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The characterization of expressed sequence tags (ESTs) generated from a cDNA library of Leishmania (Leishmania) amazonensis amastigotes is described. The ) major. The characterization of these ESTs is suitable for the genome physical mapping, as well as for the identification of genes encoding cysteine proteinases implicated with protective immune responses in leishmaniasis.Key words: Leishmania (Leishmania) amazonensis -amastigotes -cDNA library Sequencing of cDNA libraries to generate expressed sequence tags (ESTs) has been used for gene identification in Plasmodium falciparum (Chakrabarti et al. 1994), Trypanosoma brucei (El Sayed et al. 1995), Trypanosoma cruzi (Brandão et al. 1997, Verdún et al. 1998, and Leishmania (L.) major (Levick et al. 1996). The L. (L.) major genome project provided the sequence of the 36 chromosomes of the 32.8-megabase haploid genome of the parasite, predicting 911 RNA genes, 39 pseudogenes, and 8272 protein-coding genes (Ivens et al. 2005). In contrast, the genome of L. (L.) amazonensis, a causative agent of human cutaneous leishmaniasis in Brazil, is poorly studied and only 107 of its genes have been reported (http://ncbi.nlm.nih.gov). The availability of a cDNA library from amastigote forms of Leishmania can provide the characterization of genes implicated in parasite survival and host parasite interactions, opening perspectives for the development of new tools for disease control. The aim of the present work was the analysis of ESTs generated from a cDNA library of L. (L.) amazonensis amastigotes and their mapping in chromosomal bands.In our laboratory we constructed a cDNA library from the amastigote form of L. (L.) amazonensis. It is important to emphasize that the amastigotes used for RNA extraction were derived from hamster foot lesions. This provides a measure of confidence of our cDNA library, since it was demonstrated that there is an increase in the magnitude of the transcript levels in Leishmania amastigotes proceeding from axenic cultures (Holzer et al. 2006). We obtained 40,000 clones and 100 were randomly selected and sequenced generating new L. (L.) amazonensis ESTs with an average insert size of 1.3 kb. These ESTs were sequenced from the 5' and 3' ends with T7 primer (forward) and SP6 primer (reverse) originating 147 sequences with an average size of 458 bp. These ESTs were compared to database sequences using the BLAST program. Sequence homologies identified by BLAST programs were considered statiscally significant with a Poisson P value of ≤ 10 -4 . Among 100 sequences, six matched with rRNA and one with hamster gene were excluded from further analysis. From a total of 93 EST sequences, 63 (68%) showed significant identity to gene sequences available on databases, whereas 30 ESTs (32%) did not have significant matches in databases and therefore were classified as undentified. Genes from other Leishmania species were matched to 64.5% of the ESTs, 2.1% matched to L. (L.) amazonensis sequences, and 1.1% matched to genes from other organisms. Considering both t...

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