Genomic organization and chromosomal mapping of the bovine Fas/APO-1 gene.

The cell-surface protein Fas (APO-1) is a member of the tumor necrosis factor receptor (TNFR) superfamily and transduces apoptosis following binding of Fas ligand or exposure to certain anti-Fas antibodies. We have isolated the bovine Fas (bFas) gene and determined its genomic organization and chromosomal location. Our data indicate that bFas is a single-copy gene that contains 9 exons and spans approximately 31.5 kb. The 5'-flanking region lacks conventional TATA and CCAAT elements, but contains several putative regulatory elements, including multiple copies of Sp1, AP-2, E-box, and N-box consensus sites. Linkage analysis of two (CA) dinucleotide repeat microsatellites within intron 1 and physical assignment by fluorescence in situ hybridization (FISH) placed the bFas gene on bovine chromosome 26. Collectively, these data provide a basis for understanding the regulatory mechanisms that control bFas gene expression.

Chromosomal localization and molecular characterization of 53 cosmid-derived bovine microsatellites.

Gene mapping in cattle has progressed rapidly in recent years largely owing to the introduction of powerful genetic markers, such as the microsatellites, and through advances in physical mapping techniques such as synteny mapping and fluorescence in situ hybridization (FISH). Microsatellite markers are often not physically mapped because they are generally isolated from small insert plasmid libraries, which makes their chromosomal localization inefficient. In this report we describe the FISH mapping of a large group of cosmid-derived bovine microsatellite markers, as our contribution to the European mapping initiative, BovMap. One objective of BovMap is to develop a set of anchored loci for the cattle genome map. Two cosmid libraries were screened with probes corresponding to the (AC)n microsatellite motif. Positive clones were mapped by FISH, and then a subset was further analyzed by sequencing the region flanking the microsatellite repeat. In total, 58 clones were hybridized with chromosomes and identified loci on 22 of the 31 different bovine chromosomes. Three clones contained satellite DNA. Two or more markers were placed on 12 chromosomes. Sequencing of the microsatellites and flanking regions was performed directly from 43 cosmids, as previously reported (Ferretti et al. Anim. Genet. 25, 209-214, 1994). Primers were developed for 39 markers and used to describe the polymorphism associated with the corresponding loci.

Physical and linkage mapping of the bovine genome with cosmids.

We have initiated a mapping strategy using cosmid clones to chromosomally anchor a high-resolution bovine genetic linkage map. Ten cosmids containing microsatellites were assigned to bovine chromosomes by fluorescence in situ suppression hybridization (FISH). Four cosmid clones, three of which contain an informative microsatellite, were assigned to autosomes 5, 13, 24, and 28. The assignment to autosome 13 anchors bovine syntenic group U11. Two additional cosmid clones, each containing informative microsatellites, are assigned to autosomes 9 and 29, anchoring bovine linkage groups U2 and U8, respectively. Four cosmid clones, three of which contain informative microsatellites, also provide the first assignment to autosome 25, anchoring bovine syntenic group U7 and orienting the corresponding linkage group relative to the centromere.

Isolation of the human genomic brain-2/N-Oct 3 gene (POUF3) and assignment to chromosome 6q16.

N-Oct 3 is a human POU domain transcription factor that binds to the octamer sequence ATGCAAAT. The protein is expressed in the central nervous system during development and in adult brain. We have isolated and characterized genomic clones encoding the human N-Oct 3 gene (HGMW-approved symbol POUF3). Comparison of the structure of these clones with the N-Oct 3 cDNA revealed that POUF3 is an intronless gene. Sequencing of 650 bp of the promoter region showed 84% sequence identity of POUF3 with its murine homologue, the brain-2 (designated brn-2) gene. Whereas both POUF3 and brn-2 lack a TATA box, consensus sequences for AP-2, GCF, and SP1 transcription factors were identified within the highly conserved 5'-flanking region. These sequences may play a crucial role for the tissue-specific transcription activation of the POUF3 gene. Southern blotting and in situ hybridization localized the human POUF3 gene to chromosome 6q16.

Chromosomal localization and detection of DNA polymorphisms in the bovine polymeric immunoglobulin receptor gene.

Polymeric immunoglobulin receptor (PIGR) mediates transcellular transport of secretory antibodies in glandular and mucosal epithelial cells. By use of a bovine-rodent somatic cell hybrid panel the bovine PIGR locus has been assigned to syntenic group U1. Using in situ hybridization, PIGR was localized to bovine chromosome 16, segment q13, thus confirming the recent assignment of syntenic group U1 to this chromosome. Two common restriction fragment length polymorphisms (RFLPs) with the enzymes BamHI and MspI were detected using the PIGR cDNA as probe. Direct PCR sequencing of a segment in the PIGR coding region (nucleotides 162-413) from 13 bulls of Norwegian Cattle revealed single nucleotide exchanges at two positions. An efficient PCR-RFLP method for detection of these mutations was developed.

A genetic linkage map for cattle.

We report the most extensive physically anchored linkage map for cattle produced to date. Three-hundred thirteen genetic markers ordered in 30 linkage groups, anchored to 24 autosomal chromosomes (n = 29), the X and Y chromosomes, four unanchored syntenic groups and two unassigned linkage groups spanning 2464 cM of the bovine genome are summarized. The map also assigns 19 type I loci to specific chromosomes and/or syntenic groups and four cosmid clones containing informative microsatellites to chromosomes 13, 25 and 29 anchoring syntenic groups U11, U7 and U8, respectively. This map provides the skeletal framework prerequisite to development of a comprehensive genetic map for cattle and analysis of economic trait loci (ETL).

Physically mapped, cosmid-derived microsatellite markers as anchor loci on bovine chromosomes.

To identify physical and genetic anchor loci on bovine chromosomes, 13 cosmids, obtained after the screening of partial bovine cosmid libraries with the (CA)n microsatellite motif, were mapped by fluorescence in situ hybridization (FISH). Eleven cosmid probes yielded a specific signal on one of the bovine chromosomes and identified the following loci: D5S2, D5S3, D6S3, D8S1, D11S5, D13S1, D16S5, D17S2, D19S2, D19S3, D21S8. Two cosmids produced centromeric signals on many chromosomes. The microsatellite-containing regions were subcloned and sequenced. The sequence information revealed that the two centromeric cosmids were derived from bovine satellites 1.723 and 1.709, respectively. A cosmid located in the subtelomeric region of Chromosome (Chr) 17 (D17S2) had features of a chromosome-specific satellite. Primers were designed for eight of the nonsatellite cosmids, and seven of these microsatellites were polymorphic with between three and eight alleles on a set of outbred reference families. The polymorphic and chromosomally mapped loci can now be used to physically anchor other bovine polymorphic markers by linkage analysis. The microsatellite primers were also applied to DNA samples of a previously characterized panel of somatic hybrid cell lines, allowing the assignment of seven microsatellite loci to defined syntenic groups. These assignments confirmed earlier mapping results, revealed a probable case of false synteny, and placed two formerly unassigned syntenic groups on specific chromosomes.