• Journal of Microbiology and Biotechnology
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• 제20권1호
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• pp.45-53
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• 2010

We have developed an efficient and precise method for genome manipulations in Bacillus subtilis that allows rapid alteration of a gene sequence or multiple gene sequences without altering the chromosome in any other way. In our approach, the Escherichia coli toxin gene mazF, which was used as a counter-selectable marker, was placed under the control of a xylose-inducible expression system and associated with an antibiotic resistance gene to create a "mazF-cassette". A polymerase chain reaction (PCR)-generated fragment, consisting of two homology regions joined to the mazF-cassette, was integrated into the chromosome at the target locus by homologous recombination, using positive selection for antibiotic resistance. Then, the excision of the mazF-cassette from the chromosome by a single-crossover event between two short directly repeated (DR) sequences, included in the design of the PCR products, was achieved by counter-selection of mazF. We used this method efficiently and precisely to deliver a point mutation, to inactivate a specific gene, to delete a large genomic region, and to generate the in-frame deletion with minimal polar effects in the same background.

• Journal of Microbiology and Biotechnology
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• 제32권7호
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• pp.911-917
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• 2022

As valuable antibiotics, microbial natural products have been in use for decades in various fields. Among them are polyene compounds including nystatin, amphotericin, and nystatin-like Pseudonocardia polyenes (NPPs). Polyene macrolides are known to possess various biological effects, such as antifungal and antiviral activities. NPP A1, which is produced by Pseudonocardia autotrophica, contains a unique disaccharide moiety in the tetraene macrolide backbone. NPP B1, with a heptane structure and improved antifungal activity, was then developed via genetic manipulation of the NPP A1 biosynthetic gene cluster (BGC). Here, we generated a Streptomyces artificial chromosomal DNA library to isolate a large-sized NPP B1 BGC. The NPP B1 BGC was successfully isolated from P. autotrophica chromosome through the construction and screening of a bacterial artificial chromosome (BAC) library, even though the isolated 140-kb BAC clone (named pNPPB1s) lacked approximately 8 kb of the right-end portion of the NPP B1 BGC. The additional introduction of the pNPPB1s as well as co-expression of the 32-kb portion including the missing 8 kb led to a 7.3-fold increase in the production level of NPP B1 in P. autotrophica. The qRT-PCR confirmed that the transcription level of NPP B1 BGC was significantly increased in the P. autotrophica strain containing two copies of the NPP B1 BGCs. Interestingly, the NPP B1 exhibited a previously unidentified SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) inhibition activity in vitro. These results suggest that the Streptomyces BAC cloning of a large-sized, natural product BGC is a valuable approach for titer improvement and biological activity screening of natural products in actinomycetes.

• 한국가축번식학회지
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• 제13권1호
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• pp.49-55
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• 1989

The Results of colchicine and pretreatment time have shown that the cells of Coho Salmon(Oncorhynchus Kisutch) were effectively injected 6 hours prior to the harvest at a final concentration of 10$\mu\textrm{g}$/g body weight. The cell-density from 8,000 cells/㎣ (=8${\times}$106 ml) was found as ideal. The best effect of hypotonic solution in proportion to the cell is 20 : 1 and 50 minutes. The model number of diploid chromosome in this species was 2n=60. The karyotype analysis proved that the diploid complement consisted of 19 pairs of metacentric-, 4 pairs of submetacentric- and 7 paris of acrocentric-chromosomes. The diagrammatic representation proved that the diploid complement consisted of 7 pairs of metacentric-, 2 pairs of submetacentric- and 1 pair of acrocentric-chromosomes.

• 한국양식학회지
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• 제15권4호
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• pp.275-277
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• 2002

양식 산업성이 큰 메기 S. asotus를 대상으로 한 염색체 공학의 기초 자료를 얻기 위하여 본 연구를 수행하였다. 본 종의 난발생 중 핵분열(karyokinesis)을 조직학적으로 조사한 결과, 수온 24${\pm}$0.5$^{\circ}C$ 조건에서 핵분열은 수정 후 31분에 가장 현저하였으며, 이 시기는 메기에서의 제 1 난할 중기에 해당하였다.

• Fisheries and Aquatic Sciences
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• 제14권4호
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• pp.429-433
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• 2011

Eggs of Korean rose bitterling Rhodeus uyekii were collected and fertilized to observe temperature-related cleavage rates and mitotic intervals (${\tau}_0$). As the water temperature was increased, the slope of first cleavage frequency with elapsed time after fertilization increased, and approximately 30% of fertilized eggs reached first cleavage frequency at every 15 min. At higher temperatures, eggs developed faster and underwent further identical developmental processes. There were strong, negative correlations between ${\tau}_0$ and water temperatures at all temperatures studied (Y = -1.225X + 70.05, $r^2=0.988$, where Y is ${\tau}_0$ and X is temperature).

• 미생물학회지
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• 제27권2호
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• pp.92-97
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• 1989

the vector, pGUR19, for Rhizobium gene manipulation, was constructed by combining the replication and mobilization function of indigenous multicopy plasmid from Acacia(Robinia pseudoacacia L.) Rhizobia sp86 with E. coli cloning vehicle, pBR322. The vector could be efficiently mobilized by RP4 tra function incorporated into chromosome of E. coli named SM10 and efficiently transferred to various gram negative hosts including Rhizobium and Afrobacterium by transformation. Mobilization frequency of the constructed vector was ranged from $1.2\times 10^{-2}$ (E.coli HB 101) to $4.6\times 10^{-4}$ (A. tumefaciens 15955) and transformation frequency was ranged from $5.4\times 10^{-7}$(E. coli HB101) to $1.2\times 10^{-10}$ (A. tumefaciens 15955). The vector, pGUR19, was stably replicated and maintained in a variety of Rhizobium and Agrobacterium.

• 한국식물학회:학술대회논문집
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• 한국식물학회 1987년도 식물생명공학 심포지움 논문집 Proceedings of Symposia on Plant Biotechnology
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• pp.9-18
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• 1987

The plant breeding, a discipline of agricultural sciences, has greatly contributed to huan welfare in relieving food crisis by development of higher yielding, stronger resistant and better quality varieties. However, many conventional plant breeders, especially ones working for major crops, are facing exhaustion of useful genetic variability, which greatly limit the potentional of developing better cultivars. Therefore, the convectional plant breeders have been eagerly looking for new renovational methods in creating genetic varibility. It has been expected that biotechnology would provide the technique to create totally new genetic variability through gene transfer, chromosome manipulation and/or cell fusion. It is strongly suggested that very close interdisciplinary approaches between convectionla plant breeders and biotechnoligists is essentional for opening new era in developing better varieties.

• 한국양식학회지
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• 제8권2호
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• pp.141-148
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• 1995

무지개송어 전 암컷 3배체 조기난 생산을 위해 1994년 9월 30일부터 3년생 암컷 친어에 대하여 광주기를 단일 처리하여 조기 산란 및 성전환 유도와 고온 처리에 의한 염색체 조작을 실시하였다. 광주기 조절이 시작된지 1개월째 170마리의 암컷으로부터 340,000개의 난을 얻었다. 그러나, 부화율에 있어서는 자연 상태의 산란기때 부화율에 미치지 못하는 $40\~55\%$ 범위로 평균 $51.3\pm4.2\%$였다. (P<0.05). 수컷으로의 성전환을 위해 $16.5^{\circ}C$의 사육 수온 조건하에서 $17\alpha-methyltestosterone$을 사료 1kg당 3mg 섞어 첫 먹이 공급기부터 55일간 공급하여 성전환율을 조사한 결과 전부 수컷으로 판명되어 $16.5^{\circ}C$의 조건하에서도 성전환이 효율적으로 이루어짐을 알 수 있었다. $13.5^{\circ}C$의 사육수온 조건하에서 3배체를 생산하기 위해 최초 처리 시간 및 고온 처리 기간에 대해 다양한 조건으로 처리한 결과 발안기까지의 생존율에 있어 $15.0\~88.2\%$ 범위로 나타났다. 이때 3배체 유도율은 $36.7\~100\%$ 범위로 다양하였다.

• 한국양식학회지
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• 제9권1호
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• pp.101-106
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• 1996

XY 암컷에서 유도된 자성발생성 2배체 수컷 및 이의 성전환 암컷으로 부터 YY수컷(초수컷) 및 ${\Delta}YY$ 암컷(초암컷)을 선발하기 위하여 각각 정상 암컷 및 정상 수컷과 교배하여 자손 검정을 실시하였다. 교배가 이루어진 자성발생성 이배체 수컷 7마리 중 3마리가 YY 수컷으로 판정되었으며, 이들 자손의 수컷율은 $93.3\%\~100.0\%$로서 정상 교배에서 예상되는 성비인 1 : 1에 대한 $X^2$ test 결과 매우 유의하게 나타났다(P<0.01 또는 P<0.001). 자성발생성 2배체 성전환 암컷의 경우 교배가 이루어진 3마리 중 2마리가 ${\Delta}YY$ 암컷으로 판정되었으며, 이들 자손의 수컷율은 각각 $92.9\%$ 및 $86.2\%$ 그리고 $87.5\%$ 및 $95.0\%$로서 1 : 1 성비에 대해 매우 유의하였다(P<0.005). YY 수컷과 ${\Delta}YY$ 암컷간의 교배에서 전수컷 자손만이 생산되었다.

• 한국수산과학회지
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• 제47권4호
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• pp.431-434
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• 2014

Cytogenetic analysis was conducted to obtain basic information for chromosome manipulation of starry flounder Platichthys stellatus. Nuclear surface area and volume of erythrocyte were $7.60{\pm}0.93{\mu}m^2$ and $12.80{\pm}1.75{\mu}m^3$, respectively. The haploid DNA content of the species was 0.66 pg/haploid cell which correspond to 93% of olive flounder Paralichthys olivaceus. A karyotype analysis was also carried out with the species using conventional staining and Ag-NOR banding techniques. It was consisted of 48 acrocentric chromosomes and inter-sex or intra-individual polymorphism was not detected in all specimens analyzed. The NOR regions, appearing a terminal position of the short arm of the smallest acrocentric pairs.