Статті в журналах: "Hygromycine A"

1

Hussain, Iqra. "INTRODUCTION OF RICE CHITINASE GENE IN POTATO BY AGROBACTERIUM-MEDIATED TRANSFORMATION." Pakistan Journal of Agricultural Sciences 56, no. 01 (January 1, 2019): 7–13. http://dx.doi.org/10.21162/pakjas/19.8154.

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Potato is an important food crop of the world. Different viral, bacterial and fungal pathogens cause heavy economic losses of this crop every year. Potato has complex genetic makeup due to which induction of disease resistance through conventional breeding is difficult. Genetic manipulation through different transformation techniques is more precise and successful tool. In the present study different factors were investigated, which have an influence on potato transformation. The optimal dose of cefotaxime was found 500 mg/l which did not affected the growth of the potato tissues. The explants treated with Agrobacterium in the presence of acetosyringone resulted in higher frequency of transformation as compared to the explant without it. Two minutes time for co infection was found appropriate for optimum transformation efficiency. The two days cocultivation period along with 7 days preselection was found suitable for potato transformation. The putative tranformants regenerated on MS medium supplemented with 20 mg/L hygromycine and 500 mg/L cefotaxime, from nodal explants while the non-transformed tissue turned brown and gradually died after two or three sub culturing on the selection media containing selective antibiotic hygromycin. The shoots obtained on selection media shifted on root induction media supplemented with similar concentrations of hygromycin and cefotaxime, resulted complete plantlet formation after 10 days. It was observed that all the hygromycin positive plants also exhibited positive bands of desired size of 823 bp for chitinase gene, suggesting cotransformation of both genes in transformed plants

2

Chen, Grace Q. "Effective Reduction of Chimeric Tissue in Transgenics for the Stable Genetic Transformation of Lesquerella fendleri." HortScience 46, no. 1 (January 2011): 86–90. http://dx.doi.org/10.21273/hortsci.46.1.86.

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To improve the potential of Lesquerella fendleri as a valuable industrial oilseed crop, a stable genetic transformation system was developed. Genetic transformation was performed by inoculating leaf segments with an Agrobacterium tumefaciens strain AGL1 containing binary vector pCAMBIA 1301.1, which contains a β-glucuronidase gene as a reporter gene and hygromycine phosphotransferase II as a selection marker gene. Primary shoots were regenerated from the leaf segments on the half-strength Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine, 1-naphthaleneacetic acid, and hygromycin. The frequency of primary shoot generation was between 22.5% and 60%, and 81.1% to 89.3% of these shoots were chimeras. The high frequency of chimeras was probably the result of efficient protection from the hygromycin of non-transformed cells by adjacent transformed ones. The non-transformed cells were removed by multiple rounds of successive shoot regenerations. The purified isogenic shoots were subcultured and roots were induced on the MS medium plus indole-3-butyric acid. Most of the plantlets were able to establish roots and acclimate successfully in the greenhouse. The insertion of the hptII gene was confirmed by segregation analysis in T1 seeds, and the stable inheritance of the transgenes was demonstrated by the characterization transgenic lines through T2 generation. This transformation system can be used to obtain stable transgenic lines for genetic engineering of L. fendleri.

3

Prodhan, Shamsul H., K. Nagamiya, A. Komamine, and H. Morishima. "EVALUATION OF TRANSGENIC INDICA RICE (Oryza sativa L.) THROUGH REPORTER AND DESIRED GENE." Bangladesh Journal of Plant Breeding and Genetics 20, no. 2 (December 31, 2007): 11–16. http://dx.doi.org/10.3329/bjpbg.v20i2.17029.

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Rice is one of the most important food crops in the world. It is greatly affected by various abiotic stresses. Among them, to improve the complex stress salinity we introduced a desired gene katE, a catalase gene of Escherichia coli, and reporter gene GUS into the indica rice cultivar Kasalath. Plant survival under different salt water concentration was checked. Transformation was carried out using Agrobacterium tumefaciens strain EHA101 harboring a binary vector pIES6/Hm/katE and pIG121/ Hm/ GUS which contains genes for catalase katE, GUS gene, hygromycine resistance gene HPT and kanamycine resistance gene NPTII in the T-DNA region. Transformation was confirmed by PCR with katE and GUS primer. Transgenic plants at very young stage (3 days) were able to grow up to 15 days in 100 mM NaCl solution and 7 days in 250 mM NaCl solution where as non transgenic plants could not survive even up top 5 days in 100 mM condition and 7 days in 250 mM NaCl concentration. Twenty eight days matured plants could survive and were able to form iflorescence. Here a single gene introduction significantly improved the salt tolerance of this crop rice. DOI: http://dx.doi.org/10.3329/bjpbg.v20i2.17029

4

McCallum, B. D., C. C. Bernier, and L. Lamari. "Generation and utilization of chemical-resistant mutants in Pyrenophora tritici-repentis, the causal agent of tan spot of wheat." Canadian Journal of Botany 72, no. 1 (January 1, 1994): 100–105. http://dx.doi.org/10.1139/b94-014.

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Tan spot, caused by the fungal pathogen Pyrenophora tritici-repentis, is a major leaf spot disease of wheat worldwide. To facilitate genetic analysis of this homothallic fungus, mutants resistant to the fungicide iprodione or hygromycin B were created through ultraviolet light mutagenesis and used in sexual crosses. Conidia from two isolates of P. tritici-repentis, sensitive to both chemicals (iprodione-S hygromycin-S), were exposed to ultraviolet light to obtain four mutants resistant to iprodione but sensitive to hygromycin B (iprodione-R hygromycin-S) and three mutants resistant to hygromycin B but sensitive to iprodione (iprodione-S hygromycin-R). The mutants were paired in all combinations, and the markers allowed crossed progeny to be distinguished from selfed progeny. Crossed ascospore progeny from pairings between iprodione-R hygromycin-S isolates and iprodione-S hygromycin-R isolates and between iprodione-R hygromycin-R isolates and iprodione-S hygromycin-S segregated 1:1 for resistance–sensitivity to both iprodione and hygromycin B. These results indicate that one locus controls iprodione resistance and a second independent locus controls hygromycin B resistance. This study should facilitate further genetic research on the tan spot fungus by providing a simple marker system. Key words: genetics, inheritance, Drechslera, yellow spot, leaf spot.

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Sultana, Shahanaz, Chai Ling Ho, Parameswari Namasivayam, and Suhaimi Napis. "Genotypic differences in response to Hygromycin effect on untransformed calli death and rice germination." Bangladesh Rice Journal 18, no. 1-2 (April 17, 2015): 38–43. http://dx.doi.org/10.3329/brj.v18i1-2.23001.

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Hygromycin is an efficient selective agent in transformation studies of wide ranges of crop. In this study, different concentrations of hygromycin were used to observe the effect on untransformed calli death, percent germination and seedling growth of three rice varieties (Oryza sativa L.) viz BRRI dhan29, MR219 and Taipei309. Hygromycin killed the untransformed calli and inhibited the germination of tested varieties in a concentration dependent manner. Among the tested varieties, the lowest and the highest calli death was observed in MR219 and Taipei309 respectively in all the concentrations of hygromycin. Whereas, the highest and the lowest percent germination were observed in MR219 and Taipei309 respectively. The minimal inhibitory concentration (MIC) for selection of calli were calculated as 42, 40 and 47 mg/L hygromycin for BRRI dhan29, MR219 and Taipei309 respectively. During germination, 35, 62 and 32 mg/L hygromycin were suitable for the selection of BRRI dhan29, MR219 and Taipei 309 respectively. Shoot and root growth reduction after germination was increased with the increased concentration of hygromycin. Besides, root growth was more sensitive to hygromycin than the shoot. These results suggest that hygromycin increases calli death, decreases percent germination, and shoot and root growth in all varieties with the increasing rate of hygromycin. But these characteristics vary with different degrees in different genotypes as well as different stages.Bangladesh Rice j. 2014, 18(1&2): 38-43

6

Pfister, P., M. Risch, D. E. Brodersen, and E. C. Böttger. "Role of 16S rRNA Helix 44 in Ribosomal Resistance to Hygromycin B." Antimicrobial Agents and Chemotherapy 47, no. 5 (May 2003): 1496–502. http://dx.doi.org/10.1128/aac.47.5.1496-1502.2003.

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ABSTRACT Hygromycin B is an aminoglycoside antibiotic active against prokaryotic and eukaryotic ribosomes. Ribosomal alterations in bacteria conferring resistance to hygromycin B have not been described, prompting us to use a single rRNA allelic derivative of the gram-positive bacterium Mycobacterium smegmatis for investigation of the molecular mechanisms involved in ribosomal resistance to hygromycin B in eubacteria. Resistance mutations were found to localize exclusively in 16S rRNA. The mutations observed, i.e., 16S rRNA U1406C, C1496U, and U1498C (E. coli numbering), are in close proximity to the hygromycin B binding site located in conserved helix 44 of 16S rRNA. The 16S rRNA positions involved in hygromycin B resistance are highly conserved in all three domains of life, explaining the lack of specificity and general toxicity of hygromycin B.

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Mercuriani, Ixora Sartika, Aziz Purwantoro, Sukarti Moeljopawiro, Seonghoe Jang, and Endang Semiarti. "Selection of Phalaenopsis amabilis L. Blume Orchid Resistance to Hygromycin." Indonesian Journal of Biotechnology 17, no. 2 (November 9, 2015): 107. http://dx.doi.org/10.22146/ijbiotech.16000.

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Examination of Phalaenopsis amabilis orchid resistance to hygromycin antibiotic is an important step to doprior to Agrobacterium-mediated genetic transformation in this orchids using Hygromycin phosphotransferase(HPT) gene as a selection marker in the T-DNA that harboring a desired gene to be transfered. We exposedthe plant on hygromycin containing medium. The experiment was conducted using 6 weeks old P. amabilisprotocorms. These protocorms were subcultured onto NP medium supplemented with various concentrationof Hygromycin (0, 5, 10, 20, 1nd 40 mg/l). The number of survival protocorms were examined every week for4 weeks after subcultured (WAS). The resistancy of hygromycin was calculated as ratio of death protocormsper total protocorms). The result showed that 10 mg/l hygromycin with 1 weeks of application caused deathclose to LD 50. This data indicate that P. amabilis resistance to hygromycin treatment on the appropriateconcentration 10 mg/l, and this concentration can be used for other purposes in orchid system.

8

Miao, V. P., M. R. Rountree, and E. U. Selker. "Ectopic integration of transforming DNA is rare among neurospora transformants selected for gene replacement." Genetics 139, no. 4 (April 1, 1995): 1533–44. http://dx.doi.org/10.1093/genetics/139.4.1533.

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Abstract In a variety of organisms, DNA-mediated transformation experiments commonly produce transformants with multiple copies of the transforming DNA, including both selected and unselected molecules. Such "cotransformants" are much more common than expected from the individual transformation frequencies, suggesting that subpopulations of cells, or nuclei, are particularly competent for transformation. We found that Neurospora crassa transformants selected for gene replacement at the am gene had not efficiently incorporated additional DNA, suggesting that nuclei that undergo transformation by homologous recombination are not highly competent at integration of DNA by illegitimate recombination. Spheroplasts were treated with DNA fragments homologous to am and with an Escherichia coli hph plasmid. Transformants were initially selected for hph (hygromycinR), allowed to conidiate to generate homokaryons and then selected for either Am- (gene replacements) or hph. Surprisingly, most am replacement strains were hygromycinS (124/140) and carried no extraneous DNA (116/140). Most transformants selected for hph also had ectopic copies of am DNA and/or multiple copies of hph sequences (32/35), generally at multiple sites, confirming that efficient cotransformation could occur. To test the implication that cotransformation involving gene replacement and ectopic integration is rare, we compared the yields of am replacement strains with or without prior selection for hph. The initial selection did not appreciably help (or hinder) recovery of strains with replacements.

9

Dai, Qun, Zhihuan Sun, and Guido Schnabel. "Development of Spontaneous Hygromycin B Resistance in Monilinia fructicola and Its Impact on Growth Rate, Morphology, Susceptibility to Demethylation Inhibitor Fungicides, and Sporulation." Phytopathology® 93, no. 11 (November 2003): 1354–59. http://dx.doi.org/10.1094/phyto.2003.93.11.1354.

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Agrobacterium tumefaciens-mediated transformation with plasmids carrying the hygromycin B resistance gene hph frequently is being used for inserting genes into fungal spores and mycelial cells and for conducting insertional mutagenesis to identify genes connected to a particular phenotype. In this article, we report that stable hygromycin B resistance can develop spontaneously in germinating conidia from Monilinia fructicola and that the mutants exhibit altered phenotypes. One spontaneously developing hygromycin B-resistant colony developed per 2.5 × 105 germinating conidia. Mutants grew significantly slower on potato dextrose agar, were 2.4- to 3.1-fold more sensitive to demethylation inhibitor fungicides, lacked melanization, and did not produce spores. The mode of action of hygromycin B resistance in the mutants seemed to be different from the hph transgene-mediated hygromycin B resistance based on different phenotypic characters. The ability of M. fructicola and possibly other fungi to spontaneously develop hygromycin B resistance associated with an altered phenotype may interfere with the selection of true transformants if hygromycin B is used as selective agent. This is particularly confounding if the hph gene is used as selectable marker in insertional mutagenesis experiments conducted for the identification of genes involved in melanization, sporulation, or fungicide resistance.

10

Ko, Moon Kyung, Hyunchul Soh, Kyung-Moon Kim, Young Soon Kim, and Kyunghoan Im. "Stable Production of Transgenic Pepper Plants Mediated by Agrobacterium tumefaciens." HortScience 42, no. 6 (October 2007): 1425–30. http://dx.doi.org/10.21273/hortsci.42.6.1425.

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The aim of this study was to establish a stable transformation method for hot pepper using the hygromycin phosphotransferase (hpt)/hygromycin selection strategy. Explants from aseptic pepper seedlings were inoculated with Agrobacterium tumefaciens carrying pCAMBIA1301. A number of calli were developed on the medium containing hygromycin to discriminate the induction of “false-positive buds,” and then shoots were successfully regenerated from the hygromycin-resistant calli. Southern and Northern hybridization analysis indicated that the hpt gene was integrated and expressed in the transgenic pepper plants (T0) and transmitted to the progeny (T1) without genetic modification. Most T1 progenies derived from self-pollination revealed a 3:1 segregation ratio for hygromycin resistance, indicating that one copy of the T-DNA was integrated into the respective transgenic lines. Both uidA and hpt genes were stably expressed in the T1 generation and coinherited in the progenies. Finally, homozygous progenies were identified in the T1 generation of the transgenic peppers, and the homozygous state was maintained in all progenies tested (T2). The results show the reliability and stability of the hpt/hygromycin selection protocol for pepper transformation.

11

Liu, Hang, Zhongyi Zhang, Sijie Liang, Li Guo, Shiyang Sun, Kehou Pan, and Guanpin Yang. "Transcriptome Responses of Hygromycin B Resistance Gene-Transformed, Hygromycin B-Adaptive and Wild Nannochloropsis oceanica Strains to Hygromycin B." Journal of Ocean University of China 19, no. 2 (January 24, 2020): 453–58. http://dx.doi.org/10.1007/s11802-020-4252-4.

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12

McGaha, Susan M., and W. Scott Champney. "Hygromycin B Inhibition of Protein Synthesis and Ribosome Biogenesis in Escherichia coli." Antimicrobial Agents and Chemotherapy 51, no. 2 (October 16, 2006): 591–96. http://dx.doi.org/10.1128/aac.01116-06.

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ABSTRACT The aminoglycoside antibiotic hygromycin B was examined in Escherichia coli cells for inhibitory effects on translation and ribosomal-subunit formation. Pulse-chase labeling experiments were performed, which verified lower rates of ribosomal-subunit synthesis in drug-treated cells. Hygromycin B exhibited a concentration-dependent inhibitory effect on viable-cell numbers, growth rate, protein synthesis, and 30S and 50S subunit formation. Unlike other aminoglycosides, hygromycin B was a more effective inhibitor of translation than of ribosomal-subunit formation in E. coli. Examination of total RNA from treated cells showed an increase in RNA corresponding to a precursor to the 16S rRNA, while mature 16S rRNA decreased. Northern hybridization to rRNA in cells treated with hygromycin B showed that RNase II- and RNase III-deficient strains of E. coli accumulated 16S rRNA fragments upon treatment with the drug. The results indicate that hygromycin B targets protein synthesis and 30S ribosomal-subunit assembly.

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Abdulmunim ‎, Zahraa, Rabah N. Jabba, and Abdulwahid B. Al-Shaibani. "Studying the Optimum Conditions of ‎Hygromycin B Production and Detect their ‎Toxicity." JOURNAL OF UNIVERSITY OF BABYLON for Pure and Applied Sciences 26, no. 2 (December 26, 2017): 119–30. http://dx.doi.org/10.29196/jub.v26i2.480.

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Hygromycin B was extracted with ethyl acetate, which separates organic phase from aqueous phase in the broth culture filtrate, only the aqueous phase showed significant antimicrobial activity by using agar well diffusion technique. At a concentration of 25mg/ml (as crude extract), this phase excreted its activity against the test microorganisms which include; one G(+) bacteria (Staphylococcus aureus), five G(–) bacteria (Pseudomonas aeruginosa , Proteus mirabilis, Escherichia coli , Klebsiella pneumoniae, Salmonella typhi) and one yeast (Saccharomyces cerevisiae). After detecting the aminoglycoside hygromycin B by the Thin Layer Chromatography (TLC) method to ensure presence of the antibiotic, same flow rate (Rf) value (0.357) as that of the standard hygromycin B was obtained. Results of the optimization conditions showed that the highest antimicrobial activity of hygromycin B was obtained at a medium pH of 8 and incubation temperature of 35°C for 10 days. When the toxicity of hygromycin B crude extract under such conditions was examined on mice liver, a mild effects were appeared

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Waldron, C., E. B. Murphy, J. L. Roberts, G. D. Gustafson, S. L. Armour, and S. K. Malcolm. "Resistance to hygromycin B." Plant Molecular Biology 5, no. 2 (1985): 103–8. http://dx.doi.org/10.1007/bf00020092.

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15

Abedinia, M., R. J. Henry, A. B. Blakeney, and L. Lewin. "An Efficient Transformation System for the Australian Rice Cultivar, Jarrah." Functional Plant Biology 24, no. 2 (1997): 133. http://dx.doi.org/10.1071/pp96071.

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A rapid and efficient transformation system for the generation of large numbers of transformed, fertile, transgenic rice (Oryza sativa L.) plants of the Australian rice cultivar, Jarrah, is described. Embryogenic callus pieces derived from mature seeds were bombarded with gold particles coated with DNA. Two plasmids were used, one containing a gene encoding hygromycin phosphotransferase (hph, conferring hygromycin resistance) as a selectable marker and the other containing uidA (gus) as a reporter gene. The calli were selected for their resistance to hygromycin. DNA uptake, integration and expression of the hph and gus gene in selected rice were investigated by various PCR methods and dot blot and Southern analysis of genomic DNA extracted from transformed rice plants. On average one independently transformed hygromycin resistant plant was recovered from every 2.4 pieces of callus bombarded. Selection with Biolaphos using the Bar gene as selectable marker was not successful in this system.

16

Becker, Ina, Binod Prasad, Maria Ntefidou, Viktor Daiker, Peter Richter, and Michael Lebert. "Agrobacterium tumefaciens-Mediated Nuclear Transformation of a Biotechnologically Important Microalga—Euglena gracilis." International Journal of Molecular Sciences 22, no. 12 (June 11, 2021): 6299. http://dx.doi.org/10.3390/ijms22126299.

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Euglena gracilis (E. gracilis) is an attractive organism due to its evolutionary history and substantial potential to produce biochemicals of commercial importance. This study describes the establishment of an optimized protocol for the genetic transformation of E. gracilis mediated by Agrobacterium (A. tumefaciens). E. gracilis was found to be highly sensitive to hygromycin and zeocin, thus offering a set of resistance marker genes for the selection of transformants. A. tumefaciens-mediated transformation (ATMT) yielded hygromycin-resistant cells. However, hygromycin-resistant cells hosting the gus gene (encoding β-glucuronidase (GUS)) were found to be GUS-negative, indicating that the gus gene had explicitly been silenced. To circumvent transgene silencing, GUS was expressed from the nuclear genome as transcriptional fusions with the hygromycin resistance gene (hptII) (encoding hygromycin phosphotransferase II) with the foot and mouth disease virus (FMDV)-derived 2A self-cleaving sequence placed between the coding sequences. ATMT of Euglena with the hptII-2A–gus gene yielded hygromycin-resistant, GUS-positive cells. The transformation was verified by PCR amplification of the T-DNA region genes, determination of GUS activity, and indirect immunofluorescence assays. Cocultivation factors optimization revealed that a higher number of transformants was obtained when A. tumefaciens LBA4404 (A600 = 1.0) and E. gracilis (A750 = 2.0) cultures were cocultured for 48 h at 19 °C in an organic medium (pH 6.5) containing 50 µM acetosyringone. Transformation efficiency of 8.26 ± 4.9% was achieved under the optimized cocultivation parameters. The molecular toolkits and method presented here can be used to bioengineer E. gracilis for producing high-value products and fundamental studies.

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Arnaboldi, Paul Michael, and Sukanya Narasimhan. "Hygromycin A in the Lymelight." Cell Host & Microbe 29, no. 11 (November 2021): 1599–601. http://dx.doi.org/10.1016/j.chom.2021.10.007.

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18

Chida, Noritaka, Masami Ohtsuka, Keiichi Nakazawa, and Seiichiro Ogawa. "Total synthesis of hygromycin A." Journal of the Chemical Society, Chemical Communications, no. 7 (1989): 436. http://dx.doi.org/10.1039/c39890000436.

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WATSON, ADJ, and DC BURNETT. "Hygromycin B and deaf dogs." Australian Veterinary Journal 66, no. 9 (September 1989): 302–3. http://dx.doi.org/10.1111/j.1751-0813.1989.tb13960.x.

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20

Habib, El-Sayed E., J. Neel Scarsdale, and Kevin A. Reynolds. "Biosynthetic Origin of Hygromycin A." Antimicrobial Agents and Chemotherapy 47, no. 7 (July 2003): 2065–71. http://dx.doi.org/10.1128/aac.47.7.2065-2071.2003.

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ABSTRACT Hygromycin A, an antibiotic produced by Streptomyces hygroscopicus, is an inhibitor of bacterial ribosomal peptidyl transferase. The antibiotic binds to the ribosome in a distinct but overlapping manner with other antibiotics and offers a different template for generation of new agents effective against multidrug-resistant pathogens. Reported herein are the results from a series of stable-isotope-incorporation studies demonstrating the biosynthetic origins of the three distinct structural moieties which comprise hygromycin A. Incorporation of [1-13C]mannose and intact incorporation of d-[1,2-13C2]glucose into the 6-deoxy-5-keto-d-arabino-hexofuranose moiety are consistent with a pathway in which mannose is converted to an activated l-fucose, via a 4-keto-6-deoxy-d-mannose intermediate, with a subsequent unusual mutation of the pyranose to the corresponding furanose. The aminocyclitol moiety was labeled by d-[1,2-13C2]glucose in a manner consistent with formation of myo-inositol and a subsequent unprecedented oxidation and transamination of the C-2 hydroxyl group to generate neo-inosamine-2. Incorporation of [carboxy- 13C]-4-hydroxybenzoic acid and intact incorporation of [2,3-13C2]propionate are consistent with a polyketide synthase-type decarboxylation condensation to generate the 3,4-dihydroxy-α-methylcinnamic acid moiety of hygromycin A. No labeling of hygromycin A was observed when [3-13C]tyrosine, [3-13C]phenylalanine, or [carboxy- 13C]benzoic acid was used, suggesting that the 4-hydroxybenzoic acid is derived directly from chorismic acid. Consistent with this hypothesis was the observation that hygromycin A titers could be reduced by addition of N-(phosphonomethyl)-glycine (an inhibitor of chorismic acid biosynthesis) and restored by coaddition of 4-hydroxybenzoic acid. The convergent biosynthetic pathway established for hygromycin A offers significant versatility for applying the techniques of combinatorial and directed biosynthesis to production of new antibiotics which target the ribosomal peptidyl transferase activity.

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Lee, Young Seob, Dae Young Lee, Tae Jin An, Jeong Hoon Lee, Young Sup Ahn, Seon Woo Cha, Su Hyun Mun, Ok Hwa Kang, Dong Yeul Kwon, and Sin Hee Han. "Synergistic Effect of Brazilein in Combination with Hygromycin-b against Staphylococcus aureus." Korean Journal of Medicinal Crop Science 22, no. 6 (December 30, 2014): 504–9. http://dx.doi.org/10.7783/kjmcs.2014.22.6.504.

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22

Rodolosse, Annie, Alain Barbat, Isabelle Chantret, Michel Lacasa, Edith Brot-Laroche, Alain Zweibaum, and Monique Rousset. "Selecting agent hygromycin B alters expression of glucose-regulated genes in transfected Caco-2 cells." American Journal of Physiology-Gastrointestinal and Liver Physiology 274, no. 5 (May 1, 1998): G931—G938. http://dx.doi.org/10.1152/ajpgi.1998.274.5.g931.

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Incorporation into plasmids of genes conferring resistance to aminoglycoside antibiotics such as hygromycin B is currently utilized for selection in experiments involving gene transfer in eukaryotic cells. Using a subclone of Caco-2 cells stably transfected with an episomal plasmid containing the hygromycin resistance gene, we observed that transformed cells subcultured in the presence of hygromycin B exhibit, compared with the same cells subcultured in antibiotic-free medium, a sixfold increase in the rates of glucose consumption and lactic acid production and dramatic changes, at mRNA and protein level, of the expressions of sucrase-isomaltase and hexose transporter GLUT-2, which are downregulated, contrasting with an upregulation of hexose transporter GLUT-1. This occurs without significant modifications of the differentiation status of the cells, as demonstrated by the normal expression of villin, ZO-1, dipeptidyl peptidase IV, or Na+-K+-ATPase. The plasmid copy number is, however, the same, whether or not the cells are cultured in the presence of hygromycin B. These results draw attention to the need to consider antibiotic-dependent alterations of metabolism and gene expression in transfection experiments.

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Lupton, S. D., L. L. Brunton, V. A. Kalberg, and R. W. Overell. "Dominant positive and negative selection using a hygromycin phosphotransferase-thymidine kinase fusion gene." Molecular and Cellular Biology 11, no. 6 (June 1991): 3374–78. http://dx.doi.org/10.1128/mcb.11.6.3374-3378.1991.

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The hygromycin phosphotransferase gene was fused in-frame with the herpes simplex virus type 1 thymidine kinase gene. The resulting fusion gene (termed HyTK) confers hygromycin B resistance for dominant positive selection and ganciclovir sensitivity for negative selection and provides a means by which these selectable phenotypes may be expressed and regulated as a single genetic entity.

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Lupton, S. D., L. L. Brunton, V. A. Kalberg, and R. W. Overell. "Dominant positive and negative selection using a hygromycin phosphotransferase-thymidine kinase fusion gene." Molecular and Cellular Biology 11, no. 6 (June 1991): 3374–78. http://dx.doi.org/10.1128/mcb.11.6.3374.

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The hygromycin phosphotransferase gene was fused in-frame with the herpes simplex virus type 1 thymidine kinase gene. The resulting fusion gene (termed HyTK) confers hygromycin B resistance for dominant positive selection and ganciclovir sensitivity for negative selection and provides a means by which these selectable phenotypes may be expressed and regulated as a single genetic entity.

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Ong, Jun Yang, Reem Swidah, Marco Monti, Daniel Schindler, Junbiao Dai, and Yizhi Cai. "SCRaMbLE: A Study of Its Robustness and Challenges through Enhancement of Hygromycin B Resistance in a Semi-Synthetic Yeast." Bioengineering 8, no. 3 (March 23, 2021): 42. http://dx.doi.org/10.3390/bioengineering8030042.

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Recent advances in synthetic genomics launched the ambitious goal of generating the first synthetic designer eukaryote, based on the model organism Saccharomyces cerevisiae (Sc2.0). Excitingly, the Sc2.0 project is now nearing its completion and SCRaMbLE, an accelerated evolution tool implemented by the integration of symmetrical loxP sites (loxPSym) downstream of almost every non-essential gene, is arguably the most applicable synthetic genome-wide alteration to date. The SCRaMbLE system offers the capability to perform rapid genome diversification, providing huge potential for targeted strain improvement. Here we describe how SCRaMbLE can evolve a semi-synthetic yeast strain housing the synthetic chromosome II (synII) to generate hygromycin B resistant genotypes. Exploiting long-read nanopore sequencing, we show that all structural variations are due to recombination between loxP sites, with no off-target effects. We also highlight a phenomenon imposed on SCRaMbLE termed “essential raft”, where a fragment flanked by a pair of loxPSym sites can move within the genome but cannot be removed due to essentiality restrictions. Despite this, SCRaMbLE was able to explore the genomic space and produce alternative structural compositions that resulted in an increased hygromycin B resistance in the synII strain. We show that among the rearrangements generated via SCRaMbLE, deletions of YBR219C and YBR220C contribute to hygromycin B resistance phenotypes. However, the hygromycin B resistance provided by SCRaMbLEd genomes showed significant improvement when compared to corresponding single deletions, demonstrating the importance of the complex structural variations generated by SCRaMbLE to improve hygromycin B resistance. We anticipate that SCRaMbLE and its successors will be an invaluable tool to predict and evaluate the emergence of antibiotic resistance in yeast.

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Dobinson, Katherine F. "Genetic transformation of the vascular wilt fungusVerticillium dahliae." Canadian Journal of Botany 73, no. 5 (May 1, 1995): 710–15. http://dx.doi.org/10.1139/b95-076.

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To facilitate genetic analysis of pathogenicity of Verticillium dahliae, a vascular wilt pathogen, a DNA-mediated transformation system has been developed. Resistance to hygromycin B was obtained by transforming spheroplasts with the cosmid vector pAN7-2. Transformation efficiencies ranged between 3 and 5 transformants/μg vector DNA. The transforming DNA was integrated into the V. dahliae genome, in single and multiple copies and in tandem array. In several multicopy transformants, minor alterations in the integrated DNA sequences were evident following extensive vegetative growth in the absence of hygromycin B. Electrophoretic karyotype analysis also provided direct evidence of chromosome rearrangements in two transformants. The availability of a transformation system for V. dahliae will facilitate the cloning and characterization of genes that are important for pathogenicity and development. Key words: Verticillium wilt, fungal transformation, electrophoretic karyotype, hygromycin B resistance, chromosome rearrangement.

27

Nakazawa, Miki, and Minami Matsui. "Selection of Hygromycin-Resistant Arabidopsis Seedlings." BioTechniques 34, no. 1 (January 2003): 28–30. http://dx.doi.org/10.2144/03341bm02.

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28

Chida, Noritaka, Masami Ohtsuka, Keiichi Nakazawa, and Seiichiro Ogawa. "Total synthesis of antibiotic hygromycin A." Journal of Organic Chemistry 56, no. 9 (April 1991): 2976–83. http://dx.doi.org/10.1021/jo00009a009.

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29

Milojević, Jelena, Ljiljana Tubić, Vladimir Nolić, Nevena Mitić, Dušica Ćalić-Dragosavac, Branka Vinterhalter, and Snežana Zdravković-Korać. "Hygromycin promotes somatic embryogenesis in spinach." Plant Cell, Tissue and Organ Culture (PCTOC) 109, no. 3 (January 24, 2012): 573–79. http://dx.doi.org/10.1007/s11240-012-0117-x.

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30

Niu, C., Y. Akasaka-Kennedy, P. Faustinelli, M. Joshi, K. Rajasekaran, H. Yang, Y. Chu, J. Cary, and P. Ozias-Akins. "Antifungal Activity in Transgenic Peanut (Arachis hypogaea L.) Conferred by a Nonheme Chloroperoxidase Gene." Peanut Science 36, no. 2 (July 1, 2009): 126–32. http://dx.doi.org/10.3146/ps08-020.1.

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Abstract A nonheme chloroperoxidase gene (cpo-p) from Pseudomonas pyrrocinia, a growth inhibitor of mycotoxin-producing fungi, was introduced into peanut via particle bombardment. The expression of the cpo-p gene is predicted to increase pathogen defense in peanut. Embryogenic peanut tissues were bombarded with gold particles coated with plasmid pRT66 carrying the cpo-p and hygromycin phosphotransferase (hph) genes, under the control of a double CaMV 35S and a single CaMV 35S promoter, respectively. Selection for hygromycin-resistant somatic embryos was performed on a liquid medium containing 10–20 mg/L hygromycin 3–4 days after bombardment. The integration and expression of the cpo-p gene was confirmed by Southern, Northern and Western blot analyses. In vitro bioassay using crude protein extracts from transgenic T0, T1, and T4 plants showed inhibition of Aspergillus flavus hyphal growth, which could translate to a reduction in aflatoxin contamination of peanut seed.

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Smulian, A. George, Reta S. Gibbons, Jeffery A. Demland, Deborah T. Spaulding, and George S. Deepe. "Expression of Hygromycin Phosphotransferase Alters Virulence of Histoplasma capsulatum." Eukaryotic Cell 6, no. 11 (September 14, 2007): 2066–71. http://dx.doi.org/10.1128/ec.00139-07.

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ABSTRACT The Escherichia coli hygromycin phosphotransferase (hph) gene, which confers hygromycin resistance, is commonly used as a dominant selectable marker in genetically modified bacteria, fungi, plants, insects, and mammalian cells. Expression of the hph gene has rarely been reported to induce effects other than those expected. Hygromycin B is the most common dominant selectable marker used in the molecular manipulation of Histoplasma capsulatum in the generation of knockout strains of H. capsulatum or as a marker in mutant strains. hph-expressing organisms appear to have no defect in long-term in vitro growth and survival and have been successfully used to exploit host-parasite interaction in short-term cell culture systems and animal experiments. We introduced the hph gene as a selectable marker together with the gene encoding green fluorescent protein into wild-type strains of H. capsulatum. Infection of mice with hph-expressing H. capsulatum yeast cells at sublethal doses resulted in lethality. The lethality was not attributable to the site of integration of the hph construct into the genomes or to the method of integration and was not H. capsulatum strain related. Death of mice was not caused by altered cytokine profiles or an overwhelming fungal burden. The lethality was dependent on the kinase activity of hygromycin phosphotransferase. These results should raise awareness of the potential detrimental effects of the hph gene.

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McCulloch, Kathryn M., Emilianne K. McCranie, Jarrod A. Smith, Maruf Sarwar, Jeannette L. Mathieu, Bryan L. Gitschlag, Yu Du, Brian O. Bachmann, and T. M. Iverson. "Oxidative cyclizations in orthosomycin biosynthesis expand the known chemistry of an oxygenase superfamily." Proceedings of the National Academy of Sciences 112, no. 37 (August 3, 2015): 11547–52. http://dx.doi.org/10.1073/pnas.1500964112.

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Orthosomycins are oligosaccharide antibiotics that include avilamycin, everninomicin, and hygromycin B and are hallmarked by a rigidifying interglycosidic spirocyclic ortho-δ-lactone (orthoester) linkage between at least one pair of carbohydrates. A subset of orthosomycins additionally contain a carbohydrate capped by a methylenedioxy bridge. The orthoester linkage is necessary for antibiotic activity but rarely observed in natural products. Orthoester linkage and methylenedioxy bridge biosynthesis require similar oxidative cyclizations adjacent to a sugar ring. We have identified a conserved group of nonheme iron, α-ketoglutarate–dependent oxygenases likely responsible for this chemistry. High-resolution crystal structures of the EvdO1 and EvdO2 oxygenases of everninomicin biosynthesis, the AviO1 oxygenase of avilamycin biosynthesis, and HygX of hygromycin B biosynthesis show how these enzymes accommodate large substrates, a challenge that requires a variation in metal coordination in HygX. Excitingly, the ternary complex of HygX with cosubstrate α-ketoglutarate and putative product hygromycin B identified an orientation of one glycosidic linkage of hygromycin B consistent with metal-catalyzed hydrogen atom abstraction from substrate. These structural results are complemented by gene disruption of the oxygenases evdO1 and evdMO1 from the everninomicin biosynthetic cluster, which demonstrate that functional oxygenase activity is critical for antibiotic production. Our data therefore support a role for these enzymes in the production of key features of the orthosomycin antibiotics.

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Jeknic, Zoran, Stephen P. Lee, Joel Davis, Richard C. Ernst, and Tony H. H. Chen. "Genetic Transformation of Iris germanica Mediated by Agrobacterium tumefaciens." Journal of the American Society for Horticultural Science 124, no. 6 (November 1999): 575–80. http://dx.doi.org/10.21273/jashs.124.6.575.

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A protocol was developed for production of transgenic iris plants (Iris germanica L. `Skating Party') from regenerable suspension cultures via Agrobacterium-mediated transformation. We tested a series of selection agents, and identified hygromycin and geneticin as the most suitable for selecting transformed iris cells. Suspension cultures of iris were cocultured for 3 days with A. tumefaciens LBA 4404(pTOK233) carrying an intron-interrupted uidA (GUS) gene encoding β-glucuronidase, and hpt (hygromycin) and nptII (geneticin) selectable marker genes. Hygromycin- or geneticin-resistant calli having GUS enzyme activity were identified and used to induce plant regeneration. More than 300 morphologically normal transgenic iris plants were obtained in ≈6 months. About 80% of the transformants were GUS-positive and NPTII-positive (paromomycin-resistant). Integration of transgenes into the nuclear genome of iris plants was confirmed by Southern blot analysis. We have, therefore, developed an efficient A. tumefaciens-mediated transformation system for Iris germanica, which will allow future improvement of this horticulturally important ornamental monocot via genetic engineering.

34

Robledo-Paz, Alejandrina, José Luis Cabrera-Ponce, Víctor Manuel Villalobos-Arámbula, Luis Herrera-Estrella, and Alba Estela Jofre-Garfias. "Genetic Transformation of Garlic (Allium sativum L.) by Particle Bombardment." HortScience 39, no. 6 (October 2004): 1208–11. http://dx.doi.org/10.21273/hortsci.39.6.1208.

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Microprojectile bombardment was used to introduce DNA into embryogenic callus of garlic (Allium sativum L.) and produce stably transformed garlic plants. Embryogenic calluses, derived from garlic cultivar `GT96-1', were bombarded with plasmid DNA containing genes coding for hygromycin phosphotransferase and β-glucuronidase. Putatively transformed calluses were identified in the bombarded tissue after 4 months of selection on 20 mg·L-1 hygromycin B. The transgenic nature of the selected material was demonstrated by GUS histochemical assay and Southern blot hybridization analysis, and twenty transgenic plants were regenerated.

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Witrzens, Barbara, Richard I. S. Brettell, Fiona R. Murray, David McElroy, Zhongyi Li, and Elizabeth S. Dennis. "Comparison of three selectable marker genes for transformation of wheat by microprojectile bombardment." Functional Plant Biology 25, no. 1 (1998): 39. http://dx.doi.org/10.1071/pp97095.

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Three selectable marker genes were compared for their efficacy in the production of transgenic wheat plants following microprojectile bombardment of cultured immature embryos. While transformed plants were recovered using the bar (phosphinothricin acetyltransferase) gene in combination with bialaphos, and the aphA (neomycin phosphotransferase) gene in combination with geneticin or paromomycin, no transgenic material was obtained with the hpt (hygromycin phosphotransferase) gene and hygromycin B. Southern analysis revealed single copy as well as multiple copy insertions of the bar and aphA transgenes. Inheritance of these selectable marker genes was demonstrated in the T1 generation progenies.

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Nikolic, Radomirka, Nevena Mitic, Slavica Ninkovic, and Mirjana Neskovic. "Efficient genetic transformation of Lotus corniculatus L. using a direct shoot regeneration protocol, stepwise hygromycin B selection, and a super-binary Agrobacterium tumefaciens vector." Archives of Biological Sciences 59, no. 4 (2007): 311–17. http://dx.doi.org/10.2298/abs0704311n.

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Cotyledons from 6-day-old Lotus corniculatus cv. Bokor seedlings, transversally cut into two halves, were capa?ble of regenerating buds without intervening callus formation. The explants were co-cultivated with the Agrobacterium tumefaciens LBA4404/pTOK233 superbinary vector carrying the uidA-intron gene and the genes hpt and nptII. They were cultured for 14 days on a regeneration medium, then subjected to a stepwise hygromycin B selection procedure consisting of gradually increasing antibiotic concentrations (5-15 mg L-1) over 21 weeks. Transformed shoots were obtained within 5 months after co-cultivation. Out of 124 initially co-cultivated explants, 52 (42%) plants survived hygromycin B selection. The presence of transgenes in regenerated plants was verified by ?-glucuronidase histochemical assays and PCR analysis for the presence of uidA gene sequences. Hygromycin B-resistant and PCR-positive T0 plants were cultured in the greenhouse to produce flowers and seeds. The obtained data demonstrate that the reported transformation protocol could be useful for introducing agriculturally important genes into the new L. corniculatus cultivar Bokor.

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Ganoza, M. Clelia, and Michael C. Kiel. "A Ribosomal ATPase Is a Target for Hygromycin B Inhibition on Escherichia coli Ribosomes." Antimicrobial Agents and Chemotherapy 45, no. 10 (October 1, 2001): 2813–19. http://dx.doi.org/10.1128/aac.45.10.2813-2819.2001.

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ABSTRACT We demonstrate that the transfer of fully charged aminoacyl-tRNAs into peptides directed by the MS2 RNA template requires both ATP and GTP, initiation factors (IF1, IF2, and IF3), elongation factors (EF-Tu, EF-Ts, and EF-G), and the ribosomal ATPase (RbbA). The nonhydrolyzable analogue AMPPCP inhibits the reactions, suggesting that hydrolysis of ATP is required for synthesis. The RbbA protein occurs bound to ribosomes and stimulates the ATPase activity of Escherichia coli 70S and 30S particles. The gene encoding RbbA harbors four ATP binding domains; the C-terminal half of the protein bears extensive sequence similarity to EF-3, a ribosome-dependent ATPase. Here, we show that the antibiotic hygromycin B selectively inhibits the ATPase activity of RbbA. Other antibiotics with similar effects on miscoding, streptomycin and neomycin, as well as antibiotics that impair peptide bond synthesis and translocation, had little effect on the ATPase activity of RbbA on 70S ribosomes. Immunoblot analysis indicates that at physiological concentrations, hygromycin B selectively releases RbbA from 70S ribosomes. Hygromycin B protects G1494 and A1408 in the decoding region, and RbbA enhances the reactivity of A889 and G890 of the 16S rRNA switch helix region. Cross-linking and X-ray diffraction data have revealed that this helix switch and the decoding region are in close proximity. Mutations in the switch helix (889-890) region affect translational fidelity and translocation. The binding site of hygromycin B and its known dual effect on the fidelity of decoding and translocation suggest a model for the action of this drug on ribosomes.

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Mitsui, Keiji, Masafumi Matsushita, and Hiroshi Kanazawa. "Saccharomyces cerevisiae glucose signalling regulator Mth1p regulates the organellar Na+/H+ exchanger Nhx1p." Biochemical Journal 432, no. 2 (November 12, 2010): 343–52. http://dx.doi.org/10.1042/bj20100796.

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Organelle-localized NHEs (Na+/H+ exchangers) are found in cells from yeast to humans and contribute to organellar pH regulation by exporting H+ from the lumen to the cytosol coupled to an H+ gradient established by vacuolar H+-ATPase. The mechanisms underlying the regulation of organellar NHEs are largely unknown. In the present study, a yeast two-hybrid assay identified Mth1p as a new binding protein for Nhx1p, an organellar NHE in Saccharomyces cerevisiae. It was shown by an in vitro pull-down assay that Mth1p bound to the hydrophilic C-terminal half of Nhx1p, especially to the central portion of this region. Mth1p is known to bind to the cytoplasmic domain of the glucose sensor Snf3p/Rgt2p and also functions as a negative transcriptional regulator. Mth1p was expressed in cells grown in a medium containing galactose, but was lost (possibly degraded) when cells were grown in medium containing glucose as the sole carbon source. Deletion of the MTH1 gene increased cell growth compared with the wild-type when cells were grown in a medium containing galactose and with hygromycin or at an acidic pH. This resistance to hygromycin or acidic conditions was not observed for cells grown with glucose as the sole carbon source. Gene knockout of NHX1 increased the sensitivity to hygromycin and acidic pH. The increased resistance to hygromycin was reproduced by truncation of the Mth1p-binding region in Nhx1p. These results implicate Mth1p as a novel regulator of Nhx1p that responds to specific extracellular carbon sources.

39

Villanueva, M. Teresa. "Rediscovering hygromycin A for Lyme disease treatment." Nature Reviews Drug Discovery 20, no. 12 (October 29, 2021): 896. http://dx.doi.org/10.1038/d41573-021-00180-x.

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40

Carroll, Anne M., James A. Sweigard, and Barbara Valent. "Improved Vectors for Selecting Resistance to Hygromycin." Fungal Genetics Reports 41, no. 1 (January 1, 1994): 22. http://dx.doi.org/10.4148/1941-4765.1367.

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41

Pozzo, Giovanna, and John Guardiola. "Construction of hygromycin-resistant retroviral cloning vectors." Nucleic Acids Research 16, no. 23 (1988): 11372. http://dx.doi.org/10.1093/nar/16.23.11372.

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42

Severin, Klaus, and Fritz Sch�ffl. "Heat-inducible hygromycin resistance in transgenic tobacco." Plant Molecular Biology 15, no. 6 (December 1990): 827–33. http://dx.doi.org/10.1007/bf00039423.

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43

Hasegawa, Koichi, and Martin Grumet. "Trauma-induced tumorigenesis of cells implanted into the rat spinal cord." Journal of Neurosurgery 98, no. 5 (May 2003): 1065–71. http://dx.doi.org/10.3171/jns.2003.98.5.1065.

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Object. Findings in several clinical cases have suggested a correlation between tumor formation and previous injury to the central nervous system (CNS); however, the relationship between trauma and tumorigenesis has not been investigated well experimentally. In this study the authors provide evidence correlating tumorigenesis with trauma in the rat spinal cord. Methods. A glial cell line, C6R-G/H, which expresses green fluorescent protein (GFP) and hygromycin phosphotransferase (HPT), was implanted into normal and injured rat spinal cords. In all rats in which the cells were implanted into an injured site, locomotor function deteriorated and histological analysis demonstrated glioblastoma multiforme by 6 weeks; tumorigenesis was correlated with a loss of both GFP expression and resistance to hygromycin treatment. In contrast, no evidence of tumor formation was found at 6 weeks in rats in which the cells were implanted into healthy tissue. When C6R-G/H cells were treated with contused spinal cord extract in culture before implantation, they lost GFP expression and hygromycin resistance, and later formed tumors after implantation into normal spinal cord. Conclusions. The findings of this study indicate that trauma can induce tumorigenesis. Implantation of C6R-G/H cells into traumatized spinal cords resulted in their transformation, which was signaled by loss of GFP expression and hygromycin resistance accompanied by tumor formation. Exposure to extracts derived from injured spinal cord produced similar transformation and gene expression changes, as well as tumor formation after such cells were implanted into normal cords. Care, therefore, should be taken when cells are implanted into an injured CNS because of potential mutagenesis due to trauma-induced factors.

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Baumgartner, Kendra, Phillip Fujiyoshi, Gary D. Foster, and Andy M. Bailey. "Agrobacterium tumefaciens-Mediated Transformation for Investigation of Somatic Recombination in the Fungal Pathogen Armillaria mellea." Applied and Environmental Microbiology 76, no. 24 (October 15, 2010): 7990–96. http://dx.doi.org/10.1128/aem.01049-10.

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ABSTRACT Armillaria root disease is one of the most damaging timber and fruit tree diseases in the world. Despite its economic importance, many basic questions about the biology of the causal fungi, Armillaria spp., are unanswered. For example, Armillaria undergoes matings between diploid and haploid mycelia, which can result in a recombinant diploid without meiosis. Evidence of such somatic recombination in natural populations suggests that this reproductive mode may affect the pathogen's ecology. Investigations of the mechanisms and adaptive consequences of somatic recombination are, however, hampered by the lack of a method to reliably synthesize somatic recombinants. Here we report the first genetic transformation system for the genus Armillaria. We transformed A. mellea with selective markers for use in diploid-haploid matings to reliably synthesize somatic recombinants. This was accomplished with Agrobacterium tumefaciens carrying pBGgHg, which carries the hygromycin phosphotransferase gene (hph). hph was integrated into transformants, as evidenced by serial transfer to selective media, PCR, reverse transcription-PCR (RT-PCR), and Southern hybridization. Nuclear and mitochondrial markers were developed to genotype synthesized mycelia. In matings between a wild-type diploid and hygromycin-resistant haploids (transgenic), we identified recombinant, hygromycin-resistant diploids and, additionally, hygromycin-resistant triploids, all with the mitochondrial haplotype of the haploid partner. Our approach created no mycelium in which the haploid nucleus was replaced by the diploid nucleus, the typical outcome of diploid-haploid matings in Armillaria. This genetic transformation system, in combination with new markers to track chromosomal and cytoplasmic inheritance in A. mellea, will advance research aimed at characterizing the significance of somatic recombination in the ecology of this important fungus.

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Sisharmini, Atmitri, Bambang Sapta Purwoko, Nurul Khumaida, and Dan Kurniawan Rudi Trijatmiko. "Optimasi Konsentrasi Asetosiringon dan Higromisin dalam Transformasi Genetik Padi Fatmawati dengan Perantaraan Agrobacterium tumefaciens." Jurnal Agronomi Indonesia (Indonesian Journal of Agronomy) 46, no. 3 (January 21, 2019): 223–30. http://dx.doi.org/10.24831/jai.v46i3.19445.

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Protocols for genetic transformation of rice have been widely developed, however the protocols are not universal and inapplicable for all types of rice plants directly. Transformation protocol on rice cv. Fatmawati needs to be developed to generate transgenic lines. The present research was carried out to optimize genetic transformation protocol in rice cv. Fatmawati mediated by Agrobacterium tumefaciens harboring pCambia1301 construct using immature embryo as an explant. The experiment was arranged in a completely randomized design. Factors influencing efficiency of transformation, i.e., sensitivity of callus to hygromycin antibiotic, acetosyringone concentration used in cultivation medium, hygromycin concentration for transformant selection were optimized. The results showed that genetic transformation of rice cv. Fatmawati mediated by A. tumefaciens using immature embryos have been successfully carried out with several parameters. Addition of 100 µM acetosyringone in co-cultivation medium and 30 mg L-1 hygromycin for transformant callus selection were optimal for genetic transformation of rice cv. Fatmawati mediated by A. tumefaciens. Transformation efficiency was found to be 7.84% based on the lines carrying the hpt gene. This result would be a valuable reference in genetic transformation of rice cv. Fatmawati using target genes.Keywords: immature embryo, Oryza sativa, pCambia1301, transformation efficiency

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Johnson, A. P., M. Malde, N. Woodford, R. J. Cunney, and E. G. Smyth. "Urinary isolates of apramycin-resistantEscherichia coliandKlebsiella pneumoniaefrom Dublin." Epidemiology and Infection 114, no. 1 (February 1995): 105–12. http://dx.doi.org/10.1017/s0950268800051955.

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SUMMARYTwenty-two gentamicin-resistant urinary isolates ofEscherichia coliand five gentamicin-resistant urinary isolates ofKlebsiella pneumoniaefrom a Dublin hospital were examined for resistance to the veterinary aminoglycoside antibiotic apramycin. Five isolates ofE. coliand one isolate ofK. pneumoniaewere found to be resistant. The apramycin-resistant isolates, which were also resistant to the veterinary anthelmintic agent hygromycin B, hybridized with a DNA probe for the gene encoding the enzyme 3-N-aminoglycoside acetyltransferase type IV (AAC(3)IV). Resistance to apramycin and hygromycin B was co-transferable in four of the five isolates ofE. coliand the isolate ofK. pneumoniae.In one isolate ofE. coliapramycin resistance was not transferable. On the basis of their restriction enzyme digestion profiles and the antimicrobial resistance traits encoded, the transferable plasmids encoding resistance to apramycin and hygromycin B comprised three distinct types. Genetic linkage between the gene encoding AAC(3)IV and genes encoding resistance to ampicillin and either tetracycline or trimethoprim, means that the relatively widespread use of these antimicrobial agents provides a selective pressure for the persistence of resistance to apramycin and gentamicin even in the absence of bacterial exposure to aminoglycosides.

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Egelhoff, T. T., S. S. Brown, D. J. Manstein, and J. A. Spudich. "Hygromycin resistance as a selectable marker in Dictyostelium discoideum." Molecular and Cellular Biology 9, no. 5 (May 1989): 1965–68. http://dx.doi.org/10.1128/mcb.9.5.1965-1968.1989.

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We have constructed an expression cartridge which has the bacterial hygromycin resistance gene (hph) fused to the Dictyostelium discoideum actin 15 promoter, with a segment of 3'-flanking DNA from the actin 15 locus placed downstream of the hph gene to serve as a transcription terminator. The plasmid pDE109, which contained this cartridge and a Dictyostelium origin of replication, transformed D. discoideum with high efficiency under hygromycin selection. The availability of this selectable marker circumvents the previous limitation of having G418 resistance as the only selectable marker for this organism; secondary transformation can now be used to introduce DNA into previously transformed cell lines.

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Kojic, Milorad, and William K. Holloman. "Shuttle vectors for genetic manipulations in Ustilago maydis." Canadian Journal of Microbiology 46, no. 4 (April 1, 2000): 333–38. http://dx.doi.org/10.1139/w00-002.

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Shuttle vectors with new or improved features were constructed to enable facile genetic manipulations in the plant pathogen Ustilago maydis. Sets of plasmids selectable in media containing geneticin, carboxin, nourseothricin, or hygromycin, able to replicate autonomously, to transform U. maydis by integration, and to express foreign genes under control of the homologous glyceraldehyde-3-phosphate dehydrogenase promoter, were built upon a common pUC19 vector backbone. This permits a large number of choices for a cloning site, blue/white screening for recombinant plasmids, rapid transfer of a cloned DNA fragment between plasmids, and choice of several dominant drug-resistance markers for selection in U. maydis.Key words: G418, carboxin, nourseothricin, hygromycin, expression vectors.

49

Egelhoff, T. T., S. S. Brown, D. J. Manstein, and J. A. Spudich. "Hygromycin resistance as a selectable marker in Dictyostelium discoideum." Molecular and Cellular Biology 9, no. 5 (May 1989): 1965–68. http://dx.doi.org/10.1128/mcb.9.5.1965.

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We have constructed an expression cartridge which has the bacterial hygromycin resistance gene (hph) fused to the Dictyostelium discoideum actin 15 promoter, with a segment of 3'-flanking DNA from the actin 15 locus placed downstream of the hph gene to serve as a transcription terminator. The plasmid pDE109, which contained this cartridge and a Dictyostelium origin of replication, transformed D. discoideum with high efficiency under hygromycin selection. The availability of this selectable marker circumvents the previous limitation of having G418 resistance as the only selectable marker for this organism; secondary transformation can now be used to introduce DNA into previously transformed cell lines.

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Spyridonidis, Alexandros, Philipp Faber, Loizos Petrikkos, and Jurgen Finke. "Alloanatigenic Reactions after Hematopoietic Cell Transplantation Induce Genomic Alterations in Epithelial Cells as Shown in Human Studies and in an In Vitro Model." Blood 108, no. 11 (November 16, 2006): 3213. http://dx.doi.org/10.1182/blood.v108.11.3213.3213.

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Abstract Previous studies from our group demonstrated frequent genomic alterations measured by microsatellite instability (MSI) in non-neoplastic epithelial tissues of patients who underwent allogeneic hematopoietic cell transplantation (HCT) (Blood2006;107:3389–3396). These genomic alterations were found only after allogeneic but not after autologous HCT, and therefore we hypothesized that an “allogeneic” effect is substantially involved in the mutation process. We extended our previous analyses by examining 210 bucall swabs obtained from 70 patients between day (d+) 26 and d+3514 after allogeneic HCT for the presence of MSI. MSI analysis was performed by PCR and denaturing capillary electrophoresis at three tetranucleotide (THO-1, SEE33, D14S120) and three mononucleotide microsatellite (ZP3, BAT26, SRY) loci. MSI was found in the buccal smears of 38% allografted patients (median time of occurence 322 days). In a prospective trial, in which patients were followed from time before transplantation until d+365, 5 out of 14 (35%) patients exhibited MSI after transplantation although all of them showed stable microsatellites before transplantation. We are currently pefroming statistical analyses in order to identify which clinical factors influence the presence of MSI and we will present the data in the meeting. To test the hypothesis that an “alloantigenic” effect is responsible for th induction of MSI, we developed a model system in which keratinocyte (HaCaT) cells were transfected with a palsmid vector which carries a G418 (neo) selectable marker and a microsatellite repeat (CA) that places the sequence for Hygromycin Resistance (HygR) out of frame for protein translation. In this reporter system, DNA slippage mutations can restore the HygR reading frame and become detectable by hygromycin treatment as hygromycin resistant (HygR+) colonies. Pools of stably transfected (neo+) HaCaT cells were treated with supernatant (SN) of major histocompatibility complex nonmatched mixed lymphocyte cultures (MLC) and assayed for HygR+ colonies 48h later. Cells transfected with a control, in-frame hygromycin B gene construct (p12) were used as positive controls. Using this system, we found that HaCaT cells aquire hygromycin resistance after treatment with supernatatant from MLC. Treatment of cells with hydrogen hyperoxid which has been shown in a E. Coli system to induce MSI (PNAS1998, 95:12468–12473) generated HygR+ colonies at a >80% lower frequency than the SN-MLC treatment. Control p12 transfected cells were grown with high efficiency in the presence of hygromycin B. In summary, our in vivo data confirm our previous results and provide evidence of genomic alterations after allogeneic HCT and our in vitro data are compatible with the hypothesis that an “alloantigenic” factor is the driving force in producing detectable MSI in the allografted patients. Elucidating the ultimate mechanisms underlying the genomic instability following allogeneic HCT may prove to be of major therapeutic value.

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