Nuclear DNA Content Of Cells Research Articles

Endopolyploidy in Vanda Miss Joaquim (Orchidaceae)

• Occurrence of endopolyploidy in somatic tissues of the hybrid orchid Vanda Miss Joaquim (Vanda hookeriana×Vanda teres) was investigated with respect to tissue type and developmental stage. Effects of naphthaleneacetic acid (NAA) and gibberellic acid (GA3 ) on endopolyploidy during embryo development were also studied. • For the study of endopolyploidy, flow cytometric analysis was employed to determine nuclear DNA content of cells of somatic tissues. • Multiploid cells were observed in leaves, roots and column, but not in shoot apex, stem, perianth and pedicel. Furthermore, differential distribution of multiploid cells was found among different parts of leaves and roots. The degree of endopolyploidy in embryos increased with development. NAA was shown to induce endoreduplication in germinating embryos to a much larger extent than GA3 . • The pattern of endopolyploidy was characteristic of tissue type and developmental stage. The implications of endopolyploidy during differentiation and development, as well as the relevance of endopolyploidy to somaclonal variation, are discussed.

DNA content of nuclei ofActinidia deliciosavar.deliciosa‘Hayward’ (kiwifruit) analysed by image cytometry

Abstract Static image cytometry (ICM), computer-enhanced, optical microscopy was used to determine the DNA content of Feulgen-stained nuclei derived from in planta and in vitro shoots of kiwifruit (Actinidia deliciosa var. deliciosa ‘Hayward’). Nuclei in meristem tips, analysed from the same vines, showed alterations in the nuclear DNA content between successive years. These findings correlate closely with flow cytometric analysis of meristem tips of the same vines. The absolute nuclear DNA content for the samples analysed by ICM lay between 5.58 pg DNA per 2C nucleus (spring 1991) and 7.96 pg DNA per 2C nucleus (spring 1992), an increase of 42.5%. The 2C nuclear DNA content of plantlets grown in vitro for 3 years displayed no significant differences, irrespective of the duration on fresh culture medium. The 2C nuclear DNA content of cells from the meristem tips of these plants was approximately 6.71 pg DNA, lower than the high point recorded from in planta apices. These estimates were significantly higher than those obtained by flow cytometry. Nuclei of meristematic cells of in planta shoots also showed an increased nuclear DNA content between analyses 13 days prior to bud burst and again 1 day after bud burst.

Androgenetic diploids of rainbow trout (Oncorhynchus mykiss) produced by fused sperm

Androgenesis is a technique that produces individuals with all of their chromosomes from male parents. It can be useful in generating homozygous lines of fish, and in the recovery of species from cryopreserved sperm. We produced androgenetic diploids by using fused sperm to fertilize eggs. Eggs of albino "Yellow" rainbow trout (Oncorhynchus mykiss) were exposed to 60Co γ-ray irradiation to inactivate the maternally-derived nuclear DNA, and were then fertilized with wild-type rainbow trout sperm fused using an artificial seminal plasma containing 85 mM CaCl2. Embryos that reached the hatching stage (average 0.11%) were pigmented. The nuclear DNA content of cells from newly hatched fry was the same as the DNA content of erythrocyte nuclei of normal diploids.

Heterogeneity in the nuclear DNA content of cells in carcinomas of the biliary tract and pancreas

The authors investigated the frequency of aneuploidy and heterogeneity in DNA ploidy within one tumor by multiple sampling. Using flow cytometry, the authors measured the nuclear DNA content of cells in fresh surgical specimens from 24 patients with carcinoma of the extrahepatic bile duct, 8 patients with carcinoma of the gallbladder, 5 patients with carcinoma of ampulla of Vater, and 14 patients with carcinoma of the pancreas. The frequency of aneuploidy was 91.7%, 87.5%, 80.0%, and 85.7% in carcinoma of the extrahepatic bile duct, gallbladder, ampulla of Vater, and pancreas, respectively. The incidence of heterogeneity in DNA ploidy within a tumor was 79.2%, 87.5%, 80.0%, and 85.7% in carcinoma of the extrahepatic bile duct, gallbladder, ampulla of Vater, and pancreas, respectively. The DNA index ranged mostly from 1.0-2.0. Hypodiploid samples constituted 23.5% of the total, and only five (9.8%) had a DNA content above tetraploid. Many of the patients in which the DNA index was about 1.0 (including diploid cases) tended to be in the relatively early stages of the disease. It is necessary to take several fresh samples from the same tumor to accurately measure the variations in nuclear DNA content. The authors suggest that three or four samples should be studied to determine aneuploidy and that DNA ploidy heterogeneity should be analyzed in five or six samples.

Development of polysomaty in seedlings and plants of Cucumis sativus L.

The nuclear DNA content of cells from individual organs of seedlings and plants of Cucumis sativus L. (cv. Hokus) was determined during development by flow cytometry. The results revealed the presence of cells with different DNA contents (ranging from 2C to 64C) in varying proportions. This polysomaty was both specific to the developmental stage of the plant and to the individual organ. Polysomaty was already present in the hypocotyl and radicle of the embryo in the seed, and also in young secondary roots, immature leaves, flower buds and very young stem internodes. Rapid increases of the nuclear DNA contents (endopolyploidy) and the number of C-value levels occurred in all organs of the seedling and in the stem internodes and flowers during elongation. In the seedlings, these increases originated in the transition tissue between hypocotyl and radicle. In the leaves the initial pattern of polysomaty did not change during expansion. Compared to other organs, the leaves showed the highest frequency (60%) of cells with a 2C DNA content. During plant maturation, the proportion of nuclei with higher DNA contents gradually increased in the radicle, the hypocotyl and the stem internodes. The results are discussed in relation to organ elongation, to genome size and culture conditions, and to their relevance to molecular and cellular approaches for cultivar improvement.

Study of chromosomes, DNA amount, and in vitro growth in different species of Luzula

Cells of six different species of Luzula were subjected to in vitro culture and examined for callus formation. The cells of calli from the different species were analyzed for variation in chromosome number. The cells of different species showed a differential response in culture with respect to callus growth. Chromosome number variation in vitro, including both hypo- and hyper-diploidy, was recorded in all species except L. pediformis and L. luzuloides. The chromosome fragments survived as a result of a nonlocalized centromere. With prolonged culture, normal diploid cells were frequent. The nuclear DNA content of cells of these species was also measured, both in vivo and in vitro. The DNA values ranged from 6.05 to 7.03 pg per 4C nucleus. No marked change in DNA value was noted in cells even with high chromosome numbers, thereby confirming their origin through fragmentation of chromosomes. In cells of callus, the DNA amount was observed to be higher than in normal cells, indicating fragmentation and duplication of individual chromosomes. Their importance in the origin of new genotypes has been indicated.Key words: Luzula spp., in vitro growth, chromosomes, nuclear DNA content.

Diagnostic value of DNA flow cytometry combined with fine needle aspiration biopsy in lymphomas.

The nuclear DNA content of cells from 45 malignant lymphomas and from 60 benign lymph nodes obtained by fine needle aspiration was analysed to investigate the diagnostic value of DNA flow cytometry combined with routine diagnostic cytology in lymphomas. DNA aneuploidy was found in 43 per cent of lymphomas of high grade malignancy (NCI Working Formulation) but only rarely in lymphomas of intermediate- or low-grade malignancy or in Hodgkin's disease, and never in benign lymph nodes. The median percentage of proliferative cells (S + G2/M) was 22.6 per cent in diploid high-grade lymphomas, 15.3 per cent in intermediate-, and 8.1 per cent in low-grade lymphomas, as compared with 4.9 per cent in benign lymph nodes (P less than 0.0001). If the presence of DNA aneuploidy or more than 12 per cent of proliferative cells is used as a criterion for malignancy, the diagnostic accuracy of DNA flow cytometry in detecting lymphoma is 81 per cent. DNA flow cytometry suggested correct diagnosis in 10 of the 19 false positive, false negative, or indeterminate cytological findings encountered during the study. It is concluded that DNA flow cytometry combined with fine needle aspiration biopsy has diagnostic value in lymphomas, but false negative results are common especially in low-grade lymphomas; the method should therefore be used in conjunction with light microscopy.

Interaction of the Circadian Cycle with the Cell Cycle in Pyrocystis fusiformis

Dividing pairs or single cells of the large dinoflagellate, Pyrocystis fusiformis Murray, were isolated in capillary tubes and their morphology was observed over a number of days, either in a light-dark cycle or in constant darkness. Morphological stages were correlated with the first growth stage, G(1), DNA synthesis, S, the second growth stage, G(2), mitosis, M, and cytokinesis, C, segments of the cell division cycle. The S phase was identified by measuring the nuclear DNA content of cells of different morphologies by the fluorescence of 4', 6-diamidino-2-phenylindole dichloride.Cells changed from one morphological stage to the next only during the night phase of the circadian cycle, both under light-dark conditions and in continuous darkness. Cells in all segments of the cell division cycle displayed a circadian rhythm in bioluminescence. These findings are incompatible with a mechanism for circadian oscillations that invokes cycling in G(q), an hypothesized side loop from G(1). All morphological stages, not only division, appear to be phased by the circadian clock.

Detection of suspicious cells and rejection of artefacts in cervical cytology using the Leyden Television Analysis System.

In slide based automation of cervical cytology the first stage of analysis involves finding possibly suspicious cells, or areas on the slide with these types of cells. By using a television based system such as the Leyden Television Analysis System (LEYTAS), a number of detection methods can be applied to rapidly screen a large number of fields automatically for suspicious cells. In this paper, results using a parameter based on increased nuclear DNA content of cells are given and a second detection method based on a chromatin pattern feature, called chromatin contrast, is discussed. Two blind trials on 41 positive and 22 negative cervical slides, using the Leyden Television Analysis System to detect suspicious cells with an increased nuclear DNA content, were promising. In 1 of the 41 positive cases no suspicious cells were found. In the negative specimens, suspicious cells were detected in 1 of 9 cases and 1 of 13 cases, with the two detection parameters investigated. These findings are discussed and some automatic artefact rejection procedures with preliminary results are given.