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D J Porteous

Publications and source records attributed to D J Porteous.

At least 19 recordsLinked to original sources
PubMed1995

Mapping of retrotransposon sequences in the unstable region surrounding the spinal muscular atrophy locus in 5q13.

The mutation that underlies the autosomal recessive disorder spinal muscular atrophy (SMA) is located on chromosome 5q13. Recent studies show that SMA patients frequently have deletions and rearrangements in this region compared to normal controls. During the isolation of candidate cDNAs for the disease, we identified a sequence that shows high homology to the THE-1 retrotransposon gene family. Using YAC fragmentation techniques, we have refined the localization of this sequence to the domain known to show instability in SMA patients. The implication of these results for the mechanism of the mutation in SMA is discussed.

Base Sequence↗
PubMed1995

Identifying genes within microdissected genomic DNA: isolation of brain expressed genes from a translocation region associated with inherited mental illness.

An improved protocol has been developed for physical enrichment of cDNA sequences by hybridization to genomic DNA. When applied to microdissection recombinants derived from a translocation breakpoint region associated with inherited mental illness, a single cycle of the procedure permitted enriched cDNAs to be visualized directly by agarose gel electrophoresis. Hybridization screening of a library of clones derived from the enriched cDNAs, employing the genomic resource as a probe, led to the identification of six novel gene fragments. This general approach to the isolation of regionally encoded genes could be applied to any subchromosomal interval as a first step towards global transcription map construction.

Animals↗
PubMed1995

Lung disease in the cystic fibrosis mouse exposed to bacterial pathogens.

Lung disease is the major cause of death in cystic fibrosis (CF), but there is no evidence for overt lung involvement at birth. We show here that the same is true for the gene targeted cftrm1HGU mutant mouse. Furthermore, this CF mouse model demonstrates an impaired capacity to clear Staphylococcus aureus and Burkholderia (Pseudomonas) cepacia, two opportunistic lung pathogens closely associated with lung disease in CF subjects. The cftrm1HGU homozygotes display mucus retention and frank lung disease in response to repeated microbial exposure. Thus, lung disease in the cftrm1HGU mouse develops in response to bacterial infection, establishing a model to dissect the pathogenesis of CF pulmonary disease and providing a clinically relevant end point to assess the efficacy of pharmacologic or genetic interventions.

Animals↗
PubMed1995

Bioelectric characteristics of exon 10 insertional cystic fibrosis mouse: comparison with humans.

Two important issues that can be addressed by animal models are disease pathogenesis and the testing of new treatments, including gene therapy. How closely these models mimic the relevant disorder in humans will determine their usefulness. This study examines how closely the characteristic bioelectric features of cystic fibrosis (CF) are reproduced in the airways and intestinal tract of the exon 10 insertional mutant mouse (cf/cf). In agreement with CF subjects these cf/cf mutant mice demonstrate the following: 1) reduced adenosine 3',5'-cyclic monophosphate-related chloride secretion throughout the respiratory and intestinal tracts both in vivo and in vitro, 2) calcium-related chloride secretion that is preserved in the airways but reduced in the intestine, and 3) a more negative nasal potential difference and increased amiloride response compared with wild-type animals, likely to relate to increased sodium absorption. In contrast to humans, sodium absorption is not increased in the small intestine and is reduced in the trachea of the cf/cf mice. We conclude that the majority of the salient electrophysiological features of CF required for studies of pathogenesis or testing of new treatments are present in these cf/cf mice.

Animals↗
PubMed1995

Regional localisation of 19 brain expressed sequence tags to human chromosome 11 using PCR amplification of somatic cell hybrid DNAs.

Expressed sequence tags (ESTs) provide an efficient route to the identification of genes involved in normal development and in disease. PCR amplification of somatic cell hybrid DNAs was used to localise 22 brain-derived ESTs to subregions of human chromosome 11. Problems encountered with the standardised PCR conditions were overcome by optimising the annealing temperatures and the use of "touchdown" PCR. Amplification of the correct target sequence allowed the mapping of 19 ESTs, 8 to the short arm and 11 to the long arm of chromosome 11. No definitive localisation could be determined for the three remaining ESTs.

Animals↗
PubMed1995

Placement and refined mapping of established and new markers on human chromosome 11q using a small panel of somatic cell hybrids.

Constructing detailed chromosome maps relies on the ability to place new markers rapidly and accurately. Here we map two new anonymous DNA markers precisely on chromosome 11q and refine the location of established markers using a panel of just six fragmentation hybrids, with reference to four translocation chromosomes. As several of the hybrids retain multiple segments of chromosome 11, it is possible to quickly assign new markers to subintervals using this modest set of hybrid DNAs.

Base Sequence↗
PubMed1994

Alu-based vectorettes and splinkerettes. More efficient and comprehensive polymerase chain reaction amplification of human DNA from complex sources.

Alu-polymerase chain reaction (PCR) is widely used to amplify human specific fragments from complex heterologous DNAs, such as somatic cell hybrids or yeast artificial chromosome (YAC) recombinants, but the fragments amplified are limited in number and are nonrepresentative. This report describes a modified one-sided alu-PCR technique, which offers better representation of amplified sequences while maintaining human specificity. The method relies on the ligation of partially mismatched double-stranded oligonucleotides (vectorettes or splinkerettes) to endonuclease-restricted DNA and universal priming with a single alu-consensus primer, the complement to which is the unpaired region. Alu-vectorette and alu-splinkerette-PCR of two somatic cell hybrids results in a greater complexity of products than alu-PCR alone. The advantage of alu-splinkerette over alu-vectorette-PCR is the elimination of nonspecific priming owing to the presence of the vectorette primer and to an increase in the product size range, a consequence of the difference in the splinkerette design. Alu-splinkerette-PCR is a useful technique for generating new and more comprehensive markers of the human sequences contained in somatic cell hybrids and YACs.

Base Sequence↗
PubMed1994

Cloning the shared components of complex DNA resources.

The complex and repetitive nature of mammalian genomes limits the ability of conventional molecular techniques to recover sequences of interest. Here we describe a rapid and simple procedure for the direct cloning of sequences which are coincident between DNA mixtures of whole genome complexity. The system, called end ligation coincident sequence cloning (EL-CSC), can enrich coincident DNA by greater than 10(6)-fold and overcomes problems associated with repetitive elements. Applying EL-CSC to various paired DNA resources enables the facile cloning of both genomic markers and novel genes. To demonstrate the power of the method we have i) selectively purified single copy sequences from a complete genome, and ii) isolated gene fragments from 260 kb of cloned genomic DNA.

Base Sequence↗
PubMed1994

CFTR and calcium-activated chloride currents in pancreatic duct cells of a transgenic CF mouse.

We have studied the cystic fibrosis transmembrane conductance regulator (CFTR) and calcium-activated chloride currents in pancreatic duct cells isolated from a transgenic cf/cf mouse created by targeted insertional mutagenesis. Adenosine 3',5'-cyclic monophosphate (cAMP)-activated CFTR chloride currents were detected in 78% (29/37) of wild-type cells, in 81% (35/43) of heterozygote cells, and in 61% (29/47) of homozygous cf/cf duct cells (P > 0.05, cf/cf vs. wild-type and heterozygote). The CFTR current density measured at membrane potentials of +/- 60 mV averaged 22-26 pA/pF in wild-type and heterozygote groups but only 13 pA/pF in cells derived from cf/cf animals (P < 0.05, cf/cf vs. wild-type and cf/cf vs. heterozygotes). In contrast, duct cells from animals of all three genotypic groups exhibited calcium-activated chloride currents that were of similar magnitude and up to 11-fold larger than the CFTR currents. We speculate that these transgenic insertional null mice do not develop the pancreatic pathology that occurs in cystic fibrosis patients because their duct cells contain 1) some wild-type CFTR generated by exon skipping and aberrant splicing and 2) a separate anion secretory pathway mediated by calcium-activated chloride channels.

Animals↗