RESUMEN
In this review we will describe the replication of kinetoplast DNA, a subject that our lab has studied for many years. Our knowledge of kinetoplast DNA replication has depended mostly upon the investigation of the biochemical properties and intramitochondrial localisation of replication proteins and enzymes as well as a study of the structure and dynamics of kinetoplast DNA replication intermediates. We will first review the properties of the characterised kinetoplast DNA replication proteins and then describe our current model for kinetoplast DNA replication.
Asunto(s)
Crithidia fasciculata/fisiología , Replicación del ADN/fisiología , ADN de Cinetoplasto/fisiología , Animales , Crithidia fasciculata/enzimología , Crithidia fasciculata/genética , ADN de Cinetoplasto/biosíntesis , ADN de Cinetoplasto/genética , PredicciónRESUMEN
Kinetoplast DNA (kDNA), the mitochondrial DNA of the trypanosomatid Crithidia fasciculata, is a unique structure containing 5,000 DNA minicircles topologically linked into a massive network. In vivo, the network is condensed into a disk-shaped structure. Replication of minicircles initiates at unique origins that are bound by universal minicircle sequence (UMS)-binding protein (UMSBP), a sequence-specific DNA-binding protein. This protein, encoded by a nuclear gene, localizes within the cell's single mitochondrion. Using immunofluorescence, we found that UMSBP localizes exclusively to two neighboring sites adjacent to the face of the kDNA disk nearest the cell's flagellum. This site is distinct from the two antipodal positions at the perimeter of the disk that is occupied by DNA polymerase beta, topoisomerase II, and a structure-specific endonuclease. Although we found constant steady-state levels of UMSBP mRNA and protein and a constant rate of UMSBP synthesis throughout the cell cycle, immunofluorescence indicated that UMSBP localization within the kinetoplast is not static. The intramitochondrial localization of UMSBP and other kDNA replication enzymes significantly clarifies our understanding of the process of kDNA replication.
Asunto(s)
ADN de Cinetoplasto/fisiología , ADN Mitocondrial/fisiología , Proteínas de Unión al ADN/genética , Animales , Ciclo Celular/fisiología , Crithidia fasciculata , Proteínas de Unión al ADN/análisis , Técnicas Microbiológicas , Mitocondrias/química , Mitocondrias/genética , Proteínas Protozoarias/análisis , Proteínas Protozoarias/genética , ARN Mensajero/análisis , Origen de Réplica/fisiologíaRESUMEN
Kinetoplast DNA, the mitochondrial DNA of Crithidia fasciculata, is organized into a network containing 5,000 topologically interlocked minicircles. This network, situated within the mitochondrial matrix, is condensed into a disk-shaped structure located near the basal body of the flagellum. Fluorescence in situ hybridization revealed that before their replication, minicircles are released vectorially from the network face nearest the flagellum. Replication initiates in the zone between the flagellar face of the disk and the mitochondrial membrane (we term this region the kinetoflagellar zone [KFZ]). The replicating minicircles then move to two antipodal sites that flank the disk-shaped network. In later stages of replication, the number of free minicircles increases, accumulating transiently in the KFZ. The final replication events, including primer removal, repair of many of the gaps, and reattachment of the progeny minicircles to the network periphery, are thought to take place within the antipodal sites.
Asunto(s)
ADN de Cinetoplasto/fisiología , ADN Mitocondrial/fisiología , Mitocondrias/genética , Origen de Réplica/fisiología , Animales , Ciclo Celular/fisiología , Crithidia fasciculata , ADN de Cinetoplasto/análisis , ADN Mitocondrial/análisis , ADN Protozoario/análisis , ADN Protozoario/fisiología , Dosificación de Gen , Hibridación Fluorescente in SituRESUMEN
RNA interference is a powerful method for inhibition of gene expression in Trypanosoma brucei (Ngo, H., Tschudi, C., Gull, K., and Ullu, E. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 14687-14692). Here we describe a vector (pZJM) for in vivo tetracycline-inducible synthesis of double-stranded RNA (dsRNA) in stably transformed cells. The dsRNA is synthesized from opposing T7 promoters. We tested the vector with genes involved in processes such as kinetoplast DNA replication, mitochondrial mRNA synthesis, glycosyl phosphatidylinositol biosynthesis, glycosome biogenesis, and polyamine biosynthesis. In most cases the induction of dsRNA caused specific and dramatic loss of the appropriate mRNA, and in many cases there was growth inhibition or cell death. One striking phenotype was the loss of kinetoplast DNA after interference with expression of a topoisomerase II. The gene being analyzed by this procedure need not even be fully sequenced. In fact, many of the genes we tested were derived from partial sequences in the T. brucei genome data base that were identified by homology with known proteins. It takes as little as 3 weeks from identification of a gene sequence in the data base to the appearance of a phenotype.
Asunto(s)
Bacteriófago T7/genética , Regulación de la Expresión Génica/genética , Genes Protozoarios , Vectores Genéticos , Regiones Promotoras Genéticas , ARN Protozoario/antagonistas & inhibidores , Trypanosoma brucei brucei/genética , Animales , Secuencia de Bases , Cartilla de ADNRESUMEN
Purine transport is an indispensable nutritional function for protozoan parasites, since they are incapable of purine biosynthesis and must, therefore, acquire purines from the host milieu. Exploiting a mutant cell line (FBD5) of Leishmania donovani deficient in inosine and guanosine transport activity, the gene encoding this transporter (LdNT2) has been cloned by functional rescue of the mutant phenotype. LdNT2 encodes a polypeptide of 499 amino acids that shows substantial homology to other members of the equilibrative nucleoside transporter family. Molecular analysis revealed that LdNT2 is present as a single gene copy within the leishmanial genome and encodes a single transcript of 3 kilobase pairs. Transfection of FBD5 parasites with LdNT2 re-established their ability to take up inosine and guanosine with a concurrent restoration of sensitivity to the inosine analog formycin B. Kinetic analyses reveal that LdNT2 is highly specific for inosine (K(m) = 0.3 micrometer) and guanosine (K(m) = 1.7 micrometer) and does not recognize other naturally occurring nucleosides. Expression of LdNT2 cRNA in Xenopus oocytes significantly augmented their ability to take up inosine and guanosine, establishing that LdNT2 by itself suffices to mediate nucleoside transport. These results authenticate genetically and biochemically that LdNT2 is a novel nucleoside transporter with an unusual and strict specificity for inosine and guanosine.
Asunto(s)
Proteínas Portadoras/genética , Guanosina/metabolismo , Inosina/metabolismo , Leishmania donovani/genética , Proteínas de la Membrana/genética , Proteínas Protozoarias/genética , Secuencia de Aminoácidos , Animales , Transporte Biológico , Proteínas Portadoras/química , Clonación Molecular , Formicinas/farmacología , Cinética , Leishmania donovani/metabolismo , Proteínas de la Membrana/química , Datos de Secuencia Molecular , Proteínas de Transporte de Nucleósidos , Oocitos/metabolismo , Proteínas Protozoarias/química , Alineación de Secuencia , Especificidad por Sustrato , Transfección , XenopusRESUMEN
All parasitic protozoa studied to date are incapable of purine biosynthesis and must therefore salvage purine nucleobases or nucleosides from their hosts. This salvage process is initiated by purine transporters on the parasite cell surface. We have used a mutant line (TUBA5) of Leishmania donovani that is deficient in adenosine/pyrimidine nucleoside transport activity (LdNT1) to clone genes encoding these nucleoside transporters by functional rescue. Two such genes, LdNT1.1 and LdNT1.2, have been sequenced and shown to encode deduced polypeptides with significant sequence identity to the human facilitative nucleoside transporter hENT1. Hydrophobicity analysis of the LdNT1.1 and LdNT1.2 proteins predicted 11 transmembrane domains. Transfection of the adenosine/pyrimidine nucleoside transport-deficient TUBA5 parasites with vectors containing the LdNT1.1 and LdNT1.2 genes confers sensitivity to the cytotoxic adenosine analog tubercidin and concurrently restores the ability of this mutant line to take up [3H]adenosine and [3H]uridine. Moreover, expression of the LdNT1.2 ORF in Xenopus oocytes significantly increases their ability to take up [3H]adenosine, confirming that this single protein is sufficient to mediate nucleoside transport. These results establish genetically and biochemically that both LdNT1 genes encode functional adenosine/pyrimidine nucleoside transporters.
Asunto(s)
Proteínas Portadoras/genética , Genes Protozoarios , Leishmania donovani/genética , Leishmania donovani/metabolismo , Proteínas de Transporte de Nucleósidos , Proteínas Protozoarias/genética , Nucleósidos de Purina/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/metabolismo , Clonación Molecular , Resistencia a Medicamentos/genética , Tranportador Equilibrativo 1 de Nucleósido , Femenino , Expresión Génica , Humanos , Leishmania donovani/efectos de los fármacos , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Mutación , Oocitos/metabolismo , Fenotipo , Proteínas Protozoarias/metabolismo , Mapeo Restrictivo , Homología de Secuencia de Aminoácido , Tubercidina/farmacología , XenopusRESUMEN
Voltage clamp recording was used to measure steady-state and presteady-state currents mediated by a myo-inositol transporter cloned from Leishmania donovani and expressed in Xenopus oocytes. Application of myo-inositol resulted in inward currents, which did not require external sodium and which were increased by increasing the extracellular proton concentration and by membrane hyperpolarization. Alkalinization of the extracellular space occurred concomitantly with myo-inositol influx. Correlation of membrane currents with radiolabeled myo-inositol flux revealed that one positive charge is translocated with each molecule of myo-inositol, consistent with cotransport of one proton. The transport concentration dependence on both species suggested ordered binding of a proton followed by a molecule of myo-inositol. In the absence of myo-inositol, a voltage-dependent capacitance was observed that correlated with the transporter expression level. This charge movement obeyed a Boltzmann function, which was used to estimate a turnover of 0.70 +/- 0.06 s-1 at -60 mV. The pH and voltage dependence of the charge movements were simulated with a model involving alternating access of internal and external protons to sites within an occluded pore.
Asunto(s)
Proteínas Portadoras/metabolismo , Inositol/metabolismo , Leishmania donovani/metabolismo , Oocitos/fisiología , Animales , Transporte Biológico , Proteínas Portadoras/biosíntesis , Femenino , Concentración de Iones de Hidrógeno , Inositol/farmacología , Cinética , Potenciales de la Membrana/efectos de los fármacos , Modelos Biológicos , Oocitos/efectos de los fármacos , Técnicas de Placa-Clamp , Temperatura , Xenopus laevisRESUMEN
The vast majority of surface molecules in such kinetoplastid protozoa as members of the genus Leishmania contain inositol and are either glycosyl inositol phospholipids or glycoproteins that are tethered to the external surface of the plasma membrane by glycosylphosphatidylinositol anchors. We have shown that the biosynthetic precursor for these abundant glycolipids, myo-inositol, is translocated across the parasite plasma membrane by a specific transporter that is structurally related to mammalian facilitative glucose transporters. This myo-inositol transporter has been expressed and characterized in Xenopus laevis oocytes. Two-electrode voltage clamp experiments demonstrate that this protein is a sodium-independent electrogenic symporter that appears to utilize a proton gradient to concentrate myo-inositol within the cell. Immunolocalization experiments with a transporter-specific polyclonal antibody reveal the presence of this protein in the parasite plasma membrane.
Asunto(s)
Proteínas Portadoras/biosíntesis , Inositol/metabolismo , Leishmania donovani/metabolismo , Proteínas de la Membrana/biosíntesis , Proteínas Protozoarias/biosíntesis , Secuencia de Aminoácidos , Animales , Transporte Biológico Activo , Proteínas Portadoras/análisis , Proteínas Portadoras/genética , Membrana Celular/química , Genes Protozoarios/genética , Concentración de Iones de Hidrógeno , Transporte Iónico , Cinética , Proteínas de la Membrana/análisis , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Oocitos , Protones , Proteínas Protozoarias/análisis , Proteínas Protozoarias/genética , Alineación de Secuencia , Simportadores , Xenopus laevisRESUMEN
We have used expression in Xenopus oocytes to characterize a new hexose transporter from the parasitic protozoan Leishmania donovani. This transporter utilizes the hexoses glucose, fructose, and mannose as substrates. A substrate saturation curve for 2-deoxy-D-glucose reveals a very high Km, estimated to be approximately 150 mM. Immunolocalization of the protein with an antibody directed against the COOH terminus indicates that the transporter is present primarily in the parasite plasma membrane but is not detectable in the flagellar membrane. Since this protein is expressed in the insect stage promastigotes but not in the intracellular amastigotes, it may be specialized to function following an insect sugar meal when the concentrations of sugars surrounding the parasite are high.
Asunto(s)
Leishmania donovani/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Animales , Transporte Biológico Activo/efectos de los fármacos , Carbohidratos/farmacología , Desoxiglucosa/metabolismo , Femenino , Expresión Génica , Genes Protozoarios , Inmunohistoquímica , Técnicas In Vitro , Cinética , Leishmania donovani/genética , Leishmania donovani/crecimiento & desarrollo , Microscopía Inmunoelectrónica , Proteínas de Transporte de Monosacáridos/antagonistas & inhibidores , Proteínas de Transporte de Monosacáridos/genética , Nucleósidos/farmacología , Oocitos/metabolismo , Fracciones Subcelulares/metabolismo , XenopusRESUMEN
The malaria parasite Plasmodium falciparum has two extrachromosomal DNAs, a 6 kb reiterated element which appears to be the mitochondrial DNA and a 35 kb circular DNA of unknown function. Examination of relative steady-state transcript abundance during parasite development in the erythrocyte shows that transcripts of 6 kb element protein-coding genes are least abundant in the ring and early trophozoite stages and most abundant in late trophozoites and schizonts, while transcripts from the RNA polymerase subunits of the 35 kb DNA, also least abundant in ring stage, are relatively similar in abundance in succeeding stages. The fragmented rRNAs of the 6 kb element appear to be constitutively abundant except for an increase in the schizont stage, while rRNAs from the 35 kb DNA are least abundant in early trophozoites and most abundant in schizonts. Thus the relative abundance of organelle transcripts alters during the erythrocytic portion of the P. falciparum developmental cycle. These alterations may reflect the relative importance of the roles played by organelle gene products in different life cycle stages.