Monitoring dynamic expression of nuclear genes in Chlamydomonas reinhardtii by using a synthetic luciferase reporter gene

Abstract

For monitoring the expression profile of selected nuclear genes in Chlamydomonas reinhardtii in response to altered environmental parameters or during cell cycle, in the past many RNA or protein samples had to be taken and analyzed by RNA hybridization or protein immunoblotting. Here we report the synthesis of a gene that codes for the luciferase of Renilla reniformis (RLuc) and is adapted to the nuclear codon usage of C. reinhardtii . This crluc gene was expressed alone or as a fusion to the zeocin resistance gene ble under control of different promoter variants. Luciferase activity was monitored in living cells, increased with the promoter strength and paralleled the amount of expressed protein. Under control of the Lhcb-1 promoter the Luc-activity in synchronized cultures was dependent on the dark-light cycle. Additionally, crluc was placed under control of the Chop-2 promoter and activity was measured under different light conditions. Chop-2 promoter activity was found to be most pronouced under low-light and dark conditions, further supporting that channelrhodopsin-2 is most active in dark-adapted cells. We conclude that crluc is a reliable tool for convenient monitoring of nuclear gene expression in C. reinhardtii .

The dual effect of a ferredoxin-hydrogenase fusion protein in vivo: successful divergence of the photosynthetic electron flux towards hydrogen production and elevated oxygen tolerance

“…We then created expression platforms harboring a C. reinhardtii codon-optimized Fd-linker ([Gly 4 -Ser] 3 )-HydA sequence, placed under the control of (a) the hybrid Hsp70A-RbcS2 promoter [16] in the commercial vector pChlamy_1 (GeneArt); (b) the psaD promoter [17] in the pSL18 vector or (c) the endogenous C. reinhardtii hydA1 promoter (a kind gift from Prof. Maria Ghirardi of NREL) (all are schematically illustrated in Additional file 1, panels a–c). These constructs were then separately transformed into the nuclei of hyd A 1,2 algal cells.…”

A mosquito-specific protein family includes candidate receptors for malaria sporozoite invasion of salivary glands

“…Wolbachia sequences analyzed were from genome databases at GenBank (symbionts of D. melanogaster , D. ananassae , and D. simulans ), from the Pathogen Sequencing Unit at the Sanger Institute (symbionts of Culex quinquefasciatus , http://www.sanger.ac.uk/Projects/W_pipientis/, and of Onchocerca volvulus , http://www.sanger.ac.uk/Projects/Wolbachia/), and from the Wolbachia Genome Project of New England Biolabs (symbiont of Brugia malayi , http://tools.neb.com/wolbachia/). Gene concordance with genomic codon usage was measured using the Graphical Codon Usage Analyzer tool (Fuhrmann et al ., 2004) available at http://gcua.schoedl.de .…”

Optimized expression of the Starmerella bombicola lactone esterase in Pichia pastoris through temperature adaptation, codon-optimization and co-expression with HAC1

“…pastoris with the SBLE coding sequence using the online Graphical Codon Usage Analyzer tool (http://gcua.schoedl.de) [29], we indeed noticed several rare codon usages in the original SBLE gene (Supplementary files: Figure 5). These differences were most striking for arginine (CGC, CGG), glycine (GGC), leucine (CTT) and valine (GTG).…”