Electrotransformation of Saccharomyces cerevisiae protoplast by a plasmid of Escherichia coli

The experiments reported here are aimed at obtaining optimum conditions for gene transformation after electric field pulses. Saccharomyces cerevisiae DBY746 is used as the recipient strain for shuttle plasmid (YRp group). From the relationship between the optimum electric field conditions and the transformation efficiency it is discovered that the maximum transformation efficiency appears at a wide pulse length of 400 μs with an electric field strength of 4 kV/cm, yielding up to 273 transformants/μg DNA. The electroporation unit used in the experiment is a home-made set featuring simplicity, readiness and practicality.     

产黄青霉菌中细菌血红蛋白的基因表达及工程菌的应用研究

用产黄青霉HY876作受体菌,建立amds(Acetamidase)基因为选择标记的VHb表达系统.该系统构建包括原生质体的制备和再生;用于真菌表达VHb基因的质粒pVHbM和pVHBI的构建;pVHbM或pVHbI与选择标记质粒pUcamds的共转化;将VHb基因整合到产黄青霉HY876基因组中.结果表明,只有部分转化子整合有VHb基因,VHb蛋白的表达量与其在染色体上整合的位点和整合拷贝数有关,不同转化子产生不同效应.该菌株的摇瓶发酵实验表明,VHb可促进青霉素的合成,使青霉素效价提高39%.     

Genetic transformation of immobilized competent cells

This study describes the investigation of the possibility of genetic transformation of already immobilized competent cells by plasmids. The preliminary prepared competent cells were entrapped into granules of an insoluble carrier, a cryogel of poly(vinyl alcohol). The specific activity of organophosphate hydrolase and ampicillin resistance conferred by pOPf1 plasmid were used as markers of successful transformation of the immobilized competent cells. The effect of main experimental conditions of transformation usually used for free cells, i.e., time of incubation of cells with DNA solution, temperature, and time of heat shock, on the transformation efficiency of the immobilized competent cells has been studied. A num ber of important factors of preparation of immobilized transformed cells, i.e., the concentration of im mobilized competent cells inside the granules, the concentration of DNA solution used for transformation, have been shown to affect the OPH-activity of the final immobilized transformants. The possibility of transformation of the immobilized competent cells by both single- and double-stranded plasmid DNA molecules has been demonstrated.     

Gene transfection using biodegradable nanospheres: results in tissue culture and a rat osteotomy model

In this paper, we have investigated sustained release biodegradable nanospheres for the delivery of plasmid DNA. The nanospheres were formulated using a proprietary co-polymer emulsion technique to encapsulate plasmid DNA. Gene transfection with nanospheres containing reporter genes (human placental alkaline phosphatase (AP) or Luciferase) was demonstrated in tissue culture (293T and COS-7 cells), and also in vivo in a nonunion femoral fracture (osteotomy) rat model. The bone gap was filled with nanospheres and gene expression in the implantation site was measured five weeks after the initial surgery. The nanospheres had a mean diameter of 230 nm, with a DNA loading of 0.7%w/w. These nanospheres demonstrated sustained release of the encapsulated DNA under in vitro physiologic conditions with an 82% cumulative DNA release over 17 days. The transfection efficiency of the nanospheres in tissue culture was two to five orders of magnitude greater than the gene expression with the same amount of plasmid DNA in solution. In the rat studies, the mean AP activity in the tissue retrieved from the osteotomy site in the experimental group was 291.8±52.5 cpm Versus 54.1±26.5 cpm (mean±S.E.M., P=0.03) in the sham control group. In conclusion, plasmid DNA nanospheres could be used as an effective nonviral method of gene delivery. In the future, nanospheres containing therapeutic genes, such as those encoding parathyroid hormone peptide, 1–34 amino acids (PTH-34) or Bone Morphogenic Protein-4 (BMP-4), could be used for the healing of nonunion bone fracture sites.     

REPAIR OF UV-DAMAGED INCOMING PLASMID DNA IN Saccharomyces cerevisiae

A whole-cell transformation assay was used for the repair of UV-damaged plasmid DNA in highly transformable haploid strains of Saccharomyces cerevisiae having different repair capabilities. Six rad alleles were selected from the three epistasis groups: rad 1-1 and rad2-1 from the RAD3 group, rad6-1 and rad18-2 from the RAD6 group, and rad52-1 and rad54-1 from the RAD52 group. Cells carrying single, double and triple rad alleles were transformed to uracil prototrophy by centromeric plasmid DNA (YCp19) modified in vitro with UV (254 nm). Surviving fractions were calculated as the number of transformants at each fluence relative to the number of transformants with unirradiated plasmid DNA. The sensitivity of incoming DNA in single rad mutants shows that most repair is carried out by excision repair and a RAD18-dependent process. In the rad52-1 host, the sensitivity of incoming DNA was intermediate between those found in RAD+ and rad2-1 hosts, suggesting the involvement of a recombinational repair process. Non-epistatic interactions were observed between rad alleles belonging to different epistasis groups. This provides validation for the classification of the three epistasis groups concerning the repair of chromosomal DNA for UV-incoming DNA. In both rad1-1 rad6-1 and rad1-1 rad18-2 rad54-1 hosts, the mean fluence for one lethal event corresponds approximately to one pyrimidine dimer per plasmid molecule, indicating that they are absolute repairless hosts for incoming DNA. A comparison between cell and plasmid survival reveals that there are differences in the repairability of both chromosomal and incoming DNA. The large effect of rad6-1 mutation on cell survival and the small effect on incoming DNA suggest that, in the RAD+ strain, the RAD6 product may be essential for the repair processes which act on chromosomal DNA, but not for those which act on incoming DNA. It is proposed that in yeasts postreplication repair of incoming DNA is limited to supercoiled molecules with 1-2 pyrimidine dimers that can initiate replication.     

Chemical synthesis of a structure gene coding for small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from tobacco

A structural gene with 385 bp, which codes for the mature small subunit (rbcS) of ribulose-1,5-bi9phosphate carboxylase/oxygenase from tobacco, has been redesigned according to the codon usage of E.coli and chemically synthesized. To facilitate the systematic investigation of structure-functional relationship of the rbcS by site-specific mutagenesis, twenty one unique restriction sites distributed throughout the synthetic gene were designed. Gene synthesis was started from chemically synthesizing sixteen oligonucleotides each with 23-66 nucleotides, and these oligonu-cleotides were annealed to form eight duplexes from which 5'- and 3'- two half molecules were formed by stepwise T4 DNA ligase reaction, and then each half molecule was cloned into plasmid pWR13, after that, two half molecules were further confirmed by DNA sequencing, finally both half molecules excised from the cloning plasmid were recombinated to form plasmid pCOTrbcS containing the whole structural gene for tobacco rbcS.     

Expression of Cu,Zn-superoxide dismutase gene from <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> in <Emphasis Type="Italic">Pichia pastoris</Emphasis> and its resistance to oxidative stress

The gene for the Cu, Zn-superoxide dismutase (SOD) from the yeast Saccharomyces cerevisiae was cloned, characterized, and overexpressed in the methylotrophic Pichia pastoris. The sod gene sequence obtained is 465 bp and encodes 154 amino acid residues. The sod gene sequence was cloned into the pPIC9K vector, yielding pAB22. The linearized pAB22 DNA, digested with restriction enzyme SacI, was transformed into the genome of the GS115 strain of the yeast P. pastoris. The SOD was purified from the cultured yeast by ammonium sulfate precipitation and DEAE-cellulose column chromatography. This relatively simple purification method produced a single band on analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The overexpressed SOD protein was shown to have immunologically biologic activity and to be enzymatically active. The yeast overexpressing Cu, Zn-SOD appeared to be more resistant to oxidative stress such as paraquat, menadione, and heat shock.     

Use of pJANUS™-02-001 as a calibrator plasmid for Roundup Ready soybean event GTS-40-3-2 detection: an interlaboratory trial assessment

Owing to the labelling requirements of food and feed products containing materials derived from genetically modified organisms, quantitative detection methods have to be developed for this purpose, including the necessary certified reference materials and calibrator standards. To date, for most genetically modified organisms authorized in the European Union, certified reference materials derived from seed powders are being developed. Here, an assessment has been made on the feasibility of using plasmid DNA as an alternative calibrator for the quantitative detection of genetically modified organisms. For this, a dual-target plasmid, designated as pJANUS™-02-001, comprising part of a junction region of genetically modified soybean event GTS-40-3-2 and the endogenous soybean-specific lectin gene was constructed. The dynamic range, efficiency and limit of detection for the soybean event GTS-40-3-2 real-time quantitative polymerase chain reaction (Q-PCR) system described by Terry et al. (J AOAC Int 85(4):938–944, 2002) were shown to be similar for in house produced homozygous genomic DNA from leaf tissue of soybean event GTS-40-3-2 and for plasmid pJANUS™-02-001 DNA backgrounds. The performance of this real-time Q-PCR system using both types of DNA templates as calibrator standards in quantitative DNA analysis was further assessed in an interlaboratory trial. Statistical analysis and fuzzy-logic-based interpretation were performed on critical method parameters (as defined by the European Network of GMO Laboratories and the Community Reference Laboratory for GM Food and Feed guidelines) and demonstrated that the plasmid pJANUS™-02-001 DNA represents a valuable alternative to genomic DNA as a calibrator for the quantification of soybean event GTS-40-3-2 in food and feed products.     

Co-administration of Interleukin-2 Enhances Cellular and Humoral Immune Responses to HIV Vaccine DNA Prime／MVA Boost Regime

Interleukine-2 (IL-2) is a growth factor for antigen-stimulated T lymphocytes and is responsible for T-cell clonal expansion after antigen recognition. It has been demonstrated that DNA vaccine-elicited immune responses in mice could be augmented substantially by using either an IL-2 protein or a plasmid expressing IL-2. Twenty mice, divided into four experimental groups, were immunized with: (1) sham plasmid; (2) HIV-1 DNA vaccine alone; (3) HIV-1 DNA vaccine and IL-2 protein; or (4) HIV-1 DNA vaccine and IL-2 plasmid, separately. All the groups were immunized 3 times at a 2-week interval. Fourteen days after the last DNA vaccine injection, recombinant MVA was injected into all the mice except those in group 1. ELISA and ELISPOT were employed to investigate the effect of IL-2 on DNA vaccine immune responses. The obtained results strongly indicate that the efficacy of HIV vaccine can be enhanced by co-administration of a plasmid encoding IL-2.     

Effect of salt on purification of plasmid DNA using size-exclusion chromatography

In the present study, we compared the performances of size-exclusion chromatography for the purification of plasmid DNA when different concentrations (0.5M, 1M, 2M, respectively) of two types of salt (NaCl and (NH(4))(2)SO(4)) are present in running buffers. Our experiment results displayed that it is not only the resolution of RNA but also those of supercoiled plasmid DNA and host's genomic DNA were increased greatly in the presence of high concentration of water-structure salt. We deduce that two separation modes may be involved in the process: The supercoiled plasmid DNA is influenced mainly by compaction effect and eluted in the size-exclusion mode; whereas, RNA and genomic DNA are influenced mainly by hydrophobic effect due to their stretched and loose structures and eluted in the interaction mode. This method led to an improved efficiency of size-exclusion chromatography.     

Electrophoretic behavior of plasmid DNA in the presence of various intercalating dyes

In the present study, the electrophoretic behavior of linear, supercoiled and nicked circular plasmid DNA in the presence of various intercalating dyes was characterized using pGL3 plasmid DNA as a model. The enzymatic digestion of pGL3 plasmid DNA with HindIIIwas monitored by capillary electrophoresis coupled with laser-induced fluorescence detection (CE-LIF). Nicked circular plasmid DNA was found to be relatively sensitive to enzymes, and was almost digested into the linear conformer after 10-min incubation, indicating that nicked circular plasmid DNA has little chance of targeting and entering the cell nucleus. Partly digested plasmid DNA containing only linear and supercoiled conformers can be used as a standard to confirm the migration order of plasmid DNA. In methylcellulose (MC) solution with YO-PRO-1 or YOYO-1, linear plasmid DNA eluted first, followed by supercoiled and nicked plasmid DNA, and nicked plasmid DNA eluted as a broad peak. With SYBR Green 1, nicked plasmid DNA eluted first as three sharp peaks, followed by linear and supercoiled plasmid DNA. The nuclear plasmid DNA from two transfected cell lines was successfully analyzed using the present procedure. Similar results were obtained with an analysis time of seconds using microchip electrophoresis with laser-induced fluorescence detection (mu-CE-LIF). To our knowledge, these results represent the first reported analysis of nuclear plasmid DNA from transfection cells by CE-LIF or mu-CE-LIF without pre-preparation, suggesting that the present procedure is a promising alternative method for evaluating transfection efficiency of DNA delivery systems.     

Visualized investigation of yeast transformation induced with Li+ and polyethylene glycol

The effects of Li⁺ and polyethylene glycol (PEG) on the genetic transformation of Saccharomyces cerevisiae were investigated by using fluorescence microscopy (FM) to visualize the binding of plasmid DNA labeled with YOYO-1 to the surface of yeast cells, scanning electron microscopy (SEM) and atomic force microscopy (AFM) to image the change in surface topography of yeast cells, coupled with transformation frequency experiments. The results showed that under the same conditions, the transformation frequencies of yeast protoplasts were much higher than those of intact yeast cells. PEG was absolutely required for the binding of DNA to the surface of intact yeast cells or yeast protoplasts, and had no effect on the surface topography of intact yeast cells or yeast protoplasts. In the presence of PEG, Li⁺ could greatly enhance the binding of plasmid DNA to the surface of intact yeast cells, increase their transformation frequency, and affect their surface topography. On the other hand, no effect on the DNA binding to the surface of protoplasts and no increase in the number of transformants and no surface topography changes were found upon the treatment with Li⁺ to protoplasts. In the present work, the effects of Li⁺ and PEG on yeast genetic transformation were directly visualized, rather than those deduced from the results of transformation frequencies. These results indicate that cell wall might be a barrier for the uptake of plasmid DNA. Li⁺ could increase the permeability of yeast cell wall, then increase the exposed sites of DNA binding on intact yeast cells. The main role of PEG was to induce DNA binding to cell surface.     

Double-strand DNA cleavage by copper complexes of 2,2'-dipyridyl with guanidinium/ammonium pendants

Two ligands with guanidinium/ammonium groups were synthesized and their copper complexes, [Cu(L1)Cl2](ClO4)2.H2O (1) and [Cu(L2)Cl2](ClO4)2 (2) (L1 = 5,5'-di[1-(guanidyl)methyl]-2,2'-bipyridyl cation and L2 = 5,5'-di[1-(amino)methyl]-2,2'-bipyridyl cation), were prepared to serve as nuclease mimics. X-Ray analysis revealed that Cu(II) ion in 1 has a planar square CuN2Cl2-configuration. The shortest distance between the nitrogen of guanidinium and copper atoms is 6.5408(5) A, which is coincident with that of adjacent phosphodiesters in DNA (ca. 6 A). In the absence of reducing agent, supercoiled plasmid DNA cleavage by the complexes were performed and their hydrolytic mechanisms were demonstrated with radical scavengers and T4 ligase. The pseudo-Michaelis-Menten kinetic parameters (kcat, KM) were calculated to be 4.42 h(-1), 7.46 x 10(-5) M for 1, and 4.21 h(-1), 1.07 x 10(-4) M for 2, respectively. The result shows that their cleavage efficiency is about 10-fold higher than the simple analogue [Cu(bipy)Cl2] (3) (0.50 h(-1), 3.5 x 10(-4) M). The pH dependence of DNA cleavage by 1 and its hydroxide species in solution indicates that mononuclear [Cu(L1)(OH)(H2O)]3+ ion is the active species. Highly effective DNA cleavage ability of is attributed to the effective cooperation of the metal moiety and two guanidinium pendants with the phosphodiester backbone of nucleic acid.     

Synthesis and secretion of human atrial natriuretic peptide in Saccharomyces cerevisiae

A chemically synthesized alpha-hANP gene was inserted into plasmid YFD18, which was an expression-secretion vector of yeast. The recombinant then transformed in the yeast Y33. The expression level of yeast transformants was about 700 micrograms ANP/L detected by RIA. More than 99% of expression products were secreted in the culture medium. N-terminal analysis of purified product showed that the first 4 amino acid residues of alpha-hANP were deleted.     

Improvement of <Emphasis Type="Italic">Aspergillus sulphureus</Emphasis> Endo-β-1,4-Xylanase Expression in <Emphasis Type="Italic">Pichia pastoris</Emphasis> by Codon Optimization and Analysis of the Enzymic Characterization

The gene xynB from Aspergillus sulphureus encoding the endo-β-1,4-xylanase was de novo synthesized by splicing overlap extension polymerase chain reaction according to Pichia pastoris protein’s codon bias. The synthetic DNA and wild-type DNA were placed under the control of a glyceraldehyde-3-phosphate dehydrogenase gene promoter (GAP) in the constitutive expression vector plasmid pGAPzαA and electrotransformed into the P. pastoris X-33 strain, respectively. The transformants screened by Zeocin were able to constitutively secrete the xylanase in YPD liquid medium. The maximum yield of the recombinant xylanase produced by the synthetic DNA was 105 U ml⁻¹, which was about 5-fold higher than that by wild-type DNA under the flask culture at 28 °C for 3 days. The enzyme showed optimal activity at 50 °C and pH 5.0. The residual activity remained above 90% after the recombinant xylanase was pretreated in Na₂HPO₄–citric acid buffer (pH 2.4) for 2 h. The xylanase activity was significantly improved by Zn²⁺. These biochemical characteristics suggest that the recombinant xylanase has a prospective application in feed industry as an additive.     

Anion Complexation and Transport by Isophthalamide and Dipicolinamide Derivatives: DNA Plasmid Transformation in E. coli

Tris-arenes based on either isophthalic acid or 2,6-dipicolinic acid have been known for more than a decade to bind anions. Recent studies have also demonstrated their ability to transport various ions through membranes. In this report, we demonstrate two important properties of these simple diamides. First, they transport plasmid DNA into Escherichia coli about 2-fold over controls, where the ampicillin resistance gene is expressed in the bacteria. These studies were done with plasmid DNA (～2.6 kilobase (kb)) in JM109 E. coli cells. Second, known methods do not typically transport large plasmids (>15 kb). We demonstrate here that transformation of large pVIB plasmids (i.e., >20 kb) were enhanced over water controls by ～10-fold. These results are in striking contrast to the normal decrease in transformation with increasing plasmid size.     

Assessment of Factors Influencing the Agrobacterium-mediated in planta Seed Transformation of Brinjal (Solanum melongena L.)

An efficient and reproducible in planta transformation method was developed for brinjal using seed as an explant. The brinjal seeds were infected with Agrobacterium tumefaciens EHA 105 harbouring pCAMBIA 1301-bar plasmid, and the transformants were selected against BASTA®. Several parameters influencing the in planta seed transformation such as pre-culture duration, acetosyringone concentration, surfactants, duration of sonication, vacuum pressure and vacuum duration have been evaluated. The putatively transformed (T ₀) brinjal plants were screened by GUS histochemical analysis. Among the different combinations and concentrations tested, when the 18-h pre-cultured brinjal seeds were sonicated for 20 min and vacuum infiltered for 3 min at 500 mm of Hg in Agrobacterium suspension containing 100 μM acetosyringone, 0.2 % Silwett L-77 favoured the Agrobacterium infection and showed maximum transformation efficiency. Among the five brinjal varieties evaluated, Arka Samhitha showed maximum transformation efficiency at 45.66 %. The transgene was successfully transmitted to progeny plants (T ₁) which was evidenced by GUS histochemical analysis, polymerase chain reaction and Southern hybridisation. The in planta protocol developed in the present study would be beneficial to transfer the economically and nutritionally important genes into different varieties of brinjal, and the transgenic brinjal plants can be produced in less time (approximately 27 days).     

Detection of duck hepatitis B virus DNA fragments using on-column intercalating dye labeling with capillary electrophoresis-laser-induced fluorescence.

A rapid on-column DNA labeling technique is used to detect viral restriction DNA fragments by capillary electrophoresis-laser induced fluorescence detection. Intercalating dyes such as POPO3 or ethidium homodimer-2 are incorporated into the detection buffer. The cationic dyes migrate into the capillary during electrophoresis and bind to the oppositely migrating DNA fragments. A post-column sheath-flow fluorescence detector is used in the experiment. Excellent labeling efficiency is achieved at minimal background fluorescence by diluting the dyes to between 1 x 10(-7) M and 5 x 10(-7) M in a buffer with low ionic strength relative to the running buffer within the capillary. This dilute sheath-flow buffer allows stacking of dye molecules inside the capillary when an electric field is applied. Calibration curves using a series of DNA size markers (between 72 and 1353 base pairs) were linear over an order of magnitude in DNA concentration. Sensitivity also increased linearly with fragment length, and detection limits ranged from 4 x 10(-14) M to 5 x 10(-13) M for the size-standards. Analysis of cloned viral DNA using duck hepatitis B virus demonstrated a concentration detection limit of 3.9 x 10(-16) M. Last, the technique produced very high separation efficiency, 14 x 10(6) theoretical plates which is greater than 47 x 10(6) plates m-1, for the duck hepatitis B viral genome.