Sequence Organization of Maxicircle Kinetoplast DNA from Leishmania tarentolae

Abstract

The kinetoplast DNA of the hemoflagellate protozoa (Simpson 1972) consists of two molecular species, the minicircles and the maxicircles, catenated together to form a giant network of DNA situated within the single mitochondrion of the cell (Borst and Hoeijmakers 1979a; Englund 1980). The maxicircle appears to represent the homolog of the informational mtDNA found in other cells (Simpson and Simpson 1978); the role of the minicircle is unknown. The major RNA species present in a purified kinetoplast fraction from Leishmania tarentolae are the 9S and 12S RNAs, which were shown to be transcribed from the maxicircle DNA in an ethidium-bromide-sensitive process (Simpson and Simpson 1978). Similar RNAs have also been isolated from purified kinetoplast fractions of Phytomonas davidae (Cheng and Simpson 1978), Trypanosoma brucei (Simpson and Simpson 1980), and Crithidia fasciculata (unpubl.). A high level of sequence conservation of 9S and 12S RNAs has been demonstrated for several hemoflagellate species by hybridization techniques (Simpson and Simpson 1978; Borst and Hoeijmakers 1979b; Simpson and Simpson 1980). In this paper we describe several additional maxicircle transcripts and present a general method to identify mitochondrial structural genes by the use of heterologous DNA probes.

MAXICIRCLE TRANSCRIPTS: NORTHERN BLOT ANALYSIS
Total kinetoplast RNA and poly(A)⁺ kinetoplast RNA were isolated from Renografin-purified kinetoplast fractions of L. tarentolae (Simpson and Simpson 1978), separated by electrophoresis in formaldehyde-agarose gels, and blotted onto nitrocellulose filters. The filters were probed with nick-translated maxicircle DNAs, including the 6.6-kb cloned EcoRI/BamHI fragment that contains the 9S RNA and 12S RNA genes...