RESUMEN
Two Toxoplasma gondii gene fragments, which encode polypeptides that can be used as diagnostic antigens in an enzyme-linked immunosorbent assay were cloned and their nucleotide sequence was determined. One of the fragments (derived from the H4 gene) is 682 bp long. The mRNA of the single-copy H4 gene is 1300 nt long. The fragment derived from the other gene, termed H11, is 197 bp long. The mRNA of the single-copy H11 gene is 1900 nt long. The native polypeptides encoded by the H4 and H11 genes are 25 and 41 kDa, respectively. Based on computer analysis of the deduced amino acid sequences of the polypeptides encoded by the gene fragments, both appear to be very hydrophilic and that encoded by the H11 fragment has a high antigenic index profile. These results are consistent with the diagnostic usefulness of the polypeptides encoded by the gene fragments.
Asunto(s)
Antígenos de Protozoos/genética , Genes , Toxoplasma/genética , Secuencia de Aminoácidos , Animales , Antígenos de Protozoos/aislamiento & purificación , Secuencia de Bases , Northern Blotting , Western Blotting , Clonación Molecular , ADN Protozoario/análisis , ADN Protozoario/genética , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Datos de Secuencia Molecular , Embarazo , ARN Mensajero/genética , ARN Mensajero/aislamiento & purificación , Proteínas Recombinantes de Fusión/aislamiento & purificación , Toxoplasma/inmunología , Toxoplasmosis/diagnóstico , Toxoplasmosis Animal/diagnósticoRESUMEN
Toxoplasma gondii expresses high levels of an active nucleoside triphosphate hydrolase (NTPase; EC 3.6.1.3) with several unique properties. It has been detected as a circulating antigen in mice, making it an ideal candidate for diagnostic tests for toxoplasmosis. A cDNA library constructed from T. gondii poly(A)+RNA was made in lambda gt11. One hundred thousand members of this library were immunoscreened with a rabbit polyclonal antibody to the purified NTPase. Six positive clones were subcloned into the plasmid, pGEX-IN, and the inserts were restriction-mapped. All clones had identical partial restriction enzyme maps. One insert was subcloned into M13mp18 and sequencing by the deoxy/dideoxy method showed an NTPase-encoding gene (ntp) fragment of 571 bp. The insert was also purified, radiolabelled, and used to hybridize to Northern blots of tachyzoite RNA and quantitative Southern blots of tachyzoite DNA. Northern blotting revealed that the NTPase mRNA was in great abundance and had a length of about 2800 nucleotides. Southern blotting showed a gene copy number of between one and five, and the possibility that ntp is tandemly repeated over a large length of DNA. The NTPase was expressed as a glutathione S-transferase (EC 2.5.1.18) fusion protein of about 50 kDa, which reacted with polyclonal rabbit antibody on Western blotting.
Asunto(s)
Antígenos de Protozoos/genética , Genes , Monoéster Fosfórico Hidrolasas/genética , Toxoplasma/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Escherichia coli/genética , Expresión Génica , Immunoblotting , Datos de Secuencia Molecular , Nucleósido-Trifosfatasa , Poli A/genética , ARN/genética , ARN Mensajero , Mapeo RestrictivoRESUMEN
RNA was extracted from purified tachyzoites of Toxoplasma gondii (RH strain) by sequential centrifugation in guanidine hydrochloride, urea, and lithium chloride. The subunits of the RNA were characterized by denaturing and non-denaturing electrophoresis in agarose gels. Poly(A)+-RNA, purified by oligo(dT)-cellulose affinity chromatography, was translated in a rabbit reticulocyte lysate assay and the products were immunoprecipitated with an experimentally infected mouse serum and a naturally infected human serum. After sodium dodecyl sulphate-polyacrylamide gel electrophoresis, fluorography of the polypeptides confirmed that the mRNA translated specific parasite antigens.
Asunto(s)
Toxoplasma/genética , Animales , Antígenos de Protozoos/genética , Peso Molecular , Biosíntesis de Proteínas , ARN/aislamiento & purificación , ARN Mensajero/genética , ARN Mensajero/aislamiento & purificaciónRESUMEN
A method for obtaining a large proportion of the nucleotide sequence of the small subunit ribosomal RNA (srRNA) was applied to the obligate intracellular protozoon Toxoplasma gondii. The method uses reverse transcription of as little as 8 micrograms of total cellular RNA. This fast, efficient method has numerous advantages over traditional gene cloning methods when nucleotide sequences are required for evolutionary studies. A phylogenetic analysis of the srRNA sequence data showed that T. gondii is not especially closely related to any other organism for which srRNA sequences are available, including another member of the Apicomplexa.
Asunto(s)
ARN Ribosómico/genética , Toxoplasma/genética , Animales , Secuencia de Bases , Datos de Secuencia Molecular , Filogenia , Plasmodium berghei/clasificación , Plasmodium berghei/genética , Toxoplasma/clasificaciónRESUMEN
Reverse transcription of total cellular RNA was used to obtain the partial nucleotide sequence of the small subunit ribosomal RNA (srRNA) of Sarcocystis gigantea. The sequence was compared with the homologous sequences of 24 other eukaryotes. Phylogenetic analysis of the semiconserved regions by 4 different tree-building methods using bacteria as an outgroup all concur in showing monophyly of Sarcocystis gigantea and Toxoplasma gondii to the exclusion of all other taxa for which homologous sequences are available.
Asunto(s)
Filogenia , ARN Ribosómico , Sarcocystis/clasificación , Animales , Secuencia de Bases , Datos de Secuencia Molecular , Oligonucleótidos , Sarcocystis/genética , Homología de Secuencia de Ácido NucleicoRESUMEN
A mung bean nuclease genomic library of purified DNA from tachyzoites of the RH strain of Toxoplasma gondii was prepared in the bacteriophage lambda gtll and recombinants containing rRNA gene fragments were detected by hybridization with radiolabeled total RNA from the closely related coccidian Eimeria acervulina. Ten recombinants were chosen at random, and five of these were investigated further using probes for the genes of the large and small rRNA of Plasmodium berghei. An insert (called TG4) that hybridized only to the 3' end of the large rRNA coding region of P. berghei and an insert (called TG18) that hybridized only to the small rRNA coding region of P. berghei were purified by electrophoresis in low melting point agarose. Radiolabeled E. acervulina total RNA, TG4, and TG18, were then used to compare the sizes of the large and small rRNA gene fragments after DNA extracted from three strains of T. gondii, and the type strain of the closely related coccidian Hammondia hammondi were cut by one of a series of 10 restriction endonucleases. The patterns obtained for the three T. gondii isolates were identical to those obtained for H. hammondi, for each enzyme tested. In addition, the guanine plus cytosine (G + C) content of H. hammondi DNA was found to be almost identical to that obtained previously for T. gondii DNA.
Asunto(s)
Coccidios/genética , ADN/análisis , ARN Ribosómico/genética , Toxoplasma/genética , Animales , Composición de Base , Clonación Molecular , Enzimas de Restricción del ADN , ADN Recombinante , Hibridación de Ácido Nucleico , Plasmodium berghei/genéticaRESUMEN
A rapid RNA sequencing technique was used to partially sequence the small-subunit ribosomal RNA (srRNA) of four species of the amoeboid genus Naegleria. The extent of nucleotide sequence divergence between the two most divergent species was roughly similar to that found between mammals and frogs. However, the pattern of variation among the Naegleria species was quite different from that found for those species of tetrapods characterized to date. A phylogenetic analysis of the consensus Naegleria sequence showed that Naegleria was not monophyletic with either Acanthamoeba castellanii or Dictyostelium discoideum, two other amoebas for which sequences were available. It was shown that the semiconserved regions of the srRNA molecule evolve in a clocklike fashion and that the clock is time dependent rather than generation dependent.