• Journal of Microbiology and Biotechnology
• /
• v.16 no.1
• /
• pp.118-125
• /
• 2006

The region responsible for replication of the megaplasmid pAtC58 in the nopaline-type Agrobacterium tumefaciens strain C58 was determined. A derivative ofa Co1E1 vector, pBluscript SK-, incapable of autonomous replication in Agrobacterium spp, was cloned with a 7.6-kb Bg1II-HindIII fragment from a cosmid clone of pAtC58, which contains a region adjacent to the operon for the utilization of deoxyfructosyl glutamine (DFG). The resulting plasmid conferred resistance to carbenicillin on the A. tumefaciens strain UIA5 that is a plasmidfree derivative of C58. The plasmid was stably maintained in the strain even after consecutive cultures for generations. Analysis of nested deletions of the 7.6-kb fragment showed that a 4.3-kb BglII-XhoI region sufficiently confers replication of the derivative of the ColE1 vector on UIA5. The region comprises three ORFs, which have high homologies with repA, repB, and repC of plasm ids in virulent Agrobacterium spp. including pTiC58, pTiB6S3, pTi-SAKURA, and pRiA4b as well as those of symbiotic plasmids from Rhizobium spp. Phylogenie analysis showed that rep genes in pAtC58 are more closely related to those in pRiA4 than to pTi plasmids including pTiC58, suggesting that the two inborn plasmids, pTiC58 and pAtC58, harbored in C58 evolved from distinct origins.

• Food Science of Animal Resources
• /
• v.31 no.5
• /
• pp.658-662
• /
• 2011

In this study, 543 samples of press hams, sausages, processed ground meat and processed cheese acquired from retail markets in Seoul and Gyeonggi province in Korea from 2005 to 2010 were monitored using a one-step multiplex polymerase chain reaction (PCR) method that involves the amplification of specific soya or maize endogenous genes and the amplification of 35S promoter (p35S) and nopaline synthase terminator (tNOS) for GMO detection. Among the 543 samples, 477 samples were amplified for maize and/or soybean endogenous genes. Although one sausage sample collected in 2008 showed amplification of tNOS, the result was assumed to be false positive based on the results from further tests of other sausage samples of the same brand. Our results demonstrate the absence of GM soya and/or maze of livestock products in the Korean market during 2005-2010. In addition, the one-step multiplex PCR using previously constructed primer sets appears to be useful as a screening method for the detection of GMOs in processed livestock products. However, more specific methods should be established and employed to detect the event-specific GM gene for positive reaction samples by screening tests in processed livestock products.

• Journal of Microbiology and Biotechnology
• /
• v.14 no.4
• /
• pp.822-828
• /
• 2004

Nopaline-type Agrobacterium tumefaciens strain C58 cannot utilize octopine (Oct) as the sole carbon and nitrogen sources. This strain harbors two plasmids; a virulent plasmid, pTiC58, and a megaplasmid, pAtC58. From strain NT1, which is a derivative of C58 harboring only pAtC58, we isolated spontaneous mutants that utilize Oct as the sole nitrogen source. These Oct-catabolizing mutants, however, could not utilize the opine as the sole carbon source. In contrast, strain UIA5, a plasmid-free derivative of C58, could not give rise to such mutants. The mutations isolated from NT1 were mapped to socR in pAtC58, which is a negative regulator of the soc operon responsible for the uptake and catabolism of an Amadori opine, deoxyfructosyl glutamine (Dfg). A derivative of UIA5 carrying a clone of the soc operon with a transposon inserted in socR also utilizes Oct as the sole nitrogen source. However, UIA5 harboring the operon with mutations in each of the structural genes in the soc operon, socA, B, C, and D, lost the ability to generate spontaneous Oct-utilizing mutants, suggesting that soc genes in pAtC58 are required for the utilization of Oct as a nitrogen source, and that derepressed expression of these genes allows cells to utilize Oct. In contrast, Oct-catabolizing mutants derived from C58, which grew using Oct as the sole nitrogen source, could also utilize the opine as the sole carbon source. These mutants did not carry any detectable mutations in socR or the region upstream to the gene in pAtC58, suggesting that mutations occurring elsewhere in the genome, most likely in pTiC58, allow the uptake and catabolism of the opine.

• Korean Journal of Plant Tissue Culture
• /
• v.27 no.3
• /
• pp.169-173
• /
• 2000

A 1,183bp cDNA, AmA1, encoding the seed storage protein of Amaranthus hypochondriacus was isolated by reverse transcriptase-polymerase chain reaction (RT-PCR) and characterized. AmA1 gene was subcloned into plant binary vector under Cauliflower Mosaic Virus (CaMV) 35S promoter and nopaline synthase terminator (3'NOS). The recombinant binary vector was used to transform Nicotiana tabacum using Agrobacterium tumefacien -mediated transformation procedure. Shoots were induced on MS medium with 0.1 mg/L NAA, 1.0 mg/L BA, 100 mg/L kanamycin and 250 mg/L cefotaxime. Transgenic plants were selected on rooting medium based on MS medium containing 200 mg/L kanamycin and 250 mg/L cefotaxime without phytoregulators. The presence of AmA1 gene in the transgenic plants was confirmed by PCR followed by DNA hybridization. The expression of AmA1 gene in the transgenic plant was observed by RT-PCR method.

• Applied Biological Chemistry
• /
• v.41 no.1
• /
• pp.42-46
• /
• 1998

Polymerase chain reaction (PCR) and Southern hybridization analyses were carried out to identify clones of silk worm thorn (Cudraria tricuspidata) and Rose of sharon (Hibiscus syriacus) which look like one tree with two ar three, branches or two or three different trees. For PCR five different PCR primers $(17{\sim}24\;nucleotides)$ are derived from CaMV 35S promoter, nopaline synthase terminator and coding region of thylakoid membrane protein gene. In the case of silk worm thorn, about 500 bp of PCR product was produced from DNAs of one tree or branch in the presence of 35S primer alone. Southern hybridization analysis of genomic DNAs hybridized with $^{32}P$ labeled PCR product showed that the same size of DNA fragments were hybridized with different intensities. In addition, PCR analyses using 20 different primers of OPERON 10-mer kits showed that only OPA01 primer produced PCR products of different size. These results indicate that two different trees of silk worm thorn combined to one tree. In the case of the Rose of Sharon, the same size of PCR products were produced from three different samples but Southern hybridization with the above PCR product as a probe did not show any hybridized bands. PCR analyses in the presence of OPERON 10-mers showed OPA04 and OPA13 produced different products including same sizes of products. These, results indicate that three different trees of the Rose of Sharon seem to be derived from the tree.

• Journal of Korean Society of Forest Science
• /
• v.84 no.1
• /
• pp.77-86
• /
• 1995

To effectively modify tree function with genetic engineering, transgenes must be expressed at the proper level in the appropriate tissues at suitable developmental stages. Toward understanding the spatial and temporal expression of transgenes in woody plants, transgene expression was evaluated in three greenhouse-grown, transgenic lines of Populus alba ${\times}$ P. grandidentata hybrid clone 'Hansen'. All transgenic poplar lines possess constructs containing the bacterial nopaline synthase(nos) promoter linked to a neomycin phosphotransferase II(NPT II) selectable marker gene. In addition, each transgenic poplar line contains one of the following gene constructs : 1) a wound-inducible potato proteinase inhibitor II (pin2) promoter linked to a chloramphenicol acetyltransferase(CAT) reporter gene. 2) a nos promoter linked to a PIN2 structural gene : or 3) a Cauliflower Mosaic Virus 35s promoter linked to a PIN2 structural gene. Polymerase chain reaction(PCR) was used to verify the presence of foreign genes in the poplar genome. Enzyme-linked immunosorbent assays(ELISAs) were used to evaluate organ specific expression of the nos-NPT II construct. NPT II expression was detected in leaves, petioles, stems, and roots of transgenic poplar, thereby indicating that the nos promoter is potentially effective for general constitutive expression of transgenes. NPT expression varied among transgenic poplar lines and among organs for one transgenic line, Tr15. With Tr15, NPT II levels were highest in older leaves and petioles. These results indicate that screening of several transgenic lines may be required to identify lines with optimal transgene expression.

• Korean Journal of Plant Tissue Culture
• /
• v.25 no.2
• /
• pp.109-113
• /
• 1998

In this study we tried to transform an antimicrobial peptide gene (Shiva) under the promoter of tomato phenylalanine ammonia-lyase (tPAL5) into tobacco and potato plants. Antimicrobial peptide gene was isolated originally from giant silk moth (Hyalophora cecropia) and modified ie nucleotide sequence to increase antimicrobial activity. Transgenic tobacco plants were regenerated and their seeds were tested on the media containing kanamycin (500 mg/L). The results of PCR amplification and genomic Southern blot hybridization confirmed the integration of construct (tPAL5 promoter-Shiva-NOS-GUS-NOS) into chromosome. We observed that one of the transgenic tobacco plants showed chromosome rearrangement when integrated. In case of potato transformation, the efficiency of regeneration was maximized at the medium containing Zeatin 2mg/L, NAA 0.01mg/L, GA$_3$ 0.1mg/L. We also observed the high expression of GUS (${\beta}$-glucuronidase) enzyme which was located next to the terminator sequence of nopaline synthase gene (NOS) in the vascular tissue of stem, leaves of transgenic potatoes. This result suggested that a short sequence of Shiva gene (120 bp) and NOS terminator sequence might be served as a leader sequence of transcript when translated.

• Proceedings of the Korean Society of Plant Pathology Conference
• /
• 1994.06a
• /
• pp.11-26
• /
• 1994

Crown gall of stonefruit and nut trees is one of the very few plant diseases subject to efficient biological control. The disease is caused by the soil-inhabiting bacteria Agrobacterium tumefaciens and Agrobacterium rhizogenes and the original control organism was a non-pathogenic isolate of A. rhizogenes strain K84. Control is achieved by dipping planting material in a cell suspension of strain K84 which specifically inhibits pathogenic strains containing a nopaline Ti plasmid. Because the agrocin 84-encoding plasmid (pAgK84) is conjugative, it can be transmitted from the control strain to pathogenic strains which, as a result, become immune to agrocin 84 and cannot be controlled. To prevent this happening, the transfer genes on pAgK84 were located and then largely eliminated by recombinant DNA technology. The resulting construct, strain K1026, is transfer deficient but controls crown gall just as effectively as does strain K84. Field data from Spain confirm that pAgK84 can transfer to pathogenic recipients from strain K84 but not from strain K1026. The latter has been registered in Australia as a pesticide and is the first genetically engineered organism in the world to be released fro commercial use. It is recommended as a replacement for strain K84 to prevent a breakdown in the effectiveness of biological control of crown gall. Several reports indicate that both strains K84 and K1026 sometimes control crown gall pathogens that are resistant to agrocin 84. A possible reason for this is that both strains produce a second antibiotic called 434 which inhibits growth of nearly all isolates of A. rhizogenes, both pathogens and non-pathogens. Crown gall of grapevine is caused by another species, Agrobacterium vitis. It is resistant to agrocin 84 and cannot be controlled by strains K84 or K1026. It is different from other crown gall pathogens in several characteristics, including the fact that, although a rhizosphere coloniser, its also lives systemically in the vascular tissue of grapevine. Pathogen free propagating material can be obtained from tissue culture or, less surely, by heat therapy of dormant cuttings. A number of laboratories are searching for a biocontrol strain that will prevent, or at least delay, reinfection. A non-pathogenic A. vitis strain F/25 from South Africa looks very promising in this regard.