A genome assembly-integrated dog 1 Mb BAC microarray: a cytogenetic resource for canine cancer studies and comparative genomic analysis.

Molecular cytogenetic studies have been instrumental in defining the nature of numerical and structural chromosome changes in human cancers, but their significance remains to be fully understood. The emergence of high quality genome assemblies for several model organisms provides exciting opportunities to develop novel genome-integrated molecular cytogenetic resources that now permit a comparative approach to evaluating the relevance of tumor-associated chromosome aberrations, both within and between species. We have used the dog genome sequence assembly to identify a framework panel of 2,097 bacterial artificial chromosome (BAC) clones, selected at intervals of approximately one megabase. Each clone has been evaluated by multicolor fluorescence in situ hybridization (FISH) to confirm its unique cytogenetic location in concordance with its reported position in the genome assembly, providing new information on the organization of the dog genome. This panel of BAC clones also represents a powerful cytogenetic resource with numerous potential applications. We have used the clone set to develop a genome-wide microarray for comparative genomic hybridization (aCGH) analysis, and demonstrate its application in detection of tumor-associated DNA copy number aberrations (CNAs) including single copy deletions and amplifications, regional aneuploidy and whole chromosome aneuploidy. We also show how individual clones selected from the BAC panel can be used as FISH probes in direct evaluation of tumor karyotypes, to verify and explore CNAs detected using aCGH analysis. This cytogenetically validated, genome integrated BAC clone panel has enormous potential for aiding gene discovery through a comparative approach to molecular oncology.

Molecular analysis of a spontaneous dystrophin 'knockout' dog.

We have determined the molecular basis for skeletal myopathy and dilated cardiomyopathy in two male German short-haired pointer (GSHP) littermates. Analysis of skeletal muscle demonstrated a complete absence of dystrophin on Western blot analysis. PCR analysis of genomic DNA revealed a deletion encompassing the entire dystrophin gene. Molecular cytogenetic analysis of lymphocytes from the dam and both dystrophic pups confirmed a visible deletion in the p21 region of the affected canine X chromosome. Utrophin is up-regulated in the skeletal muscle, but does not appear to ameliorate the dystrophic canine phenotype. This new canine model should further our understanding of the physiological and biochemical processes in Duchenne muscular dystrophy.

Physical mapping of chromosome 6: a strategy for the rapid generation of sequence-ready contigs.

The development of radiation hybrid (RH) mapping (Cox et al., 1990) and the availability of large numbers of STS markers, together with extensive bacterial clone resources provided a means to accelerate the process of mapping a human chromosome and preparing bacterial clone contigs ready to sequence. Our aim is to construct physical clone maps covering those regions of chromosome 6 that are not currently extensively mapped, and use these to determine the DNA sequence of the whole chromosome. We report here a strategy which initially involves establishing a high density framework map using RH mapping. The framework markers are then used for the identification of bacterial genomic clones covering the chromosome. The bacterial clones are analysed by restriction enzyme fingerprinting and STS-content analysis to identify sequence-ready contigs. Contig gap closure will also be performed by clone walking.

From long range mapping to sequence-ready contigs on human chromosome 6.

Our aim is to construct physical clone maps covering those regions of chromosome 6 that are not currently extensively mapped, and use these to determine the DNA sequence of the whole chromosome. The strategy we are following involves establishing a high density framework map of the order of 15 markers per Megabase using radiation hybrid (RH) mapping. The markers are then used to identify large-insert genomic bacterial clones covering the chromosome, which are assembled into sequence-ready contigs by restriction enzyme fingerprinting and sequence tagged site (STS) content analysis. Contig gap closure is performed by walking experiments using STSs developed from the end sequences of the clone inserts.

Salmonella prevalence in crops of Ontario and Quebec broiler chickens at slaughter.

Swabs of crop contents of 635 broiler chickens were obtained from 9 Ontario and 12 Quebec processing plants and cultured for Salmonella to determine prevalence in broiler crops. Serotypes of positive cultures were determined to evaluate the serotype profile. The overall prevalence of contamination was low (4.3%). Prevalence was higher in broilers sampled in Quebec (5.8%) than in those sampled in Ontario (2.2%). In Quebec, there were differences in prevalence among the groups of broilers sampled at the various plants. These differences were believed to be attributable to differences in Salmonella prevalence among groups of flocks delivered to the plants due to the limited exposure of the chickens to the plant. The serotype profile of Salmonella isolated from the crops of broilers in this study was similar in several respects to profiles obtained from other surveys of Canadian broiler flocks using either environmental samples or cloacal swabs. Similarities included: predominance of Salmonella hadar and Salmonella heidelberg; several other common serotypes at a low prevalence; little Salmonella enteritidis isolated in other studies, and no S. enteritidis isolated in this study. Results of this field survey of Salmonella in crops of broilers are similar to those of Canadian studies of other internal and environmental sites of broilers. The similarity indicates that monitoring of Salmonella environments of flocks of live broiler chickens should define profiles of Salmonella contamination of the carcasses and would also aid in determination of Salmonella contamination status of broiler flocks. Such information would assist efforts to reduce Salmonella contamination in broiler chickens.

1p36 is a preferential target of chromosome 1 deletions in astrocytic tumours and homozygously deleted in a subset of glioblastomas.

Astrocytic, oligodendroglial and mixed gliomas are the commonest gliomas in adults. They have distinct phenotypes and clinical courses, but as they exist as a continuous histological spectrum, differentiating them can be difficult. Co-deletions of total 1p and 19q are found in the majority of oligodendrogliomas and considered as a diagnostic marker and a prognostic indicator. The 1p status of astrocytomas has not yet been thoroughly examined. Using a chromosome 1 tile path array, we investigated 108 adult astrocytic tumours for copy number alterations. Total 1p deletions were rare (2%), however partial deletions involving 1p36 were frequently identified in anaplastic astrocytomas (22%) and glioblastomas (34%). Multivariate analysis showed that patients with total 1p deletions had significantly longer survival (P=0.005). In nine glioblastomas homozygous deletions at 1p36 were identified. No somatic mutations were found among the five genes located in the homozygously deleted region. However, the CpG island of TNFRSF9 was hypermethylated in 19% of astrocytic tumours and 87% of glioma cell lines. TNFRSF9 expression was upregulated after demethylation of glioma cell lines. Akt3 amplifications were found in four glioblastomas. Our results indicate that 1p deletions are common anaplastic astrocytomas and glioblastomas but are distinct from the 1p abnormalities in oligodendrogliomas.

Array CGH profiling of favourable histology Wilms tumours reveals novel gains and losses associated with relapse.

Despite the excellent survival of Wilms tumour patients treated with multimodality therapy, approximately 15% will suffer from tumour relapse, where response rates are markedly reduced. We have carried out microarray-based comparative genomic hybridisation on a series of 76 Wilms tumour samples, enriched for cases which recurred, to identify changes in DNA copy number associated with clinical outcome. Using 1Mb-spaced genome-wide BAC arrays, the most significantly different genomic changes between favourable histology tumours that did (n = 37), and did not (n = 39), subsequently relapse were gains on 1q, and novel deletions at 12q24 and 18q21. Further relapse-associated loci included losses at 1q32.1, 2q36.3-2q37.1, and gain at 13q31. 1q gains correlated strongly with loss of 1p and/or 16q. In 3 of 11 cases with concurrent 1p(-)/1q(+), a breakpoint was identified at 1p13. Multiple low-level sub-megabase gains along the length of 1q were identified using chromosome 1 tiling-path arrays. One such recurrent region at 1q22-q23.1 included candidate genes RAB25, NES, CRABP2, HDGF and NTRK1, which were screened for mRNA expression using quantitative RT-PCR. These data provide a high-resolution catalogue of genomic copy number changes in relapsing favourable histology Wilms tumours.

The DAPI banded karyotype of the domestic dog (Canis familiaris) generated using chromosome-specific paint probes.

The domestic dog (Canis familiaris) is widely used as a model in the study of human disease. However, many of the 78 chromosomes comprising the canine karyotype are extremely difficult to identify reliably by classical cytogenetics. This has been a major hindrance to molecular cytogenetic studies of this species. The Animal Health Trust and the Sanger Centre have developed a set of canine whole chromosome-specific fluorescence in situ hybridisation (FISH) probes (chromosome paints). We have used these chromosome paints to identify unequivocally each chromosome in a metaphase spread. An increasing number of laboratories are making use of cooled CCD cameras and sophisticated software for FISH mapping. Consequently, there is a major trend towards the use of DAPI banding for concurrent chromosome identification during FISH analyses in a range of species. Here we present, for the first time, a complete DAPI banded karyotype of the dog in which each chromosome has been accurately placed, together with a 460-band DAPI ideogram. These data will facilitate the accurate assignment of FISH-mapped loci to all chromosomes comprising the karyotype and form the basis for an agreed standard of the dog karyotype.

Preparation of chromosome-specific paints and complete assignment of chromosomes in the pig flow karyotype.

Sorted chromosomes from each of the 20 clusters of the male porcine bivariate flow karyotype were amplified and biotinylated using DOP-PCR. The chromosomes comprising each cluster were identified by fluorescence in situ hybridization (FISH) of the 20 probes to R-banded male pig metaphase spreads. A standard flow karyotype for the pig is presented.

Vaccination of young Dorset lambs against haemonchosis.

Six Dorset Horn lambs were each vaccinated at age 7 weeks and 9 weeks with 50 micrograms of glycosylated integral membrane proteins, particularly enriched in the protein H11 from the intestinal brush border of adult Haemonchus. At 11 weeks of age the lambs were infected with 10,000 infective third stage Haemonchus larvae. Compared with the average for the control group the vaccinated group of lambs had a 78% reduction in parasite egg output over the patent period, with four of the six better than 93% protected. At autopsy 35 days post-infection the mean total worm burden of the vaccinated lambs was 83% reduced compared with the controls. The serum antibody titres to H11 correlated with the degree of protection.

Immune response of Clun Forest sheep to vaccination with membrane glycoproteins from Haemonchus contortus.

Clun Forest lambs were injected with a fraction containing integral membrane glycoproteins derived from the intestinal microvilli of Haemonchus contortus in two equal doses of 0.5, 5, 50 or 500 micrograms protein and then challenged with 10,000 infective larvae. The time-course of serum specific antibody responses were determined. Compared to the adjuvant control group, the animals injected with 1000 micrograms protein were better than 84% protected and those injected with 100 micrograms, better than 95% protected by all criteria. For these two groups parasite egg output was reduced 99% on average over the patent period. Two of the animals injected with 10 micrograms protein were partially protected, with 86% reduction in egg output. Two animals in the group injected with 1 microgram protein also showed partial protection. Antibody level correlated with protection.

Chromosome painting using chromosome-specific probes from flow-sorted pig chromosomes.

Biotinylated chromosome-specific probes were prepared from flow-sorted pig chromosomes 1, 13, 18, X, and Y using the degenerate oligonucleotide-primed polymerase chain reaction. Probes prepared in this way can be used to confirm the identity of chromosomes in the bivariate pig flow karyotype and in pig x mouse somatic cell hybrids.

Zoo-FISH analysis of dog chromosome 5: identification of conserved synteny with human and cat chromosomes.

Conserved segments of synteny between the human genome and chromosome 5 (CFA 5) of the domestic dog (Canis familiaris) have been identified by reciprocal chromosome painting analysis. A CFA 5 paint probe was applied to human metaphase spreads, revealing distinct hybridisation sites on human (HSA) chromosomes 1, 11, 16, and 17. Paint probes for these human chromosomes were then hybridised to dog metaphase spreads, identifying the regions of CFA 5 with which homology is shared with the corresponding human chromosome. Application of the CFA 5 paint probe to metaphase spreads of the domestic cat (Felis catus, FCA) demonstrated hybridisation to cat chromosomes C1, D1, E1, and E2. Dog PCR primers for type 1 markers known to lie in the corresponding regions of HSA 11, 16, and 17 were used to isolate dog BAC clones representing four genes. Fluorescence in situ hybridisation analysis confirmed their localisation to CFA 5 and suggested that two of the conserved segments lie in opposing orientations on CFA 5, compared to the human chromosome concerned. A third segment appears to lie in the same orientation on both human and dog chromosomes. No suitable gene markers were available for analysis of the fourth segment. The significance of these findings is discussed with reference to current and future dog genome mapping efforts.

Reciprocal chromosome painting reveals detailed regions of conserved synteny between the karyotypes of the domestic dog (Canis familiaris) and human.

The domestic dog is increasingly being recognized as a useful model for human disease. The aim of this study was to conduct the first detailed whole-genome comparison of human and dog using bidirectional heterologous chromosome painting (reciprocal Zoo-FISH) analysis. We used whole-chromosome paint probes produced from degenerate oligonucleotide-primed PCR amplification of high-resolution bivariate flow-sorted human and dog chromosomes. No fewer than 68 evolutionarily conserved segments were identified between the dog and the human karyotypes. The use of elongated metaphase chromosomes for both species allowed the boundaries of each evolutionarily conserved segment to be determined to subband resolution. The distribution of conserved segments is discussed, as are the applications of these data in refining the current status of the dog genome map.

Chromosome-specific paints from a high-resolution flow karyotype of the dog.

Using peripheral blood lymphocyte cultures and dual-laser flow cytometry, we have routinely obtained high-resolution bivariate flow karyotypes of the dog in which 32 peaks are resolved. To allow the identification of the chromosome types in each peak, chromosomes were flow sorted, amplified and labelled by polymerase chain reaction with partially degenerate primers and hybridized onto metaphase spreads of a male dog. The chromosome paints from 22 of the 32 peaks each hybridized to single homologue pairs and eight peaks each hybridized to two pairs. Paints from the remaining two peaks hybridized to only one homologue each in the male metaphase spread, thus corresponding to the sex chromosomes X and Y. All of the 38 pairs of autosomes and the two sex chromosomes of the dog could be accounted for in these painting experiments. The positions of chromosomes 1-21 were assigned to the flow karyotype (only chromosomes 1-21 have as yet been officially designated). The high-resolution flow karyotype and the chromosome paints will facilitate further standardization of the dog karyotype. The ability to sort sufficient quantities of dog chromosomes for the production of chromosome-specific DNA libraries has the potential to accelerate the physical and genetic mapping of the dog genome.

CpG island libraries from human chromosomes 18 and 22: landmarks for novel genes.

CpG islands are found at the 5' end of approximately 60% of human genes and so are important genomic landmarks. They are concentrated in early-replicating, highly acetylated gene-rich regions. With respect to CpG island content, human Chrs 18 and 22 are very different from each other: Chr 18 appears to be CpG island poor, whereas Chr 22 appears to be CpG island rich. We have constructed and validated CpG island libraries from flow-sorted Chrs 18 and 22 and used these to estimate the difference in number of CpG islands found on these two chromosomes. These libraries contain normalized collections of sequences from the 5' end of genes. Clones from the libraries were sequenced and compared with the sequence databases; one third matched ESTs, thus anchoring these ESTs at the 5' end of their gene. However, it was striking that many clones either had no match or matched only existing CpG island clones. This suggests that a significant proportion of 5' gene sequences are absent from databases, presumably either because they are difficult to clone or the gene is poorly expressed and/or has a restricted expression pattern. This point should be taken into consideration if the currently available libraries are those used for the elucidation of complete, as opposed to partial, gene sequences. The Chr 18 and 22 CpG island libraries are a sequence resource for the isolation of such 5' gene sequences from specific human chromosomes.

beta-1,3-Glucuronyltransferase-1 gene implicated as a candidate for a schizophrenia-like psychosis through molecular analysis of a balanced translocation.

We have mapped and sequenced both chromosome breakpoints of a balanced t(6;11)(q14.2;q25) chromosome translocation that segregates with a schizophrenia-like psychosis. Bioinformatics analysis of the regions revealed a number of confirmed and predicted transcripts. No confirmed transcripts are disrupted by either breakpoint. The chromosome 6 breakpoint region is gene poor, the closest transcript being the serotonin receptor 1E (HTR1E) at 625 kb telomeric to the breakpoint. The chromosome 11 breakpoint is situated close to the telomere. The closest gene, beta-1,3-glucuronyltransferase (B3GAT1 or GlcAT-P), is 299 kb centromeric to the breakpoint. B3GAT1 is the key enzyme during the biosynthesis of the carbohydrate epitope HNK-1, which is present on a number of cell adhesion molecules important in neurodevelopment. Mice deleted for the B3GAT1 gene show defects in hippocampal long-term potentiation and in spatial memory formation. We propose that the translocation causes a positional effect on B3GAT1, affecting expression levels and making it a plausible candidate for the psychosis found in this family. More generally, regions close to telomeres are highly polymorphic in both sequence and length in the general population and several studies have implicated subtelomeric deletions as a common cause of idiopathic mental retardation. This leads us to the hypothesis that polymorphic or other variation of the 11q telomere may affect the activity of B3GAT1 and be a risk factor for schizophrenia and related psychoses in the general population.

FISH mapping and identification of canine chromosomes.

The karyotype of the domestic dog (Canis familiaris) is widely accepted as one of the most difficult mammalian karyotypes to work with. The dog has a total of 78 chromosomes; all 76 autosomes are acrocentric in morphology and show only a gradual decrease in length. Standardization of the canine karyotype has been performed in two stages. The first stage dealt only with chromosomes 1-21 which can be readily identified by conventional G-banding techniques. The remaining 17 autosomal pairs have proven to be very difficult to reliably identify by banding alone. To facilitate the identification of all canine chromosomes, chromosome-specific paint probes have been produced by DOP-PCR from flow-sorted dog chromosomes. Each paint probe has been used for FISH to identify the corresponding chromosome(s), allowing precise identification of all 78 canine chromosomes. The identification of the undesignated 17 autosomal pairs has been agreed upon by the standardization committee during the second stage of their role. Cosmid clones containing microsatellite markers may now be conclusively assigned to their chromosomal origin by simultaneous dual-color FISH with the corresponding paint probe. In this way a collection of chromosome-specific cosmid clones is being constructed, comprising at least one marker per chromosome, which will allow anchoring of existing and future linkage groups to the physical map.

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.