DNA CONTENT AND HETEROCHROMATIN VARIATIONS IN VARIOUS TISSUES OF PEANUT (ARACHIS HYPOGAEA)

The average 2C DNA amount for the peanut (Arachis hypogaea L.) genome is 4.21 pg, and 73% of the dormant peanut cotyledon nuclei displayed 8C DNA amounts or higher, as compared to 0 to 4% in root-shoot apices and leaf tissue. Thermal melt profiles and heterochromatin values indicated replication of the whole genome. Cotyledon nuclear DNA declined in the percent of polyploid nuclei as well as DNA amounts within ploidy classes during germination. The presence of high DNA C levels in cotyledons generated during embryogeny is interpreted to increase the protein-synthesizing capacity and subsequently supplies a ready source of nucleosides and phosphates during early embryo growth as a result of DNA degradation. However, the later DNA decline at the onset of cotyledon senescence was age related similarly to leaf senescence. The change in proportion of heterochromatin was related to the metabolic state of the tissue and not to the DNA content, as dormant and senescing nuclei contained a higher proportion of heterochromatin as compared to nuclei from metabolically active tissue such as germinating roots. The shift in heterochromatin is interpreted to be involved in gene expression.     

Determination of ploidy level and nuclear DNA content in blueberry by flow cytometry

The technique of DNA flow cytometry was used to study variation in DNA content among different ploidy levels, as well as among diploid species, of Vaccinium section Cyanococcus. In a sample of plants of varying ploidy level, the relative fluorescence intensity (RFI) of nuclei stained with propidium iodide was a function of the number of chromosome sets (x), as represented by the linear equation RFI=3.7x-2.3 (r²=95%). The data indicated that DNA flow cytometry could be useful for the determination of ploidy level at the seedling stage in blueberry. They also suggest that conventional polyploid evolution has occurred in this section of the genus Vaccinium with an increase in nuclear DNA content concurrent with the increase in chromosome number. The nuclear DNA content of diploid species of Vaccinium section Cyanococcus was estimated from the relationship of the observed RFI to an internal known DNA standard (trout red blood cells). A nested analysis of variance indicated significant variation among species, as well as among populations within species, in nuclear DNA content, although this variation was small compared to the variation among ploidy levels. The variation in nuclear DNA content corresponded to the phylogenetic relationships among species determined from previous studies.     

Feulgen-DNA response and chromatin condensation in Malpighian tubules of Melipona rufiventris and Melipona quadrifasciata (Hymenoptera, Apoidea)

Melipona quadrifasciata and Melipona rufiventris are stingless bee species which present low and high heterochromatin content, respectively, on their mitotic chromosomes as assessed visually after a C-banding assay. However, these species do not show differences in the C-banding responses of their Malpighian tubule interphase nuclei. In the present study, the Feulgen-DNA response, which could inform on differences in DNA depurination due to differences in chromatin condensation, was compared in the cell nuclei of the Malpighian tubules of these species. It was hypothesized that differences in acid hydrolysis kinetics patterns, as assessed by Feulgen reaction and studied microspectrophotometrically, could discriminate M. quadrifasciata and M. rufiventris interphase nuclei not distinguishable with the C-banding method. Feulgen-DNA values corresponding to more than one ploidy class were found in both species; these values at the hydrolysis time corresponding to the maximal DNA depurination for each ploidy degree were higher in M. quadrifasciata, reflecting a higher DNA content in the Malpighian tubule cell nuclei of this species compared to those of M. rufiventris at the same larval instar. The maximal Feulgen-DNA values of M. quadrifasciata after short (50 min) and long (90 min) hydrolysis times were found to be closer to each other, while those of M. rufiventris occurred sharply at the long hydrolysis time, indicating that DNA depurination in M. quadrifasciata occurred faster. This result is probably related to the involvement of differences in chromatin condensation; it agrees with the idea that M. rufiventris contains more heterochromatin than M. quadrifasciata, which is supported by the analysis of results obtained with the image analysis parameter average absorption ratio. The depurination kinetics studied here with the Feulgen reaction were revealed to be more pertinent than the C-banding technique in establishing differences in levels of chromatin condensation for these cell nuclei.     

Nuclear DNA content variation associated with muscle fiber hypertrophic growth in fishes

Muscle fiber hypertrophic growth can lead to an increase in the myonuclear domain (MND), leading to greater diffusion distances within the cytoplasmic volume that each nucleus services. We tested the hypothesis that hypertrophic growth in the white muscle of fishes was associated with increases in the mean DNA content of nuclei, which may be a strategy to offset increasing diffusion constraints. DAPI-stained chicken erythrocytes standards and image analysis were used to estimate nuclear DNA content in erythrocytes and muscle fibers from 17 fish species. Mean diploid (2C) values in fish erythrocytes ranged from 0.78 to 7.2 pg. Erythrocyte 2C values were used to determine ploidy level in muscle tissue of small and large size classes of each species. Within each species, mean muscle fiber diameter was greater in the large size class than the small size class, and MND was significantly greater in larger fibers for 11 of the 17 species. Nuclear DNA content per species in muscle ranged from 2 to 64C. Fiber-size dependent increases in ploidy were observed in nine species, which is consistent with our hypothesis and indicates that endoreduplication is occurring during fiber growth. However, two species exhibited significantly lower ploidy in the larger size class, and the mechanistic basis and potential advantage of this ploidy shift is unclear. These results suggest that increases in ploidy may be a common mechanism to compensate for increases in MND associated with fiber hypertrophy in fishes, although it is likely that other factors also affect ploidy changes that occur in muscle during animal growth.     

Nuclear ultrastructure: Condensed chromatin in plants is species-specific (karyotypical), but not tissue-specific (functional)

Summary In contrast to mammalian cell nuclei those of plants display nearly an identical ultrastructure in all developmental stages and tissues. This indicates that the gross organization of chromatin is species-specific, but not tissue-specific and function-dependent. The species-specific nuclear ultrastructure is determined by the basic nuclear DNA content (2 C value). The higher the DNA content, the more the euchromatin remains in the condensed state during interphase, but to a lower coiling order than the heterochromatin.Some difficulties in the interpretation of electron micrographs of cell nuclei, and the possible role of repetitive DNA sequences in the karyotypical condensation of euchromatin in plants are discussed.     

Condensed chromatin and its underreplication during root differentiation in leguminosae

Interphase nuclear structure was studied in 15 leguminous species. Eleven species showed chromocentric interphase nuclei while the remaining 4 had reticulate nuclei. The number of chromocenters appeared to be dependent on the number of chromosomes (2n). The total proportion of condensed chromatin as determined by planimetry was found to vary from 11–24% in chromocentric nuclei and 29–62% in reticulate nuclei. The condensed chromatin amount showed a direct correlation with the nuclear DNA content (2C). Though the interphase nuclear structure remained same in differentiated cells, the amount of condensed chromatin was considerably less than that in the meristematic cells, indicating underreplication of heterochromatin during differentiation. HCl-Giemsa method seems to be the simplest method for detection of underreplication in plants.1. NCL Communication No. 35942. To whom all the correspondence should be addressed     

Estimation of nuclear DNA content of plants by flow cytometry

A rapid and simple protocol for estimation of nuclear DNA content of plants is described. Suspensions of intact nuclei are prepared either by chopping plant tissues or lysing protoplasts in a MgSO₄ buffer, mixed with DNA standards, and stained with propidium iodide in a solution containing DNAase-free RNAase. Fluorescence intensities of the stained nuclei are measured by a flow cytometer. Values for nuclear DNA content are estimated by comparing fluorescence intensities of the nuclei of the test population with those of appropriate internal DNA standards. The same procedure can also be used for rapid determination of ploidy in plant tissues.     

DNA amounts and chromatin compactness in Vicia

2C DNA amounts and areas of chromatin were determined with a M 86 Vickers microdensitometer in 56 species of Vicia (x=5, 6, 7), exhibiting large differences in chromosome size. There were significant differences between the species both in DNA content and chromatin area. The nuclear DNA amounts range from 3.85 to 27.07 pg. DNA distribution appears discontinuous; species cluster into distinct groups and the average nuclear DNA amount separating each successive pair is approximately the same (2.23 pg). The compaction of DNA in interphase nuclei increases with increasing DNA amount, which is, at least partly, due to a disproportionate increase in the heterochromatin relative to the euchromatin component of DNA. Comparisons of DNA readings at various stages of the cell cycle show that the DNA amounts are underestimated by microdensitometry in nuclei with high DNA density. Estimation of relative DNA content and area of individual chromosomes were made in twelve species. The results show that changes in DNA content within chromosomes affect the degree of metaphase coiling in an orderly fashion.     

Age-related alterations in the size of human hepatocytes

Age-related alterations in the size of human hepatocytes (both mononuclear and binucleate forms), were studied in histological sections and in separated cells and nuclei using cytophotometrical and microspectrophotometrical methods. The following results were obtained: 1. The volume of nuclear DNA increased in proportion to nuclear size. The increase occurred in a group pattern reflecting nuclear polyploidization. 2. Cell size increased in proportion to nuclear size. Tetraploid cells (4C) were roughly two times greater than diploid cells (2C). 3. In most of the binucleate cells examined, the ploidy class of the two nuclei in a binucleate cell was observed to be equal. Heterogeneity of the ploidy class among the nuclei of a binucleate cell was present in less than 1% of total binucleate cells examined. The nuclear DNA volume of individual nuclei in binucleate cells appeared to be the same as that of mononuclear cells. 4. The cell size of binucleate cells corresponded with that of mononuclear cells whose ploidy class was the same as the sum of the ploidy classes of two nuclei of a binucleate cell. 5. The incidence of binucleate cells in the lobular periphery was about 4 to 6% in the third decade, and increased slightly with age up to 5 to 7% in the tenth decade. 6. The incidence of binucleate cells in the liver at different ages followed a similar pattern to that observed in mononuclear cells whose ploidy class was half of the sum of ploidy classes of the two nuclei of the binucleate cell.     

Quantitative investigation of reproduction of condensed chromatin of sex chromosomes during trophoblast cell polyploidization and endoreduplication in the East European field vole Microtus rossiaemeridionalis

Simultaneous measurement of DNA content in cell nuclei and condensed chromatin bodies formed by heterochromatized regions of sex chromosomes (gonosomal chromatin bodies, GCB) has been performed in two trophoblast cell populations of the East-european field vole Microtus rossiaemeridionalis, namely in the proliferative population of trophoblast cells of the junctional zone of placenta and in the secondary giant trophoblast cells. One or two gonosomal chromatin bodies have been observed in trophoblast cell nuclei of all embryos studied (perhaps both male and female), In the proliferative trophoblast cell population, characterized by low ploidy levels (2c-16c), and in the highly polyploid population of secondary giant trophoblast cells (16c-256c), the total DNA content in GCB increased proportionally to the ploidy level. In separate bodies, the DNA content rose also in direct proportion with the ploidy level seen in the nuclei with both one and two GCBs in the two trophoblast cell populations. A certain increase in percentage of the nuclei with 2-3 GCBs was shown in the nuclei of the junctional zone of placenta; this may be accounted for by genome multiplication via uncompleted mitoses. In the secondary giant trophoblast cell nuclei (16c-256c), the number of GCBs did not exceed 2, and the share of nuclei with two GCBs did not increase, thus suggesting the polytene nature of sex chromosome in these cells. At different poloidy levels, the ratio of DNA content in the nucleus to the total DNA content in GCB did not change significantly giving evidence of a regular replication of sex chromosomes in each cycle of genome reproduction. In all classes of ploidy, the mean total DNA content in trophoblast cell nuclei with single heterochromatic body was less than in the nuclei with two and more GCBs. This may indicate that a single GCB in many cases does not derive from the fusion of two GCBs. To put it another way, in the nuclei with one GCB and in those with two or more GCBs, different chromosome regions may undergo heterochromatization. The regularities observed here are, most probably, associated with the peculiarities in the structure of X- and Y-chromosomes in a range of species of Microtus (M. agrestis, M. rossiaemeridionalis, M. transcaspicus). As a result, gonosomal chromatin bodies may include large blocks of both constitutive heterochromatin of X- and Y-chromosomes (in male and female embryos) and inactivated euchromatin of "lyonized" X-chromosome in female embryos. Therefore the presence of two or more GCBs in trophoblast cells of M. rossiaemeridionalis may be accounted for by both polyploidy and functional state of the nucleus, in which gonosomal constitutive heterochromatin and inactivated euchromatin form two large chromocenters rather than one. The differences in DNA content in GCBs in the nuclei with one and two GCBs seem to be an indirect indication that the two chromocenters may be formed by two different gonosomes, with the extent of their heterochromatization being higher than that in the nuclei with one GCB. GCBs in the trophoblast cells of M. rossiaemeridionalis are observed not only at the early developmental stages, as it was observed in rat at the first half of pregnancy (Zybina and Mosjan, 1967), but also at the later stages, up to the 17th day of gestation. At these stages, the nuclei with non-classical polytene chromosomes rearrange to those with a great number of endochromosomes, probably because of disintegration of chromosomes into oligotene fibrils. However, it does not seem unlikely that this process may involve heterochromatized gonosomal bodies, since only one or two large GCBs can be seen in the nuclei as before. The presence of prominent blocks of constitutive heterochromatin seems to favor a closer association of sister chromatids in polytene chromosomes, which prevents their dissociation into endochromosomes with the result that polyteny of sex chromosomes in the field vole trophoblast is probably retained during a longer period of embryonic development.     

DDP1, a single-stranded nucleic acid-binding protein of Drosophila, associates with pericentric heterochromatin and is functionally homologous to the yeast Scp160p, which is involved in the control of cell ploidy.

The centromeric dodeca-satellite of Drosophila forms altered DNA structures in vitro in which its purine-rich strand (G-strand) forms stable fold-back structures, while the complementary C-strand remains unstructured. In this paper, the purification and characterization of DDP1, a single-stranded DNA-binding protein of high molecular mass (160 kDa) that specifically binds the unstructured dodeca-satellite C-strand, is presented. In polytene chromosomes, DDP1 is found located at the chromocentre associated with the pericentric heterochromatin but its distribution is not constrained to the dodeca-satellite sequences. DDP1 also localizes to heterochromatin in interphase nuclei of larval neuroblasts. During embryo development, DDP1 becomes nuclear after cellularization, when heterochromatin is fully organized, being also associated with the condensed mitotic chromosomes. In addition to its localization at the chromocentre, in polytene chromosomes, DDP1 is also detected at several sites in the euchromatic arms co-localizing with the heterochromatin protein HP1. DDP1 is a multi-KH domain protein homologous to the yeast Scp160 protein that is involved in the control of cell ploidy. Expression of DDP1 complements a Deltascp160 deletion in yeast. These results are discussed in view of the possible contribution of DNA structure to the structural organization of pericentric heterochromatin.     

Ploidy determination in Sphagnum samples from Svalbard,Arctic Norway,by DNA image cytometry

Abstract

We measured DNA content of cell nuclei, stained with the Feulgen method, using branch tips of 11 species of Sphagnum from Svalbard, Arctic Norway, as an alternative to chromosome counting. Nine species were haploid and two were diploid, with no intraspecific variation in ploidy level. The results conformed to known chromosome numbers and/or to expectations from isozyme studies. Ploidy levels were determined for the first time in S. tundrae and S. fimbriatum ssp. concinnum (haploid) and S. arcticum and S. olafii (diploid). No mitotic divisions were observed, but unreplicated interphase nuclei still allowed precise ploidy determinations. Basic DNA contents of all Sphagnum species were very similar, and measurement of a few nuclei proved sufficient to ascertain ploidy level despite very low nuclear DNA content. Advantages of the DNA image cytometry method are: mitotic or meiotic cells are not required to be found, and only a small amount of material is required.     

Heterochromatin and histone phosphorylation

Histone phosphorylation and nuclear structure have been compared in cultured cell lines of two related species of deer mice, Peromyscus crinitus and Peromyscus eremicus, which differ greatly in their heterochromatin contents but which contain essentially the same euchromatin content. Flow microfluorometry measurements indicated that P. eremicus contained 36% more DNA than did P. crinitus, and C-band chromosome staining indicated that the extra DNA of P. eremicus existed as constitutive heterochromatin. Two striking differences in interphase nuclear structure were observed by electron microscopy. Peromyscus crinitus nuclei contained small clumps of heterochromatin and a loose, amorphous nucleolus, while P. eremicus nuclei contained large, dense clumps of heterochromatin and a densely structured, well defined, nucleolonema form of nucleolus. Incorporation of ³²PO₄ into histones indicated that the steady-state phosphorylation of H1 was identical in P. crinitus and P. eremicus cells. In contrast, the phosphorylation rate of H2a was 58% greater in the highly heterochromatic chromatin of P. eremicus cells than in the lesser heterochromatic chromatin of P. crinitus cells, suggesting an involvement of H2a phosphorylation in heterochromatin structure. It is suggested that the three histone phosphorylations related to cell growth (H1, H2a, and H3) may be associated with different levels of chromatin organization: H1 interphase phosphorylation with some submicroscopic (molecular) level of organization, H2a phosphorylation with a higher level of chromatin organization found in heterochromatin, and H3 and H1 superphosphorylation with the highest level of chromatin organization observed in condensed chromosomes.     

Flow cytometric analysis of nuclear genome of the Ethiopian cereal tef [Eragrostis tef (Zucc.) Trotter]

Flow cytometric analysis of nuclear DNA content was performed by using nuclei isolated from young leaf tissue of tef (Eragrostis tef). The method was very useful for rapid screening of ploidy levels in cultivars and lines of tef representing the phenotypic variability of this species in Ethiopia. The results of the analysis showed that all cultivars were tetraploid. Flow cytometry was also used to determine nuclear DNA content in absolute units (genome size) in four tef cultivars. Nuclei isolated from tomato (Lycopersicon esculentum, 2C=1.96 pg) were used as an internal reference standard. The 2C DNA content of individual tef cultivars ranged from 1.48 to 1.52 pg (1C genome size: 714 Mbp-733 Mbp), the differences among them being statistically nonsignificant. The fact that the nuclear genome of tef is only about 50% larger than that of rice should make it amenable for analysis and mapping at the molecular level.     

STRUCTURAL ALTERATION IN ISOLATED RAT LIVER NUCLEI AFTER REMOVAL OF TEMPLATE RESTRICTION BY POLYANIONS

Specific polyanions release DNA template restrictions for DNA synthesis in isolated rat liver nuclei. The degree to which DNA synthesis is enhanced can be correlated with a spectrum of changes in nuclear structure Each polyanion which is effective in the release of template restriction produces a characteristic alteration in nuclear ultrastructure. Polyanions which have no effect on DNA synthesis do not appear to cause any change in nuclear organization or ultrastructure. Parallel measurements of nuclear DNA release and nuclear volume changes also indicate that template-activating polyanions cause remarkable changes in the structural organization of the treated nuclei. These results indicate that DNA template activation involves direct interactions between polyanions and nuclear constituents and suggest the possibility that naturally occurring polyanions might have a role in the control of gene activity     

Genome Expression during Normal Leaf Development : 2. Direct Correlation between Ribulose Bisphosphate Carboxylase Content and Nuclear Ploidy in a Polyploid Series of Wheat

The quantitative relationships between ribulose bisphosphate carboxylase, nuclear ploidy, and plastid DNA content were examined in the nonisogenic polyploid series Triticum monococcum (2×), Triticum dicoccum (4×), and Triticum aestivum (6×). Ribulose bisphosphate carboxylase per mesophyll cell increased in step with each increase in nuclear ploidy so the ratios of ribulose bisphosphate carboxylase per mesophyll cell (picograms) to nuclear DNA per mesophyll cell (picograms) were almost identical in the three species. Ribulose bisphosphate carboxylase per plastid was 14.1, 14.7, and 16.8 picograms in the 2×, 4×, and 6× ploidy levels, respectively. Plastid area in these three species decreased with increasing nuclear ploidy so the concentration of ribulose bisphosphate carboxylase in the plastoids was 60% higher in the hexaploid compared to the diploid species. DNA levels per plastid were 64 and 67 femtograms for the diploid and tetraploid species, respectively, but were 40% less in the plastids of the hexaploid species. These relationships are discussed in terms of cellular and plastid control of ribulose bisphosphate carboxylase content.     

Improved correction of quantitative nuclear DNA (ploidy) measurements in tissue sections

OBJECTIVE: To evaluate, using a computer model, the advantages for ploidy measurements of selecting only center-containing sections of nuclei in ultrathin, very thick or relatively thick tissue sections. STUDY DESIGN: The computed corpuscle sectioning program was run on a personal computer. Its synthetic data were corrected by a variety of correction algorithms. RESULTS: When only center-containing sections of nuclei were selected in ultrathin sections, spherical nuclei could be corrected perfectly, and mildly prolate ellipsoidal nuclei with a selection bias in favor of elliptical nuclear section profiles could be corrected with high fidelity. Ultrathin sections most faithfully represented the true height of the peak of highest ploidy and showed better peak discrimination than other choices of section thickness, but small sample size, wavy sections, markedly inhomogeneous intranuclear DNA distribution and oblate ellipsoidal nuclei represented significant limitations of this approach. As nuclear prolation increased, peak definition worsened, and the peak of highest ploidy was falsely shortened. Results were unaffected by errors in the estimation of section thickness when an internal diploid standard was used. The effect of variable internuclear DNA concentration in mildly or moderately prolate ellipsoidal nuclei was nil. The choice of correction algorithm was unimportant, except that the reference curve method was better able to analyze oblate ellipsoidal nuclei, wavy sections and nuclei with inhomogeneous intranuclear DNA, and provided superior insight into nuclear and section parameters. Thick and very thick sections did not require correction and, unlike ultrathin sections, were immune to markedly inhomogeneous intranuclear DNA distribution, to nonspherical nuclear shape and to focal variation in section thickness (waviness), but (in relatively thick more than in very thick sections) the height of the peak of highest ploidy was falsely shortened, often markedly, and peak definition was worse. CONCLUSION: Choice of section thickness and selection of only center-containing nuclear sections for analysis with a bias in favor of elliptical nuclear section profiles in ultrathin sections are very important for optimal results; the choice of correction algorithm is less important.     

Cell function in the ovary of drosophila

Summary The relative DNA content of the ovarian nurse nuclei of Drosophila melanogaster has been measured by high-resolution autoradiography of DNA uniformly labelled with adenine-8-¹⁴C.The various nurse nuclei show a defined pattern of DNA classes. The posterior nuclei, i. e. those nearest to the oocyte, achieve eight reduplications of DNA by stages 8–9, thus reaching 512n, and all have lost some DNA by stage 10. The nuclei in the middle of the chamber achieve seven reduplications of DNA by stage 9, thus reaching 256n, and though there is loss of DNA in the majority of these nuclei at stage 10 some of them might enter a new reduplication cycle. The anterior nuclei, i. e. those more distant from the oocyte, achieve more than seven reduplications by stage 10 and show no loss of DNA.After stage 6 of the ovarian chambers the pattern of DNA enrichment and later degradation is clearly polarized in that there is a posterioranterior gradient for the level of ploidy, the order in time in which this is attained, and the loss of DNA. The dominant end of the gradient is towards the developing oocyte.The measured nuclear volume where DNA is present is well correlated with ploidy till stage 9. Compared with earlier stages, at stage 10 DNA shares less in the nuclear contents than other materials. The nuclear volume when calculated as a sphere is a gross overestimation, except for the earliest stages.Various possibilities likely to bring about differences in the amount of DNA among nurse nuclei within and between chambers are discussed.Research worker of the British Empire Cancer Campaign.