Rapid communication: complete nucleotide sequence of the chicken prolactin gene.

Name of Sequence. Chicken prolactin (PRL) gene. Genus and Species. Gallus gallus (chicken). Origin of Clones. Genomic DNA from Xing Hua Chinese native chickens was used as a template. Primers were designed based on exon/intron junctions of the genomic turkey PRL gene sequence (Kurima et al., 1995). Polymerase chain reaction (PCR) amplifications of the PRL gene were performed by using the following oligonucleotides (all based on cDNA sequence, GenBank accession no. E02259): forward primer PRLexon1F 5′-CAC ACA GAA TCC CTA CCA TG-3′ and reverse primer PRLexon2R 5′-ACT TGG CAG TTG ACT GAT CC-3′; forward primer PRLexon2F 5′-GAC CAA GGA AGG AGT GAC CT-3′ and reverse primer PRLexon3R 5′-CGA CCC TGA GCA TAA CGT TC-3′; forward primer PRLexon3F 5′-GAA CGT TAT GCT CAG GGT CG-3′ and reverse primer PRLexon4R 5′-CTT CAG AGG CCA GAT GGA TC-3′; forward primer PRL2F 5′AAG AGG CTT CTA GAA GGA ATG G-3′ and reverse primer PRL2R 5′-TTG CAG CCA GAA TTC ACA CAA3′. The PCR was performed with 100 ng of genomic DNA as template in a total volume of 50 L of reaction mix containing 10× PCR buffer with 15 mM MgCl2 (GIBCO BRL, Tsuen Wan, Hong Kong), 0.2 mM dNTP, 20 pmol each primer, and 1.5 U of Taq DNA polymerase (GIBCO BRL). The PCR protocol was as follows: after denaturation at 95°C for 4 min, 30 amplification cycles comprising denaturation at 94°C for 30 s, annealing at 58°C for 1 min, and extension at 72°C for 2 min 30 s, followed by an extended elongation at 72°C for 10 min. Purified PCR products (BIO 101, 1070 Joshua Way, Vista, CA) were cloned into pMosBlue vector (Amersham, Arlington Heights, IL). The 5′ and 3′ flanking regions were found by using Universal GenomeWalker kit (CLONTECH, 1020 East Meadow Circle, Palo Alto, CA). Primer PRLup2 5′-ACT GAA GTA AGA CAT TAT CCT CCC CC-3′ and primer PRLup6 5′-ATC CAC CAG ACA CTT TCC TGT GTT AC-3′ were used for amplification of the 5′ flanking region; primer PRLdown1 5′TAC CTG TGG GCT GCA TTA CTC ACT GAA A-3′