Non-histone proteins and the structure of metaphase chromosomes

Differential intensity of fluorescence corresponding to the banding patterns found in single metaphases can be obtained with isolated Chinese hamster chromosomes using the fluorochrome Hoechst 33258. Removal of histones from the chromosomes with 0.2 N HCl causes an approx. 50% increase in overall size, but does not abolish the gross metaphase morphology of the chromosomes or the ability to give their characteristic fluorescent banding patterns. In an attempt to study further the factors maintaining the characteristic metaphase structure, we have treated acid-extracted isolated chromosomes with DNase I, which was found to solubilize over 99% of the DNA content, while leaving stable ‘core’ structures which retain the basic features of metaphase chromosomes such as centromeric regions and defined chromatids. The cores appear to consist mainly of non-histone protein: they are destroyed by proteolytic action and unaffected by ribonuclease A. The structural implications of these findings are discussed.     

Role of nonhistone chromosomal proteins in determining circular dichroism spectra of chromatin.

Complexing of histone proteins, from WI-38 cells with pure DNA from WI-38 cells, causes a marked decrease in the amplitude of the positive ellipticity band and a red shift in circular dichroism spectra in the 250–300 nm region. Total nonhistone chromosomal proteins from WI-38 cells (without histones) cause an analogous effect, but of significantly reduced magnitude. However, the two effects are not additive, because, when DNA is complexed with both histones and nonhistones, the amplitude of the positive ellipticity band has an intermediate value, between the histone-DNA complex and the nonhistone-DNA complex. Removal of certain nonhistone proteins from chromatin of WI-38 cells, by extraction with 0.25–0.35 m NaCl, causes a decrease in the positive circular dichroism band in the 250–300 nm region. Removal of histones and other nonhistone proteins from chromatin by extraction with 0.75 and 1.5 m NaCl causes a strong increase in positive ellipticity. This suggests the existence of modest but definite effects of nonhistone proteins in determining DNA conformation in native chromatin. Taken as a whole, nonhistone chromosomal proteins have a weaker but analogous effect to that of histones, while the nonhistone proteins extractable with 0.25–0.35 m NaCl have an opposite effect.     

Phosphorylation of nonhistone proteins during premature chromosome condensation in a temperature-sensitive mutant, tsBN2

In tsBN2 cells, a temperature-sensitive (ts) mutant of the BHK21 cell line, with a ts-defect in its regulatory system for chromosome condensation, antigens that react with mitotic specific mouse monoclonal antibody MPM-2 were produced when premature chromosome condensation (PCC) was induced by a temperature shift. The polypeptides of antigens recognized by MPM-2 in tsBN2 cells with PCC were identical to those of antigens in mitotic cells. These antigens appeared concomitantly with chromosome condensation, which suggests that these mitotic-specific antigens may be related to chromosome condensation. As the production of mitotic-specific antigens was inhibited by W-7, a specific and potent antagonist of calmodulin, calmodulin may function in the mitotic phosphorylation of nonhistone protein.     

Higher order metaphase chromosome structure: Evidence for metalloprotein interactions

One level of DNA organization in metaphase chromosomes is brought about by a scaffolding structure that is stabilized by metalloprotein interactions. Fast-sedimenting, histone-depleted structures (4000–7000 S), derived from metaphase chromosomes by extraction of the histones, are dissociated by metal chelators or by thiol reagents. The chromosomal (scaffolding) proteins responsible for constraining the DNA in this fast-sedimenting form are solubilized under the same conditions. Chromosomes isolated in a metal-depleted form, which generate slow-sedimenting, histone-depleted structures, can be specifically and reversibly stabilized by Cu²⁺, but not by Mn²⁺, Co²⁺, Zn²⁺ or Hg²⁺. Metal-depleted chromosomes can also be stabilized by Ca²⁺ (at 37°C), but this effect is less specific than that of Cu²⁺. The scaffolding protein pattern that is reproducibly generated following treatment with Cu²⁺ is composed primarily of two high molecular weight proteins—Sc1 and Sc2 (170,000 and 135,000 daltons). The identification of this simple protein pattern has depended upon the development of new chromosome isolation methods that are highly effective in eliminating cytoskeletal contamination.     

Conservation of interphase chromatin nonhistone antigens as components of metaphase chromosomes

The degree of conservation of HeLa interphase chromatin nonhistone antigens among the nonhistones of isolated metaphase chromosomes was determined with immunological procedures. Proteins were separated on SDS-polyacrylamide gels and electrophoretically transferred to diazophenylthioether (DPT)-paper, which was then overlaid with antiserum to chromatin from interphase nuclei. The bound antibodies were detected with 125I-labeled protein A. Alternatively, polyacrylamide gels were directly overlaid with antiserum and with 125I-protein A. Densitometry of autoradiograms and stained gels revealed the degree of conservation of nonhistone antigenic determinants from interphase to metaphase to be over 90% for chromatin.     

Nuclear nonhistone proteins in mouse teratocarcinomas

Summary The nonhistone protein pattern of four murine teratocarcinomas with different capacities for differentiation were compared: a multidifferentiated teratocarcinoma OTT2289, a nondifferentiated teratocarcinoma OTT2158, a teratocarcinoma-derived rhabdomyosarcoma TDR114, and a teratocarcinoma-derived neuroblastoma TDN2151. Their nonhistone proteins (NHP) were separated by differential salt extraction and hydroxyapatite chromatography into three fractions, NHP-I, NHP-II and NHP-III. Comparison of the NHP fractions by twodimensional gel electrophoresis in combination with a sensitive silver staining method reveals that there are several tumour line specific proteins in each NHP fraction. We suggest that specific NHP, which can be used as biochemical markers for each of the four investigated tumour lines, may be involved in cell lineage specific control of gene expression.     

Degradation of phosphorylated chromosomal nonhistone proteins

In experiments with in vivo 32P-labelled nonhistone proteins of rat liver nuclei it was shown that these components are more sensitive against degradation than the mass of the nonhistone proteins. In the presence of 0.1 mM phenylmethylsulfonylfluoride and 1 mM sodium molybdate, however, they are protected against degradation.     

Changes in nonhistone chromosomal proteins in phytohemagglutininstimulated lymphocytes

Stimulation of bovine lymphocytes with phytohemagglutinin results in quantitative as well as qualitative changes in the nonhistone chromosomal proteins. Analysis of these proteins by hydroxyapatite chromatography and sodium dodecylsulfate polyacrylamide gel electrophoresis shows not only a selective increase in the amount of some nonhistone proteins but also a decrease of other nonhistone protein bands. This observation is compatible with the view that nonhistone proteins have an inhibitory as well as an activating function at the genome level.     

Torsional tension in metaphase chromosome DNA]

The torsional tension in DNA of isolated metaphase chromosomes from murine fibroblasts was measured by the microfluorescent method. The method is based on the ability of a fluorescent dye ethidium bromide to compensate for the negative torsional tension in topologically closed DNA by intercalation between DNA base pairs. The value of the relative twist difference delta Tw/Tw = -0.1 was found in a bulk (about 3/4) of unconstrained chromosomal DNA. In interphase nuclei, the torsionally stressed DNA comprises about 15%, with value of delta Tw/Tw = -0.075. We suppose that the tension in chromosomal DNA was created in the prophase stage of mitosis by condensines, the drivers of chromosomal condensation.     

Organ discrimination through organ-specific nonhistone chromosomal proteins

Prefractionation of chromosomal proteins in 5 m urea with stepwise increase in NaCl molarity has been used to facilitate the examination of nonhistone chromosomal proteins isolated from various rabbit tissues. Electrophoretic analysis on polyacrylamide gels under denaturing conditions of the protein fractions derived from brain, liver, heart, and submandibular salivary gland chromatins displays reproducible compositional differences in nonhistone chromosomal proteins. The enzymatic removal of 48% of protein-bound phosphate with alkaline phosphatase does not significantly alter the electrophoretic mobility of these proteins. With the present technique, it is estimated that chromatin polypeptides (of average M_r 100,000) occurring in greater than 3 × 10⁴ copies per genome can be detected. At this level of sensitivity, a significant fraction of total nonhistone chromosomal proteins manifests organ specificity.     

Architecture of metaphase chromosomes and chromosome scaffolds

We have developed procedures for depositing intact mitotic chromosomes and isolated residual scaffolds on electron microscope grids at controlled and reproducible levels of compaction. The chromosomes were isolated using a recently developed aqueous method. Our study has addressed two different aspects of chromosome structure. First, we present a method for improved visualization of radial chromatin loops in undisrupted mitotic chromosomes. Second, we have visualized a nonhistone protein residual scaffold isolated from nuclease-digested chromosomes under conditions of low salt protein extraction. These scaffolds, which have an extremely simple protein composition, are the size of chromosomes, are fibrous in nature, and are found to retain differentiated regions that appear to derive from the kinetochores and the chromatid axis. When our standard preparation conditions were used, the scaffold appearance was found to be very reproducible. If the ionic conditions were varied, however, the scaffold appearance underwent dramatic changes. In the presence of millimolar concentrations of Mg++ or high concentrations of NaCl, the fibrous scaffold protein network was observed to undergo a lateral aggregation or assembly into a coarse meshlike structure. The alteration of scaffold structure was apparently reversible. This observation is consistent with a model in which the scaffolding network plays a dynamic role in chromosome condensation at mitosis.     

Architecture of the Chinese hamster metaphase chromosome

The development of procedures for the isolation of unfixed metaphase chromosomes has made feasible a direct analysis of their morphology. Wholemount stereo electron microscopy was used to examine intact and partially disrupted chromosomes produced by physical shearing and extraction with salt and urea solutions. A model of chromosome architecture was developed to accommodate evidence from studies using both light and electron microscopy. In the proposed model the chromatid (anaphase chromosome) consists of two half-chromatids; each half-chromatid contains two deoxyribonucleoprotein ribbons wound into a single fiber (termed the core), with many loops of chromatin (termed epichromatin) attached along its length. The core ribbons are each about 50 Å thick by 4000 Å wide and are composed of many parallel deoxyribonucleoprotein strands. The epichromatin loops appear to be 250 Å supercoiled fibers containing about 75 per cent of the chromosomal DNA. The epichromatin can be selectively removed from the core fibers by extraction with 2.0 M NaCl or 6.0 M urea solutions.     

Phosphorylation of nonhistone proteins during the HeLa cell cycle. Relationship to DNA synthesis and mitotic chromosome condensation

Cell cycle variations in the phosphorylation of chromatin-associated nonhistones were determined. Cells were radiolabeled with [32P]orthophosphate and chromatin was obtained by mild digestion of nuclei with micrococcal nuclease. The experiments were performed in the presence of a substrate inhibitor of alkaline phosphatase, beta-glycerophosphate. The results show that, while similar molecular weight species of phosphorylated nonhistones are associated with interphase chromatin through the HeLa cell cycle, the incorporation (32P cpm/micrograms of protein) profiles of selected major phosphononhistones show substantial changes. The most prominent peaks of specific radioactivity occur in the DNA synthesis phase (S phase). The phosphorylation states of the proteins of isolated metaphase chromosomes were also determined. Nonhistone proteins of isolated metaphase chromosomes are strikingly dephosphorylated, especially in comparison to histone H1. The phosphorylation of the major phosphononhistone of chromatin, which has a molecular weight of 55,000, was further characterized by techniques that included one-dimensional peptide mapping in sodium dodecyl sulfate-polyacrylamide gels and nonequilibrium pH gradient slab gel electrophoresis. Phosphoproteins are also components of the nuclear scaffold, and cell cycle variations in these proteins were investigated. The primary phosphorylated species has a molecular weight of 119,000. As with chromatin-associated nonhistones, this nuclear scaffold protein shows substantial incorporation of 32P in S phase, and a high level of incorporation also occurs close to mitosis.