9 Mb from the C. muris genome have been made available for download from CryptoDB, of which 7.2 Mb corresponding to coding sequences. Based on these newly added genomic sequences, 7/10 (70%) of the selected putative species-specific genes appear to have orthologs in C. muris. This information, if known previously, would have decreased dramatically the number of putative species-specific genes predicted by comparative genomics. Despite this limitation, only one C. parvum and one C. hominis gene were shown experimentally by PCR to be putatively specific, the characterisation of these genes is ongoing. We considered whether the observed ubiquity of the predicted specific genes represented the closeness between C.
hominis and click here C. parvum or whether these primers would also amplify orthologous genes from
other Cryptosporidium species by testing DNA from C. andersoni, C. felis, cervine genotype, C. meleagridis and C. baileyi. Cryptosporidium meleagridis DNA amplified using 80% of the primers tested, while, C. andersoni, cervine genotype and C. felis DNA amplified with only 10% of primers. This result is in accordance with the taxonomy and evolution of Cryptosporidium species [20]. In fact, amongst the species tested, C. meleagridis is the closest species to the cluster Fedratinib molecular weight formed by C. hominis, C. parvum and C. cuniculus based on partial SSU rRNA gene [20]. Cryptosporidium meleagridis DNA did not amplify with primers of Cgd2_2430 and Chro.20156. This could be explained by either nucleotide mismatch in the primer region or that the genes were missing. PCR screening and sequencing of genes found experimentally to be common to both species provided de novo sequence information at incomplete regions of the Cryptosporidium genomes and was used to examine polymorphism in these regions. PCR product sequence analysis revealed interesting genetic variation as SNPs. In this study, 78 SNPs were detected, 78.3% (61)
of which were species-specific. The presence of species-specific SNPs was reported previously from several genetic markers and has been exploited for Cryptosporidium genotyping and subtyping [21]. PCR-RFLP of the SSU rRNA [22], COWP [23], dihydrofolate reductase (DHFR) gene [24], thrombospondin related adhesive protein of Cryptosporidium-1 (TRAP-C1) [25] and TRAP-C2 [26], polythreonine (Poly-T) repeats [27]and heat shock protein isometheptene 70 (HSP70) [28] genes allow discrimination between Cryptosporidium species from various sources. In a similar manner, the newly identified SNPs could be also used for Cryptosporidium genotyping, especially by PCR-RFLP and/or sequencing. The majority of the SNPs detected (64.2%) were synonymous. It has long been assumed that synonymous SNPs are inconsequential as the primary sequence of the protein is HDAC inhibitor mechanism preserved. However, it has been demonstrated that synonymous mutations can alter the structure, function and expression level of the protein by affecting messenger RNA splicing, stability, protein folding and structure [29].