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D N Cooper

Publications and source records attributed to D N Cooper.

194 records · Page 11Linked to original sources
PubMed1983

Unmethylated domains in vertebrate DNA.

We have detected a fraction that is rich in unmethylated HpaII and HhaI sites by end-labelling HpaII fragments of chicken DNA. The fraction is not obvious when DNA fragments are stained with ethidium bromide as it amounts to less than 2% of the genome. The average frequency of sites for HpaII is over thirteen times greater in the unmethylated fraction than in total DNA. Partial digests indicate that the unmethylated sites are clustered in the genome. Similar unmethylated fractions were detected in six other vertebrates in both somatic and germ line DNA.

Animals↗
PubMed1983

Eukaryotic DNA methylation.

Eukaryotic genomes contain 5-methylcytosine (5mC) as a rare base.5mC arises by postsynthetic modification of cytosine and occurs, at least in animals, predominantly in the dinucleotide CpG. The base is not distributed randomly in these genomes but conforms to a pattern. This pattern varies between taxa but appears to be inherited in a semi-conservative fashion. At the level of the genome, gross changes in the level of DNA methylation have been noted. This has encouraged speculation that the modification may play a role in cellular differentiation. Tissue-specific patterns of DNA methylation, predicted by various models of differentiation, have been found for most vertebrate genes so far examined. A correlation has emerged between the undermethylation of these regions and their transcription, but this is not always the case. While data for eukaryotic viral sequences are less equivocal, studies of this kind cannot in isolation distinguish between undermethylation being a cause or a consequence of gene activity. If it were a cause, it is probable that the demethylation of specific CpG sites would be a necessary yet not a sufficient condition for transcription to occur. The introduction of artificially methylated DNA sequences into individual eukaryotic cells by microinjection or transformation may provide the means to elucidate these questions in the future. In the meantime, the study of eukaryotic DNA methylation promises to contribute much to our understanding of the regulation of gene expression in these organisms.

5-Methylcytosine↗
PubMed1983

Discoidin I and discoidin II are localized differently in developing Dictyostelium discoideum.

The distribution of discoidin I and discoidin II, developmentally regulated lectins in Dictyostelium discoideum, was determined immunohistochemically at various stages of development. Discoidin I was first prominent as focal clumps in aggregating cells, then accumulated on the surface of aggregates and around them. Discoidin II became prominent later and ultimately localized in what appear to be prespore vesicles. The results indicate that discoidin I and discoidin II have different and possibly multiple functions.

Carrier Proteins↗
PubMed1983

Variation in the DNA methylation pattern of expressed and nonexpressed genes in chicken.

Using methyl-sensitive and -insensitive restriction enzymes, Hpa II and Msp I, the methylation status of various chicken genes was examined in different tissues and developmental stages. Tissue-specific differences in methylation were found for the delta-crystallin, beta-tubulin, G3PDH, rDNA, and actin genes but not for the histone genes. Developmental decreases in methylation were noted for the delta-crystallin and actin genes in chicken kidney between embryo and adult. Since most of the sequences examined were housekeeping genes, transcriptional differences are apparently not a necessary accompaniment to changes in DNA methylation at the CpG sites examined. The only exception is sperm DNA where the delta-crystallin, beta-tubulin, and actin genes are highly methylated and almost certainly not transcribed. However the G3PDH genes are no more highly methylated in sperm than in other somatic tissues. Many sequences homologous to the rDNA and histone probes used are unmethylated in all tissues examined including sperm, but a methylated rDNA subfraction is more heavily methylated in sperm than in other tissues. We speculate as to the significance of these differences in sperm DNA methylation in the light of possible requirements for early gene activation and the probable deleterious mutagenic effects of heavy methylation within coding sequences.

5-Methylcytosine↗
PubMed1983

The pattern of DNA methylation in the delta-crystallin genes in transdifferentiating neural retina cultures.

Terminally differentiated lens fibre cells are formed in the vertebrate lens throughout life. Lens fibre cells may also be obtained by an in vitro process termed transdifferentiation, from certain tissues of different developmental origin from lens, such as embryo neural retina. delta-Crystallin is the major protein in the chick embryo lens fibre cells, and also in transdifferentiated lens cells obtained from cultured embryonic neural retina. Lens crystallin proteins and mRNA are present at low levels in the intact embryonic neural retina but are no longer detectable in the early stages of neural retina cell culture. However, levels rise steeply in the later stages and crystallins become the major products in terminally transdifferentiating neural retina cultures. We have used this system to test the hypothesis that the patterns of DNA methylation in particular genes are correlated with gene expression. A number of developmentally regulated genes have been found to be undermethylated in tissues where they are expressed, and methylated in tissues where they are not. However this correspondence does not always hold true. Eight-day-old embryonic neural retina was cultured for the period of time during which crystallin gene expression increases 100-fold. DNA methylation in the delta-crystallin gene region was analysed at several stages of cell culture by using the restriction endonucleases HpaII and MspI which cleave at the sequence CCGG. The former enzyme cannot cleave internally methylated cytosine (CmCGG) while the latter cannot cleave externally methylated cytosine (mCCGG). We detect no change in the methylation of CCGG sites within the delta-crystallin gene regions during transdifferentiation. Since dramatic changes in delta-crystallin gene expression occur during this process we conclude that large scale alterations in the pattern of DNA methylation are not a necessary accompaniment to changes in gene activity.

Animals↗
PubMed1981

Isolectins from Dictyostelium purpureum. Purification and characterization of seven functionally distinct forms.

Purpurin, the lectin from Dictyostelium purpureum, has been resolved into seven tetrameric isolectins by polyacrylamide gel electrophoresis at pH 8.9. The isolectins are assembled from four distinct subunits resolved by electrophoresis in sodium dodecyl sulfate and by tryptic peptide mapping. Two of the subunits combine randomly with each other to form mixed tetramers (I4, I3II1, I2II2, I1II3, II4) in binomial proportions. The other two subunits (III and IV) form only homotetramers. The isolectins can be functionally discriminated and separated on the basis of their relative affinities for columns derivatized with complementary saccharides. On the basis of relative sensitivity to hapten inhibitors of hemagglutination, isolectins III4 and IV4 are distinct from each other and from isolectins composed of subunits I and II. However, isolectins of I and II are not distinguishable on the basis of hemagglutination inhibition. None of the subunits are glycosylated, and all form tetramers with molecular weights of approximately 88,000. The existence of multiple functionally distinct forms suggests that lectin function in cellular slime molds may be more complex than presently envisioned.

Animals↗
PubMed1981

Endogenous lectins in chickens and slime molds: transfer from intracellular to extracellular sites.

Endogenous lectins in both cellular slime molds and chicken tissues have been localized primarily intracellularly, in contrast with the predominantly extracellular localization of the glycoproteins, glycolipids, and glycosaminoglycans with which they might interact. Here we present evidence that lectins in both of these organisms may be externalized and become associated with the cell surface and/or extracellular materials. In chicken intestine, chicken-lactose-lectin-II is shown to be localized in the secretory granules of the goblet cells, along with mucin, and to be secreted onto the intestinal surface. In embryonic muscle, chicken-lactose-lectin-I is shown to be externalized with differentiation, ultimately becoming localized on the surface of myotubes and in the extracellular spaces. In a cellular slime mold, Dictyostelium purpureum, externalization of lectin is elicited by either polyvalent glycoproteins that bind the small amount of endogenous cell surface lectin, or by slime mold or plant lectins that bind unoccupied complementary cell surface oligosaccharides. These results suggest that externalization of endogenous lectin may be a response to specific external signals. We conclude that lectins are frequently held in intracellular reserves awaiting release for specific external functions.

Animals↗
PubMed1981

Relationship between asthma and gastro-oesophageal reflux.

Twenty patients with bronchial asthma who also had gastro-oesophageal reflux were investigated. The severity of their reflux was graded using symptom score of heartburn and regurgitation and by the following investigations: barium swallow and meal, fibreoptic endoscopy and biopsy, manometry and pH monitoring of the distal oesophagus, and an acid infusion test. Full lung function studies were performed and patients were entered into a double-blind crossover study using cimetidine to control their reflux in order to assess beneficial effects with respect to their respiratory problems. Eighteen patients completed the study. Significant improvements were seen in reflux and night time asthmatic symptoms, both these indices being measured on a scoring system. Home monitoring of peak flow values showed a statistical improvement for th last peak flow reading of the day. Fourteen patients felt that their chest symptoms had significantly improved during the cimetidine period.

Adult↗
PubMed1981

Efflux of radiolabeled polyethylene glycols and albumin from rat brain.

Experiments were carried out to evaluate the role of convection in the removal of large molecules from brain interstitial fluid. Radiolabeled test compounds were injected into the caudate nucleus of anesthetized rats through a guide cannula implanted 1 wk previously and the concentrations of isotope in brain and cerebrospinal fluid (CSF) determined at various times after injection. Control studies with 22Na indicate that the permeability of the blood-brain barrier is normal in tissue surrounding the intracerebral injection cannula. For 69,000 dalton serum albumin, 4,000 dalton polyethylene glycol, and 900 dalton polyethylene glycol, clearance from brain approximates a single exponential decay with half times of disappearance of 12.2, 12.6, and 14.4 h, respectively. Similarly in efflux rate, despite a fivefold difference in diffusion coefficient, is consistent with convective losses from brain, and the maximal rate of interstitial fluid removal estimated on the basis of these data is 0.11 microliter.g brain-1.min-1. Only 10-20% of total efflux is into bulk CSF withdrawn from the cisterna magna.

Animals↗
PubMedDate not supplied

The mutational spectrum of single base-pair substitutions in mRNA splice junctions of human genes: causes and consequences.

A total of 101 different examples of point mutations, which lie in the vicinity of mRNA splice junctions, and which have been held to be responsible for a human genetic disease by altering the accuracy of efficiency of mRNA splicing, have been collated. These data comprise 62 mutations at 5' splice sites, 26 at 3' splice sites and 13 that result in the creation of novel splice sites. It is estimated that up to 15% of all point mutations causing human genetic disease result in an mRNA splicing defect. Of the 5' splice site mutations, 60% involved the invariant GT dinucleotide; mutations were found to be non-randomly distributed with an excess over expectation at positions +1 and +2, and apparent deficiencies at positions -1 and -2. Of the 3' splice site mutations, 87% involved the invariant AG dinucleotide; an excess of mutations over expectation was noted at position -2. This non-randomness of mutation reflects the evolutionary conservation apparent in splice site consensus sequences drawn up previously from primate genes, and is most probably attributable to detection bias resulting from the differing phenotypic severity of specific lesions. The spectrum of point mutations was also drastically skewed: purines were significantly over-represented as substituting nucleotides, perhaps because of steric hindrance (e.g. in U1 snRNA binding at 5' splice sites). Furthermore, splice sites affected by point mutations resulting in human genetic disease were markedly different from the splice site consensus sequences. When similarity was quantified by a 'consensus value', both extremely low and extremely high values were notably absent from the wild-type sequences of the mutated splice sites. Splice sites of intermediate similarity to the consensus sequence may thus be more prone to the deleterious effects of mutation. Regarding the phenotypic effects of mutations on mRNA splicing, exon skipping occurred more frequently than cryptic splice site usage. Evidence is presented that indicates that, at least for 5' splice site mutations, cryptic splice site usage is favoured under conditions where (1) a number of such sites are present in the immediate vicinity and (2) these sites exhibit sufficient homology to the splice site consensus sequence for them to be able to compete successfully with the mutated splice site. The novel concept of a "potential for cryptic splice site usage" value was introduced in order to quantify these characteristics, and to predict the relative proportion of exon skipping vs cryptic splice site utilization consequent to the introduction of a mutation at a normal splice site.

Consensus Sequence↗