Chapter 16 Gel retardation assays and DNA footprinting

Endothelin (ET) has been shown to activate mitogen-activated protein kinase (MAPK). However, it has been unclear which of the ET receptors is coupled to MAPK activation. In the present study, we conducted experiments to determine which ET receptor is linked to MAPK activation. We found that both human ETA and ETB were coupled to the MAPK cascade in ETA or ETB cDNA-transfected Chinese hamster ovary cells. ET-1 was more potent than ET-3 in the activation of p42 MAPK, induction of MAPK kinase (MAPKK) gel retardation and uptake of [3H]thymidine in ETA-transfected cells, whereas sarafotoxin (S6c) showed no stimulatory effect on the kinases and [3H]thymidine uptake. ET-1, ET-3, and S6c had approximately the same potency to activate p42 MAPK, MAPKK gel retardation, and [3H]thymidine uptake in ETB-transfected cells. These data suggest that 1) ET isopeptides, through either ETA or ETB receptors, induce the MAPK cascade as well as cell proliferation; and 2) the different potencies of ET isopeptides for activation of the MAPK cascade and induction of cell growth are mainly due to their different affinities toward ETA and ETB.

Download Full-text

Characterization and purification of human retinoic acid receptor-γ 1 overexpressed in the baculovirus-insect cell system

Biochemical Journal ◽

10.1042/bj2870833 ◽

1992 ◽

Vol 287 (3) ◽

pp. 833-840 ◽

Cited By ~ 11

Author(s):

A P Reddy ◽

J Y Chen ◽

T Zacharewski ◽

H Gronemeyer ◽

J J Voorhees ◽

...

Keyword(s):

Retinoic Acid ◽

Dna Binding ◽

Mammalian Cells ◽

Retinoic Acid Receptor ◽

Expression System ◽

Gel Filtration ◽

Recombinant Baculovirus ◽

Sf9 Cells ◽

Acid Receptor ◽

Gel Retardation

The full-length cDNA for the human retinoic acid receptor-gamma 1 (RAR-gamma 1) has been expressed to high levels in Spodoptera frugiferda (Sf9) cells using the baculovirus expression system. Western blot analysis revealed that RAR-gamma 1 expression increased between 32 and 60 h post-infection. The recombinant receptor was expressed primarily as a nuclear protein and displayed a molecular mass of 50 kDa as determined by SDS/PAGE and gel-filtration chromatography, consistent with its cDNA-deduced size. Based on ligand binding, 2 x 10(6) RAR-gamma 1 molecules were expressed per Sf9 cell, a level approx. 2000 times greater than in mammalian cells. The receptor was partially purified 300-fold by sequential anion-exchange, gel-filtration and DNA affinity chromatographies. The overexpressed receptor specifically bound all-trans-retinoic acid (RA) and the synthetic retinoid CD367 with high affinity (Kd 0.15 nM and 0.23 nM respectively). The RA metabolites 4-hydroxy-RA and 4-oxo-RA were poor competitors for [3H]CD367 binding to recombinant RAR-gamma 1 (K(i) > 1 microM), indicating that 4-oxidation of RA greatly reduces its affinity for RAR-gamma 1. Gel-retardation analysis demonstrated that RAR-gamma 1 specifically bound the RA response element of the mouse RAR-beta gene. RAR-gamma 1 species expressed from recombinant baculovirus (in Sf9 cells) and vaccinia virus (in HeLa cells) exhibited similar affinities for RA and CD367 and had comparable DNA-binding properties in gel-retardation experiments. Moreover, a similar requirement for additional DNA-binding stimulatory factor(s) was observed in both cases. These results provide a basis for the use of baculovirus-expressed RAR-gamma 1 in further functional and structural studies.

Download Full-text

Gel Retardation Assay

Encyclopedia of Genetics, Genomics, Proteomics and Informatics ◽

10.1007/978-1-4020-6754-9_6499 ◽

2008 ◽

pp. 743-743

Keyword(s):

Gel Retardation Assay ◽

Gel Retardation

Download Full-text

Use of gel retardation to analyze protein-nucleic acid interactions.

Microbiological Reviews ◽

10.1128/mr.56.4.509-528.1992 ◽

1992 ◽

Vol 56 (4) ◽

pp. 509-528

Author(s):

D Lane ◽

P Prentki ◽

M Chandler

Keyword(s):

Nucleic Acid ◽

Gel Retardation ◽

Protein Nucleic Acid ◽

Nucleic Acid Interactions

Download Full-text

Echovirus-9 protein 2C binds single-stranded RNA unspecifically

Journal of General Virology ◽

10.1099/0022-1317-81-10-2481 ◽

2000 ◽

Vol 81 (10) ◽

pp. 2481-2484 ◽

Cited By ~ 4

Author(s):

Marcus Klein ◽

Hans J. Eggers ◽

Birgit Nelsen-Salz

Keyword(s):

Rna Binding ◽

Binding Specificity ◽

Secondary Structures ◽

Multiple Functions ◽

E Coli ◽

Linear Dna ◽

Gel Retardation ◽

Rna Fragments ◽

Poliovirus Protein

Polypeptide 2C is essential for picornavirus replication. Although many data on multiple functions of this highly conserved protein are available, the mechanism of RNA binding is still obscure. In this work, protein 2C of echovirus-9 strain Barty was expressed as a histidine-tagged protein in E. coli followed by nondenaturing purification to homogeneity. After incubation of 2C protein with different kinds of RNA fragments, binding was shown in gel retardation assays. Competition experiments revealed that 2C targets linear RNA unspecifically; however, single-stranded linear DNA does not react with this protein. In contrast to poliovirus, protein 2C of echovirus-9 only recognizes RNA with a low content of secondary structures. This may be a first hint of a different binding specificity of 2C in echo- and polioviruses.

Download Full-text