Plasmid containing a DNA ligase gene from Haemophilus influenzae

A ligase gene from Haemophilus influenzae was cloned into the shuttle vector pDM2 . Although the plasmid did not affect X-ray sensitivity, it caused an increase in UV sensitivity of the wild-type but not excision-defective H. influenzae and a decrease in UV sensitivity of the rec-1 mutant.     

Cloning of the rec-2 locus of Haemophilus influenzae

A collection of transposon mutants of Haemophilus influenzae was constructed by additive transformation with mutagenized chromosomal DNA. A rec-2::miniTn10 km mutation was cloned from a transformation-defective member of the mutant collection, followed by the reconstruction of the wild-type rec-2 locus by recombination to create pDM62. Southern blots showed that the commonly studied Rec-2 mutant, Rd(DB117)rec-, contained either a large deletion or a substitution that removed part of rec-2 locus. A collection of transposon mutations in pDM62 was used to characterize the rec-2 locus by complementation. A corresponding collection of mutants was also constructed. A single segment was required to complement the transformation defect in Rd(DB117)rec-. All of the transformation-defective transposon mutants failed to translocate donor DNA into then cell, in agreement with previous studies of Rd(DB117)rec-.     

A plasmid carrying mucA and mucB genes from pKM101 in Haemophilus influenzae and Escherichia coli.

The plasmid pMucAMucB, constructed from the Haemophilus influenzae vector pDM2, and a similar plasmid, constructed from pBR322, increased the survival after UV irradiation of Escherichia coli AB1157 with the umu-36 mutation and also caused UV-induced mutation in the E. coli strain. In H. influenzae, pMucAMucB caused a small but reproducible increase in survival after UV irradiation in wild-type cells and in a rec-1 mutant, but there was no increase in spontaneous mutation in the wild type or in the rec-1 mutant and no UV-induced mutation.     

Novobiocin resistance marker in Haemophilus influenzae that is not expressed on a plasmid.

The plasmid pNov2, carrying a cloned chromosomal marker conferring resistance to at least 2.5 micrograms of novobiocin per ml, was constructed with a new Haemophilus influenzae cloning vehicle, pDM2. The novobiocin marker of pNov2 was not normally expressed, but in Rec+ cells approximately one in 10(4) cells in a culture of a transformant became novobiocin resistant, a frequency about four orders of magnitude higher than the spontaneous mutation frequency. Variants of such cells that had lost the plasmid were also novobiocin resistant. Since Rec- cultures bearing pNov2 showed novobiocin resistance only at the normal mutation frequency, we concluded that the Rec+ novobiocin-resistant transformants arose because of a rare recombination between plasmid and chromosome in which the chromosome acquired the novobiocin marker from the plasmid. Evidence is presented that novobiocin sensitivity is dominant over this particular novobiocin resistance marker.     

Catechins are not major components responsible for anti-genotoxic effects of tea extracts against nitroarenes

A plasmid called pMucA, from a piece of the plasmid pKM101 (Mol. Gen. Genet 167 (1979) 317) cloned in the vector pDM2 (J. Bacteriol. 151 (1982) 1605), caused higher mutation in a local region of Haemophilus influenzae and caused even more mutation there in a strain also containing novC, the latter causing an increase in supercoiling (J. Bacteriol 164 (1985) 525). The novD mutation depressed supercoiling, and also depressed the mutation by pMucA in the local region of the chromosome. Thus, it is clear that supercoiling is an important phenomenon in spontaneous mutation of H. influenzae. The pMucA plasmid caused a number of other phenomena in H. influenzae, induced UV mutation (Proc. Natl. Acad. Sci. USA 82 (1985) 7753), decreased UV sensitivity of transforming DNA, but not cells, and UV-induced recombination of mutants of phage HP1c1. The effect of the MucA protein in mutagenesis of H. influenzae we consider to be due to the introduction of some of the E. coli functions from pKM101. We postulate that the localized mutation caused by the MucA plasmid also involved localization of the plasmid or its coded protein in the same area, resulting from binding to a homologous gene, probably rec-1, very close to the localized region.     

Restriction digestion monitors facilitate plasmid construction and PCR cloning

Plasmid construction by "forced" or "directional" ligation of fragments digested with two different restriction enzymes is highly efficient, except when inhibited digestion of one site favors vector recircularization. Such failures often result because incomplete double digestion is undetected in vector polylinkers or at terminal cloning sites on a PCR fragment. To test cleavage efficiency indirectly, a "monitor" plasmid is added to the digest. In a suitable monitor, the two test sites are separated by enough DNA (approximately 20% of full length) to distinguish the double digest from the failed single digest. To make this applicable to combinations of 32 popular cloning enzymes, we constructed a set of 4 monitors (pDM1, pDM2, pDM3, and pDM4). Each contains three polylinkers separated by stuffer segments of approximately 1 kb. The 32 sites are distributed in the polylinkers such that at least one plasmid in the set is diagnostic for each enzyme pair. The set is designed to be extended to up to 81 sites. A linearized version of the monitor allows for the determination of which of the two enzymes has failed in an incomplete double digest and is also useful when the target DNA is close to the size of the pDM backbone. The plasmids also serve as versatile self-monitoring cloning vectors for any site combination.     

转异源chi基因的Bt工程菌株的生防潜力评价

【目的】构建增强抑制真菌能力兼杀虫的苏云金芽胞杆菌多功能生防菌株。【方法】将含有组成型高效表达启动子、地衣芽胞杆菌chi MY基因的重组质粒p DM,转化进杀虫活性高且有一定抑菌活性的Bt519-1菌株。酶谱分析方法确认Bt519(p DM)组成型异源表达几丁质酶。室内测定工程菌株抑菌谱,计算抑菌效率,确定最敏感的植物病原真菌,进行植物盆栽病害防治的应用潜力评价。将不同浓度的Bt粗酶液灌入甜椒幼苗根部,12 h后接种辣椒疫霉孢子液,接种2 d后开始观察,记录发病株数。自7 d起调查植株发病情况统计并分析防治效果。【结果】SDS-PAGE及酶谱分析证明,Bt519(p DM)能够特异表达68 k D蛋白,该蛋白为异源几丁质酶Chi MY。抑菌谱测定证明,工程菌抑制效率达到90%以上的有5种真菌,其中最明显的是辣椒疫霉。盆栽实验证明,Bt519(p DM)7 d的防效为73.2%。工程菌株对棉铃虫的半致死浓度(LC50)为121.26 mg/L。【结论】Bt519(p DM)是一株有应用潜力的生防菌株。     

Plasmid transformation and expression of the firefly luciferase in Microbacterium testaceum type and endophytic colonizing field strains

Microbacterium testaceum is a predominant endophytic bacterial species isolated from corn and sorghum in the midwestern United States. The development of genetic transfer systems for M. testaceum may enable its use for biocontrol and other applications. The type strain (IFO 12675) and field isolates (SE017, SE034, and CE648) were grown to mid-exponential phase, concentrated (1.0 x 1011 CFU x mL(-1)), electroporated (Escherichia coli-Clavibacter shuttle plasmid pDM302), and plated on TSA with 10 microg x mL(-1) chloramphenicol. Transformation efficiencies averaged 140 CFU x microg(-1) of DNA. Restriction endonuclease analysis showed that pDM302 was not altered after extraction from transformants and re-introduction into E. coli. Transformants with pDM302 were also subjected to nonselective growth conditions, with the frequency of loss after one passage being 84% for IFO 12675 and 88% for SE034. We inserted the green fluorescent protein and the firefly luciferase (FFlux) reporter genes into pDM302, confirming the expression of FFlux in IFO 12675 and SE034. The SE034 FFlux strain was recovered from inoculated corn in greenhouse studies and found to fluoresce by luminometry. These results in M. testaceum demonstrate for the first time its transformability, pDM302 replication, FFlux gene expression, and the recovery of the FFlux recombinant strain from inoculated corn.     

Stable transformation of the gram-positive phytopathogenic bacterium Clavibacter michiganensis subsp. sepedonicus with several cloning vectors.

In this paper we describe transformation of Clavibacter michiganensis subsp. sepedonicus, the potato ring rot bacterium, with plasmid vectors. Three of the plasmids used, pDM100, pDM302, and pDM306, contain the origin of replication from pCM1, a native plasmid of C. michiganensis subsp. michiganensis. We constructed two new cloning vectors, pHN205 and pHN216, by using the origin of replication of pCM2, another native plasmid of C. michiganensis subsp. michiganensis. Plasmids pDM302, pHN205, and pHN216 were stably maintained without antibiotic selection in various strains of C. michiganensis subsp. sepedonicus. We observed that for a single plasmid, different strains of C. michiganensis subsp. sepedonicus showed significantly different transformation efficiencies. We also found unexplained strain-to-strain differences in stability with various plasmid constructions containing different arrangements of antibiotic resistance genes and origins of replication. We examined the effect of a number of factors on transformation efficiency. The best transformation efficiencies were obtained when C. michiganensis subsp. sepedonicus cells were grown on DM agar plates, harvested during the early exponential growth phase, and used fresh (without freezing) for electroporation. The maximal transformation efficiency obtained was 4.6 x 10(4) CFU/microgram of pHN216 plasmid DNA. To demonstrate the utility of this transformation system, we cloned a beta-1,4-endoglucanase-encoding gene from C. michiganensis subsp. sepedonicus into pHN216. When this construction, pHN216:C8, was electroporated into competent cells of a cellulase-deficient mutant, it restored cellulase production to almost wild-type levels.     

Structure of the complexes of distamycin type antibiotics and actinomycin D with DNA: new data on the localization of these antibiotics within the DNA narrow groove]

It is shown that antibiotics actinomycin D (AM), netropsin (Nt), distamycin A (DM) and the propyl analogue of distamycin A (pDM) being complexed with DNA are located within the narrow groove of DNA. A comparative investigation of the 3H-dimethyl sulphate methylation extent of free calf thymus DNA and its complexes with AM, Nt, DM and pDM reveals that upon DNA saturation these antibiotics decrease the methylation level of the narrow groove (AM by 30%, pDM by 50%, DM by 65% and Nt by 70%). In the triple complex of DNA+AM+DM the methylation level of the narrow groove drops by 80%. The large groove is not shielded by these antibiotics at all. However, the methylation level of the large groove decreases by 50% for T6 phage DNA due to the presence of glucosyl residues linked to 5-hydroxymethylcytosine within the large groove. The binding of AM to DNA saturated with Nt or with the analogue of distamycin A (DM2) containing the 2 N-methylpyrrole residues has been investigated by spectrophotometry. The apparent number of binding sites for AM in these 2 complexes is about half as much as observed for free DNA while the saturation level of the binding decreased only by about 20%. This proves simultaneous presence of AM and Nt (DM2) within the narrow groove of DNA.     

Introduction of Resistance to Herbicide Basta® in Savoy Cabbage

Resistance to herbicide Basta® was introduced into pure inbred lines of Savoy cabbage (Brassica oleracea L. var. sabauda) by cocultivation of cotyledon and hypocotyl explants with Agrobacterium tumefaciens strains AGL1/pDM805 and LBA4404/pGKB5 (LB5-1). Shoot regeneration occurred on Murashige and Skoog medium supplemented with 1 mg dm^–3 6-benzyladenine and 0.5 mg dm^–3 indole-3-butyric acid at 42.3 % and 71.4 % of hypocotyl explants treated with AGL1/pDM805 and LB5-1, respectively. Putative transformants that survived selection on 10 mg dm^–3 phosphinothricin (L-PPT) supplemented medium were confirmed by GUS assay and PCR analysis. The transformation rate was 58 % with AGL1/ pDM805 and 25 % with LB5-1. Rooted plantlets were acclimated and then again screened for Basta®-resistance by spraying with 15 – 60 mg dm^–3 L-PPT. Surviving plants were selfed and Basta®-resistance was demonstrated in T₁ progeny.     

Differential behavior of plasmids containing chromosomal DNA insertions of various sizes during transformation and conjugation in Haemophilus influenzae.

Plasmids with chromosomal insertions were constructed by removal of a 1.1-kilobase-pair piece from the 9.8-kilobase-pair vector plasmid pDM2 by EcoRI digestion and inserting in its place various lengths of chromosomal DNA (1.7, 3.4, and 9.0 kilobase pairs) coding for resistance to novobiocin. A fourth plasmid was constructed by insertion of the largest piece of chromosomal DNA into the SmaI site of pDM2. The plasmids without inserts were taken up poorly by competent cells and thus were considered not to contain specific DNA uptake sites. The presence of even the smallest insert of chromosomal DNA caused a large increase in transformation of Rec+ and Rec- strains. The frequency of plasmid establishment in Rec+ cells by transformation increased exponentially with increasing insert size, but in Rec- cells there was less transformation by the larger plasmids. Conjugal transfer of these plasmids was carried out with the 35-kilobase-pair mobilizing plasmid pHD147. The frequency of establishment of plasmids by this method not only was not markedly affected by the presence of the insertions, but also decreased somewhat with increase in insert size and was independent of rec-1 and rec-2 genes. Recombination between plasmid and chromosome was readily detected after transformation, but could not be detected after transconjugation even when the recipient cells were Rec+ and made competent. These data suggested that there is a special processing of plasmid DNA that enters the competent cells in transformation that makes possible recombination of homologous regions of the plasmid with the chromosome and pairing with the chromosome that aids plasmid establishment.     

Isolation of a single-stranded plasmid from Clostridium acetobutylicum NCIB 6444.

The cryptic plasmid pDM6 was isolated from late exponential-phase cells of Clostridium acetobutylicum NCIB 6444 by either alkaline lysis or electroporation. The application of high voltage during electroporation resulted in higher DNA yield than did the alkaline lysis procedure. However, electroporation-induced plasmid release generated high amounts of single-stranded DNA compared with the alkaline lysis procedure, which generated both double-stranded DNA (monomer and dimer forms) and single-stranded DNA.     

通过基因枪轰击转化获得转基因小麦植株的研究

利用ＪＱ－７００型高速基因枪将ｐＤＭ３０２质粒ＤＮＡ上的ｂａｒ基因即ＰＡＴ酶基因导入了冬小麦品种“农大１４６”的幼胚中。经过在含有的选择培养基上筛选,得到了９块具有ｐｐｔ抗性的愈伤组织。ＰＣＲ电泳检测与ＰＣＲ－Ｓｏｕｔｈｅｒｎ发要交结果显示,外源ｂａｒ基因已转化进了由其中４块愈伤组织再生出的转基因的小麦植株中。     

Mobilization of Haemophilus influenzae chromosomal markers by an Escherichia coli F'' factor.

Filter matings between E. coli K-12 strains carrying an F'::Tn5,Tn9 factor with H. influenzae Rd strains gave rise to kanamycin-chloramphenicol-resistant H. influenzae strains at a frequency of approximately 10(-6). Transfer of the F' factor to H. influenzae was verified by expression of unselected markers in H. influenzae (lac+ or cotransfer of the nonselected antibiotic resistance), physical presence of a high-molecular-weight plasmid in recipient H. influenzae cells, and detection by Southern hybridization analysis of DNA sequences specific for the F' factor replication and partition functions in recipient H. influenzae cells. H. influenzae (F' Tn5,Tn9) strains were capable of transferring kanamycin and chloramphenicol resistances to other H. influenzae strains and were capable of mobilizing H. influenzae chromosomal markers at a low frequency. Insertion of a Tn5 element in the H. influenzae genome near the novobiocin resistance gene increased the frequency of transfer of novobiocin resistance about 30-fold. Transfer of other chromosomal markers also increased, although to a lesser extent, and ordered transfer of chromosomal markers could be demonstrated. Gene transfer was insensitive to DNase I, and transfer of chromosomal (but not F' factor) markers was dependent on the H. influenzae rec-1 and rec-2 gene functions.     

Isolation of a single-stranded plasmid from Clostridium acetobutylicum NCIB 6444

The cryptic plasmid pDM6 was isolated from late exponential-phase cells of Clostridium acetobutylicum NCIB 6444 by either alkaline lysis or electroporation. The application of high voltage during electroporation resulted in higher DNA yield than did the alkaline lysis procedure. However, electroporation-induced plasmid release generated high amounts of single-stranded DNA compared with the alkaline lysis procedure, which generated both double-stranded DNA (monomer and dimer forms) and single-stranded DNA.     

Isolation of Haemophilus influenzae genes that suppress Escherichia coli polA mutations

Haemophilus influenzae was found to produce a DNA polymerase that was similar to polymerase I of Escherichia coli. E. coli polA mutants were used as backgrounds for the selection of H. influenzae polA suppressor genes. Six different H. influenzae fragments were isolated that could suppress E. coli polA mutations. None of the suppressors appeared to encode the H. influenzae equivalent of the E. coli polA gene. One type of clone, represented by pGW41, caused a polymerase I activity to appear in a suppressed polA1 mutant. Plasmids from the pGW41 class contained two genes (pol-2 and pol-3) that were both required for polA suppression. Mutated nonsuppressing derivatives of the pGW41 class were used to create H. influenzae mutants that were deficient in polymerase I.     

利用电激法转化小麦幼胚的研究

利用我们实验自制的脉冲放电式电激仪ＨＰＥＳ－３,我们将含ｂａｒ基因即ＰＡＴ酶 ｐＤＭ３０２质粒ＤＮＡ导入了一个春小品种“Ｔ２００３”的幼胚中。经过在含有ｐｐｔ的选择培养基上筛选,得到了抗ｐｐｔ的愈伤组织和再生小苗。     

Effects of plant genotype and bacterial strain on <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of <Emphasis Type="Italic">Brassica oleracea</Emphasis> L. var. <Emphasis Type="Italic">capitata</Emphasis>

Two inbred lines of Brassica oleracea L. var. capitata were transformed with two Agrobacterium tumefaciens strains harboring resistance to herbicide Basta: AGL1/pDM805 and LBA4404/pGKB5 (LB5-1). Inoculated cotyledons and hypocotyls provided equally good explants and manifested a high percentage of shoot regeneration on MS medium supplemented with 1 mg/l benzyladenine and 0.5 mg/l indole-3-butyric acid. The P₃₄I₅ genotype was superior to P₂₂I₅ in shoot regeneration (48.1 vs. 26.9%), multiplication, and acclimation in the greenhouse (76 vs. 40%). A. tumefaciens AGL1/pDM805 provided more regenerated shoots per explant, especially in the case of cotyledon explants, and the higher transformation rate (up to 35% vs. up to 12%) as compared to LB5-1. Putative transformants survived spraying with 10–30 mg/l phosphinothricin. Transformation was confirmed by GUS assay and PCR analysis in T0 and T1 generations. Published in Russian in Fiziologiya Rastenii, 2007, vol. 54, No. 5, pp. 738–743. The text was submitted by the authors in English.     

A high-throughput <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation system for the grass model species <Emphasis Type="Italic">Brachypodium distachyon</Emphasis> L.

In the ongoing process of developing Brachypodium distachyon as a model plant for temperate cereals and forage grasses, we have developed a high-throughput Agrobacterium-mediated transformation system for a diploid accession. Embryogenic callus, derived from immature embryos of the accession BDR018, were transformed with Agrobacterium tumefaciens strain AGL1 carrying two T-DNA plasmids, pDM805 and pWBV-Ds-Ubi-bar-Ds. Transient and stable transformation efficiencies were optimised by varying the pre-cultivation period, which had a strong effect on stable transformation efficiency. On average 55% of 17-day-old calli co-inoculated with Agrobacterium regenerated stable transgenic plants. Stable transformation frequencies of up to 80%, which to our knowledge is the highest transformation efficiency reported in graminaceous species, were observed. In a study of 177 transgenic lines transformed with pDM805, all of the regenerated transgenic lines were resistant to BASTA((R)), while the gusA gene was expressed in 88% of the transgenic lines. Southern blot analysis revealed that 35% of the tested plants had a single T-DNA integration. Segregation analysis performed on progenies of ten selected T(0) plants indicated simple Mendelian inheritance of the two transgenes. Furthermore, the presence of two selection marker genes, bar and hpt, on the T-DNA of pWBV-Ds-Ubi-bar-Ds allowed us to characterize the developed transformation protocol with respect to full-length integration rate. Even when not selected for, full-length integration occurred in 97% of the transformants when using bialaphos as selection agent.