• Journal of Microbiology and Biotechnology
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• v.14 no.4
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• pp.789-795
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• 2004

A promoter-probe shuttle vector pSK1Cat was constructed for the isolation of transcriptional signal sequences from Corynebacterium glutamicum. Besides conferring resistance to kanamycin in Escherichia coli and C. glutamicum, the vector carried a promoterless cat gene to confer resistance to chloramphenicol upon insertion of the appropriate transcriptional signals in the multiple cloning site. By utilizing the vector, a series of transcriptionally active fragments were isolated from the genome of C. glutamicum. The clones, ranging from 200 bp to 1 kb in size, were grouped into 3 classes of strong, medium, and weak, based on the chloramphenicol acetyltransferase (CAT) activity and sensitivity to the chloramphenicol of the clone-carrying C. glutamicum cells. C. glutamicum cells carrying the $P_{19}$ clone, a representative in the strong class, were able to grow on minimal agar plates containing over $40 mg/mell$ chloramphenicol, and showed CAT activity of 10 m㏖/mgㆍmin, performing slightly better than the cells carrying $P_{tac}$ , a strong E. coli promoter. Subcloning analysis of the $P_{19}$ clone identified a 180 bp intergenic fragment ($P_{180}$), which was located upstream of a gene encoding a hypothetical membrane protein. The expression conferred by $P_{180}$ was not affected by either the kinds of carbon sources or changes in temperature. These properties make the $P_{180}$ clone useful for the deregulated expression of biosynthetic genes in C. glutamicum during amino acid fermentation.

• The Plant Pathology Journal
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• v.19 no.2
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• pp.123-127
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• 2003

A severe disease of muskmelon (Cucumis melo cv. Alsnight) grown on rockwool in a plastic house was characterized by leaf and stem necrosis followed by death of the plants. In 2001, an isolate of Melon necrotic spot virus-MN (MNSV-MN) of the genus Camovirus was identified as the causal agent of the disease on the basis of biological reactions and nucleotide sequence analyses of coat protein (CP) gene. MNSV-MN induced necrotic local lesions on mechanically inoculated leaves and systemic necrotic spots on the upper leaves of melon cvs. Alsnight, Rui III, Party, Imperial, and Seolhang. However, the inoculated leaves of watermelon and cucumber showed only necrotic lesions. DsRNAs extracted from the melon infected with MNSV-MN were separated into three components. Molecular sizes of the dsRNAs were estimated at approximately 4.5, 1.8, and 1.6 kbp. The amplified cDNA products of CP gene for MNSV-MN by RT-PCR showed approximately 1.2 kbp. The amplified DNA was digested to three fragments by MspI treatment. The cDNA of the genomic RNA of MNSV-MN was cloned and the region deduced to encode the CP was sequenced. The CP coding region, located near 3' end of the genome, consisted of 1,170 nucleotides and had the potential to encode a 390 amino acid protein. The nucleotide and amino acid sequences of MNSV-MN CP gene were 84.0-94.6% and 90.8-94.9% identical with other MNSV isolates found in the GeneBank database, respectively. This is the first report on the occurrence of MNSV in Korea.

• The Plant Pathology Journal
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• v.36 no.1
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• pp.87-97
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• 2020

The development of reverse transcription-polymerase chain reaction using degenerate primers against conserved regions of most potyviral genomes enabled sampling of the potyvirome. However, these assays usually involve sampling potential host plants, but identifying infected plants when they are asymptomatic is challenging, and many plants, especially wild ones, contain inhibitors to DNA amplification. We used an alternative approach which utilized aphid vectors and indicator plants to identify potyviruses capable of infecting common bean (Phaseolus vulgaris). Aphids were collected from a range of asymptomatic leguminous weeds and trees in Iran, and transferred to bean seedlings under controlled conditions. Bean plants were tested serologically for potyvirus infections four-weeks postinoculation. The serological assay and symptomatology together indicated the presence of one potyvirus, and symptomology alone implied the presence of an unidentified virus. The partial genome of the potyvirus, encompassing the complete coat protein gene, was amplified using generic potyvirus primers. Sequence analysis of the amplicon confirmed the presence of an isolate of Wisteria vein mosaic virus (WVMV), a virus species not previously identified from Western Asia. Phylogenetic analyses of available WVMV sequences categorized them into five groups: East Asian-1 to 3, North American and World. The Iranian isolate clustered with those in the World group. Multiple sequence alignment indicated the presence of some genogroup-specific amino acid substitutions among the isolates studied. Chinese isolates were sister groups of other isolates and showed higher nucleotide distances as compared with the others, suggesting a possible Eastern-Asian origin of WVMV, the main region where Wisteria might have originated.

• Plant Biotechnology Reports
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• v.3 no.4
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• pp.309-315
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• 2009

Molecular markers developed from the flanking sequences of two cytoplasmic male sterility (CMS)-associated genes, orf456 and ${\Psi}atp6-2$, have been used for marker-assisted selection of CMS in pepper. However, in practice, the presence of orf456 and ${\Psi}atp6-2$ at substoichiometric levels even in maintainer lines hampers reliable selection of plants containing the CMS gene. In this study, we developed a novel CMS-specific molecular marker, accD-U, for reliable determination of CMS lines in pepper, and used the newly and previously developed markers to determine the cytoplasm types of pepper breeding lines and germplasms. This marker was developed from a deletion in a chloroplast-derived sequence in the mitochondrial genome of a CMS pepper line. CMS pepper lines could be unambiguously determined by presence or absence of the accD-U marker band. Application of orf456, ${\Psi}atp6-2$and accD-U to various pepper breeding lines and germplasms revealed that accD-U is the most reliable CMS selection marker. A wide distribution of orf456, but not ${\Psi}atp6-2$, in germplasms suggests that the pepper cytoplasm containing both orf456 and ${\Psi}atp6-2$ has been selected as CMS cytoplasm from cytoplasm containing only orf456. Furthermore, factors other than orf456 may be required for the regulation of male sterility in pepper.

• Korean Journal of Microbiology
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• v.44 no.2
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• pp.164-167
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• 2008

Recently, rolling circle amplification (RCA) technique has been widely focused in the field of gene amplification just like PCR method. We have developed RCA-mer, which is a web-based program searching for primer candidates from a given sequence. It can be applied to find primer lists in DNA amplification experiment based on RCA method. The RCA-mer compares 8-mer primer lists with user's input sequence such as vector, mitochondria, and microbial genome sequence. After calculating 8-mers existences in a given sequence, it displays matched and no-matched primer lists with their GC-contents. In addition to it, RCA-mer can search the existence of 8-mer primer lists in two sequences whether they are co-existed or not. Users can apply candidate primer lists to their researches which use RCA techniques.

• Journal of Life Science
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• v.9 no.1
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• pp.84-89
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• 1999

To determine phylogenetic relatedness of four silk-producing silkmoths (B. mori, B. mandarina, A. yamamai and A. pernyi), internal coding region of arylphorin which is a storage protein in hemolymph protein of insects were amplified by polymerase chain reaction and then sequenced and compared each other. The nucleotide composition was biased toward adenine and thymine(59% A+T) and a strong bias for use of C in the third position of codons was found for Phe and Tyr. Together TTC(Phe) and TAC(Tyr) account for about 16.8% (10 for TTC and 8 for TAC) of all codon usage. The nucleotide similarity of arylphorin gene from B. mori showed 99%, 98% and 97% homology with those of B. mandarina, A. yamamai and A. pernyi, respectively. Also, the nucleotide sequence of arylphorin gene from B. mandarina showed 98% and 97% homology with those of A. yamamai and A.pernyi, respectively. Between A. yamamai and A. pernyi, the sequence homology was 97%. The deduced amino acid sequences in B. mori, B. mandarina and A. yamamai showed almost 99% homology. Although the aryphorin gene provided insufficient variability among the four insect species, A UPGMA tree is generated that supported the monophyly of silk-producing insects, with M. sexta placed basal to it. It is suggest that silk-producing insects have a close relationship and a homogeneous genetic background from comparison with those of other insects.

• Journal of Crop Science and Biotechnology
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• v.11 no.4
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• pp.237-242
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• 2008

A liguleless mutant, which showed complete loss of lamina joint region at the junction between leaf blade and leaf sheath, was isolated from a Ds insertional mutants derived from the source cultivar, Dongjin. This mutant could not affect other developmental patterns like phyllotaxis. Southern blot analysis, using GUS as a probe, revealed that the liguleless mutant contained three Ds copies transposed in the rice genome. Among the four genomic sequences flanking the Ds, one was mapped in the intergenic region (31661640 - 31661759), and the other two predicted a protein kinase domain (12098980 - 12098667) as an original insertion site within a starter line used for massive production of Ds insertional mutant lines. Another predicted and inserted in first exon of liguleless 1 protein (OsLG1) that was mapped in coding region (LOC_Os04g56170) of chromosome 4. RT-PCR revealed that the OsLG1 gene was not expressed liguleless mutants. Structure analysis of OsLG1 protein revealed that it predicted transcription factor with a highly conserved SBP domain consisting of 79 amino acids that overlapped a nuclear localization signal (NLS). RT-PCR revealed that OsLG1 is mainly expressed in vegetative organs.

• Journal of Life Science
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• v.7 no.1
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• pp.49-58
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• 1997

Single P[en-lacZ] element including 5.7 kb of engrailed upstream sequences and the E. coli lacZ fusion gene, localized on 48A in rxyho25 strain was transposed to different sites in the Drosophila genome by the jumpstart technique. From 3315 individual genetic crosses, 113 new insertion lines carrying P[en-lacZ] inserted at different sites were obtained. $\beta$-Galactosidase expression in larval tissues of 113 insertion lines were detected by X-gal staining. & among 113 lines have been indentified to be for recessive lethal mutations. Among 7 lines, the #1119 line being lethal during embryogenesis was examined about the ${\beta}$$-Galactosidase expression, nuclear behavior and cellularization pattern during embryogenesis. The P[en-lacZ] insertion lines obtained in this study could be utilized for studying structure and function of the Drosophila development-related genes.

• Animal cells and systems
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• v.5 no.3
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• pp.243-246
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• 2001

Human endoqenous retroviral long terminal repeats (LTRs) have been found to be coexpressed with sequences of genes closely located nearby. It has been suggested that the LTR elements have contributed to structural changes or genetic variations of human genome connected to various diseases and evolution. Using cDNA library derived from placenta tissue, we performed PCR amplification and identified five new HERV-W LTR elements. Those LTR elements showed a high degree of sequence similarity (98-99%) with HERV-W LTR (AF072500). A phylogenetic tree obtained by the neighbor-joining method revealed that HERV-W LTR elements could be mainly divided into two groups through evolutionary divergence. Five new HERV-W LTR elements (pla-1, 4, 5, 6, 7) belonged to the group I with AX000960, AF072504, and AF072506 from GenBank database. The data suggest that several copy numbers of the HERV-W LTR elements are transcribed in placenta and may contribute to the understanding of biological function such as human placental morphogenesis.

• Molecules and Cells
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• v.26 no.1
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• pp.53-60
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• 2008

We characterized the human endogenous retrovirus (HERV-W) family in humans and primates. In silico expression data indicated that 22 complete HERV-W families from human chromosomes 1-3, 5-8, 10-12, 15, 19, and X are randomly expressed in various tissues. Quantitative real-time RT-PCR analysis of the HERV-W env gene derived from human chromosome 7q21.2 indicated predominant expression in the human placenta. Several copies of repeat sequences (SINE, LINE, LTR, simple repeat) were detected within the complete or processed pseudo HERV-W of the human, chimpanzee, and rhesus monkey. Compared to other regions (5'LTR, Gag, Gag-Pol, Env, 3'LTR), the repeat family has been mainly integrated into the region spanning the 5'LTRs of Gag (1398 bp) and Pol (3242 bp). FISH detected the HERV-W probe (fosWE1) derived from a gorilla fosmid library in the metaphase chromosomes of all primates (five hominoids, three Old World monkeys, two New World monkeys, and one prosimian), but not in Tupaia. This finding was supported by molecular clock and phylogeny data using the divergence values of the complete HERV-W LTR elements. The data suggested that the HERV-W family was integrated into the primate genome approximately 63 million years (Myr) ago, and evolved independently during the course of primate radiation.