• 미생물학회지
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• 제35권3호
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• pp.197-204
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• 1999

1996년 경기도, 강원도 충청북도, 전라남도, 전라북도 등 일부지역에서 채집된 들쥐들로부터 분리된 22주의 렙토스피라균의 단클론 항체에 대한 반응양상 및 genomic DNA 의 pulsed-field gel electrophoresis pattern을 분석하였다. 혈청군 Icterohemorrhagiae 내의 균주로 면역하여 제조한 7가지 단클론 항체에 대한 분리균주들의 반응은 모두 혈청형 lai 와 같은 pattern을 보였다. Not I 제한효소 절단 DNA 의 PFGE에서 JR34, JR57, JR77, JT82, JR86, JR109, NR4, NR6, NR13, CR3, KR48, NR2, NR8, NR9, NR10, NR11, NR12, JR58, 및 JR62 주 들은 모두 혈청형 lai 와 유사한 profile을 보였으며 940 kb와 63kb 사이에 13개의 절편 band를 보였다. JR89주는 혈청형 lai 및 다른 분리균주에서 관찰되지 않은 1000 kb band 와 460 kb band 가 관찰되었다.표준균주 혈청형 lai, birkini, gem, mwogolo, canicola 등은 각기 완전히 다른 pattern을 보였으며 혈청형 yeonchon 은 lai 와 같은 pattern을 보였다. UPGMA방법에 의한 dendrogram 분석결과 분리주는 lai 및 yeonchon 혈청형과 81~85%, 기타 혈청형과는 62% 이하의 유사도를 나타내어 항원분석법에 의한 혈청형 동정결과와 일치하였다. Asc I 제한요소 절단 DNA 의 PFGE에서는 JR34, JR77, JR82, JR109, NR6, NR13, CR3, KR48, 및 JR57, JR58, JR62, JR86, NR2, NR3, NR4, NR8, NR9, NR10, NR11, NR12 주들은 모두 1900 kb 에서부터 380kb 사이에 3개의 절편 band를 보였으며 이는 표준균주 lai 와 같은 pattern 이었다. JR89주는 다른 분리균주와는 달리 1640 kb 대신 650kb 의 band를 보였다. Fse I 제한효소 절단 PFGE 에서는 JR57, JR77, JR82, JR86, JR109, NR4, NR6, NR13, CR3, KR48주 및 JR34, NR2, NR3, NR9, NR10 주들은 모두 1900 kb 와 280kb 사이에 5개의 절편 band를 보였으며 이는 표준균주 lai 및 yeonchon 과 같은 pattern 이었다. 그러나 JR89주에서는 280kb 가 나타나지 않아 다른 분리균주와 구분되었다.

• Reproductive and Developmental Biology
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• 제31권2호
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• pp.83-90
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• 2007

Insulin-like growth factor II (IGF2) and H19 genes are mutually imprinted genes which may be responsible for abnormalities in the cloned fetuses and offspring. This study was performed to identify putative differentially methylated regions (DMRs) of porcine H19 locus and to explore its genomic imprinting in in vitro fertilized (IVF) and somatic cell nuclear transferred (SCNT) embryos. Based on mice genomic data, we identified DMRs on H19 and found porcine H19 DMRs that included three CTCF binding sites. Methylation patterns in IVF and SCNT embryos at the 2-, 4-, $8{\sim}16$-cells and blastocyst stages were analyzed by BS (Bisulfite Sequencing)-PCR. The CpGs in CTCF1 was significantly unmethylated in the 2-cell stage IVF embryos. However, the 4- (29.1%) and $8{\sim}16$-cell (68.2%) and blastocyst (48.2%) stages showed higher methylation levels (p<0.01). On the other hand, SCNT embryos were unmethylayted ($0{\sim}2%$) at all stages of development. The CpGs in CTCF2 showed almost unmethylation levels at the 2-,4- and $8{\sim}16$-cell and blastocyst stages of development in both IVF ($0{\sim}14.1%$) and SCNT ($0{\sim}6.4%$) embryos. At all stages of development, CTCF3 was unmethylated in IVF ($0{\sim}17.3%$) and SCNT ($0{\sim}1.2%$) embryos except at the blastocyst stage (54.5%) of IVF embryos. In conclusion, porcine SCNT embryos showed an aberrant methylation pattern comprised to IVF embryos. Therefore, we suggest that the aberrant methylation pattern of H19 loci may be a reason for increased abnormal fetus after embryo transfer of porcine SCNT embryos.

• 한국식물학회:학술대회논문집
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• 한국식물학회 1996년도 제10회 식물생명공학심포지움 고등식물 발생생물학의 최근 진보
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• pp.61-74
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• 1996

Human and monogastric animals can not synthesize 10 out of the 20 amino asids and therefor need to obtain these from their diet. The plant seed is a major source of dietary protein. It is particular important in their study to increase nutritional quality of the seed storage proteins. The low contents of lysine, asparagine and threonenein various cereal seeds and of cystein and methionine. In legume seeds is due to the low proportions of these amino acids in the major storage proteins, we have tried to apply the three strategies; (1) mutagenesis and selection of specific amino acid analogue resistance, (2) cloning and expression study of lysine biosynthesis related gene, (3) transfomation of lysine rich soybean glycinin gene. The 5-methyltryptophan (5MT) resistant cell lines, SAR1, SAR2 and SAR3 were selected from anther derived callus of rice (Oryza sativa L. "Sasanishiki"). Among these selected cell lines, two (SAR1 and SAR3) were able to grow stably at 200 mg/L of 5MT. Analysis of the freed amino acids in callus shows that 5MT resistant cells (SAR3) accumulated free tryptophan at least up to 50 times higher than those that of the higher than of SAS. These results indicated that the 5MT resistant cell lines are useful in studies of amino acid biosynthesis. Tr75, a rice (Oryza sativa L., var. Sasanishiki) mutant resistant to 5MT was segregated from the progenies of its initial mutant line, TR1. The 5MT resistant of TR75 was inherited in the M8 generations as a single dominant nuclear gene. The content of free amino acids in the TR75 homozygous seeds increased approximately 1.5 to 2.0 fold compared to wild-type seeds. Especially, the contents of tryptophan, phenylalanine and aspartic acid were 5.0, 5.3 and 2.7 times higher than those of wild-type seeds, respectively. The content of lysine is significantly low in rice. The lysine is synthesized by a complex pathway that is predominantly regulated by feedback inhibition of several enzymes including asparginase, aspatate kinase, dihydrodipicolinat synthase, etc. For understanding the regulation mechanism of lysine synthesis in rice, we try to clone the lysine biosynthetic metabolism related gene, DHPS and asparaginase, from rice. We have isolated a rice DHPS genomic clone which contains an ORF of 1044 nucleotides (347 amino acids, Mr. 38, 381 daltons), an intron of 587 nucleotides and 5'and 3'-flanking regions by screening of rice genomic DNA library. Deduced amino acid sequence of mature peptide domain of GDHPS clone is highly conserved in monocot and dicot plants whereas that of transit peptide domain is extremely different depending on plant specie. Southern blot analysis indicated that GDHPS is located two copy gene in rice genome. The transcripts of a rice GDHPS were expressed in leaves and roots but not detected in callus tissues. The transcription level of GDHPS is much higher in leaves indicating enormous chloroplast development than roots. Genomic DNA clones for asparaginase genes were screened from the rice genomic library by using plaque hybridization technique. Twelve different genomic clones were isolated from first and second screening, and 8 of 12 clones were analyzed by restriction patterns and identified by Southern Blotting, Restriction enzyme digestion patterns and Southern blot analysis of 8 clones show the different pattern for asparaginase gene. Genomic Southern blot analysis from rice were done. It is estimated that rice has at least 2-3 copy of asparaginase gene. One of 8 positive clones was subcloned into the pBluescript SK(+) vector, and was constructed the physical map. For transformation of lysine rich storage protein into tobacco, soybean glycinin genes are transformed into tobacco. To examine whether glycinin could be stably accumulated in endosperm tissue, the glycinin cDNA was transcriptionally fused to an endosperm-specific promotor of the rice storage protein glutelin gene and then introduced into tobacco genomic via Agrobacterium-mediated transformation. Consequently the glycinin gene was expressed in a seed-and developmentally-specific manner in transgenic tobacco seeds. Glycinin were targeted to vacuole-derived protein bodies in the endosperm tissue and highly accumulated in the matrix region of many transgenic plant (1-4% of total seed proteins). Synthesized glycinin was processed into mature form, and assembled into a hexamer in a similar manner as the glycinin in soybean seed. Modified glycinin, in which 4 contiguous methionine residues were inserted at the variable regions corresponding to the C - teminal regions of the acidic and basic polypeptides, were also found to be accumulated similarly as in the normal glycinin. There was no apparent difference in the expression level, processing and targeting to protein bodies, or accumulation level between normal and modified glycinin. glycinin.

• Molecules and Cells
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• 제27권6호
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• pp.635-640
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• 2009

Testis-derived germline stem (GS) cells can undergo reprogramming to acquire multipotency when cultured under appropriate culture conditions. These multipotent GS (mGS) cells have been known to differ from GS cells in their DNA methylation pattern. In this study, we examined the DNA methylation status of the H19 imprinting control region (ICR) in multipotent adult germline stem (maGS) cells to elucidate how epigenetic imprints are altered by culture conditions. DNA methylation was analyzed by bisulfite sequencing PCR of established maGS cells cultured in the presence of glial cell line-derived neurotrophic factor (GDNF) alone or both GDNF and leukemia inhibitory factor (LIF). The results showed that the H19 ICR in maGS cells of both groups was hypermethylated and had an androgenetic pattern similar to that of GS cells. In line with these data, the relative abundance of the Igf2 mRNA transcript was two-fold higher and that of H19 was three fold lower than in control embryonic stem cells. The androgenetic DNA methylation pattern of the H19 ICR was maintained even after 54 passages. Furthermore, differentiating maGS cells from retinoic acid-treated embryoid bodies maintained the androgenetic imprinting pattern of the H19 ICR. Taken together these data suggest that our maGS cells are epigenetically stable for the H19 gene during in vitro modifications. Further studies on the epigenetic regulation and chromatin structure of maGS cells are therefore necessary before their full potential can be utilized in regenerative medicine.

• Asian Pacific Journal of Cancer Prevention
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• 제15권24호
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• pp.10933-10935
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• 2015

Background: Many scientists have reported Candida species to be of great concern because of the high frequency that they colonize and infect human hosts, particularly cancer patients. Moreover, in the last decades Candida species have developed resistance to many antifungal agents. Based on this, we aimed to identify and determine the prevalence of Candida spp from blood culture bottles among cancer patients and their antifungal resistance pattern. Materials and Methods: From the blood culture bottles isolation and identification of the Candida spp were performed by conventional microbiological techniques. The in vitro antibiotic resistance pattern of the isolates was determined by CLSI guidelines. Genomic DNA was isolated and amplified. Each gene was separated by agar gel electrophoresis. Results: Identification of Candida spp was based on the presence of yeast cells in direct examination, culture and DNA extraction. Of the 68 blood samples collected during the study period (April 2013 to October 2013), five (7.35%) were positive for the presence of Candida spp, 2 (40%) of which were identified as Candida albicans and 3 (60%) were Candida non-albicans. Conclusions: High resistance to amphotricin B was observed among all the Candida non-albicans isolates. Regular investigations into antifungal resistance will help us to get an updated knowledge about their antibiotic resistance pattern which may help the physician in selecting the antibiotics for empirical therapy.

• 대한수의학회지
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• 제35권1호
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• pp.95-105
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• 1995

The human homolog of position specific element of mouse Hoxa-7 was studied using transgene. It contains a 1.1 kb human DNA (HCR)- a homolog to the intergenic region between Hoxa-7 and -9, which directs the position specific expression of Hoxa-7-, tk promoter, LacZ (${\beta}$-galactosidase) gene as a reporter, and polyadenylation signal of SV40 large T antigen. It was injected into the mice embryos, and the resulting transgenic embryos were analysed through PCR as well as genomic Southern blotting with placenta DNA. Out of 20 embryos analysed, two were transgenic. Among them, one transgenic embryo expressed transgene when stained with X-gal. The expression pattern was in analogy to that of the mouse Hoxa-7, showing spatially restricted expression pattern, Since the expression of ${\beta}$-galactosidase is regulated by the upstream human HCR sequence, it implies that the HCR is the plausible position specific regulatory element of human.

• 대한임상검사과학회지
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• 제39권3호
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• pp.183-189
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• 2007

Shigellosis is the most common bacterial gastroenteritis both in developing and developed countries, but bacteremia due to Shigella spp. is very rare. In developed countries recent shigellosis is mostly caused by S. sonnei, but S. flexeri infection is rare. We had rare cases of S. flexeri infections in a family in the Jeonbuk Province: an 8-year-old boy with bacteremic shigellosis and 10- and 12-year-old brothers with diarrhea. The isolates had identical biochemical characteristics, and were resistant to ampicillin, chloramphenicol, and co-trimoxazole. PFGE pattern of Not I-restricted genomic DNA suggested that the isolate from blood was closely related to the two strains isolated from stool which had an identical PFGE pattern.

• 대한의생명과학회지
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• 제11권2호
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• pp.121-128
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• 2005

A total of 30L monocytogenes strains from different sources including 13 strains isolated from the foreign imported meat were genotyped in order to establish their genetic relatedness and to compare them with the foreign isolates. PFGE analysis of genomic DNA showed the $11\~16$ fragments ranging in size from 38 to 504 kb. Eleven different PFGE types $(1\~11)$ were identified in the dendrogram at $75\%$ similarity, and the two major PFGE types, type 1 and 2, contained $94\%$ of domestic isolates (16/17). All isolates from domestic beef and pork carcass were grouped in each different type, however, isolates from chicken were clustered together with those from pork and beef. We also found all foreign strains were unrelated with each other, regardless of geographic criteria and that they could be differentiated from those from the domestic isolates by PFGE pattern. The PFGE pattern of one isolate from chicken wing, which the chicken meat was found to be imported from foreign country, was closely related to that of isolate from the Thailand.

• 한국미생물학회:학술대회논문집
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• 한국미생물학회 2002년도 추계학술대회
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• pp.15-21
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• 2002

Terminal-restriction fragment length polymorphism (T-RFLP) analysis has been optimized by using in vitro model community composed of genomic DNAs of known bacterial strains and has been applied to assess the bacterial community structure in marine sediments. The specific fluorescence-labeled terminal restriction fragments (T-RFs) between 39 and 839 base long specifying each strain were precisely measured for known bacterial strains. The addition of a co-solvent (dimethylsulfoxide or glycerol) into PCR reactions has reduced differential PCR amplification. Comparative bacterial community structure was investigated for pristine and polluted sediments. A complex T-RFLP pattern showing complex bacterial community structure was obtained in the pristine sediment, whereas simple T-RFLP pattern (low bacterial diversity) was shown in polluted sediments where caged aquaculture has been conducted for several years. The results of T-RFLP analysis were compared with that of cloning and sequencing 16S rDNA clones from the same sediments. Sequence analysis of 16S rDNA clones (72) of the pristine sediment revealed a diverse collection of lineages, largely of the class Proteobacteria ($6\%$ alpha subdivision, $46\%$ gamma subdivision, $13\%$ delta subdivision, and $3\%$ epsilon subdivision), Nitrospina $(8\%)$, high G+C gram positive $(8\%)$, Verrucomicrobia $(7\%)$, and Planctomycetes $(6\%)$. In the contaminated sediments, 17 $(59\%)$ of the 16S rDNA clones (29) were related to Campylobacter and symbiont of Rimicaris exoculata belonging to epsilon subdivision of Proteobacteria. The results obtained indicated that T-RFLP analysis is a rapid and precise technique for comparative bacterial community analysis.

• BMB Reports
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• 제51권1호
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• pp.1-2
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• 2018

Positive selection on a new beneficial mutation generates a characteristic pattern of DNA sequence polymorphism when it reaches an intermediate allele frequency. On genome sequences of African Drosophila melanogaster, we detected such signatures of selection at 37 candidate loci and identified "sweeping haplotypes (SHs)" that are increasing or have increased rapidly in frequency due to hitchhiking. Based on geographic distribution of SH frequencies, we could infer whether selective sweeps occurred starting from de novo beneficial mutants under simple constant selective pressure. Single SHs were identified at more than half of loci. However, at many other loci, we observed multiple independent SHs, implying soft selective sweeps due to a high beneficial mutation rate or parallel evolution across space. Interestingly, SH frequencies were intermediate across multiple populations at about a quarter of the loci despite relatively low migration rates inferred between African populations. This invokes a certain form of frequency-dependent selection such as heterozygote advantage. At one locus, we observed a complex pattern of multiple independent that was compatible with recurrent frequency-dependent positive selection on new variants. In conclusion, genomic patterns of positive selection are very diverse, with equal contributions of hard and soft sweeps and a surprisingly large proportion of frequency-dependent selection in D. melanogaster populations.