• Microbiology and Biotechnology Letters
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• v.18 no.5
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• pp.541-546
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• 1990

Bacillus sphaericus ts-Dl290 was characterized by SDS-PAGE produced by the mutant at $30^{\circ}C$ and $42^{\circ}C$. The total amount of proteins produced by the mutant at $42^{\circ}C$ decreased to one-fifth of those at $30^{\circ}C$; however, when the culture was shifted down from $42^{\circ}C$ after 4 to $30^{\circ}C$, the total amount of protein decreased to one-third and the 221 kd protein did not appear, but the 155 kd appeared remarkably. When the mutant and the wild type strain were cultured in the media containing 80$\mu g$ per ml of chloramphenicol at $42^{\circ}C$, the wild type strain synthesized half amounts of the total proteins than those at $30^{\circ}C$, and the mutant produced one-tenth of the total protein amounts. When the both strains were cultured in the media containing chloramphenicol, the 155 kd protein was produced was produced in lesser amounts than those without chloramphenicol. The 150 kd protein showed lethal activity to Culex pipiens 3rd instar larvae.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.24-25
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• 2003

The zebrafish (Danio rerio) is now the pre-eminent vertebrate model system for clarification of the roles of specific genes and signaling pathways in development. I will talk about positional cloning of two developmental mutants in zebrafish. The first mutant is headless: The vertebrate organizer can induce a complete body axis when transplanted to the ventral side of a host embryo by virtue of its distinct head and trunk inducing properties. Wingless/Wntantagonists secreted by the organizer have been identified as head inducers. Their ectopic expression can promote head formation, whereas ectopic activation of Wnt signalling during early gastrulation blocks head formation. These observations suggest that the ability of head inducers to inhibit Wntsignalling during formation of anterior structures is what distinguishes them from trunk inducers that permit the operation of posteriorizing Wnt signals. I describe the zebrafish headless (hdl) mutant and show that its severe head defects are due to a mutation in T-cell factor-3 (Tcf3), a member of the Tcf/Lef family. Loss of Tcf3 function in the hdl mutant reveals that hdl represses Wnt target genes. I provide genetic evidence that a component of the Wntsignalling pathway is essential in vertebrate head formation and patterning. Second mutant is mind bomb: Lateral inhibition, mediated by Notch signaling, leads to the selection of cells that are permitted to become neurons within domains defined by proneuralgene expression. Reduced lateral inhibition in zebrafish mib mutant embryos permits too many neural progenitors to differentiate as neurons. Positional cloning of mib revealed that it is a gene in the Notch pathway that encodes a RING ubiquitin ligase. Mib interacts with the intracellular domain of Delta to promote its ubiquitylation and internalization. Cell transplantation studies suggest that mib function is essential in the signaling cell for efficient activation of Notch in neighboring cells. (중략)

• Molecules and Cells
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• v.19 no.2
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• pp.219-222
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• 2005

The a-subunit of Escherichia coli tryptophan synthase (${\alpha}TS$), a component of the tryptophan synthase ${\alpha}_2{\beta}_2$ complex, is a monomeric 268-residues protein (Mr = 28,600). ${\alpha}TS$ by itself catalyzes the cleavage of indole-3-glycerol phosphate to glyceraldehyde-3-phosphate and indole, which is converted to tryptophan in tryptophan biosynthesis. Wild-type and P28L/Y173F double mutant ${\alpha}$-subunits were overexpressed in E. coli and crystallized at 298 K by the hanging-drop vapor-diffusion method. X-ray diffraction data were collected to $2.5{\AA}$ resolution from the wild-type crystals and to $1.8{\AA}$ from the crystals of the double mutant, since the latter produced better quality diffraction data. The wild-type crystals belonged to the monoclinic space group C2 ($a=155.64{\AA}$, $b=44.54{\AA}$, $c=71.53{\AA}$ and ${\beta}=96.39^{\circ}$) and the P28L/Y173F crystals to the monoclinic space group $P2_1$ ($a=71.09{\AA}$, b=52.70, $c=71.52{\AA}$ and ${\beta}=91.49^{\circ}$). The asymmetric unit of both structures contained two molecules of ${\alpha}TS$. Crystal volume per protein mass ($V_m$) and solvent content were $2.15{\AA}^3\;Da^{-1}$ and 42.95% for the wild-type and $2.34{\AA}^3\;Da^{-1}$ and 47.52% for the double mutant.

• International Journal of Industrial Entomology and Biomaterials
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• v.19 no.1
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• pp.199-204
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• 2009

The newly cloned Bacillus thuringiensis cry1-5 gene showed high activity to both Plutella xylostella and Spodoptera exigua, while cry1Ac only showed high activity against P. xylostella but low to S. exigua. Through the alignment of amino acid sequences between Cry1Ac and Cry1-5, we found 12 different residues in domain I (6 residues) and domain II (6 residues). In this study, the modified cry1Ac gene, which is constructed according to a crop-preferring codon usage, was used as a template to construct mutant B. thuringiensis cry1Ac genes based on cry1-5 gene through multi site-directed mutagenesis. Total 63 various mutant cry genes were obtained at 12 positions randomly. Among them, ten mutant cry genes, whose domain I was totally converted and domain II was randomly, were selected to express in baculovirus expression system as a polyhedrin fusion form. The recombinant proteins were 95 kDa in size and were stably activated as 65 kDa by trypsin. The expressed mutant Cry proteins were applied to bioassays against P. xylostella and S. exigua. All mutants showed high insecticidal activity both to P. xylostella and S. exigua similar to cry1-5. These results suggest that these mutant cry genes might be expected of desirable cry genes for introduction to transgenic crops.

• Journal of Microbiology
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• v.40 no.4
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• pp.305-312
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• 2002

The effects of two natural polyamines (putrescine and spermidine) on the synthesis of ArcA, a response regulator of the Arc two-component signal transduction system, were studied using an E. coli mutant deficient in polyamine biosynthesis. Endogenous polyamine deficiency of the mutant resulted in marked reduction in the ArcA level determined by Western blot analysis. Putrescine supplement to the growth medium effectively increased the ArcA level of the mutant in a concentration-dependent manner. Spermidine also stimulated the ArcA level in the mutant to a greater degree than putrescine. Expression of arcA'::lacZ operon fusion in the mutant was stimulated 6-fold and 10-fold by putrescine and spermidine at a 1mM concentration, respectively, indicating that the stimulatory effect of the polyamines on ArcA synthesis is due to transcriptional induction, and that spermidine is a more potent arcA inducer than putrescine. The polyamine-dependent arcA'::lacZ induction was growth-phase-dependent and independent of either arcA or fnr which are two regulators involved in anaerobic stimulation of the Arch level. These results suggested that putrescine and spermidine polyamines may be potential intracellular signal molecules in the control of arcA expression, and thereby may play an important role in cellular metabolism.

• Journal of Pharmaceutical Investigation
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• v.33 no.4
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• pp.267-272
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• 2003

Herpes simplex virus (HSV) thymidine kinase (TK) has been widely used for suicidal gene therapy in combination with nucleoside analogs such as ganciclovir (GCV). The use of HSV-TK is limited due to the side effect of GCV at high concentrations. Previous studies showed that stable transfectants of mutant HSV-TK with enhanced affinity to GCV were killed at lower GCV concentrations. In this study, we tested whether mutant HSV-TK can provide enhanced suicidal effect when transiently transfected with Epstein-Barr virus (EBV)-based plasmid. EBV-based plasmid which contains OriP and EBNA-1 sequence is well known for a stable episomal maintenance in human cells. Optimal transfection condition was assessed for SNU-638 gastric cancer cell line using polyetylnimine (PEI). Maximum transfection efficiency was achieved when DNA:PEI was 1:3 (w/v). Cytotoxicities of mutant and wild type HSV-TK were compared before and after partially selecting transfected cells. The cells were sensitive to $100\;{\mu}g/ml$ hygromycin. Following GCV treatment, more cells were killed after hygromycin selection than before selection. The mutant HSV-TK showed enhanced cytotoxicity compared with the wild type HSV-TK. Our results suggest that the EBV-based plasmid encoding mutant HSV-TK may be useful to treat the diseases caused by uncontrolled cell proliferation such as cancer and rheumatoid arthritis.

• Proceedings of the Korean Society for Bioinformatics Conference
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• 2003.10a
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• pp.277-287
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• 2003

Acetolactate synthase (ALS, EC 4.1.3.18; also referred to as acetohydroxy acid synthase) catalyzes the first common step in the biosynthesis of valine, leucine, and isoleucine in microorganisms and plants. Recently X-ray structure of yeast ALS was available. Pair-wise alignment of yeast and tobacco ALS sequences revealed 63% sequence similarity. Using Deep View and automatic modeling on Swiss model server, we have generated reliable models of tobacco ALS based on yeast ALS template with a calculated pair-wise RMSD of 0.86 Angstrom. Functional roles of four residues located on the subunit interface (H142, El43, M350, and R376) were examined by site-directed mutagenesis. Seven mutants were generated and purified, of which three mutants (H142T, M350V, and R376F) were found to be inactivated under various assay conditions. The H142k mutant showed moderately altered kinetic properties. The E143A mutant increased 10-fold in K$_m$ value while other parameters remained unchanged. The M350C mutant was strongly resistant to three tested herbicides, while the R376k mutant can bind with herbicide carder at similar affinity to that of wild type enzyme, as determined by tryptophan quenching study. Except M350V mutant, all other mutants were ate to bind with cofactor FAD. Taken together, it is likely that residues H142 and E143 are located at the active site, while residues M350 and R376 are possibly located at the overlapping region of active site and herbicide binding site of the enzyme. Our data also allows us to hypothesize that the interaction between side chains of residues M350 and R376 are probably essential for the correct conformation of the active site. It remains to be elucidated that, whether the herbicide, upon binding with enzyme, inactivates the enzyme by causing change in the active site allosterically, which is unfavorable for catalytic activity.

• Journal of Microbiology and Biotechnology
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• v.7 no.4
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• pp.215-222
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• 1997

The regulatory mechanism of the flux of acetyl-CoA in Alcaligenes eutrophus in unbalanced growth conditions was investigated using a PHB-negative mutant and transformants reintroduced PHB-biosynthesis enzymes through the transformation of cloned phbCAB genes. The PHB-negative mutant was defected absolutly in PHB synthase but partially in ${\beta}$-ketothiolase and acetoacetyl-CoA reductase, and excreted substantial amount of pyruvate to culture broth at late growth phase. The excretion was due to the inhibitory effect of acetyl-CoA on the activity of pyruvate dehydrogenase. The cloned phbC and phbCAB genes were transformed to the PHB-negative mutant strain to reintroduce PHB biosythesis enzymes. Pyruvate excretion could be decreased substantially but not completely by transformation of PHB synthase alone, while pyruvate excretion was ceased by transformation of all three PHB biosynthesis enzymes. To identify the most critical PHB biosynthesis enzyme influencing on the flux of acetyl-CoA, the effect of the variation of PHB biosynthesis enzymes on pyruvate dehydrogenase was investigated. ${\beta}$-Ketothiolase influenced the activity of pyruvate dehydrogenase more sensitively than PHB synthase. ${\beta}$-Ketothiolase, the first step enzyme of PHB biosynthesis that condense acetyl-CoA to acetoacetyl-CoA, seems to be the major enzyme determining the flux of acetyl-CoA to PHB biosynthesis or TCA cycle, and the rate of PHB biosynthesis in A. eutrophus.

• Journal of Microbiology and Biotechnology
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• v.20 no.1
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• pp.146-152
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• 2010

Streptomyces clavuligerus NRRL3585 produces a clinically important $\beta$-lactamase inhibitor, clavulanic acid (CA). In order to increase the production of CA, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene (gap) was deleted in S. clavuligerus NRRL3585 to overcome the limited glyceraldehyde-3-phosphate pool; the replicative and integrative expressions of ccaR (specific regulator of the CA biosynthetic operon) and claR (Lys-type transcriptional activator) genes were transformed together into a deletion mutant to improve clavulanic acid production. We constructed two recombinant plasmids to enhance the production of CA in the gap1 deletion mutant of S. clavuligerus NRRL3585: pHN11 was constructed for overexpression of ccaR-claR, whereas pHN12 was constructed for their chromosomal integration. Both pHN11 and pHN12 transformants enhanced the production of CA by 2.59-fold and 5.85-fold, respectively, compared with the gap1 deletion mutant. For further enhancement of CA, we fed the pHN11 and pHN12 transformants ornithine and glycerol. Compared with the gap1 deletion mutant, ornithine increased CA production by 3.24- and 6.51-fold in the pHN11 and pHN12 transformants, respectively, glycerol increased CA by 2.96- and 6.21-fold, respectively, and ornithine and glycerol together increased CA by 3.72- and 7.02-fold, respectively.

• Journal of Microbiology and Biotechnology
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• v.26 no.7
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• pp.1163-1172
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• 2016

The Ω-loop is a nonregular and flexible structure that plays an important role in molecular recognition, protein folding, and thermostability. In the present study, molecular dynamics simulation was carried out to assess the molecular stability and flexibility profile of the porcine trypsin structures. Two Ω-Loops (fragment 57-67 and fragment 78-91) were confirmed to represent the flexible region. Subsequently, glycosylation site-directed mutations (A73S, N84S, and R104S) were introduced within the Ω-loop region and its wing chain based on its potential N-glycosylation sites (Asn-Xaa-Ser/Thr consensus sequences) and structure information to improve the thermostability of trypsin. The result demonstrated that the half-life of the N84S mutant at 50℃ increased by 177.89 min when compared with that of the wild-type enzyme. Furthermore, the significant increase in the thermal stability of the N84S mutant has also been proven by an increase in the T_m values determined by circular dichroism. Additionally, the optimum temperatures of the wild-type enzyme and the N84S mutant were 75℃ and 80℃, respectively. In conclusion, we obtained the thermostability-improved enzyme N84S mutant, and the strategy used to design this mutant based on its structural information and N-linked glycosylation modification could be applied to engineer other enzymes to meet the needs of the biotechnological industry.