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P R Harrison

Publications and source records attributed to P R Harrison.

At least 37 records · Page 2Linked to original sources
PubMed1993

Transcriptional up-regulation of the mouse cytosolic glutathione peroxidase gene in erythroid cells is due to a tissue-specific 3' enhancer containing functionally important CACC/GT motifs and binding sites for GATA and Ets transcription factors.

Nuclear run-on experiments have shown that the high level of expression of the mouse cytosolic glutathione peroxidase mRNA in erythroid cells is due to up-regulation of the gene at the transcriptional level. Studies of the chromatin structure around the cytosolic glutathione peroxidase gene have revealed a series of DNase I hypersensitive sites (DHSS) in the 3' flanking region of the gene in erythroid and other high-expression tissues that are lacking in low-expression cells, in addition to a DHSS over the promoter region in both high- and low-expression tissues. Functional transfection experiments have demonstrated that one of the 3' DHSS regions functions as an enhancer in erythroid cells but not in a low-expression epithelial cell line; and site-directed mutagenesis and footprinting experiments reveal that the activity of the erythroid cell-specific enhancer requires a cluster of binding sites for the CACC/GT box factors and the GATA and Ets families of transcription factors.

Animals↗
PubMed1993

Differentiation arrest and stromal cell-independent growth of murine erythroleukemia cells are associated with elevated expression of ets-related genes but not with mutation of p53.

The ELM erythroleukemia is novel in that long-term survival of leukemic cells in culture (ELM-D cells) is dependent on contact with a bone marrow-derived stromal feeder cell layer. However, a number of stroma-independent (ELM-I) mutants that vary in their ability to differentiate in vitro in response to erythropoietin and interleukin-3 have been derived. We have attempted to define the genetic changes responsible for these different phenotypes. At the p53 locus in the primary leukemic cells, one copy of the gene has been lost whereas the other contains an 18-bp depletion, implicating its mutation as an early step in the development of the leukemia. Changes in ets gene expression have also been found. The Fli-1 gene region is rearranged in the primary tumor because of the insertion of a retrovirus inserted upstream of one Fli-1 allele, but this does not result in Fli-1 gene activation in any of the ELM-D or ELM-I cell lines except one. It seems significant that this line is the only one to have lost the ability to differentiate in response to erythropoietin. In addition, up-regulation of erg is associated with stromal cell-independent growth, since all ELM-I mutants have moderate levels of erg mRNA, whereas only low or undetectable levels are found in primary leukemic cells in vivo or in ELM-D cells in vitro. This up-regulation of erg mRNA seems to be important for stromal cell-independent growth, since ELM-D cells show elevated expression of the erg gene after separation from stromal cells. This seems to be made permanent in ELM-I mutants, since they do not down-regulate erg mRNA when grown in contact with stromal cells. We therefore propose that ets family members regulate both the survival and differentiation of erythroid cells.

Animals↗
PubMed1990

Linkage disequilibrium between DNA polymorphisms within the porphobilinogen deaminase gene.

Three restriction fragment length polymorphisms (RFLPs) (MspI, PstI, ScrFI/BstNI) within the human porphobilinogen deaminase (PBG-D) gene have been studied in 47 unrelated patients with the autosomal dominant disorder, acute intermittent porphyria (AIP), and in 92 control subjects. Each enzyme identified a two-allele polymorphism with allele frequencies close to 0.50: however, marked linkage disequilibrium limited the number of observed haplotypes to four, of which one is uncommon. No association was detected between any haplotype and AIP.

Acute Disease↗
PubMed1990

Acute intermittent porphyria caused by a C----T mutation that produces a stop codon in the porphobilinogen deaminase gene.

A mutation of the porphobilinogen (PBG) deaminase gene that produces the cross-reacting immunological material (CRIM)-negative type of acute intermittent porphyria (AIP) has been identified in one of 43 unrelated patients with this form of the disorder. The mutation is a C----T transition that abolishes a PstI recognition site in exon 9 of the gene and converts a codon for glutamine to a stop codon.

Codon↗
PubMed1990

Synergy between the NF-E1 erythroid-specific transcription factor and the CACCC factor in the erythroid-specific promoter of the human porphobilinogen deaminase gene.

A 114-base-pair promoter fragment of the human porphobilinogen deaminase gene functioned in an erythroid-specific manner in transient transfection experiments. Site-directed mutagenesis of the binding site for the erythroid-specific transcription factor (NF-E1) or an adjacent CACCC motif abolished the promoter activity. Increasing the spacing between these sites progressively reduced promoter activity, but there was no evidence that a critical alignment of the two factors on the DNA helix was required.

Ammonia-Lyases↗
PubMed1990

Molecular mechanisms involved in the regulation of gene expression during cell differentiation and development.

One general point to emerge from this review is that each step in the pathway for gene expression from DNA to final polypeptide via chromosome structure, primary transcript, mRNA processing, stability, and translation is exploited in some developmental context as a means of regulation. Moreover, there is little evidence that the regulation of different stages in this pathway is associated consistently with particular classes of genes or developmental processes: for example, homeotic and other regulatory genes, which are important in early development and in determining the overall structures of organisms, are regulated by both transcriptional and posttranscriptional mechanisms, as are many other genes encoding cell-type-specific proteins expressed late during terminal differentiation. There are also instances in which transcription factors regulating tissue-specific transcription are present constitutively but are activated only in certain cell types by posttranslational events, and in yeast, coordinate regulation of amino-acid biosynthesis genes involves the translation control of a transcription factor. Second, as far as transcriptional control is concerned, it is now clear that in most instances cell-type specificity arises from the activities of multiple cis-control elements that can act either positively or negatively: The precise way in which the gene functions depends on what combination of cis-control elements it possesses. A third somewhat negative point is that in general the mechanisms responsible for the coordinate expression of genes during cell differentiation and development are not yet understood. In view of the complexities of possible mechanisms noted above, it may be that closely related genes (such as those encoding the alpha- and beta-globins, for example) in fact come to be coordinately expressed via a complex, indirect route. Indeed, it is quite possible that such mechanisms will not be revealed simply by a reductionist approach studying relatively short regions of DNA and that some form of overall "systems analysis" will be required. Finally, what we know relates mainly to expression of genes within particular cell types. How groups of cells are organized into complex structures remains a mystery at the molecular level, although progress into the genetic principles involved in the regulation of such "compartments" is being made, mainly in insects, reviewed recently by Brower, although to some extent also in the mouse [discussed by Hogan and co-workers].

Animals↗
PubMed1990

Regulation of erythroid-specific gene expression.

The aim of our group's work has been to elucidate how the alpha- and beta-globin genes come to be co-expressed together with a set of characteristic non-globin genes during erythroid cell differentiation. Our most significant progress concerns the identification and analysis of a species-conserved transcription factor, EF1, that appears to play a general role in the regulation of erythroid-specific gene transcription. We have shown that the 4 kb of 5' flanking region of the mouse alpha-globin gene contains two erythroid-specific cis-control elements, both of which involve EF1 binding sites. We have also identified functionally active EF1 binding sites in the mouse beta-globin promoter, as well as in the erythroid-specific promoter of the gene encoding the haem biosynthetic enzyme, porphobilinogen deaminase (PBG-D). The function of the PBG-D promoter depends in part on the cooperation between an EF1 binding site and an adjacent CACCC motif, this being abolished if their spacing is increased beyond 40 nt. We have also investigated the mechanisms involved in the up-regulation in erythroid cells of two non-globin genes we have cloned, encoding the RBC-specific lipoxygenase (LOX) and glutathione peroxidase (GSHPX). As judged by the presence of tissue-specific DNAse I hypersensitive sites, the tissue-specific regulation of the GSHPX gene seems to be due to regulatory regions 3' to the gene. The level of GSHPX is also regulated by selenium and this occurs at two levels: during mRNA formation, and during translation of the mRNA due to the regulation of selenocysteine incorporation specified by a unusual use of the UGA codon.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Structure of the mRNA and of the gene coding for the rabbit erythroid 15-lipoxygenase.

The complete structure of the rabbit erythroid cell-specific 15-lipoxygenase mRNA and its gene was established by sequencing cDNA and genomic recombinants. The transcription initiation site was obtained by primer-extension sequencing. A presumptive promoter structure was characterized by sequencing 0.5 kb 5' to the transcription initiation site and by transfection experiments using constructs with the chloramphenicol transferase gene. The mRNA codes for a polypeptide of 662 amino acids. Its 3' untranslated region contains an intriguing repeated sequence of 10 copies with the consensus C4PuC3TCTTC4AAG which may be involved in its regulation during reticulocyte maturation. The transcription unit consists of 8.0 kb and is split like the gene for the leukocyte 5' lipoxygenase by 13 introns. Another interesting aspect in the structure of the 15-LOX gene is a highly conserved repeat in intron seven consisting of a unit of 54 nucleotides which is repeated eight times. Comparing the predicted amino acid sequence with those from other lipoxygenases published recently shows that lipoxygenases are a related group of enzymes which may have arisen from a common ancestral gene.

Amino Acid Sequence↗
PubMed1989

The promoter structure and complete sequence of the gene encoding the rabbit erythroid cell-specific 15-lipoxygenase.

We report the isolation and complete sequence of the gene encoding the rabbit erythroid-cell-specific 15-lipoxygenase (RBC 15-LOX), containing 14 exons spanning 8.0 kb. The transcription start point was mapped by S1 nuclease-protection experiments and comparison with the sequence of the RBC 15-LOX mRNA, as defined previously by primer extension experiments. The promoter contains a TATA-like motif, but no CCAAT motif in the canonical position, and lies within a 'CpG-rich island'. Functional analysis of the immediate 5'-flanking DNA by transfection experiments shows that a 150 nucleotide (nt) 5' fragment linked to the chloramphenicol acetyltransferase gene acts as a functional promoter in both erythroid and nonerythroid cell lines and responds in an erythroid-specific manner to the enhancer from the Friend murine leukaemia virus long terminal repeat, whereas a 40-nt fragment is inactive. Intron 7 contains eight copies of a 54-nt repeat containing a region with homology to the simian virus 40/immunoglobulin gene enhancers.

Animals↗
PubMed1989

The complete sequence of the rabbit erythroid cell-specific 15-lipoxygenase mRNA: comparison of the predicted amino acid sequence of the erythrocyte lipoxygenase with other lipoxygenases.

We report the complete sequence of the rabbit reticulocyte (RBC) 15-lipoxygenase (LOX) mRNA as deduced from (i) sequencing cDNA recombinants isolated by screening cDNA libraries or polymerase-chain-reactions, and (ii) the sequence originating from the transcription start point obtained by primer extension-sequencing reactions. Like the human leukocyte 5-LOX mRNA, the RBC 15-LOX mRNA contains a very short 5'-untranslated region with a long 3'-untranslated region. But, unlike the human leukocyte 5-LOX mRNA, the RBC 15-LOX mRNA contains an intriguing repeated sequence (ten copies with the consensus sequence C4PuC3TCTTC4AAG) just after the translational stop codon, which may be involved in its regulation during reticulocyte maturation. Comparison of the RBC 15-LOX mRNA sequence with those of the previously published human 5-LOX mRNA and the soybean 3-LOX gene shows only a few short regions of sequence similarity. However, the predicted amino acid sequences of the encoded LOX enzymes show certain conserved regions that are presumably involved in their catalytic activity, in particular a cluster of five conserved histidines that we predict chelate the iron moiety involved in the active site.

Amino Acid Sequence↗
PubMed1988

Effect of selenium status on mRNA levels for glutathione peroxidase in rat liver.

To determine the effect of Se status on the level of mRNA for Se-dependent glutathione peroxidase (EC 1.11.1.9), rats were fed either a Se-deficient torula yeast diet (less than 0.02 mg Se/kg diet) or a Se-adequate diet (+0.2 mg Se/kg as Na2SeO3) for greater than 135 d. Liver glutathione peroxidase activity was 0.025 for Se-deficient versus 0.615 EU/mg protein for Se-adequate rats. Total liver RNA and polyadenylated RNA were isolated and subjected to Northern blot analysis using a 700 bp DNA probe from cloned murine glutathione peroxidase. Autoradiography showed that Se-deficient liver had 7-17% of the mRNA for glutathione peroxidase present in Se-adequate liver, suggesting that Se status may regulate the level of mRNA for this selenoenzyme.

Actins↗
PubMed1988

Natural course of penicillamine nephropathy: a long term study of 33 patients.

To elucidate the natural course of the nephropathy associated with penicillamine and thereby facilitate its clinical management 33 patients with rheumatoid arthritis who developed proteinuria during treatment with oral penicillamine were studied in detail throughout their renal illness. Renal biopsies were performed, and creatinine clearance and proteinuria were measured serially for 74 months (range 16-148 months). Fourteen patients developed proteinuria within six months after the start of treatment and 27 within 12 months. When treatment was stopped the proteinuria reached a median peak of 4.2 g/24 h (range 0.3-15.0 g/24 h) at one month (range 0-7 months) before resolving spontaneously by six months (12 patients), 12 months (21), or 18 months (29). In all patients but one, who developed carcinoma of the renal pelvis, proteinuria resolved by 21 months and its median duration was eight months. The median first and last measurements of creatinine clearance showed no appreciable change (80 ml/min and 78 ml/min), and no patient died from or needed treatment for renal failure. The HLA-B8 or HLA-DR3 alloantigen, or both, were identified in 10 patients. Renal biopsy specimens showed membranous glomerulonephritis in 29 patients, minimal change nephropathy in two, and electron dense deposits in the mesangial regions in two. In all the patients whose nephropathy was due solely to treatment with penicillamine the proteinuria resolved completely when the drug was withdrawn; renal function did not deteriorate, and corticosteroids were unnecessary.

Adolescent↗
PubMed1988

cis and trans control of erythroid cell-specific gene expression during erythropoiesis.

The overall aim of our group's work is to investigate the molecular mechanisms regulating erythroid cell-specific gene expression during erythroid cell differentiation. We have been successful in cloning two non-globin genes of interest: the first encodes the rabbit red cell-specific lipoxygenase (LOX), which has a role in degrading mitochondrial lipids during maturation of the reticulocyte to the erythrocyte; and the second, mouse glutathione peroxidase (GSHPX), an important seleno-enzyme responsible for protection against peroxide-damage. Characterization of the GSHPX gene revealed that the seleno-cysteine residue in the active site of the enzyme is encoded by UGA, which usually functions as a translation-termination codon. This novel finding has important implications regarding the role of mRNA sequence context effects in codon recognition. In contrast with the beta-globin locus, very little is known about the mechanisms responsible for the erythroid-specific expression of the alpha-globin genes. By a combination of functional transfection assays and studies of the interactions of nuclear sequence-specific DNA-binding proteins with promoter sequences in vitro, we have recently defined two regions upstream of the mouse alpha-globin gene involved in its erythroid-specific expression: one contains a sequence motif (GATAAG) that binds to a species-conserved and erythroid-specific factor both in vitro and in vivo. Interestingly, GATAAG motifs binding the same factor are found also in the mouse and chicken adult beta-globin gene promoters, the erythroid-specific promoter of the haem pathway enzyme, porphobilinogen (PBG) deaminase and the chicken beta-globin 3' enhancer. We are now commencing purification of this erythroid-specific GATAAG-binding factor, investigating in more detail how it functions in relation to other globin gene control regions and determining whether GATAAG-like regions have a functional role in the erythroid-specific expression of other genes. We have begun to investigate the regulation of the GSHPX and red cell LOX genes. The presence of tissue-specific 3' DNAse I-hypersensitive sites (DHSS) suggests that different 3' flanking regions of the GSHPX gene may be important in its regulation in the various cell types in which it is highly expressed, i.e. erythroid cells, liver and kidney.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1988

Regulation of erythroid cell-specific gene expression during erythropoiesis.

The aim of our group's work over the past few years has been to investigate the molecular mechanisms regulating erythroid cell-specific gene expression during erythroid cell differentiation. In addition to the alpha-globin gene, we have focussed on two non-globin genes of interest encoding the rabbit red cell-specific lipoxygenase (LOX) and the mouse glutathione peroxidase (GSHPX), an important seleno-enzyme responsible for protection against peroxide-damage. Characterisation of the GSHPX gene showed that the seleno-cysteine residue in the active site of the enzyme is encoded by UGA, which usually functions as a translation-termination codon. This novel finding has important implications regarding mRNA sequence context effects affecting codon recognition. The regulation of the GSHPX and red cell LOX genes has been investigated by functional transfection experiments. The 700 bp upstream of the GSHPX promoter seems to function equally well when linked to the bacterial chloramphenicol acetyl transferase (CAT) gene and transfected into mouse erythroid or fibroblast cell lines. However, the presence of tissue-specific DNase I hypersensitive sites (DHSS) in the 3' flanking region of the GSHPX gene suggests that such sites may be important in its regulation in the various cell types in which it is highly expressed, i.e., erythroid cells, liver and kidney. The transcription unit of the RBC LOX gene has also been defined and 5' and 3' flanking regions are being investigated for erythroid-specific regulatory elements: a region upstream of the LOX gene gives increased expression of a linked CAT gene when transfected into mouse erythroid cell lines compared to non-erythroid cell lines.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute Disease↗
PubMed1987

DNA polymorphism of human porphobilinogen deaminase gene in acute intermittent porphyria.

A common two-allele MspI restriction fragment length polymorphism of the human erythroid porphobilinogen (PBG)-deaminase gene was investigated in 33 unrelated patients with acute intermittent porphyria (AIP) and 20 controls. The polymorphism was tightly linked (lod score 3.14; no recombinants) to the locus for AIP as identified by measurement of erythrocyte PBG-deaminase activity. The frequency of the polymorphism in the AIP patients did not differ significantly from that in the controls. No common polymorphisms for eight other restriction endonucleases were found in either group. In 30 of the AIP patients no crossreacting immunological material (CRIM) was produced by the mutant PBG-deaminase allele. The MspI polymorphism enabled each PBG-deaminase allele to be distinguished in subjects heterozygous for the polymorphism; thus a major gene deletion was excluded as the cause of the CRIM-negative mutation in all of the 18 families that contained an affected CRIM-negative individual heterozygous for the polymorphism. In suitable families, the MspI polymorphism provides a more certain way of identifying carriers of the AIP gene than current enzymatic methods and major gene deletions are unlikely to be present in more than a small proportion of the commonest type of AIP, the CRIM-negative form.

Acute Disease↗
PubMed1987

The natural course of gold nephropathy: long term study of 21 patients.

To clarify the natural course of gold nephropathy and thereby facilitate its clinical management 21 patients with rheumatoid arthritis who developed proteinuria during treatment with intramuscular sodium aurothiomalate were studied in detail throughout their renal illnesses. Renal biopsies were performed, and creatinine clearance and proteinuria were measured serially for 60 months (range 16-130 months). Ten patients developed proteinuria after six months' treatment, 15 after 12 months, and 18 after 24 months. When treatment was stopped the proteinuria reached a median peak of 2.1 g/day (range 0.7-30.7 g/day) at two months (range 1-13 months) before resolving spontaneously, in eight patients by six months, in 13 by 12 months, and in 18 by 24 months. All patients were free of proteinuria by 39 months, the median duration being 11 months. The median first and last measurements of creatinine clearance showed no significant change (77 ml/minute and 59 ml/minute, respectively), and no patient died from or needed treatment for renal failure. HLA-B8 or DR3 alloantigens, or both, were identified in seven patients. Renal biopsy specimens showed membranous glomerulonephritis in 15 patients, a minimal change nephropathy in two, mesangial electron dense deposits in two, and no appreciable glomerular changes in two. In these 21 patients the proteinuria of gold nephropathy resolved completely when treatment was withdrawn. Renal function did not deteriorate, corticosteroids were unnecessary, and several different renal lesions were seen.

Adult↗
PubMed1987

Cloning of a rabbit erythroid-cell-specific lipoxygenase mRNA.

We report the isolation of cDNA recombinants representing part of the rabbit reticulocyte (immature red blood cell, RBC) lipoxygenase (LOX) mRNA. One cDNA predicts an amino acid (aa) sequence matching exactly the unique N-terminal 30-aa sequence of the purified enzyme. Further, the reticulocyte mRNA, hybrid-selected by this recombinant, can be translated in vitro to give a polypeptide that comigrates with the purified reticulocyte LOX and is recognized by affinity-purified anti-RBC LOX polyclonal antibodies. Southern blotting experiments hybridising the RBC LOX cDNAs available to total rabbit genomic DNA digested with various restriction enzymes gives a fairly simple hybridisation pattern under moderate stringency conditions: moreover, the same pattern is obtained with a cloned fragment of genomic DNA containing the RBC LOX gene. This indicates that the RBC LOX gene is unique in the genome and seems not to be very closely related to the genes encoding the other tissue LOXs. We also show by Northern transfer/hybridisation experiments that the RBC LOX mRNA is expressed only in the red cell lineage but not in white blood cells (bone marrow or spleen) or in other non-erythroid cells tested (e.g., brain and lung).

Amino Acid Sequence↗