Are seeds suitable for flow cytometric estimation of plant genome size?

BACKGROUND: Nuclear DNA content in plants is commonly estimated using flow cytometry (FCM). Plant material suitable for FCM measurement should contain the majority of its cells arrested in the G0/G1 phase of the cell cycle. Usually young, rapidly growing leaves are used for analysis. However, in some cases seeds would be more convenient because they can be easily transported and analyzed without the delays and additional costs required to raise seedlings. Using seeds would be particularly suitable for species that contain leaf cytosol compounds affecting fluorochrome accessibility to the DNA. Therefore, the usefulness of seeds or their specific tissues for FCM genome size estimation was investigated, and the results are presented here. METHODS: The genome size of six plant species was determined by FCM using intercalating fluorochrome propidium iodide for staining isolated nuclei. Young leaves and different seed tissues were used as experimental material. Pisum sativum cv. Set (2C = 9.11 pg) was used as an internal standard. For isolation of nuclei from species containing compounds that interfere with propidium iodide intercalation and/or fluorescence, buffers were used supplemented with reductants. RESULTS: For Anethum graveolens, Beta vulgaris, and Zea mays, cytometrically estimated genome size was the same in seeds and leaves. For Helianthus annuus, different values for DNA amounts in seeds and in leaves were obtained when using all but one of four nuclei isolation buffers. For Brassica napus var. oleifera, none of the applied nuclei isolation buffers eliminated differences in genome size determined in the seeds and leaves. CONCLUSIONS: The genome size of species that do not contain compounds that influence fluorochrome accessibility appears to be the same when estimated using specific seed tissues and young leaves. Seeds can be more suitable than leaves, especially for species containing staining inhibitors in the leaf cytosol. Thus, use of seeds for FCM nuclear DNA content estimation is recommended, although for some species a specific seed tissue (usually the radicle) should be used. Protocols for preparation of samples from endospermic and endospermless seeds have been developed.     

Plant DNA flow cytometry and estimation of nuclear genome size

BACKGROUND: DNA flow cytometry describes the use of flow cytometry for estimation of DNA quantity in cell nuclei. The method involves preparation of aqueous suspensions of intact nuclei whose DNA is stained using a DNA fluorochrome. The nuclei are classified according to their relative fluorescence intensity or DNA content. Because the sample preparation and analysis is convenient and rapid, DNA flow cytometry has become a popular method for ploidy screening, detection of mixoploidy and aneuploidy, cell cycle analysis, assessment of the degree of polysomaty, determination of reproductive pathway, and estimation of absolute DNA amount or genome size. While the former applications are relatively straightforward, estimation of absolute DNA amount requires special attention to possible errors in sample preparation and analysis. SCOPE: The article reviews current procedures for estimation of absolute DNA amounts in plants using flow cytometry, with special emphasis on preparation of nuclei suspensions, stoichiometric DNA staining and the use of DNA reference standards. In addition, methodological pitfalls encountered in estimation of intraspecific variation in genome size are discussed as well as problems linked to the use of DNA flow cytometry for fieldwork. CONCLUSIONS: Reliable estimation of absolute DNA amounts in plants using flow cytometry is not a trivial task. Although several well-proven protocols are available and some factors controlling the precision and reproducibility have been identified, several problems persist: (1) the need for fresh tissues complicates the transfer of samples from field to the laboratory and/or their storage; (2) the role of cytosolic compounds interfering with quantitative DNA staining is not well understood; and (3) the use of a set of internationally agreed DNA reference standards still remains an unrealized goal.     

Cytochemistry and C-values: the less-well-known world of nuclear DNA amounts

BACKGROUND: In the plant sciences there are two widely applied technologies for measuring nuclear DNA content: Feulgen absorbance cytophotometry and flow cytometry (FCM). While FCM is, with good reasons, increasingly popular among plant scientists, absorbance-cytophotometric techniques lose ground. This results in a narrowing of the methodological repertoire, which is neither desirable nor beneficial. Both approaches have their advantages, but static cytophotometry seems to pose more instrumental difficulties and material-based problems than FCM, so that Feulgen-based data in the literature are often less reliable than one would expect. SCOPE: The purpose of this article is to present a selective overview of the field of nuclear DNA content measurement, and C-values in particular, with a focus on the technical difficulties imposed by the characteristics of the biological material and with some comments on the photometrical aspects of the work. For over 20 years it has been known that plant polyphenols cause problems in Feulgen DNA cytophotometry, since they act as major staining inhibitors leading to unreliable results. However, little information is available about the chemical classes of plant metabolites capable of DNA staining interference and the mechanisms of their inhibition. Plant slimes are another source of concern. CONCLUSIONS: In FCM research to uncover the effects of secondary metabolites on measurement results has begun only recently. In particular, the analysis of intraspecific genome size variation demands a stringent methodology which accounts for inhibitors. FCM tests for inhibitory effects of endogenous metabolites should become obligatory. The use of dry seeds for harvesting embryo and endosperm nuclei for FCM and Feulgen densitometry may often provide a means of circumventing staining inhibitors. The importance of internal standardization is highlighted. Our goal is a better understanding of phytochemical/cytochemical interactions in plant DNA photometry for the benefit of an ever-growing list of plant genome sizes.     

Bacterial fingerprinting by flow cytometry: bacterial species discrimination.

BACKGROUND: A flow cytometric measurement (FCM) technique has been developed to size DNA fragments. Individual fragments of a restriction digest of genomic DNA, stained with an intercalating dye, are passed through an ultrasensitive cytometer. The measured fluorescence intensity from each fragment is proportional to the fragment length. METHODS: The isolation of bacterial genomic DNA and digestion by restriction enzymes were performed inside an agarose plug. Rare cutting enzymes were employed to produce a manageable number of DNA fragments. Electroelution was used to move the DNA fragments from the agarose plug into a solution containing polyamines to protect the DNA from shear-induced breakage. The DNA was stained with the bisintercalating dye thiazole orange homodimer and introduced into our ultrasensitive flow cytometer. A histogram of the fluorescence intensities (fingerprint) was constructed. RESULTS: Gram-positive Bacillus globigii and gram-negative bacteria Escherichia coli and Erwinia herbicola were distinguished by the fingerprint pattern of restriction fragments of their genomic DNA. DNA sizes determined by FCM are in good agreement with pulsed-field gel electrophoresis (PFGE) analysis. Flow cytometry requires only picogram quantities of purified DNA and takes less than 10 min for data collection and analysis. When the total sample preparation time is included, the analysis times for PFGE and FCM are similar ( approximately 3 days). CONCLUSIONS: FCM is an attractive technique for the identification of bacterial species. It is more sensitive and potentially much faster than PFGE.     

基于流式细胞技术的灵芝基因组大小估测

以药典规定的灵芝正品来源灵芝Ganoderma lucidum作为研究对象,利用已完成全基因组测序的黑曲霉Aspergillus niger作为内标,通过机械破碎菌丝体的方法获得合适浓度的细胞核悬液,碘化丙啶荧光染色后成功应用流式细胞术进行基因组大小估测。经过优化材料培养、样品制备、上机分析等实验条件,估测得出灵芝基因组大小(48.98±0.60)Mb,为灵芝基因组学研究提供重要数据。该方法简捷稳定,在蕈菌范围内,首次得到了全基因组测序数据与光学图谱结果验证,为蕈菌基因组学研究提供重要技术平台。     

Mitochondrial DNA as a non-invasive biomarker: accurate quantification using real time quantitative PCR without co-amplification of pseudogenes and dilution bias

Circulating mitochondrial DNA (MtDNA) is a potential non-invasive biomarker of cellular mitochondrial dysfunction, the latter known to be central to a wide range of human diseases. Changes in MtDNA are usually determined by quantification of MtDNA relative to nuclear DNA (Mt/N) using real time quantitative PCR. We propose that the methodology for measuring Mt/N needs to be improved and we have identified that current methods have at least one of the following three problems: (1) As much of the mitochondrial genome is duplicated in the nuclear genome, many commonly used MtDNA primers co-amplify homologous pseudogenes found in the nuclear genome; (2) use of regions from genes such as β-actin and 18S rRNA which are repetitive and/or highly variable for qPCR of the nuclear genome leads to errors; and (3) the size difference of mitochondrial and nuclear genomes cause a “dilution bias” when template DNA is diluted. We describe a PCR-based method using unique regions in the human mitochondrial genome not duplicated in the nuclear genome; unique single copy region in the nuclear genome and template treatment to remove dilution bias, to accurately quantify MtDNA from human samples.     

Revisiting the DNA C-values of the genome size-standards used in plant flow cytometry to choose the "best primary standards"

Flow cytometry (FCM) techniques have enabled characterization of the genome size for various plant species. In order to measure the nuclear genome size of a species, reference standards with well-established DNA content are necessary. However, different 2C-values have been described for the same species used as reference standard. This fact has brought about inaccurate genome measurements, making relevant the establishment of optimal DNA reference standards for plant cytometric analyses. Our work revisited the genome size of Arabidopsis thaliana and other seven plant standards, which were denominated ??Dole?el??s standard set?? and have been widely used in plant DNA measurements. These eight plant standards were reassessed for a comparative measurement of their DNA content values, using each plant species as primary standard in a cascade-like manner, from A. thaliana to Allium cepa. The genome size values obtained here were compared to those reported in the literature by statistical analyses. As a result, Raphanus sativus and Drosophila melanogaster were considered the most inadequate primary standards, whereas A. thaliana, Solanum lycopersicum and Pisum sativum were found to be the most suitable.     

Detection of diploid males in a natural colony of the cleptobiotic bee Lestrimelitta sp (Hymenoptera, Apidae)

When working at quantifying the genome size of stingless bees, it was observed that males of Lestrimelitta sp possessed the same amount of nuclear DNA as the females. Thus, we used flow cytometry (FCM) and cytogenetic analysis to confirm the ploidy of these individuals. The males analyzed proved to be diploid, since, through cytometric analysis, it was demonstrated that the mean genome size of both males and females was the same (C = 0.463 pg), and, furthermore, cytogenetic analysis demonstrated that both had 2n = 28 chromosomes.     

Comparison of the <Emphasis Type="Italic">Coffea canephora</Emphasis> and <Emphasis Type="Italic">C. arabica</Emphasis> karyotype based on chromosomal DNA content

Nuclear genome size has been measured in various plants, seeing that knowledge of the DNA content is useful for taxonomic and evolutive studies, plant breeding programs and genome sequencing projects. Besides the nuclear DNA content, tools and protocols to quantify the chromosomal DNA content have been also applied, expanding the data about genomic structure. This study was conducted in order to calculate the Coffea canephora and Coffea arabica chromosomal DNA content, associating cytogenetic methodologies with flow cytometry (FCM) and image cytometry (ICM) tools. FCM analysis showed that the mean nuclear DNA content of C. canephora and C. arabica is 2C = 1.41 and 2.62 pg, respectively. The cytogenetic methodology provided prometaphase and metaphase cells exhibiting adequate chromosomes for the ICM measurements and karyogram assembly. Based on cytogenetic, FCM and ICM results; it was possible to calculate the chromosomal DNA content of the two species. The 1C chromosomal DNA content of C. canephora ranged from 0.09 (chromosome 1) to 0.05 pg (chromosome 11) and C. arabica from 0.09 (chromosome 1) to 0.03 pg (chromosome 22). The methodology presented in this study was suitable for DNA content measuring of each chromosome of C. canephora and C. arabica. The cytogenetic characterization and chromosomal DNA content analyses evidenced that C. arabica is a true allotetraploid originated from a cross between Coffea diploid species. Besides, the same analyses also reinforce that C. canephora is a possible progenitor of C. arabica.     

Eukaryotic non-coding DNA is functional: evidence from the differential scaling of cryptomonad genomes

Genic DNA functions are commonplace: coding for proteins and specifying non-messenger RNA structure. Yet most DNA in the biosphere is non-genic, existing in nuclei as non-coding or secondary DNA. Why so much secondary DNA exists and why its amount per genome varies over orders of magnitude (correlating positively with cell volume) are central biological problems. A novel perspective on secondary DNA function comes from natural eukaryote eukaryote chimaeras (cryptomonads and chlorarachneans) where two phylogenetically distinct nuclei have coevolved within one cell for hundreds of millions of years. By comparing cryptomonad species differing 13-fold in cell volume, we show that nuclear and nucleomorph genome sizes obey fundamentally different scaling laws. Following a more than 125-fold reduction in DNA content, nucleomorph genomes exhibit little variation in size. Furthermore, the present lack of significant amounts of nucleomorph secondary DNA confirms that selection can readily eliminate functionless nuclear DNA, refuting 'selfish' and 'junk' theories of secondary DNA. Cryptomonad nuclear DNA content varied 12-fold: as in other eukaryotes, larger cells have extra DNA, which is almost certainly secondary DNA positively selected for a volume-related function. The skeletal DNA theory explains why nuclear genome size increases with cell volume and, using new evidence on nucleomorph gene functions, why nucleomorph genomes do not.     

Rapid detection of viable yeasts and bacteria in wine by flow cytometry

The potential of using flow cytometry (FCM) in combination with fluorescent dyes for rapidly estimating counts of yeasts and malolactic bacteria in laboratory media and wines was examined. In general, there was a good correlation (regression coefficient, 0.94) between viable counts of yeasts determined by FCM and by standard plate assay. The FCM detection limit of yeasts in YPDE medium and in Pinot noir must was 10(3) cells/ml. The lowest bacterial concentration detected by FCM was 10(4) cells/ml. When yeast and malolactic bacteria populations were simultaneously analysed in wine by FCM without any previous sample treatment, difficulties were encountered in the count of bacterial cells due to their size, which is similar to natural debries present in wine. However, after the optimisation of the sample preparation, the technique appeared promising in determining the presence of such microorganisms in wine with one single measurement. Because it is rapid and easy to use, flow cytometry can be considered a useful method for microbiological quality control in wineries and for the investigation of the growth dynamics of microorganisms in wine.     

The CIC library: a large insert YAC library for genome mapping in Arabidopsis thaliana

A new Arabidopsis thaliana (ecotype Columbia) genomic library has been constructed in Yeast Artificial Chromosomes: the CIC library (for CEPH, INRA and CNRS). Optimization of plant culture conditions and protoplast preparation allowed the recovery of large amounts of viable protoplasts. Mechanical shearing of DNA was minimized by isolation of DNA from protoplasts embedded in agarose. Cloning of large inserts was favored by including two successive size fractionation steps (after partial Eco RI digestion and after ligation with the vector arms), which selected DNA fragments larger than 350 kb. The library consists of 1152 clones with an average insert size of 420 kb. Clones carrying chloroplast DNA and various nuclear repeated sequences have been identified. Twenty-one per cent of the clones are found to contain chloroplast DNA. Therefore, the library represents around four nuclear genome equivalents. The clones containing 5S rDNA genes, 18S-25S rDNA sequences and the 180 bp paracentromeric repeated element account for 3.6%, 8.9% and 5.8%, respectively. Only one clone was found to carry the 160 bp paracentromeric repeated element. Given the smaller size of clones carrying Arabidopsis repeated DNA, the average size of remaining clones is around 480 kb. The library was screened by PCR amplification using pairs of primers corresponding to sequences dispersed in the genome. Seventy out of 76 pairs of primers identified from one to seven YAC clones. Thus at least 92% of the genome is represented in the CIC library. The survey of the library for clones containing unlinked DNA sequences indicates that the proportion of chimeric clones is lower than 10%.     

Nuclear DNA Amounts in Pteridophytes

DNA amounts (C-value and genome size) are much-used biodiversitycharacters. A workshop held at Kew in 1997 identified majorgaps in our knowledge of plant DNA amounts, recommending targetsfor new work to fill them. Murray reviewed non-angiosperm plantsnoting that representation of pteridophyte species (approx.0.42%) was poor, while locating C-value data for them was verydifficult. The workshop confirmed the need to make data forother groups besides angiosperms accessible for reference purposes.This paper pools DNA C-values for 48 pteridophyte species fromeight original sources into one reference source, and fulfilsa key workshop recommendation for this group. Comparing thesedata shows that nuclear 1C-values in pteridophytes vary approx.1000-fold, from 0.055 pg in Selaginella species to about 55pg in Ophioglossum petiolatum. Genome size estimates for 25pteridophytes vary approx. 200-fold from 0.055 to 10.7 pg, andthe mean genome sizes in diploids and polyploids (5.15 and 4.59pg, respectively) are not significantly different. Wider comparisonsshow that ranges of genome sizes in the major groups of landplants are very different. Those in bryophytes and pteridophytesare narrow compared with those in gymnosperms and angiosperms.The data indicate that the origin of land plants possibly involveda first major increase in genome size in the evolution of vascularplants, while a second such increase occurred later in gymnosperms.C-values for pteridophytes remain very few, but conversely opportunitiesfor new work on them are many. Copyright 2001 Annals of BotanyCompany Pteridophyte DNA amounts, DNA C-values, nuclear genome sizes     

Analyzing the ATR-mediated checkpoint using Xenopus egg extracts

Our knowledge of cell cycle events such as DNA replication and mitosis has been advanced significantly through the use of Xenopus egg extracts as a model system. More recently, Xenopus extracts have been used to investigate the cellular mechanisms that ensure accurate and complete duplication of the genome, processes otherwise known as the DNA damage and replication checkpoints. Here we describe several Xenopus extract methods that have advanced the study of the ATR-mediated checkpoints. These include a protocol for the preparation of nucleoplasmic extract (NPE), which is a soluble extract system useful for studying nuclear events such as DNA replication and checkpoints. In addition, we describe several key assays for studying checkpoint activation as well as methods for using small DNA structures to activate ATR.     

Genome size variation in some representatives of the genus <Emphasis Type="Italic">Tripleurospermum</Emphasis>

Genome size has been estimated by flow cytometry in 14 populations belonging to eight taxa (seven species, one of them with two varieties) of the genus Tripleurospermum. 2C nuclear DNA amounts range from 4.87 to 9.22 pg, and nuclear DNA amounts per basic chromosome set from 1.99 to 2.75 pg. Statistically significant differences depending on ploidy level, life cycle or environmental factors such as altitude have been found. Also, genome size is positively correlated with total karyotype length. The presence of rhizome is related to nuclear DNA content in these species.This work was supported by project BOS2001-3041-C02-01 of the Spanish government, and one of the authors (S.G.) received a predoctoral grant from the Spanish government.     

Methods for simultaneous measurement of apoptosis and cell surface phenotype of epithelial cells in effusions by flow cytometry

We describe here a protocol for the detection of epithelial cells in effusions combined with quantification of apoptosis by flow cytometry (FCM). The procedure described consists of the following stages: culturing and induction of apoptosis by staurosporine in control ovarian carcinoma cell lines (SKOV-3 and OVCAR-8); preparation of effusion specimens and cell lines for staining; staining of cancer cells in effusions and cell lines for cell surface markers (Ber-EP4, EpCAM and CD45) and intracellular/nuclear markers of apoptosis (cleaved caspase-3 and caspase-8, and incorporated deoxyuridine triphosphates); and FCM analysis of stained cell lines and effusions. This protocol identifies a specific cell population in cytologically heterogeneous clinical specimens and applies two methods to measure different aspects of apoptosis in the cell population of interest. The cleaved caspase and deoxyuridine triphosphate incorporation FCM assays are run in parallel and require (including sample preparation, staining, instrument adjustment and data acquisition) 8 h. The culturing of cell lines requires 2-3 days and induction of apoptosis requires 16 h.     

Two new nuclear isolation buffers for plant DNA flow cytometry: a test with 37 species

Background and Aims: After the initial boom in the application of flow cytometryin plant sciences in the late 1980s and early 1990s, which wasaccompanied by development of many nuclear isolation buffers,only a few efforts were made to develop new buffer formulas.In this work, recent data on the performance of nuclear isolationbuffers are utilized in order to develop new buffers, generalpurpose buffer (GPB) and woody plant buffer (WPB), for plantDNA flow cytometry. Methods: GPB and WPB were used to prepare samples for flow cytometricanalysis of nuclear DNA content in a set of 37 plant speciesthat included herbaceous and woody taxa with leaf tissues differingin structure and chemical composition. The following parametersof isolated nuclei were assessed: forward and side light scatter,propidium iodide fluorescence, coefficient of variation of DNApeaks, quantity of debris background, and the number of particlesreleased from sample tissue. The nuclear genome size of 30 selectedspecies was also estimated using the buffer that performed betterfor a given species. Key Results: In unproblematic species, the use of both buffers resulted inhigh quality samples. The analysis of samples obtained withGPB usually resulted in histograms of DNA content with higheror similar resolution than those prepared with the WPB. In morerecalcitrant tissues, such as those from woody plants, WPB performedbetter and GPB failed to provide acceptable results in somecases. Improved resolution of DNA content histograms in comparisonwith previously published buffers was achieved in most of thespecies analysed. Conclusions: WPB is a reliable buffer which is also suitable for the analysisof problematic tissues/species. Although GPB failed with someplant species, it provided high-quality DNA histograms in speciesfrom which nuclear suspensions are easy to prepare. The resultsindicate that even with a broad range of species, either GPBor WPB is suitable for preparation of high-quality suspensionsof intact nuclei suitable for DNA flow cytometry.     

Climate and growth form: the consequences for genome size in plants

The adaptive significance of nuclear DNA variation in angiosperms is still widely debated. The discussion mainly revolves round the causative factors influencing genome size and the adaptive consequences to an organism according to its growth form and environmental conditions. Nuclear DNA values are now known for 3874 angiosperm species (including 773 woody species) from over 219 families (out of a total of 500) and 181 species of woody gymnosperms, representing all the families. Therefore, comparisons have been made on not only angiosperms, taken as a whole, but also on the subsets of data based on taxonomic groups, growth forms, and environment. Nuclear DNA amounts in woody angiosperms are restricted to less than 23.54 % of the total range of herbaceous angiosperms; this range is further reduced to 6.8 % when woody and herbaceous species of temperate angiosperms are compared. Similarly, the tropical woody dicots are restricted to less than 50.5 % of the total range of tropical herbaceous dicots, while temperate woody dicots are restricted to less than 10.96 % of the total range of temperate herbaceous dicots. In the family Fabaceae woody species account for less than 14.1 % of herbaceous species. Therefore, in the total angiosperm sample and in subsets of data, woody growth form is characterized by a smaller genome size compared with the herbaceous growth form. Comparisons between angiosperm species growing in tropical and temperate regions show highly significant differences in DNA amount and genome size in the total angiosperm sample. However, when only herbaceous angiosperms were considered, significant differences were obtained in DNA amount, while genome size showed a non-significant difference. An atypical result was obtained in the case of woody angiosperms where mean DNA amount of tropical species was almost 25.04 % higher than that of temperate species, which is because of the inclusion of 85 species of woody monocots in the tropical sample. The difference becomes insignificant when genome size is compared. Comparison of tropical and temperate species among dicots and monocots and herbaceous monocots taken separately showed significant differences both in DNA amount and genome size. In herbaceous dicots, while DNA amount showed significant differences the genome size varies insignificantly. There was a non-significant difference among tropical and temperate woody dicots. In three families, i.e., Poaceae, Asteraceae, and Fabaceae the temperate species have significantly higher DNA amount and genome size than the tropical ones. Woody gymnosperms had significantly more DNA amount and genome size than woody angiosperms, woody eudicots, and woody monocots. Woody monocots also had significantly more DNA amount and genome size than woody eudicots. Lastly, there was no significant difference between deciduous and evergreen hardwoods. The significance of these results in relation to present knowledge on the evolution of genome size is discussed.     

A short protocol for micro-purification of nuclear proteins from whole animal tissue.

Although a number of small-scale procedures have been described for the preparation of crude nuclear extracts from established cell lines, none were provided for the preparation of similar extracts from small amounts of animal tissue. In addition, no small-scale procedures contain enrichment steps that render the detection of low-abundant DNA-binding proteins easier. Here we describe a simple, efficient procedure for the rapid preparation of high-quality nuclear extracts from either whole animal tissue or established cell lines. It is based on a rapid isolation of the nuclei followed by a KCl extraction and a further micro-enrichment of the DNA binding proteins on heparin Sepharose CL-6B. Extracts prepared in such a way are suitable for the analysis of specific DNA/protein interactions by the use of gel shift assays or by DNaseI and dimethylsulfate footprinting techniques. Most importantly, the entire process can be fulfilled at minimal cost within a day on as little as one gram of fresh tissue, which renders this procedure extremely attractive for the analysis of DNA binding proteins involved in the control of gene expression.