Canine Chromosome Research Articles

Toward a framework linkage map of the canine genome.

Selective breeding to maintain specific physical and behavioral traits has made the modern dog one of the most physically diverse species on earth. One unfortunate consequence of the common breeding practices used to develop lines of dogs with the desired traits is amplification and propagation of genetic diseases within distinct breeds. To map disease loci we have constructed a first-generation framework map of the canine genome. We developed large numbers of highly polymorphic markers, constructed a panel of canine-rodent hybrid cell lines, and assigned those markers to chromosome groups using the hybrid cell lines. Finally, we determined the order and spacing of markers on individual canine chromosomes by linkage analysis using a reference panel of 17 outbred pedigrees. This article describes approaches and strategies to accomplish these goals.

Use of cosmid-derived and chromosome-specific canine microsatellites.

The majority of microsatellite markers being used to generate the emerging genetic linkage maps of the dog are derived from small-insert, random clones. While such markers are easy to generate, they have the disadvantage that they cannot easily be physically mapped by fluorescence in situ hybridization (FISH), making it difficult to assess the extent of genome coverage represented by such maps. In contrast, microsatellite markers from large-insert libraries enable the linkage groups within which they fall to be physically anchored to specific chromosomes. One aim of our work is to identify at least one microsatellite-containing cosmid clone for each canine chromosome, to ensure that linkage groups exist for all chromosomes. This is particularly important for a species with as complex a karyotype as the dog. Locating two cosmids on each chromosome would allow the orientation of the linkage groups to be established. Chromosomal locations of cosmid clones containing microsatellites have been determined by FISH and confirmed using canine chromosome-specific paints. Microsatellite sequences have been genotyped on the DogMap reference family. Microsatellites derived from flow-sorted, chromosome-specific libraries represent another source of useful markers. Initial studies have been carried out on the canine X chromosome, on which markers were underrepresented in our initial studies.

A Whole-Genome Radiation Hybrid Map of the Dog Genome

A whole genome radiation hybrid (RH) map of the canine genome was constructed by typing 400 markers, including 218 genes and 182 microsatellites, on a panel of 126 radiation hybrid cell lines. Fifty-seven RH groups have been determined with lod scores greater than 6, and 180 framework landmarks were ordered with odds greater than 1000:1. Average spacing between adjacent markers is 23 cR5000, an estimated physical distance of 3.8 Mb. Fourteen groups have been assigned to 9 of the canine chromosomes, and a comparison of RH and genetic groups allowed the successful bridging of both types of data on one map composed of 31 RH and 13 syntenic RH groups. Comparison of canine, human, mouse, and pig maps underlined regions of conserved synteny. This integrated map, covering an estimated 80% of the dog genome, should prove a powerful tool for localizing and identifiying genes implicated in pathological and phenotypical traits.

Chromosomal assignment of two putative canine keratin gene clusters.

Mutations in keratin genes account for a number of inherited keratodermas in humans. The groups of basic and acidic keratin genes are clustered on human chromosomes 12 and 17, respectively. The present authors have assigned the two putative keratin gene clusters to canine chromosomes using canine cosmid clones. Successful fluorescence in situ hybridization mapped the putative cluster of canine acidic genes to dog chromosome 20 and the putative cluster of basic keratin genes to a small autosome not yet included in the partial canine standard karyotype.

Linkage analysis and comparative mapping of canine progressive rod-cone degeneration (prcd) establishes potential locus homology with retinitis pigmentosa (RP17) in humans.

Progressive rod-cone degeneration (prcd) is the most widespread hereditary retinal disease leading to blindness in dogs and phenotypically is the canine counterpart of retinitis pigmentosa (RP) in humans. In previous efforts to identify the genetic locus for prcd, the canine homologs for many of the genes causally associated with RP in humans, such as RHO, PDE6B, and RDS/peripherin, have been excluded. In parallel with a recent undertaking to establish a framework map of the canine genome, multiple prcd-informative pedigrees have been typed with a panel of more than 100 anchor loci and microsatellite-based markers. Identification of a linkage group flanking prcd ([TK1, GALK1, prcd]-[MYL4, C09.173, C09.2263]-RARA-C09.250-C09.474-NF1) localizes prcd close to the centromeric end of canine chromosome 9 (CFA9), and excludes RARA as a candidate gene. The conserved synteny of this region of CFA9 and distal human chromosome 17q establishes the potential locus homology of prcd in the dog with RP17, a human retinitis pigmentosa locus for which no gene has yet been identified. Assignment of the prcd disease locus to an identified canine autosome represents a powerful application of the developing canine linkage map in medical genetics. The usefulness of this approach is further demonstrated by identification of the correspondence of the prcd interval to homologous human and mouse chromosomal regions. The rapid progress that is now occurring in the field of canine genetics will expedite the identification of the genes underlying many of the inherited traits and diseases that make the dog a unique asset for the study of mammalian traits.

Construction of a Panel of Canine–Rodent Hybrid Cell Lines for Use in Partitioning of the Canine Genome

We have constructed a collection of canine–rodent microcell hybrid cell lines by fusion of canine fibroblast microcell donors with immortalized rodent recipient cells. Characterization of the hybrid cell lines using a combination of fluorescencein situhybridization and PCR analysis of canine microsatellite repeat sequences allowed selection of a panel of hybrids in which most canine chromosomes are represented. Approximately 90% of genetic markers and genes that were tested could be assigned to 1 of 31 anonymous canine chromosome groups, based on common patterns of retention in the hybrid set. Many of these putative chromosome groups have now been validated by linkage analysis. This panel of cell lines provides a tool for development of genetic, physical, and comparative maps of the canine genome.

A Linkage Map of the Canine Genome

A genetic linkage map of the canine genome has been developed by typing 150 microsatellite markers using 17 three-generation pedigrees, composed of 163 F2individuals. One hundred and thirty-nine markers were linked to at least one other marker with a lod score ≥ 3.0, identifying 30 linkage groups. The largest chromosome had 9 markers spanning 106.1 cM. The average distance between markers was 14.03 cM, and the map covers an estimated 2073 cM. Eleven markers were informative on the mapping panel, but were unlinked to any other marker. These likely represent single markers located on small, distinct canine chromosomes. This map will be the initial resource for mapping canine traits of interest and serve as a foundation for development of a comprehensive canine genetic map.

Chromosomal assignment of seven genes on canine chromosomes by fluorescence in situ hybridization.

Our group has developed more than 600 DNA markers to build a map of the canine genome. Of these markers, 125 correspond to genes (anchor loci). Here we report the first six autosomal genes assigned to canine chromosomes by fluorescence in situ hybridization (FISH), using cosmid DNA: adenine phosphoribosyl transferase on Chromosome (Chr) 3; creatine kinase muscle type on Chr 4; pyruvate kinase liver and red blood cell type on Chr 2; and colony-stimulating factor-1 receptor, glucose transporter protein-2, and tumor protein p53 on Chr 5. These assignments are based on the karyotype proposed by Stone and associates (Genome 34, 407, 1991) using high-resolution techniques. In addition, we have assigned the Menkes gene to the X Chr of the dog.

Viewing the Renal Epithelium with the Atomic Force Microscope

The atomic force microscope (AFM) was invented in 1986. It created the possibility to study the surfaces of biological material at the molecular level. The AFM is not only a microscope with atomic resolution (at least when applied to suitable samples) but is also an instrument with a broad variety of other applications. We present five examples demonstating the application of the AFM technique in different ways, mainly in renal epithelium. First, it was used as a high-resolution near-field microscope which generated images of canine kidney chromosomes. Second, dynamic changes of the plasma membrane of canine kidney cells were visualized with a resolution at least 10-fold greater than that obtainable by light microscopy. Third, the AFM was used as a tool which interacted directly with the plasma membrane of canine kidney cells. Fourth, due to its excellent resolution in the third dimension (namely height), continuous measurements of changes in 'height' of multimers of cloned plasma membrane potassium channel proteins of rat kidney (ROMK1) in response to ATP were performed with a time resolution of about 100 ms in physiological buffer. Fifth, the AFM continuously followed dissociation processes of multimeric macromolecules (such as transcription factors) under physiological conditions and, on the basis of individual molecular volume measurements, allowed estimation of the number of monomers in a multimeric complex. Taken together, the AFM offers a wide spectrum of novel approaches for the experimental nephrologist.

An extended nomenclature of the canine karyotype.

In contrast to many other animals, knowledge about the canine karyotype is quite sparse. This is due in part to the rather difficult canine karyotypic pattern. Except for the X and the Y chromosome, there are only acrocentric chromosomes, which appear to be quite small and difficult to identify unambiguously. In previous reports, schematic representations of the canine karyotype have been described. However, a nomenclature comparable to that of the human karyotype or the karyotypes of sheep, cattle, or goats does not yet exist for the dog. Based on high-resolution banding of metaphase chromosomes from canine fibroblasts, we propose an ideogram of the canine karyotype with 460 numbered bands and characteristic landmarks. In addition, the centromere positions of the canine chromosomes are determined by a combined GTG-banding/FISH approach, and the R- and G-banding patterns are compared.

A PCR-RFLP marker for the erythroid aminolevulinate synthase gene (ALAS2) on canine chromosome X.

A PCR-RFLP marker for the erythroid aminolevulinate synthase gene (ALAS2) on canine chromosome X.

XLPRA: a canine retinal degeneration inherited as an X-linked trait.

Breeding studies are reported of a previously undescribed hereditary retinal degeneration identified in the Siberian Husky breed of dog. This disorder clinically resembles the previously reported autosomal recessive canine hereditary retinal degenerations collectively termed progressive retinal atrophy (PRA). However, the pedigree of the propositus, a male Siberian Husky, exhibited an X-linked pattern of transmission. This dog was outcrossed to three phenotypically normal female laboratory Beagles and two of their F1 daughters were bred to a phenotypically normal male Beagle, producing affected males in the F2 generation. Subsequent inbreedings produced further affected males and affected females as well. X-linked transmission was established by exclusion of alternative modes of inheritance and, consequently, the disease has been termed X-linked progressive retinal atrophy (XLPRA). This is the first reported X-linked retinal degeneration in an animal. Because of the many similarities of PRA in dogs to retinitis pigmentosa (RP) in humans, this new disease may not only represent the first animal model of X-linked RP (XLRP) but may well be a true homolog of one of the XLRP loci (RP2, RP3, RP6). It is the first retinal degeneration in dogs that can be assigned to an identified canine chromosome, and the first for which linkage mapping offers a realistic approach to proceed by positional cloning towards identifying the responsible gene locus.

Pure chromosome-specific PCR libraries from single sorted chromosomes.

Chromosome-specific DNA libraries can be very useful in molecular and cytogenetic genome mapping studies. We have developed a rapid and simple method for the generation of chromosome-specific DNA sequences that relies on polymerase chain reaction (PCR) amplification of a single flow-sorted chromosome or chromosome fragment. Previously reported methods for the development of chromosome libraries require larger numbers of chromosomes, with preparation of pure chromosomes sorted by flow cytometry, generation of somatic cell hybrids containing targeted chromosomes, or a combination of both procedures. These procedures are labor intensive, especially when hybrid cell lines are not already available, and this has limited the generation of chromosome-specific DNA libraries from nonhuman species. In contrast, a single sorted chromosome is a pure source of DNA for library production even when flow cytometric resolution of chromosome populations is poor. Furthermore, any sorting cytometer may be used with this technique. Using this approach, we demonstrate the generation of PCR libraries suitable for both molecular and fluorescence in situ hybridization studies from individual baboon and canine chromosomes, separate human homologues, and a rearranged marker chromosome from a transformed cell line. PCR libraries specific to subchromosomal regions have also been produced by sorting a small chromosome fragment. This simple and rapid technique will allow generation of nonhuman linkage maps and probes for fluorescence in situ hybridization and the characterization of marker chromosomes from solid tumors. In addition, allele-specific libraries generated by this strategy may also be useful for mapping genetic diseases.

Evidence that metacentric and submetacentric chromosomes in canine tumors can result from telomeric fusions

We have hypothesized that metacentric and submetacentric chromosomes frequently observed in malignant canine tumors are a result of telomeric fusions. Therefore cells from a canine mammary pleomorphic adenoma were transformed with a plasmid containing the SV40 'early region', known to cause telomeric associations. Compared with non-transformed adenoma cells, the cells had a higher proliferative capacity and expressed the large SV40-T-antigen. Karyotype studies showed the conversion from a normal to an aberrant karyotype with an increase of bi-armed chromosomes resulting from fusions of acrocentric chromosomes. In addition, the length of the telomeric repeats (TTAGGG) was determined for an early and a late passage of the transformed cells by Southern hybridization. The length of the telomeric repeats was apparently longer in the 5th than in the 38th passage. In situ hybridization with a telomere-specific DNA revealed interstitial telomeric repeats in the bi-armed chromosomes. We have concluded that these findings reflect the clonal expansion of head-to-head-telomeric fusions of canine acrocentric chromosomes leading to dicentric chromosomes with a very short distance between the two centromeres. Our results support the idea that the apparent centric fusions that have been described in some canine tumors may in fact be the cytogenetic products of head-to-head-telomeric fusions.

Bromodeoxyuridine induces chromosomal fragile sited in the canine genome

Peripheral blood lymphocytes from 3 clinically normal domestic dogs were cultured for bromodeoxyuridine (BrdU) induction of fragile site expression. BrdU induced fragile site expression in cells from all 3 dogs. The mean percent of cells with fragile sites and the mean number of fragile sites per cell were significantly increased in all BrdU incubated cultures compared to control cultures. The frequency of BrdU fragile site expression did not vary significantly among the dogs. Lymphocytes from all 3 dogs expressed BrdU induced autosomal fragile sites. Two BrdU induced fragile sites were identified on the long arm of chromosome 1, one of which was close to or coincident with a previously identified folate sensitive fragile site on this canine chromosome. Lymphocytes from the 2 female dogs also expressed BrdU induced fragile sites on the X chromosome, but BrdU failed to induce fragile sites on the X chromosome from the one male dog in the study. The 2 BrdU-induced fragile sites identified on the long arm of the X chromosome were close to, or coincident with 2 previously described folate-sensitive common fragile sites on the canine X chromosome. This is the first report of induction of BrdU-inducible fragile sites in the genome of the domestic dog.

The Giemsa banding pattern of canine chromosomes, using a cell synchronization technique

Cytogenetic investigations of the domestic dog, Canis familiaris, were performed on the Doberman pinscher and two Boxer dogs. Conventional homogeneously stained and G-banded metaphases from peripheral blood lymphocyte cultures synchronized with amethopterin and bromodeoxyuridine were studied. These procedures permitted the unequivocal identification of all canine chromosomes. A canine chromosome idiogram was constructed on the basis of the G-banding pattern at the haploid 327-band resolution level. The secondary constrictions and tapering of the telomeric regions characteristic of several canine chromosomes are described. Q-, C-, and NOR-banding were also performed and the salient features are described. This karyotype should enhance the value of the canine species in cytogenetic investigations.

The banding patterns of normal canine chromosomes.

The chromosomal banding patterns of canine normal chromosome were analyzed by G-, Q-, C- and N-banding techniques. The chromosomes of canine normal lymphocytes consisted of 38 acrocentric autosome pairs and X-, Y-metacentric sex chromosomes. Their G- and Q-banding patterns corresponded to the those reported by Manolache et al. in 1976. C-bands of chromosomes were found on the centromeric regions of the 7th, 26th, 34th, 35th, 37th and 38th pairs of chromosomes. In addition, N-bands whose location had not been confirmed until now were recognized on the telomitic regions of the 7th, 9th and 22nd pairs of chromosomes.

Characterization of the canine karyotype by counterstain-enhanced chromosome banding.

The application of the counterstain-contrasted fluorescent banding technique to canine chromosomes provided an improved capability to highlight specific heterochromatic regions and to produce well defined banding patterns both on mitotic and meiotic chromosomes. Triple staining with chromomycin A3 - distamycin A - DAPI revealed the occurrence of DA - DAPI positive heterochromatin in chromosomes 33, 36, 37, and 38. Pachytene nuclei present more favourable material for the detection of very small amounts of DA - DAPI material than mitotic division stages. Counterstain-enhanced chromomycin R-banding greatly facilitated chromosome identification. A standard R-band karyotype of Canis familiaris is proposed and described in some detail. DAPI - actinomycin D staining produced a QFH-type banding pattern and enhanced differentiation of some polymorphic regions.

A Simple Technique for the Demonstration, Post‐mortem, of Canine Chromosomes

Abstract— A simple method of demonstrating chromosomes, post mortem, in the dog is described. It is suggested that this method will be of considerable value in co‐relating multiple congenital abnormalities with chromosome anomalies.Résumé— Description d'une méhode simple pour faire apparaître les chromosomes du chien, à l'autopsie. On suggère que cette méthode pourrait avoir une valeur considérable en permettant de rattacher les anomalies congénitales multiples aux anomalies des chromosomes.Zusammenfassung— Es wird eine einfache Methode beschrieben, die Crhomosome beim Hude post mortem zu ermitteln. Es ist anzunehmen, dass diese Methode von beträchtlichem Wert für die Korrelation multipler kongenitaler Abnormitäten mit Chromosomenanomalien sien wird.