• Microbiology and Biotechnology Letters
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• v.30 no.4
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• pp.312-319
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• 2002

The Sporosarcina psychrophilia pyrB gene, which encodes aspartate transcarbamylase (ATcase), was cloned on Sau3AI restriction endonuclease fragment inserted into pUC19 plasmid vector, S. psychrophilia pyrB gene was expressed in Escherichia coli pyrB mutant for the complementation test. The sequence of 2,606 nucleotides including putative pyrB gene was determined. The region contained one full open reading frame (ORf) and two partial ORFs. The deduced amino acid sequence of the second ORF showed 59% identity with that of Bacillus caldolyticus ATCase. The first and third partial ORFs were closely related to the uracil permease (pyrP) and dihydroorotase (pyrC), respectively. Besides, potential terminator, antiterminator, and anti-antiterminator structures were found in the intergenic region between pyrP and pyrB. These results suggested that S. psychrophilia pyrimidine nucleotide biosynthesis genes are clustered as well as other Bacillus sp. Over-expressed product of pyrB encoding ATCase was purified and analyzed by the SDS-PAGE. The purified PyrB protein turned out to be molecular mass of 27 kDa and showed ATCase activity.

• The Plant Pathology Journal
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• v.19 no.4
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• pp.210-216
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• 2003

This study examined the existence of genetically diverse population of Cucumber mosaic virus (CMV), known as quasispecies, from lily, Nicotiana benthamiana and from purified virions. Based on the conserved sequences of CMV lily isolates in intergenic region (IR) on RNA3, the genetic variation of IR from three different sources was investigated by a specific restriction endonuclease hydrolysis of amplified reverse transcription-polymerase chain reaction (RT-PCR) products using virus-specific primers, and was compared with IR sequences. The IR nucleotide sequences of CMV lily isolates were highly conserved, however, quasispecies was detected from all three sources in low level, containing sub-populations of RNA3. These subpopulations of RNA3 were inoculated onto zucchini squash by in vitro transcripts from corresponding full-length cDNA clones together with Eny RNA1 and 2 transcripts. The systemic symptom of zucchini plants infected by these quasispecies was chlorotic spotting, which was milder than severe mosaic and stunt symptom caused by Eny-CMV. The severity of symptom was correlated with RNA accumulation of viruses. These results suggest that the genome of CMV lily isolates consists of quasispecies populations.

• Mycobiology
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• v.44 no.4
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• pp.314-318
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• 2016

Breeding the button mushroom requires genetic information about its strains. This study was undertaken to genetically characterize four domestically bred button mushroom strains (Saea, Saejung, Saedo, Saeyeon cultivars) and to assess the possibility of using the intergenic spacer 1 (IGS1) region of rDNA as a genetically variable region in the genetic characterization. For the experiment, 34 strains of Agaricus bisporus, two strains of A. bitorquis, and one strain of A. silvaticus, from 17 countries were used. Nucleotide sequence analysis of IGS1 rDNA in these 37 Agaricus strains confirmed that genetic variations exist, not only among the four domestic strains, but also between the four domestic strains and foreign strains. Crossing two different haploid strains of A. bisporus seems to generate genetic variation in the IGS1 region in their off-spring haploid strains. Phylogenetic analysis based on the IGS1 sequence revealed all A. bisporus strains could be differentiated from A. silvaticus and A. bitorquis strains. Five genetic groups were resolved among A. bisporus strains. Saejung and Saeyeon cultivars formed a separate genetic group. Our results suggest that IGS1 could be complementarily applied in the polymorphism analysis of button mushroom.

• Korean Journal of Environmental Biology
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• v.21 no.1
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• pp.77-85
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• 2003

We have examined the 16S-23S rRNA intergenic spacer regions (ISR) of Vibrio fluvialis. ISRs were PCR amplified, cloned into a plasmid vector and then sequenced. As results of ISR nucleotide sequence analysis, total of 6 clones were isolated depending on the size. The clones were different in both the number and the composition of the tRNA genes, and were designated ISR-A, ISR-E, ISR-El, ISR-lA, ISR-EKV, ISR-EKAV. ISR-A contains $tRNA^{Ala}$; ISR-lA, $tRNA^{Ile}$-$tRNA^{Ala}$; ISR-EKV, $tRNA^{GIu}$-$tRNA^{Lys}$-$tRNA^{Val}$;ISE-EKAV, $tRNA^{GIu}$-$tRNA^{Lys}$-$tRNA^{Ala}$-$tRNA^{Val}$; ISR -E and E1, $tRNA^{GIu}$ clusters. ISR-EKV was shown to be a minor type out of the six ISR types and showed a very limited homology between ISR-EKV from V, fluvialis and ISRa from other Vibrio species. Therefore ISR-EKV sequence was used to design species-specific primers to detect V, fiuvialis from other Vibrio species by PCR reaction. The specificity of the primers was examined using genomic DNA of other Vibrios as templates for PCR reaction. The result showed that PCR can be a useful method to detect V. fluvialis among Vibrio species in a single PCR reaction.

• Parasites, Hosts and Diseases
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• v.44 no.4 s.140
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• pp.343-353
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• 2006

Giardia intestinalis infections arise primarily from contaminated food or water Zoonotic transmission is possible, and at least 7 major assemblages including 2 assemblages recovered from humans have been identified. The determination of the genotype of G. intestinalis is useful not only for assessing the correlation of clinical symptoms and genotypes, but also for finding the infection route and its causative agent in epidemiological studies. In this study, methods to identify the genotypes more specifically than the known 2 genotypes recovered from humans have been developed using the intergenic spacer (IGS) region of rDNA. The IGS region contains varying sequences and is thus suitable for comparing isolates once they are classified as the same strain. Genomic DNA was extracted from cysts isolated from the feces of 5 Chinese, 2 Laotians and 2 Koreans infected with G. intestinalis and the trophozoites of WB, K1, and GS strains cultured in the laboratory, respectively. The rDNA containing the IGS region was amplified by PCR and cloned. The nucleotide sequence of the 3' end of IGS region was determined and examined by multiple alignment and phylogenetic analysis. Based on the nucleotide sequence of the IGS region, 13 G. intestinalis isolates were classified to assemblages A and B, and assemblage A was subdivided into A1 and A2. Then, the primers specific to each assemblage were designed, and PCR was peformed using those primers. It detected as little as 10 pg of DNA, and the PCR amplified products with the specific length to each assemblage (A1, 176bp; A2, 261 bp; B, 319 bp) were found. The PCR specific to 3 assemblages of G. intestinalis did not react with other bacteria or protozoans, and it did not react with G. intestinalis isolates obtained from dogs and rats. It was thus confirmed that by applying this PCR method amplifying the IGS region, the detection of G. intestinalis and its genotyping can be determined simultaneously.

• Journal of Microbiology
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• v.42 no.4
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• pp.365-368
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• 2004

The occurrence of strA-strB streptomycin-resistance genes within transposon Tn5393 was examined in Pseudomonas syringae pv. actinidiae, P. syringae pv. syringae, and P. marginalis, isolated from kiwifruit plants in Korea and Japan. PCR amplification with primers specific to strA-strB revealed that three of the tested Pseudomonas species harbored these genes for a streptomycin-resistance determinant. Tn5393, containing strA-strB, was also identified with PCR primers designed to amplify parts of tnpA, res, and tnpR. No IS elements were detected within tnpR, nor were they found in the intergenic region between tnpR and strA. Nucleotide sequence analysis indicated that the strA sequence of P. syringae pv. actinidiae contained a single nucleotide alteration at position 593 (CAA $\rightarrow$CGA), as compared to Tn5393a in P. syringae pv. syringae. This resulted in an amino acid change, from Gin to Arg.

• Journal of Plant Biotechnology
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• v.44 no.3
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• pp.235-242
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• 2017

Cudrania tricuspidata Bureau is a widely-used, medicinal, perennial and woody plant. Obtaining information about the genetic diversity of plant populations is highly important with regard toconservation and germplasm utilization. Although C. tricuspidata is an important medicinal plant species registered in South Korea, no molecular markers are currently available to distinguish Korean-specific ecotypes from other ecotypes from different countries. In this study, we developed single nucleotide polymorphism (SNP) markers derived from the chloroplast and nuclear genomic sequences, which serve to to identify distinct Korean-specific ecotypes of C. tricuspidata via amplification refractory mutation system (ARMS)-PCR and high resolution melting (HRM) curve analyses. We performed molecular authentication of twelve C. tricuspidata ecotypes from different regions using DNA sequences in the maturaseK (MatK) chloroplast intergenic region and nuclear ribosomal DNA internal transcribed spacer (ITS) regions. The SNP markers developed in this study are useful for rapidly identifying specific C. tricuspidata ecotypes from different regions.

• Molecules and Cells
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• v.37 no.1
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• pp.36-42
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• 2014

The tomato (Solanum lycopersicum L.) is a model plant for genome research in Solanaceae, as well as for studying crop breeding. Genome-wide single nucleotide polymorphisms (SNPs) are a valuable resource in genetic research and breeding. However, to do discovery of genome-wide SNPs, most methods require expensive high-depth sequencing. Here, we describe a method for SNP calling using a modified version of SAMtools that improved its sensitivity. We analyzed 90 Gb of raw sequence data from next-generation sequencing of two resequencing and seven transcriptome data sets from several tomato accessions. Our study identified 4,812,432 non-redundant SNPs. Moreover, the workflow of SNP calling was improved by aligning the reference genome with its own raw data. Using this approach, 131,785 SNPs were discovered from transcriptome data of seven accessions. In addition, 4,680,647 SNPs were identified from the genome of S. pimpinellifolium, which are 60 times more than 71,637 of the PI212816 transcriptome. SNP distribution was compared between the whole genome and transcriptome of S. pimpinellifolium. Moreover, we surveyed the location of SNPs within genic and intergenic regions. Our results indicated that the sufficient genome-wide SNP markers and very sensitive SNP calling method allow for application of marker assisted breeding and genome-wide association studies.

• Journal of Life Science
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• v.23 no.10
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• pp.1260-1266
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• 2013

The ribosomal DNA (rDNA) clusters of Hypsizygus marmoreus 3-10 and H. marmoreus 1-1 were analyzed in this study. The small subunit (SSU) and intergenic spacer 2 (IGS 2) was partially sequenced. The internal transcribed spacer 1 (ITS 1), 5.8S, internal transcribed spacer 2 (ITS 2), large subunit (LSU), intergenic spacer 1 (IGS 1), and 5S were completely sequenced. The rDNA clusters of H. marmoreus 3-10 and H. marmoreus 1-1 were 7,049 bp in length. The sequence of SSU rDNA, which corresponded to 18S rDNA, was 1,796 bp in length, and the sequence of LSU rDNA, which corresponded to 28S rDNA, was 3,348 bp in length. The ITS region that variable region and IGS region that non-transcribed spacer was 462 bp and 1,290 bp in length. The sequence of 5.8S rDNA and 5S rDNA was 153 bp and 43 bp in length, respectively. The 17 bp of the rDNA cluster in the H. marmoreus 3-10 strain was different to that in the H. marmoreus 1-1 strain, with 2 bp in the SSU, 3 bp in the ITS, 9 bp in the LSU, and 3 bp in the IGS. The analysis of the secondary structure revealed that the ITS regions of H. marmoreus 3-10 and H. marmoreus 1-1 have five stem-loop structures. Interestingly, among these structures, one different nucleotide sequence resulted in a different secondary structure in stem-loop V.

• Food Science of Animal Resources
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• v.33 no.6
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• pp.715-722
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• 2013

Heugu (Korea Black Cattle) is one of the indigenous cattle breeds in Korea; however there has been severe lack of genomic studies on the breed. In this study, we report the first whole genome resequencing of Heugu at higher sequence coverage using Illumina HiSeq 2000 platform. More than 153.6 Giga base pairs sequence was obtained, of which 97% of the reads were mapped to the bovine reference sequence assembly (UMD 3.1). The number of non-redundantly mapped sequence reads corresponds to approximately 28.9-fold coverage across the genome. From these data, we identified a total of over six million single nucleotide polymorphisms (SNPs), of which 29.4% were found to be novel using the single nucleotide polymorphism database build 137. Extensive annotation was performed on all the detected SNPs, showing that most of SNPs were located in intergenic regions (70.7%), which is well corresponded with previous studies. Of the total SNPs, we identified substantial numbers of non-synonymous SNPs (13,979) in 5,999 genes, which could potentially affect meat quality traits in cattle. These results provide genome-wide SNPs that can serve as useful genetic tools and as candidates in searches for phenotype-altering DNA difference implicated with meat quality traits in cattle. The importance of this study can be further pronounced with the first whole genome sequencing of the valuable local genetic resource to be used in further genomic comparison studies with diverse cattle breeds.