scholarly journals Introduction of a leaky stop codon as molecular tool in Chlamydomonas reinhardtii

2020 ◽  
Author(s):  
Oliver D Caspari
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Photosynthetic Activity ◽  
Stop Codon ◽  
Nuclear Genome ◽  
Small Subunit ◽  
Fluorescent Reporter ◽  
Molecular Tool ◽  
Organellar Genomes ◽  
Single Construct ◽  
Bicistronic Expression

AbstractExpression of proteins in the chloroplast or mitochondria of the model green alga Chlamydomonas reinhardtii can be achieved by directly inserting transgenes into organellar genomes, or through nuclear expression and post-translational import. A number of tools have been developed in the literature for achieving high expression levels from the nuclear genome despite messy genomic integration and widespread silencing of transgenes. Here, recent advances in the field are combined and two systems of bicistronic expression, based on ribosome reinitiation or ribosomal skip induced by a viral 2A sequence, are compared side-by-side. Further, the small subunit of Rubisco (RBCS) was developed as a functional nuclear reporter for successful chloroplast import and restoration of photosynthesis: To be able to combine RBCS with a Venus fluorescent reporter without compromising photosynthetic activity, a leaky stop codon is introduced as a novel molecular tool that allows the simultaneous expression of functional and fluorescently tagged versions of the protein from a single construct.

cDNA cloning and gene expression of carbonic anhydrase in Chlamydomonas reinhardtii

1991 ◽  
Vol 69 (5) ◽  
pp. 1088-1096 ◽  
Author(s):  
Hideya Fukuzawa ◽  
Sarami Ishida ◽  
Shigetoh Miyachi
Keyword(s):  
Carbonic Anhydrase ◽  
Chlamydomonas Reinhardtii ◽  
Sequence Similarity ◽  
Large Subunit ◽  
Mrna Level ◽  
Nuclear Genome ◽  
Small Subunit ◽  
Nucleotide Sequence Analysis ◽  
Cdna Clones ◽  
Upstream Gene

cDNA and genes encoding periplasmic carbonic anhydrase (CA) polypeptides of Chlamydomonas reinhardtii have been isolated and characterized. Nucleotide sequence analysis of cDNA clones revealed that the large subunit (35 kDa or 36.5 kDa) and the small subunit (4 kDa) are cotranslated as a precursor polypeptide (41 626 Da) with a NH2-terminal hydrophobic signal peptide of 20 amino acids. The amino acid sequence of Chlamydomonas CA showed 20–22% identity with animal CA isozymes (CAI, CAII, CAIII, and CAVII). Three zinc-liganded histidine residues and those forming the hydrogen-bond network to zinc-bound solvent molecules were highly conserved. No significant sequence similarity was observed between Chlamydomonas CA and chloroplast CAs of spinach and pea. Two copies of structurally related CA genes (CAH1 and CAH2) were tandemly clustered in Chlamydomonas nuclear genome and regulated by external CO2 concentration in a reverse manner. The 5′ upstream gene CAH1 encodes the major periplasmic CA whose mRNA level is induced under low CO2 condition in light. Photosynthesis is absolutely required for the accumulation of the CAH1 mRNA. The 3′ downstream gene CAH2 is possibly a gene for another periplasmic CA isozyme, which is induced under high CO2 conditions. Light has an inhibitory effect on the accumulation of the CAH2 mRNA. Key words: photosynthesis, light regulation, zinc, CO2-concentrating mechanism, intracellular processing.

The CRY1 gene in Chlamydomonas reinhardtii: structure and use as a dominant selectable marker for nuclear transformation

1994 ◽  
Vol 14 (6) ◽  
pp. 4011-4019
Author(s):  
J A Nelson ◽  
P B Savereide ◽  
P A Lefebvre
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Selectable Marker ◽  
Marker Gene ◽  
Small Subunit ◽  
Direct Selection ◽  
Nuclear Transformation ◽  
Ribulose Bisphosphate Carboxylase ◽  
Bisphosphate Carboxylase ◽  
Dominant Selectable Marker ◽  
Cry1 Gene

We have cloned and sequenced the CRY1 gene, encoding ribosomal protein S14 in Chlamydomonas reinhardtii, and found that it is highly similar to S14/rp59 proteins from other organisms, including mammals, Drosophila melanogaster, and Saccharomyces cerevisiae. We isolated a mutant strain resistant to the eukaryotic translational inhibitors cryptopleurine and emetine in which the resistance was due to a missense mutation (CRY1-1) in the CRY1 gene; resistance was dominant in heterozygous stable diploids. Cotransformation experiments using the CRY1-1 gene and the gene for nitrate reductase (NIT1) produced a low level of resistance to cryptopleurine and emetine. Resistance levels were increased when the CRY1-1 gene was placed under the control of a constitutive promoter from the ribulose bisphosphate carboxylase/oxygenase small subunit 2 (RBCS2) gene. We also found that the 5' untranslated region of the CRY1 gene was required for expression of the CRY1-1 transgene. Direct selection of emetine-resistant transformants was possible when transformed cells were first induced to differentiate into gametes by nitrogen starvation and then allowed to dedifferentiate back to vegetative cells before emetine selection was applied. With this transformation protocol, the RBCS2/CRY1-1 dominant selectable marker gene is a powerful tool for many molecular genetic applications in C. reinhardtii.

The Circadian Clock of the Unicellular Eukaryotic Model Organism Chlamydomonas reinhardtii

2003 ◽  
Vol 384 (5) ◽  
pp. 689-695 ◽  
Author(s):  
M. Mittag ◽  
V. Wagner
Keyword(s):  
Circadian Clock ◽  
Chlamydomonas Reinhardtii ◽  
Rna Binding ◽  
Outer Space ◽  
Expression Patterns ◽  
Model Organism ◽  
Nuclear Genome ◽  
Circadian System ◽  
Transcriptional Level ◽  
Circadian Expression

Abstract The green unicellular alga Chlamydomonas reinhardtii, also called 'green yeast', emerged in the past years as a model organism for specific scientific questions such as chloroplast biogenesis and function, the composition of the flagella including its basal apparatus, or the mechanism of the circadian clock. Sequencing of its chloroplast and mitochondrial genomes have already been completed and a first draft of its nuclear genome has also been released recently. In C. reinhardtii several circadian rhythms are physiologically well characterized, and one of them has even been shown to operate in outer space. Circadian expression patterns of nuclear and plastid genes have been studied. The mode of regulation of these genes occurs at the transcriptional level, although there is also evidence for posttranscriptional control. A clock-controlled, phylogenetically conserved RNA-binding protein was characterized in this alga, which interacts with several mRNAs that all contain a common cis-acting motif. Its function within the circadian system is currently under investigation. This review summarizes the current state of the knowledge about the circadian system in C. reinhardtii and points out its potential for future studies.

scholarly journals Proteomic Characterization of the Small Subunit of Chlamydomonas reinhardtii Chloroplast Ribosome

2002 ◽  
Vol 14 (11) ◽  
pp. 2957-2974 ◽  
Author(s):  
Kenichi Yamaguchi ◽  
Susana Prieto ◽  
María Verónica Beligni ◽  
Paul A. Haynes ◽  
W. Hayes McDonald ◽  
...  
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Small Subunit

scholarly journals The economics of endosymbiotic gene transfer and the evolution of organellar genomes

2020 ◽  
Author(s):  
Steven Kelly
Keyword(s):  
Gene Transfer ◽  
Host Cell ◽  
Protein Import ◽  
Nuclear Genome ◽  
Selective Advantage ◽  
Endosymbiotic Gene Transfer ◽  
Organellar Genomes ◽  
Evolution Of Life ◽  
A Cell ◽  
Life On Earth

AbstractThe endosymbiosis of the bacterial progenitors of mitochondrion and the chloroplast are landmark events in the evolution of life on earth. While both organelles have retained substantial proteomic and biochemical complexity, this complexity is not reflected in the content of their genomes. Instead, the organellar genomes encode fewer than 5% of genes found in living relatives of their ancestors. While some of the 95% of missing organellar genes have been discarded, many have been transferred to the host nuclear genome through a process known as endosymbiotic gene transfer. Here we demonstrate that the energy liberated or consumed by a cell as a result of endosymbiotic gene transfer can be sufficient to provide a selectable advantage for retention or nuclear-transfer of organellar genes in eukaryotic cells. We further demonstrate that for realistic estimates of protein abundances, organellar protein import costs, host cell sizes, and cellular investment in organelles that it is energetically favourable to transfer the majority of organellar genes to the nuclear genome. Moreover, we show that the selective advantage of such transfers is sufficiently large to enable such events to rapidly reach fixation. Thus, endosymbiotic gene transfer can be advantageous in the absence of any additional benefit to the host cell, providing new insight into the processes that have shaped eukaryotic genome evolution.One sentence summaryThe high copy number of organellar genomes renders endosymbiotic gene transfer energetically favourable for the vast majority of organellar genes.

scholarly journals An Insight Into the Mechanism of Plant Organelle Genome Maintenance and Implications of Organelle Genome in Crop Improvement: An Update

2021 ◽  
Vol 9 ◽  
Author(s):  
Kalyan Mahapatra ◽  
Samrat Banerjee ◽  
Sayanti De ◽  
Mehali Mitra ◽  
Pinaki Roy ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Crop Improvement ◽  
Nuclear Genome ◽  
Genome Maintenance ◽  
Organelle Genome ◽  
Organellar Genomes ◽  
Damage Repair ◽  
Damage Response ◽  
Repair Pathways

Besides the nuclear genome, plants possess two small extra chromosomal genomes in mitochondria and chloroplast, respectively, which contribute a small fraction of the organelles’ proteome. Both mitochondrial and chloroplast DNA have originated endosymbiotically and most of their prokaryotic genes were either lost or transferred to the nuclear genome through endosymbiotic gene transfer during the course of evolution. Due to their immobile nature, plant nuclear and organellar genomes face continuous threat from diverse exogenous agents as well as some reactive by-products or intermediates released from various endogenous metabolic pathways. These factors eventually affect the overall plant growth and development and finally productivity. The detailed mechanism of DNA damage response and repair following accumulation of various forms of DNA lesions, including single and double-strand breaks (SSBs and DSBs) have been well documented for the nuclear genome and now it has been extended to the organelles also. Recently, it has been shown that both mitochondria and chloroplast possess a counterpart of most of the nuclear DNA damage repair pathways and share remarkable similarities with different damage repair proteins present in the nucleus. Among various repair pathways, homologous recombination (HR) is crucial for the repair as well as the evolution of organellar genomes. Along with the repair pathways, various other factors, such as the MSH1 and WHIRLY family proteins, WHY1, WHY2, and WHY3 are also known to be involved in maintaining low mutation rates and structural integrity of mitochondrial and chloroplast genome. SOG1, the central regulator in DNA damage response in plants, has also been found to mediate endoreduplication and cell-cycle progression through chloroplast to nucleus retrograde signaling in response to chloroplast genome instability. Various proteins associated with the maintenance of genome stability are targeted to both nuclear and organellar compartments, establishing communication between organelles as well as organelles and nucleus. Therefore, understanding the mechanism of DNA damage repair and inter compartmental crosstalk mechanism in various sub-cellular organelles following induction of DNA damage and identification of key components of such signaling cascades may eventually be translated into strategies for crop improvement under abiotic and genotoxic stress conditions. This review mainly highlights the current understanding as well as the importance of different aspects of organelle genome maintenance mechanisms in higher plants.

scholarly journals The economics of organellar gene loss and endosymbiotic gene transfer

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Steven Kelly
Keyword(s):  
Gene Transfer ◽  
Nuclear Genome ◽  
Energy Budgets ◽  
Net Energy ◽  
Endosymbiotic Gene Transfer ◽  
Organellar Genomes ◽  
Chloroplast Genomes ◽  
Evolution Of Life ◽  
The Difference ◽  
Life On Earth

Abstract Background The endosymbiosis of the bacterial progenitors of the mitochondrion and the chloroplast are landmark events in the evolution of life on Earth. While both organelles have retained substantial proteomic and biochemical complexity, this complexity is not reflected in the content of their genomes. Instead, the organellar genomes encode fewer than 5% of the genes found in living relatives of their ancestors. While many of the 95% of missing organellar genes have been discarded, others have been transferred to the host nuclear genome through a process known as endosymbiotic gene transfer. Results Here, we demonstrate that the difference in the per-cell copy number of the organellar and nuclear genomes presents an energetic incentive to the cell to either delete organellar genes or transfer them to the nuclear genome. We show that, for the majority of transferred organellar genes, the energy saved by nuclear transfer exceeds the costs incurred from importing the encoded protein into the organelle where it can provide its function. Finally, we show that the net energy saved by endosymbiotic gene transfer can constitute an appreciable proportion of total cellular energy budgets and is therefore sufficient to impart a selectable advantage to the cell. Conclusion Thus, reduced cellular cost and improved energy efficiency likely played a role in the reductive evolution of mitochondrial and chloroplast genomes and the transfer of organellar genes to the nuclear genome.

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