FIG. 2.

Schematic representation of ptp1 and ptp2 gene clusters with positions of PCR primers (A to O) in the three Encephalitozoon species, E. cuniculi (Ec), E. intestinalis (Ei), and E. hellem (Eh). The peptide obtained by microsequencing of EcPTP2 (AVQGTDRCILAGIID) is indicated by a black square. Sense primer D, designed 186 bp upstream from the stop codon of Ecptp1, combined with antisense primer B, designed 380 bp downstream from the initiation codon of Ecptp2, were used to amplify a 1.4-kbp DNA fragment. Two primers deduced from the Ecptp2 sequence (C and E, positions 419 to 699 in the Ecptp2 ORF) were used to amplify a 280-bp DNA fragment in E. intestinalis. The corresponding sequence shared about 90% identity with that of Ecptp2 but showed some differences that were useful to determine specific oligonucleotides. Downstream and upstream regions of the 280-bp known sequence were amplified with PstI and HindIII ligation products, respectively, using two specific primers (F and G) and the reverse vector primer. Thus, SSP-PCR experiments led to 120-bp (3′ with PstI) and 1,150-bp (5′ with HindIII) amplicons, respectively. The 3′ end of Eiptp2 was completed through another SSP-PCR step with primer H, resulting in a 570-bp amplification from the ApaI ligation product. The final sequence is 1,968 bp in length with a predicted 825-bp ORF coding for EiPTP2. The Eiptp1 gene was amplified using a combination of an antisense primer in the 5′ flanking region of Eiptp2 (J) and a sense primer (I) determined from the alignment of conserved regions encoding signal peptides of EcPTP1 and EhPTP1. For E. hellem, primer K, determined in the 3′ known flanking region of Ehptp1, was combined with the reverse primer L, determined by the alignment of the highly conserved N-terminal sequence encoding the PTP2 signal peptide in E. cuniculi and E. intestinalis. The whole coding sequence of Ehptp2 and its 3′ UTR were completed by SSP-PCR amplification with primer O. The 1,696-bp sequence is from reference 21.