We are studying the importance of gene expression of the alpha subunit of eIF-2 to the induction of protein synthesis occurring during T cell activation. Using antisense DNA and antisense RNA techniques, we have been attempting to selectively block alpha gene expression in vivo to ascertain whether this increase is required for T cell activation, i.e. protein synthesis, proliferation, or induction of specific lymphokine genes. We and others have shown that there is a coordinated early increase in mRNA levels for the translation initiation factors, eIF- 2alpha, eIF-4E and eIF-4A. This ultimately results in an increased rate of protein synthesis. Initially we have focused upon one of these factors, eIF-2alpha, and have attempted to block its gene expression by using antisense DNA. Small oligonucleotides have been prepared, targeted to mainly the 5' region of the message, as well as areas in the 3'UTR. Only oligos targeted against the 3'UTR have yielded any consistent results by either Western or Northern Blot analysis. Alternatively, we have also been using antisense RNA techniques. EBV episomal vectors containing a metallothionein inducible promoter and sequences antisense to an 1.5 kb area of the eIF-2alpha promoter were constructed and used to transfected NIH 3T3 cells. We are in the process of characterizing selected clones. During our studies on the gene expression for eIF-2alpha, we have observed that although it is a single copy gene, two mRNAs, 1.6 kb and 4.5 kb, can be detected by Northern Blot analysis hybridizing to a cDNA probe. Both mRNAs are associated with large polysomes, indicating that they are being actively translated. Northern analysis of RNA from a variety of human tissues show that the ratio of these two mRNAs varies between tissues. We have isolated several cDNA clones corresponding to the 4.5 kb mRNA from a lamdagt11 library and are in the process of sequencing them. From the sequence information we hope to identify the mechanism involved with the creation of the 4.5 kb message.
