Analysis Of Chromatin Structure Research Articles

Rapid induction of rat liver S14 gene transcription by thyroid hormone

The mRNA coding for the rat liver S14 protein (Mr 17,000; pI 4.9) is rapidly induced by triiodothyronine (T3) (Jump, D. B., Narayan, P., Towle, H. C., and Oppenheimer, J. H. (1984) J. Biol. Chem. 259, 2789-2797). In an effort to define the molecular basis for the rapid increase in mRNAS14, the in vitro run-on activity and chromatin structure of the S14 gene was examined. Following injection of hypothyroid rats with a receptor-saturating dose of T3, a 5-min lag period preceded a rapid increase in S14 gene transcription. S14 transcriptional activity was induced 3.8-fold within 15 min and reached nearly 70% of the maximal 9-fold induction within 60 min of T3 administration. Hepatic mRNAS14 levels were induced 2.4-, 19-, and 24-fold within 15 min, 4 h, and 24 h, respectively. Thus, the rapid T3-mediated induction of mRNAS14 was due, in large part, to a rapid and apparent direct effect of T3 on S14 transcriptional activity. Analysis of S14 chromatin structure showed that the Hss-3 DNase I hypersensitive site located 3.3 kilobases (kb) upstream from the hepatic S14 cap site was present in both euthyroid and hyperthyroid states, but either absent or present at diminished DNase I sensitivity in the hypothyroid state. However, within 5 min of T3 administration to hypothyroid rats, the Hss-3 DNase I hypersensitive site was significantly induced. The induction of this structure preceded T3 induction of S14 gene transcription. Other DNase I hypersensitive sites located adjacent to the S14 cap site at -65 to -265 base pairs (Hss-1) or upstream at -1.3 kb (Hss-2), -2.1 kb (Hss-3'), -5.3 kb (Hss-4), and -6.2 kb (Hss-5) remained unaffected by changes in S14 gene transcription. The rapid T3 effect on the Hss-3 DNase I hypersensitive site may reflect the presence of T3 receptors in the vicinity of this chromatin locus. Modification of chromatin structure in the vicinity of the Hss-3 site may be an important antecedent event for T3-mediated induction of S14 gene transcription.

Characteristics of chromatin release during digestion of nuclei with micrococcal nuclease: Preferential solubilization of nascent rna at low enzyme concentration

1. 1. Extensive digestion of nuclei with micrococcal nuclease (MNase), commonly used in the analysis of chromatin structure, results in the production of mono- and dinucleosomal chromatin fragments. 2. 2. Digestion of nuclei from a range of cell types with low enzyme concentrations solubilized high molecular weight polynucleosomal fragments, some ⪢ 22 kb long. 3. 3. Such digestion conditions also resulted in extensive solubilization of nascent RNA which contributed considerably to the nucleic acid content of the soluble fraction. 4. 4. We conclude that the contribution of RNA to total nucleic acid content of the soluble fraction should be taken into consideration when nuclei are digested with low concentrations of MNase.

Isolation and structural characterization of the human 4F2 heavy-chain gene, an inducible gene involved in T-lymphocyte activation.

The human 4F2 cell surface antigen is a 120-kilodalton (kDa) disulfide-linked heterodimer which is composed of an 80- to 90-kDa glycosylated heavy chain (4F2HC) and a 35- to 40-kDa nonglycosylated light chain (4F2LC). 4F2 belongs to a family of inducible cell surface molecules which are involved in T-lymphocyte activation and growth. To better understand the molecular mechanism(s) that controls 4F2HC gene expression in both resting and activated T cells, a 4F2HC human genomic clone was isolated and structurally characterized. The 4F2HC gene spans 8 kilobases of chromosome 11 and is composed of nine exons. The 5' upstream region of the gene displays several properties which are characteristic of housekeeping genes. It is G+C rich and hypomethylated in peripheral blood lymphocyte DNA and contains multiple binding sites for the Sp1 transcription factor while lacking TATA or CCAAT sequences. This region of the gene also displays sequence homologies with several other inducible T-cell genes, including the interleukin-2, interleukin-2 receptor alpha chain, dihydrofolate reductase, thymidine kinase, and transferrin receptor genes. A 255-base-pair fragment of the 4F2HC gene which contains 154 base pairs of the 5' flanking sequence was able to efficiently promote expression of the bacterial chloramphenicol acetyltransferase gene in human Jurkat T cells, indicating that it contains promoter or enhancer (or both) sequences. Analyses of chromatin structure in resting and lectin-activated T cells revealed the presence of stable DNase I-hypersensitive sites within both the 5' flanking and intron 1 regions of the 4F2HC gene. Although the 4F2HC gene displayed many of the structural features characteristic of a constitutively expressed gene, lectin-mediated activation of resting peripheral blood T lymphocytes resulted in a dramatic increase in steady-state levels of 4F2HC mRNA.

Isolation and structural characterization of the human 4F2 heavy-chain gene, an inducible gene involved in T-lymphocyte activation.

The human 4F2 cell surface antigen is a 120-kilodalton (kDa) disulfide-linked heterodimer which is composed of an 80- to 90-kDa glycosylated heavy chain (4F2HC) and a 35- to 40-kDa nonglycosylated light chain (4F2LC). 4F2 belongs to a family of inducible cell surface molecules which are involved in T-lymphocyte activation and growth. To better understand the molecular mechanism(s) that controls 4F2HC gene expression in both resting and activated T cells, a 4F2HC human genomic clone was isolated and structurally characterized. The 4F2HC gene spans 8 kilobases of chromosome 11 and is composed of nine exons. The 5' upstream region of the gene displays several properties which are characteristic of housekeeping genes. It is G+C rich and hypomethylated in peripheral blood lymphocyte DNA and contains multiple binding sites for the Sp1 transcription factor while lacking TATA or CCAAT sequences. This region of the gene also displays sequence homologies with several other inducible T-cell genes, including the interleukin-2, interleukin-2 receptor alpha chain, dihydrofolate reductase, thymidine kinase, and transferrin receptor genes. A 255-base-pair fragment of the 4F2HC gene which contains 154 base pairs of the 5' flanking sequence was able to efficiently promote expression of the bacterial chloramphenicol acetyltransferase gene in human Jurkat T cells, indicating that it contains promoter or enhancer (or both) sequences. Analyses of chromatin structure in resting and lectin-activated T cells revealed the presence of stable DNase I-hypersensitive sites within both the 5' flanking and intron 1 regions of the 4F2HC gene. Although the 4F2HC gene displayed many of the structural features characteristic of a constitutively expressed gene, lectin-mediated activation of resting peripheral blood T lymphocytes resulted in a dramatic increase in steady-state levels of 4F2HC mRNA.

Relation between protoplast division, cell-cycle stage and nuclear chromatin structure

Using different sources of protoplasts and two complementary techniques, flow cytometry and image analysis, to study the cell-cycle phases, we sought to define the particular protoplast state associated with the disposition to divide. Both inPetunia and inNicotiana plumbaginifolia, tissues with a higher G2 frequency (from different aged plants) yielded protoplasts capable of increased cell division. InSorghum, the age of the plant does not modify the proportion of G2 nuclei in leaf protoplasts, and we used root protoplasts to increase G2 frequencies. InHelianthus annuus, leaf protoplasts did not divide; however, hypocotyl protoplast preparations with relatively high 4C DNA frequencies do divide. Moreover, image analysis of chromatin structure indicated that leaf nuclei were in the G0 phase, unlike those from hypocotyls which were in G1. A high frequency of protoplasts with G2 nuclei appears to be correlated with the ability of a given preparation to undergo division; conversely, the differentiated G0 state is not conducive to division.

Analysis of chromatin structure and composition

Introduction Biochemistry, like many other sciences, is currently undergoing increasing specialization which is thought to be unavoidable because of the rapid progress within this field. Obviously education in Biochemistry and Molecular Biology is also affected. Consequently, the student may lose the ability to integrate his knowledge, which should be a requirement during the training of a scientist. The solution to this problem is quite easy in the case of theoretical courses because, here, the lecturer may include several 'integrative lessons' which give a global view of previously explained facts and place them within the general context of the course. However, in practical courses it is much more difficult to find solutions to the specialization problem. Practical courses are more suitable for focusing attention on a particular technique or problem and, moreover, most laboratories are highly specialized both in equipment and human resources. A feasible solution may be the elaboration of several short practical experiments one or two sessions that offer several special features. The experiments should be based in updated topics, they should have attractive and interpretable results and use unspecialized, lowcost equipment, ie available to every laboratory, and at the same time being versatile for providing different practical experiences. Finally, the experimental approaches used should be as varied as possible. Recently we have proposed a practical experiment that combines many of these features.l In it the student is initiated into the current techniques of recombinant DNA in prokaryotes. We now suggest a new experiment, along the same lines, whose aim is to study the genetic material of eukaryotes thus providing an appropriate complement to the preceding experiment. For this reason and because basically both experiments make use of the same equipment, they may be carried out together in four or five sessions. The experiment we propose completes and updates an experiment published in this journal several years ago. e This new experiment is based on the rapid growth and interest of the chromatin research over 1000 papers per year in international journals-making it of great interest for students of Biochemistry or Molecular Biology in any university curriculum. The material used, chicken erythrocytes, is easily obtained and has a simple chromatin organization due to its low metabolic activity. This simplicity facilitates the study of the chromatin because, although it has a typical nucleosomal structure, it is easier to obtain and study than the chromatin from other more active cells. 3 When the experiment is completed the student will have performed an almost comprehensive study of a defined material, the chromatin, by using several different experimental approaches.

Analysis of the psoralen-crosslinking pattern in chromatin DNA by exonuclease digestion.

When chromatin is photoreacted with psoralen, crosslinks occur preferentially in the linker DNA between nucleosomes. The pattern of these crosslinks can be analysed by exonuclease digestion of random DNA fragments, since the exonucleases tested stop at sites of psoralen-crosslinks. Further digestion of these fragments with S1-nuclease leads to DNA fragments of nucleosomal and polynucleosomal size, which presumably carry psoralen-crosslinks at both ends. This method of analysis of chromatin structure complements the classical micrococcal nuclease digestion analysis, since it can be performed in vitro as well as in vivo, and since it is independent of pH and ionic conditions.

Human immunoglobulin kappa gene enhancer: chromatin structure analysis at high resolution.

The murine immunoglobulin kappa gene enhancer has previously been found to coincide with a region of altered chromatin structure reflected in a DNase I hypersensitivity site detectable on Southern blots of B-cell DNA. We examined the chromatin structure of the homologous region of human DNA using the high-resolution electroblotting method originally developed for genomic sequence analysis by G. Church and W. Gilbert (Proc. Natl. Acad. Sci. USA 81:1991-1995, 1984). Analysis of DNA isolated from cells treated in vivo with dimethyl sulfate revealed two B-cell-specific sites of enhanced guanine methylation. Both sites are located within perfect inverted repeats theoretically capable of forming cruciform structures; one of these repeats overlaps an enhancer core sequence. No enhancement or protection of guanine methylation was observed within sequences similar to sites of altered methylation previously described in the immunoglobulin heavy-chain enhancer. Treatment of isolated nuclei with DNase I or a variety of restriction endonucleases defined a B-cell-specific approximately 0.25-kilobase region of enhanced nuclease susceptibility similar to that observed in the murine kappa enhancer. The 130-base-pair DNA segment that shows high sequence conservation between human, mouse, and rabbit DNAs lies at the 5' end of the nuclease-susceptible region.

Human immunoglobulin kappa gene enhancer: chromatin structure analysis at high resolution.

The murine immunoglobulin kappa gene enhancer has previously been found to coincide with a region of altered chromatin structure reflected in a DNase I hypersensitivity site detectable on Southern blots of B-cell DNA. We examined the chromatin structure of the homologous region of human DNA using the high-resolution electroblotting method originally developed for genomic sequence analysis by G. Church and W. Gilbert (Proc. Natl. Acad. Sci. USA 81:1991-1995, 1984). Analysis of DNA isolated from cells treated in vivo with dimethyl sulfate revealed two B-cell-specific sites of enhanced guanine methylation. Both sites are located within perfect inverted repeats theoretically capable of forming cruciform structures; one of these repeats overlaps an enhancer core sequence. No enhancement or protection of guanine methylation was observed within sequences similar to sites of altered methylation previously described in the immunoglobulin heavy-chain enhancer. Treatment of isolated nuclei with DNase I or a variety of restriction endonucleases defined a B-cell-specific approximately 0.25-kilobase region of enhanced nuclease susceptibility similar to that observed in the murine kappa enhancer. The 130-base-pair DNA segment that shows high sequence conservation between human, mouse, and rabbit DNAs lies at the 5' end of the nuclease-susceptible region.

The expression and chromatin structure of the chicken glyceraldehyde-3-phosphate dehydrogenase gene in mouse cells.

Chicken glyceraldehyde-3-phosphate dehydrogenase gene (GAPD) and thymidine kinase gene (TK) were co-transfected into mouse LMTK- cells by the calcium phosphate precipitation technique. Four of the eight hypoxanthine/aminopterin/thymidine-containing medium-resistant, TK+ transfectants were shown to produce different amounts of chicken glyceraldehyde-3-phosphate dehydrogenase by zymogram analysis. Subcloning and further analysis revealed that the chicken GAPD was stably inherited and that its enzyme subunits randomly combined with mouse subunits in heterotetramers. Although the contribution of chicken enzyme varied from approximately 30 to approximately 90% of the total glyceraldehyde-3-phosphate dehydrogenase activity with a proportional increase in total activity in the different subclones, it did not appear to affect the expression of mouse endogenous glycolytic enzymes since there was no distinct change in the levels of either mouse glyceraldehyde-3-phosphate dehydrogenase mRNA nor mouse phosphoglycerate kinase enzyme activity. The levels of chicken GAPD copy number, mRNA, and enzyme apparently were generally correlated in the different subclones, suggesting that the chicken GAPD in the mouse cells were expressed constitutively. In situ hybridization revealed that the transfected genes were integrated into mouse chromosomes in one cluster, and the locations of these clusters were different in different clones. Chromatin structure analyses of the chicken GAPD in four different transfectants revealed three DNase I-hypersensitive sites located around 0.2, 2.0, and 3.4 kilobases upstream from the 5' side of the gene. These sites are also present in the same locations in chicken lymphoblastoid cells (Kuo, M. T., Iyer, B., and Schwartz, R. J. (1982) Nucleic Acids Res 10, 4565-4579), indicating the dominant transmission of DNase I-hypersensitive cleavage sites in the transfected gene.

DNA and chromatin structure of the human alpha 1 (I) collagen gene.

The human alpha 1 (I) collagen gene and 48 kilobase pairs of flanking DNA have been isolated on two overlapping cosmids. The alpha 1 (I) gene is 18 kilobase pairs long and contains a single repetitive element of the Alu family; at least 15 repetitive elements are present in the flanking DNA. Analysis of chromatin structure in nuclei isolated from cultured fibroblasts demonstrated a single chromatin domain greater than 65 kilobase pairs in length that contained 9 DNase I-hypersensitive sites. The pattern of hypersensitive sites was also determined in nuclei derived from placental tissue. Five of the DNase I-hypersensitive sites were observed in both placental and fibroblast chromatin including one site near the 5' end and another near the 3' end of alpha 1 (I). An additional two sites located near the 3' end of the alpha 1 (I) gene in fibroblast chromatin are associated with the tissue-specific use of different polyadenylation sites. Two DNase I-hypersensitive sites found only in fibroblast chromatin and one site found only in placental chromatin were located more than 10 kilobase pairs away from the alpha 1 (I) gene and may be related to tissue-specific expression of other genes in the domain. However, the only abundant placental mRNAs from the 65-kilobase pair domain were those transcribed from the alpha 1 (I) gene. These findings suggest that physical linkage does not play a predominant role in controlling coordinate expression of collagen genes.

Cleavage of chromatin with methidiumpropyl-EDTA . iron(II).

Methidiumpropyl-EDTA . iron(II) [MPE . Fe (II)] cleaves double-helical DNA with considerably lower sequence specificity than micrococcal nuclease. Moreover, digestions with MPE . Fe(II) can be performed in the presence of certain metal chelators, which will minimize the action of many endogenous nucleases. Because of these properties MPE . Fe(II) would appear to be a superior tool for probing chromatin structure. We have compared the patterns generated from the 1.688 g/cm3 complex satellite, 5S ribosomal RNA, and histone gene sequences of Drosophila melanogaster chromatin and protein-free DNA by MPE . Fe(II) and micrococcal nuclease cleavage. MPE . Fe(II) at low concentrations recognizes the nucleosome array, efficiently introducing a regular series of single-stranded (and some double-stranded) cleavages in chromatin DNA. Subsequent S1 nuclease digestion of the purified DNA produces a typical extended oligonucleosome pattern, with a repeating unit of ca. 190 base pairs. Under suitable conditions, relatively little other nicking is observed. Unlike micrococcal nuclease, which has a noticeable sequence preference in introducing cleavages, MPE . Fe(II) cleaves protein-free tandemly repetitive satellite and 5S DNA sequences in a near-random fashion. The spacing of cleavage sites in chromatin, however, bears a direct relationship to the length of the respective sequence repeats. In the case of the histone gene sequences a faint, but detectable, MPE . Fe(II) cleavage pattern is observed on DNA, in some regions similar to and in some regions different from the strong chromatin-specified pattern. The results indicate that MPE . Fe(II) will be very useful in the analysis of chromatin structure.

Chromatin structure differs between coding and upstream flanking sequences of the yeast 35S ribosomal genes.

Staphylococcal nuclease (EC 3.1.4.7) and DNase I (EC 3.1.4.5) digestion analysis of the nuclear chromatin structure of the yeast 35S rDNA gene shows the presence of typical and homogeneous nucleosome patterns across the coding sequence. These nucleosomal patterns change abruptly, around the site of transcription initiation and upstream in the 5'-flanking sequences, to a unique pattern with both nucleosomal and nonnucleosomal character. The mix arises, at least partly, from heterogeneity within the population of upstream regions; some regions are nucleosomal, but the majority are nonnucleosomal. The nonnucleosomal set of upstream regions appears to be nucleoprotein associated and, in fact, may be an altered nucleosome structure rather than totally restructured. The abruptness of the transition from nucleosome to other structure suggests restricted nucleosome locations in the region around the transcription initiation site of this gene.

Flow cytometric analysis of cultured cells.

The development of flow cytometry by Van Dilla et al. (21), Hulett et al. (10), Dittrich and Göhde (8) and Sprenger et al. (19) has added a new dimension to the field of cytochemistry. Flow cytometry is the rapid automated measurement of cells and cellular constituents using a device known as a flow cytometer and sorter. The flow cytometer performs fluorescence analysis on a single cell at the rate of 1000 cells per second and has the capability to sort cells from the main population. In this report I will summarize the present staining techniques and illustrate how they may be used for cell cycle and chromatin structure analyses of the cultured cells.

Role of Methylation in the Induced and Spontaneous Expression of the Avian Endogenous Virusev-1: DNA Structure and Gene Products

The endogenous avian provirus ev-1 is widespread in white leghorn chickens. Although it has no major structural defects, ev-1 has not been associated with any phenotype and is ordinarily expressed at a very low level. In this report, we describe a chicken embryo (Number 1836) cell culture containing both ev-1 and ev-6 which spontaneously expressed the ev-1 provirus. This culture released a high level of noninfectious virions containing a full complement of virion structural (gag) proteins but devoid of reverse transcriptase activity or antigen. These virions contained 70S RNA closely related to the genome of Rous-associated virus type 0, but identifiable as the ev-1 genome by oligonucleotide mapping. A fraction of the RNA molecules in the 70S complex were unusual in that they were polyadenylated 100 to 200 nucleotides downstream of the usual polyadenylation site. Eight sibling embryo cultures did not share this unusual phenotype with 1836, indicating that it was not inherited. However, an identical phenotype was inducible in the sibling cultures by treatment with 5-azacytidine, an inhibitor of DNA methylation, and the induced expression was stable for more than 10 generations. Analysis of chromatin structure and DNA methylation of the ev-1 provirus in 1836 cells revealed the presence (in a fraction of the proviruses) of both DNase I hypersensitive sites in the long terminal repeats and in gag and a pattern of cleavage sites for methyl-sensitive restriction endonuclease not found in a nonexpressing sibling. These results lend strong support to the role of DNA methylation in the control of gene expression. Additionally, they explain the lack of phenotype associated with ev-1 as due to a combination of its low expression and defectiveness in pol and env.

Role of methylation in the induced and spontaneous expression of the avian endogenous virus ev-1: DNA structure and gene products.

The endogenous avian provirus ev-1 is widespread in white leghorn chickens. Although it has no major structural defects, ev-1 has not been associated with any phenotype and is ordinarily expressed at a very low level. In this report, we describe a chicken embryo (Number 1836) cell culture containing both ev-1 and ev-6 which spontaneously expressed the ev-1 provirus. This culture released a high level of noninfectious virions containing a full complement of virion structural (gag) proteins but devoid of reverse transcriptase activity or antigen. These virions contained 70S RNA closely related to the genome of Rous-associated virus type 0, but identifiable as the ev-1 genome by oligonucleotide mapping. A fraction of the RNA molecules in the 70S complex were unusual in that they were polyadenylated 100 to 200 nucleotides downstream of the usual polyadenylation site. Eight sibling embryo cultures did not share this unusual phenotype with 1836, indicating that it was not inherited. However, an identical phenotype was inducible in the sibling cultures by treatment with 5-azacytidine, an inhibitor of DNA methylation, and the induced expression was stable for more than 10 generations. Analysis of chromatin structure and DNA methylation of the ev-1 provirus in 1836 cells revealed the presence (in a fraction of the proviruses) of both DNase I hypersensitive sites in the long terminal repeats and in gag and a pattern of cleavage sites for methyl-sensitive restriction endonuclease not found in a nonexpressing sibling. These results lend strong support to the role of DNA methylation in the control of gene expression. Additionally, they explain the lack of phenotype associated with ev-1 as due to a combination of its low expression and defectiveness in pol and env.

Analysis of chromatin structure and DNA sequence organization: use of the 1,10-phenanthroline-cuprous complex.

Limited treatment of Drosophila nuclei with the 1,10-phenanthroline-cuprous complex leads to rapid production of nucleosomal ladders indistinguishable from those obtained by micrococcal nuclease digestion. An investigation of the preferential sites of cleavage of protein-free DNA at locus 67B1 surprisingly indicated that both reagents recognized very similar features. Thus, a virtually identical pattern of preferential cleavages was generated over a 12 kb fragment encoding four transcripts at this locus. The distribution of cleavage sites was highly non-random, with major sites falling in the spacers between the genes. Both reagents cleaved certain chromatin-specific sites near the 5' ends of the genes. However, an analysis of preferential cleavages at the sequence level did not reveal the same close correspondence. We suggest that both reagents can recognize some localized secondary structural features of the DNA and that the particular distribution of sequences present at this locus results in a distinctive pattern of cleavage sites that delineates gene and spacer segments.

Quantitative analysis of chromatin structure by circular dichroism

Quantitative analysis of the circular dichroism of nucleohistones and protein-free DNA was carried out in order to determine the structure and the role of the linker region DNA in chromatin, in terms of the conformational change of chromatin as a function of the ionic strength. It is shown clearly that the circular dichroism of Hl-depleted chromatin isolated from calf thymus is determined only by the ratio of the core region to the linker region and demonstrated by the linear combination of the spectrum of protein-free DNA and that of the nucleosome core in 5 m m-Tris · HCl, 1 m m-EDTA (pH 7.8). The calculated spectrum for the linker region in the H1-depleted chromatin was in good agreement with that of protein-free DNA. From the difference spectra between nucleohistones and protein-free DNA, it is suggested that the chromatin has an additional winding of DNA other than 146 base-pairs of DNA around the histone core. By decreasing the ionic strength to values lower than 5 m m-Tris · HCl, 1 m m-EDTA, the ellipticity of H1-depleted chromatin increased greatly between 250 nm and 300 nm while the increase was small in the case of chromatin and the nucleosome core. Nucleosomes with linker region DNA but without histone H1 also show great increase in ellipticity in this range of wavelengths as the ionic strength is decreased. Therefore, the linker region in H1-depleted chromatin plays an important role in the conformational changes brought about by changes in the ionic strength, and the conformational changes caused in the DNA of chromatin by decreasing the ionic strength are suppressed by the presence of histone H1.

Effects of aurintricarboxylic acid and formaurindicarboxylic acid on nuclei and chromatin

Aurintricarboxylic acid and formaurindicarboxylic acid were used as a low molecular weight probe for the analysis of chromatin structure. These dyes induced strong nuclear swelling. The swelling reaction was monitored by the turbidity change of nuclear suspension. A stoichiometric relationship was suggested between the concentration of aluminon (ammonium aurintricarboxylic acid) at which maximal nuclear swelling occurred and the amount of nuclear DNA in the system. The swelling velocity was much slower at pH 5.4 than at pH 7.9. The swelling time course at pH 5.4 consisted of four distinct successive steps with different swelling velocities. In the presence of 1 m m Ca 2+ aluminon failed to induce nuclear swelling. The inhibition was slight, however, if Ca 2+ was added after initiation of swelling. Centrifugation of chromatin treated with formaurindicarboxylic acid in a linear gradient of sucrose revealed decreased chromatin density and dissociation of histones from the chromatin. After the dissociation H3 and H4 histones formed aggregates of various size. From the binding of formaurindicarboxylic acid to nuclei, the apparent binding parameters were estimated for intranuclear binding sites.

Chapter 13 Preparation of Chromatin and Chromosomes for Electron Microscopy

This chapter discusses some methods for preparation of chromatin and chromosomes for electron microscopy. Analysis of chromatin structure in situ must be done in sections of cells. Because chromatin fibers are usually closely packed and complexly folded, thin sections of well-fixed cells are not very informative. The situation is somewhat improved by swelling the chromatin in hypotonic fixatives. The fibers are then separated from each other and measurement of fiber thickness is greatly facilitated. In the usual thin sections, it is impossible to get information on the spatial arrangement of chromatin fibers. High-voltage electron microscopes (one million eV) have now become available for biomedical research providing a resolution of 20–50 Å with sections up to several micrometers thick. As section thickness increases, longer segments of chromatin fibers are contained in a section and the three-dimensional arrangement becomes more evident, especially in stereoscopic photographs. The practical limit to section thickness is in the overlap of structures that makes analysis difficult. The chapter also presents an overview of hypotonic fixation. Preparation of isolated chromatin suspension for electron microscopy and spreading of chromatin and chromosomes on a hypophase are discussed in the chapter.