TY - Generic T1 - Transcript expression analysis of putative Trypanosoma brucei GPI-anchored surface proteins during development in the tsetse and mammalian hosts. Y1 - 2012 A1 - Savage, Amy F A1 - Cerqueira, Gustavo C A1 - Regmi, Sandesh A1 - Wu, Yineng A1 - El Sayed, Najib M A1 - Aksoy, Serap KW - Animals KW - Computational Biology KW - Gastrointestinal Tract KW - Gene Expression Profiling KW - GPI-Linked Proteins KW - HUMANS KW - Male KW - Membrane Proteins KW - Protozoan Proteins KW - Real-Time Polymerase Chain Reaction KW - Salivary Glands KW - Trypanosoma brucei brucei KW - Trypanosomiasis, African KW - Tsetse Flies AB -

Human African Trypanosomiasis is a devastating disease caused by the parasite Trypanosoma brucei. Trypanosomes live extracellularly in both the tsetse fly and the mammal. Trypanosome surface proteins can directly interact with the host environment, allowing parasites to effectively establish and maintain infections. Glycosylphosphatidylinositol (GPI) anchoring is a common posttranslational modification associated with eukaryotic surface proteins. In T. brucei, three GPI-anchored major surface proteins have been identified: variant surface glycoproteins (VSGs), procyclic acidic repetitive protein (PARP or procyclins), and brucei alanine rich proteins (BARP). The objective of this study was to select genes encoding predicted GPI-anchored proteins with unknown function(s) from the T. brucei genome and characterize the expression profile of a subset during cyclical development in the tsetse and mammalian hosts. An initial in silico screen of putative T. brucei proteins by Big PI algorithm identified 163 predicted GPI-anchored proteins, 106 of which had no known functions. Application of a second GPI-anchor prediction algorithm (FragAnchor), signal peptide and trans-membrane domain prediction software resulted in the identification of 25 putative hypothetical proteins. Eighty-one gene products with hypothetical functions were analyzed for stage-regulated expression using semi-quantitative RT-PCR. The expression of most of these genes were found to be upregulated in trypanosomes infecting tsetse salivary gland and proventriculus tissues, and 38% were specifically expressed only by parasites infecting salivary gland tissues. Transcripts for all of the genes specifically expressed in salivary glands were also detected in mammalian infective metacyclic trypomastigotes, suggesting a possible role for these putative proteins in invasion and/or establishment processes in the mammalian host. These results represent the first large-scale report of the differential expression of unknown genes encoding predicted T. brucei surface proteins during the complete developmental cycle. This knowledge may form the foundation for the development of future novel transmission blocking strategies against metacyclic parasites.

JA - PLoS Negl Trop Dis VL - 6 CP - 6 M3 - 10.1371/journal.pntd.0001708 ER - TY - JOUR T1 - Members of a large retroposon family are determinants of post-transcriptional gene expression in Leishmania. JF - PLoS Pathog Y1 - 2007 A1 - Bringaud, Frederic A1 - Müller, Michaela A1 - Cerqueira, Gustavo Coutinho A1 - Smith, Martin A1 - Rochette, Annie A1 - el-Sayed, Najib M A A1 - Papadopoulou, Barbara A1 - Ghedin, Elodie KW - 3' Untranslated Regions KW - Animals KW - Base Sequence KW - Biological Evolution KW - Down-Regulation KW - Gene Expression Regulation KW - Genome, Protozoan KW - Leishmania KW - Leishmania major KW - Molecular Sequence Data KW - Retroelements KW - RNA, Messenger KW - sequence alignment KW - Trypanosoma brucei brucei KW - Trypanosoma cruzi AB -

Trypanosomatids are unicellular protists that include the human pathogens Leishmania spp. (leishmaniasis), Trypanosoma brucei (sleeping sickness), and Trypanosoma cruzi (Chagas disease). Analysis of their recently completed genomes confirmed the presence of non-long-terminal repeat retrotransposons, also called retroposons. Using the 79-bp signature sequence common to all trypanosomatid retroposons as bait, we identified in the Leishmania major genome two new large families of small elements--LmSIDER1 (785 copies) and LmSIDER2 (1,073 copies)--that fulfill all the characteristics of extinct trypanosomatid retroposons. LmSIDERs are approximately 70 times more abundant in L. major compared to T. brucei and are found almost exclusively within the 3'-untranslated regions (3'UTRs) of L. major mRNAs. We provide experimental evidence that LmSIDER2 act as mRNA instability elements and that LmSIDER2-containing mRNAs are generally expressed at lower levels compared to the non-LmSIDER2 mRNAs. The considerable expansion of LmSIDERs within 3'UTRs in an organism lacking transcriptional control and their role in regulating mRNA stability indicate that Leishmania have probably recycled these short retroposons to globally modulate the expression of a number of genes. To our knowledge, this is the first example in eukaryotes of the domestication and expansion of a family of mobile elements that have evolved to fulfill a critical cellular function.

VL - 3 CP - 9 M3 - 10.1371/journal.ppat.0030136 ER - TY - JOUR T1 - Trypanosoma cruzi mitochondrial maxicircles display species- and strain-specific variation and a conserved element in the non-coding region. JF - BMC Genomics Y1 - 2006 A1 - Westenberger, Scott J A1 - Cerqueira, Gustavo C A1 - El-Sayed, Najib M A1 - Zingales, Bianca A1 - Campbell, David A A1 - Sturm, Nancy R KW - Amino Acid Sequence KW - Animals KW - Animals, Inbred Strains KW - Base Composition KW - Conserved Sequence KW - DNA, Kinetoplast KW - Frameshifting, Ribosomal KW - Gene Deletion KW - Gene Order KW - Genetic Variation KW - Leishmania KW - Models, Biological KW - Molecular Sequence Data KW - Muscle Proteins KW - NADH Dehydrogenase KW - Open Reading Frames KW - Regulatory Elements, Transcriptional KW - RNA Editing KW - Sequence Homology, Amino Acid KW - Species Specificity KW - Trypanosoma brucei brucei KW - Trypanosoma cruzi KW - Ubiquitin-Protein Ligases KW - Untranslated Regions AB -

BACKGROUND: The mitochondrial DNA of kinetoplastid flagellates is distinctive in the eukaryotic world due to its massive size, complex form and large sequence content. Comprised of catenated maxicircles that contain rRNA and protein-coding genes and thousands of heterogeneous minicircles encoding small guide RNAs, the kinetoplast network has evolved along with an extreme form of mRNA processing in the form of uridine insertion and deletion RNA editing. Many maxicircle-encoded mRNAs cannot be translated without this post-transcriptional sequence modification.

RESULTS: We present the complete sequence and annotation of the Trypanosoma cruzi maxicircles for the CL Brener and Esmeraldo strains. Gene order is syntenic with Trypanosoma brucei and Leishmania tarentolae maxicircles. The non-coding components have strain-specific repetitive regions and a variable region that is unique for each strain with the exception of a conserved sequence element that may serve as an origin of replication, but shows no sequence identity with L. tarentolae or T. brucei. Alternative assemblies of the variable region demonstrate intra-strain heterogeneity of the maxicircle population. The extent of mRNA editing required for particular genes approximates that seen in T. brucei. Extensively edited genes were more divergent among the genera than non-edited and rRNA genes. Esmeraldo contains a unique 236-bp deletion that removes the 5'-ends of ND4 and CR4 and the intergenic region. Esmeraldo shows additional insertions and deletions outside of areas edited in other species in ND5, MURF1, and MURF2, while CL Brener has a distinct insertion in MURF2.

CONCLUSION: The CL Brener and Esmeraldo maxicircles represent two of three previously defined maxicircle clades and promise utility as taxonomic markers. Restoration of the disrupted reading frames might be accomplished by strain-specific RNA editing. Elements in the non-coding region may be important for replication, transcription, and anchoring of the maxicircle within the kinetoplast network.

VL - 7 M3 - 10.1186/1471-2164-7-60 ER - TY - JOUR T1 - Gene synteny and evolution of genome architecture in trypanosomatids. JF - Mol Biochem Parasitol Y1 - 2004 A1 - Ghedin, Elodie A1 - Bringaud, Frederic A1 - Peterson, Jeremy A1 - Myler, Peter A1 - Berriman, Matthew A1 - Ivens, Alasdair A1 - Andersson, Björn A1 - Bontempi, Esteban A1 - Eisen, Jonathan A1 - Angiuoli, Sam A1 - Wanless, David A1 - Von Arx, Anna A1 - Murphy, Lee A1 - Lennard, Nicola A1 - Salzberg, Steven A1 - Adams, Mark D A1 - White, Owen A1 - Hall, Neil A1 - Stuart, Kenneth A1 - Fraser, Claire M A1 - el-Sayed, Najib M A KW - Animals KW - Computational Biology KW - Evolution, Molecular KW - Gene Order KW - Genome, Protozoan KW - Genomics KW - Leishmania major KW - Multigene Family KW - Recombination, Genetic KW - Retroelements KW - Selection, Genetic KW - Synteny KW - Trypanosoma brucei brucei KW - Trypanosoma cruzi KW - Trypanosomatina AB -

The trypanosomatid protozoa Trypanosoma brucei, Trypanosoma cruzi and Leishmania major are related human pathogens that cause markedly distinct diseases. Using information from genome sequencing projects currently underway, we have compared the sequences of large chromosomal fragments from each species. Despite high levels of divergence at the sequence level, these three species exhibit a striking conservation of gene order, suggesting that selection has maintained gene order among the trypanosomatids over hundreds of millions of years of evolution. The few sites of genome rearrangement between these species are marked by the presence of retrotransposon-like elements, suggesting that retrotransposons may have played an important role in shaping trypanosomatid genome organization. A degenerate retroelement was identified in L. major by examining the regions near breakage points of the synteny. This is the first such element found in L. major suggesting that retroelements were found in the common ancestor of all three species.

VL - 134 CP - 2 M3 - 10.1016/j.molbiopara.2003.11.012 ER - TY - JOUR T1 - The ingi and RIME non-LTR retrotransposons are not randomly distributed in the genome of Trypanosoma brucei. JF - Mol Biol Evol Y1 - 2004 A1 - Bringaud, Frederic A1 - Biteau, Nicolas A1 - Zuiderwijk, Eduard A1 - Berriman, Matthew A1 - El-Sayed, Najib M A1 - Ghedin, Elodie A1 - Melville, Sara E A1 - Hall, Neil A1 - Baltz, Théo KW - Amino Acid Sequence KW - Animals KW - Base Sequence KW - Consensus Sequence KW - Genome, Protozoan KW - Molecular Sequence Data KW - Retroelements KW - Sequence Analysis KW - Trypanosoma brucei brucei AB -

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.

VL - 21 CP - 3 M3 - 10.1093/molbev/msh045 ER - TY - JOUR T1 - The sequence and analysis of Trypanosoma brucei chromosome II. JF - Nucleic Acids Res Y1 - 2003 A1 - el-Sayed, Najib M A A1 - Ghedin, Elodie A1 - Song, Jinming A1 - MacLeod, Annette A1 - Bringaud, Frederic A1 - Larkin, Christopher A1 - Wanless, David A1 - Peterson, Jeremy A1 - Hou, Lihua A1 - Taylor, Sonya A1 - Tweedie, Alison A1 - Biteau, Nicolas A1 - Khalak, Hanif G A1 - Lin, Xiaoying A1 - Mason, Tanya A1 - Hannick, Linda A1 - Caler, Elisabet A1 - Blandin, Gaëlle A1 - Bartholomeu, Daniella A1 - Simpson, Anjana J A1 - Kaul, Samir A1 - Zhao, Hong A1 - Pai, Grace A1 - Van Aken, Susan A1 - Utterback, Teresa A1 - Haas, Brian A1 - Koo, Hean L A1 - Umayam, Lowell A1 - Suh, Bernard A1 - Gerrard, Caroline A1 - Leech, Vanessa A1 - Qi, Rong A1 - Zhou, Shiguo A1 - Schwartz, David A1 - Feldblyum, Tamara A1 - Salzberg, Steven A1 - Tait, Andrew A1 - Turner, C Michael R A1 - Ullu, Elisabetta A1 - White, Owen A1 - Melville, Sara A1 - Adams, Mark D A1 - Fraser, Claire M A1 - Donelson, John E KW - Animals KW - Antigens, Protozoan KW - Chromosome mapping KW - Chromosomes KW - DNA, Protozoan KW - Gene Duplication KW - Genes, Protozoan KW - Molecular Sequence Data KW - Pseudogenes KW - Recombination, Genetic KW - Sequence Analysis, DNA KW - Trypanosoma brucei brucei AB -

We report here the sequence of chromosome II from Trypanosoma brucei, the causative agent of African sleeping sickness. The 1.2-Mb pairs encode about 470 predicted genes organised in 17 directional clusters on either strand, the largest cluster of which has 92 genes lined up over a 284-kb region. An analysis of the GC skew reveals strand compositional asymmetries that coincide with the distribution of protein-coding genes, suggesting these asymmetries may be the result of transcription-coupled repair on coding versus non-coding strand. A 5-cM genetic map of the chromosome reveals recombinational 'hot' and 'cold' regions, the latter of which is predicted to include the putative centromere. One end of the chromosome consists of a 250-kb region almost exclusively composed of RHS (pseudo)genes that belong to a newly characterised multigene family containing a hot spot of insertion for retroelements. Interspersed with the RHS genes are a few copies of truncated RNA polymerase pseudogenes as well as expression site associated (pseudo)genes (ESAGs) 3 and 4, and 76 bp repeats. These features are reminiscent of a vestigial variant surface glycoprotein (VSG) gene expression site. The other end of the chromosome contains a 30-kb array of VSG genes, the majority of which are pseudogenes, suggesting that this region may be a site for modular de novo construction of VSG gene diversity during transposition/gene conversion events.

VL - 31 CP - 16 ER - TY - JOUR T1 - Analysis of stage-specific gene expression in the bloodstream and the procyclic form of Trypanosoma brucei using a genomic DNA-microarray. JF - Mol Biochem Parasitol Y1 - 2002 A1 - Diehl, Susanne A1 - Diehl, Frank A1 - El-Sayed, Najib M A1 - Clayton, Christine A1 - Hoheisel, Jörg D KW - Animals KW - Blotting, Northern KW - Escherichia coli KW - Gene expression KW - Gene Expression Profiling KW - Genes, Protozoan KW - HUMANS KW - Life Cycle Stages KW - Molecular Sequence Data KW - Oligonucleotide Array Sequence Analysis KW - Polymerase Chain Reaction KW - Transcription, Genetic KW - Trypanosoma brucei brucei AB -

A microarray comprising 21,024 different PCR products spotted on glass slides was constructed for gene expression studies on Trypanosoma brucei. The arrayed fragments were generated from a T. brucei shotgun clone library, which had been prepared from randomly sheared and size-fractionated genomic DNA. For the identification of stage-specific gene activity, total RNA from in vitro cultures of the human, long slender form and the insect, procyclic form of the parasite was labelled and hybridised to the microarray. Approximately 75% of the genomic fragments produced a signal and about 2% exhibited significant differences between the transcript levels in the bloodstream and procyclic forms. A few results were confirmed by Northern blot analysis or reverse-transcription and PCR. Three hundred differentially regulated clones have been selected for sequencing. So far, of 33 clones that showed about 2-fold or more over-expression in bloodstream forms, 15 contained sequences similar to those of VSG expression sites and at least six others appeared non-protein-coding. Of 29 procyclic-specific clones, at least eight appeared not to be protein-coding. A surprisingly large proportion of known regulated genes was already identified in this small sample, and some new ones were found, illustrating the utility of genomic arrays.

VL - 123 CP - 2 ER - TY - JOUR T1 - A new, expressed multigene family containing a hot spot for insertion of retroelements is associated with polymorphic subtelomeric regions of Trypanosoma brucei. JF - Eukaryot Cell Y1 - 2002 A1 - Bringaud, Frederic A1 - Biteau, Nicolas A1 - Melville, Sara E A1 - Hez, Stéphanie A1 - El-Sayed, Najib M A1 - Leech, Vanessa A1 - Berriman, Matthew A1 - Hall, Neil A1 - Donelson, John E A1 - Baltz, Théo KW - Amino Acid Sequence KW - Animals KW - Base Sequence KW - Cloning, Molecular KW - DNA Primers KW - DNA, Protozoan KW - Escherichia coli KW - Genes, Protozoan KW - Molecular Sequence Data KW - Multigene Family KW - Mutagenesis, Insertional KW - Phylogeny KW - Polymorphism, Genetic KW - Protozoan Proteins KW - Pseudogenes KW - Retroelements KW - sequence alignment KW - Sequence Homology, Amino Acid KW - Telomere KW - Trypanosoma brucei brucei KW - Trypanosoma cruzi AB -

We describe a novel gene family that forms clusters in subtelomeric regions of Trypanosoma brucei chromosomes and partially accounts for the observed clustering of retrotransposons. The ingi and ribosomal inserted mobile element (RIME) non-LTR retrotransposons share 250 bp at both extremities and are the most abundant putatively mobile elements, with about 500 copies per haploid genome. From cDNA clones and subsequently in the T. brucei genomic DNA databases, we identified 52 homologous gene and pseudogene sequences, 16 of which contain a RIME and/or ingi retrotransposon inserted at exactly the same relative position. Here these genes are called the RHS family, for retrotransposon hot spot. Comparison of the protein sequences encoded by RHS genes (21 copies) and pseudogenes (24 copies) revealed a conserved central region containing an ATP/GTP-binding motif and the RIME/ingi insertion site. The RHS proteins share between 13 and 96% identity, and six subfamilies, RHS1 to RHS6, can be defined on the basis of their divergent C-terminal domains. Immunofluorescence and Western blot analyses using RHS subfamily-specific immune sera show that RHS proteins are constitutively expressed and occur mainly in the nucleus. Analysis of Genome Survey Sequence databases indicated that the Trypanosoma brucei diploid genome contains about 280 RHS (pseudo)genes. Among the 52 identified RHS (pseudo)genes, 48 copies are in three RHS clusters located in subtelomeric regions of chromosomes Ia and II and adjacent to the active bloodstream form expression site in T. brucei strain TREU927/4 GUTat10.1. RHS genes comprise the remaining sequence of the size-polymorphic "repetitive region" described for T. brucei chromosome I, and a homologous gene family is present in the Trypanosoma cruzi genome.

VL - 1 CP - 1 ER -