• Bulletin of the Korean Chemical Society
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• v.29 no.6
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• pp.1137-1140
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• 2008

M1 RNA, the catalytic subunit of Escherichia coli RNase P, is an essential ribozyme that processes the 5' leader sequence of tRNA precursors (ptRNAs). Using KS2003, an E. coli strain generating only low levels of M1 RNA, which showed growth defects, we examined whether M1 RNA is involved in polycistronic mRNA processing or degradation. Microarray analysis of total RNA from KS2003 revealed six polycistronic operon mRNAs (acpP-fabF, cysDNC, flgAMN, lepAB, phoPQ, and puuCBE) showing large differences in expression between the adjacent genes in the same mRNA transcript compared with the KS2001 wild type strain. Model substrates spanning an adjacent pair of genes for each polycistronic mRNA were tested for RNase P cleavage in vitro. Five model RNAs (cysNC, flgMN, lepAB, phoPQ, and puuBE) were cleaved by RNase P holoenzyme but not by M1 RNA alone. However, the cleavages occurred at non-ptRNA-like cleavage sites, with much less efficiency than the cleavage of ptRNA. Since cleavage products generated by RNase P from a polycistronic mRNA can have different in vivo stabilities, our results suggest that RNase P cleavage may lead to differential expression of each cistron.

• Journal of Life Science
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• v.15 no.6 s.73
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• pp.909-915
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• 2005

We investigated microRNA (miRNA) biogenesis of Epstein-Barr virus (EBV) which is the first virus shown to produce viral miRNAs. As expected, expression of all the reported EBV miRNAs were detected by Northen blot in an EBV-infected B cell line, B95-8; BHRF1-1, BHIU1-2, BHRF1-3, BART1, and BART2. The putative EBV pri-miRWAs and pre-miRNAs predicted from the known mature EBV miRNA sequences were detected by RT-PCR in B95-8 cells. Many animal miRNA genes exist as clusters of 2-7 genes and they are expressed polycistronically. As the EBV miRNAs are clustered in two regions of the EBV genome, we examined whether these clustered EBV miRNA genes are also expressed polycistronically. A long polycistronic transcript with the expected size (1602 bp) corresponding to the BHRF1-1~BHRF1-2~BHRF1-3 was amplified. However, any polycistronic transcript containing both BART1 and BART2 was detectable in B95-8. These results suggest that EBV miRNAs may be processed in a similar way with animal miRNAs and that some of the clustered EBV miRNAs can be transcribed polycistronically.

• Microbiology and Biotechnology Letters
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• v.30 no.1
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• pp.15-20
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• 2002

Diacylglycerol kinase (DGK) phosphorylates the second messenger diacylglycerol (DAG) to phosphatidic acid and it may play a role in signal transduction in Escherichia coli as well as in eukaryotic cells. In addition, DGK is important for microorganisms to adapt to several physiological stimuli. In Bacillus subtilis, the effect of stress on dgk transcription was examined by northern hybridization. The high level of dgk transcription was induced against high osmolarity, low pH value and low temperature. Transcriptional analysis revealed that the dgk gene and dgk upstream locus (ORF2, ORF3 and ORF4) were transcribed as a polycistronic mRNA to form an approximately 2.5 kb transcript.

• Journal of Plant Biotechnology
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• v.33 no.3
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• pp.185-194
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• 2006

Chloroplasts are believed to be descended from certain cyanobacteria, which were taken up by phagocytosis into a host cell and lived there in a symbiotic relationship. In contrast to the current static concept on the chloroplast genome, its dynamism has been recently demonstrated: the chloroplast genome is active in intramolecular homolgous recombination, producing subgenomic circles when it obtains homolgous sequences via genetic transformation. Chloroplast tranformation in higher plants provides many advantages over nuclear transformation that include higher expression levels of transgenes, polycistronic expression of transgenes, and maternal transmission of transgenes. Tobacco has been used as a model for chloroplast genetic transformation. However, it is recently possible to transform the chloroplasts of other major food and economic crops including rice, soybean, and cotton. Chloroplast-transformed crops will be able to replace bioreactors using microorganisms for production of value-added proteins in future.

• Molecules and Cells
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• v.40 no.4
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• pp.262-270
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• 2017

MicroRNAs (miRNAs) are single-stranded, small RNAs (21-23 nucleotides) that function in gene silencing and translational inhibition via the RNA interference mechanism. Most miRNAs originate from host genomic regions, such as intergenic regions, introns, exons, and transposable elements (TEs). Here, we focused on the palindromic structure of medium reiteration frequencies (MERs), which are similar to precursor miRNAs. Five MER consensus sequences (MER5A1, MER53, MER81, MER91C, and MER117) were matched with paralogous transcripts predicted to be precursor miRNAs in the horse genome (equCab2) and located in either intergenic regions or introns. The MER5A1, MER53, and MER91C sequences obtained from RepeatMasker were matched with the eca-miR-544b, eca-miR-1302, and eca-miR-652 precursor sequences derived from Ensembl transcript database, respectively. Each precursor form was anticipated to yield two mature forms, and we confirmed miRNA expression in six different tissues (cerebrum, cerebellum, lung, spleen, adrenal gland, and duodenum) of one thoroughbred horse. MER5A1-derived miRNAs generally showed significantly higher expression in the lung than in other tissues. MER91C-derived miRNA-5p also showed significantly higher expression in the duodenum than in other tissues (cerebellum, lung, spleen, and adrenal gland). The MER117-overlapped expressed sequence tag generated polycistronic miRNAs, which showed higher expression in the duodenum than other tissues. These data indicate that horse MER transposons encode miRNAs that are expressed in several tissues and are thought to have biological functions.

• Journal of Microbiology and Biotechnology
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• v.28 no.12
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• pp.2079-2094
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• 2018

Sunflower trypsin inhibitor (SFTI) is a 14-amino-acid bicyclic peptide that contains a single internal disulfide bond. We initially constructed chimeras of SFTI with N-terminal secretion signals from the Escherichia coli OmpA and Pseudomonas aeruginosa ToxA, but only detected small amounts of protease inhibition resulting from these constructs. A substantially higher degree of protease inhibition was detected from a C-terminal SFTI fusion with E. coli YebF, which radiated more than a centimeter from an individual colony of E. coli using a culture-based inhibitor assay. Inhibitory activity was further improved in YebF-SFTI fusions by the addition of a trypsin cleavage signal immediately upstream of SFTI, and resulted in production of a 14-amino-acid, disulfide-bonded SFTI free in the culture supernatant. To assess the potential of the secreted SFTI to protect the ability of a cytotoxic protein to kill tumor cells, we utilized a tumor-selective form of the Pseudomonas ToxA (OTG-PE38K) alone and expressed as a polycistronic construct with YebF-SFTI in the tumor-targeted Salmonella VNP20009. When we assessed the ability of toxin-containing culture supernatants to kill MDA-MB-468 breast cancer cells, the untreated OTG-PE38K was able to eliminate all detectable tumor cells, while pretreatment with trypsin resulted in the complete loss of anticancer cytotoxicity. However, when OTG-PE38K was co-expressed with YebF-SFTI, cytotoxicity was completely retained in the presence of trypsin. These data demonstrate SFTI chimeras are secreted in a functional form and that co-expression of protease inhibitors with therapeutic proteins by tumor-targeted bacteria has the potential to enhance the activity of therapeutic proteins by suppressing their degradation within a proteolytic environment.