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Isolation and Characterization of Kinetoplast DNA Networks and Minicircles from Crithidia fasciculata *
Author(s) -
SIMPSON AGDA M.,
SIMPSON LARRY
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.tb03751.x
Subject(s) - kinetoplast , minicircle , crithidia fasciculata , differential centrifugation , sedimentation coefficient , isopycnic , dna , crithidia , density gradient , biophysics , ultracentrifuge , biology , electron microscope , vesicle , monomer , biochemistry , microbiology and biotechnology , chemistry , centrifugation , genetics , physics , membrane , protozoa , organic chemistry , quantum mechanics , enzyme , polymer , optics
SYNOPSIS. Covalently closed kinetoplast DNA networks have been isolated from stationary phase Crithidia fasciculata cells by a technic involving selective pelleting of the networks at a low centrifugal field. Approximately 62% of the kinetoplast DNA of the cell was recovered free of nuclear DNA by simple differential centrifugation. Purified kinetoplast DNA networks were visualized both in the electron microscope and in the light microscope. Closed networks sedimented as a homogeneous band both in neutral and alkaline sucrose, with an s 20w in neutral sucrose of approximately 5 × 10 3 . Closed monomeric minicircles were isolated from purified networks by mild sonication and band sedimentation in alkaline sucrose. Several physical properties of closed monomeric minicircles were measured. These included molecular weight, buoyant density in CsCl, superhelix density and sedimentation coefficient.