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DNA-dependent RNA Polymerases from Drosophila melanogaster Larvae
Abstract
INTRODUCTION
Because of the extensive knowledge of its genetics, developmental biology and cytology, Drosophila melanogaster offers unique advantages for studying the properties and biological roles of eukaryotic DNA-dependent RNA polymerases. Although this realization has prompted previous attempts to isolate these enzymes from this organism (Natori 1972; Phillips and Forrest 1973; Adoutte, Clément and Hirshbein 1974), no extensive purification of Drosophila RNA polymerases has yet been reported. For our attempts to purify extensively the RNA polymerases from Drosophila melanogaster we decided to use third instar larvae because they represent the material most easily obtained in large amounts. Starting from kilogram quantities of larvae we hoped to obtain enough enzymes in pure form to establish their polypeptide compositions and to raise antipolymerase antibodies for use in studying the distribution of the polymerases in different cell types. We have succeeded in isolating in essentially pure form milligram quantities of the α-amanitin-sensitive RNA polymerase B (or II) from the larvae and in obtaining a rabbit anti-serum directed against it; we have also partially purified the larval RNA polymerase A (or I). The details of the purification and characterization of the B enzyme have been described previously (Greenleaf and Bautz 1975). In this paper we present a summary of our studies on polymerase B and a comparison of some of its enzymatic and immunological properties with those of the larval polymerase A.
Because of the extensive knowledge of its genetics, developmental biology and cytology, Drosophila melanogaster offers unique advantages for studying the properties and biological roles of eukaryotic DNA-dependent RNA polymerases. Although this realization has prompted previous attempts to isolate these enzymes from this organism (Natori 1972; Phillips and Forrest 1973; Adoutte, Clément and Hirshbein 1974), no extensive purification of Drosophila RNA polymerases has yet been reported. For our attempts to purify extensively the RNA polymerases from Drosophila melanogaster we decided to use third instar larvae because they represent the material most easily obtained in large amounts. Starting from kilogram quantities of larvae we hoped to obtain enough enzymes in pure form to establish their polypeptide compositions and to raise antipolymerase antibodies for use in studying the distribution of the polymerases in different cell types. We have succeeded in isolating in essentially pure form milligram quantities of the α-amanitin-sensitive RNA polymerase B (or II) from the larvae and in obtaining a rabbit anti-serum directed against it; we have also partially purified the larval RNA polymerase A (or I). The details of the purification and characterization of the B enzyme have been described previously (Greenleaf and Bautz 1975). In this paper we present a summary of our studies on polymerase B and a comparison of some of its enzymatic and immunological properties with those of the larval polymerase A.
METHODS AND RESULTS
Purification of Larval RNA Polymerases; Polypeptide Composition
The most effective procedure we have used for solubilizing the RNA polymerase B...
DOI: http://dx.doi.org/10.1101/0.793-801