• The Korean Journal of Mycology
• /
• v.17 no.1
• /
• pp.27-30
• /
• 1989

F. velutipes Leu 2 gene (${\beta}-isopropylmalate$ dehydrogenase gene) was used for transformation of P. florida leucine requiring auxotrophic mutant P101. Transformation frequency was very low but the transformed colony can grow on minimal medium very slowly. Transformation was identified by Southern hybridization and reverse transformation into E. coli using chromosome DNA isolated from transformed P. florida.

• Journal of Plant Biotechnology
• /
• v.6 no.2
• /
• pp.69-75
• /
• 2004

Optimal protocol for efficient genetic transformation has been defined to aid future strategies of genetic engineering in pigeon pea with agronomically important genes. Transgenic pigeonpea plants were successfully produced through Agrobacterium tumefaciens-mediated genetic transformation method using cotyledonary node explants by employing defined culture media. The explants were co-cultivated with A. tumefaciens strain C-58 harboring the binary plasmid, pCAMBIA-1301 [con-ferring $\beta$-glucuronidase(GUS) activity and resistance to hygromycin] and cultured on selection medium (regeneration medium supplemented with hygromycin) to select putatively transformed shoots. The shoots were then rooted on root induction medium and transferred to pots containing sand and soil mixture in the ratio of 1:1. About 22 putative TO transgenic plants have been produced. Stable expression and integration of the transgenes in the putative transgenics were confirmed by GUS assay, PCR and Southern blot hybridization with a transformation efficiency of over 45%. Stable integration and expression of the marker gene has been confirmed in the TO and T1 transgenics through PCR, and Southern hybridization.

• Korean Journal of Plant Tissue Culture
• /
• v.27 no.6
• /
• pp.491-496
• /
• 2000

A RAG25 gene regulating flowering time was introduced into Codonopsis lanceolata through high efficiencies (ca. 90%) of plant regeneration. The leaf explants were immersed in YEP media containing Agrobacterium tumefaciens (pGA 1209) harboring RAG25 gene, and cocultivated for 3 days. After cocultivation, they were cultured in shoot inducing media (SIM), N2B2 (NAA 2 mg/L, BA 2 mg/L and kanamycin 20 mg/L) and N2B4 (NAA 2 mg/L, BA 4 mg/L and kanamycin 20 mg/L), and the putative transformants were regenerated. The introduction of nptII and RAG25 gene into Codonopsis lanceolata was confirmed by 0.7 kb and 0.6 kb bands from polymerase chain reaction and reconfirmed by Southern hybridization using PCR product of RAG25 gene.

• Microbiology and Biotechnology Letters
• /
• v.18 no.3
• /
• pp.305-308
• /
• 1990

In order to clone the gene coding for 3-isopropylmalate dehydrogenase of Muyveromyces fragilis, a shuttle plasmid vector pHNll4 was used. It can serve as a cloning vector in Saccharomyces cerevisiae DBY746 for other Sau3AI-cleaved DNA segment of Kluyveromyces fragilis. Two cloned fragments which complement the leu2 mutation of Saccharomyces cerevisiae and E, coli were obtained. Their length was 4.4 kb an 3.5 kb, and their orientation was opposite each other. From the fact that the two recombinant plasmids were expressed in Saccharomyces cerevisiae and E, coli, probably the two inserts had the promoter of Ktuyveromyces fi-agilis and that of Kluyveromyces fiagilis was efficiently assosiated with RNA polymerase of Saccharomyces cerevisiae and E. coli. According to the result of Southern hybridization, we thought that the cloned fragment has low homology with 3-isopropylmalate dehydrogenase coding region of E. coli and Saccharomyces cerevisiae.

• Journal of Life Science
• /
• v.10 no.4
• /
• pp.333-339
• /
• 2000

To identify genes involved in the decolorization of crystal violet, we isolated random mutants generated by transponson insertion in crystal violet-declorizing bacterium, Citrobacter sp. The resulting mutant bank yielded mutants with six distinct phenotypes, and Southern hybridization with a Tn5 fragment as a probe showed a single hybridized with six distinct phenotypes, and Southern hybridization with a Tn5 fragment as a probe showed a single hybridized band in the mutants Ctg 2, 5 an 6, whereas two and three bands were detected in Ctg1, 4 and 3, respectively. Tn5-inserted genes were isolated and the DNA sequence flanking Tn5 was determined. From comparison with a sequence database, putative protein product encoded by ctg 5 was identified as E. coli maltose transproter(Mal G) homolog, whereas the deduced amino acid sequence of the other ctg genes did not show any significant similarity with any DNA or protein sequency. Therefore, these results indicate that the other ctg genes except ctg 5 encode new proteins responsible for decolorization of crystal violet.

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

• Korean Journal of Microbiology
• /
• v.31 no.1
• /
• pp.48-53
• /
• 1993

For the purpose to improve the glucoamylase productivity of Saccharomyces diastaticus, we integrated STA 1 gene into chromosomal DNA of S. diastaticus using YIp vector. After construction of Ylp-STA by the subcloning of STAI (5.3 kb) into YIp5 vector, S. diastaticus GMT-II(a. ura3. STAJ) was transformed by Ylp-STA through homologous recombination at the chromosomal STAJ gene. So we obtained the tram formants that glucoamylase productivity was increased maximum six fold. These strains transformed by the multi-copy integration of Ylp-STA in chromosomal DNA were confirmed by Southern hybridization. And the integrated Ylp-STA was maintained stably during 30 mitotic divisions.

• Journal of Microbiology and Biotechnology
• /
• v.11 no.1
• /
• pp.1-6
• /
• 2001

Ten strains of plasmid-harboring Bifidobacterium sp. were isolated from the feces of adults and children, and named as Bifidobacterium sp. GE1-GE8, ST, and SH5. These plasmids were categorized into three homologous groups (pKJ50-homologous, pKJ36-homologous, and non-homologous groups) according to Southern hybridization patterns using the formerly characterized bifidobacterial plasmids, pKJ50 and pKJ36, as probes. nine strains harboring the plasmids were shown to accumulate single-stranded DNA as a replication intermediate, based on the S1 nuclease treatment and Southern hybridization. These results suggest that the strains replicate by a rolling circle mechanism. Minimal inhibitory concentrations (MIC) of the isolated bifidobacteria against several antibiotics were determined. Two strains, GE2 and GE3, showed relatively high MiC values against tetracycline ($793.6{\mu}g/ml$) and erythromycin ($153.6{\mu}g/ml$), respectively. The tetracycline resistance of GE2 disappeared when the resident plasmid of GE2 was cured by ethidium bromide. These results show that pKJ36-homologous and pKJ50-homologous plasmids are prevalent among various Bifidobacterium strains and some Bifidobacterium plasmids appear to code for antibiotic resistance.

• Journal of Periodontal and Implant Science
• /
• v.32 no.2
• /
• pp.281-290
• /
• 2002

The purpose of this study is to develop species-specific DNA probe for detection and identification of Prevotella intermedia (P. intermedia) G8-9K-3. This study procedure includes (1) whole-genomic DNA extraction of P. intermedia G8-9K-3 (2) construction of the genomic DNA library, (3) screening of strain-specific DNA probe by reverse dot hybridization, (4) confirmation of strain-specific DNA probe by Southern blot hybridization, (5) determination of nucleotide sequences of strain-specific DNA probe. Twenty-eight recombinant plasmids containing Hind III-digested DNA fragments of P. intermedia G8-9K-3 were obtained. Reverse Dot Hybridization and Southern blot analysis data showed that one of them, Pig3, could be P. intermedia G8-9K-3-specific DNA probe. This datum indicates that this Pig3 DNA probe could be useful in detection and identification of the P. intermedia G8-9K-3 strain.

• Applied Biological Chemistry
• /
• v.40 no.4
• /
• pp.271-276
• /
• 1997

We have prepared several chimeric constructs containing the bacteriophage T7 RNA polymerase gene under control of the wound-inducible potato proteinase inhibitor II (pin2) promoter and have transformed Nicotiana tabacum plants with these constructs. Southern blot analyses indicate that either one or two copies of the gene constructs are present in the transgenic plants. Northern blot analyses indicate that mRNA encoding T7 RNA polymerase is expressed in a wound-inducible manner. We purified T7 RNA polymerase and prepared antiserum. This antiserum was used for Western blot analyses to demonstrate that a protein which is cross reactive with T7 RNA polymerase is produced. The molecular mass of this protein is 80 kDa, a size which is consistant with the nicked form of the polymerase as is often seen when expressed in E. coli. RNA polymerase assays were used to indicate that the nicked form of T7 RNA polymerase is active and capable of incorporating labeled nucleotides into transcripts in vitro. Analysis of transgenic plants did indeed show that wound-inducible activation of the T7 RNA polymerase permits the establishment of a genetic system to overexpress genes in plants using T7 RNA polymerase(Received March 20, 1997; accepted May 2, 1997)