• Journal of Genetic Medicine
• /
• 제19권2호
• /
• pp.63-75
• /
• 2022

Purpose: Osteoporosis is a common calcium and metabolic skeletal disease which is characterized by decreased bone mass, microarchitectural deterioration of bone tissue and impaired bone strength, thereby leading to enhanced risk of bone fragility. In this study, we aimed to identify novel genes for susceptibility to osteoporosis and/or bone density. Materials and Methods: To identify differentially expressed genes (DEGs) between control and osteoporosis-induced cells, annealing control primer-based differential display reverse-transcription polymerase chain reaction (RT-PCR) was carried out in pre-osteoblast MC3T3-E1 cells. Expression levels of the identified DEGs were evaluated by quantitative RT-PCR. Association studies for the quantitative bone density analysis and osteoporosis case-control analysis of single nucleotide polymorphism (SNPs) were performed in Korean women (3,570 subjects) from the Korean Association REsource (KARE) study cohort. Results: Comparison analysis of expression levels of the identified DEGs by quantitative RT-PCR found seven genes, Anxa6, Col5a1, Col6a2, Eno1, Myof, Nfib, and Scara5, that showed significantly different expression between the dexamethason-treated and untreated MC3T3-E1 cells and between the ovariectomized osteoporosis-induced mice and sham mice. Association studies revealed that there was a significant association between the SNPs in the five genes, ANXA6, COL5A1, ENO1, MYOF, and SCARA5, and bone density and/or osteoporosis. Conclusion: Using a whole-genome comparative expression analysis, gene expression evaluation analysis, and association analysis, we found five genes that were significantly associated with bone density and/or osteoporosis. Notably, the association P-values of the SNPs in the ANXA6 and COL5A1 genes were below the Bonferroni-corrected significance level.

• 한국발생생물학회:학술대회논문집
• /
• 한국발생생물학회 2003년도 제3회 국제심포지움 및 학술대회
• /
• pp.82-82
• /
• 2003

Offspring have been produced from somatic cells in a number of species. This biotechnology introduced a new phenomenon in reprogramming and differentiation of somatic cell, namely totipotency. However, birth of oversized calves and perinatal abnormalities such as increased gestation length, lack of spontaneous parturition, higher incidence of dystocia, and reduced perinatal viability of offspring are frequently observed in pregnancies of cloned bovine fetuses. Disturbance of feto-placenta has been proposed as likely causes for abnomal growth. However. Little is known the mechanism responsible for the perinatal problems. Therefore, we focused on gestation length in somatic cell nuclear recipient cows. To solve this issues, placental tissues of control and cloned bovine were obtained by a cesarean section (C-section) before 5 days of paturition. Total RNA from control and cloned bovine placenta was extractd by TRIzol reagent. GeneFishing DEG kits (Seegene) were used to identify differentially expression genes. Total RNA (3 ug) were synthesized by M-MLV reverse transcriptase (200 u/ul) with 10 uM dT-annealing control primer (ACP1) at 42C for 90 min. Then, first-strand cDNA (50 ng) was amplified using the 5 uM arbitary ACP (1-20) and 10 uM dT-ACP2 primers. Some specific expression genes were amplified, Now, we are cloning and sequencing. These finding strongly can be support to solve the problems for parturition delay in nuclear transfer cows, suggest that placenta specific proteins are key indicators for the aberration of gestation and placental function in cows.

• Molecular & Cellular Toxicology
• /
• 제1권4호
• /
• pp.230-236
• /
• 2005

Formaldehyde is a common environmental contaminant found in tobacco smoke, paint, garments, diesel and exhaust, and medical and industrial products. Formaldehyde has been considered to be potentially carcinogenic, making it a subject of major environmental concern. However, only a little information on the mechanism of immunological sensitization and asthma by this compound has been known. So, we performed with Jurkat cell line, a human T lymphocyte, to assess the induction of DNA damage and to identify the DEGs related to immune response or toxicity by formaldehyde. In this study, we investigated the induction of DNA single strand breaks by formaldehyde using single cell gel electrophoresis assay (comet assay). And we compared gene expression between control and formaldehyde treatment to identify genes that are specifically or predominantly expressed by employing annealing control primer (ACP)-based $GeneFishing^{TM}$ method. The cytotoxicity ($IC_{30}$) of formaldehyde was determined above the 0.65 mM in Jurkat cell in 48 h treatment. Based on the $IC_{30}$ value from cytotoxicity test, we performed the comet assay in this concentration. From these results, 0.65 mM of formaldehyde was not revealed significant DNA damages in the absence of S-9 metabolic activation system. And the one differentially expressed gene (DEG) of formaldehyde was identified to zinc finger protein 292 using $GeneFishing^{TM}$ method. Through further investigation, we will identify more meaningful and useful DEGs on formaldehyde, and then can get the information on the associated mechanism and pathway with immune response or other toxicity by formaldehyde exposure.

• 한국초지조사료학회지
• /
• 제34권2호
• /
• pp.114-119
• /
• 2014

염 또는 건조 스트레스 처리에 의한 톨 페스큐 종자의 발아율 변화와 유식물체 수준에서의 유전자 발현을 조사하기 위하여 in vitro 조건에서 NaCl과 PEG를 처리하여 분석하였다. NaCl 처리시 톨 페스큐 품종별 발아율은 50 mM 농도에서 발아율이 서서히 감소하기 시작하였으며 350 mM의 농도에서는 모든 품종에서 발아가 되지 않는 경향을 보였다. NaCl 처리 농도에 따른 발아율 감소율은 Fawn 품종이 가장 큰 변화를 보였으며 Kentucky-31(E-) 품종이 가장 강한 내성을 보였다. 또한, PEG 처리시 톨 페스큐 품종별 발아율의 변화도 NaCl 처리시와 유사한 경향을 보였으며 고농도인 30% PEG 처리구에서는 모든 품종에서 발아가 되지 않는 경향을 보였으며 Kentucky-31(E-) 품종이 가장 강한 내성을 보였다. 톨 페스큐 유식물체 수준에서 염해와 건조 스트레스에 의한 유전자 발현양상을 조사하기 위하여 DEGs (differentially expressed genes) 탐색을 위한 ACP-based GeneFishing$^{TM}$ PCR 분석을 통해 NaCl 또는 PEG 처리에 따른 발현량의 차이를 보이는 총 4개의 DEG를 선발하여 클로닝하고 염기서열을 분석하였다. 무처리구에 비해 NaCl 처리시 4개의 DEG가 증가하였고 감소하는 DEG는 확인 되지 않았으나, PEG 처리에서는 3개의 DEG (DEG 1, 3, 및 4)가 증가하였고 1개의 DEG가 감소하는 경향을 나타내었다. 발굴된 DEG들을 blastx 검색에 의하여 rubisco large subunit (DEG1), microsomal glutathion S-transferase (GST) 3-like isoform 1 (DEG2) 유전자로 동정되었다.

• Journal of Applied Biological Chemistry
• /
• 제51권1호
• /
• pp.1-6
• /
• 2008

Estrogens, a group of steroid compounds functioning as the primary female sex hormone, play an important role in the development and progression of breast cancer. In this study, using a novel annealing control primer-based GeneFishing PCR technology, five differentially expressed genes (DEGs), expressed using 10nM mitogenic estrogens, $17{\beta}$-estradiol (E2) and $16{\alpha}$-hydroxyestrone ($16{\alpha}$-OHE1), were selected from the estrogen receptor (ER)-positive MCF-7 human breast cancer cells. The DEGs, MRPL42, TUBA1B, SSBP1, KNCT2, and RUVBL1, were identified by comparison with the known genes via direct sequencing and sequence homology search in BLAST. Quantitative real-time PCR data showed that two DEGs, tubulin ${\alpha}1b$ and kinetochore associated 2, were greater than 2-fold upregulated by E2 or $16{\alpha}$-OHE1. Both genes could be new biomarkers for the treatment and prognosis of cancers, and further study may provide insights into the molecular mechanisms underlying development and progression of breast cancer.

• International Journal of Industrial Entomology and Biomaterials
• /
• 제31권2호
• /
• pp.79-84
• /
• 2015

A new cecropin-like antimicrobial peptide (Px-CLP) gene was isolated from the immunechallenged larvae of the swallowtail butterfly, Papilio xuthus, by employing annealing control primer (ACP)-based GeneFishing PCR. The full-length cDNA of Px-CLP is 310 nucleotides encoding a 70 amino acid precursor that contains a putative 22-residue signal peptide, a 4-residue propeptide, a presumed 37-residue mature peptide, and an uncommon 7-residue acidic pro-region at the C-terminus. The deduced amino acid sequence of Px-CLP showed significant identities with other Lepidopteran cecropin D type peptides. RT-PCR revealed that the Px-CLP transcript was detected at significant level after injection with bacterial lipopolysaccharide (LPS). The peptides with or without C-terminal acidic sequence region were synthesized on-solid phage and submitted to antibacterial activity assay. The synthetic 37-mer peptide (Px-CLPa), which removed C-terminal acidic sequence region, was showed exclusively antibacterial activity against E. coli ML35; meanwhile, a 44-mer peptide (Px-CLPb) with C-terminal acidic peptide region was not active. This result suggests that Px-CLP is produced as a larger precursor containing a C-terminal pro-region that is subsequently removed by C-terminal modification.

• 한국초지조사료학회지
• /
• 제38권3호
• /
• pp.190-195
• /
• 2018

Cold, salt and heat are the most critical factors that restrict full genetic potential, growth and development of crops globally. However, clarification of genes expression and regulation is a fundamental approach to understanding the adaptive response of plants under unfavorable environments. In this study, we applied an annealing control primer (ACP) based on the GeneFishing approach to identify differentially expressed genes (DEGs) in Italian ryegrass (cv. Kowinearly) leaves under cold, salt and heat stresses. Two-week-old seedlings were exposed to cold ($4^{\circ}C$), salt (NaCl 200 mM) and heat ($42^{\circ}C$) treatments for six hours. A total 8 differentially expressed genes were isolated from ryegrass leaves. These genes were sequenced then identified and validated using the National Center for Biotechnology Information (NCBI) database. We identified several promising genes encoding light harvesting chlorophyll a/b binding protein, alpha-glactosidase b, chromosome 3B, elongation factor 1-alpha, FLbaf106f03, Lolium multiflorum plastid, complete genome, translation initiation factor SUI1, and glyceraldehyde-3-phosphate dehydrogenase. These genes were potentially involved in photosynthesis, plant development, protein synthesis and abiotic stress tolerance in plants. However, this study provides new insight regarding molecular information about several genes in response to multiple abiotic stresses. Additionally, these genes may be useful for enhancement of abiotic stress tolerance in fodder crops as well a crop improvement under unfavorable environmental conditions.

• Journal of Microbiology and Biotechnology
• /
• 제25권12호
• /
• pp.2153-2159
• /
• 2015

Mycoplasma hyopneumoniae is known to cause porcine enzootic pneumonia (EP), an important disease in swine production. The objective of this study was to examine the effects of sonicated protein fractions of M. hyopneumoniae on inflammatory response and gene expression in the murine alveolar macrophage MH-S cell line. The effects of sonicated protein fractions and intact M. hyopneumoniae on the gene expression of cytokines and iNOS were assessed using RT-PCR. The Annealing Control Primer (ACP)-based PCR method was used to screen differentially expressed genes. Increased transcription of interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, COX-2, and iNOS mRNA was observed after exposure to the supernatant (SPT), precipitant (PPT), and intact M. hyopneumoniae protein. A time-dependent analysis of the mRNA expression revealed an upregulation after 4 h for IL-6 and iNOS and after 12 h for IL-1β and TNF-α, for both SPT and PPT; the fold change in COX-2 expression was less. A dose- and time-dependent correlation was observed in nitrite (NO) production for both protein fractions; however, there was no significant difference between the effects of the two protein fractions. In a differential gene analysis, PCR revealed differential expression for nine gene bands after 3 h of stimulation — only one gene was downregulated, while the remaining eight were upregulated. The results of this study provide insights that help improve our understanding of the mechanisms underlying the pathogenesis of and macrophage defenses against M. hyopneumoniae assault, and suggest targets for future studies on therapeutic interventions for M. hyopneumoniae infections.

• The Korean Journal of Physiology and Pharmacology
• /
• 제14권5호
• /
• pp.265-272
• /
• 2010

Follicular cystic ovary (FCO) is one of the most frequently diagnosed ovarian diseases and is a major cause of reproductive failure in mammalian species. However, the mechanism by which FCO is induced remains unclear. Genetic alterations which affect the functioning of many kinds of cells and/or tissues could be present in cystic ovaries. In this study, we performed a comparison analysis of gene expression in order to identify new molecules useful in discrimination of bovine FCO with follicular cystic follicles (FCFs). Normal follicles and FCFs were classified based on their sizes (5 to 10 mm and $\geq25mm$). These follicles had granulosa cell layer and theca interna and the hormone $17{\beta}$-estradiol ($E_2$)/ progesterone ($P_4$) ratio in follicles was greater than one. Perifollicular regions including follicles were used for the preparation of RNA or protein. Differentially expressed genes (DEG) that showed greater than a 2-fold change in expression were screened by the annealing control primer (ACP)-based PCR method using $GeneFishing^{TM}$ DEG kits in bovine normal follicles and FCFs. We identified two DEGs in the FCFs: ribosomal protein L15 (RPL15) and microtubule-associated protein 1B (MAP1B) based on BLAST searches of the NCBI GenBank. Consistent with the ACP analysis, semi-quantitative PCR data and Western blot analyses revealed an up-regulation of RPL15 and a down-regulation of MAP1B in FCFs. These results suggest that RPL15 and MAP1B may be involved in the regulation of pathological processes in bovine FCOs and may help to establish a bovine gene data-base for the discrimination of FCOs from normal ovaries.

• International Journal of Industrial Entomology and Biomaterials
• /
• 제23권2호
• /
• pp.231-238
• /
• 2011

Attacin is an insect antibacterial protein that plays an important role in immune response to injury and infection. In this report, we have isolated and characterized of cDNA encoding for the attacin from the immunized larvae of swallowtail butterfly, $Papilio$ $xuthus$. A full length cDNA of $P.$ $xuthus$ attacin was obtained by employing annealing control primer (ACP)-based differential display PCR and 5' RACE. The complete $P.$ $xuthus$ attacin cDNA was comprised of 949 bp encoding a 250 amino acid precursor. It contains a putative 18 amino acid signal peptide sequence, a 42 amino acid propeptide sequence, and a 190 amino acid mature protein with a theoretical molecular mass of 19904.01 and a pI of 9.13. The putative mature protein of $P.$ $xuthus$ attacin showed 48-52% and 24-30% identity in amino acid sequences with that of lepidopteran and dipteran insects, respectively. Semiquantitive RT-PCR results revealed that the transcript of $P.$ $xuthus$ attacin gene was up-regulated at significant levels after injection with bacterial lipopolysaccharide (LPS). We sub-cloned cDNA fragment encoding mature $P.$ $xuthus$ attacin into the expression vector, highly expressed in $E.$ $coli$ BL21 cells, and its antibacterial activity was analyzed. Recombinant $P.$ $xuthus$ attacin evidenced considerably antibacterial activity against Gram-negative bacteria, $E.$ $coli$ ML 35 and $Klebsiella$ $pneumonia$.