title: Cytosine deaminase as a negative selectable marker for the microalgal chloroplast: a strategy for the isolation of nuclear mutations that affect chloroplast gene expression.
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.
authors.affiliation: Algal Research Group, Institute of Structural and Molecular Biology, University College London, Gower Street, London, WC1E 6BT, UK.
authors.forename: Rosanna E B
authors.initials: RE
authors.lastname: Young
authors.forename: Saul
authors.initials: S
authors.lastname: Purton
mesh_terms: D020121:5' Untranslated Regions; D015415:Biomarkers / Q000032:analysis; D016825:Chlamydomonas reinhardtii / Q000187:drug effects / Q000235:genetics* / Q000528:radiation effects; D043525:Cytosine Deaminase / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics*; D005437:Flucytosine / Q000494:pharmacology; D018506:Gene Expression Regulation, Plant; D061125:Genes, Chloroplast; D016296:Mutagenesis; D009154:Mutation; D030821:Plants, Genetically Modified; D011401:Promoter Regions, Genetic; D014466:Ultraviolet Rays
chemical_list: D020121:5' Untranslated Regions; D015415:Biomarkers; D029968:Escherichia coli Proteins; D005437:Flucytosine; D043525:Cytosine Deaminase; C468827:codA protein, E coli
keywords: 5-fluorocytosine; 5-fluorouracil; Chlamydomonas reinhardtii; chloroplast; cytosine deaminase; microalgae; post-transcriptional regulation; technical advance