Cl gene cluster encoding several small nucleolar RNAs: a comparison amongst trypanosomatids

Small nucleolar RNAs (snoRNAs) are small non-coding RNAs that modify RNA molecules such as rRNA and snRNA by guiding 2'-O-ribose methylation (C/D box snoRNA family) and pseudouridylation reactions (H/ACA snoRNA family). H/ACA snoRNAs are also involved in trans-splicing in trypanosomatids. The aims of this work were to characterise the Cl gene cluster that encodes several snoRNAs in Trypanosoma rangeli and compare it with clusters from Trypanosoma cruzi, Trypanosoma brucei, Leishmania major, Leishmania infantum, Leishmania braziliensis and Leptomonas collosoma. The T. rangeli Cl gene cluster is an 801 base pair (bp) repeat sequence that encodes three C/D (Cl1, Cl2 and Cl4) and three H/ACA (Cl3, Cl5 and Cl6) snoRNAs. In contrast to T. brucei, the Cl3 and Cl5 homologues have not been annotated in the Leishmania or T. cruzi genome projects (http//:www.genedb.org). Of note, snoRNA transcribed regions have a high degree of sequence identity among all species and share gene synteny. Collectively, these findings suggest that the Cl cluster could constitute an interesting target for therapeutic (gene silencing) or diagnostic intervention strategies (PCR-derived tools).

Detection of Trypanosoma cruzi and Trypanosoma rangeli infection in triatomine vectors by amplification of the histone H2A/SIRE and the sno-RNA-C11 genes

Trypanosoma rangeli is non pathogenic for humans but of important medical and epidemiological interest because it shares vertebrate hosts, insect vectors, reservoirs and geographic areas with T. cruzi, the etiological agent of Chagas disease. Therefore, in this work, we set up two PCR reactions, TcH2AF/R and TrFR2, to distinguish T. cruzi from T. rangeli in mixed infections of vectors based on amplification of the histone H2A/SIRE and the small nucleolar RNA Cl1 genes, respectively. Both PCRs were able to appropriately detect all T. cruzi or T. rangeli experimentally infected-triatomines, as well as the S35/S36 PCR which amplifies the variable region of minicircle kDNA of T. cruzi. In mixed infections, whereas T. cruzi DNA was amplified in 100 percent of samples with TcH2AF/R and S35/S36 PCRs, T. rangeli was detected in 71 percent with TrF/R2 and in 6 percent with S35/S36. In a group of Rhodnius colombiensis collected from Coyaima (Colombia), T. cruzi was identified in 100 percent with both PCRs and T. rangeli in 14 percent with TrF/R2 and 10 percent with S35/S36 PCR. These results show that TcH2AF/R and TrF/R2 PCRs which are capable of recognizing all T. cruzi and T. rangeli strains and lineages could be useful for diagnosis as well as for epidemiological field studies of T. cruzi and T. rangeli vector infections.

Embora o Trypanosoma rangeli não seja patogênico para o homem, sua importância médica e epidemiológica reside no fato de compartilhar vetores, reservatórios e áreas geográficas com o Trypanosoma cruzi, agente causal da Doença de Chagas. Neste estudo, para distinguir T. cruzi de T. rangeli em vetores com infecções mistas, se utilizaram duas amplificações de PCR; TcH2AF/R para o gen da histona H2A/SIRE e TrFR2, para um gen repetitivo de ARN nucleolar Cl1 (sno-RNA-Cl1). Assim como a PCR S35/S36, ambas as reações foram capazes de detectar corretamente a presença de T. cruzi ou T. rangeli em triatomíneos infectados experimentalmente. Nas infecções mistas, o ADN de T. cruzi foi amplificado em 100 por cento das amostras quando se utilizaram TcH2AF/R e S35/S36, enquanto T. rangeli foi detectado em 71 por cento delas com os iniciadores TrF/R2 e em 6 por cento, com S35/S36. Adicionalmente, em um grupo de Rhodnius colombiensis coletados na região de Coyaima (Tolima), T. cruzi foi identificado em 100 por cento com ambas PCRs e T. rangeli em 14 por cento delas com os iniciadores TrF/R2 e em 10 por cento, com S35/S36. Estes resultados mostram que as reações de PCR TcH2AF/R e TrF/R2, capazes de reconhecer todas as cepas e linhagens de T. cruzi e T. rangeli, podem ser úteis no diagnóstico e também nos estudos epidemiológicos do campo com vetores infectados pelo T. cruzi e T. rangeli.

Genomic organization of nucleolin gene in carp fish: Evidence for several genes

The protein nucleolin, functionally involved in the main steps of ribosome biogenesis, is codified by a single copy gene in mammals. Here we report that at least three different genes codify for this protein in carp fish (Cyprinus carpio). This is the first description of the genomic organization of nucleolin in a teleost. The carp nucleolin gene includes 8.8 kb and contains 16 exons. Promoter cis regulatory elements are similar to constitutive genes, i.e., a putative TATA box, three G/C boxes, and three pyrimidine-rich boxes. As in other species, carp nucleolin gene introns host three snoRNA codifying sequences: U23 from the H/ACA family and two C/D box snoRNAs, U20 and U82. Both U20 and U82 span a complementary sequence with carp 18S rRNA. Additionally, we identified two cDNAs coding for nucleolin, confirming the existence of several nucleolin genes in carp. Amino acid-derived sequence from carp cDNAs differ from mammal protein because they span additional acidic domains at the amino end, whose functional significance remains unclear. We performed amino acid sequence comparison and phylogenetic analyses showing that the three isoforms of carp nucleolin, which we describe herein, cluster in two groups. cNUC1 probably diverges from cNUC2 and cNUC3 as result of ancestral fish-specific genome duplication, indeed C. carpio is a tetraploid fish.