ALBERT

Notes: Trypanosoma brucei strain 366D trypomastigotes grown at 37°C in the presence of a human fibroblast cell line formed foci underneath the feeder cells whereas trypanosomes grown in the presence of a human epithelial cell line grew only in the culture supernatant. A culture system was developed to study the differentiation of bloodstream trypomastigotes grown in the epithelial cell system into procyclic trypomastigotes at 27°C. The morphological differentiation into the procyclic form was complete by 48 h. Cell division did not occur until 30–40 h after transfer to 27°C. Various characteristics of this system were examined, including the effect of the feeder layer, the type of medium, the presence of the metabolites cis-aconitate and citrate, the preadaptation period, and the trypanosome cell concentration. The respiration of the recently differentiated procyclic cells was less sensitive to inhibition by CN-than that of established procyclic forms, implying a delayed appearance of complete mitochondrial oxidative pathways. This trypanosome differentiation system has the advantage that the animal host is not needed and the entire process is carried out in in vitro culture.

Notes: SYNOPSIS. Short pulse-labeling of log phase Crithidia fasciculata cells with [3H]thymidine allowed the autoradiographic visualization of 2 sites of replication of kinetoplast DNA situated at the periphery of the networks and separated by 180°. Longer pulse-labeling led to the previously reported total peripheral labeling pattern. Pulse-labeled networks possess an intermediate density in ethidium bromide-CsCl equilibrium gradients between the densities characteristic of closed networks and open or linear DNA. Removal of ethidium bromide by several methods and treatment of intermediate band networks with RNase and pronase had no effect on the equilibrium rebanding pattern. Closed minicircles of Leishmania tarentolae are not labeled by a short pulse of intact cells with [3H]thymidine. A chase of ∼ 3–4 hr is required for the appearance of radioactivity in closed minicircles, a time delay which implies the existence of intermediate events between replication and eventual covalent closure of the minicircles.

Notes: In early log phase cultures of several of the drug-resistant mutants of Crithidia fasciculata that we have previously obtained, a high percentage of cells attach in pairs at the base of the flagellum. This process, which we have termed “flagellar adherence,” lasts for several hours in some cases and occasionally involves changes in cell morphology. The attachment occurs optimally in gently agitated cultures. Flagellar adherent pairs can be disassociated by vigorous agitation; the pairs reappear in the culture within one to three h after disassociation. These paired forms can be clearly distinguished from the normal cell division forms. Clones of flagellar adherent-competent mutant strains are uniformly able to form these pairs in culture. A low percentage of flagellar adherent forms can be induced in wild type cells by glucose starvation.

Notes: SYNOPSIS. Covalently closed kinetoplast DNA networks have been isolated from stationary phase Crithidia fasciculata cells by a technic involving selective pelleting of the networks at a low centrifugal field. Approximately 62% of the kinetoplast DNA of the cell was recovered free of nuclear DNA by simple differential centrifugation.Purified kinetoplast DNA networks were visualized both in the electron microscope and in the light microscope. Closed networks sedimented as a homogeneous band both in neutral and alkaline sucrose, with an s20w in neutral sucrose of approximately 5 × 103.Closed monomeric minicircles were isolated from purified networks by mild sonication and band sedimentation in alkaline sucrose. Several physical properties of closed monomeric minicircles were measured. These included molecular weight, buoyant density in CsCl, superhelix density and sedimentation coefficient.

Notes: Cells of the order Kinetoplastida possess a single mitochondrion which contains a large amount of a uniquely organized DNA. This kinetoplastic DNA (K-DNA), representing 10–20% of the total cell DNA in different species, has as its major molecular component a small closed circular molecule present in large numbers. The size and thereby the amount of genetic information carried by the minicircles varies from species to species: Leishmania tarentolae and the Salivarian trypanosomes have the smallest, the Stercorarian trypanosomes Trypanosoma lewisi and Trypanosoma cruzi intermediate, and Crithidia and also Trypanosoma mega the largest minicircles. In L. tarentolae, purified minicircles, which are the size of 1 gene, have been shown by renaturation kinetics to consist of only 1 or 2 classes. L. tarentolae K-DNA also contains another molecular species—a long molecule which may represent up to 30% of the total K-DNA. The minicircles, nevertheless, represent a gene amplification of the order of 104. In all species that have been examined so far, the K-DNA consists of a single sheet of interlocked closed circular molecules which can be isolated in an intact form because of its resistance to shear forces and its high molecular weight. In addition, at least in L. tarentolae, 6–9% of the K-DNA is either free in the mitochondrion or loosely bound. The main K-DNA structure has been termed a “network” and can be seen in the light microscope after staining in solution with acridine orange or after fixing and staining with Giemsa's, or in the electron microscope. The quaternary structure of such networks in terms of the organization of minicircles and long molecules is not understood. Controlled breakdown of networks from L. tarentolae was achieved by sonication, and the release of open and closed monomeric minicircles, catenated dimers, trimers and higher oligomers, and short linear fragments was measured. A maximum of 43% of the total network DNA was released in the form of closed monomers, dimers, and trimers, thus providing a minimal estimate for the percentage of minicircles in K-DNA from this species. K-DNA replicates fairly synchronously with nuclear DNA in all species that have been examined. Replication of DNA molecules in the kinetoplast networks is limited to the periphery, as seen in autoradiographs of networks isolated from cells (L. tarentolae, Crithidia fasciculata) pulsed with 3H-thymidine. The molecular implications of this unusual replication pattern remain an open question, as does the genetic function of the K-DNA itself.

Notes: SYNOPSIS. The kinetoplast of L. tarentolae remains attached to the basal body upon cell rupture by detergent lysis, sonication, or hypotonic lysis in 0.02 M Tris buffer (pH 7.9) at 0–4 C. Hypotonic lysis in 0.02 M Tris-HCl-2 mM EDTA at 0–4 C and application of mild shearing forces bring about release of most of the swollen kinetoplasts. The kinetoplast DNA can be seen in phase contrast microscopy as a dark mass contiguous to the kinetoplast membrane directly opposite the basal body. Upon return to isotonic media, the kinetoplast shrinks; the membranes of such kinetoplasts are impermeable to added DNAase.

Notes: Attempts at continuous labeling of Crithidia fasciculata DNA with [3H]thymidine led to a pulse-chase situation due to a cell-mediated conversion of thymidine to thymine in the medium. The uptake of thymine was slow compared to that of thymidine. Neither the addition of deoxyadenosine nor the sequential addition of several aliquots of [3H]thymidine had an effect on the pattern of labeling.

Notes: SYNOPSIS Leishmania tarentolae cells in brain-heart infusion medium were partially synchronized in terms of DNA synthesis and cell division by a 10 hour period of inhibition in 200 μg/ml hydroxyurea at 27 C. Nuclear and kinetoplast DNA synthesis commenced immediately upon removal of hydroxyurea, and kinetoplast and nuclear division occurred after about 5 hr. The Index of Synchronization (3) varied from 33-41%.A moderate decay of the synchronicity was noted by the 2nd cell cycle. Hydroxyurea was selectively lethal to S-phase cells.

Notes: 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.

Notes: SYNOPSIS. The leishmania-leptomonad transformation of Leishmania donovani occurs in 20–40 hr in vitro at 27 C in the absence of cell division. The rate and extent of the process depend on the available nutrients. A source of amino adds and glucose (or sucrose) are required. Several basic or acidic amino acids can substitute for all amino acids present in the complete medium. Leishmanial forms freshly isolated from hamster spleen have a QO2 (μM O2/min/107 cells) of 0.29 ± 0.01 at 27 C in phosphate buffered saline with glucose. This respiration is sensitive to KCN, antimycin A, and Na amytal, and hence is probably cytochrome dependent. The QO2 remains constant for 5–6 hr and then increases 5–7 fold by 20–30 hr of incubation at 27 C in the proper medium. The transformation is inhibited in terms of morphogenesis and increase in respiration by Actinomycin D, puromycin and mitomycin C. Appearance of new soluble antigens during the transformation was found by gel diffusion and immunoelectrophoresis.