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Screening of a bovine yeast artificial chromosome (YAC) library revealed two clones which contain most of the class II genes of the major histocompatibility complex (MHC) known to date. The YACs were mapped by fluorescence in situ hybridization (FISH) and characterized for the class II genes they contain. We found that the classic class II genes BoLA- DQA, -DQB, -DRA, and -DRB3 are located at BTA 23q21 and the non-classic class II genes DYA, DIB, LMP2, LMP7, TAP2, BoLA-DOB, -DMA, -DMB, and -DNA are located at BTA 23q12-->q13. These two different mapping locations confirm and extend previous findings of a gross physical distance between classic and non-classic MHC class II genes in cattle.

Alleles at a microsatellite locus within the macrophage expressed lysozyme gene were shown to co-segregate with lysozyme activity in two half-sib families of Polish Black and White Lowland cattle. The bimodal distribution of lysozyme activities in both progeny groups is concordant with the occurrence of the alternative paternal alleles. The microsatellite is linked to a locus for high lysozyme activity that accounts for 70-95% of the phenotypic variation of both offspring groups considering the lysozyme activities of animals being older than 1 month.

Quantitative trait loci (QTL) influencing body weight were mapped by linkage analysis in crosses between a high body weight selected line (DU6) and a control line (DUKs). The two mouse lines differ in body weight by 106% and in abdominal fat weight by 100% at 42 days. They were generated from the same base population and maintained as outbred colonies. Determination of line-specific allele frequencies at microsatellite markers spanning the genome indicated significant changes between the lines on 15 autosomes and the X chromosome. To confirm these effects, a QTL analysis was performed using structured F2 pedigrees derived from crosses of a single male from DU6 with a female from DUKs. QTL significant at the genome-wide level were mapped for body weight on chromosome 11; for abdominal fat weight on chromosomes 4, 11, and 13; for abdominal fat percentage on chromosomes 3 and 4; and for the weights of liver on chromosomes 4 and 11, of kidney on chromosomes 2 and 9, and of spleen on chromosome 11. The strong effect on body weight of the QTL on chromosome 11 was confirmed in three independent pedigrees. The effect was additive and independent of sex, accounting for 21-35% of the phenotypic variance of body weight within the corresponding F2 populations. The test for multiple QTL on chromosome 11 with combined data from all pedigrees indicated the segregation of two loci separated by 36 cM influencing body weight.

A methodical strategy for the isolation of microsatellite markers specific for targeted regions of bovine chromosomes is presented. The procedure involves directed microdissection of one defined subchromosomal area, its DOP-PCR-amplification and cloning. With this approach, a library specific to the BTA 6q21-31 chromosomal region was constructed. Eleven unique microsatellite-containing sequences were isolated, converted into sequence-tagged microsatellite sites, and characterized concerning their species-specific origin. Seven primer pairs generated bovine-specific PCR products and provided a set of microsatellite markers that generally revealed high informativity in the HF breed. Linkage analysis assigned six of them to their predefined subchromosomal origin on BTA 6 corresponding to the specific rehybridization signal of the DOP-PCR product generated from the microdissected chromosome area 6q21-31. The results underline the usefulness of the BTA 6q21-31 library for targeted isolation of unique sequences that are specific for the dissected chromosomal region as demonstrated here by the isolation of microsatellite markers.

The mapping strategy for the bovine genome described in this paper uses large insert clones as a tool for physical mapping and as a source of highly polymorphic microsatellites for genetic typing, and was one objective of the BovMap Project funded by the European Union (UE). Eight-three cosmid and phage clones were characterized and used to physically anchor the linkage groups defining all the bovine autosomes and the X Chromosome (Chr). By combining physical and genetic mapping, clones described in this paper have led to the identification of the linkage groups corresponding to Chr 9, 12, 16, and 25. In addition, anchored loci from this study were used to orient the linkage groups corresponding to Chr 3, 7, 8, 9, 13, 16, 18, 19, and 28 as identified in previously published maps. Comparison of the estimated size of the physical and linkage maps suggests that the genetic length of the bovine genome may be around 4000 cM.

A rapid procedure for the defined isolation and characterization of single bovine chromosome fragment specific probes is described. This has been developed as a technical prerequisite for the directed generation of bovine DNA sequences. The specific regions 1q13-24, 5q21-24, 6q31-32, 7q21-22, 12q24-ter, and 20q12-ter of bovine GTG-banded metaphase chromosomes were microdissected and amplified by PCR with a degenerate oligonucleotide primer and subsequently cloned into pBluescript II SK. The DNA probes generated were characterized by gel electrophoresis, dot blot analysis and rehybridization in situ to GTG-banded metaphase spreads. The position and size of the hybridization sites on the chromosomes correspond exactly to the dissected chromosome areas and indicate the complexity and specificity of the microdissected and amplified chromosome material.

A cosmid clone containing the complete sheep inhibin subunit beta B gene (INHBB) was assigned to sheep and cattle homologous chromosome bands 2q31-q33 by fluorescence in situ hybridization. The assignment of INHBB in sheep excludes another candidate gene as the site of the FecB mutation.

Five overlapping lambda EMBL-clones, containing the complete bovine lactoferrin gene (LTF), have been used to map this gene by fluorescence in situ hybridization to bovine Chromosome (Chr) band 22q24. Primers derived from promoter and exon I sequences were applied in polymerase chain reactions (PCRs) to DNA samples of a previously characterized panel of somatic cell hybrid lines, allowing the assignment of the bovine lactoferrin locus to syntenic group U12. These results permit the assignment of syntenic group U12 to bovine Chr 22.

The first aim of the study was the molecular genetic characterization of long-term trait-selected (growth, adaptability and fertility) lines of mice using multilocus DNA fingerprinting with the simple tandem repetitive oligonucleotide probes (GAA)6 and (GACA)4. Secondly polymorphic markers were screened for association with growth performance based on DNA fingerprint analysis in reference families. Pooled DNA samples of ten unrelated mice of trait-selected mouse lines (over 40 generations) and the unselected control lines were analyzed. Resulting differences in band patterns were reanalyzed comparing individual fingerprints of the animals included in the pooled DNA samples of the different lines. Between the mouse lines about 30% of analyzed fingerprint bands were polymorphic. Individuals of long-term selected mouse lines show only a few individual-specific bands. Most polymorphic bands observed in DNA mixes appear in all animals included in the DNA mix of the corresponding line. Line-specific DNA fingerprint bands were analyzed for their inheritance and linkage with growth performance in reference families using animals with extreme growth performance of the first backcross after crossing of growth-selected with unselected mice. Scanning the distribution of line-specific bands in the reference panel few bands were identified which are associated with growth performance. They appear as useful markers for growth selection. Nevertheless most line-specific bands result from genetic drift rather than from selection.

An easy assay for quantification of Y chromosome-bearing sperm (Y-sperm) is needed, especially to monitor sperm separation techniques. In the present study a tritiated bovine male-specific DNA fragment was tested for identification of Y-sperm by in situ hybridization. A protocol for in situ hybridization to bovine sperm was developed and used to study the proportion of Y-sperm of 12 bulls. The usefulness of the method in optimization of sperm separation procedures is illustrated through analysis of fractions of sperm separated by Percoll density gradient centrifugation.

Various methods for gene transfer in cattle are described. Four vectors containing (1) the bovine papilloma virus DNA, (2) the alcohol dehydrogenase gene of Drosophila melanogaster, and the human (3) and the bovine (4) growth hormone gene were used for gene injection in bovine zygotes. The period between 78 and 82 h after prostaglandin treatment was determined as the optimum time for collection of bovine zygotes. A total of 802 eggs was obtained from 74 successfully superovulated heifers. The foreign DNA was injected into the pronuclei of 156 (Construct 1), 130 (Construct 2), 62 (Construct 3) and 250 (Construct 4) centrifuged zygotes. An average number of 12 zygotes per recipient heifer was transferred into one oviduct. The eggs were recovered after 14 days (Constructs 1 and 2) for analysis or after 7 days (Constructs 3 and 4) respectively for transfer into final recipients. After the transfer into 23 recipient heifers of 43 embryos (Construct 4), 14 calves were born and 1 fetal monster was isolated. According to dot-blot hybridization the total rate of efficiency of gene transfer was 3.2%, 6.9%, and 0.8% for Constructs 1, 2 and 4, respectively.