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Biomedical subjects

P Dickinson

Publications and source records attributed to P Dickinson.

At least 19 recordsLinked to original sources
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↗
PubMed1992

Successful targeting of the mouse cystic fibrosis transmembrane conductance regulator gene in embryonal stem cells.

We wish to construct a mouse model for the human inherited disease cystic fibrosis. We describe here the successful targeting in embryonal stem cells of the murine homologue (Cftr) of the cystic fibrosis transmembrane conductance regulator gene, as the first critical step towards this end. The targeting event precisely disrupts exon 10, the site of the major mutation in patients with cystic fibrosis. The targeted cells are pluripotent and competent to form chimaeras.

Animals↗
PubMed1991

No evidence of linkage between the locus for autosomal dominant retinitis pigmentosa and D3S47 (C17) in three Australian families.

A linkage analysis has been performed on three Australian families segregating for autosomal dominant retinitis pigmentosa (ADRP). No evidence of linkage has been found in any of the pedigrees studied between the locus D3S47 and the gene for ADRP. The D3S47 locus was found to show very close linkage with the ADRP gene in a large Irish pedigree. Our study together with a similar report on a British family indicates that there is genetic heterogeneity in this disease.

Adolescent↗
PubMed1990

Attachment of DNA to the nucleoskeleton of HeLa cells examined using physiological conditions.

Although it is widely believed that eukaryotic DNA is looped by attachment to a nucleoskeleton, there is controversy about its composition and which sequences are attached to it. As most nuclear derivatives are isolated using unphysiological conditions, the criticism that attachments seen in vitro are generated artifactually has been difficult to rebut. Therefore we have re-investigated attachments of chromatin to the skeleton using physiological conditions. HeLa cells are encapsulated in agarose microbeads and lysed using Triton in a 'physiological' buffer. Then, most chromatin can be electroeluted after treatment with a restriction enzyme to leave some at the base of the loops still attached. Analysis of the size and amounts of these residual fragments indicates that the loops are 80-90kbp long. The residual fragments are stably attached, with about 1kbp of each fragment protected from nuclease attack. This is very much longer than a typical protein-binding site of 10-20bp.

Chromatin↗
PubMed1990

Active RNA polymerase I is fixed within the nucleus of HeLa cells.

We have investigated whether active RNA polymerase I, the enzyme responsible for transcribing ribosomal RNA, is immobilized by attachment to a large subnuclear structure in HeLa cells. As unphysiological salt concentrations induce artifacts, we have used isotonic conditions throughout the preparative and analytic procedures. Cells are encapsulated in agarose microbeads and lysed in Triton and a 'physiological' buffer; then soluble proteins and RNA diffuse out through the agarose pores to leave encapsulated chromatin. This can be manipulated without aggregation but is accessible to molecular probes; it retains the replicational and transcriptional activities of the living cell. After treatment with a restriction endonuclease, most chromatin can be removed from beads by electrophoresis: then active ribosomal genes and polymerase I remain behind. Active ribosomal genes are very accessible to nuclease digestion whilst the rest are even more inaccessible than inactive globin genes. Our observations confirm the complex organization of rDNA within nucleoli and are compatible with transcription occurring at fixed sites. A model for transcription involving an attached polymerase is presented.

Cell Nucleus↗
PubMed1990

The size of chromatin loops in HeLa cells.

It is widely believed that the chromatin fibre is organized into loops during interphase, with the loop being implicated as an important unit of nuclear function. However, there remains little direct evidence for looping, with estimates of loop size varying widely. This has led to the suggestion that some loops, or even all of them, arise artefactually during isolation as chromatin aggregates so easily. We have now investigated the effect of isolation procedure on loop size using HeLa cells encapsulated in agarose to allow easy manipulation. Loop size in various derivatives (i.e. nuclei, nucleoids, matrices and scaffolds) critically depended on procedure; some (or all) of their loops are artefacts. The loop size in derivatives isolated using the most 'physiological' conditions was 86 kb; this remained unchanged throughout the cell cycle. This loop size is probably an average of a range of loops of between 5 and 200 kb.

Cell Cycle↗
PubMed1989

Non-allelic mutations in X-linked retinitis pigmentosa.

Using RFLP studies, the disease locus in two X-linked retinitis pigmentosa families was found to be centromeric to DXS7 in one family and telomeric to DXS7 in another, suggesting non-allelic heterogeneity.

Blotting, Southern↗
PubMed1989

A survey of hereditary aspects of pigmentary retinal dystrophies.

A study of 707 cases of retinitis pigmentosa and choroideraemia presenting over 12 years were classified according to their modes of inheritance--439 autosomal recessive (62%), 193 autosomal dominant (27%), 75 X-linked (10.7%). The patients with autosomal recessive transmission included 58 Usher syndrome, 12 Laurence-Moon-Bardet-Biedl syndrome and 33 Leber's congenital amaurosis. Another 37 had an early onset with macular degeneration and 31 were of late onset with pericentral dystrophy. Forty two were offspring of consanguineous parents. Of 193 individuals (78 families) with autosomal dominant inheritance, 20% had night blindness from early childhood. With X-linked transmission, 33 males and 31 female carriers comprised the retinitis pigmentosa group and eight males and three carrier females, choroideraemia. Almost all this X-linked group were of British ancestry. Of patients originating from the Mediterranean area, 94% had autosomal recessive disease.

Adolescent↗
PubMed1989

Linkage heterogeneity between X-linked retinitis pigmentosa and a map of 10 RFLP loci.

In nine families in which X-linked retinitis pigmentosa (XLRP) is segregating, the lod scores of XLRP in a map of 10 RFLP loci were obtained by multipoint linkage analysis. The XLRP locus was located telomeric to DXS7 in seven of the families and centromeric to DXS7 in two of the families. Under the hypothesis of two XLRP loci, a heterogeneity (admixture) test was performed, providing significant evidence of heterogeneity in XLRP (P less than .01). No correlation was detected between the clinical manifestations of XLRP and the two different disease loci.

Chromosome Mapping↗