• 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.

• Journal of Plant Biotechnology
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• v.40 no.1
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• pp.27-36
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• 2013

We isolated and functionally characterized a Dendrobium Actin1 (DmACT1) promoter that drives strong gene expression in the orchid genus Dendrobium. A genomic fragment containing the region 3227 bp upstream of the coding region of DmACT1 was obtained by inverse PCR. Detailed comparison of the full-length cDNA and genomic sequences revealed that DmACT1 has a 1374 bp first intron in the 5' UTR. However, the 5' flanking sequences upstream of the coding region showed no obvious sequence similarities compared to those of known promoters, including plant actin promoters. Serial deletion constructs of the 5' flanking region from the translation initiation codon were fused to the coding sequence of a GUS/luciferase fusion reporter to identify the regulatory elements necessary for promoter activity. Transient assays in the flowers of Dendrobium revealed that the 5' UTR-intron greatly enhanced promoter activity. Moreover, the DmACT1 promoter with its 5' UTR-intron yielded approximately 10-fold higher reporter activity than the rice Act1 promoter-intron. Our data suggest that the DmACT1 promoter with its 5' UTR-intron is a useful tool for strong expression of transgenes in Dendrobium orchids.

• 대한예방의학회:학술대회논문집
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• 1999.10a
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• pp.1-15
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• 1999

In a case-control study to evaluate the factors involved in the development of hepatocellular carcinoma, polymorphisms of the p53 gene were compared in 68 cases mostly infected with hepatitis C virus (HCV) and 68 controls matched for sex and age: DNA from peripheral blood leukocytes was analyzed by the polymerase chain reaction-single strand conformation polymorphism method and direct sequencing. Polymorphisms analyzed were those in exon 4 (CCC vs. CGC, Pro vs. Arg at codon 72, Al allele vs. A2 allele), intron 2 (C vs. G at nucleotide 38, Al vs. A2), intron 3 (C vs. A at nucleotide 65, Al vs. A2; absence and presence of 16 base pair repeat at nucleotides 24 to 39, Al vs. A2), intron 6 (A vs. G at nucleotide 62, Al vs. A2) and intron 7 (C and T vs. T and G at nucleotides 72 and 92, Al vs. A2). A significantly higher frequency of the allele for CCC (Pro, Al) at codon 72 of exon 4 was found in cases (39%) than in controls (26%) (p<0.05). Highly significant linkage of the polymorphisms in exon 4, intron 2, intron 3 and intron 7, and between the intron 3-16 bp duplication and polymorphism in intron 6 also was found. Matched Fair analysis showed significantly higher frequencies of certain haplotypes (1-1-1-1-2-2 or 1-1-2-1-2-1 for exon 4, intron 2, intron 3, the intron 3-16 bp duplication, intron 6 and intron 7) in cases than in controls (p=0.014, OR=2.27, 95% CI= 1.08-5.12). No preference of specific p53 polymorphisms for specific HCV genotype was detected. These findings suggest that in hepatocarcinogenesis mainly due to HCV infection, genetic factors may be involved and that genetic markers can serve as predictors of a high-risk group for hepatocarcinogenesis.

• Korean Journal of Plant Resources
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• v.23 no.6
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• pp.526-534
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• 2010

Genomes of clusters of related eukaryotes are now being sequenced at an increasing rate. In this paper, we developed an accurate, low-cost method for annotation of gene prediction and exon-intron structure. The gene prediction was adapted for delta 1-pyrroline-5-carboxylate-synthetase (p5cs) gene from China wild-type of the halophytic Leymus chinensis (Trin.), naturally adapted to highly-alkali soils. Due to complex adaptive mechanisms in halophytes, more attentions are being paid on the regulatory elements of stress adaptation in halophytes. P5CS encodes delta 1-pyrroline-5-carboxylate-synthetase, a key regulatory enzyme involved in the biosynthesis of proline, that has direct correlation with proline accumulation in vivo and positive relationship with stress tolerance. Using analysis of reverse transcription-polymerase chain reaction (RT-PCR) and PCR, and direct sequencing, 1076 base pairs (bp) of cDNA in length and 2396 bp of genomic DNA in length were obtained from direct sequencing results. Through gene prediction and exon-intron structure verification, the full-length of cDNA sequence was divided into eight parts, with seven parts of intron insertion. The average lengths of determinated coding regions and non-coding regions were 154.17 bp and 188.57 bp, respectively. Nearly all splice sites displayed GT as the donor sites at the 5' end of intron region, and 71.43% displayed AG as the acceptor sites at the 3' end of intron region. We conclude that this method is a cost-effective way for obtaining an experimentally verified genome annotation.

• Animal cells and systems
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• v.1 no.1
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• pp.87-91
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• 1997

Effects of divalent cations such as $Mg^{2+}$, $Mn^{2+}$, $Ca^{2+}$, and $Zn^2$ on splicing activity of phage T4 thymidylate synthase intron RNA have been investigated. At the concentration of 0.5 mM, $Mn^{2+}$ in the absence of $Mg^{2+}$, a very small amount of pre-RNA was cleaved into ligation products (El-E2) but no circular or linear intron was produced. As the concentration of $Mn^{2+}$ was increased from 1 to 5 mM the pre-RNA was completely hydrolyzed. In the presence of 5 mM $Mg^{2+}$, both the linear intron and circular intron were produced but no El-E2 ligation product was produced. At both 3 and 5 mM $Mn^{2+}$ the RNA was hydrolyzed completely as observed with no $Mg^2+$ being present. In the case of $Zn^{2+}$, even at 0.5 mM concentration, the pre-RNA was completely hydrolyzed. This observation suggested that $Zn^{2+}$ facilitates RNA hydrolysis more rapidly than $Mn^{2+}$ does. at 5mM $Ca^{2+}$, the RNA was not hydrolyzed and remained intact as a primary transcript.

• The Plant Pathology Journal
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• v.29 no.3
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• pp.234-241
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• 2013

Polygalacturonase (PG) gene is a typical gene family present in eukaryotes. Forty-nine PGs were mined from the genomes of Neurospora crassa and five Aspergillus species. The PGs were classified into 3 clades such as clade 1 for rhamno-PGs, clade 2 for exo-PGs and clade 3 for exo- and endo-PGs, which were further grouped into 13 sub-clades based on the polypeptide sequence similarity. In gene structure analysis, a total of 124 introns were present in 44 genes and five genes lacked introns to give an average of 2.5 introns per gene. Intron phase distribution was 64.5% for phase 0, 21.8% for phase 1, and 13.7% for phase 2, respectively. The introns varied in their sequences and their lengths ranged from 20 bp to 424 bp with an average of 65.9 bp, which is approximately half the size of introns in other fungal genes. There were 29 homologous intron blocks and 26 of those were sub-clade specific. Intron losses were counted in 18 introns in which no obvious phase preference for intron loss was observed. Eighteen introns were placed at novel positions, which is considerably higher than those of plant PGs. In an evolutionary sense both intron loss and gain must have taken place for shaping the current PGs in these fungi. Together with the small intron size, low conservation of homologous intron blocks and higher number of novel introns, PGs of fungal species seem to have recently undergone highly dynamic evolution.

• 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.

• Journal of Microbiology
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• v.33 no.2
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• pp.160-164
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• 1995

The splicing activity of T4 phage td intron RNA has been examined with various Mg$^{2+}$ ions such as MGCl$_{2}$, MgS $O_{4}$ and magnesium acetate using various splicing conditions such as different incubation time and temperature. The maximum splicing of td intron RNA occurred at the concentration of 5 mM MgCl$_{2}$. Raising the Mg$^{2+}$ concentration up to 15 mM appeared to promote P2 delection mutant to overcome the loss of some splicing activity. In both wild type and mutant, a complete hydrolysis of RNA occurred at 30 mM MgCI$_{2}$ MgS $O_{4}$ and magnesium acetate exhibited the rate and pattern of RNA splicing identical to MGCI$_{2}$. The optimal splicing conditions involve the incubation of RNA with 5 mM MgCI$_{2}$ at 58 .deg.C for 15 min. The results suggest that Mg$^{2+}$ may play a key role in the catalytic mechanism of td intron RNA.n RNA.

• Genomics & Informatics
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• v.17 no.1
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• pp.9.1-9.5
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• 2019

In previous studies, we demonstrated that some sites in the first intron likely regulate gene expression. In the present work, we sought to further confirm the functional relevance of first intron sites by estimating the quantity of rare alleles in the first intron. A basic hypothesis posited herein is that genomic regions carrying more functionally important sites will have a higher proportion of rare alleles. We estimated the proportions of rare single nucleotide polymorphisms with a minor allele frequency < 0.01 located in several histone marks in the first introns of various genes, and compared them with those in other introns and those in 2-kb upstream regions. As expected, rare alleles were found to be significantly enriched in most of the regulatory sites located in the first introns. Meanwhile, transcription factor binding sites were significantly more enriched in the 2-kb upstream regions (i.e., the regions of putative promoters of genes) than in the first introns. These results strongly support our proposal that the first intron sites of genes may have important regulatory functions in gene expression independent of promoters.

• Korean Journal of Plant Tissue Culture
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• v.26 no.1
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• pp.41-47
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• 1999

Phenylalanine ammonia-lyase (PAL; EC 4, 3, 1, 5) genomic clones were isolated from tomato(Lycopersicon esculentum L.) genomic DNA libraries using tomato PAL5 cDNA sequences as probes. The nucleotide sequences of tPAL1, tPAL4 and tPAL5 were compared. tPAL5 contains an open reading frame encoding a polypeptide of 722 amino acids, interrupted by a 710 bp intron in the codon for the amino acid 139. tPAL1 encodes a polypeptide of 249 amino acids which is much shorter than tPAL5 gene due to a premature stop codon and does not contain an intron. tPAL4 encodes a polypeptide of 357 amino acids, interrupted by a 305 bp intron in the codon for the amino acid 138. Premature stop codons observed in tPAL1 and tPAL4 gene produce a short polypeptide rather than a normal polypeptide (722 aa). tPALl shows 87.2% homology with tPAL4 and 85.3% homology with tPAL5 gene whereas tPAL4 showes 91.4% homology with tPAL5 at nucleotide level. In general, phylogenetic analysis showed that genes isolated from tomato, potato, and sweet potato were belong to the same group and another dicot plants such as parsley, bean, soybean, pea and alfalfa formed another group. PAL genes isolated from rice and yeast showed very low homology with other PAL genes and formed the other group.