• 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.

• 한국미생물·생명공학회지
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• 제34권3호
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• pp.211-215
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• 2006

S. lividans에서 클로닝된 조절유전자인 afsR2를 과발현시 나타나는 up-regulated protein과 down-regulated protein 관련 유전자들을 proteomics 방법으로 선별하였고[3], 이를 방선균 발현벡터인 pSE34를 이용하여 제작된 pPNP, pGPD를 S. avermitilis에 형질전환시켜 avermectin 및 이차대사산물의 생산량 차이를 비교 분석하였다. 그 결과 up-regulated protein으로 예상되던 PNP는 wild-type S. avermitilis에서만 제한적으로 avermetin-type 대사산물의 생산성 향상을 촉진시켰으며, 이와 반대로 down-regulated protein으로 예상되었던 GPD는 avermectin-type 대사산물의 생산량을 wild type S. avermitilis에서는 4배, over-producer S. avermitilis에서는 2.5배 증가시켰다. 본 연구결과는, 방선균 조절 단백질들이 이차대사산물의 종류 및 생합성 기작에 따라 전혀 다르게 작용될 수 있음을 암시하며, 향후 이들 조절 단백질들에 대한 보다 구체적인 분자수준에서의 연구 필요성을 제시하고 있다.

• BMB Reports
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• 제40권5호
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• pp.723-730
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• 2007

Kinesin is an ATP-driven microtubule motor protein that plays important roles in control of microtubule dynamics, intracellular transport, cell division and signal transduction. The kinesin superfamily is composed of numerous members that are classified into 14 subfamilies. Animal kinesins have been well characterized. In contrast, plant kinesins have not yet to be characterized adequately. Here, a novel plant-specific kinesin gene, GhKCH2, has been cloned from cotton (Gossypium hirsutum) fibers and biochemically identified by prokaryotic expression, affinity purification, ATPase activity assay and microtubule-binding analysis. The putative motor domain of GhKCH2, $M_{396-734}$ corresponding to amino acids Q396-N734 was fused with 6$\times$His-tag, soluble-expressed in E. coli and affinity-purified in a large amount. The biochemical analysis demonstrated that the basal ATPase activity of $M_{396-734}$ is not activated by $Ca^{2+}$, but stimulated 30-fold max by microtubules. The enzymatic activation is microtubule-concentration-dependent, and the concentration of microtubules that corresponds to half-maximum activation was about 11 ${\mu}M$, much higher than that of other kinesins reported. The cosedimentation assay indicated that $M_{396-734}$ could bind to microtubules in vitro whenever the nucleotide AMP-PNP is present or absent. As a plant-specific microtubule-dependent kinesin with a lower microtubule-affinity and a nucleotide-independent microtubule-binding ability, cotton GhKCH2 might be involved in the function of microtubules during the deposition of cellulose microfibrils in fibers or the formation of cell wall.

• Molecules and Cells
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• 제41권12호
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• pp.1033-1044
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• 2018

As sessile organisms, plants have evolved to adjust their growth and development to environmental changes. It has been well documented that the crosstalk between different plant hormones plays important roles in the coordination of growth and development of the plant. Here, we describe a novel recessive mutant, mildly insensitive to ethylene (mine), which displayed insensitivity to the ethylene precursor, ACC (1-aminocyclopropane-1-carboxylic acid), in the root under the dark-grown conditions. By contrast, mine roots exhibited a normal growth response to exogenous IAA (indole-3-acetic acid). Thus, it appears that the growth responses of mine to ACC and IAA resemble those of weak ethylene insensitive (wei) mutants. To understand the molecular events underlying the crosstalk between ethylene and auxin in the root, we identified the MINE locus and found that the MINE gene encodes the pyridoxine 5′-phosphate (PNP)/pyridoxamine 5′-phosphate (PMP) oxidase, PDX3. Our results revealed that MINE/PDX3 likely plays a role in the conversion of the auxin precursor tryptophan to indole-3-pyruvic acid in the auxin biosynthesis pathway, in which TAA1 (TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS 1) and its related genes (TRYPTOPHAN AMINOTRANSFERASE RELATED 1 and 2; TAR1 and TAR2) are involved. Considering that TAA1 and TARs belong to a subgroup of PLP (pyridoxal-5′-phosphate)-dependent enzymes, we propose that PLP produced by MINE/PDX3 acts as a cofactor in TAA1/TAR-dependent auxin biosynthesis induced by ethylene, which in turn influences the crosstalk between ethylene and auxin in the Arabidopsis root.

• 미생물학회지
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• 제45권4호
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• pp.332-338
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• 2009

꿀벌(Apis mellifera)의 장으로부터 xylan분해능이 우수한 세균 HY-20을 분리하였고, Bacillus 속으로 동정하였다. 분리균주 HY-20의 세포 외 GH11 xylanase (XylP) 유전자 (687-bp)는 25,522 Da의 분자량과 9.33의 pI 값을 갖는 228개의 아미노산으로 구성된 단백질을 인코딩하는 것으로 확인되었으며, 지금까지 특성규명이 이루어지지 않은 B. pumilus xylanase (GenBank accession no.: AY526092)의 아미노산 서열과 97%의 상동성을 보였다. pET-28a(+)/xylP를 포함하고 있는 Escherichia coli BL21 균주에서 과발현 된 His-tagged XylP (rXylP)는 cation exchange 및 gel permeation chromatography를 통해 순수하게 분리 정제 되었으며, $50^{\circ}C$의 온도와 pH 6.5 조건에서 반응할 때 birchwood xylan에 대해 가장 높은 가수분해 활성을 나타내었다. rXylP는 15분간 $55^{\circ}C$에 노출될 때 본래 활성의 약 50%를 잃어버리는 전형적인 중온성 효소의 특성을 보였으며, $Hg^{2+}$ (1mM)와 N-bromosuccinimide (5mM)에 노출될 때 완전히 불활성화 되었고, $Mn^{2+}$ (1 mM), $Fe^{2+}$ (1mM) 및 sodium azide (5 mM)에 의해서는 활성이 약 10% 증가될 수 있는 것으로 밝혀졌다. 한편, rXylP는 xylose유래 다당류는 효율적으로 분해하였지만 PNP-sugar 유도체 및 glucose 유래 다당류는 분해하지 못하였다.