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Here we describe a high-efficiency version of the lithium acetate/single-stranded carrier DNA/PEG method of transformation of Saccharomyces cerevisiae. This method currently gives the highest efficiency and yield of transformants, although a faster protocol is available for small number of transformations. The procedure takes up to 1.5 h, depending on the length of heat shock, once the yeast culture has been grown. This method is useful for most transformation requirements.

A method, using LiAc to yield competent cells, is described that increased the efficiency of genetic transformation of intact cells of Saccharomyces cerevisiae to more than 1 X 10(5) transformants per microgram of vector DNA and to 1.5% transformants per viable cell. The use of single stranded, or heat denaturated double stranded, nucleic acids as carrier resulted in about a 100 fold higher frequency of transformation with plasmids containing the 2 microns origin of replication. Single stranded DNA seems to be responsible for the effect since M13 single stranded DNA, as well as RNA, was effective. Boiled carrier DNA did not yield any increased transformation efficiency using spheroplast formation to induce DNA uptake, indicating a difference in the mechanism of transformation with the two methods.

An improved lithium acetate (LiAc)/single-stranded DNA (SS-DNA)/polyethylene glycol (PEG) protocol which yields > 1 x 10(6) transformants/micrograms plasmid DNA and the original protocol described by Schiestl and Gietz (1989) were used to investigate aspects of the mechanism of LiAc/SS-DNA/PEG transformation. The highest transformation efficiency was observed when 1 x 10(8) cells were transformed with 100 ng plasmid DNA in the presence of 50 micrograms SS carrier DNA. The yield of transformants increased linearly up to 5 micrograms plasmid per transformation. A 20-min heat shock at 42 degrees C was necessary for maximal yields. PEG was found to deposit both carrier DNA and plasmid DNA onto cells. SS carrier DNA bound more effectively to the cells and caused tighter binding of 32P-labelled plasmid DNA than did double-stranded (DS) carrier. The LiAc/SS-DNA/PEG transformation method did not result in cell fusion. DS carrier DNA competed with DS vector DNA in the transformation reaction. SS plasmid DNA transformed cells poorly in combination with both SS and DS carrier DNA. The LiAc/SS-DNA/PEG method was shown to be more effective than other treatments known to make cells transformable. A model for the mechanism of transformation by the LiAc/SS-DNA/PEG method is discussed.

Titanium dioxide (TiO2) nanoparticles (NPs) are manufactured worldwide in large quantities for use in a wide range of applications including pigment and cosmetic manufacturing. Although TiO2 is chemically inert, TiO2 NPs can cause negative health effects, like respiratory tract cancer in rats. However, the mechanisms involved in TiO2-induced genotoxicity and carcinogenicity have not been clearly defined and are poorly studied in vivo. The present study investigates TiO2 NP-induced genotoxicity, oxidative DNA damage and inflammation in a mice model. We treated wild type mice with TiO2 NPs in drinking water and determined the extent of DNA damage using the comet assay, the micronuclei assay, the γ-H2AX immuno-staining assay and by measuring 8-hydroxy-2′-deoxyguanosine (8-OHdG) levels and, as genetic instability end point, DNA deletions. We also determined mRNA levels of inflammatory cytokines in the peripheral blood. Our results show that TiO2 NPs induced 8-OHdG, γ-H2AX foci, micronuclei and DNA deletions. The formation of γ-H2AX foci, indicative of DNA double strand breaks, was the most sensitive parameter. Inflammation was also present as characterized by a moderate inflammatory response. Together these results describe the first comprehensive study of TiO2 NP induced genotoxicity in vivo in mice, possibly caused by a secondary genotoxic mechanism associated with inflammation and/or oxidative stress. Given the growing use of TiO2 NPs, these findings raise concern about potential health hazards associated with TiO2 NP exposure.

Here, we describe a quick and easy version of the lithium acetate/single-stranded carrier DNA/PEG method of transformation for Saccharomyces cerevisiae. This method can be performed when only a few transformants are needed. The procedure can take less than an hour, depending on the duration of the heat shock. It can be used to transform yeast cells from various stages of growth and storage. Cells can be transformed from freshly grown cells as well as cells stored on a plate at room temperature or in a refrigerator.

The highly efficient yeast lithium acetate transformation protocol of Schiestl and Gietz (1989) was tested for its applicability to some of the most important needs of current yeast molecular biology. The method allows efficient cloning of genes by direct transformation of gene libraries into yeast. When a random gene pool ligation reaction was transformed into yeast, the LEU2, HIS3, URA3, TRP1 and ARG4 genes were found among the primary transformants at a frequency of approximately 0.1%. The RAD4 gene, which is toxic to Escherichia coli, was also identified among the primary transformants of a ligation library at a frequency of 0.18%. Non-selective transformation using this transformation protocol was shown to increase the frequency of gene disruption three-fold. Co-transformation showed that 30-40% of the transformation-competent cells take up more than one DNA molecule which can be used to enrich for integration and deletion events 30- to 60-fold. Co-transformation was used in the construction of simultaneous double gene disruptions as well as disrupting both copies of one gene in a diploid which occurred at 2-5% the frequency of the single event.

The cause for death after lethal heat shock is not well understood. A shift from low to intermediate temperature causes the induction of heat-shock proteins in most organisms. However, except for HSP104, a convincing involvement of heat-shock proteins in the development of stress resistance has not been established in Saccharomyces cerevisiae. This paper shows that oxidative stress and antioxidant enzymes play a major role in heat-induced cell death in yeast. Mutants deleted for the antioxidant genes catalase, superoxide dismutase, and cytochrome c peroxidase were more sensitive to the lethal effect of heat than isogenic wild-type cells. Overexpression of catalase and superoxide dismutase genes caused an increase in thermotolerance. Anaerobic conditions caused a 500-to 20,000-fold increase in thermotolerance. The thermotolerance of cells in anaerobic conditions was immediately abolished upon oxygen exposure. HSP104 is not responsible for the increased resistance of anaerobically grown cells. The thermotolerance of anaerobically grown cells is not due to expression of heat-shock proteins. By using an oxidation-dependent fluorescent molecular probe a 2-to 3-fold increase in fluorescence was found upon heating. Thus, we conclude that oxidative stress is involved in heat-induced cell death.Most living cells are sensitive to sudden heat exposure. A shift in temperature from a low to an intermediate temperature induces the stress response or heat-shock response (1-3), which is considered to be an evolutionarily conserved genetic system advantageous to living organisms. After a temperature shift from 23 to 37°C in cells of the yeast Saccharomyces cerevisiae, 80 proteins were transiently induced; 20 of these proteins are now classified as major heat-shock proteins (HSPs) (2). Some of these HSPs have been characterized, but the function of many of them is still unclear (4).Initial studies suggested that HSPs play an essential role in the acquisition of stress tolerance. On the other hand, a convincing involvement of HSPs in the development of stress resistance has not been established in yeast. Except for HSP104, none of the other HSP disruption mutants show any block in the acquisition of stress resistance in yeast (5). Furthermore, a yeast strain with a temperature-sensitive mutation in the heat-shock factor (hsfl-m3) that leads to a general block in heat-shock-induced protein synthesis was not affected in the acquisition of thermotolerance (6). Therefore, HSPs may not be important for stress tolerance acquisition but rather for a rapid recovery after heat shock (4).The main factors causing death after heat exposure are still unknown. Thus, the heat-shock response may not elucidate why cells die in response to heat exposure but rather how they repair the damage afterwards. To investigate why cells die in response to heat exposure, we completely avoided the induction of the heat-shock response by exposing cells immediately to lethal heat. In particular, we investigated the possible involvement of oxidative stres...