Synthesis of Catalytically Active Polymerase α by in Vitro Translation of Calf RNA
The cell-free synthesis of DNA polymerase in translation mixtures containing calf thymus total and poly (A+) RNA was examined using activity gel analysis and immunobinding with a monoclonal antibody to calf thymus α-polymerase. Activity gel analysis indicated that functional DNA polymerase catalytic polypeptides of Mr = 110, 000 to 120, 000 and Mr ~ 68, 000 were synthesized. Sucrose density gradient centrifugation of total RNA resulted in resolution and partial purification of the mRNAs encoding these two DNA polymerase polypeptides. Immunobinding experiments with the monoclonal antibody to calf α-polymerase confirmed that an immunoreactive polypeptide of 110 to 120 kilo-daltons had been formed in vitro. This polypeptide and the 68, 000-Mr polypeptide correspond in size to α-polymerase catalytic polypeptides observed in crude extracts of calf cells and in purified calf α-polymerase.
KeywordsSodium Dodecyl Sulfate Sucrose Density Gradient Centrifugation Wheat Germ Extract Methionine Incorporation Translation Mixture
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