Rapid communication: partial nucleotide sequence of the goat stearoyl coenzyme A desaturase cDNA and gene structure.

Name of the Sequence. Stearoyl coenzyme A desaturase (SCD) cDNA. Genus and Species. Capra hircus. Origin of Clone. Total RNA was isolated from a mammary gland sample from a lactating Murciano-Granadina goat using Trizol reagent (Life Technologies, Rockville, MD) and following supplier’s instructions. The first strand of the cDNA was synthetized using the M-MuLV reverse transcriptase (Life Technologies) and two primers: an oligo-d(T)30 and E5R (5′-CCACAGCTCCCAGGGAAACC-3′) designed over the ovine SCD exon 5 (Ward et al., 1998). The cDNA was amplified by PCR using two sets of primers: E1F (5′-CAGCACAGCAGGTCGGGTCCG-3′) and E5R for the amplification of the region from exons 1 to 5, and E4F (5′-CTGATCCCCACAATTCCCGAC-3′) with 3NCR (5′-GCTCTAGAGAGAAAGGGAGCATGCTGG-3′) for the amplification from exon 4 to 6. The 3NCR primer was designed 22 bp upstream of the poly(A) tail of the sheep cDNA sequence. The cDNA bands of interest were isolated from an agarose gel and cloned using TOPO-TA cloning kit (Invitrogen, Carlsbad, CA). Several clones from different RT-PCR products were sequenced on both strands using an automated sequencer (ABI310, Applied Biosystems, Foster City, CA). For the determination of gene structure, introns were amplified using primers complementary to the flanking exons and genomic DNA from Murciano-Granadina goats. Preliminary position of exons was deduced from the sequence of the bovine SCD gene (J. F. Medrano, personal communication). The PCR was performed in a total volume of 50 L with 100 to 250 ng of genomic DNA and using 2.5 units of Long Expand PCR System (Roche Molecular Diagnosys, Indianapolis, IN). The generated fragments from multiple independent PCR reactions of each region were purified with Concert kit (Life Technologies) and directly used in sequencing reactions. Comparison with Related Sequences. Within the coding region (Figure 1), the nucleotide sequence is well conserved between the ruminant species with a similarity of 98 and 95% between the caprine SCD sequence