Intron-containing β-tubulin transcripts in Cryptosporidium parvum cultured in vitro

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Abstract
The genome ofCryptosporidium parvumcontains a relatively small number of introns, which includes theβ-tubulin gene with only a single intron. Recently, it was observed that the intron was not removed from some of theβ-tubulin transcripts in the late life cycle stages culturedin vitro. Although normally splicedβ-tubulin mRNA was detected in all parasite intracellular stages by RT-PCR (e.g. HCT-8 or Caco-2 cells infected withC. parvumfor 12–72 h), at 48–72 h post-infection unprocessedβ-tubulin transcripts containing intact introns started to appear in parasite mRNA within infected host cells. The intron-containing transcripts could be detected by fluorescencein situhybridization (FISH) using an intron-specific probe. The intron-containingβ-tubulin transcripts appeared unique to thein vitro-culturedC. parvum, since they were not detected in parasite-infected calves at 72 h. As yet, it is unclear whether the late life cycle stages ofC. parvumare partially deficient in intron-splicing or the intron-splicing processes have merely slowed, both of which would allow the detection of intron-containing transcripts. Another possible explanation is that the decay in transcript processing might simply be due to the onset of parasite death. Nonetheless, the appearance of intron-containing transcripts coincides with the arrest ofC. parvumdevelopmentin vitro. This unusual observation prompts speculation that the abnormal intron-splicing ofβ-tubulin transcripts may be one of the factors preventing complete development of this parasitein vitro. Furthermore, the presence of both processed and unprocessed introns inβ-tubulin transcriptsin vitromay provide a venue for studying overall mechanisms for intron-splicing in this parasite.

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