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Mitochondrial nucleic acids as internal standards for blot hybridization analyses.
C G Tepper1, M M Pater, A Pater
1Department of Laboratory Medicine and Pathology, UMD-New Jersey Medical School, Newark 07103.
Analytical Biochemistry
|May 15, 1992
Summary
Researchers developed a plasmid (p72) using a mitochondrial gene fragment. This tool aids in studying gene expression and standardizing nucleic acid analysis, showing consistent results across human cell lines.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Standardization of nucleic acid analysis is crucial for reliable experimental results.
- Human mitochondrial genes offer unique characteristics for molecular tools.
Purpose of the Study:
- To characterize a novel plasmid (p72) for its utility in molecular biology applications.
- To evaluate the potential of p72 as a control in nucleic acid hybridization assays.
Main Methods:
- Construction of plasmid p72 by inserting human lung carcinoma DNA into a herpes simplex virus thymidine kinase vector.
- Hybridization analysis using Southern and Northern blots with human nucleic acids.
- Assessment of gene expression stability in various human cell lines and under chemical stress.
Main Results:
- The plasmid p72, containing a 167-bp fragment of the mitochondrial 16S ribosomal RNA gene, shows strong hybridization signals with human DNA and RNA.
- Expression of the mitochondrial 16S rRNA gene is constitutive across tested human cell lines.
- Cytotoxic chemical treatments did not affect the expression of this mitochondrial gene, unlike nuclear genes.
Conclusions:
- Plasmid p72 serves as a robust tool for studying gene expression perturbations.
- The constitutive and stable expression of the mitochondrial 16S rRNA gene fragment makes p72 ideal for normalizing Southern and Northern blot analyses.