• Journal of Microbiology and Biotechnology
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• 제11권6호
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• pp.1077-1086
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• 2001

The sprA and sprB gene encoding chymotrypsin-like proteases Streptomyces griseus protease A (SGPA) and Streptomyces griseus protease B (SGPB) and the sprT gene that encodes Streptomyces griseus trypsin (SGT) were cloned from Streptomyces griseus ATCC10137 and overexpressed in Streptomyces lividans TK24 as a heterologous host. The chymotrypsin activity of tole culture broth measured with the artificial chromogenic substrate , N-succinyl-ala-ala-pro-phe-p-nitroanilide, was 10, 14 and 14 units/mg in the transformants haboring the sprA, sprB and sprD genes, respectively. The growth of S. lividans reached the maximum cell mass after 4 days of culture, yet SGPA and SGPD production started in the stationary phase of cell growth and kept increasing for up to 10 days of culture in an R2YE medium. The trypsin activity of the culture broth measured with the artificial chromogenic substrate , N-${\alpha}$-benzoyl-DL- arginine-p-nitroanilide , was 16 units/mg and SGT production started in the stationary phase of cell growth and kept increasing for up to 10 days of culture in an R2YE medium. The introduction of the sprA gene into S, lividans TK24 triggered the biosynthesis of pigmented antibiotics, actinorhodin and undecylprodigiosin, and induced significant morphological changes in the colonies in Benedict, R2YE, and R1R2 media. In addition, the introduction of the sprT gene also induced morphological changes in the colony shape without affecting the antibiotic production, thereby implying that certain proteases would appear to play very important and specific roles in secondary-metabolites formation and morphological differentiation in Streptomyces.

• 한국작물학회지
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• 제52권4호
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• pp.458-468
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• 2007

To tolerate environmentally adverse conditions such as cold, drought, and salinity, plants often synthesize and accumulate proline in cells as compatible osmolytes. ${\Delta}^1$-Pyrroline-5-carboxylate synthetase(P5CS) catalyzes the rate-limiting step of proline biosynthesis from glutamate. Two complete genes, MtP5CS1 and MtP5CS2, were isolated from the model legume Medicago truncatula by cDNA cloning and bacterial artificial chromosome library screening. Nucleotide sequence analysis showed that both genes consisted of 20 exons and 19 introns. Alignment of the predicted amino acid sequences revealed high similarities with P5CS proteins from other plant species. The two MtP5CS genes were expressed in response to high salt and low temperature treatments. Semi-quantitative reverse transcription-polymerase chain reaction showed that MtP5CS1 was expressed earlier than MtP5CS2, indicating differential regulation of the two genes. To evaluate the reverse genetic effects of nucleotide changes on MtP5CS function, a Targeting Induced Local Lesions in Genomes approach was taken. Three mutants each were isolated for MtP5CS1 and MtP5CS2, of which a P5CS2 nonsense mutant carrying a codon change from arginine to stop was expected to bring translation to premature termination. These provide a valuable genetic resource with which to determine the function of the P5CS genes in environmental stress responses of legume crops.

• 한국양식학회지
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• 제15권3호
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• pp.157-163
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• 2002

본 실험은 섭취촉진물질로 알려져 있는 사료내 13종의 아미노산들이 치어기 조피볼락에 있어 성장 및 체조성에 어떠한 영향을 미치는지 알아보고자 실시하였다 실험어는 평균무게 0.64 g인 조피볼락 치어를 사용하였으며, 기초실험 사료내에 유인효과가 있을 것으로 여겨지는 아미노산 13가지(Arg, Ala, Na-Glu, Gly, His, Ile, Lys, Met, Phe, Pro, Thr, Trp 및 Val)를 사료내 각각 0.3% 씩 첨가하여 총 13 실험구를 2반복으로 나누어 6주간 사육하였다. 6주간의 사육실험 결과, 성장률(WG)과 일간 성장률(SGR)에서는 Pro, Thr, Met 및 Gly 실험구가 다른 실험구에 비해 유의적으로 높았으나(P<0.05), 이들 실험구간에는 유의적인 차이가 없었다(P>0.05). 그리고, 사료전환효율(FE)에서는 Pro 실험구가 Met과 Gly 실험구보다 유의적으로 높았으며(P<0.05), Pro 실험구와 Thr 실험구 사이에는 유의적인 차이가 없었다(P>0.05). 그리고 Thr, Met 및 Gly 실험구간에도 유의적인 차이가 없었다(P>0.05). 단백질전화효율에 있어서 Pro 실험구가 모든 실험구에 있어서 유의적으로 가장 높았으며(P<0.05), Thr 실험구와 비교하여 유의적으로 차이가 나지 않았다(P>0.05). 그리고, 비만도(condition factor)는 Thr 실험구가 유의적으로 가장 높았으며(P<0.05), Val 실험구는 가장 낮게 나타났다(P>0.05). 간중량지수(HSI)는 모든 실험구간에서 유의적인 차이가 나지 않았다(P>0.05). 전어체의 단백질, 수분 및 회분에 있어서는 모든 실험구에 있어서 유의적인 차이가 나지 않았다(P>0.05). 지질함량은 Met, Pro 및 Thr 실험구가 Glu, Gly, Lys, Trp 및 Val 실험구보다 유의적으로 낮았다(P>0.05). 상기 결과를 토대로, 조피볼락 사료용 사료섭취촉진물질로서 아미노산을 검색한 결과 사료내 0.3% Pro, Thr, Met 및 Gly을 첨가했을 때 사료섭취촉진 효과가 나타났으며, 이것들 중 Gly을 제외하고는 전어체의 지질 함량을 감소시키는 결과를 나타내었다.

• 대한유전성대사질환학회지
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• 제2권1호
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• pp.77-79
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• 2002

The urea cycle, consisting of a series of six enzymatic reactions, plays key roles to prevent the accumulation of toxic nitrogenous compound and synthesize arginine de novo. Five well characterized diseases have been described, resulting from an enzymatic defect in the biosynthesis of one of the normally expressed enzyme. This presentation will focus on two representative diseases; ornithine transcarbamylase(OTC) deficiency and citrullinemia(argininosuccinate synthetase deficiency). OTC deficiency is one of the most common inborn error of urea cycle, which is inherited in X-linked manner. We identified 17 different mutations in 20 unrelated Korean patients with OTC deficiency; L9X, R26P, R26X, T44I, R92X, G100R, R141Q, G195R, M205T, H214Y, D249G, R277W, F281S, 853 del C, R320X, V323M and 10 bp del at nt. 796-805. These mutations occur at well conserved nucleotide sequences across species or CpG hot spot. The L9X and R26X lead to the disruption of leader sequences, required for directing mitochondrial localization of the OTC precursor. Their phenotypes are severe, and neonatal onset. The G100R, R277W and V323M mutations were uniquely identified in patients with late onset OTC deficiency. The other genotypes are associated with neonatal onset. Out of 20 patients with OTC deficiency, only 6 patients are alive; two were liver transplanted, and normal in growth and development at 2, 4 years after transplantation respectively. Citrullinemia is an autosomal recessive disease, caused by the mutations in the argininosuccinate synthetase(ASS) gene. We identified in 3 major mutations in 11 unrelated Korean patients with citrullinemia; G324S, $IVS6^{-2}$ A to G, and 67 bp ins at nt 1125-1126. Among these, the 67 base pair insertion mutation is novel. The allele frequency of each mutation is; G324S(45%), IVS6-2 A to G(32%), and 67 base pair insertion(14%). All patients are diagnosed at neonatal or infantile age. Interestingly, two patients presented with stroke like episode. Out of 11 patients, 5 patients died. Among 6 patients alive, one patient was successfully liver transplanted.