Selection of AU-rich transiently expressed sequences: reversal of cDNA abundance.
Study of early and transient response gene expression is important for understanding the mechanisms of response to growth stimuli and exogenous agents such as microbes, stress, and radiation. Many of the cytokines, proto-oncogenes, and other transiently expressed gene products are encoded by mRNAs that contain AU-rich elements (AREs) in their 3' untranslated regions (UTRs). In this article, we describe an approach to selectively synthesize ARE-containing cDNA (ARE-cDNA) using an innovative combination of culture treatment, thermostabilization of reverse transcriptase (RT) by the disaccharide trehalose, and use of optimized ARE-specific oligomers. The monocytic cell line, THP-1, was treated with cycloheximide and endotoxin to enrich for ARE-mediated gene expression followed by the RT procedure. Selection of ARE-cDNA with simultaneous suppression of abundant cDNA was made possible using the procedure as monitored by the preferential expression of IL-8, an ARE-cDNA molecule, over the abundant housekeeping cDNA, beta-actin. The use of trehalose dramatically reversed cDNA abundance, resulting in almost complete suppression of housekeeping cDNA. Finally, construction of specialized ARE-cDNA libraries confirmed the selectivity of ARE-cDNAs and the presence of rare genes. The ability to reverse the abundance of housekeeping and other highly expressed genes toward ARE genes facilitates the discovery and study of rare early response and transiently expressed genes.
Khabar
Khalid S A
KS
Interferon and Cytokine Research Unit, Department of Biological and Medical Research, Bioinformatics Section, King Faisal Specialist Hospital and Research Center, Riyadh 11211, Saudi Arabia. khabar@kfshrc.edu.sa
Dhalla
Mohammed
M
Al-Haj
Latifa
L
Bakheet
Tala
T
Sy
Cheikh
C
Naemmuddin
Mohammed
M
D020856:AT Rich Sequence / Q000502:physiology; D000818:Animals; D018076:DNA, Complementary / Q000378:metabolism; D015723:Gene Library; D006801:Humans; D016209:Interleukin-8 / Q000235:genetics; D008954:Models, Biological; D008969:Molecular Sequence Data; D008979:Moloney murine leukemia virus / Q000235:genetics; D009000:Monocytes / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism
D018076:DNA, Complementary; D016209:Interleukin-8; D012313:RNA; D012194:RNA-Directed DNA Polymerase