The chemical composition of nuclei and chromosomes isolated by the Langmuir trough technique

The pattern of staining for DNA, histone, and nonhistone protein has been studied in whole cells and in nuclei and chromosomes isolated by surface spreading. In whole interphase cells from bovine kidney tissue culture, nuclear staining for DNA and histones reveals numerous small, intensely stained clumps, surrounded by more diffusely stained material. Nuclei in whole cells stained for nonhistone proteins also contain intensely stained regions surrounded by diffuse stain. These intensely stained regions also stain for RNA, indicating that the regions contain nucleolar material. Electron microscopy of kidney cells confirms that multiple nucleoli are present. Kidney nuclei isolated by surface spreading show an even distribution of stain for DNA, histones, and nonhistone proteins, indicating that the surface forces disperse both condensed chromatin and nucleoli. DNA and protein staining was also studied in metaphase chromosomes from testes of the milkweed bug, Oncopeltus fasciatus. Staining for DNA and histones in metaphase chromosomes is essentially the same in sections of fixed and embedded testes as in preparations isolated by surface spreading. However, striking differences are noted in the distribution of nonhistone proteins. In sections, nonhistone stain is concentrated in extrachromosomal areas; metaphase chromosomes do not stain for nonhistone proteins. Chromosomes isolated by surface spreading, however, stain intensely for nonhistone proteins. This suggests that nonhistone proteins are bound to the chromosomes as a contaminant during the isolation procedure. The relationship of these findings to current work with chromosomes isolated for electron microscopy is discussed.     

A cytochemical study of the nonhistone protein content of condensed and extended chromatin

Cytochemical techniques have been used to study the distribution of nonhistone proteins in sections of interphase nuclei and mitotic chromosomes. Condensed chromatin, including the heterochromatin of interphase nuclei from frog liver, and mitotic metaphase and anaphase chromosomes from bovine kidney, show little or no staining for nonhistone protein. Regions of frog liver nuclei which contain extended chromatin (euchromatin) stain intensely for nonhistone protein. These differences in nonhistone staining of condensed and extended chromatin support the suggestion that regions of condensed chromatin contain considerably less nonhistone protein than regions of extended chromatin. The results suggest further that there may be considerably less nonhistone protein associated with chromosomes and interphase heterochromatin than has been reported in most previous analyses of isolated chromatin and chromosome preparations.     

Role of nonhistone proteins in metaphase chromosome structure

In this paper, we show that HeLa metaphase chromosomes still possess a highly organized structure retaining the familiar metaphase morphology following removal of virtually all the histones and most of the nonhistone proteins. The structure is stabilized by a relatively small number of nonhistones, which we call scaffolding proteins.These results are based on a method which allows the removal of the histones, and most of the nonhistone proteins, by competition with polyanions such as dextran sulfate and heparin.The histone-depleted chromosomes sediment in sucrose gradients as a broad peak between 4000 to 7000S. These structures are dissociated by mild trypsin or chymotrypsin treatment, or by 4 M urea, but are stable in 2 M NaCl and insensitive to treatment with RNAase A. The histone-depleted chromosomes have a DNA to protein ratio of about 6:1; gel electrophoresis reveals the presence of about 30 nonhistone proteins and the virtual absence of histones. These experiments suggest that nonhistone proteins exist in metaphase chromosomes which maintain the DNA chain in a highly folded conformation.Structural studies support this conclusion. Analysis by fluorescence microscopy of histone-depleted chromosomes stained with ethidium bromide shows that each chromatid is still paired with its sister chromatid, and consists of a central structure surrounded by a halo of DNA. The length of the central structure in each chromatid is about 2–3 times longer than the chromatid length in the original chromosome.     

The structure of histone-depleted metaphase chromosomes

We have previously shown that histone-depleted metaphase chromosomes can be isolated by treating purified HeLa chromosomes with dextran sulfate and heparin (Adolph, Cheng and Laemmli, 1977a). The chromosomes form fast-sedimenting complexes which are held together by a few nonhistone proteins.In this paper, we have studied the histone-depleted chromosomes in the electron microscope. Our results show that: the histone-depleted chromosomes consist of a scaffold or core, which has the shape characteristic of a metaphase chromosome, surrounded by a halo of DNA; the halo consists of many loops of DNA, each anchored in the scaffold at its base; most of the DNA exists in loops at least 10–30 μm long (30–90 kilobases).We also show that the same results can be obtained when the histones are removed from the chromosomes with 2 M NaCl instead of dextran sulfate. Moreover, the histone-depleted chromosomes are extraordinarily stable in 2 M NaCI, providing further evidence that they are held together by nonhistone proteins.These results suggest a scaffolding model for metaphase chromosome structure in which a backbone of nonhistone proteins is responsible for the basic shape of metaphase chromosomes, and the scaffold organizes the DNA into loops along its length.     

Immunofluorescent staining of human metaphase chromosomes with monoclonal antibody to histone H2B

A mouse monoclonal IgM antibody against the core histone H2B has been shown, by indirect immunofluorescence, to stain metaphase chromosomes from a variety of cultured cell types. Experiments carried out with human HeLa cells showed that the intensity of staining varied along the length of chromosome arms giving in some cases a rudimentary banded staining pattern. Considerable variation in staining intensity was noted between individual chromosomes and between different metaphase spreads. It was noted that chromosomes having a more swollen appearance stained more intensely than those with a more compact structure, which were often unstained. Preincubation of unfixed metaphase chromosomes in buffered salt solutions virtually eliminated the cell to cell and chromosome to chromosome variation in staining, even when no visible effect on chromosome morphology was caused by such treatment. It is concluded that the determinant recognised by antibody HBC-7 is ubiquitous but is inaccessible in some chromosomes or chromosome regions. Digestion of purified chromatin (primarily interphase) with DNAase 1 or micrococcal nuclease resulted in a several-fold increase in the binding of antibody HBC-7 measured by solid-phase radioimmunoassay. This increase was abolished by subsequent treatment with trypsin, which suggests that the antigenic determinant recognised by antibody HBC-7 lies in the trypsin-sensitive N-terminal region of nucleosomal H2B. As the cationic N-terminal regions of the core histones are involved in DNA binding, it is likely that the accessibility of the determinant recognised by antibody HBC-7 is influenced by the relationship between the core histones and their associated DNA.     

A search for protein cores in chromosomes: Is the scaffold an artifact?

A protein chromosome scaffold structure has been proposed that acts as a structural framework for attachment of chromosomal DNA. There are several troubling aspects of this concept: (1) such structures have not been seen in many previous thin-section and whole-mount electron microscopy studies of metaphase chromosomes, while they are readily seen in leptotene and zygotene chromosomes; (2) such a structure poses problems for sister chromatid exchanges; and (3) the published photographs show a marked variation in the amount of scaffold in different whole-mount preparations. An alternative explanation is that the scaffold in whole-mount preparations represents incomplete dispersion of the high concentration of chromatin in the center of chromosomes, and when the histones are removed and the DNA dispersed, the remaining nonhistone proteins (NHPs) aggregate to form a chromosome-shaped structure. Two studies were done to determine if the scaffold is real or an artifact: (1) Chinese hamster mitotic cells and isolated chromosomes were examined using two protein stains -EDTA-regressive staining and phosphotungstic acid (PTA) stain. The EDTA-regressive stain showed ribonucleoprotein particles at the periphery of the chromosomes but nothing at the center of the chromosomes. The PTA stain showed the kinetochore plates but no central structures; and (2) isolated chromosomes were partially dispersed to decrease the high concentration of chromatin in the center of the chromosome, then treated with 4 M ammonium acetate or 2 M NaCl to dehistonize them and disperse the DNA. Under these circumstances, no chromosome scaffold was seen. We conclude that the scaffold structure is an artifact resulting from incomplete dispersion of central chromatin and aggregation of NHPs in dehistonized chromosomes.     

Monoclonal antibodies specific for tight-binding human chromatin antigens reveal structural rearrangements within the nucleus during the cell cycle

The class of nonhistone chromosomal proteins that remains bound to DNA in chromatin in the presence of 2.5 M NaCl-5 M urea has proven refractile to biochemical analysis. In order to study its role in chromatin organization, we have produced monoclonal antibodies that are specific for the HeLa DNA-protein complex that remains after extraction of chromatin with high salt and urea. The antibody-producing clones were identified with an ELISA assay. Of the six clones selected, five were stabilized by limiting dilution. All clones are IgG producers. None cross-react significantly with native DNA, core histones, or the high-mobility group nonhistone proteins. All antibodies are specific for nuclear or juxtanuclear antigens. Indirect immunofluorescence shows that three antibodies, which are nonidentical, stain three different nuclear networks. Available evidence indicates that two of these networks are the nuclear matrix. A fourth antibody reveals structures reminiscent of chromocenters. A fifth antibody, AhNA-1, binds to interphase HeLa chromatin and specifically decorates metaphase chromosomes. AhNA-1 similarly recognizes rat chromosomes. Each of these monoclonal antibodies also reveals a changing pattern of nuclear staining as cells progress through the cell cycle. Presumably, this reflects the rearrangement of the cognate antigens.     

ADP-ribosylation of metaphase and interphase nonhistones using [3H]adenosine as a radioactive label

ADP-ribosylation of HeLa nonhistone proteins was investigated by using [3H]adenosine as an in vivo radioactive label. The aim was to determine basic differences in the patterns of modification of interphase and metaphase nonhistones. Fluorography revealed a relatively small number of modified proteins for isolated metaphase chromosomes. In addition to the core histones, a protein of 116 kDa, which is identified as poly-(ADP-ribose) polymerase, was a primary acceptor of [3H]adenosine. Two-dimensional gels revealed a profound difference in the modification of metaphase and interphase nonhistones. For interphase nuclei, 3H label was distributed among a large number of nonhistone acceptors.     

The three-dimensional ultrastructure of interphasic and metaphasic nucleolar argyrophilic components studied with high-voltage electron microscopy in thick sections

The three-dimensional structure of the nucleolar argyrophilic components was studied by recording stereo-pairs of tilted thick sections--0.5-2 microns thick--observed with 200 and 300 kV high-voltage electron microscopy (HVEM). Using a very specific silver staining method, the argyrophilic components were stained with a high contrast relatively to the unstained background, thus allowing their study with a high resolution within thick sections. This study was performed on compact nucleoli (of HL60 and K562 cells), on reticulated nucleoli (of human breast cancerous cells) and on metaphasic nucleolar organizer regions (NORs). In compact nucleoli argyrophilic components show a 'knotted rope-like' structure in which knots are constituted of one central fibrillar centre surrounded at some distance by loops of the dense fibrillar component and in which the rope is constituted of dense fibrillar component. In reticulated nucleoli silver deposits are confined to the surface of the nucleolonema as several strands twisted at the periphery of the fibrillar component. During metaphase some NORs get a characteristic crescent-shaped structure disposed at the periphery of some chromosomes.     

In situ factors affecting stability of the DNA helix in interphase nuclei and metaphase chromosomes

The data from earlier cytochemical studies, in which the metachromatic fluorochrome acridine orange (AO) was used to differentially stain single vs double-stranded DNA, suggested that DNA in situ in intact metaphase chromosomes or in condensed chromatin of G0 cells is more sensitive to denaturation, induced by heat or acid, than DNA in decondensed chromatin of interphase nuclei. Present studies show that, indeed, DNA in permeabilized metaphase cells, in contrast to cells in interphase, when exposed to buffers of low pH (1.5-2.8) becomes digestible with the single-strand-specific S1 or mung bean nucleases. A variety of extraction procedures and enzymatic treatments provided evidence that the presence of histones, HMG proteins, and S-S bonds in chromatin, as well as phosphorylation or poly(ADP)ribosylation of chromatin proteins, can be excluded as a factor responsible for the differential sensitivity of metaphase vs interphase DNA to denaturation. Cell treatment with NaCl at a concentration of 1.2 N and above abolished the difference between interphase and mitotic cells, rendering DNA in mitotic cells less sensitive to denaturation; such treatment also resulted in decondensation of chromatin visible by microscopy. The present data indicate that structural proteins extractable with greater than or equal to 1.2 N NaCl may be involved in anchoring DNA to the nuclear matrix or chromosome scaffold and may be responsible for maintaining a high degree of chromatin compaction in situ, such as that observed in metaphase chromosomes or in G0 cells. Following dissociation of histones, the high spatial density of the charged DNA polymer may induce topological strain on the double helix, thus decreasing its local stability; this can be detected by metachromatic staining of DNA with AO or digestion with single-strand-specific nucleases.     

Cytochemical studies of metaphase chromosomes by flow cytometry

The cytochemical properties of metaphase chromosomes from Chinese hamster and human cells were studied by flow cytometry. This technique allows precise quantitation of the fluorescence properties of individual stained chromosome types. Chromosomes were stained with the following fluorescent DNA stains: Hoechst 33258, DAPI, chromomycin A₃, ethidium bromide, and propidium iodide. The relative fluorescence of individual chromosome types varied depending on the stain used, demonstrating that individual chromosome types differ in chemical properties. Flow measurements were performed as a function of stain and chromosome concentration to characterize the number and distribution of stain binding sites. Flow analysis of double stained chromosomes show that bound stains interact by energy transfer with little or no binding competition. For most hamster chromosomes, there is a strong correlation between relative fluorescence and stain base preference suggesting that staining differences may be determined primarily by differences in average base composition. A few hamster chromosome types exhibit anomalous staining which suggests that some other property, such as repetitive DNA sequences, also may be an important determinant of chromosomal staining.     

Anti-NOR 90. A new autoantibody in scleroderma that recognizes a 90-kDa component of the nucleolus-organizing region of chromatin

We identified a patient (CAG) with scleroderma whose serum contained a high titer of IgG class antibodies that stained nucleoli in a pattern of independent tiny spots. When tested on isolated chromosomes, these antibodies selectively stained the nucleolus-organizing regions (NOR) of chromosomes 13, 14, 15, 21, and 22. These staining patterns were not altered when substrate cells and chromosomes were treated with RNase, 0.1 M HC1, or 4 M urea, but they were abolished by treatment with DNase and trypsin. Immunoblots performed with serum CAG on isolated nucleolar substrates identified a protein antigen of approximately 90 kDa. Antibodies affinity-purified from this protein selectively stained nucleoli and NOR chromosomal regions. Therefore, this protein is the antigen that accounts for the ability of serum CAG to recognize the NOR. In a search for the NOR 90-kDa specificity among 254 patients with various rheumatic diseases, we found nine additional patients whose sera stained metaphase chromosomes selectively at the NOR. Sera from five of them (three with scleroderma, two of unknown diagnosis) recognized a protein that electrophoretically co-migrated with the CAG antigen. Thus, scleroderma is present in at least four of six who appear to have this specificity. We conclude that autoantibodies to the NOR 90-kDa antigen have an association with scleroderma and may be useful diagnostically and as a probe for further studies of the biology of the cell nucleolus.     

Some remarks on the use of TaqI to detect highly repetitive DNA sequences in human chromosomes

In the attempt to conclude investigation of the action of restriction endonucleases on eukaryote chromosomes, we carried out a series of experiments digesting in situ human metaphase chromosomes with AluI/TaqI followed by Giemsa staining. We focused on the centromeric regions of chromosomes1, 2 and 16 and noted that those areas appeared as intensely stained blocks after AluI digestion, but were dramatically reduced in size or completely destroyed after subsequent TaqI treatment. These results permitted us to draw some conclusions on the highly repetitive DNA composition of these regions, in terms of alphoid and classical satellite DNAs.     

The Feulgen banded karyotype of the mouse: analysis of the mechanisms of banding

The chromosomes of the mouse have been identified by specific banding patterns revealed by the Feulgen stain. Comparison of the patterns of the Feulgen-stained karyotype with those of acetic-saline-Giemsa stain and quinacrinemustard-fluorescence demonstrates a high order of similarity among the three, with the localization of Feulgen dense bands and regions closely paralleling that of Giemsa dark and fluorescence bright bands. Since the stained substrate of the Feulgen reaction is known to be DNA, it is suggested that all three banding methods reveal the distribution of DNA or of some moiety that closely follows DNA distribution in metaphase chromosomes. The preparative procedure of the Feulgen banding method consists of a 15 to 20 minute exposure to PO₄ buffer at pH 10 and a prolonged (60–72 hrs) exposure to 12xSSC. Omission or curtailment of either step results in preparations with chromosome sets that are not karyotypable, although some stain differentiation is produced. HCl extraction prior to the preparative treatment blocks banding, but acid extraction following the preparative treatment, either that of the HCl hydrolysis of the Feulgen reaction of that of an almost fourfold extension of the standard hydrolysis time, does not obliterate bands already formed. By extrapolation from biochemical studies of chromatin, it is postulated that the localization of Feulgen dark and light stain, representing relative DNA densities, reflects the regional protein association of the DNA; the Feulgen dense regions may result from aggregation of a specific class of histones by the alkaline buffer with consequent condensation of the DNA bound to those histones; the Feulgen pale or negative regions may represent those in which non-aggregated proteins, histone and non-histone, have been solubilized in the saline incubation, rendering the DNA of those regions subject to diffusion or vulnerable to fragmentation in the Feulgen hydrolysis.     

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.     

An investigation of the mechanism of the reciprocal differential staining of BUdR-substituted and unsubstituted chromosome regions.

After treatment with hot NaH₂PO₄ at pH 9, BUdR-substituted and unsubstituted chromosome regions are palely and intensely stained with Giemsa, respectively; however, after treatment with the same solution at pH 4, the reciprocal staining patterns are produced, i.e. these chromosome regions are intensely and palely stained, respectively. The nature of the mechanisms responsible for this reciprocal differential Giemsa staining of BUdR-substituted and unsubstituted chromosome regions has been investigated by Feulgen staining, electron microscopy, and radioisotope analyses involving scintillation counting and autoradiography. The results indicate that different mechanisms are responsible for the two types of staining effect. The high pH NaH₂PO₄ treatment preferentially extracts BUdR-substituted DNA into the treatment solution, relative to unsubstituted DNA. The collective evidence from this and other work suggests that BUdR-substituted DNA in the chromosomes is partially photolysed by exposure to daylight during the harvesting procedure, and the degraded DNA is subsequently solubilized and extracted during the high pH treatment. This quantitative reduction of DNA in the BUdR-substituted chromosome regions results in pale Giemsa staining of these regions. The low pH NaH₂PO₄ treatment does not produce a significant extraction of either BUdR-substituted or unsubstituted DNA into the treatment solution; rather, there may be a redistribution of the unsubstituted DNA relative to the BUdR-substituted DNA such that the unsubstituted DNA is preferentially dispersed outside the boundaries of the chromosomes onto the surrounding area of the slide. It is suggested that the BUdR-substituted chromosome regions stain relatively intensely with Giemsa after the low pH treatment because the DNA in these regions is less dispersed than that in the unsubstituted regions.     

Relationship between secondary constrictions and nucleolus organizing regions inAllium sativum chromosomes

Summary Allium sativum L. (2 n=16) had three types of clones with regard to the number of chromosomes carrying well-defined secondary constrictions: the first type had two secondary constricted chromosomes (type I), the second had three (type II) and the third had four (type III). Silver staining was applied to these three types of cells to determine the number of nucleolus organizing regions (NORs) per cell and to study the relationship between the morphological appearance of the secondary constrictions and the ability of the chromosomes to form nucleoli. Ag-positive regions appeared on two chromosomes in type I, on three in type II and on four in type III. The comparison of Giemsa and Feulgen stained chromosomes with the silver stained ones clearly indicated that the positive reaction with silver occurred exclusively on the secondary constricted regions that responded negatively to both Giemsa and Feulgen staining, indicating that the size of the achromatic secondary constrictions directly reflects the volume of the Ag-positive materials. However, all three types of clones had a maximum of four nucleoli at interphase. Of the four nucleoli, either two or one was extremely small (less than 1 m in diameter) in types I and II respectively. The size variations of the other nucleoli seemed to be positively correlated with those of the Ag-positive regions. This and the observation that the maximum number of nucleoli per cell did not coincide with the number of Ag-positive regions on the metaphase chromosome complement suggest strongly that the NORs responsible for the minute nucleoli cannot be detected on the metaphase chromosomes. The present observations indicate that not all NORs are indicated by the morphological appearance of secondary constrictions.     

Specific silver staining of experimentally undercondensed chromosome regions

Treatment of human and mouse cell cultures with DNA binding AT-specific compounds and with some base analogues induced distinct undercondensations in several heterochromatic chromosome regions. All those heterochromatic regions undercondensed by AT-specific DNA ligands (distamycin A, DAPI, Hoechst 33258) could be heavily labeled with the silver(Ag)-staining technique; but the heterochromatic regions undercondensed with the cytidine analogue 5-azacytidine were Ag-negative. In metaphase chromosomes from BrdU-treated human cell cultures, the bifilarly substituted chromatids, which show a slight undercondensation, were also Ag-negative. Cytochemical analyses of the Ag-stained undercondensed heterochromatic regions showed that the Ag-stainable material consisted of nonhistone proteins. The mechanism of Ag staining in the undercondensed heterochromatic regions was compared with Ag staining of the nucleolus organizer regions.     

High-mobility group protein HMG-I localizes to G/Q- and C-bands of human and mouse chromosomes

Mammalian metaphase chromosomes can be identified by their characteristic banding pattern when stained with Giemsa dye after brief proteolytic digestion. The resulting G-bands are known to contain regions of DNA enriched in A/T residues and to be the principal location for the L1 (or Kpn 1) family of long interspersed repetitive sequences in human chromosomes. Here we report that antibodies raised against a highly purified and biochemically well characterized nonhistone "High-Mobility Group" protein, HMG-I, specifically localize this protein to the G-bands in mammalian metaphase chromosomes. In some preparations in which chromosomes are highly condensed, HMG-I appears to be located at the centromere and/or telomere regions of mammalian chromosomes as well. To our knowledge, this is the first well-characterized mammalian protein that localizes primarily to G-band regions of chromosomes.