Regulation of Vascular Endothelial Growth Factor Signaling by miR-200b

백광희,최영철,Sena Yoon,정용수,윤재승 한국분자세포생물학회 2011 Molecules and cells Vol.32 No.1

Vascular endothelial growth factor (VEGF) signaling plays an important role in angiogenesis. In the VEGF signaling pathway, the key components are VEGF and its receptors, Flt-1 and KDR. In this study, we show that transfection of synthetic miR-200b reduced protein levels of VEGF, Flt-1, and KDR. In A549 cells, miR-200b targeted the predicted binding sites in the 3-untranslated region (3-UTR) of VEGF, Flt-1, and KDR as revealed by a luciferase reporter assay. When transfected with miR-200b, the ability of HUVECs to form a capillary tube on Matrigel and VEGF-in-duced phosphorylation of ERK1/2 were significantly reduced. Taken together, these results suggest that miR-200b negatively regulates VEGF signaling by targeting VEGF and its receptors and that miR-200b may have therapeutic potential as an angiogenesis inhibitor.

RNA polymerase Ⅱ의 구조와 전사기작

백광희 경희대학교 유전공학연구소 1991 遺傳工學論文集 Vol.3 No.-

진핵세포의 핵내에는 원핵세포의 경우와는 달리 DNA를 주형으로 RNA를 합성할 수 있는 세 종류의 효소가 존재하여 서로 다른 종류의 RNA를 만들어 낸다. RNA Polymerase Ⅱ(or B)은 mRNA의 전구물질인 hn-RNA와 U1, U2, U4, U5 snRNA의 합성을 담당하고 있는 효소이다^1 여러 종으로부터 RNA Polymerase Ⅱ가 정제되어 그 subunits의 구조가 밝혀져 있으며, 진화하는 동안에 각 종간에 매우 보존이 잘 이루어져 saccharomyces cerevisiae의 RNA Polymerase Ⅱ와 같이 10개의 subunits로 구성되어 있다. 따라서 비교적 유전학적, 생화학적으로 다루기 쉬운 효모를 이용하여 집중적으로 연구가 진행되어 현재는 10개의 subunits에 대한 모든 유전자가 클로닝되어 각 subunits의 아미노산의 서열이 알려져 있다.^2-7. 가장 큰 세 개의 RNA Polymerase Ⅱ subunits인 RPB1. RPB2, RPB3는 procaryotic RNA Polymerase인 β', β, a subunit에 각각 해당하는 것으로 mRNA합성에 필수적인 요소이다^2^4^8^9 이들보다 작은 subunit중에 RPB5, RPB6, RPB8는 핵내의 세 RNA Polymerase가 공유하고 있다.^5

초등학교 교사들의 실과 가정 영역에 대한 인식 및 요구도

백광희,김영희 한국실과교육학회 2003 한국실과교육학회지 Vol.16 No.1

The seed sequence is necessary but insufficient for downregulation of target genes by miR-608

백광희,이광태,최영철,변유리,윤세나,정용수,윤재승 한국유전학회 2016 Genes & Genomics Vol.38 No.6

MicroRNAs are small, non-coding RNAs that inhibit gene expression posttranscriptionally through interaction with the 30 untranslated region (30 UTR) of target mRNAs. The most important factor for downregulation of target genes by miRNA is the ‘‘seed region,’’ which encompasses nucleotides 2–7 at the 50 end of the miRNA. In this study, sequence determinants for efficient downregulation of target genes were investigated by employing growth-suppressive miR-608 and miR-4651, which shares the seed sequence with miR-608. Cell growth experiments revealed that miR-4651 and miR-608-scram in which the seed sequences were mutated did not inhibit the growth of A549 cells in contrast to wild-type miR-608, which significantly inhibited cell growth. When similarity to miR-608 was increased by replacing sequences of miR- 4651 with that of miR-608, cell growth was gradually more inhibited. Similar results were obtained from a luciferase reporter assay using a reporter plasmid containing the 30 UTR of BCL2L1 and from western blot analysis of BCL2L1, CCND3, and phosphoinositide 3-kinase regulatory subunit 2. Moreover, microarray analyses revealed that overexpression of miR-4651 and miR-608-scram resulted in inefficient downregulation of target genes, and the number of downregulated genes was increased when transfected with MT-3 mimic, which differs from miR-608 by four nucleotides located in the central region. Together, our findings provide a basis for understanding the mechanism underlying target recognition and/or downregulation of target genes by miR-608 and indicate that in addition to seed sequence, central and 30 parts of miR-608 play an important role in mediating efficient downregulation of target genes.

Triple Helix Formation

Baek, Kwang-Hee 경희대학교 유전공학연구소 1989 遺傳工學論文集 Vol.1 No.-

The sequence specific recognition of double helical DNA is essential for regulation of cellular functions including transcription, replication, and cell division. Although triple stranded structures of polynucleotides were discovered decades ago, the biological function has remained unknown. Such triple helices were proposed to be involved in processes like regulation of gene expression, maintenance of folded chromosome conformations, chromosome condensation during mitosis, and induction of local conformational changes in B DNA (Morgan, 1979). Sequence specific oligonucleotide recognition of duplex DNA by forming triple helix offers powerful implications for the study of molecular biology. Oligonucleotide with efficient cleaving moiety could become useful tool in chromosome analysis. gene mapping, and isolation. Triple helix formation selectively protect the target site from restriction endonuclease and methylase, allowing specific restriction endonuclease cleavage at the triple helix target site. The fact the triple helix formation blocks the birding of specific DNA binding protein offers a new tool for analyzing protein-DNA interactions in promoter, and in some cases such oligonucleotide or their analogs might be designed to function as artificial gene specific repressor in vivo. Oligonucleotide-directed DNA binding protein can be degined to recognize specific double helical DNA sites by triple helix formation.

RBP 신호배열 돌연변이 rbsB103에 대한 RBP 아미노산 50번 위치의 복귀 돌연변이 분리

강문석,백광희 경희대학교 유전공학연구소 1993 遺傳工學論文集 Vol.5 No.-

Ribose-binding protein(RBP) of E. coli function in the periplasm as a component of a high affinity transport system for ribose and as a primary receptor for chemotaxis toward ribose. A mutant(rbsB103) in the signal sequence of RBP has a defect in the export of the protein to the periplasm. The intragenic suppressors for rbsB103 were isolated genetically. One of them had a change at 50th amino acid of RBP. In order to assess the role of the 50th amino acid in the transport of RbsB103, we substituted the valine of 50th position with various amino acids by site-directed mutagenesis. The assay of the mutants by chemotaxis indicates that the replacement of the Val by Cys, Glu, Gln, Gly, His, Ile, Leu, Gln, Ser, Thr, or Tyr can suppress the defect of protein transport.

Site-directed Mutagenesis에 의한 rbsB103의 복기 돌연변이 분리

이덕연,백광희 경희대학교 유전공학연구소 1992 遺傳工學論文集 Vol.4 No.-

Ribose-binding protein (RBP) of E. coli function in the periplasm as a component of a high affinity transport system for ribose and as a primary receptor for chemotaxis toward ribose. A mutational change(rbsB103) in the signel sequence of RBP block the export of the protein to the periplasm. One of the intragenic suppressors for rbsB103 isolated genetically carries mutation at 27th position of RBP. In order to assess the role of the 27th amino acid in protein transport, we substituted the Ala of 27th position with various amino acids by site-directed mutagenesis method. The assay of the mutants by chemotaxis indicates that the replacement of the Ala by Cys, Gly, Ser, Thr, or Val can suppress the defect of protein transport, but the replacement by Glu, Pro, or Leu cannot.

새로운 PCR 클로닝 벡터의 개발

김윤일,백광희,윤재승 경희대학교 산학협력기술연구원 1996 산학협력기술연구논문집 Vol.2 No.-

PCR (Polymerase Chain Reaction) is a powerful technique for amplifying specific DNA region of interest. The PCR was used first only in the field of molecular biology, but nowadays it is widely used in the various fields such as diagnosis of genetic disorders, identification of forensic samples, and classification of organisms. However, Taq(Thermus aquaticus)DNA polymerase used in PCR has been reported to add a single nontemplate-directed A residue to the 3' ends of amplified KNA, giving rise to the difficulties in the cloning of amplified DNA. Considering the wide applications of the PCR, the development of the efficient cloning vector for PCR-amplified DNA would be very helpful for various purposes, we have designed the PCR cloning vector using the nucleotide sequence recognized by restriction endonuclease Xcm I. The double incorporation of Xcm I recognition sites in the pUC 19 and m13 DNA can make compatible ends carrying T residue and be easily ligated to PCR products.

Site-directed Mutagenesis에 의한 RBP(Ribose Binding Protein)의 #5-Ala의 치환과 rbsB103 복귀 돌연변이의 분리

정환웅,백광희 경희대학교 유전공학연구소 1995 遺傳工學論文集 Vol.7 No.-

Ribose-binding protein (RBP) of E coli function in the periplasm as a component of a high affinity transport system for ribose and as a primary receptor for chemotaxis toward ribose. A mutant (rbsB103) in the signal sequence of RBP has a defect in the export of the protein to the periplasm. The intragenic suppressors for rbsB103 were isolated genetically. One of them had a change at 5th amino acid of RBP. In order to assess the role of the 5th amino acid in the transport of rbsB103, we substituted the alanine of 5th position with various amino acids by site-directed mutagenesis. The mutants replaced by Arg, Asp, Pro, or Ser at 5th amino acid of RBP can suppress the defect of protein transport.

초파리 norpA 돌연변이주들의 생리학적 특성 분석

이승희,백광희,윤재승 한국유전학회 1996 Genes & Genomics Vol.18 No.1

Mutations in the norpA gene which encodes the photoreceptor-specific phospholipase C affect drastically the visual signal transduction process in Drosophila melanogaster. In the present work we have grouped the 31 norpA mutants depending on the positions of their defects. For this purpose we examined the level of transcription and translation of the norpA gene in the mutants and analyzed their photoresponses by electroretinogram (ERG). The Western blot analysis using antibody aga-inst the norpA gene product revealed that 11 mutants have the point mutations within the protein coding region. The RNA dot blot analysis showed that at least 14 mutants contain the point mutations affecting significantly the transcription of the norpA gene. The ERG analysis of the norpA mutants suggests that the level of photoresponse is determined by the nature of point mutations present in the mutants, not by the amount of the gene product. Further molecular analysis of the norpA mutants will greatly help the understanding of the phospholipase C functions as well as the mechanism of the eye-specific gene expression.