Porcine PPARGC1A (peroxisome proliferative activated receptor gamma coactivator 1A): coding sequence, genomic organization, polymorphisms and mapping.

We report here the characterisation of porcine PPARGC1A. Primers based on human PPARGC1A were used to isolate two porcine BAC clones. Porcine coding sequences of PPARGC1A were sequenced together with the splice site regions and the 5' and 3' regions. Using direct sequencing nine SNPs were found. Allele frequencies were determined in unrelated animals of five different pig breeds. In the MARC Meishan-White Composite resource population, the polymorphism in exon 9 was significantly associated with leaf fat weight. PPARGC1A has been mapped by FISH to SSC8p21. A (CA)n microsatellite (SGU0001) has been localised near marker SWR1101 on chromosome 8 by RH mapping and at the same position as marker KS195 (32.5 cM) by linkage mapping. The AseI (nt857, Asn/Asn489) polymorphism in exon 8 was used to perform linkage analysis in the Hohenheim pedigrees and located the gene in the same genomic region. Transcription of the gene was detected in adipose, muscle, kidney, liver, brain, heart and adrenal gland tissues, which is in agreement with the function of PPARGC1A in adaptive thermogenesis.

A refined comparative map between porcine chromosome 13 and human chromosome 3.

We report here the localisation of BAIAP1 (13q24), HTR1F (13q45), PTPRG (13q23) and UBE1C (13q24) by fluorescence in situ hybridisation (FISH), and BAIAP1 (Swr2114; 21 cR; LOD = 11.03), GATA2 (Sw2448; 37 cR; LOD = 8.26), IL5RA (Swr2114; 64 cR; LOD = 3.85), LMCD1 (Sw2450; 61 cR; LOD = 4.73), MME (CP; 50 cR; LOD = 7.75), RYK (Swc22; 12 cR; LOD = 18.62) and SGU003 (Sw1876; 6 cR; LOD = 16.99) by radiation hybrid (RH) mapping to porcine chromosome 13 (SSC13). The mapping of these 10 different loci (all mapped to human chromosome 3; HSA3) not only confirms the extended conservation of synteny between HSA3 and SSC13, but also defines more precisely the regions with conserved linkage. The syntenic region of the centromeric part of SSC13 was determined by isolating porcine bacterial artificial chromosome (BAC) clones (842D4 and 1031H1) using primers amplifying porcine microsatellite markers S0219 and S0076 (mapped to this region). Sequence comparison of the BAC end sequences with the human genome sequence showed that the centromeric part of SSC13 is homologous with HSA3p24.

Characterization of the porcine melanocortin 2 receptor gene (MC2R ).

A porcine bacterial artificial chromosome (BAC) clone, containing the melanocortin 2 receptor gene (MC2R) was isolated. The complete coding sequence of the MC2R gene, contained in 1 exon, was determined. Polymerase chain reaction-single stranded conformational polymorphism (PCR-SSCP) was performed on a 241-bp coding fragment. An AluI polymorphism, detecting a silent mutation, was found and typed on unrelated animals of five different pig breeds. The Meishan, Piétrain and Large White breeds differ significantly in allele frequencies from the Landrace and Czech Meat Pig breeds. The melanocortin 5 receptor gene (MC5R) was detected by PCR in the same BAC clone, as could be expected from the human and porcine mapping data. PCR-SSCP was performed on a 200-bp coding of MC5R, but no polymorphisms were detected. The BAC clone was mapped to Sscr6q27 by fluorescent in situ hybridization (FISH). A (CA)n microsatellite (SGU0002), isolated from the BAC, was localized on chromosome 6 by RH mapping near marker SW1473 and by linkage mapping on the MARC reference family at the same position as the marker SW2173 (97 cM). Allele frequencies, heterozygosity and polymorphism information contents (PIC) values were calculated for the five different pig breeds examined. The transcription of both genes in porcine liver, heart, kidney, fat, brain, pancreas, stomach, bladder, ovaries, lung, spleen, skin, adrenal gland and muscle tissues was examined by reverse transcriptase-polymerase chain reaction. Transcription was detected in skin and adrenal gland tissues for MC2R, while a positive signal was detected for MC5R in kidney, fat, pancreas, skin, adrenal gland and spleen tissues.

Characterization of the porcine FABGL gene.

The porcine major histocompatibility complex, also called swine lymphocyte antigen (SLA) complex, is of particular interest not only because of its central role in the immune response, but also because of its influence on many traits such as reproduction, fatness and meat quality. The porcine FABGL (FabG (beta-ketoacyl-[acyl-carrierprotein] reductase, Escherichia coli) like) gene, coding for a 17beta-hydroxysteroid dehydrogenase (17beta-HSD), is a candidate gene for these traits. The complete gene was sequenced and compared with human and mouse FABGL sequences. The deduced amino acid sequence showed 85 and 83% sequence identity to human and mouse sequences, respectively. Polymorphicic BbvI and DdeI restriction sites were found in the porcine FABGL gene. The promoter was compared with the promoter regions of human and mouse FABGL sequence in order to identify putative regulatory elements. The transcription profile of the porcine gene was determined and showed a widespread tissue distribution.

Bovine melanocortin receptor 4: cDNA sequence, polymorphisms and mapping.

A cDNA encoding the bovine melanocortin receptor 4 (MC4R) was cloned and sequenced. Comparing human, pig and rat homologues showed a 87, 85 and 89% identity on the DNA level, respectively, and over 90% on the protein level. The bovine MC4R gene was mapped to BTU 24 by radiation hybrid mapping. Two nucleotide changes were identified by single stranded conformation polymorphism (SSCP) and sequencing. The substitutions proved to be a T to C and G (allele B) to A (allele A) resulting, respectively, in a conservative valine to alanine substitution (Val 145 Ala) and an alanine to threonine (Ala 172 Thr). Using PCR-RFLP, 13 different cattle breeds were screened for the presence of the Ala 172 Thr substitution. With the exception of one Red Pied animal, allele A could only be detected in Red Holstein animals.

Comparative mapping between human chromosome 3 and porcine chromosome 13.

Zoo-FISH and somatic cell hybrid panels have earlier demonstrated extended synteny conservation between human chromosome 3 (HSA3) and pig chromosome 13 (SSC13). In the present study, eight human genes viz., ADCY5, CASR, COL7A1, COL8A1, ITIH1, RHO, SIAT1 and XPC, spread along the length of HSA3, were chosen for expanding the comparative map between the two chromosomes. Using human and rat cDNAs, or human- and porcine-specific PCR products as probes, 8 porcine lambda clones were isolated. After subcloning and partial sequence determination, identity of the clones with regards to the specific genes was established. The eight type 1 markers thus obtained were biotin labeled and FISH mapped to pig metaphase spreads. All lambda clones localized to SSC13. In combination with the hitherto published mapping data of coding sequences on SSC13, a preliminary comparative status depicting the relative organization of this chromosome with respect to HSA3 was developed. The comparative map thus obtained bears significance in searching for candidate genes of economically important traits mapped to SSC13.

A detailed physical map of the porcine major histocompatibility complex (MHC) class III region: comparison with human and mouse MHC class III regions.

A detailed physical map of the porcine MHC class III region on Chr 7 was constructed with a panel of probes in a series of hybridizations on genomic pulsed field gel electrophoresis (PFGE) Southern blots. A precise organization of the 700-kb segment of DNA between G18 and BAT1 can now be proposed, with more than 30 genes mapped to it. Comparison of this region with homologous regions in human and mouse showed only minor differences. The biggest difference was observed in the CYP21/C4 locus with only one CYP21 gene and one C4 gene found, whereas in human and mouse these genes are duplicated. These results show the class III region is very well conserved between pig, human, and mouse, in contrast with the class I and class II regions, which seem more prone to rearrangements.

Conservation of the RD-BF-C2 organization in the pig MHC class-III region: mapping and cloning of the pig RD gene.

The RD gene, named after the arginine (R) and aspartic acid (D) repeat in the central part of its protein, was initially mapped in the mouse H-2S subregion between C4 and BF. It was later mapped in the same position in the human MHC and here we show it is also conserved in the pig MHC class III region, close to the complement BF gene. A pig RD genomic clone was isolated from a lambda-phage library. Hybridizations on genomic DNA separated with pulsed field gel electrophoresis identified common 220 kb NruI, 130 kb EagI and 200 kb MluI bands for RD, BF and C2. The RD gene has also a 17 kb Kp nI and 11 kb SacI fragment in common with BF but not with C2. The close linkage of the RD and BF genes was further established by hybridization of BF to a genomic lambda-phage clone also containing the RD gene. This genomic RD clone overlaps with a lambda-phage clone previously isolated and containing the complete BF gene and the 3' part of C2. The distance between RD and BF is about 6 kb. The junction between the two complement genes BF and C2 was sequenced and the BF 5' promoter region, overlapping the 3' noncoding region of C2, was compared with that of the human BF promoter. The overall homology was about 80% and all but one identified promoter elements were found in the same position in both genes. The results obtained demonstrate the RD-BF-C2 organization is strongly conserved between human, mouse and pig. No polymorphisms were detected in either the RD gene or in the BF promoter region using polymerase chain reaction and restriction fragment polymorphism analysis.