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.     

Characterization of Drosophila heterochromatin

The C- and N-banding patterns of D. melanogaster, D. simulans, D. virilis, D. texana, D. ezoana and D. hydei were studied in comparison with quinacrine and Hoechst banding patterns. In all these Drosophila species the C bands correspond to the heterochromatin as revealed by the positive heteropycnosis in the prometaphase chromosomes. The N bands have the following characteristics: 1) they are always localized on the heterochromatin and generally do not correspond to the C bands; 2) they do not correspond to the nucleolar organizing regions; 3) they are inversely correlated with fluorescence, i.e., they correspond to regions which are scarcely, if at all, fluorescent after Hoechst 33258 or quinacrine staining; 4) they are localized both on regions containing AT rich satellite DNA and on those containing GC rich satellite DNA.     

The effect of HCl hydrolysis on the retention of thymidine in DNA

Allium roots grown in C(14)-thymidine and H(3)-thymidine media were treated with N hydrochloric acid at 60 degrees C. as in standard Feulgen hydrolysis. The retention of the radioactive thymidine in DNA as a function of hydrolysis time was studied autoradiographically. No significant loss of label was detected until hydrolysis was extended beyond the optimal time for Feulgen staining. The data are consistent with the assumption that there is no significant loss of DNA during normal Feulgen hydrolysis in the material used.     

The relationship between repetitive DNA and chromosomal bands in man

The relationship between chromosomal bands and repetitious DNA has been investigated by means of the quinacrine fluorescence technique and in situ hybridization with c-RNA to different fractions of repetitive DNA. A comparison of the Q bands with the labelling patterns obtained showed a preferential distribution of repetitive DNA's at Cot's ranging from 0 to 5 in those regions that are Q band positive. A distinct labelling was also observed in the pericentromeric regions and in some telomeres. It is suggested that the distribution of repetitive DNA along the chromosomes plays an important role in band formation.     

Ectopic pairing of chromosome regions containing chemically similar DNA

Using genetically controlled stocks ofDrosophila melanogaster we have compared the frequency of ectopic pairing in a line showing intense quinacrine fluorescence at two sites (81F and 83E) on chromosome 3 with one showing such fluorescence at only one of these sites (81F). The frequency of ectopic pairing is an order of magnitude greater in cells from the line showing intense fluorescence in both regions than in the line showing it in only one. These data indicate that ectopic pairing is dependent upon properties of discrete chromosome regions as small as individual bands. Since A: T-rich chromatin is known to fluoresce intensely after quinacrine staining, these data further suggest that ectopic pairing is dependent on similarities of the DNA of the discrete chromosome regions involved.     

Macronuclear Duplication in the Ciliated Protozoan Euplotes

The ribbon-like macronucleus of Euplotes eurystomus pinches in half amitotically at each cell division. Several hours before the actual division two lightly staining duplication bands (reorganization bands) appear at the ends of the nucleus and approach each other slowly, finally meeting near the middle. Distal to the bands, that is, in regions through which the bands have already passed, the concentration of DNA (Feulgen) and "histone" (alkaline fast green) is greater than in the central zone. These facts suggest the hypothesis that DNA-histone synthesis takes place in a sequential fashion starting at the tips of the nucleus and proceeding to the middle. That this hypothesis is correct is shown by autoradiographic and photometric observations. Tritium-labelled thymidine is incorporated only in a limited region immediately distal to the bands. The average amount of Feulgen dye bound by the nucleus rises as the duplication bands approach each other, and is double the presynthesis value by the time the bands meet. A similar rise in the alkaline fast green dye is seen in duplicating nuclei, although no completely post-synthesis values were obtained in this study. The quantitative data are consistent with the assumption that the macronucleus contains a number of DNA-histone "units," presumably chromosomes, each of which duplicates once and only once.     

Macronuclear duplication in the ciliated protozoan Euplotes

The ribbon-like macronucleus of Euplotes eurystomus pinches in half amitotically at each cell division. Several hours before the actual division two lightly staining duplication bands (reorganization bands) appear at the ends of the nucleus and approach each other slowly, finally meeting near the middle. Distal to the bands, that is, in regions through which the bands have already passed, the concentration of DNA (Feulgen) and "histone" (alkaline fast green) is greater than in the central zone. These facts suggest the hypothesis that DNA-histone synthesis takes place in a sequential fashion starting at the tips of the nucleus and proceeding to the middle. That this hypothesis is correct is shown by autoradiographic and photometric observations. Tritium-labelled thymidine is incorporated only in a limited region immediately distal to the bands. The average amount of Feulgen dye bound by the nucleus rises as the duplication bands approach each other, and is double the presynthesis value by the time the bands meet. A similar rise in the alkaline fast green dye is seen in duplicating nuclei, although no completely post-synthesis values were obtained in this study. The quantitative data are consistent with the assumption that the macronucleus contains a number of DNA-histone "units," presumably chromosomes, each of which duplicates once and only once.     

Chromosome Banding and DNA Methylation Patterns, Chromatin Organisation and Nuclear DNA Content in Zingeria biebersteiniana

Chromosome structure and chromatin organisation of a two-chromosome model cereal Zingeria biebersteiniana (Claus) P. Smirnov were studied: nuclear DNA content was determined by microdensitometric analysis after Feulgen staining; Feulgen absorption at different thresholds of absorbance in interphase nuclei also provided evidence on the organisation of chromatin, allowing quantitative estimation of condensed chromatin within interphasic nucleus. The DNA methylation pattern of Z. biebersteiniana metaphase chromosomes was examined with a specific monoclonal antibody. 5-methyl-cytosine residues are present in several chromosome sites and differences may be present between corresponding regions of homologues. Chromosome banding pattern reveals large bands in the centromeric regions of each chromosome, showing constitutive heterochromatin; by fluorochromes staining pericentromeric blocks are evidenced. After the cold and 9-aminoacridine pre-treatments and after aceto-carmine and aceto-orceine staining, respectively, the metaphase chromosomes were analysed by image analysis system revealing a segmentation of the chromosome body that resembles Giemsa/Reverse banding in animal chromosomes. This revised version was published online in July 2006 with corrections to the Cover Date.     

Combined staining procedures for cytophotometry of protein and DNA Feulgen-Naphthol Yellow S and dinitrofluorobenzene-Feulgen

A comparison has been made between dinitrofluorobenzene (DNFB) and Naphthol Yellow S (NYS) as protein stains in combination with the pararosaniline-SO2 Feulgen procedure. Chicken erythrocytes were used as test cells. Cytophotometric measurements were made using a Zeiss scanning stage cytophotometer coupled to a PDP 11/10 minicomputer using the BICOSCAN program to obtain values for protein per cell, protein per "nuclear area' and DNA per nucleus. With 5N HCl as the Feulgen hydrolysis agent, DNFB staining, applied before the Feulgen procedure, was found to be unaffected by hydrolysis conditions required to give optimum Feulgen staining and showed only small losses after longer hydrolysis times. On the other hand measurements of NYS staining, of necessity applied after the Feulgen procedure, seem to be susceptible to the duration of Feulgen hydrolysis. This susceptibility is probably due to the interaction of the DNA phosphates with the basic amino acid residues, potential binding sites for NYS. Since the degree of this interaction may be variable, it is argued that NYS binding will measure the available basicity of proteins at the time of staining but no specific protein fraction. DNFB binding is unaffected by DNA-protein interactions and therefore can give a more reliable measure of "nuclear' protein, particularly in conjunction with Feulgen-DNA measurements.     

Factors affecting the course of DNA acid hydrolysis in carrying out the Feulgen reaction]

Literature data concerning acid hydrolysis of DNA during the Feulgen procedure are reviewed, with emphasis being made on the dependence of Schiff-apurinic acid binding on the fixation technique, the temperature of hydrolysis and acid concentration, the rate of extraction of depolymerized DNA fragments, the nucleotide composition of DNA, the chromatin state, and on the composition of nucleoprotein. Some practical considerations for optimization of the Feulgen procedure for a precise quantitative determination of DNA amount are given.     

The Feulgen reaction 75 years on

 The Feulgen reaction proposed by Feulgen and Rossenbeck 75 years ago is one of the cytohistochemical reactions most widely used in biology and medicine. It allows DNA in situ to be specifically stained based on the reaction of Schiff or Schiff-like reagents with aldehyde groups engendered in the deoxyribose molecules by HCl hydrolysis. The staining intensity is proportional to the DNA concentration. Current applications of the Feulgen reaction are mainly concerned with DNA quantification in cell nuclei by image cytometry for ploidy evaluation in tumor pathology. From the morphological point of view, specific demonstration of DNA in cell structures at the light microscopic level is very little used nowadays. On the other hand, application of the Feulgen principles to electron microscopy have recently allowed specific DNA-staining procedures to be developed for the study of the structural organization of DNA in situ. Accepted: 13 January 1999     

Exposure and removal of stainable groups during Feulgen acid hydrolysis of fixed chromatin at different temperatures

Summary Hyperdiploid Ehrlioh's ascites tumour cells grown in male mice (strain NMRI) were labeled with radioactive nucleotides. The nucleic acids were extracted from fixed, air-dried smears by fractionated hydrolysis and their radioactivity measured by liquid scintillation. The experiments showed that the exposure of aldehydes through removal of purine bases and the elimination of these aldehydes through depolymerisation of DNA were the two main processes responsible for the Feulgen hydrolysis curve. They were shown to be independent and overlapping. The depurination can be described as a simple hydrolytic reaction, while the extraction of DNA depends on a number of different factors. This entails that, in the Feulgen acid hydrolysis procedure, the part of DNA measured is dependent upon the stability of the chromatin. It was found that it is possible accurately to determine the depolymerisation process and thereby roughly correct the measured amount of Feulgen DNA.     

Feulgen Densitometry: Importance of a Stringent Hydrolysis Regime

Abstract: Feulgen densitometry is still a widely used method for DNA content measurements, but experimental procedures and results are often controversial. The present note is concerned with a recent report in the literature that optimum Feulgen staining required a remarkably longer hydrolysis time with 5 M HCI in Dactylis glomerata L. than in Hordeum vulgare L. (i.e., 62 min versus 20 min at 25 C). As this result is prone to question the usual practice of maintaining unified hydrolysis times for test material and internal standard, we established hydrolysis curves for D. glomerata, H. vulgare, Pisum sativum L. and Allium cepa L. at 20 C and 25C for 0 to 100 min. No striking differences between the species and, in particular, between Doctylis and Hordeum were found. Optimum staining occurred after 60 min with hydrolysis at 20 C and after 25 min at 25 C. It is strongly recommended to conduct the quantitative Feulgen reaction at a precisely controlled temperature instead of an inexact room temperature. The broader plateau of optimum staining at 20 C makes this regime preferable.     

Characterization of extrachromosomal DNA in the flesh fly Sarcophaga bullata

The polytene pupal foot pad cells of the flesh fly Sarcophaga bullata contain numerous extrachromosomal DNA containing granules. We have determined both the origin and the nature of the DNA sequences present in these granules. Studies done with quinacrine staining of seven day old pupal foot-pad polytene nuclei showed that the granules fluoresced very brightly while the chromosomal bands to which the granules were attached did not. The only other highly fluroescent regions of the polytene karyotype were the centromeric heterochromatin of chromosomes C and E and several bands associated with the nucleolus of Chromosome A. When polytene nuclei were hybridized in situ with cRNA made from highly repetitive DNA, many of the granules positively labeled. Most of the label on these slides was concentrated on the centromeric heterochromatin of chromosomes C and E. Quinacrine staining of the foot-pad cells at very early stages of pupal development showed that when granules were present, they were always closely associated with the same two centromeric regions, those of chromosomes C and E. Since the highly repetitive DNA located in these centromeric regions is underreplicated, we conclude that the granules result from an extrusion process which takes place early during the polytenization of these cells. The chromosomal integrity of the centromeric heterochromatin of chromosomes C and E is apparently disrupted and repetitive sequences are dissociated from the chromosomes as DNA granules which then secondarily become associated with chromosomal bands throughout the nucleus.     

Fluorescence analysis of late DNA replication in human metaphase chromosomes.

Thymidine incorporated as a terminal pulse into chromosomes otherwise substituted with 5-bromodeoxyuridine can be detected by associated bright 33258 Hoechst fluorescence. The location of metaphase chromosome regions identified by this method as last to complete DNA synthesis is consistent with the results of autoradiographic analyses with tritiated thymidine. The very late-replicating regions correspond to a subset of those which appear as bands after chromosomes are stained by quinacrine or modified Giemsa techniques. The high resolution of the 33258 Hoechst fluorescence pattern within individual cells is especially useful for revealing variations in the order of terminal replication. Both homolog asynchrony and fluctuations in the distribution of bright 33258 Hoechst fluorescence within chromosomes from different cells are apparent and localized to individual bands. The results are consistent with the possibility that these bands constitute units of chromosome replication as well as structure.     

Estimation of residual DNA in Feulgen reaction. A new correction of DNA determination per nucleus.

For the determination of the residual DNA amount after acid hydrolysis of Feulgen's method, a high salt-fluorochrome assay for DNA (5 microM Hoechst 33258 with 1 M NaCl) was effectively applied. At an optimal time length of acid hydrolysis for Feulgen reaction, the ratio of the residual DNA of non-hydrolysis to total DNA is 10% or more in hepatocyte or lymphocyte nuclei. A lot of residual DNA seems not to be negligible in Feulgen's method. A more accurate determination of DNA can be made by correcting the loss ratio of the residual DNA value to Feulgen DNA value. Thus, the combination assay of Feulgen's method with the present fluorometry is enough to measure separately both the amounts of Feulgen DNA and its residual DNA and successfully determines more accurately the total DNA per nucleus by summing both the amounts. The residual DNA, a resistant portion of the chromatin DNA against acid hydrolysis, is a possible constituent as the physiological component of nuclear structures.     

Deduction of DNA from artificial aggregates of erythrocyte nucleosomes

The nucleosomal aggregates were obtained by micrococcal nuclease treatment to chicken erythrocyte nuclei. There still leaves a bulk of nucleosomes. We used two different DNA assay methods to determine DNA in in situ nucleosomes; the Feulgen DNA assay which shows a positive apurinic acid, and the fluorometry with Hoechst 33258 which reveals only intact AT base pairs. On applying those methods to the aggregates, even in a short digestion, Feulgen DNA remains only about 1/4 of the non-digested nuclei and the fluoroassay leaves only a trace amount of AT base pairs. Thus, the nucleosomes derived from the heterochromatin of erythrocytes are not preserved as the residual DNA of Feulgen hydrolysis. This also suggests that the bulk of nucleosomal DNA is masked and sensitive to neither the Feulgen assay nor the fluorometry of AT base pairs.     

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.     

Quantitative analysis of fluorescence profiles of chromosomes : Influence of DNA base composition on banding

Chromosome banding has been analysed in terms of DNA content and base composition distribution along five human chromosomes. Three intercalative dyes (quinacrine, proflavine and ethidium bromide) whose fluorescence quantum yield in the presence of DNAs of different base compositions has been determined, have been used to examine the influence of base composition on the chromosome patterns. Considering that the amount of DNA as determined by the Feulgen reaction is almost constant along the chromosome arms and assuming that base composition is the only factor influencing the fluorescence of these dyes, a distribution of the A-T base pair content along the chromosomes has been calculated from the fluorescence intensity profiles. From the ratio of the intensity profiles obtained with quinacrine and proflavine, patterns showing the variation of the DNA content and of the A-T base pair content could also be obtained independently. The validity of these different approaches is discussed.     

Fluorometric properties of the bibenzimidazole derivative hoechst 33258, a fluorescent probe specific for AT concentration in chromosomal DNA

A new fluorescent probe of chromosomal DNA structure in situ, the bibenzimidazole derivative Hoechst 33258, shows enhanced fluorescence with both AT- and GC-rich DNA; however, enhancement by AT-rich DNA is greater than enhancement with GC-rich DNA. When this compound is used as a probe, it produces localized fluorescence which can be correlated with AT concentration in specific chromosome regions. By the use of 33258, Hilwig and Gropp (1972) were able to demonstrate the relatively AT-rich DNA present in centric regions of mouse chromosomes; these regions do not fluoresce brightly when treated with quinacrine because of the presence of guanine residues which are spaced with high periodicity and which therefore efficiently quench quinacrine fluorescence. The data obtained in this study with DNA polymers of defined structure or composition, as test model compounds, suggest that 33258 is a useful cytochemical reagent for generally identifying all types of AT-rich regions in chromosomes, including those which are not demonstrable with quinacrine.