Determination Of DNA Content Research Articles

DNA image-fluorimetry of individual human chromosomes

Mucrofluorimetric method for the determination of DNA content in individual human chromosomes has been developed. The method is based on a preliminary identification of chromosomes with Hoechst 33258, followed by staining of the chromosomes with Feulgen reaction using Schiffs reagent type ethidium bromide-SO2, then measuring the fluorescence intensity of the chromosomes using an image analyzer. The method allows to determine the DNA content of individual chromosomes with accuracy up to 4.5 fg. DNA content of individual human chromosomes, their p-and q-arms as well as homologous chromosomes were measured using the developed method. It has been shown that the DNA content in the chromosomes of normal human karyotype is unstable. Fluctuations in the DNA content in some chromosomes can vary 35-40 fg.

Karyotype analysis and DNA content in some species of Chrysolaena (Vernonieae, Asteraceae)

Cytogenetic characterization by karyotyping and determination of DNA content by flow cytometry of five species of Chrysolaena (Vernonieae, Asteraceae) was performed. This is the first study of nuclear DNA content realized in the genus. The 2C-values were compared with the ploidy level and the total karyotype length (TKL) of each species. Mitotic analysis revealed a base chromosome number x = 10 for all entities and different ploidy levels, from diploid (2n = 2x = 20) to octoploid (2n = 8x = 80). All species showed bimodal karyotypes composed of metacentric and submetacentric chromosomes. The average chromosome size (ML) varied from 1.86 μm to 2.70 μm, while the TKL ranged from 18.65 μm to 80.55 μm. The intrachromosomal asymmetry index (A1) varied from 0.27 to 0.38, while the interchromosomal asymmetry index (A2) ranged from 0.19 to 0.25. A new cytotype is reported for the first time for C. propinqua. Accessory chromosomes found in C. verbascifolia, C. cognata, C. flexuosa, and C. propinqua are also reported as new.

Insulin is essential for in vitro chondrogenesis of mesenchymal progenitor cells and influences chondrogenesis in a dose-dependent manner

Insulin is a commonly used additive in chondrogenic media for differentiating mesenchymal stem cells (MSCs). The indispensability of other bioactive factors like TGF-β or dexamethasone in these medium formulations has been shown, but the role of insulin is unclear. The purpose of this study was to investigate whether insulin is essential for MSC chondrogenesis and if there is a dose-dependent effect of insulin on MSC chondrogenesis. We cultivated human MSCs in pellet culture in serum-free chondrogenic medium with insulin concentrations between 0 and 50 μg/ml and assessed the grade of chondrogenic differentiation by histological evaluation and determination of glycosaminoglycan (GAG), total collagen and DNA content. We further tested whether insulin can be delivered in an amount sufficient for MSC chondrogenesis via a drug delivery system in insulin-free medium. Chondrogenesis was not induced by standard chondrogenic medium without insulin and the expression of cartilage differentiation markers was dose-dependent at insulin concentrations between 0 and 10 μg/ml. An insulin concentration of 50 μg/ml had no additional effect compared with 10 μg/ml. Insulin was delivered by a release system into the cell culture under insulin-free conditions in an amount sufficient to induce chondrogenesis. Insulin is essential for MSC chondrogenesis in this system and chondrogenic differentiation is influenced by insulin in a dose-dependent manner. Insulin can be provided in a sufficient amount by a drug delivery system. Therefore, insulin is a suitable and inexpensive indicator substance for testing drug release systems in vitro.

Evaluation of ploidy level and endoreduplication in carnation (Dianthus spp.)

Carnation (Dianthus caryophyllus L.) is one of the fifth most important ornamental species worldwide. Many desirable plant characteristics, such as big size, adaptation under stress, and intra or interspecific hybridization capability, are dependent on plant ploidy level. We optimized a quick flow cytometry method for DNA content determination in wild and cultivated carnation samples that allowed a systematic evaluation of ploidy levels in Dianthus species. The DNA content of different carnation cultivars and wild Dianthus species was determined using internal reference standards. The precise characterization of ploidy, endoreduplication and C-value of D. caryophyllus ‘Master’ makes it a suitable standard cultivar for ploidy level determination in other carnation cultivars. Mixoploidy was rigorously characterized in different regions of several organs from D. caryophyllus ‘Master’, which combined with a detailed morphological description suggested some distinctive developmental traits of this species. Both the number of endoreduplication cycles and the proportion of endopolyploid cells were highly variable in the petals among the cultivars studied, differently to the values found in leaves. Our results suggest a positive correlation between ploidy, cell size and petal size in cultivated carnation, which should be considered in breeding programs aimed to obtain new varieties with large flowers.

Abstract A58: High Throughput Search for a Combination Cancer Preventive Treatment

Abstract Identification of safer chemopreventive drugs is essential for the future of cancer preventive treatment. Although the feasibility for chemoprevention of estrogen receptor (ER) -positive breast cancers has been exemplified by the use of selective estrogen response modifiers (SERMs), we are still lacking preventive options for many other disease types, including breast cancers not dependent on ER status. Rexinoids are a class of agents that effectively reduce the incidence of both ER-positive and -negative tumors in animal models of breast cancer and thus may be better cancer preventive drugs than the current standard SERMs. However, significant side-effects at the effective clinical dose, including hypertriglyceridemia and weight gain, limit its generalized use. A phase II biomarker trial of the synthetic rexinoid, bexarotene (Targretin, LGD1069) demonstrated that bexarotene reduces expression of the proliferation markers Ki67 and Cyclin D1 in normal breast epithelium of postmenopausal women, suggesting that the cancer preventive effects of bexarotene are associated with its ability to elicit cell cycle blockade and inhibit proliferation of premalignant mammary epithelial cells. Here we hypothesize that the combination of repurposed drugs and some small molecule compounds with low dose rexinoid will minimize toxicity and result in effective anti-proliferative in premalignant cells. Therefore, we designed an image-based in vitro assay system to conduct high throughput screens to identify existing drugs and drug-like small molecules which enhance the growth suppressive effect of cancer preventive rexinoids. We used an automated 96 well platform to test the NIH clinical collection of drugs and a library of structurally diverse compounds (MayBridge Hitfinder) alone or in combination with 100nM bexarotene. Through multiparametric determination of cell counts, DNA content and cellular toxicity, we identified combination treatments of premalignant breast cells that fit the above criteria and report on dose-response studies and secondary screens for optimal dose and cell health. Validation of some of the candidate agents and plans for in vivo testing of their chemopreventive activity will be discussed. Citation Format: Ivan P. Uray, Powel H. Brown. High-throughput search for a combination cancer preventive treatment. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr A58.

Development of the Flow Cytometric Protocol for Ploidy Analysis and Determination of Relative Nuclear DNA Content in Cashew (Anacardium occidentale Linn.)

Development of the Flow Cytometric Protocol for Ploidy Analysis and Determination of Relative Nuclear DNA Content in Cashew (Anacardium occidentale Linn.)

Peculiarities of cytometrical methods of DNA content determination in the nucleus

This work has proposed a new theoretical approach to analysis of histograms of DNA content, which are obtained by the method of flow cytometry, in cells of Drosophila melanogaster imaginal discs. The precision of measurements of the DNA amount in G1 and G2(M) phases has been shown to be limited by precision of instrument tuning of zero of the flow cytometer. Use of the calculative zero of the flow cytometer and of dividing cells as standards of the DNA content is able to increase severalfold the precision of the DNA measurements in nuclei of the species. Comparative analysis of errors of various methods of measurement of the DNA content in cell nuclei is also performed. For methods of flow fluorescent cytometry, confocal scanning, and cytophotometry of the Feulgen-stained nuclei, it has been shown that, at present, the mean square errors of the DNA content measurements are within the interval of values considered acceptable for biological studies (0.02 < CV < 0.06).

Effects of Temperature and Water-Filtered Infrared-A Alone or in Combination on Healthy and Glyoxal-Stressed Fibroblast Cultures

Water-filtered infrared-A (wIRA) radiation has been described as supportive for tissue regeneration. We sought to investigate in detail the wIRA effect at different temperatures in 3T3 fibroblasts that were treated with glyoxal to induce formation of advanced glycation end products (AGEs). Nonirradiated and nonglyoxal-treated cells served as controls. Experiments were carried out over a range of 37°–45°C with exact temperature monitoring to distinguish between temperature and wIRA effects. Metabolic activity was assessed by resazurin assay. Mitochondrial membrane potential was assessed by JC-1 vital staining. Apoptotic changes were determined by vital staining with annexin V and YO-PRO-1 and determination of subG1 DNA content. Temperature had a dominant effect overriding effects exerted by wIRA or glyoxal treatment. The number of apoptotic cells was significantly higher at 45°C, while the percentage of healthy cells was significantly lower at 45°C. WIRA irradiation itself or in combination with glyoxal treatment exerted no damaging effects on the fibroblasts at physiological (37°–40°C) or higher (42°–45°C) temperatures compared to untreated controls. Temperatures of 45°C, which can occur during inappropriate application of infrared irradiation, damage cells even in the absence of wIRA or glyoxal application.

Comparative evaluation of different calcium phosphate‐based bone graft granules – an in vitro study with osteoblast‐like cells

Granule-shaped calcium phosphate-based bone graft materials are often required for bone regeneration especially in implant dentistry. Two newly developed bone graft materials are Ceracell(®) , an open-celled highly porous bioceramic from β-tricalcium phosphate (β-TCP) under addition of bioglass and Osseolive(®) , an open porous glass ceramic with the general formula Ca2 KNa(PO4 )2 . The goal of this study was to characterize different modifications of the two bone graft materials in vitro in comparison to already established ceramic bone grafts Cerasorb M(®) , NanoBone(®) and BONIT Matrix(®) . Adhesion and proliferation of SaOS-2 osteoblast-like cells were evaluated quantitatively by determining DNA content and lactate dehydrogenase (LDH) activity and qualitatively by scanning electron microscopy (SEM). In addition, MTT cell-vitality staining was applied to confirm the attachment of viable cells to the different materials. Osteogenic differentiation was evaluated by measurement of alkaline phosphatase (ALP) activity as well as gene expression analysis of osteogenic markers using reverse transcriptase PCR. DNA content and LDH activity revealed good cell attachment and proliferation for Ceracell and Cerasorb M. When pre-incubated with cell-culture medium, also Osseolive showed good cell attachment and proliferation. Attachment and proliferation of osteoblast-like cells on NanoBone and BONIT Matrix was very low, even after pre-incubation with cell-culture medium. Specific ALP activity on Ceracell(®) , Osseolive (®) and Cerasorb M(®) increased with time and expression of bone-related genes ALP, osteonectin, osteopontin and bone sialoprotein II was demonstrated. Ceracell as well as Osseolive granules support proliferation and osteogenic differentiation in vitro and may be promising candidates for in vivo applications.

Image cytometric determination of nuclear and chromosomal DNA contents in Allium cepa L.

Estimation of DNA content in individual chro- mosomes and their arms was carried out in the primary root meristems of Allium cepa L. var Local, (2n=16) using an Image Analysis System following Feulgen staining. Re- gression analysis revealed that fifth and seventh chromo- somes had higher DNA contents relative to their chromosomal lengths while the 1st, 2nd and 4th chromo- somes had less DNA contents than those expected on the basis of length.

Gender determination of fertilized unincubated chicken eggs by infrared spectroscopic imaging

Each year, billions of day-old layer chicks are produced in the world. Since only female chicks are reared for egg production, the chicks must be sexed and the unwanted male layer chicks are culled. The culling of male chicks is a serious problem, both in terms of animal welfare and waste disposal. The germinal disc in fertilized but unincubated eggs contains already several thousands of blastoderm cells. The cellular DNA in birds is different for male and female chicks. The difference in DNA content between male and female chicks is around 2% and is measurable by Fourier transform infrared (FT-IR) spectroscopy. In this study, small amounts of blastoderm cells from 22 chicken eggs were characterized by attenuated total reflection FT-IR spectroscopic imaging and classified by linear discriminant analysis. Polymerase chain reaction (PCR) was used as a reference method to determine the gender. The spectroscopic results demonstrate that male blastoderm cells exhibit a higher content of DNA than cells from female blastoderm. The spectroscopic-based gender determination led to the same result as the PCR analysis. FT-IR spectroscopic imaging allows the gender determination of unincubated eggs within a few seconds based on the accurate determination of the different DNA contents in blastoderm cells of both sexes.

Application of flow cytometry in pediatric hematology‐oncology

Applications of flow cytometry in pediatric cancers have expanded substantially in recent years. In acute leukemias, the commonest childhood cancer, flow cytometry can now define complex antigenic profiles that are associated with specific cytogenetic/molecular defects and can also directly identify BCR-ABL fusion protein. Flow cytometry based scoring system has been described for diagnosis of myelodysplastic syndromes. In solid tumors, flow cytometry was previously used mainly to determine DNA content for prognosis; however, recent studies in children with neuroblastoma and Ewing sarcoma have identified its diagnostic utility. In this review, we will discuss the current and future applications of flow cytometry in pediatric hematology-oncology.

Critical aspects in analysis of cellular DNA content.

This unit covers general aspects of DNA content analysis and provides introductory or complementary information to the specific protocols of DNA content assessment in this chapter. It describes principles of DNA content analysis and outlines difficulties and pitfalls common to these methods. It also reviews methods of DNA staining in live, permeabilized, and fixed cells, and in cell nuclei isolated from paraffin-embedded tissues, as well as the approaches to stain DNA concurrently with cell immunophenotype. This unit addresses factors affecting accuracy of DNA measurement, such as chromatin features restricting accessibility of fluorochromes to DNA, stoichiometry of interaction with DNA, and "mass action law" characterizing binding to DNA in relation to unbound fluorochrome concentration. It also describes controls to ensure accuracy and quality control of DNA content determination and principles of DNA ploidy assessment. Because many aspects of DNA content analysis are common to protocols in UNITS 7.3, 7.6, 7.16, 7.20, 7.23, & 7.25, certain parts of this unit provide information redundant with commentaries in these units.

Ploidy level determination within the context of in vitro breeding

Variations in genome size and chromosome complement of species provide very useful information for biosystematic studies, and also because they influence a range of ecological characteristics. They are also of utmost importance for breeding, especially when in vitro biotechnology tools are used and the need arises to assess the trueness-to-type of regenerated plants. Thus, protocols have existed for a long time for chromosome counting, and more recently also for the determination of the relative nuclear DNA content and genome size. It has also been shown that these latter traits are strongly correlated to regeneration competence and, more generally, to developmental processes in plants. This article will briefly review such approaches from a methodological and breeding-oriented viewpoint.

Anticarcinogenic Activity of Allylmercaptocaptopril Against Aflatoxin-B1 Induced Liver Carcinoma in Rats

Aim: The present study was aimed to investigate the cellular and molecular mechanisms of protective effects of allylmercaptocaptopril (AMC) against liver carcinoma induced by Aflatoxin B1, a potent inducer of liver cancer. Method: In this study we determined the protective effect of AMC on liver tissue, as well as on enzymatic liver functions by estimating glycolytic enzymes like hexokinase, phosphoisomerase and aldolase, gluconeogenic enzymes like glucose-6-phosphatase and fructose 1,6 biphosphatase. Determination of total protein, DNA and RNA content also made to elucidate its action. Result: Aflatoxin B1 treatment to rats resulted in significantly elevated levels of glycolytic enzymes like hexokinase, phosphoglucoisomerase and aldolase and along with significant decrease in serum total protein, gluconeogenic enzymes and DNA and RNA content when compared to the control rats. The administration of AMC to the hepatocellular carcinoma bearing rats resulted in restoration of most of enzymatic liver functions and also total protein content, DNA and RNA content. Conclusion: Allylmercaptocaptopril has an ability to modulate the function of glycolytic and gluconeogenic enzymes, DNA and RNA synthesis in hepatocellular carcinoma which proved its anticarcinogenic activity.

Comparative investigations on the protective effects of rhodioside, ciwujianoside‐B and astragaloside IV on radiation injuries of the hematopoietic system in mice

The aim of this study was to investigate the protective effects of three glycosides (rhodioside, ciwujianoside-B and astragaloside IV) on the hematopoietic system in the mice exposed to γ-rays, and to examine the possible mechanisms involved. Mice were pretreated with the glycosides (40 mg/kg, i.g.) daily for 7 days prior to radiation. The survival of mice pretreated with three glycosides after total body irradiation (6.0 Gy) was examined. Peripheral blood leucocytes and endogenous spleen colony counts, colony-forming unit-granulocyte macrophage assay, analysis of DNA content and apoptosis rate determination were performed to evaluate the effects of the three glycosides on hematogenesis. The fragmentation of double-stranded DNA in lymphocytes was detected by the comet assay. The changes in cell cycle were analysed by flow cytometry. Furthermore, the expression levels of Bcl-2, Bax and nuclear factor-kappa B (NF-κB) were measured by western blot and the electrophoretic mobility shift assay. The results showed that pretreatment with all of the glycosides improved survival time and increased the number of leucocytes, spleen colonies and granulocyte-macrophage colonies in mice exposed to 6.0 Gy γ-radiation. Rhodioside showed more protective efficacy than both ciwujianoside-B and astragaloside IV. All three glycosides significantly increased the proliferation abilities of bone marrow cells, and decreased the ratio of cells in G(0)/G(1) phase. Further analysis showed that these three glycosides were able to decrease DNA damage and the increment in the Bax/Bcl-2 ratio induced by radiation. In summary, the three glycosides showed radioprotective effects on the hematopoietic system in mice, which was associated with changes in the cell cycle, a reduction in DNA damage, and down-regulation of the ratio of Bax/Bcl-2 in bone marrow cells exposed to radiation.

Novel ceramic bone replacement material Osbone® in a comparative in vitro study with osteoblasts

Hydroxyapatite (HA) is a very common ceramic material for bone replacement due to its similarity in composition to the mineral phase of natural bone. A recently developed bone graft material is Osbone(®), a synthetic HA ceramic available as porous granules with different sizes and block forms. The goal of this study was to characterise Osbone(®) in vitro in comparison to the already established calcium phosphate-based bone grafts Cerasorb M(®) and Bio-Oss(®). Adhesion and proliferation of SaOS-2 osteoblasts were evaluated quantitatively by determining DNA content and lactate dehydrogenase (LDH) activity and qualitatively by scanning electron microscopy (SEM). In addition, MTT cell vitality staining was performed to confirm the attachment of viable cells to the different materials. Osteogenic differentiation of the cells was evaluated by means of alkaline phosphatase (ALP) activity quantification as well as by gene expression analysis of osteogenic markers using reverse transcriptase PCR. MTT staining after 1 day of adhesion showed viable cells on all examined materials. DNA content and LDH activity revealed proliferation of osteoblasts on Osbone(®) and Cerasorb M(®), but not on Bio-Oss(®) during cultivation over 28 days. SEM showed a well-spread morphology of cells attached to both Osbone(®) and Cerasorb M(®). We detected an increase of specific ALP activity during cultivation of osteoblasts on Osbone(®) and Cerasorb M(®) as well as expression of the bone-related genes ALP, osteonectin, osteopontin and bone sialoprotein II on both materials. Osbone(®) granules support proliferation and osteogenic differentiation in vitro and are therefore promising candidates for in vivo applications.

Abstract 2268: Intracellular delivery of RGDfk grafted docetaxel encapsulated PLGA nanoparticles for breast cancer therapy

Abstract The purpose of the study was to prepare, characterized, assess in vitro antiproliferative activity, and determine DNA content and mode of cell death of RGDfk grafted Docetaxel encapsulated PLGA nanoparticles on breast cancer cells. DC- encapsulated nanoparticles were prepared using solvent evaporation technique. RGD-conjugation was done by carbodiimide coupling method. The nanoparticles were characterized for particle size and zeta potential by zeta sizer (Nano-ZS), entrapment efficiency and in-vitro drug release profile and RGD-conjugation efficiency. To establish antiproliferative effect, the RGD-conjugated nanoparticles of DC(PLGA-DC-RGD), unconjugated nanoparticles (PLGA-DC) and equal amounts of the free drug were studied on BT-20 and MDA-MB-231 cells by CCK-8 assay method. DNA content analysis was done on flowcytometry by propadium iodide staining. The mode of cell death at different time and concentration was determined by FITC-Annexine V assay. The DC encapsulated RGD conjugated and unconjugated PLGA nanoparticles were found to be nanosized. The cytotoxicity indicated by IC50 values suggests that RGD conjugated nanoparticles at 72hrs are 2.1 and 4 times more cytotoxic than unconjugated nanoparticles and drug solution for BT-20 cell lines. Similarly, for MDA-MB-231 cells, the RGD conjugated nanoparticles are 2 and 4 time more cytotoxic than unconjugated nanoparticles and drug solution. The results shows that the RGD conjugated nanoparticles at the concentration of 5nM showed 51% G2 phase arrest as compared to 30% G2 phase arrest with unconjugated nanoparticles at the same concentration at 24hrs. Two types of mode of cell death were found during the FITC-Annexin V assay. At 24 hrs, the treatment of 5nM of drug solution with cells resulted in 13.6% &amp; 3.4 % necrotic &amp; apoptotic cell fragments respectively. While at similar drug equivalent concentration, the PLGA-DC-RGD and PLGA-DC nanoparticles showed 6.4% &amp; 28.3% and 5.1% &amp; 25.6% necrotic &amp; apoptotic cell fragments respectively. With increase in time and concentration the mode of cell death by apoptosis was found to be increasing. To conclude, RGD conjugation to nanoparticles improved antiproliferative activity when assessed in vitro in breast cancer cell lines compared to free drug and unconjugated nanoparticles. The DNA content analysis depicted the cell cycle arrest in G2 phase was more even at lower drug equivalent concentration of PLGA-RGD. The mode of cell death was found to be mainly by necrosis at low drug equivalent concentration (1nM) and by apoptosis at high drug equivalent concentration (10nM) of RGD conjugated drug encapsulated nanoparticles for breast cancer cell lines. Hence, it may be concluded that drug nanocarriers once conjugated with RGD can provide prolonged drug release to cytoplasm and can affectively target breast cancer and can probably reduce the limitations associated with breast cancer chemotherapy. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2268.

Critical Aspects in Analysis of Cellular DNA Content

This unit covers general aspects of DNA content analysis and provides introductory or complementary information to the specific protocols of DNA content assessment in this chapter. It describes principles of DNA content analysis and outlines difficulties and pitfalls common to these methods. It also reviews methods of DNA staining in live, permeabilized, and fixed cells, and in cell nuclei isolated from paraffin-embedded tissues, as well as the approaches to stain DNA concurrently with cell immunophenotype. This unit addresses factors affecting accuracy of DNA measurement, such as chromatin features restricting accessibility of fluorochromes to DNA, stoichiometry of interaction with DNA, and "mass action law" characterizing binding to DNA in relation to unbound fluorochrome concentration. It also describes controls to ensure accuracy and quality control of DNA content determination and principles of DNA ploidy assessment. Because many aspects of DNA content analysis are common to protocols in UNITS 7.3, 7.6, 7.16, 7.20, 7.23, & 7.25, certain parts of this unit provide information redundant with commentaries in these units.

Heterokaryotic nuclear conditions and a heterogeneous nuclear population are observed by flow cytometry in Phytophthora infestans

A simple and reliable method for preparation of whole nuclei of a common oomycete, Phytophthora infestans, is described for laser flow cytometry. The ease of preparation, the absence of detectable debris and aggregates, and the precision in determinations of DNA content per nucleus improve interpretation and understanding of the genetics of the organism. Phytophthora infestans is the pathogen that causes potato and tomato late blight. The genetic flexibility of P. infestans and other oomycete pathogens has complicated understanding of the mechanisms of variation contributing to shifts in race structure and virulence profiles on important agricultural crops. Significant phenotypic and genotypic changes are being reported in the apparent absence of sexual recombination in the field. Laser flow cytometry with propidium iodide is useful in investigating the nuclear condition of the somatic colony of field strains of P. infestans. The majority of the studied strains contain a single population of nuclei in nonreplicated diplophase. However, mean DNA content per nucleus varies considerably among isolates confirming the heterogeneity of the nuclear population in regard to C-value, for field isolates. Nuclear DNA content varies from 1.75x to 0.75x that of nuclei in a standard strain from central Mexico. Some strains contain two to three populations of nuclei with differing DNA contents in the mycelium and are heterokaryons. Such a range in DNA content suggests DNA-aneuploidy, but direct confirmation of aneuploidy will require microscopy of chromosomes. Heterokaryosis and populations of nuclei of differing DNA content necessarily confound standardized assays used worldwide in crop breeding programs for determination of race profiles and virulence phenotypes of this important pathogen.