SURVEY OF Eimeria SPP. TRANSCRIPTS USING OPEN READING FRAME ESTS (ORESTES)
J. Novaes1, A.Y. Kashiwabara2, L. Varuzza1; L.T.
Nagao1, A.P.S. Manha1, S. Fernandez1, A.M. Durham2, A. Gruber1 and A.M.B.N.
Madeira1,*
1Faculty of Veterinary Medicine and Zootechny, 2Institute of Mathematics and
Statistics, University of São Paulo, Brazil
*albackx@usp.br
EST sequencing of different developmental stages is a simple approach to unravel the transcriptome of a parasite. ORESTES (ORF ESTs) is a cDNA synthesis method involving low-stringency RT-PCR using arbitrary primers. Since this technique is based upon a high number of amplification cycles, even rare transcripts can be amplified. In addition, the cDNAs are biased towards the central part of the mRNAs, which represents the most informative region of the transcripts. In order to derive data complementary to the currently available Eimeria tenella sequences, we utilized ORESTES to generate cDNAs of different developmental stages of E. tenella, including sporozoites, second-generation merozoites, and unsporulated, sporulating and sporulated oocysts. In addition, for comparative purposes, we have also generated ORESTES reads for two additional and relevant species of chicken Eimeria: E. acervulina and E. maxima. All cDNA clones were sequenced in a 96-capillary DNA sequencer (ABI-3700) and submitted to an automated sequence processing pipeline (EGene system - Durham et al. – Bioinformatics 21: 2812-3, 2005). We have generated a total of 14,025 reads of E. tenella, 16,151 reads of E. acervulina and 13,667 reads of E. maxima. Clustering was performed using CAP3 assembly program. A total of 3,755, 3,310, and 3,051 clusters were obtained for E. tenella, E. acervulina and E. maxima, respectively. The E. tenella ORESTES reads were also clustered with ESTs already available for E. tenella, totaling 42,288 sequences. A total of 7,296 clusters (3,196 contigs and 4,100 singlets) were obtained, a number which is possibly not far from reflecting the transcriptome size of the parasite. EST sequencing of different developmental stages would help to clarify this point. In order to investigate differential gene expression, cluster sequences of distinct developmental stages were submitted to similarity searches against one another using BlastN. Preliminary results show that only a small fraction of the transcripts of any stage are shared by other stages. This finding suggests that any stage expresses only a small set of transcripts that is switched to another small set upon differentiation. We intend to validate this hypothesis through an extensive SAGE analysis of different developmental stages of E. tenella.
Financial support: FAPESP, CNPq, CAPES and Pró-Reitoria de Pesquisa USP