Characterization of the gene for human plasminogen, a key proenzyme in the fibrinolytic system.

T E Petersen,M R Martzen,A Ichinose,E W Davie

doi:10.1016/s0021-9258(19)39298-1

T E Petersen, M R Martzen + Show 2 more

Open Access

https://doi.org/10.1016/s0021-9258(19)39298-1

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Abstract

The organization and structure of the gene coding for plasminogen has been determined by a combination of in vitro amplification of leukocyte DNA from normal individuals and isolation of unique clones from three different human genomic libraries. These clones were characterized by restriction mapping, Southern blotting, and DNA sequencing. The gene for human plasminogen spanned about 52.5 kilobases of DNA and consisted of 19 exons separated by 18 introns. DNA sequence analysis revealed that the five kringle structures in plasminogen were coded by two exons. The nucleotides in the introns at the intron-exon boundaries were GT-AG analogous to those found in other eukaryotic genes. Three polyadenylation sites for plasminogen mRNA were also identified. When the amino acid sequences deduced from the genomic DNA and cDNAs of plasminogen were compared with that of the plasma protein determined by amino acid sequence analysis, an apparent amino acid polymorphism was observed in several positions of the polypeptide chain. Nucleotide sequence analysis of the amplified genomic DNAs and genomic clones also revealed that the plasminogen gene was very closely related to several other proteins, including apolipoprotein(a). This protein may have evolved via duplication and exon shuffling of the plasminogen gene. The presence of another plasminogen-related gene(s) in the human genomic library was also observed.

Highlights

PROCEDURESRestriction endonucleases, nuclease Bal-31, and T4 DNA ligase were purchased from Bethesda Research Laboratories or New England Biolabs
The organization and structure of the gene coding for plasminogen has been determined by a combination of in vitro amplification of leukocyte DNA from normal individuals and isolation of unique clones from three different human genomic libraries
When the amino acid sequences deduced from the genomic DNA and cDNAs of plasminogen were compared with that of the plasma protein determined by amino acid sequence analysis, an apparent amino acid polymorphism was observed in several positions of the polypeptide chain

Summary

PROCEDURES

Restriction endonucleases, nuclease Bal-31, and T4 DNA ligase were purchased from Bethesda Research Laboratories or New England Biolabs. The six overlapping X phage clones with DNA inserts coding for plasminogen are shown. To obtain genomic clones containing certain exons, appropriate restriction fragments from the cDNA or synthetic oligonucleotides were used for further screening or for identification of isolated clones by Southern blot analysis. To select the correct genomic clones coding for plasminogen and to exclude those for a plasminogen-related gene(s), the isolated phage clones were first amplified by the polymerase chain reaction, as described. The phage clones that were shown to contain the nucleotide sequences coding for exons that matched the corresponding regions of the cDNAs for plasminogen were employed for further analysis

RESULTS AND DISCUSSION

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1: II II I 0

Kringle4