Prof Prem raj Pushpakaran
@nitc.ac.in
School of Bio-Technology
National Institute of Technology, Calicut
Scopus Publications
Scopus Publications
Madhu S. Malo, Premraj Pushpakaran, and Richard A. Hodin
Elsevier BV
Madhu S. Malo, Wenying Zhang, Fuad Alkhoury, Premraj Pushpakaran, Mario A. Abedrapo, Moushumi Mozumder, Elizabeth Fleming, Aleem Siddique, Joseph W. Henderson, and Richard A. Hodin
The Endocrine Society
Thyroid hormone (T3) is a critical regulator of intestinal epithelial development and homeostasis, but its mechanism of action within the gut is not well understood. We have examined the molecular mechanisms underlying the T3 activation of the enterocyte differentiation marker intestinal alkaline phosphatase (IAP) gene. RT-PCR and Western blotting showed that thyroid hormone receptors TRalpha1 and TRbeta1 were expressed in human colorectal adenocarcinoma Caco-2 cells. Northern blotting detected expression of two IAP transcripts, which were increased approximately 3-fold in response to T3. Transient transfection studies with luciferase reporter plasmids carrying various internal and 5' deletion mutations of the IAP promoter localized a putative thyroid hormone response element (TRE) to a region approximately 620 nucleotides upstream (-620) of the ATG start codon. EMSAs using TRalpha1-retinoid X receptor alpha (RXRalpha) on sequential 5' and 3' single nucleotide deletions defined the TRE between -632 and -612 (5'-TTGAACTCAgccTGAGGTTAC-3'). Compared with the consensus TRE, the IAP-TRE is novel in that it contains an everted repeat of two nonamers (not hexamers) separated by three nucleotides. Neither TRalpha1 nor RXRalpha binds to the IAP-TRE; however, TRbeta1 binds to this TRE with minimal affinity. In the presence of TR and RXRalpha, only the TR-RXRalpha heterodimer binds to the IAP-TRE. Mutagenesis of either nonamer abolishes the biological activity of IAP promoter. We have thus identified a novel response element that appears to mediate the T3-induced activation of the enterocyte differentiation marker, intestinal alkaline phosphatase.
Madhu S. Malo, Mario Abedrapo, Alex Chen, Moushumi Mozumder, Premraj Pushpakaran, Fuad Alkhoury, Wenying Zhang, Elizabeth Fleming, and Richard A. Hodin
Future Science Ltd
Understanding gene regulation is of fundamental importance to virtually all biological systems. A gene is regulated through cis-acting regulatory DNA se-quences (elements) in conjunction with trans-acting regulatory proteins. The cis-acting regulatory elements involved in transcription include, for example, the promoter, enhancer, silencer, spe-cific activator binding sites, and spe-cific repressor binding sites, whereas the trans-acting regulatory proteins include basic transcription factors, acti-vators, repressors, coactivators, and co-repressors. For the quantitative analysis of transcriptional regulatory elements, a variety of reporter vectors have been developed based on firefly luciferase and Renilla luciferase, chlorampheni-col acetyltransferase (CAT), β-galac-tosidase, β-glucuronase, β-lactamase, alkaline phosphatase, human growth hormone (hGH), and green fluores-cent protein (GFP) (1–11). Luciferase reporter vectors have become the vec-tors of choice for most investigations because of the availability of rapid and sensitive luciferase assay systems (1). Most of the currently available eu-karyotic promoter-detection vectors carry only a single reporter gene. The use of such a vector can generate er-roneous results due to the variability in transfection efficiency (intracellular copy number) in different samples of transfected cells. Many individual factors can affect the transfection effi-ciency of a plasmid, including contami-nating nucleases, endotoxins, and salts in DNA preparations. Dispensation of variable amounts of DNA during pipet-ting of small quantities of DNA can also influence transfection efficiency. The size of a plasmid also plays a role in transfection because some smaller plas-mids can enter cells more efficiently. To minimize the error due to variable trans-fection efficiency, current protocols generally use a second control plasmid to normalize the data for the test plas-mid. The normalized value of the test plasmid data is usually expressed as a percentage of the control plasmid data. However, the use of a control plasmid cannot completely eliminate variability in transcription efficiency because of contamination of individual samples and the variable quantity of individual plasmid from sample to sample. To solve this problem, which is associated with the use of two plasmids, Park (1) developed a promoter-detection vector, pJDL