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.     

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.     

Nonhistone scaffold of mitotic chromosomes in situ

Based on the example of giant nuclei of Chironomus plumosus, we have shown (Makarov, Chentsov, 2010) that it is possible to identify the body of the polytene chromosome after its treatment in situ with 2 M NaCl and DNase in the presence of 2 mM CuCl₂. The Cu₂₊ ions stabilize bonds between the nonhistone components that form a nonhistone scaffold that repeats the general morphology and disc pattern of polytene chromosome. The goal of this work was to repeat the procedure for revealing the stabilized nonhistone scaffold in situ in the usual mitotic chromosomes of cells of the SPEV culture. After the extraction of all histones and DNA in the presence of 2 mM CuCl₂, we managed to reveal the residual body of mitotic chromosome (its nonhistone scaffold) at all stages of mitosis both in incident light and after using antibodies to proteins of topoisomerase IIα and SMC 1. Topoisomerase IIα and SMC 1 are distributed uniformly throughout the chromosome body and do not form any axial structures.     

Chromosome length and DNA loop size during early embryonic development of Xenopus laevis

The looped organization of the eukaryotic genome mediated by a skeletal framework of non-histone proteins is conserved throughout the cell cycle. The radial loop/scaffold model envisages that the higher order architecture of metaphase chromosomes relies on an axial structure around which looped DNA domains are radially arranged through stable attachment sites. In this light we investigated the relationship between the looped organization and overall morphology of chromosomes. In developing Xenopus laevis embryos at gastrulation, the bulk of the loops associated with histone-depleted nuclei exhibit a significant size increase, as visualized by fluorescence microscopy of the fully extended DNA halo surrounding high salt treated, ethidium bromide stained nuclei. This implies a reduction in the number of looped domains anchored to the supporting nucleoskeletal structure. The cytological analysis of metaphase plates from acetic acid fixed whole embryos, carried out in the absence of drugs inducing chromosome condensation, reveals a progressive thickening and shortening of metaphase chromosomes during development. We interpret these findings as a strong indication that the size and number of DNA loops influence the thickness and length of the chromosomes, respectively. The quantitative analysis of chromosome length distributions at different developmental stages suggests that the shortening is timed differently in different embryonic cells.     

A two-step scaffolding model for mitotic chromosome assembly

Topoisomerase IIalpha (topoIIalpha) and 13S condensin are both required for mitotic chromosome assembly. Here we show that they constitute the two main components of the chromosomal scaffold on histone-depleted chromosomes. The structural stability and chromosomal shape of the scaffolding toward harsh extraction procedures are shown to be mediated by ATP or its nonhydrolyzable analogs, but not ADP. TopoIIalpha and 13S condensin components immunolocalize to a radially restricted, longitudinal scaffolding in native-like chromosomes. Double staining for topoIIalpha and condensin generates a barber pole appearance of the scaffolding, where topoIIalpha- and condensin-enriched "beads" alternate; this structure appears to be generated by two juxtaposed, or coiled, chains. Cell cycle studies establish that 13S condensin appears not to be involved in the assembly of prophase chromatids; they lack this complex but contain a topoIIalpha-defined (-mediated?) scaffolding. Condensin associates only during the pro- to metaphase transition. This two-step assembly process is proposed to generate the barber pole appearance of the native-like scaffolding.     

The Middle Region of an HP1-binding Protein, HP1-BP74, Associates with Linker DNA at the Entry/Exit Site of Nucleosomal DNA

In higher eukaryotic cells, DNA molecules are present as chromatin fibers, complexes of DNA with various types of proteins; chromatin fibers are highly condensed in metaphase chromosomes during mitosis. Although the formation of the metaphase chromosome structure is essential for the equal segregation of replicated chromosomal DNA into the daughter cells, the mechanism involved in the organization of metaphase chromosomes is poorly understood. To identify proteins involved in the formation and/or maintenance of metaphase chromosomes, we examined proteins that dissociated from isolated human metaphase chromosomes by 0.4 m NaCl treatment; this treatment led to significant chromosome decondensation, but the structure retained the core histones. One of the proteins identified, HP1-BP74 (heterochromatin protein 1-binding protein 74), composed of 553 amino acid residues, was further characterized. HP1-BP74 middle region (BP74Md), composed of 178 amino acid residues (Lys⁹⁷–Lys²⁷⁴), formed a chromatosome-like structure with reconstituted mononucleosomes and protected the linker DNA from micrococcal nuclease digestion by ∼25 bp. The solution structure determined by NMR revealed that the globular domain (Met¹⁵³–Thr²³⁷) located within BP74Md possesses a structure similar to that of the globular domain of linker histones, which underlies its nucleosome binding properties. Moreover, we confirmed that BP74Md and full-length HP1-BP74 directly binds to HP1 (heterochromatin protein 1) and identified the exact sites responsible for this interaction. Thus, we discovered that HP1-BP74 directly binds to HP1, and its middle region associates with linker DNA at the entry/exit site of nucleosomal DNA in vitro.     

Interaction of metal ions with polynucleotides and related compounds. XXII. Effect of divalent metal ions on the conformational changes of polyribonucleotides

Changes in the conformation of poly(G), poly(C), poly(U), and poly(I) in the presence of divalent metal ions Mg²⁺, Ca²⁺, Mn²⁺, Co²⁺, Ni²⁺, Cu²⁺, Cd²⁺, and Zn²⁺ have been measured by means of ORD and u.v. spectra. Mg²⁺ and Ca²⁺ ions stabilize helical structures of all the polynucleotides very effectively at concentrations several orders of magnitude lower than the effective concentration of Na⁺ion. Cu²⁺ and Cd²⁺ destabilize the helical structure of polynucleotides to form random coils. Zn²⁺, Ni²⁺, Co²⁺, and Mn²⁺ions do not behave in such a clear-cut manner: they selectively stabilize some ordered structures, while destabilizing others, depending on the ligand strength of the nucleotide base as well as the preferred conformation of that polynucleotide.     

Purification of Oat Debranching Enzyme and Occurrence of Inactive Debranching Enzyme in Cereals

The interaction of some anthracycline antibiotics (adriamycin, daunomycin, aclacinomycin-A) with bacteriophage ?X174 was investigated. Adriamycin and daunomycin inactivated the infectivity of both free ?X174 phage and naked single-stranded ?X174 DNA without DNA strand scission, but aclacinomycin-A did not show this action. The phage inactivation reaction was reversibly inhibited by Superoxide dismutase, catalase or other oxygen radical scavengers. The inactivation of ?X174 by adriamycin and aclacinomycin-A was stimulated by the addition of Cu²⁺, while the ?X174 inactivation by daunomycin was inhibited by the addition of Cu²⁺. The ?X174 inactivation by adriamycin and aclacinomycin-A in the presence of Cu²⁺ was caused by degradation of DNA, and this inactivation reaction was inhibited irreversibly by oxygen radical scavengers. These results indicate that anthracycline antibiotics bind to ?X174 DNA in the form of free radicals and that during the auto-oxidation of these antibiotics in the presence of Cu²⁺, oxygen radicals were generated to cause the degradation of ?X174 DNA.     

径流雨水中溶解性有机质特征演化及其对典型污染物迁移和生物有效性的影响

地表径流污染已经逐渐成为城市面源污染的重要组成部分。其中溶解性有机质DOM (Dissolved organic matter)是有机污染物的主要组成部分。DOM中因为含有大量不饱和结构、官能团,其中包括羟基、羧基、羰基、胺基等,这些结构容易与径流中重金属结合发生络合反应,改变重金属的赋存形态,从而对其迁移转化及其生物有效性产生很大的影响。本文以北京市地表径流为研究对象,研究城市地表径流在冬、夏季不同功能区不同下垫面中溶解性有机质特征及其与典型重金属的作用机制,通过荧光淬灭滴定实验,研究夏季径流雨水中DOM的不同组分与重金属Cu~(2+)、Pb~(2+)、Zn~(2+)之间的结合机制。研究结果显示PARAFAC将获得的样品都分解成2类6个不同的组分,1种腐殖酸,1种类蛋白;类蛋白物质的荧光强度与Cu~(2+)、Pb~(2+)的淬灭率要强于腐殖酸,而Zn~(2+)则呈现相反趋势;通过使用二维相关同步光谱发现DOM对重金属Cu~(2+)、Pb~(2+)、Zn~(2+)的敏感性呈现递减趋势,二维相关异步光谱发现Cu~(2+)、Pb~(2+)会先与位于270—300nm附近类蛋白光谱带反应,Zn~(2+)则会先与330nm附近的腐殖酸光谱带反应。     

Heavy metal ions affect the activity of DNA glycosylases of the Fpg family

Prokaryotic enzymes formamidopyrimidine-DNA glycosylase (Fpg) and endonuclease VIII (Nei) and their eukaryotic homologs NEIL1, NEIL2, and NEIL3 define the Fpg family of DNA glycosylases, which initiate the process of repair of oxidized DNA bases. The repair of oxidative DNA lesions is known to be impaired in vivo in the presence of ions of some heavy metals. We have studied the effect of salts of several alkaline earth and transition metals on the activity of Fpg-family DNA glycosylases in the reaction of excision of 5,6-dihydrouracil, a typical DNA oxidation product. The reaction catalyzed by NEIL1 was characterized by values K _m = 150 nM and k _cat = 1.2 min⁻¹, which were in the range of these constants for excision of other damaged bases by this enzyme. NEIL1 was inhibited by Al³⁺, Ni²⁺, Co²⁺, Cd²⁺, Cu²⁺, Zn²⁺, and Fe²⁺ in Tris-HCl buffer and by Cd²⁺, Zn²⁺, Cu²⁺, and Fe²⁺ in potassium phosphate buffer. Fpg and Nei, the prokaryotic homologs of NEIL1, were inhibited by the same metal ions as NEIL1. The values of I₅₀ for NEIL1 inhibition were 7 μM for Cd²⁺, 16 μM for Zn²⁺, and 400 μM for Cu²⁺. The inhibition of NEIL1 by Cd²⁺, Zn²⁺, and Cu²⁺ was at least partly due to the formation of metal-DNA complexes. In the case of Cd²⁺ and Cu²⁺, which preferentially bind to DNA bases rather than phosphates, the presence of metal ions caused the enzyme to lose the ability for preferential binding to damaged DNA. Therefore, the inhibition of NEIL1 activity in removal of oxidative lesions by heavy metal ions may be a reason for their comutagenicity under oxidative stress.     

Stabilization of non-histone proteins by copper ions does not alter chromatin properties of polythene chromosomes of <Emphasis Type="Italic">Chironomus plumosus</Emphasis>

Our previous study showed that the body of polythene chromosomes can be identified even after removal of all histones and DNA in the presence of 2 mM CuCl₂; this suggested that copper ions stabilized the bonds between non-histone proteins. In this study we tried to find out if copper ions bind with non-histone proteins reversibly or irreversibly. It is shown that the bodies of normal chromosomes and chromosomes stabilized by 2 mM CuCl₂ swell with partial disappearance of the banding pattern in a hypotonic solution (0.055 M NaCl) without copper ions. The selective removal of bivalent cations by 10 mM EDTA solution resulted in decondensation of normal polythene and stabilized chromosomes. The treatment of nuclear protein matrix of polythene chromosomes preparations with 10 mM EDTA resulted in the swelling of polythene chromosome body and disappearance of the banding pattern but their morphological organization maintained.     

Nuclear protein matrix of giant nuclei of polythene chromosome organization of midge <Emphasis Type="Italic">Chironomus plumosus</Emphasis> determinates

Giant nuclei from salivary glands of the midge Chironomus plumosus were treated in situ with 2M NaCl detergent and nucleases to reveal residual nuclear matrix proteins (NMP). It was shown that, after the prestabilization of nonhistone proteins with 2 mM CuCl₂, the polythene chromosome body preserved its morphologic integrity and banding pattern, even after the extraction of all histones and DNA. The stabilized NPM of polythene chromosomes can be observed in both light and electron microscopy; no interchromatin fibrillary-granular structures are revealed in the nucleus except for peripheral lamina. Using the immunocytochemical method, in polythene chromosomes, we managed to detect major nonhistone proteins (topoisomerase IIα and SMC 1) and some RNA-components. Besides, in giant nuclei of larvae of early stages there is observed BrDU incorporation visualizing sites of DNA synthesis, which also are connected with NPM of polythene chromosomes. Thus, it can be concluded that structure of NPM of giant nuclei of Chironomus plumosus has all properties of NPMs of usual interphase nuclei; furthermore, this NPM determines specific structure of the polythene chromosome.     

Metalloregulation of Triple Helix Formation by Control of the Loop Conformation

The flexible polypyridine ligand, 2,2′:6′,2^″-terpyridine (terpy), was built into the backbone of oligonucleotides to form DNA conjugates. The terpy unit functioned as a good loop when the conjugates formed the bimolecular triplexes with complementary oligopurine. The triplex structure was destabilized by the specific interaction with divalent transition metal ions (Cu²⁺, Zn²⁺, and Fe²⁺), in particular Cu²⁺ ions. This ion destabilized one of the triplexes by 4.2 kcalmol^?1 or made the triplex formation constant less than 1/10³ at 298 K. This result is attributed to the substantial turbulence of the terminal structure of the triplexes.     

Studies on nucleic acids. VIII. Changes in the stability of DNA secondary structure by interaction with divalent metal ions

The melting temperature of a natural DNA is decreased in the presence of increasing amounts of copper ions, whereas other divalent metal ions stabilize the DNA secondary structure at low ionic strength. At 1.28 × 10^?4M, Cu²⁺ produces a decrease of T_m depending on base composition. At very low Cu²⁺ concentrations (0.5 Cu²⁺/2 DNA-P) a stabilization of the DNA conformation appears due to an interaction between Cu²⁺ and phosphate groups of the DNA molecule. In this case the normal trend of GC dependence of T_m exists similar to that with Na⁺ and Mg²⁺ as counterions. If copper ions are in excess, the observed destabilization is stronger for DNAs rich in guanine plus cytosine than for those rich in adenine plus thymine. A sharp decrease of T_m occurs between 0.5–0.8 Cu²⁺/2 DNA-P and 1.5 Cu²⁺/2 DNA-P. The breadth of the transition decreases at high Cu²⁺ concentration with further addition of copper ions. Denaturation and renaturation experiments indicate that Cu²⁺ ions exceeding the phosphate equivalents interact with the bases and reduce the forces of the DNA helix conformation. Evidence is presented, that the destabilization effect produced by Cu²⁺ is possibly due to an interaction with guanine sites of the DNA molecule.     

Experimental Visualization of Chromoneme as a Higher Level of Chromatin Compactization in the Mitotic Chromosome

We succeeded to visualize the chromoneme or a filamentous chromatin structure, with the mean thickness 0.1–0.2 μm, as a higher level of chromatin compactization in animal and plant cells at different stages of chromosome condensation at mitotic prophase and during chromatid decondensation at telophase. Under the natural conditions, chromoneme elements are not detected in the most condensed chromatin of metaphase chromosomes on ultrathin sections. We studied the ultrastructure and behavior of the chromatin of mitotic chromosomes in situ in cultured mouse L-197 cells under the conditions selectively demonstrating the chromoneme structure of the mitotic chromosomes in the presence of Ca²⁺. Loosely packaged dense chromatin bands, ca. 100 nm in diameter, chromonemes, were detected in chromosome arms in a solution containing 3 mM CaCl₂. When transferred in a hypotonic solution containing 10 mM tris-HCl, these chromosomes swelled, lost the chromoneme level of structure, and rapidly transformed in loose aggregates of elementary DNP fibrils, 30 nm in diameter. After this decondensation in the low ionic strength solution, the chromoneme structure of mitotic chromosomes was restored when they were transferred in a Ca₂₊ containing solution. The morphological characteristics of the chromoneme and pattern of its packaging in the chromosome were preserved. However, when the mitotic cells with chromosomes, in which the chromoneme structure was visualized with the help of 3 mM CaCl₂, were treated with a photosensitizer, ethidium bromide, and illuminate with a light with the wavelength 460 nm, chromatic decondensation under the hypotonic solution was not observed. The chromoneme elements in a stabilized chromatin of the mitotic chromosome preserved specific interconnection and the general pattern of their packaging in the chromatid was also preserved. The chromoneme elements in the chromosomes stabilized by light preserved their density and diameter even in a 0.6 M NaCl solution, which normally leads to chromoneme destruction. An even more rigid treatment of the stabilized chromosomes with a 2 M NaCl solution, which normally fully decondenses the chromosomes, made it possible to detect a 3D reticular skeleton devoid of any axial structures. __________ Translated from Ontogenez, Vol. 36, No. 5, 2005, pp. 323–332. Original Russian Text Copyright ? 2005 by Burakov, Tvorogova, Chentsov.     

Chiral Discrimination of Ofloxacin Enantiomers Using DNA Double Helix Regulated by Metal Ions

DNA‐based chiral selectors are constructed to discriminate ofloxacin enantiomers through metal‐ion anchoring on a special DNA double helix that contains successive GC pairs. The effects of metal ions involving Mg²⁺, Ni²⁺, Cu²⁺, Ag⁺, and Pt²⁺ were studied on the regulation of DNA chiral discrimination towards ofloxacin enantiomers. It is shown that DNA‐Cu(II) complexes exhibit the highest enantioselectivities at the [Cu²⁺]/base ratio of 0.1. The enantiomeric excess can reach 59% in R‐enantiomer after being adsorbed by the RET‐Cu(II) complex. Stereoselective recognition of ofloxacin enantiomers on the double helix is tunable via external stimulus, providing a programmable desorption process to regenerate DNA. This DNA‐based chiral selector exhibits excellent reusability without apparent loss of enantioselectivity after three cycles of adsorption and desorption. Chirality 26:249–254, 2014. © 2014 Wiley Periodicals, Inc.     

Distinct differences in metal ion specificity of RNA and DNA G-quadruplexes

RNA G-quadruplexes, as their well-studied DNA analogs, require the presence of cations to fold and remain stable. This is the first comprehensive study on the interaction of RNA quadruplexes with metal ions. We investigated the formation and stability of two highly conserved and biologically relevant RNA quadruplex-forming sequences (24nt-TERRA and 18nt-NRAS) in the presence of several monovalent and divalent metal ions, namely Li⁺, Na⁺, K⁺, Rb⁺, Cs⁺, NH₄ ⁺, Mg²⁺, Ca²⁺, Sr²⁺, and Ba²⁺. Circular dichroism was used to probe the influence of these metal ions on the folded fraction of the parallel G-quadruplexes, and UV thermal melting experiments allowed to assess the relative stability of the structures in each cationic condition. Our results show that the RNA quadruplexes are more stable than their DNA counterparts under the same buffer conditions. We have observed that the addition of mainly Na⁺, K⁺, Rb⁺, NH₄ ⁺, as well as Sr²⁺ and Ba²⁺ in water, shifts the equilibrium to the folded quadruplex form, whereby the NRAS sequence responds stronger than TERRA. However, only K⁺ and Sr²⁺ lead to a significant increase in the stability of the folded structures, which is consistent with their coordination to the O6 atoms from the G-quartet guanosines. Compared to the respective DNA motives, dNRAS and htelo, the RNA sequences are not stabilized by Na⁺ ions. Finally, the difference in response between NRAS and TERRA, as well as to the corresponding DNA sequences with respect to different metal ions, could potentially be exploited for selective targeting purposes.     

Possible mechanisms underlying copper-induced damage in biological membranes leading to cellular toxicity

It is generally accepted that copper toxicity is a consequence of the generation of reactive oxygen species (ROS) by copper ions via Fenton or Haber-Weiss reactions. Copper ions display high affinity for thiol and amino groups occurring in proteins. Thus, specialized proteins containing clusters of these groups transport and store copper ions, hampering their potential toxicity. This mechanism, however, may be overwhelmed under copper overloading conditions, in which copper ions may bind to thiol groups occurring in proteins non-related to copper metabolism. In this study, we propose that indiscriminate copper binding may lead to damaging consequences to protein structure, modifying their biological functions. Therefore, we treated liver subcellular membrane fractions, including microsomes, with Cu²⁺ ions either alone or in the presence of ascorbate (Cu²⁺/ascorbate); we then assayed both copper-binding to membranes, and microsomal cytochrome P450 oxidative system and GSH-transferase activities. All assayed sub-cellular membrane fractions treated with Cu²⁺ alone displayed Cu²⁺-binding, which was significantly increased in the presence of Zn²⁺, Hg²⁺, Cd²⁺, Ag⁺¹ and As³⁺. Treatment of microsomes with Cu²⁺ in the μM range decreased the microsomal thiol content; in the presence of ascorbate, Cu²⁺ added in the nM concentrations range induced a significant microsomal lipoperoxidation; noteworthy, increasing Cu²⁺ concentration to ≥50 μM led to non-detectable lipoperoxidation levels. On the other hand, μM Cu²⁺ led to the inhibition of the enzymatic activities tested to the same extent in either presence or absence of ascorbate. We discuss the possible significance of indiscriminate copper binding to thiol proteins as a possible mechanism underlying copper-induced toxicity.     

Identification of the microtubule-associated protein map2 and its binding sites on metaphase chromosomes from cultured cells

The microtubule-associated protein MAP₂, which binds preferentially to AT sequences of DNA, can bind to metaphase chromosomes. The binding pattern of MAP₂ to chromosomes is similar to that found for the binding of the bisbenzimidazole derivative 33258 Hoechst, which also binds preferentially to AT-rich regions.     

DNA-Replikation und Chromosomenstruktur von Mesostoma (Turbellaria)

During meiosis in M. ehrenbergi (2n=10) and M. lingua (2n=8) male certain chromosomes never pair completely. In these bivalents only terminal pairing appears, crossing over could not be proved by ³H-thymidine autoradiography. DNA amounts of the M. ehrenbergi and M. lingua genomes are in a proportion of 10∶1. The mitotic S-phase of spermatogonia in M. ehrenbergi is twice as long as in M. lingua. In metaphase of spermatogonia a differentiated DNA replication pattern can be identified in M. ehrenbergi as late-pulse-replicating segments. After incorporation of ³H-thymidine X₂-metaphase chromosomes can be found, which show single chromatid labeling, terminal and intercalary isolabeling as well as kinds of chromosome labeling, which can only result from sister strand exchange. After treating the chromosomes with low temperature, colchicine or by hydrolysis (60° C) substructures of the chromatin become visible in both spezies which however are evaluated as artefacts. — Formation of the different isolabeling types is discussed on the basis of a two-strand model of the chromosome fibril. A hypothesis is formulated that the surplusage of DNA in M. ehrenbergi is distributed over all the length of the chromatids as small parts of heterochromatin. This hypothesis is supported by investigations of the DNA replication and the contractility of the chromosomes. Furthermore, a pattern of small DNA particles can be demonstrated after partial destruction of the DNA in metaphase chromosomes of M. ehrenbergi, which could represent this intercalary heterochromatin.