5-Formylcytosine organizes nucleosomes and forms Schiff base interactions with histones in mouse embryonic stem cells.

Nucleosomes are the basic unit of chromatin that help the packaging of genetic material while controlling access to the genetic information. The underlying DNA sequence, together with transcription-associated proteins and chromatin remodelling complexes, are important factors that influence the organization of nucleosomes. Here, we show that the naturally occurring DNA modification, 5-formylcytosine (5fC) is linked to tissue-specific nucleosome organization.

Sequencing 5-Hydroxymethyluracil at Single-Base Resolution.

5-hydroxymethyluracil (5hmU) is formed through oxidation of thymine both enzymatically and non-enzymatically in various biological systems. Although 5hmU has been reported to affect biological processes such as protein-DNA interactions, the consequences of 5hmU formation in genomes have not been yet fully explored. Herein, we report a method to sequence 5hmU at single-base resolution.

Genome-wide mapping of 5-hydroxymethyluracil in the eukaryote parasite Leishmania.

Background: 5-Hydroxymethyluracil (5hmU) is a thymine base modification found in the genomes of a diverse range of organisms. To explore the functional importance of 5hmU, we develop a method for the genome-wide mapping of 5hmU-modified loci based on a chemical tagging strategy for the hydroxymethyl group.

Results: We apply the method to generate genome-wide maps of 5hmU in the parasitic protozoan Leishmania sp.

Selective Chemical Labeling of Natural T Modifications in DNA.

We present a chemical method to selectively tag and enrich thymine modifications, 5-formyluracil (5-fU) and 5-hydroxymethyluracil (5-hmU), found naturally in DNA. Inherent reactivity differences have enabled us to tag 5-fU chemoselectively over its C modification counterpart, 5-formylcytosine (5-fC). We rationalized the enhanced reactivity of 5-fU compared to 5-fC via ab initio quantum mechanical calculations.

Towards a mutant map of the mouse--new models of neurological, behavioural, deafness, bone, renal and blood disorders.

With the completion of the first draft of the human genome sequence, the next major challenge is assigning function to genes. One approach is genome-wide random chemical mutagenesis, followed by screening for mutant phenotypes of interest and subsequent mapping and identification of the mutated genes in question. We (a consortium made up of GlaxoSmithKline, the MRC Mammalian Genetics Unit and Mouse Genome Centre, Harwell, Imperial College, London, and the Royal London Hospital) have used ENU mutagenesis in the mouse for the rapid generation of novel mutant phenotypes for use as animal models of human disease and for gene function assignment (Nolan et al.

The deaf mouse mutant Jeff (Jf) is a single gene model of otitis media.

Otitis media is the most common cause of hearing impairment in children and is primarily characterized by inflammation of the middle ear mucosa. Yet nothing is known of the underlying genetic pathways predisposing to otitis media in the human population. Increasingly, large-scale mouse mutagenesis programs have undertaken systematic and genome-wide efforts to recover large numbers of novel mutations affecting a diverse array of phenotypic areas involved with genetic disease including deafness.

Defects in whirlin, a PDZ domain molecule involved in stereocilia elongation, cause deafness in the whirler mouse and families with DFNB31.

The whirler mouse mutant (wi) does not respond to sound stimuli, and detailed ultrastructural analysis of sensory hair cells in the organ of Corti of the inner ear indicates that the whirler gene encodes a protein involved in the elongation and maintenance of stereocilia in both inner hair cells (IHCs) and outer hair cells (OHCs). BAC-mediated transgene correction of the mouse phenotype and mutation analysis identified the causative gene as encoding a novel PDZ protein called whirlin. The gene encoding whirlin also underlies the human autosomal recessive deafness locus DFNB31.

Mutagenesis strategies for identifying novel loci associated with disease phenotypes.

The systematic identification of the function of all the genes in the mammalian genome is one of the major scientific challenges for the 21st century. A comprehensive insight into mammalian gene function will illuminate our understanding of the genetic bases of disease. Mouse mutagenesis is a powerful tool for the study of mammalian gene function.

A radiation hybrid map of mouse genes.

A comprehensive gene-based map of a genome is a powerful tool for genetic studies and is especially useful for the positional cloning and positional candidate approaches. The availability of gene maps for multiple organisms provides the foundation for detailed conserved-orthology maps showing the correspondence between conserved genomic segments. These maps make it possible to use cross-species information in gene hunts and shed light on the evolutionary forces that shape the genome.

The mouse slalom mutant demonstrates a role for Jagged1 in neuroepithelial patterning in the organ of Corti.

The Notch signalling pathway has recently been implicated in the development and patterning of the sensory epithelium in the cochlea, the organ of Corti. As part of an ongoing large-scale mutagenesis programme to identify new deaf or vestibular mouse mutants, we have identified a novel mouse mutant, slalom, which shows abnormalities in the patterning of hair cells in the organ of Corti and missing ampullae, structures that house the sensory epithelia of the semicircular canals. We show that the slalom mutant carries a mutation in the Jagged1 gene, implicating a new ligand in the signalling processes that pattern the inner ear neuro-epithelium.

Myosin VIIA is specifically associated with calmodulin and microtubule-associated protein-2B (MAP-2B).

Myosin VIIA is a motor molecule with a conserved head domain and tail region unique to myosin VIIA, which probably defines its unique function in vivo. In an attempt to further characterize myosin VIIA function we set out to identify molecule(s) that specifically associate with it. We demonstrate that 17 and 55 kDa proteins from mouse kidney and cochlea co-purify with myosin VIIA on affinity columns carrying immobilized anti-myosin VIIA antibody.

A systematic, genome-wide, phenotype-driven mutagenesis programme for gene function studies in the mouse.

As the human genome project approaches completion, the challenge for mammalian geneticists is to develop approaches for the systematic determination of mammalian gene function. Mouse mutagenesis will be a key element of studies of gene function. Phenotype-driven approaches using the chemical mutagen ethylnitrosourea (ENU) represent a potentially efficient route for the generation of large numbers of mutant mice that can be screened for novel phenotypes.

ENU mutagenesis and the search for deafness genes.

The availability of mouse mutant models for known human deafness loci is limited. Moreover, it is unlikely that the current mouse archives hold mutants for the full panoply of genes involved in auditory system development and transduction. A large-scale ENU mutagenesis is currently underway to increase significantly the number of mouse deafness mutants available, employing specific screens for both deafness and balance defects.

A defect of platelet aggregation associated with an abnormal distribution of glycoprotein IIb-IIIa complexes within the platelet: the cause of a lifelong bleeding disorder.

A young Italian man (A.P.) has a lifelong history of bleeding from gums and mucocutaneous tissue.

Effects of a monoclonal antibody to glycoprotein IIb/IIIa (P256) and of enzymically derived fragments of P256 on human platelets.

The anti-platelet monoclonal antibody P256 is currently undergoing development for in vivo detection of thrombus. We have examined the actions of P256 and two fragments on human platelet function. P256, and its divalent fragment, caused aggregation at concentrations of 10(-9)-3 x 10(-8) M.

Measurement of fibrinogen binding to platelets in whole blood by flow cytometry: a micromethod for the detection of platelet activation.

Platelet fibrinogen binding in whole blood has been measured in vitro by flow cytometry using a commercially available, fluorescein isothiocyanate (FITC)-conjugated polyclonal antifibrinogen antibody. Fibrinogen-antifibrinogen immune complexes were formed in experimental conditions approaching antigen-antibody equivalence, but optimal reaction conditions in which their formation was prevented or minimized could be achieved. Immune complex formation was associated with fibrinogen binding to unstimulated platelets but did not significantly affect ADP-induced fibrinogen binding.

The action of ticlopidine on human platelets. Studies on aggregation, secretion, calcium mobilization and membrane glycoproteins.

The effects on platelet function of a 5-day course of Ticlopidine (Tcl) have been studied in two groups of volunteers receiving different dosage schedules. Tcl had a relatively greater inhibitory effect on aggregation induced by ADP than by other agonists, and a greater effect, in contrast to that of an ADP receptor antagonist, on the second phase than on the initial rate of aggregation. Tcl inhibited ATP secretion in response to ADP and 0.

Potentiation by adrenaline of Ca2+ influx and mobilization in stimulated human platelets: dissociation from thromboxane generation and aggregation.

In a medium containing 1 mM extracellular Ca2+ (Ca2+o), the prior addition of 0.5 microM adrenaline to quin 2-loaded human platelets increased both the rate and amplitude of the rise in cytosolic free Ca2+ (Ca2+i) in response to sub-threshold concentrations of thrombin and PAF and these effects were not prevented by blocking either fibrinogen binding and aggregation or cyclo-oxygenase. In the presence of 2 mM EGTA [( Ca2+o] less than 100 nM), the rate, but not the extent of rise of [Ca2+i] was enhanced by adrenaline, and this was also unaffected by blockade of cyclo-oxygenase.

Intravascular platelet activation in the hemolytic uremic syndrome.

We studied intravascular platelet activation in patients with typical (epidemic) and atypical (sporadic) HUS and found defective aggregation, decreased platelet and increased plasma serotonin in both groups. The findings were present not only on admission during the thrombocytopenic stage of the disease, but persisted for several weeks after recovery of the platelet count. Reduced endothelial PGI2 production was significantly more common in plasma from atypical than typical cases.

Decreased sensitivity to heparin in vitro in steroid-responsive nephrotic syndrome.

The in vitro heparin sensitivity of 18 nephrotic children was compared with that of 10 normal children and 13 children with other renal diseases. The influence of age on the heparin sensitivity of 52 normal subjects (aged 12 to 85 years) was also studied. The heparin sensitivity was calculated from the dose-response curve obtained when increasing amounts of heparin were added to plasma and the kaolin partial thromboplastin time (KPTT) was measured.

Grey platelet syndrome: studies on platelet alpha-granules, lysosomes and defective response to thrombin.

The platelets of a young man with the grey platelet syndrome were severely depleted of all seven alpha-granule proteins assayed as well as partially deficient in alpha-mannosidase and alpha-fucosidase; four other lysosomal enzymes were present in normal concentrations. Total platelet 5-hydroxytryptamine (5HT) and adenine nucleotides were normal, and 14C-5HT uptake reached normal levels only slightly more slowly than a control. Aggregation and dense body secretion occurred normally in response to ADP, adrenaline, collagen, PAF-acether, sodium arachidonate, A23187, Ionomycin, TPA and U44069, but were very delayed in response to thrombin.