TY  - JOUR
TI  - Cytosine deaminase as a negative selectable marker for the microalgal chloroplast: a strategy for the isolation of nuclear mutations that affect chloroplast gene expression
EP  -  925
AV  - public
Y1  - 2014/12//
ID  - discovery1450681
N2  - 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.
KW  - 5-fluorocytosine
KW  -  5-fluorouracil
KW  -  Chlamydomonas reinhardtii
KW  -  chloroplast
KW  -  cytosine deaminase
KW  -  microalgae
KW  -  post-transcriptional regulation
KW  -  technical advance
SP  - 915 
VL  - 80
N1  - © 2014 The Authors. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
IS  - 5
SN  - 0960-7412
UR  - http://dx.doi.org/10.1111/tpj.12675
JF  - Plant Journal
A1  - Young, RE
A1  - Purton, S
ER  -