Underreplication during polytenization?

Summary Recent cytophotometric DNA determinations and results of labeling experiments are compared with results of biochemical experiments concerning larval polytene salivary gland nuclei of Drosophila melanogaster. Recent publications (Dennhöfer 1981; 1982 a, b) demonstrate that methodological errors both in hydrolysis of the DNA before Feulgen reaction and in interpretation of the cytophotometric values give raise to the hypothesis of heterochromatic underreplication during polytenization. It is concluded also that methodological difficulties cause the absence of polytene SAT-DNA in biochemical centrifugation experiments since, because of different solubilities of eu- and heterochromatic DNA, the latter is not resolved in DNA isolation procedures from polytene nuclei.     

Flow cytometric DNA ploidy analysis of soft tissue sarcomas. A comparative study of preoperative fine needle aspirates and postoperative fresh tissues and archival material

Flow cytometric (FCM) DNA ploidy measurements on frozen fresh samples of soft tissue sarcomas were compared with the corresponding analyses on preoperative fine needle aspirates and postoperative formalin-fixed archival tissues from the same tumors. A concordance in ploidy status (diploid versus non-diploid) was obtained for 63% of the fresh tissue-fine needle aspiration (FNA) sample comparisons and for 85% of the fresh tissue-archival material comparisons. The majority of discordances in the fresh tissue-FNA sample comparisons could be explained by FNA sampling errors. In the remaining discordant cases (3 of 27 FNA sample comparisons and 6 of 40 archival material comparisons), sampling errors could not explain the differences in ploidy status. The discordant cases were evenly distributed among the different sampling methods. Method reproducibility was not responsible for the differences in ploidy determinations; tumor heterogeneity may be an explanation for the discrepancies. This study showed that archival soft tissue sarcoma samples are as well suited for DNA ploidy analysis as are fresh frozen tissues.     

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.     

COMPARATIVE MEASUREMENTS OF NUCLEAR DNA IN A HETEROTHALLIC AND A SELF-FERTILE ISOLATE OF THE MYXOMYCETE,DIDYMIUM IRIDIS

Comparative measurements were made of the nuclear Feulgen-DNA content of a heterothallic and a self-fertile isolate of the myxomycete Didymium iridis. Plasmodial nuclei of both isolates contain the diploid amount of DNA. The replicated diploid (4C) values for the heterothallic and the self-fertile isolates are 5.66 and 5.95, respectively. Myxamoebae, however, are quite dissimilar in their nuclear DNA content. Those of the heterothallic isolates, Honduran 1–2 (A¹) and Panamanian 2–4 (A⁷), have mean values of 3.81 and 3.69, whereas myxamoebae of the self-fertile Philippine-1 isolate were found to have a mean value of 6.07. Myxamoebae of the Ph-1 isolate are, therefore, at the same ploidy level as the Ph-1 Plasmodium. Mean DNA values for Ph-1 sporangial nuclei were in category 4C. Measurement of the DNA content of mitotic metaphases in sporangia at T = 6 hr confirmed that the mean DNA content of both Ph-1 myxamoebae and plasmodial nuclei is equivalent to 4C. It is concluded that nuclear phase alternance is lacking in the Ph-1 isolate and that the Plasmodium of this isolate develops by apogamy.     

Presence of DNase γ-Like Endonuclease in Nuclei of Neuronal Differentiated PC12 Cells

DNase , which cleaves chromosomal DNA into nucleosomal units (DNA ladder formation), has been suggested to be the critical component of apoptotic machinery. Using rat pheochromocytoma PC12 cells, which are differentiated to sympathetic neurons by nerve growth factor (NGF), we investigated whether DNase -like enzyme is present in neuronal cells and is involved in neuronal cell death. The nuclear auto-digestion assay for DNase catalyzing internucleosomal DNA cleavage revealed that nuclei from neuronal differentiated PC12 cells contain acidic and neutral endonucleases, while nuclei from undifferentiated PC12 cells have only acidic endonuclease. The DNA ladder formation observed in isolated nuclei from neuronal differentiated PC12 cells at neutral pH requires both Ca²⁺ and Mg²⁺, and is sensitive to Zn²⁺. The molecular mass of the neutral endonuclease present in neuronal differentiated PC12 cell nuclei is 32000 as determined by activity gel analysis (zymography). The properties of the neuronal endonuclease present in neuronal differentiated PC12 cell nuclei were similar to those of purified DNase from rat thymocytes and splenocytes. Interestingly, in neuronal differentiated PC12 cells, internucleosomal DNA fragmentation is observed following NGF deprivation, whereas undifferentiated PC12 cells fail to exhibit DNA ladder formation during cell death by serum starvation. These results suggest that the DNase -like endonuclease present in neuronal differentiated PC12 cell nuclei is involved in internucleosomal DNA fragmentation during apoptosis, induced by NGF deprivation.     

Differential DNase I Sensitivity of the Two Complementary Nucleosomal DNA Strands in Cycloheximide-Treated Ehrlich Ascites Tumor Cells

Abstract

The accessibility of the two complementary DNA strands in newly replicated chromatin of Ehrlich ascites tumor (EAT) cells grown under conditions of cycloheximide-inhibrted protein synthesis was studied by analysis of the DNase I digestion of isolated nuclei. Bulk DNA was labeled with ¹⁴C-thymidine and the newly synthesized strands - with bromodeoxyu ridine and ³H-thymidine. The DNase I digests were fractionated in two successive CsCl density gradient centrifugations to obtain a dense fraction containing 15–20% newly replica ted DNA Analysis of the distribution of ¹⁴C-labeled parental DNA fragments complementary to the ³H-nascent strand has shown that the ¹⁴C-labeled fragments prevail in the region of 30–50 nucleotides. Simulation experiments using the rate constants for DNase I attack show that this result may be explained by an enhanced accessibility at the nucleosomal 5′-end region of the parental strands, where the H2a-H2b dimer interacts with DNA. This asymmetry seems tobe induced by interactions in the chromatin.     

Quantitative investigation of reproduction of gonosomal condensed chromatin during trophoblast cell polyploidization and endoreduplication in the east-european field vole <Emphasis Type="Italic">Microtus rossiaemeridionalis</Emphasis>

Simultaneous determinations of DNA content in cell nuclei and condensed chromatin bodies formed by heterochromatized regions of sex chromosomes (gonosomal chromatin bodies, GCB) have been performed in two trophoblast cell populations of the East-European field vole Microtus rossiaemeridionalis: in the proliferative population of trophoblast cells of the junctional zone of placenta and in the secondary giant trophoblast cells. One or two GCBs 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 (2–16c) and in the highly polyploid population of secondary giant trophoblast cells (32–256c) the total DNA content in GCB increased proportionally to the ploidy level. In individual GCBs the DNA content also rose proportionally to the ploidy level in nuclei both with one and with two GCBs in both trophoblast cell populations. Some increase in percentage of nuclei with 2–3 GCBs was shown in nuclei of the placenta junctional zone; this may be accounted for by genome multiplication via uncompleted mitoses. In nuclei of the secondary giant trophoblast cells (16–256c) the number of GCBs did not exceed 2, and the fraction of nuclei with two GCBs did not increase, which suggests the polytene nature of sex chromosomes in these cells. In all classes of ploidy the DNA content in trophoblast cell nuclei with the single GCB was lower than in nuclei with two and more GCBs. This can indicate that the single GCB in many cases does not derive from fusion of two GCBs. The measurements in individual GCBs suggest that different heterochromatized regions of the X- and Y-chromosome may contribute in GCB formation.     

CHANGES IN THE DNA CONTENT OF ADRENAL MEDULLA NUCLEI OF RATS INTERMITTENTLY EXPOSED TO COLD

In the adrenal medulla of rats exposed intermittently to cold (+4°C) for 100 and 300 hours, a considerable decrease (24 to 40 per cent) of the DNA content per nucleus was observed, followed by restoration to normal or above normal values within 10 days after the withdrawal of the stimulus. The findings were obtained with a scanning integrating histophotometer, and confirmed by microinterferometric investigations (on the basis of the measurement of total dry mass of nuclei isolated in aqueous medium before and after treatment with DNase) and by microchemical determinations, combined with the count of the nuclei in the homogenates. The observed decrease of DNA content cannot be attributed to errors of the methods used, nor to consequences of cellular degeneration. The available evidence seems to indicate a real decrease rather than a change in the state of a part of DNA in the nucleus in vivo whereby it becomes extractable by aqueous solutions. The restoration cannot be due to mitotic processes, which were actually never detected even with the use of colchicine, since the adrenal medulla cells in the adult rat are known to be irreversible, postmitotic cells. A correlation between the functional activity of the adrenal medulla cells and the content or state of DNA in their nuclei is demonstrated.     

Image analysis software for automatic DNA ploidy assessment of archival solid tumours.

BACKGROUND: Image cytometry has proved to provide a good alternative to flow cytometry for DNA ploidy measurement of archival tumors. However, when interactively done this technique is unable to give statistically valuable results within an acceptable time for clinical oncology. METHODS: An image cytometer was developed for fully automatic DNA ploidy quantitation, focusing efforts on speed and accuracy. Software functionalities include systematic acquisition of fields on a microscopic slide, detection, localization and sorting of nuclei, computation of the DNA content together with post-processing tools, for a deeper analysis of the DNA ploidy diagram. RESULTS: DNA ploidy analysis of archival breast carcinoma samples illustrates the accuracy of DNA ploidy measurements and the sensitivity in the detection of DNA ploidy abnormalities as a result of cell sorting. CONCLUSIONS: Fully automatic image cytometry is able to combine qualities of flow cytometry (automatic analysis of a statistically significant collection of cell nuclei) with additional advantages: sorting of unwanted events (debris, stromal and inflammatory cell nuclei) and facilities for an a posteriori control of the quality of cell selection. This method is well suited to DNA ploidy analysis of archival cancer samples.     

Cytophotometric DNA determinations and autoradiographic studies in salivary gland nuclei from larvae with different karyotypes in Drosophila melanogaster

Cytophotometric DNA determinations in Feulgen stained mitotic diploid chromosome sets of neuroblasts from larvae of Drosophila melanogaster stocks, which possess different karyotypes, show significant differences between the 4C values, caused by an additional or deficient X- and Y-chromosome depending on the karyotype. The ranges of polytenic DNA size classes are theoretically expected to be doublings of the corresponding 4C mean value of each karyotype. The extinction integral data of nuclei with completely duplicated 4C quantities exclusively fall into the range of the expected size classes. Not all data falling into the range of a size class necessarily originate from duplicated nuclei, because the limits of the DNA size classes cannot be determined by measurements, but must be estimated from the confidence limits of the corresponding 4C mean value. The validity of the mitotic 4C values of the karyotypes X/X and X/Y is tested using data from non-labeled interphase nuclei, where extinction integral data accumulate in two groups. The larger values (= G₂-nuclei) confirm the 4C values of mitotic chromosome sets, and the lower values (= G₁-nuclei) are just half of these. Extinction integrals from individual, ³H-thymidine non-incorporating polytene salivary gland nuclei accumulate in distinct, non-overlapping groups which are always complete doublings of the preceding smaller group. In each karyotype, the most frequent data of each group are in accord with the 4C doublings. The data from labeled nuclei alternate with those from unlabeled nuclei. The measured DNA values of individual polytene nuclei that did not incorporate any ³H-thymidine, demonstrate that all chromosomal DNA replicates completely during polytenization of the chromosomes in the larval salivary gland nuclei of Drosophila melanogaster. Specifically, this would mean that the heterochromatic Y-chromosome replicates as well as the partially heterochromatic X-chromosome along with the autosomes. There is no indication of underreplicating heterochromatin.     

Aging of chick embryo fibroblasts in vitro. V. Time course studies on polyploid nucleus accumulation

Age-dependent polyploidization of cultured chick embryo fibroblasts was quantitated using flow microfluorometry. The results confirm the previous observation that ploidy classes developing as a function of fibroblast population doubling are defined as 2ⁿC. Immediately after isolation from embryos, the proportion of 2C nuclei was 95.2–35.7%, decreasing with advancing in vitro age. The proportion of 4C nuclei was only 3.8% at the onset of culture, increasing to 34.5% in senescent cells. The proportion of nuclei 8C and greater increased during the last stage of culture, the highest ploidy class being 128C. On the basis of the polyploidization index, which indicates relative DNA content/cell, chick cells were shown to be considerably polyploidized when they stopped growing.     

Ploidy levels and DNA synthesis in fat body cells of the adult mosquito,Aedes aegypti: The role of juvenile hormone

Adult females of the mosquito Aedes aegypti showed two cycles of DNA replication in the fat body based on microspectrophotometric measurement of changes in nuclear DNA. The first cycle began after emergence and resulted in 80% of diploid fat body cells becoming tetraploid and 20% becoming octoploid by the end of the third day. The second replication cycle occurred 48–72 h after a blood meal and resulted in an increase in octoploid nuclei to 67% Topical application of juvenile hormone or methoprene to abdomens isolated at emergence stimulated an increase in ploidy levels above that normally seen in situ. Synthesis of DNA, estimated by incorporation of injected [³H]-thymidine, rose after emergence and remained high for 2 days. Synthesis increased again after a blood meal, reached a peak by 6 h, and returned to low levels by 24 h after the meal. The timing of DNA synthesis and a measurable increase in ploidy were temporally separated. The ploidy increase, but not DNA synthesis, was correlated with increases in juvenile hormone levels.     

DNA fragmentation during thymic apoptosis is catalyzed by DNase γ

The physiological and pathological importance of cell death by apoptosis has recently been recognized. One of the hallmarks of apoptosis is the enzymatic cleavage of genomic DNA into nucleosomal oligomers. The identification of an endonuclease responsible for apoptosis might help to explain how this cell suicide is regulated and why DNA is cleaved. Here, we found that γ type of DNase was retained in apoptotic rat thymocyte nuclei. Homogeneously purified DNase γ (Mr = 33 kDa) from the apoptotic nuclei was revealed to be a Ca²⁺/Mg²⁺-dependent endonuclease and inhibited by Zn²⁺. This enzyme cleaved chromosomal DNA with 3′-hydroxyl (OH) and 5′-phosphoryl (P) ends. The cleavage ends and its divalent cation dependencies match those observed in apoptotic thymocytes induced by X-irradiation or glucocorticoid treatment, indicating that this endonuclease is a central component of the thymic apoptosis machinery.     

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.     

Flow cytometric analysis of mouse hepatocyte ploidy

Preparative and mathematical procedures are presented for the investigation of the ploidy pattern of liver cells. The DNA content of enzymatically-isolated liver cells and of nuclei was measured by flow cytometry. The true DNA content could not be measured directly due to super-position of statistical coincidences (demanding "first mode correction") and incomplete separation of the nuclei in binucleate hepatocytes (demanding "second mode correction"). The statistical coincidences (caused by simultaneous measurement of two or more particles or subsequent reaggregation of particles) were corrected by splitting the "unnatural" i.e., aneuploid DNA content, and classifying it with the normal ploidy classes. In addition, the higher normal ploidy classes were reduced by the proportion of the measured coincidences in favour of the lower ones. The second mode correction applied to nuclear distributions only. It is a probability calculation based on counting nuclear pairs on microscope slides, and resulted in a 10% increase of diploid nuclei and a larger standard deviation between the age groups. 8c and 16c values were reduced. The tetraploid values were unchanged.     

On the integrity of sperm DNA

Ejaculated rabbit spermatozoa washed with buffer prior to decondensation by Triton X-100 and dithiothreitol were good templates for DNA synthesis by Escherichia coli DNA polymerase. This result agrees with the observations of Zirkin and Chang [1977], and implies that the sperm DNA is nicked. Template activity, however, was reduced if spermatozoa were extensively washed before decondensation, and if DNase inhibitors EDTA or Na₂SO₄ were present during decondensation. Template activity was also low if decondensation was induced with DNase inhibitors thioglycollic acid, Na₂SO₃ or sodium dodecylsulphate and dithiothreitol instead of with Triton X-100 and dithiothreitol. Calf thymus DNA was completely degraded when incubated with rabbit seminal plasma or buffer-washed spermatozoa, but much less degradation was observed if EDTA, Na₂SO₄, thioglycollic acid, Na₂SO₃ or sodium dodecylsulphate were also present, or if spermatozoa were extensively washed with buffer. Centrifugation of spermatozoa through 2.05 M sucrose completely removed contaminating DNase, and such spermatozoa were inactive as DNA templates after decondensation. The DNA template activity of swollen rabbit sperm nuclei thus parallels the activity of a contaminating seminal plasma DNase. This suggest that the nicks in sperm DNA enabling it to act as a template for DNA synthesis were generated by the DNase during decondensation and thus are not a natural structural feature of the DNA. The presence of breaks in the DNA of decondensed buffer-washed spermatozoa (DNase contaminated) was confirmed by their incorporation of phosphate from [γ^?32 P] ATP in the presence of the enzyme polynucleotide kinase. These spermatozoa were found to contain as few as two breaks/mole of DNA, but sucrose-washed spermatozoa (DNase free) were free of breaks. The possible use of this enzymic procedure for the assessment of sperm genome damage and the evaluation of the quality of a sperm population are discussed.     

Follicle cell polyploidy in Leucophaea maderae: Regulation by juvenile hormone

Microspectrophotometric analysis of Feulgen-stained nuclei of the terminal follicle cells in the cockroach Leucophaea maderae showed that during maturation the follicle cells became polyploid. In virgin females, the follicle cell nuclei were diploid. After mating, and during vitellogenesis, the ploidy of the follicle cells increased from 2 C to 32 C with a small percentage of 64 C nuclei. There was no further increase in the ploidy levels during the chorionic stage of development.Injections of juvenile hormone III into decapitated virgin females elevated the ploidy levels in the follicle cells. The DNA content of these nuclei at 96–120 h after injection of juvenile hormone III increased from 2 C to 4 C. Such polyploidization of nuclei was dose-dependent with the highest DNA content occurring in response to 25–50 μg juvenile hormone III. The juvenile hormone-induced increase in DNA content correlated with an increase in the rate of [³H]thymidine incorporation into DNA.Our data suggest that the role of juvenile hormone in follicle cell development during the vitellogenic period, whether direct or indirect, is to promote selectively a large increase in the DNA content of the cells. This may facilitate the next stage of follicle cell development, choriogenesis.     

Demonstration of a DNase-sensitive network associated with the nuclear pore complexes in rat liver nuclei

Rat liver nuclei have been studied by transmission electron microscopy after resuspension in a phosphate-buffered salt solution containing SO²⁻ ₄ as the quantitatively dominant anion. Owing to the high solubility of chromatin in the presence of SO²⁻ ₄ instead of Cl⁻ at isotonicity, nuclei are depleted for chromatin by DNase I digestion in this buffer, eliminating the need for high-salt extraction. This shows that at least 75% of the nuclear pore complexes are associated with fibrogranular structures, which ramify as a network throughout the nucleus, interconnecting the nuclear lamina, interchromatin granule clusters and nucleoli. Perichromatin granules are located in this material proximal to the nuclear pore complexes. Most of the chromatin is removed without major impact on the network, but below a level of 25% residual chromatin there is a considerable reduction of this material, and only about 15% of the connections to the nuclear pore complexes are resistant to digestion with DNase I or streptodornase A and B. The percentage of nuclear pore complexes connected to the network is further reduced by salt extraction and RNase treatment. These results suggest that DNA is an integral part of the network, which presumably plays a role in nucleo-cytoplasmic transport of RNA and protein. Received: 1 September 1998; in revised form: 17 December 1998 / Accepted: 17 December 1998