• 한국미생물·생명공학회지
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• 제13권3호
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• pp.297-302
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• 1985

미생물중 urease생성능이 아주 강한 B. pasteurii의 Hind III partial digest 된 chromosomal DNA를 E. coli-B. subtilis bifunctional plasmid vector pGR 71으로 E. coli RR1 균주에 cloning 하므로써 그 urease gene을 expression시킬 수 있었다. 그러나 B. subtilis에서는 insertion DNA fragment의 deletion으로 expression되지 않았다. Cloning된 E.coli RR1 균주로부터 분리 정제한 urease gene함유 Plasmid(pGU66)의 restriction map을 작성하여 본 결과 7.1 Mdal의 insertion fragment가 삽입된 12.6Mdal의 plasmid에 Hind III, Bgl II, Xba I, Sal I등 몇 개의 cleavage site 위치를 찾을 수 있었다. Cloning된 E. coli의 urease는 periplasmic space에 많은 비율로 축적되며, 그 효소학적 성질은 donor인 B.pasteurii의 그것과 매우 유사하였다.

• Journal of Microbiology and Biotechnology
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• 제31권12호
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• pp.1722-1731
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• 2021

The genus Streptomyces is intensively studied due to its excellent ability to produce secondary metabolites with diverse bioactivities. In particular, adequate precursors of secondary metabolites as well as sophisticated post modification systems make some high-yield industrial strains of Streptomyces the promising chassis for the heterologous production of natural products. However, lack of efficient genetic tools for the manipulation of industrial strains, especially the episomal vector independent tools suitable for large DNA fragment deletion, makes it difficult to remold the metabolic pathways and streamline the genomes in these strains. In this respect, we developed an efficient deletion system independent of the episomal vector for large DNA fragment deletion. Based on this system, four large segments of DNA, ranging in length from 10 kb to 200 kb, were knocked out successfully from three industrial Streptomyces strains without any marker left. Notably, compared to the classical deletion system used in Streptomyces, this deletion system takes about 25% less time in our cases. This work provides a very effective tool for further genetic engineering of the industrial Streptomyces.

• 한국수산과학회지
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• 제43권6호
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• pp.637-641
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• 2010

The antibiotics-resistance profiles of 28 strains of Vibrio parahaemolyticus isolated from seawater were investigated. All of the strains studied were resistant to ampicillin (100%), but susceptible to 12 other antibiotics. The minimum inhibitory concentration (MIC) of V. parahaemolyticus to ampicillin was as high as $1,024-2,048\;{\mu}g{\cdot}mL^{-1}$. The phenotype of strain 8 changed from ampicillin-resistant to susceptible with an in-frame deletion mutant of VPA0477, a putative ${\beta}$-lactamase gene, and the MIC for ampicillin of the mutant strain was $1{\mu}g{\cdot}mL^{-1}$. In conclusion, our findings suggest that the VPA0477 gene acts as a ${\beta}$-lactamase in ampicillin-resistant V. parahaemolyticus strains.

• 한국균학회소식:학술대회논문집
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• 한국균학회 2014년도 추계학술대회 및 정기총회
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• pp.15-15
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• 2014

Fusarium graminearum (teleomorph Gibberella zeae) is an important plant pathogen that causes head blight of major cereal crops such as wheat, barley, and rice, as well as causing ear and stalk rot on maize worldwide. Plant diseases caused by this fungus lead to severe yield losses and accumulation of harmful mycotoxins in infected cereals [1]. Fungi utilize spore production as a mean to rapidly avoid unfavorable environmental conditions and to amplify their population. Spores are produced sexually and asexually and their production is precisely controlled. Upstream developmental activators consist of fluffy genes have been known to orchestrate early induction of condiogenesis in a model filamentous fungus Aspergillus nidulans. To understand the molecular mechanisms underlying conidiogenesis in F. graminearum, we characterized functions of the F. graminearum fluffy gene homologs [2]. We found that FlbD is conserved regulatory function for conidiogenesis in both A. nidulans and F. graminearum among five fluffy gene homologs. flbD deletion abolished conidia and perithecia production, suggesting that FlbD have global roles in hyphal differentiation processes in F. graminearum. We further identified and functionally characterized the ortholog of AbaA, which is involved in differentiation from vegetative hyphae to conidia and known to be absent in F. graminearum [3]. Deletion of abaA did not affect vegetative growth, sexual development, or virulence, but conidium production was completely abolished and thin hyphae grew from abnormally shaped phialides in abaA deletion mutants. Overexpression of abaA resulted in pleiotropic defects such as impaired sexual and asexual development, retarded conidium germination, and reduced trichothecene production. AbaA localized to the nuclei of phialides and terminal cells of mature conidia. Successful interspecies complementation using A. nidulans AbaA and the conserved AbaA-WetA pathway demonstrated that the molecular mechanisms responsible for AbaA activity are conserved in F. graminearum as they are in A. nidulans. F. graminearum ortholog of Aspergillus nidulans wetA has been shown to be involved in conidiogenesis and conidium maturation [4]. Deletion of F. graminearum wetA did not alter mycelial growth, sexual development, or virulence, but the wetA deletion mutants produced longer conidia with fewer septa, and the conidia were sensitive to acute stresses, such as oxidative stress and heat stress. Furthermore, the survival rate of aged conidia from the F. graminearum wetA deletion mutants was reduced. The wetA deletion resulted in vigorous generation of single-celled conidia through autophagy-dependent microcycle conidiation, indicating that WetA functions to maintain conidia dormancy by suppressing microcycle conidiation in F. graminearum. In A. nidulans, FlbB physically interacts with FlbD and FlbE, and the resulting FlbB/FlbE and FlbB/FlbD complexes induce the expression of flbD and brlA, respectively. BrlA is an activator of the AbaA-WetA pathway. AbaA and WetA are required for phialide formation and conidia maturation, respectively [5]. In F. graminearum, the AbaA-WetA pathway is similar to that of A. nidulans, except a brlA ortholog does not exist. Amongst the fluffy genes, only fgflbD has a conserved role for regulation of the AbaA-WetA pathway.

• Journal of Veterinary Science
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• 제21권2호
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• pp.20.1-20.13
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• 2020

Actinobacillus pleuropneumoniae (APP) causes a form of porcine pleuropneumonia that leads to significant economic losses in the swine industry worldwide. The apxIBD gene is responsible for the secretion of the ApxI and ApxII toxins and the pnp gene is responsible for the adaptation of bacteria to cold temperature and a virulence factor. The apxIBD and pnp genes were deleted successfully from APP serotype 1 and 5 by transconjugation and sucrose counter-selection. The APP1ΔapxIBDΔpnp and APP5ΔapxIBDΔpnp mutants lost hemolytic activity and could not secrete ApxI and ApxII toxins outside the bacteria because both mutants lost the ApxI- and ApxII-secreting proteins by deletion of the apxIBD gene. Besides, the growth of these mutants was defective at low temperatures resulting from the deletion of pnp. The APP1ΔapxIBDΔpnp and APP5ΔapxIBDΔpnp mutants were significantly attenuated compared with wild-type ones. However, mice vaccinated intraperitoneally with APP5ΔapxIBDΔpnp did not provide any protection when challenged with a 10-times 50% lethal dose of virulent homologous (APP5) and heterologous (APP1) bacterial strains, while mice vaccinated with APP1ΔapxIBDΔpnp offered 75% protection against a homologous challenge. The ΔapxIBDΔpnp mutants were significantly attenuated and gave different protection rate against homologous virulent wild-type APP challenging.

• Reproductive and Developmental Biology
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• 제30권4호
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• pp.293-299
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• 2006

In order to generate transgenic goats expressing human growth hormone (hGH) in their mammary glands, goat ${\beta}-Casein/hGH$ hybrid gene was introduced into goat zygotes by pronuclear microinjection. DNA-injected embryos were transferred to the oviduct of recipients at 2-cell stage or to the uterus at morula/blastocyst stage after cultivation in glutathione-supplemented mSOF medium in vitro. Pregnancy and survival rate were not significantly different between 2-cell embryos and morula/blastocysts transferred to oviduct and uterus, respectively. One transgenic female goat was generated from 153 embryos survived from DNA injection. Southern blot analysis revealed that the transgenic goat harbored single-copy transgene with a partial deletion in its sequences. Despite of the partial sequence deletion, the transgene was successfully expressed hGH at the level of $72.1{\pm}15.1{\mu}g/ml$ in milk throughout lactation period, suggesting that the sequence deletion had occurred in non-essential part of the transgene for the transgene expression. Unfortunately, however, the transgene was not transmitted to her offspring during three successive breeding seasons. These results demonstrated that goat ${\beta}-casein/hGH$ gene was integrated into the transgenic goat genome in a mosaic fashion with a partial sequence deletion, which could result in a low level expression of hGH and a failure of transgene transmission.

• 미생물학회지
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• 제45권4호
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• pp.300-305
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• 2009

mRNA의 3' 말단형성 뿐만 아니라, 성숙한 mRNA의 핵에서 세포질로의 이동에 중요한 역할을 하는 출아효모 Saccharomyces cerevisiae의 폴리(A)-RNA 결합단백질인 Nab2와 유사한 분열효모 Schizosaccharomyces pombe의 단백질을 암호화하는 유전자(spNab2로 명명)의 결실돌연변이주(deletion mutant)를 제조하여 그 특성을 조사하였다. 이배체인 S. pombe 균주에 하나의 spNab2 유전자만을 결실시킨 후 4분체분석(tetrad analysis)을 수행한 결과, S. cerevisiae NAB2와는 다르게 이 유전자는 생장에 반드시 필요하지 않았다. 또한 spNab2 결실돌연변이는 mRNA의 핵에서 세포질로의 이동도 정상적으로 보였다. spNab2의 역할을 알아보기 위해, 티아민에 의해 발현이 조절되는 강력한 프러모터를 이용하여 spNab2를 과발현시켰다. spNab2 유전자가 과발현되면, 세포의 생장이 심하게 억제되었으며, 폴리(A)-RNA가 핵 안에 축적되고 세포질에서는 줄어들었다. 또한 GFP 융합단백질을 이용하여 spNab2 단백질의 세포 내 위치를 관찰한 결과, spNab2-GFP는 주로 핵 안에 존재하였지만 세포질에서도 관찰되었다. 이와 같은 결과들은 spNab2 유전자 역시 mRNA의 핵에서 세포질로의 이동에 관여하고 있음을 시사한다.

• 한국균학회소식:학술대회논문집
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• 한국균학회 2014년도 춘계학술대회 및 임시총회
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• pp.39-39
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• 2014

In a homothallic filamentous fungus Aspergillus nidulans, sexual and asexual developments are largely affected by the genetic and environmental factors. To regulate the complex subsets of genes involved in the developmental processes accurately, tight regulations of transcription factors are required. The forkhead type transcription factors are the class of regulators that function in a broad spectrum of cellular and developmental processes in many species from yeast to human. Here, we identified the fkhA and fkhB genes that encode a conserved forkhead transcription factors. The fkhA deletion resulted in the complete loss of fruiting body formation under all conditions favoring sexual development, suggesting that the fkhA gene is required for sexual development in A. nidulans. Overexpression of fkhA resulted in enhanced formation of fruiting bodies under induction condition not only in the normal condition but also in the condition of presence of 0.6 M KCl, which strongly inhibits sexual development. To know the function of the fkhB gene, we also generated fkhB knock-out strain in A. nidulans. Deletion of fkhB resulted in abnormal conidiophore formation under standard conditions and delayed sexual development process, suggesting that the fkhB gene plays an important role in conidiophore morphogenesis Taken together, these results suggest that the fkhA gene is necessary and sufficient for regulating sexual development and the fkhB gene is a transcription factor related in asexual developmental process in A. nidulans.

• The Plant Pathology Journal
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• 제24권1호
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• pp.8-16
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• 2008

Aurofusarin is a polyketide pigment produced by some Fusarium species. The PKS12 and GIP1 genes, which encode a putative type I polyketide synthase (PKS) and a fungal laccase, respectively, are known to be required for aurofusarin biosynthesis in Gibberella zeae (anamorph: Fusarium graminearum). The ten additional genes, which are located within a 30 kb region of PKS12 and GIP1 and regulated by a putative transcription factor (GIP2), organize the aurofusarin biosynthetic cluster. To determine if they are essential for aurofusarin production in G. zeae, we have employed targeted gene deletion, complementation, and chemical analyses. GIP7, which encodes O-methyltransferase, is confirmed to be required for the conversion of norrubrofusarin to rubrofusarin, an intermediate of aurofusarin. GIP1-, GIP3-, and GIP8-deleted strains accumulated rubrofusarin, indicating those gene products are essential enzymes for the conversion of rubrofusarin to aurofusarin. Based on the phenotypic changes in the gene deletion strains examined, we propose a possible pathway for aurofusarin biosynthesis in G. zeae. Our results would provide important information for better understanding of naphthoquinone biosynthesis in other fdarnentous fungi as well as the aurofusarin biosynthesis in G. zeae.

• BMB Reports
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• 제38권4호
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• pp.486-490
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• 2005

Genetic factors are important in the pathogenesis of coronary artery disease (CAD). Angiotensin converting enzyme (ACE) gene insertion(I)/deletion(D) polymorphism is one of the genetic factor found to be related with CAD. We investigated the association between I/D polymorphism of the ACE gene and the presence of CAD. Threehundred and seven patients (187 males and 120 females, aged between 35-80, mean $54.3{\pm}9.8$ years) who underwent diagnostic coronary angiography were included in the study. ACE I/D polymorphism was detected by polymerase chain reaction. Of the 307, 176 had CAD. The most frequently observed genotype in all subjects was ID (47.9 %). However, in patients with CAD the frequency of II genotype was lower whereas DD genotype was higher compared to the controls (p < 0.05). The number of D allele carrying subjects were also higher (p < 0.05) in CAD patients. The logistic regression analysis indicated that the ACE D allele is an independent risk factor (odds ratio = 1.48, 95% CI = 1.01-2.18, p < 0.05). In conclusion, the I/D polymorphism of ACE gene (carrying D allele) is an independent risk factor for CAD in the studied Turkish population.