• 한국식물학회:학술대회논문집
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• 한국식물학회 1985년도 워크샵 및 심포지엄 북한산국립공원의 식생
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• pp.7-18
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• 1985

Light-regulated translation of chloroplast mRNAs requires nuclear-encoded trans-acting factors that interact with the 5' untranslated region (UTR) of these mRNAs. A set of four proteins (60, 55, 47, and 38 kDa) that bind to the 5'-UTR of the psbA mRNA had been identified in C. reinhardtii. 47 kDa protein (RB47) was found to encode a chloroplast poly (A)-binding protein (cPABP) that specifically binds to the 5'-UTR of the psbA mRNA, and essential for translation of this mRNA, cDNA encoding 60 kDa protein (RB60) was isolated, and the amino acid sequence of the encoded protein was highly homologous to plants and mammalian protein disulfide isomerases (PDI), normally found in the endoplasmic reticulum (ER). Immunoblot analysis of C. reinhardtii proteins showed that anti-PDI recognized a distinct protein of 56 kDa in whole cell extract, whereas anti-rRB60 detected a 60 kDa protein. The ER-PDI was not retained on heparin-agarose resin whereas RB60 was retained. In vitro translation products of the RB60 cDNA can be transported into C. reinhardtii chloroplast in vitro. Immunoblot analysis of isolated pea chloroplasts indicated that higher plant also possess a RB60 homolog. In vitro RNA-binding studies showed that RB60 modulates the binding of cPABP to the 5'-UTR of the psbA mRNA by reversibly changing the redox status of cPABP using redox potential or ADP-dependent phosphorylation. Site-directed mutagenesis of -CGHC- catalytic site in thioredoxin-like domain of RB60 is an unique PDI located in the chloroplast of C. reinhardtii, and suggest that the chloroplast PDI may have evolved to utilize the redox-regulated thioredoxin like domain as a mechanism for regulating the light-activated translation of the psbA mRNA.

• BMB Reports
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• 제35권3호
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• pp.273-282
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• 2002

Sp3 is a bifunctional transcription factor that has been reported to stimulate or repress the transcription of numerous genes. Although the size of Sp3 mRNA is 4.0kb, the size of the known Sp3 cDNA sequence is 3.6kb. Thus, Sp3 functional studies have been performed with an artificially introduced start codon, and thus an amino-terminus that differs from the wild-type. Ideally, full-length cDNA expression vectors with the appropriate start codon should be utilized for these studies. Using 5'rapid amplification of cDNA ends, a full-length Sp3 cDNA clone was generated and the sequence verified in nine cell lines. No AUG initiation codon was present. However, stop codons were present in all three frames 5' to the known coding sequence. In vitro translation of this full-length cDNA clone produced the expected three isoforms-one at 100 kDa and two in the mid 60 kDa range. Electrophoretic mobility shift assays showed that the protein products had the ability to bind to the Sp1/3 consensus sequence. In vitro studies, using our Sp3 clone and site directed mutagenesis, identified the translation initiation site for the larger isoform as AUA. AUA has not been previously described as an endogenous initiation codon in eukaryotes.

• BMB Reports
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• 제53권2호
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• pp.94-99
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• 2020

Brain cytoplasmic 200 RNA (BC200 RNA) is proposed to act as a local translational modulator by inhibiting translation after being targeted to neuronal dendrites. However, the mechanism by which BC200 RNA inhibits translation is not fully understood. Although a detailed functional analysis of RNA motifs is essential for understanding the BC200 RNA-mediated translation-inhibition mechanism, there is little relevant research on the subject. Here, we performed a systematic domain-dissection analysis of BC200 RNA to identify functional RNA motifs responsible for its translational-inhibition activity. Various RNA variants were assayed for their ability to inhibit translation of luciferase mRNA in vitro. We found that the 111-200-nucleotide region consisting of part of the Alu domain as well as the A/C-rich domain (consisting of both the A-rich and C-rich domains) is most effective for translation inhibition. Surprisingly, we also found that individual A-rich, A/C-rich, and Alu domains can enhance translation but at different levels for each domain, and that these enhancing effects manifest as cap-dependent translation.

• Journal of Plant Biology
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• 제32권1호
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• pp.33-40
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• 1989

Polysomal polyadenylated mRNAs which were purified from pea leaves were fractionated by sucrose grandient sedimentation. Fractions corresponding to the peak at 11.5S were found to contain mostly mRNA encoding the precursor polypeptide to the small subunit of ribulose bisphosphate carboxylase (rbcS) by in vitro translation in wheat germ extract. Double-stranded cDNA which was synthesized from the 11.5S mRNA was cloned into Hind III site of plasmid pBR 325. A cDNA clone, H24, was identified to code for rbcS. In vitro translation product of the hybridization-selected mRNA was molecular weight 20,000, presumably the precursor of rbcS. The nucleotide sequences of the H24 showed almost complete homology with the sequences encoding the transit peptide of the rbcS-3A gene which was reported by Fluhr et al.(1986).

• Journal of Plant Biotechnology
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• 제38권1호
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• pp.87-93
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• 2011

SAMDC는 폴리아민 생합성 과정에서 주효소로 작용하며 항상성을 유지하기위해 정교하게 조절된다. 카네이션 SAMDC 유전자는 5'-leader sequence에 54개 아미노산으로 구성된 small uORF가 존재한다. Translation 과정을 조절하는 uORF의 작용기작을 연구하기 위하여 35S 프로모터에 SAMDC 유전자의 uORF 부위와 GUS 유전자를 재조합한 형질전환 담배 식물체를 이용하였다. 본 실험에서는 SAMDC uORF 염기서열 혹은 SAMDC uORF 단백질에 의해서 downstream GUS ORF의 translation이 억제되었다. 특히 translation 억제는 개시코돈이 point-mutation된 construct에서 효과적으로 이루어졌다. 따라서 이러한 결과는 ribosomal stalling이 translation 억제 과정에 관여한 것으로 사료된다. 개시 코돈과 종결코돈을 가진 SAMDC uORF의 아미노산 서열을 frame shift 시키면 GUS 활성이 증가하였는데 이는 translation inhibitor로서 작용할 때 아미노산 서열이 중요하다는 것을 의미하며, 결국은 SAMDC uORF의 단백질 구조가 중요하게 작용할 가능성을 제시한다. 또한 유식물과 담배 꽃 등의 in vivo 상에서도 GUS 발현을 조직화학적으로 분석했을 때 small uORF가 존재할 경우 GUS 염색이 크게 저하되었지만, 개시코돈이나 혹은 종결코돈이 제거되도록 point-mutation 시킨 construct가 도입된 형질전환식물체에서는 SAMDC uORF의 억제효과가 크게 완화 되었다. 또한 가장 중요한 관찰 결과로는 small uORF 염기서열로부터 in vitro 시스템에서 5.7 kDa의 단백질이 실제적으로 합성되었음을 관찰하였다. 폴리아민 처리 후 GUS 단백질이 억제된 결과는 uORF로부터 합성된 단백질이 폴리아민 뿐 만 아니라 translation 과정에 관여하는 다른 요소들과 상호작용을 이루어 조절될 수 있음을 암시한다.

• 한국동물학회지
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• 제37권2호
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• pp.267-273
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• 1994

빨간집모기가 흡혈한 후 체내에서 일어나는 total RNA 변화를 조사한 결과 흠혈 후 6시 간 이후에 RNA양이 증가하기 시작하여 18시간에서 peak를 보인 후 감소하다가 30시간째 부터 증가하기 시작하여 48시간 후에 최대의 RNA 양을 보인 후 급격히 감소하였다 난소에서의 RNA 합성을 정량한 결과 흡혈 후 24시간 이전에는 흡혈 전에 비하여 전혀 증가하지 않았고 30시간 이후부터 증가하기 시작하여 48시간에 가장 많은 양의 RNA가 난소내에 존재하였고, 그 후 급격히 감소하였다. 흡혈 후 6시간 간격으로 난소로 부터 total RNA를 추출하여 in vitro translation을 실시하고 TCA 침전법과 면역침전법으로 합성된 3H-protein과 3H-vitellogenin을 정량하였다. 그 결과 흡혈 후 36시간 이후의 난소로부터 3H-protein과 지-vitellogenin의 합성이 일어나기 시작하여 48시간된 난소에서 가장 많은 양의 3H-protein과 3H-vitellogenin이 합성되었으며, 이때 합성된 단백질의 약 45% 정도가 난황단백질인 3H-vitellogenin으로 나타났다 이상의 결과로 빨간집모기에서는 지방체에서 뿐만 아니라 난소에서도 난황단백질의 합성이 일어남을 알수 있다.

• 한국미생물·생명공학회지
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• 제38권1호
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• pp.64-69
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• 2010

본 실험은 Propolis ethanol 추출물과, chloroform, ethyl acetate, butanol 등 4가지 용매로 더 추출한 분획틀을 이용하여 DPPH radical 소거능 실험과 항진균활성을 알아보았고, 고체배지 및 액체배지에서 항진균활성을 측정하였다. 또한 luciferase mRNA를 이용한 in vitro translation으로 이들 추출물에 의한 단백질합성에의 영향을 검토하였다. 첫 번째로, 액체배지에서의 항진균 활성을 실험한 결과 Candida glabrata, Candida lusitaniae 그리고 Cryptococcos neoformans 등의 성장저해율이 chloroform 분획 존재하에서 각각 39%, 41%, 48% 이었으며 ethyl acetate 분획 존재하에서 각각 25%, 24%, 13%로 측정되었다. 이 결과는 ethyl acetate 분획에 비해 chloroform 분획에 진균 성장 저해물질이 가장 많이 존재함을 나타낸다. 두 번째로, 동일한 비율로 희석한 프로폴리스 분획물들과 합성 항산화제인 BHT의 수소공여능을 비교하였을 때 Ethanol 추출물의 수소공여능은 합성항산화제인 0.1% BHT의 수소공여능보다 높았으며, 분획들 중에서 chloroform 분획이 가장 수소공여능이 높았다. 세 번째로, luciferase mRNA를 이용한 in vitro, translation 실험에서는 Propoliis ethanol 추출물이 단백질합성을 저해하는 것으로 관찰되었다. Propolis 분획물들 중에서 chloroform 분획이 단백질 합성을 가장 많이 저해하였다. 이와 같은 결과는 chloroform 분획물이 다른 분획에 비해 수소공여능, 진균성장 저해율 및 단백질합성 저해활성이 가장 큰 것으로 보여지므로 이 분획물에 대한 생화학적인 연구가 요구된다.

• 미생물학회지
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• 제40권1호
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• pp.7-11
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• 2004

eIF5B는 단백질합성의 개시 인자로서 Met-$tRNA^{Met}$을 AUG 개시코돈에 전달하고, 리보솜의 두 소단위체 결합을 유도한다. 이 인자의 기능을 연구할 목적으로 eIF5B를 코딩하는 FUN12 유전자의 5'말단 부위를 삭제하는 연구를 수행하였다. 프로모터의 대부분을 삭제한 FUN12를 함유한 pRS효모벡터를 FUN12가 삭제되어 천천히 자라는 돌연변이주 ($fun12{\Delta}g$)에 전달하였을 때 그 표현형을 부분적으로 상보하였다. 위와 같이 제조된 벡터에서 N-말단이 상실된 eIF5B 단백질이 발현되었고, 그 양이 정상 균주에서 발현되는 eIF5B 양의 약 5%에 불과하였다. 이와 같이 부분적으로 성장을 상보한 균주에서 발현된 적은 양의 단백질합성개시 인자 eIF5B는 직접적으로 그 성장을 제한하는 요소로 작용한다. 이러한 균주에서 성장의 제한인자인 eIF5B는 in vitro 에서도 역시 전체 단백질 합성의 활성을 조절하였다.

• The Journal of Advanced Prosthodontics
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• 제9권5호
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• pp.341-349
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• 2017

PURPOSE. This study evaluated the accuracies of different bite registration techniques for implant-fixed prostheses using three dimensional file analysis. MATERIALS AND METHODS. Implant fixtures were placed on the mandibular right second premolar, and the first and second molar in a polyurethane model. Aluwax (A), Pattern Resin (P), and Blu-Mousse (B) were used as the bite registration materials on the healing abutments (H) or temporary abutments (T). The groups were classified into HA, HP, HB, TA, TP, and TB according to each combination. The group using the bite impression coping was the BC group; impression taking and bite registration were performed simultaneously. After impression and bite taking, the scan bodies were connected to the lab analogs of the casts. These casts were scanned using a model scanner. The distances between two reference points in three-dimensional files were measured in each group. One-way ANOVA and Duncan's test were used at the 5% significance level. RESULTS. The smallest distance discrepancy was observed in the TB group using the temporary abutments. The Blu-Mousse and HP groups showed the largest distance discrepancy. The TB and BC groups showed a lower distance discrepancy than the HP group (P=.001), and there was no significant difference between the groups using the temporary abutments and healing abutments (P>.05). CONCLUSION. Although this study has limitations as an in-vitro investigation, the groups using the temporary abutments to hold the Blu-Mousse record and bite impression coping showed greater accuracy than the group using the healing abutments to hold the pattern resin record.

• 한국미생물생명공학회:학술대회논문집
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• 한국미생물생명공학회 1986년도 추계학술대회
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• pp.505.1-505
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• 1986

The gene for iso-1-cytochrome c for Saccharomyces cerevisiae was recloned into a pSP65 vector containing an active bacteriophage SP6 promoter. The iso-1-cytochrome c gene was cloned as an 856 bp Xho 1-Hind III fragment. When the resulting plasmid was digested at the Hind 111 site 279 bases downstream from the termination codon of the gene and transcribed in vitro using SP6 RNA polymerase, full length transcripts were produced. The SP6 iso-1-cytochrome c mRNA was translated using a rabbit reticulocyte lysate system and the protein products analyzed on SDS polyacrylamide gels. One major band was detected by autofluorography. This band was found to have a molecular weight of 12,000 Da and coincided with the Coomassie staining band of apocytochrome c from S. cerebisiae. The product was also shown to be identical with that of standard yeast apocytochrome c on an isoelectric focusing gel. The in vitro synthesized iso-a-cytochrome c was methylated by adding partially purified S-adenosyl-L-methionine . protein-lysine N-methyltransferase (Protein methylase III; EC 2.1.1.43) from S. cerevisiae along with S-adenosyl-L-methionine to the in vitro translation mixtures. The methylation was shown to be inhibited by the addition of the methylase inhibitor S-adenosyl-L-homocysteine or the protein synthesis inhibitor pu omycin. The methyl derivatives in the protein were identified as $\varepsilon$-N-mono, di and trimethyllysine by amino acid analysis. The molar ratio of methyl groups incorporated to that of cytochrome c molecules synthesized showed that 23% of the translated cytochrome c molecules were methylated by protein methylase III.