Agda M. Simpson

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.

Kinetoplast RNA of Leishmania tarentolae

RNA has been isolated from highly purified kinetoplast-mitochondrial fractions of Leishmania tarentolae, and shown to consist of two major species that sediment at 9S and 12S in sucrose and also several additional low molecular weight species which were visualized by gel electrophoresis. The in vivo transcription of 9S and 12S RNAs was inhibited by ethidium bromide and rifampin, and was fairly insensitive to low actinomycin D and camptothecin. The 9S and 12S RNAs were isolated by acrylamide gel electrophoresis or by sedimentation in sucrose. Both RNAs contained approximately 80% A + U and did not contain long stretches of poly(A). The 9S and 12S RNAs were found to hybridize selectively to the maxicircle sequences of the kinetoplast DNA, implying that the maxicircle, and not the minicircle, represents the informational mitochondrial DNA in the kinetoplast.

Characterization of two classes of ribonucleoprotein complexes possibly involved in RNA editing from Leishmania tarentolae mitochondria

The molecular mechanism of RNA editing in trypanosomatid mitochondria is an unsolved problem. We show that two classes of ribonucleoprotein complexes exist in a mitochondrial extract from Leishmania tarentolae and appear to be involved in RNA editing. The ‘G’ class of RNP complexes consists of 170‐300 A particles which contain guide RNAs and proteins, show little terminal uridylyl transferase (TUTase) activity and exhibit an in vitro RNA editing‐like activity. The ‘T’ class consists of approximately six RNP complexes, the endogenous RNA of which can be self‐labeled with [alpha‐32P]UTP. The most abundant T complex, T‐IV, is visualized by electron microscopy as 80‐140 A particles. This complex exhibits TUTase activity in the native gel and contains guide RNAs. Both G and T complexes are possibly involved with RNA editing in vivo. These results are a starting point for the analysis of the biochemistry of RNA editing.

Native gel analysis of ribonucleoprotein complexes from a Leishmania tarentolae mitochondrial extract.

Two polypeptides of 50 and 45 kDa were adenylated by incubation of a mitochondrial extract from Leishmania tarentolae with [alpha-32P]ATP. These proteins were components of a complex that sedimented at 20S in glycerol gradients and migrated as a single band of approximately 1800 kDa in a native gel. The facts that RNA ligase activity cosedimented at 20S and that the ATP-labeled p45 and p50 polypeptides were deadenylated upon incubation with a ligatable RNA substrate suggested that these proteins may represent charged intermediates of a mitochondrial RNA ligase. Hybridization of native gel blots with guide RNA (gRNA) probes showed the presence of gRNA in the previously identified T-IV complexes that sedimented in glycerol at 10S and contained terminal uridylyl transferase (TUTase) activity, and also in a previously unidentified class of heterodisperse complexes that sedimented throughout the gradient. gRNAs were not detected in the p45 + p50-containing 1800 kDa complex. The heterodisperse gRNA-containing complexes were sensitive to incubation at 27 degrees C and appear to represent complexes of T-IV subunits with mRNA. Polyclonal antiserum to a 70 kDa protein that purified with terminal uridylyl transferase activity was generated, and the antiserum was used to show that this p70 polypeptide was a component of both the T-IV and the heterodisperse gRNA-containing complexes. We propose that the p45 + p50-containing 1800 kDa complex and the p70 + gRNA-containing heterodisperse complexes interact in the editing process. Further characterization of these various complexes should increase our knowledge of the biochemical mechanisms involved in RNA editing.

Comparison of several lizard Leishmania species and strains in terms of kinetoplast minicircle and maxicircle DNA sequences, nuclear chromosomes, and membrane lipids.

Eight strains of a lizard Leishmania species, L. tarentolae, were compared with four other saurian species [L. hoogstrali, L. adleri, L. agamae and Leishmania sp. LizS], with L. major from man and with Trypanosoma platydactyli, a putative lizard trypanosome, in terms of kinetoplast DNA minicircle and maxicircle sequences and in terms of nuclear chromosome patterns on orthogonal gel electrophoresis. The L. tarentolae strains fell into two major groups, one (group A) consisting of the L. tarentolae strains, UC, Krassner and Trager, derived from an Algerian gecko isolate and the other (group B) consisting of five L. tarentolae LEM strains isolated from geckos in southern France. T. platydactyli TPCL2, which was postulated by Wallbanks et al. to represent the lizard form of a French L. tarentolae strain, was closely related to the UC strain and not to the LEM strains, in all respects analyzed. Leishmania sp. LizS from a Mongolian gecko and L. hoogstrali from a Sudanese gecko showed some sequence similarities to the L. tarentolae strains, but the leishmanias said to be L. adleri from a Kenyan lacertid and L. agamae from an Israeli agamid showed no minicircle sequence similarities with lizard Leishmania and in fact were probably the same species. The maxicircle divergent region was larger in the group B strains than in the group A strains, but there were sequences in common with both groups, and not with L. hoogstrali and L. major. Four strains of L. tarentolae, the four other supposed saurian Leishmania species, three mammalian leishmanias, T. platydactyli and four other trypanosomes, T. cyclops (Malaysian macaque), T. conorrhini (Hawaiian reduviid bug), T. cruzi (man) and T. lewisi (feral rat) were analyzed for their contents of sterols and phosphoglyceride fatty acyl groups. T. platydactyli TPCL2 contained a sterol (5-dehydroepisterol), a phosphatidylcholine fatty acyl group (alpha-linolenic acid) and a phosphatidylethanolamine fatty acyl group (dihydrosterculic acid) characteristic of members of the genus Leishmania and not the genus Trypanosoma. The proportions of those lipids in the free sterol and phosphoglyceride fractions of T. platydactyli TPCL2 most closely resembled those seen in the Leishmania strains from Algerian, French, Mongolian and Sudanese geckos.

REPLICATION OF THE KINETOPLAST DNA OF LEISHMANIA TARENTOLAE AND CRITHIDIA FASCICULATA

Abstract 1. Replicating kinetoplast DNA networks from both Crithidia fasciculata and Leishmania tarentolae have an equilibrium density in ethidium bromide—CsCl which is less than that of covalently closed non-replicating networks. After a few hours of chase, these networks assume the covalently closed position in the gradient. 2. Pulse-labeled minicircles isolated from sonicated networks band in the upper position in ethidium bromide—CsCl equilibrium gradients. Both “free” and network minicircles incorporate [3H] thymidine at the same rate in a pulse. Labeled single stranded fragments of less than unit minicircle length are released from pulse-labeled minicircles in alkali. 3. Intact networks of Leishmania and Crithidia can be isolated in the process of replication: replication involves a doubling of the surface area of the networks as visualized by spreading the kinetoplast DNA on glass slides, staining and examining in the light microscope. 4. DNA replication within the kinetoplast DNA networks of both Leishmania and Crithidia in all parts of the S phase is restricted to the periphery of the structures. In C. fasciculata the pulse-labeled DNA remains in position as the network enlarges by peripheral growth, and then becomes redistributed throughout the network sheet by an unknown mechanism after one cell generation. 5. In the case of L. tarentolae it was demonstrated by density transfer experiments that all network minicircles replicate by an apparent semi-conservative pattern in one cell generation. This implies that there is some type of lateral mobility of molecules within the network, by which minicircles move past the peripheral locus of replication.

In vitro RNA editing-like activity in a mitochondrial extract from Leishmania tarentolae.

A mitochondrial extract from Leishmania tarentolae directs the incorporation of uridylate (U) residues within the pre‐edited domain of synthetic cytochrome b (CYb) and NADH dehydrogenase subunit 7 mRNA. This has several characteristics of an in vitro RNA editing activity, but no direct evidence for involvement of guide RNAs was obtained. Inhibition by micrococcal nuclease suggests a requirement for some type of endogenous RNA. The limitation of internal U‐incorporation to the pre‐edited region in the CYb mRNA and the inhibition by deletion or substitution of both mRNA anchor sequences for CYb gRNA‐I and ‐II could be consistent either with a gRNA‐mediated process or a secondary structure‐mediated process. A low level of incorporation of [alpha‐32P]CTP occurs at the same sites as UTP. Internal U‐incorporation activity is selectively inhibited by heterologous RNAs, suggesting an involvement of low affinity RNA‐binding proteins which can be competed by the added RNA.

Guide RNAs of the recently isolated LEM125 strain of Leishmania tarentolae: An unexpected complexity

Guide RNAs (gRNAs) are encoded both in the maxicircle and minicircle components of the mitochondrial DNA of trypanosomatid protozoa. These RNAs mediate the precise insertion and deletion of U residues in transcripts of the maxicircle DNA. We showed previously that the old UC laboratory strain of Leishmania tarentolae apparently lost more than 40 minicircle-encoded gRNAs that are present in the recently isolated LEM125 strain (Thiemann et al., EMBO J, 1994, 13:5689-5700]. We have further analyzed the population of minicircle-encoded gRNAs in the LEM125 strain. Sau3AI and MspI minicircle libraries were constructed and screened for novel gRNAs by negative colony hybridization. This search yielded 20 minicircles encoding new gRNAs that covered most of the remaining gaps in the editing cascades of the ND8, ND9, G4, and G5 genes, and in addition, more than 30 minicircles containing either unassigned or undetectable gRNA genes. We also completely sequenced 34 of the 45 minicircle sequence classes encoding previously identified gRNAs. A total of 19 pairs of redundant gRNAs, which are gRNAs of different sequences covering the same editing blocks, were identified. The gRNAs in each redundant pair generally had different relative abundances and different extents of mismatches with edited sequences. Alignments of the minicircles encoding redundant gRNAs yielded 59 to 93% matching nucleotides, suggesting an origin from duplication of ancestral minicircles and subsequent genetic drift. We propose a functional explanation for the existence of redundant gRNAs in this strain.

Kinetoplast DNA and RNA of Trypanosoma brucei.

Kinetoplast DNA (kDNA) and kinetoplast RNA (kRNA) were isolated from bloodstream and procyclic culture forms of two clonal strains of Trypanosoma brucei. No differences were observed in kDNA (maxicircle) restriction profiles between bloodstream or procyclic culture forms of the same strain. Some differences were observed in kDNA maxicircle restriction sites between the two strains. Buoyant density analysis of Pst I digested kDNA showed the release of a minor low density band representing unit length linearized maxicircle DNA. Pst I or Bam H1-linearized maxicircle DNA was isolated by the Hoechst 33258 dye--CsCl method and a restriction enzyme map of the maxicircle was constructed. Closed monomeric minicircles released from kDNA networks by sonication sedimented with a molecular size of around 1100 base pairs. A substantial minor length heterogeneity was evident in acrylamide gel electrophoresis of once cut minicircles. Several minicircle sequence classes and two Hind III maxicircle fragments representing approx. 50% of the maxicircle were cloned in the bacterial plasmid, pBR322, in Escherichia coli. A purified kinetoplast-mitochondrion fraction was isolated from procyclic culture forms by the Renografin flotation method. The major kRNA components were two small RNAs which comigrated with Leishmania tarentolae 9 and 12 S kRNAs in denaturing gels. These RNAs hybridized to the maxicircle component of the kDNA, specifically to the smaller cloned Hind III maxicircle fragment. This cloned fragment had substantial sequence homology with the cloned maxicircle fragment from L. tarentolae which contains the 9 and 12 S RNA genes, implying an evolutionary conservation of the 9 and 12 S gene sequences. Identical kRnAs were observed in cultured bloodstream forms of T. brucei.

Restriction map, partial cloning and localization of 9S and 12S kinetoplast RNA genes on the maxicircle component of the kinetoplast DNA of Leishmania tarentolae

We have constructed a restriction map of the maxicircle component of the kinetoplast DNA of Leishmania tarentolae for the enzymes EcoRI, Bam HI, HaeIII, HpaII, SalI, BglII and HindIII. The 9 and 12S kinetoplast RNAs were localized on this map. Two fragments of this maxicircle molecule were cloned in the bacterial plasmid, pBR322, including a 4.4 . 10(6) dalton EcoRI/BamHI fragment which contains the 9 and 12S RNA genes.