Application of sonication to release DNA from Bacillus cereus for quantitative detection by real-time PCR

A rapid sonication method for lysis of Gram-positive bacteria was evaluated for use in combination with quantitative real-time polymerase chain reaction (PCR) analyses for detection. Other criteria used for evaluation of lysis were microscopic cell count, colony forming units (cfu), optical density at 600 nm and total yield of DNA measured by PicoGreen fluorescence. The aim of this study was complete disruption of cellular structures and release of DNA without the need for lysing reagents and time-consuming sample preparation. The Gram-positive bacterium Bacillus cereus was used as a model organism for Gram-positive bacteria. It was demonstrated by real-time PCR that maximum yield of DNA was obtained after 3 to 5 min of sonication. The yield of DNA was affected by culture age and the cells from a 4-h-old culture in the exponential phase of growth gave a higher yield of DNA after 5 min of sonication than a 24-h-old culture in the stationary phase of growth. The 4-h-old culture was also more sensitive for lysis caused by heating. The maximum yield of DNA, evaluated by real-time PCR, from a culture of the Gram-negative bacterium Escherichia coli, was obtained after 20 s of sonication. However, the yield of target DNA from E. coli rapidly decreased after 50 s of sonication due to degradation of DNA. Plate counting (cfu), microscopic counting and absorbance at 600 nm showed that the number of viable and structurally intact B. cereus cells decreased rapidly with sonication time, whereas the yield of DNA increased as shown by PicoGreen fluorescence and real-time PCR. The present results indicate that 3-5 min of sonication is sufficient for lysis and release of DNA from samples of Gram-positive bacteria.     

Fluorometric assay of DNA in cartilage explants using Hoechst 33258

A simple two-step fluorometric assay of DNA in cartilage explants, utilizing the bisbenzimidazole dye Hoechst 33258, is described. Cartilage explants were prepared for assay by digestion with papain. Aliquots of the digest were mixed with dye solution, and the fluorescence emission measured. The enhancement in fluorescence of dye was specific for DNA, as demonstrated by 97% sensitivity to DNase and resistance to RNase. In addition, little or no interference was caused by non-DNA tissue components, since DNA caused an equal enhancement in fluorescence independent of the presence of papain-digested cartilage. By performing the assay on isolated chondrocytes, the cellular content of DNA was computed to be 7.7 pg per chondrocyte. The assay was stable for at least 2 h and sensitive to as little as 6 ng of DNA or equivalently less than 1000 cells. This procedure offers advantages over other established DNA assays of cartilage and may be especially useful in metabolic studies of cartilage explants.     

Quantification of nuclear DNA and G-C content in marine macroalgae by flow cytometry of isolated nuclei

Summary The amounts of nuclear DNA in ten species of seaweeds belonging to the Rhodophyceae, Phaeophyceae, and Chlorophyceae were determined by flow cytometric analysis of nuclei isolated from protoplasts. Genome size was determined from the fluorescence of the nuclei stained with ethidium bromide. The size of the nuclear genome ranged from 0.13 pg per cell in the 1 C population ofUlva rigida to 3.40 pg per cell in the 2 C population ofSphacelaria sp. GC% analysis was based on staining with either Hoechst 33342 or mithramycin A, two fluorochromes specific for the bases A-T and G-C, respectively. Two models were used for the estimation of the proportion of guanine plus cytosine in the nuclear genome. The first one was based on the linear relationships mithramycin A fluorescence/G-C content and ethidium bromide fluorescence/total DNA content. The second model, based on the curvilinear relationships Hoechst 33342 fluorescence/A-T content and mithramycin A fluorescence/G-C content, resulted in comparatively more homogenous and consistent data and appears more accurate. Comparison with previous reports from other methods for the physical investigation of nuclear genomes shows that flow cytometry of nuclei isolated from protoplasts is an accurate, convenient and robust technique to assay for genome sizes and base pair composition in marine macroalgae.Abbreviations A-T nucleic bases adenine and thymine - CRBC chicken red blood cell - FALS forward-angle light scatter - G-C nucleic bases guanine and cytosine - SEIM sorbitol enzymatic incubation medium - SWIM sea water incubation medium - Tm thermal denaturation temperature of DNA     

DNA amplification to enhance detection of genetically engineered bacteria in environmental samples.

The polymerase chain reaction (PCR) was performed to amplify a 1.0-kilobase (kb) probe-specific region of DNA from the herbicide-degrading bacterium Pseudomonas cepacia AC1100 in order to increase the sensitivity of detecting the organism by dot-blot analysis. The 1.0-kb region was an integral portion of a larger 1.3-kb repeat sequence which is present as 15 to 20 copies on the P. cepacia AC1100 genome. PCR was performed by melting the target DNA, annealing 24-base oligonucleotide primers to unique sequences flanking the 1.0-kb region, and performing extension reactions with DNA polymerase. After extension, the DNA was again melted, and the procedure was repeated for a total of 25 to 30 cycles. After amplification the reaction mixture was transferred to nylon filters and hybridized against radiolabeled 1.0-kb fragment probe DNA. Amplified target DNA was detectable in samples initially containing as little as 0.3 pg of target. The addition of 20 micrograms of nonspecific DNA isolated from sediment samples did not hinder amplification or detection of the target DNA. The detection of 0.3 pg of target DNA was at least a 10(3)-fold increase in the sensitivity of detecting gene sequences compared with dot-blot analysis of nonamplified samples. PCR performed after bacterial DNA was isolated from sediment samples permitted the detection of as few as 100 cells of P. cepacia AC1100 per 100 g of sediment sample against a background of 10(11) diverse nontarget organisms; that is, P. cepacia AC1100 was positively detected at a concentration of 1 cell per g of sediment. This represented a 10(3)-fold increase in sensitivity compared with nonamplified samples.     

DNA amplification to enhance detection of genetically engineered bacteria in environmental samples

The polymerase chain reaction (PCR) was performed to amplify a 1.0-kilobase (kb) probe-specific region of DNA from the herbicide-degrading bacterium Pseudomonas cepacia AC1100 in order to increase the sensitivity of detecting the organism by dot-blot analysis. The 1.0-kb region was an integral portion of a larger 1.3-kb repeat sequence which is present as 15 to 20 copies on the P. cepacia AC1100 genome. PCR was performed by melting the target DNA, annealing 24-base oligonucleotide primers to unique sequences flanking the 1.0-kb region, and performing extension reactions with DNA polymerase. After extension, the DNA was again melted, and the procedure was repeated for a total of 25 to 30 cycles. After amplification the reaction mixture was transferred to nylon filters and hybridized against radiolabeled 1.0-kb fragment probe DNA. Amplified target DNA was detectable in samples initially containing as little as 0.3 pg of target. The addition of 20 micrograms of nonspecific DNA isolated from sediment samples did not hinder amplification or detection of the target DNA. The detection of 0.3 pg of target DNA was at least a 10(3)-fold increase in the sensitivity of detecting gene sequences compared with dot-blot analysis of nonamplified samples. PCR performed after bacterial DNA was isolated from sediment samples permitted the detection of as few as 100 cells of P. cepacia AC1100 per 100 g of sediment sample against a background of 10(11) diverse nontarget organisms; that is, P. cepacia AC1100 was positively detected at a concentration of 1 cell per g of sediment. This represented a 10(3)-fold increase in sensitivity compared with nonamplified samples.     

Mitochondrial DNA copy number in bovine oocytes and somatic cells

Restriction endonuclease analysis and direct nucleotide sequencing of bovine mitochondrial DNA have revealed a high apparent rate of sequence divergence between maternally related individuals. One possible mechanism that would account for the high rate involves nonuniform amplification and/or segregation of mitochondrial DNA during development of the oocyte. We report here experiments which quantitate the amount of mitochondrial DNA in the bovine oocyte as compared to bovine somatic cells. Total DNA was isolated from purified oocytes, separated by agarose gel electrophoresis, and immobilized on nitrocellulose filters. Hybridization with the complete mitochondrial DNA genome or cloned mitochondrial DNA restriction fragments revealed a 100-fold increase in oocyte mitochondrial DNA as compared to somatic cells. Developing oocytes contained about 4.5 pg or 2.6 × 10⁵ copies per cell, whereas primary bovine tissue culture cells contained 0.045 pg or 2.6 × 10³ copies per cell. These experiments demonstrate directly the amplification of mitochondrial DNA in mammalian oocytes and are consistent with models which could generate mitochondrial DNA polymorphisms by unequal amplification of mitochondrial genomes within an animal.     

Flow cytometric analysis of RNA content in different cell populations using pyronin Y and methyl green

Pyronin Y (PY) was used, in flow cytometric (FCM) systems, to estimate the RNA content per cell in formalin fixed EL4 leukosis tumor cells, enzyme dispersed R3327-G rat prostatic adenocarcinoma cells, mouse spleen cells stimulated with concanavalin A, and human peripheral blood lymphocytes stimulated with phytohemagglutinin. Preincubation of the cells with methyl green (MG) blocked PY binding to DNA such that the intracellular fluorescence from MG-PY was due primarily to its binding to RNA. Treatment of the cells with ribonuclease resulted in a 3- to 5-fold reduction in the fluorescence intensity of intracellular MG-PY. Mitogen stimulation of either mouse or human lymphocytes resulted in an increase in DNA (propidium iodide fluorescence) and RNA (MG-PY fluorescence) content per cell over resting levels. Further, the changes in stimulated human lymphocyte DNA and RNA contents following 24, 48, and 72 hr of cell culture were monitored. The results showed that RNA levels were significantly increased prior to that of DNA. Also, the effects of different cell cycle phase specific blocking agents on lymphocyte cell cycle traverse were investigated. We found that: a) actinomycin D inhibited the increases in cellular RNA and DNA; b) hydroxyurea inhibited the increases in cellular RNA were only slightly reduced; c) tritiated thymidine caused an accumulation of cells having high DNA and RNA contents; and d) Colcemid promoted an accumulation of cells having high DNA contents while causing a reduction of cells having high RNA contents. These results were nearly identical to reports by other investigators using the metachromatic dye acridine orange to quantitate RNA per cell. Thus, the MG-PY technique described is indicated to provide a stable and accurate measure of RNA content per cell.     

Increased nuclear DNA content in developing cotton fiber cells

The nuclear DNA content of developing cotton fiber cells (Gossypium hirsutum, cv. MD51ne) increases ∼24% after 2 d postanthesis (dpa). The amount of nuclear DNA at 2 dpa is 5.4 ± 0.27 pg. At 3–4 dpa it increases to 6.7 ± 0.24 pg and by 5 dpa it is 6.8 ± 0.70 pg. These values were obtained by nuclear fluorescence after staining with Hoechst 33258. Human oral squamous cell nuclei were used as a DNA standard. Nuclear DNA content increases in fibers growing on either fertilized or unfertilized ovules. The increase also is detectable in Feulgen stained nuclei using two-wavelength cytospectrophotometry. All measurements were made on isolated fiber cell nuclei using a newly developed method tailored to cotton fiber cells. The results imply that during the early stages of development fiber cell nuclei either selectively amplify certain sequences or enter S-phase replicating a portion of their genome.     

Measurement of proviral genes in uninfected and avian myeloblastosis virus-infected cells by hybridization with 3H-labeled complementary DNA probe excess.

Viral RNA (vRNA) from avian myeloblastosis virus or DNA from virus-infected and uninfected cells was hybridized with [3H]DNA complementary to viral RNA ([3H]cDNA) under conditions of [3H]cDNA excess. When [3H]cDNA was used to drive the hybridization reaction with vRNA, a rate constant of 33.2 liters/mol-s was obtained. The same rate constant was obtained when vRNA excess was used as the driver. The specific activities of the [3H]DNA probe, estimated from kinetic measurements of the hybridization reaction and from the amount of [3H]cDNA in hybrid form at equilibrium, were 9.1 and 8.6 cpm/pg, respectively. DNA isolated from uninfected cells contained five or six copies of proviral DNA per cell genome. DNA isolated from erythrocytes infected with avian myeloblastosis virus had an additional five or six viral genes added to the cell genome, and the virus-infected target cell (myeloblasts) contained about 15 additional copies of proviral DNA per cell. The use of excess [3H]cDNA probe is an easy and accurate method to quantify the frequency of proviral DNA sequences in cell DNA and to measure a small amount (40 to 200 pg) of vRNA. Probe excess hybridization offers a number of advantages over other procedures and these are discussed.     

Integration of Proviral DNA in Chicken Cells Infected with Schmidt-Ruppin Rous Sarcoma Virus Is Not Enhanced by DNA Repair

The effect DNA repair might have on the integration of exogenous proviral DNA into host cell DNA was investigated by comparing the efficiency of proviral DNA integration in normal chicken embryonic fibroblasts and in chicken embryonic fibroblasts treated with UV or 4-nitroquinoline-1-oxide. The cells were treated with UV or 4-nitroquinoline-1-oxide at various time intervals ranging from 6 h before to 24 h after infection with Schmidt-Ruppin strain A of Rous sarcoma virus. The chicken embryonic fibroblasts were subsequently cultured for 18 to 21 days to ensure maximal integration and elimination of nonintegrated exogenous proviral DNA before DNA was extracted. Integration of proviral DNA into the cellular genome was quantitated by hybridization of denatured cellular DNA on filters with an excess of (3)H-labeled 35S viral RNA. The copy number of the integrated proviruses in normal cells and in infected cells was also determined from the kinetics of liquid RNA-DNA hybridization in DNA excess. Both RNA excess and DNA excess methods of hybridization indicate that two to three copies of the endogenous provirus appear to be present per haploid normal chicken cell genome and that two to three copies of the provirus of Schmidt-Ruppin strain A of Rous sarcoma virus become integrated per haploid cell genome after infection. The copy number of viral genome equivalents integrated per cell treated with UV or 4-nitroquinoline-1-oxide at different time intervals before or after infection did not differ from the copy number in untreated but infected cells. This finding supports our previous report that the integration of oncornavirus proviral DNA is restricted to specific sites in the host cell DNA and suggests a specific mechanism for integration.     

Nuclear DNA content and the control of chloroplast replication in wheat leaves

During development of the first leaf of breadwheat (Triticum aestivum L.) the number of chloroplasts per mesophyll cell increases between three- and four-fold. To establish if chloroplast replication is accompanied by endoreduplication, the nuclear DNA content of the cells was determined by chemical assay of isolated nuclei from mesophyll protoplasts and by microdensitometry of nuclei in mesophyll tissue. The DNA content of the nuclei was constant (27 to 32 pg) at each phase of chloroplast replication. Approximately 93% of the cells had a nuclear DNA content close to the 2C value of 32 pg. It is concluded that chloroplast replication is not dependent on nuclear endoreduplication in seedling leaves of wheat.     

Temporal relationships between DNA metabolism and the growth-inhibitory response produced by dexamethasone in rat glioma cell cultures

Summary The temporal relationships between aspects of DNA metabolism and the suppression of cell proliferation were investigated in rat glioma (strain C6) monolayer cultures exposed to 10μM dexamethasone. Cell densities (cell number per cm²), rates of DNA synthesis (dpm of [³H]thymidine incorporated per μg DNA per min), and cellular DNA (μg DNA per cm²) were measured daily in control and dexamethasone-treated cultures over a 3-day period. The percentage of cells in metaphase and the proportion of metaphases containing >2n(42) chromosomes also were determined in control and treated cultures. When log-phase C6 cultures were exposed to dexamethasone (day 0), cell densities were not significantly different from controls by day 1. Cell proliferation ceased thereafter in dexamethasone-treated cultures, whereas control cell populations continued to proliferate at log-phaserates. In contrast, cellular DNA increased exponentially in control and treated cultures over the 3-day period. On days 0 and 1, control and treated cells each contained 6 pg DNA. By day 3, the DNA content per treated cell increased to >20 pg; control cells each contained 10 pg DNA. The rates of DNA synthesis in the treated cultures did not differ significantly from controls on days 1 and 2. However, the rate in the treated cultures decreased significantly on day 3, one day after cell proliferation ceased. On day 2, the percentage of cells found in metaphase in the treated cultures was 0.32% compared to 0.64% in control cultures. By day 3, these percentages decreased to 0.20% and 0.22%, respectively. However, the proportion of metaphases containing >42 chromosomes increased 1.5-fold in the treated cultures relative to controls. These results indicate that nonproliferating dexamethasone-treated cells contain elevated amounts of DNA. Thus dexamethasone action appears to arrest the cell cycle at any point between the completion of DNA replication and mitosis. A preliminary report of this work was presented on June 8, 1977, at the 28th Annual Meeting of the Tissue Culture Association in New Orleans, Louisiana. This investigation was supported in part by grants from Merck Sharp & Dohme Research Laboratories, West Point, Pa., the American Cancer Society (IN-113), and NIH (AM 18719).     

Direct measurement of femtogram amounts of DNA in cells and chloroplasts by quantitative microspectrofluorometry

Absolute DNA amounts of individual chloroplasts were determined by measuring the fluorescence intensity of chloroplasts stained with 4',6-diamidino-2-phenylindole (DAPI) relative to that of the bacterium Pediococcus damnosus (cerevisiae) smeared on the same slide. An absolute DNA content of 7.7 X 10(15) g for a standard P. damnosus cell type was calculated by comparing the relative fluorescence values and frequency of each stage of cellular development in a culture to the average DNA content of all cell types determined by chemical methods. Chlorophyll was extracted from the chloroplasts during fixation so that chlorophyll autofluorescence was not present when DAPI fluorescence was measured. Absolute amounts of DNA could then be determined for single chloroplasts, either within cells that were individually selected from a mixed cell population or in small preparations of isolated chloroplasts. The DNA amounts of chloroplasts from mesophyll cells determined in this way were similar to the values previously determined by bulk averaging methods. Chloroplast DNA amounts from different cell types of the leaf could be measured by microspectrofluorometry, and it was found that chloroplasts from spinach epidermal cells contained about half as much DNA as chloroplasts from adjacent mesophyll cells.     

Ultramicrochemical determination of nucleic acids in individual cells using the Zeiss UMSP-I microspectrophotometer. Application to isolated rat hepatocytes of different ploidy classes

Synopsis Edström's method for the ultramicrochemical determination of RNA and DNA in individual cells was modified for the measurement of extinction in u.v. light with the aid of the Zeiss scanning microspectrophotometer UMSP-I. With this new procedure, nucleic acids down to about 3 pg RNA or about 4 pg DNA can be measured with a very high accuracy.The method was applied to enzymatically isolated rat liver parenchymal cells. A mean DNA content of 6.52 pg was found for diploid cells. The DNA content of mononuclear cells of different ploidy levels and of binuclear cells showed a close proportionality with the nuclear ploidy and the number of nuclei per cell. The RNA content of mononuclear diploid cells amounted to 33.4 pg, yielding an RNA/DNA ratio of 5.12. The RNA/DNA ratio was similar for binuclear and mononuclear cells of the same ploidy level but decreased considerably with increasing nuclear ploidy.     

Measurement of cytoplasmic pH in Dictyostelium discoideum by using a new method for introducing macromolecules into living cells

We have developed a novel method for introducing exogenous macromolecules from solution into the cytoplasm of living amoebae of the cellular slime mold Dictyostelium discoideum and have used it to measure the cytoplasmic pH of these cells. Amoebae (strain NC-4) were loaded with fluorescein-labelled dextran by sonication in a solution containing 17 mM phosphate buffer, 1 mM CaCl2, and 10 mg/ml of fluorescein-labelled dextran, pH 6.1. The recovery of living cells was approximately 40% after sonication and washing. A significant fraction (10%) of the recovered cells were loaded and contained 10(5) to 10(7) molecules of fluorescein-labelled dextran per cell as assessed by flow cytometry. The cells loaded by sonication appeared both viable and healthy, since they exhibited normal morphology and locomotion, could differentiate to form mature fruiting bodies, could chemotax in a gradient of extracellular cAMP, and could endocytose latex microspheres. The pH of single cells was estimated by using flow cytometry to measure the fluorescence ratio (fluorescein/rhodamine) in cells loaded with a mixture of the two fluorochrome-labelled dextrans. The fluorescence ratios were calibrated in situ with the flow cytometer after treatment of the cells with either weak acid or weak base to clamp the internal pH at known values. The intracellular pH measured in cells loaded with dextran in a simple salt solution was 5.9. The intracellular pH measured in cells loaded with dextran in the same solution supplemented with amino acids and glucose was 6.7. The novel sonication loading technique described may have general utility for loading diverse types of macromolecules into suspensions of living cells.     

Estimation of nuclear DNA content of various bamboo and rattan species

We determined the nuclear DNA content (genome size) of over 35 accessions each of bamboo and rattan species from Southeast Asia. The 2C DNA per nucleus was quantified by flow cytometry. The fluorescence of nuclei isolated from the leaves and stained with propidium iodide was measured. The genome size of the bamboo species examined was between 2.5 and 5.9 pg DNA per 2C nucleus. The genome size of the rattan species examined ranged from 1.8 to 10.5 pg DNA per 2C nucleus. This information will be useful for scientists working in diverse areas of plant biology such as biotechnology, biodiversity, genome analysis, plant breeding, physiology and molecular biology. Such data may be utilized to attempt to correlate the genome size with the ploidy status of bamboo species in cases where ploidy status has been reported.     

Cytofluorometric quantitation of 5-hydroxytryptamine in mast cells: an improved technique for the formaldehyde condensation reaction

A simple technique for the condensation of cellular 5-hydroxytryptamine (5-HT) with formaldehyde gas is described. The technique, which is especially suited for quantitative cytofluormetric studies, involves the generation of formaldehyde gas from dry paraformaldehyde in a closed reaction vessel with the addition of a measured quantity of water. The fluorescence yield of 5-HT was tested at various humidities. Optimal results were obtained with the addition of 100 mg water to a 1000 ml reaction vessel containing 6 g of dry paraformaldehyde. A major advantage of the method if the fact that the humidity during the reaction can be precisely controlled. The fluorescence yield of 5-HT, tested over a 50 day period showed excellent reproducibility. The stoichiometry of the reaction was tested by comparison of cytofluormetic data with that obtained by analysing the 5-HT content of pooled mast cells with an independent biochemical method. A highly satisfactory correlation (r = 0.96) was obtained within the range of 0.1 to 4 pg of 5-HT per cell. The limit of sensitivity of the cytofluorometric method was found to be of the order of 10(-13) g, and was determined by the fluorescence blank of the mast cells. This contributes to between 10 and 30 per cent of the total fluorescence emission from mast cells containing about 0.2 pg of 5-HT.     

A new method for rapid and sensitive detection of bromodeoxyuridine in DNA-replicating cells

A new flow cytometric technique, involving differential fluorescence analysis of two DNA-binding fluorochromes, was used to quantify cellular incorporation of the base analog, bromodeoxyuridine (BrdU), into DNA over short time periods. During analysis of stained cells, the blue fluorescence signal of Hoechst 33342, which is quenched by BrdU-substituted DNA, was subtracted, on a cell by cell basis, from the green-yellow fluorescence signal of mithramycin, which remained stoichiometric to cellular DNA content. Bivariate contour profiles obtained for CHO cells pulse-labeled for 30 min showed that fluorescence quenching of Hoechst 33342 in BrdU-labeled, S phase cells produced fluorescence difference signals that were significantly greater than the difference signals from G1 and G2 + M phase cells. Analysis of L1210 cells demonstrated that the amount of BrdU detected was proportional to the length of the labeling period. The novel technique is simple, rapid, and mild; it produces minimal cell loss and does not significantly affect cellular moieties such as DNA, chromatin, or RNA.     

Isolation and some chemical properties of oligodendroglia from calf brain

Abstract— The method of Norton and Poduslo (1970) for isolating brain cells has been adapted for the isolation of oligodendroglia from the white matter of calf brain. The cells were obtained in greater than 90 per cent purity, and in a yield of 11 × 10⁶ cells/g of white matter. This number of cells represented a recovery of 11 per cent of the total cells in the tissue and therefore a considerably higher recovery of the original number of oligodendroglia. The average cell contained 5, 2 pg of DNA, 2–0 pg of RNA and 6, 7 pg of lipid. The lipid comprised cholesterol, galactolipid (both cerebroside and sulphatide) and phospholipid in the molar ratio of 1:0, 45:2, 3. Gangliosides were present in a concentration similar to that found in isolated rat neurons, The myelin-specific enzyme, 2′, 3′-cyclic nucleotide 3′-phosphohydrolase, was present at a level nearly equal to that in myelin, and eight-fold higher than the levels in rat neurons or astrocytes. The isolated oligodendroglia differed considerably from isolated astrocytes in size, morphology and chemical composition.     

Multivariate flow cytometry of epidermal regeneration provoked by a skin irritant and a tumor promoter.

The DNA content and the changes in cellular and nuclear size of isolated regenerating mouse epidermal basal cells were studied after topical application of the skin irritant cantharidin and the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) to the back skin of hairless mice. The DNA and protein contents of isolated basal cells were stained with propidium iodide and fluorescein isothiocyanate, respectively, and analysed by flow cytometry using the total protein fluorescence as an estimate of cell size and the DNA fluorescence pulse width as an estimate of nuclear size. Transmission electron microscopy was used to identify cells sorted from regions in the bivariate DNA/protein distributions. The results showed that both chemicals induced an increase in cellular as well as nuclear size of the basal cells. The increase in size was higher in TPA treated than in cantharidin treated animals, and the bivariate DNA/protein distributions of TPA treated cells differed from those of cantharidin treated cells in that two subpopulations of cycling keratinocytes could be identified. These deviations are probably related to the higher proliferative response observed after TPA treatment and the possibility that proliferative subpopulations in epidermis respond differently to TPA. It may reflect mechanisms providing for a growth advantage of initiated cells, important in tumor promotion. About 8% of the cells in the suspensions from treated animals were non-cycling non-keratinocytes, probably infiltrating leukocytes. The results indicate a strong correlation between rapid regenerative cell cycle progression, i.e., reduced G1 transit time and increased cellular and nuclear size.(ABSTRACT TRUNCATED AT 250 WORDS)