• BMB Reports
• /
• 제29권2호
• /
• pp.133-136
• /
• 1996

The affinity cleavage reagent Methidiumpropyl-EDTA-Iron(II) is applied to the structural analysis of 5S rRNA. Analysis of cleavage sites induced by MPE-Fe(II) on 5S rRNA shows that MPE intercalates easily between the unstable base pairs or into the bulges, thereby it strongly cuts the nucleosides nearby. The stable helical stems A, B, D and E as well as loop d are weakly cut. Most of the single-stranded loops are not cleaved. Based on the cleavage pattern of the 5S rRNA by MPE-Fe(II) and RNase V1, we suggest that MPE-Fe(II) may be used as a potential chemical probe in searching for the unstable helical regions of RNA, and for the sequences that appear to be involved in folding and distorting 5S rRNA.

• BMB Reports
• /
• 제30권1호
• /
• pp.66-72
• /
• 1997

A mammalian DNA repair enzyme, UV endonuclease III which also functions as a ribosomal protein S3 (rpS3), was purified from mouse cells and characterized. UV endonuclease III was previously cloned and known to yield a peptide of 32 kDa upon expression in E. coli [Kim et al., (1995) J. Bioi. Chem. 270, 13620-13629]. However, biochemically purified UV endonuclease III, which has a sedimentation coefficient of 3.25, appears to have an additional peptide of 28 kDa. It appears that two bands were derived from one complex, judging from the comparison of the nuclease activity on the native and SDS-gel electrophoreses. UV endonuclease III becomes non-specific upon purification and this phenomenon is more significant in the case of pure fractions of the enzyme. Non-specific activity was not influenced by pH or any salt conditions.

• 한국동물생명공학회지
• /
• 제34권2호
• /
• pp.65-69
• /
• 2019

Since the development of the first genetically-modified mouse, transgenic animals have been utilized for a wide range of industrial applications as well as basic research. To date, these transgenic animals have been used in functional genomics studies, disease models, and therapeutic protein production. Recent advances in genome modification techniques such zinc finger nuclease (ZFN), transcription activator-like effector nucleases (TALEN), and clustered regularly interspaced short palindromic repeats (CRIPSR)-Cas9, have led to rapid advancement in the generation of genome-tailored livestock, as well as experimental animals; however, the development of genome-edited poultry has shown considerably slower progress compared to that seen in mammals. Here, we will focus primarily on the technical strategies for production of transgenic and gene-edited chickens, and their potential for future applications.

• 한국미생물·생명공학회지
• /
• 제25권5호
• /
• pp.512-519
• /
• 1997

RNA accumulating strain of Torulopsis versatilis MT-1 was cultured in molasses medium for higher contents of RNA in cell. Yeast cells were harvested at logarithmic phase on synchronous culture. Yield of cells on dry base to input sugar was 59.5%. Crude protein content was 55.1% in cell. RNA content was 13.9%. Some problems found in the process for the preparation of yeast extracts were improved by the addition of culture broth of Streptomyces faecalis MSF which secrete RNase (5' nuclease and 5' adenylic acid deaminase). When the culture broth of S. faecalis MSF was added in autolysis process 46% of RNA in cell was converted to I and G(5' inosinic acid and 5' guanylic acid) in extract. By addition of 3-7% culture broth of S.faecalis MSF in autolysis or enzymolysis process at the start or early stage, RNA in extract was converted easily to I and G and protein in cells was easily extracted and hydrolyzed to amino acid. Taste of those yeast extracts was delicious. The yeasty smell in yeast extracts was removed. And cell debris was easily removed from extract.

• 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.

• BMB Reports
• /
• 제53권7호
• /
• pp.341-348
• /
• 2020

The targeted nuclease clustered, regularly interspaced short palindromic repeats/CRISPR-associated proteins (CRISPR/Cas) system has recently emerged as a prominent gene manipulation method. Because of its ease in programming targeted DNA/protein binding through RNA in a vast range of organisms, this prokaryotic defense system is a versatile tool with many applications in the research field as well as high potential in agricultural and clinical improvements. This review will present a brief history that led to its discovery and adaptation. We also present some of its restrictions, and modifications that have been performed to overcome such restrictions, focusing specifically on the most common CRISPR/Cas9 mediated non-homologous end joint repair.

• Asian Pacific Journal of Cancer Prevention
• /
• 제17권7호
• /
• pp.3329-3334
• /
• 2016

Cancer is thought to be a direct result of transcriptional misregulation. Broad analysis of transcriptional regulatory elements in healthy and cancer cells is needed to understand cancer development. Nucleases regulatory domains are recruited to bind and manipulate a specific genomic locus with high efficacy and specificity. TALENs (transcription activator-like effector nuclease) fused to endonuclease FokI have been used widely to target specific sequences to edit several genes in healthy and cancer cells. This approach is promising to target specific cancer genes and for this purpose it is needed to pack such TALENs into viral vectors. There are some considerations which control the success of this approach, targeting appropriate sequences with efficient construction of TALENs being crucial factors. We face some obstacles in construction of TALENs; in this study we made a modification to the method of Cermk et al 2011 and added one step to make it easier and increase the availability of constructs.

• Molecules and Cells
• /
• 제37권8호
• /
• pp.569-574
• /
• 2014

As a scaffold, SLX4/FANCP interacts with multiple proteins involved in genome integrity. Although not having recognizable catalytic domains, SLX4 participates in diverse genome maintenance pathways by delivering nucleases where they are needed, and promoting their cooperative execution to prevent genomic instabilities. Physiological importance of SLX4 is emphasized by the identification of causative mutations of SLX4 genes in patients diagnosed with Fanconi anemia (FA), a rare recessive genetic disorder characterized by genomic instability and predisposition to cancers. Recent progress in understanding functional roles of SLX4 has greatly expanded our knowledge in the repair of DNA interstrand crosslinks (ICLs), Holliday junction (HJ) resolution, telomere homeostasis and regulation of DNA damage response induced by replication stress. Here, these diverse functions of SLX4 are reviewed in detail.

• Journal of Applied Biological Chemistry
• /
• 제49권3호
• /
• pp.86-89
• /
• 2006

The novel tetranuclear nickel (II) complex is a high rate accelerator in promoting hydrolysis of phosphate diesters. Nickel-bound bis-nitrophenyl phosphate (BNPP) can be $10^4$ times more reactive than the unbound BNPP. The large rate of enhancements by the complex slightly under basic condition has shown high catalytic activity in phosphate diester cleavage. The bell-shaped pH-rate profile indicated that the nickel-oxide form of the tetranuclear complex or its kinetic equivalent was the active species for cleaving BNPP. The catalytic hydrolysis between tetranuclear nickel (II) complex and phosphate diester proceeds via the formation of bidentate coordination of the anionic phosphate to the Ni (II) atom. This reveals that the complex has the possibility as artificial nuclease.

• 미생물학회지
• /
• 제22권3호
• /
• pp.183-189
• /
• 1984

In order to find specific acting site of Rec A protein in plasmic polymerization in E. coli, we randomly deleted various part of pEC-3 (a derivative of pBR322) with SI nuclease treatment. Self-ligated plasmids were introduced into E. coli WA802(Rec $A^+$). A number of colonies were analyzed if they contained monomeric or polymeric plasmids by gel electrophoresis. The plasmid (pEC-43), which was deleted the region of tetracycline gene, revealed only monomeric form in Rec $A^+$ E. coli. When two plasmids, pEC-3 and pEC-43, were co-transformed in the same E. coli, the original pEC-3 showed polymerization but pEC-43 revealed monomeric form only. These results suggest that Rec A protein requires the specific site for polymerization.