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Method and cell for detecting modulations of RGS proteins

A protein, RGS4 technology, applied in cells modified by introducing foreign genetic material, biochemical equipment and methods, fusion cells, etc., can solve problems such as low affinity

Inactive Publication Date: 2004-02-25
WYETH LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although RGS proteins have a significantly higher affinity for Gα-GTP, RGS proteins have a lower affinity for Gα-GDP

Method used

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  • Method and cell for detecting modulations of RGS proteins
  • Method and cell for detecting modulations of RGS proteins
  • Method and cell for detecting modulations of RGS proteins

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Example 1 RGS determination of FUS1-LacZ reporter gene:

[0078] GPCR receptor stimulation by alpha factor in yeast has been well characterized and characterized (see, Dohlman et al., 1996). Stimulation of this pathway normally causes cell cycle arrest in yeast. This signaling pathway is controlled by the endogenous RGS protein, Sst2, which functions as a negative regulator of GPCR signaling. The RGS protein acts to accelerate the endogenous GTPVase activity of the Gα subunit. Because deletion of the gene encoding Sst2 in yeast impairs its ability to turn off GPCR signaling, it causes hypersensitivity of the yeast cells to the ligand. The mammalian RGS protein, RGS4, is functionally complementary to the yeast Sst2.

[0079] In this experiment, reporter gene activity was measured in yeast strains lacking sst2 in the presence or absence of mammalian RGS4 protein. In this assay, we tested whether the RGS4 protein could regulate the alpha factor-induced pheromone respon...

Embodiment 2

[0102] Example 2 RGS assay of FUS1-luciferase reporter gene

[0103] Example 1 demonstrates that plasmids generated in response to the FUS1 promoter with the desired backbone and a pheromone operably linked upstream of the LacZ gene can be used to study the activity of RGS proteins. In this experiment, the luciferase gene was linked to the FUS1 promoter, not downstream of the LacZ gene. The luciferase system has several advantages over systems using the LacZ reporter, most notably the increased speed and ease of monitoring gene expression.

[0104] A FUS-1 reporter gene plasmid (Kp120) containing the luciferase gene was constructed. The FUS1 -luciferase reporter gene cassette was constructed by NcoI-XbaI digestion of plasmid pGL (Promega) to isolate the 1.7 kb fragment containing the firefly luciferase gene. This fragment was blunt-ended and purified. The Kp27 vector was used as the base vector, which was prepared by digestion with BamHI-NotI (to remove the LacZ gene), deph...

Embodiment 3

[0112] Example 3 Low copy version of the FUS1 luciferase reporter:

[0113] As a 2 [mu]m alternative to the aforementioned FUS1 luciferase reporter gene construct, a low copy number (CEN) version of the luciferase construct of Example 2 was prepared. A CEN version of the pheromone pathway responsive FUS1 luciferase reporter gene was generated by digesting plasmid Kp120 with KpnI and SacIEN to isolate a 2.8 kb fragment containing the FUS1 promoter and luciferase gene reporter cassette. Plasmids pRS414 and pRS416 (Stragagene) were digested with KpnI and SacI, respectively, and gel purified. Standard ligation was performed to generate two additional FUS1 luciferase recombinant plasmids; Kp133 (TRP1-tagged) and Kp135 / Kp131 * (URA3 tagged). Bacterial transformation and production of plasmid DNAs are performed by standard methods. Plasmids were confirmed by DNA sequencing. Plasmid Kp131 * Represents a similar construct for Kp135, but which was independently generated using a diff...

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Abstract

This application describes novel cells that respond to a pheromone comrising a heterologous nucleic acid encoding a reporter operably linked to a pheromone-responsive promoter. The cells may further comprises a heterologous nucleic acid encoding an RGS protein. Also described are methods involving the use of these cells for detecting the ability of a test sample to alter RGS protein-mediated reporter gene expression.

Description

technical field [0001] The present invention relates to novel cells that are responsive to pheromones and express a reporter gene operably linked to a pheromone responsive promoter. In some embodiments, the reporter is Renilla luciferase, Photinus luciferase, green fluorescent protein, or a derivative of green fluorescent protein. In other embodiments, the cells also express a heterologous regulator of G protein ("RGS protein"). [0002] In another embodiment, the present invention relates to a method of screening for compounds that modulate RGS proteins, wherein the method utilizes the novel cells of the present invention. Background technique [0003] The G protein signaling pathway is one of the most important signaling cascades for the transmission of extracellular signals such as neurotransmitters, hormones, odorants and light. This pathway has been identified in various organisms, including yeast and mammals. Generally, the system consists of 3 major components: G p...

Claims

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Application Information

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IPC IPC(8): C07K14/47C12N1/19C12N5/10C12N15/62C12N15/09C12N15/85C12Q1/02
CPCC07K14/4702C07K2319/00C12N2830/001C12N15/62C12N15/85C12N5/16
Inventor 凯思琳·H·杨曹健大卫·谢瓦
Owner WYETH LLC
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