• Journal of Microbiology and Biotechnology
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• 제29권1호
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• pp.127-140
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• 2019

Since 1990, many nucleic acid expression platforms consisting of DNA or RNA have been developed. However, although RNA expression platforms have been relatively neglected, several such platforms capped at the 5' end of RNA by an anti-reverse cap analog have now been developed. At the same time, the capping reaction is a bottleneck in the production of such platforms, with high cost and low efficiency. Here, we investigated several viral and eukaryotic internal ribosome entry sites (IRESs) to develop an optimal RNA expression platform, because IRES-dependent translation does not require a capping step. RNA expression platforms constructed with IRESs from the 5' untranslated regions of the encephalomyocarditis virus (EMCV) and the intergenic region of the cricket paralysis virus (CrPV) showed sufficient expression efficiency compared with cap-dependent RNA expression platforms. However, eukaryotic IRESs exhibited a lower viral IRES expression efficiency. Interestingly, the addition of a poly(A) sequence to the 5' end of the coxsackievirus B3 (CVB3) IRES (pMA-CVB3) increased the expression level compared with the CVB3 IRES without poly(A) (pCVB3). Therefore, we developed two multiexpression platforms (termed pMA-CVB3-EMCV and pCrPV-EMCV) by combining the IRESs of CVB3, CrPV, and EMCV in a single-RNA backbone. The pMA-CVB3-EMCV-derived RNA platform showed the highest expression level. Moreover, it clearly exhibited expression in mouse muscles in vivo. These RNA expression platforms prepared using viral IRESs will be useful in developing potential RNA-based prophylactic or therapeutic vaccines, because they have better expression efficiency and do not need a capping step.

• Animal cells and systems
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• 제2권1호
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• pp.113-116
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• 1998

A cDNA clone coding for ribosomal protein 46 (rp46) which is a component of 60S ribosomal large subunit has been identified from Drosophila melanogaster. A cDNA clone encoding S. cerevisiae rp46 was used as a probe to screen a Drosophila larvae cDNA library. The DNA sequence analysis revealed that the cDNA coding for Drosophils rp46 contains a complete reading frame of 153 nucleotides coding for 51 amino acids. The deduced amino acid sequence showed 71-75% homology with those of other eukaryotic organisms. Northern blot analysis showed that about 1-kb rp46 transcripts are abundant throughout fly development. Whole mount embryonic mRNA in situ hybridization also showed no preferential distribution of the transcripts to any specific region. The chromosomal in situ hybridization revealed that the identified gene is localized at position 60C on the right arm of the second polytene chromosome with a possibility of single copy.

• 대한의생명과학회지
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• 제18권3호
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• pp.313-318
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• 2012

Acanthamoeba is an opportunistic pathogen known to cause granulomatous amoebic encephalitis and amebic keratitis. Acanthamoeba exhibits life cycle consisting of trophozoite and cyst, and the cyst is highly resistant to variable antibiotics and therapeutic agents. To understand the encystation mechanism of Acanthamoeba, the expression profiles of trophozoite and cyst were compared by gene ontology (GO) analysis. Ribosomal proteins and cytoskeletal proteins were highly expressed in trophozoite. In cyst, various protease, and signal transduction - and protein turnover - related proteins were highly expressed. These results correlated with eukaryotic orthologous groups (KOG) assignment and microarray analysis of Acanthamoeba trophozoite and cyst ESTs. The information of differential expression profiles of trophozoite and cyst would provide important clues for research on encystation mechanism of cyst forming protozoa including Acanthamoeba.

• Asian-Australasian Journal of Animal Sciences
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• 제27권4호
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• pp.574-579
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• 2014

A lysozyme gene from breast of Tibetan sheep was successfully expressed by secretion using a-factor signal sequence in the methylotrophic yeast, Pichia pastoris GS115. An expression yield and specific activity greater than 500 mg/L and 4,000 U/mg was obtained. Results at optimal pH and temperature showed recombinant lysozyme has higher lytic activity at pH 6.5 and $45^{\circ}C$. This study demonstrates the successful expression of recombinant lysozyme using the eukaryotic host organism P. pastoris paving the way for protein engineering. Additionally, this study shows the feasibility of subsequent industrial manufacture of the enzyme with this expression system together with a high purity scheme for easy high-yield purification.

• Molecules and Cells
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• 제40권4호
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• pp.243-253
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• 2017

Eukaryotic cilia are organelles that project from the surface of cells to fulfill motility and sensory functions. In vertebrates, the functions of both motile and immotile cilia are critical for embryonic development and adult tissue homeostasis. Importantly, a multitude of human diseases is caused by abnormal cilia biogenesis and functions which rely on the compartmentalization of the cilium and the maintenance of its protein composition. The transition zone (TZ) is a specialized ciliary domain present at the base of the cilium and is part of a gate that controls protein entry and exit from this organelle. The relevance of the TZ is highlighted by the fact that several of its components are coded by ciliopathy genes. Here we review recent developments in the study of TZ proteomes, the mapping of individual components to the TZ structure and the establishment of the TZ as a lipid gate.

• Applied Microscopy
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• 제47권3호
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• pp.165-170
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• 2017

Gas vesicles are intracellular gas-filled protein-shelled nanocompartments. The structures are spindle or cylinder-shaped, and typically $0.1{\sim}2{\mu}m$ in length and 45~250 nm in width. A variety of prokaryotes including photosynthetic bacteria and halophilic archaea form gas vesicles in their cytoplasm. Gas vesicles provide cell buoyancy as flotation devices in aqueous habitats. They are used as nanoscale molecular reporters for ultrasound imaging for biomedical purposes. The structures in halophilic archaea are poorly resolved due to the low signal-to-noise ratio from the high salt concentration in the medium. Such a limitation can be overcome using focused ion beam-thinning or inelastically scattered electrons. As the concentric bodies (~200 nm in diameter) in fungi possess gas-filled cores, it is possible that the concept of gas vesicles could be applied to eukaryotic microbes beyond prokaryotes.

• Molecules and Cells
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• 제38권6호
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• pp.475-481
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• 2015

Programmable nucleases, which include zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and RNA-guided engineered nucleases (RGENs) repurposed from the type II clustered, regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system are now widely used for genome editing in higher eukaryotic cells and whole organisms, revolutionising almost every discipline in biological research, medicine, and biotechnology. All of these nucleases, however, induce off-target mutations at sites homologous in sequence with on-target sites, limiting their utility in many applications including gene or cell therapy. In this review, we compare methods for detecting nuclease off-target mutations. We also review methods for profiling genome-wide off-target effects and discuss how to reduce or avoid off-target mutations.

• BMB Reports
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• 제31권3호
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• pp.303-306
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• 1998

Serine proteinase cDNA fragment from protozoan parasite Acanthamoeba culbertsoni was amplified by the reverse transcription-polymerase chain reaction (RTPCR) using degenerate oligonucleotide primers derived from conserved serine proteinase sequences. The amplified DNA fragment was subcloned and sequenced. The sequence analysis and alignment showed significant sequence similarity to other eukaryotic serine proteinases and conservation of the His, Asp, and Ser residues that form the catalytic triad. The cDNA fragment was cloned into the pGEMEX-1 expression vector and expressed in Escherichia coli. A resulting fusion protein of 56 kDa had proteolytic activity. The fusion protein reacted with sera of mice immunized with purified serine proteinase of A. culbertsoni in Western blot. Immune recognition of the fusion protein by mouse antisera suggested that the fusion protein may be valuable as a diagnostic reagent.

• BMB Reports
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• 제42권12호
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• pp.823-828
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• 2009

Techniques for analyzing protein-DNA interactions on a genome-wide scale have recently established regulatory roles for distal enhancers. However, the large sizes of higher eukaryotic genomes have made identification of these elements difficult. Information regarding sequence conservation, exon annotation and repetitive regions can be used to reduce the size of the search region. However, previously developed resources are inadequate for consolidating such information. CONVIRT is a web resource for the identification of transcription factor binding sites and also features comparative genomics. Genomic information on ortholog-independent conserved regions, exons, repeats and sequences is integrated into the virtual chromosome, and statistically over-represented single or combinations of transcription factor binding sites are sought. CONVIRT provides regulatory network analysis for several organisms with long promoter regions and permits inter-species genome alignments. CONVIRT is freely available at http://biosoft.kaist.ac.kr/convirt.

• 한국해양바이오학회지
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• 제1권1호
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• pp.34-39
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• 2006

Molecular biology and microalgal biotechnology have the potential to play a major role in improving the production efficiency of a vast variety of products including functional foods, industrial chemicals, compounds with therapeutic applications and bioremediation solutions from a virtually untapped source. Microalgae are a source of natural products and have been recently studied for biotechnological applications. Efficient genetic transformation systems in microalgae are necessary to enhance their potential to be used for human health. A microalga such as Chlarella is a eukaryotic organism sharing its metabolic pathways with higher plants. This microalga is capable of expressing, glycosylating, and correctly processing proteins which normally undergo post-translational modification. Moreover, it can be cultured inexpensively because it requires only limited amount of sunlight and carbon dioxide as energy sources. Because of these advantages, Chlarella may be of great potential interest in biotechnology as a good candidate for bioreactor in the production of pharmaceutical and industrial compounds for human functional foods. Here, we briefly discuss recent progress in microalgal transgenesis that has utilized molecular biology to produce functional proteins and bioactive compounds.