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T W Brotherton

Publications and source records attributed to T W Brotherton.

12 recordsLinked to original sources
PubMed1991

Discrete regions of the avian beta-globin gene cluster have tissue-specific hypersensitivity to cleavage by sonication in nuclei.

We have analyzed the DNA released by sonication from avian nuclei, crosslinked with formaldehyde, by restriction mapping and equilibrium density gradient centrifugation. Our results indicate that regions flanking the adult beta-globin gene in adult reticulocytes have increased sensitivity to the mechanical shearing caused by sonication. These regions are near, and may overlap, the known nuclease hypersensitive regions that flank this gene. Like the hypersensitivity of these regions to nuclease digestion, the increased sensitivity to sonication is tissue-specific and appears to be due chiefly to the absence of nucleosome structures in these regions. Analysis of crosslinked chromatin fractionated by density gradient sedimentation suggests that DNA sequences near the matrix attachment region/enhancer element located 3' to the adult beta-globin gene are associated with transcriptionally-engaged chromatin fractions from both adult reticulocytes and day 5 chick embryo primitive erythroid cells. Sonication appears to be a useful tool for the study of chromatin structure.

Animals↗
PubMed1991

Postsynthetic methylation of core histones in K562 cells is associated with bulk acetylation but not with transcriptional activity.

The relationship between the postsynthetic modification of core histones by methylation and transcriptionally active chromatin was investigated in K562 erythroleukemia cells. Cells were incubated with L-[methyl-3H]methionine in the presence of cycloheximide. Under these conditions, only histones H3 and H4 were detectably methylated. Chromatin was fractionated by several methods, including low-salt elution, mononucleosome gel mobility shift using HPLC-purified HMG 17, and cesium chloride-guanidine equilibrium gradient centrifugation of formaldehyde-fixed chromatin. By these latter two methods, chromatin highly enriched for transcriptionally engaged or competent genes was isolated. A significant correlation was noted between postsynthetic modification of histones by methylation and by the slow-turnover form of acetylation. However, there was no enrichment of methylated histones in the transcriptionally competent fraction of chromatin isolated by HMG 17 binding. Moreover, only minor enrichment of methylated histone H3.1 and no enrichment of methylated histones H3.2 and H4 was detected in transcriptionally engaged chromatin isolated by gradient centrifugation. Chromatin soluble in low-salt buffer was found to be significantly enriched in methylated histones, but not in active genes. We conclude that histone methylation is associated with both transcriptionally active and transcriptionally inactive chromatin. The function of this modification, like that of bulk histone acetylation, remains to be determined.

Acetylation↗
PubMed1990

A nuclear matrix protein binds very tightly to DNA in the avian beta-globin gene enhancer.

Current evidence suggests that DNA is covalently attached to proteins in the nuclear matrix of eukaryotic cells and that specific DNA sequences are tightly associated with the nuclear matrix. However, it has not been documented that specific DNA sequences can become covalently attached to nuclear matrix protein. We have examined the binding of cloned DNA sequences that contain the avian beta-globin gene enhancer, a region previously shown to be matrix associated in erythroid cells in vivo, with nuclear matrices from several avian tissue sources to determine if covalent DNA-protein bonds are formed. Our results indicate that sequence-specific DNA-protein complexes that are resistant to denaturation by SDS, boiling, and phenol and disulfide reduction are formed. Excess protein, capable of forming very tight bonds with DNA that contains the beta-globin gene enhancer, is present in cells in which matrix attachment of this DNA sequence is not detected in vivo. Evidence is presented that suggests that the protein to which DNA forms very tight bonds is not topoisomerase II. These results are discussed in relation to current models of the nuclear matrix and the utility of in vitro assays of matrix attachment regions using cloned DNA.

Animals↗
PubMed1990

Binding of HMG 17 to mononucleosomes of the avian beta-globin gene cluster in erythroid and non-erythroid cells.

The binding of HMG 17 to stripped core mononucleosomes containing DNA from the avian beta-globin gene cluster was examined to determine whether binding in vitro in this developmentally-regulated gene domain was associated with transcriptional activity or DNaseI-sensitivity in intact nuclei. Mononucleosomes were prepared from primitive and definitive stage embryonic red blood cells of chick embryos, adult reticulocytes, adult reticulocytes in which embryonic rho-globin transcription was induced, and adult thymus cells. Preferential binding by HMG 17 to mononucleosomes containing the beta-globin gene cluster was confined to erythroid-derived mononucleosomes that contain the embryonic rho-globin gene, the adult beta-globin gene, and DNA sequences located between these two genes, but not to those that contain the embryonic epsilon-globin gene. Comparison of these results to the known patterns of transcription and DNaseI-sensitivity within the beta-globin gene cluster shows that HMG 17 binding, although tissue-specific, does not correlate directly with either DNaseI-sensitivity or active gene transcription, but is dependent on other factors present in core mononucleosomes from this active gene domain.

Animals↗
PubMed1990

Bovine pancreatic DNase I binds very tightly to DNA fragments and may be mistaken for putative endogenous nuclear proteins covalently bound to DNA.

Using published methods for the isolation of nuclear proteins tightly bound to DNA, and resistant to removal by SDS or 16-BAC detergent and urea, several new protein bands in the region of 55 kd and 62 kd on SDS gel and 43 kd and 70 kd on 16--BAC gel electrophoresis were identified in extracts of avian erythroid nuclei. These bands were radiolabelled by subjecting the DNA--protein complexes to nick--translation in the presence of [32P]--dCTP, followed by prolonged digestion with excess bovine DNase I. Amino acid sequence analysis shows that these bands contain DNase I. These results indicate that DNase I can form stable complexes with DNA, and suggest that DNase I--DNA complexes may be mistakenly identified as nuclear proteins covalently bound to DNA.

Amino Acid Sequence↗
PubMed1989

Heparin binds to intact mononucleosomes and induces a novel unfolded structure.

It has been previously shown that heparin can bind to chromatin and enhance transcriptional activity. To characterize this phenomenon further, we have studied the interaction of heparin with isolated core mononucleosomes from avian reticulocytes. The results of these studies suggest that heparin bound reversibly to intact core mononucleosomes to induce a new structure, identified by decreased electrophoretic mobility and altered circular dichroism spectra. This altered nucleosome conformation exhibits 3-5-fold increased sensitivity to digestion by the nuclease, DNase I, and allows more efficient passage of RNA polymerase. At higher concentrations of heparin, core histones were completely removed from DNA. The finding of a reversible nucleosome-heparin complex in which core DNA is readily accessible to both RNA polymerase and the nuclease DNase I is discussed in the context of transcriptionally active chromatin.

Animals↗
PubMed1986

Preferential in vitro binding of high mobility group proteins 14 and 17 to nucleosomes containing active and DNase I sensitive single-copy genes.

High mobility group (HMG) proteins 14 and 17 bind to mononucleosomes in vitro, but the exact nature of this binding has not been clearly established. A new method was developed to allow direct membrane transfer of DNA from HMG 14/17 bound and unbound nucleosomes, which have been separated by acrylamide gel electrophoresis. Hybridization analysis of membranes obtained by this method revealed that the HMG 14/17 bound nucleosomes of avian erythrocytes and rat hepatic tumor (HTC) cells were enriched, about 2-fold, in actively transcribed genes and also inactive but DNase I sensitive genes. Nucleosomes containing inactive, DNase I resistant genes were bound by HMG 14/17, but not preferentially. Several factors that have been reported to greatly influence the binding of HMG 14/17 to nucleosomes in vitro were tested and shown to not account for the preferential binding to DNase I sensitive chromatin. These factors include nucleosomal linker DNA length, single-stranded DNA nicks, and DNA bulk hypomethylation. An additional factor, histone acetylation, was preferentially associated with the HMG 14/17 bound chromatin fraction of avian erythrocytes, but it was not associated with the HMG 14/17 bound chromatin fraction of metabolically active HTC cells. The latter finding was true for all kinetic forms of histone acetylation.

Acetylation↗
PubMed1981

Only a small fraction of avian erythrocyte histone is involved in ongoing acetylation.

We have studied histone acetylation in chicken erythrocytes. We find that about 30% of the histone in these cells is acetylated, however the majority of these histones are not in a dynamic steady state typical of other chicken cells and of mammalian cells, but rather are frozen in this state of modification. A very small fraction of erythrocyte histones are being modified normally but cannot be detected as shifting to higher levels of acetylation upon treatment with butyrate because the amount of histone so modified is small. Nonetheless, chicken erythrocytes incorporate 3H-acetate into histones about 40% as well as seen in the dynamically active HTC cells. This is most likely due to the formation of very high specific activity Acetyl CoA pools in erythrocytes which have very low levels of coenzyme A. We conclude that these genetically inactive cells are involved in only a minor way with histone acetylation.

Acetates↗
PubMed1979

Hemoglobin ontogeny during normal mouse fetal development.

Pure populations of large, nucleated erythrocytes derived from yolk sac blood islands were obtained during normal fetal mouse development. Embryonic hemoglobins were present in these cells early in gestation. Later in gestation, an increasing amount of adults hemoglobin was also synthesized and accumulated in this population of primitive nucleated erythrocytes, as demonstrated by both biochemical and immunocytochemical techniques.

Animals↗
PubMed1979

Fetal erythropoiesis and hemoglobin ontogeny in tail-short (Ts/+) mutant mice.

Mutant Ts/+ fetuses are developmentally retarded as compared to normal +/+ littermates. Mutant fetuses have less total hemoglobin than do normal fetuses of the same gestational age. However, when compared to +/+ fetuses of similar body weight, Ts/+ fetuses have the appropriate amount of total hemoglobin, suggesting that the apparent anemia observed in mutant fetuses is most likely the result of delay in growth and development. Changes in proportions of embryonic hemoglobins during fetal development are similar in Ts/+ and +/+ fetuses at day 12 and later of gestation. Moreover, adult hemoglobin is detected in circulating primitive nucleated erythrocytes in the developmentally retarded Ts/+ mutant fetuses at about the same chronologic age as their +/+ normal littermates.

Aging↗