Disruption of the circadian clock, which directs rhythmic expression of numerous output genes, accelerates aging. To enquire how the circadian system protects aging organisms, here we compare circadian transcriptomes in heads of young and old Drosophila melanogaster. The core clock and most output genes remained robustly rhythmic in old flies, while others lost rhythmicity with age, resulting in constitutive over- or under-expression. Unexpectedly, we identify a subset of genes that adopted increased or de novo rhythmicity during aging, enriched for stress-response functions. These genes, termed late-life cyclers, were also rhythmically induced in young flies by constant exposure to exogenous oxidative stress, and this upregulation is CLOCK-dependent. We also identify age-onset rhythmicity in several putative primary piRNA transcripts overlapping antisense transposons. Our results suggest that, as organisms age, the circadian system shifts greater regulatory priority to the mitigation of accumulating cellular stress.

Upon exposure of Nitrosomonas europaea to chloroform (7 M, 1 h), transcripts for 175 of 2,460 genes were found at higher levels in treated cells than in untreated cells and transcripts for 501 genes were found at lower levels. With chloromethane (3.2 mM, 1 h), transcripts for 67 genes were at higher levels and transcripts for 148 genes were at lower levels. Transcripts for 37 genes were at higher levels following both treatments and included genes for heat shock proteins, -factors of the extracytoplasmic function subfamily, and toxinantitoxin loci. N. europaea has higher levels of transcripts for a variety of defense genes when exposed to chloroform or chloromethane.

The period (per) and timeless (tim) genes encode key components of the circadian oscillator in Drosophila melanogaster. The per gene is thought to encode three transcripts via differential splicing (types A, B, and C) that give rise to three proteins. Since the three per mRNA types were based on the analysis of cDNA clones, we tested whether these mRNA types were present in vivo by RNase protection assays and reverse transcriptasemediated PCR. The period (per) and timeless (tim) genes encode key components of the circadian oscillator in Drosophila melanogaster. The expression of these genes is required for circadian clock function, and an important aspect of their expression is circadian fluctuations in their mRNA and protein levels (17). These rhythms in per and tim gene products are controlled by a circadian feedback loop in which PER and TIM proteins control the expression of their own mRNAs (17,25,26). This feedback is mediated predominantly at the transcriptional level, though posttranscriptional regulation is also involved (5,16,29,31,32). The role of PER in this process is unknown, but its lack of a known DNA binding domain and inability to bind DNA indicate that it does not regulate transcription directly (17,25).Analysis of per cDNA clones uncovered three splice variants that encode three different PER isoforms (3). The most abundant of these transcripts, type A, defined both the structure of the per gene and the prototypical 1,218-amino-acid PER protein. Type B transcripts differ from type A by having two additional introns; one removes 288 nucleotides (nt) from exon 5 of type A transcripts, and the other excises 89 nt from the 3Ј untranslated region (3ЈUTR) of exon 8. After excising the intron from exon 5, type B transcripts produce a protein that is 96 amino acids shorter. The least abundant transcript (only one partial cDNA clone was isolated), type C, differs from type A by retaining introns 5, 6, and 7, thereby producing a transcript whose exon 5 spans exons 5 to 8 in type A transcripts. Due to the inclusion of these additional introns, the last 107 amino acids of the putative type C protein sequence are entirely different from the last 149 amino acids of type A protein sequence. All three per cDNAs are capable of rescuing behavioral rhythms in per 01 flies, though the type C construct may mediate behavioral rescue by generating both type A and type B transcripts (3, 4).Given the critical role that PER plays in controlling the circadian feedback loop in Drosophila, it is important to determine which isoforms contribute to the feedback loop mechanism and what impact this contribution may have on behavioral rhythms. Since the initial characterization of per mRNA splice variants was based on the structure and abundance of partial cDNA clones, we tested whether these per transcripts exist in vivo and function equally to rescue locomotor activity rhythms.Our studies failed to detect per splice variants that generate different PER isoforms. However, two per transcripts that differ by an alternatively sp...

The period (per) gene is 1 of the core elements of the circadian clock mechanism in animals from insects to mammals. In clock cells of Drosophila melanogaster, per mRNA and PER protein oscillate in daily cycles. Consistent with the molecular clock model, PER moves to cell nuclei and acts as a repressor of positive clock elements. Homologs of per are known in many insects; however, specific roles of per in generating output rhythms are not known for most species. The aim of this article was to determine whether per is functionally involved in the circadian rhythm of sperm release in the moth, Spodoptera littoralis. In this species, as in other moths, rhythmic release of sperm bundles from the testis into the upper vas deferens occurs only in the evening, and this rhythm continues in the isolated reproductive system. S. littoralis was used to investigate the expression of per mRNA and protein in the 2 types of cells involved in sperm release: the cyst cells surrounding sperm bundles in the testes, and the barrier cells separating testicular follicles from the vas deferens. In cyst cells, PER showed a nuclear rhythm in light/dark (LD) cycles but was constitutively cytoplasmic in constant darkness (DD). In barrier cells, nuclear cycling of PER was observed in both LD and DD. To determine the role of PER in rhythmic sperm release in moths, testes-sperm duct complexes were treated in vitro with double-stranded fragments of per mRNA (dsRNA). This treatment significantly lowered per mRNA and protein in cyst cells and barrier cells and caused a delay of sperm release. These data demonstrate that a molecular oscillator involving the period gene plays an essential role in the regulation of rhythmic sperm release in this species.

The authors examined patterns of spatial and temporal expression of Drosophila per gene homologue in the codling moth, Cydia pomonella. Since sperm release in moths is regulated in a circadian manner by an autonomous clock that is independent from the brain, the authors investigated per expression in male reproductive system along with its expression in moth heads. per mRNA is rhythmically expressed with the same phase and amplitude in both tissues under light-dark (LD) conditions. The levels of per mRNA are low during the day, start to increase before lights-off, reach the peak in dark, and decrease after lights-on. In constant darkness (DD), cycling of per mRNA continued in heads with severely blunted amplitude. No cycling of per mRNA was detected in testis in DD. In situ hybridization and immunocytochemistry revealed distinct spatial patterns of per expression in the moth reproductive system. There is no expression of per in cells forming the wall of testes or in sperm bundles. However, per mRNA and protein are rhythmically expressed in the epithelial cells forming the wall of the upper vas deferens (UVD) and in the cells of the terminal epithelium, which are involved in the circadian gating of sperm release. Increase in per mRNA in the UVD coincides with sperm accumulation in this part of the insect reproductive system.