S. M. Schmutz

A β-Defensin Mutation Causes Black Coat Color in Domestic Dogs

Genetic analysis of mammalian color variation has provided fundamental insight into human biology and disease. In most vertebrates, two key genes, Agouti and Melanocortin 1 receptor (Mc1r), encode a ligand-receptor system that controls pigment type-switching, but in domestic dogs, a third gene is implicated, the K locus, whose genetic characteristics predict a previously unrecognized component of the melanocortin pathway. We identify the K locus as β-defensin 103 (CBD103) and show that its protein product binds with high affinity to the Mc1r and has a simple and strong effect on pigment type-switching in domestic dogs and transgenic mice. These results expand the functional role of β-defensins, a protein family previously implicated in innate immunity, and identify an additional class of ligands for signaling through melanocortin receptors.

Association of a missense mutation in the bovine leptin gene with carcass fat content and leptin mRNA levels

Previously, we have shown that alleles of the BM1500 microsatellite, located 3.6 kb downstream of the leptin gene in cattle, were associated with carcass fat measures in a population of 154 unrelated beef bulls. Subsequently, a cytosine (C) to thymine (T) transition that encoded an amino acid change of an arginine to a cysteine was identified in exon 2 of the leptin gene. A PCR-RFLP was designed and allele frequencies in four beef breeds were correlated with levels of carcass fat. The T allele was associated with fatter carcasses and the C allele with leaner carcasses. The frequencies of the SNP alleles among breeds indicated that British breeds have a higher frequency of the T allele whereas the continental breeds have a higher occurrence of the C allele. A ribonuclease protection assay was developed to quantify leptin mRNA in a separate group of animals selected by genotype. Animals homozygous for thymine expressed higher levels of leptin mRNA. This may suggest that the T allele, which adds an extra cysteine to the protein, imparts a partial loss of biological function and hence could be the causative mutation.

Transmitochondrial differences and varying levels of heteroplasmy in nuclear transfer cloned cattle.

To assess the extent of cytoplasmic genetic variability in cloned cattle produced by nuclear transplantation procedures, we investigated 29 individuals of seven male cattle clones (sizes 2–6) from two different commercial sources. Restriction enzyme and direct sequence analysis of mitochondrial DNA (mtDNA) detected a total of 12 different haplotypes. Transmitochondrial individuals (i.e., animals which share identical nuclei but have different mitochondrial DNA) were detected in all but one of the clones, demonstrating that mtDNA variation among cloned cattle is a very common phenomenon which prevents true genetic identity. The analyses also showed that the cytoplasmic genetic status of some investigated individuals and clones is further complicated by heteroplasmy (more than one mtDNA type in an individual). The relative proportions of different mtDNA‐types in two animals with mild heteroplasmy were estimated at 2:98% and 4:96% in DNA samples derived from blood. This is in agreement with values expected from karyoplast‐cytoplast volume ratios. In contrast, the mtDNA haplotype proportions observed in six other heteroplasmic animals of two different clones ranged from 21:79% to 57:43%, reflecting a marked increase in donor blastomere mtDNA contributions. These results suggest that mtDNA type of donor embryos and recipient oocytes used in nuclear transfer cattle cloning should be controlled to obtain true clones with identical nuclear and cytoplasmic genomes. Mol. Reprod. Dev. 54:24–31, 1999.

Monomorphism and polymorphism at Mhc DRB loci in domestic and wild ruminants

Summary: Genetic polymorphism at Mhc class II DRB loci was investigated in samples of musk‐ox from Canada and Greenland; moose from Sweden, Norway, Canada, and Alaska, roe deer from Norway and Sweden; reindeer from Svalbard and Norway; fallow deer from Norway and Sweden; and red deer from Norway. The results were compared with published data on cattle, bison, goal, sheep, and red deer. Cattle‐specific primers amplified a single DRB locus in all species except fallow deer and red deer, in which two loci were found. Single strand conformation polymorphism analysis and DNA sequence analysis were employed to detect genetic polymorphism. Complete monomorphism was found in musk‐ox and fallow deer. Limited polymorphism was found in the moose, roe deer, and reindeer from Svalbard, whereas intermediate to extensive DRB diversity was present in reindeer from Norway and in bison, sheep, goat, cattle, and red deer. The restricted Mhc diversity in moose, roe deer, and fallow deer is notable in relation to the dramatic population expansion of moose and roe deer in Sweden during this century and since fallow deer is used for meat and game production with good results and without any marked disease problems. The results question the view that species or populations with restricted Mhc diversity have poor resistance to infectious diseases. A phylogenelic tree analysis revealed a clustering of DRB sequences within species rather than within allelic lineages across species. The results suggest trans‐species persistence of polymorphic sequence motifs rather than of allelic lineages.

TYRP1 and MC1R genotypes and their effects on coat color in dogs.

We used PCR amplification of cDNA prepared from skin biopsies to determine the nearly full-length, protein-coding sequence of dog TYRP1, and to define sequence variants potentially responsible for the B locus. One common variant contained a premature stop codon in exon 5 (Q331ter), and the other deleted a proline residue in exon 5 (345delP). A third variant in exon 2 (S41C) occurred less frequently. We genotyped 43 brown (including brown and white) and 34 black (including tricolor, black-and-tan, and black and white) dogs. All 43 of the brown group carried two or more of these sequence variants likely to interfere with TYRP1 function, whereas 0 of 34 in the black group carried two or more of these variants (10 carried one variant). We also genotyped 13 black-nosed and 10 brown-nosed dogs whose coat color was described as red, yellow, gold, apricot, or orange (including various degrees of white). All these dogs were homozygous for a R306X MC1R variant shown to be associated with these coat color phenotypes. The black or brown nose correlated perfectly with the absence or presence of the same three TYRP1 variants described above. TYRP1 was linkage mapped to dog chromosome 11, with a SNP in exon 7.

A form of albinism in cattle is caused by a tyrosinase frameshift mutation

We used PCR amplification of cDNA prepared from skin biopsies to determine the full-length protein-coding sequence of tyrosinase (TYR) in cattle of several coat colors. An insertion of a cytosine was detected in an albino Braunvieh calf, which resulted in a frameshift which caused a premature stop codon at residue 316. This insertion was found in the homozygous state in this calf and the genomic DNA of two related albino calves. All six parents of these calves were heterozygous for this insertion. However, an albino Holstein calf did not have this insertion, nor was any other mutation detected in the partial TYR sequence obtained from the genomic DNA available. Diagnostic genotyping tests were developed to detect this mutation in Braunvieh cattle.

A potential association between the BM 1500 microsatellite and fat deposition in beef cattle.

Abstract. The obese gene was hypothesized as a candidate gene for fat characteristics in beef cattle. The BM 1500 microsatellite, near the obese gene, was characterized in 158 purebred beef bulls for which carcass trait information was available. Four breeds were included in the analyses—Angus, Charolais, Hereford, and Simmental. Four alleles were found. Lengths were approximately 138, 147, 149, and 140 bp with genotypic frequencies of 0.47, 0.44, 0.09, and 0.003 respectively. The carcass traits %rib fat, %rib lean, average fat, and grade fat were found to be significantly associated with the different alleles. The presence of the 138-bp allele in the genotype of an animal is correlated with higher levels of fat, whereas the 147-bp allele has the opposite effect. The 149-bp allele was found in low numbers, and a homozygote was never identified. Hereford and Angus bulls had the greatest frequencies of 138-bp alleles (Hereford = 0.57, Angus = 0.59), while Charolais and Simmental had a greater proportion of 147-bp alleles (Charolais = 0.54, Simmental = 0.58). This information may aid cattle producers in selecting cattle for markets that differ in the amount of fat required.

A missense mutation in the bovine MGF gene is associated with the roan phenotype in Belgian Blue and Shorthorn cattle

Abstract. The Roan locus is responsible for the coat coloration of Belgian Blue and Shorthorn cattle. The solid-colored and white animals are homozygotes, and the roan animals, with intermingled colored and white hairs, are heterozygous. The roan phenotype was mapped to cattle Chromosome (Chr) 5 with microsatellites, and a candidate gene was proposed (Charlier et al. Mamm Genome 7, 138, 1996). PCR primers to the exons of this candidate gene, the steel locus or mast cell growth factor (MGF) were designed. Solid-colored and white animals were sequenced. A missense mutation at 654 bp (amino acid 193, Ala → Asp) was detected in these two groups. A PCR-RFLP was designed to this single base pair change, and 143 animals in total (Belgian Blue, Shorthorn, and various other breeds) were screened. In addition, the Canadian Beef Cattle Reference Herd (http://skyway.usask.ca/∼schmutz) was used to verify Mendelian inheritance of this marker with the phenotypic inheritance of roan. Our data suggest that this mutation in the bovine MGF gene is responsible for the roan phenotype.

DNA marker-assisted selection of the polled condition in Charolais cattle

Five Charolais families known to segregate for both horned and polled were selected and tested for linkage analysis by use of microsatellites and karyotyping for Robertsonian translocation 1;29. No recombinants were found between any of these markers and the polled phenotype or each other. When statistical analysis was performed, the logarithm of the odds (LOD) indicated that there was 100% linkage occurring between the markers and the phenotype (p<0.001). These microsatellite markers, TGLA49 and BM6438, can be assumed to be very close to the actual gene that determines the polled phenotype. Another linked marker, SOD1, was physically mapped, which places all of these markers within 1q12–14, very near the centromere of Chromosome (Chr) 1. A homozygous polled cow was identified in this study by following the alleles at both markers and the phenotypes in her family.

Restriction fragment length polymorphism in the mitochondrial DNA of cloned cattle

The clonal origin of 4 Holstein bulls was determined by hybridization experiments with 2 different minisatellite probes, and all 4 animals showed identical genomic DNA fingerprints, hence confirming monozygosity. Extra-chromosomal differences were observed among these 4 Holstein bulls. Mitochondrial DNA restriction fragment length polymorphisms with restriction endonucleases Avall and Hhal sites were found, and these polymorphisms can be explained by the loss of a single site for each of these 2 enzymes. Since mitochondrial DNA are maternally transmitted, all 4 bulls would produce genetically equivalent spermatozoa and offspring. The combination of embryo cloning and specific cytoplasmic markers would provide an ideal system for the study of maternal cytoplasmic effects on quantitative traits.