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Background: Several approaches can be used to determine the order of loci on chromosomes and hence develop maps of the genome. However, all mapping approaches are prone to errors either arising from technical deficiencies or lack of statistical support to distinguish between alternative orders of loci. The accuracy of the genome maps could be improved, in principle, if information from different sources was combined to produce integrated maps. The publicly available bovine genomic sequence assembly with 6x coverage (Btau_2.0) is based on whole genome shotgun sequence data and limited mapping data however, it is recognised that this assembly is a draft that contains errors. Correcting the sequence assembly requires extensive additional mapping information to improve the reliability of the ordering of sequence scaffolds on chromosomes. The radiation hybrid (RH) map described here has been contributed to the international sequencing project to aid this process., Results: An RH map for the 30 bovine chromosomes is presented. The map was built using the Roslin 3000-rad RH panel (BovGen RH map) and contains 3966 markers including 2473 new loci in addition to 262 amplified fragment-length polymorphisms (AFLP) and 1231 markers previously published with the first generation RH map. Sequences of the mapped loci were aligned with published bovine genome maps to identify inconsistencies. In addition to differences in the order of loci, several cases were observed where the chromosomal assignment of loci differed between maps. All the chromosome maps were aligned with the current 6x bovine assembly (Btau_2.0) and 2898 loci were unambiguously located in the bovine sequence. The order of loci on the RH map for BTA 5, 7, 16, 22, 25 and 29 differed substantially from the assembled bovine sequence. From the 2898 loci unambiguously identified in the bovine sequence assembly, 131 mapped to different chromosomes in the BovGen RH map., Conclusion: Alignment of the BovGen RH map with other published RH and genetic maps showed higher consistency in marker order and chromosome assignment than with the current 6x sequence assembly. This suggests that the bovine sequence assembly could be significantly improved by incorporating additional independent mapping information.

The principal components of the protein fraction in pony mare's milk have been successfully identified and partially characterized using proteomic tools. Skimmed pony mare's milk was fractionated by either reversed phase-high-performance liquid chromatography (RP-HPLC) on a C4 column or a bi-dimensional separation technique coupling RP-HPLC in the first dimension and sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) in the second dimension (two-dimensional RP-HPLC/SDS-PAGE). The fractions thus obtained were analyzed by Edman N-terminal microsequencing and mass determination, with or without tryptic digestion, on a matrix-assisted laser desorption/ionization-time of flight spectrometer. Based on the sequence and molecular mass information obtained, identifications were achieved through a protein database search using homology or pattern research algorithms. This methodological approach was shown to be rapid, efficient and reliable in identifying the principal proteins in pony mare's milk. kappa-, alpha(s1)-, alpha(s2)-, and beta-casein, lysozyme C, alpha-lactalbumin and beta-lactoglobulin I and II were thus identified. alpha(s1) and beta-caseins displayed polymorphic patterns, probably due to alternative splicing processes leading to casual exon skipping events involving exons 7 and 14 in alpha(s1)-casein and exon 5 in beta-casein. Edman N-terminal microsequencing over 35 amino acid residues, for pony alpha(s1)-casein, clearly demonstrated the occurrence, in Equidae, of a splicing pattern similar to that reported in rodents, characterized by the constitutive outsplicing of exon 5. Pony mare's milk SDS-PAGE and RP-HPLC patterns were compared with those obtained for other milks (cow, goat and human), as were the relative levels of caseins and major whey proteins in these milks. Our results provide further evidence to support the notion that Equidae milk is closer to human breast milk than milk from bovine and caprine with respect to the casein and lysozyme C contents and casein/whey proteins ratio.

A first generation clone-based physical map for the bovine genome was constructed combining, fluorescent double digestion fingerprinting and sequence tagged site (STS) marker screening. The BAC clones were selected from an Inra BAC library (105,984 clones) and a part of the CHORI-240 BAC library (26,500 clones). The contigs were anchored using the screening information for a total of 1303 markers (451 microsatellites, 471 genes, 127 EST, and 254 BAC ends). The final map, which consists of 6615 contigs assembled from 100,923 clones, will be a valuable tool for genomic research in ruminants, including targeted marker production, positional cloning or targeted sequencing of regions of specific interest.

The polymorphism of a (TA)n(CA)n repeat microsatellite present in the third intron of the bovine kappa-casein gene (CASK) has been investigated. The existence of six alleles differing only in the number of dinucleotide repeats has been established. A total of 330 animals belonging to nine different pure bred Bos taurus French breeds or to a cross-bred Bos taurus x Bos indicus population (Créole) were genotyped. The distribution of the microsatellite alleles was examined and clear breed differences were noted. Genotyping of animals by isoelectric focusing (IEF) or restriction fragment length polymorphism (RFLP) (TaqI) was performed, in order to examine the relationship of the microsatellite polymorphism to other previously described CASK polymorphisms, at the protein and DNA levels. Strong correlation was seen, indicating that evolution of the various polymorphisms was not independent, and nine CASK haplotypes were observed.

Two alleles, A and B, were previously described at the goat alpha s2-casein locus. Isoelectric focusing allowed us to subdivide the former one in two new alleles, called A and C. Although alpha s2-casein C cannot actually be distinguished from its A counterpart by starch or polyacrylamide gel electrophoresis, it differs from the previous allele by a single substitution Lys (A)/Ile (C) at position 167, which was confirmed at the nucleotide level. The frequencies of the three alpha s2-casein alleles A, B and C were estimated to be 0.85, 0.04 and 0.11 in the French dairy breeds 'Alpine' and 'Saanen'.

Analysis of elution profiles of enzymatic and CNBr digests of kappa-caseins C and E, and sequencing of most relevant peptides allowed the chemical characterization of both genetic variants. They differ from their B and A allelic counterparts by a single substitution, His97/Arg and Gly155/Ser, respectively. Electrophoretic behaviour of the investigated C and E variants was in good agreement with the observed amino acid replacements.

The extensive polymorphism of caprine alpha s1-casein, which is controlled by at least seven autosomal alleles segregating in a Mendelian fashion, was investigated by RFLP analysis. Genomic DNA from 77 lactating goats, whose genotypes had been previously determined by electrophoretic analysis of milk proteins, was digested with 11 restriction endonucleases and Southern blots were probed with a radiolabelled ovine alpha s1-casein cDNA. Three enzymes, PstI, TaqI and Rsa I, allowed the unambiguous identification of known alleles alpha s1-CnA, E and O and of the allelic pairs [alpha s1-CnD and F] and [alpha s1-CnB and C]. Evidence for a second null allele, termed alpha s1-CnO', and for an additional allele, designated alpha s1-CnF', was provided, which leads to the identification of nine alleles at the alpha s1-Cn locus, in this species. Although only 15 out of the 45 expected genotypes could be fully ascertained, this procedure allows the identification at birth of animals carrying the alpha s1-CnA, B or C alleles associated with a high alpha s1- and whole-casein content.

The sequence of caprine alpha s1-casein (199 residues) was established. The peptide chain has the same length as, and shows a 88% degree of identity with, its bovine counterpart. With the ovine alpha s1-casein, the sequence of which was deduced from that of its mRNA, the degree of identity is 97%, counting as one difference a deletion of eight residues in the ovine protein. The differences between the three genetic variants associated with a high alpha s1-casein content in milk are simple substitutions. Variant alpha s1-CnA differs from variant alpha s1-CnB by two substitutions, 16 Leu (A)----Pro (B) and 77 Gln (A)----Glu (B), the latter inducing the appearance of a phosphate group on 75 Ser. Variant alpha s1-CnC differs from alpha s1-CnB by three substitutions, 8 His (B)----Ile (C), 100 Arg (B)----Lys (C) and 195 Thr (B)----Ala (C). The original type of caprine alpha s1-casein could be another hypothetical genetic variant, having the same electrophoretic mobility as alpha s1-CnB.

Using gel electrophoresis a genetic polymorphism of alpha S2-casein (Cn) was discovered in individual milk samples from 2 bovine breeds of the eastern part of France (Vosgienne and Montbéliarde). The 3 observed phenotypes (Plate 1) are determined by 2 co-dominant alleles at an autosomal locus. The alpha S2-Cn A variant was the only one known up to now in European breeds (reference variant) and alpha S2-Cn D is a new variant, whose bands overlap the beta-casein A band at pH 8.6, and migrate faster than alpha S2-Cn A at pH 3.0. The sequence of the polypeptide chain alpha S2-Cn D differs from that of alpha S2-Cn A by the deletion of a very acidic nonapeptide, which includes a cluster of 3 phosphoseryl residues. Due to the characteristics of the reference sequence, this deletion cannot be exactly located but it involves residues 50-58, or 51-59, or 52-60. A genetic analysis shows that locus alpha S2-Cn is closely linked to the cluster alpha S1-Cn--beta-Cn--kappa-Cn. The 4 casein species are thus synthesized by 4 closely linked loci.

The A variant of bovine alpha(s1) casein is devoid of the segment of 13 amino acid residues which occupies the 14th to 26th position from the NH(2)-terminal in the polypeptide chain (198 residues) of the B and C variants.