Cytological differentiation of constitutive heterochromatin

The constitutive heterochromatin, as demonstrated by the C band technique, may be subdivided into a number of categories when other characteristics are considered. The responses to fluorochromes QM and 33258 Hoechst, the behavior following G band staining, the repetitive DNA content, and many other criteria are useful for the classification of heterochromatin. The heterochromatin patterns of three mammalian species are presented to demonstrate that within each karyotype there may be several different types of C bands. In general, a correlation may also be made between GC-rich satellite DNA and dull (or negative) Q fluorescence, and between AT-rich satellite DNA and bright Q, fluorescence.On Sabbatical leave from Department of Biology, University of North Dakota, Grand Forks, North Dakota.     

Cytological variations in the constitutive heterochromatin of Microtus agrestis

In the field vole, Microtus agrestis, most of the constitutive heterochromatin is localized in the giant chromosomes. A detailed examination of a large number of adult cell types reveals that this chromatin is actually present as a heterochromatic fiber in all interphase nuclei. Depending upon the cell types, however, the fiber shows varying degrees of condensation and folding ranging from a very long and extended fiber to a large compact chromocenter. The number of cell types with giant chromocenters was less commonly observed than those with extended fibers. This explains why some cells were previously thought to be devoid of heterochromatin.—The results of this investigation strongly indicate that constitutive heterochromatin represents a unique nuclear entity.This investigation was supported by NIH funds (Grant No. HD 1962).     

Replication of autosomal heterochromatin in man

Summary In interphase nuclei of leukocytes and oral mucosa cells of normal human males and f males, two types of heterochromatin can he distinguished according to their location in the nucleus. Firstly, nucleolus-associated heterochromatin which consists of one large mass of autosomal segments surrounding the nucleolus, or several large masses if there appears to be more than one nucleolus in the same nucleus. Secondly, scattered heterochromatin composed of a large number of positively heteropycnotic bodies scattered throughout the nucleus and not directly associated with the nucleolus. The correspondence of this type of heterochromatin with chromosome segments is obtained at late prophase where several positively heteropycnotic regions belonging to the autosomes are found scattered throughout the nucleus.In human females sex-chromatin is present in addition to these two types. In leukocytes the sex-chromatin cannot be easily identified due to the large size and number of the scattered heterochromatic bodies, but in oral mucosa cells such a distinction is more easily achieved due to the smaller amount of autosomal heterochromatin.Nucleolus-associated and scattered heterochromatin from leukocytes of both sexes synthesized their DNA at a different period of time from the euchromatin. The asynchrony of replication observed in the heterochromatin at interphase is in agreement with the asynchrony between autosomes and within autosomes described by many authors at metaphase. This does not mean, however, that every segment or chromosome found replicating asynchronously at metaphase contains necessarily heterochromatin.Dedicated to Professor H. Bauer on the occasion of his 60th birthday. — This investigation was supported by a research grant to A. Lima-de-Faria from the Swedish Natural Science Research Council.     

Proteins recognized by antibodies against isolated cytological heterochromatin from rat liver cells change their localization between cell species and between stages of mitosis (interphase vs metaphase)

Heterochromatin in the cell nucleus seems to concentrate various proteins, such as Drosophila heterochromatin protein 1, which maintain the repressed state of gene expression. However, it still remains obscure how protein composition related to chromatin structure is different between heterochromatin and euchromatin in interphase nuclei. We isolated cytological heterochromatin from sonicated interphase nuclei obtained from rat liver cells and prepared antisera against it. The dense heterochromatic bodies seen in the preparation of intact nuclei were duplicated in a relatively pure form during the preparation of heterochromatin. In the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, differences between the fractions of heterochromatin and euchromatin were noted by their protein composition. Isolated heterochromatin was then digested by DNase after partial digestion with trypsin and its dense structure changed to become highly sensitive to DNase. The prepared antibodies reacted with the heterochromatin region of rat liver cell nuclei and isolated cytological heterochromatin; however, they did not react with euchromatin. Using immunohistochemistry, the antibodies bound to each cell nucleus in all tissues observed; some cell types were distinguished by their differential stainability (e.g. staining in the cytoplasm). Staining of the mitotic cells showed that the proteins recognized by the antibodies were localized in the cytoplasm and, in part, on the chromosomes. Based on the results of molecular cloning from rat liver cDNA library using the antibodies as a probe, it seemed that the antibodies mainly recognized two proteins similar to arginase and general vesicular transport factor p115, respectively. The results obtained from these experiments reveal that some proteins located in the heterochromatin of interphase liver cell nuclei seem to play important roles in condensing a portion of the chromatin structure during interphase and suggest that proteins composing heterochromatin might be changed according to cell types or the stage of the cell cycle.     

Critical electrolyte concentration of the heterochromatin and euchromatin of Triatoma infestans

The binding of toluidine blue molecules under Mg2+ competitive staining conditions was investigated in chromocentres and the euchromatin of single- and multi-chromocentred nuclei of Triatoma infestans Malpighian tubule cells. It was demonstrated that the chromocentre of single-chromocentred nuclei exhibited the largest critical electrolyte concentration (CEC) value (0.4 M), followed by the chromocentres of multi-chromocentred nuclei (0.3 M) and the euchromatin (0.2 M). The differences in CEC values were assumed to be due to differences in availability of free DNA phosphates and in packing states of the DNA-protein complexes of these chromatin types. Differences in chromatin supra-organization were evident for the chromocentral heterochromatin of single vs multi-chromocentred nuclei. This was also valid for the chromocentral heterochromatin in some multi-chromocentred nuclei, when one of the heterochromatic bodies was especially larger than the others.     

Estimating percentage constitutive heterochromatin by flow cytometry.

Flow cytometry is a powerful method for the assessment of both plant and animal genomes. One of the most interesting aspects is the analysis of chromatin structure. By using intercalating and base pair-specific fluorochromes, the chromatin structure in various cell cultures and microorganisms has been determined. In this study, several maize lines of known heterochromatic composition were analyzed. The nuclei of each line were isolated and stained with DAPI (base pair specific) and PI (intercalator) separately. For each maize line, the PI/DAPI ratio was determined. A significant negative correlation was observed between C-band number and PI/DAPI ratio (r = 0.920) and between percentage heterochromatin and PI/DAPI ratio (r = 0.997). Flow cytometry with use of the fluorochromes DAPI and PI was found to be a rapid and efficient method of determining heterochromatin amount in maize.     

Studies in heterochromatin DNA: accessibility of late replicating heterochromatin DNA in chromatin to micrococcal nuclease digestion

Heterochromatin DNA in cactus mouse (Peromyscus eremicus) replicates in the late S phase of cell cycle. A method of obtaining cells which contain DNA preferentially labeled at heterochromatic areas by a pulse-labeling of late replicating DNA is described. When the nuclei of P. eremicus cells containing radioactively labeled DNA in heterochromatin were digested with micrococcal nuclease and the resultant nucleosomal DNA was separated by gel electrophoresis, it was found that the repeat length of nucleosomal DNA in the heterochromatin DNA is not different from that of the bulk of the genomic DNA. Furthermore, there was no significant difference in the accessibility to digestion by micrococcal nuclease between the late replicating heterochromatin DNA and the total DNA under our digestion conditions. Two dimensional gel electrophoresis patterns of nucleosomal DNAs isolated from micrococcal nuclease digested nuclei from P. eremicus, P. collatus, and P. crinitus cells in culture were very similar. Cytogenetic data showed that these three species are different in heterochromatin but similar in euchromatin.     

Characterization of Drosophila heterochromatin

A number of preliminary experiments have shown that the fluorescence pattern of Hoechst 33258, as opposed to that of quinacrine, varies with the concentration of dye. The metaphase chromosomes of D. melanogaster, D. simulans, D. virilis, D. texana, D. hydei and D. ezoana have therefore been stained with two concentrations of H 33258 (0.05 and 0.5 mug/ml in phosphate buffer at pH 7) and with a single concentration of quinacrine (0.5% in absolute alcohol). The three fluorescence patterns so obtained were shown to be somewhat different in some of the species and the coincide in others. All three stainings gave an excellent longitudinal differentiation of heterochromatin while euchromatin fluoresced homogeneously. Living ganglion cells of the six species mentioned above were treated with quinacrine and H 33258. Quinacrine induced a generalized lengthening and swelling of the chromosomes and H 33258 the decondensation of specific heterochromatic regions. A correlation of the base composition of the satellite DNAs contained in the heterochromatin of the species studied with the relative fluorescence and decondensation patterns showed that: 1) the extremely fluorochrome bright areas and those decondensed are present only in species containing AT rich satellite DNA; 2) the opposite is not true since some AT-rich satellite DNAs are neither fluorochrome bright nor decondensed; 3) there is no good correspondence between Hoechst bright areas and the decondensed ones. AT richness therefore appears to be a necessary but not sufficient condition both for bright fluorescence and decondensation. Some cytological evidence suggests that similarly AT rich satellite DNAs respond differently in fluorescence and decondensation because they are bound to different chromosomal proteins. A combination of the results of fluorescence and decondensation revealed at least 14 types of heterochromatin; 4-7 of which are simultaneously present in the same species. Since closely related species (i.e. D. melanogaster and D. simulans; D. virilis and D. texana) show marked differences in the heterochromatic types they contain, it can be suggested that within the genus Drosophila qualitative variations of heterochromatin have played an important role in speciation.     

Distribution of chromosome 18 and X centric heterochromatin in the interphase nucleus of cultured human cells

In situ hybridization of human chromosome 18 and X-specific alphoid DNA-probes was performed in combination with three dimensional (3D) and two dimensional (2D) image analysis to study the interphase distribution of the centric heterochromatin (18c and Xc) of these chromosomes in cultured human cells. 3D analyses of 18c targets using confocal laser scanning microscopy indicated a nonrandom disposition in 73 amniotic fluid cell nuclei. The shape of these nuclei resembled rather flat cylinders or ellipsoids and targets were preferentially arranged in a domain around the nuclear center, but close to or associated with the nuclear envelope. Within this domain, however, positionings of the two targets occurred independently from each other, i.e., the two targets were observed with similar frequencies at the same (upper or lower) side of the nuclear envelope as those on opposite sides. This result strongly argues against any permanent homologous association of 18c. A 2D analytical approach was used for the rapid evaluation of 18c positions in over 4000 interphase nuclei from normal male and female individuals, as well as individuals with trisomy 18 and Bloom's syndrome. In addition to epithelially derived amniotic fluid cells, investigated cell types included in vitro cultivated fibroblastoid cells established from fetal lung tissue and skin-derived fibroblasts. In agreement with the above 3D observations 18c targets were found significantly closer (P less than 0.01) to the center of the 2D nuclear image (CNI) and to each other in all these cultures compared to a random distribution derived from corresponding ellipsoid or cylinder model nuclei. For comparison, a chromosome X-specific alphoid DNA probe was used to investigate the 2D distribution of chromosome X centric heterochromatin in the same cell types. Two dimensional Xc-Xc and Xc-CNI distances fit a random distribution in diploid normal and Bloom's syndrome nuclei, as well as in nuclei with trisomy X. The different distributions of 18c and Xc targets were confirmed by the simultaneous staining of these targets in different colors within individual nuclei using a double in situ hybridization approach.     

Interactions of mercury and copper with constitutive heterochromatin and euchromatin in vivo and in vitro.

Mouse liver nuclei were fractionated into (condensed) heterochromatin and (noncondensed) euchromatin by differential centrifugation of sonicated nuclei. The fractions were subsequently characterized as unique nuclear species by thermal denaturation derivative profile analysis, which revealed the heterochromatin fraction enriched in satellite DNA and by endogenous metal content, which displayed partitioning of mercury in euchromatin over heterochromatin by a 10:1 ratio, with a comparatively uniform distribution of copper in both fractions. Fractionation of nuclei following in vivo challenge with copper showed enrichment of copper in heterochromatin, relative to euchromatin, while in vivo exposure to mercury resulted in a 20-fold accumulation of mercury in euchromatin, relative to heterochromatin. Using gel filtration and equilibrium dialysis to measure in vitro binding under relatively physiologic conditions of pH (6.0-7.0) and ionic strength (standard saline citrate or saline), the condensed and noncondensed chromatin fractions exhibited binding specificities toward mercury and copper similar to that observed in the in vivo metal challenge experiments. The level of mercury which binds to euchromatin in vitro, when measured either in physiologic [standard saline citrate (SSC)] or in dilute (1:100 SSC) salt solutions, was comparable (approximately 3 mug of Hg/mg of DNA) to that of in vivo euchromatin-bound mercury after 1 month of challenge with dietary metal. In contrast, copper showed little or no preference for the nuclear fractions in dilute salt solutions and displayed patterns which mimic in vivo binding only at higher ionic strengths (saline). Removal of proteins from the chromatin fractions resulted in a loss of binding specificity toward both metals. Therefore, the binding selectivity of condensed and noncondensed chromatin toward both mercury and copper appears to arise from protein or from protein-DNA associations. The state of chromatin condensation is especially critical in the case of copper.     

Origin of facultative heterochromatin in the endosperm ofGagea lutea (Liliaceae)

Summary Facultative heterochromatin occurs not only in certain animals in connection with sex determination but also in members of at least one plant genus,Gagea (Liliaceae s. str.), but here in the course of embryo sac development, fertilization, and endosperm formation. The present contribution intends to provide undebatable photographic and cytometric evidence, previously not available, for the events in the course of which three whole genomes in the pentaploid endosperm nuclei ofGagea lutea become heterochroma-tinized. In this plant, embryo sac formation usually follows the Fritillaria type, i.e., the embryo sac is tetrasporic, and a 1 + 3 position of the spore nuclei is followed by a mitosis in which the three chalazal spindles fuse and two triploid nuclei are formed. A triploid chalazal polar nucleus is derived from one of these, which contributes to the pentaploid endosperm. These nuclei in the chalazal part of the embryo sac show stronger condensation compared with the micropylar ones. The pycnosis of the triploid polar nucleus is maintained and even enhanced during endosperm proliferation, while the micropylar polar nucleus and the sperm nucleus maintain their euchromatic condition. The origin of the heterochromatic masses in the endosperm nuclei from the three chalazal genomes of the central cell is unambiguously evident from the distribution of heterochromatic chromosomes in the first endosperm mitosis and the following interphase. DNA content measurements confirm a 3 2 relationship of heterochromatic and euchromatic chromosome sets, which is usually maintained up to the cellularized endosperm. Pycnotic nuclei in the chalazal part of megagametophytes are characteristic of several embryo sac types, but only forGagea spp. it is documented that such nuclei can take part in fertilization and endosperm formation.Dedicated to Professor Walter Gustav Url on the occasion of his 70th birthday     

Higher-order folding of heterochromatin: protein bridges span the nucleosome arrays.

In interphase eukaryotic nuclei, chromatin is divided into two morphologically distinct types known as heterochromatin and euchromatin. It has been long suggested that the two types of chromatin differ at the level of higher-order folding. Recent studies have revealed the features of chromatin 3D architecture that distinguish the higher-order folding of repressed and active chromatin and have identified chromosomal proteins and their modifications associated with these structural transitions. This review discusses the molecular and structural determinants of chromatin higher-order folding in relation to mechanism(s) of heterochromatin formation and genetic silencing during cell differentiation and tissue development.     

A developmental study of constitutive heterochromatin in Microtus agrestis

In the vole, Microtus agrestis, the constitutive heterochromatin is largely restricted to the giant sex chromosomes but varies in its degree of condensation in various cell types. In the cleavage embryos and fibroblasts it formed one or two long and extended heterochromatic fibers, in hepatocytes it formed two large and diffuse masses and in neurons, spermatogonia and oogonia it formed two large and compact masses. The basic patterns of all differentiated cells were essentially unchanged throughout development.—At all stages of development and in cells of all types, mitotic nuclei displayed two large heteropycnotic chromosomes in prophase and persistent condensation in telophase. Apposition and delayed separation of chromatids of the giant chromosomes was also observed in metaphase and anaphase, respectively. During the first meiotic prophase of spermatocytes and oocytes, the giant chromosomes were also heteropycnotic.—The results strongly suggest that constitutive heterochromatin is localized in the same chromosomes throughout development and represents a specific entity.     

Restriction enzyme digestion of heterochromatin inDrosophila nasuta

In situ digestion of metaphase and polytene chromosomes and of interphase nuclei in different cell types ofDrosophila nasuta with restriction enzymes revealed that enzymes like AluI, EcoRI, HaeIII, Sau3a and SinI did not affect Giemsa-stainability of heterochromatin while that of euchromatin was significantly reduced; TaqI and SalI digested both heterochromatin and euchromatin in mitotic chromosomes. Digestion of genomic DNA with AluI, EcoRI, HaeIII, Sau3a and KpnI left a 23 kb DNA band undigested in agarose gels while withTaqI, no such undigested band was seen. TheAluI resistant 23 kb DNA hybridized insitu specifically with the heterochromatic chromocentre. It appears that the digestibility of heterochromatin region in genome ofDrosophila nasuta with the tested restriction enzymes is dependent on the availability of their recognition sites.     

Cytochemical heterochromatin differentiation inSinapis alba (Cruciferae) using a simple air-drying technique for producing chromosome spreads

The fluorochrome and Giemsa chromosome banding patterns and the Ag-NOR histochemical staining ofSinapis alba are described. Two major types of heterochromatin can be distinguished, one of which contains GC-rich DNA. The observations are discussed as they relate to the known satellite DNAs ofS. alba. — A simple air-drying technique for producing spreads of plant mitotic chromosomes is presented. Different materials and staining techniques were tested showing that the method has wide applications.Dedicated in gratitude to Prof. DrElisabeth Tschermak-Woess on the occasion of her 70th birthday.     

Loss of heteropycnosis of the constitutive heterochromatin in specifically activated cells of the thyroid gland of Microtus agrestis

In nuclei of the inactive thyroid gland of Microtus agrestis the constitutive heterochromatin is heteropycnotic and forms large chromocenters. After specific activation of the thyroid gland with thyroid stimulating hormone (TSH), or with the thyrostatic methylthiouracil (MTU), an enlargement of cells and nuclei, a high mitotic activity and a despiralization of the contitutive heterochromatin are observed. The facultative heterochromatin, on the contrary, remains condensed. The structure of the nuclei of the parathyroid gland is not altered by the treatment with TSH or MTU. The findings permit the assumption that constitutive heterochromatin has specific functions, e.g. as a nucleolus organizer, which are blocked in the heteropycnotic state and active in the isopycnotic state.Supported by the Bundesministerium für Bildung und Wissenschaft of the Federal Republic of Germany.     

The interphase distribution of satellite DNA-containing heterochromatin in mouse nuclei

The distribution of sites capable of binding mouse satellite-complementary RNA in the cytological hybridization reaction has been examined in mouse liver and testis interphase nuclei. The approach taken has been to combine hybridization with semi-thin sectioning and autoradiography in order to obtain a clear picture of the relationship of satellite DNA-containing structures to the rest of the interphase nucleus. In liver nuclei, hybridization occurs primarily with blocks of heterochromatin associated with the nuclear envelope. The most prominent of these, in terms both of size and intensity of hybridization, is the nucleolar stalk and the rest of the nucleolus-associated heterochromatin. The nucleolar body itself is not labeled, nor is much of the peripheral condensed chromatin ; in fact, a polarized distribution of satellite DNA is evident. In Sertoli and spematid nuclei, satellite DNA is found in a small number of large heterochromatin blocks with which the nucleolus is associated; some of this material bears a relationship to the nuclear envelope in these cells also.     

Characterization of heterochromatin in Schistocerca gregaria by C- and N-banding methods

Mitotic and meiotic chromosomes of Schistocerca gregaria were C-, mild N- and strong N-banded. After C-banding, three out of eleven autosomes show, in addition to the centromeric C-bands, a second C-band. — The mild N-banding method produces a single N-band in each of only four chromosomes. With the exception of one N-band these mild N-bands correspond to the non-centromeric, second C-bands, indicating the heterochromatic character of at least three mild N-band regions. — The strong N-banding technique produces bands both at the C- and mild N-band positions and additionally a third band in one chromosome (M₈), not present after C- or mild N-banding. — The N-bands do not correspond to the nucleolus organizer regions. Because of the mechanisms of the N-banding methods, it is concluded that the centromeric heterochromatin, as well as the non-centromeric N-band regions, contain high quantities of non-histone proteins. Presumably a specific difference exists between the non-histone proteins in the centromeric and non-centromeric N-band regions because the centromeres are banded by the strong N-banding technique, but not after mild N-banding. It is concluded that the N-band regions (two exceptions) contain a heterochromatin type which has the following features in common with the -heterochromatin of Drosophila: C- as well as N-banding positive, high nonhistone protein content, repetitive and late replicating DNA. It is discussed whether the N-banded heterochromatin regions of Schistocerca contain that DNA fraction which is, like the Drosophila -heterochromatin, underreplicated in polyploid nuclei.