Intron-containing beta-tubulin transcripts in Cryptosporidium parvum cultured in vitro.
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abstract
The genome of Cryptosporidium parvum contains a relatively small number of introns, which includes the beta-tubulin gene with only a single intron. Recently, it was observed that the intron was not removed from some of the beta-tubulin transcripts in the late life cycle stages cultured in vitro. Although normally spliced beta-tubulin mRNA was detected in all parasite intracellular stages by RT-PCR (e.g. HCT-8 or Caco-2 cells infected with C. parvum for 12-72 h), at 48-72 h post-infection unprocessed beta-tubulin transcripts containing intact introns started to appear in parasite mRNA within infected host cells. The intron-containing transcripts could be detected by fluorescence in situ hybridization (FISH) using an intron-specific probe. The intron-containing beta-tubulin transcripts appeared unique to the in vitro-cultured C. parvum, since they were not detected in parasite-infected calves at 72 h. As yet, it is unclear whether the late life cycle stages of C. parvum are 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 of C. parvum development in vitro. This unusual observation prompts speculation that the abnormal intron-splicing of beta-tubulin transcripts may be one of the factors preventing complete development of this parasite in vitro. Furthermore, the presence of both processed and unprocessed introns in beta-tubulin transcripts in vitro may provide a venue for studying overall mechanisms for intron-splicing in this parasite.
