What question did this study set out to answer?

Investigate mammalian gene expression by constructing and expressing enzymatically active hybrid proteins combining rat preproinsulin II regulatory sequences with bacterial beta-galactosidase.

September 1, 1983Proceedings of the National Academy of SciencesOpen Access

Expression of a preproinsulin-beta-galactosidase gene fusion in mammalian cells.

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Authors

DNDavid A. NielsenMichael E. DeBakey VA Medical Center JCJ ChouPrecision for Medicine (United States)AMA J MacKrellUniversity of Southern California

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Implication

In vitro study demonstrates production of active tetrameric beta-galactosidase fusions in mammalian COS-7 cells, indicating their utility as sensitive reporters for gene expression.

Key Points

Investigate mammalian gene expression by constructing and expressing enzymatically active hybrid proteins combining rat preproinsulin II regulatory sequences with bacterial beta-galactosidase.
Fused the promoters and translation initiation regions of rat preproinsulin II and simian virus 40 early genes to the Escherichia coli beta-galactosidase structural gene.
Transfected the recombinant plasmids into monkey kidney COS-7 cells under varied conditions to optimize enzymatic expression.
Characterized the hybrid protein using pH profiling, fluorogenic substrate-stained nondenaturing gel electrophoresis, and immunoprecipitation of tritium-labeled proteins in the presence of glucose, insulin, dexamethasone, and epidermal growth factor.
Transfected COS-7 cells produced fully active, tetrameric hybrid beta-galactosidase with an optimal pH of 7.0, identical to native bacterial beta-galactosidase and distinct from endogenous lysosomal acid beta-galactosidase.
Treatment with high glucose, insulin, dexamethasone, or epidermal growth factor produced no detectable regulatory changes in preproinsulin-beta-galactosidase activity.

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Nielsen et al. (1983) studied this question.

synapsesocial.com/papers/6a70a370a7fbea1e4408254e https://doi.org/10.1073/pnas.80.17.5198

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Implication

In vitro study demonstrates production of active tetrameric beta-galactosidase fusions in mammalian COS-7 cells, indicating their utility as sensitive reporters for gene expression.

Expression of a preproinsulin-beta-galactosidase gene fusion in mammalian cells.

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Implication

Key Points

Cite This Study

Expression of a preproinsulin-beta-galactosidase gene fusion in mammalian cells.

Discussion

Member takes

Implication

Key Points

Cite This Study

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