scholarly journals Molecular characterization of Chlamydomonas reinhardtii telomeres and telomerase mutants

2019 ◽  
Author(s):  
Stephan Eberhard ◽  
Sona Valuchova ◽  
Julie Ravat ◽  
Pascale Jolivet ◽  
Sandrine Bujaldon ◽  
...  
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Replicative Senescence ◽  
Model Organism ◽  
Growth Conditions ◽  
Telomeric Repeats ◽  
Gene Encoding ◽  
Unicellular Green Alga ◽  
Chromosome Integrity ◽  
Linear Chromosomes

ABSTRACTTelomeres are repeated sequences found at the end of the linear chromosomes of most eukaryotes and are required for chromosome integrity. They shorten with each cell division because of the end-replication problem. Expression of the reverse transcriptase telomerase allows for extension of telomeric repeats to counteract telomere shortening. Although Chlamydomonas reinhardtii, a photosynthetic unicellular green alga, is widely used as a model organism in photosynthesis and flagella research, and for biotechnological applications, the biology of its telomeres has not been investigated in depth. Here, we show that the C. reinhardtii (TTTTAGGG)n telomeric repeats are mostly non-degenerate and that the telomeres form a protective structure, ending with a 3′ overhang. While telomere size and length distributions are stable under various standard growth conditions, they vary substantially between 12 genetically close reference strains. Finally, we identify CrTERT, the gene encoding the catalytic subunit of telomerase and show that mutants of this gene display an “ever shortening telomere” phenotype and eventually enter replicative senescence, demonstrating that telomerase is required for long-term maintenance of telomeres in C. reinhardtii.

scholarly journals Molecular characterization of Chlamydomonas reinhardtii telomeres and telomerase mutants

2019 ◽  
Vol 2 (3) ◽  
pp. e201900315
Author(s):  
Stephan Eberhard ◽  
Sona Valuchova ◽  
Julie Ravat ◽  
Jaroslav Fulneček ◽  
Pascale Jolivet ◽  
...  
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Replicative Senescence ◽  
Model Organism ◽  
Growth Conditions ◽  
Telomeric Repeats ◽  
Gene Encoding ◽  
Unicellular Green Alga ◽  
Chromosome Integrity ◽  
Linear Chromosomes

Telomeres are repeated sequences found at the end of the linear chromosomes of most eukaryotes and are required for chromosome integrity. Expression of the reverse-transcriptase telomerase allows for extension of telomeric repeats to counteract natural telomere shortening. Although Chlamydomonas reinhardtii, a photosynthetic unicellular green alga, is widely used as a model organism in photosynthesis and flagella research, and for biotechnological applications, the biology of its telomeres has not been investigated in depth. Here, we show that the C. reinhardtii (TTTTAGGG)n telomeric repeats are mostly nondegenerate and that the telomeres form a protective structure, with a subset ending with a 3′ overhang and another subset presenting a blunt end. Although telomere size and length distributions are stable under various standard growth conditions, they vary substantially between 12 genetically close reference strains. Finally, we identify CrTERT, the gene encoding the catalytic subunit of telomerase and show that telomeres shorten progressively in mutants of this gene. Telomerase mutants eventually enter replicative senescence, demonstrating that telomerase is required for long-term maintenance of telomeres in C. reinhardtii.

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 Characterization of Chlamydomonas reinhardtii Mutants That Exhibit Strong Positive Phototaxis

Plants ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1483
Author(s):  
Jun Morishita ◽  
Ryutaro Tokutsu ◽  
Jun Minagawa ◽  
Toru Hisabori ◽  
Ken-ichi Wakabayashi
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Regulatory Mechanism ◽  
Model Organism ◽  
Negative Phototaxis ◽  
Light Conditions ◽  
Wild Type ◽  
Positive Phototaxis ◽  
Unicellular Green Alga ◽  
Cellular Factors

The most motile phototrophic organisms exhibit photo-induced behavioral responses (photobehavior) to inhabit better light conditions for photosynthesis. The unicellular green alga Chlamydomonas reinhardtii is an excellent model organism to study photobehavior. Several years ago, we found that C. reinhardtii cells reverse their phototactic signs (i.e., positive and negative phototaxis) depending on the amount of reactive oxygen species (ROS) accumulated in the cell. However, its molecular mechanism is unclear. In this study, we isolated seven mutants showing positive phototaxis, even after the induction of negative phototaxis (ap1~7: always positive) to understand the ROS-dependent regulatory mechanism for the phototactic sign. We found no common feature in the mutants regarding their growth, high-light tolerance, and photosynthetic phenotypes. Interestingly, five of them grew faster than the wild type. These data suggest that the ROS-dependent regulation of the phototactic sign is not a single pathway and is affected by various cellular factors. Additionally, the isolation and analyses of mutants with defects in phototactic-sign regulation may provide clues for their application to the efficient cultivation of algae.

scholarly journals Anaerobic Expression of the Ferredoxin-Encoding FDX5 Gene of Chlamydomonas reinhardtii Is Regulated by the Crr1 Transcription Factor

2010 ◽  
Vol 9 (11) ◽  
pp. 1747-1754 ◽  
Author(s):  
Camilla Lambertz ◽  
Anja Hemschemeier ◽  
Thomas Happe
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Binding Sites ◽  
Anaerobic Metabolism ◽  
Response Regulator ◽  
Binding Assays ◽  
Anaerobic Conditions ◽  
Gene Encoding ◽  
Content Type ◽  
Unicellular Green Alga

ABSTRACT The unicellular green alga Chlamydomonas reinhardtii has a complex anaerobic metabolism and reacts to hypoxic or anaerobic conditions with the induced expression of many genes. One gene which is upregulated particularly strongly is the FDX5 gene, encoding one of at least six ferredoxin isoforms in C. reinhardtii. Fdx5 is a typical plant-type 2Fe2S protein that is located in the chloroplast. The FDX5 promoter region contains three GTAC motifs, which are known to be the binding sites for copper response regulator 1 (Crr1) and other SQUAMOSA promoter binding proteins (SBPs). This study shows that two of these GTAC sites are essential to confer oxygen and also copper responsiveness to a reporter gene. The SBP domain of Crr1 is able to bind to both of these GTAC sites in in vitro binding assays. Moreover, in a Crr1-deficient C. reinhardtii strain, FDX5 is not expressed. These results clearly indicate that Crr1 is involved in the transcriptional regulation of the FDX5 gene in the absence of oxygen or copper.

scholarly journals To Divide or Not to Divide? How Deuterium Affects Growth and Division of Chlamydomonas reinhardtii

Biomolecules ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 861
Author(s):  
Veronika Kselíková ◽  
Vilém Zachleder ◽  
Kateřina Bišová
Keyword(s):  
Cell Cycle ◽  
Chlamydomonas Reinhardtii ◽  
Cell Cycle Progression ◽  
Model Organism ◽  
Cycle Progression ◽  
Synchronous Cultures ◽  
Multiple Fission ◽  
First Choice ◽  
High Doses

Extensive in vivo replacement of hydrogen by deuterium, a stable isotope of hydrogen, induces a distinct stress response, reduces cell growth and impairs cell division in various organisms. Microalgae, including Chlamydomonas reinhardtii, a well-established model organism in cell cycle studies, are no exception. Chlamydomonas reinhardtii, a green unicellular alga of the Chlorophyceae class, divides by multiple fission, grows autotrophically and can be synchronized by alternating light/dark regimes; this makes it a model of first choice to discriminate the effect of deuterium on growth and/or division. Here, we investigate the effects of high doses of deuterium on cell cycle progression in C. reinhardtii. Synchronous cultures of C. reinhardtii were cultivated in growth medium containing 70 or 90% D2O. We characterize specific deuterium-induced shifts in attainment of commitment points during growth and/or division of C. reinhardtii, contradicting the role of the “sizer” in regulating the cell cycle. Consequently, impaired cell cycle progression in deuterated cultures causes (over)accumulation of starch and lipids, suggesting a promising potential for microalgae to produce deuterated organic compounds.

scholarly journals Endogenous Fluctuations of DNA Topology in the Chloroplast of Chlamydomonas reinhardtii

1998 ◽  
Vol 18 (12) ◽  
pp. 7235-7242 ◽  
Author(s):  
Maria L. Salvador ◽  
Uwe Klein ◽  
Lawrence Bogorad
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Chloroplast Genome ◽  
Continuous Light ◽  
Dna Supercoiling ◽  
Dna Topology ◽  
Chloroplast Gene ◽  
Chloroplast Gene Expression ◽  
Endogenous Fluctuations ◽  
Unicellular Green Alga ◽  
In Cells

ABSTRACT DNA supercoiling in the chloroplast of the unicellular green algaChlamydomonas reinhardtii was found to change with a diurnal rhythm in cells growing in alternating 12-h dark–12-h light periods. Highest and lowest DNA superhelicities occurred at the beginning and towards the end of the 12-h light periods, respectively. The fluctuations in DNA supercoiling occurred concurrently and in the same direction in two separate parts of the chloroplast genome, one containing the genes psaB, rbcL, andatpA and the other containing the atpB gene. Fluctuations were not confined to transcribed DNA regions, indicating simultaneous changes in DNA conformation all over the chloroplast genome. Because the diurnal fluctuations persisted in cells kept in continuous light, DNA supercoiling is judged to be under endogenous control. The endogenous fluctuations in chloroplast DNA topology correlated tightly with the endogenous fluctuations of overall chloroplast gene transcription and with those of the pool sizes of most chloroplast transcripts analyzed. This result suggests that DNA superhelical changes have a role in the regulation of chloroplast gene expression in Chlamydomonas.

scholarly journals Photosymbiose — neuartige Anwendungen in der Biomedizin

BIOspektrum ◽  
2021 ◽  
Vol 27 (2) ◽  
pp. 202-204
Author(s):  
Myra N. Chávez ◽  
Benedikt Fuchs ◽  
Jörg Nickelsen
Keyword(s):  
Tissue Engineering ◽  
Chlamydomonas Reinhardtii ◽  
Green Alga ◽  
Recombinant Proteins ◽  
Biomedical Devices ◽  
Unicellular Green Alga ◽  
Surgical Sutures ◽  
Novel Strategy ◽  
Clinical Problems

AbstractWe have recently proposed a novel strategy named photosynthetic tissue engineering to overcome clinical problems due to hypoxia. The idea is based on transgenic photoautotrophic microorganisms that produce oxygen and at the same time secrete functional recombinant proteins into tissues. In particular, the unicellular green alga Chlamydomonas reinhardtii has successfully been used to boost the regenerative potential of several biomedical devices, such as dermal scaffolds and surgical sutures.

Cell-wall abnormalities of a Chlamydomonas reinhardtii mutant strain under suboptimal growth conditions

Planta ◽  
1991 ◽  
Vol 183 (1) ◽  
Author(s):  
J�rgen Voigt ◽  
Dieter Mergenhagen ◽  
Irmhild Wachholz ◽  
Elsbeth Manshard ◽  
Marianne Mix
Keyword(s):  
Cell Wall ◽  
Chlamydomonas Reinhardtii ◽  
Mutant Strain ◽  
Growth Conditions
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