An Optimized, Chemically Regulated Gene Expression System for Chlamydomonas

BACKGROUND: Chlamydomonas reinhardtii is a model system for algal and cell biology and is used for biotechnological applications, such as molecular farming or biological hydrogen production. The Chlamydomonas metal-responsive CYC6 promoter is repressed by copper and induced by nickel ions. However, induction by nickel is weak in some strains, poorly reversible by chelating agents like EDTA, and causes, at high concentrations, toxicity side effects on Chlamydomonas growth. Removal of these bottlenecks will encourage the wide use of this promoter as a chemically regulated gene expression system. METHODOLOGY: Using a codon-optimized Renilla luciferase as a reporter gene, we explored several strategies to improve the strength and reversibility of CYC6 promoter induction. Use of the first intron of the RBCS2 gene or of a modified TAP medium increases the strength of CYC6 induction up to 20-fold. In the modified medium, induction is also obtained after addition of specific copper chelators, like TETA. At low concentrations (up to 10 microM) TETA is a more efficient inducer than Ni, which becomes a very efficient inducer at higher concentrations (50 microM). Neither TETA nor Ni show toxicity effects at the concentrations used. Unlike induction by Ni, induction by TETA is completely reversible by micromolar copper concentrations, thus resulting in a transient "wave" in luciferase activity, which can be repeated in subsequent growth cycles. CONCLUSIONS: We have worked out a chemically regulated gene expression system that can be finely tuned to produce temporally controlled "waves" in gene expression. The use of cassettes containing the CYC6 promoter, and of modified growth media, is a reliable and economically sustainable system for the temporally controlled expression of foreign genes in Chlamydomonas

Expression of Somatostatin and Somatostatin Receptor Subtypes 1–5 in Human Normal and Diseased Kidney

Somatostatin mediates inhibitory functions through five G protein–coupled somatostatin receptors (sst1–5). We used immunohistochemistry, immunofluorescence, and RT-PCR to determine the presence of somatostatin receptors sst1, sst2A, sst2B, sst3, sst4, and sst5 in normal and IgA nephropathy human kidney. All somatostatin receptors were detected in the thin tubules (distal convoluted tubules and loops of Henle) and thick tubules (proximal convoluted tubules) in the tissue sections from nephrectomy and biopsy samples. Immunopositive sst1 and sst4 staining was more condensed in the cytoplasm of tubular epithelial cells. In normal kidney tissue sections, podocytes and mesangial cells in the glomeruli stained for sst1, sst2B, sst4 and sst5, and stained weakly for sst3. In IgA kidney tissue, the expression of somatostatin receptors was significantly increased with particular immmunopositive staining for sst1, sst2B, sst4, and sst5 within glomeruli. In the epithelial cells, the staining for sst2B and sst4 in proximal tubules and sst1, sst2B, and sst5 in distal tubules was increased. The mRNA expression of sst1–5 was also detected by RT-PCR. Somatostatin and all five receptor subtypes were ubiquitously distributed in normal kidney and IgA nephropathy. The increased expression of somatostatin receptors in IgA nephropathy kidney might be the potential pathogenesis of inflammatory renal disease. (J Histochem Cytochem 56:733–743, 2008