In vivo transcription of a human antithrombin III "minigene".

A 2.2-kilobase human antithrombin III (ATIII) "minigene" has been constructed which preserves many of the features of the 16-kilobase parental gene. These include approximately 300 base pairs of the 5'-flanking region containing the "TATA"-less ATIII promoter, the entire protein-coding sequence including the signal peptide, a single intervening sequence with intact donor and acceptor splice sites, and a polyadenylation signal. Unique PstI termini allow the minigene to be cloned in many commonly used plasmid vectors. The minigene has been reintroduced into heterologous cells by calcium phosphate precipitation. RNA is transcribed with heterogeneous 5'-ends and is processed by splicing either to the normal acceptor site or to an alternate site within the intervening sequence. The alternately spliced mRNA is also found in liver where it accounts for 20-40% of ATIII-specific transcripts. The alternate splicing event introduces a 42-nucleotide segment between codons -19 and -18 of the signal peptide. This segment contains an in-frame ochre termination codon such that the predicted protein product encoded by the alternately spliced mRNA would be only 19 amino acids long. The minigene is transcribed to high levels in mouse fibroblasts when present as an episome in a bovine papilloma virus-derived vector although no protein product is detected.