2C Amount Of DNA Research Articles

Nuclear DNA content of ploidy chimeras of Hosta Tratt. (Hostaceae) demonstrate three apical layers in all organs, but not in the adventitious root

A unique combination of chloroplast and ploidy chimeras is found in some Hosta Tratt. (Hostaceae) cultivars. The absence of endopolyploidy in the above–ground parts made it possible to study the fate of the apical or germ layers in all organs. Moreover, we would like to address the question of the number of apical cell layers in Hosta also to explain breeding results. Nuclear DNA content in twelve different organs of Hosta was determined by flow cytometric analysis of four chimeric cultivars with a 4-2-2 or 2-2-4 composition. The margin of the leaf, derived from the L1, can be determined separately and has a 4C amount of DNA in 4-2-2 ploidy chimeras but a 2C amount of DNA in 2-2-4 chimeras. The presence of three layers in Hosta was deduced from comparisons of the percentage of 4C nuclei in (2)-2-4 and (4)-2-2 chimeras. If Hosta organs derive only from two layers the percentage of 4C cells in the different ploidy chimeras should add up to 100%, but they do not. The absence of nuclei with a 2C amount of DNA in the roots of 2-2-4 plants shows that only the L3 participates in adventitious root formation. Gametes and consequently seedlings are mainly derived from the L2. The often found correlation between the color of the centre of the leaf and the color of the seedlings is the consequence of the way these cultivars originate. The presence of three apical layers is visualized in the tricolored leaves of Hosta cultivars like `Striptease'. The nuclear DNA amounts show unequivocally, that in all the above ground parts of Hosta the three apical layers L1, L2 and L3 are present. However, only the L3 participates in adventitious root formation.

Amounts of Nuclear DNA and Internal Morphology of Gibberellin- and Abscisic Acid-deficient Tomato (Lycopersicon esculentumMill.) Seeds During Maturation, Imbibition and Germination

Using X-ray photography and flow cytometry, the internal morphology and DNA replication activity of wild type (wt), GA- (gib-1) and ABA-deficient (sitw) tomato (Lycopersicon esculentumMill. cv. Moneymaker) mutant seeds were studied. During seed formation, from 30 to 45 d after pollination (DAP) the endosperm becomes solid and the seed starts to gain desiccation tolerance. At this time significant changes occur in the amounts of DNA in radicle tip cells. At 30 DAP, radicle tip cells of the three genotypes manifest about 60% of 2C, 30% of 4C and 10% of 8C amounts of DNA. Upon maturation (45 DAP onwards), most cells in the seeds of the three genotypes arrest in the G1phase of the cell-cycle with 2C amounts of DNA. However, a relatively high proportion of cells with 4C amounts of DNA was detected in the radicle tip cells ofsitwcompared with wild type andgib-1. At the well-matured stage (60 DAP), there were about 2% of seeds with free space in wild type andgib-1, and about 13% insitw. At the over-matured stage (75 DAP), even more seeds with free space were found insitw, whereas no increase in the proportion of the seeds with free space was detected in the other two genotypes. In −1.0 MPa PEG-6000 with or without 10 μMGA4+7, no germination occurred in well-matured wild type andgib-1seeds, whether or not they were dried after harvest. However,sitwseeds were able to germinate both in over-mature fruit and in −1.0 MPa PEG-6000. Priming of dried seeds in −1.0 MPa PEG induced a large amount of free space in almost all seeds of the three genotypes, and nuclear DNA synthesis in the radicle tip cells of wild type andsitwseeds. However, PEG priming of fresh (non-dried) seeds had no effect on the amount of free space and 2C/4C DNA ratios in wild type orgib-1seeds, but did induce free space in about 20–25% ofsitwseeds and provoked 4C signals insitwseeds. Removal of the endosperm and testa opposite the radicle tip of seeds resulted in root protrusion, the induction of free space and an increase of 4C DNA signals in the three genotypes. It is concluded that ABA is crucial for the efficient arrest of tomato embryo radicle tip cells in G1phase upon maturation, whereas GAs play an important role in re-initiating 4C DNA levels upon germination.

Alterations in the cell cycle characteristics of granulosa cells during the periovulatory period: Evidence of ovarian and oviductal influences

Granulosa cells at different stages of differentiation were collected from ovarian follicles and oviducts during the periovulatory period, and their nuclear DNA content was monitored by flow cytometry to establish their cell cycle characteristics (G0 + G1, S, G2 + M). The proportion of cells in the three phases of the cell cycle varied in characteristics patterns depending upon the time they were collected, before or following ovulation. Granulosa (cumulus) cells recovered from ovulated oocytes were mitotically inactive as shown by the large proportion of cells with a 2C amount of DNA and the absence of cells in S phase. The proportion of granulosa cells in G2 + M decreased when recovery from the oviducts was delayed. In contrast, granulosa (cumulus and/or mural) cells recovered from preovulatory follicles prior to luteinizing hormone (LH) exposure contained a considerable population of cells undergoing DNA synthesis, and a decreased proportion of cells with a 2C DNA content. Our findings indicate that granulosa cells undergo dynamic and characteristics changes in all cell cycle phases during the periovulatory period, within follicular and oviductal environments. Intrafollicular events appear to play a major role in controlling DNA synthesis, proliferation, and related cell cycle events in the granulosa cells. Flow cytometric techniques provide objective and detailed information on the cell cycle characteristics of granulosa cell populations at different stages of differentiation. Elucidation of the mechanisms regulating cell cycle parameters of granulosa cells and their physiological significance thus seems feasible.

Immune abnormalities in the diabetic New Zealand obese (NZO) mouse: Insulin treatment partially suppresses splenic hyperactivity measured by flow cytometric analysis

Immune abnormalities in both the humoral and cell-mediated immune systems were found in New Zeland obese (NZO) mice, a model of diabetes associated with autoimmune disease. Both the formation of anti-sheep red blood cell plaques and mitogen stimulation of NZO spleen cells were significantly decreased compared to normal strains of mice. In addition to perturbations of splenic lymphocyte function, the enlarged spleens of NZO mice demonstrated increased cycling cells as measured by flow cytometric analysis of the DNA content of individual cells. These abnormalities are similar but less pronounced than those found in another autoimmune model, the New Zealand black (NZB) strain. The pancreases of diabetic NZO mice contained a higher fraction of nuclei with greater than 2c amount of DNA. Chronic insulin administration decreased the percentages of splenic proliferating cells and polyploid pancreas cells in NZO mice. In addition, insulin treatment of NZO mice decreased spleen weights and serum glucose levels. Chronic insulin treatment did not alter these parameters in either nondiabetic Balb/c or autoimmune NZB mice. Thus, the features of the NZO strain which serve as both an autoimmunity model and a diabetes model can be dissociated. Insulin treatment had a unique effect on NZO traits but not on NZB traits even though many of these traits were present in both strains. In conclusion, although autoimmunity may underlie the diseases of both the NZO and the NZB strains, the amelioration of some of the features of the NZO strain by insulin may represent the beneficial effects of insulin on the metabolic control of cellular function in NZO mice.

CELL CYCLE KINETICS OF ENDOREDUPLICATION IN GAMMA‐IRRADIATED ROOT MERISTEMS OF PISUM SATIVUM

Label and mitotic indices and microspectrophotometry of unlabeled interphase cells were used to measure the proportion of root meristem cells of Pisum sativum in each cell cycle stage after exposure to protracted gamma irradiation. Three seedling types were investigated: 1) intact seedlings, 2) seedlings with cotyledons detached and treated with lanolin paste applied to the area of cotyledon excision, and 3) seedlings with detached cotyledons and treated with a G2 Factor applied to the area of cotyledon excision in lanolin paste. In intact seedling meristems, predominant cell arrest occurred with a 4C amount of DNA while 0.30 of the cells underwent endoreduplication to arrest with an 8C amount of DNA. Only 0.07 cells arrested with a 2C amount of DNA. Polyploid cells were produced several days after the start of irradiation and were derived from a diploid cell population. In seedlings exposed to lanolin only, without cotyledons, most cells arrested with a 2C amount of DNA with no polyploid cells. In seedlings exposed to a G2 Factor in lanolin after cotyledon excision, most cells arrested with a 4C amount of DNA but no cells underwent endoreduplication. These experimental results suggest that the G2 Factor derived from cotyledons of Pisum sativum was necessary for predominant cell arrest in G2 but alone was not sufficient for the polyploidization step.

Evidence for dosage compensation in parthenogenetic Hymenoptera.

Amounts of DNA-Feulgen staining in individual somatic nuclei and mature sperm of the parthenogenetic wasps, Habrobracon juglandis, H. serinopae, and Mormoniella vitripennis, were determined with a scanning microdensitometer. The haploid genome for both species of Habrobracon was estimated to be 0.15-0.16 X 10-(12) gDNA, corresponding to a molecular weight of roughly 10 X10(10) daltons. The haploid genome of M. vitripennis is approximately twice this value, 0.33-0.34 X 10-(12) g, or about 20X10(10) daltons. Measurements made on dividing nuclei from syncytial preblastoderm embryos of H. juglandis and M. viripennis showed that the chromosomes of impaternate males were present in the haploid number and contained the C amount of DNA; whereas nuclei from female preblastoderm embryos contained the diploid number of chromosomes and the 2C amount of DNA. However, hemocyte and brain cell nuclei from either male or female adult wasps contained 2C and 4C amounts of DNA. Both sexes also showed equivalent levels of polyploidy (8C, 16C, or 32C) in Malpighian tubule nuclei. Therefore, in these parthenogenetic species,, a mechanism must exist the compensates during later development for the initial two-fold difference in the chromatin content of somatic nuclei in haploid male and diploid female embryos. Hemocytes from impaternate Mormoniella diploid males and triploid females contain the 2C and 3C amounts of DNA, respectively Therefore dosage compensation involves an additional cycle of DNA replication only in hapoid cells, and it insures that a certain minimum quantity of DNA is received by each somatic cell.

The Early Action of the Floral Stimulus on Mitotic Activity and DNA Synthesis in the Apical Meristem of Xanthium strumarium

Vegetative plants of Xanthium strumarium (a short-day species) were induced to flower by exposure to a single 16-hr long night. By cutting off the induced leaf (half-expanded leaf) at various times, it was established that, by 8 hr after the end of the long night, a sufficient amount of floral stimulus had reached the meristem to induce a flowering response. The following sequence of events occurred in both the peripheral and central zones of the apical meristem of induced plants: 1) a rise in the mitotic index beginning at 28 hr after the end of the long night and culminating at 36 and 56 hr; 2) a stimulation of DNA synthesis starting at 32–36 hr and reaching a maximum at 60 hr; 3) an increase in nucleolus diameter starting at 32 hr. The cell population in the meristems of both vegetative and induced plants displayed a similar distribution, with about 80 % of the nuclei with the 2C amount of DNA. The comparison of the kinetic data concerning the mitotic index and DNA synthesis indicated that one of the early effects of the floral stimulus in the peripheral and central zones was the release in mitosis of cells whose nuclei were in the postsynthetic (G2) phase of the mitotic cycle. In the pith-rib meristem, the following events were recorded: 1) a stimulation of DNA synthesis starting at 20 hr; 2) a rise of the mitotic index beginning at 28 hr; 3) the vacuolation and elongation of cells starting at 48 hr. All these events occurred well before the initiation of bract and flower primordia, which began at 96 and 136 hr, respectively. Neither stimulation of mitotic activity nor flowering occurred in the meristems of plants subjected to a long night interrupted at its midpoint by a 5-min light break. The results are discussed in relation to the early events which are known to occur in the meristems of other photoperiodic species in transition to flowering.

THE EARLY ACTION OF THE FLORAL STIMULUS ON MITOTIC ACTIVITY AND DNA SYNTHESIS IN THE APICAL MERISTEM OF XANTHIUM STRUMARIUM

Vegetative plants of Xanthium strumarium (a short‐day species) were induced to flower by exposure to a single 16‐hr long night. By cutting off the induced leaf (half‐expanded leaf) at various times, it was established that, by 8 hr after the end of the long night, a sufficient amount of floral stimulus had reached the meristem to induce a flowering response. The following sequence of events occurred in both the peripheral and central zones of the apical meristem of induced plants: 1) a rise in the mitotic index beginning at 28 hr after the end of the long night and culminating at 36 and 56 hr; 2) a stimulation of DNA synthesis starting at 32–36 hr and reaching a maximum at 60 hr; 3) an increase in nucleolus diameter starting at 32 hr. The cell population in the meristems of both vegetative and induced plants displayed a similar distribution, with about 80 % of the nuclei with the 2C amount of DNA. The comparison of the kinetic data concerning the mitotic index and DNA synthesis indicated that one of the early effects of the floral stimulus in the peripheral and central zones was the release in mitosis of cells whose nuclei were in the postsynthetic (G2) phase of the mitotic cycle. In the pith‐rib meristem, the following events were recorded: 1) a stimulation of DNA synthesis starting at 20 hr; 2) a rise of the mitotic index beginning at 28 hr; 3) the vacuolation and elongation of cells starting at 48 hr. All these events occurred well before the initiation of bract and flower primordia, which began at 96 and 136 hr, respectively. Neither stimulation of mitotic activity nor flowering occurred in the meristems of plants subjected to a long night interrupted at its midpoint by a 5‐min light break. The results are discussed in relation to the early events which are known to occur in the meristems of other photoperiodic species in transition to flowering.

Dose Rate, Mitotic Cycle Duration, and Sensitivity of Cell Transitions from G1 → S and G2 → M to Protracted Gamma Radiation in Root Meristems

Experiments were designed to determine the relative radiosensitivity of the cell transition points of G1 $Yields$ S and G2 $Yields$ M in root meristems of several plant species. Label and mitotic indices and microspectrophotometry were used to measure the proportions of cells in each mitotic cycle stage in root meristems after protracted gamma radiation. The criterion of radiosensitivity was the dose rate needed to produce a tissue with less than 1 percent cells in S and none in M after 3 days of continuous exposure. The results show that DNA is the primary radiation target in proliferative root meristems and that the cycle duration stipulates the time interval of vulnerability. In each species, nonrandom reproducible cell proportions were established with 2C:4C:8C amounts of nuclear DNA after 3 days of exposure. Roots of Helianthus annuus, Crepis capillaris, and Tradescantia clone 02 had 80 percent cells with a 2C amount of DNA and 20 percent had a 4C amount of DNA. In these species the transition point of G1 $Yields$ S was more radiosensitive than G2 $Yields$ M. Roots of Pisum sativum and Triticum aestivum had cell proportions at 2C:4C:8C amounts of DNA in frequencies of 0.10 to 0.20:0.40 to 0.60:0.30 tomore » 0.40. In these two species 0.30 to 0.40 cells underwent radiation-induced endoreduplication that resulted from a rapid inhibition of cell transit from G2 $Yields$ M and a slower impairment of G1 $Yields$ S. Cells increased from 2C to 4C and from 4C to 8C amounts of DNA during irradiation. The proportions of nuclei with 2C:4C:8C amounts of DNA were dependent in part upon the relative radiosensitivity of the G1 $Yields$ S and G2 $Yields$ M control points. The data show the relative radiosensitivity of the transition points from G1 $Yields$ S and from G2 $Yields$ M was species specific and unrelated to the cycle duration and mean nuclear DNA content of the plant species. (auth)« less

Number and DNA content of nuclei in the free-living nematode Panagrellus silusiae at each stage during postembryonic development.

Nuclei from the four major tissues of the nematode Panagrellus silusiae were enumerated and examined using Feulgen microspectrophotometry at each stage during postembryonic development. The number of nuclei in the hypodermis, nerve, and intestine remains fairly constant during maturation, but there is a slight increase (∼57%) in the number of muscle nuclei. Thus, this organism is not stringently eutelic. The total number of somatic nuclei is about 600. DNA values of hypodermis and nerve nuclei were unimodal and adult nuclei had 2C amounts of DNA. The DNA distribution of muscle nuclei reflects the pattern expected for a tissue in which a portion of the nuclei are undergoing DNA synthesis. Intestinal nuclei accumulated DNA in the absence of nuclear division and in the adult the nuclei fall into discrete DNA classes which correspond to a geometric series of the 2C value. It is concluded that chromatin diminution does not occur in this species. In addition, the relationship in the different tissues of nuclear DNA content to nuclear volume and cell size is discussed.

THE DURATION OF THE G1 PHASE OF THE MITOTIC CYCLE AND ITS RELATION TO RADIOSENSITIVITY

SUMMARYThe duration of each phase of the mitotic cycle in four regions of the root meristem of Zea mays has been determined by pulse labelling with 3H thymidine. S, G2 and mitosis are similar in length in all the regions, but G1 differs considerably and is largely responsible for the diversity in rates of mitosis. G1 is non‐existent in the cap initials, 151 hours in the quiescent centre, and 2–4 hours in the stele.These data, together with information on sensitivity to ionizing radiation, suggest that it is the ratio of the lengths of G1 to the rest of the mitotic cycle that decides how resistant a tissue will be to radiation and they lead to a general explanation of the law that rapidly dividing cells are more susceptible than slowly dividing cells. The extreme sensitivity of the cap initials and the high resistance of the quiescent centre are correlated with the brevity of G1, in the former and the prolongation of G1 in the latter. The fraction of the mitotic cycle spent in G1 (when each nucleus has only the 2C amount of DNA) therefore probably determines both radio‐sensitivity and rates of division.

Autoradiographic and microphotometric studies of desoxyribose nucleic acid during microgametogenesis in Lilium longiflorum.

1. The technique for making autoradiographs with stripping film is described in detail. Methods for administering P32 to plants and for preparation of tissues for autoradiographs are given. 2. Two different methods, enzyme digestion and acid hydrolysis, which were used for separating RNA, DNA, and phosphoprotein in tissue sections, were compared and evaluated. Although either method is valid for quantitative work, precautions are necessary and certain limitations to the quantitative separation of RNA and DNA should be taken into account. More work is needed to properly characterize the products extracted during enzyme digestion and acid hydrolysis. 3. Incorporation of P32 into nuclei of lily anthers was studied by the autoradiographic method at all stages from premeiosis to mature pollen. A quantitative comparison of the amounts of P32 extracted by RNase, DNase, Feulgen hydrolysis and hot TCA showed that incorporation into DNA occurred at relatively short periods during interphase, namely: (1) at preleptotene before meiotic prophase in microsporocytes, (2) at late interphase preceding mitosis in the microspore and (3) during early to mid-interphase in the generative nucleus of the pollen grain (the tube nucleus incorporates P32 slowly during the same period but this P32 was not definitely identified as DNA phosphorus). Two successive mitoses occur in tapetal cells while the microsporocytes are in meiotic prophase. In the interphase preceding each division incorporation of P32 into DNA occurs, but following the second mitosis no further divisions or incorporation was detected. However, during this latter period incorporation into RNA and phosphoprotein reached the highest level detected at any stage. During the period of DNA synthesis, incorporation of P32 into RNA was low or absent, but in the interval between the cessation of DNA synthesis and prophase the incorporation of P32 into RNA of both nucleus and cytoplasm reached its peak. 4. Microphotometric determination of amounts of DNA (Feulgen stain) showed that the amount per nucleus remains constant in all stages, except for doubling during the periods when P32 is incorporated, and the reduction by half at each division of a nucleus. The 2C amount is present in sporogenous nuclei until preleptotene when values intermediate between 2C and 4C are found. By the beginning of leptotene 4C is present in each microsporocyte nucleus. The amount does not change through meiotic prophase. Each nucleus after the first meiotic division contains 2C and each nucleus of the quartet of spores has 1C. The amount per nucleus in microspores doubles toward the end of the long interphase prior to prophase. After the mitosis in the microspore, the generative nucleus attains the 2C level relatively early in interphase. The vegetative nucleus has 1C in the interphase immediately after mitosis, but could not be accurately measured at later stages. Tapetal nuclei have the 2C amount of DNA when the microsporocytes are in leptotene. The amount doubles while the microsporocytes are in zygotene and the first mitosis usually occurs while the microsporocytes are in early pachytene. Another doubling occurs during the relatively short interphase and a second mitosis occurs when the microsporocytes are in late pachytene or early diplotene. The 4C class was typical for tapetal nuclei at later stages, but this may, in some cases at least, result from fusion of nuclei in the multinucleate cells during or following the second mitosis. 5. The periods of DNA increase (measured microphotometrically) coincide very closely with the periods of incorporation of P32 into DNA. This finding supports the concept of the stability of the DNA molecule, and indicates that incorporation may be used as a criterion not only of synthesis of DNA, but also of increase in the amount of DNA per nucleus.