Patient satisfaction with a scheduled prescription-refill service.
Explore the source record for details and available documents.
Adult↗
Biomedical subjects
M Breen
Publications and source records attributed to M Breen.
Canine TCOF1; cloning, chromosome assignment and genetic analysis in dogs with different head types.
We describe the construction of a dog embryonic head/neck cDNA library and the isolation of the dog homolog of the Treacher Collins Syndrome gene, TCOF1. The protein shows a similar three-domain structure to that described for human TCOF1, but the dog gene lacks exon 10 and contains two exons not present in the human sequence. In addition, exon 19 is differentially spliced in the dog. How these structural differences relate to TCOF1 phosphorylation is discussed. Isolation of a genomic clone allowed the exon/intron boundaries to be characterized and the dog TCOF1 gene to be mapped to CF Chr 4q31, a region syntenic to human Chr 5. Genetic analysis of DNA of dogs from 13 different breeds identified nine DNA sequence variants, three of which gave rise to amino acid substitutions. Grouping dogs according to head type showed that a C396T variant, leading to a Pro117Ser substitution, is associated with skull/face shape in our dog panel. The numbers are small, but the association between the T allele and brachycephaly, broad skull/short face, was highly significant (p = 0.000024). The short period of time during which the domestic dog breeds have been established suggests that this mutation has arisen only once in the history of dog domestication.
Amino Acid Sequence↗
Canine homolog of the T-box transcription factor T; failure of the protein to bind to its DNA target leads to a short-tail phenotype.
Domestic dog breeds show a wide variety of morphologies and offer excellent opportunities to study the molecular genetics of phenotypic traits. We are interested in exploring this potential and have begun by investigating the genetic basis of a short-tail trait. Our focus has been on the T gene, which encodes a T-box transcription factor important for normal posterior mesoderm development. Haploinsufficiency of T protein underlies a short-tail phenotype in mice that is inherited in an autosomal dominant fashion. We have cloned the dog homolog of T and mapped the locus to canine Chromosome (Chr) 1q23. Full sequence analysis of the T gene from a number of different dog breeds identified several polymorphisms and a unique missense mutation in a bob-tailed dog and its bob-tailed descendants. This mutation is situated in a highly conserved region of the T-box domain and alters the ability of the T protein to bind to its consensus DNA target. Analysis of offspring from several independent bobtail x bobtail crosses indicates that the homozygous phenotype is embryonic lethal.
Amino Acid Sequence↗
An integrated cytogenetic, radiation-hybrid, and comparative map of dog chromosome 5.
The development of a detailed genome map for the domestic dog (Canis familiaris, CFA) is a prerequisite for the continued use of this species as a model system for the study of inherited traits. We present an integrated cytogenetic, radiation-hybrid, and comparative map of dog Chromosome (Chr) 5 (CFA 5). The map comprises 14 gene markers, selected from loci previously mapped within the corresponding evolutionarily conserved chromosome segments (ECCS) of the human genome. Large-insert clones representing each marker were first isolated and mapped by fluorescence in situ hybridization (FISH) analysis to determine their subchromosomal localization on CFA 5. Thirteen gene markers were subsequently mapped by using a commercially available whole genome radiation hybrid (WG-RH) panel for the dog. Nine anonymous markers were also assigned to CFA 5 by both FISH and WG-RH analysis. The 22 markers formed six RH-linkage groups, spanning each of the four ECCS comprising this 99 megabase chromosome. All cytogenetic, WG-RH, and comparative mapping data were in agreement and were combined to determine both the most likely locus order within each linkage group, and also the gross relative orientation of the corresponding ECCS. This study provides a resource for the transfer of information from the human transcript map to that of the dog, and extends existing data regarding the structural relationships between CFA 5 and its evolutionary counterparts within the human genome.
Animals↗
Mapping of 13 horse genes by fluorescence in-situ hybridization (FISH) and somatic cell hybrid analysis.
We report fluorescence in-situ hybridization (FISH) and somatic cell hybrid mapping data for 13 different horse genes (ANP, CD2, CLU, CRISP3, CYP17, FGG, IL1RN, IL10, MMP13, PRM1, PTGS2, TNFA and TP53). Primers for PCR amplification of intronic or untranslated regions were designed from horse-specific DNA or mRNA sequences in GenBank. Two different horse bacterial artificial chromosome (BAC) libraries were screened with PCR for clones containing these 13 Type I loci, nine of which were found in the libraries. BAC clones were used as probes in dual colour FISH to confirm their precise chromosomal origin. The remaining four genes were mapped in a somatic cell hybrid panel. All chromosomal assignments except one were in agreement with human-horse ZOO-FISH data and revealed new and more detailed information on the equine comparative map. CLU was mapped by synteny to ECA2 while human-horse ZOO-FISH data predicted that CLU would be located on ECA9. The assignment of IL1RN permitted analysis of gene order conservation between HSA2 and ECA15, which identified that an event of inversion had occurred during the evolution of these two homologous chromosomes.
Animals↗
The canine homeobox gene MSX2: sequence, chromosome assignment and genetic analysis in dogs of different breeds.
The MSX2 gene encodes a homeodomain transcription factor important for normal head and face morphogenesis. MSX2 is expressed in key craniofacial structures during development and mutations in the human gene give rise to various craniofacial abnormalities. We are interested in the genetic basis of non-pathogenic variation in skull and face shape. As part of this study we have analysed DNA from a panel of different dog breeds, selected for the differences they show in these traits and investigated MSX2 as a candidate gene. In this paper we describe the cloning of the canine homologue of MSX2, the determination of its structure, sequence and localization of the gene to dog chromosome 4q23. The DNAs from 11 individual domestic dogs belonging to 10 different breeds were sequenced in a search for genetic variation. Our studies show that variation in MSX2 does not contribute to the diversity of face shape observed in these domestic dogs and that the MSX2 sequence is strongly conserved between different dog breeds. The proximal promoter shows a high level of interspecies sequence conservation and several conserved transcription factor binding motifs have been identified and their significance discussed.
Amino Acid Sequence↗
Physical anchorage and orientation of equine linkage groups by FISH mapping BAC clones containing microsatellite markers.
A horse bacterial artificial chromosome (BAC) library was screened for 19 microsatellite markers from unassigned or non-oriented linkage groups. Clones containing 11 (AHT20, EB2E8, HMS45, LEX005, LEX014, LEX023, LEX044, TKY111, UCDEQ425, UCDEQ464 and VIASH21) of these were found, which were from eight different linkage groups. The BAC clones were used as probes in dual colour FISH to identify their precise chromosomal origin. The microsatellite markers are located on nine different horse chromosomes, four of which (ECA6, ECA25, ECA27 and ECA28) had no previously in situ assigned markers.
Animals↗
Chromosome assignment of six dog genes by FISH, and correlation with dog-human Zoo-FISH data.
Cross-species chromosome painting analyses have recently demonstrated the presence of regions of conserved synteny between the human and domestic dog genomes, aiding the search for candidate genes for inherited traits. Concerted efforts to subchromosomally assign substantial numbers of dog gene sequences are now needed in order to refine these comparative data, both in terms of marker density and resolution. We have developed novel PCR markers representing three dog genes (ALB, FOS, HNRPA2B1) for which no sequence or mapping data were previously available, to our knowledge. These, in addition to three gene markers previously described (ALDOA, RPE65, VCAM1), were used to isolate and chromosomally assign corresponding large insert genomic clones by fluorescence in situ hybridization (FISH). Chromosome assignments for these six dog genes are discussed in terms of those of the human orthologues, and correlated with existing comparative mapping information, identifying one apparent exception to existing Zoo-FISH data, and aiding refinement of the boundaries of conserved chromosome segments in both genomes.
Animals↗
Physical and linkage mapping of the canine phosphate carrier (SLC25A3) and apoptotic activating factor 1 (APAF1) genes to canine chromosome 15.
Explore the source record for details and available documents.
Animals↗
Chromosome-specific single-locus FISH probes allow anchorage of an 1800-marker integrated radiation-hybrid/linkage map of the domestic dog genome to all chromosomes.
We present here the first fully integrated, comprehensive map of the canine genome, incorporating detailed cytogenetic, radiation hybrid (RH), and meiotic information. We have mapped a collection of 266 chromosome-specific cosmid clones, each containing a microsatellite marker, to all 38 canine autosomes by fluorescence in situ hybridization (FISH). A 1500-marker RH map, comprising 1078 microsatellites, 320 dog gene markers, and 102 chromosome-specific markers, has been constructed using the RHDF5000-2 whole-genome radiation hybrid panel. Meiotic linkage analysis was performed, with at least one microsatellite marker from each dog autosome on a panel of reference families, allowing one meiotic linkage group to be anchored to all 38 dog autosomes. We present a karyotype in which each chromosome is identified by one meiotic linkage group and one or more RH groups. This updated integrated map, containing a total of 1800 markers, covers >90% of the dog genome. Positional selection of anchor clones enabled us, for the first time, to orientate nearly all of the integrated groups on each chromosome and to evaluate the extent of individual chromosome coverage in the integrated genome map. Finally, the inclusion of 320 dog genes into this integrated map enhances existing comparative mapping data between human and dog, and the 1000 mapped microsatellite markers constitute an invaluable tool with which to perform genome scanning studies on pedigrees of interest.
Animals↗
DNA sequence and physical mapping of the canine transglutaminase 1 gene.
The transglutaminase 1 gene (TGM1) encodes an enzyme necessary for cross-linking the structural proteins that form the cornified envelope, an essential component of the outermost layer of the skin, the stratum corneum. Reported here is the complete coding region of canine TGM1, its chromosome localization, and its map position in the integrated canine linkage-radiation hybrid map. Canine TGM1 consists of 2,448 nucleotides distributed over 15 exons. The nucleotide sequence has 90% identity to human TGM1. The deduced canine TGM1 protein is 816 amino acids long and is 92% identical to human TGM1. Using fluorescence in situ hybridization, we localized canine TGM1 to dog (Canis familiaris) chromosome 8 (CFA 8q). Canine TGM1 localized to CFA 8 on the integrated linkage-radiation hybrid map in the interval FH2149-MYH7. Characterizing the coding region of canine TGM1 is a first step in examining the role of this enzyme in normal and defective cornification in the dog.
Amino Acid Sequence↗
Characterization and chromosomal localization of five canine ATOX1 pseudogenes.
We have isolated six ATOX1 loci from the canine genome in BAC clones. Sequence analysis showed that five of these clones correspond to processed pseudogenes. Fluorescent in situ hybridization allowed us to map the genuine ATOX1 gene to CFA4q24-->q31 and the ATOX1 pseudogenes to CFA19q13.1, CFA4q24-->q31, CFA18q24-->q25, CFA9q22.1 -->q22.2 and CFA20q11-->q12.
Amino Acid Sequence↗
First comprehensive low-density horse linkage map based on two 3-generation, full-sibling, cross-bred horse reference families.
Two 3-generation full-sibling reference families have been produced and form a unique resource for genetic linkage mapping studies in the horse. The F(2) generations, now comprising 61 individuals, consist of 28- to 32-day-old embryos removed nonsurgically from two pairs of identical twin mares. The same stallion sired all F(2)s such that the two full-sibling families are half-sibling with respect to each other. The families are crossbred to maximize levels of heterozygosity and include Arabian, Thoroughbred, Welsh Cob, and Icelandic Horse breeds. Milligram quantities of DNA have been isolated from each embryo and from blood samples of the parents and grandparents. The families have been genotyped with 353 equine microsatellites and 6 biallelic markers, and 42 linkage groups were formed. In addition, the physical location of 85 of the markers is known, and this has allowed 37 linkage groups to be anchored to the physical map. The inclusion of dams in the genotyping analysis has allowed the generation of a genetic map of the X chromosome. Markers have been assigned to all 31 autosomes and the X chromosome. The average interval between markers on the map is 10.5 cM, and the linkage groups collectively span 1780 cM. The results demonstrate the benefits for horse linkage mapping studies of genotyping on these unique full-sibling families, which comprise relatively few individuals, by the generation of a comprehensive low-density map of the horse genome.
Animals↗
Cloning of the canine gene encoding transcription factor Pit-1 and its exclusion as candidate gene in a canine model of pituitary dwarfism.
Combined pituitary hormone deficiency (CPHD) is an autosomal recessive inherited disease of German shepherd dogs characterized primarily by dwarfism. In mice and humans a similar genetic disorder has been described that results from an alteration in the gene encoding the transcription factor Pit-1. In this study we characterized the canine Pit-1 gene, determined the chromosomal localization of the Pit-1 gene, and screened dwarf German shepherd dogs for the presence of mutations in this gene. The full-length canine Pit-1 cDNA contained an open reading frame encoding 291 amino acids, 92 bp of 5'-untranslated region, and 1959 bp of 3'-untranslated region. The deduced amino acid sequence was highly homologous with Pit-1 of other mammalian species. Using a Pit-1 BAC clone as probe, the Pit-1 gene was mapped by FISH to canine Chromosome (Chr) 31. In dwarf German shepherd dogs a C to A transversion was detected, causing a Phe (TTC) to Leu (TTA) substitution at codon 81. This alteration was present neither in other canine breeds analyzed nor in other mammalian species. However, healthy German shepherd dogs were also homozygous for the mutant allele, indicating that it is not the primary disease-causing mutation. In addition, linkage analysis of polymorphic DNA markers flanking the Pit-1 gene, 41K19 and 52L05, revealed no co-segregation between the Pit-1 locus and the CPHD phenotype. These findings suggest that a gene other than Pit-1 is responsible for the pituitary anomaly in dwarf German shepherd dogs.
Amino Acid Sequence↗
Molecular analysis and chromosomal assignment of the canine CALC-I/alpha-CGRP gene.
We have isolated a recombinant phage harboring the canine CALC-I/alpha-CGRP gene. The gene spans a region of approx. 5.3 kb and consists of six exons with sizes ranging from 95 bp (exon 2) and 494 bp (exon 4). By alternative splicing, two transcripts with ORFs of 390 and 384 nt are generated. These encode either the 32-amino acid-long hormone calcitonin (CALC) or the neurotransmitter calcitonin gene-related peptide (alpha-CGRP) with a length of 37 amino acids after proteolytic processing of precursor molecules. The canine calcitonin precursor consists of 130 amino acids with a molecular mass of 14.05 kDa and a statistical pI of 8.0, whereas the deduced alpha-CGRP precursor harbors 128 amino acids with a molecular mass of 13.87 kDa and a statistical pI of 8.6. Both polypeptides have a common N-terminal region of 76 amino acids that is encoded by exons 2 and 3 and separated by different eight (CALC) or six (alpha-CGRP) amino acid spacers from the biologically active polypeptide. The CALC-I/alpha-CGRP gene is a member of the calcitonin gene family and was assigned to chromosome CFA 16q25.1. A comparative analysis of different dog breeds revealed a breed-specific allelic d(CAGGAG)-hexanucleotide expansion in exon 3. This expansion results in an elongation of the common N-terminal region by two amino acids (glutamine-glutamic acid) and alters the molecular mass to 14.31 kDa (pI 7.9) and 14.13 kDa (pI 8.5) of the calcitonin and alpha-CGRP precursor, respectively.
Alternative Splicing↗
Comparative genomic hybridization (CGH) in dogs--application to the study of a canine glial tumour cell line.
Recurrent chromosome aberrations are associated with many human cancers. Detailed cytogenetic analysis of tumors has benefited enormously from the development of molecular cytogenetic techniques based on fluorescence in situ hybridization (FISH). Comparative genomic hybridization (CGH) is a recently developed FISH technique that allows a rapid and comprehensive identification of imbalanced genomic material in tumour DNA. Comparative genomic hybridisation has been used widely in human medicine to evaluate losses and gains of tumour DNA isolated from a variety of sources, including fresh samples, cell-culture material and archival specimens, and has been instrumental in identifying sites in the human genome which contain genies involved in tumour development and progression. This report describes the first application of CGH in the dog, illustrated by the analysis of DNA isolated from a canine glial tumour cell line.
Animals↗
Genomic organization of the dog dystroglycan gene DAG1 locus on chromosome 20q15.1-q15.2.
Dystroglycan is a laminin binding protein, which provides a structural link between the subsarcolemmal cytoskeleton and the extracellular matrix. It is also involved in the organization of basement membranes. So far the genomic organization of the dystroglycan gene DAG1 has not been completely investigated. Here we report the cloning and sequencing of 162 kb of dog genomic DNA containing the complete approximately 71-kb canine DAG1 gene, which consists of three exons, with the translation start codon located in exon 2. Its 2679-nucleotide ORF encodes a polypeptide of 892 amino acids, which is highly similar to human, rabbit, and bovine orthologs. To further characterize the dog DAG1 gene we determined the transcription start site and several naturally occurring polymorphisms, which partially result in amino acid substitutions of the dystroglycan protein. The dog DAG1 gene was assigned to chromosome 20q15.1-q15.2 by FISH analysis. The analysis of the entire reported sequence revealed that the genes for aminomethyltransferase (AMT), bassoon (BSN), TCTA (T-cell leukemia translocation-associated) gene, and an as yet uncharacterized protein are located very close to the DAG1 gene. Therefore, this study defines a novel syntenic region among dog chromosome 20q15, human chromosome 3p21, and murine chromosome 9F.
Animals↗
Cloning, characterization, and physical mapping of the canine Prop-1 gene (PROP1): exclusion as a candidate for combined pituitary hormone deficiency in German shepherd dogs.
Abnormalities in the genes encoding Pit-1 and Prop-1 have been reported to cause combined pituitary hormone deficiency (CPHD) in mice and humans. In dogs, a similar phenotype has been described in the German shepherd breed. We have previously reported that the Pit-1 gene (POU1F1) is not mutated in affected German shepherd dogs. In this study, we report the isolation and mapping of the canine Prop-1 gene (PROP1), and we assessed the involvement of PROP1 in German shepherd dog dwarfism. The canine PROP1 gene was found to contain three exons, encoding a 226 amino acid protein. The deduced amino acid sequence was 79% and 84% homologous with the mouse and human Prop-1 protein, respectively. Using fluorescence in situ hybridization, PROP1 was mapped to canine chromosome 11. Further mapping with a canine radiation hybrid panel showed co-localization with the polymorphic DNA marker AHT137. Sequence analysis of genomic DNA from dwarf German shepherd dogs revealed no alterations in the PROP1 gene. Moreover, linkage analysis of AHT137 revealed no co-segregation between the PROP1 locus and the CPHD phenotype, excluding this gene as candidate for canine CPHD and providing a new spontaneous model of hypopituitarism.
Amino Acid Sequence↗