Electron Microscopy of the Circular Kinetoplastic Dna From Trypanosoma Cruzi: Occurrence of Catenated Forms

Abstract

and Summary.-Circular kinetoplastic DNA was fractionated from Trypanosoma cruzi DNA by Hg-Cs2SO4 gradient. Covalantly closed circular DNA was isolated by buoyant density centrifugation in ethidium bromide CsCl density gradients. Electron We have previously described a circular DNA, 0.45 , long, extracted from the kinetoplast of Trypanosoma cruzi.1 In this paper we report the results of additional studies on this DNA: covalently closed circular duplexes and catenated forms of DNA circles sometimes associated with linear DNA. These different molecular forms of kinetoplastic DNA have been fractionated by buoyant density centrifugation with ethidium bromide in cesium chloride.Materials and Methods.-Culture of trypanosomes: Trypanosomes (Trypanosoma cruzi, strain "Institut Pasteur") were grown for 6 days at 27"C in 500-ml flasks containing 300 ml of Parker 199 medium supplemented with 10% of the liquid phase of Tobie medium; 250 IU of penicillin/ml and 50 ug of streptomycin/ml were also added to the medium.The trypanosomes (about 107 cells/ml) were washed five to six times with SSC (0.15 M NaCl-0.015 M sodium citrate) and collected by centrifugation at 1000 g.Preparation and fractionation of kinetoplastic DNA: Trypanosomes were lysed with 1% sodium dodecyl sulfate; DNA was extracted according to the method of Marmur;'0 two treatments with 50 jig RNase/ml were made under standard conditions. The material was subjected to preparative density gradient centrifugation in Cs2SO4 in the presence of mercuric ions. Kinetoplastic DNA, which has a base composition different from 210

Replicating, Covalently Closed, Circular DNA from Kinetoplasts of Trypanosoma cruzi

“…The formaldehyde vapors denature the cytochrome c film at the air-solution interface of the droplet. The molecules appear to be well extended, and their length is slightly increased as compared to our previous results obtained with other techniques (9,11 Fig. 1).…”

RNA editing in kinetoplastids

“…1 The kDNA in trypanosomes consists of thousands of small circular DNA molecules, called minicircles, which are catenated with larger circles, called maxicircles, to form a huge DNA network. [6][7][8] Besides the unique network organization, the amount of kDNA, relative to other mitochondrial DNAs, is equally unusual. In some trypanosomes up to 20% of the total DNA is organized in the kDNA, while for most other cells mitochondrial DNA is less than 1% of the total cellular DNA.…”

Trypanosoma brucei Tb927.2.6100 Is an Essential Protein Associated with Kinetoplast DNA

“…he mitochondrial DNA of kinetoplastid protozoa consists of tens of maxicircles and thousands of minicircles, all of which are intercatenated into a single network that resides within the cell's large single mitochondrion across from the flagellar basal body (1)(2)(3). Early cytologists named the highly visible region in cells stained for DNA the kinetoplast by virtue of its association with the base of the motile flagellum.…”