JBC Ideal method for primary cell transfection

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M007789200 on December 8, 2000

J. Biol. Chem., Vol. 276, Issue 10, 6937-6944, March 9, 2001
This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
276/10/6937    most recent
M007789200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Dai, B.
Right arrow Articles by Singh, P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Dai, B.
Right arrow Articles by Singh, P.

Identification of a Novel Cis Element Required for Cell Density-dependent Down-regulation of Insulin-like Growth Factor-2 P3 Promoter Activity in CaCo2 Cells*

Bosong DaiDagger , Hai WuDagger , Elly Holthuizen§, and Pomila SinghDagger

From the Dagger  Department of Anatomy and Neurosciences, The University of Texas Medical Branch, Galveston, Texas 77555-1043 and § Laboratory for Physiological Chemistry, Utrecht University, Universiteitsweg 100, 3586 CG Utrecht, The Netherlands

The activity of the exogenous, full-length insulin-like growth factor-2 (IGF-2) P3 promoter is significantly up-regulated during the logarithmic growth phase but rapidly declines in confluent CaCo2 cells undergoing differentiation. Nuclear run-on assays confirmed cell density-dependent regulation of endogenous P3 promoter. To identify regulatory elements in the P3 promoter that may be required for regulating cell density-dependent transcriptional activity, we used the methods of promoter truncation, electrophoretic mobility shift assay, DNase footprinting, and mutation analysis. The relative activity of the full-length (-1229/+140) and truncated (-1090/+140) promoter was identical, being ~19, 27, 7, and 3% of pSV-luc activity on days 3, 5, 7, and 9 of cell culture, respectively. However, truncation to -1048 resulted in complete loss of cell density-dependent down-regulation of P3 promoter activity on days 7 and 9, suggesting the presence of regulatory elements between -1091 and -1048 sequence. Further stepwise truncation to -515 did not change promoter activity. Truncation to -138/+140 resulted in complete loss of promoter activity, suggesting that the core promoter was within the -515/-138 segment. A 14-base pair footprint (-1084/-1070) was identified by DNase footprinting within the distal -1091/-1048 segment. Electrophoretic mobility shift assay with wild type and mutant probes confirmed the presence of a novel 7-base pair (CGAGGGC) (-1084/-1078) cis element (P3-D); its mutation abolished binding. Functionality of P3-D cis element was confirmed by measuring the activity of core P3 promoter ligated to distal P3 segment containing either the mutant or wild type P3-D element. We have, therefore, identified a novel cis element, P3-D, that appears to play a critical role in regulating IGF-2 P3 promoter activity in a cell density/differentiation-dependent manner.

* This work was supported by National Institutes of Health Grants CA60087 and CA72992 (to P. S.) and John Sealy Memorial Foundation Grant 454690 (to B. D.). Part of this study was published in its preliminary form as an abstract (Dai, B., Wu, P. H., Holthuizen, E., and Singh, P. (1999) in Proceedings of The Digestive Diseases Week, Abstr. 3777, American Gerontological Association, Orlando, FL).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom all reprint requests should be addressed: Dept. of Anatomy and Neurosciences, 10.138 Medical Research Bldg. 1043, University of Texas Medical Branch, Galveston, TX 77555-1043. Tel.: 409-772-4842; Fax: 409-772-1861; E-mail: posingh@utmb.edu.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.


This article has been cited by other articles:


Home page
 Cancer Res.Home page
W. Rengifo-Cam, S. Umar, S. Sarkar, and P. Singh
Antiapoptotic Effects of Progastrin on Pancreatic Cancer Cells Are Mediated by Sustained Activation of Nuclear Factor-{kappa}B
Cancer Res., August 1, 2007; 67(15): 7266 - 7274.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
R. S. Solorzano-Vargas, J. Wang, L. Jiang, H. V. Tsai, L. O. Ontiveros, M. A. Vazir, R. J. Aguilera, and M. G. Martin
Multiple transcription factors in 5'-flanking region of human polymeric Ig receptor control its basal expression
Am J Physiol Gastrointest Liver Physiol, August 1, 2002; 283(2): G415 - G425.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
M. Motwani, C. Jung, F. M. Sirotnak, Y. She, M. A. Shah, M. Gonen, and G. K. Schwartz
Augmentation of Apoptosis and Tumor Regression by Flavopiridol in the Presence of CPT-11 in Hct116 Colon Cancer Monolayers and Xenografts
Clin. Cancer Res., December 1, 2001; 7(12): 4209 - 4219.
[Abstract] [Full Text] [PDF]


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2001 by the American Society for Biochemistry and Molecular Biology.