• The Journal of Korean Physical Therapy
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• v.10 no.2
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• pp.113-125
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• 1998

The neurotropic psudorabies virus(PRV) to replicate within neurons is very useful pathogen for neuronal tracing. I carried out this study to investigate the parametric analysis on the viral infection in the rat circadian control center with two genetically engineered strains out of PRV. The two strains are isogenic with the attenuated Bartha strain of PRV ; in one strain a lacZ reporter gene was inserted into the gC locus (PRV-BaBlu ; $4.75\times10^8pfu/ml$) and the other strain contained a PRV envelope glycoprotein gene(PRV-D ; $2.5\times10^8pfu/ml$) theat is absent in PRV-BaBlu. simultaneous or temporally separated sequential injection of$2{\mu}l$ of each strain into the vetreous body of eye produced a course of transsynaptic infection of retinohypothalamic circuitry. The results were as follows; 1. PRV-BaBlu and PRV-D infected the suprachiasmatic nucleus in hypothalamus and intergeniculate leaflet in lateral geniculate nucleus of thalamus. 2. The rate of PRV infection was dependent upon PRV strain. 3. Pre-infected neurons by PRV-D were interfered with the replication of PRV-BaBlu. 4. Dual injection of PRV-D and PRV-BaBlu showed more virulent than the parental strain.

• Journal of Sericultural and Entomological Science
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• v.40 no.2
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• pp.105-110
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• 1998

The baculovirus egt gene encodes an ecdysteroid UDP-glucosyltransferase(EGT) which catalyzes the transfer of glucose from UDP-glucose to the insect moltion hormone ecdysteroid resulting in a functionally inactive ecdysteroid. In baculovirus-infected insect larvae, EGT has been shown block molting and pupation. In this study, we compared the development of 4th and 5th instar silkworm, Bombyx mori, larvae injected with either wild-type bombyx mori nucleopolyhedrovirus (BmNPV) or a mutant BmNPV(BmEGTZ) in which the egt gene was disrupted by the insertion of a lacZ gene cassette. Larvae injected with BmEGTZ died roughly 12 h more rapidly compared to indentical larvae infected with BmNPV. In addition, BmEGTZ- infected larvae prematurely stopped feeding and gain less weight compared to BmNPV-infected larvae. In order to investigate why BmEGTZ-infected larvae died more rapidly than BmNPV-infected larvae, the array of hemolymph proteins in BmEGTZ-or BmNPV-infected larvae were analyzed by SDS-PAGE. The hemolymph of BmEGTZ-infected larvae showed virus-specific proteins, including polyhedrin, about 12 h earlier than the hemolymph of BmNPV-infected larvae

• Journal of Microbiology
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• v.41 no.2
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• pp.109-114
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• 2003

A promoter that is inducible by paraquat and menadione, the superoxide generators, independently of soxRS has been found in front of the sufABCDSE operon in Escherichia coli. Based on the observation that SufA is a holomog of IscA that functions in the assembly of iron sulfur cluster and the sufA promoter (sufAp) contains a putative Fur-binding consensus, we investigated whether this gene is regulated by Fur, a ferric uptake regulator, When examined in several sufAp-lacZ chromosomal fusion strains, sufAp was induced by EDTA, an iron chelator and a well-known Fur-inducer, The basal level of sufA expression increased dramatically in fur mutant, suggesting repression of sufAp by Fur. The derepression in fur mutant and EDTA-induction of sufA expression required nucleotides up to -61, where a putative Fur box is located. Purified Fur protein bound to the DNA fragment containing the putative Fur box between -35 and -10 promoter elements. The regulation by Fur and menadione induction of sufAp acted independently. The rpoS mutation increased sufA induction by menadione, suggesting that the stationary sigma factor RpoS acts negatively on sufA induction.

• Journal of Microbiology and Biotechnology
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• v.15 no.3
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• pp.678-682
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• 2005

The promoter region of a carbon starvation gene isolated from Pseudomonas putida was cloned and analyzed for its potential use for in situ bioremediation and bioprocessing. We constructed a recombinant plasmid pMKD101 by cloning the 0.65 kb promoter region of the gene into the promoter proving vector, pMK301, which contains the lacZ for ${\beta}$-galactosidase activity as a reporter gene. pMKD101 was transformed into the wild-type P. putida MK1, resulting in P. putida RPD101, and analyzed for ${\beta}$-galactosidase activity under different culture conditions. When RPD101 was grown on the minimal medium plus $0.1\%$ glucose as a sole carbon source in batch cultures, ${\beta}$-galactosidase activity was found to be 3.2-fold higher during the stationary phase than during the exponential phase. In chemostat cultures, ${\beta}$-galactosidase activity was found to be 3.1-fold higher at the minimal growth rate (dilution rate=$0.05\;h^{-1}$) than at the maximal growth rate (dilution rate=$0.173;h^{-1}$). The results suggest that a carbon starvation promoter can be utilized to maximize the expression of a desired gene under nutrient limitation.

• Archives of Pharmacal Research
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• v.23 no.2
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• pp.139-146
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• 2000

7- Bromomethylbenz[a]anthracene is a known mutagen and carcinogen. The two major DNA adducts produced by this carcinogen, i.e., $N^2$-(benz[a]anthracen-7-yl methyl)-2'-deoxyguanosine (2, b[a]$a^2$G) and $N^6$-(benz[a]anthracen-7-ylmethyl)-2'-deoxyadenosine (4, b[a]$a^6$/A), as wel 1 as the simpler benzylated analogs,$N^2$-benzyl-2'deoxyguanosine (1, $bn^2$G) and $N^6$-benzyl-2'-deoxyadenosine (3, $bn^6$/A), were prepared by direct aralkylation of 2'-deoxyguanosine and 2'-deoxyadenosine. To determine the site-specific mutagenicity of these bulky exocyclic amino-substituted adducts, the suitably protected nucleosides were incorporated into 16-base oligodeoxyribonucleotides in place of a normal guanine or adenine residues which respectively are part of the ATG initiation codon for the lac Z' \alpha-complementation gene by using an in situ activation approach and automated phosphite triester synthetic methods. The base composition and the incorporation of the bulky adducts into synthetic oligonucleotides were characterized after purification of the modified oligonucleotides by enzymatic digestion and HPLC analysis.

• Molecules and Cells
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• v.19 no.1
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• pp.23-30
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• 2005

Spinocerebellar ataxia type 1 (SCA1) is an autosomal-dominant neurodegenerative disorder caused by expansion of the polyglutamine tract in the SCA1 gene product, ataxin-1. Using d2EGFP, a short-lived enhanced green fluorescent protein, we investigated whether polyglutamine-expanded ataxin-1 affects the function of the proteasome, a cellular multicatalytic protease that degrades most misfolded proteins and regulatory proteins. In Western blot analysis and immunofluorescence experiments, d2EGFP was less degraded in HEK 293T cells transfected with ataxin-1(82Q) than in cells transfected with lacZ or empty vector controls. To test whether the stability of the d2EGFP protein was due to aggregation of ataxin-1, we constructed a plasmid carrying $ataxin-1-{\Delta}114$, lacking the self-association region (SAR), and examined degradation of the d2EGFP. Both the level of $ataxin-1-{\Delta}114$ aggregates and the amount of d2EGFP were drastically reduced in cells containing $ataxin-1-{\Delta}114$. Furthermore, d2EGFP localization experiments showed that polyglutamine-expanded ataxin-1 inhibited the general function of the proteasome activity. Taken together, these results demonstrate that polyglutamine-expanded ataxin-1 decreases the activity of the proteasome, implying that a disturbance in the ubiquitin-proteasome pathway is directly involved in the development of spinocerebellar ataxia type1.

• Molecules and Cells
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• v.38 no.11
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• pp.1007-1012
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• 2015

EphA7 is a key molecule in regulating the development of the dien- and mesencephalon. To get insight into the mechanism of how EphA7 gene expression is regulated during the dorsal specification of the dien- and mesencephalon, we investigated the cis-acting regulatory sequence driving EphA7 to the dorsal midline of the dien- and mesencephalon. Transgenic LacZ reporter analysis, using overlapping EphA7 BACs, was used to narrow down the dorsal midline-specific enhancer, revealing the 25.3 kb genomic region as the enhancer candidate. Strikingly, this genomic DNA was located far downstream of the EphA7 transcription start site, +302.6 kb to +327.9 kb. Further enhancer mapping, using comparative genomic analysis and transgenic methods, showed that the 187 bp genomic DNA alone, approximately 305 kb downstream of the EphA7 transcription start site, was sufficient to act as the dorsal midline-specific enhancer of EphA7. Importantly, our results indicate that the 187 bp dorsal midline-specific enhancer is critically regulated by homeobox transcription factors during the development of the dien- and mesencephalon.

• Journal of Microbiology
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• v.35 no.1
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• pp.61-65
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• 1997

A novel strategy was developed for isolating suppressors from sporulation-deficient mutants. The mutation in the BCY1 gene, which codes for the regulatory subunit of cAMP-dependent protein kinase, when homozygous, results in diploids being meiosis and sporulation deficient. Two plasmids, YCp-MAT.alpha. and YEp-SPOT7-lacZ, were introduced into MAT.alpha. BCY1$\^$+/ or MAT.alpha. bcy1 haploid cells. The transformant of the BCY1$\^$+/ haploid cell produced .betha.-galactosidase under nutrient starvation, but the bcy1 transformant did not. Using this system, the mutagenesis experiment performed on the bcy1 transformant strain resulted in a number of sporulation mutants that produced .betha.-galactosidase under nutrient starvation. One complementation group, sob1, was identified from the isoalted suppressor mutants and characterized as a single recessive mutation by tetrad analysis. Genetic analysis revealed that the sob1 mutation suppressed the sporulation deficiency, the failure to arrest at the G1 phase of the cell cecle, and the sensitivity to heat or nitrogen starvation caused by the bcy1 mutation. However, the sob1 mutation did not suppress the sporulation deficiency of ime1 and of ime2 diploids. These results suggest that the sob1 mutation affects a gene which functions as a downstream regulator in both meiosis and cell cycle regulation.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 2004.06a
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• pp.236-241
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• 2004

In Escherichia coli, L-lysine biosynthetic pathway is composed of nine enzymatic reactions. It has been well established that most of the lysine biosynthetic genes are regulated by the lysine availability, even though they are all scattered around the chromosome without forming any multigenic operon structure. However, no transcriptional regulatory mechanism has been identified except for the activation of lysA gene by the LysR. In this study, changes in transcriptome profiles of wild type cells and lysR deletion mutant cells grown in the absence or presence of lysine were investigated by using DNA microarray technique. Microarray data analysis revealed three groups of genes whose expression varies depending on the availability of lysine or LysR or both. To further examine the regulatory patterns of lysine biosynthetic genes, lacZ operon fusions were constructed and their expression was measured under various conditions. Obtained results strongly suggest that there is an additional regulatory mechanism which senses the lysine availability and coordinates gene expression.

• Journal of Microbiology and Biotechnology
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• v.28 no.6
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• pp.1030-1036
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• 2018

Bacillus strains produce various types of antibiotics, and random mutagenesis has traditionally been used to overproduce these natural metabolites. However, this method leads to the accumulation of unwanted mutations in the genome. Here, we rationally designed a single nucleotide substitution in the degU gene to generate a B. subtilis strain displaying increased plipastatin production in a foreign DNA-free manner. The mutant strain (BS1028u) showed improved antifungal activity against Pythium ultimum. Notably, pps operon deletion in BS1028u resulted in complete loss of antifungal activity, suggesting that the antifungal activity strongly depends on the expression of the pps operon. Quantitative real-time PCR and lacZ assays showed that the point mutation resulted in 2-fold increased pps operon expression, which caused the increase in antifungal activity. Likewise, commercial Bacillus strains can be improved to display higher antifungal activity by rationally designed simple modifications of their genome, rendering them more efficient biocontrol agents.