Sequence heterogeneity and anomalous electrophoretic mobility of kinetoplast minicircle DNA from Leishmania tarentolae

Several unit-length minicircles from the kinetoplast DNA of Leishmania tarentolae were cloned into pBR322 and into M13 phage vectors. The complete nucleotide sequences of three different partially homologous minicircles were obtained. The molecules contained a region of approx. 80% sequence homology extending for 160–270 bp and a region unique to each minicircle. A 14-mer was found to be conserved in all kinetoplast minicircle sequences reported to date. The frequency distributions of various minicircle sequence classes in L. tarentolae were obtained by quantitative gel electrophoresis and by examination of the “T ladder” patterns of minicircles randomly cloned into M13 at several sites. By these methods we could assign approx. 50% of the total minicircle DNA into a minimum of five sequence classes. A sequence-dependent polyacrylamide gel migration abnormality was observed with several minicircle fragments both cloned and uncloned. The abnormality was dependent on the presence of a portion of the conserved region of the minicircle.     

砂鼠利什曼原虫(Leishmania gerbilli)的亚显微结构

砂鼠利什曼原虫(Leishmania gerbilli)存在于我国西北甘肃某地的砂土鼠或大砂鼠(Rhombomys opimus)体内,是中国特有的种类,王捷等60年代分离培养成功。对L.gerbilli的前鞭毛体的亚显微结构的观察表明:1.质膜下微管间距约为50nm,比别的利什曼原虫的宽;2.鞭毛基部极少观察到基板(basal plate);3.线粒体发达,从动基体所在部位伸向虫体各个部位,内有复杂的结构;4.在通常情况下,L.gerbilli的动基体呈棒状,但当细胞膨胀后则无论在光镜或电镜下动基体均呈扁环结构;5.虫体后部有发达的复片层结构。这一结构由一系列的膜卷绕成,其意义不明。     

Characterization of isolates and clones of leishmania by analysis of kinetoplast DNA

The genetic characterization of pathogenic isolates of Leishmania was attempted by analysis of the molecular properties of kinetoplast DNA (kDNA) minicircles. Unit minicircle size is not conserved during speciation of Leishmania since the minicircles of strains and clones of L t major are smaller (700 bp) than those found in certain strains of L mexicana ssp (820 bp), L donovani (850 bp) or L t tropica (900 bp). Schizodeme analysis of minicircles reveals a high degree of sequence divergence in kDNA of Leishmania with the degree of microheterogeneity varying between species. This sequence divergence allows the discrimination of species, strains, and clones of Leishmania into schizodemes. Southern blot hybridization experiments reveal that at high stringency overall minicircle sequence homology is conserved among clones and strains of one species (L t major) but not between different species. This property of minicircle DNA permits the use of kDNA probes as a species-specific diagnostic test for the identification of unknown Leishmania isolates. The properties of kDNA from an L t tropica strain LRC-L32 (a “recidiva” organism) are so diverged from those of L t major strains as to support the classification [22,23] of L t tropica and L t major as separate species of Leishmania rather than subspecies of L tropica.     

DNA Throughout the Single Mitochondrion of a Kinetoplastid Flagellate: Observations on the Ultrastructure of Cryptobia vaginalis (Hesse, 1910)

SYNOPSIS. Cryptobia vaginalis (Hesse 1910) occurs as long thin and short broad forms in the vagina of the gnathobdelliform leeches Haemopis sanguisuga (Linnaeus) and Hirudo medicinalis Linnaeus. Cytochemical staining for DNA and transmission electron microscopy of sectioned material indicate that in the thin forms the kinetoplast DNA (kDNA) is dispersed irregularly through the mitochondrial network ( pankinetoplastic condition) rather than concentrated in the adbasal region of the mitochondrion ( eukinetoplastic condition) as in trypanosomatids and most other kinetoplastid flagellates. Light-microscopic studies on the rare broad forms, however, suggest that these have conventional adbasal location of the kinetoplast. Binary fission appears to occur in the thin forms, suggesting that the dispersed kinetoplast is either highly polyenergid or lacks a genetic function. In other features of its microanatomy, C. vaginalis is a conventional kinetoplastid. The flagellate has an incomplete corset of pellicular microtubules which may have a role in the cortical contractility characteristic of the genus Cryptobia . Feeding is by pinocytosis of vaginal colloids through a microtubule-lined cytopharynx, possibly after binding to a prominent filament-coated preoral ridge. A pulsatile (contractile) vacuole is present and appears to be responsible for defecation as well as osmoregulation. Some individuals have elongate bacterial epibionts attached to the body in parallel with the cortical microtubules. All individuals have 2–8 spheroplast-like endobiotic bacteria in the prenuclear cytoplasm.     

Detection and Classification of Trypanosoma cruzi by DNA Hybridization with Nonradioactive Probes

ABSTRACT. Total or kinetoplast DNA (kDNA) from 72 isolates and clones of Trypanosoma cruzi as well as from nine related trypanosomatids were analyzed by dot hybridization using nonradioactive kDNA or cloned minicircle fragments as probes. Biotinylated-kDNA probes generated by nick-translation proved reliable for distinguishing Zymodeme 1 and Zymodeme 2bol of T. cruzi parasites. In contrast, digoxigenin-labeled kDNA obtained by random-priming did not distinguish among T. cruzi isolates but did distinguish among New World leishmanias. Cloned minicircle fragments labeled with digoxigenin gave the same results as digoxigenin-labeled kDNA, except for a 10-fold decrease in sensitivity. Digoxigenin-labeled DNA probes proved useful in unambiguously detecting T. cruzi from different geographic regions of America. However, T. rangeli and T. cruzi marinkellei were not distinguished by these probes.     

Kinetoplast morphology and segregation pattern as a marker for cell cycle progression in Leishmania donovani

Trypanosomatids are typified by uniquely configured mitochondrial DNA--the kinetoplast. The replication timing of kinetoplast DNA (kDNA) is closely linked to nuclear S phase, but nuclear and kinetoplast compartments display staggered timing of segregation, post-replication. Kinetoplast division is completed before nuclear division in Trypanosoma species while nuclear division is completed first in Crithidia species. Leishmania donovani is the causative agent of visceral leishmaniasis, a form of leishmanial infection that is often fatal. Cell cycle related studies in Leishmania are hampered by difficulties in synchronizing these cells. This report examines the replication/segregation pattern and morphology of the kinetoplast in L. donovani with the aim of determining if these traits can be used to assign cell cycle stage to individual cells. By labeling replicating cells with bromodeoxyuridine after synchronization with hydroxyurea, we find that although both nuclear and kDNA initiate replication in early S phase, nuclear division precedes kinetoplast segregation in 80% of the cells. The kinetoplast is roundish/short rod-like in G1 and in early to mid-S phase, but prominently elongated/bilobed in late S phase and early G2/M. These morphological traits and segregation pattern of the kinetoplast can be used as a marker for cell cycle stage in a population of asynchronously growing L. donovani promastigotes, in place of cell synchronization procedures or instead of using antibody staining for cell cycle stage marker proteins.     

Kinetoplast DNA from plant trypanosomatids responsible for the Hartrot disease in coconut trees

Summary— Phytomonas parasites were isolated from crude sap of coconut trees affected with Hartrot disease in French Guyana (Hart 1 and Hart 2) and Brazil (Hart 3) and cultured in vitro. Two Phytomonas isolates obtained from weeds belonging to the Euphorbiaceae family and growing in an infected coconut tree plantation were also cultured (E hys and E hir). The kinetoplast DNA (kDNA) was purified and incubated with topoisomerase II which decatenates the huge network into free minicircles of 1.6 kilobase (kb) pair for Hart 1, Hart 2 and Hart 3 and 1.3 kb for E hys and E hir. Restriction endonuclease analysis showed that more than 90% of Hart 1 and Hart 2 minicircle content was homogeneous in base sequence while minicircles from Hart 3, E hys and E hir were heterogeneous. Minicircles exhibited restriction cleavage patterns characteristic of each Phytomonas isolate allowing their identification, except for the major class of Hart I and Hart 2 minicircles whose restriction maps were identical. Cross-hybridization experiments were performed by Southern blot. A high sequence homology was found between minicircies from Hart 1, Hart 2 and Hart 3 on one hand and those from E hys and E hir on the other. In contrast, minicircles from the Hartrot Phytomonas and those from the two Euphorbiaceae Phytomonas present little sequence homology. These data showed that minicircles from Phytomonas infecting coconut trees displayed biochemical properties different from those of other Phytomonas. This could lead to the elaboration of new molecular tools aimed to help to epidemiological studies, to an early diagnosis and to a better control of the disease.