The de novo synthesis of cytochrome c oxidase subunits I, II (COI and COII), and apocytochrome b (Cyb) was investigated in kinetoplast-mitochondria of Leishmania. The organelles were isolated after breaking whole cells with nitrogen cavitation. Individual COI, COII, and Cyb polypeptides were identified by fractionation of the kinetoplast membranes, labeled with [ 35 S]methionine and cysteine, using two-dimensional (9 versus 14% and 20 versus 11%) denaturing gel electrophoresis. The reaction did not require exogenous energy sources or amino acids. On the contrary, the presence of amino acids other than methionine somewhat inhibited the labeling reaction probably by competing with the uptake of labeled amino acids. The synthesis reaction was insensitive to 100 g/ml chloramphenicol, gentamycin, paromomycin, lincomycin, hygromycin, and tetracycline, as well as cycloheximide. The process showed a linear increase in the amount of synthesized polypeptides during the first 2 h of incubation, followed by a slower accumulation of products for up to 4 h. The de novo synthesized polypeptides were stable for several additional hours. Their assembly into respiratory complexes, investigated using two-dimensional Blue Native/N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine-SDS gels, began early during the incubation and continued throughout the course of the synthesis. This work represents the first unequivocal identification of the polypeptide synthesis in kinetoplasts.The kinetoplast-mitochondrial genetic system of trypanosomes has revealed features that clearly distinguish it from animal, fungal, or plant mitochondria. The maxicircle component of kinetoplast DNA networks contains cryptic genes whose expression depends on post-transcriptional mRNA editing by uridylate insertions and deletions (1-3). This process is mediated by guide RNAs encoded in the kinetoplast DNA minicircles, which account for the bulk of DNA in these mitochondria. After the discoveries of RNA editing in 1986 (4) and guide RNAs in 1990 (5), there has been a steady progress toward elucidating the mechanism of this process, which has now been demonstrated to involve several enzymes and protein factors (6 -10).At the same time very little is known about other aspects of kinetoplast gene expression, especially at the level of translation. The very existence of a functional translation system remained unproven for almost 3 decades (summarized in Ref. 11). This question is important because kinetoplast translation must utilize templates modified or created by editing, and it is interesting to investigate interactions of these two systems. Early works demonstrated that cycloheximide at low concentrations effectively inhibited cytosolic translation in Crithidia luciliae and Trypanosoma brucei in vivo. A small fraction of the total cell protein synthesis was resistant to cycloheximide and at the same time insensitive to D-chloramphenicol and other inhibitors at concentrations that inhibit mitochondrial translation in other organisms (12, 13). However, high conce...

Establishment of the early genetic code likely required strategies to ensure translational accuracy and inevitably involved tRNA post-transcriptional modifications. One such modification, wybutosine/wyosine is crucial for translational fidelity in Archaea and Eukarya; yet it does not occur in Bacteria and has never been described in mitochondria. Here, we present genetic, molecular and mass spectromery data demonstrating the first example of wyosine in mitochondria, a situation thus far unique to kinetoplastids. We also show that these modifications are important for mitochondrial function, underscoring their biological significance. This work focuses on TyW1, the enzyme required for the most critical step of wyosine biosynthesis. Based on molecular phylogeny, we suggest that the kinetoplastids pathways evolved via gene duplication and acquisition of an FMN-binding domain now prevalent in TyW1 of most eukaryotes. These findings are discussed in the context of the extensive U-insertion RNA editing in trypanosome mitochondria, which may have provided selective pressure for maintenance of mitochondrial wyosine in this lineage.

We report here the sequences for all three segments of a novel RNA virus (LepmorLBV1) from the insect trypanosomatid parasite Leptomonas moramango. This virus belongs to a newly discovered group of bunyavirus-like elements termed Leishbunyaviruses (LBV), the first discovered from protists related to arboviruses infecting humans.

A new microsporidian Trachipleistophora extenrec n. sp. was isolated from a muscle lesion of the streaked tenrec Hemicentetes semispinosus Cuvier, 1798 (Mammalia, Tenrecidae), an insectivore endemic to Madagascar. The spores isolated from the tenrec were infectious to severe combined immunodeficient (SCID) mice by intramuscular injection. Material obtained from muscular lesions in mice was used for the parasite description. All developmental stages of the microsporidian were covered by a dense coat, which during sporogony changed into the sporophorous vesicle wall. Eight, 16, 32, or more spores were formed inside the sporophorous vesicle as the result of the division by plasmotomy and sequential fission of a multinucleate sporogonial plasmodium. Spores were ovoid, 4.7 x 2.8 microm in size, had a large posterior vacuole, and had an isofilar polar tube with 15-16 coils. Although the fine structure and the developmental pattern of the organism were in some respects similar to the genus Vavraia, molecular phylogeny based on the gene sequences of the small subunit rRNA and RNA polymerase subunit II indicated that the organism belongs to the genus Trachipleistophora. The diagnostic characters of the genera Trachipleistophora and Vavraia are discussed as well as the discrepancies between the phylogenies of these two microsporidian genera based on morphology and molecules.

We describe here a new RNA virus (PserNV1) from the plant protist parasite Phytomonas serpens (family Trypanosomatidae, Kinetoplastida, supergroup Excavata). The properties of PserNV1 permit assignment to the genus Narnavirus (Narnaviridae), the first reported from a host other than fungi or oomycetes.

Abstract. Despite its limitations, ethanol remains the most commonly used liquid for the preservation of arthropod specimens and their DnA in the field. Arthropod ecology and taxonomy have witnessed a substantial increase in the use of various trapping and molecular methods in the past two decades. However, the methods of collecting and the preservation liquids most widely used in arthropod traps do not properly preserve DNA. Trap-collected specimens are typically of limited utility for molecular studies due to the poor preservation of DNA. A stable and cheap substance that can be used to trap arthropods in and preserve their DNA is therefore needed. Here we test whether (i) 2% SDS, 100mM eDTA, (ii) 1% SDS, 50mM eDTA and (iii) 0.66% SDS, 33mM eDTA can preserve DnA of small and medium-sized beetles for one, four and eight weeks. Preservation of DnA was tested using PCr amplification of parts of the mitochondrial cytochrome c oxidase I (Cox1) and nuclear 28S rrnA genes. All the solutions tested preserved DnA for at least up to eight weeks and we recommend 2% SDS and 100mM eDTA as a cheap, stable and easily transportable alternative to ethanol for preserving specimens and their DnA collected in the field. This solution is also suitable for using as the collection and preservation liquid in arthropod traps.

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