Functional analysis of cis- and trans-regulatory elements of the lactate dehydrogenase A subunit promoter by in vitro transcription.
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.
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Department of Cellular, Molecular, and Structural Biology, Northwestern University Medical School, Chicago, Illinois 60611.
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Milkowski
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Jungmann
R A
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D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002467:Cell Nucleus / Q000502:physiology; D000242:Cyclic AMP / Q000494:pharmacology; D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D006367:HeLa Cells; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000235:genetics*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D011401:Promoter Regions, Genetic; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D015183:Restriction Mapping; D013698:Templates, Genetic; D014158:Transcription, Genetic; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D000242:Cyclic AMP; D007770:L-Lactate Dehydrogenase; D001616:beta-Galactosidase