• BMB Reports
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• 제32권1호
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• pp.20-24
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• 1999

The first step of the branch pathway in tryptophan biosynthesis is catalyzed by anthranilate synthase, which is subjected to feedback inhibition by the end product of the pathway. The $trpE^{FBR}$ gene from a mutant Escherichia coli strain coding for anthranilate synthase that was insensitive to feedback inhibition by tryptophan has been cloned. To identify the amino acid changes involved in the feedback regulation of anthranilate synthase, the nucleotide sequence of the mutant $trpE^{FBR}$ gene was determined. Sequence analysis of the $trpE^{FBR}$ gene revealed that four bases were changed in the structural gene while alteration was not found in the 5' control region. Among these base changes, only two base substitutions caused the alterations in amino acid sequences. From the results of restriction fragment exchange mapping, the 61st nucleotide, C to A substitution, that changed $Pro^{21}{\rightarrow}Ser$ was identified as the cause of the desensitization to feedback inhibition by tryptophan. Additional feedback-resistant enzymes of the E. coli anthranilate synthases were constructed by site-directed mutagenesis to examine the effect of the $Ser^{40}\;{\rightarrow}\;Arg^{40}$ change found in the $trpE^{FBR}$ gene of Brevibacterium lactofermentum. From the feedback inhibition analysis, the $Pro^{21}{\rightarrow}Ser$ and $Ser^{40}{\rightarrow}Arg$ mutants maintained about 50% and 90% of their maximal activities, respectively, even at the extreme concentration of 10 mM tryptophan. From these results, we suggest that the $Pro^{21}$ and $Ser^{40}$ residues are involved in the tryptophan binding in the E. coli enzyme.

• Journal of Plant Biology
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• 제39권4호
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• pp.243-247
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• 1996

Phosphoribosylanthranilate transferase (PAT) catalyzes the second step of the tryptophan biosynthetic pathway and is encoded by a single-copy gene that complements all the visible phenotypes of the tryptophan mutant (trp1-100) of Arabidopsis. The trp1-100 is blue fluorescent under UV light becuase it accumulates anthranilate. To obtain a plant with reduced PAT activity, PAT1 genes with several internal deletions in different promoter regions (pHS 101, pHS102, pHS104, pHS105, and pHS107) were induced into trp1-100 via Agrobacterium. Then, homozygous T3 plants were isolated and examined for blue fluorescence. Introduction of the PAT1 gene fusants results in the reversion of fluorescence phenotype except in the case of pHS105. These results prompted us to perform a parallel analysis of anthranilate synthase and PAT interms of the genetic complementation. A plant line carrying pHS105 gene fusant does not completely complement the blue fluorescence but it accumulates less anthranilate than trp1-100. The activity of PAT was reduced in the transgenic mutant as well. The plant carrying these constructs will add to the growing collection of molecular tools for the study of the indolic secondary metabolism.

• Journal of Plant Biotechnology
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• 제42권3호
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• pp.186-195
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• 2015

Anthranilate synthase (AS)는 트립토판(Trp)과 indole-3-acetic acid, indole alkaloids의 생합성 경로에서 중요한 효소로 작용한다. 트립토판 생합성 상에서 feedback inhibition에 민감하게 반응하는 AS alpha-subunit 관련 OASA2 유전자 영역의 single (F124V) 및 double (S126F/L530D) 점돌연변이로 변형된 유전자의 재조합운반체를 작성하고 이들 유전자들을 Agrobacterium 방법으로 동진벼에 도입하여 형질전환체를 육성하였다. Single 및 double 돌연변이 OsASA2 유전자가 도입된 형질전환 벼 계통들은 nos gene probe를 이용한 TaqMan PCR 방법으로 single copy를 선발하였고, intergenic 계통을 선발하기 위해서 Bfa I 제한효소를 이용하여 RB와 LB 인접서열로부터 IPCR을 통한 FST 분석을 수행하여 4 개의 intergenic 계통을 선발하였다. 도입된 유전자의 발현으로 형질전환 벼는 Trp, IAN 및 IAA가 잎에 가장 많이 축적되었고, 종자의 트립토판 함량도 증가되었다. 후대에서 tryptophan 함량이 높은 S-TG와 D-TG의 두 호모 이벤트 계통을 육성하여 트립토판 함량을 분석한 결과 대조구에 비하여 13~30배 이상 높게 나타났으며, 유리아미노산의 함량도 증가하였다. 이벤트 계통을 이용하여 microarray 분석을 수행한 결과 세포 내 이온 수송, 영양분 공급 등에 영향을 주는 유전자군들이 up-regulation 되었고, 세포 내 기능유전자의 역할을 담당하는 조효소 등이 down-regulation 된 것을 확인 할 수 있었다. 이러한 결과는 선발된 두개의 상동성 이벤트 계통들이 고함량의 유리 트립토판 생산 벼의 육종에 효과적으로 이용될 수 있음을 보여준 결과로 생각된다.

• Journal of Plant Biotechnology
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• 제2권2호
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• pp.55-60
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• 2000

Experiments initiated 30 years ago to obtain selectable markers have led to a series of studies of Trp biosynthesis and anthranilate synthase (AS) the control enzyme using largely plant tissue cultures since they have experimental properties that can be readily exploited. Enzymological and compound feeding studies provided evidence that AS is the control point in the Trp biosynthesis branch and that altering the AS feedback control by the selection of mutants resistant to the Trp analog 5-methyl-tryptophan (5MT) can lead to the overproduction of this important amino acid. Plants regenerated from these Trp overproducing lines of most species also had high free Trp levels but Nicotiana tabaum (tobacco) plants expressed the feedback altered AS only in cultured cells and not in the regenerated plants. further tests by transient and stable expression of the cloned promoter for the naturally occurring tobacco feedback-insensitive AS, denoted ASA2, confirmed the tissue culture specific nature of the expression control. The 5MT caused by the expression of a feedback-insensitive AS from tobacco has been used to select protoplast fusion hybrids with several species since the resistance is expressed dominantly. Recently the ASA2 gene has been used successfully as a selectable marker to select transformed Astragalus sinicus and Glycine max hairy roots induced by Agrobactetium rhizogenes. These results show that the ASA2y-subunit can interact with the y-subunit of another species to form active feedback-insensitive enzyme that may be useful for selecting transformed cells. Plastid DNA transformation of tobacco has also effectively expressed ASA2 in the compartment in which Trp biosynthesis is localized in the cell.

• 한국식물생명공학회:학술대회논문집
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• 한국식물생명공학회 2003년도 추계학술대회 발표논문 초록집
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• pp.127-127
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• 2003