Nuclear DNA,nuclear area and nuclear dry mass in thirteen species of Crotalaria (Angiospermae,Leguminosae)

Estimations on nuclear DNA content, nuclear area and nuclear dry mass were made on 13 species of Crotalaria, with the help of Vicker's M 85 microdensitometer and Vicker's interferometer. Highly significant differences for all three characters were found between species. DNA density (DNA/area) increased with increase in DNA content. The cultivated species, C. juncea had a lower DNA density relative to its DNA content.—Nuclear DNA made only a small fraction of nuclear dry mass (115). Although total dry mass and non-nucleolar nuclear dry mass showed significant regression on DNA content, the nucleolar dry mass gave a much wider scatter, indicating that nucleolar mass is not equally dependent on nuclear DNA content.     

Distinction between Nuclear Satellite DNAs and Chloroplast DNA in Higher Plants

Triton X-100 solubilized chloroplast DNA but not nuclear DNA from a mixture of chloroplasts and nuclei. The buoyant density of chloroplast DNA was different from that of the satellite DNA in all of the species examined (Phaseolus coccineus, Cucumis sativus, Cucumis melo, Antirrhinum majus, Vicia faba, Oenothera fruiticosa youngii). Chloroplast DNA constituted between 4.3% and 0.25% of the total leaf DNA in these species, and was present as 5 to 20 copies in each chloroplast.     

Nucleolar DNA in oocytes of Salmo irideus (Gibbons)

The amplification of ribosomal genes has been studied in oocytes from Salmo irideus. In situ nucleic acid hybridization showed that the synthesis of nucleolar DNA begins in oogonium and proceeds slowly through leptotene and zygotene when a small amount of extrachromosomal nucleolar DNA is produced. In early pachytene there is a rapid build-up of nucleolar DNA demonstrable by rapid incorporation of tritiated thymidine. Synthesis stops completely in early diplotene when nucleolar DNA becomes dispersed over the inner surface of the nuclear envelope in the form of small Feulgen-positive granules. Photometric measurements of Feulgen stained nuclei showed that the final amount of amplified nucleolar DNA synthesized in each oocyte is approximately 20 g. The amplified DNA does not form a heterochromatic mass. The buoyant density of the amplified nucleolar DNA calculated from analytical centrifuge tracings in relation to DNA from Micrococcus luteus ( = 1.731 g cm^–3) is 1.715 g cm^–3 and corresponds to a G + C content of 57%. There are indications that the buoyant density of the somatic nucleolar DNA is lower than that of amplified nucleolar DNA.Similarities and differences between ribosomal gene amplifications in oocytes of Salmo irideus and the corresponding process in Xenopus are discussed.     

Genome size and the proportion of repeated nucleotide sequence DNA in plants

The reannealing kinetics of denatured DNA fragments from 23 species of higher plants have been studied, using hydroxylapatite chromatography to distinguish reannealed from single-stranded DNA. The 2C nuclear DNA contents of the species varied between 1.7 and 98 pg. The proportions of DNA in species with a nuclear DNA mass above 5 pg that reannealed with the kinetics of sequences present in more than 100 copies were high (69–92% with a mean of 80±2.0%). For species with less than 4 pg of DNA, the mean proportion of repeated-sequence DNA was 62±2.9%. It is concluded that most of the variation in nuclear DNA mass in higher plant chromosomes can be accounted for by variation in repeated-sequence DNA. The consequences of altering the adapted DNA content of a species by the addition of families of repeated sequences are discussed in relation to the proportion of repeated-sequence DNA.     

Changes in nuclear and nucleolar protein content during the growth and differentiation of root parenchyma cells in plant species with different DNA-endoreplication dynamics

Summary Using cytophotometric procedures, we measured the nuclear and nucleolar protein content of successive zones of growth and differentiation in consecutive (1–7 mm) root segments obtained from eight species of the Angiospermae after staining the preparations with Feulgen-Naphthol Yellow S (F-NYS). In meristematic cells the nuclear and nucleolar protein content was found to double during the cell cycle. In species in which differentiation occurs at the same time as nuclear DNA endoreplication, i.e. Vicia faba subsp. minor, V. faba subsp. major, Pisum sativum, Hordeum vulgare and Amaryllis belladonna, the pool of nuclear proteins observed during the G₂ phase of the cell cycle was seen in the differentiated zone in nuclei containing 8C DNA. Species in which differentiation is not accompanied by the process of nuclear DNA endoreplication, i.e. Levisticum officinale, Tulipa kaufmanniana and Haemanthus katharinae, exhibited the highest nuclear proteins content during the G₂ phase of the cell cycle; comparably high values were not found in the differentiated zone. A decrease in nucleolar protein content was observed during the process of differentiation, this tendency being more evident in the studied species that do not exhibit endoreplication.This work was supported by the Polish Academy of Sciences as a part of project no 09.7.1.4.5     

LOCALIZATION OF RIBOSOMAL DNA WITHIN OOCYTES OF THE HOUSE CRICKET, ACHETA DOMESTICUS (ORTHOPTERA: GRYLLIDAE)

A large DNA-containing body is present in addition to the chromosomes in oocytes of the house cricket Acheta domesticus. Large masses of nucleolar material accumulate at the periphery of the DNA body during the diplotene stage of meiotic prophase I. RNA-DNA hybridization analysis demonstrates that the genes which code for 18S and 28S ribosomal RNA are amplified in the ovary. In situ hybridization indicates that the amplified genes are localized within the DNA body of early prophase cells. As the cells proceed through diplotene the DNA which hybridizes with ribosomal RNA is gradually incorporated into the developing nucleolar mass.     

The nuclear DNA content and chromatin ultrastructure of the coelacanth Latimeria chalumnae

By measurement of 731 erythrocytes by Feulgen cytophotometry, the nuclear DNA content of the coelacanth Latimeria chalumnae Smith is determined to be 7.22 picograms (pg). This value is high among fishes but is closely comparable to that of man and most other mammals. The average mass of erythrocyte nuclear chromatin, measured by quantitative electron microscopy, is 15.2 pg. This chromatin is in the form of fibers having a mean diameter of 202 Å. The average weight of the chromatin fiber is 6.75 × 10^?16 g/μm. Thus, the nucleus contains 22 500 μm of chromatin fiber. Dividing the nuclear DNA content of Latimeria by the known mass of the DNA double helix (3.26 × 10^?18 g/μm) gives a total length of 2 215 000 μm of DNA double helix. In comparing these two measurements of structural length, it is found that 98.4 lengths of double helix are packed into one length of chromatin fiber. This packing ratio is over three times greater than that of human G1 lymphocytes. The difference may be attributable to the difference between the two tissues and thus reflect a functional distinction, or it may be due to the difference between the two species and reflect an evolutionary distinction.     

Stratification within centrifuged amoeba nuclei

Summary Two species of large, fresh water amoebae were ultracentrifuged and studied with the electron microscope. Emphasis was placed on the stratification of the nucleoplasm, including nucleoli, within the confines of the nuclear envelope during interphase. Three major strata were found in the nuclei of both amoeba species, namely the centripetal nucleoplasm, the middle chromatin stratum, and the centrifugal nucleolar mass. In the highly radioresistant A. proteus, the nucleolar mass separated into a centripetal electron-opaque layer and a centrifugal electron-lucent layer. The latter layer appears to be missing from the radiosensitive P. illinoisensis. The nature of these nucleolar layers and their possible relationship to differences in radiosensitivity between the two species of amoebae is discussed. The contents of the heavier of the two nucleolar layers in A. proteus might be resistant to radiation damage and may possess radiorestorative capacity.Work supported by U.S. Atomic Energy Commission. A part of the work was reported at the 17th Meeting of the Society of Protozoologists, Boulder, Colorado in 1964.     

Chromosomal features of nucleolar dominance in hybrids between the Neotropical fish <Emphasis Type="Italic">Leporinus macrocephalus</Emphasis> and <Emphasis Type="Italic">Leporinus elongatus</Emphasis> (Characiformes,Anostomidae)

In the present study, the chromosomal mechanisms of nucleolar dominance were analyzed in the hybrid lineage “Piaupara,” which resulted from crossing the Leporinus macrocephalus female (Piauçu) and L. elongatus male (Piapara) fish. The analyses demonstrated that, in the hybrid, the nucleolar region inherited from L. elongatus presented higher activity, with expression in 100% of the cells, whereas the nucleolar region from L. macrocephalus appeared active at a frequency of 11.6%. The FISH technique with an 18S probe showed that the ribosomal DNA of the nucleolar region was not lost in the hybrid, and the results therefore demonstrated invariable marks in two chromosomes, each originating from one parent. An interesting difference between the nucleolar regions of the parental species was the association of the NOR with heterochromatic blocks (repetitive DNA) in L. elongatus, which could act as a determinative element in the establishment of this process.     

Gene pools in wild Lima bean (Phaseolus lunatus L.) from the Americas: Evidences for an Andean origin and past migrations

The aims of this research were to assess the genetic structure of wild Phaseolus lunatus L. in the Americas and the hypothesis of a relatively recent Andean origin of the species. For this purpose, nuclear and non-coding chloroplast DNA markers were analyzed in a collection of 59 wild Lima bean accessions and six allied species. Twenty-three chloroplast and 28 nuclear DNA haplotypes were identified and shown to be geographically structured. Three highly divergent wild Lima bean gene pools, AI, MI, and MII, with mostly non-overlapping geographic ranges, are proposed. The results support an Andean origin of wild Lima beans during Pleistocene times and an early divergence of the three gene pools at an age that is posterior to completion of the Isthmus of Panama and major Andean orogeny. Gene pools would have evolved and reached their current geographic distribution mainly in isolation and therefore are of high priority for conservation and breeding programs.     

DNA restriction enzyme fragment polymorphism as a tool for rapid differentiation ofAspergillus flavus fromAspergillus oryzae

Aspergillus flavus andAspergillus oryzae are difficult to differentiate using standard morphologically based characteristics. This survey, using several restriction enzymes, showed thatSmaI digestions of total DNA could be used to differentiate these two closely related species ofAspergillus. A different electrophoretic pattern was associated with each of the two species, while within a species the pattern remained constant. CsCl banding of DNA from one isolate of each species indicated the DNA that produced the species-specific pattern was associated with the nuclear DNA fraction.     

ELECTRON MICROSCOPIC STUDIES OF MITOSIS IN AMEBAE : I. Amoeba proteus

Individual organisms of Amoeba proteus have been fixed in buffered osmium tetroxide in either 0.9 per cent NaCl or 0.01 per cent CaCl₂, sectioned, and studied in the electron microscope in interphase and in several stages of mitosis. The helices typical of interphase nuclei do not coexist with condensed chromatin and thus either represent a DNA configuration unique to interphase or are not DNA at all. The membranes of the complex nuclear envelope are present in all stages observed but are discontinuous in metaphase. The inner, thick, honeycomb layer of the nuclear envelope disappears during prophase, reappearing after telophase when nuclear reconstruction is in progress. Nucleoli decrease in size and number during prophase and re-form during telophase in association with the chromatin network. In the early reconstruction nucleus, the nucleolar material forms into thin, sheet-like configurations which are closely associated with small amounts of chromatin and are closely applied to the inner, partially formed layer of the nuclear envelope. It is proposed that nucleolar material is implicated in the formation of the inner layer of the envelope and that there is a configuration of nucleolar material peculiar to this time. The plasmalemma is partially denuded of its fringe-like material during division.     

Repression of nucleolar organizer activity in an interspecific hybrid of the genus Xenopus

Nucleolar expression in reciprocal hybrids between African frogs of the species Xenopus laevis and X. mulleri has been examined throughout ontogeny. Evidence is presented for the stagespecific regulation of nucleolar expression in these hybrids, and for a maternal effect that operates during the early development. Advantage has been taken of the nucleolar organizer deletion in X. laevis to demonstrate that the uninucleolate conditions of hybrid nuclei at certain developmental stages is the result of an allelic repression of the mulleri nucleolar organizer region by the laevis genome. Differences in nucleolar expression between the reciprocal hybrids and the parental species have been put on a quantitative basis for several selected tissues.     

Fine structural organization of a non-reticulate plant cell nucleus

We studied the fine structural organization of the meristematic nucleus in roots of Lycopesicon esculentum (tomato) using ultracytochemical and immunocytochemical approaches. The nucleus has a non-reticulate (i.e. low DNA content) structure whose supramolecular organization differs in some respects from that in reticulate nuclei, principally in the organization of the chromocentres associated with the nuclear envelope, with which centromeric structures appear to be associated. The main difference at the nucleolar level is found in the fibrillar centres, which have a low amount of DNA labelling and in which inclusions of condensed chromatin are present only very rarely. The distribution of nucleolar DNA amongst the nucleolar compartments is similar to that in reticulate nucleoli as demonstrated using an anti-DNA monoclonal antibody. Tomato nuclei have nucleolus-associated bodies or karyosomes, like other plant species with a low DNA content and non-reticulate nuclear organization. The nuclear ribonucleoprotein structures in the inter- and perichromatin regions, namely inter- and perichromatin fibrils and granules, show similar ultrastructural and cytochemical characteristics in both types of nuclei.Abbreviations NAC nucleolus associated chromatin - CES centromeric structures - NOR nucleolar organizing region - NAB nucleolus associated body - IG interchromatin granules - RNP ribonucleoprotein - Mab monoclonal antibody by M.F. Trendelenburg     

contribution to the study of nuclear total proteins and DNA during the mitotic cycle in fibroblasts cultivated in vitro and in Ehrlich ascites cells

Summary The dry weight and total protein content of nuclei has been measured by interferometry in living or fixed cells cultivated in vitro (freshly prepared chick, mouse or rat embryo fibroblasts) and in fixed Ehrlich ascites tumor cells of the mouse growing in vivo. The DNA content was estimated by cytophotometry after Feulgen reaction in the same nuclei. The dry weight of nucleoli in fibroblasts and the dry weight and DNA content of chromosomes in dividing fibroblasts and Ehrlich tumor cells have also been measured.During the interphase in fibroblasts, the dry weight of the living nucleus and the nuclear total protein content as measured in fixed cells doubles during the preparation for mitosis, as the DNA content does. In chick and mammal fibroblasts and within the limits of accuracy of our measurements, the synthesis curves for nuclear proteins and DNA do not seem to be necessarily identical.In our fibroblasts, the nucleolar total dry weight per nucleus doubles during the interphase (nucleolar preparation for mitosis); it increases in proportion to the nuclear total protein content, even in polyploid nuclei.During the mitosis, the chromosomes contain all the DNA of the nucleus but some nuclear proteins (non chromosomal proteins) seem to move into the cytoplasm during the mitosis and return into the nucleus at the post-telophase.According to our observations, Ehrlich ascites mouse tumor cells are near-tetraploid as far as the number of chromosomes, nuclear total protein content and DNA content are concerned. During the preparation for mitosis, these amounts double but no necessary close time relation seems to link these premitotic syntheses. Prom this point of view, our results show no clear-cut differences between these tumor cells and the fibroblasts. Except the polyploidy, the behaviour of nuclear proteins and DNA during mitosis in the tumor cells is the same as that observed in our fibroblasts.The effects of various antimitotic agents on rat fibroblasts cultivated in vitro have also been studied with our cytochemical methods. Our measurements of nuclear protein, DNA and nucleolar material content have been made in cells in which mitosis was prevented by alkylating agents, beryllium sulphate, RNase or neutral DNase. The effects of colchicine on these cellular parameters have also been studied.     

Nuclear changes in allium cepa in response to infection by fusarium moniliforme

Fusarium moniliforme was isolated [into culture] from white onion and inoculated into holes bored in onion bulbs. After 48 hrs, the nuclear dry mass of onion cells 0–5 mm ahead of the mycelium had decreased by 19% as determined by quantitative interference microscopy as compared to nuclear dry mass decreases of 11 % caused by avirulent Aspergillus niger, 26 % by an unidentified onion pathogen, 41 % by virulent Botrytis allii, and 42 % by virulent Aspergillus niger. In the present study, the nucleolar dry mass declined by 34 % in response to F. moniliforme under the same conditions. Degradation or exit of nuclear and nucleolar macromolecules in response to fungal products secreted in advance of mycelium may be responsible for lower nuclear and nucleolar dry mass.     

Karyotype, DNA Content and Meiotic Behaviour in Five South American Species ofVicia(Fabaceae)

This paper presents the karyotype, DNA content and meiotic behaviourof five species ofViciafrom Argentina (V. macrogramineaBurk.,V.gramineaSM.,V. epetiolarisBurk.,V. pampicolaBurk. andV. nanaVog.).All the species have the same chromosome number and karyotypeformula (2n=14; 6m+4st+4t). Each species, however, displaysa characteristic number and position of the nucleolar organizerregion (NOR) and different sizes of the respective satellites,confirmed by Ag-NOR banding. Moreover, significant differenceswere found in the total chromosome volume (TCV) and DNA contentof the species. Positive correlations between DNA content andTCV, and between DNA content and type of life cycle were alsofound. TCV and DNA content are lower inV. nana(annual) and higherinV. macrograminea(biennial–perennial). The material displayedmarked karyotypic orthoselection, with similar karyotypes inall studied species, even when the overall chromosome size varied.Evolutionary changes in DNA amount are proportional to the relativelength of each chromosome arm, maintaining karyotypic uniformity.Significant differences were found between the meiotic behaviourofV. gramineaand that of the other species.V. gramineahas alower frequency of ring bivalents and chiasmata per cell, andalso has a lower interstitial chiasma frequency. In general,the results are congruent with the morphological data reportedfor these species.Copyright 1998 Annals of Botany Company. Viciaspecies, karyotype, orthoselection, nuclear DNA content, NOR banding, meiotic behaviour.     

NUCLEIC ACID AND PROTEIN METABOLISM DURING THE MITOTIC CYCLE IN VICIA FABA

In order to investigate some of the cytochemical processes involved in interphase growth and culminating in cell division, a combined autoradiographic and microphotometric study of nucleic acids and proteins was undertaken on statistically seriated cells of Vicia faba root meristems. Adenine-8-C¹⁴ and uridine-H³ were used as ribonucleic acid (RNA) precursors, thymidine-H³ as a deoxyribonucleic acid (DNA) precursor, and phenylalanine-3-C¹⁴ as a protein precursor. Stains used in microphotometry were Feulgen (DNA), azure B (RNA), pH 2.0 fast green (total protein), and pH 8.1 fast green (histone). The autoradiographic data (representing rate of incorporation per organelle) and the microphotometric data (representing changes in amounts of the various components) indicate that the mitotic cycle may be divided into several metabolic phases, three predominantly anabolic (net increase), and a fourth phase predominantly catabolic (net decrease). The anabolic periods are: 1. Telophase to post-telophase during which there are high rates of accumulation of cytoplasmic and nucleolar RNA and nucleolar and chromosomal total protein. 2. Post-telophase to preprophase characterized by histone synthesis and a diphasic synthesis of DNA with the peak of synthesis at mid-interphase and a minor peak just preceding prophase. The minor peak is coincident with a relatively localized DNA synthesis in several chromosomal regions. This period is also characterized by minimal accumulations of cytoplasmic RNA and chromosomal and nucleolar total protein and RNA. 3. Preprophase to prophase in which there are again high rates of accumulation of cytoplasmic RNA, and nucleolar and chromosomal total protein and RNA. The catabolic phase is: 4. The mitotic division during which there are marked losses of cytoplasmic RNA and chromosomal and nucleolar total protein and RNA.     

The systematic value of nuclear DNA content for all species of Narcissus L. (Amaryllidaceae)

The taxonomy of all species of Narcissus (Amaryllidaceae), an important horticultural crop, has not been investigated recently. As a new approach, genome size was determined by flow cytometry with propidium iodide from 375 accessions. The somatic nuclear DNA contents (2C) were shown to range from 14 to 38 pg for the diploids. Narcissus assoanus and N. gaditanus are, based on their nuclear DNA content, removed from section Apodanthi and placed in a new section Juncifolii. The different ploidy levels and species involved were entangled for N . “fernandesii” s.l. and a new allotetraploid form is named here. Section Pseudonarcissus was much more heterogeneous in nuclear DNA content than expected. Sixty-five accessions of N. pseudonarcissus possessed, with 23.7 pg, similar amounts of DNA. However, several species from this section were clearly distinctive in nuclear DNA content. It runs from the diploid N. primigenius with 21.7 pg to the also diploid N. nevadensis with 38.2 pg. Also N. abscissus and N. moleroi are with about 26 pg clearly different from N. pseudonarcissus. For the first time, in 11 accessions, hexaploidy was found in N. pseudonarcissus ssp. bicolor. A new section Nevadensis with 30–39 pg of nuclear DNA was split off from the section Pseudonarcissus with now 21–27 pg. A nonoploid N. dubius with 96.3 pg has by far the highest amount of nuclear DNA and can be calculated to have the highest ploidy ever reported in Narcisssus. The total number of Narcissus species was determined as 36, nine more than in Flora Europaea and they were divided up in two subgenera and 11 sections. Flow cytometry is shown to produce easily obtainable and original systematic data that lead to new insights. Genome size or C-value turns out to be one of the most salient features to define the status of the species in the genus Narcissus.