The identification of circular extrachromosomal DNA in the nuclear genome of Trypanosoma brucei

Nuclear extrachromosomal DNA elements have been identified in several kinetoplastids such as Leishmania and Trypanosoma cruzi, but never in Trypanosoma brucei. They can occur naturally or arise spontaneously as the result of sublethal drug exposure of parasites. In most cases, they are represented as circular elements and are mitotically unstable. In this study we describe the presence of circular DNA in the nucleus of Trypanosoma brucei. This novel type of DNA was termed NR-element (NlaIII repeat element). In contrast to drug-induced episomes in other kinetoplastids, the T. brucei extrachromosomal NR-element is not generated by drug selection. Furthermore, the element is stable during mitosis over many generations. Restriction analysis of tagged NR-element DNA, unusual migration patterns during pulsed field gel electrophoresis (PFGE) and CsCl/ethidium bromide equilibrium centrifugation demonstrates that the NR-element represents circular DNA. Whereas it has been found in all field isolates of the parasites we analysed, it is not detectable in some laboratory strains notably the genome reference strain 927. The DNA sequence of this element is related to a 29 bp repeat present in the subtelomeric region of VSG-bearing chromosomes of T. brucei. It has been suggested that this subtelomeric region is part of a transition zone on chromosomes separating the relatively stable telomeric repeats from the recombinationaly active region downstream of VSG genes. Therefore, we discuss a functional connection between the occurrence of this circular DNA and subtelomeric recombination events in T. brucei.     

The tubulin genes of Trypanosoma cruzi

The organization of the alpha- and beta-tubulin genes in the genome of Trypanosoma cruzi have been analysed by Southern blotting using tubulin probes derived from Trypanosoma brucei. The tubulin array appears to be more complex in this organism than in other members of the same family. Some tubulin genes are tightly clustered in an alternating (alpha-beta)n array with a basic repeat unit length of 4.3 kb. However, other pairs of alternating alpha- and beta-tubulin sequences appear to be physically separated from the basic group. This finding indicates that the tubulin gene cluster present in T. cruzi is less perfectly conserved than in T. brucei. T. (Herpetosoma) rangeli is similar to T. (Schizotrypanum) cruzi in its tubulin gene organization whereas most of these genes are tandemly clustered in the genome of T. (Trypanozoon) evansi, with a basic repeat unit length of 3.6 kb as previously described for T. (Trypanozoon) brucei. Two overlapping recombinant clones containing T. cruzi tubulin sequences have been isolated from a genomic cosmid library of T. cruzi epimastigotes using the T. brucei tubulin probes. Partial sequencing of the T. cruzi beta-tubulin gene has confirmed its identity and shows more than 70% homology with the sea urchin, chicken and T. b. rhodesiense beta-tubulin reported gene sequences. Analysis of tubulin gene organization through the parasite life cycle does not show evidence of major rearrangements within the repeat unit. Several T. cruzi strains and cloned lines whilst sharing the 4.3-kb tubulin repeat unit, exhibited very variable tubulin gene organization with tubulin probes. These striking differences in the organization of this structural gene among T. cruzi strains and cloned lines suggest that the heterogeneity previously reported in parasite populations may be related to a very dynamic, diploid genome.     

The kinetoplast DNA of Trypanosoma equiperdum

We have analyzed the kinetoplast DNA for Trypanosoma equiperdum (American Type Culture Collection 30019) and two dyskinetoplastic strains derived from it. The DNA networks from the kinetoplastic strain are made up of catenated mini-circles and maxi-circles, like the networks from the closely-related Trypanosoma brucei. The mini-circles of T. equiperdum lack the pronounced sequence heterogeneity of T. brucei mini-circles, as shown by the fragment distribution of restriction digests and by the predominance of well-matched duplexes in electron micrographs of renatured DNA. The electrophoretic analysis of kinetoplast DNA digested with various restriction endonucleases shows the maxi-circle of T. equiperdum to consist of circular DNA molecules of 8.4 x 10(6) daltons, without size or sequence heterogeneity or repetitious segments. A comparison of the sequence by restriction endonuclease fragmentation and hybridization shows extensive sequence homology. The size difference between both maxi-circles is due to the deletion of one continuous segment of 5.10(6) daltons. In the two dyskinetoplastic strains, we cannot detect DNA sequences that hybridize with kinetoplast DNA from T. brucei or from the kinetoplastic strain of T. equiperdum. In one of these strains, a 'low-density' DNA fraction contained a simple sequence DNA, cleaved by restriction endonuclease HindIII into fragments of 180 base-pairs and multimers of this. The relation of this DNA to kinetoplast DNA, if any, is unknown.     

Evolution of satellite DNAs from the genus Palorus--experimental evidence for the "library" hypothesis

Satellite DNA profiles have been characterized in the congeneric species Palorus ratzeburgii, Palorus subdepressus, Palorus genalis, and Palorus ficicola (Coleoptera, Insecta), each of which contains a single, A + T-rich satellite DNA comprising a considerable portion of the genome (20%-40%). These satellites exhibit insignificant mutual sequence similarity. Using PCR assay, it has been shown that all four sequences are present in each of the tested Palorus species: one of them is amplified into a high copy number or a major satellite, while the three others are in the form of low-copy-number repeats estimated to make up approximately 0.05% of the genome. Each of the four satellites is interspecifically high conserved concerning the sequence, monomer length, and tandem repeat organization. Major, as well as low- copy-number, satellites are colocalized in the regions of pericentromeric heterochromatin on all chromosomes of the complement. The low-copy-number satellites are dispersed between the large arrays of the major satellite over the whole heterochromatic block. Our results explain satellite DNA evolution, confirming the hypothesis that related species share a "library" of conserved satellite sequences, some of which could be amplified into a major satellite. Due to the evolutionary dynamics of satellite DNAs, the content of the "library" is variable; the elimination of some sequences parallels the creation of the new ones. Quantitative changes in satellite DNAs, induced by occasional amplification of satellite repeat from the "library", could possibly occur in the course of the speciation process, thus forming a species-specific profile of satellite DNAs.      

Evidence for gene conversion between the phosphoglycerate kinase genes of Trypanosoma brucei

Trypanosoma brucei contains a tandem array of three genes for phosphoglycerate kinase (PGKase), genes A, B and C, each coding for a different protein. We have compared allelic variants of this gene array and find evidence for gene conversion between the three genes. Near the 3' end, the different alleles and gene B contain a variable sequence that is similar to the corresponding sequence in either gene A or gene C. This sequence is flanked by glycine triplets that are conserved in all PGKases from bacteria to mammals. The triplets are encoded by (GGT)n, resulting in sequences that resemble the recombination-promoting chi-sites of Escherichia coli. Upstream of the variable sequence, there is an area of 800 base-pairs in which genes A, B and C are highly homologous; in all three genes this region ends with a sharp boundary at which gene B again shows segmental homology with both genes A and C. These results suggest that repeated gene conversion events partially erase the differences between genes A, B and C that arise in evolution and suggest that chi-like sequences may act as recombinational hotspots in protozoa such as T. brucei.     

Sequence and sequence variation within the 1.688 g/cm3 satellite DNA of Drosophila melanogaster.

We have determined the complete nucleotide sequence of the monomer repeating unit of the 1.688 g/cm³ satellite DNA from Drosophila melanogaster. This satellite DNA, which makes up 4% of the Drosophila genome and is located primarily on the sex chromosomes, has a repeat unit 359 base-pairs in length. This complex sequence is unrelated to the other three major satellite DNAs present in this species, each of which contains a very short repeated sequence only 5 to 10 base-pairs long. The repeated sequence is more similar to the complex repeating units found in satellites of mammalian origin in that it contains runs of adenylate and thymidylate residues. We have determined the nature of the sequence variations in this DNA by restriction nuclease cleavage and by direct sequence determination of (1) individual monomer units cloned in hybrid plasmids, (2) mixtures of adjacent monomers from a cloned segment of this satellite DNA, (3) mixtures of monomer units isolated by restriction nuclease cleavage of total 1.688 g/cm³ satellite DNA. Both direct sequence determination and restriction nuclease cleavage indicate that certain positions in the repeat can be highly variable with up to 50% of certain restriction sites having altered recognition sequences. Despite the high degree of variation at certain sites, most positions in the sequence are highly conserved. Sequence analysis of a mixture of 15 adjacent monomer units detected only nine variable positions out of 359 base-pairs. Total satellite DNA showed only four additional positions. While some variability would have been missed due to the sequencing methods used, we conclude that the variation from one repeat to the next is not random and that most of the satellite repeat is conserved. This conservation may reflect functional aspects of the repeated DNA, since we have shown earlier that part of this sequence serves as a binding site for a sequence-specific DNA binding protein isolated from Drosophila embryos (Hsieh &; Brutlag, 1979).     

Identification of the telomere in Trypanosoma cruzi reveals highly heterogeneous telomere lengths in different parasite strains.

Here we describe the cloning and characterisation of the Trypanosoma cruzi telomere. In the Y strain, it is formed by typical GGGTTA repeats with a mean size of approximately 500 bp. Adjacent to the telomere repeats we found a DNA sequence with significant homology to the T.cruzi 85 kDa surface antigen (gp85). Examination of the telomere in nine T.cruzi strains reveals differences in the organisation of chromosome ends. In one group of strains the size of the telomere repeat is relatively homogeneous and short (0.5-1.5 kb) as in the Y strain, while in the other, the length of the repeat is very heterogeneous and significantly longer, ranging in size from 1 to >10 kb. These different strains can be grouped similarly to previously existing classifications based on isoenzyme loci, rRNA genes, mini-exon gene sequences, randomly amplified polymorphic DNA and rRNA promoter sequences, suggesting that differential control of telomere length and organisation appeared as an early event in T. cruzi evolution. Two-dimensional pulsed field gel electrophoresis analysis shows that some chromosomes carry telomeres which are significantly larger than the mean telomere length. Importantly, the T.cruzi telomeres are organised in nucleosomal and non-nucleosomal chromatin.     

Sequence and evolution of related bovine and caprine satellite DNAs: Identification of a short DNA sequence potentially involved in satellite DNA amplification

The satellite II DNAs of the domestic ox Bos taurus and sheep Ovis aries have been sequenced, and that of the domestic goat Capra hircus partially sequenced. All three are related, and consist of repeat units of about 700 base-pairs. There is no evidence of internal repetition within these repeat units. When matched for maximum homology, the goat and sheep sequences show 83% homology, whereas the ox and sheep sequences share only 70% homology. Factors contributing to the uncertainty of the exact homology between these sequences are discussed, but the results are nevertheless consistent with their progenitor sequence being present in the common ancestor of cattle and sheep. Goat satellite II DNA is shown to contain another, unrelated, tandemly repeated sequence, which is composed of 22 base-pair repeat units. Both this sequence and a region of ox satellite II share good homology with the 11 base-pair progenitor sequence of ox 1.706 g/cm³ satellite DNA. It is suggested that this shared sequence could play a role in bovine satellite DNA amplification.     

The karyotype and ploidy of Trypanosoma cruzi.

Little is known of the number or organization of chromosomes in Trypanosoma cruzi, the protozoan parasite responsible for Chagas' disease in man in the New World. Straightforward cytogenetic analysis is precluded because trypanosome chromosomes fail to condense during the cell cycle. We have size-fractionated the chromosome-sized DNA molecules of representative T. cruzi strains by pulsed field gradient (PFG) gel electrophoresis and located several housekeeping genes by Southern blotting using cDNA probes from the related trypanosome T. brucei. We show that DNA molecules from homologous chromosomes of T. cruzi migrate differently in the PFG system and infer that T. cruzi epimastigotes are at minimum diploid. In contrast to T. brucei, mini-chromosomes are absent in T. cruzi. All the housekeeping genes studied hybridize to DNA molecules which can be resolved in the PFG system, suggesting that T. cruzi may have no chromosomes larger than a few megabase pairs.     

Isolation and characterisation of satellite DNA from Brassica juncea (L.) Czern

Nuclear DNA isolated from hypocotyls (H), proliferating callus (PC) and differentiating callus (DC) of Brassica juncea contains a satellite DNA which can be resolved in actinomycin-D/CsCl gradients. The satellite DNA undergoes changes, when an in vitro culture is raised from hypocotyl tissue and forms a higher percentage of the genome in PC and DC than in mature differentiated tissue (hypocotyl). All the three satellite DNAs are GC-rich compared to main band DNAs. Satellite DNA of H has higher Tm and GC content than that of the PC and DC satellites. A 200 bp basic repeat unit from hypocotyl nuclear DNA has been cloned and characterised.     

Trypanosoma cruzi glycosomal glyceraldehyde-3-phosphate dehydrogenase does not conform to the ''hotspot'' topogenic signal model.

The genes which encode glycosomal glyceraldehyde-phosphate dehydrogenase (gGAPDH) of Trypanosoma cruzi are arranged as a tandemly repeated pair on a single chromosome and are identical at the level of nucleotide sequence. They are separated by an intergenic region which contains a 317 base pair sequence with the properties of a retroposon. The genes express a 1.5 kb mRNA and a 38 kd protein. The amino acid sequence contains features characteristic of glycosomal enzymes such as peptide insertions and a C-terminal extension. However, T. cruzi gGAPDH lacks one of the positively charged 'hotspot' motifs which have been proposed as topogenic signals for import into the glycosome, a unique microbody-like organelle. Molecular modelling of the T. cruzi and T. brucei enzymes suggests that neither structure would fulfil the requirements of the 'hotspot' glycosomal import model.     

Characterization of a cDNA encoding a cysteine-rich cell surface protein located in the flagellar pocket of the protozoan Trypanosoma brucei.

We have characterized a cDNA encoding a cysteine-rich, acidic integral membrane protein (CRAM) of the parasitic protozoa Trypanosoma brucei and Trypanosoma equiperdum. Unlike other membrane proteins of T. brucei, which are distributed throughout the cell surface, CRAM is concentrated in the flagellar pocket, an invagination of the cell surface of the trypanosome where endocytosis has been documented. Accordingly, CRAM also locates to vesicles located underneath the pocket, providing evidence of its internalization. CRAM has a predicted molecular mass of 130 kilodaltons and has a signal peptide, a transmembrane domain, and a 41-amino-acid cytoplasmic extension. A characteristic feature of CRAM is a large extracellular domain with a roughly 66-fold acidic, cysteine-rich 12-amino-acid repeat. CRAM is conserved among different protozoan species, including Trypanosoma cruzi, and CRAM has structural similarities with eucaryotic cell surface receptors. The most striking homology of CRAM is to the human low-density-lipoprotein receptor. We propose that CRAM functions as a cell surface receptor of different trypanosome species.     

Mismatch repair in Trypanosoma brucei: heterologous expression of MSH2 from Trypanosoma cruzi provides new insights into the response to oxidative damage

Trypanosomes are unicellular eukaryotes that cause disease in humans and other mammals. Trypanosoma cruzi and Trypanosoma brucei are the causative agents, respectively, of Chagas disease in the Americas and sleeping sickness in sub-Saharan Africa. To better comprehend the interaction of these parasites with their hosts, understanding the mechanisms involved in the generation of genetic variability is critical. One such mechanism is mismatch repair (MMR), which has a crucial, evolutionarily conserved role in maintaining the fidelity of DNA replication, as well as acting in other cellular processes, such as DNA recombination. Here we have attempted to complement T. brucei MMR through the expression of MSH2 from T. cruzi. Our results show that T. brucei MSH2-null mutants are more sensitive to hydrogen peroxide (H2O2) than wild type cells, suggesting the involvement of MSH2 in the response to oxidative stress in this parasite. This phenotype is reverted by the expression of either the T. cruzi or the T. brucei MSH2 protein in the MSH2-null mutants. In contrast, MMR complementation, as assessed by resistance to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and microsatellite instability, was not achieved by the heterologous expression of T. cruzi MSH2. This finding, associated to the demonstration that mutation of MLH1, another component of the MMR system, did not affect sensitivity of T. brucei cells to H2O2, suggests an additional role of MSH2 in dealing with oxidative damage in these parasites, which may occur independently of MMR.     

Nitrosamine-induced cancer: O4-alkylthymine produces sites of DNA hyperflexibility.

The carcinogenic properties of N-nitroso compounds are associated with their ability to alkylate DNA, in particular to form O6-alkylguanine and O4-alkylthymine. DNA duplexes containing either O6-alkylguanine or O4-alkylthymine were synthesized, and each duplex was ligated to form a set of DNAs of increasing length with the alkylated base out of phase (16 base-pairs apart) or in phase (21 base-pairs apart) with the helical repeat of the DNA. The DNA contained the sequence 5' CAA 3', which is the 61st codon of the K-ras gene, because this codon is a preferred site of mutation for a number of carcinogens including the methylating carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1- butanone (NNK). O4-Methylthymine or O4-ethylthymine replaced thymine in either of the two A.T base-pairs of this codon (normally CAA), and O6-methylguanine replaced the guanine in the G.C pair. All the sequences containing O4-alkylthymine exhibited anomalous, slow, gel migration and ligated to form circles of unusually small diameter. In general, the effect was seen when the alkylated base-pair was out of phase with the helical repeat as well as when it was in phase, suggesting that the alkylated base-pair confers flexibility which is largely isotropic, i.e., has no preferred direction, rather than anisotropic flexibility or bending. However, at pH 8.3 the 21-base-pair set containing O4-alkylT.A had significantly greater anomalous migration than the 16-base-pair set, suggesting that the flexibility produced by this base-pair has a significant anisotropic component and thus resembles true bending.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stable introduction of exogenous DNA into Trypanosoma brucei.

The lack of a homologous transformation system for trypanosomes is a serious handicap to the study of gene expression in these protozoans. Attempts to develop such a system have been complicated by the lack of suitable homologous vectors and ignorance of the requirements for mRNA synthesis which is discontinuous in trypanosomes. We have found that Trypanosoma congolense, a close relative of T. brucei, contains exceptionally small chromosomes, which can be isolated whole and distinguished from those of T. brucei by the presence of a unique satellite DNA. We show here that mini-chromosomes from T. congolense can be introduced into T. brucei by electroporation and detected by hybridisation with T. congolense satellite DNA. The introduced DNA can survive through several generations in the absence of any selective pressure. These results provide the basis for the development of a transformation system for trypanosomes.     

A head-to-tail tandem organization of hsp70 genes in Trypanosoma cruzi.

We describe the isolation and characterization of the T. cruzi hsp 70 DNA coding region which was found to be formed by multigene copies organized in a tandem array in a head-to-tail manner. The restriction pattern of one of the repetition units within the largest clone obtained from the genomic library, clone Tc70.6, shows that the hsp70 coding region should be formed by at least seven identical copies of 2.5 kb. We have found, however, the presence of restriction polymorphisms (Pvu II) within these repeats. Subsequent analysis of the time course of nuclear DNA digestion has revealed that the copy number per haploid genome could be as greater as 10. The analysis of the DNA and amino acid sequence of a fragment (70%) of one of the repetition units has shown the existence of a high homology with all the hsp70 genes of other organisms. The protein sequence homology of the fragment analyzed is as high as 88% when compared with that of the T. brucei hsp70. On the other hand, there are significant restriction site variations between both. The T. cruzi hsp70 contains at the C-terminal end a tetrapeptide repeat of the structure (GMPG)9.