The biological affinities of hydrogenosomes, characteristic organelles of trichomonad flagellates, remain obscure (Miiller, 1980, Symp. Soc. Gen. Microbiol. 30: 127-142). Based on metabolic similarities they have been compared with anaerobic bacteria lacking cytochromes (Cerkasovovfi et al., 1980. In Industrial and clinical enzymology, L. Vitale and V. Simeon (eds.), pp. 257-275; Muller, 1980, loc. cit.) and it was suggested that they might be the anaerobic equivalents of mitochondria and might have a similar relationship to anaerobic bacteria as do mitochondria to aerobic bacteria and chloroplasts to cyanobacteria. This suggestion led to a search for DNA in these organelles. Cerkasovovfa et al. (1976, Folia Parasitol. Praha 23: 33-37) published electron microscopic observations on circular DNA from Tt. foetus hydrogenosomes isolated by density gradient centrifugation and subsequently treated with DNAse (Nass, 1969, J. Mol. Biol. 42: 521-528). To test this problem by an independent approach, we applied to trichomonads methods used to detect and study mitochondrial DNA (mtDNA) in various eukaryotic cells, including eukaryotic microorganisms (Williamson and Fennel, 1975. In Methods in Cell physiology, D. Prescott (ed.), 12: 235-351). Trichomonas vaginalis ATCC 30001 strain and Tt. foetus KVi strain were grown in TYM medium supplemented with 10% horse serum. Following the methods described by Williamson and Fennel (1975, loc. cit.) unfixed cells and cells fixed in formaldehyde were exposed to 4',6-diamidine2-phenylindole (DAPI), a fluorochrome strongly interacting with DNA, especially with A+T rich DNA. Under the fluorescence microscope such cells showed a clear fluorescence of the nucleus only, but none in the cytoplasm nor in any cytoplasmic organelle. Prior to extraction of DNA, cells were washed twice by gentle centrifugation with 250 mM sucrose solution containing 200 ,iM aurintricarboxylic acid (ATA). DNA was extracted either from whole, washed cells or from a large-particle fraction enriched fivefold in hydrogenosomes by differential centrifugation (Lindmark and Miiller, 1974, J. Biol. Chem. 248: 7724-7728). ATA was present in all solutions used in the fractionation procedure. Two methods were used for preparation of DNA. The first method was based on that of Cummings et al. (1979, Molec. Gen. Genet. 171: 229-238). To a pellet of whole cells or large particles of T. vaginalis resuspended in minimum volume of sucrose about 10 volumes of lysis buffer was added (250 mM Tris HC1, pH 8.0, 100 mM EDTA, 200 ,tM ATA, and 1% SDS) at 65 C, and the lysate was maintained at this temperature for about 15 min. The cells or subcellular organelles lysed within a few seconds and the suspension became viscous. The lysate was then centrifuged at 20,000 g for 15 min at 0 C and the supernatant solution retained. After addition of CsCl, the solution was recentrifuged at 20,000 g for 15 min to remove most of the remaining SDS and protein. When this method was applied to Tt. foetus, the DNA was rapidly degraded after heating to 65 C, so lysis was carried out at 0 C and within 30 sec the lysate was extracted with an equal volume of buffer-saturated phenol, followed by chloroform extraction and ethanol precipitation of the nucleic acids. The precipitate was dissolved in 250 mM Tris HC1, 100 mM EDTA (pH 8.0), and CsCl added. The second method is similar to that used previously by Mandel and Honigberg (1964, J. Protozool. 11: 114-116) to obtain high MW DNA from Trichomonas spp. To 4 ml of solution prepared by either method, 4.2 g CsCl and 500 jtg DAPI were added, and the samples were centrifuged for 24 hr at 42,000 rev min-l in the angle head of an MSE Superspeed 50 ultracentrifuge. Only one fluorescent band was observed in gradients containing DNA isolated from whole cells or hydrogenosome enriched fractions of either trichomonad species, and it probably represents nuclear DNA (Fig. 1A). A DNA species significantly lighter or
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Jul 1, 1984
G N Bowers + 1
Open Access
