Functional analysis of cis- and trans-regulatory elements of the lactate dehydrogenase A subunit promoter by in vitro transcription
| Autor: | S, Short, M L, Short, D M, Milkowski, R A, Jungmann |
|---|---|
| Rok vydání: | 1991 |
| Předmět: |
Cell Nucleus
Base Sequence L-Lactate Dehydrogenase Transcription Genetic Macromolecular Substances Recombinant Fusion Proteins Molecular Sequence Data Restriction Mapping Templates Genetic Regulatory Sequences Nucleic Acid beta-Galactosidase Gene Expression Regulation Enzymologic Cell Line Rats DNA-Binding Proteins Oligodeoxyribonucleotides Cyclic AMP Animals Humans Cyclic AMP Response Element-Binding Protein Promoter Regions Genetic HeLa Cells Plasmids |
| Zdroj: | The Journal of biological chemistry. 266(33) |
| ISSN: | 0021-9258 |
| Popis: | Using a transcription system from nuclear extracts of rat C6 glioma cells we have investigated the mechanism by which transcription from the lactate dehydrogenase A subunit (LDH) promoter is regulated via the cAMP-activated pathway. We demonstrated that the system accurately initiates transcription from the LDH promoter. Analysis of the competitive effects of linker-scanning mutants showed that the wild-type LDH promoter exhibited the highest competitive effect and reduced the rate of basal transcription, whereas LDH promoter fragments with a mutated cAMP-responsive element had little competitive activity. Cyclic AMP and the catalytic subunit of cAMP-dependent protein kinase stimulated the rate of transcription from the wild-type promoter, an effect which was inhibited by the catalytic subunit inhibitor protein. A beta-galactosidase-cAMP-responsive element binding protein fusion protein had no effect on the basal rate of transcription. Addition of beta-galactosidase-cAMP-responsive element binding protein together with cAMP or the catalytic subunit, however, enhanced the rate of transcription. The demonstrated regulatory effects indicate that the sensitivity of the transcription system makes it suitable for the functional analysis of homologous LDH and possibly heterologous transcription regulatory elements. |
| Databáze: | OpenAIRE |
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