• Journal of Veterinary Clinics
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• v.12 no.1
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• pp.905-910
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• 1995

GC 함유량(72%)이 높은 Mycobacterium paratuberculosis의 DNA의 PCR 증폭시 특이성과 생산성을 높이기 위하여 PCR 반응액에 denaturant인 DMSO, glycerol, formamide, Tween 20과 NP 40를 첨가하였다. Denaturant를 첨가하지 않은 상태의 PCR에서는 다수의 비특이적인 DNA가 관찰되었으며 표적 DNA 생산량이 낮았다. 모든 denaturant는 PCR의 특이성과 생산물의 생산량을 증가시했으며, 이들 중 DMSO, glycerol, farmamide와 NP 40는 높은 농도에서 생산량을 증가시켰다. Tween 20은 낮은 농도에서 생산량을 증가시켰다. Denaturant를 첨가하였음에도 불구하고 대부분의 반응에서 1 또는 2개의 비특이적인 DNA가 관찰되었다.

• Korean Journal of Biological Psychiatry
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• v.2 no.2
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• pp.248-251
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• 1995

정신분열병의 약물치료에서 중요한 역할을 하는 도파민의 기전은 지금까지 5종류의 수용체들이 발견되고 클론되면서, 새롭고 보다 근본적인 유전학적 접근을 통해 규명될 수 있게 되었다. 특히, 도파민 수용체 D4 (DRD4)는 막단백질의 세포질쪽 세번째굴곡에 48-bp반복 다형성배열을 가지고있다. 이러한 다형성 반복배열이 신호전달에 참여할 가능성이 높은 막단백질의 세포질쪽 굴곡에 있다는것은, 각 개인의 항정신병약물에 대한 민감성의 차이를 포함하여 정신분열병에 대한 개개인의 유전적 차이를 진단할 수 있는 가능성을 제시한다. 이러한 가능성을 검증하기 위하여 주로 손쉽고 빠른 중합효소연쇄반응(PCR)을 사용하여 DRD4의 아형들을 분류하게 되는데, DRD4의 PCR은 그 반복배열과 그 주변배열의 높은 GC함량(78% G+C) 때문에 일반적인 PCR 방법을 변형시켜 사용해야한다. DRD4의 아형을 분류하기 위해 변형된 PCR은 통상적으로 7-deaza dGTP와 10% DMSO를 사용하게된다. 이러한 DRD4 PCR은 대부분의 경우 성공하지만, 항상 모든 시료에서 PCR이 성공되는것은 아니었으며 반복적으로 시도하여 증폭시킬 수 있었다. 이러한 어려움은 대부분이 template DNA에 문제가 있을것으로 의심되며 DNA정제 또는 template DNA를 제한효소로 적절하게 무작위절단하여 성공율을 높일 수 있었다.

• The Plant Pathology Journal
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• v.18 no.4
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• pp.187-191
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• 2002

This paper describes the cloning and sequence analysis of the 5'-terminal region and full-length cDNA production of genomic RNA of Lily symptomless virus (LSV), a Species Of the genus Carlavirus. A sing1e DNA band about 600 bp harboring the 5'-end of genomic RNA of the virus was successfully amplified by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE), and was cloned for nucleotide sequence determination. Sequence analysis of selected RACE cDNA clones revealed that the LSV 5'non-translated region consists of 67 nucleotides long of AT rich stretch followed GC rich from the 5'-end. To produce full-length cDNA products for the viral genomic RNA, a set of LSV-specific primers could be designed based on the obtained sequence in this study and the known sequences of 3'-terminal region for the virus. Full-length cDNA copies of LSV, an 8.4 kb long, were directly amplified by the long-template RT-PCR technique from the purified viral genomic RNA samples. This full-length cDNA copies were analyzed by restriction mapping. The molecules produced in this study can be useful for the production of in vitro infectious cDNA clone, as well as, for the completion of genomic RNA sequence and genome structure for the virus.

• Korean Journal of Microbiology
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• v.40 no.2
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• pp.65-72
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• 2004

Instability of some eukaryotic sequence propagated in prokaryotic hosts is a frequently observed phenomenon. It is well documented that long inverted repeats, AT-rich sequences with structures like Z-DNA are extremely unstable in E. coli. These sequences may either be under-represented or even lost when cloned in E. coli. When we analyzed the polymorphic pattern for several tandom repeat (TR) in human SCKI gene, we found some TR regions were frequently deleted from plasmids and had difficult problem for their sequencing. These regions may result in non-clonability of the DNA sequence. Here we have cloned two difficult TR regions under low temperature and made two library for DNA sequencing using a nebulizer or sonicator. This study will help to determine the unstable genomic elements in complex mammalian genome.

• Archives of Pharmacal Research
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• v.24 no.5
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• pp.455-465
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• 2001

The highly potent cytotoxic DNA-DNA cross-linker consists of two cyclopropa[c]pyrrolo[3,4-3]indol-4(5H)-ones insoles [(+)-CPI-I] joined by a bisamido pyrrole (abbreviated to "Pyrrole"). The Pyrrole is a synthetic analog of Bizelesin, which is currently in phase II clinical trials due to its excellent in vivo antitumor activity. The Pyrrole has 10 times more potent cytotoxicity than Bizelesin and mostly form DNA-DNA interstrand cross-links through the N3 of adenines spaced 7 bp apart. The Pyrrole requires a centrally positioned GC base pair for high cross-linking reactivity (i.e., $5^1$-T$AT_2$A*-$3^1$), while Bizelesin prefers purely AT-rich sequences (i.e., $5^1$-T$AT_4$A*-$3^1$, where /(equation omitted) represents the cross-strand adenine alkylation and A* represents an adenine alkylation) (Park et al., 1996). In this study, the high-field $^1$H-NMR and rMD studies are conducted on the 1 1-mer DNA duplex adduct of the Pyrrole where the 5′(equation omitted)TAGTTA*-3′sequence is cross-linked by the drug. A severe structural perturbation is observed in the intervening sequences of cross-linking site, while a normal B-DNA structure is maintained in the region next to the drug-modified adenines. Based upon these observations, we propose that the interplay between the bisamido pyrrole unit of the drug and central C/C base pair (hydrogen-bonding interactions) is involved in the process of cross-linking reaction, and sequence specificity is the outcome of those interactions. This study suggests a mechanism for the sequence specific cross-linking reaction of the Pyrrole, and provides a further insight to develop new DNA sequence selective and distortive cross-linking agents.

• Parasites, Hosts and Diseases
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• v.55 no.1
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• pp.39-45
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• 2017

The present study was performed to reveal the morphological characteristics and molecular phylogenetic position of Platynosomum fastosum Kossack, 1910. A total 167 specimens of P. fastosum were collected in 8 (4.9%) out of 163 sets of gall-bladders and bile ducts of cats. The number of worms was 1-105 per infected cat. This species was characterized by having a long and slender body, slightly larger ventral sucker than the oral sucker, indistinct prepharynx, small pharynx, short esophagus, bifurcation midway between 2 suckers, and ceca extending to the posterior end of the body. The length of the partial sequences of ITS1 and 5.8S rDNA of P. fastosum were 990 bp, GC-rich. AT/GC ratio was 0.9, there were 9 polymorphic sites, and intraspecific variations ranged from 0.1% to 0.9%. Phylogenetic analyses by neighbor-joining phylogram inferred from ITS1 rDNA sequences revealed that the genetic distance between P. fastosum specimens ranged from 0.3 to 1.5% while the smallest interspecific distance among dicrocoeliid species was 20.9 %. The redescription and genetic characters of P. fastosum are taxonomically important to recognize future different species of the genus Platynosomum showing high intraspecific and morphological variability.

• Genomics & Informatics
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• v.20 no.1
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• pp.12.1-12.7
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• 2022

The two complete mitochondrial genomes (mitogenomes) of Paedocypris progenetica, the smallest fish in the world which belonged to the Cyprinidae family, were sequenced and assembled. The circular DNA molecules of mitogenomes P1-P. progenetica and S3-P. progenetica were 16,827 and 16,616 bp in length, respectively, and encoded 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes, and one control region. The gene arrangements of P. progenetica were identical to those of other Paedocypris species. BLAST and phylogenetic analyses revealed variations in the mitogenome sequences of two Paedocypris species from Perak and Selangor. The circular DNA molecule of P. progenetica yield a standard vertebrate gene arrangement and an overall nucleotide composition of A 33.0%, T 27.2%, C 23.5%, and G 15.5%. The overall AT content of this species was consistent with that of other species in other genera. The negative GC-skew and positive AT-skew of the control region in P. progenetica indicated rich genetic variability and AT nucleotide bias, respectively. The results of this study provide genomic variation information and enhance the understanding of the mitogenome of P. progenetica. They could later deliver highly valuable new insight into data for phylogenetic analysis and population genetics.

• Biomedical Science Letters
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• v.14 no.1
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• pp.47-53
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• 2008

Hox genes are known to be transcription factors controlling vertebrate pattern formation along the anteroposterior body axis by regulating many target gene expressions during vertebrate embryogenesis. In order to isolate in vivo Hox responsive target genes, ChIP-cloning technique has been applied using Hoxc8 antibody. Here murine embryo of day 11.5 post coitum (E11.5) highly expressing Hoxc8 gene was used after removing head and tail portions where Hoxc8 is rarely expressing. After fixation with formaldehyde, the chromatin DNAs harboring bound proteins were isolated. After sonication, about 0.5- to 1 Kb chromatin DNAs were immunoprecipitated with anti Hoxc8 antibody. After removing the bound proteins with proteinase K, DNAs were isolated, cloned into the pBluescsript II SK vector, and then sequenced. Total 33 random clones sequenced were anlalyzed to be located at 12 different genomic regions. Among these, 8 turned out to be introns and 4 were intergenic regions localized in random chromosomes. The base composition of total cloned genomic sequences (6608 bp) were AT-rich, i.e., 40% GC. When the Hoxc8 core binding sites, such as TAAT, ATTA, TTAT, and ATAA were analyzed total number of 55, 45, 54, and 55 were found, respectively, which are than twice as many as expected number of 26. Although this in silico analysis does not mean that the ChIP-cloned sequence is real Hoxc8 regulatory element in vivo, these results strongly imply that the DNA fragments cloned through chromatin immunoprecipitation could be very much likely the putative Hoxc8 downstream target genes.

• Korean Journal of Microbiology
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• v.49 no.1
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• pp.58-63
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• 2013

A taxonomic study was carried out to assess the phylogenetic characteristics of isolate JJM57 from marine red algae Laurencia sp. collected from intertidal zone in Jeju Island, South Korea. Comparative analysis of 16S rRNA gene sequence shows that this isolate belongs to the genus Oceanisphaera. It shows 98.02% and 97.7% sequence similarity with Oceanisphera litoralis DSM $15406^T$ and Oceanisphera donghaensis KCTC $12522^T$, respectively. Strain JJM57 is a Gram-negative, aerobic, moderately halophilic bacterium able to grow in different NaCl concentration ranges from 0.5 to 8.0% and at varying temperatures from 4 to $37^{\circ}C$. Sharing some of the physiological and biochemical properties with O. litoralis and O. donghaensis, JJM57 strain differs in the utilization of ethanol, proline, and alanine. The G+C contents of the strain JJM57 is 61.94 mol% and it is rich in $C_{16:1}$ ${\omega}7c$ and/or iso-$C_{15:0}$ 2-OH, $C_{16:0}$, and $C_{18:1}$ ${\omega}7c$ fatty acids. The DNA-DNA relatedness data separates the strain JJM57 from other species such as O. litoralis and O. donghaensis. On the basis of these polyphasic evidences, present study proposed that strain JJM57 (=KCTC 22371 =AM983543 =CCUG 60764) represents a novel bacterial species of Oceanisphaera.