• Korean Journal of Food Science and Technology
• /
• v.31 no.1
• /
• pp.110-114
• /
• 1999

The present study was conducted to investigate the preparative methods of agarose for gel electrophoresis from agar. Naturally occuring agar consists of two main polysaccharides, the neutral polysaccharide agarose and the acid sulphated polysaccharide agaropectin. The sulphate and carboxyl functions of the agar are accumulated in the agaropectin. The hydrophilic, non-ionogenic, rigid and transparent gel matrix of the agarose was found to be suitable for gel electrophoresis gel filtration and affinity chromatography. Agar was purified by chitosan treatment, cetylpyridinium chloride (CPC) treatment, and polyethylene glycol (PEG) treatment. Yields of agarose purified from agar with chitosan, CPC and PEG were 56.7%, 55.6% and 62.3%. It was proper to treat with chitosan in preparative methods of agarose for gel electrophoresis from agar.

• Korean Journal of Ecology and Environment
• /
• v.54 no.3
• /
• pp.240-246
• /
• 2021

The Chironomidae is a benthic macroinvertebrate commonly found in freshwater ecosystems, along with Ephemeroptera and Trichoptera, which can be used for environmental health assessments. There are approximately 15,000 species of Chironomidae worldwide, but there are limited studies on species identification of domestic Chironomidae larvae. In the present study, we carried out species classification of the Chironomidae larvae that found in Jeju's tap water purification plants using morphological characteristics and genetic identification based on cytochrome c oxidase subunit I (COI) gene of the mitochondrial DNA. Body shape, mentum, antenna, mandible in the head capsule, and claws were observed in the larvae for morphological classification. Analysis of 17 larvae collected from faucets and fire hydrants of domestic tap water purification plants revealed the presence of two species, including 14 Orthocladius tamarutilus and 3 Paratrichocladius tammaater. These results will aid the use of the criteria information about species classification of the Chironomidae for water quality management in water purification plants and diversity monitoring of freshwater environments.

• Journal of Life Science
• /
• v.16 no.2 s.75
• /
• pp.240-244
• /
• 2006

We present a fast and simple protocol for purification of mitochondria, mitochondrial DNA, and RNA from small amounts of tomato leaves. This method uses a high ionic strength medium to isolate mitochondria and extract mitochondrial DNA and RNA from a single preparation and is easily adaptable to other plant species. Mitochondria was confirmed by MitoTracker. The mitochondrial DNA was not contaminated by plastid DNA, was successfully used for PCR. Similarly, the isolated mitochondrial RNA was not contaminated only slightly contaminated (leaves) by plastid RNA. RNA prepared according to our method was acceptable for RT-PCR analysis

• Korean Journal of Microbiology
• /
• v.30 no.6
• /
• pp.466-471
• /
• 1992

To determine the site at which -1 ribosomal frameshifting occurs within the gag-pro overlap of HTL V-I. DNA fragment corresponding to a portion of the gene overlap was cloned into a SP6 vector. The resultant plasmid harbors the hybrid gene consisting of a synthetic gene encoding 5 amino acids derived from chick prelysozyme including the initiator methionine plus 141 nucleotides of gag-pro overlapping region followed by Staphylococcus aurcus protein A gene fragment. In vitro transcription by SP6 RNA polymerase with this DNA template made an abundant amount of single species mRNA. Cell-free translation programmed with the RNA transcribed in vitro yielded a polypeptide of 21 kDal in size. which could be purified into homogeneity by IgG-Sepharose affinity chromatography. In vitro system described in this study must be useful for rapid purification and sequencing of the Gag-Pro transframe protein. allowing to determine the exact frameshift site on mRNA and to identify the tRNA involved in frameshifting event for the expression of pro gene.

• Journal of Microbiology and Biotechnology
• /
• v.14 no.6
• /
• pp.1280-1285
• /
• 2004

Retron is a prokaryotic genetic element, producing a short single-stranded DNA covalently linked to RNA (msDNA-RNA) by a reverse transcriptase (RT). In retron EC83, msDNA is further processed at between the 4th and the $5^{th}$ nucleotides, leaving a 79 nucleotide-long single-stranded DNA as a final product. To investigate this site-specific cleavage in msDNA synthesis, we purified the RT protein of retron EC83. Initially, RT ORF was cloned under the tac promoter, but the expression was very poor largely because of poor translation. In order to facilitate translation, the nucleotide sequence for the first nine amino acids was randomized with synonymous codons. This change of downstream sequence of translational initiation codon greatly affected the efficiency of translation. We could isolate clones which greatly increased RT production, and their sequences were compared to those of the low producers. The overproduced protein was purified and was shown to have RT activity.

• Animal cells and systems
• /
• v.13 no.3
• /
• pp.351-356
• /
• 2009

Ciliates are undoubtedly one of the most diverse protozoans that play a significant role in ecology. However, molecular examination, based on comparing the DNA sequences, has been done on a limited number of the species. Because most ciliates are uncultivable and their population sizes are often too small, it is usually difficult to obtain sufficient genomic DNA required for PCR based experiments. In the present study, we evaluated the effectiveness of four commercial DNA extraction procedures that extract high quality genomic DNA from a single ciliate cell. It was discovered that RED Extract-N-$Amp^{TM}$ PCR kit is the best method for removing PCR-inhibiting substances and minimizing DNA loss during purification. This method can also amplify more than 25 reactions of PCR. In addition, this technique was applied to single cells of 19 species belonged to 7 orders under 5 classes that isolated from mixed natural populations. Their small subunit ribosomal DNA (SSU rDNA) was successfully amplified. In summary, we developed a simple technique for the high-yield extraction of purified DNA from a single ciliate cell that may be more useful for rare ciliates, such as tiny and uncultivable marine microbes.

• BMB Reports
• /
• v.33 no.2
• /
• pp.155-161
• /
• 2000

The saccharomyces cerevisiae Rad27, a structure-specific endonuclease for the okazaski fragment maturation has been known to interact genetically and biochemically with Dna2, an essential enzyme for DNA replication. In an attempt to define the significance of the interaction between the two enzymes, we expressed and purified both Dna2 and Rad27 proteins. In this report, Rad27 could not form a complex with Dna2 in the three different analyses. The analyses included glycerol gradient sedimentation, protein-column chromatography, and coinfection of baculoviruses followed by affinity purification. This is in striking contrast to the previous results that used crude extracts. These results suggest that the interaction between the two proteins is not sufficiently stable or indirect, and thus requires an additional protein(s) in order for Rad27 and Dna2 to form a stable physical complex. This result is consistent with our genetic findings that Schizosaccharomyces pombe Dna2 is capable of interacting with several proteins that include two subunits of polymerase $\delta$, DNA ligase I, as well as Fen-1. In addition, we found that the N-terminal modification of Rad27 abolished its enzymatic activity. Thus, as suspected, we found that on the basis of the structure determination, N-terminal methionine indeed plays an important role in the nucleolytic cleavage reaction.

• Proceedings of the Korean Vacuum Society Conference
• /
• 2013.08a
• /
• pp.276-276
• /
• 2013

Here we report an integrated microdevice consisting of an efficient passive mixer, a magnetic separation chamber, and a capillary electrophoretic microchannel in which DNA barcode assay, target pathogen separation, and barcode DNA capillary electrophoretic analysis were performed sequentially within 30 min for multiplex pathogen detection at the single-cell level. The intestine-shaped serpentine 3D micromixer provides a high mixing rate to generate magnetic particle-pathogenic bacteria-DNA barcode labelled AuNP complexes quantitatively. After magnetic separation and purification of those complexes, the barcode DNA strands were released and analyzed by the microfluidic capillary electrophoresis within 5 min. The size of the barcode DNA strand was controlled depending on the target bacteria (Staphylococcus aureus, Escherichia coli O157:H7, and Salmonella typhimurium), and the different elution time of the barcode DNA peak in the electropherogram allows us to recognize the target pathogen with ease in the monoplex as well as in the multiplex analysis. In addition, the quantity of the DNA barcode strand (~104) per AuNP is enough to be observed in the laser-induced confocal fluorescence detector, thereby making single-cell analysis possible. This novel integrated microdevice enables us to perform rapid, sensitive, and multiplex pathogen detection with sample-in-answer-out capability to be applied for biosafety testing, environmental screening, and clinical trials.

• YAKHAK HOEJI
• /
• v.47 no.6
• /
• pp.410-416
• /
• 2003

Autotaxin(ATX) was originally purified from conditioned media of A2058 human melanoma cells and shown to be a potent cell motility-stimulating factor, possessing a type II nucleotide pyrophosphatase/phosphodiesterase (NPP2) activity. Recombinant ATX has recently demonstrated that human plasma lysophosholipase D is identical to ATX and uses lysophosphatidylcholine as a substrate to mediate various biological functions including tumor cell growth and motility through G-protein coupled receptor. However, despite pivotal roles of ATX on physiological or pathophysiological states, the production of ATX is solely depends on complicated purification method which employs multiple column steps, but resulted in very poor yield. This limited the use of ATX for extensive analysis. We, therefore, expressed six histidine-tagged recombinant human ATX(His-ATX) in High Five TM insect cells to improve the generation of ATX and to make simple the purification of ATX. The signal sequence of the human ATX gene was truncated and replaced with sequence of insect cell secretion signal within expression vector. In addition, codons for six histidines were added to the C-termini of 120kDa ATX cDNA construct. A simple purification scheme utilizing two-step affinity column chromatography was designed to purify His-ATX to homogeneity from the culture supernatant of transfected insect cells. Homogenous His-ATX was detected and isolated from the concentrated insect cell medium using concanavalin A agarose and nickel affinity chromatography. Purified His-ATX was in full length with ATX capacity. A combination of this expression system and purification scheme would be useful for production and purification of high-quality functional ATX for research and practical application of multiple functional motogen, ATX/NPP-2.

• Asian Pacific Journal of Cancer Prevention
• /
• v.17 no.7
• /
• pp.3061-3063
• /
• 2016

Exploiting the immune system to abolish cancer growth via vaccination is a promising strategy but that is limited by many clinical issues. For DNA vaccines, viral vectors as a delivery system mediate a strong immune response due to their protein structure, which could afflect the cellular uptake of the genetic vector or even induce cytotoxic immune responses against transfected cells. Recently, synthetic DNA delivery systems have been developed and recommended as much easier and simple approaches for DNA delivery compared with viral vectors. These are based on the attraction of the positively charged cationic transfection reagents to negatively charged DNA molecules, which augments the cellular DNA uptake. In fact, there are three major cellular barriers which hinder successful DNA delivery systems: low uptake across the plasma membrane; inadequate release of DNA molecules with limited stability; and lack of nuclear targeting. Recently, a polysaccharide polymer produced by microalgae has been synthesized in a form of polymeric fiber material poly-N-acetyl glucosamine (p-GlcNAc). Due its unique properties, the F2 gel matrix was suggested as an effective delivery system for immune and gene vaccinations.