Cloning and analysis of a cDNA coding for bovine prothrombin

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Prothrombin is a crucial protein in blood coagulation.
• Understanding prothrombin gene expression requires isolating its mRNA.
• Bovine liver is a rich source of prothrombin mRNA.

Purpose of the Study:

• To isolate and clone complementary DNA (cDNA) encoding bovine prothrombin.
• To characterize the cloned prothrombin cDNA sequences.
• To facilitate further studies on prothrombin gene regulation and function.

Main Methods:

• Isolation of poly(A)-RNA enriched for prothrombin mRNA via immunoprecipitation.
• Synthesis of double-stranded cDNA using reverse transcriptase.
• Cloning of cDNA into pBR322 plasmid and transformation of Escherichia coli.
• Screening of recombinant clones using hybridization assays and DNA sequencing.

Main Results:

• Successfully isolated prothrombin mRNA, which constituted 8% of cell-free translation products.
• Generated and cloned double-stranded cDNA into E. coli, obtaining 63 tetracycline-resistant clones.
• Identified three positive clones with bovine DNA inserts of 700, 500, and 400 base pairs.
• Sequencing of the 700-base-pair insert revealed coding for the carboxyl-terminal 160 residues, a noncoding region, and a poly(A) tail.

Conclusions:

• The study successfully established a method for isolating and cloning prothrombin cDNA.
• The characterized cDNA inserts provide valuable tools for studying prothrombin gene expression.
• This work contributes to the understanding of blood coagulation at the molecular level.