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Biomedical subjects

W J Brammar

Publications and source records attributed to W J Brammar.

At least 19 recordsLinked to original sources
PubMed1992

The ontogeny of peroxisome-proliferator-activated receptor gene expression in the mouse and rat.

The expression of the gene coding for peroxisome-proliferator-activated receptor (PPAR), a novel transacting factor belonging to the steroid superfamily, has been determined in the mouse and rat throughout development using hybridization histochemistry. Messenger RNA is demonstrable in the liver and brown fat from the fetal period onwards and, additionally, in the heart, kidney and gut post-natally. It is proposed that the upregulation of transcription of peroxisomal beta oxidation genes in specific tissues follows binding of the receptor to its natural ligand. Thus PPAR may have an important role in cold adaptation and non-shivering thermogenesis as well as in detoxification.

Adipose Tissue, Brown↗
PubMed1992

Control of gene expression in tobacco cells using a bacterial operator-repressor system.

We have investigated the efficacy of using the Escherichia coli lac operator-repressor system to control plant gene expression. The lacI gene was modified to allow optimal expression in plant cells and then placed downstream of the cauliflower mosaic virus (CaMV) 35S RNA promoter. This construct was introduced into tobacco plants by leaf disc transformation. Transgenic tobacco plants synthesized significant quantities of LacI protein (up to 0.06% of total soluble protein). We have used the E.coli beta-glucuronidase gene (gus) as the reporter gene by placing it downstream of the maize chlorophyll a/b binding protein (CAB) gene promoter. Lac operators were introduced into several positions within the CAB promoter and operator-free plasmid was used as control. Repression was assessed by comparing the transient expression from CAB-operator-gus reporter constructs in protoplasts expressing lac protein, with that in control cells not expressing the repressor. Repression varied between 10 and 90% with different operator positions. Transient assays were also performed in the presence of the inducer, isopropyl-beta-D-thiogalactoside (IPTG). In lacI protoplasts the presence of IPTG manifested itself in a 4.2-fold relief of repression. The study was extended to show regulation of expression in stable transformants. Tobacco transformants harbouring a CAB-operator-gus reporter construct and the lacI gene were shown to have repressed GUS levels, but in the presence of IPTG, repression was relieved 15-fold. We conclude that the lac repressor can enter the plant cell nucleus, find its cognate operator sequence in the chromatin to form a repressor--operator complex and effectively block transcription of a downstream gene.

Bacterial Proteins↗
PubMed1992

Differential expression of the mitochondrial gene cytochrome oxidase II in benign and malignant breast tissue.

Comparative analysis of expression levels of genes in benign and malignant tumours of the breast has been performed. Differential screening of cDNA libraries identified four genes of the mitochondrial genome as being expressed at different levels in the two tissues compared, but further investigations showed that only the gene encoding subunit 2 of cytochrome c oxidase (COII) is expressed at significantly higher levels in carcinomas compared with fibroadenomas. The mitochondrial genes encoding subunits 2 and 4 of NADH dehydrogenase, and subunit 6 of F0F1ATPase, were not found to be differentially expressed in carcinomas and fibroadenomas. All four genes were expressed in the epithelium of human breast carcinomas, as shown by in situ hybridization. The expression level of the COII gene is also correlated with carcinoma grade. No gross alterations to the mitochondrial DNA from these tumours could be detected. The possible implications of these results on the behavioural differences between fibroadenomas and carcinomas are discussed.

Adenofibroma↗
PubMed1992

Differential expression of translation-associated genes in benign and malignant human breast tumours.

The human gene sequences encoding the translation-associated functions of alpha-subunit of elongation factor 1 (EF-1 alpha) and the ubiquitin carboxyl extension protein (HUBCEP80) have been isolated by differential cDNA screening, and found to have significantly higher levels of expression in fibroadenomas (benign) compared with carcinomas (malignant) of the breast. These data parallel our previous findings that the acidic ribosomal phosphoprotein P2 also has higher expression levels in the benign breast tumours (Sharp et al., 1990). In situ hybridisation has shown these genes to be expressed predominantly in the epithelium of breast tumours.

Adenofibroma↗
PubMed1992

Isolation and characterization of a novel gene with differential expression in benign and malignant human breast tumours.

We report the identification of a novel cDNA representing an mRNA showing significantly higher levels of expression in benign breast lesions than in carcinomas. This cDNA was identified by differential screening of a cDNA library generated from a breast carcinoma, and shows consistently higher expression in fibroadenomas than in carcinomas. The expression in both benign and malignant tissues is highest in epithelial cells as determined by in situ hybridization to tissue sections. The nucleotide sequence of the full-length cDNA has been determined, and the deduced protein is highly basic with no signal or transmembrane sequence, but two potential nuclear localization signals. Neither the DNA nor the protein sequence show any significant homology to sequences in current databases. The cDNA hybridizes to multiple sequences within both human and other mammalian genomes, but to single genomic sequences in Drosophila, Physarum and Schizosaccharomyces pombe. This cDNA therefore represents a highly conserved gene sequence. We have identified only one major transcript in human cells, and it seems likely that there are several pseudogenes within the human genome.

Adenofibroma↗
PubMed1991

Renal and extra-renal levels of renin mRNA in experimental hypertension.

1. Using a ribonuclease-protection assay, renin mRNA levels were compared in the kidneys, livers, brains, hearts and adrenal glands of two-kidney, one-clip Goldblatt hypertensive rats with those of age-matched control rats at 4 weeks ('early') and 20 weeks ('chronic') after clipping, and in the kidneys and adrenal glands of rats treated for 3 weeks with deoxycorticosterone and salt (deoxycorticosterone-salt hypertension) with those of control rats. 2. While marked changes were observed in kidney renin mRNA levels in all three experimental groups compared with their respective controls, in most of the extra-renal tissue studied minimal, if any, difference was seen in renin mRNA levels between the hypertensive and control rats. 3. The findings suggest that in these extra-renal tissues renin gene expression is differently regulated from that in the kidney, and particularly that it is not profoundly affected by changes in the level of circulating angiotensin II. 4. An increase in renin mRNA was observed in the adrenal glands of the 'chronic' Goldblatt rats, which may be of relevance to the maintenance of hypertension in this model.

Adrenal Glands↗
PubMed1991

Loss of chromosome 17p13 sequences and mutation of p53 in human breast carcinomas.

DNAs from ninety seven primary breast carcinoma biopsies have been examined for loss of sequences on 17p13. In addition, immunohistochemical analysis has been carried out on the majority of these cases to determine whether p53 gene expression can be detected. Detection of p53 expression is taken to indicate mutation of p53 leading to stabilisation of the protein and thus detectable levels of p53 in the cell. In 86% of breast carcinoma samples where both allele loss and expression data were available, loss of sequences on 17p13 and/or expression of p53 was detected. Alterations to p53, whether loss of one allele or mutation, are therefore by far the most common changes so far detected in primary human breast tumours. In three cases where expression of p53 could be detected by immunohistochemistry, the precise mutation to p53 was identified. All three mutations fall within the regions which are highly conserved in p53, encoded by exons 5 to 8. Two are single base changes leading to misense mutations, and the third is a single base-pair deletion. The expression of the latter gene would result in production of a truncated protein which should lack normal biological activities.

Alleles↗
PubMed1990

A sequence previously identified as metastasis-related encodes an acidic ribosomal phosphoprotein, P2.

We have used a metastasis-related human cDNA isolated from a liver metastasis from a colonic adenocarcinoma to screen a human breast carcinoma cDNA library for homologous sequences. Nucleotide sequence analysis of positive clones revealed that the cDNA represents a ribosomal phosphoprotein. P2. The expression of P2 mRNA was significantly higher (Student's t test, one tail; P less than or equal to 0.01) in seven fibroadenomas than in seven carcinomas, with an average five-fold difference. This enhanced expression level P2 mRNA in benign fibroadenomas compared with malignant carcinomas is contrary to that expected, based on earlier work with normal colonic mucosa, colorectal carcinoma and hepatic metastasis. The identification of gene transcripts which differ in abundance and correlate with the metastatic phenotype may be of considerable importance both as diagnostic aids and in defining the changes associated with tumour progression and metastasis at the molecular level. The possible role that ribosomal proteins may play in the progression of carcinoma of the breast is discussed.

Base Sequence↗
PubMed1990

Analysis of the renin gene intron A tandem repeat region of Milan and Lyon hypertensive rat strains.

The region of intron A of the rat renin gene containing a unique tandemly repeated sequence was analysed in the Milan and Lyon hypertensive rat strains and their controls, and in several Sprague-Dawley rats, using an oligonucleotide probe complementary to the tandemly repeated sequence and a renin complementary DNA probe. In the Milan rats, the size of the Bgl II DNA fragment encompassing the tandem repeat region was the same in the hypertensive (MHS) and normotensive (MNS) strains. In the Lyon model, a difference of 1.1 kilobase (equivalent to about 28 copies of the 38 basepair tandem repeat sequence) was observed in the size of the Bgl II fragment of the hypertensive (LH) and normotensive (LN) strains. However, the finding that the size of the fragment in the Lyon low-blood-pressure (LL) strain was the same as that in the LH strain rather than the LN strain suggests that the difference between the two latter strains is not by itself a major cause of the blood pressure difference between them in the intron A tandem region. An analysis of Sprague-Dawley rats, from which the Lyon strains are derived, showed that at least three different renin gene alleles, two with Bgl II fragments of the same size as those seen in the Lyon strains, are randomly segregating in this population.

Alleles↗
PubMed1990

Expression of the IGF-II/mannose-6-phosphate receptor mRNA and protein in the developing rat.

The insulin-like growth factor II (IGF-II) receptor is identical to the mannose-6-phosphate receptor (M-6-P), but its role as a somatomedin transducer is uncertain. IGF-II/M-6-P receptor expression was studied by in situ hybridization (ISH) in the developing rat. Expression occurs in extra-embryonic membranes at the time of IGF-II mRNA induction and later at paracrine/autocrine sites of IGF-II action (skeletal muscle and perichondrium) in the embryo. Highest levels of receptor mRNA occur in heart and major vessels. Postnatally transcription is strongly down-regulated. This suggests a role for the IGF-II/M-6-P receptor in IGF-II action or turnover during development distinct from its role in lysosomal transport.

Animals↗
PubMed1989

The nucleotide sequence of a mouse renin-encoding gene, Ren-1d, and its upstream region.

The renin-encoding genes have been cloned from high (Ren-1d, Ren-2d)- and low (Ren-1c)-renin-producing strains of mice (DBA/2J and C57BL/10). Each of the genes is approx. 9.6 kb in length and consists of nine exons and eight introns. The entire nucleotide sequence of the Ren-1d gene has been determined and the 5'-flanking regions of the three genes, Ren-1c, Ren-1d and Ren-2d, have been compared. The significance of several potential regulatory signals found in the DNA is discussed.

Amino Acid Sequence↗
PubMed1989

Differential extra-renal expression of the mouse renin genes.

We have used RNase-protection analyses to study renin gene expression in one- and two-gene mouse strains. The RNase-protection assay is capable of discriminating between the transcripts from the different renin genes. In a two-gene strain containing Ren-1D and Ren-2, we demonstrate transcriptional activity from Ren-1D in kidney, submandibular gland (SMG), testes, liver, brain and heart. Ren-2 is clearly expressed in kidney, SMG and testes. Similar analyses of one gene strains (containing Ren-1C only) show expression in kidney, SMG, testes, brain and heart. We cannot detect renin mRNA in the liver of these mice. Ren-1C and Ren-1D thus display quite different tissue-specificities. In order to determine whether the different tissue-specificities of the highly homologous Ren-1C and Ren-1D genes are due to different trans-acting factors in the different mouse strains or to different cis-acting DNA elements inherent to the genes, we introduced a Ren-1D transgene (Ren-1*) into a background strain containing only the Ren-1C gene. The transgene exhibits the same tissue-specificity as the Ren-1D gene of two-gene strains suggesting the presence of different cis-acting DNA elements in Ren-1C and Ren-1D.

Animals↗
PubMed1989

The pro-peptide is not necessary for active renin secretion from transfected mammalian cells.

Cultured mouse myeloma cells were transfected with expression vectors encoding the aspartyl proteinase, human renin. The full construct, encoding the renin precursor prorenin, allows transfected cells to secrete the enzymically inactive pro-protein. Activity is detectable only following trypsin treatment which mimics the physiological activation step. Accordingly, it appears that myeloma cells do not contain detectable levels of an appropriate activating proteinase. However, when these cells are transfected with a construct from which the pro-peptide coding sequence has been deleted, they secrete an apparently fully active enzyme which is indistinguishable from mature renin. We conclude that expression of the pro-peptide is not necessary to allow correct folding of the molecule and its passage through the secretory pathway.

Amino Acid Sequence↗
PubMed1989

The renin gene in patients with malignant hypertension and raised plasma renin activity.

1. We have examined the hypothesis that the raised plasma renin activity in patients with malignant hypertension without an underlying cause is the consequence of expression of a duplicate renin gene. 2. DNA extracted from leucocytes of patients with malignant hypertension and of normotensive controls was digested with the restriction endonuclease PstI and hybridized with a radioactively labelled human renin complementary DNA probe. As an internal control the DNA was concurrently hybridized with a human c-myc protooncogene probe. 3. The signals for each subject from the two probes were quantitatively compared by densitometry. 4. There was no evidence of duplication of the renin gene in the patients with malignant hypertension.

Adult↗
PubMed1989

A widespread abnormality of renin gene expression in the spontaneously hypertensive rat: modulation in some tissues with the development of hypertension.

1. Renin messenger RNA (mRNA) levels were compared in the kidneys, livers, brains, adrenals, aortae and hearts of spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats at 5 and 12 weeks of age using a ribonuclease-protection technique. 2. Relative levels of renin mRNA were increased in the kidney, liver, brain, adrenal and heart of the young SHR compared with the WKY. In the aorta, levels were similar in the two strains at 5 weeks. 3. In 12-week-old animals, while increased levels persisted in the liver, brain and adrenal of the SHR, the level in the kidney was now the same in the two strains and the levels in the heart and aorta were lower in the SHR compared with the WKY. 4. Renin mRNA levels in the kidneys of SHR and WKY were also compared by Northern blotting and confirmed the observations made with the ribonuclease-protection technique. 5. The findings indicate a widespread abnormality of renin gene expression in the SHR which is modulated in some tissues by the development of hypertension. 6. While the mechanism(s) for the abnormality remains to be determined, the increased renin mRNA levels in the SHR in several tissues concerned with blood pressure regulation suggests an important role for the renin-angiotensin system in the development and maintenance of hypertension. 7. However, the finding of increased renin mRNA in the liver also suggests abnormalities in other, as yet unknown, functions of the renin-angiotensin system in the SHR.

Adrenal Glands↗
PubMed1989

DNA fingerprinting of spontaneously hypertensive and Wistar-Kyoto rats: implications for hypertension research.

Probes to hypervariable minisatellite regions of DNA identify multiple loci scattered over the autosomal chromosomes and produce a complex Southern blot pattern of fragments termed a DNA 'fingerprint'. As concern has been raised that different stocks of spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) may not be biologically identical, we have compared the DNA of SHR and WKY from several sources using two such probes which identify different sets of minisatellite sequences. While the DNA fingerprints of SHR from the various sources were identical, variability was observed in those of WKY, indicating genetic heterogeneity between different WKY stocks. In animals from one of the commercial suppliers even inter-rat variability in DNA fingerprints was seen, suggesting genetic heterogeneity within that single colony. These observations indicate that experimental results obtained using WKY from different sources may not be directly comparable and could provide an explanation for some of the conflicting data that exist on the comparative characteristics of SHR and WKY. In separate studies, direct comparisons both of the DNA fingerprints of SHR and WKY and of SHR and stroke-prone spontaneously hypertensive rats (SHRSP) showed multiple differences between the strains. The polymorphisms seen could provide useful linkage markers in locating the chromosomal sites of the genetic loci responsible for raised blood pressure in the SHR and the propensity to strokes in the SHRSP.

Animals↗
PubMed1989

Modulation of mouse renin gene expression by dietary sodium chloride intake in one-gene, two-gene and transgenic animals.

We have studied the effect of dietary NaCl loading on renin gene expression in one-gene, two-gene and transgenic mouse strains. By Northern blotting, we found an approximate twofold reduction in renin messenger (m) RNA in the kidneys of high-NaCl-treated compared with low-NaCl-treated animals. Using an RNase-protection assay designed to discriminate between the different renin gene transcripts, we have shown that renin mRNAs derived from the Ren-1C gene of one-gene strains and the Ren-1D and Ren-2 genes of two-gene animals are all NaCl-responsive. Renin mRNA derived from a 19 kilobase Ren-1D transgene is also NaCl-responsive.

Animals↗