• Radiation Oncology Journal
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• 제23권1호
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• pp.43-50
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• 2005

목적 : 자경경부암세포에서 polymeric chain reaction (PCR)원리를 이용한 suppression subtractive hybridization (SSH) 방법으로 방사선조사 시 차등 발현되는 유전자를 동정하고자 하였다. 대상 및 방법 : 자궁경부암세포주인 HeLa세포주에 방사선조사 전과 후 총 RNA와 poly $(A)^+$ mRNA를 분리였다. SSH방법으로 forward 및 reverse-subtracted cDNA libraries를 만들었다. 차등 발현된 유전자를 screening하기 위해 reverse Northern blotting (dot blot analysis)을 이용하여 각각의 library에서 88개의 클론을 선택하였고 Nothern blotting으로 확인 후 sequencing하였다. 결과 : screening상 176개 클론 중 forward-subtracted library에서 10개의 유전자가 reverse-subtracted library에서 9개의 유전자가 동정되었다. forward-subtracted library로부터 3개의 유전자가 Northern blotting에 의하여 확인되었고 이중 telomerase catalytic subunit and sodium channel-like protein 유전자와 1개의 ESTs (expressed sequence tags) 유전자가 방사선선량에 따라 증가하쳐다. 결론 : 본 연구를 통해 자궁경부암세포주에서 방사선에 의해 유도되는 유전자를 SSH 방법을 통해 동정할 수 있었다. 그러나 이러한 유전자가 어떤 생물학적인 기능을 갖고 있는지에 대한 계속적인 연구가 필요하다

• 한국해양바이오학회지
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• 제1권3호
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• pp.186-192
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• 2006

짧은 집단 반복 요소 (Short Interspersed Repetitive Elements, SINE) 는 수백개 정도의 염기로 구성된 반복염기서열로서 LINE (Long Interspersed Nucleotide Elements)와 함께 바이러스와는 구별되는 레트로트랜스포존 (Retrotransposon)의 하나로 알려져 있다. 이들의 생체 내 역할은 정확하게 밝혀진 것은 없지만 게놈 내에서 반복염기서열의 재배열을 통해 완전히 새로운 유전자를 창조하거나 기존의 유전자를 변형시킴으로써 유전물질의 운반수단 및 진화적 변화에 있어서 중요한 역할을 할 것이라 예상되며, 질병의 원인이 된다고도 밝혀져 있다. 본 연구에서는 미꾸라지로부터 SINE의 새로운 두 그룹을 분리하였다. 두 SINE 그룹, mlSINE-L과 mlSINE-S는 각각 약 410bp와 270bp의 염기로 구성되어 있다. 두 SINE 그룹의 5'과 3'말단의 서열은 RSg-1와 SmaI SINE 의 그것과 높은 유사도를 보였다. 계통발생분석결과, mlSINE들은 미꾸라지에서 유일하였으며, dot blot hybridization의 결과는 mlSINE-L이 미꾸라지 게놈 $2{\times}10^9bp$ (2.8 pg)당 $1{\times}10^3$ copy를 가지는 것으로 추정되며, loop DNA보다 핵기질부착부위 (nuclear matrix attachment regions, MARs)에서 그 분포도가 높았다. 이런 결과는 미꾸라지의 새로운 SINE 들이 핵기질 부착부위 내에서나 혹은 가까운 주변에 우선적으로 삽입될 수 있음을 나타낸다.

• 한국산업보건학회지
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• 제21권1호
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• pp.49-54
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• 2011

In order to develop the methods for exposure assessment and find susceptibility markers for the workers who are exposed to low doses of toluene, xylene and other chemical in petroleum industries, we investigated the application of P-450 expression in human lymphocytes utilizing mouse monoclonal anti-rat CYP2B1/2, the levels of toluene and xylene in air and their metabolite levels in urine with the levels of expressed CYP2B1/2 proteins. The general characteristics such as age, smoking and drinking habit were no significant difference between the control and exposed workers, but the working durations and working hours were significantly different. Workers in exposed group were exposed to the mean of 2.1 ppm (range, 0.00-4.54) of toluene and 0.3 ppm (rang, 0.00-1.23) of xylene. The mean concentration of urinary hippuric acid was low and less than 1/5 of the biological exposure index recommended by the Ministry of Employment and Labor Korea. Methyl hippuric acid in urine was not detected in control and exposed workers. Also, there were no significant differences in the levels of the urinary metabolites between the control and exposed group. When chemiluminescence dot blottings were carried out utilizing mouse monoclonal antibody against CYP2B1/2, the strong density dots corresponding to a mouse monoclonal antibody was observed in the human lymphocytes from the exposed workers. These results suggested that the chemiluminescence dot blot assay for CYP of lymphocytes should be valuable for identifying CYP expression as biomarkers in the workers exposed to toluene and xylene.

• 한국균학회지
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• 제27권1호통권88호
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• pp.20-26
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• 1999

우리나라에서 자생하는 송이버섯의 구분 방법으로 제조된 OPO-2 primer을 사용할 때 송이에게만 특이적인 band가 나타났다. 그리고 제한효소 처리와 dot blot의 결과에서 이들 특이적인 DNA절편은 송이 genomic DNA에서 한개의 copy만 존재하는 것으로 나타났으며, 다른 외생균근 버섯의 DNA에서는 발견되지 않았다. 또한, 이들 DNA 절편의 염기서열을 분석한 결과 770bps로 나타났다. 이러한 유전자의 정보를 이용한 NCBI Blast 염기서열 분석에서 높은 상동성을 갖는 유전자는 발견할 수가 없었고, 단백질서열 분석에서도 거의 동일하게 나타났다. 따라서 translation통한 아미노산 서얼분석에서 이들 DNA절편의 유전자는 단백질에 관한 유전자이라기 보다는 다른 정보와 관련된 유전자로 생각되어진다.

• 대한수의학회지
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• 제43권4호
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• pp.667-675
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• 2003

The safety of Brucella abortus strain RB51(SRB51) was investigated in dairy cows and Korean native cattle of 4~7th month of gestation. From experimentally inoculated cattle, 18 of 25 (72.0%) dairy cows, and 3 of 10 (30.0%) Korean native cattle were aborted or delivered premature fetus. There were no significant differences in the incidence of abortion depending on the inoculation route (intramuscular and subcutaneous) and dosages ($1{\times}10^9$, $2.8{\times}10^9$, and $4.0{\times}10^9$ CFU). The antibodies to the SRB51 were measured by a dot blot enzyme-linked immunosorbent assay. The highest titers to SRB51 were detected between 5~7 weeks after inoculation and the specitic antibody could be detected up to 28 weeks after inoculation. The SRB51 was isolated from amnio-allantoic fluid, bronchial lymph node, mammary gland, and supramammary lymph node in 5 of 25 dairy cows during 4 weeks after either abortion or delivery. Although SRB51 was isolated from 4 of 24 aborted fetus or normally delivered calves at parturition time, it was not isolated during 4 weeks afterward. Eleven of twentyfive dairy cows showed the endometritis and/or necrosis until 6 weeks after delivery, no lesions were seen at 8 weeks after delivery and uterus from control dairy cows. The results of present study revealed that SRB51 might induce the clinical signs of brucellosis in the pregnant cattle at 4~7th month of gestation.

• BMB Reports
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• 제35권4호
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• pp.420-427
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• 2002

Partial nerve injury is the main cause of neuropathic pain disorders in humans. Acupuncture has long been used to relieve pain. It is known to relieve pain by controlling the activities of the autonomic nervous system. Although the mechanism of neuropathic pain and analgesic effects of electroacupuncture (EA) have been studied in a rat model system, its detailed mechanism at the molecular level remains unclear. To identify genes that might serve as either markers or explain these distinct biological functions, a cDNA microarray analysis was used to compare the expression of 8,400 genes among three sample groups. Messenger RNAs that were pooled from the spinal nerves of 7 normal. 7 neuropathic pain, and 7 EA treatment rat models were compared. Sixty-eight genes were differentially expressed more than 2-fold in the neuropathic rat model when compared to the normal, and restored to the normal expression level after the EA treatment. These genes are involved in a number of biological processes, including the signal transduction, gene expression, and nociceptive pathways. Confirmation of the differential gene expression was performed by a dot-blot analysis. Dot-blotting results showed that the opioid receptor sigma was among those genes. This indicates that opioid-signaling events are involved in neuropathic pain and the analgesic effects of EA. The potential application of these data include the identification and characterization of signaling pathways that are involved in the EA treatment, studies on the role of the opioid receptor in neuropathic pain, and further exploration on the role of selected identified genes in animal models.

• 한국축산식품학회지
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• 제34권3호
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• pp.339-345
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• 2014

This study was performed to develop a rapid immuno-assay kit, by using a specific antigen to detect Hanwoo brand meat. We selected a synthetic antigen specific to our target antibody, named BIO-TAG (Tyr-D-Ala-Phe), by utilizing a computer-based analysis and literature review. BIO-TAG tagged with adjuvant was subcutaneously injected in sheep and Hanwoo. The serum and meat juice of the immunized or non-immunized animal were then analyzed, to measure the titer of antibody by ELISA and Western blot. The amount of antibodies against the BIO-TAG increased (p<0.05) in serum by vaccination. Furthermore, meat juice from the immunized Hanwoo showed greater (p<0.05) antibody titer, compared with those from non-immunized groups. To optimze the dilution factor, we performed dot-ELISA, with various combination levels of BIO-TAG. Results from dot-ELISA showed that 2 mg/mL BIO-TAG was sufficient to distinguish the immunized meat from non-immunized groups. These results support our hypothesis that simple immunization of Hanwoo generates a sufficient amount of antibodies to be detectable in the meat juice by means of the immune-assay. Therefore, specific Hanwoo brand meat can be more precisely identified by our rapid diagnostic kit. This technology can deter possible fraud of counterfeit meat brands in the Korean domestic market with ease and rapidity; and offers a new tool that guarantees consumers high quality Hanwoo brand beef.

• Journal of Plant Biology
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• 제37권1호
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• pp.77-83
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• 1994

종자를 자연건조시킨 상태에서 ${\beta}-glucuronidase$ (GUS) 유전자와 hygromycin phosphotransferase (HPT) 유전자를 가진 plasmid DNA 용액을 imbibition시켰다. GUS 유전자의 경우 품종, vector의 종류, imbibition의 온도에 따라 표지유전자의 발현율에 차이가 있었으며 약 30-50%의 transient expression을 나타내었다. Hygromycin B (HmB)배지에서 선별된 개체의 genomic DNA를 뽑아 외부유전자의 존재를 dot 분석을 통하여 확인하였다. Inverse polymerase chain reaction 결과 만들어지는 생성물을 cloning하고 sequencing한 결과 CaMV35S promoter sequence를 찾았다. Hygromycin이 첨가된 배지에서 선별된 개체들에서 GUS 유전자의 primer를 이용하여 PCR를 수행한 결과 20개체 중 18개체에서 GUS 유전자가 안정되게 존재하여 HmB 배지에서 GUS 유전자의 존재비율은 90%였다. 본 연구의 결과로부터 두 개의 유전자를 소유한 pYJH vector system이 고등식물의 형질전환에 유용하게 이용될 수 있음을 알 수 있었다.

• Asian-Australasian Journal of Animal Sciences
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• 제17권6호
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• pp.743-749
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• 2004

The gene encoding human serum albumin (HSA) was cloned from human liver cDNA library by PCR. The HSA cDNA in size of 2,176 bp, including 1,830 bp of open reading frame, was cloned into the plasmid carried with the 5'flanking sequence of goat $\beta$-casein gene (-4,044 to +2,025 bp) to get a tissue specific expression vector in mammary gland named pGB562/HSA (12.5 kb). A 9.6 kb DNA fragment in which the sequence is in order of goat $\beta$-casein gene regulatory sequence, HSA cDNA and SV40 polyadenylation signals was isolated from the pGB562/HSA by SacI and DraIII cutting, and used to microinject into the pronuclei of mouse fertilized eggs to produce transgenic mice. Three transgenic mice (2 female and 1 male) were identified by PCR and dot Southern blot analysis. The copy numbers of integrated transgene were more than 10 copies in line #21 and #26 as well as over 50 copies in line #31 of transgenic mice. HSA protein collected from the milk of lactating transgenic mice was confirmed by immuno-detection of Western and slot blot. The concentrations of HSA in the milk were from 0.05 to 0.4 mg/ml. An obvious antigen and antibody conjugate could be observed in immunohistochemical stain of mammary gland tissue from lactating day 11 of HSA transgenic mice. The transmission of transgene and its expression was recognized according to the results of RT-PCR and sequences analyses of their progeny.

• International Journal of Oral Biology
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• 제35권1호
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• pp.13-19
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• 2010

The DNA probes Pn17 and Pn34 were evaluated for their ability to specifically detect clinical strains of P. intermedia and P. nigrescens from a Korean population by dot blot hybridization. These probes were sequenced by extension termination and their specificity was determined by Southern blot analysis. The results revealed that the Pn17 sequence (2,517 bp) partially encodes an RNA polymerase beta subunit (rpoB) and that Pn34 (1,918 bp) partially encodes both rpoB (1-169 nts) and the RNA polymerase beta subunit (rpoB'; 695-1918 nts). These probes hybridized with both HindIII- and PstI-digested genomic DNAs from the strains of P. intermedia and P. nigrescens used in this study. Interestingly, each of the hybrid bands generated from the HindIII-digested genomic DNAs of the two bacterial species could be used to distinguish between them via restriction fragment length polymorphism. These results thus indicate that Pn17 and Pn34 can simultaneously detect P. intermedia and P. nigrescens.