Mónica Olivares

The open reading frame 1 of the L1Tc retrotransposon of Trypanosoma cruzi codes for a protein with apurinic-apyrimidinic nuclease activity.

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. cruzigenome, 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.

The L1Tc non-LTR retrotransposon of Trypanosoma cruzi contains an internal RNA-pol II-dependent promoter that strongly activates gene transcription and generates unspliced transcripts

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 non-LTR (long terminal repeat) retrotransposon L1Tc from Trypanosoma cruzi codes for a protein with RNase H activity.

The deduced amino acid sequence of the region downstream of the reverse transcriptase (RT) motif of theTrypanosoma cruzi L1Tc non-LTR retrotransposon shows a significant homology with the sequence coding for proteins with RNase H activity from different organisms and retroelements. The 25-kDa His6-tagged recombinant protein bearing only the L1Tc RNase H domain, named RHL1Tc, exhibits RNase H activity as measured on the [3H]poly(rA)/poly(dT) hybrid used as substrate as well as on specific homologous and heterologous [32P]RNA/DNA hybrids. The mutation of the conserved aspartic acid at position 39 of the enzyme catalytic site, but not of the serine at position 56 (non-conservative amino acid), abolishes protein RNase H activity. The RNase H activity of the RHL1Tc protein is Mg2+-dependent, and it is also active in the presence of the Mn2+ 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.

The L1Tc, long interspersed nucleotide element from Trypanosoma cruzi, encodes a protein with 3'-phosphatase and 3'-phosphodiesterase enzymatic activities.

The presence of a long interspersed nucleotide element, named L1Tc, which is actively transcribed in the parasiteTrypanosoma cruzi, has been recently described. The open reading frame 1 of this element encodes the NL1Tc protein, which has apurinic/apyrimidinic endonuclease activity and is probably implicated in the first stage of the transposition of the element. In the present paper we show that NL1Tc effectively removes 3′-blocking groups (3′-phosphate and 3′-phosphoglycolate) from damaged DNA substrates. Thus, both 3′-phosphatase and 3′-phosphodiesterase activities are present in NL1Tc. We propose that these enzymatic activities would allow the 3′-blocking ends to function as targets for the insertion of L1Tc element, in addition to the apurinic/apyrimidinic sites previously described. The potential biological function of the NL1Tc protein has also been evidenced by its ability to repair the DNA damage induced by the methyl methanesulfonate alkylating or oxidative agents such as hydrogen peroxide and t-butyl hydroperoxide inEscherichia coli (xth andxth, nfo) mutants.

Genomic clustering of the Trypanosoma cruzi nonlong terminal L1Tc retrotransposon with defined interspersed repeated DNA elements

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 3200 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.

Characterization of reverse transcriptase activity of the L1Tc retroelement from Trypanosoma cruzi

The recombinant protein RTL1Tc, encoded by the non-LTR (long terminal repeat) retrotransposon L1Tc from Trypanosoma cruzi, has been shown to have reverse transcriptase (RT) activity using poly(rA)/oligo(dT) and poly(rC)/oligo(dG) homopolymers as template/primers. The optimal RT activity was detected at a concentration of 5 mM Mg2+, pH 8 and between 28 and 37%°C. Site-directed mutagenesis in the RT catalytic site proved that substitution of aspartic acid 313 for isoleucine (RTD313IL1Tc) practically abolishes the RT activity of the RTL1Tc protein. RT-polymerase chain reaction assays revealed that the RTL1Tc protein has the ability to use both homologous and heterologous RNA templates. Also, it is shown that the RTL1Tc protein is capable of synthesizing complementary DNA molecules by consecutive switching of the oligo molecule, which the protein uses as a template. This template switching may be involved in the retroelement integration process.

The endonuclease NL1Tc encoded by the LINE L1Tc from Trypanosoma cruzi protects parasites from daunorubicin DNA damage

In the present paper we show that the overexpression of the NL1Tc protein, encoded by the L1Tc non-LTR retrotransposon from Trypanosoma cruzi, led to a reduction of about 60% of DNA damage caused by daunorubicin treatment. This repair effect is not observed in transfected parasites overexpressing the NL1Tc mutated in the aspartic acid located in the active site of the enzyme. In addition, NL1Tc overexpression protects the parasite from the negative effect that daunorubicin has on parasites growth rate. Thus, parasites overexpressing NL1Tc show, after treatment with 4 microM of daunorubicin, growth rate two to three times higher than the growth rate observed in treated control parasites transformed with the empty vector or overexpressing the mutated NL1Tc. Likewise, parasites overexpressing the NL1Tc protein and irradiated with a single dose of gamma-radiation (6 or 9 Gy) show higher growth rates than the parasites overexpressing the mutated NL1Tc or the control transfected parasites.

The effect of temperature on the stability of the synthetic SPf(66)n malaria vaccine.

We hereby report on the effect of temperature on the physico-chemical and immunogenic characteristics and stability of the SPf(66)n synthetic malaria vaccine. The pattern of polymerization, the adsorption to aluminium hydroxide and the immunological properties of these vaccine groups were not significantly affected.