• Genomics & Informatics
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• v.6 no.2
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• pp.84-86
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• 2008

The Tetrahymena group I intron has been shown to employ a trans-splicing reaction and has been modified to specifically target and replace human telomerase reverse transcriptase (hTERT) RNA with a suicide gene transcript, resulting in the induction of selective cytotoxicity in cancer cells that express the target RNA, in animal models as well as in cell cultures. In this study, we evaluated the target RNA specificity of trans-splicing phenomena by the group I intron in mice that were intraperitoneally inoculated with hTERT-expressing human cancer cells to validate the anti-cancer therapeutic applicability of the group I intron. To this end, an adenoviral vector that encoded for the hTERT-targeting group I intron was constructed and systemically injected into the animal. 5'-end RACE-PCR and sequencing analyses of the trans-spliced cDNA clones revealed that all of the analyzed products in the tumor tissue of the virus-infected mice resulted from reactions that were generated only with the targeted hTERT RNA. This study implies the in vivo target specificity of the trans-splicing group I intron and hence suggests that RNA replacement via a trans-splicing reaction by the group I intron is a potent anti-cancer genetic approach.

• ALGAE
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• v.18 no.3
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• pp.183-190
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• 2003

Except for a group I intron in trnL-uaa occuring in eubacteria and plastids, group I introns are rarely documented in plastid genomes. Here, we report that a green alga, Caulerpa sertularioides, contains three group IA3 introns in the 16S gene (cpSSU), CS-cpSSU.i1, CS-cpSSU.i2 and CS-cpSSU.i3. Each intron has an open reading frame with LAGLIDADG motifs. CS-cpSSU.i1orf and CS-cpSSU.i3orf occur at Loop 6 in the intron secondary structure and CScpSSU. i2orf at Loop 8. CS-cpSSU.i1orf and CS-cpSSU.i2orf contain both LAGLI-DADG motifs but CS-cpSSU.i3orf has only one. CS-cpSSU.i1 and CS-cpSSU.i2 share the insetion sites and the ORFs at Loop 6 and 8 with CpSSU·1 and CpSSU·2 introns of Chlamydomonas pallidostigmatica (Chlorophyceae). In contrast, CS-cpSSU.i3, containing 28 copies of GAAATAT at Loop 6, is a novel intron found only in Caulerpa sertularioides. Possible scenarios of the evolution of the three introns and their possible use in systematic research are discussed.

• Korean Journal of Microbiology
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• v.35 no.3
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• pp.211-217
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• 1999

Effects of subsh-ate RNA configuration on the tians-splicing reactcon by group I intron ribozyme of Tetralzynzena thern\ulcornerophila were analyzed with substrate RNAs which have been generated to have very stable structures with stem-loop. RNAinapping strategy was perfo~med in vivo as well as in virro to search the mosl accessible siles to the ~irms-splicing ribozymes in the substrate RNAs. Sequences present in the loop of the target RNAs have shown to be well recognized by and reacted with group I inlron ribozymes while sequences present in the stein do not. Thesc results were confirmed with the experiments of trans-cleavage and rmnssplicing reactmn with ihe specific ribozyines recognizing those sequences. Moreover, sequence analysis of the trans-splicing products have shown that irons-splicing reaction can proceed with high fidelity. In conclusion, the secondary structure of substrate RNAs is one of the most important factors to detemine the ribozyme activity.

• Journal of Microbiology and Biotechnology
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• v.18 no.1
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• pp.23-27
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• 2008

Self-splicing group I introns in tRNA anticodon loops have been found in diverse groups of bacteria. In this work, we identified $tRNA^{fMet}$ group I introns in six strains of marine Synechococcus elongatus. Introns with sizes around 280 bp were consistently obtained in all the strains tested. In a phylogenetic analysis using the nucleotide sequence determined in this study with other cyanobacterial $tRNA^{fMet}$ and $tRNA^{Leu}$ intron sequences, the Synechococcus sequence was grouped together with the sequences from other unicellular cyanobacterial strains. Interestingly, the phylogenetic tree inferred from the intronic sequences clearly separates the different tRNA introns, suggesting that each family has its own evolutionary history.

• Korean Journal of Microbiology
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• v.35 no.4
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• pp.253-257
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• 1999

Effects of polyamines such as cadaverine, putrescine, spermidine and spermine on the self-splicig inhibition of the T4 phage thymidylate synthase(td) intron by spectinomycin have been investrigated. Without polyamine 7mM spectinomycin caused 40% reduction of the splicing rate. Cadaverine reduced the splicing rate over the concentrations of 0.1 to 5 mM. Putrescine at 0.5 mM increased the splicing rate by 13%. Spermidine at 0.5 mM enhanced the splicing rate by 11% while spermine at 0.01 mM enhanced the splicing rate by 16%. Of the all polyamines tested, spermine exhibited the maximum activation effect to counteract the splicing inhibition by spectinomycin. This effect appears to be due to the role of polyamine in stabilizing the conformation of td intron ribozyme essential for the catalytic function.

• Genomics & Informatics
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• v.3 no.4
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• pp.172-174
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• 2005

Recent anti-cancer approaches have been based to target tumor-specifically associated and/or causative molecules such as RNAs or proteins. As this specifically targeted anti-cancer modulator, we have previously described a novel human cancer gene therapeutic agent that is Tetrahymena group I intron-based trans-splicing ribozyme which can reprogram and replace human telomerase reverse transcriptase (hTERT) RNA to selectively induce tumor-specific cytotoxicity in cancer cells expressing the target RNA. Moreover, the specific ribozyme has been shown to efficiently retard tumor tissues in xenograft mice which had been inoculated with hTERT-expressing human cancer cells. In this study, we assessed specificity of trans-splicing reaction in cells to evaluate the therapeutic feasibility of the specific ribozyme. In order to analyze the trans-spliced products by the specific ribozyme in hTERT-positive cells, RT, 5'-end RACE-PCR, and sequencing reactions of the spliced RNAs were employed. Then, whole analyzed products resulted from reactions only with the hTERT RNA. This study suggested that the developed ribozyme perform highly specific RNA replacement of the target RNA in cells, hence trans-splicing ribozyme will be one of specific agents for genetic approach to revert cancer.

• Journal of Microbiology
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• v.41 no.4
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• pp.340-344
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• 2003

The group I intron from Tetrahymena thermophila has been demonstrated to employ splicing reactions with its substrate RNA in the trans configuration. Moreover, we have recently shown that the transsplicing group I ribozyme can replace HCV-specific transcripts with a new RNA that exerts anti-viral activity. In this study, we explored the potential use of RNA replacement for cancer treatment by developing trans-splicing group I ribozymes, which could replace tumor-associated RNAs with the RNA sequence attached to the 3' end of the ribozymes. Thymidine phosphorylase (TP) RNA was chosen as a target RNA because it is known as a valid cancer prognostic factor. By performing an RNA mapping strategy that is based on a trans-splicing ribozyme library, we first determined which regions of the TP RNA are accessible to ribozymes, and found that the leader sequences upstream of the AUG start codon appeared to be particularly accessible. Next, we assessed the ribozyme activities by comparing trans-splicing activities of several ribozymes that targeted different regions of the TP RNA. This assessment was performed to verify if the target site predicted to be accessible is truly the most accessible. The ribozyme that could target the most accessible site, identified by mapping studies, was the most active with high fidelity in vitro. Moreover, the specific trans-splicing ribozyme reacted with and altered the TP transcripts by transferring an intended 3' exon tag sequence onto the targeted TP RNA in mammalian cells with high fidelity. These results suggest that the Tetrahymena ribozyme can be utilized to replace TP RNAs in tumors with a new RNA harboring anti-cancer activity, which would revert the malignant phenotype.

• Journal of Microbiology and Biotechnology
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• v.15 no.6
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• pp.1408-1413
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• 2005

Elevated expression of carcinoembryonic antigen (CEA) has been implicated in various biological aspects of neoplasia such as tumor cell adhesion, metastasis, blocking of cellular immune mechanisms, and antiapoptosis function. Thus, the CEA could be an important target for anticancer therapy. In this study, we developed Tetrahymena group 1 intron-based trans-splicing ribozymes that can specifically target and replace CEA RNA. To this end, we first determined which regions of the CEA RNA were accessible to ribozymes by employing an RNA mapping strategy that was based on a trans-splicing ribozyme library. Next, we assessed the ribozyme activities by comparing the trans-splicing activities of several ribozymes that targeted different regions of the CEA RNA, and then the ribozyme that could target the most accessible site was observed to be the most active with high fidelity in vitro. Moreover, the specific trans-splicing ribozyme was found to react with and altered the target CEA transcripts in mammalian cells with high fidelity. These results suggest that the Tetrahymena ribozyme can be utilized to replace CEA RNAs in tumors with a new RNA-harboring anticancer activity, thereby hopefully reverting the malignant phenotype.

• Korean Journal of Microbiology
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• v.38 no.1
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• pp.13-18
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• 2002

Effects of divalent cations such as $Mg^{2+}$, $Mn^{2+}$ and $Zn^{2+}$ on the self-splicing inhibition of the T4 phage thymidylate synthase (td) intron by the coenzyme thiamine pyrophosphate have been investigated. The splicing activity increased in proportion to the concentration of $Mg^{2+}$ up to 30 mM. Without $Mg^{2+}$in the splicing reaction the $Zn^{2+}$ ion tested in the range of 0.1-6 mM concentration only produced the splicing activity about 20% that of the normal splicing rate. A majority of the splicing products were I-E2 and E2 but El-E2 ligation product, Cl and Ll were not detected. Similar patterns of splicing products were also observed with $Mn^{2+}$. At 6 mM $Zn^{2+}$the intron RNA was hydrolyzed. $Mn^{2+}$produced a little higher splicing activity than that of $Zn^{2+}$over the range of concentrations used and at 8 mM about 28% splicing activity was observed. In contrast, $Mn^{2+}\;and\;Zn^{2+}$ ions promoted the splicing activity about 35-40% on an average in the presence of 10 mM $Mg^{2+}$. Of all divalent cations tested, $Mg^{2+}$exhibited the maximum activation effect to counteract the splicing inhibition by thiamine pyrophosphate. This appears to be due to the stabilizing effect of td intron ribozyme structure essential for the catalytic function by $Mg^{2+}$.

• Journal of Microbiology and Biotechnology
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• v.19 no.9
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• pp.1070-1076
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• 2009

Telomerase reverse transcriptase (TERT), which prolongs the replicative life span of cells, is highly upregulated in 85-90% of human cancers, whereas most normal somatic tissues in humans express limited levels of the telomerase activity. Therefore, TERT has been a potential target for anticancer therapy. Recently, we described a new approach to human cancer gene therapy, which is based on the group I intron of Tetrahymena thermophila. This ribozyme can specifically mediate RNA replacement of human TERT (hTERT) transcript with a new transcript harboring anticancer activity through a trans-splicing reaction, resulting in selective regression of hTERT-positive cancer cells. However, to validate the therapeutic potential of the ribozyme in animal models, ribozymes targeting inherent transcripts of the animal should be developed. In this study, we developed a Tetrahymena-based trans-splicing ribozyme that can specifically target and replace the mouse TERT (mTERT) RNA. This ribozyme can trigger transgene activity not only also in mTERT-expressing cells but hTERT-positive cancer cells. Importantly, the ribozyme could selectively induce activity of the suicide gene, a herpes simplex virus thymidine kinase gene, in cancer cells expressing the TERT RNA and thereby specifically hamper the survival of these cells when treated with ganciclovir. The mTERT-targeting ribozyme will be useful for evaluation of the RNA replacement approach as a cancer gene therapeutic tool in the mouse model with syngeneic tumors.