Cloning and Transcriptional Activity of the Mouse Omi/HtrA2 Gene Promoter
Abstract
:1. Introduction
2. Results
2.1. HtrA Serine Peptidase 2 (Omi/HtrA2) is Ubiquitously Expressed but Its Protein and mRNA Levels Vary in Different Tissues
2.2. Assessment of the Activity of Mouse Omi/HtrA2 Promoter Luciferase Full-Length and Truncated Vectors
Primer Name | Sequence (5’–3’) | Amplification Region (bp) | Length (bp) |
---|---|---|---|
F1 | AGAATTTCGAGGGCAGGGCTAA | −146~+93 | 239 |
F2 | CGTGAGGTGGCGATTAAG | −379~+93 | 472 |
F3 | TGGAAATAAGGACCCTGACG | −649~+93 | 742 |
F4 | GTAGAGCAGTAGCGCGAGCA | −838~+93 | 931 |
F5 | CTGGGAAGGCGGAGTCTTT | −1205~+93 | 1298 |
R | GGCAAGCGGTCTAGGAGAA | – | – |
2.3. Bioinformatics Analysis of the Mouse Omi/HtrA2 Promoter
Prediction Area | Start | End | Promoter Sequence | Score |
---|---|---|---|---|
promoter core area 1 | −1197 | −1148 | GAGGGCAGGGCTAAAAGTGGGCAGACAGGAAAGGAACTAGGGCACCCACT | 0.98 |
promoter core area 2 | −359 | −310 | CCGGTGCGAGTCAAAGAGCCGCTCCGGCCCCGGAGCTGGGGGAGGTTCCA | 0.90 |
CpG island | −709 | +37 | Observed/Expected ratio > 0.60 | Percent C + Percent G > 50.00 |
Transcription Factors | Number | Sequence of Putative Binding Sites | Location |
---|---|---|---|
HSF1 | 1 | AAGGAACT | +83~+90 |
2 | GTGGAAGC | −46~−39 | |
3 | CTTCTTGCTTTTTCT | −172~−158 | |
4 | ATGGAAGG | −181~−174 | |
5 | GGGGAACA | −285~−278 | |
6 | AGGGAAGA | −805~−798 | |
7 | ATGGAACC | −868~−861 | |
8 | CACAGAAT | −1028~−1021 | |
SP1 | 1 | GCCTCGCCCC | −722~−713 |
2 | CACCCGCCTAT | −999~−899 | |
AP1 | 1 | CTATTGA | −944~−938 |
p53 | 1 | CGTGCCC | +65~+71 |
2 | GCGGCCC | −54~−48 | |
3 | GGGCTAG | −84~−78 | |
4 | GGGCATC | −235~−229 | |
5 | CCAGCCCAGCCC | −520~−509 | |
6 | GGGCTGG | −816~−810 | |
7 | GCAGCCC | −1103~−1197 | |
YY1 | 1 | GAAAAGACA | −867~−859 |
2 | AGGGTGACA | −843~−835 | |
3 | CTGGGGACA | −551~−543 | |
4 | TGTCGCCGC | −177~−169 |
2.4. p53 and HSF1 Directly Binds to Omi/HtrA2 Promoter with ChIP Assay
3. Discussion
4. Materials and Methods
4.1. Animals
4.2. Cell Lines and Cell Culture
4.3. Isolation of Genomic DNA
4.4. Design and Preparation of Mouse Omi/HtrA2 Gene Promoter Constructs
4.5. Transfection and Luciferase Activity Assay
4.6. Bioinformatics Analysis
4.7. Western Blotting
4.8. RNA Preparation and Quantitative Reverse Transcription-Polymerase Chain Reaction (QRT-PCR) Analysis
4.9. ChIP Assay
4.10. Statistical Analysis
5. Conclusions
Supplementary Materials
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Liu, D.; Liu, X.; Wu, Y.; Wang, W.; Ma, X.; Liu, H. Cloning and Transcriptional Activity of the Mouse Omi/HtrA2 Gene Promoter. Int. J. Mol. Sci. 2016, 17, 119. https://doi.org/10.3390/ijms17010119
Liu D, Liu X, Wu Y, Wang W, Ma X, Liu H. Cloning and Transcriptional Activity of the Mouse Omi/HtrA2 Gene Promoter. International Journal of Molecular Sciences. 2016; 17(1):119. https://doi.org/10.3390/ijms17010119
Chicago/Turabian StyleLiu, Dan, Xin Liu, Ye Wu, Wen Wang, Xinliang Ma, and Huirong Liu. 2016. "Cloning and Transcriptional Activity of the Mouse Omi/HtrA2 Gene Promoter" International Journal of Molecular Sciences 17, no. 1: 119. https://doi.org/10.3390/ijms17010119