W. Coppieters

THE PIGMAP CONSORTIUM LINKAGE MAP OF THE PIG (SUS SCROFA).

A linkage map of the porcine genome has been developed by segregation analysis of 239 genetic markers. Eighty-one of these markers correspond to known genes. Linkage groups have been assigned to all 18 autosomes plus the X Chromosome (Chr). As 69 of the markers on the linkage map have also been mapped physically (by others), there is significant integration of linkage and physical map data. Six informative markers failed to show linkage to these maps. As in other species, the genetic map of the heterogametic sex (male) was significantly shorter (∼16.5 Morgans) than the genetic map of the homogametic sex (female) (∼21.5 Morgans). The sex-averaged genetic map of the pig was estimated to be ∼18 Morgans in length. Mapping information for 61 Type I loci (genes) enhances the contribution of the pig gene map to comparative gene mapping. Because the linkage map incorporates both highly polymorphic Type II loci, predominantly microsatellites, and Type I loci, it will be useful both for large experiments to map quantitative trait loci and for the subsequent isolation of trait genes following a comparative and candidate gene approach.

Cloning and sequencing of the porcine complement factor B

A genomic clone, SSBf1, containing the complement factor B (BF), a major histocompatibility class III antigen, has been isolated from a porcine genomic library. Partial sequencing and comparison with a human BF gene has identified seven exons coding for amino acids of Ba and Bb, the two subunits of BF. The protein sequence similarity with the human BF is on the average 87%. Southern blot analysis confirmed the existence of only one BF gene per haploid genome. Restriction fragment length polymorphism typing with Taq I showed that there are at least three different porcine BF-haplotypes.

A porcine minisatellite located on chromosome 14q29.

Microsatellites or short tandem repeats (STR) have undoubtedly become the most successful class of anonymous genetic markers. They can be typed very efficiently by means of PCR (Weber and May 1989; Litt and Luty 1989) and are almost randomly distributed in mammalian genomes (Stallings et al. 1991; Winterr et al. 1992). In the pig genome, they are underrepresented in the telomeric regions (Winterr et al. 1992). Minisatellites, on the other hand, have become less attractive in linkage studies. This is not only owing to the laborious typing by Southern blot hybridization, but also because there are indications for a nonrandom distribution in the human genome towards proterminal regions (Royle et al. 1988). However, highly polymorphic minisatellites located at the telomeres could complement a microsatellite-based map, as end points for chromosomes. Thus far, only two porcine minisatellites have been described (Coppieters et al. 1990; Davies et al. 1992). Here we report the isolation, partial sequencing, and localization of a new pig minisatellite locus. A size-selected library containing genomic pig DNA fragments ranging from 3 to 4.5 kb was constructed in pUC 18:500 ~tg genomic DNA was digested to completion with MboI and separated on a 0.8% agarose gel. The 2 to 23-kb fragments were electroeluted and subsequently concentrated to 30 ~tl by ethanol precipitation. This fraction was separated a second time on a 1% Seaplaque agarose gel (FMC, Rockland, USA). The agarose was sliced and digested with Gelase (Epicentre Technologies, Madison, Wis., USA). The digested agarose slices that contain the right-sized fragments were used in a ligation reaction with BamHI-digested and dephosphorylated pUC 18 vector. The ligation mix was transformed in DH5c~ high-efficiency competent E. coli cells (BRL, Gaithersburg, Md., USA) and plated at low density on LB agar plates containing Xgal, IPTG, and ampiciline. A total of 2400 white colonies was transferred to microtiterplates and grown overnight in 150 ~tl Terrific Broth.

PIG PLASMA ALPHA-PROTEASE INHIBITORS PI2, PI3 AND PI4 ARE MEMBERS OF THE ANTICHYMOTRYPSIN FAMILY.

Three related alpha-protease inhibitors, PI2 I, PI3 C and PI4 C2, of blood serum of the pig (Sus scrofa) were isolated. PI2 I inhibited both trypsin and chymotrypsin; PI3 C and PI4 C2 strongly inhibited chymotrypsin, but did not significantly inhibit trypsin. By using SDS-PAGE, the three proteins were found to be composed of single polypeptide chains, and molecular weights were 63,000 for PI2 I, 58,000 for PI3 C and 64,000 for PI4 C2. All three proteins were shown to be glycoproteins. In PI3 C, eight sialic acid residues were found, and in PI4 C2 (similarly as in PI2 F) 10-11 residues were found. Amino acid composition as well as N-terminal sequences of the three proteins were very similar, indicating close homology. Comparison of these partial amino acid sequences with the cDNA-deduced amino acid sequence of pig alpha-antichymotrypsin (AACT; Buchman, 1989, GenBank, Accession No. M29508) revealed great similarities, the sequence of PI2 I being virtually identical with the pig AACT. On the basis of all available results, PI2 is proposed to be pig AACT, an orthologue of human AACT.

The homology between the serum proteins Po2 in pig, Xk in horse and α1B-glycoprotein in human

Abstract 1. 1. Pig serum Po2 protein and horse Xk protein were purified by FPLC, non-denaturing 2D agarose-PAGE and 2D IPG-PAGE. 2. 2. The separated fractions were electroblotted to poly (4- vinyl -N- methylpyridinium iodide ) coated GF/C glass fiber sheets. 3. 3. The partial amino acid sequences and amino acid compositions of different genetic variants of the proteins were determined. 4. 4. The results proved that previously reported polymorphic serum post-albumins in each of these species were homologous to human plasma α 1 B-glycoprotein .