Transformation of Escherichia coli with DNA from Saccharomyces cerevisiae cell lysates

We developed a system to monitor the transfer of heterologous DNA from a genetically manipulated strain of Saccharomyces cerevisiae to Escherichia coli. This system is based on a yeast strain that carries multiple integrated copies of a pUC-derived plasmid. The bacterial sequences are maintained in the yeast genome by selectable markers for lactose utilization. Lysates of the yeast strain were used to transform E. coli. Transfer of DNA was measured by determining the number of ampicillin-resistant E. coli clones. Our results show that transmission of the Amp(r) gene to E. coli by genetic transformation, caused by DNA released from the yeast, occurs at a very low frequency (about 50 transformants per microg of DNA) under optimal conditions (a highly competent host strain and a highly efficient transformation procedure). These results suggest that under natural conditions, spontaneous transmission of chromosomal genes from genetically modified organisms is likely to be rare.     

Non-B DNA conformations formed by long repeating tracts of myotonic dystrophy type 1, myotonic dystrophy type 2, and Friedreich's ataxia genes, not the sequences per se, promote mutagenesis in flanking regions

The expansions of long repeating tracts of CTG.CAG, CCTG.CAGG, and GAA.TTC are integral to the etiology of myotonic dystrophy type 1 (DM1), myotonic dystrophy type 2 (DM2), and Friedreich's ataxia (FRDA). Essentially all studies on the molecular mechanisms of this expansion process invoke an important role for non-B DNA conformations which may be adopted by these repeat sequences. We have directly evaluated the role(s) of the repeating sequences per se, or of the non-B DNA conformations formed by these sequences, in the mutagenic process. Studies in Escherichia coli and three types of mammalian (COS-7, CV-1, and HEK-293) fibroblast-like cells revealed that conditions which promoted the formation of the non-B DNA structures enhanced the genetic instabilities, both within the repeat sequences and in the flanking sequences of up to approximately 4 kbp. The three strategies utilized included: the in vivo modulation of global negative supercoil density using topA and gyrB mutant E. coli strains; the in vivo cleavage of hairpin loops, which are an obligate consequence of slipped-strand structures, cruciforms, and intramolecular triplexes, by inactivation of the SbcC protein; and by genetic instability studies with plasmids containing long repeating sequence inserts that do, and do not, adopt non-B DNA structures in vitro. Hence, non-B DNA conformations are critical for these mutagenesis mechanisms.     

Roles of double-strand breaks, nicks, and gaps in stimulating deletions of CTG.CAG repeats by intramolecular DNA repair

A series of plasmids harboring CTG.CAG repeats with double-strand breaks (DSB), single-strand nicks, or single-strand gaps (15 or 30 nucleotides) within the repeat regions were used to determine their capacity to induce genetic instabilities. These plasmids were introduced into Escherichia coli in the presence of a second plasmid containing a sequence that could support homologous recombination repair between the two plasmids. The transfer of a point mutation from the second to the first plasmid was used to monitor homologous recombination (gene conversion). Only DSBs increased the overall genetic instability. This instability took place by intramolecular repair, which was not dependent on RuvA. Double-strand break-induced instabilities were partially stabilized by a mutation in recF. Gaps of 30 nt formed a distinct 30 nt deletion product, whereas single strand nicks and gaps of 15 nt did not induce expansions or deletions. Formation of this deletion product required the CTG.CAG repeats to be present in the single-stranded region and was stimulated by E.coli DNA ligase, but was not dependent upon the RecFOR pathway. Models are presented to explain the intramolecular repair-induced instabilities and the formation of the 30 nt deletion product.     

Reciprocal and non-reciprocal homologous recombination between Escherichia coli chromosomal DNA and ultraviolet light-irradiated plasmid DNA

Plasmid DNA substrates were used to study ultraviolet (UV)-induced recombination events in Escherichia coli host cells. Plasmids derived from pBR322, containing all or part of the lac operon of E. coli, were irradiated with ultraviolet light before transformation into E. coli strains of different recA and lacY genotypes. Recombinational exchanges were identified by phenotypic changes in lactose utilization and were confirmed by restriction analysis of isolated plasmids. Ultraviolet-induced reciprocal plasmid-chromosome recombination occurred at a slightly higher frequency then non-reciprocal chromosome-to-plasmid recombination, and at a much higher frequency than non-reciprocal plasmid-to-chromosome recombination. These frequencies did not depend on segregative mechanisms. The asymmetry of non-reciprocal exchange was not due to the particular arrangement of wild-type and lacY1 alleles because the same results were observed when these were interchanged. The host recA gene was required for plasmid-chromosome recombination, and slightly enhanced plasmid survival. Evidence for plasmid replication prior to recombination was found in reciprocal recombinants, but rarely in the non-reciprocal recombinants analyzed. Irradiation of competent bacterial host cells prior to transformation did not effectively induce plasmid-chromosome recombination.     

少量制备大肠杆菌感受态细胞条件探索

目的:为了获得重复性好、转化率高的少量制备感受态细胞的方法,利用不同生长时期的大肠杆菌感受态细胞,进行转化比较。方法:根据普通实验室的实验条件,常规方法提取质粒,氯化钙法转化不同生长时期的大肠杆菌感受态细胞,比较转化率。结果:大肠杆菌感受态细胞的转化率与OD值显著相关,在OD600nm为0.39和0.55时转化率最高,在OD600nm为0.28~0.55之间均可得到理想的转化效果。结论:少量制备感受态细胞方法操作中无需添加任何保护试剂和细胞复苏培养,操作简便、重复性好,实验成本低廉。     

Fate of exogenous recombinant plasmids introduced into mouse and human cells.

We have constructed a number of plasmids selectable in both E. coli and mouse or human cells. Human DNA sequences were inserted and the recombinant plasmids were used to transfect either mouse or human cells by the Ca-phosphate precipitation technique. We have observed that: (i) competent cells uptake large amounts of plasmid DNA; (ii) input plasmids persist in transformed mammalian cells as free unreplicating circular molecules for up to 20 generations; such persistence does not depend on the presence of selective markers; (iii) plasmids incorporated into mouse L-cells undergo widespread rearrangements (in the absence of replication) entailing mostly deletions of both human and bacterial sequences which yield smaller products; the latter appear to be more stable in a subsequent transformation cycle. Surprisingly such rearrangements are almost totally absent in transformed human KB-cells. This property of human KB-cells may prove useful for the development of a vector apt at cloning and expressing human DNA sequences. Unlike what has been observed in yeast, no "autonomously replicating sequence" can be detected in mammalian cells by randomly cloning human DNA sequences into a selectable plasmid and screening for an increased transformation efficiency.     

SOS repair and DNA supercoiling influence the genetic stability of DNA triplet repeats in Escherichia coli

Molecular mechanisms responsible for the genetic instability of DNA trinucleotide sequences (TRS) account for at least 20 human hereditary disorders. Many aspects of DNA metabolism influence the frequency of length changes in such repeats. Herein, we demonstrate that expression of Escherichia coli SOS repair proteins dramatically decreases the genetic stability of long (CTG/CAG)n tracts contained in plasmids. Furthermore, the growth characteristics of the bacteria are affected by the (CTG/CAG)n tract, with the effect dependent on the length of the TRS. In an E. coli host strain with constitutive expression of the SOS regulon, the frequency of deletions to the repeat is substantially higher than that in a strain with no SOS response. Analyses of the topology of reporter plasmids isolated from the SOS+ and SOS- strains revealed higher levels of negative supercoiling in strains with the constitutively expressed SOS network. Hence, we used strains with mutations in topoisomerases to examine the effect of DNA topology upon the TRS instability. Higher levels of negative DNA supercoiling correlated with increased deletions in long (CTG/CAG)n, (CGG/CCG)n and (GAA/TTC)n. These observations suggest a link between the induction of bacterial SOS repair, changes in DNA topology and the mechanisms leading to genetic instability of repetitive DNA sequences.     

Bioassay for specific DNA sequences using a non-radioactive probe

A novel method for detecting specific DNA sequences is described. The method uses a non-radioactive DNA probe, called a probe-vector, that can transform competent Escherichia coli cells at high efficiency only when it has hybridized to a specific DNA target, thus forming a circular, double-stranded, plasmid-like molecule. The probe-vector carries a plasmid origin of replication and a gene that confers antibiotic resistance on transformed E. coli. The output of the assay--colored bacterial colonies on an agar plate--is quantitative and proportional over a wide range of target concentrations. The utility of the probe-vector method for detecting hepatitis B virus (HBV) DNA in human serum is demonstrated. The assay can detect as little as 0.1 pg HBV DNA. The presence of an internal standard monitors DNA recovery and E. coli transformation efficiency for each sample. The assay has the potential to simultaneously measure the DNA of two or more pathogens within the same clinical sample.     

Identification of two new genetically active regions associated with the osmZ locus of Escherichia coli: role in regulation of proU expression and mutagenic effect of cya, the structural gene for adenylate cyclase.

The Escherichia coli K-12 gene coding for the nucleoid-associated protein HNS was cloned together with 5.6 kb of downstream DNA in the vector pACYC184. The cloned DNA complemented a mutation in the osmZ locus of E. coli, which codes for HNS. However, the multicopy plasmid harboring the cloned sequence was found to be mutagenic and to produce at high frequency mutations that mapped to the E. coli cya gene, which codes for adenylate cyclase. Acquisition of the cya mutations was independent of RecA. These mutations were phenotypically suppressed by providing the cells with exogenous cyclic AMP and were complemented in trans by a plasmid carrying an active copy of the cya gene. A deletion analysis of the cloned sequences showed that DNA downstream of the gene coding for HNS was also required for the mutagenic effect of cya and had a role in regulating the expression of the osmZ-dependent proU locus. These sequences appear to contain at least two genetically active regions.     

An effective alternate cloning strategy for unstable mouse genomic sequences

Unstable mammalian genomic sequences frequently underwent spontaneous rearrangement during the bacterial cloning process. When the flanking sequences of an INSM1 gene comprised of 3.0 and 4.5 kb were subcloned into a targeting vector for a gene deletion study, both the genomic sequences underwent spontaneous rearrangement. Neither the usage of recombinase-free Escherichia coli competent cells nor lowering the culture incubation temperature averted the recombination events. Co-transformation of a methyltransferase vector, pAIT2, with the targeting vector had little effect in preventing recombination through methylation of the plasmid DNA. Here, we show that a single-copy cloning technique is effective to clone the unstable mouse genomic DNA into the targeting vector.     

Use of β-galactosidase (lacZ) gene α-complementation as a novel approach for assessment of titanium oxide nanoparticles induced mutagenesis

The mutagenic potential of titanium dioxide nanoparticles (TiO(2)-NPs) of an average size 30.6nm was investigated using β-galactosidase (lacZ) gene complementation in plasmid pUC19/lacZ(-)Escherichia coli DH5α system. Plasmid pUC19 was treated with varying concentrations of TiO(2)-NPs and allowed to transfect the CaCl(2)-induced competent DH5α cells. The data revealed loss in transformation efficiency of TiO(2)-NPs treated plasmids as compared to untreated plasmid DNA in DH5α host cells. Induction of multiple mutations in α-fragment of lacZ gene caused synthesis of non-functional β-galactosidase enzyme, which resulted in a significant number of white (mutant) colonies of transformed E. coli cells. Screening of mutant transformants based on blue:white colony assay and DNA sequence analysis of lacZ gene fragment clearly demonstrated TiO(2)-NPs induced mutagenesis. Multiple alignment of selectable marker lacZ gene sequences from randomly selected mutants and control cells provided a gene specific map of TiO(2)-NPs induced mutations. Mutational analysis suggested that all nucleotide changes were point mutations, predominantly transversions (TVs) and transitions (TSs). A total of 32 TVs and 6 TSs mutations were mapped within 296 nucleotides (nt) long partial sequence of lacZ gene. The region between 102 and 147nt within lacZ gene sequence was found to be most susceptible to mutations with nine detectable point mutations (8 TVs and 1 TSs). Guanine base was determined to be more prone to TiO(2)-NPs induced mutations. This study suggested the pUC19/E. coli DH5αlacZ gene α-complementation system, as a novel genetic approach for determining the mutagenic potential, and specificity of manufactured NPs and nanomaterials.     

Transformation of the Pseudonocardiaceae Amycolatopsis sp. strain HR167 is highly dependent on the physiological state of the cells

The Pseudonocardiaceae Amycolatopsis sp. strain HR167 is used in a biotransformation process to produce vanillin from ferulic acid. To make this strain accessible for genetic engineering, a direct mycelium transformation system developed for Amycolatopsis mediterranei [Madon and Hotter (1991) J Bacteriol 173: 6325-6331] was applied and optimized for Amycolatopsis sp. strain HR167. The physiological state of the cells had a major influence on the transformation rate. The highest transformation rate of about 7x10(5) transformants per microgram of DNA was obtained with mycelium harvested 6.5-7.5 h after the culture has reached the stationary growth phase. When cells were harvested outside of this time slot, the transformation rate drastically decreased. The density of the mycelium suspensions used in the transformation mixture and the methylation state of the plasmid DNA used for the transformation were also crucial parameters. With plasmid DNA isolated from Escherichia coli ET12567, transformation rates were 3,500-fold higher than those obtained with DNA isolated from E. coli XL1-Blue.     

The effect of the oligomerization of pBR322 on the level of transformation in Escherichia coli

The ability of the known Escherichia coli strain JC3881 recB recC recF sbc15 to produce oligomeric and multimeric forms of pBR322 underlies the study presented. The individual oligomeric forms of pBR322 were isolated from the agarose gel. The plasmid forms were used for electron microscopic control and also introduced into the system of E. coli competent cells. The E. coli transformation level of different forms of plasmid DNA rose from monomers to pentamers. CCC forms of the plasmid possessed high efficiency of the E. coli cell transformation. The systems of the host recombination are to be significant in the process of plasmid oligomerization.     

The molecular basis for the lack of immunostimulatory activity of vertebrate DNA

Macrophages and B cells are activated by unmethylated CpG-containing sequences in bacterial DNA. The lack of activity of self DNA has generally been attributed to CpG suppression and methylation, although the role of methylation is in doubt. The frequency of CpG in the mouse genome is 12.5% of Escherichia coli, with unmethylated CpG occurring at approximately 3% the frequency of E. coli. This suppression of CpG alone is insufficient to explain the inactivity of self DNA; vertebrate DNA was inactive at 100 micro g/ml, 3000 times the concentration at which E. coli DNA activity was observed. We sought to resolve why self DNA does not activate macrophages. Known active CpG motifs occurred in the mouse genome at 18% of random occurrence, similar to general CpG suppression. To examine the contribution of methylation, genomic DNAs were PCR amplified. Removal of methylation from the mouse genome revealed activity that was 23-fold lower than E. coli DNA, although there is only a 7-fold lower frequency of known active CpG motifs in the mouse genome. This discrepancy may be explained by G-rich sequences such as GGAGGGG, which potently inhibited activation and are found in greater frequency in the mouse than the E. coli genome. In summary, general CpG suppression, CpG methylation, inhibitory motifs, and saturable DNA uptake combined to explain the inactivity of self DNA. The immunostimulatory activity of DNA is determined by the frequency of unmethylated stimulatory sequences within an individual DNA strand and the ratio of stimulatory to inhibitory sequences.     

Release of transforming plasmid DNA from actively growing genetically engineered Escherichia coli

We studied the transforming ability of the extracellular plasmid DNA released from a genetically engineered Escherichia coli pEGFP and the culturing conditions for the release of transforming DNA. The transforming ability was evaluated by transformation of competent cells with filtrates of E. coli pEGFP cultures. The number of transformants increased with time when E. coli pEGFP cells grew exponentially in rich medium, but not in stationary phase or when inoculated in freshwater. These results suggested that crude extracellular plasmid DNA had transforming ability and this transforming DNA was mainly released by actively growing bacteria.     

外源质粒电击转入Pseudomonas putida的条件研究

以自行筛选的恶臭假单胞菌（Pseudomonas putida）(命名为Rs198,Genbank登录号为FJ788425）为受体菌,将具有卡那霉素抗性标记的大肠杆菌假单胞菌穿梭质粒PDSK519通过电转化法导入到受体菌中,对细胞生长状态、电转化温度、质粒DNA及感受态细胞浓度、电击电压及电转化介质给予转化效率的影响进行研究。结果表明,在细胞生长至OD600为0.5左右时收集菌体,在低温条件下制备浓度为 4.6×1012/ml 的感受态细胞,以0.3mol/L的蔗糖为电转化介质,在13kV/cm的场强下电击能获得较高的转化效率,最高可达1.3×107个转化子/μ g DNA。为构建恶臭假单胞的遗传转化系统,利用基因工程手段为该菌的进一步研究奠定了理论基础。     

Optimization of electrotransformation conditions for Propionibacterium acnes

Propionibacterium acnes has been known to be involved in the pathology of acne. However, the definite mechanism in the development of acne and the inflammation are unknown. For P. acnes, a transformation method has not been established, although it is believed to be a basic tool for gene manipulation. This study attempted to develop a P. acnes transformation method by using electroporation. Various parameters were used to develop and optimize the transformation of P. acnes. Among them two factors were crucial in the transformation for P. acnes: one was the E. coli strain from which the plasmid DNA had been isolated and the other the growth temperature of P. acnes-competent cells. It was essential to prepare plasmid DNA from a dam(-) E. coli strain, ET12567. When plasmid DNAs isolated from the other E. coli strains such as JM109 and HB101 were tested, transformation efficiency was extremely low. When P. acnes cells were cultivated at 24 degrees C for competent cell preparation, transformation efficiency increased considerably. When plasmid DNA isolated from a dam(-) mutant strain of E. coli was used for transformation of P. acnes which had been grown at 24 degrees C, maximum transformation efficiency of 1.5 x 10(4) transformants per mug of plasmid DNA was obtained at a field strength of 15 kV/cm with a pulse time of 3.2 ms. This is believed to be the first report on the transformation of P. acnes which can be employed for gene manipulations including knock-out of specific genes.     

Dcm methylation is detrimental to plasmid transformation in Clostridium thermocellum

ABSTRACT: BACKGROUND: Industrial production of biofuels and other products by cellulolytic microorganisms is of interest but hindered by the nascent state of genetic tools. Although a genetic system for Clostridium thermocellum DSM1313 has recently been developed, available methods achieve relatively low efficiency and similar plasmids can transform C. thermocellum at dramatically different efficiencies. RESULTS: We report an increase in transformation efficiency of C. thermocellum for a variety of plasmids by using DNA that has been methylated by Escherichia coli Dam but not Dcm methylases. When isolated from a dam+ dcm+ E. coli strain, pAMG206 transforms C. thermocellum 100-fold better than the similar plasmid pAMG205, which contains an additional Dcm methylation site in the pyrF gene. Upon removal of Dcm methylation, transformation with pAMG206 showed a four- to seven-fold increase in efficiency; however, transformation efficiency of pAMG205 increased 500-fold. Removal of the Dcm methylation site from the pAM205 pyrF gene via silent mutation resulted in increased transformation efficiencies equivalent to that of pAMG206. Upon proper methylation, transformation efficiency of plasmids bearing the pMK3 and pB6A origins of replication increased ca. three orders of magnitude. CONCLUSION: E. coli Dcm methylation decreases transformation efficiency in C. thermocellum DSM1313. The use of properly methylated plasmid DNA should facilitate genetic manipulation of this industrially relevant bacterium.     

Transformation of Escherichia coli with DNA from Saccharomyces cerevisiae Cell Lysates

We developed a system to monitor the transfer of heterologous DNA from a genetically manipulated strain of Saccharomyces cerevisiae to Escherichia coli. This system is based on a yeast strain that carries multiple integrated copies of a pUC-derived plasmid. The bacterial sequences are maintained in the yeast genome by selectable markers for lactose utilization. Lysates of the yeast strain were used to transform E. coli. Transfer of DNA was measured by determining the number of ampicillin-resistant E. coli clones. Our results show that transmission of the Amp^r gene to E. coli by genetic transformation, caused by DNA released from the yeast, occurs at a very low frequency (about 50 transformants per μg of DNA) under optimal conditions (a highly competent host strain and a highly efficient transformation procedure). These results suggest that under natural conditions, spontaneous transmission of chromosomal genes from genetically modified organisms is likely to be rare.     

Chromosomal model for analysis of a long CTG/CAG tract stability in wild-type Escherichia coli and its nucleotide excision repair mutants

Many human hereditary neurological diseases, including fragile X syndrome, myotonic dystrophy, and Friedreich's ataxia, are associated with expansions of the triplet repeat sequences (TRS) (CGG/CCG, CTG/CAG, and GAA/TTC) within or near specific genes. Mechanisms that mediate mutations of TRS include DNA replication, repair, and gene conversion and (or) recombination. The involvement of the repair systems in TRS instability was investigated in Escherichia coli on plasmid models, and the results showed that the deficiency of some nucleotide excision repair (NER) functions dramatically affects the stability of long CTG inserts. In such models in which there are tens or hundreds of plasmid molecules in each bacterial cell, repetitive sequences may interact between themselves and according to a recombination hypothesis, which may lead to expansions and deletions within such repeated tracts. Since one cannot control interaction between plasmids, it is also sometimes difficult to give precise interpretation of the results. Therefore, using modified lambda phage (lambdaInCh), we have constructed a chromosomal model to study the instability of trinucleotide repeat sequences in E. coli. We have shown that the stability of (CTG/CAG)68 tracts in the bacterial chromosome is influenced by mutations in NER genes in E. coli. The absence of the uvrC or uvrD gene products greatly enhances the instability of the TRS in the chromosome, whereas the lack of the functional UvrA or UvrB proteins causes substantial stabilization of (CTG/CAG) tracts.