• Journal of Animal Reproduction and Biotechnology
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• v.34 no.2
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• pp.111-116
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• 2019

This study was conducted to analyze the specific genes associated with sex-determination in Korean native cow. The highly organized spermatogenesis requires accurate spatial and temporal regulation of gene expression, which is governed by transcriptional, post-transcriptional, and epigenetic processes. Recently, farmers have been interested in determining the sexual identity of the calves in their farm. We analyzed the sperm of Korean native and Holstein cows, which were supplied from Hanwoo Improvement Center. We evaluated sperm motility and expression of sperm-specific genes after treating semen with both male- and female reagents. Sperm motility in Korean native cows decreased by approximately 10% in the first 30 minutes after treatment with sex-determination reagent. However, sperm motility of Holstein cows decreased to 60-70% after 15 minutes and to 20-30% after 30 minutes. We selected six specific genes expressing in the spermatozoa to analysis the gene expression level. The Real-time PCR results suggest that the selected genes (Gimap4, Tmeff1, Rac2, Abi2, Rac1, and Clu) were highly expressed in the group treated with the male reagent compared to the group treated the female reagent and to the untreated-group (control). In the present study, we suggest that the selected genes play a pivotal role in sex-determination.

• Genomics & Informatics
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• v.9 no.1
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• pp.39-43
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• 2011

MicroRNAs (miRNAs) are recently discovered small RNA molecules usually resulting in translational repression and gene silencing. Despite the fact that specific cloning of small RNA's is a method in practice, computational identification of miRNA's has been a major focus recent days, since is a rapid process following AB initio and sequence alignment methods. Here we developed new software called MiRPI that aims to identify the highly conserved miRNAs without any mismatches from given fasta formatted gene sequences by using non-repeated miRNA dataset of the user's interest. The new window embedded with the software is used to identify the targets for inputted mature miRNAs in the mRNA sequences. Also MiRPI is designed to measure the precursor miRNA statistics, majorly focusing the Adjusted Minimum Folding free Energy (AMFE) and Minimum Folding free Energy Index (MFEI), the most important parameters in miRNA confirmation. MiRPI is developed by PERL (Practical Extraction and Report Language) and Tk (Tool kit widgets) scripting languages. It is user friendly, portable offline software that works in all windows OS, sized to 3 MB.

• Asian Pacific Journal of Cancer Prevention
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• v.17 no.7
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• pp.3437-3445
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• 2016

Background: There is an increasing concern in the role of microRNA (miRNA) in the pathogenesis of bone metastasis (BM) secondary to prostate cancer (CaP). In this exploratory study, we hypothesized that the expression of vinculin (VCL) and chemokine X3C ligand 1 (CX3CL1) might be down-regulated in clinical samples, most likely due to the post-transcriptional modification by microRNAs. Targeted genes would be up-regulated upon transfection of the bone metastatic prostate cancer cell line, PC3, with specific microRNA inhibitors. Materials and Methods: MicroRNA software predicted that miR-21 targets VCL while miR-29a targets CX3CL1. Twenty benign prostatic hyperplasia (BPH) and 16 high grade CaP formalin-fixed paraffin embedded (FFPE) specimens were analysed. From the bone scan results, high grade CaP samples were further classified into CaP with no BM and CaP with BM. Transient transfection with respective microRNA inhibitors was done in both RWPE-1 (normal) and PC3 cell lines. QPCR was performed in all FFPE samples and transfected cell lines to measure VCL and CX3CL1 levels. Results: QPCR confirmed that VCL messenger RNA (mRNA) was significantly down-regulated while CX3CL1 was up-regulated in all FFPE specimens. Transient transfection with microRNA inhibitors in PC3 cells followed by qPCR of the targeted genes showed that VCL mRNA was significantly upregulated while CX3CL1 mRNA was significantly down-regulated compared to the RWPE-1 case. Conclusions: The down-regulation of VCL in FFPE specimens is most likely regulated by miR-21 based on the in vitro evidence but the exact mechanism of how miR-21 can regulate VCL is unclear. Up-regulated in CaP, CX3CL1 was found not regulated by miR-29a. More microRNA screening is required to understand the regulation of this chemokine in CaP with bone metastasis. Understanding miRNA-mRNA interactions may provide additional knowledge for individualized study of cancers.

• Journal of Periodontal and Implant Science
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• v.35 no.1
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• pp.21-30
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• 2005

Matrix metalloproteinases (MMPs) are a family of host-derived proteolytic enzymes and implicated in the remodeling and degradation of extracellular matrix under both physiological and pathological conditions. Connective tissue degradation in periodontal diseases is thought to be due to excessive MMP activities over their specific inhibitors. The effects of lipopolysaccharide (LPS) from Prevotella intermedia, one of the major putative pathogens of periodontitis, on the expression of mRNA for MMPs and tissue inhibitors of metalloproteinases (TIMPs) in human gingival and periodontal ligament fibroblasts were examined by reverse transcriptase-polymerase chain reaction (RT-PCR). The expression of mRNAs encoding MMP-1, -2, -3, -10, and -14 was increased in human gingival fibroblasts treated with p. intermedia LPS, whereas MMP-11 and TIMP-2 mRNA expression was decreased in these cells stimulated with LPS. P. intermedia LPS increased the MMP-1, -2, -10, -11, and -14 mRNA expression and decreased TIMP-1 and -2 mRNA expression in human periodontal ligament fibroblasts. These findings imply that P. intermedia LPS may play an important role in the connective tissue degradation in periodontitis.

• BMB Reports
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• v.38 no.4
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• pp.500-506
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• 2005

HOX11 encodes a homeodomain-containing transcription factor which directs the development of the spleen during embryogenesis. While HOX11 expression is normally silenced through an unknown mechanism in all tissues by adulthood, the deregulation of HOX11 expression is associated with leukemia, such as T-cell acute lymphoblastic leukemia. The elucidation of regulatory elements contributing to the molecular mechanism underlying the regulation of HOX11 gene expression is of great importance. Previous reports of HOX11 regulatory elements mainly focused on the 5'-flanking region of HOX11 on the chromosome related to transcriptional control. To expand the search of putative cis-elements involved in HOX11 regulation at the post-transcriptional level, we analyzed HOX11 mRNA 3'-untranslated region (3'UTR) and found an AU-rich region. To characterize this AU-rich region, in vitro analysis of HOX11 mRNA 3'UTR was performed with human RNA-binding protein HuR, which interacts with AU-rich element (ARE) existing in the 3'UTR of many growth factors' and cytokines' mRNAs. Our results showed that the HOX11 mRNA 3'UTR can specifically bind with human HuR protein in vitro. This specific binding could be competed effectively by typical ARE containing RNA. After the deletion of the AU-rich region present in the HOX11 mRNA 3'UTR, the interaction of HOX11 mRNA 3'UTR with HuR protein was abolished. These findings suggest that HOX11 mRNA 3'UTR contains cis-acting element which shares similarity in the action pattern with RE-HuR interactions and may involve in the post-transcriptional regulation of the HOX11 gene.

• Journal of dental hygiene science
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• v.13 no.3
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• pp.296-303
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• 2013

Lysyl oxidase (LOX), extracellular matrix enzyme, is catalyzing lysine-derived crosslinks in collagen and elastin. Recently, several LOX-like proteins (LOXL, LOXL2, LOXL3 and LOXL4) have been identified in human but their specific functions are still largely unknown. The purpose of this study was to evaluate the function of the LOX family genes during odontoblastic differentiation of human dental pulp (HDP) cells induced with odontogenic supplement (OS). The messenger RNA (mRNA) expression of LOX family genes and differentiation markers was assessed by reverse transcriptase polymerase chain reaction analysis (RT-PCR). The formation of mineralization nodules was evaluated by alrizarin red S staining. Amine oxidase activity of HDP cells was measured by peroxidase-coupled fluormetric assay. The expressions of differentiation markers, such as alkaline phosphatase (ALP), osteopontin (OPN), osteocalcin (OCN), dentin matrix protein1 (DMP1), dentin sialophosphoprotein (DSPP) in HDP cells were increased after treatment with OS media. The LOX and LOXL mRNA expression were gradually increased in OS media, whereas LOX enzyme activities were markedly detected on day 7. The mRNA expression and LOX enzyme activity of collagen type I was very similar to the pattern of LOX gene. In this study, the expression of LOX and its isoforms, and activity of LOX were highly regulated during odontoblastic differentiation. Thus, these results suggest that LOX plays a key role in odontoblastic differentiation of HDP cells.

• Journal of Periodontal and Implant Science
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• v.35 no.3
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• pp.623-634
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• 2005

Human periodontal ligament fibroblast(hPDLF) is very important to cure periodontal tissue because it can be diverged into various cells. This study examined the expression of MMP-1, TIMP-1, periodontal ligament specific PDLs22, Type I collagen, Fibronectin, TIMP-2, telomerase mRNA in a replicative senescence of hPDLF. The periodontal ligament tissue was obtained from periodontally healthy and non-carious human teeth extracted for orthodontic reasons at the Chosun University Hospital of Dentistry with the donors' informed consent. The hPDLF cells were cultured in a medium containing Dulbecco's modified Eagle medium(DMEM, Gibco BRL, USA) supplemented with 10% fetal bovine serum(FBS, Gibco BRL, USA) at 37C in humidified air with 5% $CO_2$. For the reverse transcription-polymerase chain reaction(RT-PCR) analysis, the total RNA of the 2, 4, 8, 16, 18, and 21 passage cells was extracted using a Trizol Reagent(Invitrogen, USA) in replicative hPDL cells. Two passage cells, i.e. young cells, served as the control, and ${\beta}-actin$ served as the internal control for RT-PCR The results of this study about cell morphology and gene expression according to aging of hPDLF using RT-PCR method are as follows: 1. The size of hPDLF was increased with aging and it was showed that the hPDLF was dying in the final passage. 2. PDLs22 mRNA was expressed in young hPDLF of the two, four, and six passage. 3. TIMP-1 mRNA was expressed in young hPDLF of the two and four passage. 4. There was a tendency that MMP-1 mRNA was weakly expressed over eighteen. 5. Type 1 collagen mRNA was expressed in almost all passages, but it was not expressed in the final passage. 6. Fibronectin mRNA was observed in all passages and it was weakly expressed in the final passage. 7. TIMP-2 and telomerase mRNA were not expressed in this study. Based on above results, it was observed that PDLs22, Type 1 collagen, Fibronectin, MMP-1. and TIMP-1 mRNA in hPDLF were expressed differently with aging. The study using the hPDLF that is collected from healthy patients and periodontitis patients needs in further study.

• Korean Journal of Microbiology
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• v.54 no.4
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• pp.420-427
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• 2018

The orthogonal pair of Saccharomyces cerevisiae tyrosyl-tRNA synthetase (Sc YRS) and a variant of E. coli initiator tRNA, fMam $tRNA_{CUA}$ which recognizes the amber stop codon is an effective tool for site-specific incorporation of unnatural amino acids into the protein in E. coli. To evolve the amber suppression activity of the orthogonal pair, we generated a mutant library of Sc YRS by randomizing two amino acids at 320 and 321 which involve recognition of the first base of anticodon in fMam $tRNA_{CUA}$. Two positive clones are selected from the library screening with chloramphenicol resistance mediated by amber suppression. They showed growth resistance against high concentration of chloramphenicol and their $IC_{50}$ values were approximately 1.7~2.3 fold higher than the wild type YRS. In vivo amber suppression assay reveals that mutant YRS-3 (mYRS-3) clone containing amino acid substitutions of P320A and D321A showed 6.5-fold higher activity of amber suppression compared with the wild type. In addition, in vitro aminoacylation kinetics of mYRS-3 also showed approximately 7-fold higher activity than the wild type, and the enhancement was mainly due to the increase of tRNA binding affinity. These results demonstrate that optimization of anticodon recognition by engineered aminoacyl tRNA synthetase improves the efficiency of unnatural amino acid incorporation in response to nonsense codon.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 2001.06a
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• pp.83-89
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• 2001

Recent advances in the structural and molecular biology uncovered that a set of translation factors resembles a tRNA shape and, in one case, even mimics a tRNA function for deciphering the genetic ：ode. Nature must have evolved this 'art' of molecular mimicry between protein and ribonucleic acid using different protein architectures to fulfill the requirement of a ribosome 'machine'. Termination of protein synthesis takes place on the ribosomes as a response to a stop, rather than a sense, codon in the 'decoding' site (A site). Translation termination requires two classes of polypeptide release factors (RFs)： a class-I factor, codon-specific RFs (RFI and RF2 in prokaryotes; eRFI in eukaryotes), and a class-IT factor, non-specific RFs (RF3 in prokaryotes; eRF3 in eukaryotes) that bind guanine nucleotides and stimulate class-I RF activity. The underlying mechanism for translation termination represents a long-standing coding problem of considerable interest since it entails protein-RNA recognition instead of the well-understood codon-anticodon pairing during the mRNA-tRNA interaction. Molecular mimicry between protein and nucleic acid is a novel concept in biology, proposed in 1995 from three crystallographic discoveries, one, on protein-RNA mimicry, and the other two, on protein-DNA mimicry. Nyborg, Clark and colleagues have first described this concept when they solved the crystal structure of elongation factor EF- Tu：GTP：aminoacyl-tRNA ternary complex and found its overall structural similarity with another elongation factor EF-G including the resemblance of part of EF-G to the anticodon stem of tRNA (Nissen et al. 1995). Protein mimicry of DNA has been shown in the crystal structure of the uracil-DNA glycosylase-uracil glycosylase inhibitor protein complex (Mol et al. 1995; Savva and Pear 1995) as well as in the NMR structure of transcription factor TBP-TA $F_{II}$ 230 complex (Liu et al. 1998). Consistent with this discovery, functional mimicry of a major autoantigenic epitope of the human insulin receptor by RNA has been suggested (Doudna et al. 1995) but its nature of mimic is. still largely unknown. The milestone of functional mimicry between protein and nucleic acid has been achieved by the discovery of 'peptide anticodon' that deciphers stop codons in mRNA (Ito et al. 2000). It is surprising that it took 4 decades since the discovery of the genetic code to figure out the basic mechanisms behind the deciphering of its 64 codons.

• BMB Reports
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• v.43 no.7
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• pp.474-479
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• 2010

Three assay methods for quantification of the two galactose-operon mRNAs that only differ by 5 bases in their 5'-end are presented. The 5' ends of each mRNA were extended by ligating the 3'-end of the abundant 5S rRNA. This ligation extends the 5' ends of the two gal mRNAs long enough to be distinguished by the specific PCR primers in the following quantification reactions. Quantification of the corresponding cDNAs was performed either by primer extension assay or real-time qPCR. To circumvent the problem of the RNA ligation reaction (i.e. very low ligation efficiency), we devised a new method that employs real-time qPCR directly for the quantification of the gal transcripts which differ by 5 bases in their 5'-ends.