytic enzyme glucose phosphate isomerase. Homozygous Gpi1−/− null mouse embryos die but a previous study showed that some homozygous Gpi1−/− null cells survived when combined with wild-type cells in fetal chimaeras. One adult female Gpi1−/−↔Gpi1c/c chimaera with functional Gpi1−/− null oocytes was also identified in a preliminary study. The aims were to characterise the survival of Gpi1−/− null cells in adult Gpi1−/−↔Gpi1c/c chimaeras and determine if Gpi1−/− null germ cells are functional. Analysis of adult Gpi1−/−↔Gpi1c/c chimaeras with pigment and a reiterated transgenic lineage marker showed that low numbers of homozygous Gpi1−/− null cells could survive in many tissues of adult chimaeras, including oocytes. Breeding experiments confirmed that Gpi1−/− null oocytes in one female Gpi1−/−↔Gpi1c/c chimaera were functional and provided preliminary evidence that one male putative Gpi1−/−↔Gpi1c/c chimaera produced functional spermatozoa from homozygous Gpi1−/− null germ cells. Although the male chimaera was almost certainly Gpi1−/−↔Gpi1c/c, this part of the study is considered preliminary because only blood was typed for GPI. Gpi1−/− null germ cells should survive in a chimaeric testis if they are supported by wild-type Sertoli cells. It is also feasible that spermatozoa could bypass a block at GPI, but not blocks at some later steps in glycolysis, by using fructose, rather than glucose, as the substrate for glycolysis. Although chimaera analysis proved inefficient for studying the fate of Gpi1−/− null germ cells, it successfully identified functional Gpi1−/− null oocytes and revealed that some Gpi1−/− null cells could survive in many adult tissues. PMID:27103217

Tic40, a membrane-anchored co-chaperone homolog in the chloroplast protein translocon

PubMed Central

Chou, Ming-Lun; Fitzpatrick, Lynda M.; Tu, Shuh-Long; Budziszewski, Gregory; Potter-Lewis, Sharon; Akita, Mitsuru; Levin, Joshua Z.; Keegstra, Kenneth; Li, Hsou-min

2003-01-01

The function of Tic40 during chloroplast protein import was investigated. Tic40 is an inner envelope membrane protein with a large hydrophilic domain located in the stroma. Arabidopsis null mutants of the atTic40 gene were very pale green and grew slowly but were not seedling lethal. Isolated mutant chloroplasts imported precursor proteins at a lower rate than wild-type chloroplasts. Mutant chloroplasts were normal in allowing binding of precursor proteins. However, during subsequent translocation across the inner membrane, fewer precursors were translocated and more precursors were released from the mutant chloroplasts. Cross-linking experiments demonstrated that Tic40 was part of the translocon complex and functioned at the same stage of import as Tic110 and Hsp93, a member of the Hsp100 family of molecular chaperones. Tertiary structure prediction and immunological studies indicated that the C-terminal portion of Tic40 contains a TPR domain followed by a domain with sequence similarity to co-chaperones Sti1p/Hop and Hip. We propose that Tic40 functions as a co-chaperone in the stromal chaperone complex that facilitates protein translocation across the inner membrane. PMID:12805212

Deletion of Citrate Synthase Restores Growth of Sinorhizobium meliloti 1021 Aconitase Mutants▿

PubMed Central

Koziol, Uriel; Hannibal, Luciana; Rodríguez, María Cecilia; Fabiano, Elena; Kahn, Michael L.; Noya, Francisco

2009-01-01

The symbiotic nitrogen-fixing bacterium Sinorhizobium meliloti 1021 encodes only one predicted aconitase (AcnA) in its genome. AcnA has a significant degree of similarity with other bacterial aconitases that behave as dual proteins: enzymes and posttranscriptional regulators of gene expression. Similar to the case with these bacterial aconitases, AcnA activity was reversibly labile and was regained upon reconstitution with reduced iron. The aconitase promoter was active in root nodules. acnA mutants grew very poorly, had secondary mutations, and were quickly outgrown by pseudorevertants. The acnA gene was stably interrupted in a citrate synthase (gltA) null background, indicating that the intracellular accumulation of citrate may be deleterious for survival of strain 1021. No aconitase activity was detected in this mutant, suggesting that the acnA gene encodes the only functional aconitase of strain 1021. To uncover a function of AcnA beyond its catalytic role in the tricarboxylic acid cycle pathway, the gltA acnA double mutant was compared with the gltA single mutant for differences in motility, resistance to oxidative stress, nodulation, and growth on different substrates. However, no differences in any of these characteristics were found. PMID:19820082

Drosophila Lin-52 Acts in Opposition to Repressive Components of the Myb-MuvB/dREAM Complex

PubMed Central

Lewis, Peter W.; Sahoo, Debashis; Geng, Cuiyun; Bell, Maren

2012-01-01

The Drosophila melanogaster Myb-MuvB/dREAM complex (MMB/dREAM) participates in both the activation and repression of developmentally regulated genes and origins of DNA replication. Mutants in MMB subunits exhibit diverse phenotypes, including lethality, eye defects, reduced fecundity, and sterility. Here, we used P-element excision to generate mutations in lin-52, which encodes the smallest subunit of the MMB/dREAM complex. lin-52 is required for viability, as null mutants die prior to pupariation. The generation of somatic and germ line mutant clones indicates that lin-52 is required for adult eye development and for early embryogenesis via maternal effects. Interestingly, the maternal-effect embryonic lethality, larval lethality, and adult eye defects could be suppressed by mutations in other subunits of the MMB/dREAM complex. These results suggest that a partial MMB/dREAM complex is responsible for the lethality and eye defects of lin-52 mutants. Furthermore, these findings support a model in which the Lin-52 and Myb proteins counteract the repressive activities of the other members of the MMB/dREAM complex at specific genomic loci in a developmentally controlled manner. PMID:22688510

Drosophila Torsin Protein Regulates Motor Control and Stress Sensitivity and Forms a Complex with Fragile-X Mental Retardation Protein

PubMed Central

Ahn, Hyo-Min; Koh, Young Ho

2016-01-01

We investigated unknown in vivo functions of Torsin by using Drosophila as a model. Downregulation of Drosophila Torsin (DTor) by DTor-specific inhibitory double-stranded RNA (RNAi) induced abnormal locomotor behavior and increased susceptibility to H2O2. In addition, altered expression of DTor significantly increased the numbers of synaptic boutons. One important biochemical consequence of DTor-RNAi expression in fly brains was upregulation of alcohol dehydrogenase (ADH). Altered expression of ADH has also been reported in Drosophila Fragile-X mental retardation protein (DFMRP) mutant flies. Interestingly, expression of DFMRP was altered in DTor mutant flies, and DTor and DFMRP were present in the same protein complexes. In addition, DTor and DFMRP immunoreactivities were partially colocalized in several cellular organelles in larval muscles. Furthermore, there were no significant differences between synaptic morphologies of dfmrp null mutants and dfmrp mutants expressing DTor-RNAi. Taken together, our evidences suggested that DTor and DFMRP might be present in the same signaling pathway regulating synaptic plasticity. In addition, we also found that human Torsin1A and human FMRP were present in the same protein complexes, suggesting that this phenomenon is evolutionarily conserved. PMID:27313903

An antennal carboxylesterase from Drosophila melanogaster, esterase 6, is a candidate odorant-degrading enzyme toward food odorants.

PubMed

Chertemps, Thomas; Younus, Faisal; Steiner, Claudia; Durand, Nicolas; Coppin, Chris W; Pandey, Gunjan; Oakeshott, John G; Maïbèche, Martine

2015-01-01

Reception of odorant molecules within insect olfactory organs involves several sequential steps, including their transport through the sensillar lymph, interaction with the respective sensory receptors, and subsequent inactivation. Odorant-degrading enzymes (ODEs) putatively play a role in signal dynamics by rapid degradation of odorants in the vicinity of the receptors, but this hypothesis is mainly supported by in vitro results. We have recently shown that an extracellular carboxylesterase, esterase-6 (EST-6), is involved in the physiological and behavioral dynamics of the response of Drosophila melanogaster to its volatile pheromone ester, cis-vaccenyl acetate. However, as the expression pattern of the Est-6 gene in the antennae is not restricted to the pheromone responding sensilla, we tested here if EST-6 could play a broader function in the antennae. We found that recombinant EST-6 is able to efficiently hydrolyse several volatile esters that would be emitted by its natural food in vitro. Electrophysiological comparisons of mutant Est-6 null flies and a control strain (on the same genetic background) showed that the dynamics of the antennal response to these compounds is influenced by EST-6, with the antennae of the null mutants showing prolonged activity in response to them. Antennal responses to the strongest odorant, pentyl acetate, were then studied in more detail, showing that the repolarization dynamics were modified even at low doses but without modification of the detection threshold. Behavioral choice experiments with pentyl acetate also showed differences between genotypes; attraction to this compound was observed at a lower dose among the null than control flies. As EST-6 is able to degrade various bioactive odorants emitted by food and plays a role in the response to these compounds, we hypothesize a role as an ODE for this enzyme toward food volatiles.

Linking GABA(A) receptor subunits to alcohol-induced conditioned taste aversion and recovery from acute alcohol intoxication.

PubMed

Blednov, Y A; Benavidez, J M; Black, M; Chandra, D; Homanics, G E; Rudolph, U; Harris, R A

2013-04-01

GABA type A receptors (GABA(A)-R) are important for ethanol actions and it is of interest to link individual subunits with specific ethanol behaviors. We studied null mutant mice for six different GABA(A)-R subunits (α1, α2, α3, α4, α5 and δ). Only mice lacking the α2 subunit showed reduction of conditioned taste aversion (CTA) to ethanol. These results are in agreement with data from knock-in mice with mutation of the ethanol-sensitive site in the α2-subunit (Blednov et al., 2011). All together, they indicate that aversive property of ethanol is dependent on ethanol action on α2-containing GABA(A)-R. Deletion of the α2-subunit led to faster recovery whereas absence of the α3-subunit slowed recovery from ethanol-induced incoordination (rotarod). Deletion of the other four subunits did not affect this behavior. Similar changes in this behavior for the α2 and α3 null mutants were found for flurazepam motor incoordination. However, no differences in recovery were found in motor-incoordinating effects of an α1-selective modulator (zolpidem) or an α4-selective agonist (gaboxadol). Therefore, recovery of rotarod incoordination is under control of two GABA(A)-R subunits: α2 and α3. For motor activity, α3 null mice demonstrated higher activation by ethanol (1 g/kg) whereas both α2 (-/-) and α3 (-/Y) knockout mice were less sensitive to ethanol-induced reduction of motor activity (1.5 g/kg). These studies demonstrate that the effects of ethanol at GABAergic synapses containing α2 subunit are important for specific behavioral effects of ethanol which may be relevant to the genetic linkage of the α2 subunit with human alcoholism. Copyright © 2012 Elsevier Ltd. All rights reserved.

AtHMA4 Drives Natural Variation in Leaf Zn Concentration of Arabidopsis thaliana

PubMed Central

Chen, Zi-Ru; Kuang, Lu; Gao, Yi-Qun; Wang, Ya-Ling; Salt, David E.; Chao, Dai-Yin

2018-01-01

Zinc (Zn) is an essential element for plant growth and development, and Zn derived from crop plants in the diet is also important for human health. Here, we report that genetic variation in Heavy Metal-ATPase 4 (HMA4) controls natural variation in leaf Zn content. Investigation of the natural variation in leaf Zn content in a world-wide collection of 349 Arabidopsis thaliana wild collected accessions identified two accessions, Van-0 and Fab-2, which accumulate significantly lower Zn when compared with Col-0. Both quantitative trait loci (QTL) analysis and bulked segregant analysis (BSA) identified HMA4 as a strong candidate accounting for this variation in leaf Zn concentration. Genetic complementation experiments confirmed this hypothesis. Sequence analysis revealed that a 1-bp deletion in the third exon of HMA4 from Fab-2 is responsible for the lose of function of HMA4 driving the low Zn observed in Fab-2. Unlike in Fab-2 polymorphisms in the promoter region were found to be responsible for the weak function of HMA4 in Van-0. This is supported by both an expression analysis of HMA4 in Van-0 and through a series of T-DNA insertion mutants which generate truncated HMA4 promoters in the Col-0 background. In addition, we also observed that Fab-2, Van-0 and the hma4-2 null mutant in the Col-0 background show enhanced resistance to a combination of high Zn and high Cd in the growth medium, raising the possibility that variation at HMA4 may play a role in environmental adaptation. PMID:29545819

AtHMA4 Drives Natural Variation in Leaf Zn Concentration of Arabidopsis thaliana.

PubMed

Chen, Zi-Ru; Kuang, Lu; Gao, Yi-Qun; Wang, Ya-Ling; Salt, David E; Chao, Dai-Yin

2018-01-01

Zinc (Zn) is an essential element for plant growth and development, and Zn derived from crop plants in the diet is also important for human health. Here, we report that genetic variation in Heavy Metal-ATPase 4 ( HMA4 ) controls natural variation in leaf Zn content. Investigation of the natural variation in leaf Zn content in a world-wide collection of 349 Arabidopsis thaliana wild collected accessions identified two accessions, Van-0 and Fab-2, which accumulate significantly lower Zn when compared with Col-0. Both quantitative trait loci (QTL) analysis and bulked segregant analysis (BSA) identified HMA4 as a strong candidate accounting for this variation in leaf Zn concentration. Genetic complementation experiments confirmed this hypothesis. Sequence analysis revealed that a 1-bp deletion in the third exon of HMA4 from Fab-2 is responsible for the lose of function of HMA4 driving the low Zn observed in Fab-2. Unlike in Fab-2 polymorphisms in the promoter region were found to be responsible for the weak function of HMA4 in Van-0. This is supported by both an expression analysis of HMA4 in Van-0 and through a series of T-DNA insertion mutants which generate truncated HMA4 promoters in the Col-0 background. In addition, we also observed that Fab-2, Van-0 and the hma4-2 null mutant in the Col-0 background show enhanced resistance to a combination of high Zn and high Cd in the growth medium, raising the possibility that variation at HMA4 may play a role in environmental adaptation.

Spa47 is an oligomerization-activated type three secretion system (T3SS) ATPase from Shigella flexneri.

PubMed

Burgess, Jamie L; Jones, Heather B; Kumar, Prashant; Toth, Ronald T; Middaugh, C Russell; Antony, Edwin; Dickenson, Nicholas E

2016-05-01

Gram-negative pathogens often use conserved type three secretion systems (T3SS) for virulence. The Shigella type three secretion apparatus (T3SA) penetrates the host cell membrane and provides a unidirectional conduit for injection of effectors into host cells. The protein Spa47 localizes to the base of the apparatus and is speculated to be an ATPase that provides the energy for T3SA formation and secretion. Here, we developed an expression and purification protocol, producing active Spa47 and providing the first direct evidence that Spa47 is a bona fide ATPase. Additionally, size exclusion chromatography and analytical ultracentrifugation identified multiple oligomeric species of Spa47 with the largest greater than 8 fold more active for ATP hydrolysis than the monomer. An ATPase inactive Spa47 point mutant was then engineered by targeting a conserved Lysine within the predicted Walker A motif of Spa47. Interestingly, the mutant maintained a similar oligomerization pattern as active Spa47, but was unable to restore invasion phenotype when used to complement a spa47 null S. flexneri strain. Together, these results identify Spa47 as a Shigella T3SS ATPase and suggest that its activity is linked to oligomerization, perhaps as a regulatory mechanism as seen in some related pathogens. Additionally, Spa47 catalyzed ATP hydrolysis appears to be essential for host cell invasion, providing a strong platform for additional studies dissecting its role in virulence and providing an attractive target for anti-infective agents. © 2016 The Protein Society.

Loss of plastoglobule kinases ABC1K1 and ABC1K3 causes conditional degreening, modified prenyl-lipids, and recruitment of the jasmonic acid pathway

USDA-ARS?s Scientific Manuscript database

Plastoglobules (PGs) are plastid lipid-protein particles. This study examines the function of PG-localized kinases ABC1K1 and ABC1K3 in Arabidopsis thaliana. Several lines of evidence suggested that ABC1K1 and ABC1K3 form a protein complex. Null mutants for both genes (abc1k1 and abc1k3) and the dou...

Production of Null Mutants in the Major Intestinal Esterase Gene (Ges-1) of the Nematode Caenorhabditis Elegans

PubMed Central

McGhee, J. D.; Birchall, J. C.; Chung, M. A.; Cottrell, D. A.; Edgar, L. G.; Svendsen, P. C.; Ferrari, D. C.

1990-01-01

The ges-1 gene of the nematode Caenorhabditis elegans codes for a nonspecific carboxylesterase that is expressed only in the intestinal lineage. This esterase has turned out to be a convenient biochemical marker for lineage-specific differentiation. In the present paper, we describe the production of several C. elegans strains that lack detectable activity of the ges-1 esterase. To isolate these ges-1 null strains, we first produced a strain of hermaphrodites in which the wild-type copy of the ges-1 gene was stably balanced over a previously isolated isoelectric focusing allele, ges-1(ca6); this parental strain was then mutagenized with EMS and isoelectric focusing gels were used to identify progeny populations that lacked either ges-1(+) or ges-1(ca6) esterase activity. This method is a straightforward and general approach to obtaining null mutations in any gene that has a biochemical or immunological assay. The ges-1 gene is not essential to worm survival, development or reproduction. Furthermore, lack of the ges-1 product has no obvious effect on the ability of worms (containing either normal or greatly reduced levels of acetylcholinesterases) to survive exposure to esterase inhibitors. The ges-1 gene product provides roughly half of the total esterase activity measured in crude extracts of L1 larvae or mixed worm populations. However, histochemical staining of individual ges-1(0) embryos shows that the ges-1 esterase is the first and essentially the only esterase to be produced during embryonic development, from the midproliferation phase up to at least the twofold stage of morphogenesis. These ges-1(0) strains now allow us to investigate the developmental control of the ges-1 gene by DNA-mediated transformation, in which the ges-1 gene acts as its own reporter. PMID:2379823

A gain-of-function mutation of plastidic invertase alters nuclear gene expression with sucrose treatment partially via GENOMES UNCOUPLED1-mediated signaling.

PubMed

Maruta, Takanori; Miyazaki, Nozomi; Nosaka, Ryota; Tanaka, Hiroyuki; Padilla-Chacon, Daniel; Otori, Kumi; Kimura, Ayako; Tanabe, Noriaki; Yoshimura, Kazuya; Tamoi, Masahiro; Shigeoka, Shigeru

2015-05-01

Plastid gene expression (PGE) is one of the signals that regulate the expression of photosynthesis-associated nuclear genes (PhANGs) via GENOMES UNCOUPLED1 (GUN1)-dependent retrograde signaling. We recently isolated Arabidopsis sugar-inducible cotyledon yellow-192 (sicy-192), a gain-of-function mutant of plastidic invertase, and showed that following the treatment of this mutant with sucrose, the expression of PhANGs as well as PGE decreased, suggesting that the sicy-192 mutation activates a PGE-evoked and GUN1-mediated retrograde pathway. To clarify the relationship between the sicy-192 mutation, PGE, and GUN1-mediated pathway, plastid and nuclear gene expression in a double mutant of sicy-192 and gun1-101, a null mutant of GUN1 was studied. Plastid-encoded RNA polymerase (PEP)-dependent PGE was markedly suppressed in the sicy-192 mutant by the sucrose treatment, but the suppression as well as cotyledon yellow phenotype was not mitigated by GUN1 disruption. Microarray analysis revealed that the altered expression of nuclear genes such as PhANG in the sucrose-treated sicy-192 mutant was largely dependent on GUN1. The present findings demonstrated that the sicy-192 mutation alters nuclear gene expression with sucrose treatment via GUN1, which is possibly followed by inhibiting PEP-dependent PGE, providing a new insight into the role of plastid sugar metabolism in nuclear gene expression. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

Loss of circadian clock accelerates aging in neurodegeneration-prone mutants

PubMed Central

Krishnan, Natraj; Rakshit, Kuntol; Chow, Eileen S.; Wentzell, Jill S.; Kretzschmar, Doris; Giebultowicz, Jadwiga M.

2012-01-01

Circadian clocks generate rhythms in molecular, cellular, physiological, and behavioral processes. Recent studies suggest that disruption of the clock mechanism accelerates organismal senescence and age-related pathologies in mammals. Impaired circadian rhythms are observed in many neurological diseases; however, it is not clear whether loss of rhythms is the cause or result of neurodegeneration, or both. To address this important question, we examined the effects of circadian disruption in Drosophila melanogaster mutants that display clock-unrelated neurodegenerative phenotypes. We combined a null mutation in the clock gene period (per01) that abolishes circadian rhythms, with a hypomorphic mutation in the carbonyl reductase gene sniffer (sni1), which displays oxidative stress induced neurodegeneration. We report that disruption of circadian rhythms in sni1 mutants significantly reduces their lifespan compared to single mutants. Shortened lifespan in double mutants was coupled with accelerated neuronal degeneration evidenced by vacuolization in the adult brain. In addition, per01 sni1 flies showed drastically impaired vertical mobility and increased accumulation of carbonylated proteins compared to age-matched single mutant flies. Loss of per function does not affect sni mRNA expression, suggesting that these genes act via independent pathways producing additive effects. Finally, we show that per01 mutation accelerates the onset of brain pathologies when combined with neurodegeneration-prone mutation in another gene, swiss cheese (sws1), which does not operate through the oxidative stress pathway. Taken together, our data suggest that the period gene may be causally involved in neuroprotective pathways in aging Drosophila. PMID:22227001

Fur-dependent detoxification of organic acids by rpoS mutants during prolonged incubation under aerobic, phosphate starvation conditions.

PubMed

Guillemet, Mélanie L; Moreau, Patrice L

2008-08-01

The activity of amino acid-dependent acid resistance systems allows Escherichia coli to survive during prolonged incubation under phosphate (P(i)) starvation conditions. We show in this work that rpoS-null mutants incubated in the absence of any amino acid survived during prolonged incubation under aerobic, P(i) starvation conditions. Whereas rpoS(+) cells incubated with glutamate excreted high levels of acetate, rpoS mutants grew on acetic acid. The characteristic metabolism of rpoS mutants required the activity of Fur (ferric uptake regulator) in order to decrease the synthesis of the small RNA RyhB that might otherwise inhibit the synthesis of iron-rich proteins. We propose that RpoS (sigma(S)) and the small RNA RyhB contribute to decrease the synthesis of iron-rich proteins required for the activity of the tricarboxylic acid (TCA) cycle, which redirects the metabolic flux toward the production of acetic acid at the onset of stationary phase in rpoS(+) cells. In contrast, Fur activity, which represses ryhB, and the lack of RpoS activity allow a substantial activity of the TCA cycle to continue in stationary phase in rpoS mutants, which decreases the production of acetic acid and, eventually, allows growth on acetic acid and P(i) excreted into the medium. These data may help explain the fact that a high frequency of E. coli rpoS mutants is found in nature.

Alleviation of proton toxicity by nitrate uptake specifically depends on nitrate transporter 1.1 in Arabidopsis.

PubMed

Fang, Xian Zhi; Tian, Wen Hao; Liu, Xing Xing; Lin, Xian Yong; Jin, Chong Wei; Zheng, Shao Jian

2016-07-01

Protons in acid soil are highly rhizotoxic to plants, but the mechanism of tolerance of plants to protons is largely unknown. Nitrate uptake by root cells is accompanied by the uptake of protons. Therefore, nitrate uptake transporters (NRTs) may be involved in plant tolerance to proton toxicity. We investigated the root nitrate uptake response to proton stress in Arabidopsis and its association with proton tolerance using NRT-related mutants and pharmacological methods. Lack of NRT1.1 in knockout nrt1.1 mutants led to impaired proton tolerance in nitrate-sufficient growth medium, whereas no difference was seen between wild-type plants and NRT1.2-, NRT2.1-, NRT2.2-, and NRT2.4-null mutants. Another nrt1.1 point mutant, which is defective in nitrate uptake but has a normal nitrate-sensing function, also had impaired proton tolerance compared with the wild-type plant. Furthermore, proton stress induced NRT1.1-mediated nitrate uptake. These results indicate that NRT1.1-conferred proton tolerance depends on nitrate uptake activity. In addition, the rooting medium was alkalified by wild-type plants, but not by knockout nrt1.1 mutants, and in pH-buffered medium, there were no differences in proton tolerance between wild-type plants and knockout nrt1.1 mutants. We conclude that NRT1.1-mediated nitrate uptake plays a crucial role in plant proton tolerance by alkalifying the rhizosphere. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

Reduced cell number in the hindgut epithelium disrupts hindgut left-right asymmetry in a mutant of pebble, encoding a RhoGEF, in Drosophila embryos.

PubMed

Nakamura, Mitsutoshi; Matsumoto, Kenjiroo; Iwamoto, Yuta; Muguruma, Takeshi; Nakazawa, Naotaka; Hatori, Ryo; Taniguchi, Kiichiro; Maeda, Reo; Matsuno, Kenji

2013-02-01

Animals often show left-right (LR) asymmetry in their body structures. In some vertebrates, the mechanisms underlying LR symmetry breaking and the subsequent signals responsible for LR asymmetric development are well understood. However, in invertebrates, the molecular bases of these processes are largely unknown. Therefore, we have been studying the genetic pathway of LR asymmetric development in Drosophila. The embryonic gut is the first organ that shows directional LR asymmetry during Drosophila development. We performed a genetic screen to identify mutations affecting LR asymmetric development of the embryonic gut. From this screen, we isolated pebble (pbl), which encodes a homolog of a mammalian RhoGEF, Ect2. The laterality of the hindgut was randomized in embryos homozygous for a null mutant of pbl. Pbl is a multi-functional protein required for cytokinesis and the epithelial-to-mesenchymal transition in Drosophila. Consistent with Pbl's role in cytokinesis, we found reduced numbers of cells in the hindgut epithelium in pbl homozygous embryos. The specific expression of pbl in the hindgut epithelium, but not in other tissues, rescued the LR defects and reduced cell number in embryonic pbl homozygotes. Embryos homozygous for string (stg), a mutant that reduces cell number through a different mechanism, also showed LR defects of the hindgut. However, the reduction in cell number in the pbl mutants was not accompanied by defects in the specification of hindgut epithelial tissues or their integrity. Based on these results, we speculate that the reduction in cell number may be one reason for the LR asymmetry defect of the pbl hindgut, although we cannot exclude contributions from other functions of Pbl, including regulation of the actin cytoskeleton through its RhoGEF activity. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Precocious Metamorphosis in the Juvenile Hormone–Deficient Mutant of the Silkworm, Bombyx mori

PubMed Central

Daimon, Takaaki; Kozaki, Toshinori; Niwa, Ryusuke; Kobayashi, Isao; Furuta, Kenjiro; Namiki, Toshiki; Uchino, Keiro; Banno, Yutaka; Katsuma, Susumu; Tamura, Toshiki; Mita, Kazuei; Sezutsu, Hideki; Nakayama, Masayoshi; Itoyama, Kyo; Shimada, Toru; Shinoda, Tetsuro

2012-01-01

Insect molting and metamorphosis are intricately governed by two hormones, ecdysteroids and juvenile hormones (JHs). JHs prevent precocious metamorphosis and allow the larva to undergo multiple rounds of molting until it attains the proper size for metamorphosis. In the silkworm, Bombyx mori, several “moltinism” mutations have been identified that exhibit variations in the number of larval molts; however, none of them have been characterized molecularly. Here we report the identification and characterization of the gene responsible for the dimolting (mod) mutant that undergoes precocious metamorphosis with fewer larval–larval molts. We show that the mod mutation results in complete loss of JHs in the larval hemolymph and that the mutant phenotype can be rescued by topical application of a JH analog. We performed positional cloning of mod and found a null mutation in the cytochrome P450 gene CYP15C1 in the mod allele. We also demonstrated that CYP15C1 is specifically expressed in the corpus allatum, an endocrine organ that synthesizes and secretes JHs. Furthermore, a biochemical experiment showed that CYP15C1 epoxidizes farnesoic acid to JH acid in a highly stereospecific manner. Precocious metamorphosis of mod larvae was rescued when the wild-type allele of CYP15C1 was expressed in transgenic mod larvae using the GAL4/UAS system. Our data therefore reveal that CYP15C1 is the gene responsible for the mod mutation and is essential for JH biosynthesis. Remarkably, precocious larval–pupal transition in mod larvae does not occur in the first or second instar, suggesting that authentic epoxidized JHs are not essential in very young larvae of B. mori. Our identification of a JH–deficient mutant in this model insect will lead to a greater understanding of the molecular basis of the hormonal control of development and metamorphosis. PMID:22412378

Type-f thioredoxins have a role in the short-term activation of carbon metabolism and their loss affects growth under short-day conditions in Arabidopsis thaliana.

PubMed

Naranjo, Belén; Diaz-Espejo, Antonio; Lindahl, Marika; Cejudo, Francisco Javier

2016-03-01

Redox regulation plays a central role in the adaptation of chloroplast metabolism to light. Extensive biochemical analyses in vitro have identified f-type thioredoxins (Trxs) as the most important catalysts for light-dependent reduction and activation of the enzymes of the Calvin-Benson cycle. However, the precise function of type f Trxs in vivo and their impact on plant growth are still poorly known. To address this issue we have generated an Arabidopsis thaliana double knock-out mutant, termed trxf1f2, devoid of both f1 and f2 Trxs. Despite the essential function previously proposed for f-type Trxs, the visible phenotype of the trxf1f2 double mutant was virtually indistinguishable from the wild type when grown under a long-day photoperiod. However, the Trx f-deficient plants showed growth inhibition under a short-day photoperiod which was not rescued at high light intensity. The absence of f-type Trxs led to significantly lower photosynthetic electron transport rates and higher levels of non-photochemical energy quenching. Notably, the Trx f null mutant suffered from a shortage of photosystem I electron acceptors and delayed activation of carbon dioxide fixation following a dark-light transition. Two redox-regulated Calvin-Benson cycle enzymes, fructose 1,6-bisphosphatase (FBPase) and Rubisco activase, showed retarded and incomplete reduction in the double mutant upon illumination, compared with wild-type plants. These results show that the function of f-type Trxs in the rapid activation of carbon metabolism in response to light is not entirely compensated for by additional plastid redox systems, and suggest that these Trxs have an important role in the light adjustment of photosynthetic metabolism. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

The effects of gene disruption of Kre6-like proteins on the phenotype of β-glucan-producing Aureobasidium pullulans.

PubMed

Uchiyama, Hirofumi; Iwai, Atsushi; Dohra, Hideo; Ohnishi, Toshiyuki; Kato, Tatsuya; Park, Enoch Y

2018-05-01

Killer toxin resistant 6 (Kre6) and its paralog, suppressor of Kre null 1 (Skn1), are thought to be involved in the biosynthesis of cell wall β-(1 → 6)-D-glucan in baker's yeast, Saccharomyces cerevisiae. The Δkre6Δskn1 mutant of S. cerevisiae and other fungi shows severe growth defects due to the failure to synthesize normal cell walls. In this study, two homologs of Kre6, namely, K6LP1 (Kre6-like protein 1) and K6LP2 (Kre6-like protein 2), were identified in Aureobasidium pullulans M-2 by draft genome analysis. The Δk6lp1, Δk6lp2, and Δk6lp1Δk6lp2 mutants were generated in order to confirm the functions of the Kre6-like proteins in A. pullulans M-2. The cell morphologies of Δk6lp1 and Δk6lp1Δk6lp2 appeared to be different from those of wild type and Δk6lp2 in both their yeast and hyphal forms. The productivity of the extracellular polysaccharides, mainly composed of β-(1 → 3),(1 → 6)-D-glucan (β-glucan), of the mutants was 5.1-17.3% less than that of wild type, and the degree of branching in the extracellular β-glucan of mutants was 14.5-16.8% lower than that of wild type. This study showed that the gene disruption of Kre6-like proteins affected the cell morphology, the productivity of extracellular polysaccharides, and the structure of extracellular β-glucan, but it did not have a definite effect on the cell viability even in Δk6lp1Δk6lp2, unlike in the Δkre6Δskn1 of S. cerevisiae.

p53 Mutation suppresses adult neurogenesis in medaka fish (Oryzias latipes)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Isoe, Yasuko; Okuyama, Teruhiro; Taniguchi, Yoshihito

2012-07-13

Highlights: Black-Right-Pointing-Pointer Progenitor migration is accompanied by an increase in their numbers in the adult brain. Black-Right-Pointing-Pointer p53 Mutation suppressed an increase in the number of the migrated progenitors. Black-Right-Pointing-Pointer The decreased progenitor number is not due to enhanced cell death. Black-Right-Pointing-Pointer p53 Mutation did not affect proliferation of stem cells. -- Abstract: Tumor suppressor p53 negatively regulates self-renewal of neural stem cells in the adult murine brain. Here, we report that the p53 null mutation in medaka fish (Oryzias latipes) suppressed neurogenesis in the telencephalon, independent of cell death. By using 5-bromo-29-deoxyuridine (BrdU) immunohistochemistry, we identified 18 proliferation zonesmore » in the brains of young medaka fish; in situ hybridization showed that p53 was expressed selectively in at least 12 proliferation zones. We also compared the number of BrdU-positive cells present in the whole telencephalon of wild-type (WT) and p53 mutant fish. Immediately after BrdU exposure, the number of BrdU-positive cells did not differ significantly between them. One week after BrdU-exposure, the BrdU-positive cells migrated from the proliferation zone, which was accompanied by an increased number in the WT brain. In contrast, no significant increase was observed in the p53 mutant brain. Terminal deoxynucleotidyl transferase (dUTP) nick end-labeling revealed that there was no significant difference in the number of apoptotic cells in the telencephalon of p53 mutant and WT medaka, suggesting that the decreased number of BrdU-positive cells in the mutant may be due to the suppression of proliferation rather than the enhancement of neural cell death. These results suggest that p53 positively regulates neurogenesis via cell proliferation.« less

Myosin Storage Myopathy in C. elegans and Human Cultured Muscle Cells

PubMed Central

Dahl-Halvarsson, Martin; Pokrzywa, Malgorzata; Rauthan, Manish; Pilon, Marc

2017-01-01

Myosin storage myopathy is a protein aggregate myopathy associated with the characteristic subsarcolemmal accumulation of myosin heavy chain in muscle fibers. Despite similar histological findings, the clinical severity and age of onset are highly variable, ranging from no weakness to severe impairment of ambulation, and usually childhood-onset to onset later in life. Mutations located in the distal end of the tail of slow/ß-cardiac myosin heavy chain are associated with myosin storage myopathy. Four missense mutations (L1793P, R1845W, E1883K and H1901L), two of which have been reported in several unrelated families, are located within or closed to the assembly competence domain. This location is critical for the proper assembly of sarcomeric myosin rod filaments. To assess the mechanisms leading to protein aggregation in myosin storage myopathy and to evaluate the impact of these mutations on myosin assembly and muscle function, we expressed mutated myosin proteins in cultured human muscle cells and in the nematode Caenorhabditis elegans. While L1793P mutant myosin protein efficiently incorporated into the sarcomeric thick filaments, R1845W and H1901L mutants were prone to formation of myosin aggregates without assembly into striated sarcomeric thick filaments in cultured muscle cells. In C. elegans, mutant alleles of the myosin heavy chain gene unc-54 corresponding to R1845W, E1883K and H1901L, were as effective as the wild-type myosin gene in rescuing the null mutant worms, indicating that they retain functionality. Taken together, our results suggest that the basis for the pathogenic effect of the R1845W and H1901L mutations are primarily structural rather than functional. Further analyses are needed to identify the primary trigger for the histological changes seen in muscle biopsies of patients with L1793P and E1883K mutations. PMID:28125727

Myosin Storage Myopathy in C. elegans and Human Cultured Muscle Cells.

PubMed

Dahl-Halvarsson, Martin; Pokrzywa, Malgorzata; Rauthan, Manish; Pilon, Marc; Tajsharghi, Homa

2017-01-01

Myosin storage myopathy is a protein aggregate myopathy associated with the characteristic subsarcolemmal accumulation of myosin heavy chain in muscle fibers. Despite similar histological findings, the clinical severity and age of onset are highly variable, ranging from no weakness to severe impairment of ambulation, and usually childhood-onset to onset later in life. Mutations located in the distal end of the tail of slow/ß-cardiac myosin heavy chain are associated with myosin storage myopathy. Four missense mutations (L1793P, R1845W, E1883K and H1901L), two of which have been reported in several unrelated families, are located within or closed to the assembly competence domain. This location is critical for the proper assembly of sarcomeric myosin rod filaments. To assess the mechanisms leading to protein aggregation in myosin storage myopathy and to evaluate the impact of these mutations on myosin assembly and muscle function, we expressed mutated myosin proteins in cultured human muscle cells and in the nematode Caenorhabditis elegans. While L1793P mutant myosin protein efficiently incorporated into the sarcomeric thick filaments, R1845W and H1901L mutants were prone to formation of myosin aggregates without assembly into striated sarcomeric thick filaments in cultured muscle cells. In C. elegans, mutant alleles of the myosin heavy chain gene unc-54 corresponding to R1845W, E1883K and H1901L, were as effective as the wild-type myosin gene in rescuing the null mutant worms, indicating that they retain functionality. Taken together, our results suggest that the basis for the pathogenic effect of the R1845W and H1901L mutations are primarily structural rather than functional. Further analyses are needed to identify the primary trigger for the histological changes seen in muscle biopsies of patients with L1793P and E1883K mutations.

Functional Rescue of Trafficking-Impaired ABCB4 Mutants by Chemical Chaperones

PubMed Central

Gordo-Gilart, Raquel; Andueza, Sara; Hierro, Loreto; Jara, Paloma; Alvarez, Luis

2016-01-01

Multidrug resistance protein 3 (MDR3, ABCB4) is a hepatocellular membrane protein that mediates biliary secretion of phosphatidylcholine. Null mutations in ABCB4 gene give rise to severe early-onset cholestatic liver disease. We have previously shown that the disease-associated mutations p.G68R, p.G228R, p.D459H, and p.A934T resulted in retention of ABCB4 in the endoplasmic reticulum, thus failing to target the plasma membrane. In the present study, we tested the ability of two compounds with chaperone-like activity, 4-phenylbutyrate and curcumin, to rescue these ABCB4 mutants by assessing their effects on subcellular localization, protein maturation, and phospholipid efflux capability. Incubation of transfected cells at a reduced temperature (30°C) or exposure to pharmacological doses of either 4-PBA or curcumin restored cell surface expression of mutants G228R and A934T. The delivery of these mutants to the plasma membrane was accompanied by a switch in the ratio of mature to inmature protein forms, leading to a predominant expression of the mature protein. This effect was due to an improvement in the maturation rate and not to the stabilization of the mature forms. Both mutants were also functionally rescued, displaying bile salt-dependent phospholipid efflux activity after addition of 4-PBA or curcumin. Drug-induced rescue was mutant specific, given neither 4-PBA nor curcumin had an effect on the ABCB4 mutants G68R and A934T. Collectively, these data indicate that the functionality of selected trafficking-defective ABCB4 mutants can be recovered by chemical chaperones through restoration of membrane localization, suggesting a potential treatment for patients carrying such mutations. PMID:26900700

The Flavoproteins CryD and VvdA Cooperate with the White Collar Protein WcoA in the Control of Photocarotenogenesis in Fusarium fujikuroi

PubMed Central

Castrillo, Marta; Avalos, Javier

2015-01-01

Light stimulates carotenoid biosynthesis in the ascomycete fungus Fusarium fujikuroi through transcriptional activation of the structural genes of the pathway carRA, carB, and cart, but the molecular basis of this photoresponse is unknown. The F. fujikuroi genome contains genes for different predicted photoreceptors, including the WC protein WcoA, the DASH cryptochrome CryD and the Vivid-like flavoprotein VvdA. We formerly found that null mutants of wcoA, cryD or vvdA exhibit carotenoid photoinduction under continuous illumination. Here we show that the wild type exhibits a biphasic response in light induction kinetics experiments, with a rapid increase in carotenoid content in the first hours, a transient arrest and a subsequent slower increase. The mutants of the three photoreceptors show different kinetic responses: the wcoA mutants are defective in the rapid response, the cryD mutants are affected in the slower response, while the fast and slow responses were respectively enhanced and attenuated in the vvdA mutants. Transcriptional analyses of the car genes revealed a strong reduction of dark and light-induced transcript levels in the wcoA mutants, while minor or no reductions were found in the cryD mutants. Formerly, we found no change on carRA and carB photoinduction in vvdA mutants. Taken together, our data suggest a cooperative participation of WcoA and CryD in early and late stages of photoinduction of carotenoid biosynthesis in F. fujikuroi, and a possible modulation of WcoA activity by VvdA. An unexpected transcriptional induction by red light of vvdA, cryD and carRA genes suggest the participation of an additional red light-absorbing photoreceptor. PMID:25774802

Loss of ift122, a Retrograde Intraflagellar Transport (IFT) Complex Component, Leads to Slow, Progressive Photoreceptor Degeneration Due to Inefficient Opsin Transport.

PubMed

Boubakri, Meriam; Chaya, Taro; Hirata, Hiromi; Kajimura, Naoko; Kuwahara, Ryusuke; Ueno, Akiko; Malicki, Jarema; Furukawa, Takahisa; Omori, Yoshihiro

2016-11-18

In the retina, aberrant opsin transport from cell bodies to outer segments leads to retinal degenerative diseases such as retinitis pigmentosa. Opsin transport is facilitated by the intraflagellar transport (IFT) system that mediates the bidirectional movement of proteins within cilia. In contrast to functions of the anterograde transport executed by IFT complex B (IFT-B), the precise functions of the retrograde transport mediated by IFT complex A (IFT-A) have not been well studied in photoreceptor cilia. Here, we analyzed developing zebrafish larvae carrying a null mutation in ift122 encoding a component of IFT-A. ift122 mutant larvae show unexpectedly mild phenotypes, compared with those of mutants defective in IFT-B. ift122 mutants exhibit a slow onset of progressive photoreceptor degeneration mainly after 7 days post-fertilization. ift122 mutant larvae also develop cystic kidney but not curly body, both of which are typically observed in various ciliary mutants. ift122 mutants display a loss of cilia in the inner ear hair cells and nasal pit epithelia. Loss of ift122 causes disorganization of outer segment discs. Ectopic accumulation of an IFT-B component, ift88, is observed in the ift122 mutant photoreceptor cilia. In addition, pulse-chase experiments using GFP-opsin fusion proteins revealed that ift122 is required for the efficient transport of opsin and the distal elongation of outer segments. These results show that IFT-A is essential for the efficient transport of outer segment proteins, including opsin, and for the survival of retinal photoreceptor cells, rendering the ift122 mutant a unique model for human retinal degenerative diseases. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Loss of ift122, a Retrograde Intraflagellar Transport (IFT) Complex Component, Leads to Slow, Progressive Photoreceptor Degeneration Due to Inefficient Opsin Transport*

PubMed Central

Boubakri, Meriam; Chaya, Taro; Hirata, Hiromi; Kajimura, Naoko; Kuwahara, Ryusuke; Ueno, Akiko; Malicki, Jarema; Furukawa, Takahisa; Omori, Yoshihiro

2016-01-01

In the retina, aberrant opsin transport from cell bodies to outer segments leads to retinal degenerative diseases such as retinitis pigmentosa. Opsin transport is facilitated by the intraflagellar transport (IFT) system that mediates the bidirectional movement of proteins within cilia. In contrast to functions of the anterograde transport executed by IFT complex B (IFT-B), the precise functions of the retrograde transport mediated by IFT complex A (IFT-A) have not been well studied in photoreceptor cilia. Here, we analyzed developing zebrafish larvae carrying a null mutation in ift122 encoding a component of IFT-A. ift122 mutant larvae show unexpectedly mild phenotypes, compared with those of mutants defective in IFT-B. ift122 mutants exhibit a slow onset of progressive photoreceptor degeneration mainly after 7 days post-fertilization. ift122 mutant larvae also develop cystic kidney but not curly body, both of which are typically observed in various ciliary mutants. ift122 mutants display a loss of cilia in the inner ear hair cells and nasal pit epithelia. Loss of ift122 causes disorganization of outer segment discs. Ectopic accumulation of an IFT-B component, ift88, is observed in the ift122 mutant photoreceptor cilia. In addition, pulse-chase experiments using GFP-opsin fusion proteins revealed that ift122 is required for the efficient transport of opsin and the distal elongation of outer segments. These results show that IFT-A is essential for the efficient transport of outer segment proteins, including opsin, and for the survival of retinal photoreceptor cells, rendering the ift122 mutant a unique model for human retinal degenerative diseases. PMID:27681595

Proteoglycan 4: A Dynamic Regulator of Skeletogenesis and Parathyroid Hormone Skeletal Anabolism

PubMed Central

Novince, Chad M; Michalski, Megan N; Koh, Amy J; Sinder, Benjamin P; Entezami, Payam; Eber, Matthew R; Pettway, Glenda J; Rosol, Thomas J; Wronski, Thomas J; Kozloff, Ken M; McCauley, Laurie K

2014-01-01

Proteoglycan 4 (Prg4), known for its lubricating and protective actions in joints, is a strong candidate regulator of skeletal homeostasis and parathyroid hormone (PTH) anabolism. Prg4 is a PTH-responsive gene in bone and liver. Prg4 null mutant mice were used to investigate the impact of proteoglycan 4 on skeletal development, remodeling, and PTH anabolic actions. Young Prg4 mutant and wild-type mice were administered intermittent PTH(1–34) or vehicle daily from 4 to 21 days. Young Prg4 mutant mice had decreased growth plate hypertrophic zones, trabecular bone, and serum bone formation markers versus wild-type mice, but responded with a similar anabolic response to PTH. Adult Prg4 mutant and wild-type mice were administered intermittent PTH(1–34) or vehicle daily from 16 to 22 weeks. Adult Prg4 mutant mice had decreased trabecular and cortical bone, and blunted PTH-mediated increases in bone mass. Joint range of motion and animal mobility were lower in adult Prg4 mutant versus wild-type mice. Adult Prg4 mutant mice had decreased marrow and liver fibroblast growth factor 2 (FGF-2) mRNA and reduced serum FGF-2, which were normalized by PTH. A single dose of PTH decreased the PTH/PTHrP receptor (PPR), and increased Prg4 and FGF-2 to a similar extent in liver and bone. Proteoglycan 4 supports endochondral bone formation and the attainment of peak trabecular bone mass, and appears to support skeletal homeostasis indirectly by protecting joint function. Bone- and liver-derived FGF-2 likely regulate proteoglycan 4 actions supporting trabeculae formation. Blunted PTH anabolic responses in adult Prg4 mutant mice are associated with altered biomechanical impact secondary to joint failure. PMID:21932346

Putrescine importer PlaP contributes to swarming motility and urothelial cell invasion in Proteus mirabilis.

PubMed

Kurihara, Shin; Sakai, Yumi; Suzuki, Hideyuki; Muth, Aaron; Phanstiel, Otto; Rather, Philip N

2013-05-31

Previously, we reported that the speA gene, encoding arginine decarboxylase, is required for swarming in the urinary tract pathogen Proteus mirabilis. In addition, this previous study suggested that putrescine may act as a cell-to-cell signaling molecule (Sturgill, G., and Rather, P. N. (2004) Mol. Microbiol. 51, 437-446). In this new study, PlaP, a putative putrescine importer, was characterized in P. mirabilis. In a wild-type background, a plaP null mutation resulted in a modest swarming defect and slightly decreased levels of intracellular putrescine. In a P. mirabilis speA mutant with greatly reduced levels of intracellular putrescine, plaP was required for the putrescine-dependent rescue of swarming motility. When a speA/plaP double mutant was grown in the presence of extracellular putrescine, the intracellular levels of putrescine were greatly reduced compared with the speA mutant alone, indicating that PlaP functioned as the primary putrescine importer. In urothelial cell invasion assays, a speA mutant exhibited a 50% reduction in invasion when compared with wild type, and this defect could be restored by putrescine in a PlaP-dependent manner. The putrescine analog Triamide-44 partially inhibited the uptake of putrescine by PlaP and decreased both putrescine stimulated swarming and urothelial cell invasion in a speA mutant.

Constitutive expression of ftsZ overrides the whi developmental genes to initiate sporulation of Streptomyces coelicolor.

PubMed

Willemse, Joost; Mommaas, A Mieke; van Wezel, Gilles P

2012-03-01

The filamentous soil bacteria Streptomyces undergo a highly complex developmental programme. Before streptomycetes commit themselves to sporulation, distinct morphological checkpoints are passed in the aerial hyphae that are subject to multi-level control by the whi sporulation genes. Here we show that whi-independent expression of FtsZ restores sporulation to the early sporulation mutants whiA, whiB, whiG, whiH, whiI and whiJ. Viability, stress resistance and high-resolution electron microscopy underlined that viable spores were formed. However, spores from sporulation-restored whiA and whiG mutants showed defects in DNA segregation/condensation, while spores from the complemented whiB mutant had increased stress sensitivity, perhaps as a result of changes in the spore sheath. In contrast to the whi mutants, normal sporulation of ssgB null mutants-which fail to properly localise FtsZ-could not be restored by enhancing FtsZ protein levels, forming spore-like bodies that lack spore walls. Our data strongly suggest that the whi genes control a decisive event towards sporulation of streptomycetes, namely the correct timing of developmental ftsZ transcription. The biological significance may be to ensure that sporulation-specific cell division will only start once sufficient aerial mycelium biomass has been generated. Our data shed new light on the longstanding question as to how whi genes control sporulation, which has intrigued scientists for four decades.

Mutants in the Candida glabrata Glycerol Channels Are Sensitized to Cell Wall Stress

PubMed Central

Beese-Sims, Sara E.; Pan, Shih-Jung; Lee, Jongmin; Hwang-Wong, Elizabeth; Cormack, Brendan P.

2012-01-01

Many fungal species use glycerol as a compatible solute with which to maintain osmotic homeostasis in response to changes in external osmolarity. In Saccharomyces cerevisiae, intracellular glycerol concentrations are regulated largely by the high osmolarity glycerol (HOG) response pathway, both through induction of glycerol biosynthesis and control of its flux through the plasma membrane Fps1 glycerol channel. The channel activity of Fps1 is also controlled by a pair of positive regulators, Rgc1 and Rgc2. In this study, we demonstrate that Candida glabrata, a fungal pathogen that possesses two Fps1 orthologs and two Rgc1/-2 orthologs, accumulates glycerol in response to hyperosmotic stress. We present an initial characterization of mutants with deletions in the C. glabrata FPS1 (CAGL0C03267 [www.candidagenome.org]) and FPS2 (CAGL0E03894) genes and find that a double mutant accumulates glycerol, experiences constitutive cell wall stress, and is hypersensitive to treatment by caspofungin, an antifungal agent that targets the cell wall. This mutant is cleared more efficiently in mouse infections than is wild-type C. glabrata by caspofungin treatment. Finally, we demonstrate that one of the C. glabrata RGC orthologs complements an S. cerevisiae rgc1 rgc2 null mutant, supporting the conclusion that this regulatory assembly is conserved between these species. PMID:23087370

Inactivation of a gene that is highly conserved in Gram-positive bacteria stimulates degradation of non-native proteins and concomitantly increases stress tolerance in Lactococcus lactis.

PubMed

Frees, D; Varmanen, P; Ingmer, H

2001-07-01

Exposure of cells to elevated temperatures triggers the synthesis of chaperones and proteases including components of the conserved Clp protease complex. We demonstrated previously that the proteolytic subunit, ClpP, plays a major role in stress tolerance and in the degradation of non-native proteins in the Gram-positive bacterium Lactococcus lactis. Here, we used transposon mutagenesis to generate mutants in which the temperature- and puromycin-sensitive phenotype of a lactococcal clpP null mutant was partly alleviated. In all mutants obtained, the transposon was inserted in the L. lactis trmA gene. When analysing a clpP, trmA double mutant, we found that the expression normally induced from the clpP and dnaK promoters in the clpP mutant was reduced to wild-type level upon introduction of the trmA disruption. Additionally, the degradation of puromycyl-containing polypeptides was increased, suggesting that inactivation of trmA compensates for the absence of ClpP by stimulating an as yet unidentified protease that degrades misfolded proteins. When trmA was disrupted in wild-type cells, both stress tolerance and proteolysis of puromycyl peptides was enhanced above wild-type level. Based on our results, we propose that TrmA, which is well conserved in several Gram-positive bacteria, affects the degradation of non-native proteins and thereby controls stress tolerance.