In situ PCR technique based on pricking microinjection for cDNA cloning in single cells of barley coleoptile and powdery mildew pathogen

To clone cDNAs of mRNA specifically expressed at the infection sites, we applied the polymerase chain reaction (PCR) combined with pricking microinjection to barley coleoptile epidermis inoculated with powdery mildew pathogen. In essence, first-strand cDNAs were synthesized in situ the needle-pricked epidermal cells in which fungal haustoria had formed, and were subsequently amplified by PCR with synthetic primers. The amplified DNAs were subcloned into a plasmid vector for the construction of a cDNA library. The antisense RNAs were in vitro-transcribed from subcloned DNAs, labelled, and introduced into pathogen-invaded coleoptile epidermal cells by pricking microinjection. Target cell-specific cDNAs were identified by a specific in situ hybridization in the pathogen-invaded cells. This technique was also applied to the amplification and identification of cDNAs which were reverse-transcribed from mRNAs of targeted infection structures of the powdery mildew pathogens inoculated onto barley coleoptile epidermis.     

Cadmium induced apoptotic changes in chromatin structure and subphases of nuclear growth during the cell cycle in CHO cells

CHO cells were grown in the presence of 1 M CdCl₂ and subjected to ATP-dependent replicative DNA synthesis after permeabilization. By decreasing the density of the cell culture replicative DNA synthesis was diminishing. At higher than 2 × 10⁶ cell/ml concentration Cd had virtually no effect on the rate of DNA replication. Growth at higher cell concentrations could be supressed by increasing Cd concentration. After Cd treatment cells were synchronized by counterflow centrifugal elutriation. Cadmium toxicity on cell growth in early and mid S phase led to the accumulation of enlarged cells in late S phase. Flow cytometry showed increased cellular and nuclear sizes after Cd treatment. As the cells progressed through the S phase, 11 subpopulations of nuclear sizes were distinguished. Apoptotic chromatin changes were visualized by fluorescent microscopy in a cell cycle dependent manner. In the control untreated cells the main transitory forms of chromatin corresponded to those we have published earlier (veil-like, supercoiled chromatin, fibrous, ribboned structures, chromatin strings, elongated prechromosomes, precondensed chromosomes). Cadmium treatment caused: (a) the absence of decondensed veil-like structures and premature chromatin condensation in the form of apoptotic bodies in early S phase (2.2–2.4 average C-value), (b) the absence of fibrous structures, the lack of supercoiled chromatin, the appearance of uncoiled ribboned chromatin and perichromatin semicircles, in early mid S phase (2.5–2.9 C), (c) the presence of perichromatin fibrils and chromatin bodies in mid S phase (2.9–3.2 C), (d) early intra-nuclear inclusions, elongated forms of premature chromosomes, the extrusion and rupture of nuclear membrane later in mid S phase (3.3–3.4 C), (e) the exclusion of chromatin bodies and the formation of clusters of large-sized perichromatin granules in late S phase (3.5–3.8 C) and (f) large extensive disruptions and holes in the nuclear membrane and the clumping of incompletely folded chromosomes (3.8–4. C).