Vincent G. Allfrey

Processing of newly synthesized histone molecules

deoxyribonucleic acid of the somatic cells of higher organisms occurs in association with small basic proteins called histones. In most cell types histones can be grouped into five major classes differing in size, positive charge, and amino acid composition. The primary structures of homologous histones from widely divergent species give evidence of remarkable evolutionary stability, particularly the arginine-rich histones, H3 and H4. Histones H2a and H2b (1) show slightly less evolutionary stability in their amino acid sequences, while histone H 1 varies to the greatest extent (la). The structural function of histones is to organize the long, fibrillar molecules of DNA into a more compact form. This organization is achieved, in part, by electrostatic interactions between the positively charged basic amino acid residues in the histone polypeptide chains and the negatively charged phosphate groups of DNA. Current evidence favors a cooperative interaction in which four of the five major types of histones interact with each other in specific ways and in stoichiometric proportions to form multimeric protein complexes (2). The fundamental unit of eukaryotic chromatin may be visualized as small nucleoprotein particles (3-5) [called nu bodies (4) or PS-particles (5)] along the DNA molecule, in which each histone complex is enveloped by the DNA strand (6). In the assembly of such particles and in their attachment to DNA, it is likely that particular regions of the histone polypeptide chains have different functions. Histone structures usually reveal a characteristic clustering of their basic amino acidsarginine, lysine, and histidine-thus generating regions of high positive charge (Fig. 1). This positively charged region is most

Phosphorylation of Nuclear Protein Early in the Course of Gene Activation in Lymphocytes

Human lymphocytes treated with phytohemagglutinin undergo extensive gene activation, as evidenced by augmented synthesis of ribonucleic acids. This activation is preceded by an early stimulation in the rate of phosphorylation and dephosphorylation of nuclear proteins. This finding is consistent with a hypothesized role of phosphoproteins in the modification of chromatin structure and in modulation of the template activity of DNA in vivo.

Reversible Changes in Nucleosome Structure and Histone H3 Accessibility in Transcriptionally Active and Inactive States of rDNA Chromatin

The sulfhydryl reagent iodoacetamidofluorescein (IAF) was used to probe the structure of chromatin subunits in transcribed and nontranscribed regions of Physarum rDNA. IAF labels histone H3 -SH groups in the elongated monomeric subunits (A particles) from the transcribed region, but it does not label H3 in the 11S monomers from the nontranscribed central spacer. All H3 reactivity is lost from rDNA chromatin in the inactive spherule stage of Physarum. Restriction cleavage of rDNA chromatin generates fragments from the transcription unit with reactive H3 -SH groups, whereas fragments containing nontranscribed spacer sequences are unreactive. The extended rDNA chromatin contains all four core histones and other prominent proteins. Electron microscopy shows that most of the extended subunits consist of two roughly spherical bodies connected by a 50 bp nucleoprotein bridge.

Affinity chromatographic purification of nucleosomes containing transcriptionally active DNA sequences

The unfolding of nucleosome cores in transcriptionally active chromatin uncovers the sulfhydryl groups of histone H3, making them accessible to SH-reagents. This has suggested that nucleosomes from active genes could be retained selectively on organomercurial/agarose columns. When nucleosomes released from rat liver nuclei by limited digestion with micrococcal nuclease were passed through an Hg affinity column, a run-off fraction of compact, beaded nucleosomes was separated from a retained nucleosome fraction. Although both contained monomer-length DNA and a full complement of core histones, histones in the retained fraction were hyperacetylated. Dot blot hybridizations showed the Hg-bound nucleosome fraction to be enriched in DNA sequences transcribed by hepatocytes (serum albumin and transferrin genes), while a brain-specific gene (preproenkephalin) was not retained, but appeared in the nucleosomes of the run-off fraction. The results are discussed in light of other evidence linking hyperacetylation of histones H3 and H4 to conformational changes at the middle of the nucleosome core.

Selective release of chromosomal proteins during limited DNAase 1 digestion of avian erythrocyte chromatin

Duck erythrocyte chromatin has been treated with DNAase 1 under conditions that are known to digest selectively the structural genes coding for globin mRNAs. This limited digestion releases specific sets of nonhistone chromosomal proteins that are not preferentially released during limited digestion with micrococcal nuclease, which does not selectively attack the globin sequences. Analysis of nucleosome monomer and multimer peaks separated on sucrose gradients after limited digestion with micrococcal nuclease shows that the proteins which are released by DNAase 1 digestion remain associated with the chromatin subunits and can be removed by extraction in 0.5 M NaCl. These proteins are tentatively identified as members of the high mobility group (HMG) proteins (originally described by Goodwin, Sanders and Johns, 1973) in terms of their extractability, electrophoretic characteristics and amino acid composition.

Distribution of NG, NG-dimethylarginine in nuclear protein fractions

Abstract Nuclear proteins have been fractionated into five distinct classes according to their extractability from rat liver nuclei at different pH and salt concentrations. The fractions have been analyzed for their amino acid composition which shows the presence of N G , N G -dimethylarginine, in sizable amount, in non-histone nuclear proteins (NHNP). This modification is most prominent in proteins which are found associated with rapidly-labeled heterogeneous RNA (HnRNP proteins).

Separation and analysis of histone subfractions differing in their degree of acetylation: some correlations with genetic activity in development.

Abstract Histone fractions F2A1 and F3 are modified in vivo by an acetylation of lysyl residues at specific sites in the polypeptide chain. The acetylation takes place after histone synthesis is completed; it is reversible, and it involves more than one site of substitution. The net result is a population of F2A1 (or F3) molecules which differ in the number of N ϵ -acetyllysyl residues they contain. Methods are described for the separation of various acetylated and nonacetylated forms of histone F2A1 using column Chromatographic and gel electrophoretic techniques. The electrophoretic heterogeneity of fraction F3 is also explored. The methods have been applied to a study of the relative proportions and isotopic acetyl content of the histone subtractions during development and differentiation. It has been found that fraction F2A1 is not acetylated in sperm cells of Arbacia lixula —cells which are not capable of RNA synthesis—while it exists in several acetylated forms in the Arbacia embryo at a time of extensive gene activation. The amino acid compositions of isolated subfractions of calf thymus histone F2A1 indicates that they are identical except in their degree of acetylation, and they do not show significant differences in their contents of mono- and dimethyllysine.

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.

Species-specific interactions between nuclear phosphoproteins and DNA.

Abstract A class of nuclear acidic proteins, associated with DNA and present in the chromatin of different cell types, has been isolated and resolved into separate components by electrophoresis in polyacrylamide gels. The electrophoretic patterns indicate that the fraction is highly heterogeneous and that the distribution of chromosomal proteins is tissue — specific. Many proteins in rat liver and kidney chromatin are phosphoproteins which rapidly incorporate 32 P-orthophosphate into phosphoserine and phosphothreonine residues. DNA-protein binding studies show that many nuclear phosphoproteins bind selectively to the DNA of the species of origin (rat). Binding to the DNA of a closely-related mammal (mouse) is also observed, but to a lesser extent. No binding is observed when rat phosphoproteins are mixed with the DNAs of widely-divergent species, such as calf thymus or pneumococcal DNAs.

In vitro incorporation of amino acids into the proteins of isolated nuclear ribosomes.

Abstract Ribonucleoprotein particles of diverse composition and metabolic activity can be extracted from the isolated thymus cell nucleus. If properly supplemented these ribosomes remain capable of active amino acid incorporation into their constituent proteins. This process requires adenosine triphosphate, amino acid activating enzymes, and guanosine triphosphate. Thymus nuclear ribosomes are irreversibly inactivated by brief exposure to hypertonic sucrose solutions, either before or after extraction from the nucleus. While in the nucleus they are resistant to ribonuclease digestion, but after their isolation, ribonuclease attacks the particles and destroys their activity. The addition of deoxyribonucleic acid of diverse sources stimulates amino acid incorporation by isolated nuclear ribosomes, but other polyanions may be inhibitory. This role of deoxyribonucleic acid and the influence of position within the cell nucleus upon the metabolism of nuclear ribosomes is discussed.