Use of the ptxD gene as a portable selectable marker for chloroplast transformation in Chlamydomonas reinhardtii

2019 ◽  
Vol 61 (6) ◽  
pp. 461-468 ◽  
Author(s):  
José M. Sandoval-Vargas ◽  
Luis A. Jiménez-Clemente ◽  
Karla S. Macedo-Osorio ◽  
María C. Oliver-Salvador ◽  
Luis C. Fernández-Linares ◽  
...  
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Selectable Marker ◽  
Chloroplast Transformation

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.

scholarly journals Blasticidin S Deaminase: A New Efficient Selectable Marker for Chlamydomonas reinhardtii

2020 ◽  
Vol 11 ◽  
Author(s):  
Félix de Carpentier ◽  
Jeanne Le Peillet ◽  
Nicolas D. Boisset ◽  
Pierre Crozet ◽  
Stéphane D. Lemaire ◽  
...  
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Selectable Marker ◽  
Blasticidin S

scholarly journals Manipulation of Gene Expression in the Chloroplast

2000 ◽  
Author(s):  
David Stern ◽  
Gadi Schuster
Keyword(s):  
Gene Expression ◽  
Chlamydomonas Reinhardtii ◽  
Protein Interactions ◽  
Molecular Genetic ◽  
Rna Stability ◽  
State Level ◽  
Chloroplast Transformation ◽  
Foreign Proteins ◽  
Ideal Model System ◽  
Chloroplast Mrna

The steady-state level of a given mRNA is determined by its rates of transcription and degradation. The stabilities of chloroplast mRNAs vary during plant development, in part regulating gene expression. Furthermore, the fitness of the organelle depends on its ability to destroy non-functional transcripts. In addition, there is a resurgent interest by the biotechnology community in chloroplast transformation due to the public concerns over pollen transmission of introduced traits or foreign proteins. Therefore, studies into basic gene expression mechanisms in the chloroplast will open the door to take advantage of these opportunities. This project was aimed at gaining mechanistic insights into mRNA processing and degradation in the chloroplast and to engineer transcripts of varying stability in Chlamydomonas reinhardtii cells. This research uncovered new and important information on chloroplast mRNA stability, processing, degradation and translation. In particular, the processing of the 3' untranslated regions of chloroplast mRNAs was shown to be important determinants in translation. The endonucleolytic site in the 3' untranslated region was characterized by site directed mutagensis. RNA polyadenylation has been characterized in the chloroplast of Chlamydomonas reinhardtii and chloroplast transformants carrying polyadenylated sequences were constructed and analyzed. Data obtained to date suggest that chloroplasts have gene regulatory mechanisms which are uniquely adapted to their post-endosymbiotic environment, including those that regulate RNA stability. An exciting point has been reached, because molecular genetic studies have defined critical RNA-protein interactions that participate in these processes. However, much remains to be learned about these multiple pathways, how they interact with each other, and how many nuclear genes are consecrated to overseeing them. Chlamydomonas is an ideal model system to extend our understanding of these areas, given its ease of manipulation and the existing knowledge base, some of which we have generated.

scholarly journals Robust Transgene Expression from Bicistronic mRNA in the Green Alga Chlamydomonas reinhardtii

2016 ◽  
Vol 6 (12) ◽  
pp. 4115-4125 ◽  
Author(s):  
Masayuki Onishi ◽  
John R Pringle
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Green Alga ◽  
Large Scale ◽  
Selectable Marker ◽  
Fluorescent Proteins ◽  
Model Organism ◽  
Scale Production ◽  
Entry Site ◽  
Biological Studies ◽  
Bicistronic Mrna

Abstract The unicellular green alga Chlamydomonas reinhardtii is a model organism that provides an opportunity to understand the evolution and functional biology of the lineage that includes the land plants, as well as aspects of the fundamental core biology conserved throughout the eukaryotic phylogeny. Although many tools are available to facilitate genetic, molecular biological, biochemical, and cell biological studies in Chlamydomonas, expression of unselected transgenes of interest (GOIs) has been challenging. In most methods used previously, the GOI and a selectable marker are expressed from two separate mRNAs, so that their concomitant expression is not guaranteed. In this study, we developed constructs that allow expression of an upstream GOI and downstream selectable marker from a single bicistronic mRNA. Although this approach in other systems has typically required a translation-enhancing element such as an internal ribosome entry site for the downstream marker, we found that a short stretch of unstructured junction sequence was sufficient to obtain adequate expression of the downstream gene, presumably through post-termination reinitiation. With this system, we obtained robust expression of both endogenous and heterologous GOIs, including fluorescent proteins and tagged fusion proteins, in the vast majority of transformants, thus eliminating the need for tedious secondary screening for GOI-expressing transformants. This improved efficiency should greatly facilitate a variety of genetic and cell-biological studies in Chlamydomonas and also enable new applications such as expression-based screens and large-scale production of foreign proteins.

scholarly journals The ARG9 Gene Encodes the Plastid-Resident N-Acetyl Ornithine Aminotransferase in the Green Alga Chlamydomonas reinhardtii

2009 ◽  
Vol 8 (9) ◽  
pp. 1460-1463 ◽  
Author(s):  
Claire Remacle ◽  
Sara Cline ◽  
Layla Boutaffala ◽  
Stéphane Gabilly ◽  
Véronique Larosa ◽  
...  
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Green Alga ◽  
Selectable Marker ◽  
Plastid Transformation ◽  
Ornithine Aminotransferase ◽  
Plastid Chromosome

ABSTRACT Here we report the characterization of the Chlamydomonas reinhardtii gene ARG9, encoding the plastid resident N-acetyl ornithine aminotransferase, which is involved in arginine synthesis. Integration of an engineered ARG9 cassette in the plastid chromosome of the nuclear arg9 mutant restores arginine prototrophy. This suggests that ARG9 could be used as a new selectable marker for plastid transformation.

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