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1.
PLoS Genet ; 12(5): e1005994, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-27149665

RESUMO

African trypanosomes are mammalian pathogens that must regularly change their protein coat to survive in the host bloodstream. Chronic trypanosome infections are potentiated by their ability to access a deep genomic repertoire of Variant Surface Glycoprotein (VSG) genes and switch from the expression of one VSG to another. Switching VSG expression is largely based in DNA recombination events that result in chromosome translocations between an acceptor site, which houses the actively transcribed VSG, and a donor gene, drawn from an archive of more than 2,000 silent VSGs. One element implicated in these duplicative gene conversion events is a DNA repeat of approximately 70 bp that is found in long regions within each BES and short iterations proximal to VSGs within the silent archive. Early observations showing that 70-bp repeats can be recombination boundaries during VSG switching led to the prediction that VSG-proximal 70-bp repeats provide recombinatorial homology. Yet, this long held assumption had not been tested and no specific function for the conserved 70-bp repeats had been demonstrated. In the present study, the 70-bp repeats were genetically manipulated under conditions that induce gene conversion. In this manner, we demonstrated that 70-bp repeats promote access to archival VSGs. Synthetic repeat DNA sequences were then employed to identify the length, sequence, and directionality of repeat regions required for this activity. In addition, manipulation of the 70-bp repeats allowed us to observe a link between VSG switching and the cell cycle that had not been appreciated. Together these data provide definitive support for the long-standing hypothesis that 70-bp repeats provide recombinatorial homology during switching. Yet, the fact that silent archival VSGs are selected under these conditions suggests the 70-bp repeats also direct DNA pairing and recombination machinery away from the closest homologs (silent BESs) and toward the rest of the archive.


Assuntos
Sequências Repetitivas de Ácido Nucleico/genética , Trypanosoma brucei brucei/genética , Tripanossomíase Africana/genética , Glicoproteínas Variantes de Superfície de Trypanosoma/genética , Animais , Variação Antigênica/genética , Variação Antigênica/imunologia , Antígenos de Superfície/genética , Antígenos de Superfície/imunologia , Duplicação Gênica , Genômica , Sequências Repetitivas de Ácido Nucleico/imunologia , Trypanosoma brucei brucei/imunologia , Trypanosoma brucei brucei/patogenicidade , Tripanossomíase Africana/imunologia , Tripanossomíase Africana/parasitologia , Glicoproteínas Variantes de Superfície de Trypanosoma/imunologia
2.
Genes Dev ; 23(9): 1063-76, 2009 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-19369410

RESUMO

Unusually for a eukaryote, genes transcribed by RNA polymerase II (pol II) in Trypanosoma brucei are arranged in polycistronic transcription units. With one exception, no pol II promoter motifs have been identified, and how transcription is initiated remains an enigma. T. brucei has four histone variants: H2AZ, H2BV, H3V, and H4V. Using chromatin immunoprecipitation (ChIP) and sequencing (ChIP-seq) to examine the genome-wide distribution of chromatin components, we show that histones H4K10ac, H2AZ, H2BV, and the bromodomain factor BDF3 are enriched up to 300-fold at probable pol II transcription start sites (TSSs). We also show that nucleosomes containing H2AZ and H2BV are less stable than canonical nucleosomes. Our analysis also identifies >60 unexpected TSS candidates and reveals the presence of long guanine runs at probable TSSs. Apparently unique to trypanosomes, additional histone variants H3V and H4V are enriched at probable pol II transcription termination sites. Our findings suggest that histone modifications and histone variants play crucial roles in transcription initiation and termination in trypanosomes and that destabilization of nucleosomes by histone variants is an evolutionarily ancient and general mechanism of transcription initiation, demonstrated in an organism in which general pol II transcription factors have been elusive.


Assuntos
Genoma de Protozoário/genética , Histonas/genética , Histonas/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Transcrição Gênica/genética , Trypanosoma brucei brucei/genética , Animais , Cromatina/química , Imunoprecipitação da Cromatina , DNA Polimerase II/genética , Fases de Leitura Aberta/genética , Regiões Promotoras Genéticas/genética
3.
Nature ; 459(7244): 278-81, 2009 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-19369939

RESUMO

Trypanosoma brucei is the causative agent of African sleeping sickness in humans and one of the causes of nagana in cattle. This protozoan parasite evades the host immune system by antigenic variation, a periodic switching of its variant surface glycoprotein (VSG) coat. VSG switching is spontaneous and occurs at a rate of about 10(-2)-10(-3) per population doubling in recent isolates from nature, but at a markedly reduced rate (10(-5)-10(-6)) in laboratory-adapted strains. VSG switching is thought to occur predominantly through gene conversion, a form of homologous recombination initiated by a DNA lesion that is used by other pathogens (for example, Candida albicans, Borrelia sp. and Neisseria gonorrhoeae) to generate surface protein diversity, and by B lymphocytes of the vertebrate immune system to generate antibody diversity. Very little is known about the molecular mechanism of VSG switching in T. brucei. Here we demonstrate that the introduction of a DNA double-stranded break (DSB) adjacent to the approximately 70-base-pair (bp) repeats upstream of the transcribed VSG gene increases switching in vitro approximately 250-fold, producing switched clones with a frequency and features similar to those generated early in an infection. We were also able to detect spontaneous DSBs within the 70-bp repeats upstream of the actively transcribed VSG gene, indicating that a DSB is a natural intermediate of VSG gene conversion and that VSG switching is the result of the resolution of this DSB by break-induced replication.


Assuntos
Variação Antigênica/genética , Quebras de DNA de Cadeia Dupla , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Modelos Genéticos , Proteínas de Saccharomyces cerevisiae/metabolismo , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/imunologia , Animais , Reparo do DNA/genética , Replicação do DNA , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Conversão Gênica/genética , Reação em Cadeia da Polimerase , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Análise de Sequência , Glicoproteínas Variantes de Superfície de Trypanosoma/genética , Glicoproteínas Variantes de Superfície de Trypanosoma/imunologia
4.
Mol Microbiol ; 87(1): 196-210, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23216794

RESUMO

Binding of the Origin Recognition Complex (ORC) to replication origins is essential for initiation of DNA replication, but ORC has non-essential functions outside of DNA replication, including in heterochromatic gene silencing and telomere maintenance. Trypanosoma brucei, a protozoan parasite that causes human African trypanosomiasis, uses antigenic variation as a major virulence mechanism to evade the host's immune attack by expressing its major surface antigen, the Variant Surface Glycoprotein (VSG), in a monoallelic manner. An Orc1/Cdc6 homologue has been identified in T. brucei, but its role in DNA replication has not been directly confirmed and its potential involvement in VSG repression or switching has not been thoroughly investigated. In this study, we show that TbOrc1 is essential for nuclear DNA replication in mammalian-infectious bloodstream and tsetse procyclic forms (BF and PF). Depletion of TbOrc1 resulted in derepression of telomere-linked silent VSGs in both BF and PF, and increased VSG switching particularly through the in situ transcriptional switching mechanism. TbOrc1 associates with telomere repeats but appears to do so independently of two known T. brucei telomere proteins, TbRAP1 and TbTRF. We conclude that TbOrc1 has conserved functions in DNA replication and is also required to control telomere-linked VSG expression and VSG switching.


Assuntos
Inativação Gênica , Complexo de Reconhecimento de Origem/genética , Trypanosoma brucei brucei/genética , Glicoproteínas Variantes de Superfície de Trypanosoma/genética , Variação Antigênica , Replicação do DNA , DNA de Protozoário/biossíntese , DNA de Protozoário/genética , Genes de Protozoários , Glicoproteínas de Membrana/genética , Complexo de Reconhecimento de Origem/metabolismo , Regiões Promotoras Genéticas , Trypanosoma brucei brucei/metabolismo
5.
RNA ; 18(11): 1968-83, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22966087

RESUMO

RNA-binding proteins that target mRNA coding regions are emerging as regulators of post-transcriptional processes in eukaryotes. Here we describe a newly identified RNA-binding protein, RBP42, which targets the coding region of mRNAs in the insect form of the African trypanosome, Trypanosoma brucei. RBP42 is an essential protein and associates with polysome-bound mRNAs in the cytoplasm. A global survey of RBP42-bound mRNAs was performed by applying HITS-CLIP technology, which captures protein-RNA interactions in vivo using UV light. Specific RBP42-mRNA interactions, as well as mRNA interactions with a known RNA-binding protein, were purified using specific antibodies. Target RNA sequences were identified and quantified using high-throughput RNA sequencing. Analysis revealed that RBP42 bound mainly within the coding region of mRNAs that encode proteins involved in cellular energy metabolism. Although the mechanism of RBP42's function is unclear at present, we speculate that RBP42 plays a critical role in modulating T. brucei energy metabolism.


Assuntos
Metabolismo Energético/genética , Proteínas de Protozoários/metabolismo , RNA Mensageiro/metabolismo , RNA de Protozoário/metabolismo , Proteínas de Ligação a RNA/metabolismo , Trypanosoma brucei brucei/genética , Regiões 3' não Traduzidas , Sequência de Aminoácidos , Sítios de Ligação , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Dados de Sequência Molecular , Fases de Leitura Aberta , Polirribossomos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas de Protozoários/genética , Proteínas de Protozoários/isolamento & purificação , Interferência de RNA , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/isolamento & purificação , Homologia de Sequência de Aminoácidos , Trypanosoma brucei brucei/crescimento & desenvolvimento , Trypanosoma brucei brucei/metabolismo
6.
PLoS Pathog ; 8(8): e1002900, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22952449

RESUMO

Trypanosoma brucei is a master of antigenic variation and immune response evasion. Utilizing a genomic repertoire of more than 1000 Variant Surface Glycoprotein-encoding genes (VSGs), T. brucei can change its protein coat by "switching" from the expression of one VSG to another. Each active VSG is monoallelically expressed from only one of approximately 15 subtelomeric sites. Switching VSG expression occurs by three predominant mechanisms, arguably the most significant of which is the non-reciprocal exchange of VSG containing DNA by duplicative gene conversion (GC). How T. brucei orchestrates its complex switching mechanisms remains to be elucidated. Recent work has demonstrated that an exogenous DNA break in the active site could initiate a GC based switch, yet the source of the switch-initiating DNA lesion under natural conditions is still unknown. Here we investigated the hypothesis that telomere length directly affects VSG switching. We demonstrate that telomerase deficient strains with short telomeres switch more frequently than genetically identical strains with long telomeres and that, when the telomere is short, switching preferentially occurs by GC. Our data supports the hypothesis that a short telomere at the active VSG expression site results in an increase in subtelomeric DNA breaks, which can initiate GC based switching. In addition to their significance for T. brucei and telomere biology, the findings presented here have implications for the many diverse pathogens that organize their antigenic genes in subtelomeric regions.


Assuntos
Variação Antigênica/genética , Variação Genética , Telômero/genética , Trypanosoma brucei brucei/genética , Glicoproteínas Variantes de Superfície de Trypanosoma/genética , DNA de Protozoário/genética , Conversão Gênica , Duplicação Gênica , Humanos , Fenótipo , Telomerase/genética , Telomerase/metabolismo , Telômero/metabolismo , Homeostase do Telômero/genética , Trypanosoma brucei brucei/imunologia , Trypanosoma brucei brucei/metabolismo , Glicoproteínas Variantes de Superfície de Trypanosoma/imunologia , Glicoproteínas Variantes de Superfície de Trypanosoma/metabolismo
7.
PLoS Pathog ; 6(7): e1000992, 2010 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-20628569

RESUMO

Homologous recombination (HR) mediates one of the major mechanisms of trypanosome antigenic variation by placing a different variant surface glycoprotein (VSG) gene under the control of the active expression site (ES). It is believed that the majority of VSG switching events occur by duplicative gene conversion, but only a few DNA repair genes that are central to HR have been assigned a role in this process. Gene conversion events that are associated with crossover are rarely seen in VSG switching, similar to mitotic HR. In other organisms, TOPO3alpha (Top3 in yeasts), a type IA topoisomerase, is part of a complex that is involved in the suppression of crossovers. We therefore asked whether a related mechanism might suppress VSG recombination. Using a set of reliable recombination and switching assays that could score individual switching mechanisms, we discovered that TOPO3alpha function is conserved in Trypanosoma brucei and that TOPO3alpha plays a critical role in antigenic switching. Switching frequency increased 10-40-fold in the absence of TOPO3alpha and this hyper-switching phenotype required RAD51. Moreover, the preference of 70-bp repeats for VSG recombination was mitigated, while homology regions elsewhere in ES were highly favored, in the absence of TOPO3alpha. Our data suggest that TOPO3alpha may remove undesirable recombination intermediates constantly arising between active and silent ESs, thereby balancing ES integrity against VSG recombination.


Assuntos
Variação Antigênica/genética , DNA Topoisomerases Tipo I/fisiologia , Conversão Gênica , Trypanosoma brucei brucei/genética , Glicoproteínas Variantes de Superfície de Trypanosoma/genética , Proteínas de Bactérias/genética , Proteínas Fúngicas , Humanos , Recombinação Genética , Trypanosoma brucei brucei/imunologia
8.
Nucleic Acids Res ; 38(12): 3923-35, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20215442

RESUMO

Base J is a hypermodified DNA base localized primarily to telomeric regions of the genome of Trypanosoma brucei. We have previously characterized two thymidine-hydroxylases (TH), JBP1 and JBP2, which regulate J-biosynthesis. JBP2 is a chromatin re-modeling protein that induces de novo J-synthesis, allowing JBP1, a J-DNA binding protein, to stimulate additional J-synthesis. Here, we show that both JBP2 and JBP1 are capable of stimulating de novo J-synthesis. We localized the JBP1- and JBP2-stimulated J by anti-J immunoprecipitation and high-throughput sequencing. This genome-wide analysis revealed an enrichment of base J at regions flanking polymerase II polycistronic transcription units (Pol II PTUs) throughout the T. brucei genome. Chromosome-internal J deposition is primarily mediated by JBP1, whereas JBP2-stimulated J deposition at the telomeric regions. However, the maintenance of J at JBP1-specific regions is dependent on JBP2 SWI/SNF and TH activity. That similar regions of Leishmania major also contain base J highlights the functional importance of the modified base at Pol II PTUs within members of the kinetoplastid family. The regulation of J synthesis/localization by two THs and potential biological function of J in regulating kinetoplastid gene expression is discussed.


Assuntos
DNA de Protozoário/metabolismo , Proteínas de Ligação a DNA/metabolismo , Glucosídeos/biossíntese , Oxigenases de Função Mista/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/genética , Uracila/análogos & derivados , Animais , Linhagem Celular , DNA de Protozoário/química , Genoma de Protozoário , Histonas/análise , RNA Polimerase II/metabolismo , Timidina/metabolismo , Transcrição Gênica , Trypanosoma brucei brucei/enzimologia , Uracila/biossíntese
9.
Nucleic Acids Res ; 38(15): 4946-57, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20385579

RESUMO

Transcription of protein-coding genes in trypanosomes is polycistronic and gene expression is primarily regulated by post-transcriptional mechanisms. Sequence motifs in the untranslated regions regulate mRNA trans-splicing and RNA stability, yet where UTRs begin and end is known for very few genes. We used high-throughput RNA-sequencing to determine the genome-wide steady-state mRNA levels ('transcriptomes') for approximately 90% of the genome in two stages of the Trypanosoma brucei life cycle cultured in vitro. Almost 6% of genes were differentially expressed between the two life-cycle stages. We identified 5' splice-acceptor sites (SAS) and polyadenylation sites (PAS) for 6959 and 5948 genes, respectively. Most genes have between one and three alternative SAS, but PAS are more dispersed. For 488 genes, SAS were identified downstream of the originally assigned initiator ATG, so a subsequent in-frame ATG presumably designates the start of the true coding sequence. In some cases, alternative SAS would give rise to mRNAs encoding proteins with different N-terminal sequences. We could identify the introns in two genes known to contain them, but found no additional genes with introns. Our study demonstrates the usefulness of the RNA-seq technology to study the transcriptional landscape of an organism whose genome has not been fully annotated.


Assuntos
Genoma de Protozoário , Poliadenilação , RNA Mensageiro/metabolismo , Trans-Splicing , Trypanosoma brucei brucei/genética , Animais , Linhagem Celular , Perfilação da Expressão Gênica , Genes de Protozoários , Genômica , Íntrons , Estágios do Ciclo de Vida/genética , Sítios de Splice de RNA , RNA Mensageiro/química , Análise de Sequência de RNA , Trypanosoma brucei brucei/crescimento & desenvolvimento , Regiões não Traduzidas
10.
EMBO J ; 26(23): 4856-66, 2007 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-17972917

RESUMO

The vector-borne, protistan parasite Trypanosoma brucei is the only known eukaryote with a multifunctional RNA polymerase I that, in addition to ribosomal genes, transcribes genes encoding the parasite's major cell-surface proteins-the variant surface glycoprotein (VSG) and procyclin. In the mammalian bloodstream, antigenic variation of the VSG coat is the parasite's means to evade the immune response, while procyclin is necessary for effective establishment of trypanosome infection in the fly. Moreover, the exceptionally high efficiency of mono-allelic VSG expression is essential to bloodstream trypanosomes since its silencing caused rapid cell-cycle arrest in vitro and clearance of parasites from infected mice. Here we describe a novel protein complex that recognizes class I promoters and is indispensable for class I transcription; it consists of a dynein light chain and six polypeptides that are conserved only among trypanosomatid parasites. In accordance with an essential transcriptional function of the complex, silencing the expression of a key subunit was lethal to bloodstream trypanosomes and specifically affected the abundance of rRNA and VSG mRNA. The complex was dubbed class I transcription factor A.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Drosophila/metabolismo , Transcrição Gênica , Trypanosoma brucei brucei/metabolismo , Glicoproteínas Variantes de Superfície de Trypanosoma/química , Animais , Núcleo Celular/metabolismo , Ciclinas/metabolismo , Dineínas , Inativação Gênica , Genes de Protozoários , Vetores Genéticos , Modelos Biológicos , Regiões Promotoras Genéticas , Mapeamento de Interação de Proteínas , Proteínas de Protozoários/química , RNA Mensageiro/metabolismo , Fatores de Transcrição/metabolismo , Glicoproteínas Variantes de Superfície de Trypanosoma/metabolismo
11.
PLoS Biol ; 6(7): e161, 2008 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-18597556

RESUMO

To evade the host immune system, several pathogens periodically change their cell-surface epitopes. In the African trypanosomes, antigenic variation is achieved by tightly regulating the expression of a multigene family encoding a large repertoire of variant surface glycoproteins (VSGs). Immune evasion relies on two important features: exposing a single type of VSG at the cell surface and periodically and very rapidly switching the expressed VSG. Transcriptional switching between resident telomeric VSG genes does not involve DNA rearrangements, and regulation is probably epigenetic. The histone methyltransferase DOT1B is a nonessential protein that trimethylates lysine 76 of histone H3 in Trypanosoma brucei. Here we report that transcriptionally silent telomeric VSGs become partially derepressed when DOT1B is deleted, whereas nontelomeric loci are unaffected. DOT1B also is involved in the kinetics of VSG switching: in DeltaDOT1B cells, the transcriptional switch is so slow that cells expressing two VSGs persist for several weeks, indicating that monoallelic transcription is compromised. We conclude that DOT1B is required to maintain strict VSG silencing and to ensure rapid transcriptional VSG switching, demonstrating that epigenetics plays an important role in regulating antigenic variation in T. brucei.


Assuntos
Variação Antigênica/imunologia , Regulação Enzimológica da Expressão Gênica/imunologia , Histona-Lisina N-Metiltransferase/metabolismo , Trypanosoma brucei brucei/enzimologia , Glicoproteínas Variantes de Superfície de Trypanosoma/imunologia , Animais , Variação Antigênica/genética , Inativação Gênica , Histona Metiltransferases , Histona-Lisina N-Metiltransferase/genética , Interações Hospedeiro-Parasita , Proteínas Metiltransferases , Trypanosoma brucei brucei/patogenicidade , Trypanosoma brucei brucei/fisiologia , Glicoproteínas Variantes de Superfície de Trypanosoma/genética
13.
Eukaryot Cell ; 9(1): 148-54, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19915072

RESUMO

In most eukaryotes, RNA polymerase I (Pol I) exclusively transcribes long arrays of identical rRNA genes (ribosomal DNA [rDNA]). African trypanosomes have the unique property of using Pol I to also transcribe the variant surface glycoprotein VSG genes. VSGs are important virulence factors because their switching allows trypanosomes to escape the host immune system, a mechanism known as antigenic variation. Only one VSG is transcribed at a time from one of 15 bloodstream-form expression sites (BESs). Although it is clear that switching among BESs does not involve DNA rearrangements and that regulation is probably epigenetic, it remains unknown why BESs are transcribed by Pol I and what roles are played by chromatin structure and histone modifications. Using chromatin immunoprecipitation, micrococcal nuclease digestion, and chromatin fractionation, we observed that there are fewer nucleosomes at the active BES and that these are irregularly spaced compared to silent BESs. rDNA coding regions are also depleted of nucleosomes, relative to the rDNA spacer. In contrast, genes transcribed by Pol II are organized in a more compact, regularly spaced, nucleosomal structure. These observations provide new insight on antigenic variation by showing that chromatin remodeling is an intrinsic feature of BES regulation.


Assuntos
Nucleossomos/metabolismo , RNA Polimerase I/metabolismo , Trypanosoma brucei brucei , Glicoproteínas Variantes de Superfície de Trypanosoma/metabolismo , Cromatina/química , Cromatina/metabolismo , Regulação da Expressão Gênica , Histonas/genética , Histonas/metabolismo , Conformação de Ácido Nucleico , RNA Polimerase I/genética , Transcrição Gênica , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , Glicoproteínas Variantes de Superfície de Trypanosoma/genética
14.
Mol Microbiol ; 69(4): 1054-68, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18631159

RESUMO

Chromatin modification is important for virtually all aspects of DNA metabolism but little is known about the consequences of such modification in trypanosomatids, early branching protozoa of significant medical and veterinary importance. MYST-family histone acetyltransferases in other species function in transcription regulation, DNA replication, recombination and repair. Trypanosoma brucei HAT3 was recently shown to acetylate histone H4K4 and we now report characterization of all three T. brucei MYST acetyltransferases (HAT1-3). First, GFP-tagged HAT1-3 all localize to the trypanosome nucleus. While HAT3 is dispensable, both HAT1 and HAT2 are essential for growth. Strains with HAT1 knock-down display mitosis without nuclear DNA replication and also specific de-repression of a telomeric reporter gene, a rare example of transcription control in an organism with widespread and constitutive polycistronic transcription. Finally, we show that HAT2 is responsible for H4K10 acetylation. By analogy to the situation in Saccharomyces cerevisiae, we discuss low-level redundancy of acetyltransferase function in T. brucei and suggest that two MYST-family acetyltransferases are essential due to the absence of a Gcn5 homologue. The results are also consistent with the idea that HAT1 contributes to establishing boundaries between transcriptionally active and repressed telomeric domains in T. brucei.


Assuntos
Acetiltransferases/metabolismo , Inativação Gênica , Histonas/metabolismo , Proteínas de Protozoários/metabolismo , Telômero/metabolismo , Trypanosoma brucei brucei/enzimologia , Acetilação , Acetiltransferases/classificação , Acetiltransferases/genética , Sequência de Aminoácidos , Animais , Histona Acetiltransferases , Dados de Sequência Molecular , Filogenia , Proteínas de Protozoários/classificação , Proteínas de Protozoários/genética , Telômero/genética , Trypanosoma brucei brucei/genética
15.
Mol Cell Biol ; 26(13): 4911-9, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16782879

RESUMO

In cancer cells and germ cells, shortening of chromosome ends is prevented by telomerase. Telomerase-deficient cells have a replicative life span, after which they enter senescence. Senescent cells can give rise to survivors that maintain chromosome ends through recombination-based amplification of telomeric or subtelomeric repeats. We found that in Trypanosoma brucei, critically short telomeres are stable in the absence of telomerase. Telomere stabilization ensured genomic integrity and could have implications for telomere maintenance in human telomerase-deficient cells. Cloning and sequencing revealed 7 to 27 TTAGGG repeats on stabilized telomeres and no changes in the subtelomeric region. Clones with short telomeres were used to study telomere elongation dynamics, which differed dramatically at transcriptionally active and silent telomeres, after restoration of telomerase. We propose that transcription makes the termini of short telomeres accessible for rapid elongation by telomerase and that telomere elongation in T. brucei is not regulated by a protein-counting mechanism. Many minichromosomes were lost after long-term culture in the absence of telomerase, which may reflect their different mitotic segregation properties.


Assuntos
DNA de Protozoário/metabolismo , Telomerase/genética , Telômero/genética , Telômero/metabolismo , Trypanosoma brucei brucei/enzimologia , Trypanosoma brucei brucei/genética , Animais , Cromossomos/metabolismo , Sequências Repetitivas de Ácido Nucleico/genética , Deleção de Sequência , Telomerase/metabolismo , Telômero/enzimologia , Transcrição Gênica
16.
Biochem Biophys Res Commun ; 368(4): 846-51, 2008 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-18261990

RESUMO

Some inroads have been made into characterizing histone variants and post translational modifications of histones in Trypanosoma brucei. Histone variant H2BV lysine 129 is homologous to Saccharomyces cerevisiae H2B lysine 123, whose ubiquitination is required for methylation of H3 lysines 4 and 79. We show that T. brucei H2BV K129 is not ubiquitinated, but trimethylation of H3 K4 and K76, homologs of H3 K4 and K79 in yeast, was enriched in nucleosomes containing H2BV. Mutation of H2BV K129 to alanine or arginine did not disrupt H3 K4 or K76 methylation. These data suggest that H3 K4 and K76 methylation in trypanosomes is regulated by a novel mechanism, possibly involving the replacement of H2B with H2BV in the nucleosome.


Assuntos
Histonas/genética , Histonas/metabolismo , Nucleossomos/metabolismo , Sequência de Aminoácidos , Animais , Lisina/genética , Lisina/metabolismo , Metilação , Dados de Sequência Molecular , Alinhamento de Sequência , Trypanosoma brucei brucei/genética
17.
Mol Biochem Parasitol ; 157(1): 73-82, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18006158

RESUMO

The limited repertoire of drug-resistance markers imposes a serious obstacle to genetic manipulation of Trypanosoma brucei. Here we describe experiments with a fusion protein that allows positive selection for genome integration followed by CRE recombinase-mediated excision of the marker cassette that can be selected by ganciclovir, although the excision event is so efficient that selection is not strictly necessary. We describe two variants of the tetracycline-inducible pLEW100-based CRE-expression vector that reduced its toxicity when stably integrated into the genome, and we demonstrate that transient transfection of circular pLEW100-CRE is highly efficient at catalyzing marker excision. We used this approach to delete the last two enzymes of the pyrimidine synthesis pathway, creating a cell line that is resistant to fluoroorotic acid, which would allow the same enzymes (PYR6-5) to be used as an alternative negative selectable marker.


Assuntos
Genética Microbiana/métodos , Integrases/metabolismo , Parasitologia/métodos , Recombinação Genética , Trypanosoma brucei brucei/genética , Animais , Deleção de Genes , Integrases/genética , Mutagênese Insercional , Seleção Genética
18.
Mol Biochem Parasitol ; 160(2): 171-4, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18501977

RESUMO

Trypanosoma brucei has two DNA compartments: the nucleus and the kinetoplast. DNA replication of these two compartments only partially coincides. Woodward and Gull [Woodward R, Gull K. Timing of nuclear and kinetoplast DNA replication and early morphological events in the cell cycle of Trypanosoma brucei. J Cell Sci 1990;95:49-57] comprehensively studied the relative timing of the replication and segregation of nuclear DNA (nDNA) and kinetoplast DNA (kDNA). Others have since assumed the consistency of morphological indicators of cell-cycle stage among strains and conditions. We report the use of quantitative DAPI imaging to determine the cell-cycle stage of individual procyclic cells. Using this approach, we found that kinetoplast elongation occurs mainly during nuclear S phase and not during G2, as previously assumed. We confirmed this finding by sorting cells by DNA content, followed by fluorescence microscopy. In addition, simultaneous quantitative imaging at two wavelengths can be used to determine the abundance of cell-cycle-regulated proteins during the cell cycle. We demonstrate this technique by co-staining for the non-acetylated state of lysine 4 of histone H4 (H4K4), which is enriched during nuclear S phase.


Assuntos
Ciclo Celular , Processamento de Imagem Assistida por Computador/métodos , Coloração e Rotulagem/métodos , Trypanosoma brucei brucei/fisiologia , Animais , Citometria de Fluxo , Indóis/metabolismo , Microscopia de Fluorescência
19.
Mol Cell Biol ; 25(12): 5011-21, 2005 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15923618

RESUMO

Putative TTAGGG repeat-binding factor (TRF) homologues in the genomes of Trypanosoma brucei, Trypanosoma cruzi, and Leishmania major were identified. They have significant sequence similarity to higher eukaryotic TRFs in their C-terminal DNA-binding myb domains but only weak similarity in their N-terminal domains. T. brucei TRF (tbTRF) is essential and was shown to bind to duplex TTAGGG repeats. The RNA interference-mediated knockdown of tbTRF arrested bloodstream cells at G(2)/M and procyclic cells partly at S phase. Functionally, tbTRF resembles mammalian TRF2 more than TRF1, as knockdown diminished telomere single-stranded G-overhang signals. This suggests that tbTRF, like vertebrate TRF2, is essential for telomere end protection, and this also supports the hypothesis that TRF rather than Rap1 is the more ancient DNA-binding component of the telomere protein complex. Identification of the first T. brucei telomere DNA-binding protein and characterization of its function provide a new route to explore the roles of telomeres in pathogenesis of this organism. This work also establishes T. brucei as an attractive model for telomere biology.


Assuntos
Isoformas de Proteínas/metabolismo , Telômero/metabolismo , Proteína 2 de Ligação a Repetições Teloméricas/metabolismo , Trypanosoma/genética , Sequência de Aminoácidos , Animais , Ciclo Celular/fisiologia , Humanos , Hibridização in Situ Fluorescente , Dados de Sequência Molecular , Ligação Proteica , Isoformas de Proteínas/genética , Interferência de RNA , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Proteína 2 de Ligação a Repetições Teloméricas/genética , Trypanosoma/metabolismo
20.
Mol Cell Biol ; 25(21): 9586-94, 2005 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16227607

RESUMO

mRNA maturation in Trypanosoma brucei depends upon trans splicing, and variations in trans-splicing efficiency could be an important step in controlling the levels of individual mRNAs. RNA splicing requires specific sequence elements, including conserved 5' splice sites, branch points, pyrimidine-rich regions [poly(Y) tracts], 3' splice sites (3'SS), and sometimes enhancer elements. To analyze sequence requirements for efficient trans splicing in the poly(Y) tract and around the 3'SS, we constructed a luciferase-beta-galactosidase double-reporter system. By testing approximately 90 sequences, we demonstrated that the optimum poly(Y) tract length is approximately 25 nucleotides. Interspersing a purely uridine-containing poly(Y) tract with cytidine resulted in increased trans-splicing efficiency, whereas purines led to a large decrease. The position of the poly(Y) tract relative to the 3'SS is important, and an AC dinucleotide at positions -3 and -4 can lead to a 20-fold decrease in trans splicing. However, efficient trans splicing can be restored by inserting a second AG dinucleotide downstream, which does not function as a splice site but may aid in recruitment of the splicing machinery. These findings should assist in the development of improved algorithms for computationally identifying a 3'SS and help to discriminate noncoding open reading frames from true genes in current efforts to annotate the T. brucei genome.


Assuntos
RNA de Protozoário/genética , Trans-Splicing , Trypanosoma brucei brucei/genética , Animais , Sequência de Bases , Clonagem Molecular , Genes Reporter , Luciferases/genética , Dados de Sequência Molecular , Pirimidinas/química , Sítios de Splice de RNA , Análise de Sequência , beta-Galactosidase/genética
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