Die Verteilung der Chromosomen auf die Tochterzellkerne multipolarer Mitosen in euploiden Gewebekulturen von Microtus agrestis

In kidney epithelial cultures from female Microtus agrestis, 3,55% of all mitoses are multipolar, 94% of them tripolar. Feulgen photometric measurements of 21 tripolar mitoses reveal a total DNA amount corresponding to the mitotic diploid value (4c) in 5 cases, and to the tetraploid value (8c) in 16 cases, Diploid tripolar mitoses divide into one daughter nucleus with a diploid DNA value (2c) and two nuclei each with a haploid DNA value (1c). Most tetraploid tripolar mitoses divide into one daughter nucleus with a diploid DNA value (2c) and two nuclei with a triploid DNA value (3c). Also the sex chromosomes are distributed to the daughter nuclei in the relation of 2∶3∶3. This can be seen in anaphase figures as well as in interphase nuclei presumably derived from tripolar mitoses, showing chromocenters according to the number of X-chromosomes. In two cases of tripolar tetraploid mitoses the resulting nuclei have a haploid, a triploid and a tetraploid DNA value. The DNA replication pattern is always identical in the daughter nuclei of diploid and tetraploid tripolar mitoses. — Our observations suggest segregation and distribution of haploid chromosome sets or multiples of haploid sets to the daughter nuclei of multipolar mitoses. They also show a possible way of formation of haploid and triploid cells in a basically diploid tissue. Presumably triploid nuclei (with 3 chromocenters) are capable of DNA synthesis.     

A cytophotometric analysis of the structure of hypothalamic cell populations in the frog,Rana temporaria (L.), with special reference to seasonal changes in chromatin status

Summary We used cytophotometry after the Feulgen reaction and UV cytophotometry to measure the DNA content of quiescent cells of the hypothalamic preoptic region (HPR) of adult and juvenile frogs (Rana temporaria) that had been caught in their natural habitat in winter, spring and summer. The histone-to-DNA ratio in cell nuclei was cytophotometrically determined using a combined Feulgen, heparine and alcian-blue staining procedure. The vast majority of HPR cells studied had nuclei with a diploid DNA content. However, we observed great variability in the Feulgen-DNA content of the HPR cell population, which was not detected in the diploid standard (hepatocytes). This heterogeneity in the diploid sample of the HPR cell populations was always greater in prespawning frogs and may have been due to differences in the chromatin arrangement in nuclei. About 1% of cells had a DNA content either ranging between diploid and tetraploid levels (H2C cells) or at the tetraploid level (4C and 2C x 2 cells). The proportion of these cells was not affected by the age of the animals or the annual cycle, thus suggesting that there is no age-related increase in the mean DNA content in the frog HPR. The mean DNA contents of H2C and 4C cells were much higher than those in the standard (hepatocytes). This cannot be simply attributed to the presence of different amounts of nuclear proteins, but rather indicates that at least a certain proportion of the highest DNA contents may be due to a real extra-DNA synthesis.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthday     

NUCLEAR DNA CONTENT OF MYXAMOEBAE AND PLASMODIA IN SIX NON-HETEROTHALLIC ISOLATES OF A MYXOMYCETE,DIDYMIUM IRIDIS

The nuclear DNA content of six non-heterothallic isolates of the myxomycete Didymium iridis was measured by combining the Feulgen reaction with absorption microspectrophotometry. This allowed us to distinguish between homothallic (sexual) and apogamic (non-sexual) isolates. Four of the isolates studied, Panamanian 4 and 5, California 1, and Missouri 1 are homothallic. Moreover, the average DNA content of the myxamoebal and plasmodial nuclei (0.32 and 0.61 respectively) does not differ significantly from the calculated haploid and diploid values for heterothallic isolates of D. iridis (0.34 and 0.63). Hence, it is concluded that in each of these isolates the myxamoebae are haploid and the plasmodia diploid. In two of the isolates investigated, Georgia 1 and Hawaii 1, the DNA content of the myxamoebal and plasmodial nuclei did not differ significantly. Therefore, in both of these isolates the plasmodia appear to develop apogamically. In addition the mean DNA values recorded for the Ha-1 isolate suggest that it is aneuploid.     

MICROFLUOROMETRIC MEASUREMENT OF DNA IN EUDORINA ELEGANS AND E. CALIFORNICA (CHOROPHYCEAE)1

The fluorescent dye 2,5-bis (4′-aminophenyl (1′))-1,3,4-oxidiwle (BAO) was compared to the Feulgen procedure as a method for quantitative comparison of DNA levels in two species of Eudorina. The BAO procedure proved to be specific and as quantitative as the Feulgen procedure, and was more convenient and rapid. The DNA level of the nuclei of gonidial cells of Eudorina elegans Ehrenberg and E. californica (Shaw) Goldstein doubled prior to each division. The DNA level of the somatic, usually non-dividing nuclei of E. californica did not vary from the IC (haploid) level.     

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.     

What controls haploid—diploid ratio in the red alga,Gracilaria verrucosa?

The conditions for maintenance of a haploid—diploid life cycle in the species Gracilaria verrucosa were studied. This species is a red alga, where haploid plants have separate sexes. In the two natural populations studied, male and female haploid individuals were in equal proportions, and the frequency of diploid individuals reached 0.5. A two-fold advantage in viability for diploid relative to haploid juveniles was observed in the field. This advantage can account for a frequency of 0.5 of diploid individuals in natural populations. Different types of anomalies in the reproduction of diploid individuals were observed, all of which lead to a reduction of the haploid stage.     

Comparative microphotometric studies of DNA and arginine in plant nuclei

Summary Photometric measurements have been made of the amounts of stain formed in the Feulgen (DNA) and Sakaguchi (arginine) reactions in plant nuclei of differing ploidy.In nuclei of diploid and tetraploid plants of Tradescantia ohioensis and of diploid, triploid and tetraploid plants of Ranunculus ficaria, both Feulgen and Sakaguchi values gave ratios which agreed closely with the ratios of the number of chromatids known to be present. The Feulgen/ Sakaguchi ratio for each of the different types of nuclei measured was very similar both within and between these two species.In the interphase nuclei of five different species, both Feulgen and Sakaguchi values gave bimodal distributions. In the nuclei of differentiating cells, the proportions of values falling into each of the 2C, 4C or 8C classes were the same for both stains.Measurements of the amounts of both stains were made in sequence on the same individual nuclei and a positive correlation found between the two sets of values.In nuclei from differentiating cells of Vicia faba primary roots, the Feulgen/Sakaguchi ratio decreased with increasing distance from the apex.The following suggestions were made from the results: (a) that there is some degree of quantitative constancy of nuclear arginine which parallels that of DNA; (b) that the amount of nuclear arginine, like that of DNA, is doubled during synthesis in interphase; (c) that the syntheses of DNA and arginine in interphase, if not simultaneous, at least occur within the same relatively short period; (d) that there may be a difference in the DNA/arginine ratio between the nuclei of meristematic and differentiating cells.     

Analysis of haploid mosaics in Drosophila

Adult chimeric epidermal structures were obtained following transplantation of haploid nuclei from haploid donor embryos of Drosophila into genetically marked diploid embryos. The haploid nuclei either remained haploid or became diploid. Where possible, physical measurements indicated that the haploid cells were smaller and produced smaller cuticular structures than did diploid cells. An increase in the number of pattern elements was observed in many patches which, by various criteria, were judged to be formed by haploid cells. The observation of altered pattern element spacing in haploid patches is in agreement with the conclusion, reached by L. I. Held (1979, Wilhelm Roux's Arch.187, 105–127) in triploid flies, that bristle spacing is a function of cell size.     

Is There a Relationship between Infection by Rhizobia and Occurrence of Disomatic Nuclei in Nodules of Alfalfa (Medicago sativa L.)?

Cytological examination of nodules from diploid, tetraploid, and octoploid alfalfa (Medicago sativa L.) plants revealed that the proportion of nodule cells infected by rhizobia was not significantly affected by nuclear ploidy of the host plant. Flow cytometry was used to determine the influence of host plant nuclear ploidy on the nuclear ploidy of infected cells. In nodules from diploid plants, most of the nuclei were tetraploid, whereas in nodules from tetraploid plants, about half of the nodule nuclei were tetraploid and half were octoploid; in octoploid plants, most of the nodule nuclei were octoploid. The occurrence of disomatic nuclei was independent of infection of nodule cells by rhizobia, because diploid plants had mostly disomatic nodule nuclei, and octoploid plants had mostly monosomatic nodule nuclei, whereas all nodules maintained a constant proportion of infected to uninfected cells. These results do not support the earlier hypothesis that infected nodule cells contain disomatic nuclei.     

Effects of an acridine half-mustard (ICR 191) on growth and ploidy of frog cells in culture

The effects of an acridine half-mustard, ICR 191, on the growth rate and ploidy of four haploid and two diploid lines of Rana pipiens cells in culture were studied. Growth curves indicate that the haploid and diploid cell lines were equally resistant to a 4-hour exposure of this drug (0.1 micrometer to 10 micrometer. ICR 191 treatment induced the haploid cell cultures to become diploid. The proportion of diploid cells increased progressively with respect to time after the 4-hour exposure period. The greater the concentration of ICR 191 applied, the more rapid the rate of conversion. Autoradiographic determinations of percent labelled nuclei indicate that DNA synthesis was not inhibited in haploid or in diploid cells. Therefore, the increased proportion of diploid cells did not originate from the small percentage of diploid cells in the initial population. Instead the haploid cells were converted to diploid cells. Time lapse cinematography indicated that the conversion mechanism was other than cell fusion. Conversion to higher ploidy did not occur when diploid cell cultures were exposed to ICR 191.     

Flow cytometry reveals a high degree of genomic size variation and mixoploidy in various strains of the acellular slime mold Physarum polycephalum

High-resolution flow cytometry, using avian erythrocytes as an internal standard, was employed to study constitutive genome size variation of G2-phase nuclei of Physarum polycephalum strains during the macroplasmodial stage of their life cycle. Our results document a previously unknown extent of genome size variation and mixoploidy in this organism. The unimodal diploid strain Tu 291 displayed the largest genome of the strains tested; in contrast, the Colonia strain displayed only half of the Tu 291 G2-phase fluorescence, confirming its haploid nature. An additional strain, derived from a recent cross between Lu897 and Lu898 amoebae, must have arisen by selfing (propagation of only one of the parental genomes to the macroplasmodial stage), since its nuclei display close to the haploid G2-phase DNA content. The observation of a small fraction of corresponding diploid nuclei within the haploid population of this strain, while maintained as microplasmodia, supports the notion that meiosis in haploid strains may require the presence of diploid nuclei. Two of the descendants of the prototype haploid Colonia strain, which were kept for extended periods of time in submerse culture, proved to be near diploid and mixoploid. Polyploidization and subsequent loss of DNA thus seems to contribute to the extremes of genome size variation in Physarum. In addition to unimodal fluorescence distributions, a number of diploid strains displayed bi- and even trimodal distributions within harvests of a single G2-phase macroplasmodium. Analysis of these mixoploid strains by means of gaussian curve-fitting suggests that the smaller genome size differences in Physarum may arise in step-wise diminution of DNA in approximate units of 3-5% of the original Tu 291 genome.     

Epigenetic inheritance and genome regulation: is DNA methylation linked to ploidy in haplodiploid insects?

Organisms show great variation in ploidy level. For example, chromosome copy number varies among cells, individuals and species. One particularly widespread example of ploidy variation is found in haplodiploid taxa, wherein males are typically haploid and females are typically diploid. Despite the prevalence of haplodiploidy, the regulatory consequences of having separate haploid and diploid genomes are poorly understood. In particular, it remains unknown whether epigenetic mechanisms contribute to regulatory compensation for genome dosage. To gain greater insights into the importance of epigenetic information to ploidy compensation, we examined DNA methylation differences among diploid queen, diploid worker, haploid male and diploid male Solenopsis invicta fire ants. Surprisingly, we found that morphologically dissimilar diploid males, queens and workers were more similar to one another in terms of DNA methylation than were morphologically similar haploid and diploid males. Moreover, methylation level was positively associated with gene expression for genes that were differentially methylated in haploid and diploid castes. These data demonstrate that intragenic DNA methylation levels differ among individuals of distinct ploidy and are positively associated with levels of gene expression. Thus, these results suggest that epigenetic information may be linked to ploidy compensation in haplodiploid insects. Overall, this study suggests that epigenetic mechanisms may be important to maintaining appropriate patterns of gene regulation in biological systems that differ in genome copy number.     

Methods of denaturation and renaturation of DNA in interphasic chromatin: cytochemical quantitative analysis by Methyl Green staining

Almost diploid nuclei (as judged from the microdensitometric evaluation of the Feulgen positive material) of granular and Purkinje cells of the rat cerebellar cortex, were submitted to in situ DNA denaturation and renaturation experiments. We assessed the double-strandedness of DNA, by Methyl Green staining according to Scott (1967). Under these conditions a stoichiometric ratio between bound dye and DNA exists, suitable for quantitative microdensitometric measurements. Our data show that DNA in the interphasic chromatin is never completely denatured after the treatments we used. Furthermore, the renaturation takes place in a different way in the two cell types. Owing to the unlike chromatin packing of granular and Purkinje nuclei, we suggest that nuclear proteins must interfere differently on the in situ denaturation and renaturation processes.     

PHYLOGENETIC COMPARISON OF LARGE NUCLEAR DNA CONTENTS OF DIFFERENTIATED CELLS IN THE ROOTS OF EQUISETUM,TRADESCANTIA, AND HORDEUM

Nuclear DNA of meristematic, epidermal and root cap cells from the roots of three vascular plants—the cryptogam, Equisetum hyemale L, and the phanerogams, Tradescantia Clone 02 and Hordeum vulgare L.—was measured with quantitative Feulgen microspectrophotometry. Epidermal cells of all three species and root cap cells in both phanerogams contained up to 8fold the amount of nuclear DNA found in their respective meristematic telophase nuclei. In general, the large amounts of nuclear DNA parallel development and differentiation in the epidermis regardless of phylogeny, habitat, or degree of domestication. However, comparisons of the increase in nuclear DNA contents in the various epidermal cell types among these three species suggest that the mechanisms giving rise to these increases may differ phylogenetically and may represent another character in which cryptogams and phanerogams diverged in their evolution.     

Formation and division of binucleated cells in kidney cell cultures of microtus agrestis

Summary Epithelial kidney cell cultures of Microtus agrestis contain 10 to 25% binucleated cells. Observations of living cells under the phase contrast microscope showed that binucleated cells can arise by nuclear mitosis without cytoplasmic division. When binucleated cells divide the two nuclei are highly synchronized as they enter mitosis. In mitosis the chromosomes of both nuclei combine to a common metaphase plate leading to polyploid cells. In one case a tripolar spindle was seen after formation of a metaphase by the chromosomes of the two nuclei of a binucleated cell. This tripolar mitosis resulted in one binucleated and one mononucleated cell. The DNA-content (Feulgen photometry) and the distribution of heterochromatic bodies of the nuclei were corresponding to a tetraploid, a triploid and a haploid chromosome set. This suggests the possibility of somatic segregation of complete haploid sets.Supported by the Deutsche Forschungsgemeinschaft.     

Arrest of Micronuclear DNA Replication during Genomic Exclusion in Tetrahymena Produces Haploid Strains

Diploid cells of Tetrahymena thermophila were crossed to strain A*V, whose micronucleus is defective, to induce the unilateral transfer of gametic nuclei from the diploid cells to the A*V cells (round I of genomic exclusion). These haploid nuclei presumably undergo one endomitotic cycle and then become diploid with a G1 (2C) DNA content. However, further DNA replication from 2C to 4C was transiently arrested until the pairs separated. When endomitosis was blocked by treatment with cycloheximide during 6-8 hours of conjugation, the exconjugants of round I of genomic exclusion remained haploid. Competence for diploidization is apparently limited to some period of time after nuclear transfer. Blocking of diploidization during round I of genomic exclusion can be used as an efficient way to induce haploid strains in Tetrahymena.     

Corn Smut Dikaryon in Culture

A TYPICAL smut life cycle has three phases—diploid, haploid and dikaryon¹. Diploid spores (teliospores) formed in the host tissue are a resting phase. They undergo meiosis at germination to form haploid vegetative cells which are usually yeast-like. The dikaryon is the pathogenic phase and is made up of cells with two haploid nuclei. It is initiated by the fusion of two compatible non-pathogenic haploid cells and the formation of an infection hypha by the fusion product.     

Extra DNA synthesis in the dedifferentiating cells ofVicia faba roots

Summary 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.After³H-thymidine (³H-T) feeding of the decapitated roots and autoradiography, the following results were obtained: 1. two populations of labeled nuclei, characterized by two different levels of scattered labeling occurred in dedifferentiating tissues, slightly labeled nuclei being much more numerous than heavily labeled nuclei; 2. the percentage of labeled nuclei was much greater than that of DNA presynthetic nuclei in the root tissues; 3. almost all the mitoses were labeled after a 16-hour³H-T feeding; 4. the percentage of slightly labeled nuclei paralleled that of dedifferentiating cells; 5. the duration of the DNA synthesis phase and that of the gap between completion of DNA synthesis and mitosis differed in heavily and slightly labeled nuclei; 6. all nuclei which entered DNA synthesis also entered mitosis.These results are interpreted to mean that: 1. after decapitation, two different DNA syntheses occur in the dedifferentiating root tissues ofV. faba: DNA reduplication in cells which dedifferentiate starting from a DNA presynthetic nuclear condition (heavily labeled nuclei) and extra DNA synthesis in cells which dedifferentiate starting from a DNA postsynthetic nuclear condition (slightly labeled nuclei); 2. extra DNA synthesis is required in these dedifferentiating cells for entry into mitosis.     

Proportionalitätsfehler bei der Feulgen-Hydrolyse

Zusammenfassung Beim Vergleich des Feulgen-Farbgehaltes verschiedener Zellkerne (Leberzellen, Lymphozyten, Granulozyten und Spermien) traten nach Alkoholfixierung Abweichungen der gemessenen Feulgen-Werte von den nach dem Gesetz von der DNS- Konstanz zu erwartenden DNS-Gehalt dieser Kerne auf. Verglichen mit den Feulgen-Werten der diploiden Leberzellkerne ergaben Lympho- und Granulozyten bei allen Hydrolysezeiten zu niedrige (bis zu minus 20%), haploide Spermien im postmaximalen Hydrolysebereich zu hohe Feulgen-Werte (z. T. sogar höhere Werte als die diploiden Zellkerne). Innerhalb desselben Zelltypes wurden dagegen, auch beim Vergleich der verschiedenen Ploidiestufen der Leberkerne, keine Differenzen festgestellt.Da die an Leukozyten und Spermien beobachteten Abweichungen nach Methanol-Formalin-Eisessig-Fixierung nicht mehr auftraten und auch durch UV-absorptionscytophotometrische Messungen nicht bestätigt werden konnten, muß man annehmen, daß es sich um Proportionalitätsfehler handelt, die auf Hydrolyseunterschieden beruhen.Für die quantitative Feulgen-Cytophotometrie scheint daher die Methanol-Formalin-Eisessig-Fixierung besser geeignet zu sein als die Alkoholfixierung, bei deren Verwendung es leicht zu Proportionalitätsfehlern während der Feulgen-Hydrolyse kommen kann.

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Proportionality errors during feulgen hydrolysis

Summary Comparing the Feulgen dye-content of different nuclei (liver cells, lymphocytes, granulocytes and sperms) after alcohol-fixation deviations were found between the measured Feulgen values and the DNA-content to be expected from the DNA-constancy law. The Feulgen values of lymphocytes and granulocytes proved to be lower (up to minus 20%) than those of diploid liver nuclei at all hydrolysis times, while in the postmaximal range of hydrolysis the values of the haploid sperms were too high (even higher than those of the diploid nuclei). Such differences did not appear when nuclei of the same cell type in different DNA- ploidy classes (liver nuclei) were compared.Those deviations of leucocytes and sperms were no longer found after fixation in methanol-formalin-glacial acetic acid and, in addition, could not be confirmed by UV-absorption measurements. For that reason we suppose them to be due to proportionality errors caused by variations in the hydrolytic behaviour of the different nucleoproteins.Thus fixation in methanol-formalin-glacial acetic acid seems to be more suitable for quantitative Feulgen measurements than alcohol-fixation, which may easily give rise to proportionality errors during Feulgen hydrolysis.Moreover, to avoid any false interpretation of Feulgen data we should suggest controll measurements using another independent method (f. e. UV-absorption), or — if this is impossible — to check the Feulgen values after different fixations and variant hydrolysis times (premaximal, maximal, postmaximal).

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Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Standardization of the Feulgen-Schiff technique. Staining characteristics of pure fuchsin dyes; a cytophotometric investigation

Four fuchsin analogues (Pararosaniline, Rosaniline. Magenta II and New Fuchsin) usually found in Basic Fuchsin have been applied as chemically pure dyes to the Feulgen-technique. Total nuclear absorption and wavelength of the absorption maximum were measured by microspectrophotometry in Feulgen stained cytological and plastic embedded histological liver samples, and in lymphocyte nuclei in human peripheral blood smears; absorption spectra of Feulgen stained DNA-polyacrylamide films were determined by spectrophotometry. The grey value distribution of tetraploid liver cell nuclei was calculated with an image analyzer. The staining characteristics of the pure dyes were compared to commercial fuchsin samples from various suppliers. Reverse phase thin layer chromatography was used for characterization and qualitative separation of commercial batches. Pure fuchsin analogues were all equally suitable for Feulgen staining: with respect of staining intensity all pure fuchsin dyes gave nearly identical results with a bathochromic shift of the absorption maximum from Pararosaniline to New Fuchsin of about 8 microns. Differences in staining results observed among the commercial dyes were due to varying dye content, contamination with an acridine-like fluorescent compound or simply mislabelling of samples. Pure Pararosaniline is recommended for a standard Feulgen technique.