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Biomedical subjects

P Humphries

Publications and source records attributed to P Humphries.

At least 91 records · Page 5Linked to original sources
PubMed1989

Autosomal dominant retinitis pigmentosa: exclusion of a gene from extensive regions of chromosomes 6, 13, 20, and 21.

Members of a large pedigree of Irish origin presenting with early onset Type I autosomal dominant retinitis pigmentosa (ADRP) have been typed for polymorphic DNA markers from chromosomes 6, 13, 20, and 21. For each marker close linkage to ADRP has been excluded by pairwise analyses. Using distances fixed from well-established genetic maps of these chromosomes and multipoint analyses with two or three contiguous markers, exclusion of ADRP was extended to the areas between markers, resulting in the exclusion of ADRP from extensive regions of each chromosome, totaling approximately 500 cM or 15% of the genome. The study indicates the large quantity of linkage/exclusion data obtainable using well-spaced highly polymorphic markers.

Chromosome Mapping↗
PubMed1989

Chimaerism following allogeneic bone marrow transplantation: detection of residual host cells using the polymerase chain reaction.

Chimaerism was assessed in five recipients following sex mismatched allogeneic bone marrow transplantation. Techniques included karyotyping of bone marrow cells, dot blot DNA analysis of blood and bone marrow suspensions, and in vitro amplification of DNA by the polymerase chain reaction (PCR) using blood and bone marrow suspensions and stored bone marrow slides. Results of karyotypic analysis suggested complete chimaerism in four patients, while in one patient mixed chimaerism was detected. Mixed chimaerism was also detected, however, in a second patient using PCR and confirmed by dot blot analysis on all tissues examined. PCR is a sensitive tool for investigation of chimaerism following bone marrow transplantation. Since this technique does not require radioactivity, it is an attractive method for use in a clinical laboratory. This technique represents a further development in the use of DNA methodologies in the assessment of haematological disease.

Blotting, Southern↗
PubMed1989

Autosomal dominant retinitis pigmentosa: exclusion of the gene from the short arm of chromosome 1 including the region surrounding the rhesus locus.

Members of a large Irish pedigree exhibiting early-onset autosomal dominant retinitis pigmentosa (ADRP) were typed for the rhesus blood group and nine DNA markers on chromosome 1. Close linkage between the ADRP locus and any of the marker loci was excluded using two-point analysis. With use of the sex-averaged maps of Dracopoli et al. and Donis-Keller et al. and a strategy of rolling multipoint analyses, support was gained for the exclusion of ADRP from a 224-cM region of the chromosome, including almost the entire short arm. The disease locus was significantly excluded from within at least 50 cM of the rhesus locus and, as a loose linkage between these two genes has been suggested by other studies, this result may support the possibility of genetic heterogeneity within the autosomal dominant subgroup of retinitis pigmentosa.

Chromosomes, Human, Pair 1↗
PubMed1988

Long-term dosing studies using mutagenic carcinogens indicate a highly significant correlation between elevations in the level of rat glutathione S-transferase P messenger RNA and liver tumours of hepatocellular origin.

We have investigated levels of transcript homologous with glutathione S-transferase P (GST-P; GST 7-7) in tumours and hyperplastic lesions induced in the livers of rats by long-term gavage dosing with diethylnitrosamine (DEN) and 6-p-dimethylaminophenylazobenzothiazole (6BT). Detailed histopathological examination of the livers of the 90 animals used in this study at 6-8 months after initiation of daily dosing revealed that, of the 30 animals treated with carcinogen, 15 had developed tumours or hyperplastic lesions. Of these, 11 were areas of fibrosarcoma/fibrous hyperplasia. The remaining four were hepatocellular carcinomas. Northern blotting of total RNA purified from these tissues revealed the presence of transcripts of 3 and 0.75 kb. Evidence is presented to indicate that the former is a hitherto-undetected precursor of the 3-kbp rat GST-P gene, the latter representing the previously characterized mature GST-P transcript. Large elevations of the 0.75-kb transcript (30-35-fold) were encountered in all of the hepatocellular carcinomas, but in none of the other lesions, indicating a highly significant correlation (P = less than 0.001) between high elevations in levels of GST-P mRNA and liver tumours of hepatocellular origin. Minor elevations in transcript level (less than or equal to 5-fold) were encountered in several of the non-hepatocellular lesions. In regenerating livers, small increases in the level of the 3-kb transcript (approximately 3-fold) were routinely detected in total RNA from all partial hepatectomies, a concomitant decrease of approximately similar magnitude occurring in the 0.75-kb transcript, suggesting that minor elevations in levels of GST-P transcript, where encountered in non-hepatocellular lesions, are related to pre-neoplasia rather than to the proliferative rate of hyperplastic cells per se. The data extend previous observations, carried out largely using short-term regimes, to an analysis of transcripts homologous with GST-P in hyperplastic, pre-neoplastic and neoplastic lesions induced by long-term dosing with genotoxic carcinogens, and strongly lend support to the concept that high (30-fold) elevations in GST-P transcript correlate most strikingly with tumours of hepatocellular origin.

Animals↗
PubMed1988

Transcriptional activation of the glutathione S-transferase pi gene in human ureteric and bladder carcinomas.

Using a cDNA clone derived from the rat Glutathione S-Transferase P (GST-P) gene, we have investigated homologous transcripts in a range of normal and malignant human tissues. A major transcript, of 0.75 Kb, representing human GST-pi mRNA, was present in total RNA purified from peripheral white blood cells, bladder, ureteric and prostate tissues. In total RNA derived from histopathologically benign/normal tissues, peripheral leucocytes and from hyperplastic and malignant prostates, levels of the mature transcript varied by less than 1.5-fold (n = 70). In contrast, levels of transcript were significantly elevated (up to 16-fold) in total RNA derived from bladder and ureteric carcinomas, with the highest levels of elevation approaching those previously found only for the GST-P gene in experimentally induced rodent hepatocellular carcinomas. A wide range of stage and grade of tumour has been investigated (pT1 to pT4; G1 to G3, respectively), suggesting that the transcriptional activation of the GST-pi gene is a characteristic common to many bladder/ureteric carcinomas in man.

Carcinoma↗
PubMed1988

Linkage analysis of X linked retinitis pigmentosa in the Irish population.

There is significant evidence for genetic and phenotypic heterogeneity in X linked retinitis pigmentosa (XLRP). We have studied the linkage of XLRP in four Irish families to a number of polymorphic DNA markers. We report linkage between the DXS7 (L1.28) locus and the XLRP locus (Z = 3.445, theta = 0.00). Combined with the previously published data on British and Danish families, the genetic distance between the DXS7 and XLRP loci is now estimated at 5 cM with a maximum lod score of 13.026 and a 1-lod confidence interval of 0.75 to 9.5 cM. Linkage was also observed between 754 and XLRP (Z = 3.41, theta = 0.00) and between pERT87 and XLRP (Z = 1.37, theta = 0.1). The heterogeneity of XLRP is discussed in relation to these observations.

Chromosome Mapping↗
PubMed1988

A rapid dot-blot assay to assess chimerism following sex-mismatched bone marrow transplantation.

Mixed chimerism may occur more frequently than previously thought following allogeneic bone marrow transplantation and may have implications in terms of relapse, graft-versus-host disease and immune reconstitution. DNA analysis using single or multilocus polymorphic probes cannot reliably discriminate between donor and recipient cells below a level of 10%. We used probe pHY2.1, a cloned segment of tandemly repeated DNA (2000 copies) on the long arm of chromosome Y. A dot blot procedure allowed us to immobilize DNA directly from 50 microliter of peripheral blood or bone marrow. Cross-reactivity was eliminated by hybridization at conditions of extreme stringency (65 degrees C, 50% formamide). Mixing experiments detected male DNA at a level of 0.1% after 10 h exposure. Five patients were studied serially post-bone marrow transplantation. One patient showed mixed chimerism for 12 months, one had complete autologous recovery and the remaining three showed complete engraftment. All results were verified by standard karyotyping on bone marrow cells. This technique is a simple, rapid and sensitive assay for chimerism following sex mismatched bone marrow transplantation.

Bone Marrow Transplantation↗
PubMed1987

Observations on the structure of two human 7SK pseudogenes and on homologous transcripts in vertebrate species.

A comparison of the sequence of two human 7SK RNA pseudogenes, covering approx. 190 and 240 base-pairs of the structural gene, is presented. Both repeated elements are flanked by direct repeats and begin at the 5' end of the gene. Each terminates approx. 90 base-pairs short of the 3' end, the latter representing a continuous sequence and the former carrying an internal deletion of about 40 base-pairs, this region being flanked in the progenitor gene by short repeated sequences. Southern blotting using a human 7SK pseudogene probe illuminated a series of multiple restriction fragments in mammalian genomes, with generally fewer fragments in the genomes of birds and reptiles and a single reactive fragment in DNA from terrapin (Pseudemys scripta elegans) and Xenopus laevis (South African clawed toad). In the latter case this fragment was only detectable on long exposure under the hybridization stringencies employed. 7SK transcripts were readily detectable in all mammalian, avian, reptilian and amphibian species analysed, although the gene appeared to be expressed at rather low levels in the ovaries of Xenopus laevis, possibly accounting for its failure to have become dispersed via 'retroposition' in this species.

Animals↗
PubMed1987

Analysis of transcripts homologous to acyl-CoA oxidase and enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase induced in rat liver by methylclofenapate.

Methylclofenapate is a potent peroxisome proliferating agent and liver carcinogen in rats. Animals exposed to daily oral doses (2.5 mg/kg body wt.) for a 21-day period were studied to determine the levels of mRNA homologous to peroxisomal acyl-CoA oxidase and enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme in total liver RNA. Northern blotting revealed transcripts of approximately 3.8 and 3.3 kilobases (kb), homologous to acyl-CoA oxidase and the bifunctional enzyme, respectively. Levels of these transcripts began to rise at approximately 4 h after the initial dose of the agent, and reached maximum induction (35- and 60-fold, respectively, in excess of control levels) at 2-8 days after the start of the study. The kinetics of induction for acyl-CoA oxidase mRNA resembled those of palmitoyl-CoA oxidase activity, and the induction of mRNA preceded the expression of enzyme activity, further supporting a transcriptional control model of induction of the peroxisomal enzymes. The levels of mRNA induction for the peroxisomal enzymes were higher in the present study than those reported elsewhere for single doses of peroxisome proliferating agents and probably reflect the increased tissue levels achievable in long term carcinogenesis studies.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1985

Structural and transcriptional properties associated with a member of a new family of conserved short dispersed repeated elements in human DNA.

The sequence of a 500-bp segment of human DNA containing a member of the 'A3' family of dispersed repeated elements, a family hitherto uncharacterized in human or rodent DNAs, is presented. The repeated element maps to a region of 270 bp, within which the most notable features are 17-mer direct repeats separated by 190 bp. A probe containing the repeated element, together with flanking single-copy DNA illuminates a series of multiple bands in blot transfers of restriction digests of human, mouse and hamster DNAs. However, the flanking single-copy DNA hybridizes to human, but not to rodent DNA. The same probe illuminates homologous transcripts occurring in a single size class of 0.8 kb in total RNA from a variety of human and mouse cells, levels of which vary approx. 100-fold in the tissues analyzed. Poly(A)+ RNAs from EJ-transformed 3T3 cells and mouse fibrosarcomas have been analysed and found also to contain an homologous 0.8-kb transcript.

Animals↗
PubMed1983

Isolation of a polymorphic DNA segment unique to human chromosome 7 by molecular cloning of hybrid cell DNA.

DNA isolated from a rodent-human hybrid cell line containing human chromosomes 3, 7, 9, 10, 14 and 22 was cloned in the plasmid vector pAT153. Recombinant plasmids containing inserts of human origin were identified by colony hybridization to 32P-labelled human DNA under conditions in which only repetitive sequences interact. Single- and low-copy sequences were liberated from these plasmids by restriction endonuclease digestion and used as hybridization probes against human DNA and DNA isolated from a panel of Chinese hamster-human hybrids. One single-copy probe was shown to react with a genomic sequence unique to human chromosome 7 and to recognize an apparent restriction fragment size polymorphism in human DNA.

Animals↗
PubMed1978

Structure of recombinant plasmids containing synthetic human foetal globin gene sequences.

In vitro synthesized duplex DNA complementary to human foetal globin messenger RNA was integrated into bacterial plasmids and amplified by transformation of Escherichia coli. Recombinants carrying globin DNA were identified by hybridization of foetal globin messenger RNA to bacterial DNA in situ and by liquid hybridization of purified plasmids to specific globin complementary DNA probes. Heteroduplex mapping revealed either a simple insertion loop at the position of the EcoRI site of the parental plasmid DNA. We provide evidence to suggest that these deletions are the result of a site-specific nicking activity of the EcoRI preparations used in the formation of recombinant plasmids.

DNA Restriction Enzymes↗
PubMed1978

Restriction enzyme cleavage mapping of T7 virus early region.

DNA molecules of seven T7 mutants with overlapping deletions in the early region were cleaved by restriction enzymes HindII, HpaI and II, and HaeIII. The differences in the cleavage patterns after electrophoresis have been used to generate a cleavage map of the restriction sites of this enzyme. It covers the first 9% of the T7 DNA molecule. Cleavage points for HindII are at 0.60, 1.33, 1.59, 1.76, 5.26, 6.27, 7.4 and 8.38%; for HpaI and II at 1.36, 1.62, 4.46, 6.29, 6.62, 7.56, and 8.76%, for HaeIII at 3.85, 6.98, 7.88 and 8.26%. Some fragments have been located in the region containing the early promoters, others carry the complete sequences of gene 0.3.

Coliphages↗