Related Experiment Videos
Molecular cloning of a cDNA sequence complementary to porphobilinogen deaminase mRNA from rat
Abstract:
A cDNA clone containing sequences complementary to the mRNA coding for anemic rat spleen porphobilinogen deaminase (EC 4.3.1.8) has been isolated. A cDNA library was prepared from partially purified mRNA (1% purity). This library was then screened by colony hybridization, using a cDNA probe derived from porphobilinogen deaminase mRNA further enriched (10-20% purity) by gel electrophoresis in the presence of methylmercury hydroxide. Colonies hybridizing with the probe were analyzed by hybrid-selected translation using anemic rat spleen mRNA. Four recombinant plasmids containing porphobilinogen deaminase cDNA sequences were identified by specific immunoprecipitation of the translational product from hybrid-selected mRNA. Porphobilinogen deaminase mRNA was shown to contain 1800 bases by blot hybridization analysis. The cloned cDNA sequence consists of 1500 bases. Hybridization analysis of poly(A)+ RNA from uninduced and induced mouse erythroleukemic cells indicated that induction to erythroid differentiation by dimethyl sulfoxide results in a 10-fold increase of porphobilinogen deaminase mRNA. The rat cDNA clones hybridize to the corresponding sequences encoding human porphobilinogen deaminase. This property will be useful for isolation of human gene(s) and further characterization of the molecular lesion(s) responsible for acute intermittent porphyria.
Insights
Researchers isolated porphobilinogen deaminase cDNA from anemic rat spleen. This discovery aids in understanding acute intermittent porphyria and identifying human gene mutations.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Porphobilinogen deaminase (PBG-D) is crucial for heme biosynthesis.
- Deficiency in PBG-D activity causes acute intermittent porphyria (AIP).
- Understanding PBG-D gene expression is vital for AIP research.
Purpose of the Study:
- To isolate and characterize cDNA sequences encoding rat porphobilinogen deaminase.
- To investigate the regulation of PBG-D mRNA during erythroid differentiation.
- To develop tools for studying human PBG-D and AIP.
Main Methods:
- Construction and screening of a rat spleen cDNA library using colony hybridization.
- Hybrid-selected translation and immunoprecipitation to identify positive clones.
- Blot hybridization and Northern analysis to determine mRNA size and expression levels.
Main Results:
- Isolated four recombinant plasmids containing rat PBG-D cDNA sequences.
- Determined PBG-D mRNA size to be 1800 bases, with a cloned cDNA of 1500 bases.
- Observed a 10-fold increase in PBG-D mRNA upon erythroid differentiation induction in mouse cells.
- Demonstrated cross-hybridization of rat cDNA clones with human PBG-D sequences.
Conclusions:
- Successfully cloned and characterized rat porphobilinogen deaminase cDNA.
- Established a link between erythroid differentiation and PBG-D mRNA levels.
- Rat cDNA clones serve as valuable probes for isolating human PBG-D genes and investigating AIP molecular basis.