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1.
Cell ; 150(5): 909-21, 2012 Aug 31.
Artículo en Inglés | MEDLINE | ID: mdl-22939620

RESUMEN

Some Ts in nuclear DNA of trypanosomes and Leishmania are hydroxylated and glucosylated to yield base J (ß-D-glucosyl-hydroxymethyluracil). In Leishmania, about 99% of J is located in telomeric repeats. We show here that most of the remaining J is located at chromosome-internal RNA polymerase II termination sites. This internal J and telomeric J can be reduced by a knockout of J-binding protein 2 (JBP2), an enzyme involved in the first step of J biosynthesis. J levels are further reduced by growing Leishmania JBP2 knockout cells in BrdU-containing medium, resulting in cell death. The loss of internal J in JBP2 knockout cells is accompanied by massive readthrough at RNA polymerase II termination sites. The readthrough varies between transcription units but may extend over 100 kb. We conclude that J is required for proper transcription termination and infer that the absence of internal J kills Leishmania by massive readthrough of transcriptional stops.


Asunto(s)
Glucósidos/metabolismo , Leishmania/genética , Leishmania/metabolismo , Transcripción Genética , Uracilo/análogos & derivados , Técnicas de Inactivación de Genes , ARN Polimerasa II/metabolismo , ARN Bicatenario/metabolismo , Uracilo/metabolismo
3.
Nucleic Acids Res ; 43(4): 2102-15, 2015 Feb 27.
Artículo en Inglés | MEDLINE | ID: mdl-25662217

RESUMEN

Base J (ß-D-glucosyl-hydroxymethyluracil) replaces 1% of T in the Leishmania genome and is only found in telomeric repeats (99%) and in regions where transcription starts and stops. This highly restricted distribution must be co-determined by the thymidine hydroxylases (JBP1 and JBP2) that catalyze the initial step in J synthesis. To determine the DNA sequences recognized by JBP1/2, we used SMRT sequencing of DNA segments inserted into plasmids grown in Leishmania tarentolae. We show that SMRT sequencing recognizes base J in DNA. Leishmania DNA segments that normally contain J also picked up J when present in the plasmid, whereas control sequences did not. Even a segment of only 10 telomeric (GGGTTA) repeats was modified in the plasmid. We show that J modification usually occurs at pairs of Ts on opposite DNA strands, separated by 12 nucleotides. Modifications occur near G-rich sequences capable of forming G-quadruplexes and JBP2 is needed, as it does not occur in JBP2-null cells. We propose a model whereby de novo J insertion is mediated by JBP2. JBP1 then binds to J and hydroxylates another T 13 bp downstream (but not upstream) on the complementary strand, allowing JBP1 to maintain existing J following DNA replication.


Asunto(s)
Glucósidos/análisis , Uracilo/análogos & derivados , Proteínas de Unión al ADN/metabolismo , Glucósidos/metabolismo , Leishmania/genética , Plásmidos/genética , Proteínas Protozoarias/metabolismo , Análisis de Secuencia de ADN , Uracilo/análisis , Uracilo/metabolismo
4.
Nucleic Acids Res ; 35(7): 2116-24, 2007.
Artículo en Inglés | MEDLINE | ID: mdl-17329373

RESUMEN

Base J or beta-d-glucosylhydroxymethyluracil is a DNA modification replacing a fraction of thymine in the nuclear DNA of kinetoplastid parasites and of Euglena. J is located in the telomeric sequences of Trypanosoma brucei and in other simple repeat DNA sequences. In addition, J was found in the inactive variant surface glycoprotein (VSG) expression sites, but not in the active expression site of T. brucei, suggesting that J could play a role in transcription silencing in T. brucei. We have now looked at the distribution of J in the genomes of other kinetoplastid parasites. First, we analyzed the DNA sequences immunoprecipitated with a J-antiserum in Leishmania major Friedlin. Second, we investigated the co-migration of J- and telomeric repeat-containing DNA sequences of various kinetoplastids using J-immunoblots and Southern blots of fragmented DNA. We find only approximately 1% of J outside the telomeric repeat sequences of Leishmania sp. and Crithidia fasciculata, in contrast to the substantial fraction of non-telomeric J found in T. brucei, Trypanosoma equiperdum and Trypanoplasma borreli. Our results suggest that J is a telomeric base modification, recruited for other (unknown) functions in some kinetoplastids and Euglena.


Asunto(s)
Glucósidos/análisis , Leishmania/genética , Telómero/química , Uracilo/análogos & derivados , Animales , Cromatografía en Agarosa , Crithidia fasciculata/genética , ADN Protozoario/química , Genoma de Protozoos , Immunoblotting , Inmunoprecipitación , Secuencias Repetitivas de Ácidos Nucleicos , Análisis de Secuencia de ADN , Trypanosoma cruzi/genética , Uracilo/análisis
5.
Nucleic Acids Res ; 35(7): 2107-15, 2007.
Artículo en Inglés | MEDLINE | ID: mdl-17389644

RESUMEN

Trypanosomatids contain an unusual DNA base J (beta-d-glucosylhydroxymethyluracil), which replaces a fraction of thymine in telomeric and other DNA repeats. To determine the function of base J, we have searched for enzymes that catalyze J biosynthesis. We present evidence that a protein that binds to J in DNA, the J-binding protein 1 (JBP1), may also catalyze the first step in J biosynthesis, the conversion of thymine in DNA into hydroxymethyluracil. We show that JBP1 belongs to the family of Fe(2+) and 2-oxoglutarate-dependent dioxygenases and that replacement of conserved residues putatively involved in Fe(2+) and 2-oxoglutarate-binding inactivates the ability of JBP1 to contribute to J synthesis without affecting its ability to bind to J-DNA. We propose that JBP1 is a thymidine hydroxylase responsible for the local amplification of J inserted by JBP2, another putative thymidine hydroxylase.


Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Glucósidos/biosíntesis , Oxigenasas de Función Mixta/química , Oxigenasas de Función Mixta/metabolismo , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Uracilo/análogos & derivados , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Sitios de Unión , Proteínas de Unión al ADN/clasificación , Dioxigenasas/clasificación , Glucósidos/química , Glucósidos/metabolismo , Leishmania/genética , Oxigenasas de Función Mixta/clasificación , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Proteínas Protozoarias/clasificación , Uracilo/biosíntesis , Uracilo/química , Uracilo/metabolismo
6.
Nucleic Acids Res ; 33(5): 1699-709, 2005.
Artículo en Inglés | MEDLINE | ID: mdl-15781496

RESUMEN

Attempts to inactivate an essential gene in the protozoan parasite Leishmania have often led to the generation of extra copies of the wild-type alleles of the gene. In experiments with Leishmania tarentolae set up to disrupt the gene encoding the J-binding protein 1 (JBP1), a protein binding to the unusual base beta-D-glucosyl-hydroxymethyluracil (J) of Leishmania, we obtained JBP1 mutants containing linear DNA elements (amplicons) of approximately 100 kb. These amplicons consist of a long inverted repeat with telomeric repeats at both ends and contain either the two different targeting cassettes used to inactivate JBP1, or one cassette and one JBP1 gene. Each long repeat within the linear amplicons corresponds to sequences covering the JBP1 locus, starting at the telomeres upstream of JBP1 and ending in a approximately 220 bp sequence repeated in an inverted (palindromic) orientation downstream of the JBP1 locus. We propose that these amplicons have arisen by a template switch inside a DNA replication fork involving the inverted DNA repeats and helped by the gene targeting.


Asunto(s)
ADN Protozoario/química , Proteínas de Unión al ADN/genética , Higromicina B/análogos & derivados , Leishmania/genética , Proteínas Protozoarias/genética , Secuencias Repetitivas de Ácidos Nucleicos , Alelos , Animales , Secuencia de Bases , Línea Celular , Cinamatos/farmacología , Marcación de Gen , Genes Protozoarios , Higromicina B/farmacología , Leishmania/efectos de los fármacos , Modelos Genéticos , Datos de Secuencia Molecular , Mutación , Plásmidos/química
7.
Trends Mol Med ; 21(9): 525-7, 2015 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-26254816

RESUMEN

Translational research leaves no-one indifferent and everyone expects a particular benefit. We as EU-LIFE (www.eu-life.eu), an alliance of 13 research institutes in European life sciences, would like to share our experience in an attempt to identify measures to promote translational research without undermining basic exploratory research and academic freedom.


Asunto(s)
Disciplinas de las Ciencias Biológicas/organización & administración , Investigación Biomédica/organización & administración , Investigación Biomédica Traslacional/organización & administración , Disciplinas de las Ciencias Biológicas/tendencias , Investigación Biomédica/tendencias , Europa (Continente) , Humanos , Cooperación Internacional , Investigación Biomédica Traslacional/tendencias
9.
Mol Oncol ; 4(1): 12-8, 2010 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-19819199

RESUMEN

International competition forces fundamental research organizations to assess their relative performance. We present a benchmark tool for scientific research organizations where, contrary to existing models, the group leader is placed in a central position within the organization. We used it in a pilot benchmark study involving six research institutions. Our study shows that data collection and data comparison based on this new tool can be achieved. It proved possible to compare relative performance and organizational characteristics and to generate suggestions for improvement for most participants. However, strict definitions of the parameters used for the benchmark and a thorough insight into the organization of each of the benchmark partners is required to produce comparable data and draw firm conclusions.


Asunto(s)
Benchmarking/clasificación , Toma de Decisiones en la Organización , Eficiencia Organizacional , Evaluación del Rendimiento de Empleados , Modelos Organizacionales , Innovación Organizacional , Objetivos Organizacionales , Inteligencia Artificial , Benchmarking/métodos , Benchmarking/organización & administración , Competencia Clínica , Simulación por Computador , Recolección de Datos/estadística & datos numéricos , Sistemas de Administración de Bases de Datos/organización & administración , Administración Financiera de Hospitales , Humanos , Almacenamiento y Recuperación de la Información/métodos , Liderazgo , Masculino , Modelos Estadísticos , Enfermeras Administradoras , Proyectos Piloto , Técnicas de Planificación , Control de Calidad , Gestión de la Calidad Total/estadística & datos numéricos , Estados Unidos
10.
Mol Microbiol ; 58(1): 151-65, 2005 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-16164555

RESUMEN

In its mammalian host, Trypanosoma brucei covers its iron requirements by receptor-mediated uptake of host transferrin (Tf). The Tf-receptor (Tf-R) is a heterodimeric membrane protein encoded by expression site-associated gene (ESAG) 6 and 7 located promoter-proximal in a polycistronic expression site (ES). Each of the 20 ESs encodes a slightly different Tf-R; these differences strongly affect the binding affinity for Tfs of different hosts. The Tf-R encoded in the 221 ES has a low affinity for dog Tf. Transfer of trypanosomes with an active 221 ES to dilute dog serum leads to growth arrest, which they can overcome by switching to another ES encoding a Tf-R with higher affinity for dog Tf. Here we show that trypanosomes can also adapt to dilute dog serum without switching but by replacing the ESAG7 gene in the 221 ES by one from another ES, by deleting ESAG7 from the 221 ES with concomitant upregulation of transcription of ESAG7 in 'silent' ESs, by grossly overproducing the 221 Tf-R or by combinations of these alterations. Our results illustrate the striking genetic flexibility of trypanosomes.


Asunto(s)
Regulación de la Expresión Génica , Receptores de Transferrina/genética , Transferrina/metabolismo , Trypanosoma brucei brucei/genética , Animales , Western Blotting , Bovinos , Medios de Cultivo , Perros , Eliminación de Gen , Glicoproteínas/análisis , Glicoproteínas/genética , Proteínas Protozoarias/análisis , Proteínas Protozoarias/genética , ARN Mensajero/análisis , ARN Protozoario/análisis , Receptores de Transferrina/metabolismo , Recombinación Genética
11.
J Biol Chem ; 279(39): 40690-8, 2004 Sep 24.
Artículo en Inglés | MEDLINE | ID: mdl-15263009

RESUMEN

Transfer of bloodstream-form Trypanosoma brucei variant 221a from calf serum to dog serum-based medium induces acute iron starvation, as the transferrin receptor (Tf-R) of variant 221a binds dog Tf poorly. We show here that transfer to dog serum induces a 3-5-fold increase in Tf-R mRNA and protein within one doubling time (8 h). Because iron stores are still high 8 h after transfer, we infer that the signal for Tf-R overproduction is the decreased availability of cytosolic iron when cellular iron import drops. Up to 30% of the extra Tf-R spills out of the flagellar pocket onto the pellicular surface. Because the 5-fold increase in Tf-R is accompanied by a 5-fold increase in bovine Tf uptake, the up-regulation of Tf-R levels in response to Tf starvation helps the trypanosome to compete for limiting amounts of Tf. We noted that Tf-R levels also vary in calf serum medium. Cells in dense cultures contain up to 5-fold more Tf-R mRNA and protein than in dilute cultures. Only one-tenth of the extra Tf-R reaches the pellicular surface. The increase cannot be explained by a lack of Tf or to cell density sensing but is due to pericellular hypoxia. Our results show that bloodstream-form trypanosomes can regulate the expression of the two Tf-R subunit genes and the localization of their gene products in a flexible manner. This flexibility is made possible by the promoter-proximal position of the two genes in the variant surface glycoprotein expression site.


Asunto(s)
Receptores de Transferrina/biosíntesis , Trypanosoma brucei brucei/metabolismo , Animales , Sitios de Unión , Northern Blotting , Bovinos , Medios de Cultivo/metabolismo , Citosol/metabolismo , Perros , Relación Dosis-Respuesta a Droga , Microanálisis por Sonda Electrónica , Endocitosis , Flagelos/metabolismo , Glicosilación , Hipoxia , Hierro/metabolismo , Ratones , Microscopía Fluorescente , Unión Proteica , ARN Mensajero/metabolismo , Receptores de Transferrina/metabolismo , Factores de Tiempo , Regulación hacia Arriba
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