RESUMEN
We previously purified a Crithidia fasciculata mitochondrial DNA polymerase that has unusual properties. Unlike a conventional mitochondrial DNA polymerase gamma, this enzyme is small, non-processive, deficient in 3'-exonuclease activity, and error prone (Torri, A. F., Kunkel, T. A., and Englund, P. T. (1994) J. Biol. Chem. 269, 8165-8171). In all of these characteristics, the enzyme resembles DNA polymerase beta, a nuclear enzyme thought to be involved in DNA repair. We have now cloned and sequenced the gene for this enzyme. The mitochondrial polymerase has significant homology, about 33% identity at the amino acid level, with human DNA polymerase beta. However, sequence analysis of the clone revealed the presence of a cleaved N-terminal presequence, presumably a mitochondrial import signal, which resembles presequences on other C. fasciculata mitochondrial proteins. The polymerase's function may be to repair the many gaps in newly replicated kinetoplast (mitochondrial) DNA minicircles in this parasite. This enzyme is the first example of a mitochondrial DNA polymerase beta.
Asunto(s)
Crithidia fasciculata/enzimología , ADN Polimerasa I/genética , Mitocondrias/enzimología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN Protozoario , Humanos , Datos de Secuencia Molecular , Homología de Secuencia de AminoácidoRESUMEN
Kinetoplast DNA, the mitochondrial DNA of trypanosomatid parasites, is a network containing several thousand minicircles and a few dozen maxicircles. We compared kinetoplast DNA replication in Trypanosoma brucei and Crithidia fasciculata using fluorescence in situ hybridization and electron microscopy of isolated networks. One difference is in the location of maxicircles in situ. In C. fasciculata, maxicircles are concentrated in discrete foci embedded in the kinetoplast disk; during replication the foci increase in number but remain scattered throughout the disk. In contrast, T. brucei maxicircles generally fill the entire disk. Unlike those in C. fasciculata, T. brucei maxicircles become highly concentrated in the central region of the kinetoplast after replication; then during segregation they redistribute throughout the daughter kinetoplasts. T. brucei and C. fasciculata also differ in the pattern of attachment of newly synthesized minicircles to the network. In C. fasciculata it was known that minicircles are attached at two antipodal sites but subsequently are found uniformly distributed around the network periphery, possibly due to a relative movement of the kinetoplast disk and two protein complexes responsible for minicircle synthesis and attachment. In T. brucei, minicircles appear to be attached at two antipodal sites but then remain concentrated in these two regions. Therefore, the relative movement of the kinetoplast and the two protein complexes may not occur in T. brucei.
Asunto(s)
Crithidia/genética , Replicación del ADN , ADN de Cinetoplasto/biosíntesis , Trypanosoma brucei brucei/genética , Animales , ADN de Cinetoplasto/genética , Hibridación Fluorescente in Situ , Microscopía Electrónica , Trypanosoma brucei brucei/ultraestructuraRESUMEN
The mitochondrial DNA in Crithidia fasciculata, a trypanosomatid parasite, is known as kinetoplast DNA. Kinetoplast DNA has a very unusual structure, consisting of several thousand minicircles and a few dozen maxicircles, all topologically interlocked into a giant network. There is one network within each cell's single mitochondrion. We previously purified a 43-kDa DNA polymerase from C. fasciculata mitochondria (Torri, A. F., and Englund, P. T. (1992) J. Biol. Chem. 267, 4786-4792). This enzyme has properties very different from those of a DNA polymerase gamma, the conventional mitochondrial polymerase. In addition to its small size, it is nonprocessive, has no detectable exonuclease activity, and has very low fidelity. In all of these respects, the polymerase resembles a DNA polymerase beta, a gap-filling enzyme thought to function in DNA repair in the nucleus of other eukaryotes. We speculate that this enzyme may have been specially imported into the C. fasciculata mitochondrion to repair the many gaps found in minicircles following their replication. This is the first example of a beta-like polymerase from the mitochondrion of any eukaryote.
Asunto(s)
Crithidia fasciculata/enzimología , ADN Polimerasa I/aislamiento & purificación , ADN Polimerasa I/metabolismo , Endodesoxirribonucleasas/metabolismo , Mitocondrias/enzimología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN Polimerasa I/biosíntesis , Cartilla de ADN/metabolismo , Replicación del ADN , ADN de Cinetoplasto/biosíntesis , ADN de Cinetoplasto/metabolismo , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/metabolismo , Mutación Puntual , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Eliminación de Secuencia , Especificidad por Sustrato , Moldes GenéticosRESUMEN
MCP72 is a mitochondrial hsp70 protein from the trypanosomatid Crithidia fasciculata. An MCP72 cDNA clone was isolated from a C. fasciculata cDNA library by screening with antiserum specific for the homologous protein of Trypanosoma cruzi [9]. The MCP72 cDNA encodes a polypeptide of 663 amino acids which is 84% identical to the Trypanosoma cruzi protein and 56% identical to the Escherichia coli hsp70 protein DnaK. MCP72 is less similar to other hsp70 proteins. Native MCP72 was purified to homogeneity by ATP-agarose affinity chromatography. Comparison of its N-terminal amino acid sequence with that deduced from the cDNA sequence shows that 20 amino acid residues had been cleaved from the N-terminus; this sequence probably represents a mitochondrial import signal which is cleaved during translocation into the mitochondrion. Fluorescence microscopy, using antibodies specific for MCP72, indicates that the protein is concentrated in a region of the mitochondrial matrix which surrounds the kinetoplast.
Asunto(s)
Crithidia fasciculata/metabolismo , Proteínas de Choque Térmico/biosíntesis , Mitocondrias/metabolismo , Adenosina Trifosfatasas/biosíntesis , Adenosina Trifosfatasas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Crithidia fasciculata/genética , ADN Protozoario/genética , ADN Protozoario/metabolismo , Biblioteca de Genes , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Datos de Secuencia Molecular , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Mapeo Restrictivo , Homología de Secuencia de Aminoácido , Trypanosoma cruzi/genética , Trypanosoma cruzi/metabolismoRESUMEN
The expression of cytochrome c is developmentally regulated during the life cycle of Trypanosoma brucei. The level of regulation appears to be post-transcriptional since cytochrome c mRNA is present in all life stages of the parasite. We have used RNA from each life stage to prime in vitro translation systems and found that the cytochrome c mRNAs are equally translatable. Continuous labeling experiments conducted in vivo indicate that cytochrome c is synthesized at similar rates in both bloodstream and procyclic trypanosomes. Western blots, however, confirm that steady-state levels of cytochrome c are severely depressed in bloodstream forms. In a series of pulse/chase experiments we demonstrate that the half-life of cytochrome c is approximately 1 h in the bloodstream form and no detectable turnover occurred in the procyclic form. We conclude that a major step in the developmental regulation of cytochrome c expression in T. brucei occurs post-translationally due to rapid turnover of the protein in the bloodstream trypanosomes.
Asunto(s)
Grupo Citocromo c/metabolismo , Proteínas Protozoarias/metabolismo , Trypanosoma brucei brucei/metabolismo , Animales , Secuencia de Bases , Grupo Citocromo c/genética , ADN Protozoario/genética , Datos de Secuencia Molecular , Biosíntesis de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Protozoario/genética , ARN Protozoario/metabolismo , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/crecimiento & desarrolloRESUMEN
Kinetoplast DNA is a network of interlocked minicircles and maxicircles. In situ hybridization, using probes detected by digital fluorescence microscopy, has clarified the in vivo structure and replication mechanism of the network. The probe recognizes only nicked minicircles. Hybridization reveals prereplication kinetoplasts (with closed minicircles), donut-shaped replicating kinetoplasts (with nicked minicircles on the periphery and closed minicircles in the center), and postreplication kinetoplasts (with nicked minicircles). Replicating kinetoplasts are associated with two peripheral structures containing free minicircle replication intermediates and DNA polymerase. Replication may involve release of closed minicircles from the center of the kinetoplast and their migration to the peripheral structures, replication of the free minicircles therein, and then peripheral reattachment of the progeny minicircles to the kinetoplast.
Asunto(s)
Crithidia fasciculata/química , Replicación del ADN , ADN Circular/análisis , ADN Protozoario/análisis , Mitocondrias/química , Animales , ADN de Cinetoplasto , Endopeptidasa K , Conformación Molecular , Serina EndopeptidasasRESUMEN
The steady-state levels of the mitochondrial ribosomal RNAs of Trypanosoma brucei are repressed in the early bloodstream developmental stage of the parasite and accumulate approximately 30-fold during differentiation to the stage found in the midgut of the insect vector. In order to determine the mechanism regulating this developmental process, we have examined the transcription and processing of the 9S and 12S mitochondrial rRNAs of T. brucei. A short-lived RNA was detected in pulse labeling experiments which contains the mature 12S and 9S rRNAs and at least 1200 nucleotides of RNA transcribed from upstream of the 12S rRNA gene. This putative processing precursor RNA was identified in both intact cells and in run-on experiments using isolated mitochondria. The transcripts containing the upstream sequences are unstable and reach isotopic equilibrium within 15 min. Mature rRNAs in the insect developmental stage are stable and show no detectable turnover during a 36-h chase. Comparison of rRNA synthesis in bloodstream and insect life-stages indicates that mitochondrial rRNA levels are controlled not at the transcriptional level, but rather by a mechanism which likely modulates the stability of the mature rRNAs. These results suggest that a short-lived rRNA precursor is synthesized and processed at comparable rates in both bloodstream and insect stages of the parasite. Thus, it appears that differential stability of the mature 9S and 12S rRNAs plays a major role in modulating mitochondrial gene expression during the developmental cycle of T. brucei.
Asunto(s)
ARN Protozoario/biosíntesis , ARN Ribosómico/biosíntesis , ARN/biosíntesis , Trypanosoma brucei brucei/genética , Animales , Procesamiento Postranscripcional del ARN , ARN Mitocondrial , Transcripción Genética/fisiología , Trypanosoma brucei brucei/crecimiento & desarrolloRESUMEN
The mitochondrial DNA polymerase from Crithidia fasciculata has been purified to near homogeneity. SDS-PAGE analysis of the purified enzyme reveals a single polypeptide with a molecular weight of approximately 43,000. The protein is basic, with an isoelectric point between 7.6-8.0. Its Stokes radius of 22 A and its sedimentation coefficient of 4.1 S suggest a native molecular weight of 38,000, indicating that the protein is a monomer under our experimental conditions. Western blots and immunoprecipitations of crude extracts reveal a cross-reacting protein of 48 kDa, suggesting that the purified enzyme may be an enzymatically active proteolytic product. The mitochondrial origin of the polymerase was confirmed by cell fractionation. Our results indicate that the C. fasciculata enzyme may be among the smallest known mitochondrial polymerases.
Asunto(s)
Crithidia fasciculata/enzimología , ADN Polimerasa Dirigida por ADN/aislamiento & purificación , Mitocondrias/enzimología , Animales , Secuencia de Bases , Western Blotting , Cromatografía en Gel , Reacciones Cruzadas , ADN Polimerasa Dirigida por ADN/metabolismo , Desoxirribonucleótidos/genética , Electroforesis en Gel de Poliacrilamida , Focalización Isoeléctrica , Datos de Secuencia Molecular , Peso Molecular , Pruebas de PrecipitinaRESUMEN
Trypanosoma brucei brucei is an important pathogen of domestic cattle in sub-Saharan Africa and is closely related to the human sleeping sickness parasites, Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense. However, T. b. brucei is non-infectious to humans. The restriction of the host range of T. b. brucei results from the sensitivity of the parasite to lysis by toxic human high density lipoproteins (HDL) (Rifkin, M. R. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 3450-3454). We show in this report that trypanosome lytic activity is not a universal feature of all human HDL particles but rather that it is associated with a minor subclass of HDL. We have purified the lytic activity about 8,000-fold and have identified and characterized the subspecies of HDL responsible for trypanosome lysis. This class of HDL has a relative molecular weight of 490,000, a buoyant density of 1.21-1.24 g/ml, and a particle diameter of 150-210 A. It contains apolipoproteins AI, AII, CI, CII, and CIII, and monoclonal antibodies against apo-AI and apo-AII inhibit trypanocidal activity. In addition to these common apolipoproteins, the particles also contain at least three unique proteins, as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. Treatment of the particles with dithiothreitol resulted in the disappearance of two of the proteins and abolished trypanocidal activity. Two-dimensional gel electrophoresis showed that these proteins were a disulfide-linked trimer of 45,000, 36,000, and 13,500-Da polypeptides and dimers of the 36,000- and 13,500-Da polypeptides or of 65,000- and 8,500-Da polypeptides. Studies on the lysis of T. b. brucei by the purified particle suggest that the lytic pathway may involve the uptake of the trypanocidal subspecies of HDL by endocytosis.
Asunto(s)
Antiprotozoarios/toxicidad , Lipoproteínas HDL/toxicidad , Trypanosoma brucei brucei/efectos de los fármacos , Animales , Antiprotozoarios/sangre , Antiprotozoarios/aislamiento & purificación , Apolipoproteína A-I , Apolipoproteínas A/sangre , Western Blotting , Centrifugación por Gradiente de Densidad , Humanos , Cinética , Lipoproteínas HDL/sangre , Peso MolecularRESUMEN
We examined the expression of a nucleus-encoded mitochondrial protein, cytochrome c, during the life cycle of Trypanosoma brucei. The bloodstream forms of T. brucei, the long slender and short stumpy trypanosomes, have inactive mitochondria with no detectable cytochrome-mediated respiration. The insect form of T. brucei, the procyclic trypanosomes, has fully functional mitochondria. Cytochrome c is spectrally undetectable in the bloodstream forms of trypanosomes, but during differentiation to the procyclic form, spectrally detected holo-cytochrome c accumulates rapidly. We have purified T. brucei cytochrome c and raised antibodies that react to both holo- and apo-cytochrome c. In addition, we isolated a partial cDNA to trypanosome cytochrome c. An examination of protein expression and steady-state mRNA levels in T. brucei indicated that bloodstream trypanosomes did not express cytochrome c but maintained significant steady-state levels of cytochrome c mRNA. The results suggest that in T. brucei, cytochrome c is developmentally regulated by a posttranscriptional mechanism which prevents either translation or accumulation of cytochrome c in the bloodstream trypanosomes.
Asunto(s)
Grupo Citocromo c/genética , Regulación de la Expresión Génica , Trypanosoma brucei brucei/genética , Secuencia de Aminoácidos , Animales , Apoproteínas/biosíntesis , Secuencia de Bases , Grupo Citocromo c/biosíntesis , Datos de Secuencia Molecular , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transcripción Genética , Trypanosoma brucei brucei/crecimiento & desarrollo , Trypanosoma brucei brucei/metabolismoRESUMEN
The mitochondrial DNA of trypanosomes is organized as a network of catenated circular DNA molecules called the kinetoplast. The minicircles of the kinetoplast are 1 kb circular DNA molecules present at 5,000-10,000 copies per network. The maxicircles are 20 kb circular molecules present at 50-100 copies per network. Maxicircles are transcribed and are thus analogous to mitochondrial DNAs. Here we show that, contrary to previous reports, the minicircles of T. brucei are also transcribed. A minicircle transcript of approximately 240 nucleotides is present in bloodstream and insect developmental stages of the parasite, is enriched in purified mitochondrial preparations, and is efficiently synthesized in vitro. The minicircle cDNA overlaps the conserved region of the T. brucei minicircle and is juxtaposed to a 12 base sequence common to all minicircles. These findings indicate that minicircles, in addition to their previously proposed structural role, are transcribed.
Asunto(s)
ADN Circular/genética , Transcripción Genética , Secuencia de Bases , ADN/genética , ADN de Cinetoplasto , Mitocondrias/metabolismo , Hibridación de Ácido Nucleico , ARN/genética , Trypanosoma brucei brucei/genéticaRESUMEN
The kinetoplast DNA (kDNA) of Trypanosoma equiperdum is organized as a complex structure of catenated circular DNA molecules. The major component of the kDNA network is the one kilobase minicircle that is present at about 10,000 copies per network. We have developed two assays to examine the structure of kDNA networks compacted in vitro with spermidine. Our results suggest that minicircles are arranged into a regular structure with an exposed domain which is DNAase I- and restriction-sensitive and a protected domain which is resistant to restriction endonucleases and DNAase I. This regularly packaged structure is dependent upon spermidine compaction and the circularity of the kDNA, but does not require supercoiled minicircles or catenated networks.