No abstract

L1Tc is the best represented autonomous LINE of the Trypanosoma cruzi genome, throughout which several functional copies may exist. In this study, we show that the first 77 bp of L1Tc (Pr77) (also present in the T. cruzi non-autonomous retrotransposon NARTc, in the Trypanosoma brucei RIME/ingi elements, and in the T. cruzi, T. brucei and Leishmania major degenerate L1Tc/ingi-related elements [DIREs]) behave as a promoter element that activates gene transcription. The transcription rate promoted by Pr77 is 10–14-fold higher than that mediated by sequences located upstream from the T. cruzi tandemly repeated genes KMP11 and the GAPDH. The Pr77 promoter-derived mRNAs initiate at nucleotide +1 of L1Tc, are unspliced and translated. L1Tc transcripts show a moderate half life and are RNA pol II dependent. The presence of an internal promoter at the 5′ end of L1Tc favors the production of full-length L1Tc RNAs and reinforces the hypothesis that this mobile element may be naturally autonomous in its transposition.

The deduced amino acid sequence of the open reading frame 1 (ORF1) of the L1Tc non-site-specific non-long terminal repeat retrotransposon of Trypanosoma cruzi exhibits a significant homology with the consensus sequence of the class II family of the endonuclease apurinic-apyrimidinic (AP) proteins. The analysis of the activity of the 40-kDa recombinant protein, named NL1Tc, obtained from the expression of the L1Tc ORF1 in an Escherichia coli "in vitro" expression system revealed that the sequence codes for a protein with endonuclease activity specific for apurinic-apyrimidinic (AP) sites. Data are also presented showing that in vivo expression of the NL1Tc protein conferred viability by complementation to E. coli exonuclease III deletion mutants (BW286 strain). We propose that the biological function of the AP endonuclease activity of the NL1Tc protein may be connected with the introduction into the DNA of free 3 ends that could be used as primers for the integration, along the T. cruzi genome, of the L1Tc element and that the nicking could be a general mechanism for the retrotransposition of non-site-specific non-long terminal repeat retrotransposons. Non-LTR1 retrotransposons represent a particular family of transposable elements that are present in the majority of organisms from mammals to fungi. Although they may be considered as the oldest group of the retroelements present in eukaryotes (1), their biological role is still unknown. These elements are flanked by variable length target site duplications; they lack long terminal repeats (LTRs) and have variable length poly(A) or A-rich 3Ј tails (2). Given the critical functional role that the LTRs play in the retrotransposition of retrovirus and LTR retrotransposons, it is most likely that the non-LTR retrotransposons must use an integration system different from the double-stranded linear DNA integration system proposed for retroviruses and LTR elements (3). The non-LTR integration model, originally proposed by , Finnegan (5), Bucheton (6), and developed by Eickbush (1), postulates that reverse transcription and the second strand synthesis must occur at the chromosomal insertion site using DNA free 3Ј ends as primers. In site-specific non-LTR elements that generate a specific length target site duplication, it is assumed that the free 3Ј ends in the chromosomal DNA are generated by the DNA-binding protein that, in addition, has an associated endonuclease activity (1). In fact, an endonuclease activity has been found to be coded by a site-specific non-LTR element such as the R2 from Bombyx mori (R2Bm) (7) that when expressed in Escherichia coli is able to specifically cleave the 28 S rRNA transcript (8). For integration into the genome, however, of non-site-specific non-LTR retrotransposons, the DNA-binding protein must simply associate with the presence of breaks already existing on the chromosome (1) since it has been assumed that the element does not have any associated endonuclease activity. Recently, we have described a Trypanosoma cruzi non-LTR retrotrans...

We have analyzed the genomic distribution and organization of the long interspersed nucleotide element (LINE) L1Tc, a nonlong terminal repeat (LTR) retrotransposon of Trypanosoma cruzi. The results indicate that the L1Tc element is dispersed along the parasite genome and that in some regions it is organized in tandem repeats. The data allowed us to define the existence of short direct-repeated sequences flanking the genomic L1Tc elements. Relevant is the finding that the LINE L1Tc is located in genomic regions rich in short interspersed nucleotide elements (SINE)-like sequences. In particular, the L1Tc element is found associated to E13-related sequences, redefined in this work and renamed RS13Tc, and to a newly described RS1Tc sequence. The RS1Tc sequence is present, per haploid genome, in about 3,200 copies. Northern blot analysis showed that the RS1Tc is being transcribed into RNAs of different sizes. The analysis of the chromosomal distribution of these elements in various strains of T. cruzi suggested that this type of clustering might be a common feature of the genome of these parasites.

2؉ -dependent, and it is also active in the presence of the Mn 2؉ ion. The optimal condition of RNase H activity is found at pH 8 and 37°C, although it also has significant enzymatic activity at 19°C and pH 6. However, it cannot be excluded that the RNase H activity level and its optimal conditions may be different from that of a protein containing both RT and RNase H domains.