Motif trap: a rapid method to clone motifs that can target proteins to defined subcellular localisations

1999 ◽  
Vol 112 (23) ◽  
pp. 4207-4211
Author(s):  
L.A. Bejarano ◽  
C. Gonzalez
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Eukaryotic Cells ◽  
Subcellular Localisation ◽  
Dna Fragments ◽  
Rt Pcr ◽  
Rapid Procedure ◽  
Target Proteins ◽  
Protein Motifs ◽  
Green Fluorescent

We have developed a rapid procedure termed Motif Trap (MT) to identify protein motifs that are able to target proteins to a distinct subcellular localisation in eukaryotic cells. By expressing random DNA fragments fused to green fluorescent protein (GFP), individual cells with the GFP localisation of interest are readily isolated allowing for the expressed DNA fragments to be cloned by RT-PCR. These can then be used to identify the corresponding full-length cDNAs. Using MT, we have identified patterns of GFP localisation which correspond to every major organelle and compartment. We have shown that MT is useful to identify new sequences that determine subcellular localisation as well as known targeting motifs.

Green Fluorescent Protein variants fold differentially in prokaryotic and eukaryotic cells

2001 ◽  
Vol 81 (S36) ◽  
pp. 117-128 ◽  
Author(s):  
Andrea Sacchetti ◽  
Valeria Cappetti ◽  
Pierfrancesco Marra ◽  
Roberta Dell'Arciprete ◽  
Tarek El Sewedy ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Eukaryotic Cells ◽  
Green Fluorescent ◽  
Protein Variants

scholarly journals Use of Green Fluorescent Protein and Reverse Transcription-PCR To Monitor Candida albicans Agglutinin-Like Sequence Gene Expression in a Murine Model of Disseminated Candidiasis

2005 ◽  
Vol 73 (3) ◽  
pp. 1852-1855 ◽  
Author(s):  
Clayton B. Green ◽  
Xiaomin Zhao ◽  
Lois L. Hoyer
Keyword(s):  
Candida Albicans ◽  
Green Fluorescent Protein ◽  
Reverse Transcription ◽  
Fluorescent Protein ◽  
Rt Pcr ◽  
Disseminated Candidiasis ◽  
Reverse Transcription Pcr ◽  
Gfp Reporter ◽  
Reporter Strains ◽  
Green Fluorescent

ABSTRACT Candida albicans PALS-green fluorescent protein (GFP) reporter strains were inoculated into mice in a disseminated candidiasis model, and GFP production was monitored by immunohistochemistry and reverse transcription-PCR (RT-PCR). GFP production from the ALS1 and ALS3 promoters was detected immunohistochemically. ALS1, ALS2, ALS3, ALS4, and ALS9 transcription was detected by RT-PCR, further identifying ALS genes expressed in this model.

Renal principal cell-specific expression of green fluorescent protein in transgenic mice

2002 ◽  
Vol 283 (6) ◽  
pp. F1351-F1364 ◽  
Author(s):  
Ludmilla Zharkikh ◽  
Xiaohong Zhu ◽  
Peter K. Stricklett ◽  
Donald E. Kohan ◽  
Greg Chipman ◽  
...  
Keyword(s):  
Gene Expression ◽  
Green Fluorescent Protein ◽  
Transgenic Mice ◽  
Fluorescent Protein ◽  
Pcr Analysis ◽  
Intercalated Cells ◽  
Rt Pcr ◽  
Specific Expression ◽  
Principal Cells ◽  
Green Fluorescent

The purpose of this study is to develop transgenic mice with principal cell-specific expression of green fluorescent protein (GFP). After the cloning and sequencing of the mouse aquaporin-2 (AQP2) gene, 9.5 kb of the promoter were used to drive expression of GFP in transgenic mice. In transgenic mice, GFP was selectively expressed in principal cells of the renal collecting duct and not in intercalated cells. Expression was increased by dehydration of mice. AQP2 and GFP expression was maintained in primary cultures of renal medulla that were stimulated with cAMP or vasopressin analogs. GFP-expressing cells were then isolated by fluorescence-activated cell sorting. RT-PCR analysis showed expression of AQP2, AQP3, AQP4, vasopressin type 2 receptor, and cAMP response element binding protein but not H+-ATPase B1 subunit or anion exchanger 1. After expansion of these cells in culture, RT-PCR analysis showed continued expression of the same genes. This pattern of gene expression is that of principal cells rather than intercalated cells. This transgenic mouse model can be used in future studies of gene expression during the development, differentiation, and maturation of renal principal cells.

scholarly journals Diffusion of Green Fluorescent Protein in Three Cell Environments in Escherichia Coli

2006 ◽  
Vol 188 (10) ◽  
pp. 3442-3448 ◽  
Author(s):  
Conrad W. Mullineaux ◽  
Anja Nenninger ◽  
Nicola Ray ◽  
Colin Robinson
Keyword(s):  
Escherichia Coli ◽  
Diffusion Coefficient ◽  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Membrane Protein ◽  
Diffusion Coefficients ◽  
Fluorescent Protein ◽  
Protein A ◽  
Eukaryotic Cells ◽  
Green Fluorescent

ABSTRACT Surprisingly little is known about the physical environment inside a prokaryotic cell. Knowledge of the rates at which proteins and other cell components can diffuse is crucial for the understanding of a cell as a physical system. There have been numerous measurements of diffusion coefficients in eukaryotic cells by using fluorescence recovery after photobleaching (FRAP) and related techniques. Much less information is available about diffusion coefficients in prokaryotic cells, which differ from eukaryotic cells in a number of significant respects. We have used FRAP to observe the diffusion of green fluorescent protein (GFP) in cells of Escherichia coli elongated by growth in the presence of cephalexin. GFP was expressed in the cytoplasm, exported into the periplasm using the twin-arginine translocation (Tat) system, or fused to an integral plasma membrane protein (TatA). We show that TatA-GFP diffuses in the plasma membrane with a diffusion coefficient comparable to that of a typical eukaryotic membrane protein. A previous report showed a very low rate of protein diffusion in the E. coli periplasm. However, we measured a GFP diffusion coefficient only slightly smaller in the periplasm than that in the cytoplasm, showing that both cell compartments are relatively fluid environments.

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