Solid-phase clean-up and thin-layer chromatographic detection of veterinary aminoglycosides

Chemical methods are needed to confirm the presence of antibiotics detected by microbial inhibition assays in fluids and tissues of farm animals. We have optimized the conditions for the isolation of hygromycin B with a copolymeric bonded solid-phase silica column followed by thin-layer chromatography (TLC) separation and detection of its fluorescence derivative after reaction with fluorescamine. The detection limit of the drug was 50 ng. Serum and plasma samples fortified with hygromycin B were acidified and passed through the copolymerized solid-phase columns previously conditioned with phosphate buffer. Hygromycin B was trapped in the columns and eluted with diethylamine-methanol and analyzed by TLC using acetone-ethanol-ammonium hydroxide as the developing solvent. Hygromycin B bands were derivatized at acidic pH with fluorescamine and visualized under ultraviolet light. Hygromycin B added to bovine plasma was detectable at 25, 50, 100, 250 and 500 ng/ml (ppb). Hygromycin B added to swine serum was detected at 50 ng/ml. However, the serum had to be deproteinized with trichloroacetic acid or acetonitrile prior to solid-phase extraction to gain accurate values. Neomycin and gentamicin (100 ng/ml aqueous solutions) could also be isolated with copolymeric solid-phase columns at a level of 50 ng. Gentamicin, neomycin, gentamicin, spectinomycin, hygromycin B and streptomycin could be separated by TLC, allowing multiresidue detection of these aminoglycosides. The respective R_F values of 0.64, 0.56, 0.52, 0.33 and 0.20 indicate the separation of these five compounds. This procedure provides a rapid and sensitive method for the semi-quantitative estimation of aminoglycosides.     

Development of a chromatographic method for the isolation and detection of hygromycin B in biological fluids

An affinity chromatography method was developed for the purification of hygromycin B from biological fluids. Lysozyme and α-lactalbumin were immobilized on an N-hydroxysuccinimide activated agarose support. Hygromycin B solubilized in water was bound by the proteins and subsequently eluted using 10 mM sodium citrate buffer, pH 4.0. Hygromycin B was purified from swine plasma, bovine serum and bovine milk samples using a combination of ion-exchange chromatography for initial clean-up of spiked biological samples followed by affinity chromatography. Thin layer chromatographic analysis of the isolated hygromycin B revealed one band with the same R_F value as the hygromycin B standard.     

Generation of transgenic Lolium temulentum plants by Agrobacterium tumefaciens-mediated transformation

Lolium temulentum L. (Darnel ryegrass) has been proposed to be used as a model species for functional genomics studies in forage and turf grasses, because it is a self-fertile, diploid species with a short life cycle and is closely related to other grasses. Embryogenic calluses were induced from mature embryos of a double haploid line developed through anther culture. The calluses were broken up into small pieces and used for Agrobacterium tumefaciens-mediated transformation. A. tumefaciens strain EHA105 harboring pCAMBIA1301 and pCAMBIA1305.2 vectors were used to infect embryogenic callus pieces. Hygromycin was used as a selection agent in stable transformation experiments. Hygromycin resistant calluses were obtained after 4–6 weeks of selection and transgenic plants were produced in 10–13 weeks after Agrobacterium-mediated transformation. Fertile plants were readily obtained after transferring the transgenics to the greenhouse. Transgenic nature of the regenerated plants was demonstrated by Polymerase chain reaction (PCR), Southern hybridization analysis, and GUS staining. Progeny analysis showed Mendelian inheritance of the transgenes. The transformation system provides a valuable tool for functionality tests of candidate genes in forage and turf grasses.     

A Simplified and efficient method for transformation and gene tagging of Ustilago maydis using frozen cells

Ustilago maydis is an important model fungal organism for diverse studies. Little improvement has been made in the method for its transformation since the PEG-mediated transfection of spheroplasts that was reported more than 20 years ago. We have constructed binary T-DNA vectors carrying Hygromycin and Nourseothricin resistance gene cassettes and have developed a highly efficient method for transformation of this fungus based on Agrobacterium tumefaciens-mediated transformation (ATMT). Through a series of optimization, at least 1 × 10⁴ Hygromycin B resistant colony forming units (CFU) have been achieved on each 90 mm agar plate using 10⁶ sporidia. Optimal pH value for ATMT is approximately 5.6. Approximately 96% Hygromycin B-resistant transformants contain a single-copy T-DNA inserted into the nuclear genome. Analysis of 204 T-DNA flanking sequences showed that 15.2% of them were found in the coding sequences and a further 37.25% within 0.5 kb from the coding sequences at the 5′ UTR or promoter regions. In addition, a method for preparation and preservation of transformation-ready T-DNA donor and receptor cells has been developed allowing gene tagging experiments to be performed on-demand. An initial screening of 5000 mutants resulted in the identification of a putative farnesyl transferase beta subunit and a PRE6 homologue as new players of sexual mating in U. maydis.     

Hygromycin resistance gene cassettes for vector construction and selection of transformed rice protoplasts

Hygromycin resistance gene cassettes were designed to facilitate vector construction for plant transformation. Unique EcoRI, Pstl, and Sacll sites in the coding sequence of a hygromycin B phosphotransferase gene (hph) from Escherichia coli were eliminated. The mutated hph genes were used to form gene cassettes flanked by EcoRI-Sacll-Kpnl-Hindlll sites. Hygromycin resistance of wild-type and mutated hph genes was indistinguishable in E. coli and rice protoplast growth assay.     

Agrobacterium tumefaciens-mediated genetic transformation of the white root rot ascomycete Rosellinia necatrix

Hygromycin B resistance was conferred to the mycelium of the white root rot fungus Rosellinia necatrix by transformation with the hygromycin B phosphotransferase gene (hph) of Escherichia coli under the control of the heterologous fungal Aspergillus nidulans P-gpd (glyceraldehyde 3-phosphate dehydrogenase) promoter and the trpC terminator. In all three transformants, the presence of hph and single-copy integrations of the marker gene were demonstrated by Southern analysis. This is the first report describing A. tumefaciens-mediated transformation of R. necatrix     

潮霉素A的生物合成研究进展

潮霉素A是一种从吸水链霉菌中发现的具有广谱生物学活性的抗生素。它在吸水链霉菌Streptomy-ces hygroscopicus NRRL 2388中的生物合成基因簇已被克隆并测序,其生物合成机制、遗传操作等方面的研究也取得了一定的进展。就潮霉素A的化学结构、生物合成基因簇的组织结构、生物合成和抗性机制等方面的研究进展进行综述。     

香菇对潮霉素的抗性实验

选用4株不同品系的香菇菌种,比较了香菇在固体、液体2种不同培养条件下,对潮霉素抗性的差异;研究了香菇以原生质体、原生质体再生菌丝、冷藏菌种等3种不同生理状态为实验材料,对潮霉素的抗性特点。试验表明,香菇对潮霉素极为敏感,在固定的条件下,抗性稳定;而当条件改变时,抗性有一定变化。     

Cadophora finlandia and Phialocephala fortinii: Agrobacterium-mediated transformation and functional GFP expression

Hygromycin B resistance was transferred to the sterile mycelia of Cadophora finlandia and Phialocephala fortinii by co-cultivation with Agrobacterium tumefaciens. Constitutively expressed green fluorescent protein (GFP) was also introduced using the same vector. Confocal laser scanning microscopy (CLSM) revealed strong fluorescence of transformants. Both traits were mitotically stable during one year of subculturing on non-selective growth medium. Southern blot analysis showed that the majority of the transformants contained single-copy integrations at random sites in the genome.     

Genetic transformation of golden pothos (<Emphasis Type="Italic">Epipremnum aureum</Emphasis>) mediated by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

To establish a procedure for Agrobacterium tumefaciens-mediated transformation of golden pothos (Epipremnum aureum) plants, the effects of selection antibiotics and the preculture period of stem explants before A. tumefaciens infection were examined. Explants were co-cultivated with A. tumefaciens EHA105, harboring the plasmid pGWB2/cGUS, on a somatic embryo-inducing medium supplemented with acetosyringone. Resulting transgenic somatic embryos were screened on an antibiotic selection medium, and the transgenic pothos plants were regenerated on a germination medium. Hygromycin was the optimum selection antibiotic tested. The preculture period significantly affected the transformation efficiency, with explants precultured for one-day showing the best efficiency (5–30%). Both transformed hygromycin-resistant embryos and regenerated plants showed β-glucuronidase activity. Southern blot analysis confirmed transgene integration into the pothos genome. This reproducible transformation system for golden pothos may enable the molecular breeding of this very common indoor plant.     

An improved method for direct gene transfer and subsequent regeneration ofArabidopsis thaliana Landsbergerecta and two marker lines

A protocol for PEG-mediated transformation of protoplasts is described forA. thaliana ecotype Landsbergerecta and two marker lines derived from it, M4 and M10. The optimal transformation conditions were: 14 μg plasmid DNA and 28 μg carrier DNA per 6 x 10⁵ protoplasts in 15% (w/v) PEG solution. Based on the hygromycin resistance conferred by the transgene, relative transformation frequencies of 2.5–3.2% and absolute transformation frequencies of 1–2 x 10⁻⁴, were obtained. Shoot regeneration frequencies of 40–60% were achieved, and fertile transgenic plants of the three tested lines were obtained. Southern blot hybridizations demonstrated multi-copy integration patterns in most cases. Hygromycin resistance segregation patterns of 3:1 and 15:1 were found, as well as unexpected segregation patterns, suggesting that modifications in gene expression took place and that these can progressively occur over successive generations.     

Control of Lepidopteran insect pests in transgenic Chinese cabbage (Brassica rapa ssp. pekinensis) transformed with a synthetic Bacillus thuringiensis cry1C gene

 A synthetic Bacillus thuringiensis cry1C gene was transferred to three Korean cultivars of Chinese cabbage via Agrobacterium tumefaciens-mediated transformation of hypocotyl explants. Hygromycin resistance served as an efficient selective marker. The transformation efficiency ranged from 5% to 9%. Transformation was confirmed by Southern blot analysis, PCR, Northern analysis, and progeny tests. Many transgenic plants of the closed-head types (lines Olympic and Samjin) flowered in vitro. Over 50 hygromycin-resistant plants were successfully transferred to soil. The transgenic plants and their progeny were resistant to diamondback moths (DBM, Plutella xylostella), the major insect pest of crucifers world-wide, as well as to cabbage loopers (Trichoplusia ni) and imported cabbage worms (Pieris rapae). Both susceptible Geneva DBM and a DBM population resistant to Cry1A protein were controlled by the Cry1C-transgenic plants. The efficient and reproducible transformation system described may be useful for the transfer of other agriculturally important genes into Chinese cabbage. Received: 12 June 2000 / Revision received: 21 August 2000 / Accepted: 22 August 2000     

Development of an Efficient Genome Editing Method by CRISPR/Cas9 in a Fish Cell Line

CRISPR/Cas9 system has been used widely in animals and plants to direct mutagenesis. To date, no such method exists for fish somatic cell lines. We describe an efficient procedure for genome editing in the Chinook salmon Oncorhynchus tshawytscha CHSE. This cell line was genetically modified to firstly overexpress a monomeric form of EGFP (cell line CHSE-E Geneticin resistant) and additionally to overexpress nCas9n, a nuclear version of Cas9 (cell line CHSE-EC, Hygromycin and Geneticin resistant). A pre-validated sgRNA was produced in vitro and used to transfect CHSE-EC cells. The EGFP gene was disrupted in 34.6 % of cells, as estimated by FACS and microscopy. The targeted locus was characterised by PCR amplification, cloning and sequencing of PCR products; inactivation of the EGFP gene by deletions in the expected site was validated in 25 % of clones. This method opens perspectives for functional genomic studies compatible with high-throughput screening.     

Pentalenolactone I and hygromycin A, immunosuppressants produced by Streptomyces filipinensis and Streptomyces hygroscopicus.

Immunosuppressants isolated from Streptomyces filipinensis and S. hygroscopicus were identified with pentalenolactone I and hygromycin A, respectively. The compounds as well as cyclosporin A showed immunosuppressant activity in the mixed lymphocyte reaction, and pentalenolactone I and cyclosporin A suppressed IL-2 production, however, hygromycin A did not. Hygromycin A may have immunosuppressant activity by a different mechanism from pentalenolactone I, cyclosporin A and tacrolimus.     

Shuttle vectors conferring hygromycin B resistance toE. coli and to mammalian cells

Shuttle vectors expressing resistance to hygromycin B in bothE. coli and in mammalian cells were constructed. A combination of the simian virus 40 early promoter upstream of the native bacterial promoter of theneo gene from transposon Tn5 was found to express hygromycin B resistance better in both types of host cells than a combination of the Tn5 promoter followed by the promoter of the Herpes simplex virus thymidine kinase gene. Hygromycin phosphotransferase fusion proteins with extensions at the carboxyterminus were also tested and found to be marginally less effective as selection markers in eukaryotic cells but virtually inactive inE. coli.Abbreviations HM hygromycin - hpt hygromycin phosphotransferase gene - neo neomycin (geneticin) phosphotransferase gene - DHFR dihydrofolate reductase     

Studies on the mode of action of hygromycin B, an inhibitor of translocation in eukaryotes.

Hygromycin B is an unusual aminoglycoside antibiotic active against both prokaryotic and eukaryotic cells. Hygromycin B at 0.38 mM concentration completely halts yeast cell growth in rich media, presumably by preventing protein synthesis by cytoplasmic ribosomes. Polypeptide synthesis in cell-free extracts from rabbit reticulocytes, wheat germ and yeast is strongly blocked by low concentrations of hygromycin B. The antibiotic inhibits peptide chain elongation by yeast polysomes by preventing elongation factor EF-2-dependent translocation, although it does not affect either the formation of the EF-2-GTP-ribosome complex or the EF-2- and ribosome-dependent GTP hydrolysis which takes place uncoupled from translocation. The inhibition of translocation by hygromycin B might result from the stabilization of peptidyl-tRNA bound to the ribosomal acceptor site, since the stability of [3H]Phe-tRNA-EF-1-poly(U)-ribosome and [3H]Phe-tRNA-poly(U)-ribosome complexes is increased in the presence of hygromycin B. The inhibition of polyphenylalanine synthesis by reticulocyte ribosomes and enzymic translocation of peptidyl-tRNA by yeast polysomes can be reversed by increasing concentrations of EF-2 suggesting a relationship between the binding sites of EF-2 and hygromycin B on the ribosome. Neither non-enzymic translocation, that takes place in the presence of high potassium concentrations, nor the peptide bondforming step are affected by hygromycin B.     

黑曲霉（Aspergillus niger）启动子的克隆及其序列特征

应用启动子筛选载体（Ｐｒｏｍｏｔｅｒ－ｔｒａｐｖｅｃｔｏｒ）ＰＬＸ２Ａ构建了１个黑曲霉（Ａｓｐｅｒｇｉｌｌｕｓｎｉｇｅｒ）基因组文库,这个载体含有１个潮霉素Ｂ（Ｈｙ）磷酸转移酶编码基因（ｈｐｈ）它能以启动子活性选择ＤＮＡ片段,用这个基因组文库转化大肠杆菌,获得了８００００转化子菌落,其中９４％含有插入片段,用这个基因库的质粒ＤＮＡ转化黑曲霉,产生了５３个抗潮霉素Ｂ（Ｈｙ＾Ｒ）菌落,２１个转化子的Ｓ     

The hygromycin-resistance-encoding gene as a selection marker for vaccinia virus recombinants.

Hygromycin B (Hy), an inhibitor of RNA translation, was shown to block the replication of vaccinia virus (VV) in cultured cell lines. Insertion of the Escherichia coli Hy resistance-encoding gene (hph) into the VV genome under control of early or late synthetic VV promoters could overcome inhibition of viral replication. When hph was inserted into VV in tandem with the human papillomavirus type 16 (HPV16) L1 open reading frame, hph recombinant viruses could be selected which expressed HPV16 L1.     

Measurement of hygromycin B phosphotransferase activity in crude mammalian cell extracts by a simple dot-blot assay.

Hygromycin B (Hy) resistance, encoded by the prokaryotic gene hph, is commonly used as a dominant selectable marker for gene transfer experiments in mammalian cells. We describe a simple, quantitative dot-blot assay for measuring the activity in crude mammalian cell extracts of Hy phosphotransferase, the product of the hph gene. The assay shows no cross interference with substrates for neomycin phosphotransferase II, the product of the commonly used marker gene neo; hph and neo may thus be useful as a set of two non-interfering selectable marker and reporter genes for gene transfer experiments in mammalian cells.