• Korean Journal of Ecology and Environment
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• v.54 no.3
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• pp.190-198
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• 2021

Human Aichivirus (Aichivirus A; AiV-A) is a positive-sense single-strand RNA non-enveloped virus that has been detected worldwide in various water environments including sewage, river, surface, and ground over the past decade. To develop a method with excellent sensitivity and specificity for AiV-A diagnosis from water environments such as groundwater, a combination capable of reverse transcription (RT)-nested polymerase chain reaction (PCR) was developed based on existing reported and newly designed primers. A selective method was applied to evaluate domestic drinking groundwater samples. Thus, a procedure was devised to select and subsequently identify RT-nested PCR primer sets that can successfully detect and identify AiV-A from groundwater samples. The findings will contribute to developing a better monitoring system to detect AiV-A contamination in water environments such as groundwater.

• Journal of Food Hygiene and Safety
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• v.30 no.1
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• pp.43-50
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• 2015

In this study, single PCR and multiplex PCR tests were examined for identification of four types of squid species (giant squid, cuttlefish, octopus, beka squid) purchased from fish market as well as aquatic processed products in Busan. To design the specific primers against each species, the nucleotide sequences of the mitochondrial 16s rRNA gene of Architeuthis dux, Todarodes pacificus, Enteroctopus dofleini, Enteroctopus megalocyathus, Uroteuthis chinensis, Uroteuthis duvauceli, Uroteuthis edulis groups were analyzed for the identification of each species registered in the GeneBank (www.ncbi.nlm.nih.gov) and have been used for comparative analysis. In order to obtain the size variation of amplified fragments on multiplex PCR, we designed KOJ-F, OJ-F, OCT-F, HAN-F, ALLR primers for each species. The optimal PCR conditions and primers were selected for four types of squid species to determine target base sequences in its PCR products. In the case of single PCR, giant squid was only amplified by KOJ-F/ALLR primer; cuttlefish was only amplified by OJ-F/ALLR primer; octopus was only amplified by OCT-F/ALLR primer; and beka squid was only amplified by HAN-F/ALLR primer. For multiplex PCR, the mixture of four kinds of genomic DNA (giant squid, cuttlefish, octopus, beka squid) been prepared as a template and used together with the mixture of KOJ-F/OJ-F/OCT-F/HAN-F/ALLR primers in the reaction. By the multiplex PCR, it is confirmed that four samples are correspond to multiple simultaneous amplicon. Finally, we validated the established methods of multiplex PCR in the aquatic processed products. Although the mitochondrial 16s rRNA primers used in this study was useful as a marker for detection of each species among them, the study indicated that the established multiplex PCR method can be more useful tool for monitoring the processed products.

• Asian-Australasian Journal of Animal Sciences
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• v.12 no.5
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• pp.695-701
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• 1999

The hypothalamus is the classic site of synthesis of arginine vasotocin as neurohypophyseal hormone in the chicken. However, high concentrations of arginine vasotocin were also measured in ovarian tissues by radioimmunoassay. At first, we observed specific positive signal of mRNA encoding AVT in the hypothalamus by Northern hybridization. However, we could not find any specific bands in ovarian and uterine tissues. For evidence of transcription of the arginine vasotocin gene ingonadal tissues of the chicken, this study has applied the polymerase chain reaction as a highly sensitive assay. The hypothalamus, the four largest preovulatory ovarian follicles and the shell gland (uterus) were collected at 4 h and 20 h before oviposition. The ovarian follicular tissues were separated into granulose theca interns and theca externa layers. The uterine tissues were separated into myometrium and endometrium The extracted mRNA was converted to cDNA by reverse-transcriptase using oligo-$d(T)_{15}$ primer. Then, the cDNA was amplified by Vent polymerase and arginine vasotocin specific primers. The amplification reaction was incubated by 30 cycles successively, $95^{\circ}C$, $55^{\circ}C$ and $72^{\circ}C$ earth for 1 min. Te comparisons of the mRNA levels encoding arginine vasotocin between the tissues were determined by semi-quantification methods. After amplification of the cDNA, the PCR products were detected in hypothalamus, ovarian tissues and uterine tissues. The results of semi-quantification showed that the levels of arginine vasotocin mRNA in ovarian iud uterine tissues were about from 1/50 to 1/1000 when compared to that in the hypothalamus. The very low levels of mRNA encoding arginine vasotocin in ovarian and uterine tissues probably led us to conclude that arginine vasotocin may play a role of local mediate acting autocrine and/or paracrine.

• Development and Reproduction
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• v.4 no.1
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• pp.61-66
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• 2000

This study was investigated to analyze the inactivating point mutation and expression level of follicle-stimulating hormone(FSH) receptor mRNA. In first experiment, we analyzed the point mutation. Peripheral blood was collected from each patient. To screen individuals for the C566T mutation, PCR was performed for exon 7 of the FSH receptor gene in 10 patients. No inactivating point mutation of FSH receptor gene was identified in women with premature ovarian failure. To analyze the expression level of FSH receptor, mRNA expressions were examined by RT-PCR method using specific primers for the FSH receptor. The amount of FSH receptor mRNA expressed in POF patients was lower than that in the control group. But it was not significantly different. These finding suggests that lower expression of FSH receptor in premature ovarian failure patients might be the cause of the low response to the gonadotropin during the hyperstimulation in IVF-ET cycles.

• Journal of Embryo Transfer
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• v.13 no.2
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• pp.159-172
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• 1998

사람 성장호르몬 수용체(hGHR) cDNA는 PCR방법에 의하여 fagment로서 보고되어진 바 있으나, liver cDNA로 부터 전장을 cloning한 보고는 없는 실정으로 본 연구에서는 기능을 가진 약 4.6kbp의 cDNA hGHR을 cloning 하는데 성공하였다. 먼저 cloning하기 위하여 human liver mRNA와 human breast cancer tissue로부터 회수한 mRNA를 RT-PCR방법에 의하여 human cDNA library와 cloning에 필요한 probe를 제작하였다. human library mRNA는 GT-PCR방법에 의하여 증폭하여 증폭되어진 산물은 λZAP Vector를 이용하여 cDNA library를 구축하였고,screeing을 위하여 임 보고 되어진 hGHR fragment native sequence를 기초로 N-terminal부분의 primer를 설계하여 950bp의 probe를 얻는데 성공하였다. 이 probe를 이용하여 준비된 human liver cDNA library로부터 2.5$\times$10 6개의 plaque로부터 6개의 positive clone을 획득하였고, 이들중 poly Asignal인 "AATAAA"를 포함하고 있는 가장 긴 약 3.8kbp의 clone을 sequencing한 결과 open reading frame을 포함하고 있었으나, 5'부분의 결손되어 있었다. 그리하여 이 부분은 human breast cancer tissue로 부터 회수한 mRNA를 RT-PCR에 의하여 증폭하였고, sequencing결과 이미 보고되어진 native hGHR와 비교한 결과 하나의 nucleotide가 silent mutation으로 판명되었다.한편 human liver cDNA library로부터 cloning한 3.8cp의 positive clone의 5'end의 결손된 부분에 silent mutation된 PCR 산물을 연결함으로써 native hGHR와 유사한 cDNA hGHR subcloning에 성공하였다. 이러한 cDNA hGHR의 clone이 function을 가지고 있는지를 검토하기 위하여 eukaryotic 발현 vector인 pCXN2에 의거 ligation한 후 chinese hamster ovary cell[CHO-KI]에 transfect를 실시하였다. Dexamethasone은 첨가하지 않고 hGH만의 존재하에서 이들 cell을 배양시키고 cell menbrane에서 발현 여부를 판정키 위하여 hGHR monocloual antibody를 사용하여 flow cytometery해석을 실시하는 한편 125I-hGH binding assay에 의하여 hGH binding activity를 측정하였다. 최종적으로 GH signal transduction의 target genedf으로 알려져 있는 serine protease inhibitor 2.1(Spi 2.1) gene의 promotor activity를 검토한 결과 hGHR을 transfect한 CHO Cell에 있어서 hGH의 농도에 의존적으로 증가되었다. 따라서 본 실험에서 cloning한 cDNA hGHR는 native hGHR와 같은 기능을 가지는 것으로 판명되었다.것으로 판명되었다.

• Journal of Microbiology and Biotechnology
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• v.20 no.8
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• pp.1163-1178
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• 2010

Plasmids play important roles in horizontal gene transfer among Vibrionaceae, but surprisingly little is known about their replication and incompatibility systems. In this study, we successfully developed a bioinformatics-assisted strategy of experimental identification of seven Vibrio plasmid replicons. Comparative sequences analysis of the seven Vibrio plasmid replicons obtained in this study together with eight published Vibrionaceae plasmid sequences revealed replication-participating elements involved in the ColE1 mode of replication initiation and regulation. Like plasmid ColE1, these Vibrionaceae plasmids encode two RNA species (the primer RNA and the antisense RNA) for replication initiation and regulation, and as a result, the 15 Vibrionaceae plasmids were designated as ColE1-like Vibrionaceae (CLV) plasmids. Two subgroups were obtained for the 15 CLV plasmids, based on comparison of replicon organization and phylogenetic analysis of replication regions. Coexistence of CLV plasmids were demonstrated by direct sequencing analysis and Southern hybridization, strongly suggesting that the incompatibility of CLV plasmids is determined mainly by the RNA I species like the ColE1-like plasmids. Sequences resembling the conserved Xer recombination sites were also identified on the CLV plasmids, indicating that the CLV plasmids probably use the host site-specific recombination system for multimer resolution like that used by ColE1-like plasmids. All the results indicated that the 15 plasmids form a new ColE1-like group, providing a basis for the rapid characterization and classification of Vibrionaceae plasmids.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.48 no.5
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• pp.731-738
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• 2015

Nematode infection in the epithelial tissue of cultured rockfish Sebastes schlegeli was first reported in 2012. Since then, nematode infections have caused serious economic losses in rockfish aquaculture on the west coast of Korea. Taxonomic and life cycle information for this parasite are currently unknown. In this study, 18S rRNA and cytochrome c oxidase subunit I (COI) genes were used for molecular identification and polymerase chain reaction (PCR) to detect the invisible stages of this parasite. Nucleotide sequences of the 18S rRNA of the rockfish nematode showed 98% identity with that of Philometra morii. Therefore, this rockfish nematode was classified to the Philometridae family. However, we could not identify it to genus level using 18S rRNA. Its COI nucleotide sequences shared 85% and 82% identities with those of Bursaphelenchus sinensis and Philometra overstreeti, respectively. In addition, two gene-specific primer sets were designed based on the 18S rRNA gene to detect the intermediate host and nematode larvae. These primers were specific to this rockfish nematode without cross-reacting to other pathogens. The detection limit of the PCR assay using these primers was 1,000 copies of nematoda plasmid DNA. Therefore, the PCR assay described here is suitable for the detection of nematode DNA within rockfish. In addition, this PCR assay could be used to detect nematode larvae and the intermediate host.

• Korean journal of applied entomology
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• v.42 no.1
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• pp.65-70
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• 2003

For the purpose of the protection of beneficial insects from pathogens and the development of control agent against pests, a strain of Metarhizium sp. was isolated from the infected Protaetia brevitarsis seulensis larvae in Korea. Under the scanning electron microscope, the isolate, Metarhizium sp. KMA-1, showed distinct formation of conidia on the palisade-like masse which were comprised of elongate chains and this shape is a typical feature of Metarhizium species. PCR techniques were used to identify the isolate and the primers used were designed on the basis of two kinds of rRNAs sequences, 28S rRNA and internal transcribed spacer(ITS). The specific PCR products from each primer set were amplified and the DNA sequences were determined for the similarity comparison. Sequence alignment of these fragments using GenBank database resulted in the highest homology similarity between the isolate Metarhizium sp. KMA-1 and M. anisopliae. From these results, the isolate Metarhizium sp. KMA-1 in this study was identified as M. anisopliae.

• Journal of Microbiology
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• v.45 no.5
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• pp.418-421
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• 2007

The nucleotide at position 791(G791) of E. coli 16S rRNA was previously identified as an invariant residue for ribosomal function. In order to characterize the functional role of G791, base substitutions were introduced at this position, and mutant ribosomes were analyzed with regard to their protein synthesis ability, via the use of a specialized ribosome system. These ribosomal RNA mutations attenuated the ability of ribosomes to conduct protein synthesis by more than 65%. A transition mutation (G to A) exerted a moderate effect on ribosomal function, whereas a transversion mutation (G to C or U) resulted in a loss of protein synthesis ability of more than 90%. The sucrose gradient profiles of ribosomes and primer extension analysis showed that the loss of protein-synthesis ability of mutant ribosomes harboring a base substitution from G to U at position 791 stems partially from its inability to form 70S ribosomes. These findings show the involvement of the nucleotide at position 791 in the association of ribosomal subunits and protein synthesis steps after 70S formation, as well as the possibility of using 16S rRNA mutated at position 791 for the selection of second-site revertants in order to identify ligands that interact with G791 in protein synthesis.

• The Journal of Natural Sciences
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• v.11 no.1
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• pp.95-98
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• 1999

Chinese cabbage (Brassica campestris), one of the major vegetable crops in Korea, undergoes self-incompatible pathway for reproduction. To maintain inbred lines of chinese cabbage, a method in that $CO_2$ gas is treated to the pistils to break the self-incompatibility and thereby self-pollens can successfully make germination and fertilization has been selectively used in speed company. In this study, the pistil genes induced by the $CO_2$ treatment was investigated by mRNA differential display (DD-PCR) method. The result shows PCR products amplified in a differential pattern from both $CO_2$ gas treated- and untreated-pistil mRNAs, suggesting that the pistil genes are probably regulated positively and also negatively by the $CO_2$ gas.