Skip Navigation

This Article
Right arrow Full Text Freely available
Right arrow Print PDF (576K) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (16)
Right arrowRequest Permissions
Right arrow Commercial Re-use Guidelines
for Open Access NAR Content
Google Scholar
Right arrow Articles by Yonekura, H.
Right arrow Articles by Yamamoto, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yonekura, H.
Right arrow Articles by Yamamoto, H.
Related Collections
Right arrow Miscellaneous/other
Right arrow Monitoring gene expression
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Nucleic Acids Research, Vol 27, Issue 13 2591-2600, Copyright © 1999 by Oxford University Press

ARTICLES

Antisense display--a method for functional gene screening: evaluation in a cell-free system and isolation of angiogenesis-related genes

H Yonekura, H Migita, S Sakurai, H Wang, S Harada, MJ Abedin, S Yamagishi and H Yamamoto
Department of Biochemistry, Center for Biomedical Research, Kanazawa University School of Medicine, Kanazawa 920-8640, Japan.

Presented here is an antisense-oriented method for functional gene screening, which we propose naming 'antisense display'. In principle, it consists of four steps: (i) preparation of phosphorothioate antisense repertoires that would correspond to the Kozak's consensus sequence, (ii) subgroup screening to identify active antisense molecules that could cause changes in the cellular phenotypes concerned and (iii) RT-PCR cloning of cDNA with the 5[prime] sense complement and 3[prime] anchor primers and sequence determination, followed by (iv) functional assays of candidate genes. Cell-free translation in rabbit reticulocyte lysate revealed that 10mer or longer antisense effectively halted protein synthesis. This required the presence of RNase H, and was achieved without prior heat-denaturation of the RNA templates. Then, subpools of the 10mer repertoire were administered to human microvascular endothelial cells in culture, and screened for anti- angiogenic activities. A single species having the sequence 5[prime]- GGCTCATGGT-3[prime] consistently inhibited the endothelial cell growth under hypoxia. Through RT-PCR with the corresponding sense primer, we came across three candidate cDNAs. Experiments employing longer unique antisense reproduced marked growth inhibitions in two of the three cDNAs. One encoded a mitochondrial protein and the other, which encoded a putative type-2 membrane protein containing Rab-GAP/TBC and EF-hand like domains, was a gene previously undescribed in human. The results suggest that the antisense display method is potentially useful for isolating new genes towards elucidating their functions.
Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
Y. C. Xu, R. F. Wu, Y. Gu, Y.-S. Yang, M.-C. Yang, F. E. Nwariaku, and L. S. Terada
Involvement of TRAF4 in Oxidative Activation of c-Jun N-terminal Kinase
J. Biol. Chem., July 26, 2002; 277(31): 28051 - 28057.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
H. Yonekura, S. Sakurai, X. Liu, H. Migita, H. Wang, S.-i. Yamagishi, M. Nomura, Md. J. Abedin, H. Unoki, Y. Yamamoto, et al.
Placenta Growth Factor and Vascular Endothelial Growth Factor B and C Expression in Microvascular Endothelial Cells and Pericytes. IMPLICATION IN AUTOCRINE AND PARACRINE REGULATION OF ANGIOGENESIS
J. Biol. Chem., December 3, 1999; 274(49): 35172 - 35178.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
W. Xu, Y. Takahashi, T. Sakashita, T. Iwasaki, H. Hattori, and T. Yoshimoto
Low Density Lipoprotein Receptor-related Protein Is Required for Macrophage-mediated Oxidation of Low Density Lipoprotein by 12/15-Lipoxygenase
J. Biol. Chem., September 21, 2001; 276(39): 36454 - 36459.
[Abstract] [Full Text] [PDF]


Disclaimer: Please note that abstracts for content published before 1996 were created through digital scanning and may therefore not exactly replicate the text of the original print issues. All efforts have been made to ensure accuracy, but the Publisher will not be held responsible for any remaining inaccuracies. If you require any further clarification, please contact our Customer Services Department.