• 제목/요약/키워드: Nuclease S1

검색결과 54건 처리시간 0.031초

Effective Family Shuffling Method Using Complementary DNA Fragments Produced by S1 Nuclease

  • Hong, Soon-Gyu
    • Journal of Microbiology and Biotechnology
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    • 제16권12호
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    • pp.2004-2007
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    • 2006

  • An efficient method for the in vitro reassembly of homologous DNA sequences is presented. The proposed method involves obtaining single strands of homologous genes and hybridizing them to obtain partially hybridized heteroduplex DNA; cleaving the single-stranded regions of the heteroduplex DNA using S1 nuclease to generate double-strand DNA fragments; denaturing the double-strand DNA fragments to generate single-strand DNA fragments; conducting a series of polymerase chain reactions (PCR) using the single-strand DNA fragments as internal primers and a mixture of homologous DNA as templates to obtain elongated reassembled DNA; and finally, amplifying the reassembled DNA by a PCR using terminal primers. As a result, DNA reassembly could be achieved between homologous genes with a sequence similarity as low as 78%.

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The Regulation of Alpha-Amylase Synthesis in Bacillus subtilis

  • Won, Mi-Sun
    • Journal of Microbiology and Biotechnology
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    • 제1권4호
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    • pp.256-260
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    • 1991

  • In B. subtilis, $\alpha$-amylase synthesis is regulated by amyR located directly on the upstream of amyE. Three different amyR alleles have been reported, amyR1, amyR2 and amyR3. Strains bearing the gra-10 mutation which confers derepression for catabolite repression has GlongrightarrowA transition mutation at ＋5 of amyR1. S1 nuclease mapping demonstrated that transcription initiated at 8 bases downstream from the -10 region of putative E$\sigma^{A}$ promoter P1 in amyR1 and gra-10. In amyR2, the major transcription initiatd at the same place and the minor, 10 bases downstream from -10 of P2. The transcript from P2 contributed approximately 15-20% of total amyE mRNA. S1 nuclease protection experiment indicated that amyE mRNA levels corresponded to the rate of synthesis assumed by specific activities of $\alpha$-amylase in culture supernatants, suggesting that $\alpha$-amylase synthesis is regulated at the level of transcription.n.

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$^{32}$P-Postlabelling 방법의 응용 : Azo색소 및 Flavonoid화합물에 의해 유도되는 DNA Adduct의 겸출에 관한 연구 (Application of the $^{32}$P-Postlabelling Technique : A Study on Detection of DNA Adduct Induced by Azo Dyes rind Flavonoid Compounds)

  • 김재현;박창원;박정식;홍연탁;김효정;이주한;이헌수;이동권
    • Biomolecules & Therapeutics
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    • 제1권1호
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    • pp.58-64
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    • 1993

  • DNA addicts induced by putative chemical related to carcinogenesis were detected and determined by $^{32}$P-Postlabelling assay after exposure of 4 compounds comprising two auto dyes (amaranth, new coccine) and two flavonoid compounds (rutin, quercetin) to ICR mouse. DNA was isolated from mouse liver and digested enzymatically to deoxyribonucleoside 3'-monophosphate. The postincubation of DNA digests with nuclease Pl before $^{32}$P-labelling enhanced the technique's sensitivity. Nuclease Pl cleaves deoxyribonucleoside 3'-mono-phosphates of normal nucleotides to deoxyrihonucleosides which do not serve as substrates for polynucleotide kinase, while most of addicts were found to be totally or partially resistant to the 3'-dephosphorylating action of nuclease Pl. The adducted deoxyribonucleoside 3'-monophosphate was converted to 5'-$^{32}$P-labelled deoxynucleoside 3',5'-bisphosphate by T4 polynucleotide kinase. The nucleotides were separated by anion-exchange thin layer chromatography(TLC) on polyethyleneimine cellulose by 4-dimensional or 2-dimensional TLC then detected by autoradiography. The results show that DNA addicts were detected in liver DNA of ICR mouse after administration of amaranth and quercetin by 2-dimensional and/or 4-dimensional TLC.

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Streptomyces griseus의 특이적 포자형성에 관여하는 유전자의 전사량 분석 (Transcriptional Analysis of Genes Involved in Ectopic Sporulation in Streptomyces griseus)

  • 지원재
    • 한국미생물·생명공학회지
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    • 제44권4호
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    • pp.563-570
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    • 2016

  • S. griseus wild type에서 dasA 유전자의 과발현에 의해 유도된 기저균사의 ectopic sporulation 관련 유전자를 알아보기 위해서, empty vector가 삽입된 균주와 dasA가 과발현된 균주의 전사체를 DNA microarray법으로 비교하였다. DNA microarray 결과를 토대로 dasA 유전자 과발현 균주에서 2배이상 발현량이 증가되었으며 p-value가 0.05 미만(p-value < 0.05)인 유전자들 중에서 false positive 를 제외시키는 작업을 통하여 최종적으로 4개의 유전자(SGR794, SGR2469, SGR3656, SGR3657)와 3개의 cluster (SGR795-797, SGR2377-2378, SGR6997-6998)를 선발하였다. 이들의 전사량은 low resolution Sl nuclease mapping 법을 통하여 dasA 유전자 과발현 균주에서 증가된 것을 확인하였다.

  • https://doi.org/10.4014/mbl.1607.07008 인용 PDF KSCI

시험관내에서 합성한 오이모자이크 바이러스 RNA단편을 성공적으로 절단한 ribozyme의 식물체내의 발현 (Expression of in vitro-tested ribozyme against cucumber mosaic virus RNA in tobacco plant)

  • 박상규
    • Applied Biological Chemistry
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    • 제39권5호
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    • pp.355-360
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    • 1996

  • 시험관내에서 합성한 오이모자이크 바이러스 RNA단편을 성공적으로 절단한 ribozyme (한국농화학회지 37: 56-63(1994))을 담배 식물체에 발현시켜 바이러스 저항성 식물체를 만들려고 하였다. 해당 ribozyme의 염기서열을 함유한 DNA 단편을 꽃양배추 바이러스 35S promoter와 nopaline 합성효소 terminator에 연결시키고 연결 부위 및 ribozyme의 염기서열을 확인하였다. 염기서열을 확인한 합성유전자를 Agrobacterium tumefaciens LBA4404에 합성유전자를 함유한 E. coli HB101을 E. coli HB101(pRK2073)를 helper로 Agrobacterium tumefaciens LBA4404와 함께 배양하는 tri-parental mating system을 이용하여 도입시켰다. Ribozyme 유전자를 함유한 Agrobacterium 세포를 담배잎 조각과 함께 배양한 후 항생제인 kanamycin을 함유한 MS 배지에서 자란 열개의 작은 식물체를 재분화하였다. 형질전환한 식물체내에 ribozyme 유전자가 존재하는지의 여부는 합성효소증폭반응(PCR)을 이용하였던바, 일곱개체가 예상된 570 염기쌍의 DNA 단편을 가지고 있었다. 이들 중 네 개체로부터 RNA을 분리하여 formamide를 함유한 agarose에서 전기영동한 후 35S-ribozyme-nos의 DNA 단편으로 Northern hybridization을 행하였던 바, 식물세포내의 nuclease에 의해 ribozyme RNA가 분해된 듯 감지 할 수 없었다. 따라서 ribozyme을 이용하여 바이러스 저항성 식물체를 얻으려면 nuclease에 의해 분해되지 않는 ribozyme이 필요할 것으로 사료된다.

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