Rapid communication: nucleotide sequence and physical mapping of the porcine cyclin-dependent kinase inhibitor 3 (CDKN3) gene.

Name of the Sequence. Cyclin-dependent kinase inhibitor 3 (CDKN3; CDK2-associated dual specificity phosphatase). Genus and Species. Sus scrofa. Origin of Clone. We used the differential display/reverse transcriptase PCR (DD/RT) approach to isolate potential candidate genes for congenital splay leg in piglets. A total of 16 cDNA fragments with apparent differential expression in skeletal muscle (biceps femoris) between each two healthy and affected piglets were detected in this investigation (Maak et al., 2001a,b). Among them, a 301-bp cDNA fragment from a male splay-leg piglet (German Landrace) was isolated by reverse transcription of total RNA with primer D8 and subsequent amplification with primers D8 and U17 (GeneExScreen kit, Biometra, Goettingen, Germany). We observed a more intense band in preparations from both the splay-leg piglets compared to those from healthy control animals. Homology search revealed significant similarity of the porcine cDNA to the 3′-UTR of human protein phosphatase (KAP1) gene (GenBank accession no. L27711). This gene was designated CDKN3 by the HUGO gene nomenclature committee. A primer was designed on the basis of the human sequence (forward: 5′-ATA GAC AGC CTG CGA GAC C3′) and used with a porcine primer (reverse 5′-TTG CCA ATA AAA GCT TTA GGA A-3′) for amplification of 3′UTR and partial codons. With a second primer set (human-derived forward: 5′-GTG AGA CTG CCA GCC ATG AAG-3′; reverse: 5′-TTG ATG ATA GAT GTG CAG CTA ATT T-3′) the complete coding region of the porcine gene was amplified. The PCR were performed with 100 ng porcine cDNA, 50 pmol of each primer, and a PCR bead (RTG PCR Beads, AmershamPharmacia Biotech, Freiburg, Germany) in a total volume of 25 L. Thermocycling was carried out in a TC1 machine (Perkin Elmer, Ueberlingen, Germany). Initial denaturation (94°C for 5 min) was followed by 35 cycles consisting of denaturation