• 대한환경공학회지
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• 제32권10호
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• pp.979-985
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• 2010

본 연구는 서울시에 위치한 먹는물 약수터를 대상으로 분변오염 지표미생물인 장구균 검출 특성과 염기서열을 이용한 동정 및 반코마이신 내성 장구균 특성을 평가하였다. 먹는물 약수터의 장구균은 212건의 시료 중 42건(19.8%)에서 0~110 CFU/100 mL의 범위로 검출되었고 대장균의 검출빈도와 유사(t-검정, p-값 0.268, 유의수준 0.05)하여 지표미생물로서 적용가능성을 확인하였다. 16S rDNA 염기서열을 이용한 동정에서는 표본적으로 추출한 56개 검체에서 Enterococcus faecalis (24검체), E. faecium (10검체), E. casseliflavus (10검체), E. gallinarum (3검체), E. hirae (2검체), E. durans (2검체), E. sanguinicola(1검체) 순서로 나타났으며, 가장 높은 빈도로 검출되는 E. faecalis는 국내 병원환자의 임상검체에서 분리되는 우점종 분포와 유사하였다. 반코마이신 내성 장구균은 검체 시료 53검체 중 14(26.4%)검체에서 vanB 및 vanC1/C2 형이 각각 2와 12검체가 확인 되었다. 본 연구결과는 먹는물 약수터가 분변오염 등으로 인하여 장구균 검출 가능성이 있을 것으로 판단되어 소독 등 외부오염물질 유입 차단 및 저감대책이 필요할 것을 시사하였다.

• Safety and Health at Work
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• 제7권2호
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• pp.156-160
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• 2016

Background: Workers in pesticide manufacturing industries are constantly exposed to pesticides. Genetic biomonitoring provides an early identification of potential cancer and genetic diseases in exposed populations. The objectives of this biomonitoring study were to assess DNA damage through comet assay in blood samples collected from industry workers and compare these results with those of classical analytical techniques used for complete blood count analysis. Methods: Samples from controls (n = 20) and exposed workers (n = 38) from an industrial area in Multan, Pakistan, were subjected to various tests. Malathion residues in blood samples were measured by gas chromatography. Results: The exposed workers who were employed in the pesticide manufacturing industry for a longer period (i.e., 13-25 years) had significantly higher DNA tail length ($7.04{\mu}m$) than the controls ($0.94{\mu}m$). Workers in the exposed group also had higher white blood cell and red blood cell counts, and lower levels of mean corpuscular hemoglobin (MCH), MCH concentration, and mean corpuscular volume in comparison with normal levels for these parameters. Malathion was not detected in the control group. However, in the exposed group, 72% of whole blood samples had malathion with a mean value of 0.14 mg/L (range 0.01-0.31 mg/L). Conclusion: We found a strong correlation ($R^2=0.91$) between DNA damage in terms of tail length and malathion concentration in blood. Intensive efforts and trainings are thus required to build awareness about safety practices and to change industrial workers' attitude to prevent harmful environmental and anthropogenic effects.

• Asian Pacific Journal of Cancer Prevention
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• 제15권6호
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• pp.2869-2873
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• 2014

Background: Human papillomavirus (HPV) integration within the E2 gene has been proposed as a critical event in cervical carcinogenesis. This study concerned whether HPV16 status and E2 gene intactness are predictive of radiation response in patients with cervical cancer. Materials and Methods: Biopsies of 44 patients with cervical cancer were collected before or after radiotherapy. The presence of HPV16 was assessed by polymerase chain reaction (PCR) using specific primers for the L1 region. E2 disruption was detected by amplifying the entire E2 gene. Results: HPV16 DNA was found in 54.5% of the clinical samples. Overall, 62.5% of the HPV16 positive tumors had integrated viral genome and 37.5% had episomal genome. There was a tendency of increase of HPV16 E2 negative tumors compared with HPV16 L1 ones in advanced stages (75% versus 20% in stage III respectively). Detection of E2 gene appeared influenced by the radiotherapy treatment, as the percentage of samples containing an intact HPV16 E2 was more frequent in pretreated patients compared to radiotherapy treated patients (66.6% versus 20%). The radiation therapy caused an eight-fold [OR= 8; CI=1.22-52.25; p=0.03] increase in the risk of HPV16 genome disruption. The integration status is influenced by the irradiation modalities, interestingly E2 disruption being found widely after radiotherapy treatment (75%) with a total fractioned dose of 50Gy. Conclusions: This study reveals that the status of the viral DNA may be used as a marker to optimize the radiation treatment.

• Journal of Microbiology and Biotechnology
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• 제10권6호
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• pp.764-769
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• 2000

The lipase gene(lip) from Bacillus stearothermophilus was recombined in vitro by utilizing the DNA shuffling technique. After four rounds of shuffling, transformation, and screening based on the initial rate of clear zone formation on a tricaprylin plate, a clone (M10) was isolated, the cell extract of which showed about 2.8-fold increased lipase activity. The DNA sequence of the mutant lipase gene (m10) showed 3 base changes, resulting in two cryptic mutations and one amino acid substitution: S113($AGC{\rightarrow}AGT$), L252 ($TTG{\rightarrow}TTA$), and G275E ($GGA{\rightarrow}GAA$). SDS-PAGE analysis revealed that the increased enzyme activity observed in M10 was partly caused by high expression of the m10 lipase gene. The amount of the expressed G275E lipase was estimated to comprise as much as 41% of the total soluble proteins of the cell. The maximum velocity ($V_{max}$) of the purified mutant enzyme for the hydrolysis of olive oil was measured to be 3,200 U/mg, which was 10% higher than that of the parental (WT) lipase (2,900 U/mg). Its optimum temperature for the hydrolysis of olive oil was $68^{\circ}C$ and it showed a typical $Ca^{2+}$-dependent thermostability, properties fo which were the same as those of the WT lipase. However, the mutant enzyme exhibited a high enantiospecificity towards (S)-naproxen compared with the WT lipase. In addition, it showed increased hydrolytic activity towards triolein, tricaprin, tricaprylin, and tricaproin.

• 생명과학회지
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• 제21권1호
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• pp.159-164
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• 2011

미생물 세포 바이오센서는 환경오염물질의 모니터링을 위한 좋은 분석도구가 될 수 있다. 이는 리포터유전자들(예로, lux, gfp or lacZ)을 방향족 화합물이나 중금속과 같은 오염물질에 반응하는 유도 조절유전자와 결합하여 만든다. 이러한 유전자 재조합기술을 이용하여 많은 종류의 미생물 바이오센서가 개발되었으며 환경, 의학, 식품, 농업, 및 방위등 다양한 분야에서 활용되고 있다. 또한 바이오센서의 민감도와 검출범위는 조절유전자의 변형을 통해 증가시킬 수있다. 최근에는 미생물 바이오센서 세포를 고효율 검색용 세포 에레이의 칩, 광섬유 등에 고착하여 활용하고 있다. 본 논문은 특이 오염물질의 검출을 위한 유전자 재조합으로 만든 미생물 세포 바이오센서의 현황과 미래에 대해 고찰한다.

• 생태와환경
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• 제56권1호
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• pp.1-13
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• 2023

북한강 수역에서 가장 많이 검출되는 Microcystin (MC)을 대상으로 하여 MC 생합성 유전자(mcyA gene), 남조류 세포밀도, MC 농도 사이의 관계를 분석하여 RNA-MC 환산식을 도출하고 남조류 세포 내 존재하는 MC 농도를 예측하였다. 북한강 수역에서 mcyA 유전자는 묵현천 합류 이후 북한강 하류 지점에서 주로 발견되었으며 평균적으로 다른 지점보다 높은 copy number가 발견되었다. 북한강 상류 의암호 수역의 경우, 공지천 지점에서 mcyA 유전자 copy number가 증가하였으며 9월 이후 북한강 수역 전체에서 mcyA 유전자 copy number는 감소하였다. mcyA gene expression은 상류와 하류 수역의 시·공간적 차이가 존재하였으며 여름철 짧은 시기에 집중적으로 발현하였다. mcyA gene expression 양은 MC 농도와 상관성이 매우 높을 뿐만 아니라 MC을 생합성하는 것으로 알려진 Microcystis aeruginosa와 Dolichospermum circinale의 세포밀도와도 통계적으로 유의한 상관성이 존재하였다. RNA-MC 관계를 기반으로 도출된 6개의 환산식은 통계적 유의성을 보이며(p<0.05) 0.9 이상의 높은 상관계수(r)를 나타냈다. eRNA에 존재하는 MC 생합성 유전자 발현량은 수중의 남조독소 물질 합성을 판단하고 유전자의 활성 정도를 빠르게 정량하여 MC 발생 조기경보에 충분히 활용할 수 있을 것으로 판단된다.

• Applied Biological Chemistry
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• 제48권4호
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• pp.345-351
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• 2005

난배양 미생물로부터 천연물질을 찾기 위하여 토양으로부터 직접분리 된 DNA와 cosmid vector를 이용하여 metagenomic library를 제작하고 탐색 하였다. 대장균에서 발현되는 유전자은행 초기 탐색 결과 LB배지에서 잘 자라면서 브라운 색깔을 내는 여러 개의 clone을 선발 하였다. 선발된 여러 후보 clone중 pYS85C는 돌연변이를 유도하였으며 색깔을 생산하지않는 clone 들에 대하여 염기서열을 결정 하였다. 돌연변이clone들로부터 결정된 pYS85C 염기서열 결과 아미노산이 393개이며 44.5 kDa으로 색소형성에 관여하는 4-hydroxyphenylpyruvic acid dioxygenase(HPPD) 유전자로 판명 되었다. 또한, BLAST비교 분석에서 이효소는 기존에 밝혀진 HPPD효소와 60% 정도의 identity를 보였고 C-말단에서는 많은 conserved domain이 있었다. 이러한 결과로 볼 때 천연물질을 합성 할 수 있는 유전자는 토양DNA로부터 직접 분리되어 발현될 수 있으며 이러한 기술은 새로운 물질을 찾는데 중요한 tool이 될 수 있다.

• ALGAE
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• 제37권4호
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• pp.281-291
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• 2022

Quantifying the abundance of Chattonella species is necessary to effectively manage the threats from ichthyotoxic raphidophytes, which can cause large-scale mortality of aquacultured fish in temperate waters. The identification and cell counting of Chattonella species have been conducted primarily on living cells without fixation by light microscopy because routine fixatives do not retain their morphological features. Species belonging to the Chattonella marina complex, including C. marina and C. marina var. ovata, had high genetic similarities and the lack of clear morphological delimitations between the species. To estimate the abundance of C. marina complex in marine plankton samples, we developed a protocol based on the droplet digital polymerase chain reaction (ddPCR) assay, with C. marina complex-specific primers targeting the internal transcribed spacer (ITS) region of the rDNA. Cell abundance of the C. marina complex can be determined using the ITS copy number per cell, ranging from 25 ± 1 for C. marina to 112 ± 7 for C. marina var. ovata. There were no significant differences in ITS copies estimated by the ddPCR assay between environmental DNA samples from various localities spiked with the same number of cells of culture strains. This approach can be employed to improve the monitoring efficiency of various marine protists and to support the implementation of management for harmful algal blooms, which are difficult to analyze using microscopy alone.

• Journal of Microbiology and Biotechnology
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• 제16권10호
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• pp.1561-1569
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• 2006

Denaturing gradient gel electrophoresis (DGGE) is one of the most frequently used methods for analysis of soil microbial community structure. Unbiased PCR amplification of target DNA templates is crucial for efficient detection of multiple microbial populations mixed in soil. In this study, DGGE profiles were compared using different pairs of primers targeting different hypervariable regions of thirteen representative soil bacteria and clones. The primer set (1070f-1392r) for the E. coli numbering 1,071-1,391 region could not resolve all the 16S rDNA fragments of the representative bacteria and clones, and moreover, yielded spurious bands in DGGE profiles. For the E. coli numbering 353-514 region, various forward primers were designed to investigate the efficiency of PCR amplification. A degenerate forward primer (F357IW) often yielded multiple bands for a certain single 16S rDNA fragment in DGGE analysis, whereas nondegenerate primers (338f, F338T2, F338I2) differentially amplified each of the fragments in the mixture according to the position and the number of primer-template mismatches. A forward primer (F352T) designed to have one internal mismatch commonly with all the thirteen 16S rDNA fragments efficiently produced and separated all the target DNA bands with similar intensities in the DGGE profiles. This primer set F352T-519r consistently yielded the best DGGE banding profiles when tested with various soil samples. Touchdown PCR intensified the uneven amplification, and lowering the annealing temperature had no significant effect on the DGGE profiles. These results showed that PCR amplification bias could be much improved by properly designing primers for use in fingerprinting soil bacterial communities with the DGGE technique.

• 한국환경과학회지
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• 제23권8호
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• pp.1447-1453
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• 2014

The differentiation of sex is important for species preservation. However, Fairy Pitta is sexually monomorphic and sex of an individual is indistinguishable with its external characteristics. We determined the sex of Fairy Pitta through DNA analysis and investigated the causes and time of injury and mortality and the size based on sex. We collected 21 samples at Jeju Island, Korean Peninsula from 2004 to 2013 and extracted DNA from them and amplified chromo helicase DNA-binding gene from Z and W chromosomes through Polymerase Chain Reaction (PCR). We confirmed their sex with the banding pattern through Agarose gel electrophoresis, i.e. male (ZZ): one banded and female (ZW) two banded. We distinguished the sex of 17 of 21 samples resulting in 9 males and 8 females. Most casualties were recorded in adult of both sexes. Causes of injury and mortality proved that female casualties occurred from window strikes, dehydration, car accident, predation by natural enemies, and male occurred from window strikes, car accident and dehydration. The time of injury and mortality in adults differ by sex. There was no difference between sexes in any of the six size parameters. As the time of injury and mortality differ by sex, the survey on the role and ecological nature by sex in breeding season must be carried out in the future. External measurements may not be reliable for sexing of Fairy Pitta and other traits such as vocal or characteristics are required to identify the sex of individuals in the field.