Quantification of tissue-type plasminogen activator (t-PA) mRNA in human endothelial-cell cultures by hybridization with a t-PA cDNA probe
article
We describe the construction of a recombinant DNA plasmid, consisting of the vector pBR322 and full-length tissue-type plasminogen-activator (t-PA) cDNA, by using polyadenylated RNA from cultured Bowes melanoma cells as substrate. A 1280-base-pair Pstl restriction fragment, covering the 3' untranslated region and part of the coding region for the t-PA L-chain, was used as a radiolabelled probe to determine the size and the number of t-PA mRNA molecules in cultured endothelial cells of different origin from the same individual. Northern blotting showed that in all these cells a t-PA mRNA is synthesized of about 2500 nucleotides, indicating that transcriptional initiation, splicing and polyadenylation is similar. The number of t-PA mRNA molecules per cell measured, by using a dot-blotting technique and t-PA mRNA made in vitro, with a plasmid DNA preparation harbouring a specific promotor of the Salmonella typhimurium bacteriophage SP6, t-PA cDNA and SP6 RNA polymerase as standard, is approx. 10000 in all cultured endothelial cells from adult vessels. However, the amount oft-PA antigen synthesized and/or secreted differs by a factor of 6-20. Relatively large amounts oft-PA antigen secreted were detected in conditioned medium from vena-cava-derived cells, whereas low amounts were found in conditioned medium from arteria-iliaca-derived cells.
TNO Identifier
287144
Source
Biochemical journal, 235, pp. 385-390.
Pages
385-390
Files
To receive the publication files, please send an e-mail request to TNO Repository.