Marc Mattheeuws

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.

A dual fluorescent multiprobe assay for prion protein genotyping in sheep

BackgroundScrapie and BSE belong to a group of fatal, transmissible, neurodegenerative diseases called TSE. In order to minimize the risk of natural scrapie and presumed natural BSE in sheep, breeding programmes towards TSE resistance are conducted in many countries based on resistance rendering PRNP polymorphisms at codons 136 (A/V), 154 (R/H) and 171 (R/H/Q). Therefore, a reliable, fast and cost-effective method for routine PRNP genotyping in sheep, applicable in standard equipped molecular genetic laboratories, will be a vital instrument to fulfill the need of genotyping hundreds or thousands of sheep.MethodsA dual fluorescent multiprobe assay consisting of 2 closed tube PCR reactions containing respectively 4 and 3 dual-labelled fluorescent ASO probes for the detection in real-time of the 7 allelic variants of sheep PRNP mentioned above.ResultsThe assay is succesfully performed using unpurified DNA as a template for PCR, without any post-PCR manipulations and with semi-automatic determination of the PRNP genotypes. The performance of the assay was confirmed via PCR-RFLP and sequencing in a cross-validation study with 50 sheep.ConclusionsWe report the development and validation of a robust, reliable and reproducible method for PRNP genotyping of a few to many sheep samples in a fast, simple and cost-effective way, applicable in standard equipped molecular genetic laboratories. The described primer/probe design strategy can also be applied for the detection of other polymorphisms or disease causing mutations.

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.

A novel porcine gene, α-1-antichymotrypsin 2 (SERPINA3-2): sequence, genomic organization, polymorphism and mapping

A novel porcine gene, alpha-1-antichymotrypsin 2 (SERPINA3-2), a member of the serpin superfamily, was isolated from a porcine genomic library and sequenced. The genomic organization of the approximately 9.0 kb gene was determined on the basis of the porcine liver cDNA of SERPINA3-1 and SERPINA3-2, and comprises five exons and four introns. The coding sequence of SERPINA3-2 shares 86% identity with the paralogue, SERPINA3-1. Porcine SERPINA3-2 was found to be an orthologue of human SERPINA3 (71% identity of the coding sequences) and both genes have a similar genomic organization. Polymorphisms were found in intron 4 of the porcine gene using polymerase chain reaction-restriction fragment length polymorphism. The gene was mapped by linkage analysis and radiation hybrid mapping to the distal end of chromosome 7q, to the gene cluster of the protease inhibitors including PI1 (SERPINA1), PI2, PI3, PI4 (apparently paralogues of SERPINA3), and PO1A and PO1B. SERPINA3-2 is the first porcine serpin gene whose genomic organization has been determined.

Mapping of the ATP2B2 and PCCB genes on porcine chromosome 13.

We report the cloning and mapping of two genes on porcine Chromosome (Chr) 13: the ATP2B2 gene, coding for a plasma membrane Ca2+-ATPase, and the PCCB gene, coding for the propionylCoA carboxylase beta-subunit. Plasma membrane Ca2+-ATPases play an essential role in the regulation of the free cytosolic Ca 2§ concentration in eukaryotes, by pumping free cytosolic Ca 2+ out of the cell, while hydrolyzing ATP. The pumps are encoded by a highly conserved and widely dispersed multigene family, consisting of at least four different genes (ATP2B1, ATP2B2, ATP2B3, and ATP2B4; Wang et al. 1994). Propionyl-CoA carboxylase (PCC) is a biotin-dependent, mitochondrial enzyme that catalyzes the carboxylation of propionyl-CoA to o-methylmalonyl-CoA in an ATP-dependent process. The enzyme is involved in the catabolism of branched-chain amino acids, fatty acids of odd-numbered chain lengths, and other metabolites. PCC is composed of c~and [ 3 s u b u n i t s (Os 4 o r ff61~6), encoded by the PCCA and the PCCB gene, respectively (Lamhonwah et al. 1986). cDNA clones of human ATP2B2 (phPMCA2-1) and PCCB (pPCC41A2) were obtained from the American Type Culture Collection. A 1100-bp BamHI fragment from phPMCA2-1 and a 1350-bp BamHI fragment from pPCC41A2 were labeled with 32p using the Ready To Go DNA labeling Kit (-dCTP) (Pharmacia) and hybridized to 6 x 105 tofu of a porcine phage h library (Peelman et al. 1991). The isolated hATP2B2 clone contains a 17-kb insert, and the isblated kPCCB clone contains a 15-kb insert (inserts sized using pulsed field gel electrophoresis). Southern analysis of the KATP2B2 clone, with the 1100-bp BamHI fragment from phPMCA2-1 as probe, revealed a 3100-bp and a 3300-bp BamHI fragment, and a 2800-bp EcoRI fragment. The 3100-bp BamHI fragment was cloned in pUC 18 and partially sequenced with the TTSequencingTm Kit. A 352-bp sequence was compared with the databases using BLAST and FASTA. A 91-bp fragment shows 93% sequence identity with the human phPMCA2-1 clone (X63575; from position 2387), with only one amino acid difference, whereas the sequence identity of the other part is lower than 40%. Since the 91-bp fragment is flanked by the consensus AG . . . GT splice sites, it probably forms a complete exon. This 91-bp fragment shows also 91% sequence identity with the rat ATP2B2 gene. The clone contains ATP2B2 and not the related ATP2B1, since it shows only 78% sequence identity with the porcine ATP2B 1 sequence published by De Jaegere and colleagues (1990). Southern analysis of the hPCCB clone, with the 1350-bp BamHI fragment from pPCC41A2 as probe, revealed a 1500-bp and a 3200-bp BamHI fragment, and a 2000-bp EcoRI fragment. The 1500-bp BamHI fragment was cloned in pUC18, and 452 bp respectively 460 bp from both ends was sequenced. Comparison of

Comparative analysis of a BAC contig of porcine chromosome 13q31-q32 and human chromosome 3q21-q22

BackgroundThe gene(s) encoding the ETEC F4ab/ac receptors, involved in neonatal diarrhoea in pigs (a disease not yet described in humans), is located close to the TF locus on Sscr13. In order to reveal and characterize possible candidate genes encoding these receptors, a porcine physical map of the TF region is indispensable.ResultsA contig of 33 BAC clones, covering approximately 1.35 Mb surrounding the TF locus on Sscr13q31-q32, was built by chromosome walking. A total of 22,552 bp from the BAC contig were sequenced and compared with database sequences to identify genes, ESTs and repeat sequences, and to anchor the contig to the syntenic region of the human genome sequence (Hsap3q21-q22). The contig was further annotated based on this human/porcine comparative map, and was also anchored to the Sanger porcine framework map and the integrated map of Sscr13 by RH mapping.ConclusionThe annotated contig, containing 10 genes and 2 ESTs, showed a complete conservation of linkage (gene order and orientation) with the human genome sequence, based on 46 anchor points. This underlines the importance of the human/porcine comparative map for the identification of porcine genes associated with genetic defects and economically important traits, and for assembly of the porcine genome sequence.