Molecular & Biochemical Parasitology 126 (2003) 113–117 Short communication Cloning and characterization of a gene encoding a putative protein associated with U3 small nucleolar ribonucleoprotein in Trypanosoma cruzi  Stenio P. Fragoso a,b , Claire Plazanet-Menut b , Monica A.C. Carreira b , Maria Cristina Motta c , Bruno Dallagiovana a , Marco A. Krieger a,b , Samuel Goldenberg a,b,∗ a Instituto de Biologia Molecular do Paraná, Rua Professor Algacyr Munhoz Mader 3775, Curitiba 81350-010, PR, Brazil b Fiocruz, IOC, Av. Brasil 4365, Rio de Janeiro, RJ, Brazil c Instituto de Biof´ ısica Carlos Chagas Filho, UFRJ, Rio de Janeiro, RJ, Brazil Received 7 August 2002; received in revised form 7 October 2002; accepted 7 October 2002 Keywords: Trypanosoma cruzi; Metacyclogenesis; Nucleolar protein; Transcription; Stage-specific gene The protozoan parasite Trypanosoma cruzi is the causal agent of American trypanosomiasis or Chagas disease, which afflicts millions of people in Central and South America. Its life cycle involves at least three distinct de- velopmental stages: epimastigotes, trypomastigotes and amastigotes [1]. The epimastigote forms replicate in the midgut of the insect host and develop into non-replicative metacyclic trypomastigote forms by the process of metacy- clogenesis. Metacyclic trypomastigotes are released in the excreta of insects of the Reduviidae family (triatomine in- sects) during feeding and invade the cells of the mammalian host. Within the host cells, the parasite differentiates into the replicative amastigote form, which in turn differentiates into bloodstream trypomastigotes, which infect new cells. Unlike higher eukaryotic cells, T. cruzi regulates its gene expression predominantly by a post-transcriptional pro- cess, involving either the processing of long polycistronic transcripts by trans-splicing and poly-A tail addition or mechanisms based on stage-specific changes in mRNA stability and translation. We used the representation of dif- ferential expression (RDE) technique [2] to try to isolate genes involved in the differentiation of this unicellular or- ganism. With this technique, we were able to clone several stage-specific genes of the parasite [3,4]. A T. cruzi Dm28c epimastigote culture was allowed to differentiate in vitro under chemically defined conditions  Note: Nucleotide sequence data reported in this paper is available in the GenBank database under the accession number AF092910. ∗ Corresponding author. Tel.: +55-41-316-3230; fax: +55-41-316-3267. E-mail address: sgoldenb@tecpar.br (S. Goldenberg). as previously described [10] and the RDE method was used to isolate genes differentially expressed during meta- cyclogenesis. Full-length cDNAs were obtained from the polysomal RNA fractions of epimastigote cultures (Log epi) and 24 h-adherent (differentiating) parasites by RT-PCR, using a primer corresponding to the last 22 nucleotides of the mini-exon sequence and the other complementary to the poly-A tail. We then carried out subtractive hybridiza- tion with the Log epi cDNAs, as previously described [2] to eliminate cDNAs from differentiating parasites. Three rounds of subtraction were performed and the final products were cloned in pBluescript KS+. We obtained 13 clones of different sizes (0.6–1.1kb). We analyzed the expression of each clone individually by slot blot, using it to probe polyso- mal RNA from 24 h-adherent parasites (24Ad-polysomal RNA) and Log epi polysomal RNA. We found that 30% of the clones hybridized with both RNA populations. A BLASTX search showed that the amino acid sequence of the putative protein encoded by a clone that hybridized with both mRNA populations displayed significant similar- ity (64%) to the yeast protein Imp4p, a specific component of the U3 snoRNP required for pre-18S rRNA process- ing. We named this gene TcImp4 and decided to study its expression pattern more closely. The TcImp4 protein also displayed a high level of sequence identity with putative U3 small nucleolar proteins of Arabidopsis thaliana (69%) and Caenorhabditis elegans (66%). The RDE clone was used to screen an EMBL3 genomic library of T. cruzi Dm28c, to analyze the organization of the gene in the T. cruzi genome. An 861 nt open reading frame was identified in the sequence which encodes a basic 0166-6851/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII:S0166-6851(02)00247-5