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