• Title/Summary/Keyword: Intron

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Replacement of Thymidine Phosphorylase RNA with Group I Intron of Tetrahymena thermophila by Targeted Trans-Splicing

  • Park, Young-Hee;Jung, Heung-Su;Kwon, Byung-Su;Lee, Seong-Wook
    • 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.

RRM but not the Asp/Glu domain of hnRNP C1/C2 is required for splicing regulation of Ron exon 11 pre-mRNA

  • Moon, Heegyum;Jang, Ha Na;Liu, Yongchao;Choi, Namjeong;Oh, Jagyeong;Ha, Jiyeon;Kim, Hyeon Ho;Zheng, Xuexiu;Shen, Haihong
    • BMB Reports
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    • v.52 no.11
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    • pp.641-646
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    • 2019
  • The Ron proto-oncogene is a human receptor for macrophage-stimulating protein (MSP). The exclusion of exon 11 in alternative splicing generates ${\Delta}RON$ protein that is constitutively activated. Heterogenous ribonucleaoprotein (hnRNP) $C_1/C_2$ is one of the most abundant proteins in cells. In this manuscript, we showed that both hnRNP $C_1$ and $C_2$ promoted exon 11 inclusion of Ron pre-mRNA and that hnRNP $C_1$ and hnRNP $C_2$ functioned independently but not cooperatively. Moreover, hnRNP $C_1$ stimulated exon 11 splicing through intron 10 activation but not through intron 11 splicing. Furthermore, we showed that, whereas the RRM domain was required for hnRNP $C_1$ function, the Asp/Glu domain was not. In conclusion, hnRNP $C_1/C_2$ promoted exon 11 splicing independently by stimulating intron 10 splicing through RRM but not through the Asp/Glu domain.

Gene encoding prolactin of red-spotted grouper, Epinephelus akaara, and its application as a molecular marker for grouper species identification

  • Bok-Ki Choi;Gyeong-Eon Noh;Yeo-Reum Kim;Jun-Hwan Byun;HanKyu Lim;Jong-Myoung Kim
    • Fisheries and Aquatic Sciences
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    • v.27 no.6
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    • pp.346-355
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    • 2024
  • Groupers are economically important species in the fishery and aquaculture industries in Asian countries. Various species of grouper, including hybrids, have been brought to market even without precise species identification. In this study, we analyzed the structure and expression profile of the gene encoding prolactin (PRL) in the red-spotted grouper Epinephelus akaara based on genomic DNA and cDNA templates. The results showed that the PRL gene consists of five exons encoding an open reading frame of 212 amino acids, including a putative signal peptide of 24 amino acids and a mature structural protein of 188 amino acids. It showed amino acid identities of 99% with Epinephelus coioides, 83% with Amphiprion melanopus, 82% with Acanthopagrus schlegelii, 75% with Oreochromis niloticus, 70% with Coregonus autumnalis, and 67% with Oncorhynchus mykiss, indicating its closer similarity to E. coioides and other groupers but marked distinction from non-teleost PRLs. PRL mRNA expression was detected mostly in the brain, including the pituitary gland, with little expression in other tissues. While the 5-exon structure of the PRL gene of red-spotted grouper and the exon sizes were conserved, the sizes of the introns, particularly the first intron, were markedly different among the grouper species. To examine whether these differences can be used to distinguish groupers of similar phenotypes, exon-primed intron-crossing analysis was carried out for various commercially important grouper species. The results showed clear differences in size of the amplified fragment encompassing the first intron of the PRL gene, indicating that this method could be used to develop species-specific markers capable of discriminating between grouper species and their hybrids at the molecular level.

Lack of the Association between Microsatellite Polymorphism in Toll-like Receptor 2 Gene and Development of COPD (Toll-like Receptor 2 유전자의 Microsatellite 유전자 다형성과 만성폐쇄성폐질환 발생과의 연관성 결여)

  • Lee, Hee Seok;Lee, Hye Won;Kim, Deog Kyeom;Ko, Dong Seok;Park, Gun Min;Hwang, Yong Il;Lee, Sang-Min;Yoo, Chul Gyu;Kim, Young Whan;Han, Sung Koo;Shim, Young-Soo;Yim, Jae-Joon
    • Tuberculosis and Respiratory Diseases
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    • v.58 no.4
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    • pp.367-374
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    • 2005
  • Background : The fact that only 10-20% of chronic cigarette smokers develop chronic obstructive pulmonary disease (COPD) reflects the presence of genetic factors associated with the susceptibility to COPD. Recently, it was reported that the surfactant protein A increases the secretion of matrix metalloprotease 9, which degrades extracellular matrices of the lung, through a Toll-like receptor 2 (TLR2). In this context, possible role of TLR2 in the pathogenesis of COPD was postulated, and a functional dinucleotide repeat polymorphism in intron II of TLR2 was evaluated for any association with COPD. Method : Male patients with COPD and male smokers with a normal pulmonary function were enrolled in this study. The number of Guanine-Thymine repeats in intron II of the TLR2 gene were counted. Because the distributions of the repeats were trimodal, the alleles were classified into three subclasses, 12-16 repeats: short (S) alleles; 17-22 repeats: medium length (M) alleles; and 23-27 repeats: long (L) alleles. Result : 125 male patients with COPD and 144 age- and gender-matched blood donors with a normal lung function were enrolled. There were no differences in the distribution of each allele subclass (S, M and L) between the COPD and control group (p=0.75). The frequencies of the genotypes with and without each allele subclass in the COPD and control group were similar. Conclusion : A microsatellite polymorphism in intron II of TLR2 gene was not associated with the development of COPD in Koreans.

Molecular Cloning of Glycoside Hydrolase Family 74 Genes and Analysis of Transcript Products from the Basidiomycete Phanerochaete chrysosporium (담자균 Phanerochaete chrysosporium으로부터 유래한 Glycoside Hydrolase Family 74 유전자 클로닝과 전사산물 분석)

  • Lee, Jae-Won;Samejima, Masahiro;Choi, In-Gyu
    • Journal of the Korean Wood Science and Technology
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    • v.34 no.3
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    • pp.56-63
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    • 2006
  • In order to evaluate the mechanism of cellulose hydrolysis, the complementary DNA encoding Glycoside Hydrolase Family (GHF)74 was cloned from Phanerochaete chrysosporium. Depending on the presence of Cellulose Binding Module (CBM), it can be classified as GHF74A or GHF74B. The GHF74A gene from P. chrysosporium (PcGHF74A) consists of 2163 bp encoding a protein of 721 amino acid residues. The PcGHF74A showed homology of 70~77% compared with the GHF74 from other filamentous fungi. The PcGHF74B, which contains CBM and is a member of family 1, was transcribed to various transcripts depending on the nature of carbon sources and their concentration. To study the possible presence of splice variants in GHF74B transcripts in P. chrysospoium, we carried out RT-PCR analysis using primers that designed based on the annotation data and sequenced data. Our result indicated that PcGHF74B was transcribed to several splicing variants in various culture conditions. Especially in the culture of 2% cellulose, three transcript products were observed. First transcript was presumed to be a full length ORF that contained 11th intron with stop codon at position 2562 bp. The second one consisted of 12 exons and 11 introns with stop codon at position 1187 bp with 7th exon. The shortest transcript consisted of 10 exons and 9 introns with stop codon at 910 bp in the 7th exon. These premature stop codon might prevent the synthesis of fully active GHF74 or contribute for the production of protein with distinct function depending on the ambient carbon sources.

Genomic Organization and Characterization of the Promoter Region of Bovine ADRP (Adipocyte Different Related Protein) Gene (소 Adipocyte Differentiation Related Protein (ADRP) 유전자의 Genomic Organization 및 Promoter Region의 특성 규명)

  • Jang, Y. S.;Yoon, D. H.;Kim, T. H.;Cheong, I. C.;Jo, J. K.
    • Journal of Animal Science and Technology
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    • v.45 no.2
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    • pp.169-182
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    • 2003
  • To understand the structure and regulation of bovine ADRP (Adipocyte Differentiation Related Protein) gene, we have isolated the genomic clone of bovine ADRP and determined its sequence. A genomic Southern blot analysis confirmed that ADRP gene is present as a single copy in bovine genome and the ADRP gene spans 12 kb. Bovine ADRP genomic clone, HwADRPg-1, had 8 exons and 7 introns, and all splicing sites conformed to the GT/AG rule with the exon-intron boundaries located exactly. Analysis of the upstream 649 bp of the sequence of HwADRPg-1 showed that it does not contain any canonical TATAA boxes; however Sp1 binding sites and CAAT boxes are found. The promoter contained potential binding sites for AP-1, AP-2 and several putative transcription factor binding sites. The 5'-flanking region of HwADRPg-1 contained muscle specific transcription activator Myo G and C/EBP (CCAAT/ enhancer binding protein) recognizing site. These results suppose that the Myo G transcription activator regulate the transcription of bovine ADRP gene in muscular tissue and its transcriptional activity was triggered by degree of muscular development. Our results provide the necessary analysis for other flanking sequences are needed in addition to the proximal cis elements of this promoter to confer adipocyte differentiation-dependent or growth-dependent transcriptional control.

Systematic Study on the Aconitum alboviolaceum Complex (Ranunculaceae) in Korea (한국산 줄바꽃 종집단의 분류학적 연구)

  • Lee, Soo-Rang;Park, Chong-Wook
    • Korean Journal of Plant Taxonomy
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    • v.37 no.4
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    • pp.477-502
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    • 2007
  • The Aconitum alboviolaceum Kom. complex includes four controversial species described from Korea; A. albouiolaceum, A. pseudolaeue, A. longecassidatum, and A. quelpaertense. The main objective of this study was to determine the taxonomic identities and systematic relationships among the species in the A. albouiolaceum complex based on morphology, numerical analyses and DNA sequence analysis. In the present study, variations in the principal morphological characters and chloroplast DNA noncoding region sequences (psbA-trnH IGS, trnL intron, and trnL-trnF IGS) were examined for 95 individuals from 20 populations. Also, neighbor-joining analysis was adopted to infer their relationships. Morphological variation appeared to be considerably high but not to be related to geographic distribution. These morphological results suggest that reevaluation of key morphological characters are needed for the proper taxonomic treatment of the complex. The length of psbA-trnH IGS region ranged from 241 to 250 bp, that of the trnL intron from 526 to 532 bp, and that of the trnL-trnF IGS region from 466 to 472 by in all taxa. Nine haplotypes were recognized from the analysis. Seven populations shared more than two haplotypes, while other thirteen populations shared only one haplotype. In the phylogenetic analysis, the nine haplotypes formed four groups, separated A. sibiricum, one of the sister groups of the complex. There also was no distinct grouping pattern supporting the species and populations observed. These results suggest that introgression or speciation might have been involved in the formation of taxa of A. alboviolaceum complex.

Phylogeographic study of Abies koreana and Abies nephrolepis in Korea based on mitochondrial DNA (미토콘드리아 DNA 분석을 통한 구상나무와 분비나무의 계통지리학적 연구)

  • Yang, Jong-Cheol;Yi, Dong-Keun;Joo, Min-Jeong;Choi, Kyung
    • Korean Journal of Plant Taxonomy
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    • v.45 no.3
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    • pp.254-261
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    • 2015
  • Genetic variations of Abies koreana and Abies nephrolepis were assessed using two mitochondrial DNA regions (nad5 intron 4 and nad5 intron 1) for 16 natural populations to understand their phylogeographical history. Seven polymorphic sites of the two combined regions resulted in the resolution of four haplotypes (M1-M4). The average gene diversity within the population ($H_S$) was 0.098, the total gene diversity ($H_T$) was 0.620, and the interpopulation differentiation was $G_{ST}=0.841$, $N_{ST}=0.849$. The populations were divided into three groups (northern area, central area, southern area) according to their geographic locations. The populations of the northern and southern areas were mostly fixed for M1 and M2, respectively. The populations of the central area showed the highest levels of gene diversity ($H_T=0.654$) due to introgression from the northern area and southern area. The presence of a single mtDNA haplotype in the southern area suggests that current widespread populations have expanded to the central area from a specific refugium population after the last glacial period.

Detection of p53 Common Intron Polymorphisms in Patients with Gastritis Lesions from Iran

  • Sadeghi, Rouhallah Najjar;Damavand, Behzad;Vahedi, Mohsen;Mohebbi, Seyed Reza;Zojazi, Homayon;Molaei, Mahsa;Zali, Mohamad Reza
    • Asian Pacific Journal of Cancer Prevention
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    • v.14 no.1
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    • pp.91-96
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    • 2013
  • Background: p53 alterations have been implicated in the development of many cancers, such as gastric cancer, but there is no evidence of p53 intron alterations in gastritis lesions. The aim of this study was to investigate the p53 intron alterations in gastritis along with p53 and mismatch repair protein expression and microsatellite status. Materials and Methods: PCR-sequencing was conducted for introns 2-7 on DNA extracted from 97 paired samples of gastritis lesions and normal adjacent tissue. Abnormal accumulation of p53 and mismatch repair proteins was investigated using immunohistochemistry. In addition, microsatellite status was evaluated with reference to five mononucleotide markers. Results: Gastritis cases included 41 males and 56 females in the age range of 15-83 years, 87.6% being H.pylori positive. IVS2+38, IVS3ins16 and IVS7+72 were the most polymorphic sites. Their minor allele frequency values were as follows: 0.38, 0.21 and 0.06, respectively. Samples with GG genotype at IVS2+38 and CT at IVS7+72 had no insertion. Moreover, most of the stable samples (91.9 %) had a G allele at IVS2+38. All of the samples were IHC negative for p53 protein, microsatellite stable and expressed mismatch repair proteins. p53 alterations were prominent in the H. Pylori+ group, but without statistical significance. Conclusions: According to our results, some p53 polymorphisms such as IVS2+38, IVS3ins16 and IVS7+72, because of their correlations together or with microsatellite status may contribute to gastritis development. However, so far effects on p53 expression and function remain unclear. Therefore, a comprehensive survey is needed to delineate their biological significance.

Analysis of the GCK and HNF-1α Gene Polymorphism in Korean Type 2 Diabetic Patients by PCR-DHPLC (PCR-DHPLC를 이용한 한국인 제2형 당뇨환자의 GCK와 HNF-1α의 유전자다형성 분석)

  • Nam, Youn-Hyoung;Park, Dae-Yong;Park, Sang-Bum;An, Young-Chang;Lee, Sang-Hyun;Cho, Min-Ho;Park, Su-Min;Jang, Won-Cheoul
    • Journal of the Korean Chemical Society
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    • v.51 no.6
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    • pp.543-548
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    • 2007
  • Maturity-onset diabetes of the young (MODY) is a genetically heterogeneous subtype of Type 2 diabetes characterized (non-insulin-dependent) by early onset, usually before 25 years of age, autosomal dominant inheritance and a primary defect in insulin secretion. Mutations in the glucokinase (GCK) and hepatocyte nuclear factor (HNF)-1α genes are the major causes of monogenic forms of Type 2 diabetes mellitus. Therefore it is need to study relation with these polymorphisms by diverse analysis methods. The promotor and coding regions inclusive intron exon boundaries of the GCK, HNF-1α genes were examined by PCR-DHPLC (Polymerase Chain Reaction - Denaturing High Performance Liquid Chromatography) and direct sequencing. We extracted DNA from 11 patients and 20 normals. Then we confirmed a single-nucleotide polymorphism using PCR-DHPLC. As results, we identified one mutation (R135G) in GCK gene and two mutations (I27L, S487N) in HNF-1a and at the same time detected mutation in intron 8.