| Normal human diploid fibroblast cells senesce in vitro and exhibit a finite lifespan during which time they undergo a specific number of cell doublings (Hayflick, 1965, Exp. Cell Res. 37:616-636). The molecular mechanisms leading to this loss of proliferative potential have yet to be identified. However, as cell senesce in vitro, it has been shown that an antiproliferative mRNA(s) accumulates accounting for 0.8% of the total poly A(+) RNA (Lumpkin, et al., 1986, Science 232:393-395). We have attempted to isolate this highly abundant, antiproliferative mRNA by differentially screening cDNA libraries constructed from early and late passage WI-38 human diploid fibroblast cells. From over 100 clones whose RNA exhibited changes in abundance levels during in vitro aging, one clone was selected which accounted for approximately 1% of the recombinants in the late passage library. Following plaque purification, the cDNA insert was subcloned into pUC 119 and designated as pSEN. Northern analysis of poly A(+) RNA from various intermediate populations doubling levels (PDL) shows that a 2.2 kb transcript is expressed prior to PDL-40 at very low levels. This transcript begins to accumulate at PDL-40 and is induced approximately 50 fold just prior to senescence. Cell-cycle northern blot analysis of total cellular RNA confirms the presence of the 2.2 kb species at high levels during GO of late passage cells. Another lower molecular weight species is observed at 18h following serum stimulation in early passage cells and at 9h in late passage cells (although at much lower levels). Furthermore, when total cellular RNA from 9h serum stimulated late passage WI-38 cells is analyzed by sucrose gradient fractionation, the 2.2 kb transcript is located near the top of the gradient while the second, lower molecular weight transcript is dispersed toward the bottom of the gradient, suggesting that the 2.2 kb transcript is associated with ribonuclear protein particles and the lower molecular weight transcript is loaded on polyribosomes. Nucleotide sequence analysis of the 1300 bp pSEN insert shows 100% homology to 1050 bp of human elongation factor I alpha. The pSEN cDNA lacks the 5{dollar}spprime{dollar} end of the transcript and has an additional 246 bp at the 3{dollar}spprime{dollar} end of the molecule. Comparison of the nucleotide sequence of elongation factor I alpha from other species suggests that this addition at the 3{dollar}spprime{dollar} end of the transcript is unique to pSEN. |
