Genetic studies of single gene mutations are revealing mechanisms and pathways that regulate longevity across distant species (1). One such mechanism is an alteration in histone deacetylase activity. Abolishing expression of the Rpd3 deacetylase (2) or increasing expression of the Sir2 deacetylase (1) increases life-span in yeast; Sir2 mediates increased nematode longevity as well (1). Caloric restriction is an intervention that increases life-span in mammals, insects, nematodes, and yeast (1, 3). Although the molecular pathways underlying the response to caloric restriction are yielding to genetic analysis in yeast (1), there is little information on how this response is regulated in metazoans. We investigated the relationship between histone deacetylases, caloric restriction, and longevity in Drosophila.Greatly reduced Rpd3 levels are lethal in Drosophila (4), but partial reduction of Rpd3 levels has not been evaluated for its effect on life-span. We found that males heterozygous for either a hypomorphic ( partial loss-of-function) or null mutation of rpd3 have life-span extension of 33% and 41 to 47%, respectively (Fig. 1A).Females heterozygous for the hypomorphic allele have a 52% increase in life-span, whereas females carrying the null mutation have only modest changes in life-span (maximum but not median life-spans are increased). The presence of large increases in life-span for males carrying both types of allele indicates that the effect is specific to the rpd3 locus. The different results for females may indicate a greater sensitivity to the predicted lower levels of Rpd3 in individuals carrying the null mutation compared with individuals carrying the hypomorphic allele.To further explore the parallels between life extension in yeast and Drosophila, we examined the effect of caloric restriction on normal-lived control and long-lived rpd3 mutants. Longevity is increased to approximately the same extent in control flies fed a low-calorie diet and rpd3 mutants fed a normal diet (Fig. 1B). In addition, caloric restriction of the rpd3 mutants shows no further extension of life-span (Fig. 1B). The lack of an additive increase in longevity is not due to a physiological cap for life-span extension, because rpd3 females that were kept as virgins did have a further extension of longevity [see (14) in supporting online material (SOM) (5)]. Furthermore, at least one other mutation in Drosophila, Indy, increases life-span to a greater extent (Ͼ90%) (6). It has previously been demonstrated that caloric restriction of flies leads to a moderate but significant down-regulation of Rpd3, analogous to the decreases obtained in heterozygotes carrying rpd3 mutations (7, 8).The data suggest that life-span extension by the rpd3 mutation is within a pathway related to caloric restriction.Given the evidence connecting histone deacetylases to life-span extension, we wanted to determine whether Drosophila longevity was generally responsive to changes in histone acetylation. Increased acetylation (9) was achieved by mutating an inde...

Actin, like many other proteins, is highly insoluble after expression in Escherichia coi. In order to understand the origin of insoluble aggregates, we asked whether morphological inclusions were always correlated with insolubility. The strain expressing actin was compared to one that expresses part of the myosin tail; the latter strain yields soluble protein after various cell lysis or disruption procedures.Morphological inclusions were observed in both strains, indicating there is no obligate relationship between solubility and inclusions. Studies presented here suggest that extreme insolubility results from coaggregation of the actin with bacterial outer membrane components upon bacterial lysis. The properties of the outer membrane have been exploited in the development of nondenaturing procedures that yield soluble actin. One procedure involves the disruption of coaggregates with sarkosyl detergent (N-laurylsarcosine); another prevents the formation of coaggregates by lysing in the presence of sarkosyl. These methods may be useful for other proteins that become insoluble after bacterial expression.

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The silent information regulator 2 (Sir2 or Sirtuin) family of proteins is highly conserved and has been implicated in the extension of longevity for several species. Mammalian Sirtuins have been shown to affect various aspects of physiology including metabolism, the stress response, cell survival, replicative senescence, inflammation, the circadian rhythm, neurodegeneration, and even cancer. Evidence in Drosophila implicates Sir2 in at least some of the beneficial effects of caloric restriction (CR). CR delays age-related pathology and extends life span in a wide variety of species. Here we will review the evidence linking Drosophila Sir2 (dSir2) to longevity regulation and the pathway associated with CR in Drosophila, as well as the effects of the Sir2 activator resveratrol and potential interactions between dSir2 and p53.

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