Gene Expression of eIF-2alpha The major focus of our work on the gene expression of eIF-2alpha has been on the characterization of the 4.2 kB message. eIF-2alpha is a single copy gene which produces two transcribed mRNAs, 1.6 kB and 4.2 kB in size by Northern Blot analysis. Both mRNAs are polyadenylated and located in the cytoplasm of activated T cells and other cell types. Both mRNAs are found on polysomes in total cell lysates from Jurkat cells. Northern Blot analysis of poly A+ RNA from multiple tissues (human) showed that the ratio of the 4.5 kB mRNA to 1.6 kB mRNA varied from 1:10 in the placenta to 1:3 in the brain and muscle. Ribonuclease protection assay using a probe containing sequences from the 3' end of a cDNA previously isolated and characterized, identified two protected bands which added more evidence for the presence of two distinct mRNAs for eIF-2alpha. A PCR probe based on the 3' most sequence was prepared and used to analyze activated T cell RNA. Only the 4.2 kB mRNA hybridized with this probe. This probe was then used to screen a lambda GT11 library from activated T cells, and ultimately all of the 4.2 kB cDNA was obtained and sequenced. This cDNA can be transcribed and translated in vitro. We are presently trying to determine if there is a difference in translation efficiency of the two messages. We are also trying to study the stability/degradation of the two mRNAs using both in vivo and in vitro methods. We would like to determine if the existence of the 4.2 mRNA is important for the regulation of eIF-2alpha gene expression in different tissues, and if this has an impact on protein synthesis.
