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Related Experiment Videos

Mouse ornithine decarboxylase gene: cloning, structure, and expression.

M Brabant1, L McConlogue, T van Daalen Wetters

  • 1Department of Microbiology, University of California, San Francisco 94143.

Proceedings of the National Academy of Sciences of the United States of America
|April 1, 1988
PubMed
Summary

Researchers isolated the mouse ornithine decarboxylase (OrnDCase) gene and demonstrated its functional expression. The OrnDCase promoter region shows strong activity, comparable to viral promoters, even with significant truncation.

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Area of Science:

  • Molecular Biology
  • Gene Expression
  • Biochemistry

Background:

  • Ornithine decarboxylase (OrnDCase) is a key enzyme in polyamine synthesis.
  • Understanding OrnDCase gene regulation is crucial for cellular processes.

Purpose of the Study:

  • To isolate and characterize the functional mouse OrnDCase gene.
  • To analyze the regulatory elements, particularly the promoter region, of the OrnDCase gene.

Main Methods:

  • Molecular cloning of the OrnDCase gene from amplified cell lines.
  • mRNA 5' terminus identification and sequence analysis.
  • Gene transfection into cultured cells for functional assays.
  • Transient expression assays using reporter genes (chloramphenicol acetyltransferase) to evaluate promoter strength.

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Main Results:

  • A functional mouse OrnDCase gene was successfully isolated.
  • The mRNA leader sequence is G+C rich with predicted stable secondary structure.
  • The OrnDCase gene transcription unit is approximately 6.2 kb.
  • Sequence analysis revealed a TATA box but no CAAT box near the transcription start site.
  • The cloned gene efficiently restored OrnDCase activity in deficient cells.
  • The OrnDCase promoter region, extending 3.0 kb upstream, exhibited promoter strength comparable to the Rous sarcoma virus LTR.
  • Truncation to 264 bp upstream of the transcription start site did not diminish expression levels.

Conclusions:

  • The study successfully cloned and functionally characterized the mouse OrnDCase gene.
  • The OrnDCase promoter is a strong, TATA-containing element that can drive high levels of gene expression.
  • The minimal promoter region required for strong activity is relatively short.