Carbon fiber nanoelectrode(tip diameter ca. 100 nm)-patch clamp was firstly applied to real-time monitoring dopamine release from single living vesicles of single Rat pheochromocytoma(PC12) cells with highly temporal and spatial resolution. It is first demonstrated that there are continual vesicle releases of dopamine at the same site in the active release zone, which play a main role in the exocytosis.
DNA uptake of Saccharomyces cerevisiae known as genetic transformation wasfirstly described by Gppe-noorth in 1960, and now the most commonly used efficient protocol foryeast transformation makes use of PEG and Li^+, which works well for most laboratory strains and issuitable for high-efficiency transformation of plasmid DNA. However, it is still unknown how plasmidDNA enters yeast cells and what roles Li^+ and PEG play on DNA uptake in yeast cells until now. Theelucidation of the manner in which plasmid DNA enters the intact yeast cell is beneficial foroptimization of the method of yeast transformation, and more important for general understanding ofthe processes of macromolecule uptake by cells, especially eukaryotic cells and the mechanism ofhorizontal gene transfer (HGT) in eukaryotic organisms. Previous study^(151) has supposed that DNAuptake of bacteria could be divided into three steps: DNA binding to cell surfaces, penetratingthrough cell wall/membrane into the interior of cells, and then replicating or transcripting to RNAin the cells. To investigate the relationship between DNA binding to cell surfaces and DNAtransformed into yeast cells, fluorescence microscopy was used to show the binding of pUC18 DNAlabeled with the cell-impermeable YOYO-1 to cell surfaces, and the transformation frequencies ofpAJ161 DNA in yeast cells were calculated. PEG was indispensable for the binding of plasmid DNA tothe surfaces of intact yeast cells and protoplasts (the cell wall-removed yeast cells). Without PEG,no labeled DNA was found on the surfaces of intact yeast cells and yeast protoplasts (data notshown). The main role of PEG in the transformation is supposed to induce DNA binding to cellsurfaces, and most probably to facilitate DNA to enter the cells, besides inducing membrane fusion.
