Abstract 508
Poster Session I, Saturday, 5/1 (poster 296)
As part of a continuing project aimed at prospectively identifying genes whose expression is regulated by IGF-I, we have used differential display to identify messenger RNAs whose levels are up- or down-regulated by IGF-I. Methods: Following overnight incubation in serum-free medium, virally-transformed human B- and T-lymphoblasts were treated for 4-6 with a physiological concentration (10 ng/ml) of IGF-I or control. Total RNA was DNase-treated, reverse transcribed using an oligo-dT primer, followed by PCR amplification at two different cDNA template concentrations, using (alpha-33P) dATP, "anchored" oligo-dT primers, and a second primer which binds at arbitrary but reproducible locations on the cDNA. Following long-read, temperature-controlled electrophoresis and autoradiography, cDNA fragments showing putative differential expression were eluted and reamplified for sequencing and use as probes of Northern blots prepared from the IGF-I-treated lymphoblasts. Results/Discussion: An approximately 1.1 kb cDNA fragment was cloned after differential display analysis of mRNA from IGF-I-treated B-lymphoblasts. Using this cDNA as a hybridization probe. up-regulation of the corresponding 4.7 kb mRNA was demonstrated on Northern blots of total RNA from serum-derived T-lymphoblasts treated for 4 h with IGF-I. Partial sequence analysis and sequence similarity searching using the BLAST service revealed an exact match to an expressed sequence tag (EST) fragment obtained by random sequencing of cDNA from human fetal liver and spleen. In addition, a 200-bp Alu repeat sequence was identified in the putative 3′-untranslated region. No other significant matches were found in any DNA or protein databases, suggesting that this cDNA may represent the product of a novel gene whose activity is acutely up-regulated by IGF-I treatment. In addition to obtaining full-length cDNA clones for further sequencing, we are studying the time course, dose-responsiveness, and cycloheximide sensitivity of expression of the corresponding mRNA. Identification of a novel gene whose expression changes in response to IGF-I may provide further insights into the mechanism by which this hormone controls cellular growth.
