Cloning and Characterization of the Plastid Genome and Chlorophyll α-bound D1 protein in the Apicomplexa Eimeria tenella and Cryptosporidium parvum
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The Apicomplexans Eimeria tenella and Cryptosporidium parvum, contain an element thought to be common to all apicomplexans which could account for the finding that some antibiotics have been shown effective in their treatment: a circular extrachromasomal DNA element thought to be of plastid origin. In addition, a portion of the photosynthetic reaction center of PS II, the D1 protein, has been discovered in the same organelles as the plastid genome. Using the polymerase chain reaction, the aim of this project was to amplify and clone all or parts of this genome in C. parvum and E. tenella, as well as to amplify and clone this D1 protein. Oligonucleotides primers were designed based on the sequence of the plastid genome from P. falciparum, a related apicomplexan. Primers were designed for three regions: (1) the rpoB gene; (2) the SSU rRNA gene; and (3) the region between the Ef-Tu gene and the SSU rRNA gene. In addition, primers were designed to amplify the D1 protein. The amplified fragments were ligated into a vector, transformed into competent E. coli cells, and cultured. Positive colonies were purified and sequenced. A primer designed to the rpoB gene was found to have self-primed, thus resulting in the amplification of an irrelevant fragment. The primers designed specifically to the plastid SSU rRNA gene amplified instead the nuclear SSU rRNA gene. Although a large fragment was amplified between the Ef-Tu gene and the SSU rRNA gene, it was never cloned due to lack of time. The DNA coding for the D1 protein was not amplified. Future work could include redesign of primers and further optimization of PCR conditions, as well as further attempts at cloning the uncloned fragment.