• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2002년도 국제심포지엄
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• pp.117-117
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• 2002

The oocytes from most of animal species accumulate genetic information and other necessary materials during oogenesis for the later use in the early development. Over the years oocyte maturation has been studied extensively both in vitro and in vivo. Particularly, maturation of follicular oocyte in vitro becomes one of the important tools for the studies of basic cell biology, the in vitro technology of animal production, and in particular, the somatic cell cloning by nuclear transfer. We examined meiotic maturation and cumulus expansion in the presence of translation or transcription inhibitors for varying periods of in viかo maturation (IVM) of pig oocyte. In Experiment 1, the results revealed that translation and transcription inhibitors inhibited cumulus expansion and meiotic maturation during 35h of IVM. However, 50 to 60% of the oocytes underwent nuclear maturation without cumulus expansion during 75h of IVM. The rest of the oocytes were arrested at metaphase I (40-50%) in the presence of the inhibitors. In Experiment II, the OCCs were exposed to the drugs only for 15h to examine translation and transcription inhibitors on cumulus expansion and meiotic maturation. Transcription inhibitors for 15h did not arrest meiotic maturation when the oocytes were cultured for subsequent, necessary period of IVM, whereas cumulus expansion was completely inhibited, suggesting that initial 15h is critical transcription activity far cumulus expansion. Translation inhibitors for 15h exposure did not alter cumulus expansion and meiotic maturation during subsequent culture in the absence of the drugs. In Experiment III, the OCCs were exposed to the drugs only for later 30h to examine the influence of transcription and translation inhibitors on oocyte maturation. Interestingly, all meiotic maturation underwent normally with full expansion of cumulus. Similar results were obtained from Experiment IV where 5h of exposure from 15 to 20h of IVM culture to the drugs was performed and subsequently cultured for same period in fresh medium. Taken there results together, both transcription and translation are necessary for nuclear maturation and cumulus expansion, and first 15h IVM for cumulus expansion is critical. The arrested oocytes by the drugs were still capable of undergoing nuclear maturation, although cumulus expansion was affected.

• 생명과학회지
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• 제15권5호
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• pp.779-782
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• 2005

Coupled transcription/translation cocktail을 이용하여 R. glutinis EH 유전자를 in vitro에서 합성하고 활성을 평가하였다. SDS-PAGE 및 immunoblotting을 통하여 45 kDa 크기의 EH 단백질이 발현되었음을 확인하였고, NBP assay 및 chiral GC 분석을 통해 발현된 단백질이 (R)-styrene oxide에 대한 입체선택성이 있음을 확인하였다. 따라서 무세포 단백질 합성 시스템을 이용하여 입체선택성을 유지시킨 EH 유전자 발현이 가능하며, 이러한 방법은 putative EH 유전자 탐색 등에 효율적으로 응용될 것이다.

• Molecules and Cells
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• 제40권1호
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• pp.1-9
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• 2017

Serine and arginine-rich (SR) proteins are RNA-binding proteins (RBPs) known as constitutive and alternative splicing regulators. As splicing is linked to transcriptional and post-transcriptional steps, SR proteins are implicated in the regulation of multiple aspects of the gene expression program. Recent global analyses of SR-RNA interaction maps have advanced our understanding of SR-regulated gene expression. Diverse SR proteins play partially overlapping but distinct roles in transcription-coupled splicing and mRNA processing in the nucleus. In addition, shuttling SR proteins act as adaptors for mRNA export and as regulators for translation in the cytoplasm. This mini-review will summarize the roles of SR proteins as RNA binders, regulators, and connectors from transcription in the nucleus to translation in the cytoplasm.

• BMB Reports
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• 제38권1호
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• pp.65-70
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• 2005

Choristoneura fumiferana granulovirus (ChfuGV) infection results two types of enveloped virions: Occlusion-derived virus (ODV) and budded virus (BV). Structural proteins ODVP-6E/ODV-E56 and p74 are two major conserved ODV-associated proteins that may be involved in the initiation of viral infection cycle in susceptible host insect larvae. This study presents the characterization of ChfuGV odvp-6e/odv-e56 and p74 transcription and translation as well as immunolocalization of these proteins in the occluded ChfuGV virion. Our results revealed that the transcription of odvp-6e/odv-e56 and p74 genes, both, start at 24 hours post infection (h p.i.). Using monospecific polyclonal antibodies made against ODVP-6E/ODV-E56 and p74 we demonstrated that these proteins are both expressed late in infection (24 h p.i.). Immunogold labeling using antisera against ODVP-6E/ODV-E56 and p74 proteins demonstrated that ODVP-6E/ODV-E56 and p74 proteins are both associated with the ODV envelop of ChfuGV.

• Molecules and Cells
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• 제42권7호
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• pp.523-529
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• 2019

mRNA quality is controlled by multiple RNA surveillance machineries to reduce errors during gene expression processes in eukaryotic cells. Nonsense-mediated mRNA decay (NMD) is a well-characterized mechanism that degrades error-containing transcripts during translation. The ATP-dependent RNA helicase up-frameshift 1 (UPF1) is a key player in NMD that is mostly prevalent in the cytoplasm. However, recent studies on UPF1-RNA interaction suggest more comprehensive roles of UPF1 on diverse forms of target transcripts. Here we used subcellular fractionation and immunofluorescence to understand such complex functions of UPF1. We demonstrated that UPF1 can be localized to the nucleus and predominantly associated with the chromatin. Moreover, we showed that UPF1 associates more strongly with the chromatin when the transcription elongation and translation inhibitors were used. These findings suggest a novel role of UPF1 in transcription elongation-coupled RNA machinery in the chromatin, as well as in translation-coupled NMD in the cytoplasm. Thus, we propose that cytoplasmic UPF1-centric RNA surveillance mechanism could be extended further up to the chromatin-associated UPF1 and co-transcriptional RNA surveillance. Our findings could provide the mechanistic insights on extensive regulatory roles of UPF1 for many cellular RNAs.

• Journal of Microbiology and Biotechnology
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• 제16권3호
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• pp.355-359
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• 2006

The functional stability of mRNA is one of the crucial factors affecting the efficiency of in vitro translation. As the rapid degradation of mRNA in the cell extract (S30 extract) causes early termination of the translational reactions, extending the mRNA half-life will improve the productivity of the in vitro protein synthesis. Thus, a simple PCR-based method is introduced to increase the stability of mRNA in an S30 extract. The target genes are PCR-amplified with primers designed to make the ends of the transcribed mRNA molecule anneal to each other. When compared with normal mRNA, the mRNA with the annealing sequences resulted in an approximately 2-fold increase of protein synthesis in an in vitro translation reaction. In addition, sequential transcription and translation reactions in a single tube enabled direct protein expression from the PCR-amplified genes without any separate purification of the 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.

• Journal of Microbiology
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• 제35권4호
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• pp.315-322
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• 1997

The effects of a polyamine, spermine, on the differentiation of Naegleria gruberi amebas into flagellates were tested. Addition of spermine at early stages of differentiation (until 40 min after the initiation of differentiation) completely inhibited the differentiation. To understand the inhibition mechanism, we examined the effect of spermine treatment on the transcription and translation of differentiation-specific genes during differentiation. Addition of spermine at early stages did not inhibit the accumulation of two differentiation-specific mRNAs, ${\alpha}$-tubulin and Class I mRNA, significantly, but rather prevented the rapid degradation of the mRNAs in later overall protein synthesis partially and gradually. However, translation of the ${\alpha}$-tubulin mRNA was completely inhibited. These data suggest that the inhibition of differentiation of N. gruberi by spermine treatment did not result from the inhibition of transcription of differentiation-specific genes but from the specific inhibition of translation of the mRNAs during the differentiation.

• 한국동물위생학회지
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• 제27권1호
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• pp.95-101
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• 2004

Babesia bovis rap-1 and B equi ema-1 intergenic(IG) nucleotides were analyzed and compared for identifying putative promoter sites using computer programs. The reason to initiate this research was to determine if IG nucleotides of Babesia genes that are predicted to be involved in erythrocyte invasion have functions regulating gene transcription and translation, which can be applied to functional gene knockout. Four IG sequences used included BbIG5(B bovis rap-1 5' IG), BblG3(B bovis rap-1 3' IG), BeIG5(B equi ema-1 5' IG) and BeIG3(B equi ema-1 3' IG). BbIG5 contained a putative promoter at nucleotide 197-246 with a predicted TATA-box and a transcription start site. BbIG3 had a putative promoter at nucleotide 270-320 with two predicted TATA-boxes and a transcription start site. BeIG3 had a putative promoter at nucleotide 155-205 with a predicted TATA-box and a transcription start site. Putative promoter sites in these three sequences mentioned above were identified with score cutoff 0.8, which means detection of about 40% recognized promoters with 0.1-0.4% false positives. In contrast, BeIG5 had a putative promoter at nucleotide 163-213 with score cutoff 0.8, but neither TATA-box nor transcription start site were recognized. However, BeIG5 had a putative promoter at nucleotide 388-438 with a predicted TATA-box and a transcription start site when score cutoff was decreased to 0.18, which means detection of about 70% recognized promoters with 2.2-5.3% false positives. These sequences with putative promoters can be tested if they have functions regulating gene transcription and translation.

• BMB Reports
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• 제50권4호
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• pp.194-200
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• 2017

Eukaryotic gene expression is precisely regulated at all points between transcription and translation. In this review, we focus on translational control mediated by the 3'-untranslated regions (UTRs) of mRNAs. mRNA 3'-UTRs contain cis-acting elements that function in the regulation of protein translation or mRNA decay. Each RNA binding protein that binds to these cis-acting elements regulates mRNA translation via various mechanisms targeting the mRNA cap structure, the eukaryotic initiation factor 4E (eIF4E)-eIF4G complex, ribosomes, and the poly (A) tail. We also discuss translation-mediated regulation of mRNA fate.