• Genomics & Informatics
• /
• 제1권1호
• /
• pp.55-60
• /
• 2003

The nucleotide sequence of pZMO1, a small cryptic plasmid of Zymomonas mobilis ATCC10988 was determined. Analysis of 1,680 bp of sequence revealed $69\%$ identity with Shigella sonnei plasmid, pKYM and $61\%$ identity with Nostoc sp. ss DNA replicating plasmid. Analysis of a deduced amino acid sequence of an orf of pZMO1 revealed $75\%$ identity and $90\%$ similarity with the repA gene of Synechocystis sp. plasmid pCA2.4. The upstream region of the repA gene of pZMO1 possesses six directed repeat sequences and two inverted repeat sequences at downstream of the IR consensus sequence of nick region of rolling circle replication (RCR) plasmid. A typical terminator hairpin structure was found at the downstream region of repA gene. Degradation of single-stranded plasmid DNA by S1 nuclease was detected by Southern hybridization. It suggests that pZMO1 replicates by a rolling circle mechanism in Z. mobilis ATCC10988 cells.

• 정보처리학회논문지B
• /
• 제14B권6호
• /
• pp.461-472
• /
• 2007

게놈 프로젝트 연구는 DNA사슬 내에서 생물학적 의미(예, molecule의 진화역사 또는 그 기능)를 추출하기위한 사슬분석 쪽으로 강조가 되어가고 있다. 특히, DNA사슬 내에서 상보적 또는 반복되는 패턴은 생물학적 의미를 가지고 있다. 문제는 상보적 단어가 만들어내는 흥미 있는 패턴과 단어 구성을 찾아 내는 것이다. 본 논문은 다양한 길이의 팰린드롬 쌍을 검색하는 알고리즘에 관한 연구이다. 다양한 길이의 팰린드롬 쌍 내에는 빈 공백을 또한 허용한다. 알고리즘은 팰린드롬 알고리즘이라고 명명하며 O(N)의 계산 시간을 가진다. 하나의 팰린드롬 쌍은 머리핀 형태로 구성되어 있다. 검출된 여러 팰린드롬 쌍을 활용하여 n-쌍 팰린드롬 형태를 구성하였다. 더욱이 발견된 가장 긴 팰린드롬 쌍을 DNA 사슬 원형 구조에 점으로 표현하여 가시성을 제고하였다. 본 알고리즘은 여러 게놈 상에서 실시되었으며 E.coli K12의 결과를 나타내었다. 실험결과 DNA 안에는 랜덤한 경우에는 확률상 매우 발생하기 힘든 긴 팰린드롬 패턴들이 존재 한다는 것을 발견할 수 있었다.

• Bulletin of the Korean Chemical Society
• /
• 제32권9호
• /
• pp.3223-3228
• /
• 2011

To quantify the presence of mercuric ions in aqueous solution, double-stranded DNA (dsDNA) of poly(dT) was employed using a light switch compound, $Ru(phen)_2(dppz)^{2+}$ (1) which is reported to intercalate into dsDNA of a right-handed B-form. Addition of mercuric ions induced the dehybridization of poly(dT)${\cdot}$poly(dA) duplexes to form a hairpin structure of poly(dT) at room temperature and the metal-to-ligand charge transfer emission derived from the intercalation of 1 was reduced due to the dehybridization of dsDNA. As the concentration of $Hg^{2+}$ was increased, the emission of 1 progressively decreased. This label-free emission method had a detection limit of 0.2 nM. Other metal ions, such as $K^+$, $Ag^+$, $Ca^{2+}$, $Mg^{2+}$, $Zn^{2+}$, $Mn^{2+}$, $Co^{2+}$, $Ni^{2+}$, $Cu^{2+}$, $Cd^{2+}$, $Cr^{3+}$, $Fe^{3+}$, had no significant effect on reducing emission. This emission method can differentiate matched and mismatched poly(dT) sequences based on the emission intensity of dsDNA.

• Plant Biotechnology Reports
• /
• 제4권3호
• /
• pp.201-211
• /
• 2010

We present a highly effective T-DNA inserted gene screening method as part of a reverse genetics model system using the Chinese cabbage (Brassica rapa L. spp. pekinensis). Three-step two-dimensional (2D) matrix strategies are potentially accurate and useful for the identification of specific T-DNA inserted mutants from a large population. To construct our Chinese cabbage model, we utilized a forward genetics screening approach for the abnormal phenotypes that were obtained from transgenic plants of Brassica rapa generated with Agrobacteria tumefaciens containing the pRCV2 vector. From one transgenic plant with an abnormal phenotype, we observed that the st1 gene (which is related to senescence-associated process proteins) contained a T-DNA fragment, and that its expression level was decreased. This T-DNA insert was then used as a control to construct an effective screening pool. As a result, the optimum template concentration was found to be 0.1-1 ng in our PCR strategy. For other conditions, positive changes to the Gibbs free energy prevented the formation of oligo dimers and hairpin loop structures, and autosegment extension gave better results for long fragment amplification. Using this effective reverse genetics screening method, only 23 PCR reactions were necessary to select a target gene from a pool of 100 individual DNAs. Finally, we also confirmed that the sequence we obtained from the above method was identical to the flanking sequence isolated by rescue cloning.

• Journal of Microbiology and Biotechnology
• /
• 제8권6호
• /
• pp.650-655
• /
• 1998

The chromosomal DNA fragment from Phaffia rhodozyma CBS 6938 which is able to autonomously replicate in the yeast Saccharomyces cerevisiae was cloned on an integrative URA3 plasmid. Its minimal fragment exhibiting autonomously replicating activiy in the S. cerevisiae gave a higher frequency transformation efficiency than that found for centromere-based plasmid, and enabled extrachromosoma1ly stable transmission of the plasmids in one copy per yeast cell under non-selective culture condition. The 836-bp DNA element lacked an ORF and did not contain any acceptable match to an ARS core consensus. Sequence analysis, however, displayed a cluster of three hairpin-Ioop-sequences with individual $\triangle {G_{25}}^{\circ}C$ free energy value of -10.0, -17.5, and -17.0 kcal. $mor^{-l}$as well as a 9-bp sequence with two base pair mismatches to the S. cerevisiae/E. coli gyrase-binding site. This 836-bp sequence also included one 7-bp sequence analogous to the core consensus of centromeric DNA element III (CDEIII) of S. cerevisiae, but CDEIII-like 7 bp sequence alone did not give a replicative function in this yeast.

• BMB Reports
• /
• 제46권2호
• /
• pp.92-96
• /
• 2013

The breast cancer susceptibility gene BRCA1 encodes a nuclear protein, which functions as a tumor suppressor and is involved in gene transcription and DNA repair processes. Many families with inherited breast and ovarian cancers have mutations in the BRCA1 gene. However, only a few studies have reported on the mechanism underlying the regulation of BRCA1 expression in humans. In this study, we investigated the transcriptional regulation of BRCA1 in HeLa cells treated with etoposide. We found that three Egr-1-binding sequences (EBSs) were located at -1031, -1005, and -385 within the enhancer region of the BRCA1 gene. Forced expression of Egr-1 stimulated the BRCA1 promoter activity. EMSA data showed that Egr-1 bound directly to the EBS within the BRCA1 gene. Knockdown of Egr-1 through the expression of a small hairpin RNA (shRNA) attenuated etoposide-induced BRCA1 promoter activity. We conclude that Egr-1 targets the BRCA1 gene in HeLa cells exposed to etoposide.

• Molecules and Cells
• /
• 제45권1호
• /
• pp.26-32
• /
• 2022

Living cells generate, sense, and respond to mechanical forces through their interaction with neighboring cells or extracellular matrix, thereby regulating diverse cellular processes such as growth, motility, differentiation, and immune responses. Dysregulation of mechanosensitive signaling pathways is found associated with the development and progression of various diseases such as cancer. Yet, little is known about the mechanisms behind mechano-regulation, largely due to the limited availability of tools to study it at the molecular level. The recent development of molecular tension probes allows measurement of cellular forces exerted by single ligand-receptor interaction, which has helped in revealing the hitherto unknown mechanistic details of various mechanosensitive processes in living cells. Here, we provide an introductory overview of two methods based on molecular tension probes, tension gauge tether (TGT), and molecular tension fluorescence microscopy (MTFM). TGT utilizes the irreversible rupture of double-stranded DNA tether upon application of force in the piconewton (pN) range, whereas MTFM utilizes the reversible extension of molecular springs such as polymer or single-stranded DNA hairpin under applied pN forces. Specifically, the underlying principle of how molecular tension probes measure cell-generated mechanical forces and their applications to mechanosensitive biological processes are described.

• Molecules and Cells
• /
• 제44권3호
• /
• pp.146-159
• /
• 2021

DNA methylation, and consequent down-regulation, of tumour suppressor genes occurs in response to epigenetic stimuli during cancer development. Similarly, human oncoviruses, including human papillomavirus (HPV), up-regulate and augment DNA methyltransferase (DNMT) and histone deacetylase (HDAC) activities, thereby decreasing tumour suppressor genes (TSGs) expression. Ubiquitin-like containing PHD and RING finger domain 1 (UHRF1), an epigenetic regulator of DNA methylation, is overexpressed in HPV-induced cervical cancers. Here, we investigated the role of UHRF1 in cervical cancer by knocking down its expression in HeLa cells using lentiviral-encoded short hairpin (sh)RNA and performing cDNA microarrays. We detected significantly elevated expression of thioredoxin-interacting protein (TXNIP), a known TSG, in UHRF1-knockdown cells, and this gene is hypermethylated in cervical cancer tissue and cell lines, as indicated by whole-genome methylation analysis. Up-regulation of UHRF1 and decreased TXNIP were further detected in cervical cancer by western blot and immunohistochemistry and confirmed by Oncomine database analysis. Using chromatin immunoprecipitation, we identified the inverted CCAAT domain-containing UHRF1-binding site in the TXNIP promoter and demonstrated UHRF1 knockdown decreases UHRF1 promoter binding and enhances TXNIP expression through demethylation of this region. TXNIP promoter CpG methylation was further confirmed in cervical cancer tissue by pyrosequencing and methylation-specific polymerase chain reaction. Critically, down-regulation of UHRF1 by siRNA or UHRF1 antagonist (thymoquinone) induces cell cycle arrest and apoptosis, and ubiquitin-specific protease 7 (USP7), which stabilises and promotes UHRF1 function, is increased by HPV viral protein E6/E7 overexpression. These results indicate HPV might induce carcinogenesis through UHRF1-mediated TXNIP promoter methylation, thus suggesting a possible link between CpG methylation and cervical cancer.

• BMB Reports
• /
• 제44권9호
• /
• pp.595-600
• /
• 2011

Pax 6, a member of the paired box (Pax) family, has been implicated in oncogenesis. However, its therapeutic potential has been never examined in breast cancer. To explore the role of Pax6 in breast cancer development, a lentivirus based short hairpin RNA (shRNA) delivery system was used to knockdown Pax6 expression in estrogen receptor (ER)-positive (MCF-7) and ER-negative (MDA-MB-231) breast cancer cells. Effect of Pax6 silencing on breast cancer cell proliferation and tumorigenesis was analyzed. Pax6-RNAi-lentivirus infection remarkably downregulated the expression levels of Pax6 mRNA and protein in MCF-7 and MDA-MB-231 cells. Accordingly, the cell viability, DNA synthesis, and colony formation were strongly suppressed, and the tumorigenesis in xenograft nude mice was significantly inhibited. Moreover, tumor cells were arrested at G0/G1 phase after Pax6 was knocked down. Pax6 facilitates important regulatory roles in breast cancer cell proliferation and tumor progression, and could serve as a diagnostic marker for clinical investigation.

• BMB Reports
• /
• 제40권4호
• /
• pp.577-587
• /
• 2007

Single-copy chloroplast loci are used widely to infer phylogenetic relationship at different taxonomic levels among various groups of plants. To test the utility of chloroplast loci and to provide additional data applicable to hybrid evolution in Musa, we sequenced two introns, rpl16 and ndhA, and two intergenic spacers, psaA-ycf3 and petA-psbJ-psbL-psbF and combined these data. Using these four regions, Musa acuminata Cola(A)- and M. balbisiana Colla (B)-containing genomes were clearly distinguished. Some triploid interspecific hybrids contain A-type chloroplasts (the AAB/ABB) while others contain B-type chloroplasts (the BBA/BBB). The chloroplasts of all cultivars in 'Namwa' (BBA) group came from the same wild maternal origin, but the specific parents are still unrevealed. Though, average sequence divergences in each region were little (less than 2%), we propose that petA-psbJ intergenic spacer could be developed for diversity assessment within each genome. This segment contains three single nucleotide polymorphisms (SNPs) and two indels which could distinguish diversity within A genome whereas this same region also contains one SNP and an indel which could categorize B genome. However, an inverted repeat region which could form hairpin structure was detected in this spacer and thus was omitted from the analyses due to their incongruence to other regions. Until thoroughly identified in other members of Musaceae and Zingiberales clade, utility of this inverted repeat as phylogenetic marker in these taxa are cautioned.