Jonathan D. Clayton

Circadian Cycling of the Mouse Liver Transcriptome, as Revealed by cDNA Microarray, Is Driven by the Suprachiasmatic Nucleus

BACKGROUND Genes encoding the circadian pacemaker in the hypothalamic suprachiasmatic nuclei (SCN) of mammals have recently been identified, but the molecular basis of circadian timing in peripheral tissue is not well understood. We used a custom-made cDNA microarray to identify mouse liver transcripts that show circadian cycles of abundance under constant conditions. RESULTS Using two independent tissue sampling and hybridization regimes, we show that approximately 9% of the 2122 genes studied show robust circadian cycling in the liver. These transcripts were categorized by their phase of abundance, defining clusters of day- and night-related genes, and also by the function of their products. Circadian regulation of genes was tissue specific, insofar as novel rhythmic liver genes were not necessarily rhythmic in the brain, even when expressed in the SCN. The rhythmic transcriptome in the periphery is, nevertheless, dependent on the SCN because surgical ablation of the SCN severely dampened or destroyed completely the cyclical expression of both canonical circadian genes and novel genes identified by microarray analysis. CONCLUSIONS Temporally complex, circadian programming of the transcriptome in a peripheral organ is imposed across a wide range of core cellular functions and is dependent on an interaction between intrinsic, tissue-specific factors and extrinsic regulation by the SCN central pacemaker.

Keeping time with the human genome

The cloning and characterization of ‘clock gene’ families has advanced our understanding of the molecular control of the mammalian circadian clock. We have analysed the human genome for additional relatives, and identified new candidate genes that may expand our knowledge of the molecular workings of the circadian clock. This knowledge could lead to the development of therapies for treating jet lag and sleep disorders, and add to our understanding of the genetic contribution of clock gene alterations to sleep and neuropsychiatric disorders. The human genome will also aid in the identification of output genes that ultimately control circadian behaviours.

Interaction of troponin-H and glutathione S-transferase-2 in the indirect flight muscles of Drosophila melanogaster

SummaryDrosophila indirect flight muscles (IFMs) contain a 35 kDa protein which cross-reacts with antibodies to the IFM specific protein troponin-H isoform 34 (TnH-34). Peptide fingerprinting and peptide sequencing showed that this 35 kDa protein is glutathione S-transferase-2 (GST-2). GST-2 is present in the asynchronous indirect flight muscles but not in the synchronous tergal depressor of the trochanter (jump muscle). Genetic dissection of the sarcomere showed that GST-2 is stably associated with the thin filaments but the presence of myosin is required to achieve the correct stoichiometry, suggesting that there is also an interaction with the thick filament. The two Drosophila TnHs (isoforms 33 and 34) are naturally occurring fusion proteins in which a proline-rich extension of ~250 amino acids replaces the 27 C-terminal residues of the muscle-specific tropomyosin II isoform. The proteolytic enzyme, Igase, cleaves the hydrophobic C-terminal sequence of TnH-34 at three sites and TnH-33 at one site. This results in the release of GST-2 from the myofibril. The amount of GST-2 stably bound to the myofibril is directly proportional to the total amount of undigested TnH. It is concluded that GST-2 in the thin filament is stabilized there by interaction with TnH. We speculate that the hydrophobic N-terminal region of GST-2 interacts with the hydrophobic C-terminal extension of TnH, and that both are close to a myosin cross-bridge.

Expression and function of the Drosophila ACT88F actin isoform is not restricted to the indirect flight muscles

Most higher eukaryotic genomes contain multiple actin genes, yet the sequence differences between isoforms are few. In Drosophila melanogaster it was previously established that one of the six actin genes, Act88F, is expressed only in the indirect flight muscles (IFMs). These muscles are highly specialised for oscillatory contractions to power flight. The implication was that this isoform had tissue-specific properties. In this paper we show using two reporter constructs expressing either β-galactosidase, Act88F-lacZ, or the green fluorescent protein, Act88F-GFP, that the Act88F promoter is active in a small number of other muscles, including leg (femoral) and uterine muscles. However, the levels of Act88F driven non-IFM expression are much less than in the IFMs. We have confirmed endogenous Act88F gene expression in these other muscles by in situ hybridisation studies. Using null and antimorphic mutants to show decreased walking ability and delayed/reduced oviposition we demonstrated that Act88F expression is functionally important in multiple muscle groups. Since the mutant effects are mild, this supports the expectation that other actin genes are also expressed in these muscles. The Act88F-GFP promoter–reporter also detects Act88F-driven expression in the bristle-forming cells in the pupal wings. The implications of these results for the functions and developmental expression of the Drosophila ACT88F isoform are discussed.

The Drosophila clock protein Timeless is a member of the Arm/HEAT family

This work was supported by HFSP and BBSRC grants to CPK, a Socrates studentship to NV and a MURST-British Council grant to CPK and RC.