Feulgen Absorption Research Articles

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.

Nuclear DNA contents, rDNAs, chromatin organization, and karyotype evolution inVicia sect,faba

Automated karyotype analyses, nuclear DNA contents, and sequences of rDNA internal transcribed spacers of the nine species inVicia sect.faba are reported. As karyomorphological parameters are used to evaluate the karyotype evolution, so the determination of the heterochromatin by Feulgen absorption at different thresholds of optical density provided further evidence on the chromatin organization withinVicia sect.faba. The comparison of sequences of rDNA spacers has enabled the definition of the phylogenetic relationships between the analyzed species.

Cytology ofViciaspecies. VI. Nuclear chromatin organization, karyomorphological analysis and DNA amount inVicia serratifoliaJacq.

SUMMARYKaryomorphological features of Vicia serratifolia Jacq. were studied: the nuclear DNA content was determined in meristematic root cells, karyotype and morphometric data were generated by an automated image analysis system and the distribution and staining properties of heterochromatin were assessed in metaphase chromosomes. Moreover, Feulgen absorption at different thresholds of optical density provided evidence on the organization of the chromatin in interphase nucleus. The collected data were compared with those previously gathered in V. faba used as internal standard and V. narbonensis considered the reference species of the Narbonensis complex.

Nuclear DNA changes within Helianthus annuus L.: origin and control mechanism

Previous results suggested that the amount of nuclear DNA varies in one and the same progeny of Helianthus annuus, depending on the head portion in which seeds have developed. Accordingly, cytophotometric determinations were carried out in a selfed line, after Feulgen-staining, to obtain information on the developmental stages at which DNA changes are produced and on the mechanism controlling the variation. It was found that the Feulgen absorption values of mitotic prophases in immature anthers and pistils and of meiotic prophases I are the same in any flower of the head. In contrast, the Feulgen/DNA contents of early prophases in heart-shaped embryos differ significantly, increasing from those developing at the centre of the head to those developing at its periphery, and remaining unchanged in each embryo during further development and seed germination. Variations in the number of chromosomes do not account for the differences observed in nuclear DNA contents in which sequences comprised in heterochromatic nuclear regions are involved. The Feulgen absorption values of seedlings obtained from seeds developed in different portions of single heads increase or diminish starting from those found in the mother plant. This depends on whether these latter are relatively low or high and on the gradient of seed location in the head. The variation occurring within each single progeny covers the whole range existing within the line.

Extra DNA synthesis in the dedifferentiating cells ofVicia faba roots

Cell dedifferentiation was induced inVicia faba root tissues by removing the whole root meristem (decapitation) and the behaviour of the nuclear DNA in the dedifferentiating cells was studied by means of cytophotometric and autoradiographic analyses. Cytophotometric determination after Feulgen-staining showed that: 1. the vast majority of nuclei in differentiated cells were in the DNA postsynthetic phase, but their Feulgen absorption was lower than that of DNA postsynthetic nuclei (G2, 4 C) in the meristem; 2. such a Feulgen absorption was detected in certain nuclei after root decapitation; 3. all the mitoses in the dedifferentiating tissues were diploid, fully matching the Feulgen absorption of mitoses in the meristem.

Cytochemical properties of euchromatin and heterochromatin

Two classic cytochemical tests, the Feulgen-Schiff reaction and Toluidine Blue basophilia, have been employed for investigating the differential characteristics of heterochromatin and euchromatin. Differences have been detected in the Feulgen hydrolysis kinetics, the Feulgen absorption spectrum, the image analysis of Feulgen-stained material, and the binding of Toluidine Blue under ordinary and Mg2+ competitive staining conditions. The differences are assumed to be a function of the composition and stereo-arrangement of the DNA and DNA-protein complexes present in these chromatin types and are possibly associated with physiological activities whose whole meaning is far from being clear. Differences in optical retardations in Toluidine Blue-stained material were also found. These are interpreted as being due to chromatin packing state and selective removal of histones promoted by the acetic acid-ethanol fixative.

Absorption spectrum of Feulgen-stained polytene chromosomes of Drosophila melanogaster

Hetero- and euchromatic polytene chromosome regions of early and fully grown larvae of Drosophila melanogaster had their Feulgen absorption curves determined cytophotometrically. Hydrolysis conditions adequate to induce maximal DNA depurination were used. No differences in curve shapes specially concerning the absorption shoulder at the 520 less than or equal to lambda less than or equal to 550 nm spectral range were detected with advancing polytenization or when comparing the absorption patterns for the hetero- and euchromatic regions to each other. It is considered that prominence of the Feulgen absorption shoulder could be related to the amount of Schiff molecules di-substituted with apurinic acid aldehydes and to the relatively larger resistance of these apurinic acid fragments towards solubilization with acid hydrolysis due to their binding to nuclear proteins. It is therefore assumed that no significant differences owing to the above-cited factors exist for the chromosome regions analyzed at 2 phases of the polytenization process. Consequently, the replication of reiterated sequences from the main band DNA which occurs in polytene chromosome regions of Drosophila melanogaster appears not to affect the Feulgen absorption spectrum of this material.

Feulgen-DNA absorption curves of polytene chromosome regions of Rhynchosciara americana.

Feulgen absorption spectra of polytene chromosome regions, differing in types of DNA composition and of chromatin compactness, were determined microspectrophotometrically in squashed salivary glands of Rhynchosciara americana. The absorption curves exhibited a secondary maximum at their ascending branch after the chromatins were maximally depurinated. The degree of shoulder prominence could not be specifically correlated with the packing state or even with richness in satellite repetitive DNA of the chromatins. It is assumed that other factors may exist which play a part in the phenomenon that gives rise to the Feulgen absorption shoulder characteristics. Differences in the extraction rate of the apurinic acid fragments due to their binding to specific non-histone proteins are suggested as contributing to the above-mentioned absorption patterns when comparing the various chromatin types.

Gymnosperm seedling sensitivity to gamma radiation: Its relation to seed radiosensitivity and nuclear variables

Newly germinated seedlings of Larix laricina, Picea glauca, Picea mariana, Pinus banksiana, Pinus contorta, Pinus resinosa, and Thuja occidentalis were gamma irradiated at exposures of 150, 300, 450, 600, 900, 1200, 1800, 2400 and 3600 R. Number of leaves produced and shoot dry weight were determined 50 days after irradiation and survival and shoot dry weight after 130 days. Of the 4 endpoints scored, shoot dry weight at 50 days showed greatest radiosensitivity with a D50 ranging from 195 R for Pinus resinosa to 380 R for Picea mariana. Except for shoot dry weight at 50 days, all endpoints showed a sharp response over a relatively narrow range of exposures, generally between 600 and 900 R. The 7 species differed in seedling radiosensitivity by a factor of less than 2 for all endpoints. Comparison with seed radiosensitivity of the same populations showed that seedlings are from 7 to 15 times more sensitive than seeds. Correlation analyses showed that the only relationship among the endpoints studied was between D50 for number of leaves at 50 days and shoot dry weight at 130 days: the seedlings with the greatest number of leaves at 50 days had the greatest dry weight at 130 days. No relation was found between the D50 following seedling irradiation and the D50 for irradiated seed previously reported for the same populations of the 7 species. The observed seedling radiosensitivity was not correlated with predicted mature plant sensitivity, thus adding further evidence on the variation in relative radiosensitivity patterns between stages of the life cycle among gymnosperm species suggested previously. Seedling radiosensitivity as determined from 3 of the 4 endpoints studied was related to DNA content per cell determined cytophotometrically in Feulgen absorption units, but unrelated to DNA content determined chemically or to nuclear volume.

Cytochemical Analyses on Cellular Differentiation in the Root Tip ofAllium Cepa

SUMMARYSpectrophotometry in Allium cepa root tip tissues showed that Feulgen mean absorption decreases in pericycle and protoderm cells from 500 to 1500 μ (starting from the distal end of the root cap), while in provascular cells it increases. These observations are probably consistent with the gradual accumulation of pericycle and protoderm cells in the G1 phase of the mitotic cycle and with the ability of provascular cells to undergo chromosome endoreduplication.The collected data show that fast green absorption, when compared with Feulgen absorption in the same cell, gives an indication that DNA/histone ratio is not constant among tissues and also within one single cell line during the maturation process.The significance of these observations for studies of cell metabolism is discussed.

QUANTITATIVE STUDY OF INTRASPECIFIC VARIATION OF DNA PER CELL INPICEA GLAUCAANDPINUS BANKSIANA

Intraspecific variation of DNA per cell was established chemically and cytophotometrically for 17 seed sources of Picea glauca, and cytophotometrically for 11 sources of Pinus banksiana. The DNA Feulgen absorption per cell varied from the lowest to the highest amount by factors of 1.6 and 1.5 for Picea glauca and Pinus banksiana, respectively. Intraspecific DNA also varies with intraspecific nuclear volume. Intraspecific variation of histone was similar to the observed DNA variation. A regression analysis between DNA per cell and latitude provided evidence that eastern and western population series of Picea glauca existed in the seed sources studied. Two-year seedling height growth results demonstrated that eastern seed sources are different from western seed sources. Seedling height in the western provenances varied inversely with DNA content; i.e., seed sources with small DNA per cell displayed greater growth, whereas the eastern sources did not display the inverse relationship between DNA amount and 2-year growth.

Quantitative cytophotometric studies on polyploid liver cell nuclei of frog and rat.

Nuclei were isolated from fixed liver tissue of diploid and triploid Rana pipiens and of rat. While the frog liver nuclei present a single ploidy class on the basis of Feulgen absorption measurements, rat liver contained diploid, tetraploid, and some octoploid nuclei. Nuclear areas within single ploidy classes varied over wide ranges, especially in the frog material. The mode of this variation was dependent on ploidy. Microspectrophotometric measurements of several protein components were compared to ploidy and nuclear volume. General protein methods indicated a linear relationship to nuclear volume. Protein-bound sulfhydryl and disulfide groups were not related to nuclear volume, but could be related to ploidy. Protein tyrosine values showed a partial dependence on both factors.

Quantitative cytochemical methods for the study of tumour cell populations

The general recording ultramicrospectrographic methods for quantitative cytochemical analysis are as a rule time-consuming when applied to work on especially large cell populations. A series of rapid methods are described, methods particularly suited to investigation of nucleotide and protein metabolism in large cell populations where the cells or cell elements lie free from each other. The instruments comprise: (1) A rapid scanning high-resolution microspectrograph for both the ultra-violet and the visible spectral region. This instrument is also especially suitable for measurements in ordinary cell material of the Feulgen absorption in large series with high resolution. (2) A recording high-resolution microinterferometer with an integrating device. (3) A modified microradiographic mass-determination method with a special measuring instrument for microradiograms of large cell populations ( L. Carlson). (4) A microplanimeter, designed especially for nucleolar and nuclear volume measurements, with electronic data recording on three channels. A series of examples is given of the use of these instruments in the study primarily of DNA metabolism problems in tumour cell populations.

The correlation of ultraviolet and Feulgen absorption measured in ascites tumor cells.

The correlation of ultraviolet and Feulgen absorption after ribonuclease, cold perchloric acid, or ribonuclease plus perchloric acid treatments was investigated in fixed ascites tumor cells using microspectrophotometric methods. It was concluded from the measurements that the amount of ribonucleic acid resistant to 24 hour ribonuclease digestion is proportional to the amount of desoxyribonucleic acid revealed by Feulgen staining.

Stability of the Feulgen-deoxyribonucleic acid absorption curve in situ with variation in nuclear protein content and other factors.

1. The influence of certain variables upon the shape of the Feulgen absorption curve of mouse liver nuclei has been studied in situ using cytophotometric equipment. The accuracy and reproduciblity of the cytophotometer was ascertained by various procedures. 2. The absorption curve usually is represented by a primary peak at about 585 mµ and a slight secondary peak at about 550 mµ. Reduction or removal of nuclear protein by extraction or enzymatic procedures does not alter the shape of the curve. 3. Variations in hydrolysis times do not alter the shape of the curve obtained with nuclei fixed in Zenker's fluid. There are some differences indicated around the secondary peak of formalin-fixed tissue following different hydrolysis times. 4. Comparison of absorption curves of nuclei fixed in different fixatives shows negligible differences. 5. There are no differences in the absorption curves of nuclei isolated in one per cent citric acid and in nuclei isolated in 0.88 M sucrose.