• Microbiology and Biotechnology Letters
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• v.32 no.2
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• pp.110-116
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• 2004

The over-expression in E. coli of the pHLN1-SO(＋) and pHLN2-80(－) plasmids cloned an insecticidal crystal protein (ICP) gene (crylAal type) from Bacillus thuringiensis var. kurstaki HD 1 was investigated through in part, the deletion of -80 bp promoter and an alternative change of cloning vector system. Two recombinant plasmids were constructed in an attempt to analyze the over-expression of the ICP in relations to its gene structure possessing only -14 bp ［Shine-Dalgarno (SD) sequence of -80 bp promoter］. Also, anther two recombinant plasmids similarly cloned the icp gene in a different vector system. The amounts of ICP produced from the recombinants were measured by SDS-PAGE and confirmed by Western blot analysis. One clone, pHLRBS1-14 clone in which only the SD sequence in the inverted orientation icp gene appeared, was more evident than the pHLRBS2-14 clone in which only the -14 bp SD sequence of the right orientated icp gene was shown to exist. The pHLN2-80(－) clone produced more ICP proteins than the pHLRBS1-14 clone. In the two clones, pHLNUC1-80 right-oriented icp gene and the pHLNUC2-80 clone inverted-orientation icp gene in a new different vector, the pHLNUC2-80 produced more ICP proteins in E. coli system. These results indicate that the P/ac promoter, the inverted icp gene insertion and -80 bp promoter (-66 bp part of the icp gene promoters), were concerned with the expression of the icp gene in the recombinant plasmids. In addition, the expression mechanism might result from the disruption of the transcription-suppressing regions in the promoter regions.

• Journal of Plant Biotechnology
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• v.38 no.1
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• pp.15-21
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• 2011

We developed 14 transgenic lines of Chinese cabbage (Brassica rapa) harboring the T-DNA border sequences and CryIAc1 transgene of the binary vector 416 using Agrobacterium tumefaciens-mediated DNA transfer. Six lines had single copy cryIAc1 gene and four of them contained no vector backbone DNA. Of the left border (LB) flanking sequences six nucleotides were deleted in transgenic lines 416-2 and 416-3, eleven nucleotides in line 416-9, and 65 nucleotides including the whole LB sequences in line 416-17, respectively. And we defined 499 bp of genomic DNA (gDNA) of transformed Chinese cabbage, and blast results showed 96% homology with Brassica oleracea sequences. PCR with specific primer for the right border (RB) franking sequence revealed 834 bp of PCR product sequence, and it was consisted of 3' end of cryIAc1, nosterminal region and 52 bp of Chinese cabbage genomic DNA near RB. RB sequences were not found and the 58 nucleotides including 21 bp of nos-terminator 3' end were deleted. Also, there were deletion of 10 bp of the known genomic sequences and insertion of 65 bp undefined genomic sequences of Chinese cabbage in the integration site. These results demonstrate that the integration of T-DNA can be accompanied by unusual deletions and insertions both in transgenic and genomic sequences.

• Journal of Microbiology and Biotechnology
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• v.26 no.1
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• pp.197-206
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• 2016

Listeria monocytogenes is a foodborne pathogen of considerable genetic diversity with varying pathogenicity. Initially, we found that the strain M7 was far less pathogenic than the strain Lm850658 though both are serovar 4a strains belonging to the lineage III. Comparative genomic approaches were then attempted to decipher the genetic basis that might govern the strain-dependent pathotypes. There are 2,761 coding sequences of 100% nucleotide identity between the two strains, accounting for 95.7% of the total genes in Lm850658 and 92.7% in M7. Lm850658 contains 33 specific genes, including a novel 20K prophage whereas strain M7 has 130 specific genes, including two large prophages (38K and 44K). To examine the roles of these specific prophages in pathogenicity, the 20K and 38K prophages were deleted from their respective strains. There were virtually no differences of pathogenicity between the deletion mutants and their parent strains, although some putative virulent factors like VirB4 are present in the 20K region or holin-lysin in the 38K region. In silico PCR analysis of 29 listeria genomes show that only strain SLCC2540 has the same 18 bp integration hotspot as Lm850658, whereas the sequence identity of their 20K prophages is very low (21.3%). The 38K and 44K prophages are located in two other different hotspots and are conserved in low virulent strains M7, HCC23, and L99. In conclusion, the 20K and 38K prophages of L. monocytogenes serovar 4a strains Lm850658 and M7 are not related to virulence but contribute to genetic diversity.

• Asian-Australasian Journal of Animal Sciences
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• v.22 no.6
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• pp.765-773
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• 2009

Sterol regulatory element binding factor 1 (SREBF1) and fatty acid synthase (FASN) genes play an important role in the biosynthesis of fatty acids and cholesterol, and in lipid metabolism. This study used polymorphisms in the intron 5 of bovine SREBF1 and in the thioesterase (TE) domain of FASN genes to evaluate their associations with beef fatty acid composition. A previously identified 84-bp indel (L: insertion/long type and S: deletion/short type) of the SREBF1 gene in Korean cattle had significant associations with the concentration of stearic (C18:0), linoleic (C18:2) and polyunsaturated fatty acids (PUFA). The stearic acid concentration was 6.30% lower in the SS than the LL genotype (p<0.05), but the linoleic and PUFA contents were 11.06% and 12.20% higher in SS compared to LL (p<0.05). Based on the sequence analysis, five single nucleotide polymorphisms (SNPs) g.17924G>A, g.18043C>T, g.18440G>A, g.18529G>A and g.18663C>T in the TE domain of the FASN gene were identified among the different cattle breeds studied. Among these, only g.17924 G>A and g.18663C>T SNPs were segregating in the Hanwoo population. The g.17924G>A SNP is a non-synonymous mutation (thr2264ala) and was significantly associated with the contents of palmitic (C16:0) and oleic acid (C18:1). The oleic acid concentration was 3.18% and 2.79% higher in Hanwoo with the GG genotype than the AA and AG genotypes, respectively (p<0.05), whereas the GG genotype had 3.8% and 4.01% lower palmitic acid than in those cattle with genotype AA and AG, respectively (p<0.05). Tissue expression data showed that SREBFI and FASN genes were expressed in a variety of tissues though they were expressed preferentially in different muscle tissues. In conclusion, the 84-bp indel of SREBF1 and g.17924G>A SNP of the FASN gene can be used as DNA markers to select Hanwoo breeding stock for fatty acid composition.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 2002.10a
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• pp.18-25
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• 2002

The pikromycin biosynthetic system in Streptomyces venezuleae is unique for its ability to produce two groups of antibiotics that include the 12-membered ring macrolides methymycin and neomethymycin, and the 14-membered ring macrolides narbomycin and pikromycin. The metabolic pathway also contains two post polyketide-modification enzymes, a glycosyltransferase and P450 hydroxylase that have unusually broad substrate specificities. In order to explore further the substrate flexibility of these enzymes a series of hybrid polyketide synthases were constructed and their metabolic products characterized. The plasmid-based replacement of the multifunctional protein subunits of the pikromycin PKS in S. venezuelae by the corresponding subunits from heterologous modular PKSs resulted in recombinant strains that produce both 12- and 14-membered ring macrolactones with predicted structural alterations. In all cases, novel macrolactones were produced and further modified by the DesVII glycosyltransferase and PikC hydroxylase leading to biologically active macrolide structures. These results demonstrate that hybrid PKSs in S. venezuelae can produce a multiplicity of new macrolactones that are modified further by the highly flexible DesVII glycosyltransferase and PikC hydroxylase tailoring enzymes. This work demonstrates the unique capacity of the S. venezuelae pikromycin pathway to expand the toolbox of combinatorial biosynthesis and to accelerate the creation of novel biologically active natural products. The polyketide backbone of rifamycin B is assembled through successive condensation and ${\beta}$-carbonyl processing of the extender units by the modular rifamycin PKS. The eighth module, in the RifD protein, contains nonfunctional DH domain and functional KR domain, which specify the reduction of the ${\beta}$-carbonyl group resulting in the C-21 bydroxyl of rifamycin B. A four amino acid substitution and one amino acid deletion were introduced in the putative NADPH binding motif in the proposed KR domain encoded by rifD. This strategy of mutation was based on the amino acid sequences of the corresponding motif of the KR domain of module 3 in the RifA protein, which is believed dysfunctional, so as to introduce a minimum alteration and retain the reading frame intact, yet ensure loss of function. The resulting strain produces linear polyketides, from tetraketide to octaketide, which are also produced by a rifD disrupted mutant as a consequence of premature termination of polyketide assembly. Much of the structural diversity within the polyketide superfamily of natural products is due to the ability of PKSs to vary the reduction level of every other alternate carbon atom in the backbone. Thus, the ability to introduce heterologous reductive segments such as ketoreductase (KR), dehydratase (DH), and enoylreductase (ER) into modules that naturally lack these activities would increase the power of the combinatorial biosynthetic toolbox. The dehydratase domain of module 7 of the rifamycin PKS, which is predicted to be nonfunctional in view of the sequence of the apparent active site, was replaced with its functional homolog from module 7 of rapamycin-producing polyketide synthase. The resulting mutant strain behaved like a rifC disrupted mutant, i.e., it accumulated the heptaketide intermediate and its precursors. This result points out a major difficulty we have encountered with all the Amycolatopsis mediterranei strain containing hybrid polyketide synthases: all the engineered strains prepared so far accumulate a plethora of products derived from the polyketide chain assembly intermediates as major products instead of just analogs of rifamycin B or its ansamycin precursors.

• Journal of Microbiology and Biotechnology
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• v.25 no.12
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• pp.2125-2134
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• 2015

IrrE is a highly conserved global regulator in the Deinococcus genus and contributes to survival from high doses of UV radiation, ionizing radiation, and desiccation. Drad-IrrE and Dgob-IrrE from Deinococcus radiodurans and Deinococcus gobiensis I-0 each share 66% sequence identity. However, Dgob-IrrE showed a stronger protection phenotype against UV radiation than Drad-IrrE in the D. radiodurans irrE-deletion mutant (ΔirrE), which may be due to amino acid residues differences around the DNA-binding HTH domain. Site-directed mutagenesis was used to generate a Drad-IrrE A184S single mutant, which has been characterized and compared with the ΔirrE mutant complemented strain with Drad-irrE, designated ΔirrE-E. The effects of the A184S mutation following UV radiation and mitomycin C (MMC) shock were determined. The A184S mutant displayed significantly increased resistance to UV radiation and MMC shock. The corresponding A184 site in Dgob-IrrE was inversely mutated, generating the S131A mutant, which exhibited a loss of resistance against UV radiation, MMC shock, and desiccation. qPCR analysis revealed that critical genes in the DNA repair system, such as recA, pprA, uvrA, and ddrB, were remarkably induced after UV radiation and MMC shock in the ΔirrE-IE and A184S mutants. These data suggested that A184S improves the ability against UV radiation and MMC shock, providing new insights into the modification of IrrE. We speculated that the serine residue may determine the efficiency of DNA binding, leading to the increased expression of IrrE-dependent genes important for protection against DNA damage.

• Korean Journal of Poultry Science
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• v.41 no.4
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• pp.305-311
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• 2014

Copy number variation (CNV) is a form of structural variation that shows various numbers of copies in segments of the DNA. It has been shown to account for phenotypic variations in human diseases and agricultural production traits. Currently, most of chicken breeds in the poultry industry are based on European-origin breeds that have been mostly provided from several international breeding companies. Therefore, National Institute of Animal Science, RDA has been trying to restore and improve Korean native chicken breeds (12 lines of 5 breeds) for about 20 years. Thanks to the recent advance of sequencing technologies, genome-wide CNV can be accessed in the higher resolution throughout the genome of species of interest. However, there is no systematic study available to dissect the CNV in the native chicken breed in Korea. Here, we report genome-wide copy number variations identified from a genome of Korean native chicken (Line L) by comparing between the chicken reference sequence assembly (Gallus gallus) and a de novo sequencing assembly of the Korean native chicken (Line L). Throughout all twenty eight chicken autosomes, we identified a total of 501 CNVs; defined as gain and loss of duplication and deletion respectively. Furthermore, we performed gene ontology (GO) analysis for the putative CNVs using DAVID, leading to 68 GO terms clustered independently. Of the clustered GO terms, genes related to transcription and gene regulation were mainly detected. This study provides useful genomic resource to investigate potential biological implications of CNVs with traits of interest in the Korean native chicken.

• The Korean Journal of Mycology
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• v.36 no.2
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• pp.189-195
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• 2008

When a homothallic ascomycete Aspergillus nidulans is exposed to visible light, cleistothecial development is inhibited. The light response of development in A. nidulans implies the existence of delicate regulation process including reception and translocation of light signaling and determination of development. Previously, mutants that could develop cleistothecia even in the presence of relatively intensive visible light were isolated and several complementation groups were identified. A gene that was able to complement the silA98 mutation, which was responsible for preferred cleistothecia development under visible light, was isolated from AMA-NotI genomic library. The silA gene retained in the 4.3 kb recovered genomic library DNA has an open reading frame (ORF) consisted of 2,388 bp nucleotides, interrupted by 3 introns and consequently encoding 795 amino acids. The putative SilA carries a ${Zn_2}{Cys_6}$ binuclear cluster motif at N terminus and shows high amino acid sequence similarity to Aro80p of Saccharomyces cerevisiae. Deletion mutants of silA showed a strong induction of sexual development under visible light, indicating that SilA is involved in the negative regulation of sexual development in response to the light.

• Korean Journal of Medicinal Crop Science
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• v.22 no.6
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• pp.429-434
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• 2014

This study describes the efficient method for the discrimination of 'Cheonryang' in Panax ginseng Meyer using a STS primer. A total of 208 STS primers were applied to polymerase chain reaction (PCR) amplification for discriminating Korean ginseng cultivars. Co-dominant polymorphic band patterns were generated with two primers, MFGp 0019, MFGp 0248, and successful identification of 'Cheonryang' was achieved from out of 11 Korean ginseng cultivars. Two different sizes of DNA band patterns were detected with MFGp 0019 primer. Ten Korean ginseng cultivars shared the same size of amplified DNAs (389 bp), but 'Cheonryang' showed a different size. Thus 'Cheonryang' can be efficiently distinguished from the other ten ginseng cultivars by using the MFGp 0019 primer. In the case of MFGp 0248, two different sizes of DNA band patterns were detected in the eleven ginseng cultivars. Same sized amplified DNA bands (307 bp) were shown in five cultivars (Chunpoong, Gopoong, Kumpoong, Cheongsun, Sunhyang) and 254 bp sized DNA bands were identified in the other 6 cultivars (Yunpoong, Sunpoong, Sunun, Sunone, Cheonryang, K-1). In conclusion, the two STS primers, MFGp 0019, and MFGp 0248, provide a rapid and reliable method for the specific identification of 'Cheonryang' cultivar from a large number of samples.

• Pediatric Infection and Vaccine
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• v.16 no.2
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• pp.199-204
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• 2009

Purpose : Various proteins encoded in the early region 3 (E3) of adenoviruses protect cells from being killed by cytotoxic T cells and death-inducing cytokines. We sought to find out whether the genetic heterogeneity of the E3 gene might contribute to the molecular diversity of adenoviruses. Methods : Sequences in the E3 region were analyzed for 14 adenovirus type 3 (Ad3) strains that were isolated from children with lower respiratory tract infections in the Seoul National University Children's Hospital during the period 1991-2000. Full-length adenoviral DNA was purified from the infected A549 cell lysates using a modified Hirt procedure. Results : There was 98% homology between 14 Korean Ad3 strains with a reference strain (M15952). Homology within the Korean Ad3 strains was 98.7%. Variation was found in the region of transcripts 20.1 kDa, 20.6 kDa, truncated 7.7 kDa, 10.3 kDa, 14.9 kDa, and 15.3 kDa. In particular, all 14 Korean strains showed a missense single point mutation at the start codon of the truncated 7.7 kDa. In addition, a deletion was found in the truncated 7.7 kDa region by 58 base pairs in 10 strains and 94 base pairs in 4 strains. Variations in amino acids were observed in the receptor internalization and degradation complex (10.3 kDa/14.9 kDa) which stimulates the clearance from the cell surface and subsequent degradation of the receptors for the Fas ligand and TRAIL, while no variations were observed in another immunoregulatory transcript, 19 kDa. Conclusion : Sequence analysis of the immunoregulatory region of adenovirus E3 shows that genetic heterogeneities are related to genome type patterns.