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Characterization of a complementary deoxyribonucleic acid coding for human and bovine plasminogen
Abstract:
A cDNA library was constructed in pBR322 from bovine liver mRNA that was enriched for plasminogen mRNA by polysome immunoprecipitation. A 32P-labeled single-stranded cDNA was then prepared from the enriched bovine mRNA and employed as a probe to screen the cDNA library. The screening was carried out by testing for clones that protect the hybridized 32P-labeled cDNA from S1 nuclease digestion. The longest clone that was found was 581 base pairs in length and coded for the C-terminal 107 amino acids of bovine plasminogen, a 3' noncoding region of 246 nucleotides and a poly(A) tail. The bovine cDNA clone was then used as a probe to screen a human liver cDNA library of 18 000 recombinants. Six isolates were found to contain human plasminogen sequences. The longest clone consisted of 1851 base pairs corresponding to amino acid residues 272-790, followed by a 3' noncoding region of 227 base pairs and a poly(A) tail. Restriction fragments of the human cDNA were then used as probes to screen a human genomic DNA library present in a Charon 4A lambda phage library. Approximately 50 isolates from 10(6) recombinants were identified that hybridized to varying degrees with the cDNA probe. Among these, 10 corresponding to the gene for human plasminogen have been analyzed, and 3 that overlap have been shown to extend from kringle 3 through the 3' noncoding region of the gene. A 160 base pair exon with flanking splice junctions was then characterized and shown to encode for the first half of plasminogen kringle 4, including amino acid residues 346-399.
Insights
Researchers isolated bovine and human plasminogen cDNA clones, aiding in the characterization of the human plasminogen gene. This study advances understanding of plasminogen gene structure and function.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Plasminogen is a crucial protein in the fibrinolytic system.
- Understanding plasminogen gene structure is vital for studying its role in hemostasis and disease.
Purpose of the Study:
- To isolate and characterize cDNA clones encoding bovine and human plasminogen.
- To identify and analyze the human plasminogen gene structure.
Main Methods:
- Construction and screening of bovine and human liver cDNA libraries using enriched mRNA and cDNA probes.
- Screening of a human genomic DNA library using restriction fragments of human plasminogen cDNA.
- Characterization of specific exons and splice junctions within the human plasminogen gene.
Main Results:
- Isolation of a bovine plasminogen cDNA clone encoding the C-terminal region.
- Identification of six human plasminogen cDNA clones, with the longest containing residues 272-790.
- Characterization of human genomic DNA isolates containing parts of the plasminogen gene, including an exon encoding part of kringle 4.
Conclusions:
- The study successfully isolated key cDNA fragments for bovine and human plasminogen.
- The findings provide insights into the structure of the human plasminogen gene, particularly kringle domains.
- This work facilitates further research into plasminogen regulation and function.