• Korean Journal of Plant Tissue Culture
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• v.25 no.2
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• pp.131-134
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• 1998

$\textrm{T}_{5}$ progeny of one transgenic tomato line (To9) carrying antisense polygalacturonase (PG) cDNA was generated by selfing. Five $\textrm{T}_{5}$ plants were used to analyse in detail. The PG antisense gene was stably inherited through fifth generations. In all five $\textrm{T}_{5}$ plants, expression of the antisense transcripts were detected. In consequence, it led to a reduction of the PG enzyme activity in ripe fruit to between 37% and 65% that of normal. In two plants the expression of endogenous PG gene was inhibited in ripe fruit.

• Plant Biotechnology Reports
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• v.4 no.3
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• pp.185-192
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• 2010

The Arabidopsis ${\omega}$-3 fatty acid desaturase (AtFAD7) catalyzes the synthesis of trienoic fatty acids (TA). A transgenic tobacco line, T15, was produced by a sense AtFAD7 construct and showed a cosuppression-like phenotype, namely extremely low TA levels. The sequence similarity between AtFAD7 and a tobacco ortholog gene, NtFAD7, was moderate (about 69%) in the coding sequences. AtFAD7 siRNAs accumulated at a high level, and both AtFAD7 and NtFAD7 mRNAs are degraded in T15 plants. The low-TA phenotype in T15 was dependent on a tobacco RNA-dependent RNA polymerase6 (NtRDR6). We also produced tobacco RNAi plants targeting AtFAD7 gene sequences. The AtFAD7 siRNA level was trace, which was associated with a slight reduction in leaf TA level. Unexpectedly, this RNAi plant showed an increased NtFAD7 transcript level. To investigate the effect of translational inhibition on stability of the NtFAD7 mRNAs, leaves of the wild-type tobacco plants were treated with a translational inhibitor, cycloheximide. The level of NtFAD7 mRNAs significantly increased after cycloheximde treatment. These results suggest that the translational inhibition by low levels of AtFAD7 siRNAs or by cycloheximide increased stability of NtFAD7 mRNA. The degree of silencing by an RNAi construct targeting the AtFAD7 gene was increased by co-existence of the AtFAD7 transgene, where NtRDR6-dependent amplification of siRNAs occurred. These results indicate that NtRDR6 can emphasize silencing effects in both cosuppression and RNAi.

• Environmental Engineering Research
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• v.19 no.4
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• pp.317-329
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• 2014

The diversity of cyanobacteria in Sri Lanka was studied in different water reservoirs, paddy fields, brackish water and tsunami affected areas using light microcopy, 16S rRNA sequences, followed by phylogenetic analysis. Based on light microscopy, 24 genera were identified from environmental samples belonging to the orders Chroococcales, Oscillatoriales, Pleurocapsales and Nostocales. In cultures, 33 genera were identified from all five cyanobacterial orders, including Stigonematales. Based on 16S rRNA gene sequences and their morphology, two isolates were identified up to species level, 72 to genus level, one isolate up to family and 11 up to order level. Twelve isolates couldn't be assigned to any taxonomic level. The results of 16S rRNA gene sequences along with the phylogenetic analysis indicated that some cyanobacterial isolates could be accommodated to genus or order level. The 16S rRNA sequence analysis data in this study confirmed that order Nostocales and order Pleurocapsales cyanobacteria are monophyletic while orders Chroococcales, Oscillatoriales and Stigonematales cyanobacteria are polyphyletic. Polyphasic approach including the combination of light microscopy, cultures and the analysis of 16S rRNA gene sequences provide a promising approach to ascertain the diversity of cyanobacteria in different habitats.

• Microbiology and Biotechnology Letters
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• v.21 no.5
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• pp.473-477
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• 1993

Both glycerol-phosphate acyltransferase (GPAT) and 7.2 kb mRNAs were present at the highest level in liver. Glycerol-phosphate acyltransferase and 7.2 kb mRNA levels increased dramatically when fasted mice were refed a high carbohydrate diet. In mature 3T3-L1 adipocytes, insulin increased both glycerol-phosphate acyltransferase and 7.2kb mRNA levels 2.6 to 3-fold while dibutyryl cAMP decreased mRNA levels by 50% and 80%, respectively. These results indicate positive regulation by insulin and negative regulation by dibutyryl cAMP of both glycerol-phosphate acyltransferase and 7.2 kb mRNA.

• Journal of Life Science
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• v.8 no.2
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• pp.203-207
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• 1998

NAD4 as a gene encoding NADH dehydrogenase subunit 4 in the micodhondrion from maize has been cloned using RT-PCR and sequenced for examining RNA edited sites. Analysis of mt cDNA sequences showed the typical RNA editing patterns and unusual base changes as well;RNA editing from cDNA sequences occured base change from c to U in most cases, however transitions from t to g and G to A were also observed. Even though those editings appared to be occurred randomly, RNA edited sites showed mostly in exon 1 and exon 4 regions, when compared with NAD4 cDNA from wheat, locations of edited sites did not consistent with each other suggesting that the phenomenon of RNA editing occured randomly not site-specific manner.

• Journal of Microbiology
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• v.41 no.4
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• pp.300-305
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• 2003

Intracellular expression of RNA decoys, such as TAR or RRE decoy, has been previously shown to protect immune cells from human immunodeficiency virus type 1 (HIV-1) replication by inhibiting the binding of the HIV-1 regulatory protein to the authentic HIV RNA sequence. However, HIV-1 challenge experiments of primary human T cells, which express the RNA decoy, demonstrated that the cells were only transiently protected, and hence, more improved protocols for HIV-1 inhibition with the RNA decoys need to be developed. In this report, in order to develop a more effective RNA decoy, we analyzed and compared the ability of a series of RNA decoy derivatives in inhibiting HIV-1 replication in CEM cells. Using an improved tRNA cassette to express high levels of RNA decoy transcripts in cells, we found that co-expression of both TAR and RRE decoys, in the form of an aligned sequence in a single transcription cassette, much more potently blocked cells from HIV-1 than the expression of only one kind of RNA decoy. This observation will have an important implication for experiments involving optimization of clinical applications in RNA decoy-based gene therapy against HIV-1.

• Biomedical Science Letters
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• v.13 no.2
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• pp.105-110
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• 2007

Aspartyl-tRNA synthetase (AspRS) exists in two different forms with respect to tRNA recognition. The discriminating enzyme (D-AspRS) recognizes only tRNA$^{Asp}$, while the non-discriminating one (ND-AspRS) also recognizes tRNA$^{Asn}$ and therefore forms both Asp-tRNA$^{Asn}$ and Asp-tRNA$^{Asp}$. Plus primary sequence distinguishes two general groups of AspRS. There is a predominantly bacterial-type, larger AspRS (about 580 aa) in addition to a shorter archaeal/eukaryotic type (about 430 aa). In vivo data made clear that discriminating and non-discriminating enzymes exist in both groups. The determinants in the protein sequence responsible for tRNA discrimination are not hewn. The AspRS from Acidithiobacillus ferrooxidans might be suggested ND-AspRS fur missing of AsnRS in genomic sequencing data. Therefore, we analyzed the AspRS from A. ferrooxidans with in vitro aminoacylation assay with E. coli unfractionated tRNA, in vivo missense suppression assay with tipA34 mutant and Northern hybridization with probes which were specific with tRNA$^{Asp}$ or tRNA$^{Asn}$. The AspRS from A. ferrooxidans produced more Asp-tRNA than that from E. coli. Only aspS gene from A. ferrooxidans suppressed trpA34 strain in minimal media without tryptophan. Only AspRS from A. ferrooxidans showed mischarged Asp-tRNA$^{Asn}$ band. Therefore, AspRS from A. ferrooxidans is definitely ND-AspRS.

• Korean Journal of Microbiology
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• v.50 no.1
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• pp.1-7
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• 2014

Site-specific incorporation of unnatural amino acids (SSIUA) into protein can be achieved in vivo by coexpression of an orthogonal pair of suppressor tRNA and engineered aminoacyl-tRNA synthetase (ARS) that specifically ligates an unnatural amino acid to the suppressor tRNA. As a step to develop the SSIUA technique in Escherichia coli, here we established a new 2-step screening system that can be used for selecting an ARS variant(s) that ligates an unnatural amino acid to a suppressor tRNA. A positive selection system consists of chloramphenicol acetyl transferase gene containing an amber mutation at the $27^{th}$ residue, and efficiently concentrated amber suppressible ARS with a maximum enrichment factor of $9.0{\times}10^5$. On the other hand, a negative selection system was constructed by adding multiple amber codons in front of a lethal gene encoding the control of cell death B toxin (ccdB) which acts as an inhibitory protein of bacterial topoisomerase II. Amber suppression of ccdB by an orthogonal pair of Saccharomyces cerevisiae tyrosyl-tRNA synthetase (TyrRS) and an amber suppressor tRNA significantly inhibits bacterial growth. This selection system was also able to efficiently remove amber suppressible ARS which could ligate natural amino acids to the suppressor tRNA. Thus, sequential combination of these two selection systems might be able to function as a powerful tool for selecting an ARS variant that specifically ligates an unnatural amino acid to the suppressor tRNA from an ARS mutant pool.

• The Plant Pathology Journal
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• v.21 no.4
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• pp.334-342
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• 2005

The prototype Chlorella virus PBCV-l encodes 11 tRNA genes and over 350 protein-encoding genes in its 330 kbp genome. Initial attempts to overexpress the recombinant A189/192R protein, a putative virus attachment protein, in E. coli strain BL21(DE3) SI were unsuccessful, and multiple protein bands were detected on Western blots. However, the full-length A189/192R recombinant protein or fragments derived from it were detected when they were expressed in E. coli BL21 CodonPlus (DE3) RIL, which contains extra tRNAs. Codon usage analysis of the a189/192r gene showed highly biased usage of the AGA and AVA codons compared to genes encoded by E. coli and Chlorella. In addition, there were biases of XXA/U($56\%$) and XXG/ C($44\%$) in the codons recognized by the viral tRNAs, which correspond to the codon usage bias in the PBCV-1 genome of XXA/U ($63\%$) over those ending in XXC/G ($37\%$). Analysis of the codon usage in the major capsid protein and DNA polymerase showed preferential usage of codons that can be recognized by the viral tRNAs. The Asn (AAC) and Lys (AAG) codons whose corresponding tRNA genes are duplicated in the tRNA gene cluster were the most abundant (i.e., preferred) codons in these two proteins. The tRNA genes encoded in the PBCV-l genome seem to play a very important role during the synthesis of viral proteins through supplementing the tRNAs that are frequently used in viral proteins, but are rare in the host cells. In addition, these tRNAs would help the virus to adapt to a wide range of hosts by providing tRNAs that are rare in the host cells.

• Korean Journal of Microbiology
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• v.39 no.4
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• pp.235-241
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• 2003

We have previously demonstrated that intracellular expression of an RNA aptamer termed RRE40, which was selected in vitro to bind HIV Rev 10-fold much tighter than wild-type RRE, efficiently protected human CD4+ T cell line, CEM, from HIV-1. In this study, to evaluate the efficacy of the RRE40 RNA in clinical settings, polyclonal CD4+ peripheral blood lymphocytes (PBLs) were transduced with retroviral vectors expressing RRE40 decoy RNA and then challenged with clinical isolates of HIV-1. In contrast to the control cells transduced with vectors expressing control tRNA, intracellular expression of RRE40 RNA more effectively inhibited HIV-1 replication in CD4+ PBLs. However, transient and diminished inhibition, rather than complete inhibition, of HIV-1 replication in PBLs expressing RRE40 decoys have been observed. These results suggest that RRE40 decoy RNA would be useful to inhibit HIV-1 replication in cells. However, development of more efficient gene transfer protocols and/or more effective decoy RNAs would be needed to apply RNA decoy to modulate HIV-1 patient.