• Journal of Plant Biotechnology
• /
• v.7 no.2
• /
• pp.97-103
• /
• 2005

This study was carried out to obtain basic information for gene manipulation in potent edible tobacco (Nicotiana tabacum cv. TI 516). N. tabacum cv. TI 516 is a plant for a possible candidate to use as an edible vaccine, since it contains a low level of nicotine. The effective plant regeneration system through leaf disc culture was achieved using a MS basal medium supplemented with 0.1 mg $1^{-1}$ NAA and 0.5 mg $1^{-1}$ BA. In order to transform the N. tabacum cv. TI 516 with the green fluorescent protein (GFP) gene, Agrobacterium tumefaciens LBA 4404 containing the GFP gene was used. Genomic PCR confirmed the integration of the GFP gene into nuclear genome of transgenic plants. Expression of the GFP gene was identified in callus, apical meristem and root tissue of transgenic N. tabacum cv. TI 516 plants using fluorescence microscopy. Western blot analysis revealed the expression of GFP protein in the transgenic edible tobacco plants. The amount of GFP protein detected in the transgenic tobacco plants was approximately 0.16% of the total soluble plant protein (TSP), which was determined by ELISA.

• Korean Journal Plant Pathology
• /
• v.10 no.3
• /
• pp.228-234
• /
• 1994

Genomic RNA was extracted from Cy strain of odontoglossum ringspot tobamovirus (ORSV-Cy) isolated from infected leaves of tobacco cv. Samsun. Size of the genomic RNA was about 6.6 kb in length. The genomic RNA was fractionated using Sephadex G-50 column chromatography into 2 fractions. They were polyadenylated at their 3'-end using E. coli poly(A) polymerase. Polyadenylated viral RNA was recovered by oligo (dT) primer adapter containing NotI restriction site and Moloney murine leukemia virus SuperScript reverse transcriptase (RNase H-). Second-strand cDNA was synthesized by using E. coli DNA ligase, E. coli DNA polymerase I and E. coli RNase H. Recombinant plasmids containing cDNAs for ORSV-Cy RNA ranged from about 800 bp to 3,000 bp. Among the selected 238 recombinants, pORCY-124 clone was the largest one covering 3'-terminal half of the viral RNA. This clone contained two restriction sites for EcoRI and XbaI and one site for AccI, AvaI, BglII, BstXI, HindIII, PstI, and TthIII 1. respectively. The clone contained partial viral replicase, a full-length movement protein and a complete coat protein genes followed by a 3' untranslated region of 414 nucleotides based on restriction mapping and nucleotide sequencing analyses. Clones pORCY-028, -068, -072, -187 and -224 were overlapped with the pORCY-124. Clones pORCY-014 and -095 covered 5' half upstream from the middle region of the viral RNA, which was estimated based on restriction mapping and partial sequence analysis. Constructed cDNA library covered more than 90% of the viral genome.

• International Journal of Industrial Entomology and Biomaterials
• /
• v.31 no.2
• /
• pp.62-69
• /
• 2015

The Asian cavity-nesting honeybee, Apis cerana (Hymenoptera: Apidae), has been extensively studied for its biogeography and genetic diversity, but the molecules utilized in past studies were mainly ~90 bp long mitochondrial non-coding sequences, located between $tRNA^{Leu}$ and COII. Thus, additional molecular markers may enrich our understanding of the biogeography and genetic diversity of this valuable bee species. In this study, we reviewed the public genome database to find introns of cDNA sequences, with the assumption that these introns may have less evolutionary constraints. The six introns selected were subjected to preliminary tests. Thereafter, two introns, titled White gene and MRJP9 gene, were selected. Sequencing of 552 clones from 184 individual bees showed a total of 222 and 141 sequence types in the White gene and MRJP9 gene introns, respectively. The sequence divergence ranged from 0.6% to 7.9% and from 0.26% to 17.6% in the White gene and the MRJP9 introns, respectively, indicating higher sequence divergence in both introns. Analysis of population genetic diversity for 16 populations originating from Korea, China, Vietnam, and Thailand shows that nucleotide diversity (π) ranges from 0.003117 to 0.025837 and from 0.016541 to 0.052468 in the White gene and MRJP9 introns, respectively. The highest π was found in a Vietnamese population for both intron sequences, whereas the nine Korean populations showed moderate to low sequence divergence. Considering the variability and diversity, these intron sequences can be useful as non-mitochondrial DNA-based molecular markers for future studies of population genetics.

• Journal of Microbiology and Biotechnology
• /
• v.4 no.3
• /
• pp.183-190
• /
• 1994

Cell-free extracts prepared from cultured insect cells, Spodoptera. frugiperda, were analyzed for activation of early gene transcription of an insect baculovirus, Autographa californica nuclear polyhedrosis virus (AcNPV). The template DNA used for in vitro transcription assays contained promoter sites for the baculovirus genes that have been classified as immediate early ($\alpha$) or early genes. These genes are located in the HindIII-K/Q region of the AcNPV genome. Nuclei isolated from the AcNPV-infected Spodoptera frugiperda cells were also used for in vitro transcription analysis by RNase-mapping the labeled RNA synthesized from in vitro run-on reaction in the isolated nuclei. The genes studied by this technique were p26 and pl0 genes which were classified as delayed early and late gene, respectively. We found that transcription of the genes from the HindIII-K region was accurately initiated and unique in the whole cell extract obtained from uninfected cells, although abundance of the in vitro transcripts was reverse to that of in vivo RNA. With isolated nuclei transcription of the p26 gene was inhibited by $\alpha$-amanitin suggesting that the p26 gene was transcribed by host RNA polymerase II. However, transcription of the pl0 gene in isolated nuclei was not inhibited by $\alpha$-amanitin, but rather stimulated by the inhibitor. We also found that the synthesis of $\alpha$-amanitin-resistant RNA polymerase was begun before 6 hr p.i., the time point at which the onset of viral DNA replication as well as the appearance of a-amanitin-resistant viral transcripts were detected. These studies give us strong evidence to support the previous data that early genes of AcNPV were transcribed by host RNA polymerease III, while transcription of late genes was mediated at least by a novel $\alpha$-amanitin-resistant RNA polymerase.

• Microbiology and Biotechnology Letters
• /
• v.47 no.4
• /
• pp.645-650
• /
• 2019

Despite differences in virulence between strains of Toxoplasma gondii, rapid and accurate genotyping methods are lacking. In this study, a method was developed to detect and genotype T. gondii in food and environmental samples using PCR and a novel peptide nucleic acid (PNA) melting array. An alignment of genome sequences for T. gondii type I, II, and III obtained from NCBI was generated, and a single nucleotide polymorphism analysis was performed to identify targets for PCR amplification and a PNA melting array. Prior to the PNA melting array, conventional PCR was used to amplify GRA6 of T. gondii. After amplification, the PNA melting array was performed using two different PNA hybridization probes with fluorescent labels (FAM and HEX) and quenchers. Melting curves for each probe were used to determine genotypes and identify mutations. A 214-bp region of the GRA6 gene of T. gondii was successfully amplified by PCR. For all T. gondii strains (type I, II, and III) used to evaluate specificity, the correct genotypes were determined by the PNA melting array. Non-T. gondii strains, including 14 foodborne pathogens and 3 protozoan parasites, such as Giardia lamblia, Cryptosporidium parvum, and Entamoeba histolytica, showed no signal, suggesting that the assay has a high specificity. Although this is only a proof-of-concept study, the assay is promising for the fast and reliable genotyping of T. gondii from food and environmental samples.

• Proceedings of the KSAR Conference
• /
• 2003.06a
• /
• pp.98-98
• /
• 2003

Direct gene transfer to mammalian tissues has significant potential for gene therapy and transgenesis. Liposome-mediated in vivo transfection has begun to gain attention as an alternative to viral vectors, and may also be a good mode of transfection in gene transfer. Interestingly, polymerized cationic liposomes are reported to be very stable in the bloods and efficient for in vivo gene transfer. To examine a possible gene delivery in vivo, we investigated the efficacy and safety of the liposome-mediated gene transfer using vein injection in chick or mouse as model animals. The number of injected pGFP-LacZ using either a commercial or home-made liposomes was 8 and 19 at 16 and 7 day of hatch, respectively. One of injected chick of each experiments was analyzed and the rest is being bred. In mouse, 4/22 showed expression of pGFP-LacZ but 8/22 showed no expression and the remaining animals are also being bred. After injection of liposome/pGFP-LacZ complex into wing vein of 7 or 16 day-old chick, pGFP-LacZ was detected in various tissues isolated from not only young chick but also old chick were turned out to possess. exogenous DNA. Transcripts and proteins of the transgene were also detected by RT-PCR or histochemical analysis, respectively. These results suggest that injected DNA were inserted to genome and produced mRNA and proteins in various tissues and may give an important tools for effective gene delivery in gene therapy or transgenesis.

• Journal of Animal Science and Technology
• /
• v.54 no.6
• /
• pp.411-418
• /
• 2012

KIT gene is the major causative gene for coat color variation in diverse animal species. This gene regulates melanocyte migration from the neural crest to target tissues and the mutation of this gene can affect dominant white phenotypes in animals. Because this gene has a major influence for the coat color variation, single nucleotide polymorphisms (SNPs) in 14 Korean cattle (Hanwoo) and 5 Holstein individuals were investigated. The Hanwoo DNA samples included three different colored (5 Black, 5 Yellow and 4 Stripe) animals. Total 126 polymorphisms have been identified and 23 of them are located in the exon region. Also, 5 bp (TTCTC) and 3 bp (TCT) intronic indels in intron 3 and intron 5, respectively, were identified. Out of 23 exonic polymorphisms, 15 SNPs are the missense mutations and the rest of the SNPs are silence mutations. The neighbor-joining phylogenetic tree was constructed for the different colored animals using the obtained KIT gene sequences. Holstein breed showed a clear breed-specific cluster in the phylogenetic tree which is differed from Hanwoo. Also, three colored Hanwoo animals were not discriminated among the breeds. The KIT gene polymorphisms identified in this study will possibly give some solutions for the color variations in cattle with further verifications.

• Journal of The Korean Society of Grassland and Forage Science
• /
• v.26 no.2
• /
• pp.83-90
• /
• 2006

To develop transgenic birdsfoot trefoil (Lotus corniculatus L.) plants tolerant to environmental stress, Arabidopsis NDPK gene (AtNDPK) was introduced into birdsfoot trefoil plants using Agrobacterium-mediated transformation and expressed powerfully under the control of the E35S promoter. The expression vector, pEN-K was used for introduction of AtNDPK gene into birdsfoot trefoil plaits. The transformed calli were selected on kanamycin containing medium and then regenerated. The transformed birdsfoot trefoil plants were cultivated for 4 months on BOi2Y medium. Genomic DNA PCR and Southern blot analysis confirmed the incorporation of AtNDPK into the birdsfoot trefoil genome.

• Asian-Australasian Journal of Animal Sciences
• /
• v.21 no.12
• /
• pp.1703-1709
• /
• 2008

As genetic markers, single nucleotide polymorphisms (SNP) are very appropriate for the development of genetic tests for economic traits in livestock. Several microsatellite markers have been identified as useful markers for the genetic improvement of Hanwoo. Among those markers, ILSTS035 was recently mapped at a similar position with four SNPs (AH1_11, AH1_9, 31465_446, and 12273_165) in a linkage map of EST-based SNP in BAT6. Among the four SNPs, two SNPs (31465_446 and 12273_165) were analyzed using BLAST at the NCBI web site. The sequences including the 12273_165 SNP were identified at the intron region within the LOC534614 gene on the gene sequence map (Bos taurus NCBI Map view, build 3.1). The LOC534614 gene represents a protein similar to myosin heavy chain, fat skeletal muscle, embryonic isoform 1 in the dog, and myosin_1 (Myosin heavy chain D) in Macaca mulatta. In cattle, the myosin heavy chain was associated with muscle development. The phenotypic data for growth and carcass traits in the 415 animals were analyzed by the mixed ANCOVA (analysis of covariance) linear model using PROC GLM module in SAS v9.1. By the genotyping of Hanwoo individuals (n = 415) to evaluate the association of SNP with growth and carcass traits, it was shown that the 12273_165 SNP region within LOC534614 may be a candidate marker for growth. The results of the statistical analyses suggested that the genotype of the 12273_165 SNP significantly affected birth weight, weight of the cattle at 24 months of age, average daily gain and carcass cold weight (p<0.05). Consequently, the 12273_165 SNP polymorphisms at the LOC534614 gene may be associated with growth in Hanwoo, and functional validation of polymorphisms in LOC534614 should be performed in the future.

• Journal of Microbiology and Biotechnology
• /
• v.24 no.6
• /
• pp.803-811
• /
• 2014

Concerns over drug-resistant bacteria have stimulated interest in developing alternative methods to control bacterial infections. Endolysin, a phage-encoded enzyme that breaks down bacterial peptidoglycan at the terminal stage of the phage reproduction cycle, is reported to be effective for the control of bacterial pathogenic bacteria. Bioinformatic analysis of the SPN9CC bacteriophage genome revealed a gene that encodes an endolysin with a domain structure similar to those of the endolysins produced by the P1 and P22 coliphages. The SPN9CC endolysin was purified with a C-terminal oligo-histidine tag. The endolysin was relatively stable and active over a broad temperature range (from $24^{\circ}C$ to $65^{\circ}C$). It showed maximal activity at $50^{\circ}C$, and its optimum pH range was from pH 7.5 to 8.5. The SPN9CC endolysin showed antimicrobial activity against only gram-negative bacteria and functioned by cutting the glycosidic bond of peptidoglycan. Interestingly, the SPN9CC endolysin could lyse intact gram-negative bacteria in the absence of EDTA as an outer membrane permeabilizer. The exogenous lytic activity of the SPN9CC endolysin makes it a potential therapeutic agent against gram-negative bacteria.