Isolation and characterization of kinetoplast DNA from Leishmania tarentolae

Kinetoplast DNA (? = 1.703 g/ml.) was isolated by preparative cesium chloride ultracentrifugation in a fixed-angle rotor from total cell DNA of Leishmania tarentolae and examined in terms of sedimentation properties, melting characteristics, and appearance in the electron microscope. It consisted of several molecular types, either free or bound together in associations of variable size: minicircles (molecular weight = 0.56 ± 0.03 × 10⁶), catenated minicircles, “figure 8” molecules, and long molecules. The associations seem to be held together by the long molecules threading through the smaller circles and catenanes. The large associations could be broken down by sonication, DNase II-treatment, or shear forces. Minicircles, catenated dimers, trimers, and small linear fragments were separated on preparative sucrose gradients of sonicated DNA, and S_20,w values were assigned to each molecular type by band sedimentation in the analytical ultracentrifuge.     

Selective importation of RNA into isolated mitochondria from Leishmania tarentolae

All mitochondrial tRNAs in kinetoplastid protozoa are encoded in the nucleus and imported from the cytosol. Incubation of two in vitro-transcribed tRNAs, tRNA(Ile)(UAU) and tRNA(Gln)(CUG), with isolated mitochondria from Leishmania tarentolae, in the absence of any added cytosolic fraction, resulted in a protease-sensitive, ATP-dependent importation, as measured by nuclease protection. Evidence that nuclease protection represents importation was obtained by the finding that Bacillus subtilis pre-tRNA(Asp) was protected from nuclease digestion and was also cleaved by an intramitochondrial RNase P-like activity to produce the mature tRNA. The presence of a membrane potential is not required for in vitro importation. A variety of small synthetic RNAs were also found to be efficiently imported in vitro. The data suggest that there is a structural requirement for importation of RNAs greater than approximately 17 nt, and that smaller RNAs are apparently nonspecifically imported. The signals for importation of folded RNAs have not been determined, but the specificity of the process was illustrated by the higher saturation level of importation of the mainly mitochondria-localized tRNA(Ile) as compared to the level of importation of the mainly cytosol-localized tRNA(Gln). Furthermore, exchanging the D-arm between the tRNA(Ile) and the tRNA(Gln) resulted in a reversal of the in vitro importation behavior and this could also be interpreted in terms of tertiary structure specificity.     

Isolation of a U-insertion/deletion editing complex from Leishmania tarentolae mitochondria

A multiprotein, high molecular weight complex active in both U-insertion and U-deletion as judged by a pre-cleaved RNA editing assay was isolated from mitochondrial extracts of Leishmania tarentolae by the tandem affinity purification (TAP) procedure, using three different TAP-tagged proteins of the complex. This editing- or E-complex consists of at least three protein-containing components interacting via RNA: the RNA ligase-containing L-complex, a 3' TUTase (terminal uridylyltransferase) and two RNA-binding proteins, Ltp26 and Ltp28. Thirteen approximately stoichiometric components were identified by mass spectrometric analysis of the core L-complex: two RNA ligases; homologs of the four Trypanosoma brucei editing proteins; and seven novel polypeptides, among which were two with RNase III, one with an AP endo/exonuclease and one with nucleotidyltransferase motifs. Three proteins have no similarities beyond kinetoplastids.     

Isolation and characterization of a U-specific 3'-5'-exonuclease from mitochondria of Leishmania tarentolae

We have purified a 3'-5'-exoribonuclease from mitochondrial extract of Leishmania tarentolae over 4000-fold through six column fractionations. This enzyme digested RNA in a distributive manner, showed a high level of specificity for 3'-terminal Us, and was blocked by a terminal dU; there was slight exonucleolytic activity on a 3'-terminal A or C but no activity on a 3'-terminal G residue. The enzyme preferred single-stranded 3'-oligo(U) overhangs and did not digest duplex RNA. Two other 3'-5'-exoribonuclease activities were also detected in the mitochondrial extract, one of which was stimulated by a 3'-phosphate and the other of which degraded RNAs with a 3'-OH to mononucleotides in a processive manner. The properties of the distributive U-specific 3'-5'-exoribonuclease suggest an involvement in the U-deletion RNA editing reaction that occurs in the mitochondrion of these cells.     

Effect of acriflavin on the kinetoplast of Leishmania tarentolae. Mode of action and physiological correlates of the loss of kinetoplast DNA

The loss of kinetoplast DNA in Leishmania tarentolae, which occurs in the presence of low concentrations of acriflavin, was found to be a result of selective inhibition of replication of this DNA. Nuclear DNA synthesis was relatively unaffected and cell and kinetoplast division proceeded normally for several generations. An approximately equal distribution of parental kinetoplast DNA between daughter kinetoplasts resulted in a decrease in the average amount of DNA per kinetoplast. The final disappearance of the stainable kinetoplast DNA occurred at a cell division in which all the remaining visible kinetoplast DNA was retained by one of the daughter cells. The selective inhibition of kinetoplast DNA synthesis was caused by a selective localization of acriflavin in the kinetoplast. The apparent intracellular localization of dye and the extent of uptake at a low dye concentration could be manipulated, respectively, by varying the hemin (or protoporphyrin IX) concentration in the medium and by adding red blood cell extract (or hemoglobin). Hemin and protoporphyrin IX were found to form a complex with acriflavin. During growth in acriflavin, cells exhibited an increasing impairment of colony-forming ability and rate of respiration. No change in the electrophoretic pattern of total cell soluble proteins was apparent. The data fit the working hypothesis that the loss of kinetoplast DNA leads to a respiratory defect which then leads to a decrease in biosynthetic reactions and eventual cell death. A possible use of the selective localization of acriflavin in the kinetoplast to photooxidize selectively the kinetoplast DNA is suggested.     

Sequences of six genes and several open reading frames in the kinetoplast maxicircle DNA of Leishmania tarentolae

The DNA sequence of approximately 80% of the transcribed region of the kinetoplast maxicircle DNA of Leishmania tarentolae was obtained, and structural genes were localized by comparison of the translated amino acid sequences with those of known mitochondrial genes from other organisms. By this method, the genes for cytochrome oxidase subunits I, II, and III, cytochrome b, and human mitochondrial unidentified reading frames 4 and 5 were identified. By comparing the amino acid sequences of the putative L. tarentolae genes with those of known genes, we conclude that TGA codes for tryptophan, as in most other mitochondrial systems. This is the only apparent change from the universal genetic code. The six identified structural genes show various degrees of divergence from the homologous genes in other species, with cytochrome oxidase subunit I being the most conserved and cytochrome oxidase subunit III being the least conserved. A comparison of the cytochrome b genes from L. tarentolae and Trypanosoma brucei showed that the ratio of transversions to transitions is 1:1, suggesting that these species diverged from each other more than 80 X 10(6) years ago. Several as yet unidentified open reading frames were also present in the maxicircle sequence. These data confirm that maxicircle DNA has a coding potential which typifies other mitochondrial systems.     

Characterization of a protein fraction containing cytochromes b and c1 from mitochondria of Leishmania tarentolae

A soluble red band fraction was obtained from Leishmania tarentolae cells by sucrose gradient sedimentation of a Triton X-100 lysate. Spectral analysis indicated that cytochrome b was present in the red band: the reduced minus oxidized difference spectra revealed absorption maxima at 562,527, and 431 nm at room temperature and 562, 530, and 422 nm at 77K. In addition, a 28-kDa protein was identified in this fraction which retained heme-associated peroxidase activity even after denaturation on SDS-polyacrylamide gels. The amino acid composition of this protein showed a strong similarity to cytochrome c1 of both bovine and yeast.     

Isolation of Kinetoplast-Mitochondrial Complexes from Leishmania tarentolae*

SYNOPSIS. Kinetoplast-mitochondrial complexes were liberated from Leishmania tarentolae by passing hypotonically swollen cells in dilute Tris-EDTA through a needle at 100 1bs/in². The complexes formed an equilibrium band by flotation in Renografin gradients at a density of 1.22 g/ml. The band was monitored by several mitochondrial and kinetoplastic markers: [³H]DNA, succinate-cytochrome c reductase activity, [⁵⁰Fe]hemoproteins and optical density at 600 nm. Electron microscopy showed that the sole component of the 1.22 g/ml band was the kinetoplast-mitochondrial complex.     

Specific cleavage of kinetoplast minicircle DNA from Leishmania tarentolae by mung bean nuclease and identification of several additional minicircle sequence classes

Multiple sequence classes of kinetoplast minicircle DNA from Leishmania tarentolae were cleaved by mung bean nuclease in the presence of formamide, yielding unit length linear molecules which retained the anomalous electrophoretic mobility in acrylamide characteristic of minicircle DNA. No specific cleavage site sequence common to all minicircle sequence classes was apparent, although the main region of nuclease cleavage was localized approximately 350 bp from the unique SmaI restriction site of the conserved region found in all minicircle sequence classes. Covalent closure of the minicircle substrate was not a requirement for cleavage, as linearized network-derived or cloned minicircles were also cleaved by mung bean nuclease at similar locations. The partial sequences of several new minicircle sequence classes released from the network by mung bean nuclease are also reported.     

The divergent region of the Leishmania tarentolae kinetoplast maxicircle DNA contains a diverse set of repetitive sequences.

A 2.76 kb segment of the 12 kb divergent region of the Leishmania tarentolae kinetoplast maxicircle DNA consists almost entirely of repeated sequences. The repeats can be grouped into six families, some of which are present throughout the remainder of the divergent region. The repeats are oriented in a head-to-tail fashion with the three simplest repeats clustered into large arrays. A 47 bp palindrome and two copies of a "supercluster" of three different types of repeats are also present in the sequenced region. A sequence change in the divergent region is described for a clonal strain of L. tarentolae which was passaged continuously for several years. The repetitive sequences found in the divergent region appear to be appropriate substrates for the presumed deletion/insertion/recombination events occurring in this rapidly evolving portion of the maxicircle.