No abstract

Green fluorescent protein (GFP) variants with higher expression efficiencies have been generated by mutagenesis. Favorable mutations often improve the folding of GFP. However, an effect on protein folding fails to explain the efficiency of several other GFP mutations. In this work, we demonstrate that mutations of the GFP open reading frame and untranslated regions profoundly affect mRNA transcription and translation efficiencies. The removal of the GFP 5P P untranslated region halves the transcription rate of the GFP gene, but hugely improves its translation rate. Mutations of the GFP open reading frame or the addition of peptide sequences differentially reduce the GFP mRNA transcription rate, translation efficiency and protein stability. These previously unrecognized effects are demonstrated to be critical to the efficiency of GFP mutants. These findings indicate the feasibility of generating more efficient GFP variants, with optimized mRNA transcription and translation in eukaryotic cells. ß

Better-folding Green Fluorescent Protein (GFP) mutants selected from bacterial screenings are commonly used in widely different cellular environments. However, it is unclear if the folding ef®ciency of GFPs is invariant in different cell types. In this work, we have analysed the folding properties of GFP variants in bacteria versus mammalian cells. Remarkably, S65T was found to fold at comparable levels with the wild type GFP in bacteria, but at 10-fold lower levels in mammalian cells. On the other hand, Bex1 folded 3±4 times better than the wtGFP or S65T in E. coli, and 10±20-fold or more than 95-fold better, respectively, in mammalian cells. The Vex1 mutant demonstrated similar properties to Bex1. No evidence of differential GFP unfolding in vivo or of preferential degradation of unfolded GFP molecules was found. Moreover, no relationship between GFP folding ef®ciency and expression levels, or protein stability was detected. Trivial Aconfounding factors, like GFP unfolding caused by different pH or¯uorescence quenching due to molecular crowding, were also excluded. In summary, our results demonstrate that speci®c GFP variants follow different folding trajectories in mammalian versus bacterial cells. The speci®city of this differential folding supports a role of chaperones in guiding the folding of GFP in vivo.

Trop-2 is a novel calcium signal transducer expressed at high levels by most human carcinomas. To develop an animal model to study the function of this molecule in vivo, we have cloned the murine Trop2 gene. Using human TROP2 primers, we amplified by PCR a segment of murine Trop2. This was used as a probe to clone a full-length gene by hybridization of a genomic library. The cloned murine Trop2 gene is functional, as indicated by sequencing and by expression after transfection. The murine Trop2 is 87.4% similar to its human homologue, with the highest conservation in the extracellular region between residues 86 and 157. Essentially all cysteines are conserved between the human and the murine genes, suggesting conservation of the Trop2 disulfide bridges and of its overall structure. Intriguingly, the cytoplasmic tail of Trop2 shows a highly conserved phosphatidylinositol 4,5-bisphosphate (PIP 2 )-binding sequence, which overlaps with a protein kinase C phosphorylation site. Thus, we speculate that PIP 2 might regulate the phosphorylation state of Trop2 and play a role in its signal transduction. Murine Trop2 mRNA is detected in normal kidney, lung, ovary and testis, similarly to the human gene. Interestingly, the highest levels of expression are found in immortalized keratinocytes. Since Trop2 is undetectable in undifferentiated spindle cell carcinomas, this suggests a preferential expression at early stages of tumor progression. Int.