DOCSLIB.ORG

Gene from Suaeda Monoica Enhances Photosynthesis, Carbon Assimilation, and Abiotic Stress Tolerance in a C3 Plant Under Elevated CO2 Conditions

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Gene from Suaeda Monoica Enhances Photosynthesis, Carbon Assimilation, and Abiotic Stress Tolerance in a C3 Plant Under Elevated CO2 Conditions fpls-11-00345 April 19, 2020 Time: 12:19 # 1 ORIGINAL RESEARCH published: 21 April 2020 doi: 10.3389/fpls.2020.00345 The Pyruvate-Phosphate Dikinase (C4-SmPPDK) Gene From Suaeda monoica Enhances Photosynthesis, Carbon Assimilation, and Abiotic Stress Tolerance in a C3 Plant Under Elevated CO2 Conditions Sonam Yadav, Mangal Singh Rathore and Avinash Mishra* Division of Applied Phycology and Biotechnology, CSIR-Central Salt and Marine Chemicals Research Institute, Bhavnagar, India A pyruvate-phosphate dikinase (C4-PPDK) gene was cloned from Suaeda monoica, which had a single-cell C4 photosynthesis pathway without Kranz anatomy and was functionally validated in a C3 model plant under different abiotic stress conditions in an Edited by: ambient and elevated CO2 environment. Overexpression of SmPPDK promoted growth Marouane Baslam, Niigata University, Japan of C3 transgenic plants, enhancing their photosynthesis (CO2 assimilation) by lowering Reviewed by: photorespiration under stress conditions. Transgenic plants also showed an improved Petronia Carillo, physiological status, with higher relative water content (RWC), membrane integrity, University of Campania Luigi Vanvitelli, Italy concentration of glycine betaine, total soluble sugars, free amino acids, polyphenols Parvaiz Ahmad, and antioxidant activity, and lower electrolyte leakage, lipid peroxidation, free radical Sri Pratap College Srinagar, India accumulation, and generation of reactive oxygen species (ROS), compared to control *Correspondence: plants. Moreover, SmPPDK transgenic plants exhibited earlier flowering and higher dry Avinash Mishra [email protected]; biomass compared to controls. These results suggested that the C4-PPDK gene was [email protected]; appropriate for improvement of carbon assimilation, and it also played an important role [email protected] in adaption to salinity and severe drought-induced stress. More intriguingly, an elevated Specialty section: CO2 environment alleviated the adverse effects of abiotic stress, particularly caused by This article was submitted to drought through coordination of osmoprotectants and antioxidant defense systems. The Plant Abiotic Stress, a section of the journal molecular, physiological, metabolic, and biochemical indicators ameliorated the overall Frontiers in Plant Science performance of model C3 plants overexpressing the C4-PPDK gene in an elevated Received: 09 December 2019 CO2 environment, by lowering photorespiration metabolic processes, however, further Accepted: 09 March 2020 studies are needed to confirm its precise role in C plants as protection against future Published: 21 April 2020 3 climate change. Citation: Yadav S, Rathore MS and Keywords: abiotic stress, C4 photosynthesis, carbon sequestration, halophyte, salinity, transgenic Mishra A (2020) The Pyruvate-Phosphate Dikinase (C4-SmPPDK) Gene From Suaeda INTRODUCTION monoica Enhances Photosynthesis, Carbon Assimilation, and Abiotic Global climate changes such as increasing temperature, atmospheric CO2 concentration and Stress Tolerance in a C3 Plant Under Elevated CO2 Conditions. salinization, and altered precipitation are among the primary constraints on crop improvement, Front. Plant Sci. 11:345. including biomass production and yield (Dhir, 2018). In addition, the increasing demands in doi: 10.3389/fpls.2020.00345 feeding a fast growing population also impose pressure to enhance the productivity and quality Frontiers in Plant Science| www.frontiersin.org 1 April 2020| Volume 11| Article 345 fpls-11-00345 April 19, 2020 Time: 12:19 # 2 Yadav et al. C4-SmPPDK Enhances CO2-Assimilation in C3-Plants of current crops under limited resources. Global climate changes end, effectively following the same differentiation as the Kranz result in desertification and salinization, with >6% of global type of anatomy (Dengler and Nelson, 1999; Sheen, 1999). As land area and >30% of irrigated farmland being salt affected such, engineering of a single-cell C4 organization into C3 crops (FAO, 2011). Global CO2 concentrations have increased from might provide an alternative approach to achieving Kranz type −1 a preindustrial level of 285 mmol mol to the current level C4 carbon assimilation without the need to establish a dual- of 384 mmol mol−1, and they have been predicted to reach cell system. 936 mmol mol−1 by the year 2100, with an increase in PPDK (EC 2.7.9.1) is nuclear encoded, abundantly present in global atmospheric temperature of about 6.4◦C(IPCC, 2014). the chloroplasts of mesophyll cells, an important rate-limiting These stresses will likely affect the productivity of plants, enzyme of the C4 pathway (Edwards et al., 1985; Hatch, influencing the sequestration and assimilation of atmospheric 1987), and is regulated by light-dependent gene expression CO2 through photosynthesis. (Sheen and Bogorad, 1987). It catalyzes the interconversion Various studies on elevated CO2 concentrations have shown of pyruvate to phosphoenolpyruvate (PEP), the acceptor of a close relationship between improvements to photosynthesis atmospheric CO2 in C4 plants, and C4 photosynthesis rate and increases in yield and total biomass of plants (Kellner is correlated (r = 0.96) with the activity of PPDK (Edwards et al., 2019; Pastore et al., 2019). Therefore, this provides et al., 1985). Its structural and functional properties have been an attractive approach to enhancing yield by increasing studied in the bacterium Clostridium symbiosum (Chastain photosynthesis of a particular crop species (Zhang et al., 2014; et al., 2011). The phylogenetic distribution of PPDK is Yadav et al., 2018). Among the three categories of photosynthesis confined to selected groups of bacteria, archaea, protozoa, lower in carbon-assimilating plants, productivity and biomass of fungi, and green plants (Liapounova et al., 2006; Slamovits C4 plants are not limited by atmospheric CO2; therefore, and Keeling, 2006). C4 PPDK is highly homologous to C3 they are considered the most efficient CO2 sequestrators. C4 PPDK (Agarie et al., 1997), with the regions and residues photosynthesis inhibits the oxygenation reaction and acts as involved in catalysis also being highly conserved among a CO2 pump, increasing the intracellular CO2/O2 ratio at bacteria (Fißlthaler et al., 1995). PPDK can also contribute to the active site of ribulose bisphosphate carboxylase/oxygenase the conversion of NADH to NADPH, which is an essential (Rubisco) (Hatch, 1987; Sage, 2004). It has been well documented coenzyme of antioxidant enzymes, and is also involved in that C3 plants lose about 50% of total available CO2 to nitrogen assimilation and synthesis of fatty acids and osmotically photorespiration compared to C4 plants. In addition, C4 active compounds (Doubnerová and Ryšlavá, 2011). PPDK photosynthesis has an advantage over that of C3 plants, exhibiting is critically involved during early seedling growth and seed a higher assimilation efficiency and yield of biomass under development in both C3 and C4 plants (Chastain et al., different abiotic stress conditions, including drought, high 2006; Yu et al., 2016; Chang et al., 2017). In Arabidopsis temperature, and high light intensity, by improving water thaliana, PPDK is overexpressed during leaf senescence by and nitrogen use efficiency (Hatch, 1987; Sage and Pearcy, significantly accelerating nitrogen remobilization from leaves 2000). Enzymes involved in the C4 assimilation pathway (Taylor et al., 2010; Wang et al., 2019). PPDK also plays a could also play a significant role in tolerance to abiotic and key role in host stress defense mechanisms, as it is induced biotic stress (Ku et al., 2000; Gu et al., 2013). Enhancing by various abiotic stresses, such as drought, salinity, heavy photosynthesis efficiency in C3 plants using an enzyme from metals, submergence, low oxygen and cold (Doubnerová and the C4 pathway could provide an alternative approach for Ryšlavá, 2011; Hýsková et al., 2014; Wang et al., 2018), and improving productivity, biomass, and yield (Yadav and Mishra, biotic stress, including viral infection (Spoustová et al., 2015). In 2019). Genera Suaeda and Bienertia have broken the dual- nonphotosynthetic parts of the plant, PPDK plays a significant cell paradigm that Kranz anatomy is essential for C4 carbon role in the maintenance of pH and rejuvenation of citric assimilation (Freitag and Stichler, 2002; Voznesenskaya et al., acid intermediates, which are involved in the synthesis of 2002; Akhani et al., 2005). amino acids (Hýsková et al., 2014). PPDK homologs have Suaeda species belong to the family Amaranthaceae and been reported from bacteria and unicellular parasitic protists, have a unique approach to C4 carbon assimilation within a such as Giardia lamblia, Trichomonas vaginalis, and Entamoeba single chlorenchyma cell (Voznesenskaya et al., 2002, 2004), histolytica, however, they have not been found in insects or in which chloroplasts are dimorphic. A single-cell process is vertebrates. As such, PPDK is a potential target for antiparasitic the most common in the nicotinamide adenine dinucleotide and antimicrobial drugs, as well as C4-specific herbicides phosphate-dependent malic enzyme (NADP-ME) subtype of C4 (Minges and Groth, 2017). photosynthesis species, where the chloroplasts of mesophyll cells The advantages of a C4-based physiology are observed during have well-developed grana, while the chloroplasts of bundle stress triggered by high temperatures, drought, salinity, and sheath lack grana. However, single-cell C4 photosynthesis is not elevated CO2 levels.
Load more

Recommended publications
  • Introduction of Human Telomerase Reverse Transcriptase to Normal Human Fibroblasts Enhances DNA Repair Capacity
    Vol. 10, 2551–2560, April 1, 2004 Clinical Cancer Research 2551 Introduction of Human Telomerase Reverse Transcriptase to Normal Human Fibroblasts Enhances DNA Repair Capacity Ki-Hyuk Shin,1 Mo K. Kang,1 Erica Dicterow,1 INTRODUCTION Ayako Kameta,1 Marcel A. Baluda,1 and Telomerase, which consists of the catalytic protein subunit, No-Hee Park1,2 human telomerase reverse transcriptase (hTERT), the RNA component of telomerase (hTR), and several associated pro- 1School of Dentistry and 2Jonsson Comprehensive Cancer Center, University of California, Los Angeles, California teins, has been primarily associated with maintaining the integ- rity of cellular DNA telomeres in normal cells (1, 2). Telomer- ase activity is correlated with the expression of hTERT, but not ABSTRACT with that of hTR (3, 4). Purpose: From numerous reports on proteins involved The involvement of DNA repair proteins in telomere main- in DNA repair and telomere maintenance that physically tenance has been well documented (5–8). In eukaryotic cells, associate with human telomerase reverse transcriptase nonhomologous end-joining requires a DNA ligase and the (hTERT), we inferred that hTERT/telomerase might play a DNA-activated protein kinase, which is recruited to the DNA role in DNA repair. We investigated this possibility in nor- ends by the DNA-binding protein Ku. Ku binds to hTERT mal human oral fibroblasts (NHOF) with and without ec- without the need for telomeric DNA or hTR (9), binds the topic expression of hTERT/telomerase. telomere repeat-binding proteins TRF1 (10) and TRF2 (11), and Experimental Design: To study the effect of hTERT/ is thought to regulate the access of telomerase to telomere DNA telomerase on DNA repair, we examined the mutation fre- ends (12, 13).
    [Show full text]
  • Insights Into Genomic Structure and Evolutionary Processes of Coastal Suaeda Species in East Asia Using Cpdna, Ndna, and Genome
    www.nature.com/scientificreports OPEN Insights into genomic structure and evolutionary processes of coastal Suaeda species in East Asia using cpDNA, nDNA, and genome‑wide SNPs Jong‑Soo Park, Dong‑Pil Jin & Byoung‑Hee Choi* Species in the genus Suaeda have few diagnostic characters and substantial morphological plasticity. Hence, regional foras do not provide clear taxonomic information for Suaeda spp. in East Asia. In order to assess the taxonomy of four species in the genus Suaeda (S. australis, S. maritima, S. japonica, and S. heteroptera), cpDNA (rpl32‑trnL and trnH‑psbA), nDNA (ITS), and MIG‑seq analyses were carried out. Genome‑wide SNP results indicated three lineages: (1) S. australis in Korea and S. maritima in Japan, (2) S. maritima in Korea and S. heteroptera in China, and (3) S. japionica. In phylogenetic trees and genotype analyses, cpDNA and nDNA results showed discrepancies, while S. japonica and S. maritima in Korea, and S. heteroptera in China shared the same haplotype and ribotype. We suggest that the shared haplotype may be due to chloroplast capture. Based on our results, we assume that S. japonica was formed by homoploid hybrid speciation between the two lineages. Te genus Suaeda Forssk. ex J.F. Gmel., in the Amaranthaceae family 1, comprises approximately 80–100 spe- cies of halophytic herbs or shrubs distributed worldwide in semiarid and arid regions, and along the seashores, including the Pacifc 2–5. An integrated molecular and morphological phylogenetic study of the subfamily Suae- doideae Ulbr. suggests that the genus Suaeda is comprised of two subgenera (Brezia (Moq.) Freitag & Schütze, and Suaeda) and eight sections (Brezia (Moq.) Volk.
    [Show full text]
  • Purification and Characterization of the Reverse Transcriptase Expressed In
    Volume 270, number 1,2, 76-80 FEBS 08845 September 1990 Purification and characterization of the RNase H domain of HIV-l reverse transcriptase expressed in recombinant Escherichia coli S. Patricia Becerra’, G. Marius Clore2, Angela M. Gronenbornz, Anders R. Karlstr6m3, Stephen J. Stahl‘+, Samuel H. Wilson’ and Paul T. Wingfield 1Laboratory of Biochemistry, NCI, ZLaboratory of Chemical Physics, NIDDK, 3Laboratory of Biochemistry, NHLB and 4Protein Expression Laboratory, National Institutes of Health, Bethesda, MD 20892, USA Received 2 July 1990 The ribonuclease H (RNase H) domain of human immuno-deficiency virus (HIV-l) reverse transcriptase has been produced with the aim of provid- ing sufficient amounts of protein for biophysical studies. A plasmid vector is described which directs high level expression of the RNase H domain under the control of the I PL promoter. The domain corresponds to residues 427-560 of the 66 kDa reverse transcriptase. The protein was expressed in Escherichia cob’ and was purified using ion-exchange and size exclusion chromatography. The purified protein appears to be in a native-like homogeneous conformational state as determined by iH-NMR spectroscopy and circular dichroism measurements. HIV-protease treatment of the RNase H domain resulted in cleavage between Phe-440 and Tyr-441. HIV-l reverse transcriptase; HIV-l RNase H; HIV protease; Protein expression; Protein purification; Protein conformation 1. INTRODUCTION as a separate 15 kDa protein. The purified protein ap- pears suitable for structural studies by both NMR spec- Ribonuclease H (RNase H) activity resides in the C- troscopy and X-ray analysis, thereby providing an addi- terminal region of retroviral polymerases as shown by tional target in the search for therapeutic agents against deletion experiments [ 11, linker insertion [2] and point the spread of HIV infection.
    [Show full text]
  • Rapidxfire Thermostable Reverse Transcriptase Application Note
    Application note RapiDxFire Thermostable Reverse Transcriptase Introduction RNA target, which improves cDNA yield and A reverse transcriptase (RT) is a DNA subsequent detection sensitivity from difficult polymerase enzyme that synthesises cDNA RNA targets. Thermostability also is an from an RNA template. RTs are widely indicator of general enzyme stability for storage, used to study gene expression in cells or automation applications, and lyophilisation. tissues, in next-generation sequencing Processivity, which refers to the number of (NGS) applications, and in conjunction with nucleotides incorporated into cDNA during quantitative PCR (qPCR) or isothermal a single enzyme binding event, can affect amplification methods to detect and identify cDNA length, RT efficiency, and RT reaction RNAs that are of clinical or functional time. Native RT enzymes also have RNase H significance. endonuclease activity that will cleave the RNA from a DNA-RNA duplex and limit cDNA length. RT enzymes may differ in key characteristics Some RT variants also have been engineered that affect their performance in different to reduce RNase H activity to allow longer applications. Their thermostability allows cDNA products. cDNA synthesis to be performed at higher temperatures (above 50 °C), enabling them Commercially available RTs used in molecular to melt areas of secondary structure in the biology are generally derived from Moloney Application note RapiDxFire Thermostable Reverse Transcriptase murine leukaemia virus (MMLV) or avian Skeletal Muscle Total RNA (Thermo Fisher myeloblastosis virus (AMV), which have optimal Scientific Cat No. AM7982), RNA, MS2 (Roche activity at 37-42 °C. Cloned AMV RT and Cat No. 10165948001), Zika virus ATCC® engineered variants of AMV RT and MMLV VR-1843™ (ATCC Cat No.
    [Show full text]
  • Telomere and Telomerase in Oncology
    Cell Research (2002); 12(1):1-7 http://www.cell-research.com REVIEW Telomere and telomerase in oncology JIAO MU*, LI XIN WEI International Joint Cancer Institute, Second Military Medical University, Shanghai 200433, China ABSTRACT Shortening of the telomeric DNA at the chromosome ends is presumed to limit the lifespan of human cells and elicit a signal for the onset of cellular senescence. To continually proliferate across the senescent checkpoint, cells must restore and preserve telomere length. This can be achieved by telomerase, which has the reverse transcriptase activity. Telomerase activity is negative in human normal somatic cells but can be detected in most tumor cells. The enzyme is proposed to be an essential factor in cell immortalization and cancer progression. In this review we discuss the structure and function of telomere and telomerase and their roles in cell immortalization and oncogenesis. Simultaneously the experimental studies of telomerase assays for cancer detection and diagnosis are reviewed. Finally, we discuss the potential use of inhibitors of telomerase in anti-cancer therapy. Key words: Telomere, telomerase, cancer, telomerase assay, inhibitor. Telomere and cell replicative senescence base pairs of the end of telomeric DNA with each Telomeres, which are located at the end of round of DNA replication. Hence, the continual chromosome, are crucial to protect chromosome cycles of cell growth and division bring on progress- against degeneration, rearrangment and end to end ing telomere shortening[4]. Now it is clear that te- fusion[1]. Human telomeres are tandemly repeated lomere shortening is responsible for inducing the units of the hexanucleotide TTAGGG. The estimated senescent phenotype that results from repeated cell length of telomeric DNA varies from 2 to 20 kilo division, but the mechanism how a short telomere base pairs, depending on factors such as tissue type induces the senescence is still unknown.
    [Show full text]
  • RNA-Dependent RNA Polymerase Speed and Fidelity Are Not the Only Determinants of the Mechanism Or Efficiency of Recombination
    G C A T T A C G G C A T genes Article RNA-Dependent RNA Polymerase Speed and Fidelity are not the Only Determinants of the Mechanism or Efficiency of Recombination Hyejeong Kim y, Victor D. Ellis III, Andrew Woodman, Yan Zhao, Jamie J. Arnold y and , Craig E. Cameron y * Department of Biochemistry and Molecular Biology, The Pennsylvania State University, 201 Althouse Laboratory, University Park, PA 16802, USA; [email protected] (H.K.); [email protected] (V.D.E.); [email protected] (A.W.); [email protected] (Y.Z.); [email protected] (J.J.A.) * Correspondence: [email protected]; Tel.: +1-919-966-9699 Present address: Department of Microbiology and Immunology, School of Medicine, University of North y Carolina at Chapel Hill, 125 Mason Farm Rd., Chapel Hill, NC 27599-7290, USA. Received: 15 October 2019; Accepted: 21 November 2019; Published: 25 November 2019 Abstract: Using the RNA-dependent RNA polymerase (RdRp) from poliovirus (PV) as our model system, we have shown that Lys-359 in motif-D functions as a general acid in the mechanism of nucleotidyl transfer. A K359H (KH) RdRp derivative is slow and faithful relative to wild-type enzyme. In the context of the KH virus, RdRp-coding sequence evolves, selecting for the following substitutions: I331F (IF, motif-C) and P356S (PS, motif-D). We have evaluated IF-KH, PS-KH, and IF-PS-KH viruses and enzymes. The speed and fidelity of each double mutant are equivalent. Each exhibits a unique recombination phenotype, with IF-KH being competent for copy-choice recombination and PS-KH being competent for forced-copy-choice recombination.
    [Show full text]
  • Localization of Glucokinase Gene Expression in the Rat Brain Ronald M
    Localization of Glucokinase Gene Expression in the Rat Brain Ronald M. Lynch, Linda S. Tompkins, Heddwen L. Brooks, Ambrose A. Dunn-Meynell, and Barry E. Levin The brain contains a subpopulation of glucosensing neu- rons that alter their firing rate in response to elevated glucose concentrations. In pancreatic ␤-cells, gluco- ammalian feeding behavior and general energy kinase (GK), the rate-limiting enzyme in glycolysis, medi- homeostasis appear to be regulated by circu- ates glucose-induced insulin release by regulating intra- lating levels of nutrients (glucose) and pep- cellular ATP production. A similar role for GK is pro- tides (e.g., leptin, insulin). Sensors to detect lev- posed to underlie neuronal glucosensing. Via in situ M els of these factors have been found to reside within specific hybridization, GK mRNA was localized to hypothalamic areas that are thought to contain relatively large popu- nuclei of the hypothalamus (1–8), where central regulation of lations of glucosensing neurons (the arcuate, ventrome- energy homeostasis is believed to be coordinated. For exam- dial, dorsomedial, and paraventricular nuclei and the lat- ple, large changes in blood glucose are correlated with cen- eral area). GK also was found in brain areas without trally mediated responses such as thermogenesis through known glucosensing neurons (the lateral habenula, the activation of the sympathetic nervous system. These changes bed nucleus stria terminalis, the inferior olive, the are monitored by the brain (9–11), and such responses are retrochiasmatic and medial preoptic areas, and the thal- altered in obesity-prone animals (11–13). Moreover, lesions amic posterior paraventricular, interpeduncular, oculo- of the ventromedial hypothalamus (VMH) prevent the hypo- motor, and anterior olfactory nuclei).
    [Show full text]
  • Prevalence of Rota Virus Detection by Reverse Transcriptase
    IOSR Journal of Pharmacy and Biological Sciences (IOSR-JPBS) e-ISSN:2278-3008, p-ISSN:2319-7676. Volume 12, Issue 1 Ver. III (Jan. - Feb.2017), PP 94-99 www.iosrjournals.org Prevalence of Rota Virus Detection by Reverse Transcriptase- Polymerase Chain Reaction in Stool from Children Fewer Than Five Age With Acute Diarrhea in Diyala Province in Iraq Ansam Dawod Salman Lecturer in Diyala University, Collage of science, Department of Biology Abstract: The present study was conducted for the period from 1/6/2016 to 20/1/2017 in Baquba city. The study aimed to detection of rotavirus in stool specimens of children fewer than five age and also explore the effects of certain demographic factors on the detection rates by revers transcriptase- polymerase chain reaction. The study included 49 patients with acute diarrhea, 32 were male and 17 were female. The age range was two months to 5 years. Demographic information on the patients regarding age, sex, residence, type of feeding and source of drinking water were collected from their parents. Stool specimens were collected from each patients and. Detection of rotavirus in stool specimens was done by conventional reverse transcriptase polymerase chain reaction (RT-PCR). The results of present study showed that the overall infection rate by rotavirus among patients with acute diarrhea by RT-PCR tests was 93.88%. The highest infection rate was recorded among those >10-≤15 months of age. None of the results showed significantly difference between female and male, PCR (88% vs 96.87%). Likewise, there was insignificantly difference between urban and rural residence, PCR (95.65% vs 92.30%).
    [Show full text]
  • Semi-Quantitative Detection of RNA-Dependent RNA Polymerase Activity of Human Telomerase Reverse Transcriptase Protein
    Journal of Visualized Experiments www.jove.com Video Article Semi-quantitative Detection of RNA-dependent RNA Polymerase Activity of Human Telomerase Reverse Transcriptase Protein Yoshiko Maida*1, Mami Yasukawa*1, Marco Ghilotti1, Yoshinari Ando1, Kenkichi Masutomi1 1 Division of Cancer Stem Cell, National Cancer Center Research Institute * These authors contributed equally Correspondence to: Kenkichi Masutomi at [email protected] URL: https://www.jove.com/video/57021 DOI: doi:10.3791/57021 Keywords: Genetics, Issue 136, TERT, RNA-dependent RNA polymerase, double-stranded RNA, telomerase, immunoprecipitation, radioisotope Date Published: 6/12/2018 Citation: Maida, Y., Yasukawa, M., Ghilotti, M., Ando, Y., Masutomi, K. Semi-quantitative Detection of RNA-dependent RNA Polymerase Activity of Human Telomerase Reverse Transcriptase Protein. J. Vis. Exp. (136), e57021, doi:10.3791/57021 (2018). Abstract Human telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase, and it elongates telomere through RNA-dependent DNA polymerase activity. Although TERT is named as a reverse transcriptase, structural and phylogenetic analyses of TERT demonstrate that TERT is a member of right-handed polymerases, and relates to viral RNA-dependent RNA polymerases (RdRPs) as well as viral reverse transcriptase. We firstly identified RdRP activity of human TERT that generates complementary RNA stand to a template non-coding RNA and contributes to RNA silencing in cancer cells. To analyze this non-canonical enzymatic activity, we developed RdRP assay with recombinant TERT in 2009, thereafter established in vitro RdRP assay for endogenous TERT. In this manuscript, we describe the latter method. Briefly, TERT immune complexes are isolated from cells, and incubated with template RNA and rNTPs including radioactive rNTP for RdRP reaction.
    [Show full text]
  • Introduction and Expression of PEP Synthase in Synechocystis Sp. PCC 6803
    Introduction and Expression of PEP Synthase in Synechocystis sp. PCC 6803 Paweł Piątek Degree project in applied biotechnology, Master of Science (2 years), 2013 Examensarbete i tillämpad bioteknik 45 hp till masterexamen, 2013 Biology Education Centre and the Department of Photochemistry and Molecular Science, Ångstrom, Uppsala University, Uppsala University Academic Supervisor: Prof. Peter Lindblad Laboratory Supervisors: Dr. Anja Nenninger and Elias Englund Contents Abstract 2 Introduction 3 Establishing synthetic pathways in cyanobacteria 3 Synthetic Pathway Design 5 The MOG pathway 6 Phosphoenolpyruvate Synthase 8 Pyruvate Phosphate Dikinase 8 Experimental Aims 10 Materials and Methods 10 Results 13 Construction of the pEERM[PEPS] plasmid 13 PEPS light regulation 13 Correct PEPS recombination 14 Examining overexpression of PEPS 15 Discussion 16 Plasmid Selection 16 Antibiotic Resistance Cassette 17 Light regulation of PEPS 17 RNA Extraction & cDNA Synthesis 18 Overexpression of PEPS and further experimentation 19 Conclusion 19 Appendix 20 Section 1: Colorimetric Determination of PEPS activity 20 Introduction and Theory 20 Materials and Methods 21 Results and Discussion 21 Section 2: Materials 23 Section 3: Primers 24 Acknowledgments 25 References 26 Websites/Databases 26 Literature 26 Abstract In recent years photosynthetic cyanobacteria have become the focus in high-value compound production through genetically engineered means. Through manipulation of both native and exogenous DNA, it has been shown that it is possible to construct synthetic pathways within cyanobacteria. However some of these constructed pathways are faced with natural bottle-necks found in the organism’s metabolic pathways. Therefore to produce useful compounds, the demands for the organism to fix carbon from the atmosphere greatly increase.
    [Show full text]
  • Comparative Chloroplast Genome Analysis of Single-Cell C4 Bienertia Sinuspersici with Other Amaranthaceae Genomes
    Journal of Plant Sciences 2018; 6(4): 134-143 http://www.sciencepublishinggroup.com/j/jps doi: 10.11648/j.jps.20180604.13 ISSN: 2331-0723 (Print); ISSN: 2331-0731 (Online) Comparative Chloroplast Genome Analysis of Single-Cell C4 Bienertia Sinuspersici with Other Amaranthaceae Genomes Lorrenne Caburatan, Jin Gyu Kim, Joonho Park * Department of Fine Chemistry, Seoul National University of Science and Technology, Seoul, South Korea Email address: *Corresponding author To cite this article: Lorrenne Caburatan, Jin Gyu Kim, Joonho Park. Comparative Chloroplast Genome Analysis of Single-Cell C4 Bienertia Sinuspersici with Other Amaranthaceae Genomes. Journal of Plant Sciences . Vol. 6, No. 4, 2018, pp. 134-143. doi: 10.11648/j.jps.20180604.13 Received : August 15, 2018; Accepted : August 31, 2018; Published : September 26, 2018 Abstract: Bienertia sinuspersici is a single-cell C4 (SCC 4) plant species whose photosynthetic mechanisms occur in two cytoplasmic compartments containing central and peripheral chloroplasts. The efficiency of the C4 photosynthetic pathway to suppress photorespiration and enhance carbon gain has led to a growing interest in its research. A comparative analysis of B. sinuspersici chloroplast genome with other genomes of Amaranthaceae was conducted. Results from a 70% cut off sequence identity showed that B. sinuspersici is closely related to Beta vulgaris with slight variations in the arrangement of few genes such ycf1 and ycf15; and, the absence of psbB in Beta vulgaris. B. sinuspersici has the largest 153, 472 bp while Spinacea oleracea has the largest protein-coding sequence 6,754 bp larger than B. sinuspersici. The GC contents of each of the species ranges from 36.3 to 36.9% with B.
    [Show full text]
  • Reverse Transcriptase and Cdna Synthesis - Kunitada Shimotohno
    GENETICS AND MOLECULAR BIOLOGY - Reverse Transcriptase and cDNA Synthesis - Kunitada Shimotohno REVERSE TRANSCRIPTASE AND cDNA SYNTHESIS Kunitada Shimotohno Kyoto University, Japan Keywords: retrovirus, reverse transcriptase, Rous sarcoma virus, chicken, murine, transposon, mobile genetic element, human immunodeficiency virus, human t-cell leukemia virus, copia, ty-element, cDNA, Actinomycin-D, ribonuclease H, tRNA, primer, template, inhibitor, azidothimidine, LINE, genome, hepatitis B virus, cauliflower mosaic virus, thermus thermophilus, DNA polymerase, polymerase chain reaction, avian myeloblastosis virus, RNase Contents 1. Introduction 2. Discovery of Reverse Transcriptase 3. Characteristic Features of Retroviral Reverse Transcriptases 3.1. Template and Primer Requirements for Reverse Transcriptase 3.2. Substrates for the Enzyme 3.3. Other Factors Required for Reverse Transcription 3.3. Natural Primers for cDNA Synthesis of Retrovirus Genome 3.5. Inhibitors of Reverse Transcriptases 4. Reverse Transcriptase Activity in Other Organisms 4.1. Reverse Transcriptase Activity in Retroposons 4.2. Reverse Transcripatase Activity in Pararetroviruses 4.2.1. Reverse Transcriptase Activity in Hepatitis B Virus (HBV) 4.2.2. Reverse Transcriptase Activity in Cauliflower Mosaic Virus (CaMV) 4.3. Reverse Transcriptase Activity and Telomerase 4.4. Reverse Transcriptase Activity in Thermus Thermophilus DNA Polymerase 5. Conserved Amino Acid Residues in Putative Reverse Transcriptase 6. Structure of Retroviral Reverse Transcriptases 7. cDNA Synthesis
    [Show full text]