Simultaneous localization and quantification of relative G and F actin content: optimization of fluorescence labeling methods.

Previous studies of fluorescence probes for labeling the monomeric actin pool have demonstrated lack of specificity. We have used quantitative analytical methods to assess the sensitivity and specificity of rhodamine DNAse I as a probe for monomeric (G) actin. The G-actin pool of attached or suspended fibroblasts was stabilized by ice-cold glycerol and MgCl2. Formaldehyde fixation was used to clamp the filamentous (F) actin pool. G- and F-actins were stained by rhodamine DNAse I and FITC-phalloidin, respectively. Confocal microscopy indicated that the G- and F-actins were spatially separate in substrate-attached cells. Flow cytometry and fluorescence spectrophotometry demonstrated low co-labeling of the separate actin pools, although measureable background binding of rhodamine DNAse I was detectable. Estimates of the extent of actin polymerization after trypsinization demonstrated reciprocal changes of monomeric and filamentous actins, consistent with the formation of a perinuclear array of F-actin. The labeling and quantitation methods were also sufficiently sensitive to detect cell type-dependent variations in actin content. Dual labeling of cells with rhodamine DNAse I and FITC-phalloidin may provide a simple and direct method to image and quantify actin rearrangement in individual cells.     

In situ DNAse I sensitivity assay indicates DNA conformation differences between CHO cells and the radiation-sensitive CHO mutant IRS-20

The radiosensitive mutant cell line IRS-20, its wild type counterpart CHO and a derivative of IRS-20 with a transfected YAC clone (YAC-IRS) that restores radioresistance were tested for DNAse I sensitivity. The three cell lines were cultured under the same conditions and had a mitotic index of 2-5%. One drop of fixed cells from the three lines was always spread on the same microscopic slide. After one day of ageing, slides were exposed to DNAse I and stained with DAPI. Images from every field were captured and the intensity of blue fluorescence was measured with appropriate software. For untreated cells, the fluorescence intensity was similar for all of the cell lines. After DNAse I treatment, CHO and YAC-IRS had an intensity of 85% but IRS-20 had an intensity of 60%, when compared with the controls. DNAse I sensitivity differences between the cell lines indicate that overall conformation of chromatin might contribute to radiation sensitivity of the IRS-20 cells.     

Nuclear architecture,intranuclear DNA distribution,and nuclease digestion

G0, G1, and mammalian cells and nuclei were shortly digested with either micrococcal nuclease or DNAse I, both before and after mild fixation, either before (G0) or after (G1) partial hepatectomy. Cells were Feulgen stained and examined by high resolution light microscopy. In metabolically active G1 nuclei, intranuclear DNA appears organized at least in two distinct domains, whereby, the highly dispersed one is large enough to be detected at the resolution of the light microscope and appears preferentially attacked by limited DNAse I digestion. The action of the enzyme is readily apparent only in the nuclei that are first digested and then fixed. Spectroscopic characterization of the same nuclei reveals that the fixation causes a sizeable removal of proteins, mostly in the soluble chromatin subfraction. Results are discussed in terms of two control levels for gene expression and for higher order DNA structure.     

Nuclear architecture, intranuclear DNA distribution, and nuclease digestion

G0, G1, and mammalian cells and nuclei were shortly digested with either micrococcal nuclease or DNAse I, both before and after mild fixation, either before (G0) or after (G1) partial hepatectomy. Cells were Feulgen stained and examined by high resolution light microscopy. In metabolically active G1 nuclei, intranuclear DNA appears organized at least in two distinct domains, whereby the highly dispersed one is large enough to be detected at the resolution of the light microscope and appears preferentially attacked by limited DNAse I digestion. The action of the enzyme is readily apparent only in the nuclei that are first digested and then fixed. Spectroscopic characterization of the same nuclei reveals that the fixation causes a sizeable removal of proteins, mostly in the soluble chromatin subfraction. Results are discussed in terms of two control levels for gene expression and for higher order DNA structure.     

A highly reproducible method of nucleolus organizing regions staining in plants

A method for the specific detection of the nucleolus organizing regions (NORs) of plant chromosomes has been developed employing enzymatic maceration and successive flame-drying for chromosome spreading and incubation with aqueous 50% AgNO3 at 55-60 C. When this method was applied to metaphase chromosomes the NORs were specifically discriminated as heavily stained segments in all the plant species examined. In the satisfactory results obtained by monitoring the reaction under a microscope during the course of the silver treatment, the chromosome arms were stained yellow to light brown while the NORs were dark brown to black. The present method has the advantage of yielding highly reproducible results for the specific detection of the NORs in plant materials.     

Lack of correspondence between CMA3-, Ag-positive signals and 28S rDNA loci in two Iberian minnows (Teleostei, Cyprinidae) evidenced by sequential banding

Despite the growing outcome of results that put doubt upon the reliability of silver (Ag) staining and chromomycin A3 (CMA3) fluorescent banding in the detection of major ribosomal gene sites (NORs), these methods have been widely used, especially in fishes. In order to clarify the previous patterns obtained with those techniques, we performed fluorescence in situ hybridisation (FISH) with 28S rDNA probe followed by sequential CMA3 and Ag staining in diploid non-hybrid males of the Squalius alburnoides complex and in Squalius pyrenaicus. The results from all the studied specimens revealed a lack of correlation between classical and molecular techniques. Not just some other regions besides NORs were stained with CMA3 and Ag, but also the majority of the 28S rDNA sites were not detected. Care should then be taken in considering CMA3- and Ag-stained sites as NORs since their accuracy for that purpose may not always correspond to the expectations.     

Inhibition of DNAse I activity in vivo]

Immune gamma-globulins containing antibodies to bovine DNAse I inhibit activities of bovine and mouse DNAse I both in vitro and in vivo. Bovine DNAse I was used as exogenous DNAse I in the in vivo studies and was injected to mice intraperitoneally in combination with gamma-globulins. The serous fluid of mice was used as a source of endogenous DNAse I. Both in in vitro and in vivo studies immune gamma-globulins caused a practically complete inhibition of bovine DNAse I activity, whereas the activity of mouse DNAse I (endogenous) was inhibited by 60-80%. Nonimmune gamma-globulins had no inhibitory effects whatsoever.     

Intercellular NOR-AG variability in man

Summary A new modification of the Ag I technique has been developed using human cultured blood lymphocytes, which involves ultra-violet irradiation of chromosome preparations during incubation in AgNO₃. This technique enables detection in a short incubation time all NORs capable of being stained with silver. A peculiar morphological change in Ag-stainable NORs during the incubation is described, which can be used as a criterion of the completion of Ag staining. With the refined Ag-staining procedure, acrocentric marker chromosomes were studied which showed one or two satellite stalks within the same individual. Ag staining was highly coincident with this variability.     

In situ nick translation at the electron microscopic level: a tool for studying the location of DNAse I-sensitive regions within the cell

The in situ nick translation method was adapted to the ultrastructural level, to study the location of DNAse I-sensitive sequences within the cell. Ultra-thin sections of Lowicryl-embedded cells were incubated in a medium containing DNAse I, DNA polymerase I, and all four deoxyribonucleotides, some being biotinylated. The nick-translated sites were then visualized by an indirect immunogold labeling technique. The resulting labeling pattern is closely dependent on the DNAse I concentration in the nick-translation medium. The method reveals with great precision the specific DNAse I-sensitive regions within the nucleus. This technique can be used to discriminate between active and inactive regions of interphase chromatin.     

The histological localization of DNA in rabbit medullary neurons

The nuclei of unfixed isolated rabbit neurons cleared on incubation with DNAse (10 mg/ml), but not RNAse (10 mg/ml). The nuclei stained for DNA with eight chromosomal or nuclear stains more intensely than the cytoplasm, and less intensely after treatment with DNAse (10 mg/ml). On the other hand, when the whole tissue was embedded and sectioned, DNA did not appear to be stained in the nucleus; the nucleolus and the cytoplasm were more heavily stained than the nucleoplasm. Possible explanations for this apparent anomaly are considered. It was concluded that DNA diffused out of the nucleus during embedding and sectioning, and that the colouration of the nucleolus and cytoplasm with the eight staining systems used was due to other nucleotides present.     

DNase I hypersensitivity in the gamma globin gene locus of K562 cells.

We have probed the chromatin conformation of the G gamma-A gamma-delta-beta globin gene locus of K562 cells, a human hematopoietic cell line, with the enzyme pancreatic DNAse I. This enzyme preferentially digests genes in an active configuration. We have found that in K562 cells, which produce embryonic and fetal but not adult hemoglobins, both the active gamma and inactive beta genes are DNAse I sensitive. However, only the active gamma genes have DNAse I hypersensitive regions. The hypersensitive regions have been mapped to an area approximately 100 base pairs 5' to the G gamma and A gamma genes.     

Increased susceptibility of activated rat liver chromatin to DNAse I

Rat liver chromatin activated by partial hepatectomy is more susceptible to the action of DNAse I than control chromatin isolated from intact liver. The study on the transfer of chromatin material to the acid-soluble fraction reveals a higher rate of activated chromatin degradation. Activated chromatin shows also an increased capacity for ethidium bromide (EB) binding as estimated from the isotherms of adsorption. The difference in EB binding between activated and control chromatin is abolished after DNAse I treatment. Conditions of mild digestion with DNAse I have been found under which the number of binding sites for EB per nucleotide decreases to almost the same level in activated and non-activated chromatin. The results suggest a preferential degradation of those DNA sequences in activated chromatin that are responsible for the increase in the ligand binding.     

Cytogenetics and cladistics

Chromosomal data have been underutilized in phylogenetic investigations despite the obvious potential that cytogenetic studies have to reveal both structural and functional homologies among taxa. In large part this is associated with difficulties in scoring conventional and molecular cytogenetic information for phylogenetic analysis. The manner in which chromosomal data have been used by most authors in the past was often conceptionally flawed in terms of the methods and principles underpinning modern cladistics. We present herein a review of the different methods employed, examine their relative strengths, and then outline a simple approach that considers the chromosomal change as the character, and its presence or absence the character state. We test this using one simulated and several empirical data sets. Features that are unique to cytogenetic investigations, including B-chromosomes, heterochromatic additions/deletions, and the location and number of nucleolar organizer regions (NORs), as well as the weighting of chromosomal characters, are critically discussed with regard to their suitability for phylogenetic reconstruction. We conclude that each of these classes of data have inherent problems that limit their usefulness in phylogenetic analyses and in most of these instances, inclusion should be subject to rigorous appraisal that addresses the criterion of unequivocal homology.     

Nuclease-induced DNA structural changes assessed by flow cytometry with the intercalating dye propidium iodide

A flow cytometric analysis of DNA structural changes induced by cleavage with nucleases was performed on isolated HeLa nuclei by assessing changes in stainability with the DNA-specific fluorochrome propidium iodide (PI). After mild digestion with DNAse I, micrococcal nuclease, or with the single-strand-specific S1 and Neurospora crassa nucleases, fluorescence intensity of nuclei stained with PI increased by about 15-30% above the value of undigested control samples. No significant modifications were observed with the restriction enzymes Eco RI, Alu I, and Not I. The DNAse I-induced increase in fluorescence intensity was also observed with the non-intercalating dye Hoechst 33258, but not with mithramycin. Nuclease-induced fluorescence intensity changes as determined with PI were found to be dependent on the dye concentration. A constant increase (about 20%) was measured at dye/DNA-P ratios greater than 0.11. Below this value (2 micrograms/ml PI), the fluorescence intensity of digested samples was 15-30% lower than that of undigested controls. This behaviour towards intercalating dyes is similar to that of the relaxed (nicked) vs. the supercoiled (intact) form of circular DNA. These results suggest that conformation- but not sequence-specific nucleases induce a relaxation of DNA supercoils.     

Patterns of silver staining in cells of six-day blastocyst and kidney fibroblast of the domestic rabbit

The distribution pattern of silver-NORs was studied in cells of six-day blastocysts and kidney fibroblasts of the rabbit using the Ag-AS technique. At metaphase and interphase there was a binomial distribution of the number of stained sites in both populations but blastocysts had a greater percentage of cells with larger numbers of stained sites. Up to 7 of the 8 chromosomes known to bear NORs were stained in cells from blastocysts while a maximum of 6 were stained in fibroblasts. A significant difference was found between the mean numbers of chromosomal NORs per cell in metaphases from blastocysts and fibroblasts, where they were 4.2 and 3.3 respectively. Similarly, the mean number of NORs in interphase was significantly greater in cells from blastocysts. The distribution of staining on chromosome pair 13 was related to cell type. Significantly more cells in blastocysts than fibroblasts showed staining in this chromosome pair.     

Localization of chromosomal DNA sequences homologous to ribosomal gene type I insertion DNA in Drosophila melanogaster

Summary Chromosomal sites which have DNA homology to the 1 kb (kilobase pair) BamHI restrictable fragment of the 5 kb type I insertion present in many ribosomal genes in Drosophila melanogaster, were identified by using in situ hybridization and autoradiography. XX and XY complements of polytene chromosomes showed the nucleolus and chromocenter to be heavily labeled. Of the light label over euchromatic regions, the 102C band of chromosome 4 labeled particularly intensely. In mitotic XX and XY complements, the NORs (nucleolus organizer regions) of both sex chromosomes labeled as did the centromeric heterochromatin of autosomes. Label also appeared less frequently over telomeric and euchromatic regions.     

Nucleolar organizer regions (NORs) in Chinese hamster chromosomes as visualized by Coomasie brilliant blue

Nucleolar oragnizer regions (NORs) of Chinese hamster chromosomes have been demonstrated by using a Coomasie brilliant blue dye (CBB) method. The staining procedure involved is simple and the results are reproducible. The staining process is easily controllable because over-staining of the chromosomes seldom occurs. The CBB solution is stable (pH 3) and can be used for many days at room temperature. Contrary to the silver technique, the stained material in the NORs is resistant to acid extraction. Since it is sensitive to trypsin treatment, it is suggested that the CBB stained material is protein in nature.     

Inhibition of muscle tropomyosin polymerization by DNAse I

Tropomyosin polymerization is inhibited by DNAse I, an endonuclease which also interacts with G-actin. A 1:4 molar ratio of DNAse I to adult chicken pectoralis muscle tropomyosin almost completely prevents the increased viscosity of tropomyosin under polymerizing ionic conditions. While G-actin binding to DNAse I inhibits the DNAse I hydrolysis of DNA, tropomyosin does not affect this enzymatic activity. G-actin-DNAse I interaction is also not altered by tropomyosin.     

DNA stainability with base-specific fluorochromes: dependence on the DNA topology in situ

The influence of DNA topology on stainability with the externally binding fluorochromes Hoechst 33258 (HO) and mithramycin (MI) was investigated in HeLa nuclei in comparison with the intercalating dye propidium iodide (PI). Changes in DNA topology were induced with a mild DNAse I treatment. Stainability properties of untreated and nuclease-treated nuclei were compared with those of the supercoiled-circular and the relaxed-linear forms of the plasmid pBR322. DNAse-treated nuclei stained with HO showed a higher fluorescence intensity than control samples, independently of the dye concentration, in contrast with the findings obtained with PI. Similar behaviour was observed with the relaxed-linear form of pBR322, compared with the supercoiled-circular molecule. With MI, the stainability of HeLa nuclei did not depend on the DNA topology, whereas the stainability of the plasmid was similar to that of HO. In order to assess whether this discrepancy depended on differences in the availability of DNAse-sensitive sites to the fluorochromes, fluorescence resonance energy transfer (FRET) studies were performed in nuclei stained with HO+PI, or with HO+MI dye pairs. After DNAse I digestion, the relative FRET efficiency between donor (HO) and acceptor molecules (PI or MI) was reduced significantly only when MI was the acceptor. This result may be due to greater stainability of DNAse-sensitive sites with HO than with MI. These findings indicate that DNA stainability with base-specific fluorochromes may be affected by the topology of chromatin regions.