• KSBB Journal
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• v.9 no.3
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• pp.339-345
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• 1994

Deletions between UASG and the GALI TATA box reduced glucose repression and allowed constitutive expression of the gene product in the absence of galactose. The relative inducer level (ratio of galactose/glucose concentrations) affected the extent of gene expression and glucose repression. Glucose repression was reduced by a factor of 2 to 5 as the relative inducer level increased. In the medium containing galactose only, induction of ${\beta}$-galactosidase synthesis by galactose increased with plasmid copy number. On the contrary, plasmid copy number did not affect significantly ${\beta}$-galactosidase synthesis in the medium containing both glucose and galactose (2% glucose＋2% galactose), which might be due to glucose repression caused by high glucose concentration. However, when the medium contained the relatively high inducer level (0.4% glucose＋0.8% galactose), ${\beta}$-galactosidase synthesis increased with plasmid copy number, indicating that the beneficial effect of higher galactose concentration was weaker than the repressive effect of higher glucose concentration.

• Proceedings of the Korean Society for Bioinformatics Conference
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• 2006.02a
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• pp.125-126
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• 2006

NimbleGen has developed strategies to use its high-density oligonucleotide microarray platform (385,000 probes per array) to map both promoter binding sites and copy number variation at very high-resolution in the human genome. Here we describe a genome-wide map of active promoters determined by experimentally locating the sites of transcription imitation complex binding throughout the human genome using microarrays combined with chromatin immunoprecipitation. This map defines 10,567 active promoters corresponding to 6,763 known genes and at least 1,196 un-annotated transcriptional units. Microarray-based comparative genomic hybridisation (CGH) is animportant research tool for investigating chromosomal aberrations frequently associated with complex diseases such as cancer, neuropsychiatric disorders, and congenital developmental disorders. NimbleGen array CGH is an ultra-high resolution (0.5-50 Kb) oligo array platform that can be used to detect amplifications and deletions and map the associated breakpoints on the whole-genome level or with custom fine-tiling arrays. For whole-genome array CGH, probes are tiled through genic and intergenic regions with a median probe spacing of 6 Kb, which provides a comprehensive, unbiased analysis of the genome.

• The Korean Journal of Applied Statistics
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• v.24 no.5
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• pp.847-859
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• 2011

cDNA microarray-based comparative genomic hybridization(CGH) data includes low-intensity spots and thus a statistical strategy is needed to detect subtle differences between different cancer classes. In this study, genes displaying a high frequency of alteration in one of the different classes were selected among the pre-selected genes that show relatively large variations between genes compared to total variations. Utilizing copy-number changes of the selected genes, this study suggests a statistical approach to predict patients' classes with increased performance by pre-classifying patients with similar genetic alteration scores. Two-stage logistic regression model(TLRM) was suggested to pre-classify homogeneous patients and predict patients' classes for cancer prediction; a decision tree(DT) was combined with logistic regression on the set of informative genes. TLRM was constructed in cDNA microarray-based CGH data from the Cancer Metastasis Research Center(CMRC) at Yonsei University; it predicted the patients' clinical diagnoses with perfect matches (except for one patient among the high-risk and low-risk classified patients where the performance of predictions is critical due to the high sensitivity and specificity requirements for clinical treatments. Accuracy validated by leave-one-out cross-validation(LOOCV) was 83.3% while other classification methods of CART and DT performed as comparisons showed worse performances than TLRM.

• Journal of Microbiology and Biotechnology
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• v.24 no.2
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• pp.144-151
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• 2014

Increasing the gene copy number has been commonly used to enhance the protein expression level in the yeast Pichia pastoris. However, this method has been shown to be effective up to a certain gene copy number, and a further increase of gene dosage can result in a decrease of expression level. Evidences indicate the gene dosage effect is product-dependent, which needs to be determined when expressing a new protein. Here, we describe a direct detection of the gene dosage effect on protein secretion through expressing the enhanced green fluorescent protein (EGFP) gene under the direction of the ${\alpha}$-factor preprosequence in a panel of yeast clones carrying increasing copies of the EGFP gene (from one to six copies). Directly examined under fluorescence microscopy, we found relatively lower levels of EGFP were secreted into the culture medium at one copy and two copies, substantial improvement of secretion appeared at three copies, plateau happened at four and five copies, and an apparent decrease of secretion happened at six copies. The secretion of EGFP being limiting at four and five copies was due to abundant intracellular accumulation of proteins, observed from the fluorescence image of yeast and confirmed by western blotting, which significantly activated the unfolded protein response indicated by the up-regulation of the BiP (the KAR2 gene product) and the protein disulfide isomerase. This study implies that tagging a reporter like GFP to a specific protein would facilitate a direct and rapid determination of the optimal gene copy number for high-yield expression.

• Microbiology and Biotechnology Letters
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• v.16 no.6
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• pp.476-483
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• 1988

Yeat-Escherichia coli shuttle vectors were constructed by combination of various functional segments such as autonomous replicating sequence (ARS1), centromere region (CEN3), origin of replication of 2 $\mu$m plasmid (2 $\mu$m OR). Transformation efficiency, stability and copy number of constructed vectors were analyzed in yeast strains, SHY4(cir$^+$) containing 2 $\mu$m plasmid and NNY1(cir$^{\circ}$) without it. The results showed that centromere containing plasmids were very stable and existed at one copy per cell; fused replication system (2$\mu$m OR and ARS1) containing Plasmids were more stable and higher copy number than one replicon containing plasmids ; presence of endogenous 2$\mu$m plasmid influenced on stability and copy number of 2 $\mu$m based plasmids.

• Journal of fish pathology
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• v.36 no.2
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• pp.229-238
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• 2023

In this study, the correlation between clinical changes and RSIV genome copy number was investigated to determine the quantitative criteria for the characteristics of RSIV infection. The rock bream (Oplegnathus fasciatus) was intraperitoneally injected with three different doses (1.0×10¹, 1.0×10³ and 1.0×10⁵ viral genome copies/fish) as low, medium, and high doses, respectively. The clinical signs (spleen enlargement, death) observation and real-time PCR were conducted at 5, 10 and 14 days post-injection. During the experiment, spleen index as a quantitative indicator for spleen enlargement was continuously increased in the medium- (up to 2.26) and high-dose (up to 4.99) challenge groups, respectively. Notably, when the spleen index was over 1.5, 2.0, 2.5 and 3.0, a positive correlation was revealed with average viral genome copy numbers of 2.51, 3.37, 4.97 and 5.43×10⁷ viral genome copies/mg, respectively. Moreover, the threshold of spleen index over 1.5 was 1.0×10⁶ viral genome copies/mg, while the thresholds of spleen index over 2.0 and dead was 2.51×10⁷ viral genome copies/mg and the thresholds of spleen index over 2.5 and 3 was 3.98×10⁷ viral genome copies/mg. These findings suggest the possibility of quantitatively analyzing the characteristics and development process of RSIV infection.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.1
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• pp.329-337
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• 2012

Considerable attention has been given to the accuracy of HER-2 testing and the correlation between the results of different testing methods. This interest reflects the growing importance of HER-2 status in the management of patients with breast cancer. In this study the detection of HER-2 gene and centromere 17 status was evaluated using dual-colour primed in situ labelling (PRINS) in comparison with fluorescence in situ hybridization (FISH). These two methods were evaluated on a series of 27 formalin fixed paraffin embedded breast carcinoma tumours, previously tested for protein overexpression by HercepTest (grouped into Hercep 1+/0, 2+ and 3+). HER-2 gene amplification (ratio${\geq}2.2$) by PRINS was found in 3:3, 6:21 and 0:3 in IHC 3+, 2+ and 1+/0 cases, respectively. Comparing FISH and IHC (immunohistochemistry), showed the same results as for PRINS and IHC. Chromosome 17 aneusomy was found in 10 of 21 IHC 2+ cases (47.6%), of which 1 (10%) showed hypodisomy (chromosome 17 copy number per cell${\leq}1.75$), 7 (70%) showed low polysomy (chromosome 17 copy number per cell=2.26 - 3.75) and 2 (20%) showed high polysomy (chromosome 17 copy number per cell ${\geq}3.76$). The overall concordance of detection of HER-2 gene amplification by FISH and PRINS was 100% (27:27). Furthermore, both the level of HER-2 amplification and copy number of CEN17 analysis results correlated well between the two methods. In conclusion, PRINS is a reliable, reproducible technique and in our opinion can be used as an additional test to determine HER-2 status in breast tumours.

• Genomics & Informatics
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• v.2 no.4
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• pp.174-179
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• 2004

We developed various plasmid cloning vectors that are useful in the construction of genomic and shotgun libraries. Two medium copy vectors, pCUGlblu21 (pCb21) and pAGlblu21 (pAb21), which are resistant to kanamycin ($Km^R$) and chloramphenicol ($Cam^R$), respectively, are useful for cloning DNA inserts ranging from 5kb to 15kb. Two high copy vectors, pCUGlblu31 (pCb31) and pAGlblu31 (pAb31), containing $Km^R$ and $Cam^R$, respectively, are useful for DNA inserts less than 5kb. These vectors are well adapted for large-scale genome sequencing projects by providing choice of copy number and selectable marker. The small vector size is another advantage of these vectors. All vectors contain lacZa including multicloning sites that originated from pBluscriptllsk- for easy cloning and sequencing. Two medium copy vectors contain unique and rare cutting Swal (ATTTAAAT) restriction enzyme sites for easy determination of insert size. We developed two combined vectors, pC21A31 and pC31A21, which are combinations of (pCb21 + pAb31) and (pCb31 + pAb21), respectively. These two vectors provide four choices of vectors such as $Km^R$ and medium, $Cam^R$ and high, $Cam^R$ and medium, and $Km^R$ and high copy vectors by restriction enzyme cutting, dephosphorylation, and gel purification. These vectors were successfully applied to high throughput shotgun sequencing of rice, tomato, and brassica BAC clones. With an example of extremely biased hydro sheared 3 kb shotgun library of a tomato BAC clone, which is originated from cytogenetically defined peri-centromeric region, we suggest the utility of an additional 10 kb library for sequence assembly of the difficult-to-assemble BAC clone.

• Korean Journal of Ecology and Environment
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• v.56 no.1
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• pp.1-13
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• 2023

Targeting Microcystin (MC), which is most abundantly detected in the North-Han River water area, we analyzed the relationship between the MC biosynthesis gene (mcyA gene), cyanobacteria cell density, and MC concentration, derived an RNA-MC conversion formula, and derived the cyanobacteria. The concentration of MC present in cells was predicted. In the North-Han River waters, the mcyA gene was found mainly at downstream sites of the North-Han River after Muk-Hyeon Stream junction, and higher copy numbers were found on average than other sites. In the Uiam Lake waters upstream of the North-Han River, the mcyA gene copy number increased at the Kong-Ji Stream point, and after September, the mcyA gene copy number decreased throughout the North-Han River waters. The expression of the mcyA gene was concentrated in the short period of summer due to the spatio-temporal difference between upstream and downstream water bodies. The mcyA gene expression level was not only highly correlated with MC concentration, but also correlated with the cell density of Microcystis aeruginosa and Dolichospermum circinale, which are known to biosynthesize MC. Six conversion formulas derived based on the RNA-MC relationship showed statistical significance (p<0.05) and exhibited high correlation coefficients (r) of 0.9 or higher. The expression level of MC biosynthesis gene present in eRNA determines the synthesis of cyanotoxin substances in water, quickly quantifies gene activity, and can be fully utilized for early warning of MC development.

• Korean Journal of Microbiology
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• v.29 no.2
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• pp.75-79
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• 1991

The yeast L-A virus (4.6 kb dsRNA genome) encodes the major coat protein and a "gag-pol" fusion minor coat protein that separately encapsidate itself and $M_{1}$, a 1.8 kb dsRNA satellite virus encoding a secreted protein toxin (the killer toxin). The teast chromosomal SKI genes prevent viral cytopathology by lowering the virus copy number. Thus, $ski^{-}$ mutants are ts and cs for growth. We transformed a ski2-2 virus-infested mutant with a yeast bank in a high copy cloning vector and selected the rare healthy transformants for analysis. One type of transformant segregated M-O L-A-O cells with high frequency. Elimination of the DNA clone from the ski2-2 strain eliminated this phinotype and introduction of the DNA clone recovered from such transformants into the parent ski2-2 strain, or into ski3 or ski6 mutants gave the same phenotype. This killer-curing phenotype was due to the curing of the helper L-A dsRNA virus. The 6.5 kb insert only had this activity when carried on a high copy vector and in $ski^{-}$ cells (not in $SKI^{+}$ cells). This 6.5 kb insert acts as a mutagen on L-A dsRNA producing a high rate of deletion mutations.mutations.