Investigations on the gene for human antithrombin III

Abstract

Antithrombin III is a protein synthesised mainly in the liver. Its function is to support the fluidity of the blood through the inhibition of a number of serine proteases involved in the blood coagulation cascade. It does this by means of two active sites present in its molecule, the heparin binding site and the serine proteases binding site. Human liver mRNA was subjected to in-vitro translation using rabbit reticulocyte lysate. When the product was immunoprecipitated with antithrombin III antibody, a band was observed where antithrombin III was expected to migrate. From the same mRNA, cDNA was synthesised. This cDNA was subjected to the polymerase chain reaction utilising oligonucleotide probes flanking the known antithrombin III gene. The PCR products were cloned in M13 and pKK223-3 vectors and sequenced. The nucleic acid sequences were then compared with the sequences of the cloning vectors, antithrombin III and database. No significant homologies were however observed. The PCR products did not hybridise with the single stranded DNA containing the vector and PCR product. There are two possible conclusions; either something might have gone wrong from the moment the PCR product was subjected to Klenow enzyme, cleaned and cloned in M13 or the primers used in the polymerase chain reaction could have annealed to an unknown gene.