scholarly journals Rapid and high efficiency transformation of Chlamydomonas reinhardtii by square-wave electroporation

2019 ◽  
Vol 39 (1) ◽  
Author(s):  
Liang Wang ◽  
Lijing Yang ◽  
Xin Wen ◽  
Zhuoya Chen ◽  
Qiaoying Liang ◽  
...  
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Insertional Mutagenesis ◽  
Molecular Mechanisms ◽  
High Efficiency ◽  
Model Organism ◽  
Dependent Manner ◽  
Exogenous Dna ◽  
Square Wave ◽  
Physiological Processes ◽  
Final Yield

Abstract Chlamydomonas reinhardtii, the unicellular green algae, is the model organism for studies in various physiological processes and for bioindustrial applications. To explore the molecular mechanisms underlying physiological processes or to establish engineered cell lines, the exogenous DNA needs to be integrated into the genome for the insertional mutagenesis or transgene expression. However, the amount of selected marker DNA is not seriously considered in the existing electroporation methods for mutants library construction. Here, we reported a rapid-and-high-efficiency transformation technique for cell-walled strains using square-wave electroporation system. The final yield with this electroporation method was 2–6 × 103 transformants per μg exogenous DNA for cell-walled strains in a strain-dependent manner. In general, this electroporation technique was the easy and applicable way to build a mutant library for screening phenotypes of interest.

scholarly journals Efficient Editing of the Nuclear APT Reporter Gene in Chlamydomonas reinhardtii via Expression of a CRISPR-Cas9 Module

2019 ◽  
Vol 20 (5) ◽  
pp. 1247 ◽  
Author(s):  
Daniel Guzmán-Zapata ◽  
José Sandoval-Vargas ◽  
Karla Macedo-Osorio ◽  
Edgar Salgado-Manjarrez ◽  
José Castrejón-Flores ◽  
...  
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Transient Expression ◽  
Mammalian Cells ◽  
High Efficiency ◽  
Model Organism ◽  
Nuclear Genome ◽  
Single Copy ◽  
Point Of View ◽  
Selection Marker ◽  
Gene Encoding

The clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9 (CRISPR/Cas9) technology is a versatile and useful tool to perform genome editing in different organisms ranging from bacteria and yeast to plants and mammalian cells. For a couple of years, it was believed that the system was inefficient and toxic in the alga Chlamydomonas reinhardtii. However, recently the system has been successfully implemented in this model organism, albeit relying mostly on the electroporation of ribonucleoproteins (RNPs) into cell wall deficient strains. This requires a constant source of RNPs and limits the application of the technology to strains that are not necessarily the most relevant from a biotechnological point of view. Here, we show that transient expression of the Streptococcus pyogenes Cas9 gene and sgRNAs, targeted to the single-copy nuclear apt9 gene, encoding an adenine phosphoribosyl transferase (APT), results in efficient disruption at the expected locus. Introduction of indels to the apt9 locus results in cell insensitivity to the otherwise toxic compound 2-fluoroadenine (2-FA). We have used agitation with glass beads and particle bombardment to introduce the plasmids carrying the coding sequences for Cas9 and the sgRNAs in a cell-walled strain of C. reinhardtii (CC-125). Using sgRNAs targeting exons 1 and 3 of apt9, we obtained disruption efficiencies of 3 and 30% on preselected 2-FA resistant colonies, respectively. Our results show that transient expression of Cas9 and a sgRNA can be used for editing of the nuclear genome inexpensively and at high efficiency. Targeting of the APT gene could potentially be used as a pre-selection marker for multiplexed editing or disruption of genes of interest.

scholarly journals Dopamine synergizes with caffeine to increase the heart rate of Daphnia

F1000Research ◽  
2018 ◽  
Vol 7 ◽  
pp. 254 ◽  
Author(s):  
Aman Kundu ◽  
Gyanesh Singh
Keyword(s):  
Central Nervous System ◽  
Heart Rate ◽  
Nervous System ◽  
Model Organism ◽  
Dependent Manner ◽  
Physiological Processes ◽  
Addictive Drugs ◽  
Dose Dependent

Dopamine is a key neurotransmitter, and is widely used as a central nervous system (CNS) agent.  Dopamine plays an important role in humans, including a major role in reward and motivation behaviour. Several addictive drugs are well known to increase neuronal dopamine activity. We selected Daphnia, an important model organism, to investigate the effect(s) of selected CNS agents on heart rate. Dopamine’s effects on Daphnia’s heart has not been previously reported. Caffeine is a well-known and widely consumed stimulant. Ethanol is well known for its effects on both neurological and physiological processes in mammals. We tested the effect of dopamine on the heart rate of Daphnia, and compared its effect with caffeine and ethanol alone and in combination. Both caffeine and dopamine were found to instantly increase the heart rate of Daphnia in a dose-dependent manner. Interestingly, caffeine synergized with dopamine to increase Daphnia’s heart rate. As ethanol decreased the heart rate of Daphnia and dopamine increased the heart rate of Daphnia, we wanted to test the effect of these molecules in combination. Indeed, Dopamine was able to restore the ethanol-induced decrease in the heart rate of Daphnia.  Effects of these CNS agents on Daphnia can possibly be correlated with similar effects in the case of mammals.

scholarly journals Low Dose Ionizing Radiation Strongly Stimulates Insertional Mutagenesis in a γH2AX Dependent Manner

2019 ◽  
Author(s):  
Alex N. Zelensky ◽  
Mascha Schoonakker ◽  
Inger Brandsma ◽  
Marcel Tijsterman ◽  
Dik C. van Gent ◽  
...  
Keyword(s):  
Ionizing Radiation ◽  
Insertional Mutagenesis ◽  
High Efficiency ◽  
Direct Detection ◽  
Radiation Risk ◽  
Dependent Manner ◽  
Sequence Specificity ◽  
Induced Mutations ◽  
Histone H2ax ◽  
Non Homologous End Joining

AbstractExtrachromosomal DNA can integrate into the genome with no sequence specificity producing an insertional mutation. This process, which is referred to as random integration (RI), requires a double stranded break (DSB) in the genome. Inducing DSBs by various means, including ionizing radiation, increases the frequency of integration. Here we report that non-lethal physiologically relevant doses of ionizing radiation (10-100 mGy), within the range produced by medical imaging equipment, stimulate RI of transfected and viral episomal DNA in human and mouse cells with an extremely high efficiency. Genetic analysis of stimulated RI (S-RI) revealed that it is distinct from the background RI, requires histone H2AX S139 phosphorylation (γH2AX) and is not reduced by DNA polymerase θ (Polq) inactivation. S-RI efficiency was unaffected by the main DSB repair pathway (homologous recombination and non-homologous end joining) disruptions, but double deficiency in MDC1 and 53BP1 phenocopies γH2AX inactivation. The robust responsiveness of S-RI to physiological amounts of DSBs has implications for radiation risk assessment and can be exploited for extremely sensitive, macroscopic and direct detection of DSB-induced mutations.

scholarly journals Identification of non-flagellar genes involved in swarm cell differentiation using a Bacillus thuringiensis mini-Tn10 mutant library

Microbiology ◽  
2009 ◽  
Vol 155 (3) ◽  
pp. 912-921 ◽  
Author(s):  
Sara Salvetti ◽  
Francesco Celandroni ◽  
Mara Ceragioli ◽  
Sonia Senesi ◽  
Emilia Ghelardi
Keyword(s):  
Bacillus Thuringiensis ◽  
Insertional Mutagenesis ◽  
Molecular Mechanisms ◽  
Amino Acid Permease ◽  
Active Growth ◽  
Transposon Insertion ◽  
Physiological Processes ◽  
Flagellar Assembly ◽  
Flagellar Proteins ◽  
And Function

Swarming is a social phenomenon that enables motile bacteria to move co-ordinately over solid surfaces. The molecular basis regulating this process is not completely known and may vary among species. Insertional mutagenesis of a swarming-proficient Bacillus thuringiensis strain was performed, by use of the transposon mini-Tn10, to identify novel genetic determinants of swarming that are dispensable for flagellation, swimming motility, chemotaxis and active growth. Among the 67 non-swarming mutants obtained, six were selected that showed no defect in flagellar assembly and function, chemotaxis or growth rate. Sequence analysis of DNA flanking the transposon insertion led to the identification of previously uncharacterized genes that are involved in the development of swarming colonies by B. thuringiensis and that are highly conserved in all members of the Bacillus cereus sensu lato group. These genes encode non-flagellar proteins with putative activity as sarcosine oxidase, catalase-2, amino acid permease, ATP-binding cassette transporter, dGTP triphosphohydrolase and acetyltransferase. Functional analysis of two of the isolated mutants demonstrated that swarming differentiation depends on the intracellular levels of the osmoprotectant glycine betaine and on the quantity of synthesized phenazine secondary metabolites. The finding that proteins involved in diverse physiological processes have a role in swarming motility underlines the complexity of the molecular mechanisms governing this behaviour in B. thuringiensis.

High-Efficiency Transformation of Chlamydomonas reinhardtii by Electroporation

Genetics ◽  
1998 ◽  
Vol 148 (4) ◽  
pp. 1821-1828 ◽  
Author(s):  
Kosuke Shimogawara ◽  
Shoko Fujiwara ◽  
Arthur Grossman ◽  
Hideaki Usuda
Keyword(s):  
Cell Wall ◽  
Chlamydomonas Reinhardtii ◽  
Standard Method ◽  
High Efficiency ◽  
Transformation Frequency ◽  
Argininosuccinate Lyase ◽  
Current Standard ◽  
Exogenous Dna ◽  
General Utility ◽  
Unicellular Green Alga

Abstract We have established a high-efficiency method for transforming the unicellular, green alga Chlamydomonas reinhardtii by electroporation. Electroporation of strains CC3395 and CC425, cell wall-less mutants devoid of argininosuccinate lyase (encoded by ARG7), in the presence of the plasmid pJD67 (which contains ARG7) was used to optimize conditions for the introduction of exogenous DNA. The conditions that were varied included osmolarity, temperature, concentration of exogenous DNA, voltage and capacitance. Following optimization, the maximum transformation frequency obtained was 2 × 105 transformants per μg of DNA; this frequency is two orders of magnitude higher than obtained with the current standard method using glass beads to introduce exogenous DNA. The electroporation procedure described in this article is of general utility, and makes it feasible to isolate genes by direct complementation of Chlamydomonas reinhardtii mutants.

scholarly journals Evaluation of the localization and biological effects of PAMAM G3 dendrimer-biotin/pyridoxal conjugate as HaCaT keratinocyte targeted nanocarrier

2019 ◽  
Author(s):  
Magdalena Szuster ◽  
Łukasz Stanisław Uram ◽  
Aleksandra Filipowicz-Rachwał ◽  
Stanisław Wołowiec ◽  
Elżbieta Wałajtys-Rode
Keyword(s):  
Molecular Mechanisms ◽  
Pyridoxal Phosphate ◽  
High Efficiency ◽  
Biological Effects ◽  
Normal Skin ◽  
Biological Properties ◽  
Tetrazolium Salt ◽  
Dependent Manner ◽  
Hacat Cells ◽  
Concentration Dependent Manner

Recognition of the molecular mechanisms of keratinocyte participation in normal skin homeostasis and in pathogenesis may lead to creation of more effective tools for topical application of cosmetics, cosmeceutics and drugs to a particular location within the skin for prevention and therapy of many skin disorders and diseases. For this purpose, the PAMAM G3 dendrimer with amide linkages of 9 biotin molecules and 10 molecules of pyridoxal phosphate (BC-PAMAM) was constructed, and its biological properties and cellular uptake and localization were investigated in the HaCaT keratinocytes. BC-PAMAM is nontoxic for HaCaT cells, as estimated by two assays (Neutral Red and tetrazolium salt reduction, XTT), and revealed low apoptosis induction at up to 50 µM concentration. Fluorescent labeled BC-PAMAM accumulates in HaCaT cells with high efficiency in a concentration–dependent manner. Its mitochondrial localization, estimated as Mander’s colocalization coefficient, is substantially lower than the native PAMAM, and that correlates with its cytotoxicity. The only undesirable, but significant inhibitory effect on cell mobility, evaluated by the wound healing test, was observed at 10 µM BC-PAMAM. The important anti-inflammatory action of BC-PAMAM was clearly documented by decreased production of total IL-1α, assayed with an ELISA test with unstimulated and stimulated by bacterial antigens (LPS and GroEL) HaCaT cells. Thus, it is expected that the biotin pyridoxal phosphate conjugated PAMAM may be considered as a potential carrier for safe delivery of vitamins and drugs into the epidermis.

scholarly journals Identification and molecular characterization of a novel Chlamydomonas reinhardtii mutant defective in chlorophyll biosynthesis

F1000Research ◽  
2013 ◽  
Vol 2 ◽  
pp. 138 ◽  
Author(s):  
Phillip B Grovenstein ◽  
Darryel A Wilson ◽  
Cameron G Lennox ◽  
Katherine P Smith ◽  
Alisha A Contractor ◽  
...  
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Insertional Mutagenesis ◽  
Chlorophyll Biosynthesis ◽  
Protoporphyrin Ix ◽  
Model Organism ◽  
Oxygenic Photosynthesis ◽  
Functional Roles ◽  
Protein Levels ◽  
In The Wild ◽  
Chl Biosynthesis

The green micro-alga Chlamydomonas reinhardtii is an elegant model organism to study all aspects of oxygenic photosynthesis. Chlorophyll (Chl) and heme are major tetrapyrroles that play an essential role in energy metabolism in photosynthetic organisms and are synthesized via a common branched tetrapyrrole biosynthetic pathway. One of the enzymes in the pathway is Mg chelatase (MgChel) which inserts Mg2+ into protoporphyrin IX (PPIX, proto) to form magnesium-protoporphyrin IX (MgPPIX, Mgproto), the first biosynthetic intermediate in the Chl branch. MgChel is a multimeric enzyme that consists of three subunits designated CHLD, CHLI and CHLH. Plants have two isozymes of CHLI (CHLI1 and CHLI2) which are 70%-81% identical in protein sequences. Although the functional role of CHLI1 is well characterized, that of CHLI2 is not. We have isolated a non-photosynthetic light sensitive mutant 5A7 by random DNA insertional mutagenesis that is devoid of any detectable Chl. PCR based analyses show that 5A7 is missing the CHLI1 gene and at least eight additional functionally uncharacterized genes. 5A7 has an intact CHLI2 gene. Complementation with a functional copy of the CHLI1 gene restored Chl biosynthesis, photo-autotrophic growth and light tolerance in 5A7. We have identified the first chli1 mutant of Chlamydomonas reinhardtii and in green algae. Our results show that in the wild type Chlamydomonas CHLI2 protein amount is lower than that of CHLI1 and the chli1 mutant has a drastic reduction in CHLI2 protein levels although it possesses the CHLI2 gene. Our chli1 mutant opens up new avenues to explore the functional roles of CHLI1 and CHLI2 in Chl biosynthesis and chloroplast to nucleus retrograde signaling in Chlamydomonas, which has never been studied before.

scholarly journals Identification and molecular characterization of a novel Chlamydomonas reinhardtii mutant defective in chlorophyll biosynthesis

F1000Research ◽  
2013 ◽  
Vol 2 ◽  
pp. 138
Author(s):  
Phillip B Grovenstein ◽  
Darryel A Wilson ◽  
Cameron G Lennox ◽  
Katherine P Smith ◽  
Alisha A Contractor ◽  
...  
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Insertional Mutagenesis ◽  
Chlorophyll Biosynthesis ◽  
Protoporphyrin Ix ◽  
Model Organism ◽  
Oxygenic Photosynthesis ◽  
Functional Roles ◽  
Protein Levels ◽  
In The Wild ◽  
Chl Biosynthesis

The green micro-alga Chlamydomonas reinhardtii is an elegant model organism to study all aspects of oxygenic photosynthesis. Chlorophyll (Chl) and heme are major tetrapyrroles that play an essential role in energy metabolism in photosynthetic organisms and are synthesized via a common branched tetrapyrrole biosynthetic pathway. One of the enzymes in the pathway is Mg chelatase (MgChel) which inserts Mg2+ into protoporphyrin IX (PPIX, proto) to form magnesium-protoporphyrin IX (MgPPIX, Mgproto), the first biosynthetic intermediate in the Chl branch. MgChel is a multimeric enzyme that consists of three subunits designated CHLD, CHLI and CHLH. Plants have two isozymes of CHLI (CHLI1 and CHLI2) which are 70%-81% identical in protein sequences. Although the functional role of CHLI1 is well characterized, that of CHLI2 is not. We have isolated a non-photosynthetic light sensitive mutant 5A7 by random DNA insertional mutagenesis that is devoid of any detectable Chl. PCR based analyses show that 5A7 is missing the CHLI1 gene and at least eight additional functionally uncharacterized genes. 5A7 has an intact CHLI2 gene. Complementation with a functional copy of the CHLI1 gene restored Chl biosynthesis, photo-autotrophic growth and light tolerance in 5A7. We have identified the first chli1 (chli1-1) mutant of Chlamydomonas reinhardtii and in green algae. Our results show that in the wild type Chlamydomonas CHLI2 protein amount is lower than that of CHLI1 and the chli1-1 mutant has a drastic reduction in CHLI2 protein levels although it possesses the CHLI2 gene. Our chli1-1 mutant opens up new avenues to explore the functional roles of CHLI1 and CHLI2 in Chl biosynthesis in Chlamydomonas, which has never been studied before.

The Anti-Atherogenic Properties of Sesamin are Mediated via Improved Macrophage Cholesterol Efflux Through PPARγ1-LXRα and MAPK Signaling

2014 ◽  
Vol 84 (1-2) ◽  
pp. 79-91 ◽  
Author(s):  
Amin F. Majdalawieh ◽  
Hyo-Sung Ro
Keyword(s):  
Cholesterol Efflux ◽  
Transcriptional Activity ◽  
Molecular Mechanisms ◽  
Foam Cell ◽  
Mapk Signaling ◽  
Luciferase Reporter ◽  
Foam Cell Formation ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Macrophage Cholesterol Efflux

Background: Foam cell formation resulting from disrupted macrophage cholesterol efflux, which is triggered by PPARγ1 and LXRα, is a hallmark of atherosclerosis. Sesamin and sesame oil exert anti-atherogenic effects in vivo. However, the exact molecular mechanisms underlying such effects are not fully understood. Aim: This study examines the potential effects of sesamin (0, 25, 50, 75, 100 μM) on PPARγ1 and LXRα expression and transcriptional activity as well as macrophage cholesterol efflux. Methods: PPARγ1 and LXRα expression and transcriptional activity are assessed by luciferase reporter assays. Macrophage cholesterol efflux is evaluated by ApoAI-specific cholesterol efflux assays. Results: The 50 μM, 75 μM, and 100 μM concentrations of sesamin up-regulated the expression of PPARγ1 (p< 0.001, p < 0.001, p < 0.001, respectively) and LXRα (p = 0.002, p < 0.001, p < 0.001, respectively) in a concentration-dependent manner. Moreover, 75 μM and 100 μM concentrations of sesamin led to 5.2-fold (p < 0.001) and 6.0-fold (p<0.001) increases in PPAR transcriptional activity and 3.9-fold (p< 0.001) and 4.2-fold (p < 0.001) increases in LXR transcriptional activity, respectively, in a concentration- and time-dependent manner via MAPK signaling. Consistently, 50 μM, 75 μM, and 100 μM concentrations of sesamin improved macrophage cholesterol efflux by 2.7-fold (p < 0.001), 4.2-fold (p < 0.001), and 4.2-fold (p < 0.001), respectively, via MAPK signaling. Conclusion: Our findings shed light on the molecular mechanism(s) underlying sesamin’s anti-atherogenic effects, which seem to be due, at least in part, to its ability to up-regulate PPARγ1 and LXRα expression and transcriptional activity, improving macrophage cholesterol efflux. We anticipate that sesamin may be used as a therapeutic agent for treating atherosclerosis.

The Mechanisms Behind the Biological Activity of Flavonoids

2019 ◽  
Vol 26 (39) ◽  
pp. 6976-6990 ◽  
Author(s):  
Ana María González-Paramás ◽  
Begoña Ayuda-Durán ◽  
Sofía Martínez ◽  
Susana González-Manzano ◽  
Celestino Santos-Buelga
Keyword(s):  
Gene Expression ◽  
Biological Activity ◽  
Phenolic Compounds ◽  
Intracellular Signaling ◽  
Molecular Mechanisms ◽  
Radical Scavenging ◽  
Model Organism ◽  
Stress Theory ◽  
Radical Scavenging Properties

: Flavonoids are phenolic compounds widely distributed in the human diet. Their intake has been associated with a decreased risk of different diseases such as cancer, immune dysfunction or coronary heart disease. However, the knowledge about the mechanisms behind their in vivo activity is limited and still under discussion. For years, their bioactivity was associated with the direct antioxidant and radical scavenging properties of phenolic compounds, but nowadays this assumption is unlikely to explain their putative health effects, or at least to be the only explanation for them. New hypotheses about possible mechanisms have been postulated, including the influence of the interaction of polyphenols and gut microbiota and also the possibility that flavonoids or their metabolites could modify gene expression or act as potential modulators of intracellular signaling cascades. This paper reviews all these topics, from the classical view as antioxidants in the context of the Oxidative Stress theory to the most recent tendencies related with the modulation of redox signaling pathways, modification of gene expression or interactions with the intestinal microbiota. The use of C. elegans as a model organism for the study of the molecular mechanisms involved in biological activity of flavonoids is also discussed.

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