• Journal of Microbiology and Biotechnology
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• v.9 no.3
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• pp.352-357
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• 1999

Enterobacter sp. B54 inhibited growth of the fungus Phytophthora capsici on potato dextrose agar (PDA). Three mutants with antifungal activities (denoted M54-47, M54-113, and M54-329) which were lost or increased, through Pl::Tn5 lac mutagenesis, were used to isolate genes responsible for fungal inhibition on PDA. Two clones were selected from the partially EcoR1-digested genomic library of the wild-type strain by probing with genomic flanking sequences of each mutant. We have isolated a 20-kb EcoR1 genomic DNA fragment from this strain that contains genes involved in hyphal growth inhibition of P. capsici on PDA. Subcloning and expression analysis of the above DNA fragment identified a 8-kb region which was necessary for antifungal activities. A 8-kb HindⅢDNA fragment covers three genomic loci inserted by Tn5 lac in each mutant. This suggested that all genes which are related to antifungal activities might be clustered in simple forms of at least 5-8 kb sizes.

• Microbiology and Biotechnology Letters
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• v.22 no.6
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• pp.593-598
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• 1994

A bacterium KL-57 which exhibited biosurfactant activity was isolated. This bacterium was identified as Bacillus subtilis. The biosurfactant-producing gene of B. subtilis KL-57 was cloned into R subtilis MI113 by using plasmid pTB523. The plasmid DNA from the clone was found to carry a 18 kb PstI insert. The biosurfactant-producing gene was cleaved into 4 fragments by SmaI, 3 fragments by PvulI or EcoRl, 4 fragments by PvulI and EcoRI double digestion, 5 fragments by AccI, and 2 fragments by KpnI, HindIII or BamHI. By subcloning the 18 kb Pstl insert, a 2.3 kb EcoRl fragment conferred the biosurfactant producing activity on B. subtilis cells. The 2.3 kb had one HindIII cleave site. But Two fragments, which corresponds HindIII/EcoRl termini, exhibited no biosurfactant activity.

• Journal of Food Hygiene and Safety
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• v.6 no.3
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• pp.133-137
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• 1991

In order to increase the expression of XMP aminase [EC 6.3.4.1], which catalizes the conversion of 5'-XMP to the DNA fragment containing gua A gene coding for XMP aminase from pLC 34-10 plasmid was subcloned into pBR 322, and 1.7 kb gua A gene fragment was recloned under the control of trp promoter of pDR 720, E. coli expression vector. XMP aminase activity had increased by about 17 times when compared with that of the strain earring pLC 34-10.

• Proceedings of the Zoological Society Korea Conference
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• 1996.10a
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• pp.195.2-195
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• 1996

acs gene cloning was constructed by subcloning the 2.2-kb MunI-MunI restriction fragment of 638 and 639 which include acs gene from the kohara phage into the unique EcoRI site of pUC18 and pJM9131 containing the PHA biosynthesis genes. Then recombinant E. coli fadRatoC(Con) mutants containing the polyhydroxyalkanoate(PHA) biosynthesis genes are able to incoporate s significant levels of 3-hydroxyvalerate (3HV) into the copolymer [P(3HB-co-3HV)]. Quantitative determination of PHB and P(3HB-co-3HV) was performed by gas-chromatographic analysis of extracts obtained from methanolysis of lyophilized cells.

• Journal of Plant Biology
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• v.32 no.2
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• pp.79-87
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• 1989

Southern hybridization of genomic DNAs with radioactively labeled cDNA of tomato proteinase inhibitor II revealed that proteinase inhibitor II proteins in potato plants are encoded by a family of about 10 related sequences. Screening of potato EcoRI genomic library with the cDNA resulted in isolation of 13 recombinant phage clones which carry 3 different genomic regions. Of these clones, clones 8, 18, and 39 were subjected to restriction mapping and subcloning. Further characterization of the subclones of clones 8, 18 and 39 indicated that two inhibitor II genes are present on a 8.0 kb EcoRI fragment of clone 8, one on 3.3 and 0.8 kb EcoRI fragments of clone 18 and two genes on a 13.5 kb EcoRI fragment of clone 39.

• Archives of Pharmacal Research
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• v.21 no.4
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• pp.470-474
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• 1998

A kanamycin producer, Streptomyces kanamyceticus IFO 13414 is highly resistant to kanamycin. Cloning of the kanamycin resistance genes in S. lividans 1326 with pIJ702 gave several kanamycin resistant transformants. Two transformants, S. lividans SNUS 90041 and S. lividan. SNUS 91051 showed similar resistance patterns to various aminoglycoside antibiotics. Gene mapping experiments revealed that plasmids pSJ5030 and pSJ2131 isolated from the transformants have common resistant gene fragments. Subcloning of pSJ5030 gave a 1.8 Kb gene fragment which showed resistance to kanamycin. Cell free extracts of S. lividans SNUS 90041, S. lividans SNUS 91051 and subclone a S. lividans SNUS 91064 showed kanamycin acetyltransferase activity. The detailed gene map is included.

• 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.

• The Korean Journal of Mycology
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• v.23 no.2 s.73
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• pp.129-138
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• 1995

DNA fragment being able to restore in vitro activity of ${\beta}-1,3-glucan$ synthase was cloned by transformation of the Saccharomyces cerevisiae LP353 mutant strain with genomic library constructed in the YCp50. For the selection of transformants which showed no detectable phenotype linked to recovery of the defect in ${\beta}-1,3-glucan$ synthase activity, the colony autoradiography was succesfully applied. The restriction map of the cloned DNA fragment, which is 8.5-kb in length, was constructed. Both the YEplac195 and the YCp50 carrying the 8.5-kb fragment increased ${\beta}-1,3-glucan$ synthase activity of LP353 by two fold. Neither the YEplac195 nor the YCp50 carrying the 8.5-kb DNA fragment, however, complemented the temperature-dependent osmotic sensitivity which is another distinctive phenotype of LP353. Subcloning experiments indicated that a functional region was located in 4.8-kb BglII-KpnI fragment. The 4.8-kb fragment was also able to increase the level of ${\beta}-1,3-glucan$ content in cell wall as well as the resistance of cells to cell wall lytic enzyme, ${\beta}-1,3-glucanase$. The growth rate of the LP353 with 4.8-kb fragment was almost same as that of wild type strain in liquid medium with 1.2 M sorbitol at nonpermissive temperature. Taken these results together, the 4.8-kb fragment seemed to contain the BGS2 gene for ${\beta}-1,3-glucan$ synthase activity in yeast S. cerevisiae.

• Korean Journal of Veterinary Research
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• v.36 no.3
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• pp.665-680
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• 1996

Bovine Pneumonic Pasteurellosis는 수송열(輸送熱)로 일반적으로 알려져 있는 질병으로서, 여러가지 요인의 복합적(複合的)인 작용에 의해 발병하는 것으로 알려져 있으나, Pasteurella haemolytica A1이 가장 주요(主要)한 인자(因子)로 밝혀져 있다. P haemolytica A1은 leukotoxin(LKT), lipopolysaccharide(LPS), capsular polysaccharide 등 여러가지의 병원성인자(病原性因子)을 생성한다. 이들 인자중 LKT가 가장 중요한 병원성인자로 밝혀져 있다. 이에 본 실험은 P haemolytical A1의 LKT 유전자를 Bacillus subtilis에서 발현(發現)시킴으로서 LPS에 오염(汚染)되지 않은 LKT을 대량으로 생산할 목적으로 실시되었다. 실험의 첫 단계(段階)로서 pLKT52 plasmid을 Sau3 A1의 제한효소을 이용하여 부분소화(部分消化)시킨 후 이 부분 소화(消化)된 유전자들로부터 3~5kb 크기의 유전자들을 순수분리하여 pUC18와 결합시킨 후 E coli NM522에 형질전환(形質轉換)시켰다. 이때 형질전환된 균주들은 LKT에 대한 단크론 항체인 MAb601을 이용하여 colony blot 법에 의해서 LKT 유전자 보유 및 발현여부(發現與否)을 조사하였다. 이들 양성 clone들은 제한효소분석(制限酵素分析), 염기서열분석(鹽基序列分析) 및 Western blot 등에 의해서 재확인(再確認)하였다. 총 9개의 양성 clone중 위의 방법에 의해서 한 clone을 선택(選擇)하여 lktCA insert를 재분리하여 shuttle vector에 subcloning 하였다. Subcloning된 LKT 유전자들은 shuttle vector의 종류(種類)(pHPS9, p602/20, pHPS9-Sac)와 각기(各其) 다른 종류(種類)의 B subtilis(spoO12A, BR121, WB3O, Raj1105) 숙주내(宿主內)에서 발현정도를 Western blot 법에 의해서 비교(比較)하였다. 이때 최적발현조건(最適發現條件)은 p602/20와 pBL1의 dual plasmid system을 이용하여 B subtilis spoO12A에서 2시간동안 IPTG로 발현을 유도(誘導)하는 것이었다. B subtilis에서 발현된 LKT을 visual 법과 neutral red uptake 법을 이용하여 소 폐포(肺胞) 대식구(大食求)에 대한 biological activity를 확인하였다. 발현된 LKT에 대한 LPS 오염은 LKT을 SDS-PAGE 후 silver stain에 의해서 확인하였다. 본 실험을 통해서 볼 때에 lktCA 유전자를 보유(保有)하고 있는 p602/20는 B subtilis에서 매우 불안정(不安定)하였고, 발현된 LKT는 세균자체(細菌自體)에서 생성되는 protease들에 의해서 파괴(破壞)됨으로서 농도(濃度)가 매우 낮았다. 이러한 문제점들은 다음 단계(段階)의 실험에서 해결되어야할 문제들이다.

• Korean Journal of Microbiology
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• v.41 no.4
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• pp.300-305
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• 2005

A transcriptionally active fragment $(P_{19})$ isolated by utilizing the promoter-probe shuttle vector pSK1Cat was analyzed. By subcloning analysis, the 180 bp region $(P_{180})$ responsible for the activity was determined. Transcriptional fusion of the C. glutamicum metX gene to $P_{180}\;(P_{180}-metX)$ resulted in a 24-fold increase in MetX activity in a complex medium, while a 13-fold increase was observed with the $P_{tac}$ promoter. Additionally, the expression conferred by $P_{180}$ was not affected by methionine added to the growth medium, suggesting that the $P_{180}$ clone is useful for the deregulated expression of biosynthetic genes in C. glutamicum during amino acid fermentation. Introduction of $P_{180}-metX$ into a lysine-producing C. glutamicum resulted in the production of methionine to 0.8 g/l.