Molecular approach to the characterisation of fungal communities: methods for DNA extraction, PCR amplification and DGGE analysis of painted art objects

A protocol for efficient extraction of fungal DNA from micromycetes colonising painted art objects was developed. Polymerase chain reaction (PCR) inhibitors were successfully removed by a combined application of a Chelex-100 adsorption resin and a Geneclean Kit for Ancient DNA. Universal fungal primers for PCR amplification of 28S rDNA (U1 and U2) were tested for their applicability in denaturing gradient gel electrophoresis (DGGE) analysis of fungal communities. Artificially produced mortar samples inoculated with fungal pure cultures isolated from mural paintings were used as model objects for DNA extractions and DGGE analysis. Good resolution in DGGE was achieved using 260-bp rDNA fragments amplified with U1/DGGE and U2 primers directly from model communities.     

Autoschizis: a new form of cell death for human ovarian carcinoma cells following ascorbate:menadione treatment. Nuclear and DNA degradation

Microscopic aspects, densitometric evaluation of Feulgen-stained DNA, and gel electrophoresis of total DNA have been used to elucidate the effects of 1, 2, and 3 h VC (ascorbic acid), VK3 (menadione), and combined VC:VK3 treatments on the cellular and nuclear morphology and DNA content of a human ovarian carcinoma cell line (MDAH 2774). Optical densitometry showed a significant decrease in cancer cell DNA content directly related to VC and VC:VK3 treatments while VK3 and VC:VK3 treated cells exhibited cytoskeletal changes that included self-excision of cytoplasmic pieces with no membranous organelles. Nuclei decreased in size and exhibited poor contrast consistent with progressive decondensation of their chromatin. Degraded chromatin was also detected in cytoplasmic autophagosomes. Nucleoli segregated their components and fragmented into small pieces. Gel electrophoretic analysis of total DNA revealed evidence of generalized DNA degradation specific to treated tumor cells. These results are consistent with previous observations [Scanning 20 (1998a) 564; Ultrastruct. Pathol. 25 (2001b) 183; J. Histochem. Cytochem. 49 (2001) 109] which demonstrated that the VC:VK3 combination induced autoschizic cell death by a series of cytoplasmic excisions without organelles along with specific nuclear ultrastructural damage.     

Preprophase bands of microtubules and the cell cycle: Kinetics and experimental uncoupling of their formation from the nuclear cycle in onion root-tip cells

We have studied the timing of preprophase band (PPB) development in the division cycle of onion (Allium cepa L.) root-tip cells by combinations of immunofluorescence microscopy of microtubules, microspectrophotometry of nuclear DNA, and autoradiography of [³H]thymidine incorporation during pulse-chase experiments. In normally grown onion root tips, every cell with a PPB had the G2 level of nuclear DNA. Some were in interphase, prior to chromatin condensation, and some had varying degrees of chromatin condensation, up to the stage of prophase at which the PPB-prophase spindle transition occurs. In addition, autoradiography showed that PPBs can be formed in cells which have just finished their S phase, and microspectrophotometry enabled us to detect a population of cells in G2 which had no PPBs, these presumably including cells which had left the division cycle. The effects of inhibitors of DNA synthesis showed that the formation of PPBs is not fully coupled to events of the nuclear cycle. Although the mitotic index decreased 6-10-fold to less than 0.5% when roots were kept in 20 g·ml^-1 aphidicolin for more than 8 h, the percentage of cells containing PPBs did not decrease in proportion: the number of cells in interphase with PPBs increased while the number in prophase decreased. Almost the same phenomena were observed in the presence of 100 g·ml^-1 5-aminouracil and 40 g·ml^-1 hydroxyurea. In controls, all cells with PPBs were in G2 or prophase, but in the presence of aphidicolin, 5-aminouracil or hydroxyurea, some of the interphase cells with PPBs were in the S phase or even in the G1 phase. We conclude that PPB formation normally occurs in G2 (in at least some cases very early in G2) and that this timing can be experimentally uncoupled from the timing of DNA duplication in the cell-division cycle. The result accords with other evidence indicating that the cytoplasmic events of cytokinesis are controlled in parallel to the nuclear cycle, rather than in an obligatorily coupled sequence.Abbreviations APC aphidicolin - 5-AU 5-aminouracil - DAPI 4, 6-diamidino-2phenylindole - HU hydroxyurea - MI mitotic index - MT microtubule - PMSF phenylmethyl-sulfonyl fluoride - PPB preprophase band - %PPB percentage of cells with PPBs