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Identification and functional characterization of the bovine manganous superoxide dismutase promoter
1Department of Pathology, Vanderbilt University Medical Center, Nashville, TN 36232-2650.
Abstract:
Manganous superoxide dismutase (MnSOD) gene expression is stimulated by endotoxin, tumor necrosis factor, and interleukin-1, agents thought to cause cellular damage through intracellular generation of reactive oxygen species. To study the molecular mechanisms underlying the induction of MnSOD mRNA by these stimuli, we cloned a bovine MnSOD cDNA and used it to isolate the promoter region of the bovine MnSOD gene. A 14 kb genomic DNA fragment (lambda BS1) containing the first and second exons and 5' flanking region of the gene was characterized. The transcription start site was determined by primer extension and S1 nuclease protection assays and found to be 88 bp upstream of the translation initiation codon. The sequence of approximately 1 kb of DNA upstream of the start site was determined and examined for potential regulatory elements. DNA immediately upstream of the transcription start site was GC-rich and contained two AP-2 and eight Sp-1 consensus sequences. It did not contain either a CCAAT or TATA box. A 956 bp fragment of this DNA fragment was transcriptionally active when fused to a luciferase reporter gene and transfected into both bovine pulmonary artery endothelial and hamster insulinoma tumor cells. Transfection analysis of three additional deletion mutants, whose 5' end-points were -317, -182, and -70 bp, respectively, showed a step-like reduction in transfection efficiency, suggesting the presence of regulatory elements throughout this DNA fragment that contribute to transcriptional activity of the MnSOD promoter. Despite the high homology of the bovine MnSOD cDNA to other mammalian MnSODs, the promoter sequences of bovine and rat MnSOD genes showed a virtual lack of similarity.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
This study investigated the promoter region of the bovine manganous superoxide dismutase (MnSOD) gene, identifying regulatory elements crucial for its expression in response to cellular damage signals.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Manganous superoxide dismutase (MnSOD) gene expression increases with stimuli like endotoxin, tumor necrosis factor, and interleukin-1.
- These stimuli are implicated in cellular damage via reactive oxygen species generation.
- Understanding MnSOD gene regulation is key to cellular defense mechanisms.
Purpose of the Study:
- To elucidate the molecular mechanisms controlling bovine MnSOD gene expression.
- To isolate and characterize the promoter region of the bovine MnSOD gene.
- To identify potential regulatory elements influencing MnSOD mRNA induction.
Main Methods:
- Cloning of bovine MnSOD cDNA and isolation of the gene's promoter region.
- Determination of the transcription start site using primer extension and S1 nuclease assays.
- Reporter gene assays (luciferase) with transfected DNA fragments in cell lines.
Main Results:
- A 14 kb genomic DNA fragment containing the 5' flanking region was characterized.
- The transcription start site was located 88 bp upstream of the translation initiation codon.
- GC-rich DNA upstream of the start site contained AP-2 and Sp-1 elements but lacked CCAAT/TATA boxes.
- A 956 bp promoter fragment demonstrated transcriptional activity.
- Deletion analysis revealed regulatory elements within the promoter region contributing to MnSOD activity.
- Bovine and rat MnSOD promoter sequences showed significant dissimilarity despite cDNA homology.
Conclusions:
- The characterized promoter region of the bovine MnSOD gene contains functional regulatory elements.
- GC-rich sequences and specific transcription factor binding sites (AP-2, Sp-1) are important for MnSOD gene transcription.
- The distinct promoter sequence suggests species-specific regulation of MnSOD.
- This research provides insights into the transcriptional control of MnSOD, a critical antioxidant enzyme.