• BMB Reports
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• v.33 no.6
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• pp.483-492
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• 2000

The Herpes simplex virus type 1 (HSV-1) strain F glycoprotein H (gH) gene in the pHLB-4 plasmid was recombinated into a baculovirus expression vector (lacZ-HcNPV) to construct a recombinant virus GH-HcNPV expressing gH. The sequences of gH and its expression were analyzed. The gH gene was located in the 6.41 kb BglII fragment. The open reading frame (ORF) of the gH gene was 2,517 bp and codes 838 amino acid residues. Insect cells infected with this recombinant virus synthesized a high level of the matured and gX-gH fusion protein with approximately 112 kDa. The fusion gH protein was localized on the membrane of the insect cells as seen by using immunofluorescence assay and accumulated in the cultured media by the SDS-PAGE and immunoprecipitation assays. The amino acid sequence presents additional characteristics compatible with the structure of a viral glycoprotein: signal peptide, putative glycosylation sites and a long C-terminal transmembrane sequence. Antibodies raised in mice to this recombinant protein recognized viral gH and neutralized the infectivity of HSV-1 in vitro. These results demonstrate that it is possible to produce a mature protein by gene transfer in eukaryotic cells, and indicate the utility of the HcNPV-insect cell system for producing and characterizing eukaryotic proteins. Furthermore, the neutralizing antibodies would appear to protect mice against HSV; accordingly, this particular recombinant protein may be useful in the development of a subunit vaccine.

• Microbiology and Biotechnology Letters
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• v.17 no.4
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• pp.334-342
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• 1989

The Bacillus stearothermophilus cdd gene encoding cytidine deaminase (cytidine/2'-deoxycytidine aminohydrolase; EC 3.5.4.5) was isolated through shot gun cloning by oomplementation of an E. coli cdd mutation. Primarily 3.0 kbp of the exogenote was cloned into the Pstl site of pBR322 (pJSC101). By subsequent deletion and subcloning from the insert of pJSC101 with cdd$^+$ and tetracycline resistancy, about 1.35 kbp of the EcoRI$_1$/PstI$_2$ fragment containing the cdd gene was isolated as pJSC201. The minicell experiment revealed a molecular mass of 33,000 dalton for polypeptide from the cloned DNA fragment complementing the cdd gene. From the lacZ fusion of 550 bp fragment of the EcoRI$_1$/AuaI as a putative promoter region, the transcription direction of the cdd gene on pJSC201 is from EcoRI towards the PstI sites, When the cdd gene was expressed in B. subtilis ED4O (cdd$^-$, pyr$^-$) by transformation with the E. coli-B. subtilis shuttle vector, the gene expression occured more efficiently than in E. coli and the gene appears to be stably maintained in B. subtitis as well as in E. coli.

• Journal of Life Science
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• v.14 no.2
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• pp.325-330
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• 2004

The RAD3 gene of Saccharomyces cerevisiae is required for excision repair and is essential for cell viability. RAD3 encoded protein possesses a single stranded DNA-dependent ATPase and DNA and DNA-RNA helicase activities. To examine the extent of conservation of structure and function of RAD3 during eukaryotic evolution, the RAD3 homolog gene was isolated by screening of genomic DNA library. The isolated gene was designated as HRD3 (Homologue of RAD3 gene). The over-expressed HRD3 protein was estimated to be a 75 kDa in size which is in good agreement with the estimated by the nucleotide sequence of the cloned gene. Two-dimensional gel electrophoresis showed that a number of other protein spots dramatically disappeared when the HRD3 protein was overexpressed. The overexpressed RAD3 protein showed a toxicity in E. coli host, suggesting that this protein may be involved in the inhibition of protein synthesis and/or degradation of host protein. To determine which part of HRD3 gene contributes to the toxicity in E. coli, various fusion plasmids containing a partial sequence of HRD3 and lac'Z gene were constructed. These results suggest that the C-terminal domain of HRD3 protein may be important for both toxic effect in E. coli and for its role in DNA repair in S. pombe.

• The Plant Pathology Journal
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• v.35 no.4
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• pp.351-361
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• 2019

In our previous study, pyrrolnitrin produced in Pseudomonas chlororaphis G05 plays more critical role in suppression of mycelial growth of some fungal pathogens that cause plant diseases in agriculture. Although some regulators for pyrrolnitrin biosynthesis were identified, the pyrrolnitrin regulation pathway was not fully constructed. During our screening novel regulator candidates, we obtained a white conjugant G05W02 while transposon mutagenesis was carried out between a fusion mutant $G05{\Delta}phz{\Delta}prn::lacZ$ and E. coli S17-1 (pUT/mini-Tn5Kan). By cloning and sequencing of the transposon-flanking DNA fragment, we found that a vfr gene in the conjugant G05W02 was disrupted with mini-Tn5Kan. In one other previous study on P. fluorescens, however, it was reported that the deletion of the vfr caused increased production of pyrrolnitrin and other antifungal metabolites. To confirm its regulatory function, we constructed the vfr-knockout mutant $G05{\Delta}vfr$ and $G05{\Delta}phz{\Delta}prn::lacZ{\Delta}vfr$. By quantifying ${\beta}-galactosidase$ activities, we found that deletion of the vfr decreased the prn operon expression dramatically. Meanwhile, by quantifying pyrrolnitrin production in the mutant $G05{\Delta}vfr$, we found that deficiency of the Vfr caused decreased pyrrolnitrin production. However, production of phenazine-1-carboxylic acid was same to that in the wild-type strain G05. Taken together, Vfr is required for pyrrolnitrin but not for phenazine-1-carboxylic acid biosynthesis in P. chlororaphis G05.

• Korean Journal of Microbiology
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• v.37 no.3
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• pp.228-233
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• 2001

The acid tolerance response (ATR) of log-phase Salmouella enterica seroyar Typhimurium is induced by acid adaptation below pH4.5 and will protect cells against more severe acid. Two distinctive ATR systems in thisorganism are a log-phase and stationary-phase ATR in which acid adaptations trigger the synthesis of acid shockproteins (ASPs). We found that log-phase ATR system was strongly affected by environmental factor, low tem-perature, $25^{\circ}C$. Exposure to low temperature and mild acid has been shown to increase acid survival dra-matically, and this survival rate was showed higher than $37^{\circ}C$. Especially unadapted cells at $25^{\circ}C$ presented tenthousand folds survival increasing when compared with cells at $37^{\circ}C$. The degree of acid tolerance of rpoSwhich is blown to be required for acid tolerance more increase than $37^{\circ}C$. Even though AIR pattern of rpoSbetween unadapted and adapted was showed similar at pH 3.1, rpoS-dependent ATR system also has beendetected in low temperature because rpoSAp prevents sustained acid survival at $25^{\circ}C$. Therefore the resultssuggest low temperature ATR system requires rpoS-dependent and -independent both. To investigate the basisfor low temperature related ATR system, gene that was participated for low temperature acid tolerance (lat) wasscreened in virulent S. enterica serovar Typhimurium UKl Using the technique of P22- MudJ (Km, lacZ)-directed lacZ operon fusion, LF452 latA‥‥MudJ was isolated. The latA‥‥MudJ of S. enterica Typhimurium pre-vented low temperature acid tolerance response. Therefore latA is considered one of the important genes for acidadaptation.

• BMB Reports
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• v.29 no.3
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• pp.221-224
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• 1996

To examine the growth-phase dependent control of Escherichia coli rnpB gene we used a combination of Northern analysis for RNA determination and Southern analysis for plasmid DNA determination. The relative amounts of metabolically unstable transcript derived from the internally deleted rnpB gene harbored on a multicopy plasmid as well as the relative plasmid contents were measured by Northern analysis and Southern analysis, respectively, of total nucleic acids from E coli cells containing the plasmid. The relative transcription activity of the rnB was represented by a ratio of the relative amount of the transcript to that of the plasmid DNA during bacterial growth. The rnpB transcription increased rapidly with time during exponential growth, but started to decrease before the transition period of an exponential growing cell culture into the stationary phase. Although the expression pattern was similar to the changes of ${\beta}-galactosidase$ activity expressed from the lysogenic strain carrying the chromosomal rnpB-lacZ fusion which were shown in a previous work, the present data appears to represent a more actual growth-phase control of the rnpB transcription than the previous data by the ${\beta}-galactosidase$ assay. In addition the present method described for a direct analysis of both RNA and plasmid DNA provides a rapid and efficient method that can applied to an examination of transcription control by using a multicopy plasmid.

• Journal of Microbiology
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• v.42 no.3
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• pp.233-238
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• 2004

Transcriptional regulation of the Schizosaccharomyces pombe y-glutamylcysteine synthetase (GCS) gene was examined using the two GCS-lacZ fusion plasmids pUGCS101 and pUGCS102, which harbor 607 bp and 447 bp upstream regions, respectively. The negatively-acting sequence was located in the -607 - -447 bp upstream region of the GCS gene. The upstream sequence responsible for induction by menadione(MD) and L-buthionine-(S, R)-sulfoximine (BSO) resides in the -607 - -447 bp region, whereas the sequence which codes for nitric oxide induction is located within the -447 bp region, measured from the translational initiation point. Carbon source-dependent regulation of the GCS gene appeared to be dependent on the nucleotide sequence within -447 bp region. The transcription factor Papl is involved in the induction of the GCS gene by MD and BSO, but not by nitric oxide. Induction of the GCS gene occurring due to low glucose concentration does not depend on the presence of Pap1. These data imply that induction by MD and BSO may be mediated by the Pap1 binding site, probably located in the -607 - -447 region, and also that the nitric oxide-mediated regulation of the S. pombe GCS gene may share a similar mechanism with its carbon-dependent induction.

• Journal of Life Science
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• v.16 no.4
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• pp.632-639
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• 2006

Here we examined the expression patterns and regulation of seven photosynthesis (PS) genes (puf, puc, puhA, bchC, bchE, bchF, and bchI) in the anoxygenic photosynthetic bacterium, Rhodobacter sphaeroides, based on lacZ reporter gene assay. Expression of the tested PS genes, except puhA and bchI, were strongly induced in R. sphaeroides grown under anaerobic conditions relative to that under aerobic conditions. The puhA and bchI genes appear to form the operons together with bchFNBHLM-RSP0290 and crtA, respectively. Expression of the puf, puc, and bchCXYZ operons in R. sphaeroides grown photosynthetically was proportional to the incident light intensity, whereas that of bchFNBHLM(RSP0290-puhA) was inversely related to light intensity. Expression of bchEJG was lowest under medium-light photosynthetic conditions $(10\;W/m^2)$ and highest under high light conditions $(100\;W/m^2)$. The regulation of PS genes by the three major regulatory systems involved in oxygen- and light-sensing in R. sphaeroides is as following: puf and bchC are regulated by both the PpsR repressor and the PrrBA two-component system. The puc operon is under control of PpsR, FnrL, and PrrBA system. Expression of bchE is controlled by FnrL and PrrBA two-component system, whereas bchF is regulated exclusively by PpsR. It was demonstrated that the PpsR repressor is responsible for high-light repression of bchF and that FnrL might be involved in perceiving the cellular redox state in addition to sensing $O_2$ itself.

• Journal of Life Science
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• v.18 no.2
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• pp.206-212
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• 2008

The quorum sensing (QS) regulatory network has been the subject of extensive studies during recent years and has also attracted a lot of attention because it both positively and negatively regulates various putative virulence factors, although initially considered to be a specialized system of Vibrio fischeri and related species. In this study, to identify the novel materials which inhibit QS system of microorganisms, extracts of eighteen natural products were tested by bioassay using N-(3-oxohexanoyl)-$_L$-homoserine lactone and N-(3-oxooctanoyl)-$_L$-homoserine lactone synthesized in this experiment and an Agrobacterium tumefaciens NT1 biosensor strain containing a traI::lacZ fusion. The result indicated that the extracts of cabbage, leek, and onion exhibited the QS inhibition activity. Thus, materials contained in the extracts were isolated via recycling preparative HPLC and were purified via a JAIGEL-LS255 column. The common fraction corresponding to a peak of the 83 min point of them quenched the quorum sensing of A. tumefaciens NT1 biosensor strain in ABMM containing X-gal and was designated quorum sensing inhibitor-83 min (QSI-83). The QSI-83 exhibited the heat stability and did not inhibit the growth of A. tumefaciens NTl. Furthermore, thin layer chromatography (TLC) results suggested that these novel materials may be antagonists of N-acyl homoserine lactone or may inhibit the QS autoinducer synthesis by Pseudomonas syringae pv. tabaci.