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NK-lysin is an effector protein of the innate immune system and an important component of host protection. We isolated a SNP in the NK-lysin coding sequence among different chicken breeds. This A to G substitution at the position 271 nucleotide in the ORF results in an Asn (N) to Asp (D) amino acid alteration. We synthesized two 30-aa peptides (N29N and N29D) to compare the biological activity of the helix 2-loop-helix 3 region of NK-lysin resulting from the polymorphic gene. Both peptides were found to be cytotoxic in bacteria and tumor cell cultures at micromolar concentrations. The N29N peptide, however, exhibited greater antibacterial and anticancer activity than the N29D peptide. Circular dichroism spectroscopy of the two peptides in negatively charged single unilamellar vesicles showed spectra typical of (»-helical peptides. The helical profile of N29D was reduced substantially compared with that of N29N. However, no structural change was observed in neutral vesicles. Ç-Potential measurements of liposomes incubated with increasing peptide concentrations allowed surface charge neutralization with a negatively charged lipid, but not with a zwitterionic lipid. This result suggests that a difference in electrostatic interaction between lipid membranes and the helical peptides results from the polymorphic gene and is subsequently an important factor in cell lytic activity of variant NK-lysin peptides. [ABSTRACT FROM AUTHOR]

Retinoic acid inducible gene I (RIG-I) and mitochondrial antiviral signaling (MAVS) proteins have recently been found to operate in a pathway for the detection and subsequent elimination of replicating viral genomes. Because of this innate immunity role, RIG-I and MAVS are candidates for studies of disease resistance. The objectives of this work were to (1) radiation hybrid (RH) map bovine RIG-I and MAVS and (2) perform comparative sequence analysis of partial genomic sequence from each gene. Using a bovine 5000rad RH panel, RIG-I was localized to BTA08 (LOD > 12) and MAVS was localized to BTA13 (LOD > 12). RIG-I exon 14 and partial MAVS exon five were sequenced in nine breeds and compared with available sequence from the Bovine Genome Project. RIG-I exon 14 and partial MAVS exon five were conserved in all samples examined. One T-A transversion SNP was found in intronic sequence downstream of RIG-I exon 14. [ABSTRACT FROM AUTHOR]

Approximately 3,000 cattle bacterial artificial chromosome (BAC)- end sequences were added to the Illinois-Texas 5,000-rad RH (RH, radiation hybrid) map. The BAC-end sequences selected for mapping are ≈1 Mbp apart on the human chromosomes as determined by BLASTN analysis. The map has 3,484 ordered markers, of which 3,204 are anchored in the human genome. Two hundred-and-one homologous synteny blocks (HSBs) were identified, of which 27 are previously undiscovered, 79 are extended, 26 were formed by previously unrecognized breakpoints in 18 previously defined HSBs, and 23 are the result of fusions. The comparative coverage relative to the human genome is ≈91 %, or 97% of the theoretical maximum. The positions of 64% of all cattle centromeres and telomeres were reassigned relative to their positions on the previous map, thus facilitating a more detailed comparative analysis of centromere and telomere evolution. As an example of the utility of the high-resolution map, 22 cattle BAC fingerprint contigs were directly anchored to cattle chromosome 19 (Bos taurus, (BTA) 19]. The order of markers on the cattle RH and fingerprint maps of BTA19 and the sequence-based map of human chromosome 17 (Homo sapiens, (HSA) 17] were found to be highly consistent with only two minor ordering discrepancies between the RH map and fingerprint contigs. The high-resolution Illinois-Texas 5,000-rad RH and comparative maps will facilitate identification of candidate genes for economically important traits, the phylogenomic analysis of mammalian chromosomes, proofing of the BAC fingerprint map and, ultimately, aid the assembly of cattle whole-genome sequence. [ABSTRACT FROM AUTHOR]

Genome research in animals used in agriculture has progressed rapidly in recent years, moving from rudimentary genome maps to trait maps to gene discovery. These advances are the result of animal genome projects following closely in the footsteps of the Human Genome Project, which has opened the door to genome research in farm animals. In return, genome research in livestock species is contributing to our understanding of chromosome evolution and to informing the human genome. Enhancement of these contributions plus the much anticipated application of DNA-based tools to animal health and production can be expected as livestock genomics enters its sequencing era. [ABSTRACT FROM AUTHOR]

A high-resolution (1 marker/700 kb) physically ordered radiation hybrid (RH) and comparative map of 122 loci on equine homologs of human Chromosome 19 (HSA19) shows a variant evolution of these segments in equids/Perissodactyls compared with other mammals. The segments include parts of both the long and the short arm of horse Chromosome 7 (ECA7), the proximal part of ECA21, and the entire short arm of ECA10. The map includes 93 new markers, of which 89 (64 gene-specific and 25 microsatellite) were genotyped on a 5000-rad horse × hamster RH panel, and 4 were mapped exclusively by FISH. The orientation and alignment of the map was strengthened by 21 new FISH localizations, of which 15 represent genes. The approximately sevenfold-improved map resolution attained in this study will prove extremely useful for candidate gene discovery in the targeted equine chromosomal regions. The highlight of the comparative map is the fine definition of homology between the four equine chromosomal segments and corresponding HSA19 regions specified by physical coordinates (bp) in the human genome sequence. Of particular interest are the regions on ECA7 and ECA21 that correspond to the short arm of HSA19—a genomic rearrangement discovered to date only in equids/Perissodactyls as evidenced through comparative Zoo-FISH analysis of the evolution ofancestral HSA19 segments in eight mammalian orders involving about 50 species. [ABSTRACT FROM AUTHOR]

We herein report a detailed physical map of the horse Y chromosome. The euchromatic region of the chromosome comprises ≈ 15 megabases (Mb) of the total 45- to 50-Mb size and lies in the distal one-third of the long arm, where the pseudoautosomal region (PAR) is located terminally. The rest of the chromosome is predominantly heterochromatic. Because of the unusual organization of the chromosome (common to all mammalian Y chromosomes), a number of approaches were used to crossvalidate the results. Analysis of the 5000-rad horse × hamster radiation hybrid panel produced a map Spanning 88 centirays with 8 genes and 15 sequence-tagged site (STS) markers. The map was verified by several fluorescence in situ hybridization approaches. Isolation of bacterial artificial chromosome (BAC) clones for the radiation hybrid-mapped markers, end sequencing of the BACs, STS development and bidirectional chromosome walking yielded 109 markers (100 STS and 9 genes) contained in 73 BACs. STS content mapping grouped the BACs into seven physically ordered contigs (of which one is predominantly ampliconic) that were verified by metaphase-, interphase-, and fiber-fluorescence in situ hybridization and also BAC fingerprinting. The map spans almost the entire euchromatic region of the chromosome, of which 20-25% (≈ 4 Mb) is covered by isolated BACs. The map is presently the most informative among Y chromosome maps in domesticated species, third only to the human and mouse maps. The foundation laid through the map will be critical in obtaining complete sequence of the euchromatic region of the horse Y chromosome, with an aim to identify Y specific factors governing male infertility and phenotypic sex variation. [ABSTRACT FROM AUTHOR]

Development of a dense map of the horse genome is key to efforts aimed at identifying genes controlling health, reproduction, and performance. We herein report a high-resolution gene map of the horse (Equus caballus) × chromosome (ECAX) generated by developing and typing 116 gene-specific and 12 short tandem repeat markers on the 5,000-rad horse × hamster whole-genome radiation hybrid panel and mapping 29 gene loci by fluorescence in situ hybridization. The human × chromosome sequence was used as a template to select genes at 1-Mb intervals to develop equine orthologs. Coupled with our previous data, the new map comprises a total of 175 markers (139 genes and 36 short tandem repeats, of which 53 are fluorescence in situ hybridization mapped) distributed on average at ≈880-kb intervals along the chromosome. This is the densest and most uniformly distributed chromosomal map presently available in any mammalian species other than humans and rodents. Comparison of the horse and human × chromosome maps shows remarkable conservation of gene order along the entire span of the chromosomes, including the location of the centromere. An overview of the status of the horse map in relation to mouse, livestock, and companion animal species is also provided. The map will be instrumental for analysis of × linked health and fertility traits in horses by facilitating identification of targeted chromosomal regions for isolation of polymorphic markers, building bacterial artificial chromosome contigs, or sequencing. [ABSTRACT FROM AUTHOR]

The 5′-AMP-activated protein kinase (AMPK) family is an ancient stress response system whose primary function is regulation of cellular ATP. Activation of AMPK, which is instigated by environmental and nutritional stresses, initiates energy-conserving measures that protect the cell by inhibition and phosphorylation of key enzymes in energy-consuming biochemical pathways. The seven genes that comprise the bovine AMPK family were mapped in cattle by using a radiation hybrid panel. The seven genes mapped to six different cattle chromosomes, each with a LOD score greater than 10.0. PRKAA1 mapped to BTA 20, PRKAA2 and PRKAB2 to BTA 3, PRKAB1 to BTA 17, PRKAG1 to BTA 5, PRKAG2 to BTA 4, and PRKAG3 to BTA 2. Five of the seven genes mapped to regions expected from human/cattle comparative maps. PRKAB2 and PRKAG3, however, have not been mapped in humans. We predict these genes to be located on HSA 1 and 2, respectively. Additionally, one synonymous and one non-synonymous single nucleotide polymorphism (SNP) were detected in PRKAG3 in Bos taurus cattle. In an effort to determine ancestral origins, various herds of mixed breed cattle as well as other ruminant species were characterized for sequence variation in this region of PRKAG3. Owing to the physiological importance of this gene family, we believe that its individual genes are candidate genes for conferring resistance to diseases in cattle. [ABSTRACT FROM AUTHOR]

Human BRCA1 has a genetically demonstrated role in DNA repair, and has been proposed to act as a transcriptional activator in a limited number of specialized settings. To gain insight into biologically conserved functional motifs, we isolated an ortholog of BRCA1 from cattle (Bos taurus). The predicted protein product shows 72.5% sequence identity with the human protein and conservation of amino acids involved in BRCA1 structure and function. Although the bovine C-terminus is truncated by seven amino acids as compared to human, bovine BRCA1 protein exhibited a similar cell cycle-regulated nuclear expression pattern. Expression was characteristically low and diffuse in the nucleus of G1/G0 cells, followed by increasing BRCA1-positive nuclear speckles in late S phase and G2/M phase cells. Bovine BRCA1 was phosphorylated and nuclear speckling was enhanced in response to DNA-damaging agents. Consistent with evidence from studies of human BRCA1, bovine BRCA1 was shown to interact with RNA polymerase II in vivo, an activity that was mapped to the C-terminal domain (CTD) (bBRCA1364-1849). Interestingly, when tested in the GAL4 transcriptional activation assay, full-length bovine and human BRCA1 lacked any ability to act as transcriptional activators and the CTD of bovine BRCA1 had five-fold lower activity when compared to the more acidic human C-terminus. These results provide evidence that phosphorylation and nuclear relocalization are highly conserved features of the BRCA1 response to genotoxic stress. In addition, bovine BRCA1 binds the RNA polymerase II holoenzyme, but this interaction lacks significant ability to correctly orient or recruit RNA polymerase II for transcription in the classic GAL4 transcriptional activation system.Oncogene (2003) 22, 6032-6044. doi:10.1038/sj.onc.1206515 [ABSTRACT FROM AUTHOR]

Toll-like receptor 4 (TLR4) is a cell-surface receptor that activates innate and adaptive immune responses. Because it recognizes a broad class of pathogen-associated molecular patterns presented by lipopolysaccharides and lipoteichoic acid, TLR4 is a candidate gene for resistance to a large number of diseases. In particular, mouse models suggest TLR4 as a candidate gene for resistance to major agents in bovine respiratory disease and Johne's disease. The coding sequence of bovine TLR4 is divided into three exons, with intron/exon boundaries and intron sizes similar to those of human TLR4 transcript variant 1. We amplified each exon in 40 individuals from 11 breeds and screened the sequence for singlenucleotide polymorphisms (SNPs). We identified 32 SNPs, 28 of which are in the coding sequence, for an average of one SNP per 90 bp of coding sequence. Eight SNPs were nonsynonymous and potentially alter specificity of pathogen recognition or efficiency of signaling. To evaluate the functional importance of these SNPs, we used codon-substitution models to detect diversifying selection in an extracellular region that may physically interact with ligands. One nonsynonymous SNP is located within this region, and other substitutions are in adjacent regions that may interact with coreceptor molecules. The 32 SNPs were found in 20 haplotypes that can be assigned to geographic ranges of origin. Haplotype-tagging SNP analysis indicated that 12 SNPs need to be genotyped to distinguish these 20 haplotypes. These data provide a basic understanding of bovine TLR4 sequence variation and supply haplotype markers for disease association studies. [ABSTRACT FROM AUTHOR]

Toll-like receptors are cell-surface receptors that activate innate and adaptive immune responses. We have used a 5000-rad, whole-genome radiation hybrid panel to map Toll-like receptor 4 (TLR4) to the distal end of bovine Chromosome (Chr) 8, and Toll-like receptor 2 (TLR2) to the proximal end of bovine Chr 17. To facilitate comparative mapping and contig construction, we have also used 5000- and 12,000-rad, whole-genome radiation hybrid panels to produce fine maps of the regions surrounding these genes in cattle. These fine maps triple the number of available markers in the TLR4 region and more than double the number of available markers in the TLR2 region. Comparative analyses show gene order conservation between the bovine Chr 8 region and human Chr 9, and between the bovine Chr 17 region and human Chr 4. In addition, the bovine Chr 8 region refines an evolutionary chromosomal breakpoint from a 10-megabase region to a 2.5-megabase region, and the bovine Chr 17 map suggests a new evolutionary chromosomal breakpoint. [ABSTRACT FROM AUTHOR]

Examines the role of gene, LIMBIN (Lbn) in the development of bovine chondrodysplastic dwarfism. Characterization of autosomal recessive disorder by short limbs; Expression of Lbn gene in limb buds of developing mouse embryos; Proliferation and differentiation of chondrocytes.

Examines the association between lysine-232/alanine polymorphism in a bovine gene encoding acyl-coenzyme A:diacylglycerol acyltransferase and variation at a quantitative trait locus for milk fat content. Estimation of allele frequencies on the basis of sequencing traces; Detection of genetic polymorphism; Distributions of breeding values for milk fat.

We present a comprehensive radiation hybrid map of the bovine X chromosome (Chr) containing 20 new markers, including both microsatellites and expressed genes. This study was conducted with a 5000-rad whole genome RH cell panel consisting of 90 hybrid cell lines. Retention frequencies of individual markers range from 7.8% for XIST to 31.1% for TGLA325. Statistical analysis with RHMAPPER placed all the loci into five linkage groups under a LOD score criterion of 6.0. These groups could be oriented relative to each other because they included multiple microsatellite loci from the consensus linkage map of the X Chr. Markers included in both this RH map and the bovine cytogenetic map were in a consistent order. The comparative bovine–human map thus generated consists of five blocks of genes, the order of which is conserved, although in the opposite direction when presented as ideograms with p and q arms. Inversions of three blocks account for the difference in gene order across the entirety of the two X Chrs. [ABSTRACT FROM AUTHOR]

Examines the radiation hybrid map and high resolution comparative map of Bos taurus (BTA) chromosome 18. Total length of comprehensive map; Expansion of the existing BTA 18 gene map; Chromosome evolution in cattle, humans and mice; Identification of genes for important traits.

A 5000rad whole-genome radiation hybrid (RH) panel was created for the horse. The usefulness of the panel for generating physically ordered maps of individual equine chromosomes was tested by typing 24 markers on horse Chromosome 11 (ECA11). The overall retention of markers on this chromosome was 43.6%. Almost complete retention of two of the typed markers—CA062 and AHT44—clearly indicated the location of thymidine kinase gene on the short arm of ECA11. Seven of the typed markers were FISH mapped to align the RH and cytogenetic maps. With the RH-MAPPER approach, a physically ordered map comprising four linkage groups and incorporating all the markers was obtained. The study provides the first comprehensive map for a horse chromosome that integrates all available mapping data and adds new information that spans the entire length of the equine chromosome. The map clearly underlines the resolving power and utility of the panel and emphasizes the need to have uniformly distributed cytogenetic markers for appropriate alignment of RH map with the chromosome. A comparative status of the ECA11 map in relation to the corresponding human/mouse chromosome is presented. [ABSTRACT FROM AUTHOR]

mRNA differential display was applied to identify hepatic and intestinal expressed sequence tags (ESTs) in lactating cows of different metabolic types (milk type, meat/milk type, meat type) that are potentially associated with energy turnover and involved in the regulation of these processes. Altogether, 277 ESTs (liver: 161, intestine: 116) were identified. For 150 transcripts (liver: 99, intestine: 51), the sequences showed similarity to previously described genes and ESTs. Many of these homologous sequences are reported to be involved in hepatic metabolism. Ninety-four ESTs (liver: 43, intestine: 51) did not match with any database entries. Semi-quantitative RT-PCR revealed quantitative differences in transcript represented by randomly chosen ESTs in liver samples of animals of the Holstein and Charolais breeds. One hundred twenty-two ESTs were mapped physically by using a bovine-hamster somatic cell hybrid panel (SCP) and a 5000-rad bovine whole genome radiation hybrid panel (WGRH). These ESTs were assigned to the bovine syntenic groups and positioned in the recently established RH-based ordered comparative map of the cattle and human genomes. The mapped, differentially expressed sequence tags are a useful prerequisite for cloning of genetic variation underlying economic traits. [ABSTRACT FROM AUTHOR]

The callipyge (CLPG) gene was fine-mapped by linkage analysis to a 4.6-cM chromosome interval on distal ovine OAR18q, flanked by microsatellite markers IDVGA30 and OY3. The OAR18q linkage map and human HSA14q transcript map were aligned by genotyping two bovine-hamster whole-genome radiation hybrid panels with the microsatellite markers, as well as with sequences corresponding to HSA14q genes. Using Type I loci mapping to the IDVGA30–OY3 interval as anchor points, we have constructed a 1.4-Mb bovine BAC contig containing the IDVGA30–OY3 interval. We demonstrate that the IDVGA30– OY3 interval spans approximately 770 kb and contains at least four genes: YY1, WARS, DLK1, and GTL2. [ABSTRACT FROM AUTHOR]

Comparative mapping data on evolutionary conserved coding sequences and synteny maps between human and cattle are insufficient to define the extent and distribution of conserved segments between these two species, because the order of loci is often rearranged. A 5000-rad cattle whole-genome radiation hybrid (WG-RH) panel was constructed to provide high-resolution comparative maps and also to integrate linkage maps of microsatellites with evolutionary conserved genes and transcripts in a single ordered map. We used the WG-RH panel to construct radiation hybrid maps of bovine Chromosomes (Chrs) 15 and 29 (BTA15 and BTA29), integrating microsatellites from published linkage maps with selected genes. The comprehensive map of BTA15 consists of 24 markers, 13 of which were placed in the framework map. Eleven molecular markers compose the comprehensive map of BTA29, seven of which were placed in the framework map. We identified the homologous regions between bovine Chr 15 (BTA15) and human Chrs 5 and 11 (HSA5 and HSA11), as well as between BTA29 and HSA11. The present study demonstrates that WG-RH mapping is an efficient method for integrating multiple genetic maps into one map and for incorporating monomorphic Type I loci into ordered maps for comparison between species. [ABSTRACT FROM AUTHOR]

Two major differences were detected in gene order between the radiation hybrid map and the genetic linkage map of bovine Chromosome (Chr) 5, and these were resolved by analyzing the raw radiation hybrid data by a quasi-phylogenetic method. Seventeen loci were typed on the new cattle whole genome radiation hybrid panel. Most of these loci are framework loci and include AGLA293, BM315, BM6026, BP1, BZRP, CD9, CSSM22, CSSM34, CYP2D@, ETH2, ETH10, ETH152, IGF1, LALBA, SLC2A3, SYT1, and TPI1. BP1 was found to be closer to the centromere than either BM6026 or SYT1 with two standard computer software packages for analyzing radiation hybrid panel data. This is inconsistent with any of the genetic linkage maps as well as their consensus. CYP2D@ was placed between ETH2 and BZRP, and this is also inconsistent with the genetic linkage maps, since CYP2D@ should be the most telomeric of the loci tested in this study. Resolution was reached by analyzing the raw radiation hybrid data for clones that bind some but not all of the loci, and the binding pattern was more consistent with the linkage maps and less consistent with the software-generated radiation hybrid map. The comparative mapping data confirm the relative inversion of gene order of SYT1 compared with humans and mice. A non-polymorphic fragment for CD9 indicates the conservation of gene order for three loci located on human Chr 12p. The genes of bovine Chr 5 conserved on human Chr 12p are located separately from the genes conserved on human Chr 12q. It is recommended that the raw data for radiation hybrid maps be made publicly available so that conflicts in gene order can be evaluated explicity. [ABSTRACT FROM AUTHOR]

A bovine mammary gland cDNA-library was used to characterize and map genes expressed during lactation. Fifty cDNA clones selected by differential hybridization were sequenced from both ends, and sequences were examined for similarities with database sequences. For 34 of the transcripts, the sequences showed more than 80% similarity to previously characterized genes or expressed sequence tags (ESTs). Twenty cDNAs that could be of interest as candidate genes for milk production are selected for genetic or chromosomal mapping. Twenty-three out of the 39 designed primer pairs representing 16 cDNA clones amplified the expected fragments and were used for subsequent fluorescent single-strand conformation polymorphism analysis (F-SSCP) in the International Bovine Reference Panel families (IBRP). Ten polymorphic loci could be identified and used to genotype the IBRP animals, and nine of them were subsequently genetically mapped on nine chromosomes. In addition, eight loci from the 16 cDNA clones could be mapped by somatic cell hybrids, bringing the total number of mapped genes to 16, one of which was mapped genetically as well as physically. The mapped mammary gland ESTs are potentially useful for cloning economic trait loci by a positional candidate gene cloning approach. [ABSTRACT FROM AUTHOR]

A closed YAC contig spanning the mh locus was assembled by STS content mapping with seven microsatellite markers, eight genes or EST, and nine STS corresponding to YAC ends. The contig comprises 27 YACs, has an average depth of 4.3 YACs, and spans an estimated 1.2 Mb. A linkage map was constructed based on five of the microsatellite markers anchored to the contig and shown to span 7 cM, yielding a ratio of 160 kb/1 cM for the corresponding chromosome region. Comparative mapping data indicate that the constructed contig spans an evolutionary breakpoint connecting two chromosome segments that are syntenic but not adjacent in the human. Consolidation of human gene order by means of whole genome radiation hybrids and its comparison with the bovine order as inferred from the contig confirm conservation of gene order within segments. [ABSTRACT FROM AUTHOR]

Provides an overview of the state of comparative genetics in mammals. Development of animal models of human inborn errors; Complexities of mammalian immune gene complexes; Application of hybrid somatic cells to gene mapping.