• Journal of Plant Biology
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• v.37 no.1
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• pp.77-83
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• 1994

DNA uptake in dry embryos of rice by DNA imbibition was detected by monitoring the expression of chimeric vectors. The selective markers of expression vectors used were ${\beta}-glucuronidase$ ronidase (GUS) and hygromycin phosphotransferase (HPT) genes under the control of CaMV35 S promoter. Frequency of transient expression of the foreign gene was generally 30-50% varying according to the types of vectors and rice cultivars. Dot blot analysis and DNA sequence analysis of inverse polymerase chain reaction products showed that selected rice in hygromycin B (HmB) medium had HPT gene and CaMV35S promoter DNA sequence in genomic DNA of rice. To investigate what ratio of rice having two marker genes simultaneously as rice embryos imbibed the vector DNA having two HPT and GUS gene, transform ants selected in lImB medium were subjected to PCR for GUS gene. It was shown that about 90 percentage of surviving ones in HmB medium had GUS gene.S gene.

• Applied Biological Chemistry
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• v.44 no.1
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• pp.1-6
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• 2001

To elucidate the apoptosis mechanism of Trichoplusia ni cell, fundamental studies for apoptosis induction and suppression were performed. Hygromycin B, a known inducer of apoptosis, started the inhibition of T. ni cell growth at $200\;{\mu}/ml$ concentration. Furthermore, at $400\;{\mu}/ml$ concentration, DNA fragmentation was detected on day 2 of incubation. Although both dexamethasone and sodium butyrate inhibited T. ni cell growth, DNA fragmentation was not detected by both treatments. Also, when apoptosis induced T. ni cells with $200\;{\mu}/ml$ hygromycin B were treated with caspase inhibitor (Ac-DEVD-CHO), the apoptotsis was suppressed by 36%. In addition, N-acetylcysteine, another apoptosis repressor, also inhibited the apoptosis of T. ni cells. In order to express the anti-apoptosis gene (bcl-2), T. ni cells were transiently transformed with bcl-2 and its expression was confirmed by western blot analysis. These results showed the potential of developing new insect cell lines with suppressed apoptosis.

• Korean Journal of Medicinal Crop Science
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• v.22 no.6
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• pp.504-509
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• 2014

Methicillin-resistant Staphylococcus aureus (MRSA) is a substantial contributor to morbidity and mortality. In search of a natural products capable of inhibiting this multidrug resistant bacteria, we have investigated the antimicrobial activity of brazilein (BRZ) isolated from Caesalpinia sappan L. (Leguminosae) against 8 different strains of Staphylococcus aureus (S. aureus). New antimicrobial activity was found using the minimum inhibitory concentrations (MICs), broth dilution as well as checkerboard method. Against the 8 strains, the minimum inhibitory concentrations of BRZ were in the range of $62.5-500{\mu}g/mL$. From those results we performed the checkerboard test to determine the synergism of BRZ in combination with Hygromycin-b (HgB) against 4 strains. The combined activity of BRZ and HgB against 4 strains resulted in a fractional inhibitory concentrations index (FICI) ranging from 0.18-0.5. The effect of BRZ with HgB was found to be synergistic. We found that BRZ reduced the MICs of HgB. BRZ and HgB could lead to the development of new combination antibiotics against MRSA infection.

• Microbiology and Biotechnology Letters
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• v.14 no.2
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• pp.133-137
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• 1986

17S-ribosomal RNA gene from the hygromycin resistant protozoan Tetrahymena thermophila hmr 3 was cloned on E. coli vector pBR 322 as part of study to work the 17S-rRNA structure and the mechanism of hygromycin resistance. The 17S-rDNA was inserted into the Hind 111 site of pBR 322. The clones having recombinant plasmid were selected by the method of colony hybridization with a 17S-rDNA probe of wild type B1868. The orientation of 17S-rDNA insert was located near the tetracycline resistant gene of pBR 322 in a clone 5-19 with the recombinant plasmid.

• Mycobiology
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• v.29 no.3
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• pp.132-134
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• 2001

Since it is known that Agrobacterium tumefaciens, which has long been used to transform plants, can transfer the T-DNA to yeast Saccharomyces cerevisiae during tumourigenesis, a variety of fungi were subjected to transformation to improve their transformation frequency. In this study, I report the A. tumefaciens-mediated transformation of filamentous fungus Aspergillus niger. Transfer of the binary vector pBIN9-Hg, containing the bacterial hygromycin B phosphotransferase gene under the control of the Aspergillus nidulans trpC promoter and terminator as a selectable marker, led to the selection of $50{\sim}100$ hygromycin B-resistant transformants per $1{\times}10^7$ conidia of A. niger. This efficiency is improved $10{\sim}20$ fold more than reported elsewhere. In order to avoid the difficulties in selection transformant from the over-growing non-transformant, I used top agar containing 900 ${\mu}g/ml$ of hygromycin. Genomic PCR and Southern analysis showed that all transformants contained single T-DNA insert per fungal genome. This technique offers an easier and more efficient method than that of using protoplast.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 2003.10a
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• pp.68.2-69
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• 2003

Genetic transformation carried out to induce the pathogenicity mutants from the two isolates, Elsinoe fawcettii R-34 and MUD of citrus scab fungus to hygromycin resistant by transferring plasmides (pUCATPH) that contain hygB gene. We produced protoplast for transformation by using of combinations of available enzymes including ${\beta}$-D-glucanase, ${\beta}$ -glucuronidase, Iyticase and driselase. The protoplasts regenerated at 64 $\mu\textrm{g}$/ml of hygromycin B but not 128 $\mu\textrm{g}$ in sensitivity test to identify the concentration of useful marker for the selection of transformants. Approximately 1200 and 67 hygromycin resistant isolates from strain R-34 and strain MUD, respectively, were isolated on PDA added with 200 $\mu\textrm{g}$ /ml of hygromycun B. Fifty seven and 4 of hygromycin resistant isolates from strain R-34 and MUD, respectively, did not produce necrotic lesions on the leaf in detached-leaf assay. Finally, 9 isolates were isolated from strain R-34, and these Isolates produced non or very few symptoms on seedlings of citrus in greenhouse pathogenicity test. And it's very interesting that some isolates produced melanose-like symptom on very young leaves which it was not typical symptom and somtimes produced on only expanded leaf.

• Journal of Microbiology
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• v.44 no.4
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• pp.383-395
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• 2006

Four different Trichoderma strains, T. harzianum CECT 2413, T. asperellum T53, T. atroviride T11 and T. longibrachiatum T52, which represent three of the four sections contained in this genus, were transformed by two different techniques: a protocol based on the isolation of protoplasts and a protocol based on Agrobacterium-mediated transformation. Both methods were set up using hygromycin B or phleomycin resistance as the selection markers. Using these techniques, we obtained phenotypically stable transformants of these four different strains. The highest transformation efficiencies were obtained with the T. longibrachiatum T52 strain: 65-70 $transformants/{\mu}g$ DNA when transformed with the plasmid pAN7-1 (hygromycin B resistance) and 280 $transformants/l0^7$ spores when the Agrobacterium-mediated transformation was performed with the plasmid pUR5750 (hygromycin B resistance). Overall, the genetic analysis of the transform ants showed that some of the strains integrated and maintained the transforming DNA in their genome throughout the entire transformation and selection process. In other cases, the integrated DNA was lost.

• The Korean Journal of Mycology
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• v.39 no.3
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• pp.235-238
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• 2011

Agrobacterium harboring vector pCHBs with hygromycin phosphotransferase(hph) and hepatitis B virus surface antigen(HBsAg)gene was transformed into the mycelial culture of Flammulina velutipes. In particular, mild NaOH solution was treated to the mycelia before Agrobacterium infection step. This was purposed to generate putative surface wounds in the mycelial cell walls. The results showed that hygromycin-resistant($hyg^r$) mycelia could be obtained only from NaOH-treated mycelia but not from intact mycelia. The integration of $hyg^r$ gene in fungal genome was confirmed by PCR. In addition, a single transgene integration and heterologous protein expression in F. velutipes could be verified by Southern blot hybridization and western blot analysis, respectively. This study demonstrated an efficient tool for the Agrobacterium-mediated transformation of F. velutipes mycelia.

• Mycobiology
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• v.38 no.4
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• pp.331-335
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• 2010

In this study, we developed an efficient electroporation-mediated transformation system featuring Flammulina velutipes. The flammutoxin (ftx) gene of F. velutipes was isolated by reverse transcription-PCR. pFTXHg plasmid was constructed using the partial ftx gene (410 bp) along with the hygromycin B phosphotransferase gene (hygB) downstream of the glyceraldehydes-3-phosphate dehydrogenase (gpd) promoter. The plasmid was transformed into protoplasts of monokaryotic strain 4019-20 of F. velutipes by electroporation. High transformation efficiency was obtained with an electric-pulse of 1.25 kV/cm by using 177 transformants/${\mu}g$ of DNA in $1{\times}10^7$ protoplasts. PCR and Southern blot hybridization indicated that a single copy of the plasmid DNA was inserted at different locations in the F. velutipes genome by non-homologous recombination. Therefore, this transformation system could be used as a useful tool for gene function analysis of F. velutipes.

• Microbiology and Biotechnology Letters
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• v.24 no.4
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• pp.427-429
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• 1996

We have already cloned an extracellular $\alpha$-glucosidase gene from Aspergillus niger with oligonucleotide probe synthesized on the basis of the peptide sequences determined previously. The DNA sequence revealed an open reading frame of 895 amino acids split by three introns. We are attempting to construct an A. niger strain deficient in the $\alpha$-glucosidase enzyme activity, which would be useful for the glucoamylase production without contamination by the industrially undesirable $\alpha$-glucosidase. For destruction of the $\alpha$-glucosidase gene, we try to make transformations. A cloned partial $\alpha$-glucosidase gene was introduced into Aspergillus niger, and transformants with suppressed $\alpha$-glucosidase activity were isolated. The transformants were cultured on YPD medium which contained Hygromycin B at 30$\circ$C. The activity of $\alpha$-glucosidase of the suppressed transformants was compared to that of wild type activity. As shown by southern-hybridization, we detected that the transformant was a heterocaryon.