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

Phenotypic plasticity in cell walls of maize brown midrib mutants is limited by lignin composition

PubMed Central

Vermerris, Wilfred; Sherman, Debra M.; McIntyre, Lauren M.

2010-01-01

The hydrophobic cell wall polymer lignin is deposited in specialized cells to make them impermeable to water and prevent cell collapse as negative pressure or gravitational force is exerted. The variation in lignin subunit composition that exists among different species, and among different tissues within the same species suggests that lignin subunit composition varies depending on its precise function. In order to gain a better understanding of the relationship between lignin subunit composition and the physico-chemical properties of lignified tissues, detailed analyses were performed of near-isogenic brown midrib2 (bm2), bm4, bm2-bm4, and bm1-bm2-bm4 mutants of maize. This investigation was motivated by the fact that the bm2-bm4 double mutant is substantially shorter, displays drought symptoms even when well watered, and will often not develop reproductive organs, whereas the phenotypes of the individual bm single mutants and double mutant combinations other than bm2-bm4 are only subtly different from the wild-type control. Detailed cell wall compositional analyses revealed midrib-specific reductions in Klason lignin content in the bm2, bm4, and bm2-bm4 mutants relative to the wild-type control, with reductions in both guaiacyl (G)- and syringyl (S)-residues. The cellulose content was not different, but the reduction in lignin content was compensated by an increase in hemicellulosic polysaccharides. Linear discriminant analysis performed on the compositional data indicated that the bm2 and bm4 mutations act independently of each other on common cell wall biosynthetic steps. After quantitative analysis of scanning electron micrographs of midrib sections, the variation in chemical composition of the cell walls was shown to be correlated with the thickness of the sclerenchyma cell walls, but not with xylem vessel surface area. The bm2-bm4 double mutant represents the limit of phenotypic plasticity in cell wall composition, as the bm1-bm2-bm4 and bm2-bm3-bm4 mutants did not develop into mature plants, unlike the triple mutants bm1-bm2-bm3 and bm1-bm3-bm4. PMID:20410320

Somatic cell mutations at the glycophorin A locus in erythrocytes of atomic bomb survivors: Implications for radiation carcinogenesis

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

Kyoizumi, Seishi; Akiyama, Mitoshi; Tanabe, Kazumi

To clarify the relationship between somatic cell mutations and radiation exposure, the frequency of hemizygous mutant erythrocytes at the glycophorin A (GPA) locus was measured by flow cytometry for 1,226 heterozygous atomic bomb (A-bomb) survivors in HIroshima and Nagasaki. For statistical analysis, both GPA mutant frequency and radiation dose were log-transformed to normalize skewed distributions of these variables. The GPA mutant frequency increased slightly but significantly with age at testing and with the number of cigarettes smoked. Also, mutant frequency was significantly higher in males than in females even with adjustment for smoking and was higher to Hiroshima than inmore » Nagasaki. These characteristics of background GPA mutant frequency are qualitatively similar to those of background solid cancer incidence or mortality obtained from previous epidemiological studies of survivors. An analysis of the mutant frequency dose response using a descriptive model showed that the doubling dose is about 1.20 Sv [95% confidence interval (CI): 0.95-1.56], whereas the minimum dose for detecting a significant increase in mutant frequency is about 0.24 Sv (95% CI: 0.041-0.51). No significant effects of sex, city or age at the time of exposure on the dose response were detected. Interestingly, the doubling dose of the GPA mutant frequency was similar to that of solid cancer incidence in A-bomb survivors. This observation is in line with the hypothesis that radiation-induced somatic cell mutations are the major cause of excess cancer risk after radiation. 49 refs., 6 figs., 2 tabs.« less

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.

Transcriptomic profiling-based mutant screen reveals three new transcription factors mediating menadione resistance in Neurospora crassa.

PubMed

Zhu, Jufen; Yu, Xinxu; Xie, Baogui; Gu, Xiaokui; Zhang, Zhenying; Li, Shaojie

2013-06-01

To gain insight into the regulatory mechanisms of oxidative stress responses in filamentous fungi, the genome-wide transcriptional response of Neurospora crassa to menadione was analysed by digital gene expression (DGE) profiling, which identified 779 upregulated genes and 576 downregulated genes. Knockout mutants affecting 130 highly-upregulated genes were tested for menadione sensitivity, which revealed that loss of the transcription factor siderophore regulation (SRE) (a transcriptional repressor for siderophore biosynthesis), catatase-3, cytochrome c peroxidase or superoxide dismutase 1 copper chaperone causes hypersensitivity to menadione. Deletion of sre dramatically increased transcription of the siderophore biosynthesis gene ono and the siderophore iron transporter gene sit during menadione stress, suggesting that SRE is required for repression of iron uptake under oxidative stress conditions. Contrary to its phenotype, the sre deletion mutant showed higher transcriptional levels of genes encoding reactive oxygen species (ROS) scavengers than wild type during menadione stress, which implies that the mutant suffers a higher level of oxidative stress than wild type. Uncontrolled iron uptake in the sre mutant might exacerbate cellular oxidative stress. This is the first report of a negative regulator of iron assimilation participating in the fungal oxidative stress response. In addition to SRE, eight other transcription factor genes were also menadione-responsive but their single gene knockout mutants showed wild-type menadione sensitivity. Two of them, named as mit-2 (menadione induced transcription factor-2) and mit-4 (menadione induced transcription factor-4), were selected for double mutant analysis. The double mutant was hypersensitive to menadione. Similarly, the double mutation of mit-2 and sre also had additive effects on menadione sensitivity, suggesting multiple transcription factors mediate oxidative stress resistance in an additive manner. Copyright © 2013 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

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.

The Sep1 Mutant of Saccharomyces Cerevisiae Arrests in Pachytene and Is Deficient in Meiotic Recombination

PubMed Central

Tishkoff, D. X.; Rockmill, B.; Roeder, G. S.; Kolodner, R. D.

1995-01-01

Strand exchange protein 1 (Sep1) from Saccharomyces cerevisiae promotes homologous pairing of DNA in vitro and sep1 mutants display pleiotropic phenotypes in both vegetative and meiotic cells. In this study, we examined in detail the ability of the sep1 mutant to progress through meiosis I prophase and to undergo meiotic recombination. In meiotic return-to-growth experiments, commitment to meiotic recombination began at the same time in wild type and mutant; however, recombinants accumulated at decreased rates in the mutant. Gene conversion eventually reached nearly wild-type levels, whereas crossing over reached 15-50% of wild type. In an assay of intrachromosomal pop-out recombination, the sep1, dmc1 and rad51 single mutations had only small effects; however, pop-out recombination was virtually eliminated in the sep1 dmc1 and sep1 rad51 double mutants, providing evidence for multiple recombination pathways. Analysis of meiotic recombination intermediates indicates that the sep1 mutant is deficient in meiotic double-strand break repair. In a physical assay, the formation of mature reciprocal recombinants in the sep1 mutant was delayed relative to wild type and ultimately reached only 50% of the wild-type level. Electron microscopic analysis of meiotic nuclear spreads indicates that the sep1δ mutant arrests in pachytene, with apparently normal synaptonemal complex. This arrest is RAD9-independent. We hypothesize that the Sep1 protein participates directly in meiotic recombination and that other strand exchange enzymes, acting in parallel recombination pathways, are able to substitute partially for the absence of the Sep1 protein. PMID:7713413

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.

Trapping a 96° domain rotation in two distinct conformations by engineered disulfide bridges

PubMed Central

Schultz-Heienbrok, Robert; Maier, Timm; Sträter, Norbert

2004-01-01

Engineering disulfide bridges is a common technique to lock a protein movement in a defined conformational state. We have designed two double mutants of Escherichia coli 5′-nucleotidase to trap the enzyme in both an open (S228C, P513C) and a closed (P90C, L424C) conformation by the formation of disulfide bridges. The mutant proteins have been expressed, purified, and crystallized, to structurally characterize the designed variants. The S228C, P513C is a double mutant crystallized in two different crystal forms with three independent conformers, which differ from each other by a rotation of up to 12° of the C-terminal domain with respect to the N-terminal domain. This finding, as well as an analysis of the domain motion in the crystal, indicates that the enzyme still exhibits considerable residual domain flexibility. In the double mutant that was designed to trap the enzyme in the closed conformation, the structure analysis reveals an unexpected intermediate conformation along the 96° rotation trajectory between the open and closed enzyme forms. A comparison of the five independent conformers analyzed in this study shows that the domain movement of the variant enzymes is characterized by a sliding movement of the residues of the domain interface along the interface, which is in contrast to a classical closure motion where the residues of the domain interface move perpendicular to the interface. PMID:15215524

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

Arabidopsis Fructokinases Are Important for Seed Oil Accumulation and Vascular Development.

PubMed

Stein, Ofer; Avin-Wittenberg, Tamar; Krahnert, Ina; Zemach, Hanita; Bogol, Vlada; Daron, Oksana; Aloni, Roni; Fernie, Alisdair R; Granot, David

2016-01-01

Sucrose (a disaccharide made of glucose and fructose) is the primary carbon source transported to sink organs in many plants. Since fructose accounts for half of the hexoses used for metabolism in sink tissues, plant fructokinases (FRKs), the main fructose-phosphorylating enzymes, are likely to play a central role in plant development. However, to date, their specific functions have been the subject of only limited study. The Arabidopsis genome contains seven genes encoding six cytosolic FRKs and a single plastidic FRK. T-DNA knockout mutants for five of the seven FRKs were identified and used in this study. Single knockouts of the FRK mutants did not exhibit any unusual phenotype. Double-mutants of AtFRK6 (plastidic) and AtFRK7 showed normal growth in soil, but yielded dark, distorted seeds. The seed distortion could be complemented by expression of the well-characterized tomato SlFRK1 , confirming that a lack of FRK activity was the primary cause of the seed phenotype. Seeds of the double-mutant germinated, but failed to establish on 1/2 MS plates. Seed establishment was made possible by the addition of glucose or sucrose, indicating reduced seed storage reserves. Metabolic profiling of the double-mutant seeds revealed decreased TCA cycle metabolites and reduced fatty acid metabolism. Examination of the mutant embryo cells revealed smaller oil bodies, the primary storage reserve in Arabidopsis seeds. Quadruple and penta FRK mutants showed growth inhibition and leaf wilting. Anatomical analysis revealed smaller trachea elements and smaller xylem area, accompanied by necrosis around the cambium and the phloem. These results demonstrate overlapping and complementary roles of the plastidic AtFRK6 and the cytosolic AtFRK7 in seed storage accumulation, and the importance of AtFRKs for vascular development.

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

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

eIF4E/Fmr1 double mutant mice display cognitive impairment in addition to ASD-like behaviors.

PubMed

Huynh, Thu N; Shah, Manan; Koo, So Yeon; Faraud, Kirsten S; Santini, Emanuela; Klann, Eric

2015-11-01

Autism spectrum disorder (ASD) is a group of heritable disorders with complex and unclear etiology. Classic ASD symptoms include social interaction and communication deficits as well as restricted, repetitive behaviors. In addition, ASD is often comorbid with intellectual disability. Fragile X syndrome (FXS) is the leading genetic cause of ASD, and is the most commonly inherited form of intellectual disability. Several mouse models of ASD and FXS exist, however the intellectual disability observed in ASD patients is not well modeled in mice. Using the Fmr1 knockout mouse and the eIF4E transgenic mouse, two previously characterized mouse models of fragile X syndrome and ASD, respectively, we generated the eIF4E/Fmr1 double mutant mouse. Our study shows that the eIF4E/Fmr1 double mutant mice display classic ASD behaviors, as well as cognitive dysfunction. Importantly, the learning impairments displayed by the double mutant mice spanned multiple cognitive tasks. Moreover, the eIF4E/Fmr1 double mutant mice display increased levels of basal protein synthesis. The results of our study suggest that the eIF4E/Fmr1 double mutant mouse may be a reliable model to study cognitive dysfunction in the context of ASD. Copyright © 2015 Elsevier Inc. All rights reserved.

Spire, an actin nucleation factor, regulates cell division during Drosophila heart development.

PubMed

Xu, Peng; Johnson, Tamara L; Stoller-Conrad, Jessica R; Schulz, Robert A

2012-01-01

The Drosophila dorsal vessel is a beneficial model system for studying the regulation of early heart development. Spire (Spir), an actin-nucleation factor, regulates actin dynamics in many developmental processes, such as cell shape determination, intracellular transport, and locomotion. Through protein expression pattern analysis, we demonstrate that the absence of spir function affects cell division in Myocyte enhancer factor 2-, Tinman (Tin)-, Even-skipped- and Seven up (Svp)-positive heart cells. In addition, genetic interaction analysis shows that spir functionally interacts with Dorsocross, tin, and pannier to properly specify the cardiac fate. Furthermore, through visualization of double heterozygous embryos, we determines that spir cooperates with CycA for heart cell specification and division. Finally, when comparing the spir mutant phenotype with that of a CycA mutant, the results suggest that most Svp-positive progenitors in spir mutant embryos cannot undergo full cell division at cell cycle 15, and that Tin-positive progenitors are arrested at cell cycle 16 as double-nucleated cells. We conclude that Spir plays a crucial role in controlling dorsal vessel formation and has a function in cell division during heart tube morphogenesis.

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.

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.

G Protein–Coupled Receptor-Type G Proteins Are Required for Light-Dependent Seedling Growth and Fertility in Arabidopsis[W

PubMed Central

Jaffé, Felix W.; Freschet, Gian-Enrico C.; Valdes, Billy M.; Runions, John; Terry, Matthew J.; Williams, Lorraine E.

2012-01-01

G protein–coupled receptor-type G proteins (GTGs) are highly conserved membrane proteins in plants, animals, and fungi that have eight to nine predicted transmembrane domains. They have been classified as G protein–coupled receptor-type G proteins that function as abscisic acid (ABA) receptors in Arabidopsis thaliana. We cloned Arabidopsis GTG1 and GTG2 and isolated new T-DNA insertion alleles of GTG1 and GTG2 in both Wassilewskija and Columbia backgrounds. These gtg1 gtg2 double mutants show defects in fertility, hypocotyl and root growth, and responses to light and sugars. Histological studies of shoot tissue reveal cellular distortions that are particularly evident in the epidermal layer. Stable expression of GTG1pro:GTG1-GFP (for green fluorescent protein) in Arabidopsis and transient expression in tobacco (Nicotiana tabacum) indicate that GTG1 is localized primarily to Golgi bodies and to the endoplasmic reticulum. Microarray analysis comparing gene expression profiles in the wild type and double mutant revealed differences in expression of genes important for cell wall function, hormone response, and amino acid metabolism. The double mutants isolated here respond normally to ABA in seed germination assays, root growth inhibition, and gene expression analysis. These results are inconsistent with their proposed role as ABA receptors but demonstrate that GTGs are fundamentally important for plant growth and development. PMID:23001037

The Ames dwarf gene, df, is required early in pituitary ontogeny for the extinction of Rpx transcription and initiation of lineage-specific cell proliferation.

PubMed

Gage, P J; Brinkmeier, M L; Scarlett, L M; Knapp, L T; Camper, S A; Mahon, K A

1996-12-01

Two nonallelic dwarfing mutations in mice define genes important for pituitary development and function. Mice homozygous for either the Ames (df) or Snell (Pit 1dw) dwarf mutations exhibit severe proportional dwarfism, hypothyroidism, and infertility due to the cytodifferentiation failure of three anterior pituitary cell types: thyrotropes, somatotropes, and lactotropes. Analysis of double heterozygotes and double mutants has provided evidence that the df and dw genes act sequentially in the same genetic pathway. Double heterozygotes had no reduction in growth rate or final adult size. Double homozygotes had essentially the same phenotype as the single mutants and were recovered at the predicted frequency, indicating that there are no previously unrecognized, redundant functions of the two genes. Several lines of evidence demonstrate that df acts earlier in the differentiation pathway than Pit1. The df mutants fail to extinguish expression of the homeobox gene Rpx on embryonic day 13.5 (e13.5), and the size of their nascent pituitary glands is reduced by e14.5. In contrast, Pit1dw mutants down-regulate Rpx appropriately and exhibit normal cell proliferation up to e14.5. The failure to extinguish Rpx and the concomitant hypocellularity of df pituitaries suggest the importance of Rpx repression in lineage-specific cell proliferation before the appearance of lineage-specific markers. Later, Pit-1 and hypothalamic neuropeptides act sequentially to regulate marker gene transcription and cell proliferation. These results establish the time of df action in a cascade of genes that regulate pituitary ontogeny.

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

Dicer-Like Proteins Regulate Sexual Development via the Biogenesis of Perithecium-Specific MicroRNAs in a Plant Pathogenic Fungus Fusarium graminearum

PubMed Central

Zeng, Wenping; Wang, Jie; Wang, Ying; Lin, Jing; Fu, Yanping; Xie, Jiatao; Jiang, Daohong; Chen, Tao; Liu, Huiquan; Cheng, Jiasen

2018-01-01

Ascospores act as the primary inoculum of Fusarium graminearum, which causes the destructive disease Fusarium head blight (FHB), or scab. MicroRNAs (miRNAs) have been reported in the F. graminearum vegetative stage, and Fgdcl2 is involved in microRNA-like RNA (milRNA) biogenesis but has no major impact on vegetative growth, abiotic stress or pathogenesis. In the present study, we found that ascospore discharge was decreased in the Fgdcl1 deletion mutant, and completely blocked in the double-deletion mutant of Fgdcl1 and Fgdcl2. Besides, more immature asci were observed in the double-deletion mutant. Interestingly, the up-regulated differentially expressed genes (DEGs) common to ΔFgdcl1 and ΔFgdcl1/2 were related to ion transmembrane transporter and membrane components. The combination of small RNA and transcriptome sequencing with bioinformatics analysis predicted 143 novel milRNAs in wild-type perithecia, and 138 of these milRNAs partly or absolutely depended on Fgdcl1, while only 5 novel milRNAs were still obtained in the Fgdcl1 and Fgdcl2 double-deletion mutant. Furthermore, 117 potential target genes were predicted. Overall, Fgdcl1 and Fgdcl2 genes were partly functionally redundant in ascospore discharge and perithecium-specific milRNA generation in F. graminearum, and these perithecium-specific milRNAs play potential roles in sexual development. PMID:29755439

Analysis of the Arabidopsis IRX9/IRX9-L and IRX14/IRX14-L pairs of glycosyltransferase genes reveals critical contributions to biosynthesis of the hemicellulose glucuronoxylan.

PubMed

Wu, Ai-Min; Hörnblad, Emma; Voxeur, Aline; Gerber, Lorenz; Rihouey, Christophe; Lerouge, Patrice; Marchant, Alan

2010-06-01

The hemicellulose glucuronoxylan (GX) is a major component of plant secondary cell walls. However, our understanding of GX synthesis remains limited. Here, we identify and analyze two new genes from Arabidopsis (Arabidopsis thaliana), IRREGULAR XYLEM9-LIKE (IRX9-L) and IRX14-LIKE (IRX14-L) that encode glycosyltransferase family 43 members proposed to function during xylan backbone elongation. We place IRX9-L and IRX14-L in a genetic framework with six previously described glycosyltransferase genes (IRX9, IRX10, IRX10-L, IRX14, FRAGILE FIBER8 [FRA8], and FRA8 HOMOLOG [F8H]) and investigate their function in GX synthesis. Double-mutant analysis identifies IRX9-L and IRX14-L as functional homologs of IRX9 and IRX14, respectively. Characterization of irx9 irx10 irx14 fra8 and irx9-L irx10-L irx14-L f8h quadruple mutants allows definition of a set of genes comprising IRX9, IRX10, IRX14, and FRA8 that perform the main role in GX synthesis during vegetative development. The IRX9-L, IRX10-L, IRX14-L, and F8H genes are able to partially substitute for their respective homologs and normally perform a minor function. The irx14 irx14-L double mutant virtually lacks xylan, whereas irx9 irx9-L and fra8 f8h double mutants form lowered amounts of GX displaying a greatly reduced degree of backbone polymerization. Our findings reveal two distinct sets of four genes each differentially contributing to GX biosynthesis.

Analysis of the Arabidopsis IRX9/IRX9-L and IRX14/IRX14-L Pairs of Glycosyltransferase Genes Reveals Critical Contributions to Biosynthesis of the Hemicellulose Glucuronoxylan1[C][W

PubMed Central

Wu, Ai-Min; Hörnblad, Emma; Voxeur, Aline; Gerber, Lorenz; Rihouey, Christophe; Lerouge, Patrice; Marchant, Alan

2010-01-01

The hemicellulose glucuronoxylan (GX) is a major component of plant secondary cell walls. However, our understanding of GX synthesis remains limited. Here, we identify and analyze two new genes from Arabidopsis (Arabidopsis thaliana), IRREGULAR XYLEM9-LIKE (IRX9-L) and IRX14-LIKE (IRX14-L) that encode glycosyltransferase family 43 members proposed to function during xylan backbone elongation. We place IRX9-L and IRX14-L in a genetic framework with six previously described glycosyltransferase genes (IRX9, IRX10, IRX10-L, IRX14, FRAGILE FIBER8 [FRA8], and FRA8 HOMOLOG [F8H]) and investigate their function in GX synthesis. Double-mutant analysis identifies IRX9-L and IRX14-L as functional homologs of IRX9 and IRX14, respectively. Characterization of irx9 irx10 irx14 fra8 and irx9-L irx10-L irx14-L f8h quadruple mutants allows definition of a set of genes comprising IRX9, IRX10, IRX14, and FRA8 that perform the main role in GX synthesis during vegetative development. The IRX9-L, IRX10-L, IRX14-L, and F8H genes are able to partially substitute for their respective homologs and normally perform a minor function. The irx14 irx14-L double mutant virtually lacks xylan, whereas irx9 irx9-L and fra8 f8h double mutants form lowered amounts of GX displaying a greatly reduced degree of backbone polymerization. Our findings reveal two distinct sets of four genes each differentially contributing to GX biosynthesis. PMID:20424005

A Dual Strategy to Cope with High Light in Chlamydomonas reinhardtii[W

PubMed Central

Allorent, Guillaume; Tokutsu, Ryutaro; Roach, Thomas; Peers, Graham; Cardol, Pierre; Girard-Bascou, Jacqueline; Seigneurin-Berny, Daphné; Petroutsos, Dimitris; Kuntz, Marcel; Breyton, Cécile; Franck, Fabrice; Wollman, Francis-André; Niyogi, Krishna K.; Krieger-Liszkay, Anja; Minagawa, Jun; Finazzi, Giovanni

2013-01-01

Absorption of light in excess of the capacity for photosynthetic electron transport is damaging to photosynthetic organisms. Several mechanisms exist to avoid photodamage, which are collectively referred to as nonphotochemical quenching. This term comprises at least two major processes. State transitions (qT) represent changes in the relative antenna sizes of photosystems II and I. High energy quenching (qE) is the increased thermal dissipation of light energy triggered by lumen acidification. To investigate the respective roles of qE and qT in photoprotection, a mutant (npq4 stt7-9) was generated in Chlamydomonas reinhardtii by crossing the state transition–deficient mutant (stt7-9) with a strain having a largely reduced qE capacity (npq4). The comparative phenotypic analysis of the wild type, single mutants, and double mutants reveals that both state transitions and qE are induced by high light. Moreover, the double mutant exhibits an increased photosensitivity with respect to the single mutants and the wild type. Therefore, we suggest that besides qE, state transitions also play a photoprotective role during high light acclimation of the cells, most likely by decreasing hydrogen peroxide production. These results are discussed in terms of the relative photoprotective benefit related to thermal dissipation of excess light and/or to the physical displacement of antennas from photosystem II. PMID:23424243

Superoxide-Dismutase Deficient Mutants in Common Beans (Phaseolus vulgaris L.): Genetic Control, Differential Expressions of Isozymes, and Sensitivity to Arsenic

PubMed Central

Talukdar, Dibyendu; Talukdar, Tulika

2013-01-01

Two common bean (Phaseolus vulgaris L.) mutants, sodPv 1 and sodPv 2, exhibiting foliar superoxide dismutase (SOD) activity of only 25% and 40% of their mother control (MC) cv. VL 63 were isolated in EMS-mutagenized (0.15%, 8 h) M2 progeny. Native-PAGE analysis revealed occurrence of Mn SOD, Fe SOD, Cu/Zn SOD I and Cu/Zn SOD II isozymes in MC, while Fe SOD, and Mn SOD were not formed in sodPv 1 and sodPv 2 leaves, respectively. In-gel activity of individual isozymes differed significantly among the parents. SOD deficiency is inherited as recessive mutations, controlled by two different nonallelic loci. Gene expressions using qRT PCR confirmed higher expressions of Cu/Zn SOD transcripts in both mutants and the absence of Fe SOD in sodPv 1 and Mn SOD in sodPv 2. In 50 μM arsenic, Cu/Zn SODs genes were further upregulated but other isoforms downregulated in the two mutants, maintaining SOD activity in its control level. In an F2 double mutants of sodPv 1 × sodPv 2, no Fe SOD, and Mn SOD expressions were detectable, while both Cu/Zn SODs are down-regulated and arsenic-induced leaf necrosis appeared. In contrast to both mutants, ROS-imaging study revealed overaccumulation of both superoxides and H2O2 in leaves of double mutant. PMID:24078924

Transcriptomic and molecular genetic analysis of the cell wall salvage response of Aspergillus niger to the absence of galactofuranose synthesis.

PubMed

Park, Joohae; Hulsman, Mark; Arentshorst, Mark; Breeman, Matthijs; Alazi, Ebru; Lagendijk, Ellen L; Rocha, Marina C; Malavazi, Iran; Nitsche, Benjamin M; van den Hondel, Cees A M J J; Meyer, Vera; Ram, Arthur F J

2016-09-01

The biosynthesis of cell surface-located galactofuranose (Galf)-containing glycostructures such as galactomannan, N-glycans and O-glycans in filamentous fungi is important to secure the integrity of the cell wall. UgmA encodes an UDP-galactopyranose mutase, which is essential for the formation of Galf. Consequently, the ΔugmA mutant lacks Galf-containing molecules. Our previous work in Aspergillus niger work suggested that loss of function of ugmA results in activation of the cell wall integrity (CWI) pathway which is characterized by increased expression of the agsA gene, encoding an α-glucan synthase. In this study, the transcriptional response of the ΔugmA mutant was further linked to the CWI pathway by showing the induced and constitutive phosphorylation of the CWI-MAP kinase in the ΔugmA mutant. To identify genes involved in cell wall remodelling in response to the absence of galactofuranose biosynthesis, a genome-wide expression analysis was performed using RNAseq. Over 400 genes were higher expressed in the ΔugmA mutant compared to the wild-type. These include genes that encode enzymes involved in chitin (gfaB, gnsA, chsA) and α-glucan synthesis (agsA), and in β-glucan remodelling (bgxA, gelF and dfgC), and also include several glycosylphosphatidylinositol (GPI)-anchored cell wall protein-encoding genes. In silico analysis of the 1-kb promoter regions of the up-regulated genes in the ΔugmA mutant indicated overrepresentation of genes with RlmA, MsnA, PacC and SteA-binding sites. The importance of these transcription factors for survival of the ΔugmA mutant was analysed by constructing the respective double mutants. The ΔugmA/ΔrlmA and ΔugmA/ΔmsnA double mutants showed strong synthetic growth defects, indicating the importance of these transcription factors to maintain cell wall integrity in the absence of Galf biosynthesis. © 2016 The Authors Cellular Microbiology Published by John Wiley & Sons 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

Insights into resistance mechanism of the macrolide biosensor protein MphR(A) binding to macrolide antibiotic erythromycin by molecular dynamics simulation

NASA Astrophysics Data System (ADS)

Feng, Tingting; Zhang, Yanjun; Ding, Jing-Na; Fan, Song; Han, Ju-Guang

2015-12-01

Macrolide biosensor protein MphR(A) has been known as a key regulatory protein in metabolite sensing and genetic expression regulating. MphR(A) protein binds to macrolide antibiotic erythromycin (Ery) and releases the gene operon, thus activates expression of the mphA gene and initiates Ery resistance. The two mutant amino acid residues (V66L and V126L) might potentially disrupt Ery binding to MphR(A). In these studies, the binding of macrolide antibiotic Ery to wild type (Wt) MphR(A) and double mutant (V66L/V126L) MphR(A) are explored by molecular dynamics simulations. Compared to the Apo-MphR(A) protein and Wt-MphR(A)-Ery complex, many interesting effects owing to the double mutant (V66L/V126L) are discovered. In the case of Ery, Helix I which plays an important role in transcription shows itself a right-hand α helix in Wt-MphR(A)-Ery, whereas the activated helix is broken down in double mutant-V66L/V126L-MphR(A)-Ery. The calculated results exhibit that the double mutant V66L/V126L reduces the binding affinity of the V66L/V126L-MphR(A) to Ery, resulting in the block of Ery resistance. The binding free energy decomposition analysis reveals that the decrease of the binding affinity for the variant V66L/V126L-MphR(A)-Ery is mainly attributed to the gas phase electrostatic energies. The residue Leu66, Thr154, and Arg122 enhance the binding affinity of V66L/V126L-MphR(A) to Ery. The residues Tyr103 and His147 contributes mainly to binding energies in the Wt-MphR(A)-Ery complex, whereas the two residues have no contribution to the binding free energy inV66L/V126L-MphR(A)-Ery complex. Our study gives useful insights into the nature of amino acids mutation effect, the mechanism of blocking drug resistance at the atomic level and the characteristics in binding affinity for Ery to double mutant (V66L/V126L) MphR(A), which will contribute to the design of more effective macrolide antibiotics.

Enterococcus faecalis Constitutes an Unusual Bacterial Model in Lysozyme Resistance▿

PubMed Central

Hébert, Laurent; Courtin, Pascal; Torelli, Riccardo; Sanguinetti, Maurizio; Chapot-Chartier, Marie-Pierre; Auffray, Yanick; Benachour, Abdellah

2007-01-01

Lysozyme is an important and widespread compound of the host constitutive defense system, and it is assumed that Enterococcus faecalis is one of the few bacteria that are almost completely lysozyme resistant. On the basis of the sequence analysis of the whole genome of E. faecalis V583 strain, we identified two genes that are potentially involved in lysozyme resistance, EF_0783 and EF_1843. Protein products of these two genes share significant homology with Staphylococcus aureus peptidoglycan O-acetyltransferase (OatA) and Streptococcus pneumoniae N-acetylglucosamine deacetylase (PgdA), respectively. In order to determine whether EF_0783 and EF_1843 are involved in lysozyme resistance, we constructed their corresponding mutants and a double mutant. The ΔEF_0783 mutant and ΔEF_0783 ΔEF_1843 double mutant were shown to be more sensitive to lysozyme than the parental E. faecalis JH2-2 strain and ΔEF_1843 mutant were. However, compared to other bacteria, such as Listeria monocytogenes or S. pneumoniae, the tolerance of ΔEF_0783 and ΔEF_0783 ΔEF_1843 mutants towards lysozyme remains very high. Peptidoglycan structure analysis showed that EF_0783 modifies the peptidoglycan by O acetylation of N-acetyl muramic acid, while the EF_1843 deletion has no obvious effect on peptidoglycan structure under the same conditions. Moreover, the EF_0783 and EF_1843 deletions seem to significantly affect the ability of E. faecalis to survive within murine macrophages. In all, while EF_0783 is currently involved in the lysozyme resistance of E. faecalis, peptidoglycan O acetylation and de-N-acetylation are not the main mechanisms conferring high levels of lysozyme resistance to E. faecalis. PMID:17785473

Exon skipping of AGAMOUS homolog PrseAG in developing double flowers of Prunus lannesiana (Rosaceae).

PubMed

Liu, Zhixiong; Zhang, Dandan; Liu, Di; Li, Fenglan; Lu, Hai

2013-02-01

KEY MESSAGE : Two transcript isoforms of AGAMOUS homologs, from single and double flower Prunus lannesiana, respectively, showed different functions. The Arabidopsis floral homeotic C function gene AGAMOUS (AG) confers stamen and carpel identity. Loss of AG function results in homeotic conversions of stamens into petals and formation of double flowers. In order to present a molecular dissection of a double-flower cultivar in Prunus lannesiana (Rosaceae), we isolated and identified a single-copy gene, AG homolog from two genetically cognate P. lannesiana bearing single and double flowers, respectively. Sequence analysis revealed that the AG homolog, prseag-1, from double flowers showed a 170-bp exon skipping as compared to PrseAG (Prunus serrulata AGAMOUS) from the single flowers. Genomic DNA sequence revealed that abnormal splicing resulted in mutant prseag-1 protein with the C-terminal AG motifs I and II deletions. In addition, protein sequence alignment and phylogenetic analyses revealed that the PrseAG was grouped into the euAG lineage. A semi-quantitative PCR analysis showed that the expression of PrseAG was restricted to reproductive organs of stamens and carpels in single flowers of P. lannesiana 'speciosa', while the prseag-1 mRNA was highly transcribed throughout the petals, stamens, and carpels in double flowers from 'Albo-rosea'. The transgenic Arabidopsis containing 35S::PrseAG displayed extremely early flowering, bigger stamens and carpels and homeotic conversion of petals into staminoid organs, but ectopic expression of prseag-1 could not mimic the phenotypic ectopic expression of PrseAG in Arabidopsis. In general, this study provides evidences to show that double flower 'Albo-rosea' is a putative C functional ag mutant in P. lannesiana.

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.

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.

Simultaneous ablation of prmt-1 and prmt-5 abolishes asymmetric and symmetric arginine dimethylations in Caenorhabditis elegans.

PubMed

Hirota, Keiko; Shigekawa, Chihiro; Araoi, Sho; Sha, Liang; Inagawa, Takayuki; Kanou, Akihiko; Kako, Koichiro; Daitoku, Hiroaki; Fukamizu, Akiyoshi

2017-06-01

Protein arginine methyltransferases (PRMTs) catalyze the transfer of a methyl group from S-adenosylmethionine to arginine residues and are classified into two types: type I producing asymmetric dimethylarginine (ADMA) and type II producing symmetric dimethylarginine (SDMA). PRMTs have been shown to regulate many cellular processes, including signal transduction, transcriptional regulation and RNA processing. Since the loss-of-function mutation of PRMT1 and PRMT5, each of which is the predominant type I and II, respectively, causes embryonic lethality in mice, their physiological significance at the whole-body level remains largely unknown. Here, we show the morphological and functional phenotypes of single or double null alleles of prmt-1 and prmt-5 in Caenorhabditis elegans. The prmt-1;prmt-5 double mutants are viable, and exhibit short body length and small brood size compared to N2 and each of the single mutants. The liquid chromatography-tandem mass spectrometry analysis demonstrated that the levels of ADMA and SDMA were abolished in the prmt-1;prmt-5 double mutants. Both prmt-1 and prmt-5 were required for resistance to heat and oxidative stresses, whereas prmt-5 is not involved in lifespan regulation even when prmt-1 is ablated. This mutant strain would be a useful model animal for investigating the role of asymmetric and symmetric arginine dimethylation in vivo. © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

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

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.

Genes that regulate both development and longevity in Caenorhabditis elegans

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

Larsen, P.L.; Albert, P.S.; Riddle, D.L.

1995-04-01

The nematode Caenorhabditis elegans responds to conditions of overcrowding and limited food by arresting development as a dauer larva. Genetic analysis of mutations that alter dauer larva formation (daf mutations) is presented along with an updated genetic pathway for dauer vs. nondauer development. Mutations in the daf-2 and daf-23 genes double adult life span, whereas mutations in four other dauer-constitutive genes positioned in a separate branch of this pathway (daf-1, daf-4, daf-7 and daf-8) do not. The increased life spans are suppressed completely by a daf-16 mutation and partially in a daf-2; daf-18 double mutant. A genetic pathway for determinationmore » of adult life span is presented based on the same strains and growth conditions used to characterize Daf phenotypes. Both dauer larva formation and adult life span are affected in daf-2; daf-12 double mutants in an allele-specific manner. Mutations in daf-12 do not extend adult life span, but certain combinations of daf-2 and daf-12 mutant alleles nearly quadruple it. This synergistic effect, which does not equivalently extend the fertile period, is the largest genetic extension of life span yet observed in a metazoan. 47 refs., 7 figs., 5 tabs.« less

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.

Functional Analysis of Nuclear Estrogen Receptors in Zebrafish Reproduction by Genome Editing Approach.

PubMed

Lu, Huijie; Cui, Yong; Jiang, Liwen; Ge, Wei

2017-07-01

Estrogens signal through both nuclear and membrane receptors with most reported effects being mediated via the nuclear estrogen receptors (nERs). Although much work has been reported on nERs in the zebrafish, there is a lack of direct genetic evidence for their functional roles and importance in reproduction. To address this issue, we undertook this study to disrupt all three nERs in the zebrafish, namely esr1 (ERα), esr2a (ERβII), and esr2b (ERβI), by the genome-editing technology clustered regularly interspaced short palindromic repeats and its associated nuclease (CRISPR/Cas9). Using this loss-of-function genetic approach, we successfully created three mutant zebrafish lines with each nER knocked out. In addition, we also generated all possible double and triple knockouts of the three nERs. The phenotypes of these mutants in reproduction were analyzed in all single, double, and triple nER knockouts in both females and males. Surprisingly, all three single nER mutant fish lines display normal reproductive development and function in both females and males, suggesting functional redundancy among these nERs. Further analysis of double and triple knockouts showed that nERs, especially Esr2a and Esr2b, were essential for female reproduction, and loss of these two nERs led to an arrest of folliculogenesis at previtellogenic stage II followed by sex reversal from female to male. In addition, the current study also revealed a unique role for Esr2a in follicle cell proliferation and transdifferentiation, follicle growth, and chorion formation. Taken together, this study provides the most comprehensive genetic analysis for differential functions of esr1, esr2a, and esr2b in fish reproduction. Copyright © 2017 Endocrine Society.

The barley amo1 locus is tightly linked to the starch synthase IIIa gene and negatively regulates expression of granule-bound starch synthetic genes

PubMed Central

Li, Zhongyi; Li, Dehong; Du, Xihua; Wang, Hong; Larroque, Oscar; Jenkins, Colin L. D.; Jobling, Stephen A.; Morell, Matthew K.

2011-01-01

In this study of barley starch synthesis, the interaction between mutations at the sex6 locus and the amo1 locus has been characterized. Four barley genotypes, the wild type, sex6, amo1, and the amo1sex6 double mutant, were generated by backcrossing the sex6 mutation present in Himalaya292 into the amo1 ‘high amylose Glacier’. The wild type, amo1, and sex6 genotypes gave starch phenotypes consistent with previous studies. However, the amo1sex6 double mutant yielded an unexpected phenotype, a significant increase in starch content relative to the sex6 phenotype. Amylose content (as a percentage of starch) was not increased above the level observed for the sex6 mutation alone; however, on a per seed basis, grain from lines containing the amo1 mutation (amo1 mutants and amo1sex6 double mutants) synthesize significantly more amylose than the wild-type lines and sex6 mutants. The level of granule-bound starch synthase I (GBSSI) protein in starch granules is increased in lines containing the amo1 mutation (amo1 and amo1sex6). In the amo1 genotype, starch synthase I (SSI), SSIIa, starch branching enzyme IIa (SBEIIa), and SBEIIb also markedly increased in the starch granules. Genetic mapping studies indicate that the ssIIIa gene is tightly linked to the amo1 locus, and the SSIIIa protein from the amo1 mutant has a leucine to arginine residue substitution in a conserved domain. Zymogram analysis indicates that the amo1 phenotype is not a consequence of total loss of enzymatic activity although it remains possible that the amo1 phenotype is underpinned by a more subtle change. It is therefore proposed that amo1 may be a negative regulator of other genes of starch synthesis. PMID:21813797

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

Cross-species microarray hybridization to identify developmentally regulated genes in the filamentous fungus Sordaria macrospora.

PubMed

Nowrousian, Minou; Ringelberg, Carol; Dunlap, Jay C; Loros, Jennifer J; Kück, Ulrich

2005-04-01

The filamentous fungus Sordaria macrospora forms complex three-dimensional fruiting bodies that protect the developing ascospores and ensure their proper discharge. Several regulatory genes essential for fruiting body development were previously isolated by complementation of the sterile mutants pro1, pro11 and pro22. To establish the genetic relationships between these genes and to identify downstream targets, we have conducted cross-species microarray hybridizations using cDNA arrays derived from the closely related fungus Neurospora crassa and RNA probes prepared from wild-type S. macrospora and the three developmental mutants. Of the 1,420 genes which gave a signal with the probes from all the strains used, 172 (12%) were regulated differently in at least one of the three mutants compared to the wild type, and 17 (1.2%) were regulated differently in all three mutant strains. Microarray data were verified by Northern analysis or quantitative real time PCR. Among the genes that are up- or down-regulated in the mutant strains are genes encoding the pheromone precursors, enzymes involved in melanin biosynthesis and a lectin-like protein. Analysis of gene expression in double mutants revealed a complex network of interaction between the pro gene products.

Analysis of Two Polyhydroxyalkanoate Synthases in Bradyrhizobium japonicum USDA 110

PubMed Central

Mongiardini, Elías J.; Pérez-Giménez, Julieta; Parisi, Gustavo; Lodeiro, Aníbal R.

2013-01-01

Bradyrhizobium japonicum USDA 110 has five polyhydroxyalkanoate (PHA) synthases (PhaC) annotated in its genome: bll4360 (phaC1), bll6073 (phaC2), blr3732 (phaC3), blr2885 (phaC4), and bll4548 (phaC5). All these proteins possess the catalytic triad and conserved amino acid residues of polyester synthases and are distributed into four different PhaC classes. We obtained mutants in each of these paralogs and analyzed phaC gene expression and PHA production in liquid cultures. Despite the genetic redundancy, only phaC1 and phaC2 were expressed at significant rates, while PHA accumulation in stationary-phase cultures was impaired only in the ΔphaC1 mutant. Meanwhile, the ΔphaC2 mutant produced more PHA than the wild type under this condition, and surprisingly, the phaC3 transcript increased in the ΔphaC2 background. A double mutant, the ΔphaC2 ΔphaC3 mutant, consistently accumulated less PHA than the ΔphaC2 mutant. PHA accumulation in nodule bacteroids followed a pattern similar to that seen in liquid cultures, being prevented in the ΔphaC1 mutant and increased in the ΔphaC2 mutant in relation to the level in the wild type. Therefore, we used these mutants, together with a ΔphaC1 ΔphaC2 double mutant, to study the B. japonicum PHA requirements for survival, competition for nodulation, and plant growth promotion. All mutants, as well as the wild type, survived for 60 days in a carbon-free medium, regardless of their initial PHA contents. When competing for nodulation against the wild type in a 1:1 proportion, the ΔphaC1 and ΔphaC1 ΔphaC2 mutants occupied only 13 to 15% of the nodules, while the ΔphaC2 mutant occupied 81%, suggesting that the PHA polymer is required for successful competitiveness. However, the bacteroid content of PHA did not affect the shoot dry weight accumulation. PMID:23667236

A trade off between mlo resistance to powdery mildew and increased susceptibility of barley to a newly important disease, Ramularia leaf spot

PubMed Central

McGrann, Graham R. D.; Brown, James K. M.

2014-01-01

Ramularia leaf spot (RLS), caused by the fungus Ramularia collo-cygni, is a serious, recently emerged disease of barley in Europe and other temperate regions. This study investigated the trade off between strong resistance to powdery mildew conferred by mlo mutant alleles and increased susceptibility to RLS. In field trials and seedling tests, the presence of mlo alleles increased severity of RLS. Genetic analysis of a doubled-haploid population identified one quantitative trait locus for susceptibility to RLS, colocalizing with the mlo-11 allele for mildew resistance. The effect of mlo-11 on RLS severity was environmentally sensitive. Analysis of near-isogenic lines of different mlo mutations in various genetic backgrounds confirmed that mlo alleles increased RLS severity in seedlings and adult plants. For mlo resistance to mildew to be fully effective, the genes ROR1 and ROR2 are required. RLS symptoms were significantly reduced on mlo-5 ror double mutants but fungal DNA levels remained as high as in mlo-5 single mutants, implying that ror alleles modify the transition of the fungus from endophytism to necrotrophy. These results indicate that the widespread use of mlo resistance to control mildew may have inadvertently stimulated the emergence of RLS as a major disease of barley. PMID:24399175

Arabidopsis Class I and Class II TCP Transcription Factors Regulate Jasmonic Acid Metabolism and Leaf Development Antagonistically1[C][W

PubMed Central

Danisman, Selahattin; van der Wal, Froukje; Dhondt, Stijn; Waites, Richard; de Folter, Stefan; Bimbo, Andrea; van Dijk, Aalt DJ; Muino, Jose M.; Cutri, Lucas; Dornelas, Marcelo C.; Angenent, Gerco C.; Immink, Richard G.H.

2012-01-01

TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR1 (TCP) transcription factors control developmental processes in plants. The 24 TCP transcription factors encoded in the Arabidopsis (Arabidopsis thaliana) genome are divided into two classes, class I and class II TCPs, which are proposed to act antagonistically. We performed a detailed phenotypic analysis of the class I tcp20 mutant, showing an increase in leaf pavement cell sizes in 10-d-old seedlings. Subsequently, a glucocorticoid receptor induction assay was performed, aiming to identify potential target genes of the TCP20 protein during leaf development. The LIPOXYGENASE2 (LOX2) and class I TCP9 genes were identified as TCP20 targets, and binding of TCP20 to their regulatory sequences could be confirmed by chromatin immunoprecipitation analyses. LOX2 encodes for a jasmonate biosynthesis gene, which is also targeted by class II TCP proteins that are under the control of the microRNA JAGGED AND WAVY (JAW), although in an antagonistic manner. Mutation of TCP9, the second identified TCP20 target, resulted in increased pavement cell sizes during early leaf developmental stages. Analysis of senescence in the single tcp9 and tcp20 mutants and the tcp9tcp20 double mutants showed an earlier onset of this process in comparison with wild-type control plants in the double mutant only. Both the cell size and senescence phenotypes are opposite to the known class II TCP mutant phenotype in JAW plants. Altogether, these results point to an antagonistic function of class I and class II TCP proteins in the control of leaf development via the jasmonate signaling pathway. PMID:22718775

Molecular dynamics simulations and principal component analysis on human laforin mutation W32G and W32G/K87A.

PubMed

Srikumar, P S; Rohini, K; Rajesh, Perumbilavil Kaithamanakallam

2014-06-01

Mutations in human laforin lead to an autosomal neurodegenerative disorder Lafora disease. In N-terminal carbohydrate binding domain of laforin, two mutations W32G and K87A are reported as highly disease causing laforin mutants. Experimental studies reported that mutations are responsible for the abolishment of glycogen binding which is a critical function of laforin. Our current computational study focused on the role of conformational changes in human laforin structure due to existing single mutation W32G and prepared double mutation W32G/K87A related to loss of glycogen binding. We performed 10 ns molecular dynamics (MD) simulation studies in the Gromacs package for both mutations and analyzed the trajectories. From the results, the global properties like root mean square deviation, root mean square fluctuation, radius of gyration, solvent accessible surface area and hydrogen bonds showed structural changes in atomic level observed in W32G and W32G/K87A laforin mutants. The conformational change induced by mutants influenced the loss of the overall stability of the native laforin. Moreover, the change in overall motion of protein was analyzed by principal component analysis and results showed protein clusters expanded more than native and also change in direction in case of double mutant in conformational space. Overall, our report provides theoretical information on loss of structure-function relationship due to flexible nature of laforin mutants. In conclusion, comparative MD simulation studies support the experimental data on W32G and W32G/K87A related to the lafora disease mechanism on glycogen binding.

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

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.

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.

Enhanced ε-poly-L-lysine production by inducing double antibiotic-resistant mutations in Streptomyces albulus.

PubMed

Wang, Liang; Chen, Xusheng; Wu, Guangyao; Li, Shu; Zeng, Xin; Ren, Xidong; Tang, Lei; Mao, Zhonggui

2017-02-01

ε-Poly-L-lysine (ε-PL), as a food additive, has been widely used in many countries. However, its production still needs to be improved. We successfully enhanced ε-PL production of Streptomyces albulus FEEL-1 by introducing mutations related to antibiotics, such as streptomycin, gentamicin, and rifampin. Single- and double-resistant mutants (S-88 and SG-31) were finally screened with the improved ε-PL productions of 2.81 and 3.83 g/L, 1.75- to 2.39-fold compared with that of initial strain FEEL-1. Then, the performances of mutants S-88 and SG-31 were compared with the parent strain FEEL-1 in a 5-L bioreactor under the optimal condition for ε-PL production. After 174-h fed-batch fermentation, the ε-PL production and productivity of hyper-strain SG-31 reached the maximum of 59.50 g/L and 8.21 g/L/day, respectively, which was 138 and 105% higher than that of FEEL-1. Analysis of streptomycin-resistant mutants demonstrated that a point mutation occurred in rpsL gene (encoding the ribosomal protein S12). These single and double mutants displayed remarkable increases of the activities and transcriptional levels of key enzymes in ε-PL biosynthesis pathway, which may be responsible for the enhanced mycelia viability, respiratory activity, and ε-PL productions of SG-31. These results showed that the new breeding method, called ribosome engineering, could be a novel and effective breeding strategy for the evolution of ε-PL-producing strains.

Structure-Based Alteration of Substrate Specificity and Catalytic Activity of Sulfite Oxidase from Sulfite Oxidation to Nitrate Reduction

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

Qiu, James A.; Wilson, Heather L.; Rajagopalan, K.V.

Eukaryotic sulfite oxidase is a dimeric protein that contains the molybdenum cofactor and catalyzes the metabolically essential conversion of sulfite to sulfate as the terminal step in the metabolism of cysteine and methionine. Nitrate reductase is an evolutionarily related molybdoprotein in lower organisms that is essential for growth on nitrate. In this study, we describe human and chicken sulfite oxidase variants in which the active site has been modified to alter substrate specificity and activity from sulfite oxidation to nitrate reduction. On the basis of sequence alignments and the known crystal structure of chicken sulfite oxidase, two residues are conservedmore » in nitrate reductases that align with residues in the active site of sulfite oxidase. On the basis of the crystal structure of yeast nitrate reductase, both positions were mutated in human sulfite oxidase and chicken sulfite oxidase. The resulting double-mutant variants demonstrated a marked decrease in sulfite oxidase activity but gained nitrate reductase activity. An additional methionine residue in the active site was proposed to be important in nitrate catalysis, and therefore, the triple variant was also produced. The nitrate reducing ability of the human sulfite oxidase triple mutant was nearly 3-fold greater than that of the double mutant. To obtain detailed structural data for the active site of these variants, we introduced the analogous mutations into chicken sulfite oxidase to perform crystallographic analysis. The crystal structures of the Mo domains of the double and triple mutants were determined to 2.4 and 2.1 {angstrom} resolution, respectively.« less

Neurotrophin and FGF Signaling Adapter Proteins, FRS2 and FRS3, Regulate Dentate Granule Cell Maturation and Excitatory Synaptogenesis.

PubMed

Nandi, Sayan; Alviña, Karina; Lituma, Pablo J; Castillo, Pablo E; Hébert, Jean M

2018-01-15

Dentate granule cells (DGCs) play important roles in cognitive processes. Knowledge about how growth factors such as FGFs and neurotrophins contribute to the maturation and synaptogenesis of DGCs is limited. Here, using brain-specific and germline mouse mutants we show that a module of neurotrophin and FGF signaling, the FGF Receptor Substrate (FRS) family of intracellular adapters, FRS2 and FRS3, are together required for postnatal brain development. In the hippocampus, FRS promotes dentate gyrus morphogenesis and DGC maturation during developmental neurogenesis, similar to previously published functions for both neurotrophins and FGFs. Consistent with a role in DGC maturation, two-photon imaging revealed that Frs2,3-double mutants have reduced numbers of dendritic branches and spines in DGCs. Functional analysis further showed that double-mutant mice exhibit fewer excitatory synaptic inputs onto DGCs. These observations reveal roles for FRS adapters in DGC maturation and synaptogenesis and suggest that FRS proteins may act as an important node for FGF and neurotrophin signaling in postnatal hippocampal development. Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.

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

Genetic evidence suggests that GIS functions downstream of TCL1 to regulate trichome formation in Arabidopsis.

PubMed

Zhang, Na; Yang, Li; Luo, Sha; Wang, Xutong; Wang, Wei; Cheng, Yuxin; Tian, Hainan; Zheng, Kaijie; Cai, Ling; Wang, Shucai

2018-04-13

Trichome formation in Arabidopsis is regulated by a MBW complex formed by MYB, bHLH and WD40 transcriptional factors, which can activate GLABRA2 (GL2) and the R3 MYB transcription factor genes. GL2 promotes trichome formation, whereas R3 MYBs are able to block the formation of the MBW complex. It has been reported that the C2H2 transcription factor GIS (GLABROUS INFLORESCENCE STEMS) functions upstream of the MBW activator complex to regulate trichome formation, and that the expression of TCL1 is not regulated by the MBW complex. However, gis and the R3 MYB gene mutant tcl1 (trichomeless 1) have opposite inflorescence trichome phenotypes, but their relationship in regulating trichome formation remained unknown. By generating and characterization of the gis tcl1 double mutant, we found that trichome formation in the gis tcl1double and the tcl1 single mutants were largely indistinguishable, but the trichome formation in the 35S:TCL1/gis transgenic plant was similar to that in the gis mutant. By using quantitative RT-PCR analysis, we showed that expression level of GIS was increased in the triple mutant tcl1 try cpc, but the expression level of TCL1 was not affected in the gis mutant. On the other hand, trichome morphology in both gis tcl1 and 35S:TCL1/gis plants was similar to that in the gis mutant. In summary, our results indicate that GIS may work downstream of TCL1 to regulate trichome formation, and GIS has a dominant role in controlling trichome morphology.

Molecular characterisation of four double-flowered mutants of Silene dioica representing four centuries of variation

PubMed Central

Ingle, Elizabeth K. S.; Gilmartin, Philip M.

2015-01-01

Records of double-flowered Silene dioica date from the late sixteenth century and four named varieties are grown today, as previously, for their horticultural interest. Although double-flowered mutants have been characterized in several plants, their study in dioecious species is of particular interest due to influences of the homeotic mutation on the different floral whorl configurations in males and females. We have analysed four double-flowered varieties of Silene dioica: Flore Pleno and Rosea Plena date back to the seventeenth and nineteenth centuries, Thelma Kay and Firefly were recognized in the latter part of the twentieth and early twenty-first centuries. We have analysed the floral structure of the four varieties, which have distinct floral architectures. Based on Y chromosome-specific PCR analysis we show that Firefly is male and that the other three varieties are female: Random Amplification of Polymorphic DNA (RAPD) analyses suggested a common origin for the three female varieties. The double-flowered phenotype in all four varieties is caused by mutation of the C-function MADS-box transcription factor gene SDM1. We show that Firefly carries a unique 44bp insertion into SDM1, revealing an independent origin for this variety. Comparative analysis of SDM1 cDNA and genomic sequences in Flore Pleno, Rosea Plena and Thelma Kay shows that all three are caused by the same 7bp insertion within SDM1 and therefore share a common origin. The three alleles also differ by several single nucleotide polymorphisms, which represent somatic mutations accumulated over four centuries of asexual propagation. PMID:25878355

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

The novel ER membrane protein PRO41 is essential for sexual development in the filamentous fungus Sordaria macrospora.

PubMed

Nowrousian, Minou; Frank, Sandra; Koers, Sandra; Strauch, Peter; Weitner, Thomas; Ringelberg, Carol; Dunlap, Jay C; Loros, Jennifer J; Kück, Ulrich

2007-05-01

The filamentous fungus Sordaria macrospora develops complex fruiting bodies (perithecia) to propagate its sexual spores. Here, we present an analysis of the sterile mutant pro41 that is unable to produce mature fruiting bodies. The mutant carries a deletion of 4 kb and is complemented by the pro41 open reading frame that is contained within the region deleted in the mutant. In silico analyses predict PRO41 to be an endoplasmic reticulum (ER) membrane protein, and a PRO41-EGFP fusion protein colocalizes with ER-targeted DsRED. Furthermore, Western blot analysis shows that the PRO41-EGFP fusion protein is present in the membrane fraction. A fusion of the predicted N-terminal signal sequence of PRO41 with EGFP is secreted out of the cell, indicating that the signal sequence is functional. pro41 transcript levels are upregulated during sexual development. This increase in transcript levels was not observed in the sterile mutant pro1 that lacks a transcription factor gene. Moreover, microarray analysis of gene expression in the mutants pro1, pro41 and the pro1/41 double mutant showed that pro41 is partly epistatic to pro1. Taken together, these data show that PRO41 is a novel ER membrane protein essential for fruiting body formation in filamentous fungi.

The novel ER membrane protein PRO41 is essential for sexual development in the filamentous fungus Sordaria macrospora

PubMed Central

Nowrousian, Minou; Frank, Sandra; Koers, Sandra; Strauch, Peter; Weitner, Thomas; Ringelberg, Carol; Dunlap, Jay C.; Loros, Jennifer J.; Kück, Ulrich

2013-01-01

Summary The filamentous fungus Sordaria macrospora develops complex fruiting bodies (perithecia) to propagate its sexual spores. Here, we present an analysis of the sterile mutant pro41 that is unable to produce mature fruiting bodies. The mutant carries a deletion of 4 kb and is complemented by the pro41 open reading frame that is contained within the region deleted in the mutant. In silico analyses predict PRO41 to be an endoplasmic reticulum (ER) membrane protein, and a PRO41–EGFP fusion protein colocalizes with ER-targeted DsRED. Furthermore, Western blot analysis shows that the PRO41–EGFP fusion protein is present in the membrane fraction. A fusion of the predicted N-terminal signal sequence of PRO41 with EGFP is secreted out of the cell, indicating that the signal sequence is functional. pro41 transcript levels are upregulated during sexual development. This increase in transcript levels was not observed in the sterile mutant pro1 that lacks a transcription factor gene. Moreover, microarray analysis of gene expression in the mutants pro1, pro41 and the pro1/41 double mutant showed that pro41 is partly epistatic to pro1. Taken together, these data show that PRO41 is a novel ER membrane protein essential for fruiting body formation in filamentous fungi. PMID:17501918

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

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.

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

Mice mutant for both Hoxa1 and Hoxb1 show extensive remodeling of the hindbrain and defects in craniofacial development.

PubMed

Rossel, M; Capecchi, M R

1999-11-01

The analysis of mice mutant for both Hoxa1 and Hoxb1 suggests that these two genes function together to pattern the hindbrain. Separately, mutations in Hoxa1 and Hoxb1 have profoundly different effects on hindbrain development. Hoxa1 mutations disrupt the rhombomeric organization of the hindbrain, whereas Hoxb1 mutations do not alter the rhombomeric pattern, but instead influence the fate of cells originating in rhombomere 4. We suggest that these differences are not the consequences of different functional roles for these gene products, but rather reflect differences in the kinetics of Hoxa1 and Hoxb1 gene expression. In strong support of the idea that Hoxa1 and Hoxb1 have overlapping functions, Hoxa1/Hoxb1 double mutant homozygotes exhibit a plethora of defects either not seen, or seen only in a very mild form, in mice mutant for only Hoxa1 or Hoxb1. Examples include: the loss of both rhombomeres 4 and 5, the selective loss of the 2(nd) branchial arch, and the loss of most, but not all, 2(nd) branchial arch-derived tissues. We suggest that the early role for both of these genes in hindbrain development is specification of rhombomere identities and that the aberrant development of the hindbrain in Hoxa1/Hoxb1 double mutants proceeds through two phases, the misspecification of rhombomeres within the hindbrain, followed subsequently by size regulation of the misspecified hindbrain through induction of apoptosis.

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.

Impact of distal mutations on the network of coupled motions correlated to hydride transfer in dihydrofolate reductase.

PubMed

Wong, Kim F; Selzer, Tzvia; Benkovic, Stephen J; Hammes-Schiffer, Sharon

2005-05-10

A comprehensive analysis of the network of coupled motions correlated to hydride transfer in dihydrofolate reductase is presented. Hybrid quantum/classical molecular dynamics simulations are combined with a rank correlation analysis method to extract thermally averaged properties that vary along the collective reaction coordinate according to a prescribed target model. Coupled motions correlated to hydride transfer are identified throughout the enzyme. Calculations for wild-type dihydrofolate reductase and a triple mutant, along with the associated single and double mutants, indicate that each enzyme system samples a unique distribution of coupled motions correlated to hydride transfer. These coupled motions provide an explanation for the experimentally measured nonadditivity effects in the hydride transfer rates for these mutants. This analysis illustrates that mutations distal to the active site can introduce nonlocal structural perturbations and significantly impact the catalytic rate by altering the conformational motions of the entire enzyme and the probability of sampling conformations conducive to the catalyzed reaction.

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.

Characterization and structural analysis of wild type and a non-abscission mutant at the development funiculus (Def) locus in Pisum sativum L.

PubMed

Ayeh, Kwadwo Owusu; Lee, YeonKyeong; Ambrose, Mike J; Hvoslef-Eide, Anne Kathrine

2009-06-23

In pea seeds (Pisum sativum L.), the Def locus defines an abscission event where the seed separates from the funicle through the intervening hilum region at maturity. A spontaneous mutation at this locus results in the seed failing to abscise from the funicle as occurs in wild type peas. In this work, structural differences between wild type peas that developed a distinct abscission zone (AZ) between the funicle and the seed coat and non-abscission def mutant were characterized. A clear abscission event was observed in wild type pea seeds that were associated with a distinct double palisade layers at the junction between the seed coat and funicle. Generally, mature seeds fully developed an AZ, which was not present in young wild type seeds. The AZ was formed exactly below the counter palisade layer. In contrast, the palisade layers at the junction of the seed coat and funicle were completely absent in the def mutant pea seeds and the cells in this region were seen to be extensions of surrounding parenchymatous cells. The Def wild type developed a distinct AZ associated with palisade layer and counterpalisade layer at the junction of the seed coat and funicle while the def mutant pea seed showed non-abscission and an absence of the double palisade layers in the same region. We conclude that the presence of the double palisade layer in the hilum of the wild type pea seeds plays an important structural role in AZ formation by delimiting the specific region between the seed coat and the funicle and may play a structural role in the AZ formation and subsequent detachment of the seed from the funicle.

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

Lipid transport mediated by Arabidopsis TGD proteins is unidirectional from the endoplasmic reticulum to the plastid

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

Xu, C.; Moellering, E. R., Muthan, B.; Fan, J.

2010-06-01

The transfer of lipids between the endoplasmic reticulum (ER) and the plastid in Arabidopsis involves the TRIGALACTOSYLDIACYLGLYCEROL (TGD) proteins. Lipid exchange is thought to be bidirectional based on the presence of specific lipid molecular species in Arabidopsis mutants impaired in the desaturation of fatty acids of membrane lipids in the ER and plastid. However, it was unclear whether TGD proteins were required for lipid trafficking in both directions. This question was addressed through the analysis of double mutants of tgd1-1 or tgd4-3 in genetic mutant backgrounds leading to a defect in lipid fatty acid desaturation either in the ER (fad2)more » or the plastid (fad6). The fad6 tgd1-1 and fad6 tgd4-3 double mutants showed drastic reductions in the relative levels of polyunsaturated fatty acids and of galactolipids. The growth of these plants and the development of photosynthetic membrane systems were severely compromised, suggesting a disruption in the import of polyunsaturated fatty acid-containing lipid species from the ER. Furthermore, a forward-genetic screen in the tgd1-2 dgd1 mutant background led to the isolation of a new fad6-2 allele with a marked reduction in the amount of digalactosyldiacylglycerol. In contrast, the introduction of fad2, affecting fatty acid desaturation of lipids in the ER, into the two tgd mutant backgrounds did not further decrease the level of fatty acid desaturation in lipids of extraplastidic membranes. These results suggest that the role of TGD proteins is limited to plastid lipid import, but does not extend to lipid export from the plastid to extraplastidic membranes.« less

Mutational analysis of the multicopy hao gene coding for hydroxylamine oxidoreductase in Nitrosomonas sp. strain ENI-11.

PubMed

Yamagata, A; Hirota, R; Kato, J; Kuroda, A; Ikeda, T; Takiguchi, N; Ohtake, H

2000-08-01

The ammonia-oxidizing bacterium Nitrosomonas sp. strain ENI-11 contains three copies of the hao gene (hao1, hao2, and hao3) coding for hydroxylamine oxidoreductase (HAO). Three single mutants (hao1::kan, hao2::kan, or hao3::kan) had 68 to 75% of the wild-type growth rate and 58 to 89% of the wild-type HAO activity when grown under the same conditions. A double mutant (hao1::kan and hao3::amp) also had 68% of the wild-type growth and 37% of the wild-type HAO activity.

Heat shock factors HsfB1 and HsfB2b are involved in the regulation of Pdf1.2 expression and pathogen resistance in Arabidopsis.

PubMed

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.

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.

Genetic and Developmental Analysis of Some New Color Mutants in the Goldfish, CARASSIUS AURATUS

PubMed Central

Kajishima, Takao

1977-01-01

The genotypes of three color mutants in goldfish: a depigmentation character of larval melanophores, albinism and a recessive-transparent character, were analyzed by crossing experiments. The depigmentation character in the common goldfish is controlled by two dominant multiple genes, Dp 1 and Dp2, and only fish with double recessive alleles dp1dp1 dp2dp2 can retain larval melanophores throughout life. Albinism is also controlled by double autosomal genes, p and c. The genotype of an albino fish is represented by pp cc; the non-albino fish is PP CC. Fish with either a pp CC or pp Cc genotype are albino when scored at the time of melanosome differentiation in the pigment retina, but after the time of skin melanophore differentiation, they change to the nonalbino type under the control of the C gene. The recessive-transparent character is controlled by a single autosomal gene, g. The mechanisms of gene expression of these characters were proposed as a result of observation and/or experimental data on the differentiation processes of their phenotypes, and the genotypes of these color mutant goldfish were considered in relation to the "gene duplication hypothesis in the Cyprinidae." PMID:885340

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.

Step-wise addition of disulfide bridge in firefly luciferase controls color shift through a flexible loop: a thermodynamic perspective.

PubMed

Nazari, Mahboobeh; Hosseinkhani, Saman; Hassani, Leila

2013-02-01

Multi-color bioluminescence is developed using the introduction of single/double disulfide bridges in firefly luciferase. The bioluminescence reaction, which uses luciferin, Mg(2+)-ATP and molecular oxygen to yield an electronically excited oxyluciferin, is carried out by the luciferase and emits visible light. The bioluminescence color of firefly luciferases is determined by the luciferase sequence and assay conditions. It has been proposed that the stability of a protein may increase through the introduction of a disulfide bridge that decreases the configurational entropy of unfolding. Single and double disulfide bridges are introduced into Photinus pyralis firefly luciferase to make separate mutant enzymes with a single/double bridge (C(81)-A(105)C, L(306)C-L(309)C, P(451)C-V(469)C; C(81)-A(105)C/P(451)C-V(469)C, and A(296)C-A(326)C/P(451)C-V(469)C). By introduction of disulfide bridges using site-directed mutagenesis in Photinus pyralis luciferase the color of emitted light was changed to red or kept in different extents. The bioluminescence color shift occurred with displacement of a critical loop in the luciferase structure without any change in green emitter mutants. Thermodynamic analysis revealed that among mutants, L(306)C-L(309)C shows a remarkable stability against urea denaturation and also a considerable increase in kinetic stability and a clear shift in bioluminescence spectra towards red.

Analysis of genes involved in glycogen degradation in Escherichia coli.

PubMed

Strydom, Lindi; Jewell, Jonathan; Meier, Michael A; George, Gavin M; Pfister, Barbara; Zeeman, Samuel; Kossmann, Jens; Lloyd, James R

2017-02-01

Escherichia coli accumulate or degrade glycogen depending on environmental carbon supply. Glycogen phosphorylase (GlgP) and glycogen debranching enzyme (GlgX) are known to act on the glycogen polymer, while maltodextrin phosphorylase (MalP) is thought to remove maltodextrins released by GlgX. To examine the roles of these enzymes in more detail, single, double and triple mutants lacking all their activities were produced. GlgX and GlgP were shown to act directly on the glycogen polymer, while MalP most likely catabolised soluble malto-oligosaccharides. Interestingly, analysis of a triple mutant lacking all three enzymes indicates the presence of another enzyme that can release maltodextrins from glycogen. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

MYB10 and MYB72 are required for growth under iron-limiting conditions.

PubMed

Palmer, Christine M; Hindt, Maria N; Schmidt, Holger; Clemens, Stephan; Guerinot, Mary Lou

2013-11-01

Iron is essential for photosynthesis and is often a limiting nutrient for plant productivity. Plants respond to conditions of iron deficiency by increasing transcript abundance of key genes involved in iron homeostasis, but only a few regulators of these genes have been identified. Using genome-wide expression analysis, we searched for transcription factors that are induced within 24 hours after transferring plants to iron-deficient growth conditions. Out of nearly 100 transcription factors shown to be up-regulated, we identified MYB10 and MYB72 as the most highly induced transcription factors. Here, we show that MYB10 and MYB72 are functionally redundant and are required for plant survival in alkaline soil where iron availability is greatly restricted. myb10myb72 double mutants fail to induce transcript accumulation of the nicotianamine synthase gene NAS4. Both myb10myb72 mutants and nas4-1 mutants have reduced iron concentrations, chlorophyll levels, and shoot mass under iron-limiting conditions, indicating that these genes are essential for proper plant growth. The double myb10myb72 mutant also showed nickel and zinc sensitivity, similar to the nas4 mutant. Ectopic expression of NAS4 rescues myb10myb72 plants, suggesting that loss of NAS4 is the primary defect in these plants and emphasizes the importance of nicotianamine, an iron chelator, in iron homeostasis. Overall, our results provide evidence that MYB10 and MYB72 act early in the iron-deficiency regulatory cascade to drive gene expression of NAS4 and are essential for plant survival under iron deficiency.

Genetic Analysis of mlh3 Mutations Reveals Interactions Between Crossover Promoting Factors During Meiosis in Baker’s Yeast

PubMed Central

Brown, Megan Sonntag; Lim, Elisha; Chen, Cheng; Nishant, K. T.; Alani, Eric

2013-01-01

Crossing over between homologous chromosomes occurs during the prophase of meiosis I and is critical for chromosome segregation. In baker’s yeast, two heterodimeric complexes, Msh4-Msh5 and Mlh1-Mlh3, act in meiosis to promote interference-dependent crossing over. Mlh1-Mlh3 also plays a role in DNA mismatch repair (MMR) by interacting with Msh2-Msh3 to repair insertion and deletion mutations. Mlh3 contains an ATP-binding domain that is highly conserved among MLH proteins. To explore roles for Mlh3 in meiosis and MMR, we performed a structure−function analysis of eight mlh3 ATPase mutants. In contrast to previous work, our data suggest that ATP hydrolysis by both Mlh1 and Mlh3 is important for both meiotic and MMR functions. In meiotic assays, these mutants showed a roughly linear relationship between spore viability and genetic map distance. To further understand the relationship between crossing over and meiotic viability, we analyzed crossing over on four chromosomes of varying lengths in mlh3Δ mms4Δ strains and observed strong decreases (6- to 17-fold) in crossing over in all intervals. Curiously, mlh3Δ mms4Δ double mutants displayed spore viability levels that were greater than observed in mms4Δ strains that show modest defects in crossing over. The viability in double mutants also appeared greater than would be expected for strains that show such severe defects in crossing over. Together, these observations provide insights for how Mlh1-Mlh3 acts in crossover resolution and MMR and for how chromosome segregation in Meiosis I can occur in the absence of crossing over. PMID:23316435

Arabidopsis leucine-rich repeat extensin (LRX) proteins modify cell wall composition and influence plant growth.

PubMed

Draeger, Christian; Ndinyanka Fabrice, Tohnyui; Gineau, Emilie; Mouille, Grégory; Kuhn, Benjamin M; Moller, Isabel; Abdou, Marie-Therese; Frey, Beat; Pauly, Markus; Bacic, Antony; Ringli, Christoph

2015-06-24

Leucine-rich repeat extensins (LRXs) are extracellular proteins consisting of an N-terminal leucine-rich repeat (LRR) domain and a C-terminal extensin domain containing the typical features of this class of structural hydroxyproline-rich glycoproteins (HRGPs). The LRR domain is likely to bind an interaction partner, whereas the extensin domain has an anchoring function to insolubilize the protein in the cell wall. Based on the analysis of the root hair-expressed LRX1 and LRX2 of Arabidopsis thaliana, LRX proteins are important for cell wall development. The importance of LRX proteins in non-root hair cells and on the structural changes induced by mutations in LRX genes remains elusive. The LRX gene family of Arabidopsis consists of eleven members, of which LRX3, LRX4, and LRX5 are expressed in aerial organs, such as leaves and stem. The importance of these LRX genes for plant development and particularly cell wall formation was investigated. Synergistic effects of mutations with gradually more severe growth retardation phenotypes in double and triple mutants suggest a similar function of the three genes. Analysis of cell wall composition revealed a number of changes to cell wall polysaccharides in the mutants. LRX3, LRX4, and LRX5, and most likely LRX proteins in general, are important for cell wall development. Due to the complexity of changes in cell wall structures in the lrx mutants, the exact function of LRX proteins remains to be determined. The increasingly strong growth-defect phenotypes in double and triple mutants suggests that the LRX proteins have similar functions and that they are important for proper plant development.

Petunia hybrida CAROTENOID CLEAVAGE DIOXYGENASE7 is involved in the production of negative and positive branching signals in petunia.

PubMed

Drummond, Revel S M; Martínez-Sánchez, N Marcela; Janssen, Bart J; Templeton, Kerry R; Simons, Joanne L; Quinn, Brian D; Karunairetnam, Sakuntala; Snowden, Kimberley C

2009-12-01

One of the key factors that defines plant form is the regulation of when and where branches develop. The diversity of form observed in nature results, in part, from variation in the regulation of branching between species. Two CAROTENOID CLEAVAGE DIOXYGENASE (CCD) genes, CCD7 and CCD8, are required for the production of a branch-suppressing plant hormone. Here, we report that the decreased apical dominance3 (dad3) mutant of petunia (Petunia hybrida) results from the mutation of the PhCCD7 gene and has a less severe branching phenotype than mutation of PhCCD8 (dad1). An analysis of the expression of this gene in wild-type, mutant, and grafted petunia suggests that in petunia, CCD7 and CCD8 are coordinately regulated. In contrast to observations in Arabidopsis (Arabidopsis thaliana), ccd7ccd8 double mutants in petunia show an additive phenotype. An analysis using dad3 or dad1 mutant scions grafted to wild-type rootstocks showed that when these plants produce adventitious mutant roots, branching is increased above that seen in plants where the mutant roots are removed. The results presented here indicate that mutation of either CCD7 or CCD8 in petunia results in both the loss of an inhibitor of branching and an increase in a promoter of branching.

Petunia hybrida CAROTENOID CLEAVAGE DIOXYGENASE7 Is Involved in the Production of Negative and Positive Branching Signals in Petunia1[W][OA

PubMed Central

Drummond, Revel S.M.; Martínez-Sánchez, N. Marcela; Janssen, Bart J.; Templeton, Kerry R.; Simons, Joanne L.; Quinn, Brian D.; Karunairetnam, Sakuntala; Snowden, Kimberley C.

2009-01-01

One of the key factors that defines plant form is the regulation of when and where branches develop. The diversity of form observed in nature results, in part, from variation in the regulation of branching between species. Two CAROTENOID CLEAVAGE DIOXYGENASE (CCD) genes, CCD7 and CCD8, are required for the production of a branch-suppressing plant hormone. Here, we report that the decreased apical dominance3 (dad3) mutant of petunia (Petunia hybrida) results from the mutation of the PhCCD7 gene and has a less severe branching phenotype than mutation of PhCCD8 (dad1). An analysis of the expression of this gene in wild-type, mutant, and grafted petunia suggests that in petunia, CCD7 and CCD8 are coordinately regulated. In contrast to observations in Arabidopsis (Arabidopsis thaliana), ccd7ccd8 double mutants in petunia show an additive phenotype. An analysis using dad3 or dad1 mutant scions grafted to wild-type rootstocks showed that when these plants produce adventitious mutant roots, branching is increased above that seen in plants where the mutant roots are removed. The results presented here indicate that mutation of either CCD7 or CCD8 in petunia results in both the loss of an inhibitor of branching and an increase in a promoter of branching. PMID:19846541

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

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.

Sterol Methyl Oxidases Affect Embryo Development via Auxin-Associated Mechanisms.

PubMed

Zhang, Xia; Sun, Shuangli; Nie, Xiang; Boutté, Yohann; Grison, Magali; Li, Panpan; Kuang, Susu; Men, Shuzhen

2016-05-01

Sterols are essential molecules for multiple biological processes, including embryogenesis, cell elongation, and endocytosis. The plant sterol biosynthetic pathway is unique in the involvement of two distinct sterol 4α-methyl oxidase (SMO) families, SMO1 and SMO2, which contain three and two isoforms, respectively, and are involved in sequential removal of the two methyl groups at C-4. In this study, we characterized the biological functions of members of the SMO2 gene family. SMO2-1 was strongly expressed in most tissues during Arabidopsis (Arabidopsis thaliana) development, whereas SMO2-2 showed a more specific expression pattern. Although single smo2 mutants displayed no obvious phenotype, the smo2-1 smo2-2 double mutant was embryonic lethal, and the smo2-1 smo2-2/+ mutant was dwarf, whereas the smo2-1/+ smo2-2 mutant exhibited a moderate phenotype. The phenotypes of the smo2 mutants resembled those of auxin-defective mutants. Indeed, the expression of DR5rev:GFP, an auxin-responsive reporter, was reduced and abnormal in smo2-1 smo2-2 embryos. Furthermore, the expression and subcellular localization of the PIN1 auxin efflux facilitator also were altered. Consistent with these observations, either the exogenous application of auxin or endogenous auxin overproduction (YUCCA9 overexpression) partially rescued the smo2-1 smo2-2 embryonic lethality. Surprisingly, the dwarf phenotype of smo2-1 smo2-2/+ was completely rescued by YUCCA9 overexpression. Gas chromatography-mass spectrometry analysis revealed a substantial accumulation of 4α-methylsterols, substrates of SMO2, in smo2 heterozygous double mutants. Together, our data suggest that SMO2s are important for correct sterol composition and function partially through effects on auxin accumulation, auxin response, and PIN1 expression to regulate Arabidopsis embryogenesis and postembryonic development. © 2016 American Society of Plant Biologists. All Rights Reserved.

Sterol Methyl Oxidases Affect Embryo Development via Auxin-Associated Mechanisms1

PubMed Central

Zhang, Xia; Sun, Shuangli; Nie, Xiang; Boutté, Yohann; Grison, Magali; Li, Panpan; Kuang, Susu

2016-01-01

Sterols are essential molecules for multiple biological processes, including embryogenesis, cell elongation, and endocytosis. The plant sterol biosynthetic pathway is unique in the involvement of two distinct sterol 4α-methyl oxidase (SMO) families, SMO1 and SMO2, which contain three and two isoforms, respectively, and are involved in sequential removal of the two methyl groups at C-4. In this study, we characterized the biological functions of members of the SMO2 gene family. SMO2-1 was strongly expressed in most tissues during Arabidopsis (Arabidopsis thaliana) development, whereas SMO2-2 showed a more specific expression pattern. Although single smo2 mutants displayed no obvious phenotype, the smo2-1 smo2-2 double mutant was embryonic lethal, and the smo2-1 smo2-2/+ mutant was dwarf, whereas the smo2-1/+ smo2-2 mutant exhibited a moderate phenotype. The phenotypes of the smo2 mutants resembled those of auxin-defective mutants. Indeed, the expression of DR5rev:GFP, an auxin-responsive reporter, was reduced and abnormal in smo2-1 smo2-2 embryos. Furthermore, the expression and subcellular localization of the PIN1 auxin efflux facilitator also were altered. Consistent with these observations, either the exogenous application of auxin or endogenous auxin overproduction (YUCCA9 overexpression) partially rescued the smo2-1 smo2-2 embryonic lethality. Surprisingly, the dwarf phenotype of smo2-1 smo2-2/+ was completely rescued by YUCCA9 overexpression. Gas chromatography-mass spectrometry analysis revealed a substantial accumulation of 4α-methylsterols, substrates of SMO2, in smo2 heterozygous double mutants. Together, our data suggest that SMO2s are important for correct sterol composition and function partially through effects on auxin accumulation, auxin response, and PIN1 expression to regulate Arabidopsis embryogenesis and postembryonic development. PMID:27006488

Deletion of the Candida glabrata ERG3 and ERG11 genes: effect on cell viability, cell growth, sterol composition, and antifungal susceptibility.

PubMed Central

Geber, A; Hitchcock, C A; Swartz, J E; Pullen, F S; Marsden, K E; Kwon-Chung, K J; Bennett, J E

1995-01-01

We have cloned and sequenced the structural genes encoding the delta 5,6 sterol desaturase (ERG3 gene) and the 14 alpha-methyl sterol demethylase (ERG11 gene) from Candida glabrata L5 (leu2). Single and double mutants of these genes were created by gene deletion. The phenotypes of these mutants, including sterol profiles, aerobic viabilities, antifungal susceptibilities, and generation times, were studied. Strain L5D (erg3 delta::LEU2) accumulated mainly ergosta-7,22-dien-3 beta-ol, was aerobically viable, and remained susceptible to antifungal agents but had a slower generation time than its parent strain. L5LUD (LEU2 erg11 delta::URA3) strains required medium supplemented with ergosterol and an anaerobic environment for growth. A spontaneous aerobically viable mutant, L5LUD40R (LEU erg11 delta::URA3), obtained from L5LUD (LEU2 erg11 delta::URA3), was found to accumulate lanosterol and obtusifoliol, was resistant to azole antifungal agents, demonstrated some increase in resistance to amphotericin B, and exhibited a 1.86-fold increase in generation time in comparison with L5 (leu2). The double-deletion mutant L5DUD61 (erg3 delta::LEU2 erg11 delta::URA3) was aerobically viable, produced mainly 14 alpha-methyl fecosterol, and had the same antifungal susceptibility pattern as L5LUD40R (LEU2 erg11 delta::URA3), and its generation time was threefold greater than that of L5 (leu2). Northern (RNA) analysis revealed that the single-deletion mutants had a marked increase in message for the undeleted ERG3 and ERG11 genes. These results indicate that differences in antifungal susceptibilities and the restoration of aerobic viability exist between the C. glabrata ergosterol mutants created in this study and those sterol mutants with similar genetic lesions previously reported for Saccharomyces cerevisiae. PMID:8593007

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

AGO1 controls arabidopsis inflorescence architecture possibly by regulating TFL1 expression.

PubMed

Fernández-Nohales, P; Domenech, M J; Martínez de Alba, A E; Micol, J L; Ponce, M R; Madueño, F

2014-11-01

The TERMINAL FLOWER 1 (TFL1) gene is pivotal in the control of inflorescence architecture in arabidopsis. Thus, tfl1 mutants flower early and have a very short inflorescence phase, while TFL1-overexpressing plants have extended vegetative and inflorescence phases, producing many coflorescences. TFL1 is expressed in the shoot meristems, never in the flowers. In the inflorescence apex, TFL1 keeps the floral genes LEAFY (LFY) and APETALA1 (AP1) restricted to the flower, while LFY and AP1 restrict TFL1 to the inflorescence meristem. In spite of the central role of TFL1 in inflorescence architecture, regulation of its expression is poorly understood. This study aims to expand the understanding of inflorescence development by identifying and studying novel TFL1 regulators. Mutagenesis of an Arabidopsis thaliana line carrying a TFL1::GUS (β-glucuronidase) reporter construct was used to isolate a mutant with altered TFL1 expression. The mutated gene was identified by positional cloning. Expression of TFL1 and TFL1::GUS was analysed by real-time PCR and histochemical GUS detection. Double-mutant analysis was used to assess the contribution of TFL1 to the inflorescence mutant phenotype. A mutant with both an increased number of coflorescences and high and ectopic TFL1 expression was isolated. Cloning of the mutated gene showed that both phenotypes were caused by a mutation in the ARGONAUTE1 (AGO1) gene, which encodes a key component of the RNA silencing machinery. Analysis of another ago1 allele indicated that the proliferation of coflorescences and ectopic TFL1 expression phenotypes are not allele specific. The increased number of coflorescences is suppressed in ago1 tfl1 double mutants. The results identify AGO1 as a repressor of TFL1 expression. Moreover, they reveal a novel role for AGO1 in inflorescence development, controlling the production of coflorescences. AGO1 seems to play this role through regulating TFL1 expression. © The Author 2014. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Analysis of in vivo correction of defined mismatches in the DNA mismatch repair mutants msh2, msh3 and msh6 of Saccharomyces cerevisiae.

PubMed

Lühr, B; Scheller, J; Meyer, P; Kramer, W

1998-02-01

We have analysed the correction of defined mismatches in wild-type and msh2, msh3, msh6 and msh3 msh6 mutants of Saccharomyces cerevisiae in two different yeast strain backgrounds by transformation with plasmid heteroduplex DNA constructs. Ten different base/base mismatches, two single-nucleotide loops and a 38-nucleotide loop were tested. Repair of all types of mismatches was severely impaired in msh2 and msh3 msh6 mutants. In msh6 mutants, repair efficiency of most base/base mismatches was reduced to a similar extent as in msh3 msh6 double mutants. G/T and A/C mismatches, however, displayed residual repair in msh6 mutants in one strain background, implying a role for Msh3p in recognition of base/base mismatches. Furthermore, the efficiency of repair of base/base mismatches was considerably reduced in msh3 mutants in one strain background, indicating a requirement for MSH3 for fully efficient mismatch correction. Also the efficiency of repair of the 38-nucleotide loop was reduced in msh3 mutants, and to a lesser extent in msh6 mutants. The single-nucleotide loop with an unpaired A was less efficiently repaired in msh3 mutants and that with an unpaired T was less efficiently corrected in msh6 mutants, indicating non-redundant functions for the two proteins in the recognition of single-nucleotide loops.

Essential roles for Cdx in murine primitive hematopoiesis.

PubMed

Brooke-Bisschop, Travis; Savory, Joanne G A; Foley, Tanya; Ringuette, Randy; Lohnes, David

2017-02-15

The Cdx transcription factors play essential roles in primitive hematopoiesis in the zebrafish where they exert their effects, in part, through regulation of hox genes. Defects in hematopoiesis have also been reported in Cdx mutant murine embryonic stem cell models, however, to date no mouse model reflecting the zebrafish Cdx mutant hematopoietic phenotype has been described. This is likely due, in part, to functional redundancy among Cdx members and the early lethality of Cdx2 null mutants. To circumvent these limitations, we used Cre-mediated conditional deletion to assess the impact of concomitant loss of Cdx1 and Cdx2 on murine primitive hematopoiesis. We found that Cdx1/Cdx2 double mutants exhibited defects in primitive hematopoiesis and yolk sac vasculature concomitant with reduced expression of several genes encoding hematopoietic transcription factors including Scl/Tal1. Chromatin immunoprecipitation analysis revealed that Scl was occupied by Cdx2 in vivo, and Cdx mutant hematopoietic yolk sac differentiation defects could be rescued by expression of exogenous Scl. These findings demonstrate critical roles for Cdx members in murine primitive hematopoiesis upstream of Scl. Copyright © 2017 Elsevier Inc. All rights reserved.

Strigolactones Stimulate Internode Elongation Independently of Gibberellins1[C][W

PubMed Central

de Saint Germain, Alexandre; Ligerot, Yasmine; Dun, Elizabeth A.; Pillot, Jean-Paul; Ross, John J.; Beveridge, Christine A.; Rameau, Catherine

2013-01-01

Strigolactone (SL) mutants in diverse species show reduced stature in addition to their extensive branching. Here, we show that this dwarfism in pea (Pisum sativum) is not attributable to the strong branching of the mutants. The continuous supply of the synthetic SL GR24 via the root system using hydroponics can restore internode length of the SL-deficient rms1 mutant but not of the SL-response rms4 mutant, indicating that SLs stimulate internode elongation via RMS4. Cytological analysis of internode epidermal cells indicates that SLs control cell number but not cell length, suggesting that SL may affect stem elongation by stimulating cell division. Consequently, SLs can repress (in axillary buds) or promote (in the stem) cell division in a tissue-dependent manner. Because gibberellins (GAs) increase internode length by affecting both cell division and cell length, we tested if SLs stimulate internode elongation by affecting GA metabolism or signaling. Genetic analyses using SL-deficient and GA-deficient or DELLA-deficient double mutants, together with molecular and physiological approaches, suggest that SLs act independently from GAs to stimulate internode elongation. PMID:23943865

Mutation of the Glucosinolate Biosynthesis Enzyme Cytochrome P450 83A1 Monooxygenase Increases Camalexin Accumulation and Powdery Mildew Resistance.

PubMed

Liu, Simu; Bartnikas, Lisa M; Volko, Sigrid M; Ausubel, Frederick M; Tang, Dingzhong

2016-01-01

Small secondary metabolites, including glucosinolates and the major phytoalexin camalexin, play important roles in immunity in Arabidopsis thaliana. We isolated an Arabidopsis mutant with increased resistance to the powdery mildew fungus Golovinomyces cichoracearum and identified a mutation in the gene encoding cytochrome P450 83A1 monooxygenase (CYP83A1), which functions in glucosinolate biosynthesis. The cyp83a1-3 mutant exhibited enhanced defense responses to G. cichoracearum and double mutant analysis showed that this enhanced resistance requires NPR1, EDS1, and PAD4, but not SID2 or EDS5. In cyp83a1-3 mutants, the expression of genes related to camalexin synthesis increased upon G. cichoracearum infection. Significantly, the cyp83a1-3 mutant also accumulated higher levels of camalexin. Decreasing camalexin levels by mutation of the camalexin synthetase gene PAD3 or the camalexin synthesis regulator AtWRKY33 compromised the powdery mildew resistance in these mutants. Consistent with these observations, overexpression of PAD3 increased camalexin levels and enhanced resistance to G. cichoracearum. Taken together, our data indicate that accumulation of higher levels of camalexin contributes to increased resistance to powdery mildew.

Mutation of the Glucosinolate Biosynthesis Enzyme Cytochrome P450 83A1 Monooxygenase Increases Camalexin Accumulation and Powdery Mildew Resistance

PubMed Central

Liu, Simu; Bartnikas, Lisa M.; Volko, Sigrid M.; Ausubel, Frederick M.; Tang, Dingzhong

2016-01-01

Small secondary metabolites, including glucosinolates and the major phytoalexin camalexin, play important roles in immunity in Arabidopsis thaliana. We isolated an Arabidopsis mutant with increased resistance to the powdery mildew fungus Golovinomyces cichoracearum and identified a mutation in the gene encoding cytochrome P450 83A1 monooxygenase (CYP83A1), which functions in glucosinolate biosynthesis. The cyp83a1-3 mutant exhibited enhanced defense responses to G. cichoracearum and double mutant analysis showed that this enhanced resistance requires NPR1, EDS1, and PAD4, but not SID2 or EDS5. In cyp83a1-3 mutants, the expression of genes related to camalexin synthesis increased upon G. cichoracearum infection. Significantly, the cyp83a1-3 mutant also accumulated higher levels of camalexin. Decreasing camalexin levels by mutation of the camalexin synthetase gene PAD3 or the camalexin synthesis regulator AtWRKY33 compromised the powdery mildew resistance in these mutants. Consistent with these observations, overexpression of PAD3 increased camalexin levels and enhanced resistance to G. cichoracearum. Taken together, our data indicate that accumulation of higher levels of camalexin contributes to increased resistance to powdery mildew. PMID:26973671

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

Purification, Crystallization And Preliminary X-Ray Analysis of Aminoglycoside-2 ''-Phosphotransferase-Ic [APH(2 '')-Ic] From Enterococcus Gallinarum

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

Byrnes, L.J.; /SLAC, SSRL; Badarau, A.

2009-04-30

Bacterial resistance to aminoglycoside antibiotics is primarily the result of deactivation of the drugs. Three families of enzymes are responsible for this activity, with one such family being the aminoglycoside phosphotransferases (APHs). The gene encoding one of these enzymes, aminoglycoside-2{double_prime}-phosphotransferase-Ic [APH(2{double_prime})-Ic] from Enterococcus gallinarum, has been cloned and the wild-type protein (comprising 308 amino-acid residues) and three mutants that showed elevated minimum inhibitory concentrations towards gentamicin (F108L, H258L and a double mutant F108L/H258L) were expressed in Escherichia coli and subsequently purified. All APH(2{double_prime})-Ic variants were crystallized in the presence of 14-20%(w/v) PEG 4000, 0.25 M MgCl{sub 2}, 0.1 M Tris-HClmore » pH 8.5 and 1 mM Mg{sub 2}GTP. The crystals belong to the monoclinic space group C2, with one molecule in the asymmetric unit. The approximate unit-cell parameters are a = 82.4, b = 54.2, c = 77.0 {angstrom}, {beta} = 108.8{sup o}. X-ray diffraction data were collected to approximately 2.15 {angstrom} resolution from an F108L crystal at beamline BL9-2 at SSRL, Stanford, California, USA.« less

The origin of the cooperativity in the streptavidin-biotin system: A computational investigation through molecular dynamics simulations.

PubMed

Liu, Fengjiao; Zhang, John Z H; Mei, Ye

2016-06-01

Previous experimental study measuring the binding affinities of biotin to the wild type streptavidin (WT) and three mutants (S45A, D128A and S45A/D128A double mutant) has shown that the loss of binding affinity from the double mutation is larger than the direct sum of those from two single mutations. The origin of this cooperativity has been investigated in this work through molecular dynamics simulations and the end-state free energy method using the polarized protein-specific charge. The results show that this cooperativity comes from both the enthalpy and entropy contributions. The former contribution mainly comes from the alternations of solvation free energy. Decomposition analysis shows that the mutated residues nearly have no contributions to the cooperativity. Instead, N49 and S88, which are located at the entry of the binding pocket and interact with the carboxyl group of biotin, make the dominant contribution among all the residues in the first binding shell around biotin.

The origin of the cooperativity in the streptavidin-biotin system: A computational investigation through molecular dynamics simulations

NASA Astrophysics Data System (ADS)

Liu, Fengjiao; Zhang, John Z. H.; Mei, Ye

2016-06-01

Previous experimental study measuring the binding affinities of biotin to the wild type streptavidin (WT) and three mutants (S45A, D128A and S45A/D128A double mutant) has shown that the loss of binding affinity from the double mutation is larger than the direct sum of those from two single mutations. The origin of this cooperativity has been investigated in this work through molecular dynamics simulations and the end-state free energy method using the polarized protein-specific charge. The results show that this cooperativity comes from both the enthalpy and entropy contributions. The former contribution mainly comes from the alternations of solvation free energy. Decomposition analysis shows that the mutated residues nearly have no contributions to the cooperativity. Instead, N49 and S88, which are located at the entry of the binding pocket and interact with the carboxyl group of biotin, make the dominant contribution among all the residues in the first binding shell around biotin.

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.

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

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.

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.

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

Elucidating the genomic architecture of Asian EGFR-mutant lung adenocarcinoma through multi-region exome sequencing.

PubMed

Nahar, Rahul; Zhai, Weiwei; Zhang, Tong; Takano, Angela; Khng, Alexis J; Lee, Yin Yeng; Liu, Xingliang; Lim, Chong Hee; Koh, Tina P T; Aung, Zaw Win; Lim, Tony Kiat Hon; Veeravalli, Lavanya; Yuan, Ju; Teo, Audrey S M; Chan, Cheryl X; Poh, Huay Mei; Chua, Ivan M L; Liew, Audrey Ann; Lau, Dawn Ping Xi; Kwang, Xue Lin; Toh, Chee Keong; Lim, Wan-Teck; Lim, Bing; Tam, Wai Leong; Tan, Eng-Huat; Hillmer, Axel M; Tan, Daniel S W

2018-01-15

EGFR-mutant lung adenocarcinomas (LUAD) display diverse clinical trajectories and are characterized by rapid but short-lived responses to EGFR tyrosine kinase inhibitors (TKIs). Through sequencing of 79 spatially distinct regions from 16 early stage tumors, we show that despite low mutation burdens, EGFR-mutant Asian LUADs unexpectedly exhibit a complex genomic landscape with frequent and early whole-genome doubling, aneuploidy, and high clonal diversity. Multiple truncal alterations, including TP53 mutations and loss of CDKN2A and RB1, converge on cell cycle dysregulation, with late sector-specific high-amplitude amplifications and deletions that potentially beget drug resistant clones. We highlight the association between genomic architecture and clinical phenotypes, such as co-occurring truncal drivers and primary TKI resistance. Through comparative analysis with published smoking-related LUAD, we postulate that the high intra-tumor heterogeneity observed in Asian EGFR-mutant LUAD may be contributed by an early dominant driver, genomic instability, and low background mutation rates.

High-Resolution Free-Energy Landscape Analysis of α-Helical Protein Folding: HP35 and Its Double Mutant

PubMed Central

2013-01-01

The free-energy landscape can provide a quantitative description of folding dynamics, if determined as a function of an optimally chosen reaction coordinate. Here, we construct the optimal coordinate and the associated free-energy profile for all-helical proteins HP35 and its norleucine (Nle/Nle) double mutant, based on realistic equilibrium folding simulations [Piana et al. Proc. Natl. Acad. Sci. U.S.A.2012, 109, 17845]. From the obtained profiles, we directly determine such basic properties of folding dynamics as the configurations of the minima and transition states (TS), the formation of secondary structure and hydrophobic core during the folding process, the value of the pre-exponential factor and its relation to the transition path times, the relation between the autocorrelation times in TS and minima. We also present an investigation of the accuracy of the pre-exponential factor estimation based on the transition-path times. Four different estimations of the pre-exponential factor for both proteins give k0–1 values of approximately a few tens of nanoseconds. Our analysis gives detailed information about folding of the proteins and can serve as a rigorous common language for extensive comparison between experiment and simulation. PMID:24348206

Compound Heterozygosity for Y Box Proteins Causes Sterility Due to Loss of Translational Repression

PubMed Central

Sharma, Manju; Dearth, Andrea; Smith, Benjamin; Braun, Robert E.

2015-01-01

The Y-box proteins YBX2 and YBX3 bind RNA and DNA and are required for metazoan development and fertility. However, possible functional redundancy between YBX2 and YBX3 has prevented elucidation of their molecular function as RNA masking proteins and identification of their target RNAs. To investigate possible functional redundancy between YBX2 and YBX3, we attempted to construct Ybx2 -/- ;Ybx3 -/- double mutants using a previously reported Ybx2 -/- model and a newly generated global Ybx3 -/- model. Loss of YBX3 resulted in reduced male fertility and defects in spermatid differentiation. However, homozygous double mutants could not be generated as haploinsufficiency of both Ybx2 and Ybx3 caused sterility characterized by extensive defects in spermatid differentiation. RNA sequence analysis of mRNP and polysome occupancy in single and compound Ybx2/3 heterozygotes revealed loss of translational repression almost exclusively in the compound Ybx2/3 heterozygotes. RNAseq analysis also demonstrated that Y-box protein dose-dependent loss of translational regulation was inversely correlated with the presence of a Y box recognition target sequence, suggesting that Y box proteins bind RNA hierarchically to modulate translation in a range of targets. PMID:26646932

High-Resolution Free-Energy Landscape Analysis of α-Helical Protein Folding: HP35 and Its Double Mutant.

PubMed

Banushkina, Polina V; Krivov, Sergei V

2013-12-10

The free-energy landscape can provide a quantitative description of folding dynamics, if determined as a function of an optimally chosen reaction coordinate. Here, we construct the optimal coordinate and the associated free-energy profile for all-helical proteins HP35 and its norleucine (Nle/Nle) double mutant, based on realistic equilibrium folding simulations [Piana et al. Proc. Natl. Acad. Sci. U.S.A. 2012 , 109 , 17845]. From the obtained profiles, we directly determine such basic properties of folding dynamics as the configurations of the minima and transition states (TS), the formation of secondary structure and hydrophobic core during the folding process, the value of the pre-exponential factor and its relation to the transition path times, the relation between the autocorrelation times in TS and minima. We also present an investigation of the accuracy of the pre-exponential factor estimation based on the transition-path times. Four different estimations of the pre-exponential factor for both proteins give k 0 -1 values of approximately a few tens of nanoseconds. Our analysis gives detailed information about folding of the proteins and can serve as a rigorous common language for extensive comparison between experiment and simulation.

Creation, characterization and utilization of Cryptococcus neoformans mutants sensitive to micafungin.

PubMed

Toh-E, Akio; Ohkusu, Misako; Shimizu, Kiminori; Yamaguchi, Masashi; Ishiwada, Naruhiko; Watanabe, Akira; Kamei, Katsuhiko

2017-12-01

We constructed deletion mutants of Cryptococcus neoformans var neoformans (serotype D) genes encoding late ergosterol biosynthetic pathway enzymes and found that the mutations enhanced susceptibility to various drugs including micafungin, one of the echinocandins, to which wild-type Cryptococcus strains show no susceptibility. Furthermore, through isolation of a mutant resistant to micafungin from a micafungin-sensitive erg mutant and genetic analysis of it, we found that the responsible mutation occurred in the hotspot 2 of FKS1 encoding β-1, 3-glucan synthase, indicating that micafungin inhibited the growth of the erg mutant via inhibiting Fks1 activity. Addition of ergosterol to the culture of the erg mutants recovered the resistance to micafungin, suggesting that the presence of ergosterol in membrane inhibits the accession of micafungin to its target. We found that a loss of one of genes encoding subunits of v-ATPase, VPH1, made Cryptococcus cells sensitive to micafungin. Our observation that the erg2 vph1 double mutant was more sensitive to micafungin than either single mutant suggests that these two genes act differently in becoming resistant to micafungin. The erg mutants allowed us to study the physiological significance of β-1, 3-glucan synthesis in C. neoformans; the inhibition of β-1, 3-glucan synthesis induced cell death and changes in cellular morphology. By observing the erg mutant cells recovering from the growth inhibition imposed by micafungin, we recognized β-1, 3-glucan synthesis would suppress filamentous growth in C. neoformans.

Synergistic role of curli and cellulose in cell adherence and biofilm formation of attaching and effacing Escherichia coli and identification of Fis as a negative regulator of curli.

PubMed

Saldaña, Zeus; Xicohtencatl-Cortes, Juan; Avelino, Fabiola; Phillips, Alan D; Kaper, James B; Puente, José L; Girón, Jorge A

2009-04-01

Curli are adhesive fimbriae of Escherichia coli and Salmonella enterica. Expression of curli (csgA) and cellulose (bcsA) is co-activated by the transcriptional activator CsgD. In this study, we investigated the contribution of curli and cellulose to the adhesive properties of enterohaemorragic (EHEC) O157:H7 and enteropathogenic E. coli (EPEC) O127:H6. While single mutations in csgA, csgD or bcsA in EPEC and EHEC had no dramatic effect on cell adherence, double csgAbcsA mutants were significantly less adherent than the single mutants or wild-type strains to human colonic HT-29 epithelial cells or to cow colon tissue in vitro. Overexpression of csgD (carried on plasmid pCP994) in a csgD mutant, but not in the single csgA or bscA mutants, led to significant increase in adherence and biofilm formation in EPEC and EHEC, suggesting that synchronized over-production of curli and cellulose enhances bacterial adherence. In line with this finding, csgD transcription was activated significantly in the presence of cultured epithelial cells as compared with growth in tissue culture medium. Analysis of the influence of virulence and global regulators in the production of curli in EPEC identified Fis (factor for inversion stimulation) as a, heretofore unrecognized, negative transcriptional regulator of csgA expression. An EPEC E2348/69Deltafis produced abundant amounts of curli whereas a double fis/csgD mutant yielded no detectable curli production. Our data suggest that curli and cellulose act in concert to favour host colonization, biofilm formation and survival in different environments.

CRISPR/Cas9-Induced Double-Strand Break Repair in Arabidopsis Nonhomologous End-Joining Mutants.

PubMed

Shen, Hexi; Strunks, Gary D; Klemann, Bart J P M; Hooykaas, Paul J J; de Pater, Sylvia

2017-01-05

Double-strand breaks (DSBs) are one of the most harmful DNA lesions. Cells utilize two main pathways for DSB repair: homologous recombination (HR) and nonhomologous end-joining (NHEJ). NHEJ can be subdivided into the KU-dependent classical NHEJ (c-NHEJ) and the more error-prone KU-independent backup-NHEJ (b-NHEJ) pathways, involving the poly (ADP-ribose) polymerases (PARPs). However, in the absence of these factors, cells still seem able to adequately maintain genome integrity, suggesting the presence of other b-NHEJ repair factors or pathways independent from KU and PARPs. The outcome of DSB repair by NHEJ pathways can be investigated by using artificial sequence-specific nucleases such as CRISPR/Cas9 to induce DSBs at a target of interest. Here, we used CRISPR/Cas9 for DSB induction at the Arabidopsis cruciferin 3 (CRU3) and protoporphyrinogen oxidase (PPO) genes. DSB repair outcomes via NHEJ were analyzed using footprint analysis in wild-type plants and plants deficient in key factors of c-NHEJ (ku80), b-NHEJ (parp1 parp2), or both (ku80 parp1 parp2). We found that larger deletions of >20 bp predominated after DSB repair in ku80 and ku80 parp1 parp2 mutants, corroborating with a role of KU in preventing DSB end resection. Deletion lengths did not significantly differ between ku80 and ku80 parp1 parp2 mutants, suggesting that a KU- and PARP-independent b-NHEJ mechanism becomes active in these mutants. Furthermore, microhomologies and templated insertions were observed at the repair junctions in the wild type and all mutants. Since these characteristics are hallmarks of polymerase θ-mediated DSB repair, we suggest a possible role for this recently discovered polymerase in DSB repair in plants. Copyright © 2017 Shen et al.

Genetic analysis of rice mutants responsible for narrow leaf phenotype and reduced vein number.

PubMed

Kubo, Fumika Clara; Yasui, Yukiko; Kumamaru, Toshihiro; Sato, Yutaka; Hirano, Hiro-Yuki

2017-03-17

Leaves are a major site for photosynthesis and a key determinant of plant architecture. Rice produces thin and slender leaves, which consist of the leaf blade and leaf sheath separated by the lamina joint. Two types of vasculature, the large and small vascular bundles, run in parallel, together with a strong structure, the midrib. In this paper, we examined the function of four genes that regulate the width of the leaf blade and the vein number: NARROW LEAF1 (NAL1), NAL2, NAL3 and NAL7. We backcrossed original mutants of these genes with the standard wild-type rice, Taichung 65. We then compared the effect of each mutation on similar genetic backgrounds and examined genetic interactions of these genes. The nal1 single mutation and the nal2 nal3 double mutation showed a severe effect on leaf width, resulting in very narrow leaves. Although vein number was also reduced in the nal1 and nal2 nal3 mutants, the small vein number was more strongly reduced than the large vein number. In contrast, the nal7 mutation showed a milder effect on leaf width and vein number, and both the large and small veins were similarly affected. Thus, the genes responsible for narrow leaf phenotype seem to play distinct roles. The nal7 mutation showed additive effects on both leaf width and vein number, when combined with the nal1 single or the nal2 nal3 double mutation. In addition, observations of inner tissues revealed that cell differentiation was partially compromised in the nal2 nal3 nal7 mutant, consistent with the severe reduction in leaf width in this triple mutant.

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

Consensus guided mutagenesis of Renilla luciferase yields enhanced stability and light output.

PubMed

Loening, Andreas Markus; Fenn, Timothy David; Wu, Anna M; Gambhir, Sanjiv Sam

2006-09-01

Luciferases, which have seen expansive employment as reporter genes in biological research, could also be used in applications where the protein itself is conjugated to ligands to create probes that are appropriate for use in small animal imaging. As the bioluminescence activity of commonly used luciferases is too labile in serum to permit this application, specific mutations of Renilla luciferase, selected using a consensus sequence driven strategy, were screened for their ability to confer stability of activity in serum as well as their light output. Using this information, a total of eight favorable mutations were combined to generate a mutant Renilla luciferase (RLuc8) that, compared with the parental enzyme, is 200-fold more resistant to inactivation in murine serum and exhibits a 4-fold improvement in light output. Results of the mutational analysis were also used to generate a double mutant optimized for use as a reporter gene. The double mutant had half the resistance to inactivation in serum of the native enzyme while yielding a 5-fold improvement in light output. These variants of Renilla luciferase, which exhibit significantly improved properties compared with the native enzyme, will allow enhanced sensitivity in existing luciferase-based assays as well as enable the development of novel probes labeled with the luciferase protein.

Genetic recombination induced by DNA double-strand break in bacteriophage T4: nature of the left/right bias.

PubMed

Shcherbakov, Victor P; Shcherbakova, Tamara; Plugina, Lidiya; Sizova, Svetlana; Kudryashova, Elena; Granovsky, Igor

2008-06-01

The experimental system combining double-strand breaks (DSBs), produced site-specifically by SegC endonuclease, with the famous advantages of the bacteriophage T4 rII mutant recombination analysis was used here to elucidate the origin of the recombination bias on two sides of the DSB, especially pronounced in gene 39 (topoisomerase II) and gene 59 (41-helicase loader) mutants. Three sources were found to contribute to the bias: (1) the SegC endonuclease may remain bound to the end of the broken DNA and thus protect it from exonuclease degradation; (2) in heteroduplex heterozygotes (HHs), arising as the recombinant products in the left-hand crosses, the transcribed strands are of rII mutant phenotype, so they, in contrast to the right-hand HHs, do not produce plaques on the lawn of the lambda-lysogenic host; and (3) the intrinsic polarity of T4 chromosome, reflected in transcription, may be a cause for discrimination of promoter-proximal and promoter-distal DNA sequences. It is shown that the apparent recombination bias does not imply one-sidedness of the DSB repair but just reflects a different depth of the end processing. It is inferred that the cause, underlying the "intrinsic" bias, might be interference between strand exchange and transcription. Topoisomerase and helicase functions are necessary to turn the process in favor of strand exchange. The idea is substantiated that the double-stranded to single-stranded DNA transition edge (not ss-DNA tip) serves as an actual recombinogenic element.

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.

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.

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

Ultrasensitive Quantification of Hepatitis B Virus A1762T/G1764A Mutant by a SimpleProbe PCR Using a Wild-Type-Selective PCR Blocker and a Primer-Blocker-Probe Partial-Overlap Approach ▿

PubMed Central

Nie, Hui; Evans, Alison A.; London, W. Thomas; Block, Timothy M.; Ren, Xiangdong David

2011-01-01

Hepatitis B virus (HBV) carrying the A1762T/G1764A double mutation in the basal core promoter (BCP) region is associated with HBe antigen seroconversion and increased risk of liver cirrhosis and hepatocellular carcinoma (HCC). Quantification of the mutant viruses may help in predicting the risk of HCC. However, the viral genome tends to have nucleotide polymorphism, which makes it difficult to design hybridization-based assays including real-time PCR. Ultrasensitive quantification of the mutant viruses at the early developmental stage is even more challenging, as the mutant is masked by excessive amounts of the wild-type (WT) viruses. In this study, we developed a selective inhibitory PCR (siPCR) using a locked nucleic acid-based PCR blocker to selectively inhibit the amplification of the WT viral DNA but not the mutant DNA. At the end of siPCR, the proportion of the mutant could be increased by about 10,000-fold, making the mutant more readily detectable by downstream applications such as real-time PCR and DNA sequencing. We also describe a primer-probe partial overlap approach which significantly simplified the melting curve patterns and minimized the influence of viral genome polymorphism on assay accuracy. Analysis of 62 patient samples showed a complete match of the melting curve patterns with the sequencing results. More than 97% of HBV BCP sequences in the GenBank database can be correctly identified by the melting curve analysis. The combination of siPCR and the SimpleProbe real-time PCR enabled mutant quantification in the presence of a 100,000-fold excess of the WT DNA. PMID:21562108

Construction, characterization and evaluation of the protective efficacy of the Streptococcus suis double mutant strain ΔSsPep/ΔSsPspC as a live vaccine candidate in mice.

PubMed

Hu, Jin; You, Wujin; Wang, Bin; Hu, Xueying; Tan, Chen; Liu, Jinlin; Chen, Huanchun; Bei, Weicheng

2015-01-01

Streptococcus suis serotype 2 (S. suis 2) causes sepsis and meningitis in piglets and humans, and results in one of the most serious bacterial diseases affecting the production of commercial pigs around the world. Due to the failure of the current inactivated vaccine to protect against the disease, development of a new attenuated live vaccine against S. suis 2 by deleting essential virulence factors is urgently needed. We have previously reported the construction and characterization of an SsPep single gene deletion mutant strain ΔSsPep based on S. suis 2. Our previous results have shown that SsPep plays a critical role in the pathogenesis of S. suis 2. In this study, a precisely defined double-deletion mutant ΔSsPep/ΔSsPspC of S. suis 2 without antibiotic-resistance markers was constructed based on ΔSsPep, and the levels of virulence of the wild-type (WT) and ΔSsPep/ΔSsPspC were compared in a mouse experimental infection model. We demonstrated that the double mutant ΔSsPep/ΔSsPspC was less virulent than the WT, and could induce a noticeable antibody response. Analysis of IgG subclasses (IgG1 and IgG2a) indicated that both Th1 and Th2 responses were induced by ΔSsPep/ΔSsPspC, although the IgG2a (Th1) response predominated over the IgG1 (Th2) response. Moreover, ΔSsPep/ΔSsPspC could confer 90% protective efficacy against challenge with a lethal dose of fully virulent S. suis 2. Taken together, these data demonstrate that ΔSsPep/ΔSsPspC can be used as an effective live vaccine and provide a novel strategy against infection of S. suis 2. Copyright © 2014 Elsevier GmbH. All rights reserved.

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.

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.

Genetic analysis of mouse embryonic stem cells bearing Msh3 and Msh2 single and compound mutations.

PubMed

Abuin, A; Zhang, H; Bradley, A

2000-01-01

We have previously described the use of homologous recombination and CRE-loxP-mediated marker recycling to generate mouse embryonic stem (ES) cell lines homozygous for mutations at the Msh3, Msh2, and both Msh3 and Msh2 loci (2). In this study, we describe the analysis of these ES cells with respect to processes known to be affected by DNA mismatch repair. ES cells homozygous for the Msh2 mutation displayed increased resistance to killing by the cytotoxic drug 6-thioguanine (6TG), indicating that the 6TG cytotoxic mechanism is mediated by Msh2. The mutation rate of the herpes simplex virus thymidine kinase 1 (HSV-tk1) gene was unchanged in Msh3-deficient ES cell lines but markedly elevated in Msh2-deficient and Msh3 Msh2 double-mutant cells. Notably, the HSV-tk1 mutation rate was 11-fold higher, on average, than that of the hypoxanthine-guanine phosphoribosyl transferase (Hprt) locus in Msh2-deficient cells. Sequence analysis of HSV-tk1 mutants from these cells indicated the presence of a frameshift hotspot within the HSV-tk1 coding region. Msh3-deficient cells displayed a modest (16-fold) elevation in the instability of a dinucleotide repeat, whereas Msh2-deficient and Msh2 Msh3 double-mutant cells displayed markedly increased levels of repeat instability. Targeting frequencies of nonisogenic vectors were elevated in Msh2-deficient ES cell lines, confirming the role of Msh2 in blocking recombination between diverged sequences (homeologous recombination) in mammalian cells. These results are consistent with accumulating data from other laboratories and support the current model of DNA mismatch repair in mammalian cells.

Genetic Analysis of Mouse Embryonic Stem Cells Bearing Msh3 and Msh2 Single and Compound Mutations

PubMed Central

Abuin, Alejandro; Zhang, HeJu; Bradley, Allan

2000-01-01

We have previously described the use of homologous recombination and CRE-loxP-mediated marker recycling to generate mouse embryonic stem (ES) cell lines homozygous for mutations at the Msh3, Msh2, and both Msh3 and Msh2 loci (2). In this study, we describe the analysis of these ES cells with respect to processes known to be affected by DNA mismatch repair. ES cells homozygous for the Msh2 mutation displayed increased resistance to killing by the cytotoxic drug 6-thioguanine (6TG), indicating that the 6TG cytotoxic mechanism is mediated by Msh2. The mutation rate of the herpes simplex virus thymidine kinase 1 (HSV-tk1) gene was unchanged in Msh3-deficient ES cell lines but markedly elevated in Msh2-deficient and Msh3 Msh2 double-mutant cells. Notably, the HSV-tk1 mutation rate was 11-fold higher, on average, than that of the hypoxanthine-guanine phosphoribosyl transferase (Hprt) locus in Msh2-deficient cells. Sequence analysis of HSV-tk1 mutants from these cells indicated the presence of a frameshift hotspot within the HSV-tk1 coding region. Msh3-deficient cells displayed a modest (16-fold) elevation in the instability of a dinucleotide repeat, whereas Msh2-deficient and Msh2 Msh3 double-mutant cells displayed markedly increased levels of repeat instability. Targeting frequencies of nonisogenic vectors were elevated in Msh2-deficient ES cell lines, confirming the role of Msh2 in blocking recombination between diverged sequences (homeologous recombination) in mammalian cells. These results are consistent with accumulating data from other laboratories and support the current model of DNA mismatch repair in mammalian cells. PMID:10594017

Peroxiredoxin 5 modulates immune response in Drosophila

PubMed Central

Radyuk, Svetlana N.; Michalak, Katarzyna; Klichko, Vladimir I.; Benes, Judith; Orr, William C.

2010-01-01

Background Peroxiredoxins are redox-sensing enzymes with multiple cellular functions. Previously, we reported on the potent antioxidant function of Drosophila peroxiredoxin 5 (dPrx5). Studies with mammalian and human cells suggest that peroxiredoxins can modulate immune-related signaling. Methods Survivorship studies and bacteriological analysis were used to determine resistance of flies to fungal and bacterial infections. RT-PCR and immunoblot analyses determined expression of dPrx5 and immunity factors in response to bacterial challenge. Double mutants for dprx5 gene and genes comprising the Imd/Relish and dTak1/Basket branches of the immune signaling pathways were used in epistatic analysis. Results The dprx5 mutant flies were more resistant to bacterial infection than controls, while flies overexpressing dPrx5 were more susceptible. The enhanced resistance to bacteria was accompanied by rapid induction of the Imd-dependent antimicrobial peptides, phosphorylation of the JNK kinase Basket and altered transcriptional profiling of the transient response genes, puckered, ets21C and relish, while the opposite effects were observed in flies over-expressing dPrx5. Epistatic analysis of double mutants, using attacin D and Puckered as read outs of activation of the Imd and JNK pathways, implicated dPrx5 function in the control of the dTak1-JNK arm of immune signaling. Conclusions Differential effects on fly survivorship suggested a trade-off between the antioxidant and immune functions of dPrx5. Molecular and epistatic analyses identified dPrx5 as a negative regulator in the dTak1-JNK arm of immune signaling. General significance Our findings suggest that peroxiredoxins play an important modulatory role in the Drosophila immune response. PMID:20600624

The aminoglycoside antibiotic kanamycin damages DNA bases in Escherichia coli: caffeine potentiates the DNA-damaging effects of kanamycin while suppressing cell killing by ciprofloxacin in Escherichia coli and Bacillus anthracis.

PubMed

Kang, Tina Manzhu; Yuan, Jessica; Nguyen, Angelyn; Becket, Elinne; Yang, Hanjing; Miller, Jeffrey H

2012-06-01

The distribution of mutants in the Keio collection of Escherichia coli gene knockout mutants that display increased sensitivity to the aminoglycosides kanamycin and neomycin indicates that damaged bases resulting from antibiotic action can lead to cell death. Strains lacking one of a number of glycosylases (e.g., AlkA, YzaB, Ogt, KsgA) or other specific repair proteins (AlkB, PhrB, SmbC) are more sensitive to these antibiotics. Mutants lacking AlkB display the strongest sensitivity among the glycosylase- or direct lesion removal-deficient strains. This perhaps suggests the involvement of ethenoadenine adducts, resulting from reactive oxygen species and lipid peroxidation, since AlkB removes this lesion. Other sensitivities displayed by mutants lacking UvrA, polymerase V (Pol V), or components of double-strand break repair indicate that kanamycin results in damaged base pairs that need to be removed or replicated past in order to avoid double-strand breaks that saturate the cellular repair capacity. Caffeine enhances the sensitivities of these repair-deficient strains to kanamycin and neomycin. The gene knockout mutants that display increased sensitivity to caffeine (dnaQ, holC, holD, and priA knockout mutants) indicate that caffeine blocks DNA replication, ultimately leading to double-strand breaks that require recombinational repair by functions encoded by recA, recB, and recC, among others. Additionally, caffeine partially protects cells of both Escherichia coli and Bacillus anthracis from killing by the widely used fluoroquinolone antibiotic ciprofloxacin.

A mutant of Mycobacterium smegmatis defective in the biosynthesis of mycolic acids accumulates meromycolates

PubMed Central

Liu, Jun; Nikaido, Hiroshi

1999-01-01

Mycolic acids are a major constituent of the mycobacterial cell wall, and they form an effective permeability barrier to protect mycobacteria from antimicrobial agents. Although the chemical structures of mycolic acids are well established, little is known on their biosynthesis. We have isolated a mycolate-deficient mutant strain of Mycobacterium smegmatis mc2-155 by chemical mutagenesis followed by screening for increased sensitivity to novobiocin. This mutant also was hypersensitive to other hydrophobic compounds such as crystal violet, rifampicin, and erythromycin. Entry of hydrophobic probes into mutant cells occurred much more rapidly than that into the wild-type cells. HPLC and TLC analysis of fatty acid composition after saponification showed that the mutant failed to synthesize full-length mycolic acids. Instead, it accumulated a series of long-chain fatty acids, which were not detected in the wild-type strain. Analysis by 1H NMR, electrospray and electron impact mass spectroscopy, and permanganate cleavage of double bonds showed that these compounds corresponded to the incomplete meromycolate chain of mycolic acids, except for the presence of a β-hydroxyl group. This direct identification of meromycolates as precursors of mycolic acids provides a strong support for the previously proposed pathway for mycolic acid biosynthesis involving the separate synthesis of meromycolate chain and the α-branch of mycolic acids, followed by the joining of these two branches. PMID:10097154

Aspirin increases metabolism through germline signalling to extend the lifespan of Caenorhabditis elegans

PubMed Central

Huang, Xiao-Bing; Mu, Xiao-Hui; Wan, Qin-Li; He, Xiao-Ming; Wu, Gui-Sheng

2017-01-01

Aspirin is a prototypic cyclooxygenase inhibitor with a variety of beneficial effects on human health. It prevents age-related diseases and delays the aging process. Previous research has shown that aspirin might act through a dietary restriction-like mechanism to extend lifespan. To explore the mechanism of action of aspirin on aging, we determined the whole-genome expression profile of Caenorhabditis elegans treated with aspirin. Transcriptome analysis revealed the RNA levels of genes involved in metabolism were primarily increased. Reproduction has been reported to be associated with metabolism. We found that aspirin did not extend the lifespan or improve the heat stress resistance of germline mutants of glp-1. Furthermore, Oil Red O staining showed that aspirin treatment decreased lipid deposition and increased expression of lipid hydrolysis and fatty acid β-oxidation-related genes. The effect of germline ablation on lifespan was mainly mediated by DAF-12 and DAF-16. Next, we performed genetic analysis with a series of worm mutants and found that aspirin did not further extend the lifespans of daf-12 and daf-16 single mutants, glp-1;daf-12 and glp-1;daf-16 double mutants, or glp-1;daf-12;daf-16 triple mutants. The results suggest that aspirin increase metabolism and regulate germline signalling to activate downstream DAF-12 and DAF-16 to extend lifespan. PMID:28910305

The allosteric activator ATP induces a substrate-dependent alteration of the quaternary structure of a mutant aspartate transcarbamoylase impaired in active site closure.

PubMed Central

Baker, D. P.; Fetler, L.; Vachette, P.; Kantrowitz, E. R.

1996-01-01

Aspartate transcarbamoylase from Escherichia coli shows homotropic cooperativity for aspartate as well as heterotropic regulation by nucleotides. Structurally, it consists of two trimeric catalytic subunits and three dimeric regulatory subunits, each chain being comprised of two domains. Glu-50 and Ser-171 are involved in stabilizing the closed conformation of the catalytic chain. Replacement of Glu-50 or Ser-171 by Ala in the holoenzyme has been shown previously to result in marked decreases in the maximal observed specific activity, homotropic cooperativity, and affinity for aspartate (Dembowski NJ, Newton CJ, Kantrowitz ER, 1990, Biochemistry 29:3716-3723; Newton CJ, Kantrowitz ER, 1990, Biochemistry 29:1444-1451). We have constructed a double mutant enzyme combining both mutations. The resulting Glu-50/ser-171-->Ala enzyme is 9-fold less active than the Ser-171-->Ala enzyme, 69-fold less active than the Glu-50-->Ala enzyme, and shows 1.3-fold and 1.6-fold increases in the [S]0.5Asp as compared to the Ser-171-->Ala and Glu-50-->Ala enzymes, respectively. However, the double mutant enzyme exhibits some enhancement of homotropic cooperativity with respect to aspartate, relative to the single mutant enzymes. At subsaturating concentrations of aspartate, the Glu-50/Ser-171 -->Ala enzyme is activated less by ATP than either the Glu-50-->Ala or Ser-171-->Ala enzyme, whereas CTP inhibition is intermediate between that of the two single mutants. As opposed to the wild-type enzyme, the Glu-50/Ser-171 -->Ala enzyme is activated by ATP and inhibited by CTP at saturating concentrations of aspartate. Structural analysis of the Ser-171-->Ala and Glu-50/Ser-171-->Ala enzymes by solution X-ray scattering indicates that both mutants exist in the same T quaternary structure as the wild-type enzyme in the absence of ligands, and in the same R quaternary structure in the presence of saturating N-(phosphonoacetyl)-L-aspartate. However, saturating concentrations of carbamoyl phosphate and succinate are unable to convert a significant fraction of either mutant enzyme population to the R quaternary structure, as has been observed previously for the Glu-50-->Ala enzyme. The curves for both the Ser-171-->Ala and Glu-50/Ser-171-->Ala enzymes obtained in the presence of substoichiometric amounts of PALA are linear combinations of the two extreme T and R states. The structural consequences of nucleotide binding to these two enzymes were also investigated. Most surprisingly, the direction and amplitude of the effect of ATP upon the double mutant enzyme were shown to vary depending upon the substrate analogue used. PMID:8931146

Stalk cell differentiation without polyketides in the cellular slime mold.

PubMed

Sato, Yukie G; Suarez, Teresa; Saito, Tamao

2016-07-01

Polyketides induce prestalk cell differentiation in Dictyostelium. In the double-knockout mutant of the SteelyA and B polyketide synthases, most of the pstA cells-the major part of the prestalk cells-are lost, and we show by whole mount in situ hybridization that expression of prestalk genes is also reduced. Treatment of the double-knockout mutant with the PKS inhibitor cerulenin gave a further reduction, but some pstA cells still remained in the tip region, suggesting the existence of a polyketide-independent subtype of pstA cells. The double-knockout mutant and cerulenin-treated parental Ax2 cells form fruiting bodies with fragile, single-cell layered stalks after cerulenin treatment. Our results indicate that most pstA cells are induced by polyketides, but the pstA cells at the very tip of the slug are induced in some other way. In addition, a fruiting body with a single-cell layered, vacuolated stalk can form without polyketides.

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.

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.

Xanthurenic acid 8-O-beta-D-glucoside, a novel tryptophan metabolite in eye-color mutants of Drosophila melanogaster.

PubMed

Ferré, J; Real, M D; Ménsua, J L; Jacobson, K B

1985-06-25

An unknown fluorescent metabolite has been isolated from heads of eye-color mutants of Drosophila melanogaster. Only a few mutations cause it to accumulate, viz. cardinal (cd), dark red brown (drb), Henna-recessive (Hnr), purple (pr), Punch2 (Pu2), Punch-Grape (PuGr), and scarlet (st). After purification by ion-exchange chromatography, the spectroscopic, chemical, and enzymatic analyses revealed that it is a novel quinoline derivative: xanthurenic acid 8-O-beta-D-glucoside. Feeding experiments suggest that this glucoside is synthesized from 3-hydroxykynurenine and that free xanthurenic acid is not a precursor. The results from the analysis for its occurrence in double mutants, together with the fact that xanthurenic acid 8-glucoside share the same precursor as xanthurenic acid and xanthommatin, suggest that xanthurenic acid 8-glucoside formation is closely related to the regulation of the last step in the biosynthesis of xanthommatin.

Characteristics of mutants designed to incorporate a new ion pair into the structure of a cold adapted subtilisin-like serine proteinase.

PubMed

Sigurdardóttir, Anna Gudný; Arnórsdóttir, Jóhanna; Thorbjarnardóttir, Sigrídur H; Eggertsson, Gudmundur; Suhre, Karsten; Kristjánsson, Magnús M

2009-03-01

Structural comparisons of VPR, a subtilisin-like serine proteinase from a psychrotrophic Vibrio species and a thermophilic homologue, aqualysin I, have led us to hypothesize about the roles of different residues in the temperature adaptation of the enzymes. Some of these hypotheses are now being examined by analysis of mutants of the enzymes. The selected substitutions are believed to increase the stability of the cold adapted enzyme based on structural analysis of the thermostable structure. We report here on mutants, which were designed to incorporate an ion pair into the structure of VPR. The residues Asp17 and Arg259 are assumed to form an ion pair in aqualysin I. The cold adapted VPR contains Asn (Asn15) and Lys (Lys257) at corresponding sites in its structure. In VPR, Asn 15 is located on a surface loop with its side group pointing towards the side chain of Lys257. By substituting Asn15 by Asp (N15D) it was considered feasible that a salt bridge would form between the oppositely charged groups. To mimic further the putative salt bridge from the thermophile enzyme the corresponding double mutant (N15D/K257R) was also produced. The N15D mutation increased the thermal stability of VPR by approximately 3 degrees C, both in T(50%) and T(m). Addition of the K257R mutation did not however, increase the stability of the double mutant any further. Despite this stabilization of the VPR mutants the catalytic activity (k(cat)) against the substrate Suc-AAPF-NH-Np was increased in the mutants. Molecular dynamics simulations on wild type and the two mutant proteins suggested that indeed a salt bridge was formed in both cases. Furthermore, a truncated form of the N15D mutant (N15DDeltaC) was produced, lacking a 15 residue long C-terminal extended sequence not present in the thermophilic enzyme. In wild type VPR this supposedly moveable, negatively charged arm on the protein molecule might interfere with the new salt bridge introduced as a result of the N15D mutation. Removal of the C-terminal arm improved the thermal stability (T(m) approximately +1.5 degrees C) of the truncated enzyme (VPRDeltaC) as compared to the wild type VPR. Introduction of the N15D substitution into VPRDeltaC improved the thermal stability further by about 3 degrees C, or to about the same extent as in the wild type. However, contrary to what was observed for the wild type, the introduction of the putative salt bridge did not affect the catalytic properties (k(cat)) of the C-terminal truncated enzyme.

Analysis of mutagenic DNA repair in a thermoconditional mutant of Saccharomyces cerevisiae. IV. Influence of DNA replication and excision repair on REV2 dependent UV-mutagenesis and repair.

PubMed

Siede, W; Eckardt, F

1986-01-01

A double mutant being thermoconditionally defective in mutation induction as well as in repair of pre-lethal UV-induced DNA damage (rev2ts) and deficient in excision repair (rad3-2) was studied in temperature-shift experiments. The influence of inhibitors of DNA replication (hydroxyurea, aphidicolin) was determined. Additionally, an analysis of the dose-response pattern of mutation induction ("mutation kinetics") at several ochre alleles was carried out. It was concluded that the UV-inducible REV2 dependent mutagenic repair process is not induced in excision-deficient cells. In excision-deficient cells, REV2 dependent mutation fixation is slow and mostly post-replicative though not dependent on DNA replication. The REV2 mediated mutagenic process could be separated from the repair function.

A GA-insensitive dwarf mutant of Brassica napus L. correlated with mutation in pyrimidine box in the promoter of GID1.

PubMed

Li, Huapeng; Wang, Yun; Li, Xiaocheng; Gao, Yong; Wang, Zhijun; Zhao, Yun; Wang, Maolin

2011-01-01

A dwarf mutant from Brassica napus, namely NDF-1, which was derived from a high doubled haploid (DH) line '3529'(Brassica napus L.) of which seeds were jointly treated with chemical inducers and fast neutron bombardment, was revealed that dwarfism is under the control of a major gene(designated as ndf1) with a mainly additive effect and non-significant dominance effect. The germination and hypocotyls elongation response of dwarf mutants after exogenous GA and uniconazol application showed NDF-1 was a gibberellin insensitive dwarf. We cloned the Brassica napus GID1 gene, named BnGID1, and found it was the ortholog of AtGID1a. The sequence blasting of the BnGID1 genes from NDF-1 and wild type showed there was no mutant in the gene. But the quantitative RT-PCR analysis of GID1 EST pointed out the mutation was caused by the low-level expression of BnGID1 gene. After sequenced the BnGID1 gene's upstream, we found three bases mutated in the pyrimidine box (P-box) of the BnGID1 promoter, which is linkage with the dwarf mutant.

Combinational Deletion of Three Membrane Protein-Encoding Genes Highly Attenuates Yersinia pestis while Retaining Immunogenicity in a Mouse Model of Pneumonic Plague

PubMed Central

Tiner, Bethany L.; Kirtley, Michelle L.; Erova, Tatiana E.; Popov, Vsevolod L.; Baze, Wallace B.; van Lier, Christina J.; Ponnusamy, Duraisamy; Andersson, Jourdan A.; Motin, Vladimir L.; Chauhan, Sadhana

2015-01-01

Previously, we showed that deletion of genes encoding Braun lipoprotein (Lpp) and MsbB attenuated Yersinia pestis CO92 in mouse and rat models of bubonic and pneumonic plague. While Lpp activates Toll-like receptor 2, the MsbB acyltransferase modifies lipopolysaccharide. Here, we deleted the ail gene (encoding the attachment-invasion locus) from wild-type (WT) strain CO92 or its lpp single and Δlpp ΔmsbB double mutants. While the Δail single mutant was minimally attenuated compared to the WT bacterium in a mouse model of pneumonic plague, the Δlpp Δail double mutant and the Δlpp ΔmsbB Δail triple mutant were increasingly attenuated, with the latter being unable to kill mice at a 50% lethal dose (LD50) equivalent to 6,800 LD50s of WT CO92. The mutant-infected animals developed balanced TH1- and TH2-based immune responses based on antibody isotyping. The triple mutant was cleared from mouse organs rapidly, with concurrent decreases in the production of various cytokines and histopathological lesions. When surviving animals infected with increasing doses of the triple mutant were subsequently challenged on day 24 with the bioluminescent WT CO92 strain (20 to 28 LD50s), 40 to 70% of the mice survived, with efficient clearing of the invading pathogen, as visualized in real time by in vivo imaging. The rapid clearance of the triple mutant, compared to that of WT CO92, from animals was related to the decreased adherence and invasion of human-derived HeLa and A549 alveolar epithelial cells and to its inability to survive intracellularly in these cells as well as in MH-S murine alveolar and primary human macrophages. An early burst of cytokine production in macrophages elicited by the triple mutant compared to WT CO92 and the mutant's sensitivity to the bactericidal effect of human serum would further augment bacterial clearance. Together, deletion of the ail gene from the Δlpp ΔmsbB double mutant severely attenuated Y. pestis CO92 to evoke pneumonic plague in a mouse model while retaining the required immunogenicity needed for subsequent protection against infection. PMID:25605764

Combinational deletion of three membrane protein-encoding genes highly attenuates yersinia pestis while retaining immunogenicity in a mouse model of pneumonic plague.

PubMed

Tiner, Bethany L; Sha, Jian; Kirtley, Michelle L; Erova, Tatiana E; Popov, Vsevolod L; Baze, Wallace B; van Lier, Christina J; Ponnusamy, Duraisamy; Andersson, Jourdan A; Motin, Vladimir L; Chauhan, Sadhana; Chopra, Ashok K

2015-04-01

Previously, we showed that deletion of genes encoding Braun lipoprotein (Lpp) and MsbB attenuated Yersinia pestis CO92 in mouse and rat models of bubonic and pneumonic plague. While Lpp activates Toll-like receptor 2, the MsbB acyltransferase modifies lipopolysaccharide. Here, we deleted the ail gene (encoding the attachment-invasion locus) from wild-type (WT) strain CO92 or its lpp single and Δlpp ΔmsbB double mutants. While the Δail single mutant was minimally attenuated compared to the WT bacterium in a mouse model of pneumonic plague, the Δlpp Δail double mutant and the Δlpp ΔmsbB Δail triple mutant were increasingly attenuated, with the latter being unable to kill mice at a 50% lethal dose (LD50) equivalent to 6,800 LD50s of WT CO92. The mutant-infected animals developed balanced TH1- and TH2-based immune responses based on antibody isotyping. The triple mutant was cleared from mouse organs rapidly, with concurrent decreases in the production of various cytokines and histopathological lesions. When surviving animals infected with increasing doses of the triple mutant were subsequently challenged on day 24 with the bioluminescent WT CO92 strain (20 to 28 LD50s), 40 to 70% of the mice survived, with efficient clearing of the invading pathogen, as visualized in real time by in vivo imaging. The rapid clearance of the triple mutant, compared to that of WT CO92, from animals was related to the decreased adherence and invasion of human-derived HeLa and A549 alveolar epithelial cells and to its inability to survive intracellularly in these cells as well as in MH-S murine alveolar and primary human macrophages. An early burst of cytokine production in macrophages elicited by the triple mutant compared to WT CO92 and the mutant's sensitivity to the bactericidal effect of human serum would further augment bacterial clearance. Together, deletion of the ail gene from the Δlpp ΔmsbB double mutant severely attenuated Y. pestis CO92 to evoke pneumonic plague in a mouse model while retaining the required immunogenicity needed for subsequent protection against infection. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Effective screen of CRISPR/Cas9-induced mutants in rice by single-strand conformation polymorphism.

PubMed

Zheng, Xuelian; Yang, Shixin; Zhang, Dengwei; Zhong, Zhaohui; Tang, Xu; Deng, Kejun; Zhou, Jianping; Qi, Yiping; Zhang, Yong

2016-07-01

A method based on DNA single-strand conformation polymorphism is demonstrated for effective genotyping of CRISPR/Cas9-induced mutants in rice. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) has been widely adopted for genome editing in many organisms. A large proportion of mutations generated by CRISPR/Cas9 are very small insertions and deletions (indels), presumably because Cas9 generates blunt-ended double-strand breaks which are subsequently repaired without extensive end-processing. CRISPR/Cas9 is highly effective for targeted mutagenesis in the important crop, rice. For example, homozygous mutant seedlings are commonly recovered from CRISPR/Cas9-treated calli. However, many current mutation detection methods are not very suitable for screening homozygous mutants that typically carry small indels. In this study, we tested a mutation detection method based on single-strand conformational polymorphism (SSCP). We found it can effectively detect small indels in pilot experiments. By applying the SSCP method for CRISRP-Cas9-mediated targeted mutagenesis in rice, we successfully identified multiple mutants of OsROC5 and OsDEP1. In conclusion, the SSCP analysis will be a useful genotyping method for rapid identification of CRISPR/Cas9-induced mutants, including the most desirable homozygous mutants. The method also has high potential for similar applications in other plant species.

Mis-specified cells die by an active gene-directed process, and inhibition of this death results in cell fate transformation in Drosophila

PubMed Central

Werz, Christian; Lee, Tom V.; Lee, Peter L.; Lackey, Melinda; Bolduc, Clare; Stein, David S.; Bergmann, Andreas

2009-01-01

Summary Incorrectly specified or mis-specified cells often undergo cell death or are transformed to adopt a different cell fate during development. The underlying cause for this distinction is largely unknown. In many developmental mutants in Drosophila, large numbers of mis-specified cells die synchronously, providing a convenient model for analysis of this phenomenon. The maternal mutant bicoid is particularly useful model with which to address this issue because its mutant phenotype is a combination of both transformation of tissue (acron to telson) and cell death in the presumptive head and thorax regions. We show that a subset of these mis-specified cells die through an active gene-directed process involving transcriptional upregulation of the cell death inducer hid. Upregulation of hid also occurs in oskar mutants and other segmentation mutants. In hid bicoid double mutants, mis-specified cells in the presumptive head and thorax survive and continue to develop, but they are transformed to adopt a different cell fate. We provide evidence that the terminal torso signaling pathway protects the mis-specified telson tissue in bicoid mutants from hid-induced cell death, whereas mis-specified cells in the head and thorax die, presumably because equivalent survival signals are lacking. These data support a model whereby mis-specification can be tolerated if a survival pathway is provided, resulting in cellular transformation. PMID:16280349

SalB inactivation modulates culture supernatant exoproteins and affects autolysis and viability in Enterococcus faecalis OG1RF.

PubMed

Shankar, Jayendra; Walker, Rachel G; Wilkinson, Mark C; Ward, Deborah; Horsburgh, Malcolm J

2012-07-01

The culture supernatant fraction of an Enterococcus faecalis gelE mutant of strain OG1RF contained elevated levels of the secreted antigen SalB. Using differential fluorescence gel electrophoresis (DIGE) the salB mutant was shown to possess a unique complement of exoproteins. Differentially abundant exoproteins were identified using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. Stress-related proteins including DnaK, Dps family protein, SOD, and NADH peroxidase were present in greater quantity in the OG1RF salB mutant culture supernatant. Moreover, several proteins involved in cell wall synthesis and cell division, including d-Ala-d-Lac ligase and EzrA, were present in reduced quantity in OG1RF salB relative to the parent strain. The salB mutant displayed reduced viability and anomalous cell division, and these phenotypes were exacerbated in a gelE salB double mutant. An epistatic relationship between gelE and salB was not identified with respect to increased autolysis and cell morphological changes observed in the salB mutant. SalB was purified as a six-histidine-tagged protein to investigate peptidoglycan hydrolytic activity; however, activity was not evident. High-pressure liquid chromatography (HPLC) analysis of reduced muropeptides from peptidoglycan digested with mutanolysin revealed that the salB mutant and OG1RF were indistinguishable.

Molecular basis of proton uptake in single and double mutants of cytochrome c oxidase

NASA Astrophysics Data System (ADS)

Henry, Rowan M.; Caplan, David; Fadda, Elisa; Pomès, Régis

2011-06-01

Cytochrome c oxidase, the terminal enzyme of the respiratory chain, utilizes the reduction of dioxygen into water to pump protons across the mitochondrial inner membrane. The principal pathway of proton uptake into the enzyme, the D channel, is a 2.5 nm long channel-like cavity named after a conserved, negatively charged aspartic acid (D) residue thought to help recruiting protons to its entrance (D132 in the first subunit of the S. sphaeroides enzyme). The single-point mutation of D132 to asparagine (N), a neutral residue, abolishes enzyme activity. Conversely, replacing conserved N139, one-third into the D channel, by D, induces a decoupled phenotype, whereby oxygen reduction proceeds but not proton pumping. Intriguingly, the double mutant D132N/N139D, which conserves the charge of the D channel, restores the wild-type phenotype. We use molecular dynamics simulations and electrostatic calculations to examine the structural and physical basis for the coupling of proton pumping and oxygen chemistry in single and double N139D mutants. The potential of mean force for the conformational isomerization of N139 and N139D side chains reveals the presence of three rotamers, one of which faces the channel entrance. This out-facing conformer is metastable in the wild-type and in the N139D single mutant, but predominant in the double mutant thanks to the loss of electrostatic repulsion with the carboxylate group of D132. The effects of mutations and conformational isomerization on the pKa of E286, an essential proton-shuttling residue located at the top of the D channel, are shown to be consistent with the electrostatic control of proton pumping proposed recently (Fadda et al 2008 Biochim. Biophys. Acta 1777 277-84). Taken together, these results suggest that preserving the spatial distribution of charges at the entrance of the D channel is necessary to guarantee both the uptake and the relay of protons to the active site of the enzyme. These findings highlight the interplay of long-range electrostatic forces and local structural fluctuations in the control of proton movement and provide a physical explanation for the restoration of proton pumping activity in the double mutant.

Free energy calculations on the stability of the 14-3-3ζ protein.

PubMed

Jandova, Zuzana; Trosanova, Zuzana; Weisova, Veronika; Oostenbrink, Chris; Hritz, Jozef

2018-03-01

Mutations of cysteine are often introduced to e.g. avoid formation of non-physiological inter-molecular disulfide bridges in in-vitro experiments, or to maintain specificity in labeling experiments. Alanine or serine is typically preferred, which usually do not alter the overall protein stability, when the original cysteine was surface exposed. However, selecting the optimal mutation for cysteines in the hydrophobic core of the protein is more challenging. In this work, the stability of selected Cys mutants of 14-3-3ζ was predicted by free-energy calculations and the obtained data were compared with experimentally determined stabilities. Both the computational predictions as well as the experimental validation point at a significant destabilization of mutants C94A and C94S. This destabilization could be attributed to the formation of hydrophobic cavities and a polar solvation of a hydrophilic side chain. A L12E, M78K double mutant was further studied in terms of its reduced dimerization propensity. In contrast to naïve expectations, this double mutant did not lead to the formation of strong salt bridges, which was rationalized in terms of a preferred solvation of the ionic species. Again, experiments agreed with the calculations by confirming the monomerization of the double mutants. Overall, the simulation data is in good agreement with experiments and offers additional insight into the stability and dimerization of this important family of regulatory proteins. Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.

Identification and elucidation of in vivo function of two alanine racemases from Pseudomonas putida KT2440.

PubMed

Duque, Estrella; Daddaoua, Abdelali; Cordero, Baldo F; De la Torre, Jesús; Antonia Molina-Henares, Maria; Ramos, Juan-Luis

2017-10-01

The genome of Pseudomonas putida KT2440 contains two open reading frames (ORFs), PP_3722 and PP_5269, that encode proteins with a Pyridoxal phosphate binding motif and a high similarity to alanine racemases. Alanine racemases play a key role in the biosynthesis of D-alanine, a crucial amino acid in the peptidoglycan layer. For these ORFs, we generated single and double mutants and found that inactivation of PP_5269 resulted in D-alanine auxotrophy, while inactivation of PP_3722 did not. Furthermore, as expected, the PP_3722/PP_5269 double mutant was a strict auxotroph for D-alanine. These results indicate that PP_5269 is an alr allele and that it is the essential alanine racemase in P. putida. We observed that the PP_5269 mutant grew very slowly, while the double PP_5269/PP_3722 mutant did not grow at all. This suggests that PP_3722 may replace PP_5269 in vivo. In fact, when the ORF encoding PP_3772 was cloned into a wide host range expression vector, ORF PP_3722 successfully complemented P. putida PP_5269 mutants. We purified both proteins to homogeneity and while they exhibit similar K M values, the V max of PP_5269 is fourfold higher than that of PP_3722. Here, we propose that PP_5269 and PP_3722 encode functional alanine racemases and that these genes be named alr-1 and alr-2 respectively. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

Search for methylation-sensitive amplification polymorphisms in mutant figs.

PubMed

Rodrigues, M G F; Martins, A B G; Bertoni, B W; Figueira, A; Giuliatti, S

2013-07-08

Fig (Ficus carica) breeding programs that use conventional approaches to develop new cultivars are rare, owing to limited genetic variability and the difficulty in obtaining plants via gamete fusion. Cytosine methylation in plants leads to gene repression, thereby affecting transcription without changing the DNA sequence. Previous studies using random amplification of polymorphic DNA and amplified fragment length polymorphism markers revealed no polymorphisms among select fig mutants that originated from gamma-irradiated buds. Therefore, we conducted methylation-sensitive amplified polymorphism analysis to verify the existence of variability due to epigenetic DNA methylation among these mutant selections compared to the main cultivar 'Roxo-de-Valinhos'. Samples of genomic DNA were double-digested with either HpaII (methylation sensitive) or MspI (methylation insensitive) and with EcoRI. Fourteen primer combinations were tested, and on an average, non-methylated CCGG, symmetrically methylated CmCGG, and hemimethylated hmCCGG sites accounted for 87.9, 10.1, and 2.0%, respectively. MSAP analysis was effective in detecting differentially methylated sites in the genomic DNA of fig mutants, and methylation may be responsible for the phenotypic variation between treatments. Further analyses such as polymorphic DNA sequencing are necessary to validate these differences, standardize the regions of methylation, and analyze reads using bioinformatic tools.

Conjugated Fatty Acid Synthesis

PubMed Central

Rawat, Richa; Yu, Xiao-Hong; Sweet, Marie; Shanklin, John

2012-01-01

Conjugated linolenic acids (CLNs), 18:3 Δ9,11,13, lack the methylene groups found between the double bonds of linolenic acid (18:3 Δ9,12,15). CLNs are produced by conjugase enzymes that are homologs of the oleate desaturases FAD2. The goal of this study was to map the domain(s) within the Momordica charantia conjugase (FADX) responsible for CLN formation. To achieve this, a series of Momordica FADX-Arabidopsis FAD2 chimeras were expressed in the Arabidopsis fad3fae1 mutant, and the transformed seeds were analyzed for the accumulation of CLN. These experiments identified helix 2 and the first histidine box as a determinant of conjugase product partitioning into punicic acid (18:3 Δ9cis,11trans,13cis) or α-eleostearic acid (18:3 Δ9cis,11trans,13trans). This was confirmed by analysis of a FADX mutant containing six substitutions in which the sequence of helix 2 and first histidine box was converted to that of FAD2. Each of the six FAD2 substitutions was individually converted back to the FADX equivalent identifying residues 111 and 115, adjacent to the first histidine box, as key determinants of conjugase product partitioning. Additionally, expression of FADX G111V and FADX G111V/D115E resulted in an approximate doubling of eleostearic acid accumulation to 20.4% and 21.2%, respectively, compared with 9.9% upon expression of the native Momordica FADX. Like the Momordica conjugase, FADX G111V and FADX D115E produced predominantly α-eleostearic acid and little punicic acid, but the FADX G111V/D115E double mutant produced approximately equal amounts of α-eleostearic acid and its isomer, punicic acid, implicating an interactive effect of residues 111 and 115 in punicic acid formation. PMID:22451660

Characterization of the functional role of Asp141, Asp194, and Asp464 residues in the Mn2+-L-malate binding of pigeon liver malic enzyme.

PubMed

Chou, W Y; Chang, H P; Huang, C H; Kuo, C C; Tong, L; Chang, G G

2000-02-01

Pigeon liver malic enzyme was inactivated and cleaved at Asp141, Asp194, and Asp464 by the Cu2+-ascorbate system in acidic environment. Site-specific mutagenesis was performed at these putative metal-binding sites. Three point mutants, D141N, D194N, and D464N; three double mutants, D(141,194)N, D(194,464)N, and D(141,464)N; and a triple mutant, D(141,194,464)N; as well as the wild-type malic enzyme (WT) were successfully cloned and expressed in Escherichia coli cells. All recombinant enzymes, except the triple mutant, were purified to apparent homogeneity by successive Q-Sepharose and adenosine-2',5'-bisphosphate-agarose columns. The mutants showed similar apparent Km,NADP values to that of the WT. The Km,Mal value was increased in the D141N and D194N mutants. The Km,Mn value, on the other hand, was increased only in the D141N mutant by 14-fold, corresponding to approximately 1.6 kcal/mol for the Asp141-Mn2+ binding energy. Substrate inhibition by L-malate was only observed in WT, D464N, and D(141,464)N. Initial velocity experiments were performed to derive the various kinetic parameters. The possible interactions between Asp141, Asp194, and Asp464 were analyzed by the double-mutation cycles and triple-mutation box. There are synergistic weakening interactions between Asp141 and Asp194 in the metal binding that impel the D(141,194)N double mutant to an overall specificity constant [k(cat)/(Kd,Mn Km,Mal Km,NADP)] at least four orders of magnitude smaller than the WT value. This difference corresponds to an increase of 6.38 kcal/mol energy barrier for the catalytic efficiency. Mutation at Asp464, on the other hand, has partial additivity on the mutations at Asp141 and Asp194. The overall specificity constants for the double mutants D(194,464)N and D(141,464)N or the triple mutant D(141,194,464)N were decreased by only 10- to 100-fold compared to the WT. These results strongly suggest the involvement of Asp141 in the Mn2+-L-malate binding for the pigeon liver malic enzyme. The Asp194 and Asp464, which may be oxidized by nonspecific binding of Cu2+, are involved in the Mn2+-L-malate binding or catalysis indirectly by modulating the binding affinity of Asp141 with the Mn2+.

Identification of Methylosome Components as Negative Regulators of Plant Immunity Using Chemical Genetics.

PubMed

Huang, Shuai; Balgi, Aruna; Pan, Yaping; Li, Meng; Zhang, Xiaoran; Du, Lilin; Zhou, Ming; Roberge, Michel; Li, Xin

2016-12-05

Nucleotide-binding leucine-rich repeat (NLR) proteins serve as immune receptors in both plants and animals. To identify components required for NLR-mediated immunity, we designed and carried out a chemical genetics screen to search for small molecules that can alter immune responses in Arabidopsis thaliana. From 13 600 compounds, we identified Ro 8-4304 that was able to specifically suppress the severe autoimmune phenotypes of chs3-2D (chilling sensitive 3, 2D), including the arrested growth morphology and heightened PR (Pathogenesis Related) gene expression. Further, six Ro 8-4304 insensitive mutants were uncovered from the Ro 8-4304-insensitive mutant (rim) screen using a mutagenized chs3-2D population. Positional cloning revealed that rim1 encodes an allele of AtICln (I, currents; Cl, chloride; n, nucleotide). Genetic and biochemical analysis demonstrated that AtICln is in the same protein complex with the methylosome components small nuclear ribonucleoprotein D3b (SmD3b) and protein arginine methyltransferase 5 (PRMT5), which are required for the biogenesis of small nuclear ribonucleoproteins (snRNPs) involved in mRNA splicing. Double mutant analysis revealed that SmD3b is also involved in the sensitivity to Ro 8-4304, and the prmt5-1 chs3-2D double mutant is lethal. Loss of AtICln, SmD3b, or PRMT5 function results in enhanced disease resistance against the virulent oomycete pathogen Hyaloperonospora arabidopsidis Noco2, suggesting that mRNA splicing plays a previously unknown negative role in plant immunity. The successful implementation of a high-throughput chemical genetic screen and the identification of a small-molecule compound affecting plant immunity indicate that chemical genetics is a powerful tool to study whole-organism plant defense pathways. Copyright © 2016 The Author. Published by Elsevier Inc. All rights reserved.

Modeling of Anopheles minimus Mosquito NADPH-Cytochrome P450 Oxidoreductase (CYPOR) and Mutagenesis Analysis

PubMed Central

Sarapusit, Songklod; Lertkiatmongkol, Panida; Duangkaew, Panida; Rongnoparut, Pornpimol

2013-01-01

Malaria is one of the most dangerous mosquito-borne diseases in many tropical countries, including Thailand. Studies in a deltamethrin resistant strain of Anopheles minimus mosquito, suggest cytochrome P450 enzymes contribute to the detoxification of pyrethroid insecticides. Purified A. minimus CYPOR enzyme (AnCYPOR), which is the redox partner of cytochrome P450s, loses flavin-adenosine di-nucleotide (FAD) and FLAVIN mono-nucleotide (FMN) cofactors that affect its enzyme activity. Replacement of leucine residues at positions 86 and 219 with phenylalanines in FMN binding domain increases FMN binding, enzyme stability, and cytochrome c reduction activity. Membrane-Bound L86F/L219F-AnCYPOR increases A. minimus P450-mediated pyrethroid metabolism in vitro. In this study, we constructed a comparative model structure of AnCYPOR using a rat CYPOR structure as a template. Overall model structure is similar to rat CYPOR, with some prominent differences. Based on primary sequence and structural analysis of rat and A. minimus CYPOR, C427R, W678A, and W678H mutations were generated together with L86F/L219F resulting in three soluble Δ55 triple mutants. The C427R triple AnCYPOR mutant retained a higher amount of FAD binding and increased cytochrome c reduction activity compared to wild-type and L86F/L219F-Δ55AnCYPOR double mutant. However W678A and W678H mutations did not increase FAD and NAD(P)H bindings. The L86F/L219F double and C427R triple membrane-bound AnCYPOR mutants supported benzyloxyresorufin O-deakylation (BROD) mediated by mosquito CYP6AA3 with a two-to three-fold increase in efficiency over wild-type AnCYPOR. The use of rat CYPOR in place of AnCYPOR most efficiently supported CYP6AA3-mediated BROD compared to all AnCYPORs. PMID:23325047

Functional Analysis of Arabidopsis Mutants Points to Novel Roles for Glutathione in Coupling H2O2 to Activation of Salicylic Acid Accumulation and Signaling

PubMed Central

Han, Yi; Chaouch, Sejir; Mhamdi, Amna; Queval, Guillaume; Zechmann, Bernd

2013-01-01

Abstract Aims: Through its interaction with H2O2, glutathione is a candidate for transmission of signals in plant responses to pathogens, but identification of signaling roles is complicated by its antioxidant function. Using a genetic approach based on a conditional catalase-deficient Arabidopsis mutant, cat2, this study aimed at establishing whether GSH plays an important functional role in the transmission of signals downstream of H2O2. Results: Introducing the cad2 or allelic mutations in the glutathione synthesis pathway into cat2 blocked H2O2-triggered GSH oxidation and accumulation. While no effects on NADP(H) or ascorbate were observed, and H2O2-induced decreases in growth were maintained, blocking GSH modulation antagonized salicylic acid (SA) accumulation and SA-dependent responses. Other novel double and triple mutants were produced and compared with cat2 cad2 at the levels of phenotype, expression of marker genes, nontargeted metabolite profiling, accumulation of SA, and bacterial resistance. Most of the effects of the cad2 mutation on H2O2-triggered responses were distinct from those produced by mutations for GLUTATHIONE REDUCTASE1 (GR1) or NONEXPRESSOR OF PATHOGENESIS-RELATED GENES 1 (NPR1), and were linked to compromised induction of ISOCHORISMATE SYNTHASE1 (ICS1) and ICS1-dependent SA accumulation. Innovation: A novel genetic approach was used in which GSH content or antioxidative capacity was independently modified in an H2O2 signaling background. Analysis of new double and triple mutants allowed us to infer previously undescribed regulatory roles for GSH. Conclusion: In parallel to its antioxidant role, GSH acts independently of NPR1 to allow increased intracellular H2O2 to activate SA signaling, a key defense response in plants. Antioxid. Redox Signal. 18, 2106–2121. PMID:23148658

Mycoviruses as Triggers and Targets of RNA Silencing in White Mold Fungus Sclerotinia sclerotiorum.

PubMed

Mochama, Pauline; Jadhav, Prajakta; Neupane, Achal; Lee Marzano, Shin-Yi

2018-04-22

This study aimed to demonstrate the existence of antiviral RNA silencing mechanisms in Sclerotinia sclerotiorum by infecting wild-type and RNA-silencing-deficient strains of the fungus with an RNA virus and a DNA virus. Key silencing-related genes were disrupted to dissect the RNA silencing pathway. Specifically, dicer genes ( dcl-1, dcl-2 , and both dcl-1 / dcl-2 ) were displaced by selective marker(s). Disruption mutants were then compared for changes in phenotype, virulence, and susceptibility to virus infections. Wild-type and mutant strains were transfected with a single-stranded RNA virus, SsHV2-L, and copies of a single-stranded DNA mycovirus, SsHADV-1, as a synthetic virus constructed in this study. Disruption of dcl-1 or dcl-2 resulted in no changes in phenotype compared to wild-type S. sclerotiorum ; however, the double dicer mutant strain exhibited significantly slower growth. Furthermore, the Δdcl-1/dcl-2 double mutant, which was slow growing without virus infection, exhibited much more severe debilitation following virus infections including phenotypic changes such as slower growth, reduced pigmentation, and delayed sclerotial formation. These phenotypic changes were absent in the single mutants, Δdcl-1 and Δdcl-2 . Complementation of a single dicer in the double disruption mutant reversed viral susceptibility to the wild-type state. Virus-derived small RNAs were accumulated from virus-infected wild-type strains with strand bias towards the negative sense. The findings of these studies indicate that S. sclerotiorum has robust RNA silencing mechanisms that process both DNA and RNA mycoviruses and that, when both dicers are silenced, invasive nucleic acids can greatly debilitate the virulence of this fungus.

CDPKs CPK6 and CPK3 Function in ABA Regulation of Guard Cell S-Type Anion- and Ca2+- Permeable Channels and Stomatal Closure

PubMed Central

Munemasa, Shintaro; Wang, Yong-Fei; Andreoli, Shannon; Tiriac, Hervé; Alonso, Jose M; Harper, Jeffery F; Ecker, Joseph R; Kwak, June M; Schroeder, Julian I

2006-01-01

Abscisic acid (ABA) signal transduction has been proposed to utilize cytosolic Ca2+ in guard cell ion channel regulation. However, genetic mutants in Ca2+ sensors that impair guard cell or plant ion channel signaling responses have not been identified, and whether Ca2+-independent ABA signaling mechanisms suffice for a full response remains unclear. Calcium-dependent protein kinases (CDPKs) have been proposed to contribute to central signal transduction responses in plants. However, no Arabidopsis CDPK gene disruption mutant phenotype has been reported to date, likely due to overlapping redundancies in CDPKs. Two Arabidopsis guard cell–expressed CDPK genes, CPK3 and CPK6, showed gene disruption phenotypes. ABA and Ca2+ activation of slow-type anion channels and, interestingly, ABA activation of plasma membrane Ca2+-permeable channels were impaired in independent alleles of single and double cpk3cpk6 mutant guard cells. Furthermore, ABA- and Ca2+-induced stomatal closing were partially impaired in these cpk3cpk6 mutant alleles. However, rapid-type anion channel current activity was not affected, consistent with the partial stomatal closing response in double mutants via a proposed branched signaling network. Imposed Ca2+ oscillation experiments revealed that Ca2+-reactive stomatal closure was reduced in CDPK double mutant plants. However, long-lasting Ca2+-programmed stomatal closure was not impaired, providing genetic evidence for a functional separation of these two modes of Ca2+-induced stomatal closing. Our findings show important functions of the CPK6 and CPK3 CDPKs in guard cell ion channel regulation and provide genetic evidence for calcium sensors that transduce stomatal ABA signaling. PMID:17032064

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

PubMed

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

2012-09-01

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

One-Cell Doubling Evaluation by Living Arrays of Yeast, ODELAY!

DOE PAGES

Herricks, Thurston; Dilworth, David J.; Mast, Fred D.; ...

2016-11-16

Cell growth is a complex phenotype widely used in systems biology to gauge the impact of genetic and environmental perturbations. Due to the magnitude of genome-wide studies, resolution is often sacrificed in favor of throughput, creating a demand for scalable, time-resolved, quantitative methods of growth assessment. We present ODELAY (One-cell Doubling Evaluation by Living Arrays of Yeast), an automated and scalable growth analysis platform. High measurement density and single-cell resolution provide a powerful tool for large-scale multiparameter growth analysis based on the modeling of microcolony expansion on solid media. Pioneered in yeast but applicable to other colony forming organisms, ODELAYmore » extracts the three key growth parameters (lag time, doubling time, and carrying capacity) that define microcolony expansion from single cells, simultaneously permitting the assessment of population heterogeneity. The utility of ODELAY is illustrated using yeast mutants, revealing a spectrum of phenotypes arising from single and combinatorial growth parameter perturbations.« less

One-Cell Doubling Evaluation by Living Arrays of Yeast, ODELAY!

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

Herricks, Thurston; Dilworth, David J.; Mast, Fred D.

Cell growth is a complex phenotype widely used in systems biology to gauge the impact of genetic and environmental perturbations. Due to the magnitude of genome-wide studies, resolution is often sacrificed in favor of throughput, creating a demand for scalable, time-resolved, quantitative methods of growth assessment. We present ODELAY (One-cell Doubling Evaluation by Living Arrays of Yeast), an automated and scalable growth analysis platform. High measurement density and single-cell resolution provide a powerful tool for large-scale multiparameter growth analysis based on the modeling of microcolony expansion on solid media. Pioneered in yeast but applicable to other colony forming organisms, ODELAYmore » extracts the three key growth parameters (lag time, doubling time, and carrying capacity) that define microcolony expansion from single cells, simultaneously permitting the assessment of population heterogeneity. The utility of ODELAY is illustrated using yeast mutants, revealing a spectrum of phenotypes arising from single and combinatorial growth parameter perturbations.« less

Synergic role of the two ars operons in arsenic tolerance in Pseudomonas putida KT2440.

PubMed

Fernández, Matilde; Udaondo, Zulema; Niqui, José-Luis; Duque, Estrella; Ramos, Juan-Luis

2014-10-01

The chromosome of Pseudomonas putida KT2440 carries two clusters of genes, denoted ars1 and ars2, that are annotated as putative arsenic resistance operons. In this work, we present evidence that both operons encode functional arsenic-response regulatory genes as well as arsenic extrusion systems that confer resistance to both arsenite [As(III)] and arsenate [As(V)]. Transcriptional fusions of P(ars1) and P(ars2) to lacZ revealed that expression of both operons was induced by arsenite and arsenate. We generated single mutants in ars1 and ars2, which showed lower resistance to arsenic than the wild-type strain. A double ars1/ars2 was found to be highly sensitive to arsenic. Minimum inhibitory concentrations (MICs) for single mutants decreased two- to fourfold with respect to the parental strain, while in the double mutant the MIC decreased 128-fold for arsenite and 32-fold for arsenate. Bioinformatic analysis revealed that the ars2 resistance operon is part of the core genome of P. putida, while the ars1 operon appears to only occur in the KT2440 strain, suggesting that ars1 was acquired by horizontal gene transfer. The presence of ars1 in KT2440 may explain why it exhibits higher resistance to arsenic than other P. putida strains, which bear only the ars2 operon.

Genetic inactivation of mGlu5 receptor improves motor coordination in the Grm1crv4 mouse model of SCAR13 ataxia.

PubMed

Bossi, Simone; Musante, Ilaria; Bonfiglio, Tommaso; Bonifacino, Tiziana; Emionite, Laura; Cerminara, Maria; Cervetto, Chiara; Marcoli, Manuela; Bonanno, Giambattista; Ravazzolo, Roberto; Pittaluga, Anna; Puliti, Aldamaria

2018-01-01

Deleterious mutations in the glutamate receptor metabotropic 1 gene (GRM1) cause a recessive form of cerebellar ataxia, SCAR13. GRM1 and GRM5 code for the metabotropic glutamate type 1 (mGlu1) and type 5 (mGlu5) receptors, respectively. Their different expression profiles suggest they could have distinct functional roles. In a previous study, homozygous mice lacking mGlu1 receptors (Grm1 crv4/crv4 ) and exhibiting ataxia presented cerebellar overexpression of mGlu5 receptors, that was proposed to contribute to the mouse phenotype. To test this hypothesis, we here crossed Grm1 crv4 and Grm5 ko mice to generate double mutants (Grm1 crv4/crv4 Grm5 ko/ko ) lacking both mGlu1 and mGlu5 receptors. Double mutants and control mice were analyzed for spontaneous behavior and for motor activity by rotarod and footprint analyses. In the same mice, the release of glutamate from cerebellar nerve endings (synaptosomes) elicited by 12mM KCl or by α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) was also evaluated. Motor coordination resulted improved in double mutants when compared to Grm1 crv4/crv4 mice. Furthermore, in in vitro studies, glutamate release elicited by both KCl depolarization and activation of AMPA autoreceptors resulted reduced in Grm1 crv4/crv4 mice compared to wild type mice, while it presented normal levels in double mutants. Moreover, we found that Grm1 crv4/crv4 mice showed reduced expression of GluA2/3 AMPA receptor subunits in cerebellar synaptosomes, while it resulted restored to wild type level in double mutants. To conclude, blocking of mGlu5 receptor reduced the dysregulation of glutamate transmission and improved motor coordination in the Grm1 crv4 mouse model of SCAR13, thus suggesting the possible usefulness of pharmacological therapies based on modulation of mGlu5 receptor activity for the treatment of this type of ataxia. Copyright © 2017 Elsevier Inc. All rights reserved.

Properties of uvrE mutants of Escherichia coli K12. Part 2. Construction and properties of pol and rec derivatives

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

Mattern, I.E.; Houtman, P.C.

1974-01-01

Viability and sensitivity to ultraviolet radiation and x-rays as well as frequency of spontaneous mutations was investigated for some double mutant strains of Escherichia coli and compared with parent strains. (GRA)

Pigment-dispersing factor (PDF) has different effects on Drosophila's circadian clocks in the accessory medulla and in the dorsal brain.

PubMed

Wülbeck, Corinna; Grieshaber, Eva; Helfrich-Förster, Charlotte

2008-10-01

The neuropeptide pigment-dispersing factor (PDF) is a key transmitter in the circadian clock of Drosophila melanogaster. Here we studied the rhythmic behavior of neural mutants with modified arborizations of the large PDF neurons. In sine oculis(1) (so(1)) mutants we found a higher density of PDF fibers in the fly's pacemaker center, the accessory medulla. These flies exhibited a significantly longer period (24.6 h) than control flies. When PDF levels were elevated to very high levels in the dorsal brain as true for so(mda) mutants and small optic lobes;so(1) double mutants (sol(1);so( 1)), a short-period component split off the long period in behavioral rhythmicity. The short period became shorter the higher the amount of PDF in this brain region and reached a value of approximately 21 h. The period alterations were clearly dependent on PDF, because so(1);Pdf 01 and so(mda);Pdf 01 double mutants showed a single free-running component with a period similar to Pdf 01 mutants (approximately 22.5 h) and significantly longer than the short period of so(mda) mutants. These observations indicate that PDF feeds back on the clock neurons and changes their period. Obviously, PDF lengthens the period of some clock neurons and shortens that of others.

Disruption of LACCASE4 and 17 Results in Tissue-Specific Alterations to Lignification of Arabidopsis thaliana Stems[W

PubMed Central

Berthet, Serge; Demont-Caulet, Nathalie; Pollet, Brigitte; Bidzinski, Przemyslaw; Cézard, Laurent; Le Bris, Phillipe; Borrega, Nero; Hervé, Jonathan; Blondet, Eddy; Balzergue, Sandrine; Lapierre, Catherine; Jouanin, Lise

2011-01-01

Peroxidases have been shown to be involved in the polymerization of lignin precursors, but it remains unclear whether laccases (EC 1.10.3.2) participate in constitutive lignification. We addressed this issue by studying laccase T-DNA insertion mutants in Arabidopsis thaliana. We identified two genes, LAC4 and LAC17, which are strongly expressed in stems. LAC17 was mainly expressed in the interfascicular fibers, whereas LAC4 was expressed in vascular bundles and interfascicular fibers. We produced two double mutants by crossing the LAC17 (lac17) mutant with two LAC4 mutants (lac4-1 and lac4-2). The single and double mutants grew normally in greenhouse conditions. The single mutants had moderately low lignin levels, whereas the stems of lac4-1 lac17 and lac4-2 lac17 mutants had lignin contents that were 20 and 40% lower than those of the control, respectively. These lower lignin levels resulted in higher saccharification yields. Thioacidolysis revealed that disrupting LAC17 principally affected the deposition of G lignin units in the interfascicular fibers and that complementation of lac17 with LAC17 restored a normal lignin profile. This study provides evidence that both LAC4 and LAC17 contribute to the constitutive lignification of Arabidopsis stems and that LAC17 is involved in the deposition of G lignin units in fibers. PMID:21447792

The ARG1-LIKE2 gene of Arabidopsis functions in a gravity signal transduction pathway that is genetically distinct from the PGM pathway

NASA Technical Reports Server (NTRS)

Guan, Changhui; Rosen, Elizabeth S.; Boonsirichai, Kanokporn; Poff, Kenneth L.; Masson, Patrick H.

2003-01-01

The arl2 mutants of Arabidopsis display altered root and hypocotyl gravitropism, whereas their inflorescence stems are fully gravitropic. Interestingly, mutant roots respond like the wild type to phytohormones and an inhibitor of polar auxin transport. Also, their cap columella cells accumulate starch similarly to wild-type cells, and mutant hypocotyls display strong phototropic responses to lateral light stimulation. The ARL2 gene encodes a DnaJ-like protein similar to ARG1, another protein previously implicated in gravity signal transduction in Arabidopsis seedlings. ARL2 is expressed at low levels in all organs of seedlings and plants. arl2-1 arg1-2 double mutant roots display kinetics of gravitropism similar to those of single mutants. However, double mutants carrying both arl2-1 and pgm-1 (a mutation in the starch-biosynthetic gene PHOSPHOGLUCOMUTASE) at the homozygous state display a more pronounced root gravitropic defect than the single mutants. On the other hand, seedlings with a null mutation in ARL1, a paralog of ARG1 and ARL2, behave similarly to the wild type in gravitropism and other related assays. Taken together, the results suggest that ARG1 and ARL2 function in the same gravity signal transduction pathway in the hypocotyl and root of Arabidopsis seedlings, distinct from the pathway involving PGM.

Arabidopsis myrosinases link the glucosinolate-myrosinase system and the cuticle

PubMed Central

Ahuja, Ishita; de Vos, Ric C. H.; Rohloff, Jens; Stoopen, Geert M.; Halle, Kari K.; Ahmad, Samina Jam Nazeer; Hoang, Linh; Hall, Robert D.; Bones, Atle M.

2016-01-01

Both physical barriers and reactive phytochemicals represent two important components of a plant’s defence system against environmental stress. However, these two defence systems have generally been studied independently. Here, we have taken an exclusive opportunity to investigate the connection between a chemical-based plant defence system, represented by the glucosinolate-myrosinase system, and a physical barrier, represented by the cuticle, using Arabidopsis myrosinase (thioglucosidase; TGG) mutants. The tgg1, single and tgg1 tgg2 double mutants showed morphological changes compared to wild-type plants visible as changes in pavement cells, stomatal cells and the ultrastructure of the cuticle. Extensive metabolite analyses of leaves from tgg mutants and wild-type Arabidopsis plants showed altered levels of cuticular fatty acids, fatty acid phytyl esters, glucosinolates, and indole compounds in tgg single and double mutants as compared to wild-type plants. These results point to a close and novel association between chemical defence systems and physical defence barriers. PMID:27976683

Deficiency of RgpG Causes Major Defects in Cell Division and Biofilm Formation, and Deficiency of LytR-CpsA-Psr Family Proteins Leads to Accumulation of Cell Wall Antigens in Culture Medium by Streptococcus mutans.

PubMed

De, Arpan; Liao, Sumei; Bitoun, Jacob P; Roth, Randy; Beatty, Wandy L; Wu, Hui; Wen, Zezhang T

2017-09-01

Streptococcus mutans is known to possess rhamnose-glucose polysaccharide (RGP), a major cell wall antigen. S. mutans strains deficient in rgpG , encoding the first enzyme of the RGP biosynthesis pathway, were constructed by allelic exchange. The rgpG deficiency had no effect on growth rate but caused major defects in cell division and altered cell morphology. Unlike the coccoid wild type, the rgpG mutant existed primarily in chains of swollen, "squarish" dividing cells. Deficiency of rgpG also causes significant reduction in biofilm formation ( P < 0.01). Double and triple mutants with deficiency in brpA and/or psr , genes coding for the LytR-CpsA-Psr family proteins BrpA and Psr, which were previously shown to play important roles in cell envelope biogenesis, were constructed using the rgpG mutant. There were no major differences in growth rates between the wild-type strain and the rgpG brpA and rgpG psr double mutants, but the growth rate of the rgpG brpA psr triple mutant was reduced drastically ( P < 0.001). Under transmission electron microscopy, both double mutants resembled the rgpG mutant, while the triple mutant existed as giant cells with multiple asymmetric septa. When analyzed by immunoblotting, the rgpG mutant displayed major reductions in cell wall antigens compared to the wild type, while little or no signal was detected with the double and triple mutants and the brpA and psr single mutants. These results suggest that RgpG in S. mutans plays a critical role in cell division and biofilm formation and that BrpA and Psr may be responsible for attachment of cell wall antigens to the cell envelope. IMPORTANCE Streptococcus mutans , a major etiological agent of human dental caries, produces rhamnose-glucose polysaccharide (RGP) as the major cell wall antigen. This study provides direct evidence that deficiency of RgpG, the first enzyme of the RGP biosynthesis pathway, caused major defects in cell division and morphology and reduced biofilm formation by S. mutans , indicative of a significant role of RGP in cell division and biofilm formation in S. mutans These results are novel not only in S. mutans , but also other streptococci that produce RGP. This study also shows that the LytR-CpsA-Psr family proteins BrpA and Psr in S. mutans are involved in attachment of RGP and probably other cell wall glycopolymers to the peptidoglycan. In addition, the results also suggest that BrpA and Psr may play a direct role in cell division and biofilm formation in S. mutans This study reveals new potential targets to develop anticaries therapeutics. Copyright © 2017 American Society for Microbiology.

A proteomic analysis of ferulic acid metabolism in Amycolatopsis sp. ATCC 39116.

PubMed

Meyer, Florian; Netzer, Julius; Meinert, Christina; Voigt, Birgit; Riedel, Katharina; Steinbüchel, Alexander

2018-05-16

The pseudonocardiate Amycolatopsis sp. ATCC 39116 is used for the biotechnical production of natural vanillin from ferulic acid. Our laboratory has performed genetic modifications of this strain previously, but there are still many gaps in our knowledge regarding its vanillin tolerance and the general metabolism. We performed cultivations with this bacterium and compared the proteomes of stationary phase cells before ferulic acid feeding with those during ferulic acid feeding. Thereby, we identified 143 differently expressed proteins. Deletion mutants were constructed and characterized to analyze the function of nine corresponding genes. Using these mutants, we identified an active ferulic acid β-oxidation pathway and the enzymes which constitute this pathway. A combined deletion mutant in which the β-oxidation as well as non-β-oxidation pathways of ferulic acid degradation were deleted was unable to grow on ferulic acid as the sole source of carbon and energy. This mutant differs from the single deletion mutants and was unable to grow on ferulic acid. Furthermore, we showed that the non-β-oxidation pathway is involved in caffeic acid degradation; however, its deletion is complemented even in the double deletion mutant. This shows that both pathways can complement each other. The β-oxidation deletion mutant produced significantly reduced amounts of vanillic acid (0.12 instead of 0.35 g/l). Therefore, the resulting mutant could be used as an improved production strain. The quinone oxidoreductase deletion mutant (ΔytfG) degraded ferulic acid slower at first but produced comparable amounts of vanillin and significantly less vanillyl alcohol when compared to the parent strain.

The Streptococcus mutans Serine/Threonine Kinase, PknB, Regulates Competence Development, Bacteriocin Production, and Cell Wall Metabolism ▿

PubMed Central

Banu, Liliana Danusia; Conrads, Georg; Rehrauer, Hubert; Hussain, Haitham; Allan, Elaine; van der Ploeg, Jan R.

2010-01-01

Bacteria can detect, transmit, and react to signals from the outside world by using two-component systems (TCS) and serine-threonine kinases and phosphatases. Streptococcus mutans contains one serine-threonine kinase, encoded by pknB. A gene encoding a serine-threonine phosphatase, pppL, is located upstream of pknB. In this study, the phenotypes of pknB and pppL single mutants and a pknB pppL double mutant were characterized. All mutants exhibited a reduction in genetic transformability and biofilm formation, showed abnormal cell shapes, grew slower than the wild-type strain in several complex media, and exhibited reduced acid tolerance. The mutants had reduced cariogenic capacity but no significant defects in colonization in a rat caries model. Whole-genome transcriptome analysis revealed that a pknB mutant showed reduced expression of genes involved in bacteriocin production and genetic competence. Among the genes that were differentially regulated in the pknB mutant, several were likely to be involved in cell wall metabolism. One such gene, SMU.2146c, and two genes encoding bacteriocins were shown to also be downregulated in a vicK mutant, which encodes a sensor kinase involved in the response to oxidative stress. Collectively, the results lead us to speculate that PknB may modulate the activity of the two-component signal transduction systems VicKR and ComDE. Real-time reverse transcriptase PCR (RT-PCR) showed that genes downregulated in the pknB mutant were upregulated in the pppL mutant, indicating that PppL serves to counteract PknB. PMID:20231406

The Streptococcus mutans serine/threonine kinase, PknB, regulates competence development, bacteriocin production, and cell wall metabolism.

PubMed

Banu, Liliana Danusia; Conrads, Georg; Rehrauer, Hubert; Hussain, Haitham; Allan, Elaine; van der Ploeg, Jan R

2010-05-01

Bacteria can detect, transmit, and react to signals from the outside world by using two-component systems (TCS) and serine-threonine kinases and phosphatases. Streptococcus mutans contains one serine-threonine kinase, encoded by pknB. A gene encoding a serine-threonine phosphatase, pppL, is located upstream of pknB. In this study, the phenotypes of pknB and pppL single mutants and a pknB pppL double mutant were characterized. All mutants exhibited a reduction in genetic transformability and biofilm formation, showed abnormal cell shapes, grew slower than the wild-type strain in several complex media, and exhibited reduced acid tolerance. The mutants had reduced cariogenic capacity but no significant defects in colonization in a rat caries model. Whole-genome transcriptome analysis revealed that a pknB mutant showed reduced expression of genes involved in bacteriocin production and genetic competence. Among the genes that were differentially regulated in the pknB mutant, several were likely to be involved in cell wall metabolism. One such gene, SMU.2146c, and two genes encoding bacteriocins were shown to also be downregulated in a vicK mutant, which encodes a sensor kinase involved in the response to oxidative stress. Collectively, the results lead us to speculate that PknB may modulate the activity of the two-component signal transduction systems VicKR and ComDE. Real-time reverse transcriptase PCR (RT-PCR) showed that genes downregulated in the pknB mutant were upregulated in the pppL mutant, indicating that PppL serves to counteract PknB.

Autosomal mutations in mouse kidney epithelial cells exposed to high-energy protons in vivo or in culture.

PubMed

Turker, Mitchell S; Grygoryev, Dmytro; Dan, Cristian; Eckelmann, Bradley; Lasarev, Michael; Gauny, Stacey; Kwoh, Ely; Kronenberg, Amy

2013-05-01

Proton exposure induces mutations and cancer, which are presumably linked. Because protons are abundant in the space environment and significant uncertainties exist for the effects of space travel on human health, the purpose of this study was to identify the types of mutations induced by exposure of mammalian cells to 4-5 Gy of 1 GeV protons. We used an assay that selects for mutations affecting the chromosome 8-encoded Aprt locus in mouse kidney cells and selected mutants after proton exposure both in vivo and in cell culture. A loss of heterozygosity (LOH) assay for DNA preparations from the in vivo-derived kidney mutants revealed that protons readily induced large mutational events. Fluorescent in situ hybridization painting for chromosome 8 showed that >70% of proton-induced LOH patterns resembling mitotic recombination were in fact the result of nonreciprocal chromosome translocations, thereby demonstrating an important role for DNA double-strand breaks in proton mutagenesis. Large interstitial deletions, which also require the formation and resolution of double-strand breaks, were significantly induced in the cell culture environment (14% of all mutants), but to a lesser extend in vivo (2% of all mutants) suggesting that the resolution of proton-induced double-strand breaks can differ between the intact tissue and cell culture microenvironments. In total, the results demonstrate that double-strand break formation is a primary determinant for proton mutagenesis in epithelial cell types and suggest that resultant LOH for significant genomic regions play a critical role in proton-induced cancers.

The role of loop ZA and Pro371 in the function of yeast Gcn5p bromodomain revealed through molecular dynamics and experiment.

PubMed

Pizzitutti, Francesco; Giansanti, Andrea; Ballario, Paola; Ornaghi, Prisca; Torreri, Paola; Ciccotti, Giovanni; Filetici, Patrizia

2006-01-01

Biological experiments were combined with molecular dynamics simulations to understand the importance of amino acidic residues present in the bromodomain of the yeast histone acetyltransferase Gcn5p. It was found that residue Pro371 plays an important role in the molecular recognition of the acetylated histone H4 tail by Gcn5p bromodomain. Crystallographic analysis of the complex showed that this residue does not directly interact with the histone substrate. It has been demonstrated that a double mutation Pro371Thr and Met372Ala in the Gcn5p bromodomain impairs chromatin remodeling activity. It is demonstrated here that, in this double mutant and in the fully deleted bromodomain strain, there is lower growth under amino acid deprivation conditions. By in vitro surface plasmon resonance (Biacore) experiments it is shown that the binding affinity of the double mutation to acetyl lysine 16 histone H4 peptide decreases. Molecular dynamics simulations were used to explain this loss in acetyl lysine-Gcn5p bromodomain affinity, in the double mutant. By comparing nanosecond molecular dynamics trajectories of the native as well as the single and doubly mutated bromodomain, it is concluded that the presence of Pro371 is important to the functionality of the Gcn5p bromodomain. In the simulation a point mutation involving this highly conserved residue induced an increase in the flexibility of the ZA loop, which in turn modulated the exposure of the binding pocket to the acetyl lysine. The combined double mutations (Pro371Thr-Met372Ala) not only markedly perturb the motion of the ZA loop but also destabilize the entire structure of the bromodomain. Copyright 2005 John Wiley & Sons, Ltd.

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

Dihydrofolate-Reductase Mutations in Plasmodium knowlesi Appear Unrelated to Selective Drug Pressure from Putative Human-To-Human Transmission in Sabah, Malaysia.

PubMed

Grigg, Matthew J; Barber, Bridget E; Marfurt, Jutta; Imwong, Mallika; William, Timothy; Bird, Elspeth; Piera, Kim A; Aziz, Ammar; Boonyuen, Usa; Drakeley, Christopher J; Cox, Jonathan; White, Nicholas J; Cheng, Qin; Yeo, Tsin W; Auburn, Sarah; Anstey, Nicholas M

2016-01-01

Malaria caused by zoonotic Plasmodium knowlesi is an emerging threat in Eastern Malaysia. Despite demonstrated vector competency, it is unknown whether human-to-human (H-H) transmission is occurring naturally. We sought evidence of drug selection pressure from the antimalarial sulfadoxine-pyrimethamine (SP) as a potential marker of H-H transmission. The P. knowlesi dihdyrofolate-reductase (pkdhfr) gene was sequenced from 449 P. knowlesi malaria cases from Sabah (Malaysian Borneo) and genotypes evaluated for association with clinical and epidemiological factors. Homology modelling using the pvdhfr template was used to assess the effect of pkdhfr mutations on the pyrimethamine binding pocket. Fourteen non-synonymous mutations were detected, with the most common being at codon T91P (10.2%) and R34L (10.0%), resulting in 21 different genotypes, including the wild-type, 14 single mutants, and six double mutants. One third of the P. knowlesi infections were with pkdhfr mutants; 145 (32%) patients had single mutants and 14 (3%) had double-mutants. In contrast, among the 47 P. falciparum isolates sequenced, three pfdhfr genotypes were found, with the double mutant 108N+59R being fixed and the triple mutants 108N+59R+51I and 108N+59R+164L occurring with frequencies of 4% and 8%, respectively. Two non-random spatio-temporal clusters were identified with pkdhfr genotypes. There was no association between pkdhfr mutations and hyperparasitaemia or malaria severity, both hypothesized to be indicators of H-H transmission. The orthologous loci associated with resistance in P. falciparum were not mutated in pkdhfr. Subsequent homology modelling of pkdhfr revealed gene loci 13, 53, 120, and 173 as being critical for pyrimethamine binding, however, there were no mutations at these sites among the 449 P. knowlesi isolates. Although moderate diversity was observed in pkdhfr in Sabah, there was no evidence this reflected selective antifolate drug pressure in humans.

Variations of Human DNA Polymerase Genes as Biomarkers of Prostate Cancer Progression

DTIC Science & Technology

2013-07-01

discovery , cancer genetics 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18. NUMBER OF PAGES 19a. NAME OF RESPONSIBLE PERSON USAMRMC...variations identified (including all single and double mutant combinations of the Triple mutant), and some POLK mutants • Discovery of a novel...Athens, Greece, 07/10 Makridakis N. Error-prone polymerase mutations and prostate cancer progression, COBRE /Cancer Genetics group seminar, Tulane

Double-strand break repair and genetic recombination in topoisomerase and primase mutants of bacteriophage T4.

PubMed

Shcherbakov, Victor P; Kudryashova, Elena

2014-09-01

The effects of primase and topoisomerase II deficiency on the double-strand break (DSB) repair and genetic recombination in bacteriophage T4 were studied in vivo using focused recombination. Site-specific DSBs were induced by SegC endonuclease in the rIIB gene of one of the parents. The frequency/distance relationship was determined in crosses of the wild-type phage, topoisomerase II mutant amN116 (gene 39), and primase mutant E219 (gene 61). Ordinary two-factor (i×j) and three-factor (i k×j) crosses between point rII mutations were also performed. These data provide information about the frequency and distance distribution of the single-exchange (splice) and double-exchange (patch) events. In two-factor crosses ets1×i, the topoisomerase and primase mutants had similar recombinant frequencies in crosses at ets1-i distances longer than 1000 bp, comprising about 80% of the corresponding wild-type values. They, however, differ remarkably in crosses at shorter distances. In the primase mutant, the recombinant frequencies are similar to those in the wild-type crosses at distances less than 100 bp, being a bit diminished at longer distances. In two-factor crosses ets1×i of the topoisomerase mutant, the recombinant frequencies were reduced ten-fold at the shortest distances. In three-factor crosses a6 ets1×i, where we measure patch-related recombination, the primase mutant was quite proficient across the entire range of distances. The topoisomerase mutant crosses demonstrated virtually complete absence of rII(+) recombinants at distances up to 33 bp, with the frequencies increasing steadily at longer distances. The data were interpreted as follows. The primase mutant is fully recombination-proficient. An obvious difference from the wild-type state is some shortage of EndoVII function leading to prolonged existence of HJs and thus stretched out ds-branch migration. This is also true for the topoisomerase mutant. However, the latter is deficient in the ss-branch migration step of the DSB repair pathway and partially deficient in HJ initiation. In apparent contradiction to their effects on the DSB-induced site-specific recombination, the topoisomerase and primase mutants demonstrated about 3-8-fold increase in the recombinant frequencies in the ordinary crosses, with the recombination running exclusively via patches. This implies that most of the spontaneous recombination events are not initiated by dsDNA ends in these mutants. Copyright © 2014 Elsevier B.V. All rights reserved.

Genetic Perturbation of the Maize Methylome[W

PubMed Central

Li, Qing; Hermanson, Peter J.; Zaunbrecher, Virginia M.; Song, Jawon; Wendt, Jennifer; Rosenbaum, Heidi; Madzima, Thelma F.; Sloan, Amy E.; Huang, Ji; Burgess, Daniel L.; Richmond, Todd A.; McGinnis, Karen M.; Meeley, Robert B.; Danilevskaya, Olga N.; Vaughn, Matthew W.; Kaeppler, Shawn M.; Jeddeloh, Jeffrey A.

2014-01-01

DNA methylation can play important roles in the regulation of transposable elements and genes. A collection of mutant alleles for 11 maize (Zea mays) genes predicted to play roles in controlling DNA methylation were isolated through forward- or reverse-genetic approaches. Low-coverage whole-genome bisulfite sequencing and high-coverage sequence-capture bisulfite sequencing were applied to mutant lines to determine context- and locus-specific effects of these mutations on DNA methylation profiles. Plants containing mutant alleles for components of the RNA-directed DNA methylation pathway exhibit loss of CHH methylation at many loci as well as CG and CHG methylation at a small number of loci. Plants containing loss-of-function alleles for chromomethylase (CMT) genes exhibit strong genome-wide reductions in CHG methylation and some locus-specific loss of CHH methylation. In an attempt to identify stocks with stronger reductions in DNA methylation levels than provided by single gene mutations, we performed crosses to create double mutants for the maize CMT3 orthologs, Zmet2 and Zmet5, and for the maize DDM1 orthologs, Chr101 and Chr106. While loss-of-function alleles are viable as single gene mutants, the double mutants were not recovered, suggesting that severe perturbations of the maize methylome may have stronger deleterious phenotypic effects than in Arabidopsis thaliana. PMID:25527708

The mitochondrial ribosomal protein of the large subunit, Afo1p, determines cellular longevity through mitochondrial back-signaling via TOR1.

PubMed

Heeren, Gino; Rinnerthaler, Mark; Laun, Peter; von Seyerl, Phyllis; Kössler, Sonja; Klinger, Harald; Hager, Matthias; Bogengruber, Edith; Jarolim, Stefanie; Simon-Nobbe, Birgit; Schüller, Christoph; Carmona-Gutierrez, Didac; Breitenbach-Koller, Lore; Mück, Christoph; Jansen-Dürr, Pidder; Criollo, Alfredo; Kroemer, Guido; Madeo, Frank; Breitenbach, Michael

2009-07-13

Yeast mother cell-specific aging constitutes a model of replicative aging as it occurs in stem cell populations of higher eukaryotes. Here, we present a new long-lived yeast deletion mutation,afo1 (for aging factor one), that confers a 60% increase in replicative lifespan. AFO1/MRPL25 codes for a protein that is contained in the large subunit of the mitochondrial ribosome. Double mutant experiments indicate that the longevity-increasing action of the afo1 mutation is independent of mitochondrial translation, yet involves the cytoplasmic Tor1p as well as the growth-controlling transcription factor Sfp1p. In their final cell cycle, the long-lived mutant cells do show the phenotypes of yeast apoptosis indicating that the longevity of the mutant is not caused by an inability to undergo programmed cell death. Furthermore, the afo1 mutation displays high resistance against oxidants. Despite the respiratory deficiency the mutant has paradoxical increase in growth rate compared to generic petite mutants. A comparison of the single and double mutant strains for afo1 and fob1 shows that the longevity phenotype of afo1 is independent of the formation of ERCs (ribosomal DNA minicircles). AFO1/MRPL25 function establishes a new connection between mitochondria, metabolism and aging.

CRISPR/Cas9 and TALENs generate heritable mutations for genes involved in small RNA processing of Glycine max and Medicago truncatula.

PubMed

Curtin, Shaun J; Xiong, Yer; Michno, Jean-Michel; Campbell, Benjamin W; Stec, Adrian O; Čermák, Tomas; Starker, Colby; Voytas, Daniel F; Eamens, Andrew L; Stupar, Robert M

2018-06-01

Processing of double-stranded RNA precursors into small RNAs is an essential regulator of gene expression in plant development and stress response. Small RNA processing requires the combined activity of a functionally diverse group of molecular components. However, in most of the plant species, there are insufficient mutant resources to functionally characterize each encoding gene. Here, mutations in loci encoding protein machinery involved in small RNA processing in soya bean and Medicago truncatula were generated using the CRISPR/Cas9 and TAL-effector nuclease (TALEN) mutagenesis platforms. An efficient CRISPR/Cas9 reagent was used to create a bi-allelic double mutant for the two soya bean paralogous Double-stranded RNA-binding2 (GmDrb2a and GmDrb2b) genes. These mutations, along with a CRISPR/Cas9-generated mutation of the M. truncatula Hua enhancer1 (MtHen1) gene, were determined to be germ-line transmissible. Furthermore, TALENs were used to generate a mutation within the soya bean Dicer-like2 gene. CRISPR/Cas9 mutagenesis of the soya bean Dicer-like3 gene and the GmHen1a gene was observed in the T 0 generation, but these mutations failed to transmit to the T 1 generation. The irregular transmission of induced mutations and the corresponding transgenes was investigated by whole-genome sequencing to reveal a spectrum of non-germ-line-targeted mutations and multiple transgene insertion events. Finally, a suite of combinatorial mutant plants were generated by combining the previously reported Gmdcl1a, Gmdcl1b and Gmdcl4b mutants with the Gmdrb2ab double mutant. Altogether, this study demonstrates the synergistic use of different genome engineering platforms to generate a collection of useful mutant plant lines for future study of small RNA processing in legume crops. © 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Actin dynamics involved in gravity perception in Arabidopsis inflorescense stem

NASA Astrophysics Data System (ADS)

Tasaka, Masao; Nakamura, Moritaka; Morita, Miyo T.

The amyloplasts sedimentation in the endodermal cells is important for gravity perception in Arabidopsis shoot. Our previous study suggests that SGR5(SHOOT GRAVITROPISM 5) and SGR9 are synergistically involved in regulation of amyloplast movement in these cells, and shows that sgr5 sgr9 double mutant completely loses gravitropic response. SGR5 encodes putative transcription factor and SGR9 encodes a ring finger containing protein, which surrounds amyloplasts. It has been reported that amyloplasts are surrounded by actin microfilaments (MFs), and that treatment with actin polymerization inhibitor enhances gravitropic organ curvature. However, not only the molecular link between amyolplasts and MFs, but also regulatory role of MFs in gravitropic response is still unclear. Here, we found that treatment with actin polymerization inhibitor restored gravitropic response of sgr5 sgr9 double mutant stems. The result suggests that abnormal amyloplasts movement in the double mutant could result from inhibition of MFs depolymerization, leading to abnormal gravitropism. We are investigating whether SGR5 and SGR9 are involved in amyloplasts movement by regulating actin remodeling in gravity perceptive cells.

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.

Production of isopropyl cis-6-hexadecenoate by regiospecific desaturation of isopropyl palmitate by a double mutant of a Rhodococcus strain.

PubMed

Koike, K; Takaiwa, M; Ara, K; Inoue, S; Kimura, Y; Ito, S

2000-02-01

Resting cells of a double mutant noted as KSM-MT66, derived from Rhodococcus sp. strain KSM-B-3 by UV irradiation, were found to cis-desaturate isopropyl hexadecanoate, yielding isopropyl cis-6-hexadecenoate. Addition of sodium glutamate (1.0%), Mg SO4 (2 mM), and thiamine (2 mM) increased the productivity of the unsaturated product in phosphate buffer. Optimal temperature and pH for the reaction were around 26 degrees C and 7, respectively. Under the optimized conditions, more than 50 g/l of isopropyl cis-6-hexadecenoate was produced after a 3-day incubation by resting cells of the mutant. Thus, cis-6-hexadecenoic acid, the main component of human sebaceous lipids, can be manufactured economically by the rhodococcal bioconversion.

Rational Design of Disulfide Bonds Increases Thermostability of a Mesophilic 1,3-1,4-β-Glucanase from Bacillus terquilensis

PubMed Central

Xu, Xin; Li, Qi

2016-01-01

1,3–1,4-β-glucanase is an important biocatalyst in brewing industry and animal feed industry, while its low thermostability often reduces its application performance. In this study, the thermostability of a mesophilic β-glucanase from Bacillus terquilensis was enhanced by rational design and engineering of disulfide bonds in the protein structure. Protein spatial configuration was analyzed to pre-exclude the residues pairs which negatively conflicted with the protein structure and ensure the contact of catalytic center. The changes in protein overall and local flexibility among the wild-type enzyme and the designated mutants were predicted to select the potential disulfide bonds for enhancement of thermostability. Two residue pairs (N31C-T187C and P102C-N125C) were chosen as engineering targets and both of them were proved to significantly enhance the protein thermostability. After combinational mutagenesis, the double mutant N31C-T187C/P102C-N125C showed a 48.3% increase in half-life value at 60°C and a 4.1°C rise in melting temperature (Tm) compared to wild-type enzyme. The catalytic property of N31C-T187C/P102C-N125C mutant was similar to that of wild-type enzyme. Interestingly, the optimal pH of double mutant was shifted from pH6.5 to pH6.0, which could also increase its industrial application. By comparison with mutants with single-Cys substitutions, the introduction of disulfide bonds and the induced new hydrogen bonds were proved to result in both local and overall rigidification and should be responsible for the improved thermostability. Therefore, the introduction of disulfide bonds for thermostability improvement could be rationally and highly-effectively designed by combination with spatial configuration analysis and molecular dynamics simulation. PMID:27100881

Functional diversification of Arabidopsis SEC1-related SM proteins in cytokinetic and secretory membrane fusion.

PubMed

Karnahl, Matthias; Park, Misoon; Krause, Cornelia; Hiller, Ulrike; Mayer, Ulrike; Stierhof, York-Dieter; Jürgens, Gerd

2018-06-12

Sec1/Munc18 (SM) proteins contribute to membrane fusion by interacting with Qa-SNAREs or nascent trans -SNARE complexes. Gymnosperms and the basal angiosperm Amborella have only a single SEC1 gene related to the KEULE gene in Arabidopsis However, the genomes of most angiosperms including Arabidopsis encode three SEC1-related SM proteins of which only KEULE has been functionally characterized as interacting with the cytokinesis-specific Qa-SNARE KNOLLE during cell-plate formation. Here we analyze the closest paralog of KEULE named SEC1B. In contrast to the cytokinesis defects of keule mutants, sec1b mutants are homozygous viable. However, the keule sec1b double mutant was nearly gametophytically lethal, displaying collapsed pollen grains, which suggests substantial overlap between SEC1B and KEULE functions in secretion-dependent growth. SEC1B had a strong preference for interaction with the evolutionarily ancient Qa-SNARE SYP132 involved in secretion and cytokinesis, whereas KEULE interacted with both KNOLLE and SYP132. This differential interaction with Qa-SNAREs is likely conferred by domains 1 and 2a of the two SM proteins. Comparative analysis of all four possible combinations of the relevant SEC1 Qa-SNARE double mutants revealed that in cytokinesis, the interaction of SEC1B with KNOLLE plays no role, whereas the interaction of KEULE with KNOLLE is prevalent and functionally as important as the interactions of both SEC1B and KEU with SYP132 together. Our results suggest that functional diversification of the two SEC1-related SM proteins during angiosperm evolution resulted in enhanced interaction of SEC1B with Qa-SNARE SYP132, and thus a predominant role of SEC1B in secretion.

Myosins XI-K, XI-1, and XI-2 are required for development of pavement cells, trichomes, and stigmatic papillae in Arabidopsis

PubMed Central

2012-01-01

Background The positioning and dynamics of vesicles and organelles, and thus the growth of plant cells, is mediated by the acto-myosin system. In Arabidopsis there are 13 class XI myosins which mediate vesicle and organelle transport in different cell types. So far the involvement of five class XI myosins in cell expansion during the shoot and root development has been shown, three of which, XI-1, XI-2, and XI-K, are essential for organelle transport. Results Simultaneous depletion of Arabidopsis class XI myosins XI-K, XI-1, and XI-2 in double and triple mutant plants affected the growth of several types of epidermal cells. The size and shape of trichomes, leaf pavement cells and the elongation of the stigmatic papillae of double and triple mutant plants were affected to different extent. Reduced cell size led to significant size reduction of shoot organs in the case of triple mutant, affecting bolt formation, flowering time and fertility. Phenotype analysis revealed that the reduced fertility of triple mutant plants was caused by delayed or insufficient development of pistils. Conclusions We conclude that the class XI myosins XI-K, XI-1 and XI-2 have partially redundant roles in the growth of shoot epidermis. Myosin XI-K plays more important role whereas myosins XI-1 and XI-2 have minor roles in the determination of size and shape of epidermal cells, because the absence of these two myosins is compensated by XI-K. Co-operation between myosins XI-K and XI-2 appears to play an important role in these processes. PMID:22672737

Lipidomics Characterization of Biosynthetic and Remodeling Pathways of Cardiolipins in Genetically and Nutritionally Manipulated Yeast Cells.

PubMed

Tyurina, Yulia Y; Lou, Wenjia; Qu, Feng; Tyurin, Vladimir A; Mohammadyani, Dariush; Liu, Jenney; Hüttemann, Maik; Frasso, Michael A; Wipf, Peter; Bayir, Hülya; Greenberg, Miriam L; Kagan, Valerian E

2017-01-20

Cardioipins (CLs) are unique tetra-acylated phospholipids of mitochondria and define the bioenergetics and regulatory functions of these organelles. An unresolved paradox is the high uniformity of CL molecular species (tetra-linoleoyl-CL) in the heart, liver, and skeletal muscles-in contrast to their high diversification in the brain. Here, we combined liquid chromatography-mass-spectrometry-based phospholipidomics with genetic and nutritional manipulations to explore CLs' biosynthetic vs postsynthetic remodeling processes in S. cerevisiae yeast cells. By applying the differential phospholipidomics analysis, we evaluated the contribution of Cld1 (CL-specific phospholipase A) and Taz1 (acyl-transferase) as the major regulatory mechanisms of the remodeling process. We further established that nutritional "pressure" by high levels of free fatty acids triggered a massive synthesis of homoacylated molecular species in all classes of phospholipids, resulting in the preponderance of the respective homoacylated CLs. We found that changes in molecular speciation of CLs induced by exogenous C18-fatty acids (C18:1 and C18:2) in wild-type (wt) cells did not occur in any of the remodeling mutant cells, including cld1Δ, taz1Δ, and cld1Δtaz1Δ. Interestingly, molecular speciation of CLs in wt and double mutant cells cld1Δtaz1Δ was markedly different. Given that the bioenergetics functions are preserved in the double mutant, this suggests that the accumulated MLCL-rather than the changed CL speciation-are the likely major contributors to the mitochondrial dysfunction in taz1Δ mutant cells (also characteristic of Barth syndrome). Biochemical studies of Cld1 specificity and computer modeling confirmed the hydrolytic selectivity of the enzyme toward C16-CL substrates and the preservation of C18:1-containing CL species.

Functional Analysis of the Lactobacillus casei BL23 Sortases

PubMed Central

Muñoz-Provencio, Diego; Rodríguez-Díaz, Jesús; Collado, María Carmen; Langella, Philippe; Bermúdez-Humarán, Luis G.

2012-01-01

Sortases are a class of enzymes that anchor surface proteins to the cell wall of Gram-positive bacteria. Lactobacillus casei BL23 harbors four sortase genes, two belonging to class A (srtA1 and srtA2) and two belonging to class C (srtC1 and srtC2). Class C sortases were clustered with genes encoding their putative substrates that were homologous to the SpaEFG and SpaCBA proteins that encode mucus adhesive pili in Lactobacillus rhamnosus GG. Twenty-three genes encoding putative sortase substrates were identified in the L. casei BL23 genome with unknown (35%), enzymatic (30%), or adhesion-related (35%) functions. Strains disrupted in srtA1, srtA2, srtC1, and srtC2 and an srtA1 srtA2 double mutant were constructed. The transcription of all four sortase encoding genes was detected, but only the mutation of srtA1 resulted in a decrease in bacterial surface hydrophobicity. The β-N-acetyl-glucosaminidase and cell wall proteinase activities of whole cells diminished in the srtA1 mutant and, to a greater extent, in the srtA1 srtA2 double mutant. Cell wall anchoring of the staphylococcal NucA reporter protein fused to a cell wall sorting sequence was also affected in the srtA mutants, and the percentages of adhesion to Caco-2 and HT-29 intestinal epithelial cells were reduced for the srtA1 srtA2 strain. Mutations in srtC1 or srtC2 result in an undetectable phenotype. Together, these results suggest that SrtA1 is the housekeeping sortase in L. casei BL23 and SrtA2 would carry out redundant or complementary functions that become evident when SrtA1 activity is absent. PMID:23042174

Uncoupling High Light Responses from Singlet Oxygen Retrograde Signaling and Spatial-Temporal Systemic Acquired Acclimation1[OPEN

PubMed Central

Gordon, Matthew; Havaux, Michel; Albrecht-Borth, Verónica

2016-01-01

Distinct ROS signaling pathways initiated by singlet oxygen (1O2) or superoxide and hydrogen peroxide have been attributed to either cell death or acclimation, respectively. Recent studies have revealed that more complex antagonistic and synergistic relationships exist within and between these pathways. As specific chloroplastic ROS signals are difficult to study, rapid systemic signaling experiments using localized high light (HL) stress or ROS treatments were used in this study to uncouple signals required for direct HL and ROS perception and distal systemic acquired acclimation (SAA). A qPCR approach was chosen to determine local perception and distal signal reception. Analysis of a thylakoidal ascorbate peroxidase mutant (tapx), the 1O2-retrograde signaling double mutant (ex1/ex2), and an apoplastic signaling double mutant (rbohD/F) revealed that tAPX and EXECUTER 1 are required for both HL and systemic acclimation stress perception. Apoplastic membrane-localized RBOHs were required for systemic spread of the signal but not for local signal induction in directly stressed tissues. Endogenous ROS treatments revealed a very strong systemic response induced by a localized 1 h induction of 1O2 using the conditional flu mutant. A qPCR time course of 1O2 induced systemic marker genes in directly and indirectly connected leaves revealed a direct vascular connection component of both immediate and longer term SAA signaling responses. These results reveal the importance of an EXECUTER-dependent 1O2 retrograde signal for both local and long distance RBOH-dependent acclimation signaling that is distinct from other HL signaling pathways, and that direct vascular connections have a role in spatial-temporal SAA induction. PMID:27288360

Uncoupling High Light Responses from Singlet Oxygen Retrograde Signaling and Spatial-Temporal Systemic Acquired Acclimation.

PubMed

Carmody, Melanie; Crisp, Peter A; d'Alessandro, Stefano; Ganguly, Diep; Gordon, Matthew; Havaux, Michel; Albrecht-Borth, Verónica; Pogson, Barry J

2016-07-01

Distinct ROS signaling pathways initiated by singlet oxygen ((1)O2) or superoxide and hydrogen peroxide have been attributed to either cell death or acclimation, respectively. Recent studies have revealed that more complex antagonistic and synergistic relationships exist within and between these pathways. As specific chloroplastic ROS signals are difficult to study, rapid systemic signaling experiments using localized high light (HL) stress or ROS treatments were used in this study to uncouple signals required for direct HL and ROS perception and distal systemic acquired acclimation (SAA). A qPCR approach was chosen to determine local perception and distal signal reception. Analysis of a thylakoidal ascorbate peroxidase mutant (tapx), the (1)O2-retrograde signaling double mutant (ex1/ex2), and an apoplastic signaling double mutant (rbohD/F) revealed that tAPX and EXECUTER 1 are required for both HL and systemic acclimation stress perception. Apoplastic membrane-localized RBOHs were required for systemic spread of the signal but not for local signal induction in directly stressed tissues. Endogenous ROS treatments revealed a very strong systemic response induced by a localized 1 h induction of (1)O2 using the conditional flu mutant. A qPCR time course of (1)O2 induced systemic marker genes in directly and indirectly connected leaves revealed a direct vascular connection component of both immediate and longer term SAA signaling responses. These results reveal the importance of an EXECUTER-dependent (1)O2 retrograde signal for both local and long distance RBOH-dependent acclimation signaling that is distinct from other HL signaling pathways, and that direct vascular connections have a role in spatial-temporal SAA induction. © 2016 American Society of Plant Biologists. All Rights Reserved.

shl, a New set of Arabidopsis mutants with exaggerated developmental responses to available red, far-red, and blue light.

PubMed

Pepper, A E; Seong-Kim, M; Hebst, S M; Ivey, K N; Kwak, S J; Broyles, D E

2001-09-01

The interaction of light perception with development is the subject of intensive genetic analysis in the model plant Arabidopsis. We performed genetic screens in low white light-a threshold condition in which photomorphogenetic signaling pathways are only partially active-for ethyl methane sulfonate-generated mutants with altered developmental phenotypes. Recessive mutants with exaggerated developmental responses were obtained in eight complementation groups designated shl for seedlings hyperresponsive to light. shl1, shl2, shl5, and shl3 shl4 (double mutant) seedlings showed limited or no phenotypic effects in darkness, but showed significantly enhanced inhibition of hypocotyl elongation in low-white, red, far-red, blue, and green light across a range of fluences. These results reflect developmental hyper-responsiveness to signals generated by both phytochrome and cryptochrome photoreceptors. The shl11 mutant retained significant phenotypic effects on hypocotyl length in both the phyA mutant and phyB mutant backgrounds but may be dependent on CRY1 for phenotypic expression in blue light. The shl2 phenotype was partially dependent on PHYB, PHYA, and CRY1 in red, far-red, and blue light, respectively. shl2 and, in particular, shl1 were partially dependent on HY5 activity for their light-hyperresponsive phenotypes. The SHL genes act (genetically) as light-dependent negative regulators of photomorphogenesis, possibly in a downstream signaling or developmental pathway that is shared by CRY1, PHYA, and PHYB and other photoreceptors (CRY2, PHYC, PHYD, and PHYE).

Alteration of gene conversion tract length and associated crossing over during plasmid gap repair in nuclease-deficient strains of Saccharomyces cerevisiae.

PubMed

Symington, L S; Kang, L E; Moreau, S

2000-12-01

A plasmid gap repair assay was used to assess the role of three known nucleases, Exo1, Mre11 and Rad1, in the processing of DNA ends and resolution of recombination intermediates during double-strand gap repair. In this assay, alterations in end processing or branch migration are reflected by the frequency of co-conversion of a chromosomal marker 200 bp from the gap. Gap repair associated with crossing over results in integration at the homologous chromosomal locus, whereas the plasmid remains episomal for non-crossover repair events. In mre11 strains, the frequency of gap repair was reduced 3- to 10-fold and conversion tracts were shorter than in the wild-type strain, consistent with a role for this nuclease in processing double-strand breaks. However, conversion tracts were longer in a strain containing the nuclease deficient allele, mre11-H125N, suggesting increased end processing by redundant nucleases. The frequency of gap repair was reduced 2-fold in rad1 mutants and crossing over was reduced, consistent with a role for Rad1 in cleaving recombination intermediates. The frequency of gap repair was increased in exo1 mutants with a significant increase in crossing over. In exo1 mre11 double mutants gap repair was reduced to below the mre11 single mutant level.

Combinatorial Regulation of Meiotic Holliday Junction Resolution in C. elegans by HIM-6 (BLM) Helicase, SLX-4, and the SLX-1, MUS-81 and XPF-1 Nucleases

PubMed Central

Sonneville, Remi; Jagut, Marlène; Woglar, Alexander; Blow, Julian; Jantsch, Verena; Gartner, Anton

2013-01-01

Holliday junctions (HJs) are cruciform DNA structures that are created during recombination events. It is a matter of considerable importance to determine the resolvase(s) that promote resolution of these structures. We previously reported that C. elegans GEN-1 is a symmetrically cleaving HJ resolving enzyme required for recombinational repair, but we could not find an overt role in meiotic recombination. Here we identify C. elegans proteins involved in resolving meiotic HJs. We found no evidence for a redundant meiotic function of GEN-1. In contrast, we discovered two redundant HJ resolution pathways likely coordinated by the SLX-4 scaffold protein and also involving the HIM-6/BLM helicase. SLX-4 associates with the SLX-1, MUS-81 and XPF-1 nucleases and has been implicated in meiotic recombination in C. elegans. We found that C. elegans [mus-81; xpf-1], [slx-1; xpf-1], [mus-81; him-6] and [slx-1; him-6] double mutants showed a similar reduction in survival rates as slx-4. Analysis of meiotic diakinesis chromosomes revealed a distinct phenotype in these double mutants. Instead of wild-type bivalent chromosomes, pairs of “univalents” linked by chromatin bridges occur. These linkages depend on the conserved meiosis-specific transesterase SPO-11 and can be restored by ionizing radiation, suggesting that they represent unresolved meiotic HJs. This suggests the existence of two major resolvase activities, one provided by XPF-1 and HIM-6, the other by SLX-1 and MUS-81. In all double mutants crossover (CO) recombination is reduced but not abolished, indicative of further redundancy in meiotic HJ resolution. Real time imaging revealed extensive chromatin bridges during the first meiotic division that appear to be eventually resolved in meiosis II, suggesting back-up resolution activities acting at or after anaphase I. We also show that in HJ resolution mutants, the restructuring of chromosome arms distal and proximal to the CO still occurs, suggesting that CO initiation but not resolution is likely to be required for this process. PMID:23901331

Combinatorial regulation of meiotic holliday junction resolution in C. elegans by HIM-6 (BLM) helicase, SLX-4, and the SLX-1, MUS-81 and XPF-1 nucleases.

PubMed

Agostinho, Ana; Meier, Bettina; Sonneville, Remi; Jagut, Marlène; Woglar, Alexander; Blow, Julian; Jantsch, Verena; Gartner, Anton

2013-01-01

Holliday junctions (HJs) are cruciform DNA structures that are created during recombination events. It is a matter of considerable importance to determine the resolvase(s) that promote resolution of these structures. We previously reported that C. elegans GEN-1 is a symmetrically cleaving HJ resolving enzyme required for recombinational repair, but we could not find an overt role in meiotic recombination. Here we identify C. elegans proteins involved in resolving meiotic HJs. We found no evidence for a redundant meiotic function of GEN-1. In contrast, we discovered two redundant HJ resolution pathways likely coordinated by the SLX-4 scaffold protein and also involving the HIM-6/BLM helicase. SLX-4 associates with the SLX-1, MUS-81 and XPF-1 nucleases and has been implicated in meiotic recombination in C. elegans. We found that C. elegans [mus-81; xpf-1], [slx-1; xpf-1], [mus-81; him-6] and [slx-1; him-6] double mutants showed a similar reduction in survival rates as slx-4. Analysis of meiotic diakinesis chromosomes revealed a distinct phenotype in these double mutants. Instead of wild-type bivalent chromosomes, pairs of "univalents" linked by chromatin bridges occur. These linkages depend on the conserved meiosis-specific transesterase SPO-11 and can be restored by ionizing radiation, suggesting that they represent unresolved meiotic HJs. This suggests the existence of two major resolvase activities, one provided by XPF-1 and HIM-6, the other by SLX-1 and MUS-81. In all double mutants crossover (CO) recombination is reduced but not abolished, indicative of further redundancy in meiotic HJ resolution. Real time imaging revealed extensive chromatin bridges during the first meiotic division that appear to be eventually resolved in meiosis II, suggesting back-up resolution activities acting at or after anaphase I. We also show that in HJ resolution mutants, the restructuring of chromosome arms distal and proximal to the CO still occurs, suggesting that CO initiation but not resolution is likely to be required for this process.

Gaussian decomposition of high-resolution melt curve derivatives for measuring genome-editing efficiency

PubMed Central

Zaboikin, Michail; Freter, Carl

2018-01-01

We describe a method for measuring genome editing efficiency from in silico analysis of high-resolution melt curve data. The melt curve data derived from amplicons of genome-edited or unmodified target sites were processed to remove the background fluorescent signal emanating from free fluorophore and then corrected for temperature-dependent quenching of fluorescence of double-stranded DNA-bound fluorophore. Corrected data were normalized and numerically differentiated to obtain the first derivatives of the melt curves. These were then mathematically modeled as a sum or superposition of minimal number of Gaussian components. Using Gaussian parameters determined by modeling of melt curve derivatives of unedited samples, we were able to model melt curve derivatives from genetically altered target sites where the mutant population could be accommodated using an additional Gaussian component. From this, the proportion contributed by the mutant component in the target region amplicon could be accurately determined. Mutant component computations compared well with the mutant frequency determination from next generation sequencing data. The results were also consistent with our earlier studies that used difference curve areas from high-resolution melt curves for determining the efficiency of genome-editing reagents. The advantage of the described method is that it does not require calibration curves to estimate proportion of mutants in amplicons of genome-edited target sites. PMID:29300734

[Dot1 and Set2 Histone Methylases Control the Spontaneous and UV-Induced Mutagenesis Levels in the Saccharomyces cerevisiae Yeasts].

PubMed

Kozhina, T N; Evstiukhina, T A; Peshekhonov, V T; Chernenkov, A Yu; Korolev, V G

2016-03-01

In the Saccharomyces cerevisiae yeasts, the DOT1 gene product provides methylation of lysine 79 (K79) of hi- stone H3 and the SET2 gene product provides the methylation of lysine 36 (K36) of the same histone. We determined that the dot1 and set2 mutants suppress the UV-induced mutagenesis to an equally high degree. The dot1 mutation demonstrated statistically higher sensitivity to the low doses of MMC than the wild type strain. The analysis of the interaction between the dot1 and rad52 mutations revealed a considerable level of spontaneous cell death in the double dot1 rad52 mutant. We observed strong suppression of the gamma-in- duced mutagenesis in the set2 mutant. We determined that the dot1 and set2 mutations decrease the sponta- neous mutagenesis rate in both single and d ouble mutants. The epistatic interaction between the dot1 and set2 mutations and almost similar sensitivity of the corresponding mutants to the different types of DNA damage allow one to conclude that both genes are involved in the control of the same DNA repair pathways, the ho- mologous-recombination-based and the postreplicative DNA repair.

The autophagy-related genes BbATG1 and BbATG8 have different functions in differentiation, stress resistance and virulence of mycopathogen Beauveria bassiana

PubMed Central

Ying, Sheng-Hua; Liu, Jing; Chu, Xin-Ling; Xie, Xue-Qin; Feng, Ming-Guang

2016-01-01

Autophagy-related proteins play significantly different roles in eukaryotes. In the entomopathogenic fungus Beauveria bassiana, autophagy is associated with fungal growth and development. BbATG1 (a serine/threonine protein kinase) and BbATG8 (a ubiquitin-like protein) have similar roles in autophagy, but different roles in other processes. Disruption mutants of BbATG1 and BbATG8 had impaired conidial germination under starvation stress. The mutant ΔBbATG8 exhibited enhanced sensitivity to oxidative stress, while a ΔBbATG1 mutant did not. BbATG1 and BbATG8 showed different roles in spore differentiation. The blastospore yield was reduced by 70% and 92% in ΔBbATG1 and ΔBbATG8 mutants, respectively, and the double mutant had a reduction of 95%. Conidial yield was reduced by approximately 90% and 50% in ΔBbATG1 and ΔBbATG8 mutants, respectively. A double mutant had a reduction similar to ΔBbATG1. Additionally, both BbATG1 and BbATG8 affected the levels of conidial protein BbCP15p required for conidiation. The virulence of each autophagy-deficient mutant was considerably weakened as indicated in topical and intrahemocoel injection assays, and showed a greater reduction in topical infection. However, BbATG1 and BbATG8 had different effects on fungal virulence. Our data indicate that these autophagy-related proteins have different functions in fungal stress response, asexual development and virulence. PMID:27197558

Molecular principles behind pyrazinamide resistance due to mutations in panD gene in Mycobacterium tuberculosis.

PubMed

Pandey, Bharati; Grover, Sonam; Tyagi, Chetna; Goyal, Sukriti; Jamal, Salma; Singh, Aditi; Kaur, Jagdeep; Grover, Abhinav

2016-04-25

The latest resurrection of drug resistance poses serious threat to the treatment and control of the disease. Mutations have been detected in panD gene in the Mycobacterium tuberculosis (Mtb) strains. Mutation of histidine to arginine at residue 21 (H21R) and isoleucine to valine at residue 29 (I49V) in the non-active site of panD gene has led to PZA resistance. This study will help in reconnoitering the mechanism of pyrazinamide (PZA) resistance caused due to double mutation identified in the panD gene of M. tuberculosis clinical isolates. It is known that panD gene encodes aspartate decarboxylase essential for β-alanine synthesis that makes it a potential therapeutic drug target for tuberculosis treatment. The knowledge about the molecular mechanism conferring drug resistance in M. tuberculosis is scarce, which is a significant challenge in designing successful therapeutic drug. In this study, structural and dynamic repercussions of H21R-I49V double mutation in panD complexed with PZA have been corroborated through docking and molecular dynamics based simulation. The double mutant (DM) shows low docking score and thus, low binding affinity for PZA as compared to the native protein. It was observed that the mutant protein exhibits more structural fluctuation at the ligand binding site in comparison to the native type. Furthermore, the flexibility and compactness analyses indicate that the double mutation influence interaction of PZA with the protein. The hydrogen-bond interaction patterns further supported our results. The covariance and PCA analysis elucidated that the double mutation affects the collective motion of residues in phase space. The results have been presented with an explanation for the induced drug resistance conferred by the H21R-I49V double mutation in panD gene and gain valuable insight to facilitate the advent of efficient therapeutics for combating resistance against PZA. Copyright © 2016 Elsevier B.V. All rights reserved.

The AMT1 Arginine Methyltransferase Gene Is Important for Plant Infection and Normal Hyphal Growth in Fusarium graminearum

PubMed Central

Hou, Rui; Zhou, Xiaoying; Li, Guotian; Zhang, Shijie; Xu, Jin-Rong

2012-01-01

Arginine methylation of non-histone proteins by protein arginine methyltransferase (PRMT) has been shown to be important for various biological processes from yeast to human. Although PRMT genes are well conserved in fungi, none of them have been functionally characterized in plant pathogenic ascomycetes. In this study, we identified and characterized all of the four predicted PRMT genes in Fusarium graminearum, the causal agent of Fusarium head blight of wheat and barley. Whereas deletion of the other three PRMT genes had no obvious phenotypes, the Δamt1 mutant had pleiotropic defects. AMT1 is a predicted type I PRMT gene that is orthologous to HMT1 in Saccharomyces cerevisiae. The Δamt1 mutant was slightly reduced in vegetative growth but normal in asexual and sexual reproduction. It had increased sensitivities to oxidative and membrane stresses. DON mycotoxin production and virulence on flowering wheat heads also were reduced in the Δamt1 mutant. The introduction of the wild-type AMT1 allele fully complemented the defects of the Δamt1 mutant and Amt1-GFP fusion proteins mainly localized to the nucleus. Hrp1 and Nab2 are two hnRNPs in yeast that are methylated by Hmt1 for nuclear export. In F. graminearum, AMT1 is required for the nuclear export of FgHrp1 but not FgNab2, indicating that yeast and F. graminearum differ in the methylation and nucleo-cytoplasmic transport of hnRNP components. Because AMT2 also is a predicted type I PRMT with limited homology to yeast HMT1, we generated the Δamt1 Δamt2 double mutants. The Δamt1 single and Δamt1 Δamt2 double mutants had similar defects in all the phenotypes assayed, including reduced vegetative growth and virulence. Overall, data from this systematic analysis of PRMT genes suggest that AMT1, like its ortholog in yeast, is the predominant PRMT gene in F. graminearum and plays a role in hyphal growth, stress responses, and plant infection. PMID:22693618

Axonal abnormalities in vanishing white matter.

PubMed

Klok, Melanie D; Bugiani, Marianna; de Vries, Sharon I; Gerritsen, Wouter; Breur, Marjolein; van der Sluis, Sophie; Heine, Vivi M; Kole, Maarten H P; Baron, Wia; van der Knaap, Marjo S

2018-04-01

We aimed to study the occurrence and development of axonal pathology and the influence of astrocytes in vanishing white matter. Axons and myelin were analyzed using electron microscopy and immunohistochemistry on Eif2b4 and Eif2b5 single- and double-mutant mice and patient brain tissue. In addition, astrocyte-forebrain co-culture studies were performed. In the corpus callosum of Eif2b5- mutant mice, myelin sheath thickness, axonal diameter, and G-ratio developed normally up to 4 months. At 7 months, however, axons had become thinner, while in control mice axonal diameters had increased further. Myelin sheath thickness remained close to normal, resulting in an abnormally low G-ratio in Eif2b5- mutant mice. In more severely affected Eif2b4-Eif2b5 double-mutants, similar abnormalities were already present at 4 months, while in milder affected Eif2b4 mutants, few abnormalities were observed at 7 months. Additionally, from 2 months onward an increased percentage of thin, unmyelinated axons and increased axonal density were present in Eif2b5 -mutant mice. Co-cultures showed that Eif2b5 mutant astrocytes induced increased axonal density, also in control forebrain tissue, and that control astrocytes induced normal axonal density, also in mutant forebrain tissue. In vanishing white matter patient brains, axons and myelin sheaths were thinner than normal in moderately and severely affected white matter. In mutant mice and patients, signs of axonal transport defects and cytoskeletal abnormalities were minimal. In vanishing white matter, axons are initially normal and atrophy later. Astrocytes are central in this process. If therapy becomes available, axonal pathology may be prevented with early intervention.

The Stringent Response Is Essential for Pseudomonas aeruginosa Virulence in the Rat Lung Agar Bead and Drosophila melanogaster Feeding Models of Infection▿†

PubMed Central

Vogt, Stefanie L.; Green, Christopher; Stevens, Katarzyna M.; Day, Brad; Erickson, David L.; Woods, Donald E.; Storey, Douglas G.

2011-01-01

The stringent response is a regulatory system that allows bacteria to sense and adapt to nutrient-poor environments. The central mediator of the stringent response is the molecule guanosine 3′,5′-bispyrophosphate (ppGpp), which is synthesized by the enzymes RelA and SpoT and which is also degraded by SpoT. Our laboratory previously demonstrated that a relA mutant of Pseudomonas aeruginosa, the principal cause of lung infections in cystic fibrosis patients, was attenuated in virulence in a Drosophila melanogaster feeding model of infection. In this study, we examined the role of spoT in P. aeruginosa virulence. We generated an insertion mutation in spoT within the previously constructed relA mutant, thereby producing a ppGpp-devoid strain. The relA spoT double mutant was unable to establish a chronic infection in D. melanogaster and was also avirulent in the rat lung agar bead model of infection, a model in which the relA mutant is fully virulent. Synthesis of the virulence determinants pyocyanin, elastase, protease, and siderophores was impaired in the relA spoT double mutant. This mutant was also defective in swarming and twitching, but not in swimming motility. The relA spoT mutant and, to a lesser extent, the relA mutant were less able to withstand stresses such as heat shock and oxidative stress than the wild-type strain PAO1, which may partially account for the inability of the relA spoT mutant to successfully colonize the rat lung. Our results indicate that the stringent response, and SpoT in particular, is a crucial regulator of virulence processes in P. aeruginosa. PMID:21788391

Assisted Design of Antibody and Protein Therapeutics (ADAPT)

PubMed Central

Vivcharuk, Victor; Baardsnes, Jason; Deprez, Christophe; Sulea, Traian; Jaramillo, Maria; Corbeil, Christopher R.; Mullick, Alaka; Magoon, Joanne; Marcil, Anne; Durocher, Yves; O’Connor-McCourt, Maureen D.

2017-01-01

Effective biologic therapeutics require binding affinities that are fine-tuned to their disease-related molecular target. The ADAPT (Assisted Design of Antibody and Protein Therapeutics) platform aids in the selection of mutants that improve/modulate the affinity of antibodies and other biologics. It uses a consensus z-score from three scoring functions and interleaves computational predictions with experimental validation, significantly enhancing the robustness of the design and selection of mutants. The platform was tested on three antibody Fab-antigen systems that spanned a wide range of initial binding affinities: bH1-VEGF-A (44 nM), bH1-HER2 (3.6 nM) and Herceptin-HER2 (0.058 nM). Novel triple mutants were obtained that exhibited 104-, 46- and 32-fold improvements in binding affinity for each system, respectively. Moreover, for all three antibody-antigen systems over 90% of all the intermediate single and double mutants that were designed and tested showed higher affinities than the parent sequence. The contributions of the individual mutants to the change in binding affinity appear to be roughly additive when combined to form double and triple mutants. The new interactions introduced by the affinity-enhancing mutants included long-range electrostatics as well as short-range nonpolar interactions. This diversity in the types of new interactions formed by the mutants was reflected in SPR kinetics that showed that the enhancements in affinities arose from increasing on-rates, decreasing off-rates or a combination of the two effects, depending on the mutation. ADAPT is a very focused search of sequence space and required only 20–30 mutants for each system to be made and tested to achieve the affinity enhancements mentioned above. PMID:28750054

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

Bilirubin oxidase-like proteins from Podospora anserina: promising thermostable enzymes for application in transformation of plant biomass.

PubMed

Xie, Ning; Ruprich-Robert, Gwenaël; Silar, Philippe; Chapeland-Leclerc, Florence

2015-03-01

Plant biomass degradation by fungi is a critical step for production of biofuels, and laccases are common ligninolytic enzymes envisioned for ligninolysis. Bilirubin oxidases (BODs)-like are related to laccases, but their roles during lignocellulose degradation have not yet been fully investigated. The two BODs of the ascomycete fungus Podospora anserina were characterized by targeted gene deletions. Enzymatic assay revealed that the bod1(Δ) and bod2(Δ) mutants lost partly a thermostable laccase activity. A triple mutant inactivated for bod1, bod2 and mco, a previously investigated multicopper oxidase gene distantly related to laccases, had no thermostable laccase activity. The pattern of fruiting body production in the bod1(Δ) bod2(Δ) double mutant was changed. The bod1(Δ) and bod2(Δ) mutants were reduced in their ability to grow on ligneous and cellulosic materials. Furthermore, bod1(Δ) and bod2(Δ) mutants were defective towards resistance to phenolic substrates and H2 O2 , which may also impact lignocellulose breakdown. Double and triple mutants were more affected than single mutants, evidencing redundancy of function among BODs and mco. Overall, the data show that bod1, bod2 and mco code for non-canonical thermostable laccases that participate in the degradation of lignocellulose. Thanks to their thermal stability, these enzymes may be more promising candidate for biotechnological application than canonical laccases. © 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.

Poly(ADP-ribose)polymerases are involved in microhomology mediated back-up non-homologous end joining in Arabidopsis thaliana.

PubMed

Jia, Qi; den Dulk-Ras, Amke; Shen, Hexi; Hooykaas, Paul J J; de Pater, Sylvia

2013-07-01

Besides the KU-dependent classical non-homologous end-joining (C-NHEJ) pathway, an alternative NHEJ pathway first identified in mammalian systems, which is often called the back-up NHEJ (B-NHEJ) pathway, was also found in plants. In mammalian systems PARP was found to be one of the essential components in B-NHEJ. Here we investigated whether PARP1 and PARP2 were also involved in B-NHEJ in Arabidopsis. To this end Arabidopsis parp1, parp2 and parp1parp2 (p1p2) mutants were isolated and functionally characterized. The p1p2 double mutant was crossed with the C-NHEJ ku80 mutant resulting in the parp1parp2ku80 (p1p2k80) triple mutant. As expected, because of their role in single strand break repair (SSBR) and base excision repair (BER), the p1p2 and p1p2k80 mutants were shown to be sensitive to treatment with the DNA damaging agent MMS. End-joining assays in cell-free leaf protein extracts of the different mutants using linear DNA substrates with different ends reflecting a variety of double strand breaks were performed. The results showed that compatible 5'-overhangs were accurately joined in all mutants, that KU80 protected the ends preventing the formation of large deletions and that PARP proteins were involved in microhomology mediated end joining (MMEJ), one of the characteristics of B-NHEJ.

Galactose transport in Kluyveromyces lactis: major role of the glucose permease Hgt1.

PubMed

Baruffini, Enrico; Goffrini, Paola; Donnini, Claudia; Lodi, Tiziana

2006-12-01

In Kluyveromyces lactis, galactose transport has been thought to be mediated by the lactose permease encoded by LAC12. In fact, a lac12 mutant unable to grow on lactose did not grow on galactose either and showed low and uninducible galactose uptake activity. The existence of other galactose transport systems, at low and at high affinity, had, however, been hypothesized on the basis of galactose uptake kinetics studies. Here we confirmed the existence of a second galactose transporter and we isolated its structural gene. It turned out to be HGT1, previously identified as encoding the high-affinity glucose carrier. Analysis of galactose transporter mutants, hgt1 and lac12, and the double mutant hgt1lac12, suggested that Hgt1 was the high-affinity and Lac12 was the low-affinity galactose transporter. HGT1 expression was strongly induced by galactose and insensitive to glucose repression. This could explain the rapid adaptation to galactose observed in K. lactis after a shift from glucose to galactose medium.

A CRISPR Cas9-based gene drive platform for genetic interaction analysis in Candida albicans

PubMed Central

Shapiro, Rebecca S.; Chavez, Alejandro; Porter, Caroline B. M.; Hamblin, Meagan; Kaas, Christian S.; DiCarlo, James E.; Zeng, Guisheng; Xu, Xiaoli; Revtovich, Alexey V.; Kirienko, Natalia V.; Wang, Yue; Church, George M.; Collins, James J.

2018-01-01

Candida albicans is the leading cause of fungal infections; yet, complex genetic interaction analysis remains cumbersome in this diploid pathogen. Here, we developed a CRISPR-Cas9-based ‘gene drive array’ (GDA) platform to facilitate efficient genetic analysis in C. albicans. In our system, a modified DNA donor molecule acts as a selfish genetic element, replaces the targeted site, and propagates to replace additional wild-type loci. Using mating-competent C. albicans haploids, each carrying a different gene drive disabling a gene of interest, we are able to create diploid strains that are homozygous double-deletion mutants. We generate double-gene deletion libraries to demonstrate this technology, targeting antifungal efflux and biofilm adhesion factors. We screen these libraries to identify virulence regulators and determine how genetic networks shift under diverse conditions. This platform transforms our ability to perform genetic interaction analysis in C. albicans and is readily extended to other fungal pathogens. PMID:29062088

The role of the bi-compartmental stem cell niche in delaying cancer

NASA Astrophysics Data System (ADS)

Shahriyari, Leili; Komarova, Natalia L.

2015-10-01

In recent years, by using modern imaging techniques, scientists have found evidence of collaboration between different types of stem cells (SCs), and proposed a bi-compartmental organization of the SC niche. Here we create a class of stochastic models to simulate the dynamics of such a heterogeneous SC niche. We consider two SC groups: the border compartment, S1, is in direct contact with transit-amplifying (TA) cells, and the central compartment, S2, is hierarchically upstream from S1. The S1 SCs differentiate or divide asymmetrically when the tissue needs TA cells. Both groups proliferate when the tissue requires SCs (thus maintaining homeostasis). There is an influx of S2 cells into the border compartment, either by migration, or by proliferation. We examine this model in the context of double-hit mutant generation, which is a rate-limiting step in the development of many cancers. We discover that this type of a cooperative pattern in the stem niche with two compartments leads to a significantly smaller rate of double-hit mutant production compared with a homogeneous, one-compartmental SC niche. Furthermore, the minimum probability of double-hit mutant generation corresponds to purely symmetric division of SCs, consistent with the literature. Finally, the optimal architecture (which minimizes the rate of double-hit mutant production) requires a large proliferation rate of S1 cells along with a small, but non-zero, proliferation rate of S2 cells. This result is remarkably similar to the niche structure described recently by several authors, where one of the two SC compartments was found more actively engaged in tissue homeostasis and turnover, while the other was characterized by higher levels of quiescence (but contributed strongly to injury recovery). Both numerical and analytical results are presented.

Branching patterns in leaf starches from Arabidopsis mutants deficient in diverse starch synthases.

PubMed

Zhu, Fan; Bertoft, Eric; Szydlowski, Nicolas; d'Hulst, Christophe; Seetharaman, Koushik

2015-01-12

This is the first report on the cluster structure of transitory starch from Arabidopsis leaves. In addition to wild type, the molecular structures of leaf starch from mutants deficient in starch synthases (SS) including single enzyme mutants ss1-, ss2-, or ss3-, and also double mutants ss1-ss2- and ss1-ss3- were characterized. The mutations resulted in increased amylose content. Clusters from whole starch were isolated by partial hydrolysis using α-amylase of Bacillus amyloliquefaciens. The clusters were then further hydrolyzed with concentrated α-amylase of B. amyloliquefaciens to produce building blocks (α-limit dextrins). Structures of the clusters and their building blocks were characterized by chromatography of samples before and after debranching treatment. While the mutations increased the size of clusters, the reasons were different as reflected by the composition of their unit chains and building blocks. In general, all mutants contained more of a-chains that preferentially increased the number of small building blocks with only two chains. The clusters of the double mutant ss1-ss3- were very large and possessed also more of large building blocks with four or more chains. The results from transitory starch are compared with those from agriculturally important crops in the context that to what extent the Arabidopsis can be a true biotechnological reflection for starch modifications through genetic means. Copyright © 2014 Elsevier Ltd. All rights reserved.

Generation and characterization of Kctd15 mutations in zebrafish

PubMed Central

Heffer, Alison; Marquart, Gregory D.; Aquilina-Beck, Allisan; Saleem, Nabil; Burgess, Harold A.

2017-01-01

Potassium channel tetramerization domain containing 15 (Kctd15) was previously found to have a role in early neural crest (NC) patterning, specifically delimiting the region where NC markers are expressed via repression of transcription factor AP-2a and inhibition of Wnt signaling. We used transcription activator-like effector nucleases (TALENs) to generate null mutations in zebrafish kctd15a and kctd15b paralogs to study the in vivo role of Kctd15. We found that while deletions producing frame-shift mutations in each paralog showed no apparent phenotype, kctd15a/b double mutant zebrafish are smaller in size and show several phenotypes including some affecting the NC, such as expansion of the early NC domain, increased pigmentation, and craniofacial defects. Both melanophore and xanthophore pigment cell numbers and early markers are up-regulated in the double mutants. While we find no embryonic craniofacial defects, adult mutants have a deformed maxillary segment and missing barbels. By confocal imaging of mutant larval brains we found that the torus lateralis (TLa), a region implicated in gustatory networks in other fish, is absent. Ablation of this brain tissue in wild type larvae mimics some aspects of the mutant growth phenotype. Thus kctd15 mutants show deficits in the development of both neural crest derivatives, and specific regions within the central nervous system, leading to a strong reduction in normal growth rates. PMID:29216270

Computational infrared and two-dimensional infrared photon echo spectroscopy of both wild-type and double mutant myoglobin-CO proteins.

PubMed

Choi, Jun-Ho; Kwak, Kyung-Won; Cho, Minhaeng

2013-12-12

The CO stretching mode of both wild-type and double mutant ( T67R / S92D ) MbCO (carbonmonoxymyoglobin) proteins is an ideal infrared (IR) probe for studying the local electrostatic environment inside the myoglobin heme pocket. Recently, to elucidate the conformational switching dynamics between two distinguishable states, extensive IR absorption, IR pump-probe, and two-dimensional (2D) IR spectroscopic studies for various mutant MbCO's have been performed by the Fayer group. They showed that the 2D IR spectroscopy of the double mutant, which has a peroxidase enzyme activity, reveals a rapid chemical exchange between two distinct states, whereas that of the wild-type does not. Despite the fact that a few simulation studies on these systems were already performed and reported, such complicated experimental results have not been fully reproduced nor described in terms of conformational state-to-state transition processes. Here, we first develop a distributed vibrational solvatochromic charge model for describing the CO stretch frequency shift reflecting local electric potential changes. Then, by carrying out molecular dynamic simulations of the two MbCO's and examining their CO frequency trajectories, it becomes possible to identify a proper reaction coordinate consisting of His64 imidazole ring rotation and its distance to the CO ligand. From the 2D surfaces of the resulting potential of mean forces, the spectroscopically distinguished A1 and A3 states of the wild-type as well as two more substates of the double mutant are identified and their vibrational frequencies and distributions are separately examined. Our simulated IR absorption and 2D IR spectra of the two MbCO's are directly compared with the previous experimental results reported by the Fayer group. The chemical exchange rate constants extracted from the two-state kinetic analyses of the simulated 2D IR spectra are in excellent agreement with the experimental values. On the basis of the quantitative agreement between the simulated spectra and experimental ones, we further examine the conformational differences in the heme pockets of the two proteins and show that the double mutation, T67R / S92D , suppresses the A1 population, restricts the imidazole ring rotation, and increases hydrogen-bond strength between the imidazole Nε-H and the oxygen atom of the CO ligand. It is believed that such delicate change of distal His64 imidazole ring dynamics induced by the double mutation may be responsible for its enhanced peroxidase catalytic activity as compared to the wild-type myoglobin.

The RNA-Editing Enzyme ADAR1 Controls Innate Immune Responses to RNA

PubMed Central

Mannion, Niamh M.; Greenwood, Sam M.; Young, Robert; Cox, Sarah; Brindle, James; Read, David; Nellåker, Christoffer; Vesely, Cornelia; Ponting, Chris P.; McLaughlin, Paul J.; Jantsch, Michael F.; Dorin, Julia; Adams, Ian R.; Scadden, A.D.J.; Öhman, Marie; Keegan, Liam P.; O’Connell, Mary A.

2014-01-01

Summary The ADAR RNA-editing enzymes deaminate adenosine bases to inosines in cellular RNAs. Aberrant interferon expression occurs in patients in whom ADAR1 mutations cause Aicardi-Goutières syndrome (AGS) or dystonia arising from striatal neurodegeneration. Adar1 mutant mouse embryos show aberrant interferon induction and die by embryonic day E12.5. We demonstrate that Adar1 embryonic lethality is rescued to live birth in Adar1; Mavs double mutants in which the antiviral interferon induction response to cytoplasmic double-stranded RNA (dsRNA) is prevented. Aberrant immune responses in Adar1 mutant mouse embryo fibroblasts are dramatically reduced by restoring the expression of editing-active cytoplasmic ADARs. We propose that inosine in cellular RNA inhibits antiviral inflammatory and interferon responses by altering RLR interactions. Transfecting dsRNA oligonucleotides containing inosine-uracil base pairs into Adar1 mutant mouse embryo fibroblasts reduces the aberrant innate immune response. ADAR1 mutations causing AGS affect the activity of the interferon-inducible cytoplasmic isoform more severely than the nuclear isoform. PMID:25456137

The RNA-editing enzyme ADAR1 controls innate immune responses to RNA.

PubMed

Mannion, Niamh M; Greenwood, Sam M; Young, Robert; Cox, Sarah; Brindle, James; Read, David; Nellåker, Christoffer; Vesely, Cornelia; Ponting, Chris P; McLaughlin, Paul J; Jantsch, Michael F; Dorin, Julia; Adams, Ian R; Scadden, A D J; Ohman, Marie; Keegan, Liam P; O'Connell, Mary A

2014-11-20

The ADAR RNA-editing enzymes deaminate adenosine bases to inosines in cellular RNAs. Aberrant interferon expression occurs in patients in whom ADAR1 mutations cause Aicardi-Goutières syndrome (AGS) or dystonia arising from striatal neurodegeneration. Adar1 mutant mouse embryos show aberrant interferon induction and die by embryonic day E12.5. We demonstrate that Adar1 embryonic lethality is rescued to live birth in Adar1; Mavs double mutants in which the antiviral interferon induction response to cytoplasmic double-stranded RNA (dsRNA) is prevented. Aberrant immune responses in Adar1 mutant mouse embryo fibroblasts are dramatically reduced by restoring the expression of editing-active cytoplasmic ADARs. We propose that inosine in cellular RNA inhibits antiviral inflammatory and interferon responses by altering RLR interactions. Transfecting dsRNA oligonucleotides containing inosine-uracil base pairs into Adar1 mutant mouse embryo fibroblasts reduces the aberrant innate immune response. ADAR1 mutations causing AGS affect the activity of the interferon-inducible cytoplasmic isoform more severely than the nuclear isoform. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Combined action of the major secreted exo- and endopolygalacturonases is required for full virulence of Fusarium oxysporum.

PubMed

Bravo Ruiz, Gustavo; Di Pietro, Antonio; Roncero, M Isabel G

2016-04-01

The genome of the tomato pathogen Fusarium oxysporum f. sp. lycopersici encodes eight different polygalacturonases (PGs): four endoPGs and four exoPGs. Quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) revealed that endoPGs pg1 and pg5 and exoPGs pgx4 and pgx6 are expressed at significant levels during growth on citrus pectin, polygalacturonic acid or the monomer galacturonic acid, as well as during the infection of tomato plants. The remaining PG genes exhibit low expression levels under all the conditions tested. Secreted PG activity was decreased significantly during growth on pectin in the single deletion mutants lacking either pg1 or pgx6, as well as in the double mutant. Although the single deletion mutants did not display a significant virulence reduction on tomato plants, the Δpg1Δpgx6 double mutant was significantly attenuated in virulence. The combined action of exoPGs and endoPGs is thus essential for plant infection by the vascular wilt fungus F. oxysporum. © 2015 BSPP and John Wiley & Sons Ltd.

Motor coordination in mice with hotfoot, Lurcher, and double mutations of the Grid2 gene encoding the delta-2 excitatory amino acid receptor.

PubMed

Lalonde, R; Hayzoun, K; Selimi, F; Mariani, J; Strazielle, C

2003-11-01

Grid2(ho/ho) is a loss of function gene mutation resulting in abnormal dendritic arborizations of Purkinje cells. These mutants were compared in a series of motor coordination tests requiring balance and equilibrium to nonataxic controls (Grid2(ho/+)) and to a double mutant (Grid2(ho/Lc)) with an inserted Lc mutation. The performance of Grid2(ho/ho) mutant mice was poorer than that of controls on stationary beam, coat hanger, unsteady platform, and rotorod tests. Grid2(ho/Lc) did not differ from Grid2(Lc/+) mice. However, the insertion of the Lc mutation in Grid2(ho/Lc) potentiated the deficits found in Grid2(ho/ho) in stationary beam, unsteady platform, and rotorod tests. These results indicate a deleterious effect of the Lc mutation on Grid2-deficient mice.

[Construction and stress tolerance of trehalase mutant in Saccharomyces cerevisiae].

PubMed

Lv, Ye; Xiao, Dongguang; He, Dongqin; Guo, Xuewu

2008-10-01

Accumulation of trehalose is critical in improving the stress tolerance of Saccharomyces cerevisiae. Two enzymes are capable of hydrolyzing trehalose: a neutral trehalase (NTH1) and an acidic trehalase (ATH1). We constructed trehalase disruption mutants to provide a basis for future commercial application. To retain the accumulation of trehalose in yeast cell, we constructed diploid homozygous neutral trehalase mutants (Deltanth1), acid trehalase mutants (Deltaath1) and double mutants (Deltaath1Deltanth1) by using gene disruption. We tested mutants'trehalose content and their tolerance to freezing, heat, high-sugar and ethanol concentrations. These trehalase disruption mutants were further confirmed by PCR amplification and southern blot. All mutant strains accumulated higher levels of cellular trehalose and grew to a higher cell density than the isogenic parent strain. In addition, the levels of trehalose in these mutants correlated with increased tolerance to freezing, heat, high-sugar and ethanol concentration. The improved tolerance of trehalase mutants may make them useful in commercial applications, including baking and brewing protein.

Isolation and preliminary characterization of temperature-sensitive mutants of influenza virus.

PubMed

Sugiura, A; Tobita, K; Kilbourne, E D

1972-10-01

Isolation of temperature-sensitive (ts) mutants was attempted from the WSN strain of influenza A virus which was grown and assayed in MDBK cells. After growth of wild-type virus in the presence of 5-fluorouracil, 15 ts mutants were selected for which the ratio of plaquing efficiency at 39.5 C to that at 33 C was 10(-3) or less. In pairwise crosses of ts mutants, recombination and complementation were either very efficient or undetectable. It is suggested, therefore, that the viral genome consists of physically discrete units and recombination occurs as an exchange of these units. All 15 mutants have been assigned with certainty into five recombination groups. Three mutants are suspected to be double mutants. Any two complementing mutants always recombined with each other, and noncomplementing mutants did not recombine. In physiological tests, mutants showed diverse patterns of functional defects at the nonpermissive temperature. However, it was not always possible to correlate these physiological defects with the results of genetic characterization.

A link among DNA replication, recombination, and gene expression revealed by genetic and genomic analysis of TEBICHI gene of Arabidopsis thaliana.

PubMed

Inagaki, Soichi; Nakamura, Kenzo; Morikami, Atsushi

2009-08-01

Spatio-temporal regulation of gene expression during development depends on many factors. Mutations in Arabidopsis thaliana TEBICHI (TEB) gene encoding putative helicase and DNA polymerase domains-containing protein result in defects in meristem maintenance and correct organ formation, as well as constitutive DNA damage response and a defect in cell cycle progression; but the molecular link between these phenotypes of teb mutants is unknown. Here, we show that mutations in the DNA replication checkpoint pathway gene, ATR, but not in ATM gene, enhance developmental phenotypes of teb mutants, although atr suppresses cell cycle defect of teb mutants. Developmental phenotypes of teb mutants are also enhanced by mutations in RAD51D and XRCC2 gene, which are involved in homologous recombination. teb and teb atr double mutants exhibit defects in adaxial-abaxial polarity of leaves, which is caused in part by the upregulation of ETTIN (ETT)/AUXIN RESPONSIVE FACTOR 3 (ARF3) and ARF4 genes. The Helitron transposon in the upstream of ETT/ARF3 gene is likely to be involved in the upregulation of ETT/ARF3 in teb. Microarray analysis indicated that teb and teb atr causes preferential upregulation of genes nearby the Helitron transposons. Furthermore, interestingly, duplicated genes, especially tandemly arrayed homologous genes, are highly upregulated in teb or teb atr. We conclude that TEB is required for normal progression of DNA replication and for correct expression of genes during development. Interplay between these two functions and possible mechanism leading to altered expression of specific genes will be discussed.

Biological activity of a genetically modified BMP-2 variant with inhibitory activity

PubMed Central

Klammert, Uwe; Nickel, Joachim; Würzler, Kristian; Klingelhöffer, Christoph; Sebald, Walter; Kübler, Alexander C; Reuther, Tobias

2009-01-01

Background Alterations of the binding epitopes of bone morphogenetic protein-2 (BMP-2) lead to a modified interaction with the ectodomains of BMP receptors. In the present study the biological effect of a BMP-2 double mutant with antagonistic activity was evaluated in vivo. Methods Equine-derived collagenous carriers were loaded with recombinant human BMP-2 (rhBMP-2) in a well-known dose to provide an osteoinductive stimulus. The study was performed in a split animal design: carriers only coupled with rhBMP-2 (control) were implanted into prepared cavities of lower limb muscle of rats, specimens coupled with rhBMP-2 as well as BMP-2 double mutant were placed into the opposite limb in the same way. After 28 days the carriers were explanted, measured radiographically and characterized histologically. Results As expected, the BMP-2 loaded implants showed a typical heterotopic bone formation. The specimens coupled with both proteins showed a significant decreased bone formation in a dose dependent manner. Conclusion The antagonistic effect of a specific BMP-2 double mutant could be demonstrated in vivo. The dose dependent influence on heterotopic bone formation by preventing rhBMP-2 induced osteoinduction suggests a competitive receptor antagonism. PMID:19187528

Binding affinity and adhesion force of organophosphate hydrolase enzyme with soil particles related to the isoelectric point of the enzyme.

PubMed

Islam, Shah Md Asraful; Yeasmin, Shabina; Islam, Md Saiful; Islam, Md Shariful

2017-07-01

The binding affinity of organophosphate hydrolase enzyme (OphB) with soil particles in relation to the isoelectric point (pI) was studied. Immobilization of OphB with soil particles was observed by confocal microscopy, Fourier transform infrared spectroscopy (FT-IR), and Atomic force microscopy (AFM). The calculated pI of OphB enzyme was increased from 8.69 to 8.89, 9.04 and 9.16 by the single, double and triple mutant of OphB enzyme, respectively through the replacement of negatively charged aspartate with positively charged histidine. Practically, the binding affinity was increased to 5.30%, 11.50%, and 16.80% for single, double and triple mutants, respectively. In contrast, enzyme activity of OphB did not change by the mutation of the enzyme. On the other hand, adhesion forces were gradually increased for wild type OphB enzyme (90 pN) to 96, 100 and 104 pN for single, double and triple mutants of OphB enzyme, respectively. There was an increasing trend of binding affinity and adhesion force by the increase of isoelectric point (pI) of OphB enzyme. Copyright © 2017 Elsevier Inc. All rights reserved.

The characteristics of the (alpha V371C)3(beta R337C)3 gamma double mutant subcomplex of the TF1-ATPase indicate that the catalytic site at the alpha TP-beta TP interface with bound MgADP in crystal structures of MF1 represents a catalytic site containing inhibitory MgADP.

PubMed

Bandyopadhyay, Sanjay; Muneyuki, Eiro; Allison, William S

2005-02-22

In the MF(1) crystal structure with the MgADP-fluoroaluminate complex bound to two catalytic sites [Menz, R. I., Walker, J. E., and Leslie, A. G. W. (2001) Cell 106, 331-341], the guanidinium of betaR(337) is within 2.9 A of the alpha-oxygen of alphaS(370) and 3.7 A of a methyl group of alphaV(371) at the alpha(E)-beta(HC) interface. To examine the functional role of this contact, the (alphaV(371)C)(3)(betaR(337)C)(3)gamma subcomplex of the TF(1)-ATPase was prepared and characterized. Steady state ATPase activity of the reduced double-mutant is 30% of that of the wild type. Inactivation of the double mutant containing empty catalytic sites or MgADP bound to one catalytic site with CuCl(2) cross-linked two alpha-beta pairs, whereas a single alpha-beta pair cross-linked when at least two catalytic sites contained MgADP. The reduced double mutant hydrolyzed substoichiometric ATP 100-fold more rapidly than the enzyme containing two cross-linked alpha-beta pairs. Addition of AlCl(3) and NaF to the reduced double mutant after incubation with stoichiometric MgADP or 200 microM MgADP irreversibly inactivated the steady state ATPase activity with rate constants of 1.5 x10(-2) and 4.1 x 10(-2) min(-1), respectively. In contrast, addition of AlCl(3) and NaF to the cross-linked enzyme after incubation with stoichiometric or 200 microM MgADP irreversibly inactivated ATPase activity with a common rate constant of approximately 10(-4) min(-1). Correlation of these results with crystal structures of MF(1) suggests that the catalytic site at the alpha(TP)-beta(TP) interface is loaded first upon addition of nucleotides to nucleotide-depleted F(1)-ATPases and that the catalytic site at the alpha(TP)-beta(TP) interface with bound MgADP in crystal structures represents a catalytic site containing inhibitory MgADP.

Arabidopsis thaliana VOZ (Vascular plant One-Zinc finger) transcription factors are required for proper regulation of flowering time

PubMed Central

Celesnik, Helena; Ali, Gul S.; Robison, Faith M.; Reddy, Anireddy S. N.

2013-01-01

Summary Transition to flowering in plants is tightly controlled by environmental cues, which regulate the photoperiod and vernalization pathways, and endogenous signals, which mediate the autonomous and gibberellin pathways. In this work, we investigated the role of two Zn2+-finger transcription factors, the paralogues AtVOZ1 and AtVOZ2, in Arabidopsis thaliana flowering. Single atvoz1-1 and atvoz2-1 mutants showed no significant phenotypes as compared to wild type. However, atvoz1-1 atvoz2-1 double mutant plants exhibited several phenotypes characteristic of flowering-time mutants. The double mutant displayed a severe delay in flowering, together with additional pleiotropic phenotypes. Late flowering correlated with elevated expression of FLOWERING LOCUS C (FLC), which encodes a potent floral repressor, and decreased expression of its target, the floral promoter FD. Vernalization rescued delayed flowering of atvoz1-1 atvoz2-1 and reversed elevated FLC levels. Accumulation of FLC transcripts in atvoz1-1 atvoz2-1 correlated with increased expression of several FLC activators, including components of the PAF1 and SWR1 chromatin-modifying complexes. Additionally, AtVOZs were shown to bind the promoter of MOS3/SAR3 and directly regulate expression of this nuclear pore protein, which is known to participate in the regulation of flowering time, suggesting that AtVOZs exert at least some of their flowering regulation by influencing the nuclear pore function. Complementation of atvoz1-1 atvoz2-1 with AtVOZ2 reversed all double mutant phenotypes, confirming that the observed morphological and molecular changes arise from the absence of functional AtVOZ proteins, and validating the functional redundancy between AtVOZ1 and AtVOZ2. PMID:23616927

2-cysteine peroxiredoxins and thylakoid ascorbate peroxidase create a water-water cycle that is essential to protect the photosynthetic apparatus under high light stress conditions.

PubMed

Awad, Jasmin; Stotz, Henrik U; Fekete, Agnes; Krischke, Markus; Engert, Cornelia; Havaux, Michel; Berger, Susanne; Mueller, Martin J

2015-04-01

Different peroxidases, including 2-cysteine (2-Cys) peroxiredoxins (PRXs) and thylakoid ascorbate peroxidase (tAPX), have been proposed to be involved in the water-water cycle (WWC) and hydrogen peroxide (H2O2)-mediated signaling in plastids. We generated an Arabidopsis (Arabidopsis thaliana) double-mutant line deficient in the two plastid 2-Cys PRXs (2-Cys PRX A and B, 2cpa 2cpb) and a triple mutant deficient in 2-Cys PRXs and tAPX (2cpa 2cpb tapx). In contrast to wild-type and tapx single-knockout plants, 2cpa 2cpb double-knockout plants showed an impairment of photosynthetic efficiency and became photobleached under high light (HL) growth conditions. In addition, double-mutant plants also generated elevated levels of superoxide anion radicals, H2O2, and carbonylated proteins but lacked anthocyanin accumulation under HL stress conditions. Under HL conditions, 2-Cys PRXs seem to be essential in maintaining the WWC, whereas tAPX is dispensable. By comparison, this HL-sensitive phenotype was more severe in 2cpa 2cpb tapx triple-mutant plants, indicating that tAPX partially compensates for the loss of functional 2-Cys PRXs by mutation or inactivation by overoxidation. In response to HL, H2O2- and photooxidative stress-responsive marker genes were found to be dramatically up-regulated in 2cpa 2cpb tapx but not 2cpa 2cpb mutant plants, suggesting that HL-induced plastid to nucleus retrograde photooxidative stress signaling takes place after loss or inactivation of the WWC enzymes 2-Cys PRX A, 2-Cys PRX B, and tAPX. © 2015 American Society of Plant Biologists. All Rights Reserved.

Distinct Roles of the DmNav and DSC1 Channels in the Action of DDT and Pyrethroids

PubMed Central

Rinkevich, Frank D.; Du, Yuzhe; Tolinski, Josh; Ueda, Atsushi; Wu, Chun-Fang; Zhorov, Boris S.; Dong, Ke

2015-01-01

Voltage-gated sodium channels (Nav channels) are critical for electrical signaling in the nervous system and are the primary targets of the insecticides DDT and pyrethroids. In Drosophila melanogaster, besides the canonical Nav channel, Para (also called DmNav), there is a sodium channel-like cation channel called DSC1 (Drosophila sodium channel 1). Temperature-sensitive paralytic mutations in DmNav (parats) confer resistance to DDT and pyrethroids, whereas DSC1 knockout flies exhibit enhanced sensitivity to pyrethroids. To further define the roles and interaction of DmNav and DSC1 channels in DDT and pyrethroid neurotoxicology, we generated a DmNav/DSC1 double mutant line by introducing a parats1 allele (carrying the I265N mutation) into a DSC1 knockout line. We confirmed that the I265N mutation reduced the sensitivity to two pyrethroids, permethrin and deltamethrin of a DmNav variant expressed in Xenopus oocytes. Computer modeling predicts that the I265N mutation confers pyrethroid resistance by allosterically altering the second pyrethroid receptor site on the DmNav channel. Furthermore, we found that I265N-mediated pyrethroid resistance in parats1 mutant flies was almost completely abolished in parats1;DSC1−/− double mutant flies. Unexpectedly, however, the DSC1 knockout flies were less sensitive to DDT, compared to the control flies (w1118A), and the parats1;DSC1−/− double mutant flies were even more resistant to DDT compared to the DSC1 knockout or parats1 mutant. Our findings revealed distinct roles of the DmNav and DSC1 channels in the neurotoxicology of DDT vs. pyrethroids and implicate the exciting possibility of using DSC1 channel blockers or modifiers in the management of pyrethroid resistance. PMID:25687544

Induction of endoplasmic reticulum stress by deletion of Grp78 depletes Apc mutant intestinal epithelial stem cells.

PubMed

van Lidth de Jeude, J F; Meijer, B J; Wielenga, M C B; Spaan, C N; Baan, B; Rosekrans, S L; Meisner, S; Shen, Y H; Lee, A S; Paton, J C; Paton, A W; Muncan, V; van den Brink, G R; Heijmans, J

2017-06-15

Intestinal epithelial stem cells are highly sensitive to differentiation induced by endoplasmic reticulum (ER) stress. Colorectal cancer develops from mutated intestinal epithelial stem cells. The most frequent initiating mutation occurs in Apc, which results in hyperactivated Wnt signalling. This causes hyperproliferation and reduced sensitivity to chemotherapy, but whether these mutated stem cells are sensitive to ER stress induced differentiation remains unknown. Here we examined this by generating mice in which both Apc and ER stress repressor chaperone Grp78 can be conditionally deleted from the intestinal epithelium. For molecular studies, we used intestinal organoids derived from these mice. Homozygous loss of Apc alone resulted in crypt elongation, activation of the Wnt signature and accumulation of intestinal epithelial stem cells, as expected. This phenotype was however completely rescued on activation of ER stress by additional deletion of Grp78. In these Apc-Grp78 double mutant animals, stem cells were rapidly lost and repopulation occurred by non-mutant cells that had escaped recombination, suggesting that Apc-Grp78 double mutant stem cells had lost self-renewal capacity. Although in Apc-Grp78 double mutant mice the Wnt signature was lost, these intestines exhibited ubiquitous epithelial presence of nuclear β-catenin. This suggests that ER stress interferes with Wnt signalling downstream of nuclear β-catenin. In conclusion, our findings indicate that ER stress signalling results in loss of Apc mutated intestinal epithelial stem cells by interference with the Wnt signature. In contrast to many known inhibitors of Wnt signalling, ER stress acts downstream of β-catenin. Therefore, ER stress poses a promising target in colorectal cancers, which develop as a result of Wnt activating mutations.

BMP-Mediated Functional Cooperation between Dlx5;Dlx6 and Msx1;Msx2 during Mammalian Limb Development

PubMed Central

Vieux-Rochas, Maxence; Bouhali, Kamal; Mantero, Stefano; Garaffo, Giulia; Provero, Paolo; Astigiano, Simonetta; Barbieri, Ottavia; Caratozzolo, Mariano F.; Tullo, Apollonia; Guerrini, Luisa; Lallemand, Yvan; Robert, Benoît

2013-01-01

The Dlx and Msx homeodomain transcription factors play important roles in the control of limb development. The combined disruption of Msx1 and Msx2, as well as that of Dlx5 and Dlx6, lead to limb patterning defects with anomalies in digit number and shape. Msx1;Msx2 double mutants are characterized by the loss of derivatives of the anterior limb mesoderm which is not observed in either of the simple mutants. Dlx5;Dlx6 double mutants exhibit hindlimb ectrodactyly. While the morphogenetic action of Msx genes seems to involve the BMP molecules, the mode of action of Dlx genes still remains elusive. Here, examining the limb phenotypes of combined Dlx and Msx mutants we reveal a new Dlx-Msx regulatory loop directly involving BMPs. In Msx1;Dlx5;Dlx6 triple mutant mice (TKO), beside the expected ectrodactyly, we also observe the hallmark morphological anomalies of Msx1;Msx2 double mutants suggesting an epistatic role of Dlx5 and Dlx6 over Msx2. In Msx2;Dlx5;Dlx6 TKO mice we only observe an aggravation of the ectrodactyly defect without changes in the number of the individual components of the limb. Using a combination of qPCR, ChIP and bioinformatic analyses, we identify two Dlx/Msx regulatory pathways: 1) in the anterior limb mesoderm a non-cell autonomous Msx-Dlx regulatory loop involves BMP molecules through the AER and 2) in AER cells and, at later stages, in the limb mesoderm the regulation of Msx2 by Dlx5 and Dlx6 occurs also cell autonomously. These data bring new elements to decipher the complex AER-mesoderm dialogue that takes place during limb development and provide clues to understanding the etiology of congenital limb malformations. PMID:23382810

BMP-mediated functional cooperation between Dlx5;Dlx6 and Msx1;Msx2 during mammalian limb development.

PubMed

Vieux-Rochas, Maxence; Bouhali, Kamal; Mantero, Stefano; Garaffo, Giulia; Provero, Paolo; Astigiano, Simonetta; Barbieri, Ottavia; Caratozzolo, Mariano F; Tullo, Apollonia; Guerrini, Luisa; Lallemand, Yvan; Robert, Benoît; Levi, Giovanni; Merlo, Giorgio R

2013-01-01

The Dlx and Msx homeodomain transcription factors play important roles in the control of limb development. The combined disruption of Msx1 and Msx2, as well as that of Dlx5 and Dlx6, lead to limb patterning defects with anomalies in digit number and shape. Msx1;Msx2 double mutants are characterized by the loss of derivatives of the anterior limb mesoderm which is not observed in either of the simple mutants. Dlx5;Dlx6 double mutants exhibit hindlimb ectrodactyly. While the morphogenetic action of Msx genes seems to involve the BMP molecules, the mode of action of Dlx genes still remains elusive. Here, examining the limb phenotypes of combined Dlx and Msx mutants we reveal a new Dlx-Msx regulatory loop directly involving BMPs. In Msx1;Dlx5;Dlx6 triple mutant mice (TKO), beside the expected ectrodactyly, we also observe the hallmark morphological anomalies of Msx1;Msx2 double mutants suggesting an epistatic role of Dlx5 and Dlx6 over Msx2. In Msx2;Dlx5;Dlx6 TKO mice we only observe an aggravation of the ectrodactyly defect without changes in the number of the individual components of the limb. Using a combination of qPCR, ChIP and bioinformatic analyses, we identify two Dlx/Msx regulatory pathways: 1) in the anterior limb mesoderm a non-cell autonomous Msx-Dlx regulatory loop involves BMP molecules through the AER and 2) in AER cells and, at later stages, in the limb mesoderm the regulation of Msx2 by Dlx5 and Dlx6 occurs also cell autonomously. These data bring new elements to decipher the complex AER-mesoderm dialogue that takes place during limb development and provide clues to understanding the etiology of congenital limb malformations.

The Campylobacter jejuni MarR-like transcriptional regulators RrpA and RrpB both influence bacterial responses to oxidative and aerobic stresses.

PubMed

Gundogdu, Ozan; da Silva, Daiani T; Mohammad, Banaz; Elmi, Abdi; Mills, Dominic C; Wren, Brendan W; Dorrell, Nick

2015-01-01

The ability of the human intestinal pathogen Campylobacter jejuni to respond to oxidative stress is central to bacterial survival both in vivo during infection and in the environment. Re-annotation of the C. jejuni NCTC11168 genome revealed the presence of two MarR-type transcriptional regulators Cj1546 and Cj1556, originally annotated as hypothetical proteins, which we have designated RrpA and RrpB (regulator of response to peroxide) respectively. Previously we demonstrated a role for RrpB in both oxidative and aerobic (O2) stress and that RrpB was a DNA binding protein with auto-regulatory activity, typical of MarR-type transcriptional regulators. In this study, we show that RrpA is also a DNA binding protein and that a rrpA mutant in strain 11168H exhibits increased sensitivity to hydrogen peroxide oxidative stress. Mutation of either rrpA or rrpB reduces catalase (KatA) expression. However, a rrpAB double mutant exhibits higher levels of resistance to hydrogen peroxide oxidative stress, with levels of KatA expression similar to the wild-type strain. Mutation of either rrpA or rrpB also results in a reduction in the level of katA expression, but this reduction was not observed in the rrpAB double mutant. Neither the rrpA nor rrpB mutant exhibits any significant difference in sensitivity to either cumene hydroperoxide or menadione oxidative stresses, but both mutants exhibit a reduced ability to survive aerobic (O2) stress, enhanced biofilm formation and reduced virulence in the Galleria mellonella infection model. The rrpAB double mutant exhibits wild-type levels of biofilm formation and wild-type levels of virulence in the G mellonella infection model. Together these data indicate a role for both RrpA and RrpB in the C. jejuni peroxide oxidative and aerobic (O2) stress responses, enhancing bacterial survival in vivo and in the environment.

Leptotene/Zygotene Chromosome Movement Via the SUN/KASH Protein Bridge in Caenorhabditis elegans

PubMed Central

Baudrimont, Antoine; Penkner, Alexandra; Woglar, Alexander; Machacek, Thomas; Wegrostek, Christina; Gloggnitzer, Jiradet; Fridkin, Alexandra; Klein, Franz; Gruenbaum, Yosef; Pasierbek, Pawel; Jantsch, Verena

2010-01-01

The Caenorhabditis elegans inner nuclear envelope protein matefin/SUN-1 plays a conserved, pivotal role in the process of genome haploidization. CHK-2–dependent phosphorylation of SUN-1 regulates homologous chromosome pairing and interhomolog recombination in Caenorhabditis elegans. Using time-lapse microscopy, we characterized the movement of matefin/SUN-1::GFP aggregates (the equivalent of chromosomal attachment plaques) and showed that the dynamics of matefin/SUN-1 aggregates remained unchanged throughout leptonene/zygotene, despite the progression of pairing. Movement of SUN-1 aggregates correlated with chromatin polarization. We also analyzed the requirements for the formation of movement-competent matefin/SUN-1 aggregates in the context of chromosome structure and found that chromosome axes were required to produce wild-type numbers of attachment plaques. Abrogation of synapsis led to a deceleration of SUN-1 aggregate movement. Analysis of matefin/SUN-1 in a double-strand break deficient mutant revealed that repair intermediates influenced matefin/SUN-1 aggregate dynamics. Investigation of movement in meiotic regulator mutants substantiated that proper orchestration of the meiotic program and effective repair of DNA double-strand breaks were necessary for the wild-type behavior of matefin/SUN-1 aggregates. PMID:21124819

Roles for the Rad27 Flap Endonuclease in Mitochondrial Mutagenesis and Double-Strand Break Repair in Saccharomyces cerevisiae.

PubMed

Nagarajan, Prabha; Prevost, Christopher T; Stein, Alexis; Kasimer, Rachel; Kalifa, Lidza; Sia, Elaine A

2017-06-01

The structure-specific nuclease, Rad27p/FEN1, plays a crucial role in DNA repair and replication mechanisms in the nucleus. Genetic assays using the rad27-∆ mutant have shown altered rates of DNA recombination, microsatellite instability, and point mutation in mitochondria. In this study, we examined the role of Rad27p in mitochondrial mutagenesis and double-strand break (DSB) repair in Saccharomyces cerevisiae Our findings show that Rad27p is essential for efficient mitochondrial DSB repair by a pathway that generates deletions at a region flanked by direct repeat sequences. Mutant analysis suggests that both exonuclease and endonuclease activities of Rad27p are required for its role in mitochondrial DSB repair. In addition, we found that the nuclease activities of Rad27p are required for the prevention of mitochondrial DNA (mtDNA) point mutations, and in the generation of spontaneous mtDNA rearrangements. Overall, our findings underscore the importance of Rad27p in the maintenance of mtDNA, and demonstrate that it participates in multiple DNA repair pathways in mitochondria, unlinked to nuclear phenotypes. Copyright © 2017 by the Genetics Society of America.

Spontaneous Chloroplast Mutants Mostly Occur by Replication Slippage and Show a Biased Pattern in the Plastome of Oenothera[OPEN

PubMed Central

Massouh, Amid; Schubert, Julia; Yaneva-Roder, Liliya; Ulbricht-Jones, Elena S.; Johnson, Marc T.J.; Wright, Stephen I.; Pellizzer, Tommaso; Sobanski, Johanna; Greiner, Stephan

2016-01-01

Spontaneous plastome mutants have been used as a research tool since the beginning of genetics. However, technical restrictions have severely limited their contributions to research in physiology and molecular biology. Here, we used full plastome sequencing to systematically characterize a collection of 51 spontaneous chloroplast mutants in Oenothera (evening primrose). Most mutants carry only a single mutation. Unexpectedly, the vast majority of mutations do not represent single nucleotide polymorphisms but are insertions/deletions originating from DNA replication slippage events. Only very few mutations appear to be caused by imprecise double-strand break repair, nucleotide misincorporation during replication, or incorrect nucleotide excision repair following oxidative damage. U-turn inversions were not detected. Replication slippage is induced at repetitive sequences that can be very small and tend to have high A/T content. Interestingly, the mutations are not distributed randomly in the genome. The underrepresentation of mutations caused by faulty double-strand break repair might explain the high structural conservation of seed plant plastomes throughout evolution. In addition to providing a fully characterized mutant collection for future research on plastid genetics, gene expression, and photosynthesis, our work identified the spectrum of spontaneous mutations in plastids and reveals that this spectrum is very different from that in the nucleus. PMID:27053421

Immunization by intrabronchial administration to 1-week-old foals of an unmarked double gene disruption strain of Rhodococcus equi strain 103+.

PubMed

Pei, Yanlong; Nicholson, Vivian; Woods, Katharine; Prescott, John F

2007-11-15

Rhodococcus equi causes fatal granulomatous pneumonia in foals and immunocompromised animals and humans. However, there is no effective vaccine against this infection. In this study, the chromosomal genes isocitrate lyase (icl) and cholesterol oxidase (choE) were chosen as targets for mutation and assessment of the double mutant as an intrabronchial vaccine in 1-week-old foals. Using a modification of a suicide plasmid previously developed in this laboratory, we developed a choE-icl unmarked deletion mutant of R. equi strain 103+. Five 1-week-old foals were infected intrabronchially with the mutant and challenged intrabronchially with the parent, virulent, strain 2 weeks later. Three of the foals were protected against pneumonia caused by the virulent strain, but the other two foals developed pneumonia caused by the mutant strain during the post-challenge period. Since infection of 3-week-old foals by an icl mutant in an earlier study had shown complete attenuation of the strain, we conclude that a proportion of foals in the 1st week or so of life are predisposed to developing R. equi pneumonia because of an inability to mount an effective immune response. This has been suspected previously but this is the first time that this has been demonstrated experimentally.

The parkin Mutant Phenotype in the Fly Is Largely Rescued by Metal-Responsive Transcription Factor (MTF-1) ▿ †

PubMed Central

Saini, Nidhi; Georgiev, Oleg; Schaffner, Walter

2011-01-01

The gene for Parkin, an E3 ubiquitin ligase, is mutated in some familial forms of Parkinson's disease, a severe neurodegenerative disorder. A homozygous mutant of the Drosophila ortholog of human parkin is viable but results in severe motoric impairment including an inability to fly, female and male sterility, and a decreased life span. We show here that a double mutant of the genes for Parkin and the metal-responsive transcription factor 1 (MTF-1) is not viable. MTF-1, which is conserved from insects to mammals, is a key regulator of heavy metal homeostasis and detoxification and plays additional roles in other stress conditions, notably oxidative stress. In contrast to the synthetic lethality of the double mutant, elevated expression of MTF-1 dramatically ameliorates the parkin mutant phenotype, as evidenced by a prolonged life span, motoric improvement including short flight episodes, and female fertility. At the cellular level, muscle and mitochondrial structures are substantially improved. A beneficial effect is also seen with a transgene encoding human MTF-1. We propose that Parkin and MTF-1 provide complementary functions in metal homeostasis, oxidative stress and other cellular stress responses. Our findings also raise the possibility that MTF-1 gene polymorphisms in humans could affect the severity of Parkinson's disease. PMID:21383066

Improving the activity of the subtilisin nattokinase by site-directed mutagenesis and molecular dynamics simulation.

PubMed

Weng, Meizhi; Deng, Xiongwei; Bao, Wei; Zhu, Li; Wu, Jieyuan; Cai, Yongjun; Jia, Yan; Zheng, Zhongliang; Zou, Guolin

2015-09-25

Nattokinase (NK), a bacterial serine protease from Bacillus subtilis var. natto, is a potential cardiovascular drug exhibiting strong fibrinolytic activity. To broaden its commercial and medical applications, we constructed a single-mutant (I31L) and two double-mutants (M222A/I31L and T220S/I31L) by site-directed mutagenesis. Active enzymes were expressed in Escherichia coli with periplasmic secretion and were purified to homogeneity. The kinetic parameters of enzymes were examined by spectroscopy assay and isothermal titration calorimetry (ITC), and their fibrinolytic activities were determined by fibrin plate method. The substitution of Leu(31) for Ile(31) resulted in about 2-fold enhancement of catalytic efficiency (Kcat/KM) compared with wild-type NK. The specific activities of both double-mutants (M222A/I31L and T220S/I31L) were significantly increased when compared with the single-mutants (M222A and T220S) and the oxidative stability of M222A/I31L mutant was enhanced with respect to wild-type NK. This study demonstrates the feasibility of improving activity of NK by site-directed mutagenesis and shows successful protein engineering cases to improve the activity of NK as a potent therapeutic agent. Copyright © 2015 Elsevier Inc. All rights reserved.

Investigating the Molecular Mechanism of TSO1 Function in Arabidopsis cell division and meristem development

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

Zhongchi Liu

2004-10-01

Unlike animals, plants are constantly exposed to environmental mutagens including ultraviolet light and reactive oxygen species. Further, plant cells are totipotent with highly plastic developmental programs. An understanding of molecular mechanisms underlying the ability of plants to monitor and repair its DNA and to eliminate damaged cells are of great importance. Previously we have identified two genes, TSO1 and TSO2, from a flowering plant Arabidopsis thaliana. Mutations in these two genes cause callus-like flowers, fasciated shoot apical meristems, and abnormal cell division, indicating that TSO1 and TSO2 may encode important cell cycle regulators. Previous funding from DOE led to themore » molecular cloning of TSO1, which was shown to encode a novel nuclear protein with two CXC domains suspected to bind DNA. This DOE grant has allowed us to characterize and isolate TSO2 that encodes the small subunit of the ribonucleotide reductase (RNR). RNR comprises two large subunits (R1) an d two small subunits (R2), catalyzes a rate-limiting step in the production of deoxyribonucleotides needed for DNA replication and repair. Previous studies in yeast and mammals indicated that defective RNR often led to cell cycle arrest, growth retardation and p53-dependent apoptosis while abnormally elevated RNR activities led to higher mutation rates. Subsequently, we identified two additional R2 genes, R2A and R2B in the Arabidopsis genome. Using reverse genetics, mutations in R2A and R2B were isolated, and double and triple mutants among the three R2 genes (TSO2, R2A and R2B) were constructed and analyzed. We showed that Arabidopsis tso2 mutants, with reduced dNTP levels, were more sensitive to UV-C. While r2a or r2b single mutants did not exhibit any phenotypes, tso2 r2b double mutants were embryonic lethal and tso2 r2a double mutants were seedling lethal indicating redundant functions among the three R2 genes. Furthermore, tso2 r2a double mutants exhibited increased DNA dam age, massive programmed cell death, and the release of transcriptional gene silencing. Our data suggests that plants can initiate programmed cell death to eliminate damaged cells despite the absence of p53 in plant genome.« less

Functional characterization of GPC-1 genes in hexaploid wheat.

PubMed

Avni, Raz; Zhao, Rongrong; Pearce, Stephen; Jun, Yan; Uauy, Cristobal; Tabbita, Facundo; Fahima, Tzion; Slade, Ann; Dubcovsky, Jorge; Distelfeld, Assaf

2014-02-01

In wheat, monocarpic senescence is a tightly regulated process during which nitrogen (N) and micronutrients stored pre-anthesis are remobilized from vegetative tissues to the developing grains. Recently, a close connection between senescence and remobilization was shown through the map-based cloning of the GPC (grain protein content) gene in wheat. GPC-B1 encodes a NAC transcription factor associated with earlier senescence and increased grain protein, iron and zinc content, and is deleted or non-functional in most commercial wheat varieties. In the current research, we identified 'loss of function' ethyl methanesulfonate mutants for the two GPC-B1 homoeologous genes; GPC-A1 and GPC-D1, in a hexaploid wheat mutant population. The single gpc-a1 and gpc-d1 mutants, the double gpc-1 mutant and control lines were grown under field conditions at four locations and were characterized for senescence, GPC, micronutrients and yield parameters. Our results show a significant delay in senescence in both the gpc-a1 and gpc-d1 single mutants and an even stronger effect in the gpc-1 double mutant in all the environments tested in this study. The accumulation of total N in the developing grains showed a similar increase in the control and gpc-1 plants until 25 days after anthesis (DAA) but at 41 and 60 DAA the control plants had higher grain N content than the gpc-1 mutants. At maturity, GPC in all mutants was significantly lower than in control plants while grain weight was unaffected. These results demonstrate that the GPC-A1 and GPC-D1 genes have a redundant function and play a major role in the regulation of monocarpic senescence and nutrient remobilization in wheat.

Functional characterization of GPC-1 genes in hexaploid wheat

PubMed Central

Pearce, Stephen; Jun, Yan; Uauy, Cristobal; Tabbita, Facundo; Fahima, Tzion; Slade, Ann; Dubcovsky, Jorge; Distelfeld, Assaf

2016-01-01

In wheat, monocarpic senescence is a tightly regulated process during which nitrogen (N) and micronutrients stored pre-anthesis are remobilized from vegetative tissues to the developing grains. Recently, a close connection between senescence and remobilization was shown through the map-based cloning of the GPC (Grain Protein Content) gene in wheat. GPC-B1 encodes a NAC transcription factor associated with earlier senescence and increased grain protein, iron and zinc content, and is deleted or non-functional in most commercial wheat varieties. In the current research, we identified 'loss of function' ethyl methane sulphonate mutants for the two GPC-B1 homoeologous genes; GPC-A1 and GPC-D1, in a hexaploid wheat mutant population. The single gpc-a1 and gpc-d1 mutants, the double gpc-1 mutant and control lines were grown under field conditions at four locations and were characterized for senescence, GPC, micronutrients and yield parameters. Our results show a significant delay in senescence in both the gpc-a1 and gpc-d1 single mutants and an even stronger effect in the gpc-1 double mutant in all the environments tested in this study. The accumulation of total N in the developing grains showed a similar increase in the control and gpc-1 plants until 25 days after anthesis (DAA) but at 41 and 60 DAA the control plants had higher Grain N content than the gpc-1 mutants. At maturity, GPC in all mutants was significantly lower than in control plants while grain weight was unaffected. These results demonstrate that theGPC-A1 and GPC-D1 genes have a redundant function and play a major role in the regulation of monocarpic senescence and nutrient remobilization in wheat. PMID:24170335

Further enhanced production of heterologous proteins by double-gene disruption (ΔAosedD ΔAovps10) in a hyper-producing mutant of Aspergillus oryzae.

PubMed

Zhu, Lin; Maruyama, Jun-ichi; Kitamoto, Katsuhiko

2013-07-01

The filamentous fungus Aspergillus oryzae is used as one of the most favored hosts for heterologous protein production due to its ability to secrete large amounts of proteins into the culture medium. We previously generated a hyper-producing mutant strain of A. oryzae, AUT1, which produced 3.2- and 2.6-fold higher levels of bovine chymosin (CHY) and human lysozyme (HLY), respectively, compared with the wild-type strain. However, further enhancement of heterologous protein production by multiple gene disruption is difficult because of the low gene-targeting efficiency in strain AUT1. Here, we disrupted the ligD gene, which is involved in nonhomologous recombination, and the pyrG gene to create uridine/uracil auxotrophy in strain AUT1, to generate a hyper-producing mutant applicable to pyrG marker recycling with highly efficient gene targeting. We generated single and double disruptants of the tripeptidyl peptidase gene AosedD and vacuolar sorting receptor gene Aovps10 in the hyper-producing mutant background, and found that all disruptants showed significant increases in heterologous protein production. Particularly, double disruption of the Aovps10 and AosedD genes increased the production levels of CHY and HLY by 1.6- and 2.1-fold, respectively, compared with the parental strain. Thus, we successfully generated a fungal host for further enhancing the heterologous protein production ability by combining mutational and molecular breeding techniques.

Mutations Abrogating VP35 Interaction with Double-Stranded RNA Render Ebola Virus Avirulent in Guinea Pigs

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

Prins, Kathleen C.; Delpeut, Sebastien; Leung, Daisy W.

2010-10-11

Ebola virus (EBOV) protein VP35 is a double-stranded RNA (dsRNA) binding inhibitor of host interferon (IFN)-{alpha}/{beta} responses that also functions as a viral polymerase cofactor. Recent structural studies identified key features, including a central basic patch, required for VP35 dsRNA binding activity. To address the functional significance of these VP35 structural features for EBOV replication and pathogenesis, two point mutations, K319A/R322A, that abrogate VP35 dsRNA binding activity and severely impair its suppression of IFN-{alpha}/{beta} production were identified. Solution nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography reveal minimal structural perturbations in the K319A/R322A VP35 double mutant and suggest that lossmore » of basic charge leads to altered function. Recombinant EBOVs encoding the mutant VP35 exhibit, relative to wild-type VP35 viruses, minimal growth attenuation in IFN-defective Vero cells but severe impairment in IFN-competent cells. In guinea pigs, the VP35 mutant virus revealed a complete loss of virulence. Strikingly, the VP35 mutant virus effectively immunized animals against subsequent wild-type EBOV challenge. These in vivo studies, using recombinant EBOV viruses, combined with the accompanying biochemical and structural analyses directly correlate VP35 dsRNA binding and IFN inhibition functions with viral pathogenesis. Moreover, these studies provide a framework for the development of antivirals targeting this critical EBOV virulence factor.« less

A loss-of-function mutation in the nucleoporin AtNUP160 indicates that normal auxin signalling is required for a proper ethylene response in Arabidopsis

PubMed Central

Robles, Linda M.; Deslauriers, Stephen D.; Alvarez, Ashley A.; Larsen, Paul B.

2012-01-01

As part of a continuing effort to elucidate mechanisms that regulate the magnitude of ethylene signalling, an Arabidopsis mutant with an enhanced ethylene response was identified. Subsequent characterization of this loss-of-function mutant revealed severe hypocotyl shortening in the presence of saturating ethylene along with increased expression in leaves of a subset of ethylene-responsive genes. It was subsequently determined by map-based cloning that the mutant (sar1-7) represents a loss-of-function mutation in the previously described nucleoporin AtNUP160 (At1g33410, SAR1). In support of previously reported results, the sar1-7 mutant partially restored auxin responsiveness to roots of an rce1 loss-of-function mutant, indicating that AtNUP160/SAR1 is required for proper expression of factors responsible for the repression of auxin signalling. Analysis of arf7-1/sar1-7 and arf19-1/sar1-7 double mutants revealed that mutations affecting either ARF7 or ARF19 function almost fully blocked manifestation of the sar1-7-dependent ethylene hypersensitivity phenotype, suggesting that ARF7- and ARF19-mediated auxin signalling is responsible for regulating the magnitude of and/or competence for the ethylene response in Arabidopsis etiolated hypocotyls. Consistent with this, addition of auxin to ethylene-treated seedlings resulted in severe hypocotyl shortening, reminiscent of that seen for other eer (enhanced ethylene response) mutants, suggesting that auxin functions in part synergistically with ethylene to control hypocotyl elongation and other ethylene-dependent phenomena. PMID:22238449

A gain-of-function mutation of plastidic invertase alters nuclear gene expression with sucrose treatment partially via GENOMES UNCOUPLED1-mediated signaling.

PubMed

Maruta, Takanori; Miyazaki, Nozomi; Nosaka, Ryota; Tanaka, Hiroyuki; Padilla-Chacon, Daniel; Otori, Kumi; Kimura, Ayako; Tanabe, Noriaki; Yoshimura, Kazuya; Tamoi, Masahiro; Shigeoka, Shigeru

2015-05-01

Plastid gene expression (PGE) is one of the signals that regulate the expression of photosynthesis-associated nuclear genes (PhANGs) via GENOMES UNCOUPLED1 (GUN1)-dependent retrograde signaling. We recently isolated Arabidopsis sugar-inducible cotyledon yellow-192 (sicy-192), a gain-of-function mutant of plastidic invertase, and showed that following the treatment of this mutant with sucrose, the expression of PhANGs as well as PGE decreased, suggesting that the sicy-192 mutation activates a PGE-evoked and GUN1-mediated retrograde pathway. To clarify the relationship between the sicy-192 mutation, PGE, and GUN1-mediated pathway, plastid and nuclear gene expression in a double mutant of sicy-192 and gun1-101, a null mutant of GUN1 was studied. Plastid-encoded RNA polymerase (PEP)-dependent PGE was markedly suppressed in the sicy-192 mutant by the sucrose treatment, but the suppression as well as cotyledon yellow phenotype was not mitigated by GUN1 disruption. Microarray analysis revealed that the altered expression of nuclear genes such as PhANG in the sucrose-treated sicy-192 mutant was largely dependent on GUN1. The present findings demonstrated that the sicy-192 mutation alters nuclear gene expression with sucrose treatment via GUN1, which is possibly followed by inhibiting PEP-dependent PGE, providing a new insight into the role of plastid sugar metabolism in nuclear gene expression. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

Heterotrimeric G proteins-mediated resistance to necrotrophic pathogens includes mechanisms independent of salicylic acid-, jasmonic acid/ethylene- and abscisic acid-mediated defense signaling.

PubMed

Trusov, Yuri; Sewelam, Nasser; Rookes, James Edward; Kunkel, Matt; Nowak, Ekaterina; Schenk, Peer Martin; Botella, José Ramón

2009-04-01

Heterotrimeric G proteins are involved in the defense response against necrotrophic fungi in Arabidopsis. In order to elucidate the resistance mechanisms involving heterotrimeric G proteins, we analyzed the effects of the Gβ (subunit deficiency in the mutant agb1-2 on pathogenesis-related gene expression, as well as the genetic interaction between agb1-2 and a number of mutants of established defense pathways. Gβ-mediated signaling suppresses the induction of salicylic acid (SA)-, jasmonic acid (JA)-, ethylene (ET)- and abscisic acid (ABA)-dependent genes during the initial phase of the infection with Fusarium oxysporum (up to 48 h after inoculation). However, at a later phase it enhances JA/ET-dependent genes such as PDF1.2 and PR4. Quantification of the Fusarium wilt symptoms revealed that Gβ- and SA-deficient mutants were more susceptible than wild-type plants, whereas JA- and ET-insensitive and ABA-deficient mutants demonstrated various levels of resistance. Analysis of the double mutants showed that the Gβ-mediated resistance to F. oxysporum and Alternaria brassicicola was mostly independent of all of the previously mentioned pathways. However, the progressive decay of agb1-2 mutants was compensated by coi1-21 and jin1-9 mutations, suggesting that at this stage of F. oxysporum infection Gβ acts upstream of COI1 and ATMYC2 in JA signaling. © 2008 The Authors. Journal compilation © 2008 Blackwell Publishing Ltd.

Glycosyltransfer in mutants of putative catalytic residue Glu303 of the human ABO(H) A and B blood group glycosyltransferases GTA and GTB proceeds through a labile active site.

PubMed

Blackler, Ryan J; Gagnon, Susannah M L; Polakowski, Robert; Rose, Natisha L; Zheng, Ruixiang B; Letts, James A; Johal, Asha R; Schuman, Brock; Borisova, Svetlana N; Palcic, Monica M; Evans, Stephen V

2017-04-01

The homologous glycosyltransferases α-1,3-N-acetylgalactosaminyltransferase (GTA) and α-1,3-galactosyltransferase (GTB) carry out the final synthetic step of the closely related human ABO(H) blood group A and B antigens. The catalytic mechanism of these model retaining enzymes remains under debate, where Glu303 has been suggested to act as a putative nucleophile in a double displacement mechanism, a local dipole stabilizing the intermediate in an orthogonal associative mechanism or a general base to stabilize the reactive oxocarbenium ion-like intermediate in an SNi-like mechanism. Kinetic analysis of GTA and GTB point mutants E303C, E303D, E303Q and E303A shows that despite the enzymes having nearly identical sequences, the corresponding mutants of GTA/GTB have up to a 13-fold difference in their residual activities relative to wild type. High-resolution single crystal X-ray diffraction studies reveal, surprisingly, that the mutated Cys, Asp and Gln functional groups are no more than 0.8 Å further from the anomeric carbon of donor substrate compared to wild type. However, complicating the analysis is the observation that Glu303 itself plays a critical role in maintaining the stability of a strained "double-turn" in the active site through several hydrogen bonds, and any mutation other than E303Q leads to significantly higher thermal motion or even disorder in the substrate recognition pockets. Thus, there is a remarkable juxtaposition of the mutants E303C and E303D, which retain significant activity despite disrupted active site architecture, with GTB/E303Q, which maintains active site architecture but exhibits zero activity. These findings indicate that nucleophilicity at position 303 is more catalytically valuable than active site stability and highlight the mechanistic elasticity of these enzymes. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Deep sequencing reveals double mutations in cis of MPL exon 10 in myeloproliferative neoplasms.

PubMed

Pietra, Daniela; Brisci, Angela; Rumi, Elisa; Boggi, Sabrina; Elena, Chiara; Pietrelli, Alessandro; Bordoni, Roberta; Ferrari, Maurizio; Passamonti, Francesco; De Bellis, Gianluca; Cremonesi, Laura; Cazzola, Mario

2011-04-01

Somatic mutations of MPL exon 10, mainly involving a W515 substitution, have been described in JAK2 (V617F)-negative patients with essential thrombocythemia and primary myelofibrosis. We used direct sequencing and high-resolution melt analysis to identify mutations of MPL exon 10 in 570 patients with myeloproliferative neoplasms, and allele specific PCR and deep sequencing to further characterize a subset of mutated patients. Somatic mutations were detected in 33 of 221 patients (15%) with JAK2 (V617F)-negative essential thrombocythemia or primary myelofibrosis. Only one patient with essential thrombocythemia carried both JAK2 (V617F) and MPL (W515L). High-resolution melt analysis identified abnormal patterns in all the MPL mutated cases, while direct sequencing did not detect the mutant MPL in one fifth of them. In 3 cases carrying double MPL mutations, deep sequencing analysis showed identical load and location in cis of the paired lesions, indicating their simultaneous occurrence on the same chromosome.

Conjugated fatty acid synthesis: residues 111 and 115 influence product partitioning of Momordica charantia conjugase.

PubMed

Rawat, Richa; Yu, Xiao-Hong; Sweet, Marie; Shanklin, John

2012-05-11

Conjugated linolenic acids (CLNs), 18:3 Δ(9,11,13), lack the methylene groups found between the double bonds of linolenic acid (18:3 Δ(9,12,15)). CLNs are produced by conjugase enzymes that are homologs of the oleate desaturases FAD2. The goal of this study was to map the domain(s) within the Momordica charantia conjugase (FADX) responsible for CLN formation. To achieve this, a series of Momordica FADX-Arabidopsis FAD2 chimeras were expressed in the Arabidopsis fad3fae1 mutant, and the transformed seeds were analyzed for the accumulation of CLN. These experiments identified helix 2 and the first histidine box as a determinant of conjugase product partitioning into punicic acid (18:3 Δ(9cis,11trans,13cis)) or α-eleostearic acid (18:3 Δ(9cis,11trans,13trans)). This was confirmed by analysis of a FADX mutant containing six substitutions in which the sequence of helix 2 and first histidine box was converted to that of FAD2. Each of the six FAD2 substitutions was individually converted back to the FADX equivalent identifying residues 111 and 115, adjacent to the first histidine box, as key determinants of conjugase product partitioning. Additionally, expression of FADX G111V and FADX G111V/D115E resulted in an approximate doubling of eleostearic acid accumulation to 20.4% and 21.2%, respectively, compared with 9.9% upon expression of the native Momordica FADX. Like the Momordica conjugase, FADX G111V and FADX D115E produced predominantly α-eleostearic acid and little punicic acid, but the FADX G111V/D115E double mutant produced approximately equal amounts of α-eleostearic acid and its isomer, punicic acid, implicating an interactive effect of residues 111 and 115 in punicic acid formation.

[Molecular mechanism of AtGA3OX1 and AtGA3OX2 genes affecting secondary wall thickening in stems in Arabidopsis].

PubMed

Wang, Zeng-Guang; Chai, Guo-Hua; Wang, Zhi-Yao; Tang, Xian-Feng; Sun, Chang-Jiang; Zhou, Gong-Ke; Ma, San-Mei

2013-05-01

Bioactive gibberellins (GAs) are a type of important plant growth regulators, which play the key roles in multiple processes, such as seed germination, leaf expansion, flowering, fruit bearing, and stem development. Its biosynthesis is regulated by a variety of enzymes including gibberellin 3-oxidase that is a key rate-limiting enzyme. In Arabidopsis, gibberellin 3-oxidase consists of four members, of which AtGA3OX1 and AtGA3OX2 are highly expressed in stems, suggesting the potential roles in the stem development played by the two genes. To date, there are few studies on AtGA3OX1 and AtGA3OX2 regulating secondary wall thickening in stems. In this study, we used the atga3ox1atga3ox2 double mutant as the materials to study the effects of AtGA3OX1 and AtGA3OX2 genes on secondary wall thickening in stems. The results indicated that simulations repression of AtGA3OX1 and AtGA3OX2 genes resulted in significantly reduction of secondary wall thickening of fiber cells, but not that of vessel cells. Three main components (cellulose, hemicelluloses, and lignin) were also dramatically suppressed in the double mutants. qRT-PCR analysis demonstrated that the expressions of secondary wall biosynthetic genes and the associated transcription factors were obviously affected in AtGA3OX1 and AtGA3OX2 double mutant. Therefore, we presume that Arabidopsis AtGA3OX1 and AtGA3OX2 genes might activate the expression of these transcription factors, thus regulate secondary wall thickening in stems. Together, our results provide a theoretical basis for enhancing the lodging resistance of food crops and improving the biomass of energy plants by genetically engineering Arabidopsis AtGA3OX homologs.

Proton conduction within the reaction centers of Rhodobacter capsulatus: the electrostatic role of the protein.

PubMed

Maróti, P; Hanson, D K; Baciou, L; Schiffer, M; Sebban, P

1994-06-07

Light-induced charge separation in the photosynthetic reaction center results in delivery of two electrons and two protons to the terminal quinone acceptor QB. In this paper, we have used flash-induced absorbance spectroscopy to study three strains that share identical amino acid sequences in the QB binding site, all of which lack the protonatable amino acids Glu-L212 and Asp-L213. These strains are the photosynthetically incompetent site-specific mutant Glu-L212/Asp-L213-->Ala-L212/Ala-L213 and two different photocompetent derivatives that carry both alanine substitutions and an intergenic suppressor mutation located far from QB (class 3 strain, Ala-Ala + Arg-M231-->Leu; class 4 strain, Ala-Ala + Asn-M43-->Asp). At pH 8 in the double mutant, we observe a concomitant decrease of nearly 4 orders of magnitude in the rate constants of second electron and proton transfer to QB compared to the wild type. Surprisingly, these rates are increased to about the same extent in both types of suppressor strains but remain > 2 orders of magnitude smaller than those of the wild type. In the double mutant, at pH 8, the loss of Asp-L213 and Glu-L212 leads to a substantial stabilization (> or = 60 meV) of the semiquinone energy level. Both types of compensatory mutations partially restore, to nearly the same level, the original free energy difference for electron transfer from primary quinone QA to QB. The pH dependence of the electron and proton transfer processes in the double-mutant and the suppressor strains suggests that when reaction centers of the double mutant are shifted to lower pH (1.5-2 units), they function like those of the suppressor strains at physiological pH. Our data suggest that the main effect of the compensatory mutations is to partially restore the negative electrostatic environment of QB and to increase an apparent "functional" pK of the system for efficient proton transfer to the active site. This emphasizes the role of the protein in tuning the electrostatic environment of its cofactors and highlights the possible long-range electrostatic effects.

Unusual Δ7,12,19 C35:3 Alkenone Produced by the Mutant Emiliania huxleyi strain CCMP2758 in Culture

NASA Astrophysics Data System (ADS)

Zheng, Y.; Huang, Y.; Zhang, Y.; Dillon, J. T.

2015-12-01

Alkenones with chain length ranging from C37 to C40 are highly specific biomarkers for certain haptophyte algae in ocean and lake sediments and have been widely used for paleoclimate studies. Short chain alkenones (e.g., C35 and C36) have been found in environmental and culture samples but the origin and structures of these compounds are not fully understood. The benchmark marine alkenone producer, Emiliania huxleyi CCMP2758 strain (the mutant of strain CCMP1742, NEPCC55a) was reported to make 35:2 alkenone when cultured at 15 °C (Prahl et al., 2006). Here we show, when this strain is cultured at lower temperatures (e.g., 4°C), CCMP2758 produces large amount of 35:3 alkenone with unusual double bond positions of Δ7,12,19. We determined the double bond positions of the C35:3 methyl ketonee based on GC-MS analysis of cyclobutylimine derivatives and dimethyl disulfide derivatives respectively, and provide the first temperature calibrations based on the unsaturation ratios of C35 alkenones. Previous studies have found 35:2 alkenone with three methylene interruption in the Black Sea sediment, but it is the first time that an alkenone with a mixed three and five methylene interruption is found. The discovery of short chain alkenones with unusual double bond positions may shed new light to alkenone biosynthesis.

Arabidopsis STERILE APETALA, a multifunctional gene regulating inflorescence, flower, and ovule development

PubMed Central

Byzova, Marina V.; Franken, John; Aarts, Mark G.M.; de Almeida-Engler, Janice; Engler, Gilbert; Mariani, Celestina; Van Lookeren Campagne, Michiel M.; Angenent, Gerco C.

1999-01-01

A recessive mutation in the Arabidopsis STERILE APETALA (SAP) causes severe aberrations in inflorescence and flower and ovule development. In sap flowers, sepals are carpelloid, petals are short and narrow or absent, and anthers are degenerated. Megasporogenesis, the process of meiotic divisions preceding the female gametophyte formation, is arrested in sap ovules during or just after the first meiotic division. More severe aberrations were observed in double mutants between sap and mutant alleles of the floral homeotic gene APETALA2 (AP2) suggesting that both genes are involved in the initiation of female gametophyte development. Together with the organ identity gene AGAMOUS (AG) SAP is required for the maintenance of floral identity acting in a manner similar to APETALA1. In contrast to the outer two floral organs in sap mutant flowers, normal sepals and petals develop in ag/sap double mutants, indicating that SAP negatively regulates AG expression in the perianth whorls. This supposed cadastral function of SAP is supported by in situ hybridization experiments showing ectopic expression of AG in the sap mutant. We have cloned the SAP gene by transposon tagging and revealed that it encodes a novel protein with sequence motifs, that are also present in plant and animal transcription regulators. Consistent with the mutant phenotype, SAP is expressed in inflorescence and floral meristems, floral organ primordia, and ovules. Taken together, we propose that SAP belongs to a new class of transcription regulators essential for a number of processes in Arabidopsis flower development. PMID:10215627

The Role of Zic Genes in Inner Ear Development in the Mouse: Exploring Mutant Mouse Phenotypes

PubMed Central

Chervenak, Andrew P.; Bank, Lisa M.; Thomsen, Nicole; Glanville-Jones, Hannah C; Skibo, Jonathan; Millen, Kathleen J.; Arkell, Ruth M.; Barald, Kate F.

2014-01-01

Background Murine Zic genes (Zic1-5) are expressed in the dorsal hindbrain and in periotic mesenchyme (POM) adjacent to the developing inner ear. Zic genes are involved in developmental signaling pathways in many organ systems, including the ear, although their exact roles haven't been fully elucidated. This report examines the role of Zic1, Zic2, and Zic4 during inner ear development in mouse mutants in which these Zic genes are affected Results Zic1/Zic4 double mutants don't exhibit any apparent defects in inner ear morphology. By contrast, inner ears from Zic2kd/kd and Zic2Ku/Ku mutants have severe but variable morphological defects in endolymphatic duct/sac and semicircular canal formation and in cochlear extension in the inner ear. Analysis of otocyst patterning in the Zic2Ku/Ku mutants by in situ hybridization showed changes in the expression patterns of Gbx2 and Pax2. Conclusions The experiments provide the first genetic evidence that the Zic genes are required for morphogenesis of the inner ear. Zic2 loss-of-function doesn't prevent initial otocyst patterning but leads to molecular abnormalities concomitant with morphogenesis of the endolymphatic duct. Functional hearing deficits often accompany inner ear dysmorphologies, making Zic2 a novel candidate gene for ongoing efforts to identify the genetic basis of human hearing loss. PMID:25178196

Analysis of the role of the two flagella of Bradyrhizobium japonicum in competition for nodulation of soybean.

PubMed

Althabegoiti, Maria Julia; Covelli, Julieta M; Pérez-Giménez, Julieta; Quelas, Juan Ignacio; Mongiardini, Elías J; López, Maria Florencia; López-García, Silvina L; Lodeiro, Aníbal R

2011-06-01

Bradyrhizobium japonicum has two types of flagella. One has thin filaments consisting of the 33-kDa flagellins FliCI and FliCII (FliCI-II) and the other has thick filaments consisting of the 65-kDa flagellins FliC1, FliC2, FliC3, and FliC4 (FliC1-4). To investigate the roles of each flagellum in competition for nodulation, we obtained mutants deleted in fliCI-II and/or fliC1-4 in the genomic backgrounds of two derivatives from the reference strain USDA 110: the streptomycin-resistant derivative LP 3004 and its more motile derivative LP 3008. All mutations diminished swimming motility. When each mutant was co-inoculated with the parental strain on soybean plants cultivated in vermiculite either at field capacity or flooded, their competitiveness differed according to the flagellin altered. ΔfliCI-II mutants were more competitive, occupying 64-80% of the nodules, while ΔfliC1-4 mutants occupied 45-49% of the nodules. Occupation by the nonmotile double mutant decreased from 55% to 11% as the water content of the vermiculite increased from 85% to 95% field capacity to flooding. These results indicate that the influence of motility on competitiveness depended on the water status of the rooting substrate. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Role of tryptophan 95 in substrate specificity and structural stability of Sulfolobus solfataricus alcohol dehydrogenase.

PubMed

Pennacchio, Angela; Esposito, Luciana; Zagari, Adriana; Rossi, Mosè; Raia, Carlo A

2009-09-01

A mutant of the thermostable NAD(+)-dependent (S)-stereospecific alcohol dehydrogenase from Sulfolobus solfataricus (SsADH) which has a single substitution, Trp95Leu, located at the substrate binding pocket, was fully characterized to ascertain the role of Trp95 in discriminating between chiral secondary alcohols suggested by the wild-type SsADH crystallographic structure. The Trp95Leu mutant displays no apparent activity with short-chain primary and secondary alcohols and poor activity with aromatic substrates and coenzyme. Moreover, the Trp --> Leu substitution affects the structural stability of the archaeal ADH, decreasing its thermal stability without relevant changes in secondary structure. The double mutant Trp95Leu/Asn249Tyr was also purified to assist in crystallographic analysis. This mutant exhibits higher activity but decreased affinity toward aliphatic alcohols, aldehydes as well as NAD(+) and NADH compared to the wild-type enzyme. The crystal structure of the Trp95Leu/Asn249Tyr mutant apo form, determined at 2.0 A resolution, reveals a large local rearrangement of the substrate site with dramatic consequences. The Leu95 side-chain conformation points away from the catalytic metal center and the widening of the substrate site is partially counteracted by a concomitant change of Trp117 side chain conformation. Structural changes at the active site are consistent with the reduced activity on substrates and decreased coenzyme binding.

Assignment of simian rotavirus SA11 temperature-sensitive mutant groups B and E to genome segments

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

Gombold, J.L.; Estes, M.K.; Ramig, R.F.

1985-05-01

Recombinant (reassortant) viruses were selected from crosses between temperature-sensitive (ts) mutants of simian rotavirus SA11 and wild-type human rotavirus Wa. The double-stranded genome RNAs of the reassortants were examined by electrophoresis in Tris-glycine-buffered polyacrylamide gels and by dot hybridization with a cloned DNA probe for genome segment 2. Analysis of replacements of genome segments in the reassortants allowed construction of a map correlating genome segments providing functions interchangeable between SA11 and Wa. The reassortants revealed a functional correspondence in order of increasing electrophoretic mobility of genome segments. Analysis of the parental origin of genome segments in ts+ SA11/Wa reassortants derivedmore » from the crosses SA11 tsB(339) X Wa and SA11 tsE(1400) X Wa revealed that the group B lesion of tsB(339) was located on genome segment 3 and the group E lesion of tsE(1400) was on segment 8.« less

Phosphorylation Regulates myo-Inositol-3-phosphate Synthase

PubMed Central

Deranieh, Rania M.; He, Quan; Caruso, Joseph A.; Greenberg, Miriam L.

2013-01-01

myo-Inositol-3-phosphate synthase (MIPS) plays a crucial role in inositol homeostasis. Transcription of the coding gene INO1 is highly regulated. However, regulation of the enzyme is not well defined. We previously showed that MIPS is indirectly inhibited by valproate, suggesting that the enzyme is post-translationally regulated. Using 32Pi labeling and phosphoamino acid analysis, we show that yeast MIPS is a phosphoprotein. Mass spectrometry analysis identified five phosphosites, three of which are conserved in the human MIPS. Analysis of phosphorylation-deficient and phosphomimetic site mutants indicated that the three conserved sites in yeast (Ser-184, Ser-296, and Ser-374) and humans (Ser-177, Ser-279, and Ser-357) affect MIPS activity. Both S296A and S296D yeast mutants and S177A and S177D human mutants exhibited decreased enzymatic activity, suggesting that a serine residue is critical at that location. The phosphomimetic mutations S184D (human S279D) and S374D (human S357D) but not the phosphodeficient mutations decreased activity, suggesting that phosphorylation of these two sites is inhibitory. The double mutation S184A/S374A caused an increase in MIPS activity, conferred a growth advantage, and partially rescued sensitivity to valproate. Our findings identify a novel mechanism of regulation of inositol synthesis by phosphorylation of MIPS. PMID:23902760

Function and evolution of a Lotus japonicus AP2/ERF family transcription factor that is required for development of infection threads

PubMed Central

Yano, Koji; Aoki, Seishiro; Liu, Meng; Umehara, Yosuke; Suganuma, Norio; Iwasaki, Wataru; Sato, Shusei; Soyano, Takashi; Kouchi, Hiroshi

2017-01-01

Abstract Legume-rhizobium symbiosis is achieved by two major events evolutionarily acquired: root hair infection and organogenesis. Infection thread (IT) development is a distinct element for rhizobial infection. Through ITs, rhizobia are efficiently transported from infection foci on root hairs to dividing meristematic cortical cells. To unveil this process, we performed genetic screening using Lotus japonicus MG-20 and isolated symbiotic mutant lines affecting nodulation, root hair morphology, and IT development. Map-based cloning identified an AP2/ERF transcription factor gene orthologous to Medicago truncatula ERN1. LjERN1 was activated in response to rhizobial infection and depended on CYCLOPS and NSP2. Legumes conserve an ERN1 homolog, ERN2, that functions redundantly with ERN1 in M. truncatula. Phylogenetic analysis showed that the lineages of ERN1 and ERN2 genes originated from a gene duplication event in the common ancestor of legume plants. However, genomic analysis suggested the lack of ERN2 gene in the L. japonicus genome, consistent with Ljern1 mutants exhibited a root hair phenotype that is observed in ern1/ern2 double mutants in M. truncatula. Molecular evolutionary analysis suggested that the nonsynonymous/synonymous rate ratios of legume ERN1 genes was almost identical to that of non-legume plants, whereas the ERN2 genes experienced a relaxed selective constraint. PMID:28028038

Genetic Interaction Mapping in Schizosaccharomyces pombe Using the Pombe Epistasis Mapper (PEM) System and a ROTOR HDA Colony Replicating Robot in a 1536 Array Format.

PubMed

Roguev, Assen; Xu, Jiewei; Krogan, Nevan

2018-02-01

This protocol describes an optimized high-throughput procedure for generating double deletion mutants in Schizosaccharomyces pombe using the colony replicating robot ROTOR HDA and the PEM (pombe epistasis mapper) system. The method is based on generating high-density colony arrays (1536 colonies per agar plate) and passaging them through a series of antidiploid and mating-type selection (ADS-MTS) and double-mutant selection (DMS) steps. Detailed program parameters for each individual replication step are provided. Using this procedure, batches of 25 or more screens can be routinely performed. © 2018 Cold Spring Harbor Laboratory Press.

Role of Arabidopsis ABF1/3/4 during det1 germination in salt and osmotic stress conditions.

PubMed

Fernando, V C Dilukshi; Al Khateeb, Wesam; Belmonte, Mark F; Schroeder, Dana F

2018-05-01

Arabidopsis det1 mutants exhibit salt and osmotic stress resistant germination. This phenotype requires HY5, ABF1, ABF3, and ABF4. While DE-ETIOLATED 1 (DET1) is well known as a negative regulator of light development, here we describe how det1 mutants also exhibit altered responses to salt and osmotic stress, specifically salt and mannitol resistant germination. LONG HYPOCOTYL 5 (HY5) positively regulates both light and abscisic acid (ABA) signalling. We found that hy5 suppressed the det1 salt and mannitol resistant germination phenotype, thus, det1 stress resistant germination requires HY5. We then queried publically available microarray datasets to identify genes downstream of HY5 that were differentially expressed in det1 mutants. Our analysis revealed that ABA regulated genes, including ABA RESPONSIVE ELEMENT BINDING FACTOR 3 (ABF3), are downregulated in det1 seedlings. We found that ABF3 is induced by salt in wildtype seeds, while homologues ABF4 and ABF1 are repressed, and all three genes are underexpressed in det1 seeds. We then investigated the role of ABF3, ABF4, and ABF1 in det1 phenotypes. Double mutant analysis showed that abf3, abf4, and abf1 all suppress the det1 salt/osmotic stress resistant germination phenotype. In addition, abf1 suppressed det1 rapid water loss and open stomata phenotypes. Thus interactions between ABF genes contribute to det1 salt/osmotic stress response phenotypes.

Characterization of a wheat mutant missing low-molecular-weight glutenin subunits encoded by the B-genome

USDA-ARS?s Scientific Manuscript database

DH20, a new wheat mutant missing low-molecular weight glutenin subunits encoded by the Glu-B3 locus, was discovered among double haploid lines obtained from a cross between the Korean wheat cultivars Keumkang and Olgeuru. Absence of the Glu-B3 LMW-GS proteins was determined by one-dimensional gel e...

Bearded-Ear Encodes a MADS-box Transcription Factor Critical for Maize Floral Development

USDA-ARS?s Scientific Manuscript database

We cloned bde by positional cloning and found that it encodes zag3, a MADS-box transcription factor in the conserved AGL6 clade. Mutants in the maize homolog of AGAMOUS, zag1, have a subset of bde floral defects. bde; zag1 double mutants have a severe ear phenotype, not observed in either single m...

Roles of brca2 (fancd1) in oocyte nuclear architecture, gametogenesis, gonad tumors, and genome stability in zebrafish.

PubMed

Rodríguez-Marí, Adriana; Wilson, Catherine; Titus, Tom A; Cañestro, Cristian; BreMiller, Ruth A; Yan, Yi-Lin; Nanda, Indrajit; Johnston, Adam; Kanki, John P; Gray, Erin M; He, Xinjun; Spitsbergen, Jan; Schindler, Detlev; Postlethwait, John H

2011-03-01

Mild mutations in BRCA2 (FANCD1) cause Fanconi anemia (FA) when homozygous, while severe mutations cause common cancers including breast, ovarian, and prostate cancers when heterozygous. Here we report a zebrafish brca2 insertional mutant that shares phenotypes with human patients and identifies a novel brca2 function in oogenesis. Experiments showed that mutant embryos and mutant cells in culture experienced genome instability, as do cells in FA patients. In wild-type zebrafish, meiotic cells expressed brca2; and, unexpectedly, transcripts in oocytes localized asymmetrically to the animal pole. In juvenile brca2 mutants, oocytes failed to progress through meiosis, leading to female-to-male sex reversal. Adult mutants became sterile males due to the meiotic arrest of spermatocytes, which then died by apoptosis, followed by neoplastic proliferation of gonad somatic cells that was similar to neoplasia observed in ageing dead end (dnd)-knockdown males, which lack germ cells. The construction of animals doubly mutant for brca2 and the apoptotic gene tp53 (p53) rescued brca2-dependent sex reversal. Double mutants developed oocytes and became sterile females that produced only aberrant embryos and showed elevated risk for invasive ovarian tumors. Oocytes in double-mutant females showed normal localization of brca2 and pou5f1 transcripts to the animal pole and vasa transcripts to the vegetal pole, but had a polarized rather than symmetrical nucleus with the distribution of nucleoli and chromosomes to opposite nuclear poles; this result revealed a novel role for Brca2 in establishing or maintaining oocyte nuclear architecture. Mutating tp53 did not rescue the infertility phenotype in brca2 mutant males, suggesting that brca2 plays an essential role in zebrafish spermatogenesis. Overall, this work verified zebrafish as a model for the role of Brca2 in human disease and uncovered a novel function of Brca2 in vertebrate oocyte nuclear architecture.

Roles of brca2 (fancd1) in Oocyte Nuclear Architecture, Gametogenesis, Gonad Tumors, and Genome Stability in Zebrafish

PubMed Central

Rodríguez-Marí, Adriana; Wilson, Catherine; Titus, Tom A.; Cañestro, Cristian; BreMiller, Ruth A.; Yan, Yi-Lin; Nanda, Indrajit; Johnston, Adam; Kanki, John P.; Gray, Erin M.; He, Xinjun; Spitsbergen, Jan; Schindler, Detlev; Postlethwait, John H.

2011-01-01

Mild mutations in BRCA2 (FANCD1) cause Fanconi anemia (FA) when homozygous, while severe mutations cause common cancers including breast, ovarian, and prostate cancers when heterozygous. Here we report a zebrafish brca2 insertional mutant that shares phenotypes with human patients and identifies a novel brca2 function in oogenesis. Experiments showed that mutant embryos and mutant cells in culture experienced genome instability, as do cells in FA patients. In wild-type zebrafish, meiotic cells expressed brca2; and, unexpectedly, transcripts in oocytes localized asymmetrically to the animal pole. In juvenile brca2 mutants, oocytes failed to progress through meiosis, leading to female-to-male sex reversal. Adult mutants became sterile males due to the meiotic arrest of spermatocytes, which then died by apoptosis, followed by neoplastic proliferation of gonad somatic cells that was similar to neoplasia observed in ageing dead end (dnd)-knockdown males, which lack germ cells. The construction of animals doubly mutant for brca2 and the apoptotic gene tp53 (p53) rescued brca2-dependent sex reversal. Double mutants developed oocytes and became sterile females that produced only aberrant embryos and showed elevated risk for invasive ovarian tumors. Oocytes in double-mutant females showed normal localization of brca2 and pou5f1 transcripts to the animal pole and vasa transcripts to the vegetal pole, but had a polarized rather than symmetrical nucleus with the distribution of nucleoli and chromosomes to opposite nuclear poles; this result revealed a novel role for Brca2 in establishing or maintaining oocyte nuclear architecture. Mutating tp53 did not rescue the infertility phenotype in brca2 mutant males, suggesting that brca2 plays an essential role in zebrafish spermatogenesis. Overall, this work verified zebrafish as a model for the role of Brca2 in human disease and uncovered a novel function of Brca2 in vertebrate oocyte nuclear architecture. PMID:21483806

Two O-linked N-acetylglucosamine transferase genes of Arabidopsis thaliana L. Heynh. have overlapping functions necessary for gamete and seed development.

PubMed Central

Hartweck, Lynn M; Scott, Cheryl L; Olszewski, Neil E

2002-01-01

The Arabidopsis SECRET AGENT (SEC) and SPINDLY (SPY) proteins are similar to animal O-linked N-acetylglucosamine transferases (OGTs). OGTs catalyze the transfer of N-acetylglucosamine (GlcNAc) from UDP-GlcNAc to Ser/Thr residues of proteins. In animals, O-GlcNAcylation has been shown to affect protein activity, stability, and/or localization. SEC protein expressed in Escherichia coli had autocatalytic OGT activity. To determine the function of SEC in plants, two tDNA insertional mutants were identified and analyzed. Although sec mutant plants did not exhibit obvious phenotypes, sec and spy mutations had a synthetic lethal interaction. This lethality was incompletely penetrant in gametes and completely penetrant postfertilization. The rate of both female and male sec spy gamete transmission was higher in plants heterozygous for both mutations than in plants heterozygous for sec and homozygous for spy. Double-mutant embryos aborted at various stages of development and no double-mutant seedlings were obtained. These results indicate that OGT activity is required during gametogenesis and embryogenesis with lethality occurring when parentally derived SEC, SPY, and/or O-GlcNAcylated proteins become limiting. PMID:12136030

Hypolipidemic effects of starch and γ-oryzanol from wx/ae double-mutant rice on BALB/c.KOR-Apoe(shl) mice.

PubMed

Nakaya, Makoto; Shojo, Aiko; Hirai, Hiroaki; Matsumoto, Kenji; Kitamura, Shinichi

2013-01-01

waxy/amylose-extender (wx/ae) double-mutant japonica rice (Oryza sativa L.) produces resistant starch (RS) and a large amount of γ-oryzanol. Our previous study has shown the hypolipidemic effect of wx/ae brown rice on mice. To identify the functional constituents of the hypolipidemic activity in wx/ae rice, we prepared pure wx/ae starch and γ-oryzanol from wx/ae rice and investigated their effect on the lipid metabolism in BALB/c.KOR/Stm Slc-Apoe(shl) mice. The mice were fed for 3 weeks a diet containing non-mutant rice starch, non-mutant rice starch plus γ-oryzanol, wx/ae starch, or wx/ae starch plus γ-oryzanol. γ-Oryzanol by itself had no effect on the lipid metabolism, and wx/ae starch prevented an accumulation of triacylglycerol (TAG) in the liver. Interestingly, the combination of wx/ae starch plus γ-oryzanol not only prevented a TAG accumulation in the liver, but also partially suppressed the rise in plasma TAG concentration, indicating that wx/ae starch and γ-oryzanol could have a synergistic effect on the lipid metabolism.

A Role for Single-Stranded Exonucleases in the Use of DNA as a Nutrient▿

PubMed Central

Palchevskiy, Vyacheslav; Finkel, Steven E.

2009-01-01

Nutritional competence is the ability of bacterial cells to utilize exogenous double-stranded DNA molecules as a nutrient source. We previously identified several genes in Escherichia coli that are important for this process and proposed a model, based on models of natural competence and transformation in bacteria, where it is assumed that single-stranded DNA (ssDNA) is degraded following entry into the cytoplasm. Since E. coli has several exonucleases, we determined whether they play a role in the long-term survival and the catabolism of DNA as a nutrient. We show here that mutants lacking either ExoI, ExoVII, ExoX, or RecJ are viable during all phases of the bacterial life cycle yet cannot compete with wild-type cells during long-term stationary-phase incubation. We also show that nuclease mutants, alone or in combination, are defective in DNA catabolism, with the exception of the ExoX− single mutant. The ExoX− mutant consumes double-stranded DNA better than wild-type cells, possibly implying the presence of two pathways in E. coli for the processing of ssDNA as it enters the cytoplasm. PMID:19329645

Analysis of Loss-of-Function Mutants in Aspartate Kinase and Homoserine Dehydrogenase Genes Points to Complexity in the Regulation of Aspartate-Derived Amino Acid Contents1[OPEN

PubMed Central

2015-01-01

Biosynthesis of aspartate (Asp)-derived amino acids lysine (Lys), methionine (Met), threonine (Thr), and isoleucine involves monofunctional Asp kinases (AKs) and dual-functional Asp kinase-homoserine dehydrogenases (AK-HSDHs). Four-week-old loss-of-function Arabidopsis (Arabidopsis thaliana) mutants in the AK-HSDH2 gene had increased amounts of Asp and Asp-derived amino acids, especially Thr, in leaves. To explore mechanisms behind this phenotype, we obtained single mutants for other AK and AK-HSDH genes, generated double mutants from ak-hsdh2 and ak mutants, and performed free and protein-bound amino acid profiling, transcript abundance, and activity assays. The increases of Asp, Lys, and Met in ak-hsdh2 were also observed in ak1-1, ak2-1, ak3-1, and ak-hsdh1-1. However, the Thr increase in ak-hsdh2 was observed in ak-hsdh1-1 but not in ak1-1, ak2-1, or ak3-1. Activity assays showed that AK2 and AK-HSDH1 are the major contributors to overall AK and HSDH activities, respectively. Pairwise correlation analysis revealed positive correlations between the amount of AK transcripts and Lys-sensitive AK activity and between the amount of AK-HSDH transcripts and both Thr-sensitive AK activity and total HSDH activity. In addition, the ratio of total AK activity to total HSDH activity negatively correlates with the ratio of Lys to the total amount of Met, Thr, and isoleucine. These data led to the hypothesis that the balance between Lys-sensitive AKs and Thr-sensitive AK-HSDHs is important for maintaining the amounts and ratios of Asp-derived amino acids. PMID:26063505

Improving thermostability of phosphatidylinositol-synthesizing Streptomyces phospholipase D.

PubMed

Damnjanović, Jasmina; Takahashi, Rie; Suzuki, Atsuo; Nakano, Hideo; Iwasaki, Yugo

2012-08-01

Aimed to produce thermostable phosphatidylinositol (PI)-synthesizing phospholipase D (PLD), we initiated site-directed combinatorial mutagenesis followed by high-throughput screening. Previous site-directed combinatorial mutagenesis of wild-type Streptomyces PLD produced a mutant, DYR (W187D/Y191Y/Y385R) with PI-synthesizing ability. Deriving PI as a product of transphosphatidylation between phosphatidylcholine and myo-inositol, with myo-inositol in excess at high-temperature reaction conditions can increase yield due to enhanced solubility of this substrate. Thus, we improved DYR's thermostability by introduction of random mutations into selected amino acid positions having high B-factor. Screening of the libraries under restricted conditions yielded single-point mutants, specifically D40H, T291Y and R329G. Combinations of these point mutations yielded double (D40H/T291Y, D40H/R329G and T291Y/R329G) and triple (D40H/T291Y/R329G) mutants. PI synthesis at elevated temperatures pointed at D40H/T291Y as the most efficient enzyme. Circular dichroism analysis revealed D40H/T291Y to have increased melting temperature and postponed onset of thermal unfolding compared with DYR. Thermal tolerance study at 65°C confirmed D40H/T291Y's thermostability as its half-inactivation time was 8.7 min longer compared with DYR. This mutant had significantly less root-mean-square deviation change compared with DYR and showed no change in root-mean-square fluctuation when temperature shifts from 40 to 60°C, as determined by molecular dynamics analysis. Acquired different degrees of thermostability were also observed for several other DYR mutants.

Division-induced DNA double strand breaks in the chromosome terminus region of Escherichia coli lacking RecBCD DNA repair enzyme

PubMed Central

Durand, Adeline; Desfontaines, Jean-Michel; Iurchenko, Ielyzaveta; Auger, Hélène; Leach, David R. F.

2017-01-01

Marker frequency analysis of the Escherichia coli recB mutant chromosome has revealed a deficit of DNA in a specific zone of the terminus, centred on the dif/TerC region. Using fluorescence microscopy of a marked chromosomal site, we show that the dif region is lost after replication completion, at the time of cell division, in one daughter cell only, and that the phenomenon is transmitted to progeny. Analysis by marker frequency and microscopy shows that the position of DNA loss is not defined by the replication fork merging point since it still occurs in the dif/TerC region when the replication fork trap is displaced in strains harbouring ectopic Ter sites. Terminus DNA loss in the recB mutant is also independent of dimer resolution by XerCD at dif and of Topo IV action close to dif. It occurs in the terminus region, at the point of inversion of the GC skew, which is also the point of convergence of specific sequence motifs like KOPS and Chi sites, regardless of whether the convergence of GC skew is at dif (wild-type) or a newly created sequence. In the absence of FtsK-driven DNA translocation, terminus DNA loss is less precisely targeted to the KOPS convergence sequence, but occurs at a similar frequency and follows the same pattern as in FtsK+ cells. Importantly, using ftsIts, ftsAts division mutants and cephalexin treated cells, we show that DNA loss of the dif region in the recB mutant is decreased by the inactivation of cell division. We propose that it results from septum-induced chromosome breakage, and largely contributes to the low viability of the recB mutant. PMID:28968392

Replication factor C1 (RFC1) is required for double-strand break repair during meiotic homologous recombination in Arabidopsis.

PubMed

Liu, Yang; Deng, Yingtian; Li, Gang; Zhao, Jie

2013-01-01

Replication factor C1 (RFC1), which is conserved in eukaryotes, is involved in DNA replication and checkpoint control. However, a RFC1 product participating in DNA repair at meiosis has not been reported in Arabidopsis. Here, we report functional characterization of AtRFC1 through analysis of the rfc1-2 mutant. The rfc1-2 mutant displayed normal vegetative growth but showed silique sterility because the male gametophyte was arrested at the uninucleus microspore stage and the female at the functional megaspore stage. Expression of AtRFC1 was concentrated in the reproductive organ primordia, meiocytes and developing gametes. Chromosome spreads showed that pairing and synapsis were normal, and the chromosomes were broken when desynapsis began at late prophase I, and chromosome fragments remained in the subsequent stages. For this reason, homologous chromosomes and sister chromatids segregated unequally, leading to pollen sterility. Immunolocalization revealed that the AtRFC1 protein localized to the chromosomes during zygotene and pachytene in wild-type but were absent in the spo11-1 mutant. The chromosome fragmentation of rfc1-2 was suppressed by spo11-1, indicating that AtRFC1 acted downstream of AtSPO11-1. The similar chromosome behavior of rad51 rfc1-2 and rad51 suggests that AtRFC1 may act with AtRAD51 in the same pathway. In summary, AtRFC1 is required for DNA double-strand break repair during meiotic homologous recombination of Arabidopsis. © 2012 The Authors The Plant Journal © 2012 Blackwell Publishing Ltd.

Hox11 paralogous genes are essential for metanephric kidney induction

PubMed Central

Wellik, Deneen M.; Hawkes, Patrick J.; Capecchi, Mario R.

2002-01-01

The mammalian Hox complex is divided into four linkage groups containing 13 sets of paralogous genes. These paralogous genes have retained functional redundancy during evolution. For this reason, loss of only one or two Hox genes within a paralogous group often results in incompletely penetrant phenotypes which are difficult to interpret by molecular analysis. For example, mice individually mutant for Hoxa11 or Hoxd11 show no discernible kidney abnormalities. Hoxa11/Hoxd11 double mutants, however, demonstrate hypoplasia of the kidneys. As described in this study, removal of the last Hox11 paralogous member, Hoxc11, results in the complete loss of metanephric kidney induction. In these triple mutants, the metanephric blastema condenses, and expression of early patterning genes, Pax2 and Wt1, is unperturbed. Eya1 expression is also intact. Six2 expression, however, is absent, as is expression of the inducing growth factor, Gdnf. In the absence of Gdnf, ureteric bud formation is not initiated. Molecular analysis of this phenotype demonstrates that Hox11 control of early metanephric induction is accomplished by the interaction of Hox11 genes with the pax-eya-six regulatory cascade, a pathway that may be used by Hox genes more generally for the induction of multiple structures along the anteroposterior axis. PMID:12050119

Hox11 paralogous genes are essential for metanephric kidney induction.

PubMed

Wellik, Deneen M; Hawkes, Patrick J; Capecchi, Mario R

2002-06-01

The mammalian Hox complex is divided into four linkage groups containing 13 sets of paralogous genes. These paralogous genes have retained functional redundancy during evolution. For this reason, loss of only one or two Hox genes within a paralogous group often results in incompletely penetrant phenotypes which are difficult to interpret by molecular analysis. For example, mice individually mutant for Hoxa11 or Hoxd11 show no discernible kidney abnormalities. Hoxa11/Hoxd11 double mutants, however, demonstrate hypoplasia of the kidneys. As described in this study, removal of the last Hox11 paralogous member, Hoxc11, results in the complete loss of metanephric kidney induction. In these triple mutants, the metanephric blastema condenses, and expression of early patterning genes, Pax2 and Wt1, is unperturbed. Eya1 expression is also intact. Six2 expression, however, is absent, as is expression of the inducing growth factor, Gdnf. In the absence of Gdnf, ureteric bud formation is not initiated. Molecular analysis of this phenotype demonstrates that Hox11 control of early metanephric induction is accomplished by the interaction of Hox11 genes with the pax-eya-six regulatory cascade, a pathway that may be used by Hox genes more generally for the induction of multiple structures along the anteroposterior axis.

Mapping DNA damage-dependent genetic interactions in yeast via party mating and barcode fusion genetics.

PubMed

Díaz-Mejía, J Javier; Celaj, Albi; Mellor, Joseph C; Coté, Atina; Balint, Attila; Ho, Brandon; Bansal, Pritpal; Shaeri, Fatemeh; Gebbia, Marinella; Weile, Jochen; Verby, Marta; Karkhanina, Anna; Zhang, YiFan; Wong, Cassandra; Rich, Justin; Prendergast, D'Arcy; Gupta, Gaurav; Öztürk, Sedide; Durocher, Daniel; Brown, Grant W; Roth, Frederick P

2018-05-28

Condition-dependent genetic interactions can reveal functional relationships between genes that are not evident under standard culture conditions. State-of-the-art yeast genetic interaction mapping, which relies on robotic manipulation of arrays of double-mutant strains, does not scale readily to multi-condition studies. Here, we describe barcode fusion genetics to map genetic interactions (BFG-GI), by which double-mutant strains generated via en masse "party" mating can also be monitored en masse for growth to detect genetic interactions. By using site-specific recombination to fuse two DNA barcodes, each representing a specific gene deletion, BFG-GI enables multiplexed quantitative tracking of double mutants via next-generation sequencing. We applied BFG-GI to a matrix of DNA repair genes under nine different conditions, including methyl methanesulfonate (MMS), 4-nitroquinoline 1-oxide (4NQO), bleomycin, zeocin, and three other DNA-damaging environments. BFG-GI recapitulated known genetic interactions and yielded new condition-dependent genetic interactions. We validated and further explored a subnetwork of condition-dependent genetic interactions involving MAG1 , SLX4, and genes encoding the Shu complex, and inferred that loss of the Shu complex leads to an increase in the activation of the checkpoint protein kinase Rad53. © 2018 The Authors. Published under the terms of the CC BY 4.0 license.

Free energy simulations reveal a double mutant avian H5N1 virus hemagglutinin with altered receptor binding specificity.

PubMed

Das, Payel; Li, Jingyuan; Royyuru, Ajay K; Zhou, Ruhong

2009-08-01

Historically, influenza pandemics have been triggered when an avian influenza virus or a human/avian reassorted virus acquires the ability to replicate efficiently and become transmissible in the human population. Most critically, the major surface glycoprotein hemagglutinin (HA) must adapt to the usage of human-like (alpha-2,6-linked) sialylated glycan receptors. Therefore, identification of mutations that can switch the currently circulating H5N1 HA receptor binding specificity from avian to human might provide leads to the emergence of pandemic H5N1 viruses. To define such mutations in the H5 subtype, here we provide a computational framework that combines molecular modeling with extensive free energy simulations. Our results show that the simulated binding affinities are in good agreement with currently available experimental data. Moreover, we predict that one double mutation (V135S and A138S) in HA significantly enhances alpha-2,6-linked receptor recognition by the H5 subtype. Our simulations indicate that this double mutation in H5N1 HA increases the binding affinity to alpha-2,6-linked sialic acid receptors by 2.6 +/- 0.7 kcal/mol per HA monomer that primarily arises from the electrostatic interactions. Further analyses reveal that introduction of this double mutation results in a conformational change in the receptor binding pocket of H5N1 HA. As a result, a major rearrangement occurs in the hydrogen-bonding network of HA with the human receptor, making the human receptor binding pattern of double mutant H5N1 HA surprisingly similar to that observed in human H1N1 HA. These large scale molecular simulations on single and double mutants thus provide new insights into our understanding toward human adaptation of the avian H5N1 virus. 2009 Wiley Periodicals, Inc.

Spontaneous Chloroplast Mutants Mostly Occur by Replication Slippage and Show a Biased Pattern in the Plastome of Oenothera.

PubMed

Massouh, Amid; Schubert, Julia; Yaneva-Roder, Liliya; Ulbricht-Jones, Elena S; Zupok, Arkadiusz; Johnson, Marc T J; Wright, Stephen I; Pellizzer, Tommaso; Sobanski, Johanna; Bock, Ralph; Greiner, Stephan

2016-04-01

Spontaneous plastome mutants have been used as a research tool since the beginning of genetics. However, technical restrictions have severely limited their contributions to research in physiology and molecular biology. Here, we used full plastome sequencing to systematically characterize a collection of 51 spontaneous chloroplast mutants in Oenothera (evening primrose). Most mutants carry only a single mutation. Unexpectedly, the vast majority of mutations do not represent single nucleotide polymorphisms but are insertions/deletions originating from DNA replication slippage events. Only very few mutations appear to be caused by imprecise double-strand break repair, nucleotide misincorporation during replication, or incorrect nucleotide excision repair following oxidative damage. U-turn inversions were not detected. Replication slippage is induced at repetitive sequences that can be very small and tend to have high A/T content. Interestingly, the mutations are not distributed randomly in the genome. The underrepresentation of mutations caused by faulty double-strand break repair might explain the high structural conservation of seed plant plastomes throughout evolution. In addition to providing a fully characterized mutant collection for future research on plastid genetics, gene expression, and photosynthesis, our work identified the spectrum of spontaneous mutations in plastids and reveals that this spectrum is very different from that in the nucleus. © 2016 American Society of Plant Biologists. All rights reserved.

Attenuated Actinobacillus pleuropneumoniae double-deletion mutant S-8∆clpP/apxIIC confers protection against homologous or heterologous strain challenge.

PubMed

Xie, Fang; Li, Gang; Zhou, Long; Zhang, Yanhe; Cui, Ning; Liu, Siguo; Wang, Chunlai

2017-01-06

Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, which leads to large economic losses to the swine industry worldwide. In this study, S-8△clpP△apxIIC, a double-deletion mutant of A. pleuropneumoniae was constructed, and its safety and protective efficacy were evaluated in pigs. The S-8△clpP△apxIIC mutant exhibited attenuated virulence in a murine (BALB/c) model, and caused no detrimental effects on pigs even at a dose of up to 1.0 × 10 9  CFU. Furthermore, the S-8△clpP△apxIIC mutant was able to induce a strong immune response in pigs, which included high levels of IgG1 and IgG2, stimulated gamma interferon (IFN-γ), interleukin 12 (IL-12), and interleukin 4 (IL-4) production, and conferred effective protection against the lethal challenge with A. pleuropneumoniae serovars 7 or 5a. The pigs in the S-8△clpP△apxIIC immunized groups have no lesions and reduced bacterial loads in the lung tissue after challenge. The data obtained in this study suggest that the S-8△clpP△apxIIC mutant can serve as a highly immunogenic and potential live attenuated vaccine candidate against A. pleuropneumoniae infection.

Loss of RNA-directed DNA Methylation in Maize Chromomethylase and DDM1-type Nucleosome Remodeler Mutants.

PubMed

Fu, Fang-Fang; Dawe, R Kelly; Gent, Jonathan I

2018-06-08

Plants make use of distinct types of DNA methylation characterized by their DNA methyltransferases and modes of regulation. One type, RNA-directed DNA methylation (RdDM), is guided by small interfering RNAs (siRNAs) to the edges of transposons that are close to genes, areas called mCHH islands in maize (Zea mays). Another type, chromomethylation, is guided by histone H3 lysine 9 methylation to heterochromatin across the genome. We examined DNA methylation and small RNA expression in plant tissues that were mutant for both copies of the genes encoding chromomethylases as well as mutants for both copies of the genes encoding DECREASED DNA METHYLATION1 (DDM1)-type nucleosome remodelers, which facilitate chromomethylation. Both sets of double mutants were nonviable but produced embryos and endosperm. RdDM was severely compromised in the double mutant embryos, both in terms of DNA methylation and siRNAs. Loss of 24-nt siRNA from mCHH islands was coupled with a gain of 21-, 22-, and 24-nt siRNAs in heterochromatin. These results reveal a requirement for both chromomethylation and DDM1-type nucleosome remodeling for RdDM in mCHH islands, which we hypothesize is due to dilution of RdDM components across the genome when heterochromatin is compromised. © 2018 American Society of Plant Biologists. All rights reserved.

Methylation of Gibberellins by Arabidopsis GAMT1 and GAMT2[W

PubMed Central

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

2007-01-01

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

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

PubMed

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

2002-07-19

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

Localization and roles of Ski8p protein in Sordaria meiosis and delineation of three mechanistically distinct steps of meiotic homolog juxtaposition.

PubMed

Tessé, Sophie; Storlazzi, Aurora; Kleckner, Nancy; Gargano, Silvana; Zickler, Denise

2003-10-28

Ski8p is implicated in degradation of non-poly(A) and double-stranded RNA, and in meiotic DNA recombination. We have identified the Sordaria macrospora SKI8 gene. Ski8p is cytoplasmically localized in all vegetative and sexual cycle cells, and is nuclear localized, specifically in early-mid-meiotic prophase, in temporal correlation with Spo11p, the meiotic double-strand break (DSB) transesterase. Localizations of Ski8p and Spo11p are mutually interdependent. ski8 mutants exhibit defects in vegetative growth, entry into the sexual program, and sporulation. Diverse meiotic defects, also seen in spo11 mutants, are diagnostic of DSB absence, and they are restored by exogenous DSBs. These results suggest that Ski8p promotes meiotic DSB formation by acting directly within meiotic prophase chromosomes. Mutant phenotypes also divide meiotic homolog juxtaposition into three successive, mechanistically distinct steps; recognition, presynaptic alignment, and synapsis, which are distinguished by their differential dependence on DSBs.

Localization and roles of Ski8p protein in Sordaria meiosis and delineation of three mechanistically distinct steps of meiotic homolog juxtaposition

PubMed Central

Tessé, Sophie; Storlazzi, Aurora; Kleckner, Nancy; Gargano, Silvana; Zickler, Denise

2003-01-01

Ski8p is implicated in degradation of non-poly(A) and double-stranded RNA, and in meiotic DNA recombination. We have identified the Sordaria macrospora SKI8 gene. Ski8p is cytoplasmically localized in all vegetative and sexual cycle cells, and is nuclear localized, specifically in early-mid-meiotic prophase, in temporal correlation with Spo11p, the meiotic double-strand break (DSB) transesterase. Localizations of Ski8p and Spo11p are mutually interdependent. ski8 mutants exhibit defects in vegetative growth, entry into the sexual program, and sporulation. Diverse meiotic defects, also seen in spo11 mutants, are diagnostic of DSB absence, and they are restored by exogenous DSBs. These results suggest that Ski8p promotes meiotic DSB formation by acting directly within meiotic prophase chromosomes. Mutant phenotypes also divide meiotic homolog juxtaposition into three successive, mechanistically distinct steps; recognition, presynaptic alignment, and synapsis, which are distinguished by their differential dependence on DSBs. PMID:14563920

Structure- and reactivity-based development of covalent inhibitors of the activating and gatekeeper mutant forms of the epidermal growth factor receptor (EGFR).

PubMed

Ward, Richard A; Anderton, Mark J; Ashton, Susan; Bethel, Paul A; Box, Matthew; Butterworth, Sam; Colclough, Nicola; Chorley, Christopher G; Chuaqui, Claudio; Cross, Darren A E; Dakin, Les A; Debreczeni, Judit É; Eberlein, Cath; Finlay, M Raymond V; Hill, George B; Grist, Matthew; Klinowska, Teresa C M; Lane, Clare; Martin, Scott; Orme, Jonathon P; Smith, Peter; Wang, Fengjiang; Waring, Michael J

2013-09-12

A novel series of small-molecule inhibitors has been developed to target the double mutant form of the epidermal growth factor receptor (EGFR) tyrosine kinase, which is resistant to treatment with gefitinib and erlotinib. Our reported compounds also show selectivity over wild-type EGFR. Guided by molecular modeling, this series was evolved to target a cysteine residue in the ATP binding site via covalent bond formation and demonstrates high levels of activity in cellular models of the double mutant form of EGFR. In addition, these compounds show significant activity against the activating mutations, which gefitinib and erlotinib target and inhibition of which gives rise to their observed clinical efficacy. A glutathione (GSH)-based assay was used to measure thiol reactivity toward the electrophilic functionality of the inhibitor series, enabling both the identification of a suitable reactivity window for their potency and the development of a reactivity quantitative structure-property relationship (QSPR) to support design.

Peroxisome proliferator-activated receptor gamma agonist troglitazone induces colon tumors in normal C57BL/6J mice and enhances colonic carcinogenesis in Apc1638 N/+ Mlh1+/- double mutant mice.

PubMed

Yang, Kan; Fan, Kun-Hua; Lamprecht, Sergio A; Edelmann, Winfried; Kopelovich, Levy; Kucherlapati, Raju; Lipkin, Martin

2005-09-10

The role of the nuclear peroxisome proliferator-activated receptor-gamma (PPAR-gamma) in colon tumorigenesis remains controversial. Notwithstanding evidence that PPAR-gamma ligands impede murine colorectal carcinogenesis, PPAR-gamma agonists have been shown to enhance in vivo tumor formation in mouse models of human colon cancer. Our study was designed to determine whether troglitazone (TGZ) induces colonic tumor formation in normal C57BL/6J mice and enhances colorectal carcinogenesis in double mutant Apc1638N/+ Mlh1+/- mice fed a standard AIN-76A diet. We report herein that not only does TGZ enhance carcinogenesis in the large intestine of mutant mice predisposed to intestinal carcinogenesis but TGZ also induces colonic tumors in normal mice without gene targeting or carcinogen administration. This observation indicates that preexisting mutational events are not necessary for induction of colonic tumors by activated PPAR-gamma in vivo. (c) 2005 Wiley-Liss, Inc.

Impact of resistance mutations on inhibitor binding to HIV-1 integrase

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

Chen, Qi; Buolamwini, John K.; Smith, Jeremy C.

2013-11-08

Here, HIV-1 integrase (IN) is essential for HIV-1 replication, catalyzing two key reaction steps termed 3' processing and strand transfer. Therefore, IN has become an important target for antiviral drug discovery. However, mutants have emerged, such as E92Q/N155H and G140S/Q148H, which confer resistance to raltegravir (RAL), the first IN strand transfer inhibitor (INSTI) approved by the FDA, and to the recently approved elvitegravir (EVG). To gain insights into the molecular mechanisms of ligand binding and drug resistance, we performed molecular dynamics (MD) simulations of homology models of the HIV-1 IN and four relevant mutants complexed with viral DNA and RAL.more » The results show that the structure and dynamics of the 140s loop, comprising residues 140 to 149, are strongly influenced by the IN mutations. In the simulation of the G140S/Q148H double mutant, we observe spontaneous dissociation of RAL from the active site, followed by an intrahelical swing-back of the 3' -OH group of nucleotide A17, consistent with the experimental observation that the G140S/Q148H mutant exhibits the highest resistance to RAL compared to other IN mutants. An important hydrogen bond between residues 145 and 148 is present in the wild-type IN but not in the G140S/Q148H mutant, accounting for the structural and dynamical differences of the 140s' loop and ultimately impairing RAL binding in the double mutant. End-point free energy calculations that broadly capture the experimentally known RAL binding profiles elucidate the contributions of the 140s' loop to RAL binding free energies and suggest possible approaches to overcoming drug resistance.« less

Mutation and virulence assessment of chromosomal genes of Rhodococcus equi 103

PubMed Central

Pei, Yanlong; Parreira, Valeria; Nicholson, Vivian M.; Prescott, John F.

2007-01-01

Rhodococcus equi can cause severe or fatal pneumonia in foals as well as in immunocompromised animals and humans. Its ability to persist in macrophages is fundamental to how it causes disease, but the basis of this is poorly understood. To examine further the general application of a recently developed system of targeted gene mutation and to assess the importance of different genes in resistance to innate immune defenses, we disrupted the genes encoding high-temperature requirement A (htrA), nitrate reductase (narG), peptidase D (pepD), phosphoribosylaminoimidazole-succinocarboxamide synthase (purC), and superoxide dismutase (sodC) in strain 103 of R. equi using a double-crossover homologous recombination approach. Virulence testing by clearance after intravenous injection in mice showed that the htrA and narG mutants were fully attenuated, the purC and sodC mutants were unchanged, and the pepD mutant was slightly attenuated. Complementation with the pREM shuttle plasmid restored the virulence of the htrA and pepD mutants but not that of the narG mutant. A single-crossover mutation approach was simpler and faster than the double-crossover homologous recombination technique and was used to obtain mutations in 6 other genes potentially involved in virulence (clpB, fadD8, fbpB, glnA1, regX3, and sigF). These mutants were not attenuated in the mouse clearance assay. We were not able to obtain mutants for genes furA, galE, and sigE using the single-crossover mutation approach. In summary, the targeted-mutation system had general applicability but was not always completely successful, perhaps because some genes are essential under the growth conditions used or because the success of mutation depends on the target genes. PMID:17193875

Mutation and virulence assessment of chromosomal genes of Rhodococcus equi 103.

PubMed

Pei, Yanlong; Parreira, Valeria; Nicholson, Vivian M; Prescott, John F

2007-01-01

Rhodococcus equi can cause severe or fatal pneumonia in foals as well as in immunocompromised animals and humans. Its ability to persist in macrophages is fundamental to how it causes disease, but the basis of this is poorly understood. To examine further the general application of a recently developed system of targeted gene mutation and to assess the importance of different genes in resistance to innate immune defenses, we disrupted the genes encoding high-temperature requirement A (htrA), nitrate reductase (narG), peptidase D (pepD), phosphoribosylaminoimidazole-succinocarboxamide synthase (purC), and superoxide dismutase (sodC) in strain 103 of R. equi using a double-crossover homologous recombination approach. Virulence testing by clearance after intravenous injection in mice showed that the htrA and narG mutants were fully attenuated, the purC and sodC mutants were unchanged, and the pepD mutant was slightly attenuated. Complementation with the pREM shuttle plasmid restored the virulence of the htrA and pepD mutants but not that of the narG mutant. A single-crossover mutation approach was simpler and faster than the double-crossover homologous recombination technique and was used to obtain mutations in 6 other genes potentially involved in virulence (clpB, fadD8, fbpB, glnA1, regX3, and sigF). These mutants were not attenuated in the mouse clearance assay. We were not able to obtain mutants for genesfurA, galE, and sigE using the single-crossover mutation approach. In summary, the targeted-mutation system had general applicability but was not always completely successful, perhaps because some genes are essential under the growth conditions used or because the success of mutation depends on the target genes.

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.

Activity of Gemifloxacin against Quinolone-Resistant Streptococcus pneumoniae Strains In Vitro and in a Mouse Pneumonia Model

PubMed Central

Azoulay-Dupuis, E.; Bédos, J. P.; Mohler, J.; Moine, P.; Cherbuliez, C.; Peytavin, G.; Fantin, B.; Köhler, T.

2005-01-01

Gemifloxacin is a novel fluoronaphthyridone quinolone with enhanced in vitro activity against Streptococcus pneumoniae. We investigated the activities of gemifloxacin and trovafloxacin, their abilities to select for resistance in vitro and in vivo, and their efficacies in a mouse model of acute pneumonia. Immunocompetent Swiss mice were infected with 105 CFU of a virulent, encapsulated S. pneumoniae strain, P-4241, or its isogenic parC, gyrA, parC gyrA, and efflux mutant derivatives (serotype 3); and leukopenic mice were infected with 107 CFU of two poorly virulent clinical strains (serotype 11A) carrying either a parE mutation or a parC, gyrA, and parE triple mutation. The drugs were administered six times every 12 h, starting at either 3 or 18 h postinfection. In vitro, gemifloxacin was the most potent agent against strains with and without acquired resistance to fluoroquinolones. While control mice died within 6 days, gemifloxacin at doses of 25 and 50 mg/kg of body weight was highly effective (survival rates, 90 to 100%) against the wild-type strain and against mutants harboring a single mutation, corresponding to area under the time-versus-serum concentration curve at 24 h (AUC24)/MIC ratios of 56.5 to 113, and provided a 40% survival rate against a mutant with a double mutation (parC and gyrA). A total AUC24/MIC ratio of 28.5 was associated with poor efficacy and the emergence of resistant mutants. Trovafloxacin was as effective as gemifloxacin against mutants with single mutations but did not provide any protection against the mutant with double mutations, despite treatment with a high dose of 200 mg/kg. Gemifloxacin preferentially selected for parC mutants both in vitro and in vivo. PMID:15728901

Actinobacillus pleuropneumoniae Iron Transport and Urease Activity: Effects on Bacterial Virulence and Host Immune Response

PubMed Central

Baltes, Nina; Tonpitak, Walaiporn; Gerlach, Gerald-F.; Hennig-Pauka, Isabel; Hoffmann-Moujahid, Astrid; Ganter, Martin; Rothkötter, Hermann-J.

2001-01-01

Actinobacillus pleuropneumoniae, a porcine respiratory tract pathogen, has been shown to express transferrin-binding proteins and urease during infection. Both activities have been associated with virulence; however, their functional role for infection has not yet been elucidated. We used two isogenic A. pleuropneumoniae single mutants (ΔexbB and ΔureC) and a newly constructed A. pleuropneumoniae double (ΔureC ΔexbB) mutant in aerosol infection experiments. Neither the A. pleuropneumoniae ΔexbB mutant nor the double ΔureC ΔexbB mutant was able to colonize sufficiently long to initiate a detectable humoral immune response. These results imply that the ability to utilize transferrin-bound iron is required for multiplication and persistence of A. pleuropneumoniae in the porcine respiratory tract. The A. pleuropneumoniae ΔureC mutant and the parent strain both caused infections that were indistinguishable from one another in the acute phase of disease; however, 3 weeks postinfection the A. pleuropneumoniae ΔureC mutant, in contrast to the parent strain, could not be isolated from healthy lung tissue. In addition, the local immune response—as assessed by fluorescence-activated cell sorter and enzyme-linked immunosorbent spot analyses—revealed a significantly higher number of A. pleuropneumoniae-specific B cells in the bronchoalveolar lavage fluid (BALF) of pigs infected with the A. pleuropneumoniae ΔureC mutant than in the BALF of those infected with the parent strain. These results imply that A. pleuropneumoniae urease activity may cause sufficient impairment of the local immune response to slightly improve the persistence of the urease-positive A. pleuropneumoniae parent strain. PMID:11119539

nana plant2 Encodes a Maize Ortholog of the Arabidopsis Brassinosteroid Biosynthesis Gene DWARF1, Identifying Developmental Interactions between Brassinosteroids and Gibberellins1[OPEN

PubMed Central

Budka, Josh; Fujioka, Shozo; Johal, Gurmukh

2016-01-01

A small number of phytohormones dictate the pattern of plant form affecting fitness via reproductive architecture and the plant’s ability to forage for light, water, and nutrients. Individual phytohormone contributions to plant architecture have been studied extensively, often following a single component of plant architecture, such as plant height or branching. Both brassinosteroid (BR) and gibberellin (GA) affect plant height, branching, and sexual organ development in maize (Zea mays). We identified the molecular basis of the nana plant2 (na2) phenotype as a loss-of-function mutation in one of the two maize paralogs of the Arabidopsis (Arabidopsis thaliana) BR biosynthetic gene DWARF1 (DWF1). These mutants accumulate the DWF1 substrate 24-methylenecholesterol and exhibit decreased levels of downstream BR metabolites. We utilized this mutant and known GA biosynthetic mutants to investigate the genetic interactions between BR and GA. Double mutants exhibited additivity for some phenotypes and epistasis for others with no unifying pattern, indicating that BR and GA interact to affect development but in a context-dependent manner. Similar results were observed in double mutant analyses using additional BR and GA biosynthetic mutant loci. Thus, the BR and GA interactions were neither locus nor allele specific. Exogenous application of GA3 to na2 and d5, a GA biosynthetic mutant, also resulted in a diverse pattern of growth responses, including BR-dependent GA responses. These findings demonstrate that BR and GA do not interact via a single inclusive pathway in maize but rather suggest that differential signal transduction and downstream responses are affected dependent upon the developmental context. PMID:27288361

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

PubMed Central

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

2016-01-01

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

Electrostatic dominoes: long distance propagation of mutational effects in photosynthetic reaction centers of Rhodobacter capsulatus.

PubMed

Sebban, P; Maróti, P; Schiffer, M; Hanson, D K

1995-07-04

Two point mutants from the purple bacterium Rhodobacter capsulatus, both modified in the M protein of the photosynthetic reaction center, have been studied by flash-induced absorbance spectroscopy. These strains carry either the M231Arg --> Leu or M43ASN --> Asp mutations, which are located 9 and 15 A, respectively, from the terminal electron acceptor QB. In the wild-type Rb. sphaeroides structure, M231Arg is involved in a conserved salt bridge with H125Glu and H232Glu and M43Asn is located among several polar residues that form or surround the QB binding site. These substitutions were originally uncovered in phenotypic revertants isolated from the photosynthetically incompetent L212Glu-L213Asp --> Ala-Ala site-specific double mutant. As second-site suppressor mutations, they have been shown to restore the proton transfer function that is interrupted in the L212Ala-L213Ala double mutant. The electrostatic effects that are induced in reaction centers by the M231Arg --> Leu and M43Asn --> Asp substitutions are roughly the same in either the double-mutant or wild-type backgrounds. In a reaction center that is otherwise wild type in sequence, they decrease the free energy gap between the QA- and QB- states by 24 +/- 5 and 45 +/- 5 meV, respectively. The pH dependences of K2, the QA-QB <--> QAQB- equilibrium constant, are altered in reaction centers that carry either of these substitutions, revealing differences in the pKas of titratable groups compared to the wild type.(ABSTRACT TRUNCATED AT 250 WORDS)

Endosomal/Lysosomal Processing of Gangliosides Affects Neuronal Cholesterol Sequestration in Niemann-Pick Disease Type C

PubMed Central

Zhou, Sharon; Davidson, Cristin; McGlynn, Robert; Stephney, Gloria; Dobrenis, Kostantin; Vanier, Marie T.; Walkley, Steven U.

2011-01-01

Niemann-Pick disease type C (NPC) is a severe neurovisceral lysosomal storage disorder caused by defects in NPC1 or NPC2 proteins. Although numerous studies support the primacy of cholesterol storage, neurons of double-mutant mice lacking both NPC1 and an enzyme required for synthesis of all complex gangliosides (β1,4GalNAc transferase) have been reported to exhibit dramatically reduced cholesterol sequestration. Here we show that NPC2-deficient mice lacking this enzyme also exhibit reduced cholesterol, but that genetically restricting synthesis to only a-series gangliosides fully restores neuronal cholesterol storage to typical disease levels. Examining the subcellular locations of sequestered compounds in neurons lacking NPC1 or NPC2 by confocal microscopy revealed that cholesterol and the two principal storage gangliosides (GM2 and GM3) were not consistently co-localized within the same intracellular vesicles. To determine whether the lack of GM2 and GM3 co-localization was due to differences in synthetic versus degradative pathway expression, we generated mice lacking both NPC1 and lysosomal β-galactosidase, and therefore unable to generate GM2 and GM3 in lysosomes. Double mutants lacked both gangliosides, indicating that each is the product of endosomal/lysosomal processing. Unexpectedly, GM1 accumulation in double mutants increased compared to single mutants consistent with a direct role for NPC1 in ganglioside salvage. These studies provide further evidence that NPC1 and NPC2 proteins participate in endosomal/lysosomal processing of both sphingolipids and cholesterol. PMID:21708114

Arabidopsis Chloroplast Mini-Ribonuclease III Participates in rRNA Maturation and Intron Recycling

PubMed Central

Hotto, Amber M.; Castandet, Benoît; Gilet, Laetitia; Higdon, Andrea; Condon, Ciarán; Stern, David B.

2015-01-01

RNase III proteins recognize double-stranded RNA structures and catalyze endoribonucleolytic cleavages that often regulate gene expression. Here, we characterize the functions of RNC3 and RNC4, two Arabidopsis thaliana chloroplast Mini-RNase III-like enzymes sharing 75% amino acid sequence identity. Whereas rnc3 and rnc4 null mutants have no visible phenotype, rnc3/rnc4 (rnc3/4) double mutants are slightly smaller and chlorotic compared with the wild type. In Bacillus subtilis, the RNase Mini-III is integral to 23S rRNA maturation. In Arabidopsis, we observed imprecise maturation of 23S rRNA in the rnc3/4 double mutant, suggesting that exoribonucleases generated staggered ends in the absence of specific Mini-III-catalyzed cleavages. A similar phenotype was found at the 3′ end of the 16S rRNA, and the primary 4.5S rRNA transcript contained 3′ extensions, suggesting that Mini-III catalyzes several processing events of the polycistronic rRNA precursor. The rnc3/4 mutant showed overaccumulation of a noncoding RNA complementary to the 4.5S-5S rRNA intergenic region, and its presence correlated with that of the extended 4.5S rRNA precursor. Finally, we found rnc3/4-specific intron degradation intermediates that are probable substrates for Mini-III and show that B. subtilis Mini-III is also involved in intron regulation. Overall, this study extends our knowledge of the key role of Mini-III in intron and noncoding RNA regulation and provides important insight into plastid rRNA maturation. PMID:25724636

Double nanohole optical tweezers visualize protein p53 suppressing unzipping of single DNA-hairpins

PubMed Central

Kotnala, Abhay; Gordon, Reuven

2014-01-01

Here we report on the use of double-nanohole (DNH) optical tweezers as a label-free and free-solution single-molecule probe for protein–DNA interactions. Using this approach, we demonstrate the unzipping of individual 10 base pair DNA-hairpins, and quantify how tumor suppressor p53 protein delays the unzipping. From the Arrhenius behavior, we find the energy barrier to unzipping introduced by p53 to be 2 × 10−20 J, whereas cys135ser mutant p53 does not show suppression of unzipping, which gives clues to its functional inability to suppress tumor growth. This transformative approach to single molecule analysis allows for ultra-sensitive detection and quantification of protein–DNA interactions to revolutionize the fight against genetic diseases. PMID:24940547

A mutational analysis of the yeast proliferating cell nuclear antigen indicates distinct roles in DNA replication and DNA repair.

PubMed Central

Ayyagari, R; Impellizzeri, K J; Yoder, B L; Gary, S L; Burgers, P M

1995-01-01

The saccharomyces cerevisiae proliferating cell nuclear antigen (PCNA), encoded by the POL30 gene, is essential for DNA replication and DNA repair processes. Twenty-one site-directed mutations were constructed in the POL30 gene, each mutation changing two adjacently located charged amino acids to alanines. Although none of the mutant strains containing these double-alanine mutations as the sole source of PCNA were temperature sensitive or cold sensitive for growth, about a third of the mutants showed sensitivity to UV light. Some of those UV-sensitive mutants had elevated spontaneous mutation rates. In addition, several mutants suppressed a cold-sensitive mutation in the CDC44 gene, which encodes the large subunit of replication factor C. A cold-sensitive mutant, which was isolated by random mutagenesis, showed a terminal phenotype at the restrictive temperature consistent with a defect in DNA replication. Several mutant PCNAs were expressed and purified from Escherichia coli, and their in vitro properties were determined. The cold-sensitive mutant (pol30-52, S115P) was a monomer, rather than a trimer, in solution. This mutant was deficient for DNA synthesis in vitro. Partial restoration of DNA polymerase delta holoenzyme activity was achieved at 37 degrees C but not at 14 degrees C by inclusion of the macromolecular crowding agent polyethylene glycol in the assay. The only other mutant (pol30-6, DD41,42AA) that showed a growth defect was partially defective for interaction with replication factor C and DNA polymerase delta but completely defective for interaction with DNA polymerase epsilon. Two other mutants sensitive to DNA damage showed no defect in vitro. These results indicate that the latter mutants are specifically impaired in one or more DNA repair processes whereas pol30-6 and pol30-52 mutants show their primary defects in the basic DNA replication machinery with probable associated defects in DNA repair. Therefore, DNA repair requires interactions between repair-specific protein(s) and PCNA, which are distinct from those required for DNA replication. PMID:7623835

The deltaA gene of zebrafish mediates lateral inhibition of hair cells in the inner ear and is regulated by pax2.1.

PubMed

Riley, B B; Chiang, M; Farmer, L; Heck, R

1999-12-01

Recent studies of inner ear development suggest that hair cells and support cells arise within a common equivalence group by cell-cell interactions mediated by Delta and Notch proteins. We have extended these studies by analyzing the effects of a mutant allele of the zebrafish deltaA gene, deltaA(dx2), which encodes a dominant-negative protein. deltaA(dx2/dx2 )homozygous mutants develop with a 5- to 6-fold excess of hair cells and a severe deficiency of support cells. In addition, deltaA(dx2/dx2) mutants show an increased number of cells expressing pax2.1 in regions where hair cells are normally produced. Immunohistological analysis of wild-type and deltaA(dx2/dx2) mutant embryos confirmed that pax2.1 is expressed during the initial stages of hair cell differentiation and is later maintained at high levels in mature hair cells. In contrast, pax2.1 is not expressed in support cells. To address the function of pax2.1, we analyzed hair cell differentiation in no isthmus mutant embryos, which are deficient for pax2.1 function. no isthmus mutant embryos develop with approximately twice the normal number of hair cells. This neurogenic defect correlates with reduced levels of expression of deltaA and deltaD in the hair cells in no isthmus mutants. Analysis of deltaA(dx2/dx2); no isthmus double mutants showed that no isthmus suppresses the deltaA(dx2) phenotype, probably by reducing levels of the dominant-negative mutant protein. This interpretation was supported by analysis of T(msxB)(b220), a deletion that removes the deltaA locus. Reducing the dose of deltaA(dx2) by generating deltaA(dx2)/T(msxB)(b220 )trans-heterozygotes weakens the neurogenic effects of deltaA(dx2), whereas T(msxB)(b220) enhances the neurogenic defects of no isthmus. mind bomb, another strong neurogenic mutation that may disrupt reception of Delta signals, causes a 10-fold increase in hair cell production and is epistatic to both no isthmus and deltaA(dx2). These data indicate that deltaA expressed by hair cells normally prevents adjacent cells from adopting the same cell fate, and that pax2.1 is required for normal levels of Delta-mediated lateral inhibition.

Metabolomic signature associated with reproduction-regulated aging in Caenorhabditis elegans.

PubMed

Wan, Qin-Li; Shi, Xiaohuo; Liu, Jiangxin; Ding, Ai-Jun; Pu, Yuan-Zhu; Li, Zhigang; Wu, Gui-Sheng; Luo, Huai-Rong

2017-02-06

In Caenorhabditis elegans (C. elegans) , ablation of germline stem cells (GSCs) leads to infertility, which extends lifespan. It has been reported that aging and reproduction are both inextricably associated with metabolism. However, few studies have investigated the roles of polar small molecules metabolism in regulating longevity by reproduction. In this work, we combined the nuclear magnetic resonance (NMR) and ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) to profile the water-soluble metabolome in C. elegans . Comparing the metabolic fingerprint between two physiological ages among different mutants, our results demonstrate that aging is characterized by metabolome remodeling and metabolic decline. In addition, by analyzing the metabolic profiles of long-lived germline-less glp-1 mutants, we discovered that glp-1 mutants regulate the levels of many age-variant metabolites to attenuate aging, including elevated concentrations of the pyrimidine and purine metabolism intermediates and decreased concentrations of the citric acid cycle intermediates. Interestingly, by analyzing the metabolome of daf-16;glp-1 double mutants, our results revealed that some metabolic exchange contributing to germline-mediated longevity was mediated by transcription factor FOXO/DAF-16, including pyrimidine metabolism and the TCA cycle. Based on a comprehensive metabolic analysis, we provide novel insight into the relationship between longevity and metabolism regulated by germline signals in C. elegans .

Metabolomic signature associated with reproduction-regulated aging in Caenorhabditis elegans

PubMed Central

Wan, Qin-Li; Shi, Xiaohuo; Liu, Jiangxin; Ding, Ai-Jun; Pu, Yuan-Zhu; Li, Zhigang; Wu, Gui-Sheng; Luo, Huai-Rong

2017-01-01

In Caenorhabditis elegans (C. elegans), ablation of germline stem cells (GSCs) leads to infertility, which extends lifespan. It has been reported that aging and reproduction are both inextricably associated with metabolism. However, few studies have investigated the roles of polar small molecules metabolism in regulating longevity by reproduction. In this work, we combined the nuclear magnetic resonance (NMR) and ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) to profile the water-soluble metabolome in C. elegans. Comparing the metabolic fingerprint between two physiological ages among different mutants, our results demonstrate that aging is characterized by metabolome remodeling and metabolic decline. In addition, by analyzing the metabolic profiles of long-lived germline-less glp-1 mutants, we discovered that glp-1 mutants regulate the levels of many age-variant metabolites to attenuate aging, including elevated concentrations of the pyrimidine and purine metabolism intermediates and decreased concentrations of the citric acid cycle intermediates. Interestingly, by analyzing the metabolome of daf-16;glp-1 double mutants, our results revealed that some metabolic exchange contributing to germline-mediated longevity was mediated by transcription factor FOXO/DAF-16, including pyrimidine metabolism and the TCA cycle. Based on a comprehensive metabolic analysis, we provide novel insight into the relationship between longevity and metabolism regulated by germline signals in C. elegans PMID:28177875

Molecular dynamics simulation studies of the wild type and E92Q/N155H mutant of Elvitegravir-resistance HIV-1 integrase.

PubMed

Chen, Qi; Cheng, Xiaolin; Wei, Dongqing; Xu, Qin

2015-03-01

Although Elvitegravir (EVG) is a newly developed antiretrovirals drug to treat the acquired immunodeficiency syndrome (AIDS), drug resistance has already been found in clinic, such as E92Q/N155H and Q148H/G140S. Several structural investigations have already been reported to reveal the molecular mechanism of the drug resistance. As full length crystal structure for HIV-1 integrase is still unsolved, we herein use the crystal structure of the full length prototype foamy virus (PFV) in complex with virus DNA and inhibitor Elvitegravir as a template to construct the wild type and E92Q/N155H mutant system of HIV-1 integrase. Molecular dynamic simulations was used to revel the binding mode and the drug resistance of the EVG ligand in E92Q/N155H. Several important interactions were discovered between the mutated residues and the residues in the active site of the E92Q/N155H double mutant pattern, and cross correlation and clustering methods were used for detailed analysis. The results from the MD simulation studies will be used to guide the experimental efforts of developing novel inhibitors against drug-resistant HIV integrase mutants.

The Starch Granule-Associated Protein EARLY STARVATION1 Is Required for the Control of Starch Degradation in Arabidopsis thaliana Leaves[OPEN

PubMed Central

Feike, Doreen; Seung, David; Graf, Alexander; Bischof, Sylvain; Ellick, Tamaryn; Coiro, Mario; Soyk, Sebastian; Eicke, Simona; Mettler-Altmann, Tabea; Lu, Kuan Jen; Trick, Martin; Zeeman, Samuel C.

2016-01-01

To uncover components of the mechanism that adjusts the rate of leaf starch degradation to the length of the night, we devised a screen for mutant Arabidopsis thaliana plants in which starch reserves are prematurely exhausted. The mutation in one such mutant, named early starvation1 (esv1), eliminates a previously uncharacterized protein. Starch in mutant leaves is degraded rapidly and in a nonlinear fashion, so that reserves are exhausted 2 h prior to dawn. The ESV1 protein and a similar uncharacterized Arabidopsis protein (named Like ESV1 [LESV]) are located in the chloroplast stroma and are also bound into starch granules. The region of highest similarity between the two proteins contains a series of near-repeated motifs rich in tryptophan. Both proteins are conserved throughout starch-synthesizing organisms, from angiosperms and monocots to green algae. Analysis of transgenic plants lacking or overexpressing ESV1 or LESV, and of double mutants lacking ESV1 and another protein necessary for starch degradation, leads us to propose that these proteins function in the organization of the starch granule matrix. We argue that their misexpression affects starch degradation indirectly, by altering matrix organization and, thus, accessibility of starch polymers to starch-degrading enzymes. PMID:27207856

Two phloem nitrate transporters, NRT1.11 and NRT1.12, are important for redistributing xylem-borne nitrate to enhance plant growth.

PubMed

Hsu, Po-Kai; Tsay, Yi-Fang

2013-10-01

This study of the Arabidopsis (Arabidopsis thaliana) nitrate transporters NRT1.11 and NRT1.12 reveals how the interplay between xylem and phloem transport of nitrate ensures optimal nitrate distribution in leaves for plant growth. Functional analysis in Xenopus laevis oocytes showed that both NRT1.11 and NRT1.12 are low-affinity nitrate transporters. Quantitative reverse transcription-polymerase chain reaction and immunoblot analysis showed higher expression of these two genes in larger expanded leaves. Green fluorescent protein and β-glucuronidase reporter analyses indicated that NRT1.11 and NRT1.12 are plasma membrane transporters expressed in the companion cells of the major vein. In nrt1.11 nrt1.12 double mutants, more root-fed (15)NO3(-) was translocated to mature and larger expanded leaves but less to the youngest tissues, suggesting that NRT1.11 and NRT1.12 are required for transferring root-derived nitrate into phloem in the major veins of mature and larger expanded leaves for redistributing to the youngest tissues. Distinct from the wild type, nrt1.11 nrt1.12 double mutants show no increase of plant growth at high nitrate supply. These data suggested that NRT1.11 and NRT1.12 are involved in xylem-to-phloem transfer for redistributing nitrate into developing leaves, and such nitrate redistribution is a critical step for optimal plant growth enhanced by increasing external nitrate.

Human mitochondrial NDUFS3 protein bearing Leigh syndrome mutation is more prone to aggregation than its wild-type.

PubMed

Jaokar, Tulika M; Patil, Deepak P; Shouche, Yogesh S; Gaikwad, Sushama M; Suresh, C G

2013-12-01

NDUFS3 is an integral subunit of the Q module of the mitochondrial respiratory Complex-I. The combined mutation (T145I + R199W) in the subunit is reported to cause optic atrophy and Leigh syndrome accompanied by severe Complex-I deficiency. In the present study, we have cloned and overexpressed the human NDUFS3 subunit and its double mutant in a soluble form in Escherichia coli. The wild-type (w-t) and mutant proteins were purified to homogeneity through a serial two-step chromatographic purification procedure of anion exchange followed by size exclusion chromatography. The integrity and purity of the purified proteins was confirmed by Western blot analysis and MALDI-TOF/TOF. The conformational transitions of the purified subunits were studied through steady state as well as time resolved fluorescence and CD spectroscopy under various denaturing conditions. The mutant protein showed altered polarity around tryptophan residues, changed quenching parameters and also noticeably altered secondary and tertiary structure compared to the w-t protein. Mutant also exhibited a higher tendency than the w-t protein for aggregation which was examined using fluorescent (Thioflavin-T) and spectroscopic (Congo red) dye binding techniques. The pH stability of the w-t and mutant proteins varied at extreme acidic pH and the molten globule like structure of w-t at pH1 was absent in case of the mutant protein. Both the w-t and mutant proteins showed multi-step thermal and Gdn-HCl induced unfolding. Thus, the results provide insight into the alterations of NDUFS3 protein structure caused by the mutations, affecting the overall integrity of the protein and finally leading to disruption of Complex-I assembly. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Different mechanisms of radiation-induced loss of heterozygosity in two human lymphoid cell lines from a single donor

NASA Technical Reports Server (NTRS)

Wiese, C.; Gauny, S. S.; Liu, W. C.; Cherbonnel-Lasserre, C. L.; Kronenberg, A.

2001-01-01

Allelic loss is an important mutational mechanism in human carcinogenesis. Loss of heterozygosity (LOH) at an autosomal locus is one outcome of the repair of DNA double-strand breaks (DSBs) and can occur by deletion or by mitotic recombination. We report that mitotic recombination between homologous chromosomes occurred in human lymphoid cells exposed to densely ionizing radiation. We used cells derived from the same donor that express either normal TP53 (TK6 cells) or homozygous mutant TP53 (WTK1 cells) to assess the influence of TP53 on radiation-induced mutagenesis. Expression of mutant TP53 (Met 237 Ile) was associated with a small increase in mutation frequencies at the hemizygous HPRT (hypoxanthine phosphoribosyl transferase) locus, but the mutation spectra were unaffected at this locus. In contrast, WTK1 cells (mutant TP53) were 30-fold more susceptible than TK6 cells (wild-type TP53) to radiation-induced mutagenesis at the TK1 (thymidine kinase) locus. Gene dosage analysis combined with microsatellite marker analysis showed that the increase in TK1 mutagenesis in WTK1 cells could be attributed, in part, to mitotic recombination. The microsatellite marker analysis over a 64-cM region on chromosome 17q indicated that the recombinational events could initiate at different positions between the TK1 locus and the centromere. Virtually all of the recombinational LOH events extended beyond the TK1 locus to the most telomeric marker. In general, longer LOH tracts were observed in mutants from WTK1 cells than in mutants from TK6 cells. Taken together, the results demonstrate that the incidence of radi-ation-induced mutations is dependent on the genetic background of the cell at risk, on the locus examined, and on the mechanisms for mutation available at the locus of interest.

Escalating Plasmodium falciparum antifolate drug resistance mutations in Macha, rural Zambia

PubMed Central

Mkulama, Mtawa AP; Chishimba, Sandra; Sikalima, Jay; Rouse, Petrica; Thuma, Philip E; Mharakurwa, Sungano

2008-01-01

Background In Zambia the first-line treatment for uncomplicated malaria is artemisinin combination therapy (ACT), with artemether-lumefantrine currently being used. However, the antifolate regimen, sulphadoxine-pyrimethamine (SP), remains the treatment of choice in children weighing less than 5 kg and also in expectant mothers. SP is also the choice drug for intermittent preventive therapy in pregnancy and serves as stand-by treatment during ACT stock outs. The current study assessed the status of Plasmodium falciparum point mutations associated with antifolate drug resistance in the area around Macha. Methods A representative sample of 2,780 residents from the vicinity of Macha was screened for malaria by microscopy. At the same time, blood was collected onto filter paper and dried for subsequent P. falciparum DNA analysis. From 188 (6.8%) individuals that were thick film-positive, a simple random sub-set of 95 P. falciparum infections were genotyped for DHFR and DHPS antifolate resistance mutations, using nested PCR and allele-specific restriction enzyme digestion. Results Plasmodium falciparum field samples exhibited a high prevalence of antifolate resistance mutations, including the DHFR triple (Asn-108 + Arg-59 + Ile-51) mutant (41.3%) and DHPS double (Gly-437 + Glu-540) mutant (16%). The quintuple (DHFR triple + DHPS double) mutant was found in 4 (6.5%) of the samples. Levels of mutated parasites showed a dramatic escalation, relative to previous surveys since 1988. However, neither of the Val-16 and Thr-108 mutations, which jointly confer resistance to cycloguanil, was detectable among the human infections. The Leu-164 mutation, associated with high grade resistance to both pyrimethamine and cycloguanil, as a multiple mutant with Asn-108, Arg-59 and (or) Ile-51, was also absent. Conclusion This study points to escalating levels of P. falciparum antifolate resistance in the vicinity of Macha. Continued monitoring is recommended to ensure timely policy revisions before widespread resistance exacts a serious public health toll. PMID:18495008

Escalating Plasmodium falciparum antifolate drug resistance mutations in Macha, rural Zambia.

PubMed

Mkulama, Mtawa A P; Chishimba, Sandra; Sikalima, Jay; Rouse, Petrica; Thuma, Philip E; Mharakurwa, Sungano

2008-05-21

In Zambia the first-line treatment for uncomplicated malaria is artemisinin combination therapy (ACT), with artemether-lumefantrine currently being used. However, the antifolate regimen, sulphadoxine-pyrimethamine (SP), remains the treatment of choice in children weighing less than 5 kg and also in expectant mothers. SP is also the choice drug for intermittent preventive therapy in pregnancy and serves as stand-by treatment during ACT stock outs. The current study assessed the status of Plasmodium falciparum point mutations associated with antifolate drug resistance in the area around Macha. A representative sample of 2,780 residents from the vicinity of Macha was screened for malaria by microscopy. At the same time, blood was collected onto filter paper and dried for subsequent P. falciparum DNA analysis. From 188 (6.8%) individuals that were thick film-positive, a simple random sub-set of 95 P. falciparum infections were genotyped for DHFR and DHPS antifolate resistance mutations, using nested PCR and allele-specific restriction enzyme digestion. Plasmodium falciparum field samples exhibited a high prevalence of antifolate resistance mutations, including the DHFR triple (Asn-108 + Arg-59 + Ile-51) mutant (41.3%) and DHPS double (Gly-437 + Glu-540) mutant (16%). The quintuple (DHFR triple + DHPS double) mutant was found in 4 (6.5%) of the samples. Levels of mutated parasites showed a dramatic escalation, relative to previous surveys since 1988. However, neither of the Val-16 and Thr-108 mutations, which jointly confer resistance to cycloguanil, was detectable among the human infections. The Leu-164 mutation, associated with high grade resistance to both pyrimethamine and cycloguanil, as a multiple mutant with Asn-108, Arg-59 and (or) Ile-51, was also absent. This study points to escalating levels of P. falciparum antifolate resistance in the vicinity of Macha. Continued monitoring is recommended to ensure timely policy revisions before widespread resistance exacts a serious public health toll.

The Arabidopsis DELAY OF GERMINATION 1 gene affects ABSCISIC ACID INSENSITIVE 5 (ABI5) expression and genetically interacts with ABI3 during Arabidopsis seed development.

PubMed

Dekkers, Bas J W; He, Hanzi; Hanson, Johannes; Willems, Leo A J; Jamar, Diaan C L; Cueff, Gwendal; Rajjou, Loïc; Hilhorst, Henk W M; Bentsink, Leónie

2016-02-01

The seed expressed gene DELAY OF GERMINATION (DOG) 1 is absolutely required for the induction of dormancy. Next to a non-dormant phenotype, the dog1-1 mutant is also characterized by a reduced seed longevity suggesting that DOG1 may affect additional seed processes as well. This aspect however, has been hardly studied and is poorly understood. To uncover additional roles of DOG1 in seeds we performed a detailed analysis of the dog1 mutant using both transcriptomics and metabolomics to investigate the molecular consequences of a dysfunctional DOG1 gene. Further, we used a genetic approach taking advantage of the weak aba insensitive (abi) 3-1 allele as a sensitized genetic background in a cross with dog1-1. DOG1 affects the expression of hundreds of genes including LATE EMBRYOGENESIS ABUNDANT and HEAT SHOCK PROTEIN genes which are affected by DOG1 partly via control of ABI5 expression. Furthermore, the content of a subset of primary metabolites, which normally accumulate during seed maturation, was found to be affected in the dog1-1 mutant. Surprisingly, the abi3-1 dog1-1 double mutant produced green seeds which are highly ABA insensitive, phenocopying severe abi3 mutants, indicating that dog1-1 acts as an enhancer of the weak abi3-1 allele and thus revealing a genetic interaction between both genes. Analysis of the dog1 and dog1 abi3 mutants revealed additional seed phenotypes and therefore we hypothesize that DOG1 function is not limited to dormancy but that it is required for multiple aspects of seed maturation, in part by interfering with ABA signalling components. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

Reactive oxygen species and transcript analysis upon excess light treatment in wild-type Arabidopsis thaliana vs a photosensitive mutant lacking zeaxanthin and lutein

PubMed Central

2011-01-01

Background Reactive oxygen species (ROS) are unavoidable by-products of oxygenic photosynthesis, causing progressive oxidative damage and ultimately cell death. Despite their destructive activity they are also signalling molecules, priming the acclimatory response to stress stimuli. Results To investigate this role further, we exposed wild type Arabidopsis thaliana plants and the double mutant npq1lut2 to excess light. The mutant does not produce the xanthophylls lutein and zeaxanthin, whose key roles include ROS scavenging and prevention of ROS synthesis. Biochemical analysis revealed that singlet oxygen (1O2) accumulated to higher levels in the mutant while other ROS were unaffected, allowing to define the transcriptomic signature of the acclimatory response mediated by 1O2 which is enhanced by the lack of these xanthophylls species. The group of genes differentially regulated in npq1lut2 is enriched in sequences encoding chloroplast proteins involved in cell protection against the damaging effect of ROS. Among the early fine-tuned components, are proteins involved in tetrapyrrole biosynthesis, chlorophyll catabolism, protein import, folding and turnover, synthesis and membrane insertion of photosynthetic subunits. Up to now, the flu mutant was the only biological system adopted to define the regulation of gene expression by 1O2. In this work, we propose the use of mutants accumulating 1O2 by mechanisms different from those activated in flu to better identify ROS signalling. Conclusions We propose that the lack of zeaxanthin and lutein leads to 1O2 accumulation and this represents a signalling pathway in the early stages of stress acclimation, beside the response to ADP/ATP ratio and to the redox state of both plastoquinone pool. Chloroplasts respond to 1O2 accumulation by undergoing a significant change in composition and function towards a fast acclimatory response. The physiological implications of this signalling specificity are discussed. PMID:21481232

Regulation of drought tolerance by the F-box protein MAX2 in Arabidopsis.

PubMed

Bu, Qingyun; Lv, Tianxiao; Shen, Hui; Luong, Phi; Wang, Jimmy; Wang, Zhenyu; Huang, Zhigang; Xiao, Langtao; Engineer, Cawas; Kim, Tae Houn; Schroeder, Julian I; Huq, Enamul

2014-01-01

MAX2 (for MORE AXILLARY GROWTH2) has been shown to regulate diverse biological processes, including plant architecture, photomorphogenesis, senescence, and karrikin signaling. Although karrikin is a smoke-derived abiotic signal, a role for MAX2 in abiotic stress response pathways is least investigated. Here, we show that the max2 mutant is strongly hypersensitive to drought stress compared with wild-type Arabidopsis (Arabidopsis thaliana). Stomatal closure of max2 was less sensitive to abscisic acid (ABA) than that of the wild type. Cuticle thickness of max2 was significantly thinner than that of the wild type. Both of these phenotypes of max2 mutant plants correlate with the increased water loss and drought-sensitive phenotype. Quantitative real-time reverse transcription-polymerase chain reaction analyses showed that the expression of stress-responsive genes and ABA biosynthesis, catabolism, transport, and signaling genes was impaired in max2 compared with wild-type seedlings in response to drought stress. Double mutant analysis of max2 with the ABA-insensitive mutants abi3 and abi5 indicated that MAX2 may function upstream of these genes. The expression of ABA-regulated genes was enhanced in imbibed max2 seeds. In addition, max2 mutant seedlings were hypersensitive to ABA and osmotic stress, including NaCl, mannitol, and glucose. Interestingly, ABA, osmotic stress, and drought-sensitive phenotypes were restricted to max2, and the strigolactone biosynthetic pathway mutants max1, max3, and max4 did not display any defects in these responses. Taken together, these results uncover an important role for MAX2 in plant responses to abiotic stress conditions.

Mutants of Escherichia coli defective in membrane phospholipid synthesis: macromolecular synthesis in an sn-glycerol 3-phosphate acyltransferase Km mutant.

PubMed

Bell, R M

1974-03-01

sn-Glycerol 3-phosphate (G3P) auxotrophs of Escherichia coli have been selected from a strain which cannot aerobically catabolize G3P. The auxotrophy resulted from loss of the biosynthetic G3P dehydrogenase (EC 1.1.1.8) or from a defective membranous G3P acyltransferase. The apparent K(m) of the acyltransferase for G3P was 11- to 14-fold higher (from about 90 mum to 1,000 to 1,250 mum) in membrane preparations from the mutants than those of the parent. All extracts prepared from revertants of the G3P dehydrogenase mutants showed G3P dehydrogenase activity, but most contained less than 10% of the wild-type level. Membrane preparations from revertants of the acyltransferase mutants had apparent K(m)'s for G3P similar to that of the parent. Strains have been derived in which the G3P requirement can be satisfied with glycerol in the presence of glucose, presumably because the glycerol kinase was desensitized to inhibition by fructose 1,6-diphosphate. Investigations on the growth and macromolecular synthesis in a G3P acyltransferase K(m) mutant revealed that upon glycerol deprivation, net phospholipid synthesis stopped immediately; growth continued for about one doubling; net ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and protein nearly doubled paralleling the growth curve; the rate of phospholipid synthesis assessed by labeling cells with (32)P-phosphate, (14)C-acetate, or (3)H-serine was reduced greater than 90%; the rates of RNA and DNA synthesis increased as the cells grew and then decreased as the cells stopped growing; the rate of protein synthesis showed no increase and declined more slowly than the rates of RNA and DNA synthesis when the cells stopped growing. The cells retained and gained in the capacity to synthesize phospholipids upon glycerol deprivation. These data indicate that net phospholipid synthesis is not required for continued macromolecular synthesis for about one doubling, and that the rates of these processes are not coupled during this time period.

Degradation of Glucan Primers in the Absence of Starch Synthase 4 Disrupts Starch Granule Initiation in Arabidopsis*

PubMed Central

Lu, Kuan-Jen; Stettler, Michaela; Streb, Sebastian

2016-01-01

Arabidopsis leaf chloroplasts typically contain five to seven semicrystalline starch granules. It is not understood how the synthesis of each granule is initiated or how starch granule number is determined within each chloroplast. An Arabidopsis mutant lacking the glucosyl-transferase, STARCH SYNTHASE 4 (SS4) is impaired in its ability to initiate starch granules; its chloroplasts rarely contain more than one large granule, and the plants have a pale appearance and reduced growth. Here we report that the chloroplastic α-amylase AMY3, a starch-degrading enzyme, interferes with granule initiation in the ss4 mutant background. The amy3 single mutant is similar in phenotype to the wild type under normal growth conditions, with comparable numbers of starch granules per chloroplast. Interestingly, the ss4 mutant displays a pleiotropic reduction in the activity of AMY3. Remarkably, complete abolition of AMY3 (in the amy3 ss4 double mutant) increases the number of starch granules produced in each chloroplast, suppresses the pale phenotype of ss4, and nearly restores normal growth. The amy3 mutation also restores starch synthesis in the ss3 ss4 double mutant, which lacks STARCH SYNTHASE 3 (SS3) in addition to SS4. The ss3 ss4 line is unable to initiate any starch granules and is thus starchless. We suggest that SS4 plays a key role in granule initiation, allowing it to proceed in a way that avoids premature degradation of primers by starch hydrolases, such as AMY3. PMID:27458017

Differential effects of the mismatch repair genes MSH2 and MSH3 on homeologous recombination in Saccharomyces cerevisiae.

PubMed

Selva, E M; Maderazo, A B; Lahue, R S

1997-12-01

The products of the yeast mismatch repair genes MSH2 and MSH3 participate in the inhibition of genetic recombination between homeologous (divergent) DNA sequences. In strains deficient for these genes, homeologous recombination rates between repeated elements are elevated due to the loss of this inhibition. In this study, the effects of these mutations were further analyzed by quantitation of mitotic homeologous recombinants as crossovers, gene conversions or exceptional events in wild-type, msh2, msh3 and msh2 msh3 mutant strains. When homeologous sequences were present as a direct repeat in one orientation, crossovers and gene conversions were elevated in msh2, msh3 and msh2 msh3 strains. The increases were greater in the msh2 msh3 double mutant than in either single mutant. When the order of the homeologous sequences was reversed, the msh2 mutation again yielded increased rates of crossovers and gene conversions. However, in an msh3 strain, gene conversions occurred at higher levels but interchromosomal crossovers were not increased and intrachromosomal crossovers were reduced relative to wild type. The msh2 msh3 double mutant behaved like the msh2 single mutant in this orientation. Control strains harboring homologous duplications were largely but not entirely unaffected in mutant strains, suggesting specificity for the mismatched intermediates of homeologous recombination. In all strains, very few (< 10%) recombinants could be attributed to exceptional events. These results suggest that MSH2 and MSH3 can function differentially to control homeologous exchanges.

Identification and functional analysis of two Golgi-localized UDP-galactofuranose transporters with overlapping functions in Aspergillus niger.

PubMed

Park, Joohae; Tefsen, Boris; Heemskerk, Marc J; Lagendijk, Ellen L; van den Hondel, Cees A M J J; van Die, Irma; Ram, Arthur F J

2015-11-02

Galactofuranose (Galf)-containing glycoconjugates are present in numerous microbes, including filamentous fungi where they are important for morphology, virulence and maintaining cell wall integrity. The incorporation of Galf-residues into galactomannan, galactomannoproteins and glycolipids is carried out by Golgi-localized Galf transferases. The nucleotide sugar donor used by these transferases (UDP-Galf) is produced in the cytoplasm and has to be transported to the lumen of the Golgi by a dedicated nucleotide sugar transporter. Based on homology with recently identified UDP-Galf-transporters in A. fumigatus and A. nidulans, two putative UDP-Galf-transporters in A. niger were found. Their function and localization was determined by gene deletions and GFP-tagging studies, respectively. The two putative UDP-Galf-transporters in A. niger are homologous to each other and are predicted to contain eleven transmembrane domains (UgtA) or ten transmembrane domains (UgtB) due to a reduced length of the C-terminal part of the UgtB protein. The presence of two putative UDP-Galf-transporters in the genome was not unique for A. niger. From the twenty Aspergillus species analysed, nine species contained two additional putative UDP-Galf-transporters. Three of the nine species were outside the Aspergillus section nigri, indication an early duplication of UDP-Galf-transporters and subsequent loss of the UgtB copy in several aspergilli. Deletion analysis of the single and double mutants in A. niger indicated that the two putative UDP-Galf-transporters (named UgtA and UgtB) have a redundant function in UDP-Galf-transport as only the double mutant displayed a Galf-negative phenotype. The Galf-negative phenotype of the double mutant could be complemented by expressing either CFP-UgtA or CFP-UgtB fusion proteins from their endogenous promoters, indicating that both CFP-tagged proteins are functional. Both Ugt proteins co-localize with each other as well as with the GDP-mannose nucleotide transporter, as was demonstrated by fluorescence microscopy, thereby confirming their predicted localization in the Golgi. A. niger contains two genes encoding UDP-Galf-transporters. Deletion and localization studies indicate that UgtA and UgtB have redundant functions in the biosynthesis of Galf-containing glycoconjugates.

The ASK1 gene regulates development and interacts with the UFO gene to control floral organ identity in Arabidopsis.

PubMed

Zhao, D; Yang, M; Solava, J; Ma, H

1999-09-01

Normal flower development likely requires both specific and general regulators. We have isolated an Arabidopsis mutant ask1-1 (for -Arabidopsis skp1-like1-1), which exhibits defects in both vegetative and reproductive development. In the ask1-1mutant, rosette leaf growth is reduced, resulting in smaller than normal rosette leaves, and internodes in the floral stem are shorter than normal. Examination of cell sizes in these organs indicates that cell expansion is normal in the mutant, but cell number is reduced. In the mutant, the numbers of petals and stamens are reduced, and many flowers have one or more petals with a reduced size. In addition, all mutant flowers have short stamen filaments. Furthermore, petal/stamen chimeric organs are found in many flowers. These results indicate that the ASK1 gene affects the size of vegetative and floral organs. The ask1 floral phenotype resembles somewhat that of the Arabidopsis ufo mutants in that both genes affect whorls 2 and 3. We therefore tested for possible interactions between ASK1 and UFO by analyzing the phenotypes of ufo-2 ask1-1 double mutant plants. In these plants, vegetative development is similar to that of the ask1-1 single mutant, whereas the floral defects are more severe than those in either single mutant. Interior to the first whorl, the double mutant flowers have more sepals or sepal-like organs than are found in ufo-2, and less petals than ask1-1. Our results suggest that ASK1 interacts with UFO to control floral organ identity in whorls 2 and 3. This is very intriguing because ASK1 is very similar in sequence to the yeast SKP1 protein and UFO contains an F-box, a motif known to interact with SKP1 in yeast. Although the precise mechanism of ASK1 and UFO action is unknown, our results support the hypothesis that these two proteins physically interact in vivo. Copyright 1999 Wiley-Liss, Inc.

Distinct roles of the DmNav and DSC1 channels in the action of DDT and pyrethroids.

PubMed

Rinkevich, Frank D; Du, Yuzhe; Tolinski, Josh; Ueda, Atsushi; Wu, Chun-Fang; Zhorov, Boris S; Dong, Ke

2015-03-01

Voltage-gated sodium channels (Nav channels) are critical for electrical signaling in the nervous system and are the primary targets of the insecticides DDT and pyrethroids. In Drosophila melanogaster, besides the canonical Nav channel, Para (also called DmNav), there is a sodium channel-like cation channel called DSC1 (Drosophila sodium channel 1). Temperature-sensitive paralytic mutations in DmNav (para(ts)) confer resistance to DDT and pyrethroids, whereas DSC1 knockout flies exhibit enhanced sensitivity to pyrethroids. To further define the roles and interaction of DmNav and DSC1 channels in DDT and pyrethroid neurotoxicology, we generated a DmNav/DSC1 double mutant line by introducing a para(ts1) allele (carrying the I265N mutation) into a DSC1 knockout line. We confirmed that the I265N mutation reduced the sensitivity to two pyrethroids, permethrin and deltamethrin of a DmNav variant expressed in Xenopus oocytes. Computer modeling predicts that the I265N mutation confers pyrethroid resistance by allosterically altering the second pyrethroid receptor site on the DmNav channel. Furthermore, we found that I265N-mediated pyrethroid resistance in para(ts1) mutant flies was almost completely abolished in para(ts1);DSC1(-/-) double mutant flies. Unexpectedly, however, the DSC1 knockout flies were less sensitive to DDT, compared to the control flies (w(1118A)), and the para(ts1);DSC1(-/-) double mutant flies were even more resistant to DDT compared to the DSC1 knockout or para(ts1) mutant. Our findings revealed distinct roles of the DmNav and DSC1 channels in the neurotoxicology of DDT vs. pyrethroids and implicate the exciting possibility of using DSC1 channel blockers or modifiers in the management of pyrethroid resistance. Copyright © 2015 Elsevier Inc. All rights reserved.

Brn3a/Pou4f1 Regulates Dorsal Root Ganglion Sensory Neuron Specification and Axonal Projection into the Spinal Cord

PubMed Central

Zou, Min; Li, Shengguo; Klein, William H.; Xiang, Mengqing

2012-01-01

The sensory neurons of the dorsal root ganglia (DRG) must project accurately to their central targets to convey proprioceptive, nociceptive and mechanoreceptive information to the spinal cord. How these different sensory modalities and central connectivities are specified and coordinated still remains unclear. Given the expression of the POU homeodomain transcription factors Brn3a/Pou4f1 and Brn3b/Pou4f2 in DRG and spinal cord sensory neurons, we determined the subtype specification of DRG and spinal cord sensory neurons as well as DRG central projections in Brn3a and Brn3b single and double mutant mice. Inactivation of either or both genes causes no gross abnormalities in early spinal cord neurogenesis; however, in Brn3a single and Brn3a;Brn3b double mutant mice, sensory afferent axons from the DRG fail to form normal trajectories in the spinal cord. The TrkA+ afferents remain outside the dorsal horn and fail to extend into the spinal cord, while the projections of TrkC+ proprioceptive afferents into the ventral horn are also impaired. Moreover, Brn3a mutant DRGs are defective in sensory neuron specification, as marked by the excessive generation of TrkB+ and TrkC+ neurons as well as TrkA+/TrkB+ and TrkA+/TrkC+ double positive cells at early embryonic stages. At later stages in the mutant, TrkB+, TrkC+ and parvalbumin+ neurons diminish while there is a significant increase of CGRP+ and c-ret+ neurons. In addition, Brn3a mutant DRGs display a dramatic down-regulation of Runx1 expression, suggesting that the regulation of DRG sensory neuron specification by Brn3a is mediated in part by Runx1. Our results together demonstrate a critical role for Brn3a in generating DRG sensory neuron diversity and regulating sensory afferent projections to the central targets. PMID:22326227

Novel Synthesis and Phenotypic Analysis of Mutant Clouds for Hepatitis E Virus Genotype 1.

PubMed

Agarwal, Shubhra; Baccam, Prasith; Aggarwal, Rakesh; Veerapu, Naga Suresh

2018-02-15

Many RNA viruses exist as an ensemble of genetically diverse, replicating populations known as a mutant cloud. The genetic diversity (cloud size) and composition of this mutant cloud may influence several important phenotypic features of the virus, including its replication capacity. We applied a straightforward, bacterium-free approach using error-prone PCR coupled with reverse genetics to generate infectious mutant RNA clouds with various levels of genetic diversity from a genotype 1 strain of hepatitis E virus (HEV). Cloning and sequencing of a genomic fragment encompassing 70% of open reading frame 1 ( ORF1 ) or of the full genome from variants in the resultant clouds showed the occurrence of nucleotide mutations at a frequency on the order of 10 -3 per nucleotide copied and the existence of marked genetic diversity, with a high normalized Shannon entropy value. The mutant clouds showed transient replication in cell culture, while wild-type HEV did not. Cross-sectional data from these cell cultures supported the existence of differential effects of clouds of various sizes and compositions on phenotypic characteristics, such as the replication level of (+)-RNA progeny, the amounts of double-stranded RNA (a surrogate for the rate of viral replication) and ORF1 protein, and the expression of interferon-stimulated genes. Since mutant cloud size and composition influenced the viral phenotypic properties, a better understanding of this relationship may help to provide further insights into virus evolution and prediction of emerging viral diseases. IMPORTANCE Several biological or practical limitations currently prevent the study of phenotypic behavior of a mutant cloud in vitro We developed a simple and rapid method for synthesizing mutant clouds of hepatitis E virus (HEV), a single-stranded (+)-RNA [ss(+) RNA] virus, with various and controllable levels of genetic diversity, which could then be used in a cell culture system to study the effects of cloud size and composition on viral phenotype. In a cross-sectional analysis, we demonstrated that a particular mutant cloud which had an extremely high genetic diversity had a replication rate exceeding that of wild-type HEV. This method should thus provide a useful model for understanding the phenotypic behavior of ss(+) RNA viruses. Copyright © 2018 American Society for Microbiology.

Point mutations in the post-M2 region of human alpha-ENaC regulate cation selectivity.

PubMed

Ji, H L; Parker, S; Langloh, A L; Fuller, C M; Benos, D J

2001-07-01

We tested the hypothesis that an arginine-rich region immediately following the second transmembrane domain may constitute part of the inner mouth of the epithelial Na+ channel (ENaC) pore and, hence, influence conduction and/or selectivity properties of the channel by expressing double point mutants in Xenopus oocytes. Double point mutations of arginines in this post-M2 region of the human alpha-ENaC (alpha-hENaC) led to a decrease and increase in the macroscopic conductance of alphaR586E,R587Ebetagamma- and alphaR589E,R591Ebetagamma-hENaC, respectively, but had no effect on the single-channel conductance of either double point mutant. However, the apparent equilibrium dissociation constant for Na+ was decreased for both alphaR586E,R587Ebetagamma- and alphaR589E,R591Ebetagamma-hENaC, and the maximum amiloride-sensitive Na+ current was decreased for alphaR586E,R587Ebetagamma-hENaC and increased for alphaR589E,R591Ebetagamma-hENaC. The relative permeabilities of Li+ and K+ vs. Na+ were increased 11.25- to 27.57-fold for alphaR586E,R587Ebetagamma-hENaC compared with wild type. The relative ion permeability of these double mutants and wild-type ENaC was inversely related to the crystal diameter of the permeant ions. Thus the region of positive charge is important for the ion permeation properties of the channel and may form part of the pore itself.

Arabidopsis double-stranded RNA binding protein DRB3 participates in methylation-mediated defense against geminiviruses.

PubMed

Raja, Priya; Jackel, Jamie N; Li, Sizhun; Heard, Isaac M; Bisaro, David M

2014-03-01

Arabidopsis encodes five double-stranded RNA binding (DRB) proteins. DRB1 and DRB2 are involved in microRNA (miRNA) biogenesis, while DRB4 functions in cytoplasmic posttranscriptional small interfering RNA (siRNA) pathways. DRB3 and DRB5 are not involved in double-stranded RNA (dsRNA) processing but assist in silencing transcripts targeted by DRB2-associated miRNAs. The goal of this study was to determine which, if any, of the DRB proteins might also participate in a nuclear siRNA pathway that leads to geminivirus genome methylation. Here, we demonstrate that DRB3 functions with Dicer-like 3 (DCL3) and Argonaute 4 (AGO4) in methylation-mediated antiviral defense. Plants employ repressive viral genome methylation as an epigenetic defense against geminiviruses, using an RNA-directed DNA methylation (RdDM) pathway similar to that used to suppress endogenous invasive DNAs such as transposons. Chromatin methylation inhibits virus replication and transcription, and methylation-deficient host plants are hypersusceptible to geminivirus infection. Using a panel of drb mutants, we found that drb3 plants uniquely exhibit a similar hypersensitivity and that viral genome methylation is substantially reduced in drb3 compared to wild-type plants. In addition, like dcl3 and ago4 mutants, drb3 plants fail to recover from infection and cannot accomplish the viral genome hypermethylation that is invariably observed in asymptomatic, recovered tissues. Small RNA analysis, bimolecular fluorescence complementation, and coimmunoprecipitation experiments show that DRB3 acts downstream of siRNA biogenesis and suggest that it associates with DCL3 and AGO4 in distinct subnuclear compartments. These studies reveal that in addition to its previously established role in the miRNA pathway, DRB3 also functions in antiviral RdDM. Plants use RNA-directed DNA methylation (RdDM) as an epigenetic defense against geminiviruses. RNA silencing pathways in Arabidopsis include five double-stranded RNA binding proteins (DRBs) related to Drosophila R2D2 and mammalian TRBP and PACT. While DRB proteins have defined roles in miRNA and cytoplasmic siRNA pathways, a role in nuclear RdDM was elusive. Here, we used the geminivirus system to show that DRB3 is involved in methylation-mediated antiviral defense. Beginning with a panel of Arabidopsis drb mutants, we demonstrated that drb3 plants uniquely show enhanced susceptibility to geminiviruses. Further, like dcl3 and ago4 mutants, drb3 plants fail to hypermethylate the viral genome, a requirement for host recovery. We also show that DRB3 physically interacts with the RdDM pathway components DCL3 and AGO4 in the nucleus. This work highlights the utility of geminiviruses as models for de novo RdDM and places DRB3 protein in this fundamental epigenetic pathway.

Inactivation of dhaD and dhaK abolishes by-product accumulation during 1,3-propanediol production in Klebsiella pneumoniae.

PubMed

Horng, Yu-Tze; Chang, Kai-Chih; Chou, Ta-Chung; Yu, Chung-Jen; Chien, Chih-Ching; Wei, Yu-Hong; Soo, Po-Chi

2010-07-01

1,3-Propanediol (1,3-PD) can be used for the industrial synthesis of a variety of compounds, including polyesters, polyethers, and polyurethanes. 1,3-PD is generated from petrochemical and microbial sources. 1,3-Propanediol is a typical product of glycerol fermentation, while acetate, lactate, 2,3-butanediol, and ethanol also accumulate during the process. Substrate and product inhibition limit the final concentration of 1,3-propanediol in the fermentation broth. It is impossible to increase the yield of 1,3-propanediol by using the traditional whole-cell fermentation process. In this study, dhaD and dhaK, the genes for glycerol dehydrogenase and dihydroxyacetone kinase, respectively, were inactivated by homologous recombination in Klebsiella pneumoniae. The dhaD/dhaK double mutant (designated TC100), selected from 5,000 single or double cross homologous recombination mutants, was confirmed as a double cross by using polymerase chain reaction. Analysis of the cell-free supernatant with high-performance liquid chromatography revealed elimination of lactate and 2,3-butanediol, as well as ethanol accumulation in TC100, compared with the wild-type strain. Furthermore, 1,3-propanediol productivity was increased in the TC100 strain expressing glycerol dehydratase and 1,3-PDO dehydrogenase regulated by the arabinose P(BAD) promoter. The genetic engineering and medium formulation approaches used here should aid in the separation of 1,3-propanediol from lactate, 2,3-butanediol, and ethanol and lead to increased production of 1,3-propanediol in Klebsiella pneumoniae.

Stamina pistilloida, the Pea ortholog of Fim and UFO, is required for normal development of flowers, inflorescences, and leaves.

PubMed

Taylor, S; Hofer, J; Murfet, I

2001-01-01

Isolation and characterization of two severe alleles at the Stamina pistilloida (Stp) locus reveals that Stp is involved in a wide range of developmental processes in the garden pea. The most severe allele, stp-4, results in flowers consisting almost entirely of sepals and carpels. Production of ectopic secondary flowers in stp-4 plants suggests that Stp is involved in specifying floral meristem identity in pea. The stp mutations also reduce the complexity of the compound pea leaf, and primary inflorescences often terminate prematurely in an aberrant sepaloid flower. In addition, stp mutants were shorter than their wild-type siblings due to a reduction in cell number in their internodes. Fewer cells were also found in the epidermis of the leaf rachis of stp mutants. Examination of the effects of stp-4 in double mutant combinations with af, tl, det, and veg2-2-mutations known to influence leaf, inflorescence, and flower development in pea-suggests that Stp function is independent of these genes. A synergistic interaction between weak mutant alleles at Stp and Uni indicated that these two genes act together, possibly to regulate primordial growth. Molecular analysis revealed that Stp is the pea homolog of the Antirrhinum gene Fimbriata (Fim) and of UNUSUAL FLORAL ORGANS (UFO) from Arabidopsis. Differences between Fim/UFO and Stp mutant phenotypes and expression patterns suggest that expansion of Stp activity into the leaf was an important step during evolution of the compound leaf in the garden pea.

The MADS-box transcription factor FgMcm1 regulates cell identity and fungal development in Fusarium graminearum.

PubMed

Yang, Cui; Liu, Huiquan; Li, Guotian; Liu, Meigang; Yun, Yingzi; Wang, Chenfang; Ma, Zhonghua; Xu, Jin-Rong

2015-08-01

In eukaryotic cells, MADS-box genes are known to play major regulatory roles in various biological processes by combinatorial interactions with other transcription factors. In this study, we functionally characterized the FgMCM1 MADS-box gene in Fusarium graminearum, the causal agent of wheat and barley head blight. Deletion of FgMCM1 resulted in the loss of perithecium production and phialide formation. The Fgmcm1 mutant was significantly reduced in virulence, deoxynivalenol biosynthesis and conidiation. In yeast two-hybrid assays, FgMcm1 interacted with Mat1-1-1 and Fst12, two transcription factors important for sexual reproduction. Whereas Fgmcm1 mutants were unstable and produced stunted subcultures, Fgmcm1 mat1-1-1 but not Fgmcm1 fst12 double mutants were stable. Furthermore, spontaneous suppressor mutations occurred frequently in stunted subcultures to recover growth rate. Ribonucleic acid sequencing analysis indicated that a number of sexual reproduction-related genes were upregulated in stunted subcultures compared with the Fgmcm1 mutant, which was downregulated in the expression of genes involved in pathogenesis, secondary metabolism and conidiation. We also showed that culture instability was not observed in the Fvmcm1 mutants of the heterothallic Fusarium verticillioides. Overall, our data indicate that FgMcm1 plays a critical role in the regulation of cell identity, sexual and asexual reproduction, secondary metabolism and pathogenesis in F. graminearum. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

Conditional immortalization of human thyroid epithelial cells: a tool for analysis of oncogene action.

PubMed Central

Wynford-Thomas, D; Bond, J A; Wyllie, F S; Burns, J S; Williams, E D; Jones, T; Sheer, D; Lemoine, N R

1990-01-01

To overcome the difficulty of assessing oncogene action in human epithelial cell types, such as thyroid, which have limited proliferative potential in culture, we have explored the use of temperature-sensitive (ts) mutants of simian virus 40 (SV40) early region to create conditionally immortalized epithelial cell lines. Normal primary cultures of human thyroid follicular cells were transfected with a plasmid containing the SV40 early region from mutant tsA58. Expanding epithelial colonies were observed after 2 to 3 months, all of which grew to greater than 200 population doublings without crisis. All showed tight temperature dependence for growth. After switch-up to the restrictive temperature (40.5 degrees C), no further increase in cell number was seen after 1 to 2 days. However, DNA synthesis declined much more slowly; the dissociation from cell division led to marked polyploidy. Viability was maintained for up to 2 weeks. Introduction of an inducible mutant ras gene into ts thyroid cells led, as expected, to morphological transformation at the permissive temperature when ras was induced. Interestingly, this was associated with a marked reduction in net growth rate. At the restrictive temperature, induction of mutant ras caused rapid cell death. These results demonstrate the utility of a ts SV40 mutant to permit the study of oncogene action in an otherwise nonproliferative target cell and reveal important differences in the interaction between ras and SV40 T in these epithelial cells compared with previously studied cell types. Images PMID:1697930

Convergence of developmental mutants into a single tomato model system: 'Micro-Tom' as an effective toolkit for plant development research

PubMed Central

2011-01-01

Background The tomato (Solanum lycopersicum L.) plant is both an economically important food crop and an ideal dicot model to investigate various physiological phenomena not possible in Arabidopsis thaliana. Due to the great diversity of tomato cultivars used by the research community, it is often difficult to reliably compare phenotypes. The lack of tomato developmental mutants in a single genetic background prevents the stacking of mutations to facilitate analysis of double and multiple mutants, often required for elucidating developmental pathways. Results We took advantage of the small size and rapid life cycle of the tomato cultivar Micro-Tom (MT) to create near-isogenic lines (NILs) by introgressing a suite of hormonal and photomorphogenetic mutations (altered sensitivity or endogenous levels of auxin, ethylene, abscisic acid, gibberellin, brassinosteroid, and light response) into this genetic background. To demonstrate the usefulness of this collection, we compared developmental traits between the produced NILs. All expected mutant phenotypes were expressed in the NILs. We also created NILs harboring the wild type alleles for dwarf, self-pruning and uniform fruit, which are mutations characteristic of MT. This amplified both the applications of the mutant collection presented here and of MT as a genetic model system. Conclusions The community resource presented here is a useful toolkit for plant research, particularly for future studies in plant development, which will require the simultaneous observation of the effect of various hormones, signaling pathways and crosstalk. PMID:21714900

Spectroelectrochemical insights into structural and redox properties of immobilized endonuclease III and its catalytically inactive mutant

NASA Astrophysics Data System (ADS)

Moe, Elin; Rollo, Filipe; Silveira, Célia M.; Sezer, Murat; Hildebrandt, Peter; Todorovic, Smilja

2018-01-01

Endonuclease III is a Fe-S containing bifunctional DNA glycosylase which is involved in the repair of oxidation damaged DNA. Here we employ surface enhanced IR spectroelectrochemistry and electrochemistry to study the enzyme from the highly radiation- and desiccation-resistant bacterium Deinococcus radiodurans (DrEndoIII2). The experiments are designed to shed more light onto specific parameters that are currently proposed to govern damage search and recognition by endonucleases III. We demonstrate that electrostatic interactions required for the redox activation of DrEndoIII2 may result in high electric fields that alter its structural and thermodynamic properties. Analysis of inactive DrEndoIII2 (K132A/D150A double mutant) interacting with undamaged DNA, and the active enzyme interacting with damaged DNA also indicate that the electron transfer is modulated by subtle differences in the protein-DNA complex.

Spectroelectrochemical insights into structural and redox properties of immobilized endonuclease III and its catalytically inactive mutant.

PubMed

Moe, Elin; Rollo, Filipe; Silveira, Célia M; Sezer, Murat; Hildebrandt, Peter; Todorovic, Smilja

2018-01-05

Endonuclease III is a Fe-S containing bifunctional DNA glycosylase which is involved in the repair of oxidation damaged DNA. Here we employ surface enhanced IR spectroelectrochemistry and electrochemistry to study the enzyme from the highly radiation- and desiccation-resistant bacterium Deinococcus radiodurans (DrEndoIII 2 ). The experiments are designed to shed more light onto specific parameters that are currently proposed to govern damage search and recognition by endonucleases III. We demonstrate that electrostatic interactions required for the redox activation of DrEndoIII 2 may result in high electric fields that alter its structural and thermodynamic properties. Analysis of inactive DrEndoIII 2 (K132A/D150A double mutant) interacting with undamaged DNA, and the active enzyme interacting with damaged DNA also indicate that the electron transfer is modulated by subtle differences in the protein-DNA complex. Copyright © 2017 Elsevier B.V. All rights reserved.

The Transcription Factor Foxg1 Promotes Optic Fissure Closure in the Mouse by Suppressing Wnt8b in the Nasal Optic Stalk

PubMed Central

Smith, Rowena; Huang, Yu-Ting; Tian, Tian; Vojtasova, Dominika; Mesalles-Naranjo, Oscar; Price, David J.

2017-01-01

During vertebrate eye morphogenesis, a transient fissure forms at its inferior part, known as the optic fissure. This will gradually close, giving rise to a healthy, spherical optic cup. Failure of the optic fissure to close gives rise to an ocular disorder known as coloboma. During this developmental process, Foxg1 is expressed in the optic neuroepithelium, with highest levels of expression in the nasal optic stalk. Foxg1−/− mutant mice have microphthalmic eyes with a large ventral coloboma. We found Wnt8b expression upregulated in the Foxg1−/− optic stalk and hypothesized that, similar to what is observed in telencephalic development, Foxg1 directs development of the optic neuroepithelium through transcriptional suppression of Wnt8b. To test this, we generated Foxg1−/−;Wnt8b−/− double mutants of either sex and found that the morphology of the optic cup and stalk and the closure of the optic fissure were substantially rescued in these embryos. This rescue correlates with restored Pax2 expression in the anterior tip of the optic fissure. In addition, although we do not find evidence implicating altered proliferation in the rescue, we observe a significant increase in apoptotic cell density in Foxg1−/−;Wnt8b−/− double mutants compared with the Foxg1−/− single mutant. Upregulation of Wnt/β-catenin target molecules in the optic cup and stalk may underlie the molecular and morphological defects in the Foxg1−/− mutant. Our results show that proper optic fissure closure relies on Wnt8b suppression by Foxg1 in the nasal optic stalk to maintain balanced apoptosis and Pax2 expression in the nasal and temporal edges of the fissure. SIGNIFICANCE STATEMENT Coloboma is an ocular disorder that may result in a loss of visual acuity and accounts for ∼10% of childhood blindness. It results from errors in the sealing of the optic fissure (OF), a transient structure at the bottom of the eye. Here, we investigate the colobomatous phenotype of the Foxg1−/− mutant mouse. We identify upregulated expression of Wnt8b in the optic stalk of Foxg1−/− mutants before OF closure initiates. Foxg1−/−;Wnt8b−/− double mutants show a substantial rescue of the Foxg1−/− coloboma phenotype, which correlates with a rescue in molecular and cellular defects of Foxg1−/− mutants. Our results unravel a new role of Foxg1 in promoting OF closure providing additional knowledge about the molecules and cellular mechanisms underlying coloboma formation. PMID:28729440

The Role of Transition Metal Transporters for Iron, Zinc, Manganese, and Copper in the Pathogenesis of Yersinia pestis

PubMed Central

Perry, Robert D.; Bobrov, Alexander G.; Fetherston, Jacqueline D.

2015-01-01

Yersinia pestis, the causative agent of bubonic, septicemic and pneumonic plague, encodes a multitude of Fe transport systems. Some of these are defective due to frameshift or IS element insertions, while others are functional in vitro but have no established role in causing infections. Indeed only 3 Fe transporters (Ybt, Yfe and Feo) have been shown to be important in at least one form of plague. The yersiniabactin (Ybt) system is essential in the early dermal/lymphatic stages of bubonic plague, irrelevant in the septicemic stage, and critical in pneumonic plague. Two Mn transporters have been characterized (Yfe and MntH). These two systems play a role in bubonic plague but the double yfe mntH mutant is fully virulent in a mouse model of pneumonic plague. The same in vivo phenotype occurs with a mutant lacking two (Yfe and Feo) of four ferrous transporters. A role for the Ybt siderophore in Zn acquisition has been revealed. Ybt-dependent Zn acquisition uses a transport system completely independent of the Fe-Ybt uptake system. Together Ybt components and ZnuABC play a critical role in Zn acquisition in vivo. Single mutants in either system retain high virulence in a mouse model of septicemic plague while the double mutant is completely avirulent. PMID:25891079

The role of transition metal transporters for iron, zinc, manganese, and copper in the pathogenesis of Yersinia pestis.

PubMed

Perry, Robert D; Bobrov, Alexander G; Fetherston, Jacqueline D

2015-06-01

Yersinia pestis, the causative agent of bubonic, septicemic and pneumonic plague, encodes a multitude of Fe transport systems. Some of these are defective due to frameshift or IS element insertions, while others are functional in vitro but have no established role in causing infections. Indeed only 3 Fe transporters (Ybt, Yfe and Feo) have been shown to be important in at least one form of plague. The yersiniabactin (Ybt) system is essential in the early dermal/lymphatic stages of bubonic plague, irrelevant in the septicemic stage, and critical in pneumonic plague. Two Mn transporters have been characterized (Yfe and MntH). These two systems play a role in bubonic plague but the double yfe mntH mutant is fully virulent in a mouse model of pneumonic plague. The same in vivo phenotype occurs with a mutant lacking two (Yfe and Feo) of four ferrous transporters. A role for the Ybt siderophore in Zn acquisition has been revealed. Ybt-dependent Zn acquisition uses a transport system completely independent of the Fe-Ybt uptake system. Together Ybt components and ZnuABC play a critical role in Zn acquisition in vivo. Single mutants in either system retain high virulence in a mouse model of septicemic plague while the double mutant is completely avirulent.

Function and evolution of a Lotus japonicus AP2/ERF family transcription factor that is required for development of infection threads.

PubMed

Yano, Koji; Aoki, Seishiro; Liu, Meng; Umehara, Yosuke; Suganuma, Norio; Iwasaki, Wataru; Sato, Shusei; Soyano, Takashi; Kouchi, Hiroshi; Kawaguchi, Masayoshi

2017-04-01

Legume-rhizobium symbiosis is achieved by two major events evolutionarily acquired: root hair infection and organogenesis. Infection thread (IT) development is a distinct element for rhizobial infection. Through ITs, rhizobia are efficiently transported from infection foci on root hairs to dividing meristematic cortical cells. To unveil this process, we performed genetic screening using Lotus japonicus MG-20 and isolated symbiotic mutant lines affecting nodulation, root hair morphology, and IT development. Map-based cloning identified an AP2/ERF transcription factor gene orthologous to Medicago truncatula ERN1. LjERN1 was activated in response to rhizobial infection and depended on CYCLOPS and NSP2. Legumes conserve an ERN1 homolog, ERN2, that functions redundantly with ERN1 in M. truncatula. Phylogenetic analysis showed that the lineages of ERN1 and ERN2 genes originated from a gene duplication event in the common ancestor of legume plants. However, genomic analysis suggested the lack of ERN2 gene in the L. japonicus genome, consistent with Ljern1 mutants exhibited a root hair phenotype that is observed in ern1/ern2 double mutants in M. truncatula. Molecular evolutionary analysis suggested that the nonsynonymous/synonymous rate ratios of legume ERN1 genes was almost identical to that of non-legume plants, whereas the ERN2 genes experienced a relaxed selective constraint. © The Author 2016. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

Wing 1 of protein HOP2 is as important as helix 3 in DNA binding by MD simulation.

PubMed

Moktan, Hem; Zhou, Donghua H

2018-05-01

The repair of programmed DNA double-strand breaks through recombination is required for proper association and disjunction of the meiotic homologous chromosomes. Meiosis-specific protein HOP2 plays essential roles in recombination by promoting recombinase activities. The N-terminal domain of HOP2 interacts with DNA through helix 3 (H3) and wing 1 (W1). Mutations in wing 1 (Y65A/K67A/Q68A) slightly weakened the binding but mutations in helices 2 and 3 (Q30A/K44A/K49A) nearly abolished the binding. To better understand such differential effects at atomic level, molecular dynamics simulations were employed. Despite losing some hydrogen bonds, the W1-mutant DNA complex was rescued by stronger hydrophobic interactions. For the wild type and W1-mutant, the protein was found to slide along the DNA grooves as the DNA rolls along its double-helix axis. This motion could be functionally important to facilitate the precise positioning of the single-stranded DNA with the homologous double-stranded DNA. The sliding motion was reduced in the W1-mutant. The H-mutant nearly lost all intermolecular interactions. Moreover, an additional mutation in wing 1 (Y65A/K67A/Q68A/K69A) also caused complete complex dissociation. Therefore, both wing 1 and helix 3 make important contribution to the DNA binding, which could be important to the strand invasion function of HOP2 homodimer and HOP2-MND1 heterodimer. Similar to cocking a medieval crossbow with the archer's foot placed in the stirrup, wing 1 may push the minor groove to cause distortion while helix 3 grabs the major groove.

Calcium-dependent protein kinases regulate polarized tip growth in pollen tubes.

PubMed

Myers, Candace; Romanowsky, Shawn M; Barron, Yoshimi D; Garg, Shilpi; Azuse, Corinn L; Curran, Amy; Davis, Ryan M; Hatton, Jasmine; Harmon, Alice C; Harper, Jeffrey F

2009-08-01

Calcium signals are critical for the regulation of polarized growth in many eukaryotic cells, including pollen tubes and neurons. In plants, the regulatory pathways that code and decode Ca(2+) signals are poorly understood. In Arabidopsis thaliana, genetic evidence presented here indicates that pollen tube tip growth involves the redundant activity of two Ca(2+)-dependent protein kinases (CPKs), isoforms CPK17 and -34. Both isoforms appear to target to the plasma membrane, as shown by imaging of CPK17-yellow fluorescent protein (YFP) and CPK34-YFP in growing pollen tubes. Segregation analyses from two independent sets of T-DNA insertion mutants indicate that a double disruption of CPK17 and -34 results in an approximately 350-fold reduction in pollen transmission efficiency. The near sterile phenotype of homozygous double mutants could be rescued through pollen expression of a CPK34-YFP fusion. In contrast, a transgene rescue was blocked by mutations engineered to disrupt the Ca(2+)-activation mechanism of CPK34 (CPK34-YFP-E465A,E500A), providing in vivo evidence linking Ca(2+) activation to a biological function of a CPK. While double mutant pollen tubes displayed normal morphology, relative growth rates for the most rapidly growing tubes were reduced by more than three-fold compared with wild type. In addition, while most mutant tubes appeared to grow far enough to reach ovules, the vast majority (>90%) still failed to locate and fertilize ovules. Together, these results provide genetic evidence that CPKs are essential to pollen fitness, and support a mechanistic model in which CPK17 and -34 transduce Ca(2+) signals to increase the rate of pollen tube tip growth and facilitate a response to tropism cues.

Arabidopsis fhl/fhy1 double mutant reveals a distinct cytoplasmic action of phytochrome A

PubMed Central

Rösler, Jutta; Klein, Ilse; Zeidler, Mathias

2007-01-01

Phytochrome A (phyA) plays an important role during germination and early seedling development. Because phyA is the primary photoreceptor for the high-irradiance response and the very-low-fluence response, it can trigger development not only in red and far-red (FR) light but also in a wider range of light qualities. Although phyA action is generally associated with translocation to the nucleus and regulation of transcription, there is evidence for additional cytoplasmic functions. Because nuclear accumulation of phyA has been shown to depend on far-red-elongated hypocotyl 1 (FHY1) and FHL (FHY1-like), investigation of phyA function in a double fhl/fhy1 mutant might be valuable in revealing the mechanism of phyA translocation and possible cytoplasmic functions. In fhl/fhy1, the FR-triggered nuclear translocation of phyA could no longer be detected but could be restored by transgenic expression of CFP:FHY1. Whereas the fhl/fhy1 mutant showed a phyA phenotype in respect to hypocotyl elongation and cotyledon opening under high-irradiance response conditions as well as a typical phyA germination phenotype under very-low-fluence response conditions, fhl/fhy1 showed no phenotype with respect to the phyA-dependent abrogation of negative gravitropism in blue light and in red-enhanced phototropism, demonstrating clear cytoplasmic functions of phyA. Disturbance of phyA nuclear import in fhl/fhy1 led to formation of FR-induced phyA:GFP cytoplasmic foci resembling the sequestered areas of phytochrome. FHY1 and FHL play crucial roles in phyA nuclear translocation and signaling. Thus the double-mutant fhl/fhy1 allows nuclear and cytoplasmic phyA functions to be separated, leading to the novel identification of cytoplasmic phyA responses. PMID:17566111

GammaM23K, gammaM232K, and gammaL77K single substitutions in the TF1-ATPase lower ATPase activity by disrupting a cluster of hydrophobic side chains.

PubMed

Bandyopadhyay, Sanjay; Allison, William S

2004-07-27

In crystal structures of the bovine F(1)-ATPase (MF(1)), the side chains of gammaMet(23), gammaMet(232), and gammaLeu(77) interact in a cluster. Substitution of the corresponding residues in the alpha(3)beta(3)gamma subcomplex of TF(1) with lysine lowers the ATPase activity to 2.3, 11, and 15%, respectively, of that displayed by wild-type. In contrast, TF(1) subcomplexes containing the gammaM(23)C, gammaM(232)C, and gammaL(77)C substitutions display 36, 36, and 130%, respectively, of the wild-type ATPase activity. The ATPase activity of the gammaM(23)C/gammaM(232)C double mutant subcomplex is 36% that of the wild-type subcomplex before and after cross-linking the introduced cysteines, whereas the ATPase activity of the gammaM(23)C/L(77)C double mutant increased from 50 to 85% that of wild-type after cross-linking the introduced cysteines. Only beta-beta cross-links formed when the alpha(3)(betaE(395)C)(3)gammaM(23)C double mutant was inactivated with CuCl(2). The overall results suggest that the attenuated ATPase of the mutant subcomplexes containing the gammaM(23)K, gammaL(77)K, and gammaM(232)K substitutions is caused by disruption of the cluster of hydrophobic amino acid side chains and that the midregion of the coiled-coil comprised of the amino- and carboxyl-terminal alpha helices of the gamma subunit does not undergo unwinding or major displacement from the side chain of gammaLeu(77) during ATP-driven rotation of the gamma subunit.

Effects of three different nucleoid-associated proteins encoded on IncP-7 plasmid pCAR1 on host Pseudomonas putida KT2440.

PubMed

Suzuki-Minakuchi, Chiho; Hirotani, Ryusuke; Shintani, Masaki; Takeda, Toshiharu; Takahashi, Yurika; Matsui, Kazuhiro; Vasileva, Delyana; Yun, Choong-Soo; Okada, Kazunori; Yamane, Hisakazu; Nojiri, Hideaki

2015-04-01

Nucleoid-associated proteins (NAPs), which fold bacterial DNA and influence gene transcription, are considered to be global transcriptional regulators of genes on both plasmids and the host chromosome. Incompatibility P-7 group plasmid pCAR1 carries genes encoding three NAPs: H-NS family protein Pmr, NdpA-like protein Pnd, and HU-like protein Phu. In this study, the effects of single or double disruption of pmr, pnd, and phu were assessed in host Pseudomonas putida KT2440. When pmr and pnd or pmr and phu were simultaneously disrupted, both the segregational stability and the structural stability of pCAR1 were markedly decreased, suggesting that Pmr, Pnd, and Phu act as plasmid-stabilizing factors in addition to their established roles in replication and partition systems. The transfer frequency of pCAR1 was significantly decreased in these double mutants. The segregational and structural instability of pCAR1 in the double mutants was recovered by complementation of pmr, whereas no recovery of transfer deficiency was observed. Comprehensive phenotype comparisons showed that the host metabolism of carbon compounds, which was reduced by pCAR1 carriage, was restored by disruption of the NAP gene(s). Transcriptome analyses of mutants indicated that transcription of genes for energy production, conversion, inorganic ion transport, and metabolism were commonly affected; however, how their products altered the phenotypes of mutants was not clear. The findings of this study indicated that Pmr, Pnd, and Phu act synergistically to affect pCAR1 replication, maintenance, and transfer, as well as to alter the host metabolic phenotype. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Effects of Three Different Nucleoid-Associated Proteins Encoded on IncP-7 Plasmid pCAR1 on Host Pseudomonas putida KT2440

PubMed Central

Suzuki-Minakuchi, Chiho; Hirotani, Ryusuke; Shintani, Masaki; Takeda, Toshiharu; Takahashi, Yurika; Matsui, Kazuhiro; Vasileva, Delyana; Yun, Choong-Soo; Okada, Kazunori; Yamane, Hisakazu

2015-01-01

Nucleoid-associated proteins (NAPs), which fold bacterial DNA and influence gene transcription, are considered to be global transcriptional regulators of genes on both plasmids and the host chromosome. Incompatibility P-7 group plasmid pCAR1 carries genes encoding three NAPs: H-NS family protein Pmr, NdpA-like protein Pnd, and HU-like protein Phu. In this study, the effects of single or double disruption of pmr, pnd, and phu were assessed in host Pseudomonas putida KT2440. When pmr and pnd or pmr and phu were simultaneously disrupted, both the segregational stability and the structural stability of pCAR1 were markedly decreased, suggesting that Pmr, Pnd, and Phu act as plasmid-stabilizing factors in addition to their established roles in replication and partition systems. The transfer frequency of pCAR1 was significantly decreased in these double mutants. The segregational and structural instability of pCAR1 in the double mutants was recovered by complementation of pmr, whereas no recovery of transfer deficiency was observed. Comprehensive phenotype comparisons showed that the host metabolism of carbon compounds, which was reduced by pCAR1 carriage, was restored by disruption of the NAP gene(s). Transcriptome analyses of mutants indicated that transcription of genes for energy production, conversion, inorganic ion transport, and metabolism were commonly affected; however, how their products altered the phenotypes of mutants was not clear. The findings of this study indicated that Pmr, Pnd, and Phu act synergistically to affect pCAR1 replication, maintenance, and transfer, as well as to alter the host metabolic phenotype. PMID:25681185

Re-engineering of CYP2C9 to probe acid-base substrate selectivity.

PubMed

Tai, Guoying; Dickmann, Leslie J; Matovic, Nicholas; DeVoss, James J; Gillam, Elizabeth M J; Rettie, Allan E

2008-10-01

A common feature of many CYP2C9 ligands is their weak acidity. As revealed by crystallography, the structural basis for this behavior involves a charge-pairing interaction between an anionic moiety on the substrate and an active site R108 residue. In the present study we attempted to re-engineer CYP2C9 to better accept basic ligands by charge reversal at this key residue. We expressed and purified the R108E and R108E/D293N mutants and compared their ability with that of native CYP2C9 to interact with (S)-warfarin, diclofenac, pyrene, propranolol, and ibuprofen amine. As expected, the R108E mutant maintained all the native enzyme's pyrene 1-hydroxylation activity, but catalytic activity toward diclofenac and (S)-warfarin was abrogated. In contrast, the double mutant displayed much less selectivity in its behavior toward these control ligands. Neither of the mutants displayed significant enhancement of propranolol metabolism, and all three preparations exhibited a type II (inhibitor) rather than type I (substrate) spectrum with ibuprofen amine, although binding became progressively weaker with the single and double mutants. Collectively, these data underscore the importance of the amino acid at position 108 in the acid substrate selectivity of CYP2C9, highlight the accommodating nature of the CYP2C9 active site, and provide a cautionary note regarding facile re-engineering of these complex cytochrome P450 active sites.

Re-engineering of CYP2C9 to Probe Acid-Base Substrate Selectivity

PubMed Central

Tai, Guoying; Dickmann, Leslie J.; Matovic, Nicholas; DeVoss, James J.; Gillam, Elizabeth M. J.; Rettie, Allan E.

2009-01-01

A common feature of many CYP2C9 ligands is their weak acidity. As revealed by crystallography, the structural basis for this behavior involves a charge-pairing interaction between an anionic moiety on the substrate and an active site R108 residue. In the present study we attempted to re-engineer CYP2C9 to better accept basic ligands by charge reversal at this key residue. We expressed and purified the R108E and R108E/D293N mutants and compared their ability with that of native CYP2C9 to interact with (S)-warfarin, diclofenac, pyrene, propranolol, and ibuprofen amine. As expected, the R108E mutant maintained all the native enzyme's pyrene 1-hydroxylation activity, but catalytic activity toward diclofenac and (S)-warfarin was abrogated. In contrast, the double mutant displayed much less selectivity in its behavior toward these control ligands. Neither of the mutants displayed significant enhancement of propranolol metabolism, and all three preparations exhibited a type II (inhibitor) rather than type I (substrate) spectrum with ibuprofen amine, although binding became progressively weaker with the single and double mutants. Collectively, these data underscore the importance of the amino acid at position 108 in the acid substrate selectivity of CYP2C9, highlight the accommodating nature of the CYP2C9 active site, and provide a cautionary note regarding facile re-engineering of these complex cytochrome P450 active sites. PMID:18606741

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

Thompson, J.; Chassy, B.M.; Egan, W.

A mutant of Streptococcus lactis 133 has been isolated that lacks both glucokinase and phosphoenolpyruvate-dependent mannose- phosphotransferase (mannose-PTS) activities. The double mutant S. lactis 133 mannose-PTSd GK- is unable to utilize either exogenously supplied or intracellularly generated glucose for growth. Fluorographic analyses of metabolites formed during the metabolism of (/sup 14/C)lactose labeled specifically in the glucose or galactosyl moiety established that the cells were unable to phosphorylate intracellular glucose. However, cells of S. lactis 133 mannose-PTSd GK- readily metabolized intracellular glucose 6-phosphate, and the growth rates and cell yield of the mutant and parental strains on sucrose were the same.more » During growth on lactose, S. lactis 133 mannose-PTSd GK- fermented only the galactose moiety of the disaccharide, and 1 mol of glucose was generated per mol of lactose consumed. For an equivalent concentration of lactose, the cell yield of the mutant was 50% that of the wild type. The specific rate of lactose utilization by growing cells of S. lactis 133 mannose-PTSd GK- was ca. 50% greater than that of the wild type, but the cell doubling times were 70 and 47 min, respectively. High-resolution /sup 31/P nuclear magnetic resonance studies of lactose transport by starved cells of S. lactis 133 and S. lactis 133 mannose-PTSd GK- showed that the latter cells contained elevated lactose-PTS activity. Throughout exponential growth on lactose, the mutant maintained an intracellular steady-state glucose concentration of 100 mM.« less

Role of the plastidic glucose translocator in the export of starch degradation products from the chloroplasts in Arabidopsis thaliana.

PubMed

Cho, Man-Ho; Lim, Hyemin; Shin, Dong Ho; Jeon, Jong-Seong; Bhoo, Seong Hee; Park, Youn-Il; Hahn, Tae-Ryong

2011-04-01

In higher plants, the plastidic glucose translocator (pGlcT) is assumed to play a role in the export of starch degradation products, but this has not yet been studied in detail. To elucidate the role of pGlcT in the leaves of Arabidopsis thaliana, we generated single and double mutants lacking three plastidic sugar transporters, pGlcT, the triose-phosphate/phosphate translocator (TPT), and the maltose transporter (MEX1), and analyzed their growth phenotypes, photosynthetic properties and metabolite contents. In contrast to the pglct-1 and pglct-2 single mutants lacking a visible growth phenotype, the double mutants pglct-1/mex1 and tpt-2/mex1 displayed markedly inhibited plant growth. Notably, pglct-1/mex1 exhibited more severe growth retardation than that seen for the other mutants. In parallel, the most severe reductions in sucrose content and starch turnover were observed in the pglct-1/mex1 mutant. The concurrent loss of pGlcT and MEX1 also resulted in severely reduced photosynthetic activities and extreme chloroplast abnormalities. These findings suggest that pGlcT, together with MEX1, contributes significantly to the export of starch degradation products from chloroplasts in A. thaliana leaves, and that this starch-mediated pathway for photoassimilate export via pGlcT and MEX1 is essential for the growth and development of A. thaliana. © 2010 The Authors. New Phytologist © 2010 New Phytologist Trust.

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.

The orphan nuclear receptor small heterodimer partner is required for thiazolidinedione effects in leptin-deficient mice.

PubMed

Tseng, Hsiu-Ting; Park, Young Joo; Lee, Yoon Kwang; Moore, David D

2015-05-08

Small heterodimer partner (SHP, NR0B2) is involved in diverse metabolic pathways, including hepatic bile acid, lipid and glucose homeostasis, and has been implicated in effects on the peroxisome proliferator-activated receptor γ (PPARγ), a master regulator of adipogenesis and the receptor for antidiabetic drugs thiazolidinediones (TZDs). In this study, we aim to investigate the role of SHP in TZD response by comparing TZD-treated leptin-deficient (ob/ob) and leptin-, SHP-deficient (ob/ob;Shp(-/-)) double mutant mice. Both ob/ob and double mutant ob/ob;Shp(-/-) mice developed hyperglycemia, insulin resistance, and hyperlipidemia, but hepatic fat accumulation was decreased in the double mutant ob/ob;Shp(-/-) mice. PPARγ2 mRNA levels were markedly lower in ob/ob;Shp(-/-) liver and decreased to a lesser extent in adipose tissue. The TZD troglitazone did not reduce glucose or circulating triglyceride levels in ob/ob;Shp(-/-) mice. Expression of the adipocytokines, such as adiponectin and resistin, was not stimulated by troglitazone treatment. Expression of hepatic lipogenic genes was also reduced in ob/ob;Shp(-/-) mice. Moreover, overexpression of SHP by adenovirus infection increased PPARγ2 mRNA levels in mouse primary hepatocytes. Our results suggest that SHP is required for both antidiabetic and hypolipidemic effects of TZDs in ob/ob mice through regulation of PPARγ expression.

A homozygous nonsense CEP250 mutation combined with a heterozygous nonsense C2orf71 mutation is associated with atypical Usher syndrome.

PubMed

Khateb, Samer; Zelinger, Lina; Mizrahi-Meissonnier, Liliana; Ayuso, Carmen; Koenekoop, Robert K; Laxer, Uri; Gross, Menachem; Banin, Eyal; Sharon, Dror

2014-07-01

Usher syndrome (USH) is a heterogeneous group of inherited retinitis pigmentosa (RP) and sensorineural hearing loss (SNHL) caused by mutations in at least 12 genes. Our aim is to identify additional USH-related genes. Clinical examination included visual acuity test, funduscopy and electroretinography. Genetic analysis included homozygosity mapping and whole exome sequencing (WES). A combination of homozygosity mapping and WES in a large consanguineous family of Iranian Jewish origin revealed nonsense mutations in two ciliary genes: c.3289C>T (p.Q1097*) in C2orf71 and c.3463C>T (p.R1155*) in centrosome-associated protein CEP250 (C-Nap1). The latter has not been associated with any inherited disease and the c.3463C>T mutation was absent in control chromosomes. Patients who were double homozygotes had SNHL accompanied by early-onset and severe RP, while patients who were homozygous for the CEP250 mutation and carried a single mutant C2orf71 allele had SNHL with mild retinal degeneration. No ciliary structural abnormalities in the respiratory system were evident by electron microscopy analysis. CEP250 expression analysis of the mutant allele revealed the generation of a truncated protein lacking the NEK2-phosphorylation region. A homozygous nonsense CEP250 mutation, in combination with a heterozygous C2orf71 nonsense mutation, causes an atypical form of USH, characterised by early-onset SNHL and a relatively mild RP. The severe retinal involvement in the double homozygotes indicates an additive effect caused by nonsense mutations in genes encoding ciliary proteins. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Characterization of a Weak Allele of Zebrafish cloche Mutant

PubMed Central

Ma, Ning; Huang, Zhibin; Chen, Xiaohui; He, Fei; Wang, Kun; Liu, Wei; Zhao, Linfeng; Xu, Xiangmin; Liao, Wangjun; Ruan, Hua; Luo, Shenqiu; Zhang, Wenqing

2011-01-01

Hematopoiesis is a complicated and dynamic process about which the molecular mechanisms remain poorly understood. Danio rerio (zebrafish) is an excellent vertebrate system for studying hematopoiesis and developmental mechanisms. In the previous study, we isolated and identified a cloche 172 (clo 172) mutant, a novel allele compared to the original cloche (clo) mutant, through using complementation test and initial mapping. Here, according to whole mount in-situ hybridization, we report that the endothelial cells in clo 172 mutant embryos, although initially developed, failed to form the functional vascular system eventually. In addition, further characterization indicates that the clo 172 mutant exhibited weaker defects instead of completely lost in primitive erythroid cells and definitive hematopoietic cells compared with the clo s5 mutant. In contrast, primitive myeloid cells were totally lost in clo 172 mutant. Furthermore, these reappeared definitive myeloid cells were demonstrated to initiate from the remaining hematopoietic stem cells (HSCs) in clo 172 mutant, confirmed by the dramatic decrease of lyc in clo 172 runx1w84x double mutant. Collectively, the clo 172 mutant is a weak allele compared to the clo s5 mutant, therefore providing a model for studying the early development of hematopoietic and vascular system, as well as an opportunity to further understand the function of the cloche gene. PMID:22132109

A mutant of the major apple allergen, Mal d 1, demonstrating hypo-allergenicity in the target organ by double-blind placebo-controlled food challenge.

PubMed

Bolhaar, S T H P; Zuidmeer, L; Ma, Y; Ferreira, F; Bruijnzeel-Koomen, C A F M; Hoffmann-Sommergruber, K; van Ree, R; Knulst, A C

2005-12-01

Allergen-specific immunotherapy for food allergy has been hindered by severe side-effects in the past. Well-characterized hypo-allergenic recombinant food allergens potentially offer a safe solution. To demonstrate hypo-allergenicity of a mutated major food allergen from apple, Mal d 1, in vitro and in vivo. A mutant of the major apple allergen, Mal d 1, was obtained by site-directed mutagenesis exchanging five amino acid residues. Fourteen patients with combined birch pollen-related apple allergy were included in the study. Hypo-allergenicity of the mutant rMal d 1 (rMal d 1mut) compared with rMal d 1 was assessed by in vitro methods, i.e. RAST (inhibition), immunoblotting and basophil histamine release (BHR) and in vivo by skin prick test and double-blind placebo-controlled food challenge (DBPCFC). RAST analysis (n = 14) revealed that IgE reactivity to rMal d 1mut was twofold lower than that of the wild-type molecule (95% confidence interval (CI): 1.7-2.4). RAST inhibition (n = 6) showed a 7.8-fold decrease in IgE-binding potency (95% CI: 3.0-12.6). In contrast to this moderate decrease in IgE-binding potency, the biological activity of rMal d 1mut assessed by SPT and BHR decreased 10-200-fold. Hypo-allergenicity was confirmed by DBPCFC (n = 2) with both recombinant molecules. A moderate decrease in IgE-binding potency translates into a potent inhibition of biological activity. This is the first study that confirms by DBPCFC that a mutated recombinant major food allergen is clinically hypo-allergenic. This paves the way towards safer immunotherapy for the treatment of food-allergic patients.

[Construction of Plasmodium falciparum signal peptide peptidase-GFP mutant and its expression analysis in the malaria parasite].

PubMed

Li, Xue-rong; Wu, Yin-juan; Shang, Mei; Li, Ye; Xu, Jin; Yu, Xin-bing; Athar, Chishti

2014-08-01

To construct recombinant plasmid pSPPcGT which contains signal peptide peptidase gene of Plasmodium falciparum (PJSPP) and GFP, and transfect into P. falciparum (3D7 strain) to obtain mutant parasites which can express PJSPP-GFP. Plasmodium falciparum(3D7 strain) genomic DNA was extracted from cultured malaria parasites. The C-terminal region of PJSPP, an 883 bp gene fragment was amplified by PCR, and then cloned into pPM2GT vector to get recombinant vector pSPPcGT. The recombinant vectors were identified by PCR, double restriction enzyme digestion and DNA sequencing. pSPPcGT vector was transfected into malaria parasites. The positive clones were selected by adding inhibitor of Plasmodium falciparum dihydrofolate reductase WR99210 to the culture medium. The pSPP-GFP-transfected parasites were fixed with methanol, stained with DAPI, and observed under immunofluorescence microscope. The PJSPP-GFP expression in P. falciparum was identified by SDS-PAGE and Western blotting. The C-terminal region of PJSPP was amplified from P.falciparum (3D7 strain) genomic DNA by PCR with the length of 883 bp. The constructed recombinant vectors were identified by PCR screening, double restriction enzyme digestion and DNA sequencing. The pSPPcGT vector was transfected into P. falciparum and the positive clones were selected by WR99210. GFP fluorescence was observed in transfected parasites by immunofluorescence microscopy, and mainly located in the cytoplasm. The PJSPP-GFP expression in malaria parasites was confirmed by Western blotting with a relative molecular mass of Mr 64,000. Recombinant vector PJSPP-GFP is constructed and transfected into P. falciparum to obtain P. falciparum mutant clone which can express PfSPP-GFP.

Novel mutations in β-tubulin gene in Trichoderma harzianum mutants resistant to methyl benzimidazol-2-yl carbamate.

PubMed

Li, M; Zhang, H Y; Liang, B

2013-01-01

Twelve-low resistant (LR) mutants of Trichoderma harzianum with the capability of grow fast at 0.8 μg/mL methyl benzimidazol-2-yl carbamate (MBC) were obtained using UV mutagenesis. MR and HR mutants which could grow fast at 10 and 100 μg/mL MBC, respectively, were isolated by step-up selection protocols in which UV-treated mutants were induced and mycelial sector screening was made in plates with growth medium. Subsequently, β-tubulin genes of 14 mutants were cloned to describe-the molecular lesion likely to be responsible-for MBC resistance. Comparison of the β-tubulin sequences of the mutant and sensitive strains of T. harzianum revealed 2 new MBC-binding sites differed from those in other plant pathogens. A single mutation at-amino acid 168, having Phe (TTC) instead of Ser (TCC)', was demonstrated for the HR mutant; a double mutation in amino acid 13 resulting in the substitution of Gly (GGC) by Val (GTG) was observed in β-tubulin gene of MR mutant. On the other hand, no substitutions were identified in the β-tubulin gene and its 5'-flanking regions in 12 LR mutants of T. harzianum.

Life without complex I: proteome analyses of an Arabidopsis mutant lacking the mitochondrial NADH dehydrogenase complex

PubMed Central

Fromm, Steffanie; Senkler, Jennifer; Eubel, Holger; Peterhänsel, Christoph; Braun, Hans-Peter

2016-01-01

The mitochondrial NADH dehydrogenase complex (complex I) is of particular importance for the respiratory chain in mitochondria. It is the major electron entry site for the mitochondrial electron transport chain (mETC) and therefore of great significance for mitochondrial ATP generation. We recently described an Arabidopsis thaliana double-mutant lacking the genes encoding the carbonic anhydrases CA1 and CA2, which both form part of a plant-specific ‘carbonic anhydrase domain’ of mitochondrial complex I. The mutant lacks complex I completely. Here we report extended analyses for systematically characterizing the proteome of the ca1ca2 mutant. Using various proteomic tools, we show that lack of complex I causes reorganization of the cellular respiration system. Reduced electron entry into the respiratory chain at the first segment of the mETC leads to induction of complexes II and IV as well as alternative oxidase. Increased electron entry at later segments of the mETC requires an increase in oxidation of organic substrates. This is reflected by higher abundance of proteins involved in glycolysis, the tricarboxylic acid cycle and branched-chain amino acid catabolism. Proteins involved in the light reaction of photosynthesis, the Calvin cycle, tetrapyrrole biosynthesis, and photorespiration are clearly reduced, contributing to the significant delay in growth and development of the double-mutant. Finally, enzymes involved in defense against reactive oxygen species and stress symptoms are much induced. These together with previously reported insights into the function of plant complex I, which were obtained by analysing other complex I mutants, are integrated in order to comprehensively describe ‘life without complex I’. PMID:27122571

The Tomato (Solanum Lycopersicum cv. Micro-Tom) Natural Genetic Variation Rg1 and the DELLA Mutant Procera Control the Competence Necessary to Form Adventitious Roots and Shoots

PubMed Central

Peres, Lázaro Eustáquio Pereira

2012-01-01

Despite the wide use of plant regeneration for biotechnological purposes, the signals that allow cells to become competent to assume different fates remain largely unknown. Here, it is demonstrated that the Regeneration1 (Rg1) allele, a natural genetic variation from the tomato wild relative Solanum peruvianum, increases the capacity to form both roots and shoots in vitro; and that the gibberellin constitutive mutant procera (pro) presented the opposite phenotype, reducing organogenesis on either root-inducing medium (RIM) or shoot-inducing medium (SIM). Mutants showing alterations in the formation of specific organs in vitro were the auxin low-sensitivity diageotropica (dgt), the lateral suppresser (ls), and the KNOX-overexpressing Mouse ears (Me). dgt failed to form roots on RIM, Me increased shoot formation on SIM, and the high capacity for in vitro shoot formation of ls contrasted with its recalcitrance to form axillary meristems. Interestingly, Rg1 rescued the in vitro organ formation capacity in proRg1 and dgtRg1 double mutants and the ex vitro low lateral shoot formation in pro and ls. Such epistatic interactions were also confirmed in gene expression and histological analyses conducted in the single and double mutants. Although Me phenocopied the high shoot formation of Rg1 on SIM, it failed to increase rooting on RIM and to rescue the non-branching phenotype of ls. Taken together, these results suggest REGENERATION1 and the DELLA mutant PROCERA as controlling a common competence to assume distinct cell fates, rather than the specific induction of adventitious roots or shoots, which is controlled by DIAGEOTROPICA and MOUSE EARS, respectively. PMID:22915742

Genetic interactions of the unfinished flower development (ufd) mutant support a significant role of the tomato UFD gene in regulating floral organogenesis.

PubMed

Poyatos-Pertíñez, Sandra; Quinet, Muriel; Ortíz-Atienza, Ana; Bretones, Sandra; Yuste-Lisbona, Fernando J; Lozano, Rafael

2016-09-01

Genetic interactions of UFD gene support its specific function during reproductive development of tomato; in this process, UFD could play a pivotal role between inflorescence architecture and flower initiation genes. Tomato (Solanum lycopersicum L.) is a major vegetable crop that also constitutes a model species for the study of plant developmental processes. To gain insight into the control of flowering and floral development, a novel tomato mutant, unfinished flower development (ufd), whose inflorescence and flowers were unable to complete their normal development was characterized using double mutant and gene expression analyses. Genetic interactions of ufd with mutations affecting inflorescence fate (uniflora, jointless and single flower truss) were additive and resulted in double mutants displaying the inflorescence structure of the non-ufd parental mutant and the flower phenotype of the ufd mutant. In addition, ufd mutation promotes an earlier inflorescence meristem termination. Taken together, both results indicated that UFD is not involved in the maintenance of inflorescence meristem identity, although it could participate in the regulatory system that modulates the rate of meristem maturation. Regarding the floral meristem identity, the falsiflora mutation was epistatic to the ufd mutation even though FALSIFLORA was upregulated in ufd inflorescences. In terms of floral organ identity, the ufd mutation was epistatic to macrocalyx, and MACROCALYX expression was differently regulated depending on the inflorescence developmental stage. These results suggest that the UFD gene may play a pivotal role between the genes required for flowering initiation and inflorescence development (such as UNIFLORA, FALSIFLORA, JOINTLESS and SINGLE FLOWER TRUSS) and those required for further floral organ development such as the floral organ identity genes.

Galactose-depleted xyloglucan is dysfunctional and leads to dwarfism in Arabidopsis.

PubMed

Kong, Yingzhen; Peña, Maria J; Renna, Luciana; Avci, Utku; Pattathil, Sivakumar; Tuomivaara, Sami T; Li, Xuemei; Reiter, Wolf-Dieter; Brandizzi, Federica; Hahn, Michael G; Darvill, Alan G; York, William S; O'Neill, Malcolm A

2015-04-01

Xyloglucan is a polysaccharide that has important roles in the formation and function of the walls that surround growing land plant cells. Many of these plants synthesize xyloglucan that contains galactose in two different side chains (L and F), which exist in distinct molecular environments. However, little is known about the contribution of these side chains to xyloglucan function. Here, we show that Arabidopsis (Arabidopsis thaliana) mutants devoid of the F side chain galactosyltransferase MURUS3 (MUR3) form xyloglucan that lacks F side chains and contains much less galactosylated xylose than its wild-type counterpart. The galactose-depleted xyloglucan is dysfunctional, as it leads to mutants that are dwarfed with curled rosette leaves, short petioles, and short inflorescence stems. Moreover, cell wall matrix polysaccharides, including xyloglucan and pectin, are not properly secreted and instead accumulate within intracellular aggregates. Near-normal growth is restored by generating mur3 mutants that produce no detectable amounts of xyloglucan. Thus, cellular processes are affected more by the presence of the dysfunctional xyloglucan than by eliminating xyloglucan altogether. To identify structural features responsible for xyloglucan dysfunction, xyloglucan structure was modified in situ by generating mur3 mutants that lack specific xyloglucan xylosyltransferases (XXTs) or that overexpress the XYLOGLUCAN L-SIDE CHAIN GALACTOSYLTRANSFERASE2 (XLT2) gene. Normal growth was restored in the mur3-3 mutant overexpressing XLT2 and in mur3-3 xxt double mutants when the dysfunctional xyloglucan was modified by doubling the amounts of galactosylated side chains. Our study assigns a role for galactosylation in normal xyloglucan function and demonstrates that altering xyloglucan side chain structure disturbs diverse cellular and physiological processes. © 2015 American Society of Plant Biologists. All Rights Reserved.

The tomato (Solanum lycopersicum cv. Micro-Tom) natural genetic variation Rg1 and the DELLA mutant procera control the competence necessary to form adventitious roots and shoots.

PubMed

Lombardi-Crestana, Simone; da Silva Azevedo, Mariana; e Silva, Geraldo Felipe Ferreira; Pino, Lílian Ellen; Appezzato-da-Glória, Beatriz; Figueira, Antonio; Nogueira, Fabio Tebaldi Silveira; Peres, Lázaro Eustáquio Pereira

2012-09-01

Despite the wide use of plant regeneration for biotechnological purposes, the signals that allow cells to become competent to assume different fates remain largely unknown. Here, it is demonstrated that the Regeneration1 (Rg1) allele, a natural genetic variation from the tomato wild relative Solanum peruvianum, increases the capacity to form both roots and shoots in vitro; and that the gibberellin constitutive mutant procera (pro) presented the opposite phenotype, reducing organogenesis on either root-inducing medium (RIM) or shoot-inducing medium (SIM). Mutants showing alterations in the formation of specific organs in vitro were the auxin low-sensitivity diageotropica (dgt), the lateral suppresser (ls), and the KNOX-overexpressing Mouse ears (Me). dgt failed to form roots on RIM, Me increased shoot formation on SIM, and the high capacity for in vitro shoot formation of ls contrasted with its recalcitrance to form axillary meristems. Interestingly, Rg1 rescued the in vitro organ formation capacity in proRg1 and dgtRg1 double mutants and the ex vitro low lateral shoot formation in pro and ls. Such epistatic interactions were also confirmed in gene expression and histological analyses conducted in the single and double mutants. Although Me phenocopied the high shoot formation of Rg1 on SIM, it failed to increase rooting on RIM and to rescue the non-branching phenotype of ls. Taken together, these results suggest REGENERATION1 and the DELLA mutant PROCERA as controlling a common competence to assume distinct cell fates, rather than the specific induction of adventitious roots or shoots, which is controlled by DIAGEOTROPICA and MOUSE EARS, respectively.

Roles of BOR2, a Boron Exporter, in Cross Linking of Rhamnogalacturonan II and Root Elongation under Boron Limitation in Arabidopsis1[W

PubMed Central

Miwa, Kyoko; Wakuta, Shinji; Takada, Shigeki; Ide, Koji; Takano, Junpei; Naito, Satoshi; Omori, Hiroyuki; Matsunaga, Toshiro; Fujiwara, Toru

2013-01-01

Boron (B) is required for cross linking of the pectic polysaccharide rhamnogalacturonan II (RG-II) and is consequently essential for the maintenance of cell wall structure. Arabidopsis (Arabidopsis thaliana) BOR1 is an efflux B transporter for xylem loading of B. Here, we describe the roles of BOR2, the most similar paralog of BOR1. BOR2 encodes an efflux B transporter localized in plasma membrane and is strongly expressed in lateral root caps and epidermis of elongation zones of roots. Transfer DNA insertion of BOR2 reduced root elongation by 68%, whereas the mutation in BOR1 reduced it by 32% under low B availability (0.1 µm), but the reduction in shoot growth was not as obvious as that in the BOR1 mutant. A double mutant of BOR1 and BOR2 exhibited much more severe growth defects in both roots and shoots under B-limited conditions than the corresponding single mutants. All single and double mutants grew normally under B-sufficient conditions. These results suggest that both BOR1 and BOR2 are required under B limitation and that their roles are, at least in part, different. The total B concentrations in roots of BOR2 mutants were not significantly different from those in wild-type plants, but the proportion of cross-linked RG-II was reduced under low B availability. Such a reduction in RG-II cross linking was not evident in roots of the BOR1 mutant. Thus, we propose that under B-limited conditions, transport of boric acid/borate by BOR2 from symplast to apoplast is required for effective cross linking of RG-II in cell wall and root cell elongation. PMID:24114060

Symmetric vs. Asymmetric Stem Cell Divisions: An Adaptation against Cancer?

PubMed Central

Shahriyari, Leili; Komarova, Natalia L.

2013-01-01

Traditionally, it has been held that a central characteristic of stem cells is their ability to divide asymmetrically. Recent advances in inducible genetic labeling provided ample evidence that symmetric stem cell divisions play an important role in adult mammalian homeostasis. It is well understood that the two types of cell divisions differ in terms of the stem cells' flexibility to expand when needed. On the contrary, the implications of symmetric and asymmetric divisions for mutation accumulation are still poorly understood. In this paper we study a stochastic model of a renewing tissue, and address the optimization problem of tissue architecture in the context of mutant production. Specifically, we study the process of tumor suppressor gene inactivation which usually takes place as a consequence of two “hits”, and which is one of the most common patterns in carcinogenesis. We compare and contrast symmetric and asymmetric (and mixed) stem cell divisions, and focus on the rate at which double-hit mutants are generated. It turns out that symmetrically-dividing cells generate such mutants at a rate which is significantly lower than that of asymmetrically-dividing cells. This result holds whether single-hit (intermediate) mutants are disadvantageous, neutral, or advantageous. It is also independent on whether the carcinogenic double-hit mutants are produced only among the stem cells or also among more specialized cells. We argue that symmetric stem cell divisions in mammals could be an adaptation which helps delay the onset of cancers. We further investigate the question of the optimal fraction of stem cells in the tissue, and quantify the contribution of non-stem cells in mutant production. Our work provides a hypothesis to explain the observation that in mammalian cells, symmetric patterns of stem cell division seem to be very common. PMID:24204602

The fast-folding HP35 double mutant has a substantially reduced primary folding free energy barrier

NASA Astrophysics Data System (ADS)

Lei, Hongxing; Deng, Xiaojian; Wang, Zhixiang; Duan, Yong

2008-10-01

The LYS24/29NLE double mutant of villin headpiece subdomain (HP35) is the fastest folding protein known so far with a folding time constant of 0.6μs. In this work, the folding mechanism of the mutant has been investigated by both conventional and replica exchange molecular dynamics (CMD and REMD) simulations with AMBER FF03 force field and a generalized-Born solvation model. Direct comparison to the ab initio folding of the wild type HP35 enabled a close examination on the mutational effect on the folding process. The mutant folded to the native state, as demonstrated by the 0.50Å Cα-root mean square deviation (RMSD) sampled in both CMD and REMD simulations and the high population of the folded conformation compared with the denatured conformations. Consistent with experiments, the significantly reduced primary folding free energy barrier makes the mutant closer to a downhill folder than the wild type HP35 that directly leads to the faster transition and higher melting temperature. However, unlike the proposed downhill folding which envisages a smooth shift between unfolded and folded states without transition barrier, we observed a well-defined folding transition that was consistent with experiments. Further examination of the secondary structures revealed that the two mutated residues have higher intrinsic helical preference that facilitated the formation of both helix III and the intermediate state which contains the folded segment helix II/III. Other factors contributing to the faster folding include the more favorable electrostatic interactions in the transition state with the removal of the charged NH3+ groups from LYS. In addition, both transition state ensemble and denatured state ensemble are shifted in the mutant.

[Double mutant alleles in the EXT1 gene not previously reported in a teenager with hereditary multiple exostoses].

PubMed

Cammarata-Scalisi, Francisco; Cozar, Mónica; Grinberg, Daniel; Balcells, Susana; Asteggiano, Carla G; Martínez-Domenech, Gustavo; Bracho, Ana; Sánchez, Yanira; Stock, Frances; Delgado-Luengo, Wilmer; Zara-Chirinos, Carmen; Chacín, José Antonio

2015-04-01

Hereditary forms of multiple exostoses, now called EXT1/EXT2-CDG within Congenital Disorders of Glycosylation, are the most common benign bone tumors in humans and clinical description consists of the formation of several cartilage-capped bone tumors, usually benign and localized in the juxta-epiphyseal region of long bones, although wide body dissemination in severe cases is not uncommon. Onset of the disease is variable ranging from 2-3 years up to 13-15 years with an estimated incidence ranging from 1/18,000 to 1/50,000 cases in European countries. We present a double mutant alleles in the EXT1 gene not previously reported in a teenager and her family with hereditary multiple exostoses.

Dissecting hematopoietic and renal cell heterogeneity in adult zebrafish at single-cell resolution using RNA sequencing.

PubMed

Tang, Qin; Iyer, Sowmya; Lobbardi, Riadh; Moore, John C; Chen, Huidong; Lareau, Caleb; Hebert, Christine; Shaw, McKenzie L; Neftel, Cyril; Suva, Mario L; Ceol, Craig J; Bernards, Andre; Aryee, Martin; Pinello, Luca; Drummond, Iain A; Langenau, David M

2017-10-02

Recent advances in single-cell, transcriptomic profiling have provided unprecedented access to investigate cell heterogeneity during tissue and organ development. In this study, we used massively parallel, single-cell RNA sequencing to define cell heterogeneity within the zebrafish kidney marrow, constructing a comprehensive molecular atlas of definitive hematopoiesis and functionally distinct renal cells found in adult zebrafish. Because our method analyzed blood and kidney cells in an unbiased manner, our approach was useful in characterizing immune-cell deficiencies within DNA-protein kinase catalytic subunit ( prkdc ), interleukin-2 receptor γ a ( il2rga ), and double-homozygous-mutant fish, identifying blood cell losses in T, B, and natural killer cells within specific genetic mutants. Our analysis also uncovered novel cell types, including two classes of natural killer immune cells, classically defined and erythroid-primed hematopoietic stem and progenitor cells, mucin-secreting kidney cells, and kidney stem/progenitor cells. In total, our work provides the first, comprehensive, single-cell, transcriptomic analysis of kidney and marrow cells in the adult zebrafish. © 2017 Tang et al.

Dissecting hematopoietic and renal cell heterogeneity in adult zebrafish at single-cell resolution using RNA sequencing

PubMed Central

Iyer, Sowmya; Lobbardi, Riadh; Chen, Huidong; Hebert, Christine; Shaw, McKenzie L.; Neftel, Cyril; Suva, Mario L.; Bernards, Andre; Aryee, Martin; Drummond, Iain A.

2017-01-01

Recent advances in single-cell, transcriptomic profiling have provided unprecedented access to investigate cell heterogeneity during tissue and organ development. In this study, we used massively parallel, single-cell RNA sequencing to define cell heterogeneity within the zebrafish kidney marrow, constructing a comprehensive molecular atlas of definitive hematopoiesis and functionally distinct renal cells found in adult zebrafish. Because our method analyzed blood and kidney cells in an unbiased manner, our approach was useful in characterizing immune-cell deficiencies within DNA–protein kinase catalytic subunit (prkdc), interleukin-2 receptor γ a (il2rga), and double-homozygous–mutant fish, identifying blood cell losses in T, B, and natural killer cells within specific genetic mutants. Our analysis also uncovered novel cell types, including two classes of natural killer immune cells, classically defined and erythroid-primed hematopoietic stem and progenitor cells, mucin-secreting kidney cells, and kidney stem/progenitor cells. In total, our work provides the first, comprehensive, single-cell, transcriptomic analysis of kidney and marrow cells in the adult zebrafish. PMID:28878000

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

Evidence for the Critical Role of Sucrose Synthase for Anoxic Tolerance of Maize Roots using a Double Mutant

PubMed Central

Ricard, Bérénice; Toai, Tara Van; Chourey, Prem; Saglio, Pierre

1998-01-01

The induction of the sucrose synthase (SuSy) gene (SuSy) by low O2, low temperature, and limiting carbohydrate supply suggested a role in carbohydrate metabolism under stress conditions. The isolation of a maize (Zea mays L.) line mutant for the two known SuSy genes but functionally normal showed that SuSy activity might not be required for aerobic growth and allowed the possibility of investigating its importance during anaerobic stress. As assessed by root elongation after return to air, hypoxic pretreatment improved anoxic tolerance, in correlation with the number of SuSy genes and the level of SuSy expression. Furthermore, root death in double-mutant seedlings during anoxic incubation could be attributed to the impaired utilization of sucrose (Suc). Collectively, these data provide unequivocal evidence that Suc is the principal C source and that SuSy is the main enzyme active in Suc breakdown in roots of maize seedlings deprived of O2. In this situation, SuSy plays a critical role in anoxic tolerance. PMID:9536049

Actin cables and the exocyst form two independent morphogenesis pathways in the fission yeast

PubMed Central

Bendezú, Felipe O.; Martin, Sophie G.

2011-01-01

Cell morphogenesis depends on polarized exocytosis. One widely held model posits that long-range transport and exocyst-dependent tethering of exocytic vesicles at the plasma membrane sequentially drive this process. Here, we describe that disruption of either actin-based long-range transport and microtubules or the exocyst did not abolish polarized growth in rod-shaped fission yeast cells. However, disruption of both actin cables and exocyst led to isotropic growth. Exocytic vesicles localized to cell tips in single mutants but were dispersed in double mutants. In contrast, a marker for active Cdc42, a major polarity landmark, localized to discreet cortical sites even in double mutants. Localization and photobleaching studies show that the exocyst subunits Sec6 and Sec8 localize to cell tips largely independently of the actin cytoskeleton, but in a cdc42 and phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2)–dependent manner. Thus in fission yeast long-range cytoskeletal transport and PIP2-dependent exocyst represent parallel morphogenetic modules downstream of Cdc42, raising the possibility of similar mechanisms in other cell types. PMID:21148300

Inviability of a DNA2 deletion mutant is due to the DNA damage checkpoint.

PubMed

Budd, Martin E; Antoshechkin, Igor A; Reis, Clara; Wold, Barbara J; Campbell, Judith L

2011-05-15

Dna2 is a dual polarity exo/endonuclease, and 5' to 3' DNA helicase involved in Okazaki Fragment Processing (OFP) and Double-Strand Break (DSB) Repair. In yeast, DNA2 is an essential gene, as expected for a DNA replication protein. Suppression of the lethality of dna2Δ mutants has been found to occur by two mechanisms: overexpression of RAD27 (scFEN1) , encoding a 5' to 3' exo/endo nuclease that processes Okazaki fragments (OFs) for ligation, or deletion of PIF1, a 5' to 3' helicase involved in mitochondrial recombination, telomerase inhibition and OFP. Mapping of a novel, spontaneously arising suppressor of dna2Δ now reveals that mutation of rad9 and double mutation of rad9 mrc1 can also suppress the lethality of dna2Δ mutants. Interaction of dna2Δ and DNA damage checkpoint mutations provides insight as to why dna2Δ is lethal but rad27Δ is not, even though evidence shows that Rad27 (ScFEN1) processes most of the Okazaki fragments, while Dna2 processes only a subset.

Molecular crowding overcomes the destabilizing effects of mutations in a bacterial ribozyme

DOE PAGES

Lee, Hui-Ting; Kilburn, D.; Behrouzi, R.; ...

2014-12-05

The native structure of the Azoarcus group I ribozyme is stabilized by the cooperative formation of tertiary interactions between double helical domains. Thus, even single mutations that break this network of tertiary interactions reduce ribozyme activity in physiological Mg2+ concentrations. Here, we report that molecular crowding comparable to that in the cell compensates for destabilizing mutations in the Azoarcus ribozyme. Small angle X-ray scattering, native polyacrylamide gel electrophoresis and activity assays were used to compare folding free energies in dilute and crowded solutions containing 18% PEG1000. Crowder molecules allowed the wild-type and mutant ribozymes to fold at similarly low Mg2+more » concentrations and stabilized the active structure of the mutant ribozymes under physiological conditions. This compensation helps explains why ribozyme mutations are often less deleterious in the cell than in the test tube. Nevertheless, crowding did not rescue the high fraction of folded but less active structures formed by double and triple mutants. We conclude that crowding broadens the fitness landscape by stabilizing compact RNA structures without improving the specificity of self-assembly.« less

Molecular crowding overcomes the destabilizing effects of mutations in a bacterial ribozyme

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

Lee, Hui-Ting; Kilburn, D.; Behrouzi, R.

The native structure of the Azoarcus group I ribozyme is stabilized by the cooperative formation of tertiary interactions between double helical domains. Thus, even single mutations that break this network of tertiary interactions reduce ribozyme activity in physiological Mg2+ concentrations. Here, we report that molecular crowding comparable to that in the cell compensates for destabilizing mutations in the Azoarcus ribozyme. Small angle X-ray scattering, native polyacrylamide gel electrophoresis and activity assays were used to compare folding free energies in dilute and crowded solutions containing 18% PEG1000. Crowder molecules allowed the wild-type and mutant ribozymes to fold at similarly low Mg2+more » concentrations and stabilized the active structure of the mutant ribozymes under physiological conditions. This compensation helps explains why ribozyme mutations are often less deleterious in the cell than in the test tube. Nevertheless, crowding did not rescue the high fraction of folded but less active structures formed by double and triple mutants. We conclude that crowding broadens the fitness landscape by stabilizing compact RNA structures without improving the specificity of self-assembly.« less

Hot-spot analysis to dissect the functional protein-protein interface of a tRNA-modifying enzyme.

PubMed

Jakobi, Stephan; Nguyen, Tran Xuan Phong; Debaene, François; Metz, Alexander; Sanglier-Cianférani, Sarah; Reuter, Klaus; Klebe, Gerhard

2014-10-01

Interference with protein-protein interactions of interfaces larger than 1500 Ų by small drug-like molecules is notoriously difficult, particularly if targeting homodimers. The tRNA modifying enzyme Tgt is only functionally active as a homodimer. Thus, blocking Tgt dimerization is a promising strategy for drug therapy as this protein is key to the development of Shigellosis. Our goal was to identify hot-spot residues which, upon mutation, result in a predominantly monomeric state of Tgt. The detailed understanding of the spatial location and stability contribution of the individual interaction hot-spot residues and the plasticity of motifs involved in the interface formation is a crucial prerequisite for the rational identification of drug-like inhibitors addressing the respective dimerization interface. Using computational analyses, we identified hot-spot residues that contribute particularly to dimer stability: a cluster of hydrophobic and aromatic residues as well as several salt bridges. This in silico prediction led to the identification of a promising double mutant, which was validated experimentally. Native nano-ESI mass spectrometry showed that the dimerization of the suggested mutant is largely prevented resulting in a predominantly monomeric state. Crystal structure analysis and enzyme kinetics of the mutant variant further support the evidence for enhanced monomerization and provide first insights into the structural consequences of the dimer destabilization. © 2014 Wiley Periodicals, Inc.

Structural and mechanistic analysis of engineered trichodiene synthase enzymes from Trichoderma harzianum: towards higher catalytic activities empowering sustainable agriculture.

PubMed

Kumari, Indu; Chaudhary, Nitika; Sandhu, Padmani; Ahmed, Mushtaq; Akhter, Yusuf

2016-06-01

Trichoderma spp. are well-known bioagents for the plant growth promotion and pathogen suppression. The beneficial activities of the fungus Trichoderma spp. are attributed to their ability to produce and secrete certain secondary metabolites such as trichodermin that belongs to trichothecene family of molecules. The initial steps of trichodermin biosynthetic pathway in Trichoderma are similar to the trichothecenes from Fusarium sporotrichioides. Trichodiene synthase (TS) encoded by tri5 gene in Trichoderma catalyses the conversion of farnesyl pyrophosphate to trichodiene as reported earlier. In this study, we have carried out a comprehensive comparative sequence and structural analysis of the TS, which revealed the conserved residues involved in catalytic activity of the protein. In silico, modelled tertiary structure of TS protein showed stable structural behaviour during simulations. Two single-substitution mutants, i.e. D109E, D248Y and one double-substitution mutant (D109E and D248Y) of TS with potentially higher activities are screened out. The mutant proteins showed more stability than the wild type, an increased number of electrostatic interactions and better binding energies with the ligand, which further elucidates the amino acid residues involved in the reaction mechanism. These results will lead to devise strategies for higher TS activity to ultimately enhance the trichodermin production by Trichoderma spp. for its better exploitation in the sustainable agricultural practices.

Regulation of Drought Tolerance by the F-Box Protein MAX2 in Arabidopsis1[C][W][OPEN

PubMed Central

Bu, Qingyun; Lv, Tianxiao; Shen, Hui; Luong, Phi; Wang, Jimmy; Wang, Zhenyu; Huang, Zhigang; Xiao, Langtao; Engineer, Cawas; Kim, Tae Houn; Schroeder, Julian I.; Huq, Enamul

2014-01-01

MAX2 (for MORE AXILLARY GROWTH2) has been shown to regulate diverse biological processes, including plant architecture, photomorphogenesis, senescence, and karrikin signaling. Although karrikin is a smoke-derived abiotic signal, a role for MAX2 in abiotic stress response pathways is least investigated. Here, we show that the max2 mutant is strongly hypersensitive to drought stress compared with wild-type Arabidopsis (Arabidopsis thaliana). Stomatal closure of max2 was less sensitive to abscisic acid (ABA) than that of the wild type. Cuticle thickness of max2 was significantly thinner than that of the wild type. Both of these phenotypes of max2 mutant plants correlate with the increased water loss and drought-sensitive phenotype. Quantitative real-time reverse transcription-polymerase chain reaction analyses showed that the expression of stress-responsive genes and ABA biosynthesis, catabolism, transport, and signaling genes was impaired in max2 compared with wild-type seedlings in response to drought stress. Double mutant analysis of max2 with the ABA-insensitive mutants abi3 and abi5 indicated that MAX2 may function upstream of these genes. The expression of ABA-regulated genes was enhanced in imbibed max2 seeds. In addition, max2 mutant seedlings were hypersensitive to ABA and osmotic stress, including NaCl, mannitol, and glucose. Interestingly, ABA, osmotic stress, and drought-sensitive phenotypes were restricted to max2, and the strigolactone biosynthetic pathway mutants max1, max3, and max4 did not display any defects in these responses. Taken together, these results uncover an important role for MAX2 in plant responses to abiotic stress conditions. PMID:24198318

Loss of protein phosphatase 6 in mouse keratinocytes enhances K-rasG12D -driven tumor promotion.

PubMed

Kurosawa, Koreyuki; Inoue, Yui; Kakugawa, Yoichiro; Yamashita, Yoji; Kanazawa, Kosuke; Kishimoto, Kazuhiro; Nomura, Miyuki; Momoi, Yuki; Sato, Ikuro; Chiba, Natsuko; Suzuki, Mai; Ogoh, Honami; Yamada, Hidekazu; Miura, Koh; Watanabe, Toshio; Tanuma, Nobuhiro; Tachi, Masahiro; Shima, Hiroshi

2018-05-14

Here, we address the function of protein phosphatase 6 (PP6) loss on K-ras-initiated tumorigenesis in keratinocytes. To do so, we developed tamoxifen-inducible double mutant (K-ras G12D -expressing and Ppp6c-deficient) mice in which K-ras G12D expression is driven by the cytokeratin 14 (K14) promoter. Doubly-mutant mice showed early onset tumor formation in lip, nipples, external genitalia, anus and palms, and had to be sacrificed by three weeks after induction by tamoxifen, while comparably-treated K-ras G12D -expressing mice did not. HE-staining of lip tumors before euthanasia revealed that all were papillomas, some containing focal squamous cell carcinoma. Immunohistochemical analysis of lip of doubly-mutant versus K-ras G12D mice revealed that cell proliferation and cell size increased approximately two-fold relative to K-ras G12D -expressing mutants, and epidermal thickness of lip tissue greatly increased relative to that seen in K-ras G12D only mice. Moreover, AKT phosphorylation increased in K-ras G12D -expressing/Ppp6c-deficient cells, as did phosphorylation of the downstream effectors 4EBP1, S6, and GSK3, suggesting that protein synthesis and survival signals are enhanced in lip tissues of doubly-mutant mice. Finally, increased numbers of K14-positive cells were present in the suprabasal layer of doubly-mutant mice, indicating abnormal keratinocyte differentiation, and γH2AX-positive cells accumulated, indicating perturbed DNA repair. Taken together, Ppp6c deficiency enhances K-ras G12D -dependent tumor promotion. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Theoretical Analysis of Allosteric and Operator Binding for Cyclic-AMP Receptor Protein Mutants

NASA Astrophysics Data System (ADS)

Einav, Tal; Duque, Julia; Phillips, Rob

2018-02-01

Allosteric transcription factors undergo binding events both at their inducer binding sites as well as at distinct DNA binding domains, and it is often difficult to disentangle the structural and functional consequences of these two classes of interactions. In this work, we compare the ability of two statistical mechanical models - the Monod-Wyman-Changeux (MWC) and the Koshland-N\\'emethy-Filmer (KNF) models of protein conformational change - to characterize the multi-step activation mechanism of the broadly acting cyclic-AMP receptor protein (CRP). We first consider the allosteric transition resulting from cyclic-AMP binding to CRP, then analyze how CRP binds to its operator, and finally investigate the ability of CRP to activate gene expression. In light of these models, we examine data from a beautiful recent experiment that created a single-chain version of the CRP homodimer, thereby enabling each subunit to be mutated separately. Using this construct, six mutants were created using all possible combinations of the wild type subunit, a D53H mutant subunit, and an S62F mutant subunit. We demonstrate that both the MWC and KNF models can explain the behavior of all six mutants using a small, self-consistent set of parameters. In comparing the results, we find that the MWC model slightly outperforms the KNF model in the quality of its fits, but more importantly the parameters inferred by the MWC model are more in line with structural knowledge of CRP. In addition, we discuss how the conceptual framework developed here for CRP enables us to not merely analyze data retrospectively, but has the predictive power to determine how combinations of mutations will interact, how double mutants will behave, and how each construct would regulate gene expression.

Rational design of Bacillus stearothermophilus US100 L-arabinose isomerase: potential applications for D-tagatose production.

PubMed

Rhimi, Moez; Aghajari, Nushin; Juy, Michel; Chouayekh, Hichem; Maguin, Emmanuelle; Haser, Richard; Bejar, Samir

2009-05-01

L-arabinose isomerases catalyze the bioconversion of D-galactose into D-tagatose. With the aim of producing an enzyme optimized for D-tagatose production, three Bacillus stearothermophilus US100 L-arabinose isomerase mutants were constructed, purified and characterized. Our results indicate that mutant Q268K was significantly more acidotolerant and more stable at acidic pH than the wild-type enzyme. The N175H mutant has a broad optimal temperature range from 50 to 65 degrees C. With the aim of constructing an acidotolerant mutant working at relatively low temperatures we generated the Q268K/N175H construct. This double mutant displays an optimal pH in the range 6.0-7.0 and an optimal activity around 50-65 degrees C, temperatures at which the enzyme was stable without addition of metal ions.

Genetic separation of phototropism and blue light inhibition of stem elongation

NASA Technical Reports Server (NTRS)

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

1992-01-01

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

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

PubMed

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

2005-02-28

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

Drosophila Kette coordinates myoblast junction dissolution and the ratio of Scar-to-WASp during myoblast fusion

PubMed Central

Hamp, Julia; Löwer, Andreas; Dottermusch-Heidel, Christine; Beck, Lothar; Moussian, Bernard; Flötenmeyer, Matthias

2016-01-01

ABSTRACT The fusion of founder cells and fusion-competent myoblasts (FCMs) is crucial for muscle formation in Drosophila. Characteristic events of myoblast fusion include the recognition and adhesion of myoblasts, and the formation of branched F-actin by the Arp2/3 complex at the site of cell–cell contact. At the ultrastructural level, these events are reflected by the appearance of finger-like protrusions and electron-dense plaques that appear prior to fusion. Severe defects in myoblast fusion are caused by the loss of Kette (a homolog of Nap1 and Hem-2, also known as NCKAP1 and NCKAP1L, respectively), a member of the regulatory complex formed by Scar or WAVE proteins (represented by the single protein, Scar, in flies). kette mutants form finger-like protrusions, but the electron-dense plaques are extended. Here, we show that the electron-dense plaques in wild-type and kette mutant myoblasts resemble other electron-dense structures that are known to function as cellular junctions. Furthermore, analysis of double mutants and attempts to rescue the kette mutant phenotype with N-cadherin, wasp and genes of members of the regulatory Scar complex revealed that Kette has two functions during myoblast fusion. First, Kette controls the dissolution of electron-dense plaques. Second, Kette controls the ratio of the Arp2/3 activators Scar and WASp in FCMs. PMID:27521427

C2cd3 is required for cilia formation and Hedgehog signaling in mouse

PubMed Central

Hoover, Amber N.; Wynkoop, Aaron; Zeng, Huiqing; Jia, Jinping; Niswander, Lee A.; Liu, Aimin

2011-01-01

Cilia are essential for mammalian embryonic development as well as for the physiological activity of various adult organ systems. Despite the multiple crucial roles that cilia play, the mechanisms underlying ciliogenesis in mammals remain poorly understood. Taking a forward genetic approach, we have identified Hearty (Hty), a recessive lethal mouse mutant with multiple defects, including neural tube defects, abnormal dorsal-ventral patterning of the spinal cord, a defect in left-right axis determination and severe polydactyly (extra digits). By genetic mapping, sequence analysis of candidate genes and characterization of a second mutant allele, we identify Hty as C2cd3, a novel gene encoding a vertebrate-specific C2 domain-containing protein. Target gene expression and double-mutant analyses suggest that C2cd3 is an essential regulator of intracellular transduction of the Hedgehog signal. Furthering a link between Hedgehog signaling and cilia function, we find that cilia formation and proteolytic processing of Gli3 are disrupted in C2cd3 mutants. Finally, we observe C2cd3 protein at the basal body, consistent with its essential function in ciliogenesis. Interestingly, the human ortholog for this gene lies in proximity to the critical regions of Meckel-Gruber syndrome 2 (MKS2) and Joubert syndrome 2 (JBTS2), making it a potential candidate for these two human genetic disorders. PMID:19004860

The nexin link and B-tubule glutamylation maintain the alignment of outer doublets in the ciliary axoneme.

PubMed

Alford, Lea M; Stoddard, Daniel; Li, Jennifer H; Hunter, Emily L; Tritschler, Douglas; Bower, Raqual; Nicastro, Daniela; Porter, Mary E; Sale, Winfield S

2016-06-01

We developed quantitative assays to test the hypothesis that the N-DRC is required for integrity of the ciliary axoneme. We examined reactivated motility of demembranated drc cells, commonly termed "reactivated cell models." ATP-induced reactivation of wild-type cells resulted in the forward swimming of ∼90% of cell models. ATP-induced reactivation failed in a subset of drc cell models, despite forward motility in live drc cells. Dark-field light microscopic observations of drc cell models revealed various degrees of axonemal splaying. In contrast, >98% of axonemes from wild-type reactivated cell models remained intact. The sup-pf4 and drc3 mutants, unlike other drc mutants, retain most of the N-DRC linker that interconnects outer doublet microtubules. Reactivated sup-pf4 and drc3 cell models displayed nearly wild-type levels of forward motility. Thus, the N-DRC linker is required for axonemal integrity. We also examined reactivated motility and axoneme integrity in mutants defective in tubulin polyglutamylation. ATP-induced reactivation resulted in forward swimming of >75% of tpg cell models. Analysis of double mutants defective in tubulin polyglutamylation and different regions of the N-DRC indicate B-tubule polyglutamylation and the distal lobe of the linker region are both important for axonemal integrity and normal N-DRC function. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

SLX-1 Is Required for Maintaining Genomic Integrity and Promoting Meiotic Noncrossovers in the Caenorhabditis elegans Germline

PubMed Central

Meyer, Katherine; Harper, J. Wade; Colaiácovo, Monica P.

2012-01-01

Although the SLX4 complex, which includes structure-specific nucleases such as XPF, MUS81, and SLX1, plays important roles in the repair of several kinds of DNA damage, the function of SLX1 in the germline remains unknown. Here we characterized the endonuclease activities of the Caenorhabditis elegans SLX-1-HIM-18/SLX-4 complex co-purified from human 293T cells and determined SLX-1 germline function via analysis of slx-1(tm2644) mutants. SLX-1 shows a HIM-18/SLX-4–dependent endonuclease activity toward replication forks, 5′-flaps, and Holliday junctions. slx-1 mutants exhibit hypersensitivity to UV, nitrogen mustard, and camptothecin, but not gamma irradiation. Consistent with a role in DNA repair, recombination intermediates accumulate in both mitotic and meiotic germ cells in slx-1 mutants. Importantly, meiotic crossover distribution, but not crossover frequency, is altered on chromosomes in slx-1 mutants compared to wild type. This alteration is not due to changes in either the levels or distribution of double-strand breaks (DSBs) along chromosomes. We propose that SLX-1 is required for repair at stalled or collapsed replication forks, interstrand crosslink repair, and nucleotide excision repair during mitosis. Moreover, we hypothesize that SLX-1 regulates the crossover landscape during meiosis by acting as a noncrossover-promoting factor in a subset of DSBs. PMID:22927825

The ribokinases of Arabidopsis thaliana and Saccharomyces cerevisiae are required for ribose recycling from nucleotide catabolism, which in plants is not essential to survive prolonged dark stress.

PubMed

Schroeder, Rebekka Y; Zhu, Anting; Eubel, Holger; Dahncke, Kathleen; Witte, Claus-Peter

2018-01-01

Nucleotide catabolism in Arabidopsis thaliana and Saccharomyces cerevisiae leads to the release of ribose, which requires phosphorylation to ribose-5-phosphate mediated by ribokinase (RBSK). We aimed to characterize RBSK in plants and yeast, to quantify the contribution of plant nucleotide catabolism to the ribose pool, and to investigate whether ribose carbon contributes to dark stress survival of plants. We performed a phylogenetic analysis and determined the kinetic constants of plant-expressed Arabidopsis and yeast RBSKs. Using mass spectrometry, several metabolites were quantified in AtRBSK mutants and double mutants with genes of nucleoside catabolism. Additionally, the dark stress performance of several nucleotide metabolism mutants and rbsk was compared. The plant PfkB family of sugar kinases forms nine major clades likely representing distinct biochemical functions, one of them RBSK. Nucleotide catabolism is the dominant ribose source in plant metabolism and is highly induced by dark stress. However, rbsk cannot be discerned from the wild type in dark stress. Interestingly, the accumulation of guanosine in a guanosine deaminase mutant strongly enhances dark stress symptoms. Although nucleotide catabolism contributes to carbon mobilization upon darkness and is the dominant source of ribose, the contribution appears to be of minor importance for dark stress survival. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

Effect of impaired twitching motility and biofilm dispersion on performance of Pseudomonas aeruginosa-powered microbial fuel cells.

PubMed

Shreeram, Devesh D; Panmanee, Warunya; McDaniel, Cameron T; Daniel, Susan; Schaefer, Dale W; Hassett, Daniel J

2018-02-01

Pseudomonas aeruginosa is a metabolically voracious bacterium that is easily manipulated genetically. We have previously shown that the organism is also highly electrogenic in microbial fuel cells (MFCs). Polarization studies were performed in MFCs with wild-type strain PAO1 and three mutant strains (pilT, bdlA and pilT bdlA). The pilT mutant was hyperpiliated, while the bdlA mutant was suppressed in biofilm dispersion chemotaxis. The double pilT bdlA mutant was expected to have properties of both mutations. Polarization data indicate that the pilT mutant showed 5.0- and 3.2-fold increases in peak power compared to the wild type and the pilT bdlA mutant, respectively. The performance of the bdlA mutant was surprisingly the lowest, while the pilT bdlA electrogenic performance fell between the pilT mutant and wild-type bacteria. Measurements of biofilm thickness and bacterial viability showed equal viability among the different strains. The thickness of the bdlA mutant, however, was twice that of wild-type strain PAO1. This observation implicates the presence of dead or dormant bacteria in the bdlA mutant MFCs, which increases biofilm internal resistance as confirmed by electrochemical measurements.

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

Chen, Qi; Cheng, Xiaolin; Univ. of Tennessee, Knoxville, TN

Although Elvitegravir (EVG) is a newly developed antiretrovirals drug to treat the acquired immunodeficiency syndrome (AIDS), drug resistance has already been found in clinic, such as E92Q/N155H and Q148H/G140S. Several structural investigations have already been reported to reveal the molecular mechanism of the drug resistance. As full length crystal structure for HIV-1 integrase is still unsolved, we use in this paper the crystal structure of the full length prototype foamy virus (PFV) in complex with virus DNA and inhibitor Elvitegravir as a template to construct the wild type and E92Q/N155H mutant system of HIV-1 integrase. Molecular dynamic simulations was usedmore » to revel the binding mode and the drug resistance of the EVG ligand in E92Q/N155H. Several important interactions were discovered between the mutated residues and the residues in the active site of the E92Q/N155H double mutant pattern, and cross correlation and clustering methods were used for detailed analysis. The results from the MD simulation studies will be used to guide the experimental efforts of developing novel inhibitors against drug-resistant HIV integrase mutants.« less

GATA-3 is required for early T lineage progenitor development

PubMed Central

Hosoya, Tomonori; Kuroha, Takashi; Moriguchi, Takashi; Cummings, Dustin; Maillard, Ivan; Lim, Kim-Chew

2009-01-01

Most T lymphocytes appear to arise from very rare early T lineage progenitors (ETPs) in the thymus, but the transcriptional programs that specify ETP generation are not completely known. The transcription factor GATA-3 is required for the development of T lymphocytes at multiple late differentiation steps as well as for the development of thymic natural killer cells. However, a role for GATA-3 before the double-negative (DN) 3 stage of T cell development has to date been obscured both by the developmental heterogeneity of DN1 thymocytes and the paucity of ETPs. We provide multiple lines of in vivo evidence through the analysis of T cell development in Gata3 hypomorphic mutant embryos, in irradiated mice reconstituted with Gata3 mutant hematopoietic cells, and in mice conditionally ablated for the Gata3 gene to show that GATA-3 is required for ETP generation. We further show that Gata3 loss does not affect hematopoietic stem cells or multipotent hematopoietic progenitors. Finally, we demonstrate that Gata3 mutant lymphoid progenitors exhibit neither increased apoptosis nor diminished cell-cycle progression. Thus, GATA-3 is required for the cell-autonomous development of the earliest characterized thymic T cell progenitors. PMID:19934022

Choline catabolism to glycine betaine contributes to Pseudomonas aeruginosa survival during murine lung infection.

PubMed

Wargo, Matthew J

2013-01-01

Pseudomonas aeruginosa can acquire and metabolize a variety of molecules including choline, an abundant host-derived molecule. In P. aeruginosa, choline is oxidized to glycine betaine which can be used as an osmoprotectant, a sole source of carbon and nitrogen, and as an inducer of the virulence factor, hemolytic phospholipase C (PlcH) via the transcriptional regulator GbdR. The primary objective was to determine the contribution of choline conversion to glycine betaine to P. aeruginosa survival during mouse lung infection. A secondary objective was to gain insight into the relative contributions of the different roles of glycine betaine to P. aeruginosa survival during infection. Using a model of acute murine pneumonia, we determined that deletion of the choline oxidase system (encoded by betBA) decreased P. aeruginosa survival in the mouse lung. Deletion of the glycine betaine demethylase genes (gbcA-B), required for glycine betaine catabolism, did not impact P. aeruginosa survival in the lung. Thus, the defect of the betBA mutant was not due to a requirement for glycine betaine catabolism or dependence on a downstream metabolite. Deletion of betBA decreased the abundance of plcH transcript during infection, which suggested a role for PlcH in the betBA survival defect. To test the contribution of plcH to the betBA mutant phenotype a betBAplcHR double deletion mutant was generated. The betBA and betBAplcHR double mutant had a small but significant survival defect compared to the plcHR single mutant, suggesting that regulation of plcH expression is not the only role for glycine betaine during infection. The conclusion was that choline acquisition and its oxidation to glycine betaine contribute to P. aeruginosa survival in the mouse lung. While defective plcH induction can explain a portion of the betBA mutant phenotype, the exact mechanisms driving the betBA mutant survival defect remain unknown.

Choline Catabolism to Glycine Betaine Contributes to Pseudomonas aeruginosa Survival during Murine Lung Infection

PubMed Central

Wargo, Matthew J.

2013-01-01

Pseudomonas aeruginosa can acquire and metabolize a variety of molecules including choline, an abundant host-derived molecule. In P. aeruginosa, choline is oxidized to glycine betaine which can be used as an osmoprotectant, a sole source of carbon and nitrogen, and as an inducer of the virulence factor, hemolytic phospholipase C (PlcH) via the transcriptional regulator GbdR. The primary objective was to determine the contribution of choline conversion to glycine betaine to P. aeruginosa survival during mouse lung infection. A secondary objective was to gain insight into the relative contributions of the different roles of glycine betaine to P. aeruginosa survival during infection. Using a model of acute murine pneumonia, we determined that deletion of the choline oxidase system (encoded by betBA) decreased P. aeruginosa survival in the mouse lung. Deletion of the glycine betaine demethylase genes (gbcA-B), required for glycine betaine catabolism, did not impact P. aeruginosa survival in the lung. Thus, the defect of the betBA mutant was not due to a requirement for glycine betaine catabolism or dependence on a downstream metabolite. Deletion of betBA decreased the abundance of plcH transcript during infection, which suggested a role for PlcH in the betBA survival defect. To test the contribution of plcH to the betBA mutant phenotype a betBAplcHR double deletion mutant was generated. The betBA and betBAplcHR double mutant had a small but significant survival defect compared to the plcHR single mutant, suggesting that regulation of plcH expression is not the only role for glycine betaine during infection. The conclusion was that choline acquisition and its oxidation to glycine betaine contribute to P. aeruginosa survival in the mouse lung. While defective plcH induction can explain a portion of the betBA mutant phenotype, the exact mechanisms driving the betBA mutant survival defect remain unknown. PMID:23457628

Deletion of Braun lipoprotein and plasminogen-activating protease-encoding genes attenuates Yersinia pestis in mouse models of bubonic and pneumonic plague.

PubMed

van Lier, Christina J; Sha, Jian; Kirtley, Michelle L; Cao, Anthony; Tiner, Bethany L; Erova, Tatiana E; Cong, Yingzi; Kozlova, Elena V; Popov, Vsevolod L; Baze, Wallace B; Chopra, Ashok K

2014-06-01

Currently, there is no FDA-approved vaccine against Yersinia pestis, the causative agent of bubonic and pneumonic plague. Since both humoral immunity and cell-mediated immunity are essential in providing the host with protection against plague, we developed a live-attenuated vaccine strain by deleting the Braun lipoprotein (lpp) and plasminogen-activating protease (pla) genes from Y. pestis CO92. The Δlpp Δpla double isogenic mutant was highly attenuated in evoking both bubonic and pneumonic plague in a mouse model. Further, animals immunized with the mutant by either the intranasal or the subcutaneous route were significantly protected from developing subsequent pneumonic plague. In mice, the mutant poorly disseminated to peripheral organs and the production of proinflammatory cytokines concurrently decreased. Histopathologically, reduced damage to the lungs and livers of mice infected with the Δlpp Δpla double mutant compared to the level of damage in wild-type (WT) CO92-challenged animals was observed. The Δlpp Δpla mutant-immunized mice elicited a humoral immune response to the WT bacterium, as well as to CO92-specific antigens. Moreover, T cells from mutant-immunized animals exhibited significantly higher proliferative responses, when stimulated ex vivo with heat-killed WT CO92 antigens, than mice immunized with the same sublethal dose of WT CO92. Likewise, T cells from the mutant-immunized mice produced more gamma interferon (IFN-γ) and interleukin-4. These animals had an increasing number of tumor necrosis factor alpha (TNF-α)-producing CD4(+) and CD8(+) T cells than WT CO92-infected mice. These data emphasize the role of TNF-α and IFN-γ in protecting mice against pneumonic plague. Overall, our studies provide evidence that deletion of the lpp and pla genes acts synergistically in protecting animals against pneumonic plague, and we have demonstrated an immunological basis for this protection.

Deletion of Braun Lipoprotein and Plasminogen-Activating Protease-Encoding Genes Attenuates Yersinia pestis in Mouse Models of Bubonic and Pneumonic Plague

PubMed Central

van Lier, Christina J.; Sha, Jian; Kirtley, Michelle L.; Cao, Anthony; Tiner, Bethany L.; Erova, Tatiana E.; Cong, Yingzi; Kozlova, Elena V.; Popov, Vsevolod L.; Baze, Wallace B.

2014-01-01

Currently, there is no FDA-approved vaccine against Yersinia pestis, the causative agent of bubonic and pneumonic plague. Since both humoral immunity and cell-mediated immunity are essential in providing the host with protection against plague, we developed a live-attenuated vaccine strain by deleting the Braun lipoprotein (lpp) and plasminogen-activating protease (pla) genes from Y. pestis CO92. The Δlpp Δpla double isogenic mutant was highly attenuated in evoking both bubonic and pneumonic plague in a mouse model. Further, animals immunized with the mutant by either the intranasal or the subcutaneous route were significantly protected from developing subsequent pneumonic plague. In mice, the mutant poorly disseminated to peripheral organs and the production of proinflammatory cytokines concurrently decreased. Histopathologically, reduced damage to the lungs and livers of mice infected with the Δlpp Δpla double mutant compared to the level of damage in wild-type (WT) CO92-challenged animals was observed. The Δlpp Δpla mutant-immunized mice elicited a humoral immune response to the WT bacterium, as well as to CO92-specific antigens. Moreover, T cells from mutant-immunized animals exhibited significantly higher proliferative responses, when stimulated ex vivo with heat-killed WT CO92 antigens, than mice immunized with the same sublethal dose of WT CO92. Likewise, T cells from the mutant-immunized mice produced more gamma interferon (IFN-γ) and interleukin-4. These animals had an increasing number of tumor necrosis factor alpha (TNF-α)-producing CD4+ and CD8+ T cells than WT CO92-infected mice. These data emphasize the role of TNF-α and IFN-γ in protecting mice against pneumonic plague. Overall, our studies provide evidence that deletion of the lpp and pla genes acts synergistically in protecting animals against pneumonic plague, and we have demonstrated an immunological basis for this protection. PMID:24686064

Site-directed mutagenesis of mouse glutathione transferase P1-1 unlocks masked cooperativity, introduces a novel mechanism for 'ping pong' kinetic behaviour, and provides further structural evidence for participation of a water molecule in proton abstraction from glutathione.

PubMed

McManus, Gavin; Costa, Marta; Canals, Albert; Coll, Miquel; Mantle, Timothy J

2011-01-01

Mouse liver glutathione transferase P1-1 has three cysteine residues at positions 14, 47 and 169. We have constructed the single, double and triple cysteine to alanine mutants to define the behaviour of all three thiols. We confirm that C47 is the 'fast' thiol (pK 7.4), and define C169 as the alkaline reactive residue with a pK(a) of 8.6. Only a small proportion of C14 is reactive with 5,5'-dithiobis-(2-nitrobenoic acid) (DTNB) at pH 9 in the C47A/C169A double mutant. The native enzyme and the C169A mutant exhibited Michaelis-Menten kinetics, but all other thiol to alanine mutants exhibited sigmoidal kinetics to varying degrees. The C169A mutant exhibited 'ping pong' kinetics, consistent with a mechanism whereby liberation of a proton from a reduced enzyme-glutathione (GSH) complex to form an enzyme-GS(-) (unprotonated) complex is essentially irreversible. Intriguingly, similar behaviour has recently been reported for a mutant of the yeast prion Ure2p. This cooperative behaviour is 'mirrored' in the crystal structure of the C47A mutant, which binds the p-nitrobenzyl moiety of p-nitrobenzyglutathione in distinct orientations in the two crystallographic subunits. The asymmetry seen in this structure for product binding is associated with absence of a water molecule W0 in the standard wild-type conformation of product binding that is clearly identifiable in the new structure, which may represent a structural model for binding of incoming GSH prior to displacement of W0. Elimination of W0 as a hydroxonium ion may be the mechanism for the initial proton extrusion from the active site. © 2010 The Authors Journal compilation © 2010 FEBS.

Structural and functional characterization of the product of disease-related factor H gene conversion.

PubMed

Herbert, Andrew P; Kavanagh, David; Johansson, Conny; Morgan, Hugh P; Blaum, Bärbel S; Hannan, Jonathan P; Barlow, Paul N; Uhrín, Dušan

2012-03-06

Numerous complement factor H (FH) mutations predispose patients to atypical hemolytic uremic syndrome (aHUS) and other disorders arising from inadequately regulated complement activation. No unifying structural or mechanistic consequences have been ascribed to these mutants beyond impaired self-cell protection. The S1191L and V1197A mutations toward the C-terminus of FH, which occur in patients singly or together, arose from gene conversion between CFH encoding FH and CFHR1 encoding FH-related 1. We show that neither single nor double mutations structurally perturbed recombinant proteins consisting of the FH C-terminal modules, 19 and 20 (FH19-20), although all three FH19-20 mutants were poor, compared to wild-type FH19-20, at promoting hemolysis of C3b-coated erythrocytes through competition with full-length FH. Indeed, our new crystal structure of the S1191L mutant of FH19-20 complexed with an activation-specific complement fragment, C3d, was nearly identical to that of the wild-type FH19-20:C3d complex, consistent with mutants binding to C3b with wild-type-like affinity. The S1191L mutation enhanced thermal stability of module 20, whereas the V1197A mutation dramatically decreased it. Thus, although mutant proteins were folded at 37 °C, they differ in conformational rigidity. Neither single substitutions nor double substitutions increased measurably the extent of FH19-20 self-association, nor did these mutations significantly affect the affinity of FH19-20 for three glycosaminoglycans, despite critical roles of module 20 in recognizing polyanionic self-surface markers. Unexpectedly, FH19-20 mutants containing Leu1191 self-associated on a heparin-coated surface to a higher degree than on surfaces coated with dermatan or chondroitin sulfates. Thus, potentially disease-related functional distinctions between mutants, and between FH and FH-related 1, may manifest in the presence of specific glycosaminoglycans.

Effects of light and the regulatory B-subunit composition of protein phosphatase 2A on the susceptibility of Arabidopsis thaliana to aphid (Myzus persicae) infestation

PubMed Central

Rasool, Brwa; Karpinska, Barbara; Konert, Grzegorz; Durian, Guido; Denessiouk, Konstantin; Kangasjärvi, Saijaliisa; Foyer, Christine H.

2014-01-01

The interactions between biotic and abiotic stress signaling pathways are complex and poorly understood but protein kinase/phosphatase cascades are potentially important components. Aphid fecundity and susceptibility to Pseudomonas syringae infection were determined in the low light-grown Arabidopsis thaliana wild type and in mutant lines defective in either the protein phosphatase (PP)2A regulatory subunit B'γ (gamma; pp2a-b'γ) or B'ζ (zeta; pp2a-b'ζ1-1 and pp2a-b'ζ 1-2) and in gamma zeta double mutants (pp2a-b'γζ) lacking both subunits. All the mutants except for pp2a-b'ζ 1-1 had significantly lower leaf areas than the wild type. Susceptibility to P. syringae was similar in all genotypes. In contrast, aphid fecundity was significantly decreased in the pp2a-b'γ mutant relative to the wild type but not in the pp2a-b'γζ double mutant. A high light pre-treatment, which led to a significant increase in rosette growth in all mutant lines but not in the wild type, led to a significant decrease in aphid fecundity in all genotypes. The high light pre-treatment abolished the differences in aphid resistance observed in the pp2a-b'γ mutant relative to the wild type. The light and CO2 response curves for photosynthesis were changed in response to the high light pre-treatment, but the high light effects were similar in all genotypes. These data demonstrate that a pre-exposure to high light and the composition of B-subunits on the trimeric PP2A holoenzymes are important in regulating plant resistance to aphids. The functional specificity for the individual regulatory B-subunits may therefore limit aphid colonization, depending on the prevailing abiotic stress environment. PMID:25191331

Genetic diversity of Plasmodium vivax metacaspase 1 and Plasmodium vivax multi-drug resistance 1 genes of field isolates from Mauritania, Sudan and Oman.

PubMed

Sow, Fatimata; Bonnot, Guillaume; Ahmed, Bilal Rabah; Diagana, Sidi Mohamed; Kebe, Hachim; Koita, Mohamedou; Samba, Ba Malado; Al-Mukhaini, Said K; Al-Zadjali, Majed; Al-Abri, Seif S; Ali, Osama A M; Samy, Abdallah M; Hamid, Muzamil Mahdi Abdel; Ali Albsheer, Musab M; Simon, Bruno; Bienvenu, Anne-Lise; Petersen, Eskild; Picot, Stéphane

2017-02-02

Plasmodium vivax is the second most important human malaria parasite, widely spread across the world. This parasite is associated with important issues in the process toward malaria elimination, including potential for relapse and increased resistance to chloroquine. Plasmodium vivax multi-drug resistant (pvmdr1) is suspected to be a marker of resistance although definitive evidence is lacking. Progress has been made in knowledge of biological factors affecting parasite growth, including mechanisms of regulated cell death and the suspected role of metacaspase. Plasmodium vivax metacaspase1 (PvMCA1-cd) has been described with a catalytic domain composed of histidine (H372) and cysteine (C428) residues. The aim of this study was to test for a link between the conserved histidine and cysteine residues in PvMCA1-cd, and the polymorphism of the P. vivax multi-drug resistant gene (pvmdr1). Thirty P. vivax isolates were collected from Mauritania, Sudan, and Oman. Among the 28 P. vivax isolates successfully sequenced, only 4 samples showed the conserved His (372)-Cys (428) residues in PvMCA1-cd. Single nucleotide polymorphisms observed were H372T (46.4%), H372D (39.3%), and C428R (85.7%). A new polymorphic catalytic domain was observed at His (282)-Cys (305) residues. Sequences alignment analysis of pvmdr1 showed SNP in the three codons 958, 976 and 1076. A single SNP was identified at the codon M958Y (60%), 2 SNPs were found at the position 976: Y976F (13%) and Y976V (57%), and 3 SNPs were identified at the position 1076: F1076L (40%), F1076T (53%) and F1076I (3%). Only one isolate was wildtype in all three codons (MYF), 27% were single MYL mutants, and 10% were double MFL mutants. Three new haplotypes were also identified: the triple mutant YVT was most prevalent (53.3%) distributed in the three countries, while triple YFL and YVI mutants (3%), were only found in samples from Sudan and Mauritania. Triple or quadruple mutants for metacaspase genes and double or triple mutants for Pvmdr1 were observed in 24/28 and 19/28 samples. There was no difference in the frequency of mutations between PvMCA1-cd and Pvmdr1 (P > 0.2). Histidine and cysteine residues in PvMCA1-cd are highly polymorphic and linkage disequilibrium with SNPs of Pvmdr1 gene may be expected from these three areas with different patterns of P. vivax transmission.

Sickle Cell Hemoglobin with Mutation at αHis-50 Has Improved Solubility.

PubMed

Tam, Ming F; Tam, Tsuey Chyi S; Simplaceanu, Virgil; Ho, Nancy T; Zou, Ming; Ho, Chien

2015-08-28

The unliganded tetrameric Hb S has axial and lateral contacts with neighbors and can polymerize in solution. Novel recombinants of Hb S with single amino acid substitutions at the putative axial (recombinant Hb (rHb) (βE6V/αH20R) and rHb (βE6V/αH20Q)) or lateral (rHb (βE6V/αH50Q)) or double amino acid substitutions at both the putative axial and lateral (rHb (βE6V/αH20R/αH50Q) and rHb (βE6V/αH20Q/αH50Q)) contact sites were expressed in Escherichia coli and purified for structural and functional studies. The (1)H NMR spectra of the CO and deoxy forms of these mutants indicate that substitutions at either αHis-20 or αHis-50 do not change the subunit interfaces or the heme pockets of the proteins. The double mutants show only slight structural alteration in the β-heme pockets. All mutants have similar cooperativity (n50), alkaline Bohr effect, and autoxidation rate as Hb S. The oxygen binding affinity (P50) of the single mutants is comparable with that of Hb S. The double mutants bind oxygen with slightly higher affinity than Hb S under the acidic conditions. In high salt, rHb (βE6V/αH20R) is the only mutant that has a shorter delay time of polymerization and forms polymers more readily than Hb S with a dextran-Csat value of 1.86 ± 0.20 g/dl. Hb S, rHb (βE6V/αH20Q), rHb (βE6V/αH50Q), rHb (βE6V/αH20R/αH50Q), and rHb (βE6V/αH20Q/αH50Q) have dextran-Csat values of 2.95 ± 0.10, 3.04 ± 0.17, 11.78 ± 0.59, 7.11 ± 0.66, and 10.89 ± 0.83 g/dl, respectively. rHb (βE6V/αH20Q/αH50Q) is even more stable than Hb S under elevated temperature (60 °C). © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Molecular determinants for the high constitutive activity of the human histamine H4 receptor: functional studies on orthologues and mutants

PubMed Central

Wifling, D; Löffel, K; Nordemann, U; Strasser, A; Bernhardt, G; Dove, S; Seifert, R; Buschauer, A

2015-01-01

Background and Purpose Some histamine H4 receptor ligands act as inverse agonists at the human H4 receptor (hH4R), a receptor with exceptionally high constitutive activity, but as neutral antagonists or partial agonists at the constitutively inactive mouse H4 receptor (mH4R) and rat H4 receptor (rH4R). To study molecular determinants of constitutive activity, H4 receptor reciprocal mutants were constructed: single mutants: hH4R-F169V, mH4R-V171F, hH4R-S179A, hH4R-S179M; double mutants: hH4R-F169V+S179A, hH4R-F169V+S179M and mH4R-V171F+M181S. Experimental Approach Site-directed mutagenesis with pVL1392 plasmids containing hH4 or mH4 receptors were performed. Wild-type or mutant receptors were co-expressed with Gαi2 and Gβ1γ2 in Sf9 cells. Membranes were studied in saturation and competition binding assays ([3H]-histamine), and in functional [35S]-GTPγS assays with inverse, partial and full agonists of the hH4 receptor. Key Results Constitutive activity decreased from the hH4 receptor via the hH4R-F169V mutant to the hH4R-F169V+S179A and hH4R-F169V+S179M double mutants. F169 alone or in concert with S179 plays a major role in stabilizing a ligand-free active state of the hH4 receptor. Partial inverse hH4 receptor agonists like JNJ7777120 behaved as neutral antagonists or partial agonists at species orthologues with lower or no constitutive activity. Some partial and full hH4 receptor agonists showed decreased maximal effects and potencies at hH4R-F169V and double mutants. However, the mutation of S179 in the hH4 receptor to M as in mH4 receptor or A as in rH4 receptor did not significantly reduce constitutive activity. Conclusions and Implications F169 and S179 are key amino acids for the high constitutive activity of hH4 receptors and may also be of relevance for other constitutively active GPCRs. Linked Articles This article is part of a themed issue on Histamine Pharmacology Update published in volume 170 issue 1. To view the other articles in this issue visit http://onlinelibrary.wiley.com/doi/10.1111/bph.2013.170.issue-1/issuetoc PMID:24903527

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.

Contribution of the tricarboxylic acid (TCA) cycle and the glyoxylate shunt in Saccharomyces cerevisiae to succinic acid production during dough fermentation.

PubMed

Rezaei, Mohammad N; Aslankoohi, Elham; Verstrepen, Kevin J; Courtin, Christophe M

2015-07-02

Succinic acid produced by yeast during bread dough fermentation can significantly affect the rheological properties of the dough. By introducing mutations in the model S288C yeast strain, we show that the oxidative pathway of the TCA cycle and the glyoxylate shunt contribute significantly to succinic acid production during dough fermentation. More specifically, deletion of ACO1 and double deletion of ACO1 and ICL1 resulted in a 36 and 77% decrease in succinic acid levels in fermented dough, respectively. Similarly, double deletion of IDH1 and IDP1 decreased succinic acid production by 85%, while also affecting the fermentation rate. By contrast, double deletion of SDH1 and SDH2 resulted in a two-fold higher succinic acid accumulation compared to the wild-type. Deletion of fumarate reductase activity (FRD1 and OSM1) in the reductive pathway of the TCA cycle did not affect the fermentation rate and succinic acid production. The changes in the levels of succinic acid produced by mutants Δidh1Δidp1 (low level) and Δsdh1Δsdh2 (high level) in fermented dough only resulted in small pH differences, reflecting the buffering capacity of dough at a pH of around 5.1. Moreover, Rheofermentometer analysis using these mutants revealed no difference in maximum dough height and gas retention capacity with the dough prepared with S288C. The impact of the changed succinic acid profile on the organoleptic or antimicrobial properties of bread remains to be demonstrated. Copyright © 2015 Elsevier B.V. All rights reserved.

A second gene for type I signal peptidase in Bradyrhizobium japonicum, sipF, is located near genes involved in RNA processing and cell division.

PubMed

Bairl, A; Müller, P

1998-11-01

The TnphoA-induced Bradyrhizobium japonicum mutant 184 shows slow growth and aberrant colonization of soybean nodules. Using a DNA fragment adjacent to the transposon insertion site as a probe, a 3.4-kb BglII fragment of B. japonicum 110spc4 DNA was identified and cloned. Sequence analysis indicated that two truncated ORFs and three complete ORFs were encoded on this fragment. A database search revealed homologies to several other prokaryotic proteins: PdxJ (an enzyme involved in vitamin B6 biosynthesis), AcpS (acyl carrier protein synthase), Lep or Sip (prokaryotic type I signal peptidase), RNase III (an endoribonuclease which processes double-stranded rRNA precursors and mRNA) and Era (a GTP-binding protein required for cell division). The mutation in strain 184 was found to lie within the signal peptidase gene, which was designated sipF. Therefore, sipF is located in a region that encodes gene products involved in posttranscriptional and posttranslational processing processes. By complementation of the lep(ts) E. coli mutant strain IT41 it was demonstrated that sipF indeed encodes a functional signal peptidase, and genetic complementation of B. japonicum mutant 184 by a 2.8-kb SalI fragment indicated that sipF is expressed from a promoter located directly upstream of sipF. Using a non-polar kanamycin resistance cassette, a specific sipF mutant was constructed which exhibited defects in symbiosis similar to those of the original mutant 184.

Of the Nine Cytidine Deaminase-Like Genes in Arabidopsis, Eight Are Pseudogenes and Only One Is Required to Maintain Pyrimidine Homeostasis in Vivo1

PubMed Central

2016-01-01

CYTIDINE DEAMINASE (CDA) catalyzes the deamination of cytidine to uridine and ammonia in the catabolic route of C nucleotides. The Arabidopsis (Arabidopsis thaliana) CDA gene family comprises nine members, one of which (AtCDA) was shown previously in vitro to encode an active CDA. A possible role in C-to-U RNA editing or in antiviral defense has been discussed for other members. A comprehensive bioinformatic analysis of plant CDA sequences, combined with biochemical functionality tests, strongly suggests that all Arabidopsis CDA family members except AtCDA are pseudogenes and that most plants only require a single CDA gene. Soybean (Glycine max) possesses three CDA genes, but only two encode functional enzymes and just one has very high catalytic efficiency. AtCDA and soybean CDAs are located in the cytosol. The functionality of AtCDA in vivo was demonstrated with loss-of-function mutants accumulating high amounts of cytidine but also CMP, cytosine, and some uridine in seeds. Cytidine hydrolysis in cda mutants is likely caused by NUCLEOSIDE HYDROLASE1 (NSH1) because cytosine accumulation is strongly reduced in a cda nsh1 double mutant. Altered responses of the cda mutants to fluorocytidine and fluorouridine indicate that a dual specific nucleoside kinase is involved in cytidine as well as uridine salvage. CDA mutants display a reduction in rosette size and have fewer leaves compared with the wild type, which is probably not caused by defective pyrimidine catabolism but by the accumulation of pyrimidine catabolism intermediates reaching toxic concentrations. PMID:27208239

Functional Redundancy and Divergence within the Arabidopsis RETICULATA-RELATED Gene Family1[W][OA

PubMed Central

Pérez-Pérez, José Manuel; Esteve-Bruna, David; González-Bayón, Rebeca; Kangasjärvi, Saijaliisa; Caldana, Camila; Hannah, Matthew A.; Willmitzer, Lothar; Ponce, María Rosa; Micol, José Luis

2013-01-01

A number of Arabidopsis (Arabidopsis thaliana) mutants exhibit leaf reticulation, having green veins that stand out against paler interveinal tissues, fewer cells in the interveinal mesophyll, and normal perivascular bundle sheath cells. Here, to examine the basis of leaf reticulation, we analyzed the Arabidopsis RETICULATA-RELATED (RER) gene family, several members of which cause leaf reticulation when mutated. Although transcripts of RE, RER1, and RER3 were mainly detected in the bundle sheath cells of expanded leaves, functional RER3:GREEN FLUORESCENT PROTEIN was visualized in the chloroplast membranes of all photosynthetic cells. Leaf reticulation in the re and rer3 loss-of-function mutants occurred, along with accumulation of reactive oxygen species, in a photoperiod-dependent manner. A comparison of re and rer3 leaf messenger RNA expression profiles showed more than 200 genes were similarly misexpressed in both mutants. In addition, metabolic profiles of mature leaves revealed that several biosynthetic pathways downstream of pyruvate are altered in re and rer3. Double mutant analysis showed that only re rer1 and rer5 rer6 exhibited synergistic phenotypes, indicating functional redundancy. The redundancy between RE and its closest paralog, RER1, was confirmed by overexpressing RER1 in re mutants, which partially suppressed leaf reticulation. Our results show that RER family members can be divided into four functional modules with divergent functions. Moreover, these results provide insights into the origin of the reticulated phenotype, suggesting that the RER proteins functionally interconnect photoperiodic growth, amino acid homeostasis, and reactive oxygen species metabolism during Arabidopsis leaf growth. PMID:23596191

Stamina pistilloida, the Pea Ortholog of Fim and UFO, Is Required for Normal Development of Flowers, Inflorescences, and Leaves

PubMed Central

Taylor, Scott; Hofer, Julie; Murfet, Ian

2001-01-01

Isolation and characterization of two severe alleles at the Stamina pistilloida (Stp) locus reveals that Stp is involved in a wide range of developmental processes in the garden pea. The most severe allele, stp-4, results in flowers consisting almost entirely of sepals and carpels. Production of ectopic secondary flowers in stp-4 plants suggests that Stp is involved in specifying floral meristem identity in pea. The stp mutations also reduce the complexity of the compound pea leaf, and primary inflorescences often terminate prematurely in an aberrant sepaloid flower. In addition, stp mutants were shorter than their wild-type siblings due to a reduction in cell number in their internodes. Fewer cells were also found in the epidermis of the leaf rachis of stp mutants. Examination of the effects of stp-4 in double mutant combinations with af, tl, det, and veg2-2—mutations known to influence leaf, inflorescence, and flower development in pea—suggests that Stp function is independent of these genes. A synergistic interaction between weak mutant alleles at Stp and Uni indicated that these two genes act together, possibly to regulate primordial growth. Molecular analysis revealed that Stp is the pea homolog of the Antirrhinum gene Fimbriata (Fim) and of UNUSUAL FLORAL ORGANS (UFO) from Arabidopsis. Differences between Fim/UFO and Stp mutant phenotypes and expression patterns suggest that expansion of Stp activity into the leaf was an important step during evolution of the compound leaf in the garden pea. PMID:11158527

A SAL1 Loss-of-Function Arabidopsis Mutant Exhibits Enhanced Cadmium Tolerance in Association with Alleviation of Endoplasmic Reticulum Stress.

PubMed

Xi, Hongmei; Xu, Hua; Xu, Wenxiu; He, Zhenyan; Xu, Wenzhong; Ma, Mi

2016-06-01

SAL1, as a negative regulator of stress response signaling, has been studied extensively for its role in plant response to environmental stresses. However, the role of SAL1 in cadmium (Cd) stress response and the underlying mechanism is still unclear. Using an Arabidopsis thaliana loss-of-function mutant of SAL1, we assessed Cd resistance and further explored the Cd toxicity mechanism through analysis of the endoplasmic reticulum (ER) stress response. The loss of SAL1 function greatly improved Cd tolerance and significantly attenuated ER stress in Arabidopsis. Exposure to Cd induced an ER stress response in Arabidopsis as evidenced by unconventional splicing of AtbZIP60 and up-regulation of ER stress-responsive genes. Damage caused by Cd was markedly reduced in the ER stress response double mutant bzip28 bzip60 or by application of the ER stress-alleviating chemical agents, tauroursodeoxycholic acid (TUDCA) and 4-phenyl butyric acid (4-PBA), in wild-type plants. The Cd-induced ER stress in Arabidopsis was also alleviated by loss of function of SAL1. These results identified SAL1 as a new component mediating Cd toxicity and established the role of the ER stress response in Cd toxicity. Additionally, the attenuated ER stress in the sal1 mutant might also shed new light on the mechanism of diverse abiotic stress resistance in the SAL1 loss-of-function mutants. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Establishment of an immortalized cell line derived from the prairie vole via lentivirus-mediated transduction of mutant cyclin-dependent kinase 4, cyclin D, and telomerase reverse transcriptase

PubMed Central

Katayama, Masafumi; Kiyono, Tohru; Horie, Kengo; Hirayama, Takashi; Eitsuka, Takahiro; Kuroda, Kengo; Donai, Kenichiro; Hidema, Shizu; Nishimori, Katsuhiko; Fukuda, Tomokazu

2015-01-01

The prairie vole (Microtus ochrogaster) shows social behaviors such as monogamy and parenting of infants with pair bonding. These social behaviors are specific to the prairie vole and have not been observed in other types of voles, such as mountain voles. Although the prairie vole has several unique characteristics, an in vitro cell culture system has not been established for this species. Furthermore, establishment of cultured cells derived from the prairie vole may be beneficial based on the three Rs (i.e., Replacement, Reduction, and Refinement) concept. Therefore, in this study, we attempted to establish an immortalized cell line derived from the prairie vole. Our previous research has shown that transduction with mutant forms of cyclin-dependent kinase 4 (CDK4), cyclin D, and telomerase reverse transcriptase (TERT) could efficiently immortalize cells from multiple species, including humans, cattle, pigs, and monkeys. Here, we introduced these three genes into prairie vole-derived muscle fibroblasts. The expression of mutant CDK4 and cyclin D proteins was confirmed by western blotting, and telomerase activity was detected in immortalized vole muscle-derived fibroblasts (VMF-K4DT cells or VMFs) by stretch PCR. Population doubling analysis showed that the introduction of mutant CDK4, cyclin D, and TERT extended the lifespan of VMFs. To the best of our knowledge, this is the first report describing the establishment of an immortalized cell line derived from the prairie vole through the expression of mutant CDK4, cyclin D, and human TERT. PMID:26496927

Establishment of an immortalized cell line derived from the prairie vole via lentivirus-mediated transduction of mutant cyclin-dependent kinase 4, cyclin D, and telomerase reverse transcriptase.

PubMed

Katayama, Masafumi; Kiyono, Tohru; Horie, Kengo; Hirayama, Takashi; Eitsuka, Takahiro; Kuroda, Kengo; Donai, Kenichiro; Hidema, Shizu; Nishimori, Katsuhiko; Fukuda, Tomokazu

2016-01-01

The prairie vole (Microtus ochrogaster) shows social behaviors such as monogamy and parenting of infants with pair bonding. These social behaviors are specific to the prairie vole and have not been observed in other types of voles, such as mountain voles. Although the prairie vole has several unique characteristics, an in vitro cell culture system has not been established for this species. Furthermore, establishment of cultured cells derived from the prairie vole may be beneficial based on the three Rs (i.e., Replacement, Reduction, and Refinement) concept. Therefore, in this study, we attempted to establish an immortalized cell line derived from the prairie vole. Our previous research has shown that transduction with mutant forms of cyclin-dependent kinase 4 (CDK4), cyclin D, and telomerase reverse transcriptase (TERT) could efficiently immortalize cells from multiple species, including humans, cattle, pigs, and monkeys. Here, we introduced these three genes into prairie vole-derived muscle fibroblasts. The expression of mutant CDK4 and cyclin D proteins was confirmed by western blotting, and telomerase activity was detected in immortalized vole muscle-derived fibroblasts (VMF-K4DT cells or VMFs) by stretch PCR. Population doubling analysis showed that the introduction of mutant CDK4, cyclin D, and TERT extended the lifespan of VMFs. To the best of our knowledge, this is the first report describing the establishment of an immortalized cell line derived from the prairie vole through the expression of mutant CDK4, cyclin D, and human TERT.

Hydrogen is a preferred intermediate in the energy-conserving electron transport chain of Methanosarcina barkeri.

PubMed

Kulkarni, Gargi; Kridelbaugh, Donna M; Guss, Adam M; Metcalf, William W

2009-09-15

Methanogens use an unusual energy-conserving electron transport chain that involves reduction of a limited number of electron acceptors to methane gas. Previous biochemical studies suggested that the proton-pumping F(420)H(2) dehydrogenase (Fpo) plays a crucial role in this process during growth on methanol. However, Methanosarcina barkeri Delta fpo mutants constructed in this study display no measurable phenotype on this substrate, indicating that Fpo plays a minor role, if any. In contrast, Delta frh mutants lacking the cytoplasmic F(420)-reducing hydrogenase (Frh) are severely affected in their ability to grow and make methane from methanol, and double Delta fpo/Delta frh mutants are completely unable to use this substrate. These data suggest that the preferred electron transport chain involves production of hydrogen gas in the cytoplasm, which then diffuses out of the cell, where it is reoxidized with transfer of electrons into the energy-conserving electron transport chain. This hydrogen-cycling metabolism leads directly to production of a proton motive force that can be used by the cell for ATP synthesis. Nevertheless, M. barkeri does have the flexibility to use the Fpo-dependent electron transport chain when needed, as shown by the poor growth of the Delta frh mutant. Our data suggest that the rapid enzymatic turnover of hydrogenases may allow a competitive advantage via faster growth rates in this freshwater organism. The mutant analysis also confirms the proposed role of Frh in growth on hydrogen/carbon dioxide and suggests that either Frh or Fpo is needed for aceticlastic growth of M. barkeri.

JAZ7 negatively regulates dark-induced leaf senescence in Arabidopsis

PubMed Central

Yu, Juan; Zhang, Yixiang; Di, Chao; Zhang, Qunlian; Zhang, Kang; Wang, Chunchao; You, Qi; Yan, Hong; Dai, Susie Y.; Yuan, Joshua S; Xu, Wenying; Su, Zhen

2016-01-01

JASMONATE ZIM-domain (JAZ) proteins play important roles in plant defence and growth by regulating jasmonate signalling. Through data mining, we discovered that the JAZ7 gene was up-regulated in darkness. In the dark, the jaz7 mutant displayed more severe leaf yellowing, quicker chlorophyll degradation, and higher hydrogen peroxide accumulation compared with wild-type (WT) plants. The mutant phenotype of dark-induced leaf senescence could be rescued in the JAZ7-complemented and -overexpression lines. Moreover, the double mutants of jaz7 myc2 and jaz7 coi1 exhibited delayed leaf senescence. We further employed GeneChip analysis to study the molecular mechanism. Some key genes down-regulated in the triple mutant myc2 myc3 myc4 were up-regulated in the jaz7 mutant under darkness. The Gene Ontology terms ‘leaf senescence’ and ‘cell death’ were significantly enriched in the differentially expressed genes. Combining the genetic and transcriptomic analyses together, we proposed a model whereby darkness can induce JAZ7, which might further block MYC2 to suppress dark-induced leaf senescence. In darkness, the mutation of JAZ7 might partially liberate MYC2/MYC3/MYC4 from suppression, leading the MYC proteins to bind to the G-box/G-box-like motifs in the promoters, resulting in the up-regulation of the downstream genes related to indole-glucosinolate biosynthesis, sulphate metabolism, callose deposition, and JA-mediated signalling pathways. In summary, our genetic and transcriptomic studies established the JAZ7 protein as an important regulator in dark-induced leaf senescence. PMID:26547795

UNUSUAL FLORAL ORGANS Controls Meristem Identity and Organ Primordia Fate in Arabidopsis.

PubMed

Wilkinson, M. D.; Haughn, G. W.

1995-09-01

A novel gene that is involved in regulating flower initiation and development has been identified in Arabidopsis. This gene has been designated UNUSUAL FLORAL ORGANS (UFO), with five corresponding nuclear recessive alleles designated ufo[middot]1 to ufo[middot]5. Under short day-length conditions, ufo homozygotes generate more coflorescences than do the wild type, and coflorescences often appear apical to the first floral shoot, resulting in a period of inflorescence development in which regions of floral and coflorescence shoots are produced alternately. ufo enhances the phenotype of weak leafy alleles, and the double mutant Ufo-1 Apetala1-1 produces only coflorescence-like shoots, suggesting that these two genes control different aspects of floral initiation. Floral development was also altered in Ufo plants. Ufo flowers have an altered organ number in all whorls, and organs in the first, second, and third whorls exhibit variable homeotic transformations. Ufo single and double mutant phenotypes suggest that the floral changes result from reduction in class B floral homeotic gene expression and fluctuations in the expression boundaries of class C function and FLO10. Surprisingly, in situ hybridization analysis revealed no obvious differences in expression pattern or level in developing Ufo flowers compared with that of the wild type for any class B or C gene studied. We propose that UFO acts in concert with known floral initiation genes and regulates the domains of floral homeotic gene function.

ETHYLENE INSENSITIVE3 and ETHYLENE INSENSITIVE3-LIKE1 repress SALICYLIC ACID INDUCTION DEFICIENT2 expression to negatively regulate plant innate immunity in Arabidopsis.

PubMed

Chen, Huamin; Xue, Li; Chintamanani, Satya; Germain, Hugo; Lin, Huiqiong; Cui, Haitao; Cai, Run; Zuo, Jianru; Tang, Xiaoyan; Li, Xin; Guo, Hongwei; Zhou, Jian-Min

2009-08-01

Pathogen/microbe-associated molecular patterns (PAMPs/MAMPs) trigger plant immunity that forms the first line inducible defenses in plants. The regulatory mechanism of MAMP-triggered immunity, however, is poorly understood. Here, we show that Arabidopsis thaliana transcription factors ETHYLENE INSENSITIVE3 (EIN3) and ETHYLENE INSENSITIVE3-LIKE1 (EIL1), previously known to mediate ethylene signaling, also negatively regulate PAMP-triggered immunity. Plants lacking EIN3 and EIL1 display enhanced PAMP defenses and heightened resistance to Pseudomonas syringae bacteria. Conversely, plants overaccumulating EIN3 are compromised in PAMP defenses and exhibit enhanced disease susceptibility to Pseudomonas syringae. Microarray analysis revealed that EIN3 and EIL1 negatively control PAMP response genes. Further analyses indicated that SALICYLIC ACID INDUCTION DEFICIENT2 (SID2), which encodes isochorismate synthase required for pathogen-induced biosynthesis of salicylic acid (SA), is a key target of EIN3 and EIL1. Consistent with this, the ein3-1 eil1-1 double mutant constitutively accumulates SA in the absence of pathogen attack, and a mutation in SID2 restores normal susceptibility in the ein3 eil1 double mutant. EIN3 can specifically bind SID2 promoter sequence in vitro and in vivo. Taken together, our data provide evidence that EIN3/EIL1 directly target SID2 to downregulate PAMP defenses.

GOLDEN2-LIKE transcription factors coordinate the tolerance to Cucumber mosaic virus in Arabidopsis

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

Han, Xue-Ying; Li, Peng-Xu; Zou, Li-Juan

Arabidopsis thaliana GOLDEN2-LIKE (GLKs) transcription factors play important roles in regulation of photosynthesis-associated nuclear genes, as well as participate in chloroplast development. However, the involvement of GLKs in plants resistance to virus remains largely unknown. Here, the relationship between GLKs and Cucumber mosaic virus (CMV) stress response was investigated. Our results showed that the Arabidopsis glk1glk2 double-mutant was more susceptible to CMV infection and suffered more serious damages (such as higher oxidative damages, more compromised in PSII photochemistry and more reactive oxygen species accumulation) when compared with the wild-type plants. Interestingly, there was little difference between single mutant (glk1 ormore » glk2) and wild-type plants in response to CMV infection, suggesting GLK1 and GLK2 might function redundant in virus resistance in Arabidopsis. Furthermore, the induction of antioxidant system and defense-associated genes expression in the double mutant were inhibited when compared with single mutant or wild-type plants after CMV infection. Further evidences showed that salicylic acid (SA) and jasmonic acid (JA) might be involved in GLKs-mediated virus resistance, as SA or JA level and synthesis-related genes transcription were impaired in glk1glk2 mutant. Taken together, our results indicated that GLKs played a positively role in virus resistance in Arabidopsis. - Highlights: • GLKs play a positive role in CMV resistance in Arabidopsis. • Defective of GLKs suffered more ROS accumulation. • Arabidopsis lacking GLKs have damaged photosynthesis. • Arabidopsis lacking GLKs show low SA and JA accumulation.« less

Irradiation of Yarrowia lipolytica NRRL YB-567 creating novel strains with enhanced ammonia and oil production on protein and carbohydrate substrates.

PubMed

Lindquist, Mitch R; López-Núñez, Juan Carlos; Jones, Marjorie A; Cox, Elby J; Pinkelman, Rebecca J; Bang, Sookie S; Moser, Bryan R; Jackson, Michael A; Iten, Loren B; Kurtzman, Cletus P; Bischoff, Kenneth M; Liu, Siqing; Qureshi, Nasib; Tasaki, Kenneth; Rich, Joseph O; Cotta, Michael A; Saha, Badal C; Hughes, Stephen R

2015-11-01

Increased interest in sustainable production of renewable diesel and other valuable bioproducts is redoubling efforts to improve economic feasibility of microbial-based oil production. Yarrowia lipolytica is capable of employing a wide variety of substrates to produce oil and valuable co-products. We irradiated Y. lipolytica NRRL YB-567 with UV-C to enhance ammonia (for fertilizer) and lipid (for biodiesel) production on low-cost protein and carbohydrate substrates. The resulting strains were screened for ammonia and oil production using color intensity of indicators on plate assays. Seven mutant strains were selected (based on ammonia assay) and further evaluated for growth rate, ammonia and oil production, soluble protein content, and morphology when grown on liver infusion medium (without sugars), and for growth on various substrates. Strains were identified among these mutants that had a faster doubling time, produced higher maximum ammonia levels (enzyme assay) and more oil (Sudan Black assay), and had higher maximum soluble protein levels (Bradford assay) than wild type. When grown on plates with substrates of interest, all mutant strains showed similar results aerobically to wild-type strain. The mutant strain with the highest oil production and the fastest doubling time was evaluated on coffee waste medium. On this medium, the strain produced 0.12 g/L ammonia and 0.20 g/L 2-phenylethanol, a valuable fragrance/flavoring, in addition to acylglycerols (oil) containing predominantly C16 and C18 residues. These mutant strains will be investigated further for potential application in commercial biodiesel production.

LIL3, a light-harvesting-like protein, plays an essential role in chlorophyll and tocopherol biosynthesis

PubMed Central

Tanaka, Ryouichi; Rothbart, Maxi; Oka, Seiko; Takabayashi, Atsushi; Takahashi, Kaori; Shibata, Masaru; Myouga, Fumiyoshi; Motohashi, Reiko; Shinozaki, Kazuo; Grimm, Bernhard

2010-01-01

The light-harvesting chlorophyll-binding (LHC) proteins are major constituents of eukaryotic photosynthetic machinery. In plants, six different groups of proteins, LHC-like proteins, share a conserved motif with LHC. Although the evolution of LHC and LHC-like proteins is proposed to be a key for the diversification of modern photosynthetic eukaryotes, our knowledge of the evolution and functions of LHC-like proteins is still limited. In this study, we aimed to understand specifically the function of one type of LHC-like proteins, LIL3 proteins, by analyzing Arabidopsis mutants lacking them. The Arabidopsis genome contains two gene copies for LIL3, LIL3:1 and LIL3:2. In the lil3:1/lil3:2 double mutant, the majority of chlorophyll molecules are conjugated with an unsaturated geranylgeraniol side chain. This mutant is also deficient in α-tocopherol. These results indicate that reduction of both the geranylgeraniol side chain of chlorophyll and geranylgeranyl pyrophosphate, which is also an essential intermediate of tocopherol biosynthesis, is compromised in the lil3 mutants. We found that the content of geranylgeranyl reductase responsible for these reactions was severely reduced in the lil3 double mutant, whereas the mRNA level for this enzyme was not significantly changed. We demonstrated an interaction of geranylgeranyl reductase with both LIL3 isoforms by using a split ubiquitin assay, bimolecular fluorescence complementation, and combined blue-native and SDS polyacrylamide gel electrophoresis. We propose that LIL3 is functionally involved in chlorophyll and tocopherol biosynthesis by stabilizing geranylgeranyl reductase. PMID:20823244

Salmonella typhimurium IroN and FepA Proteins Mediate Uptake of Enterobactin but Differ in Their Specificity for Other Siderophores

PubMed Central

Rabsch, Wolfgang; Voigt, Wolfgang; Reissbrodt, Rolf; Tsolis, Renée M.; Bäumler, Andreas J.

1999-01-01

Salmonella typhimurium possesses two outer membrane receptor proteins, IroN and FepA, which have been implicated in the uptake of enterobactin. To determine whether both receptors have identical substrate specificities, fepA and iroN mutants and a double mutant were characterized. While both receptors transported enterobactin, the uptake of corynebactin and myxochelin C was selectively mediated by IroN and FepA, respectively. PMID:10348879

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.

Cytochrome oxidase assembly does not require catalytically active cytochrome C.

PubMed

Barrientos, Antoni; Pierre, Danielle; Lee, Johnson; Tzagoloff, Alexander

2003-03-14

Cytochrome c oxidase (COX), the terminal enzyme of the mitochondrial respiratory chain, catalyzes the transfer of electrons from reduced cytochrome c to molecular oxygen. COX assembly requires the coming together of nuclear- and mitochondrial-encoded subunits and the assistance of a large number of nuclear gene products acting at different stages of maturation of the enzyme. In Saccharomyces cerevisiae, expression of cytochrome c, encoded by CYC1 and CYC7, is required not only for electron transfer but also for COX assembly through a still unknown mechanism. We have attempted to distinguish between a functional and structural requirement of cytochrome c in COX assembly. A cyc1/cyc7 double null mutant strain was transformed with the cyc1-166 mutant gene (Schweingruber, M. E., Stewart, J. W., and Sherman, F. (1979) J. Biol. Chem. 254, 4132-4143) that expresses stable but catalytically inactive iso-1-cytochrome c. The COX content of the cyc1/cyc7 double mutant strain harboring non-functional iso-1-cytochrome c has been characterized spectrally, functionally, and immunochemically. The results of these studies demonstrate that cytochrome c plays a structural rather than functional role in assembly of cytochrome c oxidase. In addition to its requirement for COX assembly, cytochrome c also affects turnover of the enzyme. Mutants containing wild type apocytochrome c in mitochondria lack COX, suggesting that only the folded and mature protein is able to promote COX assembly.

Ion Exchangers NHX1 and NHX2 Mediate Active Potassium Uptake into Vacuoles to Regulate Cell Turgor and Stomatal Function in Arabidopsis[W][OA

PubMed Central

Barragán, Verónica; Leidi, Eduardo O.; Andrés, Zaida; Rubio, Lourdes; De Luca, Anna; Fernández, José A.; Cubero, Beatriz; Pardo, José M.

2012-01-01

Intracellular NHX proteins are Na+,K+/H+ antiporters involved in K+ homeostasis, endosomal pH regulation, and salt tolerance. Proteins NHX1 and NHX2 are the two major tonoplast-localized NHX isoforms. Here, we show that NHX1 and NHX2 have similar expression patterns and identical biochemical activity, and together they account for a significant amount of the Na+,K+/H+ antiport activity in tonoplast vesicles. Reverse genetics showed functional redundancy of NHX1 and NHX2 genes. Growth of the double mutant nhx1 nhx2 was severely impaired, and plants were extremely sensitive to external K+. By contrast, nhx1 nhx2 mutants showed similar sensitivity to salinity stress and even greater rates of Na+ sequestration than the wild type. Double mutants had reduced ability to create the vacuolar K+ pool, which in turn provoked greater K+ retention in the cytosol, impaired osmoregulation, and compromised turgor generation for cell expansion. Genes NHX1 and NHX2 were highly expressed in guard cells, and stomatal function was defective in mutant plants, further compromising their ability to regulate water relations. Together, these results show that tonoplast-localized NHX proteins are essential for active K+ uptake at the tonoplast, for turgor regulation, and for stomatal function. PMID:22438021

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

Ultraviolet-Sensitive Mutator Strain of Escherichia coli K-12

PubMed Central

Siegel, Eli C.

1973-01-01

An ultraviolet (UV)-sensitive mutator gene, mutU, was identified in Escherichia coli K-12. The mutation mutU4 is very close to uvrD, between metE and ilv, on the E. coli chromosome. It was recessive as a mutator and as a UV-sensitive mutation. The frequency of reversion of trpA46 on an F episome was increased by mutU4 on the chromosome. The mutator gene did not increase mutation frequencies in virulent phages or in lytically grown phage λ. The mutU4 mutation predominantly induced transitional base changes. Mutator strains were normal for recombination and host-cell reactivation of UV-irradiated phage T1. They were normally resistant to methyl methanesulfonate and were slightly more sensitive to gamma irradiation than Mut+ strains. UV irradiation induced mutations in a mutU4 strain, and phage λ was UV-inducible. Double mutants containing mutU4 and recA, B, or C were extremely sensitive to UV irradiation; a mutU4 uvrA6 double mutant was only slightly more sensitive than a uvrA6 strain. The mutU4 uvrA6 and mutU4 recA, B, or C double mutants had mutation rates similar to that of a mutU4 strain. Two UV-sensitive mutators, mut-9 and mut-10, isolated by Liberfarb and Bryson in E. coli B/UV, were found to be co-transducible with ilv in the same general region as mutU4. PMID:4345920

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.

Oncogenic ras-driven cancer cell vesiculation leads to emission of double-stranded DNA capable of interacting with target cells

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

Lee, Tae Hoon; Chennakrishnaiah, Shilpa; Audemard, Eric

2014-08-22

Highlights: • Oncogenic H-ras stimulates emission of extracellular vesicles containing double-stranded DNA. • Vesicle-associated extracellular DNA contains mutant N-ras sequences. • Vesicles mediate intercellular transfer of mutant H-ras DNA to normal fibroblasts where it remains for several weeks. • Fibroblasts exposed to vesicles containing H-ras DNA exhibit increased proliferation. - Abstract: Cell free DNA is often regarded as a source of genetic cancer biomarkers, but the related mechanisms of DNA release, composition and biological activity remain unclear. Here we show that rat epithelial cell transformation by the human H-ras oncogene leads to an increase in production of small, exosomal-like extracellularmore » vesicles by viable cancer cells. These EVs contain chromatin-associated double-stranded DNA fragments covering the entire host genome, including full-length H-ras. Oncogenic N-ras and SV40LT sequences were also found in EVs emitted from spontaneous mouse brain tumor cells. Disruption of acidic sphingomyelinase and the p53/Rb pathway did not block emission of EV-related oncogenic DNA. Exposure of non-transformed RAT-1 cells to EVs containing mutant H-ras DNA led to the uptake and retention of this material for an extended (30 days) but transient period of time, and stimulated cell proliferation. Thus, our study suggests that H-ras-mediated transformation stimulates vesicular emission of this histone-bound oncogene, which may interact with non-transformed cells.« less

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.

Increased metabolite production by deletion of an HDA1-type histone deacetylase in the phytopathogenic fungi, Magnaporthe oryzae (Pyricularia oryzae) and Fusarium asiaticum.

PubMed

Maeda, K; Izawa, M; Nakajima, Y; Jin, Q; Hirose, T; Nakamura, T; Koshino, H; Kanamaru, K; Ohsato, S; Kamakura, T; Kobayashi, T; Yoshida, M; Kimura, M

2017-11-01

Histone deacetylases (HDACs) play an important role in the regulation of chromatin structure and gene expression. We found that dark pigmentation of Magnaporthe oryzae (anamorph Pyricularia oryzae) ΔMohda1, a mutant strain in which an orthologue of the yeast HDA1 was disrupted by double cross-over homologous recombination, was significantly stimulated in liquid culture. Analysis of metabolites in a ΔMohda1 mutant culture revealed that the accumulation of shunt products of the 1,8-dihydroxynaphthalene melanin and ergosterol pathways were significantly enhanced compared to the wild-type strain. Northern blot analysis of the ΔMohda1 mutant revealed transcriptional activation of three melanin genes that are dispersed throughout the genome of M. oryzae. The effect of deletion of the yeast HDA1 orthologue was also observed in Fusarium asiaticum from the Fusarium graminearum species complex; the HDF2 deletion mutant produced increased levels of nivalenol-type trichothecenes. These results suggest that histone modification via HDA1-type HDAC regulates the production of natural products in filamentous fungi. Natural products of fungi have significant impacts on human welfare, in both detrimental and beneficial ways. Although HDA1-type histone deacetylase is not essential for vegetative growth, deletion of the gene affects the expression of clustered secondary metabolite genes in some fungi. Here, we report that such phenomena are also observed in physically unlinked genes required for melanin biosynthesis in the rice blast fungus. In addition, production of Fusarium trichothecenes, previously reported to be unaffected by HDA1 deletion, was significantly upregulated in another Fusarium species. Thus, the HDA1-inactivation strategy may be regarded as a general approach for overproduction and/or discovery of fungal metabolites. © 2017 The Society for Applied Microbiology.

Membrane-localized extra-large G proteins and Gbg of the heterotrimeric G proteins form functional complexes engaged in plant immunity in Arabidopsis.

PubMed

Maruta, Natsumi; Trusov, Yuri; Brenya, Eric; Parekh, Urvi; Botella, José Ramón

2015-03-01

In animals, heterotrimeric G proteins, comprising Ga, Gb, and Gg subunits, are molecular switches whose function tightly depends on Ga and Gbg interaction. Intriguingly, in Arabidopsis (Arabidopsis thaliana), multiple defense responses involve Gbg, but not Ga. We report here that the Gbg dimer directly partners with extra-large G proteins (XLGs) to mediate plant immunity. Arabidopsis mutants deficient in XLGs, Gb, and Gg are similarly compromised in several pathogen defense responses, including disease development and production of reactive oxygen species. Genetic analysis of double, triple, and quadruple mutants confirmed that XLGs and Gbg functionally interact in the same defense signaling pathways. In addition, mutations in XLG2 suppressed the seedling lethal and cell death phenotypes of BRASSINOSTEROID INSENSITIVE1-associated receptor kinase1-interacting receptor-like kinase1 mutants in an identical way as reported for Arabidopsis Gb-deficient mutants. Yeast (Saccharomyces cerevisiae) three-hybrid and bimolecular fluorescent complementation assays revealed that XLG2 physically interacts with all three possible Gbg dimers at the plasma membrane. Phylogenetic analysis indicated a close relationship between XLGs and plant Ga subunits, placing the divergence point at the dawn of land plant evolution. Based on these findings, we conclude that XLGs form functional complexes with Gbg dimers, although the mechanism of action of these complexes, including activation/deactivation, must be radically different form the one used by the canonical Ga subunit and are not likely to share the same receptors. Accordingly, XLGs expand the repertoire of heterotrimeric G proteins in plants and reveal a higher level of diversity in heterotrimeric G protein signaling.

Molecular synergy underlies the co-occurrence patterns and phenotype of NPM1-mutant acute myeloid leukemia

PubMed Central

Dovey, Oliver M.; Cooper, Jonathan L.; Mupo, Annalisa; Grove, Carolyn S.; Lynn, Claire; Conte, Nathalie; Andrews, Robert M.; Pacharne, Suruchi; Tzelepis, Konstantinos; Vijayabaskar, M. S.; Green, Paul; Rad, Roland; Arends, Mark; Wright, Penny; Yusa, Kosuke; Bradley, Allan; Varela, Ignacio

2017-01-01

NPM1 mutations define the commonest subgroup of acute myeloid leukemia (AML) and frequently co-occur with FLT3 internal tandem duplications (ITD) or, less commonly, NRAS or KRAS mutations. Co-occurrence of mutant NPM1 with FLT3-ITD carries a significantly worse prognosis than NPM1-RAS combinations. To understand the molecular basis of these observations, we compare the effects of the 2 combinations on hematopoiesis and leukemogenesis in knock-in mice. Early effects of these mutations on hematopoiesis show that compound Npm1cA/+;NrasG12D/+ or Npm1cA;Flt3ITD share a number of features: Hox gene overexpression, enhanced self-renewal, expansion of hematopoietic progenitors, and myeloid differentiation bias. However, Npm1cA;Flt3ITD mutants displayed significantly higher peripheral leukocyte counts, early depletion of common lymphoid progenitors, and a monocytic bias in comparison with the granulocytic bias in Npm1cA/+;NrasG12D/+ mutants. Underlying this was a striking molecular synergy manifested as a dramatically altered gene expression profile in Npm1cA;Flt3ITD, but not Npm1cA/+;NrasG12D/+, progenitors compared with wild-type. Both double-mutant models developed high-penetrance AML, although latency was significantly longer with Npm1cA/+;NrasG12D/+. During AML evolution, both models acquired additional copies of the mutant Flt3 or Nras alleles, but only Npm1cA/+;NrasG12D/+ mice showed acquisition of other human AML mutations, including IDH1 R132Q. We also find, using primary Cas9-expressing AMLs, that Hoxa genes and selected interactors or downstream targets are required for survival of both types of double-mutant AML. Our results show that molecular complementarity underlies the higher frequency and significantly worse prognosis associated with NPM1c/FLT3-ITD vs NPM1/NRAS-G12D-mutant AML and functionally confirm the role of HOXA genes in NPM1c-driven AML. PMID:28835438

Impact of mutation on proton transfer reactions in ketosteroid isomerase: insights from molecular dynamics simulations.

PubMed

Chakravorty, Dhruva K; Hammes-Schiffer, Sharon

2010-06-02

The two proton transfer reactions catalyzed by ketosteroid isomerase (KSI) involve a dienolate intermediate stabilized by hydrogen bonds with Tyr14 and Asp99. Molecular dynamics simulations based on an empirical valence bond model are used to examine the impact of mutating these residues on the hydrogen-bonding patterns, conformational changes, and van der Waals and electrostatic interactions during the proton transfer reactions. While the rate constants for the two proton transfer steps are similar for wild-type (WT) KSI, the simulations suggest that the rate constant for the first proton transfer step is smaller in the mutants due to the significantly higher free energy of the dienolate intermediate relative to the reactant. The calculated rate constants for the mutants D99L, Y14F, and Y14F/D99L relative to WT KSI are qualitatively consistent with the kinetic experiments indicating a significant reduction in the catalytic rates along the series of mutants. In the simulations, WT KSI retained two hydrogen-bonding interactions between the substrate and the active site, while the mutants typically retained only one hydrogen-bonding interaction. A new hydrogen-bonding interaction between the substrate and Tyr55 was observed in the double mutant, leading to the prediction that mutation of Tyr55 will have a greater impact on the proton transfer rate constants for the double mutant than for WT KSI. The electrostatic stabilization of the dienolate intermediate relative to the reactant was greater for WT KSI than for the mutants, providing a qualitative explanation for the significantly reduced rates of the mutants. The active site exhibited restricted motion during the proton transfer reactions, but small conformational changes occurred to facilitate the proton transfer reactions by strengthening the hydrogen-bonding interactions and by bringing the proton donor and acceptor closer to each other with the proper orientation for proton transfer. Thus, these calculations suggest that KSI forms a preorganized active site but that the structure of this preorganized active site is altered upon mutation. Moreover, small conformational changes due to stochastic thermal motions are required within this preorganized active site to facilitate the proton transfer reactions.

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

PubMed

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

2017-06-01

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

Decreases in activation energy and substrate affinity in cold-adapted A4-lactate dehydrogenase: evidence from the Antarctic notothenioid fish Chaenocephalus aceratus.

PubMed

Fields, Peter A; Houseman, Daniel E

2004-12-01

Enzyme function is strongly affected by temperature, and orthologs from species adapted to different thermal environments often show temperature compensation in kinetic properties. Antarctic notothenioid fishes live in a habitat of constant, extreme cold (-1.86 +/- 2 degrees C), and orthologs of the enzyme A4-lactate dehydrogenase (A4-LDH) in these species have adapted to this environment through higher catalytic rates, lower Arrhenius activation energies (Ea), and increases in the apparent Michaelis constant for the substrate pyruvate (Km(PYR)). Here, site-directed mutagenesis was used to determine which amino acid substitutions found in A4-LDH of the notothenioid Chaenocephalus aceratus, with respect to orthologs from warm-adapted teleosts, are responsible for these adaptive changes in enzyme function. Km(PYR) was measured in eight single and two double mutants, and Ea was tested in five single and two double mutants in the temperature range 0 degrees C-20 degrees C. Of the four mutants that had an effect on these parameters, two increased Ea but did not affect Km(PYR) (Gly224Ser, Ala310Pro), and two increased both Ea and Km(PYR) (Glu233Met, Gln317Val). The double mutants Glu233Met/Ala310Pro and Glu233Met/Gln317Val increased Km(PYR) and Ea to levels not significantly different from the A4-LDH of a warm temperate fish (Gillichthys mirabilis, habitat temperature 10 degrees C-35 degrees C). The four single mutants are associated with two alpha-helices that move during the catalytic cycle; those that affect Ea but not Km(PYR) are further from the active site than those that affect both parameters. These results provide evidence that (1) cold adaptation in A4-LDH involves changes in mobility of catalytically important molecular structures; (2) these changes may alter activation energy alone or activation energy and substrate affinity together; and (3) the extent to which these parameters are affected may depend on the location of the substitutions within the mobile alpha-helices, perhaps due to differences in proximity to the active site.

Involvement of two glycoside hydrolase family 19 members in colony morphotype and virulence in Flavobacterium columnare

NASA Astrophysics Data System (ADS)

Zhang, Xiaolin; Li, Nan; Qin, Ting; Huang, Bei; Nie, Pin

2017-11-01

Flavobacterium columnare is the pathogenic agent of columnaris disease in aquaculture. Using a recently developed gene deletion strategy, two genes that encode the Glyco_hydro_19 domain (GH19 domain) containing proteins, ghd-1 and ghd-2, were deleted separately and together from the F. columnare G4 wild type strain. Surprisingly, the single-, Δ ghd-1 and Δ ghd-2, and double-gene mutants, Δ ghd-1 Δghd -2, all had rhizoid and non-rhizoid colony morphotypes, which we named Δ ghd-1, Δ ghd-2, Δ ghd-1 Δ ghd-2, and NΔ ghd-1, NΔ ghd-2, and NΔ ghd-1 Δ ghd-2. However, chitin utilization was not detected in either these mutants or in the wild type. Instead, skimmed milk degradation was observed for the mutants and the wild type; the non-rhizoid strain NΔ ghd-2 exhibited higher degradation activity as revealed by the larger transparent circle on the skimmed milk plate. Using zebrafish as the model organism, we found that non-rhizoid mutants had higher LD50 values and were less virulent because zebrafish infected with these survived longer. Transcriptome analysis between the non-rhizoid and rhizoid colony morphotypes of each mutant, i.e., NΔ ghd -1 versus (vs) Δ ghd-1, NΔ ghd-2 vs Δ ghd-2, and NΔ ghd-1 Δ ghd-2 vs Δ ghd-1 Δ ghd-2, revealed a large number of differentially expressed genes, among which 39 genes were common in three of the pairs compared. Although most of these genes encode hypothetical proteins, a few molecules such as phage tail protein, rhs element Vgr protein, thiol-activated cytolysin, and TonB-dependent outer membrane receptor precursor, expression of which was down-regulated in non-rhizoid mutants but up-regulated in rhizoid mutants, may play a role F. columnare virulence.

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.

Neurotactin functions in concert with other identified CAMs in growth cone guidance in Drosophila.

PubMed

Speicher, S; García-Alonso, L; Carmena, A; Martín-Bermudo, M D; de la Escalera, S; Jiménez, F

1998-02-01

We have isolated and characterized mutations in Drosophila neurotactin, a gene that encodes a cell adhesion protein widely expressed during neural development. Analysis of both loss and gain of gene function conditions during embryonic and postembryonic development revealed specific requirements for neurotactin during axon outgrowth, fasciculation, and guidance. Furthermore, embryos of some double mutant combinations of neurotactin and other genes encoding adhesion/signaling molecules, including neuroglian, derailed, and kekkon1, displayed phenotypic synergy. This result provides evidence for functional cooperativity in vivo between the adhesion and signaling pathways controlled by neurotactin and the other three genes.

A eukaryotic-type signalling system of Pseudomonas aeruginosa contributes to oxidative stress resistance, intracellular survival and virulence

PubMed Central

2011-01-01

Background The genome of Pseudomonas aeruginosa contains at least three genes encoding eukaryotic-type Ser/Thr protein kinases, one of which, ppkA, has been implicated in P. aeruginosa virulence. Together with the adjacent pppA phosphatase gene, they belong to the type VI secretion system (H1-T6SS) locus, which is important for bacterial pathogenesis. To determine the biological function of this protein pair, we prepared a pppA-ppkA double mutant and characterised its phenotype and transcriptomic profiles. Results Phenotypic studies revealed that the mutant grew slower than the wild-type strain in minimal media and exhibited reduced secretion of pyoverdine. In addition, the mutant had altered sensitivity to oxidative and hyperosmotic stress conditions. Consequently, mutant cells had an impaired ability to survive in murine macrophages and an attenuated virulence in the plant model of infection. Whole-genome transcriptome analysis revealed that pppA-ppkA deletion affects the expression of oxidative stress-responsive genes, stationary phase σ-factor RpoS-regulated genes, and quorum-sensing regulons. The transcriptome of the pppA-ppkA mutant was also analysed under conditions of oxidative stress and showed an impaired response to the stress, manifested by a weaker induction of stress adaptation genes as well as the genes of the SOS regulon. In addition, expression of either RpoS-regulated genes or quorum-sensing-dependent genes was also affected. Complementation analysis confirmed that the transcription levels of the differentially expressed genes were specifically restored when the pppA and ppkA genes were expressed ectopically. Conclusions Our results suggest that in addition to its crucial role in controlling the activity of P. aeruginosa H1-T6SS at the post-translational level, the PppA-PpkA pair also affects the transcription of stress-responsive genes. Based on these data, it is likely that the reduced virulence of the mutant strain results from an impaired ability to survive in the host due to the limited response to stress conditions. PMID:21880152

A eukaryotic-type signalling system of Pseudomonas aeruginosa contributes to oxidative stress resistance, intracellular survival and virulence.

PubMed

Goldová, Jana; Ulrych, Aleš; Hercík, Kamil; Branny, Pavel

2011-08-31

The genome of Pseudomonas aeruginosa contains at least three genes encoding eukaryotic-type Ser/Thr protein kinases, one of which, ppkA, has been implicated in P. aeruginosa virulence. Together with the adjacent pppA phosphatase gene, they belong to the type VI secretion system (H1-T6SS) locus, which is important for bacterial pathogenesis. To determine the biological function of this protein pair, we prepared a pppA-ppkA double mutant and characterised its phenotype and transcriptomic profiles. Phenotypic studies revealed that the mutant grew slower than the wild-type strain in minimal media and exhibited reduced secretion of pyoverdine. In addition, the mutant had altered sensitivity to oxidative and hyperosmotic stress conditions. Consequently, mutant cells had an impaired ability to survive in murine macrophages and an attenuated virulence in the plant model of infection. Whole-genome transcriptome analysis revealed that pppA-ppkA deletion affects the expression of oxidative stress-responsive genes, stationary phase σ-factor RpoS-regulated genes, and quorum-sensing regulons. The transcriptome of the pppA-ppkA mutant was also analysed under conditions of oxidative stress and showed an impaired response to the stress, manifested by a weaker induction of stress adaptation genes as well as the genes of the SOS regulon. In addition, expression of either RpoS-regulated genes or quorum-sensing-dependent genes was also affected. Complementation analysis confirmed that the transcription levels of the differentially expressed genes were specifically restored when the pppA and ppkA genes were expressed ectopically. Our results suggest that in addition to its crucial role in controlling the activity of P. aeruginosa H1-T6SS at the post-translational level, the PppA-PpkA pair also affects the transcription of stress-responsive genes. Based on these data, it is likely that the reduced virulence of the mutant strain results from an impaired ability to survive in the host due to the limited response to stress conditions.

In Vivo Analysis of Lrig Genes Reveals Redundant and Independent Functions in the Inner Ear

PubMed Central

del Rio, Tony; Nishitani, Allison M.; Yu, Wei-Ming; Goodrich, Lisa V.

2013-01-01

Lrig proteins are conserved transmembrane proteins that modulate a variety of signaling pathways from worm to humans. In mammals, there are three family members – Lrig1, Lrig2, and Lrig3 – that are defined by closely related extracellular domains with a similar arrangement of leucine rich repeats and immunoglobulin domains. However, the intracellular domains show little homology. Lrig1 inhibits EGF signaling through internalization and degradation of ErbB receptors. Although Lrig3 can also bind ErbB receptors in vitro, it is unclear whether Lrig2 and Lrig3 exhibit similar functions to Lrig1. To gain insights into Lrig gene functions in vivo, we compared the expression and function of the Lrigs in the inner ear, which offers a sensitive system for detecting effects on morphogenesis and function. We find that all three family members are expressed in the inner ear throughout development, with Lrig1 and Lrig3 restricted to subsets of cells and Lrig2 expressed more broadly. Lrig1 and Lrig3 overlap prominently in the developing vestibular apparatus and simultaneous removal of both genes disrupts inner ear morphogenesis. This suggests that these two family members act redundantly in the otic epithelium. In contrast, although Lrig1 and Lrig2 are frequently co-expressed, Lrig1−/−;Lrig2−/− double mutant ears show no enhanced structural abnormalities. At later stages, Lrig1 expression is sustained in non-sensory tissues, whereas Lrig2 levels are enhanced in neurons and sensory epithelia. Consistent with these distinct expression patterns, Lrig1 and Lrig2 mutant mice exhibit different forms of impaired auditory responsiveness. Notably, Lrig1−/−;Lrig2−/− double mutant mice display vestibular deficits and suffer from a more severe auditory defect that is accompanied by a cochlear innervation phenotype not present in single mutants. Thus, Lrig genes appear to act both redundantly and independently, with Lrig2 emerging as the most functionally distinct family member. PMID:24086156

Abnormal Septation and Inhibition of Sporulation by Accumulation of l-α-Glycerophosphate in Bacillus subtilis Mutants

PubMed Central

Oh, Yong K.; Freese, Elisabeth B.; Freese, Ernst

1973-01-01

Accumulation of l-α-glycerophosphate, in cells of Bacillus subtilis mutants lacking the nicotinamide adenine dinucleotide-independent glycerophosphate dehydrogenase activity, suppresses both growth and sporulation. After growth has stopped, the cells slowly develop one and later more asymmetric septa that are thicker than normal prespore septa and apparently contain too much cell wall material to allow further membrane development into forespores or spores. l-Malate prevents accumulation of glycerophosphate and restores sporulation of the mutant. Glucose or gluconate cannot resotre sporulation, because they still effect glycerophosphate accumulation via de novo synthesis. If that accumulation is blocked in a double mutant, which is unable to make glycerophosphate from or to metabolize it into Embden-Meyerhof compounds, then nonsuppressing amounts of glucose or gluconate can restore sporulation. Images PMID:4632310

Salmonella typhimurium gyrA mutations associated with fluoroquinolone resistance.

PubMed Central

Reyna, F; Huesca, M; González, V; Fuchs, L Y

1995-01-01

Spontaneous quinolone-resistant mutants obtained from Salmonella typhimurium Su694 were screened for mutations by direct DNA sequencing of an amplified PCR gyrA fragment. Substitutions Ser-83-->Phe (Ser83Phe), Ser83Tyr, Asp87Tyr, and Asp87Asn and double mutation Ala67Pro-Gly81Ser, which resulted in decreased sensitivities to ciprofloxacin, enoxacin, pefloxacin, norfloxacin, ofloxacin, and nalidixic acid, were found. The levels of resistance to quinolones for each mutant were determined. PMID:7492118

Probing transcription-specific outputs of β-catenin in vivo

PubMed Central

Valenta, Tomas; Gay, Max; Steiner, Sarah; Draganova, Kalina; Zemke, Martina; Hoffmans, Raymond; Cinelli, Paolo; Aguet, Michel; Sommer, Lukas; Basler, Konrad

2011-01-01

β-Catenin, apart from playing a cell-adhesive role, is a key nuclear effector of Wnt signaling. Based on activity assays in Drosophila, we generated mouse strains where the endogenous β-catenin protein is replaced by mutant forms, which retain the cell adhesion function but lack either or both of the N- and the C-terminal transcriptional outputs. The C-terminal activity is essential for mesoderm formation and proper gastrulation, whereas N-terminal outputs are required later during embryonic development. By combining the double-mutant β-catenin with a conditional null allele and a Wnt1-Cre driver, we probed the role of Wnt/β-catenin signaling in dorsal neural tube development. While loss of β-catenin protein in the neural tube results in severe cell adhesion defects, the morphology of cells and tissues expressing the double-mutant form is normal. Surprisingly, Wnt/β-catenin signaling activity only moderately regulates cell proliferation, but is crucial for maintaining neural progenitor identity and for neuronal differentiation in the dorsal spinal cord. Our model animals thus allow dissecting signaling and structural functions of β-catenin in vivo and provide the first genetic tool to generate cells and tissues that entirely and exclusively lack canonical Wnt pathway activity. PMID:22190459

Deletion of genes involved in glutamate metabolism to improve poly-gamma-glutamic acid production in B. amyloliquefaciens LL3.

PubMed

Zhang, Wei; He, Yulian; Gao, Weixia; Feng, Jun; Cao, Mingfeng; Yang, Chao; Song, Cunjiang; Wang, Shufang

2015-02-01

Here, we attempted to elevate poly-gamma-glutamic acid (γ-PGA) production by modifying genes involved in glutamate metabolism in Bacillus amyloliquefaciens LL3. Products of rocR, rocG and gudB facilitate the conversion from glutamate to 2-oxoglutarate in Bacillus subtillis. The gene odhA is responsible for the synthesis of a component of the 2-oxoglutarate dehydrogenase complex that catalyzes the oxidative decarboxylation of 2-oxoglutarate to succinyl coenzyme A. In-frame deletions of these four genes were performed. In shake flask experiments the gudB/rocG double mutant presented enhanced production of γ-PGA, a 38 % increase compared with wild type. When fermented in a 5-L fermenter with pH control, the γ-PGA yield of the rocR mutant was increased to 5.83 g/L from 4.55 g/L for shake flask experiments. The gudB/rocG double mutant produced 5.68 g/L γ-PGA compared with that of 4.03 g/L for the wild type, a 40 % increase. Those results indicated the possibility of improving γ-PGA production by modifying glutamate metabolism, and identified potential genetic targets to improve γ-PGA production.

Regulatory Mutants at the his1 Locus of Yeast

PubMed Central

Lax, Carol; Fogel, Seymour; Cramer, Carole

1979-01-01

The his1 gene in Saccharomyces cerevisiae codes for phosphoribosyl transferase, an allosteric enzyme that catalyzes the initial step in histidine biosynthesis. Mutants that specifically alter the feedback regulatory function were isolated by selecting his1 prototrophic revertants that overproduce and excrete histidine. The prototrophs were obtained from diploids homoallelic for his1–7 and heterozygous for the flanking markers thr3 and arg6. Among six independently derived mutant isolates, three distinct levels of histidine excretion were detected. The mutants were shown to be second-site alterations mapping at the his1 locus by recovery of the original auoxtrophic parental alleles. The double mutants, HIS1–7e, are dominant with respect to catalytic function but recessive in regulatory function. When removed from this his1–7 background, the mutant regulatory site (HIS1–e) still confers prototrophy but not histidine excretion. To yield the excretion phenotype, the primary and altered secondary sites are required in cis array. Differences in histidine excretion levels correlate with resistance to the histidine analogue, triazoalanine. PMID:385447

Phytochromes A and C cooperatively regulate early and transient gene expression after red-light irradiation in rice seedlings.

PubMed

Kiyota, Seiichiro; Xie, Xianzhi; Takano, Makoto

2012-02-01

Phytochromes are red/far-red photoreceptors encoded by a small gene family in higher plants. Differences in phenotype among mutants suggest distinct functions among phytochrome subfamilies. We attempted to find distinct functions among phytochromes by oligo-microarray analysis of single, double, and triple mutants in rice. In most cases, gene expression was redundantly regulated by phytochromes A and B after irradiation by a red light pulse in etiolated rice shoots. However, we found that several genes were specifically regulated by phytochromes A and C. Most of them were expressed immediately after the red light pulse in a transient manner. They are stress-related genes that may be involved in resistance to light stress when etiolated seedlings are exposed to light. These genes were not expressed in green leaves after the red light pulse, suggesting that they have a function specific to etiolated seedlings. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

Function and Regulation of the Formate Dehydrogenase Genes of the Methanogenic Archaeon Methanococcus maripaludis

PubMed Central

Wood, Gwendolyn E.; Haydock, Andrew K.; Leigh, John A.

2003-01-01

Methanococcus maripaludis is a mesophilic species of Archaea capable of producing methane from two substrates: hydrogen plus carbon dioxide and formate. To study the latter, we identified the formate dehydrogenase genes of M. maripaludis and found that the genome contains two gene clusters important for formate utilization. Phylogenetic analysis suggested that the two formate dehydrogenase gene sets arose from duplication events within the methanococcal lineage. The first gene cluster encodes homologs of formate dehydrogenase α (FdhA) and β (FdhB) subunits and a putative formate transporter (FdhC) as well as a carbonic anhydrase analog. The second gene cluster encodes only FdhA and FdhB homologs. Mutants lacking either fdhA gene exhibited a partial growth defect on formate, whereas a double mutant was completely unable to grow on formate as a sole methanogenic substrate. Investigation of fdh gene expression revealed that transcription of both gene clusters is controlled by the presence of H2 and not by the presence of formate. PMID:12670979

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

Hoekstra, M.F.; Ou, A.C.; DeMaggio, A.J.

In simple eukaryotes, protein kinases regulate mitotic and meiotic cell cycles, the response to polypeptide pheromones, and the initiation of nuclear DNA synthesis. The protein HRR25 from the budding yeast Saccharomyces cerevisiae was defined by the mutation hrr25-1. This mutation resulted in sensitivity to continuous expression of the HO double-strand endonuclease, to methyl methanesulfonate, and to x-irradiation. Homozygotes of hrr25-1 were unable to sporulate and disruption and deletion of HRR25 interfered with mitotic and meiotic cell division. Sequence analysis revealed two distinctive regions in the protein. The NH{sub 2}-terminus of HRR25 contains the hallmark features of protein kinases, whereas themore » COOH-terminus is rich in proline and glutamine. Mutations in HRR25 at conserved residues found in all protein kinases inactivated the gene, and these mutants exhibited the hrr25 null phenotypes. Taken together, the hrr25 mutant phenotypes and the features of the gene product indicate that HRR25 is a distinctive member of the protein kinase superfamily.« less

Stress Tolerance in Doughs of Saccharomyces cerevisiae Trehalase Mutants Derived from Commercial Baker’s Yeast

PubMed Central

Shima, Jun; Hino, Akihiro; Yamada-Iyo, Chie; Suzuki, Yasuo; Nakajima, Ryouichi; Watanabe, Hajime; Mori, Katsumi; Takano, Hiroyuki

1999-01-01

Accumulation of trehalose is widely believed to be a critical determinant in improving the stress tolerance of the yeast Saccharomyces cerevisiae, which is commonly used in commercial bread dough. To retain the accumulation of trehalose in yeast cells, we constructed, for the first time, diploid homozygous neutral trehalase mutants (Δnth1), acid trehalase mutants (Δath1), and double mutants (Δnth1 ath1) by using commercial baker’s yeast strains as the parent strains and the gene disruption method. During fermentation in a liquid fermentation medium, degradation of intracellular trehalose was inhibited with all of the trehalase mutants. The gassing power of frozen doughs made with these mutants was greater than the gassing power of doughs made with the parent strains. The Δnth1 and Δath1 strains also exhibited higher levels of tolerance of dry conditions than the parent strains exhibited; however, the Δnth1 ath1 strain exhibited lower tolerance of dry conditions than the parent strain exhibited. The improved freeze tolerance exhibited by all of the trehalase mutants may make these strains useful in frozen dough. PMID:10388673

Molecular Determinants of Sporulation in Ashbya gossypii

PubMed Central

Wasserstrom, Lisa; Lengeler, Klaus B.; Walther, Andrea; Wendland, Jürgen

2013-01-01

Regulation of development and entry into sporulation is critical for fungi to ensure survival of unfavorable environmental conditions. Here we present an analysis of gene sets regulating sporulation in the homothallic ascomycete Ashbya gossypii. Deletion of components of the conserved pheromone/starvation MAP kinase cascades, e.g., STE11 and STE7, results in increased sporulation. In kar3 mutants sporulation is severely reduced, while deletion of KAR4 as well as of homologs of central Saccharomyces cerevisiae regulators of sporulation, IME1, IME2, IME4, and NDT80, abolishes sporulation in A. gossypii. Comparison of RNAseq transcript profiles of sporulation-deficient mutants identified a set of 67 down-regulated genes, most of which were up-regulated in the oversporulating ste12 mutant. One of these differentially expressed genes is an endoglucanase encoded by ENG2. We found that Eng2p promotes hyphal fragmentation as part of the developmental program of sporulation, which generates single-celled sporangia. Sporulation-deficient strains are arrested in their development but form sporangia. Supply of new nutrients enabled sporangia to return to hyphal growth, indicating that these cells are not locked in meiosis. Double-strand break (DSB) formation by Spo11 is apparently not required for sporulation; however, the absence of DMC1, which repairs DSBs in S. cerevisiae, results in very poor sporulation in A. gossypii. We present a comprehensive analysis of the gene repertoire governing sporulation in A. gossypii and suggest an altered regulation of IME1 expression compared to S. cerevisiae. PMID:23833180

Molecular determinants of sporulation in Ashbya gossypii.

PubMed

Wasserstrom, Lisa; Lengeler, Klaus B; Walther, Andrea; Wendland, Jürgen

2013-09-01

Regulation of development and entry into sporulation is critical for fungi to ensure survival of unfavorable environmental conditions. Here we present an analysis of gene sets regulating sporulation in the homothallic ascomycete Ashbya gossypii. Deletion of components of the conserved pheromone/starvation MAP kinase cascades, e.g., STE11 and STE7, results in increased sporulation. In kar3 mutants sporulation is severely reduced, while deletion of KAR4 as well as of homologs of central Saccharomyces cerevisiae regulators of sporulation, IME1, IME2, IME4, and NDT80, abolishes sporulation in A. gossypii. Comparison of RNAseq transcript profiles of sporulation-deficient mutants identified a set of 67 down-regulated genes, most of which were up-regulated in the oversporulating ste12 mutant. One of these differentially expressed genes is an endoglucanase encoded by ENG2. We found that Eng2p promotes hyphal fragmentation as part of the developmental program of sporulation, which generates single-celled sporangia. Sporulation-deficient strains are arrested in their development but form sporangia. Supply of new nutrients enabled sporangia to return to hyphal growth, indicating that these cells are not locked in meiosis. Double-strand break (DSB) formation by Spo11 is apparently not required for sporulation; however, the absence of DMC1, which repairs DSBs in S. cerevisiae, results in very poor sporulation in A. gossypii. We present a comprehensive analysis of the gene repertoire governing sporulation in A. gossypii and suggest an altered regulation of IME1 expression compared to S. cerevisiae.

Selective gene dosage by CRISPR-Cas9 genome editing in hexaploid Camelina sativa.

PubMed

Morineau, Céline; Bellec, Yannick; Tellier, Frédérique; Gissot, Lionel; Kelemen, Zsolt; Nogué, Fabien; Faure, Jean-Denis

2017-06-01

In many plant species, gene dosage is an important cause of phenotype variation. Engineering gene dosage, particularly in polyploid genomes, would provide an efficient tool for plant breeding. The hexaploid oilseed crop Camelina sativa, which has three closely related expressed subgenomes, is an ideal species for investigation of the possibility of creating a large collection of combinatorial mutants. Selective, targeted mutagenesis of the three delta-12-desaturase (FAD2) genes was achieved by CRISPR-Cas9 gene editing, leading to reduced levels of polyunsaturated fatty acids and increased accumulation of oleic acid in the oil. Analysis of mutations over four generations demonstrated the presence of a large variety of heritable mutations in the three isologous CsFAD2 genes. The different combinations of single, double and triple mutants in the T3 generation were isolated, and the complete loss-of-function mutants revealed the importance of delta-12-desaturation for Camelina development. Combinatorial association of different alleles for the three FAD2 loci provided a large diversity of Camelina lines with various lipid profiles, ranging from 10% to 62% oleic acid accumulation in the oil. The different allelic combinations allowed an unbiased analysis of gene dosage and function in this hexaploid species, but also provided a unique source of genetic variability for plant breeding. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Analysis of purified glabra3-shapeshifter trichomes reveals a role for NOECK in regulating early trichome morphogenic events.

PubMed

Gilding, Edward K; Marks, M David

2010-10-01

Transcriptome analysis using the Affymetrix ATH1 platform has been completed on purified trichomes from the gl3-sst mutant. These trichomes display immature features, such as glassy cell walls and blunted branches. The gl3-sst trichome transcriptome was greatly enriched for genes involved in lipid biosynthesis, including those mediating the synthesis of fatty acids and wax. In addition, gl3-sst trichomes displayed reduced expression of the R3 MYBs TRY and CPC, which normally function to limit trichome development. The expression of the MIXTA-like MYB gene NOK was elevated. Members of the MIXTA-like family promote conical cell outgrowth, and in some cases, trichome initiation in diverse plant species. In contrast, NOK limits trichome outgrowth in wild-type Arabidopsis plants. Similar to other MIXTA-like genes, NOK was required for the expansion of gl3-sst trichomes, as the gl3-sst nok double mutant trichomes were greatly reduced in size. Expression of NOK in nok mutants reduced branch formation, whereas in gl3-sst nok, NOK expression promoted trichome cell outgrowth, illustrating duel roles for NOK in both promoting and limiting trichome development. MIXTA-like genes from phylogenetically diverse plant species could substitute for NOK in both nok and gl3-sst nok backgrounds. These findings suggest that certain aspects of NOK and MIXTA-like gene function have been conserved. © 2010 The Authors. Journal compilation © 2010 Blackwell Publishing Ltd.

Rhizobium meliloti lipooligosaccharide nodulation factors: different structural requirements for bacterial entry into target root hair cells and induction of plant symbiotic developmental responses.

PubMed

Ardourel, M; Demont, N; Debellé, F; Maillet, F; de Billy, F; Promé, J C; Dénarié, J; Truchet, G

1994-10-01

Rhizobium meliloti produces lipochitooligosaccharide nodulation NodRm factors that are required for nodulation of legume hosts. NodRm factors are O-acetylated and N-acylated by specific C16-unsaturated fatty acids. nodL mutants produce non-O-acetylated factors, and nodFE mutants produce factors with modified acyl substituents. Both mutants exhibited a significantly reduced capacity to elicit infection thread (IT) formation in alfalfa. However, once initiated, ITs developed and allowed the formation of nitrogen-fixing nodules. In contrast, double nodF/nodL mutants were unable to penetrate into legume hosts and to form ITs. Nevertheless, these mutants induced widespread cell wall tip growth in trichoblasts and other epidermal cells and were also able to elicit cortical cell activation at a distance. NodRm factor structural requirements are thus clearly more stringent for bacterial entry than for the elicitation of developmental plant responses.

Interrelationships between mitochondrial fusion, energy metabolism and oxidative stress during development in Caenorhabditis elegans.

PubMed

Yasuda, Kayo; Hartman, Philip S; Ishii, Takamasa; Suda, Hitoshi; Akatsuka, Akira; Shoyama, Tetsuji; Miyazawa, Masaki; Ishii, Naoaki

2011-01-21

Mitochondria are known to be dynamic structures with the energetically and enzymatically mediated processes of fusion and fission responsible for maintaining a constant flux. Mitochondria also play a role of reactive oxygen species production as a byproduct of energy metabolism. In the current study, interrelationships between mitochondrial fusion, energy metabolism and oxidative stress on development were explored using a fzo-1 mutant defective in the fusion process and a mev-1 mutant overproducing superoxide from mitochondrial electron transport complex II of Caenorhabditis elegans. While growth and development of both single mutants was slightly delayed relative to the wild type, the fzo-1;mev-1 double mutant experienced considerable delay. Oxygen sensitivity during larval development, superoxide production and carbonyl protein accumulation of the fzo-1 mutant were similar to wild type. fzo-1 animals had significantly lower metabolism than did N2 and mev-1. These data indicate that mitochondrial fusion can profoundly affect energy metabolism and development. Copyright © 2010 Elsevier Inc. All rights reserved.

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.

Joint Molecule Resolution Requires the Redundant Activities of MUS-81 and XPF-1 during Caenorhabditis elegans Meiosis

PubMed Central

O'Neil, Nigel J.; Martin, Julie S.; Youds, Jillian L.; Ward, Jordan D.; Petalcorin, Mark I. R.; Rose, Anne M.; Boulton, Simon J.

2013-01-01

The generation and resolution of joint molecule recombination intermediates is required to ensure bipolar chromosome segregation during meiosis. During wild type meiosis in Caenorhabditis elegans, SPO-11-generated double stranded breaks are resolved to generate a single crossover per bivalent and the remaining recombination intermediates are resolved as noncrossovers. We discovered that early recombination intermediates are limited by the C. elegans BLM ortholog, HIM-6, and in the absence of HIM-6 by the structure specific endonuclease MUS-81. In the absence of both MUS-81 and HIM-6, recombination intermediates persist, leading to chromosome breakage at diakinesis and inviable embryos. MUS-81 has an additional role in resolving late recombination intermediates in C. elegans. mus-81 mutants exhibited reduced crossover recombination frequencies suggesting that MUS-81 is required to generate a subset of meiotic crossovers. Similarly, the Mus81-related endonuclease XPF-1 is also required for a subset of meiotic crossovers. Although C. elegans gen-1 mutants have no detectable meiotic defect either alone or in combination with him-6, mus-81 or xpf-1 mutations, mus-81;xpf-1 double mutants are synthetic lethal. While mus-81;xpf-1 double mutants are proficient for the processing of early recombination intermediates, they exhibit defects in the post-pachytene chromosome reorganization and the asymmetric disassembly of the synaptonemal complex, presumably triggered by crossovers or crossover precursors. Consistent with a defect in resolving late recombination intermediates, mus-81; xpf-1 diakinetic bivalents are aberrant with fine DNA bridges visible between two distinct DAPI staining bodies. We were able to suppress the aberrant bivalent phenotype by microinjection of activated human GEN1 protein, which can cleave Holliday junctions, suggesting that the DNA bridges in mus-81; xpf-1 diakinetic oocytes are unresolved Holliday junctions. We propose that the MUS-81 and XPF-1 endonucleases act redundantly to process late recombination intermediates to form crossovers during C. elegans meiosis. PMID:23874209

Isolation and characterization of Candida albicans morphological mutants derepressed for the formation of filamentous hypha-type structures

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

Gil, C.; Pomes, R.; Nombela, C.

1990-05-01

Several Candida albicans morphological mutants were obtained by a procedure based on a combined treatment with nitrous acid plus UV irradiation and a double-enrichment step to increase the proportion of mutants growing as long filamentous structures. Altered cell morphogenesis in these mutants correlated with an altered colonial phenotype. Two of these mutants, C. albicans NEL102 and NEL103, were selected and characterized. Mutant blastoconidia initiated budding but eventually gave rise to filamentous hypha-type formations. These filaments were long and septate, and they branched very regularly at positions near septa. Calcofluor white (which is known to bind chitin-rich areas) stained septa, branchingmore » zones, and filament tips very intensely, as observed under the fluorescence microscope. Wild-type hybrids were obtained by fusing protoplasts of strain NEL102 with B14, another morphological mutant previously described as being permanently pseudomycelial, indicating that genetic determinants responsible for the two altered phenotypes are different. The mutants characterized in this work seemed to sequentially express the morphogenic characteristics of C. albicans, from blastoconidia to hyphae, in the absence of any inducer. Further characterization of these strains could be relevant to gain understanding of the genetic control of dimorphism in this species.« less

Oncolytic reovirus preferentially induces apoptosis in KRAS mutant colorectal cancer cells, and synergizes with irinotecan

PubMed Central

Maitra, Radhashree; Seetharam, Raviraja; Tesfa, Lydia; Augustine, Titto A.; Klampfer, Lidija; Coffey, Matthew C.; Mariadason, John M.; Goel, Sanjay

2014-01-01

Reovirus is a double stranded RNA virus, with an intrinsic preference for replication in KRAS mutant cells. As 45% of human colorectal cancers (CRC) harbor KRAS mutations, we sought to investigate its efficacy in KRAS mutant CRC cells, and examine its impact in combination with the topoisimerase-1 inhibitor, irinotecan. Reovirus efficacy was examined in the KRAS mutant HCT116, and the isogenic KRAS WT Hke3 cell line, and in the non-malignant rat intestinal epithelial cell line. Apoptosis was determined by flow cytometry and TUNEL staining. Combination treatment with reovirus and irintoecan was investigated in 15 CRC cell lines, including the HCT116 p21 isogenic cell lines. Reovirus preferentially induced apoptosis in KRAS mutant HCT116 cells compared to its isogenic KRAS WT derivative, and in KRAS mutant IEC cells. Reovirus showed a greater degree of caspase 3 activation with PARP 1 cleavage, and preferential inhibition of p21 protein expression in KRAS mutant cells. Reovirus synergistically induced growth inhibition when combined with irinotecan. This synergy was lost upon p21 gene knock out. Reovirus preferentially induces apoptosis in KRAS mutant colon cancer cells. Reovirus and irinotecan combination therapy is synergistic, p21 mediated, and represents a novel potential treatment for patients with CRC. PMID:24798549

The Arabidopsis NAC Transcription Factor ANAC096 Cooperates with bZIP-Type Transcription Factors in Dehydration and Osmotic Stress Responses[W

PubMed Central

Xu, Zheng-Yi; Kim, Soo Youn; Hyeon, Do Young; Kim, Dae Heon; Dong, Ting; Park, Youngmin; Jin, Jing Bo; Joo, Se-Hwan; Kim, Seong-Ki; Hong, Jong Chan; Hwang, Daehee; Hwang, Inhwan

2013-01-01

Multiple transcription factors (TFs) play essential roles in plants under abiotic stress, but how these multiple TFs cooperate in abiotic stress responses remains largely unknown. In this study, we provide evidence that the NAC (for NAM, ATAF1/2, and CUC2) TF ANAC096 cooperates with the bZIP-type TFs ABRE binding factor and ABRE binding protein (ABF/AREB) to help plants survive under dehydration and osmotic stress conditions. ANAC096 directly interacts with ABF2 and ABF4, but not with ABF3, both in vitro and in vivo. ANAC096 and ABF2 synergistically activate RD29A transcription. Our genome-wide gene expression analysis revealed that a major proportion of abscisic acid (ABA)–responsive genes are under the transcriptional regulation of ANAC096. We found that the Arabidopsis thaliana anac096 mutant is hyposensitive to exogenous ABA and shows impaired ABA-induced stomatal closure and increased water loss under dehydration stress conditions. Furthermore, we found the anac096 abf2 abf4 triple mutant is much more sensitive to dehydration and osmotic stresses than the anac096 single mutant or the abf2 abf4 double mutant. Based on these results, we propose that ANAC096 is involved in a synergistic relationship with a subset of ABFs for the transcriptional activation of ABA-inducible genes in response to dehydration and osmotic stresses. PMID:24285786

The SaeRS Two-Component System Controls Survival of Staphylococcus aureus in Human Blood through Regulation of Coagulase

PubMed Central

Guo, Haiyong; Hall, Jeffrey W.; Yang, Junshu; Ji, Yinduo

2017-01-01

The SaeRS two-component system plays important roles in regulation of key virulence factors and pathogenicity. In this study, however, we found that the deletion mutation of saeRS enhanced bacterial survival in human blood, whereas complementation of the mutant with SaeRS returned survival to wild-type levels. Moreover, these phenomena were observed in different MRSA genetic background isolates, including HA-MRSA WCUH29, CA-MRSA 923, and MW2. To elucidate which gene(s) regulated by SaeRS contribute to the effect, we conducted a series of complementation studies with selected known SaeRS target genes in trans. We found coagulase complementation abolished the enhanced survival of the SaeRS mutant in human blood. The coa and saeRS deletion mutants exhibited a similar survival phenotype in blood. Intriguingly, heterologous expression of coagulase decreased survival of S. epidermidis in human blood. Further, the addition of recombinant coagulase to blood significantly decreased the survival of S. aureus. Further, analysis revealed staphylococcal resistance to killing by hydrogen peroxide was partially dependent on the presence or absence of coagulase. Furthermore, complementation with coagulase, but not SaeRS, returned saeRS/coa double mutant survival in blood to wild-type levels. These data indicate SaeRS modulates bacterial survival in blood in coagulase-dependent manner. Our results provide new insights into the role of staphylococcal SaeRS and coagulase on bacterial survival in human blood. PMID:28611950

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

PubMed Central

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

2016-01-01

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

The NosX and NirX Proteins of Paracoccus denitrificans Are Functional Homologues: Their Role in Maturation of Nitrous Oxide Reductase

PubMed Central

Saunders, Neil F. W.; Hornberg, Jorrit J.; Reijnders, Willem N. M.; Westerhoff, Hans V.; de Vries, Simon; van Spanning, Rob J. M.

2000-01-01

The nos (nitrous oxide reductase) operon of Paracoccus denitrificans contains a nosX gene homologous to those found in the nos operons of other denitrifiers. NosX is also homologous to NirX, which is so far unique to P. denitrificans. Single mutations of these genes did not result in any apparent phenotype, but a double nosX nirX mutant was unable to reduce nitrous oxide. Promoter-lacZ assays and immunoblotting against nitrous oxide reductase showed that the defect was not due to failure of expression of nosZ, the structural gene for nitrous oxide reductase. Electron paramagnetic resonance spectroscopy showed that nitrous oxide reductase in cells of the double mutant lacked the CuA center. A twin-arginine motif in both NosX and NirX suggests that the NosX proteins are exported to the periplasm via the TAT translocon. PMID:10960107

Escaping Underground Nets: Extracellular DNases Degrade Plant Extracellular Traps and Contribute to Virulence of the Plant Pathogenic Bacterium Ralstonia solanacearum

PubMed Central

Tran, Tuan Minh; MacIntyre, April; Hawes, Martha; Allen, Caitilyn

2016-01-01

Plant root border cells have been recently recognized as an important physical defense against soil-borne pathogens. Root border cells produce an extracellular matrix of protein, polysaccharide and DNA that functions like animal neutrophil extracellular traps to immobilize pathogens. Exposing pea root border cells to the root-infecting bacterial wilt pathogen Ralstonia solanacearum triggered release of DNA-containing extracellular traps in a flagellin-dependent manner. These traps rapidly immobilized the pathogen and killed some cells, but most of the entangled bacteria eventually escaped. The R. solanacearum genome encodes two putative extracellular DNases (exDNases) that are expressed during pathogenesis, suggesting that these exDNases contribute to bacterial virulence by enabling the bacterium to degrade and escape root border cell traps. We tested this hypothesis with R. solanacearum deletion mutants lacking one or both of these nucleases, named NucA and NucB. Functional studies with purified proteins revealed that NucA and NucB are non-specific endonucleases and that NucA is membrane-associated and cation-dependent. Single ΔnucA and ΔnucB mutants and the ΔnucA/B double mutant all had reduced virulence on wilt-susceptible tomato plants in a naturalistic soil-soak inoculation assay. The ΔnucA/B mutant was out-competed by the wild-type strain in planta and was less able to stunt root growth or colonize plant stems. Further, the double nuclease mutant could not escape from root border cells in vitro and was defective in attachment to pea roots. Taken together, these results demonstrate that extracellular DNases are novel virulence factors that help R. solanacearum successfully overcome plant defenses to infect plant roots and cause bacterial wilt disease. PMID:27336156

The impact of the IGF-1 system of cancer cells on radiation response - An in vitro study.

PubMed

Venkatachalam, Senthiladipan; Mettler, Esther; Fottner, Christian; Miederer, Matthias; Kaina, Bernd; Weber, Matthias M

2017-12-01

Overexpression of the insulin-like growth factor-1 receptor (IGF-1R) is associated with increased cell proliferation, differentiation, transformation, and tumorigenicity. Additionally, signaling involved in the resistance of cancer cells to radiotherapy originates from IGF-1R. The purpose of this study was to investigate the role of the IGF-1 system in the radiation response and further evaluate its effect on the expression of DNA repair pathway genes. To inhibit the IGF-1 system, we stably transfected the Caco-2 cell line to express a kinase-deficient IGF-1R mutant. We then studied the effects of this mutation on cell growth, the response to radiation, and clonogenic survival, as well as using a cell viability assay to examine DNA damage and repair. Finally, we performed immunofluorescence for γ-H2AX to examine double-strand DNA breaks and evaluated the expression of 84 key genes involved in DNA repair with a real-time PCR array. Mutant IGF-1R cells exhibited significantly blunted cell growth and viability, compared to wild-type cells, as well as reduced clonogenic survival after γ-irradiation. However, mutant IGF-1R cells did not show any significant delays in the repair of radiation-induced DNA double-strand breaks. Furthermore, expression of mutant IGF-1R significantly down-regulated the mRNA levels of BRCA2, a major protein involved in homologous recombination DNA repair. These results indicate that blocking the IGF-1R-mediated signaling cascade, through the expression of a kinase-deficient IGF-1R mutant, reduces cell growth and sensitizes cancer cells to ionizing radiation. Therefore, the IGF-1R system could be a potential target to enhance radio-sensitivity and the efficacy of cancer treatments.

Identification of Independent Streptococcus gordonii SspA and SspB Functions in Coaggregation with Actinomyces naeslundii

PubMed Central

Egland, Paul G.; Dû, Laurence D.; Kolenbrander, Paul E.

2001-01-01

The initial stages of dental plaque formation involve the adherence of early colonizing organisms such as Streptococcus gordonii and Actinomyces naeslundii to the saliva-coated tooth surface and to each other. The S. gordonii surface proteins SspA and SspB are known to play a role in adherence to salivary proteins and mediate coaggregation with other bacteria. Coaggregation is the adhesin receptor-mediated interaction between genetically distinct cell types and appears to be ubiquitous among oral isolates. To define the function of SspA and SspB separately on the surface of their natural host, we constructed and analyzed the coaggregation properties of an isogenic sspB mutant of S. gordonii DL1, an sspAB double mutant, and a previously described sspA mutant. A. naeslundii strains have been previously classified into six coaggregation groups based on the nature of their coaggregations with S. gordonii DL1 and other oral streptococci. Coaggregation assays with the sspA and sspB mutants showed that SspA and SspB are the streptococcal proteins primarily responsible for defining these coaggregation groups and, thus, are highly significant in the establishment of early dental plaque. SspA exhibited two coaggregation-specific functions. It participated in lactose-inhibitable and -noninhibitable interactions, while SspB mediated only lactose-noninhibitable coaggregations. Accordingly, the sspAB double mutant lacked these functions and allowed us to detect a third coaggregation interaction with one of these organisms. These proteins may play an important role in development of S. gordonii-A. naeslundii communities in early dental plaque. Understanding these adhesin proteins will aid investigations of complex microbial communities that characterize periodontal diseases. PMID:11705927

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

PubMed Central

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

2013-01-01

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

A quadruple mutant of Arabidopsis reveals a β-carotene hydroxylation activity for LUT1/CYP97C1 and a regulatory role of xanthophylls on determination of the PSI/PSII ratio

PubMed Central

2012-01-01

Background Xanthophylls are oxygenated carotenoids playing an essential role as structural components of the photosynthetic apparatus. Xanthophylls contribute to the assembly and stability of light-harvesting complex, to light absorbance and to photoprotection. The first step in xanthophyll biosynthesis from α- and β-carotene is the hydroxylation of ε- and β-rings, performed by both non-heme iron oxygenases (CHY1, CHY2) and P450 cytochromes (LUT1/CYP97C1, LUT5/CYP97A3). The Arabidopsis triple chy1chy2lut5 mutant is almost completely depleted in β-xanthophylls. Results Here we report on the quadruple chy1chy2lut2lut5 mutant, additionally carrying the lut2 mutation (affecting lycopene ε-cyclase). This genotype lacks lutein and yet it shows a compensatory increase in β-xanthophylls with respect to chy1chy2lut5 mutant. Mutant plants show an even stronger photosensitivity than chy1chy2lut5, a complete lack of qE, the rapidly reversible component of non-photochemical quenching, and a peculiar organization of the pigment binding complexes into thylakoids. Biochemical analysis reveals that the chy1chy2lut2lut5 mutant is depleted in Lhcb subunits and is specifically affected in Photosystem I function, showing a deficiency in PSI-LHCI supercomplexes. Moreover, by analyzing a series of single, double, triple and quadruple Arabidopsis mutants in xanthophyll biosynthesis, we show a hitherto undescribed correlation between xanthophyll levels and the PSI-PSII ratio. The decrease in the xanthophyll/carotenoid ratio causes a proportional decrease in the LHCII and PSI core levels with respect to PSII. Conclusions The physiological and biochemical phenotype of the chy1chy2lut2lut5 mutant shows that (i) LUT1/CYP97C1 protein reveals a major β-carotene hydroxylase activity in vivo when depleted in its preferred substrate α-carotene; (ii) xanthophylls are needed for normal level of Photosystem I and LHCII accumulation. PMID:22513258

Effect of apoA-I Mutations in the Capacity of Reconstituted HDL to Promote ABCG1-Mediated Cholesterol Efflux.

PubMed

Daniil, Georgios; Zannis, Vassilis I; Chroni, Angeliki

2013-01-01

ATP binding cassette transporter G1 (ABCG1) mediates the cholesterol transport from cells to high-density lipoprotein (HDL), but the role of apolipoprotein A-I (apoA-I), the main protein constituent of HDL, in this process is not clear. To address this, we measured cholesterol efflux from HEK293 cells or J774 mouse macrophages overexpressing ABCG1 using as acceptors reconstituted HDL (rHDL) containing wild-type or various mutant apoA-I forms. It was found that ABCG1-mediated cholesterol efflux was severely reduced (by 89%) when using rHDL containing the carboxyl-terminal deletion mutant apoA-I[Δ(185-243)]. ABCG1-mediated cholesterol efflux was not affected or moderately decreased by rHDL containing amino-terminal deletion mutants and several mid-region deletion or point apoA-I mutants, and was restored to 69-99% of control by double deletion mutants apoA-I[Δ(1-41)Δ(185-243)] and apoA-I[Δ(1-59)Δ(185-243)]. These findings suggest that the central helices alone of apoA-I associated to rHDL can promote ABCG1-mediated cholesterol efflux. Further analysis showed that rHDL containing the carboxyl-terminal deletion mutant apoA-I[Δ(185-243)] only slightly reduced (by 22%) the ABCG1-mediated efflux of 7-ketocholesterol, indicating that depending on the sterol type, structural changes in rHDL-associated apoA-I affect differently the ABCG1-mediated efflux of cholesterol and 7-ketocholesterol. Overall, our findings demonstrate that rHDL-associated apoA-I structural changes affect the capacity of rHDL to accept cellular cholesterol by an ABCG1-mediated process. The structure-function relationship seen here between rHDL-associated apoA-I mutants and ABCG1-mediated cholesterol efflux closely resembles that seen before in lipid-free apoA-I mutants and ABCA1-dependent cholesterol efflux, suggesting that both processes depend on the same structural determinants of apoA-I.

Pharmacodynamics of levofloxacin against characterized ciprofloxacin-resistant Streptococcus pneumoniae.

PubMed

Lister, Philip D

2008-09-01

In a previous study, levofloxacin 750 mg eradicated 4 ciprofloxacin-resistant isolates of Streptococcus pneumoniae from an in vitro pharmacodynamic model (IVPM). However, quinolone resistance-determining region (QRDR) mutations were not detected among those isolates. This study further evaluates levofloxacin 500 mg and 750 mg against S pneumoniae strains with characterized QRDR mutations. Six isolates with levofloxacin minimum inhibitory concentrations (MICs) of 2 to 4 microg/mL were selected for this study. Strains 5401, 5409, and 5437 contained only parC mutations. Three additional strains contained 2 mutations each: strain 5429 (parC and parE ), strain 5442 (parC and gyrA), and strain 5445 (parC and gyrB). Logarithmic-phase cultures (approximately 1 x 10(7) CFU/mL) were inoculated into the peripheral compartment of the IVPM and exposed to peak free-drug concentrations achieved with levofloxacin 500 mg and 750 mg (PO) and ciprofloxacin 750 mg (PO). Elimination pharmacokinetics were simulated and changes in viable counts were measured over 30 h. Ciprofloxacin exhibited very little antibacterial activity against the 6 strains, while levofloxacin 750 mg rapidly killed and eradicated the 3 parC mutant strains and the dual parC/parE mutant strains. Although levofloxacin 500 mg initially decreased viable counts by 4.5 to 6 logs, inoculum regrowth was observed between 12 and 24 h for the 6 strains. Regrowth was not due to the selection of mutant subpopulations. The pharmacodynamics of both levofloxacin doses were substantially diminished against the 2 strains with dual mutations in both parC and gyrA/B. The rapid eradication of single parC and dual parC/parE mutants with levofloxacin 750 mg demonstrates that this dose may slow the emergence of resistance due to these mutations. The decreased efficacy of both levofloxacin doses against the double parC and gyrA/B mutants highlights the importance of preventing the development and spread of double mutants.

Effects of HA and NA glycosylation pattern changes on the transmission of avian influenza A(H7N9) virus in guinea pigs.

PubMed

Park, Sehee; Lee, Ilseob; Kim, Jin Il; Bae, Joon-Yong; Yoo, Kirim; Kim, Juwon; Nam, Misun; Park, Miso; Yun, Soo-Hyeon; Cho, Woo In; Kim, Yeong-Su; Ko, Yun Young; Park, Man-Seong

2016-10-14

Avian influenza H7N9 virus has posed a concern of potential human-to-human transmission by resulting in seasonal virus-like human infection cases. To address the issue of sustained human infection with the H7N9 virus, here we investigated the effects of hemagglutinin (HA) and neuraminidase (NA) N-linked glycosylation (NLG) patterns on influenza virus transmission in a guinea pig model. Based on the NLG signatures identified in the HA and NA genetic sequences of H7N9 viruses, we generated NLG mutant viruses using either HA or NA gene of a H7N9 virus, A/Anhui/01/2013, by reverse genetics on the 2009 pandemic H1N1 virus backbone. For the H7 HA NLG mutant viruses, NLG pattern changes appeared to reduce viral transmissibility in guinea pigs. Intriguingly, however, the NLG changes in the N9 NA protein, such as a removal from residue 42 or 66 or an addition at residue 266, increased transmissibility of the mutant viruses by more than 33%, 50%, and 16%, respectively, compared with a parental N9 virus. Given the effects of HA-NA NLG changes with regard to viral transmission, we then generated the HA-NA NLG mutant viruses harboring the H7 HA of double NLG addition and the N9 NA of various NLG patterns. As seen in the HA NLG mutants above, the double NLG-added H7 HA decreased viral transmissibility. However, when the NA NLG changes occurred by a removal of residue 66 and an addition at 266 were additionally accompanied, the HA-NA NLG mutant virus recovered the transmissibility of its parental virus. These demonstrate the effects of specific HA-NA NLG changes on the H7N9 virus transmission by highlighting the importance of a HA-NA functional balance. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Implication of Proteins Containing Tetratricopeptide Repeats in Conditional Virulence Phenotypes of Legionella pneumophila

PubMed Central

Bandyopadhyay, Purnima; Sumer, Eren U.; Jayakumar, Deepak; Liu, Shuqing; Xiao, Huifang

2012-01-01

Legionella pneumophila, the causative agent of Legionnaires' disease, is a ubiquitous freshwater bacterium whose virulence phenotypes require a type IV secretion system (T4SS). L. pneumophila strain JR32 contains two virulence-associated T4SSs, the Dot/Icm and Lvh T4SSs. Defective entry and phagosome acidification phenotypes of dot/icm mutants are conditional and reversed by incubating broth-grown stationary-phase cultures in water (WS treatment) prior to infection, as a mimic of the aquatic environment of Legionella. Reversal of dot/icm virulence defects requires the Lvh T4SS and is associated with a >10-fold induction of LpnE, a tetratricopeptide repeat (TPR)-containing protein. In the current study, we demonstrated that defective entry and phagosome acidification phenotypes of mutants with changes in LpnE and EnhC, another TPR-containing protein, were similarly reversed by WS treatment. In contrast to dot/icm mutants for which the Lvh T4SS was required, reversal for the ΔlpnE or the ΔenhC mutant required that the other TPR-containing protein be present. The single and double ΔlpnE and ΔenhC mutants showed a hypersensitivity to sodium ion, a phenotype associated with dysfunction of the Dot/Icm T4SS. The ΔlpnE single and the ΔlpnE ΔenhC double mutant showed 3- to 9-fold increases in translocation of Dot/Icm T4SS substrates, LegS2/SplY and LepB. Taken together, these data identify TPR-containing proteins in a second mechanism by which the WS mimic of a Legionella environmental niche can reverse virulence defects of broth-grown cultures and implicate LpnE and EnhC directly or indirectly in translocation of Dot/Icm T4SS protein substrates. PMID:22563053

Understanding the loss-of-function in a triple missense mutant of DNA polymerase β found in prostate cancer.

PubMed

An, Changlong; Beard, William A; Chen, Desheng; Wilson, Samuel H; Makridakis, Nick M

2013-10-01

Human DNA polymerase (pol) β is essential for base excision repair. We previously reported a triple somatic mutant of pol β (p.P261L/T292A/I298T) found in an early onset prostate tumor. This mutation abolishes polymerase activity, and the wild-type allele was not present in the tumor, indicating a complete deficiency in pol β function. The effect on polymerase activity is unexpected because the point mutations that comprise the triple mutant are not part of the active site. Herein, we demonstrate the mechanism of this loss-of-function. In order to understand the effect of the individual point mutations we biochemically analyzed all single and double mutants that comprise the triple mutant. We found that the p.I298T mutation is responsible for a marked instability of the triple mutant protein at 37˚C. At room temperature the triple mutant's low efficiency is also due to a decrease in the apparent binding affinity for the dNTP substrate, which is due to the p.T292A mutation. Furthermore, the triple mutant displays lower fidelity for transversions in vitro, due to the p.T292A mutation. We conclude that distinct mutations of the triple pol β mutant are responsible for the loss of activity, lower fidelity, and instability observed in vitro.

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

A non-Mendelian MAPK-generated hereditary unit controlled by a second MAPK pathway in Podospora anserina.

PubMed

Lalucque, Hervé; Malagnac, Fabienne; Brun, Sylvain; Kicka, Sébastien; Silar, Philippe

2012-06-01

The Podospora anserina PaMpk1 MAP kinase (MAPK) signaling pathway can generate a cytoplasmic and infectious element resembling prions. When present in the cells, this C element causes the crippled growth (CG) cell degeneration. CG results from the inappropriate autocatalytic activation of the PaMpk1 MAPK pathway during growth, whereas this cascade normally signals stationary phase. Little is known about the control of such prion-like hereditary units involved in regulatory inheritance. Here, we show that another MAPK pathway, PaMpk2, is crucial at every stage of the fungus life cycle, in particular those controlled by PaMpk1 during stationary phase, which includes the generation of C. Inactivation of the third P. anserina MAPK pathway, PaMpk3, has no effect on the development of the fungus. Mutants of MAPK, MAPK kinase, and MAPK kinase kinase of the PaMpk2 pathway are unable to present CG. This inability likely relies upon an incorrect activation of PaMpk1, although this MAPK is normally phosphorylated in the mutants. In PaMpk2 null mutants, hyphae are abnormal and PaMpk1 is mislocalized. Correspondingly, stationary phase differentiations controlled by PaMpk1 are defective in the mutants of the PaMpk2 cascade. Constitutive activation of the PaMpk2 pathway mimics in many ways its inactivation, including an effect on PaMpk1 localization. Analysis of double and triple mutants inactivated for two or all three MAPK genes undercover new growth and differentiation phenotypes, suggesting overlapping roles. Our data underscore the complex regulation of a prion-like element in a model organism.

Gene editing rescue of a novel MPL mutant associated with congenital amegakaryocytic thrombocytopenia.

PubMed

Cleyrat, Cédric; Girard, Romain; Choi, Eun H; Jeziorski, Éric; Lavabre-Bertrand, Thierry; Hermouet, Sylvie; Carillo, Serge; Wilson, Bridget S

2017-09-26

Thrombopoietin (Tpo) and its receptor (Mpl) are the principal regulators of early and late thrombopoiesis and hematopoietic stem cell maintenance. Mutations in MPL can drastically impair its function and be a contributing factor in multiple hematologic malignancies, including congenital amegakaryocytic thrombocytopenia (CAMT). CAMT is characterized by severe thrombocytopenia at birth, which progresses to bone marrow failure and pancytopenia. Here we report unique familial cases of CAMT that presented with a previously unreported MPL mutation: T814C (W272R) in the background of the activating MPL G117T (K39N or Baltimore) mutation. Confocal microscopy, proliferation and surface biotinylation assays, co-immunoprecipitation, and western blotting analysis were used to elucidate the function and trafficking of Mpl mutants. Results showed that Mpl protein bearing the W272R mutation, alone or together with the K39N mutation, lacks detectable surface expression while being strongly colocalized with the endoplasmic reticulum (ER) marker calreticulin. Both WT and K39N-mutated Mpl were found to be signaling competent, but single or double mutants bearing W272R were unresponsive to Tpo. Function of the deficient Mpl receptor could be rescued by using 2 separate approaches: (1) GRASP55 overexpression, which partially restored Tpo-induced signaling of mutant Mpl by activating an autophagy-dependent secretory pathway and thus forcing ER-trapped immature receptors to traffic to the cell surface; and (2) CRISPR-Cas9 gene editing used to repair MPL T814C mutation in transfected cell lines and primary umbilical cord blood-derived CD34 + cells. We demonstrate proof of principle for rescue of mutant Mpl function by using gene editing of primary hematopoietic stem cells, which indicates direct therapeutic applications for CAMT patients.

Gene editing rescue of a novel MPL mutant associated with congenital amegakaryocytic thrombocytopenia

PubMed Central

Girard, Romain; Choi, Eun H.; Jeziorski, Éric; Lavabre-Bertrand, Thierry; Hermouet, Sylvie; Carillo, Serge; Wilson, Bridget S.

2017-01-01

Thrombopoietin (Tpo) and its receptor (Mpl) are the principal regulators of early and late thrombopoiesis and hematopoietic stem cell maintenance. Mutations in MPL can drastically impair its function and be a contributing factor in multiple hematologic malignancies, including congenital amegakaryocytic thrombocytopenia (CAMT). CAMT is characterized by severe thrombocytopenia at birth, which progresses to bone marrow failure and pancytopenia. Here we report unique familial cases of CAMT that presented with a previously unreported MPL mutation: T814C (W272R) in the background of the activating MPL G117T (K39N or Baltimore) mutation. Confocal microscopy, proliferation and surface biotinylation assays, co-immunoprecipitation, and western blotting analysis were used to elucidate the function and trafficking of Mpl mutants. Results showed that Mpl protein bearing the W272R mutation, alone or together with the K39N mutation, lacks detectable surface expression while being strongly colocalized with the endoplasmic reticulum (ER) marker calreticulin. Both WT and K39N-mutated Mpl were found to be signaling competent, but single or double mutants bearing W272R were unresponsive to Tpo. Function of the deficient Mpl receptor could be rescued by using 2 separate approaches: (1) GRASP55 overexpression, which partially restored Tpo-induced signaling of mutant Mpl by activating an autophagy-dependent secretory pathway and thus forcing ER-trapped immature receptors to traffic to the cell surface; and (2) CRISPR-Cas9 gene editing used to repair MPL T814C mutation in transfected cell lines and primary umbilical cord blood–derived CD34+ cells. We demonstrate proof of principle for rescue of mutant Mpl function by using gene editing of primary hematopoietic stem cells, which indicates direct therapeutic applications for CAMT patients. PMID:29296828