Cerevisiae Spheroplasts Research Articles

Direct Transfer of a Mycoplasma mycoides Genome to Yeast Is Enhanced by Removal of the Mycoides Glycerol Uptake Factor Gene glpF.

We previously discovered that intact bacterial chromosomes can be directly transferred to a yeast host cell where they can propagate as centromeric plasmids by fusing bacterial cells with S accharomyces cerevisiae spheroplasts. Inside the host any desired number of genetic changes can be introduced into the yeast centromeric plasmid to produce designer genomes that can be brought to life using a genome transplantation protocol. Earlier research demonstrated that the removal of restriction-systems from donor bacteria, such as Mycoplasma mycoides, Mycoplasma capricolum, or Haemophilus influenzae increased successful genome transfers. These findings suggested that other genetic factors might also impact the bacteria-to-yeast genome transfer process. In this study, we demonstrated that the removal of a particular genetic factor, the glycerol uptake facilitator protein gene glpF from M. mycoides, significantly increased direct genome transfer by up to 21-fold. Additionally, we showed that intact bacterial cells were endocytosed by yeast spheroplasts producing organelle-like structures within these yeast cells. These might lead to the possibility of creating novel synthetic organelles.

Trans-packaging of human immunodeficiency virus type 1 genome into Gag virus-like particles in Saccharomyces cerevisiae

BackgroundYeast is recognized as a generally safe microorganism and is utilized for the production of pharmaceutical products, including vaccines. We previously showed that expression of human immunodeficiency virus type 1 (HIV-1) Gag protein in Saccharomyces cerevisiae spheroplasts released Gag virus-like particles (VLPs) extracellularly, suggesting that the production system could be used in vaccine development. In this study, we further establish HIV-1 genome packaging into Gag VLPs in a yeast cell system.ResultsThe nearly full-length HIV-1 genome containing the entire 5′ long terminal repeat, U3-R-U5, did not transcribe gag mRNA in yeast. Co-expression of HIV-1 Tat, a transcription activator, did not support the transcription. When the HIV-1 promoter U3 was replaced with the promoter for the yeast glyceraldehyde-3-phosphate dehydrogenase gene, gag mRNA transcription was restored, but no Gag protein expression was observed. Co-expression of HIV-1 Rev, a factor that facilitates nuclear export of gag mRNA, did not support the protein synthesis. Progressive deletions of R-U5 and its downstream stem-loop-rich region (SL) to the gag start ATG codon restored Gag protein expression, suggesting that a highly structured noncoding RNA generated from the R-U5-SL region had an inhibitory effect on gag mRNA translation. When a plasmid containing the HIV-1 genome with the R-U5-SL region was coexpressed with an expression plasmid for Gag protein, the HIV-1 genomic RNA was transcribed and incorporated into Gag VLPs formed by Gag protein assembly, indicative of the trans-packaging of HIV-1 genomic RNA into Gag VLPs in a yeast cell system. The concentration of HIV-1 genomic RNA in Gag VLPs released from yeast was approximately 500-fold higher than that in yeast cytoplasm. The deletion of R-U5 to the gag gene resulted in the failure of HIV-1 RNA packaging into Gag VLPs, indicating that the packaging signal of HIV-1 genomic RNA present in the R-U5 to gag region functions similarly in yeast cells.ConclusionsOur data indicate that selective trans-packaging of HIV-1 genomic RNA into Gag VLPs occurs in a yeast cell system, analogous to a mammalian cell system, suggesting that yeast may provide an alternative packaging system for lentiviral RNA.

Poly(A)-tail-promoted translation in yeast: implications for translational control.

The cap structure and the poly(A) tail synergistically activate mRNA translation in vivo. Recent work using Saccharomyces cerevisiae spheroplasts and a yeast cell-free translation system revealed that the poly(A) tail can function as an independent promotor for ribosome recruitment, to internal initiation sites within an mRNA. This raises the question of how regulatory upstream open reading frames and translational repressor proteins binding to the 5'UTR can function, as well as how regulated polyadenylation can support faithful activation of protein synthesis. We investigated the function of the regulatory upstream open reading frame 4 from the yeast GCN 4 gene and the effect of IRP-1 binding to an iron-responsive element introduced into the 5' UTR of reporter mRNAs. Both manipulations effectively block cap-dependent translation, whereas ribosome recruitment promoted by the poly(A) tail under non-competitive conditions can efficiently bypass both blocks. We show that the synergistic use of both, the cap structure and the poly-A tail enforced by mRNA competition reinstates the full extent of translational control by both types of 5' UTR regulatory elements. With a view towards regulated polyadenylation, we studied the function of poly(A) tails of defined length on the translation of capped mRNAs. We find that poly(A) tail elongation increases translational efficiency, particularly under competitive conditions. Our results integrate recent findings on the function of the poly(A) tail into an understanding of translational control.

Direct Transfer of Plasmid DNA from Intact Yeast Spheroplasts into Plant Protoplasts

We developed a polyethylene glycol (PEG)-mediated direct DNA transfer method from intact Saccharomyces cerevisiae spheroplasts into Arabidopsis thaliana protoplasts. To monitor the DNA transfer from yeast to plant cells, beta-glucuronidase (GUS) reporter gene in which a plant intron was inserted was used as a reporter. This intron-GUS reporter gene on a 2 microns-based plasmid vector was not expressed in yeast transformants, while it expressed GUS activity when the plasmid DNA was introduced into plant cells. When a mixture of 1 x 10(8) of S. cerevisiae spheroplasts harboring the plasmid and 2 x 10(6) of A. thaliana protoplasts was treated with PEG and high pH-high Ca2+ solution (0.4 M mannitol, 50 mM CaCl2, 50 mM glycine-NaOH pH 10.5), GUS activity was detected in the extract of the plant cells after a three-day culture. The GUS activity was higher than that of a reconstitution experiment in which the mixture of 1 x 10(8) of S. cerevisiae spheroplasts which did not carry the reporter gene, 2 x 10(6) of A. thaliana protoplasts and the same amount of the reporter plasmid DNA as that contained in 1 x 10(8) of S. cerevisiae spheroplasts, was treated with PEG and high pH-high Ca2+ solution. Moreover, the GUS gene expression was resistant to micrococcal nuclease treatment before and during PEG treatment. From these results, we concluded that plasmid DNA can be directly transferred from intact yeast spheroplasts to plant protoplasts by a nuclease-resistant process, possibly by the cell fusion.

Transformation of yeast spheroplasts without cell fusion

The efficiency of genetic transformation of Saccharomyces cerevisiae spheroplasts has been increased 10- to 100-fold over previously published procedures. Optimal transformation frequencies for single-stranded and double-stranded replicating plasmids are 2 × 10 7 and 5 × 10 6 transformants/μg, respectively. At saturating DNA concentrations, 12 and 3%, respectively, of the viable spheroplasts contain plasmid DNA. The percentage of transformants that have undergone nuclear fusion varies from 0.1 to 3%, indicating that fusion is not required for the uptake of DNA by yeast spheroplasts.

Characterization of the fusion of enveloped viruses with the plasma membrane of Saccharomyces cerevisiae spheroplasts.

Vesicular stomatitis virus (VSV) was associated at low pH with Saccharomyces cerevisiae spheroplasts. In the cold, the association was characterized as reversible binding to the spheroplast surface. At 37 degrees C, the association became irreversible due to fusion of the viral envelope with the yeast plasma membrane according to the following data. Proteinase K digestion degraded the viral envelope glycoprotein G but left the internal N and M proteins of VSV intact and associated with the spheroplasts. The plasma membrane could be stained by indirect immunofluorescent labeling using antiserum against VSV. By immunoelectron microscopy, no VSV particles could be detected at the spheroplast surface. Instead, the G protein could be visualized at the external aspect of the plasma membrane using specific antiserum and protein A-gold. Fusion of VSV with spheroplasts occurred below pH 4.75 at temperatures of 30-42 degrees C. It was strictly dependent on the prior removal of the yeast cell wall. The fusion process was fast, calcium-independent, and nonleaky, leaving the spheroplasts viable for at least 4 h. On the average, less than 100 VSV particles could be fused per one spheroplast. Similar data were obtained with Semliki Forest virus.

Endocytosis in Saccharomyces cerevisiae: internalization of enveloped viruses into spheroplasts.

When vesicular stomatitis virus was incubated with Saccharomyces cerevisiae spheroplasts at 37 degrees C, part of the virus was internalized by the spheroplasts as shown by the following criteria. (i) The spheroplast-associated virus was protected from proteinase K digestion, which releases surface-bound virus by degrading the envelope glycoproteins. (ii) The spheroplast-associated virus was resistant to mild Triton X-100 treatment, which readily solubilizes the virus. The same results were obtained with Semliki Forest virus. Internalization of the two viruses followed linear kinetics up to 90 min at 37 degrees C. Internalization was concentration- and temperature-dependent. At 11 degrees C no uptake could be detected for at least 2 h. Homogenization and organelle fractionation protocols were designed for the S. cerevisiae spheroplasts to study the compartments into which the virions were internalized. Three compartments containing both marker viruses could be separated in density gradients. One coincided with vacuole markers, one banded at a slightly higher and one at a similar density to the plasma membrane markers. Thus, S. cerevisiae spheroplasts appear to have the capability of endocytosing particulate markers like viruses. The companion paper describes internalization of two soluble macromolecules, alpha-amylase and fluorescent dextran, into intact cells.

Endocytosis in Saccharomyces cerevisiae: internalization of alpha-amylase and fluorescent dextran into cells.

In the preceding paper I reported that Saccharomyces cerevisiae spheroplasts were able to internalize particulate markers, enveloped viruses, into intracellular organelles. Here the internalization of soluble macromolecules into cells having an intact cell wall is described. alpha-Amylase was taken up into cells in a temperature- and concentration-dependent way. The kinetics of accumulation were linear for the first 20-40 min at 37 degrees C and then started to level off. Internalization of alpha-amylase into spheroplasts displayed similar characteristics, but the accumulation rate was about four times higher than into cells. Fluorescent dextran was used to mark morphologically the compartment into which internalization occurred. This marker was accumulated into the vacuole of the cells in a time-, temperature- and concentration-dependent way. A temperature-sensitive mutant deficient in exocytosis was found to be defective in intracellular accumulation of alpha-amylase and dextran. At the restrictive temperature, very little alpha-amylase accumulated into the cells and only faint staining of intracellular organelles with fluorescent dextran could be detected. At the permissive temperatures, accumulation of alpha-amylase and dextran into the mutant cells was comparable with accumulation into wild-type cells. I conclude that alpha-amylase and fluorescent dextran were internalized into S. cerevisiae cells and directed into the vacuoles.

Transformation of Saccharomyces cerevisiae spheroplasts by high electric pulse

An electric pulse was applied to transformation of yeast cell. Saccharomyces cerevisiae D13-1A ( a his3-532 trpl gal2) was used as a recipient strain for plasmid YRp7. Spheroplasts of S.cerevisiae D13-1A were placed under an electric pulse in the presence of YRp7. Transformants were observed when the pulse height was above 5 kV cm . The number of transformants increased with increasing pulse height and 945 transformants per ng DNA were obtained at 10 kV cm . Introduction of DNA might be caused by breakdown of the cell membrane under high electric voltage. Transformation Saccharomyces cerevisiae Spheroplast Plasmid Electric pulse

Saccharomyces cerevisiae spheroplasts are sensitive to the action of diphtheria toxin.

Diphtheria toxin kills spheroplasts of Saccharomyces cerevisiae but not the intact yeast cells. After 2 h of exposure to ca. 10(-7) M toxin, less than 1% of spheroplasts were able to regenerate into intact cells. The same high levels of toxin inhibited the rate of protein synthesis by more than 90% within 1 h, whereas RNA and DNA synthesis were not inhibited until 4 h or exposure. Both killing and protein synthesis inhibition were dependent on toxin concentration. The nature of the toxin-cell interaction was also studied by using fragments of intact toxin and mutant toxin proteins. Neither toxin fragment A nor CRM45 nor CRM197 affected spheroplasts, but CRM197 and ATP prevented the inhibitory action of intact toxin. These results suggest that toxin acts on S. cerevisiae spheroplasts in much the same manner as it acts on sensitive mammalian cells.

Saccharomyces cerevisiae spheroplasts are sensitive to the action of diphtheria toxin.

Diphtheria toxin kills spheroplasts of Saccharomyces cerevisiae but not the intact yeast cells. After 2 h of exposure to ca. 10(-7) M toxin, less than 1% of spheroplasts were able to regenerate into intact cells. The same high levels of toxin inhibited the rate of protein synthesis by more than 90% within 1 h, whereas RNA and DNA synthesis were not inhibited until 4 h or exposure. Both killing and protein synthesis inhibition were dependent on toxin concentration. The nature of the toxin-cell interaction was also studied by using fragments of intact toxin and mutant toxin proteins. Neither toxin fragment A nor CRM45 nor CRM197 affected spheroplasts, but CRM197 and ATP prevented the inhibitory action of intact toxin. These results suggest that toxin acts on S. cerevisiae spheroplasts in much the same manner as it acts on sensitive mammalian cells.

Papulacandin B: an Inhibitor of Glucan Synthesis in Yeast Spheroplasts

Papulacandin B, the main component of a series of antibiotics produced by the deuteromycete Papularia sphaerosperma, inhibits the growth of yeasts. It is a highly amphophilic substance containing residues of glucose, galactose and two long‐chain unsaturated fatty acids. It does not cause the release of potassium ions from yeast cells and therefore differs in its mode of action from the polyene antibiotics. Papulacandin B, at concentrations slightly below the minimal inhibitory concentration, partially but selectively inhibited the incorporation of glucose into cells of Saccharomyces cerevisiae and Candida albicans. A much clearer effect was observed on spheroplasts, obtained by digestion of the yeast cell wall with snail digestive enzyme. Incubation of spheroplasts in a minimal medium stabilised by sorbitol led to the incorporation of glucose into alkali‐insoluble glucan, mannan and a fraction containing mainly alkali‐soluble 1,3‐glucan together with some glycogen. Papulacandin B inhibited the synthesis of the alkali‐insoluble fraction, while causing a slight stimulation of glucose incorporation into the other two polysaccharide fractions. The 50% inhibitory concentrations of papulacandin B for glucan synthesis in S. cerevisiae spheroplasts and C. albicans spheroplasts were respectively 0.16 μg/ml and 0.03 μg/ml. C. albicans cells were irradiated with ultraviolet light and selected for maximum resistance to papulacandin B. The 50% inhibitory concentration for glucan synthesis in spheroplasts prepared from this mutant was 2.5 μg/ml. Echinocandin B, a polypeptide antibiotic containing a long‐chain fatty acid, also inhibited the synthesis of glucan in spheroplasts. It is concluded that papulacandin B and probably also echinocandin B inhibit glucan synthesis during cell‐wall synthesis, and thus cause lysis of cells by osmotic rupture.