RESUMEN
The promoter region of the ribosomal RNA (rRNA) genes of Leishmania amazonensis was characterised and the transcription start point, defined by primer extension, was shown to be a T residue, 1048 nucleotides upstream of the beginning of the 18S sequence. A repetitive element of 60 bp was identified in the intergenic spacer. This element did not show sequence similarity with the region around the transcription start point. Conserved sequences were found in the external transcribed spacer of L. amazonensis, Trypanosoma cruzi and Crithidia fasciculata rRNA genes, 150 nucleotides downstream of the transcription start point. These sequences might be involved in processing events of the rRNA precursor molecule. The general organisation of the gene resembles the pattern observed for the ribosomal cistron in eukaryotic cells. Constructs containing the L. amazonensis promoter region upstream of the chloramphenicol acetyltransferase (cat) gene were able to drive the expression of the reporter gene in transient transfection experiments. CAT expression could be detected even when no trans-splicing acceptor sequence was added to the constructs, although its presence enhanced 5-fold the level of CAT activity. Species-specificity of the RNA polymerase I promoter activity was also demonstrated since constructs containing the L. amazonensis promoter region were unable to drive CAT expression when transfected into the related trypanosomatids, T. cruzi, C. fasciculata and Endotrypanum schaudini.
Asunto(s)
Leishmania mexicana/genética , Regiones Promotoras Genéticas , ARN Protozoario , ARN Ribosómico , Animales , Secuencia de Bases , Cloranfenicol O-Acetiltransferasa/genética , Mapeo Cromosómico , Crithidia fasciculata/genética , Expresión Génica , Datos de Secuencia Molecular , Homología de Secuencia , Activación Transcripcional , Transfección , Trypanosoma cruzi/genéticaRESUMEN
A method for discriminating among Leishmania is described, based upon small subunit ribosomal DNA sequence differences. The method was to amplify the entire 2.2 kb small subunit rDNA by polymerase chain reaction using conserved primers specific for the 5' and 3' termini of the small subunit ribosomal RNA, and then hybridize the product dotted onto nylon membranes with labeled oligonucleotides. The design of the hybridization probes was based upon complete small subunit rDNA sequences from L. amazonensis, L. major and L. guyanensis and partial sequences of L. mexicana, L. braziliensis, L. tropica and L. chagasi. A high degree of sequence similarity (> 99%) among species was found. However, sufficient sequence divergence occurred to permit the design of internal oligonucleotide probes specific for species complexes. This procedure successfully discriminated amongst a wide range of Leishmania isolates. The method detected as few as 10 cultured organisms and detected parasites in tissue samples from experimentally infected animals. Non-radioactive labeling showed the same specificity and sensitivity as radioactive probes.
Asunto(s)
ADN Protozoario/análisis , Leishmania/aislamiento & purificación , Leishmaniasis/diagnóstico , Reacción en Cadena de la Polimerasa/métodos , Animales , Secuencia de Bases , Clonación Molecular , Cricetinae , ADN Ribosómico/análisis , ADN Ribosómico/genética , Electroforesis en Gel de Agar , Humanos , Leishmania/clasificación , Leishmania/genética , Leishmaniasis/parasitología , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Sondas de OligonucleótidosRESUMEN
Fifty-four species or isolates of insect trypanosomatids were examined for the presence of selected restriction enzyme sites in the small (SSU) and large (LSU) rRNA coding units of ribosomal genes. In the SSU, sites for Eco RI, Bgl II, Pst I, and Hind III were found to occur at the same location for all species examined, thus displaying a universal distribution among trypanosomatids. In the LSU, a site for Bgl II in the 24S-alpha sequence and sites for Hind III and Pst I in the 24S-beta sequence were found in all species examined. In contrast, a site for Pvu II in the SSU exhibited a genus-related distribution, being present in Crithidia and Herpetomonas but absent in Phytomonas. A site for Hind III in the 24S-alpha sequence of the LSU also exhibited genus-restricted distribution. The site was present in Crithidia but absent in Phytomonas and Herpetomonas. These findings were confirmed by dot hybridization with a synthetic oligonucleotide complementary to the 18S rRNA sequence containing the Pvu II site. Results point to the usefulness of restriction markers as diagnostic tools for distinguishing the lower trypanosomatid genera Crithidia, Herpetomonas, and Phytomonas at the same time revealing a marked complexity within the genus Leptomonas.
Asunto(s)
ADN Ribosómico/genética , ARN Ribosómico 18S/genética , Trypanosomatina/genética , Amidohidrolasas/genética , Animales , Anticuerpos Monoclonales , Arginasa/genética , Secuencia de Bases , Southern Blotting , Enzimas de Restricción del ADN , ADN Protozoario/genética , Datos de Secuencia Molecular , Sondas de Oligonucleótidos , ARN Ribosómico/genética , Trypanosomatina/clasificaciónRESUMEN
The analysis of PvuII restriction patterns of Leishmania spp. and Trypanosoma spp. genomic DNA showed genus distinctive profiles. A specific PvuII site was detected in the 5' domain of 18S ribosomal DNA of Leishmania. A 20-mer oligonucleotide encompassing this PvuII region was synthesized. This sequence, when utilized as probe, on short exposures of dot tests, detected 10(3) whole promastigotes of all Leishmania species analyzed but did not hybridize with T. cruzi or human nucleic acids. Two other oligonucleotides were synthesized to be used as primers for amplification through polymerase chain reaction of the 18S ribosomal DNA region containing the PvuII site. The probes described may be useful for the detection of Leishmania spp. under clinical and epidemiological trials.