Biomedical subjects
D J Porteous
Publications and source records attributed to D J Porteous.
Gene therapy for cystic fibrosis--where and when?
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Dinucleotide repeat polymorphism at the human olfactory marker protein (OMP) locus on chromosome 11q13.5 near tyrosinase (TYR).
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High frequency gene targeting using insertional vectors.
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Schizophrenia-associated chromosome 11q21 translocation: identification of flanking markers and development of chromosome 11q fragment hybrids as cloning and mapping resources.
Genetic linkage, molecular analysis, and in situ hybridization have identified TYR and D11S388 as markers flanking the chromosome 11 breakpoint in a large pedigree where a balanced translocation, t(1;11)(q43;q21), segregates with schizophrenia and related affective disorders. Somatic cell hybrids, separating the two translocation chromosomes from each other and from the normal homologues, have been produced with the aid of immunomagnetic sorting for chromosome 1- and chromosome 11-encoded cell-surface antigens. The genes for two of these antigens map on either side of the 11q breakpoint. Immunomagnetic bead sorting was also used to isolate two stable X-irradiation hybrids for each cell-surface antigen. Each hybrid carries only chromosome 11 fragments. Translocation and X-irradiation hybrids were analyzed, mainly by PCR, for the presence of 19 chromosome 11 and 4 chromosome 1 markers. Ten newly designed primers are reported. The X-irradiation hybrids were also studied cytogenetically, for human DNA content, by in situ Cot1 DNA hybridization and by painting the Alu-PCR products from these four lines back onto normal human metaphases. The generation of the translocation hybrids and of the chromosome 11q fragment hybrids is a necessary preliminary to determining whether a schizophrenia-predisposition gene SCZD2 is encoded at this site.
Cystic fibrosis mice with disease-related changes in lung and reproductive tract.
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Distribution of Alu and L1 repeats in human YAC recombinants.
Evidence is accumulating that the two major families of interspersed repeated human DNA sequences, Alu and L1, are not randomly distributed. However, only limited information is available on their relative long-range distribution. We have analyzed a set of randomly selected, human Chromosome (Chr) 11-specific YAC recombinants constituting a total length of about 2 Mbp for the local and global distribution of Alu and L1 repeats: the data show a strong asymmetry in the distribution of these two repeat classes and give weight, at the long-range molecular level, to previous studies indicating their partition in the human genome; they also suggest a strong tendency for L1 repeats to cluster, with a higher proportion of full-length elements than expected.
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.
Polyacrylamide-agarose composite gels for high resolution of fingerprints.
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YAC mapping by FISH using Alu-PCR-generated probes.
Human genomic mapping has been greatly advanced by the independent development of three new methods: large DNA fragment cloning in yeast artificial chromosomes, amplification from complex DNAs of human specific segments by Alu-PCR, and high-resolution localization of complex DNA probes by fluorescent in situ hybridization. We describe here the combination of these three analytical tools for efficient and accurate localization of randomly screened or especially selected human YAC recombinants to chromosome 11. We map a YAC clone encompassing the pepsinogen A (PGA) locus to 11q13.1-11q13.3.
High-resolution localization of 69 potential human zinc finger protein genes: a number are clustered.
In this study, we describe the identification and partial characterization of 101 potential human zinc finger protein genes (ZnFPs). These sequences were isolated by hybridization of cosmids, obtained from mouse-human cell lines enriched for chromosome 11p, with an oligonucleotide specific for the "link" sequence between contiguous zinc fingers. Sixty-nine of these cosmids were regionally localized to human prometaphase chromosomes by in situ hybridization. The localization of these cosmids suggests that a number of finger protein genes occur in linked clusters. Their assignment to chromosomes 3p, 11p, 19p, 19qter, 20p, and 21q makes them valuable as markers or "candidate" genes for diseases associated with these chromosome regions.
Gene targeting for somatic cell manipulation: rapid analysis of reduced chromosome hybrids by Alu-PCR fingerprinting and chromosome painting.
The techniques of reverse genetics rely heavily on parasexual methods for manipulating the human genome. However, the application of somatic cell genetics is severely limited by the availability of suitable endogenous selectable markers in the genome. We have addressed this problem by targeting a universally selectable marker into a predetermined region of the genome, using a stringent selection for homologous recombination. Correct gene targeting to human chromosome 7q11 was screened for by Southern blotting and confirmed by fluorescent in situ hybridization. Reduced chromosome 7 hybrids were generated by chromosome mediated gene transfer and selection for the neo gene. The resultant transgenomes were characterized by a combination of L1 fingerprinting, locus specific marker analysis, Alu-PCR and chromosome 'painting'. Alu-PCR and L1 'fingerprints' are complementary and mutually consistent. Chromosome 'painting' reflects and extends the results obtained for specific marker co-transfer. Thus Alu-PCR 'fingerprinting' and 'painting' combine to rapidly provide an accurate picture of transgenome content and complexity. Gene targeting, chromosome tagging and subsequent isolation can be applied to any region of the genome for which a molecular probe is available.
Coincident sequence cloning.
We describe a novel method, Coincident Sequence Cloning (CSC), which permits the selective recovery of common sequences shared between two complex and partially coincident DNA mixtures. We evaluate this method by integrating human DNA with DNA from a mouse-human somatic cell hybrid, and we recover exclusively human DNA products which are all represented in the hybrid genome. CSC strategies should be useful in addressing many highly complex problems in genome analysis.
Construction of a library enriched for human chromosome 11 and Xp YAC recombinants.
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Cystic fibrosis--the way forward from the gene.
Cloning of the cystic fibrosis (CF) gene and elucidation of the physiological functions of the encoded protein is a triumph, not only for molecular biology, but also for people affected by CF. For them, not only is there now the possibility of screening for the commonest mutations, but they may also look forward to the prospect of improved therapies being developed.
Cystic fibrosis. 3. Cloning the cystic fibrosis gene: implications for diagnosis and treatment.
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Multiple DNA sample processing by agarose gel electrophoresis.
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Molecular cloning of a rearranged HRAS1 oncogene in chromosome mediated gene transfer associated with elevated tumorigenicity.
We describe the molecular cloning of the rearranged HRAS1 oncogene found in association with the increased tumorigenic potential of the chromosome mediated gene transfectant E65.5. The rearrangement occurs immediately 3' to the c-Ha-ras coding region, removing the variable numbered tandem repeat (VNTR) but not altering the HRAS1 transcription unit. The novel 3' DNA sequence contains a short open reading frame but shows no homology to any previously cloned elements. Sequence analysis identifies a number of short DNA motifs consistent with the activity of an aberrant recombinogenic mechanism.