Feulgen Microdensitometry Research Articles

The quest for suitable reference standards in genome size research

in the molecular genomics era, as the flood gates opento deliver a deluge of DNA sequence data, knowledge of thetotal amount of DNA that comprises an organism’s genome(5 genome size) remains vital for many diverse fields of biol-ogy. Indeed, it may be considered to be a key biodiversitycharacter with both practical and biological consequences forthe organism. Knowledge of genome size is important, forexample, in ongoing and planned sequencing programs toassess costs and to know how much DNA to sequence, or fordetermining how many large insert clones (e.g., bacterial arti-ficial chromosomes [BACs] or fosmids) are needed for con-structing genomic libraries. In addition, various genetic finger-printing tools (e.g., microsatellites and AFLPs) have beenshown to be sensitive to genome size so knowledge of thischaracter is important before embarking on such studies.From a biological perspective variation in genome size hasbeen shown to have diverse yet predictable consequences foran organism, influencing, for example, how it will respondto changes in carbon dioxide, rising temperatures, andpollution (1).While people have been estimating genome sizes in plantsand animals for over 50 years, the last decade has seen a hugegrowth in the number of estimates published, not only to pro-vide data for molecular studies but also for large scale compar-ative analyses seeking to understand the biological and evolu-tionary significance of the 40,000-fold range of genome sizesencountered across eukaryotes (2). Indeed, data are now avail-able for over 10,000 species, accessible through the internetin three databases (Animal Genome Size Database—www.genomesize.com; Fungal Genome Size Database—www.zbi.ee/fungal-genomesize; and the Plant DNA C-values Database—http://data.kew.org/cvalues).

Natural polyploidy inVanilla planifolia(Orchidaceae)

Vanilla planifolia accessions cultivated in Reunion Island display important phenotypic variation, but little genetic diversity is demonstrated by AFLP and SSR markers. This study, based on analyses of flow cytometry data, Feulgen microdensitometry data, chromosome counts, and stomatal length measurements, was performed to determine whether polyploidy could be responsible for some of the intraspecific phenotypic variation observed. Vanilla planifolia exhibited an important variation in somatic chromosome number in root cells, as well as endoreplication as revealed by flow cytometry. Nevertheless, the 2C-values of the 50 accessions studied segregated into three distinct groups averaging 5.03 pg (for most accessions), 7.67 pg (for the 'Stérile' phenotypes), and 10.00 pg (for the 'Grosse Vanille' phenotypes). For the three groups, chromosome numbers varied from 16 to 32, 16 to 38, and 22 to 54 chromosomes per cell, respectively. The stomatal length showed a significant variation from 37.75 microm to 48.25 microm. Given that 2C-values, mean chromosome numbers, and stomatal lengths were positively correlated and that 'Stérile' and 'Grosse Vanille' accessions were indistinguishable from 'Classique' accessions using molecular markers, the occurrence of recent autotriploid and autotetraploid types in Reunion Island is supported. This is the first report showing evidence of a recent autopolyploidy in V. planifolia contributing to the phenotypic variation observed in this species.

First nuclear DNA C-values for 18 eudicot families.

A key target set at the second Plant Genome Size Workshop, held at the Royal Botanic Gardens, Kew in 2003, was to produce first DNA C-value data for an additional 1 % of angiosperm species, and, within this, to achieve 75 % familial coverage overall (up from approx. 50 %) by 2009. The present study targeted eudicot families for which representation in 2003 (42.5 %) was much lower than monocot (72.8 %) and basal angiosperm (69.0 %) families. Flow cytometry or Feulgen microdensitometry were used to estimate nuclear DNA C-values, and chromosome counts were obtained where possible. First nuclear DNA C-values are reported for 20 angiosperm families, including 18 eudicots. This substantially increases familial representation to 55.2 % for angiosperms and 48.5 % for eudicots. The importance of targeting specific plant families to improve familial nuclear DNA C-value representation is reconfirmed. International collaboration will be increasingly essential to locate and obtain material of unsampled plant families, if the target set by the second Plant Genome Size Workshop is to be met.

From Pixels to Picograms

The study of genome size variation is important from a number of practical and theoretical perspectives. For example, the long-standing "C-value enigma" relating to the more than 200,000-fold range in eukaryotic genome sizes is best studied from a broad comparative standpoint. Genome size data are also required in detailed analyses of genome structure and evolution. The choice of future genome sequencing projects will be dependent on knowledge regarding the sizes of genomes to be sequenced, and so on. To date, genome size data have been acquired primarily by Feulgen microdensitometry or flow cytometry. Each has several advantages but also important limitations. In this review, we provide a practical guide to the new technique of Feulgen image analysis densitometry. The review is designed for those interested in genome size measurements but not extensively experienced in histochemistry, densitometry, or microscopy. Therefore, relevant historical and technical background information is included. For easy reference, we provide recipes for required reagents, guidelines for cell staining, and a checklist of steps for successful image analysis. We hope that the accuracy, rapidity, and cost-effectiveness of Feulgen image analysis demonstrated here will stimulate further surveys of genome sizes in a variety of taxa.

Determinação da quantidade de DNA nuclear em plantas

O valor C de DNA é um caráter de significado biológico fundamental e o conhecimento da quantidade de DNA nuclear de um grupo de organismos pode ser útil em vários campos da ciência. Do ponto de vista prático, a determinação da quantidade de DNA nuclear, que é mais comumente realizada por microdensitometria de Feulgen e citometria de fluxo, pode substituir a contagem de cromossomos, especialmente quando se trabalha com um número muito grande de indivíduos. A microdensitometria por Feulgen baseia-se na ligação específica do DNA a esse corante, havendo uma proporcionalidade entre a quantidade de DNA existente e a quantidade de corante que o núcleo incorporou. A citometria de fluxo envolve a análise das propriedades óticas de partículas em fluxo. Em plantas, basicamente consiste no isolamento dos núcleos, coloração destes com um fluorocromo e leitura da fluorescência emitida. As grandes vantagens desta técnica, em relação à microdensitometria de Feulgen, são a relativa facilidade e a rapidez da preparação das amostras, o grande número de núcleos que podem ser analisados, a necessidade de pequenas quantidades de tecido e a possibilidade de detecção de pequenas diferenças na quantidade de DNA. Relatos de resultados conflitantes entre determinações de diferentes autores mostram a necessidade de uma criteriosa padronização das técnicas, para que problemas metodológicos não venham a ser interpretados como eventuais diferenças reais na quantidade de DNA.

Chromosome number, karyotype and DNA C-value of the Wollemi Pine (Wollemia nobilis, Araucariaceae)

The Wollemi Pine (Wollemia nobilis, Araucariaceae) was discovered in 1994 in a remote area of the Wollemi National Park (New South Wales, Australia). With only around 40 adult trees known to be growing in the wild, it is one of the world's rarest plant species. The results of the first cytological study are reported, including a chromosome count (2n =2x=26), karyotype illustration and nuclear DNA C-value (4C=55.74 pg), obtained using Feulgen microdensitometry. The results are compared with data available on species in the other two genera of the Araucariaceae,Araucaria and Agathis, and with other gymnosperms.

Chromosome number, karyotype and DNA C-value of the Wollemi Pine (Wollemia nobilis, Araucariaceae)

The Wollemi Pine (Wollemia nobilis, Araucariaceae) was discovered in 1994 in a remote area of the Wollemi National Park (New South Wales, Australia). With only around 40 adult trees known to be growing in the wild, it is one of the world's rarest plant species. The results of the first cytological study are reported, including a chromosome count (2n =2x=26), karyotype illustration and nuclear DNA C-value (4C=55.74 pg), obtained using Feulgen microdensitometry. The results are compared with data available on species in the other two genera of the Araucariaceae,Araucaria and Agathis, and with other gymnosperms.

Genome Size is Negatively Correlated with Altitude in Natural Populations of Dactylis glomerata

Previously, we found a significant negative correlation between DNA C-value and altitude among eight natural populations of Dactylis glomerataL. (Creber et al., New Phytologist 128: 555–561, 1994). We have examined the extent to which similar negative relationships exist in other altitudinal transects, one in southern France and the other in Italy. Using Feulgen microdensitometry, C-values were negatively correlated with altitude both for the French and Italian populations. A combined plot of DNA C-values against altitude for all of the transects (representing C-values for 17 natural populations), exhibited a highly significant negative relationship; there was a 1.3-fold variation in DNA C-value from the largest genome-lowest altitude to the smallest genome-highest altitude natural population. Such a consistent marked altitudinal cline suggests strong nucleotypic selection acting upon these populations with increasing altitude. Preliminary examination of amplified fragment length polymorphisms between populations selected from the upper and lower limits of the French and Italian transects has shown that these populations are genetically distinct. The extent to which this genetic separation is related to altitude or genome size, or both, is discussed.

Genome size and karyotype evolution in the slipper orchids (Cypripedioideae: Orchidaceae)

Nuclear DNA contents (4C) were estimated by Feulgen microdensitometry in 27 species of slipper orchids. These data and recent information concerning the molecular systematics of Cypripedioideae allow an interesting re-evaluation of karyotype and genome size variation among slipper orchids in a phylogenetic context. DNA amounts differed 5.7-fold, from 24.4 pg in Phragmipedium longifolium to 138.1 pg in Paphiopedilum wardii. The most derived clades of the conduplicate-leaved slipper orchids have undergone a radical process of genome fragmentation that is most parsimoniously explained by Robertsonian changes involving centric fission. This process seems to have occurred independently of genome size variation. However, it may reflect environmental or selective pressures favoring higher numbers of linkage groups in the karyotype.

Nuclear DNA content of ten rice species as determined by flow cytometry.

Accurate estimations of nuclear DNA content of rice are important because this crop is being used in many types of molecular studies. The main objective of this study was to estimate the nuclear DNA content in ten rice species by flow cytometry. Most of the values obtained were lower than those obtained in earlier studies using Feulgen microdensitometry. Significant differences in genome size among rice species were found. Oryza glaberrima had the smallest genome (0.73-0.76 pg/2C), while O. minuta and O. latifolia had the largest values (ca. 2.33 pg/2C). Tetraploid species had more nuclear DNA than diploid ones. The AA genome had less DNA (0.86-0.96 pg/2C) than the CC (1.14-1.17 pg/2C) and EE (1.99 pg/2C) genomes. The AA genome of O. saliva was larger than the AgA g genome of O. glaberrima and the A1A 1 genome of O. longistaminata, which had mean values of 0.73-0.76 and 0.78 pg/2C, respectively. Indica (IR36) and japonica (Yukihikari) cultivars of O. saliva also showed significant differences. Three different nuclear DNA levels (1.93, 1.85, and 1.31 pg/2C) were found among five O. ridleyi plants analyzed. Flow cytometry also allowed rapid and reliable determination of the ploidy level of anther culture-derived plants.

Isolation and characterization of microprotoplasts from APM-treated suspension cells ofNicotiana plumbaginifolia

Subprotoplasts with a DNA content of less than the G1 level (microprotoplasts) were isolated from micronucleated cells of transformedNicotiana plumbaginifolia ('Doba' line resistant to kanamycin) and characterized cytologically as well as by flow cytometry and Feulgen microdensitometry. Micronuclei were induced upon treatment of the suspension cells with the anti-microtubule drug amiprophos-methyl (APM). Protoplasts were fractionated on a continuous iso-osmotic gradient of Percoll; this resulted in several visible bands. Flow cytometric analysis of fluorescein and nuclear DNA contents after staining with fluorescein and DAPI respectively showed that the main band contained mostly evacuolated, intact (sub)protoplasts. Microprotoplasts contained one or a few micronuclei surrounded by a thin rim of cytoplasm and an intact plasma membrane. A maximum of 40% of the microprotoplasts in the fraction just below the main band had a DNA content less than the G1 level, in other fractions this maximum was 20%. Some of these contained an amount equivalent to that of one or a few chromosomes. The application of microprotoplasts for chromosome-mediated gene transfer in plants is indicated.

Cell population kinetics and ploidy rate of early focal lesions during hepatocarcinogenesis in the rat.

We have studied the changes in cell population kinetics and DNA-content of cycling parenchymal cells during the very early steps of rat hepatocarcinogenesis in Faber's protocol. Adult rats were initiated by a single dose of diethylnitrosamine (DENA, 200 mg kg-1), followed 2 weeks later by a 2-week diet of 0.03% 2-acetylaminofluorene (2-AAF) as selection phase. In the middle of selection time, a single necrogenic dose of carbon tetrachloride (CCl4, 2 ml kg-1) was administered by gavage. Twenty four hours thereafter, radiolabelled thymidine (3H-TdR, 1.5 microCi g-1) was given by repeated injections during 24 h. An emergence of small, pyroninophilic ('tigroid') foci was observed at the second, fifth and eighth days after the proliferative stimulus. The focal putative precancerous cells presented a significant higher labelling index (L1) than the non-affected parenchymal cells for all exposure times. However, the labelling intensity decreased from the second to the eighth day after CCl4, suggesting a dilution of the radiolabelled DNA by repeated divisions within the foci. The nuclei of the same foci were analysed for DNA-content by feulgen microdensitometry on neighbouring sections. A gradual reduction of nuclear DNA-content was observed in 66% of the foci at the fifth day and in 100% of foci at the eight day, as compared to surrounding tissue and untreated animals, where labelling and DNA-content remain in the same ratio.

Parasexual Crosses by Hyphal Anastomosis and Protoplast Fusion in the Entomopathogen Verticillium lecanii

SUMMARY: Sixty-two ‘crosses’ were attempted using mixed conidial suspensions and a ‘hanging drop’ technique involving 21 auxotrophic mutants obtained from seven isolates of Verticillium lecanil. Frequency of heterokaryon formation varied from 0 to 100%, with apparently complete incompatibility being expressed between some isolates, although this was influenced by the particular auxotrophic marker used. Protoplast fusion between compatible isolates usually resulted in prototrophic colonies, but was often unsuccessful when testing apparently incompatible isolates, suggesting that incompatibility was due mainly to nuclear or cytoplasmic factors, rather than to the wall. Evidence for the formation of prototrophic heterozygous diploids was provided by nutritional studies, and DNA content per conidiospore as measured by Feulgen microdensitometry. Conidiospore volume, determined by Coulter counter analysis, was not a reliable indicator of ploidy when investigating parasexual recombination between isolates of differing conidiospore size. Heterozygous diploids generally tended to be unstable, breaking down to yield recombinant haploids; however, four diploids were isolated which remained stable for 3-4 weeks on artificial media.

Isolation and culture of a tetraploid subpopulation of smooth muscle cells from the normal rat aorta.

Smooth muscle cells with 4C (double diploid) DNA content have been found in major arteries. The proportion of 4C cells increases with normal aging and with hypertension. These cells may represent a state of arrest at the G2 phase of the cell cycle or may be examples of true tetraploidy. Flow cytometric cell sorting was used to isolate 4C smooth muscle cells from the rat aorta, and the cells were cultured. Flow cytometry, Feulgen microdensitometry, and karyotyping of the progeny of the 4C cells established the presence of true tetraploid cells. These findings demonstrate the presence of reproductively viable tetraploid cells in a normal mammalian tissue.

Polyploid nuclei in human artery wall smooth muscle cells.

Although polyploid nuclei have long been known to be present in many adult human tissues, the ploidy of smooth muscle cells in human artery wall has never been determined. We measured DNA content in individual smooth muscle cell nuclei of artery wall specimens by two means: Feulgen microdensitometry and flow microfluorimetry. A significant percentage of nuclei were polyploid; most of these were tetraploid, although higher levels were also found. The frequency of polyploidy varied with age from less than 1% at birth to a mean of 7% in adult aortic, carotid, and iliac vessels. Atherosclerotic plaques had a lower tetraploid content than the underlying media, whereas normal intima was similar to the corresponding media. The increase in frequency of hyperploid smooth muscle cell nuclei correlates with the normal growth, development, and aging of human artery wall. We suggest that the regular existence of a subset of polyploid smooth muscle cells may indicate an important functional role for this phenotype.

Studies on the control of the cell cycle in cultured plant cells

When suspension-cultured sycamore (Acer pseudoplatanus L.) cells are deprived of the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) cell division activity ceases after a brief period of exponential growth. Feulgen microdensitometry analyses showed that sycamore cultures starved of 2,4-D do not exhibit exclusive arrest in any particular cell cycle compartment. Regrowth, following readdition of 2,4-D to 2,4-D-starved cells, produced periodic oscillations in mitotic index and discontinuous increases in cell number. Detailed characteristics of the regrowth kinetics indicated involvement of periodic, rather than synchronous, growth. The results of this study suggest that the stimulation of cell division by 2,4-D is indirect and not due to the activation of a cell cycle control point to commit cells to division.

Flow Cytometric Analysis of Megakaryocyte Ploidy. Comparison With Feulgen Microdensitometry and Discovery That 8N is the Predominant Ploidy Class in Guinea Pig and Monkey Marrow

Flow cytometry (FCM) was adapted to measure ploidy values in megakaryocytes isolated from normal guinea pigs and African green monkeys. The FCM approach was validated by microscopic identification of electronically sorted 8N and higher ploidy cells and by comparisons with Feulgen microdensitometry on the same samples. Sorted 8N populations contained only megakaryocytes and were completely free of cell clumps; clumping was also absent in cell suspensions examined prior to FCM. Discrete megakaryocyte peaks were found at 8N, 16N, and 32N but not at intermediate values. The ploidy patterns of isolated megakaryocytes were similar by both methods (approximately 15% 8N, 58% 16N, and 27% 32N in guinea pigs). Unexpectedly, FCM analysis of unseparated marrow cell suspensions found that the most frequent ploidy class of megakaryocytes was 8N (50%). In contrast, by microdensitometry, the 8N class accounted for only 21% of the megakaryocytes in unseparated marrow cell suspensions. Since the traditional method depends on measurement only of easily recognizable megakaryocyte nuclei, a microdensitometric analysis of unselected marrow cells was carried out. Many more smaller 8N megakaryocytes (48%) were found by this approach, confirming the FCM data and the bias of the traditional sampling method. The decrease in 8N megakaryocytes (50%–15%) with enrichment was accounted for by the consistent finding that cells lost during isolation were predominantly 8N. with relative sparing of the 32N class. FCM provides more rapid and objective analysis of ploidy patterns in much larger samples of megakaryocytes than previous methods.

Flow cytometric analysis of megakaryocyte ploidy. Comparison with Feulgen microdensitometry and discovery that 8N is the predominant ploidy class in guinea pig and monkey marrow

Flow cytometry (FCM) was adapted to measure ploidy values in megakaryocytes isolated from normal guinea pigs and African green monkeys. The FCM approach was validated by microscopic identification of electronically sorted 8N and higher ploidy cells and by comparisons with Feulgen microdensitometry on the same samples. Sorted 8N populations contained only megakaryocytes and were completely free of cell clumps; clumping was also absent in cell suspensions examined prior to FCM. Discrete megakaryocyte peaks were found at 8N, 16N, and 32N but not at intermediate values. The ploidy patterns of isolated megakaryocytes were similar by both methods (approximately 15% 8N, 58% 16N, and 27% 32N in guinea pigs). Unexpectedly, FCM analysis of unseparated marrow cell suspensions found that the most frequent ploidy class of megakaryocytes was 8N (50%). In contrast, by microdensitometry, the 8N class accounted for only 21% of the megakaryocytes in unseparated marrow cell suspensions. Since the traditional method depends on measurement only of easily recognizable megakaryocyte nuclei, a microdensitometric analysis of unselected marrow cells was carried out. Many more smaller 8N megakaryocytes (48%) were found by this approach, confirming the FCM data and the bias of the traditional sampling method. The decrease in 8N megakaryocytes (50%–15%) with enrichment was accounted for by the consistent finding that cells lost during isolation were predominantly 8N. with relative sparing of the 32N class. FCM provides more rapid and objective analysis of ploidy patterns in much larger samples of megakaryocytes than previous methods.

The gross and cellular response of a rat mammary tumour to single doses of cyclophosphamide

The response of a transplanted mammary carcinoma of Wistar rats, to single intraperitoneal doses of cyclophosphamide ( 25–250 mg kg −1 ) has been assayed by measurement of; delay in growth of the tumour between 9 and 25 mm; cell survival in vitro, 3 and 24 hr after in situ treatment; tritiated thymidine autoradiography and Feulgen microdensitometry of histological material. All three methods of assay indicate that at 9 mm diameter the tumour contains a subpopulation of malignant cells relatively resistant to cyclophosphamide doses in excess of 50 mg kg −1 . The in vitro cell survival assay suggests that these cells comprise 40–50% of the clonogenic population and are capable of considerable in situ repair of potentially lethal damage within 24 hr of treatment. Cellular repopulation from this resistant component, after 150 mg kg −1 of drug, is delayed for 6 or more days and restoration to near pre-treatment status only achieved by 10 days, in this rapidly-growing tumour.

Feulgen microdensitometry and analysis of S-phase cells in cervical tumour biopsies.

Tissue specimens from the cervical tumours of 70 patients undergoing radiotherapy were examined by Feulgen microdensitometry. Twenty-five of the 70 specimens were also subjected to in-vitro tritiated thymidine autoradiography to determine the proportion of DNA synthesising cells they contained. A spectrum of frequency distributions of nuclear DNA content was obtained from Feulgen microdensitometry, but by inspecting the data the basic DNA content of malignant cells could be established in most cases. Fifty-nine per cent of the tumours were 'diploid', 10% 'tetraploid', 13% 'diploid to tetraploid', and the remaining 18% 'aneuploid'. Grafical analysis of DNA frequencies from 48 'diploid' or 'tetraploid' tumours enabled the proportion of DNA synthesising (S) cells to be estimated by frequency distribution analysis. Estimates of the S component ranged from nil to 30%, were log normally distributed, and comparable to direct measurements of cells in S determined by autoradiography for 25 cases (range 1.2-28.7%). For all paired data the mean difference was 1.2 +/- 1.45% (confidence limits), suggesting that overall Feulgen microdensitometry analysis may be an equally valid technique in providing cellular kinetic information with human tumour material.