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A screen of apoptosis and senescence regulatory genes for life span effects when over-expressed in Drosophila.

Shen J, Curtis C, Tavaré S, Tower J - Aging (Albany NY) (2009)

Bottom Line: Both wingless and Ras activated form transgenes were lethal when expressed in larvae, and reduced life span when expressed in adults, consistent with results from other model systems indicating that the wingless and Ras pathways can promote senescence.Over-expression of the caspase inhibitor baculovirus p35 during larval development reduced the mean life span of male and female adults, and also produced a subset of females with increased life span.These experiments suggest that baculovirus p35 and the wingless and Ras pathways can have sex-specific and developmental stage-specific effects on adult Drosophila life span, and these reagents should be useful for the further analysis of the role of these conserved pathways in aging.

View Article: PubMed Central - PubMed

Affiliation: Molecular and Computational Biology Program, Department of Biological Sciences, University of Southern California, Los Angeles, CA 90089, USA.

ABSTRACT
Conditional expression of transgenes in Drosophila was produced using the Geneswitch system, wherein feeding the drug RU486/Mifepristone activates the artificial transcription factor Geneswitch. Geneswitch was expressed using the Actin5C promoter and this was found to yield conditional, tissue-general expression of a target transgene (UAS-GFP) in both larvae and adult flies. Nervous system-specific (Elav-GS) and fat body-specific Geneswitch drivers were also characterized using UAS-GFP. Fourteen genes implicated in growth, apoptosis and senescence regulatory pathways were over-expressed in adult flies or during larval development, and assayed for effects on adult fly life span. Over-expression of a dominant p53 allele (p53-259H) in adult flies using the ubiquitous driver produced increased life span in females but not males, consistent with previous studies. Both wingless and Ras activated form transgenes were lethal when expressed in larvae, and reduced life span when expressed in adults, consistent with results from other model systems indicating that the wingless and Ras pathways can promote senescence. Over-expression of the caspase inhibitor baculovirus p35 during larval development reduced the mean life span of male and female adults, and also produced a subset of females with increased life span. These experiments suggest that baculovirus p35 and the wingless and Ras pathways can have sex-specific and developmental stage-specific effects on adult Drosophila life span, and these reagents should be useful for the further analysis of the role of these conserved pathways in aging.

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Effect of Baculovirus p35 over-expression on survival of adult flies.                                        Baculovirus p35                                            transgenes inserted on the X chromosome, chromosome 2, and chromosome 3                                            were over-expressed during larval development or adult stage, as indicated.                                            The life span assays were performed at 25°C. Open circles represent the no-drug control ("-"). Solid squares                                            represent adults treated with drug ("A"). Grey triangles represent larvae                                            on drug ("L"). Survival curves are plotted as a function of adult age in                                            days. Median life span of each cohort is presented along with p                                            value for log rank tests  (in parentheses). (A, C, E, G) male                                            flies.  (B, D, F, H) female flies.  (A,B) Control flies                                            containing the driver and no target transgene. (C, D) Baculovirus                                                    p35 transgene on X chromosome.  (E, F) Baculovirus p35 transgene                                            on second chromosome. (G, H) Baculovirus p35 transgene on                                            third chromosome.
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Figure 4: Effect of Baculovirus p35 over-expression on survival of adult flies. Baculovirus p35 transgenes inserted on the X chromosome, chromosome 2, and chromosome 3 were over-expressed during larval development or adult stage, as indicated. The life span assays were performed at 25°C. Open circles represent the no-drug control ("-"). Solid squares represent adults treated with drug ("A"). Grey triangles represent larvae on drug ("L"). Survival curves are plotted as a function of adult age in days. Median life span of each cohort is presented along with p value for log rank tests (in parentheses). (A, C, E, G) male flies. (B, D, F, H) female flies. (A,B) Control flies containing the driver and no target transgene. (C, D) Baculovirus p35 transgene on X chromosome. (E, F) Baculovirus p35 transgene on second chromosome. (G, H) Baculovirus p35 transgene on third chromosome.

Mentions: To control for any possible effects of the Geneswitch system and the RU486 drug itself, life span was assayed in flies that were the progeny of Act-GS-2555B driver crossed to either Oregon-R (Or-R) wild-type strain or to the w1118 control strain, to produce progeny containing only the driver. In these control flies, treatment with drug produced small, but statistically significant reductions in life span in both male and female adults: treatment during adulthood reduced mean life span by -4% to -10%, while treatment in larval stages reduced adult life span by -8% to -16% (Figure 3A, B; Figure 4A, B; Tables 2, 3). There were no significant increases in life span in control flies treated with RU486 in any of the replicate experiments. These data indicate that in these experiments, when the Act-GS-255B driver is present, the RU486 can cause small but significant reductions in adult life span, and this effect must be taken into account when interpreting the effects of transgene over-expression. Other studies [22], including ones from our own laboratory using the Act-GS-255B driver [36], found no negative effects of RU486 on adult fly life span. We conclude that the small negative effects observed here result from differences in the lot of RU486 drug, and/or small differences in effective concentrations due to specifics of media preparation. To confirm that the Act-GS-255B driver can produce increased life span, it was used to drive over-expression of the dominant p53 allele (p53-259H). Over-expression of p53-259H in adult flies using the ubiquitous Act-GS-255B driver produced increased median life span in females (+8%) but not males (-2.8%), and no life span increase when expressed in larvae (Table 3). These results are consistent with previous studies showing that expression of p53-259H in the adult nervous system with the Elav-GS driver can cause increased life span in females [14], and confirms that the Act-GS-255B driver can indeed produce increased life span when combined with an appropriate target gene.


A screen of apoptosis and senescence regulatory genes for life span effects when over-expressed in Drosophila.

Shen J, Curtis C, Tavaré S, Tower J - Aging (Albany NY) (2009)

Effect of Baculovirus p35 over-expression on survival of adult flies.                                        Baculovirus p35                                            transgenes inserted on the X chromosome, chromosome 2, and chromosome 3                                            were over-expressed during larval development or adult stage, as indicated.                                            The life span assays were performed at 25°C. Open circles represent the no-drug control ("-"). Solid squares                                            represent adults treated with drug ("A"). Grey triangles represent larvae                                            on drug ("L"). Survival curves are plotted as a function of adult age in                                            days. Median life span of each cohort is presented along with p                                            value for log rank tests  (in parentheses). (A, C, E, G) male                                            flies.  (B, D, F, H) female flies.  (A,B) Control flies                                            containing the driver and no target transgene. (C, D) Baculovirus                                                    p35 transgene on X chromosome.  (E, F) Baculovirus p35 transgene                                            on second chromosome. (G, H) Baculovirus p35 transgene on                                            third chromosome.
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Figure 4: Effect of Baculovirus p35 over-expression on survival of adult flies. Baculovirus p35 transgenes inserted on the X chromosome, chromosome 2, and chromosome 3 were over-expressed during larval development or adult stage, as indicated. The life span assays were performed at 25°C. Open circles represent the no-drug control ("-"). Solid squares represent adults treated with drug ("A"). Grey triangles represent larvae on drug ("L"). Survival curves are plotted as a function of adult age in days. Median life span of each cohort is presented along with p value for log rank tests (in parentheses). (A, C, E, G) male flies. (B, D, F, H) female flies. (A,B) Control flies containing the driver and no target transgene. (C, D) Baculovirus p35 transgene on X chromosome. (E, F) Baculovirus p35 transgene on second chromosome. (G, H) Baculovirus p35 transgene on third chromosome.
Mentions: To control for any possible effects of the Geneswitch system and the RU486 drug itself, life span was assayed in flies that were the progeny of Act-GS-2555B driver crossed to either Oregon-R (Or-R) wild-type strain or to the w1118 control strain, to produce progeny containing only the driver. In these control flies, treatment with drug produced small, but statistically significant reductions in life span in both male and female adults: treatment during adulthood reduced mean life span by -4% to -10%, while treatment in larval stages reduced adult life span by -8% to -16% (Figure 3A, B; Figure 4A, B; Tables 2, 3). There were no significant increases in life span in control flies treated with RU486 in any of the replicate experiments. These data indicate that in these experiments, when the Act-GS-255B driver is present, the RU486 can cause small but significant reductions in adult life span, and this effect must be taken into account when interpreting the effects of transgene over-expression. Other studies [22], including ones from our own laboratory using the Act-GS-255B driver [36], found no negative effects of RU486 on adult fly life span. We conclude that the small negative effects observed here result from differences in the lot of RU486 drug, and/or small differences in effective concentrations due to specifics of media preparation. To confirm that the Act-GS-255B driver can produce increased life span, it was used to drive over-expression of the dominant p53 allele (p53-259H). Over-expression of p53-259H in adult flies using the ubiquitous Act-GS-255B driver produced increased median life span in females (+8%) but not males (-2.8%), and no life span increase when expressed in larvae (Table 3). These results are consistent with previous studies showing that expression of p53-259H in the adult nervous system with the Elav-GS driver can cause increased life span in females [14], and confirms that the Act-GS-255B driver can indeed produce increased life span when combined with an appropriate target gene.

Bottom Line: Both wingless and Ras activated form transgenes were lethal when expressed in larvae, and reduced life span when expressed in adults, consistent with results from other model systems indicating that the wingless and Ras pathways can promote senescence.Over-expression of the caspase inhibitor baculovirus p35 during larval development reduced the mean life span of male and female adults, and also produced a subset of females with increased life span.These experiments suggest that baculovirus p35 and the wingless and Ras pathways can have sex-specific and developmental stage-specific effects on adult Drosophila life span, and these reagents should be useful for the further analysis of the role of these conserved pathways in aging.

View Article: PubMed Central - PubMed

Affiliation: Molecular and Computational Biology Program, Department of Biological Sciences, University of Southern California, Los Angeles, CA 90089, USA.

ABSTRACT
Conditional expression of transgenes in Drosophila was produced using the Geneswitch system, wherein feeding the drug RU486/Mifepristone activates the artificial transcription factor Geneswitch. Geneswitch was expressed using the Actin5C promoter and this was found to yield conditional, tissue-general expression of a target transgene (UAS-GFP) in both larvae and adult flies. Nervous system-specific (Elav-GS) and fat body-specific Geneswitch drivers were also characterized using UAS-GFP. Fourteen genes implicated in growth, apoptosis and senescence regulatory pathways were over-expressed in adult flies or during larval development, and assayed for effects on adult fly life span. Over-expression of a dominant p53 allele (p53-259H) in adult flies using the ubiquitous driver produced increased life span in females but not males, consistent with previous studies. Both wingless and Ras activated form transgenes were lethal when expressed in larvae, and reduced life span when expressed in adults, consistent with results from other model systems indicating that the wingless and Ras pathways can promote senescence. Over-expression of the caspase inhibitor baculovirus p35 during larval development reduced the mean life span of male and female adults, and also produced a subset of females with increased life span. These experiments suggest that baculovirus p35 and the wingless and Ras pathways can have sex-specific and developmental stage-specific effects on adult Drosophila life span, and these reagents should be useful for the further analysis of the role of these conserved pathways in aging.

Show MeSH