The use of antinucleoside antibodies to probe the organization of chromosomes denatured by ultraviolet irradiation

Ultraviolet irradiation of methanol: acetic acid-fixed human and mouse metaphase chromosomes rendered them capable of binding antibodies specific for purine or pyrimidine bases. Since these antibodies react with single-stranded but not with native DNA, our results indicate that UV irradiation generated single-stranded regions in chromosomal DNA. Using an indirect immuno-fluorescence technique to detect antibody binding, highly characteristic, nonrandom patterns of antibody binding were observed. Antibodies to adenosine (anti-A) and thymidine (anti-T) produced identical patterns of binding which in most respects matched the chromosome banding patterns produced by quinacrine. However, additional foci of intense fluorescence were seen in the paracentromeric regions of constitutive heterochromatin on chromosomes 1, 9 and 16, regions which had been shown by in situ DNA-RNA hybridization to be the locations of AT-rich human satellite DNA. Antibodies to cytidine also bound to the same region of chromosome 9. In mouse chromosome preparations, both anti-A and anti-T produced bright fluorescence of the region containing centromeric heterochromatin, which had been shown to be the location of the AT-rich satellite DNA of this species.     

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.     

Regional differences in immunostainability of isolated metaphase chromosomes of Indian muntjac with anti-Z-DNA antibody

The distribution of Z-form DNA along the length of metaphase chromosomes of Indian muntjac was studied by indirect immunofluorescence procedures using an antibody specific to the Z-DNA conformation. Several fixation conditions were compared for reproducible detection of Z-DNA in isolated metaphase chromosomes. Fixation of chromosomes with 45% acetic acid alone gave reproducible reactivity with the antibody. When fixation was done either with Carnoy's solution (3:1 methanol:acetic acid) or with 75% alcohol alone, the antibody binding was at background level. Acetic acid-fixed chromosomes exhibited intense fluorescence both at C-band heterochromatin and at nucleolus organizer regions (NORs). The euchromatic regions had weakly, but clearly, stained bands, which were quite similar to the chromomycin A₃ R-bands. After treatment with topoisomerase I, the immunofluorescence at NORs and R-bands disappeared, but only a slight decrease in immunofluorescence intensity was observed at C-band regions. We suggest that this difference in the immunoreactivity of NORs and R-bands from C-bands reflects a difference in gene activity among these regions. Possible molecular mechanisms involved in Z-DNA immunoreactivity are discussed, based on SDS-polyacrylamide gel electrophoretic analysis of chromosomal proteins after extraction of metaphase chromosomes with different fixative solutions.Abbreviations PI propidium iodide - NOR(s) nucleolus organizer region(s) - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis Deceased, April 23, 1988     

Immunological detection of left-handed Z DNA in isolated polytene chromosomes. Effects of ionic strength, pH, temperature and topological stress

We have searched for the presence of left-handed Z DNA in unfixed polytene chromosomes isolated from the salivary glands of Chironomus thummi larvae. Physiological as well as fixation conditions were explored to assess the effects of a variety of factors known to influence the B-Z equilibrium. At neutral pH and physiological ionic strength, a weak immunofluorescence staining confined to the periphery of chromosomal bands is elicited but only by using high concentrations of anti-Z DNA immunoglobulin (IgG). The accessibility of internal highly condensed structures, as monitored with antibodies against core histones, is very limited under these conditions. Increasing the ionic strength exposes core histone determinants but results in a decondensation of the bands. The staining for Z DNA is still weak and primarily restricted to regions resisting decondensation or undergoing collapse. Dramatic changes in anti-Z DNA immunofluorescence intensities occur upon short exposure to low pH. Adjustment of the pH between 2.5 and 2.0 leads to an abrupt large increase in antibody binding, at first confined to a few specific bands and then generalized to bands throughout the chromosomes in a pattern very similar to that elicited in classical acid-fixed squash preparations. The acid-mediated effects are influenced by ionic strength, temperature and prior removal of histones; they can be mimicked by exposure to high temperature at neutral pH. The 'transition pH' assessed with a monoclonal IgG specific for left-handed d(G-C)n sequences is slightly lower than in the case of polyclonal antibodies which also recognize d(A-C)n X d(G-T)n.(ABSTRACT TRUNCATED AT 400 WORDS)     

An immunofluorescent analysis of Drosophila polytene chromosomes with antisera directed against H3, H4, and a native H3–H4 complex

Fixed polytene chromosome preparations have been stained by indirect immunofluorescence. Anti-H3 serum and anti-H4 serum cause very intense and highly specific staining of chromosomes. Anti-(H3–H4 complex) serum did not produce staining of chromosomes at a level above background. The results obtained in these staining experiments are in direct contrast to serological results obtained with soluble chromatin. It appears that a unique structure exists within the H3–H4 complex that is not present on the individual histone components. This structure is apparently destroyed or obscured by acetic acid fixation during the preparation of polytene chromosome spreads.     

Nuclear protein matrix in giant nuclei from salivary glands of <Emphasis Type="Italic">Chironomus plumosus</Emphasis>

Polythene chromosomes from salivary glands of Chironomus plumosus were treated in situ in order to reveal residual nuclear protein matrix (NPM). It was shown that after the removal of H1-histones by 0.6 M NaCl the general morphology of chromosomes is preserved, revealing distinct banding pattern. Further treatment of chromosomes with 2 M NaCl and DNase completely disorganized the structure of chromosome bodies and patterns of banding. Preliminary treatment of salivary glands with 2 mM CuCl₂ resulted in stabilization of the structure of polythene chromosome in every stage of histone and DNA extractions. Stabilized chromosomes maintained their morphology and banding patterns observed by phase contrast or after the staining with Brilliant blue. Thus, after the removal of histones and DNA, stabilized chromosomes retain their morphological features, which depend on the presence of NMP. In stabilized polythene chromosomes, in spite of the absence of histones and DNA, topoisomerase IIα retains its localization, typical for untreated chromosomes.     

T antigen banding on chromosomes of simian virus 40 infected muntjac cells.

Chromosomes were prepared from mitotic munjac cells 48 to 72 h after infection with SV40 virus. When stained for SV40 T antigen by indirect immunofluorescence, all chromosomes within an infected cell were fluorescent, indicating the presence of T antigen. Furthermore, the chromosomes were not uniformly stained but appeared to have regions of high and low fluorescence intensity. A variety of controls showed that the banding patterns are specific and highly reproducible and may indeed reflect the binding sites of T antigen. The bright, fluorescent bands T antigen were found to correspond to bands visualized by trypsin-Giesma staining (G-bands) and also by quinacrine staining (Q-bands). Current knowledge of chromosome banding indicates that Q-bands reflect the distribution of AT-rich regions along the chromosome. From the DNA sequence of SV40, it is known that one of the T antigen binding sites contains AT-rich sequences; thus, T antigen banding might be due to the base-specific binding of T antigen to chromatin. In addition, these bands have been implicated as centers for chromosome condensation and units in control of DNA replication. While the functional significance of T antigen binding has yet to be determined, the SV40-muntjac system provides an unusual opportunity to study the interaction of a known regulatory protein with mammalian chromosomes.     

Immunofluorescent studies of human chromosomes with antibodies against phosphorylated H1 histone

One of the prominent cell cycle-related modifications of histone proteins whose function remains unresolved is the phosphorylation of linker histone H1. In this work we have used indirect immunofluorescence on human cells with antibodies that are specific for phosphorylated histone H1 to examine the cellular distribution and chromosome association patterns of this protein. With confocal microscopy on whole cells, strong immunofluorescence was seen in association with mitotic chromosomes as well as a prominent punctate pattern of labeling throughout the mitotic cell, whereas interphase cells showed very little, if any, specific fluorescence. Multiple patterns of fluorescence distribution were detected with metaphase chromosomes, ranging from apparent tight colocalization with the DNA to expanded ”puffy” mitotic figures to an amorphous network of staining. It was also shown that the ability to label chromosomes could vary drastically with different fixation procedures, adding further complications to interpretation of the potentially complex role of phosphorylated histone H1 in chromatin condensation or decondensation. Received: 8 September 1999; in revised form: 14 September 1999 / Accepted: 17 September 1999     

Subnuclear distribution of the entire complement of linker histone variants in Arabidopsis thaliana

Linker histones (e.g. H1, H5, H1°) are thought to exert control on chromatin function by restricting nucleosomal dynamics. All higher eukaryotes possess a diverse family of linker histones, which may exhibit functional specialization. Arabidopsis thaliana apparently contains a minimal complement of linker histone structural variants and therefore is an ideal model for investigating functional differentiation among linker histones. Histones H1-1 and H1-2 are relatively similar proteins that are expressed in a wide variety of tissues and make up the majority of linker histone while H1-3 is a highly divergent minor variant protein that is induced by drought stress. We are interested in determining whether the in vivo distribution of each of these proteins also differs. To this end, we have produced subtype-specific antibodies and have localized each of the three proteins at the intranuclear and DNA sequence levels by indirect immunofluorescence and immunoprecipitation, respectively. Antibodies against linker histones H1-1 and H1-2 decorate nuclei in patterns very similar to 4’,6-diamidino-2-phenylindole (DAPI) staining, but different than the staining pattern of total histones. In contrast, antibodies made against two regions of H1-3 bind to chromatin in a diffuse pattern distinct from the DAPI-staining pattern. We also describe a technique to determine the localization of plant linker histone variants along regions of chromatin, employing in vivo chemical DNA-protein cross-linking to preserve native associations followed by immunoprecipitation with subtype-specific antibodies. We use this technique to demonstrate that, in contrast to the major linker histones, H1-3 does not bind the repetitive sequences pAL1 and 5S rDNA. In addition, we show that linker histones are bound to the compacted nucleosomal arrays at the telomere but with reduced stoichiometry. Taken together, our results suggest that plants, as has been shown for animals, possess a variant linker histone that is differentially localized. Received: 15 April 1999 / Accepted: 1 Mai 1999     

Immunofluorescent study of the basic chromosomal protein set of green algae and Euglena]

Using antisera to fractions H1, H2a, H3 and H4 of the calf thymus histones, a comparative immunofluorescent investigation of these proteins in the nuclei of Chlamydomonas reinhardii, Haematococcus pluvialis, Dunaliella salina and Euglena gracilis was carried out. It has been shown that according to the immunofluorescent test, the nuclei of these algae contain proteins close to fractions H2a, H3 and H4 of the calf thymus histones. H1 fraction in these algae is either absent or can be considered as a protein immunochemically non-related to H1 fraction of the calf thymus histone. For quantitative evaluation (in units of the immunological distance) of the difference between histones of the algae and of the calf thymus in situ by indirect immunofluorescence, it was suggested to use the ultimate dilutions of antisera to histones. It was shown that the ultimate dilutions were correlated with titres of antisera in the reaction of microcomplement fixation. Such an approach and the data obtained are of interest for studying into the evolution of nucleosome histones in unicellular and multicellular eukaryotes.     

The effect of chromosome banding techniques on the proteins of isolated chromosomes

Experiments were undertaken to determine the effect of various chromosome banding treatments on the histone and nonhistone proteins of isolated, fixed, air-dried metaphase chromosomes. Chromosome preparations were exposed to G-banding (SSC, urea, NaCl-urea, or trypsin), R-banding (Earle's balanced salt solution), and C-banding (NaOH or Ba(OH)₂) treatments, and the extracted and residual proteins were examined by SDS polyacrylamide gel electrophoresis. The results indicate that each of the banding treatments induce characteristic alterations in the chromosomal proteins. The residual proteins left in chromosomes after the diverse G-banding treatments were generally similar to one another, indicating that treatments inducing the same type of banding have similar effects on the chromosomal proteins. This was also true for the two different C-banding treatments. On the other hand, the residual protein patterns seen after the G-banding treatments were strikingly different from those seen after R-banding, which in turn differed from those seen after C-banding. The treatments inducing different types of banding therefore produce markedly different effects on the chromosomal proteins. These protein alterations may have an important influence on the induction of chromosome bands.     

Three dimensional analysis of histone methylation patterns in normal and tumor cell nuclei

Histone modifications represent an important epigenetic mechanism for the organization of higher order chromatin structure and gene regulation. Methylation of position-specific lysine residues in the histone H3 and H4 amino termini has linked with the formation of constitutive and facultative heterochromatin as well as with specifically repressed single gene loci. Using an antibody, directed against dimethylated lysine 9 of histone H3 and several other lysine methylation sites, we visualized the nuclear distribution pattern of chromatin flagged by these methylated lysines in 3D preserved nuclei of normal and malignant cell types. Optical confocal serial sections were used for a quantitative evaluation. We demonstrate distinct differences of these histone methylation patterns among nuclei of different cell types after exit of the cell cycle. Changes in the pattern formation were also observed during the cell cycle. Our data suggest an important role of methylated histones in the reestablishment of higher order chromatin arrangements during telophase/early G1. Cell type specific histone methylation patterns are possibly casually involved in the formation of cell type specific heterochromatin compartments, composed of (peri)centromeric regions and chromosomal subregions from neighboring chromosomes territories, which contain silent genes.     

Clearly differentiated and stable chromosome bands produced by a spermine bis-acridine, a bifunctional intercalating analogue of quinacrine

Characteristic fluorescent banding patterns on human metaphase chromosomes are produced by treating chromosome preparations directly with a spermine bis-acridine fluorochrome (CMA)₂S. The clearly differentiated bands are similar to those produced by quinacrine (Q-banding), but show enhanced definition between bright and dull regions as compared with the banding patterns obtained by the quinacrine technique. In addition, the bands on chromosomes produced by (CMA)₂S show insignificant fluorescence fading over extended periods of excitation. Solution interactions between DNA and (CMA)₂S showed a greater fluorescence differential between fluorescence enhancement by the alternating polymers poly d(A-T) · poly d(A-T) and fluorescence quenching by the polynucleotide poly d(G-C) · poly d(G-C) for this fluorochrome than was observed for quinacrine. The increased definition in Q-type bands produced by the spermine bis-intercalating derivative and the lack of fluorescence fading make this fluorochrome an excellent one for routine clinical cytogenetic analysis.     

Fixation-dependent organization of core histones following DNA fluorescent in situ hybridization

We have evaluated the effects of different DNA denaturation protocols commonly used in DNA fluorescent in situ hybridization (FISH) experiments on chromatin structure using indirect immunofluorescence. The use of antibodies to acetylated histones H3 and H4 demonstrates that the different procedures differ considerably in their extent of histone displacement. Procedures involving paraformaldehyde fixation were found to be compatible with the structural preservation of acetylated chromatin organization by indirect immunofluorescence. These results provide a basis for interpreting DNA FISH experiments aimed at determining chromatin organization of individual loci. Received: 19 November 1996; in revised form: 10 January 1997 / Accepted: 10 January 1997     

Antisera to individual histone fractions of the calf thymus. II. A study of the immunochemical specificity of the histones by an indirect immunofluorescence method]

Immune antisera to 5 fractions (H1, H2a, H2b, H3, H4) of calf thymus histone were assayed using indirect immunofluorescence (IIF). The analysis of such sera by this technique, as well as the data on complement fixation obtained previously, show that these antisera are highly active and specific for various test-objects: thymys, liver nuclei of rat, chicken, and calf, chicken erythrocytes, metaphase chromosomes of mouse fibroblasts. These antisera are of importance for the evaluation of species- and tissue-specificity of different histone fractions. Using the IIF reaction, a comparison was made between the nucleosome fraction H3, which is evolutionary stable, and fraction HI from calf thymus, rat and chicken liver, and chicken erythrocytes.