Identification of promoter elements required for in vitro transcription of hamster 3-hydroxy-3-methylglutaryl coenzyme A reductase gene

T. F. Osborne, G. Gil, M. S. Brown, R. C. Kowal, J. L. Goldstein

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Abstract

The 5'-flanking region of the gene for hamster 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) is shown to contain promoter sequences that drive transcription in vitro in the presence of a HeLa whole-cell extract. DNase I protection studies revealed at least six different regions within the 277-base-pair (bp) promoter that bind nuclear proteins and produce 'footprints'. The functional significance of these sequences was determined through transcriptional analysis of a series of substitution mutations that scrambled short sequences throughout this region. Two of the footprint sequences were crucial for transcription in vitro; one of these contains a match in 6 of 6 bp, with a sequence in the adenovirus type 2 major late promoter that is known to be required for transcription. Scrambling a 26-bp sequence in a third footprint led to a consistent 2-fold increase in transcription, suggesting that this sequence might be a site for negative regulation. These studies define three regions that play a role in regulating transcription of the gene for HMG-CoA reductase, a negatively regulated enzyme in the cholesterol biosynthetic pathway.

Original languageEnglish (US)
Pages (from-to)3614-3618
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume84
Issue number11
DOIs
StatePublished - 1987

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Base Pairing
Cricetinae
Oxidoreductases
Protein Footprinting
Genes
5' Flanking Region
Deoxyribonuclease I
Biosynthetic Pathways
Coenzyme A
Nuclear Proteins
Cell Extracts
HeLa Cells
Adenoviridae
Cholesterol
Mutation
Enzymes
In Vitro Techniques
3-hydroxy-3-methylglutaryl-coenzyme A

ASJC Scopus subject areas

  • Genetics
  • General

Cite this

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title = "Identification of promoter elements required for in vitro transcription of hamster 3-hydroxy-3-methylglutaryl coenzyme A reductase gene",
abstract = "The 5'-flanking region of the gene for hamster 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) is shown to contain promoter sequences that drive transcription in vitro in the presence of a HeLa whole-cell extract. DNase I protection studies revealed at least six different regions within the 277-base-pair (bp) promoter that bind nuclear proteins and produce 'footprints'. The functional significance of these sequences was determined through transcriptional analysis of a series of substitution mutations that scrambled short sequences throughout this region. Two of the footprint sequences were crucial for transcription in vitro; one of these contains a match in 6 of 6 bp, with a sequence in the adenovirus type 2 major late promoter that is known to be required for transcription. Scrambling a 26-bp sequence in a third footprint led to a consistent 2-fold increase in transcription, suggesting that this sequence might be a site for negative regulation. These studies define three regions that play a role in regulating transcription of the gene for HMG-CoA reductase, a negatively regulated enzyme in the cholesterol biosynthetic pathway.",
author = "Osborne, {T. F.} and G. Gil and Brown, {M. S.} and Kowal, {R. C.} and Goldstein, {J. L.}",
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T1 - Identification of promoter elements required for in vitro transcription of hamster 3-hydroxy-3-methylglutaryl coenzyme A reductase gene

AU - Osborne, T. F.

AU - Gil, G.

AU - Brown, M. S.

AU - Kowal, R. C.

AU - Goldstein, J. L.

PY - 1987

Y1 - 1987

N2 - The 5'-flanking region of the gene for hamster 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) is shown to contain promoter sequences that drive transcription in vitro in the presence of a HeLa whole-cell extract. DNase I protection studies revealed at least six different regions within the 277-base-pair (bp) promoter that bind nuclear proteins and produce 'footprints'. The functional significance of these sequences was determined through transcriptional analysis of a series of substitution mutations that scrambled short sequences throughout this region. Two of the footprint sequences were crucial for transcription in vitro; one of these contains a match in 6 of 6 bp, with a sequence in the adenovirus type 2 major late promoter that is known to be required for transcription. Scrambling a 26-bp sequence in a third footprint led to a consistent 2-fold increase in transcription, suggesting that this sequence might be a site for negative regulation. These studies define three regions that play a role in regulating transcription of the gene for HMG-CoA reductase, a negatively regulated enzyme in the cholesterol biosynthetic pathway.

AB - The 5'-flanking region of the gene for hamster 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) is shown to contain promoter sequences that drive transcription in vitro in the presence of a HeLa whole-cell extract. DNase I protection studies revealed at least six different regions within the 277-base-pair (bp) promoter that bind nuclear proteins and produce 'footprints'. The functional significance of these sequences was determined through transcriptional analysis of a series of substitution mutations that scrambled short sequences throughout this region. Two of the footprint sequences were crucial for transcription in vitro; one of these contains a match in 6 of 6 bp, with a sequence in the adenovirus type 2 major late promoter that is known to be required for transcription. Scrambling a 26-bp sequence in a third footprint led to a consistent 2-fold increase in transcription, suggesting that this sequence might be a site for negative regulation. These studies define three regions that play a role in regulating transcription of the gene for HMG-CoA reductase, a negatively regulated enzyme in the cholesterol biosynthetic pathway.

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U2 - 10.1073/pnas.84.11.3614

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JO - Proceedings of the National Academy of Sciences of the United States of America

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