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Biomedical subjects

P Dickinson

Publications and source records attributed to P Dickinson.

At least 19 recordsLinked to original sources
PubMed2006

Time-resolved FRET and FLIM of four-way DNA junctions.

Conformational transitions in a 4-way DNA junction when titrated with ionic solutions are studied using time-resolved fluorescence resonance energy transfer. Parameters characterising the transition in terms of critical ion concentration (c1/2) and the Hill coefficient for ion binding are obtained by fitting a simple two-state model using steady-state spectra. Data obtained from a fluorescence lifetime plate reader and analysed by fitting a single exponential to donor fluorescence lifetime decays are shown to be in good agreement with the parameters obtained from steady-state measurements. Fluorescence lifetimes, however, offer advantages, particularly in being independent of fluorophore concentration, output intensity, inhomogeneity in the excitation source and output wavelength. We demonstrate preliminary FRET-FLIM images of DNA junction solutions obtained using a picosecond gated CCD which are in agreement with results from a fluorescence lifetime plate reader. The results suggest that time-resolved FRET-FLIM is sensitive to subtle structural changes and may be useful in assays based on 4-way DNA junctions.

DNA, Cruciform↗
PubMed2006

The stability and characteristics of a DNA Holliday junction switch.

A Holliday junction (HJ) consists of four DNA double helices, with a branch point discontinuity at the intersection of the component strands. At low ionic strength, the HJ adopts an open conformation, with four widely spaced arms, primarily due to strong electrostatic repulsion between the phosphate groups on the backbones. At high ionic strength, screening of this repulsion induces a switch to a more compact (closed) junction conformation. Fluorescent labelling with dyes placed on the HJ arms allows this conformational switch to be detected optically using fluorescence resonance energy transfer (FRET), producing a sensitive fluorescent output of the switch state. This paper presents a systematic and quantitative survey of the switch characteristics of such a labelled HJ. A short HJ (arm length 8 bp) is shown to be prone to dissociation at low switching ion concentration, whereas an HJ of arm length 12 bp is shown to be stable over all switching ion concentrations studied. The switching characteristics of this HJ have been systematically and quantitatively studied for a variety of switching ions, by measuring the required ion concentration, the sharpness of the switching transition and the fluorescent output intensity of the open and closed states. This stable HJ is shown to have favourable switch characteristics for a number of inorganic switching ions, making it a promising candidate for use in nanoscale biomolecular switch devices.

DNA, Cruciform↗
PubMed2001

Gene targeting in primary fetal fibroblasts from sheep and pig.

Nuclear transfer offers a new cell-based route for introducing precise genetic modifications in a range of animal species. However, significant challenges, such as establishment of somatic gene targeting techniques, must be overcome before the technology can be applied routinely. In this report, we describe targeted deletion at the GGTA1 (alpha 1,3-galactosyl transferase) and PrP (prion protein) loci in primary fibroblasts from livestock. We place particular emphasis on the growth characteristics of the primary cell cultures, since these are key to determining success.

Animals↗
PubMed2001

The effect of families on the process of outpatient visits in family practice.

OBJECTIVES: Our goal was to describe how physician knowledge of patients' families affects the processes of patient care in family practices. STUDY DESIGN: Using a multimethod comparative case study design, detailed dictated field notes were recorded after direct observation of patient encounters and the office environment as part of the Prevention and Competing Demands in Primary Care Study. We identified domains of outpatient visits in which patients were accompanied by a family member or in which family-oriented content was discussed. POPULATION: Outpatient encounters with 1637 patients presenting in 18 family practices in the Midwest were analyzed using an editing style. OUTCOMES: We developed a typology for ways in which family context affects outpatient visits. RESULTS: Patients were accompanied during 35% of all outpatient visits, the vast majority of these visits involving children. Family history or a family member's problems were discussed during 35% of visits during which no family member was present. An analysis of these "family-oriented" visits resulted in a typology of 6 ways that family context informs and affects the outpatient visit: (1) using family social context to illuminate patient disease, illness, and health; (2) using family to discover the source of an illness; (3) discussing and managing the health and illness of family members; (4) family concern for patient's health; (5) using the family as a care resource and care collaborator; and, (6) giving family members unscheduled care. CONCLUSION: Family context is an important feature of family practice that influences the processes of patient care. Since family-oriented care is an essential feature of family practice, outcomes of this largely hidden part of care deserve further study.

Adult↗
PubMed2000

Enhancing the efficiency of introducing precise mutations into the mouse genome by hit and run gene targeting.

The creation of precise clinical mutations by targeting is important in elucidating disease pathogenesis using mouse models. 'Hit and run' gene targeting is an elegant method to achieve this goal. This uses first a positive selection to introduce the targeting vector carrying the required mutation and then a negative selection to identify clones which have removed vector and wild-type sequences by intrachromosomal recombination. However, this approach has only been successfully used in a handful of cases. We used this procedure to introduce precise clinical mutations into the exon 10 region of the cystic fibrosis transmembrane conductance regulator (Cftr) gene. Using a CMV promoter driven hygromycin/thymidine kinase (hyg/tk) fusion gene as both our dominant and negative selectable marker, we targeted the Cftr locus very efficiently but only identified false runs after the negative selection step. This defect in thymidine kinase induced toxicity to gancyclovir correlated with methylation of the transgene. Consequently we devised a stringent screening procedure to select only true 'run' clones. Unfortunately these 'run' clones had lost the mutation so we altered the vector design to bias the run step to retain the mutation and used a different tk selection cassette with a HSVtk promoter sequence. This new vector design allowed both efficient 'hit and run' for two cystic fibrosis (CF) mutations with no false positives and successful germline transmission of the novel G480C missense mutation.

Animals↗
PubMed2000

Gene targeting in livestock: a preview.

Until recently genetically modified livestock could only be generated by pronuclear injection. The discovery that animals can be cloned by nuclear transfer from cultured somatic cells means that it will now be possible to achieve gene targeting in these species. We discuss current developments in NT, the prospects and technical challenges for introducing targeted changes into the germline by this route, and the types of application for which this new technology will be used.

Animals↗
PubMed1999

The early phenotype associated with the jimpy mutation of the proteolipid protein gene.

The jimpy mutation of the X-linked proteolipid protein (Plp) gene causes dysmyelination and premature death of the mice. The established phenotype is characterised by severe hypomyelination, increased numbers of dead oligodendrocytes and astrocytosis. The purpose of this study was to define the earliest cellular abnormalities in the cervical spinal cord. We find that on the first and third postnatal days the amount of myelin in jimpy spinal cord is approximately 20% of wild-type. However, the total glial cell density, the number of dead glial cells and the number and distribution of Plp-positive cells, as assessed by in situ hybridization, are similar to wild-type during the first week of life. Immunostaining of cryosections has identified that jimpy spinal cords express on schedule, a variety of antigens associated with mature oligodendrocytes. Dissociated oligodendrocytes, cultured for 18 hours to reflect their in vivo differentiation, express MBP and surface myelin-associated glycoprotein at the same frequency as wild-type. By comparison, the proportion of jimpy oligodendrocytes expressing surface myelin/oligodendrocyte glycoprotein is reduced by approximately 34%. In vivo, however, only a small minority of axons is surrounded by a collar of myelin-associated glycoprotein, suggesting that the majority of jimpy oligodendrocytes fail to make appropriate ensheathment of axons. Although the DM20 isoform is expressed in the embryonic CNS prior to myelin formation, the cellular abnormalities appear to correspond to the time at which the Plp isoform becomes predominant. The results suggest that the primary abnormality in jimpy is the inability of oligodendrocytes to properly associate with, and then ensheath, axons and that oligodendrocyte death compounds, rather than initiates, the established phenotype.

Age Factors↗
PubMed1998

A new yeast artificial chromosome vector designed for gene transfer into mammalian cells.

This report describes the construction of a new yeast artificial chromosome (YAC) vector designed for gene transfer into mammalian cells. For ease of use, the two arms of the vector were cloned separately. The vector harbours the Neo and Hyg genes for dominant selection in mammalian cells, a putative human origin of replication, a synthetic matrix attachment region and two loxP sites (one on each arm). The cloning ability of the vector was demonstrated by successful propagation of the cDNA of the cystic fibrosis gene, CFTR, as a YAC in Saccharomyces cerevisiae. A YAC containing the entire CFTR gene was also constructed by retrofitting the two arms of a pre-existing clone (37AB12) with the two arms of the novel vector. Both the cDNA and entire gene containing YACs were circularized in yeast by inducible expression of the Cre recombinase. Recombination occurred very specifically at the loxP sequences present on the two arms of the YAC. Applications of the vector to gene transfer are discussed.

Animals↗
PubMed1995

Modelling cystic fibrosis in the mouse.

Animal models of human diseases are of vital importance, both for understanding basic disease mechanisms, and for developing potential treatments. Recent advances in molecular biology have helped identify the lesions underlying many genetic diseases. In conjunction with the development of techniques for manipulating the mouse embryo the possibility of mimicking these diseases in vivo has been raised. The example of cystic fibrosis illustrates this potential very well, demonstrating the usefulness of mouse models for gaining an improved understanding of the disease in humans and for the testing of new treatments.

Animals↗
PubMed1995

The proteolipid protein gene.

Proteolipid protein (PLP) is the major myelin protein of the CNS and is believed to have a structural role in maintaining the intraperiod line of compact myelin. An isoform, DM-20, produced by alternative splicing of exon 3B is expressed earlier than PLP in the CNS and may be involved in glial cell development. DM-20 is also present in myelin-forming and non-myelin-forming Schwann cells, olfactory nerve ensheathing cells, some glial cell lines and cardiac myocytes. Molecular studies suggest the existence of a PLP gene family with sequence similarities between molecules of different species. Such studies also lend credence to the suggestion that PLP and/or DM-20 may function as a membrane pore. Mutations in the PLP gene occur in several animal species and cause severe pleiotropic effects on myelination. In man this presents as Pelizaeus-Merzbacher disease (PMD). The phenotype of such mutants is characterized by dysmyelination with myelin of abnormal periodicity, paucity of mature oligodendrocytes and astrocytosis. Duplication of the PLP gene in transgenic animals or in one form of PMD also results in dysmyelination. X-linked spastic paraplegia (SPG2) is allelic to PMD and is associated with PLP mutations in which the levels of the DM-20 isoform are probably relatively normal. The effects of PLP gene dosage on CNS myelination can be compared in many ways to the variety of phenotypes in the PNS in hereditary neuropathies of the Charcot-Marie-Tooth type in which the peripheral myelin-22 gene is mutated.

Animals↗
PubMed1995

Expression of the proteolipid protein gene in glial cells of the post-natal peripheral nervous system of rodents.

The proteolipid protein (PLP) gene encodes for two proteins, PLP and DM-20, which are produced by alternative splicing of exon 3B. PLP is the major CNS myelin protein and is postulated to play a structural role at the intraperiod line. Its developmental expression mirrors that of CNS myelination. DM-20 predominates in the embryo and prior to myelination of the CNS and may be involved in glial cell development. The PLP gene is expressed in the PNS in which DM-20 is the predominant isoform at all ages. In this study we describe the localization of the two isoforms in the post-natal rodent PNS using immunostaining and reverse transcriptase PCR. DM-20 is present in relatively high abundance in non-myelin-forming Schwann cells and within cytoplasmic regions of myelinated internodes, particularly the paranodes and Schmidt-Lanterman incisures and also the outer Schwann cytoplasm and perinuclear cytoplasm. DM-20 is also located in the perineuronal satellite cells of spinal, cranial and autonomic ganglia and in the ensheathing cells of the olfactory nerve layer of the olfactory bulb. PLP was detected by immunocytochemistry in the perinuclear region of myelinated internodes; PCR analysis indicated small amounts of PLP mRNA in the other locations but protein was not detected by immunostaining. Neither protein was identified in compact myelin of the PNS. DM-20 is the predominant product of the PLP gene expressed in a wide variety of peripheral glia. Its presence is not correlated to a myelin-forming state. Other studies have demonstrated early embryonic expression of the PLP gene throughout the PNS and all these features support the hypothesis that any putative role for DM-20 is unrelated to myelination but may involve glial cell development.

Animals↗
PubMed1995

Evaluation in vitro and in vivo of cationic liposome-expression construct complexes for cystic fibrosis gene therapy.

We have tested the cationic liposome N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethyl-ammoniummethylsul phate, (DOTAP), for gene delivery in vitro and in vivo with a view to clinical use in gene therapy for cystic fibrosis. Delivery of lacZ cDNA-DOTAP complexes via aerosol showed promoter-dependent differences in the pattern and longevity of expression. Repeated administration was well tolerated. The potential for the transfer of foreign genes into reproductive tissue was investigated by intravenous injection of DNA-DOTAP into female mice. Foreign DNA was undetectable in the ovaries by Southern blot analysis at 1 and 7 days after injection. Our results suggest that DOTAP merits testing in cystic fibrosis patients for delivery of the cystic fibrosis transmembrane conductance regulator (CFTR) gene to the respiratory tract and that substitution of the cytomegalovirus (CMV) promoter for the simian virus (SV) promoter may improve on the transitory response reported previously.

Aerosols↗
PubMed1994

Long-term survival of the exon 10 insertional cystic fibrosis mutant mouse is a consequence of low level residual wild-type Cftr gene expression.

Recently we have created a mouse model of cystic fibrosis (CF) by insertional gene targeting to exon 10. In common with CF subjects, this model displays a low incidence of meconium ileus. This contrasts strikingly with the very high level of fatal intestinal obstruction in the three other CF mouse models so far described. We investigate here the molecular basis of this difference in phenotype. We show that the partial duplication consequent upon insertional gene targeting allows exon skipping and aberrant splicing to produce normal Cftr mRNA, but at levels greatly reduced compared with wild-type mice. Furthermore, instead of the predicted mutant Cftr transcript, a novel mRNA is produced that utilizes cryptic splice sites in the disrupting plasmid sequence. However, we have previously shown that these mice display the ion transport defect characteristic of CF, and mutant animals can be distinguished from their normal littermates on this basis. Consistent with this, residual CFTR function has recently been observed for several "mild" mutations in CF individuals who display pancreatic sufficiency but still develop lung disease. We conclude that (i) residual wild-type mRNA in the exon 10 insertional mutant mouse ameliorates the severity of the intestinal phenotype observed in the absolute "null" CF mice, (ii) the presence of low-level residual wild-type Cftr mRNA does not correct the CF ion transport defect, and (iii) the long-term survival of this insertional mutant mouse provides the opportunity to address the factors important in development of lung disease.

Animals↗
PubMed1993

A new codon 15 rhodopsin gene mutation in autosomal dominant retinitis pigmentosa is associated with sectorial disease.

OBJECTIVE: To ascertain and characterize rhodopsin gene mutations in autosomal dominant retinitis pigmentosa and to correlate these mutations with the clinical phenotypes. METHODS: DNA was extracted from leukocytes, and the rhodopsin gene was amplified and analyzed using molecular-biological methods. Clinical and electrophysiological data were collected from patient charts. RESULTS: We found a disease-causing mutation that was previously undescribed, to our knowledge, for autosomal dominant retinitis pigmentosa within codon 15 of exon 1 of the rhodopsin gene. It was a single base-pair transversion (AAT to AGT) leading to a serine-for-asparagine substitution. This altered a glycosylation site in the intradiscal portion of the rhodopsin molecule. The pedigree examined demonstrated an inferior distribution of retinal pigmentary changes and predominantly superior visual field loss with relative preservation of electroretinographic amplitudes and good vision, which is consistent with sectorial or sectorial-like retinitis pigmentosa. CONCLUSIONS: A codon 15 rhodopsin gene mutation caused retinitis pigmentosa in the pedigree studied. There may be an association between intradiscal rhodopsin gene mutations and sectorial forms of retinitis pigmentosa.

Adult↗
PubMed1992

Cystic fibrosis in the mouse by targeted insertional mutagenesis.

Cystic fibrosis is a fatal genetic disorder which afflicts 50,000 people worldwide. A viable animal model would be invaluable for investigating and combating this disease. The mouse cystic fibrosis transmembrane conductance regulator gene was disrupted in embryonal stem cells using an insertional gene targeting vector. Germ-line chimaeras were derived and the offspring of heterozygous crosses studied. These homozygous mutant mice survive beyond weaning. In vivo electrophysiology demonstrates the predicted defect in chloride ion transport in these mice and can distinguish between each genotype. Histological analysis detects important hallmarks of human disease pathology, including abnormalities of the colon, lung and vas deferens. This insertional mouse mutation provides a valid model system for the development and testing of therapies for cystic fibrosis patients.

Animals↗