The identification of hydroxymethyluracil in DNA of Trypanosoma brucei.

We have previously reported the detection of two unusual nucleotides, pdJ and pdV, in the DNA of Trypanosoma brucei (Gommers-Ampt et al., 1991). pdJ was found to be a novel nucleotide and is possibly involved in the regulation of variant specific surface antigen gene expression in trypanosomes. Recent evidence suggests that V could be a precursor of J, making V a key compound in the study of the biosynthesis and function of J. We have therefore determined the structure of V and here we present proof that V is HOMeU. The identity is based on a detailed comparison of dV(p) with authentic HOMedU(p), showing: I) co-migration in three different liquid chromatography analyses II) identical UV absorbance characteristics III) identical behavior in acetyl-pentafluorobenzyl derivatization and subsequent Gas chromatography/Mass spectrometry (GC/MS). The GC/MS technique has not been used before to analyse HOMedU purified from biological material. Because of its high sensitivity, it may also be useful for the detection of the low amounts of HOMedU resulting from oxidative damage of DNA.     

Nuclear and genome organization of Trypanosoma brucei

In this article, Klaus Ersfeld, Sara Melville and Keith Gull review current understanding of the structural organization of the nucleus of Trypanosoma brucei, and summarize recent data pertinent to the organization of its genome. Until recently, the cell biology of the trypanosome nucleus and issues of DNA organization and gene expression have often been treated as separate themes. However, recent work emphasizes the need for a more holistic approach to understanding these aspects of the biology of this parasite.     

The mitochondrial tRNAs of Trypanosoma brucei are nuclear encoded

The mitochondrial DNA of Trypanosoma brucei is organized as a catenated network of maxicircles and minicircles. The maxicircles are equivalent to the typical mitochondrial genome except that the genes for the mitochondrial tRNAs have not been identified by sequence analysis of the maxicircle DNA. The apparent absence of tRNA genes in the maxicircle DNA suggests that the mitochondrial tRNAs are encoded by either the minicircle or the nuclear DNA. In order to determine their genomic origin, we isolated and identified the mitochondrial tRNAs of T. brucei. We show that these mitochondrial tRNAs are truly mitochondrially located in vivo and that they are free from detectable contamination by cytosolic RNAs. By hybridization analysis, using mitochondrial tRNAs as the probe, we determined that the mitochondrial tRNAs are encoded by nuclear DNA. This implies that RNAs, like proteins, are imported into the mitochondria. We investigated the relationship between the cytosolic and the mitochondrial tRNA genes and show that there are unique cytosolic tRNA genes, unique mitochondrial tRNA genes, and tRNA genes which appear to be shared and whose products are therefore targeted to both the cytosol and the mitochondrion.     

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.     

Mitochondrial DNA ligases of Trypanosoma brucei

The mitochondrial DNA of Trypanosoma brucei, termed kinetoplast DNA or kDNA, consists of thousands of minicircles and a small number of maxicircles catenated into a single network organized as a nucleoprotein disk at the base of the flagellum. Minicircles are replicated free of the network but still contain nicks and gaps after rejoining to the network. Covalent closure of remaining discontinuities in newly replicated minicircles after their rejoining to the network is delayed until all minicircles have been replicated. The DNA ligase involved in this terminal step in minicircle replication has not been identified. A search of kinetoplastid genome databases has identified two putative DNA ligase genes in tandem. These genes (LIG k alpha and LIG k beta) are highly diverged from mitochondrial and nuclear DNA ligase genes of higher eukaryotes. Expression of epitope-tagged versions of these genes shows that both LIG k alpha and LIG k beta are mitochondrial DNA ligases. Epitope-tagged LIG k alpha localizes throughout the kDNA, whereas LIG k beta shows an antipodal localization close to, but not overlapping, that of topoisomerase II, suggesting that these proteins may be contained in distinct structures or protein complexes. Knockdown of the LIG k alpha mRNA by RNA interference led to a cessation of the release of minicircles from the network and resulted in a reduction in size of the kDNA networks and rapid loss of the kDNA from the cell. Closely related pairs of mitochondrial DNA ligase genes were also identified in Leishmania major and Crithidia fasciculata.     

An analysis of cosmid clones of nuclear DNA from Trypanosoma brucei shows that the genes for variant surface glycoproteins are clustered in the genome

Trypanosoma brucei contains more than a hundred genes coding for the different variant surface glycoproteins (VSGs). Activation of some of these genes involves the duplication of the gene (the basic copy or BC) and transposition of the duplicate to an expression site (yielding the expression-linked copy or ELC). We have cloned large fragments of genomic DNA in cosmid vectors in Escherichia coli. Cosmids containing the BCs of genes 117, 118 and 121 were readily obtained, but DNA containing the ELCs was strongly selected against in the cosmid and plasmid cloning systems used. We have analysed the distribution of VSG genes in the genome using probes for the sequences at the edges of the transposed segment which are partially homologous among these genes. In genomic cosmid clone banks, about 9% of all colonies hybridize with probes from the 5'- and 3'-edges of the transposed segment, showing that these sequences are linked in the genome. Moreover, the 117 and 118 BC cosmids contain several additional putative VSG genes in tandem, as deduced from hybridization and sequence analyses. We conclude that the VSG genes are highly clustered and share common sequences at the borders of the transposed segment.     

The configuration of DNA replication sites within the Trypanosoma brucei kinetoplast

The kinetoplast is a concatenated network of circular DNA molecules found in the mitochondrion of many trypanosomes. This mass of DNA is replicated in a discrete "S" phase in the cell cycle. We have tracked the incorporation of the thymidine analogue 5-bromodeoxyuridine into newly replicated DNA by immunofluorescence and novel immunogold labeling procedures. This has allowed the detection of particular sites of replicated DNA in the replicating and segregating kinetoplast. These studies provide a new method for observing kinetoplast DNA (kDNA) replication patterns at high resolution. The techniques reveal that initially the pattern of replicated DNA is antipodal and can be detected both on isolated complexes and in replicating kDNA in vivo. In Trypanosoma brucei the opposing edges of replicating kDNA never extend around the complete circumference of the network, as seen in other kinetoplastids. Furthermore, crescent-shaped labeling patterns are formed which give way to labeling of most of the replicating kDNA except the characteristic midzone. The configuration of these sites of replicated DNA molecules is different to previous studies on organisms such as Crithidia fasciculata, suggesting differences in the timing of replication of mini and maxicircles and/or organization of the replicative apparatus in the kinetoplast of the African trypanosome.     

Ab initio identification of novel regulatory elements in the genome of Trypanosoma brucei by Bayesian inference on sequence segmentation

Background

The rapid increase in the availability of genome information has created considerable demand for both comparative and ab initio predictive bioinformatic analyses. The biology laid bare in the genomes of many organisms is often novel, presenting new challenges for bioinformatic interrogation. A paradigm for this is the collected genomes of the kinetoplastid parasites, a group which includes Trypanosoma brucei the causative agent of human African trypanosomiasis. These genomes, though outwardly simple in organisation and gene content, have historically challenged many theories for gene expression regulation in eukaryotes.

Methodology/Principle Findings

Here we utilise a Bayesian approach to identify local changes in nucleotide composition in the genome of T. brucei. We show that there are several elements which are found at the starts and ends of multicopy gene arrays and that there are compositional elements that are common to all intergenic regions. We also show that there is a composition-inversion element that occurs at the position of the trans-splice site.

Conclusions/Significance

The nature of the elements discovered reinforces the hypothesis that context dependant RNA secondary structure has an important influence on gene expression regulation in Trypanosoma brucei.     

Base J, found in nuclear DNA of Trypanosoma brucei, is not a target for DNA glycosylases

Base excision repair (BER) is an evolutionarily conserved system which removes altered bases from DNA. The initial step in BER is carried out by DNA glycosylases which recognize altered bases and cut the N-glycosylic bond between the base and the DNA backbone. In kinetoplastid flagellates, such as Trypanosoma brucei, the modified base beta-D-glucosyl-hydroxymethyluracil (J) replaces a small percentage of thymine residues, predominantly in repetitive telomeric sequences. Base J is synthesized at the DNA level via the precursor 5-hydroxymethyluracil (5-HmU). We have investigated whether J in DNA can be recognized by DNA glycosylases from non-kinetoplastid origin, and whether the presence of J and 5-HmU in DNA has required modifications of the trypanosome BER system. We tested the ability of 15 different DNA glycosylases from various origins to excise J or 5-HmU paired to A from duplex oligonucleotides. No excision of J was found, but 5-HmU was excised by AlkA and Mug from Escherichia coli and by human SMUG1 and TDG, confirming previous reports. In a combination of database searches and biochemical assays we identified several DNA glycosylases in T. brucei, but in trypanosome extracts we detected no excision activity towards 5-HmU or ethenocytosine, a product of oxidative DNA damage and a substrate for Mug, TDG and SMUG1. Our results indicate that trypanosomes have a BER system similar to that of other organisms, but might be unable to excise certain forms of oxidatively damaged bases. The presence of J in DNA does not require a specific modification of the BER system, as this base is not recognized by any known DNA glycosylase.     

Identification of a telomeric DNA sequence in Trypanosoma brucei

A simple repetitive DNA sequence in the nuclear genome of Trypanosoma brucei, consisting of tandem repeats of the hexanucleotide 5' CCCTAA 3', was identified as being telomeric by several criteria. This sequence was specifically labeled with T. brucei genomic DNA as the template for in vitro nick translation by DNA polymerase I, and was present in Bal 31 nuclease sensitive, genomic restriction fragments of the large sizes expected in this organism for at least some telomeric regions. The same repeated sequence was found in six other flagellates tested. A segment of DNA from T. brucei including this telomeric sequence was cloned in pBR322 and characterized. The cloned segment contained a sequence highly homologous to the 3' ends of several variant surface glycoprotein mRNAs, upstream of the tandemly repeated hexanucleotide sequence.     

Genomic organization of Trypanosoma brucei kinetoplast DNA minicircles

The sequences of seven new Trypanosoma brucei kinetoplast DNA minicircles were obtained. A detailed comparative analysis of these sequences and those of the 18 complete kDNA minicircle sequences from T. brucei available in the database was performed. These 25 different minicircles contain 86 putative gRNA genes. The number of gRNA genes per minicircle varies from 2 to 5. In most cases, the genes are located between short imperfect inverted repeats, but in several minicircles there are inverted repeat cassettes that did not contain identifiable gRNA genes. Five minicircles contain single gRNA genes not surrounded by identifiable repeats. Two pairs of closely related minicircles may have recently evolved from common ancestors: KTMH1 and KTMH3 contained the same gRNA genes in the same order, whereas KTCSGRA and KTCSGRB contained two gRNA genes in the same order and one gRNA gene specific to each. All minicircles could be classified into two classes on the basis of a short substitution within the highly conserved region, but the minicircles in these two classes did not appear to differ in terms of gRNA content or gene organization. A number of redundant gRNAs containing identical editing information but different sequences were present. The alignments of the predicted gRNAs with the edited mRNA sequences varied from a perfect alignment without gaps to alignments with multiple mismatches. Multiple gRNAs overlapped with upstream gRNAs, but in no case was a complete set of overlapping gRNAs covering an entire editing domain obtained. We estimate that a minimum set of approximately 65 additional gRNAs would be required for complete overlapping sets. This analysis should provide a basis for detailed studies of the evolution and role in RNA editing of kDNA minicircles in this species.     

Duplicated paralogous genes subject to positive selection in the genome of Trypanosoma brucei

Background

Whole genome studies have highlighted duplicated genes as important substrates for adaptive evolution. We have investigated adaptive evolution in this class of genes in the human parasite Trypanosoma brucei, as indicated by the ratio of non-synonymous (amino-acid changing) to synonymous (amino acid retaining) nucleotide substitution rates.

Methodology/Principal Findings

We have identified duplicated genes that are most rapidly evolving in this important human parasite. This is the first attempt to investigate adaptive evolution in this species at the codon level. We identify 109 genes within 23 clusters of paralogous gene expansions to be subject to positive selection.

Conclusions/Significance

Genes identified include surface antigens in both the mammalian and insect host life cycle stage suggesting that competitive interaction is not solely with the adaptive immune system of the mammalian host. Also surface transporters related to drug resistance and genes related to developmental progression are detected. We discuss how adaptive evolution of these genes may highlight lineage specific processes essential for parasite survival. We also discuss the implications of adaptive evolution of these targets for parasite biology and control.     

Measure of molecular diversity within the Trypanosoma brucei subspecies Trypanosoma brucei brucei and Trypanosoma brucei gambiense as revealed by genotypic characterization

We have evaluated whether sequence polymorphisms in the rRNA intergenic spacer region can be used to study the relatedness of two subspecies of Trypanosoma brucei. Thirteen T. brucei isolates made up of 6 T. b. brucei and 7 T. b. gambiense were analyzed using restriction fragment length polymorphism (RFLP). By PCR-based restriction mapping of the ITS1-5.8S-ITS2 ribosomal repeat unit, we found a fingerprint pattern that separately identifies each of the two subspecies analyzed, with unique restriction fragments observed in all but 1 of the T. b. gambiense "human" isolates. Interestingly, the restriction profile for a virulent group 2 T. b. gambiense human isolate revealed an unusual RFLP pattern different from the profile of other human isolates. Sequencing data from four representatives of each of the two subspecies indicated that the intergenic spacer region had a conserved ITS-1 and a variable 5.8S with unique transversions, insertions, or deletions. The ITS-2 regions contained a single repeated element at similar positions in all isolates examined, but not in 2 of the human isolates. A unique 4-bp [C(3)A] sequence was found within the 5.8S region of human T. b. gambiense isolates. Phylogenetic analysis of the data suggests that their common ancestor was a nonhuman animal pathogen and that human pathogenicity might have evolved secondarily. Our data show that cryptic species within the T. brucei group can be distinguished by differences in the PCR-RFLP profile of the rDNA repeat.     

The ingi and RIME non-LTR retrotransposons are not randomly distributed in the genome of Trypanosoma brucei

The ingi (long and autonomous) and RIME (short and nonautonomous) non--long-terminal repeat retrotransposons are the most abundant mobile elements characterized to date in the genome of the African trypanosome Trypanosoma brucei. These retrotransposons were thought to be randomly distributed, but a detailed and comprehensive analysis of their genomic distribution had not been performed until now. To address this question, we analyzed the ingi/RIME sequences and flanking sequences from the ongoing T. brucei genome sequencing project (TREU927/4 strain). Among the 81 ingi/RIME elements analyzed, 60% are complete, and 7% of the ingi elements (approximately 15 copies per haploid genome) appear to encode for their own transposition. The size of the direct repeat flanking the ingi/RIME retrotransposons is conserved (i.e., 12-bp), and a strong 11-bp consensus pattern precedes the 5'-direct repeat. The presence of a consensus pattern upstream of the retroelements was confirmed by the analysis of the base occurrence in 294 GSS containing 5'-adjacent ingi/RIME sequences. The conserved sequence is present upstream of ingis and RIMEs, suggesting that ingi-encoded enzymatic activities are used for retrotransposition of RIMEs, which are short nonautonomous retroelements. In conclusion, the ingi and RIME retroelements are not randomly distributed in the genome of T. brucei and are preceded by a conserved sequence, which may be the recognition site of the ingi-encoded endonuclease.     

Survey of extrachromosomal circular DNA derived from plant satellite repeats

Background  

Satellite repeats represent one of the most dynamic components of higher plant genomes, undergoing rapid evolutionary changes of their nucleotide sequences and abundance in a genome. However, the exact molecular mechanisms driving these changes and their eventual regulation are mostly unknown. It has been proposed that amplification and homogenization of satellite DNA could be facilitated by extrachromosomal circular DNA (eccDNA) molecules originated by recombination-based excision from satellite repeat arrays. While the models including eccDNA are attractive for their potential to explain rapid turnover of satellite DNA, the existence of satellite repeat-derived eccDNA has not yet been systematically studied in a wider range of plant genomes.