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In vitro Transcription of Kinetoplast and Nuclear DNA in Kinetoplastida * †
Author(s) -
HILL GEORGE C.,
BONILLA CARLOS A.
Publication year - 1974
Publication title -
the journal of protozoology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.067
H-Index - 77
eISSN - 1550-7408
pISSN - 0022-3921
DOI - 10.1111/j.1550-7408.1974.tb03717.x
Subject(s) - crithidia fasciculata , kinetoplast , biology , microbiology and biotechnology , dna , rna , ethidium bromide , dna polymerase , rna dependent rna polymerase , polymerase , rna polymerase , biochemistry , transcription (linguistics) , gene , linguistics , philosophy
SYNOPSIS. DNA‐dependent RNA polymerases have been solubilized from homogenates of Crithidia fasciculata using gentle extraction procedures. RNA polymerase I and II are separated on DEAE cellulose at 0.07M (NH 4 ) 2 SO 4 and 0.13M (NH 4 ) 2 SO 4 respectively. RNA polymerase II is inhibited 80% by α‐amanitin (25 μg/ml). Both RNA polymerases require DNA as a template, ribonucleoside triphosphates and Mn 2+ . The synthesis of RNA as a product is inhibited by DNase. RNase, pronase and actinomycin D. Purified kinetoplast and nuclear DNA can serve as templates for the RNA polymerases. Denatured DNA templates are preferred. The synthesis of RNA continues for at least an hour and is inhibited by trypanocidal drugs including suramin. antrycide, acriflavine, ethidium bromide and berenil. Complementary RNA synthesized in vitro from C. fasciculata kinetoplast DNA hybridizes with C. fasciculata kinetoplast DNA but not with C. fasciculata nuclear DNA or Blastocrithidia culicis kinetoplast DNA, Escherichia coli , T4 or calf thymus DNAs. The complementary RNA synthesized in vitro from C.fasciculata kinetoplast DNA sediments at 4–5S.