Protein synthesis elongation factor EF-1 alpha expression and longevity in Drosophila melanogaster.

Abstract

It has been proposed that the decline in protein synthesis observed in aging organisms may result from a decrease in elongation factor EF-la. Transgenic Drosophila melanogaster flies carrying an additional copy of the EF-la gene under control of a heat-inducible promoter have an extended lifespan, further indicating that the EF-la gene may play an important role in determining longevity. To test this hypothesis, we have quantitated EF-1amRNA, EF-la protein, and the EF-la complex-formation activity in these transgenic flies. Furthermore, we have tested whether the transgene construct is functiona-i.e., whether transgenic mRNA is induced when flies are grown at higher temperature. The results show that although there is a clear difference in mean lifespan between the EF-la transgenic (E) flies and the control transgenic (C) flies, E flies do not express more EF-la protein or mRNA than C flies kept at the same experimental conditions. Although the transgene can be induced when E flies are heat-shocked at 3rC, transgenic mRNA is not detectable in E flies aged at 29°C. In both lines, the loss in catalytic activity with age is the same. We conclude that the E flies examined here do not live longer because of overexpressing the EF-1a gene.scription of the EF-la gene. The eukaryotic polypeptidechain-elongation factor EF-la is a GTP-binding protein that catalyzes the binding of aminoacyl-tRNA to the ribosome (13). Based on Webster's experiments, Shepherd et al. (14) addressed the question of whether expression of the EF-la gene may be directly involved in determining the lifespan of the fruit fly. They transformed Drosophila with a P-element vector containing a cDNA copy of the EF-la gene under control of the inducible hsp7O (70-kDa heat shock protein) gene promoter. At 250C the flies carrying the EF-la P element (E flies) lived significantly longer than the flies carrying a control P element (C flies). This difference in mean lifespan was increased when flies were kept at 29.50C. The authors concluded that the heat-induced overexpression of the EF-la gene led to increased lifespan of the flies and thus that there might be a positive correlation between the expression of the EF-la gene and longevity. Their results suggested that an important aging control might be exerted at the level of transcription of the EF-la gene. We have tested this hypothesis by measuring the expression of EF-la in these transgenic E and C flies both at the mRNA and at the protein level. The lifespan of each species, including man, is different, but genetically defined. According to the program theory of aging, the longevity of animal species, as well as the lifespan of their cells in culture, is determined by genetically controlled processes similar to those that control early development. Several attempts have been made to demonstrate the existence oflongevity-determining genes (1-5), but nothing is known about possible targets for such genes at the cellular or at the molecular level. Most higher organisms contain mitotic as well as p...

Multiple-stress analysis for isolation ofDrosophilalongevity genes

“…Elongation factor 1␣ (ef-1␣) plays a role in protein synthesis, which decreases during aging (42). Transgenic flies of overexpressing ef-1␣ were reported to extend lifespan in Drosophila (43), but whether ef-1␣ was overexpressed in these flies is controversial (44). In our screening, the level of ef-1␣ was up-regulated by all three stresses.…”

P-element inserts in transgenic flies: a cautionary tale

“…Expression was tested using the Si nuclease protection assay described in Shikama et al (1994). Line T was used by Shepherd et al (1989) and has been shown to express the transgenic EF-tz message at 37°C, but not at 29°C (1994).…”

“…Independently, Shikama et al (1994) investigated the EF-lcc transgenic flies of Shepherd et a!. (1989) at the molecular level.…”

EFFECTS ON FITNESS COMPONENTS OF P-ELEMENT INSERTS INDROSOPHILA MELANOGASTER: ANALYSIS OF TRADE-OFFS

“…Until 1993, these experiments, which were done at two temperatures, 25°C and 29.5°C, were interpreted on the assumption that the gene for elongation factor was more strongly expressed at the higher temperature (a design used to control for position effects). We now know that the additional copy of the gene was not expressed at the temperatures used, neither in Shepherd et al's (1989) experiment (Shikama et al 1994) nor in ours (Kaiser et al, unpubl.). That changed the interpretation of the results from a study of overexpression of a known gene to a study of proper controls in experiments on genetic engineering of phenotypes.…”

“…Before describing the experiments, we summarize the evidence that the gene was not expressed. Expression was tested using the S 1 nuclease protection assay (Shikama et al 1994). Line T, which was used by Shepherd et al (1989) and which expresses the transgenic EF-lex message at 3rC but not at 29°C (Shikama et al 1994), served as a positive control.…”