Analysis of cellular DNA distributions

In the fluorescent-flow cytophotometric measurement of cellular DNA content the DNA distributions usually have two peaks. The second peak, which corresponds to the 4C DNA content of G2 and M cells, is often positioned at lower values of DNA content than twice that of the 2C DNA peak which contains G1 cells. Computerized numerical analyses were performed on artificial DNA distributions in which the proportion of S-phase cells was varied. It was demonstrated that the contribution of late S-phase cells to the 4C DNA peak in the histogram shifts the second peak to a position below twice the 2C DNA value. Also, increasing the coefficient of variation of the DNA measurement shifts the second peak position to lower values. A group of 33 DNA distribution histograms was found to have an average G2/G1 peak position ratio of 1.90, in keeping with typical values obtained from the numerical analysis of the artificial populations.     

Dna Ploidy Studies of Benign and Malignant Tumours: Comparison of Flow Cytometry and Image Analysis Techniques Using Two Types of Cytological Specimen

DNA ploidy studies were carried out on Feulgen stained smears and cytocentrifuge preparations from 35 malignant tumours and four benign neoplasms using the CAS image analyser. The smears were prepared from scrapings from fresh tumour tissue whereas the cytocentrifuge preparations were prepared from single nuclear suspensions from paraffin-embedded cell blocks from the same tumour. Histograms obtained by image analysis of the tumour scrapes were compared with those obtained on the cytocentrifuge preparations. Concordant results were obtained in four benign tumours (100%) and 32 malignant tumours (91%). The results obtained by image analysis were also compared with results obtained by flow cytometry of the tumour tissue. Discordant results were obtained for three malignant tumours. Possible reasons for the discrepancy include sampling error, tumour heterogeneity and selective loss of cell populations during processing.     

Analysis of DNA distributions from flow cytometry. Validation of an automatic procedure against simulated data

A recently proposed automatic procedure for analyzing DNA distribution from flow cytometric data is extensively tested against simulated data. After a discussion of the procedure itself and of the simulation program, the results obtained are reported. They are evidence of the reliability of the procedure in extracting the proper underlying DNA distribution from sets of data obtained under various simulated instances. The different sources of error are then analyzed, along with their quantitative effects on the fit of the fluorescence histogram.     

Intra- and interspecific variation in genome size in Lathyrus (Leguminosae)

2C nuclear DNA amounts for 24 species of Lathyrus (section Lathyrus ) were determined using flow cytometry. A greater than two-fold variation was observed, ranging from 10.2 pg in L. basalticus to 24.2 pg in L. latifolius. In general, the perennial species had greater DNA amounts than the annuals. Significant intraspecific variation was observed in five species of Lathyrus (from 10.1% in L. annuus to 28% in L. tingitanus). A positive correlation was observed between DNA values obtained by flow cytometry and those previously determined by microdensitometry. Finally, the distribution of DNA amounts in species within section Lathyrus appears to be continuous.     

Computation of identity by descent probabilities conditional on DNA markers via a Monte Carlo Markov Chain method

The accurate estimation of the probability of identity by descent (IBD) at loci or genome positions of interest is paramount to the genetic study of quantitative and disease resistance traits. We present a Monte Carlo Markov Chain method to compute IBD probabilities between individuals conditional on DNA markers and on pedigree information. The IBDs can be obtained in a completely general pedigree at any genome position of interest, and all marker and pedigree information available is used. The method can be split into two steps at each iteration. First, phases are sampled using current genotypic configurations of relatives and second, crossover events are simulated conditional on phases. Internal track is kept of all founder origins and crossovers such that the IBD probabilities averaged over replicates are rapidly obtained. We illustrate the method with some examples. First, we show that all pedigree information should be used to obtain line origin probabilities in F2 crosses. Second, the distribution of genetic relationships between half and full sibs is analysed in both simulated data and in real data from an F2 cross in pigs.     

Monoparametric models of flow cytometric karyotypes with spreadsheet software

Summary Theoretical flow karyotypes from both plant and mammalian species have been simply modelled using computer spreadsheet software. The models are based upon published values of relative DNA content or relative lengths of each of the chromosomes. From such data, the histograms of chromosome distribution have been simulated for both linear and logarithmic modules of a flow cytometer, and as a function of the coefficient of variation. Simulated and experimental histograms are compard for Nicotiana plumbaginifolia. This readily accessible exercise facilitates the planning and execution of flow cytometric analysis and sorting of chromosomes.     

A computer-aided measuring system for the characterization of yeast populations combining 2D-image analysis, electronic particle counter, and flow cytometry

An integrated measuring system was developed that directly compares the shape of size distributions of Saccharomyces cerevisiae populations obtained from either microscopic measurements, electronic particle counter, or flow cytometer. Because of its asymmetric mode of growth, a yeast population consists of two different subpopulations, parents and daughters. Although electronic particle counter and flow cytometer represent fast methods to assess the growth state of the population as a whole, the determination of important cell cycle parameters like the fraction of daughters or budded cells requires microscopic observation. We therefore adapted a semiautomatic and interactive 2D-image processing program for rapid and accurate determination of volume distributions of the different sub-populations. The program combines the capacity of image processing and volume calculation by contour-rotation, with the potential of visual evaluation of the cells. High-contrast images from electron micrographs are well suited for image analysis, but the necessary air drying caused the cells to shrink to 35% of their hydrated volume. As an alternative, hydrated cells overstained with the fluorochrome calcofluor and visualized by fluorescence light microscopy were used. Cell volumes calculated from length, and diameter measurements with the assumption of an ellipsoid cell shape were underestimated as compared to volumes derived from 2D-image analysis and contour rotation, because of a deviating cell shape, especially in the older parent cells with more than one bud scar. The bimodal volume distribution obtained from microscopic measurements was identical to the protein distribution measured with the flow cytometer using cells stained with dansylchloride, but differed significantly from the size distribution measured with the electronic particle counter. Compared with the flow cytometer, 2-D image analysis can thus provide accurate distributions with important additional information on, for instance, the distributions of subpopulations like parents, daughters, or budded cells.     

Subclassification of murine T cells by computerized microphotometric analysis

Splenocytes separated by physical means and classified as T cells by immunologic tests and computerized microphotometric analysis are differentiated into subgroups by analysis of the distribution patterns of Feulgen-positive nuclear DNA. In like fashion T cells obtained as purified preparations after separation on a nylon column, and accepted as T cells by micromorphometric analysis were subjected to further computerized morphometric analysis of nuclear DNA to form subgroups of cells. In each case, the number and composition of the detected subgroups were consistent. The classification does not appear to reflect any obvious phases of the cell cycle and is not dependent upon the sex and strain of mice from which the cells were obtained.     

Increased variability in nuclear DNA content of testis cells and spermatozoa from mice with irregular meiotic segregation.

Variability in DNA content to testis cells and sperm from F₁ hybrids between the laboratory mouse (M. muscullus) and the tobacco mouse (M. poschiavinus), has been determined by flow cytometry (FMC). The F₁ hybrid mouse is known to be heterozygous for seven metacentric chromosomes produced by Robertsonian fusion. Enriched populations of nuclei from late pachytene spermatocytes and round spermatids were obtained by velocity sedimentation. These nuclei, as well as epididymal sperm nuclei and spleen cells, were stained by the acriflavin-Feulgen technique for DNA and measured by FCM. Peaks in the fluorescence intensity frequency distributions resulting from these measurements were analyzed to determine their mean fluorescence intensities and their widths (coefficients of variation). Because mean intensities of corresponding cell types from M. musculus and the F₁ hybrids were identical, the average DNA contents were taken to be the same. The average coefficients of variation of the peaks to fluorescence from the pachytene, spermatid, and sperm nuclei and spleen cells from M. muscullus animals were about 5%. While the peaks of fluorescence from spleen cells and pachytene nuclei from f₁ hybrids also had average coefficients of variation of 5%, post-meiotic nuclei from spermatids and spermatozoa had coefficients of variationof 8%. From these results we conclude that, in these F₁ hybrids, abnormal meiotic segregation causes an increased variability of 6% in the amount of DNA in the spermatozoa.     

CHARACTERIZATION OF OCEANIC PHOTOSYNTHETIC PICOEUKARYOTES BY FLOW CYTOMETRY1

To interpret flow cytometric data that are routinely obtained on natural oceanic communities, 23 strains of photosynthetic picoeukaryotes belonging to four classes (Prasinophyceae, Chlorophyceae, Pelagophyceae, and Prymnesiophyceae) and six pigment types were investigated for their light scattering in the forward and right-angle directions, chlorophyll fluorescence, and DNA content as measured by flow cytometry. Cell she was assessed by Coulter counter, and pigment composition was measured by reverse-phase high-performance liquid chromatography. The size and GC% of the nuclear genome of cultured picoeukaryotes was measured from the fluorescence of DNA-specific dyes. Using these two parameters, we could discriminate species within pigment groups. DNA staining of preserved natural samples may also prove useful in discriminating cooccurring populations in situ as long as the communities are not too complex. Using the relationships that we established between size and light-scattering properties of the cells, we estimated equivalent diameters of picoeukaryotes in natural populations to be between 1.3 and 2 μm. Chlorophyll a content was between 6 and 16 fg·cel^?1 as calculated from relationships that we established between chlorophyll a content and red fluorescence of the cultured strains. With respect to size, chlorophyll a content, and pigment composition, Pelagomonas sp. strains (Pelagophyceae) appeared to be the most representative of the natural communities in subtropical ocean waters. In contrast, green coccoid strains, which often outcompete other strains in culture, might only be minor contributors to these communities.     

Comparison of three DNA fluorochromes for flow cytometric estimation of nuclear DNA content in plants

Flow cytometric estimation of nuclear DNA content was performed in six plant species employing three fluorochromes showing different DNA base preferences: propidium iodide (no base preference), 4',6-diamidino-2-phenylindole (DAPI; AT preference), and mithramycin (GC preference). Nuclei isolated from human leukocytes were used as a primary reference standard. While nuclear DNA contents estimated using propidium iodide were in agreement with published data obtained using other techniques, the values obtained using fluorochromes showing base preference were significantly different. It was found that the differences were caused by the differences in overall AT/GC ratios, and by the species-specific differences in binding of these fluorochromes to DNA. It was concluded that nuclear DNA content estimations performed with fluorochromes showing base preference should be interpreted with caution even when AT/GC ratios of the reference and the sample are equal. The use of intercalting dyes (e.g. propidium iodide) is recommended for this purpose. On the other hand, comparison of the staining behaviour of intercalating dyes with that of dyes showing base preference may give additional information on chromatin structural differences and arrangement of molecule pairs in DNA.     

On the inverse problem in flow cytometry recovering DNA distribution from FMF data

The problem of recovering DNA distribution from cytofluorometric experimental data was investigated. Theoretical analysis led to a convenient formulation of the problem and to uniqueness results for its solution. A minimization algorithm has been implemented to get the optimal estimate of G₁, S, G₂, and M phase percentages. This algorithm was tested in some experimental cases.     

Synchronization of 9L rat brain tumor cells by centrifugal elutriation

Asynchronous 9L cells were separated into relatively homogeneously-sized populations using centrifugal elutriation with both a conventional collection method and a long collection method. A substantial increase in the homogeneity of the volume distributions and in the degree of synchrony of the separated fractions was obtained using the long collection method. Autoradiographic data indicated that fractions containing ≥97% G₁ cells, ≥80% S cells, and 70–75% G₂ cells could be routinely recovered with this procedure. Recovery in these fractions varied from 5 to 8% of the total number of cells elutriated. The colony forming efficiency (CFE) of cells from fractions representing each phase of the cell cycle was a constant 60–70%, which was comparable to the 60–80% usually found for asynchronous 9L cells. The percentage of cells in the G₁, S, and G₂ phases in the elutriated fractions was more accurately determined from the volume distribution than from computer fits of the DNA histogram obtained from flow cytometry. In general, the degree of synchrony was related to the coefficient of variation (CV) of the volume distributions of the elutriated fractions. The CV was about 14% for all elutriated fractions. When the ≥97% G₁ population was allowed to progress to S and G₂, the CVs were about 17 and 20.2%, respectively. Thus, the best nonperturbing method for obtaining synchronous 9L cells in the S or G₂ phases was direct elutriation with the long collection method.     

Preparation of HMW DNA from Plant Nuclei and Chromosomes Isolated from Root Tips

Simple, fast and cost-effective method for preparation of DNA with high molecular weight (HMW DNA) from plant nuclei and mitotic chromosomes has been developed. The technique involves mechanical homogenization of formaldehyde-fixed root tips, purification of nuclei and/or chromosomes on sucrose gradient, embedding in low-melting-point agarose, and DNA isolation in agarose plugs. Alternatively, nuclei and chromosomes may be purified using flow cytometry. Majority of DNA obtained is megabase-sized and well digestible by restriction endonucleases. The method is highly efficient as microgram amounts of DNA can be obtained from only several milligrams of plant tissue. Handling negligible amounts of plant material reduces the consumption of chemicals. Furthermore, the use of root tips makes it possible to obtain high-quality DNA even from plant species with leaves that are rigid or rich in secondary metabolites such as polyphenols. It is expected that preparation of HMW DNA from root tip nuclei will facilitate long-range mapping and construction of large-insert DNA libraries also in these species. Successful isolation of HMW DNA from flow-sorted chromosomes opens a way for construction of chromosome-specific large-insert libraries in plants. This revised version was published online in July 2006 with corrections to the Cover Date.     

Simultaneous Measurement of DAPI-Sulforhodamine 101 Stained Nuclear DNA and Protein in Higher Plants by Flow Cytometry

A 2-step staining procedure is presented for simultaneous measurement of nuclear DNA and protein content in higher plants by flow cytometry. To release nuclei, plant tissues were chopped and stirred in the presence of the DNA specific fluorochrome 4', 6-diamidino-2-phenylindole (DAPI) and the nonionic detergent Triton X-100. Plant protoplasts were stirred in the DAPI dye solution with the detergent. After a short incubation period a second dye solution containing DAPI and the protein fluorochrome sulforhodamine 101 (SR 101) without detergent was added. Following another incubation, and after filtration through nylon gauze, the highly fluorescent nuclei were analyzed with an impulse cytophotometer. Accurate bivariate DNA-protein histograms were obtained with CV-values of about 2% or less for the 2C-peak of the univariate DNA parameter. The method presented here can be used for basic and applied cytogenetic studies of higher plants, for characterization of subcompartments of the cell cycle phases, or for examination of heterogeneity in plant tissues.     

Quantitation of spermatogenesis by DNA flow cytometry: Comparative study among six species of mammals

Suspensions of testicular germ cells from six species of mammals were prepared and stained for the DNA content with a fluorochrome (ethidium bromide) adopting a common technique and subjected to DNA flow cytometry. While uniform staining of the germ cells of the mouse, hamster, rat and monkey could be obtained by treating with 0.5% pepsin for 60 min followed by staining with ethidium bromide for 30 min, that of the guinea pig and rabbit required for optimal staining pepsinization for 90 min and treatment with ethidium bromide for 60 min. The procedure adopted here provided a uniform recovery of over 80% of germ cells with each one of the species tested and the cell population distributed itself according to the DNA content (expressed as C values) into 5 major classes-spermatogonia (2C), cells in S-phase, primary spermatocytes (4C), round spermatids (1C), and elongating/elongated spermatids (HC). Comparison of the DNA distribution pattern of the germ cell populations between species revealed little variation in the relative quantities of cells with 2C (8–11%), S-phase (6–9%), and 4C (6–9%) amount of DNA. Though the spermatid cell populations exhibited variations (1C:31–46%, HCl:7–20% and and HC2:11–25%) they represented the bulk of germ cells (70–80%). The overall conversion of 2C to 1C (1C:2C ratio) and meiotic transformation of 4C cells to 1C (1C:4C ratio) kinetics were relatively constant between the species studied. The present study clearly demonstrates that DNA flow cytometry can be adopted with ease and assurance to quantify germ cell transformation and as such spermatogenesis by analysing a large number of samples with consistency both within and across the species barrier. Any variation from the norms in germ cell proportions observed following treatment, fore.g. hormonal stimulation or deprivation can then be ascribed due to a specific effect of the hormone/drug on single/multiple steps in germ cell transformation     

Flow sorting of X and Y chromosome-bearing spermatozoa into two populations

The only established difference on which to base the separation of X and Y chromosome-bearing spermatozoa is chromosomal constitution. This difference is quantifiable both from chromosome morphology (karyotype) and from DNA content. Flow cytometric techniques were used to measure relative DNA content of the X and Y populations and to flow-sort spermatozoa from Chinchilla laniger. Epididymal spermatozoa were recovered in PBS, fixed in 80% ethanol, treated with papain and dithioerythritol, and stained for DNA with Hoechst 33342. Sperm nuclei were analyzed and sorted on an EPICS V flow cytometer/cell sorter, modified specifically for spermatozoa. Two clearly resolved peaks (coefficient of variation < 1.5%) with approximately 7.5% difference in DNA content between X and Y chromosome-bearing spermatozoa were evident. Sperm nuclei were sorted from a portion of the X and Y peaks at a rate of 55 nuclei/sec for each population. Purities of individual X and Y populations averaged 95% as determined by reanalysis of the sorted populations. Successful sorting of Chinchilla X and Y chromosome-bearing spermatozoa into separate populations may aid in the identification of a biochemical marker that could be used to discriminate between the two sperm populations and lead to a practical procedure for sexing spermatozoa.     

Microcloning of maize chromosome 9 by using a flow-sorting technique

We constructed a chromosome 9 lambda DNA library from flow-sorted maize chromosomes. Approximately 3 million maize chromosome 9 were collected with high purity by flow cytometric sorting of chromosomes isolated from an oat-maize chromosome 9 addition line based on the cytogram of fluorescent pulse area versus fluorescent pulse width. Chromosome 9 DNA was partially digested withBamH I, dephosphorylated, and ligated with arms ofBamH I-digested lambda DASH vector (Stratagene). A total of 2.0×10⁶ independent recombinants with an average insert size of 15 kb were obtained. For a 99% probability that every sequence of chromosome 9 is represented in at least one chimeric phage, 5.6×10⁴ cloned fragments are needed. This library covers the entire maize chromosome 9. Hybridizing cloned fragments with labeled maize genomic DNA showed that the high, middle, or low copy number DNA sequences presented in the different phage clones. This individual chromosome library is useful in plant genome mapping and gene isolation.     

Detection of DNA in Ancient Skeletal Remains Using DNA Flow Cytometry

Cortical bone samples were removed from individual burials from Tomb Dk31 in the Dakhleh Oasis, Egypt. The tissue was disaggregated, stained with the DNA specific fluorescent dye DAPI and analyzed using the flow cytom-eter. DNA flow cytometry measures the cellular DNA content and this is correlated with modal chromosome content. When DNA is present in skeletal remains further investigations such as extracting, amplifying and sequencing may then be carried out. The method offers a relatively rapid and inexpensive means of pinpointing samples of skeletal DNA that can be further analyzed.     

Genome Size and Pollen Viability as Taxonomic Criteria: Application to the Genus Hosta

Abstract: Genome size (C values) and pollen viability staining were applied as new criteria to investigate the taxonomy of the genus Hosta Tratt. (Hostaceae). Nearly all species of the genus Hosta have the same basic chromosome number (2n = 2x = 60). However, the nuclear DNA contents, as measured by flow cytometry with propidium iodide, could be demonstrated to range between 17.2 to 26.6 pg. This implies that the largest genome contains roughly 10¹⁰ more base pairs than the smallest. Therefore, nuclear DNA content is a very relevant taxonomic trait that can be measured simply by flow cytometry. In addition, differences in overall DNA composition were demonstrated by comparing to DAPI fluorescence. In general, genome size data confirmed the division into three subgenera. The geographical distribution of genome sizes indicates the migration pattern of Hosta throughout East Asia. The species belonging to the mainly Korean subgenus Bryocles, with a low nuclear DNA content (17.2 - 19.3 pg), can now largely be distinguished from the mainly Japanese species of the subgenus Giboshi (21.3 - 26.5 pg). The exception is H. longissima, that with only 19.6 pg provides a nice example of a decrease in DNA content. On the mainland, as well as on Honshu, species with increased and decreased DNA content have evolved independently. The usefulness of pollen viability to detect hybrids in Hosta was demonstrated in a large series of artificial crosses between bona fide species. Consequently, pollen viability was measured in all available Hosta described as species. Several had low pollen viability and were concluded to be hybrids. Morphology and DNA content confirmed this in most cases. The resulting 23 species approximate the number of Hosta species that follows from the combined studies by Fujita (1976¹⁸) on the Japanese species and Chung (1991 a¹¹) on the Korean species.