Cytosine deaminase as a negative selectable marker for the microalgal chloroplast: a strategy for the isolation of nuclear mutations that affect chloroplast gene expression. (25th October 2014)

• Record Type: Journal Article
• Authors: Young, Rosanna E. B.; Purton, Saul
• Is Part Of: Plant journal. Volume 80:Number 5(2014:Dec.)
• Page Start: 915
• Page End: 925
• Publication Date: 2014-10-25
• Subjects: Plant molecular biology -- Periodicals; Plant cells and tissues -- Periodicals; Botany -- Periodicals; 580
• DOI: 10.1111/tpj.12675
• Languages: English
• ISSNs: 0960-7412

Abstract

<abstract abstract-type="main" id="tpj12675-abs-0001"> <title>Summary</title> <p>Negative selectable markers are useful tools for forward‐genetic screens aimed at identifying <italic>trans</italic>‐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 <italic>Escherichia coli</italic> cytosine deaminase gene <italic>codA</italic> as a conditional negative selectable marker for use in the model green alga <italic>Chlamydomonas reinhardtii</italic>. We show that a codon‐optimized variant of <italic>codA</italic> 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 <italic>psaA</italic> or <italic>petA</italic>. UV mutagenesis of the <italic>psaA</italic> 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 <italic>psaA</italic> translation. Similarly, the <italic>petA</italic> line was used to isolate mutants of the <italic>petA</italic> mRNA stability factor MCA1 and the translation factor TCA1. The <italic>codA</italic> 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.</p> </abstract>