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Eimeria tenella microneme protein EtMIC4: capture of the full-length transcribed sequence and comparison with other microneme proteins

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Abstract

EtMIC4 is a microneme protein of Eimeria tenella, an intracellular apicomplexan protozoan that can cause severe enteritis in chickens. The EtMIC4 gene has been partially characterised, and in this study, we used a combined strategy of rapid amplification of cDNA ends (5′RACE) and reverse transcription-polymerase chain reaction to identify the authentic 5′ end of the transcribed sequence (accession number AJ306453.2). Comparison of the predicted EtMIC4 transcription start site with predicted start sites for EtMIC1, 2 and 3 genes identified comparable initiator regions that each conform to the consensus sequence for a transcriptional initiator element. The EtMIC4 gene is organised over 11 exons and analysis of the full-length predicted protein identified a new N-terminal region that comprises a hydrophobic signal peptide followed by four thrombospondin-type 1 modules that are similar to those previously described further downstream in the protein. Best-fit analysis shows that EtMIC4 shares high homology with the Eimeria maxima protein EmTFP250 and with TgMIC12, a predicted Toxoplasma gondii microneme protein. EtMIC4 and EmTFP250 share 70% amino acid identity and all predicted structural domains are conserved between the two. EtMIC4 and TgMIC12 share 48% identity and they have very similar domain organisation and conservation of intron/exon boundaries.

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Acknowledgements

This research was supported by an IAH studentship to J.P. and BBSRC funding to F.M.T. These experiments were completed within the UK and complied with current UK law.

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Author notes

  1. Javier Periz

    Present address: Nuffield Department of Obstetrics and Gynaecology, University of Oxford, John Radcliffe Hospital, Oxford, OX3 9DU, UK

  2. Rachael Ryan

    Present address: Academic Unit of Pathology, School of Medicine and Biomedical Sciences, University of Sheffield, Beech Hill Rd, Sheffield, S10 2RX, UK

Authors and Affiliations

  1. Institute for Animal Health, Compton, Newbury, Berkshire, RG20 7NN, UK

    Javier Periz, Rachael Ryan, Damer P. Blake & Fiona M. Tomley

Authors
  1. Javier Periz
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  2. Rachael Ryan
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  3. Damer P. Blake
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  4. Fiona M. Tomley
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Corresponding author

Correspondence to Fiona M. Tomley.

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Table S1

Oligonucleotide primers used for 5′RACE of E. tenella microneme transcribed sequences EtMIC1, -2 and -3 (DOC 29.5 KB)

Fig. S1

Strategy used to capture the EtMIC4 5′ cDNA. A putative translation initiation site and signal peptide were identified in the upstream genomic sequence of EtMIC4 using the Signal P server (Nielsen et al. 1997) to screen this region from the database of the E. tenella genome project at http://www.sanger.ac.uk/projects/E_tenella. Forward primers were designed upstream of this putative signal peptide and reverse primers designed downstream of the 5′ end of the previously sequenced EtMIC4 cDNA. A product was successfully amplified by RT-PCR from sporozoite mRNA, cloned, sequenced and confirmed to derive from this region. The novel sequence was used to design gene specific primers (GSP1-3) for 5′RACE to capture the EtMIC4 5′UTR (GIF 990 KB)

High resolution image file (TIF 416 KB)

Fig. S2

5′ UTR sequence and N terminus of EtMIC4. 5′RACE and RT-PCR were used to amplify the region spanning the initiation transcription site and the known 5′ end of the cDNA of EtMIC4 (accession number AJ306453.2). The putative transcription initiation site is in bold and italics, and the predicted signal peptide is underlined. TSP-1-like domains are in bold. The fourth of four TSP-1-like domains partially corresponds to the previously suggested signal peptide (nucleotide 477+, AJ306453.1; Tomley et al. 2001) (GIF 122 KB)

High resolution image file (TIF 42.9 KB)

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Periz, J., Ryan, R., Blake, D.P. et al. Eimeria tenella microneme protein EtMIC4: capture of the full-length transcribed sequence and comparison with other microneme proteins. Parasitol Res 104, 717–721 (2009). https://doi.org/10.1007/s00436-008-1301-0

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  • DOI: https://doi.org/10.1007/s00436-008-1301-0

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