• Applied Biological Chemistry
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• v.38 no.3
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• pp.224-231
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• 1995

To increase the expression of a foreign protein in transgenic plant, the benefits of 5'-untranslated leader sequences of tobacco mosaic virus (TMV) RNA or soybean glycinin gene, Gy2, fused to a protein coding sequence were exploited. pGA643-derived plasmid contains 355 promoter of cauliflower mosaic virus, protein coding sequence of maize 10 kDa zein (10kZ) and Gy2 terminator. The leader from Gy2 or TMV RNA was inserted between the promoter and the coding sequence in each construct. The recombinant DNAs were introduced into tobacco plants by Agrobacterium mediated leaf disc transformation method. Although the transgene without the leader had more transcripts than the others, mRNAs containing the leader were translated more efficiently. It might be due to difference in the length of 5'-untranslated sequence and context surrounding the AUG codon, but could be sequence specific rather. These results suggest that the leader sequences of Gy2 and TMV play important roles as an enhancer in translational control of foreign gene in transgenic tobacco plant.

• Journal of fish pathology
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• v.34 no.1
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• pp.17-22
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• 2021

Eukaryotic translation is initiated by either cap-dependent or cap-independent way, and the cap-independent translation can be initiated by the internal ribosomal entry site (IRES). In this study, to know whether the 5'UTR leader sequence of marine birnavirus (MABV) segment A and segment B can act as IRES, bicistronic vectors harboring a CMV promoter-driven red fluorescent gene (mCherry) and poliovirus IRES- or MABV's leader sequence-driven green fluorescent gene (eGFP) were constructed, then, transfected into a mammalian cell line (BHK-21 cells) and a fish cell line (CHSE-214 cells). The results showed that the poliovirus IRES worked well in BHK-21 cells, but did not work in CHSE-214 cells. In the evaluation of MABV's leader sequences, the reporter eGFP gene under the 5'UTR leader sequence of MABV's segment A was well-translated in CHSE-214 cells, indicating 5'UTR of MABV's segment A initiates translation in the cap-independent way and can be used as a fish-specific IRES system. However, the 5'UTR leader sequence of MABV's segment B did not initiate translation in CHSE-214 cells. As the precise mechanism of birnavirid IRES-mediated translation is not known, more elaborate investigations are needed to uncover why the leader sequence of segment B could not initiate translation in the present study. In addition, further studies on the host species range of MABV's segment A IRES and on the screening of other fish-specific IRESs are needed.

• BMB Reports
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• v.30 no.1
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• pp.80-84
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• 1997

The synthesis and secretion of human angiogenin in E. coli by the natural leader sequence has been studied. We constructed a recombinant plasmid containing human angiogenin cDNA which encompassed all the coding region including leader sequence required for secretion. The recombinant plasmid was introduced into a suitable E. coli host. The angiogenin was detected in the culture medium and periplasm upon the induction of gene expression. The molecular weight of the secreted angiogenin was identical to that of authentic angiogenin purfied from human plasma when estimated by SDS-PAGE and immunoblotting. showing that the natural leader sequence was recognized and processed by the secretion machinery of E. coli. The angiogenin concentration in the culture medium reached a maximum within 2 h when expressed at $37^{\circ}C$ with 0.02~2 mM IPTG. In contrast, the expression level increased gradually over time up to 11 h at $23^{\circ}C$ with 0.002~2 mM IPTG and at $37^{\circ}C$ with 0.002 mM IPTG.

• ALGAE
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• v.26 no.3
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• pp.229-235
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• 2011

Spliced leader (SL) trans-splicing is a mRNA processing mechanism in dinoflagellate nuclear genes. Although studies have identified a short, conserved dinoflagellate SL (dinoSL) sequence (22-nt) in their nuclear-encoded transcripts, whether the majority of nuclear-coded transcripts in dinoflagellates have the dinoSL sequence remains doubtful. In this study, we investigated dinoSL-containing gene transcripts using 454 pyrosequencing data (Cochlodinium polykrikoides, 93 K sequence reads, 31 Mb; Prorocentrum minimum, 773 K sequence reads, 291 Mb). After making comparisons and performing local BLAST searches, we identified dinoSL for one C. polykrikoides gene transcript and eight P. minimum gene transcripts. This showed transcripts containing the dinoSL sequence were markedly fewer in number than the total expressed sequence tag (EST) transcripts. In addition, we found no direct evidence to prove that most dinoflagellate nuclear-coded transcripts have this dinoSL sequence.

• Biotechnology and Bioprocess Engineering:BBE
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• v.6 no.4
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• pp.306-308
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• 2001

A recombinant human growth hormone(hGH) was expressed as a secretory product in the yeast Saccharomyuces cerevisiae. There different leader sequences derived from the mating fac-tor $\alpha$1(MF$\alpha$1) inulinase and invertase were used to direct the secretion of hGH into the extracel-lular medium. Among three leader sequences tested, the inulinase leader sequence was found to be the most efficient in the secretory expression of hGH. In contrast, no hGH was detected in the ex-tracellular medium with the invertase leader sequence. After 48 h shake-flask culture, the yields of hGH secreted into th emedium by the invertase. MF$\alpha$1 inulinase and invertase leader sequences were approximately 0, 0.3 and 0.9 mg/L, respectively. The secretion efficiencies were also found to be 0, 3.8 and 13% for the invertase , MG$\alpha$1 and inulinase leader sequences, respectively.

• Archives of Pharmacal Research
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• v.29 no.12
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• pp.1154-1157
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• 2006

The ermK gene from Bacillus lichenformis encodes an inducible rRNA methylase that confers resistance to the macrolide-lincosamide-streptogramin B antibiotics. The ermK mRNA leader sequence has a total length of 357 nucleotides and encodes a 14-amino acid leader peptide together with its ribosome binding site. The secondary structure of ermK leader mRNA and a leader peptide sequence have been reported as the elements that control expression. In this study, the contribution of specific leader peptide amino acid residues to induction of ermK was studied using the PCR-based megaprimer mutation method. ermK methylases with altered leader peptide codons were translationally fused to E. coli ${\beta}-galactosidase$ reporter gene. The deletion of the codons for Thr-2 through Ser-4 reduced inducibility by erythromycin, whereas that for Thr-2 and His-3 was not. The replacement of the individual codons for Ser-4, Met-5 and Arg-6 with termination codon led to loss of inducibility, but stop mutation of codon Phe-9 restored inducibility by erythromycin. Collectively, these findings suggest that the codons for residue 4, 5 and 6 comprise the critical region for induction. The stop mutation at Leu-7 expressed constitutively ermK gene. Thus, ribosome stalling at codon 7 appears to be important for ermK induction.

• Genomics & Informatics
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• v.15 no.4
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• pp.142-146
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• 2017

More effective production of human insulin is important, because insulin is the main medication that is used to treat multiple types of diabetes and because many people are suffering from diabetes. The current system of insulin production is based on recombinant DNA technology, and the expression vector is composed of a preproinsulin sequence that is a fused form of an artificial leader peptide and the native proinsulin. It has been reported that the sequence of the leader peptide affects the production of insulin. To analyze how the leader peptide affects the maturation of insulin structurally, we adapted several in silico simulations using 13 artificial proinsulin sequences. Three-dimensional structures of models were predicted and compared. Although their sequences had few differences, the predicted structures were somewhat different. The structures were refined by molecular dynamics simulation, and the energy of each model was estimated. Then, protein-protein docking between the models and trypsin was carried out to compare how efficiently the protease could access the cleavage sites of the proinsulin models. The results showed some concordance with experimental results that have been reported; so, we expect our analysis will be used to predict the optimized sequence of artificial proinsulin for more effective production.

• Journal of Microbiology and Biotechnology
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• v.9 no.2
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• pp.196-200
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• 1999

A recombinant human $\alpha_{s1}$-casein was expressed as a secretory product in the yeast Saccharomyces cerevisiae. Three different leader sequences derived from the mating factor $\alpha$l (MF$\alpha$l), inulinase, and human $\alpha_{s1}$-casein were used to direct the secretion of human $\alpha_{s1}$-casein into the extracellular medium. Among the three leader sequences tested, the native leader sequence of human $\alpha_{s1}$-casein was found to be the most efficient in the secretory expression of human $\alpha_{s1}$-casein, which implies that the native leader sequence of human $\alpha_{s1}$-casein might be used very efficiently for the secretory production of other heterologous proteins in yeast. The recombinant human $\alpha_{s1}$-casein was proteolytically cleaved as the culture proceeded. Therefore, an attempt was made to produce human $\alpha_{s1}$-casein using a S. cerevisiae mutant in which the YAP3 gene encoding yeast aspartic protease 3 (YAP3) was disrupted. After 72 h of culture, most of the human $\alpha_{s1}$-casein secreted by the wild type was cleaved, whereas more than 70% of the human $\alpha_{s1}$-casein secreted by yap3-disruptant remained intact. The results suggest that YAP3 might be involved in the internal cleavage of human $\alpha_{s1}$-casein expressed in yeast

• Microbiology and Biotechnology Letters
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• v.31 no.1
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• pp.36-41
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• 2003

In order to increase the production and secretion rate of mouse salivary $\alpha$-amylase from Saccharomyces cerevisiae, various experiments were attempted. A plasmid pCNNinv (AMY) was constructed by the substitution of ADCl promoter and native signal sequence of mouse salivary $\alpha$-amylase cDNA gene with PRBI promoter and yeast invertase leader sequence, which resulted in 25% increase in the production of $\alpha$-amylase in the culture medium. The respiratory deficient transformant carrying pCNNinv (AMY) were obtained by treating yeast cells with ethidium bromide, and the $\alpha$-amylase activities in the culture brothes of the respiratory-deficient transformants were 5-8 times higher than that of parental wild type strain. $\alpha$-Amylase activity was also increased 3 times when the 0.015% (w/v) of 2-mercaptoethanol was added to the culture medium.

• Journal of Microbiology and Biotechnology
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• v.1 no.1
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• pp.31-36
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• 1991

Yeast expression plasmids containing an appropriate leader sequence and bovine $\beta$-casein cDNA were constructed to produce $\beta$-casein for the study of its functional characteristics. Two kinds of expression systems for $\beta$-casein were constructed using pCGYl444 as a precursor plasmid. This plasmid is a yeast-E. coli shuttle vector which contains the chelatin promoter. The plasmid pISB202 contains the invertase leader sequence and $\beta$-casein gene. The plasmid pDEB303 contains the original bovine $\beta$-casein leader sequence gene. These two plasmids were introduced into S. cerevisiae AB116 which is a strain deficient in the major yeast proteinases. Each clone was grown in minimal media for 24 h before induction by $CuSO_4$. The cells were thus grown under expression conditions. Both strains harbouring pISB202 and pDEB303 expressed bovine $\beta$-casein. The $\beta$-casein was detected using immunochemical staining after western blot. Secretion of $\beta$-casein was detected in the culture broth. The estimated amount of secreted $\beta$-casein was approximately 50 ${\MU}g$/l.