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Purification of antithrombin III mRNA and cloning of its cDNA
Abstract:
Antithrombin III mRNA was enriched from a baboon liver by specific polysome immunoprecipitation. The partially purified antithrombin III mRNA preparation was used for cDNA synthesis and cloning. Candidate antithrombin III cDNA clones were identified by differential hybridization using as probes [32P]cDNAs synthesized from the polysome-enriched and -depleted RNA fractions, respectively. The candidate clones were further analyzed by hybrid-selected translation. The authenticity of a cDNA clone positive to both tests was unambiguously confirmed by matching its nucleotide sequence with the known amino acid sequence of human antithrombin III. The baboon antithrombin III cDNA clone hybridized well with human antithrombin III mRNA and can be used as a probe to isolate the corresponding human gene.
Insights
Researchers successfully isolated and sequenced baboon antithrombin III (ATIII) complementary DNA (cDNA). This baboon ATIII cDNA clone can serve as a valuable tool for isolating the human ATIII gene.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Antithrombin III (ATIII) is a crucial protein in the blood coagulation cascade.
- Understanding ATIII gene structure is vital for research into thrombotic disorders.
Purpose of the Study:
- To isolate and characterize the complementary DNA (cDNA) for baboon antithrombin III.
- To develop a tool for isolating the human antithrombin III gene.
Main Methods:
- Enrichment of antithrombin III mRNA from baboon liver using polysome immunoprecipitation.
- cDNA synthesis and cloning, followed by differential hybridization screening.
- Hybrid-selected translation and nucleotide sequencing for clone verification.
Main Results:
- A baboon antithrombin III cDNA clone was successfully isolated and identified.
- The nucleotide sequence of the baboon cDNA matched the known human ATIII amino acid sequence.
- The baboon ATIII cDNA clone showed strong hybridization with human ATIII mRNA.
Conclusions:
- The isolated baboon antithrombin III cDNA is authentic and accurately represented.
- This baboon cDNA clone is a useful probe for isolating the homologous human gene.
- Facilitates further research into antithrombin III function and related genetic studies.