Detection in soil of a deletion in an engineered DNA sequence by using DNA probes.

Two Pseudomonas strains were engineered to contain the nptII gene and plasmid vector sequences in their chromosomes. After incubation of these strains in nonsterile soil, total bacterial DNA was isolated and analyzed by Southern blot hybridization with the nptII gene and the plasmid vector as probes. In addition to the expected bands of hybridization, a new band corresponding to the loss of vector sequences from the chromosome while retaining the nptII gene was observed for one of the strains. The more stressful conditions encountered in soil appeared to increase the frequency of loss of the vector sequences from this strain.     

The natural transformation of the soil bacteria Pseudomonas stutzeri and Acinetobacter sp. by transgenic plant DNA strictly depends on homologous sequences in the recipient cells

The nptII(+) gene present in the genome of transgenic potato plants transforms naturally competent cells of the soil bacteria Pseudomonas stutzeri and Acinetobacter BD413 (both harboring a plasmid with an nptII gene containing a small deletion) with the same high efficiency as nptII(+) genes on plasmid DNA (3x10(-5)-1x10(-4) transformants per nptII(+)) despite the presence of a more than 10(6)-fold excess of plant DNA. However, in the absence of homologous sequences in the recipient cells the transformation by nptII(+) dropped by at least about 10(8)-fold in P. stutzeri and 10(9)-fold in Acinetobacter resulting in the latter strain in < or =1x10(-13) transformants per nptII(+). This indicated a very low probability of non-homologous DNA fragments to be integrated by illegitimate recombination events during transformation.     

Detection of Tn5-like sequences in kanamycin-resistant stream bacteria and environmental DNA.

Resistance to kanamycin and neomycin in the bacterial assemblage of a coastal plain stream was detected by growth of colonies on media containing antibiotics. Three of 184 kanamycin-resistant colonies hybridized with a probe containing the nptII gene from transposon Tn5; the nptII gene encodes the enzyme neomycin phosphotransferase and conveys resistance to kanamycin and neomycin. In one of these isolates, the homologous gene was cloned and shown to confer resistance to a kanamycin-sensitive Escherichia coli strain. Since enumeration of bacteria by acridine orange direct counts revealed that less than 0.2% of the bacteria present were cultivated, direct examination of environmental DNA was used to assess abundance of sequences that hybridize to the nptII gene. To examine the resistance potential of bacteria that were not cultured, total DNA was extracted from environmental samples and hybridized with specific probes. The relative amount of eubacterial DNA in each sample was determined by using a eubacterial specific rDNA probe. Then, the abundance of sequences that hybridize to the eubacterial neomycin phosphotransferase gene was determined by hybridization and expressed relative to the total eubacterial DNA in the assemblage. Relative gene abundance was significantly different among assemblages from different habitats (leaves, midchannel sediments, and bank sediments) but did not differ among stream sites.     

Identification and cloning of a plasmid-encoded erythromycin resistance determinant from Lactobacillus reuteri

Plasmid analysis, plasmid curing, cloning, and hybridization experiments were used to study four Lactobacillus reuteri strains showing high resistance to erythromycin. Plasmid curing with acriflavine resulted in a loss of erythromycin resistance in a frequency of 1-10%. For three of the strains this was accompanied by a loss of a 6.9-MDa plasmid, which was shown to be identical for the different strains and designated pLUL631. The erythromycin (erm) gene was located on a 5.5-MDa plasmid in the fourth strain. A restriction map of pLUL631 was constructed and the location of the erm gene on the plasmid was identified by cloning in Escherichia coli. By using a Streptococcus lactis-E. coli shuttle vector, the erm gene was also transformed to S. lactis and expressed. The erm gene from L. reuteri was shown to be related to the erm gene from pIP501 (Streptococcus agalactiae) by DNA-DNA hybridization.     

Tn 5 to assess soil fate of genetically marked bacteria: screening for aminoglycoside-resistance advantage and labelling specificity

Abstract The reliability of Tn 5 as labelling tool was investigated in soil microcosm. The occurence of a selective in soil microcosm. The occurence of resistances encoded by Tn 5 nptII gene was assesed by kanamycin and neomycin amendment. The bioassay developed to monitor the persistence of the soil-added kanamycin did not detect the antibiotic activity in soil extract. A nptII -engineered Escherichia coli strain showed no enhanced survival in aminoglycoside amended soil. Tn 5-marker properties were investigated within indigenous bacteria to determine the specificity of labelling to follow the fate of recombinant DNA. Kanamycin and neomycin resistant population levels made Tn 5 aminoglycoside-resistance phenotype non-sensitive enough to select a soil dissemination of the labelled DNA. The unexpected occurrence of homologous sequences among soil organisms also prevented Tn 5 from being a specific DNA marker. By contrast, colony hybridization did not reveal homology to nptII suggesting its use as a reliable gene transfer indicator.     

Heat-shock-mediated elimination of the nptII marker gene in transgenic apple (Malus×domestica Borkh.)

Production of marker-free genetically modified (GM) plants is one of the major challenges of molecular fruit breeding. Employing clean vector technologies, allowing the removal of undesired DNA sequences from GM plants, this goal can be achieved. The present study describes the establishment of a clean vector system in apple Malus×domestica Borkh., which is based on the use of the neomycin phosphotransferase II gene (nptII) as selectable marker gene and kanamycin/paramomycin as selective agent. The nptII gene can be removed after selection of GM shoots via site-specific excision mediated by heat-shock-inducible expression of the budding yeast FLP recombinase driven by the soybean Gmhsp17.5-E promoter. We created a monitoring vector containing the nptII and the flp gene as a box flanked by two direct repeats of the flp recognition target (FRT) sites. The FRT-flanked box separates the gusA reporter gene from the Cauliflower Mosaic Virus 35S (CaMV 35S) promoter. Consequently, GUS expression does only occur after elimination of the FRT-flanked box. Transformation experiments using the monitoring vector resulted in a total of nine transgenic lines. These lines were investigated for transgenicity by PCR, RT-PCR and Southern hybridization. Among different temperature regimes tested, exposure to 42 °C for 3.5 to 4h led to efficient induction of FLP-mediated recombination and removal of the nptII marker gene. A second round of shoot regeneration from leaf explants led to GM apple plants completely free of the nptII gene.     

Transformation of Acinetobacter sp. BD413 with DNA from commercially available genetically modified potato and papaya

AIM: To estimate the likelihood of transfer of kanamycin-resistance gene (nptII) from commercially available genetically modified (GM) plants. METHODS AND RESULTS: Acinetobacter sp. BD413 carrying a plasmid containing an inactivated nptII gene was treated with DNA derived from GM potato and GM papaya. Kanamycin-resistant transformants were obtained at a frequency of 10-30 microg(-1) DNA. Calculation of the results suggested that 6-9 x 10(4) molecules of genomic DNA from GM plants were needed to obtain one transformant. However, such transformation events were not detectable in the absence of the plasmid in the host strain. CONCLUSIONS: Acinetobacter sp. BD413 was transformed with DNA derived from GM potato and GM papaya, in the presence of an inactivated nptII gene on a plasmid. However, the frequency of such events in the natural environment on wild-type strains, while evidently low, remains unknown. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results may help to evaluate potential risks associated with the use of antibiotic-resistance determinants as genetic markers in GM plants. Complete risk assessment must consider factors other than transformation frequency alone, including the natural background of antibiotic resistance present in bacterial populations, and the spectrum and clinical use of the antimicrobial agents in question.     

Homology between bacterial DNA and bovine mitochondrial DNA encoding cytochrome c oxidase subunit III

A segment of mitochondrial DNA encoding the bovine cytochrome c oxidase subunit III gene was isolated and inserted into an Escherichia coli plasmid vector. A 556 base pair fragment of the insert DNA representing about 70% of the 3'-end of the subunit III gene was used to search for homology with bacterial DNA from strains that contain heme aa3-type cytochrome c oxidases. Bacillus subtilis, Thermus thermophilus, and PS3 DNAs all showed strong hybridization to the probe, whereas Paracoccus denitrificans and Rhodopseudomonas sphaeroides DNAs showed only weak hybridization to the probe, even under low stringency conditions.     

Molecular and cellular evidence of chimaeric tissues in primary transgenics and elimination of chimaerism through improved selection protocols

Transgenic plants of strawberry cultivar Totem were developed by Agrobacterium-mediated transformation using a plasmid vector containing gus and nptII genes. Parallel experiments were carried out with and without repeated subculturing (iterative cultures) for generation of transgenic shoots on selection medium. The selection levels in the non-iterative pathway were kept constant, while in the iterative protocol, stepwise increase of selection pressure was applied at different stages of tissue growth. Rooted transgenic plants obtained via both protocols were outplanted in soil. Random leaf samples of greenhouse-grown transgenics were analysed for the presence of gus gene sequences by Southern hybridization as well as gus expression on leaf and petiole tissues by X-Gluc histological assay. Random leaf samples analysed from individual transgenic events developed under iterative culture were positive for the gus insert as verified by Southern analysis confirming the presence of transgenes and lack of chimaeras. Leaf samples of the transgenic events from the non-iterative protocol were either positive or negative on Southern analysis indicating the chimaeric nature of the transgenic plants. The absence of gus sequences in the transgenic plants grown under the non-iterative protocol reinforced the necessity of iterative cultures along with stepwise increase in selection levels for generating non-chimaeric transgenics in strawberry. The gus expression was highly variable, irrespective of the iterative or non-iterative protocol used for transformation. We conclude that strawberry is highly prone to develop chimaeric transgenics if derived from primary regenerants and that the iterative culture technique effectively converts chimaeras to pure line transgenic plants     

An improved procedure for production of white spruce (Picea glauca) transgenic plants using Agrobacterium tumefaciens.

An efficient and reproducible procedure for the transformation of white spruce (Picea glauca [Moench] Voss) embryogenic tissues was developed using A. tumefaciens-mediated gene transfer. Rapidly dividing white spruce embryogenic tissues were co-cultivated with disarmed A. tumefaciens strains containing additional copies of the virulence regions from plasmid PToK47. The plasmid pBi121, containing the neomycin phosphotransferase II (nptII) gene providing kanamycin resistance as a selectable marker and the beta-glucuronidase (uidA) reporter gene, was used as binary vector. The highest frequency of transformation (15 transformed tissues g(-1) FW of treated embryogenic tissue) was obtained with 5-d-old tissues grown in liquid medium and co-cultivated with Agrobacterium for 2 d in the same medium but containing 50 microM acetosyringone. Recovery of kanamycin-resistant tissues was improved when tissues were first grown for 10 d on a timentin-containing medium (400 mg l(-1)), to prevent bacterial overgrowth, before application of the selection pressure. After 6 weeks on kanamycin-selection medium, resistant tissues were obtained and showed stable uidA expression. The presence of the transgenes was demonstrated by PCR analysis and their integration into the genome was confirmed by Southern hybridization. Transgenic plants were regenerated from transformed tissues within 4 months after co-culture.     

Transferable Streptomyces DNA amplification and coamplification of foreign DNA sequences.

The 8.8-kb amplifiable unit of DNA of Streptomyces achromogenes subsp. rubradiris, AUD-Sar 1, which carries 0.8-kb terminal direct repeats and a spectinomycin resistance determinant, can mediate high-level amplification of an AUD-Sar 1-derived 8.0-kb DNA sequence not only in S. achromogenes but also in the heterologous host Streptomyces lividans. This was seen upon introduction of AUD-Sar 1 into chloramphenicol-sensitive strains of S. lividans via the temperature-sensitive (39 degrees C) plasmid pMT660, which contains the thiostrepton resistance gene tsr. Following the cultivation of transformants at 39 degrees C on media containing spectinomycin, a number of strains which were unable to grow on thiostrepton and which carried the amplified 8.0-kb DNA sequence as arrays of 200 to 300 copies of tandem 8.0-kb repeats were found. Chloramphenicol-resistant strains of S. lividans did not yield amplified sequences under similar conditions. Studies with plasmids carrying inserted antibiotic resistance genes at two sites of AUD-Sar 1 yielded coamplified sequences which contain the inserted DNA. Transformation with a plasmid carrying a 1.0-kb deletion in AUD-Sar 1 followed by growth under similar conditions yielded a 7.0-kb repeated DNA sequence. Southern analysis revealed the absence of vector sequences located on the right side of AUD-Sar 1 in the input plasmids in all examined DNA samples of amplified strains. In contrast, a majority of the samples revealed the presence at unit copy level of AUD-Sar 1 left-adjacent sequences which are part of the input plasmids and in several samples the presence of certain vector sequences located near them. The results suggest input plasmid integration into the S. lividans chromosome prior to the generation of the amplified sequences and the deletion of AUD-Sar 1 adjacent sequences.     

Cloning ofCandida boidinii DNA fragments promoting autonomous replication of plasmids inSaccharomyces cerevisiae

Fragments of Candida boidinii chromosomal DNA were inserted into the integrative vector YIp-kanr and examined for the presence of sequences promoting autonomous replication of plasmids in Saccharomyces cerevisiae. Restriction maps of two plasmids, designated S6/4 and S6/5, originating from the same S. cerevisiae transformant, were constructed. Southern hybridization data confirmed that the plasmids carry sequences from the C. boidinii chromosome. Both plasmids transform S. cerevisiae strains at 4-5-fold higher frequency than cloning vectors based on the replication origin of the 2 microns plasmid. Mitotic stability of the constructed plasmids is similar to that of the 2 mu-based vector pNF2 in S. cerevisiae.     

Persistence of Free Plasmid DNA in Soil Monitored by Various Methods, Including a Transformation Assay

The persistence and stability of free plasmid pUC8-ISP DNA introduced into 10-g samples of various soils and kept at 23°C were monitored over a period of 60 days. The soils were sampled at a plant science farm and included a loamy sand soil (no. 1), a clay soil (no. 2), and a silty clay soil (no. 3). Four different methods allowed monitoring of (i) the production of acid-soluble radioactive material from [³H]thymidine-labeled plasmid DNA, (ii) the decrease of hybridizing nucleotide sequences in slot blot analysis, (iii) the loss of plasmid integrity measured by Southern hybridization, and (iv) the decay of the biological activity as determined by transformation of Ca²⁺-treated Escherichia coli cells with the DNA extracted from soil. Acid-soluble material was not produced within the first 24 h but then increased to 45% (soil no. 1), 27% (soil no. 2), and 77% (soil no. 3) until the end of incubation. A quite parallel loss of material giving a slot blot hybridization signal was observed. Southern hybridization indicated that after 1 h in the soils, plasmid DNA was mostly in the form of circular and full-length linear molecules but that, depending on the soil type, after 2 to 5 days full-length plasmid molecules were hardly detectable. The transforming activity of plasmid DNA reextracted from the soils followed inactivation curves over 2 to 4 orders of magnitude and dropped below the detection limit after 10 days. The inactivation was slower in soil no. 2 (28.2-h half-life time of the transforming activity of a plasmid molecule) than in soils no. 3 (15.1 h) and no. 1 (9.1 h). The studies provide data on the persistence of free DNA molecules in natural bacterial soil habitats. The data suggest that plasmid DNA may persist long enough to be available for uptake by competent recipient cells in situ.     

DNA Polymorphisms in Lentinula edodes, the Shiitake Mushroom

DNA restriction fragment length polymorphisms (RFLPs) were examined in Lentinula edodes strains. Genomic DNA from strain 70 was cloned in plasmid vector pUC19, and 18 random clones containing low-copy DNA sequences were used to probe seven strains in Southern DNA-DNA hybridizations. Each cloned fragment revealed DNA polymorphism. An RFLP genotype was determined for each strain and the genetic relatedness was assessed. The coefficients of genetic similarity among the seven strains ranged from 0.43 to 0.90. The inheritance of RFLP markers was examined in single spore isolates. Homokaryons displayed a loss of polymorphic bands compared with the parent dikaryon. Hybrids constructed by crossing compatible homokaryons displayed the inheritance of RFLP markers from each parent homokaryon.     

华癸中生根瘤菌质粒复制基因repC的克隆与鉴定

摘要：【目的】repC为质粒复制必需的起始蛋白基因。本研究旨在对华癸中生根瘤菌菌株HN3015及其质粒消除突变株进行repC基因的克隆和鉴定。【方法】采用通用引物RC1和RC3进行repC基因的PCR扩增,扩增产物克隆到载体pMD-18T,然后测序。利用Southern 杂交对repC基因定位。利用在线软件分析基因的序列特征,BLAST 工具进行同源性搜索;ExPASy推断其氨基酸的序列;ClustalW进行同源核苷酸和氨基酸序列的多重比较分析;PredictProtein 进行蛋白二级结构分析。【结果】     

Characterization of the Salmonella typhimurium phoE gene and development of Salmonella-specific DNA probes.

In Escherichia coli K-12, the phoE gene, encoding a phosphate-limitation-inducible outer membrane pore protein (PhoE), is closely linked to the genes proA and proB. When the corresponding fragment of the Salmonella typhimurium chromosome was transferred to E. coli K-12 using an RP4::miniMu plasmid, pULB113, no production of S. typhimurium PhoE could be detected. Nevertheless, DNA hybridization studies revealed that the corresponding plasmid did contain S. typhimurium phoE. Production of S. typhimurium PhoE in E. coli was detected only after subcloning the gene in a multicopy vector. Nucleotide (nt) sequence analysis showed extensive homology of S. typhimurium phoE to the E. coli gene and suggested possible explanations for the low expression of S. typhimurium phoE in E. coli. In addition, the sequence information was used to develop Salmonella-specific DNA probes. Two oligodeoxyribonucleotides were synthesized based on nt sequences encoding the fifth and eighth cell-surface-exposed regions of PhoE. When used in polymerase chain reactions, these probes turned out to be specific, i.e., no crossreactions occurred with the non-Salmonella strains, whereas 132 out of 133 tested Salmonella strains were recognized.