• BMB Reports
• /
• v.34 no.1
• /
• pp.59-65
• /
• 2001

In subtractive hybridization, target sequences in the tester are enriched by hybridizing with an excess amount of driver, followed by removing the tester hybridized with the driver. All of existing subtractive cloning methods are designed to remove the tester/driver hybrid. The removal of hybrid, however, is often unsatisfactory For various reasons. In this study we developed a subtractive enrichment protocol in which the tester/driver can be completely removed by selecting only the tester/tester after hybridization. In this protocol both the tester and driver DNAs are ligated with same linker DNAs and amplified by polymerase chain reaction (PCR). The tester DNA is then digested with two different enzymes and used in subsequent hybridization with an excess driver. After hybridization, the DNA is ligated with the adaptor that is only compatible with the tester/tester. Since only the tester/tester can have the new adaptor, no tester/driver can be amplified by PCR in this protocol. Unlike other methods, a 100% subtraction efficiency can be achieved even though the enzymatic treatments used in the enrichment procedure are incomplete. Furthermore, only the hybridized tester DNA can have the new adaptor and be amplified by PCR, resulting in 100% denaturation in effect. The efficacy of this novel method was verified with the model system in which a known amount of the target sequence is included.

• Bulletin of the Korean Chemical Society
• /
• v.31 no.12
• /
• pp.3623-3631
• /
• 2010

We prepared several novel molecular transporters built on myo- and scyllo-inositol scaffolds with variations in the number of guanidine residues, linker chain lengths and patterns. Some of these transporters were found to localize in mitochondria, and the mitochondrial affinity seems to be substantially related to the scaffold stereochemistry.

• Korean Journal of Microbiology
• /
• v.31 no.4
• /
• pp.279-285
• /
• 1993

Alterations of the p53 gene arc among the most frequent genetic changes in human cancer and often result in increased levels of p53 protein within the malignant cells. Detection of accumulated p53 protein can be a useful prognostic tool in human cancer. In order to make polyclonal antibodies for immunohistochemical screening. human p53 gene was expressed in E. coli in the form of GST (glutathione S-transfi.:rase) fusion proteins. Two p53 gene fragments. which were N('()I small fragment encoding amino acid residues of 1-151-: and Ncol large fragment of 159-393. were subeloned into the unique BamHI site present within the pGEX-2T vector using BamHI linker and recombinant plasmids pGTNS and pGTNL were constructed. respectively. The p53 cDNA fragment (from pC53-$SN_3$,) encoding amino acid 38-145 (proline at residue 72) was amplified by polymerase chain reaction(PCR). The amplified DNA was digested with BamHI and Prull and inserted into the BamHI-Smal sites of pG EX-2T and recombinant plasmid pGTBP was constructed. After IPTG induction of these plasmids for 4 hours. fusion proteins were purified from E. coli extracts with glutathione Sepharose beads. The bound proteins were resolved by 10% SDS-polyacrylamide gel electrophoresis and the molecular weights were 54 kDa. 53 kDa and 40 kDa. respectively. Approximately one milligram of fusion proteins were purified from 1 -liter cultures.

• Applied Chemistry for Engineering
• /
• v.26 no.4
• /
• pp.445-451
• /
• 2015

Semi-flexible liquid crystalline polymers containing a mesogenic group and an octamethylene flexible spacer in the main chain were synthesized by solution polycondensation. The mesogenic group in the polymer consists of four aromatic rings connected by ester and ketone, ether, sulfide, methylene, sulfone, or isopropylidene linkage groups. This paper discusses effects of the central linker of the mesogenic group on polymer properties. The structures and properties of synthesized polymers were investigated by $^1H$-NMR, FT-IR, differential scanning calorimeter (DSC), thermogravimetric analyzer (TGA), X-ray diffractometer (XRD), and polarizing optical microscope (POM). Polymers having bent linkage groups exhibited low thermal transition temperatures, narrow mesophase temperature ranges, low liquid crystallinity, and good solubilities in organic solvents, while those having bulky linkage groups were amorphous and exhibited high glass transition temperatures.

• Korean Journal of Environmental Biology
• /
• v.29 no.4
• /
• pp.379-387
• /
• 2011

Copepod nauplii plays an important role as a linker between the microbial food web and classical food chain in marine ecosystem and is an essential food source for early stage of many larval fishes. Study on the influencing factors on the growth of copepod naupliar stages has been rarely carried out in despite of these ecological significances. Many studies have shown that food availability and temperature are major factors to influence copepod growth. However, due to the complicated environment parameters in coastal ecosystem, the relationships between growth of copepods and influencing factors are still unclear under the natural condition. Growth rates of the copepod $Acartia$ $hongi$ nauplii were measured in Kyeonggi Bay from February to December 2001. $Acartia$ $hongi$ is numerically abundant and widespread predominant species in the coastal regions of the Yellow Sea and occurs continuously throughout the year, with a maximum peak in late spring. The naupliar growth rates of $Acartia$ $hongi$ by the artificial cohort method varied from 0.03 to 0.18 $day^{-1}$, with a mean of 0.09 $day^{-1}$. The overall naupliar growth rates showed a significantly positive relationship with the variation in water temperature. However, Previous study reported that the growth rates of adult $Acartia$ $hongi$ were primarily influenced by the variation in chlorophyll-$a$. Therefore, these differences demonstrated that the influencing factors of growth did not correspond with the developmental stages. The results of this study suggest that the dissimilarity of growth between nauplius and adult female resulted from the size-dependant difference in food availability and the growth of older developmental stages containing adults are more food-dependent than juveniles.

• IMMUNE NETWORK
• /
• v.3 no.2
• /
• pp.126-135
• /
• 2003

Background: One of the important factors in the prognosis of chronic hepatitis B patient is the degree of replication of hepatitis B virus (HBV). It has been known that HBV DNA polymerase plays the essential role in the replication of HBV. HBV DNA polymerase is composed of four domains, TP (Terminal protein), spacer, RT (Reverse transcriptase) and RNaseH. Among these domains, tyrosine, the $65^{th}$ residue of TP is an important residue in protein-priming reaction that initiates reverse transcription. If monoclonal antibody that recognizes around tyrosine residue were selected, it could be applied to further study of HBV replication. Methods: To produce TP-specific scFv (single-chain Fv) by phage display, mice were immunized using synthetic TP-peptide contains $57{\sim}80^{th}$ amino acid residues of TP domain. After isolation of mRNA of heavy-variable region ($V_H$) and light-chain variable region ($V_L$) from the spleen of the immunized mouse, DNA of $V_H$ and $V_L$ were obtained by RT-PCR and joined by a DNA linker encoding peptide (Gly4Ser)3 as a scFv DNA fragments. ScFv DNA fragments were cloned into a phagemid vector. scFv was expressed in E.coli TG1 as a fusion protein with E tag and phage gIII. To select the scFv that has specific affinity to TP-peptide from the phage-antibody library, we used two cycles of panning and colony lift assay. Results: The TP-peptide-specific scFv was isolated by selection process using TP-peptide as an antigen. Selected scFv had 30 kDa of protein size and its nucleotide sequences were analyzed. Indirect- and competitive-ELISA revealed that the selected scFv specifically recognized both TP-peptide and the HBV DNA polymerase. Conclusion: The scFv that recognizes the TP domain of the HBV DNA polymerase was isolated by phage display.

• Journal of the Korea Academia-Industrial cooperation Society
• /
• v.17 no.2
• /
• pp.528-535
• /
• 2016

Two types of oleophilic acrylic prepolymers were prepared by the solution copolymerization of either ethyl acrylate (EA) or lauryl acrylate (LA) with hydroxy ethyl acrylate (HEA). For the formation of oil-absorbent materials, a mixed solution of the prepolymer and hexamethylene diisocyanate (HDI) as a cross-linker in toluene was applied to polypropylene knit velvet fabric through the conventional pad-dry-cure procedure. The gel fraction of the crosslinked resin, EA-HEA-HDI, increased with increasing feed ratio of HEA to total acrylate or HDI concentration. The oil absorbancy and retention ratio of the prepared materials were compared according to the add-on ratio of resin to fabric, and were assessed with n-decane, toluene, soybean oil, lubricant and bunker C oil as test oils. The optimal oil absorbancy of the materials were observed at around 6% of the add-on ratio for all these oils except for soybean oil. On the other hand, the oil retention ratio increased as the add-on ratio increased. Futhermore, heavier and more viscous oil generally showed higher oil retention ratios. In addition, the oil absorbancy of the materials treated with LA-HEA-HDI resin was higher than that treated with EA-HEA-HDI resin, which showed that the acrylic resins are more absorptive with increasing length of their side alkyl chain.

• Applied Chemistry for Engineering
• /
• v.18 no.1
• /
• pp.10-16
• /
• 2007

In this study, we examined the preparation and hydrolysis property of immobilized urease membrane to decompose harmful urea in the body and remove ammonia which was produced by its decomposition. Urease immobilized membrane was prepared by introducing anion-exchange group DEA into porous hollow-fiber membrane by radiation graft polymerization method, and immobilization of urease. When urease was immobilized at membrane introduced with anion-exchange group, the more increasing grafting rate, the more increasing immobilization amount. The result originates from the fact that a greater amount of protein was immobilized by forming a multilayer on the longer grafted chain. Meanwhile, the addition of the cross-linker was possible not only to suppress separation phenomenon produced during a washing process of immobilized urease membrane but also to enable the recycling of membrane. Urease Immobilized membrane with no separation phenomenon was prepared by cross-linking reaction for 5 h, and the hydrolysis rate of prepared urease immobilized membrane was over 98% and 50%, respectively, in 1 mol and 4 mol urea solutions.