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Messenger RNA for human tissue plasminogen activator
Abstract:
A human melanoma cell line (Bowes), which secretes extrinsic (tissue-type) plasminogen activator, was used as a source for the preparation of mRNA for extrinsic plasminogen activator. The cells were lysed and total RNA was extracted with the phenol method. A poly(A)-rich RNA fraction was isolated by affinity chromatography on oligo(dT)-cellulose. This preparation was translated by oocytes into (a) protein(s) that had biological activity in the assay for extrinsic plasminogen activator. On sucrose gradient centrifugation the translatable fraction of the RNA migrated with a sedimentation coefficient of approximately 19 S, a value compatible with the molecular weight of extrinsic plasminogen activator (70 000). The translation product was characterized as being similar to or identical with authentic extrinsic plasminogen activator by the following criteria: (a) serological cross-reactivity with purified extrinsic plasminogen activator in neutralization and immunoprecipitation reactions; (b) plasminogen dependency of fibrinolytic activity and (c) apparent molecular weight of 70 000 on sodium dodecyl sulphate/polyacrylamide gel electrophoresis.
Insights
Researchers isolated mRNA from melanoma cells to produce extrinsic plasminogen activator (t-PA). The resulting protein demonstrated biological activity and characteristics identical to authentic t-PA.
Area of Science:
- Molecular Biology
- Biochemistry
Background:
- Human melanoma cells (Bowes) secrete extrinsic plasminogen activator (t-PA).
- t-PA plays a crucial role in fibrinolysis.
Purpose of the Study:
- To isolate and characterize the mRNA encoding extrinsic plasminogen activator (t-PA).
- To produce biologically active t-PA from the isolated mRNA.
Main Methods:
- Extraction of total RNA from melanoma cells.
- Isolation of poly(A)-rich RNA using oligo(dT)-cellulose chromatography.
- In vitro translation of RNA by oocytes.
- Sucrose gradient centrifugation to determine sedimentation coefficient.
- Characterization of the translation product using serological assays, fibrinolytic activity assays, and SDS-PAGE.
Main Results:
- A translatable mRNA fraction with a sedimentation coefficient of approximately 19 S was isolated.
- Oocyte translation produced a protein with extrinsic plasminogen activator biological activity.
- The translation product exhibited serological cross-reactivity, plasminogen dependency, and an apparent molecular weight of 70,000 Da, consistent with authentic t-PA.
Conclusions:
- The study successfully isolated mRNA encoding functional extrinsic plasminogen activator (t-PA).
- The characterized translation product is identical or highly similar to authentic t-PA, confirming the methodology.