RESUMO
Vitamin D is a potent steroid hormone that induces widespread changes in gene expression and controls key biological pathways. Here we review pathophysiology of vitamin D with particular reference to COVID-19 and pancreatic cancer. Utility as a therapeutic agent is limited by hypercalcemic effects and attempts to circumvent this problem have used vitamin D superagonists, with increased efficacy and reduced calcemic effect. A further caveat is that vitamin D mediates multiple diverse effects. Some of these (anti-fibrosis) are likely beneficial in patients with COVID-19 and pancreatic cancer, whereas others (reduced immunity), may be beneficial through attenuation of the cytokine storm in patients with advanced COVID-19, but detrimental in pancreatic cancer. Vitamin D superagonists represent an untapped resource for development of effective therapeutic agents. However, to be successful this approach will require agonists with high cell-tissue specificity.
Assuntos
Tratamento Farmacológico da COVID-19 , Carcinoma Ductal Pancreático/tratamento farmacológico , Neoplasias Pancreáticas/tratamento farmacológico , Vitamina D/agonistas , Vitaminas/agonistas , Síndrome da Liberação de Citocina/tratamento farmacológico , Humanos , Vitamina D/fisiologiaRESUMO
Type II topoisomerase enzymes are essential for resolving DNA topology problems arising through various aspects of DNA metabolism. In vertebrates two isoforms are present, one of which (TOP2A) accumulates on chromatin during mitosis. Moreover, TOP2A targets the mitotic centromere during prophase, persisting there until anaphase onset. It is the catalytically-dispensable C-terminal domain of TOP2 that is crucial in determining this isoform-specific behaviour. In this study we show that, in addition to the recently identified chromatin tether domain, several other features of the alpha-C-Terminal Domain (CTD). influence the mitotic localisation of TOP2A. Lysine 1240 is a major SUMOylation target in cycling human cells and the efficiency of this modification appears to be influenced by T1244 and S1247 phosphorylation. Replacement of K1240 by arginine results in fewer cells displaying centromeric TOP2A accumulation during prometaphase-metaphase. The same phenotype is displayed by cells expressing TOP2A in which either of the mitotic phosphorylation sites S1213 or S1247 has been substituted by alanine. Conversely, constitutive modification of TOP2A by fusion to SUMO2 exerts the opposite effect. FRAP analysis of protein mobility indicates that post-translational modification of TOP2A can influence the enzyme's residence time on mitotic chromatin, as well as its subcellular localisation.
Assuntos
Anáfase/fisiologia , Cromatina/metabolismo , DNA Topoisomerases Tipo II/metabolismo , Metáfase/fisiologia , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Linhagem Celular , Centrômero/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células HEK293 , Humanos , Fosforilação/fisiologia , Processamento de Proteína Pós-Traducional/fisiologia , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Sumoilação/fisiologiaRESUMO
In addition to its roles in transcription and replication, topoisomerase 2 (topo 2) is crucial in shaping mitotic chromosomes and in ensuring the orderly separation of sister chromatids. As well as its recruitment throughout the length of the mitotic chromosome, topo 2 accumulates at the primary constriction. Here, following cohesin release, the enzymatic activity of topo 2 acts to remove residual sister catenations. Intriguingly, topo 2 does not bind and cleave all sites in the genome equally; one preferred site of cleavage is within the core centromere. Discrete topo 2-centromeric cleavage sites have been identified in α-satellite DNA arrays of active human centromeres and in the centromere regions of some protozoans. In this study, we show that topo 2 cleavage sites are also a feature of the centromere in Schizosaccharomyces pombe, the metazoan Drosophila melanogaster and in another vertebrate species, Gallus gallus (chicken). In vertebrates, we show that this site-specific cleavage is diminished by depletion of CENP-I, an essential constitutive centromere protein. The presence, within the core centromere of a wide range of eukaryotes, of precise sites hypersensitive to topo 2 cleavage suggests that these mark a fundamental and conserved aspect of this functional domain, such as a non-canonical secondary structure.
Assuntos
Centrômero/genética , DNA Topoisomerases Tipo II/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Centrômero/metabolismo , Proteína Centromérica A/metabolismo , Galinhas , Proteínas Cromossômicas não Histona/metabolismo , DNA Satélite/genética , Drosophila melanogaster , Humanos , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , CoesinasRESUMO
As proliferating cells transit from interphase into M-phase, chromatin undergoes extensive reorganization, and topoisomerase (topo) IIα, the major isoform of this enzyme present in cycling vertebrate cells, plays a key role in this process. In this study, a human cell line conditional null mutant for topo IIα and a derivative expressing an auxin-inducible degron (AID)-tagged version of the protein have been used to distinguish real mitotic chromosome functions of topo IIα from its more general role in DNA metabolism and to investigate whether topo IIß makes any contribution to mitotic chromosome formation. We show that topo IIß does contribute, with endogenous levels being sufficient for the initial stages of axial shortening. However, a significant effect of topo IIα depletion, seen with or without the co-depletion of topo IIß, is the failure of chromosomes to hypercompact when delayed in M-phase. This requires much higher levels of topo II protein and is impaired by drugs or mutations that affect enzyme activity. A prolonged delay at the G2/M border results in hyperefficient axial shortening, a process that is topo IIα-dependent. Rapid depletion of topo IIα has allowed us to show that its function during late G2 and M-phase is truly required for shaping mitotic chromosomes.
Assuntos
Antígenos de Neoplasias/fisiologia , Cromossomos Humanos , DNA Topoisomerases Tipo II/fisiologia , Proteínas de Ligação a DNA/fisiologia , Mitose/genética , Antígenos de Neoplasias/genética , Domínio Catalítico , Linhagem Celular , DNA Topoisomerases Tipo II/genética , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Fase G2/genética , Humanos , Mutação , Isoformas de Proteínas/antagonistas & inibidores , Quinolinas/farmacologia , Tiazóis/farmacologiaRESUMO
Mitotic chromosome formation involves a relatively minor condensation of the chromatin volume coupled with a dramatic reorganization into the characteristic "X" shape. Here we report results of a detailed morphological analysis, which revealed that chromokinesin KIF4 cooperated in a parallel pathway with condensin complexes to promote the lateral compaction of chromatid arms. In this analysis, KIF4 and condensin were mutually dependent for their dynamic localization on the chromatid axes. Depletion of either caused sister chromatids to expand and compromised the "intrinsic structure" of the chromosomes (defined in an in vitro assay), with loss of condensin showing stronger effects. Simultaneous depletion of KIF4 and condensin caused complete loss of chromosome morphology. In these experiments, topoisomerase IIα contributed to shaping mitotic chromosomes by promoting the shortening of the chromatid axes and apparently acting in opposition to the actions of KIF4 and condensins. These three proteins are major determinants in shaping the characteristic mitotic chromosome morphology.
Assuntos
Adenosina Trifosfatases/metabolismo , Antígenos de Neoplasias/metabolismo , Cromossomos/metabolismo , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA/metabolismo , Cinesinas/metabolismo , Mitose , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismo , Adenosina Trifosfatases/genética , Animais , Galinhas , Cromátides/metabolismo , Proteínas de Ligação a DNA/genética , Cinesinas/genética , Complexos Multiproteicos/genética , Mutação , Proteínas Nucleares/genética , Células Tumorais CultivadasRESUMO
Human centromeres are multi-megabase regions of highly ordered arrays of alpha satellite DNA that are separated from chromosome arms by unordered alpha satellite monomers and other repetitive elements. Complexities in assembling such large repetitive regions have limited detailed studies of centromeric chromatin organization. However, a genomic map of the human X centromere has provided new opportunities to explore genomic architecture of a complex locus. We used ChIP to examine the distribution of modified histones within centromere regions of multiple X chromosomes. Methylation of H3 at lysine 4 coincided with DXZ1 higher order alpha satellite, the site of CENP-A localization. Heterochromatic histone modifications were distributed across the 400-500 kb pericentromeric regions. The large arrays of alpha satellite and gamma satellite DNA were enriched for both euchromatic and heterochromatic modifications, implying that some pericentromeric repeats have multiple chromatin characteristics. Partial truncation of the X centromere resulted in reduction in the size of the CENP-A/Cenp-A domain and increased heterochromatic modifications in the flanking pericentromere. Although the deletion removed approximately 1/3 of centromeric DNA, the ratio of CENP-A to alpha satellite array size was maintained in the same proportion, suggesting that a limited, but defined linear region of the centromeric DNA is necessary for kinetochore assembly. Our results indicate that the human X centromere contains multiple types of chromatin, is organized similarly to smaller eukaryotic centromeres, and responds to structural changes by expanding or contracting domains.
Assuntos
Centrômero , Cromossomos Humanos X , Histonas/metabolismo , Animais , Sequência de Bases , Metilação de DNA , Primers do DNA , Humanos , CamundongosRESUMO
DT40 is a B-cell lymphoma-derived avian cell line widely used to study cell autonomous gene function because of the high rates with which DNA constructs are homologously recombined into its genome. Here, we demonstrate that the power of the DT40 system can be extended yet further through the use of RNA interference as an alternative to gene targeting. We have generated and characterized stable DT40 transfectants in which both topo 2 genes have been in situ tagged using gene targeting, and from which the mRNA of both topoisomerase 2 isoforms can be conditionally depleted through the tetracycline-induced expression of short hairpin RNAs. The cell cycle phenotype of topo 2-depleted DT40 cells has been compared with that previously reported for other vertebrate cells depleted either of topo 2alpha through gene targeting, or depleted of both isoforms simultaneously by transient RNAi. In addition, the DT40 knockdown system has been used to explore whether excess catenation arising through topo 2 depletion is sufficient to trigger the G2 catenation (or decatenation) checkpoint, proposed to exist in differentiated vertebrate cells.
Assuntos
DNA Topoisomerases Tipo II/genética , Técnicas de Silenciamento de Genes , Interferência de RNA , Animais , Ciclo Celular , Morte Celular , Linhagem Celular Tumoral , Proliferação de Células , Galinhas/genética , Genes Letais , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Índice Mitótico , Fenótipo , Ploidias , Inibidores da Topoisomerase II , TransgenesRESUMO
The telomere end-protection complex prevents the ends of linear eukaryotic chromosomes from degradation or inappropriate DNA repair. The homodimeric double-stranded DNA-binding protein, Trf1, is a component of this complex and is essential for mouse embryonic development. To define the requirement for Trf1 in somatic cells, we deleted Trf1 in chicken DT40 cells by gene targeting. Trf1-deficient cells proliferated as rapidly as control cells and showed telomeric localization of Trf2, Rap1, and Pot1. Telomeric G-strand overhang lengths were increased in late-passage Trf1-deficient cells, although telomere lengths were unaffected by Trf1 deficiency, as determined by denaturing Southern and quantitative FISH analysis. Although we observed some clonal variation in terminal telomere fragment lengths, this did not correlate with cellular Trf1 levels. Trf1 was not required for telomere seeding, indicating that de novo telomere formation can proceed without Trf1. The Pin2 isoform and a novel exon 4, 5-deleted isoform localized to telomeres in Trf1-deficient cells. Trf1-deficient cells were sensitive to DNA damage induced by ionizing radiation. Our data demonstrate that chicken DT40 B cells do not require Trf1 for functional telomere structure and suggest that Trf1 may have additional, nontelomeric roles involved in maintaining genome stability.
Assuntos
Telômero/metabolismo , Proteína 1 de Ligação a Repetições Teloméricas/metabolismo , Animais , Linhagem Celular , Proliferação de Células/efeitos da radiação , Galinhas , Marcação de Genes , Hibridização in Situ Fluorescente , Fenótipo , Splicing de RNA/efeitos da radiação , Radiação Ionizante , Telomerase/metabolismo , Proteína 1 de Ligação a Repetições Teloméricas/deficiência , TransfecçãoRESUMO
When chromosomes are aligned and bioriented at metaphase, the elastic stretch of centromeric chromatin opposes pulling forces exerted on sister kinetochores by the mitotic spindle. Here we show that condensin ATPase activity is an important regulator of centromere stiffness and function. Condensin depletion decreases the stiffness of centromeric chromatin by 50% when pulling forces are applied to kinetochores. However, condensin is dispensable for the normal level of compaction (rest length) of centromeres, which probably depends on other factors that control higher-order chromatin folding. Kinetochores also do not require condensin for their structure or motility. Loss of stiffness caused by condensin-depletion produces abnormal uncoordinated sister kinetochore movements, leads to an increase in Mad2(+) kinetochores near the metaphase plate and delays anaphase onset.
Assuntos
Adenosina Trifosfatases/metabolismo , Centrômero/metabolismo , Proteínas de Ligação a DNA/metabolismo , Complexos Multiproteicos/metabolismo , Vertebrados/metabolismo , Animais , Autoantígenos/metabolismo , Linhagem Celular , Centrômero/ultraestrutura , Proteína Centromérica A , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Inativação Gênica , Proteínas de Fluorescência Verde/metabolismo , Humanos , Cinetocoros/metabolismo , Cinetocoros/ultraestrutura , Microtúbulos/ultraestrutura , Mitose , Proteínas Recombinantes de Fusão/metabolismo , Fuso Acromático/metabolismo , Fuso Acromático/ultraestruturaRESUMO
We mapped regulatory loci for nearly all protein-coding genes in mammals using comparative genomic hybridization and expression array measurements from a panel of mouse-hamster radiation hybrid cell lines. The large number of breaks in the mouse chromosomes and the dense genotyping of the panel allowed extremely sharp mapping of loci. As the regulatory loci result from extra gene dosage, we call them copy number expression quantitative trait loci, or ceQTLs. The -2log10P support interval for the ceQTLs was <150 kb, containing an average of <2-3 genes. We identified 29,769 trans ceQTLs with -log10P > 4, including 13 hotspots each regulating >100 genes in trans. Further, this work identifies 2,761 trans ceQTLs harboring no known genes, and provides evidence for a mode of gene expression autoregulation specific to the X chromosome.
Assuntos
Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Genes/fisiologia , Locos de Características Quantitativas , Mapeamento de Híbridos Radioativos , Animais , Cricetinae , Mecanismo Genético de Compensação de Dose , Genoma , Genótipo , Células Híbridas , Camundongos , Hibridização de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Cromossomo X/genéticaRESUMO
Telomere repeat sequences are added to linear chromosome ends by telomerase, an enzyme comprising a reverse transcriptase (TERT) and an RNA template component (TR). We aimed to investigate TR in the DT40 B-cell tumour line using gene targeting, but were unable to generate TR nulls, suggesting a requirement for TR in DT40 proliferation. Disruption of one TR allele reduced telomerase activity and caused a progressive decline in telomere and G-strand overhang length. We then examined the interactions between TR and cellular DNA double-strand break (DSB) repair. Deletion in TR+/- cells of the gene encoding the non-homologous end-joining protein, Ku70, caused rapid loss of G-strand overhangs. Ku70-/-TR+/- cells proliferated more slowly than either single mutant and showed frequent mitotic aberrations. Activation of the DNA damage response was observed in TR-deficient cells and was exacerbated by Ku deficiency, although frequent telomeric DNA damage signals were not observed until late passages. This activation of the DNA damage response was suppressed by deletion of Rad54, a key homologous recombination gene. These findings suggest that Ku and telomerase cooperate to block homologous recombination from acting on telomeres.
Assuntos
Antígenos Nucleares/metabolismo , Galinhas/genética , Galinhas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Instabilidade Genômica/genética , RNA/genética , Telomerase/genética , Animais , Linhagem Celular Tumoral , Proliferação de Células , Aberrações Cromossômicas , Dano ao DNA , Heterozigoto , Autoantígeno Ku , RNA/metabolismo , Telomerase/metabolismo , Telômero/genética , Proteínas de Ligação a Telômeros/metabolismoRESUMO
Topoisomerase II (topo II) is a major component of mitotic chromosomes, and its unique decatenating activity has been implicated in many aspects of chromosome dynamics, of which chromosome segregation is the most seriously affected by loss of topo II activity in living cells. There is considerable evidence that topo II plays a role at the centromere including: the centromere-specific accumulation of topo II protein; cytogenetic/molecular mapping of the catalytic activity of topo II to active centromeres; the influence of sumoylated topo II on sister centromere cohesion; and its involvement in the activation of a Mad2-dependent spindle checkpoint. By using a human cell line with a conditional-lethal mutation in the gene encoding DNA topoisomerase IIalpha, we find that depletion of topo IIalpha, while leading to a disorganised metaphase plate, does not have any overt effect on general assembly of kinetochores. Fluorescence in situ hybridisation suggested that centromeres segregate normally, most segregation errors being chromatin bridges involving longer chromosome arms. Strikingly, a linear human X centromere-based minichromosome also displayed a significantly increased rate of missegregation. This sensitivity to depletion of topo IIalpha might be linked to structural alterations within the centromere domain, as indicated by a significant shortening of the distance across metaphase sister centromeres and the abnormal persistence of PICH-coated connections between segregating chromatids.
Assuntos
Anáfase , DNA Helicases/metabolismo , DNA Topoisomerases Tipo II/deficiência , Proteínas de Ligação a DNA/deficiência , Cinetocoros/metabolismo , Metáfase , Antígenos de Neoplasias , Segregação de Cromossomos , Cromossomos Humanos/metabolismo , Humanos , Isoenzimas/metabolismoRESUMO
The reversible condensation of chromosomes during cell division remains a classic problem in cell biology. Condensation requires the condensin complex in certain experimental systems, but not in many others. Anaphase chromosome segregation almost always fails in condensin-depleted cells, leading to the formation of prominent chromatin bridges and cytokinesis failure. Here, live-cell analysis of chicken DT40 cells bearing a conditional knockout of condensin subunit SMC2 revealed that condensin-depleted chromosomes abruptly lose their compact architecture during anaphase and form massive chromatin bridges. The compact chromosome structure can be preserved and anaphase chromosome segregation rescued by preventing the targeting subunit Repo-Man from recruiting protein phosphatase 1 (PP1) to chromatin at anaphase onset. This study identifies an activity critical for mitotic chromosome structure that is inactivated by Repo-Man-PP1 during anaphase. This activity, provisionally termed 'regulator of chromosome architecture' (RCA), cooperates with condensin to preserve the characteristic chromosome architecture during mitosis.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromossomos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Mitose , Complexos Multiproteicos/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Anáfase , Animais , Células Cultivadas , Galinhas , Cromatina/metabolismo , Segregação de Cromossomos , Cromossomos/química , Humanos , Proteínas Nucleares , Proteína Fosfatase 1 , Fuso Acromático/metabolismoAssuntos
Cromossomos/genética , Cromossomos/metabolismo , Animais , Evolução Biológica , Centrômero , Cromossomos/ultraestrutura , Cromossomos Artificiais , DNA/genética , DNA/metabolismo , DNA/ultraestrutura , Replicação do DNA/genética , Humanos , Origem de Replicação , Telômero/química , Telômero/genética , Telômero/metabolismo , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismoRESUMO
Chromosome engineering has allowed the generation of an extensive and well-defined series of linear human X centromere-based minichromosomes, which has been used to investigate the influence of size and structure on chromosome segregation in vertebrate cells. A clear relationship between overall chromosome size and mitotic stability was detected, with decreasing size associated with increasing loss rates. In chicken DT40, the lower size limit for prolonged mitotic stability is approximately 550 kb: at 450 kb, there was a dramatic increase in chromosome loss, while structures of approximately 200 kb could not be recovered. In human HT1080 cells, the size threshold for mitotic stability is approximately 1.6 Mb. Minichromosomes of 0.55-1.0 Mb can be recovered, but display high loss rates. However, all minichromosomes examined exhibited more segregation errors than normal chromosomes in HT1080 cells. This error rate increases with decreased size and correlates with reduced levels of CENP-A and Aurora B. In mouse LA-9 and Indian muntjac FM7 cells, the size requirements for mitotic stability are much greater. In mouse, a human 2.7-Mb minichromosome is rarely able to propagate a kinetochore and behaves acentrically. In Indian muntjac, CENP-C associates with the human minichromosome, but the mitotic apparatus appears unable to handle its segregation.
Assuntos
Cromossomos , Vertebrados/genética , Animais , Sequência de Bases , Southern Blotting , Centrômero , Galinhas , Cromossomos Humanos X , Primers do DNA , DNA Recombinante , Eletroforese em Gel de Campo Pulsado , Humanos , Mitose , Hibridização de Ácido NucleicoRESUMO
Topoisomerase II (Topo II) is a major component of mitotic chromosomes and its unique decatenating activity has been implicated in many aspects of chromosome dynamics including DNA replication, transcription, recombination, chromosome condensation and segregation. Of these, chromosome segregation is the most seriously affected by loss of Topo II, most probably because of residual catenations between sister chromatids. At metaphase, vertebrate chromatids are attached principally through their centromeric regions. Intriguingly, evidence has recently been presented for Topo II cleavage activity within the centromeric alpha-satellite DNA arrays of the human X and Y chromosomes. In this report we extend these observations by mapping distinct sites of Topo II cleavage activity within the alpha-satellite array of human chromosome 11. A single major site of cleavage has been assigned within the centromeric DNA of each of three independently derived, and active, 11 centromeres. Unlike the X and Y centromeres, where cleavage sites mapped close to (within 150 kb of) the short arm edge of the arrays, on chromosome 11, the cleavage sites lie many hundreds of kilobases into each alpha-satellite array. We also demonstrate that catalytically active Topo II is concentrated within the centromere domain through an extended period of G2 and M, with levels declining in G1 and S.
Assuntos
DNA Topoisomerases Tipo II/metabolismo , DNA Satélite/genética , Sequência de Bases , Cromossomos Humanos Par 11 , Primers do DNA , Eletroforese em Gel de Campo Pulsado , Humanos , Células Híbridas , Hidrólise , Metáfase , Reação em Cadeia da PolimeraseRESUMO
Topoisomerase II (topo II) is a major component of mitotic chromosomes and its unique decatenating activity has been implicated in many aspects of chromosome dynamics including DNA replication, transcription, recombination, chromosome condensation and segregation. Of these, chromosome segregation is the most seriously affected by loss of topo II expression or activity in living cells, most likely because of residual catenations between sister chromatids. At metaphase, vertebrate chromatids are attached to each other principally through their centromeric regions, and we review here evidence that topo II has a specific role at the centromere. Despite strong evidence for the centromere-specific accumulation of topo II protein and the cytogenetic and molecular mapping of topo II catalytic activity to active centromeres, there is so far relatively little evidence for an overt role in centromere function (as judged by the effects of topo II inactivation on kinetochore assembly, bipolar microtubule attachment and chromosome separation). Nevertheless, recent data linking the post-translational modification of topo II to the regulation of sister centromere cohesion suggest that topo II may indeed contribute to the timely separation of centromeres at anaphase.
Assuntos
Centrômero/enzimologia , Segregação de Cromossomos , DNA Topoisomerases Tipo II/fisiologia , Animais , Centrômero/metabolismo , DNA Topoisomerases Tipo II/metabolismo , HumanosAssuntos
Cromossomos , Vetores Genéticos , Mamíferos/genética , Animais , Terapia Genética , Humanos , TransgenesRESUMO
Chromosome (chr) X is under-represented in current maps of the genome of the domestic dog ( Canis familiaris). To address this problem, we have constructed a small-insert, genomic DNA library in pBluescript from flow-sorted canine Chr X DNA. Fluorescence in situ hybridization (FISH) studies confirmed that the library was highly enriched for Chr X. Clones containing microsatellites were identified and sequenced. Database searches detected significant sequence identity between four X-derived clones and genes previously characterized in other species. Thirty-seven markers derived from these clones were mapped on Chr X by FISH, and of these, 28 were mapped by using the female-derived T72 whole-genome radiation hybrid (RH) panel (Research Genetics). Four X-linked canine genes from publicly available data were also mapped. Eight RH linkage groups with LOD >4.0 were identified, and FISH data were used to locate the groups on the chromosome; four groups could be unambiguously orientated by FISH data. In each case, the FISH and RH data were mutually consistent. The data suggest strongly conserved synteny between canine and human X Chrs. The pseudoautosomal region has been further characterized, and the putative or actual locations of nine genes of clinical relevance have been suggested.