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.     

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.     

Analysis of the DNA and RNA changes associated with the expression of isotypic variant-specific antigens of trypanosomes.

Using specific (32P) labelled cDNA probes, we compared the mRNAs and the genomic DNA sequences coding for the synthesis of two pairs of serologically related variant-specific antigens (VSAs) of trypanosomes: AnTat 1.1 and AnTat 1.1b, both from the strain 1125 of T.b.brucei and AnTat 1.8 and LiTat 1.6 from T.b.brucei and T.b. gambiense, respectively. Within each pair, large similarities were observed in the coding sequence, except in the 3' region which appears to be highly variable. However, a low level of cross-hybridization can be detected between all sequences, in the 3' region only. The expression of these VSAs is linked to a similar duplication-transposition mechanism. The insertion locus of the transposition unit is the same both in AnTat 1.1 and AnTat 1.1b DNAs. In both pairs, the transposition unit seems to comprise at least about 200 bp upstream of the 5' extremity of the coding sequence. The significance of these results, regarding the structure and synthesis of the VSAs, is discussed.     

The four ribosomal DNA units of the malaria parasite Plasmodium berghei. Identification, restriction map, and copy number analysis

The four ribosomal RNA genes (rDNA units) of the rodent malaria parasite, Plasmodium berghei, were identified and mapped by restriction enzyme analysis and Southern blot hybridization of genomic DNA. Although the four genes share common characteristics, they appear to be internally different from each other in expanse and sequence. One HindIII site near the 3' end of the coding region for the large rRNA is the only restriction site which we have detected that is conserved in all of the genes. The distance between the conserved HindIII site and the coding region for the small rRNA is at least 1-2 kilobases longer in two of the rDNA units than in a third unit. None of the four rDNA units were linked by restriction mapping where about 150 kilobases of the genome were accounted for. The copy number of two of the rDNA units was determined to be approximately 1 per haploid genome by quantitative analysis of cloned (plasmid) DNA versus genomic DNA digests on Southern blots. The other two genes also appear to be present in 1 copy. Restriction analysis confirms both the low copy number and that these genes are not in an easily recognizable tandem array. The low number of rDNA units requires that new copies of the genome produced during intraerythrocytic growth be active in RNA synthesis soon after their replication.     

Genomic organization and context of a trypanosome variant surface glycoprotein gene family.

We have defined the genomic organization and genomic context of a Trypanosoma brucei brucei gene family encoding variant surface glycoproteins (VSGs). This gene family is neither tandemly repeated nor closely linked in the genome, and is not located on small or intermediate size chromosomes. Two dispersed repeated sequence elements, RIME-ingi and the upstream repeat sequence, are linked to members of this gene family; however, the upstream repeat sequences are closely linked only to the basic copy. In other isolates of T.b. brucei this gene family appears conserved with some variation; a restriction fragment length polymorphism found among these isolates suggests the hypothesis that VSG genes may occasionally be diploid. A model accounting for both the generation of dispersed families of VSG genes, and for the interstrain variability of VSG genes, is proposed.     

The African trypanosome genome

The haploid nuclear genome of the African trypanosome, Trypanosoma brucei, is about 35 Mb and varies in size among different trypanosome isolates by as much as 25%. The nuclear DNA of this diploid organism is distributed among three size classes of chromosomes: the megabase chromosomes of which there are at least 11 pairs ranging from 1 Mb to more than 6 Mb (numbered I-XI from smallest to largest); several intermediate chromosomes of 200-900 kb and uncertain ploidy; and about 100 linear minichromosomes of 50-150 kb. Size differences of as much as four-fold can occur, both between the two homologues of a megabase chromosome pair in a specific trypanosome isolate and among chromosome pairs in different isolates. The genomic DNA sequences determined to date indicated that about 50% of the genome is coding sequence. The chromosomal telomeres possess TTAGGG repeats and many, if not all, of the telomeres of the megabase and intermediate chromosomes are linked to expression sites for genes encoding variant surface glycoproteins (VSGs). The minichromosomes serve as repositories for VSG genes since some but not all of their telomeres are linked to unexpressed VSG genes. A gene discovery program, based on sequencing the ends of cloned genomic DNA fragments, has generated more than 20 Mb of discontinuous single-pass genomic sequence data during the past year, and the complete sequences of chromosomes I and II (about 1 Mb each) in T. brucei GUTat 10.1 are currently being determined. It is anticipated that the entire genomic sequence of this organism will be known in a few years. Analysis of a test microarray of 400 cDNAs and small random genomic DNA fragments probed with RNAs from two developmental stages of T. brucei demonstrates that the microarray technology can be used to identify batteries of genes differentially expressed during the various life cycle stages of this parasite.     

Ribosomal DNA genes of Bombyx mori: a minor fraction of the repeating units contain insertions

We have analyzed multiple recombinant DNA clones containing ribosomal RNA repeat units of the silkmoth, Bombyx mori. In combination with genomic DNA blots, analysis of these clones indicated that the rDNA repeat of B. mori is 10.8 kilobase pair in length and tandemly repeated in the genome, as reported by Manning et al. (18). However, contrary to that report, approximately 12% of the rDNA cistrons are interrupted by insertions of non-ribosomal DNA. Two classes of DNA insertions were identified. In one class the insertions are positioned in a region of the 28S coding sequence similar to that of the predominant rDNA insertions found in a variety of Dipteran and Tetrahymena species. In the second class, probable insertions are found close to the 3' terminus of the 28S coding sequence. Restriction enzyme analysis indicates that the two classes of insertions are not related.     

Maxicircle DNA and edited mRNA sequences of closely related trypanosome species: implications of kRNA editing for evolution of maxicircle genomes.

kRNA editing produces functional mRNAs by uridine insertion and deletion. We analyzed portions of the apocytochrome b and NADH dehydrogenase subunits 7 and 8 (ND7 and 8) genes and their edited mRNAs in Trypanosoma congolense and compared these to the corresponding sequences in T.brucei. We find that these genes are highly diverged between the two species, especially in the positions of thymidines and in nucleotide transitions. Editing eliminates differences in encoded uridines producing edited mRNAs that are identical except for the nucleotide substitutions. The resulting predicted proteins are identical since all nucleotide substitutions are silent. A T.congolense minicircle-encoded gRNA which can specify editing of ND8 mRNA was identified. This gRNA can basepair with both T.congolense and T.brucei ND8 mRNA despite nucleotide transitions due to the flexibility of G:U base-pairing. These results illustrate how editing affects the characteristics of maxicircle sequence divergence and allows protein sequence conservation despite a level of DNA sequence divergence which would be predicted to be intolerable in the absence of editing.     

Rhodanobacter fulvus sp. nov., a beta-galactosidase-producing gammaproteobacterium

A taxonomic study was carried out on a bacterial strain designated as Jip2T isolated from a soil sample mixed with rotten rice straw. It was a Gram-negative, aerobic, motile, and rod-shaped bacterium. It grew well on nutrient agar medium and utilized a fairly narrow spectrum of carbon source. The G+C content of the genomic DNA was 65.3 mol%. The major ubiquinone was Q-8. The major fatty acids were branched fatty acids, especially large amounts of iso C15:0 and iso C17:1 w9c were detected in the cells grown on TSA agar for 24 h. Comparative 16S rDNA study showed a clear affiliation of this bacterium to the genus Rhodanobacter. The 16S rDNA sequence of strain Jip2T showed 96.4% sequence similarity to that of Rhodanobacter lindaniclasticus RP5575T. On the basis of phenotypic characteristics and 16S rDNA sequence analysis, strain Jip2T is clearly distinct from Rhodanobacter lindaniclasticus. We propose the name Rhodanobacter fulvus sp. nov. for strain Jip2T (=IAM 15025T=KCTC 12098T).     

Comparison of the maxicircle (mitochondrial) genomes of Leishmania tarentolae and Trypanosoma brucei at the level of nucleotide sequence

The entire 16.7-kilobase (kb) transcribed region of the Leishmania tarentolae maxicircle was compared to the entire 15-kb transcribed region of the Trypanosoma brucei maxicircle at the nucleotide sequence level by dot matrix analysis and by alignments of individual genes. The L. tarentolae NADH dehydrogenase subunit 1 (ND1) gene was identified in a newly obtained 2.9-kb sequence. All but two regions which flank the cytochrome b gene are highly conserved in both species. One 3.1-kb region in L. tarentolae that contains the cytochrome oxidase subunit III (COIII) gene and several open reading frames corresponds to a 2-kb sequence in T. brucei with limited sequence homology that lacks the COIII gene. Another 0.6-kb region that comprises an unidentified open reading frame (open reading frame 12) in L. tarentolae is substituted by a nonhomologous 0.4-kb open reading frame in T. brucei. A short intergenic region between the ND1 gene and the maxicircle unidentified reading frame 1 gene shows limited sequence homology, and the regions between the ND4 and ND5 genes and between the COI and ND4 genes are not conserved. All of the intergenic regions share G + C richness and a similar pattern of G versus C strand bias. 1.8 kb of the L. tarentolae divergent region (DV) and around 3 kb of the T. brucei DV were also obtained. The T. brucei DV sequences were not homologous to the L. tarentolae DV sequence but were organized in a similar fashion with tandem repeats of varying complexity.     

Coordinate transcription of variant surface glycoprotein genes and an expression site associated gene family in Trypanosoma brucei

Trypanosoma brucei variant surface glycoprotein (VSG) genes are activated either by duplicative (DA) transposition of the gene to a pre-activated expression site or by nonduplicative (NDA) activation of a previously silent telomeric gene. We have obtained a recombinant clone spanning the 5' barren region of the expression linked copy of the duplicated VSG gene 117a. By DNA sequence and hybridization analyses we have identified a pleomorphic family of 14-25 non-VSG genes that lie upstream of both DA and NDA VSG expression sites. These expression site associated genes (ESAGs) encode 1.2 kb poly(A)+ mRNAs that are specifically transcribed from the active VSG expression telomere in mammalian bloodstream stages of T. brucei but, in common with VSG genes, are not transcribed in procyclic culture forms. cDNA and genomic sequences predict open reading frames that are conserved in the two ESAGs examined.     

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.     

A gene encoding a novel extremely thermostable 1,4-beta-xylanase isolated directly from an environmental DNA sample

Small-subunit (SSU) rRNA genes (rDNA) were amplified by PCR from a hot pool environmental DNA sample using Bacteria- or Archaea-specific rDNA primers. Unique rDNA types were identified by restriction fragment length polymorphism (RFLP) analysis and representative sequences were determined. Family 10 glycoside hydrolase consensus PCR primers were used to explore the occurrence and diversity of xylanase genes in the hot pool environmental DNA sample. Partial sequences for three different xylanases were obtained and genomic walking PCR (GWPCR), in combination with nested primer pairs, was used to obtained a unique 1,741-bp nucleotide sequence. Analysis of this sequence identified a putative XynA protein encoded by the xynA open reading frame. The single module novel xylanase shared sequence similarity to the family 10 glycoside hydrolases. The purified recombinant enzyme, XynA expressed in E. coli exhibited optimum activity at 100 degrees C and pH 6.0, and was extremely thermostable at 90 degrees C. The enzyme showed high specificity toward different xylans and xylooligosaccharides.     

Glossina dynamics in and around the sleeping sickness endemic Serengeti ecosystem of northwestern Tanzania

We investigated the dynamics of Glossina spp. and their role in the transmission of trypanosomiasis in the sleeping sickness endemic Serengeti ecosystem, northwestern Tanzania. The study investigated Glossina species composition, trap density, trypanosome infection rates, and the diversity of trypanosomes infecting the species. Tsetse were trapped using monopyramidal traps in the mornings between 06:00 to 11:00 and transported to the veterinary laboratory in Serengeti National Park where they were sorted into species and sex, and dissected microscopically to determine trypanosome infection rates. Age estimation of dissected flies was also conducted concurrently. Tsetse samples positive for trypanosomes were subjected to PCR to determine the identity of the detected trypanosomes. Out of 2,519 tsetse trapped, 1,522 (60.42%) were G. swynnertoni, 993 (39.42%) were G. pallidipes, three (0.12%) were G. m. morsitans, and one (0.04%) was G. brevipalpis. The trap density for G. swynnertoni was between 1.40 and 14.17 while that of G. pallidipes was between 0.23 and 9.70. Out of 677 dissected G. swynnertoni, 63 flies (9.3%) were infected, of which 62 (98.4%) were females. A total of 199 G. pallidipes was also dissected but none was infected. There was no significant difference between the apparent densities of G. swynnertoni compared to that of G. pallidipes (t = 1.42, p = 0.18). Molecular characterization of the 63 infected G. swynnertoni midguts showed that 19 (30.2%) were trypanosomes associated with suid animals while nine (14.3%) were trypanosomes associated with bovid animals and five samples (7.9%) had T. brucei s.l genomic DNA. Thirty (47.6%) tsetse samples could not be identified. Subsequent PCR to differentiate between T. b. brucei and T. b. rhodesiense showed that all five samples that contained the T. brucei s.l genomic DNA were positive for the SRA molecular marker indicating that they were T. b. rhodesiense. These results indicate that G. swynnertoni plays a major role in the transmission of trypaniosomiasis in the area and that deliberate and sustainable control measures should be initiated and scaled up.