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Isolation and identification of a cDNA clone coding for rat uroporphyrinogen decarboxylase
Abstract:
We have cloned and identified a DNA sequence complementary to the mRNA of uroporphyrinogen decarboxylase ( UroDCase ) from rat. This mRNA is a minor species (0.1%) of the total mRNA from anemic rat spleen. Poly(A)+ mRNA was enriched for UroDCase mRNA to 20% purity by a very efficient procedure involving two successive steps of preparative gel electrophoresis under various denaturing conditions. cDNA prepared from partially purified UroDCase mRNA (1% purity) was cloned in the Pst I site of pBR322 by using the homopolymeric G-C tailing method. Primary screening of 500 clones from this cDNA library was performed with a cDNA probe complementary to highly purified mRNA for UroDCase (20% purity) and UroDCase cDNA clones were finally identified by hybrid-selected translation. The rat cDNA clones obtained hybridize to human UroDCase mRNA. This will permit the isolation of the corresponding human gene and molecular analysis of porphyria cutanea tarda, the commonest type of porphyria.
Insights
Researchers cloned rat uroporphyrinogen decarboxylase (UroDCase) DNA. This breakthrough facilitates studying porphyria cutanea tarda by enabling human gene isolation and analysis.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Uroporphyrinogen decarboxylase (UroDCase) is crucial for heme biosynthesis.
- Deficiency in UroDCase activity is linked to porphyria cutanea tarda.
- A molecular understanding of UroDCase is essential for disease research.
Purpose of the Study:
- To clone and identify the DNA sequence for rat UroDCase.
- To develop a tool for isolating the human UroDCase gene.
- To enable molecular analysis of porphyria cutanea tarda.
Main Methods:
- Enrichment of UroDCase mRNA from anemic rat spleen using preparative gel electrophoresis.
- cDNA synthesis and cloning into the pBR322 vector using G-C tailing.
- Screening of cDNA library and identification of clones via hybrid-selected translation.
Main Results:
- Successfully cloned and identified rat UroDCase cDNA.
- Developed an efficient mRNA enrichment and cDNA cloning procedure.
- Confirmed that rat UroDCase cDNA hybridizes to human UroDCase mRNA.
Conclusions:
- The cloned rat UroDCase cDNA serves as a probe for human UroDCase mRNA.
- This facilitates the isolation and characterization of the human UroDCase gene.
- Enables further molecular investigation into the pathogenesis of porphyria cutanea tarda.