ArticleThe Plant journal : for cell and molecular biology2014
Cytosine deaminase as a negative selectable marker for the microalgal chloroplast: a strategy for the isolation of nuclear mutations that affect chloroplast gene expression.
Article in The Plant journal : for cell and molecular biology, 2014. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 21 papers.
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Who cites it
21 citing papers in PubMed, 52 citations in OpenAlex.
- Recombinant bovine chymosin expression in microalgae Chlamydomonas reinhardtii chloroplast: A step towards algal biomanufacturing of dairy enzymes.World journal of microbiology & biotechnology · 2026Article
- Article
- An RNA thermometer in the chloroplast genome of Chlamydomonas facilitates temperature-controlled gene expression.Nucleic acids research · 2023Article
- Current Nuclear Engineering Strategies in the Green MicroalgaLife (Basel, Switzerland) · 2023Review
- A PETase enzyme synthesised in the chloroplast of the microalga Chlamydomonas reinhardtii is active against post-consumer plastics.Scientific reports · 2023Article
- Cloning and expression of an anti-cancerous cytokine: human IL-29 gene in Chlamydomonas reinhardtii.AMB Express · 2023Article
- Over-expression of a cyanobacterial gene for 1-deoxy-d-xylulose-5-phosphate synthase in the chloroplast ofJournal of King Saud University. Science · 2022Article
- Development of a Biotechnology Platform for the Fast-Growing CyanobacteriumBiomolecules · 2022Article
- The Chloroplast ofInternational journal of molecular sciences · 2021Review
- Multigenic engineering of the chloroplast genome in the green algaMicrobiology (Reading, England) · 2020Article
- The phosphite oxidoreductase gene, ptxD as a bio-contained chloroplast marker and crop-protection tool for algal biotechnology using Chlamydomonas.Applied microbiology and biotechnology · 2020Article
- Article
- Use of the ptxD gene as a portable selectable marker for chloroplast transformation in Chlamydomonas reinhardtii.Molecular biotechnology · 2019Article
- CITRIC: cold-inducible translational readthrough in the chloroplast of Chlamydomonas reinhardtii using a novel temperature-sensitive transfer RNA.Microbial cell factories · 2018Article
- Review
- Synthesis of bacteriophage lytic proteins against Streptococcus pneumoniae in the chloroplast of Chlamydomonas reinhardtii.Plant biotechnology journal · 2017Article
- New tools for chloroplast genetic engineering allow the synthesis of human growth hormone in the green alga Chlamydomonas reinhardtii.Applied microbiology and biotechnology · 2016Article
- Codon reassignment to facilitate genetic engineering and biocontainment in the chloroplast of Chlamydomonas reinhardtii.Plant biotechnology journal · 2016Article
- Improving recombinant protein production in the Chlamydomonas reinhardtii chloroplast using vivid Verde Fluorescent Protein as a reporter.Biotechnology journal · 2015Article
- Establishing Chlamydomonas reinhardtii as an industrial biotechnology host.The Plant journal : for cell and molecular biology · 2015Review
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Authors and funding
2 authors at 1 institution in 1 country.
Funding
Abstract
Negative selectable markers are useful tools for forward-genetic screens aimed at identifying trans-acting factors that are required for expression of specific genes. Transgenic lines harbouring the marker fused to a gene element, such as a promoter, may be mutagenized to isolate loss-of-function mutants able to survive under selection. Such a strategy allows the molecular dissection of factors that are essential for expression of the gene. Expression of individual chloroplast genes in plants and algae typically requires one or more nuclear-encoded factors that act at the post-transcriptional level, often through interaction with the 5' UTR of the mRNA. To study such nuclear control further, we have developed the Escherichia coli cytosine deaminase gene codA as a conditional negative selectable marker for use in the model green alga Chlamydomonas reinhardtii. We show that a codon-optimized variant of codA with three amino acid substitutions confers sensitivity to 5-fluorocytosine (5-FC) when expressed in the chloroplast under the control of endogenous promoter/5' UTR elements from the photosynthetic genes psaA or petA. UV mutagenesis of the psaA transgenic line allowed recovery of 5-FC-resistant, photosynthetically deficient lines harbouring mutations in the nuclear gene for the factor TAA1 that is required for psaA translation. Similarly, the petA line was used to isolate mutants of the petA mRNA stability factor MCA1 and the translation factor TCA1. The codA marker may be used to identify critical residues in known nuclear factors and to aid the discovery of additional factors required for expression of chloroplast genes.
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