Analysis of human metaphase chromosomes using antibodies to double-stranded and single-stranded DNA: staining patterns are related to DNA conformation

Fresh and 6-day-old fixed chromosome spreads, both untreated and treated with various banding techniques and nucleases, were stained using monoclonal antibodies to double-stranded and single-stranded DNA. DNA in fixed chromosome preparations became progressively denatured with ageing. The staining pattern of untreated chromosomes with anti-double-stranded DNA antibodies (which resembles G-banding) was determined by the conformation of the chromosomal DNA.     

Immunofluorescent staining of mammalian nuclei and chromosomes with a monoclonal antibody to triplex DNA

Triplex DNA is an unusual conformation of DNA formed when two pyrimidine nucleotide strands share a common purine strand. A monoclonal antibody, demonstrated by numerous criteria to be specific for triplex DNA, was used to investigate the presence and distribution of this unique DNA configuration in nuclei and chromosomes of mouse LM cells and human lymphocytes. Indirect immunofluorescence microscopy revealed that constitutive heterochromatin in acetic-methanol fixed mouse nuclei was usually, but not always immunofluorescent, suggesting possible cell cycle related variations in the amount of triplex DNA or its accessibility in this condensed chromatin. In fixed mouse and human chromosomes, there was a positive correlation between immunofluorescent staining patterns, Hoechst 33258 banding, and G- and/or C-banding patterns. Unfixed, isolated mouse chromosomes also reacted positively with the antibody, particularly when they were gently decondensed by exposure to low ionic conditions at neutral pH. This result indicates that fixation is not mandatory for antibody staining, suggesting that some mammalian chromosomal DNA may be naturally organized in a triplex configuration. However, there is a possibility that fixation may facilitate the formation of additional triplex DNA complexes in potential sequences or expose previously inaccessible triplex DNA. The precise correspondence between the immunofluorescent patterns produced by anti-triplex DNA antibodies and G- and C-bands known to represent regions of chromatin condensation, suggests a potential role of triplex DNA in chromosome structure and regional chromatin condensation.     

Reversible differential decondensation of unfixed Chinese hamster chromosomes induced by change in calcium ion concentration of the medium

Differential decondensation of isolated unfixed Chinese hamster metaphase chromosomes was obtained by decreasing the calcium ion concentration in the surrounding medium. A banded appearance of the swollen chromosomes could be observed either directly by phase contrast microscopy or after glutaraldehyde fixation and staining. There was a gradual transition from homogeneously dense to banded and finally to extensively decondensed chromosomes. The patterns induced at different stages were similar to those observed on fixed chromosomes after standard banding procedures (i.e., G-, C-, C_d–, Ag-NOR-staining). Chromosome decondensation could be reversed by the addition of calcium ions to the medium. Ca⁺⁺-dependent reversible differential chromosome decondensation was not observed if the chromosomes were previously treated with 0.35 M NaCl. Chromosome regions which had incorporated BrdU into their DNA were more resistant to a decrease in calcium ion concentration than BrdU non-substituted regions.     

Visualization of R-bands in human metaphase chromosomes by the restriction endonuclease MseI

Human metaphase chromosomes were treated with the restriction endonuclease MseI, which cuts DNA at TTAA sequences. This enzyme preferentially cuts and extracts DNA from G-bands and thus is the first restriction endonuclease allowing direct R-band visualization. Specific patterns ranging from R+C-like to C-like banding can be induced, depending on the concentration of the enzyme. At intermediate concentrations, only a subset of R-bands are produced, corresponding to GC-rich bands that are especially resistant to heat denaturation (so-called T-bands). These results suggest that compositional differences between chromosomal regions determine the different rates of cleavage by MseI, not only between R- and G-bands but also among different R-bands.     

Dependence of heterochromatin differential staining on the time of its reduplication and the degree of condensation]

A comparison of the chromosomes banding pattern after G-and C-staining with the time of DNA reduplication and the degree of chromosome condensation, was carried out using Chinese hamster metaphase chromosomes. Chromosome condensation was studied under 5-bromodeoxyuridine and 5-bromodeoxycytidine treatment. All the chromosomal segments stained with C-technique are also stainable with G-technique, while only some G-positive segments are capable to be C-bands. C-bands are heterochromatic segments characterized by extremely late replication and great delay in condensation under the analog action, while G-bands are segments with earlier labelling and irregular decondensation. The data obtained suggest a close correlation between the capability of chromosomal region of G- and C- staining and the degree of its heterochromatinization.     

Nucleotide sequence of an heterochromatic segment recognized by the antibodies to Z-DNA in fixed metaphase chromosomes.

The purpose of this work was to analyse at the molecular level the DNA recognized by the antibodies to Z-DNA in in situ experiments. Antibodies to Z-DNA interact strongly with R-band positive heterochromatic segments of fixed metaphase chromosomes of Cebus (Viegas-Pequignot et al., 1983). These segments are constituted of a satellite DNA the repeat unit of which is about 1520 base pairs long. The base sequence of the repeat unit has been determined. It contains a (AC)n rich region which, in vitro, adopts the Z conformation under topological constraints. Experiments with nuclei suggest that this sequence is not predominantly in the Z conformation in vivo. The polymorphic structure of the (AC)n rich region argues for an active recombination sequence.     

Identification of left-handed Z-DNA by indirect immunomethods in metaphasic chromosomes of a mammal, Gerbillus nigeriae (Gerbillidae, Rodentia)

Antisera and antibodies against Z-DNA were used on metaphasic fixed chromosomes of a mammal, Gerbillus nigeriae (Gerbillidae, Rodentia). By indirect immunofluorescence and indirect immunoperoxidase labelling a heavy staining was detected in a fraction of the R-band positive heterochromatic segments, which are presumed to be rich in G-C base pairs. A weak and non homogeneous staining was also observed on euchromatic segments. The presence of a left-handed Z-DNA in mammalian chromosomes is discussed.     

Influence of bromodeoxyuridine on the stability and function of polytene chromosomes

Polytene chromosomes of Drosophila hydei have been employed to study the effect of bromodeoxyuridine (BrdU) on chromosome function and structure. Two media have been tested for their capacity to permit substitution of thymidine by BrdU in larval salivary glands. In medium L up to 80% of the polytene DNA is replicated with maximum substitution of 80% of the thymidine in one strand by BrdU. The degree of substitution achieved with this medium is uniform within individual nuclei but varies between nuclei. An alternate medium (S) results in more complete substitution but less total DNA synthesis. Autoradiographic analyses reveal that extensive BrdU substitution is correlated with an inhibition of chromosomal RNA synthesis. Nucleolar RNA synthesis is almost totally suppressed by exposure to BrdU. Chromosomes which exhibit extensive BrdU incorporation produce puffs in response to a temperature shock or in vitro incubation. Preliminary studies suggest that puffing at some loci is anomalous. The data, therefore, indicate that moderate BrdU incorporation completely suppresses nucleolar RNA synthesis and reduces chromosomal RNA synthesis but does not generally prevent the induction of new puffs. — Exposure of larvae to BrdU also stabilizes the morphology of their polytene chromosomes in the presence of high pH and high ionic strength. Chromosome morphology that is lost at 0.6 M NaCl is regained at 0.15 M NaCl in isolated nuclei derived from BrdU-fed larvae. No recovery is observed in chromosomes of unsubstituted control larvae.     

Immunocytochemical analysis of human metaphase chromosome methylation status

The present paper describes a distribution of 5-methylcytosine-rich DNA in human metaphase chromosomes from PHA-stimulated lymphocytes. Immunocytochemical detection of 5-methylcytosine was carried out with monoclonal antibodies. Fluorescent signals were preferentially localized in certain chromosomal regions, corresponding to R-, some T-bands, pricentromeric heterochromatin, and short arms of acrocentric chromosomes. Specificity of fluorescent signals distribution along chromosomes allowed to describe a new type of human metaphase chromosomes banding pattern, which we call M-banding. Specific M-markers of landmarks were identified for each chromosome pair. The analysis of M-bands methylation status was carried out taking into account data available in literature on their nucleotide structure features, namely GC-rich H3 isochore content and CpG-islands concentration. According to our results, a high level of methylation is typical for the majority of GC-rich regions. However, certain bands of 6, 9, 10, 13, 15 chromosomes (6q15, 6q21, 6q23, 9p13, 9p22, 9p32, 10q24, 13q22, 15q15, 15q24) were shown to be hypomethylated, suggesting their special functional status in lymphocytes.     

Compositional properties of telomeric regions from human chromosomes.

We have investigated the GC levels of third codon position of genes localized in G- (Giemsa), R-(reverse) and T-(telomeric) bands of human metaphase chromosomes, as well as the hybridization of telomeric probes on fractionated human DNA. The first set of results shows much higher GC levels for genes localized in T-bands than in G- or R-bands (the latter being higher than the former). The second set of data shows that telomeric probes corresponding to T-bands hybridize on the GC-richest family (H3) of isochores, whereas telomeric probes corresponding to R-bands hybridize on GC-rich families H1 and H2; in agreement with these findings, the telomeric repeat common to all chromosomes hybridized on isochore families H1, H2 and H3.     

S1 nuclease removes DNA from eukaryotic metaphase chromosomes: cytological evidence

Fixed and unfixed human chromosomes, as well as fixed rye chromosomes were treated with S1 nuclease, which specifically cleaves single stranded DNA. Subsequent staining with either acridine orange, ethidium bromide or Giemsa revealed that, contrary to what has previously been reported, S1 digestion extensively altered chromosomal morphology and staining intensity, although the alteration was more pronounced in fixed as compared to unfixed metaphases. A number of mechanisms, which may account for our findings, have been invoked: a) the presence in metaphase chromatin of B-DNA/Z-DNA transitional junctions, b) the induction, by alcohol: acid fixation procedure, of nicks within regular B-DNA conformation and c) the induction of sites available to S1 by torsional stress due to metaphase high condensation degree.     

BrdU related direct revelation of typical dynamic R- and G-banding with the use of monoclonal anti-BrdU antibody.

Pulse 5-bromodeoxyuridine (5-BrdU) incorporation during the last S-phase is known to produce R- or G-banded chromosomes after photolysis-plus-Giemsa (FPG) staining. The authors applied an immunological staining with monoclonal anti-BrdU antibody instead of the FPG protocol. The results offered banded chromosomes with an immunological typical R-banding (RBI) on the GBG cultivated cells (early pulse incorporation), and an immunological G-banding (GBI) on the RBG cultivated ones (late pulse incorporation). After a further FPG protocol following an immunological treatment, an inverted banding pattern became evident whereas a faint immunological staining remained. Thus the method superimposed a GBG-banding on the RBI-staining or a RBG on the GBI one. This allows a rapid and easy R and G double chromosomal identification on the same metaphase cell, using first the immunological banding then the classical FPG staining. The method allows a reproducible dynamic G-banding with an easy monitored late 5-BrdU pulse incorporation specially attractive in spontaneous dividing cells from bone marrow. This dynamic G-banding protocol should be extended to chorionic villi and malignant cells. Our data are in agreement with a connection between dynamic banding and chromosomal portions containing or not BrdU. The lack of an immunological staining after the FPG protocol has been noticed and assume the photolysis degradation-elution of the DNA in BrdU-substituted areas.     

Chromosome banding in Amphibia. XXVII. DNA replication banding patterns in three anuran species with greatly differing genome sizes

The mitotic chromosomes of three anuran species, Scaphiopus holbrooki, Litoria infrafrenata and Odontophrynus americanus, were analyzed by means of the 5-bromodeoxyuridine/deoxythymidine (BrdU/dT) replication banding technique. These species exhibit large differences in their genome sizes: S. holbrooki possesses one of the smallest genomes among vertebrates, L. infrafrenata has a genome size near the modal DNA value of most Amphibia, whereas O. americanus is a tetraploid species. BrdU/dT labeling induces reproducible and reliable R- and G-replication bands along the metaphase chromosomes of all three species. Irrespective of the genome size of the species considered, the number of early (R-) and late (G-) replicating bands per haploid karyotype is nearly the same. The chromosomes of the autotetraploid O. americanus can be arranged into sets of four homologous chromosomes (quartets). C-bands and BrdU/dT replication bands reveal heterogeneity within the quartets 1, 3 and 4 that are interpreted as the initiation of a diploidization process.     

Characterisation of a very complex constitutive heterochromatin in two Gerbillus species (Rodentia)

A large amount of heterochromatin is observed in two species of the genus Gerbillus, G. nigeriae and G. hesperinus. The C-band material represents about one-half of the total karyotype length in the former species, and about one-third in the latter. Several banding techniques and various 5-bromodeoxyuridine (BrdU) treatments were used to characterise these heterochromatic segments. After applying the R-banding technique, three different staining responses of the heterochromatin can be distinguished. In G. nigeriae, strongly stained segments (R-band positive) appear in most chromosomes and, in particular, constitute the short arms of all the larger chromosomes. Palely staining heterochromatic segments (R-band negative) are less abundant in G. nigeriae but predominate in G. hesperinus. In addition, in both species an intermediate staining of heterochromatin is observed near the centromere or in the heterochromatic short arms of some acrocentric and small submetacentric chromosomes. Very short BrdU treatment during the end of the last cell cycle results in asymmetrical staining of chromatids in heterochromatic segments after applying the acridine orange or FPG (fluorescence plus Giemsa) technique. The alternating location of strongly staining segments in one or the other chromatid simulates sister chromatid exchanges (pseudo-SCE). This pattern persists after longer BrdU treatment during different stages of the last cell cycle and is independent of the R-staining properties of the heterochromatin. The lateral asymmetric appearance of the large heterochromatic segments in Gerbillus is interpreted as reflecting an uneven distribution of adenine and thymidine between the two strands of DNA.     

Analysis of Z-DNA in fixed polytene chromosomes with monoclonal antibodies that show base sequence-dependent selectivity in reactions with supercoiled plasmids and polynucleotides

Five monoclonal anti-Z-DNA antibodies were characterized with respect to their binding of synthetic nucleic acid polymers and of supercoiled circular plasmid DNA. All of the antibodies reacted only with DNA in the Z-conformation; however, they fell into two classes on the basis of sequence specificity. One class, with broad specificity, reacted well with all sequences in the Z-form, including poly(dG-dC), poly(dG-dm5C), and poly (dG-dBr5C) in linear polymers and poly(dG-dC)n and poly[(dC-dA)n.(dT-dG)n] sequences in supercoiled plasmids. The other class bound only Z-DNA formed by poly(dG-dC). Binding of the monoclonal antibodies specifically to inserts of Z-DNA-forming sequences in plasmids was mapped directly by cross-linking of antibody to the DNA, digestion with restriction nuclease, and electrophoretic analysis of both the unbound fragments and the bound fragments recovered from immune complexes. The monoclonal antibodies were used for indirect immunofluorescence staining of Drosophila polytene chromosomes fixed by two procedures. One procedure yielded chromosomes with Z-specific antibody binding in many interbands, a few specific bands, and parts of some puffs. On chromosomes fixed by the second procedure, antibody staining appeared to follow the DNA concentration, staining all bands brightly. For each fixation procedure, chromosomes showed the same staining pattern with each of the broad specificity monoclonal antibodies that had been seen with polyclonal antibodies. The antibodies that reacted only with poly(dG-dC) and poly (dG-dC)n plasmid inserts did not stain chromosomes fixed by either protocol. We conclude that stretches of poly(dG-dC)n sequences do not contribute significantly to the presence of Z-DNA in fixed polytene chromosomes of Drosophila melanogaster.     

Factors influencing the frequency of mitomycin C-induced sister-chromatid exchanges in 5-bromodeoxyuridine-substituted human lymphocytes in culture.

Newly developed techniques for the detection of sister-chromatid exchanges (SCE) require the substitution of 5-bromodeoxyuridine (BrdU) for thymidine in DNA. We investigated the possibility of interactions between BrdU and one mutagen--carcinogen, mitomycin C (MMC) for the induction of both chromosomal aberrations and SCE in human peripheral lymphocytes in culture. No effect on aberration yield was found. Neither comparisons between the yields of SCE by BrdU substitution and differential staining and those detected by tritiated thymidine incorporation and autoradiography nor between the yields of SCE for different levels of BrdU incorporation provided any evidence of synergism. It was found, however, that MMC persists in cultures and continues to increase SCE frequencies for about 30 h. It was also observed that some MMC-induced DNA lesions persist long enough so that some of those present prior to S phase of the first cell cycle cause additional SCE in the third cycle.     

A structural basis for R- and T-banding: a scanning electron microscopy study

The structure of reverse (R)-banded and telomeric (T)-banded chromosomes was studied by examination of the same chromosomes first in the light microscope (LM) followed by the scanning electron microscope (SEM). This procedure demonstrated a structural basis to both the R- and T-banding techniques. A direct correlation was shown between the LM staining patterns and the structural patterns observed in the SEM. In the R-banded chromosomes the positively stained R-bands, viewed by LM, corresponded to highly fibrous three-dimensional regions in the SEM. The negatively stained R-interbands corresponded to flatter regions from which material appeared to have been extracted. These structural observations strongly support the suggestion that chromosomal material is preferentially lost from the R-interbands with aggregation of fibres in the R-bands. T-banded chromosomes showed a similar structure to the R-banded chromosomes. The positively stained T-bands located at the telomeres corresponded to regions of highly aggregated fibres. The remainder of the chromosome, corresponding to the negatively stained area, had a flattened and extracted appearance. These similarities in morphology between the T- and R-banded chromosomes support the view that T-bands result from a progressive breakdown of the R-banded chromosome structure.     

Identification of left-handed Z-DNA by indirect immunofluorescence in polytene chromosomes of Chironomus thummi thummi

In this work, antibodies against Z-DNA were used to stain polytene chromosomes of Chironomus thummi thummi. By indirect immunofluorescence we report the first identification of left-handed conformation of DNA in a band region. The Chironomus pattern also contrasts with the general staining observed in Drosophila. In Chironomus the antibodies to Z-DNA bind to one interband region of the chromosome II and two bands regions of the chromosome IV.     

A relation between G-, C-, and N-band patterns as revealed by progressive oxidation of chromosomes and a note on the nature of N-bands

Near-ultraviolet irradiation of chromosome preparations mounted in a hydrogen peroxide solution resulted in an oxidative disintegration of the structure of fixed metaphase chromosomes with concomitant production of various band patterns appearing after staining with Giemsa. Neither irradiation nor hydrogen peroxide alone could produce banding. After irradiation in the presence of hydrogen peroxide the gradually increasing effect of oxidation on the chromosomes along the gradient of light intensities from the periphery of the slide towards the radiation focus in the centre of the slide became visible as G-, C-, and N-banding, respectively. Close to the centre only contours of chromosomes were left after this treatment. Although G-banding and differential DNA-extraction often went together, extraction of DNA was not an absolute requirement to obtain a G-band pattern. N-bands appeared to be the chromosomal regions that were most resistant to destruction. Staining methods specific for DNA failed to demonstrate these bands, although with Giemsa an intense staining reaction occurred. On the analogy of the staining behaviour of model protein preparations with Giemsa a phosphoprotein nature is suggested for the N-band material in the chromosomes.     

Specific modification of Q and R bands by 5-bromodeoxyuridine and actinomycin D]

When introduced into human lymphocyte culture, 5-bromodeoxyuridine (BUdR) and actinomycin D (AMD) induce chromosome differentiation by lack of condensation of segments corresponding to Q-bands (BUdR) and R-bands (BUdR and AMD). The total amount of DNA per cell is not modified by these treatments. The non-condensed segments partly keep their properties of R- or Q-banding after heat treatment or staining with quinacrine mustard. On the other hand, they lose their properties after ASG treatment (G-bands), and emit modified fluorescence after staining with acridine orange. With heat treatment or QM staining, it seems that BUdR or AMD elongate the R or Q segments in several ways—homogeneous repartition or fragmentation of various types. On the other hand, this elongation seems homogeneous after Feulgen staining. This suggests that the relation between Feulgen-revealed DNA and substratum of the R- and Q-bands might not be direct.