Isolation and characterization of kinetoplast DNA and RNA of Phytomonas davidi

Kinetoplast DNA networks were isolated from stationary-phase culture forms of Phytomonas davidi. The networks banded in CsCl at a density of 1.699 g/ml and consisted of covalently closed circular molecules. The networks were sensitive to shear forces and exhibited several discrete sedimenting components in neutral and alkaline sucrose. Closed monomeric minicircles were isolated from sonicated networks by alkaline band sedimentation. Closed monomers showed a heterogeneous banding pattern on electrophoresis in acrylamide-agarose gels and had sedimentation coefficients of 20.5 S in alkaline sucrose and 11 S in neutral sucrose. The mean minicircle molecular weight as measured by cospreading with φXRF II was 0.70 × 10⁶ or 1064 nucleotide pairs. Minicircles exhibited a sequence microheterogeneity as evidenced by restriction enzyme analysis, melting analysis, and renaturation kinetics. Network maxicircles were evidenced by the appearance of high molecular weight fragments after restriction with several enzymes and by the existence of supertwisted “edge loops” extending out from the periphery of networks. The maxicircle molecular weight was estimated to be approximately 24 × 10⁶. A purified kinetoplast-mitochondrion fraction was found to contain 9 and 12 S RNA species that comigrated with L. tarentolae 9 and 12 S kinetoplast RNAs.     

Isolation of the Kinetoplast DNA of Leishmania tarentolae in the Form of a Network*

The major kinetoplast DNA complement of Leishmania tarentolae promastigotes has been isolated as a single sheet of interconnected molecules on the basis of its relative stability to shear forces and its high sedimentation coefficient. Two successive differential centrifugations were sufficient to recover 50 ± 10% of the total kinetoplast DNA free of nuclear DNA contamination. We use the term “network” to describe this unusual type of DNA configuration. Leishmania networks have a molecular weight of ～10¹⁰ daltons and an S_20,W in neutral sucrose gradients of 1729 7plusmn; 189 [n = 19] and exhibit an extremely low specific viscosity due to the compactness of packing of the DNA. The networks were visualized in the electron microscope, and in the light microscope either by fluorescence in solution after staining with acridine orange or in dried smears after staining with Giemsa. Purified networks from stationary phase cells banded in the position characteristic of closed monomeric minicircles in ethidum bromide-CsCl equilibrium gradients, and were stable in alkaline sucrose. Treatment of the closed networks with RNase and pronase had no effect on the ethidium bromide-CsCl banding pattern. However, treatment of closed networks with DNase I or II, X-irradiation or γ-irradiation changed the banding pattern by introducing single strand and double strand breaks, yielding an upper band and in some cases a intermediate band.     

Pulse-Labeling of Kinetoplast DNA: Localization of 2 Sites of Synthesis Within the Networks and Kinetics of Labeling of Closed Minicircles*

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.     

Characterization by sedimentation analysis of kinetoplast DNA from Trypanosoma cruzi at different stages of culture.

The kinetoplast DNA networks of Trypanosoma cruzi exist under two forms which have been studied by equilibrium density centrifugation in CsCl gradients containing ethidium bromide and by band sedimentation analysis. The relative proportion of the two forms has been measured and varies significantly between the exponential and stationary phase of growth, suggesting that one of these forms is a replicative intermediate. Both forms exhibit very high sedimentation coefficients. The sedimentation velocity ethidium titration was used to measure the superhelix density of the kinetoplast DNA after having established the validity of the method with in vitro closed DNA networks. The superhelix density of the native form of the kinetoplast DNA minicircles is very low and varies according to the physiological state of the trypanosomes. Furthermore, we observed a significant increase of the superhelix density of the kinetoplast DNA of trypanosomes grown in the presence of ethidium.     

The replication of kinetoplast DNA networks in Crithidia fasciculata

Kinetoplast DNA from the mitochondria of Crithidia is in the form of a two-dimensional network of thousands of minicircles each containing about 2.5 kb, and a small number of maxicircles each containing about 40 kb. Fractionation of kinetoplast DNA by equilibrium centrifugation in a CsCl-propidium dilodide gradient resolves it into three types of networks. Form I networks band at high density and contain minicircles which are covalently closed; form II networks band at low density and contain minicircles which are nicked or gapped; and replicating networks band at intermediate density and contain some minicircles of each type. Form I networks contain about 5000 minicircles; form II networks contain about 11,000; and replicating networks contain an intermediate number. When cells are pulse-labeled with ³H-thymidine, radioactivity in mitochondrial DNA is preferentially incorporated into replicating networks, but after a chase it appears first in form II networks and finally in form I. Examination of replicating networks by electron microscopy in the presence of ethidium bromide reveals that minicircles in the central region of the network are twisted and therefore covalently closed, whereas those in the peripheral region are not twisted and therefore must be nicked or gapped. The pulse-label is incorporated into the nicked or gapped minicircles of the replicating networks. These results indicate that replication of form I networks begins in peripheral minicircles and that progeny minicircles remain nicked or gapped. As replication proceeds, the size of the network increases, and the peripheral zone of nicked or gapped minicircles enlarges. Finally, when all minicircles have replicated, the network, now form II, is double the size of form I and contains only nicked or gapped minicircles. The final step in replication presumably includes both the cleavage of the network into two form I species and the covalent closure of all the minicircles.     

A nicking enzyme from trypanosomatids which specifically affects the topological linking of duplex DNA circles. Purification and characterization

Newly replicated duplex DNA minicircles of trypanosomal kinetoplast DNA are nicked in both their monomeric and catenated topological states, whereas mature ones are covalently sealed. The possibility that nicking may play a role during kinetoplast DNA replication by affecting the topological interconversions of monomeric DNA minicircles and catenane networks was studied here in vitro using Crithidia fasciculata DNA topoisomerase. An enzyme that catalyzes the nicking of duplex DNA circles has been purified to apparent homogeneity from C. fasciculata cell extracts. The native enzyme has a sedimentation coefficient of 6.8 S and was found to be a dimer with a protomer Mr = 60,000. Nicking of kinetoplast DNA networks by the purified enzyme inhibits their decatenation by the Crithidia DNA topoisomerase but has no effect on the catenation of monomeric DNA minicircles into networks. This differential effect on decatenation versus catenation is specific to the purified nicking enzyme. Random nicking of interlocked DNA minicircles has no detectable effect on the reversibility of the topological reaction. The potential role of Crithidia nicking enzyme in the replication of kinetoplast DNA networks in trypanosomatids is discussed.     

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.     

Structure-Function Relationships in Cellular Organelles Introductory Remarks by the Convener

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.     

The structure of kinetoplast DNA. I. Properties of the intact multi-circular complex from Crithidia luciliae.

We have developed a modified isolation procedure that yields kinetoplast DNA networks containing more than 90% closed circular DNA, as judged by two criteria: (a) In 0.15 M NaCl/0.015 M sodium citrate (pH 7.0), less than 10% of the intact kinetoplast DNA melts in the temperature region of sonicated kinetoplast DNA. In 7.2 M NaCl04 the kinetoplast DNA melts with a Tm 26 degrees C higher than sonicated kinetoplast DNA. Even after complete melting in 7.2 M NaClO4 at 90 degrees C, the network remains intact, as judged by regain of hypochromicity on cooling and analysis in CsCl containing propidium dixodide. (b) In alkaline sucrose gradients more than 90% of the kinetoplast DNA sediments in a single peak. 2. In CsCl gradients containing ethidium bromide of propidium diiodide intact kinetoplast DNA gives a single uni-modal band showing an extremely restricted dye uptake. From the position of the bank relative to the bands of PM2 DNA, the superhelix density of these networks is calculated to be +3.9 twists per 1000 base pairs. The superhelix density of closed mini-circles, efficiently liberated from the networks by shear in a French press, is -0.5 twists per 1000 base pairs. We attribute the high superhelix density (the highest yet observed in any DNA) of intact networks to their compact, highly catenated structure, leading to an additional constraint on dye uptake, superimposed on the restriction due to closed circularity.     

Interlocked DNA rings. II. Physicochemical studies

Several species of topologically interlocked λb2b5c DNA rings (catenanes) have been prepared in vitro. The sedimentation behavior of dimeric catenanes containing 0, 1, or 2 covalently closed duplex rings has been studied as a function of the superhelical density of the covalently closed ring or rings. In general, if XtpY represents rings X and Y topologically interlocked, the sedimentation coefficient of this species, ^SXtpY, is related to the sedimentation coefficients ^SX and ^SY of the component rings by the empirical relationship ^SXtpY/^SY = [(M_X + M_Y)/M_Y] [1 + (M_X/M_Y)^1.78(^SY/^SX)^1.78]^?0.56 This equation can also be extended to the case where three monomeric rings are topologically linked in a chain. For two topologically interlocked monomeric rings with neither ring covalently closed, the sedimentation coefficient is 1.35 times that of the monomeric ring. This is different from results reported for mitochondrial and P22 catenanes by others. Several possibilities are discussed to account for this discrepancy. The sedimentation coefficient of a species containing one covalently closed duplex ring and a single-stranded ring was also measured in an alkaline medium. This species, which can be easily derived from a dimeric catenane containing two covalently closed duplex rings, is particularly useful for the identification of covalently closed dimeric catenanes. Some electron microscopic studies of these interlocked rings are presented in an accompanying paper.     

Chemical and physical properties of adeno-associated satellite virus DNA produced during coinfection with herpes simplex virus

Infectious DNA from adeno-associated satellite virus (ASV) has been isolated from cells coinfected with a temperature-sensitive mutant of herpes simplex virus (HSV) type 1 in the absence of contaminating HSV DNA. This satellite virus DNA does not appear to differ in its physical, chemical and biological properties from DNA isolated directly from virions or from cells co-infected with adenovirus. The DNA is double-stranded with a buoyant density of 1.718 gm/cm³. It sediments at 16S in both neutral and alkaline sucrose gradients. Single-stranded DNA from alkaline sucrose gradients has a modal length of 1.5 μm and demonstrates evidence of internal redundancies in the electron microscope.     

Evidence for the attachment of RNA to pulse-labeled DNA in the slime mold, Physarum polycephalum

When $\text{Physarum}\text{polycephalum}$ is pulse-labeled for up to 20 minutes with ³H-thymidine and the shortest labeled DNA strands are partially purified by sedimentation through a neutral aqueous sucrose gradient and then through a formamide-sucrose gradient, these short strands band in Cs₂SO₄ isopycnic density gradients at a density greater than that of bulk single-stranded DNA. Their density is brought partially or nearly completely back to that of single-stranded DNA by hydrolysis with pancreatic RNase A or alkali, respectively. Therefore the dense material attached to the short pulse-labeled DNA strands consists at least partially of RNA.     

Kinetoplast DNA of Trypanosoma brucei: Physical map of the maxicircle

Trypanosoma brucei maxicircle DNA in kinetoplast DNA (kDNA) networks was characterized with restriction endonucleases. The data allow the construction of a circular map of a 22.2-kb molecule. Based on these and previous data each T. brucei kDNA network contains about 45 maxicircles which probably have the same sequence. The maxicircle of strain 164 used in this study was slightly larger and had three EcoRI sites compared to two found in other strains. Fragments generated by digestion with BamHI were largely singly cleaved maxicircles that had a density of 1.681 g/cm³ compared to 1.693 g/cm³ for the intact network. This suggests that maxicircles have a higher A + T content than minicircles. Minicircles in the kDNA network were also characterized with restriction endonucleases. Each enzyme cleaved a specific subset of minicircles from the network. However, no single restriction endonuclease or combination of up to three of these enzymes cleaved all molecules in the network. These results are consistent with earlier results of renaturation kinetic experiments and indicate that there are many different sequence classes of mini-circle DNA.     

The segregation of kinetoplast DNA networks in Trypanosoma brucei

The kinetoplast DNA of Trypanosoma brucei consists of 10⁴ minicircles (0.3 μm) and 10² maxicircles (6 μm) held together by catenation in a complex network. In electron micrographs of kinetoplast DNA spread in a protein monolayer we have identified four types of network with the appearance of different stages in network replication and segregation. We show that each network type has characteristic properties with respect to shape, size, number, and location of maxicircle loops and nicked or covalently closed character of minicircles and maxicircles. We propose a detailed model for network segregation that involves a gradual elongation of the network, followed by network cleavage. During this process the basic network structure remains unaltered, implying a complicated mechanism of minicircle rearrangements.     

Effect of heparin on the porcine lymphocyte chromatin—II. comparative study of sedimentation of chromatin dna and isolated dna

• 1.1. Sedimentation of chromatin DNA and isolated deproteinized DNA was compared in neutral and alkaline sucrose density gradients after incubation of chromatin or DNA with various concentrations of heparin.
• 2.2. Irrespective of the molecular weight of DNA, an increase in the sedimentation constant of DNA was found with increasing concentration of the polyanion employed.

     

Characterization of mycoplasmatales virus DNA

The DNA of the group L1 $\text{Mycoplasmatales}$ virus, MVL51, was analyzed using alkaline sucrose velocity sedimentation, neutral and alkaline CsCl isopycnic sedimentation, and treatment of the DNA with nucleases. These treatments show that the viral chromosome is a covalently linked single-stranded DNA circle of molecular weight 2×10⁶ daltons.     

Effects of Ethidium Bromide and Several Acridine Dyes on the Kinetoplast DNA of Leishmania tropica*†

Whole cell DNA from Leishmania tropica has 2 peaks when banded by CsCl equilibrium density centrifugation. The main band has a buoyant density of 1.721 and the satellite band a buoyant density of 1.705, with Clostridium perfringens DNA (ρ= 1.6915) used as a reference. The satellite band has been identified as the kinetoplast DNA by purifying DNA from isolated kinetoplasts. L. tropica has the highest G + C content of both nuclear and kinetoplastic DNA thus far reported for trypanosomatids. The effects of ethidium bromide, acriflavin, proflavin, and 5-aminoacridine on the kinetoplast of L. tropica have been compared. Ethidium bromide and acriflavin, but not proflavin or 5-aminoacridine, induce dyskinetoplasty. L. tropica is one of the most sensitive trypanosomatids to ethidium bromide and acriflavin. Examination of the DNA from drug-treated cells in CsCl gradients revealed a loss of the satellite band after ethidium bromide or acriflavin treatment, but not after proflavin or 5-aminoacridine treatment. Cell division was required to produce these effects on the kinetoplast.     

Structure of replicating DNA molecules of Bacillus subtilis bacteriophage phi 29.

We isolated phi 29 DNA replicative intermediates from extracts of phage-infected Bacillus subtilis, pulsed-labeled with [3H]thymidine, by velocity sedimentation in neutral sucrose followed by CsCl equilibrium density gradient centrifugation. During a chase, the DNA with a higher sedimentation coefficient in neutral sucrose and a lower sedimentation rate in alkaline sucrose than that of viral phi 29 DNA was converted into mature DNA. The material with a density higher than that of mature phi 29 DNA consisted of replicative intermediates, as analyzed with an electron microscope. We found two major types of molecules. One consisted of unit-length duplex DNA with one single-stranded branch at a random position. The length of the single-stranded branches was similar to that of one of the double-stranded regions. The other type of molecules was unit-length DNA with one double-stranded region and one single-stranded region extending a variable distance from one end. Partial denaturation of the latter molecules showed that replication was initiated with a similar frequency from either DNA end. These findings suggest that phi 29 DNA replication occurs by a mechanism of strand displacement and that replication starts non-simultaneously from either DNA end, as in the case of adenovirus.     

In situ hybridization to the Crithidia fasciculata kinetoplast reveals two antipodal sites involved in kinetoplast DNA replication.

Kinetoplast DNA is a network of interlocked minicircles and maxicircles. In situ hybridization, using probes detected by digital fluorescence microscopy, has clarified the in vivo structure and replication mechanism of the network. The probe recognizes only nicked minicircles. Hybridization reveals prereplication kinetoplasts (with closed minicircles), donut-shaped replicating kinetoplasts (with nicked minicircles on the periphery and closed minicircles in the center), and postreplication kinetoplasts (with nicked minicircles). Replicating kinetoplasts are associated with two peripheral structures containing free minicircle replication intermediates and DNA polymerase. Replication may involve release of closed minicircles from the center of the kinetoplast and their migration to the peripheral structures, replication of the free minicircles therein, and then peripheral reattachment of the progeny minicircles to the kinetoplast.