The bovine cocaine and amphetamine‐regulated transcript locus: gene characterization and SNP discovery

Source/description: The cocaine and amphetamine-regulated transcript (CART) protein is an endogenous inhibitor of feed intake, and is produced by the hypothalamus in normal animals.1 Mice with disrupted leptin signalling show very low levels of CART mRNA, while CART injection inhibits the normal and starvation-induced feeding responses. The CART protein blocks the neuropeptide Y-induced feeding response when administered systemically. However, the effect of CART depends on the site of administration, however, and several recent reports have demonstrated an orexigenic role for CART when injected directly into the arcuate nucleus of the hypothalamus, suggesting that the gene product plays a role in adaptation to cold2 mediated by its effect on uncoupling proteins.3 In this study, we report: (i) the DNA sequence of the complete coding region of the bovine CART gene; (ii) genomic localization of the gene using whole genome radiation hybrid mapping (WGRH); (iii) the results of a single nucleotide polymorphism (SNP) discovery experiment using a beef cattle reference panel; and (iv) the genotypes of beef cattle reference panel bulls with respect to an embedded microsatellite. Genomic DNA amplification: CART 1.1B: 5′-CCG AGC CCT GGA CAT CTA CTC-3′ CART 1.2D: 5′-GGG GAC AGT CAC ACA GCT TC-3′ SNP discovery: CART 1.1G: 5′-CTG GAC ATC TAC TCC GCC G-3′ CART 1.2H: 5′-GAG CTT CTT CAG AAC TTC CTG-3′ C-1 microsatellite analysis: CART 1.1F: 5′-GCC TTC TGA AGC ACT GCC AGT-3′ CART 1.2R: 5′-GTA CCT TTC CTG GGT CCT CC-3′ Cloning and characterization of the bovine CART gene: We used information on the gene structure of the human homologue and the available sequence data from bovine expressed sequence tags (ESTs; BM288295, AW335960, CB537060 and CB535122) to design primers for the polymerase chain reaction (PCR) amplification of a genomic DNA fragment spanning the bovine CART coding region. The sequence of the resulting amplicon (1497 bp) was extended with available bovine EST sequence information in the upstream and downstream directions, and the resulting 1769 bp contig submitted to GenBank (accession number AY603972). Like its human counterpart, bovine CART is a compact gene consisting of three exons separated by two introns of 425 bp and 859 bp, respectively. Alignment of the PCR-amplified genomic DNA fragments with bovine CART-specific EST sequences revealed three putative SNP (A/G at position 645; A/G at position 654; T/C at position 1041) as well as a CA-repeat microsatellite within intron 2 of the CART gene. WGRH mapping: The WGRH was carried out on a 3000 rad radiation hybrid panel consisting of 94 cell lines4 using primers CART 1.1G and CART 1.2H (see above). Two-point and multipoint analyses were performed using carthagene software.5 Results indicated that the CART gene is located on bovine chromosome 20, with strongest linkage to ILSTS085 (two-point distance of 60.4 cR; LOD score 6.1) and BM1225 (two-point distance of 66.7 cR; LOD score 5.4). An optimized map suggests that the gene is probably located between ILSTS085 (distance 62.4 cR) and TGLA126 (distance 139 cR), despite insignificant linkage to the latter marker (LOD score 1.0). SNP discovery: The SNP discovery was performed on a DNA minipanel consisting of three males each of a variety of predominantly beef cattle breeds of Bos taurus; Brahman (Bo. indicus) and American Bison (Bison bison) were included as outliers (see Table 1). DNA was extracted from semen (Bos samples) and liver (Bison samples) using the PUREGENE DNA Isolation kit (Gentra Systems, Minneapolis, MN, USA). The SNP discovery was performed on a fragment of 506 bp extending from positions 136–641 in GenBank accession number AY603972. The PCR amplifications were carried out using the Amplitaq Gold Kit (Roche Diagnostics, Laval, QC, Canada) and primers CART 1.1G and CART 1.2H (see above). Amplified products were purified using the QIAquick PCR Purification kit (Qiagen Inc., Mississauga, ON, Canada) and both strands sequenced directly using the BigDye Terminator Cycle Sequencing method (Applied Biosystems, Foster City, CA, USA). A total of nine SNP were uncovered: a single SNP was polymorphic in all taurine breeds (C/T at position 258), three were diagnostic for bison (T, A, and T at positions 261, 395, and 579, respectively), one was shared by bison and Brahman (G at position 396), and four SNP are polymorphic in Brahman (A/G, C/T, A/G, and A/T at positions 416, 420, 513 and 521, respectively; see Table 1). Microsatellite analysis: Sequence analysis revealed a CA-repeat microsatellite within intron 2 of the bovine CART gene (microsatellite C-1). We designed primers (see above) for the amplification and fluorescent dye-tagged size analysis of this microsatellite on the DNA minipanel used for SNP discovery. Based on the published sequence, we expected an amplicon of 258 bp. The observed amplicon sizes were as follows: (i) all three bison samples yielded an amplified fragment size of 238 bp which was not found in any of the other samples; (ii) two of the Brahman bulls yielded a fragment of 250 bp which was not found in any other breeds, and a fragment of 262 bp which was rare in other breeds, i.e. detected only in a single Hereford bull; (iii) a fragment of 248 bp was also rare and found only in one bull of each of the heavily muscled Charolais and Limousin breeds as well as a single Wagyu bull; (iv) a 258 bp fragment was found in the Holstein and Wagyu breeds only; (v) common fragment sizes included 256 bp (22 of 54) and 260 bp (11 of 54). Studies are currently underway to test for possible associations between C-1 microsatellite genotype and growth and feed efficiency traits in beef cattle resource populations. Acknowledgement: Funding for this research was provided by the Canada/Alberta Beef Industry Development Fund.

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