Bioconjugate Chem. 2005, 16, 250−254
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ARTICLES Yeast Transformation Process Studied by Fluorescence Labeling Technique Hu-Zhi Zheng,† Hui-Hui Liu,‡ Shao-Xing Chen,‡ Zhe-Xue Lu,† Zhi-Ling Zhang,† Dai-Wen Pang,*,† Zhi-Xiong Xie,*,‡ and Ping Shen‡ College of Chemistry & Molecular Sciences and College of Life Sciences, Wuhan University, Wuhan 430072, P. R. China. Received July 14, 2004; Revised Manuscript Received February 1, 2005
A new method based on fluorescence imaging and flow cytometry was developed to investigate the transformation process of Saccharomyces cerevisiae AY. Yeast and fluorescent-labeled plasmid pUC18 were used as models of cells and DNA molecules, respectively. Binding of DNA molecules to yeast cell surfaces was observed. Factors influencing DNA binding to cell surfaces were investigated. It has been found that poly(ethylene glycol) (PEG) could induce DNA binding to yeast surfaces, while Li+ showed a weak effect on the binding. When both Li+ and PEG were used, synergetic effect occurred, resulting in the binding of pUC18 to the surface of more yeast cells compared with that in the presence of PEG or Li+ only. It was also confirmed that heat shock, Li+, and PEG all can increase the permeability of yeast cells. This simple method is helpful for understanding the process of yeast transformation and can be used to investigate the interaction of DNA with cell surfaces.
1. INTRODUCTION
The term “transformation” is used to describe DNA uptake in both prokaryotes and eukaryotes, as detected by consequent changes in phenotype (1). Genetic transformation of Saccharomyces cerevisiae was first reported in 1960 (2). Several methods for yeast transformation, such as spheroplast (3), Li+ intact yeast cell, electroporation (4), glass beads (5), and biolistic (6), were developed. Li+ intact yeast cell transformation, which is a simple method with no special equipment required and with relatively high transformation efficiency, was first established by Ito in 1983 (7) and subsequently improved (8-11) and standardized (12). So, this method has been widely used. The natural transformation process of bacteria usually includes three steps: first, DNA is bound to cell surfaces; second, the uptake of DNA by cells occurs; last, DNA molecules are integrated into chromosomal DNA or replicated as extrachromosomal genetic elements (13). Although the details in the yeast transformation process are unknown, it can be supposed that the process involves the contact of DNA to yeast cells, the uptake of DNA by yeast cells, and the integration of DNA. Factors influencing these three steps will affect transformation efficiency. Some reports suggested that DNA could bind to yeast cell surfaces (11). It can be believed that factors facilitating DNA binding to cell surfaces will increase * Corresponding authors. Dai-Wen Pang: phone, 86-2768756759; fax, 86-27-68754067; e-mail address, dwpang@ whu.edu.cn. Zhi-Xiong Xie: e-mail address,
[email protected]. † College of Chemistry and Molecular Sciences, Wuhan University. ‡ College of Life Sciences, Wuhan University.
transformation efficiency. However, strong binding of DNA to the cell surfaces will result in reduced transformation efficiency (11). Increasing cell wall permeability to DNA enhanced transformation efficiency also (14). Herein, DNA binding sites on yeast cell surfaces were observed, and factors that affect DNA binding to cell surfaces and cell permeability were explored in detail. Nevertheless, the integration of DNA molecules into chromosomal DNA cannot be studied by the current method. It is evident that this new method is helpful to understand the transformation process and can be promising in studying the transformation of other cells and the interaction of DNA with cells. 2. MATERIALS AND METHODS
Lithium acetate (LiAc), PEG4000, yeast extract, polypepton, Tris buffer, glucose, glass slides, and cover glass were all purchased from Sigma. YOYO-1 was a product of Molecular Probes. Plasmid pUC18 was obtained from Sino-American Biotechnology Co. Ultrapure water (18.2 MΩ) was prepared with a Labconco system and used throughout. Saccharomyces cerevisiae AY was obtained from Prof. Ping Shen’s lab and used as a cell model. Before use, it was dispersed on a YPD plate (1.0% yeast extract, 2.0% polypepton, 2.0% glucose, 1.5% agar) and cultured for 2-3 days at 30 °C. Subsequently, a single colony was selected and dispersed on another YPD plate and cultured and then kept at 4 °C. Plasmid pUC18 was labeled with YOYO-1 at a ratio of 50:1 (base pairs to YOYO-1) in the dark for 90 min at 4 °C immediately before use (15). At such a ratio of base pairs to YOYO-1, there is almost no free YOYO-1 because of its complete binding to DNA. A titration experiment demonstrated that addition of excess unlabeled DNA did not result in increase in fluorescence (16).
10.1021/bc049833v CCC: $30.25 © 2005 American Chemical Society Published on Web 02/19/2005
Yeast Transformation Process Studied
The Li+ intact yeast cell transformation protocol was as previously reported (1). Yeast was precultured overnight in 5 mL of YPD (1.0% yeast extract, 2.0% polypepton, 2.0% glucose). A suitable volume of precultured cell suspension was added in 50 mL of YPD and incubated at 30 °C with shaking until cell growth reached the late logarithm phase. The cells were then harvested by centrifugation at 5000 rpm for 5 min, washed twice with ultrapure water, and resuspended in ultrapure water at 109 cells/mL. One hundred microliters of sample was centrifuged for 5 min at 5000 rpm, and the supernatant was discarded. The pellets were resuspended in 360 µL of transformation mixture (240 µL of 50% PEG4000 (w/ v), 36 µL of 1.0 M LiAc, 50 µL of 2.0 mg/mL singlestranded carrier DNA, YOYO-1-labeled pUC18 DNA diluted to 34 µL with water). The cells in transformation mixture were incubated at 30 °C in the dark with shaking for 30 min, and then heat shocked at 42 °C in dark for 15 min. Cells for experiments were dropped on glass slides and covered with a cover glass and then observed with a fluorescence microscope (FM) (Axiovert 200M, Zeiss) equipped with an oil-immersion objective (100× objective, Zeiss, N.A. ) 1.25) or a confocal laser scanning microscope (CLSM) (TCS SP, Leica) equipped with an oil immersion objective (Leica, N.A. ) 1.40). Cell samples were filtered with a 300 mesh filter and then diluted by several hundred times with 0.1 M Li+-containing solution for cells in solution containing Li+ or with ultrapure water for cells in Li+-free solution before being analyzed with a flow cytometer (Beckman Coulter Epics Altra) coupled with a 488 nm Ar+ laser and a 525 nm bandpass filter. The ultrapure-water-washed yeast cells were used as the negative control sample. To examine yeast cell permeability, 10 µL of 10 µM YOYO-1 was added into 100 µL of water-washed cells, and then, Li+, PEG, or both were added. The mixture was incubated at 30 °C with shaking in the dark for 30 min and then heat shocked at 42 °C in the dark for 15 min. At last, it was diluted and analyzed with a flow cytometer. Atomic force microscopic (AFM) images of cell surfaces were obtained using an atomic force microscope (Picoscan, Molecular Imaging) in contact mode with a commercial MAClever type II probe (Molecular Imaging). 3. RESULTS AND DISCUSSION
3.1. Binding of DNA to Yeast Cell Surfaces. A widely used cell-impermeable fluorescent probe, YOYO1, was used to label pUC18 plasmid DNA. After intercalation into double-stranded DNA, fluorescence intensity of YOYO-1 is enhanced by more than 1000 times (17). So, a very high ratio of signal-to-noise can be obtained using YOYO-1 to label DNA. A commercial plasmid DNA, pUC18, was used to investigate the interaction of DNA with yeast cell surfaces, although it cannot be translated in yeast. Because pUC18 is short in strand length (2686 base pairs), each pUC18 molecule can only combine with a small number of YOYO-1 molecules (no more than 60 molecules) under our experimental conditions, the fluorescence of which is too weak to observe. When pUC18 molecules labeled with YOYO-1 are bound to cell surfaces, the fluorescence intensity is strong enough to observe since more labeled pUC18 molecules are concentrated on the surfaces. The yeast cell wall acts as a barrier to DNA (1), and most DNA molecules are bound to the cell wall (11). So, all the fluorescence signals arose from DNA binding to
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Figure 1. Fluorescence images of DNA binding to yeast cell surfaces: (a) a big field of view obtained by using a FM; (b) a small field of view containing two cells obtained by using a CLSM. Conditions were as follows: ca. 108 yeast cells treated with 20 ng of pUC18 stained with YOYO-1; image size ca. 80 × 80 µm2 (a) and 25 × 25 µm2 (b).
cell surfaces, and the fluorescence of cells will indicate DNA binding to their surfaces. It was confirmed that the binding of YOYO-1 to DNA was of high affinity, thereby preventing the migration of YOYO-1 label from one DNA molecule to another (17, 18). The addition of excessive single-stranded DNA does not affect the fluorescence of YOYO-1-labeled plasmid DNA. Because the cell wall of dead cells is not intact, YOYO1-labeled DNA might diffuse into dead cells and make them fluoresce. So, the presence of dead cells will make difficult the judgment of DNA binding to cell surfaces. To ensure the reliability of experimental results, cells pretreated with the transformation mixture were labeled with methylene blue to determine whether the cells were dead (19). It was found that no more than 5% of cells were stained blue; that is, no more than 5% cells were dead. Binding sites of DNA molecules on yeast cell surfaces were found (Figure 1). As the concentration of pUC18 increased, more pUC18 molecules would be bound to the cell surface. When 80 ng of pUC18 DNA was added into the mixture containing ca. 108 cells (the ratio of DNA molecules to cells was about 200:1), the DNA would be bound to the whole cell surface (Figure 2a). These results indicated that DNA binding sites on the surface of one yeast cell could be saturated in the presence of excessive DNA. Excessive pUC18 on the cell surfaces can be washed off (Figure 2b). 3.2. Factors Influencing DNA Binding to Yeast Cell Surfaces. Li+ and PEG can facilitate DNA binding
252 Bioconjugate Chem., Vol. 16, No. 2, 2005
Zheng et al. Table 2. Transformation Frequencya transformation frequency control 0 a
Figure 2. Fluorescence images of saturation of DNA binding to yeast cells. Conditions were as follows: ca. 108 yeast cells treated with 80 ng of pUC18 stained with YOYO-1 without washing (a) and with washing (b); 100× oil-immersion objective (N.A. ) 1.40) (a) and 40× objective (N.A. ) 0.70) (b); image size 25 × 25 µm2 (a) and 40 × 40 µm2 (b). Table 1. Factors Influencing DNA Binding to Cell Surfaces percentage of cells with bound DNA (%) conditiona
YOYO-1 labeled DNA
Li+
PEG
PEG and Li+
PEG, Li+, and ss-DNA
a b c
1.2 3.9 10.6
6.1 8.3 23.1
9.8 44.9 61.7
44.8 95.5 98.2
27.9 69.9
a Yeast cells (ca. 108) were treated with 4 (a), 20 (b), and 80 (c) ng of pUC18 stained with YOYO-1. Negative control sample was water-washed yeast cells.
to the yeast cell surface. Table 1 shows the percentage of fluorescent yeast cells treated with Li+, PEG, or both in the presence of pUC18 of different concentrations. As mentioned above, the fluorescence of yeast cells was from labeled pUC18 on their surfaces; hence the percentage of fluorescent cells suggests the percentage of the cells with pUC18 bound on their surfaces. Li+ can induce DNA binding to yeast cell surfaces, but its inducing effect was weak. PEG can induce DNA binding to yeast cell surfaces more strongly than Li+. In our experiments, it was found that the use of both PEG and Li+ in the transformation mixture could most strongly induce the binding. The percentage of cells with bound pUC18 was 1.2%, 3.9%, and 10.6% in the presence of three different concentrations of YOYO-1-labeled pUC18 as listed in Table 1. But in the presence of Li+, the percentage increased to 6.1%, 8.3%, and 23.1%, showing that Li+ can induce DNA binding to yeast cell surfaces. As known,
Li+ 0
PEG 5.12 ×
10-5
Li+ and PEG 3.57 × 10-4
For details, see Supporting Information.
both DNA and the yeast cell surface are partly negatively charged (20); they will electrostatically repel. Li+ can weaken such repulsion, inducing DNA molecules to be bound to yeast cell surfaces. Other cations, such as Na+ and Cs+, have been also used for yeast cell transformation (7, 8), most probably due to their reduction of the repulsion between DNA and cell surfaces. PEG has a strong effect on inducing DNA binding to yeast cell surfaces. In the presence of PEG, the percentages of cells with bound pUC18 on their surfaces increased to 9.8%, 44.9%, and 61.7% at three different labeled pUC18 concentrations (Table 1). It has been reported that PEG facilitates DNA deposition onto yeast cells (11). The effect of PEG may be attributed to its changing charges of cell surfaces (7). Such changes may induce both conformational changes and aggregation of cells (7). And it was also found that PEG could induce a drastic change in the conformation of plasmid DNA molecules (8). Perhaps both the conformational changes of DNA molecules and cell surfaces were propitious to the binding of DNA molecules to cell surfaces. The use of both PEG and Li+ induced DNA binding to yeast cell surfaces more effectively than PEG or Li+ only. At three different concentrations of labeled pUC18, the percentage of fluorescent cells was 44.8%, 95.5%, and 98.2%, which might be attributed to a synergetic effect. These results are exactly consistent with the transformation results (Table 2 and Supporting Information): in the presence of Li+, PEG, or PEG plus Li+, the transformation frequency was 0, 5.12 × 10-5, and 3.57 × 10-4, respectively. Single-stranded carrier DNA can be bound more effectively to the yeast cell surfaces and occupy the DNA binding sites. It was reported that 500-fold excess singlestranded DNA decreased the strong binding of DNA to cell surfaces so that most of the plasmid was available for uptake, resulting in high transformation efficiency (11). Our results are in agreement with this. Singlestranded DNA decreased the percentage of cells with pUC18 bound to their surfaces from ca. 95% down to 28% in the case of ca. 20 ng of pUC18 in the transform mixture containing ca. 108 cells (Table 1). 3.3. Factors Influencing Cell Permeability. As mentioned above, the permeability of cells will affect the transformation for it can influence the cell uptake of DNA. Increasing cell permeability will result in high transformation efficiency. The factors influencing cell permeability were investigated with YOYO-1 by flow cytometry. As a cell-impermeable reagent, YOYO-1 cannot pass through intact cell membranes, so it has been used to distinguish dead from living cells (21). Intact living yeast cells treated with YOYO-1 will not be fluorescent, but dead cells will be fluorescent. For our experiments, it was found that 19.1% (incubated at 30 °C), 73.1% (treated with Li+), 83.1% (with PEG), and 99.4% (with Li+ and PEG) of yeast cells were fluorescent (Table 3). However, methylene blue staining experiments showed that almost all of the fluorescent cells were alive. With increasing permeability of living cells, YOYO-1 can get into cells and be bound to the in-cell DNA, making living cells fluorescent. The cells that fluoresce should be permeable.
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Yeast Transformation Process Studied Table 3. Factors Increasing Cell Permeabilitya percentage of permeable cells incubation at 30 °C
heat shock
Li+
PEG
PEG and Li+
19.1
36.1
73.1
83.1
99.4
a
Negative control sample was water-washed yeast cells.
taminates AFM probe tips seriously, cells treated with PEG cannot be studied by AFM. The use of both PEG and Li+ can largely increase cell permeability probably due to synergetic effect. This result partly coincides with the transformation results. Although Li+ can strongly increasing cell permeability, the transformation frequency was zero when only Li+ was used to treat cells, which may be due to its weak effect on inducing DNA binding to cell surfaces. Heat shock plays a significant role in transformation. It was reported that heat-shock proteins acted as molecular chaperones, protecting important structural proteins and enzymes from stress-induced degradation (22). Heatshock-induced changes in the level of activities of specific proteins might affect either the transport of plasmid DNA from the cell membrane to the nucleus or elimination of cellular components that can degrade plasmid DNA (11). It was found, in our research, that heat shock caused 36.1% of cells to be permeable (19.1% for samples not treated with heat shock) (Table 3), suggesting that heat shock can also increase cell permeability. 4. CONCLUSIONS
A new way to study of yeast transformation process has been explored based on various techniques: fluorescence imaging to examine the DNA binding sites on yeast cell surfaces, flow cytometry to investigate the factors influencing DNA binding to cell surfaces and cell permeability, and AFM to observe cell surfaces to provide complementary information on the cell permeability. Our research confirmed that there are DNA binding sites on yeast cell surfaces. At a high concentration of DNA, DNA binding sites will be saturated, and DNA molecules will adhere to the whole yeast cell surface. Heat shock can apparently increase cell permeability. Li+ is able to weakly promote DNA binding to yeast cell surfaces and strongly increase cell permeability. PEG can strongly induce DNA binding to cell surfaces and increase cell permeability, which are perhaps responsible for the increase of transformation efficiency. PEG can more effectively promote DNA binding to cell surfaces than Li+. Use of both Li+ and PEG will result in synergetic effect, which extremely strongly induces DNA binding to cell surfaces and increases cell permeability. Single-stranded carrier DNA is able to decrease strong binding of plasmid DNA to cell surfaces. ACKNOWLEDGMENT
Figure 3. AFM images of some yeast cells (a), Li+-treated yeast cells (b), and the cell wall of a Li+-treated single yeast cell (c). Cell sample preparation was as follows: after being cultured and harvested, yeast cells were suspended at ca. 109 cells/mL in ultrapure water (a) or 0.1 M Li+ solution (b, c) and subsequently incubated at 30 °C with shaking for 30 min, and then ca. 50 µL of cell sample was cast onto freshly cleaned slides (1.5 × 1.5 cm2).
Treating with Li+ can increase yeast cell permeability (14). Our results from AFM imaging (Figure 3) showed that cells treated with Li+ had rougher surfaces than cells not treated with Li+, indicating that the increase in cell permeability was perhaps attributed to the change of the cell surface structure. PEG is essential in yeast transformation (7, 8). Many authors suggested that PEG was responsible for the adsorption of the DNA on the yeast cell surfaces. But our results revealed that PEG could also increase the cell permeability. Because PEG con-
This work was supported by the National Science Fund for Distinguished Young Scholars (Grant No. 20025311), National Natural Science Foundation of China (Grant Nos. 20305011, 20299034, 20207005, and 30170268), and the Ministry of Education of China (Grant No. 200065). The authors wish to thank Ms. Jing Zhang (Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China), Prof. Ming-Sheng Zu, and Prof. Yi-Peng Qi (College of Life Sciences, Wuhan University). Supporting Information Available: Description of the transformation experiment with a table of the transformation frequencies. This material is available free of charge via the Internet at http://pubs.acs.org. LITERATURE CITED (1) Gietz, R. D., and Woods, R. A. (2001) Genetic transformation of yeast. BioTechniques 30, 816-831. (2) Oppenoorth, W. F. F. (1960) Modification of the Hereditary Character of Yeast by Ingestion of Cell-Free Extracts. J. Microbiol. Serol. 26, 129-147.
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