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Biomedical subjects

V G Allfrey

Publications and source records attributed to V G Allfrey.

At least 55 records · Page 3Linked to original sources
PubMed1980

Incorporation of exogenous pyrene-labeled histone into Physarum chromatin: a system for studying changes in nucleosomes assembled in vivo.

Treatment of Physarum histone with iodoacetoxypyrene selectively derivatizes a single H3 cysteine with acetoxypyrene. Microplasmodia can incorporate this AP-H3 into nucleosomes. The distinction between blue monomeric pyrene fluorescence and green excimer pyrene fluorescence allows detection of changes in distance between the closely positioned H3 cysteines in nucleosomes. Fluorescence of nucleosomes labeled in vivo with AP-H3 is almost exclusively of the excimer form, indicating that H3 cysteines are within a few angstroms of each other in the nucleosome core. In histones recovered from these nucleosomes all detectable pyrene is covalently bound to H3. When Physarum is exposed sequentially to labeled followed by unlabeled histone, there is a rapid appearance of green excimer emission in nucleosomes after addition of labeled histone and no apparent switch from excimer to monomer fluorescence after several replications of the genome in the presence of unlabeled histone. These experiments provide evidence in favor of a model for conservative distribution of nucleosomal histones during chromatin replication.

Biological Transport↗
PubMed1979

Different nucleosome structures on transcribing and nontranscribing ribosomal gene sequences.

Monomeric DNA lengths from Physarum nuclear chromatin occur in two subunit forms which differ from each other and from higher oligomers of nucleosomes in content of transcribed ribosomal DNA sequences. Labeled DNA restriction fragments from ribosomal RNA coding regions reanneal most rapidly with DNA from a monomeric subunit fraction. A particles, isolated from growing plasmodia and containing 144 base pairs of DNA in an extended conformation. Higher oligomers of nucleosomes are depleted in sequences from transcribing gene regions but are enriched in sequences from the nontranscribed central spacer of the ribosomal DNA palindrome. Nucleosome configuration on two 26S gene intervening sequences resembles that on adjacent coding regions.

Cell Nucleolus↗
PubMed1979

Phosphorlyation of H1 and H5 histones by cyclic AMP-dependent protein kinase reduces DNA binding.

Phosphorylation of H1 histones by cyclic AMP-dependent protein kinase may be an important transcriptional control mechanism. We have used affinity chromatography to examine the effect of phosphorylation by this enzyme on the DNA-binding properties of calf thymus H1 histones and two highly basic H1 homologues from condensed and transcriptionally silent nuclei: duck erythrocyte H5 and Strongylocentrotus purpuratus sperm H1. Without in vitro phosphorylation, all three histones were eluted from native DNA-Sephadex G-25 columns at salt concentrations which closely resembled those required to extract these histones from nuclei or chromatin. When a small portion of radioactively phosphorylated histone was chromatographed with untreated carrier histone, the phosphorylated species was consistently eluted from the DNA column at slightly lower salt concentrations than the main histone peak. Rechromatography experiments showed that in vitro phosphorylation of H1 can shift its elution position to lower salt concentrations.

Amino Acids↗
PubMed1979

Unique sequence arrangement of ribosomal genes in the palindromic rDNA molecule of Physarum polycephalum.

R-loop and restriction mapping procedures reveal the organization of coding regions at each end of the giant rDNA palindrome of Physarum polycephalum. A 19S coding region of 2.10 +/- 0.21 kb is located at each end of a very long central spacer (35.64 +/- 2.08 kb). An internal spacer of 1.66 +/- 0.12 kb lies distal to the 19S gene. The 5.8S rRNA coding region is located in this spacer. The 26S gene lies distal to the internal spacer. The 26S gene is unusual among those of eukaryotes in that it consists of 3 coding regions (alpha, beta and gamma) interrupted by 2 intervening sequences. The 26S alpha (most central) coding segment of 2.41 +/- 0.33 kb is separated from the 26S beta segment by an intervening sequence of 0.68 +/- 0.13 kb. The 26S beta segment (0.70 +/- 0.11 kb) is separated from the most distal 26S gamma segment (0.59 +/- 0.14 kb) by an intervening sequence of 1.21 +/- 0.14 kb. The 2 intervening sequences are present in at least 88% of ribsomal genes from active plasmodia, indicating that genes containing these sequences are transcribed. The rDNA termini contain a heterogeneous region which varies in length by +/- 300 base pairs.

Base Sequence↗
PubMed1978

Postsynthetic modification of high mobility group proteins. Evidence that high mobility group proteins are acetylated.

High mobility group proteins were isolated from calf thymus and duck erythrocyte nuclei and the possibility was investigated that these proteins undergo acetylation similar to that occurring in some histones. Dinitrophenylation of the proteins followed by acid hydrolysis and amino acid analysis indicated that 2 to 3% of the lysine residues present were unavailable for reaction with fluorodinitrobenzene. Extensive enzymatic degradation with trypsin and pronase and subsequent amino acid analysis showed a significant amount of material eluting at the position of epsilon-N-acetyllysine. Recovery and acid hydrolysis of this material generated a peak eluting in the lysine position. In vitro radioactive labeling of calf thymus nuclei with [3H]acetate yielded labeled high mobility group proteins. All of these findings are in accord with the conclusion that high mobility group proteins are acetylated and that acetylation occurs as a postsynthetic modification of these proteins.

Acetylation↗
PubMed1978

Suppression of histone deacetylation in vivo and in vitro by sodium butyrate.

In HeLa cells which have been exposed to 5 mM sodium butyrate for 21 h, the level of histone acetylation is greatly increased as compared to control cells (Riggs, M.G., Whittaker, R.G., Neumann, J.R., and Ingram, V.R. (1977) Nature 268, 462-464). Our experiments indicate that the increase in the relative amounts of multiacetylated forms of histones H4 and H3 following butyrate treatment is the result of an inhibition of histone deacetylase activity.

Butyrates↗
PubMed1978

The use of netropsin with CsCl gradients for the analysis of DNA and its application to restriction nuclease fragments of ribosomal DNA from Physarum polycephalum.

Netropsin binds to DNA in caesium chloride density gradients and reduces the density of the DNA. The DNA is saturated at a netropsin/DNA weight ratio of about 6 and the change in density, deltarho, at saturation is given by deltarho = -109 (dA + dT content)1.87 mg/ml for the six DNAs tested covering dA + dT contents from 0.28 to 0.69. At lower netropsin/DNA ratios the observed density shifts are consistent with a two-site model for netropsin binding to DNA. Netropsin approximately doubles the resolution of Physarum polycephalum nucleolar satellite DNA from main-band DNA. The fragments of P. polycephalum nucleolar satellite DNA obtained with the restriction endonuclease HindIII do not separate on CsCl gradients, even in the presence of netropsin, which shows that the transcribed and non-transcribed sequences in this DNA have similar nucleotide compositions.

Centrifugation, Density Gradient↗
PubMed1978

Altered nucleosome structure containing DNA sequences complementary to 19S and 26S ribosomal RNA in Physarum polycephalum.

The localization of DNA sequences coding for ribosomal RNA was studied by hybridization of purified ribosomal RNA to DNA from chromatin fragments prepared by limited digestion of Physarum nuclei with staphylococcal nuclease. The 32P-labeled 19S and 26S RNA hybridized to DNA from nucleosome monomers, dimers, trimers, and higher oligomers, separated by sucrose gradient centrifugation, although the level of hybridization to DNA from nucleosome fractions was less than the level of hybridization to undigested nuclear DNA. The distribution of 19S and 26S rDNA sequences in the nucleosome fractions differed from the distribution of bulk DNA in that the rDNA sequences were recovered primarily in two fractions containing monomer-sized DNA lengths (140-160 base pairs). The percentage of DNA hybridizing to 19S plus 26S RNA was greater in peak A, the more slowly sedimenting monomer peak, than in any other chromatin fraction at all stages of digestion. Peak A and monomer particles differed in protein content and distribution. The presence of ribosomal cistrons in an altered nucleosome configuration may be related to changes in functional states of rDNA chromatin.

Chromatin↗
PubMed1978

Butyrate suppression of histone deacetylation leads to accumulation of multiacetylated forms of histones H3 and H4 and increased DNase I sensitivity of the associated DNA sequences.

Exposure of HeLa cells to Na butyrate leads to an accumulation of multiacetylated forms of histones H3 and H4. Our studies of histone acetylation in HeLa S-3 cells show that 7 mM butyrate suppresses the deacetylation of histones without influencing the rate of radioactive acetate incorporation. An alteration in nucleosome structure in highly acetylated chromatin is indicated by an increased rate of DNA degradation by DNase I. A close association of acetylated histones with the DNase I-sensitive sequences is confirmed by the finding that histones remaining after limited DNase I digestion are depleted in the multiacetylated forms of histones H3 and H4. DNase I treatment has also been found to selectively release [3H]acetyl-labeled H3 and H4 from avian erythrocyte nuclei under conditions previously shown to preferentially degrade the globlin genes in erthyrocyte chromatin. Our results are consistent with the view that histone acetylation provides a key to the mechanism for altering chromatin structure at the nucleosomal level, and that this may explain the selective DNase I sensitivity of transcriptionally active DNA sequences in different cell types.

Acetylation↗