• Journal of Microbiology and Biotechnology
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• 제5권3호
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• pp.132-137
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• 1995

The lambda integrase (lnt) is believed to bind to several arm and core sites of attP DNA in order to facilitate intasome formation. We have done systematic mutagenic analysis on all 5 arm sites and found that P1 is absolutely required for integration while P2 is not. We also found that all 3 P' arm sites(P'1, P'2, and P'3) are required for efficient integrative recombination. P'1, which is an important binding site for excision, also seems to be crucial for integration when preincubation of attP DNA with Int and IHF is performed before recombination. Preincubation assay revealed that preincubation with Int and IHF improved the efficiency of recombination of wild type attP DNA and demolished recombinations of P'1 mutant attP DNAs.

• Journal of Microbiology and Biotechnology
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• 제17권2호
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• pp.342-347
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• 2007

Bacteriophage ${\Phi}FC1$ integrase (MJ1) was previously shown to perform a site-specific recombination between a phage attachment site (attP) and a host attachment site (attB) in its host, Enterococcus faecalis, and also in a non-host bacterium, Escherichia coli. Here, we investigated biochemical features of MJ1 integrase. First, MJ1 integrase could perform in vitro recombination between attP and attB in the absence of additional factors. Second, MJ1 integrase interacted with att sites. Electrophoretic mobility shift assays and DNase I footprinting revealed that MJ1 integrase could efficiently bind to all the att sites and that MJ1 integrase recognized relatively short sequences (${\sim}50bp$) containing an overlapping region within attB and attP. These results demonstrate that MJ1 integrase indeed catalyzes an integrative recombination between attP and attB, the mechanism of which might be simple and unidirectional, as found in serine integrases.

• BMB Reports
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• 제29권5호
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• pp.448-454
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• 1996

Bacteriophage ${\phi}FC1$ is a temperate phage which was identified as a prophage in the Enterococcus faecalis KBL703 chromosome. Phage ${\phi}FC1$ integrates into the host chromosome by site-specific recombination. The phage attachment site P (attP) was localized within the 0.65-kb XhoI-HindIII fragment and the nucleotide sequence of the region was determined. An open reading frame (mj1) which adjoined the phage attachment site encoded a deduced protein related to the site-specific recombinase family. The organization of this region was comparable to other site-specific recombination systems. The molecular weight of the expressed MJ1 in E. coli was in good agreement with the predicted 53,537 Da of the mj1 gene product. Elucidation of the phage-specific integration process in this study would provide useful genetic tools such as a chromosomal integration system.

• 한국미생물·생명공학회지
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• 제32권4호
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• pp.356-361
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• 2004

The ethionine resisconferring gene (ERCI) was constitutively expressed under the control of the alcohol dehydrogenase gene promoter (ADClp) and introduced into the chromosomes of an industrial polyploid strain of Saccharocerevisiae by using the 8-sequences of the Tyl retrotransposon as the recombination site. 8-Integrative cassette devoid of bacterial DNA sequences containing the ampicillin resistance gene was constructed that had the aureobasidin A resistance gene (AURl-C) as the selection marker and ERCl gene. The ERCl gene was also employed as the selection marker in the 8-integrative cassette lacking the A URl-C gene. Industrial Saccerevisiae transformed with these integrative cassettes exhibited strong resistance to DL-ethioncompared with nontransformants.

• 미생물학회지
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• 제41권2호
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• pp.146-151
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• 2005

전분 이용이 가능한 산업용 Saccharomyces cerevisiae균주를 개발하기 위해 alcohol dehydrogenase 유전자 프로모터(ADClp)의 조절하에 발현되는 Aspergillus awamori glucoamylase cDNA 유전자(GA1)를 산업용 S. cerevisiae의 염색체에 도입하였다. 산업적 이용에 적합한 효모균주를 얻기 위해 세균 ampicillin 저항성 유전자가 제거되고 GA1 유전자와 선별 표지유전자로 S. cerevisiae aureobasidin A 저항성 유전자(AUR1-C)와 재조합 부위로 Tyretrotransposon $\delta$-서열이 포함된 integrative cassette를제조하였다. 이 $\delta-integrative$ cassette로 형질전환된 산업용 S. cerevisiae는 배지상에 glucoamylase를 생산 분비하였고 전환을 유일한 탄소원으로 하여 생장하였다. 형질전환체를 비선택배지에서 배양했을 매 삽입된 GA1유전자가 100세대까지 안정되게 유지되었다.

• Animal cells and systems
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• 제1권4호
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• pp.619-627
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• 1997

Maintenance of polymorphism in a two-locus system with two alleles under stabilizing selection has been tested by Monte-Carlo simulation. The effect of each allele was additive. Only gene x environment interactions and degree of genetic linkage between loci were considered. There were no other evolutionary forces acting except stabilizing selection. Fixation rates were influenced by the extent of environmental change and the degree of genetic linkage. In most cases, stabilizing selection depleted genetic variability when two loci have a lower degree of linkage (10 cM). When two loci are closely linked (0.1 cM), however, stabilizing selection promoted balanced heterozygotes in changing environments. Thus, environment-dependent selection and recombination rate are important parameters which should be incorporated into mechanisms of maintenance of genetic variability.

• Journal of Microbiology and Biotechnology
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• 제16권9호
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• pp.1472-1476
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• 2006

Previous study demonstrated that the restriction barrier of Streptomyces griseus is almost completely bypassed by the Streptomyces-E. coli shuttle vectors passed through the E. coli GM161 strain and methylated with AluI and HpaII methyltransferases. The same DNA methylation of the genomic DNA fragments cloned the nonreplicative vectors generated integrative transformation and gene disruption of their chromosomal counterparts at high efficiencies in S. griseus. This result indicated that the efficiency of gene disruption depends on the efficient transfer of the incoming DNA into bacterial hosts.

• Journal of Microbiology and Biotechnology
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• 제24권11호
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• pp.1503-1509
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• 2014

With the purpose of facilitating the process of stable strain generation, a shuttle vector for integration of genes via a double recombination event into two ectopic sites on the Sulfolobus acidocaldarius chromosome was constructed. The novel chromosomal integration and expression vector pINEX contains a pyrE gene from S. solfataricus P2 ($pyrE_{sso}$) as an auxotrophic selection marker, a multiple cloning site with histidine tag, the internal sequences of malE and malG for homologous recombination, and the entire region of pGEM-T vector, except for the multiple cloning region, for propagation in E. coli. For stable expression of the target gene, an ${\alpha}$-glucosidase-producing strain of S. acidocaldarius was generated employing this vector. The malA gene (saci_1160) encoding an ${\alpha}$-glucosidase from S. acidocaldarius fused with the glutamate dehydrogenase ($gdhA_{saci}$) promoter and leader sequence was ligated to pINEX to generate pINEX_malA. Using the "pop-in" and "pop-out" method, the malA gene was inserted into the genome of MR31 and correct insertion was verified by colony PCR and sequencing. This strain was grown in YT medium without uracil and purified by His-tag affinity chromatography. The ${\alpha}$-glucosidase activity was confirmed by the hydrolysis of $pNP{\alpha}G$. The pINEX vector should be applicable in delineating gene functions in this organism.

• Food Science and Biotechnology
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• 제17권6호
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• pp.1221-1227
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• 2008

The application of the cellulase gene (celA) as a selection marker of food-grade integration system was investigated in Lactobacillus (Lb.) casei, Lactococcus lactis, and Leuconostoc (Leu.) mesenteroides. The 6.0-kb vector pOC13 containing celA from Clostridium thermocellum with an integrase gene and a phage attachment site originating from bacteriophage A2 was used for site-specific recombination into chromosomal DNA of lactic acid bacteria (LAB). pOC13 was also equipped with a broad host range plus replication origin from the lactococcal plasmid pWV01, and a controllable promoter of nisA ($P_{nisA}$) for the production of foreign proteins. pOC13 was integrated successfully into Lb. casei EM116, and pOC13 integrants were easily detectable by the formation of halo zone on plates containing cellulose. Recombinant Lb. casei EM 116::pOC13 maintained these traits in the absence of selection pressure during 100 generations. pOC13 was integrated into the chromosome of L. lactis and Leu. mesenteroides, and celA acted as an efficient selection marker. These results show that celA can be used as a food-grade selection marker, and that the new integrative vector could be used for the production of foreign proteins in LAB.

• 한국동물학회지
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• 제37권2호
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• pp.232-239
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• 1994

RecA protein plans a central role in homologous recombination and DNA repair in Escherichia cofi (E. colD. The function 8nd structure of this protein are universal in prokarvotes and also conserved in eukaryotes such as yeast. The RecA-like protein with 74 lInDa in size has already been identified and purified from a fission yeast Schizosaccharomyces pombe (5. pommel (Lee, 19911. From this study it was revealed that the RecA-like protein of 5. pombe was highly inducible to various DNA damaging agents and inhibitors of nucleotide pool svnthesizins enzymes. The cellular level of the 5. pombe RecA-like protein wi,u markedly increased, upto 5- to 10-fold, by treatment with various DNA-damains agents including ultraviolet (UV) light, methyl methanesulfonate WS),4-nitroquinoline-1-oxide (4-NQO), and mitomycin-C (MMC), similar to E. cofi RecA protein. Interestingly, the protein level was also increased by inhibitors of nucleotide pool forming enzlwnes such as methotrexate (MTX) and hvdroxvurea (HU). The most effective doses for the inducibility of 4-NQO, MMS, W, MMC, MTX, and HU were 0.2 Ug/ml, 30 mM, 200 J/ma, 0.4 $\mus/ml,$ 1 Ug/ml, and 100 mM, respectively. The range of effective duration time for the inducibilitv of RecA-like protein was from 270 to 450 mins. These results suggest that the 5. pombe RecA-like protein also platys an imortant role in cellular responses to DNA damage as in E. coli system.