Variations in maxi-circle and mini-circle sequences in kinetoplast DNAs from different Trypanosoma brucei strains

We have compared a total of 30 recognition sites for eight restriction endonucleases on the 20-kilobase-pair maxi-circle of kinetoplast DNAs from five different Trypanosoma brucei strains. In addition to three polymorphic sites were have found a 5 kilobase-pair region that is not cleaved by any of the eight enzymes and that varies in size over 1 kilobase pair in the strains analysed. Mini-circles from these five strains, digested with endonuclease TaqI or MboII, yield very complex fragment patterns, showing that extensive mini-circle sequence heterogeneity is a common characteristic of these T. brucei strains. The size distribution of mini-circle fragments in these digests was identical for different clones of the 427 strain, but very different for mini-circles from different strains. These results show that maxi-circle sequence is conserved, whereas mini-circle sequence is not. Restriction digests of maxi-circles could be useful in determining how closely two Trypanosoma strains are related, whereas mini-circle digests can serve as sensitive tags for individual strains.     

Characterization of the molecular components in kinetoplast- mitochondrial DNA of Trypanosoma equiperdum. Comparative study of the dyskinetoplastic and wild strains

The structure of the kinetoplast DNA of Trypanosoma equiperdum has been studied and compared to the structure of the circular mitochondrial DNA extracted from a dyskinetoplastic strain of T. equiperdum. In T. equiperdum wild type, the kinetoplast DNA constitutes approximately 6% of the total cellular DNA and is composed of approximately 3,000 supercoiled minicircles of 6.4 x 10(5) daltons and approximately 50 circular supercoiled molecules of 15.4 x 10(6) daltons topologically interlocked; The buoyant density in CsCl of the minicircles is 1.691 g/cm 3. The large circles have a buoyant density of 1.684 g/cm 3, are homogeneous in size and are selectively cleaved by several restriction endonucleases which do not cleave the minicircles. The cleavage sites of six different restriction endonucleases have been mapped on the large circle. The minicircles are cleaved by two other restriction endonucleases, and their cleavage sites have been mapped. The mitochondrial DNA extracted from the dyskinetoplastic strain of T. equiperdum represents 7% of the total DNA of the cell and is composed of supercoiled circles, heterogeneous in size, and topologically associated in catenated oligomers. Its buoyant density in CsCl is 1.688 g/cm 3. These molecules are not cleaved by any of the eight restriction endonucleases tested. The reassociation kinetics of in vitro labeled kDNA minicircles and large circles has been studied. The results indicate that the minicircles as well as the large circles are homogeneous in sequence and that the circular DNA of the dyskinetoplastic strain has no sequence in common with the kDNA of the wild strain.     

Isolation and characterization of kinetoplast DNA from bloodstream form of Trypanosoma brucei

We have used restriction endonucleases PstI, EcoRI, HapII, HhaI, and S1 nuclease to demonstrate the presence of a large complex component, the maxi-circle, in addition to the major mini-circle component in kinetoplast DNA (kDNA) networks of Trypanosoma brucei (East African Trypanosomiasis Research Organization [EATRO] 427). Endonuclease PstI and S1 nuclease cut the maxi-circle at a single site, allowing its isolation in a linear form with a mol wt of 12.2 x 10(6), determined by electron microscopy. The other enzymes give multiple maxi-circle fragments, whose added mol wt is 12-13 x 10(6), determined by gel electrophoresis. The maxi-circle in another T. brucei isolate (EATRO 1125) yields similar fragments but appears to contain a deletion of about 0.7 x 10(6) daltons. Electron microscopy of kDNA shows the presence of DNA considerably longer than the mini-circle contour length (0.3 micron) either in the network or as loops extending from the edge. This long DNA never exceeds the maxi-circle length (6.3 microns) and is completely removed by digestion with endonuclease PstI. 5-10% of the networks are doublets with up to 40 loops of DNA clustered between the two halves of the mini-circle network and probably represent a division stage of the kDNA. Digestion with PstI selectively removes these loops without markedly altering the mini-circle network. We conclude that the long DNA in both single and double networks represents maxi-circles and that long tandemly repeated oligomers of mini-circles are (virtually) absent. kDNA from Trypanosoma equiperdum, a trypanosome species incapable of synthesizing a fully functional mitochondrion, contains single and double networks of dimensions similar to those from T. brucei but without any DNA longer than mini-circle contour length. We conclude that the maxi-circle of trypanosomes is the genetic equivalent of the mitochondrial DNA (mtDNA) of other organisms.     

EVIDENCE FOR THE RETENTION OF KINETOPLAST DNA IN AN ACRIFLAVINE-INDUCED DYSKINETOPLASTIC STRAIN OF TRYPANOSOMA BRUCEI WHICH REPLICATES THE ALTERED CENTRAL ELEMENT OF THE KINETOPLAST

A pleomorphic dyskinetoplastic strain of Trypanosoma brucei was produced by repeated acriflavine treatment. No kinetoplastic cells reappeared after 2 yr of maintenance in the absence of acriflavine. These dyskinetoplastic cells retained and therefore replicated the central element of the kinetoplast. This element was present in the "condensed" state typical of acriflavine-treated cells rather than the normal fibrillar state. Whole-cell DNA extracted from both normal and dyskinetoplastic strains revealed three bands upon isopycnic sedimentation, and there was no detectable alteration in buoyant density of any of these DNA components in the dyskinetoplastic strain. It seems likely that the dyskinetoplastic strain has retained its kinetoplast DNA but in an altered state.     

Natural and induced dyskinetoplastic trypanosomatids: how to live without mitochondrial DNA

Salivarian trypanosomes are the causative agents of several diseases of major social and economic impact. The most infamous parasites of this group are the African subspecies of the Trypanosoma brucei group, which cause sleeping sickness in humans and nagana in cattle. In terms of geographical distribution, however, Trypanosoma equiperdum and Trypanosoma evansi have been far more successful, causing disease in livestock in Africa, Asia, and South America. In these latter forms the mitochondrial DNA network, the kinetoplast, is altered or even completely lost. These natural dyskinetoplastic forms can be mimicked in bloodstream form T. brucei by inducing the loss of kinetoplast DNA (kDNA) with intercalating dyes. Dyskinetoplastic T. brucei are incapable of completing their usual developmental cycle in the insect vector, due to their inability to perform oxidative phosphorylation. Nevertheless, they are usually as virulent for their mammalian hosts as parasites with intact kDNA, thus questioning the therapeutic value of attempts to target mitochondrial gene expression with specific drugs. Recent experiments, however, have challenged this view. This review summarises the data available on dyskinetoplasty in trypanosomes and revisits the roles the mitochondrion and its genome play during the life cycle of T. brucei.     

Genetic relatedness among Trypanosoma evansi stocks by random amplification of polymorphic DNA and evaluation of a synapomorphic DNA fragment for species-specific diagnosis.

In this study we employed randomly amplified polymorphic DNA patterns to assess the genetic relatedness among 14 Brazilian Trypanosoma evansi stocks from domestic and wild hosts, which are known to differ in biological characteristics. These akinetoplastic stocks were compared with one another, to three Old World (Ethiopia, China and Philippines) dyskinetoplastic stocks of T. evansi, and also with Trypanosoma equiperdum, Trypanosoma brucei brucei, Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense. Randomly amplified polymorphic DNA analysis showed limited heterogeneity in T. evansi stocks from different hosts and geographical regions of the world, or in other species of the subgenus Trypanozoon. However, minor variations generated random amplification of polymorphic DNA analysis disclosed a pattern consisting of a unique synapomorphic DNA fragment (termed Te664) for the T. evansi cluster that was not detected in any other trypanosome species investigated. Pulsed field gel electrophoresis analysis demonstrated that the Te664 fragment is a repetitive sequence, dispersed in intermediate and minichromosomes of T. evansi. Based on this sequence, we developed a conventional PCR assay for the detection of T. evansi using crude preparations of blood collected either on glass slides or on filter paper as template DNA. Our results showed that this assay may be useful as a diagnostic tool for field-epidemiological studies of T. evansi.     

Isolation and characterization of circular DNA molecules heterogeneous in size from a dyskinetoplastic strain of Trypanosoma equiperdum

In a naturally occuring dyskinetoplastic mutant strain of $\text{T. equiperdum}$, covalently closed circular DNA molecules of assumed mitochondrial origin were isolated. These molecules, heterogeneous in size, represent 6–9 % of total DNA and are essentially organized in catenated oligomers composed of molecules of different length. The typical molecular organization of the kinetoplast DNA from kinetoplastic trypanosomes, the network, was not observed.     

Characteristics of kinetoplastic DNA from Leptomonas pessoai

The kinetoplast (mitochondrial) DNA from trypanosomatid Leptomonas pessoai represents a network, composed of mini-circles heterogeneous in base sequence and homogeneous maxi-circles and thus has the main structural features in common with DNAs from kinetoplasts of other Trypanosomatidae. The size of mini-circular molecules of DNA is 1,35 kilobase pairs (kbp) and that of maxicircular molecules-30,9 kbp. Based on the data of single and double restriction cleavages the physical map of the maxi-circular molecules was constructed for the endonucleases BamHI, BglII, BspI , HindIII, MspI, SalGI and PstI.     

The structure of kinetoplast DNA. 2. Characterization of a novel component of high complexity present in the kinetoplast DNA network of Crithidia luciliae.

1. Degradation of highly purified kinetoplast DNA (kDNA) networks with restriction endonucleases yields "extra" bands in agarose gels that are absent from digests of mini-circles. Each of the five endonucleases tested, i.e. AluI, HapII, EcoRI, Hsu and HindII + III, yields a unique set of "extra" bands. The "extra" bands consist of linear DNA; they are not mini-circle oligomers and their added molecular weight, calculated from mobility in gels, are around 2 X 10(7). Double digests with two restriction endonucleases yield a new set of "extra" bands, showing that the "extra" bands obtained with different enzymes are all derived from the same complex component of kDNA. In digests of 32P-labelled kDNA an average of 2.3% of the radioactivity is recovered in the "extra" bands. 2. Treatment of kDNA networks with the single-strand-specific S1 nuclease of Aspergillus oryzae preferentially releases a linear DNA with a molecular weight of 26 X 10(6), calculated from mobility in gels. We present evidence that the 'extra' bands obtained with restriction endonucleases are derived from this component. 3. DNA-DNA renaturation analysis of fragmented kDNA shows the presence of a minor complex component with a complexity of about 3 X 10(7), making up less than 10% of the total kDNA. 4. From these results we conclude that 3--5% of the kDNA consists of a homogeneous class of maxi-circles catenated in the mini-circle network. The molecular weight of these maxi-circles is about 26 X 10(6) and they contain a unique, non-repetitive, non-mini-circle nucleotide sequence. This component is a prime candidate for the true mitochondrial DNA of trypanosomes.     

Trypanosoma equiperdum: master of disguise or historical mistake?

After 100 years of research, only a small number of laboratory strains of Trypanosoma equiperdum exists, and the history of most of the strains is unknown. No definitive diagnosis of dourine can be made at the serological or molecular level. Only clinical signs are pathognomonic and international screening relies on an outdated cross-reactive serological test (the complement-fixation test) from 1915, resulting in serious consequences at the practical level. Despite many characterization attempts, no clear picture has emerged of the position of T. equiperdum within the Trypanozoon group. In this article, we highlight the controversies that exist regarding T. equiperdum, and the overlap that occurs with Trypanosoma evansi and Trypanosoma brucei brucei. By revisiting the published data, from the early decades of discovery to the recent serological- and molecular-characterization studies, a new hypothesis arises in which T. equiperdum no longer exists as a separate species and in which current strains can be divided into T. evansi (the historical mistake) and Trypanosoma brucei equiperdum (the master of disguise). Hence, dourine is a disease caused by specific host immune responses to a T. b. equiperdum or T. evansi infection.     

Sequence heterogeneity of the mini-circles of kinetoplast DNA of Crithidia luciliae and evidence for the presence of a component more complex than mini-circle DNA in the kinetoplast network.

Exhaustive digestion of the 0.76 mum mini-circles of the kinetoplast DNA from Crithidia luciliae with endonuclease HapII yields at least 37 fragments with an added molecular weight of at least 24-10(6), i.e. about 16 times that of the mini-circle. The DNA isolated from cloned cells yields the same digestion pattern. Endonuclease EcoRI cuts only part of the mini-circles in each network. This proves that mini-circles are not homogeneous in sequence. Digestion of total kinetoplast DNA with HapII yields, in addition to the mini-circle fragments, 7 fragments with an added molecular weight of 16-10(6). We conclude that these are derived from a minor component of the network, with a higher sequence complexity than the mini-circles.     

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 structure of kinetoplast DNA. 1. The mini-circles of Crithidia lucilae are heterogeneous in base sequence.

We have analysed limit digests of mini-circles from kinetoplast DNA of Crithidia luciliae by gel electrophoresis. Endonucleases HapII and AluI cut the circles into at least 37 and 21 fragments, respectively, and leave no circles intact. In both cases the added molecular weights of the fragments, estimated from mobility in gels, exceeds 18 X 10(6), i.e. more than 12 times the molecular weight of the mini-circle DNA. Endonucleases HindII + III, EcoRI and HpaI cut only part of the circles. These results show that the mini-circles are heterogeneous in base sequence. Different sequence classes are present in different amounts. DNA-DNA renaturation analysis of mini-circle DNA yields a complexity of about 3 X 10(6), i.e. twice the molecular weight on one mini-circle. The delta tm of native and renatured duplexes is about 1 degree C, showing that the sequence heterogeneity is a micro-heterogeneity. Electron microscopy, gel electrophoresis and sedimentation analysis show that the circles that are not cut by endonucleases HindII + III remain catenated in very large associations. These associations lack the 'rosette' structures and the long edge loops characteristic of intact kinetoplast DNA. This suggests that the mini-circle classes cut by endonucleases HindII + III are present throughout the network and that the maxi-circle component of the network (see accompanying paper) is not essential to hold the network together. Prolonged electrophoresis on 1.5% or 2% agarose gels resolves the open mini-circles into three and linearized mini-circles into four bands, present in different amounts. We conclude that the mini-circles are also heterogeneous in size, the difference in size between the two extreme size classes being 4% of the contour length. Digestion with endonuclease HapII shows that at least three out of these four bands differ in sequence. Possible mechanisms that could account for the micro-heterogeneity in sequence of mini-circles are discussed.     

Analysis by electron microscopy of the variable segment in the maxi-circle of kinetoplast DNA from Trypanosoma brucei

The 20.5-kbp maxi-circle from the kinetoplast DNA of Trypanosoma brucei contains a 5-kbp segment which is not cut by most restriction endonucleases and which varies in size in closely-related trypanosome strains (Borst, P., Fase-Fowler, F., Hoeijmakers, J.H.J. and Frasch, A.C.C. (1980) Biochim. Biophys. Acta 610, 197–210). We have now analysed partial denaturation maps of the linearized maxi-circles by electron microscopy and find that the variable segment is not more AT-rich than the remainder of the maxi-circle. Early denaturation begins at two separate regions of the maxi-circle outside the variable region and one of these corresponds with the position of the gene for the large (12 S) ribosomal RNA. Denaturation-renaturation of maxi-circles leads to the formation of partially mismatched duplexes that look like underwound loops in electron micrographs. These loops are only found in the variable region and they vary in size and appearance. Under our renaturation conditions single-stranded maxi-circle DNA is devoid of secondary structure and this suggests that the underwound loops arise by misalignment of straight tandem repeats in the DNA. We have also analysed heteroduplexes between maxi-circles from two closely related T. brucei strains that differ by 1 kbp in the size of their variable segment. Most molecules had no underwound loops and contained mismatched regions in the variable segment only. The appearance of these regions is diverse, varying from fully duplex with two single-stranded loops to molecules with a heterogeneous array of smaller loops. The total size of single-stranded DNA in the heteroduplexes may be as high as 1.2 μm, i.e., a factor 4 higher than the size difference between the heteroduplex partners. We conclude that the variable region consists of imperfect tandem repeats of a sequence that evolves rapidly. This region might contain the origin of maxi-circle replication.     

The kinetoplast duplication cycle in Trypanosoma brucei is orchestrated by cytoskeleton-mediated cell morphogenesis

The mitochondrial DNA of Trypanosoma brucei is organized in a complex structure called the kinetoplast. In this study, we define the complete kinetoplast duplication cycle in T. brucei based on three-dimensional reconstructions from serial-section electron micrographs. This structural model was enhanced by analyses of the replication process of DNA maxi- and minicircles. Novel insights were obtained about the earliest and latest stages of kinetoplast duplication. We show that kinetoplast S phase occurs concurrently with the repositioning of the new basal body from the anterior to the posterior side of the old flagellum. This emphasizes the role of basal body segregation in kinetoplast division and suggests a possible mechanism for driving the rotational movement of the kinetoplast during minicircle replication. Fluorescence in situ hybridization with minicircle- and maxicircle-specific probes showed that maxicircle DNA is stretched out between segregated minicircle networks, indicating that maxicircle segregation is a late event in the kinetoplast duplication cycle. This new view of the complexities of kinetoplast duplication emphasizes the dependencies between the dynamic remodelling of the cytoskeleton and the inheritance of the mitochondrial genome.     

Organized packaging of kinetoplast DNA networks

The kinetoplast DNA (kDNA) of Trypanosoma equiperdum is organized as a complex structure of catenated circular DNA molecules. The major component of the kDNA network is the one kilobase minicircle that is present at about 10,000 copies per network. We have developed two assays to examine the structure of kDNA networks compacted in vitro with spermidine. Our results suggest that minicircles are arranged into a regular structure with an exposed domain which is DNAase I- and restriction-sensitive and a protected domain which is resistant to restriction endonucleases and DNAase I. This regularly packaged structure is dependent upon spermidine compaction and the circularity of the kDNA, but does not require supercoiled minicircles or catenated networks.     

Trypanosoma cruzi populations: more clonal than sexual

The ancient question of trypanosome sexuality has recently been reactivated in view of important observations in the African species Trypanosoma brucie, in which Mendelian sexuality has been proposed as a working hypothesis on the basis o f indirect isozyme evidence. Subsequent experiments have confirmed that recombination can occur in T. brucei under defined experimental conditions and suggest that this parasite undergoes meiosis. In this article, Michel Tibayrenc and Francisco Ayala discuss the intraspecific variability of another species, Tyapanosoma cruzi - causative agent of american trypanosomiasis or Chagas disease. They interpret the variation revealed by extensive isozyme analysis and restriction endonuclease analysis of kinetoplast DNA, to suggest that T. cruzi is diploid, genetically very polymorphic, and has a clonal structure that manifests a lack of (or very restricted) sexuality.     

Kinetoplast DNA replication: mechanistic differences between Trypanosoma brucei and Crithidia fasciculata

Kinetoplast DNA, the mitochondrial DNA of trypanosomatid parasites, is a network containing several thousand minicircles and a few dozen maxicircles. We compared kinetoplast DNA replication in Trypanosoma brucei and Crithidia fasciculata using fluorescence in situ hybridization and electron microscopy of isolated networks. One difference is in the location of maxicircles in situ. In C. fasciculata, maxicircles are concentrated in discrete foci embedded in the kinetoplast disk; during replication the foci increase in number but remain scattered throughout the disk. In contrast, T. brucei maxicircles generally fill the entire disk. Unlike those in C. fasciculata, T. brucei maxicircles become highly concentrated in the central region of the kinetoplast after replication; then during segregation they redistribute throughout the daughter kinetoplasts. T. brucei and C. fasciculata also differ in the pattern of attachment of newly synthesized minicircles to the network. In C. fasciculata it was known that minicircles are attached at two antipodal sites but subsequently are found uniformly distributed around the network periphery, possibly due to a relative movement of the kinetoplast disk and two protein complexes responsible for minicircle synthesis and attachment. In T. brucei, minicircles appear to be attached at two antipodal sites but then remain concentrated in these two regions. Therefore, the relative movement of the kinetoplast and the two protein complexes may not occur in T. brucei.