A. M. Ryan

A genetic linkage map of the bovine genome

A cattle genetic linkage map was constructed which marks about 90% of the expected length of the cattle genome. Over 200 DNA polymorphisms were genotyped in cattle families which comprise 295 individuals in full sibling pedigrees. One hundred and seventy–one loci were found linked to one other locus. Twenty nine of the 30 chromosome pairs are represented by at least one of the 36 linkage groups. Less than a 50 cM difference was found in the male and female genetic maps. The conserved loci on this map show as many differences in gene order compared to humans as is found between humans and mice. The conservation is consistent with the patterns of karyotypic evolution found in the rodents, primates and artiodactyls. This map will be important for localizing quantitative trait loci and provides a basis for further mapping.

A genetic map of DNA loci on bovine chromosome 1

We constructed a genetic map of most of the length of bovine chromosome 1 using the CSIRO and the Texas A&M University cattle reference families. Twelve loci are in a single linkage group, 9 of which are highly polymorphic loci. Four loci are of known biochemical function, alpha-1 crystallin (CRYA1), gamma-s crystallin (CRYG8), superoxide dismutase 1 (SOD1), and uridine monophosphate synthase (UMPS), and these have also been previously mapped in humans. The loci CRYA 1, CSRD 1613, GMBT 7, RM 95, SOD1, and UMPS had been previously assigned to bovine syntenic group U10, while CSRD 1613 and UMPS had also been assigned to chromosome 1 by in situ hybridization. All of the loci show statistically significant linkage to at least one other locus. The conserved loci indicate that there have been major rearrangements during the evolution of bovine chromosome 1 compared to other mammalian chromosomes. The estimate of the total length of the linkage group is 168 cM, which accords well with the predicted length based on chiasmata frequencies for the bovine genome and the relative size of chromosome 1 in the bovine genome.

Somatic cell mapping of conglutinin (CGN1) to cattle syntenic group U29 and fluorescence in situ localization to Chromosome 28

A 260-bp genomic PstI fragment, which encodes a portion of the carbohydrate recognition domain, was used along with hybrid somatic cells to map the conglutinin gene (CGN1) to domestic cow (Bos taurus) syntenic group U29. In turn, a cosmid containing the entire bovine CGN1 was used with fluorescence in situ hybridization to sublocalize this gene to cattle chromosome (Chr) (BTA) 28 band 18. Since BTA 28 and several of the other small acrocentric autosomes of cattle are difficult to discriminate, we have also chromosomally sublocalized CGN1 to the p arm of the lone biarmed autosome of the gaur (Bos gaurus). The use of the gaur 2/28 Robertsonian as a marker chromosome and our assignment of CGN1 to BTA 28 should help resolve some of the nomenclatural questions involving this cattle chromosome.

Validation of rat reference genes for improved quantitative gene expression analysis using low density arrays

Real-time PCR has become increasingly important in gene expression profiling research, and it is widely agreed that normalized data are required for accurate estimates of messenger RNA (mRNA) expression. With increased gene expression profiling in preclinical research and toxicogenomics, a need for reference genes in the rat has emerged, and the studies in this area have not yet been thoroughly evaluated. The purpose of our study was to evaluate a panel of rat reference genes for variation of gene expression in different tissue types. We selected 48 known target genes based on their putative invariability. The gene expression of all targets was examined in 11 types of rat tissues using TaqMan low density array (LDA) technology. The variability of each gene was assessed using a two-step statistical model. The analysis of mean expression using multiple reference genes was shown to provide accurate and reliable normalized expression data. The least five variable genes from each specific tissue were recommended for future tissue-specific studies. Finally, a subset of investigated rat reference genes showing the least variation is recommended for further evaluation using the LDA platform. Our work should considerably enhance a researchers ability to simply and efficiently identify appropriate reference genes for given experiments.

Somatic cell mapping of β-defensin genes to cattle syntenic group U25 and fluorescence in situ localization to Chromosome 27

Antimicrobial peptides have been isolated from a diversity of animal species including insects, lower vertebrates, and mammals (Boman et al. 1994). These peptides are classified into molecular families on the basis of structural features (Boman 1995) and have a proposed role in innate host defense (Zasloff 1992). These peptides exhibit broad-spectrum antimicrobial activity in vitro, owing to their ability to disrupt membranes (Boman 1995). In mammals, two families of cysteine-rich antimicrobial peptides have been recognized. The defensins have been identified in circulating phagocytic cells and in specialized epithelial cells of the small intestine of several species (Lehrer et al. 1993). More recently, a second family of structurally related peptides was discovered. Tracheal antimicrobial peptide (TAP) was the first of these peptides to be isolated and characterized (Diamond et al. 1991) from the bovine tracheal mucosa. Subsequently, 13 additional peptides were isolated from the neutrophils of the cow by Selsted and colleagues (1993), who named this family ~-defensins. Although peptides of both defensins and ~-defensins are similar in size, in vitro antibiotic activity, and structural features, they are clearly distinguish, able by consensus sequences at the gene and protein levels (Bevins 1994; Diamond et al. 1993), and by their disulfide array (Selsted and Harwig 1989; Tang and Selsted 1993). The ~-defensins are encoded by a large family (Diamond et al. 1993). During the isolation of the TAP gene (clone G-3), several additional cross-hybridizing clones were isolated (Diamond et al. 1993). Upon sequence characterization, several of these clones were predicted to encode other ~-defensins. Three of these clones have been further characterized. The clone designated G-I was determined by Northern blot analysis to be expressed exclusively in bone marrow (A. Tarver, G. Diamond, and C.L. Bevins, unpublished observations). Clone G-6 encodes a ~-defensin, BNBD-4 (Yount et al. 1994), found in bovine neutrophils (Selsted et al. 1993), and clone G-11 is expressed in enteric epithelial cells (A. Tarver, G. Diamond, and C.L. Bevins, manuscript in preparation). Sequence similarity among the [3-defensins strongly suggests that this molecular family is derived by duplication of an ancestral gene. To further study the evolutionary history and tissue-specific

Chromosomal localization of omega and trophoblast interferon genes in cattle and river buffalo by sequential R-banding and fluorescent in situ hybridization

Fluorescent in situ hybridization (FISH) of the cattle cDNA probe bTP-509 to RBA-banded cattle (Bos taurus L.) chromosomes confirmed the assignment of the omega (IFNW) and trophoblast (IFNT) interferon genes to chromosome 8q15. Using the same probe, these genes were also localized to river buffalo (Bubalus bubalis L.) chromosome 3q15 following sequential RBA-banding and FISH. The extensive G- and R-banding homology observed between cattle chromosome 8 and river buffalo chromosome arm 3q supports the conserved chromosomal location of the IFNW and IFNT genes in these two species.

Physical mapping of the lysozyme gene family in cattle

Amplification of an ancestral lysozyme gene in artiodactyls is associated with the evolution of foregut fermetation in the ruminant lineage and has resulted in about ten lysozyme genes in true ruminants. Hybridization of a cow stomach lysozyme 2 cDNA clone to restricted DNAs of a panel of cowxhamster hybrid cell lines revealed that all but one of the multiple bovine-specific bands segregate concordantly with the marker for bovine syntenic group U3 [Chromosome (Chr) 5]. The anomalous band was subsequently mapped to bovine syntenic group U22 (Chr 7) with a second panel of hybrids representing all 31 bovine syntenic groups. By two-dimensional pulsed-field gel electrophoresis the lysozyme genes on cattle Chr 5 were shown to be clustered on a 2- to 3-Mb DNA fragment, while the lactalbumin gene and pseudogenes that are paralogous and syntenic with the lysozymes were outside the lysozyme gene cluster. Chromosomal fluorescence in situ hybridization of a cocktail of lysozyme genomic clones localized the lysozyme gene cluster to cattle Chr 5 band 23, corroborating the somatic cell assignment.

Syntenic mapping and chromosomal localization of bovine ? and ? interferon genes

The previous assignment of bovine α-(IFNA) and β-(IFNB) interferon gene families to syntenic group U18 was confirmed with additional cDNA probes and a bovine-rodent hybrid somatic cell panel representing all 29 bovine autosomal syntenic groups. Fluorescent in situ hybridization (FISH) localized these genes to bovine Chromosome (Chr) 8 band 15 and demonstrates that with biotinylated plasmids, as few as five tandemly arrayed sequences can be detected by conventional fluorescent microscopy. This technique can be applied to physical mapping of other multicopy genes in domestic animals.

Somatic cell mapping of omega and trophoblast interferon genes to bovine syntenic group U18 and in situ localization to chromosome 8.

Bovine omega (IFNW) and trophoblast (IFNT) interferon genes were assigned to bovine syntenic group U18 by somatic cell genetics. Fluorescent in situ hybridization subsequently localized these genes to bovine chromosome 8 band 15. This assignment conflicts with a previous assignment of U17 genes to the same chromosome.

Chromosomal localization of uroplakin genes of cattle and mice

The asymmetric unit membrane (AUM) of the apical surface of mammalian urinary bladder epithelium contains several major integral membrane proteins, including uroplakins IA and IB (both 27 kDa), II (15 kDa), and III (47 kDa). These proteins are synthesized only in terminally differentiated bladder epithelial cells. They are encoded by separate genes and, except for uroplakins IA and IB, appear to be unrelated in their amino acid sequences. The genes encoding these uroplakins were mapped to chromosomes of cattle through their segregation in a panel of bovine x rodent somatic cell hybrids. Genes for uroplakins IA, IB, and II were mapped to bovine (BTA) Chromosomes (Chrs) 18 (UPK1A), 1 (UPK1B), and 15 (UPK2), respectively. Two bovine genomic DNA sequences reactive with a uroplakin III cDNA probe were identified and mapped to BTA 6 (UPK3A) and 5 (UPK3B). We have also mapped genes for uroplakins 1A and II in mice, to the proximal regions of mouse Chr 7 (Upk1a) and 9 (Upk2), respectively, by analyzing the inheritance of restriction fragment length variants in recombinant inbred mouse strains. These assignments are consistent with linkage relationships known to be conserved between cattle and mice. The mouse genes for uroplakins IB and III were not mapped because the mouse genomic DNA fragments reactive with each probe were invariant among the inbred strains tested. Although the stoichiometry of AUM proteins is nearly constant, the fact that the uroplakin genes are unlinked indicates that their expression must be independently regulated. Our results also suggest likely positions for two human uroplakin genes and should facilitate further analysis of their possible involvement in disease.