• Asian Pacific Journal of Cancer Prevention
• /
• v.13 no.2
• /
• pp.451-457
• /
• 2012

One decade early onset of the breast cancer in Iranian females was reported but the basis of the observed difference has remained unclear and difference in gene silencing by epigenetic processes is suggested. Hence, this study was sought to map the methylation status of estrogen receptor (ER) gene CpG islands and its impact on clinicopathological factors of triple negative and non-triple negative ductal cell carcinoma of the breast in Iranian females. Surgically resected formalin-fixed paraffin-embedded breast tissues from sixty Iranian women with confirmed invasive ductal carcinoma were assessed by methylation-specific PCR using primer sets encompassing some of the 29 CpGs across the ER gene CpG island. The estrogen and progesterone receptors, Her-$2^+$ overexpression, and nuclear accumulation of P53 were examined using immunohistochemistry (IHC). Methylated ER3, ER4, and ER5 were found in 41.7, 11.3, and 43.3% of the samples, respectively. Significantly higher methylation of ER4 was found in the tumors with nuclear accumulation of P53, and significantly higher methylation of ER5 was found in patients with lymph node involvement and tumor with bigger size or higher grades. Furthermore, significantly higher rate of ER5 methylation was found in patients with Her-$2^+$ tumors and in postmenopausal patients with $ER^-$, $PgR^-$, or $ER^-/PgR^-$ tumors. However, no significant difference in ERs methylation status was found between triple negative and non-triple negative tumors in pre- and postmenopausal patients. Findings revealed that aberrant hypermethylation of the ER-alpha gene frequently occurs in Iranian women with invasive ductal cell carcinoma of the breast. However, methylation of different CpG islands produced a diverse impact on the prognosis of breast cancer, and ER5 was found to be the most frequently methylated region in the Iranian women, and could serve as a marker of poor prognosis.

• Microbiology and Biotechnology Letters
• /
• v.31 no.2
• /
• pp.111-116
• /
• 2003

The xylA gene of Bacillus stearothermophilus No. 236 encoding $\beta$-xylosidase was cloned and sequenced previously. The transcriptional start site of the xylA gene cloned in E. coli was identified to be the guanine (G) by primer extension analysis. This supports that the expression of xylA gene is also directed in the E. coli cells by the previously determined transcription initiation signals, -10 sequence (CATAAT) and -35 sequence (TTGTTA) separated by 12 bp. To increase the expression of $\beta$-xylosidase, firstly the spacer region of xylA promoter was extended from 12 to 17 bp, and then the -10 and -35 elements were converted into their respective consensus sequences. The mutant promoters thus obtained were tested for their activities in both the E. coli and B. subtilis host cells. The change of the length of the spacer region from 12 to 17 bp resulted in a 1.6- and 2.5-fold increase in promoter strength in comparison with the wild type promoter in E. coli and B. subtilis cells, respectively. Also, strength of the promoter with the fourth T to A transversion on its -35 element increased in the transcription level by about 35 times compared with that of wild-type promoter. However, surprisingly the 5' end C-to-T transition of the -10 hexamer showed a 5- to 15-fold reduction in $\beta$-xylosidase activity in both E. coli and B. subtilis. Together, the present data demonstrated that the 5' end nucleotide C of the -10 sequence CATAAT and the fourth nucleotide A of the -35 hexamer are two most critical nucleotides for the promoter activity in the context of the xylA promoter.

• Journal of Veterinary Science
• /
• v.22 no.6
• /
• pp.87.1-87.12
• /
• 2021

Background: African swine fever virus (ASFV), classical swine fever virus (CSFV), and porcine reproductive and respiratory syndrome virus (PRRSV) are still prevalent in many regions of China. Co-infections make it difficult to distinguish their clinical symptoms and pathological changes. Therefore, a rapid and specific method is needed for the differential detection of these pathogens. Objectives: The aim of this study was to develop a multiplex real-time quantitative reverse transcription polymerase chain reaction (multiplex qRT-PCR) for the simultaneous differential detection of ASFV, CSFV, and PRRSV. Methods: Three pairs of primers and TaqMan probes targeting the ASFV p72 gene, CSFV 5' untranslated region, and PRRSV ORF7 gene were designed. After optimizing the reaction conditions, including the annealing temperature, primer concentration, and probe concentration, multiplex qRT-PCR for simultaneous and differential detection of ASFV, CSFV, and PRRSV was developed. Subsequently, 1,143 clinical samples were detected to verify the practicality of the assay. Results: The multiplex qRT-PCR assay could specifically and simultaneously detect the ASFV, CSFV, and PRRSV with a detection limit of 1.78 × 10⁰ copies for the ASFV, CSFV, and PRRSV, but could not amplify the other major porcine viruses, such as pseudorabies virus, porcine circovirus type 1 (PCV1), PCV2, PCV3, foot-and-mouth disease virus, porcine parvovirus, atypical porcine pestivirus, and Senecavirus A. The assay had good repeatability with coefficients of variation of intra- and inter-assay of less than 1.2%. Finally, the assay was used to detect 1,143 clinical samples to evaluate its practicality in the field. The positive rates of ASFV, CSFV, and PRRSV were 25.63%, 9.36%, and 17.50%, respectively. The co-infection rates of ASFV+CSFV, ASFV+PRRSV, CSFV+PRRSV, and ASFV+CSFV+PRRSV were 2.45%, 2.36%, 1.57%, and 0.17%, respectively. Conclusions: The multiplex qRT-PCR developed in this study could provide a rapid, sensitive, specific diagnostic tool for the simultaneous and differential detection of ASFV, CSFV, and PRRSV.

• Horticultural Science & Technology
• /
• v.28 no.6
• /
• pp.1025-1038
• /
• 2010

Carrot ($Daucus$ $carota$ L. var. $sativa$) is one of the most widely used crops in the world. Moreover it is an important crop because of its high content of ${\beta}$-carotene, well-known as the precursor of vitamin A carotenoid. However, seed-hair which is generated in epidermal cell of seeds inhibits absorption and germination. For that reason, carrot seeds are commercialized after mechanical hair removal process. To overcome such cumbersome weaknesses, new breeding program for developing hairless-seed carrot cultivar has been needed. Therefore, in this study, cDNA libraries from seeds of short-hair seed phenotype CT-ATR615 OP 666-13line and hairy seed CT-ATR615 OP-CK1-9 line were constructed and expression patterns related to generation of seed-hair were analyzed by comparison of EST sequences. Differential EST sequence results between two lines were classified into FunCat functional categories based on the results of BlastX search. Higher expression quantities belonging to metabolic category were shown on short-hair seed line than hairy-seed one. Differential expression quantities between those two lines in the protein folding and stabilization, subcellular localization categories were supposed to contribute variously on the generation of seed-hair. We confirmed 50 and 59 SSR sites, and 2 SNP sites by analyzing EST sequences in two lines; thereafter, we designed SNP and SSR primer sets from these EST sequence information as a molecular marker. These markers are thought to be used in research of molecular markers for classification of carrot family and related to various traits, as well as seed-hair characteristic.

• Journal of Animal Science and Technology
• /
• v.45 no.5
• /
• pp.703-710
• /
• 2003

The purpose of this study was to detect association between genetic variation and economic trait in the porcine heart type fatty acid-binding protein gene as a candidate gene for the traits related with growth and meat quality in pigs. The H-FABP is a 15-kDa protein expressed in several tissues with high demand for fat metabolism such as cardiac and skeletal muscle and lactating mammary gland. H-FABP is small intracellular protein involved in fatty acid transport from the plasma membrane to the site of $\beta$-oxidation and/or triacylglycerol or phospholipid synthesis. In this study, H-FABP PCR-RFLP was performed in F$_2$ population composed of 214 individuals from an intercross between Korean Native Boars and Landrace sows. PCR products from two primer sets within H-FABP gene were amplified in 850bp and 700bp. Digestion of PCR products with the restriction digestion enzymes HaeⅢ and HinfⅠ, revealed fragment length polymorphisms(RFLPs). The genotype frequencies from H-FABP/HaeⅢ was .29 for genotype DD, .53 for genotype Dd, and .15 for genotype dd, respectively. The genotype frequencies of HH, Hh, and hh from H-FABP/HinfⅠ was .38, .41 and .20, respectively, in the population. Relationships between their genotypes and economic traits were estimated. In H-FABP/HaeⅢ locus, there were specific genotypes(Dd and dd) associated with economic traits such as body weights at 3, 5, 12, and 30 week of age (p〈.05 to .001). The ‘d’ allele was associated with gaining of body weight. In H-FABP/HinfⅠ locus, Genotypes of HH and Hh associated with growth traits such as body weights at 5, 12, and 30 week of age (p〈.05 or p〈.001) and back fat thickness, body fat including abdominal and trimmed fat (p〈.001) and intramuscular fat(p〈.05) The ‘H’ allele was positively associated with gaining of body weight and fatness deposition. In conclusion, a significant association of the H-FABP gene from its genetic variation was found on body weight, intramuscular fat and backfat thickness.

• Journal of Life Science
• /
• v.27 no.8
• /
• pp.857-863
• /
• 2017

The FBJ murine osteosarcoma viral oncogene homolog B (FosB) gene is located at chromosome 19, and encodes 43 Kda protein. Functionally, the FosB gene is important for differentiation, development, and pathogenesis. Furthermore, the FosB gene is suggested as possible biomarker for tracing disease prognosis. In this study, we constructed plasmid containing a FosB promoter region and evaluate its promoter activity. We analyzed the putative promoter region in FosB genomic DNA using bioinformatics program, and we found important regulatory elements in 1 Kb upstream from transcription start site (TSS). Therefore, we performed polymerase chain reaction (PCR) amplification on region from-1,555 upstream to +73 of the FosB genomic DNA, and PCR product was inserted into TA vector to create the $TA-1^{st}FosBp$ plasmid. We then prepared the primer sets, which contain a restriction enzyme site for Kpn1 and Nhe1, in order to reinsert into the TA vector to prepare $TA-2^{nd}FosBp$ plasmid. It was finally subcloned into pGL3-luc vector after enzyme cutting. To evaluate whether the cloned plasmid is useful in cell based experiment, we performed luciferase assay with pGL3-FosBp-luctransfection. FosB promoter activity was increased compared to empty vector, and this activity was significantly increased by treatment of doxorubicin and taxol. We obtained consistent data on regulation of FosB gene expression after anticancer drug treatment using Western blot analysis. The results suggest that promoter cloning of the human FosB gene is very useful for studying gene expression and analyzing biomarkers.

• Journal of Food Hygiene and Safety
• /
• v.32 no.3
• /
• pp.199-205
• /
• 2017

Although many PCR-based assays have been developed, the majority of rapid detection of Toxoplasma gondii in animal and their meat product has been dependent on immunogenic assays. Thus, there is still a need for more reliable PCR based detection method for T. gondii in retail meats. Recently, a 529-bp repeat element that exists in 200-300 copies per genome of T. gondii genome had been spotlighted for its usefulness as potential detection targers. In this study, the 529-bp repeat element was selected for real-time PCR to detect three types of T. gondii (type I, II and III). A primer pair targeting 82-bp of the 529-bp element detected all three types of T. gondii and showed high level of specificity against 14 different food-borne pathogens as well as 3 protozoan parasites such as Giardia lamblia, Cryptosporidium parvum and Entamoeba histolytica. Application of the new real-time PCR assay in meat samples showed improved detection sensitivity compared to the B1-gene targeted method suggesting potential new target for Toxoplasma gondii screening in retail meats.

• The Korean Journal of Malacology
• /
• v.26 no.2
• /
• pp.185-188
• /
• 2010

The Web-based the genus Haliotis SNP database was constructed on the basis of Intel Server Platform ZSS130 dual Xeon 3.2 GHz cpu and Linux-based (Cent OS) operating system. Haliotis related sequences (2,830 nucleotide sequences, 9,102 EST sequences) were downloaded through NCBI taxonomy browser. In order to eliminate vector sequences, we conducted vector masking step using cross match software with vector sequence database. In addition, poly-A tails were removed using Trimmest software from EMBOSS package. The processed sequences were clustered and assembled by TGICL package (TIGR tools) equipped with CAP3 software. A web-based interface (Haliotis SNP Database, http://www.haliotis.or.kr) was developed to enable optimal use of the clustered assemblies. The Clustering Res. menu shows the contig sequences from the clustering, the alignment results and sequences from each cluster. And also we can compare any sequences with Haliotis related sequences in BLAST menu. The search menu is equipped with its own search engine so that it is possible to search all of the information in the database using the name of a gene, accession number and/or species name. Taken together, the Web-based SNP database for Haliotis will be valuable to develop SNPs of Haliotis in the future.

• ALGAE
• /
• v.34 no.2
• /
• pp.111-126
• /
• 2019

The phototrophic dinoflagellate Biecheleriopsis adriatica is a small suessioid species characterized by a fragile thin wall. Although the morphology of this dinoflagellate is well established, there is currently little information available on its distribution and the environmental factors that influence this distribution. Thus, to investigate the spatial and seasonal distributions of the vegetative cells of B. adriatica in Korean waters, surface water samples were collected on a seasonal basis from 28 stations in the East, West, and South Sea of Korea and Jeju Island from April 2015 to October 2018, and abundances of the vegetative cells of B. adriatica were quantified using quantitative real-time polymerase chain reactions, for which we developed the species-specific primer and probe set. Simultaneously, major environmental parameters, including temperature, salinity, nutrient concentrations, and dissolved oxygen concentrations were measured. The vegetative cells of B. adriatica were detected at 20 of the 28 sampling stations: 19 stations in summer and 6 in autumn, although from no stations in either spring or winter. The ranges of water temperature and salinity at sites where this species was detected were $17.7-26.4^{\circ}C$ and 9.9-34.3, respectively, whereas those of nitrate and phosphate concentrations were not detectable-96.2 and $0.18-2.66{\mu}M$, respectively. Thus, the sites at which this species is found are characterized by a narrow range of temperature, but wide ranges of salinity and concentrations of nitrate and phosphate. The highest abundance of the vegetative cells of B. adriatica was $41.7cells\;mL^{-1}$, which was recorded in Jinhae Bay in July 2018. In Jinhae Bay, the abundance of vegetative cells was significantly positively correlated with the concentration of nitrate, but was negatively correlated with salinity. On the basis of these findings, it appears that the abundance of B. adriatica vegetative cells shows strong seasonality, and in Jinhae Bay, could be affected by the concentrations of nitrate.

• Microbiology and Biotechnology Letters
• /
• v.40 no.4
• /
• pp.339-347
• /
• 2012

Viral safety is an important prerequisite for clinical preparations of mammalian cell culture-derived biopharmaceuticals, because numerous adventitious viruses have been contaminated during the manufacturing process. In particular, Chinese hamster ovary (CHO) cells are highly susceptible to several RNA viruses including reovirus (Reo), bovine viral diarrhea virus (BVDV), and bovine parainfluenza virus (BPIV) and there have been reports of such viral contaminations. Therefore, viral detection during the CHO cell process is necessary to ensure the safety of biopharmaceuticals against viruses. In this study, a multiplex reverse transcription (RT)-PCR assay was developed and subsequently evaluated for its effectiveness as a means to simultaneously detect Reo, BVDV, and BPIV during the manufacture of cell culture-derived biopharmaceuticals. Specific primers for Reo, BVDV, and BPIV were selected, and a multiplex RT-PCR was optimized. The sensitivity of the assay for simultaneous amplification of all viral target RNAs was $7.76{\times}10^2\;TCID_{50}/ml$ for Reo, $7.44{\times}10^1\;TCID_{50}/ml$ for BVDV, and $6.75{\times}10^1\;TCID_{50}/ml$ for BPIV. The multiplex RT-PCR was proven to be very specific to Reo, BVDV, and BPIV and was subsequently applied to the validation of CHO cells artificially infected with each virus. It could detect each viral RNA from CHO cells as well as culture supernatants. Therefore, it was concluded that the multiplex RT-PCR assay can be applied to detection of the adventitious viruses during the manufacture of cell culture-derived biopharmaceuticals.