Biomedical subjects

Kwanghee Baek

Publications and source records attributed to Kwanghee Baek.

8 recordsLinked to original sources

PubMed2004

A DNA replication-related element downstream from the initiation site of Drosophila selenophosphate synthetase 2 gene is essential for its transcription.

Selenophosphate synthetase catalyzes the synthesis of selenophosphate which is a selenium donor for Sec biosynthesis. In Drosophila melanogaster, there are two types of selenophosphate synthetases designated dSPS1 and dSPS2, where dSPS2 is a selenoprotein. The mechanism of gene expression of dSPS2 as well as other selenoproteins in Drosophila has not been elucidated. Herein, we report an essential regulator system that regulates the transcription of the dSPS2 gene (dsps2). Through deletion/substitution mutagenesis, the downstream DNA replication-related element (DRE) located at +71 has been identified as an essential element for dsps2 promoter activity. Furthermore, double-stranded RNA interference (dsRNAi) experiments were performed to ablate transcription factors such as TBP, TRF1, TRF2 and DREF in Schneider cells. The dsRNAi experiments showed that dsps2 promoter activities in DREF- and TRF2-depleted cells were significantly decreased by 90% and 50%, respectively. However, the depletion of TBP or TRF1 did not affect the expression level of dsps2 even though there is a putative TATA box at -20. These results strongly suggest that the DRE/DREF system controls the basal level of transcription of dsps2 by interacting with TRF2.

5' Flanking Region↗

PubMed2004

Improved recombinant gene expression in CHO cells using matrix attachment regions.

The use of animal cells such as Chinese hamster ovary (CHO) cells for recombinant gene expression provides many advantageous features such as proper folding and post-translational modification of the recombinant protein. However, recombinant genes introduced into animal cells are often expressed at low levels mainly due to position effects from the neighboring chromatin context. The tedious and time-consuming selection and amplification procedure has been the major hurdle for using animal cell line such as CHO cells. To improve mammalian cell expression systems, we screened a variety of matrix/scaffold attachment region (MAR/SAR) elements for their ability to insulate transgene expression from the position effects in CHO cells. We found that the human beta-globin MAR element is particularly effective as the frequency of beta-Gal positive colonies was increased by up to 80%. The expression levels of these colonies were also enhanced about seven-fold. These improvements appear to be related to the increased copy numbers and a higher efficiency of expression of the integrated genes. When this element was used to express soluble TGF-beta type II receptor (sTbetaRII) through the gene amplification system, the frequency of colonies expressing detectable amounts of sTbetaRII was much higher than that of the control vector. We could also generate high sTbetaRII producers with uniform growth properties by a simple two-step amplification process involving two concentrations of methotrexate. This eliminates the need to isolate individual colonies followed by multi-step treatments of methotrexate and thereby greatly simplifies this mammalian expression system.

Animals↗

PubMed2004

Specific molecular alterations in the norpA-encoded phospholipase C of Drosophila and their effects on electrophysiological responses in vivo.

A large number of mutants in the norpA gene, which encodes the phospholipase C (PLC) involved in Drosophila phototransduction, is available for the investigation of the effects of specific amino acid substitutions in PLC on biochemical and electrophysiological properties of these mutants. Of the 47 norpA mutants screened for PLC protein content, all but one (H43) displayed drastically decreased amounts of the protein suggesting that almost any mutational alteration has a deleterious effect on the integrity of the protein. Three new amino acids were identified in the catalytic domains X and Y that are important for PLC catalytic activity and the generation of photoreceptor responses (ERG). One of them was found substituted in H43, which showed a low specific PLC activity, a pronounced decrease in ERG sensitivity, and a wild-type-like response termination time. The response termination times obtained from three mutants was found to be approximately inversely proportional to the amount of PLC. In addition, we show that (i) the specific PLC activity is a key factor determining the photoreceptor sensitivity; (ii) the catalytic activity and response termination are separable functions of PLC; and (iii) a mutation in the putative G alpha-interacting C2 domain causes a preferentially strong defect in latency.

Amino Acid Sequence↗

PubMed2003

Molecular characterization of a gene encoding the Drosophila melanogaster phospholipase A2.

A gene encoding Drosophila melanogaster secretory phospholipase A2 (sPLA2) has been cloned and characterized(.) The coding region of the sPLA2 gene was interrupted by a short intron, and codes for a signal peptide of 18 amino acids, followed by a mature protein of 168 amino acids, containing the structural features of group III sPLA2. From a Northern blot analysis, about a 1.0-kb Drosophila sPLA2 transcript was found to be expressed throughout its development and in both the adult bodies and heads. The recombinant Drosophila sPLA2 expressed and purified in Escherichia coli was found to be Ca(+2)-dependent and maximally active at pH 5.

Amino Acid Sequence↗

PubMed2002

Single amino acid change in the fifth transmembrane segment of the TRP Ca2+ channel causes massive degeneration of photoreceptors.

The trp gene encodes subunits of a highly Ca(2+)-permeable class of light-activated channels of Drosophila photoreceptors. The recently characterized mutation in this gene, Trp(P365), is semidominant and causes massive degeneration of photoreceptors by making the TRP channel constitutively active. We show that a single amino acid change, Phe-550 to Ile, near the beginning of the fifth transmembrane domain of TRP channel subunits is necessary to induce, and sufficient to closely mimic, the original mutant phenotypes of Trp(P365). Hypotheses are presented as to why the amino acid residues at position 550 and its immediate vicinity might be important in influencing the regulation of the TRP channel and why the substitution of Phe for Ile at this position, in particular, could result in constitutive activity of the channel.

Amino Acid Sequence↗

PubMed2002

Transactivation of the mouse sulfonylurea receptor I gene by BETA2/NeuroD.

The sulfonylurea receptor 1 (SUR1) plays a key role in regulation of insulin secretion in pancreatic beta-cells. In this study we investigated the mechanism for tissue-specific expression of the SUR1 gene. A -138/-20 fragment exhibited basal promoter activity while the -660/-20 fragment contained a regulatory element for tissue-specific expression of the mouse SUR1 gene. A pancreatic beta-cell-specific transcription factor, BETA2 (beta-cell E box transcription factor)/NeuroD, enhanced the promoter activity of the -660/-20 fragment in cooperation with E47. Coexpression of a dominant negative mutant of BETA2/NeuroD, BETA2(1-233), repressed the promoter activity of the -660/-20 fragment. BETA2/NeuroD bound specifically to the E3 element located at -141. The E3 sequence in a heterologous context conferred transactivation by BETA2/NeuroD in HeLa and HIT cells. Mutation of E3 eliminated the stimulatory effect of BETA2/NeuroD. Unlike BETA2/NeuroD, neurogenin 3 (ngn3) could not activate the E3 element in HeLa cells. Overexpression of ngn3 concomitantly increased expression of BETA2/NeuroD and SUR1 in HIT cells but not in HeLa cells. These results indicate that BETA2/NeuroD induces tissue-specific expression of the SUR1 gene through the E3 element. These results also suggest that E3 is specific for BETA2/NeuroD, and the stimulatory effect of ngn3 in HIT cells may require factors specifically expressed in HIT cells.

ATP-Binding Cassette Transporters↗

PubMedDate not supplied

Improved mammalian expression systems by manipulating transcriptional termination regions.

Here we show that the transcriptional terminator element of human gastrin gene, which is the only element characterized to date in terms of its function in transcriptional termination, increases the transient expression levels of recombinant proteins. The expression of the beta-galactosidase gene was enhanced 3-4-fold in HeLa cells by inserting the terminator element of human gastrin gene at the 3'-side of the SV40 polyadenylation signal/cleavage site of the control vector (pSV-beta-gal). This effect of the terminator element is orientation-dependent but not cell-specific since a similar enhancement of beta-galactosidase gene expression was detected in COS.M6 and CHO DG44 cells. The increased level of beta-galactosidase gene expression by the transcriptional terminator element of human gastrin gene turned out to arise from elevated cellular mRNA levels, suggesting that the terminator element stabilizes mRNA by enhancing proper 3'-end processing of mRNA.

Animals↗

PubMedDate not supplied

Efficient selection of stable chinese hamster ovary (CHO) cell lines for expression of recombinant proteins by using human interferon beta SAR element.

We describe the development of an efficient expression system suitable for the stable expression of recombinant genes in Chinese hamster ovary (CHO) cells using the human interferon beta SAR element. The insertion of two copies of the human interferon beta SAR element at the 5' and 3' flanking regions of the beta-galactosidase reporter gene increased the frequency of beta-galactosidase positive colonies by up to 75% and enhanced beta-galactosidase expression by 15- to 20-fold after G418 selection or 30- to 40-fold at the initial stage of the MTX selection procedure. Deletion analysis showed that the whole DNA regions of the human interferon beta SAR element are required for beta-galactosidase expression enhancement. The developed expression system was also highly effective at enhancing the stable expression of two therapeutically important proteins, namely, erythropoietin (EPO) and hepatocyte growth factor (HGF). We isolated stable colonies with expression levels of 47 microg/10(6) cells/day for EPO and 13 microg/10(6) cells/day for HGF, suggesting that the developed expression system based on the human beta SAR element is suitable for expressing high levels of recombinant proteins in CHO cells.

Animals↗