DNA TRANSFECTION TO STUDY TRANSLATIONAL CONTROL IN MAMMALIAN-CELLS

Mammalian cells respond to changes in their environment by rapid and r eversible covalent modification of the translational machinery. In mos t cases, these modifications involve the phosphorylation and dephospho rylation of translation initiation factors (for review see Ref. 1). Th e modification of translation initiation factors may affect translatio nal activity of either specific mRNAs or general cellular mRNAs. To st udy the effect of a particular factor or its modification on the trans lational capacity of an mRNA, there are a number of potential approach es that include in vitro translation reactions as well as in vivo expe riments. Generally, experiments initially report a covalent modificati on that correlates with altered translational capacity of either a spe cific or a general class of mRNAs. The modification and the particular amino acid residue involved are then identified. Then mutations are m ade at the modified residue to prevent modification (for example, a se rine-to-alanine mutation to prevent phosphorylation) and the effect of the mutant factor on the translation of a target mRNA is tested. The most convenient method for monitoring the effect of a mutant translati on factor on translation is the use of transient DNA transfection. How ever, in certain situations it is desirable to isolate stably transfec ted cell lines to study the effect of overexpression, underexpression, or expression of a particular mutant translation factor. This article reviews two methods that are routinely used to study translational co ntrol that involve either transient or stable DNA transfection. (C) 19 97 Academic Press.