Roles of HAUSP-mediated p53 regulation in central nervous system development.

PubMed

Kon, N; Zhong, J; Kobayashi, Y; Li, M; Szabolcs, M; Ludwig, T; Canoll, P D; Gu, W

2011-08-01

The deubiquitinase HAUSP (herpesvirus-associated ubiquitin-specific protease; also called USP7) has a critical role in regulating the p53-Mdm2 (murine double minute 2) pathway. By using the conventional knockout approach, we previously showed that hausp inactivation leads to early embryonic lethality. To fully understand the physiological functions of hausp, we have generated mice lacking hausp specifically in the brain and examined the impacts of this manipulation on brain development. We found that deletion of hausp in neural cells resulted in neonatal lethality. The brains from these mice displayed hypoplasia and deficiencies in development, which were mainly caused by p53-mediated apoptosis. Detailed analysis also showed an increase of both p53 levels and p53-dependent transcriptional activation in hausp knockout brains. Notably, neural cell survival and brain development of hausp-mutant mice can largely be restored in the p53-null background. Nevertheless, in contrast to the case of mdm2- and mdm4 (murine double minute 4)-mutant mice, inactivation of p53 failed to completely rescue the neonatal lethality of these hausp-mutant mice. These results indicate that HAUSP-mediated p53 regulation is crucial for brain development, and also suggest that both the p53-dependent and the p53-independent functions of HAUSP contribute to the neonatal lethality of hausp-mutant mice.

Transcription factors WRKY11 and WRKY17 are involved in abiotic stress responses in Arabidopsis.

PubMed

Ali, Muhammad Amjad; Azeem, Farrukh; Nawaz, Muhammad Amjad; Acet, Tuba; Abbas, Amjad; Imran, Qari Muhammad; Shah, Kausar Hussain; Rehman, Hafiz Mamoon; Chung, Gyuhwa; Yang, Seung Hwan; Bohlmann, Holger

2018-04-17

Plant WRKY transcription factors play a vital role in abiotic stress tolerance and regulation of plant defense responses. This study examined AtWRKY11 and AtWRKY17 expression under ABA, salt, and osmotic stress at different developmental stages in Arabidopsis. We used reverse transcriptase PCR, quantitative real-time PCR, and promoter:GUS lines to analyze expression. Both genes were upregulated in response to abiotic stress. Next, we applied the same stressors to seedlings of T-DNA insertion wrky11 and 17 knock-out mutants (single and double). Under stress, the mutants exhibited slower germination and compromised root growth compared with the wild type. In most cases, double-mutant seedlings were more affected than single mutants. These results suggest that wrky11 and wrky17 are not strictly limited to plant defense responses but are also involved in conferring stress tolerance. Copyright © 2018 Elsevier GmbH. All rights reserved.

Pheromones and Pheromone Receptors Are Required for Proper Sexual Development in the Homothallic Ascomycete Sordaria macrospora

PubMed Central

Mayrhofer, Severine; Weber, Jan M.; Pöggeler, Stefanie

2006-01-01

The homothallic, filamentous ascomycete Sordaria macrospora is self-fertile and produces sexual fruiting bodies (perithecia) without a mating partner. Even so, S. macrospora transcriptionally expresses two pheromone-precursor genes (ppg1 and ppg2) and two pheromone-receptor genes (pre1 and pre2). The proteins encoded by these genes are similar to α-factor-like and a-factor-like pheromones and to G-protein-coupled pheromone receptors of the yeast Saccharomyces cerevisiae. It has been suggested that in S. macrospora, PPG1/PRE2 and PPG2/PRE1 form two cognate pheromone–receptor pairs. To investigate their function, we deleted (Δ) pheromone-precursor genes (Δppg1, Δppg2) and receptor genes (Δpre1, Δpre2) and generated single- as well as double-knockout strains. No effect on vegetative growth, fruiting-body, and ascospore development was seen in the single pheromone-mutant and receptor-mutant strains, respectively. However, double-knockout strains lacking any compatible pheromone-receptor pair (Δpre2/Δppg2, Δpre1/Δppg1) and the double-pheromone mutant (Δppg1/Δppg2) displayed a drastically reduced number of perithecia and sexual spores, whereas deletion of both receptor genes (Δpre1/Δpre2) completely eliminated fruiting-body and ascospore formation. The results suggest that pheromones and pheromone receptors are required for optimal sexual reproduction of the homothallic S. macrospora. PMID:16387884

Pheromones and pheromone receptors are required for proper sexual development in the homothallic ascomycete Sordaria macrospora.

PubMed

Mayrhofer, Severine; Weber, Jan M; Pöggeler, Stefanie

2006-03-01

The homothallic, filamentous ascomycete Sordaria macrospora is self-fertile and produces sexual fruiting bodies (perithecia) without a mating partner. Even so, S. macrospora transcriptionally expresses two pheromone-precursor genes (ppg1 and ppg2) and two pheromone-receptor genes (pre1 and pre2). The proteins encoded by these genes are similar to alpha-factor-like and a-factor-like pheromones and to G-protein-coupled pheromone receptors of the yeast Saccharomyces cerevisiae. It has been suggested that in S. macrospora, PPG1/PRE2 and PPG2/PRE1 form two cognate pheromone-receptor pairs. To investigate their function, we deleted (delta) pheromone-precursor genes (delta ppg1, delta ppg2) and receptor genes (delta pre1, delta pre2) and generated single- as well as double-knockout strains. No effect on vegetative growth, fruiting-body, and ascospore development was seen in the single pheromone-mutant and receptor-mutant strains, respectively. However, double-knockout strains lacking any compatible pheromone-receptor pair (delta pre2/delta ppg2, delta pre1/delta ppg1) and the double-pheromone mutant (delta ppg1/delta ppg2) displayed a drastically reduced number of perithecia and sexual spores, whereas deletion of both receptor genes (delta pre1/delta pre2) completely eliminated fruiting-body and ascospore formation. The results suggest that pheromones and pheromone receptors are required for optimal sexual reproduction of the homothallic S. macrospora.

Functional modeling identifies paralogous solanesyl-diphosphate synthases that assemble the side chain of plastoquinone-9 in plastids.

PubMed

Block, Anna; Fristedt, Rikard; Rogers, Sara; Kumar, Jyothi; Barnes, Brian; Barnes, Joshua; Elowsky, Christian G; Wamboldt, Yashitola; Mackenzie, Sally A; Redding, Kevin; Merchant, Sabeeha S; Basset, Gilles J

2013-09-20

It is a little known fact that plastoquinone-9, a vital redox cofactor of photosynthesis, doubles as a precursor for the biosynthesis of a vitamin E analog called plastochromanol-8, the physiological significance of which has remained elusive. Gene network reconstruction, GFP fusion experiments, and targeted metabolite profiling of insertion mutants indicated that Arabidopsis possesses two paralogous solanesyl-diphosphate synthases, AtSPS1 (At1g78510) and AtSPS2 (At1g17050), that assemble the side chain of plastoquinone-9 in plastids. Similar paralogous pairs were detected throughout terrestrial plant lineages but were not distinguished in the literature and genomic databases from mitochondrial homologs involved in the biosynthesis of ubiquinone. The leaves of the atsps2 knock-out were devoid of plastochromanol-8 and displayed severe losses of both non-photoactive and photoactive plastoquinone-9, resulting in near complete photoinhibition at high light intensity. Such a photoinhibition was paralleled by significant damage to photosystem II but not to photosystem I. In contrast, in the atsps1 knock-out, a small loss of plastoquinone-9, restricted to the non-photoactive pool, was sufficient to eliminate half of the plastochromanol-8 content of the leaves. Taken together, these results demonstrate that plastochromanol-8 originates from a subfraction of the non-photoactive pool of plastoquinone-9. In contrast to other plastochromanol-8 biosynthetic mutants, neither the single atsps knock-outs nor the atsps1 atsps2 double knock-out displayed any defects in tocopherols accumulation or germination.

Functional Modeling Identifies Paralogous Solanesyl-diphosphate Synthases That Assemble the Side Chain of Plastoquinone-9 in Plastids*

PubMed Central

Block, Anna; Fristedt, Rikard; Rogers, Sara; Kumar, Jyothi; Barnes, Brian; Barnes, Joshua; Elowsky, Christian G.; Wamboldt, Yashitola; Mackenzie, Sally A.; Redding, Kevin; Merchant, Sabeeha S.; Basset, Gilles J.

2013-01-01

It is a little known fact that plastoquinone-9, a vital redox cofactor of photosynthesis, doubles as a precursor for the biosynthesis of a vitamin E analog called plastochromanol-8, the physiological significance of which has remained elusive. Gene network reconstruction, GFP fusion experiments, and targeted metabolite profiling of insertion mutants indicated that Arabidopsis possesses two paralogous solanesyl-diphosphate synthases, AtSPS1 (At1g78510) and AtSPS2 (At1g17050), that assemble the side chain of plastoquinone-9 in plastids. Similar paralogous pairs were detected throughout terrestrial plant lineages but were not distinguished in the literature and genomic databases from mitochondrial homologs involved in the biosynthesis of ubiquinone. The leaves of the atsps2 knock-out were devoid of plastochromanol-8 and displayed severe losses of both non-photoactive and photoactive plastoquinone-9, resulting in near complete photoinhibition at high light intensity. Such a photoinhibition was paralleled by significant damage to photosystem II but not to photosystem I. In contrast, in the atsps1 knock-out, a small loss of plastoquinone-9, restricted to the non-photoactive pool, was sufficient to eliminate half of the plastochromanol-8 content of the leaves. Taken together, these results demonstrate that plastochromanol-8 originates from a subfraction of the non-photoactive pool of plastoquinone-9. In contrast to other plastochromanol-8 biosynthetic mutants, neither the single atsps knock-outs nor the atsps1 atsps2 double knock-out displayed any defects in tocopherols accumulation or germination. PMID:23913686

Homology-dependent repair is involved in 45S rDNA loss in plant CAF-1 mutants

PubMed Central

Muchová, Veronika; Amiard, Simon; Mozgová, Iva; Dvořáčková, Martina; Gallego, Maria E; White, Charles; Fajkus, Jiří

2015-01-01

Arabidopsis thaliana mutants in FAS1 and FAS2 subunits of chromatin assembly factor 1 (CAF1) show progressive loss of 45S rDNA copies and telomeres. We hypothesized that homology-dependent DNA damage repair (HDR) may contribute to the loss of these repeats in fas mutants. To test this, we generated double mutants by crossing fas mutants with knock-out mutants in RAD51B, one of the Rad51 paralogs of A. thaliana. Our results show that the absence of RAD51B decreases the rate of rDNA loss, confirming the implication of RAD51B-dependent recombination in rDNA loss in the CAF1 mutants. Interestingly, this effect is not observed for telomeric repeat loss, which thus differs from that acting in rDNA loss. Involvement of DNA damage repair in rDNA dynamics in fas mutants is further supported by accumulation of double-stranded breaks (measured as γ-H2AX foci) in 45S rDNA. Occurrence of the foci is not specific for S-phase, and is ATM-independent. While the foci in fas mutants occur both in the transcribed (intranucleolar) and non-transcribed (nucleoplasmic) fraction of rDNA, double fas rad51b mutants show a specific increase in the number of the intranucleolar foci. These results suggest that the repair of double-stranded breaks present in the transcribed rDNA region is RAD51B dependent and that this contributes to rDNA repeat loss in fas mutants, presumably via the single-stranded annealing recombination pathway. Our results also highlight the importance of proper chromatin assembly in the maintenance of genome stability. PMID:25359579

Heat Shock Factors HsfB1 and HsfB2b Are Involved in the Regulation of Pdf1.2 Expression and Pathogen Resistance in Arabidopsis

PubMed Central

Kumar, Mukesh; Busch, Wolfgang; Birke, Hannah; Kemmerling, Birgit; Nürnberger, Thorsten; Schöffl, Friedrich

2009-01-01

In order to assess the functional roles of heat stress-induced class B-heat shock factors in Arabidopsis, we investigated T-DNA knockout mutants of AtHsfB1 and AtHsfB2b. Micorarray analysis of double knockout hsfB1/hsfB2b plants revealed as strong an up-regulation of the basal mRNA-levels of the defensin genes Pdf1.2a/b in mutant plants. The Pdf expression was further enhanced by jasmonic acid treatment or infection with the necrotrophic fungus Alternaria brassicicola. The single mutant hsfB2b and the double mutant hsfB1/B2b were significantly improved in disease resistance after A. brassicicola infection. There was no indication for a direct interaction of Hsf with the promoter of Pdf1.2, which is devoid of perfect HSE consensus Hsf-binding sequences. However, changes in the formation of late HsfA2-dependent HSE binding were detected in hsfB1/B2b plants. This suggests that HsfB1/B2b may interact with class A-Hsf in regulating the shut-off of the heat shock response. The identification of Pdf genes as targets of Hsf-dependent negative regulation is the first evidence for an interconnection of Hsf in the regulation of biotic and abiotic responses. PMID:19529832

Bacillus subtilis Mutants with Knockouts of the Genes Encoding Ribonucleases RNase Y and RNase J1 Are Viable, with Major Defects in Cell Morphology, Sporulation, and Competence

PubMed Central

Figaro, Sabine; Durand, Sylvain; Gilet, Laetitia; Cayet, Nadège; Sachse, Martin

2013-01-01

The genes encoding the ribonucleases RNase J1 and RNase Y have long been considered essential for Bacillus subtilis cell viability, even before there was concrete knowledge of their function as two of the most important enzymes for RNA turnover in this organism. Here we show that this characterization is incorrect and that ΔrnjA and Δrny mutants are both viable. As expected, both strains grow relatively slowly, with doubling times in the hour range in rich medium. Knockout mutants have major defects in their sporulation and competence development programs. Both mutants are hypersensitive to a wide range of antibiotics and have dramatic alterations to their cell morphologies, suggestive of cell envelope defects. Indeed, RNase Y mutants are significantly smaller in diameter than wild-type strains and have a very disordered peptidoglycan layer. Strains lacking RNase J1 form long filaments in tight spirals, reminiscent of mutants of the actin-like proteins (Mre) involved in cell shape determination. Finally, we combined the rnjA and rny mutations with mutations in other components of the degradation machinery and show that many of these strains are also viable. The implications for the two known RNA degradation pathways of B. subtilis are discussed. PMID:23504012

Deoxynucleoside stress exacerbates the phenotype of a mouse model of mitochondrial neurogastrointestinal encephalopathy

PubMed Central

Garcia-Diaz, Beatriz; Garone, Caterina; Barca, Emanuele; Mojahed, Hamed; Gutierrez, Purification; Pizzorno, Giuseppe; Tanji, Kurenai; Arias-Mendoza, Fernando; Quinzii, Caterina M.

2014-01-01

Balanced pools of deoxyribonucleoside triphosphate precursors are required for DNA replication, and alterations of this balance are relevant to human mitochondrial diseases including mitochondrial neurogastrointestinal encephalopathy. In this disease, autosomal recessive TYMP mutations cause severe reductions of thymidine phosphorylase activity; marked elevations of the pyrimidine nucleosides thymidine and deoxyuridine in plasma and tissues, and somatic multiple deletions, depletion and site-specific point mutations of mitochondrial DNA. Thymidine phosphorylase and uridine phosphorylase double knockout mice recapitulated several features of these patients including thymidine phosphorylase activity deficiency, elevated thymidine and deoxyuridine in tissues, mitochondrial DNA depletion, respiratory chain defects and white matter changes. However, in contrast to patients with this disease, mutant mice showed mitochondrial alterations only in the brain. To test the hypothesis that elevated levels of nucleotides cause unbalanced deoxyribonucleoside triphosphate pools and, in turn, pathogenic mitochondrial DNA instability, we have stressed double knockout mice with exogenous thymidine and deoxyuridine, and assessed clinical, neuroradiological, histological, molecular, and biochemical consequences. Mutant mice treated with exogenous thymidine and deoxyuridine showed reduced survival, body weight, and muscle strength, relative to untreated animals. Moreover, in treated mutants, leukoencephalopathy, a hallmark of the disease, was enhanced and the small intestine showed a reduction of smooth muscle cells and increased fibrosis. Levels of mitochondrial DNA were depleted not only in the brain but also in the small intestine, and deoxyribonucleoside triphosphate imbalance was observed in the brain. The relative proportion, rather than the absolute amount of deoxyribonucleoside triphosphate, was critical for mitochondrial DNA maintenance. Thus, our results demonstrate that stress of exogenous pyrimidine nucleosides enhances the mitochondrial phenotype of our knockout mice. Our mouse studies provide insights into the pathogenic role of thymidine and deoxyuridine imbalance in mitochondrial neurogastrointestinal encephalopathy and an excellent model to study new therapeutic approaches. PMID:24727567

AtSIG6 and other members of the sigma gene family jointly but differentially determine plastid target gene expression in Arabidopsis thaliana

PubMed Central

Bock, Sylvia; Ortelt, Jennifer; Link, Gerhard

2014-01-01

Plants contain a nuclear gene family for plastid sigma factors, i.e., proteins that associate with the “bacterial-type” organellar RNA polymerase and confer the ability for correct promoter binding and transcription initiation. Questions that are still unresolved relate to the “division of labor” among members of the sigma family, both in terms of their range of target genes and their temporal and spatial activity during development. Clues to the in vivo role of individual sigma genes have mainly come from studies of sigma knockout lines. Despite its obvious strengths, however, this strategy does not necessarily trace-down causal relationships between mutant phenotype and a single sigma gene, if other family members act in a redundant and/or compensatory manner. We made efforts to reduce the complexity by genetic crosses of Arabidopsis single mutants (with focus on a chlorophyll-deficient sig6 line) to generate double knockout lines. The latter typically had a similar visible phenotype as the parental lines, but tended to be more strongly affected in the transcript patterns of both plastid and sigma genes. Because triple mutants were lethal under our growth conditions, we exploited a strategy of transformation of single and double mutants with RNAi constructs that contained sequences from the unconserved sigma region (UCR). These RNAi/knockout lines phenotypically resembled their parental lines, but were even more strongly affected in their plastid transcript patterns. Expression patterns of sigma genes revealed both similarities and differences compared to the parental lines, with transcripts at reduced or unchanged amounts and others that were found to be present in higher (perhaps compensatory) amounts. Together, our results reveal considerable flexibility of gene activity at the levels of both sigma and plastid gene expression. A (still viable) “basal state” seems to be reached, if 2–3 of the 6 Arabidopsis sigma genes are functionally compromised. PMID:25505479

Reduced starch granule number per chloroplast in the dpe2/phs1 mutant is dependent on initiation of starch degradation

PubMed Central

Malinova, Irina

2017-01-01

An Arabidopsis double knock-out mutant lacking cytosolic disproportionating enzyme 2 (DPE2) and the plastidial phosphorylase (PHS1) revealed a dwarf-growth phenotype, reduced starch content, an uneven distribution of starch within the plant rosette, and a reduced number of starch granules per chloroplast under standard growth conditions. In contrast, the wild type contained 5–7 starch granules per chloroplast. Mature and old leaves of the double mutant were essentially starch free and showed plastidial disintegration. Several analyses revealed that the number of starch granules per chloroplast was affected by the dark phase. So far, it was unclear if it was the dark phase per se or starch degradation in the dark that was connected to the observed decrease in the number of starch granules per chloroplast. Therefore, in the background of the double mutant dpe2/phs1, a triple mutant was generated lacking the initial starch degrading enzyme glucan, water dikinase (GWD). The triple mutant showed improved plant growth, a starch-excess phenotype, and a homogeneous starch distribution. Furthermore, the number of starch granules per chloroplast was increased and was similar to wild type. However, starch granule morphology was only slightly affected by the lack of GWD as in the triple mutant and, like in dpe2/phs1, more spherical starch granules were observed. The characterized triple mutant was discussed in the context of the generation of starch granules and the formation of starch granule morphology. PMID:29155859

Reduced starch granule number per chloroplast in the dpe2/phs1 mutant is dependent on initiation of starch degradation.

PubMed

Malinova, Irina; Fettke, Joerg

2017-01-01

An Arabidopsis double knock-out mutant lacking cytosolic disproportionating enzyme 2 (DPE2) and the plastidial phosphorylase (PHS1) revealed a dwarf-growth phenotype, reduced starch content, an uneven distribution of starch within the plant rosette, and a reduced number of starch granules per chloroplast under standard growth conditions. In contrast, the wild type contained 5-7 starch granules per chloroplast. Mature and old leaves of the double mutant were essentially starch free and showed plastidial disintegration. Several analyses revealed that the number of starch granules per chloroplast was affected by the dark phase. So far, it was unclear if it was the dark phase per se or starch degradation in the dark that was connected to the observed decrease in the number of starch granules per chloroplast. Therefore, in the background of the double mutant dpe2/phs1, a triple mutant was generated lacking the initial starch degrading enzyme glucan, water dikinase (GWD). The triple mutant showed improved plant growth, a starch-excess phenotype, and a homogeneous starch distribution. Furthermore, the number of starch granules per chloroplast was increased and was similar to wild type. However, starch granule morphology was only slightly affected by the lack of GWD as in the triple mutant and, like in dpe2/phs1, more spherical starch granules were observed. The characterized triple mutant was discussed in the context of the generation of starch granules and the formation of starch granule morphology.

Functional characterization of double-knockout mouse sperm lacking SPAM1 and ACR or SPAM1 and PRSS21 in fertilization.

PubMed

Zhou, Chong; Kang, Woojin; Baba, Tadashi

2012-01-01

Mammalian fertilization requires sperm to penetrate the cumulus to reach the oocyte. Although sperm hyaluronidase has long been believed to participate in the penetration process, our previous works revealed that neither of two sperm hyaluronidases, SPAM1 and HYAL5, are essential for fertilization. In this study, we have produced double-knockout mice lacking SPAM1 and either one of two sperm serine proteases, ACR and PRSS21, and characterized the mutant sperm. The SPAM1/ACR- and SPAM1/PRSS21-deficient males were fertile, whereas epididymal sperm of the mutant mice exhibited a reduced capacity to fertilize the oocytes in vitro. Despite normal motility, the ability of sperm to traverse the cumulus matrix was more severely impaired by the loss of SPAM1 and ACR or SPAM1 and PRSS21 than by the loss of only SPAM1. Moreover, SPAM1/ACR- and SPAM1/PRSS21-deficient sperm accumulated on the surface (outer edge) of the cumulus more abundantly than SPAM1-deficient sperm. These results suggest that ACR or PRSS21 or both may function cooperatively with SPAM1 in sperm/cumulus penetration.

GeneKnockout by Targeted Mutagenesis in a Hemimetabolous Insect, the Two-Spotted Cricket Gryllus bimaculatus, using TALENs.

PubMed

Watanabe, Takahito; Noji, Sumihare; Mito, Taro

2016-01-01

Hemimetabolous, or incompletely metamorphosing, insects are phylogenetically basal. These insects include many deleterious species. The cricket, Gryllus bimaculatus, is an emerging model for hemimetabolous insects, based on the success of RNA interference (RNAi)-based gene-functional analyses and transgenic technology. Taking advantage of genome-editing technologies in this species would greatly promote functional genomics studies. Genome editing using transcription activator-like effector nucleases (TALENs) has proven to be an effective method for site-specific genome manipulation in various species. TALENs are artificial nucleases that are capable of inducing DNA double-strand breaks into specified target sequences. Here, we describe a protocol for TALEN-based gene knockout in G. bimaculatus, including a mutant selection scheme via mutation detection assays, for generating homozygous knockout organisms.

Knockout of glial channel ACD-1 exacerbates sensory deficits in a C. elegans mutant by regulating calcium levels of sensory neurons

PubMed Central

Wang, Ying; D'Urso, Giulia

2012-01-01

Degenerin/epithelial Na+ channels (DEG/ENaCs) are voltage-independent Na+ or Na+/Ca2+ channels expressed in many tissues and are needed for a wide range of physiological functions, including sensory perception and transepithelial Na+ transport. In the nervous system, DEG/ENaCs are expressed in both neurons and glia. However, the role of glial vs. neuronal DEG/ENaCs remains unclear. We recently reported the characterization of a novel DEG/ENaC in Caenorhabditis elegans that we named ACD-1. ACD-1 is expressed in glial amphid sheath cells. The glial ACD-1, together with the neuronal DEG/ENaC DEG-1, is necessary for acid avoidance and attraction to lysine. We report presently that knockout of acd-1 in glia exacerbates sensory deficits caused by another mutant: the hypomorphic allele of the cGMP-gated channel subunit tax-2. Furthermore, sensory deficits caused by mutations in Gi protein odr-3 and guanylate cyclase daf-11, which regulate the activity of TAX-2/TAX-4 channels, are worsened by knockout of acd-1. We also show that sensory neurons of acd-1 tax-2(p694) double mutants fail to undergo changes in intracellular Ca2+ when animals are exposed to low concentrations of attractant. Finally, we show that exogenous expression of TRPV1 in sensory neurons and exposure to capsaicin rescue sensory deficits of acd-1 tax-2(p694) mutants, suggesting that sensory deficits of these mutants are bypassed by increasing neuronal excitability. Our data suggest a role of glial DEG/ENaC channel ACD-1 in supporting neuronal activity. PMID:21994266

Knockout of glial channel ACD-1 exacerbates sensory deficits in a C. elegans mutant by regulating calcium levels of sensory neurons.

PubMed

Wang, Ying; D'Urso, Giulia; Bianchi, Laura

2012-01-01

Degenerin/epithelial Na(+) channels (DEG/ENaCs) are voltage-independent Na(+) or Na(+)/Ca(2+) channels expressed in many tissues and are needed for a wide range of physiological functions, including sensory perception and transepithelial Na(+) transport. In the nervous system, DEG/ENaCs are expressed in both neurons and glia. However, the role of glial vs. neuronal DEG/ENaCs remains unclear. We recently reported the characterization of a novel DEG/ENaC in Caenorhabditis elegans that we named ACD-1. ACD-1 is expressed in glial amphid sheath cells. The glial ACD-1, together with the neuronal DEG/ENaC DEG-1, is necessary for acid avoidance and attraction to lysine. We report presently that knockout of acd-1 in glia exacerbates sensory deficits caused by another mutant: the hypomorphic allele of the cGMP-gated channel subunit tax-2. Furthermore, sensory deficits caused by mutations in G(i) protein odr-3 and guanylate cyclase daf-11, which regulate the activity of TAX-2/TAX-4 channels, are worsened by knockout of acd-1. We also show that sensory neurons of acd-1 tax-2(p694) double mutants fail to undergo changes in intracellular Ca(2+) when animals are exposed to low concentrations of attractant. Finally, we show that exogenous expression of TRPV1 in sensory neurons and exposure to capsaicin rescue sensory deficits of acd-1 tax-2(p694) mutants, suggesting that sensory deficits of these mutants are bypassed by increasing neuronal excitability. Our data suggest a role of glial DEG/ENaC channel ACD-1 in supporting neuronal activity.

Gene knockout by targeted mutagenesis in a hemimetabolous insect, the two-spotted cricket Gryllus bimaculatus, using TALENs.

PubMed

Watanabe, Takahito; Noji, Sumihare; Mito, Taro

2014-08-15

Hemimetabolous, or incompletely metamorphosing, insects are phylogenetically basal. These insects include many deleterious species. The cricket, Gryllus bimaculatus, is an emerging model for hemimetabolous insects, based on the success of RNA interference (RNAi)-based gene-functional analyses and transgenic technology. Taking advantage of genome-editing technologies in this species would greatly promote functional genomics studies. Genome editing using transcription activator-like effector nucleases (TALENs) has proven to be an effective method for site-specific genome manipulation in various species. TALENs are artificial nucleases that are capable of inducing DNA double-strand breaks into specified target sequences. Here, we describe a protocol for TALEN-based gene knockout in G. bimaculatus, including a mutant selection scheme via mutation detection assays, for generating homozygous knockout organisms. Copyright © 2014 Elsevier Inc. All rights reserved.

Cross-Regulation between the phz1 and phz2 Operons Maintain a Balanced Level of Phenazine Biosynthesis in Pseudomonas aeruginosa PAO1

PubMed Central

Jiang, Bei; Xiao, Bo; Liu, Linde; Ge, Yihe; Hu, Xiaomei

2016-01-01

Gene duplication often provides selective advantages for the survival of microorganisms in adapting to varying environmental conditions. P. aeruginosa PAO1 possesses two seven-gene operons [phz1 (phzA1B1C1D1E1F1G1) and phz2 (phzA2B2C2D2E2F2G2)] that are involved in the biosynthesis of phenazine-1-carboxylic acid and its derivatives. Although the two operons are highly homologous and their functions are well known, it is unclear how the two phz operons coordinate their expressions to maintain the phenazine biosynthesis. By constructing single and double deletion mutants of the two phz operons, we found that the phz1-deletion mutant produced the same or less amount of phenazine-1-carboxylic acid and pyocyanin in GA medium than the phz2-knockout mutant while the phz1-phz2 double knockout mutant did not produce any phenazines. By generating phzA1 and phzA2 translational and transcriptional fusions with a truncated lacZ reporter, we found that the expression of the phz1 operon increased significantly at the post-transcriptional level and did not alter at the transcriptional level in the absence of the phz2 operon. Surprisingly, the expression the phz2 operon increased significantly at the post-transcriptional level and only moderately at the transcriptional level in the absence of the phz1 operon. Our findings suggested that a complex cross-regulation existed between the phz1 and phz2 operons. By mediating the upregulation of one phz operon expression while the other was deleted, this crosstalk would maintain the homeostatic balance of phenazine biosynthesis in P. aeruginosa PAO1. PMID:26735915

How do the effects of mutations add up?

NASA Astrophysics Data System (ADS)

Velenich, Andrea; Dai, Mingjie; Gore, Jeff

2011-03-01

Genetic mutations affect the fitness of any organism and provide the variability necessary for natural selection to occur. Given the fitness of a wild type organism and the fitness of mutants A and B which differ from the wild type by a single mutation, predicting the fitness of the double mutant AB is a fundamental problem with broad implications in many fields, from evolutionary theory to medicine. Analysis of millions of double gene knockouts in yeast reveals that, on average, the fitness of AB is the product of the fitness of A and the fitness of B. However, most pairs of mutations deviate from this mean behavior in a way that challenges existing theoretical models. We propose a natural generalization of the geometric Fisher's model which accommodates the experimentally observed features and allows us to characterize the fitness landscape of yeast.

Calpains are involved in asexual and sexual development, cell wall integrity and pathogenicity of the rice blast fungus

PubMed Central

Liu, Xiao-Hong; Ning, Guo-Ao; Huang, Lu-Yao; Zhao, Ya-Hui; Dong, Bo; Lu, Jian-Ping; Lin, Fu-Cheng

2016-01-01

Calpains are ubiquitous and well-conserved proteins that belong to the calcium-dependent, non-lysosomal cysteine protease family. In this study, 8 putative calpains were identified using Pfam domain analysis and BlastP searches in M. oryzae. Three single gene deletion mutants (ΔMocapn7, ΔMocapn9 and ΔMocapn14) and two double gene deletion mutants (ΔMocapn4ΔMocapn7 and ΔMocapn9ΔMocapn7) were obtained using the high-throughput gene knockout system. The calpain disruption mutants showed defects in colony characteristics, conidiation, sexual reproduction and cell wall integrity. The mycelia of the ΔMocapn7, ΔMocapn4ΔMocapn7 and ΔMocapn9ΔMocapn7 mutants showed reduced pathogenicity on rice and barley. PMID:27502542

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.

The role of the Serratia marcescens SdeAB multidrug efflux pump and TolC homologue in fluoroquinolone resistance studied via gene-knockout mutagenesis.

PubMed

Begic, Sanela; Worobec, Elizabeth A

2008-02-01

Serratia marcescens is a prominent opportunistic nosocomial pathogen resistant to several classes of antibiotics. The major mechanism for fluoroquinolone resistance in various Gram-negative pathogens is active efflux. Our group previously identified SdeAB, a resistance-nodulation-cell division (RND) efflux pump complex, and a TolC-like outer-membrane protein (HasF), which together mediate energy-dependent fluoroquinolone efflux. In addition, a regulatory protein-encoding gene in the upstream region of sdeAB was identified (sdeR) and found to be 40 % homologous to MarA, an Escherichia coli transcriptional regulator. To provide conclusive evidence as to the role of these components in S. marcescens, sdeB, hasF and sdeR deletion mutants were constructed. Suicide vectors were created and introduced via triparental mating into S. marcescens UOC-67 (wild-type) and, for sdeB and hasF, T-861 (clinical isolate). We have analysed these genetically altered strains using minimal inhibitory concentration (MIC) assays for a wide range of compounds (fluoroquinolones, SDS, novobiocin, ethidium bromide and chloramphenicol). Intracellular accumulation of a variety of fluoroquinolones was measured fluorospectroscopically. The sdeB, hasF and sdeR knockout strains were consistently more susceptible to antibiotics than the parent strains, with the sdeB/hasF double knockout strain showing the highest susceptibility. A marked increase in fluoroquinolone (ciprofloxacin) accumulation was observed for strains deficient in either the sdeB or hasF genes when compared to the parental strains, with the highest ciprofloxacin accumulation observed for the sdeB/hasF double knockout. Antibiotic accumulation assays for the sdeB knockout mutant strains performed in the presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP), a proton-motive-force inhibitor, demonstrated that SdeAB-mediated efflux is proton-motive-force dependent. Due to the comparable susceptibility of the sdeB and the hasF individual knockouts, we conclude that S. marcescens HasF is the sole outer-membrane component of the SdeAB pump. In addition, MIC data for sdeR-deficient and overexpressing strains confirm that SdeR is an activator of sdeAB and acts to enhance the overall multidrug resistance of S. marcescens.

A Phex Mutation in a Murine Model of X-linked Hypophosphatemia Alters Phosphate Responsiveness of Bone Cells

PubMed Central

Ichikawa, Shoji; Austin, Anthony M.; Gray, Amie K.; Econs, Michael J.

2011-01-01

Mutations in the PHEX gene cause X-linked hypophosphatemia (XLH). Hypophosphatemia in XLH results from increased circulating levels of a phosphaturic hormone, fibroblast growth factor 23 (FGF23), which inhibits renal phosphate reabsorption and 1,25-dihydroxyvitamin D (calcitriol) synthesis. The current standard therapy for XLH – high dose phosphate and calcitriol – further increases FGF23 concentrations, suggesting that patients with XLH may have an altered response to extracellular phosphate. To test for the presence of abnormal phosphate responsiveness, we compared serum biochemistries and femoral Fgf23 mRNA expression between wild-type mice, murine models of XLH (PhexK496X) and hyperphosphatemic tumoral calcinosis (Galnt3 -/-), and Galnt3/Phex double mutant mice. Phex mutant mice had not only increased Fgf23 expression, but also reduced proteolytic cleavage of intact Fgf23 protein, resulting in markedly elevated intact Fgf23 levels and consequent hypophosphatemia. In contrast, despite markedly increased Fgf23 expression, Galnt3 knockout mice had significantly high proteolytic cleavage of Fgf23 protein, leading to low intact Fgf23 concentrations and hyperphosphatemia. Galnt3/Phex double mutant mice had an intermediate biochemical phenotype between wild-type and Phex mutant mice, including slightly elevated intact Fgf23 concentrations with milder hypophosphatemia. Despite the hypophosphatemia, double mutant mice attempted to reduce serum phosphate back to the level of Phex mutant mice by up-regulating Fgf23 expression as much as 24 fold higher than Phex mutant mice. These data suggest that Phex mutations alter the responsiveness of bone cells to extracellular phosphate concentrations and may create a lower set point for “normal” phosphate levels. PMID:22006791

A Phex mutation in a murine model of X-linked hypophosphatemia alters phosphate responsiveness of bone cells.

PubMed

Ichikawa, Shoji; Austin, Anthony M; Gray, Amie K; Econs, Michael J

2012-02-01

Mutations in the PHEX gene cause X-linked hypophosphatemia (XLH). Hypophosphatemia in XLH results from increased circulating levels of a phosphaturic hormone, fibroblast growth factor 23 (FGF23), which inhibits renal phosphate reabsorption and 1,25-dihydroxyvitamin D (calcitriol) synthesis. The current standard therapy for XLH--high-dose phosphate and calcitriol--further increases FGF23 concentrations, suggesting that patients with XLH may have an altered response to extracellular phosphate. To test for the presence of abnormal phosphate responsiveness, we compared serum biochemistries and femoral Fgf23 mRNA expression between wild-type mice, murine models of XLH (Phex(K496X)) and hyperphosphatemic tumoral calcinosis (Galnt3(-/-)), and Galnt3/Phex double-mutant mice. Phex mutant mice had not only increased Fgf23 expression but also reduced proteolytic cleavage of intact Fgf23 protein, resulting in markedly elevated intact Fgf23 levels and consequent hypophosphatemia. In contrast, despite markedly increased Fgf23 expression, Galnt3 knockout mice had significantly high proteolytic cleavage of Fgf23 protein, leading to low intact Fgf23 concentrations and hyperphosphatemia. Galnt3/Phex double-mutant mice had an intermediate biochemical phenotype between wild-type and Phex mutant mice, including slightly elevated intact Fgf23 concentrations with milder hypophosphatemia. Despite the hypophosphatemia, double-mutant mice attempted to reduce serum phosphate back to the level of Phex mutant mice by upregulating Fgf23 expression as much as 24-fold higher than Phex mutant mice. These data suggest that Phex mutations alter the responsiveness of bone cells to extracellular phosphate concentrations and may create a lower set point for "normal" phosphate levels.

The gene for a lectin-like protein is transcriptionally activated during sexual development, but is not essential for fruiting body formation in the filamentous fungus Sordaria macrospora.

PubMed

Nowrousian, Minou; Cebula, Patricia

2005-11-03

The filamentous fungus Sordaria macrospora forms complex three-dimensional fruiting bodies called perithecia that protect the developing ascospores and ensure their proper discharge. In previous microarray analyses, several genes have been identified that are downregulated in sterile mutants compared to the wild type. Among these genes was tap1 (transcript associated with perithecial development), a gene encoding a putative lectin homolog. Analysis of tap1 transcript levels in the wild type under conditions allowing only vegetative growth compared to conditions that lead to fruiting body development showed that tap1 is not only downregulated in developmental mutants but is also upregulated in the wild type during fruiting body development. We have cloned and sequenced a 3.2 kb fragment of genomic DNA containing the tap1 open reading frame and adjoining sequences. The genomic region comprising tap1 is syntenic to its homologous region in the closely related filamentous fungus Neurospora crassa. To determine whether tap1 is involved in fruiting body development in S. macrospora, a knockout construct was generated in which the tap1 open reading frame was replaced by the hygromycin B resistance gene hph under the control of fungal regulatory regions. Transformation of the S. macrospora wild type with this construct resulted in a tap1 deletion strain where tap1 had been replaced by the hph cassette. The knockout strain displayed no phenotypic differences under conditions of vegetative growth and sexual development when compared to the wild type. Double mutants carrying the Deltatap1 allele in several developmental mutant backgrounds were phenotypically similar to the corresponding developmental mutant strains. The tap1 transcript is strongly upregulated during sexual development in S. macrospora; however, analysis of a tap1 knockout strain shows that tap1 is not essential for fruiting body formation in S. macrospora.

The gene for a lectin-like protein is transcriptionally activated during sexual development, but is not essential for fruiting body formation in the filamentous fungus Sordaria macrospora

PubMed Central

Nowrousian, Minou; Cebula, Patricia

2005-01-01

Background The filamentous fungus Sordaria macrospora forms complex three-dimensional fruiting bodies called perithecia that protect the developing ascospores and ensure their proper discharge. In previous microarray analyses, several genes have been identified that are downregulated in sterile mutants compared to the wild type. Among these genes was tap1 (transcript associated with perithecial development), a gene encoding a putative lectin homolog. Results Analysis of tap1 transcript levels in the wild type under conditions allowing only vegetative growth compared to conditions that lead to fruiting body development showed that tap1 is not only downregulated in developmental mutants but is also upregulated in the wild type during fruiting body development. We have cloned and sequenced a 3.2 kb fragment of genomic DNA containing the tap1 open reading frame and adjoining sequences. The genomic region comprising tap1 is syntenic to its homologous region in the closely related filamentous fungus Neurospora crassa. To determine whether tap1 is involved in fruiting body development in S. macrospora, a knockout construct was generated in which the tap1 open reading frame was replaced by the hygromycin B resistance gene hph under the control of fungal regulatory regions. Transformation of the S. macrospora wild type with this construct resulted in a tap1 deletion strain where tap1 had been replaced by the hph cassette. The knockout strain displayed no phenotypic differences under conditions of vegetative growth and sexual development when compared to the wild type. Double mutants carrying the Δtap1 allele in several developmental mutant backgrounds were phenotypically similar to the corresponding developmental mutant strains. Conclusion The tap1 transcript is strongly upregulated during sexual development in S. macrospora; however, analysis of a tap1 knockout strain shows that tap1 is not essential for fruiting body formation in S. macrospora. PMID:16266439

Disruption of both chloroplastic and cytosolic FBPase genes results in a dwarf phenotype and important starch and metabolite changes in Arabidopsis thaliana.

PubMed

Rojas-González, José A; Soto-Súarez, Mauricio; García-Díaz, Ángel; Romero-Puertas, María C; Sandalio, Luisa M; Mérida, Ángel; Thormählen, Ina; Geigenberger, Peter; Serrato, Antonio J; Sahrawy, Mariam

2015-05-01

In this study, evidence is provided for the role of fructose-1,6-bisphosphatases (FBPases) in plant development and carbohydrate synthesis and distribution by analysing two Arabidopsis thaliana T-DNA knockout mutant lines, cyfbp and cfbp1, and one double mutant cyfbp cfbp1 which affect each FBPase isoform, cytosolic and chloroplastic, respectively. cyFBP is involved in sucrose synthesis, whilst cFBP1 is a key enzyme in the Calvin-Benson cycle. In addition to the smaller rosette size and lower rate of photosynthesis, the lack of cFBP1 in the mutants cfbp1 and cyfbp cfbp1 leads to a lower content of soluble sugars, less starch accumulation, and a greater superoxide dismutase (SOD) activity. The mutants also had some developmental alterations, including stomatal opening defects and increased numbers of root vascular layers. Complementation also confirmed that the mutant phenotypes were caused by disruption of the cFBP1 gene. cyfbp mutant plants without cyFBP showed a higher starch content in the chloroplasts, but this did not greatly affect the phenotype. Notably, the sucrose content in cyfbp was close to that found in the wild type. The cyfbp cfbp1 double mutant displayed features of both parental lines but had the cfbp1 phenotype. All the mutants accumulated fructose-1,6-bisphosphate and triose-phosphate during the light period. These results prove that while the lack of cFBP1 induces important changes in a wide range of metabolites such as amino acids, sugars, and organic acids, the lack of cyFBP activity in Arabidopsis essentially provokes a carbon metabolism imbalance which does not compromise the viability of the double mutant cyfbp cfbp1. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

E3 Ubiquitin Ligase CHIP and NBR1-Mediated Selective Autophagy Protect Additively against Proteotoxicity in Plant Stress Responses

PubMed Central

Qi, Jingxia; Chi, Yingjin; Fan, Baofang; Yu, Jing-Quan; Chen, Zhixiang

2014-01-01

Plant stress responses require both protective measures that reduce or restore stress-inflicted damage to cellular structures and mechanisms that efficiently remove damaged and toxic macromolecules, such as misfolded and damaged proteins. We have recently reported that NBR1, the first identified plant autophagy adaptor with a ubiquitin-association domain, plays a critical role in plant stress tolerance by targeting stress-induced, ubiquitinated protein aggregates for degradation by autophagy. Here we report a comprehensive genetic analysis of CHIP, a chaperone-associated E3 ubiquitin ligase from Arabidopsis thaliana implicated in mediating degradation of nonnative proteins by 26S proteasomes. We isolated two chip knockout mutants and discovered that they had the same phenotypes as the nbr1 mutants with compromised tolerance to heat, oxidative and salt stresses and increased accumulation of insoluble proteins under heat stress. To determine their functional interactions, we generated chip nbr1 double mutants and found them to be further compromised in stress tolerance and in clearance of stress-induced protein aggregates, indicating additive roles of CHIP and NBR1. Furthermore, stress-induced protein aggregates were still ubiquitinated in the chip mutants. Through proteomic profiling, we systemically identified heat-induced protein aggregates in the chip and nbr1 single and double mutants. These experiments revealed that highly aggregate-prone proteins such as Rubisco activase and catalases preferentially accumulated in the nbr1 mutant while a number of light-harvesting complex proteins accumulated at high levels in the chip mutant after a relatively short period of heat stress. With extended heat stress, aggregates for a large number of intracellular proteins accumulated in both chip and nbr1 mutants and, to a greater extent, in the chip nbr1 double mutant. Based on these results, we propose that CHIP and NBR1 mediate two distinct but complementary anti-proteotoxic pathways and protein's propensity to aggregate under stress conditions is one of the critical factors for pathway selection of protein degradation. PMID:24497840

Differential Requirements for c-Myc in Chronic Hematopoietic Hyperplasia and Acute Hematopoietic Malignancies in Pten-null Mice

PubMed Central

Zhang, Jun; Xiao, Yechen; Guo, Yinshi; Breslin, Peter; Zhang, Shubin; Wei, Wei; Zhang, Zhou; Zhang, Jiwang

2011-01-01

Myeloproliferative disorders (MPDs), lymphoproliferative disorders (LPDs), acute T-lymphocytic or myeloid leukemia and T-lymphocytic lymphoma were developed in inducible Pten-knockout (Pten−/−) mice. The appearance of these multiple diseases in one animal model provides an opportunity to study the pathogenesis of multiple diseases simultaneously. To study whether Myc function is required for the development of these hematopoietic disorders in Pten−/− mice, we generated inducible Pten/Myc double-knockout mice (Pten−/−/Myc−/−). By comparing the hematopoietic phenotypes of these double-knockout mice with those of Pten−/− mice, we found that both sets of animals developed MPDs and LPDs. However, none of the compound-mutant mice developed acute leukemia or lymphoma. Interestingly, in contrast to the MPDs which developed in Pten−/− mice which are dominated by granulocytes, megakaryocytes predominate in the MPDs of Pten−/−/Myc−/− mice. Our study suggests that the deregulation of PI3K/Akt signaling in Pten−/− hematopoietic cells protects these cells from apoptotic cell death, resulting in chronic proliferative disorders. But due to the differential requirement for Myc in granulocyte as compared to megakaryocyte proliferation, Myc deletion converts Pten−/− MPDs from granulocyte-dominated to megakaryocyte-dominated conditions. Myc is absolutely required for the development of acute hematopoietic malignancies. PMID:21926961

The Mysterious Rescue of adg1-1/tpt-2 – an Arabidopsis thaliana Double Mutant Impaired in Acclimation to High Light – by Exogenously Supplied Sugars

PubMed Central

Heinrichs, Luisa; Schmitz, Jessica; Flügge, Ulf-Ingo; Häusler, Rainer E.

2012-01-01

An Arabidopsis thaliana double mutant (adg1-1/tpt-2) defective in the day- and night-path of photoassimilate export from the chloroplast due to a knockout in the triose phosphate/phosphate translocator (TPT; tpt-2) and a lack of starch [mutation in ADP glucose pyrophosphorylase (AGPase); adg1-1] exhibits severe growth retardation, a decrease in the photosynthetic capacity, and a high chlorophyll fluorescence (HCF) phenotype under high light conditions. These phenotypes could be rescued when the plants were grown on sucrose (Suc) or glucose (Glc). Here we address the question whether Glc-sensing hexokinase1 (HXK1) defective in the Glc insensitive 2 (gin2-1) mutant is involved in the sugar-dependent rescue of adg1-1/tpt-2. Triple mutants defective in the TPT, AGPase, and HXK1 (adg1-1/tpt-2/gin2-1) were established as homozygous lines and grown together with Col-0 and Landsberg erecta (Ler) wild-type plants, gin2-1, the adg1-1/tpt-2 double mutant, and the adg1-1/tpt-2/gpt2-1 triple mutant [additionally defective in the glucose 6-phosphate/phosphate translocator 2 (GPT2)] on agar in the presence or absence of 50 mM of each Glc, Suc, or fructose (Fru). The growth phenotype of the double mutant and both triple mutants could be rescued to a similar extent only by Glc and Suc, but not by Fru. All three sugars were capable of rescuing the HCF and photosynthesis phenotype, irrespectively of the presence or absence of HXK1. Quantitative RT-PCR analyses of sugar-responsive genes revealed that plastidial HXK (pHXK) was up-regulated in adg1-1/tpt-2 plants grown on sugars, but showed no response in adg1-1/tpt-2/gin2-1. It appears likely that soluble sugars are directly taken up by the chloroplasts and enter further metabolism, which consumes ATP and NADPH from the photosynthetic light reaction and thereby rescues the photosynthesis phenotype of the double mutant. The implication of sugar turnover and probably signaling inside the chloroplasts for the concept of retrograde signaling is discussed. PMID:23233856

The Cytoplasmic Carbonic Anhydrases βCA2 and βCA4 Are Required for Optimal Plant Growth at Low CO2.

PubMed

DiMario, Robert J; Quebedeaux, Jennifer C; Longstreth, David J; Dassanayake, Maheshi; Hartman, Monica M; Moroney, James V

2016-05-01

Carbonic anhydrases (CAs) are zinc metalloenzymes that interconvert CO2 and HCO3 (-) In plants, both α- and β-type CAs are present. We hypothesize that cytoplasmic βCAs are required to modulate inorganic carbon forms needed in leaf cells for carbon-requiring reactions such as photosynthesis and amino acid biosynthesis. In this report, we present evidence that βCA2 and βCA4 are the two most abundant cytoplasmic CAs in Arabidopsis (Arabidopsis thaliana) leaves. Previously, βCA4 was reported to be localized to the plasma membrane, but here, we show that two forms of βCA4 are expressed in a tissue-specific manner and that the two proteins encoded by βCA4 localize to two different regions of the cell. Comparing transfer DNA knockout lines with wild-type plants, there was no reduction in the growth rates of the single mutants, βca2 and βca4 However, the growth rate of the double mutant, βca2βca4, was reduced significantly when grown at 200 μL L(-1) CO2 The reduction in growth of the double mutant was not linked to a reduction in photosynthetic rate. The amino acid content of leaves from the double mutant showed marked reduction in aspartate when compared with the wild type and the single mutants. This suggests the cytoplasmic CAs play an important but not previously appreciated role in amino acid biosynthesis. © 2016 American Society of Plant Biologists. All Rights Reserved.

Rapid construction of a whole-genome transposon insertion collection for Shewanella oneidensis by Knockout Sudoku.

PubMed

Baym, Michael; Shaket, Lev; Anzai, Isao A; Adesina, Oluwakemi; Barstow, Buz

2016-11-10

Whole-genome knockout collections are invaluable for connecting gene sequence to function, yet traditionally, their construction has required an extraordinary technical effort. Here we report a method for the construction and purification of a curated whole-genome collection of single-gene transposon disruption mutants termed Knockout Sudoku. Using simple combinatorial pooling, a highly oversampled collection of mutants is condensed into a next-generation sequencing library in a single day, a 30- to 100-fold improvement over prior methods. The identities of the mutants in the collection are then solved by a probabilistic algorithm that uses internal self-consistency within the sequencing data set, followed by rapid algorithmically guided condensation to a minimal representative set of mutants, validation, and curation. Starting from a progenitor collection of 39,918 mutants, we compile a quality-controlled knockout collection of the electroactive microbe Shewanella oneidensis MR-1 containing representatives for 3,667 genes that is functionally validated by high-throughput kinetic measurements of quinone reduction.

Genome Editing in Mice Using TALE Nucleases.

PubMed

Wefers, Benedikt; Brandl, Christina; Ortiz, Oskar; Wurst, Wolfgang; Kühn, Ralf

2016-01-01

Gene engineering for generating targeted mouse mutants is a key technology for biomedical research. Using TALENs as sequence-specific nucleases to induce targeted double-strand breaks, the mouse genome can be directly modified in zygotes in a single step without the need for embryonic stem cells. By embryo microinjection of TALEN mRNAs and targeting vectors, knockout and knock-in alleles can be generated fast and efficiently. In this chapter we provide protocols for the application of TALENs in mouse zygotes.

Analysis of a Range of Catabolic Mutants Provides Evidence That Phytanoyl-Coenzyme A Does Not Act as a Substrate of the Electron-Transfer Flavoprotein/Electron-Transfer Flavoprotein:Ubiquinone Oxidoreductase Complex in Arabidopsis during Dark-Induced Senescence1[W][OA

PubMed Central

Araújo, Wagner L.; Ishizaki, Kimitsune; Nunes-Nesi, Adriano; Tohge, Takayuki; Larson, Tony R.; Krahnert, Ina; Balbo, Ilse; Witt, Sandra; Dörmann, Peter; Graham, Ian A.; Leaver, Christopher J.; Fernie, Alisdair R.

2011-01-01

The process of dark-induced senescence in plants is not fully understood, however, the functional involvement of an electron-transfer flavoprotein/electron-transfer flavoprotein:ubiquinone oxidoreductase (ETF/ETFQO), has been demonstrated. Recent studies have revealed that the enzymes isovaleryl-coenzyme A (CoA) dehydrogenase and 2-hydroxyglutarate dehydrogenase act as important electron donors to this complex. In addition both enzymes play a role in the breakdown of cellular carbon storage reserves with isovaleryl-CoA dehydrogenase being involved in degradation of the branched-chain amino acids, phytol, and lysine while 2-hydroxyglutarate dehydrogenase is exclusively involved in lysine degradation. Given that the chlorophyll breakdown intermediate phytanoyl-CoA accumulates dramatically both in knockout mutants of the ETF/ETFQO complex and of isovaleryl-CoA dehydrogenase following growth in extended dark periods we have investigated the direct importance of chlorophyll breakdown for the supply of carbon and electrons during this process. For this purpose we isolated three independent Arabidopsis (Arabidopsis thaliana) knockout mutants of phytanoyl-CoA 2-hydroxylase and grew them under the same extended darkness regime as previously used. Despite the fact that these mutants accumulated phytanoyl-CoA and also 2-hydroxyglutarate they exhibited no morphological changes in comparison to the other mutants previously characterized. These results are consistent with a single entry point of phytol breakdown into the ETF/ETFQO system and furthermore suggest that phytol is not primarily metabolized by this pathway. Furthermore analysis of isovaleryl-CoA dehydrogenase/2-hydroxyglutarate dehydrogenase double mutants generated here suggest that these two enzymes essentially account for the entire electron input via the ETF complex. PMID:21788362

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.

Brn3a and Islet1 act epistatically to regulate the gene expression program of sensory differentiation

PubMed Central

Dykes, Iain M.; Tempest, Lynne; Lee, Su-In; Turner, Eric E.

2011-01-01

The combinatorial expression of transcription factors frequently marks cellular identity in the nervous system, yet how these factors interact to determine specific neuronal phenotypes is not well understood. Sensory neurons of the trigeminal (TG) and dorsal root ganglia (DRG) co-express the homeodomain transcription factors Brn3a and Islet1, and past work has revealed partially overlapping programs of gene expression downstream of these factors. Here we examine sensory development in Brn3a/Islet1 double knockout mice (DKO mice). Sensory neurogenesis and the formation of the TG and DRG occur in DKO embryos, but the DRG are dorsally displaced, and the peripheral projections of the ganglia are markedly disturbed. Sensory neurons in DKO embryos show a profound loss of all early markers of sensory subtypes, including the Ntrk neurotrophin receptors, and the runt-family transcription factors Runx1 and Runx3. Examination of global gene expression in the E12.5 DRG of single and double mutant embryos shows that Brn3a and Islet1 are together required for nearly all aspects of sensory-specific gene expression, including several newly identified sensory markers. On a majority of targets Brn3a and Islet1 exhibit negative epistasis, in which the effects of the individual knockout alleles are less than additive in the DKO. Smaller subsets of targets exhibit positive epistasis, or are regulated exclusively by one factor. Brn3a/Islet1 double mutants also fail to developmentally repress neurogenic bHLH genes, and in vivo chromatin immunoprecipitation shows that Islet1 binds to a known Brn3a -regulated enhancer in the neurod4 gene, suggesting a mechanism of interaction between these genes. PMID:21734270

The groEL2 gene, but not groEL1, is required for biosynthesis of the secondary metabolite myxovirescin in Myxococcus xanthus DK1622.

PubMed

Wang, Yan; Li, Xi; Zhang, Wenyan; Zhou, Xiuwen; Li, Yue-zhong

2014-03-01

Myxococcus xanthus DK1622 possesses two copies of the groEL gene: groEL1, which participates in development, and groEL2, which is involved in the predatory ability of cells. In this study, we determined that the groEL2 gene is required for the biosynthesis of the secondary metabolite myxovirescin (TA), which plays essential roles in predation. The groEL2-knockout mutant strain was defective in producing a zone of inhibition and displayed decreased killing ability against Escherichia coli, while the groEL1-knockout mutant strain exhibited little difference from the wild-type strain DK1622. HPLC revealed that deletion of the groEL2 gene blocked the production of TA, which was present in the groEL1-knockout mutant. The addition of exogenous TA rescued the inhibition and killing abilities of the groEL2-knockout mutant against E. coli. Analysis of GroEL domain-swapping mutants indicated that the C-terminal equatorial domain of GroEL2 was essential for TA production, while the N-terminal equatorial or apical domains of GroEL2 were not sufficient to rescue TA production of the groEL2 knockout.

Male Fertility Defect Associated with Disrupted BRCA1-PALB2 Interaction in Mice*

PubMed Central

Simhadri, Srilatha; Peterson, Shaun; Patel, Dharm S.; Huo, Yanying; Cai, Hong; Bowman-Colin, Christian; Miller, Shoreh; Ludwig, Thomas; Ganesan, Shridar; Bhaumik, Mantu; Bunting, Samuel F.; Jasin, Maria; Xia, Bing

2014-01-01

PALB2 links BRCA1 and BRCA2 in homologous recombinational repair of DNA double strand breaks (DSBs). Mono-allelic mutations in PALB2 increase the risk of breast, pancreatic, and other cancers, and biallelic mutations cause Fanconi anemia (FA). Like Brca1 and Brca2, systemic knock-out of Palb2 in mice results in embryonic lethality. In this study, we generated a hypomorphic Palb2 allele expressing a mutant PALB2 protein unable to bind BRCA1. Consistent with an FA-like phenotype, cells from the mutant mice showed hypersensitivity and chromosomal breakage when treated with mitomycin C, a DNA interstrand crosslinker. Moreover, mutant males showed reduced fertility due to impaired meiosis and increased apoptosis in germ cells. Interestingly, mutant meiocytes showed a significant defect in sex chromosome synapsis, which likely contributed to the germ cell loss and fertility defect. Our results underscore the in vivo importance of the PALB2-BRCA1 complex formation in DSB repair and male meiosis. PMID:25016020

WASP family proteins and formins compete in pseudopod- and bleb-based migration

PubMed Central

2018-01-01

Actin pseudopods induced by SCAR/WAVE drive normal migration and chemotaxis in eukaryotic cells. Cells can also migrate using blebs, in which the edge is driven forward by hydrostatic pressure instead of actin. In Dictyostelium discoideum, loss of SCAR is compensated by WASP moving to the leading edge to generate morphologically normal pseudopods. Here we use an inducible double knockout to show that cells lacking both SCAR and WASP are unable to grow, make pseudopods or, unexpectedly, migrate using blebs. Remarkably, amounts and dynamics of actin polymerization are normal. Pseudopods are replaced in double SCAR/WASP mutants by aberrant filopods, induced by the formin dDia2. Further disruption of the gene for dDia2 restores cells’ ability to initiate blebs and thus migrate, though pseudopods are still lost. Triple knockout cells still contain near-normal F-actin levels. This work shows that SCAR, WASP, and dDia2 compete for actin. Loss of SCAR and WASP causes excessive dDia2 activity, maintaining F-actin levels but blocking pseudopod and bleb formation and migration. PMID:29191847

The Ovary Is an Alternative Site of Origin for High-Grade Serous Ovarian Cancer in Mice

PubMed Central

Coffey, Donna M.; Ma, Lang; Matzuk, Martin M.

2015-01-01

Although named “ovarian cancer,” it has been unclear whether the cancer actually arises from the ovary, especially for high-grade serous carcinoma (HGSC), also known as high-grade serous ovarian cancer, the most common and deadliest ovarian cancer. In addition, the tumor suppressor p53 is the most frequently mutated gene in HGSC. However, whether mutated p53 can cause HGSC remains unknown. In this study, we bred a p53 mutation, p53R172H, into conditional Dicer-Pten double-knockout (DKO) mice, a mouse model duplicating human HGSC, to generate triple-mutant (TKO) mice. Like DKO mice, these TKO mice develop metastatic HGSCs originating from the fallopian tube. Unlike DKO mice, however, even after fallopian tubes are removed in TKO mice, ovaries alone can develop metastatic HGSCs, indicating that a p53 mutation can drive HGSC arising from the ovary. To confirm this, we generated p53R172H-Pten double-mutant mice, one of the genetic control lines for TKO mice. As anticipated, these double-mutant mice also develop metastatic HGSCs from the ovary, verifying the HGSC-forming ability of ovaries with a p53 mutation. Our study therefore shows that ovaries harboring a p53 mutation, as well as fallopian tubes, can be a distinct tissue source of high-grade serous ovarian cancer in mice. PMID:25815421

The ovary is an alternative site of origin for high-grade serous ovarian cancer in mice.

PubMed

Kim, Jaeyeon; Coffey, Donna M; Ma, Lang; Matzuk, Martin M

2015-06-01

Although named "ovarian cancer," it has been unclear whether the cancer actually arises from the ovary, especially for high-grade serous carcinoma (HGSC), also known as high-grade serous ovarian cancer, the most common and deadliest ovarian cancer. In addition, the tumor suppressor p53 is the most frequently mutated gene in HGSC. However, whether mutated p53 can cause HGSC remains unknown. In this study, we bred a p53 mutation, p53(R172H), into conditional Dicer-Pten double-knockout (DKO) mice, a mouse model duplicating human HGSC, to generate triple-mutant (TKO) mice. Like DKO mice, these TKO mice develop metastatic HGSCs originating from the fallopian tube. Unlike DKO mice, however, even after fallopian tubes are removed in TKO mice, ovaries alone can develop metastatic HGSCs, indicating that a p53 mutation can drive HGSC arising from the ovary. To confirm this, we generated p53(R172H)-Pten double-mutant mice, one of the genetic control lines for TKO mice. As anticipated, these double-mutant mice also develop metastatic HGSCs from the ovary, verifying the HGSC-forming ability of ovaries with a p53 mutation. Our study therefore shows that ovaries harboring a p53 mutation, as well as fallopian tubes, can be a distinct tissue source of high-grade serous ovarian cancer in mice.

NBS1 plays a synergistic role with telomerase in the maintenance of telomeres in Arabidopsis thaliana.

PubMed

Najdekrova, Lucie; Siroky, Jiri

2012-09-17

Telomeres, as elaborate nucleo-protein complexes, ensure chromosomal stability. When impaired, the ends of linear chromosomes can be recognised by cellular repair mechanisms as double-strand DNA breaks and can be healed by non-homologous-end-joining activities to produce dicentric chromosomes. During cell divisions, particularly during anaphase, dicentrics can break, thus producing naked chromosome tips susceptible to additional unwanted chromosome fusion. Many telomere-building protein complexes are associated with telomeres to ensure their proper capping function. It has been found however, that a number of repair complexes also contribute to telomere stability. We used Arabidopsis thaliana to study the possible functions of the DNA repair subunit, NBS1, in telomere homeostasis using knockout nbs1 mutants. The results showed that although NBS1-deficient plants were viable, lacked any sign of developmental aberration and produced fertile seeds through many generations upon self-fertilisation, plants also missing the functional telomerase (double mutants), rapidly, within three generations, displayed severe developmental defects. Cytogenetic inspection of cycling somatic cells revealed a very early onset of massive genome instability. Molecular methods used for examining the length of telomeres in double homozygous mutants detected much faster telomere shortening than in plants deficient in telomerase gene alone. Our findings suggest that NBS1 acts in concert with telomerase and plays a profound role in plant telomere renewal.

FUS/TLS acts as an aggregation-dependent modifier of polyglutamine disease model mice.

PubMed

Kino, Yoshihiro; Washizu, Chika; Kurosawa, Masaru; Yamada, Mizuki; Doi, Hiroshi; Takumi, Toru; Adachi, Hiroaki; Katsuno, Masahisa; Sobue, Gen; Hicks, Geoffrey G; Hattori, Nobutaka; Shimogori, Tomomi; Nukina, Nobuyuki

2016-10-14

FUS/TLS is an RNA/DNA-binding protein associated with neurodegenerative diseases including amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Previously, we found that a prion-like domain in the N-terminus of FUS/TLS mediates co-aggregation between FUS/TLS and mutant huntingtin, the gene product of Huntington's disease (HD). Here, we show that heterozygous knockout of FUS/TLS worsened the phenotypes of model mice of (HD, but not spinal and bulbar muscular atrophy (SBMA). This difference was correlated with the degree of pathological association between disease proteins and FUS/TLS. Co-aggregation between FUS/TLS and mutant huntingtin resulted in the depletion of free FUS/TLS protein in HD mice that was detected as a monomer in SDS-PAGE analysis. Recently, we found that FUS/TLS paralogs, TAF15 and EWS, were up-regulated in homozygous FUS/TLS knockout mice. These two proteins were up-regulated in both HD and FUS/TLS heterozygote mice, and were further elevated in HD-TLS +/- double mutant mice, consistent with the functional impairment of FUS/TLS. These results suggest that FUS/TLS sequestration by co-aggregation is a rate-limiting factor of disease phenotypes of HD and that inclusions may have an adverse aspect, rather than being simply benign or protective. In addition, our results highlight inclusions as repositories of potential modifiers of neurodegeneration.

Hypothalamic-pituitary cytokine network.

PubMed

Kariagina, Anastasia; Romanenko, Dmitry; Ren, Song-Guang; Chesnokova, Vera

2004-01-01

Cytokines expressed in the brain and involved in regulating the hypothalamus-pituitary-adrenal (HPA) axis contribute to the neuroendocrine interface. Leukemia inhibitory factor (LIF) and LIF receptors are expressed in human pituitary cells and murine hypothalamus and pituitary. LIF potently induces pituitary proopiomelanocortin (POMC) gene transcription and ACTH secretion and potentiates CRH induction of POMC. In vivo, LIF, along with CRH, enhances POMC expression and ACTH secretion in response to emotional and inflammatory stress. To further elucidate specific roles for both CRH and LIF in activating the inflammatory HPA response, double-knockout mice (CRH/LIFKO) were generated by breeding the null mutants for each respective single gene. Inflammation produced by ip injection of lipopolysaccharide (1 microg/mouse) to double CRH and LIF-deficient mice elicited pituitary POMC induction similar to wild type and markedly higher than in single null animals (P<0.0.01). Double-knockout mice also demonstrated robust corticosterone response to inflammation. High pituitary POMC mRNA levels may reflect abundant TNFalpha, IL-1beta, and IL-6 activation observed in the hypothalamus and pituitary of these animals. Our results suggest that increased central proinflammatory cytokine expression can compensate for the impaired HPA axis function and activates inflammatory ACTH and corticosterone responses in mice-deficient in both CRH and LIF.

Improving the production of acetyl-CoA-derived chemicals in Escherichia coli BL21(DE3) through iclR and arcA deletion.

PubMed

Liu, Min; Ding, Yamei; Chen, Hailin; Zhao, Zhe; Liu, Huizhou; Xian, Mo; Zhao, Guang

2017-01-07

Acetyl-CoA-derived chemicals are suitable for multiple applications in many industries. The bio-production of these chemicals has become imperative owing to the economic and environmental problems. However, acetate overflow is the major drawback for acetyl-CoA-derived chemicals production. Approaches for overcoming acetate overflow may be beneficial for the production of acetyl-CoA-derived chemicals. In this study, a transcriptional regulator iclR was knocked out in E.coli BL21(DE3) to overcome acetate overflow and improve the chemicals production. Two important acetyl-CoA-derived chemicals, phloroglucinol (PG) and 3-hydroxypropionate (3HP) were used to evaluate it. It is revealed that knockout of iclR significantly increased expressions of aceBAK operon. The cell yields and glucose utilization efficiencies were higher than those of control strains. The acetate concentrations were decreased by more than 50% and the productions of PG and 3HP were increased more than twice in iclR mutants. The effects of iclR knockout on cell physiology, cell metabolism and production of acetyl-CoA-derived chemicals were similar to those of arcA knockout in our previous study. However, the arcA-iclR double mutants couldn't gain higher productions of PG and 3HP. The mechanisms are unclear and needed to be resolved in future. Knockout of iclR significantly increased gene expression of aceBAK operon and concomitantly activated glyoxylate pathway. This genetic modification may be a good way to overcome acetate overflow, and improve the production of a wide range of acetyl-CoA-derived chemicals.

Synergistic function of Smad4 and PTEN in suppressing forestomach squamous cell carcinoma in the mouse.

PubMed

Teng, Yan; Sun, An-Na; Pan, Xiao-Chen; Yang, Guan; Yang, Lei-Lei; Wang, Ming-Rong; Yang, Xiao

2006-07-15

The genetic bases underlying esophageal tumorigenesis are poorly understood. Our previous studies have shown that coordinated deletion of the Smad4 and PTEN genes results in accelerated hair loss and skin tumor formation in mice. Herein, we exemplify that the concomitant inactivation of Smad4 and PTEN accelerates spontaneous forestomach carcinogenesis at complete penetrance during the first 2 months of age. All of the forestomach tumors were invasive squamous cell carcinomas (SCCs), which recapitulated the natural history and pathologic features of human esophageal SCCs. A small population of the SCC lesions was accompanied by adenocarcinomas at the adjacent submucosa region in the double mutant mice. The rapid progression of forestomach tumor formation in the Smad4 and PTEN double knockout mice corresponded to a dramatic increase in esophageal and forestomach epithelial proliferation. The decreased expression of p27, p21, and p16 together with the overexpression of cyclin D1 contributed cooperatively to the accelerated forestomach tumorigenesis in the double mutant mice. Our results point strongly to the crucial relevance of synergy between Smad4 and PTEN to suppress forestomach tumorigenesis through the cooperative induction of cell cycle inhibitors.

The chloroplast NADPH thioredoxin reductase C, NTRC, controls non-photochemical quenching of light energy and photosynthetic electron transport in Arabidopsis.

PubMed

Naranjo, Belén; Mignée, Clara; Krieger-Liszkay, Anja; Hornero-Méndez, Dámaso; Gallardo-Guerrero, Lourdes; Cejudo, Francisco Javier; Lindahl, Marika

2016-04-01

High irradiances may lead to photooxidative stress in plants, and non-photochemical quenching (NPQ) contributes to protection against excess excitation. One of the NPQ mechanisms, qE, involves thermal dissipation of the light energy captured. Importantly, plants need to tune down qE under light-limiting conditions for efficient utilization of the available quanta. Considering the possible redox control of responses to excess light implying enzymes, such as thioredoxins, we have studied the role of the NADPH thioredoxin reductase C (NTRC). Whereas Arabidopsis thaliana plants lacking NTRC tolerate high light intensities, these plants display drastically elevated qE, have larger trans-thylakoid ΔpH and have 10-fold higher zeaxanthin levels under low and medium light intensities, leading to extremely low linear electron transport rates. To test the impact of the high qE on plant growth, we generated an ntrc-psbs double-knockout mutant, which is devoid of qE. This double mutant grows faster than the ntrc mutant and has a higher chlorophyll content. The photosystem II activity is partially restored in the ntrc-psbs mutant, and linear electron transport rates under low and medium light intensities are twice as high as compared with plants lacking ntrc alone. These data uncover a new role for NTRC in the control of photosynthetic yield. © 2015 John Wiley & Sons Ltd.

Phosphoglycerate Kinases Are Co-Regulated to Adjust Metabolism and to Optimize Growth.

PubMed

Rosa-Téllez, Sara; Anoman, Armand Djoro; Flores-Tornero, María; Toujani, Walid; Alseek, Saleh; Fernie, Alisdair R; Nebauer, Sergio G; Muñoz-Bertomeu, Jesús; Segura, Juan; Ros, Roc

2018-02-01

In plants, phosphoglycerate kinase (PGK) converts 1,3-bisphosphoglycerate into 3-phosphoglycerate in glycolysis but also participates in the reverse reaction in gluconeogenesis and the Calvin-Benson cycle. In the databases, we found three genes that encode putative PGKs. Arabidopsis ( Arabidopsis thaliana ) PGK1 was localized exclusively in the chloroplasts of photosynthetic tissues, while PGK2 was expressed in the chloroplast/plastid of photosynthetic and nonphotosynthetic cells. PGK3 was expressed ubiquitously in the cytosol of all studied cell types. Measurements of carbohydrate content and photosynthetic activities in PGK mutants and silenced lines corroborated that PGK1 was the photosynthetic isoform, while PGK2 and PGK3 were the plastidial and cytosolic glycolytic isoforms, respectively. The pgk1.1 knockdown mutant displayed reduced growth, lower photosynthetic capacity, and starch content. The pgk3.2 knockout mutant was characterized by reduced growth but higher starch levels than the wild type. The pgk1.1 pgk3.2 double mutant was bigger than pgk3.2 and displayed an intermediate phenotype between the two single mutants in all measured biochemical and physiological parameters. Expression studies in PGK mutants showed that PGK1 and PGK3 were down-regulated in pgk3.2 and pgk1.1 , respectively. These results indicate that the down-regulation of photosynthetic activity could be a plant strategy when glycolysis is impaired to achieve metabolic adjustment and optimize growth. The double mutants of PGK3 and the triose-phosphate transporter ( pgk3.2 tpt3) displayed a drastic growth phenotype, but they were viable. This implies that other enzymes or nonspecific chloroplast transporters could provide 3-phosphoglycerate to the cytosol. Our results highlight both the complexity and the plasticity of the plant primary metabolic network. © 2018 American Society of Plant Biologists. All Rights Reserved.

A miniature mechanical ventilator for newborn mice.

PubMed

Kolandaivelu, K; Poon, C S

1998-02-01

Transgenic/knockout mice with pre-defined mutations have become increasingly popular in biomedical research as models of human diseases. In some instances, the resulting mutation may cause cardiorespiratory distress in the neonatal or adult animals and may necessitate resuscitation. Here we describe the design and testing of a miniature and versatile ventilator that can deliver varying ventilatory support modes, including conventional mechanical ventilation and high-frequency ventilation, to animals as small as the newborn mouse. With a double-piston body chamber design, the device circumvents the problem of air leakage and obviates the need for invasive procedures such as endotracheal intubation, which are particularly important in ventilating small animals. Preliminary tests on newborn mice as early as postnatal day O demonstrated satisfactory restoration of pulmonary ventilation and the prevention of respiratory failure in mutant mice that are prone to respiratory depression. This device may prove useful in the postnatal management of transgenic/knockout mice with genetically inflicted respiratory disorders.

Silencing of AtRAP, a target gene of a bacteria-induced small RNA, triggers antibacterial defense responses through activation of LSU2 and down-regulation of GLK1

PubMed Central

Wang, Huan; Seo, Jang-Kyun; Gao, Shang; Cui, Xinping; Jin, Hailing

2017-01-01

Summary Plants fine-tune their sophisticated immunity systems in response to pathogen infections. We previously showed that AtlsiRNA-1, a bacteria-induced plant endogenous small interfering RNA, silences the AtRAP gene, which encodes a putative RNA binding protein.In this study, we demonstrate that AtRAP functions as a negative regulator in plant immunity by characterizing molecular and biological responses of the knockout mutant and overexpression lines of AtRAP upon bacterial infection.AtRAP is localized in chloroplasts and physically interacts with Low Sulfur Upregulated 2 (LSU2), which positively regulates plant defense. Our results suggest that AtRAP negatively regulates defense responses by suppressing LSU2 through physical interaction. We also detected downregulation of the transcription factor GOLDEN2-LIKE 1 (GLK1) in atrap-1 using microarray analysis. The glk1 glk2 double mutant showed enhanced resistance to Pseudomonas syringae pv. tomato, which is consistent with a previous study showing enhanced resistance of a glk1 glk2 double mutant to Hyaloperonospora arabidopsidis.Taken together, our data suggest that silencing of AtRAP by AtlsiRNA-1 upon bacterial infection triggers defense responses through regulation of LSU2 and GLK1. PMID:28656601

Evolutionary, Molecular and Genetic Analyses of Tic22 Homologues in Arabidopsis thaliana Chloroplasts

PubMed Central

Kasmati, Ali Reza; Patel, Ramesh; Ling, Qihua; Karim, Sazzad; Aronsson, Henrik; Jarvis, Paul

2013-01-01

The Tic22 protein was previously identified in pea as a putative component of the chloroplast protein import apparatus. It is a peripheral protein of the inner envelope membrane, residing in the intermembrane space. In Arabidopsis, there are two Tic22 homologues, termed atTic22-III and atTic22-IV, both of which are predicted to localize in chloroplasts. These two proteins defined clades that are conserved in all land plants, which appear to have evolved at a similar rates since their separation >400 million years ago, suggesting functional conservation. The atTIC22-IV gene was expressed several-fold more highly than atTIC22-III, but the genes exhibited similar expression profiles and were expressed throughout development. Knockout mutants lacking atTic22-IV were visibly normal, whereas those lacking atTic22-III exhibited moderate chlorosis. Double mutants lacking both isoforms were more strongly chlorotic, particularly during early development, but were viable and fertile. Double-mutant chloroplasts were small and under-developed relative to those in wild type, and displayed inefficient import of precursor proteins. The data indicate that the two Tic22 isoforms act redundantly in chloroplast protein import, and that their function is non-essential but nonetheless required for normal chloroplast biogenesis, particularly during early plant development. PMID:23675512

Activating Akt1 mutations alter DNA double strand break repair and radiosensitivity

PubMed Central

Oeck, S.; Al-Refae, K.; Riffkin, H.; Wiel, G.; Handrick, R.; Klein, D.; Iliakis, G.; Jendrossek, V.

2017-01-01

The survival kinase Akt has clinical relevance to radioresistance. However, its contributions to the DNA damage response, DNA double strand break (DSB) repair and apoptosis remain poorly defined and often contradictory. We used a genetic approach to explore the consequences of genetic alterations of Akt1 for the cellular radiation response. While two activation-associated mutants with prominent nuclear access, the phospho-mimicking Akt1-TDSD and the clinically relevant PH-domain mutation Akt1-E17K, accelerated DSB repair and improved survival of irradiated Tramp-C1 murine prostate cancer cells and Akt1-knockout murine embryonic fibroblasts in vitro, the classical constitutively active membrane-targeted myrAkt1 mutant had the opposite effects. Interestingly, DNA-PKcs directly phosphorylated Akt1 at S473 in an in vitro kinase assay but not vice-versa. Pharmacological inhibition of DNA-PKcs or Akt restored radiosensitivity in tumour cells expressing Akt1-E17K or Akt1-TDSD. In conclusion, Akt1-mediated radioresistance depends on its activation state and nuclear localization and is accessible to pharmacologic inhibition. PMID:28209968

Metabolic engineering of Clostridium autoethanogenum for selective alcohol production.

PubMed

Liew, Fungmin; Henstra, Anne M; Kӧpke, Michael; Winzer, Klaus; Simpson, Sean D; Minton, Nigel P

2017-03-01

Gas fermentation using acetogenic bacteria such as Clostridium autoethanogenum offers an attractive route for production of fuel ethanol from industrial waste gases. Acetate reduction to acetaldehyde and further to ethanol via an aldehyde: ferredoxin oxidoreductase (AOR) and alcohol dehydrogenase has been postulated alongside the classic pathway of ethanol formation via a bi-functional aldehyde/alcohol dehydrogenase (AdhE). Here we demonstrate that AOR is critical to ethanol formation in acetogens and inactivation of AdhE led to consistently enhanced autotrophic ethanol production (up to 180%). Using ClosTron and allelic exchange mutagenesis, which was demonstrated for the first time in an acetogen, we generated single mutants as well as double mutants for both aor and adhE isoforms to confirm the role of each gene. The aor1+2 double knockout strain lost the ability to convert exogenous acetate, propionate and butyrate into the corresponding alcohols, further highlighting the role of these enzymes in catalyzing the thermodynamically unfavourable reduction of carboxylic acids into alcohols. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Evidence that tRNA modifying enzymes are important in vivo targets for 5-fluorouracil in yeast

PubMed Central

Gustavsson, Marie; Ronne, Hans

2008-01-01

We have screened a collection of haploid yeast knockout strains for increased sensitivity to 5-fluorouracil (5-FU). A total of 138 5-FU sensitive strains were found. Mutants affecting rRNA and tRNA maturation were particularly sensitive to 5-FU, with the tRNA methylation mutant trm10 being the most sensitive mutant. This is intriguing since trm10, like many other tRNA modification mutants, lacks a phenotype under normal conditions. However, double mutants for nonessential tRNA modification enzymes are frequently temperature sensitive, due to destabilization of hypomodified tRNAs. We therefore tested if the sensitivity of our mutants to 5-FU is affected by the temperature. We found that the cytotoxic effect of 5-FU is strongly enhanced at 38°C for tRNA modification mutants. Furthermore, tRNA modification mutants show similar synthetic interactions for temperature sensitivity and sensitivity to 5-FU. A model is proposed for how 5-FU kills these mutants by reducing the number of tRNA modifications, thus destabilizing tRNA. Finally, we found that also wild-type cells are temperature sensitive at higher concentrations of 5-FU. This suggests that tRNA destabilization contributes to 5-FU cytotoxicity in wild-type cells and provides a possible explanation why hyperthermia can enhance the effect of 5-FU in cancer therapy. PMID:18314501

Hip1-related Mutant Mice Grow and Develop Normally but Have Accelerated Spinal Abnormalities and Dwarfism in the Absence of HIP1†

PubMed Central

Hyun, Teresa S.; Li, Lina; Oravecz-Wilson, Katherine I.; Bradley, Sarah V.; Provot, Melissa M.; Munaco, Anthony J.; Mizukami, Ikuko F.; Sun, Hanshi; Ross, Theodora S.

2004-01-01

In mice and humans, there are two known members of the Huntingtin interacting protein 1 (HIP1) family, HIP1 and HIP1-related (HIP1r). Based on structural and functional data, these proteins participate in the clathrin trafficking network. The inactivation of Hip1 in mice leads to spinal, hematopoietic, and testicular defects. To investigate the biological function of HIP1r, we generated a Hip1r mutant allele in mice. Hip1r homozygous mutant mice are viable and fertile without obvious morphological abnormalities. In addition, embryonic fibroblasts derived from these mice do not have gross abnormalities in survival, proliferation, or clathrin trafficking pathways. Altogether, this demonstrates that HIP1r is not necessary for normal development of the embryo or for normal adulthood and suggests that HIP1 or other functionally related members of the clathrin trafficking network can compensate for HIP1r absence. To test the latter, we generated mice deficient in both HIP1 and HIP1r. These mice have accelerated development of abnormalities seen in Hip1 -deficient mice, including kypholordosis and growth defects. The severity of the Hip1r/Hip1 double-knockout phenotype compared to the Hip1 knockout indicates that HIP1r partially compensates for HIP1 function in the absence of HIP1 expression, providing strong evidence that HIP1 and HIP1r have overlapping roles in vivo. PMID:15121852

Hip1-related mutant mice grow and develop normally but have accelerated spinal abnormalities and dwarfism in the absence of HIP1.

PubMed

Hyun, Teresa S; Li, Lina; Oravecz-Wilson, Katherine I; Bradley, Sarah V; Provot, Melissa M; Munaco, Anthony J; Mizukami, Ikuko F; Sun, Hanshi; Ross, Theodora S

2004-05-01

In mice and humans, there are two known members of the Huntingtin interacting protein 1 (HIP1) family, HIP1 and HIP1-related (HIP1r). Based on structural and functional data, these proteins participate in the clathrin trafficking network. The inactivation of Hip1 in mice leads to spinal, hematopoietic, and testicular defects. To investigate the biological function of HIP1r, we generated a Hip1r mutant allele in mice. Hip1r homozygous mutant mice are viable and fertile without obvious morphological abnormalities. In addition, embryonic fibroblasts derived from these mice do not have gross abnormalities in survival, proliferation, or clathrin trafficking pathways. Altogether, this demonstrates that HIP1r is not necessary for normal development of the embryo or for normal adulthood and suggests that HIP1 or other functionally related members of the clathrin trafficking network can compensate for HIP1r absence. To test the latter, we generated mice deficient in both HIP1 and HIP1r. These mice have accelerated development of abnormalities seen in Hip1 -deficient mice, including kypholordosis and growth defects. The severity of the Hip1r/Hip1 double-knockout phenotype compared to the Hip1 knockout indicates that HIP1r partially compensates for HIP1 function in the absence of HIP1 expression, providing strong evidence that HIP1 and HIP1r have overlapping roles in vivo.

Genetic analysis of the roles of agaA, agaI, and agaS genes in the N-acetyl-D-galactosamine and D-galactosamine catabolic pathways in Escherichia coli strains O157:H7 and C

PubMed Central

2013-01-01

Background The catabolic pathways of N-acetyl-D-galactosamine (Aga) and D-galactosamine (Gam) in E. coli were proposed from bioinformatic analysis of the aga/gam regulon in E. coli K-12 and later from studies using E. coli C. Of the thirteen genes in this cluster, the roles of agaA, agaI, and agaS predicted to code for Aga-6-P-deacetylase, Gam-6-P deaminase/isomerase, and ketose-aldolase isomerase, respectively, have not been experimentally tested. Here we study their roles in Aga and Gam utilization in E. coli O157:H7 and in E. coli C. Results Knockout mutants in agaA, agaI, and agaS were constructed to test their roles in Aga and Gam utilization. Knockout mutants in the N-acetylglucosamine (GlcNAc) pathway genes nagA and nagB coding for GlcNAc-6-P deacetylase and glucosamine-6-P deaminase/isomerase, respectively, and double knockout mutants ΔagaA ΔnagA and ∆agaI ∆nagB were also constructed to investigate if there is any interplay of these enzymes between the Aga/Gam and the GlcNAc pathways. It is shown that Aga utilization was unaffected in ΔagaA mutants but ΔagaA ΔnagA mutants were blocked in Aga and GlcNAc utilization. E. coli C ΔnagA could not grow on GlcNAc but could grow when the aga/gam regulon was constitutively expressed. Complementation of ΔagaA ΔnagA mutants with either agaA or nagA resulted in growth on both Aga and GlcNAc. It was also found that ΔagaI, ΔnagB, and ∆agaI ΔnagB mutants were unaffected in utilization of Aga and Gam. Importantly, ΔagaS mutants were blocked in Aga and Gam utilization. Expression analysis of relevant genes in these strains with different genetic backgrounds by real time RT-PCR supported these observations. Conclusions Aga utilization was not affected in ΔagaA mutants because nagA was expressed and substituted for agaA. Complementation of ΔagaA ΔnagA mutants with either agaA or nagA also showed that both agaA and nagA can substitute for each other. The ∆agaI, ∆nagB, and ∆agaI ∆nagB mutants were not affected in Aga and Gam utilization indicating that neither agaI nor nagB is involved in the deamination and isomerization of Gam-6-P. We propose that agaS codes for Gam-6-P deaminase/isomerase in the Aga/Gam pathway. PMID:23634833

Functional Specialization amongst the Arabidopsis Toc159 Family of Chloroplast Protein Import ReceptorsW⃞

PubMed Central

Kubis, Sybille; Patel, Ramesh; Combe, Jonathan; Bédard, Jocelyn; Kovacheva, Sabina; Lilley, Kathryn; Biehl, Alexander; Leister, Dario; Ríos, Gabino; Koncz, Csaba; Jarvis, Paul

2004-01-01

The initial stages of preprotein import into chloroplasts are mediated by the receptor GTPase Toc159. In Arabidopsis thaliana, Toc159 is encoded by a small gene family: atTOC159, atTOC132, atTOC120, and atTOC90. Phylogenetic analysis suggested that at least two distinct Toc159 subtypes, characterized by atToc159 and atToc132/atToc120, exist in plants. atTOC159 was strongly expressed in young, photosynthetic tissues, whereas atTOC132 and atTOC120 were expressed at a uniformly low level and so were relatively prominent in nonphotosynthetic tissues. Based on the albino phenotype of its knockout mutant, atToc159 was previously proposed to be a receptor with specificity for photosynthetic preproteins. To elucidate the roles of the other isoforms, we characterized Arabidopsis knockout mutants for each one. None of the single mutants had strong visible phenotypes, but toc132 toc120 double homozygotes appeared similar to toc159, indicating redundancy between atToc132 and atToc120. Transgenic complementation studies confirmed this redundancy but revealed little functional overlap between atToc132/atToc120 and atToc159 or atToc90. Unlike toc159, toc132 toc120 caused structural abnormalities in root plastids. Furthermore, when proteomics and transcriptomics were used to compare toc132 with ppi1 (a receptor mutant that is specifically defective in the expression, import, and accumulation of photosynthetic proteins), major differences were observed, suggesting that atToc132 (and atToc120) has specificity for nonphotosynthetic proteins. When both atToc159 and the major isoform of the other subtype, atToc132, were absent, an embryo-lethal phenotype resulted, demonstrating the essential role of Toc159 in the import mechanism. PMID:15273297

THE EFFECT OF TARGETED KNOCKOUT MUTATION ON THE TRANSCRIPTIONAL PROFILE OF THE KIDNEY IN TSC2 MUTANT LONG-EVANS (EKER) RATS.

EPA Science Inventory

The effect of a targeted knockout mutation on the transcriptional profile of the kidney in
Tsc2 mutant Long-Evans (Eker) rats.

Renal cell carcinoma (RCC) is the most common tumor of the adult kidney, accounting
for up to 80% of malignant renal neoplasms. Hereditary...

Studies on Arabidopsis athak5, atakt1 double mutants disclose the range of concentrations at which AtHAK5, AtAKT1 and unknown systems mediate K uptake.

PubMed

Rubio, Francisco; Alemán, Fernando; Nieves-Cordones, Manuel; Martínez, Vicente

2010-06-01

The high-affinity K(+) transporter AtHAK5 and the inward-rectifier K(+) channel AtAKT1 have been described to contribute to K(+) uptake in Arabidopsis thaliana. Studies with T-DNA insertion lines showed that both systems participate in the high-affinity range of concentrations and only AtAKT1 in the low-affinity range. However the contribution of other systems could not be excluded with the information and plant material available. The results presented here with a double knock-out athak5, atakt1 mutant show that AtHAK5 is the only system mediating K(+) uptake at concentrations below 0.01 mM. In the range between 0.01 and 0.05 mM K(+) AtHAK5 and AtAKT1 are the only contributors to K(+) acquisition. At higher K(+) concentrations, unknown systems come into operation and participate together with AtAKT1 in low-affinity K(+) uptake. These systems can supply sufficient K(+) to promote plant growth even in the absence of AtAKT1 or in the presence of 10 mM K(+) where AtAKT1 is not essential.

Morphological observation of the stria vascularis in midkine and pleiotrophin knockout mice.

PubMed

Sone, Michihiko; Muramatsu, Hisako; Muramatsu, Takashi; Nakashima, Tsutomu

2011-02-01

Midkine and Pleiotrophin are low molecular weight basic proteins with closely related structures and serve as growth/differentiation factors. They have been reported to be expressed in the cochlea during the embryonic and perinatal periods. In the present study, we focused on the roles of midkine and pleiotrophin in the stria vascularis and investigated morphological changes using mice deficient in these genes. Midkine knockout, pleiotrophin knockout, and double knockout mice were used and compared to wild-type mice. Auditory brain stem responses (ABRs) and cochlear blood flows were measured in each type of mice. Pathological changes in the stria vascularis were examined by light microscopy, including immunohistochemical staining with anti-Kir4.1 antibody, and electron microscopy. Hearing thresholds examined by ABRs were significantly higher in midkine knockout and pleiotrophin knockout mice than in wild-type mice. Double knockout mice showed higher thresholds compared to midkine knockout and pleiotrophin knockout mice. Blood flow in the lateral walls did not significantly differ and light microscopy examination showed an almost normal appearance of the stria vascularis in these knockout mice. However, the expression of Kir4.1 was weak in the knockout mice and severe vacuolar degeneration was observed by electron microscopy in the intermediate cells of the double knockout mice. The present study demonstrates that midkine and pleiotrophin play some roles for the morphological maintenance of intermediate cell in the stria vascularis. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

Impaired eye-blink conditioning in waggler, a mutant mouse with cerebellar BDNF deficiency.

PubMed

Bao, S; Chen, L; Qiao, X; Knusel, B; Thompson, R F

1998-01-01

In addition to their trophic functions, neurotrophins are also implicated in synaptic modulation and learning and memory. Although gene knockout techniques have been used widely in studying the roles of neurotrophins at molecular and cellular levels, behavioral studies using neurotrophin knockouts are limited by the early-onset lethality and various sensory deficits associated with the gene knockout mice. In the present study, we found that in a spontaneous mutant mouse, waggler, the expression of brain-derived neurotrophic factor (BDNF) was selectively absent in the cerebellar granule cells. The cytoarchitecture of the waggler cerebellum appeared to be normal at the light microscope level. The mutant mice exhibited no sensory deficits to auditory stimuli or heat-induced pain. However, they were massively impaired in classic eye-blink conditioning. These results suggest that BDNF may have a role in normal cerebellar neuronal function, which, in turn, is essential for classic eye-blink conditioning.

Precision-engineering the Pseudomonas aeruginosa genome with two-step allelic exchange

PubMed Central

Hmelo, Laura R.; Borlee, Bradley R.; Almblad, Henrik; Love, Michelle E.; Randall, Trevor E.; Tseng, Boo Shan; Lin, Chuyang; Irie, Yasuhiko; Storek, Kelly M.; Yang, Jaeun Jane; Siehnel, Richard J.; Howell, P. Lynne; Singh, Pradeep K.; Tolker-Nielsen, Tim; Parsek, Matthew R.; Schweizer, Herbert P.; Harrison, Joe J.

2016-01-01

Allelic exchange is an efficient method of bacterial genome engineering. This protocol describes the use of this technique to make gene knockouts and knockins, as well as single nucleotide insertions, deletions and substitutions in Pseudomonas aeruginosa. Unlike other approaches to allelic exchange, this protocol does not require heterologous recombinases to insert or excise selective markers from the target chromosome. Rather, positive and negative selection are enabled solely by suicide vector-encoded functions and host cell proteins. Here, mutant alleles, which are flanked by regions of homology to the recipient chromosome, are synthesized in vitro and then cloned into allelic exchange vectors using standard procedures. These suicide vectors are then introduced into recipient cells by conjugation. Homologous recombination then results in antibiotic resistant single-crossover mutants in which the plasmid has integrated site-specifically into the chromosome. Subsequently, unmarked double-crossover mutants are isolated directly using sucrose-mediated counter-selection. This two-step process yields seamless mutations that are precise to a single base pair of DNA. The entire procedure requires ~2 weeks. PMID:26492139

Characterization of Arabidopsis sterol glycosyltransferase TTG15/UGT80B1 role during freeze and heat stress

PubMed Central

Mishra, Manoj K; Singh, Gaurav; Tiwari, Shalini; Singh, Ruchi; Kumari, Nishi; Misra, Pratibha

2015-01-01

Sterol glycosyltransferases regulate the properties of sterols by catalyzing the transfer of carbohydrate molecules to the sterol moiety for the synthesis of steryl glycosides and acyl steryl glycosides. We have analyzed the functional role of TTG15/UGT80B1 gene of Arabidopsis thaliana in freeze/thaw and heat shock stress using T-DNA insertional sgt knockout mutants. Quantitative study of spatial as well as temporal gene expression showed tissue-specific and dynamic expression patterns throughout the growth stages. Comparative responses of Col-0, TTG15/UGT80B1 knockout mutant and p35S:TTG15/UGT80B1 restored lines were analyzed under heat and freeze stress conditions. Heat tolerance was determined by survival of plants at 42°C for 3 h, MDA analysis and chlorophyll fluorescence image (CFI) analysis. Freezing tolerance was determined by survival of the plants at -1°C temperature in non-acclimatized (NA) and cold acclimatized (CA) conditions and also by CFI analysis, which revealed that, p35S:TTG15/UGT80B1 restored plants were more adapted to freeze stress than TTG15/UGT80B1 knockout mutant under CA condition. HPLC analysis of the plants showed reduced sterol glycoside in mutant seedlings as compared to other genotypes. Following CA condition, both β-sitosterol and sitosterol glycoside quantity was more in Col-0 and p35S:TTG15/UGT80B1 restored lines, whereas it was significantly less in TTG15/UGT80B1 knockout mutants. From these results, it may be concluded that due to low content of free sterols and sterol glycosides, the physiology of mutant plants was more affected during both, the chilling and heat stress. PMID:26382564

Nup82 functions redundantly with Nup136 in a salicylic acid-dependent defense response of Arabidopsis thaliana.

PubMed

Tamura, Kentaro; Fukao, Yoichiro; Hatsugai, Noriyuki; Katagiri, Fumiaki; Hara-Nishimura, Ikuko

2017-05-04

The nuclear pore complex (NPC) comprises more than 30 nucleoporins (Nups). NPC mediates macromolecular trafficking between the nucleoplasm and the cytoplasm, but specific roles of individual Nups are poorly understood in higher plants. Here, we show that the novel nucleoporin unique to angiosperm plants (designated as Nup82) functions in a salicylic acid-dependent defense in a redundant manner with Nup136, which is a component of the nuclear basket in the NPC. Arabidopsis thaliana Nup82 had a similar amino acid sequence to the N-terminal half of Nup136 and a Nup82-GFP fusion was localized on the nuclear envelope. Immunoprecipitation and bimolecular fluorescence complementation analyses revealed that Nup82 interacts with the NPC components Nup136 and RAE1. The double knockout mutant nup82 nup136 showed severe growth defects, while the single knockout mutant nup82 did not, suggesting that Nup82 functions redundantly with Nup136. nup82 nup136 impaired benzothiadiazole (an analog of salicylic acid)-induced resistance to the virulent bacteria Pseudomonas syringae pv. tomato DC3000. Furthermore, transcriptome analysis of nup82 nup136 indicates that deficiency of Nup82 and Nup136 causes noticeable downregulation of immune-related genes. These results suggest that Nup82 and Nup136 are redundantly involved in transcriptional regulation of salicylic acid-responsive genes through nuclear transport of signaling molecules.

Generating double knockout mice to model genetic intervention for diabetic cardiomyopathy in humans.

PubMed

Chavali, Vishalakshi; Nandi, Shyam Sundar; Singh, Shree Ram; Mishra, Paras Kumar

2014-01-01

Diabetes is a rapidly increasing disease that enhances the chances of heart failure twofold to fourfold (as compared to age and sex matched nondiabetics) and becomes a leading cause of morbidity and mortality. There are two broad classifications of diabetes: type1 diabetes (T1D) and type2 diabetes (T2D). Several mice models mimic both T1D and T2D in humans. However, the genetic intervention to ameliorate diabetic cardiomyopathy in these mice often requires creating double knockout (DKO). In order to assess the therapeutic potential of a gene, that specific gene is either overexpressed (transgenic expression) or abrogated (knockout) in the diabetic mice. If the genetic mice model for diabetes is used, it is necessary to create DKO with transgenic/knockout of the target gene to investigate the specific role of that gene in pathological cardiac remodeling in diabetics. One of the important genes involved in extracellular matrix (ECM) remodeling in diabetes is matrix metalloproteinase-9 (Mmp9). Mmp9 is a collagenase that remains latent in healthy hearts but induced in diabetic hearts. Activated Mmp9 degrades extracellular matrix (ECM) and increases matrix turnover causing cardiac fibrosis that leads to heart failure. Insulin2 mutant (Ins2+/-) Akita is a genetic model for T1D that becomes diabetic spontaneously at the age of 3-4 weeks and show robust hyperglycemia at the age of 10-12 weeks. It is a chronic model of T1D. In Ins2+/- Akita, Mmp9 is induced. To investigate the specific role of Mmp9 in diabetic hearts, it is necessary to create diabetic mice where Mmp9 gene is deleted. Here, we describe the method to generate Ins2+/-/Mmp9-/- (DKO) mice to determine whether the abrogation of Mmp9 ameliorates diabetic cardiomyopathy.

Embryonic Lethality Due to Arrested Cardiac Development in Psip1/Hdgfrp2 Double-Deficient Mice.

PubMed

Wang, Hao; Shun, Ming-Chieh; Dickson, Amy K; Engelman, Alan N

2015-01-01

Hepatoma-derived growth factor (HDGF) related protein 2 (HRP2) and lens epithelium-derived growth factor (LEDGF)/p75 are closely related members of the HRP2 protein family. LEDGF/p75 has been implicated in numerous human pathologies including cancer, autoimmunity, and infectious disease. Knockout of the Psip1 gene, which encodes for LEDGF/p75 and the shorter LEDGF/p52 isoform, was previously shown to cause perinatal lethality in mice. The function of HRP2 was by contrast largely unknown. To learn about the role of HRP2 in development, we knocked out the Hdgfrp2 gene, which encodes for HRP2, in both normal and Psip1 knockout mice. Hdgfrp2 knockout mice developed normally and were fertile. By contrast, the double deficient mice died at approximate embryonic day (E) 13.5. Histological examination revealed ventricular septal defect (VSD) associated with E14.5 double knockout embryos. To investigate the underlying molecular mechanism(s), RNA recovered from ventricular tissue was subjected to RNA-sequencing on the Illumina platform. Bioinformatic analysis revealed several genes and biological pathways that were significantly deregulated by the Psip1 knockout and/or Psip1/Hdgfrp2 double knockout. Among the dozen genes known to encode for LEDGF/p75 binding factors, only the expression of Nova1, which encodes an RNA splicing factor, was significantly deregulated by the knockouts. However the expression of other RNA splicing factors, including the LEDGF/p52-interacting protein ASF/SF2, was not significantly altered, indicating that deregulation of global RNA splicing was not a driving factor in the pathology of the VSD. Tumor growth factor (Tgf) β-signaling, which plays a key role in cardiac morphogenesis during development, was the only pathway significantly deregulated by the double knockout as compared to control and Psip1 knockout samples. We accordingly speculate that deregulated Tgf-β signaling was a contributing factor to the VSD and prenatal lethality of Psip1/Hdgfrp2 double-deficient mice.

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.

Arabidopsis serotonin N-acetyltransferase knockout mutant plants exhibit decreased melatonin and salicylic acid levels resulting in susceptibility to an avirulent pathogen.

PubMed

Lee, Hyoung Yool; Byeon, Yeong; Tan, Dun-Xian; Reiter, Russel J; Back, Kyoungwhan

2015-04-01

Serotonin N-acetyltransferase (SNAT) is the penultimate enzyme in the melatonin biosynthesis pathway in plants. We examined the effects of SNAT gene inactivation in two Arabidopsis T-DNA insertion mutant lines. After inoculation with the avirulent pathogen Pseudomonas syringe pv. tomato DC3000 harboring the elicitor avrRpt2 (Pst-avrRpt2), melatonin levels in the snat knockout mutant lines were 50% less than in wild-type Arabidopsis Col-0 plants. The snat knockout mutant lines exhibited susceptibility to pathogen infection that coincided with decreased induction of defense genes including PR1, ICS1, and PDF1.2. Because melatonin acts upstream of salicylic acid (SA) synthesis, the reduced melatonin levels in the snat mutant lines led to decreased SA levels compared to wild-type, suggesting that the increased pathogen susceptibility of the snat mutant lines could be attributed to decreased SA levels and subsequent attenuation of defense gene induction. Exogenous melatonin treatment failed to induce defense gene expression in nahG Arabidopsis plants, but restored the induction of defense gene expression in the snat mutant lines. In addition, melatonin caused translocation of NPR1 (nonexpressor of PR1) protein from the cytoplasm into the nucleus indicating that melatonin-elicited pathogen resistance in response to avirulent pathogen attack is SA-dependent in Arabidopsis. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Arabidopsis scaffold protein RACK1A modulates rare sugar D-allose regulated gibberellin signaling.

PubMed

Fennell, Herman; Olawin, Abdulquadri; Mizanur, Rahman M; Izumori, Ken; Chen, Jin-Gui; Ullah, Hemayet

2012-11-01

As energy sources and structural components, sugars are the central regulators of plant growth and development. In addition to the abundant natural sugars in plants, more than 50 different kinds of rare sugars exist in nature, several of which show distinct roles in plant growth and development. Recently, one of the rare sugars, D-allose, an epimer of D-glucose at C3, is found to suppress plant hormone gibberellin (GA) signaling in rice. Scaffold protein RACK1A in the model plant Arabidopsis is implicated in the GA pathway as rack1a knockout mutants show insensitivity to GA in GA-induced seed germination. Using genetic knockout lines and a reporter gene, the functional role of RACK1A in the D-allose pathway was investigated. It was found that the rack1a knockout seeds showed hypersensitivity to D-allose-induced inhibition of seed germination, implicating a role for RACK1A in the D-allose mediated suppression of seed germination. On the other hand, a functional RACK1A in the background of the double knockout mutations in the other two RACK1 isoforms, rack1b/rack1c, showed significant resistance to the D-allose induced inhibition of seed germination. The collective results implicate the RACK1A in the D-allose mediated seed germination inhibition pathway. Elucidation of the rare sugar signaling mechanism will help to advance understanding of this less studied but important cellular signaling pathway.

Arabidopsis scaffold protein RACK1A modulates rare sugar D-allose regulated gibberellin signaling

PubMed Central

Fennell, Herman; Olawin, Abdulquadri; Mizanur, Rahman M.; Izumori, Ken; Chen, Jin-Gui; Ullah, Hemayet

2012-01-01

As energy sources and structural components, sugars are the central regulators of plant growth and development. In addition to the abundant natural sugars in plants, more than 50 different kinds of rare sugars exist in nature, several of which show distinct roles in plant growth and development. Recently, one of the rare sugars, D-allose, an epimer of D-glucose at C3, is found to suppress plant hormone gibberellin (GA) signaling in rice. Scaffold protein RACK1A in the model plant Arabidopsis is implicated in the GA pathway as rack1a knockout mutants show insensitivity to GA in GA-induced seed germination. Using genetic knockout lines and a reporter gene, the functional role of RACK1A in the D-allose pathway was investigated. It was found that the rack1a knockout seeds showed hypersensitivity to D-allose-induced inhibition of seed germination, implicating a role for RACK1A in the D-allose mediated suppression of seed germination. On the other hand, a functional RACK1A in the background of the double knockout mutations in the other two RACK1 isoforms, rack1b/rack1c, showed significant resistance to the D-allose induced inhibition of seed germination. The collective results implicate the RACK1A in the D-allose mediated seed germination inhibition pathway. Elucidation of the rare sugar signaling mechanism will help to advance understanding of this less studied but important cellular signaling pathway. PMID:22951405

Neurodegeneration in a Drosophila model of adrenoleukodystrophy: the roles of the Bubblegum and Double bubble acyl-CoA synthetases

PubMed Central

Sivachenko, Anna; Gordon, Hannah B.; Kimball, Suzanne S.; Gavin, Erin J.; Bonkowsky, Joshua L.; Letsou, Anthea

2016-01-01

ABSTRACT Debilitating neurodegenerative conditions with metabolic origins affect millions of individuals worldwide. Still, for most of these neurometabolic disorders there are neither cures nor disease-modifying therapies, and novel animal models are needed for elucidation of disease pathology and identification of potential therapeutic agents. To date, metabolic neurodegenerative disease has been modeled in animals with only limited success, in part because existing models constitute analyses of single mutants and have thus overlooked potential redundancy within metabolic gene pathways associated with disease. Here, we present the first analysis of a very-long-chain acyl-CoA synthetase (ACS) double mutant. We show that the Drosophila bubblegum (bgm) and double bubble (dbb) genes have overlapping functions, and that the consequences of double knockout of both bubblegum and double bubble in the fly brain are profound, affecting behavior and brain morphology, and providing the best paradigm to date for an animal model of adrenoleukodystrophy (ALD), a fatal childhood neurodegenerative disease associated with the accumulation of very-long-chain fatty acids. Using this more fully penetrant model of disease to interrogate brain morphology at the level of electron microscopy, we show that dysregulation of fatty acid metabolism via disruption of ACS function in vivo is causal of neurodegenerative pathologies that are evident in both neuronal cells and their supporting cell populations, and leads ultimately to lytic cell death in affected areas of the brain. Finally, in an extension of our model system to the study of human disease, we describe our identification of an individual with leukodystrophy who harbors a rare mutation in SLC27a6 (encoding a very-long-chain ACS), a human homolog of bgm and dbb. PMID:26893370

Switching of dominant retrotransposon silencing strategies from posttranscriptional to transcriptional mechanisms during male germ-cell development in mice

PubMed Central

Inoue, Kota; Fukuda, Kei; Sasaki, Hiroyuki

2017-01-01

Mammalian genomes harbor millions of retrotransposon copies, some of which are transpositionally active. In mouse prospermatogonia, PIWI-interacting small RNAs (piRNAs) combat retrotransposon activity to maintain the genomic integrity. The piRNA system destroys retrotransposon-derived RNAs and guides de novo DNA methylation at some retrotransposon promoters. However, it remains unclear whether DNA methylation contributes to retrotransposon silencing in prospermatogonia. We have performed comprehensive studies of DNA methylation and polyA(+) RNAs (transcriptome) in developing male germ cells from Pld6/Mitopld and Dnmt3l knockout mice, which are defective in piRNA biogenesis and de novo DNA methylation, respectively. The Dnmt3l mutation greatly reduced DNA methylation levels at most retrotransposons, but its impact on their RNA abundance was limited in prospermatogonia. In Pld6 mutant germ cells, although only a few retrotransposons exhibited reduced DNA methylation, many showed increased expression at the RNA level. More detailed analysis of RNA sequencing, nascent RNA quantification, profiling of cleaved RNA ends, and the results obtained from double knockout mice suggest that PLD6 works mainly at the posttranscriptional level. The increase in retrotransposon expression was larger in Pld6 mutants than it was in Dnmt3l mutants, suggesting that RNA degradation by the piRNA system plays a more important role than does DNA methylation in prospermatogonia. However, DNA methylation had a long-term effect: hypomethylation caused by the Pld6 or Dnmt3l mutation resulted in increased retrotransposon expression in meiotic spermatocytes. Thus, posttranscriptional silencing plays an important role in the early stage of germ cell development, then transcriptional silencing becomes important in later stages. In addition, intergenic and intronic retrotransposon sequences, in particular those containing the antisense L1 promoters, drove ectopic expression of nearby genes in both mutant spermatocytes, suggesting that retrotransposon silencing is important for the maintenance of not only genomic integrity but also transcriptomic integrity. PMID:28749988

The Rice Receptor-Like Kinases DWARF AND RUNTISH SPIKELET1 and 2 Repress Cell Death and Affect Sugar Utilization during Reproductive Development

PubMed Central

Song, Feng-Yan; Zhao, Ying; Wang, Chun-Yan; Zhang, Yong-Cun; Yang, Qian; Wang, Jiao; Bu, Shuo-Lei; Sun, Li-Jing; Zhang, Sheng-Wei; Zhang, Su-Qiao; Sun, Da-Ye

2017-01-01

Cell-to-cell communication precisely controls the creation of new organs during reproductive growth. However, the sensor molecules that mediate developmental signals in monocot plants are poorly understood. Here, we report that DWARF AND RUNTISH SPIKELET1 (DRUS1) and DRUS2, two closely related receptor-like kinases (RLKs), redundantly control reproductive growth and development in rice (Oryza sativa). A drus1-1 drus2 double knockout mutant, but not either single mutant, showed extreme dwarfism and barren inflorescences that harbored sterile spikelets. The gibberellin pathway was not impaired in this mutant. A phenotypic comparison of mutants expressing different amounts of DRUS1 and 2 revealed that reproductive growth requires a threshold level of DRUS1/2 proteins. DRUS1 and 2 maintain cell viability by repressing protease-mediated cell degradation and likely by affecting sugar utilization or conversion. In the later stages of anther development, survival of the endothecium requires DRUS1/2, which may stimulate expression of the UDP-glucose pyrophosphorylase gene UGP2 and starch biosynthesis in pollen. Unlike their Arabidopsis thaliana ortholog FERONIA, DRUS1 and 2 mediate a fundamental signaling process that is essential for cell survival and represents a novel biological function for the CrRLK1L RLK subfamily. PMID:28082384

Class XI Myosins Move Specific Organelles in Pollen Tubes and Are Required for Normal Fertility and Pollen Tube Growth in Arabidopsis1[OPEN

PubMed Central

Madison, Stephanie L.; Buchanan, Matthew L.; Glass, Jeremiah D.; McClain, Tarah F.; Park, Eunsook; Nebenführ, Andreas

2015-01-01

Pollen tube growth is an essential aspect of plant reproduction because it is the mechanism through which nonmotile sperm cells are delivered to ovules, thus allowing fertilization to occur. A pollen tube is a single cell that only grows at the tip, and this tip growth has been shown to depend on actin filaments. It is generally assumed that myosin-driven movements along these actin filaments are required to sustain the high growth rates of pollen tubes. We tested this conjecture by examining seed set, pollen fitness, and pollen tube growth for knockout mutants of five of the six myosin XI genes expressed in pollen of Arabidopsis (Arabidopsis thaliana). Single mutants had little or no reduction in overall fertility, whereas double mutants of highly similar pollen myosins had greater defects in pollen tube growth. In particular, myo11c1 myo11c2 pollen tubes grew more slowly than wild-type pollen tubes, which resulted in reduced fitness compared with the wild type and a drastic reduction in seed set. Golgi stack and peroxisome movements were also significantly reduced, and actin filaments were less organized in myo11c1 myo11c2 pollen tubes. Interestingly, the movement of yellow fluorescent protein-RabA4d-labeled vesicles and their accumulation at pollen tube tips were not affected in the myo11c1 myo11c2 double mutant, demonstrating functional specialization among myosin isoforms. We conclude that class XI myosins are required for organelle motility, actin organization, and optimal growth of pollen tubes. PMID:26358416

Cytosolic Glutamine Synthetase Gln1;2 Is the Main Isozyme Contributing to GS1 Activity and Can Be Up-Regulated to Relieve Ammonium Toxicity1[OPEN

PubMed Central

Pedersen, Carsten

2016-01-01

Cytosolic GS1 (Gln synthetase) is central for ammonium assimilation in plants. High ammonium treatment enhanced the expression of the GS1 isogene Gln-1;2 encoding a low-affinity high-capacity GS1 protein in Arabidopsis (Arabidopsis thaliana) shoots. Under the same conditions, the expression of the high-affinity low-capacity isoform Gln-1;1 was reduced. The expression of Gln-1;3 did not respond to ammonium treatment while Gln-1;4 and Gln-1;5 isogenes in all cases were expressed at a very low level. Gln-2 was highly expressed in shoots but only at a very low level in roots. To investigate the specific functions of the two isogenes Gln-1;1 and Gln-1;2 in shoots for ammonium detoxification, single and double knock-out mutants were grown under standard N supply or with high ammonium provision. Phenotypes of the single mutant gln1;1 were similar to the wild type, while growth of the gln1;2 single mutant and the gln1;1:gln1;2 double mutant was significantly impaired irrespective of N regime. GS1 activity was significantly reduced in both gln1;2 and gln1;1:gln1;2. Along with this, the ammonium content increased while that of Gln decreased, showing that Gln-1;2 was essential for ammonium assimilation and amino acid synthesis. We conclude that Gln-1;2 is the main isozyme contributing to shoot GS1 activity in vegetative growth stages and can be up-regulated to relieve ammonium toxicity. This reveals, to our knowledge, a novel shoot function of Gln-1;2 in Arabidopsis shoots. PMID:27231101

Morphological and genetic characterization of group I Clostridium botulinum type B strain 111 and the transcriptional regulator spoIIID gene knockout mutant in sporulation.

PubMed

Hosomi, Koji; Kuwana, Ritsuko; Takamatsu, Hiromu; Kohda, Tomoko; Kozaki, Shunji; Mukamoto, Masafumi

2015-06-01

Clostridium botulinum is a heat-resistant spore-forming bacterium that causes the serious paralytic illness botulism. Heat-resistant spores may cause food sanitation hazards and sporulation plays a central role in the survival of C. botulinum. We observed morphological changes and investigated the role of the transcriptional regulator SpoIIID in the sporulation of C. botulinum type B strain 111 in order to elucidate the molecular mechanism in C. botulinum. C. botulinum type B formed heat-resistant spores through successive morphological changes corresponding to those of Bacillus subtilis, a spore-forming model organism. An analysis of the spoIIID gene knockout mutant revealed that the transcriptional regulator SpoIIID contributed to heat-resistant spore formation by C. botulinum type B and activated the transcription of the sigK gene later during sporulation. Transcription of the spoIIID gene, which differed from that in B. subtilis and Clostridium difficile, was observed in the sigE gene knockout mutant of C. botulinum type B. An analysis of the sigF gene knockout mutant showed that the sporulation-specific sigma factor SigF was essential for transcription of the spoIIID gene in C. botulinum type B. These results suggest that the regulation of sporulation in C. botulinum is not similar to that in B. subtilis and other clostridia. Copyright © 2015 Elsevier Ltd. All rights reserved.

A Novel Occulta-Type Spina Bifida Mediated by Murine Double Heterozygotes EphA2 and EphA4 Receptor Tyrosine Kinases.

PubMed

Abdullah, Nor Linda; Mohd-Zin, Siti W; Ahmad-Annuar, Azlina; Abdul-Aziz, Noraishah M

2017-01-01

Members of the Eph receptor tyrosine kinase have previously been implicated in cranial neural tube development. Failure of neural tube closure leads to the devastating conditions known as anencephaly and spina bifida. EphA2 and EphA4 are expressed at the tips of the closing spinal neural folds prior and during neural tube closure. We investigated the possible role of murine EphA2 and EphA4 during the last step of primary neural tube closure, which is adhesion and fusion. The individual mouse knockouts of EphA2 and EphA4 per se do not exhibit neural tube defects (NTDs). The embryos generated by the crossing of double heterozygotes Epha2 tm1Jrui/+ Epha4 rb-2J/+ displayed NTDs with a wide degree of severity including close exencephaly and close spina bifida (spina bifida occulta). Interestingly, mutants displaying NTDs had skin covering the underlying lesion. The tissue sections revealed the elevated neural folds had not adhered and fused. The phenotypes seen in Epha2 tm1Jrui/+ Epha4 rb-2J/+ double heterozygous embryos suggest both genes play a compensatory role with each other in the adhesion and fusion of the neural tube. In this study, there exists a >50% penetrance of NTDs in the mouse mutants, which genetically have a single allele each of EphA2 and EphA4 absent.

A Novel Occulta-Type Spina Bifida Mediated by Murine Double Heterozygotes EphA2 and EphA4 Receptor Tyrosine Kinases

PubMed Central

Abdullah, Nor Linda; Mohd-Zin, Siti W.; Ahmad-Annuar, Azlina; Abdul-Aziz, Noraishah M.

2017-01-01

Members of the Eph receptor tyrosine kinase have previously been implicated in cranial neural tube development. Failure of neural tube closure leads to the devastating conditions known as anencephaly and spina bifida. EphA2 and EphA4 are expressed at the tips of the closing spinal neural folds prior and during neural tube closure. We investigated the possible role of murine EphA2 and EphA4 during the last step of primary neural tube closure, which is adhesion and fusion. The individual mouse knockouts of EphA2 and EphA4 per se do not exhibit neural tube defects (NTDs). The embryos generated by the crossing of double heterozygotes Epha2tm1Jrui/+Epha4rb-2J/+ displayed NTDs with a wide degree of severity including close exencephaly and close spina bifida (spina bifida occulta). Interestingly, mutants displaying NTDs had skin covering the underlying lesion. The tissue sections revealed the elevated neural folds had not adhered and fused. The phenotypes seen in Epha2tm1Jrui/+Epha4rb-2J/+ double heterozygous embryos suggest both genes play a compensatory role with each other in the adhesion and fusion of the neural tube. In this study, there exists a >50% penetrance of NTDs in the mouse mutants, which genetically have a single allele each of EphA2 and EphA4 absent. PMID:29312933

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.

True-breeding targeted gene knock-out in barley using designer TALE-nuclease in haploid cells.

PubMed

Gurushidze, Maia; Hensel, Goetz; Hiekel, Stefan; Schedel, Sindy; Valkov, Vladimir; Kumlehn, Jochen

2014-01-01

Transcription activator-like effector nucleases (TALENs) are customizable fusion proteins able to cleave virtually any genomic DNA sequence of choice, and thereby to generate site-directed genetic modifications in a wide range of cells and organisms. In the present study, we expressed TALENs in pollen-derived, regenerable cells to establish the generation of instantly true-breeding mutant plants. A gfp-specific TALEN pair was expressed via Agrobacterium-mediated transformation in embryogenic pollen of transgenic barley harboring a functional copy of gfp. Thanks to the haploid nature of the target cells, knock-out mutations were readily detected, and homozygous primary mutant plants obtained following genome duplication. In all, 22% of the TALEN transgenics proved knocked out with respect to gfp, and the loss of function could be ascribed to the deletions of between four and 36 nucleotides in length. The altered gfp alleles were transmitted normally through meiosis, and the knock-out phenotype was consistently shown by the offspring of two independent mutants. Thus, here we describe the efficient production of TALEN-mediated gene knock-outs in barley that are instantaneously homozygous and non-chimeric in regard to the site-directed mutations induced. This TALEN approach has broad applicability for both elucidating gene function and tailoring the phenotype of barley and other crop species.

Trpc2 Depletion Protects RBC from Oxidative Stress-Induced Hemolysis

PubMed Central

Hirschler-Laszkiewicz, Iwona; Zhang, Wenyi; Keefer, Kerry; Conrad, Kathleen; Tong, Qin; Chen, Shu-jen; Bronson, Sarah; Cheung, Joseph Y.; Miller, Barbara A.

2011-01-01

Transient receptor potential channels Trpc2 and Trpc3 are expressed on normal murine erythroid precursors, and erythropoietin stimulates an increase in intracellular calcium ([Ca2+]i) through TRPC2 and TRPC3. Because modulation of [Ca2+]i is an important signaling pathway in erythroid proliferation and differentiation, Trpc2, Trpc3, and Trpc2/Trpc3 double knockout mice were utilized to explore the roles of these channels in erythropoiesis. Trpc2, Trpc3, and Trpc2/Trpc3 double knockout mice were not anemic, and had similar red blood cell counts, hemoglobins, and reticulocyte counts as wild type littermate controls. Although the erythropoietin induced increase in [Ca2+]i was reduced, these knockout mice showed no defects in red cell production. The major phenotypic difference at steady state was that the mean corpuscular volume, mean corpuscular hemoglobin, and hematocrit of red cells were significantly greater in Trpc2 and Trpc2/Trpc3 double knockout mice, and mean corpuscular hemoglobin concentration was significantly reduced. All hematological parameters in Trpc3 knockout mice were similar to controls. When exposed to phenyhydrazine, unlike the Trpc3 knockouts, Trpc2 and Trpc2/Trpc3 double knockout mice showed significant resistance to hemolysis. This was associated with significant reduction in hydrogen peroxide-induced calcium influx in erythroblasts. While erythropoietin induced calcium influx through TRPC2 or TRPC3 is not critical for erythroid production, these data demonstrate that TRPC2 plays an important role in oxidative stress-induced hemolysis which may be related to reduced calcium entry in red cells in the presence of Trpc2 depletion. PMID:21924222

The Pseudomonas aeruginosa pirA gene encodes a second receptor for ferrienterobactin and synthetic catecholate analogues.

PubMed

Ghysels, Bart; Ochsner, Urs; Möllman, Ute; Heinisch, Lothar; Vasil, Michael; Cornelis, Pierre; Matthijs, Sandra

2005-05-15

Actively secreted iron chelating agents termed siderophores play an important role in the virulence and rhizosphere competence of fluorescent pseudomonads, including Pseudomonas aeruginosa which secretes a high affinity siderophore, pyoverdine, and the low affinity siderophore, pyochelin. Uptake of the iron-siderophore complexes is an active process that requires specific outer membrane located receptors, which are dependent of the inner membrane-associated protein TonB and two other inner membrane proteins, ExbB and ExbC. P. aeruginosa is also capable of using a remarkable variety of heterologous siderophores as sources of iron, apparently by expressing their cognate receptors. Illustrative of this feature are the 32 (of which 28 putative) siderophore receptor genes observed in the P. aeruginosa PAO1 genome. However, except for a few (pyoverdine, pyochelin, enterobactin), the vast majority of P. aeruginosa siderophore receptor genes still remain to be characterized. Ten synthetic iron chelators of catecholate type stimulated growth of a pyoverdine/pyochelin deficient P. aeruginosa PAO1 mutant under condition of severe iron limitation. Null mutants of the 32 putative TonB-dependent siderophore receptor encoding genes engineered in the same genetic background were screened for obvious deficiencies in uptake of the synthetic siderophores, but none showed decreased growth stimulation in the presence of the different siderophores. However, a double knock-out mutant of ferrienterobactin receptor encoding gene pfeA (PA 2688) and pirA (PA0931) failed to be stimulated by 4 of the tested synthetic catecholate siderophores whose chemical structures resemble enterobactin. Ferric-enterobactin also failed to stimulate growth of the double pfeA-pirA mutant although, like its synthetic analogues, it stimulated growth of the corresponding single mutants. Hence, we confirmed that pirA represents a second P. aeruginosa ferric-enterobactin receptor. The example of these two enterobactin receptors probably illustrates a more general phenomenon of siderophore receptor redundancy in P. aeruginosa.

Aptamer redesigned tRNA is nonfunctional and degraded in cells

PubMed Central

LEE, DENNIS; MCCLAIN, WILLIAM H.

2004-01-01

An RNA aptamer derived from tRNAGln isolated in vitro and a rationally redesigned tRNAGln were used to address the relationship between structure and function of tRNAGln aminoacylation in Escherichia coli. Two mutant tRNAGln sequences were studied: an aptamer that binds 26-fold tighter to glutaminyl-tRNA synthetase than wild-type tRNAGln in vitro, redesigned in the variable loop, and a mutant with near-normal aminoacylation kinetics for glutamine, redesigned to contain a long variable arm. Both mutants were tested in a tRNAGln knockout strain of E. coli, but neither supported knockout cell growth. It was later found that both mutant tRNAs were present in very low amounts in the cell. These results reveal the difference between in vitro and in vivo studies, demonstrating the complexities of in vivo systems that have not been replicated in vitro. PMID:14681579

The Role of Beta-Adrenergic Receptors in the Regulation of Circadian Intraocular Pressure Rhythm in Mice.

PubMed

Tsuchiya, Shunsuke; Higashide, Tomomi; Toida, Kazunori; Sugiyama, Kazuhisa

2017-07-01

To investigate whether the elimination of β1- and β2-adrenergic receptors alters the diurnal intraocular pressure (IOP) rhythm in mice. β1-/β2-adrenergic receptor double-knockout and C57BL/6J mice were anesthetized intraperitoneally, with their IOPs measured via microneedle method. After entrainment to a 12-h light-dark (LD) cycle (light phase 6:00-18:00), IOPs were measured every 3 h from 9:00 to 24:00 (group 1, β1-/β2-adrenergic receptor double-knockout mice, n = 11; C57BL/6J, n = 15). The IOP measurements at 15:00 and 24:00 under a 12-h LD cycle and in the constant darkness (1 day and 8 days after exposure to darkness, respectively) were performed in another group of β1-/β2-adrenergic receptor double-knockout mice (group 2, n = 12). IOP variance throughout the day and mean IOP differences among time points were evaluated using a linear mixed model. β1-/β2-adrenergic receptor double-knockout and C57BL/6J mice showed biphasic IOP curves, low during the light phase and high during the dark phase; the fluctuation was significant (P < 0.001). The peak IOP (18.7 ± 1.4 mmHg) occurred at 24:00 and the trough IOP (13.5 ± 1.5 mmHg) occurred at 15:00 in β1-/β2-adrenergic receptor double-knockout mice group. IOP curves of β1-/β2-adrenergic receptor double-knockout and C57BL/6J were nearly parallel, and the IOPs of β1-/β2-adrenergic receptor double-knockout mice were significantly higher than those of C57BL/6J mice (P < 0.001). Under constant dark (DD) conditions, IOP at 24:00 (18.1 ± 1.5 mmHg) was significantly higher than that at 15:00 (13.3 ± 1.2 mmHg) (P < 0.001). The transition from the LD cycle to DD environment produced no significant change in IOP (P = 0.728). Elimination of both β1- and β2-adrenergic receptors did not disturb the biphasic diurnal IOP rhythm in mice.

Directed mutagenesis of the Rickettsia prowazekii pld gene encoding phospholipase D.

PubMed

Driskell, Lonnie O; Yu, Xue-jie; Zhang, Lihong; Liu, Yan; Popov, Vsevolod L; Walker, David H; Tucker, Aimee M; Wood, David O

2009-08-01

Rickettsia prowazekii, the causative agent of epidemic typhus, is an obligately intracytoplasmic bacterium, a lifestyle that imposes significant barriers to genetic manipulation. The key to understanding how this unique bacterium evades host immunity is the mutagenesis of selected genes hypothesized to be involved in virulence. The R. prowazekii pld gene, encoding a protein with phospholipase D activity, has been associated with phagosomal escape. To demonstrate the feasibility of site-directed knockout mutagenesis of rickettsial genes and to generate a nonrevertible vaccine strain, we utilized homologous recombination to generate a pld mutant of the virulent R. prowazekii strain Madrid Evir. Using linear DNA for transformation, a double-crossover event resulted in the replacement of the rickettsial wild-type gene with a partially deleted pld gene. Linear DNA was used to prevent potentially revertible single-crossover events resulting in plasmid insertion. Southern blot and PCR analyses were used to confirm the presence of the desired mutation and to demonstrate clonality. While no phenotypic differences were observed between the mutant and wild-type strains when grown in tissue culture, the pld mutant exhibited attenuated virulence in the guinea pig model. In addition, animals immunized with the mutant strain were protected against subsequent challenge with the virulent Breinl strain, suggesting that this transformant could serve as a nonrevertible, attenuated vaccine strain. This study demonstrates the feasibility of generating site-directed rickettsial gene mutants, providing a new tool for understanding rickettsial biology and furthering advances in the prevention of epidemic typhus.

Deletion of Numb/Numblike in glutamatergic neurons leads to anxiety-like behavior in mice.

PubMed

Qian, Wenyu; Hong, Yang; Zhu, Minyan; Zhou, Liang; Li, Hongchang; Li, Huashun

2017-06-15

Endocytic adaptor protein Numb is the first identified cell fate determinant in Drosophila melanogaster. It has been implicated in Notch signaling pathway and regulation of neural stem cells proliferation in the central nervous system. Numb is also expressed in postmitotic neurons, in vitro studies showed that Numb is involved in neuronal morphologic development, such as neurite growth, axonal growth and spine development. However, in vivo functions of Numb in the postmitotic neurons are largely unknown. Here we show that deletion of Numb/Numblike in glutamatergic neurons causes anxiety-like behavior in mouse. In this study, we conditionally deleted Numb and its homologous gene Numblike in the glutamatergic neurons in dorsal forebrain, and thoroughly characterized the behavioral phenotypes of mutant mice. On a battery of tests for anxiety-like behavior, the conditional double knockout mice showed increased anxiety-like behavior on light/dark exploration and novel open field tests, but not on elevated zero maze tests. The conditional double knockout mice also displayed novelty induced hyperactivity in novel open field test. Control measures of general health, motor functions, startle response, sensorimotor gating, depression-related behaviors did not show differences between genotypes. Our present findings provide new insight into the indispensable functions of Numb/Numblike in the brain and behavior, and suggest that Numb/Numblike may play a role in mediating neuronal functions that underlie behaviors related to anxiety. Copyright © 2017. Published by Elsevier B.V.

Mild deficits in mice lacking pituitary adenylate cyclase-activating polypeptide receptor type 1 (PAC1) performing on memory tasks.

PubMed

Sauvage, M; Brabet, P; Holsboer, F; Bockaert, J; Steckler, T

2000-12-08

Pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptor subtype 1 (PAC1) have been suggested to play a role in the modulation of learning and memory. However, behavioral evidence for altered mnemonic function due to altered PAC1 activity is missing. Therefore, the role of PAC1 in learning and memory was studied in mouse mutants lacking this receptor (PAC1 knock-out mice), tested in water maze two-choice spatial discrimination, one-trial contextual and cued fear conditioning, and multiple-session contextual discrimination. Water maze spatial discrimination was unaffected in PAC1 mutants, while a mild deficit was observed in multiple session contextual discrimination in PAC1 knock-out mice. Furthermore, PAC1 knock-out mice were able to learn the association between context and shock in one-trial contextual conditioning, but showed faster return to baseline than wild-type mice. Thus, the effects of PAC1 knock-out on modulating performance in these tasks were subtle and suggest that PAC1 only plays a limited role in learning and memory.

Characterization and proteomic analysis of the Pseudomonas sp. HK-6 xenB knockout mutant under RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) stress.

PubMed

Lee, Bheong-Uk; Choi, Moon-Seop; Oh, Kye-Heon

2015-01-01

Pseudomonas sp. HK-6 is able to utilize RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) as its sole nitrogen source. The role of the xenB gene, encoding xenobiotic reductase B, was investigated using HK-6 xenB knockout mutants. The xenB mutant degraded RDX to a level that was 10-fold less than that obtained with the wild-type HK-6 strain. After 60 days of culture with 25 or 50 μM RDX, no residual RDX was detected in the supernatants of the wild-type aerobically grown cultures, whereas approximately 90 % of the RDX remained in the xenB mutant cultures. The xenB mutant bacteria exhibited a 10(2)-10(4)-fold decrease in survival rate compared to the wild-type. The expression of DnaK and GroEL proteins, two typical stress shock proteins (SSPs), in the xenB mutant increased after immediate exposure to RDX, yet dramatically decreased after 4 h of exposure. In addition, DnaK and GroEL were more highly expressed in the cultures with 25 μM RDX in the medium but showed low expression in the cultures with 50 or 75 μM RDX. The expression levels of the dnaK and groEL genes measured by RT-qPCR were also much lower in the xenB genetic background. Analyses of the proteomes of the HK-6 and xenB mutant cells grown under conditions of RDX stress showed increased induction of several proteins, such as Alg8, alginate biosynthesis sensor histidine kinase, and OprH in the xenB mutants when compared to wild-type. However, many proteins, including two SSPs (DnaK and GroEL) and proteins involved in metabolism, exhibited lower expression levels in the xenB mutant than in the wild-type HK-6 strain. The xenB knockout mutation leads to reduced RDX degradation ability, which renders the mutant more sensitive to RDX stress and results in a lower survival rate and an altered proteomic profile under RDX stress.

Deficiency of Arabidopsis thaliana frataxin alters activity of mitochondrial Fe-S proteins and induces oxidative stress.

PubMed

Busi, Maria V; Maliandi, María V; Valdez, Hugo; Clemente, Marina; Zabaleta, Eduardo J; Araya, Alejandro; Gomez-Casati, Diego F

2006-12-01

Frataxin, a protein crucial for the biogenesis of mitochondria in different organisms, was recently identified in Arabidopsis thaliana. To investigate the role of frataxin in higher plants, we analyze two knock-out and one knock-down T-DNA insertion mutants. The knock-out mutants present an embryo-lethal phenotype, indicating an essential role for frataxin. The knock-down mutant has reduced frataxin mRNA and protein levels. This mutant also presents retarded growth, reduced fresh weight of fruits and reduced number of seeds per fruit. Surprisingly, transcription of aconitase and the Fe-S subunit of succinate dehydrogenase (SDH2-1) are increased in mutant plants; however, the activity of these proteins is reduced, indicating a role for frataxin in Fe-S cluster assembly or insertion of Fe-S clusters into proteins. Mutant plants also have increased CO(2) assimilation rates, exhibit increased formation of reactive oxygen species (ROS) and have increased levels of transcripts for proteins known to be involved in the ROS stress responses. These results indicate that frataxin is an essential protein in plants, required for full activity of mitochondrial Fe-S proteins and playing a protective role against oxidative damage.

EMMA—mouse mutant resources for the international scientific community

PubMed Central

Wilkinson, Phil; Sengerova, Jitka; Matteoni, Raffaele; Chen, Chao-Kung; Soulat, Gaetan; Ureta-Vidal, Abel; Fessele, Sabine; Hagn, Michael; Massimi, Marzia; Pickford, Karen; Butler, Richard H.; Marschall, Susan; Mallon, Ann-Marie; Pickard, Amanda; Raspa, Marcello; Scavizzi, Ferdinando; Fray, Martin; Larrigaldie, Vanessa; Leyritz, Johan; Birney, Ewan; Tocchini-Valentini, Glauco P.; Brown, Steve; Herault, Yann; Montoliu, Lluis; de Angelis, Martin Hrabé; Smedley, Damian

2010-01-01

The laboratory mouse is the premier animal model for studying human disease and thousands of mutants have been identified or produced, most recently through gene-specific mutagenesis approaches. High throughput strategies by the International Knockout Mouse Consortium (IKMC) are producing mutants for all protein coding genes. Generating a knock-out line involves huge monetary and time costs so capture of both the data describing each mutant alongside archiving of the line for distribution to future researchers is critical. The European Mouse Mutant Archive (EMMA) is a leading international network infrastructure for archiving and worldwide provision of mouse mutant strains. It operates in collaboration with the other members of the Federation of International Mouse Resources (FIMRe), EMMA being the European component. Additionally EMMA is one of four repositories involved in the IKMC, and therefore the current figure of 1700 archived lines will rise markedly. The EMMA database gathers and curates extensive data on each line and presents it through a user-friendly website. A BioMart interface allows advanced searching including integrated querying with other resources e.g. Ensembl. Other resources are able to display EMMA data by accessing our Distributed Annotation System server. EMMA database access is publicly available at http://www.emmanet.org. PMID:19783817

Efficient PRNP deletion in bovine genome using gene-editing technologies in bovine cells

PubMed Central

Choi, WooJae; Kim, Eunji; Yum, Soo-Young; Lee, ChoongIl; Lee, JiHyun; Moon, JoonHo; Ramachandra, Sisitha; Malaweera, Buddika Oshadi; Cho, JongKi; Kim, Jin-Soo; Kim, SeokJoong; Jang, Goo

2015-01-01

abstract Even though prion (encoded by the PRNP gene) diseases like bovine spongiform encephalopathy (BSE) are fatal neurodegenerative diseases in cattle, their study via gene deletion has been limited due to the absence of cell lines or mutant models. In this study, we aim to develop an immortalized fibroblast cell line in which genome-engineering technology can be readily applied to create gene-modified clones for studies. To this end, this study is designed to 1) investigate the induction of primary fibroblasts to immortalization by introducing Bmi-1 and hTert genes; 2) investigate the disruption of the PRNP in those cells; and 3) evaluate the gene expression and embryonic development using knockout (KO) cell lines. Primary cells from a male neonate were immortalized with Bmi-1and hTert. Immortalized cells were cultured for more than 180 days without any changes in their doubling time and morphology. Furthermore, to knockout the PRNP gene, plasmids that encode transcription activator-like effector nuclease (TALEN) pairs were transfected into the cells, and transfected single cells were propagated. Mutated clonal cell lines were confirmed by T7 endonuclease I assay and sequencing. Four knockout cell lines were used for somatic cell nuclear transfer (SCNT), and the resulting embryos were developed to the blastocyst stage. The genes (CSNK2A1, FAM64A, MPG and PRND) were affected after PRNP disruption in immortalized cells. In conclusion, we established immortalized cattle fibroblasts using Bmi-1 and hTert genes, and used TALENs to knockout the PRNP gene in these immortalized cells. The efficient PRNP KO is expected to be a useful technology to develop our understanding of in vitro prion protein functions in cattle. PMID:26217959

Conformational co-dependence between Plasmodium berghei LCCL proteins promotes complex formation and stability.

PubMed

Saeed, Sadia; Tremp, Annie Z; Dessens, Johannes T

2012-10-01

Malaria parasites express a conserved family of LCCL-lectin adhesive-like domain proteins (LAPs) that have essential functions in sporozoite transmission. In Plasmodium falciparum all six family members are expressed in gametocytes and form a multi-protein complex. Intriguingly, knockout of P. falciparum LCCL proteins adversely affects expression of other family members at protein, but not at mRNA level, a phenomenon termed co-dependent expression. Here, we investigate this in Plasmodium berghei by crossing a PbLAP1 null mutant parasite with a parasite line expressing GFP-tagged PbLAP3 that displays strong fluorescence in gametocytes. Selected and validated double mutants show normal synthesis and subcellular localization of PbLAP3::GFP. However, GFP-based fluorescence is dramatically reduced without PbLAP1 present, indicating that PbLAP1 and PbLAP3 interact. Moreover, absence of PbLAP1 markedly reduces the half-life of PbLAP3, consistent with a scenario of misfolding. These findings unveil a potential mechanism of conformational interdependence that facilitates assembly and stability of the functional LCCL protein complex. Copyright © 2012 Elsevier B.V. All rights reserved.

Inactivation of Thioredoxin Reductases Reveals a Complex Interplay between Thioredoxin and Glutathione Pathways in Arabidopsis Development[W

PubMed Central

Reichheld, Jean-Philippe; Khafif, Mehdi; Riondet, Christophe; Droux, Michel; Bonnard, Géraldine; Meyer, Yves

2007-01-01

NADPH-dependent thioredoxin reductases (NTRs) are key regulatory enzymes determining the redox state of the thioredoxin system. The Arabidopsis thaliana genome has two genes coding for NTRs (NTRA and NTRB), both of which encode mitochondrial and cytosolic isoforms. Surprisingly, plants of the ntra ntrb knockout mutant are viable and fertile, although with a wrinkled seed phenotype, slower plant growth, and pollen with reduced fitness. Thus, in contrast with mammals, our data demonstrate that neither cytosolic nor mitochondrial NTRs are essential in plants. Nevertheless, in the double mutant, the cytosolic thioredoxin h3 is only partially oxidized, suggesting an alternative mechanism for thioredoxin reduction. Plant growth in ntra ntrb plants is hypersensitive to buthionine sulfoximine (BSO), a specific inhibitor of glutathione biosynthesis, and thioredoxin h3 is totally oxidized under this treatment. Interestingly, this BSO-mediated growth arrest is fully reversible, suggesting that BSO induces a growth arrest signal but not a toxic accumulation of activated oxygen species. Moreover, crossing ntra ntrb with rootmeristemless1, a mutant blocked in root growth due to strongly reduced glutathione synthesis, led to complete inhibition of both shoot and root growth, indicating that either the NTR or the glutathione pathway is required for postembryonic activity in the apical meristem. PMID:17586656

Inactivation of thioredoxin reductases reveals a complex interplay between thioredoxin and glutathione pathways in Arabidopsis development.

PubMed

Reichheld, Jean-Philippe; Khafif, Mehdi; Riondet, Christophe; Droux, Michel; Bonnard, Géraldine; Meyer, Yves

2007-06-01

NADPH-dependent thioredoxin reductases (NTRs) are key regulatory enzymes determining the redox state of the thioredoxin system. The Arabidopsis thaliana genome has two genes coding for NTRs (NTRA and NTRB), both of which encode mitochondrial and cytosolic isoforms. Surprisingly, plants of the ntra ntrb knockout mutant are viable and fertile, although with a wrinkled seed phenotype, slower plant growth, and pollen with reduced fitness. Thus, in contrast with mammals, our data demonstrate that neither cytosolic nor mitochondrial NTRs are essential in plants. Nevertheless, in the double mutant, the cytosolic thioredoxin h3 is only partially oxidized, suggesting an alternative mechanism for thioredoxin reduction. Plant growth in ntra ntrb plants is hypersensitive to buthionine sulfoximine (BSO), a specific inhibitor of glutathione biosynthesis, and thioredoxin h3 is totally oxidized under this treatment. Interestingly, this BSO-mediated growth arrest is fully reversible, suggesting that BSO induces a growth arrest signal but not a toxic accumulation of activated oxygen species. Moreover, crossing ntra ntrb with rootmeristemless1, a mutant blocked in root growth due to strongly reduced glutathione synthesis, led to complete inhibition of both shoot and root growth, indicating that either the NTR or the glutathione pathway is required for postembryonic activity in the apical meristem.

Arabidopsis Heterotrimeric G-Proteins Play a Critical Role in Host and Nonhost Resistance against Pseudomonas syringae Pathogens

PubMed Central

Lee, Seonghee; Rojas, Clemencia M.; Ishiga, Yasuhiro; Pandey, Sona; Mysore, Kirankumar S.

2013-01-01

Heterotrimeric G-proteins have been proposed to be involved in many aspects of plant disease resistance but their precise role in mediating nonhost disease resistance is not well understood. We evaluated the roles of specific subunits of heterotrimeric G-proteins using knock-out mutants of Arabidopsis Gα, Gβ and Gγ subunits in response to host and nonhost Pseudomonas pathogens. Plants lacking functional Gα, Gβ and Gγ1Gγ2 proteins displayed enhanced bacterial growth and disease susceptibility in response to host and nonhost pathogens. Mutations of single Gγ subunits Gγ1, Gγ2 and Gγ3 did not alter bacterial disease resistance. Some specificity of subunit usage was observed when comparing host pathogen versus nonhost pathogen. Overexpression of both Gα and Gβ led to reduced bacterial multiplication of nonhost pathogen P. syringae pv. tabaci whereas overexpression of Gβ, but not of Gα, resulted in reduced bacterial growth of host pathogen P. syringae pv. maculicola, compared to wild-type Col-0. Moreover, the regulation of stomatal aperture by bacterial pathogens was altered in Gα and Gβ mutants but not in any of the single or double Gγ mutants. Taken together, these data substantiate the critical role of heterotrimeric G-proteins in plant innate immunity and stomatal modulation in response to P. syringae. PMID:24349286

Synapse-associated protein 102/dlgh3 couples the NMDA receptor to specific plasticity pathways and learning strategies.

PubMed

Cuthbert, Peter C; Stanford, Lianne E; Coba, Marcelo P; Ainge, James A; Fink, Ann E; Opazo, Patricio; Delgado, Jary Y; Komiyama, Noboru H; O'Dell, Thomas J; Grant, Seth G N

2007-03-07

Understanding the mechanisms whereby information encoded within patterns of action potentials is deciphered by neurons is central to cognitive psychology. The multiprotein complexes formed by NMDA receptors linked to synaptic membrane-associated guanylate kinase (MAGUK) proteins including synapse-associated protein 102 (SAP102) and other associated proteins are instrumental in these processes. Although humans with mutations in SAP102 show mental retardation, the physiological and biochemical mechanisms involved are unknown. Using SAP102 knock-out mice, we found specific impairments in synaptic plasticity induced by selective frequencies of stimulation that also required extracellular signal-regulated kinase signaling. This was paralleled by inflexibility and impairment in spatial learning. Improvement in spatial learning performance occurred with extra training despite continued use of a suboptimal search strategy, and, in a separate nonspatial task, the mutants again deployed a different strategy. Double-mutant analysis of postsynaptic density-95 and SAP102 mutants indicate overlapping and specific functions of the two MAGUKs. These in vivo data support the model that specific MAGUK proteins couple the NMDA receptor to distinct downstream signaling pathways. This provides a mechanism for discriminating patterns of synaptic activity that lead to long-lasting changes in synaptic strength as well as distinct aspects of cognition in the mammalian nervous system.

Pathogenicty and immune prophylaxis of cag pathogenicity island gene knockout homogenic mutants

PubMed Central

Lin, Huan-Jian; Xue, Jing; Bai, Yang; Wang, Ji-De; Zhang, Ya-Li; Zhou, Dian-Yuan

2004-01-01

AIM: To clarify the role of cag pathogenicity island (cagPAI) of Helicobacter pylori (H pylori) in the pathogenicity and immune prophylaxis of H pylori infection. METHODS: Three pairs of H pylori including 3 strains of cagPAI positive wildtype bacteria and their cagPAI knockout homogenic mutants were utilized. H pylori binding to the gastric epithelial cells was analyzed by flow cytometry assays. Apoptosis of gastric epithelial cells induced by H pylori was determined by ELISA assay. Prophylaxis effect of the wildtype and mutant strains was compared by immunization with the sonicate of the bacteria into mice model. RESULTS: No difference was found in the apoptasis between cagPAI positive and knockout H pylori strains in respective of the ability in the binding to gastric epithelial cells as well as the induction of apoptosis. Both types of the bacteria were able to protect the mice from the infection of H pylori after immunization, with no difference between them regarding to the protection rate as well as the stimulation of the proliferation of splenocytes of the mice. CONCLUSION: The role of cagPAI in the pathogenicity and prophylaxis of H pylori infection remains to be cleared. PMID:15484302

Improvement of constraint-based flux estimation during L-phenylalanine production with Escherichia coli using targeted knock-out mutants.

PubMed

Weiner, Michael; Tröndle, Julia; Albermann, Christoph; Sprenger, Georg A; Weuster-Botz, Dirk

2014-07-01

Fed-batch production of the aromatic amino acid L-phenylalanine was studied with recombinant Escherichia coli strains on a 15 L-scale using glycerol as carbon source. Flux Variability Analysis (FVA) was applied for intracellular flux estimation to obtain an insight into intracellular flux distribution during L-phenylalanine production. Variability analysis revealed great flux uncertainties in the central carbon metabolism, especially concerning malate consumption. Due to these results two recombinant strains were genetically engineered differing in the ability of malate degradation and anaplerotic reactions (E. coli FUS4.11 ΔmaeA pF81kan and E. coli FUS4.11 ΔmaeA ΔmaeB pF81kan). Applying these malic enzyme knock-out mutants in the standardized L-phenylalanine production process resulted in almost identical process performances (e.g., L-phenylalanine concentration, production rate and byproduct formation). This clearly highlighted great redundancies in central metabolism in E. coli. Uncertainties of intracellular flux estimations by constraint-based analyses during fed-batch production of L-phenylalanine were drastically reduced by application of the malic enzyme knock-out mutants. © 2014 Wiley Periodicals, Inc.

Essential but partially redundant roles for POU4F1/Brn-3a and POU4F2/Brn-3b transcription factors in the developing heart

PubMed Central

Maskell, Lauren J; Qamar, Kashif; Babakr, Aram A; Hawkins, Thomas A; Heads, Richard J; Budhram-Mahadeo, Vishwanie S

2017-01-01

Congenital heart defects contribute to embryonic or neonatal lethality but due to the complexity of cardiac development, the molecular changes associated with such defects are not fully understood. Here, we report that transcription factors (TFs) Brn-3a (POU4F1) and Brn-3b (POU4F2) are important for normal cardiac development. Brn-3a directly represses Brn-3b promoter in cardiomyocytes and consequently Brn-3a knockout (KO) mutant hearts express increased Brn-3b mRNA during mid-gestation, which is linked to hyperplastic growth associated with elevated cyclin D1, a known Brn-3b target gene. However, during late gestation, Brn-3b can cooperate with p53 to enhance transcription of pro-apoptotic genes e.g. Bax, thereby increasing apoptosis and contribute to morphological defects such as non-compaction, ventricular wall/septal thinning and increased crypts/fissures, which may cause lethality of Brn-3a KO mutants soon after birth. Despite this, early embryonic lethality in e9.5 double KO (Brn-3a−/− : Brn-3b−/−) mutants indicate essential functions with partial redundancy during early embryogenesis. High conservation between mammals and zebrafish (ZF) Brn-3b (87%) or Brn-3a (76%) facilitated use of ZF embryos to study potential roles in developing heart. Double morphant embryos targeted with morpholino oligonucleotides to both TFs develop significant cardiac defects (looping abnormalities and valve defects) suggesting essential roles for Brn-3a and Brn-3b in developing hearts. PMID:28594399

Mapping Mammary Epithelial Cell Transformation in BRCA1 Mutant Mice

DTIC Science & Technology

2006-07-01

Transformation in BRCA1 Mutant Mice PRINCIPAL INVESTIGATOR: Gerburg M. Wulf CONTRACTING ORGANIZATION: Beth Israel Deaconess Medical...REPORT NUMBER Beth Israel Deaconess Medical Center Boston, MA 02215 9. SPONSORING / MONITORING AGENCY NAME(S) AND ADDRESS(ES...and whether it allowed us to analyze the early steps of tumor formation. For this purpose transgenic and conditional knock-out mice (mutant p53 or

An activating mutant of Rac1 that fails to interact with Rho GDP-dissociation inhibitor stimulates membrane ruffling in mammalian cells.

PubMed Central

Gandhi, Payal N; Gibson, Richard M; Tong, Xiaofeng; Miyoshi, Jun; Takai, Yoshimi; Konieczkowski, Martha; Sedor, John R; Wilson-Delfosse, Amy L

2004-01-01

Rac1, a member of the Rho family of small GTP-binding proteins, is involved in the regulation of the actin cytoskeleton via activation of lamellipodia and membrane ruffle formation. RhoGDI (Rho-family-specific GDP-dissociation inhibitor) forms a complex with Rho proteins in the cytosol of mammalian cells. It not only regulates guanine nucleotide binding to Rho proteins, but may also function as a molecular shuttle to carry Rho proteins from an inactive cytosolic pool to the membrane for activation. These studies tested if RhoGDI is necessary for the translocation of Rac1 from the cytosol to the plasma membrane for the formation of membrane ruffles. We describe a novel mutant of Rac1, R66E (Arg66-->Glu), that fails to bind RhoGDI. This RhoGDI-binding-defective mutation is combined with a Rac1-activating mutation G12V, resulting in a double-mutant [Rac1(G12V/R66E)] that fails to interact with RhoGDI in COS-7 cells, but remains constitutively activated. This double mutant stimulates membrane ruffling to a similar extent as that observed after epidermal growth factor treatment of non-transfected cells. To confirm that Rac1 can signal ruffle formation in the absence of interaction with RhoGDI, Rac1(G12V) was overexpressed in cultured mesangial cells derived from a RhoGDI knockout mouse. Rac1-mediated membrane ruffling was indistinguishable between the RhoGDI(-/-) and RhoGDI(+/+) cell lines. In both the COS-7 and cultured mesangial cells, Rac1(G12V) and Rac1(G12V/R66E) co-localize with membrane ruffles. These findings suggest that interaction with RhoGDI is not essential in the mechanism by which Rac1 translocates to the plasma membrane to stimulate ruffle formation. PMID:14629200

Beta-carbonic anhydrases play a role in fruiting body development and ascospore germination in the filamentous fungus Sordaria macrospora.

PubMed

Elleuche, Skander; Pöggeler, Stefanie

2009-01-01

Carbon dioxide (CO(2)) is among the most important gases for all organisms. Its reversible interconversion to bicarbonate (HCO(3) (-)) reaches equilibrium spontaneously, but slowly, and can be accelerated by a ubiquitous group of enzymes called carbonic anhydrases (CAs). These enzymes are grouped by their distinct structural features into alpha-, beta-, gamma-, delta- and zeta-classes. While physiological functions of mammalian, prokaryotic, plant and algal CAs have been extensively studied over the past years, the role of beta-CAs in yeasts and the human pathogen Cryptococcus neoformans has been elucidated only recently, and the function of CAs in multicellular filamentous ascomycetes is mostly unknown. To assess the role of CAs in the development of filamentous ascomycetes, the function of three genes, cas1, cas2 and cas3 (carbonic anhydrase of Sordaria) encoding beta-class carbonic anhydrases was characterized in the filamentous ascomycetous fungus Sordaria macrospora. Fluorescence microscopy was used to determine the localization of GFP- and DsRED-tagged CAs. While CAS1 and CAS3 are cytoplasmic enzymes, CAS2 is localized to the mitochondria. To assess the function of the three isoenzymes, we generated knock-out strains for all three cas genes (Deltacas1, Deltacas2, and Deltacas3) as well as all combinations of double mutants. No effect on vegetative growth, fruiting-body and ascospore development was seen in the single mutant strains lacking cas1 or cas3, while single mutant Deltacas2 was affected in vegetative growth, fruiting-body development and ascospore germination, and the double mutant strain Deltacas1/2 was completely sterile. Defects caused by the lack of cas2 could be partially complemented by elevated CO(2) levels or overexpression of cas1, cas3, or a non-mitochondrial cas2 variant. The results suggest that CAs are required for sexual reproduction in filamentous ascomycetes and that the multiplicity of isoforms results in redundancy of specific and non-specific functions.

Loss of the two major leaf isoforms of sucrose-phosphate synthase in Arabidopsis thaliana limits sucrose synthesis and nocturnal starch degradation but does not alter carbon partitioning during photosynthesis

PubMed Central

Volkert, Kathrin; Debast, Stefan; Voll, Lars M.; Voll, Hildegard; Schießl, Ingrid; Hofmann, Jörg; Schneider, Sabine; Börnke, Frederik

2014-01-01

Sucrose (Suc)-phosphate synthase (SPS) catalyses one of the rate-limiting steps in the synthesis of Suc in plants. The Arabidopsis genome contains four annotated SPS genes which can be grouped into three different families (SPSA1, SPSA2, SPSB, and SPSC). However, the functional significance of this multiplicity of SPS genes is as yet only poorly understood. All four SPS isoforms show enzymatic activity when expressed in yeast although there is variation in sensitivity towards allosteric effectors. Promoter–reporter gene analyses and quantitative real-time reverse transcription–PCR studies indicate that no two SPS genes have the same expression pattern and that AtSPSA1 and AtSPSC represent the major isoforms expressed in leaves. An spsa1 knock-out mutant showed a 44% decrease in leaf SPS activity and a slight increase in leaf starch content at the end of the light period as well as at the end of the dark period. The spsc null mutant displayed reduced Suc contents towards the end of the photoperiod and a concomitant 25% reduction in SPS activity. In contrast, an spsa1/spsc double mutant was strongly impaired in growth and accumulated high levels of starch. This increase in starch was probably not due to an increased partitioning of carbon into starch, but was rather caused by an impaired starch mobilization during the night. Suc export from excised petioles harvested from spsa1/spsc double mutant plants was significantly reduced under illumination as well as during the dark period. It is concluded that loss of the two major SPS isoforms in leaves limits Suc synthesis without grossly changing carbon partitioning in favour of starch during the light period but limits starch degradation during the dark period. PMID:24994761

MYB5 and MYB14 Play Pivotal Roles in Seed Coat Polymer Biosynthesis in Medicago truncatula1[W][OPEN

PubMed Central

Liu, Chenggang; Jun, Ji Hyung; Dixon, Richard A.

2014-01-01

In Arabidopsis (Arabidopsis thaliana), the major MYB protein regulating proanthocyanidin (PA) biosynthesis is TT2, named for the transparent testa phenotype of tt2 mutant seeds that lack PAs in their coats. In contrast, the MYB5 transcription factor mainly regulates seed mucilage biosynthesis and trichome branching, with only a minor role in PA biosynthesis. We here characterize MYB5 and MYB14 (a TT2 homolog) in the model legume Medicago truncatula. Overexpression of MtMYB5 or MtMYB14 strongly induces PA accumulation in M. truncatula hairy roots, and both myb5 and myb14 mutants of M. truncatula exhibit darker seed coat color than wild-type plants, with myb5 also showing deficiency in mucilage biosynthesis. myb5 mutant seeds have a much stronger seed color phenotype than myb14. The myb5 and myb14 mutants accumulate, respectively, about 30% and 50% of the PA content of wild-type plants, and PA levels are reduced further in myb5 myb14 double mutants. Transcriptome analyses of overexpressing hairy roots and knockout mutants of MtMYB5 and MtMYB14 indicate that MtMYB5 regulates a broader set of genes than MtMYB14. Moreover, we demonstrate that MtMYB5 and MtMYB14 physically interact and synergistically activate the promoters of anthocyanidin reductase and leucoanthocyanidin reductase, the key structural genes leading to PA biosynthesis, in the presence of MtTT8 and MtWD40-1. Our results provide new insights into the complex regulation of PA and mucilage biosynthesis in M. truncatula. PMID:24948832

Dictyostelium myosin I double mutants exhibit conditional defects in pinocytosis.

PubMed

Novak, K D; Peterson, M D; Reedy, M C; Titus, M A

1995-12-01

The functional relationship between three Dictyostelium myosin Is, myoA, myoB, and myoC, has been examined through the creation of double mutants. Two double mutants, myoA-/B- and myoB-/C-, exhibit similar conditional defects in fluid-phase pinocytosis. Double mutants grown in suspension culture are significantly impaired in their ability to take in nutrients from the medium, whereas they are almost indistinguishable from wild-type and single mutant strains when grown on a surface. The double mutants are also found to internalize gp126, a 116-kD membrane protein, at a slower rate than either the wild-type or single mutant cells. Ultrastructural analysis reveals that both double mutants possess numerous small vesicles, in contrast to the wild-type or myosin I single mutants that exhibit several large, clear vacuoles. The alterations in fluid and membrane internalization in the suspension-grown double mutants, coupled with the altered vesicular profile, suggest that these cells may be compromised during the early stages of pinocytosis, a process that has been proposed to occur via actin-based cytoskeletal rearrangements. Scanning electron microscopy and rhodamine-phalloidin staining indicates that the myosin I double mutants appear to extend a larger number of actin-filled structures, such as filopodia and crowns, than wild-type cells. Rhodamine-phalloidin staining of the F-actin cytoskeleton of these suspension-grown cells also reveals that the double mutant cells are delayed in the rearrangement of cortical actin-rich structures upon adhesion to a substrate. We propose that myoA, myoB, and myoC play roles in controlling F-actin filled membrane projections that are required for pinosome internalization in suspension.

Role of AMACR (α-methylacyl-CoA racemase) and MFE-1 (peroxisomal multifunctional enzyme-1) in bile acid synthesis in mice.

PubMed

Autio, Kaija J; Schmitz, Werner; Nair, Remya R; Selkälä, Eija M; Sormunen, Raija T; Miinalainen, Ilkka J; Crick, Peter J; Wang, Yuqin; Griffiths, William J; Reddy, Janardan K; Baes, Myriam; Hiltunen, J Kalervo

2014-07-01

Cholesterol is catabolized to bile acids by peroxisomal β-oxidation in which the side chain of C27-bile acid intermediates is shortened by three carbon atoms to form mature C24-bile acids. Knockout mouse models deficient in AMACR (α-methylacyl-CoA racemase) or MFE-2 (peroxisomal multifunctional enzyme type 2), in which this β-oxidation pathway is prevented, display a residual C24-bile acid pool which, although greatly reduced, implies the existence of alternative pathways of bile acid synthesis. One alternative pathway could involve Mfe-1 (peroxisomal multifunctional enzyme type 1) either with or without Amacr. To test this hypothesis, we generated a double knockout mouse model lacking both Amacr and Mfe-1 activities and studied the bile acid profiles in wild-type, Mfe-1 and Amacr single knockout mouse line and Mfe-1 and Amacr double knockout mouse lines. The total bile acid pool was decreased in Mfe-1-/- mice compared with wild-type and the levels of mature C24-bile acids were reduced in the double knockout mice when compared with Amacr-deficient mice. These results indicate that Mfe-1 can contribute to the synthesis of mature bile acids in both Amacr-dependent and Amacr-independent pathways.

Cch1 and Mid1 Are Functionally Required for Vegetative Growth under Low-Calcium Conditions in the Phytopathogenic Ascomycete Botrytis cinerea

PubMed Central

Harren, Karin

2013-01-01

In the filamentous phytopathogen Botrytis cinerea, the Ca2+/calcineurin signaling cascade has been shown to play an important role in fungal growth, differentiation, and virulence. This study deals with the functional characterization of two components of this pathway, the putative calcium channel proteins Cch1 and Mid1. The cch1 and mid1 genes were deleted, and single and double knockout mutants were analyzed during different stages of the fungal life cycle. Our data indicate that Cch1 and Mid1 are functionally required for vegetative growth under conditions of low extracellular calcium, since the growth of both deletion mutants is strongly impaired when they are exposed to the Ca2+-chelating agents EGTA and 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA). The impact of external Ca2+ was investigated by supplementing with CaCl2 and the ionophore A23187, both of which resulted in elevated growth for all mutants. However, deletion of either gene had no impact on germination, sporulation, hyphal morphology, or virulence. By use of the aequorin reporter system to measure intracellular calcium levels, no differences between the mutant strains and the wild type were obtained. Localization studies revealed a subcellular distribution of the Mid1–green fluorescent protein (GFP) fusion protein in network-like filaments, probably the endoplasmic reticulum (ER) membranes, indicating that Mid1 is not a plasma membrane-located calcium channel in B. cinerea. PMID:23475703

Knock-out of a mitochondrial sirtuin protects neurons from degeneration in Caenorhabditis elegans.

PubMed

Sangaletti, Rachele; D'Amico, Massimo; Grant, Jeff; Della-Morte, David; Bianchi, Laura

2017-08-01

Sirtuins are NAD⁺-dependent deacetylases, lipoamidases, and ADP-ribosyltransferases that link cellular metabolism to multiple intracellular pathways that influence processes as diverse as cell survival, longevity, and cancer growth. Sirtuins influence the extent of neuronal death in stroke. However, different sirtuins appear to have opposite roles in neuronal protection. In Caenorhabditis elegans, we found that knock-out of mitochondrial sirtuin sir-2.3, homologous to mammalian SIRT4, is protective in both chemical ischemia and hyperactive channel induced necrosis. Furthermore, the protective effect of sir-2.3 knock-out is enhanced by block of glycolysis and eliminated by a null mutation in daf-16/FOXO transcription factor, supporting the involvement of the insulin/IGF pathway. However, data in Caenorhabditis elegans cell culture suggest that the effects of sir-2.3 knock-out act downstream of the DAF-2/IGF-1 receptor. Analysis of ROS in sir-2.3 knock-out reveals that ROS become elevated in this mutant under ischemic conditions in dietary deprivation (DD), but to a lesser extent than in wild type, suggesting more robust activation of a ROS scavenging system in this mutant in the absence of food. This work suggests a deleterious role of SIRT4 during ischemic processes in mammals that must be further investigated and reveals a novel pathway that can be targeted for the design of therapies aimed at protecting neurons from death in ischemic conditions.

Key Enzymes of the Semiphosphorylative Entner-Doudoroff Pathway in the Haloarchaeon Haloferax volcanii: Characterization of Glucose Dehydrogenase, Gluconate Dehydratase, and 2-Keto-3-Deoxy-6-Phosphogluconate Aldolase.

PubMed

Sutter, Jan-Moritz; Tästensen, Julia-Beate; Johnsen, Ulrike; Soppa, Jörg; Schönheit, Peter

2016-08-15

The halophilic archaeon Haloferax volcanii has been proposed to degrade glucose via the semiphosphorylative Entner-Doudoroff (spED) pathway. So far, the key enzymes of this pathway, glucose dehydrogenase (GDH), gluconate dehydratase (GAD), and 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase (KDPGA), have not been characterized, and their functional involvement in glucose degradation has not been demonstrated. Here we report that the genes HVO_1083 and HVO_0950 encode GDH and KDPGA, respectively. The recombinant enzymes show high specificity for glucose and KDPG and did not convert the corresponding C4 epimers galactose and 2-keto-3-deoxy-6-phosphogalactonate at significant rates. Growth studies of knockout mutants indicate the functional involvement of both GDH and KDPGA in glucose degradation. GAD was purified from H. volcanii, and the encoding gene, gad, was identified as HVO_1488. GAD catalyzed the specific dehydration of gluconate and did not utilize galactonate at significant rates. A knockout mutant of GAD lost the ability to grow on glucose, indicating the essential involvement of GAD in glucose degradation. However, following a prolonged incubation period, growth of the Δgad mutant on glucose was recovered. Evidence is presented that under these conditions, GAD was functionally replaced by xylonate dehydratase (XAD), which uses both xylonate and gluconate as substrates. Together, the characterization of key enzymes and analyses of the respective knockout mutants present conclusive evidence for the in vivo operation of the spED pathway for glucose degradation in H. volcanii The work presented here describes the identification and characterization of the key enzymes glucose dehydrogenase, gluconate dehydratase, and 2-keto-3-deoxy-6-phosphogluconate aldolase and their encoding genes of the proposed semiphosphorylative Entner-Doudoroff pathway in the haloarchaeon Haloferax volcanii The functional involvement of the three enzymes was proven by analyses of the corresponding knockout mutants. These results provide evidence for the in vivo operation of the semiphosphorylative Entner-Doudoroff pathway in haloarchaea and thus expand our understanding of the unusual sugar degradation pathways in the domain Archaea. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Role of plnB gene in the regulation of bacteriocin production in Lactobacillus paraplantarum L-XM1.

PubMed

Zhang, Xiangmei; Shang, Nan; Zhang, Xu; Gui, Meng; Li, Pinglan

2013-06-12

Homologues of plnB gene have been shown to participate in regulation of bacteriocin production through quorum sensing system in other organisms, to investigate the possible role of plnB gene in Lactobacillus paraplantarum L-XM1, we cloned and insertionally inactivated the plnB gene. The plnB knockout mutant ΔplnB21 showed loss of bacteriocin production, its Bac⁺ phenotype could not be restored even after the addition of PlnA. Furthermore, reverse transcription-PCR analysis from total RNA preparations showed that the bacteriocin structural genes of the plnEF and plnJK were not transcribed in the plnB knockout mutant compared with the wild-type strain. It was therefore concluded that plnB is invovled in a quorum sensing based bacteriocin production. This is the first demonstration of a role for plnB by gene knockout in L. paraplantarum. Copyright © 2012 Elsevier GmbH. All rights reserved.

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

RNA sequencing of an nsdC mutant reveals global regulation of secondary metabolic gene clusters in Aspergillus flavus

USDA-ARS?s Scientific Manuscript database

The zinc finger transcription factor nsdC is required for both sexual development and aflatoxin production in the saprophytic fungus Aspergillus flavus. While previous work with an nsdC knockout mutant was conducted in Aspergillus nidulans and A. flavus strain 3357, here we demonstrate perturbations...

Overexpression of 3β-hydroxysteroid dehydrogenases/C-4 decarboxylases causes growth defects possibly due to abnormal auxin transport in Arabidopsis.

PubMed

Kim, Bokyung; Kim, Gyusik; Fujioka, Shozo; Takatsuto, Suguru; Choe, Sunghwa

2012-07-01

Sterols play crucial roles as membrane components and precursors of steroid hormones (e.g., brassinosteroids, BR). Within membranes, sterols regulate membrane permeability and fluidity by interacting with other lipids and proteins. Sterols are frequently enriched in detergent-insoluble membranes (DIMs), which organize molecules involved in specialized signaling processes, including auxin transporters. To be fully functional, the two methyl groups at the C-4 position of cycloartenol, a precursor of plant sterols, must be removed by bifunctional 3β-hydroxysteroid dehydrogenases/C-4 decarboxylases (3βHSD/D). To understand the role of 3βHSD/D in Arabidopsis development, we analyzed the phenotypes of knock-out mutants and overexpression lines of two 3βHSD/D genes (At1g47290 and At2g26260). Neither single nor double knock-out mutants displayed a noticeable phenotype; however, overexpression consistently resulted in plants with wrinkled leaves and short inflorescence internodes. Interestingly, the internode growth defects were opportunistic; even within a plant, some stems were more severely affected than others. Endogenous levels of BRs were not altered in the overexpression lines, suggesting that the growth defect is not primarily due to a flaw in BR biosynthesis. To determine if overexpression of the sterol biosynthetic genes affects the functions of membrane-localized auxin transporters, we subjected plants to the auxin efflux carrier inhibitor, 1-N-naphthylphthalamic acid (NPA). Where-as the gravity vectors of wild-type roots became randomly scattered in response to NPA treatment, those of the overexpression lines continued to grow in the direction of gravity. Overexpression of the two Arabidopsis 3βHSD/D genes thus appears to affect auxin transporter activity, possibly by altering sterol composition in the membranes.

Translation Initiation Factor AteIF(iso)4E Is Involved in Selective mRNA Translation in Arabidopsis Thaliana Seedlings

PubMed Central

Martínez-Silva, Ana Valeria; Aguirre-Martínez, César; Flores-Tinoco, Carlos E.; Alejandri-Ramírez, Naholi D.; Dinkova, Tzvetanka D.

2012-01-01

One of the most regulated steps of translation initiation is the recruitment of mRNA by the translation machinery. In eukaryotes, this step is mediated by the 5′end cap-binding factor eIF4E bound to the bridge protein eIF4G and forming the eIF4F complex. In plants, different isoforms of eIF4E and eIF4G form the antigenically distinct eIF4F and eIF(iso)4F complexes proposed to mediate selective translation. Using a microarray analysis of polyribosome- and non-polyribosome-purified mRNAs from 15 day-old Arabidopsis thaliana wild type [WT] and eIF(iso)4E knockout mutant [(iso)4E-1] seedlings we found 79 transcripts shifted from polyribosomes toward non-polyribosomes, and 47 mRNAs with the opposite behavior in the knockout mutant. The translationally decreased mRNAs were overrepresented in root-preferentially expressed genes and proteins from the endomembrane system, including several transporters such as the phosphate transporter PHOSPHATE1 (PHO1), Sucrose transporter 3 (SUC3), ABC transporter-like with ATPase activity (MRP11) and five electron transporters, as well as signal transduction-, protein modification- and transcription-related proteins. Under normal growth conditions, eIF(iso)4E expression under the constitutive promoter 35 S enhanced the polyribosomal recruitment of PHO1 supporting its translational preference for eIF(iso)4E. Furthermore, under phosphate deficiency, the PHO1 protein increased in the eIF(iso)4E overexpressing plants and decreased in the knockout mutant as compared to wild type. In addition, the knockout mutant had larger root, whereas the 35 S directed expression of eIF(iso)4E caused shorter root under normal growth conditions, but not under phosphate deficiency. These results indicate that selective translation mediated by eIF(iso)4E is relevant for Arabidopsis root development under normal growth conditions. PMID:22363683

Production of α1,3-galactosyltransferase and cytidine monophosphate-N-acetylneuraminic acid hydroxylase gene double-deficient pigs by CRISPR/Cas9 and handmade cloning.

PubMed

Gao, Hanchao; Zhao, Chengjiang; Xiang, Xi; Li, Yong; Zhao, Yanli; Li, Zesong; Pan, Dengke; Dai, Yifan; Hara, Hidetaka; Cooper, David K C; Cai, Zhiming; Mou, Lisha

2017-02-16

Gene-knockout pigs hold great promise as a solution to the shortage of organs from donor animals for xenotransplantation. Several groups have generated gene-knockout pigs via clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) and somatic cell nuclear transfer (SCNT). Herein, we adopted a simple and micromanipulator-free method, handmade cloning (HMC) instead of SCNT, to generate double gene-knockout pigs. First, we applied the CRISPR/Cas9 system to target α1,3-galactosyltransferase (GGTA1) and cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) genes simultaneously in porcine fetal fibroblast cells (PFFs), which were derived from wild-type Chinese domestic miniature Wuzhishan pigs. Cell colonies were obtained by screening and were identified by Surveyor assay and sequencing. Next, we chose the GGTA1/CMAH double-knockout (DKO) cells for HMC to produce piglets. As a result, we obtained 11 live bi-allelic GGTA1/CMAH DKO piglets with the identical phenotype. Compared to cells from GGTA1-knockout pigs, human antibody binding and antibody-mediated complement-dependent cytotoxicity were significantly reduced in cells from GGTA1/CMAH DKO pigs, which demonstrated that our pigs would exhibit reduced humoral rejection in xenotransplantation. These data suggested that the combination of CRISPR/Cas9 and HMC technology provided an efficient and new strategy for producing pigs with multiple genetic modifications.

The two Dictyostelium discoideum autophagy 8 proteins have distinct autophagic functions.

PubMed

Meßling, Susanne; Matthias, Jan; Xiong, Qiuhong; Fischer, Sarah; Eichinger, Ludwig

2017-06-01

Autophagy is a highly conserved cellular degradation pathway which is crucial for various cellular processes. The autophagic process is subdivided in the initiation, autophagosome maturation and lysosomal degradation phases and involves more than forty core and accessory autophagy-related (ATG) proteins. Autophagy 8 (ATG8, in mammals LC3) is a well-established marker of autophagy and is linked to the autophagic membrane from initiation until fusion with the lysosome. We generated single and double knock-out mutants of the two Dictyostelium paralogues, ATG8a and ATG8b, as well as strains that expressed RFP-ATG8a and/or GFP-ATG8b, RFP-ATG8b, RFP-GFP-ATG8a or RFP-GFP-ATG8b in different knock-out mutants. The ATG8b¯ mutant displayed only subtle phenotypic changes in comparison to AX2 wild-type cells. In contrast, deletion of ATG8a resulted in a complex phenotype with delayed development, reduced growth, phagocytosis and cell viability, an increase in ubiquitinylated proteins and a concomitant decrease in proteasomal activity. The phenotype of the ATG8a¯/b¯ strain was, except for cell viability, in all aforementioned aspects more severe, showing that both proteins function in parallel during most analysed cellular processes. Immunofluorescence analysis of knock-out strains expressing either RFP-GFP-ATG8a or RFP-GFP-ATG8b suggests a crucial function for ATG8b in autophagosome-lysosome fusion. Quantitative analysis of strains expressing RFP-ATG8a, RFP-ATG8b, or RFP-ATG8a and GFP-ATG8b revealed that ATG8b generally localised to small and large vesicles, whereas ATG8a preferentially co-localised with ATG8b on large vesicles, indicating that ATG8b associated with nascent autophagosomes before ATG8a, which is supported by previous results (Matthias et al., 2016). Deconvoluted confocal fluorescence images showed that ATG8b localised around ATG8a and was presumably mainly present on the outer membrane of the autophagosome while ATG8a appears to be mainly associated with the inner membrane. In summary, our data show that ATG8a and ATG8b have distinct functions and are involved in canonical as well as non-canonical autophagy. The data further suggest that ATG8b predominantly acts as adapter for the autophagy machinery at the outer and ATG8a as cargo receptor at the inner membrane of the autophagosome. Copyright © 2017 The Authors. Published by Elsevier GmbH.. All rights reserved.

Absence of Wip1 partially rescues Atm deficiency phenotypes in mice

PubMed Central

Darlington, Yolanda; Nguyen, Thuy-Ai; Moon, Sung-Hwan; Herron, Alan; Rao, Pulivarthi; Zhu, Chengming; Lu, Xiongbin; Donehower, Lawrence A.

2011-01-01

Wildtype p53-Induced Phosphatase 1 (WIP1) is a serine/threonine phosphatase that dephosphorylates proteins in the ataxia telangiectasia mutated (ATM)-initiated DNA damage response pathway. WIP1 may play a homeostatic role in ATM signaling by returning the cell to a normal pre-stress state following completion of DNA repair. To better understand the effects of WIP1 on ATM signaling, we crossed Atm-deficient mice to Wip1-deficient mice and characterized phenotypes of the double knockout progeny. We hypothesized that the absence of Wip1 might rescue Atm deficiency phenotypes. Atm null mice, like ATM-deficient humans with the inherited syndrome ataxia telangiectasia, exhibit radiation sensitivity, fertility defects, and are T-cell lymphoma prone. Most double knockout mice were largely protected from lymphoma development and had a greatly extended lifespan compared to Atm null mice. Double knockout mice had increased p53 and H2AX phosphorylation and p21 expression compared to their Atm null counterparts, indicating enhanced p53 and DNA damage responses. Additionally, double knockout splenocytes displayed reduced chromosomal instability compared to Atm null mice. Finally, doubly null mice were partially rescued from infertility defects observed in Atm null mice. These results indicate that inhibition of WIP1 may represent a useful strategy for cancer treatment in general and A-T patients in particular. PMID:21765465

Earlier onset of motor deficits in mice with double mutations in Dyt1 and Sgce.

PubMed

Yokoi, Fumiaki; Yang, Guang; Li, Jindong; DeAndrade, Mark P; Zhou, Tong; Li, Yuqing

2010-10-01

DYT1 early-onset generalized torsion dystonia is an inherited movement disorder caused by mutations in DYT1 coding for torsinA with ∼30% penetrance. Most of the DYT1 dystonia patients exhibit symptoms during childhood and adolescence. On the other hand, DYT1 mutation carriers without symptoms during these periods mostly do not exhibit symptoms later in their life. Little is known about what controls the timing of the onset, a critical issue for DYT1 mutation carriers. DYT11 myoclonus-dystonia is caused by mutations in SGCE coding for ε-sarcoglycan. Two dystonia patients from a single family with double mutations in DYT1 and SGCE exhibited more severe symptoms. A recent study suggested that torsinA contributes to the quality control of ε-sarcoglycan. Here, we derived mice carrying mutations in both Dyt1 and Sgce and found that these double mutant mice showed earlier onset of motor deficits in beam-walking test. A novel monoclonal antibody against mouse ε-sarcoglycan was developed by using Sgce knock-out mice to avoid the immune tolerance. Western blot analysis suggested that functional deficits of torsinA and ε-sarcoglycan may independently cause motor deficits. Examining additional mutations in other dystonia genes may be beneficial to predict the onset in DYT1 mutation carriers.

Discovery of rice essential genes by characterizing a CRISPR-edited mutation of closely related rice MAP kinase genes.

PubMed

Minkenberg, Bastian; Xie, Kabin; Yang, Yinong

2017-02-01

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) system depends on a guide RNA (gRNA) to specify its target. By efficiently co-expressing multiple gRNAs that target different genomic sites, the polycistronic tRNA-gRNA gene (PTG) strategy enables multiplex gene editing in the family of closely related mitogen-activated protein kinase (MPK) genes in Oryza sativa (rice). In this study, we identified MPK1 and MPK6 (Arabidopsis AtMPK6 and AtMPK4 orthologs, respectively) as essential genes for rice development by finding the preservation of MPK functional alleles and normal phenotypes in CRISPR-edited mutants. The true knock-out mutants of MPK1 were severely dwarfed and sterile, and homozygous mpk1 seeds from heterozygous parents were defective in embryo development. By contrast, heterozygous mpk6 mutant plants completely failed to produce homozygous mpk6 seeds. In addition, the functional importance of specific MPK features could be evaluated by characterizing CRISPR-induced allelic variation in the conserved kinase domain of MPK6. By simultaneously targeting between two and eight genomic sites in the closely related MPK genes, we demonstrated 45-86% frequency of biallelic mutations and the successful creation of single, double and quadruple gene mutants. Indels and fragment deletion were both stably inherited to the next generations, and transgene-free mutants of rice MPK genes were readily obtained via genetic segregation, thereby eliminating any positional effects of transgene insertions. Taken together, our study reveals the essentiality of MPK1 and MPK6 in rice development, and enables the functional discovery of previously inaccessible genes or domains with phenotypes masked by lethality or redundancy. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

Exacerbated febrile responses to LPS, but not turpentine, in TNF double receptor-knockout mice.

PubMed

Leon, L R; Kozak, W; Peschon, J; Kluger, M J

1997-02-01

We examined the effects of injections of systemic [lipopolysaccharide (LPS), 2.5 mg/kg or 50 pg/kg ip] or local (turpentine, 100 microl sc) inflammatory stimuli on fever, motor activity, body weight, and food intake in tumor necrosis factor (TNF) double receptor (TNFR)-knockout mice. A high dose of LPS resulted in exacerbated fevers in TNFR-knockout mice compared with wild-type mice for the early phase of fever (3-15 h); the late phase of fever (16-24 h) and fevers to a low dose of LPS were similar in both groups. Motor activity, body weight, and food intake were similarly reduced in both groups of mice after LPS administration. In response to turpentine, TNFR-knockout and wild-type mice developed virtually identical responses to all variables monitored. These results suggest that 1) TNF modulates fevers to LPS dose dependently, 2) TNF does not modulate fevers to a subcutaneous injection of turpentine, and 3) knockout mice may develop cytokine redundancy in the regulation of the acute phase response to intraperitoneally injected LPS or subcutaneously injected turpentine.

Role of the glyoxylate pathway in acetic acid production by Acetobacter aceti.

PubMed

Sakurai, Kenta; Yamazaki, Shoko; Ishii, Masaharu; Igarashi, Yasuo; Arai, Hiroyuki

2013-01-01

Wild-type Acetobacter aceti NBRC 14818 possesses genes encoding isocitrate lyase (aceA) and malate synthase (glcB), which constitute the glyoxylate pathway. In contrast, several acetic acid bacteria that are utilized for vinegar production lack these genes. Here, an aceA-glcB knockout mutant of NBRC 14818 was constructed and used for investigating the role of the glyoxylate pathway in acetate productivity. In medium containing ethanol as a carbon source, the mutant grew normally during ethanol oxidation to acetate, but exhibited slower growth than that of the wild-type strain as the accumulated acetate was oxidized. The mutant grew similarly to that of the wild-type strain in medium containing glucose as a carbon source, indicating that the glyoxylate pathway was not necessary for glucose utilization. However, in medium containing both ethanol and glucose, the mutant exhibited significantly poorer growth and lower glucose consumption compared to the wild-type strain. Notably, the mutant oxidized ethanol nearly stoichiometrically to acetate, which was retained in the medium for a longer period of time than the acetate produced by wild-type strain. The features of the aceA-glcB knockout mutant revealed here indicate that the lack of the glyoxylate pathway is advantageous for industrial vinegar production by A. aceti. Copyright © 2012 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Knock-out of a mitochondrial sirtuin protects neurons from degeneration in Caenorhabditis elegans

PubMed Central

Sangaletti, Rachele; Grant, Jeff; Della-Morte, David

2017-01-01

Sirtuins are NAD⁺-dependent deacetylases, lipoamidases, and ADP-ribosyltransferases that link cellular metabolism to multiple intracellular pathways that influence processes as diverse as cell survival, longevity, and cancer growth. Sirtuins influence the extent of neuronal death in stroke. However, different sirtuins appear to have opposite roles in neuronal protection. In Caenorhabditis elegans, we found that knock-out of mitochondrial sirtuin sir-2.3, homologous to mammalian SIRT4, is protective in both chemical ischemia and hyperactive channel induced necrosis. Furthermore, the protective effect of sir-2.3 knock-out is enhanced by block of glycolysis and eliminated by a null mutation in daf-16/FOXO transcription factor, supporting the involvement of the insulin/IGF pathway. However, data in Caenorhabditis elegans cell culture suggest that the effects of sir-2.3 knock-out act downstream of the DAF-2/IGF-1 receptor. Analysis of ROS in sir-2.3 knock-out reveals that ROS become elevated in this mutant under ischemic conditions in dietary deprivation (DD), but to a lesser extent than in wild type, suggesting more robust activation of a ROS scavenging system in this mutant in the absence of food. This work suggests a deleterious role of SIRT4 during ischemic processes in mammals that must be further investigated and reveals a novel pathway that can be targeted for the design of therapies aimed at protecting neurons from death in ischemic conditions. PMID:28820880

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.

Amyloid precursor protein modulates Nav1.6 sodium channel currents through a Go-coupled JNK pathway.

PubMed

Li, Shao; Wang, Xi; Ma, Quan-Hong; Yang, Wu-Lin; Zhang, Xiao-Gang; Dawe, Gavin S; Xiao, Zhi-Cheng

2016-12-23

Amyloid precursor protein (APP), commonly associated with Alzheimer's disease, also marks axonal degeneration. In the recent studies, we demonstrated that APP aggregated at nodes of Ranvier (NORs) in myelinated central nervous system (CNS) axons and interacted with Nav1.6. However, the physiological function of APP remains unknown. In this study, we described reduced sodium current densities in APP knockout hippocampal neurons. Coexpression of APP or its intracellular domains containing a VTPEER motif with Na v 1.6 sodium channels in Xenopus oocytes resulted in an increase in peak sodium currents, which was enhanced by constitutively active Go mutant and blocked by a dominant negative mutant. JNK and CDK5 inhibitor attenuated increases in Nav1.6 sodium currents induced by overexpression of APP. Nav1.6 sodium currents were increased by APPT668E (mutant Thr to Glu) and decreased by T668A (mutant Thr to ALa) mutant, respectively. The cell surface expression of Nav1.6 sodium channels in the white matter of spinal cord and the spinal conduction velocity is decreased in APP, p35 and JNK3 knockout mice. Therefore, APP modulates Nav1.6 sodium channels through a Go-coupled JNK pathway, which is dependent on phosphorylation of APP at Thr668.

Amyloid precursor protein modulates Nav1.6 sodium channel currents through a Go-coupled JNK pathway

PubMed Central

Li, Shao; Wang, Xi; Ma, Quan-Hong; Yang, Wu-lin; Zhang, Xiao-Gang; Dawe, Gavin S.; Xiao, Zhi-Cheng

2016-01-01

Amyloid precursor protein (APP), commonly associated with Alzheimer’s disease, also marks axonal degeneration. In the recent studies, we demonstrated that APP aggregated at nodes of Ranvier (NORs) in myelinated central nervous system (CNS) axons and interacted with Nav1.6. However, the physiological function of APP remains unknown. In this study, we described reduced sodium current densities in APP knockout hippocampal neurons. Coexpression of APP or its intracellular domains containing a VTPEER motif with Nav1.6 sodium channels in Xenopus oocytes resulted in an increase in peak sodium currents, which was enhanced by constitutively active Go mutant and blocked by a dominant negative mutant. JNK and CDK5 inhibitor attenuated increases in Nav1.6 sodium currents induced by overexpression of APP. Nav1.6 sodium currents were increased by APPT668E (mutant Thr to Glu) and decreased by T668A (mutant Thr to ALa) mutant, respectively. The cell surface expression of Nav1.6 sodium channels in the white matter of spinal cord and the spinal conduction velocity is decreased in APP, p35 and JNK3 knockout mice. Therefore, APP modulates Nav1.6 sodium channels through a Go-coupled JNK pathway, which is dependent on phosphorylation of APP at Thr668. PMID:28008944

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.

ATF6α/β-mediated adjustment of ER chaperone levels is essential for development of the notochord in medaka fish

PubMed Central

Ishikawa, Tokiro; Okada, Tetsuya; Ishikawa-Fujiwara, Tomoko; Todo, Takeshi; Kamei, Yasuhiro; Shigenobu, Shuji; Tanaka, Minoru; Saito, Taro L.; Yoshimura, Jun; Morishita, Shinichi; Toyoda, Atsushi; Sakaki, Yoshiyuki; Taniguchi, Yoshihito; Takeda, Shunichi; Mori, Kazutoshi

2013-01-01

ATF6α and ATF6β are membrane-bound transcription factors activated by regulated intramembrane proteolysis in response to endoplasmic reticulum (ER) stress to induce various ER quality control proteins. ATF6α- and ATF6β single-knockout mice develop normally, but ATF6α/β double knockout causes embryonic lethality, the reason for which is unknown. Here we show in medaka fish that ATF6α is primarily responsible for transcriptional induction of the major ER chaperone BiP and that ATF6α/β double knockout, but not ATF6α- or ATF6β single knockout, causes embryonic lethality, as in mice. Analyses of ER stress reporters reveal that ER stress occurs physiologically during medaka early embryonic development, particularly in the brain, otic vesicle, and notochord, resulting in ATF6α- and ATF6β-mediated induction of BiP, and that knockdown of the α1 chain of type VIII collagen reduces such ER stress. The absence of transcriptional induction of several ER chaperones in ATF6α/β double knockout causes more profound ER stress and impaired notochord development, which is partially rescued by overexpression of BiP. Thus ATF6α/β-mediated adjustment of chaperone levels to increased demands in the ER is essential for development of the notochord, which synthesizes and secretes large amounts of extracellular matrix proteins to serve as the body axis before formation of the vertebra. PMID:23447699

ATF6α/β-mediated adjustment of ER chaperone levels is essential for development of the notochord in medaka fish.

PubMed

Ishikawa, Tokiro; Okada, Tetsuya; Ishikawa-Fujiwara, Tomoko; Todo, Takeshi; Kamei, Yasuhiro; Shigenobu, Shuji; Tanaka, Minoru; Saito, Taro L; Yoshimura, Jun; Morishita, Shinichi; Toyoda, Atsushi; Sakaki, Yoshiyuki; Taniguchi, Yoshihito; Takeda, Shunichi; Mori, Kazutoshi

2013-05-01

ATF6α and ATF6β are membrane-bound transcription factors activated by regulated intramembrane proteolysis in response to endoplasmic reticulum (ER) stress to induce various ER quality control proteins. ATF6α- and ATF6β single-knockout mice develop normally, but ATF6α/β double knockout causes embryonic lethality, the reason for which is unknown. Here we show in medaka fish that ATF6α is primarily responsible for transcriptional induction of the major ER chaperone BiP and that ATF6α/β double knockout, but not ATF6α- or ATF6β single knockout, causes embryonic lethality, as in mice. Analyses of ER stress reporters reveal that ER stress occurs physiologically during medaka early embryonic development, particularly in the brain, otic vesicle, and notochord, resulting in ATF6α- and ATF6β-mediated induction of BiP, and that knockdown of the α1 chain of type VIII collagen reduces such ER stress. The absence of transcriptional induction of several ER chaperones in ATF6α/β double knockout causes more profound ER stress and impaired notochord development, which is partially rescued by overexpression of BiP. Thus ATF6α/β-mediated adjustment of chaperone levels to increased demands in the ER is essential for development of the notochord, which synthesizes and secretes large amounts of extracellular matrix proteins to serve as the body axis before formation of the vertebra.

Absence of Tec Family Kinases Interleukin-2 Inducible T cell Kinase (Itk) and Bruton's Tyrosine Kinase (Btk) Severely Impairs FcϵRI-dependent Mast Cell Responses*

PubMed Central

Iyer, Archana S.; Morales, J. Luis; Huang, Weishan; Ojo, Folake; Ning, Gang; Wills, Elizabeth; Baines, Joel D.; August, Avery

2011-01-01

Mast cells are critical effector cells in the pathophysiology of allergic asthma and other IgE-mediated diseases. The Tec family of tyrosine kinases Itk and Btk serve as critical signal amplifiers downstream of antigen receptors. Although both kinases are expressed and activated in mast cells following FcϵRI stimulation, their individual contributions are not clear. To determine whether these kinases play unique and/or complementary roles in FcϵRI signaling and mast cell function, we generated Itk and Btk double knock-out mice. Analyses of these mice show decreased mast cell granularity and impaired passive systemic anaphylaxis responses. This impaired response is accompanied by a significant elevation in serum IgE in Itk/Btk double knock-out mice. In vitro analyses of bone marrow-derived mast cells (BMMCs) indicated that Itk/Btk double knock-out BMMCs are defective in degranulation and cytokine secretion responses downstream to FcϵRI activation. These responses were accompanied by a significant reduction in PLCγ2 phosphorylation and severely impaired calcium responses in these cells. This defect also results in altered NFAT1 nuclear localization in double knock-out BMMCs. Network analysis suggests that although they may share substrates, Itk plays both positive and negative roles, while Btk primarily plays a positive role in mast cell FcϵRI-induced cytokine secretion. PMID:21212279

Random transposon mutagenesis of the Saccharopolyspora erythraea genome reveals additional genes influencing erythromycin biosynthesis

PubMed Central

Fedashchin, Andrij; Cernota, William H.; Gonzalez, Melissa C.; Leach, Benjamin I.; Kwan, Noelle; Wesley, Roy K.; Weber, J. Mark

2015-01-01

A single cycle of strain improvement was performed in Saccharopolyspora erythraea mutB and 15 genotypes influencing erythromycin production were found. Genotypes generated by transposon mutagenesis appeared in the screen at a frequency of ∼3%. Mutations affecting central metabolism and regulatory genes were found, as well as hydrolases, peptidases, glycosyl transferases and unknown genes. Only one mutant retained high erythromycin production when scaled-up from micro-agar plug fermentations to shake flasks. This mutant had a knockout of the cwh1 gene (SACE_1598), encoding a cell-wall-associated hydrolase. The cwh1 knockout produced visible growth and morphological defects on solid medium. This study demonstrated that random transposon mutagenesis uncovers strain improvement-related genes potentially useful for strain engineering. PMID:26468041

Genome-wide essential gene identification in Streptococcus sanguinis

PubMed Central

Xu, Ping; Ge, Xiuchun; Chen, Lei; Wang, Xiaojing; Dou, Yuetan; Xu, Jerry Z.; Patel, Jenishkumar R.; Stone, Victoria; Trinh, My; Evans, Karra; Kitten, Todd; Bonchev, Danail; Buck, Gregory A.

2011-01-01

A clear perception of gene essentiality in bacterial pathogens is pivotal for identifying drug targets to combat emergence of new pathogens and antibiotic-resistant bacteria, for synthetic biology, and for understanding the origins of life. We have constructed a comprehensive set of deletion mutants and systematically identified a clearly defined set of essential genes for Streptococcus sanguinis. Our results were confirmed by growing S. sanguinis in minimal medium and by double-knockout of paralogous or isozyme genes. Careful examination revealed that these essential genes were associated with only three basic categories of biological functions: maintenance of the cell envelope, energy production, and processing of genetic information. Our finding was subsequently validated in two other pathogenic streptococcal species, Streptococcus pneumoniae and Streptococcus mutans and in two other gram-positive pathogens, Bacillus subtilis and Staphylococcus aureus. Our analysis has thus led to a simplified model that permits reliable prediction of gene essentiality. PMID:22355642

β-Carbonic Anhydrases Play a Role in Fruiting Body Development and Ascospore Germination in the Filamentous Fungus Sordaria macrospora

PubMed Central

Elleuche, Skander; Pöggeler, Stefanie

2009-01-01

Carbon dioxide (CO2) is among the most important gases for all organisms. Its reversible interconversion to bicarbonate (HCO3 −) reaches equilibrium spontaneously, but slowly, and can be accelerated by a ubiquitous group of enzymes called carbonic anhydrases (CAs). These enzymes are grouped by their distinct structural features into α-, β-, γ-, δ- and ζ-classes. While physiological functions of mammalian, prokaryotic, plant and algal CAs have been extensively studied over the past years, the role of β-CAs in yeasts and the human pathogen Cryptococcus neoformans has been elucidated only recently, and the function of CAs in multicellular filamentous ascomycetes is mostly unknown. To assess the role of CAs in the development of filamentous ascomycetes, the function of three genes, cas1, cas2 and cas3 (carbonic anhydrase of Sordaria) encoding β-class carbonic anhydrases was characterized in the filamentous ascomycetous fungus Sordaria macrospora. Fluorescence microscopy was used to determine the localization of GFP- and DsRED-tagged CAs. While CAS1 and CAS3 are cytoplasmic enzymes, CAS2 is localized to the mitochondria. To assess the function of the three isoenzymes, we generated knock-out strains for all three cas genes (Δcas1, Δcas2, and Δcas3) as well as all combinations of double mutants. No effect on vegetative growth, fruiting-body and ascospore development was seen in the single mutant strains lacking cas1 or cas3, while single mutant Δcas2 was affected in vegetative growth, fruiting-body development and ascospore germination, and the double mutant strain Δcas1/2 was completely sterile. Defects caused by the lack of cas2 could be partially complemented by elevated CO2 levels or overexpression of cas1, cas3, or a non-mitochondrial cas2 variant. The results suggest that CAs are required for sexual reproduction in filamentous ascomycetes and that the multiplicity of isoforms results in redundancy of specific and non-specific functions. PMID:19365544

Independent Origin of Plasmodium falciparum Antifolate Super-Resistance, Uganda, Tanzania, and Ethiopia

PubMed Central

Alifrangis, Michael; Schousboe, Mette L.; Ishengoma, Deus; Lusingu, John; Pota, Hirva; Kavishe, Reginald A.; Pearce, Richard; Ord, Rosalynn; Lynch, Caroline; Dejene, Seyoum; Cox, Jonathan; Rwakimari, John; Minja, Daniel T.R.; Lemnge, Martha M.; Roper, Cally

2014-01-01

Super-resistant Plasmodium falciparum threatens the effectiveness of sulfadoxine–pyrimethamine in intermittent preventive treatment for malaria during pregnancy. It is characterized by the A581G Pfdhps mutation on a background of the double-mutant Pfdhps and the triple-mutant Pfdhfr. Using samples collected during 2004–2008, we investigated the evolutionary origin of the A581G mutation by characterizing microsatellite diversity flanking Pfdhps triple-mutant (437G+540E+581G) alleles from 3 locations in eastern Africa and comparing it with double-mutant (437G+540E) alleles from the same area. In Ethiopia, both alleles derived from 1 lineage that was distinct from those in Uganda and Tanzania. Uganda and Tanzania triple mutants derived from the previously characterized southeastern Africa double-mutant lineage. The A581G mutation has occurred multiple times on local Pfdhps double-mutant backgrounds; however, a novel microsatellite allele incorporated into the Tanzania lineage since 2004 illustrates the local expansion of emergent triple-mutant lineages. PMID:25061906

A knock-in/knock-out mouse model of HSPB8-associated distal hereditary motor neuropathy and myopathy reveals toxic gain-of-function of mutant Hspb8.

PubMed

Bouhy, Delphine; Juneja, Manisha; Katona, Istvan; Holmgren, Anne; Asselbergh, Bob; De Winter, Vicky; Hochepied, Tino; Goossens, Steven; Haigh, Jody J; Libert, Claude; Ceuterick-de Groote, Chantal; Irobi, Joy; Weis, Joachim; Timmerman, Vincent

2018-01-01

Mutations in the small heat shock protein B8 gene (HSPB8/HSP22) have been associated with distal hereditary motor neuropathy, Charcot-Marie-Tooth disease, and recently distal myopathy. It is so far not clear how mutant HSPB8 induces the neuronal and muscular phenotypes and if a common pathogenesis lies behind these diseases. Growing evidence points towards a role of HSPB8 in chaperone-associated autophagy, which has been shown to be a determinant for the clearance of poly-glutamine aggregates in neurodegenerative diseases but also for the maintenance of skeletal muscle myofibrils. To test this hypothesis and better dissect the pathomechanism of mutant HSPB8, we generated a new transgenic mouse model leading to the expression of the mutant protein (knock-in lines) or the loss-of-function (functional knock-out lines) of the endogenous protein Hspb8. While the homozygous knock-in mice developed motor deficits associated with degeneration of peripheral nerves and severe muscle atrophy corroborating patient data, homozygous knock-out mice had locomotor performances equivalent to those of wild-type animals. The distal skeletal muscles of the post-symptomatic homozygous knock-in displayed Z-disk disorganisation, granulofilamentous material accumulation along with Hspb8, αB-crystallin (HSPB5/CRYAB), and desmin aggregates. The presence of the aggregates correlated with reduced markers of effective autophagy. The sciatic nerve of the homozygous knock-in mice was characterized by low autophagy potential in pre-symptomatic and Hspb8 aggregates in post-symptomatic animals. On the other hand, the sciatic nerve of the homozygous knock-out mice presented a normal morphology and their distal muscle displayed accumulation of abnormal mitochondria but intact myofiber and Z-line organisation. Our data, therefore, suggest that toxic gain-of-function of mutant Hspb8 aggregates is a major contributor to the peripheral neuropathy and the myopathy. In addition, mutant Hspb8 induces impairments in autophagy that may aggravate the phenotype.

Genetic deletion of CB1 receptors improves non-associative learning.

PubMed

Degroot, Aldemar; Salhoff, Craig; Davis, Richard J; Nomikos, George G

2005-07-01

Habituation (a form of non-associative learning) was measured by assessing locomotion in novel activity monitors in CB1 receptor knockout mice and juxtaposed to habituation measured in muscarinic M2, M4, and double M2/M4 receptor knockout mice. M2 and M2/M4, but not M4, receptor knockout mice appeared to have an impaired ability to habituate, whereas CB1 receptor knockout mice showed enhanced habituation compared to wild-type animals. We conclude that CB1 receptor gene invalidation improves habituation tentatively through an increase in cholinergic neurotransmission.

Deletion of the Braun Lipoprotein-Encoding Gene and Altering the Function of Lipopolysaccharide Attenuate the Plague Bacterium

PubMed Central

Sha, Jian; Kirtley, Michelle L.; van Lier, Christina J.; Wang, Shaofei; Erova, Tatiana E.; Kozlova, Elena V.; Cao, Anthony; Cong, Yingzi; Fitts, Eric C.; Rosenzweig, Jason A.

2013-01-01

Braun (murein) lipoprotein (Lpp) and lipopolysaccharide (LPS) are major components of the outer membranes of Enterobacteriaceae family members that are capable of triggering inflammatory immune responses by activating Toll-like receptors 2 and 4, respectively. Expanding on earlier studies that demonstrated a role played by Lpp in Yersinia pestis virulence in mouse models of bubonic and pneumonic plague, we characterized an msbB in-frame deletion mutant incapable of producing an acyltransferase that is responsible for the addition of lauric acid to the lipid A moiety of LPS, as well as a Δlpp ΔmsbB double mutant of the highly virulent Y. pestis CO92 strain. Although the ΔmsbB single mutant was minimally attenuated, the Δlpp single mutant and the Δlpp ΔmsbB double mutant were significantly more attenuated than the isogenic wild-type (WT) bacterium in bubonic and pneumonic animal models (mouse and rat) of plague. These data correlated with greatly reduced survivability of the aforementioned mutants in murine macrophages. Furthermore, the Δlpp ΔmsbB double mutant was grossly compromised in its ability to disseminate to distal organs in mice and in evoking cytokines/chemokines in infected animal tissues. Importantly, mice that survived challenge with the Δlpp ΔmsbB double mutant, but not the Δlpp or ΔmsbB single mutant, in a pneumonic plague model were significantly protected against a subsequent lethal WT CO92 rechallenge. These data were substantiated by the fact that the Δlpp ΔmsbB double mutant maintained an immunogenicity comparable to that of the WT strain and induced long-lasting T-cell responses against heat-killed WT CO92 antigens. Taken together, the data indicate that deletion of the msbB gene augmented the attenuation of the Δlpp mutant by crippling the spread of the double mutant to the peripheral organs of animals and by inducing cytokine/chemokine responses. Thus, the Δlpp ΔmsbB double mutant could provide a new live-attenuated background vaccine candidate strain, and this should be explored in the future. PMID:23275092

Long G2 accumulates recombination intermediates and disturbs chromosome segregation at dysfunction telomere in Schizosaccharomyces pombe

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

Habib, Ahmed G.K.; Masuda, Kenta; Yukawa, Masashi

Protection of telomere (Pot1) is a single-stranded telomere binding protein which is essential for chromosome ends protection. Fission yeast Rqh1 is a member of RecQ helicases family which has essential roles in the maintenance of genomic stability and regulation of homologous recombination. Double mutant between fission yeast pot1Δ and rqh1 helicase dead (rqh1-hd) maintains telomere by homologous recombination. In pot1Δ rqh1-hd double mutant, recombination intermediates accumulate near telomere which disturb chromosome segregation and make cells sensitive to microtubule inhibitors thiabendazole (TBZ). Deletion of chk1{sup +} or mutation of its kinase domain shortens the G2 of pot1Δ rqh1-hd double mutant andmore » suppresses both the accumulation of recombination intermediates and the TBZ sensitivity of that double mutant. In this study, we asked whether the long G2 is the reason for the TBZ sensitivity of pot1Δ rqh1-hd double mutant. We found that shortening the G2 of pot1Δ rqh1-hd double mutant by additional mutations of wee1 and mik1 or gain of function mutation of Cdc2 suppresses both the accumulation of recombination intermediates and the TBZ sensitivity of pot1Δ rqh1-hd double mutant. Our results suggest that long G2 of pot1Δ rqh1-hd double mutant may allow time for the accumulation of recombination intermediates which disturb chromosome segregation and make cells sensitive to TBZ. - Ηighlights: • We show link between long G2 and accumulation of toxic recombination intermediates. • Accumulation of recombination intermediates at telomere results in TBZ sensitivity. • Activation of DNA damage checkpoint worsens cells' viability in presence of TBZ.« less

Heat shock factor-1 knockout enhances cholesterol 7α-hydroxylase (CYP7A1) and multidrug transporter (MDR1) gene expressions to attenuate atherosclerosis

PubMed Central

Krishnamurthy, Karthikeyan; Glaser, Shannon; Alpini, Gianfranco D.; Cardounel, Arturo J.; Liu, Zhenguo; Ilangovan, Govindasamy

2016-01-01

Aims Stress response, in terms of activation of stress factors, is known to cause obesity and coronary heart disease such as atherosclerosis in human. However, the underlying mechanism(s) of these pathways are not known. Here, we investigated the effect of heat shock factor-1 (HSF-1) on atherosclerosis. Methods and results HSF-1 and low-density lipoprotein receptor (LDLr) double knockout (HSF-1−/−/LDLr−/−) and LDLr knockout (LDLr−/−) mice were fed with atherogenic western diet (WD) for 12 weeks. WD-induced weight gain and atherosclerotic lesion in aortic arch and carotid regions were reduced in HSF-1−/−/LDLr−/− mice, compared with LDLr−/− mice. Also, repression of PPAR-γ2 and AMPKα expression in adipose tissue, low hepatic steatosis, and lessened plasma adiponectins and lipoproteins were observed. In HSF-1−/−/LDLr−/− liver, higher cholesterol 7α-hydroxylase (CYP7A1) and multidrug transporter [MDR1/P-glycoprotein (P-gp)] gene expressions were observed, consistent with higher bile acid transport and larger hepatic bile ducts. Luciferase reporter gene assays with wild-type CYP7A1 and MDR1 promoters showed lesser luminescence than with mutant promoters (HSF-1 binding site deleted), indicating that HSF-1 binding is repressive of CYP7A1 and MDR1 gene expressions. Conclusion HSF-1 ablation not only eliminates heat shock response, but it also transcriptionally up-regulates CYP7A1 and MDR1/P-gp axis in WD-diet fed HSF-1−/−/LDLr−/− mice to reduce atherosclerosis. PMID:27131506

High affinity kainate receptor subunits are necessary for ionotropic but not metabotropic signaling

PubMed Central

Fernandes, Herman B.; Catches, Justin S.; Petralia, Ronald S.; Copits, Bryan A.; Xu, Jian; Russell, Theron A.; Swanson, Geoffrey T.; Contractor, Anis

2009-01-01

Summary Kainate receptors are atypical members of the glutamate receptor family which are able to signal through both ionotropic and metabotropic pathways. Of the five individual kainate receptor subunits the high-affinity subunits, GluK4 (KA1) and GluK5 (KA2), are unique in that they do not form functional homomeric receptors in recombinant expression systems, but combine with the primary subunits GluK1-3 (GluR5-7) to form heteromeric assemblies. Here we generated a GluK4 mutant mouse by disrupting the Grik4 gene locus. We found that loss of the GluK4 subunit leads to a significant reduction in synaptic kainate receptor currents. Moreover, ablation of both high-affinity subunits in GluK4/GluK5 double knockout mice leads to a complete loss of pre- and postsynaptic ionotropic function of synaptic kainate receptors. The principal subunits remain at the synaptic plasma membrane, but are distributed away from postsynaptic densities and presynaptic active zones. There is also an alteration in the properties of the remaining kainate receptors, as kainic acid application fails to elicit responses in GluK4/GluK5 knockout neurons. Despite the lack of detectable ionotropic synaptic receptors, the kainate receptor-mediated inhibition of the slow afterhyperpolarization current (IsAHP), which is dependent on metabotropic pathways, was intact in GluK4/GluK5 knockout mice. These results uncover a previously unknown critical role for the high-affinity kainate receptor subunits as obligatory components of ionotropic kainate receptor function, and further, demonstrate that kainate receptor participation in metabotropic signaling pathways does not require their classic role as ion channels. PMID:19778510

Semaphorin 6A knockout mice display abnormalities across ethologically-based topographies of exploration and in motor learning.

PubMed

Håkansson, Kerstin; Runker, Annette E; O'Sullivan, Gerard J; Mitchell, Kevin J; Waddington, John L; O'Tuathaigh, Colm M P

2017-02-22

Semaphorins are secreted or membrane-bound proteins implicated in neurodevelopmental processes of axon guidance and cell migration. Exploratory behaviour and motor learning was examined ethologically in Semaphorin 6A (Sema6A) mutant mice. The ethogram of initial exploration in Sema6A knockout mice was characterised by increased rearing to wall with decreased sifting; over subsequent habituation, locomotion, sniffing and rearing to wall were increased, with reduced habituation of rearing seated. Rotarod analysis indicated delayed motor learning in Sema6A heterozygous mutants. Disruption to the axonal guidance and cell migration processes regulated by Sema6A is associated with topographically specific disruption to fundamental aspects of behaviour, namely the ethogram of initial exploration and subsequent habituation to the environment, and motor learning. Copyright © 2017 Elsevier B.V. All rights reserved.

An episomal vector-based CRISPR/Cas9 system for highly efficient gene knockout in human pluripotent stem cells.

PubMed

Xie, Yifang; Wang, Daqi; Lan, Feng; Wei, Gang; Ni, Ting; Chai, Renjie; Liu, Dong; Hu, Shijun; Li, Mingqing; Li, Dajin; Wang, Hongyan; Wang, Yongming

2017-05-24

Human pluripotent stem cells (hPSCs) represent a unique opportunity for understanding the molecular mechanisms underlying complex traits and diseases. CRISPR/Cas9 is a powerful tool to introduce genetic mutations into the hPSCs for loss-of-function studies. Here, we developed an episomal vector-based CRISPR/Cas9 system, which we called epiCRISPR, for highly efficient gene knockout in hPSCs. The epiCRISPR system enables generation of up to 100% Insertion/Deletion (indel) rates. In addition, the epiCRISPR system enables efficient double-gene knockout and genomic deletion. To minimize off-target cleavage, we combined the episomal vector technology with double-nicking strategy and recent developed high fidelity Cas9. Thus the epiCRISPR system offers a highly efficient platform for genetic analysis in hPSCs.

Construction and phenotypic characterization of M68, an RruI quorum sensing knockout mutant of the photosynthetic alphaproteobacterium Rhodospirillum rubrum.

PubMed

Condori, Sandra; Atkinson, Steve; Leys, Natalie; Wattiez, Ruddy; Mastroleo, Felice

2016-06-01

Many bacterial species communicate using a complex system known as quorum sensing (QS) in which gene expression is controlled in response to cell density. In this study an N-acylhomoserine lactone (AHL) synthase (Rru_A3396) knockout mutant (M68) of Rhodospirillum rubrum S1H (WT) was constructed and characterized phenotypically under light anaerobic conditions. Results showed that R. rubrum WT produces unsubstituted, 3-OH and 3-oxo-substituted AHLs with acyl chains ranging from 4 to 14 carbons, with 3-OH-C8 being the most abundant. Growth, pigment content and swimming motility were found to be under the control of this LuxI-type QS system. In addition, cultivation in a low shear environment put forward the aggregative phenotype of M68 and linked biofilm formation to QS in R. rubrum S1H. Interestingly, QS-mutant M68 continued to produce decreased levels of 3-OH-C8-HSL, probably due to the presence of an extra HdtS-type AHL synthase. Copyright © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Differential sensitivity of Pak5, Pak6, and Pak5/Pak6 double-knockout mice to the stimulant effects of amphetamine and exercise-induced alterations in body weight.

PubMed

Furnari, Melody A; Jobes, Michelle L; Nekrasova, Tanya; Minden, Audrey; Wagner, George C

2014-04-01

PAK5 and PAK6 are protein kinases highly expressed in the brain. Previously, we observed that Pak6 knockout mice gained significantly more weight during development than Pak5 knockout mice as well as wild-type controls and double-knockout mice lacking both Pak5 and Pak6. In this study, we assessed the effects of exercise on food intake and weight gain of these mice as well as their sensitivity to the stimulant effects of amphetamine. Mice of each genotype were placed in cages with free access to run wheel exercise or in cages without run wheels for a total of 74 days. Food and fluid intake as well as body weight of each mouse were measured on a weekly basis. Finally, mice were given a high dose of amphetamine and activity levels were observed immediately thereafter for 90 minutes. Brains and testes of mice were assayed for protein levels of the estrogen alpha and progesterone receptors. While run wheel mice consumed significantly more food, they weighed less than non-run wheel mice. In addition, although Pak6 knockout mice consumed the same amount of food as wild-type mice, they were significantly heavier regardless of run wheel condition. Pak5 knockout mice were found to be more active than other genotypes after amphetamine treatment. Finally, protein levels of the progesterone and estrogen alpha receptors were altered in brain and testes of the Pak6 knockout mice. Collectively, these data suggest that PAK6 play a role in weight gain unrelated to exercise and caloric intake and that Pak5 knockout mice are more sensitive to the stimulant effects of amphetamine.

Knockout Mice for Dyslexia Susceptibility Gene Homologs KIAA0319 and KIAA0319L have Unaffected Neuronal Migration but Display Abnormal Auditory Processing

PubMed Central

Guidi, Luiz G; Mattley, Jane; Martinez-Garay, Isabel; Monaco, Anthony P; Linden, Jennifer F; Velayos-Baeza, Antonio

2017-01-01

Abstract Developmental dyslexia is a neurodevelopmental disorder that affects reading ability caused by genetic and non-genetic factors. Amongst the susceptibility genes identified to date, KIAA0319 is a prime candidate. RNA-interference experiments in rats suggested its involvement in cortical migration but we could not confirm these findings in Kiaa0319-mutant mice. Given its homologous gene Kiaa0319L (AU040320) has also been proposed to play a role in neuronal migration, we interrogated whether absence of AU040320 alone or together with KIAA0319 affects migration in the developing brain. Analyses of AU040320 and double Kiaa0319;AU040320 knockouts (dKO) revealed no evidence for impaired cortical lamination, neuronal migration, neurogenesis or other anatomical abnormalities. However, dKO mice displayed an auditory deficit in a behavioral gap-in-noise detection task. In addition, recordings of click-evoked auditory brainstem responses revealed suprathreshold deficits in wave III amplitude in AU040320-KO mice, and more general deficits in dKOs. These findings suggest that absence of AU040320 disrupts firing and/or synchrony of activity in the auditory brainstem, while loss of both proteins might affect both peripheral and central auditory function. Overall, these results stand against the proposed role of KIAA0319 and AU040320 in neuronal migration and outline their relationship with deficits in the auditory system. PMID:29045729

The Multiple Functions of Common Microbial Carbon Polymers, Glycogen and PHB, during Stress Responses in the Non-Diazotrophic Cyanobacterium Synechocystis sp. PCC 6803.

PubMed

Damrow, Ramon; Maldener, Iris; Zilliges, Yvonne

2016-01-01

Classical microbial carbon polymers such as glycogen and polyhydroxybutyrate (PHB) have a crucial impact as both a sink and a reserve under macronutrient stress conditions. Most microbial species exclusively synthesize and degrade either glycogen or PHB. A few bacteria such as the phototrophic model organism Synechocystis sp. PCC 6803 surprisingly produce both physico-chemically different polymers under conditions of high C to N ratios. For the first time, the function and interrelation of both carbon polymers in non-diazotrophic cyanobacteria are analyzed in a comparative physiological study of single- and double-knockout mutants (ΔglgC; ΔphaC; ΔglgC/ΔphaC), respectively. Most of the observed phenotypes are explicitly related to the knockout of glycogen synthesis, highlighting the metabolic, energetic, and structural impact of this process whenever cells switch from an active, photosynthetic 'protein status' to a dormant 'glycogen status'. The carbon flux regulation into glycogen granules is apparently crucial for both phycobilisome degradation and thylakoid layer disassembly in the presence of light. In contrast, PHB synthesis is definitely not involved in this primary acclimation response. Moreover, the very weak interrelations between the two carbon-polymer syntheses indicate that the regulation and role of PHB synthesis in Synechocystis sp. PCC 6803 is different from glycogen synthesis.

Effect of sporophytic PIRL9 genotype on post-meiotic expression of the Arabidopsis pirl1;pirl9 mutant pollen phenotype.

PubMed

Forsthoefel, Nancy R; Vernon, Daniel M

2011-02-01

Plant intracellular ras-group-related leucine-rich repeat proteins (PIRLs) are a novel class of plant leucine-rich repeat (LRR) proteins structurally related to animal ras-group LRRs involved in cell signaling and gene regulation. Gene knockout analysis has shown that two members of the Arabidopsis thaliana PIRL gene family, PIRL1 and PIRL9, are redundant and essential for pollen development and viability: pirl1;pirl9 microspores produced by pirl1/PIRL1;pirl9 plants consistently abort just before pollen mitosis I. qrt1 tetrad analysis demonstrated that the genes become essential after meiosis, during anther stage 10. In this study, we characterized the phenotype of pirl1;pirl9 pollen produced by plants heterozygous for pirl9 (pirl1;pirl9/PIRL9). Alexander's staining, scanning electron microscopy, and fluorescence microscopy indicated that pirl1;pirl9 double mutants produced by pirl9 heterozygotes have a less severe phenotype and more variable morphology than pirl1;pirl9 pollen from pirl1/PIRL1;pirl9 plants. Mutant pollen underwent developmental arrest with variable timing, often progressing beyond pollen mitosis I and arresting at the binucleate stage. Thus, although the pirl1 and pirl9 mutations act post-meiosis, the timing and expressivity of the pirl1;pirl9 pollen phenotype depends on the pirl9 genotype of the parent plant. These results suggest a continued requirement for PIRL1 and PIRL9 beyond the initiation of pollen mitosis. Furthermore, they reveal a modest but novel sporophytic effect in which parent plant genotype influences a mutant phenotype expressed in the haploid generation.

The contribution of NADPH thioredoxin reductase C (NTRC) and sulfiredoxin to 2-Cys peroxiredoxin overoxidation in Arabidopsis thaliana chloroplasts.

PubMed

Puerto-Galán, Leonor; Pérez-Ruiz, Juan M; Guinea, Manuel; Cejudo, Francisco Javier

2015-05-01

Hydrogen peroxide is a harmful by-product of photosynthesis, which also has important signalling activity. Therefore, the level of hydrogen peroxide needs to be tightly controlled. Chloroplasts harbour different antioxidant systems including enzymes such as the 2-Cys peroxiredoxins (2-Cys Prxs). Under oxidizing conditions, 2-Cys Prxs are susceptible to inactivation by overoxidation of their peroxidatic cysteine, which is enzymatically reverted by sulfiredoxin (Srx). In chloroplasts, the redox status of 2-Cys Prxs is highly dependent on NADPH-thioredoxin reductase C (NTRC) and Srx; however, the relationship of these activities in determining the level of 2-Cys Prx overoxidation is unknown. Here we have addressed this question by a combination of genetic and biochemical approaches. An Arabidopsis thaliana double knockout mutant lacking NTRC and Srx shows a phenotype similar to the ntrc mutant, while the srx mutant resembles wild-type plants. The deficiency of NTRC causes reduced overoxidation of 2-Cys Prxs, whereas the deficiency of Srx has the opposite effect. Moreover, in vitro analyses show that the disulfide bond linking the resolving and peroxidatic cysteines protects the latter from overoxidation, thus explaining the dominant role of NTRC on the level of 2-Cys Prx overoxidation in vivo. The overoxidation of chloroplast 2-Cys Prxs shows no circadian oscillation, in agreement with the fact that neither the NTRC nor the SRX genes show circadian regulation of expression. Additionally, the low level of 2-Cys Prx overoxidation in the ntrc mutant is light dependent, suggesting that the redox status of 2-Cys Prxs in chloroplasts depends on light rather than the circadian clock. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Use of Heme Compounds as Iron Sources by Pathogenic Neisseriae Requires the Product of the hemO Gene

PubMed Central

Zhu, Wenming; Hunt, Desiree J.; Richardson, Anthony R.; Stojiljkovic, Igor

2000-01-01

Heme compounds are an important source of iron for neisseriae. We have identified a neisserial gene, hemO, that is essential for heme, hemoglobin (Hb), and haptoglobin-Hb utilization. The hemO gene is located 178 bp upstream of the hmbR Hb receptor gene in Neisseria meningitidis isolates. The product of the hemO gene is homologous to enzymes that degrade heme; 21% of its amino acid residues are identical, and 44% are similar, to those of the human heme oxygenase-1. DNA sequences homologous to hemO were ubiquitous in commensal and pathogenic neisseriae. HemO genetic knockout strains of Neisseria gonorrhoeae and N. meningitidis were unable to use any heme source, while the assimilation of transferrin-iron and iron-citrate complexes was unaffected. A phenotypic characterization of a conditional hemO mutant, constructed by inserting an isopropyl-β-d-thiogalactopyranoside (IPTG)-regulated promoter upstream of the ribosomal binding site of hemO, confirmed the indispensability of the HemO protein in heme utilization. The expression of HemO also protected N. meningitidis cells against heme toxicity. hemO mutants were still able to transport heme into the cell, since both heme and Hb could complement an N. meningitidis hemA hemO double mutant for growth. The expression of the HmbR receptor was reduced significantly by the inactivation of the hemO gene, suggesting that hemO and hmbR are transcriptionally linked. The expression of the unlinked Hb receptor, HpuAB, was not altered. Comparison of the polypeptide patterns of the wild type and the hemO mutant led to detection of six protein spots with an altered expression pattern, suggesting a more general role of HemO in the regulation of gene expression in Neisseriae. PMID:10629191

Differential expression of glutamate transporters EAAT1 and EAAT2 in mice deficient for PACAP-type I receptor.

PubMed

Zink, M; Schmitt, A; Henn, F A; Gass, P

2004-12-01

Pituitary adenylate cyclase-activating polypeptide (PACAP) modulates glutamatergic neurotransmission and induces the expression of glutamate transporters EAAT1 and EAAT2 in newborn mouse astroglial cell cultures. Since nanomolar concentrations of PACAP exert this effect, signal transduction via the high affinity PACAP-type I-receptor PAC1 was assumed. To test this hypothesis and to assess the importance of PAC1-signalling in vivo, we analyzed glutamate transporter expression in mice with a PAC1 knockout. EAAT1 and EAAT2 expression was investigated in the hippocampus and the cerebral cortex of PAC1 mutant mice and wildtype littermates by semiquantitative in-situ-hybridization. PAC1-knockout mice show a subtle but significant reduction of EAAT1 expression in the dentate gyrus. In contrast, reduced expression levels of EAAT1 in the cerebral cortex did not reach statistical significance and EAAT2 expression was unchanged in CA3 and cerebral cortex of PAC1 mutant mice. Our data confirm the previously reported in-vitro-regulation of EAAT1 in the adult nervous system in vivo. EAAT2 expression, however, is unchanged in PAC1 knockout mice, most likely due to counterbalancing factors.

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

The filamentous ascomycete Sordaria macrospora can survive in ambient air without carbonic anhydrases.

PubMed

Lehneck, Ronny; Elleuche, Skander; Pöggeler, Stefanie

2014-06-01

The rapid interconversion of carbon dioxide and bicarbonate (hydrogen carbonate) is catalysed by metalloenzymes termed carbonic anhydrases (CAs). CAs have been identified in all three domains of life and can be divided into five evolutionarily unrelated classes (α, β, γ, δ and ζ) that do not share significant sequence similarities. The function of the mammalian, prokaryotic and plant α-CAs has been intensively studied but the function of CAs in filamentous ascomycetes is mostly unknown. The filamentous ascomycete Sordaria macrospora codes for four CAs, three of the β-class and one of the α-class. Here, we present a functional analysis of CAS4, the S. macrospora α-class CA. The CAS4 protein was post-translationally glycosylated and secreted. The knockout strain Δcas4 had a significantly reduced rate of ascospore germination. To determine the cas genes required for S. macrospora growth under ambient air conditions, we constructed double and triple mutations of the four cas genes in all possible combinations and a quadruple mutant. Vegetative growth rate of the quadruple mutant lacking all cas genes was drastically reduced compared to the wild type and invaded the agar under normal air conditions. Likewise the fruiting bodies were embedded in the agar and completely devoid of mature ascospores. © 2014 John Wiley & Sons Ltd.

Abnormal cerebellar development and Purkinje cell defects in Lgl1-Pax2 conditional knockout mice.

PubMed

Hou, Congzhe; Ding, Lingcui; Zhang, Jian; Jin, Yecheng; Sun, Chen; Li, Zhenzu; Sun, Xiaoyang; Zhang, Tingting; Zhang, Aizhen; Li, Huashun; Gao, Jiangang

2014-11-01

Lgl1 was initially identified as a tumour suppressor in flies and is characterised as a key regulator of epithelial polarity and asymmetric cell division. A previous study indicated that More-Cre-mediated Lgl1 knockout mice exhibited significant brain dysplasia and died within 24h after birth. To overcome early neonatal lethality, we generated Lgl1 conditional knockout mice mediated by Pax2-Cre, which is expressed in almost all cells in the cerebellum, and we examined the functions of Lgl1 in the cerebellum. Impaired motor coordination was detected in the mutant mice. Consistent with this abnormal behaviour, homozygous mice possessed a smaller cerebellum with fewer lobes, reduced granule precursor cell (GPC) proliferation, decreased Purkinje cell (PC) quantity and dendritic dysplasia. Loss of Lgl1 in the cerebellum led to hyperproliferation and impaired differentiation of neural progenitors in ventricular zone. Based on the TUNEL assay, we observed increased apoptosis in the cerebellum of mutant mice. We proposed that impaired differentiation and increased apoptosis may contribute to decreased PC quantity. To clarify the effect of Lgl1 on cerebellar granule cells, we used Math1-Cre to specifically delete Lgl1 in granule cells. Interestingly, the Lgl1-Math1 conditional knockout mice exhibited normal proliferation of GPCs and cerebellar development. Thus, we speculated that the reduction in the proliferation of GPCs in Lgl1-Pax2 conditional knockout mice may be secondary to the decreased number of PCs, which secrete the mitogenic factor Sonic hedgehog to regulate GPC proliferation. Taken together, these findings suggest that Lgl1 plays a key role in cerebellar development and folia formation by regulating the development of PCs. Copyright © 2014. Published by Elsevier Inc.

Target sequencing and CRISPR/Cas editing reveal simultaneous loss of UTX and UTY in urothelial bladder cancer.

PubMed

Ahn, Jinwoo; Kim, Kwang Hyun; Park, Sanghui; Ahn, Young-Ho; Kim, Ha Young; Yoon, Hana; Lee, Ji Hyun; Bang, Duhee; Lee, Dong Hyeon

2016-09-27

UTX is a histone demethylase gene located on the X chromosome and is a frequently mutated gene in urothelial bladder cancer (UBC). UTY is a paralog of UTX located on the Y chromosome. We performed target capture sequencing on 128 genes in 40 non-metastatic UBC patients. UTX was the most frequently mutated gene (30%, 12/40). Of the genetic alterations identified, 75% were truncating mutations. UTY copy number loss was detected in 8 male patients (22.8%, 8/35). Of the 9 male patients with UTX mutations, 6 also had copy number loss (66.7%). To evaluate the functional roles of UTX and UTY in tumor progression, we designed UTX and UTY single knockout and UTX-UTY double knockout experiments using a CRISPR/Cas9 lentiviral system, and compared the proliferative capacities of two UBC cell lines in vitro. Single UTX or UTY knockout increased cell proliferation as compared to UTX-UTY wild-type cells. UTX-UTY double knockout cells exhibited greater proliferation than single knockout cells. These findings suggest both UTX and UTY function as dose-dependent suppressors of UBC development. While UTX escapes X chromosome inactivation in females, UTY may function as a male homologue of UTX, which could compensate for dosage imbalances.

Disruption of tetR type regulator adeN by mobile genetic element confers elevated virulence in Acinetobacter baumannii.

PubMed

Saranathan, Rajagopalan; Pagal, Sudhakar; Sawant, Ajit R; Tomar, Archana; Madhangi, M; Sah, Suresh; Satti, Annapurna; Arunkumar, K P; Prashanth, K

2017-10-03

Acinetobacter baumannii is an important human pathogen and considered as a major threat due to its extreme drug resistance. In this study, the genome of a hyper-virulent MDR strain PKAB07 of A. baumannii isolated from an Indian patient was sequenced and analyzed to understand its mechanisms of virulence, resistance and evolution. Comparative genome analysis of PKAB07 revealed virulence and resistance related genes scattered throughout the genome, instead of being organized as an island, indicating the highly mosaic nature of the genome. Many intermittent horizontal gene transfer events, insertion sequence (IS) element insertions identified were augmenting resistance machinery and elevating the SNP densities in A. baumannii eventually aiding in their swift evolution. ISAba1, the most widely distributed insertion sequence in A. baumannii was found in multiple sites in PKAB07. Out of many ISAba1 insertions, we identified novel insertions in 9 different genes wherein insertional inactivation of adeN (tetR type regulator) was significant. To assess the significance of this disruption in A. baumannii, adeN mutant and complement strains were constructed in A. baumannii ATCC 17978 strain and studied. Biofilm levels were abrogated in the adeN knockout when compared with the wild type and complemented strain of adeN knockout. Virulence of the adeN knockout mutant strain was observed to be high, which was validated by in vitro experiments and Galleria mellonella infection model. The overexpression of adeJ, a major component of AdeIJK efflux pump observed in adeN knockout strain could be the possible reason for the elevated virulence in adeN mutant and PKB07 strain. Knocking out of adeN in ATCC strain led to increased resistance and virulence at par with the PKAB07. Disruption of tetR type regulator adeN by ISAba1 consequently has led to elevated virulence in this pathogen.

Tracing the tracks of genotoxicity by trivalent and hexavalent chromium in Drosophila melanogaster.

PubMed

Mishra, Manish; Sharma, Anurag; Negi, M P S; Dwivedi, U N; Chowdhuri, D Kar

2011-05-18

Mutagen sensitive strains (mus) in Drosophila are known for their hypersensitivity to mutagens and environmental carcinogens. Accordingly, these mutants were grouped in pre- and post-replication repair pathways. However, studying mutants belonging to one particular repair pathway may not be adequate for examining chemical-induced genotoxicity when other repair pathways may neutralize its effect. To test whether both pre-and post-replication pathways are involved and effect of Cr(III)- and Cr(VI)-induced genotoxicity in absence or presence of others, we used double mutant approach in D. melanogaster. We observed DNA damage as evident by changes in Comet assay DNA migration in cells of larvae of Oregon R(+) and single mutants of pre- (mei-9, mus201 and mus210) and post- (mei-41, mus209 and mus309) replication repair pathways and also in double mutants of different combinations (pre-pre, pre-post and post-post replication repair) exposed to increasing concentrations of Cr(VI) (0.0, 5.0, 10.0 and 20.0 μg/ml) for 48 h. The damage was greater in pre-replication repair mutants after exposure to 5.0 μg/ml Cr(VI), while effects on Oregon R(+) and post replication repair mutants were insignificant. Post-replication repair mutants revealed significant DNA damage after exposure to 20.0 μg/ml Cr(VI). Further, double mutants generated in the above repair categories were examined for DNA damage following Cr(VI) exposure and a comparison of damage was studied between single and double mutants. Combinations of double mutants generated in the pre-pre replication repair pathways showed an indifferent interaction between the two mutants after Cr(VI) exposure while a synergistic interaction was evident in exposed post-post replication repair double mutants. Cr(III) (20.0 μg/ml) exposure to these strains did not induce any significant DNA damage in their cells. The study suggests that both pre- and post-replication pathways are affected in Drosophila by Cr(VI) leading to genotoxicity, which may have consequences for metal-induced carcinogenesis. 2011 Elsevier B.V. All rights reserved.

Knockout of the Gnrh genes in zebrafish: effects on reproduction and potential compensation by reproductive and feeding-related neuropeptides.

PubMed

Marvel, Miranda; Spicer, Olivia Smith; Wong, Ten-Tsao; Zmora, Nilli; Zohar, Yonathan

2018-04-04

Gonadotropin-releasing hormone (GnRH) is known as a pivotal upstream regulator of reproduction in vertebrates. However, reproduction is not compromised in the hypophysiotropic Gnrh3 knockout line in zebrafish (gnrh3-/-). In order to determine if Gnrh2, the only other Gnrh isoform in zebrafish brains, is compensating for the loss of Gnrh3, we generated a double Gnrh knockout zebrafish line. Surprisingly, the loss of both Gnrh isoforms resulted in no major impact on reproduction, indicating that a compensatory response, outside of the Gnrh system, was evoked. A plethora of factors acting along the reproductive hypothalamus-pituitary axis were evaluated as possible compensators based on neuroanatomical and differential gene expression studies. In addition, we also examined the involvement of feeding factors in the brain as potential compensators for Gnrh2, which has known anorexigenic effects. We found that the double knockout fish exhibited upregulation of several genes in the brain, specifically gonadotropin-inhibitory hormone (gnih), secretogranin 2 (scg2), tachykinin 3a (tac3a), and pituitary adenylate cyclase-activating peptide 1 (pacap1), and downregulation of agouti-related peptide 1 (agrp1), indicating the compensation occurs outside of Gnrh cells and therefore is a non-cell autonomous response to the loss of Gnrh. While the differential expression of gnih and agrp1 in the double knockout line was confined to the periventricular nucleus and hypothalamus, respectively, the upregulation of scg2 corresponded with a broader neuronal redistribution in the lateral hypothalamus and hindbrain. In conclusion, our results demonstrate the existence of a redundant reproductive regulatory system that comes into play when Gnrh2 and Gnrh3 are lost.

Connexin-deficiency affects expression levels of glial glutamate transporters within the cerebrum.

PubMed

Unger, Tina; Bette, Stefanie; Zhang, Jiong; Theis, Martin; Engele, Jürgen

2012-01-06

The glial glutamate transporter subtypes, GLT-1/EAAT-2 and GLAST/EAAT-1 clear the bulk of extracellular glutamate and are severely dysregulated in various acute and chronic brain diseases. Despite the previous identification of several extracellular factors modulating glial glutamate transporter expression, our knowledge of the regulatory network controlling glial glutamate transport in health and disease still remains incomplete. In studies with cultured cortical astrocytes, we previously obtained evidence that glial glutamate transporter expression is also affected by gap junctions/connexins. To assess whether gap junctions would likewise control the in vivo expression of glial glutamate transporters, we have now assessed their expression levels in brains of conditional Cx43 knockout mice, total Cx30 knockouts, as well as Cx43/Cx30 double knockouts. We found that either knocking out Cx30, Cx43, or both increases GLT-1/EAAT-2 protein levels in the cerebral cortex to a similar extent. By contrast, GLAST/EAAT-1 protein levels maximally increased in cerebral cortices of Cx30/Cx43 double knockouts, implying that gap junctions differentially affect the expression of GLT-1/EAAT-2 and GLAST/EAAT-1. Quantitative PCR analysis further revealed that increases in glial glutamate transporter expression are brought about by transcriptional and translational/posttranslational processes. Moreover, GLT-1/EAAT-2- and GLAST/EAAT-1 protein levels remained unchanged in the hippocampi of Cx43/Cx30 double knockouts when compared to Cx43fl/fl controls, indicating brain region-specific effects of gap junctions on glial glutamate transport. Since astrocytic gap junction coupling is affected in various forms of brain injuries, our findings point to gap junctions/connexins as important regulators of glial glutamate turnover in the diseased cerebral cortex. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Functional analysis of chloroplast early light inducible proteins (ELIPs)

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

Wetzel, Carolyn M

The objectives of this project were to characterize gene expression patterns of early light inducible protein (ELIP) genes in Arabidopsis thaliana and in Lycopersicon esculentum, to identify knock mutants of the 2 ELIP genes in Arabidopsis, and to characterize the effects of the knockouts. Expression in Arabidopsis was studied in response to thylakoid electron transport chain (PETC) capacity, where it was found that there is a signal for expression associated with reduction of the PETC. Expression in response to salt was also studied, with different responses of the two gene copies. Knockout lines for ELIP1 and ELIP2 have been identifiedmore » and are being characterized. In tomato, it was found that the single-copy ELIP gene is highly expressed in ripening fruit during the chloroplast-to-chromoplast transition. Studies of expression in tomato ripening mutants are ongoing.« less

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

PubMed Central

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

2015-01-01

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

PAC1- and VPAC2 receptors in light regulated behavior and physiology: Studies in single and double mutant mice.

PubMed

Hannibal, Jens; Georg, Birgitte; Fahrenkrug, Jan

2017-01-01

The two sister peptides, pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) and their receptors, the PAC1 -and the VPAC2 receptors, are involved in regulation of the circadian timing system. PACAP as a neurotransmitter in the retinohypothalamic tract (RHT) and VIP as a neurotransmitter, involved in synchronization of SCN neurons. Behavior and physiology in VPAC2 deficient mice are strongly regulated by light most likely as a result of masking. Consequently, we used VPAC2 and PAC1/VPAC2 double mutant mice in comparison with PAC1 receptor deficient mice to further elucidate the role of PACAP in the light mediated regulation of behavior and physiology of the circadian system. We compared circadian rhythms in mice equipped with running wheels or implanted radio-transmitter measuring core body temperature kept in a full photoperiod ((FPP)(12:12 h light dark-cycles (LD)) and skeleton photo periods (SPP) at high and low light intensity. Furthermore, we examined the expression of PAC1- and VPAC2 receptors in the SCN of the different genotypes in combination with visualization of PACAP and VIP and determined whether compensatory changes in peptide and/or receptor expression in the reciprocal knockouts (KO) (PAC1 and VPAC2) had occurred. Our data demonstrate that in although being closely related at both ligand and receptor structure/sequence, PACAP/PAC1 receptor signaling are independent of VIP/VPAC2 receptor signaling and vice versa. Furthermore, lack of either of the receptors does not result in compensatory changes at neither the physiological or anatomical level. PACAP/PAC1 signaling is important for light regulated behavior, VIP/VPAC2signaling for stable clock function and both signaling pathways may play a role in shaping diurnality versus nocturnality.

Genetic evidence that thyroid hormone is indispensable for prepubertal insulin-like growth factor-I expression and bone acquisition in mice.

PubMed

Xing, Weirong; Govoni, Kristen E; Donahue, Leah Rae; Kesavan, Chandrasekhar; Wergedal, Jon; Long, Carlin; Bassett, J H Duncan; Gogakos, Apostolos; Wojcicka, Anna; Williams, Graham R; Mohan, Subburaman

2012-05-01

Understanding how bone growth is regulated by hormonal and mechanical factors during early growth periods is important for optimizing the attainment of peak bone mass to prevent or postpone the occurrence of fragility fractures later in life. Using genetic mouse models that are deficient in thyroid hormone (TH) (Tshr(-/-) and Duox2(-/-)), growth hormone (GH) (Ghrhr(lit/lit)), or both (Tshr(-/-); Ghrhr(lit/lit)), we demonstrate that there is an important period prior to puberty when the effects of GH are surprisingly small and TH plays a critical role in the regulation of skeletal growth. Daily administration of T3/T4 during days 5 to 14, the time when serum levels of T3 increase rapidly in mice, rescued the skeletal deficit in TH-deficient mice but not in mice lacking both TH and GH. However, treatment of double-mutant mice with both GH and T3/T4 rescued the bone density deficit. Increased body fat in the TH-deficient as well as TH/GH double-mutant mice was rescued by T3/T4 treatment during days 5 to 14. In vitro studies in osteoblasts revealed that T3 in the presence of TH receptor (TR) α1 bound to a TH response element in intron 1 of the IGF-I gene to stimulate transcription. In vivo studies using TRα and TRβ knockout mice revealed evidence for differential regulation of insulin-like growth factor (IGF)-I expression by the two receptors. Furthermore, blockade of IGF-I action partially inhibited the biological effects of TH, thus suggesting that both IGF-I-dependent and IGF-I-independent mechanisms contribute to TH effects on prepubertal bone acquisition. Copyright © 2012 American Society for Bone and Mineral Research.

The phytochelatin transporters AtABCC1 and AtABCC2 mediate tolerance to cadmium and mercury.

PubMed

Park, Jiyoung; Song, Won-Yong; Ko, Donghwi; Eom, Yujin; Hansen, Thomas H; Schiller, Michaela; Lee, Tai Gyu; Martinoia, Enrico; Lee, Youngsook

2012-01-01

Heavy metals such as cadmium (Cd) and mercury (Hg) are toxic pollutants that are detrimental to living organisms. Plants employ a two-step mechanism to detoxify toxic ions. First, phytochelatins bind to the toxic ion, and then the metal-phytochelatin complex is sequestered in the vacuole. Two ABCC-type transporters, AtABCC1 and AtABCC2, that play a key role in arsenic detoxification, have recently been identified in Arabidopsis thaliana. However, it is unclear whether these transporters are also implicated in phytochelatin-dependent detoxification of other heavy metals such as Cd(II) and Hg(II). Here, we show that atabcc1 single or atabcc1 atabcc2 double knockout mutants exhibit a hypersensitive phenotype in the presence of Cd(II) and Hg(II). Microscopic analysis using a Cd-sensitive probe revealed that Cd is mostly located in the cytosol of protoplasts of the double mutant, whereas it occurs mainly in the vacuole of wild-type cells. This suggests that the two ABCC transporters are important for vacuolar sequestration of Cd. Heterologous expression of the transporters in Saccharomyces cerevisiae confirmed their role in heavy metal tolerance. Over-expression of AtABCC1 in Arabidopsis resulted in enhanced Cd(II) tolerance and accumulation. Together, these results demonstrate that AtABCC1 and AtABCC2 are important vacuolar transporters that confer tolerance to cadmium and mercury, in addition to their role in arsenic detoxification. These transporters provide useful tools for genetic engineering of plants with enhanced metal tolerance and accumulation, which are desirable characteristics for phytoremediation. © 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.

Adenosine Deaminase Acting on RNA 1 (ADAR1) Suppresses the Induction of Interferon by Measles Virus

PubMed Central

Li, Zhiqun; Okonski, Kristina M.

2012-01-01

ADAR1, the interferon (IFN)-inducible adenosine deaminase acting on RNA, catalyzes the C-6 deamination of adenosine (A) to produce inosine (I) in RNA substrates with a double-stranded character. Because double-stranded RNA is a known inducer of IFN, we tested the role of ADAR1 in IFN induction following virus infection. HeLa cells made stably deficient in ADAR1 (ADAR1kd) were compared to vector control (CONkd) and protein kinase PKR-deficient (PKRkd) cells for IFN-β induction following infection with either parental (wild-type [WT]) recombinant Moraten vaccine strain measles virus (MV) or isogenic knockout mutants deficient for either V (Vko) or C (Cko) protein expression. We observed potent IFN-β transcript induction in ADAR1kd cells by all three viruses; in contrast, in ADAR1-sufficient CONkd cells, only the Cko mutant virus was an effective inducer and the IFN-β RNA induction was amplified by PKR. The enhanced IFN-β transcript-inducing capacity of the WT and Vko viruses seen in ADAR1-deficient cells correlated with the enhanced activation of PKR, IFN regulatory factor IRF3, and activator of transcription ATF2, reaching levels similar to those seen in Cko virus-infected cells. However, the level of IFN-β protein produced was not proportional to the level of IFN-β RNA but rather correlated inversely with the level of activated PKR. These results suggest that ADAR1 functions as an important suppressor of MV-mediated responses, including the activation of PKR and IRF3 and the induction of IFN-β RNA. Our findings further implicate a balanced interplay between PKR and ADAR1 in modulating IFN-β protein production following virus infection. PMID:22278222

Bone Is a Major Target of PTH/PTHrP Receptor Signaling in Regulation of Fetal Blood Calcium Homeostasis

PubMed Central

Hirai, Takao; Kobayashi, Tatsuya; Nishimori, Shigeki; Karaplis, Andrew C.; Goltzman, David

2015-01-01

The blood calcium concentration during fetal life is tightly regulated within a narrow range by highly interactive homeostatic mechanisms that include transport of calcium across the placenta and fluxes in and out of bone; the mechanisms of this regulation are poorly understood. Our findings that endochondral bone-specific PTH/PTHrP receptor (PPR) knockout (KO) mice showed significant reduction of fetal blood calcium concentration compared with that of control littermates at embryonic day 18.5 led us to focus on bone as a possibly major determinant of fetal calcium homeostasis. We found that the fetal calcium concentration of Runx2 KO mice was significantly higher than that of control littermates, suggesting that calcium flux into bone had a considerable influence on the circulating calcium concentration. Moreover, Runx2:PTH double mutant fetuses showed calcium levels similar to those of Runx2 KO mice, suggesting that part of the fetal hypocalcemia in PTH KO mice was caused by the increment of the mineralized bone mass allowed by the formation of osteoblasts. Finally, Rank:PTH double mutant mice had a blood calcium concentration even lower than that of the either Rank KO or PTH KO mice alone at embryonic day 18.5. These observations in our genetic models suggest that PTH/PTHrP receptor signaling in bones has a significant role of the regulation of fetal blood calcium concentration and that both placental transport and osteoclast activation contribute to PTH's hypercalcemic action. They also show that PTH-independent deposition of calcium in bone is the major controller of fetal blood calcium level. PMID:26052897

Loss of Adult Cardiac Myocyte GSK-3 Leads to Mitotic Catastrophe Resulting in Fatal Dilated Cardiomyopathy.

PubMed

Zhou, Jibin; Ahmad, Firdos; Parikh, Shan; Hoffman, Nichole E; Rajan, Sudarsan; Verma, Vipin K; Song, Jianliang; Yuan, Ancai; Shanmughapriya, Santhanam; Guo, Yuanjun; Gao, Erhe; Koch, Walter; Woodgett, James R; Madesh, Muniswamy; Kishore, Raj; Lal, Hind; Force, Thomas

2016-04-15

Cardiac myocyte-specific deletion of either glycogen synthase kinase (GSK)-3α and GSK-3β leads to cardiac protection after myocardial infarction, suggesting that deletion of both isoforms may provide synergistic protection. This is an important consideration because of the fact that all GSK-3-targeted drugs, including the drugs already in clinical trial target both isoforms of GSK-3, and none are isoform specific. To identify the consequences of combined deletion of cardiac myocyte GSK-3α and GSK-3β in heart function. We generated tamoxifen-inducible cardiac myocyte-specific mice lacking both GSK-3 isoforms (double knockout). We unexpectedly found that cardiac myocyte GSK-3 is essential for cardiac homeostasis and overall survival. Serial echocardiographic analysis reveals that within 2 weeks of tamoxifen treatment, double-knockout hearts leads to excessive dilatative remodeling and ventricular dysfunction. Further experimentation with isolated adult cardiac myocytes and fibroblasts from double-knockout implicated cardiac myocytes intrinsic factors responsible for observed phenotype. Mechanistically, loss of GSK-3 in adult cardiac myocytes resulted in induction of mitotic catastrophe, a previously unreported event in cardiac myocytes. Double-knockout cardiac myocytes showed cell cycle progression resulting in increased DNA content and multinucleation. However, increased cell cycle activity was rivaled by marked activation of DNA damage, cell cycle checkpoint activation, and mitotic catastrophe-induced apoptotic cell death. Importantly, mitotic catastrophe was also confirmed in isolated adult cardiac myocytes. Together, our findings suggest that cardiac myocyte GSK-3 is required to maintain normal cardiac homeostasis, and its loss is incompatible with life because of cell cycle dysregulation that ultimately results in a severe fatal dilated cardiomyopathy. © 2016 American Heart Association, Inc.

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.

Independent effects of apolipoprotein AV and apolipoprotein CIII on plasma triglyceride concentrations

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

Baroukh, Nadine N.; Bauge, Eric; Akiyama, Jennifer

2003-08-15

Both the apolipoprotein A5 and C3 genes have repeatedly been shown to play an important role in determining plasma triglyceride concentrations in humans and mice. In mice, transgenic and knockout experiments indicate that plasma triglyceride levels are negatively and positively correlated with APOA5 and APOC3 expression, respectively. In humans, common polymorphisms in both genes have also been associated with plasma triglyceride concentrations. The evolutionary relationship among these two apolipoprotein genes and their close proximity on human chromosome 11q23 have largely precluded the determination of their relative contribution to altered Both the apolipoprotein A5 and C3 genes have repeatedly been shownmore » to play an important role in determining plasma triglyceride concentrations in humans and mice. In mice, transgenic and knockout experiments indicate that plasma triglyceride levels are negatively and positively correlated with APOA5 and APOC3 expression, respectively. In humans, common polymorphisms in both genes have also been associated with plasma triglyceride concentrations. The evolutionary relationship among these two apolipoprotein genes and their close proximity on human chromosome 11q23 have largely precluded the determination of their relative contribution to altered triglycerides. To overcome these confounding factors and address their relationship, we generated independent lines of mice that either over-expressed (''double transgenic'') or completely lacked (''double knockout'') both apolipoprotein genes. We report that both ''double transgenic'' and ''double knockout'' mice display intermedia tetriglyceride concentrations compared to over-expression or deletion of either gene alone. Furthermore, we find that human ApoAV plasma protein levels in the ''double transgenic'' mice are approximately 500-fold lower than human ApoCIII levels, supporting ApoAV is a potent triglyceride modulator despite its low concentration. Together, these data indicate that APOA5 and APOC3 independently influence plasma triglyceride concentrations but in an opposing manner.« less

Insights from the Study of Animals Lacking Functional Estrogen Receptor

NASA Astrophysics Data System (ADS)

Korach, Kenneth S.

1994-12-01

Estrogen hormones produce physiological actions within a variety of target sites in the body and during development by activating a specific receptor protein. Hormone responsiveness for the estrogen receptor protein was investigated at different stages of development with the use of gene knockout techniques because no natural genetic mutants have been described. A mutant mouse line without a functional estrogen receptor was created and is being used to assess estrogen responsiveness. Both sexes of these mutant animals are infertile and show a variety of phenotypic changes, some of which are associated with the gonads, mammary glands, reproductive tracts, and skeletal tissues.

Cytoprotective role of autophagy against BH3 mimetic gossypol in ATG5 knockout cells generated by CRISPR-Cas9 endonuclease.

PubMed

Kim, Na-Yeon; Han, Byeal-I; Lee, Michael

2016-01-01

Previously, we demonstrated the association between autophagy and gossypol-induced growth inhibition of mutant BRAF melanoma cells. Here, we investigate the role of autophagy in ATG5 knockout cell lines generated by the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas-mediated genome editing. The MTT assay revealed that the inhibitory effect of gossypol was weaker on ATG5 knockout cells than that on the wild type (WT) cells. The conversion of non-autophagic LC3-I to autophagic LC3-II and RT-PCR confirmed the functional gene knockout. However, Cyto-ID autophagy assay revealed that gossypol induced ATG5- and LC3-independent autophagy in ATG5 knockout cells. Moreover, gossypol acts as an autophagy inducer in ATG5 knockout cells while blocking the later stages of the autophagy process in WT cells, which was determined by measuring autophagic flux after co-treatment of gossypol with chloroquine (late-stage autophagy inhibitor). On the other hand, inhibition of autophagy with 3-MA or Beclin-1 siRNA caused a partial increase in the sensitivity to gossypol in ATG5 knockout cells, but not in the WT cells. Together, our findings suggest that the resistance to gossypol in ATG5 knockout cells is associated with increased cytoprotective autophagy, independent of ATG5. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Effect of IAA on in vitro growth and colonization of Nostoc in plant roots

PubMed Central

Hussain, Anwar; Shah, Syed T.; Rahman, Hazir; Irshad, Muhammad; Iqbal, Amjad

2015-01-01

Nostoc is widely known for its ability to fix atmospheric nitrogen and the establishment of symbiotic relationship with a wide range of plants from various taxonomic groups. Several strains of Nostoc produce phytohormones that promote growth of its plant partners. Nostoc OS-1 was therefore selected for study because of the presence of putative ipdC gene that encodes a key enzyme to produce Indole-3-acetic acid (IAA). The results indicated that both cellular and released IAA was found high with increasing incubation time and reached to a peak value (i.e., 21 pmol mg-1ch-a) on the third week as determined by UPLC-ESI-MS/MS. Also the Nostoc OS-1 strain efficiently colonized the roots and promoted the growth of rice as well as wheat under axenic conditions and induced ipdC gene that suggested the possible involvement of IAA in these phenotypes. To confirm the impact of IAA on root colonization efficiency and plant promoting phenotypes of Nostoc OS-1, an ipdC knockout mutant was generated by homologous recombinant method. The amount of releasing IAA, in vitro growth, root colonization, and plant promoting efficiency of the ipdC knockout mutant was observed significantly lower than wild type strain under axenic conditions. Importantly, these phenotypes were restored to wild-type levels when the ipdC knockout mutant was complemented with wild type ipdC gene. These results together suggested that ipdC and/or synthesized IAA of Nostoc OS-1 is required for its efficient root colonization and plant promoting activity. PMID:25699072

Murine GRPR and Stathmin Control in Opposite Directions both Cued Fear Extinction and Neural Activities of the Amygdala and Prefrontal Cortex

PubMed Central

Martel, Guillaume; Hevi, Charles; Wong, Alexandra; Zushida, Ko; Uchida, Shusaku; Shumyatsky, Gleb P.

2012-01-01

Extinction is an integral part of normal healthy fear responses, while it is compromised in several fear-related mental conditions in humans, such as post-traumatic stress disorder (PTSD). Although much research has recently been focused on fear extinction, its molecular and cellular underpinnings are still unclear. The development of animal models for extinction will greatly enhance our approaches to studying its neural circuits and the mechanisms involved. Here, we describe two gene-knockout mouse lines, one with impaired and another with enhanced extinction of learned fear. These mutant mice are based on fear memory-related genes, stathmin and gastrin-releasing peptide receptor (GRPR). Remarkably, both mutant lines showed changes in fear extinction to the cue but not to the context. We performed indirect imaging of neuronal activity on the second day of cued extinction, using immediate-early gene c-Fos. GRPR knockout mice extinguished slower (impaired extinction) than wildtype mice, which was accompanied by an increase in c-Fos activity in the basolateral amygdala and a decrease in the prefrontal cortex. By contrast, stathmin knockout mice extinguished faster (enhanced extinction) and showed a decrease in c-Fos activity in the basolateral amygdala and an increase in the prefrontal cortex. At the same time, c-Fos activity in the dentate gyrus was increased in both mutant lines. These experiments provide genetic evidence that the balance between neuronal activities of the amygdala and prefrontal cortex defines an impairment or facilitation of extinction to the cue while the hippocampus is involved in the context-specificity of extinction. PMID:22312434

Murine GRPR and stathmin control in opposite directions both cued fear extinction and neural activities of the amygdala and prefrontal cortex.

PubMed

Martel, Guillaume; Hevi, Charles; Wong, Alexandra; Zushida, Ko; Uchida, Shusaku; Shumyatsky, Gleb P

2012-01-01

Extinction is an integral part of normal healthy fear responses, while it is compromised in several fear-related mental conditions in humans, such as post-traumatic stress disorder (PTSD). Although much research has recently been focused on fear extinction, its molecular and cellular underpinnings are still unclear. The development of animal models for extinction will greatly enhance our approaches to studying its neural circuits and the mechanisms involved. Here, we describe two gene-knockout mouse lines, one with impaired and another with enhanced extinction of learned fear. These mutant mice are based on fear memory-related genes, stathmin and gastrin-releasing peptide receptor (GRPR). Remarkably, both mutant lines showed changes in fear extinction to the cue but not to the context. We performed indirect imaging of neuronal activity on the second day of cued extinction, using immediate-early gene c-Fos. GRPR knockout mice extinguished slower (impaired extinction) than wildtype mice, which was accompanied by an increase in c-Fos activity in the basolateral amygdala and a decrease in the prefrontal cortex. By contrast, stathmin knockout mice extinguished faster (enhanced extinction) and showed a decrease in c-Fos activity in the basolateral amygdala and an increase in the prefrontal cortex. At the same time, c-Fos activity in the dentate gyrus was increased in both mutant lines. These experiments provide genetic evidence that the balance between neuronal activities of the amygdala and prefrontal cortex defines an impairment or facilitation of extinction to the cue while the hippocampus is involved in the context-specificity of extinction.

Phenotype detection in morphological mutant mice using deformation features.

PubMed

Roy, Sharmili; Liang, Xi; Kitamoto, Asanobu; Tamura, Masaru; Shiroishi, Toshihiko; Brown, Michael S

2013-01-01

Large-scale global efforts are underway to knockout each of the approximately 25,000 mouse genes and interpret their roles in shaping the mammalian embryo. Given the tremendous amount of data generated by imaging mutated prenatal mice, high-throughput image analysis systems are inevitable to characterize mammalian development and diseases. Current state-of-the-art computational systems offer only differential volumetric analysis of pre-defined anatomical structures between various gene-knockout mice strains. For subtle anatomical phenotypes, embryo phenotyping still relies on the laborious histological techniques that are clearly unsuitable in such big data environment. This paper presents a system that automatically detects known phenotypes and assists in discovering novel phenotypes in muCT images of mutant mice. Deformation features obtained from non-linear registration of mutant embryo to a normal consensus average image are extracted and analyzed to compute phenotypic and candidate phenotypic areas. The presented system is evaluated using C57BL/10 embryo images. All cases of ventricular septum defect and polydactyly, well-known to be present in this strain, are successfully detected. The system predicts potential phenotypic areas in the liver that are under active histological evaluation for possible phenotype of this mouse line.

[Construction of Corynebacterium crenatum AS 1.542 δ argR and analysis of transcriptional levels of the related genes of arginine biosynthetic pathway].

PubMed

Chen, Xuelan; Tang, Li; Jiao, Haitao; Xu, Feng; Xiong, Yonghua

2013-01-04

ArgR, coded by the argR gene from Corynebacterium crenatum AS 1.542, acts as a negative regulator in arginine biosynthetic pathway. However, the effect of argR on transcriptional levels of the related biosynthetic genes has not been reported. Here, we constructed a deletion mutant of argR gene: C. crenatum AS 1.542 Delta argR using marker-less knockout technology, and compared the changes of transcriptional levels of the arginine biosynthetic genes between the mutant strain and the wild-type strain. We used marker-less knockout technology to construct C. crenatum AS 1.542 Delta argR and analyzed the changes of the relate genes at the transcriptional level using real-time fluorescence quantitative PCR. C. crenatum AS 1.542 Delta argR was successfully obtained and the transcriptional level of arginine biosynthetic genes in this mutant increased significantly with an average of about 162.1 folds. The arginine biosynthetic genes in C. crenatum are clearly controlled by the negative regulator ArgR. However, the deletion of this regulator does not result in a clear change in arginine production in the bacteria.

Impact of CCR1 silencing on the assembly of lignified secondary walls in Arabidopsis thaliana.

PubMed

Ruel, Katia; Berrio-Sierra, Jimmy; Derikvand, Mohammad Mir; Pollet, Brigitte; Thévenin, Johanne; Lapierre, Catherine; Jouanin, Lise; Joseleau, Jean-Paul

2009-01-01

A cinnamoyl-CoA reductase 1 knockout mutant in Arabidopsis thaliana was investigated for the consequences of lignin synthesis perturbation on the assembly of the cell walls. The mutant displayed a dwarf phenotype and a strong collapse of its xylem vessels corresponding to lower lignin content and a loss of lignin units of the noncondensed type. Transmission electron microscopy revealed that the transformation considerably impaired the capacity of interfascicular fibers and vascular bundles to complete the assembly of cellulose microfibrils in the S(2) layer, the S(1) layer remaining unaltered. Such disorder in cellulose was correlated with X-ray diffraction showing altered organization. Semi-quantitative immunolabeling of lignins showed that the patterns of distribution were differentially affected in interfascicular fibers and vascular bundles, pointing to the importance of noncondensed lignin structures for the assembly of a coherent secondary wall. The use of laser capture microdissection combined with the microanalysis of lignins and polysaccharides allowed these polymers to be characterized into specific cell types. Wild-type A. thaliana displayed a two-fold higher syringyl to guaiacyl ratio in interfascicular fibers compared with vascular bundles, whereas this difference was less marked in the cinnamoyl-CoA reductase 1 knockout mutant.

PtAUREO1a and PtAUREO1b knockout mutants of the diatom Phaeodactylum tricornutum are blocked in photoacclimation to blue light.

PubMed

Mann, Marcus; Serif, Manuel; Jakob, Torsten; Kroth, Peter G; Wilhelm, Christian

2017-10-01

Aureochromes are blue light receptors specifically found in photosynthetic Stramenopiles (algae). Four different Aureochromes have been identified in the marine diatom Phaeodactylum tricornutum (PtAUREO 1a, 1b, 1c, and 2). Since blue light is necessary for high light acclimation in diatoms, it has been hypothesized that Aureochromes might play an important role in the light acclimation capacity of diatoms. This hypothesis was supported by an RNAi knockdown line of PtAUREO1a, which showed a phenotype different from wild type cells when grown in either blue or red light. Here, we show for the first time the phenotype and the photoacclimation reaction of TALEN-mediated knockout mutants of PtAUREO1a and PtAUREO1b, clearly proving the necessity of Aureochromes for light acclimation under blue light. However, both mutants do also show specific differences in their respective phenotypes. Hence, PtAUREO1a and 1b are not functionally redundant in photoacclimation to blue light, and their specific contribution needs to be clarified further. Copyright © 2017 Elsevier GmbH. All rights reserved.

Prevalence of sexual dimorphism in mammalian phenotypic traits.

PubMed

Karp, Natasha A; Mason, Jeremy; Beaudet, Arthur L; Benjamini, Yoav; Bower, Lynette; Braun, Robert E; Brown, Steve D M; Chesler, Elissa J; Dickinson, Mary E; Flenniken, Ann M; Fuchs, Helmut; Angelis, Martin Hrabe de; Gao, Xiang; Guo, Shiying; Greenaway, Simon; Heller, Ruth; Herault, Yann; Justice, Monica J; Kurbatova, Natalja; Lelliott, Christopher J; Lloyd, K C Kent; Mallon, Ann-Marie; Mank, Judith E; Masuya, Hiroshi; McKerlie, Colin; Meehan, Terrence F; Mott, Richard F; Murray, Stephen A; Parkinson, Helen; Ramirez-Solis, Ramiro; Santos, Luis; Seavitt, John R; Smedley, Damian; Sorg, Tania; Speak, Anneliese O; Steel, Karen P; Svenson, Karen L; Wakana, Shigeharu; West, David; Wells, Sara; Westerberg, Henrik; Yaacoby, Shay; White, Jacqueline K

2017-06-26

The role of sex in biomedical studies has often been overlooked, despite evidence of sexually dimorphic effects in some biological studies. Here, we used high-throughput phenotype data from 14,250 wildtype and 40,192 mutant mice (representing 2,186 knockout lines), analysed for up to 234 traits, and found a large proportion of mammalian traits both in wildtype and mutants are influenced by sex. This result has implications for interpreting disease phenotypes in animal models and humans.

Multiple strategies to prevent oxidative stress in Arabidopsis plants lacking the malate valve enzyme NADP-malate dehydrogenase

PubMed Central

Hebbelmann, Inga; Selinski, Jennifer; Wehmeyer, Corinna; Goss, Tatjana; Voss, Ingo; Mulo, Paula; Kangasjärvi, Saijaliisa; Aro, Eva-Mari; Oelze, Marie-Luise; Dietz, Karl-Josef; Nunes-Nesi, Adriano; Do, Phuc T.; Fernie, Alisdair R.; Talla, Sai K.; Raghavendra, Agepati S.; Linke, Vera; Scheibe, Renate

2012-01-01

The nuclear-encoded chloroplast NADP-dependent malate dehydrogenase (NADP-MDH) is a key enzyme controlling the malate valve, to allow the indirect export of reducing equivalents. Arabidopsis thaliana (L.) Heynh. T-DNA insertion mutants of NADP-MDH were used to assess the role of the light-activated NADP-MDH in a typical C3 plant. Surprisingly, even when exposed to high-light conditions in short days, nadp-mdh knockout mutants were phenotypically indistinguishable from the wild type. The photosynthetic performance and typical antioxidative systems, such as the Beck–Halliwell–Asada pathway, were barely affected in the mutants in response to high-light treatment. The reactive oxygen species levels remained low, indicating the apparent absence of oxidative stress, in the mutants. Further analysis revealed a novel combination of compensatory mechanisms in order to maintain redox homeostasis in the nadp-mdh plants under high-light conditions, particularly an increase in the NTRC/2-Cys peroxiredoxin (Prx) system in chloroplasts. There were indications of adjustments in extra-chloroplastic components of photorespiration and proline levels, which all could dissipate excess reducing equivalents, sustain photosynthesis, and prevent photoinhibition in nadp-mdh knockout plants. Such metabolic flexibility suggests that the malate valve acts in concert with other NADPH-consuming reactions to maintain a balanced redox state during photosynthesis under high-light stress in wild-type plants. PMID:22140244

Neisseria gonorrhoeae PIII has a role on NG1873 outer membrane localization and is involved in bacterial adhesion to human cervical and urethral epithelial cells.

PubMed

Leuzzi, Rosanna; Nesta, Barbara; Monaci, Elisabetta; Cartocci, Elena; Serino, Laura; Soriani, Marco; Rappuoli, Rino; Pizza, Mariagrazia

2013-11-09

Protein PIII is one of the major outer membrane proteins of Neisseria gonorrhoeae, 95% identical to RmpM (reduction modifiable protein M) or class 4 protein of Neisseria meningitidis. RmpM is known to be a membrane protein associated by non-covalent bonds to the peptidoglycan layer and interacting with PorA/PorB porin complexes resulting in the stabilization of the bacterial membrane. The C-terminal domain of PIII (and RmpM) is highly homologous to members of the OmpA family, known to have a role in adhesion/invasion in many bacterial species. The contribution of PIII in the membrane architecture and its role in the interaction with epithelial cells has never been investigated. We generated a ΔpIII knock-out mutant strain and evaluated the effects of the loss of PIII expression on bacterial morphology and on outer membrane composition. Deletion of the pIII gene does not cause any alteration in bacterial morphology or sensitivity to detergents. Moreover, the expression profile of the main membrane proteins remains the same for the wild-type and knock-out strains, with the exception of the NG1873 which is not exported to the outer membrane and accumulates in the inner membrane in the ΔpIII knock-out mutant strain.We also show that purified PIII protein is able to bind human cervical and urethral cells and that the ΔpIII knock-out mutant strain has a lower ability to adhere to human cervical and urethral cells. Here we demonstrated that the PIII protein does not play a key structural role in the membrane organization of gonococcus and does not induce major effects on the expression of the main outer membrane proteins. However, in the PIII knock-out strain, the NG1873 protein is not localized in the outer membrane as it is in the wild-type strain suggesting a possible interaction of PIII with NG1873. The evidence that PIII binds to human epithelial cells derived from the female and male genital tract highlights a possible role of PIII in the virulence of gonococcus and suggests that the structural homology to OmpA is conserved also at functional level.

AmyR Is a Novel Negative Regulator of Amylovoran Production in Erwinia amylovora

PubMed Central

Wang, Dongping; Korban, Schuyler S.; Pusey, P. Lawrence; Zhao, Youfu

2012-01-01

In this study, we attempted to understand the role of an orphan gene amyR in Erwinia amylovora, a functionally conserved ortholog of ybjN in Escherichia coli, which has recently been characterized. Amylovoran, a high molecular weight acidic heteropolymer exopolysaccharide, is a virulent factor of E. amylovora. As reported earlier, amylovoran production in an amyR knockout mutant was about eight-fold higher than that in the wild type (WT) strain of E. amylovora. When a multicopy plasmid containing the amyR gene was introduced into the amyR mutant or WT strains, amylovoran production was strongly inhibited. Furthermore, amylovoran production was also suppressed in various amylovoran-over-producing mutants, such as grrSA containing multicopies of the amyR gene. Consistent with amylovoran production, an inverse correlation was observed between in vitro expression of amyR and that of amylovoran biosynthetic genes. However, both the amyR knockout mutant and over-expression strains showed reduced levan production, another exopolysaccharide produced by E. amylovora. Virulence assays demonstrated that while the amyR mutant was capable of inducing slightly greater disease severity than that of the WT strain, strains over-expressing the amyR gene did not incite disease on apple shoots or leaves, and only caused reduced disease on immature pear fruits. Microarray studies revealed that amylovoran biosynthesis and related membrane protein-encoding genes were highly expressed in the amyR mutant, but down-regulated in the amyR over-expression strains in vitro. Down-regulation of amylovoran biosynthesis genes in the amyR over-expression strain partially explained why over-expression of amyR led to non-pathogenic or reduced virulence in vivo. These results suggest that AmyR plays an important role in regulating exopolysaccharide production, and thus virulence in E. amylovora. PMID:23028751

AmyR is a novel negative regulator of amylovoran production in Erwinia amylovora.

PubMed

Wang, Dongping; Korban, Schuyler S; Pusey, P Lawrence; Zhao, Youfu

2012-01-01

In this study, we attempted to understand the role of an orphan gene amyR in Erwinia amylovora, a functionally conserved ortholog of ybjN in Escherichia coli, which has recently been characterized. Amylovoran, a high molecular weight acidic heteropolymer exopolysaccharide, is a virulent factor of E. amylovora. As reported earlier, amylovoran production in an amyR knockout mutant was about eight-fold higher than that in the wild type (WT) strain of E. amylovora. When a multicopy plasmid containing the amyR gene was introduced into the amyR mutant or WT strains, amylovoran production was strongly inhibited. Furthermore, amylovoran production was also suppressed in various amylovoran-over-producing mutants, such as grrSA containing multicopies of the amyR gene. Consistent with amylovoran production, an inverse correlation was observed between in vitro expression of amyR and that of amylovoran biosynthetic genes. However, both the amyR knockout mutant and over-expression strains showed reduced levan production, another exopolysaccharide produced by E. amylovora. Virulence assays demonstrated that while the amyR mutant was capable of inducing slightly greater disease severity than that of the WT strain, strains over-expressing the amyR gene did not incite disease on apple shoots or leaves, and only caused reduced disease on immature pear fruits. Microarray studies revealed that amylovoran biosynthesis and related membrane protein-encoding genes were highly expressed in the amyR mutant, but down-regulated in the amyR over-expression strains in vitro. Down-regulation of amylovoran biosynthesis genes in the amyR over-expression strain partially explained why over-expression of amyR led to non-pathogenic or reduced virulence in vivo. These results suggest that AmyR plays an important role in regulating exopolysaccharide production, and thus virulence in E. amylovora.

Rice mutants deficient in ω-3 fatty acid desaturase (FAD8) fail to acclimate to cold temperatures.

PubMed

Tovuu, Altanzaya; Zulfugarov, Ismayil S; Wu, Guangxi; Kang, In Soon; Kim, Choongrak; Moon, Byoung Yong; An, Gynheung; Lee, Choon-Hwan

2016-12-01

To investigate the role of ω-3 fatty acid (FA) desaturase (FAD8) during cold acclimation in higher plants, we characterized three independent T-DNA insertional knock-out mutants of OsFAD8 from rice (Oryza sativa L.). At room temperature (28 °C), osfad8 plants exhibited significant alterations in fatty acid (FA) unsaturation for all four investigated plastidic lipid classes. During a 5-d acclimation period at 4 °C, further changes in FA unsaturation in both wild-type (WT) and mutant plants varied according to the type of lipid. We also monitored the fluidity of the thylakoid membrane using a threshold temperature to represent the change in fluorescence. The values were altered significantly by both FAD8 mutation and cold acclimation, suggesting that factors other than FAD8 are involved in C18 FA unsaturation and fluctuations in membrane fluidity. Similarly, significant changes were noted for both the mutant and WT samples in terms of their FA compositions as well as activities related to photosystem (PS) I, PSII, and photoprotection. This included the development of non-photochemical quenching and increased zeaxanthin accumulation. Despite the relatively small changes in FA composition during cold acclimation, cold-inducible FAD8 knock-out mutants displayed strong differences in photoprotective activities and a further drop in membrane fluidity. The mutants were more sensitive than WT to short-term low-temperature stress that resulted in increased production of reactive oxygen species after 5 d of chilling. Taken together, our findings suggest that FA unsaturation by OsFAD8 is crucial for the acclimation of higher plants to low-temperature stress. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Dnmt1 and Dnmt3a are required for the maintenance of DNA methylation and synaptic function in adult forebrain neurons

PubMed Central

Feng, Jian; Zhou, Yu; Campbell, Susan L.; Le, Thuc; Li, En; Sweatt, J. David; Silva, Alcino J.; Fan, Guoping

2011-01-01

Dnmt1 and Dnmt3a, two major DNA methyltransferases, are expressed in postmitotic neurons, but their function in the central nervous system (CNS) is unclear. We generated conditional mutant mice that lack either Dnmt1, or Dnmt3a, or both exclusively in forebrain excitatory neurons and found only double knockout (DKO) mice exhibited abnormal hippocampal CA1 long-term plasticity and deficits of learning and memory. While no neuronal loss was found, the size of hippocampal neurons in DKO was smaller; furthermore, DKO neurons showed a deregulation of gene expression including class I MHC and Stat1 that are known to play a role in synaptic plasticity. In addition, we observed a significant decrease in DNA methylation in DKO neurons. We conclude that Dnmt1 and Dnmt3a are required for synaptic plasticity, learning and memory through their overlapping roles in maintaining DNA methylation and modulating neuronal gene expression in adult CNS neurons. PMID:20228804

Decreased APOE-containing HDL subfractions and cholesterol efflux capacity of serum in mice lacking Pcsk9

PubMed Central

2013-01-01

Background Studies in animals showed that PCSK9 is involved in HDL metabolism. We investigated the molecular mechanism by which PCSK9 regulates HDL cholesterol concentration and also whether Pcsk9 inactivation might affect cholesterol efflux capacity of serum and atherosclerotic fatty streak volume. Methods Mass spectrometry and western blot were used to analyze the level of apolipoprotein E (APOE) and A1 (APOA1). A mouse model overexpressing human LDLR was used to test the effect of high levels of liver LDLR on the concentration of HDL cholesterol and APOE-containing HDL subfractions. Pcsk9 knockout males lacking LDLR and APOE were used to test whether LDLR and APOE are necessary for PCSK9-mediated HDL cholesterol regulation. We also investigated the effects of Pcsk9 inactivation on cholesterol efflux capacity of serum using THP-1 and J774.A1 macrophage foam cells and atherosclerotic fatty streak volume in the aortic sinus of Pcsk9 knockout males fed an atherogenic diet. Results APOE and APOA1 were reduced in the same HDL subfractions of Pcsk9 knockout and human LDLR transgenic male mice. In Pcsk9/Ldlr double-knockout mice, HDL cholesterol concentration was lower than in Ldlr knockout mice and higher than in wild-type controls. In Pcsk9/Apoe double-knockout mice, HDL cholesterol concentration was similar to that of Apoe knockout males. In Pcsk9 knockout males, THP-1 macrophage cholesterol efflux capacity of serum was reduced and the fatty streak lesion volume was similar to wild-type controls. Conclusions In mice, LDLR and APOE are important factors for PCSK9-mediated HDL regulation. Our data suggest that, although LDLR plays a major role in PCSK9-mediated regulation of HDL cholesterol concentration, it is not the only mechanism and that, regardless of mechanism, APOE is essential. Pcsk9 inactivation decreases the HDL cholesterol concentration and cholesterol efflux capacity in serum, but does not increase atherosclerotic fatty streak volume. PMID:23883163

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.

An evolutionarily conserved NIMA-related kinase directs rhizoid tip growth in the basal land plant Marchantia polymorpha.

PubMed

Otani, Kento; Ishizaki, Kimitsune; Nishihama, Ryuichi; Takatani, Shogo; Kohchi, Takayuki; Takahashi, Taku; Motose, Hiroyasu

2018-03-01

Tip growth is driven by turgor pressure and mediated by the polarized accumulation of cellular materials. How a single polarized growth site is established and maintained is unclear. Here, we analyzed the function of NIMA-related protein kinase 1 (MpNEK1) in the liverwort Marchantia polymorpha In the wild type, rhizoid cells differentiate from the ventral epidermis and elongate through tip growth to form hair-like protrusions. In Mp nek1 knockout mutants, rhizoids underwent frequent changes in growth direction, resulting in a twisted and/or spiral morphology. The functional MpNEK1-Citrine protein fusion localized to microtubule foci in the apical growing region of rhizoids. Mp nek1 knockouts exhibited increases in both microtubule density and bundling in the apical dome of rhizoids. Treatment with the microtubule-stabilizing drug taxol phenocopied the Mp nek1 knockout. These results suggest that MpNEK1 directs tip growth in rhizoids through microtubule organization. Furthermore, MpNEK1 expression rescued ectopic outgrowth of epidermal cells in the Arabidopsis thaliana nek6 mutant, strongly supporting an evolutionarily conserved NEK-dependent mechanism of directional growth. It is possible that such a mechanism contributed to the evolution of the early rooting system in land plants. © 2018. Published by The Company of Biologists Ltd.

Gli function is essential for motor neuron induction in zebrafish.

PubMed

Vanderlaan, Gary; Tyurina, Oksana V; Karlstrom, Rolf O; Chandrasekhar, Anand

2005-06-15

The Gli family of zinc-finger transcription factors mediates Hedgehog (Hh) signaling in all vertebrates. However, their roles in ventral neural tube patterning, in particular motor neuron induction, appear to have diverged across species. For instance, cranial motor neurons are essentially lost in zebrafish detour (gli1(-)) mutants, whereas motor neuron development is unaffected in mouse single gli and some double gli knockouts. Interestingly, the expression of some Hh-regulated genes (ptc1, net1a, gli1) is mostly unaffected in the detour mutant hindbrain, suggesting that other Gli transcriptional activators may be involved. To better define the roles of the zebrafish gli genes in motor neuron induction and in Hh-regulated gene expression, we examined these processes in you-too (yot) mutants, which encode dominant repressor forms of Gli2 (Gli2(DR)), and following morpholino-mediated knockdown of gli1, gli2, and gli3 function. Motor neuron induction at all axial levels was reduced in yot (gli2(DR)) mutant embryos. In addition, Hh target gene expression at all axial levels except in rhombomere 4 was also reduced, suggesting an interference with the function of other Glis. Indeed, morpholino-mediated knockdown of Gli2(DR) protein in yot mutants led to a suppression of the defective motor neuron phenotype. However, gli2 knockdown in wild-type embryos generated no discernable motor neuron phenotype, while gli3 knockdown reduced motor neuron induction in the hindbrain and spinal cord. Significantly, gli2 or gli3 knockdown in detour (gli1(-)) mutants revealed roles for Gli2 and Gli3 activator functions in ptc1 expression and spinal motor neuron induction. Similarly, gli1 or gli3 knockdown in yot (gli2(DR)) mutants resulted in severe or complete loss of motor neurons, and of ptc1 and net1a expression, in the hindbrain and spinal cord. In addition, gli1 expression was greatly reduced in yot mutants following gli3, but not gli1, knockdown, suggesting that Gli3 activator function is specifically required for gli1 expression. These observations demonstrate that Gli activator function (encoded by gli1, gli2, and gli3) is essential for motor neuron induction and Hh-regulated gene expression in zebrafish.

High Levels of S100A8/A9 Proteins Aggravate Ventilator-Induced Lung Injury via TLR4 Signaling

PubMed Central

Aslami, Hamid; Jongsma, Geartsje; van den Berg, Elske; Vlaar, Alexander P. J.; Roelofs, Joris J. T. H.; Juffermans, Nicole P.; Schultz, Marcus J.; van der Poll, Tom; Roth, Johannes; Wieland, Catharina W.

2013-01-01

Background Bacterial products add to mechanical ventilation in enhancing lung injury. The role of endogenous triggers of innate immunity herein is less well understood. S100A8/A9 proteins are released by phagocytes during inflammation. The present study investigates the role of S100A8/A9 proteins in ventilator-induced lung injury. Methods Pulmonary S100A8/A9 levels were measured in samples obtained from patients with and without lung injury. Furthermore, wild-type and S100A9 knock-out mice, naive and with lipopolysaccharide-induced injured lungs, were randomized to 5 hours of spontaneously breathing or mechanical ventilation with low or high tidal volume (VT). In addition, healthy spontaneously breathing and high VT ventilated mice received S100A8/A9, S100A8 or vehicle intratracheal. Furthermore, the role of Toll-like receptor 4 herein was investigated. Results S100A8/A9 protein levels were elevated in patients and mice with lung injury. S100A8/A9 levels synergistically increased upon the lipopolysaccharide/high VT MV double hit. Markers of alveolar barrier dysfunction, cytokine and chemokine levels, and histology scores were attenuated in S100A9 knockout mice undergoing the double-hit. Exogenous S100A8/A9 and S100A8 induced neutrophil influx in spontaneously breathing mice. In ventilated mice, these proteins clearly amplified inflammation: neutrophil influx, cytokine, and chemokine levels were increased compared to ventilated vehicle-treated mice. In contrast, administration of S100A8/A9 to ventilated Toll-like receptor 4 mutant mice did not augment inflammation. Conclusion S100A8/A9 proteins increase during lung injury and contribute to inflammation induced by HVT MV combined with lipopolysaccharide. In the absence of lipopolysaccharide, high levels of extracellular S100A8/A9 still amplify ventilator-induced lung injury via Toll-like receptor 4. PMID:23874727

Active site-directed double mutants of dihydrofolate reductase.

PubMed

Ercikan-Abali, E A; Mineishi, S; Tong, Y; Nakahara, S; Waltham, M C; Banerjee, D; Chen, W; Sadelain, M; Bertino, J R

1996-09-15

Variants of dihydrofolate reductase (DHFR), which confer resistance to antifolates, are used as dominant selectable markers in vitro and in vivo and may be useful in the context of gene therapy. To identify improved mutant human DHFRs with increased catalytic efficiency and decreased binding to methotrexate, we constructed by site-directed mutagenesis four variants with substitutions at both Leu22 and Phe31 (i.e., Phe22-Ser31, Tyr22-Ser31, Phe22-Gly31, and Tyr22-Gly31). Antifolate resistance has been observed previously when individual changes are made at these active-site residues. Substrate and antifolate binding properties of these "double" mutants revealed that each have greatly diminished affinity for antifolates (> 10,000-fold) yet only slightly reduced substrate affinity. Comparison of in vitro measured properties with those of single-residue variants indicates that double mutants are indeed significantly superior. This was verified for one of the double mutants that provided high-level methotrexate resistance following retrovirus-mediated gene transfer in NIH3T3 cells.

pKAMA-ITACHI Vectors for Highly Efficient CRISPR/Cas9-Mediated Gene Knockout in Arabidopsis thaliana

PubMed Central

2017-01-01

The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) system is widely used as a tool for genome engineering in various organisms. A complex consisting of Cas9 and single guide RNA (sgRNA) induces a DNA double-strand break in a sequence-specific manner, resulting in knockout. Some binary vectors for CRISPR/Cas9 in plants have been reported, but there is a problem with low efficiency. Here, we present a newly developed, highly efficient CRISPR/Cas9 vector for Arabidopsis thaliana, pKAMA-ITACHI Red (pKIR), harboring the RIBOSOMAL PROTEIN S5 A (RPS5A) promoter to drive Cas9. The RPS5A promoter maintains high constitutive expression at all developmental stages starting from the egg cell and including meristematic cells. Even in the T1 generation, pKIR induced null phenotypes in some genes: PHYTOENE DESATURASE 3 (PDS3), AGAMOUS (AG) and DUO POLLEN 1 (DUO1). Mutations induced by pKIR were carried in the germ cell line of the T1 generation. Surprisingly, in some lines, 100% of the T2 plants had the adh1 (ALCOHOL DEHYDROGENASE 1) null phenotype, indicating that pKIR strongly induced heritable mutations. Cas9-free T2 mutant plants were obtained by removing T2 seeds expressing a fluorescent marker in pKIR. Our results suggest that the pKIR system is a powerful molecular tool for genome engineering in Arabidopsis. PMID:27856772

Multigene knockout utilizing off-target mutations of the CRISPR/Cas9 system in rice.

PubMed

Endo, Masaki; Mikami, Masafumi; Toki, Seiichi

2015-01-01

The clustered regularly interspaced short palindromic repeat (CRISPR)-associated endonuclease 9 (CRISPR/Cas9) system has been demonstrated to be a robust genome engineering tool in a variety of organisms including plants. However, it has been shown that the CRISPR/Cas9 system cleaves genomic DNA sequences containing mismatches to the guide RNA strand. We expected that this low specificity could be exploited to induce multihomeologous and multiparalogous gene knockouts. In the case of polyploid plants, simultaneous modification of multiple homeologous genes, i.e. genes with similar but not identical DNA sequences, is often needed to obtain a desired phenotype. Even in diploid plants, disruption of multiparalogous genes, which have functional redundancy, is often needed. To validate the applicability of the CRISPR/Cas9 system to target mutagenesis of paralogous genes in rice, we designed a single-guide RNA (sgRNA) that recognized 20 bp sequences of cyclin-dependent kinase B2 (CDKB2) as an on-target locus. These 20 bp possess similarity to other rice CDK genes (CDKA1, CDKA2 and CDKB1) with different numbers of mismatches. We analyzed mutations in these four CDK genes in plants regenerated from Cas9/sgRNA-transformed calli and revealed that single, double and triple mutants of CDKA2, CDKB1 and CDKB2 can be created by a single sgRNA. © The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.

Pyviko: an automated Python tool to design gene knockouts in complex viruses with overlapping genes.

PubMed

Taylor, Louis J; Strebel, Klaus

2017-01-07

Gene knockouts are a common tool used to study gene function in various organisms. However, designing gene knockouts is complicated in viruses, which frequently contain sequences that code for multiple overlapping genes. Designing mutants that can be traced by the creation of new or elimination of existing restriction sites further compounds the difficulty in experimental design of knockouts of overlapping genes. While software is available to rapidly identify restriction sites in a given nucleotide sequence, no existing software addresses experimental design of mutations involving multiple overlapping amino acid sequences in generating gene knockouts. Pyviko performed well on a test set of over 240,000 gene pairs collected from viral genomes deposited in the National Center for Biotechnology Information Nucleotide database, identifying a point mutation which added a premature stop codon within the first 20 codons of the target gene in 93.2% of all tested gene-overprinted gene pairs. This shows that Pyviko can be used successfully in a wide variety of contexts to facilitate the molecular cloning and study of viral overprinted genes. Pyviko is an extensible and intuitive Python tool for designing knockouts of overlapping genes. Freely available as both a Python package and a web-based interface ( http://louiejtaylor.github.io/pyViKO/ ), Pyviko simplifies the experimental design of gene knockouts in complex viruses with overlapping genes.

Prevalence of sexual dimorphism in mammalian phenotypic traits

PubMed Central

Karp, Natasha A.; Mason, Jeremy; Beaudet, Arthur L.; Benjamini, Yoav; Bower, Lynette; Braun, Robert E.; Brown, Steve D.M.; Chesler, Elissa J.; Dickinson, Mary E.; Flenniken, Ann M.; Fuchs, Helmut; Angelis, Martin Hrabe de; Gao, Xiang; Guo, Shiying; Greenaway, Simon; Heller, Ruth; Herault, Yann; Justice, Monica J.; Kurbatova, Natalja; Lelliott, Christopher J.; Lloyd, K.C. Kent; Mallon, Ann-Marie; Mank, Judith E.; Masuya, Hiroshi; McKerlie, Colin; Meehan, Terrence F.; Mott, Richard F.; Murray, Stephen A.; Parkinson, Helen; Ramirez-Solis, Ramiro; Santos, Luis; Seavitt, John R.; Smedley, Damian; Sorg, Tania; Speak, Anneliese O.; Steel, Karen P.; Svenson, Karen L.; Obata, Yuichi; Suzuki, Tomohiro; Tamura, Masaru; Kaneda, Hideki; Furuse, Tamio; Kobayashi, Kimio; Miura, Ikuo; Yamada, Ikuko; Tanaka, Nobuhiko; Yoshiki, Atsushi; Ayabe, Shinya; Clary, David A.; Tolentino, Heather A.; Schuchbauer, Michael A.; Tolentino, Todd; Aprile, Joseph Anthony; Pedroia, Sheryl M.; Kelsey, Lois; Vukobradovic, Igor; Berberovic, Zorana; Owen, Celeste; Qu, Dawei; Guo, Ruolin; Newbigging, Susan; Morikawa, Lily; Law, Napoleon; Shang, Xueyuan; Feugas, Patricia; Wang, Yanchun; Eskandarian, Mohammad; Zhu, Yingchun; Nutter, Lauryl M. J.; Penton, Patricia; Laurin, Valerie; Clarke, Shannon; Lan, Qing; Sohel, Khondoker; Miller, David; Clark, Greg; Hunter, Jane; Cabezas, Jorge; Bubshait, Mohammed; Carroll, Tracy; Tondat, Sandra; MacMaster, Suzanne; Pereira, Monica; Gertsenstein, Marina; Danisment, Ozge; Jacob, Elsa; Creighton, Amie; Sleep, Gillian; Clark, James; Teboul, Lydia; Fray, Martin; Caulder, Adam; Loeffler, Jorik; Codner, Gemma; Cleak, James; Johnson, Sara; Szoke-Kovacs, Zsombor; Radage, Adam; Maritati, Marina; Mianne, Joffrey; Gardiner, Wendy; Allen, Susan; Cater, Heather; Stewart, Michelle; Keskivali-Bond, Piia; Sinclair, Caroline; Brown, Ellen; Doe, Brendan; Wardle-Jones, Hannah; Grau, Evelyn; Griggs, Nicola; Woods, Mike; Kundi, Helen; Griffiths, Mark N. D.; Kipp, Christian; Melvin, David G.; Raj, Navis P. S.; Holroyd, Simon A.; Gannon, David J.; Alcantara, Rafael; Galli, Antonella; Hooks, Yvette E.; Tudor, Catherine L.; Green, Angela L.; Kussy, Fiona L.; Tuck, Elizabeth J.; Siragher, Emma J.; Maguire, Simon A.; Lafont, David T.; Vancollie, Valerie E.; Pearson, Selina A.; Gates, Amy S.; Sanderson, Mark; Shannon, Carl; Anthony, Lauren F. E.; Sumowski, Maksymilian T.; McLaren, Robbie S. B.; Swiatkowska, Agnieszka; Isherwood, Christopher M.; Cambridge, Emma L; Wilson, Heather M.; Caetano, Susana S.; Mazzeo, Cecilia Icoresi; Dabrowska, Monika H.; Lillistone, Charlotte; Estabel, Jeanne; Maguire, Anna Karin B.; Roberson, Laura-Anne; Pavlovic, Guillaume; Birling, Marie-Christine; Marie, Wattenhofer-Donze; Jacquot, Sylvie; Ayadi, Abdel; Ali-Hadji, Dalila; Charles, Philippe; André, Philippe; Le Marchand, Elise; El Amri, Amal; Vasseur, Laurent; Aguilar-Pimentel, Antonio; Becker, Lore; Treise, Irina; Moreth, Kristin; Stoeger, Tobias; Amarie, Oana V.; Neff, Frauke; Wurst, Wolfgang; Bekeredjian, Raffi; Ollert, Markus; Klopstock, Thomas; Calzada-Wack, Julia; Marschall, Susan; Brommage, Robert; Steinkamp, Ralph; Lengger, Christoph; Östereicher, Manuela A.; Maier, Holger; Stoeger, Claudia; Leuchtenberger, Stefanie; Yildrim, AliÖ; Garrett, Lillian; Hölter, Sabine M; Zimprich, Annemarie; Seisenberger, Claudia; Bürger, Antje; Graw, Jochen; Eickelberg, Oliver; Zimmer, Andreas; Wolf, Eckhard; Busch, Dirk H; Klingenspor, Martin; Schmidt-Weber, Carsten; Gailus-Durner, Valérie; Beckers, Johannes; Rathkolb, Birgit; Rozman, Jan; Wakana, Shigeharu; West, David; Wells, Sara; Westerberg, Henrik; Yaacoby, Shay; White, Jacqueline K.

2017-01-01

The role of sex in biomedical studies has often been overlooked, despite evidence of sexually dimorphic effects in some biological studies. Here, we used high-throughput phenotype data from 14,250 wildtype and 40,192 mutant mice (representing 2,186 knockout lines), analysed for up to 234 traits, and found a large proportion of mammalian traits both in wildtype and mutants are influenced by sex. This result has implications for interpreting disease phenotypes in animal models and humans. PMID:28650954

Postsynaptic P2X3-containing receptors in gustatory nerve fibres mediate responses to all taste qualities in mice

PubMed Central

Vandenbeuch, Aurelie; Larson, Eric D; Anderson, Catherine B; Smith, Steven A; Ford, Anthony P; Finger, Thomas E; Kinnamon, Sue C

2015-01-01

Abstract Taste buds release ATP to activate ionotropic purinoceptors composed of P2X2 and P2X3 subunits, present on the taste nerves. Mice with genetic deletion of P2X2 and P2X3 receptors (double knockout mice) lack responses to all taste stimuli presumably due to the absence of ATP-gated receptors on the afferent nerves. Recent experiments on the double knockout mice showed, however, that their taste buds fail to release ATP, suggesting the possibility of pleiotropic deficits in these global knockouts. To test further the role of postsynaptic P2X receptors in afferent signalling, we used AF-353, a selective antagonist of P2X3-containing receptors to inhibit the receptors acutely during taste nerve recording and behaviour. The specificity of AF-353 for P2X3-containing receptors was tested by recording Ca2+ transients to exogenously applied ATP in fura-2 loaded isolated geniculate ganglion neurons from wild-type and P2X3 knockout mice. ATP responses were completely inhibited by 10 μm or 100 μm AF-353, but neither concentration blocked responses in P2X3 single knockout mice wherein the ganglion cells express only P2X2-containing receptors. Furthermore, AF-353 had no effect on taste-evoked ATP release from taste buds. In wild-type mice, i.p. injection of AF-353 or simple application of the drug directly to the tongue, inhibited taste nerve responses to all taste qualities in a dose-dependent fashion. A brief access behavioural assay confirmed the electrophysiological results and showed that preference for a synthetic sweetener, SC-45647, was abolished following i.p. injection of AF-353. These data indicate that activation of P2X3-containing receptors is required for transmission of all taste qualities. Key points Acute inhibition of purinergic receptors with a selective P2X3 antagonist prevents transmission of information from taste buds to sensory nerves. The P2X3 antagonist has no effect on taste-evoked release of ATP, confirming the effect is postsynaptic. The results confirm previous results with P2X2/3 double knockout mice that ATP is required for transmission of all taste qualities, including sour and salty. Previously, ATP was confirmed to be required for bitter, sweet and umami tastes, but was questioned for salty and sour tastes due to pleomorphic deficits in the double knockout mice. The geniculate ganglion in mouse contains two populations of ganglion cells with different subunit composition of P2X2 and P2X3 receptors making them differently susceptible to pharmacological block and, presumably, desensitization. PMID:25524179

An ABA-regulated and Golgi-localized protein phosphatase controls water loss during leaf senescence in Arabidopsis.

PubMed

Zhang, Kewei; Xia, Xiuying; Zhang, Yanyan; Gan, Su-Sheng

2012-02-01

It is known that a senescing leaf loses water faster than a non-senescing leaf and that ABA has an important role in promoting leaf senescence. However, questions such as why water loss is faster, how water loss is regulated, and how ABA functions in leaf senescence are not well understood. Here we report on the identification and functional analysis of a leaf senescence associated gene called SAG113. The RNA blot and GUS reporter analyses all show that SAG113 is expressed in senescing leaves and is induced by ABA in Arabidopsis. The SAG113 expression levels are significantly reduced in aba2 and abi4 mutants. A GFP fusion protein analysis revealed that SAG113 protein is localized in the Golgi apparatus. SAG113 encodes a protein phosphatase that belongs to the PP2C family and is able to functionally complement a yeast PP2C-deficient mutant TM126 (ptc1Δ). Leaf senescence is delayed in the SAG113 knockout mutant compared with that in the wild type, stomatal movement in the senescing leaves of SAG113 knockouts is more sensitive to ABA than that of the wild type, and the rate of water loss in senescing leaves of SAG113 knockouts is significantly reduced. In contrast, inducible over-expression of SAG113 results in a lower sensitivity of stomatal movement to ABA treatment, more rapid water loss, and precocious leaf senescence. No other aspects of growth and development, including seed germination, were observed. These findings suggest that SAG113, a negative regulator of ABA signal transduction, is specifically involved in the control of water loss during leaf senescence. © 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.

A Negative Regulator of Cellulose Biosynthesis, bcsR, Affects Biofilm Formation, and Adhesion/Invasion Ability of Cronobacter sakazakii.

PubMed

Gao, Jian-Xin; Li, Ping; Du, Xin-Jun; Han, Zhong-Hui; Xue, Rui; Liang, Bin; Wang, Shuo

2017-01-01

Cronobacter sakazakii is an important foodborne pathogen that causes neonatal meningitis and sepsis, with high mortality in neonates. However, very little information is available regarding the pathogenesis of C. sakazakii at the genetic level. In our previous study, a cellulose biosynthesis-related gene ( bcsR ) was shown to be involved in C. sakazakii adhesion/invasion into epithelial cells. In this study, the detailed functions of this gene were investigated using a gene knockout technique. A bcsR knockout mutant (Δ bcsR ) of C. sakazakii ATCC BAA-894 showed decreased adhesion/invasion (3.9-fold) in human epithelial cell line HCT-8. Biofilm formation by the mutant was reduced to 50% of that exhibited by the wild-type (WT) strain. Raman spectrometry was used to detect variations in biofilm components caused by bcsR knockout, and certain components, including carotenoids, fatty acids, and amides, were significantly reduced. However, another biofilm component, cellulose, was increased in Δ bcsR , suggesting that bcsR negatively affects cellulose biosynthesis. This result was also verified via RT-PCR, which demonstrated up-regulation of five crucial cellulose synthesis genes ( bcsA, B, C, E, Q ) in Δ bcsR . Furthermore, the expression of other virulence or biofilm-related genes, including flagellar assembly genes ( fliA, C, D ) and toxicity-related genes ( ompA, ompX, hfq ), was studied. The expression of fliC and ompA in the Δ bcsR mutant was found to be remarkably reduced compared with that in the wild-type and the others were also affected excepted ompX . In summary, bcsR is a negative regulator of cellulose biosynthesis but positively regulates biofilm formation and the adhesion/invasion ability of C. sakazakii .

P2X2 knockout mice and P2X2/P2X3 double knockout mice reveal a role for the P2X2 receptor subunit in mediating multiple sensory effects of ATP

PubMed Central

Cockayne, Debra A; Dunn, Philip M; Zhong, Yu; Rong, Weifang; Hamilton, Sara G; Knight, Gillian E; Ruan, Huai-Zhen; Ma, Bei; Yip, Ping; Nunn, Philip; McMahon, Stephen B; Burnstock, Geoffrey; Ford, Anthony PDW

2005-01-01

Extracellular ATP plays a role in nociceptive signalling and sensory regulation of visceral function through ionotropic receptors variably composed of P2X2 and P2X3 subunits. P2X2 and P2X3 subunits can form homomultimeric P2X2, homomultimeric P2X3, or heteromultimeric P2X2/3 receptors. However, the relative contribution of these receptor subtypes to afferent functions of ATP in vivo is poorly understood. Here we describe null mutant mice lacking the P2X2 receptor subunit (P2X2−/−) and double mutant mice lacking both P2X2 and P2X3 subunits (P2X2/P2X3Dbl−/−), and compare these with previously characterized P2X3−/− mice. In patch-clamp studies, nodose, coeliac and superior cervical ganglia (SCG) neurones from wild-type mice responded to ATP with sustained inward currents, while dorsal root ganglia (DRG) neurones gave predominantly transient currents. Sensory neurones from P2X2−/− mice responded to ATP with only transient inward currents, while sympathetic neurones had barely detectable responses. Neurones from P2X2/P2X3Dbl−/− mice had minimal to no response to ATP. These data indicate that P2X receptors on sensory and sympathetic ganglion neurones involve almost exclusively P2X2 and P2X3 subunits. P2X2−/− and P2X2/P2X3Dbl−/− mice had reduced pain-related behaviours in response to intraplantar injection of formalin. Significantly, P2X3−/−, P2X2−/−, and P2X2/P2X3Dbl−/− mice had reduced urinary bladder reflexes and decreased pelvic afferent nerve activity in response to bladder distension. No deficits in a wide variety of CNS behavioural tests were observed in P2X2−/− mice. Taken together, these data extend our findings for P2X3−/− mice, and reveal an important contribution of heteromeric P2X2/3 receptors to nociceptive responses and mechanosensory transduction within the urinary bladder. PMID:15961431

Nppa and Nppb act redundantly during zebrafish cardiac development to confine AVC marker expression and reduce cardiac jelly volume.

PubMed

Grassini, Daniela R; Lagendijk, Anne K; De Angelis, Jessica E; Da Silva, Jason; Jeanes, Angela; Zettler, Nicole; Bower, Neil I; Hogan, Benjamin M; Smith, Kelly A

2018-05-11

Atrial natriuretic peptide ( nppa/anf ) and brain natriuretic peptide ( nppb/bnp ) form a gene cluster with expression in the chambers of the developing heart. Despite restricted expression, a function in cardiac development has not been demonstrated by mutant analysis. This is attributed to functional redundancy however their genomic location in cis has impeded formal analysis. Using genome-editing, we generated mutants for nppa and nppb and found single mutants indistinguishable from wildtype whereas nppa / nppb double mutants display heart morphogenesis defects and pericardial oedema. Analysis of atrioventricular canal (AVC) markers show expansion of bmp4 , tbx2b, has2 and versican expression into the atrium of double mutants. This expanded expression correlates with increased extracellular matrix in the atrium. Using a biosensor for Hyaluronic acid to measure the cardiac jelly (cardiac extracellular matrix), we confirm cardiac jelly expansion in nppa / nppb double mutants. Finally, bmp4 knockdown rescues the expansion of has2 expression and cardiac jelly in double mutants. This definitively shows that nppa and nppb function redundantly during cardiac development to restrict gene expression to the AVC, preventing excessive cardiac jelly synthesis in the atrial chamber. © 2018. Published by The Company of Biologists Ltd.

Transforming Growth Factor Beta (TGFβ1, TGFβ2 and TGFβ3) Null-Mutant Phenotypes in Embryonic Gonadal Development

PubMed Central

Memon, Mushtaq A.; Anway, Matthew D.; Covert, Trevor R.; Uzumcu, Mehmet; Skinner, Michael K.

2008-01-01

The role transforming growth factor beta (TGFb) isoforms TGFb1, TGFb2 and TGFb3 have in the regulation of embryonic gonadal development was investigated with the use of null-mutant (i.e. knockout) mice for each of the TGFb isoforms. Late embryonic gonadal development was investigated because homozygote TGFb null-mutant mice generally die around birth, with some embryonic loss as well. In the testis, the TGFb1 null-mutant mice had a decrease in the number of germ cells at birth, postnatal day 0 (P0). In the testis, the TGFb2 null-mutant mice had a decrease in the number of seminiferous cords at embryonic day 15 (E15). In the ovary, the TGFb2 null-mutant mice had an increase in the number of germ cells at P0. TGFb isoforms appear to have a role in gonadal development, but interactions between the isoforms is speculated to compensate in the different TGFb isoform null-mutant mice. PMID:18790002

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.

MRE11 and RAD50, but not NBS1, are essential for gene targeting in the moss Physcomitrella patens.

PubMed

Kamisugi, Yasuko; Schaefer, Didier G; Kozak, Jaroslav; Charlot, Florence; Vrielynck, Nathalie; Holá, Marcela; Angelis, Karel J; Cuming, Andrew C; Nogué, Fabien

2012-04-01

The moss Physcomitrella patens is unique among plant models for the high frequency with which targeted transgene insertion occurs via homologous recombination. Transgene integration is believed to utilize existing machinery for the detection and repair of DNA double-strand breaks (DSBs). We undertook targeted knockout of the Physcomitrella genes encoding components of the principal sensor of DNA DSBs, the MRN complex. Loss of function of PpMRE11 or PpRAD50 strongly and specifically inhibited gene targeting, whilst rates of untargeted transgene integration were relatively unaffected. In contrast, disruption of the PpNBS1 gene retained the wild-type capacity to integrate transforming DNA efficiently at homologous loci. Analysis of the kinetics of DNA-DSB repair in wild-type and mutant plants by single-nucleus agarose gel electrophoresis revealed that bleomycin-induced fragmentation of genomic DNA was repaired at approximately equal rates in each genotype, although both the Ppmre11 and Pprad50 mutants exhibited severely restricted growth and development and enhanced sensitivity to UV-B and bleomycin-induced DNA damage, compared with wild-type and Ppnbs1 plants. This implies that while extensive DNA repair can occur in the absence of a functional MRN complex; this is unsupervised in nature and results in the accumulation of deleterious mutations incompatible with normal growth and development.

MRE11 and RAD50, but not NBS1, are essential for gene targeting in the moss Physcomitrella patens

PubMed Central

Kamisugi, Yasuko; Schaefer, Didier G.; Kozak, Jaroslav; Charlot, Florence; Vrielynck, Nathalie; Holá, Marcela; Angelis, Karel J.; Cuming, Andrew C.; Nogué, Fabien

2012-01-01

The moss Physcomitrella patens is unique among plant models for the high frequency with which targeted transgene insertion occurs via homologous recombination. Transgene integration is believed to utilize existing machinery for the detection and repair of DNA double-strand breaks (DSBs). We undertook targeted knockout of the Physcomitrella genes encoding components of the principal sensor of DNA DSBs, the MRN complex. Loss of function of PpMRE11 or PpRAD50 strongly and specifically inhibited gene targeting, whilst rates of untargeted transgene integration were relatively unaffected. In contrast, disruption of the PpNBS1 gene retained the wild-type capacity to integrate transforming DNA efficiently at homologous loci. Analysis of the kinetics of DNA-DSB repair in wild-type and mutant plants by single-nucleus agarose gel electrophoresis revealed that bleomycin-induced fragmentation of genomic DNA was repaired at approximately equal rates in each genotype, although both the Ppmre11 and Pprad50 mutants exhibited severely restricted growth and development and enhanced sensitivity to UV-B and bleomycin-induced DNA damage, compared with wild-type and Ppnbs1 plants. This implies that while extensive DNA repair can occur in the absence of a functional MRN complex; this is unsupervised in nature and results in the accumulation of deleterious mutations incompatible with normal growth and development. PMID:22210882

Bile Acid Sodium Symporter BASS6 Can Transport Glycolate and Is Involved in Photorespiratory Metabolism in Arabidopsis thaliana[OPEN

PubMed Central

Badger, Murray

2017-01-01

Photorespiration is an energy-intensive process that recycles 2-phosphoglycolate, a toxic product of the Rubisco oxygenation reaction. The photorespiratory pathway is highly compartmentalized, involving the chloroplast, peroxisome, cytosol, and mitochondria. Though the soluble enzymes involved in photorespiration are well characterized, very few membrane transporters involved in photorespiration have been identified to date. In this work, Arabidopsis thaliana plants containing a T-DNA disruption of the bile acid sodium symporter BASS6 show decreased photosynthesis and slower growth under ambient, but not elevated CO2. Exogenous expression of BASS6 complemented this photorespiration mutant phenotype. In addition, metabolite analysis and genetic complementation of glycolate transport in yeast showed that BASS6 was capable of glycolate transport. This is consistent with its involvement in the photorespiratory export of glycolate from Arabidopsis chloroplasts. An Arabidopsis double knockout line of both BASS6 and the glycolate/glycerate transporter PLGG1 (bass6, plgg1) showed an additive growth defect, an increase in glycolate accumulation, and reductions in photosynthetic rates compared with either single mutant. Our data indicate that BASS6 and PLGG1 partner in glycolate export from the chloroplast, whereas PLGG1 alone accounts for the import of glycerate. BASS6 and PLGG1 therefore balance the export of two glycolate molecules with the import of one glycerate molecule during photorespiration. PMID:28351992

Hesr1 and Hesr3 are essential to generate undifferentiated quiescent satellite cells and to maintain satellite cell numbers

PubMed Central

Fukada, So-ichiro; Yamaguchi, Masahiko; Kokubo, Hiroki; Ogawa, Ryo; Uezumi, Akiyoshi; Yoneda, Tomohiro; Matev, Miroslav M.; Motohashi, Norio; Ito, Takahito; Zolkiewska, Anna; Johnson, Randy L.; Saga, Yumiko; Miyagoe-Suzuki, Yuko; Tsujikawa, Kazutake; Takeda, Shin’ichi; Yamamoto, Hiroshi

2011-01-01

Satellite cells, which are skeletal muscle stem cells, divide to provide new myonuclei to growing muscle fibers during postnatal development, and then are maintained in an undifferentiated quiescent state in adult skeletal muscle. This state is considered to be essential for the maintenance of satellite cells, but their molecular regulation is unknown. We show that Hesr1 (Hey1) and Hesr3 (Heyl) (which are known Notch target genes) are expressed simultaneously in skeletal muscle only in satellite cells. In Hesr1 and Hesr3 single-knockout mice, no obvious abnormalities of satellite cells or muscle regenerative potentials are observed. However, the generation of undifferentiated quiescent satellite cells is impaired during postnatal development in Hesr1/3 double-knockout mice. As a result, myogenic (MyoD and myogenin) and proliferative (Ki67) proteins are expressed in adult satellite cells. Consistent with the in vivo results, Hesr1/3-null myoblasts generate very few Pax7+ MyoD– undifferentiated cells in vitro. Furthermore, the satellite cell number gradually decreases in Hesr1/3 double-knockout mice even after it has stabilized in control mice, and an age-dependent regeneration defect is observed. In vivo results suggest that premature differentiation, but not cell death, is the reason for the reduced number of satellite cells in Hesr1/3 double-knockout mice. These results indicate that Hesr1 and Hesr3 are essential for the generation of adult satellite cells and for the maintenance of skeletal muscle homeostasis. PMID:21989910

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

PubMed

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

2015-09-01

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

Directed evolution to re-adapt a co-evolved network within an enzyme.

PubMed

Strafford, John; Payongsri, Panwajee; Hibbert, Edward G; Morris, Phattaraporn; Batth, Sukhjeet S; Steadman, David; Smith, Mark E B; Ward, John M; Hailes, Helen C; Dalby, Paul A

2012-01-01

We have previously used targeted active-site saturation mutagenesis to identify a number of transketolase single mutants that improved activity towards either glycolaldehyde (GA), or the non-natural substrate propionaldehyde (PA). Here, all attempts to recombine the singles into double mutants led to unexpected losses of specific activity towards both substrates. A typical trade-off occurred between soluble expression levels and specific activity for all single mutants, but many double mutants decreased both properties more severely suggesting a critical loss of protein stability or native folding. Statistical coupling analysis (SCA) of a large multiple sequence alignment revealed a network of nine co-evolved residues that affected all but one double mutant. Such networks maintain important functional properties such as activity, specificity, folding, stability, and solubility and may be rapidly disrupted by introducing one or more non-naturally occurring mutations. To identify variants of this network that would accept and improve upon our best D469 mutants for activity towards PA, we created a library of random single, double and triple mutants across seven of the co-evolved residues, combining our D469 variants with only naturally occurring mutations at the remaining sites. A triple mutant cluster at D469, E498 and R520 was found to behave synergistically for the specific activity towards PA. Protein expression was severely reduced by E498D and improved by R520Q, yet variants containing both mutations led to improved specific activity and enzyme expression, but with loss of solubility and the formation of inclusion bodies. D469S and R520Q combined synergistically to improve k(cat) 20-fold for PA, more than for any previous transketolase mutant. R520Q also doubled the specific activity of the previously identified D469T to create our most active transketolase mutant to date. Our results show that recombining active-site mutants obtained by saturation mutagenesis can rapidly destabilise critical networks of co-evolved residues, whereas beneficial single mutants can be retained and improved upon by randomly recombining them with natural variants at other positions in the network. Copyright © 2011 Elsevier B.V. All rights reserved.

Genetic Rescue of Glycosylation-deficient Fgf23 in the Galnt3 Knockout Mouse

PubMed Central

Gray, Amie K.; Padgett, Leah R.; Allen, Matthew R.; Clinkenbeard, Erica L.; Sarpa, Nicole M.; White, Kenneth E.; Econs, Michael J.

2014-01-01

Fibroblast growth factor 23 (FGF23) is a hormone that inhibits renal phosphate reabsorption and 1,25-dihydroxyvitamin D biosynthesis. The FGF23 subtilisin-like proprotein convertase recognition sequence (176RHTR179↓) is protected by O-glycosylation through ppGalNAc-T3 (GALNT3) activity. Thus, inactivating GALNT3 mutations render FGF23 susceptible to proteolysis, thereby reducing circulating intact hormone levels and leading to hyperphosphatemic familial tumoral calcinosis. To further delineate the role of glycosylation in the Fgf23 function, we generated an inducible FGF23 transgenic mouse expressing human mutant FGF23 (R176Q and R179Q) found in patients with autosomal dominant hypophosphatemic rickets (ADHR) and bred this animal to Galnt3 knockout mice, a model of familial tumoral calcinosis. Due to the low intact Fgf23 level, Galnt3 knockout mice with wild-type Fgf23 alleles were hyperphosphatemic. In contrast, carriers of the mutant FGF23 transgene, regardless of Galnt3 mutation status, had significantly higher serum intact FGF23, resulting in severe hypophosphatemia. Importantly, serum phosphorus and FGF23 were comparable between transgenic mice with or without normal Galnt3 alleles. To determine whether the presence of the ADHR mutation could improve biochemical and skeletal abnormalities in Galnt3-null mice, these mice were also mated to Fgf23 knock-in mice, carrying heterozygous or homozygous R176Q ADHR Fgf23 mutations. The knock-in mice with functional Galnt3 had normal Fgf23 but were slightly hypophosphatemic. The stabilized Fgf23 ADHR allele reversed the Galnt3-null phenotype and normalized total Fgf23, serum phosphorus, and bone Fgf23 mRNA. However, the skeletal phenotype was unaffected. In summary, these data demonstrate that O-glycosylation by ppGaINAc-T3 is only necessary for proper secretion of intact Fgf23 and, once secreted, does not affect Fgf23 function. Furthermore, the more stable Fgf23 ADHR mutant protein could normalize serum phosphorus in Galnt3 knockout mice. PMID:25051439

Genetic rescue of glycosylation-deficient Fgf23 in the Galnt3 knockout mouse.

PubMed

Ichikawa, Shoji; Gray, Amie K; Padgett, Leah R; Allen, Matthew R; Clinkenbeard, Erica L; Sarpa, Nicole M; White, Kenneth E; Econs, Michael J

2014-10-01

Fibroblast growth factor 23 (FGF23) is a hormone that inhibits renal phosphate reabsorption and 1,25-dihydroxyvitamin D biosynthesis. The FGF23 subtilisin-like proprotein convertase recognition sequence ((176)RHTR(179)↓) is protected by O-glycosylation through ppGalNAc-T3 (GALNT3) activity. Thus, inactivating GALNT3 mutations render FGF23 susceptible to proteolysis, thereby reducing circulating intact hormone levels and leading to hyperphosphatemic familial tumoral calcinosis. To further delineate the role of glycosylation in the Fgf23 function, we generated an inducible FGF23 transgenic mouse expressing human mutant FGF23 (R176Q and R179Q) found in patients with autosomal dominant hypophosphatemic rickets (ADHR) and bred this animal to Galnt3 knockout mice, a model of familial tumoral calcinosis. Due to the low intact Fgf23 level, Galnt3 knockout mice with wild-type Fgf23 alleles were hyperphosphatemic. In contrast, carriers of the mutant FGF23 transgene, regardless of Galnt3 mutation status, had significantly higher serum intact FGF23, resulting in severe hypophosphatemia. Importantly, serum phosphorus and FGF23 were comparable between transgenic mice with or without normal Galnt3 alleles. To determine whether the presence of the ADHR mutation could improve biochemical and skeletal abnormalities in Galnt3-null mice, these mice were also mated to Fgf23 knock-in mice, carrying heterozygous or homozygous R176Q ADHR Fgf23 mutations. The knock-in mice with functional Galnt3 had normal Fgf23 but were slightly hypophosphatemic. The stabilized Fgf23 ADHR allele reversed the Galnt3-null phenotype and normalized total Fgf23, serum phosphorus, and bone Fgf23 mRNA. However, the skeletal phenotype was unaffected. In summary, these data demonstrate that O-glycosylation by ppGaINAc-T3 is only necessary for proper secretion of intact Fgf23 and, once secreted, does not affect Fgf23 function. Furthermore, the more stable Fgf23 ADHR mutant protein could normalize serum phosphorus in Galnt3 knockout mice.

Contributions of β2-microglobulin–dependent molecules and lymphocytes to iron regulation: insights from HfeRag1−/− and β2mRag1−/− double knock-out mice

PubMed Central

Miranda, Carlos J.; Makui, Hortence; Andrews, Nancy C.; Santos, Manuela M.

2010-01-01

Genetic causes of hereditary hemochromatosis (HH) include mutations in the HFE gene, coding for a β2-microglobulin (β2m)–associated major histocompatibility complex class I-like protein. However, iron accumulation in patients with HH can be highly variable. Previously, analysis of β2mRag1−/− double-deficient mice, lacking all β2m-dependent molecules and lymphocytes, demonstrated increased iron accumulation in the pancreas and heart compared with β2m single knock-out mice. To evaluate whether the observed phenotype in β2mRag1−/− mice was due solely to the absence of Hfe or to other β2m-dependent molecules, we generated HfeRag1−/− double-deficient mice. Our studies revealed that introduction of Rag1 deficiency in Hfe knock-out mice leads to heightened iron overload, mainly in the liver, whereas the heart and pancreas are relatively spared compared with β2mRag1−/− mice. These results suggest that other β2m-interacting protein(s) may be involved in iron regulation and that in the absence of functional Hfe molecules lymphocyte numbers may influence iron overload severity. PMID:14656877

Leptin receptor-deficient (knockout) medaka, Oryzias latipes, show chronical up-regulated levels of orexigenic neuropeptides, elevated food intake and stage specific effects on growth and fat allocation.

PubMed

Chisada, Shin-ichi; Kurokawa, Tadahide; Murashita, Koji; Rønnestad, Ivar; Taniguchi, Yoshihito; Toyoda, Atsushi; Sakaki, Yoshiyuki; Takeda, Shunichi; Yoshiura, Yasutoshi

2014-01-01

The first studies that identified leptin and its receptor (LepR) in mammals were based on mutant animals that displayed dramatic changes in body-weight and regulation of energy homeostasis. Subsequent studies have shown that a deficiency of leptin or LepR in homoeothermic mammals results in hyperphagia, obesity, infertility and a number of other abnormalities. The physiological roles of leptin-mediated signaling in ectothermic teleosts are still being explored. Here, we produced medaka with homozygous LepR gene mutation using the targeting induced local lesions in a genome method. This knockout mutant had a point mutation of cysteine for stop codon at the 357th amino acid just before the leptin-binding domain. The evidence for loss of function of leptin-mediated signaling in the mutant is based on a lack of response to feeding in the expression of key appetite-related neuropeptides in the diencephalon. The mutant lepr−/− medaka expressed constant up-regulated levels of mRNA for the orexigenic neuropeptide Ya and agouti-related protein and a suppressed level of anorexigenic proopiomelanocortin 1 in the diencephalon independent of feeding, which suggests that the mutant did not possess functional LepR. Phenotypes of the LepR-mutant medaka were analyzed in order to understand the effects on food intake, growth, and fat accumulation in the tissues. The food intake of the mutant medaka was higher in post-juveniles and adult stages than that of wild-type (WT) fish. The hyperphagia led to a high growth rate at the post-juvenile stage, but did not to significant alterations in final adult body size. There was no additional deposition of fat in the liver and muscle in the post-juvenile and adult mutants, or in the blood plasma in the adult mutant. However, adult LepR mutants possessed large deposits of visceral fat, unlike in the WT fish, in which there were none. Our analysis confirms that LepR in medaka exert a powerful influence on the control on food intake. Further analyses using the mutant will contribute to a better understanding of the role of leptin in fish. This is the first study to produce fish with leptin receptor deficiency.

A Mutation in the Dmp1 Gene Alters Phosphate Responsiveness in Mice

PubMed Central

Gerard-O'Riley, Rita L.; Acton, Dena; McQueen, Amie K.; Strobel, Isabel E.; Witcher, Phillip C.; Feng, Jian Q.; Econs, Michael J.

2017-01-01

Mutations in the dentin matrix protein 1 (DMP1) gene cause autosomal recessive hypophosphatemic rickets (ARHR). Hypophosphatemia in ARHR results from increased circulating levels of the phosphaturic hormone, fibroblast growth factor 23 (FGF23). Similarly, elevated FGF23, caused by mutations in the PHEX gene, is responsible for the hypophosphatemia in X-linked hypophosphatemic rickets (XLH). Previously, we demonstrated that a Phex mutation in mice creates a lower set point for extracellular phosphate, where an increment in phosphorus further stimulates Fgf23 production to maintain low serum phosphorus levels. To test the presence of the similar set point defect in ARHR, we generated 4- and 12-week-old Dmp1/Galnt3 double knockout mice and controls, including Dmp1 knockout mice (a murine model of ARHR), Galnt3 knockout mice (a murine model of familial tumoral calcinosis), and phenotypically normal double heterozygous mice. Galnt3 knockout mice had increased proteolytic cleavage of Fgf23, leading to low circulating intact Fgf23 levels with consequent hyperphosphatemia. In contrast, Dmp1 knockout mice had little Fgf23 cleavage and increased femoral Fgf23 expression, resulting in hypophosphatemia and low femoral bone mineral density (BMD). However, introduction of the Galnt3 null allele to Dmp1 knockout mice resulted in a significant increase in serum phosphorus and normalization of BMD. This increased serum phosphorus was accompanied by markedly elevated Fgf23 expression and circulating Fgf23 levels, an attempt to reduce serum phosphorus in the face of improving phosphorus levels. These data indicate that a Dmp1 mutation creates a lower set point for extracellular phosphate and maintains it through the regulation of Fgf23 cleavage and expression. PMID:28005411

A Mutation in the Dmp1 Gene Alters Phosphate Responsiveness in Mice.

PubMed

Ichikawa, Shoji; Gerard-O'Riley, Rita L; Acton, Dena; McQueen, Amie K; Strobel, Isabel E; Witcher, Phillip C; Feng, Jian Q; Econs, Michael J

2017-03-01

Mutations in the dentin matrix protein 1 (DMP1) gene cause autosomal recessive hypophosphatemic rickets (ARHR). Hypophosphatemia in ARHR results from increased circulating levels of the phosphaturic hormone, fibroblast growth factor 23 (FGF23). Similarly, elevated FGF23, caused by mutations in the PHEX gene, is responsible for the hypophosphatemia in X-linked hypophosphatemic rickets (XLH). Previously, we demonstrated that a Phex mutation in mice creates a lower set point for extracellular phosphate, where an increment in phosphorus further stimulates Fgf23 production to maintain low serum phosphorus levels. To test the presence of the similar set point defect in ARHR, we generated 4- and 12-week-old Dmp1/Galnt3 double knockout mice and controls, including Dmp1 knockout mice (a murine model of ARHR), Galnt3 knockout mice (a murine model of familial tumoral calcinosis), and phenotypically normal double heterozygous mice. Galnt3 knockout mice had increased proteolytic cleavage of Fgf23, leading to low circulating intact Fgf23 levels with consequent hyperphosphatemia. In contrast, Dmp1 knockout mice had little Fgf23 cleavage and increased femoral Fgf23 expression, resulting in hypophosphatemia and low femoral bone mineral density (BMD). However, introduction of the Galnt3 null allele to Dmp1 knockout mice resulted in a significant increase in serum phosphorus and normalization of BMD. This increased serum phosphorus was accompanied by markedly elevated Fgf23 expression and circulating Fgf23 levels, an attempt to reduce serum phosphorus in the face of improving phosphorus levels. These data indicate that a Dmp1 mutation creates a lower set point for extracellular phosphate and maintains it through the regulation of Fgf23 cleavage and expression. Copyright © 2017 by the Endocrine Society.

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.

Different roles of axon guidance cues and patterned spontaneous activity in establishing receptive fields in the mouse superior colliculus.

PubMed

Liu, Mingna; Wang, Lupeng; Cang, Jianhua

2014-01-01

Visual neurons in the superior colliculus (SC) respond to both bright (On) and dark (Off) stimuli in their receptive fields. This receptive field property is due to proper convergence of On- and Off-centered retinal ganglion cells to their target cells in the SC. In this study, we have compared the receptive field structure of individual SC neurons in two lines of mutant mice that are deficient in retinotopic mapping: the ephrin-A knockouts that lack important retinocollicular axonal guidance cues and the nAChR-β2 knockouts that have altered activity-dependent refinement of retinocollicular projections. We find that even though the receptive fields are much larger in the ephrin-A knockouts, their On-Off overlap remains unchanged. These neurons also display normal level of selectivity for stimulus direction and orientation. In contrast, the On-Off overlap is disrupted in the β2 knockouts. Together with the previous finding of disrupted direction and orientation selectivity in the β2 knockout mice, our results indicate that molecular guidance cues and activity-dependent processes play different roles in the development of receptive field properties in the SC.

Novel In Vivo Model for Combinatorial Fluorescence Labeling in Mouse Prostate

PubMed Central

Fang, Xiaolan; Gyabaah, Kenneth; Nickkholgh, Bita; Cline, J. Mark; Balaji, K.C.

2015-01-01

BACKGROUND The epithelial layer of prostate glands contains several types of cells, including luminal and basal cells. Yet there is paucity of animal models to study the cellular origin of normal or neoplastic development in the prostate to facilitate the treatment of heterogenous prostate diseases by targeting individual cell lineages. METHODS We developed a mouse model that expresses different types of fluorescent proteins (XFPs) specifically in prostatic cells. Using an in vivo stochastic fluorescent protein combinatorial strategy, XFP signals were expressed specifically in prostate of Protein Kinase D1 (PKD1) knock-out, K-RasG12D knock-in, and Phosphatase and tensin homolog (PTEN) and PKD1 double knock-out mice under the control of PB-Cre promoter. RESULTS In vivo XFP signals were observed in prostate of PKD1 knock-out, K-RasG12D knock-in, and PTEN PKD1 double knock-out mice, which developed normal, hyperplastic, and neoplastic prostate, respectively. The patchy expression pattern of XFPs in neoplasia tissue indicated the clonal origin of cancer cells in the prostate. CONCLUSIONS The transgenic mouse models demonstrate combinatorial fluorescent protein expression in normal and cancerous prostatic tissues. This novel prostate-specific fluorescent labeled mouse model, which we named Prorainbow, could be useful in studying benign and malignant pathology of prostate. PMID:25753731

Novel In Vivo model for combinatorial fluorescence labeling in mouse prostate.

PubMed

Fang, Xiaolan; Gyabaah, Kenneth; Nickkholgh, Bita; Cline, J Mark; Balaji, K C

2015-06-15

The epithelial layer of prostate glands contains several types of cells, including luminal and basal cells. Yet there is paucity of animal models to study the cellular origin of normal or neoplastic development in the prostate to facilitate the treatment of heterogenous prostate diseases by targeting individual cell lineages. We developed a mouse model that expresses different types of fluorescent proteins (XFPs) specifically in prostatic cells. Using an in vivo stochastic fluorescent protein combinatorial strategy, XFP signals were expressed specifically in prostate of Protein Kinase D1 (PKD1) knock-out, K-Ras(G) (12) (D) knock-in, and Phosphatase and tensin homolog (PTEN) and PKD1 double knock-out mice under the control of PB-Cre promoter. In vivo XFP signals were observed in prostate of PKD1 knock-out, K-Ras(G) (12) (D) knock-in, and PTEN PKD1 double knock-out mice, which developed normal, hyperplastic, and neoplastic prostate, respectively. The patchy expression pattern of XFPs in neoplasia tissue indicated the clonal origin of cancer cells in the prostate. The transgenic mouse models demonstrate combinatorial fluorescent protein expression in normal and cancerous prostatic tissues. This novel prostate-specific fluorescent labeled mouse model, which we named Prorainbow, could be useful in studying benign and malignant pathology of prostate. © 2015 Wiley Periodicals, Inc.

ALS-associated mutant FUS induces selective motor neuron degeneration through toxic gain of function

PubMed Central

Sharma, Aarti; Lyashchenko, Alexander K.; Lu, Lei; Nasrabady, Sara Ebrahimi; Elmaleh, Margot; Mendelsohn, Monica; Nemes, Adriana; Tapia, Juan Carlos; Mentis, George Z.; Shneider, Neil A.

2016-01-01

Mutations in FUS cause amyotrophic lateral sclerosis (ALS), including some of the most aggressive, juvenile-onset forms of the disease. FUS loss-of-function and toxic gain-of-function mechanisms have been proposed to explain how mutant FUS leads to motor neuron degeneration, but neither has been firmly established in the pathogenesis of ALS. Here we characterize a series of transgenic FUS mouse lines that manifest progressive, mutant-dependent motor neuron degeneration preceded by early, structural and functional abnormalities at the neuromuscular junction. A novel, conditional FUS knockout mutant reveals that postnatal elimination of FUS has no effect on motor neuron survival or function. Moreover, endogenous FUS does not contribute to the onset of the ALS phenotype induced by mutant FUS. These findings demonstrate that FUS-dependent motor degeneration is not due to loss of FUS function, but to the gain of toxic properties conferred by ALS mutations. PMID:26842965

Forebrain-Specific Loss of BMPRII in Mice Reduces Anxiety and Increases Object Exploration.

PubMed

McBrayer, Zofeyah L; Dimova, Jiva; Pisansky, Marc T; Sun, Mu; Beppu, Hideyuki; Gewirtz, Jonathan C; O'Connor, Michael B

2015-01-01

To investigate the role of Bone Morphogenic Protein Receptor Type II (BMPRII) in learning, memory, and exploratory behavior in mice, a tissue-specific knockout of BMPRII in the post-natal hippocampus and forebrain was generated. We found that BMPRII mutant mice had normal spatial learning and memory in the Morris water maze, but showed significantly reduced swimming speeds with increased floating behavior. Further analysis using the Porsolt Swim Test to investigate behavioral despair did not reveal any differences in immobility between mutants and controls. In the Elevated Plus Maze, BMPRII mutants and Smad4 mutants showed reduced anxiety, while in exploratory tests, BMPRII mutants showed more interest in object exploration. These results suggest that loss of BMPRII in the mouse hippocampus and forebrain does not disrupt spatial learning and memory encoding, but instead impacts exploratory and anxiety-related behaviors.

Forebrain-Specific Loss of BMPRII in Mice Reduces Anxiety and Increases Object Exploration

PubMed Central

McBrayer, Zofeyah L.; Dimova, Jiva; Pisansky, Marc T.; Sun, Mu; Beppu, Hideyuki; Gewirtz, Jonathan C.; O’Connor, Michael B.

2015-01-01

To investigate the role of Bone Morphogenic Protein Receptor Type II (BMPRII) in learning, memory, and exploratory behavior in mice, a tissue-specific knockout of BMPRII in the post-natal hippocampus and forebrain was generated. We found that BMPRII mutant mice had normal spatial learning and memory in the Morris water maze, but showed significantly reduced swimming speeds with increased floating behavior. Further analysis using the Porsolt Swim Test to investigate behavioral despair did not reveal any differences in immobility between mutants and controls. In the Elevated Plus Maze, BMPRII mutants and Smad4 mutants showed reduced anxiety, while in exploratory tests, BMPRII mutants showed more interest in object exploration. These results suggest that loss of BMPRII in the mouse hippocampus and forebrain does not disrupt spatial learning and memory encoding, but instead impacts exploratory and anxiety-related behaviors. PMID:26444546

ALS-associated mutant FUS induces selective motor neuron degeneration through toxic gain of function.

PubMed

Sharma, Aarti; Lyashchenko, Alexander K; Lu, Lei; Nasrabady, Sara Ebrahimi; Elmaleh, Margot; Mendelsohn, Monica; Nemes, Adriana; Tapia, Juan Carlos; Mentis, George Z; Shneider, Neil A

2016-02-04

Mutations in FUS cause amyotrophic lateral sclerosis (ALS), including some of the most aggressive, juvenile-onset forms of the disease. FUS loss-of-function and toxic gain-of-function mechanisms have been proposed to explain how mutant FUS leads to motor neuron degeneration, but neither has been firmly established in the pathogenesis of ALS. Here we characterize a series of transgenic FUS mouse lines that manifest progressive, mutant-dependent motor neuron degeneration preceded by early, structural and functional abnormalities at the neuromuscular junction. A novel, conditional FUS knockout mutant reveals that postnatal elimination of FUS has no effect on motor neuron survival or function. Moreover, endogenous FUS does not contribute to the onset of the ALS phenotype induced by mutant FUS. These findings demonstrate that FUS-dependent motor degeneration is not due to loss of FUS function, but to the gain of toxic properties conferred by ALS mutations.

Further characterization of a highly attenuated Yersinia pestis CO92 mutant deleted for the genes encoding Braun lipoprotein and plasminogen activator protease in murine alveolar and primary human macrophages.

PubMed

van Lier, Christina J; Tiner, Bethany L; Chauhan, Sadhana; Motin, Vladimir L; Fitts, Eric C; Huante, Matthew B; Endsley, Janice J; Ponnusamy, Duraisamy; Sha, Jian; Chopra, Ashok K

2015-03-01

We recently characterized the Δlpp Δpla double in-frame deletion mutant of Yersinia pestis CO92 molecularly, biologically, and immunologically. While Braun lipoprotein (Lpp) activates toll-like receptor-2 to initiate an inflammatory cascade, plasminogen activator (Pla) protease facilitates bacterial dissemination in the host. The Δlpp Δpla double mutant was highly attenuated in evoking bubonic and pneumonic plague, was rapidly cleared from mouse organs, and generated humoral and cell-mediated immune responses to provide subsequent protection to mice against a lethal challenge dose of wild-type (WT) CO92. Here, we further characterized the Δlpp Δpla double mutant in two murine macrophage cell lines as well as in primary human monocyte-derived macrophages to gauge its potential as a live-attenuated vaccine candidate. We first demonstrated that the Δpla single and the Δlpp Δpla double mutant were unable to survive efficiently in murine and human macrophages, unlike WT CO92. We observed that the levels of Pla and its associated protease activity were not affected in the Δlpp single mutant, and, likewise, deletion of the pla gene from WT CO92 did not alter Lpp levels. Further, our study revealed that both Lpp and Pla contributed to the intracellular survival of WT CO92 via different mechanisms. Importantly, the ability of the Δlpp Δpla double mutant to be phagocytized by macrophages, to stimulate production of tumor necrosis factor-α and interleukin-6, and to activate the nitric oxide killing pathways of the host cells remained unaltered when compared to the WT CO92-infected macrophages. Finally, macrophages infected with either the WT CO92 or the Δlpp Δpla double mutant were equally efficient in their uptake of zymosan particles as determined by flow cytometric analysis. Overall, our data indicated that although the Δlpp Δpla double mutant of Y. pestis CO92 was highly attenuated, it retained the ability to elicit innate and subsequent acquired immune responses in the host similar to that of WT CO92, which are highly desirable in a live-attenuated vaccine candidate. Copyright © 2015 Elsevier Ltd. All rights reserved.

Cellular responses during morphological transformation in Azospirillum brasilense and Its flcA knockout mutant.

PubMed

Hou, Xingsheng; McMillan, Mary; Coumans, Joëlle V F; Poljak, Anne; Raftery, Mark J; Pereg, Lily

2014-01-01

FlcA is a response regulator controlling flocculation and the morphological transformation of Azospirillum cells from vegetative to cyst-like forms. To understand the cellular responses of Azospirillum to conditions that cause morphological transformation, proteins differentially expressed under flocculation conditions in A. brasilense Sp7 and its flcA knockout mutant were investigated. Comparison of 2-DE protein profiles of wild-type (Sp7) and a flcA deletion mutant (Sp7-flcAΔ) revealed a total of 33 differentially expressed 2-DE gel spots, with 22 of these spots confidently separated to allow protein identification. Analysis of these spots by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and MASCOT database searching identified 48 proteins (≥10% emPAI in each spot). The functional characteristics of these proteins included carbon metabolism (beta-ketothiolase and citrate synthase), nitrogen metabolism (Glutamine synthetase and nitric oxide synthase), stress tolerance (superoxide dismutase, Alkyl hydroperoxidase and ATP-dependent Clp protease proteolytic subunit) and morphological transformation (transducer coupling protein). The observed differences between Sp7 wild-type and flcA- strains enhance our understanding of the morphological transformation process and help to explain previous phenotypical observations. This work is a step forward in connecting the Azospirillum phenome and genome.

Cellular Responses during Morphological Transformation in Azospirillum brasilense and Its flcA Knockout Mutant

PubMed Central

Coumans, Joëlle V. F.; Poljak, Anne; Raftery, Mark J.; Pereg, Lily

2014-01-01

FlcA is a response regulator controlling flocculation and the morphological transformation of Azospirillum cells from vegetative to cyst-like forms. To understand the cellular responses of Azospirillum to conditions that cause morphological transformation, proteins differentially expressed under flocculation conditions in A. brasilense Sp7 and its flcA knockout mutant were investigated. Comparison of 2-DE protein profiles of wild-type (Sp7) and a flcA deletion mutant (Sp7-flcAΔ) revealed a total of 33 differentially expressed 2-DE gel spots, with 22 of these spots confidently separated to allow protein identification. Analysis of these spots by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and MASCOT database searching identified 48 proteins (≥10% emPAI in each spot). The functional characteristics of these proteins included carbon metabolism (beta-ketothiolase and citrate synthase), nitrogen metabolism (Glutamine synthetase and nitric oxide synthase), stress tolerance (superoxide dismutase, Alkyl hydroperoxidase and ATP-dependent Clp protease proteolytic subunit) and morphological transformation (transducer coupling protein). The observed differences between Sp7 wild-type and flcA − strains enhance our understanding of the morphological transformation process and help to explain previous phenotypical observations. This work is a step forward in connecting the Azospirillum phenome and genome. PMID:25502569

Genetic resources offer efficient tools for rice functional genomics research.

PubMed

Lo, Shuen-Fang; Fan, Ming-Jen; Hsing, Yue-Ie; Chen, Liang-Jwu; Chen, Shu; Wen, Ien-Chie; Liu, Yi-Lun; Chen, Ku-Ting; Jiang, Mirng-Jier; Lin, Ming-Kuang; Rao, Meng-Yen; Yu, Lin-Chih; Ho, Tuan-Hua David; Yu, Su-May

2016-05-01

Rice is an important crop and major model plant for monocot functional genomics studies. With the establishment of various genetic resources for rice genomics, the next challenge is to systematically assign functions to predicted genes in the rice genome. Compared with the robustness of genome sequencing and bioinformatics techniques, progress in understanding the function of rice genes has lagged, hampering the utilization of rice genes for cereal crop improvement. The use of transfer DNA (T-DNA) insertional mutagenesis offers the advantage of uniform distribution throughout the rice genome, but preferentially in gene-rich regions, resulting in direct gene knockout or activation of genes within 20-30 kb up- and downstream of the T-DNA insertion site and high gene tagging efficiency. Here, we summarize the recent progress in functional genomics using the T-DNA-tagged rice mutant population. We also discuss important features of T-DNA activation- and knockout-tagging and promoter-trapping of the rice genome in relation to mutant and candidate gene characterizations and how to more efficiently utilize rice mutant populations and datasets for high-throughput functional genomics and phenomics studies by forward and reverse genetics approaches. These studies may facilitate the translation of rice functional genomics research to improvements of rice and other cereal crops. © 2015 John Wiley & Sons Ltd.

Impaired phloem loading in zmsweet13a,b,c sucrose transporter triple knock-out mutants in Zea mays.

PubMed

Bezrutczyk, Margaret; Hartwig, Thomas; Horschman, Marc; Char, Si Nian; Yang, Jinliang; Yang, Bing; Frommer, Wolf B; Sosso, Davide

2018-04-01

Crop yield depends on efficient allocation of sucrose from leaves to seeds. In Arabidopsis, phloem loading is mediated by a combination of SWEET sucrose effluxers and subsequent uptake by SUT1/SUC2 sucrose/H + symporters. ZmSUT1 is essential for carbon allocation in maize, but the relative contribution to apoplasmic phloem loading and retrieval of sucrose leaking from the translocation path is not known. Here we analysed the contribution of SWEETs to phloem loading in maize. We identified three leaf-expressed SWEET sucrose transporters as key components of apoplasmic phloem loading in Zea mays L. ZmSWEET13 paralogues (a, b, c) are among the most highly expressed genes in the leaf vasculature. Genome-edited triple knock-out mutants were severely stunted. Photosynthesis of mutants was impaired and leaves accumulated high levels of soluble sugars and starch. RNA-seq revealed profound transcriptional deregulation of genes associated with photosynthesis and carbohydrate metabolism. Genome-wide association study (GWAS) analyses may indicate that variability in ZmSWEET13s correlates with agronomical traits, especifically flowering time and leaf angle. This work provides support for cooperation of three ZmSWEET13s with ZmSUT1 in phloem loading in Z. mays. © 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.

RNaseT2 knockout rats exhibit hippocampal neuropathology and deficits in memory.

PubMed

Sinkevicius, Kerstin W; Morrison, Thomas R; Kulkarni, Praveen; Caffrey Cagliostro, Martha K; Iriah, Sade; Malmberg, Samantha; Sabrick, Julia; Honeycutt, Jennifer A; Askew, Kim L; Trivedi, Malav; Ferris, Craig F

2018-06-27

RNASET2 deficiency in humans is associated with infant cystic leukoencephalopathy, which causes psychomotor impairment, spasticity and epilepsy. A zebrafish mutant model suggests that loss of RNASET2 function leads to neurodegeneration due to the accumulation of non-degraded RNA in the lysosomes. The goal of this study was to characterize the first rodent model of RNASET2 deficiency. The brains of 3- and 12-month-old RNaseT2 knockout rats were studied using multiple magnetic resonance imaging modalities and behavioral tests. While T1- and T2-weighted images of RNaseT2 knockout rats exhibited no evidence of cystic lesions, the prefrontal cortex and hippocampal complex were enlarged in knockout animals. Diffusion-weighted imaging showed altered anisotropy and putative gray matter changes in the hippocampal complex of the RNaseT2 knockout rats. Immunohistochemistry for glial fibrillary acidic protein (GFAP) showed the presence of hippocampal neuroinflammation. Decreased levels of lysosome-associated membrane protein 2 (LAMP2) and elevated acid phosphatase and β-N-acetylglucosaminidase (NAG) activities indicated that the RNASET2 knockout rats likely had altered lysosomal function and potential defects in autophagy. Object recognition tests confirmed that RNaseT2 knockout rats exhibited memory deficits. However, the Barnes maze, and balance beam and rotarod tests indicated there were no differences in spatial memory or motor impairments, respectively. Overall, patients with RNASET2 deficiency exhibited a more severe neurodegeneration phenotype than was observed in the RNaseT2 knockout rats. However, the vulnerability of the knockout rat hippocampus as evidenced by neuroinflammation, altered lysosomal function and cognitive defects indicates that this is still a useful in vivo model to study RNASET2 function. © 2018. Published by The Company of Biologists Ltd.

SUCROSE TRANSPORTER 5 supplies Arabidopsis embryos with biotin and affects triacylglycerol accumulation

PubMed Central

Pommerrenig, Benjamin; Popko, Jennifer; Heilmann, Mareike; Schulmeister, Sylwia; Dietel, Katharina; Schmitt, Bianca; Stadler, Ruth; Feussner, Ivo; Sauer, Norbert

2013-01-01

The Arabidopsis SUC5 protein represents a classical sucrose/H+ symporter. Functional analyses previously revealed that SUC5 also transports biotin, an essential co-factor for fatty acid synthesis. However, evidence for a dual role in transport of the structurally unrelated compounds sucrose and biotin in plants was lacking. Here we show that SUC5 localizes to the plasma membrane, and that the SUC5 gene is expressed in developing embryos, confirming the role of the SUC5 protein as substrate carrier across apoplastic barriers in seeds. We show that transport of biotin but not of sucrose across these barriers is impaired in suc5 mutant embryos. In addition, we show that SUC5 is essential for the delivery of biotin into the embryo of biotin biosynthesis-defective mutants (bio1 and bio2). We compared embryo and seedling development as well as triacylglycerol accumulation and fatty acid composition in seeds of single mutants (suc5, bio1 or bio2), double mutants (suc5 bio1 and suc5 bio2) and wild-type plants. Although suc5 mutants were like the wild-type, bio1 and bio2 mutants showed developmental defects and reduced triacylglycerol contents. In suc5 bio1 and suc5 bio2 double mutants, developmental defects were severely increased and the triacylglycerol content was reduced to a greater extent in comparison to the single mutants. Supplementation with externally applied biotin helped to reduce symptoms in both single and double mutants, but the efficacy of supplementation was significantly lower in double than in single mutants, showing that transport of biotin into the embryo is lower in the absence of SUC5. PMID:23031218

Arabidopsis DNA polymerase lambda mutant is mildly sensitive to DNA double strand breaks but defective in integration of a transgene

PubMed Central

Furukawa, Tomoyuki; Angelis, Karel J.; Britt, Anne B.

2015-01-01

The DNA double-strand break (DSB) is a critical type of damage, and can be induced by both endogenous sources (e.g., errors of oxidative metabolism, transposable elements, programmed meiotic breaks, or perturbation of the DNA replication fork) and exogenous sources (e.g., ionizing radiation or radiomimetic chemicals). Although higher plants, like mammals, are thought to preferentially repair DSBs via nonhomologous end joining (NHEJ), much remains unclear about plant DSB repair pathways. Our reverse genetic approach suggests that DNA polymerase λ is involved in DSB repair in Arabidopsis. The Arabidopsis T-DNA insertion mutant (atpolλ-1) displayed sensitivity to both gamma-irradiation and treatment with radiomimetic reagents, but not to other DNA damaging treatments. The atpolλ-1 mutant showed a moderate sensitivity to DSBs, while Arabidopsis Ku70 and DNA ligase 4 mutants (atku70-3 and atlig4-2), both of which play critical roles in NHEJ, exhibited a hypersensitivity to these treatments. The atpolλ-1/atlig4-2 double mutant exhibited a higher sensitivity to DSBs than each single mutant, but the atku70/atpolλ-1 showed similar sensitivity to the atku70-3 mutant. We showed that transcription of the DNA ligase 1, DNA ligase 6, and Wee1 genes was quickly induced by BLM in several NHEJ deficient mutants in contrast to wild-type. Finally, the T-DNA transformation efficiency dropped in NHEJ deficient mutants and the lowest transformation efficiency was scored in the atpolλ-1/atlig4-2 double mutant. These results imply that AtPolλ is involved in both DSB repair and DNA damage response pathway. PMID:26074930

Describing the role of Drosophila melanogaster ABC transporters in insecticide biology using CRISPR-Cas9 knockouts.

PubMed

Denecke, Shane; Fusetto, Roberto; Batterham, Philip

2017-12-01

ABC transporters have a well-established role in drug resistance, effluxing xenobiotics from cells and tissues within the organism. More recently, research has been dedicated to understanding the role insect ABC transporters play in insecticide toxicity, but progress in understanding the contribution of specific transporters has been hampered by the lack of functional genetic tools. Here, we report knockouts of three Drosophila melanogaster ABC transporter genes, Mdr49, Mdr50, and Mdr65, that are homologous to the well-studied mammalian ABCB1 (P-glycoprotein). Each knockout mutant was created in the same wild type background and tested against a panel of insecticides representing different chemical classes. Mdr65 knockouts were more susceptible to all neuroactive insecticides tested, but Mdr49 and Mdr50 knockouts showed increased susceptibility or resistance depending on the insecticide used. Mdr65 was chosen for further analysis. Calculation of LC 50 values for the Mdr65 knockout allowed the substrate specificity of this transporter to be examined. No obvious distinguishing structural features were shared among MDR65 substrates. A role for Mdr65 in insecticide transport was confirmed by testing the capacity of the knockout to synergize with the ABC inhibitor verapamil and by measuring the levels of insecticide retained in the body of knockout flies. These data unambiguously establish the influence of ABC transporters on the capacity of wild type D. melanogaster to tolerate insecticide exposure and suggest that both tissue and substrate specificity underpin this capacity. Copyright © 2017 Elsevier Ltd. All rights reserved.

Characterization of multiple SPS knockout mutants reveals redundant functions of the four Arabidopsis sucrose phosphate synthase isoforms in plant viability, and strongly indicates that enhanced respiration and accelerated starch turnover can alleviate the blockage of sucrose biosynthesis.

PubMed

Bahaji, Abdellatif; Baroja-Fernández, Edurne; Ricarte-Bermejo, Adriana; Sánchez-López, Ángela María; Muñoz, Francisco José; Romero, Jose M; Ruiz, María Teresa; Baslam, Marouane; Almagro, Goizeder; Sesma, María Teresa; Pozueta-Romero, Javier

2015-09-01

We characterized multiple knock-out mutants of the four Arabidopsis sucrose phosphate synthase (SPSA1, SPSA2, SPSB and SPSC) isoforms. Despite their reduced SPS activity, spsa1/spsa2, spsa1/spsb, spsa2/spsb, spsa2/spsc, spsb/spsc, spsa1/spsa2/spsb and spsa2/spsb/spsc mutants displayed wild type (WT) vegetative and reproductive morphology, and showed WT photosynthetic capacity and respiration. In contrast, growth of rosettes, flowers and siliques of the spsa1/spsc and spsa1/spsa2/spsc mutants was reduced compared with WT plants. Furthermore, these plants displayed a high dark respiration phenotype. spsa1/spsb/spsc and spsa1/spsa2/spsb/spsc seeds poorly germinated and produced aberrant and sterile plants. Leaves of all viable sps mutants, except spsa1/spsc and spsa1/spsa2/spsc, accumulated WT levels of nonstructural carbohydrates. spsa1/spsc leaves possessed high levels of metabolic intermediates and activities of enzymes of the glycolytic and tricarboxylic acid cycle pathways, and accumulated high levels of metabolic intermediates of the nocturnal starch-to-sucrose conversion process, even under continuous light conditions. Results presented in this work show that SPS is essential for plant viability, reveal redundant functions of the four SPS isoforms in processes that are important for plant growth and nonstructural carbohydrate metabolism, and strongly indicate that accelerated starch turnover and enhanced respiration can alleviate the blockage of sucrose biosynthesis in spsa1/spsc leaves. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Separating genetic and hemodynamic defects in neuropilin 1 knockout embryos.

PubMed

Jones, Elizabeth A V; Yuan, Li; Breant, Christine; Watts, Ryan J; Eichmann, Anne

2008-08-01

Targeted inactivation of genes involved in murine cardiovascular development frequently leads to abnormalities in blood flow. As blood fluid dynamics play a crucial role in shaping vessel morphology, the presence of flow defects generally prohibits the precise assignment of the role of the mutated gene product in the vasculature. In this study, we show how to distinguish between genetic defects caused by targeted inactivation of the neuropilin 1 (Nrp1) receptor and hemodynamic defects occurring in homozygous knockout embryos. Our analysis of a Nrp1 null allele bred onto a C57BL/6 background shows that vessel remodeling defects occur concomitantly with the onset of blood flow and cause death of homozygous mutants at E10.5. Using mouse embryo culture, we establish that hemodynamic defects are already present at E8.5 and continuous circulation is never established in homozygous mutants. The geometry of yolk sac blood vessels is altered and remodeling into yolk sac arteries and veins does not occur. To separate flow-induced deficiencies from those caused by the Nrp1 mutation, we arrested blood flow in cultured wild-type and mutant embryos and followed their vascular development. We find that loss of Nrp1 function rather than flow induces the altered geometry of the capillary plexus. Endothelial cell migration, but not replication, is altered in Nrp1 mutants. Gene expression analysis of endothelial cells isolated from freshly dissected wild-type and mutants and after culture in no-flow conditions showed down-regulation of the arterial marker genes connexin 40 and ephrin B2 related to the loss of Nrp1 function. This method allows genetic defects caused by loss-of-function of a gene important for cardiovascular development to be isolated even in the presence of hemodynamic defects.

Impaired pH homeostasis in Arabidopsis lacking the vacuolar dicarboxylate transporter and analysis of carboxylic acid transport across the tonoplast.

PubMed

Hurth, Marco Alois; Suh, Su Jeoung; Kretzschmar, Tobias; Geis, Tina; Bregante, Monica; Gambale, Franco; Martinoia, Enrico; Neuhaus, H Ekkehard

2005-03-01

Arabidopsis (Arabidopsis thaliana) mutants lacking the tonoplastic malate transporter AttDT (A. thaliana tonoplast dicarboxylate transporter) and wild-type plants showed no phenotypic differences when grown under standard conditions. To identify putative metabolic changes in AttDT knock-out plants, we provoked a metabolic scenario connected to an increased consumption of dicarboxylates. Acidification of leaf discs stimulated dicarboxylate consumption and led to extremely low levels of dicarboxylates in mutants. To investigate whether reduced dicarboxylate concentrations in mutant leaf cells and, hence, reduced capacity to produce OH(-) to overcome acidification might affect metabolism, we measured photosynthetic oxygen evolution under conditions where the cytosol is acidified. AttDT::tDNA protoplasts showed a much stronger inhibition of oxygen evolution at low pH values when compared to wild-type protoplasts. Apparently citrate, which is present in higher amounts in knock-out plants, is not able to replace dicarboxylates to overcome acidification. To raise more information on the cellular level, we performed localization studies of carboxylates. Although the total pool of carboxylates in mutant vacuoles was nearly unaltered, these organelles contained a lower proportion of malate and fumarate and a higher proportion of citrate when compared to wild-type vacuoles. These alterations concur with the observation that radioactively labeled malate and citrate are transported into Arabidopsis vacuoles by different carriers. In addition, wild-type vacuoles and corresponding organelles from AttDT::tDNA mutants exhibited similar malate channel activities. In conclusion, these results show that Arabidopsis vacuoles contain at least two transporters and a channel for dicarboxylates and citrate and that the activity of AttDT is critical for regulation of pH homeostasis.

Impaired pH Homeostasis in Arabidopsis Lacking the Vacuolar Dicarboxylate Transporter and Analysis of Carboxylic Acid Transport across the Tonoplast1

PubMed Central

Hurth, Marco Alois; Suh, Su Jeoung; Kretzschmar, Tobias; Geis, Tina; Bregante, Monica; Gambale, Franco; Martinoia, Enrico; Neuhaus, H. Ekkehard

2005-01-01

Arabidopsis (Arabidopsis thaliana) mutants lacking the tonoplastic malate transporter AttDT (A. thaliana tonoplast dicarboxylate transporter) and wild-type plants showed no phenotypic differences when grown under standard conditions. To identify putative metabolic changes in AttDT knock-out plants, we provoked a metabolic scenario connected to an increased consumption of dicarboxylates. Acidification of leaf discs stimulated dicarboxylate consumption and led to extremely low levels of dicarboxylates in mutants. To investigate whether reduced dicarboxylate concentrations in mutant leaf cells and, hence, reduced capacity to produce OH− to overcome acidification might affect metabolism, we measured photosynthetic oxygen evolution under conditions where the cytosol is acidified. AttDT::tDNA protoplasts showed a much stronger inhibition of oxygen evolution at low pH values when compared to wild-type protoplasts. Apparently citrate, which is present in higher amounts in knock-out plants, is not able to replace dicarboxylates to overcome acidification. To raise more information on the cellular level, we performed localization studies of carboxylates. Although the total pool of carboxylates in mutant vacuoles was nearly unaltered, these organelles contained a lower proportion of malate and fumarate and a higher proportion of citrate when compared to wild-type vacuoles. These alterations concur with the observation that radioactively labeled malate and citrate are transported into Arabidopsis vacuoles by different carriers. In addition, wild-type vacuoles and corresponding organelles from AttDT::tDNA mutants exhibited similar malate channel activities. In conclusion, these results show that Arabidopsis vacuoles contain at least two transporters and a channel for dicarboxylates and citrate and that the activity of AttDT is critical for regulation of pH homeostasis. PMID:15728336

Functional characterization of Autographa californica multiple nucleopolyhedrovirus gp16 (ac130)

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

Yang, Ming; Huang, Cui; Qian, Duo-Duo

2014-09-15

To investigate the function of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) gp16, multiple gp16-knockout and repair mutants were constructed and characterized. No obvious difference in productivity of budded virus, DNA synthesis, late gene expression and morphogenesis was observed between gp16-knockout and repair viruses, but gp16 deletion resulted in six hours of lengthening in ST{sub 50} to the third instar Spodoptera exigua larvae in bioassays. GP16 was fractionated mainly in the light membrane fraction, by subcellular fractionation. A GP16-EGFP fusion protein was predominantly localized close around the nuclear membrane in infected cells, being coincident with formation of the vesicles associated with themore » nuclear membrane, which hosted nucleocapsids released from the nucleus. These data suggest that gp16 is not required for viral replication, but may be involved in membrane trafficking associated with the envelopment/de-envelopment of budded viruses when they cross over the nuclear membrane and pass through cytoplasm. - Highlights: • gp16 knockout and repair mutants of AcMNPV were constructed and characterized. • AcMNPV gp16 is not essential to virus replication. • Deletion of gp16 resulted in time lengthening to kill S. exigua larvae. • GP16 was localized close around the nuclear membrane of infected cells. • GP16 was fractionated in the light membrane fraction in subcellular fractionation.« less

Strong morphological defects in conditional Arabidopsis abp1 knock-down mutants generated in absence of functional ABP1 protein.

PubMed

Michalko, Jaroslav; Glanc, Matouš; Perrot-Rechenmann, Catherine; Friml, Jiří

2016-01-01

The Auxin Binding Protein 1 (ABP1) is one of the most studied proteins in plants. Since decades ago, it has been the prime receptor candidate for the plant hormone auxin with a plethora of described functions in auxin signaling and development. The developmental importance of ABP1 has recently been questioned by identification of Arabidopsis thaliana abp1 knock-out alleles that show no obvious phenotypes under normal growth conditions. In this study, we examined the contradiction between the normal growth and development of the abp1 knock-outs and the strong morphological defects observed in three different ethanol-inducible abp1 knock-down mutants ( abp1-AS, SS12K, SS12S). By analyzing segregating populations of abp1 knock-out vs. abp1 knock-down crosses we show that the strong morphological defects that were believed to be the result of conditional down-regulation of ABP1 can be reproduced also in the absence of the functional ABP1 protein. This data suggests that the phenotypes in  abp1 knock-down lines are due to the off-target effects and asks for further reflections on the biological function of ABP1 or alternative explanations for the missing phenotypic defects in the abp1 loss-of-function alleles.

Enhanced rhamnolipid production in Burkholderia thailandensis transposon knockout strains deficient in polyhydroxyalkanoate (PHA) synthesis.

PubMed

Funston, Scott J; Tsaousi, Konstantina; Smyth, Thomas J; Twigg, Matthew S; Marchant, Roger; Banat, Ibrahim M

2017-12-01

Microbially produced rhamnolipids have significant commercial potential; however, the main bacterial producer, Pseudomonas aeruginosa, is an opportunistic human pathogen, which limits biotechnological exploitation. The non-pathogenic species Burkholderia thailandensis produces rhamnolipids; however, yield is relatively low. The aim of this study was to determine whether rhamnolipid production could be increased in Burkholderia thailandensis through mutation of genes responsible for the synthesis of the storage material polyhydroxyalkanoate (PHA), thereby increasing cellular resources for the production of rhamnolipids. Potential PHA target genes were identified in B. thailandensis through comparison with known function genes in Pseudomonas aeruginosa. Multiple knockout strains for the phbA, phbB and phbC genes were obtained and their growth characteristics and rhamnolipid and PHA production determined. The wild-type strain and an rhamnolipid (RL)-deficient strain were used as controls. Three knockout strains (ΔphbA1, ΔphbB1 and ΔphbC1) with the best enhancement of rhamnolipid production were selected for detailed study. ΔphbB1 produced the highest level of purified RL (3.78 g l -1 ) compared to the wild-type strain (1.28 g l -1 ). In ΔphbB1, the proportion of mono-rhamnolipid was also increased compared to the wild-type strain. The production of PHA was reduced by at least 80% in all three phb mutant strains, although never completely eliminated. These results suggest that, in contrast to Pseudomonas aeruginosa, knockout of the PHA synthesis pathway in Burkholderia thailandensis could be used to increase rhamnolipid production. The evidence of residual PHA production in the phb mutant strains suggests B. thailandensis possesses a secondary unelucidated PHA synthesis pathway.

The Legionella pneumophila Dot/Icm-secreted Effector PlcC/CegC1 Together with PlcA and PlcB Promotes Virulence and Belongs to a Novel Zinc Metallophospholipase C Family Present in Bacteria and Fungi*

PubMed Central

Aurass, Philipp; Schlegel, Maren; Metwally, Omar; Harding, Clare R.; Schroeder, Gunnar N.; Frankel, Gad; Flieger, Antje

2013-01-01

Legionella pneumophila is a water-borne bacterium that causes pneumonia in humans. PlcA and PlcB are two previously defined L. pneumophila proteins with homology to the phosphatidylcholine-specific phospholipase C (PC-PLC) of Pseudomonas fluorescens. Additionally, we found that Lpg0012 shows similarity to PLCs and has been shown to be a Dot/Icm-injected effector, CegC1, which is designated here as PlcC. It remained unclear, however, whether these L. pneumophila proteins exhibit PLC activity. PlcC expressed in Escherichia coli hydrolyzed a broad phospholipid spectrum, including PC, phosphatidylglycerol (PG), and phosphatidylinositol. The addition of Zn2+ ions activated, whereas EDTA inhibited, PlcC-derived PLC activity. Protein homology search revealed that the three Legionella enzymes and P. fluorescens PC-PLC share conserved domains also present in uncharacterized fungal proteins. Fifteen conserved amino acids were essential for enzyme activity as identified via PlcC mutagenesis. Analysis of defined L. pneumophila knock-out mutants indicated Lsp-dependent export of PG-hydrolyzing PLC activity. PlcA and PlcB exhibited PG-specific activity and contain a predicted Sec signal sequence. In line with the reported requirement of host cell contact for Dot/Icm-dependent effector translocation, PlcC showed cell-associated PC-specific PLC activity after bacterial growth in broth. A PLC triple mutant, but not single or double mutants, exhibited reduced host killing in a Galleria mellonella infection model, highlighting the importance of the three PLCs in pathogenesis. In summary, we describe here a novel Zn2+-dependent PLC family present in Legionella, Pseudomonas, and fungi with broad substrate preference and function in virulence. PMID:23457299

Novel features of a PIWI-like protein homolog in the parasitic protozoan Leishmania.

PubMed

Padmanabhan, Prasad K; Dumas, Carole; Samant, Mukesh; Rochette, Annie; Simard, Martin J; Papadopoulou, Barbara

2012-01-01

In contrast to nearly all eukaryotes, the Old World Leishmania species L. infantum and L. major lack the bona fide RNAi machinery genes. Interestingly, both Leishmania genomes code for an atypical Argonaute-like protein that possesses a PIWI domain but lacks the PAZ domain found in Argonautes from RNAi proficient organisms. Using sub-cellular fractionation and confocal fluorescence microscopy, we show that unlike other eukaryotes, the PIWI-like protein is mainly localized in the single mitochondrion in Leishmania. To predict PIWI function, we generated a knockout mutant for the PIWI gene in both L. infantum (Lin) and L. major species by double-targeted gene replacement. Depletion of PIWI has no effect on the viability of insect promastigote forms but leads to an important growth defect of the mammalian amastigote lifestage in vitro and significantly delays disease pathology in mice, consistent with a higher expression of the PIWI transcript in amastigotes. Moreover, amastigotes lacking PIWI display a higher sensitivity to apoptosis inducing agents than wild type parasites, suggesting that PIWI may be a sensor for apoptotic stimuli. Furthermore, a whole-genome DNA microarray analysis revealed that loss of LinPIWI in Leishmania amastigotes affects mostly the expression of specific subsets of developmentally regulated genes. Several transcripts encoding surface and membrane-bound proteins were found downregulated in the LinPIWI((-/-)) mutant whereas all histone transcripts were upregulated in the null mutant, supporting the possibility that PIWI plays a direct or indirect role in the stability of these transcripts. Although our data suggest that PIWI is not involved in the biogenesis or the stability of small noncoding RNAs, additional studies are required to gain further insights into the role of this protein on RNA regulation and amastigote development in Leishmania.

Extracellular ammonia at sites of pulmonary infection with Coccidioides posadasii contributes to severity of the respiratory disease.

PubMed

Wise, Hua Zhang; Hung, Chiung-Yu; Whiston, Emily; Taylor, John W; Cole, Garry T

2013-01-01

Coccidioides is the causative agent of a potentially life-threatening respiratory disease of humans. A feature of this mycosis is that pH measurements of the microenvironment of pulmonary abscesses are consistently alkaline due to ammonia production during the parasitic cycle. We previously showed that enzymatically active urease is partly responsible for elevated concentrations of extracellular ammonia at sites of lung infection and contributes to both localized host tissue damage and exacerbation of the respiratory disease in BALB/c mice. Disruption of the urease gene (URE) of Coccidioides posadasii only partially reduced the amount of ammonia detected during in vitro growth of the parasitic phase, suggesting that other ammonia-producing pathways exist that may also contribute to the virulence of this pathogen. Ureidoglycolate hydrolase (Ugh) expressed by bacteria, fungi and higher plants catalyzes the hydrolysis of ureidoglycolate to yield glyoxylate and the release CO2 and ammonia. This enzymatic pathway is absent in mice and humans. Ureidoglycolate hydrolase gene deletions were conducted in a wild type (WT) isolate of C. posadasii as well as the previously generated Δure knock-out strain. Restorations of UGH in the mutant stains were performed to generate and evaluate the respective revertants. The double mutant revealed a marked decrease in the amount of extracellular ammonia without loss of reproductive competence in vitro compared to both the WT and Δure parental strains. BALB/c mice challenged intranasally with the Δugh/Δure mutant showed 90% survival after 30 days, decreased fungal burden, and well-organized pulmonary granulomas. We conclude that loss of both Ugh and Ure activity significantly reduced the virulence of this fungal pathogen. Copyright © 2013 Elsevier Ltd. All rights reserved.

Suppressing Farnesyl Diphosphate Synthase Alters Chloroplast Development and Triggers Sterol-Dependent Induction of Jasmonate- and Fe-Related Responses1[OPEN

PubMed Central

Andrade, Paola; Caudepón, Daniel; Arró, Montserrat

2016-01-01

Farnesyl diphosphate synthase (FPS) catalyzes the synthesis of farnesyl diphosphate from isopentenyl diphosphate and dimethylallyl diphosphate. Arabidopsis (Arabidopsis thaliana) contains two genes (FPS1 and FPS2) encoding FPS. Single fps1 and fps2 knockout mutants are phenotypically indistinguishable from wild-type plants, while fps1/fps2 double mutants are embryo lethal. To assess the effect of FPS down-regulation at postembryonic developmental stages, we generated Arabidopsis conditional knockdown mutants expressing artificial microRNAs devised to simultaneously silence both FPS genes. Induction of silencing from germination rapidly caused chlorosis and a strong developmental phenotype that led to seedling lethality. However, silencing of FPS after seed germination resulted in a slight developmental delay only, although leaves and cotyledons continued to show chlorosis and altered chloroplasts. Metabolomic analyses also revealed drastic changes in the profile of sterols, ubiquinones, and plastidial isoprenoids. RNA sequencing and reverse transcription-quantitative polymerase chain reaction transcriptomic analysis showed that a reduction in FPS activity levels triggers the misregulation of genes involved in biotic and abiotic stress responses, the most prominent one being the rapid induction of a set of genes related to the jasmonic acid pathway. Down-regulation of FPS also triggered an iron-deficiency transcriptional response that is consistent with the iron-deficient phenotype observed in FPS-silenced plants. The specific inhibition of the sterol biosynthesis pathway by chemical and genetic blockage mimicked these transcriptional responses, indicating that sterol depletion is the primary cause of the observed alterations. Our results highlight the importance of sterol homeostasis for normal chloroplast development and function and reveal important clues about how isoprenoid and sterol metabolism is integrated within plant physiology and development. PMID:27382138

Suppressing Farnesyl Diphosphate Synthase Alters Chloroplast Development and Triggers Sterol-Dependent Induction of Jasmonate- and Fe-Related Responses.

PubMed

Manzano, David; Andrade, Paola; Caudepón, Daniel; Altabella, Teresa; Arró, Montserrat; Ferrer, Albert

2016-09-01

Farnesyl diphosphate synthase (FPS) catalyzes the synthesis of farnesyl diphosphate from isopentenyl diphosphate and dimethylallyl diphosphate. Arabidopsis (Arabidopsis thaliana) contains two genes (FPS1 and FPS2) encoding FPS. Single fps1 and fps2 knockout mutants are phenotypically indistinguishable from wild-type plants, while fps1/fps2 double mutants are embryo lethal. To assess the effect of FPS down-regulation at postembryonic developmental stages, we generated Arabidopsis conditional knockdown mutants expressing artificial microRNAs devised to simultaneously silence both FPS genes. Induction of silencing from germination rapidly caused chlorosis and a strong developmental phenotype that led to seedling lethality. However, silencing of FPS after seed germination resulted in a slight developmental delay only, although leaves and cotyledons continued to show chlorosis and altered chloroplasts. Metabolomic analyses also revealed drastic changes in the profile of sterols, ubiquinones, and plastidial isoprenoids. RNA sequencing and reverse transcription-quantitative polymerase chain reaction transcriptomic analysis showed that a reduction in FPS activity levels triggers the misregulation of genes involved in biotic and abiotic stress responses, the most prominent one being the rapid induction of a set of genes related to the jasmonic acid pathway. Down-regulation of FPS also triggered an iron-deficiency transcriptional response that is consistent with the iron-deficient phenotype observed in FPS-silenced plants. The specific inhibition of the sterol biosynthesis pathway by chemical and genetic blockage mimicked these transcriptional responses, indicating that sterol depletion is the primary cause of the observed alterations. Our results highlight the importance of sterol homeostasis for normal chloroplast development and function and reveal important clues about how isoprenoid and sterol metabolism is integrated within plant physiology and development. © 2016 American Society of Plant Biologists. All rights reserved.

The Legionella pneumophila Dot/Icm-secreted effector PlcC/CegC1 together with PlcA and PlcB promotes virulence and belongs to a novel zinc metallophospholipase C family present in bacteria and fungi.

PubMed

Aurass, Philipp; Schlegel, Maren; Metwally, Omar; Harding, Clare R; Schroeder, Gunnar N; Frankel, Gad; Flieger, Antje

2013-04-19

Legionella pneumophila is a water-borne bacterium that causes pneumonia in humans. PlcA and PlcB are two previously defined L. pneumophila proteins with homology to the phosphatidylcholine-specific phospholipase C (PC-PLC) of Pseudomonas fluorescens. Additionally, we found that Lpg0012 shows similarity to PLCs and has been shown to be a Dot/Icm-injected effector, CegC1, which is designated here as PlcC. It remained unclear, however, whether these L. pneumophila proteins exhibit PLC activity. PlcC expressed in Escherichia coli hydrolyzed a broad phospholipid spectrum, including PC, phosphatidylglycerol (PG), and phosphatidylinositol. The addition of Zn(2+) ions activated, whereas EDTA inhibited, PlcC-derived PLC activity. Protein homology search revealed that the three Legionella enzymes and P. fluorescens PC-PLC share conserved domains also present in uncharacterized fungal proteins. Fifteen conserved amino acids were essential for enzyme activity as identified via PlcC mutagenesis. Analysis of defined L. pneumophila knock-out mutants indicated Lsp-dependent export of PG-hydrolyzing PLC activity. PlcA and PlcB exhibited PG-specific activity and contain a predicted Sec signal sequence. In line with the reported requirement of host cell contact for Dot/Icm-dependent effector translocation, PlcC showed cell-associated PC-specific PLC activity after bacterial growth in broth. A PLC triple mutant, but not single or double mutants, exhibited reduced host killing in a Galleria mellonella infection model, highlighting the importance of the three PLCs in pathogenesis. In summary, we describe here a novel Zn(2+)-dependent PLC family present in Legionella, Pseudomonas, and fungi with broad substrate preference and function in virulence.

AtMYB44 regulates WRKY70 expression and modulates antagonistic interaction between salicylic acid and jasmonic acid signaling.

PubMed

Shim, Jae Sung; Jung, Choonkyun; Lee, Sangjoon; Min, Kyunghun; Lee, Yin-Won; Choi, Yeonhee; Lee, Jong Seob; Song, Jong Tae; Kim, Ju-Kon; Choi, Yang Do

2013-02-01

The role of AtMYB44, an R2R3 MYB transcription factor, in signaling mediated by jasmonic acid (JA) and salicylic acid (SA) is examined. AtMYB44 is induced by JA through CORONATINE INSENSITIVE 1 (COI1). AtMYB44 over-expression down-regulated defense responses against the necrotrophic pathogen Alternaria brassicicola, but up-regulated WRKY70 and PR genes, leading to enhanced resistance to the biotrophic pathogen Pseudomonas syringae pv. tomato DC3000. The knockout mutant atmyb44 shows opposite effects. Induction of WRKY70 by SA is reduced in atmyb44 and npr1-1 mutants, and is totally abolished in atmyb44 npr1-1 double mutants, showing that WRKY70 is regulated independently through both NPR1 and AtMYB44. AtMYB44 over-expression does not change SA content, but AtMYB44 over-expression phenotypes, such as retarded growth, up-regulated PR1 and down-regulated PDF1.2 are reversed by SA depletion. The wrky70 mutation suppressed AtMYB44 over-expression phenotypes, including up-regulation of PR1 expression and down-regulation of PDF1.2 expression. β-estradiol-induced expression of AtMYB44 led to WRKY70 activation and thus PR1 activation. AtMYB44 binds to the WRKY70 promoter region, indicating that AtMYB44 acts as a transcriptional activator of WRKY70 by directly binding to a conserved sequence element in the WRKY70 promoter. These results demonstrate that AtMYB44 modulates antagonistic interaction by activating SA-mediated defenses and repressing JA-mediated defenses through direct control of WRKY70. © 2012 The Authors The Plant Journal © 2012 Blackwell Publishing Ltd.

Cold Shock as a Screen for Genes Involved in Cold Acclimatization in Neurospora crassa

PubMed Central

Watters, Michael K.; Manzanilla, Victor; Howell, Holly; Mehreteab, Alexander; Rose, Erik; Walters, Nicole; Seitz, Nicholas; Nava, Jacob; Kekelik, Sienna; Knuth, Laura; Scivinsky, Brianna

2018-01-01

When subjected to rapid drops of temperature (cold shock), Neurospora responds with a temporary shift in its morphology. This report is the first to examine this response genetically. We report here the results of a screen of selected mutants from the Neurospora knockout library for alterations in their morphological response to cold shock. Three groups of knockouts were selected to be subject to this screen: genes previously suspected to be involved in hyphal development as well as knockouts resulting in morphological changes; transcription factors; and genes homologous to E. coli genes known to alter their expression in response to cold shock. A total of 344 knockout strains were subjected to cold shock. Of those, 118 strains were identified with altered responses. We report here the cold shock morphologies and GO categorizations of strains subjected to this screen. Of strains with knockouts in genes associated with hyphal growth or morphology, 33 of 131 tested (25%) showed an altered response to cold shock. Of strains with knockouts in transcription factor genes, 30 of 145 (20%) showed an altered response to cold shock. Of strains with knockouts in genes homologous to E. coli genes which display altered levels of transcription in response to cold shock, a total of 55 of 68 tested (81%) showed an altered cold shock response. This suggests that the response to cold shock in these two organisms is largely shared in common. PMID:29563189

Effect of the luxS gene on biofilm formation and antibiotic resistance by Salmonella serovar Dublin.

PubMed

Ju, Xiangyu; Li, Junjie; Zhu, Mengjiao; Lu, Zhaoxin; Lv, Fengxia; Zhu, Xiaoyu; Bie, Xiaomei

2018-05-01

Biofilms are communities of bacterial cells that serve to protect them from external adverse influences and enhance bacterial resistance to antibiotics and sanitizers. Here, we studied the regulatory effects of glucose and sodium chloride on biofilm formation in Salmonella serovar Dublin (S. Dublin). To analyze expression levels of the quorum sensing gene luxS, we created a luxS knockout mutant. Also, antimicrobial resistance, hydrophobicity and autoinducer-2 (AI-2) activity of both the wild-type (WT) and the mutant strain were investigated. Our results revealed that glucose was not essential for S. Dublin biofilm formation but had an inhibitory effect on biofilm formation when the concentration was over 0.1%. NaCl was found to be indispensable in forming biofilm, and it also exerted an inhibitory effect at high concentrations (>1.0%). Both the WT and the mutant strains displayed significant MIC growth after biofilm formation. An increase of up to 32,768 times in the resistance of S. Dublin in biofilm phonotype against antibiotic (ampicillin) compared to its planktonic phonotype was observed. However, S. Dublin luxS knockout mutant only showed slight differences compared to the WT strain in the antimicrobial tests although it displayed better biofilm-forming capacity than the WT strain. The mutant strain also exhibited higher hydrophobicity than the WT strain, which was a feature related to biofilm formation. The production of the quorum sensing autoinducer-2 (AI-2) was significantly lower in the mutant strain than in the WT strain since the LuxS enzyme, encoded by the luxS gene, plays an essential role in AI-2 synthesis. However, the limited biofilm-forming ability in the WT strain indicated AI-2 was not directly related to S. Dublin biofilm formation. Furthermore, gene expression analysis of the WT and mutant strains revealed upregulation of genes related to biofilm stress response and enhanced resistance in the luxS mutant strain, which may provide evidence for the regulatory role of the luxS gene in biofilm formation. Copyright © 2018 Elsevier Ltd. All rights reserved.

Lesions in two Escherichia coli type 1 pilus genes alter pilus number and length without affecting receptor binding.

PubMed Central

Russell, P W; Orndorff, P E

1992-01-01

We describe the characterization of two genes, fimF and fimG (also called pilD), that encode two minor components of type 1 pili in Escherichia coli. Defined, in-frame deletion mutations were generated in vitro in each of these two genes. A double mutation that had deletions identical to both single lesions was also constructed. Examination of minicell transcription and translation products of parental and mutant plasmids revealed that, as predicted from the nucleotide sequence and previous reports, the fimF gene product was a protein of ca. 16 kDa and that the fimG gene product was a protein of ca. 14 kDa. Each of the constructions was introduced, via homologous recombination, into the E. coli chromosome. All three of the resulting mutants produced type 1 pili and exhibited hemagglutination of guinea pig erythrocytes. The latter property was also exhibited by partially purified pili isolated from each of the mutants. Electron microscopic examination revealed that the fimF mutant had markedly reduced numbers of pili per cell, whereas the fimG mutant had very long pili. The double mutant displayed the characteristics of both single mutants. However, pili in the double mutant were even longer than those seen in the fimG mutant, and the numbers of pili were even fewer than those displayed by the fimF mutant. All three mutants could be complemented in trans with a single-copy-number plasmid bearing the appropriate parental gene or genes to give near-normal parental piliation. On the basis of the phenotypes exhibited by the single and double mutants, we believe that the fimF gene product may aid in initiating pilus assembly and that the fimG product may act as an inhibitor of pilus polymerization. In contrast to previous studies, we found that neither gene product was required for type 1 pilus receptor binding. Images PMID:1355769

Role of aromatic interactions in amyloid formation by islet amyloid polypeptide.

PubMed

Tu, Ling-Hsien; Raleigh, Daniel P

2013-01-15

Aromatic-aromatic and aromatic-hydrophobic interactions have been proposed to play a role in amyloid formation by a range of polypeptides, including islet amyloid polypeptide (IAPP or amylin). IAPP is responsible for amyloid formation in patients with type 2 diabetes. The polypeptide is 37 residues long and contains three aromatic residues, Phe-15, Phe-23, and Tyr-37. The ability of all single aromatic to leucine mutants, all double aromatic to leucine mutants, and the triple leucine mutant to form amyloid were examined. Amyloid formation was almost twice as rapid for the F15L mutant as for the wild type but was almost 3-fold slower for the Y37L mutant and almost 2-fold slower for the F23L mutant. Amyloid fibrils formed from each of the single mutants were effective at seeding amyloid formation by wild-type IAPP, implying that the fibril structures are similar. The F15L/F23L double mutant has a larger effect than the F15L/Y37L double mutant on the rate of amyloid formation, even though a Y37L substitution has more drastic consequences in the wild-type background than does the F23L mutation, suggesting nonadditive effects between the different sites. The triple leucine mutant and the F23L/Y37L double mutant are the slowest to form amyloid. F15 has been proposed to make important contacts early in the aggregation pathway, but the data for the F15L mutant indicate that they are not optimal. A set of variants containing natural and unnatural amino acids at position 15, which were designed to conserve hydrophobicity, but alter α-helix and β-sheet propensity, were analyzed to determine the properties of this position that control the rate of amyloid formation. There is no correlation between β-sheet propensity at this position and the rate of amyloid formation, but there is a correlation with α-helical propensity.

Fertility: purinergic receptors and the male contraceptive pill.

PubMed

Dunn, P M

2000-04-20

Knockout mice lacking the P2X(1) receptor appear normal, but fail to breed. Analysis of these mutant mice clearly shows that purinergic co-transmission has a physiological role in the was deferens. These findings also raise the possibility of developing non-hormonal ways of regulating male fertility.

Uterine Dysfunction in Biglycan and Decorin Deficient Mice Leads to Dystocia during Parturition

PubMed Central

Wu, Zhiping; Aron, Abraham W.; Macksoud, Elyse E.; Iozzo, Renato V.; Hai, Chi-Ming; Lechner, Beatrice E.

2012-01-01

Cesarean birth rates are rising. Uterine dysfunction, the exact mechanism of which is unknown, is a common indication for Cesarean delivery. Biglycan and decorin are two small leucine-rich proteoglycans expressed in the extracellular matrix of reproductive tissues and muscle. Mice deficient in biglycan display a mild muscular dystrophy, and, along with mice deficient in decorin, are models of Ehlers-Danlos Syndrome, a connective tissue anomaly associated with uterine rupture. As a variant of Ehlers-Danlos Syndrome is caused by a genetic mutation resulting in abnormal biglycan and decorin secretion, we hypothesized that biglycan and decorin play a role in uterine function. Thus, we assessed wild-type, biglycan, decorin and double knockout pregnancies for timing of birth and uterine function. Uteri were harvested at embryonic days 12, 15 and 18. Nonpregnant uterine samples of the same genotypes were assessed for tissue failure rate and spontaneous and oxytocin-induced contractility. We discovered that biglycan/decorin mixed double-knockout dams displayed dystocia, were at increased risk of delayed labor onset, and showed increased tissue failure in a predominantly decorin-dependent manner. In vitro spontaneous uterine contractile amplitude and oxytocin-induced contractile force were decreased in all biglycan and decorin knockout genotypes compared to wild-type. Notably, we found no significant compensation between biglycan and decorin using quantitative real time PCR or immunohistochemistry. We conclude that the biglycan/decorin mixed double knockout mouse is a model of dystocia and delayed labor onset. Moreover, decorin is necessary for uterine function in a dose-dependent manner, while biglycan exhibits partial compensatory mechanisms in vivo. Thus, this model is poised for use as a model for testing novel targets for preventive or therapeutic manipulation of uterine dysfunction. PMID:22253749

Importance of the alternative oxidase (AOX) pathway in regulating cellular redox and ROS homeostasis to optimize photosynthesis during restriction of the cytochrome oxidase pathway in Arabidopsis thaliana

PubMed Central

Vishwakarma, Abhaypratap; Tetali, Sarada Devi; Selinski, Jennifer; Scheibe, Renate; Padmasree, Kollipara

2015-01-01

Background and Aims The importance of the alternative oxidase (AOX) pathway, particularly AOX1A, in optimizing photosynthesis during de-etiolation, under elevated CO2, low temperature, high light or combined light and drought stress is well documented. In the present study, the role of AOX1A in optimizing photosynthesis was investigated when electron transport through the cytochrome c oxidase (COX) pathway was restricted at complex III. Methods Leaf discs of wild-type (WT) and aox1a knock-out mutants of Arabidopsis thaliana were treated with antimycin A (AA) under growth-light conditions. To identify the impact of AOX1A deficiency in optimizing photosynthesis, respiratory O2 uptake and photosynthesis-related parameters were measured along with changes in redox couples, reactive oxygen species (ROS), lipid peroxidation and expression levels of genes related to respiration, the malate valve and the antioxidative system. Key Results In the absence of AA, aox1a knock-out mutants did not show any difference in physiological, biochemical or molecular parameters compared with WT. However, after AA treatment, aox1a plants showed a significant reduction in both respiratory O2 uptake and NaHCO3-dependent O2 evolution. Chlorophyll fluorescence and P700 studies revealed that in contrast to WT, aox1a knock-out plants were incapable of maintaining electron flow in the chloroplastic electron transport chain, and thereby inefficient heat dissipation (low non-photochemical quenching) was observed. Furthermore, aox1a mutants exhibited significant disturbances in cellular redox couples of NAD(P)H and ascorbate (Asc) and consequently accumulation of ROS and malondialdehyde (MDA) content. By contrast, WT plants showed a significant increase in transcript levels of CSD1, CAT1, sAPX, COX15 and AOX1A in contrast to aox1a mutants. Conclusions These results suggest that AOX1A plays a significant role in sustaining the chloroplastic redox state and energization to optimize photosynthesis by regulating cellular redox homeostasis and ROS generation when electron transport through the COX pathway is disturbed at complex III. PMID:26292995

A mental retardation gene, motopsin/neurotrypsin/prss12, modulates hippocampal function and social interaction

PubMed Central

Mitsui, Shinichi; Osako, Yoji; Yokoi, Fumiaki; Dang, Mai T.; Yuri, Kazunari; Li, Yuqing; Yamaguchi, Nozomi

2010-01-01

Motopsin is a mosaic serine protease secreted from neuronal cells in various brain regions including the hippocampus. The loss of motopsin function causes nonsyndromic mental retardation in humans and impairs long-term memory formation in Drosophila. To understand motopsin’s function in the mammalian brain, motopsin knockout mice were generated. Motopsin knockout mice did not have significant deficit in memory formation, as was tested using in the Morris water maze, passive avoidance, and Y-maze tests. A social recognition test showed that the motopsin knockout mice had the ability to recognize two stimulator mice, suggesting normal social memory. In a social novelty test, motopsin knockout mice spent a longer time investigating a familiar mouse than wild-type mice did. In a resident-intruder test, motopsin knockout mice showed prolonged social interaction compared to wild-type mice. Consistent with the behavioral deficit, spine density was significantly decreased on apical dendrites, but not on basal dendrites, of hippocampal pyramidal neurons of motopsin knockout mice. In contrast, pyramidal neurons at the cingulate cortex showed normal spine density. Spatial learning and social interaction induced the phosphorylation of cAMP responsive element binding protein (CREB) in hippocampal neurons of wild-type mice, whereas the phosphorylation of CREB was markedly decreased in mutant mouse brains. Our results indicate that an extracellular protease, motopsin, preferentially affects social behaviors, and modulates the functions of hippocampal neurons. PMID:20092579

Investigation of gene effects and epistatic interactions between Akt1 and neuregulin 1 in the regulation of behavioral phenotypes and social functions in genetic mouse models of schizophrenia

PubMed Central

Huang, Ching-Hsun; Pei, Ju-Chun; Luo, Da-Zhong; Chen, Ching; Chen, Yi-Wen; Lai, Wen-Sung

2015-01-01

Accumulating evidence from human genetic studies has suggested several functional candidate genes that might contribute to susceptibility to schizophrenia, including AKT1 and neuregulin 1 (NRG1). Recent findings also revealed that NRG1 stimulates the PI3-kinase/AKT signaling pathway, which might be involved in the functional outcomes of some schizophrenic patients. The aim of this study was to evaluate the effect of Akt1-deficiency and Nrg1-deficiency alone or in combination in the regulation of behavioral phenotypes, cognition, and social functions using genetically modified mice as a model. Male Akt1+/−, Nrg1+/−, and double mutant mice were bred and compared with their wild-type (WT) littermate controls. In Experiment 1, general physical examination revealed that all mutant mice displayed a normal profile of body weight during development and a normal brain activity with microPET scan. In Experiment 2, no significant genotypic differences were found in our basic behavioral phenotyping, including locomotion, anxiety-like behavior, and sensorimotor gating function. However, both Nrg1+/− and double mutant mice exhibited impaired episodic-like memory. Double mutant mice also had impaired sociability. In Experiment 3, a synergistic epistasis between Akt1 and Nrg1 was further confirmed in double mutant mice in that they had impaired social interaction compared to the other 3 groups, especially encountering with a novel male or an ovariectomized female. Double mutant and Nrg1+/− mice also emitted fewer female urine-induced ultrasonic vocalization calls. Collectively, our results indicate that double deficiency of Akt1 and Nrg1 can result in the impairment of social cognitive functions, which might be pertinent to the pathogenesis of schizophrenia-related social cognition. PMID:25688191

Investigation of gene effects and epistatic interactions between Akt1 and neuregulin 1 in the regulation of behavioral phenotypes and social functions in genetic mouse models of schizophrenia.

PubMed

Huang, Ching-Hsun; Pei, Ju-Chun; Luo, Da-Zhong; Chen, Ching; Chen, Yi-Wen; Lai, Wen-Sung

2014-01-01

Accumulating evidence from human genetic studies has suggested several functional candidate genes that might contribute to susceptibility to schizophrenia, including AKT1 and neuregulin 1 (NRG1). Recent findings also revealed that NRG1 stimulates the PI3-kinase/AKT signaling pathway, which might be involved in the functional outcomes of some schizophrenic patients. The aim of this study was to evaluate the effect of Akt1-deficiency and Nrg1-deficiency alone or in combination in the regulation of behavioral phenotypes, cognition, and social functions using genetically modified mice as a model. Male Akt1 (+/-), Nrg1 (+/-), and double mutant mice were bred and compared with their wild-type (WT) littermate controls. In Experiment 1, general physical examination revealed that all mutant mice displayed a normal profile of body weight during development and a normal brain activity with microPET scan. In Experiment 2, no significant genotypic differences were found in our basic behavioral phenotyping, including locomotion, anxiety-like behavior, and sensorimotor gating function. However, both Nrg1 (+/-) and double mutant mice exhibited impaired episodic-like memory. Double mutant mice also had impaired sociability. In Experiment 3, a synergistic epistasis between Akt1 and Nrg1 was further confirmed in double mutant mice in that they had impaired social interaction compared to the other 3 groups, especially encountering with a novel male or an ovariectomized female. Double mutant and Nrg1 (+/-) mice also emitted fewer female urine-induced ultrasonic vocalization calls. Collectively, our results indicate that double deficiency of Akt1 and Nrg1 can result in the impairment of social cognitive functions, which might be pertinent to the pathogenesis of schizophrenia-related social cognition.

Acetolactate synthase regulatory subunits play divergent and overlapping roles in branched-chain amino acid synthesis and Arabidopsis development.

PubMed

Dezfulian, Mohammad H; Foreman, Curtis; Jalili, Espanta; Pal, Mrinal; Dhaliwal, Rajdeep K; Roberto, Don Karl A; Imre, Kathleen M; Kohalmi, Susanne E; Crosby, William L

2017-04-07

Branched-chain amino acids (BCAAs) are synthesized by plants, fungi, bacteria, and archaea with plants being the major source of these amino acids in animal diets. Acetolactate synthase (ALS) is the first enzyme in the BCAA synthesis pathway. Although the functional contribution of ALS to BCAA biosynthesis has been extensively characterized, a comprehensive understanding of the regulation of this pathway at the molecular level is still lacking. To characterize the regulatory processes governing ALS activity we utilized several complementary approaches. Using the ALS catalytic protein subunit as bait we performed a yeast two-hybrid (Y2H) screen which resulted in the identification of a set of interacting proteins, two of which (denoted as ALS-INTERACTING PROTEIN1 and 3 [AIP1 and AIP3, respectively]) were found to be evolutionarily conserved orthologues of bacterial feedback-regulatory proteins and therefore implicated in the regulation of ALS activity. To investigate the molecular role AIPs might play in BCAA synthesis in Arabidopsis thaliana, we examined the functional contribution of aip1 and aip3 knockout alleles to plant patterning and development and BCAA synthesis under various growth conditions. Loss-of-function genetic backgrounds involving these two genes exhibited differential aberrant growth responses in valine-, isoleucine-, and sodium chloride-supplemented media. While BCAA synthesis is believed to be localized to the chloroplast, both AIP1 and AIP3 were found to localize to the peroxisome in addition to the chloroplast. Analysis of free amino acid pools in the mutant backgrounds revealed that they differ in the absolute amount of individual BCAAs accumulated and exhibit elevated levels of BCAAs in leaf tissues. Despite the phenotypic differences observed in aip1 and aip3 backgrounds, functional redundancy between these loci was suggested by the finding that aip1/aip3 double knockout mutants are severely developmentally compromised. Taken together the data suggests that the two regulatory proteins, in conjunction with ALS, have overlapping but distinct functions in BCAA synthesis, and also play a role in pathways unrelated to BCAA synthesis such as sodium-ion homeostasis, extending to broader aspects of patterning and development.

Overexpression of IRM1 Enhances Resistance to Aphids in Arabidopsis thaliana

PubMed Central

Chen, Xi; Zhang, Zhao; Visser, Richard G. F.; Broekgaarden, Colette; Vosman, Ben

2013-01-01

Aphids are insects that cause direct damage to crops by the removal of phloem sap, but more importantly they spread devastating viruses. Aphids use their sophisticated mouthpart (i.e. stylet) to feed from the phloem sieve elements of the host plant. To identify genes that affect host plant resistance to aphids, we previously screened an Arabidopsis thaliana activation tag mutant collection. In such mutants, tagged genes are overexpressed by a strong 35S enhancer adjacent to the natural promoter, resulting in a dominant gain-of-function phenotype. We previously identified several of these mutants on which the aphid Myzus persicae showed a reduced population development compared with wild type. In the present study we show that the gene responsible for the phenotype of one of the mutants is At5g65040 and named this gene Increased Resistance to Myzus persicae 1 (IRM1). Overexpression of the cloned IRM1 gene conferred a phenotype identical to that of the original mutant. Conversely, an IRM1 knockout mutant promoted aphid population development compared to the wild type. We performed Electrical Penetration Graph analysis to investigate how probing and feeding behaviour of aphids was affected on plants that either overexpressed IRM1 or contained a knockout mutation in this gene. The EPG results indicated that the aphids encounter resistance factors while reaching for the phloem on the overexpressing line. This resistance mechanism also affected other aphid species and is suggested to be of mechanical nature. Interestingly, genetic variation for IRM1 expression in response to aphid attack was observed. Upon aphid attack the expression of IRM1 was initially (after 6 hours) induced in ecotype Wassilewskija followed by suppression. In Columbia-0, IRM1 expression was already suppressed six hours after the start of the infestation. The resistance conferred by the overexpression of IRM1 in A. thaliana trades off with plant growth. PMID:23951039

New approach for fish breeding by chemical mutagenesis: establishment of TILLING method in fugu (Takifugu rubripes) with ENU mutagenesis.

PubMed

Kuroyanagi, Miwa; Katayama, Takashi; Imai, Tadashi; Yamamoto, Yoshihisa; Chisada, Shin-ichi; Yoshiura, Yasutoshi; Ushijima, Tomokazu; Matsushita, Tomonao; Fujita, Masashi; Nozawa, Aoi; Suzuki, Yuzuru; Kikuchi, Kiyoshi; Okamoto, Hiroyuki

2013-11-13

In fish breeding, it is essential to discover and generate fish exhibiting an effective phenotype for the aquaculture industry, but screening for natural mutants by only depending on natural spontaneous mutations is limited. Presently, reverse genetics has become an important tool to generate mutants, which exhibit the phenotype caused by inactivation of a gene. TILLING (Targeting Induced Local Lesions IN Genomes) is a reverse genetics strategy that combines random chemical mutagenesis with high-throughput discovery technologies for screening the induced mutations in target genes. Although the chemical mutagenesis has been used widely in a variety of model species and also genetic breeding of microorganisms and crops, the application of the mutagenesis in fish breeding has been only rarely reported. In this study, we developed the TILLING method in fugu with ENU mutagenesis and high-resolution melting (HRM) analysis to detect base pair changes in target sequences. Fugu males were treated 3 times at weekly intervals with various ENU concentrations, and then the collected sperm after the treatment was used to fertilize normal female for generating the mutagenized population (F1). The fertilization and the hatching ratios were similar to those of the control and did not reveal a dose dependency of ENU. Genomic DNA from the harvested F1 offspring was used for the HRM analysis. To obtain a fish exhibiting a useful phenotype (e.g. high meat production and rapid growth), fugu myostatin (Mstn) gene was examined as a target gene, because it has been clarified that the mstn deficient medaka exhibited double-muscle phenotype in common with MSTN knockout mice and bovine MSTN mutant. As a result, ten types of ENU-induced mutations were identified including a nonsense mutation in the investigated region with HRM analysis. In addition, the average mutation frequency in fugu Mstn gene was 1 mutant per 297 kb, which is similar to values calculated for zebrafish and medaka TILLING libraries. These results demonstrate that the TILLING method in fugu was established. We anticipate that this TILLING approach can be used to generate a wide range of mutant alleles, and be applicable to many farmed fish that can be chemically mutagenized.

High-throughput discovery of novel developmental phenotypes.

PubMed

Dickinson, Mary E; Flenniken, Ann M; Ji, Xiao; Teboul, Lydia; Wong, Michael D; White, Jacqueline K; Meehan, Terrence F; Weninger, Wolfgang J; Westerberg, Henrik; Adissu, Hibret; Baker, Candice N; Bower, Lynette; Brown, James M; Caddle, L Brianna; Chiani, Francesco; Clary, Dave; Cleak, James; Daly, Mark J; Denegre, James M; Doe, Brendan; Dolan, Mary E; Edie, Sarah M; Fuchs, Helmut; Gailus-Durner, Valerie; Galli, Antonella; Gambadoro, Alessia; Gallegos, Juan; Guo, Shiying; Horner, Neil R; Hsu, Chih-Wei; Johnson, Sara J; Kalaga, Sowmya; Keith, Lance C; Lanoue, Louise; Lawson, Thomas N; Lek, Monkol; Mark, Manuel; Marschall, Susan; Mason, Jeremy; McElwee, Melissa L; Newbigging, Susan; Nutter, Lauryl M J; Peterson, Kevin A; Ramirez-Solis, Ramiro; Rowland, Douglas J; Ryder, Edward; Samocha, Kaitlin E; Seavitt, John R; Selloum, Mohammed; Szoke-Kovacs, Zsombor; Tamura, Masaru; Trainor, Amanda G; Tudose, Ilinca; Wakana, Shigeharu; Warren, Jonathan; Wendling, Olivia; West, David B; Wong, Leeyean; Yoshiki, Atsushi; MacArthur, Daniel G; Tocchini-Valentini, Glauco P; Gao, Xiang; Flicek, Paul; Bradley, Allan; Skarnes, William C; Justice, Monica J; Parkinson, Helen E; Moore, Mark; Wells, Sara; Braun, Robert E; Svenson, Karen L; de Angelis, Martin Hrabe; Herault, Yann; Mohun, Tim; Mallon, Ann-Marie; Henkelman, R Mark; Brown, Steve D M; Adams, David J; Lloyd, K C Kent; McKerlie, Colin; Beaudet, Arthur L; Bućan, Maja; Murray, Stephen A

2016-09-22

Approximately one-third of all mammalian genes are essential for life. Phenotypes resulting from knockouts of these genes in mice have provided tremendous insight into gene function and congenital disorders. As part of the International Mouse Phenotyping Consortium effort to generate and phenotypically characterize 5,000 knockout mouse lines, here we identify 410 lethal genes during the production of the first 1,751 unique gene knockouts. Using a standardized phenotyping platform that incorporates high-resolution 3D imaging, we identify phenotypes at multiple time points for previously uncharacterized genes and additional phenotypes for genes with previously reported mutant phenotypes. Unexpectedly, our analysis reveals that incomplete penetrance and variable expressivity are common even on a defined genetic background. In addition, we show that human disease genes are enriched for essential genes, thus providing a dataset that facilitates the prioritization and validation of mutations identified in clinical sequencing efforts.

High-throughput discovery of novel developmental phenotypes

PubMed Central

Dickinson, Mary E.; Flenniken, Ann M.; Ji, Xiao; Teboul, Lydia; Wong, Michael D.; White, Jacqueline K.; Meehan, Terrence F.; Weninger, Wolfgang J.; Westerberg, Henrik; Adissu, Hibret; Baker, Candice N.; Bower, Lynette; Brown, James M.; Caddle, L. Brianna; Chiani, Francesco; Clary, Dave; Cleak, James; Daly, Mark J.; Denegre, James M.; Doe, Brendan; Dolan, Mary E.; Edie, Sarah M.; Fuchs, Helmut; Gailus-Durner, Valerie; Galli, Antonella; Gambadoro, Alessia; Gallegos, Juan; Guo, Shiying; Horner, Neil R.; Hsu, Chih-wei; Johnson, Sara J.; Kalaga, Sowmya; Keith, Lance C.; Lanoue, Louise; Lawson, Thomas N.; Lek, Monkol; Mark, Manuel; Marschall, Susan; Mason, Jeremy; McElwee, Melissa L.; Newbigging, Susan; Nutter, Lauryl M.J.; Peterson, Kevin A.; Ramirez-Solis, Ramiro; Rowland, Douglas J.; Ryder, Edward; Samocha, Kaitlin E.; Seavitt, John R.; Selloum, Mohammed; Szoke-Kovacs, Zsombor; Tamura, Masaru; Trainor, Amanda G; Tudose, Ilinca; Wakana, Shigeharu; Warren, Jonathan; Wendling, Olivia; West, David B.; Wong, Leeyean; Yoshiki, Atsushi; MacArthur, Daniel G.; Tocchini-Valentini, Glauco P.; Gao, Xiang; Flicek, Paul; Bradley, Allan; Skarnes, William C.; Justice, Monica J.; Parkinson, Helen E.; Moore, Mark; Wells, Sara; Braun, Robert E.; Svenson, Karen L.; de Angelis, Martin Hrabe; Herault, Yann; Mohun, Tim; Mallon, Ann-Marie; Henkelman, R. Mark; Brown, Steve D.M.; Adams, David J.; Lloyd, K.C. Kent; McKerlie, Colin; Beaudet, Arthur L.; Bucan, Maja; Murray, Stephen A.

2016-01-01

Approximately one third of all mammalian genes are essential for life. Phenotypes resulting from mouse knockouts of these genes have provided tremendous insight into gene function and congenital disorders. As part of the International Mouse Phenotyping Consortium effort to generate and phenotypically characterize 5000 knockout mouse lines, we have identified 410 lethal genes during the production of the first 1751 unique gene knockouts. Using a standardised phenotyping platform that incorporates high-resolution 3D imaging, we identified novel phenotypes at multiple time points for previously uncharacterized genes and additional phenotypes for genes with previously reported mutant phenotypes. Unexpectedly, our analysis reveals that incomplete penetrance and variable expressivity are common even on a defined genetic background. In addition, we show that human disease genes are enriched for essential genes identified in our screen, thus providing a novel dataset that facilitates prioritization and validation of mutations identified in clinical sequencing efforts. PMID:27626380

Mutagenesis and phenotyping resources in zebrafish for studying development and human disease

PubMed Central

Varshney, Gaurav Kumar

2014-01-01

The zebrafish (Danio rerio) is an important model organism for studying development and human disease. The zebrafish has an excellent reference genome and the functions of hundreds of genes have been tested using both forward and reverse genetic approaches. Recent years have seen an increasing number of large-scale mutagenesis projects and the number of mutants or gene knockouts in zebrafish has increased rapidly, including for the first time conditional knockout technologies. In addition, targeted mutagenesis techniques such as zinc finger nucleases, transcription activator-like effector nucleases and clustered regularly interspaced short sequences (CRISPR) or CRISPR-associated (Cas), have all been shown to effectively target zebrafish genes as well as the first reported germline homologous recombination, further expanding the utility and power of zebrafish genetics. Given this explosion of mutagenesis resources, it is now possible to perform systematic, high-throughput phenotype analysis of all zebrafish gene knockouts. PMID:24162064

Down-Regulation of p53 by Double-Stranded RNA Modulates the Antiviral Response

PubMed Central

Marques, Joao T.; Rebouillat, Dominique; Ramana, Chilakamarti V.; Murakami, Junko; Hill, Jason E.; Gudkov, Andrei; Silverman, Robert H.; Stark, George R.; Williams, Bryan R. G.

2005-01-01

p53 has been well characterized as a tumor suppressor gene, but its role in antiviral defense remains unclear. A recent report has demonstrated that p53 can be induced by interferons and is activated after vesicular stomatitis virus (VSV) infection. We observed that different nononcogenic viruses, including encephalomyocarditis virus (EMCV) and human parainfluenza virus type 3 (HPIV3), induced down-regulation of p53 in infected cells. Double-stranded RNA (dsRNA) and a mutant vaccinia virus lacking the dsRNA binding protein E3L can also induce this effect, indicating that dsRNA formed during viral infection is likely the trigger for down-regulation of p53. The mechanism of down-regulation of p53 by dsRNA relies on translation inhibition mediated by the PKR and RNase L pathways. In the absence of p53, the replication of both EMCV and HPIV3 was retarded, whereas, conversely, VSV replication was enhanced. Cell cycle analysis indicated that wild-type (WT) but not p53 knockout (KO) fibroblasts undergo an early-G1 arrest following dsRNA treatment. Moreover, in WT cells the onset of dsRNA-induced apoptosis begins after p53 levels are down-regulated, whereas p53 KO cells, which lack the early-G1 arrest, rapidly undergo apoptosis. Hence, our data suggest that the down-regulation of p53 facilitates apoptosis, thereby limiting viral replication. PMID:16103161

Simian virus 40 small t antigen is not required for the maintenance of transformation but may act as a promoter (cocarcinogen) during establishment of transformation in resting rat cells.

PubMed Central

Seif, R; Martin, R G

1979-01-01

Simian virus 40 deletion mutants affecting the 20,000-dalton (20K) t antigen and tsA mutants rendering the 90K T antigen temperature sensitive, as well as double mutants containing both mutations, induced host DNA synthesis in resting rat cells at the restrictive temperature. Nonetheless, the deletion mutants and double mutants did not induce transformation in resting cells even at the permissive temperature. On the other hand, the deletion mutants did induce full transformants when actively growing rat cells were infected; the transformants grew efficiently in agar and to high saturation densities on platic. The double mutants did not induce T-antigen-independent (temperature-insensitive) transformants which were shown previously to arise preferentially from resting cells. Thus, small t antigen was dispensable for the maintenance of the transformed phenotype in T-antigen-dependent rat transformants (transformants derived from growing cells) and may play a role in the establishment of T-antigen-independent transformants. We attempt to establish a parallel between transformation induced by chemical carcinogens and simian virus 40-induced transformation. Images PMID:229274

Simian virus 40 small t antigen is not required for the maintenance of transformation but may act as a promoter (cocarcinogen) during establishment of transformation in resting rat cells.

PubMed

Seif, R; Martin, R G

1979-12-01

Simian virus 40 deletion mutants affecting the 20,000-dalton (20K) t antigen and tsA mutants rendering the 90K T antigen temperature sensitive, as well as double mutants containing both mutations, induced host DNA synthesis in resting rat cells at the restrictive temperature. Nonetheless, the deletion mutants and double mutants did not induce transformation in resting cells even at the permissive temperature. On the other hand, the deletion mutants did induce full transformants when actively growing rat cells were infected; the transformants grew efficiently in agar and to high saturation densities on platic. The double mutants did not induce T-antigen-independent (temperature-insensitive) transformants which were shown previously to arise preferentially from resting cells. Thus, small t antigen was dispensable for the maintenance of the transformed phenotype in T-antigen-dependent rat transformants (transformants derived from growing cells) and may play a role in the establishment of T-antigen-independent transformants. We attempt to establish a parallel between transformation induced by chemical carcinogens and simian virus 40-induced transformation.

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

Fan, J.; Xu, C.

The biogenesis of photosynthetic membranes in plants relies largely on lipid import from the endoplasmic reticulum (ER) and this lipid transport process is mediated by TGD proteins in Arabidopsis. Such a dependency of chloroplast biogenesis on ER-to-plastid lipid transport was recently exemplified by analyzing double mutants between tgd1-1 or tgd4-3 and fad6 mutants. The fad6 mutants are defective in the desaturation of membrane lipids in chloroplasts and therefore dependent on import of polyunsaturated lipid precursors from the ER for constructing a competent thylakoid membrane system. In support of a critical role of TGD proteins in ER-to-plastid lipid trafficking, we showedmore » that the introduction of the tgd mutations into fad6 mutant backgrounds led to drastic reductions in relative amounts of thylakoid lipids. Moreover, the tgd1-1 fad6 and tgd4-3 fad6 double mutants were deficient in polyunsaturated fatty acids in chloroplast membrane lipids, and severely compromised in the biogenesis of photosynthetic membrane systems. Here we report that these double mutants are severely impaired in chloroplast division. The possible role of membrane lipids in chloroplast division is discussed.« less

pKAMA-ITACHI Vectors for Highly Efficient CRISPR/Cas9-Mediated Gene Knockout in Arabidopsis thaliana.

PubMed

Tsutsui, Hiroki; Higashiyama, Tetsuya

2017-01-01

The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) system is widely used as a tool for genome engineering in various organisms. A complex consisting of Cas9 and single guide RNA (sgRNA) induces a DNA double-strand break in a sequence-specific manner, resulting in knockout. Some binary vectors for CRISPR/Cas9 in plants have been reported, but there is a problem with low efficiency. Here, we present a newly developed, highly efficient CRISPR/Cas9 vector for Arabidopsis thaliana, pKAMA-ITACHI Red (pKIR), harboring the RIBOSOMAL PROTEIN S5 A (RPS5A) promoter to drive Cas9. The RPS5A promoter maintains high constitutive expression at all developmental stages starting from the egg cell and including meristematic cells. Even in the T1 generation, pKIR induced null phenotypes in some genes: PHYTOENE DESATURASE 3 (PDS3), AGAMOUS (AG) and DUO POLLEN 1 (DUO1). Mutations induced by pKIR were carried in the germ cell line of the T1 generation. Surprisingly, in some lines, 100% of the T2 plants had the adh1 (ALCOHOL DEHYDROGENASE 1) null phenotype, indicating that pKIR strongly induced heritable mutations. Cas9-free T2 mutant plants were obtained by removing T2 seeds expressing a fluorescent marker in pKIR. Our results suggest that the pKIR system is a powerful molecular tool for genome engineering in Arabidopsis. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.

Myostatin knockout using zinc-finger nucleases promotes proliferation of ovine primary satellite cells in vitro.

PubMed

Salabi, Fatemeh; Nazari, Mahmood; Chen, Qing; Nimal, Jonathan; Tong, Jianming; Cao, Wen G

2014-12-20

Myostatin (MSTN) has previously been shown to negatively regulate the proliferation and differentiation of skeletal muscle cells. Satellite cells are quiescent muscle stem cells that promote muscle growth and repair. Because the mechanism of MSTN in the biology of satellite cells is not well understood, this study was conducted to generate MSTN mono-allelic knockout satellite cells using the zinc-finger nuclease mRNA (MSTN-KO ZFN mRNA) and also to investigate the effect of this disruption on the proliferation and differentiation of sheep primary satellite cells (PSCs). Nineteen biallelic and four mono-allelic knockout cell clones were obtained after sequence analysis. The homologous mono-allelic knockout cells with 5-bp deletion were used to further evaluations. The results demonstrated that mono-allelic knockout of MSTN gene leads to translation inhibition. Real-time quantitative PCR results indicated that knockout of MSTN contributed to an increase in CDK2 and follistatin and a decrease in p21 at the transcript level in proliferation conditions. Moreover, MSTN knockout significantly increased the proliferation of mutant clones (P < 0.01). Consistent with the observed increase in CDK2 and decrease in p21 in cells lacking MSTN, cell cycle analysis showed that MSTN negatively regulated the G1 to S progression. In addition, knockout of myostatin resulted in a remarkable increase in MyoD and MyoG expression under differentiating conditions but had no effect on Myf5 expression. These results expanded our understanding of the regulation mechanism of MSTN. Furthermore, the MSTN-KO ZFN mRNA system in PSCs could be used to generate transgenic sheep in the future.

The Role of mDia1 in the Aberrant Innate Immune Signaling in del(5q) Myelodysplastic Syndromes

DTIC Science & Technology

2016-10-01

Myeloproliferative Neoplasms The goal of this project will be to identify transcriptional pathways that are dysregulated in PMF megakaryocytes and... myeloproliferative phenotype, as previously reported in miR-146 knockout mice. Here we propose that the mDia1/miR-146a double knockout mice phenocopy...ineffective erythropoiesis and represent a model of anemia that is commonly seen MDS. However, the possibility of a myeloproliferative phenotype cannot be

Mutations in Arabidopsis yellow stripe-like1 and yellow stripe-like3 reveal their roles in metal ion homeostasis and loading of metal ions in seeds.

PubMed

Waters, Brian M; Chu, Heng-Hsuan; Didonato, Raymond J; Roberts, Louis A; Eisley, Robynn B; Lahner, Brett; Salt, David E; Walker, Elsbeth L

2006-08-01

Here, we describe two members of the Arabidopsis (Arabidopsis thaliana) Yellow Stripe-Like (YSL) family, AtYSL1 and AtYSL3. The YSL1 and YSL3 proteins are members of the oligopeptide transporter family and are predicted to be integral membrane proteins. YSL1 and YSL3 are similar to the maize (Zea mays) YS1 phytosiderophore transporter (ZmYS1) and the AtYSL2 iron (Fe)-nicotianamine transporter, and are predicted to transport metal-nicotianamine complexes into cells. YSL1 and YSL3 mRNAs are expressed in both root and shoot tissues, and both are regulated in response to the Fe status of the plant. Beta-glucuronidase reporter expression, driven by YSL1 and YSL3 promoters, reveals expression patterns of the genes in roots, leaves, and flowers. Expression was highest in senescing rosette leaves and cauline leaves. Whereas the single mutants ysl1 and ysl3 had no visible phenotypes, the ysl1ysl3 double mutant exhibited Fe deficiency symptoms, such as interveinal chlorosis. Leaf Fe concentrations are decreased in the double mutant, whereas manganese, zinc, and especially copper concentrations are elevated. In seeds of double-mutant plants, the concentrations of Fe, zinc, and copper are low. Mobilization of metals from leaves during senescence is impaired in the double mutant. In addition, the double mutant has reduced fertility due to defective anther and embryo development. The proposed physiological roles for YSL1 and YSL3 are in delivery of metal micronutrients to and from vascular tissues.

A novel regulation mechanism of DNA repair by damage-induced and RAD23-dependent stabilization of xeroderma pigmentosum group C protein

PubMed Central

Ng, Jessica M.Y.; Vermeulen, Wim; van der Horst, Gijsbertus T.J.; Bergink, Steven; Sugasawa, Kaoru; Vrieling, Harry; Hoeijmakers, Jan H.J.

2003-01-01

Primary DNA damage sensing in mammalian global genome nucleotide excision repair (GG-NER) is performed by the xeroderma pigmentosum group C (XPC)/HR23B protein complex. HR23B and HR23A are human homologs of the yeast ubiquitin-domain repair factor RAD23, the function of which is unknown. Knockout mice revealed that mHR23A and mHR23B have a fully redundant role in NER, and a partially redundant function in embryonic development. Inactivation of both genes causes embryonic lethality, but appeared still compatible with cellular viability. Analysis of mHR23A/B double-mutant cells showed that HR23 proteins function in NER by governing XPC stability via partial protection against proteasomal degradation. Interestingly, NER-type DNA damage further stabilizes XPC and thereby enhances repair. These findings resolve the primary function of RAD23 in repair and reveal a novel DNA-damage-dependent regulation mechanism of DNA repair in eukaryotes, which may be part of a more global damage-response circuitry. PMID:12815074

Scaling laws and universality for the strength of genetic interactions in yeast

NASA Astrophysics Data System (ADS)

Velenich, Andrea; Dai, Mingjie; Gore, Jeff

2012-02-01

Genetic interactions provide a window to the organization of the thousands of biochemical reactions in living cells. If two mutations affect unrelated cellular functions, the fitness effects of their combination can be easily predicted from the two separate fitness effects. However, because of interactions, for some pairs of mutations their combined fitness effect deviates from the naive prediction. We study genetic interactions in yeast cells by analyzing a publicly available database containing experimental growth rates of 5 million double mutants. We show that the characteristic strength of genetic interactions has a simple power law dependence on the fitness effects of the two interacting mutations and that the probability distribution of genetic interactions is a universal function. We further argue that the strength of genetic interactions depends only on the fitness effects of the interacting mutations and not on their biological origin in terms of single point mutations, entire gene knockouts or even more complicated physiological perturbations. Finally, we discuss the implications of the power law scaling of genetic interactions on the ruggedness of fitness landscapes and the consequent evolutionary dynamics.

PAC1- and VPAC2 receptors in light regulated behavior and physiology: Studies in single and double mutant mice

PubMed Central

Georg, Birgitte; Fahrenkrug, Jan

2017-01-01

The two sister peptides, pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) and their receptors, the PAC1 –and the VPAC2 receptors, are involved in regulation of the circadian timing system. PACAP as a neurotransmitter in the retinohypothalamic tract (RHT) and VIP as a neurotransmitter, involved in synchronization of SCN neurons. Behavior and physiology in VPAC2 deficient mice are strongly regulated by light most likely as a result of masking. Consequently, we used VPAC2 and PAC1/VPAC2 double mutant mice in comparison with PAC1 receptor deficient mice to further elucidate the role of PACAP in the light mediated regulation of behavior and physiology of the circadian system. We compared circadian rhythms in mice equipped with running wheels or implanted radio-transmitter measuring core body temperature kept in a full photoperiod ((FPP)(12:12 h light dark-cycles (LD)) and skeleton photo periods (SPP) at high and low light intensity. Furthermore, we examined the expression of PAC1- and VPAC2 receptors in the SCN of the different genotypes in combination with visualization of PACAP and VIP and determined whether compensatory changes in peptide and/or receptor expression in the reciprocal knockouts (KO) (PAC1 and VPAC2) had occurred. Our data demonstrate that in although being closely related at both ligand and receptor structure/sequence, PACAP/PAC1 receptor signaling are independent of VIP/VPAC2 receptor signaling and vice versa. Furthermore, lack of either of the receptors does not result in compensatory changes at neither the physiological or anatomical level. PACAP/PAC1 signaling is important for light regulated behavior, VIP/VPAC2signaling for stable clock function and both signaling pathways may play a role in shaping diurnality versus nocturnality. PMID:29155851

Lack of genetic interaction between Tbx20 and Tbx3 in early mouse heart development.

PubMed

Gavrilov, Svetlana; Harvey, Richard P; Papaioannou, Virginia E

2013-01-01

Members of the T-box family of transcription factors are important regulators orchestrating the complex regionalization of the developing mammalian heart. Individual mutations in Tbx20 and Tbx3 cause distinct congenital heart abnormalities in the mouse: Tbx20 mutations result in failure of heart looping, developmental arrest and lack of chamber differentiation, while hearts of Tbx3 mutants progress further, loop normally but show atrioventricular convergence and outflow tract defects. The two genes have overlapping areas of expression in the atrioventricular canal and outflow tract of the heart but their potential genetic interaction has not been previously investigated. In this study we produced compound mutants to investigate potential genetic interactions at the earliest stages of heart development. We find that Tbx20; Tbx3 double heterozygous mice are viable and fertile with no apparent abnormalities, while double homozygous mutants are embryonic lethal by midgestation. Double homozygous mutant embryos display abnormal cardiac morphogenesis, lack of heart looping, expression patterns of cardiac genes and time of death that are indistinguishable from Tbx20 homozygous mutants. Prior to death, the double homozygotes show an overall developmental delay similar to Tbx3 homozygous mutants. Thus the effects of Tbx20 are epistatic to Tbx3 in the heart but Tbx3 is epistatic to Tbx20 with respect to developmental delay.

Increased production of biomass-degrading enzymes by double deletion of creA and creB genes involved in carbon catabolite repression in Aspergillus oryzae.

PubMed

Ichinose, Sakurako; Tanaka, Mizuki; Shintani, Takahiro; Gomi, Katsuya

2018-02-01

In a previous study, we reported that a double gene deletion mutant for CreA and CreB, which constitute the regulatory machinery involved in carbon catabolite repression, exhibited improved production of α-amylase compared with the wild-type strain and single creA or creB deletion mutants in Aspergillus oryzae. Because A. oryzae can also produce biomass-degrading enzymes, such as xylolytic and cellulolytic enzymes, we examined the production levels of those enzymes in deletion mutants in this study. Xylanase and β-glucosidase activities in the wild-type were hardly detected in submerged culture containing xylose as the carbon source, whereas those enzyme activities were significantly increased in the single creA deletion (ΔcreA) and double creA and creB deletion (ΔcreAΔcreB) mutants. In particular, the ΔcreAΔcreB mutant exhibited >100-fold higher xylanase and β-glucosidase activities than the wild-type. Moreover, in solid-state culture, the β-glucosidase activity of the double deletion mutant was >7-fold higher than in the wild-type. These results suggested that deletion of both creA and creB genes could also efficiently improve the production levels of biomass-degrading enzymes in A. oryzae. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Draft Genome Sequence of Xylella fastidiosa subsp. fastidiosa Strain Stag's Leap.

PubMed

Chen, J; Wu, F; Zheng, Z; Deng, X; Burbank, L P; Stenger, D C

2016-04-21

ITALIC! Xylella fastidiosasubsp. ITALIC! fastidiosacauses Pierce's disease of grapevine. Presented here is the draft genome sequence of the Stag's Leap strain, previously used in pathogenicity/virulence assays to evaluate grapevine germplasm bearing Pierce's disease resistance and a phenotypic assessment of knockout mutants to determine gene function. Copyright © 2016 Chen et al.

Knockout-Rescue Embryonic Stem Cell-Derived Mouse Reveals Circadian-Period Control by Quality and Quantity of CRY1.

PubMed

Ode, Koji L; Ukai, Hideki; Susaki, Etsuo A; Narumi, Ryohei; Matsumoto, Katsuhiko; Hara, Junko; Koide, Naoshi; Abe, Takaya; Kanemaki, Masato T; Kiyonari, Hiroshi; Ueda, Hiroki R

2017-01-05

To conduct comprehensive characterization of molecular properties in organisms, we established an efficient method to produce knockout (KO)-rescue mice within a single generation. We applied this method to produce 20 strains of almost completely embryonic stem cell (ESC)-derived mice ("ES mice") rescued with wild-type and mutant Cry1 gene under a Cry1 -/- :Cry2 -/- background. A series of both phosphorylation-mimetic and non-phosphorylation-mimetic CRY1 mutants revealed that multisite phosphorylation of CRY1 can serve as a cumulative timer in the mammalian circadian clock. KO-rescue ES mice also revealed that CRY1-PER2 interaction confers a robust circadian rhythmicity in mice. Surprisingly, in contrast to theoretical predictions from canonical transcription/translation feedback loops, the residues surrounding the flexible P loop and C-lid domains of CRY1 determine circadian period without changing the degradation rate of CRY1. These results suggest that CRY1 determines circadian period through both its degradation-dependent and -independent pathways. Copyright © 2017 Elsevier Inc. All rights reserved.

Methyl Farnesoate Plays a Dual Role in Regulating Drosophila Metamorphosis

PubMed Central

Wen, Di; Rivera-Perez, Crisalejandra; Abdou, Mohamed; Jia, Qiangqiang; He, Qianyu; Liu, Xi; Zyaan, Ola; Xu, Jingjing; Bendena, William G.; Tobe, Stephen S.; Noriega, Fernando G.; Palli, Subba R.; Wang, Jian; Li, Sheng

2015-01-01

Corpus allatum (CA) ablation results in juvenile hormone (JH) deficiency and pupal lethality in Drosophila. The fly CA produces and releases three sesquiterpenoid hormones: JH III bisepoxide (JHB3), JH III, and methyl farnesoate (MF). In the whole body extracts, MF is the most abundant sesquiterpenoid, followed by JHB3 and JH III. Knockout of JH acid methyl transferase (jhamt) did not result in lethality; it decreased biosynthesis of JHB3, but MF biosynthesis was not affected. RNAi-mediated reduction of 3-hydroxy-3-methylglutaryl CoA reductase (hmgcr) expression in the CA decreased biosynthesis and titers of the three sesquiterpenoids, resulting in partial lethality. Reducing hmgcr expression in the CA of the jhamt mutant further decreased MF titer to a very low level, and caused complete lethality. JH III, JHB3, and MF function through Met and Gce, the two JH receptors, and induce expression of Kr-h1, a JH primary-response gene. As well, a portion of MF is converted to JHB3 in the hemolymph or peripheral tissues. Topical application of JHB3, JH III, or MF precluded lethality in JH-deficient animals, but not in the Met gce double mutant. Taken together, these experiments show that MF is produced by the larval CA and released into the hemolymph, from where it exerts its anti-metamorphic effects indirectly after conversion to JHB3, as well as acting as a hormone itself through the two JH receptors, Met and Gce. PMID:25774983

p53 and TAp63 Promote Keratinocyte Proliferation and Differentiation in Breeding Tubercles of the Zebrafish

PubMed Central

Fischer, Boris; Metzger, Manuel; Richardson, Rebecca; Knyphausen, Philipp; Ramezani, Thomas; Franzen, Rainer; Schmelzer, Elmon; Bloch, Wilhelm; Carney, Thomas J.; Hammerschmidt, Matthias

2014-01-01

p63 is a multi-isoform member of the p53 family of transcription factors. There is compelling genetic evidence that ΔNp63 isoforms are needed for keratinocyte proliferation and stemness in the developing vertebrate epidermis. However, the role of TAp63 isoforms is not fully understood, and TAp63 knockout mice display normal epidermal development. Here, we show that zebrafish mutants specifically lacking TAp63 isoforms, or p53, display compromised development of breeding tubercles, epidermal appendages which according to our analyses display more advanced stratification and keratinization than regular epidermis, including continuous desquamation and renewal of superficial cells by derivatives of basal keratinocytes. Defects are further enhanced in TAp63/p53 double mutants, pointing to partially redundant roles of the two related factors. Molecular analyses, treatments with chemical inhibitors and epistasis studies further reveal the existence of a linear TAp63/p53->Notch->caspase 3 pathway required both for enhanced proliferation of keratinocytes at the base of the tubercles and their subsequent differentiation in upper layers. Together, these studies identify the zebrafish breeding tubercles as specific epidermal structures sharing crucial features with the cornified mammalian epidermis. In addition, they unravel essential roles of TAp63 and p53 to promote both keratinocyte proliferation and their terminal differentiation by promoting Notch signalling and caspase 3 activity, ensuring formation and proper homeostasis of this self-renewing stratified epithelium. PMID:24415949

Cell surface fucosylation does not affect development of colon tumors in mice with germline Smad3 mutation

PubMed Central

Domino, Steven E.; Karnak, David M.; Hurd, Elizabeth A.

2006-01-01

Background/Aims: Neoplasia-related alterations in cell surface α(1,2)fucosylated glycans have been reported in multiple tumors including colon, pancreas, endometrium, cervix, bladder, lung, and choriocarcinoma. Spontaneous colorectal tumors from mice with a germline null mutation of transforming growth factor-β signaling gene Smad3 (Madh3) were tested for α(1,2)fucosylated glycan expression. Methods: Ulex Europaeus Agglutinin-I lectin staining, fucosyltransferase gene northern blot analysis, and a cross of mutant mice with Fut2 and Smad3 germline mutations were performed. Results: Spontaneous colorectal tumors from Smad3 (-/-) homozygous null mice were found to express α(1,2)fucosylated glycans in an abnormal pattern compared to adjacent nonneoplastic colon. Northern blot analysis of α(1,2)fucosyltransferase genes Fut1 and Fut2 revealed that Fut2, but not Fut1, steady-state mRNA levels were significantly increased in tumors relative to adjacent normal colonic mucosa. Mutant mice with a Fut2-inactivating germline mutation were crossed with Smad3 targeted mice. In Smad3 (-/-)/Fut2 (-/-) double knock-out mice, UEA-I lectin staining was eliminated from colon and colon tumors, however, the number and size of tumors present by 24 weeks of age did not vary regardless of the Fut2 genotype. Conclusions: In this model of colorectal cancer, cell surface α(1,2)fucosylation does not affect development of colon tumors. PMID:17264540

The Fanconi anemia pathway limits the severity of mutagenesis.

PubMed

Hinz, John M; Nham, Peter B; Salazar, Edmund P; Thompson, Larry H

2006-08-13

Fanconi anemia (FA) is a developmental and cancer predisposition disorder in which key, yet unknown, physiological events promoting chromosome stability are compromised. FA cells exhibit excess metaphase chromatid breaks and are universally hypersensitive to DNA interstrand crosslinking agents. Published mutagenesis data from single-gene mutation assays show both increased and decreased mutation frequencies in FA cells. In this review we discuss the data from the literature and from our isogenic fancg knockout hamster CHO cells, and interpret these data within the framework of a molecular model that accommodates these seemingly divergent observations. In FA cells, reduced rates of recovery of viable X-linked hypoxanthine phosphoribosyltransferase (hprt) mutants are characteristically observed for diverse mutagenic agents, but also in untreated cultures, indicating the relevance of the FA pathway for processing assorted DNA lesions. We ascribe these reductions to: (1) impaired mutagenic translesion synthesis within hprt during DNA replication and (2) lethality of mutant cells following replication fork breakage on the X chromosome, caused by unrepaired double-strand breaks or large deletions/translocations encompassing essential genes flanking hprt. These findings, along with studies showing increased spontaneous mutability of FA cells at two autosomal loci, support a model in which FA proteins promote both translesion synthesis at replication-blocking lesions and repair of broken replication forks by homologous recombination and DNA end joining. The essence of this model is that the FANC protein pathway serves to restrict the severity of mutational outcome by favoring base substitutions and small deletions over larger deletions and chromosomal rearrangements.

Exogenous and evoked oxytocin restores social behavior in the Cntnap2 mouse model of autism

PubMed Central

Peñagarikano, Olga; Lázaro, María T.; Lu, Xiao-Hong; Gordon, Aaron; Dong, Hongmei; Lam, Hoa A.; Peles, Elior; Maidment, Nigel T.; Murphy, Niall P.; Yang, X. William; Golshani, Peyman; Geschwind, Daniel H.

2015-01-01

Mouse models of neuropsychiatric diseases provide a platform for mechanistic understanding and development of new therapies. We previously demonstrated that knockout of the mouse homologue of CNTNAP2, in which mutant forms cause Cortical Dysplasia and Focal Epilepsy syndrome (CDFE), displays many features parallel to the human disorder. Since CDFE has high penetrance for autism spectrum disorder (ASD) we performed an in vivo screen for drugs that treat abnormal social behavior in Cntnap2 mutant mice and found that acute administration of the neuropeptide oxytocin improved social deficits. We found a decrease in the number of oxytocin immunoreactive neurons in the paraventricular nucleus (PVN) of the hypothalamus in mutant mice and an overall decrease in brain oxytocin levels. Administration of a selective melanocortin receptor 4 agonist, which causes endogenous oxytocin release, also acutely rescued the social deficits, an effect blocked by an oxytocin antagonist. We confirmed that oxytocin neurons mediated the behavioral improvement by activating endogenous oxytocin neurons in the paraventricular hypothalamus with Designer Receptors Exclusively Activated by Designer Drugs (DREADD). Last, we showed that chronic early postnatal treatment with oxytocin led to more lasting behavioral recovery and restored oxytocin immunoreactivity in the PVN. These data demonstrate dysregulation of the oxytocin system in Cntnap2 knockout mice and suggest that there may be critical developmental windows for optimal treatment. PMID:25609168

Swimming in Light: A Large-Scale Computational Analysis of the Metabolism of Dinoroseobacter shibae

PubMed Central

Rex, Rene; Bill, Nelli; Schmidt-Hohagen, Kerstin; Schomburg, Dietmar

2013-01-01

The Roseobacter clade is a ubiquitous group of marine α-proteobacteria. To gain insight into the versatile metabolism of this clade, we took a constraint-based approach and created a genome-scale metabolic model (iDsh827) of Dinoroseobacter shibae DFL12T. Our model is the first accounting for the energy demand of motility, the light-driven ATP generation and experimentally determined specific biomass composition. To cover a large variety of environmental conditions, as well as plasmid and single gene knock-out mutants, we simulated 391,560 different physiological states using flux balance analysis. We analyzed our results with regard to energy metabolism, validated them experimentally, and revealed a pronounced metabolic response to the availability of light. Furthermore, we introduced the energy demand of motility as an important parameter in genome-scale metabolic models. The results of our simulations also gave insight into the changing usage of the two degradation routes for dimethylsulfoniopropionate, an abundant compound in the ocean. A side product of dimethylsulfoniopropionate degradation is dimethyl sulfide, which seeds cloud formation and thus enhances the reflection of sunlight. By our exhaustive simulations, we were able to identify single-gene knock-out mutants, which show an increased production of dimethyl sulfide. In addition to the single-gene knock-out simulations we studied the effect of plasmid loss on the metabolism. Moreover, we explored the possible use of a functioning phosphofructokinase for D. shibae. PMID:24098096

SYN2 is an autism predisposing gene: loss-of-function mutations alter synaptic vesicle cycling and axon outgrowth

PubMed Central

Corradi, Anna; Fadda, Manuela; Piton, Amélie; Patry, Lysanne; Marte, Antonella; Rossi, Pia; Cadieux-Dion, Maxime; Gauthier, Julie; Lapointe, Line; Mottron, Laurent; Valtorta, Flavia; Rouleau, Guy A.; Fassio, Anna; Benfenati, Fabio; Cossette, Patrick

2014-01-01

An increasing number of genes predisposing to autism spectrum disorders (ASDs) has been identified, many of which are implicated in synaptic function. This ‘synaptic autism pathway’ notably includes disruption of SYN1 that is associated with epilepsy, autism and abnormal behavior in both human and mice models. Synapsins constitute a multigene family of neuron-specific phosphoproteins (SYN1-3) present in the majority of synapses where they are implicated in the regulation of neurotransmitter release and synaptogenesis. Synapsins I and II, the major Syn isoforms in the adult brain, display partially overlapping functions and defects in both isoforms are associated with epilepsy and autistic-like behavior in mice. In this study, we show that nonsense (A94fs199X) and missense (Y236S and G464R) mutations in SYN2 are associated with ASD in humans. The phenotype is apparent in males. Female carriers of SYN2 mutations are unaffected, suggesting that SYN2 is another example of autosomal sex-limited expression in ASD. When expressed in SYN2  knockout neurons, wild-type human Syn II fully rescues the SYN2 knockout phenotype, whereas the nonsense mutant is not expressed and the missense mutants are virtually unable to modify the SYN2 knockout phenotype. These results identify for the first time SYN2  as a novel predisposing gene for ASD and strengthen the hypothesis that a disturbance of synaptic homeostasis underlies ASD. PMID:23956174

Autographa californica multiple nucleopolyhedrovirus PK-1 is essential for nucleocapsid assembly

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

Liang, Changyong, E-mail: cyliang@yzu.edu.cn; Li, Min; Dai, Xuejuan

2013-09-01

PK-1 (Ac10) is a baculovirus-encoded serine/threonine kinase and its function is unclear. Our results showed that a pk-1 knockout AcMNPV failed to produce infectious progeny, while the pk-1 repair virus could rescue this defect. qPCR analysis demonstrated that pk-1 deletion did not affect viral DNA replication. Analysis of the repaired recombinants with truncated pk-1 mutants demonstrated that the catalytic domain of protein kinases of PK-1 was essential to viral infectivity. Moreover, those PK-1 mutants that could rescue the infectious BV production defect exhibited kinase activity in vitro. Therefore, it is suggested that the kinase activity of PK-1 is essential inmore » regulating viral propagation. Electron microscopy revealed that pk-1 deletion affected the formation of normal nucleocapsids. Masses of electron-lucent tubular structures were present in cell transfected with pk-1 knockout bacmid. Therefore, PK-1 appears to phosphorylate some viral or cellular proteins that are essential for DNA packaging to regulate nucleocapsid assembly. - Highlights: • A pk-1 knockout AcMNPV failed to produce infectious progeny. • The pk-1 deletion did not affect viral DNA replication. • The catalytic domain of protein kinases (PKc) of PK-1 was essential to viral infectivity. • The kinase activity of PK-1 is essential in regulating viral propagation. • PK-1 appears to phosphorylate some viral proteins that are essential for DNA packaging to regulate nucleocapsid assembly.« less

Primary Coenzyme Q Deficiency in Pdss2 Mutant Mice Causes Isolated Renal Disease

PubMed Central

Haase, Volker H.; King, Rhonda; Polyak, Erzsebet; Selak, Mary; Yudkoff, Marc; Hancock, Wayne W.; Meade, Ray; Saiki, Ryoichi; Lunceford, Adam L.; Clarke, Catherine F.; Gasser, David L.

2008-01-01

Coenzyme Q (CoQ) is an essential electron carrier in the respiratory chain whose deficiency has been implicated in a wide variety of human mitochondrial disease manifestations. Its multi-step biosynthesis involves production of polyisoprenoid diphosphate in a reaction that requires the enzymes be encoded by PDSS1 and PDSS2. Homozygous mutations in either of these genes, in humans, lead to severe neuromuscular disease, with nephrotic syndrome seen in PDSS2 deficiency. We now show that a presumed autoimmune kidney disease in mice with the missense Pdss2kd/kd genotype can be attributed to a mitochondrial CoQ biosynthetic defect. Levels of CoQ9 and CoQ10 in kidney homogenates from B6.Pdss2kd/kd mutants were significantly lower than those in B6 control mice. Disease manifestations originate specifically in glomerular podocytes, as renal disease is seen in Podocin/cre,Pdss2loxP/loxP knockout mice but not in conditional knockouts targeted to renal tubular epithelium, monocytes, or hepatocytes. Liver-conditional B6.Alb/cre,Pdss2loxP/loxP knockout mice have no overt disease despite demonstration that their livers have undetectable CoQ9 levels, impaired respiratory capacity, and significantly altered intermediary metabolism as evidenced by transcriptional profiling and amino acid quantitation. These data suggest that disease manifestations of CoQ deficiency relate to tissue-specific respiratory capacity thresholds, with glomerular podocytes displaying the greatest sensitivity to Pdss2 impairment. PMID:18437205

AINTEGUMENTA-LIKE genes have partly overlapping functions with AINTEGUMENTA but make distinct contributions to Arabidopsis thaliana flower development

PubMed Central

Krizek, Beth A.

2015-01-01

AINTEGUMENTA (ANT) is an important regulator of Arabidopsis flower development that has overlapping functions with the related AINTEGUMENTA-LIKE6 (AIL6) gene in floral organ initiation, identity specification, growth, and patterning. Two other AINTEGUMENTA-LIKE (AIL) genes, AIL5 and AIL7, are expressed in developing flowers in spatial domains that partly overlap with those of ANT. Here, it is shown that AIL5 and AIL7 also act in a partially redundant manner with ANT. The results demonstrate that AIL genes exhibit unequal genetic redundancy with roles for AIL5, AIL6, and AIL7 only revealed in the absence of ANT function. ant ail5 and ant ail7 double mutant flowers show alterations in floral organ positioning and growth, sepal fusion, and reductions in petal number. In ant ail5, petals are often replaced by filaments or dramatically reduced in size. ant ail7 double mutants produce increased numbers of carpels, which have defects in valve fusion and a loss of apical tissues. The distinct phenotypes of ant ail5, ant ail7 and the previously characterized ant ail6 indicate that AIL5, AIL6, and AIL7 make unique contributions to flower development. These distinct roles are also supported by genetic analyses of ant ail triple mutants. While ant ail5 ail6 triple mutants closely resemble ant ail6 double mutants, ant ail5 ail7 triple mutants exhibit more severe deviations from the wild type than either ant ail5 or ant ail7 double mutants. Furthermore, it is shown that AIL5, AIL6, and AIL7 act in a dose dependent manners in ant and other mutant backgrounds. PMID:25956884

Maize endosperm-specific transcription factors O2 and PBF network the regulation of protein and starch synthesis

PubMed Central

Zhang, Zhiyong; Zheng, Xixi; Yang, Jun; Messing, Joachim; Wu, Yongrui

2016-01-01

The maize endosperm-specific transcription factors opaque2 (O2) and prolamine-box binding factor (PBF) regulate storage protein zein genes. We show that they also control starch synthesis. The starch content in the PbfRNAi and o2 mutants was reduced by ∼5% and 11%, respectively, compared with normal genotypes. In the double-mutant PbfRNAi;o2, starch was decreased by 25%. Transcriptome analysis reveals that >1,000 genes were affected in each of the two mutants and in the double mutant; these genes were mainly enriched in sugar and protein metabolism. Pyruvate orthophosphate dikinase 1 and 2 (PPDKs) and starch synthase III (SSIII) are critical components in the starch biosynthetic enzyme complex. The expression of PPDK1, PPDK2, and SSIII and their protein levels are further reduced in the double mutants as compared with the single mutants. When the promoters of these genes were analyzed, we found a prolamine box and an O2 box that can be additively transactivated by PBF and O2. Starch synthase IIa (SSIIa, encoding another starch synthase for amylopectin) and starch branching enzyme 1 (SBEI, encoding one of the two main starch branching enzymes) are not directly regulated by PBF and O2, but their protein levels are significantly decreased in the o2 mutant and are further decreased in the double mutant, indicating that o2 and PbfRNAi may affect the levels of some other transcription factor(s) or mRNA regulatory factor(s) that in turn would affect the transcript and protein levels of SSIIa and SBEI. These findings show that three important traits—nutritional quality, calories, and yield—are linked through the same transcription factors. PMID:27621432

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

CYP79F1 and CYP79F2 have distinct functions in the biosynthesis of aliphatic glucosinolates in Arabidopsis.

PubMed

Chen, Sixue; Glawischnig, Erich; Jørgensen, Kirsten; Naur, Peter; Jørgensen, Bodil; Olsen, Carl-Erik; Hansen, Carsten H; Rasmussen, Hasse; Pickett, John A; Halkier, Barbara A

2003-03-01

Cytochromes P450 of the CYP79 family catalyze the conversion of amino acids to oximes in the biosynthesis of glucosinolates, a group of natural plant products known to be involved in plant defense and as a source of flavor compounds, cancer-preventing agents and bioherbicides. We report a detailed biochemical analysis of the substrate specificity and kinetics of CYP79F1 and CYP79F2, two cytochromes P450 involved in the biosynthesis of aliphatic glucosinolates in Arabidopsis thaliana. Using recombinant CYP79F1 and CYP79F2 expressed in Escherichia coli and Saccharomyces cerevisiae, respectively, we show that CYP79F1 metabolizes mono- to hexahomomethionine, resulting in both short- and long-chain aliphatic glucosinolates. In contrast, CYP79F2 exclusively metabolizes long-chain elongated penta- and hexahomomethionines. CYP79F1 and CYP79F2 are spatially and developmentally regulated, with different gene expression patterns. CYP79F2 is highly expressed in hypocotyl and roots, whereas CYP79F1 is strongly expressed in cotyledons, rosette leaves, stems, and siliques. A transposon-tagged CYP79F1 knockout mutant completely lacks short-chain aliphatic glucosinolates, but has an increased level of long-chain aliphatic glucosinolates, especially in leaves and seeds. The level of long-chain aliphatic glucosinolates in a transposon-tagged CYP79F2 knockout mutant is substantially reduced, whereas the level of short-chain aliphatic glucosinolates is not affected. Biochemical characterization of CYP79F1 and CYP79F2, and gene expression analysis, combined with glucosinolate profiling of knockout mutants demonstrate the functional role of these enzymes. This provides valuable insights into the metabolic network leading to the biosynthesis of aliphatic glucosinolates, and into metabolic engineering of altered aliphatic glucosinolate profiles to improve nutritional value and pest resistance.

Characterization of the Serratia marcescens SdeCDE multidrug efflux pump studied via gene knockout mutagenesis.

PubMed

Begic, Sanela; Worobec, Elizabeth A

2008-05-01

Serratia marcescens is an important nosocomial agent having high antibiotic resistance. A major mechanism for S. marcescens antibiotic resistance is active efflux. To ascertain the substrate specificity of the S. marcescens SdeCDE efflux pump, we constructed pump gene deletion mutants. sdeCDE knockout strains showed no change in antibiotic susceptibility in comparison with the parental strains for any of the substrates, with the exception of novobiocin. In addition, novobiocin was the only antibiotic to be accumulated by sdeCDE-deficient strains. Based on the substrates used in our study, we conclude that SdeCDE is a Resistance-Nodulation-Cell Division family pump with limited substrate specificity.

Adaptive bi-level programming for optimal gene knockouts for targeted overproduction under phenotypic constraints

PubMed Central

2013-01-01

Background Optimization procedures to identify gene knockouts for targeted biochemical overproduction have been widely in use in modern metabolic engineering. Flux balance analysis (FBA) framework has provided conceptual simplifications for genome-scale dynamic analysis at steady states. Based on FBA, many current optimization methods for targeted bio-productions have been developed under the maximum cell growth assumption. The optimization problem to derive gene knockout strategies recently has been formulated as a bi-level programming problem in OptKnock for maximum targeted bio-productions with maximum growth rates. However, it has been shown that knockout mutants in fact reach the steady states with the minimization of metabolic adjustment (MOMA) from the corresponding wild-type strains instead of having maximal growth rates after genetic or metabolic intervention. In this work, we propose a new bi-level computational framework--MOMAKnock--which can derive robust knockout strategies under the MOMA flux distribution approximation. Methods In this new bi-level optimization framework, we aim to maximize the production of targeted chemicals by identifying candidate knockout genes or reactions under phenotypic constraints approximated by the MOMA assumption. Hence, the targeted chemical production is the primary objective of MOMAKnock while the MOMA assumption is formulated as the inner problem of constraining the knockout metabolic flux to be as close as possible to the steady-state phenotypes of wide-type strains. As this new inner problem becomes a quadratic programming problem, a novel adaptive piecewise linearization algorithm is developed in this paper to obtain the exact optimal solution to this new bi-level integer quadratic programming problem for MOMAKnock. Results Our new MOMAKnock model and the adaptive piecewise linearization solution algorithm are tested with a small E. coli core metabolic network and a large-scale iAF1260 E. coli metabolic network. The derived knockout strategies are compared with those from OptKnock. Our preliminary experimental results show that MOMAKnock can provide improved targeted productions with more robust knockout strategies. PMID:23368729

Adaptive bi-level programming for optimal gene knockouts for targeted overproduction under phenotypic constraints.

PubMed

Ren, Shaogang; Zeng, Bo; Qian, Xiaoning

2013-01-01

Optimization procedures to identify gene knockouts for targeted biochemical overproduction have been widely in use in modern metabolic engineering. Flux balance analysis (FBA) framework has provided conceptual simplifications for genome-scale dynamic analysis at steady states. Based on FBA, many current optimization methods for targeted bio-productions have been developed under the maximum cell growth assumption. The optimization problem to derive gene knockout strategies recently has been formulated as a bi-level programming problem in OptKnock for maximum targeted bio-productions with maximum growth rates. However, it has been shown that knockout mutants in fact reach the steady states with the minimization of metabolic adjustment (MOMA) from the corresponding wild-type strains instead of having maximal growth rates after genetic or metabolic intervention. In this work, we propose a new bi-level computational framework--MOMAKnock--which can derive robust knockout strategies under the MOMA flux distribution approximation. In this new bi-level optimization framework, we aim to maximize the production of targeted chemicals by identifying candidate knockout genes or reactions under phenotypic constraints approximated by the MOMA assumption. Hence, the targeted chemical production is the primary objective of MOMAKnock while the MOMA assumption is formulated as the inner problem of constraining the knockout metabolic flux to be as close as possible to the steady-state phenotypes of wide-type strains. As this new inner problem becomes a quadratic programming problem, a novel adaptive piecewise linearization algorithm is developed in this paper to obtain the exact optimal solution to this new bi-level integer quadratic programming problem for MOMAKnock. Our new MOMAKnock model and the adaptive piecewise linearization solution algorithm are tested with a small E. coli core metabolic network and a large-scale iAF1260 E. coli metabolic network. The derived knockout strategies are compared with those from OptKnock. Our preliminary experimental results show that MOMAKnock can provide improved targeted productions with more robust knockout strategies.

Improved α-amylase production by Aspergillus oryzae after a double deletion of genes involved in carbon catabolite repression.

PubMed

Ichinose, Sakurako; Tanaka, Mizuki; Shintani, Takahiro; Gomi, Katsuya

2014-01-01

In filamentous fungi, the expression of secretory glycoside hydrolase encoding genes, such as those for amylases, cellulases, and xylanases, is generally repressed in the presence of glucose. CreA and CreB have been observed to be regulating factors for carbon catabolite repression. In this study, we generated single and double deletion creA and/or creB mutants in Aspergillus oryzae. The α-amylase activities of each strain were compared under various culture conditions. For the wild-type strain, mRNA levels of α-amylase were markedly decreased in the later stage of submerged culture under inducing conditions, whereas this reduced expression was not observed for single creA and double creA/creB deletion mutants. In addition, α-amylase activity of the wild-type strain was reduced in submerged culture containing high concentrations of inducing sugars, whereas all constructed mutants showed higher α-amylase activities. In particular, the α-amylase activity of the double deletion mutant in a medium containing 5% starch was >10-fold higher than that of the wild-type strain under the same culture conditions. In solid-state cultures using wheat bran as a substrate, the α-amylase activities of single creA and double deletion mutants were >2-fold higher than that of the wild-type strain. These results suggested that deleting both creA and creB resulted in dramatic improvements in the production of secretory glycoside hydrolases in filamentous fungi.

Mutations in Arabidopsis Yellow Stripe-Like1 and Yellow Stripe-Like3 Reveal Their Roles in Metal Ion Homeostasis and Loading of Metal Ions in Seeds1

PubMed Central

Waters, Brian M.; Chu, Heng-Hsuan; DiDonato, Raymond J.; Roberts, Louis A.; Eisley, Robynn B.; Lahner, Brett; Salt, David E.; Walker, Elsbeth L.

2006-01-01

Here, we describe two members of the Arabidopsis (Arabidopsis thaliana) Yellow Stripe-Like (YSL) family, AtYSL1 and AtYSL3. The YSL1 and YSL3 proteins are members of the oligopeptide transporter family and are predicted to be integral membrane proteins. YSL1 and YSL3 are similar to the maize (Zea mays) YS1 phytosiderophore transporter (ZmYS1) and the AtYSL2 iron (Fe)-nicotianamine transporter, and are predicted to transport metal-nicotianamine complexes into cells. YSL1 and YSL3 mRNAs are expressed in both root and shoot tissues, and both are regulated in response to the Fe status of the plant. β-Glucuronidase reporter expression, driven by YSL1 and YSL3 promoters, reveals expression patterns of the genes in roots, leaves, and flowers. Expression was highest in senescing rosette leaves and cauline leaves. Whereas the single mutants ysl1 and ysl3 had no visible phenotypes, the ysl1ysl3 double mutant exhibited Fe deficiency symptoms, such as interveinal chlorosis. Leaf Fe concentrations are decreased in the double mutant, whereas manganese, zinc, and especially copper concentrations are elevated. In seeds of double-mutant plants, the concentrations of Fe, zinc, and copper are low. Mobilization of metals from leaves during senescence is impaired in the double mutant. In addition, the double mutant has reduced fertility due to defective anther and embryo development. The proposed physiological roles for YSL1 and YSL3 are in delivery of metal micronutrients to and from vascular tissues. PMID:16815956

Catabolic regulation analysis of Escherichia coli and its crp, mlc, mgsA, pgi and ptsG mutants

PubMed Central

2011-01-01

Background Most bacteria can use various compounds as carbon sources. These carbon sources can be either co-metabolized or sequentially metabolized, where the latter phenomenon typically occurs as catabolite repression. From the practical application point of view of utilizing lignocellulose for the production of biofuels etc., it is strongly desirable to ferment all sugars obtained by hydrolysis from lignocellulosic materials, where simultaneous consumption of sugars would benefit the formation of bioproducts. However, most organisms consume glucose prior to consumption of other carbon sources, and exhibit diauxic growth. It has been shown by fermentation experiments that simultaneous consumption of sugars can be attained by ptsG, mgsA mutants etc., but its mechanism has not been well understood. It is strongly desirable to understand the mechanism of metabolic regulation for catabolite regulation to improve the performance of fermentation. Results In order to make clear the catabolic regulation mechanism, several continuous cultures were conducted at different dilution rates of 0.2, 0.4, 0.6 and 0.7 h-1 using wild type Escherichia coli. The result indicates that the transcript levels of global regulators such as crp, cra, mlc and rpoS decreased, while those of fadR, iclR, soxR/S increased as the dilution rate increased. These affected the metabolic pathway genes, which in turn affected fermentation result where the specific glucose uptake rate, the specific acetate formation rate, and the specific CO2 evolution rate (CER) were increased as the dilution rate was increased. This was confirmed by the 13C-flux analysis. In order to make clear the catabolite regulation, the effect of crp gene knockout (Δcrp) and crp enhancement (crp+) as well as mlc, mgsA, pgi and ptsG gene knockout on the metabolism was then investigated by the continuous culture at the dilution rate of 0.2 h-1 and by some batch cultures. In the case of Δcrp (and also Δmlc) mutant, TCA cycle and glyoxylate were repressed, which caused acetate accumulation. In the case of crp+ mutant, glycolysis, TCA cycle, and gluconeogenesis were activated, and simultaneous consumption of multiple carbon sources can be attained, but the glucose consumption rate became less due to repression of ptsG and ptsH by the activation of Mlc. Simultaneous consumption of multiple carbon sources could be attained by mgsA, pgi, and ptsG mutants due to increase in crp as well as cyaA, while glucose consumption rate became lower. Conclusions The transcriptional catabolite regulation mechanism was made clear for the wild type E. coli, and its crp, mlc, ptsG, pgi, and mgsA gene knockout mutants. The results indicate that catabolite repression can be relaxed and crp as well as cyaA can be increased by crp+, mgsA, pgi, and ptsG mutants, and thus simultaneous consumption of multiple carbon sources including glucose can be made, whereas the glucose uptake rate became lower as compared to wild type due to inactivation of ptsG in all the mutants considered. PMID:21831320

AtrbohD and AtrbohF negatively regulate lateral root development by changing the localized accumulation of superoxide in primary roots of Arabidopsis.

PubMed

Li, Ning; Sun, Lirong; Zhang, Liyue; Song, Yalin; Hu, Panpan; Li, Cui; Hao, Fu Shun

2015-03-01

NADPH oxidase AtrbohD an d AtrbohF negatively modulate lateral root development by changing the peroxidase activity and increasing the local generation of superoxide in primary roots of Arabidopsis in an auxin-independent manner. NADPH oxidase subunits AtrbohD and AtrbohF play pivotal roles in regulating growth, development and stress responses in Arabidopsis. However, whether they modulate lateral root (LR) formation has not yet been addressed, and the detailed mechanisms underlying the process remain unanswered. Here, we show that two null double mutants atrbohD1/F1 and atrbohD2/F2, in which both AtrbohD and AtrbohF genes are disrupted, had remarkably higher LR density than wild-type (WT), or the single mutant atrbohD1 and atrbohF1. Compared to WT, the double mutants exhibited early emerged LRs and enhanced density of lateral root primordia (LRP). Unexpectedly, the production of superoxide (O2 (-)), but not hydrogen peroxide, in the mature area of the primary root containing LRs significantly increased in the double mutants relative to that in WT. Further experiments revealed that the local accumulation of O2 (-) led to the enhancement of LR density in the double mutants. Moreover, the deficiency of AtrbohD and AtrbohF caused a marked increase in peroxidase activity in the mature root zone, which contributed to the localized accumulation of O2 (-) and the elevated LR density in the double mutants. Furthermore, the double mutants were not sensitive to exogenous auxin naphthalene acetic acid or auxin transport inhibitor 1-N-naphthylphthalamic acid in terms of LR formation. The auxin response of LRP in vivo in atrbohD1/F1 was also similar to that in WT. Taken together, these results suggest that AtrbohD and AtrbohF negatively modulate LR development by controlling the local generation of superoxide in an auxin-independent manner. These findings provide new insights into the mechanisms of NADPH oxidase-mediated regulation of LR branching in Arabidopsis.

The Meckel syndrome- associated protein MKS1 functionally interacts with components of the BBSome and IFT complexes to mediate ciliary trafficking and hedgehog signaling

PubMed Central

Barrington, Chloe L.; Katsanis, Nicholas

2017-01-01

The importance of primary cilia in human health is underscored by the link between ciliary dysfunction and a group of primarily recessive genetic disorders with overlapping clinical features, now known as ciliopathies. Many of the proteins encoded by ciliopathy-associated genes are components of a handful of multi-protein complexes important for the transport of cargo to the basal body and/or into the cilium. A key question is whether different complexes cooperate in cilia formation, and whether they participate in cilium assembly in conjunction with intraflagellar transport (IFT) proteins. To examine how ciliopathy protein complexes might function together, we have analyzed double mutants of an allele of the Meckel syndrome (MKS) complex protein MKS1 and the BBSome protein BBS4. We find that Mks1; Bbs4 double mutant mouse embryos exhibit exacerbated defects in Hedgehog (Hh) dependent patterning compared to either single mutant, and die by E14.5. Cells from double mutant embryos exhibit a defect in the trafficking of ARL13B, a ciliary membrane protein, resulting in disrupted ciliary structure and signaling. We also examined the relationship between the MKS complex and IFT proteins by analyzing double mutant between Mks1 and a hypomorphic allele of the IFTB component Ift172. Despite each single mutant surviving until around birth, Mks1; Ift172avc1 double mutants die at mid-gestation, and exhibit a dramatic failure of cilia formation. We also find that Mks1 interacts genetically with an allele of Dync2h1, the IFT retrograde motor. Thus, we have demonstrated that the MKS transition zone complex cooperates with the BBSome to mediate trafficking of specific trans-membrane receptors to the cilium. Moreover, the genetic interaction of Mks1 with components of IFT machinery suggests that the transition zone complex facilitates IFT to promote cilium assembly and structure. PMID:28291807

Efficient modification of the myostatin gene in porcine somatic cells and generation of knockout piglets.

PubMed

Rao, Shengbin; Fujimura, Tatsuya; Matsunari, Hitomi; Sakuma, Tetsushi; Nakano, Kazuaki; Watanabe, Masahito; Asano, Yoshinori; Kitagawa, Eri; Yamamoto, Takashi; Nagashima, Hiroshi

2016-01-01

Myostatin (MSTN) is a negative regulator of myogenesis, and disruption of its function causes increased muscle mass in various species. Here, we report the generation of MSTN-knockout (KO) pigs using genome editing technology combined with somatic-cell nuclear transfer (SCNT). Transcription activator-like effector nuclease (TALEN) with non-repeat-variable di-residue variations, called Platinum TALEN, was highly efficient in modifying genes in porcine somatic cells, which were then used for SCNT to create MSTN KO piglets. These piglets exhibited a double-muscled phenotype, possessing a higher body weight and longissimus muscle mass measuring 170% that of wild-type piglets, with double the number of muscle fibers. These results demonstrate that loss of MSTN increases muscle mass in pigs, which may help increase pork production for consumption in the future. © 2015 Wiley Periodicals, Inc.

AmyR is a novel negative regulator of amylovoran production in Erwinia amylovora

USDA-ARS?s Scientific Manuscript database

We have previously reported the characterization of an orphan gene ybjN from Escherichia coli. In this study, we attempted to understand the role of amyR in Erwinia amylovora, a functionally conserved homolog of E. coli ybjN. As reported earlier, amylovoran production in the amyR knockout mutant is ...

L166P MUTANT DJ-1, CAUSATIVE FOR RECESSIVE PARKINSON'S DISEASE IS DEGRADED THROUGH THE UBIQUITIN-PROTEASOME SYSTEM

EPA Science Inventory

Abstract

Mutations in a gene on chromosome 1, DJ-1, have been reported recently to be associated with recessive, early-onset Parkinson's disease. Whilst one mutation is a large deletion that is predicted to produce an effective knockout of the gene, the second is a point ...

THE EFFECT OF A TARGETED KNOCKOUT MUTATION ON THE TRANSCRIPTIONAL PROFILE OF THE KIDNEY IN TSC2 MUTANT (EKER) RATS.

EPA Science Inventory

Renal cell carcinoma (RCC) is the most common tumor of the adult kidney, accounting for up to 80% of malignant renal neoplasms. Hereditary RCC in the Eker rat, which bear a number of cellular, molecular and phenotypic similarities to human RCC, results from an inherited insertion...

Efficient production of infectious viruses requires enzymatic activity of Epstein-Barr virus protein kinase.

PubMed

Murata, Takayuki; Isomura, Hiroki; Yamashita, Yoriko; Toyama, Shigenori; Sato, Yoshitaka; Nakayama, Sanae; Kudoh, Ayumi; Iwahori, Satoko; Kanda, Teru; Tsurumi, Tatsuya

2009-06-20

The Epstein-Barr virus (EBV) BGLF4 gene product is the only protein kinase encoded by the virus genome. In order to elucidate its physiological roles in viral productive replication, we here established a BGLF4-knockout mutant and a revertant virus. While the levels of viral DNA replication of the deficient mutant were equivalent to those of the wild-type and the revertant, virus production was significantly impaired. Expression of the BGLF4 protein in trans fully complemented the low yield of the mutant virus, while expression of a kinase-dead (K102I) form of the protein failed to restore the virus titer. These results demonstrate that BGLF4 plays a significant role in production of infectious viruses and that the kinase activity is crucial.

Generation and characterisation of a parkin-Pacrg knockout mouse line and a Pacrg knockout mouse line.

PubMed

Stephenson, Sarah E M; Aumann, Timothy D; Taylor, Juliet M; Riseley, Jessica R; Li, Ruili; Mann, Jeffrey R; Tomas, Doris; Lockhart, Paul J

2018-05-14

Mutations in PARK2 (parkin) can result in Parkinson's disease (PD). Parkin shares a bidirectional promoter with parkin coregulated gene (PACRG) and the transcriptional start sites are separated by only ~200 bp. Bidirectionally regulated genes have been shown to function in common biological pathways. Mice lacking parkin have largely failed to recapitulate the dopaminergic neuronal loss and movement impairments seen in individuals with parkin-mediated PD. We aimed to investigate the function of PACRG and test the hypothesis that parkin and PACRG function in a common pathway by generating and characterizing two novel knockout mouse lines harbouring loss of both parkin and Pacrg or Pacrg alone. Successful modification of the targeted allele was confirmed at the genomic, transcriptional and steady state protein levels for both genes. At 18-20 months of age, there were no significant differences in the behaviour of parental and mutant lines when assessed by openfield, rotarod and balance beam. Subsequent neuropathological examination suggested there was no gross abnormality of the dopaminergic system in the substantia nigra and no significant difference in the number of dopaminergic neurons in either knockout model compared to wildtype mice.

Rapid and efficient galactose fermentation by engineered Saccharomyces cerevisiae.

PubMed

Quarterman, Josh; Skerker, Jeffrey M; Feng, Xueyang; Liu, Ian Y; Zhao, Huimin; Arkin, Adam P; Jin, Yong-Su

2016-07-10

In the important industrial yeast Saccharomyces cerevisiae, galactose metabolism requires energy production by respiration; therefore, this yeast cannot metabolize galactose under strict anaerobic conditions. While the respiratory dependence of galactose metabolism provides benefits in terms of cell growth and population stability, it is not advantageous for producing fuels and chemicals since a substantial fraction of consumed galactose is converted to carbon dioxide. In order to force S. cerevisiae to use galactose without respiration, a subunit (COX9) of a respiratory enzyme was deleted, but the resulting deletion mutant (Δcox9) was impaired in terms of galactose assimilation. Interestingly, after serial sub-cultures on galactose, the mutant evolved rapidly and was able to use galactose via fermentation only. The evolved strain (JQ-G1) produced ethanol from galactose with a 94% increase in yield and 6.9-fold improvement in specific productivity as compared to the wild-type strain. (13)C-metabolic flux analysis demonstrated a three-fold reduction in carbon flux through the TCA cycle of the evolved mutant with redirection of flux toward the fermentation pathway. Genome sequencing of the JQ-G1 strain revealed a loss of function mutation in a master negative regulator of the Leloir pathway (Gal80p). The mutation (Glu348*) in Gal80p was found to act synergistically with deletion of COX9 for efficient galactose fermentation, and thus the double deletion mutant Δcox9Δgal80 produced ethanol 2.4 times faster and with 35% higher yield than a single knockout mutant with deletion of GAL80 alone. When we introduced a functional COX9 cassette back into the JQ-G1 strain, the JQ-G1-COX9 strain showed a 33% reduction in specific galactose uptake rate and a 49% reduction in specific ethanol production rate as compared to JQ-G1. The wild-type strain was also subjected to serial sub-cultures on galactose but we failed to isolate a mutant capable of utilizing galactose without respiration. We concluded that the metabolic "death valley" (i.e. no galactose utilization by the Δcox9 mutant) is a necessary intermediate phenotype to facilitate galactose utilization without respiration in yeast. The results in this study demonstrate a promising approach for directing adaptive evolution toward fermentative metabolism and for generating evolved yeast strains with improved phenotypes under anaerobic conditions. Copyright © 2016 Elsevier B.V. All rights reserved.

Contribution of chloride channel permease to fluoride resistance in Streptococcus mutans.

PubMed

Murata, Takatoshi; Hanada, Nobuhiro

2016-06-01

Genes encoding fluoride transporters have been identified in bacterial and archaeal species. The genome sequence of the cariogenic Streptococcus mutans bacteria suggests the presence of a putative fluoride transporter, which is referred to as a chloride channel permease. Two homologues of this gene (GenBank locus tags SMU_1290c and SMU_1289c) reside in tandem in the genome of S. mutans The aim of this study was to determine whether the chloride channel permeases contribute to fluoride resistance. We constructed SMU_1290c- and SMU_1289c-knockout S. mutans UA159 strains. We also constructed a double-knockout strain lacking both genes. SMU_1290c or SMU_1289c was transformed into a fluoride transporter- disrupted Escherichia coli strain. All bacterial strains were cultured under appropriate conditions with or without sodium fluoride, and fluoride resistance was evaluated. All three gene-knockout S. mutans strains showed lower resistance to sodium fluoride than did the wild-type strain. No significant changes in resistance to other sodium halides were recognized between the wild-type and double-knockout strains. Both SMU_1290c and SMU_1289c transformation rescued fluoride transporter-disrupted E. coli cell from fluoride toxicity. We conclude that the chloride channel permeases contribute to fluoride resistance in S. mutans. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Cryptococcus gattii urease as a virulence factor and the relevance of enzymatic activity in cryptococcosis pathogenesis.

PubMed

Feder, Vanessa; Kmetzsch, Lívia; Staats, Charley Christian; Vidal-Figueiredo, Natalia; Ligabue-Braun, Rodrigo; Carlini, Célia Regina; Vainstein, Marilene Henning

2015-04-01

Ureases (EC 3.5.1.5) are Ni(2+) -dependent metalloenzymes produced by plants, fungi and bacteria that hydrolyze urea to produce ammonia and CO2 . The insertion of nickel atoms into the apo-urease is better characterized in bacteria, and requires at least three accessory proteins: UreD, UreF, and UreG. Our group has demonstrated that ureases possess ureolytic activity-independent biological properties that could contribute to the pathogenicity of urease-producing microorganisms. The presence of urease in pathogenic bacteria strongly correlates with pathogenesis in some human diseases. Some medically important fungi also produce urease, including Cryptococcus neoformans and Cryptococcus gattii. C. gattii is an etiological agent of cryptococcosis, most often affecting immunocompetent individuals. The cryptococcal urease might play an important role in pathogenesis. It has been proposed that ammonia produced via urease action might damage the host endothelium, which would enable yeast transmigration towards the central nervous system. To analyze the role of urease as a virulence factor in C. gattii, we constructed knockout mutants for the structural urease-coding gene URE1 and for genes that code the accessory proteins Ure4 and Ure6. All knockout mutants showed reduced multiplication within macrophages. In intranasally infected mice, the ure1Δ (lacking urease protein) and ure4Δ (enzymatically inactive apo-urease) mutants caused reduced blood burdens and a delayed time of death, whereas the ure6Δ (enzymatically inactive apo-urease) mutant showed time and dose dependency with regard to fungal burden. Our results suggest that C. gattii urease plays an important role in virulence, in part possibly through enzyme activity-independent mechanism(s). © 2015 FEBS.

Mice mutant for glucokinase regulatory protein exhibit decreased liver glucokinase: A sequestration mechanism in metabolic regulation

PubMed Central

Farrelly, Dennis; Brown, Karen S.; Tieman, Aaron; Ren, Jianming; Lira, Sergio A.; Hagan, Deborah; Gregg, Richard; Mookhtiar, Kasim A.; Hariharan, Narayanan

1999-01-01

The importance of glucokinase (GK; EC 2.7.1.12) in glucose homeostasis has been demonstrated by the association of GK mutations with diabetes mellitus in humans and by alterations in glucose metabolism in transgenic and gene knockout mice. Liver GK activity in humans and rodents is allosterically inhibited by GK regulatory protein (GKRP). To further understand the role of GKRP in GK regulation, the mouse GKRP gene was inactivated. With the knockout of the GKRP gene, there was a parallel loss of GK protein and activity in mutant mouse liver. The loss was primarily because of posttranscriptional regulation of GK, indicating a positive regulatory role for GKRP in maintaining GK levels and activity. As in rat hepatocytes, both GK and GKRP were localized in the nuclei of mouse hepatocytes cultured in low-glucose-containing medium. In the presence of fructose or high concentrations of glucose, conditions known to relieve GK inhibition by GKRP in vitro, only GK was translocated into the cytoplasm. In the GKRP-mutant hepatocytes, GK was not found in the nucleus under any tested conditions. We propose that GKRP functions as an anchor to sequester and inhibit GK in the hepatocyte nucleus, where it is protected from degradation. This ensures that glucose phosphorylation is minimal when the liver is in the fasting, glucose-producing phase. This also enables the hepatocytes to rapidly mobilize GK into the cytoplasm to phosphorylate and store or metabolize glucose after the ingestion of dietary glucose. In GKRP-mutant mice, the disruption of this regulation and the subsequent decrease in GK activity leads to altered glucose metabolism and impaired glycemic control. PMID:10588736

Assessing stress responses to atmospheric cold plasma exposure using Escherichia coli knock-out mutants.

PubMed

Han, L; Boehm, D; Patil, S; Cullen, P J; Bourke, P

2016-08-01

This study investigated the effect of atmospheric cold plasma (ACP) exposure-induced stress on microbial inactivation patterns and the regulation of genes involved in the microbial stress response in conjunction with key processing parameters of exposure time and post-treatment storage time. Cell suspensions of Escherichia coli BW 25113 and its isogenic knock-out mutants in rpoS, soxR, soxS, oxyR and dnaK genes were treated with high-voltage ACP in a sealed package for 1, 3 and 5 min followed by 0-, 1- and 24-h post-treatment storage. Reactive oxygen species (ROS) densities and colony formation were determined. ΔrpoS strain showed higher microbial reduction and greater cell permeability than other mutants, while ΔoxyR only showed this effect after 5 min of treatment. With increased post-treatment storage time, ΔsoxS and ΔsoxR had increased sensitivity and resistance respectively. ΔdnaK cell suspensions had much higher ROS than other strains and showed increased sensitivity with 24 h post-treatment storage. RpoS and oxyR genes have both short-term and long-term regulatory effects under plasma stress. However, knocking out dnaK gene had an immediate response on ROS scavenging and long-term repairing mechanisms. ΔsoxR and ΔsoxS had different responses to ACP treatment with the increase in post-treatment time in relation to clearance of reactive species implying the different characteristics and functions as subunits. By comparing the response of mutants under ACP exposure to key processing parameters, the mechanism of microbial inactivation was partly revealed with respect to cellular regulation and repairing genes. © 2016 The Society for Applied Microbiology.

DNA Double Strand Break Response and Limited Repair Capacity in Mouse Elongated Spermatids.

PubMed

Ahmed, Emad A; Scherthan, Harry; de Rooij, Dirk G

2015-12-16

Spermatids are extremely sensitive to genotoxic exposures since during spermiogenesis only error-prone non homologous end joining (NHEJ) repair pathways are available. Hence, genomic damage may accumulate in sperm and be transmitted to the zygote. Indirect, delayed DNA fragmentation and lesions associated with apoptotic-like processes have been observed during spermatid elongation, 27 days after irradiation. The proliferating spermatogonia and early meiotic prophase cells have been suggested to retain a memory of a radiation insult leading later to this delayed fragmentation. Here, we used meiotic spread preparations to localize phosphorylate histone H2 variant (γ-H2AX) foci marking DNA double strand breaks (DSBs) in elongated spermatids. This technique enabled us to determine the background level of DSB foci in elongated spermatids of RAD54/RAD54B double knockout (dko) mice, severe combined immunodeficiency SCID mice, and poly adenosine diphosphate (ADP)-ribose polymerase 1 (PARP1) inhibitor (DPQ)-treated mice to compare them with the appropriate wild type controls. The repair kinetics data and the protein expression patterns observed indicate that the conventional NHEJ repair pathway is not available for elongated spermatids to repair the programmed and the IR-induced DSBs, reflecting the limited repair capacity of these cells. However, although elongated spermatids express the proteins of the alternative NHEJ, PARP1-inhibition had no effect on the repair kinetics after IR, suggesting that DNA damage may be passed onto sperm. Finally, our genetic mutant analysis suggests that an incomplete or defective meiotic recombinational repair of Spo11-induced DSBs may lead to a carry-over of the DSB damage or induce a delayed nuclear fragmentation during the sensitive programmed chromatin remodeling occurring in elongated spermatids.

Functional census of mutation sequence spaces: The example of p53 cancer rescue mutants

PubMed Central

Danziger, Samuel A.; Swamidass, S. Joshua; Zeng, Jue; Dearth, Lawrence R.; Lu, Qiang; Chen, Jonathan H.; Cheng, Jainlin; Hoang, Vinh P.; Saigo, Hiroto; Luo, Ray; Baldi, Pierre; Brachmann, Rainer K.; Lathrop, Richard H.

2009-01-01

Many biomedical problems relate to mutant functional properties across a sequence space of interest, e.g., flu, cancer, and HIV. Detailed knowledge of mutant properties and function improves medical treatment and prevention. A functional census of p53 cancer rescue mutants would aid the search for cancer treatments from p53 rescue. We devised a general methodology for conducting a functional census of a mutation sequence space, and conducted a double-blind predictive test on the functional rescue property of 71 novel putative p53 cancer rescue mutants iteratively predicted in sets of 3. Double-blind predictive accuracy (15-point moving window) rose from 47% to 86% over the trial (r = 0.74). Code and data are available upon request1. PMID:17048398

The RAD24 (= Rs1) Gene Product of Saccharomyces cerevisiae Participates in Two Different Pathways of DNA Repair

PubMed Central

Eckardt-Schupp, Friederike; Siede, Wolfram; Game, John C.

1987-01-01

The moderately UV- and X-ray-sensitive mutant of Saccharomyces cerevisiae originally designated rs1 complements all rad and mms mutants available. Therefore, the new nomination rad24-1 according to the RAD nomenclature is suggested. RAD24 maps on chromosome V, close to RAD3 (1.3 cM). In order to associate the RAD24 gene with one of the three repair pathways, double mutants of rad24 and various representative genes of each pathway were constructed. The UV and X-ray sensitivities of the double mutants compared to the single mutants indicate that RAD24 is involved in excision repair of UV damage (RAD3 epistasis group), as well as in recombination repair of UV and X-ray damage (RAD52 epistasis group). Properties of the mutant are discussed which hint at the control of late steps in the pathways. PMID:3549445

Erythropoiesis and Blood Pressure Are Regulated via AT1 Receptor by Distinctive Pathways.

PubMed

Kato, Hideki; Ishida, Junji; Matsusaka, Taiji; Ishimaru, Tomohiro; Tanimoto, Keiji; Sugiyama, Fumihiro; Yagami, Ken-Ichi; Nangaku, Masaomi; Fukamizu, Akiyoshi

2015-01-01

The renin-angiotensin system (RAS) plays a central role in blood pressure regulation. Although clinical and experimental studies have suggested that inhibition of RAS is associated with progression of anemia, little evidence is available to support this claim. Here we report that knockout mice that lack angiotensin II, including angiotensinogen and renin knockout mice, exhibit anemia. The anemia of angiotensinogen knockout mice was rescued by angiotensin II infusion, and rescue was completely blocked by simultaneous administration of AT1 receptor blocker. To genetically determine the responsible receptor subtype, we examined AT1a, AT1b, and AT2 knockout mice, but did not observe anemia in any of them. To investigate whether pharmacological AT1 receptor inhibition recapitulates the anemic phenotype, we administered AT1 receptor antagonist in hypotensive AT1a receptor knockout mice to inhibit the remaining AT1b receptor. In these animals, hematocrit levels barely decreased, but blood pressure further decreased to the level observed in angiotensinogen knockout mice. We then generated AT1a and AT1b double-knockout mice to completely ablate the AT1 receptors; the mice finally exhibited the anemic phenotype. These results provide clear evidence that although erythropoiesis and blood pressure are negatively controlled through the AT1 receptor inhibition in vivo, the pathways involved are complex and distinct, because erythropoiesis is more resistant to AT1 receptor inhibition than blood pressure control.

Importance of the alternative oxidase (AOX) pathway in regulating cellular redox and ROS homeostasis to optimize photosynthesis during restriction of the cytochrome oxidase pathway in Arabidopsis thaliana.

PubMed

Vishwakarma, Abhaypratap; Tetali, Sarada Devi; Selinski, Jennifer; Scheibe, Renate; Padmasree, Kollipara

2015-09-01

The importance of the alternative oxidase (AOX) pathway, particularly AOX1A, in optimizing photosynthesis during de-etiolation, under elevated CO2, low temperature, high light or combined light and drought stress is well documented. In the present study, the role of AOX1A in optimizing photosynthesis was investigated when electron transport through the cytochrome c oxidase (COX) pathway was restricted at complex III. Leaf discs of wild-type (WT) and aox1a knock-out mutants of Arabidopsis thaliana were treated with antimycin A (AA) under growth-light conditions. To identify the impact of AOX1A deficiency in optimizing photosynthesis, respiratory O2 uptake and photosynthesis-related parameters were measured along with changes in redox couples, reactive oxygen species (ROS), lipid peroxidation and expression levels of genes related to respiration, the malate valve and the antioxidative system. In the absence of AA, aox1a knock-out mutants did not show any difference in physiological, biochemical or molecular parameters compared with WT. However, after AA treatment, aox1a plants showed a significant reduction in both respiratory O2 uptake and NaHCO3-dependent O2 evolution. Chlorophyll fluorescence and P700 studies revealed that in contrast to WT, aox1a knock-out plants were incapable of maintaining electron flow in the chloroplastic electron transport chain, and thereby inefficient heat dissipation (low non-photochemical quenching) was observed. Furthermore, aox1a mutants exhibited significant disturbances in cellular redox couples of NAD(P)H and ascorbate (Asc) and consequently accumulation of ROS and malondialdehyde (MDA) content. By contrast, WT plants showed a significant increase in transcript levels of CSD1, CAT1, sAPX, COX15 and AOX1A in contrast to aox1a mutants. These results suggest that AOX1A plays a significant role in sustaining the chloroplastic redox state and energization to optimize photosynthesis by regulating cellular redox homeostasis and ROS generation when electron transport through the COX pathway is disturbed at complex III. © The Author 2015. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Characterization of miR-122-independent propagation of HCV

PubMed Central

Motooka, Daisuke; Nakamura, Shota; Yamamoto, Satomi; Mori, Hiroyuki; Sato, Asuka; Uemura, Kentaro; Fauzyah, Yuzy; Suda, Takahiro; Nishio, Akira; Hmwe, Su Su; Okamoto, Toru; Tatsumi, Tomohide; Takehara, Tetsuo; Chayama, Kazuaki; Wakita, Takaji; Koike, Kazuhiko

2017-01-01

miR-122, a liver-specific microRNA, is one of the determinants for liver tropism of hepatitis C virus (HCV) infection. Although miR-122 is required for efficient propagation of HCV, we have previously shown that HCV replicates at a low rate in miR-122-deficient cells, suggesting that HCV-RNA is capable of propagating in an miR-122-independent manner. We herein investigated the roles of miR-122 in both the replication of HCV-RNA and the production of infectious particles by using miR-122-knockout Huh7 (Huh7-122KO) cells. A slight increase of intracellular HCV-RNA levels and infectious titers in the culture supernatants was observed in Huh7-122KO cells upon infection with HCV. Moreover, after serial passages of HCV in miR-122-knockout Huh7.5.1 cells, we obtained an adaptive mutant, HCV122KO, possessing G28A substitution in the 5’UTR of the HCV genotype 2a JFH1 genome, and this mutant may help to enhance replication complex formation, a possibility supported by polysome analysis. We also found the introduction of adaptive mutation around miR-122 binding site in the genotype 1b/2a chimeric virus, which originally had an adenine at the nucleotide position 29. HCV122KO exhibited efficient RNA replication in miR-122-knockout cells and non-hepatic cells without exogenous expression of miR-122. Competition assay revealed that the G28A mutant was dominant in the absence of miR-122, but its effects were equivalent to those of the wild type in the presence of miR-122, suggesting that the G28A mutation does not confer an advantage for propagation in miR-122-rich hepatocytes. These observations may explain the clinical finding that the positive rate of G28A mutation was higher in miR-122-deficient PBMCs than in the patient serum, which mainly included the hepatocyte-derived virus from HCV-genotype-2a patients. These results suggest that the emergence of HCV mutants that can propagate in non-hepatic cells in an miR-122-independent manner may participate in the induction of extrahepatic manifestations in chronic hepatitis C patients. PMID:28494029

Random phenotypic variation of yeast (Saccharomyces cerevisiae) single-gene knockouts fits a double pareto-lognormal distribution.

PubMed

Graham, John H; Robb, Daniel T; Poe, Amy R

2012-01-01

Distributed robustness is thought to influence the buffering of random phenotypic variation through the scale-free topology of gene regulatory, metabolic, and protein-protein interaction networks. If this hypothesis is true, then the phenotypic response to the perturbation of particular nodes in such a network should be proportional to the number of links those nodes make with neighboring nodes. This suggests a probability distribution approximating an inverse power-law of random phenotypic variation. Zero phenotypic variation, however, is impossible, because random molecular and cellular processes are essential to normal development. Consequently, a more realistic distribution should have a y-intercept close to zero in the lower tail, a mode greater than zero, and a long (fat) upper tail. The double Pareto-lognormal (DPLN) distribution is an ideal candidate distribution. It consists of a mixture of a lognormal body and upper and lower power-law tails. If our assumptions are true, the DPLN distribution should provide a better fit to random phenotypic variation in a large series of single-gene knockout lines than other skewed or symmetrical distributions. We fit a large published data set of single-gene knockout lines in Saccharomyces cerevisiae to seven different probability distributions: DPLN, right Pareto-lognormal (RPLN), left Pareto-lognormal (LPLN), normal, lognormal, exponential, and Pareto. The best model was judged by the Akaike Information Criterion (AIC). Phenotypic variation among gene knockouts in S. cerevisiae fits a double Pareto-lognormal (DPLN) distribution better than any of the alternative distributions, including the right Pareto-lognormal and lognormal distributions. A DPLN distribution is consistent with the hypothesis that developmental stability is mediated, in part, by distributed robustness, the resilience of gene regulatory, metabolic, and protein-protein interaction networks. Alternatively, multiplicative cell growth, and the mixing of lognormal distributions having different variances, may generate a DPLN distribution.

Two Hydroxyproline Galactosyltransferases, GALT5 and GALT2, Function in Arabinogalactan-Protein Glycosylation, Growth and Development in Arabidopsis

PubMed Central

Basu, Debarati; Showalter, Allan M.

2015-01-01

Hydroxyproline-O-galactosyltransferase (GALT) initiates O-glycosylation of arabinogalactan-proteins (AGPs). We previously characterized GALT2 (At4g21060), and now report on functional characterization of GALT5 (At1g74800). GALT5 was identified using heterologous expression in Pichia and an in vitro GALT assay. Product characterization showed GALT5 specifically adds galactose to hydroxyproline in AGP protein backbones. Functions of GALT2 and GALT5 were elucidated by phenotypic analysis of single and double mutant plants. Allelic galt5 and galt2 mutants, and particularly galt2 galt5 double mutants, demonstrated lower GALT activities and reductions in β-Yariv-precipitated AGPs compared to wild type. Mutant plants showed pleiotropic growth and development phenotypes (defects in root hair growth, root elongation, pollen tube growth, flowering time, leaf development, silique length, and inflorescence growth), which were most severe in the double mutants. Conditional mutant phenotypes were also observed, including salt-hypersensitive root growth and root tip swelling as well as reduced inhibition of pollen tube growth and root growth in response to β-Yariv reagent. These mutants also phenocopy mutants for an AGP, SOS5, and two cell wall receptor-like kinases, FEI1 and FEI2, which exist in a genetic signaling pathway. In summary, GALT5 and GALT2 function as redundant GALTs that control AGP O-glycosylation, which is essential for normal growth and development. PMID:25974423

Two Hydroxyproline Galactosyltransferases, GALT5 and GALT2, Function in Arabinogalactan-Protein Glycosylation, Growth and Development in Arabidopsis.

PubMed

Basu, Debarati; Wang, Wuda; Ma, Siyi; DeBrosse, Taylor; Poirier, Emily; Emch, Kirk; Soukup, Eric; Tian, Lu; Showalter, Allan M

2015-01-01

Hydroxyproline-O-galactosyltransferase (GALT) initiates O-glycosylation of arabinogalactan-proteins (AGPs). We previously characterized GALT2 (At4g21060), and now report on functional characterization of GALT5 (At1g74800). GALT5 was identified using heterologous expression in Pichia and an in vitro GALT assay. Product characterization showed GALT5 specifically adds galactose to hydroxyproline in AGP protein backbones. Functions of GALT2 and GALT5 were elucidated by phenotypic analysis of single and double mutant plants. Allelic galt5 and galt2 mutants, and particularly galt2 galt5 double mutants, demonstrated lower GALT activities and reductions in β-Yariv-precipitated AGPs compared to wild type. Mutant plants showed pleiotropic growth and development phenotypes (defects in root hair growth, root elongation, pollen tube growth, flowering time, leaf development, silique length, and inflorescence growth), which were most severe in the double mutants. Conditional mutant phenotypes were also observed, including salt-hypersensitive root growth and root tip swelling as well as reduced inhibition of pollen tube growth and root growth in response to β-Yariv reagent. These mutants also phenocopy mutants for an AGP, SOS5, and two cell wall receptor-like kinases, FEI1 and FEI2, which exist in a genetic signaling pathway. In summary, GALT5 and GALT2 function as redundant GALTs that control AGP O-glycosylation, which is essential for normal growth and development.

Biochemical Analysis of Two Single Mutants that Give Rise to a Polymorphic G6PD A-Double Mutant

PubMed Central

Ramírez-Nava, Edson Jiovany; González-Valdez, Abigail; Vanoye-Carlo, America; Hernández-Ochoa, Beatriz; Sierra-Palacios, Edgar; Hernández-Pineda, Jessica; Rodríguez-Bustamante, Eduardo; Arreguin-Espinosa, Roberto; Oria-Hernández, Jesús; Reyes-Vivas, Horacio; Marcial-Quino, Jaime

2017-01-01

Glucose-6-phosphate dehydrogenase (G6PD) is a key regulatory enzyme that plays a crucial role in the regulation of cellular energy and redox balance. Mutations in the gene encoding G6PD cause the most common enzymopathy that drives hereditary nonspherocytic hemolytic anemia. To gain insights into the effects of mutations in G6PD enzyme efficiency, we have investigated the biochemical, kinetic, and structural changes of three clinical G6PD variants, the single mutations G6PD A+ (Asn126AspD) and G6PD Nefza (Leu323Pro), and the double mutant G6PD A− (Asn126Asp + Leu323Pro). The mutants showed lower residual activity (≤50% of WT G6PD) and displayed important kinetic changes. Although all Class III mutants were located in different regions of the three-dimensional structure of the enzyme and were not close to the active site, these mutants had a deleterious effect over catalytic activity and structural stability. The results indicated that the G6PD Nefza mutation was mainly responsible for the functional and structural alterations observed in the double mutant G6PD A−. Moreover, our study suggests that the G6PD Nefza and G6PD A− mutations affect enzyme functions in a similar fashion to those reported for Class I mutations. PMID:29072585

Tissue-Specific Profiling Reveals Transcriptome Alterations in Arabidopsis Mutants Lacking Morphological Phenotypes[C][W

PubMed Central

Simon, Marissa; Bruex, Angela; Kainkaryam, Raghunandan M.; Zheng, Xiaohua; Huang, Ling; Woolf, Peter J.; Schiefelbein, John

2013-01-01

Traditional genetic analysis relies on mutants with observable phenotypes. Mutants lacking visible abnormalities may nevertheless exhibit molecular differences useful for defining gene function. To examine this, we analyzed tissue-specific transcript profiles from Arabidopsis thaliana transcription factor gene mutants with known roles in root epidermis development, but lacking a single-gene mutant phenotype due to genetic redundancy. We discovered substantial transcriptional changes in each mutant, preferentially affecting root epidermal genes in a manner consistent with the known double mutant effects. Furthermore, comparing transcript profiles of single and double mutants, we observed remarkable variation in the sensitivity of target genes to the loss of one or both paralogous genes, including preferential effects on specific branches of the epidermal gene network, likely reflecting the pathways of paralog subfunctionalization during evolution. In addition, we analyzed the root epidermal transcriptome of the transparent testa glabra2 mutant to clarify its role in the network. These findings provide insight into the molecular basis of genetic redundancy and duplicate gene diversification at the level of a specific gene regulatory network, and they demonstrate the usefulness of tissue-specific transcript profiling to define gene function in mutants lacking informative visible changes in phenotype. PMID:24014549

Insilico modeling and molecular dynamic simulation of claudin-1 point mutations in HCV infection.

PubMed

Vipperla, Bhavaniprasad; Dass, J Febin Prabhu; Jayanthi, S

2014-01-01

Claudin-1 (CLDN1) in association with envelope glycoprotein (CD81) mediates the fusion of HCV into the cytosol. Recent studies have indicated that point mutations in CLDN1 are important for the entry of hepatitis C virus (HCV). To validate these findings, we employed a computational platform to investigate the structural effect of two point mutations (I32M and E48K). Initially, three-dimensional co-ordinates for CLDN1 receptor sequence were generated. Then, three mutant models were built using the point mutation including a double mutant (I32M/E48K) model from the native model structure. Finally, all the four model structures including the native and three mutant models were subjected to molecular dynamics (MD) simulation for a period of 25 ns to appreciate their dynamic behavior. The MD trajectory files were analyzed using cluster and principal component method. The analysis suggested that either of the single mutation has negligible effect on the overall structure of CLDN1 compared to the double mutant form. However, the double mutant model of CLDN1 shows significant negative impact through the impairment of H-bonds and the simultaneous increase in solvent accessible surface area. Our simulation results are visibly consistent with the experimental report suggesting that the CLDN1 receptor distortion is prominent due to the double mutation with large surface accessibility. This increase in accessible surface area due to the coexistence of double mutation may be presumed as one of the key factor that results in permissive action of HCV attachment and infection.

Metabolic adaptation of two in silico mutants of Mycobacterium tuberculosis during infection.

PubMed

López-Agudelo, Víctor A; Baena, Andres; Ramirez-Malule, Howard; Ochoa, Silvia; Barrera, Luis F; Ríos-Estepa, Rigoberto

2017-11-21

Up to date, Mycobacterium tuberculosis (Mtb) remains as the worst intracellular killer pathogen. To establish infection, inside the granuloma, Mtb reprograms its metabolism to support both growth and survival, keeping a balance between catabolism, anabolism and energy supply. Mtb knockouts with the faculty of being essential on a wide range of nutritional conditions are deemed as target candidates for tuberculosis (TB) treatment. Constraint-based genome-scale modeling is considered as a promising tool for evaluating genetic and nutritional perturbations on Mtb metabolic reprogramming. Nonetheless, few in silico assessments of the effect of nutritional conditions on Mtb's vulnerability and metabolic adaptation have been carried out. A genome-scale model (GEM) of Mtb, modified from the H37Rv iOSDD890, was used to explore the metabolic reprogramming of two Mtb knockout mutants (pfkA- and icl-mutants), lacking key enzymes of central carbon metabolism, while exposed to changing nutritional conditions (oxygen, and carbon and nitrogen sources). A combination of shadow pricing, sensitivity analysis, and flux distributions patterns allowed us to identify metabolic behaviors that are in agreement with phenotypes reported in the literature. During hypoxia, at high glucose consumption, the Mtb pfkA-mutant showed a detrimental growth effect derived from the accumulation of toxic sugar phosphate intermediates (glucose-6-phosphate and fructose-6-phosphate) along with an increment of carbon fluxes towards the reductive direction of the tricarboxylic acid cycle (TCA). Furthermore, metabolic reprogramming of the icl-mutant (icl1&icl2) showed the importance of the methylmalonyl pathway for the detoxification of propionyl-CoA, during growth at high fatty acid consumption rates and aerobic conditions. At elevated levels of fatty acid uptake and hypoxia, we found a drop in TCA cycle intermediate accumulation that might create redox imbalance. Finally, findings regarding Mtb-mutant metabolic adaptation associated with asparagine consumption and acetate, succinate and alanine production, were in agreement with literature reports. This study demonstrates the potential application of genome-scale modeling, flux balance analysis (FBA), phenotypic phase plane (PhPP) analysis and shadow pricing to generate valuable insights about Mtb metabolic reprogramming in the context of human granulomas.

Improved motor performance in Dyt1 ΔGAG heterozygous knock-in mice by cerebellar Purkinje-cell specific Dyt1 conditional knocking-out

PubMed Central

Yokoi, Fumiaki; Dang, Mai Tu; Li, Yuqing

2012-01-01

Early-onset generalized torsion dystonia (dystonia 1) is an inherited movement disorder caused by mutations in DYT1 (TOR1A), which codes for torsinA. Most patients have a 3-base pair deletion (ΔGAG) in one allele of DYT1, corresponding to a loss of a glutamic acid residue (ΔE) in the C-terminal region of the protein. Functional alterations in basal ganglia circuits and the cerebellum have been reported in dystonia. Pharmacological manipulations or mutations in genes that result in functional alterations of the cerebellum have been reported to have dystonic symptoms and have been used as phenotypic rodent models. Additionally, structural lesions in the abnormal cerebellar circuits, such as cerebellectomy, have therapeutic effects in these models. A previous study has shown that the Dyt1 ΔGAG heterozygous knock-in (KI) mice exhibit motor deficits in the beam-walking test. Both Dyt1 ΔGAG heterozygous knock-in (KI) and Dyt1 Purkinje cell-specific knockout (Dyt1 pKO) mice exhibit dendritic alterations of cerebellar Purkinje cells. Here, Dyt1 pKO mice exhibited significantly less slip numbers in the beam-walking test, suggesting better motor performance than control littermates, and normal gait. Furthermore, Dyt1 ΔGAG KI/Dyt1 pKO double mutant mice exhibited significantly lower numbers of slips than Dyt1 ΔGAG heterozygous KI mice, suggesting Purkinje-cell specific knockout of Dyt1 wild-type (WT) allele in Dyt1 ΔGAG heterozygous KI mice rescued the motor deficits. The results suggest that molecular lesions of torsinA in Purkinje cells by gene therapy or intervening in the signaling pathway downstream of the cerebellar Purkinje cells may rescue motor symptoms in dystonia 1. PMID:22391119

The actin-binding proteins eps8 and gelsolin have complementary roles in regulating the growth and stability of mechanosensory hair bundles of mammalian cochlear outer hair cells.

PubMed

Olt, Jennifer; Mburu, Philomena; Johnson, Stuart L; Parker, Andy; Kuhn, Stephanie; Bowl, Mike; Marcotti, Walter; Brown, Steve D M

2014-01-01

Sound transduction depends upon mechanosensitive channels localized on the hair-like bundles that project from the apical surface of cochlear hair cells. Hair bundles show a stair-case structure composed of rows of stereocilia, and each stereocilium contains a core of tightly-packed and uniformly-polarized actin filaments. The growth and maintenance of the stereociliary actin core are dynamically regulated. Recently, it was shown that the actin-binding protein gelsolin is expressed in the stereocilia of outer hair cells (OHCs) and in its absence they become long and straggly. Gelsolin is part of a whirlin scaffolding protein complex at the stereocilia tip, which has been shown to interact with other actin regulatory molecules such as Eps8. Here we investigated the physiological effects associated with the absence of gelsolin and its possible overlapping role with Eps8. We found that, in contrast to Eps8, gelsolin does not affect mechanoelectrical transduction during immature stages of development. Moreover, OHCs from gelsolin knockout mice were able to mature into fully functional sensory receptors as judged by the normal resting membrane potential and basolateral membrane currents. Mechanoelectrical transducer current in gelsolin-Eps8 double knockout mice showed a profile similar to that observed in the single mutants for Eps8. We propose that gelsolin has a non-overlapping role with Eps8. While Eps8 is mainly involved in the initial growth of stereocilia in both inner hair cells (IHCs) and OHCs, gelsolin is required for the maintenance of mature hair bundles of low-frequency OHCs after the onset of hearing.

Phosphatase and Tensin Homolog Is a Growth Repressor of Both Rhizoid and Gametophore Development in the Moss Physcomitrella patens1[OPEN

PubMed Central

Saavedra, Laura; Heilmann, Ingo

2015-01-01

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a lipid phosphatase implicated in cellular proliferation and survival. In animal cells, loss of PTEN leads to increased levels of phosphatidylinositol (3,4,5)-trisphosphate, stimulation of glucose and lipid metabolism, cellular growth, and morphological changes (related to adaptation and survival). Intriguingly, in plants, phosphatidylinositol (3,4,5)-trisphosphate has not been detected, and the enzymes that synthesize it were never reported. In this study we performed a genetic, biochemical, and functional characterization of the moss Physcomitrella patens PTEN gene family. P. patens has four PTENs, which are ubiquitously expressed during the entire moss life cycle. Using a knock-in approach, we show that all four genes are expressed in growing tissues, namely caulonemal and rhizoid cells. At the subcellular level, PpPTEN-green fluorescent protein fusions localized to the cytosol and the nucleus. Analysis of single and double knockouts revealed no significant phenotypes at different developmental stages, indicative of functional redundancy. However, compared with wild-type triple and quadruple pten knockouts, caulonemal cells grew faster, switched from the juvenile protonemal stage to adult gametophores earlier, and produced more rhizoids. Furthermore, analysis of lipid content and quantitative real-time polymerase chain reaction data performed in quadruple mutants revealed altered phosphoinositide levels [increase in phosphatidylinositol (3,5)-bisphosphate and decrease in phosphatidylinositol 3-phosphate] and up-regulation of marker genes from the synthesis phase of the cell cycle (e.g. P. patens proliferating cell nuclear antigen, ribonucleotide reductase, and minichromosome maintenance) and of the retinoblastoma-related protein gene P. patens retinoblastoma-related protein1. Together, these results suggest that PpPTEN is a suppressor of cell growth and morphogenic development in plants. PMID:26463087

Phosphatase and Tensin Homolog Is a Growth Repressor of Both Rhizoid and Gametophore Development in the Moss Physcomitrella patens.

PubMed

Saavedra, Laura; Catarino, Rita; Heinz, Tobias; Heilmann, Ingo; Bezanilla, Magdalena; Malhó, Rui

2015-12-01

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a lipid phosphatase implicated in cellular proliferation and survival. In animal cells, loss of PTEN leads to increased levels of phosphatidylinositol (3,4,5)-trisphosphate, stimulation of glucose and lipid metabolism, cellular growth, and morphological changes (related to adaptation and survival). Intriguingly, in plants, phosphatidylinositol (3,4,5)-trisphosphate has not been detected, and the enzymes that synthesize it were never reported. In this study we performed a genetic, biochemical, and functional characterization of the moss Physcomitrella patens PTEN gene family. P. patens has four PTENs, which are ubiquitously expressed during the entire moss life cycle. Using a knock-in approach, we show that all four genes are expressed in growing tissues, namely caulonemal and rhizoid cells. At the subcellular level, PpPTEN-green fluorescent protein fusions localized to the cytosol and the nucleus. Analysis of single and double knockouts revealed no significant phenotypes at different developmental stages, indicative of functional redundancy. However, compared with wild-type triple and quadruple pten knockouts, caulonemal cells grew faster, switched from the juvenile protonemal stage to adult gametophores earlier, and produced more rhizoids. Furthermore, analysis of lipid content and quantitative real-time polymerase chain reaction data performed in quadruple mutants revealed altered phosphoinositide levels [increase in phosphatidylinositol (3,5)-bisphosphate and decrease in phosphatidylinositol 3-phosphate] and up-regulation of marker genes from the synthesis phase of the cell cycle (e.g. P. patens proliferating cell nuclear antigen, ribonucleotide reductase, and minichromosome maintenance) and of the retinoblastoma-related protein gene P. patens retinoblastoma-related protein1. Together, these results suggest that PpPTEN is a suppressor of cell growth and morphogenic development in plants. © 2015 American Society of Plant Biologists. All Rights Reserved.

Outer membrane protein e of Escherichia coli K-12 is co-regulated with alkaline phosphatase.

PubMed

Tommassen, J; Lugtenberg, B

1980-07-01

Outer membrane protein e is induced in wild-type cells, just like alkaline phosphatase and some other periplasmic proteins, by growth under phosphatase limitation. nmpA and nmpB mutants, which synthesize protein e constitutively, are shown also to produce the periplasmic enzyme alkaline phosphatase constitutively. Alternatively, individual phoS, phoT, and phoR mutants as well as pit pst double mutants, all of which are known to produce alkaline phosphatase constitutively, were found to be constitutive for protein e. Also, the periplasmic space of most nmpA mutants and of all nmpB mutants grown in excess phosphate was found to contain, in addition to alkaline phosphatase, at least two new proteins, a phenomenon known for individual phoT and phoR mutants as well as for pit pst double mutants. The other nmpA mutants as well as phoS mutants lacked one of these extra periplasmic proteins, namely the phosphate-binding protein. From these data and from the known positions of the mentioned genes on the chromosomal map, it is concluded that nmpB mutants are identical to phoR mutants. Moreover, some nmpA mutants were shown to be identical to phoS mutants, whereas other nmpA mutants are likely to contain mutations in one of the genes phoS, phoT, or pst.

Linking Yeast Gcn5p Catalytic Function and Gene Regulation Using a Quantitative, Graded Dominant Mutant Approach

PubMed Central

Lanza, Amanda M.; Blazeck, John J.; Crook, Nathan C.; Alper, Hal S.

2012-01-01

Establishing causative links between protein functional domains and global gene regulation is critical for advancements in genetics, biotechnology, disease treatment, and systems biology. This task is challenging for multifunctional proteins when relying on traditional approaches such as gene deletions since they remove all domains simultaneously. Here, we describe a novel approach to extract quantitative, causative links by modulating the expression of a dominant mutant allele to create a function-specific competitive inhibition. Using the yeast histone acetyltransferase Gcn5p as a case study, we demonstrate the utility of this approach and (1) find evidence that Gcn5p is more involved in cell-wide gene repression, instead of the accepted gene activation associated with HATs, (2) identify previously unknown gene targets and interactions for Gcn5p-based acetylation, (3) quantify the strength of some Gcn5p-DNA associations, (4) demonstrate that this approach can be used to correctly identify canonical chromatin modifications, (5) establish the role of acetyltransferase activity on synthetic lethal interactions, and (6) identify new functional classes of genes regulated by Gcn5p acetyltransferase activity—all six of these major conclusions were unattainable by using standard gene knockout studies alone. We recommend that a graded dominant mutant approach be utilized in conjunction with a traditional knockout to study multifunctional proteins and generate higher-resolution data that more accurately probes protein domain function and influence. PMID:22558379

Experimental Infections with Mycoplasma agalactiae Identify Key Factors Involved in Host-Colonization

PubMed Central

Baranowski, Eric; Bergonier, Dominique; Sagné, Eveline; Hygonenq, Marie-Claude; Ronsin, Patricia; Berthelot, Xavier; Citti, Christine

2014-01-01

Mechanisms underlying pathogenic processes in mycoplasma infections are poorly understood, mainly because of limited sequence similarities with classical, bacterial virulence factors. Recently, large-scale transposon mutagenesis in the ruminant pathogen Mycoplasma agalactiae identified the NIF locus, including nifS and nifU, as essential for mycoplasma growth in cell culture, while dispensable in axenic media. To evaluate the importance of this locus in vivo, the infectivity of two knock-out mutants was tested upon experimental infection in the natural host. In this model, the parental PG2 strain was able to establish a systemic infection in lactating ewes, colonizing various body sites such as lymph nodes and the mammary gland, even when inoculated at low doses. In these PG2-infected ewes, we observed over the course of infection (i) the development of a specific antibody response and (ii) dynamic changes in expression of M. agalactiae surface variable proteins (Vpma), with multiple Vpma profiles co-existing in the same animal. In contrast and despite a sensitive model, none of the knock-out mutants were able to survive and colonize the host. The extreme avirulent phenotype of the two mutants was further supported by the absence of an IgG response in inoculated animals. The exact role of the NIF locus remains to be elucidated but these data demonstrate that it plays a key role in the infectious process of M. agalactiae and most likely of other pathogenic mycoplasma species as many carry closely related homologs. PMID:24699671

Arabidopsis Mutants Deleted in the Light-Harvesting Protein Lhcb4 Have a Disrupted Photosystem II Macrostructure and Are Defective in Photoprotection[C][W

PubMed Central

de Bianchi, Silvia; Betterle, Nico; Kouril, Roman; Cazzaniga, Stefano; Boekema, Egbert; Bassi, Roberto; Dall’Osto, Luca

2011-01-01

The role of the light-harvesting complex Lhcb4 (CP29) in photosynthesis was investigated in Arabidopsis thaliana by characterizing knockout lines for each of the three Lhcb4 isoforms (Lhcb4.1/4.2/4.3). Plants lacking all isoforms (koLhcb4) showed a compensatory increase of Lhcb1 and a slightly reduced photosystem II/I ratio with respect to the wild type. The absence of Lhcb4 did not result in alteration in electron transport rates. However, the kinetic of state transition was faster in the mutant, and nonphotochemical quenching activity was lower in koLhcb4 plants with respect to either wild type or mutants retaining a single Lhcb4 isoform. KoLhcb4 plants were more sensitive to photoinhibition, while this effect was not observed in knockout lines for any other photosystem II antenna subunit. Ultrastructural analysis of thylakoid grana membranes showed a lower density of photosystem II complexes in koLhcb4. Moreover, analysis of isolated supercomplexes showed a different overall shape of the C2S2 particles due to a different binding mode of the S-trimer to the core complex. An empty space was observed within the photosystem II supercomplex at the Lhcb4 position, implying that the missing Lhcb4 was not replaced by other Lhc subunits. This suggests that Lhcb4 is unique among photosystem II antenna proteins and determinant for photosystem II macro-organization and photoprotection. PMID:21803939

Characterization of the first knock-out aldh7a1 zebrafish model for pyridoxine-dependent epilepsy using CRISPR-Cas9 technology

PubMed Central

Zabinyakov, Nikita; Bullivant, Garrett; Cao, Feng; Fernandez Ojeda, Matilde; Jia, Zheng Ping; Wen, Xiao-Yan; Dowling, James J.; Salomons, Gajja S.

2017-01-01

Pyridoxine dependent epilepsy (PDE) is caused by likely pathogenic variants in ALDH7A1 (PDE-ALDH7A1) and inherited autosomal recessively. Neurotoxic alpha-amino adipic semialdehyde (alpha-AASA), piperideine 6-carboxylate and pipecolic acid accumulate in body fluids. Neonatal or infantile onset seizures refractory to anti-epileptic medications are clinical features. Treatment with pyridoxine, arginine and lysine-restricted diet does not normalize neurodevelopmental outcome or accumulation of neurotoxic metabolites. There is no animal model for high throughput drug screening. For this reason, we developed and characterized the first knock-out aldh7a1 zebrafish model using CRISPR-Cas9 technology. Zebrafish aldh7a1 mutants were generated by using a vector free method of CRISPR-Cas9 mutagenesis. Genotype analysis of aldh7a1 knock-out zebrafish was performed by high resolution melt analysis, direct sequencing and QIAxcel system. Electroencephalogram was performed. Alpha-AASA, piperideine 6-carboxylate and pipecolic acid, were measured by liquid chromatography-tandem mass spectrometry. Our knock-out aldh7a1 zebrafish has homozygous 5 base pair (bp) mutation in ALDH7A1. Knock-out aldh7a1 embryos have spontaneous rapid increase in locomotion and a rapid circling swim behavior earliest 8-day post fertilization (dpf). Electroencephalogram revealed large amplitude spike discharges compared to wild type. Knock-out aldh7a1 embryos have elevated alpha-AASA, piperideine 6-carboxylate and pipecolic acid compared to wild type embryos at 3 dpf. Knock-out aldh7a1 embryos showed no aldh7a1 protein by western blot compared to wild type. Our knock-out aldh7a1 zebrafish is a well characterized model for large-scale drug screening using behavioral and biochemical features and accurately recapitulates the human PDE-ALDH7A1 disease. PMID:29053735

Characterization of the first knock-out aldh7a1 zebrafish model for pyridoxine-dependent epilepsy using CRISPR-Cas9 technology.

PubMed

Zabinyakov, Nikita; Bullivant, Garrett; Cao, Feng; Fernandez Ojeda, Matilde; Jia, Zheng Ping; Wen, Xiao-Yan; Dowling, James J; Salomons, Gajja S; Mercimek-Andrews, Saadet

2017-01-01

Pyridoxine dependent epilepsy (PDE) is caused by likely pathogenic variants in ALDH7A1 (PDE-ALDH7A1) and inherited autosomal recessively. Neurotoxic alpha-amino adipic semialdehyde (alpha-AASA), piperideine 6-carboxylate and pipecolic acid accumulate in body fluids. Neonatal or infantile onset seizures refractory to anti-epileptic medications are clinical features. Treatment with pyridoxine, arginine and lysine-restricted diet does not normalize neurodevelopmental outcome or accumulation of neurotoxic metabolites. There is no animal model for high throughput drug screening. For this reason, we developed and characterized the first knock-out aldh7a1 zebrafish model using CRISPR-Cas9 technology. Zebrafish aldh7a1 mutants were generated by using a vector free method of CRISPR-Cas9 mutagenesis. Genotype analysis of aldh7a1 knock-out zebrafish was performed by high resolution melt analysis, direct sequencing and QIAxcel system. Electroencephalogram was performed. Alpha-AASA, piperideine 6-carboxylate and pipecolic acid, were measured by liquid chromatography-tandem mass spectrometry. Our knock-out aldh7a1 zebrafish has homozygous 5 base pair (bp) mutation in ALDH7A1. Knock-out aldh7a1 embryos have spontaneous rapid increase in locomotion and a rapid circling swim behavior earliest 8-day post fertilization (dpf). Electroencephalogram revealed large amplitude spike discharges compared to wild type. Knock-out aldh7a1 embryos have elevated alpha-AASA, piperideine 6-carboxylate and pipecolic acid compared to wild type embryos at 3 dpf. Knock-out aldh7a1 embryos showed no aldh7a1 protein by western blot compared to wild type. Our knock-out aldh7a1 zebrafish is a well characterized model for large-scale drug screening using behavioral and biochemical features and accurately recapitulates the human PDE-ALDH7A1 disease.

Functional Angucycline-Like Antibiotic Gene Cluster in the Terminal Inverted Repeats of the Streptomyces ambofaciens Linear Chromosome

PubMed Central

Pang, Xiuhua; Aigle, Bertrand; Girardet, Jean-Michel; Mangenot, Sophie; Pernodet, Jean-Luc; Decaris, Bernard; Leblond, Pierre

2004-01-01

Streptomyces ambofaciens has an 8-Mb linear chromosome ending in 200-kb terminal inverted repeats. Analysis of the F6 cosmid overlapping the terminal inverted repeats revealed a locus similar to type II polyketide synthase (PKS) gene clusters. Sequence analysis identified 26 open reading frames, including genes encoding the β-ketoacyl synthase (KS), chain length factor (CLF), and acyl carrier protein (ACP) that make up the minimal PKS. These KS, CLF, and ACP subunits are highly homologous to minimal PKS subunits involved in the biosynthesis of angucycline antibiotics. The genes encoding the KS and ACP subunits are transcribed constitutively but show a remarkable increase in expression after entering transition phase. Five genes, including those encoding the minimal PKS, were replaced by resistance markers to generate single and double mutants (replacement in one and both terminal inverted repeats). Double mutants were unable to produce either diffusible orange pigment or antibacterial activity against Bacillus subtilis. Single mutants showed an intermediate phenotype, suggesting that each copy of the cluster was functional. Transformation of double mutants with a conjugative and integrative form of F6 partially restored both phenotypes. The pigmented and antibacterial compounds were shown to be two distinct molecules produced from the same biosynthetic pathway. High-pressure liquid chromatography analysis of culture extracts from wild-type and double mutants revealed a peak with an associated bioactivity that was absent from the mutants. Two additional genes encoding KS and CLF were present in the cluster. However, disruption of the second KS gene had no effect on either pigment or antibiotic production. PMID:14742212

Metabolic engineering of a diazotrophic bacterium improves ammonium release and biofertilization of plants and microalgae.

PubMed

Ambrosio, Rafael; Ortiz-Marquez, Juan Cesar Federico; Curatti, Leonardo

2017-03-01

The biological nitrogen fixation carried out by some Bacteria and Archaea is one of the most attractive alternatives to synthetic nitrogen fertilizers. However, with the exception of the symbiotic rhizobia-legumes system, progress towards a more extensive realization of this goal has been slow. In this study we manipulated the endogenous regulation of both nitrogen fixation and assimilation in the aerobic bacterium Azotobacter vinelandii. Substituting an exogenously inducible promoter for the native promoter of glutamine synthetase produced conditional lethal mutant strains unable to grow diazotrophically in the absence of the inducer. This mutant phenotype could be reverted in a double mutant strain bearing a deletion in the nifL gene that resulted in constitutive expression of nif genes and increased production of ammonium. Under GS non-inducing conditions both the single and the double mutant strains consistently released very high levels of ammonium (>20mM) into the growth medium. The double mutant strain grew and excreted high levels of ammonium under a wider range of concentrations of the inducer than the single mutant strain. Induced mutant cells could be loaded with glutamine synthetase at different levels, which resulted in different patterns of extracellular ammonium accumulation afterwards. Inoculation of the engineered bacteria into a microalgal culture in the absence of sources of C and N other than N 2 and CO 2 from the air, resulted in a strong proliferation of microalgae that was suppressed upon addition of the inducer. Both single and double mutant strains also promoted growth of cucumber plants in the absence of added N-fertilizer, while this property was only marginal in the parental strain. This study provides a simple synthetic genetic circuit that might inspire engineering of optimized inoculants that efficiently channel N 2 from the air into crops. Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Involvement of arginine-specific cysteine proteinase (Arg-gingipain) in fimbriation of Porphyromonas gingivalis.

PubMed Central

Nakayama, K; Yoshimura, F; Kadowaki, T; Yamamoto, K

1996-01-01

Arginine-specific cysteine proteinase (Arg-gingipain [RGP], a major proteinase secreted from the oral anaerobic bacterium Porphyromonas gingivalis, is encoded by two separate genes (rgpA and rgpB) on the P. gingivalis chromosome and widely implicated as an important virulence factor in the pathogenesis of periodontal disease (K. Nakayama, T. Kadowaki, K. Okamoto, and K. Yamamoto, J. Biol. Chem. 270:23619-23626, 1995). In this study, we investigated the role of RGP in the formation of P. gingivalis fimbriae which are thought to mediate adhesion of the organism to the oral surface by use of the rgp mutants. Electron microscopic observation revealed that the rgpA rgpB double (RGP-null) mutant possessed very few fimbriae on the cell surface, whereas the number of fimbriae of the rgpA or rgpB mutant was similar to that of the wild-type parent strain. The rgpB+ revertants that were isolated from the double mutant and recovered 20 to 40% of RGP activity of the wild-type parent possessed as many fimbriae as the wild-type parent, indicating that RGP significantly contributes to the fimbriation of P. gingivalis as well as to the degradation of various host proteins, disturbance of host defense mechanisms, and hemagglutination. Immunoblot analysis of cell extracts of these mutants with antifimbrilin antiserum revealed that the rgpA rgpB double mutant produced small amounts of two immunoreactive proteins with molecular masses of 45 and 43 kDa, corresponding to those of the precursor and mature forms of fimbrilin, respectively. The result suggests that RGP may function as a processing proteinase for fimbrilin maturation. In addition, a precursor form of the 75-kDa protein, one of the major outer membrane proteins of P. gingivalis, was accumulated in the rgpA rgpB double mutant but not in the single mutants and the revertants, suggesting an extensive role for RGP in the maturation of some of the cell surface proteins. PMID:8631669

A Novel Knock-Out Animal Model to Analyze Transcriptional Signaling by p53 Tumor Suppressor Protein in Breast Cancer

DTIC Science & Technology

2002-05-01

homozygous for the pcna and p21 mutant genes will be accomplised with the help of Gene Targeting and Transgenic Facility at the Rosewel Park Cancer Institute...screening of BAC library was performed with the help of the DNA Microarray Facility Facility at the Rosewel Park Cancer Institute. Sequence of mouse

Characterization of Bombyx mori nucleopolyhedrovirus with a knockout of Bm17.

PubMed

Shen, Hongxing; Zhou, Yang; Zhang, Wen; Nin, Bin; Wang, Hua; Wang, Xiaochun; Shao, Shihe; Chen, Huiqing; Guo, Zhongjian; Liu, Xiaoyong; Yao, Qin; Chen, Keping

2012-12-01

Open reading frame 17 (Bm17) gene of Bombyx mori nucleopolyhedrovirus is a highly conserved gene in lepidopteran nucleopolyhedroviruses, but its function remains unknown. In this report, transient-expression and superinfection assays indicated that BM17 localized in the nucleus and cytoplasm of infected BmN cells. To determine the role of Bm17 in baculovirus life cycle, we constructed a Bm17 knockout virus and characterized its properties in cells. Analysis of the production and infection of budded virions, the level of viral DNA replication revealed showed that there was no significant difference among the mutant, the control, and the Bm17 repaired virus strains. These results suggest that BM17 is not essential for virus replication in cultured cells.

Inactivation of Phaeodactylum tricornutum urease gene using transcription activator-like effector nuclease-based targeted mutagenesis

DOE PAGES

Weyman, Philip D.; Beeri, Karen; Lefebvre, Stephane C.; ...

2014-10-10

Diatoms are unicellular photosynthetic algae with promise for green production of fuels and other chemicals. Recent genome-editing techniques have greatly improved the potential of many eukaryotic genetic systems, including diatoms, to enable knowledge-based studies and bioengineering. Using a new technique, transcription activator-like effector nucleases (TALENs), the gene encoding the urease enzyme in the model diatom, Phaeodactylum tricornutum, was targeted for interruption. The knockout cassette was identified within the urease gene by PCR and Southern blot analyses of genomic DNA. The lack of urease protein was confirmed by Western blot analyses in mutant cell lines that were unable to grow onmore » urea as the sole nitrogen source. Untargeted metabolomic analysis revealed a build-up of urea, arginine and ornithine in the urease knockout lines. All three intermediate metabolites are upstream of the urease reaction within the urea cycle, suggesting a disruption of the cycle despite urea production. Numerous high carbon metabolites were enriched in the mutant, implying a breakdown of cellular C and N repartitioning. The presented method improves the molecular toolkit for diatoms and clarifies the role of urease in the urea cycle.« less

Investigations of Salmonella enterica serovar newport infections of oysters by using immunohistochemistry and knockout mutagenesis.

PubMed

Morrison, Christopher M; Dial, Sharon M; Day, William A; Joens, Lynn A

2012-04-01

The consumption of raw oysters is an important risk factor in the acquisition of food-borne disease, with Salmonella being one of a number of pathogens that have been found in market oysters. Previous work by our lab found that Salmonella was capable of surviving in oysters for over 2 months under laboratory conditions, and this study sought to further investigate Salmonella's tissue affinity and mechanism of persistence within the oysters. Immunohistochemistry was used to show that Salmonella was capable of breaching the epithelial barriers, infecting the deeper connective tissues of the oysters, and evading destruction by the oysters' phagocytic hemocytes. To further investigate the mechanism of these infections, genes vital to the function of Salmonella's two main type III secretion systems were disrupted and the survivability of these knockout mutants within oysters was assayed. When the Salmonella pathogenicity island 1 and 2 mutant strains were exposed to oysters, there were no detectable deficiencies in their abilities to survive, suggesting that Salmonella's long-term infection of oysters does not rely upon these two important pathogenicity islands and must be due to some other, currently unknown, mechanism.

Investigations of Salmonella enterica Serovar Newport Infections of Oysters by Using Immunohistochemistry and Knockout Mutagenesis

PubMed Central

Morrison, Christopher M.; Dial, Sharon M.; Day, William A.

2012-01-01

The consumption of raw oysters is an important risk factor in the acquisition of food-borne disease, with Salmonella being one of a number of pathogens that have been found in market oysters. Previous work by our lab found that Salmonella was capable of surviving in oysters for over 2 months under laboratory conditions, and this study sought to further investigate Salmonella's tissue affinity and mechanism of persistence within the oysters. Immunohistochemistry was used to show that Salmonella was capable of breaching the epithelial barriers, infecting the deeper connective tissues of the oysters, and evading destruction by the oysters' phagocytic hemocytes. To further investigate the mechanism of these infections, genes vital to the function of Salmonella's two main type III secretion systems were disrupted and the survivability of these knockout mutants within oysters was assayed. When the Salmonella pathogenicity island 1 and 2 mutant strains were exposed to oysters, there were no detectable deficiencies in their abilities to survive, suggesting that Salmonella's long-term infection of oysters does not rely upon these two important pathogenicity islands and must be due to some other, currently unknown, mechanism. PMID:22307286

Generation of knockout rabbits using transcription activator-like effector nucleases.

PubMed

Wang, Yu; Fan, Nana; Song, Jun; Zhong, Juan; Guo, Xiaogang; Tian, Weihua; Zhang, Quanjun; Cui, Fenggong; Li, Li; Newsome, Philip N; Frampton, Jon; Esteban, Miguel A; Lai, Liangxue

2014-01-01

Zinc-finger nucleases and transcription activator-like effector nucleases are novel gene-editing platforms contributing to redefine the boundaries of modern biological research. They are composed of a non-specific cleavage domain and a tailor made DNA-binding module, which enables a broad range of genetic modifications by inducing efficient DNA double-strand breaks at desired loci. Among other remarkable uses, these nucleases have been employed to produce gene knockouts in mid-size and large animals, such as rabbits and pigs, respectively. This approach is cost effective, relatively quick, and can produce invaluable models for human disease studies, biotechnology or agricultural purposes. Here we describe a protocol for the efficient generation of knockout rabbits using transcription activator-like effector nucleases, and a perspective of the field.

Role of thin descending limb urea transport in renal urea handling and the urine concentrating mechanism

PubMed Central

Lei, Tianluo; Zhou, Lei; Layton, Anita T.; Zhou, Hong; Zhao, Xuejian; Bankir, Lise

2011-01-01

Urea transporters UT-A2 and UT-B are expressed in epithelia of thin descending limb of Henle's loop and in descending vasa recta, respectively. To study their role and possible interaction in the context of the urine concentration mechanism, a UT-A2 and UT-B double knockout (UT-A2/B knockout) mouse model was generated by targeted deletion of the UT-A2 promoter in embryonic stem cells with UT-B gene knockout. The UT-A2/B knockout mice lacked detectable UT-A2 and UT-B transcripts and proteins and showed normal survival and growth. Daily urine output was significantly higher in UT-A2/B knockout mice than that in wild-type mice and lower than that in UT-B knockout mice. Urine osmolality in UT-A2/B knockout mice was intermediate between that in UT-B knockout and wild-type mice. The changes in urine osmolality and flow rate, plasma and urine urea concentration, as well as non-urea solute concentration after an acute urea load or chronic changes in protein intake suggested that UT-A2 plays a role in the progressive accumulation of urea in the inner medulla. These results suggest that in wild-type mice UT-A2 facilitates urea absorption by urea efflux from the thin descending limb of short loops of Henle. Moreover, UT-A2 deletion in UT-B knockout mice partially remedies the urine concentrating defect caused by UT-B deletion, by reducing urea loss from the descending limbs to the peripheral circulation; instead, urea is returned to the inner medulla through the loops of Henle and the collecting ducts. PMID:21849488

Role of thin descending limb urea transport in renal urea handling and the urine concentrating mechanism.

PubMed

Lei, Tianluo; Zhou, Lei; Layton, Anita T; Zhou, Hong; Zhao, Xuejian; Bankir, Lise; Yang, Baoxue

2011-12-01

Urea transporters UT-A2 and UT-B are expressed in epithelia of thin descending limb of Henle's loop and in descending vasa recta, respectively. To study their role and possible interaction in the context of the urine concentration mechanism, a UT-A2 and UT-B double knockout (UT-A2/B knockout) mouse model was generated by targeted deletion of the UT-A2 promoter in embryonic stem cells with UT-B gene knockout. The UT-A2/B knockout mice lacked detectable UT-A2 and UT-B transcripts and proteins and showed normal survival and growth. Daily urine output was significantly higher in UT-A2/B knockout mice than that in wild-type mice and lower than that in UT-B knockout mice. Urine osmolality in UT-A2/B knockout mice was intermediate between that in UT-B knockout and wild-type mice. The changes in urine osmolality and flow rate, plasma and urine urea concentration, as well as non-urea solute concentration after an acute urea load or chronic changes in protein intake suggested that UT-A2 plays a role in the progressive accumulation of urea in the inner medulla. These results suggest that in wild-type mice UT-A2 facilitates urea absorption by urea efflux from the thin descending limb of short loops of Henle. Moreover, UT-A2 deletion in UT-B knockout mice partially remedies the urine concentrating defect caused by UT-B deletion, by reducing urea loss from the descending limbs to the peripheral circulation; instead, urea is returned to the inner medulla through the loops of Henle and the collecting ducts.

Identification of SACE_7040, a member of TetR family related to the morphological differentiation of Saccharopolyspora erythraea.

PubMed

Han, Shu; Song, Ping; Ren, Ting; Huang, Xunduan; Cao, Cheng; Zhang, Buchang

2011-08-01

SACE_7040 is presumed to be a member of the TetR family of transcriptional regulators in Saccharopolyspora erythraea, but its biological function is unknown. It was shown that the SACE_7040 gene knockout mutant formed aerial mycelium earlier than its original strain, and this phenotype could be restored by complementation of a single copy of SACE_7040 gene, demonstrating that SACE_7040 is an important regulator of the morphological differentiation of Sac. erythraea. When SACE_7040 gene was disrupted in the bldD mutant, we intriguingly found that the defect in aerial development exhibited by the bldD mutant could be overcome, suggesting a crosstalk between SACE_7040 and BldD in Sac. erythraea morphogenesis. These findings provide novel insights toward the Sac. erythraea developmental biology.

Probucol prevents early coronary heart disease and death in the high-density lipoprotein receptor SR-BI/apolipoprotein E double knockout mouse

PubMed Central

Braun, Anne; Zhang, Songwen; Miettinen, Helena E.; Ebrahim, Shamsah; Holm, Teresa M.; Vasile, Eliza; Post, Mark J.; Yoerger, Danita M.; Picard, Michael H.; Krieger, Joshua L.; Andrews, Nancy C.; Simons, Michael; Krieger, Monty

2003-01-01

Mice with homozygous null mutations in the high-density lipoprotein receptor SR-BI (scavenger receptor class B, type I) and apolipoprotein E genes fed a low-fat diet exhibit a constellation of pathologies shared with human atherosclerotic coronary heart disease (CHD): hypercholesterolemia, occlusive coronary atherosclerosis, myocardial infarctions, cardiac dysfunction (heart enlargement, reduced systolic function and ejection fraction, and ECG abnormalities), and premature death (mean age 6 weeks). They also exhibit a block in RBC maturation and abnormally high plasma unesterified-to-total cholesterol ratio (0.8) with associated abnormal lipoprotein morphology (lamellar/vesicular and stacked discoidal particles reminiscent of those in lecithin/cholesterol acyltransferase deficiency and cholestasis). Treatment with the lipid-lowering, antiatherosclerosis, and antioxidation drug probucol extended life to as long as 60 weeks (mean 36 weeks), and at 5–6 weeks of age, virtually completely reversed the cardiac and most RBC pathologies and corrected the unesterified to total cholesterol ratio (0.3) and associated distinctive abnormal lipoprotein morphologies. Manipulation of the timing of administration and withdrawal of probucol could control the onset of death and suggested that critical pathological changes usually occurred in untreated double knockout mice between ≈3 (weaning) and 5 weeks of age and that probucol delayed heart failure even after development of substantial CHD. The ability of probucol treatment to modulate pathophysiology in the double knockout mice enhances the potential of this murine system for analysis of the pathophysiology of CHD and preclinical testing of new approaches for the prevention and treatment of cardiovascular disease. PMID:12771386

Ssb1 and Ssb2 cooperate to regulate mouse hematopoietic stem and progenitor cells by resolving replicative stress.

PubMed

Shi, Wei; Vu, Therese; Boucher, Didier; Biernacka, Anna; Nde, Jules; Pandita, Raj K; Straube, Jasmin; Boyle, Glen M; Al-Ejeh, Fares; Nag, Purba; Jeffery, Jessie; Harris, Janelle L; Bain, Amanda L; Grzelak, Marta; Skrzypczak, Magdalena; Mitra, Abhishek; Dojer, Norbert; Crosetto, Nicola; Cloonan, Nicole; Becherel, Olivier J; Finnie, John; Skaar, Jeffrey R; Walkley, Carl R; Pandita, Tej K; Rowicka, Maga; Ginalski, Krzysztof; Lane, Steven W; Khanna, Kum Kum

2017-05-04

Hematopoietic stem and progenitor cells (HSPCs) are vulnerable to endogenous damage and defects in DNA repair can limit their function. The 2 single-stranded DNA (ssDNA) binding proteins SSB1 and SSB2 are crucial regulators of the DNA damage response; however, their overlapping roles during normal physiology are incompletely understood. We generated mice in which both Ssb1 and Ssb2 were constitutively or conditionally deleted. Constitutive Ssb1/Ssb2 double knockout (DKO) caused early embryonic lethality, whereas conditional Ssb1/Ssb2 double knockout (cDKO) in adult mice resulted in acute lethality due to bone marrow failure and intestinal atrophy featuring stem and progenitor cell depletion, a phenotype unexpected from the previously reported single knockout models of Ssb1 or Ssb2 Mechanistically, cDKO HSPCs showed altered replication fork dynamics, massive accumulation of DNA damage, genome-wide double-strand breaks enriched at Ssb-binding regions and CpG islands, together with the accumulation of R -loops and cytosolic ssDNA. Transcriptional profiling of cDKO HSPCs revealed the activation of p53 and interferon (IFN) pathways, which enforced cell cycling in quiescent HSPCs, resulting in their apoptotic death. The rapid cell death phenotype was reproducible in in vitro cultured cDKO-hematopoietic stem cells, which were significantly rescued by nucleotide supplementation or after depletion of p53. Collectively, Ssb1 and Ssb2 control crucial aspects of HSPC function, including proliferation and survival in vivo by resolving replicative stress to maintain genomic stability. © 2017 by The American Society of Hematology.

Ssb1 and Ssb2 cooperate to regulate mouse hematopoietic stem and progenitor cells by resolving replicative stress

PubMed Central

Vu, Therese; Boucher, Didier; Biernacka, Anna; Nde, Jules; Pandita, Raj K.; Straube, Jasmin; Boyle, Glen M.; Al-Ejeh, Fares; Jeffery, Jessie; Harris, Janelle L.; Bain, Amanda L.; Grzelak, Marta; Skrzypczak, Magdalena; Mitra, Abhishek; Dojer, Norbert; Crosetto, Nicola; Cloonan, Nicole; Becherel, Olivier J.; Finnie, John; Skaar, Jeffrey R.; Walkley, Carl R.; Pandita, Tej K.; Rowicka, Maga; Ginalski, Krzysztof

2017-01-01

Hematopoietic stem and progenitor cells (HSPCs) are vulnerable to endogenous damage and defects in DNA repair can limit their function. The 2 single-stranded DNA (ssDNA) binding proteins SSB1 and SSB2 are crucial regulators of the DNA damage response; however, their overlapping roles during normal physiology are incompletely understood. We generated mice in which both Ssb1 and Ssb2 were constitutively or conditionally deleted. Constitutive Ssb1/Ssb2 double knockout (DKO) caused early embryonic lethality, whereas conditional Ssb1/Ssb2 double knockout (cDKO) in adult mice resulted in acute lethality due to bone marrow failure and intestinal atrophy featuring stem and progenitor cell depletion, a phenotype unexpected from the previously reported single knockout models of Ssb1 or Ssb2. Mechanistically, cDKO HSPCs showed altered replication fork dynamics, massive accumulation of DNA damage, genome-wide double-strand breaks enriched at Ssb-binding regions and CpG islands, together with the accumulation of R-loops and cytosolic ssDNA. Transcriptional profiling of cDKO HSPCs revealed the activation of p53 and interferon (IFN) pathways, which enforced cell cycling in quiescent HSPCs, resulting in their apoptotic death. The rapid cell death phenotype was reproducible in in vitro cultured cDKO-hematopoietic stem cells, which were significantly rescued by nucleotide supplementation or after depletion of p53. Collectively, Ssb1 and Ssb2 control crucial aspects of HSPC function, including proliferation and survival in vivo by resolving replicative stress to maintain genomic stability. PMID:28270450

Functional Analysis of Glycosylation of Zika Virus Envelope Protein.

PubMed

Fontes-Garfias, Camila R; Shan, Chao; Luo, Huanle; Muruato, Antonio E; Medeiros, Daniele B A; Mays, Elizabeth; Xie, Xuping; Zou, Jing; Roundy, Christopher M; Wakamiya, Maki; Rossi, Shannan L; Wang, Tian; Weaver, Scott C; Shi, Pei-Yong

2017-10-31

Zika virus (ZIKV) infection causes devastating congenital abnormities and Guillain-Barré syndrome. The ZIKV envelope (E) protein is responsible for viral entry and represents a major determinant for viral pathogenesis. Like other flaviviruses, the ZIKV E protein is glycosylated at amino acid N154. To study the function of E glycosylation, we generated a recombinant N154Q ZIKV that lacks the E glycosylation and analyzed the mutant virus in mammalian and mosquito hosts. In mouse models, the mutant was attenuated, as evidenced by lower viremia, decreased weight loss, and no mortality; however, knockout of E glycosylation did not significantly affect neurovirulence. Mice immunized with the mutant virus developed a robust neutralizing antibody response and were completely protected from wild-type ZIKV challenge. In mosquitoes, the mutant virus exhibited diminished oral infectivity for the Aedes aegypti vector. Collectively, the results demonstrate that E glycosylation is critical for ZIKV infection of mammalian and mosquito hosts. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Lipoprotein lipase regulates hematopoietic stem progenitor cell maintenance through DHA supply.

PubMed

Liu, Chao; Han, Tianxu; Stachura, David L; Wang, Huawei; Vaisman, Boris L; Kim, Jungsu; Klemke, Richard L; Remaley, Alan T; Rana, Tariq M; Traver, David; Miller, Yury I

2018-04-03

Lipoprotein lipase (LPL) mediates hydrolysis of triglycerides (TGs) to supply free fatty acids (FFAs) to tissues. Here, we show that LPL activity is also required for hematopoietic stem progenitor cell (HSPC) maintenance. Knockout of Lpl or its obligatory cofactor Apoc2 results in significantly reduced HSPC expansion during definitive hematopoiesis in zebrafish. A human APOC2 mimetic peptide or the human very low-density lipoprotein, which carries APOC2, rescues the phenotype in apoc2 but not in lpl mutant zebrafish. Creating parabiotic apoc2 and lpl mutant zebrafish rescues the hematopoietic defect in both. Docosahexaenoic acid (DHA) is identified as an important factor in HSPC expansion. FFA-DHA, but not TG-DHA, rescues the HSPC defects in apoc2 and lpl mutant zebrafish. Reduced blood cell counts are also observed in Apoc2 mutant mice at the time of weaning. These results indicate that LPL-mediated release of the essential fatty acid DHA regulates HSPC expansion and definitive hematopoiesis.

Targeted Mutants of Cochliobolus carbonum Lacking the Two Major Extracellular Polygalacturonases

PubMed Central

Scott-Craig, John S.; Cheng, Yi-Qiang; Cervone, Felice; De Lorenzo, Giulia; Pitkin, John W.; Walton, Jonathan D.

1998-01-01

The filamentous fungus Cochliobolus carbonum produces endo-α1,4-polygalacturonase (endoPG), exo-α1,4-polygalacturonase (exoPG), and pectin methylesterase when grown in culture on pectin. Residual activity in a pgn1 mutant (lacking endoPG) was due to exoPG activity, and the responsible protein has now been purified. After chemical deglycosylation, the molecular mass of the purified protein decreased from greater than 60 to 45 kDa. The gene that encodes exoPG, PGX1, was isolated with PCR primers based on peptide sequences from the protein. The product of PGX1, Pgx1p, has a predicted molecular mass of 48 kDa, 12 potential N-glycosylation sites, and 61% amino acid identity to an exoPG from the saprophytic fungus Aspergillus tubingensis. Strains of C. carbonum mutated in PGX1 were constructed by targeted gene disruption and by gene replacement. Growth of pgx1 mutant strains on pectin was reduced by ca. 20%, and they were still pathogenic on maize. A double pgn1/pgx1 mutant strain was constructed by crossing. The double mutant grew as well as the pgx1 single mutant on pectin and was still pathogenic despite having less than 1% of total wild-type PG activity. Double mutants retained a small amount of PG activity with the same cation-exchange retention time as Pgn1p and also pectin methylesterase and a PG activity associated with the mycelium. Continued growth of the pgn1/pgx1 mutant on pectin could be due to one or more of these residual activities. PMID:9546185

Cyclic adenosine monophosphate metabolism in synaptic growth, strength, and precision: neural and behavioral phenotype-specific counterbalancing effects between dnc phosphodiesterase and rut adenylyl cyclase mutations.

PubMed

Ueda, Atsushi; Wu, Chun-Fang

2012-03-01

Two classic learning mutants in Drosophila, rutabaga (rut) and dunce (dnc), are defective in cyclic adenosine monophosphate (cAMP) synthesis and degradation, respectively, exhibiting a variety of neuronal and behavioral defects. We ask how the opposing effects of these mutations on cAMP levels modify subsets of phenotypes, and whether any specific phenotypes could be ameliorated by biochemical counter balancing effects in dnc rut double mutants. Our study at larval neuromuscular junctions (NMJs) demonstrates that dnc mutations caused severe defects in nerve terminal morphology, characterized by unusually large synaptic boutons and aberrant innervation patterns. Interestingly, a counterbalancing effect led to rescue of the aberrant innervation patterns but the enlarged boutons in dnc rut double mutant remained as extreme as those in dnc. In contrast to dnc, rut mutations strongly affect synaptic transmission. Focal loose-patch recording data accumulated over 4 years suggest that synaptic currents in rut boutons were characterized by unusually large temporal dispersion and a seasonal variation in the amount of transmitter release, with diminished synaptic currents in summer months. Experiments with different rearing temperatures revealed that high temperature (29-30°C) decreased synaptic transmission in rut, but did not alter dnc and wild-type (WT). Importantly, the large temporal dispersion and abnormal temperature dependence of synaptic transmission, characteristic of rut, still persisted in dnc rut double mutants. To interpret these results in a proper perspective, we reviewed previously documented differential effects of dnc and rut mutations and their genetic interactions in double mutants on a variety of physiological and behavioral phenotypes. The cases of rescue in double mutants are associated with gradual developmental and maintenance processes whereas many behavioral and physiological manifestations on faster time scales could not be rescued. We discuss factors that could contribute to the effectiveness of counterbalancing interactions between dnc and rut mutations for phenotypic rescue.

Cyclic-AMP metabolism in synaptic growth, strength and precision: Neural and behavioral phenotype-specific counterbalancing effects between dnc PDE and rut AC mutations

PubMed Central

Ueda, Atsushi; Wu, Chun-Fang

2012-01-01

Two classic learning mutants in Drosophila, rutabaga (rut) and dunce (dnc), are defective in cAMP synthesis and degradation, respectively, exhibiting a variety of neuronal and behavioral defects. We ask how the opposing effects of these mutations on cAMP levels modify subsets of phenotypes, and whether any specific phenotypes could be ameliorated by biochemical counter balancing effects in dnc rut double mutants. Our study at larval neuromuscular junctions (NMJs) demonstrate that dnc mutations caused severe defects in nerve terminal morphology, characterized by unusually large synaptic boutons and aberrant innervation patterns. Interestingly, a counterbalancing effect led to rescue of the aberrant innervation patterns but the enlarged boutons in dnc rut double mutant remained as extreme as those in dnc. In contrast to dnc, rut mutations strongly affect synaptic transmission. Focal loose-patch recording data accumulated over 4 years suggest that synaptic currents in rut boutons were characterized by unusually large temporal dispersion and a seasonal variation in the amount of transmitter release, with diminished synaptic currents in summer months. Experiments with different rearing temperatures revealed that high temperature (29–30 °C) decreased synaptic transmission in rut, but did not alter dnc and WT. Importantly, the large temporal dispersion and abnormal temperature dependence of synaptic transmission, characteristic of rut, still persisted in dnc rut double mutants. To interpret these results in a proper perspective, we reviewed previously documented differential effects of dnc and rut mutations and their genetic interactions in double mutants on a variety of physiological and behavioral phenotypes. The cases of rescue in double mutants are associated with gradual developmental and maintenance processes whereas many behavioral and physiological manifestations on faster time scales could not be rescued. We discuss factors that could contribute to the effectiveness of counter balancing interactions between dnc and rut mutations for phenotypic rescue. PMID:22380612

Trichoderma-Plant Root Colonization: Escaping Early Plant Defense Responses and Activation of the Antioxidant Machinery for Saline Stress Tolerance

PubMed Central

Brotman, Yariv; Landau, Udi; Cuadros-Inostroza, Álvaro; Takayuki, Tohge; Fernie, Alisdair R.; Chet, Ilan; Viterbo, Ada; Willmitzer, Lothar

2013-01-01

Trichoderma spp. are versatile opportunistic plant symbionts which can colonize the apoplast of plant roots. Microarrays analysis of Arabidopsis thaliana roots inoculated with Trichoderma asperelloides T203, coupled with qPCR analysis of 137 stress responsive genes and transcription factors, revealed wide gene transcript reprogramming, proceeded by a transient repression of the plant immune responses supposedly to allow root colonization. Enhancement in the expression of WRKY18 and WRKY40, which stimulate JA-signaling via suppression of JAZ repressors and negatively regulate the expression of the defense genes FMO1, PAD3 and CYP71A13, was detected in Arabidopsis roots upon Trichoderma colonization. Reduced root colonization was observed in the wrky18/wrky40 double mutant line, while partial phenotypic complementation was achieved by over-expressing WRKY40 in the wrky18 wrky40 background. On the other hand increased colonization rate was found in roots of the FMO1 knockout mutant. Trichoderma spp. stimulate plant growth and resistance to a wide range of adverse environmental conditions. Arabidopsis and cucumber (Cucumis sativus L.) plants treated with Trichoderma prior to salt stress imposition show significantly improved seed germination. In addition, Trichoderma treatment affects the expression of several genes related to osmo-protection and general oxidative stress in roots of both plants. The MDAR gene coding for monodehydroascorbate reductase is significantly up-regulated and, accordingly, the pool of reduced ascorbic acid was found to be increased in Trichoderma treated plants. 1-Aminocyclopropane-1-carboxylate (ACC)-deaminase silenced Trichoderma mutants were less effective in providing tolerance to salt stress, suggesting that Trichoderma, similarly to ACC deaminase producing bacteria, can ameliorate plant growth under conditions of abiotic stress, by lowering ameliorating increases in ethylene levels as well as promoting an elevated antioxidative capacity. PMID:23516362

HRS1 acts as a negative regulator of abscisic acid signaling to promote timely germination of Arabidopsis seeds.

PubMed

Wu, Chongming; Feng, Juanjuan; Wang, Ran; Liu, Hong; Yang, Huixia; Rodriguez, Pedro L; Qin, Huanju; Liu, Xin; Wang, Daowen

2012-01-01

In this work, we conducted functional analysis of Arabidopsis HRS1 gene in order to provide new insights into the mechanisms governing seed germination. Compared with wild type (WT) control, HRS1 knockout mutant (hrs1-1) exhibited significant germination delays on either normal medium or those supplemented with abscisic acid (ABA) or sodium chloride (NaCl), with the magnitude of the delay being substantially larger on the latter media. The hypersensitivity of hrs1-1 germination to ABA and NaCl required ABI3, ABI4 and ABI5, and was aggravated in the double mutant hrs1-1abi1-2 and triple mutant hrs1-1hab1-1abi1-2, indicating that HRS1 acts as a negative regulator of ABA signaling during seed germination. Consistent with this notion, HRS1 expression was found in the embryo axis, and was regulated both temporally and spatially, during seed germination. Further analysis showed that the delay of hrs1-1 germination under normal conditions was associated with reduction in the elongation of the cells located in the lower hypocotyl (LH) and transition zone (TZ) of embryo axis. Interestingly, the germination rate of hrs1-1 was more severely reduced by the inhibitor of cell elongation, and more significantly decreased by the suppressors of plasmalemma H(+)-ATPase activity, than that of WT control. The plasmalemma H(+)-ATPase activity in the germinating seeds of hrs1-1 was substantially lower than that exhibited by WT control, and fusicoccin, an activator of this pump, corrected the transient germination delay of hrs1-1. Together, our data suggest that HRS1 may be needed for suppressing ABA signaling in germinating embryo axis, which promotes the timely germination of Arabidopsis seeds probably by facilitating the proper function of plasmalemma H(+)-ATPase and the efficient elongation of LH and TZ cells.

HRS1 Acts as a Negative Regulator of Abscisic Acid Signaling to Promote Timely Germination of Arabidopsis Seeds

PubMed Central

Wang, Ran; Liu, Hong; Yang, Huixia; Rodriguez, Pedro L.; Qin, Huanju; Liu, Xin; Wang, Daowen

2012-01-01

In this work, we conducted functional analysis of Arabidopsis HRS1 gene in order to provide new insights into the mechanisms governing seed germination. Compared with wild type (WT) control, HRS1 knockout mutant (hrs1-1) exhibited significant germination delays on either normal medium or those supplemented with abscisic acid (ABA) or sodium chloride (NaCl), with the magnitude of the delay being substantially larger on the latter media. The hypersensitivity of hrs1-1 germination to ABA and NaCl required ABI3, ABI4 and ABI5, and was aggravated in the double mutant hrs1-1abi1-2 and triple mutant hrs1-1hab1-1abi1-2, indicating that HRS1 acts as a negative regulator of ABA signaling during seed germination. Consistent with this notion, HRS1 expression was found in the embryo axis, and was regulated both temporally and spatially, during seed germination. Further analysis showed that the delay of hrs1-1 germination under normal conditions was associated with reduction in the elongation of the cells located in the lower hypocotyl (LH) and transition zone (TZ) of embryo axis. Interestingly, the germination rate of hrs1-1 was more severely reduced by the inhibitor of cell elongation, and more significantly decreased by the suppressors of plasmalemma H+-ATPase activity, than that of WT control. The plasmalemma H+-ATPase activity in the germinating seeds of hrs1-1 was substantially lower than that exhibited by WT control, and fusicoccin, an activator of this pump, corrected the transient germination delay of hrs1-1. Together, our data suggest that HRS1 may be needed for suppressing ABA signaling in germinating embryo axis, which promotes the timely germination of Arabidopsis seeds probably by facilitating the proper function of plasmalemma H+-ATPase and the efficient elongation of LH and TZ cells. PMID:22545134

OsHAC1;1 and OsHAC1;2 Function as Arsenate Reductases and Regulate Arsenic Accumulation1

PubMed Central

Wang, Tao; Tang, Zhong; Wu, Zhongchang; Salt, David E.; Chao, Dai-Yin

2016-01-01

Rice is a major dietary source of the toxic metalloid arsenic (As). Reducing its accumulation in rice (Oryza sativa) grain is of critical importance to food safety. Rice roots take up arsenate and arsenite depending on the prevailing soil conditions. The first step of arsenate detoxification is its reduction to arsenite, but the enzyme(s) catalyzing this reaction in rice remains unknown. Here, we identify OsHAC1;1 and OsHAC1;2 as arsenate reductases in rice. OsHAC1;1 and OsHAC1;2 are able to complement an Escherichia coli mutant lacking the endogenous arsenate reductase and to reduce arsenate to arsenite. OsHAC1:1 and OsHAC1;2 are predominantly expressed in roots, with OsHAC1;1 being abundant in the epidermis, root hairs, and pericycle cells while OsHAC1;2 is abundant in the epidermis, outer layers of cortex, and endodermis cells. Expression of the two genes was induced by arsenate exposure. Knocking out OsHAC1;1 or OsHAC1;2 decreased the reduction of arsenate to arsenite in roots, reducing arsenite efflux to the external medium. Loss of arsenite efflux was also associated with increased As accumulation in shoots. Greater effects were observed in a double mutant of the two genes. In contrast, overexpression of either OsHAC1;1 or OsHAC1;2 increased arsenite efflux, reduced As accumulation, and enhanced arsenate tolerance. When grown under aerobic soil conditions, overexpression of either OsHAC1;1 or OsHAC1;2 also decreased As accumulation in rice grain, whereas grain As increased in the knockout mutants. We conclude that OsHAC1;1 and OsHAC1;2 are arsenate reductases that play an important role in restricting As accumulation in rice shoots and grain. PMID:27702843

Molecular evidence of Zn chelation of the procaspase activating compound B-PAC-1 in B cell lymphoma.

PubMed

Sarkar, Aloke; Balakrishnan, Kumudha; Chen, Jefferson; Patel, Viralkumar; Neelapu, Sattva S; McMurray, John S; Gandhi, Varsha

2016-01-19

The resistance of apoptosis in cancer cells is pivotal for their survival and is typically ruled by mutations or dysregulation of core apoptotic cascade. Mantle cell lymphoma (MCL) is a non-Hodgkin's B-cell malignancy expressing higher anti-apoptotic proteins providing survival advantage. B-PAC-1, a procaspase activating compound, induces apoptosis by sequestering Zn bound to procaspase-3, but the amino acids holding Zn in Caspase-3 is not known. Here we show that reintroduction of WT caspase-3 or 7 in Caspase3-7 double knock-out (DKO) mouse embryonic fibroblasts (MEF) promoted B-PAC-1 to induce apoptosis (27-43%), but not in DKO MEFs or MEFs expressing respective Casp3-7 catalytic mutants (12-13%). Using caspase-6 and -9 exosite analysis, we identified and mutated predicted Zn-ligands in caspase-3 (H108A, C148S and E272A) and overexpressed into DKO MEFs. Mutants carrying E272A abrogated Zn-reversal of apoptosis induced by B-PAC-1 via higher XIAP and smac expressions but not in H108A or C148S mutants. Co-immunoprecipitation analysis revealed stronger XIAP-caspase-3 interaction suggesting a novel mechanism of impulsive apoptosis resistance by disrupting predicted Zn-ligands in caspase-3. B-PAC-1 sponsored apoptosis in MCL cell lines (30-73%) via caspase-3 and PARP cleavages accompanied by loss of Mcl-1 and IAPs including XIAP while Zn substantially abrogated B-PAC-1-driven apoptosis (18-36%). In contrary, Zn is dispensable to inhibit staurosporin, bendamustine, ABT199 or MK206-induced apoptosis. Consistent to cell lines, B-PAC-1 stimulated cell death in primary B-lymphoma cells via caspase-3 cleavage with decline in both Mcl-1 and XIAP. This study underscores the first genetic evidence that B-PAC-1 driven apoptosis is mediated via Zn chelation.

Prediction of reaction knockouts to maximize succinate production by Actinobacillus succinogenes

PubMed Central

Nag, Ambarish; St. John, Peter C.; Crowley, Michael F.

2018-01-01

Succinate is a precursor of multiple commodity chemicals and bio-based succinate production is an active area of industrial bioengineering research. One of the most important microbial strains for bio-based production of succinate is the capnophilic gram-negative bacterium Actinobacillus succinogenes, which naturally produces succinate by a mixed-acid fermentative pathway. To engineer A. succinogenes to improve succinate yields during mixed acid fermentation, it is important to have a detailed understanding of the metabolic flux distribution in A. succinogenes when grown in suitable media. To this end, we have developed a detailed stoichiometric model of the A. succinogenes central metabolism that includes the biosynthetic pathways for the main components of biomass—namely glycogen, amino acids, DNA, RNA, lipids and UDP-N-Acetyl-α-D-glucosamine. We have validated our model by comparing model predictions generated via flux balance analysis with experimental results on mixed acid fermentation. Moreover, we have used the model to predict single and double reaction knockouts to maximize succinate production while maintaining growth viability. According to our model, succinate production can be maximized by knocking out either of the reactions catalyzed by the PTA (phosphate acetyltransferase) and ACK (acetyl kinase) enzymes, whereas the double knockouts of PEPCK (phosphoenolpyruvate carboxykinase) and PTA or PEPCK and ACK enzymes are the most effective in increasing succinate production. PMID:29381705

Prediction of reaction knockouts to maximize succinate production by Actinobacillus succinogenes.

PubMed

Nag, Ambarish; St John, Peter C; Crowley, Michael F; Bomble, Yannick J

2018-01-01

Succinate is a precursor of multiple commodity chemicals and bio-based succinate production is an active area of industrial bioengineering research. One of the most important microbial strains for bio-based production of succinate is the capnophilic gram-negative bacterium Actinobacillus succinogenes, which naturally produces succinate by a mixed-acid fermentative pathway. To engineer A. succinogenes to improve succinate yields during mixed acid fermentation, it is important to have a detailed understanding of the metabolic flux distribution in A. succinogenes when grown in suitable media. To this end, we have developed a detailed stoichiometric model of the A. succinogenes central metabolism that includes the biosynthetic pathways for the main components of biomass-namely glycogen, amino acids, DNA, RNA, lipids and UDP-N-Acetyl-α-D-glucosamine. We have validated our model by comparing model predictions generated via flux balance analysis with experimental results on mixed acid fermentation. Moreover, we have used the model to predict single and double reaction knockouts to maximize succinate production while maintaining growth viability. According to our model, succinate production can be maximized by knocking out either of the reactions catalyzed by the PTA (phosphate acetyltransferase) and ACK (acetyl kinase) enzymes, whereas the double knockouts of PEPCK (phosphoenolpyruvate carboxykinase) and PTA or PEPCK and ACK enzymes are the most effective in increasing succinate production.

Cytotoxic Mechanisms Employed by Mouse T Cells to Destroy Pancreatic β-Cells

PubMed Central

Varanasi, Vineeth; Avanesyan, Lia; Schumann, Desiree M.; Chervonsky, Alexander V.

2012-01-01

Several cytotoxic mechanisms have been attributed to T cells participating in β-cell death in type 1 diabetes. However, sensitivity of β-cells to these mechanisms in vitro and in vivo is likely to be different. Moreover, CD4+ and CD8+ T cells may use distinct mechanisms to cause β-cell demise that possibly involve activation of third-party cytotoxic cells. We used the transfer of genetically modified diabetogenic T cells into normal, mutant, and bone marrow chimeric recipients to test the contribution of major cytotoxic mechanisms in β-cell death. We found that 1) the killing of β-cells by CD4+ T cells required activation of the recipient’s own cytotoxic cells via tumor necrosis factor-α (TNF-α); 2) CD8+ T-cell cytotoxic mechanisms destroying β-cells were limited to perforin and Fas ligand, as double knockouts of these molecules abrogated the ability of T cells to cause diabetes; and 3) individual CD8+ T-cell clones chose their cytotoxic weaponry by a yet unknown mechanism and destroyed their targets via either Fas-independent or Fas-dependent (∼40% of clones) pathways. Fas-dependent destruction was assisted by TNF-α. PMID:22773667

FGF-dependent metabolic control of vascular development

PubMed Central

Yu, Pengchun; Alves, Tiago C.; Fang, Jennifer S.; Xie, Yi; Zhu, Jie; Chen, Zehua; De Smet, Frederik; Zhang, Jiasheng; Jin, Suk-Won; Sun, Lele; Sun, Hongye; Kibbey, Richard G.; Hirschi, Karen K.; Hay, Nissim; Carmeliet, Peter; Chittenden, Thomas W.; Eichmann, Anne; Potente, Michael; Simons, Michael

2017-01-01

Blood and lymphatic vasculatures are intimately involved in tissue oxygenation and fluid homeostasis maintenance. Assembly of these vascular networks involves sprouting, migration and proliferation of endothelial cells. Recent studies have suggested that changes in cellular metabolism are of importance to these processes1. While much is known about vascular endothelial growth factor (VEGF)-dependent regulation of vascular development and metabolism2,3, little is understood about the role of fibroblast growth factors (FGFs) in this context4. Here we identify FGF receptor (FGFR) signaling as a critical regulator of vascular development. This is achieved by FGF-dependent control of c-MYC (MYC) expression that, in turn, regulates expression of the glycolytic enzyme hexokinase 2 (HK2). A decrease in HK2 levels in the absence of FGF signaling inputs results in decreased glycolysis leading to impaired endothelial cell proliferation and migration. Pan-endothelial- and lymphatic-specific Hk2 knockouts phenocopy blood and/or lymphatic vascular defects seen in Fgfr1/r3 double mutant mice while HK2 overexpression partially rescues the defects caused by suppression of FGF signaling. Thus, FGF-dependent regulation of endothelial glycolysis is a pivotal process in developmental and adult vascular growth and development. PMID:28467822

FGF-dependent metabolic control of vascular development.

PubMed

Yu, Pengchun; Wilhelm, Kerstin; Dubrac, Alexandre; Tung, Joe K; Alves, Tiago C; Fang, Jennifer S; Xie, Yi; Zhu, Jie; Chen, Zehua; De Smet, Frederik; Zhang, Jiasheng; Jin, Suk-Won; Sun, Lele; Sun, Hongye; Kibbey, Richard G; Hirschi, Karen K; Hay, Nissim; Carmeliet, Peter; Chittenden, Thomas W; Eichmann, Anne; Potente, Michael; Simons, Michael

2017-05-11

Blood and lymphatic vasculatures are intimately involved in tissue oxygenation and fluid homeostasis maintenance. Assembly of these vascular networks involves sprouting, migration and proliferation of endothelial cells. Recent studies have suggested that changes in cellular metabolism are important to these processes. Although much is known about vascular endothelial growth factor (VEGF)-dependent regulation of vascular development and metabolism, little is understood about the role of fibroblast growth factors (FGFs) in this context. Here we identify FGF receptor (FGFR) signalling as a critical regulator of vascular development. This is achieved by FGF-dependent control of c-MYC (MYC) expression that, in turn, regulates expression of the glycolytic enzyme hexokinase 2 (HK2). A decrease in HK2 levels in the absence of FGF signalling inputs results in decreased glycolysis, leading to impaired endothelial cell proliferation and migration. Pan-endothelial- and lymphatic-specific Hk2 knockouts phenocopy blood and/or lymphatic vascular defects seen in Fgfr1/Fgfr3 double mutant mice, while HK2 overexpression partly rescues the defects caused by suppression of FGF signalling. Thus, FGF-dependent regulation of endothelial glycolysis is a pivotal process in developmental and adult vascular growth and development.

Regulatory function of Arabidopsis lipid transfer protein 1 (LTP1) in ethylene response and signaling.

PubMed

Wang, Honglin; Sun, Yue; Chang, Jianhong; Zheng, Fangfang; Pei, Haixia; Yi, Yanjun; Chang, Caren; Dong, Chun-Hai

2016-07-01

Ethylene as a gaseous plant hormone is directly involved in various processes during plant growth and development. Much is known regarding the ethylene receptors and regulatory factors in the ethylene signal transduction pathway. In Arabidopsis thaliana, REVERSION-TO-ETHYLENE SENSITIVITY1 (RTE1) can interact with and positively regulates the ethylene receptor ETHYLENE RESPONSE1 (ETR1). In this study we report the identification and characterization of an RTE1-interacting protein, a putative Arabidopsis lipid transfer protein 1 (LTP1) of unknown function. Through bimolecular fluorescence complementation, a direct molecular interaction between LTP1 and RTE1 was verified in planta. Analysis of an LTP1-GFP fusion in transgenic plants and plasmolysis experiments revealed that LTP1 is localized to the cytoplasm. Analysis of ethylene responses showed that the ltp1 knockout is hypersensitive to 1-aminocyclopropanecarboxylic acid (ACC), while LTP1 overexpression confers insensitivity. Analysis of double mutants etr1-2 ltp1 and rte1-3 ltp1 demonstrates a regulatory function of LTP1 in ethylene receptor signaling through the molecular association with RTE1. This study uncovers a novel function of Arabidopsis LTP1 in the regulation of ethylene response and signaling.

Synapsis-defective mutants reveal a correlation between chromosome conformation and the mode of double-strand break repair during Caenorhabditis elegans meiosis.

PubMed

Smolikov, Sarit; Eizinger, Andreas; Hurlburt, Allison; Rogers, Eric; Villeneuve, Anne M; Colaiácovo, Mónica P

2007-08-01

SYP-3 is a new structural component of the synaptonemal complex (SC) required for the regulation of chromosome synapsis. Both chromosome morphogenesis and nuclear organization are altered throughout the germlines of syp-3 mutants. Here, our analysis of syp-3 mutants provides insights into the relationship between chromosome conformation and the repair of meiotic double-strand breaks (DSBs). Although crossover recombination is severely reduced in syp-3 mutants, the production of viable offspring accompanied by the disappearance of RAD-51 foci suggests that DSBs are being repaired in these synapsis-defective mutants. Our studies indicate that once interhomolog recombination is impaired, both intersister recombination and nonhomologous end-joining pathways may contribute to repair during germline meiosis. Moreover, our studies suggest that the conformation of chromosomes may influence the mode of DSB repair employed during meiosis.

CDK10 Mutations in Humans and Mice Cause Severe Growth Retardation, Spine Malformations, and Developmental Delays.

PubMed

Windpassinger, Christian; Piard, Juliette; Bonnard, Carine; Alfadhel, Majid; Lim, Shuhui; Bisteau, Xavier; Blouin, Stéphane; Ali, Nur'Ain B; Ng, Alvin Yu Jin; Lu, Hao; Tohari, Sumanty; Talib, S Zakiah A; van Hul, Noémi; Caldez, Matias J; Van Maldergem, Lionel; Yigit, Gökhan; Kayserili, Hülya; Youssef, Sameh A; Coppola, Vincenzo; de Bruin, Alain; Tessarollo, Lino; Choi, Hyungwon; Rupp, Verena; Roetzer, Katharina; Roschger, Paul; Klaushofer, Klaus; Altmüller, Janine; Roy, Sudipto; Venkatesh, Byrappa; Ganger, Rudolf; Grill, Franz; Ben Chehida, Farid; Wollnik, Bernd; Altunoglu, Umut; Al Kaissi, Ali; Reversade, Bruno; Kaldis, Philipp

2017-09-07

In five separate families, we identified nine individuals affected by a previously unidentified syndrome characterized by growth retardation, spine malformation, facial dysmorphisms, and developmental delays. Using homozygosity mapping, array CGH, and exome sequencing, we uncovered bi-allelic loss-of-function CDK10 mutations segregating with this disease. CDK10 is a protein kinase that partners with cyclin M to phosphorylate substrates such as ETS2 and PKN2 in order to modulate cellular growth. To validate and model the pathogenicity of these CDK10 germline mutations, we generated conditional-knockout mice. Homozygous Cdk10-knockout mice died postnatally with severe growth retardation, skeletal defects, and kidney and lung abnormalities, symptoms that partly resemble the disease's effect in humans. Fibroblasts derived from affected individuals and Cdk10-knockout mouse embryonic fibroblasts (MEFs) proliferated normally; however, Cdk10-knockout MEFs developed longer cilia. Comparative transcriptomic analysis of mutant and wild-type mouse organs revealed lipid metabolic changes consistent with growth impairment and altered ciliogenesis in the absence of CDK10. Our results document the CDK10 loss-of-function phenotype and point to a function for CDK10 in transducing signals received at the primary cilia to sustain embryonic and postnatal development. Copyright © 2017 American Society of Human Genetics. All rights reserved.

An FBXO40 knockout generated by CRISPR/Cas9 causes muscle hypertrophy in pigs without detectable pathological effects.

PubMed

Zou, Yunlong; Li, Zhiyuan; Zou, Yunjing; Hao, Haiyang; Li, Ning; Li, Qiuyan

2018-04-15

The regulatory function of Fbxo40 has been well characterized in mice. As a key component of the SCF-E3 ubiquitin ligase complex, Fbxo40 induces IRS1 ubiquitination, thus inactivating the IGF1/Akt pathway. The expression of Fbxo40 is restricted to muscle, and mice with an Fbxo40 null mutation exhibit muscle hypertrophy. However, the function of FBXO40 has not been elucidated in pigs, and it is not known whether FBXO40 mutations affect their health. We therefore generated FBXO40 knockout pigs using somatic cell nuclear transfer (SCNT) technology. CRISPR/Cas9 technology was combined with G418 selection, making it possible to generate donor cells at an efficiency of 75.86%. In muscle from FBXO40 knockout pigs, IRS1 levels were higher, and the IGF1/Akt pathway was stimulated. Mutant animals also had approximately 4% more muscle mass compared to WT controls. The knockout pigs developed normally and no pathological changes were found in major organs. These results demonstrate that FBXO40 is a promising candidate gene for improving production traits in agricultural livestock and for developing therapeutic interventions for muscle diseases. Copyright © 2018. Published by Elsevier Inc.

Stress Marker Signatures in Lesion Mimic Single and Double Mutants Identify a Crucial Leaf Age-Dependent Salicylic Acid Related Defense Signal.

PubMed

Kaurilind, Eve; Brosché, Mikael

2017-01-01

Plants are exposed to abiotic and biotic stress conditions throughout their lifespans that activates various defense programs. Programmed cell death (PCD) is an extreme defense strategy the plant uses to manage unfavorable environments as well as during developmentally induced senescence. Here we investigated the role of leaf age on the regulation of defense gene expression in Arabidopsis thaliana. Two lesion mimic mutants with misregulated cell death, catalase2 (cat2) and defense no death1 (dnd1) were used together with several double mutants to dissect signaling pathways regulating defense gene expression associated with cell death and leaf age. PCD marker genes showed leaf age dependent expression, with the highest expression in old leaves. The salicylic acid (SA) biosynthesis mutant salicylic acid induction deficient2 (sid2) had reduced expression of PCD marker genes in the cat2 sid2 double mutant demonstrating the importance of SA biosynthesis in regulation of defense gene expression. While the auxin- and jasmonic acid (JA)- insensitive auxin resistant1 (axr1) double mutant cat2 axr1 also led to decreased expression of PCD markers; the expression of several marker genes for SA signaling (ISOCHORISMATE SYNTHASE 1, PR1 and PR2) were additionally decreased in cat2 axr1 compared to cat2. The reduced expression of these SA markers genes in cat2 axr1 implicates AXR1 as a regulator of SA signaling in addition to its known role in auxin and JA signaling. Overall, the current study reinforces the important role of SA signaling in regulation of leaf age-related transcript signatures.

A Pseudomonas putida double mutant deficient in butanol assimilation: a promising step for engineering a biological biofuel production platform.

PubMed

Cuenca, María Del Sol; Molina-Santiago, Carlos; Gómez-García, María R; Ramos, Juan L

2016-03-01

Biological production in heterologous hosts is of interest for the production of the C4 alcohol (butanol) and other chemicals. However, some hurdles need to be overcome in order to achieve an economically viable process; these include avoiding the consumption of butanol and maintaining tolerance to this solvent during production. Pseudomonas putida is a potential host for solvent production; in order to further adapt P. putida to this role, we generated mini-Tn5 mutant libraries in strain BIRD-1 that do not consume butanol. We analyzed the insertion site of the mini-Tn5 in a mutant that was deficient in assimilation of butanol using arbitrary PCR followed by Sanger sequencing and found that the transposon was inserted in the malate synthase B gene. Here, we show that in a second round of mutagenesis a double mutant unable to take up butanol had an insertion in a gene coding for a multisensor hybrid histidine kinase. The genetic context of the histidine kinase sensor revealed the presence of a set of genes potentially involved in butanol assimilation; qRT-PCR analysis showed induction of this set of genes in the wild type and the malate synthase mutant but not in the double mutant. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Programming the quorum sensing-based AND gate in Shewanella oneidensis for logic gated-microbial fuel cells.

PubMed

Hu, Yidan; Yang, Yun; Katz, Evgeny; Song, Hao

2015-03-11

An AND logic gate based on a synthetic quorum-sensing (QS) module was constructed in a Shewanella oneidensis MR-1 mtrA knockout mutant. The presence of two input signals activated the expression of a periplasmic decaheme cytochrome MtrA to regenerate the extracellular electron transfer conduit, enabling the construction of AND-gated microbial fuel cells.

Quorum-sensing contributes to virulence, twitching motility, seed attachment and biofilm formation in the wild type strain Aac-5 of Acidovorax citrulli

USDA-ARS?s Scientific Manuscript database

Acidovorax citrulli is a seed-borne pathogen that causes bacterial fruit blotch of cucurbits including melon and watermelon. We investigated the roles of quorum sensing in the wild-type group II strain Aac-5 of A. citrulli by generating aacR and aacI knockout mutants and their complementation strain...

Contribution of Egr1/zif268 to Activity-Dependent Arc/Arg3.1 Transcription in the Dentate Gyrus and Area CA1 of the Hippocampus

PubMed Central

Penke, Zsuzsa; Chagneau, Carine; Laroche, Serge

2011-01-01

Egr1, a member of the Egr family of transcription factors, and Arc are immediate early genes known to play major roles in synaptic plasticity and memory. Despite evidence that Egr family members can control Arc transcriptional regulation, demonstration of a selective role of Egr1 alone is lacking. We investigated the extent to which activity-dependent Arc expression is dependent on Egr1 by analyzing Arc mRNA expression using fluorescence in situ hybridization in the dorsal dentate gyrus and CA1 of wild-type (WT) and Egr1 knockout mice. Following electroconvulsive shock, we found biphasic expression of Arc in area CA1 in mice, consisting in a rapid (30 min) and transient wave followed by a second late-phase of expression (8 h), and a single but prolonged wave of expression in the dentate gyrus. Egr1 deficiency abolished the latest, but not the early wave of Arc expression in CA1, and curtailed that of the dentate gyrus. Since the early wave of Arc expression was not affected in Egr1 mutant mice, we next analyzed behaviorally induced Arc expression patterns as an index of neural ensemble activation in the dentate gyrus and area CA1 of WT and Egr1 mutant mice. Spatial exploration of novel or familiar environments induced in mice a single early and transient wave of Arc expression in the dentate gyrus and area CA1, which were not affected in Egr1 mutant mice. Analyses of Arc-expressing cells revealed that exploration recruits similar size dentate gyrus and CA1 neural ensembles in WT and Egr1 knockout mice. These findings suggest that hippocampal neural ensembles are normally activated immediately following spatial exploration in Egr1 knockout mice, indicating normal hippocampal encoding of information. They also provide evidence that in condition of strong activation Egr1 alone can control late-phases of activity-dependent Arc transcription in the dentate gyrus and area CA1 of the hippocampus. PMID:21887136

Postsynaptic P2X3-containing receptors in gustatory nerve fibres mediate responses to all taste qualities in mice.

PubMed

Vandenbeuch, Aurelie; Larson, Eric D; Anderson, Catherine B; Smith, Steven A; Ford, Anthony P; Finger, Thomas E; Kinnamon, Sue C

2015-03-01

Taste buds release ATP to activate ionotropic purinoceptors composed of P2X2 and P2X3 subunits, present on the taste nerves. Mice with genetic deletion of P2X2 and P2X3 receptors (double knockout mice) lack responses to all taste stimuli presumably due to the absence of ATP-gated receptors on the afferent nerves. Recent experiments on the double knockout mice showed, however, that their taste buds fail to release ATP, suggesting the possibility of pleiotropic deficits in these global knockouts. To test further the role of postsynaptic P2X receptors in afferent signalling, we used AF-353, a selective antagonist of P2X3-containing receptors to inhibit the receptors acutely during taste nerve recording and behaviour. The specificity of AF-353 for P2X3-containing receptors was tested by recording Ca(2+) transients to exogenously applied ATP in fura-2 loaded isolated geniculate ganglion neurons from wild-type and P2X3 knockout mice. ATP responses were completely inhibited by 10 μm or 100 μm AF-353, but neither concentration blocked responses in P2X3 single knockout mice wherein the ganglion cells express only P2X2-containing receptors. Furthermore, AF-353 had no effect on taste-evoked ATP release from taste buds. In wild-type mice, i.p. injection of AF-353 or simple application of the drug directly to the tongue, inhibited taste nerve responses to all taste qualities in a dose-dependent fashion. A brief access behavioural assay confirmed the electrophysiological results and showed that preference for a synthetic sweetener, SC-45647, was abolished following i.p. injection of AF-353. These data indicate that activation of P2X3-containing receptors is required for transmission of all taste qualities. © 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.

Dietary phosphate restriction normalizes biochemical and skeletal abnormalities in a murine model of tumoral calcinosis.

PubMed

Ichikawa, Shoji; Austin, Anthony M; Gray, Amie K; Allen, Matthew R; Econs, Michael J

2011-12-01

Mutations in the GALNT3 gene cause tumoral calcinosis characterized by ectopic calcifications due to persistent hyperphosphatemia. We recently developed Galnt3 knockout mice in a mixed background, which had hyperphosphatemia with increased bone mineral density (BMD) and infertility in males. To test the effect of dietary phosphate intake on their phenotype, Galnt3 knockout mice were generated in the C57BL/6J strain and fed various phosphate diets: 0.1% (low), 0.3% (low normal), 0.6% (normal), and 1.65% (high). Sera were analyzed for calcium, phosphorus, alkaline phosphatase, creatinine, blood urine nitrogen, 1,25-dihydroxyvitamin D, osteocalcin, tartrate-resistant acid phosphatase 5b, and fibroblast growth factor 23 (Fgf23). Femurs were evaluated by dual-energy x-ray absorptiometry, dynamic histomorphometry, and/or microcomputed tomography. Galnt3 knockout mice in C57BL/6J had the same biochemical phenotype observed in our previous study: hyperphosphatemia, inappropriately normal 1,25-dihydroxyvitamin D level, decreased alkaline phosphatase activity, and low intact Fgf23 concentration but high Fgf23 fragments. Skeletal analyses of their femurs revealed significantly high BMD with increased cortical bone area and trabecular bone volume. On all four phosphate diets, Galnt3 knockout mice had consistently higher phosphorus levels and lower alkaline phosphatase and intact Fgf23 concentrations than littermate controls. The low-phosphate diet normalized serum phosphorus, alkaline phosphatase, and areal BMD but failed to correct male infertility in Galnt3 knockout mice. The high-phosphate diet did not increase serum phosphorus concentration in either mutant or control mice due to a compensatory increase in circulating intact Fgf23 levels. In conclusion, dietary phosphate restriction normalizes biochemical and skeletal phenotypes of Galnt3 knockout mice and, thus, can be an effective therapy for tumoral calcinosis.

Dietary Phosphate Restriction Normalizes Biochemical and Skeletal Abnormalities in a Murine Model of Tumoral Calcinosis

PubMed Central

Austin, Anthony M.; Gray, Amie K.; Allen, Matthew R.; Econs, Michael J.

2011-01-01

Mutations in the GALNT3 gene cause tumoral calcinosis characterized by ectopic calcifications due to persistent hyperphosphatemia. We recently developed Galnt3 knockout mice in a mixed background, which had hyperphosphatemia with increased bone mineral density (BMD) and infertility in males. To test the effect of dietary phosphate intake on their phenotype, Galnt3 knockout mice were generated in the C57BL/6J strain and fed various phosphate diets: 0.1% (low), 0.3% (low normal), 0.6% (normal), and 1.65% (high). Sera were analyzed for calcium, phosphorus, alkaline phosphatase, creatinine, blood urine nitrogen, 1,25-dihydroxyvitamin D, osteocalcin, tartrate-resistant acid phosphatase 5b, and fibroblast growth factor 23 (Fgf23). Femurs were evaluated by dual-energy x-ray absorptiometry, dynamic histomorphometry, and/or microcomputed tomography. Galnt3 knockout mice in C57BL/6J had the same biochemical phenotype observed in our previous study: hyperphosphatemia, inappropriately normal 1,25-dihydroxyvitamin D level, decreased alkaline phosphatase activity, and low intact Fgf23 concentration but high Fgf23 fragments. Skeletal analyses of their femurs revealed significantly high BMD with increased cortical bone area and trabecular bone volume. On all four phosphate diets, Galnt3 knockout mice had consistently higher phosphorus levels and lower alkaline phosphatase and intact Fgf23 concentrations than littermate controls. The low-phosphate diet normalized serum phosphorus, alkaline phosphatase, and areal BMD but failed to correct male infertility in Galnt3 knockout mice. The high-phosphate diet did not increase serum phosphorus concentration in either mutant or control mice due to a compensatory increase in circulating intact Fgf23 levels. In conclusion, dietary phosphate restriction normalizes biochemical and skeletal phenotypes of Galnt3 knockout mice and, thus, can be an effective therapy for tumoral calcinosis. PMID:22009723

A homozygous Keap1-knockout human embryonic stem cell line generated using CRISPR/Cas9 mediates gene targeting.

PubMed

Kim, So-Jung; Habib, Omer; Kim, Jin-Soo; Han, Hyo-Won; Koo, Soo Kyung; Kim, Jung-Hyun

2017-03-01

Kelch-like ECH-associated protein 1 (keap1) is a cysteine-rich protein that interacts with transcription factor Nrf2 in a redox-sensitive manner, leading to the degradation of Nrf2 (Kim et al., 2014a). Disruption of Keap1 results in the induction of Nrf2-related signaling pathways involving the expression of a set of anti-oxidant and anti-inflammatory genes. We generated biallelic mutants of the Keap1 gene using a CRISPR-Cas9 genome editing method in the H9 human embryonic stem cell (hESC). The Keap1 homozygous-knockout H9 cell line retained normal morphology, gene expression, and in vivo differentiation potential. Copyright © 2016 The Author(s). Published by Elsevier B.V. All rights reserved.

Double knockout of pendrin and Na-Cl cotransporter (NCC) causes severe salt wasting, volume depletion, and renal failure.

PubMed

Soleimani, Manoocher; Barone, Sharon; Xu, Jie; Shull, Gary E; Siddiqui, Faraz; Zahedi, Kamyar; Amlal, Hassane

2012-08-14

The Na-Cl cotransporter (NCC), which is the target of inhibition by thiazides, is located in close proximity to the chloride-absorbing transporter pendrin in the kidney distal nephron. Single deletion of pendrin or NCC does not cause salt wasting or excessive diuresis under basal conditions, raising the possibility that these transporters are predominantly active during salt depletion or in response to excess aldosterone. We hypothesized that pendrin and NCC compensate for loss of function of the other under basal conditions, thereby masking the role that each plays in salt absorption. To test our hypothesis, we generated pendrin/NCC double knockout (KO) mice by crossing pendrin KO mice with NCC KO mice. Pendrin/NCC double KO mice displayed severe salt wasting and sharp increase in urine output under basal conditions. As a result, animals developed profound volume depletion, renal failure, and metabolic alkalosis without hypokalemia, which were all corrected with salt replacement. We propose that the combined inhibition of pendrin and NCC can provide a strong diuretic regimen without causing hypokalemia for patients with fluid overload, including patients with congestive heart failure, nephrotic syndrome, diuretic resistance, or generalized edema.

Double knockout of pendrin and Na-Cl cotransporter (NCC) causes severe salt wasting, volume depletion, and renal failure

PubMed Central

Soleimani, Manoocher; Barone, Sharon; Xu, Jie; Shull, Gary E.; Siddiqui, Faraz; Zahedi, Kamyar; Amlal, Hassane

2012-01-01

The Na-Cl cotransporter (NCC), which is the target of inhibition by thiazides, is located in close proximity to the chloride-absorbing transporter pendrin in the kidney distal nephron. Single deletion of pendrin or NCC does not cause salt wasting or excessive diuresis under basal conditions, raising the possibility that these transporters are predominantly active during salt depletion or in response to excess aldosterone. We hypothesized that pendrin and NCC compensate for loss of function of the other under basal conditions, thereby masking the role that each plays in salt absorption. To test our hypothesis, we generated pendrin/NCC double knockout (KO) mice by crossing pendrin KO mice with NCC KO mice. Pendrin/NCC double KO mice displayed severe salt wasting and sharp increase in urine output under basal conditions. As a result, animals developed profound volume depletion, renal failure, and metabolic alkalosis without hypokalemia, which were all corrected with salt replacement. We propose that the combined inhibition of pendrin and NCC can provide a strong diuretic regimen without causing hypokalemia for patients with fluid overload, including patients with congestive heart failure, nephrotic syndrome, diuretic resistance, or generalized edema. PMID:22847418

Functional PAK-2 knockout and replacement with a caspase cleavage-deficient mutant in mice reveals differential requirements of full-length PAK-2 and caspase-activated PAK-2p34.

PubMed

Marlin, Jerry W; Chang, Yu-Wen E; Ober, Margaret; Handy, Amy; Xu, Wenhao; Jakobi, Rolf

2011-06-01

p21-Activated protein kinase 2 (PAK-2) has both anti- and pro-apoptotic functions depending on its mechanism of activation. Activation of full-length PAK-2 by the monomeric GTPases Cdc42 or Rac stimulates cell survival, whereas caspase activation of PAK-2 to the PAK-2p34 fragment is involved in the apoptotic response. In this study we use functional knockout of PAK-2 and gene replacement with the caspase cleavage-deficient PAK-2D212N mutant to differentiate the biological functions of full-length PAK-2 and caspase-activated PAK-2p34. Knockout of PAK-2 results in embryonic lethality at early stages before organ development, whereas replacement with the caspase cleavage-deficient PAK-2D212N results in viable and healthy mice, indicating that early embryonic lethality is caused by deficiency of full-length PAK-2 rather than lack of caspase activation to the PAK-2p34 fragment. However, deficiency of caspase activation of PAK-2 decreased spontaneous cell death of primary mouse embryonic fibroblasts and increased cell growth at high cell density. In contrast, stress-induced cell death by treatment with the anti-cancer drug cisplatin was not reduced by deficiency of caspase activation of PAK-2, but switched from an apoptotic to a nonapoptotic, caspase-independent mechanism. Homozygous PAK-2D212N primary mouse embryonic fibroblasts that lack the ability to generate the proapoptotic PAK-2p34 show less activation of the effector caspase 3, 6, and 7, indicating that caspase activation of PAK-2 amplifies the apoptotic response through a positive feedback loop resulting in more activation of effector caspases.

Inactivation of p53 by Human T-Cell Lymphotropic Virus Type 1 Tax Requires Activation of the NF-κB Pathway and Is Dependent on p53 Phosphorylation

PubMed Central

Pise-Masison, Cynthia A.; Mahieux, Renaud; Jiang, Hua; Ashcroft, Margaret; Radonovich, Michael; Duvall, Janet; Guillerm, Claire; Brady, John N.

2000-01-01

p53 plays a key role in guarding cells against DNA damage and transformation. We previously demonstrated that the human T-cell lymphotropic virus type 1 (HTLV-1) Tax can inactivate p53 transactivation function in lymphocytes. The present study demonstrates that in T cells, Tax-induced p53 inactivation is dependent upon NF-κB activation. Analysis of Tax mutants demonstrated that Tax inactivation of p53 function correlates with the ability of Tax to induce NF-κB but not p300 binding or CREB transactivation. The Tax-induced p53 inactivation can be overcome by overexpression of a dominant IκB mutant. Tax-NF-κB-induced p53 inactivation is not due to p300 squelching, since overexpression of p300 does not recover p53 activity in the presence of Tax. Further, using wild-type and p65 knockout mouse embryo fibroblasts (MEFs), we demonstrate that the p65 subunit of NF-κB is critical for Tax-induced p53 inactivation. While Tax can inactivate endogenous p53 function in wild-type MEFs, it fails to inactivate p53 function in p65 knockout MEFs. Importantly, Tax-induced p53 inactivation can be restored by expression of p65 in the knockout MEFs. Finally, we present evidence that phosphorylation of serines 15 and 392 correlates with inactivation of p53 by Tax in T cells. This study provides evidence that the divergent NF-κB proliferative and p53 cell cycle arrest pathways may be cross-regulated at several levels, including posttranslational modification of p53. PMID:10779327

Enhanced hexose fermentation by Saccharomyces cerevisiae through integration of stoichiometric modeling and genetic screening.

PubMed

Quarterman, Josh; Kim, Soo Rin; Kim, Pan-Jun; Jin, Yong-Su

2015-01-20

In order to determine beneficial gene deletions for ethanol production by the yeast Saccharomyces cerevisiae, we performed an in silico gene deletion experiment based on a genome-scale metabolic model. Genes coding for two oxidative phosphorylation reactions (cytochrome c oxidase and ubiquinol cytochrome c reductase) were identified by the model-based simulation as potential deletion targets for enhancing ethanol production and maintaining acceptable overall growth rate in oxygen-limited conditions. Since the two target enzymes are composed of multiple subunits, we conducted a genetic screening study to evaluate the in silico results and compare the effect of deleting various portions of the respiratory enzyme complexes. Over two-thirds of the knockout mutants identified by the in silico study did exhibit experimental behavior in qualitative agreement with model predictions, but the exceptions illustrate the limitation of using a purely stoichiometric model-based approach. Furthermore, there was a substantial quantitative variation in phenotype among the various respiration-deficient mutants that were screened in this study, and three genes encoding respiratory enzyme subunits were identified as the best knockout targets for improving hexose fermentation in microaerobic conditions. Specifically, deletion of either COX9 or QCR9 resulted in higher ethanol production rates than the parental strain by 37% and 27%, respectively, with slight growth disadvantages. Also, deletion of QCR6 led to improved ethanol production rate by 24% with no growth disadvantage. The beneficial effects of these gene deletions were consistently demonstrated in different strain backgrounds and with four common hexoses. The combination of stoichiometric modeling and genetic screening using a systematic knockout collection was useful for narrowing a large set of gene targets and identifying targets of interest. Copyright © 2014 Elsevier B.V. All rights reserved.

Metalloproteins and phytochelatin synthase may confer protection against zinc oxide nanoparticle induced toxicity in Caenorhabditis elegans.

PubMed

Polak, Natasa; Read, Daniel S; Jurkschat, Kerstin; Matzke, Marianne; Kelly, Frank J; Spurgeon, David J; Stürzenbaum, Stephen R

2014-03-01

Zinc oxide nanoparticles (ZnONPs) are used in large quantities by the cosmetic, food and textile industries. Here we exposed Caenorhabditis elegans wild-type and a metal sensitive triple knockout mutant (mtl-1;mtl-2;pcs-1) to ZnONPs (0-50mg/L) to study strain and exposure specific effects on transcription, reactive oxygen species generation, the biomolecular phenotype (measured by Raman microspectroscopy) and key endpoints of the nematode life cycle (growth, reproduction and lifespan). A significant dissolution effect was observed, where dissolved ZnO constituted over 50% of total Zn within a two day exposure to the test medium, suggesting that the nominal exposure to pure ZnONPs represents in vivo, at best, a mixture exposure of ionic zinc and nanoparticles. Nevertheless, the analyses provided evidence that the metallothioneins (mtl-1 and mtl-2), the phytochelatin synthase (pcs-1) and an apoptotic marker (cep-1) were transcriptionally activated. In addition, the DCFH-DA assay provided in vitro evidence of the oxidative potential of ZnONPs in the metal exposure sensitive triple mutant. Raman spectroscopy highlighted that the biomolecular phenotype changes significantly in the mtl-1;mtl-2;pcs-1 triple knockout worm upon ZnONP exposure, suggesting that these metalloproteins are instrumental in the protection against cytotoxic damage. Finally, ZnONP exposure was shown to decrease growth and development, reproductive capacity and lifespan, effects which were amplified in the triple knockout. By combining diverse toxicological strategies, we identified that individuals (genotypes) housing mutations in key metalloproteins and phytochelatin synthase are more susceptible to ZnONP exposure, which underlines their importance to minimize ZnONP induced toxicity. Copyright © 2013 Elsevier Inc. All rights reserved.

Non-Aggregating Tau Phosphorylation by Cyclin-Dependent Kinase 5 Contributes to Motor Neuron Degeneration in Spinal Muscular Atrophy

PubMed Central

Miller, Nimrod; Feng, Zhihua; Edens, Brittany M.; Yang, Ben; Shi, Han; Sze, Christie C.; Hong, Benjamin Taige; Su, Susan C.; Cantu, Jorge A.; Topczewski, Jacek; Crawford, Thomas O.; Ko, Chien-Ping; Sumner, Charlotte J.; Ma, Long

2015-01-01

Mechanisms underlying motor neuron degeneration in spinal muscular atrophy (SMA), the leading inherited cause of infant mortality, remain largely unknown. Many studies have established the importance of hyperphosphorylation of the microtubule-associated protein tau in various neurodegenerative disorders, including Alzheimer's and Parkinson's diseases. However, tau phosphorylation in SMA pathogenesis has yet to be investigated. Here we show that tau phosphorylation on serine 202 (S202) and threonine 205 (T205) is increased significantly in SMA motor neurons using two SMA mouse models and human SMA patient spinal cord samples. Interestingly, phosphorylated tau does not form aggregates in motor neurons or neuromuscular junctions (NMJs), even at late stages of SMA disease, distinguishing it from other tauopathies. Hyperphosphorylation of tau on S202 and T205 is mediated by cyclin-dependent kinase 5 (Cdk5) in SMA disease condition, because tau phosphorylation at these sites is significantly reduced in Cdk5 knock-out mice; genetic knock-out of Cdk5 activating subunit p35 in an SMA mouse model also leads to reduced tau phosphorylation on S202 and T205 in the SMA;p35−/− compound mutant mice. In addition, expression of the phosphorylation-deficient tauS202A,T205A mutant alleviates motor neuron defects in a zebrafish SMA model in vivo and mouse motor neuron degeneration in culture, whereas expression of phosphorylation-mimetic tauS202E,T205E promotes motor neuron defects. More importantly, genetic knock-out of tau in SMA mice rescues synapse stripping on motor neurons, NMJ denervation, and motor neuron degeneration in vivo. Altogether, our findings suggest a novel mechanism for SMA pathogenesis in which hyperphosphorylation of non-aggregating tau by Cdk5 contributes to motor neuron degeneration. PMID:25878277

Knock-out of the magnesium protoporphyrin IX methyltransferase gene in Arabidopsis. Effects on chloroplast development and on chloroplast-to-nucleus signaling

PubMed Central

Pontier, Dominique; Albrieux, Catherine; Joyard, Jacques; Lagrange, Thierry; Block, Maryse

2007-01-01

Protoporphyrin IX is the last common intermediate between the haem and chlorophyll biosynthesis pathways. The addition of Mg directs this molecule toward chlorophyll biosynthesis. The first step downstream from the branchpoint is catalyzed by the Mg chelatase and is a highly regulated process. The corresponding product, Mg protoporphyrin IX, has been proposed to play an important role as a signaling molecule implicated in plastid-to-nucleus communication. In order to get more information on the chlorophyll biosynthesis pathway and on Mg protoporphyrin IX derivative functions, we have identified an Mg protoporphyrin IX methyltransferase (CHLM) knock-out mutant in Arabidopsis in which the mutation induces a blockage downstream from Mg protoporphyrin IX and an accumulation of this chlorophyll biosynthesis intermediate. Our results demonstrate that the CHLM gene is essential for the formation of chlorophyll and subsequently for the formation of photosystems I and II and cyt b6f complexes. Analysis of gene expression in the chlm mutant provides an independent indication that Mg protoporphyrin IX is a negative effector of nuclear photosynthetic gene expression, as previously reported. Moreover, it suggests the possible implication of Mg protoporphyrin IX methylester, the product of CHLM, in chloroplast-to-nucleus signaling. Finally, post-transcriptional up-regulation of the level of the CHLH subunit of the Mg chelatase has been detected in the chlm mutant and most likely corresponds to specific accumulation of this protein inside plastids. This result suggests that the CHLH subunit might play an important regulatory role when the chlorophyll biosynthetic pathway is disrupted at this particular step. PMID:17135235

Rac1 modulates cardiomyocyte adhesion during mouse embryonic development

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

Abu-Issa, Radwan, E-mail: rabuissa@umich.edu

2015-01-24

Highlights: • Conditional knockout of Rac1 using Nkx2.5 Cre line is lethal at E13.5. • The myocardium of the mutant is thin and disorganized. • The phenotype is not due to cardiomyocyte low proliferation or apoptosis. • The phenotype is due to specific defect in cardiomyocyte adhesion. - Abstract: Rac1, a member of the Rho subfamily of small GTPases, is involved in morphogenesis and differentiation of many cell types. Here we define a role of Rac1 in cardiac development by specifically deleting Rac1 in the pre-cardiac mesoderm using the Nkx2.5-Cre transgenic driver line. Rac1-conditional knockout embryos initiate heart development normallymore » until embryonic day 11.5 (E11.5); their cardiac mesoderm is specified, and the heart tube is formed and looped. However, by E12.5-E13.5 the mutant hearts start failing and embryos develop edema and hemorrhage which is probably the cause for the lethality observed soon after. The hearts of Rac1-cKO embryos exhibit disorganized and thin myocardial walls and defects in outflow tract alignment. No significant differences of cardiomyocyte death or proliferation were found between developing control and mutant embryos. To uncover the role of Rac1 in the heart, E11.5 primary heart cells were cultured and analyzed in vitro. Rac1-deficient cardiomyocytes were less spread, round and loosely attached to the substrate and to each other implying that Rac1-mediated signaling is required for appropriate cell–cell and/or cellmatrix adhesion during cardiac development.« less

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

ADP/ATP mitochondrial carrier MD simulations to shed light on the structural-dynamical events that, after an additional mutation, restore the function in a pathological single mutant.

PubMed

Di Marino, Daniele; Oteri, Francesco; Morozzo Della Rocca, Blasco; Chillemi, Giovanni; Falconi, Mattia

2010-12-01

Molecular dynamics simulations of the wild type bovine ADP/ATP mitochondrial carrier, and of the single Ala113Pro and double Ala113Pro/Val180Met mutants, embedded in a lipid bilayer, have been carried out for 30ns to shed light on the structural-dynamical changes induced by the Val180Met mutation restoring the carrier function in the Ala113Pro pathologic mutant. Principal component analysis indicates that, for the three systems, the protein dynamics is mainly characterized by the motion of the matrix loops and of the odd-numbered helices having a conserved proline in their central region. Analysis of the motions shows a different behaviour of single pathological mutant with respect of the other two systems. The single mutation induces a regularization and rigidity of the H3 helix, lost upon the introduction of the second mutation. This is directly correlated to the salt bridge distribution involving residues Arg79, Asp134 and Arg234, hypothesized to interact with the substrate. In fact, in the wild type simulation two stable inter-helices salt bridges, crucial for substrate binding, are present almost over all the simulation time. In line with the impaired ADP transport, one salt interaction is lost in the single mutant trajectory but reappears in the double mutant simulation, where a salt bridge network matching the wild type is restored. Other important structural-dynamical properties, such as the trans-membrane helices mobility, analyzed via the principal component analysis, are similar for the wild type and double mutant while are different for the single mutant, providing a mechanistic explanation for their different functional properties. Copyright © 2010 Elsevier Inc. All rights reserved.

Lovastatin synergizes with itraconazole against planktonic cells and biofilms of Candida albicans through the regulation on ergosterol biosynthesis pathway.

PubMed

Zhou, Yujie; Yang, Hong; Zhou, Xuedong; Luo, Hongke; Tang, Fan; Yang, Jin; Alterovitz, Gil; Cheng, Lei; Ren, Biao

2018-06-01

The increase of fungal infectious diseases and lack of safe and efficacious antifungal drugs result in the urgent need of new therapeutic strategies. Here, we repurposed the lovastatin (LOV) as a synergistic antifungal potentiator to itraconazole (ITZ) against Candida albicans planktonic cells and biofilms in vitro for the first time. Mutants from ergosterol biosynthesis pathway were employed and key gene expression profiles of ergosterol pathway were also measured. LOV single treatment was unable to inhibit C. albicans strains except the ERG3 and ERG11 double mutant. LOV and ITZ combination was capable of inhibiting the C. albicans planktonic cells and biofilms synergistically including the ITZ resistant mutants. The synergistic antifungal ability was stronger in either ERG11 or ERG3 dysfunctional mutants compared to wild type. The combination lost the synergistic activities in the ERG11 and ERG3 double mutant, while it was sensitive to LOV single treatment. The expression of HMG1, encoding HMG-CoA the target of LOV, was significantly upregulated in ERG11 and ERG3 double mutant strain by the treatment of the combination at 1.5 and 3 h. The combination also significantly increased the HMG1 expression in mutants from ergosterol pathway compared with wild type. The ERG11 and ERG3 gene expressions were upregulated by ITZ and its combination with LOV, but seemingly not by LOV single treatment after 1.5 and 3 h. The combination of LOV and ITZ on C. albicans planktonic cells and biofilms highlights its potential clinical practice especially against the azole drug-resistant mutants.

Generation of astaxanthin mutants in Xanthophyllomyces dendrorhous using a double recombination method based on hygromycin resistance.

PubMed

Niklitschek, Mauricio; Baeza, Marcelo; Fernández-Lobato, María; Cifuentes, Víctor

2012-01-01

Generally two selection markers are required to obtain homozygous mutations in a diploid background, one for each gene copy that is interrupted. In this chapter is described a method that allows the double gene deletions of the two copies of a gene from a diploid organism, a wild-type strain of the Xanthophyllomyces dendrorhous yeast, using hygromycin B resistance as the only selection marker. To accomplish this, in a first step, a heterozygous hygromycin B-resistant strain is obtained by a single process of transformation (carrying the inserted hph gene). Following, the heterozygous mutant is grown in media with increasing concentrations of the antibiotic. In this way, the strains that became homozygous (by mitotic recombination) for the antibiotic marker would able to growth at higher concentration of the antibiotic than the heterozygous. The method can be potentially applied for obtaining double mutants of other diploid organisms.

d-myo-Inositol-3-Phosphate Affects Phosphatidylinositol-Mediated Endomembrane Function in Arabidopsis and Is Essential for Auxin-Regulated Embryogenesis[W][OA

PubMed Central

Luo, Yu; Qin, Genji; Zhang, Jun; Liang, Yuan; Song, Yingqi; Zhao, Meiping; Tsuge, Tomohiko; Aoyama, Takashi; Liu, Jingjing; Gu, Hongya; Qu, Li-Jia

2011-01-01

In animal cells, myo-inositol is an important regulatory molecule in several physiological and biochemical processes, including signal transduction and membrane biogenesis. However, the fundamental biological functions of myo-inositol are still far from clear in plants. Here, we report the genetic characterization of three Arabidopsis thaliana genes encoding d-myo-inositol-3-phosphate synthase (MIPS), which catalyzes the rate-limiting step in de novo synthesis of myo-inositol. Each of the three MIPS genes rescued the yeast ino1 mutant, which is defective in yeast MIPS gene INO1, and they had different dynamic expression patterns during Arabidopsis embryo development. Although single mips mutants showed no obvious phenotypes, the mips1 mips2 double mutant and the mips1 mips2 mips3 triple mutant were embryo lethal, whereas the mips1 mips3 and mips1 mips2+/− double mutants had abnormal embryos. The mips phenotypes resembled those of auxin mutants. Indeed, the double and triple mips mutants displayed abnormal expression patterns of DR5:green fluorescent protein, an auxin-responsive fusion protein, and they had altered PIN1 subcellular localization. Also, membrane trafficking was affected in mips1 mips3. Interestingly, overexpression of PHOSPHATIDYLINOSITOL SYNTHASE2, which converts myo-inositol to membrane phosphatidylinositol (PtdIns), largely rescued the cotyledon and endomembrane defects in mips1 mips3. We conclude that myo-inositol serves as the main substrate for synthesizing PtdIns and phosphatidylinositides, which are essential for endomembrane structure and trafficking and thus for auxin-regulated embryogenesis. PMID:21505066

Intraflagellar transporter protein (IFT27), an IFT25 binding partner, is essential for male fertility and spermiogenesis in mice.

PubMed

Zhang, Yong; Liu, Hong; Li, Wei; Zhang, Zhengang; Shang, Xuejun; Zhang, David; Li, Yuhong; Zhang, Shiyang; Liu, Junpin; Hess, Rex A; Pazour, Gregory J; Zhang, Zhibing

2017-12-01

Intraflagellar transport (IFT) is an evolutionarily conserved mechanism essential for the assembly and maintenance of most eukaryotic cilia and flagella. In mice, mutations in IFT proteins have been shown to cause several ciliopathies including retinal degeneration, polycystic kidney disease, and hearing loss. However, little is known about its role in the formation of the sperm tail, which has the longest flagella of mammalian cells. IFT27 is a component of IFT-B complex and binds to IFT25 directly. In mice, IFT27 is highly expressed in the testis. To investigate the role of IFT27 in male germ cells, the floxed Ift27 mice were bred with Stra8-iCre mice so that the Ift27 gene was disrupted in spermatocytes/spermatids. The Ift27: Stra8-iCre mutant mice did not show any gross abnormalities, and all of the mutant mice survived to adulthood. There was no difference between testis weight/body weight between controls and mutant mice. All adult homozygous mutant males examined were completely infertile. Histological examination of the testes revealed abnormally developed germ cells during the spermiogenesis phase. The epididymides contained round bodies of cytoplasm. Sperm number was significantly reduced compared to the controls and only about 2% of them remained significantly reduced motility. Examination of epididymal sperm by light microscopy and SEM revealed multiple morphological abnormalities including round heads, short and bent tails, abnormal thickness of sperm tails in some areas, and swollen tail tips in some sperm. TEM examination of epididymal sperm showed that most sperm lost the "9+2″ axoneme structure, and the mitochondria sheath, fibrous sheath, and outer dense fibers were also disorganized. Some sperm flagella also lost cell membrane. Levels of IFT25 and IFT81 were significantly reduced in the testis of the conditional Ift27 knockout mice, and levels of IFT20, IFT74, and IFT140 were not changed. Sperm lipid rafts, which were disrupted in the conditional Ift25 knockout mice, appeared to be normal in the conditional Ift27 knockout mice. Our findings suggest that like IFT25, IFT27, even though not required for ciliogenesis in somatic cells, is essential for sperm flagella formation, sperm function, and male fertility in mice. IFT25 and IFT27 control sperm formation/function through many common mechanisms, but IFT25 has additional roles beyond IFT27. Published by Elsevier Inc.

Intraflagellar Transporter Protein (IFT27), an IFT25 binding partner, Is Essential For Male Fertility and Spermiogenesis In Mice

PubMed Central

Zhang, Yong; Liu, Hong; Li, Wei; Zhang, Zhengang; Shang, Xuejun; Zhang, David; Li, Yuhong; Zhang, Shiyang; Liu, Junpin; Hess, Rex A; Pazour, Gregory J; Zhang, Zhibing

2017-01-01

Intraflagellar transport (IFT) is an evolutionarily conserved mechanism essential for the assembly and maintenance of most eukaryotic cilia and flagella. In mice, mutations in IFT proteins have been shown to cause several ciliopathies including retinal degeneration, polycystic kidney disease, and hearing loss. However, little is known about its role in the formation of the sperm tail, which has the longest flagella of mammalian cells. IFT27 is a component of IFT-B complex and binds to IFT25 directly. In mice, IFT27 is highly expressed in the testis. To investigate the role of IFT27 in male germ cells, the floxed Ift27 mice were bred with Stra8-iCre mice so that the Ift27 gene was disrupted in spermatocytes/spermatids. The Ift27:Stra8-iCre mutant mice did not show any gross abnormalities, and all of the mutant mice survive to adulthood. There was no difference between testis weight/body weight between controls and mutant mice. All adult homozygous mutant males examined were completely infertile. Histological examination of the testes revealed abnormally developed germ cells during the spermiogenesis phase. The epididymis contained round bodies of cytoplasm. Sperm number was significantly reduced compared to the controls and only about 2% of them remained significantly reduced motility. Examination of epididymal sperm by light microscopy and SEM revealed multiple morphological abnormalities including round heads, short and bent tails, abnormal thickness of sperm tails in some areas, and swollen tail tips in some sperm. TEM examination of epididymal sperm showed that most sperm lost the “9+2” axoneme structure, and the mitochondria sheath, fibrous sheath, and outer dense fibers were also disorganized. Some sperm flagella also lost cell membrane. Levels of IFT25 and IFT81 were significantly reduced in the testis of the conditional Ift27 knockout mice, and levels of IFT20, IFT74, and IFT140 were not changed. Sperm lipid rafts, which were disrupted in the conditional Ift25 knockout mice, appeared to be normal in the conditional Ift27 knockout mice. Our findings suggest that like IFT25, IFT27, even though not required to ciliogenesis in somatic cells, is essential for sperm flagella formation, sperm function, and male fertility in mice. IFT25 and IFT27 control sperm formation/function through many common mechanisms, but IFT25 has additional roles beyond IFT27. PMID:28964737

TRPV2 KNOCKOUT MICE ARE SUSCEPTIBLE TO PERINATAL LETHALITY BUT DISPLAY NORMAL THERMAL AND MECHANICAL NOCICEPTION

PubMed Central

Park, Una; Vastani, Nisha; Guan, Yun; Raja, Srinivasa N.; Koltzenburg, Martin; Caterina, Michael J.

2011-01-01

TRPV2 is a nonselective cation channel expressed prominently in medium- to large-diameter sensory neurons that can be activated by extreme heat (>52°C). These features suggest that TRPV2 might be a transducer of noxious heat in vivo. TRPV2 can also be activated by hypoosmolarity or cell stretch, suggesting potential roles in mechanotransduction. To address the physiological functions of TRPV2 in somatosensation, we generated TRPV2 knockout mice and examined their behavioral and electrophysiological responses to heat and mechanical stimuli. TRPV2 knockout mice showed reduced embryonic weight and perinatal viability. As adults, surviving knockout mice also exhibited a slightly reduced body weight. TRPV2 knockout mice showed normal behavioral responses to noxious heat over a broad range of temperatures and normal responses to punctate mechanical stimuli, both in the basal state and under hyperalgesic conditions such as peripheral inflammation and L5 spinal nerve ligation. Moreover, behavioral assays of TRPV1/TRPV2 double knockout mice or of TRPV2 knockout mice treated with resiniferatoxin to desensitize TRPV1-expressing afferents revealed no thermosensory consequences of TRPV2 absence. In line with behavioral findings, electrophysiological recordings from skin afferents showed that C-fiber responses to heat and C- and Aδ-fiber responses to noxious mechanical stimuli were unimpaired in the absence of TRPV2. The prevalence of thermosensitive Aδ-fibers was too low to permit comparison between genotypes. Thus, TRPV2 is important for perinatal viability but is not essential for heat or mechanical nociception or hypersensitivity in the adult mouse. PMID:21832173

In Vitro and in Vivo Analysis of the Binding of the C Terminus of the HDL Receptor Scavenger Receptor Class B, Type I (SR-BI), to the PDZ1 Domain of Its Adaptor Protein PDZK1*

PubMed Central

Kocher, Olivier; Birrane, Gabriel; Tsukamoto, Kosuke; Fenske, Sara; Yesilaltay, Ayce; Pal, Rinku; Daniels, Kathleen; Ladias, John A. A.; Krieger, Monty

2010-01-01

The PDZ1 domain of the four PDZ domain-containing protein PDZK1 has been reported to bind the C terminus of the HDL receptor scavenger receptor class B, type I (SR-BI), and to control hepatic SR-BI expression and function. We generated wild-type (WT) and mutant murine PDZ1 domains, the mutants bearing single amino acid substitutions in their carboxylate binding loop (Lys14-Xaa4-Asn19-Tyr-Gly-Phe-Phe-Leu24), and measured their binding affinity for a 7-residue peptide corresponding to the C terminus of SR-BI (503VLQEAKL509). The Y20A and G21Y substitutions abrogated all binding activity. Surprisingly, binding affinities (Kd) of the K14A and F22A mutants were 3.2 and 4.0 μm, respectively, similar to 2.6 μm measured for the WT PDZ1. To understand these findings, we determined the high resolution structure of WT PDZ1 bound to a 5-residue sequence from the C-terminal SR-BI (505QEAKL509) using x-ray crystallography. In addition, we incorporated the K14A and Y20A substitutions into full-length PDZK1 liver-specific transgenes and expressed them in WT and PDZK1 knock-out mice. In WT mice, the transgenes did not alter endogenous hepatic SR-BI protein expression (intracellular distribution or amount) or lipoprotein metabolism (total plasma cholesterol, lipoprotein size distribution). In PDZK1 knock-out mice, as expected, the K14A mutant behaved like wild-type PDZK1 and completely corrected their hepatic SR-BI and plasma lipoprotein abnormalities. Unexpectedly, the 10–20-fold overexpressed Y20A mutant also substantially, but not completely, corrected these abnormalities. The results suggest that there may be an additional site(s) within PDZK1 that bind(s) SR-BI and mediate(s) productive SR-BI-PDZK1 interaction previously attributed exclusively to the canonical binding of the C-terminal SR-BI to PDZ1. PMID:20739281

In vitro and in vivo Analysis of the Binding of the C Terminus of the HDL Receptor Scavenger Receptor Class B type I (SR-BI) to the PDZ1 Domain of its Cytoplasmic Adaptor Protein PDZK1

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

O Kocher; G Birrane; K Tsukamoto

2011-12-31

The PDZ1 domain of the four PDZ domain-containing protein PDZK1 has been reported to bind the C terminus of the HDL receptor scavenger receptor class B, type I (SR-BI), and to control hepatic SR-BI expression and function. We generated wild-type (WT) and mutant murine PDZ1 domains, the mutants bearing single amino acid substitutions in their carboxylate binding loop (Lys(14)-Xaa(4)-Asn(19)-Tyr-Gly-Phe-Phe-Leu(24)), and measured their binding affinity for a 7-residue peptide corresponding to the C terminus of SR-BI ((503)VLQEAKL(509)). The Y20A and G21Y substitutions abrogated all binding activity. Surprisingly, binding affinities (K(d)) of the K14A and F22A mutants were 3.2 and 4.0 ?M,more » respectively, similar to 2.6 ?M measured for the WT PDZ1. To understand these findings, we determined the high resolution structure of WT PDZ1 bound to a 5-residue sequence from the C-terminal SR-BI ((505)QEAKL(509)) using x-ray crystallography. In addition, we incorporated the K14A and Y20A substitutions into full-length PDZK1 liver-specific transgenes and expressed them in WT and PDZK1 knock-out mice. In WT mice, the transgenes did not alter endogenous hepatic SR-BI protein expression (intracellular distribution or amount) or lipoprotein metabolism (total plasma cholesterol, lipoprotein size distribution). In PDZK1 knock-out mice, as expected, the K14A mutant behaved like wild-type PDZK1 and completely corrected their hepatic SR-BI and plasma lipoprotein abnormalities. Unexpectedly, the 10-20-fold overexpressed Y20A mutant also substantially, but not completely, corrected these abnormalities. The results suggest that there may be an additional site(s) within PDZK1 that bind(s) SR-BI and mediate(s) productive SR-BI-PDZK1 interaction previously attributed exclusively to the canonical binding of the C-terminal SR-BI to PDZ1.« less

Synergistic Action of FOXP3 and TSC1 Pathways During Tumor Progression

DTIC Science & Technology

2015-10-01

invasive carcinoma and, ultimately, metastatic disease [1-3]. Mouse models of PIN (mPIN) generated by a single- mutant gene in prostate do not progress...downstream target) is sufficient to significantly reduce the initiation of prostate cancer in the Pten conditional knockout mouse model [19-21...the possibility that these two genetic hits cooperate to promote tumor progression, and mouse models show that this cooperation accelerates

Double Knockout of the Na+-Driven Cl-/HCO3- Exchanger and Na+/Cl- Cotransporter Induces Hypokalemia and Volume Depletion.

PubMed

Sinning, Anne; Radionov, Nikita; Trepiccione, Francesco; López-Cayuqueo, Karen I; Jayat, Maximilien; Baron, Stéphanie; Cornière, Nicolas; Alexander, R Todd; Hadchouel, Juliette; Eladari, Dominique; Hübner, Christian A; Chambrey, Régine

2017-01-01

We recently described a novel thiazide-sensitive electroneutral NaCl transport mechanism resulting from the parallel operation of the Cl - /HCO 3 - exchanger pendrin and the Na + -driven Cl - /2HCO 3 - exchanger (NDCBE) in β-intercalated cells of the collecting duct. Although a role for pendrin in maintaining Na + balance, intravascular volume, and BP is well supported, there is no in vivo evidence for the role of NDCBE in maintaining Na + balance. Here, we show that deletion of NDCBE in mice caused only subtle perturbations of Na + homeostasis and provide evidence that the Na + /Cl - cotransporter (NCC) compensated for the inactivation of NDCBE. To unmask the role of NDCBE, we generated Ndcbe/Ncc double-knockout (dKO) mice. On a normal salt diet, dKO and single-knockout mice exhibited similar activation of the renin-angiotensin-aldosterone system, whereas only dKO mice displayed a lower blood K + concentration. Furthermore, dKO mice displayed upregulation of the epithelial sodium channel (ENaC) and the Ca 2+ -activated K + channel BKCa. During NaCl depletion, only dKO mice developed marked intravascular volume contraction, despite dramatically increased renin activity. Notably, the increase in aldosterone levels expected on NaCl depletion was attenuated in dKO mice, and single-knockout and dKO mice had similar blood K + concentrations under this condition. In conclusion, NDCBE is necessary for maintaining sodium balance and intravascular volume during salt depletion or NCC inactivation in mice. Furthermore, NDCBE has an important role in the prevention of hypokalemia. Because NCC and NDCBE are both thiazide targets, the combined inhibition of NCC and the NDCBE/pendrin system may explain thiazide-induced hypokalemia in some patients. Copyright © 2016 by the American Society of Nephrology.

Multimodal nonlinear optical imaging of cartilage development in mouse model

NASA Astrophysics Data System (ADS)

He, Sicong; Xue, Wenqian; Sun, Qiqi; Li, Xuesong; Huang, Jiandong; Qu, Jianan Y.

2017-02-01

Kinesin-1 is a kind of motor protein responsible for intracellular transportation and has been studied in a variety of tissues. However, its roles in cartilage development are not clear. In this study, a kinesin-1 heavy chain (Kif5b) knockout mouse model is used to study the functions of kinesin-1 in the cartilage development. We developed a multimodal nonlinear optical (NLO) microscope system integrating stimulated Raman scattering (SRS), second harmonic generation (SHG) and two-photon excited fluorescence (TPEF) to investigate the morphological and biomedical characteristics of fresh tibial cartilage from normal and mutant mice at different developmental stages. The combined forward and backward SHG imaging resolved the fine structure of collagen fibrils in the extracellular matrix of cartilage. Meanwhile, the chondrocyte morphology in different zones of cartilage was visualized by label-free SRS and TPEF images. The results show that the fibrillar collagen in the superficial zone of cartilage in postnatal day 10 and 15 (P10 and P15) knockout mice was significantly less than that of control mice. Moreover, we observed distorted morphology and disorganization of columnar arrangement of chondrocytes in the growth plate cartilage of mutant mice. This study reveals the significant roles of kinesin-1 in collagen formation and chondrocyte morphogenesis.

Sulfate Assimilation Mediates Tellurite Reduction and Toxicity in Saccharomyces cerevisiae▿†

PubMed Central

Ottosson, Lars-Göran; Logg, Katarina; Ibstedt, Sebastian; Sunnerhagen, Per; Käll, Mikael; Blomberg, Anders; Warringer, Jonas

2010-01-01

Despite a century of research and increasing environmental and human health concerns, the mechanistic basis of the toxicity of derivatives of the metalloid tellurium, Te, in particular the oxyanion tellurite, Te(IV), remains unsolved. Here, we provide an unbiased view of the mechanisms of tellurium metabolism in the yeast Saccharomyces cerevisiae by measuring deviations in Te-related traits of a complete collection of gene knockout mutants. Reduction of Te(IV) and intracellular accumulation as metallic tellurium strongly correlated with loss of cellular fitness, suggesting that Te(IV) reduction and toxicity are causally linked. The sulfate assimilation pathway upstream of Met17, in particular, the sulfite reductase and its cofactor siroheme, was shown to be central to tellurite toxicity and its reduction to elemental tellurium. Gene knockout mutants with altered Te(IV) tolerance also showed a similar deviation in tolerance to both selenite and, interestingly, selenomethionine, suggesting that the toxicity of these agents stems from a common mechanism. We also show that Te(IV) reduction and toxicity in yeast is partially mediated via a mitochondrial respiratory mechanism that does not encompass the generation of substantial oxidative stress. The results reported here represent a robust base from which to attack the mechanistic details of Te(IV) toxicity and reduction in a eukaryotic organism. PMID:20675578

Zinc Detoxification Is Required for Full Virulence and Modification of the Host Leaf Ionome by Xylella fastidiosa.

PubMed

Navarrete, Fernando; De La Fuente, Leonardo

2015-04-01

Zinc (Zn) is an essential element for all forms of life because it is a structural or catalytic cofactor of many proteins, but it can have toxic effects at high concentrations; thus, microorganisms must tightly regulate its levels. Here, we evaluated the role of Zn homeostasis proteins in the virulence of the xylem-limited bacterium Xylella fastidiosa, causal agent of Pierce's disease of grapevine, among other diseases. Two mutants of X. fastidiosa 'Temecula' affected in genes which regulate Zn homeostasis (zur) and Zn detoxification (czcD) were constructed. Both knockouts showed increased sensitivity to Zn at physiologically relevant concentrations and increased intracellular accumulation of this metal compared with the wild type. Increased Zn sensitivity was correlated with decreased growth in grapevine xylem sap, reduced twitching motility, and downregulation of exopolysaccharide biosynthetic genes. Tobacco plants inoculated with either knockout mutant showed reduced foliar symptoms and a much reduced (czcD) or absent (zur) modification of the leaf ionome (i.e., the mineral nutrient and trace element composition), as well as reduced bacterial populations. The results show that detoxification of Zn is crucial for the virulence of X. fastidiosa and verifies our previous findings that modification of the host leaf ionome correlates with bacterial virulence.

Inactivation of Phaeodactylum tricornutum urease gene using transcription activator-like effector nuclease-based targeted mutagenesis.

PubMed

Weyman, Philip D; Beeri, Karen; Lefebvre, Stephane C; Rivera, Josefa; McCarthy, James K; Heuberger, Adam L; Peers, Graham; Allen, Andrew E; Dupont, Christopher L

2015-05-01

Diatoms are unicellular photosynthetic algae with promise for green production of fuels and other chemicals. Recent genome-editing techniques have greatly improved the potential of many eukaryotic genetic systems, including diatoms, to enable knowledge-based studies and bioengineering. Using a new technique, transcription activator-like effector nucleases (TALENs), the gene encoding the urease enzyme in the model diatom, Phaeodactylum tricornutum, was targeted for interruption. The knockout cassette was identified within the urease gene by PCR and Southern blot analyses of genomic DNA. The lack of urease protein was confirmed by Western blot analyses in mutant cell lines that were unable to grow on urea as the sole nitrogen source. Untargeted metabolomic analysis revealed a build-up of urea, arginine and ornithine in the urease knockout lines. All three intermediate metabolites are upstream of the urease reaction within the urea cycle, suggesting a disruption of the cycle despite urea production. Numerous high carbon metabolites were enriched in the mutant, implying a breakdown of cellular C and N repartitioning. The presented method improves the molecular toolkit for diatoms and clarifies the role of urease in the urea cycle. © 2014 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Construction of "Toxin Complex" in a Mutant Serotype C Strain of Clostridium botulinum Harboring a Defective Neurotoxin Gene.

PubMed

Suzuki, Tomonori; Nagano, Thomas; Niwa, Koichi; Uchino, Masataka; Tomizawa, Motohiro; Sagane, Yoshimasa; Watanabe, Toshihiro

2017-01-01

A non-toxigenic mutant of the toxigenic serotype C Clostridium botulinum strain Stockholm (C-St), C-N71, does not produce the botulinum neurotoxin (BoNT). However, the original strain C-St produces botulinum toxin complex, in which BoNT is associated with non-toxic non-hemagglutinin (NTNHA) and three hemagglutinin proteins (HA-70, HA-33, and HA-17). Therefore, in this study, we aimed to elucidate the effects of bont gene knockout on the formation of the "toxin complex." Nucleotide sequence analysis revealed that a premature stop codon was introduced in the bont gene, whereas other genes were not affected by this mutation. Moreover, we successfully purified the "toxin complex" produced by C-N71. The "toxin complex" was identified as a mixture of NTNHA/HA-70/HA-17/HA-33 complexes with intact NTNHA or C-terminally truncated NTNHA, without BoNT. These results indicated that knockout of the bont gene does not affect the formation of the "toxin complex." Since the botulinum toxin complex has been shown to play an important role in oral toxin transport in the human and animal body, a non-neurotoxic "toxin complex" of C-N71 may be valuable for the development of an oral drug delivery system.

Heparan Sulfate Biosynthesis Enzyme, Ext1, Contributes to Outflow Tract Development of Mouse Heart via Modulation of FGF Signaling.

PubMed

Zhang, Rui; Cao, Peijuan; Yang, Zhongzhou; Wang, Zhenzhen; Wu, Jiu-Lin; Chen, Yan; Pan, Yi

2015-01-01

Glycosaminoglycans are important regulators of multiple signaling pathways. As a major constituent of the heart extracellular matrix, glycosaminoglycans are implicated in cardiac morphogenesis through interactions with different signaling morphogens. Ext1 is a glycosyltransferase responsible for heparan sulfate synthesis. Here, we evaluate the function of Ext1 in heart development by analyzing Ext1 hypomorphic mutant and conditional knockout mice. Outflow tract alignment is sensitive to the dosage of Ext1. Deletion of Ext1 in the mesoderm induces a cardiac phenotype similar to that of a mutant with conditional deletion of UDP-glucose dehydrogenase, a key enzyme responsible for synthesis of all glycosaminoglycans. The outflow tract defect in conditional Ext1 knockout(Ext1f/f:Mesp1Cre) mice is attributable to the reduced contribution of second heart field and neural crest cells. Ext1 deletion leads to downregulation of FGF signaling in the pharyngeal mesoderm. Exogenous FGF8 ameliorates the defects in the outflow tract and pharyngeal explants. In addition, Ext1 expression in second heart field and neural crest cells is required for outflow tract remodeling. Our results collectively indicate that Ext1 is crucial for outflow tract formation in distinct progenitor cells, and heparan sulfate modulates FGF signaling during early heart development.

Membrane-localized ubiquitin ligase ATL15 functions in sugar-responsive growth regulation in Arabidopsis.

PubMed

Aoyama, Shoki; Terada, Saki; Sanagi, Miho; Hasegawa, Yoko; Lu, Yu; Morita, Yoshie; Chiba, Yukako; Sato, Takeo; Yamaguchi, Junji

2017-09-09

Ubiquitin ligases play important roles in regulating various cellular processes by modulating the protein function of specific ubiquitination targets. The Arabidopsis Tóxicos en Levadura (ATL) family is a group of plant-specific RING-type ubiquitin ligases that localize to membranes via their N-terminal transmembrane-like domains. To date, 91 ATL isoforms have been identified in the Arabidopsis genome, with several ATLs reported to be involved in regulating plant responses to environmental stresses. However, the functions of most ATLs remain unknown. This study, involving transcriptome database analysis, identifies ATL15 as a sugar responsive ATL gene in Arabidopsis. ATL15 expression was rapidly down-regulated in the presence of sugar. The ATL15 protein showed ubiquitin ligase activity in vitro and localized to plasma membrane and endomembrane compartments. Further genetic analyses demonstrated that the atl15 knockout mutants are insensitive to high glucose concentrations, whereas ATL15 overexpression depresses plant growth. In addition, endogenous glucose and starch amounts were reciprocally affected in the atl15 knockout mutants and the ATL15 overexpressors. These results suggest that ATL15 protein plays a significant role as a membrane-localized ubiquitin ligase that regulates sugar-responsive plant growth in Arabidopsis. Copyright © 2017 Elsevier Inc. All rights reserved.

Knocking out the MFE-2 gene of Candida bombicola leads to improved medium-chain sophorolipid production.

PubMed

Van Bogaert, Inge N A; Sabirova, Julia; Develter, Dirk; Soetaert, Wim; Vandamme, Erick J

2009-06-01

The nonpathogenic yeast Candida bombicola synthesizes sophorolipids. These biosurfactants are composed of the disaccharide sophorose linked to a long-chain hydroxy fatty acid and have potential applications in the food, pharmaceutical, cosmetic and cleaning industries. In order to expand the range of application, a shift of the fatty acid moiety towards medium-chain lengths would be recommendable. However, the synthesis of medium-chain sophorolipids by C. bombicola is a challenging objective. First of all, these sophorolipids can only be obtained by fermentations on unconventional carbon sources, which often have a toxic effect on the cells. Furthermore, medium-chain substrates are partially metabolized in the beta-oxidation pathway. In order to redirect unconventional substrates towards sophorolipid synthesis, the beta-oxidation pathway was blocked on the genome level by knocking out the multifunctional enzyme type 2 (MFE-2) gene. The total gene sequence of the C. bombicola MFE-2 (6033 bp) was cloned (GenBank accession number EU371724), and the obtained nucleotide sequence was used to construct a knock-out cassette. Several knock-out mutants with the correct geno- and phenotype were evaluated in a fermentation on 1-dodecanol. All mutants showed a 1.7-2.9 times higher production of sophorolipids, indicating that in those strains the substrate is redirected towards the sophorolipid synthesis.

Relative axial myopia in Egr-1 (ZENK) knockout mice.

PubMed

Schippert, Ruth; Burkhardt, Eva; Feldkaemper, Marita; Schaeffel, Frank

2007-01-01

Experiments in chickens have implicated the transcription factor ZENK (also known as Egr-1, NGFI-A, zif268, tis8, cef5, and Krox24) in the feedback mechanisms for visual control of axial eye growth and myopia development. ZENK is upregulated in retinal glucagon amacrine cells when axial eye growth is inhibited by positive spectacle lens wear and is downregulated when it is enhanced by negative spectacle lens wear, suggesting that ZENK may be linked to an inhibitory signal for axial eye growth. This study was undertaken to determine whether a Egr-1(-/-) knockout mouse mutant, lacking ZENK completely, has longer eyes and more myopic refraction, than do Egr-1(+/)(-) heterozygous and Egr-1(+/+) wild-type mice with near-identical genetic backgrounds. Eye growth and refractive development were tracked from day P28 to P98. Corneal radius of curvature was measured with infrared photokeratometry, refractive state with infrared photoretinoscopy, and ocular dimensions with low-coherence interferometry. As a functional vision test, grating acuity was determined in an automated optomotor task. The abundance of ZENK protein in the retina was quantified by immunohistochemistry. Egr-1 knockout mice had longer eyes and a relative myopic shift in refraction, with additional minor effects on anterior chamber depth and corneal radius of curvature. Paraxial schematic eye modeling suggested changes in the optics of the crystalline lens as well. With increasing age, the differences between mutant and wild-type mice declined, although the differences in refraction persisted over the observation period. Grating acuity was not affected by the lack of the Egr-1 protein during development. Although it has been shown that different mouse strains may have differently large eyes, the present study shows that a specific gene knockout can produce relative myopia, compared with the wild-type with near-identical genetic background. Further experiments are needed to determine whether the observed effects of Egr-1 deletion are due to changes in function within the retina or other ocular tissues or to changes of function in other systems that may affect ocular growth from outside the eye.

The role of nuclear factor E2-Related factor 2 and uncoupling protein 2 in glutathione metabolism: Evidence from an in vivo gene knockout study.

PubMed

Chen, Yanyan; Xu, Yuanyuan; Zheng, Hongzhi; Fu, Jingqi; Hou, Yongyong; Wang, Huihui; Zhang, Qiang; Yamamoto, Masayuki; Pi, Jingbo

2016-09-09

Nuclear factor E2-related factor 2 (NRF2) and uncoupling protein 2 (UCP2) are indicated to protect from oxidative stress. They also play roles in the homeostasis of glutathione. However, the detailed mechanisms are not well understood. In the present study, we found Nrf2-knockout (Nrf2-KO) mice exhibited altered glutathione homeostasis and reduced expression of various genes involved in GSH biosynthesis, regeneration, utilization and transport in the liver. Ucp2-knockout (Ucp2-KO) mice exhibited altered glutathione homeostasis in the liver, spleen and blood, as well as increased transcript of cystic fibrosis transmembrane conductance regulator in the liver, a protein capable of mediating glutathione efflux. Nrf2-Ucp2-double knockout (DKO) mice showed characteristics of both Nrf2-KO and Ucp2-KO mice. But no significant difference was observed in DKO mice when compared with Nrf2-KO or Ucp2-KO mice, except in blood glutathione levels. These data suggest that ablation of Nrf2 and Ucp2 leads to disrupted GSH balance, which could result from altered expression of genes involved in GSH metabolism. DKO may not evoke more severe oxidative stress than the single gene knockout. Copyright © 2016 Elsevier Inc. All rights reserved.

Honey-sensitive Pseudomonas aeruginosa mutants are impaired in catalase A.

PubMed

Bolognese, Fabrizio; Bistoletti, Michela; Barbieri, Paola; Orlandi, Viviana Teresa

2016-09-01

The antimicrobial power of honey seems to be ascribable to several factors, including oxidative and osmotic stress. The aim of this study was to find genetic determinants involved in the response to honey stress in the opportunistic pathogen Pseudomonas aeruginosa, chosen as model micro-organism. A library of transposon mutants of P. aeruginosa PAO1 was constructed and only four mutants unable to grow in presence of fir honeydew honey were selected. All four mutants were impaired in the major H2O2-scavenging enzyme catalase A (KatA). The knockout of katA gene caused sensitivity, as expected, not only to hydrogen peroxide but also to different types of honey including Manuka GMO 220 honey. Genetic complementation, as well as the addition of PAO1 supernatant containing extracellular catalase, restored tolerance to honey stress in all the mutants. As P. aeruginosa PAO1 catalase KatA copes with H2O2 stress, it is conceivable that the antimicrobial activity of honey is, at least partially, due to the presence of hydrogen peroxide in honey or the ability of honey to induce production of hydrogen peroxide. The katA-deficient mutants could be used as tester micro-organisms to compare the power of different types of natural and curative honeys in eliciting oxidative stress mediated by hydrogen peroxide.

In Vitro Effect of Porphyromonas gingivalis Methionine Gamma Lyase on Biofilm Composition and Oral Inflammatory Response.

PubMed

Stephen, Abish S; Millhouse, Emma; Sherry, Leighann; Aduse-Opoku, Joseph; Culshaw, Shauna; Ramage, Gordon; Bradshaw, David J; Burnett, Gary R; Allaker, Robert P

2016-01-01

Methanethiol (methyl mercaptan) is an important contributor to oral malodour and periodontal tissue destruction. Porphyromonas gingivalis, Prevotella intermedia and Fusobacterium nucleatum are key oral microbial species that produce methanethiol via methionine gamma lyase (mgl) activity. The aim of this study was to compare an mgl knockout strain of P. gingivalis with its wild type using a 10-species biofilm co-culture model with oral keratinocytes and its effect on biofilm composition and inflammatory cytokine production. A P. gingivalis mgl knockout strain was constructed using insertion mutagenesis from wild type W50 with gas chromatographic head space analysis confirming lack of methanethiol production. 10-species biofilms consisting of Streptococcus mitis, Streptococcus oralis, Streptococcus intermedius, Fusobacterium nucleatum ssp polymorphum, Fusobacterium nucleatum ssp vincentii, Veillonella dispar, Actinomyces naeslundii, Prevotella intermedia and Aggregatibacter actinomycetemcomitans with either the wild type or mutant P. gingivalis were grown on Thermanox cover slips and used to stimulate oral keratinocytes (OKF6-TERT2), under anaerobic conditions for 4 and 24 hours. Biofilms were analysed by quantitative PCR with SYBR Green for changes in microbial ecology. Keratinocyte culture supernatants were analysed using a multiplex bead immunoassay for cytokines. Significant population differences were observed between mutant and wild type biofilms; V. dispar proportions increased (p<0.001), whilst A. naeslundii (p<0.01) and Streptococcus spp. (p<0.05) decreased in mutant biofilms. Keratinocytes produced less IL-8, IL-6 and IL-1α when stimulated with the mutant biofilms compared to wild type. Lack of mgl in P. gingivalis has been shown to affect microbial ecology in vitro, giving rise to a markedly different biofilm composition, with a more pro-inflammatory cytokine response from the keratinocytes observed. A possible role for methanethiol in biofilm formation and cytokine response with subsequent effects on oral malodor and periodontitis is suggested.

Electrical phenotypes of calcium transport mutant strains of a filamentous fungus, Neurospora crassa.

PubMed

Hamam, Ahmed; Lew, Roger R

2012-05-01

We characterized the electrical phenotypes of mutants with mutations in genes encoding calcium transporters-a mechanosensitive channel homolog (MscS), a Ca(2+)/H(+) exchange protein (cax), and Ca(2+)-ATPases (nca-1, nca-2, nca-3)-as well as those of double mutants (the nca-2 cax, nca-2 nca-3, and nca-3 cax mutants). The electrical characterization used dual impalements to obtain cable-corrected current-voltage measurements. Only two types of mutants (the MscS mutant; the nca-2 mutant and nca-2-containing double mutants) exhibited lower resting potentials. For the nca-2 mutant, on the basis of unchanged conductance and cyanide-induced depolarization of the potential, the cause is attenuated H(+)-ATPase activity. The growth of the nca-2 mutant-containing strains was inhibited by elevated extracellular Ca(2+) levels, indicative of lesions in Ca(2+) homeostasis. However, the net Ca(2+) effluxes of the nca-2 mutant, measured noninvasively with a self-referencing Ca(2+)-selective microelectrode, were similar to those of the wild type. All of the mutants exhibited osmosensitivity similar to that of the wild type (the turgor of the nca-2 mutant was also similar to that of the wild type), suggesting that Ca(2+) signaling does not play a role in osmoregulation. The hyphal tip morphology and tip-localized mitochondria of the nca-2 mutant were similar to those of the wild type, even when the external [Ca(2+)] was elevated. Thus, although Ca(2+) homeostasis is perturbed in the nca-2 mutant (B. J. Bowman et al., Eukaryot. Cell 10:654-661, 2011), the phenotype does not extend to tip growth or to osmoregulation but is revealed by lower H(+)-ATPase activity.

Electrical Phenotypes of Calcium Transport Mutant Strains of a Filamentous Fungus, Neurospora crassa

PubMed Central

Hamam, Ahmed

2012-01-01

We characterized the electrical phenotypes of mutants with mutations in genes encoding calcium transporters—a mechanosensitive channel homolog (MscS), a Ca2+/H+ exchange protein (cax), and Ca2+-ATPases (nca-1, nca-2, nca-3)—as well as those of double mutants (the nca-2 cax, nca-2 nca-3, and nca-3 cax mutants). The electrical characterization used dual impalements to obtain cable-corrected current-voltage measurements. Only two types of mutants (the MscS mutant; the nca-2 mutant and nca-2-containing double mutants) exhibited lower resting potentials. For the nca-2 mutant, on the basis of unchanged conductance and cyanide-induced depolarization of the potential, the cause is attenuated H+-ATPase activity. The growth of the nca-2 mutant-containing strains was inhibited by elevated extracellular Ca2+ levels, indicative of lesions in Ca2+ homeostasis. However, the net Ca2+ effluxes of the nca-2 mutant, measured noninvasively with a self-referencing Ca2+-selective microelectrode, were similar to those of the wild type. All of the mutants exhibited osmosensitivity similar to that of the wild type (the turgor of the nca-2 mutant was also similar to that of the wild type), suggesting that Ca2+ signaling does not play a role in osmoregulation. The hyphal tip morphology and tip-localized mitochondria of the nca-2 mutant were similar to those of the wild type, even when the external [Ca2+] was elevated. Thus, although Ca2+ homeostasis is perturbed in the nca-2 mutant (B. J. Bowman et al., Eukaryot. Cell 10:654–661, 2011), the phenotype does not extend to tip growth or to osmoregulation but is revealed by lower H+-ATPase activity. PMID:22408225

Predicting effects of structural stress in a genome-reduced model bacterial metabolism

NASA Astrophysics Data System (ADS)

Güell, Oriol; Sagués, Francesc; Serrano, M. Ángeles

2012-08-01

Mycoplasma pneumoniae is a human pathogen recently proposed as a genome-reduced model for bacterial systems biology. Here, we study the response of its metabolic network to different forms of structural stress, including removal of individual and pairs of reactions and knockout of genes and clusters of co-expressed genes. Our results reveal a network architecture as robust as that of other model bacteria regarding multiple failures, although less robust against individual reaction inactivation. Interestingly, metabolite motifs associated to reactions can predict the propagation of inactivation cascades and damage amplification effects arising in double knockouts. We also detect a significant correlation between gene essentiality and damages produced by single gene knockouts, and find that genes controlling high-damage reactions tend to be expressed independently of each other, a functional switch mechanism that, simultaneously, acts as a genetic firewall to protect metabolism. Prediction of failure propagation is crucial for metabolic engineering or disease treatment.

Calorie restriction ameliorates neurodegenerative phenotypes in forebrain-specific presenilin-1 and presenilin-2 double knockout mice.

PubMed

Wu, Pu; Shen, Qian; Dong, Suzhen; Xu, Zhiliang; Tsien, Joe Z; Hu, Yinghe

2008-10-01

Conditional double knockout of presenilin-1 and presenilin-2 (cDKO) in forebrain of mice led to brain atrophy, tau hyperphosphorylation, synaptic dysfunction and cognitive deficit. These brain changes recapitulated most of the neurodegenerative phenotypes of Alzheimer's disease (AD). In this report, we have investigated the effects of 4-month calorie restriction (CR) regimen on different phenotypes in cDKO mice. We found that CR improved novel object recognition and contextual fear conditioning memory in the cDKO mice. Histological and biochemical analysis showed that CR attenuated ventricle enlargement, caspase-3 activation and astrogliosis. In addition, the induction of tau hyperphosphorylation in the cDKO mice was reduced by CR, possibly through reduction of p25 accumulation and aberrant CDK5 activation. Finally, DNA microarray analysis demonstrated that CR could increase the expression of neurogenesis related genes and decrease the expression of inflammation related genes in the hippocampus of cDKO mice. The possible molecular mechanisms of the CR effects on alleviating AD pathogenesis have been discussed.

Construction of a large-scale Burkholderia cenocepacia J2315 transposon mutant library

NASA Astrophysics Data System (ADS)

Wong, Yee-Chin; Pain, Arnab; Nathan, Sheila

2014-09-01

Burkholderia cenocepacia, a pathogenic member of the Burkholderia cepacia complex (Bcc), has emerged as a significant threat towards cystic fibrosis patients, where infection often leads to the fatal clinical manifestation known as cepacia syndrome. Many studies have investigated the pathogenicity of B. cenocepacia as well as its ability to become highly resistant towards many of the antibiotics currently in use. In addition, studies have also been undertaken to understand the pathogen's capacity to adapt and survive in a broad range of environments. Transposon based mutagenesis has been widely used in creating insertional knock-out mutants and coupled with recent advances in sequencing technology, robust tools to study gene function in a genome-wide manner have been developed based on the assembly of saturated transposon mutant libraries. In this study, we describe the construction of a large-scale library of B. cenocepacia transposon mutants. To create transposon mutants of B. cenocepacia strain J2315, electrocompetent bacteria were electrotransformed with the EZ-Tn5 transposome. Tetracyline resistant colonies were harvested off selective agar and pooled. Mutants were generated in multiple batches with each batch consisting of ˜20,000 to 40,000 mutants. Transposon insertion was validated by PCR amplification of the transposon region. In conclusion, a saturated B. cenocepacia J2315 transposon mutant library with an estimated total number of 500,000 mutants was successfully constructed. This mutant library can now be further exploited as a genetic tool to assess the function of every gene in the genome, facilitating the discovery of genes important for bacterial survival and adaptation, as well as virulence.

A Cold-Inducible DEAD-Box RNA Helicase from Arabidopsis thaliana Regulates Plant Growth and Development under Low Temperature.

PubMed

Liu, Yuelin; Tabata, Daisuke; Imai, Ryozo

2016-01-01

DEAD-box RNA helicases comprise a large family and are involved in a range of RNA processing events. Here, we identified one of the Arabidopsis thaliana DEAD-box RNA helicases, AtRH7, as an interactor of Arabidopsis COLD SHOCK DOMAIN PROTEIN 3 (AtCSP3), which is an RNA chaperone involved in cold adaptation. Promoter:GUS transgenic plants revealed that AtRH7 is expressed ubiquitously and that its levels of the expression are higher in rapidly growing tissues. Knockout mutant lines displayed several morphological alterations such as disturbed vein pattern, pointed first true leaves, and short roots, which resemble ribosome-related mutants of Arabidopsis. In addition, aberrant floral development was also observed in rh7 mutants. When the mutants were germinated at low temperature (12°C), both radicle and first leaf emergence were severely delayed; after exposure of seedlings to a long period of cold, the mutants developed aberrant, fewer, and smaller leaves. RNA blots and circular RT-PCR revealed that 35S and 18S rRNA precursors accumulated to higher levels in the mutants than in WT under both normal and cold conditions, suggesting the mutants are partially impaired in pre-rRNA processing. Taken together, the results suggest that AtRH7 affects rRNA biogenesis and plays an important role in plant growth under cold.

Mutation in Rice Abscisic Acid2 Results in Cell Death, Enhanced Disease-Resistance, Altered Seed Dormancy and Development

PubMed Central

Liao, Yongxiang; Bai, Que; Xu, Peizhou; Wu, Tingkai; Guo, Daiming; Peng, Yongbin; Zhang, Hongyu; Deng, Xiaoshu; Chen, Xiaoqiong; Luo, Ming; Ali, Asif; Wang, Wenming; Wu, Xianjun

2018-01-01

Lesion mimic mutants display spontaneous cell death, and thus are valuable for understanding the molecular mechanism of cell death and disease resistance. Although a lot of such mutants have been characterized in rice, the relationship between lesion formation and abscisic acid (ABA) synthesis pathway is not reported. In the present study, we identified a rice mutant, lesion mimic mutant 9150 (lmm9150), exhibiting spontaneous cell death, pre-harvest sprouting, enhanced growth, and resistance to rice bacterial and blast diseases. Cell death in the mutant was accompanied with excessive accumulation of H2O2. Enhanced disease resistance was associated with cell death and upregulation of defense-related genes. Map-based cloning identified a G-to-A point mutation resulting in a D-to-N substitution at the amino acid position 110 of OsABA2 (LOC_Os03g59610) in lmm9150. Knock-out of OsABA2 through CRISPR/Cas9 led to phenotypes similar to those of lmm9150. Consistent with the function of OsABA2 in ABA biosynthesis, ABA level in the lmm9150 mutant was significantly reduced. Moreover, exogenous application of ABA could rescue all the mutant phenotypes of lmm9150. Taken together, our data linked ABA deficiency to cell death and provided insight into the role of ABA in rice disease resistance. PMID:29643863

Mutation in Rice Abscisic Acid2 Results in Cell Death, Enhanced Disease-Resistance, Altered Seed Dormancy and Development.

PubMed

Liao, Yongxiang; Bai, Que; Xu, Peizhou; Wu, Tingkai; Guo, Daiming; Peng, Yongbin; Zhang, Hongyu; Deng, Xiaoshu; Chen, Xiaoqiong; Luo, Ming; Ali, Asif; Wang, Wenming; Wu, Xianjun

2018-01-01

Lesion mimic mutants display spontaneous cell death, and thus are valuable for understanding the molecular mechanism of cell death and disease resistance. Although a lot of such mutants have been characterized in rice, the relationship between lesion formation and abscisic acid (ABA) synthesis pathway is not reported. In the present study, we identified a rice mutant, lesion mimic mutant 9150 ( lmm9150 ), exhibiting spontaneous cell death, pre-harvest sprouting, enhanced growth, and resistance to rice bacterial and blast diseases. Cell death in the mutant was accompanied with excessive accumulation of H 2 O 2 . Enhanced disease resistance was associated with cell death and upregulation of defense-related genes. Map-based cloning identified a G-to-A point mutation resulting in a D-to-N substitution at the amino acid position 110 of OsABA2 (LOC_Os03g59610) in lmm9150 . Knock-out of OsABA2 through CRISPR/Cas9 led to phenotypes similar to those of lmm9150 . Consistent with the function of OsABA2 in ABA biosynthesis, ABA level in the lmm9150 mutant was significantly reduced. Moreover, exogenous application of ABA could rescue all the mutant phenotypes of lmm9150 . Taken together, our data linked ABA deficiency to cell death and provided insight into the role of ABA in rice disease resistance.

Arabidopsis thaliana gonidialess A/Zuotin related factors (GlsA/ZRF) are essential for maintenance of meristem integrity.

PubMed

Guzmán-López, José Alfredo; Abraham-Juárez, María Jazmín; Lozano-Sotomayor, Paulina; de Folter, Stefan; Simpson, June

2016-05-01

Observation of a differential expression pattern, including strong expression in meristematic tissue of an Agave tequilana GlsA/ZRF ortholog suggested an important role for this gene during bulbil formation and developmental changes in this species. In order to better understand this role, the two GlsA/ZFR orthologs present in the genome of Arabidopsis thaliana were functionally characterized by analyzing expression patterns, double mutant phenotypes, promoter-GUS fusions and expression of hormone related or meristem marker genes. Patterns of expression for A. thaliana show that GlsA/ZFR genes are strongly expressed in SAMs and RAMs in mature plants and developing embryos and double mutants showed multiple changes in morphology related to both SAM and RAM tissues. Typical double mutants showed stunted growth of aerial and root tissue, formation of multiple ectopic meristems and effects on cotyledons, leaves and flowers. The KNOX genes STM and BP were overexpressed in double mutants whereas CLV3, WUSCHEL and AS1 were repressed and lack of AtGlsA expression was also associated with changes in localization of auxin and cytokinin. These results suggest that GlsA/ZFR is an essential component of the machinery that maintains the integrity of SAM and RAM tissue and underline the potential to identify new genes or gene functions based on observations in non-model plants.

Histone H3 and the histone acetyltransferase Hat1p contribute to DNA double-strand break repair.

PubMed

Qin, Song; Parthun, Mark R

2002-12-01

The modification of newly synthesized histones H3 and H4 by type B histone acetyltransferases has been proposed to play a role in the process of chromatin assembly. The type B histone acetyltransferase Hat1p and specific lysine residues in the histone H3 NH(2)-terminal tail (primarily lysine 14) are redundantly required for telomeric silencing. As many gene products, including other factors involved in chromatin assembly, have been found to participate in both telomeric silencing and DNA damage repair, we tested whether mutations in HAT1 and the histone H3 tail were also sensitive to DNA-damaging agents. Indeed, mutations both in specific lysine residues in the histone H3 tail and in HAT1 resulted in sensitivity to methyl methanesulfonate. The DNA damage sensitivity of the histone H3 and HAT1 mutants was specific for DNA double-strand breaks, as these mutants were sensitive to the induction of an exogenous restriction endonuclease, EcoRI, but not to UV irradiation. While histone H3 mutations had minor effects on nonhomologous end joining, the primary defect in the histone H3 and HAT1 mutants was in the recombinational repair of DNA double-strand breaks. Epistasis analysis indicates that the histone H3 and HAT1 mutants may influence DNA double-strand break repair through Asf1p-dependent chromatin assembly.

Trehalose Biosynthesis Promotes Pseudomonas aeruginosa Pathogenicity in Plants

PubMed Central

Djonović, Slavica; Urbach, Jonathan M.; Drenkard, Eliana; Bush, Jenifer; Feinbaum, Rhonda; Ausubel, Jonathan L.; Traficante, David; Risech, Martina; Kocks, Christine; Fischbach, Michael A.; Priebe, Gregory P.; Ausubel, Frederick M.

2013-01-01

Pseudomonas aeruginosa strain PA14 is a multi-host pathogen that infects plants, nematodes, insects, and vertebrates. Many PA14 factors are required for virulence in more than one of these hosts. Noting that plants have a fundamentally different cellular architecture from animals, we sought to identify PA14 factors that are specifically required for plant pathogenesis. We show that synthesis by PA14 of the disaccharide trehalose is required for pathogenesis in Arabidopsis, but not in nematodes, insects, or mice. In-frame deletion of two closely-linked predicted trehalose biosynthetic operons, treYZ and treS, decreased growth in Arabidopsis leaves about 50 fold. Exogenously co-inoculated trehalose, ammonium, or nitrate, but not glucose, sulfate, or phosphate suppressed the phenotype of the double ΔtreYZΔtreS mutant. Exogenous trehalose or ammonium nitrate does not suppress the growth defect of the double ΔtreYZΔtreS mutant by suppressing the plant defense response. Trehalose also does not function intracellularly in P. aeruginosa to ameliorate a variety of stresses, but most likely functions extracellularly, because wild-type PA14 rescued the in vivo growth defect of the ΔtreYZΔtreS in trans. Surprisingly, the growth defect of the double ΔtreYZΔtreS double mutant was suppressed by various Arabidopsis cell wall mutants that affect xyloglucan synthesis, including an xxt1xxt2 double mutant that completely lacks xyloglucan, even though xyloglucan mutants are not more susceptible to pathogens and respond like wild-type plants to immune elicitors. An explanation of our data is that trehalose functions to promote the acquisition of nitrogen-containing nutrients in a process that involves the xyloglucan component of the plant cell wall, thereby allowing P. aeruginosa to replicate in the intercellular spaces in a leaf. This work shows how P. aeruginosa, a multi-host opportunistic pathogen, has repurposed a highly conserved “house-keeping” anabolic pathway (trehalose biosynthesis) as a potent virulence factor that allows it to replicate in the intercellular environment of a leaf. PMID:23505373

ABA-deficiency results in reduced plant and fruit size in tomato.

PubMed

Nitsch, L; Kohlen, W; Oplaat, C; Charnikhova, T; Cristescu, S; Michieli, P; Wolters-Arts, M; Bouwmeester, H; Mariani, C; Vriezen, W H; Rieu, I

2012-06-15

Abscisic acid (ABA) deficient mutants, such as notabilis and flacca, have helped elucidating the role of ABA during plant development and stress responses in tomato (Solanum lycopersicum L.). However, these mutants have only moderately decreased ABA levels. Here we report on plant and fruit development in the more strongly ABA-deficient notabilis/flacca (not/flc) double mutant. We observed that plant growth, leaf-surface area, drought-induced wilting and ABA-related gene expression in the different genotypes were strongly correlated with the ABA levels and thus most strongly affected in the not/flc double mutants. These mutants also had reduced fruit size that was caused by an overall smaller cell size. Lower ABA levels in fruits did not correlate with changes in auxin levels, but were accompanied by higher ethylene evolution rates. This suggests that in a wild-type background ABA stimulates cell enlargement during tomato fruit growth via a negative effect on ethylene synthesis. Copyright © 2012 Elsevier GmbH. All rights reserved.

The Anti-Methicillin-Resistant Staphylococcus aureus Quinolone WCK 771 Has Potent Activity against Sequentially Selected Mutants, Has a Narrow Mutant Selection Window against Quinolone-Resistant Staphylococcus aureus, and Preferentially Targets DNA Gyrase▿ †

PubMed Central

Bhagwat, Sachin S.; Mundkur, Lakshmi A.; Gupte, Shrikant V.; Patel, Mahesh V.; Khorakiwala, Habil F.

2006-01-01

WCK 771 is a broad-spectrum fluoroquinolone with enhanced activity against quinolone-resistant staphylococci. To understand the impact of the target-level interactions of WCK 771 on its antistaphylococcal pharmacodynamic properties, we determined the MICs for genetically defined mutants and studied the mutant prevention concentrations (MPCs), the frequency of mutation, and the cidality against the wild type and double mutants. There was a twofold increase in the MICs of WCK 771 for single gyrA mutants, indicating that DNA gyrase is its primary target. All first- and second-step mutants selected by WCK 771 revealed gyrA and grlA mutations, respectively. The MICs of WCK 771 and clinafloxacin were found to be superior to those of other quinolones against strains with double and triple mutations. WCK 771 was also cidal for high-density double mutants at low concentrations. WCK 771 and clinafloxacin showed narrow mutant selection windows compared to those of the other quinolones. Against a panel of 50 high-level quinolone-resistant clinical isolates of staphylococci (ciprofloxacin MIC ≥ 16 μg/ml), the WCK 771 MPCs were ≤2 μg/ml for 68% of the strains and ≤4 μg/ml for 28% of the strains. Our results demonstrate that gyrA is the primary target of WCK 771 and that it has pharmacodynamic properties remarkably different from those of quinolones with dual targets (garenoxacin and moxifloxacin) and topoisomerase IV-specific quinolones (trovafloxacin). WCK 771 displayed an activity profile comparable to that of clinafloxacin, a dual-acting quinolone with a high affinity to DNA gyrase. Overall, the findings signify the key role of DNA gyrase in determining the optimal antistaphylococcal features of quinolones. PMID:16940059

Prediction of reaction knockouts to maximize succinate production by Actinobacillus succinogenes

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

Nag, Ambarish; St. John, Peter C.; Crowley, Michael F.

Succinate is a precursor of multiple commodity chemicals and bio-based succinate production is an active area of industrial bioengineering research. One of the most important microbial strains for bio-based production of succinate is the capnophilic gram-negative bacterium Actinobacillus succinogenes, which naturally produces succinate by a mixed-acid fermentative pathway. To engineer A. succinogenes to improve succinate yields during mixed acid fermentation, it is important to have a detailed understanding of the metabolic flux distribution in A. succinogenes when grown in suitable media. To this end, we have developed a detailed stoichiometric model of the A. succinogenes central metabolism that includes themore » biosynthetic pathways for the main components of biomass - namely glycogen, amino acids, DNA, RNA, lipids and UDP-N-Acetyl-a-D-glucosamine. We have validated our model by comparing model predictions generated via flux balance analysis with experimental results on mixed acid fermentation. Moreover, we have used the model to predict single and double reaction knockouts to maximize succinate production while maintaining growth viability. According to our model, succinate production can be maximized by knocking out either of the reactions catalyzed by the PTA (phosphate acetyltransferase) and ACK (acetyl kinase) enzymes, whereas the double knockouts of PEPCK (phosphoenolpyruvate carboxykinase) and PTA or PEPCK and ACK enzymes are the most effective in increasing succinate production.« less

Prediction of reaction knockouts to maximize succinate production by Actinobacillus succinogenes

DOE PAGES

Nag, Ambarish; St. John, Peter C.; Crowley, Michael F.; ...

2018-01-30

Succinate is a precursor of multiple commodity chemicals and bio-based succinate production is an active area of industrial bioengineering research. One of the most important microbial strains for bio-based production of succinate is the capnophilic gram-negative bacterium Actinobacillus succinogenes, which naturally produces succinate by a mixed-acid fermentative pathway. To engineer A. succinogenes to improve succinate yields during mixed acid fermentation, it is important to have a detailed understanding of the metabolic flux distribution in A. succinogenes when grown in suitable media. To this end, we have developed a detailed stoichiometric model of the A. succinogenes central metabolism that includes themore » biosynthetic pathways for the main components of biomass - namely glycogen, amino acids, DNA, RNA, lipids and UDP-N-Acetyl-a-D-glucosamine. We have validated our model by comparing model predictions generated via flux balance analysis with experimental results on mixed acid fermentation. Moreover, we have used the model to predict single and double reaction knockouts to maximize succinate production while maintaining growth viability. According to our model, succinate production can be maximized by knocking out either of the reactions catalyzed by the PTA (phosphate acetyltransferase) and ACK (acetyl kinase) enzymes, whereas the double knockouts of PEPCK (phosphoenolpyruvate carboxykinase) and PTA or PEPCK and ACK enzymes are the most effective in increasing succinate production.« less

Heteromeric Canonical Transient Receptor Potential 1 and 4 Channels Play a Critical Role in Epileptiform Burst Firing and Seizure-Induced Neurodegeneration

PubMed Central

Phelan, Kevin D.; Mock, Matthew M.; Kretz, Oliver; Shwe, U. Thaung; Kozhemyakin, Maxim; Greenfield, L. John; Dietrich, Alexander; Birnbaumer, Lutz; Freichel, Marc; Flockerzi, Veit

2012-01-01

Canonical transient receptor potential channels (TRPCs) are receptor-operated cation channels that are activated in response to phospholipase C signaling. Although TRPC1 is ubiquitously expressed in the brain, TRPC4 expression is the most restrictive, with the highest expression level limited to the lateral septum. The subunit composition of neuronal TRPC channels remains uncertain because of conflicting data from recombinant expression systems. Here we report that the large depolarizing plateau potential that underlies the epileptiform burst firing induced by metabotropic glutamate receptor agonists in lateral septal neurons was completely abolished in TRPC1/4 double-knockout mice, and was abolished in 74% of lateral septal neurons in TRPC1 knockout mice. Furthermore, neuronal cell death in the lateral septum and the cornu ammonis 1 region of hippocampus after pilocarpine-induced severe seizures was significantly ameliorated in TRPC1/4 double-knockout mice. Our data suggest that both TRPC1 and TRPC4 are essential for an intrinsic membrane conductance mediating the plateau potential in lateral septal neurons, possibly as heteromeric channels. Moreover, excitotoxic neuronal cell death, an underlying process for many neurological diseases, is not mediated merely by ionotropic glutamate receptors but also by heteromeric TRPC channels activated by metabotropic glutamate receptors. TRPC channels could be an unsuspected but critical molecular target for clinical intervention for excitotoxicity. PMID:22144671

Heteromeric canonical transient receptor potential 1 and 4 channels play a critical role in epileptiform burst firing and seizure-induced neurodegeneration.

PubMed

Phelan, Kevin D; Mock, Matthew M; Kretz, Oliver; Shwe, U Thaung; Kozhemyakin, Maxim; Greenfield, L John; Dietrich, Alexander; Birnbaumer, Lutz; Freichel, Marc; Flockerzi, Veit; Zheng, Fang

2012-03-01

Canonical transient receptor potential channels (TRPCs) are receptor-operated cation channels that are activated in response to phospholipase C signaling. Although TRPC1 is ubiquitously expressed in the brain, TRPC4 expression is the most restrictive, with the highest expression level limited to the lateral septum. The subunit composition of neuronal TRPC channels remains uncertain because of conflicting data from recombinant expression systems. Here we report that the large depolarizing plateau potential that underlies the epileptiform burst firing induced by metabotropic glutamate receptor agonists in lateral septal neurons was completely abolished in TRPC1/4 double-knockout mice, and was abolished in 74% of lateral septal neurons in TRPC1 knockout mice. Furthermore, neuronal cell death in the lateral septum and the cornu ammonis 1 region of hippocampus after pilocarpine-induced severe seizures was significantly ameliorated in TRPC1/4 double-knockout mice. Our data suggest that both TRPC1 and TRPC4 are essential for an intrinsic membrane conductance mediating the plateau potential in lateral septal neurons, possibly as heteromeric channels. Moreover, excitotoxic neuronal cell death, an underlying process for many neurological diseases, is not mediated merely by ionotropic glutamate receptors but also by heteromeric TRPC channels activated by metabotropic glutamate receptors. TRPC channels could be an unsuspected but critical molecular target for clinical intervention for excitotoxicity.

Growth of Chlamydomonas reinhardtii in acetate-free medium when co-cultured with alginate-encapsulated, acetate-producing strains of Synechococcus sp. PCC 7002.

PubMed

Therien, Jesse B; Zadvornyy, Oleg A; Posewitz, Matthew C; Bryant, Donald A; Peters, John W

2014-01-01

The model alga Chlamydomonas reinhardtii requires acetate as a co-substrate for optimal production of lipids, and the addition of acetate to culture media has practical and economic implications for algal biofuel production. Here we demonstrate the growth of C. reinhardtii on acetate provided by mutant strains of the cyanobacterium Synechococcus sp. PCC 7002. Optimal growth conditions for co-cultivation of C. reinhardtii with wild-type and mutant strains of Synechococcus sp. 7002 were established. In co-culture, acetate produced by a glycogen synthase knockout mutant of Synechococcus sp. PCC 7002 was able to support the growth of a lipid-accumulating mutant strain of C. reinhardtii defective in starch production. Encapsulation of Synechococcus sp. PCC 7002 using an alginate matrix was successfully employed in co-cultures to limit growth and maintain the stability. The ability of immobilized strains of the cyanobacterium Synechococcus sp. PCC 7002 to produce acetate at a level adequate to support the growth of lipid-accumulating strains of C. reinhartdii offers a potentially practical, photosynthetic alternative to providing exogenous acetate into growth media.

Identification of a response regulator involved in surface attachment, cell-cell aggregation, exopolysaccharide production and virulence in the plant pathogen Xylella fastidiosa.

PubMed

Voegel, Tanja M; Doddapaneni, Harshavardhan; Cheng, Davis W; Lin, Hong; Stenger, Drake C; Kirkpatrick, Bruce C; Roper, M Caroline

2013-04-01

Xylella fastidiosa, the causal agent of Pierce's disease of grapevine, possesses several two-component signal transduction systems that allow the bacterium to sense and respond to changes in its environment. Signals are perceived by sensor kinases that autophosphorylate and transfer the phosphate to response regulators (RRs), which direct an output response, usually by acting as transcriptional regulators. In the X. fastidiosa genome, 19 RRs were found. A site-directed knockout mutant in one unusual RR, designated XhpT, composed of a receiver domain and a histidine phosphotransferase output domain, was constructed. The resulting mutant strain was analysed for changes in phenotypic traits related to biofilm formation and gene expression using microarray analysis. We found that the xhpT mutant was altered in surface attachment, cell-cell aggregation, exopolysaccharide (EPS) production and virulence in grapevine. In addition, this mutant had an altered transcriptional profile when compared with wild-type X. fastidiosa in genes for several biofilm-related traits, such as EPS production and haemagglutinin adhesins. © 2012 BSPP AND BLACKWELL PUBLISHING LTD.

A genome-wide nanotoxicology screen of Saccharomyces cerevisiae mutants reveals the basis for cadmium sulphide quantum dot tolerance and sensitivity.

PubMed

Marmiroli, M; Pagano, L; Pasquali, F; Zappettini, A; Tosato, V; Bruschi, C V; Marmiroli, N

2016-01-01

The use of cadmium sulphide quantum dots (CdS QDs) is increasing, particularly in the electronics industry. Their size (1-10 nm in diameter) is, however, such that they can be taken up by living cells. Here, a bakers' yeast (Saccharomyces cerevisiae) deletion mutant collection has been exploited to provide a high-throughput means of revealing the genetic basis for tolerance/susceptibility to CdS QD exposure. The deletion of 112 genes, some associated with the abiotic stress response, some with various metabolic processes, some with mitochondrial organization, some with transport and some with DNA repair, reduced the level of tolerance to CdS QDs. A gene ontology analysis highlighted the role of oxidative stress in determining the cellular response. The transformation of sensitive mutants with centromeric plasmids harbouring DNA from a wild type strain restored the wild type growth phenotype when the complemented genes encoded either HSC82, DSK2 or ALD3. The use of these simple eukaryote knock-out mutants for functional toxicogenomic analysis will inform studies focusing on higher organisms.

Targeted Disruption of the Basic Krüppel-Like Factor Gene (Klf3) Reveals a Role in Adipogenesis ▿ †

PubMed Central

Sue, Nancy; Jack, Briony H. A.; Eaton, Sally A.; Pearson, Richard C. M.; Funnell, Alister P. W.; Turner, Jeremy; Czolij, Robert; Denyer, Gareth; Bao, Shisan; Molero-Navajas, Juan Carlos; Perkins, Andrew; Fujiwara, Yuko; Orkin, Stuart H.; Bell-Anderson, Kim; Crossley, Merlin

2008-01-01

Krüppel-like factors (KLFs) recognize CACCC and GC-rich sequences in gene regulatory elements. Here, we describe the disruption of the murine basic Krüppel-like factor gene (Bklf or Klf3). Klf3 knockout mice have less white adipose tissue, and their fat pads contain smaller and fewer cells. Adipocyte differentiation is altered in murine embryonic fibroblasts from Klf3 knockouts. Klf3 expression was studied in the 3T3-L1 cellular system. Adipocyte differentiation is accompanied by a decline in Klf3 expression, and forced overexpression of Klf3 blocks 3T3-L1 differentiation. Klf3 represses transcription by recruiting C-terminal binding protein (CtBP) corepressors. CtBPs bind NADH and may function as metabolic sensors. A Klf3 mutant that does not bind CtBP cannot block adipogenesis. Other KLFs, Klf2, Klf5, and Klf15, also regulate adipogenesis, and functional CACCC elements occur in key adipogenic genes, including in the C/ebpα promoter. We find that C/ebpα is derepressed in Klf3 and Ctbp knockout fibroblasts and adipocytes from Klf3 knockout mice. Chromatin immunoprecipitations confirm that Klf3 binds the C/ebpα promoter in vivo. These results implicate Klf3 and CtBP in controlling adipogenesis. PMID:18391014

Apoptosis-inducing Factor (AIF) and Its Family Member Protein, AMID, Are Rotenone-sensitive NADH:Ubiquinone Oxidoreductases (NDH-2)*

PubMed Central

Elguindy, Mahmoud M.; Nakamaru-Ogiso, Eiko

2015-01-01

Apoptosis-inducing factor (AIF) and AMID (AIF-homologous mitochondrion-associated inducer of death) are flavoproteins. Although AIF was originally discovered as a caspase-independent cell death effector, bioenergetic roles of AIF, particularly relating to complex I functions, have since emerged. However, the role of AIF in mitochondrial respiration and redox metabolism has remained unknown. Here, we investigated the redox properties of human AIF and AMID by comparing them with yeast Ndi1, a type 2 NADH:ubiquinone oxidoreductase (NDH-2) regarded as alternative complex I. Isolated AIF and AMID containing naturally incorporated FAD displayed no NADH oxidase activities. However, after reconstituting isolated AIF or AMID into bacterial or mitochondrial membranes, N-terminally tagged AIF and AMID displayed substantial NADH:O2 activities and supported NADH-linked proton pumping activities in the host membranes almost as efficiently as Ndi1. NADH:ubiquinone-1 activities in the reconstituted membranes were highly sensitive to 2-n-heptyl-4-hydroxyquinoline-N-oxide (IC50 = ∼1 μm), a quinone-binding inhibitor. Overexpressing N-terminally tagged AIF and AMID enhanced the growth of a double knock-out Escherichia coli strain lacking complex I and NDH-2. In contrast, C-terminally tagged AIF and NADH-binding site mutants of N-terminally tagged AIF and AMID failed to show both NADH:O2 activity and the growth-enhancing effect. The disease mutant AIFΔR201 showed decreased NADH:O2 activity and growth-enhancing effect. Furthermore, we surprisingly found that the redox activities of N-terminally tagged AIF and AMID were sensitive to rotenone, a well known complex I inhibitor. We propose that AIF and AMID are previously unidentified mammalian NDH-2 enzymes, whose bioenergetic function could be supplemental NADH oxidation in cells. PMID:26063804

Compounds from Sichuan and Melegueta peppers activate, covalently and non-covalently, TRPA1 and TRPV1 channels

PubMed Central

Riera, CE; Menozzi-Smarrito, C; Affolter, M; Michlig, S; Munari, C; Robert, F; Vogel, H; Simon, SA; le Coutre, J

2009-01-01

Background and purpose: Oily extracts of Sichuan and Melegueta peppers evoke pungent sensations mediated by different alkylamides [mainly hydroxy-α-sanshool (α-SOH)] and hydroxyarylalkanones (6-shogaol and 6-paradol). We assessed how transient receptor potential ankyrin 1 (TRPA1) and TRP vanilloid 1 (TRPV1), two chemosensory ion channels, participate in these pungent sensations. Experimental approach: The structure–activity relationships of these molecules on TRPA1 and TRPV1 was measured by testing natural and synthetic analogues using calcium and voltage imaging on dissociated dorsal root ganglia neurons and human embryonic kidney 293 cells expressing the wild-type channels or specific cysteine mutants using glutathione trapping as a model to probe TRPA1 activation. In addition, using Trpv1 knockout mice, the compounds' aversive responses were measured in a taste brief-access test. Key results: For TRPA1 activation, the cis C6 double bond in the polyenic chain of α-SOH was critical, whereas no structural specificity was required for activation of TRPV1. Both 6-shogaol and 6-paradol were found to activate TRPV1 and TRPA1 channels, whereas linalool, an abundant terpene in Sichuan pepper, activated TRPA1 but not TRPV1 channels. Alkylamides and 6-shogaol act on TRPA1 by covalent bonding whereas none of these compounds activated TRPV1 through such interactions. Finally, TRPV1 mutant mice retained sensitivity to 6-shogaol but were not responsive to α-SOH. Conclusions and implications: The pungent nature of components of Sichuan and Melegueta peppers was mediated via interactions with TRPA1 and TRPV1 channels and may explain the aversive properties of these compounds. PMID:19594761

Elongation factor P is dispensable in Escherichia coli and Pseudomonas aeruginosa.

PubMed

Balibar, Carl J; Iwanowicz, Dorothy; Dean, Charles R

2013-09-01

Elongation factor P (EF-P) is a highly conserved ribosomal initiation factor responsible for stimulating formation of the first peptide bond. Its essentiality has been debated and may differ depending on the organism. Here, we demonstrate that EF-P is dispensable in Escherichia coli and Pseudomonas aeruginosa under laboratory growth conditions. Although knockouts are viable, growth rates are diminished compared with wild-type strains. Despite this cost in fitness, these mutants are not more susceptible to a wide range of antibiotics; including ribosome targeting antibiotics, such as lincomycin, chloramphenicol, and streptomycin, which have been shown previously to disrupt EF-P function in vitro. In Pseudomonas, knockout of efp leads to an upregulation of mexX, a phenotype previously observed with other genetic lesions affecting ribosome function and that can be induced by the treatment with antibiotics affecting protein synthesis.

Hydrophobins contribute to root colonization and stress responses in the rhizosphere-competent insect pathogenic fungus Beauveria bassiana.

PubMed

Moonjely, Soumya; Keyhani, Nemat O; Bidochka, Michael J

2018-04-01

The hyd1/hyd2 hydrophobins are important constituents of the conidial cell wall of the insect pathogenic fungus Beauveria bassiana. This fungus can also form intimate associations with several plant species. Here, we show that inactivation of two Class I hydrophobin genes, hyd1 or hyd2, significantly decreases the interaction of B. bassiana with bean roots. Curiously, the ∆hyd1/∆hyd2 double mutant was less impaired in root association than Δhyd1 or Δhyd2. Loss of hyd genes affected growth rate, conidiation ability and oosporein production. Expression patterns for genes involved in conidiation, cell wall integrity, insect virulence, signal transduction, adhesion, hydrophobicity and oosporein production were screened in the deletion mutants grown in different conditions. Repression of the major MAP-Kinase signal transduction pathways (Slt2 MAPK pathway) was observed that was more pronounced in the single versus double hyd mutants under certain conditions. The ∆hyd1/∆hyd2 double mutant showed up-regulation of the Hog1 MAPK and the Msn2 transcription factor under certain conditions when compared to the wild-type or single hyd mutants. The expression of the bad2 adhesin and the oosporein polyketide synthase 9 gene was severely reduced in all of the mutants. On the other hand, fewer changes were observed in the expression of key conidiation and cell wall integrity genes in hyd mutants compared to wild-type. Taken together, the data from this study indicated pleiotropic consequences of deletion of hyd1 and hyd2 on signalling and stress pathways as well as the ability of the fungus to form stable associations with plant roots.

Mutational Evidence for the Critical Role of CBF Transcription Factors in Cold Acclimation in Arabidopsis1

PubMed Central

Zhang, Zhengjing; Li, Yuanya

2016-01-01

The three tandemly arranged CBF genes, CBF1, CBF2, and CBF3, are involved in cold acclimation. Due to the lack of stable loss-of-function Arabidopsis (Arabidopsis thaliana) mutants deficient in all three CBF genes, it is still unclear whether the CBF genes are essential for freezing tolerance and whether they may have other functions besides cold acclimation. In this study, we used the CRISPR/Cas9 system to generate cbf single, double, and triple mutants. Compared to the wild type, the cbf triple mutants are extremely sensitive to freezing after cold acclimation, demonstrating that the three CBF genes are essential for cold acclimation. Our results show that the three CBF genes also contribute to basal freezing tolerance. Unexpectedly, we found that the cbf triple mutants are defective in seedling development and salt stress tolerance. Transcript profiling revealed that the CBF genes regulate 414 cold-responsive (COR) genes, of which 346 are CBF-activated genes and 68 are CBF-repressed genes. The analysis suggested that CBF proteins are extensively involved in the regulation of carbohydrate and lipid metabolism, cell wall modification, and gene transcription. Interestingly, like the triple mutants, cbf2 cbf3 double mutants are more sensitive to freezing after cold acclimation compared to the wild type, but cbf1 cbf3 double mutants are more resistant, suggesting that CBF2 is more important than CBF1 and CBF3 in cold acclimation-dependent freezing tolerance. Our results not only demonstrate that the three CBF genes together are required for cold acclimation and freezing tolerance, but also reveal that they are important for salt tolerance and seedling development. PMID:27252305

Riboflavin Is an Active Redox Cofactor in the Na+-pumping NADH:Quinone Oxidoreductase (Na+-NQR) from Vibrio cholerae*

PubMed Central

Juárez, Oscar; Nilges, Mark J.; Gillespie, Portia; Cotton, Jennifer; Barquera, Blanca

2008-01-01

Here we present new evidence that riboflavin is present as one of four flavins in Na+-NQR. In particular, we present conclusive evidence that the source of the neutral radical is not one of the FMNs and that riboflavin is the center that gives rise to the neutral flavosemiquinone. The riboflavin is a bona fide redox cofactor and is likely to be the last redox carrier of the enzyme, from which electrons are donated to quinone. We have constructed a double mutant that lacks both covalently bound FMN cofactors (NqrB-T236Y/NqrC-T225Y) and have studied this mutant together with the two single mutants (NqrB-T236Y and NqrC-T225Y) and a mutant that lacks the noncovalently bound FAD in NqrF (NqrF-S246A). The double mutant contains riboflavin and FAD in a 0.6:1 ratio, as the only flavins in the enzyme; noncovalently bound flavins were detected. In the oxidized form, the double mutant exhibits an EPR signal consistent with a neutral flavosemiquinone radical, which is abolished on reduction of the enzyme. The same radical can be observed in the FAD deletion mutant. Furthermore, when the oxidized enzyme reacts with ubiquinol (the reduced form of the usual electron acceptor) in a process that reverses the physiological direction of the electron flow, a single kinetic phase is observed. The kinetic difference spectrum of this process is consistent with one-electron reduction of a neutral flavosemiquinone. The presence of riboflavin in the role of a redox cofactor is thus far unique to Na+-NQR. PMID:18832377

mei-41 and bub1 block mitosis at two distinct steps in response to incomplete DNA replication in Drosophila embryos.

PubMed

Garner, M; van Kreeveld, S; Su, T T

2001-10-16

Drosophila double park encodes a homolog of Cdt1 that functions in initiation of DNA replication in fission yeast and Xenopus. dup mutants complete the first 15 embryonic cell cycles, presumably via maternal dup products, and show defects in the 16(th) S phase (S16). Cells carrying dup(a1) allele forgo S16 altogether but enter mitosis 16 (M16). We find that the timing of entry into M16 is similar in dup(a1) and heterozygous or wild-type (wt) controls. In contrast, we find that mutant cells carrying another allele, dup(a3), undergo a partial S16 and delay the entry into M16. Thus, initiation of S16 appears necessary for delaying M16. This delay is absent in double mutants of dup(a3) and mei-41 (Drosophila ATR), indicating that a mei-41-dependent checkpoint acts to delay the entry into mitosis in response to incomplete DNA replication. dup(a3) and dup(a1) mutant cells that enter M16 become arrested in M16. We find that mitotic cyclins are stabilized and that a spindle checkpoint protein, Bub1, localizes onto chromosomes during mitotic arrest in dup mutants. These features suggest an arrest prior to metaphase-anaphase transition. dup(a3) bub1 double mutant cells exit M16, indicating that a bub1-mediated checkpoint acts to block mitotic exit in dup mutants. To our knowledge, this is the first report of (1) incomplete DNA replication affecting both the entry into and the exit from mitosis in a single cell cycle via different mechanisms and (2) the role of bub1 in regulating mitotic exit in response to incomplete DNA replication.

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.

Acquisition of a Circular Dichroism Spectrometer to Study Biological Molecules at Interfaces

DTIC Science & Technology

2016-02-10

H133C double mutant) was immobilized by itself and co-immobilized with poly- sorbitol methacrylate on maleimide SAM surfaces. The purpose of this...work is to see whether the hydromimetic poly- sorbitol methacrylate can protect protein secondary structure when the co-immobilized protein-polymer...partially lost its secondary structure after the sample was exposed to air for 1 day. The co-immobilized NsfB-H360C-H133C double mutant and poly- sorbitol

Immunization against Genital Herpes with a Vaccine Virus That has Defects in Productive and Latent Infection

NASA Astrophysics Data System (ADS)

da Costa, Xavier J.; Jones, Cheryl A.; Knipe, David M.

1999-06-01

An effective vaccine for genital herpes has been difficult to achieve because of the limited efficacy of subunit vaccines and the safety concerns about live viruses. As an alternative approach, mutant herpes simplex virus strains that are replication-defective can induce protective immunity. To increase the level of safety and to prove that replication was not needed for immunization, we constructed a mutant herpes simplex virus 2 strain containing two deletion mutations, each of which eliminated viral replication. The double-mutant virus induces protective immunity that can reduce acute viral shedding and latent infection in a mouse genital model, but importantly, the double-mutant virus shows a phenotypic defect in latent infection. This herpes vaccine strain, which is immunogenic but has defects in both productive and latent infection, provides a paradigm for the design of vaccines and vaccine vectors for other sexually transmitted diseases, such as AIDS.

Histochemical and cellular changes accompanying the appearance of lung fibrosis in an experimental mouse model for Hermansky Pudlak syndrome

PubMed Central

Lyerla, Timothy

2010-01-01

Hermansky Pudlak syndrome (HPS) is a heterogeneous recessive genetic disease with a tendency to develop lung fibrosis with aging. A mouse strain with two mutant HPS genes affecting separate vesicle trafficking pathways, C57BL/6-Hps1ep-Ap3b1pe, exhibits severe lung abnormalities at young ages, including enlarged alveolar type II (ATII) cells with giant lamellar bodies and foamy alveolar macrophages (AMs), which are readily identified histologically. In this study, the appearance of lung fibrosis in older animals was studied using classical histological and biochemical methods. The HPS double mutant mice, but not Chediak Higashi syndrome (C57BL/6-Lystbg-J-J, CHS) or C57BL/6J black control (WT) mice, were found to develop lung fibrosis at about 17 months of age using Masson trichrome staining, which was confirmed by hydroxyproline analysis. TGF β1 levels were elevated in bronchial alveolar lavage samples at all ages tested in the double mutant, but not WT or CHS mice, indicative of a prefibrotic condition in this experimental strain; and AMs were highly positive for this cytokine using immunohistochemistry staining. Prosurfactant protein C staining for ATII cells showed redistribution and dysmorphism of these cells with aging, but there was no evidence for epithelial-mesenchymal transition of ATII cells by dual staining for prosurfactant C protein and α-smooth muscle actin. This investigation showed that the HPS double mutant mouse strain develops interstitial pneumonia (HPSIP) past 1 year of age, which may be initiated by abnormal ATII cells and exacerbated by AM activation. With prominent prefibrotic abnormalities, this double mutant may serve as a model for interventive therapy in HPS. PMID:20603711

Different sets of ER-resident J-proteins regulate distinct polar nuclear-membrane fusion events in Arabidopsis thaliana.

PubMed

Maruyama, Daisuke; Yamamoto, Masaya; Endo, Toshiya; Nishikawa, Shuh-ichi

2014-11-01

Angiosperm female gametophytes contain a central cell with two polar nuclei. In many species, including Arabidopsis thaliana, the polar nuclei fuse during female gametogenesis. We previously showed that BiP, an Hsp70 in the endoplasmic reticulum (ER), was essential for membrane fusion during female gametogenesis. Hsp70 function requires partner proteins for full activity. J-domain containing proteins (J-proteins) are the major Hsp70 functional partners. A. thaliana ER contains three soluble J-proteins, AtERdj3A, AtERdj3B, and AtP58(IPK). Here, we analyzed mutants of these proteins and determined that double-mutant ovules lacking AtP58(IPK) and AtERdj3A or AtERdj3B were defective in polar nuclear fusion. Electron microscopy analysis identified that polar nuclei were in close contact, but no membrane fusion occurred in mutant ovules lacking AtP58(IPK) and AtERdj3A. The polar nuclear outer membrane appeared to be connected via the ER remaining at the inner unfused membrane in mutant ovules lacking AtP58(IPK) and AtERdj3B. These results indicate that ER-resident J-proteins, AtP58(IPK)/AtERdj3A and AtP58(IPK)/AtERdj3B, function at distinct steps of polar nuclear-membrane fusion. Similar to the bip1 bip2 double mutant female gametophytes, the aterdj3a atp58(ipk) double mutant female gametophytes defective in fusion of the outer polar nuclear membrane displayed aberrant endosperm proliferation after fertilization with wild-type pollen. However, endosperm proliferated normally after fertilization of the aterdj3b atp58(ipk) double mutant female gametophytes defective in fusion of the inner membrane. Our results indicate that the polar nuclear fusion defect itself does not cause an endosperm proliferation defect. © The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

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.

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

Mu opioid receptors on primary afferent nav1.8 neurons contribute to opiate-induced analgesia: insight from conditional knockout mice.

PubMed

Weibel, Raphaël; Reiss, David; Karchewski, Laurie; Gardon, Olivier; Matifas, Audrey; Filliol, Dominique; Becker, Jérôme A J; Wood, John N; Kieffer, Brigitte L; Gaveriaux-Ruff, Claire

2013-01-01

Opiates are powerful drugs to treat severe pain, and act via mu opioid receptors distributed throughout the nervous system. Their clinical use is hampered by centrally-mediated adverse effects, including nausea or respiratory depression. Here we used a genetic approach to investigate the potential of peripheral mu opioid receptors as targets for pain treatment. We generated conditional knockout (cKO) mice in which mu opioid receptors are deleted specifically in primary afferent Nav1.8-positive neurons. Mutant animals were compared to controls for acute nociception, inflammatory pain, opiate-induced analgesia and constipation. There was a 76% decrease of mu receptor-positive neurons and a 60% reduction of mu-receptor mRNA in dorsal root ganglia of cKO mice. Mutant mice showed normal responses to heat, mechanical, visceral and chemical stimuli, as well as unchanged morphine antinociception and tolerance to antinociception in models of acute pain. Inflammatory pain developed similarly in cKO and controls mice after Complete Freund's Adjuvant. In the inflammation model, however, opiate-induced (morphine, fentanyl and loperamide) analgesia was reduced in mutant mice as compared to controls, and abolished at low doses. Morphine-induced constipation remained intact in cKO mice. We therefore genetically demonstrate for the first time that mu opioid receptors partly mediate opiate analgesia at the level of Nav1.8-positive sensory neurons. In our study, this mechanism operates under conditions of inflammatory pain, but not nociception. Previous pharmacology suggests that peripheral opiates may be clinically useful, and our data further demonstrate that Nav1.8 neuron-associated mu opioid receptors are feasible targets to alleviate some forms of persistent pain.

Mu Opioid Receptors on Primary Afferent Nav1.8 Neurons Contribute to Opiate-Induced Analgesia: Insight from Conditional Knockout Mice

PubMed Central

Karchewski, Laurie; Gardon, Olivier; Matifas, Audrey; Filliol, Dominique; Becker, Jérôme A. J.; Wood, John N.; Kieffer, Brigitte L.; Gaveriaux-Ruff, Claire

2013-01-01

Opiates are powerful drugs to treat severe pain, and act via mu opioid receptors distributed throughout the nervous system. Their clinical use is hampered by centrally-mediated adverse effects, including nausea or respiratory depression. Here we used a genetic approach to investigate the potential of peripheral mu opioid receptors as targets for pain treatment. We generated conditional knockout (cKO) mice in which mu opioid receptors are deleted specifically in primary afferent Nav1.8-positive neurons. Mutant animals were compared to controls for acute nociception, inflammatory pain, opiate-induced analgesia and constipation. There was a 76% decrease of mu receptor-positive neurons and a 60% reduction of mu-receptor mRNA in dorsal root ganglia of cKO mice. Mutant mice showed normal responses to heat, mechanical, visceral and chemical stimuli, as well as unchanged morphine antinociception and tolerance to antinociception in models of acute pain. Inflammatory pain developed similarly in cKO and controls mice after Complete Freund’s Adjuvant. In the inflammation model, however, opiate-induced (morphine, fentanyl and loperamide) analgesia was reduced in mutant mice as compared to controls, and abolished at low doses. Morphine-induced constipation remained intact in cKO mice. We therefore genetically demonstrate for the first time that mu opioid receptors partly mediate opiate analgesia at the level of Nav1.8-positive sensory neurons. In our study, this mechanism operates under conditions of inflammatory pain, but not nociception. Previous pharmacology suggests that peripheral opiates may be clinically useful, and our data further demonstrate that Nav1.8 neuron-associated mu opioid receptors are feasible targets to alleviate some forms of persistent pain. PMID:24069332

Use of a cryptic splice site for the expression of huntingtin interacting protein 1 in select normal and neoplastic tissues.

PubMed

Graves, Chiron W; Philips, Steven T; Bradley, Sarah V; Oravecz-Wilson, Katherine I; Li, Lina; Gauvin, Alice; Ross, Theodora S

2008-02-15

Huntingtin interacting protein 1 (HIP1) is a 116-kDa endocytic protein, which is necessary for the maintenance of several tissues in vivo as its deficiency leads to degenerative adult phenotypes. HIP1 deficiency also inhibits prostate tumor progression in mice. To better understand how deficiency of HIP1 leads to such phenotypes, we analyzed tumorigenic potential in mice homozygous for a Hip1 mutant allele, designated Hip1(Delta 3-5), which is predicted to result in a frame-shifted, nonsense mutation in the NH(2) terminus of HIP1. In contrast to our previous studies using the Hip1 null allele, an inhibition of tumorigenesis was not observed as a result of the homozygosity of the nonsense Delta 3-5 allele. To further examine the contrasting results from the prior Hip1 mutant mice, we cultured tumor cells from homozygous Delta 3-5 allele-bearing mice and discovered the presence of a 110-kDa form of HIP1 in tumor cells. Upon sequencing of Hip1 DNA and message from these tumors, we determined that this 110-kDa form of HIP1 is the product of splicing of a cryptic U12-type AT-AC intron. This event results in the insertion of an AG dinucleotide between exons 2 and 6 and restoration of the reading frame. Remarkably, this mutant protein retains its capacity to bind lipids, clathrin, AP2, and epidermal growth factor receptor providing a possible explanation for why tumorigenesis was not altered after this knockout mutation. Our data show how knowledge of the transcript that is produced by a knockout allele can lead to discovery of novel types of molecular compensation at the level of splicing.

Use of a Cryptic Splice Site for the Expression of Huntingtin Interacting Protein 1 in Select Normal and Neoplastic Tissues

PubMed Central

Graves, Chiron W.; Philips, Steven T.; Bradley, Sarah V.; Oravecz-Wilson, Katherine I.; Li, Lina; Gauvin, Alice; Ross, Theodora S.

2011-01-01

Huntingtin interacting protein 1 (HIP1) is a 116-kDa endocytic protein, which is necessary for the maintenance of several tissues in vivo as its deficiency leads to degenerative adult phenotypes. HIP1 deficiency also inhibits prostate tumor progression in mice. To better understand how deficiency of HIP1 leads to such phenotypes, we analyzed tumorigenic potential in mice homozygous for a Hip1 mutant allele, designated Hip1Δ3-5, which is predicted to result in a frame-shifted, nonsense mutation in the NH2 terminus of HIP1. In contrast to our previous studies using the Hip1 null allele, an inhibition of tumorigenesis was not observed as a result of the homozygosity of the nonsense Δ3-5 allele. To further examine the contrasting results from the prior Hip1 mutant mice, we cultured tumor cells from homozygous Δ3-5 allele–bearing mice and discovered the presence of a 110-kDa form of HIP1 in tumor cells. Upon sequencing of Hip1 DNA and message from these tumors, we determined that this 110-kDa form of HIP1 is the product of splicing of a cryptic U12-type AT-AC intron. This event results in the insertion of an AG dinucleotide between exons 2 and 6 and restoration of the reading frame. Remarkably, this mutant protein retains its capacity to bind lipids, clathrin, AP2, and epidermal growth factor receptor providing a possible explanation for why tumorigenesis was not altered after this knockout mutation. Our data show how knowledge of the transcript that is produced by a knockout allele can lead to discovery of novel types of molecular compensation at the level of splicing. PMID:18281481

Generation of α1,3-galactosyltransferase and cytidine monophospho-N-acetylneuraminic acid hydroxylase gene double-knockout pigs

PubMed Central

MIYAGAWA, Shuji; MATSUNARI, Hitomi; WATANABE, Masahito; NAKANO, Kazuaki; UMEYAMA, Kazuhiro; SAKAI, Rieko; TAKAYANAGI, Shuko; TAKEISHI, Toki; FUKUDA, Tooru; YASHIMA, Sayaka; MAEDA, Akira; EGUCHI, Hiroshi; OKUYAMA, Hiroomi; NAGAYA, Masaki; NAGASHIMA, Hiroshi

2015-01-01

Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) are new tools for producing gene knockout (KO) animals. The current study reports produced genetically modified pigs, in which two endogenous genes were knocked out. Porcine fibroblast cell lines were derived from homozygous α1,3-galactosyltransferase (GalT) KO pigs. These cells were subjected to an additional KO for the cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) gene. A pair of ZFN-encoding mRNAs targeting exon 8 of the CMAH gene was used to generate the heterozygous CMAH KO cells, from which cloned pigs were produced by somatic cell nuclear transfer (SCNT). One of the cloned pigs obtained was re-cloned after additional KO of the remaining CMAH allele using the same ZFN-encoding mRNAs to generate GalT/CMAH-double homozygous KO pigs. On the other hand, the use of TALEN-encoding mRNAs targeting exon 7 of the CMAH gene resulted in efficient generation of homozygous CMAH KO cells. These cells were used for SCNT to produce cloned pigs homozygous for a double GalT/CMAH KO. These results demonstrate that the combination of TALEN-encoding mRNA, in vitro selection of the nuclear donor cells and SCNT provides a robust method for generating KO pigs. PMID:26227017

Generation of α1,3-galactosyltransferase and cytidine monophospho-N-acetylneuraminic acid hydroxylase gene double-knockout pigs.

PubMed

Miyagawa, Shuji; Matsunari, Hitomi; Watanabe, Masahito; Nakano, Kazuaki; Umeyama, Kazuhiro; Sakai, Rieko; Takayanagi, Shuko; Takeishi, Toki; Fukuda, Tooru; Yashima, Sayaka; Maeda, Akira; Eguchi, Hiroshi; Okuyama, Hiroomi; Nagaya, Masaki; Nagashima, Hiroshi

2015-01-01

Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) are new tools for producing gene knockout (KO) animals. The current study reports produced genetically modified pigs, in which two endogenous genes were knocked out. Porcine fibroblast cell lines were derived from homozygous α1,3-galactosyltransferase (GalT) KO pigs. These cells were subjected to an additional KO for the cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) gene. A pair of ZFN-encoding mRNAs targeting exon 8 of the CMAH gene was used to generate the heterozygous CMAH KO cells, from which cloned pigs were produced by somatic cell nuclear transfer (SCNT). One of the cloned pigs obtained was re-cloned after additional KO of the remaining CMAH allele using the same ZFN-encoding mRNAs to generate GalT/CMAH-double homozygous KO pigs. On the other hand, the use of TALEN-encoding mRNAs targeting exon 7 of the CMAH gene resulted in efficient generation of homozygous CMAH KO cells. These cells were used for SCNT to produce cloned pigs homozygous for a double GalT/CMAH KO. These results demonstrate that the combination of TALEN-encoding mRNA, in vitro selection of the nuclear donor cells and SCNT provides a robust method for generating KO pigs.

Global deletion of glutathione S-Transferase A4 exacerbates developmental nonalcoholic steatohepatitis

USDA-ARS?s Scientific Manuscript database

We established a mouse model of developmental nonalcoholic steatohepatitis (NASH) by feeding a high polyunsaturated fat liquid diet to female glutathione-S-transferase 4-4 (Gsta4-/-)/peroxisome proliferator activated receptor a (Ppara-/-) double knockout 129/SvJ mice for 12 weeks from weaning. We us...

Growth and sporulation of a pyrimidine spore color mutant of Sordaria fimicola.

PubMed

el-Ani, A S

1967-04-07

A nonautonomous spore color mutant of Sordaria fimicola is a pyrimidine auxotroph that produces hyaline nonviable ascospores. Uracil, uridine, and cytidine are more effective growth factors than cytosine and thymine and, in high concentrations, render the mutant self-fertile by inducing the ascospores to resume development and maturation. Crosses with the unlinked arginine non-autonomus spore color mutant st-59 yielded the double mutant st-59 pyr that requires both arginine and a pyrimidine for growth, which indicates a lack of suppression of the pyrimidine requirement by the arginine locus.

A novel aminoacid determinant of HIV-1 restriction in the TRIM5α variable 1 region isolated in a random mutagenic screen.

PubMed

Pham, Quang Toan; Veillette, Maxime; Brandariz-Nuñez, Alberto; Pawlica, Paulina; Thibert-Lefebvre, Caroline; Chandonnet, Nadia; Diaz-Griffero, Felipe; Berthoux, Lionel

2013-05-01

Human-derived antiretroviral transgenes are of great biomedical interest and are actively pursued. HIV-1 is efficiently inhibited at post-entry, pre-integration replication stages by point mutations in the variable region 1 (v1) of the human restriction factor TRIM5α. Here we use a mutated megaprimer approach to create a mutant library of TRIM5αHu v1 and to isolate a mutation at Gly330 (G330E) that inhibits transduction of an HIV-1 vector as efficiently as the previously described mutants at positions Arg332 and Arg335. As was the case for these other mutations, modification of the local v1 charge toward increased acidity was key to inhibiting HIV-1. G330E TRIM5αHu also disrupted replication-competent HIV-1 propagation in a human T cell line. Interestingly, G330E did not enhance restriction of HIV-1 when combined with mutations at Arg332 or Arg335. Accordingly, the triple mutant G330E-R332G-R335G bound purified recombinant HIV-1 capsid tubes less efficiently than the double mutant R332G-R335G did. In a structural model of the TRIM5αHu PRYSPRY domain, the addition of G330E to the double mutant R332G-R335G caused extensive changes to the capsid-binding surface, which may explain why the triple mutant was no more restrictive than the double mutant. The HIV-1 inhibitory potential of Gly330 mutants was not predicted by examination of natural TRIM5α orthologs that are known to strongly inhibit HIV-1. This work underlines the potential of random mutagenesis to isolate novel variants of human proteins with antiviral properties. Copyright © 2013 Elsevier B.V. All rights reserved.

Co-regulation of intragenic microRNA miR-153 and its host gene Ia-2 β: identification of miR-153 target genes with functions related to IA-2β in pancreas and brain.

PubMed

Mandemakers, W; Abuhatzira, L; Xu, H; Caromile, L A; Hébert, S S; Snellinx, A; Morais, V A; Matta, S; Cai, T; Notkins, A L; De Strooper, B

2013-07-01

We analysed the genomic organisation of miR-153, a microRNA embedded in genes that encode two of the major type 1 diabetes autoantigens, islet-associated protein (IA)-2 and IA-2β. We also identified miR-153 target genes that correlated with IA-2β localisation and function. A bioinformatics approach was used to identify miR-153's genomic organisation. To analyse the co-regulation of miR-153 and IA-2β, quantitative PCR analysis of miR-153 and Ia-2β (also known as Ptprn2) was performed after a glucose stimulation assay in MIN6B cells and isolated murine pancreatic islets, and also in wild-type Ia-2 (also known as Ptprn), Ia-2β single knockout and Ia-2/Ia-2β double knockout mouse brain and pancreatic islets. Bioinformatics identification of miR-153 target genes and validation via luciferase reporter assays, western blotting and quantitative PCR were also carried out. Two copies of miR-153, miR-153-1 and miR-153-2, are localised in intron 19 of Ia-2 and Ia-2β, respectively. In rodents, only miR-153-2 is conserved. We demonstrated that expression of miR-153-2 and Ia-2β in rodents is partially co-regulated as demonstrated by a strong reduction of miR-153 expression levels in Ia-2β knockout and Ia-2/Ia-2β double knockout mice. miR-153 levels were unaffected in Ia-2 knockout mice. In addition, glucose stimulation, which increases Ia-2 and Ia-2β expression, also significantly increased expression of miR-153. Several predicted targets of miR-153 were reduced after glucose stimulation in vitro, correlating with the increase in miR-153 levels. This study suggests the involvement of miR-153, IA-2β and miR-153 target genes in a regulatory network, which is potentially relevant to insulin and neurotransmitter release.

Arabidopsis genomes uncoupled 5 (GUN5) mutant reveals the involvement of Mg-chelatase H subunit in plastid-to-nucleus signal transduction

PubMed Central

Mochizuki, Nobuyoshi; Brusslan, Judy A.; Larkin, Robert; Nagatani, Akira; Chory, Joanne

2001-01-01

A plastid-derived signal plays an important role in the coordinated expression of both nuclear- and chloroplast-localized genes that encode photosynthesis-related proteins. Arabidopsis GUN (genomes uncoupled) loci have been identified as components of plastid-to-nucleus signal transduction. Unlike wild-type plants, gun mutants have nuclear Lhcb1 expression in the absence of chloroplast development. We observed a synergistic phenotype in some gun double-mutant combinations, suggesting there are at least two independent pathways in plastid-to-nucleus signal transduction. There is a reduction of chlorophyll accumulation in gun4 and gun5 mutant plants, and a gun4gun5 double mutant shows an albino phenotype. We cloned the GUN5 gene, which encodes the ChlH subunit of Mg-chelatase. We also show that gun2 and gun3 are alleles of the known photomorphogenic mutants, hy1 and hy2, which are required for phytochromobilin synthesis from heme. These findings suggest that certain perturbations of the tetrapyrrole biosynthetic pathway generate a signal from chloroplasts that causes transcriptional repression of nuclear genes encoding plastid-localized proteins. The comparison of mutant phenotypes of gun5 and another Mg-chelatase subunit (ChlI) mutant suggests a specific function for ChlH protein in the plastid-signaling pathway. PMID:11172074

Selective aliphatic carbon-hydrogen bond activation of protected alcohol substrates by cytochrome P450 enzymes.

PubMed

Bell, Stephen G; Spence, Justin T J; Liu, Shenglan; George, Jonathan H; Wong, Luet-Lok

2014-04-21

Protected cyclohexanol and cyclohex-2-enol substrates, containing benzyl ether and benzoate ester moieties, were designed to fit into the active site of the Tyr96Ala mutant of cytochrome P450cam. The protected cyclohexanol substrates were efficiently and selectively hydroxylated by the mutant enzyme at the trans C-H bond of C-4 on the cyclohexyl ring. The selectivity of oxidation of the benzoate ester protected cyclohexanol could be altered by making alternative amino acid substitutions in the P450cam active site. The addition of the double bond in the cyclohexyl ring of the benzoate ester protected cyclohex-2-enol has a debilitative effect on the activity of the Tyr96Ala mutant with this substrate. However, the Phe87Ala/Tyr96Phe double mutant, which introduces space at a different location in the active site than the Tyr96Ala mutant, was able to efficiently hydroxylate the C-H bonds of 1-cyclohex-2-enyl benzoate at the allylic C-4 position. Mutations at Phe87 improved the selectivity of the oxidation of 1-phenyl-1-cyclohexylethylene to trans-4-phenyl-ethenylcyclohexanol (92%) when compared to single mutants at Tyr96 of P450cam.

Fractionating spatial memory with glutamate receptor subunit-knockout mice.

PubMed

Bannerman, David M

2009-12-01

In recent years, the contribution that different glutamate receptor subtypes and subunits make to spatial learning and memory has been studied extensively using genetically modified mice in which key proteins are knocked out. This has revealed dissociations between different aspects of spatial memory that were not previously apparent from lesion studies. For example, studies with GluA1 AMPAR [AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) receptor] subunit-knockout mice have revealed the presence of a GluA1-dependent, non-associative short-term memory mechanism that is important for performance on spatial working memory tasks, and a GluA1-independent, long-term associative memory mechanism which underlies performance on spatial reference memory tasks. Within this framework we have also studied the contributions of different GluN2-containing NMDARs [NMDA (N-methyl-D-aspartate) receptors] to spatial memory. Studies with GluN2 NMDAR mutants have revealed different contributions from GluN2A- and GluN2B-containing NMDARs to spatial learning. Furthermore, comparison of forebrain- and hippocampus-specific GluN2B-knockout mice has demonstrated that both hippocampal and extra-hippocampal NMDARs make important contributions to spatial memory performance.

Characterization of a Bacillus subtilis surfactin synthetase knockout and antimicrobial activity analysis.

PubMed

Liu, Hongxia; Qu, Xiaoxu; Gao, Ling; Zhao, Shengming; Lu, Zhaoxin; Zhang, Chong; Bie, Xiaomei

2016-11-10

Gene knockout is an important approach to improve the production of antimicrobial compounds. B. subtilis PB2-LS10, derived from B. subtilis PB2-L by a surfactin synthetase (srf) genes knockout, exhibits stronger inhibitory action than its parental strain against all tested pathogenic bacteria and fungi. The antimicrobial extracts produced by B. subtilis PB2-L and B. subtilis PB2-LS10 respectively were characterized by the high-resolution LC-ESI-MS. To provide further insight into the distinct antimicrobial activities, we investigated the impact of the srf genes deletion on the growth and gene transcriptional profile of the strains. The mutant strain grew quickly and reached stationary phase 2h earlier than the wild-type. Prominent expression changes in the modified strain involved genes that were essential to metabolic pathways and processes. Genes related to amino acid transport, ATP-binding cassette (ABC) transporters and protein export were up-regulated in strain PB2-LS10. However, amino acid metabolism, carbohydrate metabolism and fatty acid metabolism were repressed. Because of its excellent antimicrobial activity, strain PB2-LS10 has potential for use in food preservation. Copyright © 2016 Elsevier B.V. All rights reserved.

Relevance of the two-component sensor protein CiaH to acid and oxidative stress responses in Streptococcus pyogenes.

PubMed

Tatsuno, Ichiro; Isaka, Masanori; Okada, Ryo; Zhang, Yan; Hasegawa, Tadao

2014-03-28

The production of virulence proteins depends on environmental factors, and two-component regulatory systems are involved in sensing these factors. We previously established knockout strains in all suspected two-component regulatory sensor proteins of the emm1 clinical strain of S. pyogenes and examined their relevance to acid stimuli in a natural atmosphere. In the present study, their relevance to acid stimuli was re-examined in an atmosphere containing 5% CO2. The spy1236 (which is identical to ciaHpy) sensor knockout strain showed significant growth reduction compared with the parental strain in broth at pH 6.0, suggesting that the Spy1236 (CiaHpy) two-component sensor protein is involved in acid response of S. pyogenes. CiaH is also conserved in Streptococcus pneumoniae, and it has been reported that deletion of the gene for its cognate response regulator (ciaRpn) made the pneumococcal strains more sensitive to oxidative stress. In this report, we show that the spy1236 knockout mutant of S. pyogenes is more sensitive to oxidative stress than the parental strain. These results suggest that the two-component sensor protein CiaH is involved in stress responses in S. pyogenes.

Efficient CRISPR/Cas9-based gene knockout in watermelon.

PubMed

Tian, Shouwei; Jiang, Linjian; Gao, Qiang; Zhang, Jie; Zong, Mei; Zhang, Haiying; Ren, Yi; Guo, Shaogui; Gong, Guoyi; Liu, Fan; Xu, Yong

2017-03-01

CRISPR/Cas9 system can precisely edit genomic sequence and effectively create knockout mutations in T0 generation watermelon plants. Genome editing offers great advantage to reveal gene function and generate agronomically important mutations to crops. Recently, RNA-guided genome editing system using the type II clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) has been applied to several plant species, achieving successful targeted mutagenesis. Here, we report the genome of watermelon, an important fruit crop, can also be precisely edited by CRISPR/Cas9 system. ClPDS, phytoene desaturase in watermelon, was selected as the target gene because its mutant bears evident albino phenotype. CRISPR/Cas9 system performed genome editing, such as insertions or deletions at the expected position, in transfected watermelon protoplast cells. More importantly, all transgenic watermelon plants harbored ClPDS mutations and showed clear or mosaic albino phenotype, indicating that CRISPR/Cas9 system has technically 100% of genome editing efficiency in transgenic watermelon lines. Furthermore, there were very likely no off-target mutations, indicated by examining regions that were highly homologous to sgRNA sequences. Our results show that CRISPR/Cas9 system is a powerful tool to effectively create knockout mutations in watermelon.

Immunological Development and Cardiovascular Function Are Normal in Annexin VI Null Mutant Mice

PubMed Central

Hawkins, Tim E.; Roes, Jürgen; Rees, Daryl; Monkhouse, Jayne; Moss, Stephen E.

1999-01-01

Annexins are calcium-binding proteins of unknown function but which are implicated in important cellular processes, including anticoagulation, ion flux regulation, calcium homeostasis, and endocytosis. To gain insight into the function of annexin VI, we performed targeted disruption of its gene in mice. Matings between heterozygous mice produced offspring with a normal Mendelian pattern of inheritance, indicating that the loss of annexin VI did not interfere with viability in utero. Mice lacking annexin VI reached sexual maturity at the same age as their normal littermates, and both males and females were fertile. Because of interest in the role of annexin VI in cardiovascular function, we examined heart rate and blood pressure in knockout and wild-type mice and found these to be identical in the two groups. Similarly, the cardiovascular responses of both sets of mice to septic shock were indistinguishable. We also examined components of the immune system and found no differences in thymic, splenic, or bone marrow lymphocyte levels between knockout and wild-type mice. This is the first study of annexin knockout mice, and the lack of a clear phenotype has broad implications for current views of annexin function. PMID:10567528

Endoplasmic reticulum stress sensor protein kinase R-like endoplasmic reticulum kinase (PERK) protects against pressure overload-induced heart failure and lung remodeling.

PubMed

Liu, Xiaoyu; Kwak, Dongmin; Lu, Zhongbing; Xu, Xin; Fassett, John; Wang, Huan; Wei, Yidong; Cavener, Douglas R; Hu, Xinli; Hall, Jennifer; Bache, Robert J; Chen, Yingjie

2014-10-01

Studies have reported that development of congestive heart failure is associated with increased endoplasmic reticulum stress. Double stranded RNA-activated protein kinase R-like endoplasmic reticulum kinase (PERK) is a major transducer of the endoplasmic reticulum stress response and directly phosphorylates eukaryotic initiation factor 2α, resulting in translational attenuation. However, the physiological effect of PERK on congestive heart failure development is unknown. To study the effect of PERK on ventricular structure and function, we generated inducible cardiac-specific PERK knockout mice. Under unstressed conditions, cardiac PERK knockout had no effect on left ventricular mass, or its ratio to body weight, cardiomyocyte size, fibrosis, or left ventricular function. However, in response to chronic transverse aortic constriction, PERK knockout mice exhibited decreased ejection fraction, increased left ventricular fibrosis, enhanced cardiomyocyte apoptosis, and exacerbated lung remodeling in comparison with wild-type mice. PERK knockout also dramatically attenuated cardiac sarcoplasmic reticulum Ca(2+)-ATPase expression in response to aortic constriction. Our findings suggest that PERK is required to protect the heart from pressure overload-induced congestive heart failure. © 2014 American Heart Association, Inc.

Nutrient-regulated Phosphorylation of ATG13 Inhibits Starvation-induced Autophagy*

PubMed Central

Puente, Cindy; Hendrickson, Ronald C.; Jiang, Xuejun

2016-01-01

Autophagy is a conserved catabolic process that utilizes a defined series of membrane trafficking events to generate a de novo double-membrane vesicle termed the autophagosome, which matures by fusing to the lysosome. Subsequently, the lysosome facilitates the degradation and recycling of the cytoplasmic cargo. In yeast, the upstream signals that regulate the induction of starvation-induced autophagy are clearly defined. The nutrient-sensing kinase Tor inhibits the activation of autophagy by regulating the formation of the Atg1-Atg13-Atg17 complex, through hyperphosphorylation of Atg13. However, in mammals, the ortholog complex ULK1-ATG13-FIP200 is constitutively formed. As such, the molecular mechanism by which mTOR regulates mammalian autophagy is unknown. Here we report the identification and characterization of novel nutrient-regulated phosphorylation sites on ATG13: Ser-224 and Ser-258. mTOR directly phosphorylates ATG13 on Ser-258 while Ser-224 is modulated by the AMPK pathway. In ATG13 knock-out cells reconstituted with an unphosphorylatable mutant of ATG13, ULK1 kinase activity is more potent, and amino acid starvation induced more rapid ATG13 and ULK1 translocation. These events culminated in a more rapid starvation-induced autophagy response. Therefore, ATG13 phosphorylation plays a crucial role in autophagy regulation. PMID:26801615

ABI1 and PP2CA Phosphatases Are Negative Regulators of Snf1-Related Protein Kinase1 Signaling in Arabidopsis[C][W

PubMed Central

Rodrigues, Américo; Adamo, Mattia; Crozet, Pierre; Margalha, Leonor; Confraria, Ana; Martinho, Cláudia; Elias, Alexandre; Rabissi, Agnese; Lumbreras, Victoria; González-Guzmán, Miguel; Antoni, Regina; Rodriguez, Pedro L.; Baena-González, Elena

2013-01-01

Plant survival under environmental stress requires the integration of multiple signaling pathways into a coordinated response, but the molecular mechanisms underlying this integration are poorly understood. Stress-derived energy deprivation activates the Snf1-related protein kinases1 (SnRK1s), triggering a vast transcriptional and metabolic reprogramming that restores homeostasis and promotes tolerance to adverse conditions. Here, we show that two clade A type 2C protein phosphatases (PP2Cs), established repressors of the abscisic acid (ABA) hormonal pathway, interact with the SnRK1 catalytic subunit causing its dephosphorylation and inactivation. Accordingly, SnRK1 repression is abrogated in double and quadruple pp2c knockout mutants, provoking, similarly to SnRK1 overexpression, sugar hypersensitivity during early seedling development. Reporter gene assays and SnRK1 target gene expression analyses further demonstrate that PP2C inhibition by ABA results in SnRK1 activation, promoting SnRK1 signaling during stress and once the energy deficit subsides. Consistent with this, SnRK1 and ABA induce largely overlapping transcriptional responses. Hence, the PP2C hub allows the coordinated activation of ABA and energy signaling, strengthening the stress response through the cooperation of two key and complementary pathways. PMID:24179127

Loss of Cbl and Cbl-b ubiquitin ligases abrogates hematopoietic stem cell quiescence and sensitizes leukemic disease to chemotherapy

PubMed Central

An, Wei; Nadeau, Scott A.; Mohapatra, Bhopal C.; Feng, Dan; Zutshi, Neha; Storck, Matthew D.; Arya, Priyanka; Talmadge, James E.; Meza, Jane L.; Band, Vimla; Band, Hamid

2015-01-01

Cbl and Cbl-b are tyrosine kinase-directed RING finger type ubiquitin ligases (E3s) that negatively regulate cellular activation pathways. E3 activity-disrupting human Cbl mutations are associated with myeloproliferative disorders (MPD) that are reproduced in mice with Cbl RING finger mutant knock-in or hematopoietic Cbl and Cbl-b double knockout. However, the role of Cbl proteins in hematopoietic stem cell (HSC) homeostasis, especially in the context of MPD is unclear. Here we demonstrate that HSC expansion and MPD development upon combined Cbl and Cbl-b deletion are dependent on HSCs. Cell cycle analysis demonstrated that DKO HSCs exhibit reduced quiescence associated with compromised reconstitution ability and propensity to undergo exhaustion. We show that sustained c-Kit and FLT3 signaling in DKO HSCs promotes loss of colony-forming potential, and c-Kit or FLT3 inhibition in vitro protects HSCs from exhaustion. In vivo, treatment with 5-fluorouracil hastens DKO HSC exhaustion and protects mice from death due to MPD. Our data reveal a novel and leukemia therapy-relevant role of Cbl and Cbl-b in the maintenance of HSC quiescence and protection against exhaustion, through negative regulation of tyrosine kinase-coupled receptor signaling. PMID:25871390

High-throughput transformation of Saccharomyces cerevisiae using liquid handling robots.

PubMed

Liu, Guangbo; Lanham, Clayton; Buchan, J Ross; Kaplan, Matthew E

2017-01-01

Saccharomyces cerevisiae (budding yeast) is a powerful eukaryotic model organism ideally suited to high-throughput genetic analyses, which time and again has yielded insights that further our understanding of cell biology processes conserved in humans. Lithium Acetate (LiAc) transformation of yeast with DNA for the purposes of exogenous protein expression (e.g., plasmids) or genome mutation (e.g., gene mutation, deletion, epitope tagging) is a useful and long established method. However, a reliable and optimized high throughput transformation protocol that runs almost no risk of human error has not been described in the literature. Here, we describe such a method that is broadly transferable to most liquid handling high-throughput robotic platforms, which are now commonplace in academic and industry settings. Using our optimized method, we are able to comfortably transform approximately 1200 individual strains per day, allowing complete transformation of typical genomic yeast libraries within 6 days. In addition, use of our protocol for gene knockout purposes also provides a potentially quicker, easier and more cost-effective approach to generating collections of double mutants than the popular and elegant synthetic genetic array methodology. In summary, our methodology will be of significant use to anyone interested in high throughput molecular and/or genetic analysis of yeast.

Dictyostelium discoideum Dgat2 Can Substitute for the Essential Function of Dgat1 in Triglyceride Production but Not in Ether Lipid Synthesis

PubMed Central

Du, Xiaoli; Herrfurth, Cornelia; Gottlieb, Thomas; Kawelke, Steffen; Feussner, Kristin; Rühling, Harald; Feussner, Ivo

2014-01-01

Triacylglycerol (TAG), the common energy storage molecule, is formed from diacylglycerol and a coenzyme A-activated fatty acid by the action of an acyl coenzyme A:diacylglycerol acyltransferase (DGAT). In order to conduct this step, most organisms rely on more than one enzyme. The two main candidates in Dictyostelium discoideum are Dgat1 and Dgat2. We show, by creating single and double knockout mutants, that the endoplasmic reticulum (ER)-localized Dgat1 enzyme provides the predominant activity, whereas the lipid droplet constituent Dgat2 contributes less activity. This situation may be opposite from what is seen in mammalian cells. Dictyostelium Dgat2 is specialized for the synthesis of TAG, as is the mammalian enzyme. In contrast, mammalian DGAT1 is more promiscuous regarding its substrates, producing diacylglycerol, retinyl esters, and waxes in addition to TAG. The Dictyostelium Dgat1, however, produces TAG, wax esters, and, most interestingly, also neutral ether lipids, which represent a significant constituent of lipid droplets. Ether lipids had also been found in mammalian lipid droplets, but the role of DGAT1 in their synthesis was unknown. The ability to form TAG through either Dgat1 or Dgat2 activity is essential for Dictyostelium to grow on bacteria, its natural food substrate. PMID:24562909

Detection of Iss and Bor on the surface of Escherichia coli.

PubMed

Lynne, A M; Skyberg, J A; Logue, C M; Nolan, L K

2007-03-01

To confirm the presence of Iss and Bor on the outer membrane of Escherichia coli using Western blots of outer membrane protein (OMP) preparations and fluorescence microscopy, and explore the use of fluorescence microscopy for the detection of avian pathogenic E. coli (APEC) and diagnosis of avian colibacillosis. Knockout mutants of iss and bor were created using a one-step recombination of target genes with PCR-generated antibiotic resistance cassettes. Anti-Iss monoclonal antibodies (Mabs) that cross-react with Bor protein were used to study the mutants relative to the wild-type organism. These Mabs were used as reagents to study OMP preparations of the mutants with Western blotting and intact E. coli cells with fluorescence microscopy. Iss and Bor were detected in Western blots of OMP preparations of the wild type. Also, Iss was detected on Deltabor mutants, and Bor was detected on Deltaiss mutants. Iss and Bor were also detected on the surface of the intact, wild-type cells and mutants using fluorescence microscopy. These results demonstrate that Bor and Iss are exposed on E. coli's outer membrane where they may be recognized by the host's immune system. To our knowledge, this is the first report confirming Iss' location in the outer membrane of an E. coli isolate. Such surface exposure has implications for the use of these Mabs for APEC detection and colibacillosis control.

A Metagenome-Wide Association Study and Arrayed Mutant Library Confirm Acetobacter Lipopolysaccharide Genes Are Necessary for Association with Drosophila melanogaster.

PubMed

White, K Makay; Matthews, Melinda K; Hughes, Rachel C; Sommer, Andrew J; Griffitts, Joel S; Newell, Peter D; Chaston, John M

2018-03-28

A metagenome wide association (MGWA) study of bacterial host association determinants in Drosophila predicted that LPS biosynthesis genes are significantly associated with host colonization. We were unable to create site-directed mutants for each of the predicted genes in Acetobacter , so we created an arrayed transposon insertion library using Acetobacter fabarum DsW_054 isolated from Drosophila Creation of the A. fabarum DsW_054 gene knock-out library was performed by combinatorial mapping and Illumina sequencing of random transposon insertion mutants. Transposon insertion locations for 6,418 mutants were successfully mapped, including hits within 63% of annotated genes in the A. fabarum DsW_054 genome. For 45/45 members of the library, insertion sites were verified by arbitrary PCR and Sanger sequencing. Mutants with insertions in four different LPS biosynthesis genes were selected from the library to validate the MGWA predictions. Insertion mutations in two genes biosynthetically upstream of Lipid-A formation, lpxC and lpxB , show significant differences in host association, whereas mutations in two genes encoding LPS biosynthesis functions downstream of Lipid-A biosynthesis had no effect. These results suggest an impact of bacterial cell surface molecules on the bacterial capacity for host association. Also, the transposon insertion mutant library will be a useful resource for ongoing research on the genetic basis for Acetobacter traits. Copyright © 2018 White et al.

Ascl1 (Mash1) Knockout Perturbs Differentiation of Nonneuronal Cells in Olfactory Epithelium

PubMed Central

Jang, Woochan; Wildner, Hendrik; Schwob, James E.

2012-01-01

The embryonic olfactory epithelium (OE) generates only a very few olfactory sensory neurons when the basic helix-loop-helix transcription factor, ASCL1 (previously known as MASH1) is eliminated by gene mutation. We have closely examined the structure and composition of the OE of knockout mice and found that the absence of neurons dramatically affects the differentiation of multiple other epithelial cell types as well. The most prominent effect is observed within the two known populations of stem and progenitor cells of the epithelium. The emergence of horizontal basal cells, a multipotent progenitor population in the adult epithelium, is anomalous in the Ascl1 knockout mice. The differentiation of globose basal cells, another multipotent progenitor population in the adult OE, is also aberrant. All of the persisting globose basal cells are marked by SOX2 expression, suggesting a prominent role for SOX2 in progenitors upstream of Ascl1. However, NOTCH1-expressing basal cells are absent from the knockout; since NOTCH1 signaling normally acts to suppress Ascl1 via HES1 and drives sustentacular (Sus) cell differentiation during adult epithelial regeneration, its absence suggests reciprocity between neurogenesis and the differentiation of Sus cells. Indeed, the Sus cells of the mutant mice express a markedly lower level of HES1, strengthening that notion of reciprocity. Duct/gland development appears normal. Finally, the expression of cKIT by basal cells is also undetectable, except in those small patches where neurogenesis escapes the effects of Ascl1 knockout and neurons are born. Thus, persistent neurogenic failure distorts the differentiation of multiple other cell types in the olfactory epithelium. PMID:23284756

Exchangers man the pumps: Functional interplay between proton pumps and proton-coupled Ca exchangers.

PubMed

Barkla, Bronwyn J; Hirschi, Kendal D; Pittman, Jon K

2008-05-01

Tonoplast-localised proton-coupled Ca(2+) transporters encoded by cation/H(+)exchanger (CAX) genes play a critical role in sequestering Ca(2+) into the vacuole. These transporters may function in coordination with Ca(2+) release channels, to shape stimulus-induced cytosolic Ca(2+) elevations. Recent analysis of Arabidopsis CAX knockout mutants, particularly cax1 and cax3, identified a variety of phenotypes including sensitivity to abiotic stresses, which indicated that these transporters might play a role in mediating the plant's stress response. A common feature of these mutants was the perturbation of H(+)-ATPase activity at both the tonoplast and the plasma membrane, suggesting a tight interplay between the Ca(2+)/H(+) exchangers and H(+) pumps. We speculate that indirect regulation of proton flux by the exchangers may be as important as the direct regulation of Ca(2+) flux. These results suggest cautious interpretation of mutant Ca(2+)/H(+) exchanger phenotypes that may be due to either perturbed Ca(2+) or H(+) transport.

Exchangers man the pumps

PubMed Central

Barkla, Bronwyn J; Hirschi, Kendal D

2008-01-01

Tonoplast-localised proton-coupled Ca2+ transporters encoded by cation/H+ exchanger (CAX) genes play a critical role in sequestering Ca2+ into the vacuole. These transporters may function in coordination with Ca2+ release channels, to shape stimulus-induced cytosolic Ca2+ elevations. Recent analysis of Arabidopsis CAX knockout mutants, particularly cax1 and cax3, identified a variety of phenotypes including sensitivity to abiotic stresses, which indicated that these transporters might play a role in mediating the plant's stress response. A common feature of these mutants was the perturbation of H+-ATPase activity at both the tonoplast and the plasma membrane, suggesting a tight interplay between the Ca2+/H+ exchangers and H+ pumps. We speculate that indirect regulation of proton flux by the exchangers may be as important as the direct regulation of Ca2+ flux. These results suggest cautious interpretation of mutant Ca2+/H+ exchanger phenotypes that may be due to either perturbed Ca2+ or H+ transport. PMID:19841670

RNAi assisted genome evolution unveils yeast mutants with improved xylose utilization.

PubMed

HamediRad, Mohammad; Lian, Jiazhang; Li, Hejun; Zhao, Huimin

2018-06-01

Xylose is a major component of lignocellulosic biomass, one of the most abundant feedstocks for biofuel production. Therefore, efficient and rapid conversion of xylose to ethanol is crucial in the viability of lignocellulosic biofuel plants. In this study, RNAi Assisted Genome Evolution (RAGE) was used to improve the xylose utilization rate in SR8, one of the most efficient publicly available xylose utilizing Saccharomyces cerevisiae strains. To identify gene targets for further improvement, we created a genome-scale library consisting of both genetic over-expression and down-regulation mutations in SR8. Followed by screening in media containing xylose as the sole carbon source, yeast mutants with 29% faster xylose utilization, and 45% higher ethanol productivity were obtained relative to the parent strain. Two known and two new effector genes were identified in these mutant strains. Notably, down-regulation of CDC11, an essential gene, resulted in faster xylose utilization, and this gene target cannot be identified in genetic knock-out screens. © 2018 Wiley Periodicals, Inc.

The rpoE operon regulates heat stress response in Burkholderia pseudomallei.

PubMed

Vanaporn, Muthita; Vattanaviboon, Paiboon; Thongboonkerd, Visith; Korbsrisate, Sunee

2008-07-01

Burkholderia pseudomallei is a gram-negative bacterium and the causative agent of melioidosis, one of the important lethal diseases in tropical regions. In this article, we demonstrate the crucial role of the B. pseudomallei rpoE locus in the response to heat stress. The rpoE operon knockout mutant exhibited growth retardation and reduced survival when exposed to a high temperature. Expression analysis using rpoH promoter-lacZ fusion revealed that heat stress induction of rpoH, which encodes heat shock sigma factor (sigma(H)), was abolished in the B. pseudomallei rpoE mutant. Analysis of the rpoH promoter region revealed sequences sharing high homology to the consensus sequence of sigma(E)-dependent promoters. Moreover, the putative heat-induced sigma(H)-regulated heat shock proteins (i.e. GroEL and HtpG) were also absent in the rpoE operon mutant. Altogether, our data suggest that the rpoE operon regulates B. pseudomallei heat stress response through the function of rpoH.

Relationships between starch synthase I and branching enzyme isozymes determined using double mutant rice lines

PubMed Central

2014-01-01

Background Starch is the most important carbohydrate in plant storage tissues. Multiple isozymes in at least four enzyme classes are involved in starch biosynthesis. Some of these isozymes are thought to interact and form complexes for efficient starch biosynthesis. Of these enzyme classes, starch synthases (SSs) and branching enzymes (BEs) play particularly central roles. Results We generated double mutant lines (ss1/be1 and ss1 L /be2b) between SSI (the largest component of total soluble SS activity) and BEI or BEIIb (major BEs in developing rice endosperm) to explore the relationships among these isozymes. The seed weight of ss1/be1 was comparable to that of wild type, although most ss1/be2b seeds were sterile and no double recessive plants were obtained. The seed weight of the double recessive mutant line ss1 L /be2b, derived from the leaky ss1 mutant (ss1 L ) and be2b, was higher than that of the single be2b mutant. Analyses of the chain-length distribution of amylopectin in ss1/be1 endosperm revealed additive effects of SSI and BEI on amylopectin structure. Chain-length analysis indicated that the BEIIb deficiency significantly reduced the ratio of short chains in amylopectin of ss1 L /be2b. The amylose content of endosperm starch of ss1/be1 and ss1 L /be2b was almost the same as that of wild type, whereas the endosperm starch of be2b contained more amylose than did that of wild type. SSI, BEI, and BEIIb deficiency also affected the extent of binding of other isozymes to starch granules. Conclusions Analysis of the chain-length distribution in amylopectin of the double mutant lines showed that SSI and BEI or BEIIb primarily function independently, and branching by BEIIb is followed by SSI chain elongation. The increased amylose content in be2b was because of reduced amylopectin biosynthesis; however, the lower SSI activity in this background may have enhanced amylopectin biosynthesis as a result of a correction of imbalance between the branching and elongation found in the single mutant. The fact that a deficiency of SSI, BEI, or BEIIb affected the affinity of other starch biosynthetic isozymes for the starch granule implies that there is a close interaction among SSI, BEI and BEIIb during amylopectin biosynthesis in rice endosperm. PMID:24670252

Relationships between starch synthase I and branching enzyme isozymes determined using double mutant rice lines.

PubMed

Abe, Natsuko; Asai, Hiroki; Yago, Hikari; Oitome, Naoko F; Itoh, Rumiko; Crofts, Naoko; Nakamura, Yasunori; Fujita, Naoko

2014-03-26

Starch is the most important carbohydrate in plant storage tissues. Multiple isozymes in at least four enzyme classes are involved in starch biosynthesis. Some of these isozymes are thought to interact and form complexes for efficient starch biosynthesis. Of these enzyme classes, starch synthases (SSs) and branching enzymes (BEs) play particularly central roles. We generated double mutant lines (ss1/be1 and ss1L/be2b) between SSI (the largest component of total soluble SS activity) and BEI or BEIIb (major BEs in developing rice endosperm) to explore the relationships among these isozymes. The seed weight of ss1/be1 was comparable to that of wild type, although most ss1/be2b seeds were sterile and no double recessive plants were obtained. The seed weight of the double recessive mutant line ss1L/be2b, derived from the leaky ss1 mutant (ss1L) and be2b, was higher than that of the single be2b mutant. Analyses of the chain-length distribution of amylopectin in ss1/be1 endosperm revealed additive effects of SSI and BEI on amylopectin structure. Chain-length analysis indicated that the BEIIb deficiency significantly reduced the ratio of short chains in amylopectin of ss1L/be2b. The amylose content of endosperm starch of ss1/be1 and ss1L/be2b was almost the same as that of wild type, whereas the endosperm starch of be2b contained more amylose than did that of wild type. SSI, BEI, and BEIIb deficiency also affected the extent of binding of other isozymes to starch granules. Analysis of the chain-length distribution in amylopectin of the double mutant lines showed that SSI and BEI or BEIIb primarily function independently, and branching by BEIIb is followed by SSI chain elongation. The increased amylose content in be2b was because of reduced amylopectin biosynthesis; however, the lower SSI activity in this background may have enhanced amylopectin biosynthesis as a result of a correction of imbalance between the branching and elongation found in the single mutant. The fact that a deficiency of SSI, BEI, or BEIIb affected the affinity of other starch biosynthetic isozymes for the starch granule implies that there is a close interaction among SSI, BEI and BEIIb during amylopectin biosynthesis in rice endosperm.

Improved motor performance in Dyt1 ΔGAG heterozygous knock-in mice by cerebellar Purkinje-cell specific Dyt1 conditional knocking-out.

PubMed

Yokoi, Fumiaki; Dang, Mai Tu; Li, Yuqing

2012-05-01

Early-onset generalized torsion dystonia (dystonia 1) is an inherited movement disorder caused by mutations in DYT1 (TOR1A), which codes for torsinA. Most patients have a 3-base pair deletion (ΔGAG) in one allele of DYT1, corresponding to a loss of a glutamic acid residue (ΔE) in the C-terminal region of the protein. Functional alterations in basal ganglia circuits and the cerebellum have been reported in dystonia. Pharmacological manipulations or mutations in genes that result in functional alterations of the cerebellum have been reported to have dystonic symptoms and have been used as phenotypic rodent models. Additionally, structural lesions in the abnormal cerebellar circuits, such as cerebellectomy, have therapeutic effects in these models. A previous study has shown that the Dyt1 ΔGAG heterozygous knock-in (KI) mice exhibit motor deficits in the beam-walking test. Both Dyt1 ΔGAG heterozygous knock-in (KI) and Dyt1 Purkinje cell-specific knockout (Dyt1 pKO) mice exhibit dendritic alterations of cerebellar Purkinje cells. Here, Dyt1 pKO mice exhibited significantly less slip numbers in the beam-walking test, suggesting better motor performance than control littermates, and normal gait. Furthermore, Dyt1 ΔGAG KI/Dyt1 pKO double mutant mice exhibited significantly lower numbers of slips than Dyt1 ΔGAG heterozygous KI mice, suggesting Purkinje-cell specific knockout of Dyt1 wild-type (WT) allele in Dyt1 ΔGAG heterozygous KI mice rescued the motor deficits. The results suggest that molecular lesions of torsinA in Purkinje cells by gene therapy or intervening in the signaling pathway downstream of the cerebellar Purkinje cells may rescue motor symptoms in dystonia 1. Copyright © 2012 Elsevier B.V. All rights reserved.

Using a Fluorescent PCR-capillary Gel Electrophoresis Technique to Genotype CRISPR/Cas9-mediated Knockout Mutants in a High-throughput Format.

PubMed

Ramlee, Muhammad Khairul; Wang, Jing; Cheung, Alice M S; Li, Shang

2017-04-08

The development of programmable genome-editing tools has facilitated the use of reverse genetics to understand the roles specific genomic sequences play in the functioning of cells and whole organisms. This cause has been tremendously aided by the recent introduction of the CRISPR/Cas9 system-a versatile tool that allows researchers to manipulate the genome and transcriptome in order to, among other things, knock out, knock down, or knock in genes in a targeted manner. For the purpose of knocking out a gene, CRISPR/Cas9-mediated double-strand breaks recruit the non-homologous end-joining DNA repair pathway to introduce the frameshift-causing insertion or deletion of nucleotides at the break site. However, an individual guide RNA may cause undesirable off-target effects, and to rule these out, the use of multiple guide RNAs is necessary. This multiplicity of targets also means that a high-volume screening of clones is required, which in turn begs the use of an efficient high-throughput technique to genotype the knockout clones. Current genotyping techniques either suffer from inherent limitations or incur high cost, hence rendering them unsuitable for high-throughput purposes. Here, we detail the protocol for using fluorescent PCR, which uses genomic DNA from crude cell lysate as a template, and then resolving the PCR fragments via capillary gel electrophoresis. This technique is accurate enough to differentiate one base-pair difference between fragments and hence is adequate in indicating the presence or absence of a frameshift in the coding sequence of the targeted gene. This precise knowledge effectively precludes the need for a confirmatory sequencing step and allows users to save time and cost in the process. Moreover, this technique has proven to be versatile in genotyping various mammalian cells of various tissue origins targeted by guide RNAs against numerous genes, as shown here and elsewhere.

Prostaglandin E2 Activates YAP and a Positive-Signaling Loop to Promote Colon Regeneration After Colitis but Also Carcinogenesis in Mice.

PubMed

Kim, Han-Byul; Kim, Minchul; Park, Young-Soo; Park, Intae; Kim, Tackhoon; Yang, Sung-Yeun; Cho, Charles J; Hwang, DaeHee; Jung, Jin-Hak; Markowitz, Sanford D; Hwang, Sung Wook; Yang, Suk-Kyun; Lim, Dae-Sik; Myung, Seung-Jae

2017-02-01

Prostaglandin E 2 (PGE 2 ) is mediator of inflammation that regulates tissue regeneration, but its continual activation has been associated with carcinogenesis. Little is known about factors in the PGE 2 signaling pathway that contribute to tumor formation. We investigated whether yes-associated protein 1 (YAP1), a transcriptional co-activator in the Hippo signaling pathway, mediates PGE 2 function. DLD-1 and SW480 colon cancer cell lines were transfected with vectors expressing transgenes or small hairpin RNAs and incubated with recombinant PGE 2 , with or without pharmacologic inhibitors of signaling proteins, and analyzed by immunoblot, immunofluorescence, quantitative reverse-transcription polymerase chain reaction, transcriptional reporter, and proliferation assays. Dextran sodium sulfate (DSS) was given to induce colitis in C57/BL6 (control) mice, as well as in mice with disruption of the hydroxyprostaglandin dehydrogenase 15 gene (15-PGDH-knockout mice), Yap1 gene (YAP-knockout mice), and double-knockout mice. Some mice also were given indomethacin to block PGE 2 synthesis. 15-PGDH knockout mice were crossed with mice with intestine-specific disruption of the salvador family WW domain containing 1 gene (Sav1), which encodes an activator of Hippo signaling. We performed immunohistochemical analyses of colon biopsy samples from 26 patients with colitis-associated cancer and 51 age-and sex-matched patients with colorectal cancer (without colitis). Incubation of colon cancer cell lines with PGE 2 led to phosphorylation of cyclic adenosine monophosphate-responsive element binding protein 1 and increased levels of YAP1 messenger RNA, protein, and YAP1 transcriptional activity. This led to increased transcription of the prostaglandin-endoperoxide synthase 2 gene (PTGS2 or cyclooxygenase 2) and prostaglandin E-receptor 4 gene (PTGER4 or EP4). Incubation with PGE 2 promoted proliferation of colon cancer cell lines, but not cells with knockdown of YAP1. Control mice developed colitis after administration of DSS, but injection of PGE 2 led to colon regeneration in these mice. However, YAP-knockout mice did not regenerate colon tissues and died soon after administration of DSS. 15-PGDH-knockout mice regenerated colon tissues more rapidly than control mice after withdrawal of DSS, and had faster recovery of body weight, colon length, and colitis histology scores. These effects were reversed by injection of indomethacin. SAV1-knockout or 15-PGDH-knockout mice did not develop spontaneous tumors after colitis induction, but SAV1/15-PGDH double-knockout mice developed polyps that eventually progressed to carcinoma in situ. Administration of indomethacin to these mice prevented spontaneous tumor formation. Levels of PGE 2 correlated with those of YAP levels in human sporadic colorectal tumors and colitis-associated tumors. PGE 2 signaling increases the expression and transcriptional activities of YAP1, leading to increased expression of cyclooxygenase 2 and EP4 to activate a positive signaling loop. This pathway promotes proliferation of colon cancer cell lines and colon tissue regeneration in mice with colitis. Constitutive activation of this pathway led to formation of polyps and colon tumors in mice. Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.

Knockout of Foxp2 disrupts vocal development in mice.

PubMed

Castellucci, Gregg A; McGinley, Matthew J; McCormick, David A

2016-03-16

The FOXP2 gene is important for the development of proper speech motor control in humans. However, the role of the gene in general vocal behavior in other mammals, including mice, is unclear. Here, we track the vocal development of Foxp2 heterozygous knockout (Foxp2+/-) mice and their wildtype (WT) littermates from juvenile to adult ages, and observe severe abnormalities in the courtship song of Foxp2+/- mice. In comparison to their WT littermates, Foxp2+/- mice vocalized less, produced shorter syllable sequences, and possessed an abnormal syllable inventory. In addition, Foxp2+/- song also exhibited irregular rhythmic structure, and its development did not follow the consistent trajectories observed in WT vocalizations. These results demonstrate that the Foxp2 gene is critical for normal vocal behavior in juvenile and adult mice, and that Foxp2 mutant mice may provide a tractable model system for the study of the gene's role in general vocal motor control.