15b] The preparation of RNA-directed DNA polymerase from ovaries of Xenopus laevis

Abstract

Publisher Summary This chapter describes the preparation of RNA-Directed DNA polymerase from ovaries of Xenopus laevi . Young oocytes of Xenopus laevis synthesize DNA that corresponds to a selected portion of the chromosome—the genes coding for ribosomal RNA. Several assays have been used during the isolation of this RNA-directed DNA polymerase activity. First, the synthesis of DNA using native calf thymus DNA as a template has been routinely used during the enzyme purification. Second, enzyme fractions have been assayed for their ability to use the artificial homopolymers poly rA:dT 10 and poly dA:dT 10 , as templates. It has been suggested that the preferential use of poly rA:dT 10 as a template is a characteristic trait of RNA-directed DNA-polymerases. Third, as only very small amounts of the supposed natural template can be isolated, an indirect assay has been developed that measures the use of the long transcript as a template by its binding to Millipore filters.