2.3. Gene Cloning

In order to search P. longiseta ParaHox genes, degenerate PCR with asexual reproducing worms’ cDNA was performed. All primers are given in the Supplementary Materials (Table S1). The extended gene fragments were amplified by 5′-RACE and 3′-RACE PCR with gene-specific primers and cDNA prepared with SMARTer RACE cDNA Amplification Kit (Clontech, Cat. #634923). To validate overlapping of the P. longiseta 5′-RACE and 3′-RACE gene fragments inside the homeobox, as well as to isolate the N. communis ParaHox gene fragments, PCRs with gene-specific primers and cDNAs were carried out. These amplified gene fragments were cloned and used for RNA probe synthesis. The PCR products were inserted in pCRII vectors by using TOPO-TA cloning kit (Invitrogen, Cat. #K4600-01) and used in the transformation of chemically competent E. coli (One Shot^® TOP10; Invitrogen, Cat. #K4600-01). The obtained colonies with the correct insert were checked by sequencing. As a result, except for Nco-Gsx2 and Plo-Gsx2, all amplified fragments include complete CDS, 5′ and 3′UTR. Sequences of Nco-Gsx1 (1440 bp), Nco-Gsx2 (1223 bp), Nco-Xlox (1834 bp), Nco-Cdx1 (2100 bp), Nco-Cdx2 (1746 bp), Plo-Gsx1 (1074 bp), Plo-Gsx2 (1061 bp), Plo-Xlox (1089 bp), Plo-Cdx1 (1243 bp), and Plo-Cdx2 (1667 bp) are deposited in GenBank with the accession numbers {"type":"entrez-nucleotide-range","attrs":{"text":"OR050790-OR050799","start_term":"OR050790","end_term":"OR050799","start_term_id":"2556705626","end_term_id":"2556705644"}}OR050790-OR050799. A 5′ partial CDS of Plo-Cdx2 cDNA (1507 bp) was cloned previously (GenBank {"type":"entrez-nucleotide","attrs":{"text":"JQ685130","term_id":"387625264","term_text":"JQ685130"}}JQ685130).